Synthesis of novel ribavirin hydrazone derivatives and anti-proliferative activity against A549 lung cancer cells

Article abstract of DOI:10.1016/j.carres.2009.05.017

A series of novel ribavirin hydrazone derivatives were synthesized by the reaction of ribavirin hydrazone with benzaldehyde or acetophenone derivatives. The structures of the compounds were determined by IR, ¹H NMR, and HRESIMS. Preliminary bio

Full text of DOI:10.1016/j.carres.2009.05.017

Carbohydrate Research 344 (2009) 1270–1275
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of novel ribavirin hydrazone derivatives and anti-proliferative
activity against A549 lung cancer cells
c
a
b,
Wei-Yong Liu a, , Hai-Ying Li b, , Bao-Xiang Zhao a, , Dong-Soo Shin , Song Lian , Jun-Ying Miao
*
*
a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China
b Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China
c Department of Chemistry, Changwon National University, Changwon 641-773, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel ribavirin hydrazone derivatives were synthesized by the reaction of ribavirin hydrazone
with benzaldehyde or acetophenone derivatives. The structures of the compounds were determined by
IR, 1H NMR, and HRESIMS. Preliminary biological evaluation showed that one compound (7h) inhibits
Received 2 March 2009
Received in revised form 7 May 2009
Accepted 13 May 2009
the growth of A549 cells at 20 lM.
Available online 22 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Ribavirin hydrazone
Biological evaluation
A549 cells
1. Introduction
triazole-3-carboxamidine hydrochloride). Another strategy is to re-
place the furanose oxygen in ribavirin with a methylene group,
which gives the carbocyclic version of ribavirin.6
Lung cancer is one of the leading causes of death worldwide.1
Our understanding of the biology of cancer has undoubtedly im-
proved in the last decade. One characteristic of cancer cells is their
highly proliferative nature. Consequently, inhibition of prolifera-
tive pathways is considered an effective strategy to fight cancer,
and much attention has recently been paid to the discovery and
development of new, more selective anticancer agents.2–4
In the search for more effective antiviral and antitumor agents,
various modifications of nucleotides have been proposed. Ribavirin
(1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a nucleoside
analog that has demonstrated efficacy in treating viral diseases
both as monotherapy (respiratory syncytial virus) and in combina-
tion therapy with interferon alpha (hepatitis C virus) since it was
initially synthesized in 1970.5
Structural modification of ribavirin represents a promising ap-
proach in the search for new antiviral agents. Much effort has been
made to develop new ribavirin analogs with safer profiles. How-
ever, in the majority of the studies reported, the investigators fo-
cused on compounds having biologic nucleobase moieties with
A
number of hydrazide–hydrazone derivatives have been
claimed to possess interesting bioactivity such as antibacterial–
antifungal, anticonvulsant, antiinflammatory, antimalarial, analge-
sic, antiplatelets, antituberculosis, and anticancer activities.7–12
Aroylhydrazide–hydrazones containing a heterocycle such as pyr-
idine or an indole ring have attracted special attention.13 A few
pyrazole carbohydrazide hydrazone derivatives have also been re-
ported.14 In our effort to discover and develop apoptosis inducers
as potential new anticancer agents, we recently synthesized a no-
vel 3-aryl-1-arylmethyl-1H-pyrazole-5-carbohydrazide hydrazone
and found that these compounds could inhibit proliferation of
A549 cells.15,16
Based on a fragmented approach, we proposed that ribavirin
hydrazone derivatives might have interesting bioactivities such
as anticancer activity. Thus, we synthesized ribavirin hydrazone
derivatives and carried out the preliminary biological evaluation.
2. Results and discussion
altered sugars, such as levovirin (1-b-
L-ribofuranosyl-1,2,4-tria-
zole-3-carboxamide) and viramidine (1-b-
D
-ribofuranosyl-1,2,4-
2.1. Synthesis of ribavirin hydrazone derivatives
* Corresponding authors. Tel.: +86 531 88366425; fax: +86 531 88564464
(B.-X.Z.).
E-mail addresses: bxzhao@sdu.edu.cn, sduzhao@hotmail.com (B.-X. Zhao),
miaojy@sdu.edu.cn (J.-Y. Miao).
Our synthetic approach for the preparation of ribavirin
hydrazone derivatives is shown in Scheme 1. The key interme-
diate nucleoside, (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(3-(ethoxy-
carbonyl)-1H-1,2,4-triazol-1-yl)tetrahydrofuran-3,4-diyl diacetate
Equal contribution.
