2-Hydroxyimino-6-aza-pyrimidine nucleosides: Synthesis, DFT calculations, and antiviral evaluations

Article abstract of DOI:10.1039/d0nj04154h

The global public health concerns and economic impact caused by emerging outbreaks of RNA viruses call for the search for new direct acting antiviral agents. Herein, we describe the synthesis, DFT calculations, and antiviral evaluation of a series of novel 2-hydroxyimino-6-aza-pyrimidine ribonucleosides. DFT//B3LYP/6-311+G?? calculations of the tautomeric distributions of the 2-hydroxyimino nucleosides 7, 8, and 9 in aqueous environments indicate a predominance of the canonical 2-(E)-hydroxyimino structure, where the hydroxyl group points away from the sugar moiety. The conformer distributions of the latter geometrical isomers of 7, 8, and 9 support the formation of five membered rings via hydrogen bonding between the (E)-C2N-O-H moiety and N3-H of 7 and 8 and between (E)-C2N-O-H and N3 of 9, creating purine shaped nucleosides with the glycosidic linkage at the pyrimidine ring. The newly synthesized nucleosides were screened against an RNA viral panel, of which moderate antiviral activity was observed against Zika virus (ZIKV) and human respiratory syncytial virus (HRSV). 6-Aza-2-hydroxyimino-5-methyluridine derivative 18 showed activity against ZIKV (EC50 3.2 μM), while its peracetylated derivative 19 showed activity against HRSV (EC50 5.2 μM). The corresponding 4-thiono-2-hydroxyimino derivative 8 showed activity against HRSV (EC50 6.1 μM) and against ZIKA (EC50 2.4 μM). This study shows that the 6-aza-2-hydroxyimino-5-methyluracil derived nucleosides can be further optimized to provide potent antiviral agents. This journal is

Full text of DOI:10.1039/d0nj04154h

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DOI: 10.1039/D0NJ04154H.
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2-Hydroxyimino-6-aza-pyrimidine Nucleosides: Synthesis, DFT Calculations, and Antiviral
Evaluations
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9
Reham A. I Abou-Elkhair1,*, Abdalla A. Wasfy1,2, Song Mao3, Jinxi Du3,
Sobhy ElAdl2, Kamel Metwally2, Abdalla E. A. Hassan1, Jia Sheng3,*
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1Applied Nucleic Acids Research Center & Chemistry, Faculty of Science, Zagazig University.
Email: Riham31@yahoo.com
2Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt.
3Department of Chemistry and The RNA Institute, University at Albany, State University of
New York, 1400 Washington Avenue, Albany, NY, 12222 (USA), Email: jsheng@albany.edu
Abstract.
The global public health concerns and economic impact caused by emerged outbreaks of RNA
viruses call for the search for new direct acting antiviral agents. Herein, we describe the synthesis,
DFT calculations, and antiviral evaluation of a series of novel 2-hydroxyimino-6-aza-pyrimidine
ribonucleosides. DFT//B3LYP/6-311+G** calculations of the tautomeric distributions of the 2-
hydroxyimino nucleosides 7, 8, and 9 in aqueous environment indicate a predominance of the
canonical 2-(E)-hydroxyimino structure, where the hydroxyl group points away from the sugar
moiety. Conformer distribution of the latter geometrical isomers of 7, 8, and 9 support the
formation of five membered rings via hydrogen bonding between the (E)-C2=N-O-H moiety and
N3-H of 7 and 8 and between (E)-C2=N-O-H and N3 with 9, creating purine shaped nucleosides
with the glycosidic linkage at the pyrimidine ring. The newly synthesized nucleosides were
screened against RNA viral panel, of which moderate antiviral activity was observed against Zika
virus (ZIKV) and human respiratory syncytial virus (HRSV). 6-Aza-2-hydroxylimino-5-
methyluridine derivative 18 showed activity against ZIKV (EC50 3.2 M), while its peracetylated
derivative 19 showed activity against HRSV (EC50 5.2 M). The corresponding 4-thiono-2-
hydroxylimino derivative 8 showed activity against HRSV (EC50 6.1 M) and against ZIKA
(EC50 2.4 M). This study shows that the 6-aza-2-hydroxyimino-5-methyluracil derived
nucleosides can be further optimized to provide potent antiviral agents.
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Keywords: 6-Aza-5-methyluridine, Nucleosides, anti-HRSV, Anti-MERS-CoV, anti-ZIKV,
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6
Hepatitis C virus.
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Introduction
Recent outbreaks of RNA viruses such as Zika virus (ZIKV)1, 2, Middle East respiratory syndrome
coronavirus (MERS-CoV)3, Influenza A viruses (IAVs) of human, avian, and swine origins are
causing global public health concerns and associated with large economic impact. ZIKV is a
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positive-stranded RNA (+RNA) flavivirus4, 5, transmitted by mosquitoes and sexually as well .
ZIKV is associated with severe neural disorders, such as microcephaly in fetuses and newborns
and Guillain-Barr’s syndrome in adults. MERS-CoV can cause serious respiratory diseases with
38% fatality rate of infected patients 7-9. Human respiratory syncytial virus (HRSV), a member
of Paramyxoviridae family, is a single stranded RNA (-RNA) virus 10. HRSV infection causes
acute upper and lower respiratory tract infection in infants, children, elderly, and immune
compromised adults, with significant morbidity and mortality. Severe HRSV infection causes
around 160K deaths per year in children under 5 years old worldwide 11, 12. Ribavirin (1), a broad-
spectrum antiviral agent, is the only small molecule approved for the treatment of HRSV infection
(Fig. 1). Inhibiting inosine monophosphate dehydrogenase (IMPDH) and inducing viral
mutagenesis are the most two important mechanisms by which ribavirin induces its antiviral
activity 13-15. However, toxicity liabilities and limited efficacy undermine the clinical use of
ribavirin 16, 17. Also, there are no effective drugs or vaccines available for the treatment or
prevention of ZIKV and MERS-CoV, infections. These unmet medical needs call for the search
HO
O
N
O
N
NH
NH2
NH
N
N
N
O
O
N
N
HO
HO
HO
O
O
O
HO
HO
OH
OH
HO
OH
3
NHC, 2
1
HRSV: EC50 = 0.69
SI = 394
M

ZIKA: EC50 = 20.8
CC50 >100
M
M


ZIKA: EC50 = 0.32 g/mL

CC50 >100 g/mL

HRSV: EC50 = 5.6 g/mL

CC50 >320 g/mL

Fig. 1. Structures of base modified nucleosides with activity against Zika and RSV viruses.
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for new agents that combat these viruses. N4-Hydroxycytidine (NHC, 2), a cytidine/uridine
analogue, has been reported to inhibit HRSV, influenza B virus, and Influenza A viruses (IAVs)
of human, avian, and swine origins, and severe acute respiratory syndrome-associated coronavirus
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9
18
19
(SARS-CoV) (Fig. 1). NHC has shown inhibitory activity against Ebola virus replication ,
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Norovirus , bovine diarrhea virus (BVDV) and Hepatitis C (HCV) virus , anti-Venezuelan
22
equine encephalitis virus (VEEV) . Recently, NHC has been reported to have activity both in
vitro and im mice against SARS-CoV-2 23. The antiviral activity of NHC relates primarily to the
incorporation of NHC-triphosphate derivative into viral genome in place of uridine, in which case,
NHC behaves both as cytosine and uracil, resulting in high mutation frequencies and, ultimately,
error catastrophe.24
Azapyrimidine nucleosides with triazine as a nucleobase are associated with antitumor 25
and antiviral activities. 6-Azacytidine (6-azaCyd) has demonstrated anti-human adenoviral
activity in vitro and in vivo 26-31. 6-Azauridine (6-azaUrd, 3), a pyrimidine synthesis inhibitor, has
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33
been reported to have anti-ZIKV , anti-Lassa, anti-Ebola , and anti-Chikungunya (CHIKV)
viral activity 34. Furthermore, 6-azaUrd, 6-azauridine triacetate, and 2-thio-6-azaurdine were also
reported to have anti-West Nile virus (New York isolate) activity 35.
In this study, we envisioned that replacing the C2=O moiety of 6-aza-pyrimidin-1-yl
nucleobase with C2=N-OH and C2=S would maintain the geometrical C2 exocyclic double bond
required for enzyme recognition, while altering the steric and electronic effects on the ring. Herein,
we wish to report on the synthesis of 5-methyl-6-azapyrimidine nucleosides 4-9 (Fig. 2), Density
Functional Theory (DFT) calculations of their tautomeric and conformer distributions, and the
evaluation of their activity and that of their intermediates 11-19 against RNA viral panel.
R1
X
N
NH2
H3C
H3C
H3C
N
N
NH
OH
OH
N
N
N
N
N
X
N
N
HO
HO
HO
O
O
O
HO
HO
HO
OH
OH
OH
4; R1 = OH; X = S
5; R1 = NH2, X = S
6; R1 = OH; X = O
7; X= O
8; X= S
9
Fig. 2. Known 6-azanucleosides 4, 5, 7 and additional nucleosides 6, 8, 9 investigated in this
study.
