Anti-norovirus activity of C7-modified 4-amino-pyrrolo[2,1-f][1,2,4]triazine C-nucleosides

Article abstract of DOI:10.1016/j.ejmech.2020.112198

Synthetic nucleoside analogues characterized by a C–C anomeric linkage form a family of promising therapeutics against infectious and malignant diseases. Herein, C-nucleosides comprising structural variations at the sugar and nucleobase moieties were exam

Full text of DOI:10.1016/j.ejmech.2020.112198

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Anti-norovirus activity of C7-modified 4-amino-pyrrolo[2,1-f][1,2,4]triazine C-
nucleosides
Qingfeng Li, Elisabetta Groaz, Joana Rocha-Pereira, Johan Neyts, Piet Herdewijn
PII:
S0223-5234(20)30165-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.112198
EJMECH 112198
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 November 2019
Revised Date: 24 December 2019
Accepted Date: 28 February 2020
Please cite this article as: Q. Li, E. Groaz, J. Rocha-Pereira, J. Neyts, P. Herdewijn, Anti-norovirus
activity of C7-modified 4-amino-pyrrolo[2,1-f][1,2,4]triazine C-nucleosides, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112198.
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X
Virus
MNV
HuNoV
MNV
Cell lines
EC50 (µM)
0.007
RAW 264.7 cells
HGT-1 cells
RAW 264.7 cells
H
H
Cl
0.015
0.007

Anti-norovirus activity of C7-modified 4-amino-pyrrolo[2,1-f][1,2,4]triazine
C-nucleosides
Qingfeng Li,a Elisabetta Groaz, a,* Joana Rocha-Pereira,b Johan Neyts,b and Piet Herdewijna,*
a KU Leuven, Rega Institute for Medical Research, Medicinal Chemistry, Herestraat 49 - Box 1041, Leuven
3000, Belgium
b KU Leuven, Rega Institute for Medical Research, Department of Microbiology and Immunology, Laboratory of
Virology and Chemotherapy, Herestraat 49 - Box 1041, Leuven 3000, Leuven, Belgium
ABSTRACT
Synthetic nucleoside analogues characterized by a C-C anomeric linkage form a family of
promising therapeutics against infectious and malignant diseases. Herein, C-nucleosides
comprising structural variations at the sugar and nucleobase moieties were examined for their
ability to inhibit both murine and human norovirus RNA-dependent RNA polymerase (RdRp).
We have found that the combination of 4-amino-pyrrolo[2,1-f][1,2,4]triazine and its
7-halogenated congeners with either a
D-ribose or 2’-C-methyl-D-ribose unit resulted in
analogues with good antiviral activity against murine norovirus (MNV), albeit coupled with a
significant cytotoxicity. Among this series, 4-aza-7,9-dideazaadenosine notably retained a
strong antiviral effect in a human norovirus (HuNoV) replicon assay with an EC50 = 0.015
µM. This study demonstrates that C-nucleosides can be used as viable starting scaffolds for
further optimization towards the development of nucleoside-based inhibitors of norovirus
replication.
Keywords: C-Nucleosides, Structure-activity relationship, Norovirus, Antiviral activity
1. Introduction
Noroviruses are positive-sense single-stranded RNA (+ssRNA) viruses responsible for
large outbreaks of acute gastroenteritis around the world.1,2 Typically, symptoms including
vomiting, diarrhea, abdominal cramps, and nausea may last about two or three days in healthy
adults. However, this infection can be prolonged and severe (even life-threatening) in young
children, elderly and immunocompromised persons, who can develop chronic
grastroenteritis.3,4 The current therapy for norovirus infections relies on electrolyte
replenishment for dehydrated individuals along with measures for outbreak control and
prevention that are restricted to the often inefficient use of antiseptics and hand-sanitizers.
Despite the clear need for medical intervention, no approved vaccine or small molecule
antiviral treatment is currently available. This is partly due to the fact that only recently
human norovirus (HuNoV) was successfully cultivated in vitro.5,6 Hence, there is an urgency
to develop therapeutic agents that can either directly inhibit norovirus RNA replication or
interfere with the function of structural and non-structural proteins encoded by the norovirus
genome.7,8 Selected examples of molecules recognized as inhibitors of the activity of

norovirus RNA-dependent RNA polymerase (RdRp) are given in Fig.1. From a structural
standpoint, they include both nucleosides and non-nucleoside compounds.9,10
Several small heterocycles exhibiting anti-norovirus inhibitory activity in the micromolar
range, such as the phenylthiazole (NIC02) and triazole (NIC10) derivatives shown in Fig. 1,11
were identified as potential scaffolds for further drug development. On the other hand,
ribavirin was one of the first nucleosides found to effectively inhibit norovirus replication
(EC50 = 43 µM).12 2’-C-Methyl-cytidine (2CM-C) was initially developed as a HCV
polymerase inhibitor, but later proven to inhibit also murine norovirus (MNV) polymerase
(EC50 = 2 µM)13 and HuNoV replication in the human B cell BJAB cell line (EC50 = 0.3
µM).14 Both 2CM-C and its fluorinated analogue 2’-fluoro-2’-C-methyl-cytidine
(2’-F-2’-CM-C) displayed comparable antiviral activity against both MNV and HuNoV in cell
based assays.10 Among non-nucleoside anti-norovirus agents, it is worth mentioning
polyanionic naphthalene analogues such as suramin15,16 and NF023.16
In recent years, much effort has been devoted to the chemical synthesis and biological
evaluation of C-nucleoside analogues as potential antiviral agents.17,18 In particular, the
coupling of 4-amino-pyrrolo[2,1-f][1,2,4]triazine (or 4-aza-7,9-dideazaadenine) to various
sugar moieties has delivered modified C-nucleosides with a broad-spectrum activity against
viruses belonging to the Flaviviridae (HCV), Orthomyxoviridae, Paramyxoviridae, and
Coronaviridae families. In an early study, 2’-C-methyl-4-aza-7,9-dideazaadenosine (Fig. 2, 1)
was identified as a selective HCV polymerase inhibitor in cell cultures (EC50 = 1.98 µM).19
Base-modified derivatives of 1 have also shown promising anti-HCV properties in vitro.
Specifically, the 7-carboamido analogue 2 exhibited a remarkable 40-fold improvement in
potency relative to the parent compound 1, while the 7-fluoro pyrrolotriazine analogue 3
showed good anti-HCV activity (EC50 = 3.1 µM) without concomitant cytotoxicity
(CC50 >100 µM).20 We too became interested in these analogues and synthesized ribose
containing C-nucleosides 4-7 (Fig. 2) with either a hydrogen atom or halogen group at the
7-position of the nucleobase.21
S
NH2
N
NH2
N
O
O
N
N
N
O
S
N
N
HO
NH
NH
Me
O
O
O
N
N
N
HO
HO
HO
NH2
O
O
Me
Me
OH
Ribavirin
OH
F
N
HO
HO
HO
NIC02
NIC10
2CM-C
2'F-2'C-Me-C
HuNoV EC50 = 12.7/3.2 µM
CC50 > 100 µM
HuNoVpol IC50 = 5.0 µM
MNV EC50 = 4.8 µM
HuNoVpol IC50 = 9.2 µM
MNV EC50 = 34.5 µM
HuNoV EC50 = 43 µM
CC50 > 100 µM
HuNoV EC50 = 8.2 µM
CC50 > 100 µM
O
H
H
N
N
N
N
H
H
-O3S
SO3
-O3S
SO3
-
-
O
O
O
H
H
N
N
-
-O3S
SO3
HN
NH
N
N
H
H
O
O
-O3S
SO3
-
-
-O3S
SO3
-
-
SO3
SO3
Suramin
NF023
HuNoVpol IC50 = 0.025 µM
MNVpol IC50 = 0.07 µM
HuNoVpol IC50 = 0.071 µM
MNVpol EC50 = 0.2 µM
Fig. 1. Nucleoside and non-nucleoside inhibitors of norovirus replication.
Given the broad-spectrum activity of pyrrolotriazine containing C-nucleosides against RNA
viruses, we compared a series of sugar- and base-modified C-nucleosides as potential

