Microwave assisted synthesis and antimicrobial activity of 2-quinoxalinone-3-hydrazone derivatives

Article abstract of DOI:10.1016/j.bmc.2009.10.064

A simple and efficient method has been developed for the synthesis of various 2-quinoxalinone-3-hydrazone derivatives using microwave irradiation technique. The series of 2-quinoxalinone-3-hydrazone derivatives synthesized, were structurally confirmed by analytical and spectral data and evaluated for their antimicrobial activities. The results showed that this skeletal framework exhibited marked potency as antimicrobial agents. The most active antibacterial agent was 3-{2-[1-(6-chloro-2-oxo-2H-chromen-3-yl)ethylidene]hydrazinyl}quinoxalin-2(1H)-one, 7 while 3-[2-(propan-2-ylidene)hydrazinyl]quinoxalin-2(1H)-one, 2 appeared to be the most active antifungal agent.

Full text of DOI:10.1016/j.bmc.2009.10.064

Bioorganic & Medicinal Chemistry 18 (2010) 214–221
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Microwave assisted synthesis and antimicrobial activity
of 2-quinoxalinone-3-hydrazone derivatives
b
c
d
Olayinka O. Ajani a, , Craig A. Obafemi , Obinna C. Nwinyi , David A. Akinpelu
*
a Chemistry Department, Covenant University, Canaanland, P.M.B. 1023, Ota, Ogun State, Nigeria
b Chemistry Department, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria
c Department of Biological Science, Covenant University, Canaanland, P.M.B. 1023, Ota, Ogun State, Nigeria
d Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and efficient method has been developed for the synthesis of various 2-quinoxalinone-3-hydra-
zone derivatives using microwave irradiation technique. The series of 2-quinoxalinone-3-hydrazone
derivatives synthesized, were structurally confirmed by analytical and spectral data and evaluated for
their antimicrobial activities. The results showed that this skeletal framework exhibited marked potency
as antimicrobial agents. The most active antibacterial agent was 3-{2-[1-(6-chloro-2-oxo-2H-chromen-3-
yl)ethylidene]hydrazinyl}quinoxalin-2(1H)-one, 7 while 3-[2-(propan-2-ylidene)hydrazinyl]quinoxalin-
2(1H)-one, 2 appeared to be the most active antifungal agent.
Received 29 October 2009
Accepted 30 October 2009
Available online 6 November 2009
Keywords:
3-Hydrazinoquinoxalin-2(1H)-one
Antibacterial activity
Gram positive bacteria
Microwave irradiation
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Scedosporium apiospernum,40 among others, had been reported41 as
a major threat to human health. In view of this occurrence of micro-
Day by day, the chemistry of carbon–nitrogen double bond of
hydrazone is fast becoming the backbone of condensation reaction
in benzo-fused N-heterocycles.1 Hydrazone containing azomethine
–NHN@CH protons constitute an important class of compounds for
new drug development.2 Many researchers have synthesized these
compounds2 as target structuresandevaluated their biologicalactiv-
ities. Hydrazones have been reported to possess, among others,
antimicrobial,3 antitubercular,4,5 anticonvulsant,6 analgesic,7 anti-
inflammatory,8,9 antiplatelet,10 anticancer,11,12 antifungal,13 antivi-
ral,14 antitumoral,15,16 antibacterial17 and antimalarial18 activities.
Reactions of phenylhydrazine, the first hydrazine derivative charac-
terized by Fischer in 1875,19 with isatin,20–22 coumarin-3-carboxyl-
organisms’ resistance to drugs42 currently in use and emergence of
new diseases,43 there is a continuous need for the synthesis of new
organic compounds as potential antimicrobial agents using a fast
and efficient approach. Thus, it is conceivable to develop a series of
quinoxalinylhydrazones by microwave assisted approach with the
aim of investigating their antimicrobial properties.
2. Results and discussion
2.1. Chemistry
In the continuation of our effort on the quinoxaline moieties,44
the hydrazone derivatives were synthesized in order to evaluate
their antimicrobial activity. Firstly, the 1,2,3,4-tetrahydroquinoxa-
line-2,3-dione earlier synthesized by the modified procedure of
Obafemi and Pfeiferer,45 was made to react with hydrazine hydrate
according to a known procedure46 to afford 3-hydrazinoquinoxa-
lin-2(1H)-one, 1 which is the main precursor in this present study
(Scheme 1). The treatment of 1 with some acyclic ketones under
microwave irradiation afforded the hydrazones 2–4. The prelimin-
ary structure activity relationship studies were performed by vary-
ing the hydrazone side chain in order to determine how these
subunits affect the antibacterial activity of the entire template.
Based on these, we made related modification to the benzofused
side chain of 3-acetylcoumarin and isatin by changing the substit-
uents at 6- and 5-positions, respectively before condensing them
ate,23
2-bromobenzaldehyde,24
4-azido-3-acyl-2-quinolone,25
mercaptoacetic acid,26 and 1,2-benzothiazole derivatives27 were re-
ported to afford the corresponding hydrazones in moderate to high
yields. Hydrazone has been recently established as a good precursor
for one pot synthesis of C-4 functionalized 1,2,3,4-tetrahydroquinol-
ones containing a quaternary stereocenter.28 Diflunisal hydrazide-
hydrazone was also prepared as possible dual acting antimicrobial/
antituberculosis agents with anti-inflammatory properties.29 The
upsurge of widespread multi-drug resistance microorganisms such
as Staphylococcus epidermidis,30,31 Staphylococcus aureus,32,33 Pseudo-
monas aeruginosa,34,35 Escherichia coli,36,37 Enterococcus faecium,38,39
* Corresponding author. Tel.: +234 8061670254.
E-mail address: wajanfresh@yahoo.com (O.O. Ajani).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.064

O. O. Ajani et al. / Bioorg. Med. Chem. 18 (2010) 214–221
215
H
N
NH2
NH2
N
N
OH
OH
O
O
COOH
COOH
HCl
H2O
+
N
H
H
H
N
N
O
O
O
NH2NH2.H2O
N
H
N
NH-NH2
1
Scheme 1. Synthesis of 3-hydrazinoquinoxalin-2(1H)-one, 1.
with 3-hydrazinoquinoxalin-2(1H)-one, 1 precursor. Hence, the
condensation of 1 with 6-substituted acetylcoumarin and 5-substi-
tuted isatin (R = H, Br, Cl, CH3) led to the formation of hydrazones
5–8 and 10–13, respectively (Scheme 2). The preparation of 3-[2-
(2-substitutedcyclohexylidene)hydrazinyl]quinoxalin-2(1H)-one
17–19 was achieved by microwave irradiation of an equimolar
amount of 3-hydrazinoquinoxalin-2(1H)-one, 1 and 2-substituted
cyclohexanone (R = H, Br, Cl, CH3). Rapid treatment of 1 with cyclic
ketones; cyclopentanone, anthrone and camphor produced 2-qui-
noxalinone-3-hydrazone derivatives 9, 14 and 15, respectively.