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Results and Discussion
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Chemistry
We have previously reported the synthesis of 2-selenothymidine and 2-selenouridine derivatives
via NaSeH nucleophilic substitution of C2-methylthiothymidine and uridine derivatives 36, 37. We
have adopted this strategy for modifying the C2 position of 6-aza-5-methyluridine with =NOH
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moiety. The precursor for the corresponding 2-methylthio derivative, 2-thio-6-azauridine
38
derivative have been synthesized via Vorbrüggen-type glycosylation
of 6-aza-5-methyl-2-
thiouracil (12) with 1-O-acetyl-2,3,5-tri-O-benzoyl--D-ribofuranose (13) 39. The synthesis of 12
is outlined in scheme 1. Reaction of ethyl pyruvate and thiosemicarbazide provided the
corresponding carbazone derivative 11 in quantitative yield. Cyclization of the carbazone
derivative 11 under basic conditions (NaOH/EtOH/reflux) 40, however did not provide the cyclized
product, 2-thio-6-azathymine (12) in a satisfactory yield. Conducting the reaction in the presence
of anhydrous NaOAc/AcOH under fusion conditions gave 12 in 87% yield (Table 1, entry 1).
Performing the reaction using NaOAc/AcOH in absolute EtOH gave 12 in quantitative yield as a
pale-yellow crystals (table 1, entry 2, 3).
Scheme 1a
O
O
O
BzO
O
OAc
OBz
H2N
H2N
O
NH
N
NH
a
O
+
b
NH
+
N
O
S
S
N
H
H2N
S
BzO
11
12
10
c
13
NH2
N
O
N
NH2
N
O
N
NH
NH
N
O
N
N
N
S
N
S
S
BzO
N
BzO
X
HO
HO
O
O
O
e
e
d
BzO
OBz
OBz
BzO
HO
OH
HO
OH
15
5
14
4; X = S
6; X = O
f
aReagents and conditions. a) EtOH, reflux, 2 h; b) NaOAc. AcOH, EtOH, reflux 2.5 h; c) i) BSA, DCE, rt, 30
min; ii) SnCl4, -10, 3 h, then DMSO, rt, 1 h; d) TBSCl, DMAP, CH3CN., TEA, 12 h, then NH3 gas, 0 ºC, 30 min.;
e) NaOMe, MeOH, rt, 3 h.
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Table 1. Cyclization conditions of 11 to 6-aza-2-thiothymine (12).
Entry
Additive, No. equiv.
NaOAc, 8 / AcOH, 8
NaOAc, 8 / AcOH, 8
NaOAc, 3 / AcOH, 3
Solventa
Time
2.5 h
3 h
Yield %
87b
6
7
8
9
1
2
3
EtOH
EtOH
90c
3.5 h
96c
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12
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All reactions were performed at 120 °C; b) Compound 12 was isolated as dark brown solid; c) Compound 12 was
isolated as faint shiny yellow crystals.
Coupling of 6-aza-2-thiothymine (12) with the protected D-ribofuranosyl derivative 13
under Vorbrüggen conditions (SnCl4/DCE) provided the 2-thio-6-aza-5-methyluracil derivative 14
in 89% yield. The N1--configuration of the 2-thionucleoside derivative 14 was confirmed by 1H,
13
and C-NMR. Treatment of 20 with TBSCl/DMAP/Et3N, and then NH3 in CH3CN gave the
corresponding cytidine derivative 21 in 73.5% yield. Deprotection of the hydroxyl groups of 14
and 15 with NaOMe/MeOH gave the corresponding free nucleosides 441 and 5, respectively in
good yields (scheme 1). Treatment of 4 with H2O2 gave the corresponding 2-oxo derivative 6 42
in good yield.
Scheme 2a
O
N
O
N
O
N
CH3
NH
N
N
N
N
N
S
SCH3
S
BzO
BzO
BzO
a
O
O
O
+
BzO
OBz
16
OBz
BzO
BzO
OBz
17
14
b
O
N
O
N
O
N
NH
NH
NH
OH
OAc
N
N
N
N
OH
N
BzO
BzO
N
HO
O
O
d
c
O
OBz
BzO
OBz
BzO
OH
HO
19
7
18
Reagents and conditions a) MeI, DBU, DMF, 0 °C-rt, 2 h; b) NH2OH·HCl, dry pyridine, 40 ºC, 48 h; c) NaOMe,
MeOH, , rt, 3 h; d) Ac2O, pyridine, 0 °C - rt, 3 h.
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The synthesis of the 2-hydroxylimino derivative 7 is outlined in Scheme 2. Treatment of
14 with MeI in DMF in the presence of DBU gave 2-methylthio derivative 16 along with the
corresponding N3-methyl derivative 17 in 74.71% and 8.04% yields respectively. H-1’ NMR signal
of the 2-methylthio derivative 16 was shifted upfield at 6.32 ppm compared with that of the N3-
methyl derivative 17 at 7.33 ppm. Treatment of 16 with hydroxylamine hydrochloride in dry
pyridine gave the corresponding 2-hydroxylimino derivative 18 in 83% yield. Treatment of 18
with Ac2O/dry pyridine gave the corresponding mono-acetate derivative 19 in 78% yield. The
protection of hydroxyl group in 19 as an acetate ester provides more lipophilicity as well as
chemical stability at the C2 position of the nucleoside. Treatment of 18 with NaOMe/MeOH gave
the corresponding free nucleoside 7 in 85% yield. Selective 1D-NOSEY (SELNOGP, supporting
information) of 7 showed medium correlation between N3-H and C2-hydoxylimino moieties, and
between 5’-OH and C2=NOH moieties, supporting the E-configuration around the C2=NOH side
chain (Fig. 3).
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O
S
NH
NH
OH
OH
HO
N
OH
N
N
N
OH
HO
N
N
O
7
O
8
OH
OH
Fig. 3. Selective 1D-NOESY of compounds 7 and 8 in (DMSO-d6). The observed NOE correlation
between N3-H and C2=NOH, and the later with 5’-OH shown with arrows.
The synthesis of 4-thiono and 4-amino derivatives of 8 and 9 is shown in scheme 3. First,
7 was treated with TBDMSCl/Im/DMF to give the fully protected nucleoside 20 in 87.4% yield.
Compound 20 in turn was treated with Lawesson’s reagent to give the 4-thiono derivative 22 in
65.3% yield, which was then treated with TBAF/THF to give 8. To make target 9, compound 20
was treated with TPSCl/DMAP/Et3N, and then with NH3 gas to give the 4-amino derivative 21;
and the latter was finally deprotected by TBAF. Although the TBDMS group at the C2-
hydroxyimino of 20 underwent partial aminolysis to give the oxime derivative 23 as a side product,
it is worth noting that no 2-amino derivative was formed due to the aminolysis.
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Scheme 3a
6
7
8
9
O
S
O
N
NH
NH
NH
OTBDMS
OTBDMS
N
OH
N
N
N
N
N
N
N
TBDMSO
TBDMSO
HO
10
11
12
13
14
15
16
17
18
19
20
21
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O
O
O
a
b
OTBDMS
20
OTBDMS
TBDMSO
TBDMSO
OH
HO
7
22
c
d
NH2
N
NH2
S
N
NH
OH
N
OR
OH
N
N
N
N
N
N
N
HO
N
TBDMSO
HO
O
O
O
c
OH
HO
OTBDMS
OH
TBDMSO
HO
9
21: R = TBDMS
23: R = H
8
aReagents and conditions. a) TBDMSCl, Im., DMF, , rt, 24 h; b) Lawesson’s reagent, toluene, reflux,
overnight; c)TBAF, AcOH, THF, , rt, 3 h; d) TBSCl, DMAP, CH3CN., TEA, rt, 12 h, then NH3 gas, 0
ºC 30 min.
Selective 1D-NOSEY (SELNOGP, supporting information) of 8 showed a medium correlation
between the N3-H and C2-hydoxylimino moieties, and between the 5’-OH and the C2=NOH
moieties, supporting the E-configuration around the C2=NOH structure (Fig. 3).
DFT calculations
It has been reported that NHC-triphosphate derivative is incorporated into HRSV’s viral RNA in
place of uridine, implying that NHC’s nucleobase has a dual base pairing character (cytosine and
uracil) as a result of placing a hydroxylamine moiety at the C4-position of the nucleoside. To
investigate whether placing the NHOH moiety at the C2-position could results in a nucleoside with
dual base pairing (uridine and isocytidine) characteristics, the tautomeric distributions of 7, 8, and
9 were calculated, in parallel with those of NHC, using DFT implemented in Spartan 18, at
B3LYP/6-311+G∗∗ basis set 43-45. Tautomer distribution results for NHC in aqueous environment
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showed close Boltzmann weights for the uridine like tautomer (NHC-I) and cytidine like tautomer
(NHC-II, 0.373) (Fig. 4) (see supporting information).
