A novel synthesis of acyclonucleosides via allylation of 3-[1-(phenylhydrazono)-L-threo-2,3,4-trihydroxybut-1-yl]quinoxalin-2(1H)one

Article abstract of DOI:10.1016/S0008-6215(03)00376-8

The allylation of 3-[1-(phenylhydrazono)-L-threo-2,3,4-trihydroxybut-1-yl] quinoxalin-2(1H)one (1) gave, in addition to the anticipated 1-N-allyl derivative (2), a dehydrative cyclized product, 1-N-allyl-3-[5-(hydroxymethyl)- 1-phenylpyrazol-3-yl]quinoxal

Full text of DOI:10.1016/S0008-6215(03)00376-8

Carbohydrate Research 338 (2003) 2301ꢀ2309
/
www.elsevier.com/locate/carres
A novel synthesis of acyclonucleosides via allylation of 3-[1-
(phenylhydrazono)-
L
-threo-2,3,4-trihydroxybut-1-yl]quinoxalin-
2(1H)one
Hamida Mohamed Abdel Hamid
Department of Chemistry, Faculty of Science, Alexandria University, Alexandria, Egypt
Received 28 February 2003; accepted 20 June 2003
Dedicated to Professor Hassan S. El Khadem on the occasion of his 80th birthday and to Professor El Sayed H. El Ashry on his 60th birthday
Abstract
The allylation of 3-[1-(phenylhydrazono)-L-threo-2,3,4-trihydroxybut-1-yl]quinoxalin-2(1H)one (1) gave, in addition to the
anticipated 1-N-allyl derivative (2), a dehydrative cyclized product, 1-N-allyl-3-[5-(hydroxymethyl)-1-phenylpyrazol-3-yl]quinox-
alin-2-one (4) and its isomeric O-allyl derivative 3. The O-allyl group in 3 underwent acetolysis under acetylation conditions, in
addition to the acetylation of the hydroxyl group, to afford 2-acetoxy-3-[5-(acetoxymethyl)-1-phenylpyrazol-3-yl]quinoxaline (8)
instead of the O-acetyl derivative of 3. Allylation of the tri-O-acetyl derivative of 1 caused the elimination of a molecule of acetic
acid in addition to N-allylation to give 1-N-allyl-3-[3,4-di-O-acetyl-2-deoxy-1-(phenylhydrazono)but-2-en-1-yl]quinoxalin-2-one
(11). Hydroxylation of the allyl group gave a glycerol-1-yl acyclonucleoside which can be alternatively obtained by a displacement
reaction of the tosyloxy group in 2,3-O-isopropylidene-1-O-(p-tolylsulfonyl)glycerol (14), followed by deisopropylidenation. 1-N-
(2,3-Dibromopropyl)-3-[5-(hydroxymethyl)-1-(4-bromophenyl)pyrazol-3-yl]quinoxalin-2-one (15) underwent azidolysis to give a
2,3-diazido derivative. The assigned structures were based on spectral analysis. The activity of compounds 2, 4, 6, and 15 against
hepatitis B virus was studied.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Allyl bromide; NaH/DMF; Pyrazolylquinoxaline; Acyclonucleosides; Allylation
1. Introduction
Consequently, the foregoing aspects have attracted
our attention towards the synthesis of acyclonucleoside
analogues carrying a pyrazolylquinoxaline moiety as a
base, in order to have a combination of quinoxaline and
pyrazole rings with an acyclic side-chain. This investiga-
tion has shown a new transformation from an acyclic C-
Various pharmacological properties such as hypnotic,1
antiinflammatory,2 analgesic,3 tranquilizing, antidepres-
sant,4 fungicidal,5 and anthelmintic6 effects have been
reported for quinoxalin-2-ones. Similarily, pyrazole
derivatives have various biological activities.7ꢀ14 The
biological activities of acyclonucleosides, as a conse-
quence of the potent activity reported for some of their
members after the discovery of acyclovir, has led to
synthesis of a diversity of structures of either the lead
compounds or their analogues, with many variations in
order to enhance biological activity and selectivity, or to
lower their toxicity.15ꢀ29
nucleoside of the seco type, 3-[1-(phenylhydrazono)-L-
threo-2,3,4-trihydroxybut-1-yl]quinoxalin-2(1H)one, to
an acyclic N-nucleosides of the tetra seco type, a
classification recently used for acyclonucleosides.20ꢀ22
2. Results and discussion
The starting material 3-[1-(phenylhydrazono)-L-threo-
2,3,4-trihydroxybut-1-yl]quinoxalin-2-(1H)one (1) was
prepared as reported in the literature.25 Attempted
allylation of 1 with allyl bromide in the presence of
E-mail address: habdelhamid3@yahoo.com (H.M.A.
Hamid).
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00376-8

2302
H.M.A. Hamid / Carbohydrate Research 338 (2003) 2301ꢀ2309
/
potassium carbonate in N,N-dimethylformamide gave a
mixture of three products (2ꢀ4). Structural elucidation
of these products indicated that compound 2 is the
expected N-allylated derivative, isolated as an orange
crystalline product, 1-N-allyl-3-[1-(phenylhydrazono)-L-
threo-2,3,4-trihydroxybut-1-yl]quinoxalin-2-one (2).