Generally speaking, the infrared spectra of the compounds (1–
19) showed absorption bands due to the stretching vibrations of
In addition, the chemical shift and multiplicity patterns corre-
lated well with the proposed structures. Thus, the 1H NMR of 2
showed a singlet corresponding to resonance of N–H of hydrazone
at d 6.13 while that of amide was observed at d 13.55 downfield of
TMS scale and it was exchangeable with D2O. The multiplet at d
7.37–7.83 confirmed the presence of four aromatic protons. Two
highly intense sharp singlets at d 2.28 and 2.39 established the
presence of six alkyl protons of two -CH3 substituents attached
to the hydrazone carbon. The 13C NMR of 2 revealed eleven carbon
atoms with C@O having highest signal at 158.0 ppm while the two
–CH3 carbon atoms appeared lowest signals at 25.0 ppm and
19.0 ppm. The remaining eight carbon atoms were sp2 hybridized
with their signals ranging between 157.6 and 115.2 ppm. The mass
spectral data of 2 showed molecular ion peak at m/z 216 (25%)
which was in agreement with the molecular mass of the compound
(C11H12N4O) while base peak was observed at m/z 160 (100%).
Other peaks appeared at m/z 146, and 76 with relative intensities
of 82% and 12%, respectively due to some fragmentation processes.
The result of elemental analysis did not only correlate well with
the molecular masses of all compounds but also showed a consis-
tent minimum difference of not more than 0.39 between % calcu-
lated and % found for the carbon, hydrogen and nitrogen of
compound 2 as well as other compounds.
N–H, C@O, C@C and C@N at 3412–3118 cmÀ1, 1742–1648 cmÀ1
,
1620–1600 cmÀ1 and 1588–1531 cmÀ1, respectively while the elec-
tronic transition in UV–vis spectra of 1–19 gave rise to wavelength
(kmax) ranging from 210 nm to 425 nm. Compound 2 was used as a
representative for the spectroscopic study of the hyrazone tem-
plates. Hence, in the infrared spectrum of 2 for instance, the stretch-
ing vibrational frequencies of N–H and C@O appeared at 3241 cmÀ1
and 1685 cmÀ1, respectively while the absorption band of CH ali-
phatic occurred at 2928 cmÀ1. The C@C of aromatic and C@N of
hydrazone were responsible for the band at 1612 cmÀ1 and
1563 cmÀ1, respectively. Similarly, The UV–vis absorption spectrum
of 2 showed a peak at kmax = 216 nm (log
value for the confirmation of presence of phenyl ring due to
transition while the bathochromic shift observed at kmax = 304 nm
(log max = 3.23) was due to the presence of C@N of hydrazone; a
noticeable peak were also seen at kmax = 348 nm (log max = 3.03)
emax = 3.76) which was the
p?
p*
2.2. Antibacterial activities
e
The antimicrobial sensitivity testing of the nineteen synthe-
sized compounds were assayed using agar diffusion technique
against the test organisms (clinical isolates). Prior to the assay,
e
while a shoulder was observed at kmax = 330 nm (log
e
max = 3.13 s).
H
R
16
: R=H
R
N
O
17: R=Br
10
: R=H
11: R=Br
12
H
N
18
: R=Cl
N
O
N
N
H
19: R=CH3
: R=Cl
13: R=CH3
N
v
N
N
H
NH
H
N
iv
NH2
H
N
O
O
O
i
2
: R=R'=CH3
3: R=R'=C2H5
4:
N
R
N
N
H
N
1
N
H
R=CH3, R'=C2H5
R'
iii
ii
H
N
O
H
N
N
5
O
O
O
: R=H
N
9
N
H
6: R=Br
N
7
: R=Cl
N
N
H
R
8: R=CH3
CH3
(i) Acyclic ketone (ii) 6-substituted-3-acetylcoumarin (iii) cyclopentanone (iv) 5-substituted isatin
(v) 2-substituted cyclohexanone. Reaction Condition: Microwave Irradiation (MWI) at 140ºC
Scheme 2. Synthesis of some 2-quinoxalinone-3-hydrazone derivatives.

216
O. O. Ajani et al. / Bioorg. Med. Chem. 18 (2010) 214–221
Table 1
6349), S. aureus (NCIB 8588) and five gram negative bacteria; E. coli
(NCIB 86), Pseudomonas fluorescence (NCIB 3756), Klebsiella pneu-
monia (NCIB 418), Shigella dysenteriae (LIO), P. aeruginosa (NCIB
950). The results of their antibacterial sensitivity testing measured
in mm were reported in Table 1. The result of antifungal properties
of these synthesized compounds on Candida albicans (LIO) was rep-
resented in histogram as shown in Figure 1.
Results of antimicrobial screening on gram positive and negative bacteria with zones
of inhibition in mm
Compd No. B. a B. s B.su B. c S. a E. c P. f K. p S. d P. a C.a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Str.
Nys.
24
14
0
15
30
25
17
28
29
30
15
14
13
22
24
13
18
14
15
13
14
25
23
20
30
20
16
32
30
33
16
12
14
13
20
15
12
16
16
20
16
12
20
28
24
28
25
20
15
30
25
20
0
15
28
10
25
10
25
28
12
13
28
24
12
10
19
15
14
32
19
0
19
20
25
19
14
11
19
13
12
19
21
25
30
15
21
30
18
30
15
17
10
13
19
14
20
18
11
13
12
12
0
0
0
0
0
0
0
10
12
15
19
20
18
22
13
0
0
0
15
0
0
0
0
18
31
18
22
29
21
14
10
29
20
15
18
30
9
21
21
19
15
14
NA
12
18
16
25
14
10
11
8
20
0
14
5
The choice of use of Streptomycin as clinical standards is based
on the fact that at low concentrations, Streptomycin only inhibits
growth of the bacteria through induction of prokaryotic ribosomes
to misread mRNA.47 Streptomycin also prevents initiation of pro-
tein synthesis and leads to death of microbial cells. Also in humans,
they have structurally different ribosomes from bacteria, thereby
allowing the selectivity of this antibiotic for bacteria. The choice
of Nystatin is that is a polyene antifungal antibiotic which is both
fungistatic and fungicidal against a wide variety of yeasts. From the
antibacterial screening, compounds 3, 9, 10, 13 and 16 showed
activities on most of the five gram positive bacterial strains except
B. anthracis, (3, 13, 16), S. aureus (9), B. cereus (10) while other syn-
thesized compounds as well as Streptomycin standard were active
on all the gram positive bacteria with zones of inhibition ranging
from 5 mm to 33 mm (Table 1). Compared to Streptomycin, com-
pounds 1, 7 and 12 showed larger zones of inhibition against B.
anthracis (i.e., >18 mm) and S. aureus (i.e., >21 mm) while 1, 3, 12
and 17 showed nearly similar zones of inhibition as Streptomycin
upon B. subtilis (20 mm) and B. cereus (28 mm). The result of anti-
microbial screening (sensitivity testing) on five gram negative is
also shown in Table 1. As a general observation, E. coli and K. pneu-
moniae developed resistance against Streptomycin whereas all the
synthesized compounds 1–19 were active on these two organisms
with the zones of inhibition ranging from 10 mm to 32 mm with 7
showing largest zone of inhibition (32 mm).
These significant antibacterial activities of the synthesized com-
pounds may be explained with clue of the site of action of hydra-
zones, where it interacts with bases of DNA of the organisms,
and thus inserts (intercalates) between the stacked bases of helix.
