Emimycin and its nucleoside derivatives: Synthesis and antiviral activity

Accepted Manuscript Emimycin and its nucleoside derivatives: Synthesis and antiviral activity Elzbieta Plebanek, Eveline Lescrinier, Graciela Andrei, Robert Snoeck, Piet Herdewijn, Steven De Jonghe PII:

S0223-5234(17)31024-3

DOI:

10.1016/j.ejmech.2017.12.018

Reference:

EJMECH 9996

To appear in:

European Journal of Medicinal Chemistry

Received Date: 20 October 2017 Revised Date:

4 December 2017

Accepted Date: 5 December 2017

Please cite this article as: E. Plebanek, E. Lescrinier, G. Andrei, R. Snoeck, P. Herdewijn, S. De Jonghe, Emimycin and its nucleoside derivatives: Synthesis and antiviral activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract

ACCEPTED MANUSCRIPT

Emimycin and its nucleoside derivatives : synthesis and antiviral activity Elzbieta Plebanek,a Eveline Lescrinier,a Graciela Andrei,b Robert Snoeck,b Piet Herdewijn,a Steven De Jongheb* a

Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49 – bus 1041,

b

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3000 Leuven, Belgium

Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven,

Herestraat 49 - bus 1043, 3000 Leuven, Belgium

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*Corresponding author. E-mail: [email protected] ; Phone: +32 16 32 26 62

Abstract

The synthesis of emimycin, 5-substituted emimycin analogues and the corresponding ribo- and 2’-

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deoxyribonucleoside derivatives is described. Emimycin, its 5-substituted congeners and the ribonucleoside derivatives are completely devoid of antiviral activity against RNA viruses. In contrast, some of the 2’-deoxyribosyl emimycin derivatives are potent inhibitors of the replication of herpes simplex virus-1 and varicella-zoster virus, lacking cytotoxicity.

Keywords

1.

Introduction

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Emimycin, nucleoside, antiviral, pyrazine

Structural modification of the nucleobase moiety of natural pyrimidine nucleosides is an established

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strategy for the discovery of biologically active nucleoside analogues (Figure 1) [1]. A prominent class are the 5-substituted pyrimidine congeners. 5-Fluoro-2'-deoxyuridine (floxuridine) is an inhibitor of

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thymidylate synthase and is mainly used for the treatment of hepatic metastases which are a frequent complication of colorectal cancer [2]. (E)-5-(2-bromovinyl)-2’-deoxyuridine (BVDU) is a highly potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) infections [3]. DNA methyl transferase inhibitors, such as 5-azacytidine and 5-aza-2′-deoxycytidine (decitabine), are approved as chemotherapeutics for the treatment of myelodysplastic syndrome [4]. 6Azauridine inhibits the replication of different pathogenic flaviviruses, but is also rather cytostatic, ultimately leading to low selectivity indexes [5].

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Figure 1. Pyrimidine modified nucleoside analogues

The pyrazine ring resembles the pyrimidine moiety of the natural nucleobases uracil and thymine. In contrast to pyrimidine modifications, the pyrazine ring is not very well explored as potential isoster of natural pyrimidine nucleobases. A well-known example is favipiravir (Figure 2), a broad spectrum

the treatment of influenza virus infected patients [6].

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antiviral agent with activity against many RNA viruses, that received marketing approval in Japan for

Emimycin (or 1,2-dihydro-2-oxo-pyrazine 4-oxide) is a pyrazine analogue which structurally

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resembles uracil (Figure 2). It is endowed with antibacterial activity against Streptococcus faecium and Escherichia coli, yielding EC50 values of 8 µM and 10 µM, respectively. The corresponding ribonucleoside derivative has the same level of activity against these bacteria, suggesting that emimycin exerts its antibacterial activity via a glycosylation reaction, whereby intracellularly the corresponding ribosyl nucleoside derivative is formed, acting as the biologically active species [7]. The corresponding 2’-deoxyribo analogue is much more potent than emimycin with EC50 values of

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0.05 nM (against S. faecium) and 0.04 nM (against E. coli), suggesting a different molecular target [8].

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Figure 2. Pyrazine-based nucleoside analogues

Despite these promising biological data and the potential of nucleoside analogues to serve as antiviral drugs, no systematic antiviral evaluation of emimycin and its analogues has been performed. In this manuscript, the synthesis and antiviral evaluation of these compounds is described.

2.

Chemistry

2.1. Synthesis of (5-(m)ethyl)emimycin The synthesis of emimycine followed a literature procedure [9]. N-oxidation of 2-chloropyrazine 1 by reaction with 3-chloroperoxybenzoic acid (mCPBA) afforded 2-chloropyrazine 4-oxide 2. Treatment

ACCEPTED MANUSCRIPT of 2 with an aqueous sodium hydroxide solution afforded emimycin (compound 3) in excellent yield (Scheme 1). The position of the N-oxide functionality was unambiguously established by comparison of the 13C NMR spectral data with those previously described [9].

a

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Scheme 1. Synthesis of emimycina

Reagents and conditions: (a) mCPBA, DCE, 65°C, 30 h; (b) NaOH, H2O, 100°C, 2h 30 min,

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quantitative over 2 steps.

5-Methylemimycin 13a was prepared as shown in Scheme 2. Commercially available methylglyoxal 5

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was condensed with glycine amide hydrochloride under alkaline conditions [10], furnishing a mixture 7a/b of isomeric 5- and 6-substituted 2-hydroxypyrazines (in a ratio of 45:55), which could not be separated by flash chromatography on silica gel.

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Scheme 2. Synthesis of 5-methyl- and 5-ethylemimycina

a

Reagents and conditions: a) SeO2, dioxane, reflux, 6 h (for 6); b) glycine amide.HCl, MeOH, H2O,

NaOH, -30°C 30 min, rt, 3 h; c) BzCl, pyridine, rt, o/n; d) mCPBA, DCE, 60°C, o/n; e) 2M NaOCH3, MeOH.

In order to assign the correct regiochemistry, a heteronuclear multiple bond correlation (HMBC) spectrum of the mixture of regioisomers 7a/b was recorded (Figure 3). For the 5-substituted congener

ACCEPTED MANUSCRIPT 7a, HMBC correlations of C-7 with H-3 and H-6 were observed. In contrast, for the regioisomeric 6methylpyrazine analogue 7b, only a HMBC cross-peak of C-7 with H-5 was observed.

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Figure 3. Key HMBC correlations for compounds 7a, 7b and 8a.

The synthesis of 5-ethylemimycin followed a similar strategy. Oxidation of methyl ethyl ketone 4 with

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SeO2 yielded ethylglyoxal 6 [11]. The pyrazine ring was assembled by reaction of crude 6 with glycine amide. In contrast to the 5-methyl series, in case of the 5-ethylemimycin analogues, the major isomer

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8a was isolated in pure form after careful flash chromatography. The HMBC NMR spectrum of 8a indicated HMBC connectivities between C-5 and H-3 and between C-2 and H-6, indicating that the major isomer was indeed the 5-ethylpyrazine analogue 8a (Figure 3). Although both isomers 8a/8b could be separated by careful silica gel based flash chromatography, for further chemistry, the mixture was used as such.

The resulting 2-oxopyrazine analogues (the mixtures 7a/b and 8a/b, respectively) were protected with

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benzoyl chloride [7] to furnish the corresponding 2-benzoyloxy derivatives 9a/b and 10a/b. Both mixtures were used for an oxidation reaction with m-chloroperoxybenzoic acid affording pyrazine-Noxides 11a/b and 12a/b. At this stage, for the methyl-, as well as for the ethyl-substituted pyrazines, both regioisomers were separated by flash chromatography. The exact position of the methyl or ethyl

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group was determined based on 2D HMBC correlation spectra as previously discussed. For the 5substituted congeners 11a, two correlations of C-7 with H-3 and H-6 are observed. On the other hand, for the 6-substituted congener 11b, only a correlation of C-7 with H-5 is evident. For 5-ethylpyrazine

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4-oxide 12a, correlations of C-2 with H-3 and H-6 are visible, whereas for the 6-ethylpyrazine 4-oxide 12b only one HMBC connectivity of C-2 with H-3 was noticed. For the methyl substituted pyrazine analogues, the mixture 11a/b was used for further reaction. Cleavage of the benzoyl protecting groups of 11a/b under alkaline conditions gave the 2-oxo-1,2dihydropyrazine 4-oxides 13a/b. Both regioisomers were separated by silica gel flash chromatography and further purified by reversed phase HPLC, but only the desired isomer 13a was obtained in pure form. In the ethyl substituted pyrazine series, deprotection of the benzoyl ester started from pure 12a, affording the desired 5-ethylpyrazine 4-oxide 14a. The identified HMBC correlations that allowed us to determine the exact regiochemistry of 13a and 14a are indicated in Figure 4.

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2.2.

Synthesis of ribosyl derivatives of (5-(m)ethyl)emimycin

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Figure 4. Key HMBC correlations of compounds 13a and 14a.

