Emimycin and its nucleoside derivatives: Synthesis and antiviral activity

Article abstract of DOI:10.1016/j.ejmech.2017.12.018

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

Full text of DOI:10.1016/j.ejmech.2017.12.018

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
10.1016/j.ejmech.2017.12.018
EJMECH 9996
DOI:
Reference:
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.
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ACCEPTED MANUSCRIPT
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*
aMedicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49 – bus 1041,
3000 Leuven, Belgium
bLaboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven,
Herestraat 49 - bus 1043, 3000 Leuven, Belgium
*Corresponding author. E-mail: steven.dejonghe@kuleuven.be ; Phone: +32 16 32 26 62
Abstract
The synthesis of emimycin, 5-substituted emimycin analogues and the corresponding ribo- and 2’-
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
Emimycin, nucleoside, antiviral, pyrazine
1.
Introduction
Structural modification of the nucleobase moiety of natural pyrimidine nucleosides is an established
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
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]. 6-
Azauridine 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
antiviral agent with activity against many RNA viruses, that received marketing approval in Japan for
the treatment of influenza virus infected patients [6].
Emimycin (or 1,2-dihydro-2-oxo-pyrazine 4-oxide) is a pyrazine analogue which structurally
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
0.05 nM (against S. faecium) and 0.04 nM (against E. coli), suggesting a different molecular target [8].
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

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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].
Scheme 1. Synthesis of emimycina
aReagents and conditions: (a) mCPBA, DCE, 65°C, 30 h; (b) NaOH, H2O, 100°C, 2h 30 min,
quantitative over 2 steps.
5-Methylemimycin 13a was prepared as shown in Scheme 2. Commercially available methylglyoxal 5
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.
Scheme 2. Synthesis of 5-methyl- and 5-ethylemimycina
aReagents 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

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7a, HMBC correlations of C-7 with H-3 and H-6 were observed. In contrast, for the regioisomeric 6-
methylpyrazine analogue 7b, only a HMBC cross-peak of C-7 with H-5 was observed.
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
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
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
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-N-
oxides 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
group was determined based on 2D HMBC correlation spectra as previously discussed. For the 5-
substituted 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
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-1,2-
dihydropyrazine 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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Figure 4. Key HMBC correlations of compounds 13a and 14a.
2.2.
Synthesis of ribosyl derivatives of (5-(m)ethyl)emimycin
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-
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
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
this final stage, each pair of anomers was separated and isolated by reversed phase HPLC (Scheme 3).
Scheme 3. Synthesis of (5-(m)ethyl)emimycin ribosidesa
aReagents 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.

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Table 1. Glycosylation reaction conditions and yields.
Entry
Nucleobase
Methoda
Products
16a: 30%
16b: 70%
16a
Yieldb
31%
A
B
A
1
2
3
3
3
42%
17a: 85%
17b: 15%
17a
11%
13a
B
B
79%
80%
4
5
13a
14a
18a
aMethod A: (i) BSA, DCE, 70 min, rt; (ii) TiCl4, reflux ; Method B: (i) BSA, DCE, 70 min, rt; (ii) TiCl4, rt.
bCombined yield of both anomers.
The assignment of the α/β anomers was done based on ROESY NMR spectroscopy of the mixture. As
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.
Figure 5. Key ROESY correlations of 16a and 16b
2.3.
Synthesis of 2’-deoxyribosyl derivatives of (5-(m)ethyl)emimycin
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%

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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
1
proton at C-1’ in the H 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)
the characteristic “pseudo-triplet” is evident [12]. This is in complete agreement with literature data
for 2’-deoxyribosylemimycin [8].
Scheme 4. Synthesis of 2’-deoxyriboside-(5-(m)ethyl)emimycin
aReagents and conditions: a) DBU, acetonitrile, rt; b) NaOMe, MeOH, rt, 20 min.
3.
Antiviral evaluation
It has been demonstrated that favipiravir acts as a prodrug that is intracellularly phosphoribosylated
yielding the ribose-5’-monophosphate metabolite. Further phosphorylation yields the ribose-
triphosphate 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).