norovirus inhibitors. Our planned structure-activity relationship study comprehended three
types of sugar modifications, i.e., -ribose, 2’-C-methyl- -ribose, and -ribose, in
D
D
L
combination with the presence of H, Cl, Br, or I atoms at the 7 position of the pyrrolotriazine
scaffold (Fig. 2). In addition, N-nucleoside analogue 8 featuring 7-deaza-7-chloroadenine as
nucleobase was also included in our study. The synthesis of
10 and 11 was motivated by the generally lower toxicity of nucleoside antivirals bearing a
-sugar moiety compared to their -counterparts, as demonstrated by the clinical use of
lamivudine ( -3TC), clevudine, and telbivudine (
-dT).22
L-ribose type of analogues such as
L
D
L
L
NH2
R1
N
N
O
R1 = H
R1 = CONH2
1
2
3
N
HO
Me
OH
R1
=
F
HO
NH2
N
NH2
N
NH2
N
NH2
R3
R2
Cl
Cl
N
N
N
N
O
O
O
O
N
N
N
N
N
OH
HO
HO
HO
Me
OH
HO
OH
OH
OH
HO
HO
HO
R2 = H
2 = Cl
R2 = Br
4
5
6
7
8
9
R3 = H 10
R3 = Cl 11
R
R2 = I
Fig. 2. Known C-nucleosides 1-7 and additional structural analogues (8-11) investigated in this study.
2. Results and discussion
2.1. Chemistry
Suitable synthetic procedures for the preparation of compounds 4-7 were previously
described.21 Compound 8 was synthesized according to a literature method reported by Seela
et al.23 As shown in Scheme 1, nucleobase 13 was first silylated under standard conditions
[bis(trimethylsilyl)acetamide (BSA) in anhydrous MeCN] at room temperature, and then
reacted with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose 12 to afford glycosylated
intermediate 13 with a 1’-β-configuration. Compound 14 was then converted into 7-chloro
tubercidin derivative 8 upon treatment with methanolic ammonia in a sealed tube (120 °C, 24
h).
Scheme 1. Synthesis of 7-chloro tubercidin analogue 8. Reagents and conditions: (a) BSA, TMSOTf, MeCN,
50 °C, 1 h, 70%; (b) 7 M NH3 in MeOH, 120 °C, 24 h, 84%.

4-Amino-pyrrolo[2,1-f][1,2,4]triazine containing C-nucleoside analogue 1 was previously
obtained using a linear synthetic route by building the bicyclic heterocyclic nucleobase at the
1-position of a 2-methyl modified sugar moiety.24 However, the 3,5-bis-dichlorobenzyl
protected 2-C-Me-riboside used as starting material is not easily available. Moreover, in the
initial stage of the nucleobase construction an anomeric mixture of pyrrolo nucleoside
intermediates was obtained, which needed to be further separated. Therefore, it was deemed
preferable to follow a convergent synthetic approach to obtain this compound or its analogues
such
3,5-di-O-benzyl-2-keto-1-α-O-methyl-
yield over four steps starting from
as
compound
9.
D
As
shown
in
Scheme
2,
-ribofuranose 15 was synthesized in a 43% overall
D
-ribose and by employing a similar procedure as that
reported by Li et al.25 Subsequent stereospecific addition of a 2’-methyl group at the β-face of
the ribofuranose moiety was accomplished by reacting 15 with methylmagnesium bromide in
THF to give di-O-benzyl-2-C-β-methoxyl-1-α-O-methyl-D-ribofuranose 16 in 70% yield. At
this stage of the synthesis, the [S]- and [R]-configuration of 1-C and 2-C, respectively, were
confirmed by 2D NMR analysis based on the NOE interactions between H-1 and H-3,
2-C-Me as well as OMe with H-4 (see Supporting Information).
OMe
OMe
Me
OMe
Me
O
O
O
O
O
O
OH
BnO
4 steps
Ref. 25
a
c
HO
BnO
BnO
b
BnO
Me
HO
OH
BnO
O
BnO
OH
BnO
OBn
BnO OBn
D-Ribose
15
16
17
18
SCH3
N
N
d
19
N
NH2
N
NH2
SCH3
SCH3
Cl
e
N
N
N
f,g
N
h
N
N
N
N
O
O
O
O
N
N
N
BnO
HO
HO
BnO
OH
Me
Me
HO OH
Me
Me
HO OH
BnO OBn
BnO OBn
9
1
21
20
Scheme 2. Synthesis of 2’-C-methyl-4-aza-7-chloro-7,9-dideazaadenosine C-nucleoside 9. Reagents and
conditions: (a) CH3MgBr, THF, 3 h, 92%; (b) NaH, BnBr, TBAI, THF, 70 °C, on, 73%; (c) (i) AcOH, 1 N HCl in
H2O, 80 °C, 4 h; (ii) DMSO, Ac2O, rt, on, 76% over two steps; (d) Lithium diisopropylamide (LDA), THF,
-78 °C, 3 h, 32%; (e) BF3.OEt2, Et3SiH, DCM, 0 °C, 40 min, 71%; (f) 7 M NH3 in MeOH, 100 °C, 24 h, 59%; (g)
Pd(OH)2/C, H2, EtOH:cyclohexene = 4:1, rt, 24 h, 80%; (h) N-chlorosuccinimide (NCS), DMF, 0 °C to rt, 2 days,
32%.
Subsequently, compound 16 underwent a benzylation reaction to afford fully protected
sugar analogue 17. This step was found to proceed sluggishly at room temperature, however, a
satisfactory 73% yield could be obtained when the reaction was performed at elevated
temperature (70 °C). Hydrolysis of compound 17 by treatment with 1 N HCl, followed by
oxidation of the resulting 1-OH group in the presence of DMSO/Ac2O26 afforded lactone 18
in 76% yield over two steps. For the C-nucleoside construction, we employed a similar
synthetic strategy as that previously used for the synthesis of 4.21 The lithium salt of
thiomethyl substituted heterocycle 19 was coupled with protected ribonolactone 18, affording

