Suzanne Peyrottes

Anne-Laure Villard

Anne-Marie Aubertin

Christian Périgaud
email: christian.perigaud@umontpellier.fr

An original pronucleotide strategy for the simultaneous delivery of two bioactive drugs

Keywords: mononucleotide, prodrug, antiviral, drug combination

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Introduction

During the last decade, the most significant advances in the treatment of viral infections have probably been associated with the development of 5'-mononucleotide prodrugs (pronucleotides), notably phosphoramidate pronucleotides [START_REF] De Clercq | Fifty Years in Search of Selective Antiviral Drugs[END_REF][START_REF] Yates | The evolution of antiviral nucleoside analogues: A review for chemists and non-chemists. Part II: Complex modifications to the nucleoside scaffold[END_REF] . Thus, Sofosbuvir [START_REF] Keating | Sofosbuvir: a review of its use in patients with chronic hepatitis C[END_REF] and Tenofovir alafenamide [START_REF] Scott | Tenofovir Alafenamide: A Review in Chronic Hepatitis B[END_REF] (Fig. 1) were approved by the United States Food and Drug Administration (FDA) on December 2013 and November 2016 for the treatment of chronic hepatitis C (HCV) and B (HBV) infections, respectively. More recently, the potential of Remdesivir (Fig. 1), a phosphoramidate pronucleotide with broad-spectrum activities against RNA viruses , has been proposed for the treatment of coronavirus disease-2019 [START_REF] Eastman | Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19[END_REF] . First disclosed by McGuigan et al. [START_REF] Peyrottes | Mononucleotide Prodrug Synthetic Strategies[END_REF] , such kind of pronucleotides (i.e. mononucleoside aryl phosphoramidate diesters) were developed to address not only bioavailability issues but also poor in vivo conversion of the parent nucleoside into its corresponding 5'-monophosphorylated form (5'-mononucleotide). Their structure typically consists in a 5'-mononucleotide analogue where the phosphate (or phosphonate) group is masked by appending an aryloxy group (usually a phenol) and a α-amino acid ester (Fig. 1). This type of pronucleotides is commonly named McGuigan ProTides. As illustrated in the Scheme 1 for the Sofosbuvir decomposition pathway [START_REF] Summers | Sofosbuvir, a novel nucleotide analogue inhibitor used for the treatment of hepatitis C virus[END_REF] , the phenyl phosphoramidate diester is hydrolyzed by a sequence involving enzymatic and chemical steps requiring either carboxyesterase or cathepsin A to cleave the terminal amino acid ester, then intramolecular displacement of the phenol substituent and the enzymatic cleavage of the amino acid moiety by a phosphoramidase or histidine triad nucleotide-binding protein 1 (HINT 1).

Scheme 1. Sofosbuvir decomposition pathway.

As part of our work in the field of pronucleotides, we have previously studied [START_REF] Egron | S-Acyl-2thioethyl Phosphoramidate Diester Derivatives as Mononucleotide Prodrugs[END_REF] the potential of series of phosphoramidate diesters incorporating a S-acyl-2-thioethyl (SATE) phosphate protecting group as an alternative to the aryl substituent, and aliphatic (or aromatic) amino residues (Fig. 2). In cell culture experiments, such pronucleotides lead to the selective and efficient intracellular release of the parent 5'-mononucleotide, through a mechanism involving successively an esterase and a phosphoramidase activation step. Various amino residues could be introduced without affecting the biological activity of the corresponding prodrugs indicating a less restrictive intracellular decomposition process than previously reported in the literature. One of the most promising SATE phosphoramidate diester derivative was IDX 184 (Fig. 2), a 2′-methylguanosine pronucleotide, which demonstrated to be effective in patients with chronic HCV infections in phase II clinical trials [START_REF] Sizun | Design, synthesis and antiviral evaluation of 2′-C-methyl branched guanosine pronucleotides: the discovery of IDX184, a potent liver-targeted HCV polymerase inhibitor[END_REF][START_REF] Dousson | Current and future use of nucleo(s)tide prodrugs in the treatment of hepatitis C virus infection[END_REF] . Unfortunately, severe cardiotoxicity complications observed during phase III trials of INX 189, a McGuigan ProTide sharing the same nucleoside analogue parent, led to the withdrawal of IDX 184 from further clinical developments [START_REF] Dousson | Current and future use of nucleo(s)tide prodrugs in the treatment of hepatitis C virus infection[END_REF] .

The image of the Trojan horse has often been used to illustrate strategies aiming to the selective mononucleotide-delivery based on enzymatic and/or chemically induced cascade mechanisms. To pursue the comparison with Greek mythology, we report herein like Orthrus, the two-headed dog who guarded Geryon's cattle, an unprecedented phosphoramidate pronucleotide series designed to deliver two potent and different biologically active metabolites into cells (Scheme 2). In such constructs, the amino function bound to the phosphorus atom is the one present on the nucleobase of the second nucleoside analogue. Their expected decomposition pathway could lead to the simultaneous release of a 5'-mononucleotide and of another nucleoside analogue. Thus, in addition to the benefit of the previously reported pronucleotide approach, developed to overcome the undesirable metabolism of a nucleoside analogue (Nu1 Scheme 2), the concomitant delivery of another (properly selected) bioactive nucleoside (Nu2) could increase the potency and the selectivity of the biological effect, as already observed in current combination antiretroviral therapy [START_REF] Cihlar | Nucleoside and nucleotide HIV reverse transcriptase inhibitors: 25 years after zidovudine[END_REF] . Thus, we report the synthesis and biological evaluation of two SATE dinucleoside phosphoramidate diester models (Fig. 3, compounds 1 and 2) incorporating a S-pivaloyl-2thioethyl (tBuSATE) phosphate protecting group and characterized by the presence of the 2',3'dideoxycytidine (ddC) linked to the phosphorus atom through its exocyclic amino group. In addition, pronucleotide 1 combines 3'-azido-2',3'-dideoxythymidine (AZT) while 2',3'dideoxyadenosine (ddA) is used for derivative 2.

Results and discussion

Chemistry

The pronucleotide 1 was first synthesized according to the hydrogenphosphonate approach (Scheme 3), previously reported for the preparation of various phosphoramidate diester derivatives of AZT [START_REF] Egron | S-Acyl-2thioethyl Phosphoramidate Diester Derivatives as Mononucleotide Prodrugs[END_REF] . Oxidative coupling of the H-phosphonate diester 3 [START_REF] Egron | Synthesis, Anti-HIV Activity, and Stability Studies of 5′-Phosphorofluoridate Derivatives of AZT[END_REF] with the protected nucleoside 4 [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF] , prepared beforehand according to a published procedure [START_REF] Hayakawa | Facile Synthesis of Oligodeoxyribonucleotides via the Phosphoramidite Method without Nucleoside Base Protection[END_REF] , afforded the desired phosphorylated intermediate 5 in low yield (9%). Amidative oxidation reactions generally lead to satisfactory yields. Nevertheless, in the case of weakly basic amines (such as arylamines) the addition of a tertiary amine to the reaction medium is required [START_REF] Atherton | Studies on phosphorylation. Part II. The reaction of dialkyl phosphites with polyhalogen compounds in presence of bases. A new method for the phosphorylation of amines[END_REF] . In our case, the optimized reaction is using an excess of nucleoside 4 (4 eq.) in presence of 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU, 3 eq.). Finally, acid treatment of 5 afforded the desired tBuSATE dinucleoside phosphoramidate 1 in 80% yield. The silylated nucleoside 6 [START_REF] Okabe | Synthesis of the dideoxynucleosides "ddC" and "CNT" from glutamic acid, ribonolactone, and pyrimidine bases[END_REF] was phosphitylated with phosphoramidite 7, previously prepared from S-pivaloyl-2-thioethyl N,N-bis(diisopropylamino)phosphine [START_REF] Egron | Synthesis, Anti-HIV Activity, and Stability Studies of 5′-Phosphorofluoridate Derivatives of AZT[END_REF] , upon tetrazole activation and in presence of N,N-diisopropylamine. The resulting phosphoramidite was oxidized in situ with tert-butyl hydroperoxide to give rise to the 4-N-phosphoramidate 8 in 38% yield. The key step in this multi-step procedure results in the introduction of the second nucleoside analogue. The dinucleoside phosphoramidate 9 was obtained following a one-pot two steps procedure.

Thus, the β-cyanoethyl group of 8 was removed by using DBU to obtain a phosphoroamidate monoester intermediate, which is directly condensed without purification with AZT in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl) and affording the phosphorylated intermediate 9 in 83% yield over two steps. Subsequent acidic treatment gave rise to the desired pronucleotide 1 in 80% yield.

Then, we extended this dinucleoside pronucleotide approach to the study of a phosphoramidate derivative in which a purine nucleoside analogue replaces . AZT and we selected 2',3'-dideoxyadenosine (ddA). Indeed, we previously demonstrated that applied to this anti-human immunodeficiency virus (anti-HIV) drug, a pronucleotide approach led to an enhanced in vitro antiviral efficiency [START_REF] Peyrottes | SATE pronucleotide approaches: an overview[END_REF] . The synthesis of the resulting pronucleotide 2 (Fig. 3) was carried out using a strategy based on phosphoramidite intermediates (Scheme 5). The latter could not be used for the preparation of pronucleotide 1 because of the reactivity of phosphoramidites in presence of the azide group of AZT [START_REF] Gololobov | Sixty years of staudinger reaction[END_REF] . Protected nucleoside 10, synthesized according to a previously described procedure [START_REF] Ti | Transient protection: efficient one-flask syntheses of protected deoxynucleosides[END_REF] , was reacted with S-pivaloyl-2-thioethyl N,N-bis(diisopropylamino)phosphine [START_REF] Egron | Synthesis, Anti-HIV Activity, and Stability Studies of 5′-Phosphorofluoridate Derivatives of AZT[END_REF] to afford phosphoramidite 11 in 78% yield. Coupling this intermediate with nucleoside 4 in presence of lH-tetrazole, followed by in situ oxidation with tert-butyl hydroperoxide, gave rise to the fully protected phosphoramidate 12 in 41% yield. Finally, a controlled acidic treatment (in order to avoid depurination) led to the desired pronucleotide 2 in 67% yield. In some cases, distinct diastereoisomeric signals were also noted on the proton-decoupled [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF] C NMR spectra, from 1 H NMR spectroscopy and analytical HPLC studies. Characteristic features of 1 H NMR spectra of the phosphorylated prodrugs were downfield shifts of the cytosine 5-H and 6-H protons that may be attributed to the electron-withdrawing effect of the 4-N-phosphorylation. The same effect has already been reported for 4-N-phosphorylated [START_REF] Wada | Synthesis and Properties of N-Phosphorylated Ribonucleosides[END_REF] and acylated 21 cytidine derivatives. No significant effect of N-substitution on the chemical shifts of the sugar moiety of ddC was observed.

Similarly, there were no significant effects of the N-phosphorylation on the chemical shifts of the sugar carbons of ddC. [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF] C NMR spectra of phosphoramidate derivatives showed an upfield effect for chemical shifts of the cytosine 2-C and 4-C carbons. Determination of P-C couplings is made difficult due to the complexity generated by the presence of diastereoisomers.

Antiviral activity

The inhibitory effects on the replication of HIV-1 of the tBuSATE dinucleoside phosphoramidate diesters 1 and 2 were evaluated, as diastereoisomeric mixtures, in three cell culture systems in comparison to AZT, ddC and ddA as well as an equimolar mixture of each constitutive nucleosides. The related cytotoxicity (CC50) was also determined in parallel in uninfected cells. These results are summarized in Table 1. With 50% effective concentration (EC50) about nanomolar concentration range, pronucleotide 1 appeared to be most potent than AZT, ddC, as well as an equimolar mixture of the two nucleoside analogues in human T4-lymphoblastoid cells, CEM-SS and MT-4. In addition, the antiviral effect observed was not correlated with an increased cytotoxicity. Thus, no cytotoxicity was observed in MT-4 cells at the highest concentration tested (up to 500 µM), leading to a selectivity index (SI) more than five times higher than the one of the mixture of the parent nucleosides (AZT and ddC), whereas in CEM cells, the SI of pronucleotide 1 is two hundred times higher than the same mixture.

Table 1

Anti-HIV activity (µM) a in three cell culture systems of pronucleotides 1 and 2 compared to the parent nucleosides as well as the equimolar mixture of each constitutive nucleoside analogues. ddA/ddC f 0.01 46 4600 1.2 >100 >83 a All data represent average values for at least three separate experiments. The variation of these results under standard operating procedures is below ± 10%. b EC50: effective concentration or concentration required to inhibit the replication of HIV-1 by 50%. c CC50: cytotoxic concentration or concentration required to reduce the viability of uninfected cells by 50%. d SI: selectivity index, ratio CC50/EC50. e Equimolar mixture of AZT and ddC. f Equimolar mixture of AZT and ddA.

Compound

CEM-SS MT-4 CEM/TK - EC50 b CC50 c SI d EC50 b CC50 c SI d EC50 b CC50 c SI d 1 0.
The CEM/TK -cell line, highly deficient in thymidine kinase, should be considered as a predictive model to investigate the efficiency of nucleotide prodrugs of AZT [START_REF] Périgaud | Anti-HIV phosphotriester pronucleotides: basis for the rational design of biolabile phosphate protecting groups[END_REF] . In contrast to AZT, the SATE dinucleoside phosphoramidate 1 exhibited significant anti-HIV effects in CEM/TK -cells, similar to that observed in infected CEM-SS cells. This result demonstrates the successful release of the corresponding 5′-mononucleotide (i.e. AZT 5'-monophosphate) inside cells. Using an already published method [START_REF] Lefebvre | Mononucleoside Phosphotriester Derivatives with S-Acyl-2thioethyl Bioreversible Phosphate-Protecting Groups: Intracellular Delivery of 3'-Azido-2',3'dideoxythymidine 5'-Monophosphate[END_REF][START_REF] Valette | Decomposition pathways and in vitro HIV inhibitory effects of isoddA pronucleotides: toward a rational approach for intracellular delivery of nucleoside 5'-monophosphates[END_REF] , the stability of pronucleotide 1 was studied in culture medium (RPMI 1640 containing 10% heat-inactivated fetal calf serum) and in total cell extracts from CEM-SS cells in order to mimic its behavior inside the cells (see supplementary data). Thus, pronucleotide 1 decomposed faster in cell extracts (t1/2 = 2.3 h) than in culture medium (t1/2 = 165 h). In both media, the formation of the corresponding phosphoramidate monoester was observed after a first step mediated by esterases (loss of the SATE group, Scheme 2). Then, this metabolite appeared to be stable in the culture medium and was very slowly hydrolyzed in cell extracts. These data are in agreement with the literature concerning the enzymatic stability of McGuigan ProTides and SATE phosphoramidate pronucleotides in these biological media used as models and may be associated with the partial denaturation of the enzymatic content during the preparation of the cell extracts [START_REF] Egron | S-Acyl-2thioethyl Phosphoramidate Diester Derivatives as Mononucleotide Prodrugs[END_REF][START_REF] Beltran | Rational design of a new series of pronucleotide[END_REF] .