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.05.017

W.-Y. Liu et al. / Carbohydrate Research 344 (2009) 1270–1275
1271
O
O
OEt
NH2
N
N
O
AcOH2C
OAc
O
N
N
AcOH2C
HOH2C
OEt
N
N
b
N
O
a
O
+
N
N
AcO OAc
H
AcO
OAc
HO OH
4
2
1
3
c
c
R
O
O
N
NH2
N
Ar
N
N
N
H
H
N
HOH2C
N
HOH2C
N
O
d
N
O
O
HO
OH
HO
OH
R
Ar
6
7
5
Compound
a
b
c
d
e
f
g
h
i
H
∗
H
H
H
∗
H
H
Me
Me
Me
R
∗
∗
Cl
∗
∗
∗
∗
∗
Me
Cl
O
O
O
Ar
HO
HO
HO
HO
Cl
N
OMe
Scheme 1. Synthesis of ribavirin hydrazone derivatives. Reagents and conditions: (a) bis(4-nitrophenyl) hydrogen phosphate, 165–170 °C, vacuum (15–20 mmHg), 20 min,
60% yield; (b) MeOH, NH3, 25 °C, 48 h, 90% yield; (c) 80% hydrazine hydrate, EtOH, reflux for 5 h; and (d) HOAc, EtOH, 6, 0.5 h, 80% yield (for c and d, two steps).
(3), was synthesized from commercially available tetra-O-acetyl-
b- -ribofuranose (1) and ethyl 1,2,4-triazole-3-carboxylate (2)
2.3. Biological activities
D
that was prepared according to the literature procedure.17 Con-
densation of 1 with ethyl 1,2,4-triazole-3-carboxylate (2) in the
presence of a catalytic amount of bis(p-nitrophenyl)phosphate
at 165–170 °C for 20 min under vacuum, followed by workup
provided the fully protected nucleoside (3) in 60% yield. Stirring
a solution of 3 in methanolic ammonia in a flask at room tem-
The cytotoxicity of the compounds against A549 lung cancer
cells was examined using the MTT assay and the results are shown
in Figure 1. Compound 7h showed cytotoxicity at 20
lM, although
other compounds showed no cytotoxicity at 40 M. Results from
l
LDH assays showed that there was no significant difference in
LDH release between the control group and the treatment groups
perature for 48 h, gave ribavirin, 1-b-
D
-ribofuranosyl-1,2,4-tria-
with compounds 7 at 40 lM (except compound 7h at 20 lM) for
zole-3-carboxamide (4) in 90% yield. Treatment of 3 or 4 with
hydrazine hydrate in ethanol for 5 h at reflux under nitrogen
afforded 5 (see Experimental Section for details of purification).
The reaction of compound 5 with benzaldehyde or acetophe-
none derivatives 6 in ethanol furnished products 7 in 34–79%
yields.
48 h (Fig. 2), indicating that compounds 7 did not cause necrosis
in A549 cells.
3. Conclusions
A series of novel ribavirin hydrazone derivatives have been
synthesized by the reaction of ribavirin hydrazone with benzal-
dehyde or acetophenone derivatives. The anti-proliferative activ-
ity of these compounds against A549 lung cancer cells was
examined by the MTT assay. Only compound 7h showed inhibi-
tion of the growth of A549 lung cancer cells at a concentration
2.2. Structural characterization of the compounds
The structures of the products 7 were determined by the anal-
yses of their spectral data including 1H NMR, IR, and HRESIMS.
For example, compound 7h, obtained as yellow crystals, gave a
[M+H]-ion peak at m/z 412.1016 in the HRESIMS, in accordance
with the molecular formula C16H19ClN5O6. In the IR spectra, the
carbonyl group absorptions in the hydrazide moiety and NH
bands in CONH were observed in the 1694 cmꢀ1 and 3096–
2921 cmꢀ1 region, respectively. The 1H NMR chemical shift of
the NH proton recorded as a singlet peak at d 11.32 indicated a
strong deshielding, which can be explained by hydrogen-bond
formation with the phenol group of the aromatic moiety of 7h.
The signal of the hydroxyl group at d 13.05 is also characteristic
for the intramolecular bond formation. All other signals are con-
sistent with the structure of 7h.
of 20 lM, whereas other compounds were not active at a con-
centration of 40 lM.
4. Experimental
Thin-layer chromatography (TLC) was conducted on Silica Gel
60 F254 plates (E. Merck KgaA). 1H NMR and 13C NMR spectra were
recorded on a Bruker Avance 400 (400 MHz) spectrometer, using
DMSO-d6 as solvent and tetramethylsilane (TMS) as internal stan-
dard. Melting points were determined on an XD-4 digital micro
melting point apparatus. IR spectra were recorded with a VERTEX
70 FTIR spectrometer (Bruker Optics). MS spectra were recorded
on a Trace DSQ mass spectrometer.

