[1,2,4]Triazolo[4,3-a]quinoxaline: Synthesis, antiviral, and antimicrobial activities

Article abstract of DOI:10.1007/s00044-011-9753-7

A series of novel [1,2,4]triazolo[4,3-a]quinoxaline derivatives and their isosteres, pyrimido-quinoxaline, were synthesized as potential antiviral and antimicrobial agents. The new compounds were synthesized via aromatic nucleophilic substitution of 4-chloro-8-methyl[1,2,4]triazolo[ 4,3-a]quinoxaline-1-amine with different amines and triazole-2-thiol. Some of the synthesized compounds were subjected to antiviral and cytotoxicity screening using plaque-reduction assay. Most of the tested compounds exhibited cytotoxicity at concentration 160 μg/ml and compound 8b showed promising antiviral activity. In vitro antimicrobial screening against different pathogenic organisms using agar diffusion method demonstrated that compounds 4d, 6c, 7b, and 8a exhibit antibacterial and/or antifungal activities. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9753-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-011-9753-7
ORIGINAL RESEARCH
[1,2,4]Triazolo[4,3-a]quinoxaline: synthesis, antiviral,
and antimicrobial activities
•
Morkos A. Henen Serry A. A. El Bialy
•
•
Fatma E. Goda Magda N. A. Nasr
•
Hassan M. Eisa
Received: 9 March 2011 / Accepted: 18 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of novel [1,2,4]triazolo[4,3-a]quinoxa-
line derivatives and their isosteres, pyrimido-quinoxaline,
were synthesized as potential antiviral and antimicrobial
agents. The new compounds were synthesized via aromatic
nucleophilic substitution of 4-chloro-8-methyl[1,2,4]triaz-
olo[4,3-a]quinoxaline-1-amine with different amines and
triazole-2-thiol. Some of the synthesized compounds were
subjected to antiviral and cytotoxicity screening using pla-
que-reduction assay. Most of the tested compounds exhibited
cytotoxicity at concentration 160 lg/ml and compound 8b
showed promising antiviral activity. In vitro antimicrobial
screening against different pathogenic organisms using agar
diffusion method demonstrated that compounds 4d, 6c, 7b,
and 8a exhibit antibacterial and/or antifungal activities.
combating fungal infections. In addition, several literatures
have pointed out the value of triazoles as antimicrobial
agents (Suresh Kumar et al., 2010; Creaven et al., 2010;
Sangshetti and Shinde, 2010) On the other hand, quinoxa-
lines show broad spectrum antibacterial activity (Khan et al.,
2007). These findings encouraged us for further exploration
of [1,2,4]triazolo[4,3-a]quinoxaline as antimicrobial agents.
It was reported that the presence of piperazine subunit or
its isosteres enhances the antimicrobial activity of the fused
triazoles ring systems (Wei et al., 2006). Furthermore, the
highly potent fluoroquinolone antibiotics, such as norflox-
acin and ciprofloxacin contain piperazine moiety (Fig. 1).
Based on this hypothesis, [1,2,4]triazolo[4,3-a]quinoxaline
2a, b and 4a–d were deliberated to contain piperazine or
piperidine in order to investigate their antimicrobial
properties.
Keywords [1,2,4]-triazolo[4,3-a]quinoxaline ꢀ
Pyrimidotriazoloquinoxaline ꢀ Antiviral agents ꢀ
Antimicrobial agents
Moreover, to increase the bioactivity of [1,2,4]triazol-
o[4,3-a]quinoxaline as antiviral agents, thioamide group is
another structural modification that was achieved in this
study (Zachariadis et al., 2003). Therefore, compounds 8a,
b were designed and synthesized as potential antiviral
agents.
Introduction
The antifungal drugs that have triazole moiety, such as vo-
riconazole and posaconazole, play the leading role in
Furthermore, it was reported that [1,3,4]oxadiazole
moiety enhances the antimicrobial activity (Abbas et al.,
1994). Therefore, We proposed to incorporate different
[1,3,4]-oxadiazole and [1,2,4]-triazole subunits onto the
[1,2,4]-triazolo[4,3-a]quinoxaline ring. Finally, it is known
that the arylidene derivatives have antimicrobial properties
(Periyasami et al., 2008; Pandeya et al., 1998). Therefore,
certain Schiff’s bases were synthesized by the reaction of
amino[1,2,4]triazolo[4,3-a]quinoxalines with certain aro-
matic aldehydes. Therefore, this study was aimed at syn-
thesizing [1,2,4]triazolo[4,3-a]quinoxalines substituted
with piperazine, piperidine, thioamide, [1,3,4]oxadiazole,
and [1,2,4]triazole as potential antimicrobial agents.
M. A. Henen ꢀ S. A. A. El Bialy ꢀ F. E. Goda ꢀ
M. N. A. Nasr ꢀ H. M. Eisa
Department of Pharmaceutical Organic Chemistry,
Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt
S. A. A. El Bialy (&)
Department of Chemistry and Center for Biotechnology
and Drug Design, Georgia State University, Atlanta,
GA 30303-3083, USA
e-mail: sbialy65@yahoo.com
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Med Chem Res
Fig. 1 Fluoroquinolones
antibiotics and antifungal drugs
N
N
N
N
N
N
N
O
HO
N
N
F
F
F
F
O
OH
O
N
N
N
O
N
F
posaconazole
voriconazole
O
N
O
N
O
O
F
F
OH
O
OH
N
N
N
NH
O
O
N
O
N
HN
HN
norfloxacin
ciprofloxacin
nitrofurantoin
Chemistry
in the presence of Et3N as a base and xylene, respectively.
Treatment of compound 3b with p-aminoacetophenone in
the presence of Et3N in toluene afforded compound 5
(Scheme 1).