We previously demonstrated the efficacy of the SATE pronucleotide approach applied to various anti-HIV nucleoside analogues that are hampered by the first phosphorylation step, through a dependence on kinase-mediated phosphorylation, or by a rate limiting-step in the anabolism pathway. Thus, the bis(SATE)phosphotriester derivatives of ddA emerged as very potent and selective inhibitors of HIV and simian immunodeficiency virus (SIV) replications in vitro in various human infected cells, stimulated and unstimulated primary cultured human cell lines such as peripheral blood mononuclear (PBM) cells or monocyte-derived macrophages [START_REF] Peyrottes | SATE pronucleotide approaches: an overview[END_REF] . The inhibitory effects of pronucleotide 2 on the replication of HIV-1 was evaluated in CEM-SS and MT-4 cells (Table 1). This derivative appeared to be most potent than its parent nucleoside ddA. As observed with analogue 1, the SI of pronucleotide 2 is higher than that of the equimolar mixture of each constitutive nucleosides (ddA and ddC).As suggested by our previous works, the putative phosphoramidase activity that could be involved in the bioconversion of the dinucleoside phosphoramidate monoester intermediate into the corresponding 5′-mononucleotide (Scheme 2) seems to exhibit a broad substrate specificity, hydrolyzing phosphoramidates bearing arylamino substituents as well as pyrimidine aglycones.

Conclusion

The synthesis of novel SATE dinucleoside phosphoramidate derivatives has been performed using different phosphorus chemistries. Such constructs are characterized by the combination of two different anti-HIV nucleoside analogues linked to the phosphorus atom respectively by the sugar residue (5'-hydroxyl function) and the exocyclic amino function presents on the pyrimidine nucleobase. Comparative anti-HIV evaluation in three culture cell lines indicates that these original constructs are able to deliver efficiently a 5'-mononucleotide and another nucleoside analogue within infected cells. On the basis of the literature data, the proposed decomposition pathway may successively involve an esterase (hydrolysis of SATE phosphate protecting group), followed by another bioactivation step carried out by a putative phosphoramidase activity. These promising results encourage us to pursue the study of this new series of pronucleotides in order to evaluate their pharmacological potential with regard to the current use of drug combinations.

Experimental section 4.1. General Information

Unless otherwise stated, 1 H NMR spectra were recorded at 300 MHz and [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF] C NMR spectra at 100 MHz with proton decoupling at 25 °C on a Bruker 300 Avance or DRX 400. Chemical shifts are given in  values referenced to the residual solvent peak (CDCl3 at 7.26 ppm and 77 ppm, DMSO-d6 at 2.49 ppm and 39.5 ppm) relative to TMS. Deuterium exchange, decoupling and COSY experiments were performed in order to confirm proton assignments. Coupling constants, J, are reported in Hertz. 2D 1 H- [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF] C heteronuclear COSY were recorded for the attribution of [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF] C signals. 31 P NMR spectra were recorded at ambient temperature at 121 MHz with proton decoupling. Chemical shifts are reported relative to external H3PO4. FAB mass spectra were recorded in the positive-ion or negative-ion mode on a JEOL JMS DX 300 using thioglycerol/glycerol (1:1, v/v, G-T) as matrix. HRMS using electrospray ionization-mass spectrometry (ESI-MS) were performed using a SSQ 7000 single quadrupole mass spectrometer (Finnigan, San Jose, California, USA) in the negative-ion mode with a spray voltage at -4.5 kV. The capillary temperature was maintained at 250 °C. Nitrogen served both as sheath gas (operating pressure of 80 psi) and as auxiliary gas with a flow rate of 15 units. Under these conditions, full scan data acquisition was performed from m/z 200 to 800 in centroïd mode and using a cycle time of 1.0 second. UV spectra were recorded on an Uvikon 931 (Kontron). Elemental analyses were carried out by the Service de Microanalyses du CNRS, Division de Vernaison (France). Analytical HPLC experiments were carried out on an Alliance 2690 system (Waters, Milford, Massachusetts, USA) equipped with 996-photodiode detector and a Millennium data workstation. A reverse-phase analytical column (Nucleosil, C18, 150  4.6 mm, 5 m) equipped with a prefilter, and a precolumn (Nucleosil, C18, 5 m) were used. Detection was monitored at 267 nm. Nucleotidic derivatives were eluted using a linear gradient of 0 to 80 % acetonitrile in 20 mM triethylammonium acetate buffer (pH 7) over 30 min at 1 mL/min flow rate. TLC was performed on precoated aluminum sheets of silica gel 60 F254 (Merck, Art. 9385), visualization of products being accomplished by UV absorbance followed by charring with 5% ethanolic sulphuric acid with heating for nucleotides. Flash chromatography was carried out using 63 -100 µm silica gel (Merck Art. N°115101) otherwise 40 -63 µm silica gel (Merck Art. N°109385) was used. Solvents were reagent grade or purified by distillation prior to use, and solids were dried over P2O5 under reduced pressure at rt. Moisture sensitive reactions were performed under argon atmosphere using oven-dried glassware. All aqueous (aq.) solutions were saturated with the specified salt unless otherwise indicated. AZT was from Brantford Chemicals Inc.; ddC was purchased from Carbosynth; S-pivaloyl-2thioethyl N,N-bis(diisopropylamino)phosphine and H-phosphonate diester 3 were obtained following a previously published procedure [START_REF] Egron | Synthesis, Anti-HIV Activity, and Stability Studies of 5′-Phosphorofluoridate Derivatives of AZT[END_REF] .

1-(2,3-Dideoxy-5-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl) cytosine (4)

According to a published procedure 14 , 2',3'-dideoxycytidine (ddC, 1.05 g, 4.97 mmol) was co-evaporated (3x) with pyridine and suspended in the same solvent (6 mL). To this suspension were added first triethylamine (0.69 mL, 4.97 mmol) and then dichloroacetic acid (0.41 mL, 4.97 mmol). After 15 minutes stirring at room temperature, 4,4'-dimethoxytrityl chloride (2.02 g, 5.96 mmol) was added portion wise. After 2 hours at room temperature, methanol (1 mL) was added. Then, the reaction mixture was diluted with ethyl acetate (50 mL), washed with saturated NaHCO3 (30 mL) and finally with water (30 mL). The resulting organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude was co-evaporated with toluene (3x) and purified by silica gel column chromatography (gradient methanol 0-5% in ethyl acetate/ 0.2% NEt3) to afford 4 as white foam (2.14 g, 84%). Rf (CH2Cl2/CH3OH/NEt3, 89/10/1, v/v/v): 0.48; NMR 1 H (CDCl3) data were in accordance with literature [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF] ; NMR [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF] C (CDCl3, 100 MHz)  165.5 (1s, C-4), 158.5 (1s, CH3OPh ipso), 155.9 (1s, C-2), 144.6 (1s, Ph ipso), 141.9 (1s, C-6), 135.7-135.6-130.1 (3s, CH3OPh), 128.2-127.9-126.9 (3s, Ph), 113.2 (1s, CH3OPh), 92.9 (1s, C-5), 87.

4.1.2. O-(3'-Azido-2',3'-dideoxythymidin-5'-yl)-O'-(S-pivaloyl-2-thioethyl)-4-N-[2',3'- dideoxy-5'-O-(4,4'-dimethoxytrityl)cytidine] phosphoramidate (5)
The protected nucleoside 4 (2.24 g, 4.36 mmol) was dissolved in pyridine (6 mL) and 1,8-diazabicyclo[5.4.0]undec-7-en (DBU, 0.49 mL, 3.27 mmol) was added. After 30 minutes stirring at room temperature, the mixture was added to a solution of H-phosphonate diester 3 (0.52 g, 1.09 mmol), previously co-evaporated with pyridine (3x), in carbon tetrachloride (10 mL). After 3 hours stirring at room temperature, the mixture was diluted with chloroform (50 mL) and washed with saturated NaHCO3 (25 mL) then with water (25 mL). The resulting organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. After co-evaporation with toluene, column chromatography on silica gel (gradient ethyl acetate 60-100% in hexane/ 0.1% NEt3, then methanol 0-2% in ethyl acetate/ 0.1% NEt3) gave a diastereoisomeric mixture of the desired phosphoramidate 5 (0.097 g, 9%). Rf (AcOEt/NEt3, 99/1, v/v): 0.37; NMR 1 H (CDCl3, 400 MHz)  10.4 (bs, 1H, Thy-NH), 8.6 (bs, 1H, P-NH), 7.98 (m, 1H, H-6ddC), 7.48-7.46 (2d, 1H, J = 1.0 Hz, H-6AZT), 7.34 (m, 2H, Ph), 7.21 (m, 7H, CH3OPh meta, Ph), 6.78 (d, 4H, J = 8.8 Hz, CH3OPh), 6.20 (m, 1H, H-1'AZT), 5.97 (dd, 1H, J = 6.3 Hz, J = 1.5 Hz, H-1'ddC), 5.50-5.48 (2d, 1H, J = 3.3 Hz, H-5ddC), 4.37 (m, 1H, H-3'AZT), 4.16 (m, 3H, H-5'AZT, H-5''AZT, H-4'AZT), 3.98 (m, 3H, H-4'ddC, OCH2CH2), 3.73 (s, 6H, CH3OPh), 3.47 (dd, 1H, J = 10.8 Hz, J = 2.4 Hz, H-5'ddC), 3.26 (m, 1H, H-5''ddC), 3.06-3.05 (2t, 2H, J = 6.8 Hz, OCH2CH2S), 2.32 (m, 3H, H-2'AZT, H-2''AZT, H-2'ddC ), 1.98 (m, 3H, H-2''ddC, H-3'ddC, H-3''ddC), 1.87-1.85 (2d, 3H, J = 0.7 Hz, Thy-CH3), 1.15-1.14 (2s, 9H, (CH3)3CSATE); NMR 31 P (CDCl3, 81 MHz)  8.07-7.90; MS FAB>0 (GT) m/z 987 (M+H) + , 961 (M-N2+3H) + , 738 (M-AZT+2H) + . FAB<0 (GT) m/z 985 (M-H) -, 959 (M-N2+H) -, 736 (M-AZT) -.

1-(2,3-Dideoxy-5-O-tert-butyldimethylsilyl-β-D-ribofuranosyl) cytosine (6)

To a suspension of 2',3'-dideoxycytidine (ddC, 1.50 g, 7.10 mmol) in pyridine (30 mL) was added tert-butyldimethylsilyl chloride (1.61 g, 2.25 mmol). After stirring overnight at room temperature, the mixture was diluted in dichloromethane (70 mL) and washed with water. The resulting organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The purification of the crude was performed by column chromatography on silica gel (gradient methanol 3-10% in dichloromethane) affording the desired nucleoside 6 as a white powder (2.22 g, 96%). Rf (CH2Cl2/CH3OH, 9/1, v/v): 0.30; NMR 1 H (CDCl3) data were in accordance with literature [START_REF] Okabe | Synthesis of the dideoxynucleosides "ddC" and "CNT" from glutamic acid, ribonolactone, and pyrimidine bases[END_REF] ; NMR 1 H (DMSO-d6, 400 MHz)  7.88 (d, 1H, J = 7.4 Hz, H-6), 7.10 (bl, 2H, J = 14.1 Hz, Cyt-NH2), 5.92 (m, 1H, H-1'), 5.64 (d, 1H, J = 7.4 Hz, H-5), 4.04 (m, 1H, H-4'), 3.91 (dd, 1H, J = 11.5 Hz, J = 2.8 Hz, H-5'), 3.70 (dd, 1H, J = 11.5 Hz, J = 3.1 Hz, H-5''), 2.27 (m, 1H, H-2'), 1.81 (m, 3H, H-2'', H-3', H-3''), 0.88 (s, 9H, (CH3)3CSi), 0.07-0.06 (2s, 6H, (CH3)2Si); NMR [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF]

O-(2-Cyanoethyl)-O'-(S-pivaloyl-2-thioethyl)-N,N-diisopropylaminophosphoramidite (7)

To a solution of 2-hydroxypropionitrile (0.38 µL, 5.61 mmol) in acetonitrile (55 mL) were added S-pivaloyl-2-thioethyl N,N-bis(diisopropylamino)phosphine (3.30 g, 8.41 mmol), N,N-diisopropylamine (1.57 mL, 11.22 mmol) and 1H-tetrazole (0.714 g, 11.22 mmol). The mixture was stirred at room temperature for 2 hours and then diluted in ethyl acetate (200 mL). This solution was washed with satured aq. NaHCO3. The resulting organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. After co-evaporation with cyclohexane/ 1% NEt3, silica gel column flash-chromatography (gradient ethyl acetate 0-10% in cyclohexane/ 1% NEt3) gave the desired phosphoramidite 7 as colorless oil (1.85 g, 84%). Rf (cyclohexane/AcOEt/NEt3, 80/19/1, v/v/v): 0.58; NMR 1 H (DMSO-d6, 400 MHz)  3.66 (m, 6H, OCH2CH2S, OCH2CH2CN, CH(CH3)2), 3.04 (t, 2H, J = 6.3 Hz, OCH2CH2S), 1.17 (1s, 9H, C(CH3)3), 1.13-1.11 (2d, 12H, J = 6.8 Hz, CH(CH3)2); NMR [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF]

O-(2-Cyanoethyl)-O'-(S-pivaloyl-2-thioethyl)-4-N-[2',3'-dideoxy-5'-O-tertbutyldimethylsilyl] cytidine phosphoramidate (8)

The silylated nucleoside 6 was co-evaporated with pyridine and acetonitrile (3x), and then solubilized in dichloromethane (32 mL). To this solution, kept under argon atmosphere, were added phosphoramidite 7 (1.771 g, 4.89 mmol) in dichloromethane (5 mL) and then 1Htetrazole (0.571 g, 8.15 mmol). After 2 hours stirring, the mixture was cooled to -40°C and a solution of tert-butyl hydroperoxide in toluene (3 M, 5.43 mL) was added. The reaction mixture was stirred for 1 hour and allowed to warm to 0°C. To reduce the excess of oxidant, sodium hydrogenosulphite (10%) aqueous solution (3 mL) was added. This mixture was diluted with dichloromethane (70 mL) and washed with the previous reducing solution (30 mL) and then water (50 mL). After decantation, the organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The purification of the crude was performed by column chromatography on silica gel (gradient methanol 0-2.5% in dichloromethane/ 0.2% acetic acid) to afford a diastereoisomeric mixture of the desired phosphoramidate 8 as colorless oil (0.747 g, 38%). Rf (CH2Cl2/CH3OH, 95/5, v/v): 0.33; NMR [START_REF] De Clercq | Fifty Years in Search of Selective Antiviral Drugs[END_REF]

'-O-tert-butyldimethylsilyl)cytidine phosphoramidate (9)