1272
W.-Y. Liu et al. / Carbohydrate Research 344 (2009) 1270–1275
Figure 1. Viability of A549 cells treated with compounds 7a–i and ribavirin. Cells were seeded in 96-well plates at the density of 6250/cm2. Cells were treated with the
compounds at concentrations of 40 M (except compound 7h at 20
M) for 24 and 48 h. The cell viability was determined by MTT assay. (*p < 0.05 and **p < 0.01 vs control, n = 3).
l
l
Figure 2. Effects of the compounds 7a–i and ribavirin on the release of LDH from A549 cells. The culture media from the cells were treated with the compounds at 40
lM
(except compound 7h at 20 M) for 48 h. Light absorption was analyzed at 340 nm using a model Cintra 5 UV–vis spectrometer. There was no significant difference in LDH
l
release. (p > 0.05 vs control group, n = 3).
4.1. (2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(3-(ethoxycarbonyl)-
1H-1,2,4-triazol-1-yl)tetrahydrofuran-3,4-diyl diacetate (3)
diacetate (9 g, 22.5 mmol) in 90 mL of MeOH was saturated with
NH3 for about 30 min in an ice bath, then stirred at rt for 48 h.
The reaction mixture was condensed, and the residue was washed
with 15 mL of MeOH, filtered, and dried to afford 4 as a white solid
product: 4.9 g (90%); mp 177–179 °C (lit.18 mp 174–176 °C);
1HNMR (DMSO-d6, 400 MHz): d 8.86 (s, 1H, CH@N triazole), 7.80
(s, 1H, CONH2), 7.59 (s, 1H, CONH2), 5.81 (d, J 3.8 Hz, 1H), 5.55
(d, J 5.7 Hz, 1H, OH), 5.17 (d, J 5.6 Hz, 1H, OH), 4.89 (t, J 5.5 Hz,
1H, OH), 4.35 (q, J 4.8 Hz, 1H), 4.13 (q, J 5.2 Hz, 1H), 3.95 (q, J
4.6 Hz, 1H), 3.60–3.65 (m, 1H, CH2), 3.48–3.53 (m, 1H, CH2).
(2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(3-(ethoxycarbonyl)-1H-1,
2,4-triazol-1-yl)tetrahydrofuran-3,4-diyl diacetate (3) was pre-
pared according to the procedure for the preparation of
(2R,3R,4R,5R)-2-(acetoxymethyl)-5-(3-(methoxycarbonyl)-1H-1,2,
4-triazol-1-yl)tetrahydrofuran-3,4-diyl diacetate.18 Briefly, tetra-
O-acetyl-b-D-ribofuranose (1) (14.5 g, 45.5 mmol) and ethyl 1H-
1,2,4-triazole-3-carboxylate (2) (6.7 g, 47.5 mmol) were added to
a flask in an oil bath at 170 °C, and stirred until the sugar had
melted. Then bis(4-nitrophenyl) hydrogen phosphate (0.12 g,
0.35 mmol) was added to the reaction mixture. The mixture was
heated at 165–170 °C under vacuum at 15–20 mmHg for about
20 min. The crude product was dissolved in 100 mL of EtOH, and
125 mL of petroleum ether was added. The product was allowed
to crystallize at 0 °C, and the first crop was filtered off. The mother
liquor was then concentrated, and recrystallized again. The solid
thus obtained was dried to give the target product: 11.3 g, 60%,
4.3. 1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetra-
hydrofuran-2-yl)-1H-1,2,4-triazole-3-carbohydrazide (5)
Method A: 1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)
tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-carboxamide (4) (0.5 g,
2.05 mmol), 80% hydrazine hydrate (1.3 g, 20.7 mmol), and 5 mL
of EtOH were added to a flask. The mixture was stirred and heated
at reflux for 5 h. The solvent was removed under reduced pressure,
and the residue was then heated under reduced pressure at 100 °C
to remove the remaining hydrazine. The product was used directly
in the next step.
mp 85–87 °C; IR (KBr):
m ;
3489, 3103, 2984, 1749 cmꢀ1 1HNMR
(DMSO-d6, 400 MHz): d 8.92 (s, 1H, CH@N triazole), 6.35 (d, J
3.5 Hz, 1H), 5.69 (t, J 4.4 Hz, 1H), 5.53 (t, J 5.4 Hz, 1H), 4.43 (q, J
5.4 Hz, 1H), 4.39 (dd, J 3.4 Hz, J 12 Hz, 1H, CH2), 4.35 (q, J 7.2 Hz,
2H, CH2), 4.11 (dd, J 4.0 Hz, J 12 Hz, 1H, CH2), 2.09, 2.08, 2.03 (s,
3 ꢁ 3H, CH3C@O), 1.31 (t, J 7.2 Hz, 3H, CH3); HRESIMS: calcd for
[M+Na]+ C16H21N3NaO9: 422.1175; found: 442.1180.