We initiated this study by the reaction of 4-chloro-8-
methyl[1,2,4]triazolo[4,3-a]quinoxaline-1-amine (1) (Sarges
et al., 1990; Sastry et al., 1988) with 1-(o-tolyl)piperazine or
piperidine in the presence of Et3N as a base and xylene as a
solvent to afford [1,2,4]-triazolo[4,3-a]quinoxaline (2a, b),
respectively. Acetylation of compound 1 was achieved by a
mixture of acetic anhydride and acetic acid to give com-
pound 3a. Furthermore, benzoylation of compound 1 was
carried out using benzoyl chloride in pyridine to give the
compound 3b.
Several methods were reported for the preparation of
thioethers from the reaction of aromatic halides and mer-
capto derivatives in EtOH/KOH (Muhi-Eldeen et al., 1991),
CHCl3/TEA (Hirai et al., 1991), or DMF/K2CO3 (De La
Rosa et al., 2006). In this investigation, the synthesis of
compounds 6a–c was achieved by reacting compound 1 with
the appropriate mercapto-1,3,4-oxadiazoles and mercapto-
1,2,4-triazole in DMF containing anhydrous K2CO3. In a
similar way, the title compounds 7a, b were prepared by
reaction of compound 1 with the appropriate 3-(substituted
Compounds 2a, b and 4a–d were prepared by refluxing
compounds 3a, b with 1-(o-tolyl)piperazine or piperidine
H2N
N
R
4
X
(Yield %)
N
X
HN
Me
N
a
b
c
d
N-(2-Me)Ph
N-(2-Me)Ph
CH2
Me
Ph
Me
Ph
60
56
40
50
N
N
Xylene/TEA
(65-70%)
X
2a, X= CH2
2b, X= N-(o-Me)Ph
CH2
H2N
N
N
Me
N
N
ROCHN
N
X
HN
N
Me
N
Cl
1
Xylene/TEA
ROCHN
N
N
(CH3CO)2O/
CH3COOH
or C6H5COCl
N
N
X
4
Me
N
N
pyridine
(60-70%)
Cl
NH2
PhOCHN
3a, R= Me
3b
N
N
, R= Ph
Me
N
COMe
COCH3
Toluene
(60%)
N
N
H
5
Scheme 1 N-substitution products of 4-chloro-8-methyl[1,2,4]triazolo[4,3-a]quinoxaline-1-amine
123

Med Chem Res
benzylideneamino)-5-mercapto-1H-1,2,4-triazoles in DMF/
K2CO3 (Scheme 2).
Biology
The synthesis of the thioamide derivatives 8a, b was
accomplished by the reaction of the amine 1 with the
appropriate isothiocyanate derivatives in ethanol. The for-
mation of Schiff’s bases 9a–c was carried out by the
reaction of the amine 1 and aromatic aldehydes in EtOH
(Love and Ren, 1993; Khalil, 2006; Novikova et al., 2005),
containing catalytic amount of acetic acid (Scheme 3).
Cyclocondensation reaction of amino[1,2,4]triazolo[4,
3-a]quinoxaline with dimethyl acetylene dicarboxylate
(DMAD) in methanol gave the corresponding methyl 4-oxo-
4H-pyrimido[20,10:5,1][1,2,4]triazolo[4,3-a]quinoxaline-2-car-
boxylate (10). Finally, the title compound 11 was prepared
via reacting 1,2,4-triazolo[4,3-a]quinoxaline-1-amine(1)
with diethyl ethoxymethylenemalonate (DEEM) in glacial
acetic acid (Scheme 4).
Antiviral and cytotoxicity screening
Potential antiviral activity and cytotoxicity using an
improved plaque-reduction assay against Herpes simplex
virus grown on Vero African monkey kidney cells
(Schmidtke et al., 2001; Badria et al., 2003) were tested for
eleven [1,2,4]triazolo[4,3-a]quinoxaline derivatives. The
antiviral and cytotoxicity data for these compounds are
summarized in Table 1. The greatest antiviral activity
among the [1,2,4]triazolo[4,3-a]quinoxalines compounds
was found for 8b which showed reduction of the number of
the plaques by 25% at 20 mg/ml. On the other hand, the
other ten compounds reduced the number of plaques by
less than 25% at relatively high concentrations. Moreover,
compound 4a showed the lowest cytotoxicity among the
H2N
R
(Yield %)
6
X
N
N
N
N
Me
N
N
R
HS
X
a
b
c
O
O
(2-OH)Ph
3-pyridyl
60
77
80
N
N
K2CO3/DMF
R
S
X
N-C6H5 2-thienyl
H2N
6
N
N
Me
N
R
R'
R
N
Cl
R'
H2N
N
N
HC
(Yield %)
N
R'
7
R
1
N
HS
N
N
N
H
Me
N
N
60
55
N
a
b
H
2-OH
K2CO3/DMF
N
OMe 3-OMe
S
N
H
7
Scheme 2 S-substitution products of 4-chloro-8-methyl[1,2,4]triazolo[4,3-a]quinoxaline-1-amine
RHNSCHN
N
RNCS
(Yield %)
R
8
C2H5OH
N
Me
N
60
65
a
b
C2H5
C6H5
H2N
N
Cl
N
N
8
R1
Me
N
R
N
1
Cl
R1
(Yield %)
9
R
C
N
R1
H
N
N
OHC
R
64
60
60
a
b
c
OH
H
Me
N
OMe
OMe
H
CH3COOH
C2H5OH
(60-64%)
N
Cl
OMe
9
Scheme 3 Thioamides and Schiff’s base formation
123

Med Chem Res
CO2Et
O
MeO2C
H2N
O
N
N
N
N
N
N
N
N
DMAD
CH3OH
DEEM
AcOH
Me
N
Me
N
Me
N
( 55 %)
N
Cl
(56 %)
N
Cl
N
Cl
1
11
10
Scheme 4 Formation 4-oxo-4H-pyrimido[1,2,4]triazolo[4,3-a]quinoxaline-2-carboxylate
tested compounds. It can be concluded that the antiviral
activity may be due to thiourea moiety and this indicates
the importance of the selected moieties for the develop-
ment of antiviral activity (Table 1).