To a solution of phosphoramidate 8 (0.184 g, 0.305 mmol) in tetrahydrofuran (3 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-en (DBU, 0.073 mL, 0.488 mmol). After stirring overnight at room temperature, the mixture was concentrated under reduced pressure, and coevaporated with pyridine (3x). After addition of 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl, 2.713 g, 8.96 mmol), the resulting mixture was co-evaporated with pyridine (2x), then solubilized in the same solvent (5 mL). 3'-Azido-2',3'-dideoxythymidine (AZT, 0.40 g, 1.49 mmol) was added and the resulting solution was stirred under argon atmosphere at room temperature for 24 hours. The reaction was quenched by addition of aqueous NaHCO3 (5%; 3 mL). After 15 minutes stirring, the mixture was diluted with chloroform (50 mL) and washed with aqueous NaHCO3 (5%; 25mL), then water (25 mL). The organic layer was dried, filtered and concentrated under reduced pressure. The crude was co-evaporated with toluene and purified by column chromatography on silica gel (gradient methanol 0-3% in dichloromethane) to afford a diastereoisomeric mixture of the dinucleoside phosphoramidate diester 9 (0.66 g, 83%) as colorless oil. Rf (CH2Cl2/CH3OH, 95/5, v/v) : 0.26; NMR 1 H (CDCl3, 400 MHz)  10.6 (bs, 1H, Thy-NH), 8.6 (bs, 1H, P-NH), 8.18 (m, 1H, H-6ddC), 7.56-7.55 (2d, 1H, J = 0.9 Hz, H-6AZT), 6.27 (q, 1H, H-1'AZT), 6.03 (m, 1H, H-1'ddC), 5.83 (d, 1H, J = 7.9 Hz, H-5ddC), 4.43 (m, 1H, H-3'AZT), 4.26 (m, 3H, H-4'AZT, H-5'AZT, H-5''AZT), 4.17 (m, 1H, H-4'ddC), 4.09 (m, 3H, H-5'ddC, OCH2CH2S), 3.71 (m, 1H, H-5''ddC), 3.12 (t, 2H, J = 6.8 Hz, OCH2CH2S), 2.44 (m, 1H, H-2'ddC ), 2.34 (m, 2H, H-2'AZT, H-2''AZT), 1.98 (m, 6H, H-2''ddC, H-3'ddC, H-3''ddC, Thy-CH3), 1.21-1.20 (2s, 9H, (CH3)3CSATE), 0.92 (s, 9H, (CH3)3CSi), 0.10--0.10 (2s, 6H, (CH3)2Si); NMR

4.1.7. O-(3'-Azido-2',3'-dideoxythymidin-5'-yl)-O'-(S-pivaloyl-2-thioethyl)-4-N-(2',3'- dideoxycytidine) phosphoramidate (1)
From the phosphoramidate 5. The tritylated phosphoramidate 5 (100 mg, 0.10 mmol) was treated by a solution of trichloroacetic acid (3%) in dichloromethane (2 mL). After 15 minutes stirring at room temperature, the mixture was neutralized by addition of triethylammonium carbonate buffer (TEAB, 1M, pH=7), then diluted in dichloromethane (50 mL). The organic layer was washed successively with aqueous NaHCO3 (10%) and water, then filtered and finally concentrated under reduced pressure. Silica gel column chromatography of the residue (gradient 0-5% methanol in dichloromethane) gave rise to a diastereoisomeric mixture of the expected tBuSATE dinucleoside phosphoramidate diester 1 as colorless oil (0.056 g, 80%).

From the phosphoramidate 9. The silylated phosphoramidate 9 (339 mg, 0.50 mmol) was treated by a solution of hydrochloric acid (1%) in ethanol 95 (12.5 mL). After 20 minutes stirring at room temperature, the mixture was neutralized, at 0°C, by addition of TEAB (7 mL, 1M, pH=8.5), then diluted with dichloromethane (70 mL) and washed with water (30 mL). The resulting organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. Purification by column chromatography on silica gel (gradient methanol 2-5% in dichloromethane) gave rise to a diastereoisomeric mixture of the expected tBuSATE dinucleoside phosphoramidate diester 1 as colorless oil (0.274 g, 80%). Rf (CH2Cl2/CH3OH, 9/1, v/v): 0.26; NMR 1 H (CDCl3, 400 MHz)  10.2 (bs, 1H, Thy-NH), 8.8 (bs, 1H, P-NH), 7.95 (m, 1H, H-6ddC), 7.51-7.49 (2d, 1H, J = 1.2/1.1 Hz, H-6AZT), 6.24-6.20 (2t, 1H, J = 6.5/6.4 Hz, H-1'AZT), 6.00 (m, 2H, H-1'ddC, H-5ddC), 4.45 (m, 1H, H-3'AZT), 4.31 (m, 3H, H-5'AZT, H-5''AZT, H-4'ddC), 4.06 (m, 4H, H-4'AZT, H-5'ddC, OCH2CH2), 3.72 (m, 1H, H-5''ddC), 3.13-3.12 (2t, 2H, J = 6.9/6.7 Hz, OCH2CH2), 2.40 (m, 3H, H-2'AZT, H-2''AZT, H-2'ddC ), 2.13 (m, 1H, H-2''ddC), 1.97 (m, 5H, H-3'ddC, H-3''ddC, Thy-CH3), 1.22-1.21 (2s, 9H, (CH3)3CSATE); NMR (10) To a solution of 2',3'-dideoxyadenosine (ddA, 1.25 g, 5.31 mmol) in pyridine (27 mL) was added at 0°C trimethylsilyl chloride (1.35 mL, 10.62 mmol). The reaction mixture was stirred for 1 hour at room temperature before the addition of 4,4'-dimethoxytrityl chloride (2.16 g, 6.37 mmol). The mixture was stirred overnight under argon atmosphere and then NH4OH (28%, 5 mL) was added. After 20 minutes stirring, the mixture was diluted in dichloromethane (100 mL) and washed with water (50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The crude was co-evaporated with toluene and purified by column chromatography on silica gel (gradient methanol 0-2% in dichloromethane/ 0.2% NEt3) to afford the protected nucleoside 10 as a white powder (2.74 g, 96%). Rf (CH2Cl2/CH3OH/NEt3, 97/2/1, v/v/v): 0.27; NMR 1 H (CDCl3, 400 MHz)  7.92 (s, 1H, H-2), 7.70 (s, 1H, H-8), 7.17 To a solution of tritylated nucleoside 11 (2.40 g, 4.46 mmol) in dichloromethane (35 mL) and acetonitrile (9 mL) were added, under argon atmosphere, S-pivaloyl-2-thioethyl N,Nbis(diisopropylamino)phosphine (2.63 g, 6.69 mmol), N,N-diisopropylamine (1.25 mL, 8.92 mmol) and 1H-tetrazole (0.626 g, 8.92 mmol). After 1 hour stirring at room temperature, aqueous NaHCO3 (10%, 5 mL) was added and the resulting mixture was diluted with ethyl acetate (100 mL) and washed with the same aqueous solution (30 mL). The organic layer was dried over Na2SO4, filtered, concentrated under reduced pressure and co-evaporated with cyclohexane/ 1% NEt3. Purification of the crude was performed by silica gel column flash-chromatography (gradient ethyl acetate 10-25% in cyclohexane/ 1% NEt3) and afforded a diastereoisomeric mixture of the desired phosphoramidite 11 (2.88 A mixture containing tritylated nucleoside 4 (0.77 g, 1.50 mmol) and phosphoramidite 11 (1.87 g, 2.25 mmol) was co-evaporated with anhydrous toluene (3x) and dissolved in dichloromethane (15 mL). To the resulting solution was added 1H-tetrazole (0.263 g, 3.75 mmol). After 1h30 stirring at room temperature under argon atmosphere, the reaction mixture was cooled at -40°C and a solution of tert-butyl hydroperoxide in toluene (3 M, 2.50 mL, 7.50 mmol) was added dropwise. The reaction mixture was stirred for 1 hour at room temperature, and then to reduce the excess of oxidant sodium hydrogen sulfite (10%) aqueous solution (5 mL) was added at 0°C. The mixture was diluted in dichloromethane (100 mL), washed with the previous aqueous solution (50 mL) and water (50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. Column chromatography of the crude on silica gel (gradient acetone in ethyl acetate/ 0.2% NEt3) gave a diastereoisomeric mixture of the titled phosphoramidate 12 (0.773 g, 41%) obtained as white foam. Rf (CH2Cl2/CH3OH/NEt3, 97/3/0. Phosphoramidate 12 (0.754 g, 0.60 mmol) was dissolved in an acid mixture (pH 2.5) containing water, dioxane and acetic acid (120 mL, 25/25/17, v/v/v). After 24 hours stirring at room temperature, the reaction mixture was freeze-dried and purification of the crude was performed by column chromatography, first on silica gel (gradient methanol 0-10% in dichloromethane) then on reverse phase (gradient methanol 0-60% in water). After freezedrying, a diastereoisomeric mixture of the desired tBuSATE dinucleoside phosphoramidate diester 2 was obtained as a white powder (255 mg, 67%). Rf (CH2Cl2/CH3OH, 85/15, v/v) : 0.5; NMR 1 H (DMSO-d6, 400 MHz)  11.2 (bs, 1H, P-NH), 8.27-8.26 (2s, 1H, H-8ddA), 8.12 (1s, 1H, H-2ddA), 8.02 (1d, 1H, J = 7.0 Hz, H-6ddC), 7.21 (s, 2H, NH2,ddA), 6.23 (m, 1H, H-1'ddA), 6.20 (bs, 1H, H-5ddC), 5.90 (dd, 1H, J = 6.7 Hz, J = 3.0 Hz, H-1'ddC), 5.00 (bs, 1H, OH), 4.27 (m, 1H, H-4'ddA), 4.07 (m, 1H, H-4'ddC ), 4.00 (m, 2H, H-5'ddA, H-5''ddA), 3.88 (m, 2H, OCH2CH2S), 3.68 (m, 1H, H-5'ddC), 3.53 (m, 1H, H-5''ddC), 3.01 (m, 2H, OCH2CH2S), 2.44 (m, 2H, H-2'ddA, H-2''ddA), 2.28 (m, 1H, H-2'ddC), 2.12 (m, 2H, H-3'ddA, H-3''ddA), 1.95 (m, 1H, H-2''ddC), 1.81 (m, 2H, H-3'ddC, H-3''ddC), 1.13-1.12 (2s, 9H, C(CH3)3); NMR [START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF]

Biological methods

The origin of the viruses and the techniques used for measuring inhibition of virus multiplication were as previously described [START_REF] Lefebvre | Mononucleoside Phosphotriester Derivatives with S-Acyl-2thioethyl Bioreversible Phosphate-Protecting Groups: Intracellular Delivery of 3'-Azido-2',3'dideoxythymidine 5'-Monophosphate[END_REF] .

2 Fig. 1 .

 21 Fig. 1. Chemical structures of Sofosbuvir, Tenofovir alafenamide and Remdesivir.

Fig. 2 .

 2 Fig. 2. Structures of SATE phosphoramidates diesters and IDX 184.

2 Scheme 2 .

 22 Scheme 2. Expected decomposition pathway of the studied pronucleotides.

Fig. 3 .

 3 Fig. 3. Structures of the tBuSATE dinucleoside phosphoramidate diesters investigated in this study.

1 b 3 .d 4 .

 134 Scheme 3. Synthesis of the pronucleotide 1 following a hydrogenphosphonate strategy. Reagents and conditions: (a) CCl4, 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU), pyridine, rt, 3h, 9%; (b) 3% CCl3CO2H in CH2Cl2, 15min, 80%.

Scheme 5 .

 5 Scheme 5. Synthesis of the pronucleotide 2 using a phosphoramidite strategy. Reagents and conditions: (a) S-pivaloyl-2-thioethyl N,N-bis(diisopropylamino)phosphine, 1H-tetrazole, HN(iPr)2, CH2Cl2/CH3CN, rt, 1h, 78%; (b) 4, 1H-tetrazole, CH2Cl2/CH3CN, rt, 1h30, then tBuOOH (3M in toluene), -40°C, 41%; (c) AcOH/H2O/dioxane (pH 2.5), rt, 24h, 67%.

  2 (1s, C-1'), 86.5 (1s, C[(CH3OPh)2Ph]), 81.2 (1s, C-4'), 63.7 (1s, C-5'), 55.2 (1s, CH3OPh), 33.6 (1s, C-2'), 24.9 (1s, C-3'); MS FAB>0 (GT) m/z 514 (M+H) + , 112 (BH2) + ; MS FAB<0 (GT) m/z 512 (M-H) -, 110 (B) -; UV (ethanol 95) λmax 275 nm ( 11 000), 233 ( 25 000), λmin 258 nm ( 8 300); HPLC tR 29.1 min; Anal. Calculated for C30H31N3O5 (MW: 513.58): C, 70.16; H, 6.08; N, 8.18. Found: C, 69.90; H, 6.10; N, 7.85.

  C (CDCl3, 100 MHz)  165.6 (1s, C-4), 156.0 (1s, C-2), 141.8 (1s, C-6), 93.0 (1s, C-5), 87.0 (1s, C-1'), 82.1 (1s, C-4'), 63.5 (1s, C-5'), 33.7 (1s, C-2'), 25.9 (1s, (CH3)3CSi), 24.0 (1s, C-3'), 18.4 (1s, (CH3)3CSi), -5.4--5.5 (2s, (CH3)2Si); (DMSO-d6, 100 MHz)  165.6 (1s, C-2), 155.0 (1s, C-4), 140.6 (1s, C-6), 93.0 (1s, C-5), 85.6 (1s, C-1'), 81.0 (1s, C-4'), 63.5 (1s, C-5'), 32.7 (1s, C-2'), 25.8 (1s, (CH3)3CSi), 24.2 (1s, C-3'), 18.0 (1s, (CH3)3CSi), -5.5 -5.6 (2s, (CH3)2Si); MS FAB>0 (GT) m/z 651 (2M+H) + , 326 (M+H) + , 112 (BH2) + ; FAB<0 (GT) m/z 324 (M-H) -, 110 (B) -; HRMS calculated for C15H28N3O3Si (M+H) + : 326.1900. Found: 326.1900; UV (ethanol 95) λmax 275 nm ( 10 700), λmin 253 nm ( 8 000); HPLC tR 26.2 min.

  C (DMSO-d6, 100 MHz)  205.4 (1s, CO), 118.9 (1s, CN), 61.6 (1d, JP-C = 17.4 Hz, OCH2CH2S), 58.3 (1d, JP-C = 18.2 Hz, OCH2CH2CN), 45.9 (1s, C(CH3)3), 42.5 (1d, JP-C = 12.3 Hz, CH(CH3)2), 30.3 (1d, JP-C = 7.1 Hz, OCH2CH2S), 26.9 (1s, C(CH3)3), 24.3 (1d, JP-C = 7.3 Hz, CH(CH3)2), 19.8 (1d, JP-C = 6.8 Hz, OCH2CH2CN); NMR 31 P (DMSO-d6, 81 MHz)  148.53; MS FAB>0 (GT) m/z 379 (M+O+H) + , 363 (M+H) + ; Anal. Calculated for C16H31N2O3PS (MW: 362.47): C, 53.02; H, 8.62; N, 7.73; S: 8.85. Found: C, 52.77; H, 8.52; N, 7.61; S: 8.85.