nucleoside lactol 20 as a mixture of anomers. Anomeric reduction of hemiacetal 20 using
triethylsilane and boron trifluoride etherate afforded stereoselectively C-nucleoside 21 as a
β-anomer. Next, amine replacement of the thiomethyl group furnished a benzyl protected
C-nucleoside intermediate, which underwent O-debenzylation upon hydrogenolysis catalyzed
by palladium hydroxide on carbon (Pd(OH)2/C) affording C-nucleoside 1. Chlorine was then
readily introduced at the 7-position of the nucleobase by treatment with N-chlorosuccinimide
to provide 9 in 32% yield.
The same general strategy was used to synthesize
in Scheme 3. In this case, the synthetic route started from
with methanol under Fischer glycosylation conditions to give 23 in 91% yield. A subsequent
perbenzylation reaction yielded methyl-2,3,5-tri-O-benzyl-β/α- -riboside 24, which was then
L-C-nucleosides 10 and 11, as illustrated
L-ribose, which was first reacted
L
subjected to acid hydrolysis and oxidation in the presence of DMSO/Ac2O26 to afford lactone
24 in 50% yield over two steps. Compound 25 was then reacted with its aglycone coupling
partner under the conditions described above to provide 26 as a mixture of anomers in 60%
yield. Upon reduction, only β-anomer 27 was isolated in 70% yield. Subsequent protecting
group manipulation led to 10, which smoothly underwent further chlorination at the
nucleobase to afford 11.
SCH3
N
O
HO
O
N
O
O
MeO
O
d
MeO
OH
O
a
c
b
N
OBn
OBn
OH
OBn
HO
BnO
HO
OH
BnO
OBn
OBn
HO
OH
BnO
OBn
25
L-Ribose
23
24
22
e
NH2
NH2
NH2
SCH3
Cl
N
N
N
N
h
g
f
N
N
N
N
N
O
O
O
O
N
N
N
OH
OH
OBn
OBn
HO
OH
HO
OH
BnO
OBn
BnO
OBn
11
10
27
26
Scheme 3. Synthesis of
L-sugar modified 7-chloro pyrrolotriazine nucleosides 10 and 11. Reagents and
conditions: (a) conc. H2SO4, MeOH, rt, 5 h, 91%; (b) NaH, BnBr, THF, 0 °C to rt, on, 75%; (c) (i) AcOH, 1 N
HCl in H2O, 80 °C, 4 h; (ii) DMSO, Ac2O, rt, on, 60% over two steps; (d) 19, Lithium diisopropylamide (LDA),
THF, -78 °C, 3 h, 60%; (e) BF3.OEt2, Et3SiH, DCM, 0 °C, 40 min, 70%; (f) 7 M NH3 in MeOH, 100 °C, 24 h,
80%; (g) Pd(OH)2/C, H2, CH3COOH, rt, 48 h, 80%; (h) N-Chlorosuccinimide (NCS), DMF, 0 °C to rt, 2 days,
25%.
In addition, encouraged by previous literature examples,27,28 a selected prodrug of
compound 5, which emerged as the most active congener against MNV but did not retain its
activity against HuNoV (see below), was also prepared (Scheme 4). Benzyl esters are
commonly used as prodrug moieties to improve the potency of nucleoside prodrugs due to the
fact that they are good substrates for the esterase involved in phosphoramidate conversion.27-28
In this study, bisphosphoramide 28 was specifically proposed in order to avoid the
introduction of a chiral phosphorus atom in the parent compound.

NH2
N
Cl
NH2
N
Cl
O
P
O
a
N
O
N
N
N
O
O
H
N
BnO
NH
HO
OH
HO
OH
HO
BnO
O
5
28
Scheme 4. Synthesis of bisphosphoramide prodrug 28. Reagents and conditions: (a) L-alanine benzyl ester
hydrochloride, POCl3, NaHCO3, PO(OMe)3, 0 °C to rt, 11%.
2.2. Antiviral activity
At first, compounds 1, 4-11, and 28 were screened for their ability to inhibit the in vitro
replication of genogroup V (GV) MNV, which is a surrogate for the HuNoV. To this aim, a
cytopathic effect (CPE)-based assay was performed in the murine macrophage cell line RAW
264.7 with a readout at 72 h post-infection. All compounds were evaluated by performing
dose response experiments at 8 different concentrations in order to determine their EC50
values. The related cytotoxicity (CC50) was also determined in parallel in uninfected cells. All
results obtained from these tests are summarized in Table 1.
Compound 4 along with its halogenated derivatives 5-6 and 2’-β-Me-C-nucleoside 1
exhibited good to potent inhibitory activity against norovirus, yet also displayed substantial
cytotoxicity. In particular, 2’-β-Me C-nucleoside 1 inhibited MNV replication with an EC50 of
~11 µM and CC50 of ~24 µM. Compound
4
bearing an unmodified
pyrrolo[2,1-f][triazin-4-amino]adenine base and its 7-chlorinated analogue 5 emerged as the
most potent compounds with a similar EC50 of 0.007 µM. The cytotoxic effect increased upon
replacement of the 7-H (4) with a Cl atom (5), as demonstrated by the corresponding decrease
in CC50 from 0.14 to 0.01 along with a diminishing SI. Bromo and iodo analogues 6 and 7
were slightly less active and cytotoxic with an EC50 of 0.024 and 0.035 µM and CC50 of 0.45
and 0.78 µM, respectively.
In a previous study, cellular toxicity against specific immortalized cell lines was also
observed for compounds 4-7.21 On the other hand, the N-nucleoside congener 8 and the base
chlorinated derivative of 2’-β-Me C-nucleoside 1 (compound 9) were found to be devoid of
any significant antiviral activity, while exhibiting a CC50 of 2.53 and 4.78 µM, respectively. It
should be noted that the 7-chloro modified 2’-β-Me C-nucleoside 9 was more toxic than 1.
No antiviral activity or cytotoxicity was observed at the highest concentration tested for
compounds 10 and 11 featuring a L-sugar moiety. Surprisingly, the introduction of a prodrug
moiety at the 5’-position of compound 5 led to the complete loss of both antiviral and
cytotoxic effects, as shown in Table 1 for compound 28 (entry 10). This is unusual as
generally phosphoramidate prodrugs are more potent than the parent drugs; however, a similar
case was recently reported29 and could most likely be justified by the fact that the prodrug
cannot be readily cleaved to release the parent nucleoside inside the cell.
Table 1
In vitro inhibitory activity of compounds 1, 4-11, and 28 against murine norovirus (MNV).
Entry
Compound
EC50 (µM)a
CC50 (µM)b
SI

11.1
23.7
0.14
>0.01
0.45
0.78
2.53
4.78
>100c
>100c
>10
2
1
2
3
4
5
6
7
8
1
4
5
6
7
8
9
10
11
28
0.007
0.007
0.024
0.035
>2c
20
>1
19
22
–
–
–
–
>4c
>100c
>100c
>10c
9
10
–
aEC50 is the effective concentration to inhibit the replication of the virus by 50%; CC50 is the cytotoxic
b
c
concentration that reduces the number of viable cells by 50%. EC50> indicates that no antiviral activity was
observed at the highest concentration tested.
Furthermore, compounds 4-7, which emerged as the most active of this series in the MNV
assay, were also evaluated against HuNoV using a human gastric tumor-1 (HGT-1) cell line
that stably expresses the genome of genogroup I (GI.1) HuNoV. This HuNoV GI.1
replicon-bearing cell line is the standardly used model for studying the antiviral effect of
small molecules.30 In this system, the intracellular GI.1 virus replicon RNA was quantified by
quantitative reverse transcription-PCR [qRT-PCR] using β-actin mRNA as reference gene.31
Since the gene encoding for the major capsid protein is replaced by a neomycin resistance
gene, no new virus particles are produced, however the non-structural proteins are expressed
and the replication of the genomic RNA can be analyzed.30,32
As shown in Table 2, it can be seen that compound 4 reduced the levels of HuNoV virus
RNA with an EC50 of 0.015 µM. However, at concentrations higher than the EC50, a toxic
effect was observed on the host cells. Compound 5-7 did not show activity to reduce level of
HuNoV virus RNA but displayed cytotoxicity with a CC50 ranges from 3.1 µM to 6.3 µM.
The differences in EC50 values between the antiviral assays used in this study could be due
to the fact that the corresponding viruses belong to two different norovirus genogroups.
Despite the great similarities between the RdRps of GI and GV noroviruses, at the amino acid
level they are ~70% alike. Another factor could be attributed to the technical details of the
assays that use very different readouts.
Table 2
In vitro inhibitory activity of compounds 4-7 against human norovirus (HuNoV).
Entry
Compound
EC50 (µM)a
0.015
>10
>10
>10
CC50 (µM)b
0.031
4.7
3.1
6.3
1
2
3
4
4
5
6
7
aEC50 is defined as the compound concentration that resulted in a 50% reduction of the relative HuNoV
replicon RNA levels (when compared to a housekeeping gene, β-actin). In addition, a potential toxic effect of the
compounds in host cells was scored microscopically at the same time point (72 hours pi). bCC50 is defined as the
compound concentration that reduced the number of cells with a normal morphology by 50%.