Method B: (2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(3-(ethoxycar-
bonyl)-1H-1,2,4-triazol-1-yl) tetrahydrofuran-3,4-diyl diacetate
(3) (150 mg, 0.375 mmol), 80% hydrazine hydrate (470 mg,
7.5 mmol), and 4 mL of EtOH were added to a flask, stirred, and
heated to reflux for 5 h. The solvent was removed under reduced
pressure, and then the residue was heated under reduced pressure
at 140–150 °C to remove the remaining hydrazine and acetyl
hydrazine. The product was used directly in the next step.
4.2. Preparation of 1-((2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-
carboxamide (4)
IR (KBr):
m ;
3416, 2930, 1668, 1494, 1265, 1103, 862, 659 cmꢀ1
A
stirred solution of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(3-
1HNMR (DMSO-d6, 400 MHz): d 9.71 (s, 1H, NH), 8.85 (s, 1H, CH@N
triazole), 5.81 (d, J 2.9 Hz, 1H), 5.56 (s, 1H, OH), 5.20 (s, 1H, OH),
(ethoxycarbonyl)-1H-1,2,4-triazol-1-yl) tetrahydrofuran-3,4-diyl

W.-Y. Liu et al. / Carbohydrate Research 344 (2009) 1270–1275
1273
4.89 (s, 1H, OH), 4.35 (s, 1H), 4.15 (q, J 4.3 Hz, 1H), 3.95 (q, J 4.1 Hz,
1H), 3.61–3.64 (m, 1H, CH2), 3.49–3.52 (m, 1H, CH2), 3.31 (s, 2H,
NH2); HRESIMS: calcd for [M+Na]+ C8H13N5NaO5: 282.0814; found:
282.0804.
(d, J 8.7 Hz, 2 ꢁ 1H, ArH), 5.86 (d, J 4.0 Hz, 1H), 5.54 (d, J 5.6 Hz, 1H,
OH), 5.15 (d, J 5.6 Hz, 1H, OH), 4.89 (t, J 5.5 Hz, 1H, OH), 4.41 (q, J
4.8 Hz, 1H), 4.16 (q, J 5.1 Hz, 1H), 3.98 (q, J 4.6 Hz, 1H), 3.82 (s,
3H, OCH3), 3.63–3.68 (m, 1H, CH2), 3.50–3.56 (m, 1H, CH2); 13C
NMR (DMSO-d6, 100 MHz): d 161.4, 156.8, 155.4, 149.3, 145.5,
129.3 (2 ꢁ C), 127.2, 114.8 (2 ꢁ C), 92.4, 86.0, 75.0, 70.3, 61.6,
55.8; HRESIMS: calcd for [M+H]+ C16H20N5O6: 378.1414; found:
378.1404.
4.4. General procedure for the synthesis of ribavirin hydrazone
derivatives
4.4.1. (Z)-N0-Benzylidene-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-
carbohydrazide (7a)
4.4.4. (Z)-1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-N0-(4-(dimethylamino)benzylidene)-1H-
1,2,4-triazole-3-carbohydrazide (7d)
1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-1H-1,2,4-triazole-3-carbohydrazide
(5)
(530 mg,
1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydro-
2.04 mmol, theoretical quantity) was added to 7 mL of HOAc, with
stirring and heating until 5 dissolved. Then PhCHO (350 mg,
3.3 mmol) and 12 mL of EtOH were added. The mixture was then
heated to reflux for 0.5 h, at the end of which time it was filtered,
and the solid was washed with 10 mL of EtOH and dried to afford
furan-2-yl)-1H-1,2,4-triazole-3-carbohydrazide
(5)
(530 mg,
2.04 mmol, theoretical quantity) was added to 7 mL of HOAc, with
stirringand heating until 5 dissolved. Then 4-(dimethylamino)benz-
aldehyde (458 mg, 3.07 mmol) and 12 mL of EtOH were added. The
reaction mixture was heated to reflux for 1 h, and then the solvent
was removed on a rotary evaporator. The crude residue was washed
with 1:3 EtOAc–petroleum ether and subsequently purified by col-
umn chromatography (silica gel; 1:2 EtOH–EtOAc) to afford 7d as a
yellow solid: 342 mg (42.8%); mp 228–236 °C; IR (KBr):
3402, 3351, 3322, 3288, 3107, 2934, 2903, 2854, 2820, 1681, 1667,
1599, 1365, 1215, 1181, 1093, 1032, 824, 607, 532 cmꢀ1 1H NMR
(DMSO-d6, 400 MHz): d 11.57 (s, 1H, CONH–N), 8.95 (s, 1H, CH@N
triazole), 8.39 (s, 1H, ArCH@N), 7.52 (d, J 8.7 Hz, 2 ꢁ 1H, ArH), 6.76
(d, J 8.8 Hz, 2 ꢁ 1H, ArH), 5.85 (d, J 3.8 Hz, 1H), 5.53 (d, J 5.5 Hz, 1H,
OH), 5.14 (d, J 5.6 Hz, 1H, OH), 4.89 (t, J 5.5 Hz, 1H, OH), 4.41 (q, J
4.8 Hz, 1H), 4.16 (q, J 5.1 Hz, 1H), 3.98 (q, J 4.5 Hz, 1H), 3.63–3.68
(m, 1H, CH2), 3.50–3.56 (m, 1H, CH2), 2.98 (s, 6H, N(CH3)2); 13C
NMR (DMSO-d6, 100 MHz): d 157.0, 155.1, 152.1, 150.2, 145.4,
129.0 (2 ꢁ C), 121.8, 112.2 (2 ꢁ C), 92.4, 86.0, 75.0, 70.3, 61.6, 40.2
(2 ꢁ C); HRESIMS: calcd for [M+H]+ C17H23N6O5: 391.1730; found:
391.1720.