  H (CDCl3, 400 MHz)  8.14 (d, 1H, J = 7.4 Hz, H-6), 6.00 (d, 1H, J = 5.4 Hz, H-1'), 5.87 (m, 1H, H-5), 4.19 (m, 3H, OCH2CH2CN, H-4'), 4.06 (m, 3H, OCH2CH2S, H-5'), 3.70 (dd, 1H, J = 11.5 Hz, J = 1.5 Hz, H-5''), 3.12 (t, 2H, OCH2CH2S), 2.75 (m, 2H, OCH2CH2CN), 2.39 (m, 1H, H-2'), 2.05 (m, 1H, H-2''), 1.91 (m, 2H, H-3', H-3''), 1.20 (s, 9H, (CH3)3CSATE), 0.90 (s, 9H, (CH3)3CSi), 0.11-0.08 (2s, 6H, (CH3)2Si); NMR 13 C (CDCl3, 100 MHz)  205.7 (1s, COSATE), 160.0 (1s, C-4), 148.2 (1s, C-2), 140.9 (1s, C-6), 116.5 (1d, JP-C = 1.7 Hz, CN), 101.3 (1d, JP-C = 18.9 Hz, C-5), 86.6 (1s, C-1'), 82.3 (1s, C-4'), 65.3 (1d, JP-C = 5.6 Hz, OCH2CH2S), 63.3 (1s, C-5'), 60.9 (m, OCH2CH2CN), 46.4 (1s, (CH3)3CSATE), 33.6 (1s, C-2'), 28.6 (1d, JP-C = 7.5 Hz, OCH2CH2S), 27.2-27.1 (2s, (CH3)3CSATE), 25.8-25.7 (2s, (CH3)3CSi), 23.9 (1s, C-3'), 19.6 (1d, JP-C = 6.9 Hz, OCH2CH2CN), 18.4 (1s, (CH3)3CSi), -5.4--5.5 (2s, (CH3)2Si); NMR 31 P (CDCl3, 81 MHz)  7.22-7.18; MS FAB>0 (GT) m/z 603 (M+H) + , 112 (BH2) + ; FAB<0 (GT) m/z 1203 (2M-H) -, 601 (M-H) -, 110 (B) -; UV (ethanol 95) λmax 284 nm ( 7 250), λmin 254 nm ( 2 800); HPLC tR 31.4 min; Anal. Calculated for C25H43N4O7PSSi (MW: 602.76): C, 49.82; H, 7.19; N, 9.30. Found: C, 50.04; H, 7.34; N, 9.00. 4.1.6. O-(3'-Azido-2',3'-dideoxythymidin-5'-yl)-O'-(S-pivaloyl-2-thioethyl)-4-N-(2',3'dideoxy-5

13 C

 13 (CDCl3, 100 MHz)  205.8-205.7 (2s, COSATE), 163.5 (1s, C-4AZT), 160.3 (1d, JP-C = 4.7 Hz, C-4ddC), 150.2-150.1 (2s, C-2AZT), 148.1 (1s, C-2ddC), 141.1 (1s, C-6ddC), 135.4 (m, C-6AZT), 111.6-111.4 (2s, C-5AZT), 101.5 (1d, J = 23.2 Hz, C-5ddC), 86.6 (1s, C-1'ddC), 84.6-84.5 (2s, C-1'AZT), 82.6-82.5 (2d, JP-C = 7.9/7.8 Hz, C-4'AZT), 82.4 (1s, C-4'ddC), 65.3 (m, C-5'AZT, OCH2CH2S), 63.3 (1s, C-5'ddC), 60.3-60.2 (2s, C-3'AZT), 46.5 (1s, (CH3)3CSATE), 37.7-37.6 (2s, C-2'AZT), 33.7 (1s, C-2'ddC), 28.6 (1d, JP-C = 7.5 Hz, OCH2CH2S), 27.3 (1s, (CH3)3CSATE), 25.9 (1s, (CH3)3CSi), 23.9 (1s, C-3'ddC), 18.4 (1s, (CH3)3CSi), 12.5-12.4 (2s, Thy-CH3), -5.5--5.6 (2s, (CH3)2Si); NMR 31 P (CDCl3, 81 MHz)  8.22-8.01; MS FAB>0 (GT) m/z 799 (M+H) + , 773 (M-N2+3H) + , . FAB<0 (GT) m/z 797 (M-H) -, 771 (M-N2+H)-; HRMS calculated for C32H52N8O10PSSi (M+H) + : 799.3034. Found: 799.3173; UV (ethanol 95) λmax 277 nm ( 20 700), λmin 245 nm ( 11 150); HPLC tR 32.6 min; Anal. Calculated for C32H51N8O10PSSi (MW: 798.92): C, 48.11; H, 6.43; N, 14.03. Found: C, 48.27; H, 6.64; N, 13.91.

13 C

 13 (CDCl3, 100 MHz)  205.9-205.8 (2s, COSATE), 164.1 (1s, C-4AZT), 160.5 (m, C-4ddC), 150.4-150.3 (2s, C-2AZT), 149.0 (m, C-2ddC), 141.2-141.1 (2s, C-6ddC), 136.0 (m, C-6AZT), 111.1-111.0 (2s, C-5AZT), 100.6 (m, C-5ddC), 86.9 (1s, C-1'ddC), 85.0-84.7 (2s, C-1'AZT), 82.6 (m, C-4'AZT), 82.3 (1s, C-4'ddC), 65.3 (m, C-5'AZT, OCH2CH2S), 62.6 (1s, C-5'ddC), 60.2 (1s, C-3'AZT), 46.4 (1s, (CH3)3CSATE), 37.6-37.3 (2s, C-2'AZT), 33.1 (1s, C-2'ddC), 28.5 (1d, JP-C = 7.2 Hz, OCH2CH2S), 27.2 (1s, (CH3)3CSATE), 24.4 (1s, C-3'ddC), 12.4 (1s, Thy-CH3); NMR 31 P (CDCl3, 81 MHz)  7.28-6.97; MS FAB>0 (GT) m/z 685 (M+H) + , 659 (M-N2+3H) + , 112 (BH2ddC) + ; FAB<0 (GT) m/z 1365 (2M-H) -, 683 (M-H) -, 125 (BAZT) -; HRMS calculated for C26H38N8O10PS (M+H) + : 685.2169. Found: 685.2198; UV (ethanol 95) λmax 276 nm ( 21 200), 212 ( 24 950), λmin 246 nm ( 10 900); HPLC tR 23.2 min; Anal. Calculated for C26H37N8O10PS (MW: 684.66): C, 45.61; H, 5.45; N, 16.37. Found: C, 45.34; H, 5.67; N, 16.16. 4.1.8. 1-(2',3'-Dideoxy-β-D-ribofuranosyl)-6-N-(4,4'-dimethoxytrityl) adenine

  (m, 9H, CH3OPh, Ph), 6.88 (s, 1H, NH),6.69 (d, 4H, J = 8.9 Hz, CH3OPh), 5.96 (dd, 1H, J = 7.6 Hz, J = 6.2 Hz, H-1'), 5.89 (d, 1H, J = 9.4 Hz, OH), 4.24 (m, 1H, H-4'), 3.89 (d, 1H, J = 12.6 Hz, H-5'), 3.68 (s, 6H, CH3OPh), 3.49 (m, 1H, H-5''), 2.69 (m, 1H, H-2'), 2.33 (m, 1H, H-3'), 2.23 (m, 2H, H-2''), 2.10 (m, 1H, H-3''); NMR 13 C (CDCl3, 100 MHz)  158.3 (1s, CH3OPh ipso), 154.5 (1s, C-6), 151.7 (1s, C-2), 147.7 (1s, C-4), 145.2 (1s, Ph ipso), 139.2 (1s, C-8), 137.3-137.2-130.0 (3s, CH3OPh), 128.3-127.9-126.8 (3s, Ph), 122.3 (1s, C-5), 113.1 (1s, CH3OPh), 88.3 (1s, C-1'), 81.6 (1s, C-4'), 70.7 (1s, C[(CH3OPh)2Ph]), 65.1 (1s, C-5'), 55.2 (1s, CH3OPh), 32.2 (1s, C-2'), 26.2 (1s, C-3'); MS FAB>0 (GT) m/z 538 (M+H) + . FAB<0 (GT) m/z 1073 (2M-H) -, 536 (M-H) -, 436 (B) -; UV (ethanol 95) λmax 276 nm ( 22 300), λmin 249 nm ( 10 800); HPLC tR 28.3 min; Anal. Calculated for C31H31N5O4 (MW: 537.61): C, 69.26; H, 5.81; N, 13.03. Found: C, 69.26; H, 5.92; N, 12.62.4.1.9. O-[2',3'-Dideoxy-6-N-(4,4'-dimethoxytrityl)adenosin-5'-yl]-O'-(S-pivaloyl-2-thioethyl)-N,N-disopropylaminophosphoramidite(11)

4. 1 .

 1 10. O-[2',3'-Dideoxy-6-N-(4,4'-dimethoxytrityl)adenosin-5'-yl]-O'-(S-pivaloyl-2thioethyl)-4-N-[2',3'-dideoxy-5'-O-(4,4'-dimethoxytrityl)cytidine]phosphoramidate(12)

  , 4.05 (m, 2H, CH2CH2O), 3.79-3.78 (2s, 6H, CH3OPh), 3.77-3.76 (2s, 6H, CH3OPh), 3.51 (dd, 1H, J = 10.8 Hz, J = 2.5 Hz, H-5'ddC ), 3.32 (m, 2H, H-5''ddC), 3.11 (m, 2H, SCH2CH2), 2.54 (m, 2H, H-2'ddA, H-2''ddA), 2.33 (m, 2H, H-2'ddC, H-2''ddC), 2.12 (m, 2H, H-3'ddA, H-3''ddA), 1.98 (m, 2H, H-3'ddC, H-3''ddC), 1.20-1.19 (2s, 9H, C(CH3)3); NMR[START_REF] Ikeda | Specific 3′-Terminal Modification of DNA with a Novel Nucleoside Analogue that Allows a Covalent Linkage of a Nuclear Localization Signal and Enhancement of DNA Stability[END_REF] C (CDCl3, 100 MHz)  205.7 (1s, COSATE), 159.8 (1s, C-4ddC), 158.6-158.2 (2s, CH3OPh ipso), 154.0 (1s, C-6ddA), 152.0 (1s, C-2ddA), 148.5 (1s, C-2ddC), 148.1 (1s, C-4ddA), 145.5-145.4 (2s, Ph ipso), 140.3 (1s, C-6ddC), 138.6-138.5 (2s, C-8ddA), 137.5-135.5-135.3-130.2-130.1 (5s, CH3OPh), 128.1-128.0-127.7-127.1-126.6 (5s, Ph), 121.2 (1s, C-5ddA), 113.2-113.0 (2s, CH3OPh), 101.4 (1d, JP-C = 22.9 Hz, C-5ddC), 86.7 (1s, C[(CH3OPh)2Ph]), 86.6 (1s, C-1'ddC), 85.6-85.5 (2s, C-1'ddA), 81.3 (1s, C-4'ddC), 79.6 (1d, JP-C = 7.7 Hz, C-4'ddA ), 70.5 (1s, C[(CH3OPh)2Ph]), 67.2-67.1 (2d, JP-C = 5.6/5.7 Hz, C-5'ddA), 64.9-64.8 (2d, JP-C = 5.7/5.6 Hz, OCH2CH2S), 63.6 (1s, C-5'ddC), 55.2-55.1 (2s, CH3OPh), 46.4 (1s, (CH3)3CSATE), 33.4 (1s, C-2'ddC), 32.5 (1s, C-2'ddA), 28.7 (1d, JP-C = 7.5 Hz, OCH2CH2S), 27.3 (1s, (CH3)3CSATE), 26.1-26.0 (2s, C-3'ddA), 25.0 (1s, C-3'ddC); NMR 31 P (CDCl3, 81 MHz)  7.36-7.30; MS FAB>0 (GT) m/z 1257 (M+H) + , 738 (M-B+2H) + ; FAB<0 (GT) m/z 1255 (M-H) -, 736 (M-B) -. HPLC tR 38.6 min (λmax 275.6 nm).

4. 1 .

 1 11. O-(2',3'-Dideoxyadenosin-5'-yl)-O'-(S-pivaloyl-2-thioethyl)-4-N-(2',3'dideoxycytidine) phosphoramidate(2)

  C (DMSO-d6, 100 MHz)  205.1 (1s, COSATE), 162.0 (1s, C-4ddC), 156.0 (1s, C-6ddA), 152.5 (1s, C-2ddA), 148.9 (1s, C-4ddA), 140.9 (m, C-6ddC), 138.9-138.8 (2s, C-8ddA), 119.0 (1s, C-5ddA), 98.5 (m, C-5ddC), 85.8 (1s, C-1'ddC), 84.2 (1s, C-1'ddA), 82.1 (1s, C-4'ddC), 79.0 (1d, JP-C = 7.2 Hz, C-4'ddA), 67.3 (1s, C-5'ddA), 64.1 (1s, OCH2CH2S), 61.8 (1s, C-5'ddC), 45.9 (1s, (CH3)3CSATE), 32.2 (1s, C-2'ddC), 31.0 (1s, C-2'ddA), 28.3 (1d, JP-C = 7.3 Hz, OCH2CH2S), 26.9 (1s, (CH3)3CSATE), 26.1-26.0 (2s, C-3'ddA), 24.5 (1s, C-3'ddC); NMR 31 P (DMSO-d6, 81 MHz)  5.82 (bs); MS FAB>0 (GT) m/z 653 (M+H) + ; FAB<0 (GT) m/z 651 (M-H) -, 434 (M-ddA) -; HRMS C26H38N8O8PS (M+H) + : calculated: 653.2271. Found: 653.2294; UV (ethanol 95) λmax 264 nm ( 17 400), λmin 240 nm ( 10 500); HPLC tR 20.6 min; Anal. Calculated for C26H37N8O8PS + 1.0 H2O: C, 46.56; H, 5.86; N, 16.71. Found: C, 46.50; H, 5.77; N, 16.56.

  g, 78%). Rf (AcOEt/hexane/NEt3,12/ 8/1, v/v/v): 0.39; NMR 1 H (CDCl3, 400 MHz)  8.27-8.20-8.10 (3s, 2H, H-2, H-8),7.40 (m, 2H, Ph), 7.30 (m, 7H, CH3OPh meta, Ph), 6.98 (s, 1H, NH), 6.84 (d, 4H, J = 8.8 Hz, CH3OPh), 6.33 (m, 1H, H-1'), 4.39 (m, 1H, H-4'), 3.83 (m, 4H, H-5', H-5'',