Elemental analysis of 2 agreed with the molecular
formula C21H22N4O4. Its IR spectrum showed bands
at 1648 and 3403 cmꢁ1 for the OCN and OH groups,
respectively. The 1H NMR spectrum showed two
doublets of doublets at d 3.53 and 3.90 due to H-4,4?,
a doublet at d 4.15 due to H-2, and a multiplet at d 4.97
due to H-3 of the side-chain, confirming the presence of
the trihydroxybutyl group. The presence of one allyl
group was indicated by the presence of a doublet (d
4.78) and two multiplets (d 5.30 and 6.47). Moreover,
the phenylhydrazone residue remained unaffected, as
the spectrum showed the presence of a singlet at d 9.77
due to NH of the hydrazone moiety.
presence of an CH2OH group. The elemental analysis
agreed with the molecular formula C21H18N4O2, which
upon combination with the spectral data, led to the
conclusion that the allyl group had been introduced on
the oxygen atom of the quinoxalinone ring, and the
trihydroxybutyl hydrazone residue had undergone de-
hydrative cyclization by losing two molecules of water.
The presence of the allyl group on oxygen rather than on
the nitrogen of the quinoxalinone can be explained as
being due to rearrangement of the N-allyl group via a
Claisen rearrangement to give the O-allyl derivative.
/
Attempted acetylation of compound 3 by Ac2Oꢀ
/
pyridine did not afford the expected product 7. Its IR
spectrum showed no absorption for an amide group and
1
showed a band at 1740 cmꢁ1. Its H NMR spectrum
showed a methylene group, which appeared downfield
(d 5.23) as a singlet. However, no signals were present
corresponding to the allyl group, but an extra acetyl
group appeared, with two singlets at d 1.57 and 2.12.
The second product was assigned the structure 4
based on the following data. It showed the presence of a
band in its IR spectrum at 1645 cmꢁ1, indicating the
presence of an OCN group. Its 1H NMR spectrum
showed the presence of one hydroxyl group giving a
broad singlet at d 3.00 and a methylene group as a
singlet at d 4.51. This indicated that a dehydrative
cyclization of the glycerol-1-yl moiety and the hydra-
zone residue in the quinoxalinone 2, during the allyla-
tion, had taken place and the allyl group was introduced
on the nitrogen of the quinoxalinone ring, as indicated
from the 1H NMR spectrum which showed as a doublet
at d 4.81 and two multiplets at d 5.18 and 5.63. Also the
elemental analysis agreed with the molecular formula
C21H18N4O2. The structure of 4 was chemically con-
firmed by an unequivocal synthesis of its acetyl deriva-
tive by the allylation of 3-[5-(acetoxymethyl)-1-
Moreover, deacetylation of 8 gave 5 (RꢂH). These data
/
led to assignment of structure 8 for this product. The
presence of the acetoxy group in place of the allyloxy
group would result from an acetolysis process.
Acetylation of compound 1 afforded 9,14 whose
allylation with allyl bromide in presence of K2CO3 in
DMF gave a product whose spectral data ruled out the
expected structure 1-N-allyl-3-[L-threo-2,3,4-triacetoxy-
1-(phenylhydrazono)but-1-yl]quinoxalin-2-one (10) or
even a product resulting from the eliminative cyclization
process, such as 6. Its IR spectrum showed acetoxy and
amide carbonyl absorption bands at 1748, 1733, and
1
1645 cmꢁ1, respectively. These data as well as its H
NMR spectrum established its acyclic nature. The two
signals at d 2.08 and 2.19 were assigned as acetyl groups
and two singlets at d 5.17 (2H) and 6.23 (1H) were
attributed, respectively to methylene protons and a
methine proton, each without coupling. In addition,
the methine proton appeared at a lower field indicating
its linkage to a double bond. Moreover, the expected
signals for the allyl, aromatic, and NH protons were
present. These data indicated that, under the conditions
of allylation, an elimination of one molecule of acetic
acid from the side-chain of the expected product 10 had
taken place, to give 1-N-allyl-3-[3,4-di-O-acetyl-2-
deoxy-1-(phenylhydrazono)but-2-en-1-yl]quinoxalin-2-
one (11).
phenylpyrazol-3-yl]quinoxalin-2(1H)one (5)25 (Rꢂ
Ac)
/
with allyl bromide in presence of K2CO3 in DMF to give
1-N -allyl-3-[5-(acetoxymethyl)-1-phenylpyrazol-3-
yl]quinoxalin-2-one (6), which was found to be identical
with the product of acetylation of 4. This confirmed the
basic skeleton of the product as a pyrazolyl quinoxali-
none derivative. Its IR spectrum showed, in addition to
the band of the OCN group, a band at 1730 cmꢁ1 for
1
the acetyl group. Its H NMR spectrum confirmed the
presence of a CH2OAc group and one allyl group.-
Schemes 1 and 2.
The allyl group is an excellent precursor for various
chemical modifications. Thus, the reaction of 6 with
bromine in CHCl3 gave a product whose combustion
analysis and mass spectrum indicated the presence of
three bromine atoms. Its IR spectrum showed, in
addition to the amide band, a band at 3425 cmꢁ1 due
to hydroxyl group; the acetyl band of 6 was absent. This
indicated that a deacetylation process had taken place
The third product, 3, showed no absorption in the
range 1660 cmꢁ1, indicating absence of the OCN group.