This insertion possibly causes a stretching of the DNA duplex and
the DNA polymerase is fooled into inserting an extra base opposite
an intercalated molecule thereby results to frame shifts. The frame
shifts invariably will affect the physiological activity by not array-
ing the right bases that confer resistance on the organisms. Thus,
the organism becomes susceptible to the influence of the synthe-
sized compounds. On the other hand, most of the synthesized
0
0
10 32
19
10 14
12
11 15
19
12
12
0
11
10
14
0
14
10
0
17
0
0
0
15
13
0
0
0
0
0
0
0
20
17
21
16
0
0
12 17
0
9
13 15
30
10
15
0
0
22
19
14
16
18
12
13
0
0
0
0
0
NA NA NA
NA NA NA NA NA
NA NA 29
B. a = Bacillus anthracis (LIO)G+, B. s = Bacillus stearothermophilus (NCIB 8222)G+, B.
su = Bacillus subtilis (NCIB 3610)G+, B. c = Bacillus cereus (NCIB 6349)G+, S. a = Staph-
ylococcus aureus (NCIB 8588)G+, E. c = Escherichia coli (NCIB 86)GÀ, P. f = Pseudomonas
fluorescence (NCIB 3756)GÀ, K. p = Klebsiella pneumonia (NCIB 418)GÀ, S. d = S higella
dysenteriae (LIO)GÀ, Candid albican (LIO), P. a = Pseudomonas aeruginosa (NCIB 950),
G+ = Gram positive, GÀ = Gram negative, NA = not applicable, Str. = Streptomycin,
Nys. = Nystatin.
the clinical isolates were tested for viability by resuscitating the
organisms in buffered peptone broth, after which it was sub-cul-
tured into nutrient agar medium and incubated at 37 °C for 24 h.
The isolates were sub-cultured in a nutrient broth at 37 °C for
8 h prior to antibacterial testing. The Mueller Hinton agar was
inoculated with the test organisms and solutions of the different
synthesized compounds dissolved in DMSO were aseptically intro-
duced into the bored holes. Streptomycin and Nystatin were used
as reference clinical standards for the antibacterial and antifungal
activities, respectively. The clinical isolates used include five gram
positive bacteria; Bacillus anthracis (LIO), Bacillus stearothermophi-
lus (NCIB 8222), Bacillus subtilis (NCIB 3610), Bacillus cereus (NCIB
35
30
25
20
15
10
5
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19 Nsy.
Synthesized Compounds and Nystatin Standard
Figure 1. Result of the screening on fungus, Candida albican.

O. O. Ajani et al. / Bioorg. Med. Chem. 18 (2010) 214–221
217
Table 2
Results of minimum inhibitory concentrations (MIC) on some selected bacteria in
l
g/ml
B. cereus
Compd No.
B. anthracis
B. stearotherm.
B. subtilis
S. Aureus
E. coli
K. pneumonia
S. dysenteriea
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Str.
15.6
62.5
R
62.5
62.5
62.5
7.8
62.5
125
125
125
15.6
R
31.3
125
R
15.6
7.8
7.8
31.3
31.3
7.8
15.6
7.8
31.3
31.3
7.8
15.6
15.6
7.8
125
62.5
62.5
15.6
31.3
62.5
15.6
15.6
R
31.3
15.6
7.8
15.6
15.6
62.5
15.6
31.3
62.5
7.8
15.6
7.8
125
15.6
31.3
62.5
31.3
62.5
7.8
31.3
15.6
62.5
31.3
7.8
125
31.3
R
R
R
31.3
31.3
31.3
62.5
7.8
7.8
31.3
62.5
7.8
7.8
15.6
R
62.5
7.8
62.5
7.8
62.5
15.6
7.8
31.3
15.6
7.8
7.8
7.8
R
7.8
15.6
62.5
62.5
15.6
125
62.5
31.3
R
31.3
62.5
62.5
15.6
7.8
62.5
15.6
31.3
31.3
31.3
31.3
7.8
31.3
31.3
62.5
62.5
15.6
31.3
31.3
15.6
31.3
7.8
62.5
31.3
125
62.5
15.6
31.3
15.6
15.6
62.5
31.3
62.5
31.3
R
15.6
7.8
31.3
15.6
31.3
31.3
7.8
R
125
15.6
R
R
15.6
15.6
31.3
15.6
31.3
15.6
62.5
15.6
7.8
7.8
R
R = resistance.
hydrazone compounds showed either low or no zones of inhibition
against P. fluorescence and S. dysenteriae while Streptomycin
showed high activity on these two organisms with large zone of
inhibition of 30 mm and 22 mm, respectively. From the result, it
indicated that though Streptomycin had broad spectrum of activ-
ity, the synthesized compounds (i.e., the synthesized hydrazones
2–19 and the precursor 1 also showed reasonable level of activity,
most especially on E. coli and K. pneumoniae. Large zones of inhibi-
tion and broad spectrum of activities were observed for all the
tested organisms except P. fluorescence and P. aeruginosa which
were sensitive to only six out of 19 compounds screened. The little
or no zones of inhibition exhibited by P. fluorescence, P. aeruginosa
and S. dysenteriae may be explained by the hardy cell wall of the
organisms which contains porins and efflux pumps called ABC
transporters, which pump out some antibiotics before they are able
to act. It may also be due to the protective biofilms formed by these
organisms.48
tween 9 mm and 22 mm. From this comparative study, compound
2 emerged as the most active antifungal agent.
3. Conclusion
It wasdiscoveredthatmicrowaveassistedapproachis highlyeffi-
cient procedure for the preparation of various 2(1H)-quinoxalinone-
3-hydrazone derivatives in moderate to excellent yield. The reac-
tions occurred remarkably fast, under mild condition using highly
inexpensive reagents and microwave oven as the irradiation source.
3-{2-[1-(6-Chloro-2-oxo-2H-chromen-3-yl)ethylidene]hydrazinyl}
quinoxalin-2(1H)-one, 7, emerged as the most active antibacterial
agent while 3-[2-(propan-2-ylidene)hydrazinyl]quinoxalin-2(1H)-
one, 2appearedtobe themostactiveantifungalagentuponitsaction
onC. albican. Thus, this work will be very useful for further studies in
terms of toxicity effect and Structural Activity Relationship (SAR) to
improve their biological and pharmacological properties.
Furthermore, minimum inhibitory concentration (MIC) test was
carried out on eight organisms (five gram positive and three gram
negative bacteria) using Russell and Furr method (1977).49 The re-
sult is shown in Table 2. The MIC values of 1, 9, 10, 11, 15 varied
4. Experimental
4.1. General conditions
between 15.6
for 13, 16, 18, the MIC values varied between 15.6
62.5 g/ml; for 2, 3, 4, 5, 6, 8, 12, 19 the value varied between
7.8 g/ml and 62.5 g/ml and between 7.8 g/ml and 31.3 g/ml
l
g/ml and 125
l
g/ml for all bacterial strains and also
lg/ml and
Melting points were determined with open capillary tube on a
Gallenkamp (variable heater) melting point apparatus and were
uncorrected. Infra red spectra were recorded as KBr pellets on a
Buck Spectrometer while UV–vis spectra were recorded on a He-
l
l
l
l
l
for 14, 17 and the Streptomycin while 7 stood out by inhibiting
the growth of the organisms at the lowest concentration ranging
between 7.8 lg/ml and 15.6 lg/ml. This result indicated that 7
kios
a
UV–vis Spectrometer v2.05. The 1H and 13C NMR were run
on a Jeol-EX 300 spectrometer (d in ppm relative to Me4Si). Mass
spectra were run on Finnigan MAT 312 machine. All compounds
were routinely checked by TLC on silical gel G plates using ace-
tone/benzene (9:1, v/v) solvent system and the developed plates
were visualized by UV light. The elemental analysis (C, H, N) of
compounds were performed using a Carlo Erba-1108 elemental
analyzer. Results were found to be in good agreement with the cal-
culated values (Table 1). The microwave assisted syntheses were
carried out using a CEM Discover monomode oven operating at
2450 MHz monitored by a PC computer and temperature main-
tained at a constant value (140 °C) within the power modulation
of 300 W. Stirring was provided by an in situ magnetic stirrer while
reactions were performed in open glass vessels within a ramp time
of 75 s to 3 min. All reagents used were obtained from Sigma–Al-
drich Chemicals, except acetone, ethanol, oxalic acid and vanillin
which were obtained from BDH Chemical Limited. Solvents used
had the highest activity while 12 and 19 competed favorably with
the Streptomycin. From the overview of the SAR study, it was dis-
covered that the nature of substitutent on 5-position and 6-posi-
tions of the phenyl group of isatin and coumarin moieties,
respectively had positive effect on the antibacterial activity in
which the order include Cl > Br > CH3 > H. In like manner, all the
compounds were screened for their in vitro antifungal activity
against C. albican with the zone of inhibition duly reported in
mm (Fig. 1) using a standard procedure.50 Upon the inoculation
of C. albican with the solution of compound 5, 9 and Nystatin,
the observed zone of inhibition was 29 mm while better growth
inhibition was noticed when compounds 2 and 14 were used and
the zones of inhibition were 31 mm and 30 mm, respectively.