7a as a white solid: 568 mg (79.8%); mp 245–249 °C; IR (KBr):
m
3457, 3368, 3326, 3129, 2930, 1694, 1545, 1229, 1084 cmꢀ1 1H
;
NMR (DMSO-d6, 400 MHz): d 11.91 (s, 1H, CONH–N), 8.98 (s, 1H,
CH@N triazole), 8.57 (s, 1H, ArCH@N), 7.70–7.72 (2 ꢁ 1H, ArH),
7.45–7.48 (3 ꢁ 1H, ArH), 5.87 (d, J 4.0 Hz, 1H), 5.55 (d, J 5.6 Hz,
1H, OH), 5.15 (d, J 5.5 Hz, 1H, OH), 4.89 (t, J 5.5 Hz, 1H, OH), 4.41
(q, J 4.8 Hz, 1H), 4.16 (q, J 5.1 Hz, 1H), 3.98 (q, J 4.6 Hz, 1H),
3.64–3.69 (m, 1H, CH2), 3.51–3.56 (m, 1H, CH2); 13C NMR
(DMSO-d6, 100 MHz): d 156.1, 155.0, 148.9, 144.9, 134.1, 130.1,
128.7 (2 ꢁ C), 127.1 (2 ꢁ C), 91.9, 85.4, 74.4, 69.7, 61.0. HRESIMS:
calcd for [M+H]+ C15H18N5O5: 348.1308; found: 348.1304.
m 3463,
;
4.4.2. (Z)-N0-(Benzo[d][1,3]dioxol-5-ylmethylene)-1-((2R,3R,4S,-
5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-1,
2,4-triazole-3-carbohydrazide (7b)
1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-1H-1,2,4-triazole-3-carbohydrazide (5) (514 mg, 1.98
mmol, theoretical quantity) was added to 6 mL of HOAc, with stir-
ring and heating until 5 dissolved. Then benzo[d][1,3]dioxole-5-
carbaldehyde (358 mg, 2.38 mmol) and 25 mL of EtOH were added.
The reaction mixture was heated to reflux for 2 h, and then the sol-
vent was removed on a rotary evaporator. The crude residue was
washed with 1:3 EtOAc–petroleum ether and subsequently,
purified by column chromatography (silica gel; 1:2 EtOH–EtOAc)
to afford 7b as a white solid: 357 mg (61.7%); mp 190–199 °C; IR
4.4.5. (Z)-1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-N0-(furan-2-ylmethylene)-1H-1,2,4-triazole-
3-carbohydrazide (7e)
Compound 7e was prepared from furan-2-carbaldehyde
(318 mg, 3.3 mmol) using the same procedure as described for
7d. Purification by column chromatography resulted in 7e as a
white solid: 461 mg (66.8%); mp 243–246 °C; IR (KBr):
3258, 3222, 3127, 2951, 2881, 1670, 1621, 1328, 1250, 1126,
1078, 1031, 766, 649, 596 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d
m 3419,
(KBr):
m
3451, 3354, 3327, 3284, 3122, 2936, 2910, 1765,
;
;
1689 cmꢀ1
1H NMR (DMSO-d6, 400 MHz): d 11.87 (s, 1H, CONH–
11.93 (s, 1H, CONH–N), 8.97 (s, 1H, CH@N triazole), 8.46 (s, 1H,
ArCH@N), 7.84 (s, 1H, ArH), 6.92 (d, J 3.0 Hz, 1H, ArH), 6.63 (s,
1H, ArH), 5.86 (d, J 3.7 Hz, 1H), 5.54 (d, J 5.6 Hz, 1H, OH), 5.15 (d,
J 5.5 Hz, 1H, OH), 4.89 (t, J 5.2 Hz, 1H, OH), 4.40 (q, J 4.6 Hz, 1H),
4.16 (q, J 5.1 Hz, 1H), 3.98 (q, J 4.4 Hz, 1H), 3.64–3.69 (m, 1H,
CH2), 3.50–3.56 (m, 1H, CH2); 13C NMR (DMSO-d6, 100 MHz): d
156.6, 155.5, 149.8, 145.8, 145.5, 139.0, 114.2, 112.7, 92.4, 86.0,
75.0, 70.3, 61.6; HRESIMS: calcd for [M+H]+ C13H16N5O6:
338.1101; found: 338.1098.
