Novel 1,2,4-triazole and imidazole derivatives of l-ascorbic and imino-ascorbic acid: Synthesis, anti-HCV and antitumor activity evaluations

Article abstract of DOI:10.1016/j.bmc.2012.01.054

Several novel 1,2,4-triazole and imidazole l-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7 and 8) derivatives were prepared and evaluated for their inhibitory activity against hepatitis C virus (HCV) replication and human tumour cell proliferation. Compounds 6 and 9 exerted the most pronounced cytostatic effects in all tumour cell lines tested, and were highly selective for human T-cell acute lymphoblastic leukaemia cells (CEM/0) with IC₅₀s of 10 ± 4 and 7.3 ± 0.1 μM, respectively. Unlike compound 9, compound 6 showed no toxicity in human diploid fibroblasts. One of the possible mechanisms of action of compound 6 accounting for observed cytostatic activity towards haematological malignancies might be inhibition of inosine monophosphate dehydrogenase (IMPDH) activity, a key enzyme of de novo purine nucleotide biosynthesis providing the cells with precursors for DNA and RNA synthesis indispensable for cell growth and division, which has emerged as an important target for antileukemic therapy. In addition, this compound proved to be the most potent inhibitor of the hepatitis C virus replication as well. However, observed antiviral effect was most likely associated with the effect that the compound exerted on the host cell rather than with selective effect on the replication of the virus itself. In conclusion, results of this study put forward compound 6 as a potential novel antitumor agent (IMPDH inhibitor) for treating leukaemia. Its significant biological activity and low toxicity in human diploid fibroblasts encourage further development of this compound as a lead.

Full text of DOI:10.1016/j.bmc.2012.01.054

Bioorganic & Medicinal Chemistry 20 (2012) 3675–3685
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 1,2,4-triazole and imidazole derivatives of L-ascorbic and
imino-ascorbic acid: Synthesis, anti-HCV and antitumor activity evaluations
a
a
d
Karlo Wittine , Maja Stipkovic Babic , Damjan Makuc b,c, Janez Plavec b,c, Sandra Kraljevic Pavelic ,
´
´
´
´
´
d
d
e
e
e
a,
⇑
´
Mirela Sedic , Krešimir Pavelic , Pieter Leyssen , Johan Neyts , Jan Balzarini , Mladen Mintas
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruli´cev trg 20, HR-10000 Zagreb, Croatia
b Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, PO Box 660, SI-1001 Ljubljana, Slovenia
c EN?FIST Centre of Excellence, Dunajska 156, SI-1001 Ljubljana, Slovenia
d
ˇ
´
´
Department of Biotechnology, University of Rijeka, Trg brace Mazuranica 10, HR-51000 Rijeka, Croatia
e Katholieke Universiteit Leuven, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Several novel 1,2,4-triazole and imidazole L-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7
Received 14 December 2011
Revised 24 January 2012
Accepted 25 January 2012
Available online 15 February 2012
and 8) derivatives were prepared and evaluated for their inhibitory activity against hepatitis C virus
(HCV) replication and human tumour cell proliferation. Compounds 6 and 9 exerted the most pronounced
cytostatic effects in all tumour cell lines tested, and were highly selective for human T-cell acute lympho-
blastic leukaemia cells (CEM/0) with IC50s of 10 4 and 7.3 0.1 lM, respectively. Unlike compound 9,
compound 6 showed no toxicity in human diploid fibroblasts. One of the possible mechanisms of action
of compound 6 accounting for observed cytostatic activity towards haematological malignancies might
be inhibition of inosine monophosphate dehydrogenase (IMPDH) activity, a key enzyme of de novo
purine nucleotide biosynthesis providing the cells with precursors for DNA and RNA synthesis indispens-
able for cell growth and division, which has emerged as an important target for antileukemic therapy. In
addition, this compound proved to be the most potent inhibitor of the hepatitis C virus replication as
well. However, observed antiviral effect was most likely associated with the effect that the compound
exerted on the host cell rather than with selective effect on the replication of the virus itself. In conclu-
sion, results of this study put forward compound 6 as a potential novel antitumor agent (IMPDH inhib-
itor) for treating leukaemia. Its significant biological activity and low toxicity in human diploid fibroblasts
encourage further development of this compound as a lead.
Keywords:
1,2,4-Triazole
Imidazole
L
-ascorbic acid
Anti-HCV activity
Antitumor activity
IMPDH
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
mice bearing glioblastoma xenografts, and significantly decreased
growth rates of ovarian, pancreatic, and glioblastoma tumours
Ascorbic acid is an essential micronutrient and is considered to
have an antioxidant function in living systems. For the past several
decades, ascorbic acid has risen to prominence as anticancer agent,
although the mechanism underlying this biological effect still
awaits to be unveiled. Recent studies showed that pharmacological
concentrations of ascorbic acid selectively killed cancer but not
normal cells by inducing programmed cell death (apoptosis) and
pyknosis/necrosis, and that ascorbate-mediated cell death was
due to protein-dependent extracellular H2O2 generation, via ascor-
bate radical formation from ascorbate as the electron donor.1
Further in vivo studies provided evidence that single pharmacolog-
ical dose of ascorbate produced sustained ascorbate radical and
H2O2 formation selectively within interstitial fluids of tumours of
established in mice.2 Additional confirmation of antitumor poten-
tial of ascorbic acid came from the study demonstrating that phar-
macological doses of ascorbic acid suppress tumour growth and
metastases in hormone-refractory prostate cancer.3 In addition to
anticancer properties, vitamin C and its derivatives have been as-
cribed antiviral activity as well.4–6 In combination with several
other bioactive compounds, ascorbic acid was shown to contribute
to notable improvement of oxidative stress and immunological
parameters in patients with chronic hepatitis C.7 Standard therapy
for treating hepatitis C is based on the combination of pegylated
interferon alpha (pIFN-a D-ribofuranosyl-
) with ribavirin8 (1-b-
1H-1,2,4-triazole-3-carboxamide), a nucleoside analogue whose
one of the many mechanisms of action also includes inhibition of
inosine monophosphate dehydrogenase (IMPDH), a key enzyme
of de novo purine nucleotide biosynthesis. Though standard ther-
apy is effective in eradicating the hepatitis C virus in more than
half of patients,9 there is currently little prospect of an effective
⇑
Corresponding author. Tel.: +385 1 4597 214; fax: +385 1 4597 250.
E-mail address: mladen.mintas@fkit.hr (M. Mintas).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.054

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K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
Figure 1. The novel 1,2,4-triazole-3-carboxamide and 4,5-disubstituted-imidazole L-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7 and 8) derivatives.
vaccine for hepatitis C virus, which urges the need for developing
more efficient antiviral drugs.10
zyl-L
-ascorbic acid (EDAA)11 in dry acetonitrile, using trimethyl-
silyl trifluoromethanesulfonate (TMSOTf) as a catalyst, yielded
compound 1 with a free hydroxy group at the C-5 of the ethylenic
spacer. Treatment of silylated methyl 1H-1,2,4-triazole-3-carbox-
In spite of long-standing research on ascorbic acid, its clinical
utility has been limited by relatively low potency and in vivo
efficacy therefore necessitating the need for development of novel
derivatives with improved biological activity. Additionally, 1,2,4-
triazole represents a unique template that is associated with
antiviral, antibacterial, antifungal, anti-inflammatory and central
nervous system (CNS) activity. Compounds incorporating a 1,2,4-
triazol ring have also been shown to be antitumor agents.11 Imid-
azole is an entity incorporated into many important biological
molecules with a wide range of pharmacological activity. In the
drug discovery the imidazole is the most important drug strategy
and imidazoles are generally well known as anticancer agents.12
Governed by this idea and in line with our previous study,5 we effi-
ciently synthesized a series of new molecules containing 1,2,4-tri-
azole-3-carboxamide moiety (1–5), heterocyclic constituent of the
ribavirin, or 4,5-disubstituted-imidazole ring (6–9) linked via eth-
ylenic spacer to lactone or lactam moiety (Fig. 1). Newly prepared
compounds were evaluated for their anti-HCV and anti-cancer
properties, and their effect on IMPDH activity in mouse and human
leukaemia cell lines was assessed.
ylate with 2,3-di-O-benzyl-5,6-di-O-acetyl-L-ascorbic acid (dAd-
BA)13 in the same manner produced compound 2 as a mixture of
(Z) and (E) isomers in 2:1 ratio.