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Table 1. Antiviral and cytotoxic properties of emimycin and ribo-emimycin analogues in Madin
Darby canine kidney (MDCK) cells.
b
EC50 (µM)a
CC50
MCCc
Cmpd
Influenza A/H1N1
A/Ned/378/05
Influenza A/H3N2
A/HK/7/87
Influenza B
MDCKd MDCKd
B/Ned/537/05
>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
>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
>100
>100
>100
>100
>100
>100
3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
13a
14a
19a
19b
20a
20b
21a
21b
a50% 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.
b50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-
based MTS assay.
cMinimum compound concentration that causes a microscopically detectable alteration of normal cell
morphology.
dMDCK cells: Madin Darby canine kidney cells
The 2’-deoxyribose emimycine nucleosides 26a/b, 27a/b and 28a/b were assayed for their antiviral
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,
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 5-
methylemimycin (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

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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
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
embryonic lung (HEL) cells.
EC50 (µM)a
VZV
Cytotoxicity
Cmpd
b
HCMV
HSV
HSV-1
(KOS
ACVr)
>438
>438
59.44
>412
>390
>390
88.81
20.14
1.56
CC50
MCCc
TK+
OKA
strain
>438
438
TK- 07-
AD-169 Davis
HSV-1
(KOS)
HSV-2
(G)
1.
HEL
HEL
strain
strain
strain
>438
>438
6.91
>412
138
>438
>87
>438
>438
>412
>412
>78
>438
>438
1.79
>412
8.55
174
>438
>438
17.48
>412
145
>438
>438
>412
>412
>390
>390
>444
>300
>358
NDd
NDd
169
NDd
NDd
NDd
>444
>300
>358
26a
26b
>412
>82
0.99
328
27a
27b
>78
7.77
142
28a
>78
>78
174
204
28b
NDd
NDd
1.04
NDd
NDd
1.58
5.31
0.035
NDd
53.51
6.66
NDd
0.38
0.068
2.81
0.29
30.02
1.97
Acyclovir
BVDU
Cidofovir
aEffective concentration required to reduce virus plaque formation by 50%. Data are the mean of two
independent experiments.
bCytotoxic concentration required to reduce cell growth by 50%. Data are the mean of two independent
experiments.
cMinimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology. Data
are the mean of two independent experiments.
dNot 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

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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
Avance 300 MHz (1H NMR, 300 MHz, 13C NMR, 75 MHz), 500 MHz (1H NMR, 500 MHz, 13C
13
NMR, 125 MHz) or 600 MHz (1H NMR, 600 MHz, C 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
13
(49.00 ppm) for C NMR spectra. Abbreviations used are: s = singlet, d = doublet, t = triplet, q =
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
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%
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
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
(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
1
solution afforded the title compound as a creamy solid (0.34 g, 100%). H NMR (500 MHz, DMSO-
d6) 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.