N), 8.98 (s, 1H, CH@N triazole), 8.46 (s, 1H, ArCH@N), 7.28 (d, J
1.2 Hz, 1H, ArH), 7.14 (dd, J 8.1 Hz, 1.6 Hz, 1H, ArH), 6.99 (d, J
8 Hz, 1H, ArH), 6.09 (s, 2H, O–CH2–O), 5.85 (d, J 3.9 Hz, 1H), 5.60
(d, J 5.5 Hz, 1H, OH), 5.21 (d, J 5.5 Hz, 1H, OH), 4.94 (t, J 5.5 Hz,
1H, OH), 4.40 (q, J 4.8 Hz, 1H), 4.15 (q, J 5.0 Hz, 1H), 3.97 (q, J
4.6 Hz, 1H), 3.63–3.68 (m, 1H, CH2), 3.50–3.55 (m, 1H, CH2); 13C
NMR (DMSO-d6, 100 MHz): d 156.8, 155.5, 149.7, 149.3, 148.5,
145.5, 129.0, 124.1, 108.9, 105.5, 102.1, 92.5, 86.0, 72.0, 70.3,
61.6; HRESIMS: calcd for [M+H]+ C16H18N5O7: 392.1206; found:
392.1205.
4.4.6. (Z)-1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-N0-(2-hydroxybenzylidene)-1H-1,2,4-
triazole-3-carbohydrazide (7f)
4.4.3. (Z)-1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-N’-(4-methoxybenzylidene)-1H-1,2,4-
triazole-3-carbohydrazide (7c)
Compound 7c was prepared from 4-methoxybenzaldehyde
(427 mg, 3.13 mmol) using the same procedure as described for
7a. Purification of the solid resulted in 7c as a white solid:
1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-1H-1,2,4-triazole-3-carbohydrazide (5) (530 mg, 2.04
mmol, theoretical quantity) was added to 7 mL of HOAc, with stir-
ring and heating until 5 dissolved. Then 2-hydroxybenzaldehyde
(542 mg, 4.43 mmol) and 12 mL of EtOH were added. The reaction
mixture was heated to reflux for 4 h, and then the solvent was re-
moved on a rotary evaporator. The crude residue was washed with
1:3 EtOAc–petroleum ether and subsequently purified by column
chromatography (silica gel; 1:2 EtOH–EtOAc) to afford 7f as a
608 mg (78.7%); mp 241–244 °C; IR (KBr):
m
3445, 3345, 3134,
2964, 2928, 1698, 1604, 1254, 1081, 850, 682 cmꢀ1
;
1H NMR
(DMSO-d6, 400 MHz): d 11.76 (s, 1H, CONH–N), 8.96 (s, 1H, CH@N
triazole), 8.49 (s, 1H, ArCH@N), 7.65 (d, J 8.7 Hz, 2 ꢁ 1H, ArH), 7.02

1274
W.-Y. Liu et al. / Carbohydrate Research 344 (2009) 1270–1275
yellow solid: 430 mg (57.8%); mp 216–222 °C; IR (KBr):
2925, 2874, 1685, 1613, 1549, 1490, 1453, 1344, 1272, 1211,
1083, 1033, 980, 915, 870, 755, 652, 521 cmꢀ1 1H NMR (DMSO-
m
3443,
4.4.9. (Z)-N0-(1-(3,5-Dichloro-2-hydroxyphenyl)ethylidene)-1-
((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-1H-1,2,4-triazole-3-carbohydrazide (7i)
;
d6, 400 MHz): d 12.32 (s, 1H, ArOH), 11.20 (s, 1H, CONH–N), 9.01
(s, 1H, CH@N triazole), 8.76 (s, 1H, ArCH@N), 7.50 (d, J 8.4 Hz,
1H, ArH), 7.31 (t, J 7.9 Hz, 1H, ArH), 6.92–6.95 (2 ꢁ 1H, ArH), 5.87
(d, J 3.8 Hz, 1H), 5.61 (d, J 5.4 Hz, 1H, OH), 5.21 (d, J 5.5 Hz, 1H,
OH), 4.94 (t, J 5.5 Hz, 1H, OH), 4.40 (q, J 4.8 Hz, 1H), 4.16 (q, J
5.2 Hz, 1H), 3.98 (q, J 4.6 Hz, 1H), 3.63–3.69 (m, 1H, CH2), 3.50–
3.56 (m, 1H, CH2); 13C NMR (DMSO-d6, 100 MHz): d 157.1, 155.5,
154.6, 149.1, 144.7, 131.2, 129.2, 118.9, 118.2, 116.0, 91.6, 85.2,
74.2, 69.5, 60.7; HRESIMS: calcd for [M+H]+ C15H18N5O6:
364.1257; found: 392.1255.