3. Conclusions
In summary, a series of C-nucleoside derivatives obtained by combining diversely modified
sugar and nucleobase moieties were evaluated for antiviral activity against both murine and
human norovirus. Synthetically, all compounds were prepared using a direct coupling method
based on the nucleophilic addition of a heterocyclic base to suitably protected modified
furanolactone intermediates. A prodrug (compound 28) was also prepared in the expectation
that the delivery of the monophosphate of the parent modified nucleoside to the cell could
lead to enhanced biological activity. Among these compounds, 4-aza-7,9-dideazaadenosine,
its 7-halogenated analogue as well as
a
2’-β-Me-C-nucleoside bearing
4-aza-7,9-dideazaadenine displayed good inhibitory activity in the MVN assay with EC50
values ranging from 0.007 to 11 µM. Despite the activity being coupled with significant
cytotoxicity, it was interesting to note that 4-aza-7,9-dideazaadenosine retained its antiviral
activity also in a HuNoV GI.1 replicon system. Owing to its dual activity against both murine
and human norovirus, compound 4 could function as reference structure to further elaborate
the structure-activity relationship of C-nucleosides as selective anti-norovirus agents.
4. Experimental section
4.1. General information
All reagents and solvents were purchased from commercial sources and used as obtained.
Moisture sensitive reactions were carried out using oven-dried glassware under a nitrogen or
1
13
31
argon atmosphere. H NMR, C NMR, and P NMR spectra were recorded on a Bruker
Avance 300 or 600 MHz spectrometer using tetramethylsilane as internal standard or
referenced to the residual solvent signal. The following abbreviations were used to indicate
multiplicities: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), and dd
(doublet of doublets). Coupling constants are expressed in hertz (Hz). High-resolution mass
spectra (HRMS) were obtained 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 ionization mode with a resolution of 15000 (fwhm)
using leucine enkephalin as lock mass. Pre-coated aluminum sheets (254 nm) were used for
thin layer chromatography (TLC), and spots were visualized with UV light. All intermediate
products were purified by silica gel column chromatography on silica gel (40-60 µ, 60 Å).
Final compounds were purified by preparative RP-HPLC (C18 Phenomenex Gemini column,
110 Å, 10 µm, 21.2 mm × 250 mm) using a linear gradient of CH3CN or MeOH and H2O.
A detailed account of the synthesis and characterization of compounds 4-7 was previously
reported.21
4.1.1. 7-Deaza-7-chloroadenosine (8). A solution of compound 1423 (1.0 g, 1.58 mmol) in 7 N

methanolic NH3 (60 mL) was stirred for 24 h at 120 °C. After removal of all the volatiles in
vacuo, the crude residue was purified by silica gel column chromatography (CH2Cl2/MeOH =
1
10:1, v/v) to afford title compound 8 as a white solid (400 mg, 84% yield). H NMR (300
MHz, CD3OD) δ 8.08 (s, 1H, H2), 7.56 (s, 1H, H8), 6.06 (d, J1’,2’ = 6.0 Hz, 1H, H1’), 4.32 (dd,
J2’3’ = 4.9 Hz, J2’1’ = 6.0 Hz, 1H, H2’), 4.06 (dd, J3’,4’ = 3.4 Hz, J3’,2’ = 4.9 Hz, 1H, H3’), 3.88
(ddd, J4’,3’ = 3.4 Hz, J4’,5’ = 3.8, J4’,5’’ = 3.7 Hz, 1H, H4’), 3.63-3.47 (2 × dd, Jgem = 11.8 Hz, J5’,4’
13
= 3.8 Hz, J5’’,4’ = 3.7 Hz, 2H, H5’, H5’’); C{1H} NMR (75 MHz, CD3OD) δ 156.8 (C-2),
152.7 (C-4), 149, 3 (C-5), 119.2 (C-6), 102.7 (C-7), 100.0 (C-8), 86.9 (C-1’), 85.2 (C-4’), 73.9
(C-2’), 70.5 (C-3’), 61.5 (C-5’); HRMS (ESI-TOF) m/z: calcd for C11H13Cl1N4O5 ([M+Na]+),
301.0704, found 301.0703.
4.1.2. Methyl 3,5-di-O-benzyl-2-C-β-methyl-α-
D-ribofuranoside (16). To a solution of methyl
3,5-di-O-benzyl-2-keto-α-
D
-ribofuranoside 1525 (8.0 g, 23.4 mmol) in dry THF (50.0 mL) at 0
ºC was added methylmagnesium bromide (3.0 M in THF, 19.5 mL, 58.4 mmol) under an
argon atmosphere. After stirring at room temperature for 4 h, the reaction mixture was
quenched with saturated aq. NH4Cl. It was then extracted with dichloromethane, and the
organic layer was washed with brine. After removal of all the volatiles in vacuo, the residue
was purified by silica gel column chromatography (heptane/EtOAc, 2:1, v/v) to give
compound 16 as an oil (7.8 g, 92% yield). 1H NMR (600 MHz, CDCl3) δ 7.33-7.25 (m, 10H,
Ar-H), 4.76 (d, J = 12.0 Hz, 1H, OCH2Ph), 4.50-4.41 (m, 4H, H1, OCH2Ph), 4.12 (ddd, J4,3
=
4.2 Hz, J4,5a = 4.2 Hz, J4,5b = 3.2, 1H, H4), 3.51-3.43 (2 × dd, Jgem = 11.3 Hz, J5b,4 = 3.2 Hz,
J5a,4 = 4.2 Hz, 2H, H5a, H5b), 3.41 (s, 3H, OCH3), 3.39 (s, 1H, OH), 3.33 (d, J3,4 = 4.1 Hz, 1H,
H3); 13C{1H} NMR (150 MHz, CDCl3) δ 138.2, 128.7, 128.2, 128.1, 127.9, 107.8, 82.6, 81.9,
76.9, 73.7, 73.3, 70.2, 55.5, 24.9; HRMS (ESI-TOF) m/z: calcd for C21H26O5 ([M+Na]+),
381.1678, found 381.1670.
4.1.3. Methyl 2,3,5-di-O-benzyl-2-C-β-methyl-α-D-ribofuranoside (17). Sodium hydride (60%,
17 g, 87.0 mmol) was added to a solution of 16 (7.8 g, 21.8 mmol) in dry THF (100 mL) at
room temperature. After hydrogen generation ceased, benzyl bromide (7.8 mL, 65.3 mmol)
and tetrabutylammonium iodide (241 mg, 0.653 mmol) were added. After stirring overnight at
70 ºC, the reaction mixture was slowly poured into cold saturated aq. ammonium chloride
(100 mL) at 0 ºC. The mixture was then extracted with EtOAc (3 × 200 mL), and the
combined organic layers were washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The resulting crude residue was purified by silica gel
column chromatography (gradient heptane/EtOAc, 20:1 to 5:1, v/v) to afford 17 as an oil (7.2
1
g, 73%). H NMR (300 MHz, CDCl3) δ 7.43 (d, J = 7.0 Hz, 2H, Ar-H), 7.35-7.21 (m, 13H,
Ar-H), 4.79 (d, J = 12.0 Hz, 1H, OCH2Ph), 4.69 (s, 1H, H1), 4.63-4.42 (m, 4H, H3 and
OCH2Ph), 4.27 (ddd, J4,5a = 4.5 Hz, J4,5b = 3.6 Hz, J4,3 = 4.2 Hz, 1H, H4), 3.58-3.50 (2 × dd,
Jgem = 13.2 Hz, J5b,4 = 3.6 Hz, J5a,4 = 4.5 Hz, 2H, H5a, H5b), 3.45 (s, 3H, OCH3), 1.33 (s, 3H,
13
CH3); C{1H} NMR (75 MHz, CDCl3) δ 139.7, 138.8, 138.3, 128.7, 128.6, 128.5, 128.0,
127.9, 127.6, 127.5, 107.7, 82.5, 82.1, 82.0, 73.8, 73.0, 70.2, 66.8, 55.6, 22.9; HRMS
(ESI-TOF) m/z: calcd for C28H32O5 ([M+Na]+), 471.2147, found 471.2135.
4.1.4. 2,3,5-Tri-O-benzyl-2-C-β-methyl-D-ribono-1,4-lactone (18). To a solution of 17 (7.2 g,
16.1 mmol) in AcOH (50 mL) was added 1 N aq. HCl (12.5 mL), and the mixture was stirred
for 4 h at 80 °C. After removal of all the volatiles in vacuo, the residue was redissolved in
EtOAc. The organic layer was washed with saturated aq. NaHCO3 and brine, dried over