Experimental
Chemistry
Melting points (°C) were recorded using Fisher-John
apparatus and are uncorrected. Elemental analyses were
performed at the microanalytical unit, Cairo University,
Egypt. IR spectra were recorded on Mattason 5000FT-IR
spectrometer (t in cm-1) using KBr disk. The 1H NMR and
13C NMR spectrums were recorded on Bruker Ac 250 FT
NMR spectrometer (250 MHz) in DMSO-d6, microana-
lytical unit, Cairo University. The chemical shifts in ppm
are expressed in d units using DMSO-d6 as solvent and
tetramethylsilane (TMS) as internal reference. Mass spec-
tra were performed on JOEL JMS-600H spectrometer at
Cairo University, Egypt. 4-Chloro-8-methyl-1,2,4-triazol-
o[4,3-a]quinoxaline-1-amine (1) was prepared previously
in the literature (Sarges et al., 1990; Sastry et al., 1988).
Antimicrobial screening
[1,2,4]Triazolo[4,3-a]quinoxaline derivatives were screened
for their potential in vitro antimicrobial activity against Gram-
positive (Bacillus subtilis and Staphylococcus aureus), Gram-
negative bacteria (Escherichia coli), and fungi (Candida
albicans) using disk diffusion assay (Smith et al., 2006;
Espinel-Ingroff, 2007). Ciprofloxacin (50 lg/ml) was used as
a reference standard for antibacterial screening, while clo-
trimazole (1000 lg/ml) was used as a reference standard for
antifungal screening. Compounds exhibited moderate activity
(inhibition zone[ 15 mm) were subjected to further quanti-
tative assay to determine their minimum inhibitory concen-
trations (MICs) using the agar dilution method (Table 1)
(Wiegand et al., 2008) against B. subtilis, S. aureus, E. coli,
and C. albicans, respectively. The antimicrobial data for these
compounds are summarized in Table 1.
Synthesis of 4-chloro-8-methyl-[1,2,4]triazolo-[4,3-
a]quinoxaline-1-amine (1)
The greatest antibacterial activity among the [1,2,4]triaz-
olo[4,3-a]quinoxaline compounds was found for 4d, and 6c
which showed strong in vitro activity against S. aureus and
E. coli, respectively. Compounds 6c, 7b, and 8a showed the
greatest activity against C. albicans (20–25 mm) with MIC
from 78 to 112 lg/l. It could be concluded that compound 8b
which showed the highest antiviral activity did not have good
activity as antibacterial or antifungal agent which is expected
because of the unique structure of viruses.
A mixture of 2,3-dichloro-6-methylquinoxaline (1.1 g,
0.005 mol) and thiosemicarbazide (0.9 g, 0.01 mol) in
n-butanol (20 ml) was heated under reflux for 6 h. The
solid product was filtered, washed with water, dried, and
recrystallized from ethanol to give 1. Yield, 75%, mp:
1
225–230 °C, IR (KBr): mmax, cm-1: 3100 (NH2), H NMR
(DMSO-d6): d 2.3 (s, 3H, CH3), 6.6 (s, 2H, NH2, D2O-
exchangeable), 7.4–7.8 (m, 3H, Ar–H). MS FAB: m/z 234
(31, M??1), 151 (16.5), 82 (100), Anal. Calcd for
C10H8ClN5 (233.66): C, 51.40; H, 3.45; N, 29.97. Found:
C, 51.32; H, 3.72; N, 30.20.
In addition, among the [1,2,4]triazolo[4,3-a]quinoxaline
compounds, five compounds (2b, 5, 6c, 7b, and 8a) showed
a good activity. Compound 6c has comparable activity as
ciprofloxacin against B. subtilis at MIC levels of 91 lg/ml.
Good activity was also demonstrated by the compounds 5,
6c, and 8a against S. aureus at MIC from 94 to 124 lg/ml.
Compounds 2b, 5, 7b, and 8a displayed good activity
against E. coli at MIC from 67 to 102 lg/ml. Finally, it is
of interest to note that the piperidine (2a, 4d) or thio-
triazole (6c, 7b) substituted [1,2,4]triazolo[4,3-a]quinoxa-
line exhibited the greatest antimicrobial activity.
General method of nucleophilic substitution reaction
of 4-chloro-8-methyl-[1,2,4]-triazolo-[4,3-a]-
quinoxaline-1-amine.
A mixture of 1-amino-4-chloro-8-methyl-[1,2,4]-triazolo-
[4,3-a]-quinoxaline (1) (2.3 g, 0.01 mol), 1-o-Tolylpiper-
azine or piperidine (0.01 mol), and Et3N (1 ml) in xylene
(15 ml) was heated under reflux for 24 h. The precipitate
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Med Chem Res
was collected by filtration, washed with water, dried, and
30 min. The mixture was extracted with chloroform, and
the organic layer was washed with water and 5% HCl. The
chloroform layer was dried over anhydrous magnesium
sulfate and evaporated to afford 3b. Yield, 60%, mp:
110°C, IR (KBr): mmax, cm-1: 1640 (CO, amide), 3320
recrystallized from EtOH/DMF mixture to afford 2a, b.
Synthesis of 8-methyl-4-(piperidin-1-yl)-[1,2,4]-
triazolo[4,3-a]quinoxaline-1-amine (2a)
1
(NH, amide), H NMR (DMSO-d6): d 2.5 (s, 3H, CH3),
Yield, 65%, mp: 237–239°C; IR (KBr): mmax, cm-1: 2950
7.4–7.6 (m, 3H, Ar–H), 7.6–8.1 (m, 5H, Ar–H), 9.4 (s, 1H,
NH, D2O-exchangeable), 13C NMR (DMSO-d6): d 21.7,
125.7, 127.5, 132, 134.9, 138.6, 145.4, 149.3, 157, 165.1.
MS FAB: m/z 339 (41, M??2), 217 (100), 120 (21), Anal.