1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-1H-1,2,4-triazole-3-carbohydrazide (5) (424 mg, 1.63
mmol, theoretical quantity) was added to 6 mL of HOAc, with stir-
ring and heating until 5 dissolved. Then 1-(5-chloro-2-hydroxy-
phenyl)ethanone (503 mg, 2.45 mmol) and 10 mL of EtOH were
added. The reaction mixture was heated to reflux for 1 h, and then
stirred at rt overnight. The solvent was removed on a rotary evap-
orator. The crude residue was washed with 1:3 EtOAc–petroleum
ether and subsequently purified by column chromatography (silica
gel; 1:2 EtOH–EtOAc) to afford 7i as a yellow solid: 283 mg
(38.7%); mp 192–199 °C; IR (KBr):
1535, 1441, 1366, 1245, 1189, 1094, 1038, 906, 859, 739, 662,
584, 552 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d 14.21 (s, 1H,
m 3426, 3090, 2926, 1700,
4.4.7. (Z)-1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-N0-(1-(2-hydroxy-5-methylphenyl)-
ethylidene)-1H-1,2,4-triazole-3-carbohydrazide (7g)
;
ArOH), 11.54 (s, 1H, CONH–N), 9.02 (s, 1H, CH@N triazole), 7.68
(d, J 1.6 Hz, 1H, ArH), 7.62 (d, J 1.7 Hz, 1H, ArH), 5.89 (d, J 3.9 Hz,
1H), 5.57 (d, J 5.3 Hz, 1H, OH), 5.18 (d, J 5.3 Hz, 1H, OH), 4.90 (t, J
5.3 Hz, 1H, OH), 4.41 (q, J 4.6 Hz, 1H), 4.17 (q, J 5.0 Hz, 1H), 4.00
(q, J 4.6 Hz, 1H), 3.65–3.70 (m, 1H, CH2), 3.52–3.57 (m, 1H, CH2);
13C NMR (DMSO-d6, 100 MHz): d 157.8, 156.2, 155.6, 153.6,
145.1, 130.5, 126.9, 122.0, 121.7, 121.1, 92.1, 85.5, 74.5, 69.7,
61.0, 14.1; HRESIMS: calcd for [M+H]+ C16H18Cl2N5O6: 446.0634;
found: 446.0633.
1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-1H-1,2,4-triazole-3-carbohydrazide
(5)
(424 mg,
1.63 mmol, theoretical quantity) was added to 6 mL of HOAc,
with stirring and heating until 5 dissolved. Then 1-(2-hydro-
xy-5-methylphenyl)ethanone (369 mg, 2.45 mmol) and 10 mL
of EtOH were added. The reaction mixture was heated to reflux
for 1 h, and then the solvent was removed on a rotary evapora-
tor. The crude residue was washed with 1:3 EtOAc–petroleum
ether and subsequently purified by column chromatography
(silica gel; 1:2 EtOH–EtOAc) to afford 7g as a light yellow
4.5. Biological activity assays
solid: 220 mg (34.3%); mp 228–247 °C; IR (KBr):
3089, 2914, 1713, 1692, 1542, 1494, 1212, 1106, 1052, 1020,
980, 514 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d 12.73 (s, 1H,
ArOH), 11.19 (s, 1H, CONH–N), 9.00 (s, 1H, CH@N triazole),
7.44 (s, 1H, ArH), 7.13 (d, J 8.1 Hz, 1H, ArH), 6.81 (d, J 8.2 Hz,
m 3406, 3359,
4.5.1. Cell culture
A549 lung cancer cells were cultured in RPMI 1640 medium at
37 °C with 5% CO2 and 95% air, supplemented with 10% (v/v) bo-
vine calf serum and 80 U/mL gentamicin. The cells were seeded
onto 96-well plates or other appropriate dishes containing the
medium at a density of 6250/cm2.