The synthesis of emimycin-riboside 19a has been described before by glycosylation of persilylated emimycin with 1,2,3,5-tetra-O-acetyl β-D-ribofuranose and titanium tetrachloride in anhydrous 1,2-

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dichloroethane at reflux temperature. The authors claimed to isolate the β-anomer in low yield, although no structural proof that allows the exact assignment of the stereochemistry at the anomeric centre is provided [7]. However, when applying these reaction conditions in our hands (using both

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emimycin 3 and 5-methylemimycin 13a as coupling partner), the α-anomers 16b and 17b were isolated as the major products (entries 1 and 3 in Table 1). In contrast, when the same reaction was run at room temperature (entries 2,4 and 5 in Table 1), the desired β-anomers were isolated in greatly improved yield. Although pure anomers were isolated after flash chromatography, for the final deprotection step, we started from roughly purified mixtures. Hence, cleavage of the acetyl groups was achieved by treatment of 16a/b, 17a/b and 18a/b with sodium methoxide in anhydrous methanol. At

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this final stage, each pair of anomers was separated and isolated by reversed phase HPLC (Scheme 3).

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Scheme 3. Synthesis of (5-(m)ethyl)emimycin ribosidesa

a

Reagents and conditions: a) (i) BSA, DCE, 70 min, rt; (ii) Method A : TiCl4, reflux ; Method B: TiCl4, room temperature ; b) NaOMe, MeOH, rt, 30 min.

ACCEPTED MANUSCRIPT Table 1. Glycosylation reaction conditions and yields. Nucleobase

Methoda

1

3

A

2

3

B

3

13a

A

4

13a

B

17a

5

14a

B

18a

Yieldb

Products 16a: 30%

31%

16b: 70%

42%

16a 17a: 85% 17b: 15%

11%

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a

Entry

79% 80%

Method A: (i) BSA, DCE, 70 min, rt; (ii) TiCl4, reflux ; Method B: (i) BSA, DCE, 70 min, rt; (ii) TiCl4, rt. Combined yield of both anomers.

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b

The assignment of the α/β anomers was done based on ROESY NMR spectroscopy of the mixture. As

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a representative example, the key correlations observed for compounds 16a and 16b are shown in Figure 5. The existence of ROESY correlations between H-6 and H-4’ is indicative for the α-anomer 16b, and the correlations of H-6 with H-1’, H-2’ and H-3’ allowed to identify the β-anomer 16a.

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Figure 5. Key ROESY correlations of 16a and 16b

Synthesis of 2’-deoxyribosyl derivatives of (5-(m)ethyl)emimycin

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The synthesis of 1,2-dihydro-1-(2-deoxy-β-D-ribofuranose)-2-oxopyrazine 4-oxide 26a has been described previously [8]. According to this procedure, 2-(trimethylsilyl)oxypyrazine-4-oxide was condensed with 2-deoxy-3,5-di-O-p-chlorobenzoyl-α-D-erythro-pentofuranosyl chloride in benzene at room temperature for 3 days. However, this procedure was hard to reproduce in our hands and therefore an alternative procedure was worked out. The nucleobases 3, 13a or 14a were reacted first with the strong base DBU, followed by the addition of the Hoffer’s chlorosugar 22. This procedure allowed to isolate the desired compounds 23a/b, 24a/b and 25a/b. Noteworthy is the drastically shortened reaction time (2 hours versus 3 days for the published procedure). These mixtures were quickly purified by flash chromatography and characterized by mass spectroscopy. No efforts were made to separate both anomers or to run NMR spectra. Finally, alkaline removal of the toluoyl protecting groups furnished the 2’-deoxyribose emimycin analogues 26a/b, 27a/b and 28a/b in 20%

ACCEPTED MANUSCRIPT yield over two steps. The anomers were obtained in pure form after purification of the anomeric mixture by RP-HPLC. In all three cases, the major isomer was the β-anomer (60-80% of the β-anomer and 20-40% of the α-anomer). The anomeric configuration of the 2’-deoxyribose analogues 26a/b, 27a/b and 28a/b was determined based on the apparent multiplicity of the signal corresponding to the proton at C-1’ in the 1H NMR spectra. The H-1’ of the α-anomers (compounds 26b, 27b and 28b) show the characteristic doublet of doublets, whereas for the β-anomers (compounds 26a, 27a and 28a)

for 2’-deoxyribosylemimycin [8].

Reagents and conditions: a) DBU, acetonitrile, rt; b) NaOMe, MeOH, rt, 20 min.

3.

Antiviral evaluation

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Scheme 4. Synthesis of 2’-deoxyriboside-(5-(m)ethyl)emimycin

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the characteristic “pseudo-triplet” is evident [12]. This is in complete agreement with literature data

It has been demonstrated that favipiravir acts as a prodrug that is intracellularly phosphoribosylated

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yielding the ribose-5’-monophosphate metabolite. Further phosphorylation yields the ribosetriphosphate analogue which is the pharmacologically active compound. These findings and the structural resemblance of favipiravir and emimycin (Figure 2) prompted us to investigate the antiviral activity of emimycin 3, its 5-substituted analogues 13a and 14a, as well as the corresponding ribonucleoside congeners 19a/b, 20a/b and 21a/b. No antiviral activity against different influenza strains, nor any cytotoxicity was observed, even at the highest tested concentration of 100 µM (Table 1). Besides its anti-influenza activity, favipiravir blocks the replication of many other RNA viruses, including

arenaviruses,

phleboviruses,

hantaviruses,

flaviviruses,

enteroviruses,

alphavirus,

paramyxovirus and noroviruses. Therefore, as representative example, antiviral activity against the respiratory syncytial virus (RSV) in HeLa cells was also evaluated, but similarly no antiviral activity or cytotoxicity was seen at 100 µM (data not shown).

ACCEPTED MANUSCRIPT Table 1. Antiviral and cytotoxic properties of emimycin and ribo-emimycin analogues in Madin Darby canine kidney (MDCK) cells. EC50 (µM)a

Cmpd

Influenza A/H3N2

Influenza B

A/Ned/378/05

A/HK/7/87

B/Ned/537/05

>100

>100

>100

>100

>100

13a

>100

>100

>100

>100

>100

14a

>100

>100

>100

>100

>100

19a

>100

>100

>100

>100

>100

19b

>100

>100

>100

>100

>100

20a

>100

>100

>100

>100

>100

20b

>100

>100

>100

>100

>100

21a

>100

>100

>100

>100

21b

>100

>100

>100

>100

>100

MCCc

MDCKd

MDCKd

>100

>100

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3

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

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>100

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a

Influenza A/H1N1

CC50b

50% Effective concentration or concentration producing 50% inhibition of virus-induced cytopathic effect, as

determined by visual scoring of the CPE, or by measuring the cell viability with the colorimetric formazan-based MTS assay. b

50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-

based MTS assay.

Minimum compound concentration that causes a microscopically detectable alteration of normal cell

morphology. d

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c

MDCK cells: Madin Darby canine kidney cells

The 2’-deoxyribose emimycine nucleosides 26a/b, 27a/b and 28a/b were assayed for their antiviral

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activity against different DNA viruses from the Herpesviridae family: herpes simplex virus 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV) and human cytomegalovirus (HCMV). Acyclovir,

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BVDU and cidofovir were included as positive controls. As can be derived from the data in Table 3, all 2’-deoxyribose emimycine nucleosides completely lacked antiviral activity against HCMV. The 2’deoxyribonucleosides 26a and 26b with an emimycin nucleobase (structurally similar to uracil) were also completely devoid of inhibitory activity against VZV, HSV-1 and HSV-2. The presence of 5methylemimycin (related to the natural thymidine nucleobase) in compound 27a conferred potent antiviral activity against VZV (EC50 = 0.99 µM) and HSV-1 (EC50 = 1.79 µM), with a selectivity index (SI, ratio CC50/EC50) of more than 416 (for VZV) and 230 (for HSV-1). Compound 27a showed a much weaker activity against HSV-2 (EC50 = 17.48 µM) with a SI of 24. Compound 27a was 7-fold less active when tested against the mutant TK- VZV strain (EC50 = 6.91 µM) and 30-fold less active against the mutant TK- HSV-1 strain (EC50 = 59.44 µM). 5-Ethylemimycin 2’-deoxyribose 28a has a very similar profile as its 5-methyl congener 27a, but its antiviral activity is less pronounced. It is

ACCEPTED MANUSCRIPT endowed with an EC50 value of 7.77 µM against VZV (SI = 50) and 8.55 µM against HSV (SI = 46), whereas it lacks activity for HSV-2 and for TK- HSV-1 and VZV strains. As expected, the α-anomers (27b and 28b) were completely devoid of antiviral activity. The fact that compounds 27a and 28a are much less active against the TK- VZV and HSV mutants when compared to the wild type strains, demonstrate that they need to undergo phosphorylation before being antivirally active. Therefore, most likely, the compounds folllow the classical mode of action of

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nucleoside analogues, by being incorporated in viral DNA, leading to chain termination.