	OCH2CH2S), 3.83 (s, 6H, CH3OPh), 3.65 (m, 2H, (CH3)2CHN), 3.17 (m, 2H, OCH2CH2S), 2.54
	(m, 2H, H-2', H-2''), 2.19 (m, 2H, H-3', H-3''), 1.25 (m, 21H, (CH3)2CHN, (CH3)3CSATE); NMR
	13 C (CDCl3, 100 MHz)  206.3-206.2 (2s, CO), 158.2 (1s, CH3OPh ipso), 154.0 (1s, C-6),
	152.1-152.0 (2s, C-2), 148.1-148.0 (2s, C-4), 145.5 (1s, Ph ipso), 138.3-138.2 (2s, C-8), 137.5-
	130.1 (2s, CH3OPh), 128.8-127.8-126.7 (3s, Ph), 121.3-121.2 (2s, C-5), 113.0 (1s, CH3OPh),
	88.5-88.4 (2s, C-1'), 80.9-80.7 (m, C-4'), 70.5 (1s, C[(CH3OPh)2Ph]), 64.8-64.4 (2d, JP-C =
	16.1/15.5 Hz, C-5'), 62.3-62.2 (2d, JP-C = 18.8/18.6 Hz, OCH2CH2S), 55.2 (2s, CH3OPh), 46.4
	(1s, (CH3)3CSATE), 42.9 (2d, JP-C = 12.5 Hz, (CH3)2CHN), 33.0-32.8 (2s, C-2'), 30.0-29.9 (2d,
	JP-C = 7.0/7.3 Hz, OCH2CH2S), 27.3-27.2 (2s, (CH3)3CSATE), 26.3-26.1 (2s, C-3'), 24.7 (m,
	(CH3)2CHN); NMR 31 P (CDCl3, 81 MHz)  149.24-149.06; MS FAB>0 (GT) m/z 829 (M+H) + ,
	746 (M-N(iPr)2+2H) + ; UV (ethanol 95) λmax 274 nm ( 21 700), λmin 249 nm ( 10 900); Anal.
	Calculated for C44H57N6O6PS (MW: 829.00): C, 63.75; H, 6.93; N, 10.14. Found: C, 63.39; H,
	6.95; N, 9.80.

  H-1'ddC), 5.69-5.68 (2d, 1H, J = 7.8/7.9 Hz, H-5ddC), 4.37 (m, 1H, H-4'ddA), 4.23 (m, 3H, H-5'ddA, H-5''ddA, H-4'ddC)

	5, v/v/v) : 0.23; NMR 1 H (CDCl3, 400 MHz)  8.22-8.21 (2s,
	1H, H-8ddA), 8.04 (1s, 1H, H-2ddA), 7.95-7.94 (2d, 1H, J = 7.9 Hz, H-6ddC), 7.71-7.52 (2m, 1H,
	P-NH), 7.40 (m, 4H, Ph), 7.25 (m, 14H, CH3OPh meta, Ph), 6.96 (s, 1H, NHddA), 6.84-6.83 (2d,
	4H, J = 8.8/8.5 Hz, CH3OPh), 6.77 (d, 4H, J = 8.8 Hz, CH3OPh), 6.26 (m, 1H, H-1'ddA), 6.00
	(m, 1H,

Acknowledgments

We thank "Agence Nationale de Recherches sur le SIDA » (ANRS, France) for funding. A-L V. is also grateful to ANRS for a PhD grant.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data (NMR spectra for intermediates and final compounds) to this article can be found online at …


Sarah Couturier

Suzanne Peyrottes

Christian Périgaud
email: christian.perigaud@umontpellier.fr

2'-Derivatisation of 3'-C-methyl pyrimidine nucleosides

Keywords: Nucleoside analogues, Fluoration, Azidation, Pyrimidines, Antivirals

The stereoselective synthesis of some 3'-deoxy-3'-Cmethyl pyrimidine nucleosides is reported. The studied modifications concern the inversion of the configuration in 2'-position as well as the introduction of fluoro and azido substituents. The corresponding arabinonucleoside and 2'-substituted ribonucleoside analogues of uracil and cytosine have been obtained and fully characterized. Attempts to introduce fluoro and azido substituents with inversion at C-2' are also presented.

Introduction

The current pandemic associated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its health, economic, environmental and social consequences highlight the need for therapeutic treatments in parallel to the development of prophylactic strategies. In the search for efficient antiviral smallmolecules against this disease, drug repurposing (also called repositioning, redirecting or reprofiling) was first extensively studied. [1] This has some advantages over new drug discovery since chemical synthesis steps, manufacturing processes, reliable safety, and pharmacological properties in early clinical developmental phases are already available. Nucleoside and nucleotide analogues, which have a long and rich history in the field of medicinal chemistry, are in consequence well represented in this race to arsenal COVID-19 therapeutics. During the last decades, extensive modifications of the nucleoside/tide endogenous scaffolds have been reported. [2] Among them, the introduction of a methyl group on the osidic residue has led to new molecules endowed with a wide range of biological activities. As example, (2'S)-2'-deoxy-2'-Cmethylcytidine [3] (SMDC, Figure 1) as well as 3'-Cmethyladenosine [4] (Figure 1) exhibited potent activities against various human cancer cell lines. In addition, 2'-C-methyl series is known to include potent inhibitors of hepatitis C virus (HCV) replication and Sofosbuvir (Figure 1) was approved by the United States Food and Drug Administration (FDA) for the treatment of this disease. [5] As part of our work on this topic, we previously reported preliminary studies in the 3'-C-methyl nucleoside series [6] with the synthesis of 3'-deoxy and 2',3'-dideoxy-3'-C-methyl nucleosides bearing naturally occurring nucleobases and various aglycones of pharmacological interest (Figure 2). We pursue our investigations in this series by the study of modifications in the 2'-position such as inversion of configuration, introduction of fluoro, azido and amino substituents. Thus, the stereoselective synthesis of the resulting 3'-deoxy-3'-C-methyl nucleosides bearing uracil and cytosine is reported herein (Figure 2).

Results and Discussion

3'-Deoxy-3'-C-methyl arabinonucleoside analogues. Anhydronucleosides are useful starting materials for the stereochemically-controlled introduction of functional groups into the secondary positions of the sugar moiety. [7] In this respect, the anhydronucleoside 2 (Scheme 1) appeared as a suitable candidate for various modifications with 2'-inversion of configuration. This last was obtained from the benzoylated nucleoside 1 [6a] under Mitsunobu conditions. Its treatment with an aqueous sodium hydroxyde solution gave the arabino derivative of uracile 3. This nucleoside was previously published according to a non regiospecific reaction involving opening of a 2',3'epoxide. [8] Conversion of the uracil aglycone of 3 into the cytosine counterpart required acetylation to obtain the fully protected nucleoside 4. Then, the desired arabino derivative of cytosine 5 was obtained in two steps from 4, via the reaction with Lawesson's reagent, [9] followed by the treatment of 4-thioamide intermediate with methanolic ammonia at 100 °C in a stainlesssteel bomb. The proposed structures of these arabinonucleosides were based on UV, 1 H and 13 C NMR and mass spectral data. The 1 H spectrum shows H-1' as doublet with coupling constants (J 1'-2' = 6.1 and 5.8 Hz for 3 and 5, respectively) in agreement with the arabinose configuration of the 2'-hydroxyl function. [10] In addition, the signal of the 3'-methyl substituent (δ ≃ 1.0 ppm) appeared as doublet and the CH 3 -H 3' coupling constants were 6.6 and 6.7 Hz for 3 and 5 respectively, similar to those observed for the 3'-C-methyl-β-Dribo-and 2'-deoxy-β-D-ribonucleoside analogues. [6] 2'-Fluoro-3'-deoxy-3'-C-methyl ribonucleoside analogues. Several routes for the synthesis of 2′-deoxy-2′-fluoro-β-Dribonucleosides have been previously reported, including the opening of 2,2′-anhydronucleosides with a fluorinating agent (HF/dioxane or KF/crown ether), [11] the coupling of a suitably blocked 2-deoxy-2-fluororibofuranoside with appropriate heterocyclic bases, [12] an enzyme-catalyzed transglycosylation, [13] the nucleophilic displacement of 2′-O-trifluoromethanesulfonyl arabinonucleosides by tetra-n-butylammonium fluoride (TBAF), [14] and the direct introduction of fluorine atom by using diethylaminosulfur trifluoride (DAST) and an arabinonucleoside. [15] We selected this last methodology for the preparation of the fluoro derivative 8 (Scheme 2). Thus, the previously obtained arabinonucleoside 3 reacted with 4monomethoxytrityl chloride to afford the corresponding 5'-Oprotected nucleoside 6, which was treated with DAST to give the intermediate 7. Then, acidic treatment of 7 led to the desired fluorinated ribonucleoside 8 which was crystallized in methanol. The uracil derivative 8 was converted to 10 using the same methodology as for the nucleoside analogue 5 (Scheme 1). The structures of fluoronucleosides 8 and 10 were confirmed by UV, 1 H, 13 C, 19 F NMR and mass spectra analysis. 1 H NMR spectra of these derivatives exhibited a downfield shift of the H-2' due to the electron-withdrawing effect of fluorine. Illustrated with NMR data observed for 8, the assignments of H-1' (δ 5.79 ppm), H-2' (δ 5.00 ppm), and H-3' (δ 2.12 ppm) are consistent with the vicinal and geminal H-F coupling values found: 17.5, 52.3, and 35.9 Hz respectively. [16] The large proton-fluorine coupling constants allow ready assignments of the protons but the spectrum was insufficiently resolved in the used solvent to determine the magnitude of J 1'-2' (known to be particularly low for 1',2'-trans stereoisomery) [17] and J 3'-4'. The (2'R) configuration of the fluorinated nucleosides 8 and 10 was also assigned from 13 C NMR spectra analysis. For the two compounds, the measured J C1'-F (37 Hz) is similar to that observed for other 2'-deoxy-2'fluoronucleoside analogues characterized by a 1',2'-trans stereoisomery. [18] In addition, we measured a coupling constant between the 3'-methyl group (δ ≃ 7.5 ppm, d) and the fluorine atom (J CH3-F = 7.6 and 8.3 Hz for 8 and 10, respectively) which is only observed when these two substituents are in a cis position. [19] 2'-Azido-and 2'-amino-3'-deoxy-3'-C-methyl ribonucleoside analogues. The synthesis of the 2'-azido derivative 11 (Scheme 3) was carried out according to published procedure involving the in situ generation of lithium azide from lithium fluoride and azidotrimethylsilane in the presence of N,N,N',N'tetramethylethylenediamine (TMEDA) in DMF. [20] In these conditions, the introduction of the azido group on the 2'-position was obtained with satisfactory yield from the previously described anhydronucleoside 2. The treatment of the intermediate 11 with methanolic ammonia afforded the desired 2'-azido-ribonucleoside 12. The corresponding cytosine analogue 13 cannot be obtained by a thiation procedure using Lawesson's reagent, [9b] and we selected for its preparation the Sung's methodology. [21] Thus, the reaction of the protected nucleoside 11 with 4chlorophenyl phosphorodichloridate and 1,2,4-triazole in pyridine at room temperature led to the corresponding 4triazolylpyrimidinone derivative (not shown) which was purified on silica gel column chromatography. This intermediate was treated with aqueous ammonia in dioxane, followed by methanolic ammonia, to give rise to the desired nucleoside 13. Various synthetic protocols have been described for converting azides to amines, [22] among them the Staudinger procedure [23] appeared to be the most promising in the nucleoside field. Thus, 2'-azido nucleoside 12 was converted in presence of triphenylphosphine into a phosphinimine intermediate, the latter being hydrolyzed with aqueous ammonia to afford the 2'aminoribonucleoside analogue 14. The structures of nucleosides 12-14 were in accordance with UV, IR, 1 H, 13 C NMR and mass spectral data. The infrared spectra of compounds 11-13 exhibited a strong absorption band at 2110 cm -1 , characteristic of the azido function. Compared to 1 H NMR spectra of fluorinated nucleosides 8 and 10, the chemical shifts and coupling constants measured for the derivatives 12-14 reflect the relationship between the electronegativity of the 2'substitutents and the conformation of the sugar residue. The assignments of H-1'/H-2' for 8 (δ 5.83/5.12 ppm), 12 (δ 5.73/4.42 ppm), and 14 (δ 5.50/3.30 ppm) are consistent with the electronegativity of fluorine, azido and amino functions. It is well known that coupling constants J 1'-2' is primarily conformation dependent. Sugar puckerings of natural ribo-and deoxyribonucleosides exist in dynamic equilibria between two major conformers: the North (N) and the South (S). [24] NMR studies of 2'-substituted ribonucleotide analogues demonstrated that the contribution of the N form (2'-exo-3'-exo) increases with the electronegativity of the 2'-substituent. [25] In addition, it seems reasonable that the bulky methyl group in 3'-position adopt an equatorial position of the sugar plane resulting in an increase of the N conformer population. Thus, as observed for 2'-fluoro derivatives, the anomeric protons of 2'-azido-ribonucleosides 12 and 13 appeared as a singlet in accordance with the Karplus equation. In the case of the 2'-amino derivative 14, the measured J 1'-2 (2.5 Hz) seems to indicate that the equilibrium of the canonical conformers would be less in favor of the N form. This result was already observed for other 2'-amino pyrimidine and purine nucleosides. [25,26] In addition, the (2'R) configuration of the 2'azido nucleoside 12 was corroborated by the NOE effects observed between H-6, H-2' and H-3' protons. Finally, the 13 C NMR analysis showed a downfield C2' chemical shifts correlated to the electronegativity of the 2'-substituent for 14 (δ 59.8 ppm), 12 (δ 69.8 ppm) and 10 (δ 98.5 ppm).