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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,
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,
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
(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
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
g, 89%). HRMS (ESI) m/z [M+H]+ calcd for C12H11N2O2 215.0815, found 215.0823.
5.1.5. 2-Benzoyloxy-5-methylpyrazine 4-oxide (11a) and 2-benzoyloxy-6-methylpyrazine 4-oxide
(11b)
mCPBA (0.94 g, 2.85 mmol) was added to a solution of 9a/b (0.61 g, 2.85 mmol) in 1,2-
dichloroethane (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
1H 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
13
(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). C
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
1H 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
C12H10N2O3Na 253.0584, found 253.0586.
5.1.6. 5-Methyl-2-oxo-1,2-dihydropyrazine 4-oxide (13a)
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
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
1
13
g, 79%). H NMR (300 MHz, MeOD) δ 7.66 (s, 1H, H-3), 7.50 (s, 1H, H-6), 2.17 (s, 3H, CH3). C
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
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,
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)
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
13C 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
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%).
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
(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).
Compound 12a
1H 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
13
(t, J = 7.5 Hz, 3H, CH3). C NMR (151 MHz, CDCl3) δ 163.7 (C9), 156.1 (C2), 147.1 (C5), 143.2
(C6), 134.5 (C13), 130.5 (C11, C15, C10), 128.8 (C12, C14), 127.9 (C3), 21.0 (CH2), 10.1 (CH3).
Compound 12b
1H 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 –
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
1
washed with diethylether to give the title compound as a white solid (0.112 g, 97%). H 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-
1-oxide (16a) and 4-((2S,3R,4R,5R)-3,4-diacetoxy-5-
dihydropyrazine
(acetoxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4-dihydropyrazine 1-oxide (16b)
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
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
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).
1H 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’),
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
(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
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
1H 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
13
(m, 6H, CH3). C 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
(C5), 20.9 (CH3), 20.6 (CH3), 20.6 (CH3). HRMS (ESI) m/z [M+H]+ calcd for C15H19N2O9 371.1085,
found 371.1077.
5.1.13. 4-((2R,3R,4R,5R)-3,4-diacetoxy-5-(acetoxymethyl)tetrahydrofuran-2-yl)-6-methyl-3-oxo-
3,4-dihydropyrazine
1-oxide
(17a)
and
4-((2S,3R,4R,5R)-3,4-diacetoxy-5-
(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
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
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%).
1H 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
(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
13
(s, 3H, α CH3-Ac), 1.94 (s, 3H, α CH3-Ac). 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%).
1H 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’),
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),
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),
20.5 (CH3 Ac), 13.8 (CH3).
5.1.14. 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-
(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
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%).
1H 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 –
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
(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-
1-oxide (19a) and 4-((2S,3R,4S,5R)-3,4-dihydroxy-5-
dihydropyrazine
(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 RP-
HPLC, yielding the pure compounds 19a and 19b, respectively.
Compound 19a
1H 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’,
13
H-4’), 4.06 – 3.82 (m, 2H, H-5’). C 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
1H 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),
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, H-
13
5’). C NMR (126 MHz, D2O) δ 158.1 (C2), 130.1 (C6), 130.0 (C3), 121.0 (C5), 87.5 (C1’), 83.7
(C4’), 70.5 (C2’), 70.2 (C3’), 60.8 (C5’). HRMS (ESI) m/z [M+Na]+ calcd for C9H12N2O6Na
267.0588, found 267.0591.
5.1.16.
4-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-methyl-3-
1-oxide (20a) and 4-((2S,3R,4S,5R)-3,4-dihydroxy-5-
oxo-3,4-dihydropyrazine
(hydroxymethyl)tetrahydrofuran-2-yl)-6-methyl-3-oxo-3,4-dihydropyrazine 1-oxide (20b)
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
RP-HPLC, yielding compounds 20a and 20b, respectively.
Compound 20a
1H 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
(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,
13
3H, CH3). C 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
1H 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
13
(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). C
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.

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5.1.17. 4-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-ethyl-3-oxo-
3,4-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
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%).
1H 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
13
= 13.1, 2.9 Hz, 1H, H-5’), 2.73 – 2.62 (m, 2H, CH2), 1.20 (t, J = 7.4 Hz, 3H, CH3). C 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
295.0901, found 295.0901.
5.1.18.
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)tetrahydrofuran-
2-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
mmol). The resulting mixture was stirred for 30 minutes at room temperaturę. Then, a solution of 1-
chloro-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
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.
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
1H 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,

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13
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’). C 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
1H 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,
13
1H, H3’), 3.80 – 3.63 (m, 2H, H5’), 2.84 – 2.73 (m, 1H, H2’), 2.32 – 2.22 (m, 1H, H2’). C 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.
5.1.19. 4-((2R,4S,5R)-4-Hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-methyl-3-oxo-3,4-
dihydropyrazine 1-oxide (27a) and 4-((2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-
2-yl)-6-methyl-3-oxo-3,4-dihydropyrazine 1-oxide (27b)
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-chloro-
2-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
(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
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
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
1H 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
1H 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.
5.1.20.
6-Ethyl-4-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4-
1-oxide (28a) and 6-ethyl-4-((2S,4S,5R)-4-hydroxy-5-
dihydropyrazine
(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,
0.83 mmol). The mixture was stirred for 30 minutes at room temperaturę and then a solution of 1-
chloro-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
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
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).
Compound 28a
1H 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,
13
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). C 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
1H 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.

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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
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
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
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%
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
morphology.
Acknowledgements
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-for-
profit sectors.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://
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Highlights
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-
-
The synthesis of emimycin and 5-substituted emimycin analogues was carried out.
The synthesis of the corresponding ribo- and 2’-deoxyribonucleosides was also effected.
2’-Deoxyribo-5-methylemimycin was a potent inhibitor of HSV-1 and VZV replication.