However, all other compounds displayed lower activity compared
to Nystatin because of lower zone of inhibition in the range be-

218
O. O. Ajani et al. / Bioorg. Med. Chem. 18 (2010) 214–221
were of analytical grade and, when necessary, were purified and
dried by standard methods.
3H, CH3), 7.00(s, 1H, NH, D2O, exchangeable). 7.09–8.27(m, 4H, Ar-
H), 8.00(s, 1H, NH, D2O, exchangeable). 13C NMR [300 MHz,
CH3OH-d4]: 158.1(C@O), 158.0, 157.6, 142.7, 131.7, 129.1, 125.9,
123.5, 115.2, 26.9(CH2), 13.9(CH3), 11.1(CH3). MS: in m/z[rel.%]:
230[M+, 40%], 202[100%], 103[58%], 29[5%]. Anal. Calcd for
C12H14N4O: C, 62.61; H, 6.09; N, 24.35. Found: C, 62.58; H, 6.06;
N, 24.30.
4.2. Synthesis
4.2.1. 3-Hydrazinoquinoxalin-2(1H)-one, 1
To
a
solution of 1,2,3,4-tetrahydroquinoxaline-2,3-dione
(20.1 g, 124.0 mmol) in hydrazine hydrate (100.0 ml, 2.2 mol),
water (50 ml) was added and the resulting mixture was refluxed
for 3 h. The mixture was allowed to cool and the formed precipitate
was filtered, recrystallized from ethanol to give 1 19.8 g (90%) as a
yellow solid: mp >360 °C, (mp >360 °C, Lit.).46 IR (cmÀ1, KBr): mmax
3412(N–H), 3280(N–H), 3175(N–H), 1679(C@O), 1620(C@C). kmax
4.2.3. General procedure of 3-{2-[1-(6-substituted-2-oxo-2H-
chromen-3-yl)ethylidene]hydrazinyl}quinoxalin-2(1H)-one 5–8
To a ground mixture of 3-hydrazinoquinoxalin-2(1H)-one, 1
(1.0 g, 5.7 mmol) and unsubstituted or 6-substituted 3-acetylcou-
marin (5.7 mmol), was added dry DMF (20 ml) in a 250 ml
round-bottomed flask. The reaction mixture was irradiated in a
microwave oven at 400 W for 1 min. and the solvent was distilled
off. The solid product obtained was filtered, dried and recrystal-
lized from methanol to afford 5–8.
(log
e
max): 216(4.34), 247(3.75s), 327(3.61s). 1H NMR (300 MHz,
5.81(s-br, 2H, NH2; D2O exchangeable), 7.49–
DMSO-d6):
d
7.96(m, 4H, Ar-H), 8.14(s, 1H, NH; D2O exchangeable), 12.55(s,
1H, NH; D2O exchangeable). 13C NMR (300 MHz, DMSO-d6): d
190.5(C@O), 141.9, 134.2, 125.7, 119.6, 117.0, 115.4, 110.4 ppm.
MS: in m/z[rel.%]: 176[55.5%], 161[92.3%], 146[85.5%], 118[100%],
106[80.1%], 78[40.5%]. Anal. Calcd for C8H8N4O: C, 54.55; H, 4.55;
N, 31.82. Found: C, 54.52; H, 4.57; N, 31.83.
4.2.3.1. 3-{2-[1-(2-Oxo-2H-chromen-3-yl)ethylidene]hydrazinyl}
quinoxalin-2(1H)-one, 5. Reagents: Compound
5.7 mmol), 3-acetylcoumarin (1.07 g, 5.7 mmol), DMF (20 ml),
ethanol (20 ml). Yield: 1.6 g (84.0%). Mp 238–241 °C. IR (cmÀ1
KBr): mmax 3240(N–H), 1740(C@O ester), 1685(C@O), 1606(C@C),
1563(C@N), 1381(C–O). kmax (log max): 212(4.58), 327(3.65s),
1
(1.0 g,
,
4.2.2. General procedure of 3-[2-(alkylidene)hydrazinyl]qui-
noxalin-2(1H)-one 2–4
e
To a mixture of 3-hydrazinoquinoxalin-2(1H)-one, 1 (1.0 g,
5.7 mmol) and acyclic ketones (5.7 mmol), was added ethanol
(20 ml). The reaction mixture was taken in round-bottomed flask
placed in a microwave oven and irradiated at 400 W for 3 min
and then the solvent was removed by vacuum distillation. The so-
lid product was filtered, dried and recrystallized from ethanol to
afford 3-[2-(alkylidene)hydrazinyl]quinoxalin-2(1H)-one 2–4.
344(3.68), 365(3.32s). 1H NMR [300 MHz, CH3OH-d4]: 2.07(s, 3H,
CH3), 7.00(s, 1H, NH, D2O, exchangeable). 7.09–8.27(m, 4H, Ar-H),
7.54(s, 1H, C@CH), 7.42–7.84(m, 4H, Chrom-H), 8.00(s, 1H, NH,
D2O, exchangeable). 13C NMR [300 MHz, CH3OH-d4]: 159.4(C@O),
158.0(C@O), 157.6, 155.6, 153.0, 142.7, 133.5, 131.7, 129.1, 128.3,
127.9, 125.9, 125.4, 123.5, 123.3, 118.1, 116.1, 115.2, 4.0. MS: in
m/z[rel.%]: 346[M+, 35%], 331[53%], 201[13%], 186[82%],
161[100%], 15[3%]. Anal. Calcd for C19H14N4O3: C, 65.90; H, 4.05;
N, 16.18. Found: C, 65.94; H, 4.08; N, 16.21.