Na2SO4, filtered, and evaporated under reduced pressure. The resulting residue was dissolved
in DMSO (25 mL), and then Ac2O (15 mL) was slowly added at room temperature. The
mixture was stirred at room temperature overnight. It was then extracted with EtOAc (3 × 200
mL), and the combined organic layers were washed with brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude residue was purified by silica gel column
chromatography (heptane/EtOAc, 5:1, v/v) to afford 18 as an oil (5.3 g, 76%). 1H NMR (300
MHz, CDCl3) δ 7.40-7.22 (m, 15H, Ar-H), 4.81-4.45 (m, 4H, H3, H4 and OCH2Ph), 4.02 (d, J
= 7.4 Hz, 1H, OCH2Ph), 3.76-3.53 (2 × dd, Jgem = 11.6 Hz, J5b,4 = 2.3 Hz, J5a,4 = 3.7 Hz, 2H,
13
H5a, H5b); C{1H} NMR (75 MHz, CDCl3) δ 173.7, 138.3, 137.9, 137.7, 128.9, 128.8, 128.7,
128.5, 128.2, 128.0, 80.7, 80.4, 77.7, 73.8, 73.6, 67.8, 20.3; HRMS (ESI-TOF) m/z: calcd for
C27H28O5 ([M+Na]+), 455.1834, found 455.1825.
4.1.5. 2’,3’,5’-O-Tribenzyl-1’-hydroxy-2’-C-methyl-6-methylthio-4-aza-7,9-dideazadenosine
(20). To a solution of 4-(methylthio)pyrrolo[2,1-f][1,2,4]triazine 19 (1.2 g, 7.26 mmol) in
THF (20 mL) was added lithium diisopropylamide (2 M in THF, 5.4 mL, 10.9 mmol) at -78
°C under an argon atmosphere. The resulting mixture was stirred for 30 min at -78 °C. Then, a
solution of compound 18 (3.2 g, 7.40 mmol) in THF (10 mL) was added at -78 °C and the
resulting reaction mixture was stirred for 3 h at -78 °C. The reaction was quenched by adding
saturated aq. NH4Cl and then extracted with ethyl acetate. The organic layer was washed with
brine, dried over Na2SO4, and concentrated in vacuo. The crude residue was purified by silica
gel silica gel column chromatography (heptane/ EtOAc = 5:1, v/v) to give the title compound
as an oil (1.2 g, 32% yield). HRMS (ESI-TOF) m/z: calcd for C33H35N3O5S1 ([M+H]+),
598.2376, found 598.2339.
4.1.6. 2’,3’,5’-O-Tribenzyl-2’-C-methyl-6-methylthio-4-aza-7,9-dideazadenosine (21). To a
solution of compound 20 (1.3 g, 2.17 mmol) in dichloromethane (50 mL) was added
triethylsilane (1.01 g, 8.70 mmol) and trifluoroborane (552.0 mg, 4.35 mmol) at 0 °C under an
argon atmosphere. The resulting solution was stirred for 40 min at 0 °C and then quenched
with saturated aq. Na2CO3. The mixture was extracted with ethyl acetate. The organic layer
was washed with water, brine, dried over Na2SO4, and concentrated in vacuo. The crude
residue was purified by silica gel column chromatography (heptane/EtOAc = 10:1, v/v) to
1
give the title compound as an oil (900 mg, 71%). H NMR (300 MHz, CDCl3) δ 8.17 (s, 1H,
H2), 7.47-7.20 (m, 15H, Ar-H), 7.04 (d, J = 4.5 Hz, 1H, H7), 6.70 (d, J = 4.5 Hz, H8), 5.92 (s,
1H, H1’), 4.85 (q, J = 11.5 Hz, 2H, OCH2Ph), 4.71-4.39 (m, 5H, H3’, H4’ and OCH2Ph), 4.08
(d, J = 8.3 Hz, 1H, OCH2Ph), 3.94-3.68 (2 × dd, Jgem = 11.2 Hz, J5’,4 = 2.2 Hz, J5’’,4 = 3.4 Hz,
2H, H5’, H5’’), 2.62 (s, 3H, SCH3), 1.00 (s, 3H, CH3); 13C{1H} NMR (75 MHz, CDCl3) δ 164.6,
145.3, 140.0, 138.5, 128.7, 128.6, 128.5, 128.2, 128.1, 128.0, 127.5, 127.4, 121.9, 112.6,
102.5, 85.3, 82.3, 79.6, 78.1, 73.8, 69.4, 66.1, 17.7, 11.7; HRMS (ESI-TOF) m/z: calcd for
C34H35N3O4S1 ([M+H]+), 582.2427, found 582.2441.
4.1.7. 2’-C-Methyl-4-aza-7,9-dideazaadenosine (1). A solution of compound 21 (900 mg, 2.11
mmol) in 7 N methanolic ammonia (60 mL) was stirred for 24 h at 100 °C. After removal of
all the volatiles under reduced pressure, the remaining crude residue was purified by silica gel
column chromatography (heptane/EtOAc = 5:1, v/v) to afford the debenzylated product as an
1
oil (500 mg, 59% yield). H NMR (300 MHz, CDCl3) δ 7.84 (s, 1H, H2), 7.45-7.22 (m, 15H,
Ar-H), 6.87 (d, J = 4.5 Hz, H7), 6.52 (d, J = 4.5 Hz, H8), 6.14 (br, 2H, NH2), 5.90 (s, 1H, H1’),
4.85 (q, J = 12.0 Hz, 2H, OCH2Ph), 4.70-4.37 (m, 5H, H4’, H3’, and OCH2Ph), 4.07 (d, J = 8.5