Attempts to obtain 2'-modified-3'-deoxy-3'-C-methyl arabinonucleoside analogues. Fluoration. The introduction of a substituent at the C-2'-β (arabino) position in pyrimidine nucleosides by nucleophilic reactions cannot be achieved due to the neighboring-group participation of the carbonyl function of the aglycon. [7] It is well known that the intra-molecular attack of the 2carbonyl group of the nucleobase on C-2' of the sugar moiety supercedes the inter-molecular nucleophilic substitution. To circumvent this competition and to limit direct S N 2 reaction at the 2'-position of uridine derivatives, the protection of N 3 -imide function has attracted attention and various protecting groups have been reported. [27] Among them, we selected pmethoxybenzyl (PMB). [28] The PBM protecting group is often used in nucleoside chemistry due to its compatibility with a wide range of reaction conditions and its stability in acid and alkaline media. [29] Thus, the 5'-O-benzoylated precursor 1 was treated with 4methoxylbenzyl chloride in presence of DBU to afford the N 3protected nucleoside 15 in 78% yield (Scheme 4). This compound submitted to usual fluorination conditions led to the unexpected 2'-fluoro-ribonucleoside analogue (see supporting information). Unfortunately, the retention of configuration of C-2' sugar residue demonstrate that this strategy cannot circumvent the neighboring group participation by the respective O 2 -atom in fluoration conditions. Using N 3 -benzyl uridine analogues, similar results have just been reported during the writing of this article. [30] Azidation. The N 3 -protected nucleoside 15 reacted under the conditions used for the preparation of nucleoside 11 (Scheme 3) to give a complex mixture of at least three compounds (see supporting information). A chromatography separation allows to separate one of them, which turns out to be again the unexpected 2'-azido-ribonucleoside analogue presenting a retention of the configuration. The other mixture fractions are collected, treated with methanolic ammonia to provide after purification the 2',3'insaturated nucleoside analogue and the target nucleoside 16 in low yield (23%). Finally, starting from the purified intermediate 16, the PBM group is removed under mildly oxidizing conditions using ceric ammonium nitrate (CAN) to afford the desired 2'-azido-3'deoxy-3'-C-methyl arabinonucleoside 17. The (2'S) configuration of the azidonucleoside 17 was assigned in comparison to its stereoisomer 12 on the basis of NMR spectra and NOE experiments. In the 1 H NMR spectrum of 17 the signal for the anomeric proton appeared at a lower field resonance (δ 6.13 ppm) than that of the isomer 12 (δ 5.73 ppm). The larger coupling constant (6.5 Hz) for the H-1' proton of 17 while the anomeric proton of 12 appeared as a singlet providing additional support to the 1',2-cis configuration of 17. These typical 1 H NMR patterns are in agreement with the abundant literature in the field, especially with the empirically rules used for the determination of the anomeric configuration. Finally, irradiations of the 3'-methyl group of the nucleoside 17 gave large NOE effects (5.0%) on H-2' and H-4' protons (Scheme 4). A similar effect was also observed between the same protons.

Conclusion

In summary, the stereoselective synthesis of 3'-deoxy-3'-Cmethyl nucleoside analogues of uracil and cytosine bearing several modifications at the 2'-position of the sugar residue were undertaken with the hope of discovering new compounds endowed with potential activities. The corresponding arabinonucleoside and 2'-substituted ribonucleoside analogues were relatively straightforward to obtain, whereas attempts to introduce a substituent at C-2'-β (arabino) position of uracil nucleosides by nucleophilic reactions have failed (fluoration) or led to complex mixtures (azidation). These results are due to the neighboring-group participation of the carbonyl function of the aglycon despite the protection of N 3 -imide function. The biological evaluations of these compounds are currently in progress.

Experimental Section

General Information: Melting points (m.p.) were determined in open capillary tubes on a Gallenkamp MFB-595-010 M apparatus and are uncorrected. 1 H NMR and 13 C NMR spectra were recorded at 300 MHz on a Bruker 300 Advance, 19 F spectra at 250 MHz on Bruker 250. Chemical shifts are quoted in parts per million (ppm) using residual non deuterated solvents as internal references. Deuterium exchange, decoupling and COSY experiments were performed in order to confirm proton assignments. Coupling constants, J, are reported in Hertz. 2D 1 H- 13 C heteronuclear COSY were recorded for the attribution of 13 C signals. UV spectra were recorded on an Uvikon 931 (Kontron). IR spectra were recorded on an Perkin Elmer Paragon 1000 FT-IR. FAB mass spectra were recorded in the positive-ion or negative-ion mode on a JEOL JMS DX 300 using thioglycerol/glycerol (1:1, v/v, G-T) as matrix. ESI HRMS were recorded in the positive mode on a Micromass Q-TOF Waters. TLC was performed on precoated aluminium sheets of silica gel 60 F254 (Merck, Art. 9385), visualization of products being accomplished by UV absorbance followed by charring with 5% ethanolic sulphuric acid with heating. Solvents were reagent grade or purified by distillation prior to use, and solids were dried over P2O5 under reduced pressure at rt. Moisture sensitive reactions were performed under argon atmosphere using ovendried glassware. All aqueous solutions were saturated with the specified salt unless otherwise indicated. The nucleoside analogue 1 was obtained following a previously published procedure. [6a] 2,2'-Anhydro-1-( 5

1-(3-Deoxy-3-C-methyl-β-D-arabinofuranosyl)uracil (3):

The 2,2'-Oanhydronucleoside 2 (0.74 g, 2.25 mmol) was dissolved in methanol/water (1/1: v/v, 22.5 mL) and treated with 1 M NaOH (22.5 mL) for 30 min, then neutralized with glacial AcOH and evaporated to dryness. The crude material was purified by silica gel column chromatography using a stepwise gradient of methanol (0-10%) in dichloromethane to afford the title compound 3 (0.49 g, 90% yield) as white solid, which was crystallized from MeOH. m.p. 213-214°C [litt.: 211-213°C [8c] ; 208-210°C (MeOH/toluene/petroleum ether) [8b]

1-(2,5-Di-O-acetyl-3-deoxy-3-C-methyl-β-D-arabinofuranosyl)uracil (4):

To a solution of the arabinonucleoside 3 (94.2 mg, 0.39 mmol) in anhydrous pyridine (2.3 mL) were added acetic anhydride (0.54 mL) and a catalytic amount of 4-dimethylaminopyridine (DMAP). The solution was stirred 30 min at room temperature, neutralized with solid NaHCO3. The reaction mixture was extracted with diethyl ether and this organic layer was evaporated to dryness. The crude material was purified by silica gel column chromatography using a stepwise gradient of methanol (0-2%) in dichloromethane to afford the title compound 4 (75.0 mg, 59% yield) as white solid. 1

1-(3-Deoxy-3-C-methyl-β-D-arabinofuranosyl)cytosine

(5): Lawesson's reagent (64.8 mg, 0.16 mmol) was added to a solution of the protected nucleoside 4 (75.0 mg, 0.23 mmol) in anhydrous 1,2dichloroethane (7.3 mL) and the reaction mixture was refluxed for 2 h. The solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography using a stepwise gradient of methanol (0-1%) in dichloromethane affording the corresponding 4thioamide intermediate as a orange foam. The foam was dissolved in methanolic ammonia (7 mL, saturated beforehand at -10°C and stoppered tightly) and heated overnight at 100°C in a stainless-steel bomb. After cooling to room temperature, the solution was evaporated under reduced pressure and the residue was subjected to silica gel column chromatography using as eluent a stepwise gradient of methanol (0-15%) in dichloromethane to afford the title compound 5 (51.0 mg, 92% yield) as white solid. 1 6): 4-Monomethoxytrityl chloride (372.0 mg, 1.21 mmol) was added to a solution of the protected nucleoside 3 (265.3 mg, 1.10 mmol) in anhydrous pyridine (4.9 mL) and the reaction mixture was refluxed for 4 h. After cooling to room temperature, methanol (4 mL) was added and the reaction mixture was evaporated under reduced pressure. The resulting residue was subjected to silica gel column chromatography using as eluent a stepwise gradient of methanol (0-5%) in dichloromethane to afford the title compound 6 (472.2 mg, 84% yield) as foam. 1 7): To a solution of the protected arabinonucleoside 6 (435.0 mg, 0.85 mmol) in a mixture of dry dichloromethane and pyridine (5.1 mL, 92/8: v/v) at 0°C was added DAST (0.166 mL, 1.35 mmol). The reaction mixture was stirred at room temperature one day, neutralized with saturated NaHCO3 and diluted with dichloromethane (30 mL). The organic layer was washed with water, dried over sodium sulfate and evaporated to dryness. The residue was purified by silica gel column chromatography using as eluent a stepwise gradient of methanol (0-2%) in dichloromethane to afford the title compound 7 (398.7 mg, 91 % yield) as white foam. 1 H NMR (CDCl3) δ 10.55 (bs, 1H, NH), 8.16 (d, J = 8.1, 1H, H-6), 7.46-7.22 (m, 12H, Ph), 6.87 (m, 2H, Ph)

1-(3-Deoxy-3-C-methyl-5-O-monomethoxytrityl-β-D-arabinofuranosyl)uracil (

1-(2,3-Dideoxy-2-fluoro-3-C-methyl-5-O-monomethoxytrityl-β-Dribofuranosyl)uracil (

1-(5-O-Acetyl-2,3-dideoxy-2-fluoro-3-C-methyl-β-D-ribofuranosyl)uracil (9):

To a solution of the nucleoside 8 (94.6 mg, 0.39 mmol) in anhydrous pyridine (3.9 mL) were added acetic anhydride (0.467 mL, 5.42 mmol) and a catalytic amount of DMAP. After stirring 3 h at room temperature, EtOH 95 was added and the reaction mixture was evaporated to dryness. The crude material was purified by silica gel column chromatography using a stepwise gradient of methanol (0-5%) in dichloromethane to afford the title compound 9 (107.4 mg, 96% yield) as white foam. 1

1-(2,3-Dideoxy-2-fluoro-3-C-methyl-β-D-ribofuranosyl)cytosine (10):

Lawesson's reagent (80.5 mg, 0.20 mmol) was added to a solution of the protected nucleoside 9 (81.4 mg, 0.28 mmol) in anhydrous 1,2dichloroethane (9 mL) and the reaction mixture was refluxed for 2 h. The solvent was evaporated under reduced pressure and the residue was purified using silica gel column chromatography using a stepwise gradient of methanol (0-1%) in dichloromethane to give the corresponding 4thioamide intermediate as a yellow foam. The foam was dissolved in methanolic ammonia (10 mL, saturated beforehand at -10°C and stoppered tightly) and heated overnight at 100°C in a stainless-steel bomb. After cooling to room temperature, the solution was evaporated under reduced pressure and the residue was subjected to silica gel column chromatography using as eluent a stepwise gradient of methanol (0-15%) in dichloromethane to afford the title compound 10 (50.0 mg, 72% yield) as a white solid. 1 H NMR (D2O) δ 7.84 (d, J = 7.5, 1H, H-6), 5.89 (d, J = 7.5, 1H, H-5), 5.86 (d, J = 17.7, 1H, H-1'), 4.96 (dd, J = 4.0, J = 51.8, 1H, H-2'), 3.97 (m, 1H, H-4), 3.92 (m, 1H, H-5'), 3.69 (dd, J = 4.0, J = 13.0, 1H, H-5"), 2.13 (dm, J = 35.2, 1H, H-3'), 0.98 (d, J = 6.8, 3H, CH3); 13 11): Lithium fluoride (10.96 mg, 0.42 mmol) was suspended in anhydrous DMF (0.26 mL) at room temperature. To the stirred suspension was added N,N,N',N'-tetramethylethylenediamine (TMEDA, 0.23 mL, 1.54 mmol) followed by azidotrimethylsilane (65 µL, 0.49 mmol). The reaction mixture was heated 3 h at 110°C and the 2,2'-O-anhydronucleoside 2 (84.4 mg, 0.26 mmol) was added. After stirring 48 h at 110°C, the solvent was evaporated under reduced pressure and the residue was subjected to silica gel column chromatography using as eluent a stepwise gradient of methanol (0-2%) in dichloromethane to afford the title compound 11

1-(2-Azido-2,3-dideoxy-3-C-methyl-β-D-ribofuranosyl)cytosine (13):

To a solution of the protected nucleoside 11 (49.3 mg, 0.13 mmol) in dry pyridine (820 µL) was added 1,2,4-triazole (120.6 mg, 1.75 mmol) followed by the dropwise addition at 0°C of 4-chlorophenylphosphorodichloridate (95 µL, 0.59 mmol). The reaction mixture was stirred 24h at room temperature, and the solvent was evaporated to dryness. The residue was dissolved en dichloromethane (8.7 mL) and washed with water. After decantation, the organic layer was dried (Na2SO4), concentrated to dryness and the residue was purified by silica gel column chromatography using as eluent a stepwise gradient of methanol (0-3%) in dichloromethane to afford the 4-triazolyl intermediate. This last was then dissolved in NH4OH (28%)/dioxane (1.85 mL, 1/3:v/v) and the reaction mixture was stirred overnight at room temperature. After evaporation to dryness, the resulting residue was subjected to silica gel column chromatography using as eluent a stepwise gradient of methanol (0-10%) in dichloromethane to afford the title compound 13 (23.7 mg, 67% yield) as white solid. 1 H NMR (DMSO-d6) δ 8.07 (d, J = 7.5, 1H, H-6), 7.15 (bs, 2H, NH2) 5.71 (s, 1H, H-1'), 5.69 (d, J = 7.5, 1H, H-5), 5.19 (pt, 1H, OH-5'), 4.24 (d, J = 5.5, 1H, H-2'), 3.80 (m, 1H, H-5'), 3.69 (m, 1H, H-4'), 3.55 (m, 1H, H-5"), 2.20 (m, 1H, H-3'), 0.96 (d, J = 6.7, 3H, CH3); 13 14): A solution of the nucleoside analogue 12 (213.1 mg, 0.80 mmol) and triphenylphosphine (315.7 mg, 1.20 mmol) in anhydrous pyridine (10 mL) was stirred 1 h at room temperature, then NH4OH (20%, 6.2 mL) was added. After stirring overnight at room temperature, the solvent was evaporated under reduced pressure and the residue was subjected to silica gel column chromatography using as eluent a stepwise gradient of methanol (0-15%) in dichloromethane to afford the title compound 14 (142.0 mg, 74% yield) which was lyophilized in water. 15): To a suspension of nucleoside analogue 1 (780 mg, 2.25 mmol) in anhydrous acetonitrile (22.5 mL) were added pmethoxybenzyl chloride (550 µL, 4.05 mmol) and DBU (673 µL, 4.05 mmol). The reaction mixture was refluxed for 2 h then, after cooling to 0°C, hydrolyzed with a KHSO4 (5%) solution and diluted with dichloromethane. After decantation, the organic layer was dried (Na2SO4), concentrated to dryness and the residue was purified by silica gel column chromatography using as eluent a stepwise gradient of methanol (0-2%) in dichloromethane to afford the title compound 15 (819 mg, 78% yield) as white foam. 1 16): To a solution of the protected nucleoside 15 (0.64 g, 1.37 mmol) in dry THF (13.5 mL) was added triphenylphosphine (1.07 g, 4.10 mmol). The reaction mixture was cooled at -10°C and a solution of diethyl azodicarboxylate (DEAD, 0.64 mL, 4.105 mmol) and diphenylphosphorylazide (DPPA, 0.89 mL, 4.10 mmol) in dry THF was added dropwise. After stirring 10 min at -10°C then 2h at room temperature, the solvent was evaporated under reduced pressure and the residue was subjected to silica gel column chromatography using as eluent a stepwise gradient of methanol (0-1%) in dichloromethane. The collected fractions containing products with Rf = 0.3 (diethyl ether/petroleum ether, 8/2:v/v) were evaporated and the residue was subjected to silica gel column chromatography using an isocratic elution of diethyl ether/petroleum ether (1/1:v/v). The appropriate fractions were evaporated under reduced pressure and the residue (0.42 g, 0.90 mmol) was dissolved in methanolic ammonia (30 mL, saturated beforehand at -10°C and stoppered tightly) and the resulting solution was stirred overnight at room temperature. The solution was evaporated under reduced pressure and the residue was subjected to reverse phase column chromatography using as eluent a stepwise gradient of acetonitrile (15-20%) 17): To a solution of the nucleoside 16 (134.6 mg, 0.35 mmol) in a mixture of acetonitrile and water (11 mL, 8/2:v/v) was added ceric ammonium nitrate (CAN, 1.14 g, 2.09 mmol). The reaction mixture was stirred 2 h at room temperature, then evaporated under reduced pressure. The crude residue was dissolved in dichloromethane (10 mL) and washed with water. After decantation, the organic layer was dried (Na2SO4), concentrated to dryness and the residue was purified by silica gel column chromatography using as eluent a stepwise gradient of methanol (0-5%) in dichloromethane to afford the title compound 17 (62.3 mg, 67% yield) which was lyophilized in water. 1

Entry for the Table of Contents

Insert graphic for The study of nucleoside and nucleotide analogues has been the subject of an extensive development during these last decades. In this field, additions or substitutions on the sugar residue had led to potent drugs in antiviral and anticancer chemotherapies. Herein, we reported the stereoselective synthesis of a new series of pyrimidine nucleosides bearing a double modification in the 2' and 3' positions of the sugar scaffold.