4.2.2.1. 3-[2-(Propan-2-ylidene)hydrazinyl]quinoxalin-2(1H)-
one, 2. Reagents: Compound 1 (1.0 g, 5.7 mmol), acetone (0.42 ml,
5.7 mmol), ethanol (20 ml). Yield 0.8 g (68.3%) as a creamy solid:
mp 256 °C (sharp). IR (cmÀ1, KBr): mmax 3241(N–H), 2928(CH
4.2.3.2. 3-{2-[1-(6-Bromo-2-oxo-2H-chromen-3-yl)ethylidene]
hydrazinyl}quinoxalin-2(1H)-one, 6. Reagents: Compound
1
aliphatic), 1685(C@O), 1612(C@C), 1563(C@N). kmax (log
e
max):
(1.0 g, 5.7 mmol), 6-bromo-3-acetylcoumarin (1.52 g, 5.7 mmol),
DMF (20 ml), ethanol (20 ml). Yield: 2.08 g (86.0%). Mp 265–
266 °C. IR (cmÀ1, KBr): mmax 3135(N–H), 1740(C@O), 1665(C@O),
216(3.76), 304(3.23), 330(3.13s), 348(3.03). 1H NMR [300 MHz,
CH3OH-d4]: 2.28(s, 3H, CH3), 2.39(s, 3H, CH3), 6.13(s, 1H, NH, D2O
exchangeable), 7.37–7.83(m, 4H, Ar-H), 13.55(s-br, 1H, NH, D2O
exchangeable). 13C NMR [300 MHz, CH3OH-d4]: 158.0(C@O), 157.6,
151.4, 142.7, 131.7, 125.9, 123.5, 119.0, 115.2, 25.0, 19.0. MS: in m/
z[rel.%]: 216[M+, 25%], 160[100%], 146[82%], 76[12%]. Anal. Calcd
for C11H12N4O: C, 61.11; H, 5.56; N, 25.93. Found: C, 61.40; H, 5.53;
N, 25.90.
1575(C@N), 1288(m), 1130(m). kmax (log emax): 210(4.10),
325(3.51), 345(3.41), 414(3.32). 1H NMR [300 MHz, CH3OH-d4]:
2.07(s, 3H, CH3), 7.00(s, 1H, NH, D2O, exchangeable). 7.09–
8.27(m, 4H, Ar-H), 7.31–8.19(m, 3H, Chrom-H), 7.54(s, 1H, C@CH),
8.00(s, 1H, NH, D2O, exchangeable). 13C NMR [300 MHz, CH3OH-
d4]: 159.4(C@O), 158.0(C@O), 157.6, 155.6, 152.0, 142.7, 134.2,
133.5, 131.7, 130.3, 129.1, 125.9, 124.4, 123.5, 123.3, 119.8,
118.2, 115.2, 4.0. MS: in m/z[rel.%]: 425[M+, 38%], 345[12%],
161[100%], 155[63%], 75[25%]. Anal. Calcd for C19H13N4O3Br: C,
53.65; H, 3.06; N, 13.18. Found: C, 53.66; H, 3.08; N, 13.21.
4.2.2.2. 3-[2-(Pentan-3-ylidene)hydrazinyl]quinoxalin-2(1H)-
one, 3. Reagents: Compound 1 (1.0 g, 5.7 mmol), pentan-3-one
(0.60 ml, 5.7 mmol), ethanol (20 ml). Yield 0.79 g (56.8%). Mp 277–
278 °C. IR (cmÀ1, KBr): mmmax 3270(N–H), 1675(C@O), 1600(C@C),
1570(C@N), 1270(m). kmax (log
e
max): 220(3.71), 345(3.41s). 1H
4.2.3.3. 3-{2-[1-(6-Chloro-2-oxo-2H-chromen-3-yl)ethylidene]
NMR [300 MHz, CH3OH-d4]: 0.86(t, 6H, 2 Â CH3), 1.57(q, 4H,
2 Â CH2), 7.00(s, 1H, NH, D2O exchangeable), 7.09–8.28(m, 4H, Ar-
H), 8.00(s-br, 1H, NH, D2O, exchangeable). 13C NMR [300 MHz,
CH3OH-d4]: 163.4(C@O), 158.0, 157.6, 142.7, 131.7, 129.1, 125.9,
123.5, 115.2, 24.4(CH2), 18.4(CH2), 11.4(CH3), 11.4(CH3). MS: in
m/z[rel.%]: 244[M+, 10%], 188[65%], 161[100%], 146[85%], 118[75%],
99[15%], 71[17%], 43[8%], 15[3%]. Anal. Calcd for C13H16N4O: C,
63.93; H, 6.56; N, 22.95. Found: C, 63.95; H, 6.54; N, 22.93.
hydrazinyl}quinoxalin-2(1H)-one, 7. Reagents: Compound
1
(1.0 g, 5.7 mmol), 6-chloro-3-acetylcoumarin (1.27 g, 5.7 mmol),
DMF (20 ml), ethanol (20 ml). Yield: 1.97 g (91.0%). Mp 245–
246 °C. IR (cmÀ1
,
KBr): mmax 3387(N–H), 1740(C@O ester),
1648(C@O), 1612(C@C), 1570(C@N), 1375(C–O ester), 1290(m).
kmax (log max): 224(4.11), 310(3.71s), 332(3.41), 360(3.97s),
e
396(4.22). 1H NMR [300 MHz, CH3OH-d4]: 2.07(s, 3H, CH3),
7.00(s, 1H, NH, D2O, exchangeable), 7.09–8.27(m, 4H, Ar-H),
7.54(s, 1H, C@CH), 7.36–8.02(m, 3H, Chrom-H), 8.00(s, 1H, NH,
D2O, exchangeable). 13C NMR [300 MHz, CH3OH-d4]: 159.4(C@O),
158.0(C@O), 157.6, 155.6, 151.1, 142.7, 133.5, 131.7, 131.0, 129.5,
129.1, 126.8, 125.9, 123.6, 123.5, 123.3, 118.0, 115.2, 4.0. MS: in
m/z[rel.%]: 380.5[M+, 40%], 345[15%], 160[100%], 15[5%]. Anal.
Calcd for C19H13N4O3Cl: C, 59.92; H, 3.42; N, 14.72. Found: C,
59.90; H, 3.39; N, 14.70.
4.2.2.3. 3-[2-(Butan-2-ylidene)hydrazinyl]quinoxalin-2(1H)-one,
4. Reagents: Compound 1 (1.0 g, 5.7 mmol), butan-2-one (0.51 ml,
5.7 mmol), ethanol (20 ml). Yield 0.68 g (51.9%). Mp 268–269 °C.
IR (cmÀ1, KBr): mmax 3275(N–H), 1690(C@O), 1570(C@N), 1220(m).
kmax (log
e
max): 220(3.41), 348(3.62s), 355(3.37). 1H NMR
[300 MHz, CH3OH-d4]: 0.86(t, 3H, CH3), 1.57(q, 2H, CH2), 1.94(s,

O. O. Ajani et al. / Bioorg. Med. Chem. 18 (2010) 214–221
219
4.2.3.4. 3-{2-[1-(6-Methyl-2-oxo-2H-chromen-3-yl)ethyli-
dene]hydrazinyl}quinoxalin-2(1H)-one, 8. Reagents: Compound
1 (1.0 g, 5.7 mmol), 6-methyl-3-acetylcoumarin (1.15 g, 5.7 mmol),
DMF (20 ml), ethanol (20 ml). Yield: 1.5 g (73.2%). Mp 183–185 °C.