1
Its H NMR spectrum showed a doublet at d 4.42 and
two multiplets at d 5.20 and 5.90 characteristic for the
presence of one allyl group. The spectrum indicated the
absence of the glycerol-1-yl side-chain and showed
instead a doublet at d 5.00 and a triplet at d 5.56;
upon deuteration the former doublet was transformed
into a singlet and the triplet disappeared, indicating the
1
during the bromination. Its H NMR spectrum showed
the presence of the CH2O signal of the starting material

H.M.A. Hamid / Carbohydrate Research 338 (2003) 2301ꢀ
/
2309
2303
Scheme 1.
at d 4.64. The two doublets of doublets, at d 3.48, and
3.61, the multiplet at d 4.17, and the doublet at d 4.35
indicated the addition of bromine to the double bond,
along with bromination of the phenyl group. The
introduction of an azide group in nucleoside analogues
is of potential significance from the biological point of
view. Thus, nucleophilic displacement of the bromine in
compound 15 by sodium azide afforded the diazide 16.
Its IR spectrum showed the azide band at 2103 cmꢁ1
.
Another type of acyclonucleoside of the tetra seco
type, possessing a glycerol-1-yl side-chain was prepared.
Hydroxylation of compound 6 with alkaline KMnO4

2304
H.M.A. Hamid / Carbohydrate Research 338 (2003) 2301ꢀ
/
2309
Scheme 2.
afforded compound 12, which could alternatively be
obtained by treatment of compound 5 (RꢂAc) with
data. The IR spectrum of 13 showed a band at 3411
1
/
(OH) and a band at 1664 cmꢁ1 (OCN). Its H NMR
NaH in dry DMF followed by 2,3-O-isopropylidene-1-
O-(p-tolylsulfonyl)-glycerol (14)30 to give 13, deprotec-
tion of which with 80% acetic acid at reflux temperature
gave 1-(2,3-dihydroxypropyl)-3-[5-(hydroxymethyl)-1-
phenylpyrazol-3-yl]quinoxalin-2-one (12), whose reac-
tion with acetone in sulfuric acid gave back compound
13. The structures were confirmed from their spectral
showed two singlets at d 1.36 and 1.42 for the two
methyl groups of the isopropylidene groups, whose Dd
was 0.06, in agreement with a terminal isopropylidene
group according to the shift rule of El Ashry.31,32
Viral screening against HBV of selected compounds
indicated that compound 15 was the most effective one
in this series against HBV, with a selective index ꢀ
/

H.M.A. Hamid / Carbohydrate Research 338 (2003) 2301ꢀ
/
2309
2305
476.2. This compound showed high inhibition with high
cytotoxicity, while compounds 2 and 6 showed moderate
inhibition of viral replication and mild cytotoxicity, with
bromide (1.2 mL, 10 mmol) was added, and the mixture
was stirred for 4 h at room temperature (rt). The mixture
was diluted with water and the product collected by
filtration, washed with water and EtOH. TLC showed
the presence of three products. The crude product was
purified by fractional crystallization from absolute
EtOH to give, the following 3 products.
selective index as ꢀ
/
154 and ꢀ303, respectively. The
/
effective concentration of compound 4 was 10.0 mM,
which showed low inhibition and low cytotoxicity with
the selective index ꢀ91 (Tables 1 and 2)
/
3.3. 1-N-Allyl-3-[1-(phenylhydrazono)-L-threo-2,3,4-
trihydroxybut-1-yl]quinoxalin-2-one (2)
3. Experimental
3.1. General methods
Orange crystals, Rf 0.184, yield 0.7 g (36%), mp 152ꢀ
/
154 8C; IR (KBr) 3403 (OH), 1648 (OCN) and 1600
cmꢁ1 (CÄ
3.90 (2 dd, 2 H, J2,3? 6.0, J2,3 4, J3,3? 12 Hz, H-4,4?); 4.15
(d, 1 H, J 4.5 Hz, H-2); 4.78 (d, 2 H, J 7.5 Hz, NÃCH2);
4.97 (m, 1 H, H-3); 5.30 (m, 2 H, ÄCH2); 6.47 (m, 1 H, Ä
CH); 6.79ꢀ7.63 (m, 9 H, ArÃH) and 9.77 (s, 1 H, D2O
/
N and CÄ/C); H NMR (Me2SO-d6): d 3.53,
1
Melting points were determined with a Mel-Temp
apparatus and are uncorrected. IR spectra were re-
corded for the compounds in a KBr matrix with a
/
/
/
1
Unicam SP 1025 spectrophotometer. H NMR spectra
/
/
were recorded on EM-390 and a Bruker AC 300 MHz
spectrometers using Me4Si as the standard. Chemical
shifts are given on the d scale. Mass spectra were
recorded using electron ionization (EI) on a Jeol JMS
AX-500 spectrometer. Thin layer chromatography
(TLC) was performed on Baker-Flex silica gel IB-F
exchangeable, NH). Anal. Calcd for C21H22N4O4
(394.42): C, 63.95; H, 5.62; N, 14.21. Found: C, 63.72;
H, 5.50; N, 14.0.
3.4. 1-N-Allyl-3-[5-(hydroxymethyl)-1-phenylpyrazol-3-
yl]quinoxalin-2-one (4)
plates using 2:3 EtOAcꢀhexane as developing solvent.