Hz, 1H, OCH2Ph), 3.92-3.69 (2 × dd, Jgem = 10.8 Hz, J5’,4 = 2.2 Hz, J5’’,4 = 3.4 Hz, 2H, H5’,
H5’’), 1.04 (s, 3H, CH3); 13C{1H} NMR (75 MHz, CDCl3) δ 155.6, 146.9, 140.0, 138.5, 131.1,
128.7, 128.6, 128.5, 128.3, 128.1, 127.9, 127.8, 127.5, 127.4, 114.3, 110.9, 100.9, 85.2, 82.7,
79.5, 78.3, 73.7, 69.6, 66.0, 17.9; HRMS (ESI-TOF) m/z: calcd for C33H34N4O4 ([M+H]+),
551.2658, found 551.2661. Such intermediate (500 mg, 0.908 mmol) was dissolved in
cyclohexene/ethanol = 4/1 (10 mL), followed by the addition of Pd(OH)2 (10%) (500 mg).
The reaction mixture was stirred under a hydrogen atmosphere at 85 °C for 12 h, cooled, and
then filtered through a pad of Celite. After evaporation under reduced pressure, the remaining
crude residue was purified by silica gel column chromatography (CH2Cl2/MeOH = 10:1, v/v)
affording compound 1 as a white solid (200 mg, 80% yield). 1H NMR (300 MHz, CD3OD) δ
7.79 (s, 1H, H2), 6.88 (d, J = 4.5 Hz, H7), 6.77 (d, J = 4.5 Hz, H8), 5.57 (s, 1H, H1’), 4.02-3.78
(m, 3H, H4’, H5’, H5’’), 3.64 (s, 1H, H3’), 0.94 (s, 3H, CH3); 13C{1H} NMR (75 MHz, CD3OD)
δ 155.3 (C-2), 146.0 (C-5), 129.9 (C-6), 113.8 (C-9), 109.65 (C-7), 101.27 (C-8), 81.9 (C-1’),
78.8 (C-4’), 74.4 (C-2’), 69.6 (C-3’), 61.2 (C-5’), 19.6 (2-C-CH3); HRMS (ESI-TOF) m/z:
calcd for C12H16N4O4 ([M+H]+), 281.1250, found 281.1243.
4.1.8. 2’-C-Methyl-7-chloro-4-aza-7,9-dideazaadenosine (9). A solution of compound 1 (85
mg, 0.303 mmol) in anhydrous DMF (5 mL) was cooled to 0 °C. N-Chlorosuccinimide (32
mg, 0.242 mmol) was added and the reaction mixture was stirred for 2 days at room
temperature. After evaporation under reduced pressure, the crude residue was purified by
silica gel column chromatography (CH2Cl2/MeOH = 10:1, v/v) furnishing the title compound
as a white solid (30 mg, 32% yield). 1H NMR (300 MHz, CD3OD) δ 7.74 (s, 1H, H2), 6.83 (s,
1H, H8), 5.53 (s, 1H, H1’), 4.00-3.75 (m, 3H, H4’, H5’, H5’’), 3.64 (s, 1H, H3’), 0.96 (s, 3H,
CH3); 13C{1H} NMR (75 MHz, CD3OD) δ 154.9 (C-2), 146.9 (C-5), 130.0 (C-6), 109.5 (C-9),
104.6 (C-8), 81.9 (C-7), 80.6 (C-1’), 78.8 (C-4’), 74.0 (C-2’), 69.8 (C-3’), 60.8 (C-5’), 19.4
(2-C-CH3); HRMS (ESI-TOF) m/z: calcd for C12H15Cl1N4O4 ([M+H]+), 315.0860, found
315.0869.
4.1.9. Methyl α/β-L-ribofuranoside (22). A solution of L-ribose (5.0 g, 33.3 mmol) in
anhydrous methanol (100 mL) at 0 °C was treated with concentrated sulfuric acid (0.6 mL).
The reaction mixture was stirred at room temperature for 4.5 h. The solution was then
neutralized with solid Na2CO3 to pH > 7. The solid was filtered and washed with methanol (3
× 10 mL). After evaporation of the filtrate, methyl α/β-L-ribofuranoside 22 was obtained as an
oil (5.0 g, 91% yield). 1H NMR analysis showed a ratio of α-22/β-22 ~1/4. Data for α: 13C{1H}
NMR (75 MHz, CD3OD) δ 102.9 (C-1), 85.3 (C-4), 71.1 (C-2), 69.4 (C-3), 61.6 (C-5), 53.9
(OCH3); HRMS: calcd for C6H12O5 ([M+Na]+), 187.0582, found 187.0576. Data for β-22: 1H
NMR (300 MHz, CD3OD) δ 4.78 (s, 1H, H1), 4.06 (d, J2,3 = 4.5 Hz, 1H, H2), 3.98 (dd, J4,5b
=
3.4 Hz, J4,5a = 6.1 Hz, 1H, H4), 3.92 (d, J3,2 = 4.5 Hz, H3), 3.74-3.54 (2 × dd, Jgem = 11.8 Hz,
13
J5a,4 = 6.1 Hz, J5b,4 = 3.6 Hz, 2H, H5a, H5b), 3.36 (s, 3H, OCH3); C{1H} NMR (75 MHz,
CD3OD) δ 108.1, 83.0, 74.4, 71.0, 63.3, 53.8.
4.1.10. Methyl 2,3,5-tri-O-benzyl-α/β-
152.3 mmol) was added to a solution of methyl α/β-
L
-ribofuranoside (23). Sodium hydride (60%, 13.0 g,
-ribofuranoside 22 (5.0 g, 30.5 mmol) in
L
dry DMF (100 mL) at room temperature under an argon atmosphere. After hydrogen
generation ceased, benzyl bromide (19.0 mL, 121.8 mmol) was added, and the mixture was
stirred at 0 °C to rt for 24 h. It was then slowly poured into cold saturated aq. NH4Cl (100 mL)
at 0 °C, and further diluted and extracted with EtOAc (3 × 200 mL). The combined organic

layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced
pressure. The crude residue was purified by silica gel column chromatography (gradient
heptane/EtOAc, 20:1 to 5:1, v/v) to afford α-23/β-23 (10.0 g, 75% yield). Data for β: 1H NMR
(300 MHz, CDCl3) δ 8.18-7.99 (m, 15H, Ar-H), 5.74 (s, 1H, H1), 5.47-5.17 (m, 7H, H2, 3 ×
OCH2Ph), 4.86 (m, 1H, H3), 4.66 (m, 1H, H4), 4.44-4.32 (m, 2H, H5a, H5b), 4.09 (s, 3H,
13
OCH3); C{1H} NMR (75 MHz, CDCl3) δ 138.8, 138.4, 138.3, 128.8, 128.4, 128.3, 128.2,
128.0, 127.9, 106.8, 80.9, 80.2, 78.9, 73.5, 72.8, 72.7, 71.8, 55.4; HRMS (ESI-TOF) m/z:
calcd for C27H30O5 ([M+Na]+), 457.1991, found 457.1978.
4.1.11. 2,3,5-Tri-O-benzyl-L-ribono-1,4-lactone (24). To a solution of 23 (10.0 g, 23.0 mmol)
in AcOH (100 mL) was added 1 N aq. HCl (25 mL), and the mixture was stirred for 4 h at
80 °C. After removal of all the volatiles under reduced pressure, the residue was dissolved in
EtOAc and washed with aq. saturated NaHCO3 and brine. The organic layer was then dried
over Na2SO4, filtered, and evaporated under reduced pressure. The resulting residue was
dissolved in DMSO (55 mL), then Ac2O (33 mL) was slowly added at room temperature. The
mixture was stirred at room temperature overnight, evaporated under reduced pressure, and
the residue was diluted and extracted with EtOAc (3 × 200 mL). The combined organic layers
were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude
residue was purified by silica gel column chromatography (heptane/EtOAc, 5:1, v/v) to afford
24 as an oil (6.0 g, 60%). 1H NMR (300 MHz, CDCl3) δ 7.36-7.21 (m, 13H, Ar-H), 7.15-7.08
(m, 2H, Ar-H), 4.87 (d, J = 12.0 Hz, 1H, OCH2Ph), 4.67 (t, J = 12.3 Hz, 1H, OCH2Ph),
4.66-4.29 (m, 6H, H2, OCH2Ph), 4.07 (d, J3,2 = 5.2 Hz, 1H, H3), 3.53 (m, 2H, H5a, H5b); 13C{1H}
NMR (75 MHz, CDCl3) δ 174.2, 137.7, 137.6, 137.4, 128.9, 128.9, 128.6, 128.5, 128.4, 128.3,
127.9, 82.2, 75.8, 74.3, 73.9, 73.1, 72.70, 69.2; HRMS (ESI-TOF) m/z: calcd for C26H26O5
([M+Na]+), 441.1678, found 441.1669.
4.1.12. 2’,3’,5’-O-Tribenzyl-1’-hydroxy-6-methylthio-4-aza-7,9-dideaza-L-adenosine (25). To
a solution of 4-(methylthio)pyrrolo[2,1-f][1,2,4]triazine 19 (800 mg, 4.78 mmol) in dry THF
(20 mL) was added lithium diisopropylamide (2 M in THF, 3.6 mL, 7.17 mmol) at -78 °C
under an argon atmosphere, and the resulting mixture was stirred for 30 min at this
temperature. Then, a solution of compound 24 (3.0 g, 7.17 mmol) in dry THF (10 mL) was
added and the resulting reaction mixture was stirred for further 3 h at -78 °C. The reaction
was quenched with saturated aq. NH4Cl and then extracted with ethyl acetate. The organic
layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The crude
residue was purified by silica gel column chromatography (heptane/EtOAc = 5:1, v/v) to give
the title compound as a yellow oil (1.6 g, 60% yield). HRMS (ESI-TOF) m/z: calcd for
C33H35N3O5S1 ([M+H]+), 584.2219, found 584.2186.
4.1.13. 2’,3’,5’-O-Tribenzyl-6-methylthio-4-aza-7,9-dideaza-L-adenosine (26). To a solution
of compound 25 (1.60 g, 2.74 mmol) in dichloromethane (50 mL) was added triethylsilane
(1.27 g, 11.0 mmol) and trifluoroborane (778.1 mg, 5.48 mmol) at 0 °C under an argon
atmosphere. The resulting solution was stirred for 40 min at 0 °C and then quenched with a
saturated aq. Na2CO3 solution. The mixture was extracted with ethyl acetate, and the organic
layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The crude residue was purified by silica gel column chromatography (heptane/EtOAc
1
= 10:1, v/v) to give the title compound as an oil (1.2 g, 80% yield). H NMR (300 MHz,
CDCl3) δ 8.17 (s, 1H, H2), 7.32-7.20 (m, 15H, Ar-H), 6.80 (d, J = 4.6 Hz, 1H, H7), 6.66 (d, J =