7
8
9
10
11
12
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NHC-III
NHC-I
NHC-II
Boltzmann Weight = 0.000
Boltzmann Weight= 0.627
Boltzmann Weight = 0.373
Fig. 4. Optimized structures of NHC tautomers at B3LYP/6-311+G∗∗ basis set
The tautomeric distribution for both geometrical isomers; (E) and (Z)-C2=NOH of 7, 8, and 9 were
calculated under the same conditions used with NHC (supporting information). A predominance
of the (E)-C2=NOH tautomer was observed with Boltzmann weight for 7 (EO-I; 0.997), 8 (ES-I,
0.986), and 9 (EA-I, 1.000) (Fig. 5). These results come in accordance with the observed NOE
data (Fig. 5).
7 (EO-I)
8 (ES-I)
9 (EA-I)
Fig. 5. Optimized tautomeric structures of 7, 8, and 9 at B3LYP/6-311+G∗∗ basis set.
Conformational distribution of the most stable tautomers for 7 (EO-I), 8 (ES-I), and 9 (EA-I) were
assessed using a restricted hybrid HF-DFT SCF calculation at WB97X-D 6-31G* basis set (see
supporting information). A preference of zigzag like conformation around the C2=N-O-H bond
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was shown for the most stable tautomers for 7 (EO-I-Conf. A), and 8 (ES-I-Conf. A), where the
hydrogen atom of the C2=N-O-H moiety is pointed away from the ring N3-H, allowing hydrogen
bonding between the oxygen’s lone pair of electrons of the C2=N-O-H moiety and the N3-H (Fig.
6). A conformational preference around the N-O-H of the C2=N-O-H moiety of 9 (EA-I-Conf. A)
enables hydrogen bonding between the N3 lone pair of electrons and the hydrogen atom of C2=N-
O-H moiety, thereby forming a five membered ring (Fig. 6).
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7: EO-I-Conf. A
9: EA-I-Conf. A
8: ES-I-Conf. A
Fig. 6. Optimized conformers of tautomers 7 (E-OI), 8 (ES-I), and 9 (EA-I)
using DFT at WB97X-D 6-31G* basis set.
Overall, DFT calculations concluded that placing a hydroxylamine moiety at the C4 position of a
pyrimidine nucleoside, as in NHC, results in a dual nucleobase behavior, cytosine-1-yl and uracil-
1-yl, thereby supporting the mutagenic characteristic of the nucleoside. On the other hand, placing
the hydroxylamine moiety at the C2 position of a pyrimidine nucleoside supports its existence
predominantly in the (E)-C2=NOH form forming a purine like nucleoside through hydrogen
bonding between the (E)-C2=N-O-H moiety and the N3-H or N3-lone pair of electrons.
Antiviral activity
The novel free nucleoside analogs 4-9 and their intermediates 11, 12, 14, and 16-19 were screened
against several single stranded RNA viruses at Institute for Antiviral Research, Utah State
University. The compounds were tested for their antiviral activity at four different concentrations by
visual and neutral red (cytopathic effect/toxicity) assays. Nucleoside analogs that showed antiviral
activity in the primary screening were further tested in dose response experiments at 8 different
concentrations to determine the EC50. Cytotoxicity (CC50) was determined in parallel in uninfected cells.
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9
Compounds were tested against the following viral strains: MR766 (Zika Virus) in Vero76 cell line;
CIN-1, genotype 1 (hepatitis C) in Huh-7 cell line; EMC (MERS coronavirus) in Vero76 cell line; A2
(respiratory syncytial virus) in MA-104 cell line; California/07/2009 (Influenza A virus H1N1) in
MDCK cell line; Kern 5156, WNo2 (west Nile Virus) in Vero76 cell line; 17D (Yellow fever Virus )
in Vero 76 cell line. DMSO was used as vehicle in testing the in vitro antiviral activity of the tested
compounds. Screening results for 4, 5, 7-9 and the intermediates 11, 12, 14, 16-19 against respiratory
viruses; MERS-CoV, HRSV and Flu A are shown in Table 2. Among the tested compounds, 4-
thiono-2-hydroxylimino derivative 8 showed a significant activity against HRSV (EC50 = 6.1 M,
CC50 > 100 M, SI >18) and was inactive against the other two respiratory viruses (Table 2). The
observed activity of 8 against HRSV could be attributed to its potential mutagenic activity as a
result of its existence in two forms, uridine-like (ES-I) and hydroxyisocytidine-like (ES-II)
tautomers (Fig. 1, supporting information). On the other hand, the corresponding 4-oxo-2-
hydroxylimino derivative 7 was lacking activity and cytotoxicity at the highest tested
concentration.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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Table 2. Anti-respiratory viral activity of nucleosides 5, 6, 8-12, 17-19.
Compd.
MERS-CoVd
HRSVd
Flu -Ad (H1N1)e
aEC50 bCC50 cSI50 EC50 CC50 SI50 EC50 CC50 SI50
M M M M M M
11
12
4
5
7
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
6.1 >100 >16 >100 >100
>100 >100
>4.2 4.2
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
8
9
>100 >100
>6.6 6.6
>100 >100
>100 >100
14
16
17
18
19
>4.2
>56
4.2
56
>48
48
>100 >100
>100 >100
>32
>24
32
24
32
5.2
32
33
>34
>12
34
12
6
aEC50: effective concentration that inhibit the replication of the virus by 50%.
bCC50: cytotoxic concentration that inhibit the replication of normal cells by 50%.
cSI = CC50/EC50. dEC50 > 100 indicates no antiviral activity was observed at the
tested highest concentration. e California/07/2009 isolate. Ribavirin was used as a
control for HRSV.
.
The carbazone derivative 11, its cyclized form 12, and the 2-thio-nucleosides 4, 5 were devoid of
antirespiratory viral activity and cytotoxicity at the highest tested concentration. Interestingly, the
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9
2’,3’,5’-tri-O-benzoyl-2-hydroxylimino derivative 18 showed higher activity against HRSV (EC50
= 23 M), compared with the free nucleoside 7 (EC50 = >100 M), and the activity was further
increased with the acetyl protected 2-hydroxylimino derivative 19 (EC50 = 5.3 M, CC50 = 33 M,
SI = 6). The benzoyl protected 2-thio-6-aza-5-methyluridine derivative 14 showed activity against
MERS-CoV, HRSV, and Flu-A viruses at the same cytotoxic concentration (Table 2). The
activity/toxicity of the latter compound was lashed out upon methylation at the N-3 position, as in
compound 17. On the other hand, the activity/toxicity reappeared upon methylation at the 2-
position of 14 as in 16 (Table 2), suggesting that 14 might act as a nucleoside analogue, once it
gets into cells in high concentration. The lipophilic characteristics of the benzoyl groups in 14, 16,
18, 19, and the acetyl group in the latter compound could enhance the uptake of the compounds,
and consequently their biological activity. Whether those protected nucleosides act as enzyme
allosteric inhibitor(s) or as nucleoside analogues, that are activated to the corresponding nucleoside
triphosphates is under investigation and will be reported in due course.
10
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12
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14
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16
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18
19
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Compounds 4-9 and 11, 12, 14, 16-19 were also screened against selected members of
flaviviruses: ZIKA, WNV, YFV, HCV, and DENV-2 (Table 3). It is notable that the benzoyl
protected nucleosides 14, 18, and 19 were also active against ZIKA, WNV, and YFV with SI50 in
the range of 8.8-7.2 (Table 3).
Compd.
ZIKAd
WNVd
YFVd
HCV
aEC50 bCC50 cSI50 EC50 CC50 SI50 EC50 CC50 SI50 EC50 CC50 SI50
M
M
M
M
M
M
M M
ND
ND
>20 >20
ND
ND
11
12
4
5
6
>100 >100
>100 >100
ND
>100 >100
>100 >100
>100 >100
>100 >100
ND
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
ND
>100 >100
>100 >100
>100 >100
>100 >100
1
3
>100
2.4
>100
7.74 >20
ND
7
8
>100 >24 >100 >100
9
>100 >100
>100 >100
>100 >100
ND
ND
ND
ND
ND
ND
3.2
>100
>100
3.2
24
>100
>100
28
7.2
>3.2
>28
>42
>3.2
>3.2
3.2
28
42
3.2
3.2
>3.2
>56
>52
>3.2
>2.4
3.2
56
52
3.2
2.4
14
16
17
18
19
8.8
8.8
3.2
28
aEC50: effective concentration that inhibit the replication of the virus by 50%. bCC50: cytotoxic
concentration that inhibit the replication of normal cells by 50%. cSI = CC50/EC50. dEC50 > 100
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indicates no antiviral activity was observed at the tested highest concentration. 6-Azauridine was used
as a control for Zika virus.
6
7
Table 3. Activity of nucleosides 4, 5, 7-9, 14, 16-19 against some flaviviruses.
8
9
Interestingly, the 4-thiono-2-hydroxylimino derivative 8 showed selective activity against ZIKA
(EC50 = 2.4 M, CC50 = >100 M). The 2-hydroxylimino-6-aza-5-methyluridine (7) showed a
moderate activity against HCV (EC50 = 7.74 M) while it was inactive against the rest of the tested
flaviviruses (Table 3). The cytidine analogue 5, 9 were inactive against all tested viruses at the
highest tested concentration.