/
The spots were detected by their characteristic color and
by UV absorption. The biological testing was carried
out at the Liver Institute, Menoufia University, Egypt.
Microanalyses were performed at the microanalytical
laboratory at the Faculty of Science, Cairo University.
Pale yellow crystals Rf 0.553, yield 0.8 g (41%), mp 176ꢀ
178 8C; IR (KBr) 3310 (OH); 1642 (OCN) and 1600
cmꢁ1 (CÄ C); 1H NMR (CDCl3): d 3.00 (bs, 1
N and CÄ
H, OH); 4.51 (s, 2 H, CH2OH); 4.81 (d, 2 H, J 7.5 Hz,
NÃCH2); 5.18 (m, 2 H, ÄCH2); 5.63 (m, 1 H, ÄCH);
7.13ꢀ7.59, 7.89 (m, d, 10 H, ArÃHꢃpyrazole proton).
/
/
/
/
/
/
/
/
/
3.2. Reaction of 3-[1-(phenylhydrazono)-L-threo-2,3,4-
trihydroxybut-1-yl]quinoxalin-2(1H)one with allyl
bromide
Anal. Calcd for C21H18N4O2 (358.38): C, 70.38; H, 5.06;
N, 15.63. Found: C, 70.50; H, 4.90; N, 15.50.
3.5. 2-O-Allyl-3-[5-(hydroxymethyl)-1-phenylpyrazol-3-
yl]quinoxaline (3)
A solution of 3-[1-(phenylhydrazono)-L-threo-2,3,4-tri-
hydroxybut-1-yl]quinoxalin-2(1H)one (1) (1.77 g, 5
mmol) in dry DMF (10 mL) was treated with anhyd
K2CO3 (1.38 g, 10 mmol) and the mixture was heated at
80 8C for 2 h. The resulting mixture was cooled, allyl
Pale yellow product, Rf 0.58, yield 0.4 g (20%), mp 105ꢀ
108 8C; IR (KBr) 3414 (OH) and 1623 cmꢁ1 (CÄ
N and
C); H NMR (Me2SO-d6): d 4.42 (d, 2 H, J 9.0 Hz,
CH2); 5.00 (s, 2 H, CH2OH); 5.20 (m, 2 H, ÄCH2);
5.56 (t, 1 H, OH); 5.90 (m, 1 H, ÄCH); 7.13ꢀ7.59, 7.89
(m, d, 10 H, ArÃH, pyrazole proton). Anal. Calcd for
/
/
1
CÄ
/
OÃ
/
/
/
/
/
C21H18N4O2 (358.38): C, 70.38; H, 5.06; N, 15.63.
Found: C, 70.20; H, 5.00; N, 15.70.
Table 1
Cytotoxic effect (CC50), inhibitory concentration (IC50) and
selective index (SI) of selected compounds
3.6. 1-N-Allyl-3-[5-(acetoxymethyl)-1-phenylpyrazol-3-
yl]quinoxalin-2-one (6)
Compound
HBV DNA
IC50 (mM)
Hep G2 2.2.15
CC50 (mM)
SI
3.6.1. Method (a). A cold solution of 1-N-allyl-3-[5-
hydroxymethyl)-1-phenylpyrazol-3-yl]quinoxalin-2-one
(4) (0.3 g, 0.84 mmol) in dry pyridine (5 mL) was treated
with Ac2O (5 mL). The mixture was kept overnight at rt,
diluted with ice-cold water, and the product was filtered
off, washed repeatedly with water, and dried, yield 0.24
g (71%). The crude product was recrystallized from
Lamivudine
B
/
0.1
ꢀ
/
100
100
100
ꢀ
/
1000
153.9
90.9
2
4
0.65
1.10
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
6
15
0.33
0.21
ꢀ
/
100
ꢀ
/
303.0
ꢀ
/
100
ꢀ
/
476.2

2306
H.M.A. Hamid / Carbohydrate Research 338 (2003) 2301ꢀ2309
/
Table 2
Results of inhibition of HBV replication by selected compounds
Compound
Concentration (mM)
HBV DNA in supernatant (pg/mL)