Calcd for C17H12ClN5O (337.76): C, 60.45; H, 3.58; N,
20.73. Found: C, 60.23; H, 3.90; N, 21.01.
1
(CH), 3400 (NH2), H NMR (DMSO-d6): d 2.2 (s, 3H,
CH3), 3.2–3.7 (m, 10H, piperidine-H), 6.3 (s, 2H, NH2,
D2O-exchangeable), 7.1–7.6 (m, 3H, Ar–H). 13C NMR
(DMSO-d6): d 17.9, 21.2, 48.6, 49.3, 118.6, 121.7, 135.4,
151, 155, 167.1. MS FAB: m/z 283 (16, M??1), 198 (100),
175 (34), Anal. Calcd for C21H23N7 (282.34): C, 63.81; H,
6.43; N, 29.77. Found: C, 64.11; H, 6.26; N, 29.97.
General method of nucleophilic substitution reaction
of N-acyl-4-chloro-8-methyl-[1,2,4]-triazolo[4,3-
a]quinoxaline-1-amine (3a, b)
Synthesis of 8-methyl-4-(4-o-tolylpiperazin-1-yl)-[1,2,4]-
triazolo-[4,3-a]-quinoxaline-1-amine (2b)
A mixture of N-acyl-4-chloro-8-methyl[1,2,4]triazolo[4,3-
a]quinoxaline-1-amine (3a, b) (0.005 mol), 1-o-Tolylpip-
erazine or piperidine (0.005 mol), and Et3N (0.5 ml) in
xylene (10 ml) was refluxed for 24 h. The solid product
was collected by filtration, washed with water, dried, and
recrystallized from EtOH to furnish 4a–d.
Yield, 70%, mp: 215–217°C, IR (KBr): mmax, cm-1: 2920
(CH), 3450 (NH2), H NMR (DMSO-d6): d 1.9 (s, 3H,
1
tolyl-CH3), 2.5 (s, 3H, CH3), 3.2–3.6 (m, 8H, piperazine-
H), 6.8 (s, 2H, NH2, D2O-exchangeable), 6.9–7.2 (m, 4H,
Ar–H), 7.8–8.1 (m, 3H, Ar–H). 13C NMR (DMSO-d6): d
21.3, 24.5, 25.3, 52, 127.4, 129, 135.1, 135.8, 155.9. MS
FAB: m/z 273 (36, M?), 198 (100), 175 (25), Anal. Calcd
for C21H23N7 (373.45): C, 67.54; H, 6.21; N, 26.25. Found:
C, 67.83; H, 6.50; N, 25.97.
Synthesis of N-[8-methyl-4-(4-o-tolylpiperazin-1-
yl)[1,2,4]triazolo[4,3-a]quinoxalin-1-yl]acetamide (4a)
Yield, 60%, mp: 250–252°C, IR (KBr): mmax, cm-1: 1640
(CO, amide), 2910 (C–H), 3315 (NH, amide), H NMR
Synthesis of N-(4-chloro-8-methyl[1,2,4]triazolo-[4,3-a]-
quinoxalin-1-yl)acetamide (3a)
1
(DMSO-d6): d 2.1 (s, 3H, COCH3), 2.2 (s, 3H, tolyl-CH3),
2.5 (s, 3H, CH3), 3.1 (m, 8H, piperazine-H), 6.9–7.2 (m,
4H, Ar–H), 7.7–7.9 (m, 3H, Ar–H), 9.5 (s, 1H, NH, D2O-
exchangeable). 13C NMR (DMSO-d6): d 17.9, 21.3, 24.9,
52.6, 121.9, 128.4, 135.4, 142.6, 167.1. MS FAB: m/z 416
(1.54, M??1), 341 (100), 325 (8.71), 240 (2.30), 175
(4.26), 91 (24.43), 58 (3.57). Anal. Calcd for C23H25N7O
(415.49): C, 66.49; H, 6.06; N, 23.60. Found: C, 66.53; H,
5.91; N, 23.56.
4-Chloro-8-methyl-[1,2,4]-triazolo-[4,3-a]-quinoxaline-1-
amine (1) (2.3 g, 0.01 mol) was added to a mixture of
acetic anhydride (20 ml) and acetic acid (10 ml). The
mixture was refluxed for 2 h, cooled, and poured onto ice-
water. The formed solid was collected by filtration, dried,
and recrystallized from absolute EtOH to yield compound
3a. Yield, 70%, mp: 122–125°C, IR (KBr): mmax, cm-1
:
1
1652 (CO, amide), 3600 (NH, amide), H NMR (DMSO-
d6): d 2.1 (s, 3H, COCH3), 2.4 (s, 3H, CH3), 7.4–7.8 (m,
3H, Ar–H), 9.2 (s, 1H, NH, D2O-exchangeable), 13C NMR
(DMSO-d6): d 21.3, 24, 125.7, 127.4, 132.8, 135.1, 145.9,
149.7, 157.2. MS FAB: m/z 277 (58, M??2), 151 (11), 124
(100), Anal. Calcd for C12H10ClN5O (275.69): C, 52.28; H,
3.66; N, 25.40. Found: C, 52.73; H, 3.60; N, 25.20.
Synthesis of N-[8-methyl-4-(4-o-tolylpiperazin-1-
yl)[1,2,4]triazolo[4,3-a]quinoxalin-1-yl]benzamide (4b)
Yield, 56%, mp:[300°C, IR (KBr): mmax, cm-1: 1630 (CO,
amide), 2920 (C–H), 3315 (NH, amide), 1H NMR (DMSO-
d6): d 2.3 (s, 3H, tolyl-CH3), 2.4 (s, 3H, CH3), 3.4 (m, 8H,
piperazine-H), 6.8–7.2 (m, 4H, Ar–H), 7.4–7.6 (m, 5H,
Ar–H), 7.7–7.9 (m, 3H, Ar–H), 9.1 (s, 1H, NH, D2O-
exchangeable), 13C NMR (DMSO-d6): d 17.2, 24, 48.9,
49.9, 121.4, 135.7, 142.6, 151.3, 157.8. MS FAB: m/z 477
(27.8, M ?), 241 (100), 174 (13), Anal. Calcd for
C28H27N7O (477.56): C, 70.42; H, 5.70; N, 20.53. Found:
C, 70.52; H, 5.32; N, 20.96.