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Figure 1 .

 1 Figure 1. Examples of C-methyl nucleoside/tide analogues with biological activities.

Figure 2 .

 2 Figure 2. Previously reported 3'-C-nucleosides and additional structural analogues investigated in this study.

Scheme 1 .

 1 Scheme 1. Synthesis of 3'-deoxy-3'-C-methyl arabinonucleoside analogues 3 and 5.

Scheme 4 .

 4 Scheme 3. Synthesis of 2'-azido-and 2'-amino-3'-deoxy-3'-C-methyl ribonucleoside analogues 12-14.

  -O-benzoyl-3-deoxy-3-C-methyl-β-D-erythro-pentofuranosyl)uracil (2): To a solution of the protected nucleoside 1 (1.81 g, 5.23 mmol) in anhydrous toluene (93 mL) were added triphenylphosphine (2.73 g, 10.46 mmol) and diethyl azodicarboxylate (DEAD, 1.61 mL, 10.46 mmol). After stirring 30 min at room temperature, the reaction mixture was evaporated under reduced pressure and the resulting residue was subjected to silica gel column chromatography using as eluent a stepwise gradient of methanol (0-5%) in dichloromethane to afford the title compound 2 (1.55 g, 91% yield) as white solid. 1 H NMR (CDCl3) δ 7.87-7.34 (m, 5H, C6H5CO), 7.29 (d, J = 7.5, 1H, H-6), 6.11 (d, J = 5.6, 1H, H-1'), 5.98 (d, J = 7.5, 1H, H-5), 5.06 (dd, J•= 5.6, J = 2.7, 1H, H-2'), 4.31 (m, 1H, H-5'), 4.20 (m, 2H, H-4', H-5"), 2.67 (m, 1H, H-3'), 1.27 (d, J = 7.3, 3H, CH3); 13 C NMR (CDCl3) δ 171.8 (CO), 165.9 (C-4), 159.6 (C-2), 134.9 (C-6), 133.5-128.6 (Ph), 110.4 (C-5), 90.3 (C-1'), 89.2 (C-2'), 85.7 (C-4'), 64.0 (C-5'), 41.5 (C-3'), 16.4 (CH3); UV (EtOH 95) λmax= 259 nm (ɛ = 10 700); MS: m/z (FAB > 0) 657 (2M+H) + , 329 (M+H) + ; m/z (FAB < 0) 327 (M-H) -.

  ; 1 H NMR (DMSO-d6) δ 11.20 (s, 1H, NH), 7.88 (d, J = 8.1, 1H, H-6), 5.98 (d, J = 6.1, 1H, H-1'), 5.54 (d, J = 8.1, 1H, H-5), 5.1 (bs, 1H, OH-5'), 3.98 (pt, 1H, H-2'), 3.68 (m, 1H, H-5'), 3.57 (m, 2H, H-5", H-4'), 3.38 (bs, 1H, OH-2'), 1.93 (1H, m, H-3'), 1.00 (d, J = 6.6, 3H, CH3). 13 C NMR (DMSO-d6) δ 163.3 (C-4), 150.6 (C-2), 142.1 (C-6), 100.0 (C-5), 83.5 (C-1'), 83.4 (C-4'), 76.6 (C-2'), 60.0 (C-5'), 38.6 (C-3'), 14.0 (CH3); UV (EtOH 95) λmax= 262 nm (ɛ = 9 500); MS: m/z (FAB > 0) 243 (M+H) + ; HRMS (ESI + ): m/z calcd. for C10H15N2O5 (M+H) + : 243.0981; found: 243.0978; elemental analysis calcd (%) for C10H14N2O5: C, 49.58;H, 5.83; N, 11.56; found: C, 49.89; H, 5.76; N, 11.52.

  H NMR (CDCl3) δ 8.50 (s, 1H, NH), 7.50 (d, J = 8.2, 1H, H-6), 6.11 (d, J = 5.1, 1H, H-1'), 5.66 (dd, J = 8.1, J = 2.2, 1H, H-5), 5.11 (pt, 1H, H-2'), 4.28 (dd, J = 5.5, J = 12.3, 1H, H-5"), 4.23 (dd, J = 3.7, J = 12.3, 1H, H-5'), 3.81 (m, 1H, H-4'), 2.13 (m, 1H, H-3'), 2.07 (s, 3H, CH3CO), 1.94 (s, 3H, CH3CO), 1.15 (d, J = 7.0, 3H, CH3); 13 C NMR (CDCl3) δ 170.5 (CO), 169.3 (CO), 162.7 (C-4), 149.7 (C-2), 140.6 (C-6), 101.4 (C-5), 83.9 (C-1'), 81.5 (C-4'), 77.5 (C-2'), 63.7 (C-5'), 39.9 (C-3'), 20.8 (CH3CO), 20.6 (CH3CO), 15.3 (CH3); UV (EtOH 95) λmax= 259 nm (ɛ = 9 900) ; MS: m/z (FAB > 0) 653 (2M+H) + , 327 (M+H) + , 215 (S) + , 113 (BH2) + ; m/z (FAB < 0) 651 (2M-H) -, 325 (M-H) -, 111 (B) -.

  H NMR (DMSO-d6) δ 7.76 (d, J = 7.4, 1H, H-6), 7.01 (pd, 2H, NH2), 6.02 (d, J = 5.8, 1H, H-1'), 5.65 (d, J = 7.4, 1H, H-5), 5.29 (d, J = 5.4, 1H, OH-2'), 5.02 (pt, 1H, OH-5'), 3.92 (m, 1H, H-2'), 3.65 (m, 1H, H-5'), 3.56 (m, 1H, H-5"), 3.49 (m, 1H, H-4'), 1.92 (m, 1H, H-3'), 1.01 (d, J = 6.7, 3H, CH3); 13 C NMR (DMSO-d6) δ 165.4 (C-4), 155.5 (C-2), 142.7 (C-6), 92.5 (C-5), 84.1 (C-1'), 83.2 (C-4'), 76.5 (C-2'), 60.7 (C-5'), 39.5 (C-3'), 14.6 (CH3); UV (EtOH 95) λmax= 272 nm (ɛ = 8 000) ; MS: m/z (FAB > 0) 242 (M+H) + , 112 (BH2) + ; m/z (FAB < 0) 240 (M-H) -; HRMS (ESI + ): m/z calcd. for C10H16N3O4 (M+H) + : 242.1141; found: 242.1138.

  H NMR (CDCl3) δ 9.76 (bs, 1H, NH), 8.21 (d, J = 8.1, 1H, H-6), 7.37-7.09 (m, 12H, Ph), 6.78 (m, 2H, Ph), 6.00 (d, J = 5.8, 1H, H-1'), 5.31 (dd, J = 8.1, J = 1.3, 1H, H-5), 4.37 (bs, 1H, OH-2'), 4.12 (m, 1H, H-2'), 3.73 (s, 3H, CH3O), 3.57 (m, 1H,H-4'), 3.51 (dd, J = 1.7, J = 11.2, 1H, H-5'), 3.27 (dd, J = 2.4, J = 11.2, 1H, H-5"), 2.26 (m, 1H, H-3'), 0.97 (d, J = 6.5, 3H, CH3); 13 C NMR (CDCl3) δ 164.4 (C-4), 158.8 (Ph), 151.6 (C-2), 143.9-125.3 (Ph), 141.8 (C-6), 113.3 (Ph), 101.5 (C-5), 87.2 (CPh3), 85.4 (C-1'), 83.3 (C-4'), 78.1 (C-2'), 61.3 (C-5'), 55.3 (OCH3), 37.3 (C-3'), 13.6 (CH3); UV (EtOH 95) λmax = 264 nm (ɛ =10 600), 230 nm (ɛ = 15 900); MS: m/z (FAB > 0) 515 (M+H) + , 113 (BH2) + ; m/z (FAB < 0) 1027 (2M-H) -, 513 (M-H) -, 241(M-mMTr) -,111 (B) -.

  , 6.05 (d, J = 16.3, 1H, H-1'), 5.40 (d, J = 8.1, 1H, H-5), 5.00 (dd, J' = 3.7, J = 51.4, 1H, H-2'), 4.02 (m, 1H, H-4'), 3.78 (s, 3H, CH3O), 3.73 (dd, J = 1.6, J = 11.5, 1H, H-5'), 3.36 (dd, J = 2.3, J•= 11.5, 1H, H-5"), 2.56 (dm, J = 34.1, 1H, H-3'), 1.01 (d, J = 6.7, 3H, CH3); 13 C NMR (CDCl3) δ 164.1 (C-4), 158.8 (Ph), 150.5 (C-2), 143.8-123.9 (Ph), 140.0 (C-6), 113.7 (Ph), 102.0 (C-5), 98.2 (d, J = 185.7, C-2'), 89.3 (d, J = 38.0, C-1'), 87.2 (CPh3), 85.0 (C-4'), 60.6 (C-5'), 55.2 (OCH3), 35.6 (d, J = 19.6, C-3'), 8.0 (d, J = 7.6, CH3); 19 F NMR (CDCl3) δ -196,5 (m, J = 16.3, J = 51.4, J = 34.1, F-2'); UV (EtOH 95) λmax = 260 nm (ɛ =11 300), 231 nm (ɛ = 17 500) ; MS: m/z (FAB > 0) 517 (M+H) + , 113 (BH2) + ; m/z (FAB < 0) 1031 (2M-H) -, 515 (M-H) -, 243 (M-mMTr) -, 111 (B) -. 1-(2,3-Dideoxy-2-fluoro-3-C-methyl-β-D-ribofuranosyl)uracil (8): A solution of the protected nucleoside 7 (454.9 mg, 0.88 mmol) in a mixture of AcOH, methanol and water (26 mL, 8/1/1: v/v/v) was stirred overnight at room temperature. The solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography using a stepwise gradient of methanol (0-10%) in dichloromethane to afford the title compound 8 (180.1 mg, 84% yield), which was crystallized from MeOH. m.p. 214-215°C; 1 H NMR (DMSO-d6) δ 11.4 (bs, 1H, NH), 8.06 (d, J = 8.1, 1H, H-6), 5.83 (d, J = 17.5, 1H, H-1'), 5.58 (d, J = 8.1, 1H, H-5), 5.26 (pt, 1H, OH-5'), 5.12 (dd, J = 4.0, J = 52.3, 1H, H-2'), 3.85 (m, 1H, H-5'), 3.82 (m, 1H, H-4'), 3.60 (m, 1H, H-5"), 2.29 (dm, J = 35.9, 1H, H-3'), 1.00 (d, J = 6.7, 3H, CH3); 13 C NMR (DMSO-d6) δ 163.3 (C-4), 150.2 (C-2), 139.9 (C-6), 100.8 (C-5), 98.5 (d, J = 181.1, C-2'), 88.5 (d, J = 37.0, C-1'), 85.8 (C-4'), 58.8 (C-5'), 34.4 (d, J = 19.6, C-3'), 7.9 (d, J = 7.6, CH3); 19 F NMR (DMSO-d6) δ -195,6 (m, J = 17.5, J = 52.3, J = 35.9, F-2'); UV (EtOH 95) λmax = 259 nm (ɛ = 10 300); MS: m/z (FAB > 0) 489 (2M+H) + , 245 (M+H) + , 113 (BH2) + ; m/z (FAB < 0) 487 (2M-H) -, 243 (M-H) -, 111 (B) -; HRMS (ESI + ): m/z calcd. for C10H14FN2O4 (M+H) + : 245.0938; found: 245.0938.

  H NMR (CDCl3) δ 8.84 (bs, 1H, NH), 7.60 (d, J = 8.2, 1H, H-6), 5.79 (d, J = 17.3, 1H, H-1'), 5.65 (d, J = 8.2, 1H, H-5), 5.00 (dd, J = 4.2, J = 51.5, 1H, H-2'), 4.33 (m, 2H, H-5', H-5"), 4.10 (m, 1H, H-4'), 2.12 (m, 1H, H-3'), 2.06 (s, 3H, CH3O), 1.08 (dd, J = 6.8, J = 0.8, 3H, CH3); 13 C NMR (CDCl3) δ 170.3 (CO), 162.9 (C-4), 149.8 (C-2), 139.6 (C-6), 101.9 (C-5), 97.5 (d, J = 186.4, C-2'), 90.9 (d, J = 37.7, C-1'), 83.5 (C-4'), 62.4 (C-5'), 36.5 (d, J = 20.4, C-3'), 20.7 (CH3CO), 8.1 (d, J = 8.3, CH3); 19 F NMR (CDCl3) δ -195,4 (m, J = 17.3, J = 51.5, J = 33.9, J = 0.8, F-2'); UV (EtOH 95) λmax = 258 nm (ɛ = 9 700); MS: m/z (FAB > 0) 573 (2M+H) + , 287 (M+H) + , 175 (S) + , 113 (BH2) + ; m/z (FAB < 0) 571 (2M-H) -, 285 (M-H) -, 111 (B) -.

  C NMR (D2O) δ 171.0 (C-4), 166.3 (C-2), 141.1 (C-6), 98.7 (d, J = 182.6, C-2'), 95.5 (C-5), 90.0 (d, J = 37.7, C-1'), 86.0 (C-4'), 60.0 (C-5'), 35.4 (d, J = 19.6, C-3'), 7.2 (d, J = 8.3, CH3); 19 F NMR (D2O) δ -196.1 (m, J = 17.7, J = 51.8, J = 33.9, F-2'); UV (EtOH 95) λmax = 272 nm (ɛ = 8 700) ; MS m/z (FAB > 0) 244 (M+H) + , 112 (BH2) + ; HRMS (ESI + ): m/z calcd. for C10H15FN3O3 (M+H) + : 244.1097; found: 244.1098.