exchangeable), 7.09–8.27(m, 4H, Ar-H), 7.56–8.10(m, 3H, In-H),
8.00(s-br, 2H, 2 Â NH, D2O exchangeable). 13C NMR [300 MHz,
CH3OH-d4]: 168.5(C@O), 158.0(C@O), 157.9, 142.7, 140.2, 136.9,
134.5, 132.9, 131.7, 129.1, 125.9, 123.5, 119.9, 118.8, 118.0,
115.2. MS: in m/z[rel.%]: 384[M+, 55%], 304[65%], 160[97%],
90[13%]. Anal. Calcd for C16H10N5O2Br: C, 50.00; H, 2.60; N,
18.23. Found: C, 50.04; H, 2.65; N, 18.23.
IR (cmÀ1, KBr):
N), 1273(m). kmax (log
m
max 3118(N–H), 1742(C@O), 1665(C@O), 1571(C@
e
max): 210(3.11), 320(3.67), 342(3.17),
350(3.84), 360(3.51), 408(3.57). 1H NMR [300 MHz, CH3OH-d4]:
2.07(s, 3H, CH3), 2.34(s, 3H, CH3), 7.00(s, 1H, NH, D2O, exchangeable),
7.09–8.27(m, 7H, 2 Â Ar-H), 7.54(s, 1H, C@CH), 8.00(s, 1H, NH, D2O,
exchangeable). 13C NMR [300 MHz, CH3OH-d4]: 159.4(C@O),
158.0(C@O), 157.6, 155.6, 150.0, 142.7, 135.1, 133.5, 132.0, 131.7,
129.1, 127.0, 125.9, 123.5, 123.3, 122.1, 116.9, 115.2, 21.7, 5.0. MS:
in m/z[rel.%]: 360[M+, 35%], 346[20%], 161[100%], 158[60%],
72[18%], 15[7%]. Anal. Calcd for C20H16N4O3: C, 66.67; H, 4.44; N,
15.56. Found: C, 66.68; H, 4.47; N, 15.59.
4.2.5.3. 3-[2-(5-Chloro-2-oxoindolin-3-ylidene)hydrazinyl]qui-
noxalin-2(1H)-one, 12. Reagents: Compound 1 (1.0 g, 5.7 mmol),
5-chloroisatin (1.03 g, 5.7 mmol), 1:5 of DMF/ethanol (30 ml).
Yield: 1.70 g (93.4%). Mp 326 °C (dec.). IR (cmÀ1, KBr): mmax
3145(N–H), 1705(C@O), 1670(C@O), 1533(C@N), 1349, 1188,
1139 cmÀ1
. kmax (log emax): 210(3.74), 265(3.91), 340(3.46),
421(4.01). 1H NMR [300 MHz, DMSO-d6]: 7.00(s, 1H, NH, D2O,
exchangeable), 7.09–8.27(m, 4H, Ar-H), 7.50–7.89(m, 3H, In-H),
8.00(s-br, 2H, 2 Â NH, D2O exchangeable). 13C NMR [300 MHz,
CH3OH-d4]: 168.5(C@O), 158.0(C@O), 157.6, 142.7, 139.3, 134.5,
131.7, 131.3, 130.0, 129.4, 129.1, 125.9, 125.2, 123.5, 119.2,
115.2. MS: in m/z[rel.%]: 339.5[M+, 63%], 304[60%], 161[100%],
125.5[18%]. Anal. Calcd for C16H10N5O2Cl: C, 56.55; H, 2.95; N,
20.62. Found: C, 56.51; H, 2.93; N, 20.58.
4.2.4. 3-(2-Cyclopentylidenehydrazinyl)quinoxalin-2(1H)-one, 9
To a solution of 3-hydrazinoquinoxalin-2(1H)-one, 1 (1.0 g,
5.7 mmol) in cyclopentanone (0.50 ml, 5.7 mmol), was added 50%
aqueous ethanol (20 ml) in an open beaker over heated alumina.
The mixture was irradiated in microwave oven at an emitted
power of 400 W for 3 min to get a clear solution. The clear solution
formed was left to stand at room temperature to crystallize. The
solid formed was recrystallized from ethanol to afford 9 1.08 g
4.2.5.4. 3-[2-(5-Methyl-2-oxoindolin-3-ylidene)hydrazinyl]qui-
noxalin-2(1H)-one, 13. Reagents: Compound 1 (1.0 g, 5.7 mmol),
5-methylisatin (0.92 g, 5.7 mmol), 1:5 of DMF/ethanol (30 ml).
(78.3%). Mp 249–250 °C. IR (cmÀ1
, KBr): mmax 3120(N–H),
1675(C@O). kmax (log
e
max): 220(3.11), 344(3.89), 350(3.35s). 1H
NMR [300 MHz, CH3OH-d4]: 1.30(quintet, 4H, 2 Â CH2), 2.35(t,
4H, 2 Â CH2), 7.00(s, 1H, NH, D2O, exchangeable), 7.09–8.27(m,
4H, Ar-H), 8.00(s, 1H, NH, D2O, exchangeable). 13C NMR
[300 MHz, CH3OH-d4]: 187.7(C@N Cp), 158.0(C@O), 157.6, 142.7,
131.7, 129.1, 125.9, 123.5, 115.2, 35.7, 29.7, 24.8, 24.8. MS: in m/
z[rel.%]: 242[M+, 75%], 161[100%], 118[68%], 78[10%]. Anal. Calcd
for C13H14N4O: C, 64.46; H, 5.79; N, 23.14. Found: C, 64.41; H,
5.74; N, 23.10.
Yield: 1.61 g (88.4%). Mp 257–259 °C. IR (cmÀ1
,
KBr): mmax
3120(N–H), 1690(C@O), 1665(C@O), 1570(C@N), 1322, 1175. kmax
(log
max): 223(3.11), 267(3.78), 344(3.88), 425(4.12). 1H NMR
e
[300 MHz, CH3OH-d4]: 2.34(s, 3H, CH3), 6.66–8.21(m, 3H, In-H),
7.00(s, 1H, NH, D2O, exchangeable), 7.09–8.27(m, 4H, Ar-H),
8.00(s-br, 2H, 2 Â NH, D2O exchangeable). 13C NMR [300 MHz,
CH3OH-d4]: 168.5(C@O), 158.0(C@O), 157.6, 142.7, 138.2, 134.5,
134.1, 131.7, 131.5, 129.6, 129.1, 125.9, 123.5, 121.6, 117.6,
115.2, 21.3(CH3). MS: in m/z[rel.%]: 319[M+, 73%], 304[51%],
161[100%], 145[29%], 90[10%]. Anal. Calcd for C17H13N5O2: C,
63.95; H, 4.08; 21.94. Found: C, 63.99; H, 4.04; N, 21.90.
4.2.5. General procedure of 5-substituted-1H-indole-2,3-dione
3-[(3-oxo-3,4-dihydroquinoxalin-2-yl)hydrazone], 10–13
To a ground mixture of 3-hydrazinoquinoxalin-2(1H)-one, 1
(1.0 g, 5.7 mmol) and isatin or 5-substituted-isatin (5.7 mmol),
1:5 of DMF/ethanol (30 mL) was added dropwise in a 250 ml
round-bottomed flask. The reaction mixture was irradiated in
microwave oven at an emitted power of 400 W for 2 min. Then,
it was poured into crushed ice and the product was filtered and
recrystallized from DMF/water to give 10–13.
4.2.5.5. 3-[2-(Anthracen-9(10H)-ylidene)hydrazinyl]quinoxalin-
2(1H)-one, 14. To a ground mixture of 3-hydrazinoquinoxalin-
2(1H)-one, 1 (1.0 g, 5.7 mmol) and anthrone (1.1 g, 5.7 mmol),
was added 1:4 of DMF/ethanol (20 ml). The reaction mixture was
irradiated in microwave oven over heated alumina at an emitted
power of 400 W for 75 s to give a clear solution. The crude product
obtained was recrystallized from ethanol to afford 14 2.0 g (99.5%).