Synthesis of N-(4-chloro-8-methyl-[1,2,4]-triazolo[4,3-
a]quinoxalin-1-yl)benzamide (3b)
To a mixture of 4-chloro-8-methyl-[1,2,4]-triazolo[4,3-
a]quinoxaline-1-amine (1) (2.3 g, 0.01 mol) and pyridine
(0.79 g, 0.01 mol), benzoyl chloride (1.4 g, 0.01 mol) was
added and the mixture was stirred at 0°C over a period of
123

Med Chem Res
Synthesis of N-[8-methyl-4-(piperidin-1-
yl)[1,2,4]triazolo[4,3-a]quinoxalin-1-yl]acetamide (4c)
anhydrous K2CO3 (1.4 g, 0.01 mol) in DMF (30 ml) was
stirred for overnight. The reaction mixture was poured onto
ice-water. The solid was collected by filtration, washed with
water, and recrystallized from DMF/water mixture afforded
6a–c.
Yield, 40%, mp: 231–233°C, IR (KBr): mmax, cm-1: 1650
(CO, amide), 2910 (C–H), 3300 (NH, amide), H NMR
1
(DMSO-d6): d 2.2 (s, 3H, COCH3), 2.6 (s, 3H, CH3), 3.8
(m, 10H, piperidine-H), 7.5–7.7 (m, 3H, Ar–H), 9.3 (s, 1H,
NH, D2O-exchangeable), 13C NMR (DMSO-d6): d 24.5,
25.5, 52.7, 121.5, 127.4, 128.2, 132.4, 135.8, 142.6, 157.6,
167.1. MS FAB: m/z 326 (4.13, M??2), 325 (4.39,
M??1), 170 (5.68), 104 (100), 84 (7.24), 58 (5.68). Anal.
Calcd for C17H20N6O (324.38): C, 62.95; H, 6.21; N,
25.91. Found: C, 62.52; H, 6.05; N, 26.11.
Synthesis of 1-amino-8-methyl-4-(5-(2-hydroxyphenyl)-
1,3,4-oxadiazol-2-yl)-1,2,4-triazolo[4,3-a]quinoaxalines
(6a)
Yield, 60%, mp: 230–232°C, IR (KBr): mmax, cm-1:3224
1
(OH); 3502 (NH2), H NMR (DMSO-d6): d 2.3 (s, 3H,
CH3), 6.9 (s, 2H, NH2, D2O-exchangeable), 7.1–7.3 (m,
4H, Ar–H), 7.5–7.8 (m, 3H, Ar–H), 10.5 (s, 1H, OH, D2O-
exchangeable). 13C NMR (DMSO-d6): d 21.4, 108, 117.8,
121.4, 126.8, 138.7, 142, 150.7, 156.9, 164.7. MS FAB:
m/z 393 (12, M??2), 198 (100), 197 (31). Anal. Calcd for
C18H13N7O2S (391.41): C, 55.23; H, 3.35; N, 25.05.
Found: C, 54.96; H, 3.80; N, 25.32.
Synthesis of N-[8-methyl-4-(piperidin-1-
yl)[1,2,4]triazolo[4,3-a]quinoxalin-1-yl]benzamide (4d)
Yield, 50%, mp: 240–242°C, IR (KBr): mmax, cm-1: 1653
(CO, amide), 2910 (C–H), 3304 (NH, amide), H NMR
1
(DMSO-d6): d 2.7 (s, 3H, CH3), 3.1–3.3 (m, 10H, piperi-
dine-H), 7.4–7.6 (m, 3H, Ar–H), 7.7–8.2 (m, 5H, Ar–H),
9.5 (s, 1H, NH, D2O-exchangeable), 13C NMR (DMSO-
d6): d 21.3, 48.2, 49.4, 118.7, 123.1, 128.4, 129, 132.4,
135.6, 165.7, 167. MS FAB: m/z 386 (3.55, M?), 264
(5.67), 120 (5.51), 84 (34.20), 69 (100). Anal. Calcd for
C22H22N6O (386.45): C, 68.38; H, 5.74; N, 21.75. Found:
C, 68.56; H, 5.71; N, 21.94.
Synthesis of 8-methyl-4-[5-(pyridin-3-yl)-1,3,4-oxadiazol-
2-ylthio)[1,2,4]triazolo[4,3-a]quinoaxaline-1-amine (6b)
Yield, 77%, mp: 250–252°C, IR (KBr): mmax, cm-1: 3513
1
(NH2), H NMR (DMSO-d6): d 2.5 (s, 3H, CH3), 6.9 (s,
2H, NH2, D2O-exchangeable), 7.6–7.8 (m, 3H, Ar–H),
8.1–8.5 (m, 4H, pyridine-H). 13C NMR (DMSO-d6): d
21.4, 108.2, 117, 121.7, 126.2, 138.7, 140, 150.2, 157.4,
164.9. MS FAB: m/z 378 (24, M??2), 300 (100), 198 (33),
180 (23), 78 (12.3). Anal. Calcd for C17H12N8OS (376.40):
C, 54.25; H, 3.21; N, 29.77. Found: C, 54.21; H, 3.56; N,
30.12.
Synthesis of N-[4-(4-actophenylamino)-8-
methyl[1,2,4]triazolo[4,3-a]quinoxalin-1-yl]benzamide (5)
A
mixture of N-(4-chloro-8-methyl[1,2,4]triazolo[4,3-
a]quinoxalin-1-yl)benzamide (3b) (1.7 g, 0.005 mol),
4-Aminoacetophenone (0.7 g, 0.005 mol), and Et3N
(0.5 ml) in toluene (12 ml) was heated under reflux for 7 h.