Hep G2 viable cells
Lamivudine
1.0
10.0
0.25
0.18
0.15
1.03
1.01
1.07
100.0
2
1.0
10.0
0.41
0.36
0.27
0.04
0.03
0.03
100.0
4
1.0
10.0
0.16
0.13
0.10
0.02
0.01
0.01
100.0
6
1.0
10.0
0.25
0.24
0.18
0.05
0.04
0.03
100.0
15
1.0
10.0
0.75
0.61
0.40
0.81
0.70
0.61
100.0
A value of 1.0 pg HBV DNA /mL corresponds to approximately 3ꢄ
103 HBV virions/mL.
/
EtOH to give colorless needles; mp 195ꢀ
/
196 8C; IR
3.8. 2-Acetoxy-3-[5-(acetoxymethyl)-1-phenylpyrazol-3-
yl]quinoxaline (8)
(KBr) 1730 (OAc) and 1640 cmꢁ1 (OCN); H NMR
(CDCl3): d 2.03 (s, 3 H, CH3CO); 4.96 (d, 2 H, J 7.5 Hz,
1
NÃ
5.89 (m, 1 H, Ä
HꢃpyrazoleÃH). Anal. Calcd for C23H20N4O3 (400.42):
/
CH2); 5.06 (s, 2 H, CH2OAc); 5.23 (m, 2 H, Ä
/
CH2);
A solution of 3 (0.2 g, 0.56 mmol) in pyridine (5 mL)
was cooled in an ice-bath, then treated with Ac2O (5
mL) with stirring. The mixture was kept at rt overnight,
then poured onto crushed ice. The product that sepa-
rated, was filtered off, washed with water, and dried. It
was crystallized from EtOH as colorless crystals, yield
/
CH); 7.16ꢀ7.57, 7.92 (m, d, 10 H, ArÃ
/
/
/
/
C, 68.99; H, 5.04; N, 13.99. Found: C, 69.00; H, 5.00; N,
13.80.
0.2 g (89%), mp 200ꢀ
(OAc); 1H NMR (CDCl3): d 1.57, 2.12 (2s, 6 H, 2
CH3CO); 5.23 (s, 2 H, CH2OAc); 7.51ꢀ7.89, 8.03, 8.42
and 8.64 (m, s, 2 d, 10 H, ArÃH, pyrazoleÃH). Anal.
/
203 8C; IR (KBr) 1740 cmꢁ1
3.6.2. Method (b). A stirred solution of 3-[5-(acetoxy-
methyl)-1-phenylpyrazol-3-yl]quinoxalin-2-(1H)-one (5)
/
(Rꢂ
Ac)25 (0.40 g; 1.1 mmol) in dry DMF (5 mL) was
/
/
/
treated with anhyd K2CO3 (0.15 g; 1.1 mmol) and allyl
bromide (0.30 mL; 2.5 mmol) at rt for 4 h. The mixture
was diluted with water and the product collected by
Calcd for C22H18N4O4 (402.39): C, 65.66; H, 4.50; N,
13.92. Found: C, 65.45; H, 4.30; N, 14.00.
filtration and crystallized from EtOH as colorless
1
needles, yield 0.3 g (68%), mp 195ꢀ
/
196 8C; IR and H
NMR spectra were superimposable with the spectra of
3.9. 2-Hydroxy-3-[5-(hydroxymethyl)-1-phenylpyrazol-
3-yl]quinoxaline (5)(RꢀH)
/
the product from method (a).
A solution of 8 (0.4, 1.0 mmol) in EtOH (10 mL) was
treated with 1M NaOH solution, (10 mL). The mixture
was heated under reflux for 4 h. The clear solution
obtained was neutralized with AcOH and left to cool, a
colorless product separated out. The product was
filtered off, washed with water, and crystallized from
3.7. 1-N-Allyl-3-[5-(hydroxymethyl)-1-phenylpyrazol-3-
yl]quinoxalin-2-one (4)
A solution of 5 (RꢂAc) (0.36 g, 1.0 mmol) and NaH
/
(0.024 g, 1.0 mmol) in dry DMF (10 mL) was stirred at
rt for 2 h. Then allyl bromide (0.30 mL, 2.5 mmol) was
added and the mixture was stirred for 4 h. The mixture
was diluted with water, the product obtained was
filtered off, washed with water, and dried. It was
crystallized from EtOH as pale yellow crystals, yield
0.30 g (84%). This compound was identical to that
obtained from the reaction of 1 with allyl bromide in
EtOH, mp 251 8C [Lit.25 250ꢀ
251 8C].
/
3.10. 1-N-Allyl-3-[3,4-di-O-acetyl-2-deoxy-1-
(phenylhydrazono)but-2-en-1-yl]quinoxalin-2-one (11)
A solution of 914 (0.5 g, 1.0 mmol) in dry DMF (5 mL)
was treated with anhyd K2CO3 (0.28 g, 2.0 mmol) and
allyl bromide (0.24 mL, 2.0 mmol) and processed as
K2CO3ꢀDMF.
/

H.M.A. Hamid / Carbohydrate Research 338 (2003) 2301ꢀ
/
2309
2307
before to give 11, yield 0.3 g (63%), mp 144ꢀ
(KBr) 1748, 1733 (OAc); 1645 (OCN) and 1599 cmꢁ1
(CÄ
N); 1H NMR (CDCl3): d 2.08, 2.19 (2s, 6 H, 2
CH3CO); 4.42, 4.91 (dd, 2 H, J 1.5, 3.6 Hz, NÃCH2);
5.17 (s, 2 H, CH2OAc); 5.23 (m, 2 H, ÄCH2); 5.97 (m, 1
H, ÄCH); 6.23 (s, 1 H, CHOAc); 7.24ꢀ7.80 (m, 9 H,
ArÃH) and 9.13 (s, 1 H, D2O exchangeable NH). Anal.
/
146 8C; IR
cooled to rt, whereupon a white product separated out.