Mp 229–231 °C. IR (cmÀ1, KBr): mmax 3411(N–H), 1705(C@O),
4.2.5.1. 3-[2-(2-Oxoindolin-3-ylidene)hydrazinyl]quinoxalin-
2(1H)-one, 10. Reagents: Compound 1 (1.0 g, 5.7 mmol), isatin
(0.8 g, 5.7 mmol), 1:5 of DMF/ethanol (30 ml). Yield: 1.6 g (90.8%).
Mp 323–324 °C. IR (cmÀ1, KBr): mmax 3138(N–H), 1705(C@O),
1665(C@O), 1618(C@C), 1533(C@N), 1338, 1290, 1180, 1132. kmax
1575(C@N). kmax (log emax): 212(3.73), 276(3.50), 352(3.38s),
376(3.14s), 398(3.11s). 1H NMR [300 MHz, CH3OH-d4]: 3.81(s, 2H,
CH2), 7.00(s, 1H, NH, D2O, exchangeable), 7.09–8.27(m, 4H, Ar-H),
7.20–7.85(m, 8H, Ar-H), 8.00(s, 1H, NH, D2O exchangeable). 13C
NMR [300 MHz, CH3OH-d4]: 158.0(C@O), 157.6, 142.7, 139.4,
139.4, 133.0, 133.0, 131.7, 131.5, 131.5, 129.7, 129.7, 129.1, 128.3,
128.3, 127.5, 126.3, 126.3, 125.9, 123.5, 115.2, 32.5(CH2). MS: in
m/z[rel.%]: 351[M+, 85%], 174[100%], 76[23%]. Anal. Calcd for
C22H15N4O: C, 75.21; H, 4.27; N, 15.95. Found: C, 75.17; H, 4.22;
N, 15.91.
(log emax): 228(4.22), 272(4.13), 348(3.18s), 370(3.49s), 424(3.56).
1H NMR [300 MHz, CH3OH-d4]: 7.00(s, 1H, NH, D2O, exchangeable),
7.09–8.27(m, 8H, 2 Â Ar-H), 8.00(s-br, 2H, 2 Â NH, D2O exchange-
able). 13C NMR [300 MHz, CH3OH-d4]: 168.5(C@O), 158.0(C@O),
157.9, 142.7, 141.2, 134.5, 131.7, 131.2, 129.4, 129.1, 125.9, 124.4,
123.5, 119.4, 117.7, 115.2. MS: in m/z[rel.%]: 305[M+, 68%],
161[100%], 145[35%], 91[15%]. Anal. Calcd for C16H11N5O2: C,
62.95; H, 3.61; N, 22.95. Found: C, 62.95; H, 3.64; N, 22.90.
4.2.5.6. 3-{2-(1,7,7-Trimethylbicyclo[2.2.1]heptan-2-ylidene)hy-
drazinyl}quinoxalin-2(1H)-one, 15. To a solution of Camphor
(0.9 g, 5.7 mmol) in ethanol (15 ml) was added homogenous mix-
ture of 3-hydrazinoquinoxalin-2(1H)-one, 1 (1.0 g, 5.7 mmol) and
1:5 of DMF/ethanol (30 ml) in an open beaker. The resulting mix-
ture was irradiated in microwave oven at 400 W for 1 min. and al-
lowed to cool at room temperature. The crude product obtained
was recrystallized from ethanol to afford 15 1.0 g (56.0%). Mp
4.2.5.2. 3-[2-(5-Bromo-2-oxoindolin-3-ylidene)hydrazinyl]qui-
noxalin-2(1H)-one, 11. Reagents: Compound 1 (1.0 g, 5.7 mmol),
5-bromoisatin (1.29 g, 5.7 mmol), 1:5 of DMF/ethanol (30 ml).
Yield: 2.01 g (91.7%). Mp 328–330 °C. IR (cmÀ1
,
KBr): mmax
3140(N–H), 1690(C@O), 1665(C@O), 1531(C@N), 1344, 1298,
1185, 1137. kmax (log max): 225(4.10), 270(4.01), 345(3.21),
420(3.71). 1H NMR [300 MHz, CH3OH-d4]: 7.00(s, 1H, NH, D2O,
e
331–333 °C. IR (cmÀ1
,
KBr): mmax 3272(N–H), 1685(C@O),

220
O. O. Ajani et al. / Bioorg. Med. Chem. 18 (2010) 214–221
1588(C@N). kmax (log
e
max): 216(4.31), 328(3.59), 350(3.56s). 1H
4.2.6.4. 3-[2-(2-Methylcyclohexylidene)hydrazinyl]quinoxalin-
2(1H)-one, 19. Reagents: Compound 1 (1.0 g, 5.7 mmol), 2-methyl-
cyclohexanone (0.69 ml, 5.7 mmol), ethanol (30 ml). Yield: 0.85 g
NMR [300 MHz, CH3OH-d4]: 0.99(s, 6H, 2 Â CH3), 1.10(s, 3H, CH3),
1.32(m, 2H, CH2), 1.50(m, 1H, CH), 1.60(m, 2H, CH2), 2.61–2.86(m,
2H, CH2), 7.00(s, 1H, NH, D2O, exchangeable), 7.09–8.27(m, 4H,
Ar-H), 8.00(s, 1H, NH, D2O exchangeable). 13C NMR [300 MHz,
CH3OH-d4]: 166.5(C@O), 158.0, 157.6, 142.7, 131.7, 129.1, 125.9,
123.5, 115.2, 51.2, 47.0, 43.7, 35.1, 31.8, 27.5, 19.6, 19.6, 11.2. MS:
in m/z[rel.%]: 310[M+, 89%], 160[95%], 150[100%], 72[15%], 15[2%].
Anal. Calcd for C18H22N4O: C, 69.68; H, 7.10; N, 18.06. Found: C,
69.71; H, 7.14; N, 18.09.
(55.2%). Mp 282–284 °C. IR (cmÀ1
,
KBr): mmax 3205(N–H),
1663(C@O), 1570(C@N). kmax (log
emax): 230(3.73), 238(3.41),
350(3.73s). 1H NMR [300 MHz, CH3OH-d4]: 0.91(d, 3H, CH3),
1.18–1.40(m, 8H, 4 Â CH2), 1.36(m, 1H, CH), 7.00(s, 1H, NH, D2O,
exchangeable), 7.09–8.27(m, 4H, Ar-H), 8.00(s, 1H, NH, D2O
exchangeable). 13C NMR [300 MHz, CH3OH-d4]: 158.5(C@O),
158.0, 157.6, 142.7, 131.7, 129.1, 125.9, 123.5, 115.2, 33.1, 31.8,
27.3, 24.1, 20.2, 17.6(CH3). MS: in m/z(rel.%): 271[M+, 66%],
256[36%], 125[100%], 84[15%], 15[7%]. Anal. Calcd for C15H18N4O:
C, 66.67; H, 6.67; N, 20.74. Found: C, 66.69; H, 6.68; N, 20.79.
4.2.6. General procedure of 3-[2-(2-substitutedcyclohexylidene)
hydrazinyl]quinoxalin-2(1H)-one 16–19
Pure and dry 3-hydrazinoquinoxalin-2(1H)-one,
1 (1.0 g,
5.7 mmol) was added in small portion to cyclohexanone or 2-sub-
stitutedcyclohexanone (5.7 mmol) with continuous stirring, fol-
lowed by the addition of ethanol (30 ml). The reaction mixture
was irradiated in microwave oven at an emitted power of 400 W
for 2 min over bentonite support. The resulting solution was cooled
over crushed ice to give crude product. The crude product obtained
was recrystallized from ethanol to afford 16–19.