The solvent was evaporated, and the remaining solid was
washed with water, dried, and recrystallized from EtOH to
Synthesis of 8-methyl-4-[(4-phenyl-5-(thien-2-yl)-4H-1,2,4-
triazol-3-yl)thio][1,2,4]triazolo[4,3-a] quinoxaline-1-
amine (6c)
afford 5. Yield, 60%, mp: [300°C, IR (KBr): mmax, cm-1
:
Yield, 80%, mp: 210–212°C, IR (KBr): mmax, cm-1: 3514
1653 (CO, amide), 3214 (NH), 3304 (NH, amide), 1H
NMR (DMSO-d6): d 2.5 (s, 3H, CH3), 2.6 (s, 3H, COCH3),
7.5–7.9 (m, 12H, Ar–H), 8.3 (s, 1H, NH, D2O-exchange-
able), 8.4 (s, 1H, NH, D2O-exchangeable). 13C NMR
(DMSO-d6): d 21.2, 26.4, 111.8, 125.3, 127.4, 131, 133.8,
140.1, 145.9, 157.1, 165, 196.7. MS FAB: m/z 437 (32,
M??1), 304 (42), 133 (100). Anal. Calcd for C25H20N6O2
(436.47): C, 68.80; H, 4.62; N, 19.25. Found: C, 68.54; H,
4.50; N, 19.41.
1
(NH2), H NMR (DMSO-d6): d 2.3 (s, 3H, CH3), 6.7 (s,
2H, NH2, D2O-exchangeable), 7.5–7.9 (m, 8H, Ar–H; 3H,
thiophen-H). 13C NMR (DMSO-d6): d 21.7, 125.6, 128.3,
129.6, 138.4, 140, 143.8, 150.4, 152.1, 155.9, 161.1. MS
FAB: m/z 458 (24, M??2), 260 (100), 199 (32.5). Anal.
Calcd for C22H16N8S2 (456.55): C, 57.88; H, 3.53; N,
24.54. Found: C, 57.98; H, 3.77; N, 24.90.
General method of thioether formation (7a–b)
General method of thioether formation (6a–c)
A mixture of 4-chloro-8-methyl-1,2,4-triazolo[4,3-a]quin-
oxaline-1-amine (1) (2.3 g, 0.01 mol), the appropriate
A
mixture of 4-chloro-8-methyl-[1,2,4]-triazolo[4,3-a]
quinoxaline-1-amine (1) (2.3 g, 0.01 mol), the appropriate
3-(substituted
benzylideneamino)-5-mercapto-1H-1,2,4-
triazoles (0.01 mol), and anhydrous potassium carbonate
(1.4 g, 0.01 mol) in DMF (50 ml) was stirred overnight.
mercapto oxadiazole or mercapto triazole (0.01 mol), and
123

Med Chem Res
The reaction mixture was poured onto ice-water. The
formed solid was collected by filtration, washed with
water, and recrystallized from DMF/water mixture to fur-
nish 7a, b.
(0.53, M??2), 232 (0.56), 217 (0.92), 205 (100), 103
(7.38), 88 (5.40). Anal. Calcd for C13H13ClN6S (320.80):
C, 48.67; H, 4.08; N, 26.20. Found: C, 48.54; H, 3.96; N,
26.71.
Synthesis of 8-methyl-4-[(3-(2-hydroxy)benzylideneamino)-
1H-[1,2,4]-triazolo-5-yl)thio]-1,2,4-triazolo[4,3-
a]quinoxaline-1-amine (7a)
Synthesis of 1-(4-chloro-8-methyl[1,2,4]triazolo[4,3-
a]quinoxalin-1-yl)-3-phenyl thiourea (8b)
Yield, 65%, mp: 192–193°C, IR (KBr): mmax, cm-1: 1050
Yield, 60%, mp: 295–298°C, IR (KBr): mmax, cm-1:1665
(C=N), 2690 (=CH), 3200 (OH), 3415 (NH2), H NMR
(C=S), 3435, 3512 (NH), H NMR (DMSO-d6): d 2.4 (s,
1
1
3H, CH3), 3.5 (s, 1H, NH), 6.8–7.2 (m, 5H, Ar–H), 7.4–7.8
(m, 3H, Ar–H), 8.2 (s, 1H, NH), 13C NMR (DMSO-d6): d
22, 126.4, 127.4, 128.9, 129.4, 133.1, 138.1, 138.9, 145.2,
149.4, 179.4. MS FAB: m/z 368 (3.34, M?), 217 (32.56),
191 (2.03), 151 (100), 136 (12.78). Anal. Calcd for
C17H13ClN6S (368.84): C, 55.36; H, 3.55; N, 22.78. Found:
C, 55.76; H, 3.92; N, 22.20.
(DMSO-d6): d 2.9 (s, 3H, CH3), 6.8 (s, 2H, NH2, D2O-
exchangeable), 7.2–7.9 (m, 7H, Ar–H), 8.6 (s, 1H, N=CH),
12.2 (s, 1H, OH, D2O-exchangeable), 12.5 (s, 1H, triazole-
H). 13C NMR (DMSO-d6): d 21.1, 117.4, 121, 127.4,
128.3, 132.1, 132.7, 134.4, 138.7, 150.8, 155.9, 158.1,
159.3, 160.8. MS FAB: m/z 419 (31, M??2), 221 (15), 198
(100). Calcd for C19H15N9OS (417.45): C, 54.67; H, 3.62;
N, 30.20. Found: C, 54.97; H, 3.55; N, 30.23.
General method of Schiff’s base formation (9a–c)
Synthesis of 8-methyl-4-{[3-(3,4-
dimethoxy)benzylideneamino]-1H-[1,2,4]-triazolo-5-
yl)thio]-[1,2,4]-triazolo[4,3-a]quinoxaline-1-amine (7b)
A mixture of 4-chloro-8-methyl[1,2,4]triazolo[4,3-a]quin-
oxaline-1-amine (1) (0.01 mol) and the appropriate benz-
aldehyde derivatives (0.01 mol) were dissolved in EtOH
(30 ml) containing catalytic amount of acetic acid (1 ml).