;
1H, ArH), 5.89 (d,
J 3.7 Hz, 1H), 5.55 (d, J 5.5 Hz, 1H,
OH), 5.16 (d, J 5.5 Hz, 1H, OH), 4.89 (t, J 5.4 Hz, 1H, OH), 4.41
(q, J 4.8 Hz, 1H), 4.17 (q, J 5.1 Hz, 1H), 3.99 (q, J 4.6 Hz, 1H),
3.65–3.69 (m, 1H, CH2), 3.53–3.57 (m, 1H, CH2); 13C NMR
4.5.2. Cell viability assay
As described in the previous report, cells were seeded onto 96-
well plates and treated with compounds 7 and ribavirin at 40
(DMSO-d6, 100 MHz):
d 160.5, 156.9, 156.3, 156.2, 145.6,
lM
132.7, 129.2, 127.6, 119.3, 117.6, 92.5, 86.0, 75.1, 70.3, 61.5,
20.6, 14.5; HRESIMS: calcd for [M+H]+ C17H22N5O6: 392.1570;
found: 392.1573.
(except compound 7h at 20 M) for 24 and 48 h, respectively. Cell
l
viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium) assay according to Price et al.19 The light
absorption was measured at 570 nm using a Spectra MAX 190
microplate spectrophotometer (GMI Co., USA).
4.4.8. (Z)-N0-(1-(5-Chloro-2-hydroxyphenyl)ethylidene)-1-((2R,
3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-
yl)-1H-1,2,4-triazole-3-carbohydrazide (7h)
4.5.3. LDH assay
1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydro-
furan-2-yl)-1H-1,2,4-triazole-3-carbohydrazide (5) (530 mg, 2.04
mmol, theoretical quantity) was added to 7 mL of HOAc, with stir-
ring and heating until 5 dissolved. Then 1-(5-chloro-2-hydroxy-
phenyl)ethanone (422 mg, 2.47 mmol) and 12 mL of EtOH were
added. The mixture was heated to reflux for 1 h and then stirred
at rt for 10 h. The reaction mixture was then filtered, and the solid
was washed with 20 mL of EtOH and dried to afford 7h as a
Lactate dehydrogenase (LDH) assay was performed on cells
treated with 40 lM compounds (except compound 7h at 20 lM)
for 48 h using a LDH kit (Nanjing Jiancheng, China) according to
the manufacturer’s protocol. Light absorption was measured at
340 nm using
Australia).
a model Cintra 5 UV–vis spectrometer (GBC,
4.5.4. Statistical analyses
yellow solid: 549 mg (65.1%); mp 256–264 °C; IR (KBr):
3356, 3096, 2921, 1694, 1542, 1485, 1367, 1282, 1230, 1213,
1108, 1052, 1021, 904, 825, 750, 648, 590, 516 cmꢀ1 1H NMR
(DMSO-d6, 400 MHz): 13.05 (s, 1H, ArOH), 11.32 (s, 1H,
m 3406,
Data were presented as means SE and analyzed by SPSS
software. Pictures were processed with Photoshop software.
Mean values were derived from at least three independent
experiments. Differences at p < 0.05 were considered statistically
significant.
;
d
CONH–N), 9.01 (s, 1H, CH@N triazole), 7.65 (d, J 1.9 Hz, 1H, ArH),
7.35 (dd, J 8.7 Hz, 1.9 Hz, 1H, ArH), 6.95 (d, J 8.7 Hz, 1H, ArH),
5.89 (d, J 3.7 Hz, 1H), 5.55 (d, J 5.6 Hz, 1H, OH), 5.16 (d, J 5.5 Hz,
1H, OH), 4.89 (t, J 5.4 Hz, 1H, OH), 4.41 (q, J 4.8 Hz, 1H), 4.16 (q, J
5.2 Hz, 1H), 3.99 (q, J 4.6 Hz, 1H), 3.64–3.69 (m, 1H, CH2), 3.51–
3.56 (m, 1H, CH2); 13C NMR (DMSO-d6, 100 MHz): d 158.4, 157.2,
155.8, 155.6, 145.1, 131.0, 127.9, 122.1, 120.6, 119.0, 92.0, 85.5,
74.5, 69.7, 60.9, 14.1; HRESIMS: calcd for [M+H]+ C16H19ClN5O6:
412.1024; found: 412.1016.
Acknowledgments
This study was supported by the Natural Science Foundation
of Shandong Province (Z2008B10) and the Science and Technol-
ogy Developmental Project of Shandong Province (2008GG1
0002034).

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1275
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