Table 3. Antiviral and cytotoxic properties of 2’-deoxyribose emimycin analogues in human

EC50 (µM)a HCMV

VZV

AD-169

Davis

strain

strain

26a

>438

26b

TK+

HSV

TK- 07-

OKA

HSV-1

HSV-1

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Cmpd

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embryonic lung (HEL) cells.

1.

(KOS)

(KOS

(G)

CC50b

MCCc

HEL

HEL

strain

>438

>438

>438

>438

>438

>438

>438

NDd

>87

>438

438

>438

>438

>438

>438

>438

NDd

27a

>412

>412

0.99

6.91

1.79

59.44

17.48

>412

169

27b

>82

>412

328

>412

>412

>412

>412

>412

NDd

28a

>78

>78

7.77

138

8.55

>390

145

>390

NDd

28b

>78

>78

142

174

174

>390

204

>390

NDd

Acyclovir

NDd

NDd

5.31

53.51

0.38

88.81

0.29

>444

>444

d

d

0.035

6.66

0.068

20.14

30.02

>300

>300

2.81

1.56

1.97

>358

>358

ND

a

1.04

1.58

ND

d

ND

d

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Cidofovir

ND

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strain

BVDU

ACVr)

HSV-2

Cytotoxicity

Effective concentration required to reduce virus plaque formation by 50%. Data are the mean of two

independent experiments.

Cytotoxic concentration required to reduce cell growth by 50%. Data are the mean of two independent

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b

experiments. c

Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. Data

are the mean of two independent experiments. d

Not determined

4.

Conclusion

The synthesis of emimycin, 5-substituted emimycin analogues and the corresponding ribo- and 2’deoxyribonucleoside derivatives was undertaken and compounds were evaluated for antiviral activity. Because of the structural resemblance of the emimycin analogues and their ribosyl derivatives to favipiravir, they were tested for antiviral activity against two representative RNA viruses (influenza virus and RSV), but were found to be completely inactive. In contrast, the 2’-deoxyribose derivatives

ACCEPTED MANUSCRIPT with either 5-methylemimycin or 5-ethylemimycin as nucleobase displayed selective antiviral activity against HSV-1 and VZV.

5. Experimental section 5.1. Chemistry For all reactions, analytical grade solvents were used. NMR spectra were recorded on a Bruker 13

C NMR, 75 MHz), 500 MHz (1H NMR, 500 MHz,

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Avance 300 MHz (1H NMR, 300 MHz,

13

C

NMR, 125 MHz) or 600 MHz (1H NMR, 600 MHz, 13C NMR, 150 MHz) with tetramethylsilane as internal standard for 1H NMR spectra and (D6)-DMSO (39.52 ppm) or CDCl3 (77.16 ppm) or CD3OD (49.00 ppm) for 13C NMR spectra. Abbreviations used are: s = singlet, d = doublet, t = triplet, q =

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quartet, m = multiplet, br = broad. Coupling constants are expressed in Hz. High resolution mass spectrometry spectra were measured on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Samples were infused at 3 µL/min and

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spectra were obtained in positive or negative ionization mode with a resolution of 15000 (FWHM) using leucine encephalin as lock mass. Pre-coated aluminum sheets (Fluka Silica Gel/TLC-cards, 254 nm) were used for TLC. Flash column chromatography was performed on ICN silica gel 63-200 60 Å. Preparative RP-HPLC purifications were performed on an X-Bridge Prep C18 OBD column (5 µm, 19 mm × 150 mm) using a mixture of acetonitrile and water as mobile phase (starting from 1%

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acetonitrile, gradually increasing to 99% acetonitrile).

5.1.1. 2-Chloropyrazine 4-oxide (2)

To a solution of 2-chloropyrazine 1 (0.8 mL, 8.96 mmol) in dry 1,2-dichloroethane (15 mL) was added 3-chloroperbenzoic acid (mCPBA, 2.47 g, 14.34 mmol, 1.6 equiv). The reaction mixture was stirred at

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65°C for 30 hours and then cooled to room temperature. The mixture was washed with a 1M NaOH solution, a saturated Na2S2O3 solution, water and then dried over Na2SO4. The filtrate was concentrated under reduced pressure, to give the title compound as white solid (0.95 g, 81%). 1H NMR

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(300 MHz, CDCl3) δ 8.24 (d, J = 4.1 Hz, 1H, H-6), 8.18 – 8.10 (m, 1H, H-3), 8.01 (dd, J = 4.1, 1.5 Hz, 1H, H-5). 13C NMR (75 MHz, CDCl3) δ 151.9 (C-2), 146.1 (C-6), 133.6 (C-3), 133.3 (C-5).

5.1.2. 2-Pyrazinone 4-oxide (3) A solution of 2 (0.40 g, 3.06 mmol, 1 equiv) in 1M NaOH (10 mL) was stirred for 2.5 hours at 100°C in a sealed tube. After cooling down to room temperature, the solution was passed through the resin “Dowex 50WX8 hydrogen form” and eluted with distilled water. Concentration of collected aqueous solution afforded the title compound as a creamy solid (0.34 g, 100%). 1H NMR (500 MHz, DMSOd6) δ 7.56 (d, J = 5.5 Hz, 1H, H-6), 7.52 (bs, 1H, H-3), 7.16 (dd, J = 5.5, 1.8 Hz, 1H, H-5). 13C NMR (75 MHz, DMSO) δ 158.4 (C-2), 130.7 (C-6), 128.8 (C-3), 121.0 (C-5). HRMS (ESI) m/z [M+H]+ calcd for C4H5N2O2 113.0346, found 113.0342.

ACCEPTED MANUSCRIPT 5.1.3. 2-Hydroxy-5-methylpyrazine (7a) and 2-hydroxy-6-methylpyrazine (7b) A solution of glycine amide hydrochloride (6.0 g, 54 mmol) in MeOH (100 mL) and H2O (10 mL) was cooled at -30/-40°C. Pyruvic aldehyde (13.8 mL, 65 mmol, 1.2 equiv) was rapidly added. Following the dropwise addition of 12.5 M NaOH solution (10.8 mL, 135 mmol), the mixture was stirred at 30°C for 30 minutes and then at room temperature for 3 hours. The reaction mixture was cooled down in an ice bath and then HCl (20 mL) and K2CO3 (till neutral pH) were added. The mixture was filtered,

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washed with MeOH and the filtrate was concentrated in vacuo. The residue was solubilized in CHCl3 and H2O. The phases were separated and the aqueous phase was extracted with CHCl3. The combined organic phases were dried over Na2SO4 and concentrated to furnish the title compounds as a silverish black solid, being a mixture of two regioisomers (2.33 g, 84%). 1H NMR (600 MHz, CDCl3) δ 8.21 (d,

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J = 1.4 Hz, 1H, H-3, 7b), 8.04 (bs, 1H, H-3, 7a), 7.34 (bs, 1H, H-6, 7a), 7.08 – 7.04 (m, H-5, 7b), 2.35 – 2.33 (m, 3H, CH3, 7a), 2.33 – 2.32 (m, 2H, CH3, 7b). 13C NMR (151 MHz, CDCl3) δ 159.0 (C2, 7a), 157.4 (C2, 7b), 148.0 (C3, 7b), 145.4 (C3, 7a), 137.2 (C5, 7a), 134.3 (C6, 7b), 124.7 (C6, 7a), 123.1

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(C5, 7b), 19.7 (CH3, 7b), 16.4 (CH3, 7b). HRMS (ESI) m/z [M+H]+ calcd for C5H7N2O 111.0553, found 111.0556.

5.1.4. 2-Benzoyloxy-5-methylpyrazine (9a) and 2-benzoyloxy-6-methylpyrazine (9b) To a solution of 7a/b (0.47 g, 4.27 mmol, 1 equiv) in dry pyridine (8 mL) was dropwise added benzoyl chloride (0.55 mL, 4.70 mmol, 1.1 equiv). The resulting mixture was stirred overnight at room

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temperature and then poured into ice cold water. The phases were separated and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with water (2 times), dried over Na2SO4 and concentrated. The crude residue was purified by silica gel flash column chromatography (Hexane/EtOAc from 9:1 to 7:3) to afford the title compound as a yellowish oil (0.81

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g, 89%). HRMS (ESI) m/z [M+H]+ calcd for C12H11N2O2 215.0815, found 215.0823.

(11b)

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5.1.5. 2-Benzoyloxy-5-methylpyrazine 4-oxide (11a) and 2-benzoyloxy-6-methylpyrazine 4-oxide

mCPBA (0.94 g, 2.85 mmol) was added to a solution of 9a/b (0.61 g, 2.85 mmol) in 1,2dichloroethane (10 mL). The reaction mixture was stirred overnight at 60°C and then cooled to room temperature. The mixture was washed with a saturated NaHCO3 solution and water, dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by silica gel flash column chromatography, allowing the separation of both isomers (0.39 g, 59%). Compound 11a 1

H NMR (600 MHz, CDCl3) δ 8.24 – 8.17 (m, 2H, H-10, H-14), 8.09 – 8.04 (m, 1H, H-3), 8.00 – 7.94

(m, 1H, H-6), 7.72 – 7.66 (m, 1H, H-12), 7.56 – 7.51 (m, 2H, H-11, H-13), 2.52 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3) δ 163.6 (C-8), 157.3 (C-2), 155.7 (C-5), 134.7 (C-12), 131.5 (C-6), 130.7 (C-10, C-14), 128.9 (C-11, C-13), 128.0 (C-9), 125.8 (C-3), 21.7 (CH3).