  (95.4 mg, 72% yield) which was crystallized from EtOH 100. m.p. 153-154°C; 1 H NMR (CDCl3) δ 9.25 (bs, 1H, NH), 7.94 (m, 2H, Ph), 7.71 (d, J = 8.2, 1H, H-6), 7.58-7.39 (m, 3H, Ph), 5.76 (s, 1H, H-1'), 5.39 (d, J = 8.2, 1H, H-5), 4.62 (dd, J = 2.4, J = 13.0, 1H, H-5'), 4.57 (dd, J = 3.1, J = 13.0, 1H, H-5"), 4.15 (d, J = 5.5, 1H, H-2'), 4.11 (m, 1H, H-4'), 2.17 (m, 1H, H-3'), 1.10 (d, J = 6.7, 3H, CH3); 13 C NMR (CDCl3) δ 166.0 (CO), 162.9 (C-4), 149.9 (C-2), 138.7 (C-6), 133.8-128.7 (Ph), 101.7 (C-5), 90.4 (C-1'), 84.2 (C-4'), 70.1 (C-2'), 62.0 (C-5'), 35.4 (C-3'), 9.3 (CH3); UV (EtOH 95) λmax = 261 nm (ɛ = 10 400), 228 nm (ɛ = 15 500); IR υ = 2112 (s) cm -1 (N3); MS: m/z (FAB > 0) 743 (2M+H) + , 372 (M+H) + , 260 (S) + , 113 (BH2) + ; m/z (FAB > 0) 370 (M-H) -, 111 (B) -. 1-(2-Azido-2,3-dideoxy-3-C-methyl-β-D-ribofuranosyl)uracil (12): The protected nucleoside 11 (604.8 mg, 1.63 mmol) was dissolved in methanolic ammonia (40.7 mL, saturated beforehand at -10°C and stoppered tightly) and the resulting solution was stirred overnight at room temperature. The solution was evaporated under reduced pressure and the residue was subjected to silica gel column chromatography using as eluent a stepwise gradient of methanol (0-5%) in dichloromethane to afford the title compound 12 (385 mg, 89% yield) which was crystallized from EtOH 100. m.p. 167-168°C; 1 H NMR (DMSO-d6) δ 11.4 (bs, 1H, NH), 8.10 (d, J = 8.1, 1H, H-6), 5.73 (s, 1H, H-1'), 5.58 (dd, J = 8.1, J = 1.9, 1H, H-5), 5.23 (pt, 1H, OH-5'), 4.42 (d, J' = 5.6, 1H, H-2'), 3.80 (ddd, J = 2.2, J = 5.5, J = 12.7, 1H, H-5'), 3.69 (m, 1H, H-4'), 3.55 (ddd, J = 2.4, J = 4.8, J = 12.7, 1H, H-5"), 2.30 (m, 1H, H-3'), 0.98 (d, J = 6.7, 3H, CH3); 13 C NMR (DMSO-d6) δ 163.3 (C-4), 150.3 (C-2), 139.6 (C-6), 100.6 (C-5), 88.7 (C-1'), 86.3 (C-4'), 69.8 (C-2'), 58.8 (C-5'), 33.7 (C-3'), 9.4 (CH3); UV (EtOH 95) λmax = 261nm (ɛ = 10 400); IR υ = 2110 (s) cm -1 (N3); MS: m/z (FAB > 0) 268 (M+H) + , 113 (BH2) + ; HRMS (ESI + ): calcd. for C10H14N5O4 (M+H) + : 268.1046; found: 268.1050.

  C NMR (DMSO-d6) δ 166.9 (C-4), 155.0 (C-2), 140.2 (C-6), 93.0 (C-5), 89.2 (C-1'), 86.1 (C-4'), 70.1 (C-2'), 58.9 (C-5'), 33.4(C-3'), 9.3 (CH3); UV (EtOH 95) λmax = 272 nm (ɛ = 8 900) ; IR υ = 2110 (s) cm -1 (N3); MS: m/z (FAB > 0) 533 (2M+H) + , 267 (M+H) + , 112 (BH2) + ; m/z (FAB < 0) 531 (2M-H) -, 265 (M-H) -; HRMS (ESI + ): calcd. for C10H15N6O3 (M+H) + : 267.1206; found: 267.1206. 1-(2-Amino-2,3-dideoxy-3-C-methyl-β-D-ribofuranosyl)uracil (

  1 H NMR (DMSO-d6) δ 11.3 (bs, 1H, NH), 8.06 (d, J = 8.1, 1H, H-6), 5.55 (d, J = 8.1, 1H, H-5), 5.50 (d, J = 2.5, 1H, H-1'), 5.07 (pt, 1H, OH-5'), 3.80 (m, 1H, H-4'), 3.73 (ddd, J = 2.0, J = 4.8, J = 12.3, 1H, H-5'), 3.52 (ddd, J = 3.6, J = 7.4, J = 12.3, 1H, H-5"), 3.30 (dd, J = 2.5, J = 6.4, 1H, H-2'), 2.10 (m, 1H, H-3'), 0.98 (d, J = 6.7, 3H, CH3); 13 C NMR (DMSO-d6) δ 163.3 (C-4), 150.6 (C-2), 140.5 (C-6), 100.5 (C-5), 91.3 (C-1'), 85.7 (C-4'), 60.4 (C-5'), 59.8 (C-2'), 34.7 (C-3'), 10.1 (CH3); UV (EtOH 95) λmax = 262 nm (ɛ = 10 200); MS: m/z (FAB > 0) 242 (M+H) + , 130 (S) + , 113 (BH2) + ; m/z (FAB < 0) 240 (M-H) -, 111 (B) -; HRMS (ESI + ): calcd. for C10H16N3O4 (M+H) + : 242.1141; found: 242.1141.

  H NMR (CDCl3) δ 7.94 (m, 2H, Ph), 7.66 (d, J = 8.1, 1H, H-6), 7.57-7.33 (m, 5H, Ph), 6.75 (m, 2H, Ph), 5.58 (s, 1H, H-1'), 5.47 (d, J = 8.1, 1H, H-5), 4.95 (d, J = 13.6, 2H, CH2Ph), 4.63 (dd, J = 2.4, J = 12.9, 1H, H-5'), 4.55 (dd, J = 3.3, J = 12.9, 1H, H-5"), 4.25 (m, 1H, H-4'), 4.15 (d, J = 5.1, 1H, H-2'), 3.70 (s, 3H, CH3O), 2,85 (bs, 1H, OH-2'), 2.04 (m, 1H, H-3'), 1.07 (d, J = 6.8, 3H, CH3); 13 C NMR (CDCl3) δ 167.6 (CO), 164.0 (C-4), 160.6 (CH2Ph), 152.6 (C-2), 138.2 (C-6), 135.1-130.1 (Ph), 115.2 (CH2Ph), 102.7 (C-5), 95.1 (C-1'), 85.5 (C-4'), 80.2 (C-2'), 64.2 (C-5'), 56.7 (OCH3), 44.9 (CH2Ph), 37.7 (C-3'), 10.0 (CH3); UV (EtOH 95) λmax = 264 nm (ɛ = 11 500), 225 nm (ɛ = 27 900); MS: m/z (FAB > 0) 933 (2M+H) + , 467 (M+H) + ; m/z (FAB < 0) 465 (M-H) -, 231 (B) -.

  in water to afford the title compound 16 (121.8 mg, 23% yield) which was crystallized from EtOH 100. m.p. 156-157°C; 1 H NMR (CDCl3) δ 7.73 (d, J = 8.1 Hz, 1H, H-6), 7.40-7.33 (m, 2H, Ph), 6.74 (m, 2H, Ph), 6.16 (d, J = 6.1, 1H, H-1'), 5.68 (d, J = 8.1, 1H, H-5), 4.99 (m, 2H, CH2Ph), 3.91 (m, 2H, H-5', H-2'), 3.70 (s, 3H, CH3O), 3,67 (s, 1H, H-5"), 3.60 (m, 1H, H-4'), 2.13 (m, 1H, H-3'), 1.10 (d, J = 6.7, 3H, CH3); 13 C NMR (CDCl3) δ 162.8 (CO), 159.0 (C-4), 151.1 (C-2), 138.3 (C-6), 133.2-130.3 (Ph), 113.7 (Ph), 101.5 (C-5), 84.6 (C-4' ), 84.5 (C-1'), 69.5 (C-2'), 60.8 (C-5'), 55.2 (CH3O), 43.6 (CH2Ph), 37.5 (C-3'), 14.6 (CH3); UV (EtOH 95) λmax = 263 nm (ɛ =10 500), 221 nm (ɛ =15 200) ; IR υ = 2112 (s) cm -1 (N3); MS: m/z (FAB > 0) 388 (M+H) + ; m/z (FAB < 0) 231 (B) -. 1-(2-Azido-2,3-dideoxy-3-C-methyl-1-β-D-arabinofuranosyl)uracil (

  H NMR (DMSO-d6) δ 11.40 (bs, 1H, NH), 8.04 (d, J = 8.1, 1H, H-6), 6.13 (d, J = 6.5, 1H, H-1'), 5.61 (d, J = 8.1, 1H, H-5), 5.24 (pt, 1H, OH-5'), 4.33 (dd, J = 6.5, J = 10.2, 1H, H-2'), 3.75 (m, 1H, H-5'), 3.61 (m, 2H, H-4', H-5"), 2.03 (m, 1H, H-3'), 1.07 (d, J = 6.4, 3H, CH3); 13 C NMR (DMSO-d6) δ 163.0 (C-4), 150.5 (C-2), 140.5 (C-6), 100.9 (C-5), 84.4 (C-4'), 82.8 (C-1'), 68.4 (C-2'), 58.9 (C-5'), 36.0 (C-3'), 13.2 (CH3); IR υ = 2112 (s) cm -1 (N3); UV (EtOH 95) λmax = 261 nm (E = 10 000) ; MS: m/z (FAB > 0) 535 (2M+H) + , 268 (M+H) + , 113 (BH2) + ; m/z (FAB < 0) 533 (2M-H) -, 266 (M-H) -, 111 (B) -; HRMS (ESI + ): calcd. for C10H14N5O4 (M+H) + : 268.1046; found: 268.1046.

  text for Table of Contents here. ((The Table of Contents text should give readers a short preview of the main theme of the research and results included in the paper to attract their attention into reading the paper in full. The Table of Contents text should be different from the abstract and should be no more than 450 characters including spaces.))

  Table of Contents here.

Acknowledgements S.C. is particularly grateful to the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation, France, for a doctoral fellowship. This work was supported by Grants from the European Economic Community program 'Flavitherapeutics' (QLK3-CT-2001-00506).


Simon Mitton

Mohamed Ali

M A Bouhifd

From Crust to Core -A Chronicle of Deep Carbon Science by

Carbon is the fourth most abundant element in the solar system. Its presence significantly affects chemical and physical processes in the Earth's atmosphere, mantle and core. The importance of carbon also lies in the fact that it has been essential for the origin and evolution of life, that it is involved in an important fraction of the energy we use, and that it is assumed to play a fundamental role in the Earth's climate system. In From Crust to Core -A Chronicle of deep Carbon Science, Simon Mitton explores several centuries of philosophical and scientific inquiry on the nature of the Earth's interior, as based on the stories of over hundred scientists, and sheds light on the particular roles of deep carbon. The author is a Life Fellow at St. Edmund's College at University of Cambridge. His current research discipline is history of science, and his field of expertise is the history of astronomy and planetary science in the nineteenth and twentieth centuries. He is a member of the Deep Carbon Observatory (Carnegie Institution for Science, Washington DC) network of scientists whose mission is to understand better the element carbon in Earth.

Composed of 15 chapters, the book starts with why carbon in Earth matters, follows wit the origin of carbon and its delivery to Earth. In chapters 4-7, the author presents scientific adventures behind the determination of the age and of the physical and chemical interior of the Earth. In chapters 8-10, the reader will meet a number of remarkable scientists whose discoveries were to have major implications for understanding not only the dynamics of the Earth's interiors, but also the genesis of a new interdisciplinary field: oceanography. In chapters 12-14 the focus is about the cycles, reservoirs and the fluxes of carbon between the main Earth reservoirs, and also on the history of high-pressure physics and chemistry of carbon-bearing minerals under the Earth's mantle conditions. In the final chapter, research on deep life is told from the studies by the pioneer Alexander von Humboldt to our days. For every chapter, the author provides a very interesting list of references reporting some remarkable discoveries that changed our understanding of Earth-system science as a whole.

In summary, the book introduces the history of Earth's carbon all the way from it synthesis in the first generation of stars in our universe, to its incorporation in the solar nebula, where the Sun and planets formed nearly 4.5 billion years ago, to its final delivery to Earth with chondrites. The book takes the reader on a captivating trip with an excellent narrative style. Many discoveries, which led to enormous advances in broadening our understanding of the history of science in general, are told. For Earth and the nature of deep life. In a final note, the author reaches the philosophical conclusion that there is a great deal still to be discovered about the microbial environment of Earth's interiors that is highly relevant to the search for life elsewhere. Therefore, it seems entirely plausible that, right beneath our feet, deep-carbon science could yet uncover important clues on the origin of life in the universe. This is an excellent book for everyone interested in knowing the stories behind the key discoveries in Deep Carbon Science that includes (but not restricted to) the Big Bang, origin of carbon in the universe, formation of rocky planets, diamonds in the Earth's mantle and the signs of deep life. Additionally, From Crust to Core -A Chronicle of deep Carbon Science is well affordable for all "amateurs" and also professionals who are looking for the fascinating story of the evolution of Earth system science in general, and deep-carbon science in particular.

  Book review: « From Crust to Core -A Chronicle of deep Carbon Science » by Simon Mitton. Published at History of Geo-and Space Sciences, 12, 95-96, 2021 M. Ali Bouhifd Laboratoire Magmas et Volcans, CNRS & Université Clermont Auvergne Campus universitaire des Cézeaux, 6 Avenue Blaise Pascal, 63170 Aubière, France

  example, the discovery by Hans Bethe (Physics Nobel prize in 1967) who made a breakthrough regarding the synthesis of elements in stars, by showing the carbon role in the astrophysical conversion of mass into energy, validating Einstein's theory, E = mc 2 . The book also includes the history of the discovery of radioactivity, a process for which Becquerel, Pierre and Marie Curie were awarded the Noble prize in physics in 1903. This discovery would have a huge impact on the Earth's internal dynamics over its 4.5 billion years of history owing to the important amount of thermal energy produced by radioactive elements present within the Earth. The discovery of radioactivity provided a novel and robust way to measure the age of rock samples that made most other geological dating methods obsolete. The book includes the fascinating history of the theory of plate tectonics, and a history of research on deep diamonds, which can keep the chemical message of their formation billion or million of years ago. It also includes the history of the pioneering research on deep life during the last half a century of extraordinary progress on questions about the limits of life on