The reaction mixture was refluxed for 4 h, and the organic
solvent was evaporated. The resulted solid was dried and
recrystallized from EtOH to afford 9a–c.
Yield, 55%, mp: 281–283°C, IR (KBr): mmax, cm-1: 1659
1
(C=N), 2693 (=CH), 3510 (NH2), H NMR (DMSO-d6): d
2.5 (s, 3H, CH3), 3.4 (s, 6H, OCH3), 6.9 (s, 2H, NH2, D2O-
exchangeable), 7.2–7.7 (m, 6H, Ar–H), 8.4 (s, 1H,
N = CH), 12.3 (s, 1H, triazole-H). 13C NMR (DMSO-d6):
d 21.7, 56.1, 56.4, 109.6, 11, 125.1, 128.3, 128.9, 130.1,
138.7, 150.1, 155.8, 158.1, 159.6, 161. MS FAB: m/z 462
(9.5, M??1), 232 (0.36), 230 (100). Anal. Calcd for
C21H19N9O2S (461.50): C, 54.65; H, 4.15; N, 27.32.
Found: C, 55.05; H, 4.10; N, 27.43.
Synthesis of 4-chloro-1-(4-hydroxybenzylideneamino)-8-
methyl[1,2,4]triazolo[4,3-a]quinoxalines (9a)
Yield, 64%, mp: 194–196°C, IR (KBr): mmax, cm-1: 1675
1
(C=N), 2709 (=CH), 3224 (OH), H NMR (DMSO-d6): d
2.4 (s, 3H, CH3), 5.3 (s, 1H, OH), 6.8–7.4 (m, 4H, Ar–H),
7.5–7.8 (m, 3H, Ar–H), 8.6 (s, 1H, N=CH), 13C NMR
(DMSO-d6): d 21.7, 116.7, 127.4, 128.3, 129.6, 130.9,
135.5, 138.1, 145.3, 149, 153.5, 160.6. MS FAB: m/z 337
(22.02, M?), 217 (9.01), 168 (0.23), 146 (100), 120
(21.60). Anal. Calcd for C17H12ClN5O (337.76): C, 60.45;
H, 3.58; N, 20.73. Found: C, 60.23; H, 3.67; N, 20.79.
General method of preparation of thioureas derivatives
(8a, b)
A mixture of 4-chloro-8-methyl[1,2,4]triazolo[4,3-a]quin-
oxaline-1-amine (1) (2.3 g, 0.01 mol) and appropriate
isothiocyante derivatives (0.01 mol) in EtOH (15 ml) was
refluxed for 6 h. On cooling, the separated solid was col-
lected by filtration, dried, and recrystallized.
Synthesis of 4-chloro-8-methyl-1-(4-
methoxybenzylideneamino)[1,2,4]triazolo[4,3-
a]quinoxalines (9b)
Synthesis of 1-(4-chloro-8-methyl[1,2,4]triazolo[4,3-
a]quinoxalin-1-yl)-3-ethyl thiourea (8a)
Yield, 60%, mp: 215–217°C, IR (KBr): mmax, cm-1: 1663
(C=N), 2686 (=CH), H NMR (DMSO-d6): d 2.5 (s, 3H,
1
Yield, 60%, mp: 160–162°C, IR (KBr): mmax, cm-1: 1050
CH3), 3.3 (s, 3H, OCH3), 7.1–7.7 (m, 7H, Ar–H), 9.8 (s,
N=CH). 13C NMR (DMSO-d6): d 20.9, 55.7, 113, 125.6,
128.4, 135.7, 132, 145.6, 154. MS FAB: m/z 351 (22, M?),
218 (1.2), 134 (100), Anal. Calcd for C18H14ClN5O
(351.79): C, 61.46; H, 4.01; N, 19.91. Found: C, 61.35; H,
3.97; N, 19.47.
1
(C=S), 3415,3490 (NH), H NMR (DMSO-d6): d 1.2 (t,
3H, CH3-ethyl), 2.0 (s, 1H, NH), 2.3 (s, 3H, CH3), 4.1 (s,
1H, NH), 4.3 (q, 2H, CH2-ethyl), 7.4–7.8 (m, 3H, Ar–H),
13C NMR (DMSO-d6): d 15.2, 21.7, 40.6, 127.5, 128.8,
132.4, 136.1, 145.3, 148.9, 157.9, 179.2. MS FAB: m/z 323
123

Med Chem Res
Synthesis of 4-chloro-8-methyl-1-(3,4-
dimethoxybenzylideneamino)-[1,2,4]-triazolo[4,3-
a]quinoxalines (9c)
C16H12ClN5O3 (357.75): C, 53.72; H, 3.38; N, 19.58.
Found: C, 53.63; H, 3.12; N, 19.91.
Biology
Yield, 60%, mp: [300°C, IR (KBr): mmax, cm-1: 1673
(C=N), 2680 (=CH), H NMR (DMSO-d6): d 2.2 (s, 3H,
1
Antiviral and cytotoxicity screening
CH3), 3.1 (s, 6H, OCH3), 7.1–7.4 (m, 3H, Ar–H), 7.6–7.9
(m, 3H, Ar–H), 9.5 (s, N=CH). 13C NMR (DMSO-d6): d
21.4, 56.4, 56.8, 109.1, 111.1, 128, 131.1, 132.7, 135.4,
138.4, 145.1, 149.2, 152.3. MS FAB: m/z 381 (40.7, M?),
217 (3), 164 (100), Anal. Calcd for C19H16ClN5O2
(381.82): C, 59.77; H, 4.22; N, 18.34. Found: C, 60.06; H,
4.25; N, 18.01.
Preparation of synthetic drug solutions Samples were
prepared by dissolving in 50 ll of DMSO and diluting
aliquots into sterile culture medium. These solutions were
sub-diluted in sterile medium and the two solutions used as
stocks to test samples at 2–200 lg/ml in triplicate in the
wells of micotiter plates.