ACCEPTED MANUSCRIPT Compound 11b 1

H NMR (500 MHz, CDCl3) δ 8.31 – 8.27 (m, 1H, H-5), 8.28 – 8.22 (m, 1H, H-3), 8.23 – 8.17 (m, 2H,

H-10, H-14), 7.71 – 7.65 (m, 1H, H-12), 7.57 – 7.49 (m, 2H, H-11, H-13), 2.47 (s, 3H, CH3).13C NMR (126 MHz, CDCl3) δ 163.8 (C-8), 156.4 (C-2), 144.5 (C-5), 143.0 (C-6), 134.6 (C-12), 130.7 (C-10, C-14), 128.9 (C-11, C-13), 128.0 (C-3), 14.2 (CH3). HRMS (ESI) m/z [M+Na]+ calcd for

5.1.6. 5-Methyl-2-oxo-1,2-dihydropyrazine 4-oxide (13a)

RI PT

C12H10N2O3Na 253.0584, found 253.0586.

To a solution of 11a/b (0.146 g, 0.63 mmol) in MeOH (5 mL) was added dropwise a 2 M NaOMe solution in MeOH until pH 9-10. Then, Dowex H+ was added until neutral pH. The mixture was

SC

filtered and washed with methanol. The filtrate was concentrated. The crude solid was washed with a mixture of hexane and ethylacetate (in a ratio of 6:4) to give the title compound as white solid (0.062 g, 79%). 1H NMR (300 MHz, MeOD) δ 7.66 (s, 1H, H-3), 7.50 (s, 1H, H-6), 2.17 (s, 3H, CH3). 13C

M AN U

NMR (126 MHz, MeOD) δ 161.0 (C2), 132.1 (C5), 130.2 (C3), 130.1 (C6), 13.1 (CH3). HRMS (ESI) m/z [M+H]+ calcd for C5H7N2O2 127.0502, found 127.0500. 5.1.7. 2-Oxobutanal (6)

To a solution of methyl ethyl ketone 4 (10.0 mL, 112 mmol) in dioxane (20 mL) was added selenium(IV)oxide (30 g, 269 mmol). The reaction mixture was refluxed for 6 hours, cooled down to

TE D

room temperature and diluted with a mixture of chloroform and methanol (in a ratio of 1:1). This solution was filtered through a pad of Celite and washed with a chloroform/methanol mixture. The filtrate was concentrated to give the title compound (12 g), which was directly used in the next step,

EP

without further purification.

5.1.8. 2-Hydroxy-5-ethylpyrazine (8a) and 2-hydroxy-6-ethylpyrazine (8b) A solution of glycine amide hydrochloride (10.2 g, 92 mmol) in MeOH (150 mL) and H2O (18 mL)

AC C

was cooled at -30/-40°C. A solution of crude 6 (112 mmol, 1.2 equiv) in MeOH (25 mL) was quickly added. A 12.5 M NaOH solution (18.3 mL, 230 mmol, 2.5 equiv) was added dropwise and the resulting mixture was stirred at -30°C for 30 minutes and then at room temperature for 3 hours. The reaction was cooled in an ice bath. Then, HCl (28 mL) and NaHCO3 (22.5 g) were added. The mixture was filtered, washed with MeOH and the filtrate was concentrated in vacuo. The crude residue was solubilized in CHCl3 and a small volume of H2O. The phases were separated and the aqueous phase was extracted with CHCl3. The combined organic phases were dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (CH2Cl2/MeOH from 1:0 to 94:6) to give pure 8a as a beige solid (1.01 g, 7% over 2 steps). 1H NMR (500 MHz, CDCl3) δ 8.25 – 8.17 (m, 1H, H-3), 7.08 – 6.99 (m, 1H, H-6), 2.67 – 2.56 (m, 2H, CH2), 1.23 (t, J = 7.5 Hz, 3H, CH3).

ACCEPTED MANUSCRIPT 13

C NMR (126 MHz, CDCl3) δ 157.8 (C-2), 148.1 (C-3), 139.6 (C-5), 122.3 (C-6), 26.9 (CH2), 13.43

(CH3). HRMS (ESI) m/z [M+H]+ calcd for C6H9N2O 125.0709, found 125.0707. 5.1.9. 2-Benzoyloxy-5-ethylpyrazine (10a) and 2-benzoyloxy-6-ethylpyrazine (10b) Benzoyl chloride (0.61 mL, 5.23 mmol) was added dropwise to a solution of 8a/b (0.59 g, 4.75 mmol, 1 equiv) in dry pyridine (10 mL). The resulting mixture was stirred overnight at room temperature and

RI PT

then poured into ice cold water. The phases were separated and the aqueous phase was extracted with dichloromethane. The combined organic phases were washed with water (2 times), dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (Hexane/EtOAc from 95:5 to 80:20) to give the title compounds as a yellowish oil (0.63 g, 58%).

SC

HRMS (ESI) m/z [M+H]+ calcd for C13H13N2O2 229.0971, found 229.0970.

5.1.10. 2-Benzoyloxy-5-ethylpyrazine 4-oxide (12a) and 2-benzoyloxy-6-ethylpyrazine 4-oxide

M AN U

(12b)

mCPBA (0.63 g, 1.9 mmol, 1 equiv) was added to a solution of 10a/b (0.435 g, 2.9 mmol, 1 equiv) in 1,2-dichloroethane (7 mL). The reaction mixture was stirring overnight at 60°C and then cooled to room temperature. The mixture was washed with a saturated NaHCO3 solution and water, dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography yielding the pure title compounds (0.338 g, 73% combined yield, 12a = 0.207 g, 12b = 0.131 g). 1

TE D

Compound 12a

H NMR (600 MHz, CDCl3) δ 8.21 (bs, 1H, H-6), 8.19 (bs, 1H, H-3), 8.17 – 8.09 (m, 2H, H-11, H-

15), 7.68 – 7.58 (m, 1H, H-13), 7.54 – 7.41 (m, 2H, H-12, H-14), 2.85 (q, J = 7.5 Hz, 2H, CH2), 1.30 (t, J = 7.5 Hz, 3H, CH3). 13C NMR (151 MHz, CDCl3) δ 163.7 (C9), 156.1 (C2), 147.1 (C5), 143.2 Compound 12b 1

EP

(C6), 134.5 (C13), 130.5 (C11, C15, C10), 128.8 (C12, C14), 127.9 (C3), 21.0 (CH2), 10.1 (CH3). H NMR (600 MHz, CDCl3) δ 8.22 – 8.12 (m, 2H, H-11, H-15), 8.04 (d, J = 1.4 Hz, 1H, H-3), 8.01 –

AC C

7.92 (m, 1H, H-5), 7.70 – 7.58 (m, 1H, H-13), 7.56 – 7.43 (m, 2H, H-12, H-14), 2.75 (q, J = 7.6 Hz, 2H, CH2), 1.29 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (151 MHz, CDCl3) δ 163.5 (C9), 160.6 (C2), 157.2 (C6), 134.6 (C13), 130.7 (C5), 130.6 (C11, C15), 128.8 (C12, C14), 127.8 (C10), 125.7 (C3), 28.6 (CH2), 12.6 (CH3). HRMS (ESI) m/z [M+Na]+ calcd for C13H12N2O3Na 267.0740, found 267.0738. 5.1.11. 5-Ethyl-2-oxo-1,2-dihydropyrazine 4-oxide (14a) To a solution of 12a (0.20 g, 0.8 mmol) in methanol (8 mL) was added dropwise a 2M NaOMe in methanol solution until pH 9-10. Then, Dowex H+ was added until neutral pH. The resulting mixture was filtered and washed with methanol. The filtrate was concentrated in vacuo. The crude solid was washed with diethylether to give the title compound as a white solid (0.112 g, 97%). 1H NMR (600 MHz, MeOD) δ 7.67 (s, 1H, H-3), 7.46 (s, 1H, H-6), 2.67 (q, J = 7.4 Hz, 2H, CH2), 1.21 (t, J = 7.4 Hz,

ACCEPTED MANUSCRIPT 3H, CH3). 13C NMR (151 MHz, MeOD) δ 161.11 (C2), 136.77 (C5), 130.26 (C3), 129.71 (C6), 21.25 (CH2), 11.64 (CH3). HRMS (ESI) m/z [M+H]+ calcd for C6H9N2O2 141.0658, found 141.0663. 5.1.12.