4.3. Antibacterial activity assays
Most of the organisms used were standard bacteria of National
Collection for Industrial Bacteria (NCIB) while few others were Lo-
cally Isolated Organisms (LIO). The organisms were B. cereus (NCIB
6349), B. stearothermophilus (NCIB 8222), B. subtilis (NCIB 3610), B.
anthracis (LIO), Bacillus polymyxa (LIO), Corynebacterium pyogenes
(LIO), Streptococcus faecalis (NCIB775), S. aureus (NCIB 8588), Clos-
tridium sporogenes (LIO), E. coli (NCIB 86), P. fluorescence (NCIB
3756), K. pneumonia (NCIB 418), S. dysenteriae (LIO), P. aeruginosa
(NCIB 950) and C. albican (LIO).
4.2.6.1. 3-(2-Cyclohexylidenehydrazinyl)quinoxalin-2(1H)-one,
16. Reagents: Compound
(0.60 ml, 5.7 mmol), ethanol (30 ml). Yield: 0.90 g (61.4%). Mp
201–203 °C. IR (cmÀ1
KBr): mmax 3225(N–H), 1667(C@O),
1575(C@N). kmax (log
max): 220(3.81), 233(4.10), 345(3.52s). 1H
1 (1.0 g, 5.7 mmol), cyclohexanone
,
4.3.1. Antibacterial sensitivity testing of compounds, 1–19
All the synthesized compounds (1–19) and Streptomycin were
screened for antibacterial activity on nine gram positive and five
gram negative bacterial strains using agar well diffusion method.49
The medium employed was diagnostic sensitivity test agar (Bio-
tech Ltd). With the aid of a sterile 1 ml pipette, about 0.2 ml of
the broth culture of test organism was added to 18 ml sterile mol-
ten diagnostic sensitivity test agar (Biotech Ltd) which had already
cooled down to 45 °C. This was well mixed and poured into previ-
ously sterilized petri dishes, which had been properly labeled
according to the test organisms. The medium was then allowed
to set. With the aid of a sterile cork borer, the required numbers
of holes were bored into the medium. The wells were made of
about 5 mm to the edge of the plate. The wells were then filled
up aseptically with the solution of the compound in DMSO using
Pasteur pipettes. Streptomycin was used as the standard antibacte-
e
NMR [300 MHz, CH3OH-d4]: 1.60–1.67(m, 6H, 3 Â CH2), 2.34(t,
4H, 2 Â CH2), 7.00(s, 1H, NH, D2O, exchangeable), 7.09–8.27(m,
4H, Ar-H), 8.00(s, 1H, NH, D2O exchangeable). 13C NMR
[300 MHz, CH3OH-d4]: 161.7(C@O), 158.0, 157.6, 142.7, 131.7,
129.1, 125.9, 123.5, 115.2, 34.1, 28.1, 27.1, 27.1, 25.5. MS: in m/
z[rel.%]: 257[M+, 37%], 161[92%], 111[100%], 70[28%]. Anal. Calcd
for C14H16N4O: C, 65.63; H, 6.25; N, 21.88. Found: C, 65.60; H,
6.22; N, 21.84.
4.2.6.2. 3-[2-(2-Bromocyclohexylidene)hydrazinyl]quinoxalin-
2(1H)-one, 17. Reagents: Compound 1 (1.0 g, 5.7 mmol), 2-bromo-
cyclohexanone (0.84 ml, 5.7 mmol), ethanol (30 ml). Yield: 1.42 g
(73.9%). Mp 176–178 °C. IR (cmÀ1
,
KBr): mmax 3235(N–H),
1670(C@O), 1577(C@N). kmax (log
emax): 225(3.62), 235(3.96),
346(3.48s). 1H NMR [300 MHz, CH3OH-d4]: 1.18–1.34(m, 6H,
3 Â CH2), 1.70–1.90(m, 2H, CH2), 4.34(t, 1H, CH–Br), 7.00(s, 1H,
NH, D2O, exchangeable), 7.09–8.27(m, 4H, Ar-H), 8.00(s, 1H, NH,
D2O exchangeable). 13C NMR [300 MHz, CH3OH-d4]: 158.0(C@O),
157.6, 155.6, 142.7, 131.7, 129.1, 125.9, 123.5, 115.2, 41.6(C–Br),
38.2, 26.4, 24.3, 23.2. MS: in m/z[rel.%]: 336[M+, 49%], 256[40%],
190[100%], 175[70%]. Anal. Calcd for C14H15N4OBr: C, 50.15; H,
4.48; N, 16.72. Found: C, 50.18; H, 4.50; N, 16.76.
rial agent at a concentration of 1000 lg/ml. The plates were al-
lowed to stand for about 1 h on the bench for proper diffusion of
the antibacterial agents into the medium and then incubated up-
rightly at 37 °C for 24 h. Care was taken not to stockpile the plates.
Clear zones of inhibition in millimetres indicated the relative sus-
ceptibility of the bacteria to the compounds (1–19) and Streptomy-
cin standard.
4.3.2. Determination ofminimum inhibitory concentration (MIC)
The Minimum Inhibitory Concentration (MIC) was done using
the method of Russell and Furr.49 Based on the level of resistance
of some organisms and large zones of inhibition experienced in
others, Minimum Inhibitory Concentration (MIC) was selectively
done for five gram positive and five gram negative bacterial strains.
4.2.6.3. 3-[2-(2-Chlorocyclohexylidene)hydrazinyl]quinoxalin-
2(1H)-one, 18. Reagents: Compound 1 (1.0 g, 5.7 mmol), 2-chlo-
rocyclohexanone (0.66 ml, 5.7 mmol), ethanol (30 ml). Yield:
1.47 g (88.3%). Mp 162–163 °C. IR (cmÀ1, KBr): mmax 3241(N–H),
1685(C@O), 1577(C@N). kmax (log emax): 225(3.41), 238(3.18),
340(3.73s). 1H NMR [300 MHz, CH3OH-d4]: 1.17–1.32(m, 6H,
3 Â CH2), 1.40–1.70(m, 2H, CH2), 3.50(t, 1H, CH–Cl), 7.00(s, 1H,
NH, D2O, exchangeable), 7.09–8.27(m, 4H, Ar-H), 8.00(s, 1H,
NH, D2O exchangeable). 13C NMR [300 MHz, CH3OH-d4]:
158.0(C@O), 157.6, 155.6, 142.7, 131.7, 129.1, 125.9, 123.5,
115.2, 53.7(C-Cl), 30.4, 26.6, 23.0, 21.9. MS: in m/z[rel.%]:
291.5[M+, 53%], 256[39%], 145.5[100%], 130.5[73%]. Anal. Calcd
for C14H15N4OCl: C, 57.83; H, 5.16; N, 19.27. Found: C, 57.88;
H, 5.20; N, 19.31.
Different concentrations (7.8 lg/ml and 1000.0 lg/ml) of the com-
pounds and standard were prepared using a twofold dilution
which was prepared in a sterile plate with the aid of sterile pipette
and then mixed with 18 ml of molten nutrient agar. This was then
allowed to set. The surface of the nutrient agar plate was allowed
to dry before streaking with overnight broth cultures of the bacte-
rial strains. The plates were then labeled accordingly and incu-
bated at 37 °C for up to 72 h. They were subsequently examined
for the presence or absence of growth. The lowest concentration

O. O. Ajani et al. / Bioorg. Med. Chem. 18 (2010) 214–221
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