4.6 Hz, 1H, H8), 5.71 (d, J1’,2’ = 4.2 Hz, 1H, H1’), 4.72-4.37 (m, 7H, 3 × OCH2Ph, H2’), 4.25
(m, 1H, H3’), 4.12 (m, H4’), 3.81-3.60 (2 × dd, J5’’,4’ = 3.3 Hz, J5’’,4’ = 3.9 Hz, Jgem = 10.8 Hz,
13
2H, H5’, H5’’), 2.59 (s, 3H, SCH3); C{1H} NMR (75 MHz, CDCl3) δ 164.8, 145.4, 138.6,
138.2, 129.6, 128.7, 128.6, 128.4, 128.2, 128.1, 127.9, 122.6, 112.6, 102.2, 81.0, 79.4, 77.6,
76.3, 73.7, 72.4, 72.0, 70.0, 11.7; HRMS (ESI-TOF) m/z: calcd for C33H33N3O4S1 ([M+H]+),
568.2270, found 568.2258.
4.1.14. 2’,3’,5’-O-Tribenzyl-4-aza-7,9-dideaza-L-adenosine (27). A solution of compound 26
(1.2 g, 2.11 mmol) in 7 N methanolic ammonia (60 mL) was stirred for 24 h at 100 °C. After
removal of all the volatiles in vacuo, the crude residue was purified by silica gel column
chromatography (heptane/EtOAc = 5:1, v/v) to afford the title compound as a yellow oil (1.0
g, 80% yield). 1H NMR (300 MHz, CDCl3) δ 7.86 (s, 1H, H2), 7.41-7.19 (m, 15H, Ar-H), 6.60
(d, J = 4.6 Hz, 1H, H7), 6.49 (d, J = 4.6 Hz, 1H, H8), 6.16 (br, 2H, NH2), 5.68 (d, J1’,2’ = 4.2
Hz, 1H, H1’), 4.74-4.35 (m, 7H, 3 × OCH2Ph, H2’), 4.25 (m, 1H, H3’), 4.12 (m, 1H, H4’),
13
3.81-3.60 (m, 2H, H5’, H5’’); C{1H} NMR (75 MHz, CDCl3) δ 155.7, 147.2, 138.5, 138.2,
129.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9, 1278; HRMS (ESI-TOF) m/z: calcd for
C32H32N4O4 ([M+H]+), 537.2502, found 537.2512.
4.1.15. 4-Aza-7,9-dideaza-L-adenosine (10). To a solution of compound 27 (1.0 g, 1.86 mmol)
in AcOH (10 mL) was added Pd(OH)2 (10%) (1.0 g) and the reaction mixture was stirred
under a hydrogen atmosphere at room temperature for 48 h. It was then filtered through a pad
of Celite, and the filtrate was concentrated under reduced pressure. The resulting crude
residue was purified by silica gel column chromatography (CH2Cl2/MeOH = 8:1, v/v) to
afford the title compound as a white solid (400 mg, 80% yield). 1H NMR (300 MHz, CD3OD)
δ 7.78 (s, 1H, H2), 6.88 (d, J = 4.6 Hz, 1H, H7), 6.76 (d, J = 4.6 Hz, 1H, H8), 5.25 (d, J1’,2’
=
6.3 Hz, 1H, H1’), 4.53 (dd, J2’,1’ = 6.3 Hz, J2’,3’ = 5.4, 1H, H2’), 4.20 (dd, J3’,4’ = 3.9 Hz, J3’,2’=
5.4 Hz, 1H, H3’), 4.06 (ddd, J4’,5’’ = 3.2 Hz, J4’,5’ = 3.9 Hz, J4’,3’ = 3.9 Hz, 1H, H4’), 3.85-3.67
(2 × dd, J5’’,4’ = 3.2 Hz, J5’,4’ = 3.9 Hz, Jgem = 12.1 Hz, 2H, H5’, H5’’); 13C{1H} NMR (75 MHz,
CD3OD-d4) δ 155.6 (C-2), 146.2 (C-5), 128.3 (C-6), 114.9 (C-9), 110.4 (C-7), 100.9 (C-8),
84.7 (C-1’), 76.9 (C-4’), 73.2 (C-2’), 71.6 (C-3’), 62.1 (C-5’); HRMS (ESI-TOF) m/z: calcd
for C11H14N4O5 ([M+H]+), 267.1093, found 267.1091.
4.1.16. 7-Chloro-4-aza-7,9-dideaza-L-adenosine (11). A solution of compound 10 (92 mg,
0.345 mmol) in anhydrous DMF (6 mL) was cooled to 0 °C. N-Chlorosuccinimide (51 mg,
0.345 mmol) was added and the reaction mixture was stirred for 2 days at room temperature.
The reaction mixture was concentrated and the crude residue was purified by silica gel
column chromatography (CH2Cl2/MeOH = 10:1, v/v) furnishing the title compound as a white
solid (30 mg, 29% yield). 1H NMR (300 MHz, DMSO-d6) δ 7.85 (s, 1H), 6.83 (s, 1H), 5.12 (d,
J1’,2’ = 6.0 Hz, 1H, H1’), 4.16 (dd, J2’,1’ = 6.1 Hz, J2’,3’ = 5.6 Hz, 1H, H2’), 3.94 (dd, J3’,2’ = 5.6
Hz, J3’,4’= 4.6 Hz, H3’), 3.78 (ddd, J4’,3’ = 4.6 Hz, J4’,5’’ = 3.8 Hz, J4’,5’ = 4.1 Hz, H4’), 3.60-3.41
13
(2xdd, J5’,4’ = 4.1 Hz, J5’’,4’ = 3.8 Hz, Jgem = 11.3 Hz, H5’, H5’’); C{1H} NMR (75 MHz,
DMSO-d6) δ 155.0 (C-2), 148.1 (C-5), 129.5 (C-6), 110.5 (C-9), 109.8 (C-8), 103.1 (C-7),
84.6 (C-1’), 75.0 (C-4’), 74.2 (C-2’), 71.1 (C-3’), 61.8 (C-5’); HRMS (ESI-TOF) m/z: calcd
for C17H13Cl1N4O4 ([M+H]+), 301.0704, found 301.0703.
4.1.17. 7-Chloro-5'-bis(l-alanine benzyl ester)phosphate-4-aza-7,9-dideazaadenosine (28). To
a suspension of compound 5 (50.0 mg, 0.16 mmol) and solid sodium bicarbonate (50 mg) in
trimethyl phosphate (1.5 mL) at 0 °C was added POCl3 (117 mg, 0.78 mmol). The mixture