4-((2R,3R,4R,5R)-3,4-diacetoxy-5-(acetoxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4-

dihydropyrazine

1-oxide

(16a)

and

4-((2S,3R,4R,5R)-3,4-diacetoxy-5-

(acetoxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4-dihydropyrazine 1-oxide (16b)

RI PT

Method A

To a suspension of 3 (0.50 g, 4.46 mmol) and β-D-ribofuranose 1,2,3,5-tetraacetate 15 (1.29 g, 4.05 mmol) in dry 1,2-dichloroethane (15 mL) was added bis(trimethylsilyl)acetamide (BSA, 1.09 mL, 4.46 mmol) under an argon atmosphere. The mixture was stirred for 70 minutes at room temperature

SC

and then TiCl4 (1.20 mL, 10.94 mmol) was added. The resulting solution was refluxed for 4 hours and stirred at room temperature overnight. The solution was poured into a saturated NaHCO3 solution, filtered through Celite and washed with dichloromethane. The combined organic layers were washed

M AN U

with water, dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by silica gel flash column chromatography (CH2Cl2/acetone from 10:1 to 7:1) yielding an anomeric mixture (16a: 30%; 16b: 70%) of the title compounds as a yellowish oil (0.39 g, 31% combined yield). 1

H NMR (600 MHz, CDCl3) δ 7.56 – 7.51 (m, 1.44 H, H-3), 7.47 (d, J = 6.1 Hz, 0.44 H, β H-6), 7.44

(d, J = 6.1 Hz, 1H, α H-6), 7.11 (dd, J = 6.1, 2.0 Hz, 1H, α H-5), 7.09 (dd, J = 6.1, 2.0 Hz, 1H, β H-5), 6.43 (d, J = 4.9 Hz, 1H, α H-1’), 6.14 (d, J = 4.6 Hz, 0.44H, β H-1’), 5.82 (t, J = 4.9 Hz, 1H, αH-2’),

TE D

5.44 (t, J = 5.1 Hz, 1H, αH-3’), 5.39 – 5.36 (m, 0.44 H, βH-2’), 5.31 – 5.29 (m, 0.44 H, βH-3’),4.62 – 4.57 (m, 1H, αH-4’), 4.45 – 4.39 (m, 0.88 H, βH-4’, H-5’), 4.38 – 4.32 (m, 1.44 H, H5’), 4.23 – 4.16 (m, 1H, H-5’), 2.18 – 1.96 (m, 13H, CH3).13C NMR (151 MHz, CDCl3) δ 170.4 (C=O Ac), 170.2 (C=O Ac), 169.7 (C=O Ac), 169.6 (C=O Ac), 169.2 (C=O Ac), 168.5 (C=O Ac), 157.0 (βC2), 156.8

EP

(αC2), 129.4 (βC3), 129.0 (αC3), 127.1 (αC6), 126.3 (βC6), 122.2 (βC5), 121.1 (αC5), 87.4 (βC1’), 84.5 (αC1’), 80.5 (αC4’), 80.3 (βC4’), 73.5 (βC2’), 71.0 (αC3’), 70.0 (αC2’), 69.9 (βC3’), 63.0 (αC5’), 62.8 (βC5’), 20.9 (CH3), 20.6 (CH3), 20.5 (CH3), 20.4 (CH3). HRMS (ESI) m/z [M+H]+ calcd for

AC C

C15H19N2O9 371.1085, found 371.1092. Method B

To a suspension of 3 (0.20 g, 1.8 mmol) and β-D-ribofuranose 1,2,3,5-tetraacetate (0.51 g, 1.6 mmol) in dry 1,2-dichloroethane (6 mL) was added bis(trimethylsilyl)acetamide (BSA, 0.59 mL, 2.4 mmol) under an argon atmosphere. The mixture was stirred for 70 minutes at room temperature and then cooled on ice. TiCl4 (0.47 mL, 4.3 mmol) was added. The solution was stirred at room temperature overnight. The solution was poured into a saturated NaHCO3 solution, filtered through Celite and washed with dichloromethane. The combined organic layers were washed with water, dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by column chromatography (CH2Cl2/acetone from 10:1 to 7:1) to afford pure 16a as a yellowish oil (0.25 g, 42% yield).

ACCEPTED MANUSCRIPT 1

H NMR (600 MHz, CDCl3) δ 7.53 (d, J = 2.0 Hz, 1H, H-3), 7.49 (d, J = 6.1 Hz, 1H, H-6), 7.09 (dd, J

= 6.1, 1.9 Hz, 1H, H-5), 6.14 (d, J = 4.5 Hz, 1H, H-1’), 5.37 (t, J = 5.0 Hz, 1H, H-2’), 5.31 – 5.24 (m, 1H, H-3’), 4.47 – 4.39 (m, 2H, H-4’, H-5’), 4.37 – 4.30 (m, 1H, H-5’), 2.13 (s, 3H, CH3), 2.12 – 2.09 (m, 6H, CH3). 13C NMR (151 MHz, CDCl3) δ 170.2 (C=O Ac), 169.7 (C=O Ac), 169.6 (C=O Ac), 157.0 (C2), 129.4 (C3), 126.3 (C6), 122.1 (C5), 87.4 (C1’), 80.2 (C4’), 73.5 (C2), 69.7 (C3), 62.7

found 371.1077.

RI PT

(C5), 20.9 (CH3), 20.6 (CH3), 20.6 (CH3). HRMS (ESI) m/z [M+H]+ calcd for C15H19N2O9 371.1085,

5.1.13. 4-((2R,3R,4R,5R)-3,4-diacetoxy-5-(acetoxymethyl)tetrahydrofuran-2-yl)-6-methyl-3-oxo3,4-dihydropyrazine

1-oxide

(17a)

and

4-((2S,3R,4R,5R)-3,4-diacetoxy-5-

SC

(acetoxymethyl)tetrahydrofuran-2-yl)-6-methyl-3-oxo-3,4-dihydropyrazine 1-oxide (17b) Method A

To a suspension of 13a (0.22 g, 1.73 mmol) and β-D-ribofuranose 1,2,3,5-tetraacetate (0.50 g, 1.57

M AN U

mmol) in dry 1,2-dichloroethane (6 mL) was added bis(trimethylsilyl)acetamide (BSA, 0.42 mL, 1.73 mmol) and the mixture was stirred for 70 min at room temperaturę. Then, TiCl4 (0.77 mL, 4.24 mmol) was added. The resulting solution was heating at reflux for 4 hours and then stirred at room temperature overnight. The solution was poured into a saturated NaHCO3 solution, filtered through Celite and washed with dichloromethane. The combined collected organic phases were washed with water, dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by silica gel flash

TE D

chromatography (CH2Cl2/Acetone from 1:0 to 8:2) to give the title compound as an anomeric mixture (0.068 g, 11% combined yield, 17a: 15%, 17b: 85%). 1

H NMR (500 MHz, CDCl3) δ 7.60 (s, 1H, α/β H-3), 7.35 (s, 0.17H, β H-6), 7.34 (s, 1H, α H-6), 6.43

(d, J = 4.9 Hz, 1H, α H-1’), 6.16 (d, J = 4.7 Hz, 0.17 H, β H-1’), 5.79 (t, J = 4.9 Hz, 1H, α H-2’), 5.42

EP

(t, J = 5.1 Hz, 1H, α H-3’), 5.37 – 5.34 (m, 0.17 H, β H-2’), 5.31 – 5.27 (m, 0.17 H, β H-3’), 4.65 – 4.53 (m, 1H, α H-4’), 4.41 – 4.30 (m, 1.53 H, β H-4’, β H-5’, α H-5’), 4.21 – 4.15 (m, 1H, α H-5’), 2.23 (s, 3H, α CH3), 2.19 (s, 0.59H, β CH3), 2.15 – 2.05 (m, 4.53 H, 3 x (β CH3-Ac), α CH3-Ac), 2.01

AC C

(s, 3H, α CH3-Ac), 1.94 (s, 3H, α CH3-Ac).

13

C NMR (75 MHz, CDCl3) δ 170.3 (C=O Ac), 169.0

(C=O Ac), 168.4 (C=O Ac), 157.0 (β C2), 156.8 (α C2), 130.4 (β C5), 129.1 (α C5),129.0 (β C3), 128.6 (α C3), 124.3 (α C6), 123.5 (β C6), 87.1 (β C1’), 84.3 (α C1’), 80.4 (α C4’), 80.2 (β C4’), 73.5 (β C2’), 71.1 (α C3’), 70.0 (α C2’, β C3’), 63.0 (α C5’), 62.9 (β C5’), 20.8 (CH3 Ac), 20.4 (CH3 Ac), 20.3 (CH3 Ac), 13.7 (β CH3), 13.6 (α CH3). HRMS (ESI) m/z [M+H]+ calcd for C16H21N2O9 385.1241, found 385.1240. Method B To a suspension of 13a (0.20 g, 1.6 mmol) and β-D-ribofuranose 1,2,3,5- tetraacetate (0.48 g, 1.5 mmol) in dry 1,2-dichloroethane (6 mL) was added bis(trimethylsilyl)acetamide (BSA, 0.56 mL, 2.3 mmol) and the mixture was stirred for 70 minutes at room temperaturę. The reaction was then cooled on ice and TiCl4 (0.45 mL, 4.1 mmol) was added. The resulting solution was stirred at room

ACCEPTED MANUSCRIPT temperature overnight. The solution was poured into a saturated NaHCO3 solution, filtered through Celite and washed with dichloromethane. The combined organic phases were washed with water, dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (CH2Cl2/Acetone from 1:0 to 8:2) to give compound 17a as a yellowish solid (0.46 g, 79%). 1

H NMR (500 MHz, CDCl3) δ 7.62 (s, 1H, H-3), 7.35 (bs, 1H, H-6), 6.19 (d, J = 4.7 Hz, 1H, H-1’),

RI PT

5.36 (dd, J = 5.7, 4.7 Hz, 1H, H-2’), 5.33 – 5.25 (m, 1H, H-3’), 4.47 – 4.32 (m, 3H, H-4’, H-5’)), 2.22 (s, 3H, CH3), 2.14 (s, 3H, CH3-Ac), 2.11 (s, 3H, CH3-Ac), 2.10 (s, 3H, CH3-Ac). 13C NMR (126 MHz, CDCl3) δ 170.2 (C=O Ac), 169.7 (C=O Ac), 169.6 (C=O Ac), 157.0 (C2), 130.4 (C5), 129.1 (C3), 20.5 (CH3 Ac), 13.8 (CH3).