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Conclusions
We have synthesized for the first time a series of 6-azauridine analogues probing the replacement of
the oxygen atom at the 2-position with hydroxylimin moiety on the anti-RNA viral activity. DFT
results support a dual base pairing character for NHC while supporting a purine-like nucleobase
for 2-hydroxyimino nucleosides. A significant activity against HRSV (EC50 6.1 M and SI >16)
and ZIKA (EC50 2.4 M, CC50 >100 M) were observed with 2-hydroxylimino-4-thionouridine
derivative 8. Unexpectedly, the fully benzoyl protected 2-hydroxylimino-6-azauridine derivative
19 showed significant activity against HRSV (EC50= 5.2 M) and ZIKA (EC50 24 M). Moderate
activity against HCV (EC50 = 7.1 M) was observed with 2-hydroxyliminouridine derivative 7.
The mechanisms of antiviral activity of the sugar protected nucleosides 14-16 and 18-19 is under
investigation and will be reported in due course. This study shows that the 2-hydroxylimino-6-
aza-5-methyluracil derived nucleosides provide lead compounds for further development of broad
spectrum antiviral agents.
Acknowledgments.
Applied Nucleic Acids Research Center, Zagazig University utilized the non-clinical and pre-
clinical services program offered by the National Institute of Allergy and Infectious Diseases. This
work is supported by STDF Grants No. 2698/ 4603/5904 and by USA National Science Foundaiton
CHE-1845486.
Protocols and experimental results.
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Material, instruments and general considerations. Starting materials and reagents were purchased
from Acros-Organics, Alpha Aeser, and Sigma-Aldrich. Reaction progress was monitored by TLC
analysis using aluminum-backed plates pre-coated with Merck silica gel 60-F254. Column
chromatography was carried out on Agela Technologies Flash silica 40-60 mesh. Melting points
recorded on Electrothermal IA 9100 apparatus and were uncorrected. IR spectra (ATR) were
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1
recorded on Bruker alpha spectrometer. UV-vis spectra were recorded on Agilent Cary 60. H
and 13C-NMR spectra were recorded on Bruker 400 MHz spectrometer. Elemental analysis were
recorded on Vario MICRO Cube elemental analyzer. Computational details: all the structures of
the compounds were built using Wavefunction Spartan 18 V2.0.7 and subjected to energy
minimization to remove their strain energies. DFT was adopted in calculating the equilibrium
geometry and conformational distribution using B3LYP/6-311+G** and WB97X-D 6-31G* basis
sets, respectively.
Antiviral protocols.
Primary CPE test method. Four-concentration CPE inhibition assays are performed on confluent
or near-confluent cell culture monolayers in 96-well plates. Cells are maintained in MEM or
DMEM supplemented with FBS as required for each cell line. For antiviral assays the same
medium is used but with FBS reduced to 2% or less and supplemented with 50 µg/ml gentamicin
(10 g/mL EDTA and 1 IU/mL trypsin are added for influenza virus). The test compound is
prepared at four final concentrations, usually 0.1, 1.0, 10, and 100 µg/ml or µM. Five microwells
are used per dilution: three for infected cultures and two for uninfected toxicity evaluation run in
parallel. Controls for the experiment consist of six microwells that are infected but not treated
(virus controls) and six that are untreated (cell controls). The virus control and cell control wells
are on every microplate. Growth medium is removed from the 96-well plates of cells, then the test
compound is applied in 0.1 ml volume to wells at 2x concentration. Virus, normally <100 CCID50
(50% cell culture infectious doses) in 0.1 ml volume, is added to wells designated for virus
infection. Medium devoid of virus is placed in toxicity control wells and cell control wells. Plates
are incubated at 37oC (33C for rhinovirus and enterovirus-D68) with 5% CO2 until maximum
CPE is observed microscopically in virus control wells. Plates are then stained with 0.011% neutral
red for approximately two hours at 37oC with 5% CO2. The neutral red medium is removed by
complete aspiration, and the cells rinsed with phosphate buffered saline (PBS) to remove residual
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dye. The PBS is completely removed and the incorporated neutral red is eluted with 50%
Sorensen’s citrate buffer/50% ethanol for at least 30 minutes. Neutral red dye penetrates into living
cells, thus, the more intense the red color, the larger the number of viable cells present. The dye
content in each well is quantified by optical density (OD) on a spectrophotometer at 540 nm
wavelength. The OD for each set of wells is converted to a percentage compared to untreated
control wells using a Microsoft Excel™ computer-based spreadsheet. Infected wells are
normalized to the virus control. The 50% effective (EC50, virus-inhibitory) concentrations and 50%
cytotoxic (CC50, cell-inhibitory) concentrations are then calculated by regression analysis. The
quotient of CC50 divided by EC50 gives the selectivity index (SI50) value. The percent CPE in each
well may also be read microscopically, calculated as above, and reported as a second data set from
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the same plate for verification 46-48
.
Secondary assay method: CPE and virus yield reduction (VYR). The secondary assay confirms
the CPE assay result, with the principal assessment being virus yield reduction (VYR). It employs
a similar method as described for the primary assay in section 2D.8.1, with the differences noted
in this section. The secondary assay is run independently of the primary test with cells, culture
media, infectious virus solution, and test compound dilutions all newly prepared for this assay.
Eight half-log10 concentrations are tested for antiviral activity and cytotoxicity. After sufficient
virus replication occurs (normally 3 days post infection), a sample of supernatant is taken from
each infected well (three replicate wells are pooled) and titrated immediately (as with HRSV) or
frozen and stored for virus titration at a later time. Alternately, a separate plate may be prepared
and the plate may be frozen and then thawed to release intracellular or cell-associated virus to test
the cell lysate rather than supernatant fluid. After maximum CPE is observed, the plates are stained
with neutral red dye as described above to generate the neutral red EC50, CC50, and SI50 values.
Uninfected wells are tested in parallel for compound toxicity as in the primary assay explained
above. The positive control compound is evaluated in parallel with each test. The second step is to
determine the virus titer in the supernatant or lysate samples. Samples from triplicate wells,
collected as described above, are pooled and tested by endpoint dilution 49. This is a direct
determination virus produced in the presence of the test compound compared to virus from
untreated, infected controls. This is accomplished by making log10 dilutions of the pooled samples
and plating each dilution on 3 or 4 microwells with fresh monolayers of cells in 96-well plates.
Plates are incubated until maximum CPE is observed, then each well is scored for presence (+) or
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absence (-) of virus and the virus titer calculated using the Reed-Meunch method 50. Plotting the
log of the inhibitor concentration versus log10 of virus produced at each concentration allows
calculation of the 90% (one log10 reduction) effective concentration by linear regression. Dividing
EC90 by the CC50 toxicity obtained in the CPE assay gives the SI90 value (so named because it is
derived from a 90% virus-inhibitory value).
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Chemistry
(Z)-Ethyl 2-(2-carbamothioylhydrazono)propanoate (11). To a solution of 10 (10 g, 0.11 mol)
in absolute ethanol (30 mL), was added ethyl pyruvate (15.3 mL). Then the solution was stirred
for 2 hours at reflux temperature. The reaction mixture was cooled to room temperature and the
precipitate was filtered off, dried under vacuum to give compound 1151 (19.08 g, 95.4% yield) as
1
a white solid. M. p.,149-151 ºC; UV-vis (MeOH) max 300 nm; H NMR (400 MHz, DMSO-d6)
 ppm:10.66 s,1H, NH), 8.66 (s, 1H, NH), 7.64 (s, 1H, NH) 4.18 (q, J = 6.8 Hz, 2H, CH2CH3),
2.09 (s, 3H, CH3), 1.24 (t, J = 6.8 Hz, 3H, CH2CH3); 13C APT NMR (100 MHz, DMSO-d6) 
ppm:  C=S), 164.20 (C=O), 139.02 (C=N), 61.11 (CH2CH3) 14.00 (CH3), 13.04 (CH3); IR
(ATR, cm-1): 3434.71 (N-H), 3175 (NH2), 1702 (C=O), 1594.44 (C=S); Anal cald For
C6H11N3O2S: C, 38.08; H, 5.86; N, 22.21. Found: C, 37.98; H, 5.74; N, 22.08.
6-Aza-5-methyl-2-thiouracil (12). A solution of compound 11 (16.51 g, 0.087 mol) and
anhydrous sodium acetate (57.27 g, 0.698 mol) in (1/1 v/v glacial acetic acid/absolute ethanol, 80
mL) was heated for 2.5 hours at 110 ºC. The reaction was cooled to room temperature, quenched
with cold saturated sodium bicarbonate solution, and then extracted with ethyl acetate. The organic
layers were dried over anhydrous sodium sulfate, filtered, and evaporated under vacuum. The
residue was purified by silica gel column chromatography (eluate: ethyl acetate) to give compound
1251 (11.99 g, 96% yield) as a yellow crystal: M. p., 222-224 ºC; UV-vis (MeOH, nm) max 269.98,
214.98 (shoulder); 1H NMR (400 MHz, DMSO-d6)  ppm: 13.25 s,  NH), 13.00 (s, 1H, NH),
2.06 (s, 3H, CH3); 13C APT NMR (100 MHz, DMSO-d6)  ppm: 173.36 C=S), 153.43 (C=O),
148.42 (C=N), 16.03 (CH3). IR (ATR, cm-1): 3041.60 (N-H), 2892.47 (N-H), 1676.04 (C=O)
1599.58 (C=S); Anal cald For C4H5N3OS: C, 33.56; H, 3.52; N, 29.35. Found: C,33.66 ; H, 3.58;
N, 29.18.