Reaction of 1-[2-(3,4-bis-benzyloxy-5-oxo-5H-furan-2-ylidene)-
ethyl]-1H-1,2,4-triazole-3-carboxylic acid methyl ester (2) with
ammonia in anhydrous methanol gave compound 3 bearing a
methoxy group at the C-4 of the lactone moiety, while the same
reaction of 2 in dry dioxane yielded 4 with the hydroxyl group
at C-4 of the lactam moiety (Fig. 2). We have found that in this
reaction lactone ring is being rearranged to lactam ring. Such a
rearrangement proceeds via nucleophilic addition of the ammonia
to the carbonyl group with concomitant ring-opening of the lac-
tone to the keto-enol-amide and subsequent intramolecular ring
closure to give lactam as noted previously.14 The structure of
this compound and others obtained by reaction of ammonolysis
was unambiguously confirmed by correlation 2D 1H–15N NMR
spectroscopy indicating the presence of the imino group of the
lactam ring.
Removal of benzyl protecting groups in compound 2 was
achieved by using boron-trichloride to yield 5 bearing the free
enolic hydroxyl groups at C-2 and C-3 of the lactone moiety as a
mixture of (Z) and (E) isomers in 10:1 ratio.
2. Results and discussion
2.1. Chemistry
Reactionof4,5-dicarboxymethyl-(DMI)or4,5-dicyanoimidazole
(DCI) with 5,6-di-O-acetyl-2,3-di-O-benzyl-L-ascorbic acid (dAdBA)
and 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) afforded 4,5-dicarb-
oxymethyl-(6) (Z/E = 10:1) and 4,5-dicyano-1,3-imidazole deriva-
The novel 1,2,4-triazole and 4,5-disubstituted-imidazole L-
ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7
and 8) derivatives are shown in Figure 1.
Compounds 1 and 2 were synthesized by standard Vorbrüggen
condensation reaction conditions as depicted in Figure 2. Treat-
ment of commercially available methyl 1H-1,2,4-triazol-3-carbox-
ylate (MT) with excess hexamethyldisilazane (HMDS) and catalytic
amount of ammonium sulphate resulted in the formation of the
silylated derivative TMS-1,2,4-triazol-3-carboxylic methyl ester,
which in subsequent reaction with 4-(5,6-epoxy)-2,3-di-O-ben-
tives of L-ascorbic acid (9) (Z/E = 3:1) containing a double bond at
the C4–C5 conjugated with the enone system of the lactone moiety.
Introduction of gaseous ammonia in the solution of 6 in methanol
and dioxane resulted in the addition of ammonia at the carbonyl
group of the lactone moiety with concomitant ring opening and clo-
sure to lactam with free hydroxyl group at C-4 (7). Compound 8, with
the amido groups at the C-4 and C-5 of imidazole ring and hydroxyl

K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
3677
Figure 2. Synthesis of 1,2,4-triazole and 4,5-disubstituted-imidazole
L-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7 and 8) derivatives. Reagents and
conditions: (i) HMDS/(NH4)2SO4/argon/reflux 12 h; TMS-triflate/dry acetonitrile/55-70 °C/12 h; (ii) NH3/MeOH-dioxane/rt/24 h; (iii) BCl3/CH2Cl2/ꢀ78 °C/2 h.
0
0
group at the C-4 of the lactam moiety, broke forth from prolonged
reaction time (96 h) at room temperature.
J-HMBC experiment. The 3JC3 H5 coupling constant of 2.4 Hz corre-
sponded to (Z) geometry along the C40@C50 double bond, while lar-
ger coupling constant of 6.2 Hz indicated (E) geometry (Fig. 3a).
Weak NOE enhancements between H-5 and H-50 suggested
predominance of an orientation, where H-5 and H-50 protons are
spatially close in both isomers (Fig. 3a). Likewise, Z and E isomers
for compound 9 were distinguished with the use of long-range
It is interesting to note that removal of O-benzyl protective
groups in triazole (4) and imidazole (7 and 8) 3-pyrroline-2-on
derivatives containing a free hydroxyl group at C-4 of the lactam
moiety by using either Lewis acid (BCl3)-catalyzed hydrolysis,
catalytic hydrogenation with Pd/H2 or microwave-assisted hydro-
genation with PdENC didn’t yield the desired derivatives with free
enolic hydroxyl groups which may be due to instability of the lac-
tam moiety caused by the presence of proton on nitrogen and the
hydroxyl group at the position C-4.
proton–carbon coupling constants. 3JC3 H5 of 3.0 Hz is in agreement
with (Z) configuration, whereas coupling constant of 6.5 Hz sug-
gested (E) geometry along the C40@C50 double bond.
0
0
Major chemical shift differences of geminal 5-CONH2 protons
(DdH = 3 ppm) were observed in 8. These observations suggested
2.2. Structural and conformational properties
involvement of more deshielded proton in hydrogen bond leading
to stabilization of conformer presented in Figure 3b. Such confor-
mation is supported by NOE enhancements (Fig. 3b).
The chemical identities of compounds 1–9 were confirmed by
1H, 13C and 15N NMR measurements. Proton and carbon NMR
chemical shifts are reported in the Experimental section. Notewor-
thy, the correlation signals observed in 1H–15N HSQC and HMBC
spectra confirmed the presence of the lactam functionality in 8.
NH proton at dH 8.15 ppm showed cross-peak at dN 127.0 ppm
(Fig. S1). The exchangeable proton at dH 6.13 ppm showed no cor-
2.3. Biological results
2.3.1. Anti-HCV activity
The novel 1,2,4-triazole and 4,5-disubstituted-imidazole
L-
ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7
and 8) derivatives were evaluated for their inhibition of the hepa-
titis C virus replication in the Huh 5-2 replicon system. The 4,5-
15
relation signal in N HSQC and was assigned to the C-40-hydroxyl
group. The remaining cross-peaks observed in 15N HSQC spectrum
were attributed to the two amide groups in the imidazole ring (dN
109.4 and 114.0 ppm, Fig. S1). Additionally, the presence of the lac-
tam ring in 8 was supported by more shielded C40 atom (dC
81.85 ppm) in comparison with the chemical shift of C40 atom in
lactone derivative 3 (dC 102.24 ppm). In a similar manner, the lac-
tam functionalities were confirmed by 2D 15N NMR experiments in
compounds 4 and 7 (Figs. S2 and S3, respectively).