It was filtered off, washed with EtOH, and recrystallized
from EtOH to give colorless crystals of 13, yield 1.5 g
/
/
(63%), mp 190ꢀ192 8C; IR (KBr) 3411 (OH) and 1664
/
1
/
cmꢁ1 (OCN); H NMR (CDCl3): d 1.36, 1.42 (2s, 6 H,
2CH3); 1.67 (bs, 1 H, D2O-exchangable OH); 3.57 (m, 2
H, H-1?a, H-3?a); 3.65 (dd, 1 H, J 2.8, 6.6 Hz, H-1?b);
4.01 (m, 1 H, H-3?b); 4.35 (m, 1 H, H-2?); 4.73 (d, 2 H, J
/
/
/
Calcd for C25H24N4O5 (460.47): C, 65.21; H, 5.25; N,
12.17. Found: C, 65.00; H, 5.10; N, 11.9.
29.0 Hz, CH2OH) and 7.26ꢀ
/
8.08 (m, 10 H, ArÃ
/
Hꢃ
/
pyrazoleÃH). Anal. Calcd for C24H24N4O4 (432.46): C,
/
3.10.1. Effect of boiling acetic anhydride on compound
11. A suspension of compound 11 (0.1 g, 0.2 mmol) in
Ac2O (10 mL) was heated under reflux. The reflux was
continued for 30 min after the solid had dissolved. The
66.65; H, 5.60; N, 12.96. Found: C, 66.40; H, 5.50; N,
13.10.
3.12.2. Method (b). Compound 12 (0.1 g, 0.25 mmol)
was stirred vigorously with dry acetone (10 mL) and
96% H2SO4 (3 drops) for 2 h, and then kept overnight at
rt. The resulting mixture was neutralized by the addition
of solid anhyd Na2CO3, and filtered, and the inorganic
salts were well washed with dry acetone. The acetone
resulting solution was cooled and then poured into iceꢀ
/
water. The product obtained was filtered off, washed
with water, and dried. It was crystallized from EtOH to
give product identical with compound 6 (0.05 g, 57%):
mp 194ꢀ196 8C.
/
solution was evaporated in vacuo at 30ꢀ40 8C, and the
/
3.11. 1-(2,3-Dihydroxypropyl)-3-[5-(hydroxymethyl)-1-
phenylpyrazol-3-yl]quinoxalin-2-one (12)
resulting product dissolved in EtOH, the product that
separated out was filtered off, and dried, yield 0.12 g
(51%). This product was identical with the product from
Method (a).
3.11.1. Method (a). A suspension of compound 6 (0.2 g,
0.5 mmol) in a solution of Na2CO3 (0.1 g, 0.9 mmol) in
H2O (5 mL) was stirred at rt. Alkaline KMnO4 was then
added dropwise, until the color of KMnO4 persisted.
The mixture was stirred for 2 h and then kept overnight.
The product that separated out was filtered off, washed
with water and crystallized from EtOH as colorless
3.13. 1-N-(2,3-Dibromopropyl)-3-[5-(hydroxymethyl)-1-
(4-bromophenyl)pyrazol-3-yl]quinoxaline-2-one (15)
To suspension of 6 (0.8 g, 2.0 mmol) in CHCl3 (10 mL)
was added dropwise bromine (2 mL), during 2 h with
occasional shaking. The mixture was kept overnight at
rt diluted with water, whereupon a yellow product
separated out. It was filtered off, washed with water,
dried, and crystallized from EtOH to give a yellow
crystals, yield 0.1 g (51%), mp 97ꢀ
(OH); 1640 (OCN) and 1600 cmꢁ1 (CÄ
(Me2SO-d6ꢀD2O): d 4.08 (dd, 1 H, J 5.4, 12.6 Hz; H-
/
98 8C; IR (KBr) 3439
1
/
N); H NMR
/
3?a); 4.15 (dd, 1 H, J 5.1 Hz, H-3?b); 4.41 (m, 2 H, H-1?a,
H-2?); 4.57 (d, 1 H, J 10.2 Hz, H-1?b); 5.15 (s, 2 H,
crystals of 15, yield 0.9 g (76%), mp 118ꢀ
/
120 8C; IR
N);
CH2OH); 7.37ꢀ
/
7.86 and 7.88 (m, d, 10 H, ArÃ
/
Hꢃ
/
(KBr) 3425 (OH); 1658 (OCN) and 1597 cmꢁ1 (CÄ
/
PyrazoleÃH). Anal. Calcd for C21H20N4O4 (392.4): C,
/
1H NMR (Me2SO-d6): d 3.48, and 3.61 (2dd, 2 H, J 5.4,
6.0, 10.2, 21.9 Hz, CH2Br); 4.17 (m, 1 H, CHBr); 4.35 (d,
64.27; H, 5.14; N, 14.28. Found: C, 64.00; H, 4.90; N,
14.00.
2 H, J 6.6 Hz, NÃ
H, D2O exchangeable, OH); 7.42ꢀ
H, Arà and pyrazoleÃH). Anal. Calcd for
/CH2); 4.64 (s, 2 H, CH2OH); 4.90 (s, 1
/7.92 and 8.22 (m, d, 9
3.11.2. Method (b). Compound 13 (0.2 g, 0.46 mmol)
was refluxed in 80% AcOH (10 mL) for 2 h the solvent
was evaporated under diminished pressure to give a
/H
/
C21H17Br3N4O2 (597.13): C, 42.24; H, 2.87; N, 9.38.
Found: C, 41.90; H, 2.50; N, 9.00.
colorless product, yield 0.12 g (67%), mp 96ꢀ98 8C. This
/
product was identical with the product from method a.