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1-O-Acetyl-2,3,5-tri-O-benzoyl--D-ribofuranose (13)39. Sulfuric acid (0.5 mL) in methanol (1
mL) was added dropwise to a suspension of D(-)ribose (5 g; 33.3 mmol) in methanol (100 mL).
The mixture was stirred for 5 hours at room temperature, and then the reaction was quenched with
sodium carbonate (10 g). The reaction mixture was filtered and the filtrate was concentrated under
reduced pressure to afford 7.79 g of crude 1-O-methyl-D-ribofuranose as a yellowish syrup. The
crude 1-O-methyl-D-ribofuranose (5.46 g) was dissolved dry pyridine (16 mL) and the solution
was cooled to 0 ºC and benzoyl chloride (15.56 mL) was added dropwise, the reaction mixture
was stirred for 12 hours at room temperature. The volatiles were evaporated and co-evaporated
with toluene. The residue was diluted with ethyl acetate, washed with saturated solution of
NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated under
reduced pressure to give of 2,3,5-tri-O-benzoyl-1-O-methyl-D-ribofuranose (11 g) as a colorless
syrup. To a solution of 7.55 g of the crude 2,3,5-tri-O-benzoyl-1-O-methyl-D-ribofuranose in
acetic acid (13.2 mL) and acetic anhydride (2.25 mL), conc. H2SO4 (0.85 mL) was added dropwise
at 0 ºC. The reaction mixture was stirred for 5 hours at room temperature, and then reaction was
quenched with saturated solution NaHCO3. The whole was partitioned between with ethyl acetate
and water. The organic layer was separated, dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The residue was crystallized from iso-propanol to give a white crystal: 1H NMR
(400 MHz, CDCl3)  ppm: 8.90-8.07 (m, 2H, Ar), 8.02-7.99 (m, 2H, Ar), 7.90-7.88 (m, 2H, Ar),7.61-
7.51 (m, 3H, Ar), 7.45-7.32 (m, 6H, Ar), 6.43 (s, 1H, H-1 ), 5.92-5.89 (m, 1H, H-2 ), 5.79-7.78
(m, 1H, H-3 ), 4.81-7.75 (m, 2H, H-4 and H-5), 4.54-4.49 (m, 1H, H-5), 2.00 (s, 1H, CH3); APT
13C NMR (100 MHz, CDCl3)  ppm: 169.23 (C=O), 166.14 (C=O), 165.52 (C=O),165.18 (C=O),
133.83 (Ar),133.72 (Ar), 133.42 (Ar), 130.01 (Ar), 129.92 (Ar), 129.74 (Ar), 128.98 (Ar), 128.82
(Ar), 128.70 (Ar), 128.57 (Ar), 128.56 (Ar), 98.56 (C-1), 80.13 (C-2), 75.15 (C-3),71.53 (C-4),
63.88 (C-5), 21.05 (CH3); IR (ATR, cm-1): 1720 (C=O).
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1-(2’,3’,5’-tri-O-benzoyl--D-ribofuranosyl)-6-aza-5-methyl-2-thiouracil
(14).
To
a
suspension of 12 (2 g, 13.9 mmol) and 13 (7.39 g 14 mmol) in dry DCE (200 mL) was added BSA
(4.15 mL, 16.7 mmol) dropwise at room temperature. The solution was stirred for 30 minutes until
complete dissolution. Then SnCl4 (4.91 mL, 16.7 mmol) was added dropwise at -10 ºC. The
reaction mixture was stirred 3 hours at 0 ºC, then the reaction was quenched by DMSO (5.9 mL),
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and the formed solid was filtered off. The filtrate was neutralized with saturated aqueous NaHCO3
solution. The whole was extracted with DCM and the combined organic phases were dried over
anhydrous Na2SO4, and evaporated under vacuum. The residue was purified with silica gel column
chromatography (eluate: 2% ethyl acetate / DCM),to give compound 14 (7.298 g, 89% yield) as a
white solid: M. p., 159-161 ºC; UV-vis (MeOH) max 275 nm; 1H NMR (400 MHz, DMSO-d6) 
ppm: 13.35 (s, 1H, NH), 7.98-7.39(m, 15H, Bz), 7.35 (d, J = 2.8Hz, 1H, H-1’), 5.98 (dd, J = 2.8,
J = 5.6 Hz, 1H, H-2'), 5.92 (dd, J = 5.6, J = 6.8 Hz, 1H H-3'), 4.83 (m, 1H, H-4'), 4.70 (dd, J = 4.7,
12.4 Hz, 1H, H-5'a), 4.58 (dd, ,J = 4.8, 12.4 Hz, 1H, H-5'b), 2.02 (s, 3H, CH3); APT 13C NMR (100
MHz, DMSO - d6)  ppm:174.55 (C=S), 165.34 (C=O), 164.56 (C=O), 164.50 (C=O), 152.11
(C=O), 150.03 (C=N), 134 (Ar), 133.82 (Ar), 133.55 (Ar), 129.41 (Ar), 129.21 (Ar), 128.87 (Ar),
128.74 (Ar), 128.70 (Ar), 128.51 (Ar), 128.39 (Ar), 91.10 (C-1'), 78.98 (C-2'), 74.11 (C-3'),
70.96(C-4'), 63.31 (C-5'), 16.25 (CH3); IR (ATR, cm-1): 3215.85 (N-H), 1735.65 (C=O),
1707.34 (C=N), 1600.94 (C=S); EI-Mass m/z 587 [M+]; Anal cald For C30H25N3O8S: C, 61.32;
H, 4.29; N, 7.15. Found: C, 61.49; H, 4.36; N, 6.98.
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1-(2’,3’,5’-Tri-O-benzoyl--D-ribofuranosyl)6-aza-5-methyl-2-thiocytosine (15). A mixture
compound 14 (0.5 g, 0.85 mmol), TBSCl (0.64 g, 2.128 mmol) and DMAP (0.259 g, 2.13 mmol)
were dissolved in dry acetonitrile (10 mL), then TEA (0.295 mL) was added dropwise at 0 ºC. The
mixture was stirred for 12 hours at room temperature, then ammonia gas was bubbled for 30 min.
at ºC and the mixture was stirred for 30 minutes at room temperature. The solvents were evaporated
under vacuum and the residue was purified by silica gel column chromatography (eluate: 5%
methanol / DCM) to give compound 15 (0.374 g, 75% yield) as a pale yellow crystals: M. p., 106-
111 ºC; UV-vis (MeOH) max 275, 230 nm;1H NMR (400 MHz, DMSO-d6)  ppm: 7.95-7.90 (m,
6H, Ar), 7.67-7.65 (m, 3H, Ar), 7.51-7.44 (m, 8H, Ar and 4-NH2, 2H were exchangeable with
D2O), 6.35 (br s , 1H, H-1'), 6.07 (br d, 1H, H-2'), 5.92 (dd, J = 5.6, J = 6.4 Hz, 1H, H-3'), 4.78 (br
d, 1H, H-4'), 4.69 (br dd, J = 12.4 Hz, 1H, H-5a'), 4.58 (br dd, J = 12.4 Hz, 1H, H-5b'), 1.91 (s,
3H, CH3); APT 13C NMR (100 MHz, DMSO-d6)  ppm: 165.32 (C=S), 164.68 (C4), 164.52
(C=O), 162.11 (C=O), 155.48 (C=N), 148.11 (C=N), 133.97 (Ar), 133.83 (Ar), 133.54 (Ar),
129.34 (Ar), 129.32 (Ar), 129.21 (Ar), 129.13 (Ar), 128.82 (Ar), 128.74 (Ar), 128.69 (Ar), 128.57
(Ar), 128.53 (Ar), 89.49 (C-1'), 78.91 (C-2'), 73.70 (C-3'), 70.76 (C-4'), 62.92 (C-5'), 16.80 (CH3);
IR (ATR, cm-1): 3327.39 (NH2), 1721.95 (C=O), 1621.92 (C=N), 1515.81 (C=S).
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1-(-D-Ribofuranosyl)6-aza-5-methyl-2-thiocytosine (5). A solution of compound 15 (0.35 g,
0.596 mmol) in dry methanol (5 mL) was treated with sodium methoxide (1M, 5.6 mL) at 0 ºC.