dicarboxymethyl-imidazole
to be the most potent compound (Table 1) with EC50 of 13.3
mL as deduced from the dose–response curve. Based upon the
measurement of its antimetabolic effect (CC50 of 70.5 g/mL), a
L-ascorbic acid derivative (6) proved
l
g/
l
selectivity index of 5.3 could be calculated. However, bearing in
mind poor antimetabolic effect of this compound at concentrations
that induce observed antiviral effect, the latter might be ascribed
to non-specific pleiotropic effects that the compound exerts on
the host cells rather than to selective effect on the multiplication
Conformational properties of compound 2 were assessed using
long range JCH coupling constants, which were established by

3678
K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
3
Figure 3. (a) (Z) and (E) isomers of 2 with the corresponding JCH coupling constants and key NOE enhancements. (b) Predominant conformation of 8 established by NOE
enhancements and chemical shifts of amino protons suggesting stabilization through O. . .H–N hydrogen bonds.
of the replicon itself. Therefore, it seems implausible to perceive
this compound as a selective inhibitor of the hepatitis C virus rep-
lication in the Huh 5-2 replicon system.
is then converted to guanosine 50-monophosphate (GMP) by GMP
synthase. IMP also serves as a substrate for the biosynthesis of
adenosine 50-monophosphate (AMP). Due to its both, antiprolifera-
tive and differentiation-inducing effects, IMPDH inhibitors repre-
sent a promising class of antileukemic agents.15 In order to
determine whether newly prepared compounds exert any inhibi-
tory effect on IMPDH activity in L1210 and CEM leukaemia cell
lines, we measured tritium release from [2,8-3H]hypoxanthine in
the absence (control) or presence of different concentrations of
these compounds. Ribavirin, a well-known IMPDH inhibitor, was
included as a reference drug. As expected, this compound mark-
edly inhibited IMPDH activity in a dose-dependent manner in both
2.3.2. Cytostatic activity
The synthesized compounds 1–2 and 4–9 were evaluated
in vitro for their cytostatic effect against murine leukaemia
(L1210) and human tumour cell lines including human T-cell acute
lymphoblastic leukaemia (CEM), cervical carcinoma (HeLa), meta-
static breast epithelial adenocarcinoma (MCF-7), hepatocellular
carcinoma (HepG2), colorectal adenocarcinoma (SW620) and pan-
creatic carcinoma (MiaPaCa-2) along with human normal diploid
fibroblasts (WI38) (Table 2). Compounds 6 and 9 exerted the most
pronounced cytostatic effects in all tumour cell lines tested, and
were highly selective for human T-cell acute lymphoblastic leukae-
tumour cell lines (Fig. 4). Its IC50 was approximately 1–2.5 lM,
regardless of measurement time points (20, 40 or 60 min). Among
the tested ascorbic acid derivatives, compounds 2, and 8 as well as
compound 6 at a lower extent, showed inhibitory potential in both
leukaemia cell lines, albeit at concentrations that were approxi-
mately 50- to 100-fold higher than those of ribavirin effective
mia cells (CEM/0) with IC50s of 10 4 and 7.3 0.1
l
M, respec-
tively. However, unlike compound 9, compound
6 was not
cytotoxic to human fibroblasts. In addition, compound 4 contain-
ing 3-carboxyamide-1,2,4-triazole ring connected via ethylenic
spacer to lactam moiety bearing C-4 substituted free hydroxyl
group showed selective but rather weak antiproliferative activity
concentrations (IC50: 100–250 lM) (Fig. 4), pointing to their ability
to interfere with purine biosynthesis. It should be noted that
IMPDH exists in two isoforms, namely type I (‘housekeeping’) that
is constitutively expressed in normal and neoplastic cells, and type
II whose expression is up-regulated in human neoplastic cell lines
and in leukaemic cells from patients with chronic granulocytic,
lymphocytic and acute myeloid leukaemias.15 Moreover, inhibitors
of IMPDH can alternatively suppress the enzyme activity by bind-
ing at the cofactor (NAD) site as well. For example, ribavirin is con-
verted in vivo into its 50-monophosphate, which mimics IMP and
binds at the substrate site. Although our data clearly speak in
in MCF-7 and HepG2 cells with IC50s of 61 0.2 and 66 0.6
respectively, and no cytotoxicity in WI38.
lM,
2.3.3. IMPDH activity assays
Inosine 50-monophosphate dehydrogenase (IMPDH, E.C.1.1.
1.205), a NAD dependent enzyme that controls de novo synthesis
of purine nucleotides, catalyzes the oxidation of inosine 50-mono-
phosphate (IMP) to xanthosine 50-monophosphate (XMP), which

K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
3679
Table 1
Antiviral effect of compounds 1–2 and 4–9 on hepatitis C virus replication in the Huh 5-2 replicon system
Compd
Antimetabolic and antiviral effect
a
b
c
CC50
EC50
EC90
Unit
O
N
OH
H3CO
N
N
O
>100
62.1
NDd
lg/mL
O
BnO
OBn
1
O
N
H3CO
N
N
>100
78.5
>100
l
l
l
g/mL
O
O
2
BnO
OBn
O
N
H2N
H
N
N
N
>100
>100
>100
g/mL
O
HO
BnO
OBn
4
O
N
H3CO
N
N
>100
61.3
ND
g/mL
O
O
HO
OH
5
6
O
N
H3CO
H3CO
N
70.5
13.3
22.7
lg/mL
O
O
O
OBn
BnO
O
N
H
N
H3CO
H3CO
N
>100
>100
36.6
81.3
l
g/mL
g/mL
O
HO
O
BnO
OBn
7
O
N
H
N
H2N
H2N
N
>100
>100
l
O
HO
BnO
O
OBn
8
(continued on next page)

3680
K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
Table 1 (continued)
Compd
Antimetabolic and antiviral effect
a
b
c
CC50
EC50
EC90
Unit
NC
NC
N
N
>100
>100
>100
lg/mL
O
O
9
BnO
OBn
a
b
c
CC50 = 50% cytostatic/cytotoxic concentration (concentration at which 50% adverse effect is observed on the host cell).
EC50 = 50% effective concentration (concentration at which 50% inhibition of virus replication is observed).
EC90 = 90% effective concentration (concentration at which 90% inhibition of virus replication is observed).
ND = not determined.
d
Table 2
Inhibitory effects of compounds 1–2 and 4–9 on the growth of malignant tumour cell lines in comparison with their effect on normal diploid human fibroblasts (WI38)
a
Compd
Tumour cell growth IC50
(l
M)
SW620
L1210/0
>100
CEM/0
>100
HeLa
>100
MCF-7
HepG2
MiaPaCa-2
>100
WI38
>100
O
N
OH
H3CO
N
N
O
>100
>100
>100
O
BnO
OBn
1
O
N
H3CO
N
N
103 0.3
44 0.4
97 0.4
54 0.4
70 0.7
>100
80 0.5
>100
O
O
BnO
OBn
2
O
N
H2N
H
N
N
N
P500
445 77
>100
61 0.2
66 0.6
>100
>100
>100
O
HO
BnO
OBn
4
O
N
H3CO
N
N
370 183 >500
>100
>100
>100
>100
>100
>100
O
O
5
HO
OH
O
N
H3CO
H3CO
N
32 11
10
4
34 0.9
52 0.8
55 1.3
49 0.9
33 1.1
>100
O
O
O
BnO
OBn
6

K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
3681
Table 2 (continued)
a
Compd
Tumour cell growth IC50
(l
M)
SW620
L1210/0
CEM/0
HeLa
MCF-7
HepG2
MiaPaCa-2
47 0.6
WI38
O
N
H
N
H3CO
H3CO
N
46
6
35
1
81 0.4
>100
>100
>100
>100
52 0.4
O
HO
O
BnO
OBn
7
O
N
H
N
H2N
H2N
N
198
1
154
9
>100
>100
>100
>100
98 0.3
O
HO
BnO
O
OBn
8
NC
N
N
NC
21
6
7.3 0.1
59 0.6
63 0.6
92 0.6
73 0.7
36 1.0
73 0.5
O
O
OBn
BnO
9
Doxorubicin
1.3 0.1
22
0.39 0.21 0.06 0.01 0.06 0.01
63 14 14.93 1.0 66.91 1.16 56.2 1.39
0.05 0.002 0.04 0.005 0.03 0.004 0.04 0.006
6.68 0.12 11.67 0.53 24.66 3.37
Ribavirin (for L1210 and CEM) 5-Fluoruracil (for
5
other cell lines)
a
IC50; 50% inhibitory concentration, or compound concentration required to inhibit tumour cell proliferation by 50%.
favour of compound 6 as a specific IMPDH inhibitor in human leu-
kaemia cells, the precise binding mode of this compound to IMPDH
remains speculative until additional crystallographic studies will
be performed.
covalently modify the active cysteine site on IMPDH as another
IMPDH inhibitor, EICAR does in the IMP binding pocket.