4-((2R,3R,4R,5R)-3,4-diacetoxy-5-(acetoxymethyl)tetrahydrofuran-2-yl)-6-ethyl-3-oxo-

3,4-dihydropyrazine

1-oxide

(18a)

and

4-((2S,3R,4R,5R)-3,4-diacetoxy-5-

M AN U

5.1.14.

SC

123.4 (C6), 87.1 (C1’), 80.2 (C4’), 73.6 (C2’), 69.9 (C3’), 62.9 (C5’), 20.9 (CH3 Ac), 20.6 (CH3 Ac),

(acetoxymethyl)tetrahydrofuran-2-yl)-6-ethyl-3-oxo-3,4-dihydropyrazine 1-oxide (18b) To a suspension of 14a (0.24 g, 1.73 mmol) and β-D-ribofuranose 1,2,3,5- tetraacetate (0.50 g, 1.57 mmol) in dry 1,2-dichloroethane (6 mL) was added bis(trimethylsilyl)acetamide (BSA, 0.42 mL, 1.73 mmol) and the mixture was stirred for 70 minutes at room temperaturę. The mixture was then cooled on ice and TiCl4 (0.77 mL, 4.24 mmol) was added. The resulting solution was stirred at room

TE D

temperature overnight and was then was poured into a saturated NaHCO3 solution, filtered through Celite and washed with dichloromethane. The combined organic phases were washed with water, dried over MgSO4 and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (CH2Cl2/Acetone from 10:1 to 8:2) yielding pure compound 18a (0.50 g, 80%). H NMR (500 MHz, CDCl3) δ 7.61 (s, 1H, H3), 7.23 (s, 1H, H6), 6.20 (d, J = 5.0 Hz, 1H, H1’), 5.39 –

EP

1

5.36 (m, 1H, H2’), 5.36 – 5.30 (m, 1H, H3’), 4.45 (dd, J = 12.1, 4.1 Hz, 1H, H5’), 4.42 – 4.39 (m, 1H, H4’), 4.36 (dd, J = 12.1, 2.4 Hz, 1H, H5’), 2.66 (q, J = 7.3 Hz, 2H, CH2), 2.14 (s, 3H, CH3 Ac), 2.12

AC C

(s, 3H, CH3 Ac), 2.10 (s, 3H, CH3 Ac), 1.21 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (126 MHz, CDCl3) δ 170.2 (C=O Ac), 169.7 (C=O Ac), 169.6 (C=O Ac), 156.9 (C2), 135.5 (C5), 129.2 (C3), 122.8 (C6), 87.1 (C1’), 80.4 (C4’), 73.5 (C2’), 70.1 (C3’), 63.0 (C5’), 20.9 (CH3 Ac), 20.7 (CH2), 20.6 (CH3 Ac), 20.6 (CH3 Ac), 11.5 (CH3). HRMS (ESI) m/z [M+H]+ calcd for C17H23N2O9 399.1398, found 399.1393.

5.1.15.

4-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4-

dihydropyrazine

1-oxide

(19a)

and

4-((2S,3R,4S,5R)-3,4-dihydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4-dihydropyrazine 1-oxide (19b) To a solution of 16a/b (0.316g, 0.85 mmol) in MeOH (20 mL) was added a catalytic amount of NaOMe (0.03 mL, 0.12 mmol). The mixture was stirred for 30 minutes at room temperature and was

ACCEPTED MANUSCRIPT then neutralized by the addition of the resin “Dowex 50WX8 hydrogen form”. After filtration and washing with methanol, the filtrate was concentrated in vacuo. The crude residue was purified by RPHPLC, yielding the pure compounds 19a and 19b, respectively. Compound 19a 1

H NMR (600 MHz, D2O) δ 8.17 (d, J = 6.1 Hz, 1H, H-6), 7.86 – 7.78 (m, 1H, H-3), 7.43 (dd, J = 6.1,

2.1 Hz, 1H, H-5), 6.01 (d, J = 2.4 Hz, 1H, H-1’), 4.37 – 4.32 (m, 1H, H-2’), 4.26 – 4.18 (m, 2H, H-3’,

RI PT

H-4’), 4.06 – 3.82 (m, 2H, H-5’). 13C NMR (151 MHz, D2O) δ 158.2 (C2), 130.3 (C3), 128.7 (C6), 121.4 (C5), 90.4 (C1’), 83.8 (C4’), 74.5 (C2’), 68.4 (C3’), 59.9 (C5’). HRMS (ESI) m/z [M+Na]+ calcd for C9H12N2O6Na 267.0588, found 267.0589. Compound 19b

H NMR (500 MHz, D2O) δ 7.96 (d, J = 4.9 Hz, 1H, H-6), 7.83 (s, 1H, H-3), 7.52 – 7.42 (m, 1H, H-5),

SC

1

6.31 (bs, 1H, H-1’), 4.62 (bs, 1H, H-2’), 4.37 (bs, 2H, H-3’, H-4’), 3.86 (dd, J = 96.3, 12.6 Hz, 2H, H5’).

13

C NMR (126 MHz, D2O) δ 158.1 (C2), 130.1 (C6), 130.0 (C3), 121.0 (C5), 87.5 (C1’), 83.7

267.0588, found 267.0591.

5.1.16.

M AN U

(C4’), 70.5 (C2’), 70.2 (C3’), 60.8 (C5’). HRMS (ESI) m/z [M+Na]+ calcd for C9H12N2O6Na

4-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-methyl-3-

oxo-3,4-dihydropyrazine

1-oxide

(20a)

and

4-((2S,3R,4S,5R)-3,4-dihydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-yl)-6-methyl-3-oxo-3,4-dihydropyrazine 1-oxide (20b)

TE D

To a solution of 17a/b (0.068 g, 0.2 mmol) in MeOH (1 mL) was added a catalytic amount of NaOMe (0.02 mL, 0.17 mmol) to obtain a 0.1 M solution. The reaction mixture was stirred for 30 minutes and then neutralized by the addition of the resin “Dowex 50WX8 hydrogen form”. After filtration and washing with methanol, the filtrate was concentrated in vacuo (0.057 g) and then further purified by

Compound 20a 1

EP

RP-HPLC, yielding compounds 20a and 20b, respectively.

H NMR (600 MHz, D2O) δ 8.07 (s, 1H, H-6), 7.76 (s, 1H, H-3), 5.93 (d, J = 2.3 Hz, 1H, H-1’), 4.22

AC C

(dd, J = 4.4, 2.4 Hz, 1H, H-2’), 4.14 – 4.08 (m, 2H, H-3’, H-4’), 3.98 – 3.76 (m, 2H, H-5’), 2.14 (s, 3H, CH3). 13C NMR (126 MHz, D2O) δ 158.0 (C2), 130.9 (C5), 129.9 (C3), 126.3 (C6), 90.2 (C1’), 83.8 (C4’), 74.6 (C2’), 68.3 (C3’), 59.7 (C5’), 12.8 (CH3). HRMS (ESI) m/z [M+Na]+ calcd for C10H14N2O6Na 281.0744, found 281.0747. Compound 20b 1

H NMR (500 MHz, D2O) δ 7.93 – 7.83 (m, 2H, H-3, H-6), 6.31 (d, J = 4.1 Hz, 1H, H-1’), 4.67 – 4.55

(m, 1H, H-2’), 4.42 – 4.31 (m, 2H, H-3’, H-4’), 4.04 – 3.71 (m, 2H, H-5’), 2.29 (s, 3H, CH3).

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NMR (126 MHz, D2O) δ 157.7 (C2), 130.4 (C5), 129.4 (CHpyr), 127.6 (CHpyr), 87.5 (C1’), 83.4 (C4’), 70.3 (C2’), 70.1 (C3’), 60.7 (C5’), 12.8 (CH3). HRMS (ESI) m/z [M+H]+ calcd for C10H15N2O6 259.0925, found 259.0925.

ACCEPTED MANUSCRIPT 5.1.17. 4-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-ethyl-3-oxo3,4-dihydropyrazine 1-oxide (21a) To a solution of 18a (0.487g, 1.2 mmol) in MeOH (6 mL) was added a catalytic amount of NaOMe (0.12 mL, 0.24 mmol) to obtain a 0.1 M solution. The mixture was stirred for 30 minutes and then neutralized by the addition of the resin “Dowex 50WX8 hydrogen form”. After filtration and washing with MeOH, the filtrate was concentrated in vacuo. The crude residue was purified by silica gel flash

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chromatography (using a mixture of dichloromethane and methanol as mobile phase, gradually increasing from 0 to 6% methanol) to yield the pure title compound (0.29 g, 88%). 1

H NMR (500 MHz, D2O) δ 8.15 (s, 1H, H-6), 7.86 (s, 1H, H-3), 6.05 (d, J = 2.1 Hz, 1H, H-1’), 4.33

(dd, J = 4.6, 2.2 Hz, 1H, H-2’), 4.29 – 4.18 (m, 2H, H-3’, H-4’), 4.13 – 4.03 (m, 1H, H-5’), 3.90 (dd, J

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= 13.1, 2.9 Hz, 1H, H-5’), 2.73 – 2.62 (m, 2H, CH2), 1.20 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (151 MHz, D2O) δ 157.8 (C2), 135.8 (C5), 129.8 (C3), 125.5 (C6), 90.4 (C1’), 83.5 (C4’), 74.7 (C2’), 68.0 (C3’), 59.2 (C5’), 20.1 (CH2), 10.1 (CH3). HRMS (ESI) m/z [M+H]+ calcd for C11H16N2O6Na

5.1.18.

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295.0901, found 295.0901.

4-((2R,4S,5R)-4-Hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4-

dihydropyrazine 1-oxide (26a) and 4-((2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran2-yl)-3-oxo-3,4-dihydropyrazine 1-oxide (26b)

To a suspension of 3 (0.135 g, 1.2 mmol) in dry acetonitrile (20 mL) was added DBU (0.18 mL, 1.2

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mmol). The resulting mixture was stirred for 30 minutes at room temperaturę. Then, a solution of 1chloro-2-deoxy-3,5-di-O-toluoyl-β-D-ribofuranose (0.50 g, 1.3 mmol) in dry acetonitrile (4 mL) was added. The resulting reaction mixture was stirred for 2 hours, then filtered and concentrated in vacuo. The residue was redissolved in EtOAc and washed with water (2x), dried over MgSO4 and

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concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (CH2Cl2/EtOAc from 9:1 to 7:3), affording the anomeric mixture 23a/b, which was used as such in the next step. HRMS (ESI) m/z [M+Na]+ calcd for C25H25N2O7Na 465.1656, found 465.1655.

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To a solution of 23a/b (0.52g, 1.12 mmol) in methanol (5.6 mL) was added a catalytic amount of NaOMe (0.1 mL, 0.56 mmol) to obtain a 0.1 M solution. The mixture was stirred for 30 minutes and then neutralized by the addition of the resin “Dowex 50WX8 hydrogen form”. After filtration and washing with methanol, the collected solution was concentrated. The crude residue was purified by silica gel flash chromatography (using a mixture of methanol and dichloromethane as mobile phase, in a gradient gradually raising from 0% to 10% methanol), affording compounds 26a/b as an anomeric mixture (0.06 g, 22% over 2 steps, combined yield of both anomers). The anomeric mixture was separated by RP-HPLC. Compound 26a 1

H NMR (600 MHz, D2O) δ 8.12 (d, J = 6.1 Hz, 1H, H-6), 7.81 (dd, J = 2.1, 0.6 Hz, 1H, H-3), 7.44

(dd, J = 6.0, 2.0 Hz, 1H, H-5), 6.33 (t, J = 6.2 Hz, 1H, H1’), 4.50 – 4.37 (m, 1H. H-3’), 4.25 – 4.13 (m,

ACCEPTED MANUSCRIPT 1H, H-4’), 3.94 – 3.72 (m, 2H, H5’), 2.69 – 2.56 (m, 1H, H2’), 2.45 – 2.31 (m, 1H, H-2’). 13C NMR (151 MHz, D2O) δ 158.1 (C2), 130.2 (C3), 128.9 (C6), 121.4 (C5), 87.4 (C4’), 86.6 (C1’), 69.9 (C3’), 60.7 (C5’), 39.7 (C2’). HRMS (ESI) m/z [M+Na]+ calcd for C9H12N2O5Na 251.0639 found 251.0632. Compound 26b 1

H NMR (600 MHz, D2O) δ 8.06 (d, J = 6.0 Hz, 1H, H-6), 7.80 (d, J = 2.1 Hz, 1H, H-3), 7.45 (dd, J =

6.0, 2.1 Hz, 1H, H-5), 6.26 (dd, J = 7.0, 1.7 Hz, 1H, H1’), 4.63 – 4.54 (m, 1H, H4’), 4.51 – 4.43 (m,

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1H, H3’), 3.80 – 3.63 (m, 2H, H5’), 2.84 – 2.73 (m, 1H, H2’), 2.32 – 2.22 (m, 1H, H2’). 13C NMR (151 MHz, D2O) δ 158.1 (C2), 130.0 (C3), 129.4 (C6), 121.0 (C5), 90.2 (C4’), 89.0 (C1’), 70.8 (C3’), 61.5 (C5’), 39.9 (C2’). HRMS (ESI) m/z [M+Na]+ calcd for C9H12N2O5Na 251.0639, found 251.0622.

4-((2R,4S,5R)-4-Hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-methyl-3-oxo-3,4-

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5.1.19.

dihydropyrazine 1-oxide (27a) and 4-((2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran2-yl)-6-methyl-3-oxo-3,4-dihydropyrazine 1-oxide (27b)

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To a suspension of 13a (0.119 g, 0.94 mmol) in dry acetonitrile (16 mL) was added DBU (0.14 mL, 0.94 mmol). The mixture was stirred for 30 minutes at room temperaturę. Then, a solution of 1-chloro2-deoxy-3,5-di-O-toluoyl-β-D-ribofuranose 22 (0.40 g, 1.03 mmol, 1.1 equiv) in dry acetonitrile (3 mL) was added. The resulting reaction mixture was stirred for 2 hours than filtered and concentrated in vacuo. The crude residue was redissolved in EtOAc and washed with water (2x), dried over MgSO4 and concentrated in vacuo. The crude residue was purified by flash column chromatography

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(CH2Cl2/EtOAc from 9:1 to 7:3) affording an anomeric mixture of 24a/b (0.09 g, 20%). HRMS (ESI) m/z [M+Na]+ calcd for C26H26N2O7Na 501.1632 found 501.1315. To a solution of 24a/b (0.143 g, 0.3 mmol) in MeOH (1.5 mL) was added NaOMe (0.03 mL, 0.15 mmol) to obtain a 0.1 M solution. The mixture was stirred for 40 minutes and then neutralized by the

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addition of the resin “Dowex 50WX8 hydrogen form”. After filtration and washing with MeOH, the collected solution was concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (using a mixture of methanol and dichloromethane as mobile phase in a ratio

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gradually increasing from 0 to 10% methanol) to give the title compound as an anomeric mixture 27a/b (0.078 g, 80% yield over 2 steps; α and β mixture 8:2). The anomeric mixture 27a/b was separated by RP-HPLC. Compound 27a 1

H NMR (300 MHz, D2O) δ 8.05 (s, 1H, H-6), 7.83 (s, 1H, H-3), 6.31 (t, J = 6.2 Hz, 1H, H-1’), 4.50 –

4.38 (m, 1H, H-3’), 4.19 – 4.07 (m, 1H, H-4’), 3.96 – 3.72 (m, 2H, H-5’), 2.66 – 2.51 (m, 1H, H-2’), 2.39 – 2.28 (m, 1H, H-2’), 2.24 (s, 3H, CH3).13C NMR (126 MHz, D2O) δ 157.9 (C2), 131.0 (C5), 129.8 (C3), 126.5 (C6), 87.3 (C4’), 86.3 (C1’), 69.8 (C3’), 60.6 (C5’), 39.8 (C2’), 12.8 (CH3). HRMS (ESI) m/z [M+Na]+ calcd for C10H14N2O5Na 265.0795, found 265.0803. Compound 27b

ACCEPTED MANUSCRIPT 1

H NMR (300 MHz, D2O) δ 7.96 (s, 1H, H-6), 7.82 (s, 1H, H-3), 6.24 (dd, J = 7.1, 1.9 Hz, 1H, H1’),

4.62 – 4.51 (m, 1H, H-4’), 4.49 – 4.36 (m, 1H, H-3’), 3.80 – 3.58 (m, 2H, H-5’), 2.87 – 2.70 (m, 1H, H-2’), 2.26 (s, 3H, CH3), 2.25 – 2.17 (m, 1H, H-2’).13C NMR (126 MHz, D2O) δ 157.7 (C2), 130.6 (C5), 129.5 (C3), 127.0 (C6), 90.0 (C4’), 88.9 (C1’), 70.8 (C3’), 61.6 (C5’), 40.2 (C2’), 12.8 (CH3). HRMS (ESI) m/z [M+Na]+ calcd for C10H14N2O5Na 265.0795, found 265.0799. 6-Ethyl-4-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4-

dihydropyrazine

1-oxide

(28a)

and

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5.1.20.