The reaction mixture was stirred for 2 hours at room temperature, and then was neutralized with
acetic acid. The solvents were removed under vacuum and the residue was purified by silica gel
column chromatography (eluate: 5% methanol/DCM), then (8:1:1:1, EtOAc : EtOH : Acetone :
H2O) to give compound 5 (0.139 g, 85% yield) as a white solid: M. p., 237-238 ºC; UV-vis
(MeOH) max 245 nm; 1H NMR (400 MHz, DMSO-d6)  ppm: 7.32 (br s, 2H, NH2, exchangeable
with D2O), 5.62 (d, J = 3.6 Hz, 1H, H-1'), 5.33 (br s, 1H, 2’-OH, exchangeable with D2O), 5.06
(br s, 1H, 3'-OH, exchangeable with D2O), 4.90 (br t, 1H, 5'-OH, exchangeable with D2O), 4.37
(br s, 1H, H-2'), 4.04 (br s, 1H, H-3'), 3.85 (br d, 1H, H-4'), 3.56-3.40 (m, 2H, H-5'a and H5'b),
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2.01 (s, 3H, CH3); APT C NMR (100 MHz, DMSO-d6)  ppm: 162.45 (C=S), 155.78 (C-4),
147.05 (C=N), 93.03 (C-1'), 85.02 (C-2'), 71.91 (C-3'), 70.29 (C-4'), 61.80 (C-5'), 16.89 (CH3); IR
(ATR, cm-1): 3430.08 (O-H), 3346.57 ( OH), 3181.48 ( OH), 2957.75-2933.87 ( N-H2),
1726.77 (C4=N), 1668.49 (C5=N), 1622.60 (C=S); EI-Mass m/z 274.44 [M+]; Anal cald For
C9H14N4O4S: C, 39.41; H, 5.14; N, 20.43. Found: C, 39.37; H, 5.29; N, 20.22.
1-(-D-Ribofuranosyl)6-aza-5-methyl-2-thiouracil (4). Sodium methoxide (1M, 7.99 mL) was
added to a solution of compound 14 (0.5 g, 0.85 mmol) in dry methanol (5 mL) at 0 ºC. The
reaction mixture was stirred for 3 hours at room temperature, neutralized with acetic acid, and
evaporated under vacuum. The residue was purified by silica gel column chromatography (eluate:
10% methanol / DCM) to give compound 4 (0.1991 g, 85% yield) as a white solid granules: M. p.,
220-224 ºC; (C=S) ; UV-vis (MeOH) max 275 nm; 1H NMR (400 MHz, DMSO-d6)  ppm: 13.19
(s, 1H, NH, exchangeable with D2O), 6.79 (br d, 1H, H-1'), 5.28 (d, J = 4.8 Hz, 1H, 2'-OH,
exchangeable with D2O), 5.00 (d, J = 6.4 Hz, 1H, 3'-OH, exchangeable with D2O), 4.59 (t, J = 5.6
Hz, 1H, 5'-OH, exchangeable with D2O), 4.17 (m, 1H, H-2'), 4.06 (m, 1H, H-3'), 3.81 (m, 1H, H-
4'), 3.57 (m, 1H, H-5'a), 3.44 (m, 1H, H-5'b), 2.1 (s, 3H, CH3); APT 13C NMR (100 MHz, DMSO-
d6)  ppm: 174.44 (C=S), 152.18 (C=O), 149.08 (C=N), 93.67 (C-1'), 84.78 (C-2'), 73.4 (C-3'),
70.4 (C-4'), 62.04 (C-5'), 16.34 (CH3); IR (ATR, cm-1): 3472.02 ( OH), 3426.22 ( OH), 3178.14
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( OH), 2886.77 (N-H), 1694.17 (C=O), 1493.75; EI-Mass m/z 275.27 [M+]. Anal cald For
5
6
C9H13N3O5S: C, 39.27; H, 4.76; N, 15.26. Found: C, 39.41; H, 4.97; N, 15.41.
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1-(-D-Ribofuranosyl)-6-aza-5-methyluracil (6). A solution of compound 4 (50 mg, 0.1816
mmol) was dissolved in distilled water (2 mL) and treated with H2O2 (50% in H2O, 74 L, 1.0897
mmol), and NaOH (43.58 mg, 1.0897 mmol) at 0 ºC. The mixture was stirred for 1 hour at room
temperature and the solvent was evaporated to dryness. The residue was purified by silica gel
column chromatography (eluate: EtOAc) to give compound 6 (38 mg, 80.73% yield) as a white
foam: UV-vis (MeOH) max 210, 265 nm; 1H NMR (400 MHz, DMSO-d6)  ppm: 5.90 (d, J = 4
Hz, 1H, H-1'), 5.04 (d, J = 5.2 Hz, 1H, 2'-OH, exchangeable with D2O), 4.84 (d, J = 5.6 Hz, 1H,
3'-OH, exchangeable with D2O), 4.66 (t, 1H, J = 5.2 Hz, 5'-OH, exchangeable with D2O), 4.20 (m,
1H, H-2'), 3.99 (m, 1H, H-3’), 3.72 (m, 1H, H-4'), 3.51 (dd, J = 4.4, 12 Hz, 1H, H-5'a), 3.38 (dd,
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J = 6, 11.6 Hz, 1H, H-5'b), 1.95 (s, 3H, CH3); C NMR (100 MHz, DMSO-d6)  ppm: 163.88
(C=O), 155.44 (C=O), 143.22 (C=N), 90.21 (C-1'), 84.56 (C-2'), 72.78 (C-3'), 71.20 (C-4'), 62.99
(C-5'), 17.43 (CH3); IR (ATR) (cm-1): 3255 cm-1 ( OH), 1701 cm-1 ( C=O), 1608cm-1(C=S),
1530 (C=N); Anal cald For C9H13N3O6: C, 41.70; H, 5.06; N, 16.21. Found: C, 41.81; H, 5.13;
N, 16.08.
1-(2,3,5-Tri-O-benzoyl--D-ribo-furanosyl)-6-aza-5-methyl-2-methylthiouracil (16) and 1-
(2,3,5-tri-O-benzoyl--D-ribofuranosyl)-6-aza-N3-methyl-5-methyl-2-thiouracil (17). To a
solution of compound 14 (1.7 g, 2.89 mmol) in dry DMF (100 mL) and CH3I (0.54 mL, 8.679
mmol), DBU (0.648 mL, 4.34 mmol) was added dropwise at 0 ºC and the reaction mixture was
stirred for 2 hours at the same temperature. The mixture was diluted with ethyl acetate then washed
with H2O and brine. The combined organic phases were dried over anhydrous sodium sulfate,
filtered off and evaporated under vacuum. The residue was purified by silica gel column
chromatography (eluate: 4% ethyl acetate/DCM) to give compound 17 (0.139 g, 8.04% yield) as
a white solid, then eluted with 10% ethyl acetate in DCM to give 16 (1.3 g, 74.71% yield) as a
white glassy semi solid. Analytical data for 17: M. p.,136-137 ºC; UV-vis (MeOH) max 225 nm
(shoulder), 275 nm; 1H NMR(400MHz, CDCl3)  ppm: 8.04-7.92 (m, 6H, Ar), 7.58-7.52 (m, 4H,
Ar), 7.42-7.33 (m, 6H, Ar and H-1'), 6.09 (dd, J = 2.8, 5.6 Hz, 1H, H-2'), 5.97 (br dd, 1H, H-3'),
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4.81-4.75 (m, 2H, H-4'and H-5'a), 4.60 (m, 1H, H-5'b), 2.22 (s, 3H, 5-CH3), 1.57 (s, 3H, N3-CH3);
APT 13C NMR (100 MHz, CHCl3-d6)  ppm: 175.97 (C=S), 166.20 (C=O), 165.47 (C=O), 165.31
(C=O), 152.35 (C=O), 146.95 (C=N) 133.83 (Ar), 133.73 (Ar), 133.43 (Ar), 130.03 (Ar), 129.91
(Ar), 129.87 (Ar), 129.69 (Ar), 128.89 (Ar), 128.67 (Ar), 128.60 (Ar), 128.58 (Ar), 93.81 (C-1'),
79.98 (C-2'), 74.43 (C-3'), 71.61 (C-4'), , 63.64 (C-5'), 34.10 (N3-CH3), 17.29 (5-CH3);IR (ATR,
cm-1): 1722.43 (C=O), 1695.70 (C=N) 1601.21 (C=S); Anal cald For C31H27N3O8S: C, 61.89;
H, 4.52; N, 6.98. Found: C, 61.84; H, 4.48; N, 6.89. Analytical data for Compound 16: UV-vis
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(MeOH) max 230 nm; H NMR (400MHz, DMSO-d6)  ppm: 7.96-7.85 (m, 6H, Ar), 7.68-7.62
(m, 3H, Ar), 7.51-7.42 (m, 6H, Ar), 6.32 (d, J = 2.4 Hz, 1H, H-1'), 6.08 (dd, J = 2.4, 5.6 Hz, 1H,
H-2'), 5.96 (dd, J = 5.6, 7.2 Hz, 1H, H-3'), 4.88 (m, 1H, H-4'), 4.70 (dd, J = 3.2, 12.4 Hz, 1H, H-
5'a), 4.57 (dd, J = 4.0, 12.4 Hz, 1H, H-5'b), 2.5 (s, 3H, SCH3), 2.1 (s, 3H, 5-CH3); 13C APT NMR
(100 MHz, DMSO-d6)  ppm: 165.28 (C=O), 164.59 (C=O), 164.47 (C=O), 158.94 (C=O), 149.98
(C=N), 133.99 (Ar), 133.85 (Ar), 133.57 (Ar), 129.45 (Ar), 129.27 (Ar), 129.12 (Ar), 128.81 (Ar),
128.76 (Ar), 128.71 (Ar), 128.48 (Ar), 128.42 (Ar), 91.44 (C-1'), 79.52 (C-2'), 73.94 (C-3'), 70.55
(C-4'), , 62.67 (C-5'), 16.65 (5-CH3), 13.84 (5-CH3); IR (ATR, cm-1): 1718.09 (C=O), 1666.39
(C=N), 1598.85 (C=S); Anal cald For C31H27N3O8S: C, 61.89; H, 4.52; N, 6.98. Found: C,
61.92; H, 4.59; N, 6.84
1-(2,3,5-Tri-O-benzoyl--D-ribofuranosyl)6-aza-2-hydroxylimino-5-methyluracil (18). A
mixture of compound 16 (1.3 g, 2.1 mmol) and hydroxylamine hydrochloride (3 g, 43.2 mmol)
was dissolved in dry pyridine (25 mL). The reaction mixture was stirred for 48 hours at 40 ºC.
Then pyridine was evaporated and co-evaporated with toluene. The residue was purified by silica
gel column chromatography (eluate: ethyl acetate /DCM).to give compound 18 (1.051 g, 82.98%
yield) as a green fluffy foam; UV-vis (MeOH) max 330 nm (broad), 275 (shoulder), 230 nm; 1H
NMR (400MHz, DMSO-d6)  ppm: 11.01 (s , 1H, OH, exchangeable with D2O), 9.58 (s, 1H, NH,
exchangeable with D2O), 8.00 -7.42 (m, 15H, Bz), 6.20 (d, J = 3.2Hz, 1H, H-1'), 6.02 (dd, J = 3.2,
5.2 Hz, 1H, H-2'), 5.86 (dd, J = 5.6, 6.0 Hz, 1H, H-3’), 4.72 (m, 1H, H-4'), 4.66 (dd, J = 3.6, 12
Hz, 1H, H-5'a), 4.54 (dd, J = 4.4, 12 Hz, 1H, H-5'b), 1.85 (s, 3H, CH3); APT 13C NMR (100 MHz,
DMSO-d6)  ppm: 165.37 (C=O ), 164.63 (C=O) , 164.58 (C=O), 153.70 (C=O), 143.41 (C=N),
138.77 (C=N), 133.95 (Ar), 133.81 (Ar), 133.55 (Ar), 129.36 (Ar) , 129.28 (Ar), 129.21 (Ar),
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128.84 (Ar), 128.74 (Ar), 128.72 (Ar), 128.57 (Ar), 128.45 (Ar), 88.14 (C-1'), 78.36 (C-2'), 72.91
(C-3'), 71.00 (C-4'), 63.38 (C-5'), 15.94 (CH3); IR (ATR, cm-1): 3268.55 (O-H), 3064.31(NH
oxime), 1717.26 (C=O), 1657.59 (C=N), 1600.74 (C=N); EI-Mass m/z 587.13 [M+]; Anal cald
For C30H26N4O9: C, 61.43; H, 4.47; N, 9.55. Found: C, 61.55; H, 4.89; N, 9.32.
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1-(2,3,5-Tri-O-benzoyl--D-ribofuranosyl)6-aza-2-O-acetylhydroxyimino-5-methyluracil
(19). Acetic anhydride (0.288 mL, 3.1 mmol) was added dropwise at 0 ºC to a solution of
compound 18 (0.6 g, 1.02 mmol) in dry pyridine (15 mL). The reaction mixture was stirred for 3
hours at room temperature. The volatiles were evaporated and co-evaporated with toluene. The
residue was purified by silica gel column chromatography (eluate: 10 %ethyl acetate /DCM).to
give compound 19 (0.5 g, 77.7% yield) as a white foam: UV-vis (MeOH) max 234, 274.98 (broad),
310 nm (broad ); 1H NMR (400 MHz, DMSO-d6)  ppm: 11.99 (s, 1H, N-H, exchangeable with
D2O), 7.99 -7.39 (m, 15H, Bz), 6.28 (d, J = 3.2 Hz, 1H, H-1'), 6.06 (dd, J = 3.2, 5.6 Hz, 1H, H-2'),
5.92 (br dd, 1H, H-3’), 4.77 (m, 1H, H-4'), 4.67 (dd, J = 3.2, 12.4 Hz, 1H, H-5'a), 4.56 (dd, J = 4.4,
12.4 Hz, 1H, H-5'b), 2.1 (s, 3H, COCH3); 1.92(s, 3H, CH3); APT 13C NMR (100 MHz, DMSO-
d6)  ppm: 167.48 (C=O), 165.36 (C=O), 164.61 (C=O), 164.56 (C=O), 153.73 (C=O), 148.88
(C=N),141.45(C=N) 133.97-133.55 (Ar), 129.43-128.42 (Ar), 89 (C-1'), 78.62 (C-2'), 73.13 (C-
3'), 70.84 (C-4'), 63.35 (C-5'), 15.92 (CH3), 14.08(CH3); IR (ATR, cm-1): 1769.9 (C=O), 1717.41
(C=O), 1631.9 (C=N), 1600.07 (C=N); Anal cald For C32H28N4O10: C, 61.14; H, 4.49; N, 8.91;.
Found: C, 61.34; H, 4.51; N, 8.89.
1-(-D-Ribofuranosyl)-6-aza-2-hydroxyimino-5-methyluracil (7). Sodium methoxide (1M,
64.1 mL) was added a solution of compound 18 (4 g, 6.82 mmol) in dry methanol (20 mL) at 0 ºC
and the mixture was stirred for 3 hours at room temperature. The reaction mixture was neutralized
with acetic acid, and then the solvents were evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (eluate: 8:1:1:1 ethyl acetate/acetone/ethanol/H2O)
to give compound 7 (1.588 g, 85% yield) as a green solid: M. p.,171-173ºC; UV-vis (MeOH) max
330 , 230 nm (shoulder); 1H NMR (400 MHz, DMSO-d6)  ppm: 10.76 (s, 1H, NH, exchangeable
with D2O), 9.43 (s, 1H, N=OH, exchangeable with D2O), 5.66 (d, J = 3.8 Hz, 1H, H-1'), 5.15 (d, J
= 5.3 Hz, 1H, 2’-OH, exchangeable with D2O), 4.93 (d, J = 5.8 Hz, 1H, 3’-OH, exchangeable with
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D2O), 4.63 (br t, 1H, 5’-OH, exchangeable with D2O), 4.23 (m, 1H, H-2'), 3.98 (d, 1H, H-3'), 3.74
(m, 1H, H-4'), 3.49 (m, 1H, H-5'a), 3.38 (m, 1H, H-5'b), 1.98 (s, 3H, CH3); APT 13C NMR (100
MHz, DMSO-d6)  ppm:153.95 (C=O) 144.16 (C=N), 137.03 (C=N), 90.26 (C-1'), 84.25 (C-2'),
71.92 (C-3'), 70.37 (C-4'), 62.21 (C-5'), 16.08 (CH3); IR (ATR, cm-1): 3374.72 (N-H), 3262.42
( OH), 1691.18 (C=O), 1652.82 (C=N); EI-Mass m/z 274.18 [M+]; Anal cald For C9H14N4O6:
C, 39.42; H, 5.15; N, 20.43; found: C, 39.13; H, 5.27; N, 20.33.
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2,2’,3’,5’-Tetra-O-tert-butyldimethylsilyl--D-ribofuranosyl-6-aza-2-hydoxyimino-5-
methyluridine (20). A mixture of compound 7 (1.5 g, 5.47 mmol), TBDMSCl (11.543 g, 76.59
mmol) and imidazole (10.428 g, 153.1 mol) were dissolved in dry DMF (10 mL) and stirred for
24 hours at room temperature. The mixture was quenched with ice-H2O, diluted with ethyl acetate
and then washed with water. The organic phases were dried over anhydrous sodium sulfate,
filtered off, and evaporated under vacuum. The residue was purified by silica gel column
chromatography (eluate: 5% ethyl acetate /hexane) to give compound 20 (3.497 g, 87.4% yield)
as a greenish syrup: UV-vis (MeOH) max 335, 235 nm (shoulder); 1H NMR (400 MHz, CDCl3) 
ppm: 8.74 (s, 1H, NH), 5.88 (d, J = 4.8 Hz, 1H, H-1'), 4.42 (dd, J = 4.4 Hz, 1H, H-2'), 4.28 (dd, J
= 4.0 Hz, 1H, H-3'), 3.93 (m, 1H, H-4'), 3.72-3.63 (m, 2H, H-5'a,b), 2.13 (s, 3H, 5-CH3), 0.89 (m,
36H, t-Bu-Si), 0.1(m, 24H, CH3-Si); APT 13C NMR (100 MHz, CDCl3-d6)  ppm: 153.58 (C=O),
146.85 (C=N), 137.76 (C=N), 89.04 (C-1'), 84.16 (C-2'), 73.49 (C-3'), 71.90 (C-4'), 62.84 (C-5'),
26.36 (t-Bu-Si), 26.06 (t-Bu-Si), 25.99 (t-Bu-Si), 25.88 (t-Bu-Si), 25.79 (t-Bu-Si), 18.56 (t-Bu-Si),
18.45 (t-Bu-Si), 18.21 (t-Bu-Si),16.29 (5-CH3), 0.14 (CH3-Si), -3.43 (CH3Si), -4.25 (CH3-Si), -
4.37 (CH3-Si), -4.43 (CH3-Si), -4.58 (CH3-Si), -5.18 (CH3-Si), -5.21(CH3-Si), -5.25 (CH3-Si). IR
(ATR, cm-1): 2952.77 cm-1 (N-H), 1697.88 (C=O), 1649.62 (C=N).
2,2’,3’,5’-Tetra-O-tert-butyldimethylsilyl--D-ribo-furanosyl-6-aza-2-hydoxyimino-5-
methylcytidine (21) and 2’,3’,5’-tri-O-tert-butyldimethylsilyl--D-ribo-furanosyl-6-aza-2-
hydoxyimino-5-methylcytidine (23). A mixture of compound 20 (1.5 g, 2.05 mmol), TPSCl
(1.55g, 5.13 mmol) and DMAP (0.626 g 5.13 mmol) were dissolved in dry acetonitrile (25 mL),
then cooled to 0 ºC. TEA (0.71 mL) was added dropwise and the reaction mixture was stirred for
12 hours at room temperature. Ammonia gas was bubbled into the reaction mixture for 30 min at
0 °C, then the mixture was stirred for 15 minutes at room temperature. The volatiles were removed
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under vacuum and the residue was partitioned between EtOAc and H2O. The organic phase was
dried over anhydrous Na2SO4 and evaporated. The residue was purified by silica gel column
chromatography (5% EA / DCM) to give a mixture of 21 its partially deprotected derivative 23 as
a pale-yellow foam (1.1 g, 73.48% yield). Compounds 21 and 23 were partially separated for
analytical purposes. Analytical data for compound 21: 1H NMR (400 MHz, CDCl3)  ppm: 5.71
(d, J = 4.0 Hz, 1H, H-1'), 4.53 (br dd, J = 4.0, 8 Hz, 1H, H-2'), 4.29 (br dd, J = 4. 8 Hz, 1H, H-3'),
3.97 (m, 1H, H-4'), 3.73-3.65 (m, 2H, H-5'a and H-5'b), 2.16 (s, 3H, 5-CH3), 0.92-0.86 (4s, 36H,
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tert-BuSi), 0.09-00 (8s, 24H, Si-CH3). Analytical data for compound 23: H NMR(400 MHz,
DMSO-d6)  ppm: 7.87 (s, 1H, N-OH), 7.79 (br s, 1H, NH), 7.18 (br s, 1H, NH), 5.70 (d, J = 5.2
Hz, 1H, H-1'), 4.47 (br dd, J = 4.8 Hz, 1H, H-2'), 4.29 (br dd, J = 4.0 Hz, 1H, H-3') , 3.76 (m, 1H,
H-4'), 3.64-3.56 (m, 2H, H-5'a, H-5'b), 2.01 (s, 3H, 5-CH3), 0.88 (m, 28H, tert-BuSi), 0.08-0.01
(6s, 18H, Si-CH3).
1-(-D-Ribofuranosyl)-2-hydroxylimino-6-aza-5-methylcytosine (9). 1M solution of TBAF in
THF (6 mL) was added dropwise to a solution of the mixture of 21 and 23 (1g, 1.369 mmol) in
dry THF (15 mL) and the reaction mixture was stirred for 3 hours at room temperature. The
solvent was removed under reduced pressure and the residue was purified by DOWEX 50
RESIN to give compound 9 ( 0.22 g, 59.29% yield) as a yellow foam; UV-vis (MeOH) max
325nm, 240 nm; 1H NMR (400 MHz, DMSO-d6)  ppm: 7.82 (br s, 1H, NH, exchangeable with
D2O) 5.75 (br s, 2H, NH and OH, exchangeable with D2O), 5.63 (d, J = 4.0 Hz, 1H, H-1'), 4.17
(m, 1H, H-2'), 3.92 (m, 1H, H-3'), 3.72 (m, 1H, H-4’), 3.51-3.38. (m, 2H, H-5'a, and H-5'b), 2.01
(s, 3H, CH3);APT 13C NMR (100 MHz, DMSO- d6)  ppm: 152.77 (C-4), 149.01 (C-2), 130.56
(C-5), 90.97 (C-1'), 84.11 (C-2'), 71.95 (C-3'), 70.82 (C-4'), 62.58 (C-5'), 19.19 (CH3); Anal cald
For C9H15N5O5: C, 39.56; H, 5.53; N, 25.63. Found: C, 39.44; H, 5.65; N, 25.74.
2,2’,3’,5’-Tetra-O-tert-butyldimethylsilyl--D-ribo-furanosyl-6-aza-2-hydoxyimino -5-
methyl-4-thiouridine (22). To a mixture of compound 20 (1.5g, 2.05 mmol), and Lawesson
reagent (0.829 g, 2.1 mmol) in dry toluene (50 mL) was heated at reflux temperature overnight.
The mixture was cooled down to room temperature and the volatiles were removed under
vacuum. The residue was purified by silica gel column chromatography (eluate: 1% ethyl
acetate/hexane) to give compound 22 (1 g, 65.27% yield) as a dark yellow syrup: UV-vis
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(MeOH) max 230 (shoulder), 275, 320 nm; 1H NMR (400 MHz, CDCl3)  ppm: 10.04 (s, 1H,
NH), 5.83 (d, J = 4.4 Hz, 1H, H-1'), 4.44 (br dd, J = 4.4Hz, 1H, H-2'), 4.30 (br dd, 1H, H-3'),
3.94 (m, 1H, H-4'), 3.73-3.64 (m, 2H, H-5'a,b), 2.28 (s, 3H, 5-CH3), 0.90 (m, 36H, t-Bu-Si),
0.05-0.2 (m, 24H, Si-CH3); 13C NMR (100 MHz, CDCl3)  ppm: 180.10 (C=S), 143.41 (C=N),
140.85 (C=N), 89.34 (C-1'), 84.20 (C-2'), 73.65 (C-3'), 71.88 (C-4'), 62.76 (C-5'), 53.57 (t-Bu-
Si), 51.05 (t-Bu-Si), 29.85 (t-Bu-Si), 26.40 (t-Bu-Si), 26.05 (t-Bu-Si), 26.00 (t-Bu-Si), 25.88 (t-
Bu-Si), 20.06 (5-CH3), 18.56 (Si-CMe3), 18.21 (Si-CMe3), 1.17 (CH3-Si), 0.14 (CH3-Si), -4.27
(CH3-Si), -4.36 (CH3-Si), -4.46 (CH3-Si), -4.57 (CH3-Si), -5.13 (CH3-Si), -5.14 (CH3-Si), -5.27
(CH3-Si).
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1-(-D-Ribofuranosyl)-6-aza-2-hydoxyimino-5-methyl-4-thiouracil (8). 1M solution of TBAF
in THF (6.4 mL) was added dropwise to a solution of compound 22 (1g, 1.338 mmol) in dry THF
(15 mL) and the reaction mixture was stirred for 3 hours at room temperature. The solvent was
removed under reduced pressure and the residue was purified by silica gel column chromatography
(eluate: 8:1:1:1 ethyl acetate/ ethanol / acetone/ H2O) to give compound 8 (0.302 g, 77.92% yield)
as a yellow solid: M. p.,160-162 ºC; UV-vis (MeOH) max 325 nm; 1H NMR (400 MHz, DMSO-
d6)  ppm 11.66 (s, 1H, NH, exchangeable with D2O), 9.79 (s, 1H, N=OH, exchangeable with
D2O), 5.62 (d, J = 3.6 Hz, 1H, H-1'), 5.24 (br s, 1H, 2'-OH, exchangeable with D2O), 4.98 (br d,
1H, 3’-OH, exchangeable with D2O), 4.66.( br s, 1H, 5'-OH, exchangeable with D2O), 4.26 (br s,
1H, H-2'), 4.01 (br dd, 1H, H-3'), 3.77 (m, 1H, H-4'), 3.51 (m, 1H, H-5'a), 3.39 (br m, 1H, H-5'b,)
2.17 (s, 3H, CH3); APT 13C NMR (100 MHz, DMSO- d6)  ppm: 180.93 (C=S), 140.62 (C=N),
139.91 (C=N), 90.70 (C-1'), 84.69 (C-2'), 72.08 (C-3'), 70.53 (C-4'), 62.22 (C-5'), 20.05 (CH3); IR
(ATR, cm-1): 3375.17 (OH), 3225.83 ( OH), 2952. 91 ( OH), 2908.38 ( NH), 1628.64
(C=N), 1551.45 (C=S); EI-Mass m/z 290.88 [M+]; Anal cald for C9H14N4O5S: C, 37.24; H, 4.86;
N, 19.30. Found: C, 37.39; H, 4.94; N, 19.21.
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