4. Experimental section
3. Conclusion
4.1. Materials and general methods
We have synthesized new 1H-1,2,4-triazole and imidazole
-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7
All commercially available chemicals were purchased from
Sigma Aldrich (Germany) and used without purification. All sol-
vents were analytical grade purity and dried. Acetonitrile (CH3CN)
was refluxed over calcium hydride (CaH2), distilled and stored over
3 Å molecular sieves. Methanol (CH3OH) was stored over 3 Å
molecular sieves. Dioxane was dried with sodium and stored over
4 Å molecular sieves. Dichloromethane (CH2Cl2) was refluxed over
phosphorus pentoxide (P2O5), distilled and stored over 4 Å molec-
ular sieves.
Merck silica gel 60 F254 plates were used for thin-layer chroma-
tography. Column chromatography was performed with Merck sil-
ica gel (0.063–0.200 mm), with petroleum ether/ethylacetate (1:1)
and dichloromethane/methanol (70:1, 50:1, 20:1) as eluents.
Melting points were determined on a Kofler micro hot-stage
instrument (Reichter, Wien) and were uncorrected. 1H and 13C
L
and 8) derivatives and assed their antiviral and anticancer proper-
ties. Among those, compounds 9 and 6 were the most potent anti-
tumor agents with high selectivity towards human T-cell acute
lymphoblastic leukaemia cells (CEM). However, compound 6 is
preferred over compound 9 due to its non-toxic effects on human
diploid fibroblasts. Its growth inhibitory activity might be partially
ascribed to inhibition of IMPDH activity, a key enzyme in providing
an adequate pool of purine nucleotides for cell proliferation, cell
signalling, and as an energy source. Consequently, inhibition of
IMPDH causes a variety of biological responses, and this enzyme
has emerged as a major target for antiviral, antileukemic and
immunosuppressive therapies. One of the most potent inhibitors
of tumour cell proliferation, namely compound 6, ranked also
among the most potent inhibitors of the hepatitis C virus replica-
tion, and was comparable in its cytostatic potency to ribavirin, a
standard agent in treating chronic hepatitis C virus infection. How-
ever, observed antiviral effect is most likely associated with the ef-
fect the compound exerts on the host cell rather than with
selective effect on the replication of the virus itself. In conclusion,
several interesting properties including highly specific growth
inhibitory activity towards tumour cells, in particular towards hae-
matological malignancies and low cytotoxicity make compound 6
attractive drug candidate to be further investigated from therapeu-
tic viewpoint. Next challenge would be to synthesize 5-ethynylim-
NMR spectra were recorded on a Varian Gemini 300 spectrometer
-
´
(Institute Ruder Boškovic, Zagreb) and Varian NMR System 600 and
Varian Unity Inova 300 and Agilent Technologies DD2 300 MHz
NMR spectrometers (National Institute of Chemistry, Ljubljana,
Slovenia) Samples were measured in CDCl3 and DMSO-d6 solutions
at 25 °C in 5 mm NMR tubes. Chemical shifts (d) in ppm were re-
ferred to TMS. High performance LC was performed on Agilent
1100 series system with UV detection (photodiode array detector)
using Zorbax C18 reverse-phase analytical column (2.1 ꢁ 30 mm;
3.5 lm). All compound used for biological evaluation showed
>95% purity in this HPLC system. The electron impact mass spectra
idazole derivatives of L-ascorbic acid to reveal whether they can
and the purity of compounds were assessed by using Agilent

3682
K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
Figure 4. IMP dehydrogenase activity measurements in L1210 and CEM cell cultures in the presence of ribavirin and the test compounds.
Technologies 6410 Triple Quad LC/MS instrument equipped with
electrospray interface and triple quadrupole analyzer (LC–MS/MS).
a silica gel column chromatography using dichloromethane/meth-
anol = 70:1, gave 1 as a white oil (22.5 mg; 3.95%); MS m/z 450.5
[M+].
4.2. Synthesis
1H NMR (DMSO-d6): d 3.88 (OCH3, 3H, s), 4.08 (H50, 1H, m), 4.36
(H60a, 2 ꢁ 1H, 2 ꢁ d, J = 13.8, 10.0), 5.01 (H40, 1H, d, J = 1.0), 4.59
(H60b, 2 ꢁ 1H, 2 ꢁ d, J = 13.8, 2.6), 4.95/4.97 (20-OCH2, 2 ꢁ 1H,
2 ꢁ d, J = 11.2/11.2), 5.18/5.27 (30-OCH2, 2 ꢁ 1H, 2 ꢁ d, J = 11.7/
11.7), 5.62 (50-OH, 1H, d, J = 6.9), 7.29–7.41 (Ph, 10H, m),
8.74 ppm (H5, 1H, s).
4.2.1. 1-[2-(3,4-Bis-benzyloxy-5-oxo-2,5-dihydro-furan-2-yl)-2-
hydroxy-ethyl]-1H-1,2,4-triazole-3-carboxylic acid methyl ester
(1)
1H-1,2,4-triazol-3-carboxylate (643.25 mg; 5.06 mmol), (NH4)2-
SO4 (38.64 mg; 0.34 mmol) and hexamethyldisilazane (HMDS;
18.20 mL) were heated under reflux in argon atmosphere
overnight. Excess HMDS was evaporated under reduced pressure.
Silylated 1H-1,2,4-triazol-3-carboxylate, 4-(5,6-epoxy)-2,3-di-
13C NMR (DMSO): d 49.15 (C60), 53.10 (OCH3), 67.27 (C50), 73.27
(20-OCH2), 74.11 (30-OCH2), 76.22 (C40), 121.46 (C20), 128.26–
129.15 (C6H5), 136.06 (C6H5), 136.60 (C6H5), 141.36 (C30), 144.83
(C3), 147.35 (C5), 157.70 (3-CO), 169.46 ppm (C-10).
Elemental Anal. Calcd for C24H23N3O7: C 61.93, H 4.98, N 9.03,
Found: C 62.05, H 4.99, N 9.05.