3.14. 1-N-(2,3-Diazidopropyl)-3-[5-(hydroxymethyl)-1-
(4-bromophenyl)pyrazol-3-yl]quinoxaline (16)
3.12. 3-[5-(Hydroxymethyl)-1-(2,3-O-
isopropylidenedioxypropyl-1-phenylpyrazol-3-
yl]quinoxaline-2-one (13)
A mixture of 15 (0.5 g, 0.84 mmol) and NaN3 (0.13 g,
2.0 mmol) in dry DMF (5 mL) was heated for 3 h at
80 8C. The solvent was evaporated in vacuo, and the
resulting product was dissolved in EtOH, decolorized
with charcoal, and concentrated. The product yield 0.3 g
(68%) that separated out was filtered off and recrystal-
3.12.1. Method (a). A stirred solution of 5 (RÄ
/
Ac) (2.0
g, 5.6 mmol) in anhyd DMF (10 mL) was added NaH
(0.14 g, 6.0 mmol) and after evolution of H2, was almost
complete the mixture was heated to 100 8C for 1 h. Then
compound 1430 (1.52 g, 6.0 mmol) was added, and the
lized from EtOH to give pale yellow crystals, mp 130ꢀ
/
132 8C; IR (KBr) 3389 (OH); 2103 (azide) and 1646
mixture was stirred for additional 8ꢀ10 h at 100 8C, and
/
cmꢁ1 (OCN); 1H NMR (CDCl3): d 3.46 (dd, 1 H, J 1.8,

2308
H.M.A. Hamid / Carbohydrate Research 338 (2003) 2301ꢀ2309
/
9.9 Hz, H- 3?a); 3.49 (dd, 1 H, J 8.1 Hz, H-3?b); 4.27 (bs,
1 H, H-1?a); 4.69 (m, 2 H, H-1?b, H-2?); 4.71 (s, 2 H,
References
1. St Clair, R. L.; Thibault, T. D. Ger. Offen. 2, 459, 453
(1975) [C.A., 83, 164237 (1975)].
CH2OH); 5.20 (bs, 1 H, D2O exchangeable, OH); 7.39ꢀ
/
7.62 and 8.03 (m, d, 9 H, ArÃ
/
Hꢃ H). Anal.
/
pyrazoleÃ
/
2. Yamamoto, H. Jap. Pat. 69, 17, 136. (1969) [C.A., 71,
124505d (1969)].
Calcd for C21H17BrN10O2 (521.35): C, 48.38; H, 3.29; N,
26.87, Found: C, 48.00; H, 3.00; N, 26.60.
3. Zeliner, H.; Pailer, M.; Prukmayr, G., Austrian Pat. 228,
204 (1963) [C.A., 59, 11514 (1963)].
3.15. Preparation and culture of Hep G2 2.2.15 cells
4. Yellin, T. O., U. S. Pat. 3, 635, 971 (1972) [C. A., 76,
99708r (1972)].
5. Fisons Pest Control Ltd., Belg. Pat. 631, 044 (1963) [C.A.,
60, 15891 (1964)].
6. Higgins ans, J.; Menon, C. S. J. Polym. Sci. (B) 1972, 10,
The required cell line was made by transfection of Hep
G2-cells with a plasmid containing multiple tandem
copies of HBV genome (subtype ayw).33 The 2.2.15 cell
line was maintained in RPMI-1640 (Glutamax) culture
129ꢀ
7. Biere, H.; Bottcher, I.; Kapp, J. F. Arch. Pharm. 1983,
316, 588ꢀ597.
/
131.
media containing 100 IU/mL nystatinꢃ
G418 (geneticin). The transfected Hep G2-2.2.15 cell
line was kept in a tissue culture flask at 37 8Cꢃ5% CO2.
/
380 mg/mL
/
8. Georgescu, C.; Dumitrescu, C.; Danau, M.; Spandonidis,
/
S.; Costache, G. Med. Intern. 1986, 24, 147.
9. Muller, P.; Dammann, H. G.; Simon, B. Arzneim. -Forsch.
Subcultures were set up after a week by aspiration of the
media from culture flask and washing the cells twice by
1986, 36, 1399ꢀ
10. Vinge, E.; Bjorkman, S. Acta Pharmacol. Toxicol. (Co-
penh.) 1986, 59, 165ꢀ174.
/
1400.
PBS. A 10% verseneꢀtrypsin was added and the cells
/
were incubated for 1 min at 37 8C.
/
The drug Lamivudine, which is a potent selective
inhibitor of HBV replication,34 was used as a standard
for the comparative studies.
11. El Ashry, E. S. H., in P. A. Seib, B. M. Tolbert (Eds.),
Ascorbic Acid Chemistry, Metabolism and Uses. vol. 200,
American Chemical Society Washington, DC, 1982, pp.
179ꢀ
12. El Ashry, E. S. H.; Mousaad, A.; Rashed, N. Adv.
Heterocycl. Chem. 1992, 53, 233ꢀ312.
13. Rashed, N.; Abdel Hamid, H.; El Ashry, E. S. H.
Carbohydr. Res. 1993, 243, 399ꢀ405.
/
186.