6-ethyl-4-((2S,4S,5R)-4-hydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4-dihydropyrazine 1-oxide (28b)

To a suspension of 14a (0.116 g, 0.83 mmol) in dry acetonitrile (14 mL) was added DBU (0.12 mL,

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0.83 mmol). The mixture was stirred for 30 minutes at room temperaturę and then a solution of 1chloro-2-deoxy-3,5-di-O-toluoyl-β-D-ribofuranose (0.35 g, 0.91 mmol) in dry acetonitrile (3 mL) was added. The resulting reaction mixture was stirred for 3 hours and then filtered and concentrated in

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vacuo. The crude residue was redissolved in EtOAc and washed with water (2x), dried over MgSO4 and concentrated in vacuo. The crude residue was purified by flash column chromatography (CH2Cl2/EtOAc from 9:1 to 7:3), yielding an anomeric mixture of 25a/b. HRMS (ESI) m/z [M+Na]+ calcd for C27H28N2O7Na 515.1789, found 515.1780.

To a solution of 25a/b (0.35 g, 0.7 mmol) in MeOH (3.5 mL) was added NaOMe (0.07 mL, 0.35 mmol) to obtain a 0.1 M solution. The mixture was stirred for 30 minutes and then neutralized by the

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addition of the resin “Dowex 50WX8 hydrogen form”. After filtration and washing with MeOH, the collected solution was concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (CH2Cl2/MeOH from 1:0 to 9:1) yielding the title compounds 28a/b as an anomeric mixture (0.042 g, 21% over 2 steps, combined yield of α and β anomers). 1

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Compound 28a

H NMR (600 MHz, D2O) δ 8.05 (s, 1H, H-3), 7.84 (s, 1H, H-6), 6.36 (t, J = 6.1 Hz, 1H, H1’), 4.53 –

4.42 (m, 1H, H-3’), 4.23 – 4.12 (m, 1H, H-4’), 3.98 – 3.74 (m, 2H, H-5’), 2.69 (q, J = 7.4 Hz, 2H,

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CH2), 2.65 – 2.56 (m, 1H, H-2’), 2.43 – 2.33 (m, 1H, H-2’), 1.20 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (151 MHz, D2O) δ 157.7 (C2), 135.8 (C5), 129.8 (C6), 125.7 (C3), 87.2 (C4’), 86.4 (C1’), 69.5 (C3’), 60.3 (C5’), 39.9 (C2’), 20.1 (CH2), 10.2 (CH3). HRMS (ESI) m/z [M+Na]+ calcd for C11H16N2O5Na 279.0952, found 279.0953. Compound 28b 1

H NMR (600 MHz, D2O) δ 7.90 (s, 1H, H-3), 7.83 (s, 1H, H-6), 6.31 (dd, J = 7.2, 1.8 Hz, 1H, H-1’),

4.61 – 4.55 (m, 1H, H-4’), 4.50 – 4.45 (m, 1H, H-3’), 3.77 – 3.64 (m, 2H, H-5’), 2.84 – 2.76 (m, 1H, H-2’), 2.76 – 2.67 (m, 2H, CH2), 2.29 – 2.22 (m, 1H, H-2’), 1.23 – 1.19 (m, 3H, CH3). 13C NMR (151 MHz, D2O) δ 157.6 (C2), 135.3 (C5), 129.7 (C6), 126.5 (C3), 90.2 (C4’), 88.7 (C1’), 70.9 (C3’), 61.5 (C5’), 40.0 (C2’), 20.1 (CH2), 10.5 (CH3). HRMS (ESI) m/z [M+Na]+ calcd for C11H16N2O5Na 279.0952, found 279.0950.

ACCEPTED MANUSCRIPT 5.2. Antiviral evaluation The compounds were evaluated against the following viruses: herpes simplex virus 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK−) HSV-1 KOS strain resistant to ACV (ACVr), herpes simplex virus 2 (HSV-2) strains Lyons and G, varicella-zoster virus (VZV) strain Oka, TK− VZV strain 07−1, human cytomegalovirus (HCMV) strains AD-169 and Davis. The antiviral assays are based on

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inhibition of virus-induced cytopathicity (HSV and HCMV) or plaque formation (VZV) in human embryonic lung (HEL) fibroblasts. Confluent cell cultures in microtiter 96-well plates are inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU) (VZV). After 2 hours of adsorption, the viral inoculum is removed and

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the cultures are further incubated in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation is recorded after 2-3 (HSV), 5 (VZV) or 6-7 (HCMV) days post-infection. Antiviral activity is expressed as the EC50 or compound concentration required

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inhibiting virus-induced cytopathicity or viral plaque formation by 50%.

The cytostatic activity measurements are based on the inhibition of cell growth. HEL cells are seeded at a rate of 5 x 103 cells/well into 96-well microtiter plates and allow proliferating for 24 hours. Then, medium containing different concentrations of the test compounds is added. After 3 days of incubation at 37 °C, the cell number is determined with a Coulter counter. The cytostatic concentration is calculated as the CC50, or the compound concentration required reducing cell proliferation by 50%

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relative to the number of cells in the untreated controls. CC50 values are estimated from graphic plots of the number of cells (percentage of control) as a function of the concentration of the test compounds. Alternatively, cytotoxicity of the test compounds is expressed as the minimum cytotoxic concentration (MCC) or the compound concentration that causes a microscopically detectable alteration of cell

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morphology.

Acknowledgements

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We thank Leentje Persoons (KU Leuven, Rega Institute, Laboratory of Virology and Chemotherapy) for the biological evaluation of the compounds against the influenza virus and RSV. This research grant did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://

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ACCEPTED MANUSCRIPT [2] J. Homsi, C.R. Garrett, Hepatic arterial infusion of chemotherapy for hepatic metastases from colorectal cancer, Cancer Control 13 (2006) 42–47. [3] E. De Clercq, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), Med. Res. Rev. 25 (2005) 1-20. [4] J.K. Christman, 5-Azacytidine and 5-aza-2’-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy, Oncogene 21 (2002) 5483-5495. [5] J.M. Crance, N. Scaramozzino, A. Jouan, D. Garin, Interferon, ribavirin, 6-azauridine and

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glycyrrhizin: antiviral compounds active against pathogenic flaviviruses, Antiviral Res. 58 (2003) 7379.

[6] a) Y. Furuta, B.B. Gowen, K. Takahashi, K. Shiraki, D.F. Smee, D.L. Barnard, Favipiravir (T705), a novel viral RNA polymerase inhibitor, Antiviral Res. 100 (2013) 446–454. b) D. Jochmans, S.

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van Nieuwkoop, S.L. Smits, J. Neyts, R.A.M. Fouchier, B.G. van den Hoogen, Antiviral activity of favipiravir (T-705) against a broad range of paramyxoviruses in vitro and against human metapneumovirus in hamsters, Antimicrob. Agents Chemother. 60 (2016) 4620–4629.

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[7] M. Bobek, A. Bloch, Synthesis and biological activity of pyrazines and pyrazine ribonucleosides as pyrimidine analogs, J. Med. Chem. 15 (1972) 164-168.

[8] P.T. Berkowitz, T.J. Bardos, A. Bloch, Synthesis of 1,2-dihydro-1-(2-deoxy-β-D-erythropentofuranosyl)-2-oxopyrazine 4-oxide, a potent analog of deoxyuridine, J. Med. Chem. 16 (1973) 183-184.

[9] M. Butler, G.M. Cabrera, Determination of the position of the N-O function in substituted pyrazine

(2013) 37-42.

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N-oxides by chemometric analysis of carbon-13 nuclear magnetic resonance data, J. Mol. Struct. 1043

[10] G. Karms, P.E. Spoerrt, The preparation of hydroxypyrazines and derived chloropyrazines, J. Am. Chem. Soc. 74 (1952) 1580-1584.

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[11] U.S. Gi, W. Baltes, Model Reactions on Roast Aroma Formation. 15. Investigations on the formation of pyrido[3,4-d]imidazoles during the Maillard Reactions, J. Agric. Food Chem. 43 (1995) 2226–2230.

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[12] M.J. Robins, R.K. Robins, Purine nucleosides. XI. The synthesis of 2'-deoxy-9-α- and -β-Dribofuranosylpurines and the correlation of their anomeric structure with proton magnetic resonance spectra, J. Am. Chem. Soc. 87 (1965) 4934–4940.

ACCEPTED MANUSCRIPT Highlights The synthesis of emimycin and 5-substituted emimycin analogues was carried out.

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The synthesis of the corresponding ribo- and 2’-deoxyribonucleosides was also effected.

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2’-Deoxyribo-5-methylemimycin was a potent inhibitor of HSV-1 and VZV replication.

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