O,O-benzyl-L-ascorbic acid (427.67 mg; 1.26 mmol) and TMSOTf
(0.64 mL; 4 ꢁ 0.16 mL) were heated at 55 °C in anhydrous acetoni-
trile (6.40 mL) under argon atmosphere. The progress of the reac-
tion was monitored by TLC [CH2Cl2/MeOH = 50:1] and after
stirring overnight, reaction mixture was concentrated in vacuum
and the resulting residue was extracted with dichloromethane
and saturated solution of NaHCO3. Organic layer was dried over
MgSO4 and concentrated under reduced pressure. Purification on
4.2.2. 1-[2-(3,4-Bis-benzyloxy-5-oxo-5H-furan-2-ylidene)-
ethyl]-1H-1,2,4-triazole-3-carboxylic acid methyl ester (2)
Mixture of 1H-1,2,4-triazol-3-carboxylate (2.00 g; 15.74 mol),
(NH4)2SO4 (246 mg; 15.74 mol) and HMDS (50 mL) was heated
under reflux for 3 h under argon atmosphere. Excess HMDS was

K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
3683
evaporated under reduced pressure and the resulting yellow oil of
silylated 1H-1,2,4-triazol-3-carboxylate, 2,3-di-O,O-benzyl-5,6-di-
O,O-acetyl-L-ascorbic acid (4.6 g; 10.44 mmol) and TMSOTf
2 ꢁ 1H, 2 ꢁ d, J = 11.9/11.9 Hz), 6.18 (OH, 1H, s), 7.3–7.4 (Ph, 10H,
m), 7.84/8.22 (3-CONH2, 2 ꢁ 1H, 2 ꢁ s), 8.18 (NH, 1H, s),
8.56 ppm (H5, 1H, s).
(4.00 mL; 2 ꢁ 2.00 mL) was heated at 55 °C in dry acetonitril
(30 mL) under argon atmosphere overnight. Progress of the
reaction was monitored by TLC [petroleum/ethylacetate = 1:1].
Reaction mixture was concentrated by evaporation and the crude
product thus obtained extracted with dichloromethane and satu-
rated NaHCO3 solution. Organic layer was dried over MgSO4 and
concentrated in vacuo. Crude product was purified by silica gel col-
umn chromatography using petroleum ether/ethylacetate = 1:1 as
eluent to give 2 as a mixture of (Z)- and (E)-isomers (2:1), as a
dark-yellow oil (625.3 mg; 13.4%); MS m/z 447.8 [M+].
13C NMR (DMSO-d6): d 37.52 (C50), 41.10 (C60), 71.99 (30-OCH2),
73.29 (20-OCH2), 81.71 (C40), 123.09 (C20), 127.7–128.5 (C6H5),
136.33 (C6H5), 136.73 (C6H5), 146.11 (C3), 146.83 (C5), 153.30
(C30), 159.01 (3-CO), 167.86 ppm (C10).
Elemental Anal. Calcd For C23H23N5O5: C 61.46, H 5.16, N 15.58,
Found: C 61.37, H 5.17, N 15.62.
4.2.5. 1-[2-(3,4-Dihydroxy-5-oxo-5H-furan-2-ylidene)-ethyl]-
1H-1,2,4-triazole-3-carboxylic acid methyl ester (5)
To a solution of compound 2 (512.8 mg; 1.146 mmol) in
anhydrous dichloromethane (30 mL) under the argon atmo-
sphere, BCl3 (3.2 mL; 2 ꢁ 1.6 mL) was added at ꢀ70 °C. The reac-
tion mixture was stirred for 30 minutes. The temperature was
then raised gradually to room temperature and reaction mixture
stirred further one hour. Reaction was quenched by adding
10 mL of dichloromethane/methanol (1:1). The crude product
was purified by silica gel column chromatography (CH2Cl2/
MeOH = 20:1), to give 5 in a mixture of (Z)- and (E)-isomers
(10:1) as a yellow powder (77.3 mg; 25.2%; mp = 145–148 °C);
MS m/z 265.9 [Mꢀ1].
1H NMR (CDCl3): (Z)-2: d 4.00 (OCH3, 3H, s), 5.18 (H60, 2H, d,
J = 7.6 Hz), 5.21 (20-OCH2, 2H, s), 5.23 (30-OCH2, 2H, s), 5.49 (H50,
1H, t, J = 7.6 Hz), 7.2–7.4 (Ph, 10H+10H (E)-2, m), 8.31 (H5, 1H, s).
(E)-2: d 3.97 (OCH3, 3H, s), 5.21 (H60, 2H, d, J = 8.4 Hz), 5.30 (20-
OCH2 and 30-OCH2, 4H, s), 5.76 (H50, 1H, t, J = 8.4 Hz), 7.2–7.4 (Ph,
10H+10H (Z)-2, m), 7.87 (H5, 1H, s).
13C NMR (CDCl3): (Z)-2: d 40.96 (C60), 53.24 (OCH3), 73.69 (30-
OCH2), 74.25 (20-OCH2), 100.31 (C50), 123.99 (C20), 127.8–129.8
(C6H5), 135.21 (C6H5), 135.61 (C6H5), 144.29 (C3), 145.40 (C40),
146.37 (C5), 147.62 (C30), 158.45 (3-CO), 163.59 (C10). (E)-2: d
41.27 (C60), 53.18 (OCH3), 74.23/74.67 (20-OCH2/30-OCH2), 105.46
(C50), 125.90 (C20), 127.8–129.8 (C6H5), 134.92 (C6H5), 135.54
(C6H5), 144.21 (C3), 145.11 (C40), 145.85 (C5), 147.83 (C30),
158.35 (3-CO), 163.54 (C10).
1H NMR (DMSO-d6): d 3.91 (OCH3, 3H, s), 5.15 (H60, 2H, d,
J = 14.2 Hz), 5.49 (H50, 1H, t, J = 14.2 Hz), 8.83 (H5, 1H, s), 10.18
(20-OH), 10.96 ppm (30-OH).
13C NMR (DMSO-d6): d 41.50 (C60), 53.12 (OCH3), 100.08 (C50),
122.21 (C20), 143.49 (C40), 144.62 (C3), 145.27 (C30), 146.98 (C5),
158.36 (3-CO), 165.11 ppm (C10).
Elemental Anal. Calcd For C24H21N3O6: C 64.42, H 4.73, N 9.39,
Found: C 64.27, H 4.74, N 9.37.
Elemental Anal. Calcd For C10H9N3O6: C 44.95, H 3.40, N 15.73,
Found: C 45.04, H 3.41, N 15.77.
4.2.3. 1-[2-(3,4-Bis-benzyloxy-2-methoxy-5-oxo-2,5-dihydro-
furan-2-yl)-ethyl]-1H-[1,2,4]triazole-3-carboxylic acid amide
(3)
4.2.6. 1-[2-(3,4-Bis-benzyloxy-5-oxo-5H-furan-2-ylidene)-
ethyl]-1H-imidazole-4,5-dicarboxylic acid dimethyl ester (6)
4,5-Dimethyl-imidazole carboxylate (DMI) (1 g; 5.43 mmol) and
Ammonia was introduced in a stirred solution of compound 2
(145 mg; 0.324 mmol) in anhydrous methanol (10 mL) at 0 °C.
After saturation the reaction mixture was stirred overnight at room
temperature. Methanol and ammonia were removed in vacuum.
Purification of crude product by silica gel column chromatography
using dichloromethane/methanol = 50:1 gave desired compound 3
as a yellow oil (46.6 mg; 31.1%); MS m/z 465.2 [M+1].
1H NMR (CDCl3): d 2.18/2.42 (H50, 2 ꢁ 1H, 2 ꢁ m), 2.96 (40-
OCH3, 3H, s), 4.48/4.50 (H60, 2 ꢁ 1H, 2 ꢁ m), 5.14/5.27 (20-OCH2,
2 ꢁ 1H, 2 ꢁ d, J = 11.4/11.4 Hz), 5.20 (30-OCH2, 2H, s), 7.2–7.4 (Ph,
10H, m), 7.83/8.21 (3-CONH2) 8.10 ppm (H5, 1H, s).
2,3-di-O-benzyl-5,6-di-O-acetyl-L-ascorbic acid (2.17 g; 4.98 mmol)
were dissolved with stirring in dry CH3CN at room temperature.