Synthesis of methyl 7-chloro-11-methyl-4-oxo-4H-
pyrimido[20,10:5,1][1,2,4]triazolo[4,3-a]quinoxaline-2-
carboxylate (10)
Virus and cell culture H. simplex type I (HS-I) stock was
prepared as aliquots of culture medium from Vero cells
infected at multiplicity of 1 virion per 10 cells and cultured
3 days. They were stored at -80°C.
Dimethyl acetylenedicarboxylate (1.4 g, 0.01 mol) was
added dropwise to a stirred solution of 4-chloro-8-methyl-
Working stocks were prepared by titrating virus by
serial dilution in culture medium and assayed in triplicate
on Vero monolayers in the wells of microtiter trays. Virus
suspensions that gave about 30 plaques per well were
stored at 4°C until used. Vero African green monkey kid-
ney cells were purchased from Viromed Laboratories,
Minnetonka, MN, and grew in Dulbeccois modified
Eagle’s medium supplemented with 10% (v/v) calf serum
(HyClone Laboratories, Ogden, UT), 60 lg/ml streptomy-
cin sulfate maintained at 37°C in a humidified atmosphere
containing about 15% (v/v) CO2 in air.
1,2,4-triazolo[4,3-a]quinoxaline-1-amine
(1)
(2.3 g,
0.01 mol) in methanol (20 ml). The reaction mixture was
refluxed for 5 h, and the solvent was evaporated. The
product was dried and recrystallized from EtOH to give 10.
Yield, 55%, mp: 160–162°C, IR (KBr): mmax, cm-1: 1760
(C=O, ester), 1731 (C=O; ketone), 1H NMR (DMSO-d6): d
2.3 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 6.9–7.1 (m, 3H, Ar–
H), 8.8 (s, 1H, pyrimidine-H), 13C NMR (DMSO-d6): d
21.7, 52.4, 102.4, 117.2, 134, 137.1, 139, 142.6, 148.8,
153.1, 169, 174.1. MS FAB: m/z 344 (18, M??1), 179
(100), 169 (41). Anal. Calcd for C15H10ClN5O3 (343.72):
C, 52.41; H, 2.93; N, 20.37. Found: C, 52.75; H, 2.67; N,
20.17.
All medium components were obtained from Sigma
Chemical Co., St. Louis, MO, unless otherwise indicated.
Vero stocks were maintained at 34°C in culture flasks filled
with medium supplemented with 1% (v/v) calf serum.
Subculture for virus titration or antiviral screening were
grown in the wells of microtiter trays (Falcon Microtest III
96-wells tray, Becton–Dickinson Labware, Lincolin Park,
NJ) by suspending Vero cells in medium following trypsin–
EDTA treatment, counting the suspension with a hemo-
cyto-meter, diluting in medium containing 10% calf serum
to 2 9 104 cells per 200 ml culture, aliquoting into each
well of a tray, and culturing until confluent.
Synthesis of ethyl 7-chloro-11-methyl-4-oxo-4H-
pyrimido[20,1’:5,1][1,2,4]triazolo[4,3-a]quinoxaline-3-
carboxylate (11)
Diethyl ethoxymethylenemalonate (2.1 g, 0.01 mol) was
added dropwise to a stirred solution of 4-chloro-8-methyl-
1,2,4-triazolo[4,3-a]quinoxaline-1-amine
(1)
(2.3 g,
0.01 mol) in glacial acetic acid (25 ml). The reaction
mixture was refluxed for 3 h and left to attain room tem-
perature. The precipitated solid was filtered, dried, and
recrystallized from acetic acid to afford the desired com-
Antiviral screening procedures Microtiter trays with
confluent monolayer culture of Vero cells were inverted.
The medium was shaken out and replaced with serial
dilutions of sterile extracts of samples in triplicate in
100 ll medium followed by tittered virus in 100 ll med-
ium containing 10% (v/v) calf serum in each well. Aphi-
dicolin was used as positive control.
pound 11. Yield, 56%, mp: 210–213°C, IR (KBr): mmax
,
1
cm-1: 1767 (C=O, ester), 1742 (C=O; ketone), H NMR
(DMSO-d6): d 1.4 (t, 3H, CH3-ethyl), 2.3 (s, 3H, CH3), 4.2
(q, 2H, CH2-ethyl), 6.9–7.2 (m, 3H, Ar–H), 9.2 (s, 1H,
pyrimidine-H), 13C NMR (DMSO-d6): d 14.1, 21.6, 61.9,
113, 118.1, 135.1, 136.4, 137.2, 141, 143.1, 150.1, 152.8,
163.4, 167.1. MS FAB: m/z 357 (32.02, M?), 284 (16.09),
219 (7.17), 138 (100), 73 (34.17). Anal. Calcd for
In each tray, the last row of wells was reserved for
controls that were not treated with compounds nor with
virus. The trays were cultured for 66 h then inverted into a
pad of paper towels. The remaining cells rinsed carefully
123

Med Chem Res
with medium and fixed with 3.7% (v/v) formaldehyde in
saline for at least 20 min. The fixed cells were rinsed with
water and examined visually.
the test substance exhibiting no visible growth. Data are
shown in Table 1.
Acknowledgments The authors are grateful to Dr. Shady K. George
for performing the antimicrobial screening and to Prof. Dr. Farid A.
Badria for performing the antiviral and cytotoxic screening at
Microbiology Department, and Liver Research unit, respectively,
Faculty of Pharmacy, Mansoura University. H. simplex type I (HS-I)
was the gift of Dr. R.G.Hughes, Roswell Park Memorial Institute,
Buffalo, NY.
Antiviral activity is identified as confluent, relatively
unaltered monolayers of stained Vero cells treated with
HS-1. Furthermore, cytotoxicity was estimated as the
concentration that caused approximately 50% loss of the
monolayer present around the plaques caused by HSV-1.
Observed data are shown in Table 1.
Antimicrobial screening
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MIC
MIC of the compounds was determined by agar dilution
method. Stock solutions of the synthesized compounds
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and Sabouraud dextrose agar medium for anti-fungal
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37°C for 24 and 48 h for bacteria and fungi, respectively.
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