3.15.1. PCR-ELISA. The PCR reaction mixture con-
tained 14 mL of extracted supernatant, 4 mmol/L MgCl2,
10 mmol/L DIG-11-dUTP, 190 mmol/L dTTP, 200 mmol/
L dATP, dGTP, dCTP, 1.5 U Taq polymerase, 20
mmol/L HCl (pH 8.4), 50 mmol/L KCl, 1 mmol/L
HCID-1 primer (5?GGA AAG AAG TCA GAA GGC
A3?) and 1 mmol/L HCID-2 (5?TTG GGG GAG GAG
ATT AGG TT3?), in a total volume 50 mL. PCR
reaction conditions were 32 cycles of 1 min at 94 8C,
/
/
14. Amer, A.; El Massry, A. M.; Awad, L.; Rashed, N.; El
Ashry, E. S. H.; HO, D. M. J. Chem. Soc. Perkin Trans. 1
1990, 2513ꢀ
15. Chu, C. K.; Cutler, S. J. J. Heterocylc. Chem. 1986, 23,
289ꢀ319.
/2518.
/
16. MacCoss, M.; Tolman, R. L.; Ashton, W. T.; Wagner, A.
F.; Hannah, J.; Field, A. K.; Karkas, J. D.; Germershau-
sen, J. I. Chem. Scr. 1986, 26, 113.
30 s at 58 8C and 30 s at 72 8Cꢃ3 s for each cycle in a
/
thermal circler as described in literature.35
17. Yokoyama, M.; S. Watanabe, Yuki Gosei Kagaku Kyo-
kaishi 47, 694 (1989)[C.A. 11, 98984c (1990)].
18. Balzarini, J.; De Clercq, E. Pharmaco. Chem. Libr. 1990,
3.15.2. Cytotoxicity assay. A colorimetric assay for
living cells should utilize a colorless substrate that is
modified to a colored product by any living cells, but not
by dead cells or tissue-culture medium. 3-(3,5-Di-
14, 175ꢀ194.
/
19. Chu, C. K.; Baker, D. C. Nucleosides and Nucleotides as
Antitumor and Antiviral Agents; Plenum Press: New York,
1993.
methylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
20. El Ashry, E. S. H.; El Kilany, Y. Adv. Heterocycl. Chem.
(MTT) is the attractive candidate for this purpose. The
cytotoxic effect of the compounds was accessed by
culturing the Hep G2-2.2.15 cells in the presence of
compounds, using a MTT-assay.36
1998, 69, 129ꢀ
21. El Ashry, E. S. H.; El Kilany, Y. Adv. Heterocycl. Chem.
1997, 68, 1ꢀ88.
22. El Ashry, E. S. H.; El Kilany, Y. Adv. Heterocycl. Chem.
1996, 67, 391ꢀ438.
23. El Ashry, E. S. H.; Rashed, N.; Abdel Hamid, H.;
Ramadan, E. S. Z. Naturforsch 1997, 52b, 873ꢀ882.
24. Rashed, N.; Abdel Hamid, H.; Ramadan, E. S.; El Ashry,
E. S. H. Nucleosides Nucleotides 1998, 17 (8), 1373ꢀ1384.
25. Henseke, G.; Dittrich, K. Chem. Ber. 1959, 92, 1550ꢀ
1558.
26. Henseke, G. Z. Chem. 1966, 6, 329ꢀ
27. El Ashry, E. S. H.; El Kholy, I. E.; El Kilany, Y.
Carbohydr. Res. 1978, 60, 303ꢀ314.
/
215.
/
/
3.15.3. Calculation of IC50, CC50, and SI. The 50%
inhibitory concentration of antiviral drugs (IC50) was
determined by interpolation from the plots of amount of
DNA copies versus antiviral drug concentrations. The
50% cytotoxic effect (CC50) was calculated from the
average viability of the cells with concentration of drugs.
The selective index (SI) could be calculated as CC50/
IC50.
/
/
/
/336.
/

H.M.A. Hamid / Carbohydrate Research 338 (2003) 2301ꢀ
/
2309
2309
28. El Ashry, E. S. H.; El Kholy, I. E.; El Kilany, Y.
Carbohydr. Res. 1978, 60, 396ꢀ399.
29. El Ashry, E. S. H.; El Kholy, I. E.; El Kilany, Y.
Carbohydr. Res. 1978, 67, 495ꢀ499.
30. Baldwin, J. J.; Raab, A. W.; Mensler, K.; Arison, B. H.;
Mcclure, D. E. J. Org. Chem. 1978, 43, 4876ꢀ4878.
31. El Ashry, E. S. H. J. Chem. Soc. Chem. Commun. 1986,
1024ꢀ1026.
32. El Ashry, E. S. H.; Abdel Hamid, H.; El Habrouk, M.
Carbohydr. Res. 1995, 267, 177ꢀ186.
33. Sells, M. A.; Chen, M. L.; Acs, G. Proc. Natl. Acad. Sci.
USA 1987, 84, 1005.
34. Doong, S. L.; Tsai, C. H.; Schinazi, R. F.; Liotta, D. C.;
/
/
Cheng, Y. C. Proc. Natl. Acad. Sci. USA 1991, 88, 8495ꢀ
/
8499.
/
35. Korba, B. E.; Gerin, J. L. Antiviral Res. 1992, 19, 55ꢀ
/
70.
/
36. Fouad, T.; Nielsen, C.; Brunn, L.; Pedersen, E. B. Sc. J.
Az. Med. Fac. (GIRLS)
1187.
1998, 19, 1173ꢀ
/
/