To a reaction mixture 1,8-diazobicyclo[5.4.0]undec-7-en (DBU)
(2.23 mL; 14.94 mmol) was added. The temperature was lowered
to 0 °C and TMSOTf (4 ꢁ 0.9 mL) was added in portions. Reaction
mixture was then stirred for 24 h at 60 °C. The solvent was removed
under the reduced pressure and the crude extracted with CH2Cl2
(30 mL) and saturated aq NaHCO3. Organic layer was dried over
MgSO4, evaporated and the residue submitted to silica gel column
chromatography (CH2Cl2/CH3OH = 70:1) to yield 6 in a mixture of
(Z)- and (E)-isomers (2:1) as yellow oil (705 mg; 28.06%); HRMS
m/z 505.1605 [M+1].
13C NMR (CDCl3): d 37.44 (C50), 41.29 (C60), 50.69 (40-OCH3),
73.82 (30-OCH2), 73.93 (20-OCH2), 102.24 (C40), 121.92 (C20),
128.1–129.6 (C6H5), 135.24 (C6H5), 135.74 (C6H5), 145.77 (C3),
147.26 (C5), 154.00 (C30), 158.84 (3-CO), 166.26 ppm (C10).
Elemental Anal. Calcd For C24H24N4O6: C 62.02, H 5.21, N 12.06,
Found: C 62.19, H 5.21, N 12.09.
1H NMR (DMSO-d6): (Z)-6: d 3.77 (OCH3, 2 ꢁ 3H, s), 5.03 (H60,
2H, d, J = 7.2 Hz), 5.15 (20-OCH2, 2H, s), 5.31 (30-OCH2, 2H, s), 5.54
(H50, 1H, t, J = 7.2 Hz), 7.3–7.5 (Ph, 10H+10H (E)-6, m), 7.99 ppm
(H2, 1H, s). (E)-6: d 3.72 (OCH3, 3H, s), 3.77 (OCH3, 3H, s), 5.14
(H60, 2H, d, J = 7.6 Hz), 5.19 (20-OCH2, 2H, s), 5.41 (30-OCH2, 2H,
s), 5.84 (H50, 1H, t, J = 7.6 Hz), 7.3–7.5 (Ph, 10H+10H (Z)-6, m),
7.84 ppm (H5, 1H, s).
4.2.4. 1-[2-(3,4-Bis-benzyloxy-2-hydroxy-5-oxo-2,5-dihydro-1H-
pyrrol-2-yl)-ethyl]-1H-1,2,4-triazole-3-carboxylic acid amide
(4)
Compound 2 (512.5 mg; 1.145 mmol) was dissolved in dry
dioxane (25 mL) at 0 °C and ammonia was introduced until satura-
tion. Reaction mixture was then stirred at room temperature over-
night. Anhydrous methanol (25 mL) was added to a stirred solution
at 0 °C and ammonia again introduced until saturation. After stir-
ring overnight at room temperature, solvents and ammonia were
removed under reduced pressure. The resulting residue was puri-
fied by silica gel column chromatography (CH2Cl2/MeOH = 50:1)
to give 4 as a white crystals (193.1 mg; 37.4%, mp = 156–158 °C);
MS m/z 449.9 [M+].
13C NMR (DMSO-d6): (Z)-6: d 41.21 (C60), 51.90 (OCH3), 52.50
(OCH3), 73.00 (30-OCH2), 73.93 (20-OCH2), 103.01 (C50), 123.16
(C20), 124.82 (C5), 127.8–128.8 (C6H5), 135.35 (C6H5), 135.66
(C4), 140.82 (C2), 142.74 (C40), 147.91 (C30), 159.84 (COOCH3),
162.73 (COOCH3), 163.51 ppm (C10). (E)-6: d 41.53 (C60), 51.93
(OCH3), 52.44 (OCH3), 73.51 (30-OCH2), 73.88 (20-OCH2), 108.33
(C50), 127.8–128.8 (C6H5), 135.35 (C6H5), 140.31 (C2), 142.45
(C40), 148.62 (C30), 159.75 (COOCH3), 162.76 (COOCH3),
163.53 ppm (C10).
1H NMR (DMSO-d6): d 2.13 (H50, 2H, m), 4.33 (H60, 2H, m), 4.99/
5.01 (20-OCH2, 2 ꢁ 1H, 2 ꢁ d, J = 11.2/11.2 Hz), 5.05/5.14 (30-OCH2,
Elemental Anal. Calcd For C27H24N2O8: C 64.28, H 4.80, N 5.55,
Found: C 64.28, H 4.81, N 5.56.

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K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
4.2.7. 1-[2-(3,4-Bis-benzyloxy-2-hydroxy-5-oxo-2,5-dihydro-1H-
pyrrol-2-yl)-ethyl]-1H-imidazole-4,5-dicarboxylic acid dimethyl
ester (7)
13C NMR (DMSO-d6): (Z)-9: d 42.22 (C60), 73.08 (30-OCH2), 74.00
(20-OCH2), 100.85 (C50), 108.29 (CN), 112.41 (C5), 121.37 (C4),
123.48 (C20), 127.8–128.8 (C6H5), 135.29 (C6H5), 135.58 (C6H5),
143.51 (C2), 143.87 (C40), 147.73 (C30), 163.33 ppm (C10). (E)-9: d
42.46 (C60), 73.50 (30-OCH2), 73.97 (20-OCH2), 105.97 (C50),
108.33 (CN), 112.36 (C5), 121.46 (C4), 125.08 (C20), 127.8–128.8
(C6H5), 143.13 (C2), 143.73 (C40), 148.41 (C30), 163.37 ppm (C10).
Elemental Anal. Calcd For C25H18N4O4: C 68.49, H 4.14, N 12.78,
Found: C 68.33, H 4.15, N 12.80.
To a solution of 6 (334 mg; 0.66 mmol) in dioxane (7.5 mL) and
methanol (7.5 mL) ammonia (g) was introduced at 0 °C until satu-
ration. The reaction mixture was stirred at room temperature for
24 h, then concentrated under reduced pressure and the crude
product purified by silica gel column chromatography (CH2Cl2/
CH3OH = 20:1) to give 7 as a yellow oil (150 mg; 43.58%); MS m/z
505.1 [MꢀNH3]+.
1H NMR (DMSO-d6): d 2.08 (H50, 2H, m), 3.77 (C4-COCH3, 3H, s),
3.78 (C5-COCH3, 3H, s), 4.13 (H60, 2H, m), 4.99/5.05 (20-OCH2,
2 ꢁ 1H, 2 ꢁ d, J = 11.1/11.1 Hz), 5.07/5.15 (30-OCH2, 2 ꢁ 1H, 2 ꢁ d,
J = 11.7/11.7 Hz), 6.25 (OH, 1H, s), 7.3–7.4 (Ph, 10H, m), 7.85 (H2,
1H, s), 8.21 ppm (NH, 1H, s).
4.3. Biological methods
4.3.1. Cell culturing
The suspension cell lines L1210 (murine lymphocytic leukaemia
cells), CEM (human T-lymphocyte leukaemia cell line) were cul-
tured in RPMI-1640 medium supplemented with 10% foetal bovine
13C NMR (DMSO-d6): d 37.72 (C50), 41.96 (C60), 51.88 (C4-
COCH3), 52.44 (C5-COCH3), 72.05 (30-OCH2), 73.24 (20-OCH2),
81.59 (C40), 123.10 (C20), 124.16 (C5), 127.6–128.6 (C6H5), 136.15
(C4), 136.31 (C6H5), 136.70 (C6H5), 140.73 (C2), 153.14 (C30),
159.93 (5-CO), 162.89 (4-CO), 167.86 ppm (C10).
serum, 2 mM L-glutamine and 0.075% NaHCO3 in a humidified
atmosphere with 5% CO2 at 37 °C. Cell lines HeLa (cervical
carcinoma), SW620 (colorectal adenocarcinoma, metastatic), Mia-
PaCa-2 (pancreatic carcinoma), MCF-7 (breast epithelial adenocar-
cinoma, metastatic), HepG2 (hepatocellular carcinoma) and WI 38
(normal diploid human fibroblasts) were cultured as monolayers
and maintained in Dulbecco0s modified Eagle medium (DMEM)
Elemental Anal. Calcd For C27H27N3O8: C 62.18, H 5.22, N 8.06,
Found: C 62.29, H 5.24, N 8.08.
4.2.8. 1-[2-(3,4-Bis-benzyloxy-2-hydroxy-5-oxo-2,5-dihydro-1H-
pyrrol-2-yl)-ethyl]-1H-imidazole-4,5-dicarboxylic acid diamide
(8)
supplemented with 10% foetal bovine serum (FBS), 2 mM
L-gluta-
mine, 100 U/mL penicillin and 100 g/mL streptomycin in
l
a
humidified atmosphere with 5% CO2 at 37 °C.
To a solution of 6 (284 mg; 0.56 mmol) in dioxane (6 mL) and
methanol (6 mL) ammonia (g) was introduced at 0 °C until satura-
tion. The reaction mixture was stirred at room temperature for
96 h, then concentrated under reduced pressure and the crude
product purified by silica gel column chromatography (CH2Cl2/
CH3OH = 20:1) to yield 8 as a yellow oil (81 mg; 29.07%); MS m/z
475.1 [MꢀNH3]+.
4.3.2. Proliferation assays
The panel cell lines were inoculated onto a series of standard 96-
well microtiter plates on day 0, at 3000 cells to 6000 cells per well
according to the doubling times of specific cell line. Test agents
were then added in five, 10-fold dilutions (1 ꢁ 10ꢀ8 to 1 ꢁ 10ꢀ4 M)
and incubated for further 72 h. Working dilutions were freshly
prepared on the day of testing in the growth medium. The solvent
(DMSO) was also tested for eventual inhibitory activity by adjusting
its concentration to be the same as in the working concen-
trations (DMSO concentration never exceeded 0.1%). After 72 h of
incubation, the cell growth rate was evaluated by performing the
MTT assay: experimentally determined absorbance values were
transformed into a cell percentage growth (PG) using the formulas
proposed by NIH and described previously.16 This method directly
relies on control cells behaving normally at the day of assay because
it compares the growth of treated cells with the growth of untreated
cells in control wells on the same plate—the results are therefore a
percentile difference from the calculated expected value.
1H NMR (DMSO-d6): d 2.10 (H50, 2H, m), 4.39 (H60, 2H, t,
J = 7.0 Hz), 4.99/5.03 (20-OCH2, 2 ꢁ 1H, 2 ꢁ d, J = 11.1/11.1 Hz),
5.06/5.14 (30-OCH2, 2 ꢁ 1H, 2 ꢁ d, J = 11.9/11.9 Hz), 6.13 (OH, 1H,
s), 7.3–7.4 (Ph, 10H, m), 7.59/10.67 (5-CONH2, 2 ꢁ 1H, 2 ꢁ d,
J = 2.1/2.1 Hz), 7.75 (H2, 1H, s), 7.78/7.98 (4-CONH2, 2 ꢁ 1H,
2 ꢁ d, J = 2.0/2.0 Hz), 8.15 ppm (NH, 1H, s).
13C NMR (DMSO-d6): d 38.06 (C50), 42.90 (C60), 72.00 (30-OCH2),
73.30 (20-OCH2), 81.85 (C40), 123.05 (C20), 126.30 (C5), 127.7–128.5
(C6H5), 135.08 (C4), 136.33 (C6H5), 136.75 (C6H5), 139.95 (C2),
153.47 (C30), 160.44/165.72 (5-CO/4-CO), 167.94 ppm (C10).
Elemental Anal. Calcd For C25H25N5O6: C 61.09, H 5.13, N 14.25,
Found: C 61.25, H 5.14, N 14.25.
The IC50 and LC50 values for each compound were calculated
from dose-response curves using linear regression analysis by fit-
ting the mean test concentrations that give PG values above and
below the reference value. If, however, all of the tested concentra-
tions produce PGs exceeding the respective reference level of effect
(e.g., PG value of 50) for a given cell line, the highest tested concen-
tration is assigned as the default value (in the screening data report
that default value is preceded by a ‘>’ sign). Each test point was
performed in quadruplicate in three individual experiments. The
results were statistically analyzed (ANOVA, Tukey post-hoc test
at p <0.05). Finally, the effects of the tested substances were eval-
uated by plotting the mean percentage growth for each cell type in
comparison to control on dose response graphs.
4.2.9. 1-[2-(3,4-Bis-benzyloxy-5-oxo-5H-furan-2-ylidene)-
ethyl]-1H-imidazole-4,5-dicarbonitrile (9)
4,5-Dicyano imidazole (500 mg; 4.23 mmol) and 2,3-di-O-ben-
zyl-5,6-di-O-acetyl-L-ascorbic acid (2.17 g; 4.98 mmol) were dis-
solved with stirring in dry CH3CN at room temperature. To a
reaction mixture DBU (1.94 mL; 12.69 mmol) was added. The tem-
perature was lowered to 0 °C and TMSOTf (4 ꢁ 0.76 mL; 16.92
mmol) was added in portions. Reaction mixture was stirred for
24 h at 60 °C. The solvent was removed under reduced pressure
and the crude product extracted with CH2Cl2 (30 mL) and sat. aq
NaHCO3. Organic layer was dried over MgSO4 and the solvent evap-
orated. The residue was submitted to silica gel column chromatog-
raphy (CH2Cl2/CH3OH=80:1) to afford 9 in a mixture of (Z)- and (E)-
isomers (4:1) as yellow oil (400 mg; 21.57%); MS m/z 439.1 [M+1].
1H NMR (DMSO-d6): (Z)-9: d 5.09 (H60, 2H, d, J = 7.3 Hz), 5.16
(20-OCH2, 2H, s), 5.31 (30-OCH2, 2H, s), 5.66 (H50, 1H, t, J = 7.3 Hz),
7.3–7.4 (Ph, 10H+10H (E)-9, m), 8.34 ppm (H2, 1H, s). (E)-9: d
5.16 (20-OCH2, 2H, s), 5.18 (H60, 2H, d, J = 7.8 Hz), 5.38 (30-OCH2,
2H, s), 5.95 (H50, 1H, t, J = 7.8 Hz), 7.3–7.4 (Ph, 10H+10H (Z)-9,
m), 8.27 ppm (H5, 1H, s).
4.3.3. Cytostatic assay
To each well of a 96-well microtiter plate were added 5–
7.5 ꢁ 104 cells and a given amount of the test compound. The cells
were allowed to proliferate for 48 h (L1210) or 72 h (CEM) at 37 °C
in a humidified CO2-controlled atmosphere. At the end of the incu-
bation period, the cells were counted in a Coulter Counter (Coulter
Electronics Ltd, Harpenden Herts, United Kingdom). The IC50 (50%

K. Wittine et al. / Bioorg. Med. Chem. 20 (2012) 3675–3685
3685
inhibitory concentration) was defined as the concentration of com-
pound that reduced the number of viable cells by 50%.
The EC50 and EC90 (values calculated from the dose-response
curve) represent the concentrations at which 50% and 90% inhibi-
tion, respectively, of viral replication is achieved. The CC50 (value
calculated from the dose-response curve) represents the concen-
tration at which the metabolic activity of the cells is reduced by
50% as compared to untreated cells.
A concentration of compound is considered to elicit a genuine
antiviral effect in the HCV replicon system when the anti-replicon
effect is well above the 70% threshold at concentrations where no
antimetabolic activity is observed.
4.3.4. Inhibition of IMP dehydrogenase by the test compounds
in intact tumour cells
The activity of IMP dehydrogenase in the intact L1210 and CEM
tumour cells was measured by estimation of 3H release from
[2,8-3H]IMP that had been formed in the cells from [2,8-3H]hypo-
xanthine in the reaction catalyzed by IMP dehydrogenase. Briefly,
300
l
L of a cell suspension were mixed with 60
L (4 Ci) of radiolabelled Hx.
L of the reaction mixture were withdrawn
L of a freshly prepared cold suspension of
lL of RPMI 1640
medium or test compound and 40
At various times, 100
and mixed with 250
l
l
l
l
Acknowledgments
carbon black in 5% trichloroacetic acid. After centrifugation at
Support for this study was provided by the Ministry of Science
of the Republic of Croatia (Projects #125-098-2464-2922, #335-
0982464-2393, #335-0000000-3532). We thank Kristien Minner
and Lizette van Berckelaer for excellent technical assistance. The
research was supported by the K.U. Leuven (GOA No. 10/014).
1100ꢁg for 10 min, 200-lL samples of the supernatants were
analyzed for radioactivity. The amount of free 3H in the reaction
mixture (before incubation with the cells) was less than 10% of
the amount of labelled Hx added.
4.3.5. Antiviral assay for hepatitis C virus
Supplementary data
Huh 5.2 cells containing the hepatitis C virus genotype 1b
I389luc-ubi-neo/NS3-3’/5.1 replicon17 were sub-cultured in DMEM
supplemented with 10% FCS, 1% non-essential amino acids, 1% pen-
icillin/streptomycin and 2% Geneticin at a ratio of 1:3 to 1:4, and
grown for 3–4 days in 75 cm2 tissue culture flasks. One day before
addition of the compound, cells were harvested and seeded in as-
say medium (DMEM, 10% FCS, 1% non-essential amino acids, 1%
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.01.054.
References and notes
1. Chen, Q.; Espey, M. G.; Krishna, M. C.; Mitchell, J. B.; Corpe, C. P.; Buettner, G. R.;
Shacter, E.; Levine, M. PNAS 2005, 102, 13604.
2. Chen, Q.; Espey, M. G.; Sun, A. Y.; Pooput, C.; Kirk, K. L.; Krishna, M. C.; Khosh, D.
B.; Drisko, J.; Levine, M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 11105.
3. Pollard, H. B.; Levine, M. A.; Eidelman, O.; Pollard, M. In Vivo 2010, 24, 249.
4. Rawal, B. D.; Bartolini, F.; Vyas, G. Biologicals 1995, 23, 75.
penicillin/streptomycin) at a density of 6 500 cells/well (100 lL/
well) in 96-well tissue culture microtiter plates for evaluation of
antimetabolic effect and CulturPlate (Perkin Elmer) for evaluation
of antiviral effect. The microtiter plates were incubated overnight
(37 °C, 5% CO2, 95–99% relative humidity), yielding a non-confluent
cell monolayer.
The evaluation of antimetabolic as well as antiviral effect of
each compound was performed in parallel. Four-step, 1-to-5 com-
pound dilution series were prepared. Following assay setup, the
microtiter plates were incubated for 72 h (37 °C, 5% CO2, 95–99%
relative humidity). For the evaluation of antimetabolic effects,
ˇ
´
´
5. Gazivoda, T.; Šokcevic, M.; Kralj, M.; Šuman, L.; Pavelic, K.; De Clercq, E.; Andrei,
´
´
G.; Snoeck, R.; Balzarini, J.; Mintas, M.; Raic-Malic, S. J. Med. Chem. 2007, 50,
4105.
6. Madhusudana, S. N.; Shamsundar, R.; Seetharaman, S. Int. J. Infect. Dis. 2004, 8, 21.
7. Gomez, E. V.; Perez, Y. M.; Sanchez, H. V.; Forment, G. R.; Soler, E. A.; Bertot, L.
C.; Garcia, A. Y.; Vazquez, M. del R. A.; Fabian, L. G. World J. Gastroenterol. 2010,
16, 2638.
8. Manns, M. P.; McHutchison, J. G.; Gordon, S. C.; Rustgi, V. K.; Shiffman, M.;
Reindollar, R.; Goodman, Z. D.; Koury, K.; Ling, M.; Albrecht, J. K. Lancet 2001,
358, 958.
9. Benhamou, Y.; Afdhal, N. H.; Nelson, D. R.; Shiffman, M. L.; Halliman, D. G.;
Heise, J.; Chun, E.; Pockros, P. J. Hepatology 2009, 50, 717.
10. De Clercq, E. Nat. Rev. Drug Disc. 2007, 6, 1001.
11. Singhal, N.; Sharma, P. K.; Dudhe, R.; Kumar, N. J. Chem. Pharm. Res. 2011, 3,
126.
the assay medium was aspirated, replaced with 75 lL of a 5%
MTS solution in phenol red-free medium and incubated for 1.5 h
(37 °C, 5% CO2, 95–99% relative humidity). Absorbance was
measured at a wavelength of 498 nm (Safire2, Tecan), and optical
densities (OD values) were converted to percentage of untreated
controls.
12. Bhatnagar, A.; Sharma, P. K.; Kumar, N. Int. J. PharmTech. Res. 2011, 3, 268.
ˇ ´
´
´
13. Raic´-Malic´, S.; Svedruzic, D.; Gazivoda, T.; Marunovic, A.; Hergold Brundic, A.;
Nagl, A.; Balzarini, J.; DeClercq, E.; Mintas, M. J. Med. Chem. 2000, 43, 4806.
14. (a) Singh, B. K.; Verma, S. S.; Dwivedi, N.; Tripathi, R. P. Tetrahedron Lett. 2006,
47, 2219; (b) Dallacker, F.; Sanders, J. Chem. Ztg. 1985, 109, 277.
For the evaluation of antiviral effects, assay medium was aspi-
rated and the cell monolayers were washed with PBS. The wash
15. Pankiewicz, K. W.; Goldstein, B. M. Inosine monophosphate dehydrogenase
and its inhibitors: an overview. ACS Symposium Series (2003), 839 (Inosine
Monophosphate Dehydrogenase) pp 1–17.
16. Gazivoda, T.; Raic´-Malic´, S.; Krištafor, V.; Makuc, D.; Plavec, J.; Bratulic´, S.;
Kraljevic´-Pavelic´, S.; Pavelic´, K.; Naesens, L.; Andrei, G.; Snoeck, R.; Balzarini, J.;
Mintas, M. Bioorg. Med. Chem. 2008, 16, 5624.
buffer was aspirated, and 25
was added allowing for the lysis to proceed for 5 min at room
temperature. Subsequently, 50 L of Luciferase Assay System (Pro-
lL of Glo Lysis Buffer (Promega)
l
mega) was added, and the luciferase luminescence signal was
quantified immediately (1000 ms integration time/well, Safire2,
Tecan). Relative luminescence units were converted into percent-
age of untreated controls.
17. Vrolijk, J. M.; Kaul, A.; Hansen, B. E.; Lohmann, V.; Haagmans, B. L.; Schalm, S.
W.; Bartenschlager, R. J. Virol. Methods 2003, 110, 201.