was stirred at 0 °C for 3 h, and a solution of L-alanine benzyl ester hydrochloride (900 mg,
4.16 mmol) was then added. The reaction mixture was stirred at 0 °C for 0.5 h, then
triethylamine (0.1 mL) was added and stirred at room temperature for 0.5 h. The reaction
mixture was diluted with ethyl acetate (10 mL), washed with water (10 mL), brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified first by
silica gel column chromatography (CH2Cl2/MeOH = 10:1, v/v) and then reverse phase HPLC
(5-60% MeCN in water) to afford compound 28 as a white foam (12 mg, 11%). 1H NMR (300
MHz, CD3OD) δ 7.78 (s, 1H, H2), 7.38-7.28 (m, 10H, Ar-H), 6.77 (s, 1H, H8), 5.32 (d, J1’,2’
=
5.4 Hz, 1H, H1’), 5.16-5.05 (m, 4H, 2 × OCH2Ph), 4.34 (dd, J2’,1’ = 5.4 Hz, 1H, J2’,3’= 4.8 Hz,
H2’), 4.20-3.84 (m, 6H, 2 × CH(CH3)NH), H3’, H4’, H5’, H5’’), 1.29 (d, J = 7.2 Hz, 3H, CH3),
1.25 (d, J = 7.2 Hz, 3H, CH3); 13C{1H} NMR (300 MHz, CD3OD) δ 177.3, 177.2 (q, J = 4.8
Hz, CO), 156.6, 148.8, 137.2 (d, J = 5.5 Hz), 130.0, 129.5, 129.2, 112.2, 111.1, 106.0, 83.6,
83.5 (d, J = 7.4 Hz), 77.8, 75.3, 72.3, 67.8, 67.9 (d, J = 3.7 Hz), 66.5, 66.4 (d, J = 5.7 Hz),
31
51.0, 20.7 (d, J = 5.9 Hz), 20.5 (d, J = 6.3 Hz); P NMR (121 MHz, CD3OD) δ = 13.70;
HRMS (ESI-TOF) m/z: calcd for C31H36Cl1N6O9P1 ([M+H]+), 703.2048, found 703.2062.
4.2. Biological assays
4.2.1. Cells, viruses, compounds
Murine norovirus (strain MNV-1.CW1) was propagated in RAW 264.7 cells grown in DMEM
supplemented with 10 or 2% FBS, 2 mM L-glutamine, 20 mM HEPES, 0.075 g/L sodium
bicarbonate, 1 mM sodium pyruvate, 100 U penicillin/mL, 100 µg/mL streptomycin (Thermo
Fisher, Gent, Belgium) at 37 °C in a humidified atmosphere of 5% CO2.
For in vitro assays, a stock solution of each compound was prepared either in dimethyl
sulfoxide (DMSO, VWR Chemicals, Haasrode, Belgium) or MilliQ water.
4.2.2. Antiviral assay for murine norovirus (MNV)
The antiviral activity of the synthesized compounds was initially determined using an MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
]-based cytopathic effect (CPE) reduction assay. RAW 264.7 cells (1 × 104 cells/well) were
seeded in a 96-well plate and infected with MNV (MOI of 0.001) in the presence (or absence)
of a dilution series of compounds (0.023–50 or 100 µg/mL). Cells were incubated for 3 days,
i.e., until complete CPE was observed in infected untreated cells. Then, a
MTS-phenazinemethosulfate (MTS/PMS) stock solution [(2 mg/mL MTS (Promega, Leiden,
The Netherlands) and 46 g/mL PMS (Sigma–Aldrich, Bornem, Belgium) in PBS at pH 6–6.5)]
was diluted 1/20 in MEM (Thermo Fisher, Gent, Belgium) and 75 µL were added to each well.
After 2 h, the optical density (OD) was read at 498 nm. The %CPE reduction was calculated
as [(ODtreated
)
- ODVC ]/[ODCC - ODVC ] × 100, where ODCC represents the OD of the
MNV
uninfected untreated cells, while ODVC and (ODtreated
)
represent the OD of infected
MNV
untreated cells and virus-infected cells treated with a compound concentration, respectively.
The 50% effective concentration (EC50) was defined as the compound concentration that
protected 50% of the cells from virus-induced CPE.
4.2.3. Antiviral assay for human norovirus (HuNoV)
The in vitro antiviral activity of the compounds was evaluated based on the determination of

intracellular GI.1 virus replicon RNA by quantitative reverse transcription-PCR [qRT-PCR])
using β-actin mRNA as reference (housekeeping) gene. HGT-1 cells (5,000/well) were seeded
into the wells of 96-well plates in complete DMEM without G418. After 24 h of incubation,
serial dilutions of compounds were added. Cells were further incubated for 72 h, after which
cell culture supernatant was removed and monolayers were washed with phosphate-buffered
saline (PBS). Cell monolayers were collected for RNA load quantification by qRT-PCR. To
determine the relative levels of GI.1 virus replicon RNA, β-actin was used as a normalizer and
ratios were calculated by the Pfaffl method.29 The GI.1 virus replicon/β-actin ratio was
calculated as follows: Ratio = EGI.1 CT, GI.1 (CC - TC)/Eβ-actin CT, β-actin (CC - TC), where EGI.1 and
β-actin represent the amplification efficiencies (E = 10-1/slope) of the GI.1 virus replicon and
△
△
E
β-actin qRT-PCRs, respectively, △CT, GI.1 virus (CC - TC) is the CT (threshold cycle) of
untreated control cells (CC) minus the CT of cells treated with a compound concentration (TC)
obtained with GI.1 virus replicon primers and probe; and △CT, β-actin (CC - TC) is the CT of
untreated control cells (CC) minus the CT of cells treated with a compound concentration (TC)
obtained with β-actin primers and probe. Efficiency values (EGI.1 and Eβ-actin) were determined
for each qRT-PCR. The 50% effective concentration (EC50) was defined as the compound
concentrations that resulted in 50% reductions of the relative GI.1 virus replicon RNA levels.
4.2.4. Cytotoxicity assay
The cytotoxicity of the compounds was evaluated by the MTS-method, by exposing
uninfected cells to the same concentrations of compounds for 3 days. The % cell viability was
calculated as (ODtreated/ODCC) × 100, where ODCC is the OD of uninfected untreated cells and
OD treated are uninfected cells treated with compound. The CC50 was defined as the
compound concentration that reduces the number of viable cells by 50%. The selectivity
index (SI) was calculated as CC50/EC50.
AUTHOR INFORMATION
Corresponding Author
*
E-mail
addresses:
Piet.Herdewijn@kuleuven.be
(P.
Herdewijn);
E-mail:
elisabetta.groaz@kuleuven.be (E. Groaz).
Notes
The authors declare no competing financial interest.
Acknowledgments
Q.L. thanks the China Scholarship Council (CSC) for funding (grant 201506890014). We
thank Jasper Rymenants and Lindsey Bervoets (KU Leuven) for excellent technical assistance.
We are grateful to Ian Goodfellow (University of Cambridge) for kindly providing the
HuNoV GI replicon.
Appendix A. Supplementary data
Supplementary data related to this article was uploaded.
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• C-Nucleosides comprising structural variations at the sugar and nucleobase were prepared.
• Their ability to inhibit both murine and human norovirus was examined.
• Compounds 4 and 5 resulted in good antiviral activity against murine norovirus.
• Compound 4 retained its antiviral effect in a human replicon assay (EC50 = 0.015 µM).
• Phosphonodiamidate prodrug 28 was devoid of antiviral activity.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: