Synthesis, antimicrobial activities and cytogenetic studies of newer diazepino quinoline derivatives via Vilsmeier-Haack reaction

Article abstract of DOI:10.1016/j.ejmech.2007.01.004

The study of the Vilsmeier-Haack reagent on 4-hydroxyquinaldines resulted in a new versatile intermediate 4-chloro-3-formyl-2-(2-hydroxy-ethene-1-yl)quinolines, which on further treatment with hydrazine hydrate yielded the desired diazepino quinoline derivatives. All the synthesized diazepino quinoline derivatives are screened for their antibacterial and antifungal activities. Cytogenetic analysis of the samples is also reported.

Full text of DOI:10.1016/j.ejmech.2007.01.004

European Journal of Medicinal Chemistry 42 (2007) 1128e1136
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, antimicrobial activities and cytogenetic studies of newer
diazepino quinoline derivatives via VilsmeiereHaack reaction
b
c
d
R. Nandhakumar a, , T. Suresh , A.L. Calistus Jude , V. Rajesh kannan , P.S. Mohan
*
b,**
a Bio-Chiral Laboratory, Department of Chemistry, Ewha Womans University, 11-1, Daehyun dong, Seodaemun gu, Seoul 120-750, South Korea
b Department of Chemistry, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India
c Department of Zoology, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India
d Department of microbiology, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India
Received 11 November 2006; received in revised form 26 December 2006; accepted 2 January 2007
Available online 20 January 2007
Abstract
The study of the VilsmeiereHaack reagent on 4-hydroxyquinaldines resulted in a new versatile intermediate 4-chloro-3-formyl-2-(2-hy-
droxy-ethene-1-yl)quinolines, which on further treatment with hydrazine hydrate yielded the desired diazepino quinoline derivatives. All the
synthesized diazepino quinoline derivatives are screened for their antibacterial and antifungal activities. Cytogenetic analysis of the samples
is also reported.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: VilsmeiereHaack reagent; 4-Chloro-3-formyl-2-(2-hydroxy-ethene-1-yl)quinolines; Diazepino[4,5-b]quinolines; Antibacterial activities; Antifungal
activities; Cytogenetic studies
1. Introduction
biologically active molecules by virtue of the presence of
some critical structural features. Certain small heterocyclic
molecules act as highly functional scaffolds and are known
pharmacophores of a number of biologically active and medic-
inally useful molecules [9]. In the interest of the above, we
planned to synthesize a system which combines these two
biolabile components together to give a compact structure
like the title compounds via VilsmeiereHaack reaction.
The VilsmeiereHaack reagent is an efficient, economical
and mild reagent for the formylation of reactive aromatic
and hetroaromatic substrates [10e12]. Nevertheless, its impor-
tance in various synthetic methodologies [13e20] including
the microwave chemistry [21e27] is remarkable and inspiring.
The classical VilsmeiereHaack reaction, however, involves
electrophilic substitution of an activated aromatic ring with
a halomethyleniminium salt to yield the corresponding imi-
nium species, which facilitates easy entry into large number
of novel and biologically potential heterocyclic systems.
Recently, we have been involved in the synthesis and chemis-
try of novel quinoline derivatives via the VilsmeiereHaack
reaction with a prospect to incorporate diverse bioactive
Quinolines and their derivatives are important constituents
of pharmacologically active synthetic compounds, as these
systems have been associated with a wide spectrum of biolog-
ical activities [1e4] such as DNA binding capability [5],
antitumor activities [6,7], DNA-intercalating carrier [8] etc.
The quinoline nucleus can also be frequently recognized in
the structure of numerous naturally occurring alkaloids and
synthetic compounds with interesting pharmacological proper-
ties. As a consequence, the development of general methods
for the synthesis and biological evaluation of new agents,
retaining the ‘core’ quinoline moiety has been the subject of
considerable synthetic effort. An essential component of the
search for new leads in the drug designing program is the
synthesis of molecules, which are novel yet resemble known
* Corresponding author.
** Corresponding author.
E-mail addresses: rajunandhakumar@yahoo.com (R. Nandhakumar),
ps_mohan_in@yahoo.com (P.S. Mohan).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.01.004

R. Nandhakumar et al. / European Journal of Medicinal Chemistry 42 (2007) 1128e1136
1129
heterocyclic nucleus intact for evaluating their antibacterial
and antifungal significance and also as a reagent for effective
functional group inter conversion [28e35]. Herein, we wish to
report the synthesis, antimicrobial activities and cytogenetic
studies of the title compounds.
8 (derived from phosphorus oxychlorideedimethylformamide)
is responsible for the formylation, which reacts with the active
methyl group of 4-hydroxyquinaldines 4 to yield 9. The sec-
ond formylation occurs at the C3-H moiety of the quinaldine
leading to an iminium species 11. However, these iminium
salts having the special capability to replace the hydroxyl
group at aromatic C4 by the nucleophiles [37,38] like chlorine,
bromine, etc. that might have led to the formation of 4-chlor-
oquinaldine 7 in minor yields and also in the conversion of
hydroxy moiety to the chloro moiety in case of 4-chloro-3-for-
myl-2-(2-hydroxy-ethene-1-yl)quinoline 5 (Scheme 2).
Despite the success in obtaining a potential quinoline
intermediate, our further endeavors towards the formylation
of 2,4-dihydroxy quinolines [39] with the classical Vils-
meiereHaack reagent under the similar conditions as the
above yielded only 2,4-dichloro quinolines 14 and not the
expected 2,4-dichloro-3-formyl quinolines 15 (Scheme 3).
Furthermore, as a continuation of the reaction sequence, the
VilsmeiereHaack reaction performed on 2,4-dichloro quino-
lines 14, under identical conditions as above had absolutely
no reaction. This may be either due to steric hindrance of
the bulky groups at C2 and C4 positions or the electron with-
drawing nature of chloro groups at same positions which re-
stricts the electrophilic attack of the iminium species at C3-H.
Finally, having realized the above vital intermediate 5 our
efforts turned towards the synthesis of novel and biologically
potential annulated quinolines. Hence, the intermediate 5 is
treated with hydrazine hydrate in absolute ethanol at reflux
temperature for 3 h. The removal of the solvent by reduced
pressure and recrystallization of the residual mass by chloro-
form afforded the pure product. The analytical and spectral
data verified the compound as 11-chloro-(3H )[1,2]diazepino
[5,4-b]quinoline 17(aee) (Scheme 4). As per our investiga-
tion, the reaction may proceed via the formation of the Schiff’s
base between the aldehyde and amino group and subsequent
2. Chemistry
The VilsmeiereHaack reaction on 4-hydroxyquinaldines 4
(prepared from aniline 1 and ethyl acetoacetate 2 and cycliza-
tion of the b-anilino crotonates 3) provided a vital and
efficient intermediate for the synthesis of several newer
substituted [b] and [c] annelated heterocyclic compounds.
The reaction is performed at 100 ꢀC for 15e20 h, using the
typical VilsmeiereHaack reagent derived from phosphorus
oxychlorideedimethylformamide [36]. Although, the reaction
proceeded uneventfully, the products formed are isolated
using silica gel column chromatography. The analytical and
spectroscopic data confirmed the products as 4-chloro-3-
formyl-2-(2-hydroxy-ethene-1-yl)quinolines 5, 4-hydroxy-3-
formyl quinaldine 6 and 4-chloroquinaldine 7. Interestingly,
1
the two singlets around d 9.5 in the H NMR spectra and
(M þ 2) peak with 3:1 intensity compared to the molecular
ion peak in the mass spectra, advocates the presence of the
vinyl protons and a chlorine atom for compounds 5(aee)
respectively, and are obtained in moderate to high yields
whereas compounds 6(aee) and 7(aee) are formed in good
to low yields. Therefore, the VilsmeiereHaack reaction on
4-hydroxyquinaldines 4 at 100 ꢀC provided a facile method
for the generation of an efficient and novel quinoline vinyl
alcohol. The time taken for each reaction and their yields
are shown in Scheme 1 and Table 1.
The VilsmeiereHaack reagents are normally applied for
the formylation of aromatic and heteroaromatic compounds.
The in situ formation of the chloromethyleniminium species
O
O
R4
R4
R4
R1
R4
R1
OH
R3
R2
R3
R2
R3
R2
COOEt
CH3
OEt
2
i
ii
NH2
N
H
N
CH3
R1
R1
4
3
1
iii
Cl
N
OH
R4
R4
Cl
N
CHO
CH3
R3
R3
R3
R2
CHO
+
+
CH3
N
R2
R2
OH
R1
R1
5
6
7
Scheme 1. (5e7)a: R1 ¼ R2 ¼ R3 ¼ R4 ¼ H, 15 h; b: R1 ¼ CH3; R2 ¼ R3 ¼ R4 ¼ H, 12.5 h; c: R2 ¼ CH3; R1 ¼ R3 ¼ R4 ¼ H, 16 h; d: R3 ¼ Cl; R1 ¼ R2 ¼ R4 ¼ H,
17.5 h; e: R1 ¼ R4 ¼ CH3; R2 ¼ R3 ¼ H, 20 h. (i) dil. HCl (1:1), rt; (ii) Ph2O, 240 ꢀC; (iii) VilsmeiereHaack reagent (DMFePOCl3), 100 ꢀC.

1130
R. Nandhakumar et al. / European Journal of Medicinal Chemistry 42 (2007) 1128e1136
Table 1
VilsmeiereHaack reaction of 4-hydroxyquinaldinesa
used as inoculum for this study. Bacterial cells were swabbed
on to nutrient agar medium [prepared from NaCl (5.0 g),
peptone (5.0 g), beef extract powder (3.0 g), yeast extract pow-
der (3.0 g), agar (20.0 g) in 100 mL distilled water; pH ¼
7.5 ꢁ 0.2)] in Petri plates. The compounds to be tested were
dissolved in chloroform to a final concentration (weight/volume)
of 0.125%, 0.25% and 0.5% and soaked in filter paper discs of
5 mm diameter and 1 mm thickness. These discs were placed
on the already seeded plates and incubated at 35 ꢁ 2 ꢀC for
24 h. To avoid the activity of the solvent that is used in the
test solutions, a solvent only treated plate was maintained,
which showed a 2 mm diameter zone of inhibition. Finally,
the diameter (mm) of the inhibition zone around each disc
was measured after 24 h and subtracting the diameter of inhibi-
tion zone formed by the solvent only treated plate, the results
are listed in Table 3. Streptomycin was used as standard.
Entry Substrates R1
R2
R3 R4
Reaction Yieldsb (%)
time (h)
5
6
7
1
2
3
4
5
5a
5b
5c
5d
5e
H
H
H
H
H
Cl
H
H
H
H
H
15
70
73.5 18
15 10
5
CH3
H
H
12.5
16
CH3
H
55
78
65
12 17
12
10 15
H
CH3
17.5
5
H
CH3 20
a
All reactions were carried out at 100 ꢀC.
Isolated yields after column chromatography on silica gel.
b
aromatization lead to the formation of the diazepino quinoline
system. The yields, reaction time taken and the temperature at
which the reaction carried are shown in Table 2.
3. Pharmacology
3.2. Antifungal studies
3.1. Antibacterial studies
Antifungal activities of all the synthesized compounds were
preliminarily screened for the in vitro growth inhibitory activity
against Aspergillus flavus, Penicillium funiculosum, Alternaria
macrospora and Fusarium oxysporum by using the disc diffu-
sion method [40]. The fungi were cultured in potato dextrose
agar medium. Potato dextrose agar medium (prepared from
Antibacterial activities of all the synthesized compounds
were preliminarily screened for the in vitro growth inhibitory
activity against Aeromonas hydrophila, Escherichia coli and
Salmonella typhi by using the disc diffusion method [40,41].
The bacteria were cultured in nutrient agar medium and
The Mechanism
N
N
O
Cl
N
Cl
OPOCl2
OH
POCl3
OPOCl2
+
H
H
H
8
N
CH3
1
N
Cl
OH
OPOCl2
OH
N
OH
N
Cl
CH-N
H
H
H
CH=CH-N
N
CH=CH-N
CH=CH-N
10
9
11
N
Cl
OPOCl2
H
Cl
O
CH=N
CH=N
OH
N
Cl
CHO
CHO
CH=CH-N
CH=CH-OH
N
N
OH
5
12
Scheme 2.

R. Nandhakumar et al. / European Journal of Medicinal Chemistry 42 (2007) 1128e1136
1131
Cl
3.3. Cytogenetic analysis
OH
N
R4
R4
R3
R2
R3
R2
DMF / POCl3
100°C
3.3.1. Cytogenetic analysis of compounds 17(aee)
in human peripheral blood leukocyte culture (PBLC)
N
Cl
OH
Cultures of leukocytes were obtained from peripheral blood
set-up following the method of Hungerford [30,42]. The chro-
mosomal preparations obtained were stained with Giemsa and
were processed to obtain G-bands. About 2 mL of venous
blood was drawn into a sterile, heparinized syringe. This blood
of 0.5 mL (about 30 drops) was inoculated under aseptic con-
ditions into a culture vial containing 5.0 mL of the culture
medium (McCoy’s 5a, Himedia), 2 mL of AB serum and
0.2 mL of phytohaemagglutinin (PHA, prepared in the labora-
tory from red kidney bean, Phaseolus vulgaris). Three exper-
iments were carried out using three non-smoking healthy male
donors’ aged 23, 24, and 28 years. The compound was
dissolved in 1% acetone. Four different concentrations of
0.02, 0.2, 2 and 20 mg/mL were added to the culture medium
(0.1 mL solution of the compounds per 8 mL of the medium),
at 0, 24, 48 h after culture initiation, indicated as treatment for
72, 48 and 24 h, respectively. Triplicate cultures for each dose
from three donors were maintained for the study of chromo-
somal aberrations. Control-I was given an equal volume of
distilled water. Control-II was given an equal volume of 1%
acetone. Acetone solution (0.1 mL) was added to 8 mL of
the medium at 0, 24 and 48 h after the culture initiation, indi-
cated as treatment for 72, 48 and 24 h, respectively. The cul-
tures were incubated at 37 ꢀC for a period of 72 h.
R1
R1
14
13
DMF / POCl3
100°C
DMF / POCl3
100°C
Cl
N
R4
R3
R2
CHO
Cl
R1
15
Scheme 3. (13e15)a: R1 ¼ R2 ¼ R3 ¼ R4 ¼ H; b: R1 ¼ CH3; R2 ¼ R3 ¼ R4 ¼ H;
c: R2 ¼ CH3; R1 ¼ R3 ¼ R4 ¼ H; d: R3 ¼ Cl; R1 ¼ R2 ¼ R4 ¼ H; e: R1 ¼ R4 ¼
CH3; R2 ¼ R3 ¼ H.
potato 150 g; dextrose 5 g and agar 2 g in 200 mL of distilled
water) was poured in the sterilized Petri plates and allowed to so-
lidify. The plates were inoculated with a spore suspension of A.
flavus, P. funiculosum, A. macrospora and F. oxysporum
(106 spores’/mL of medium). The compounds to be tested
were dissolved in acetone to a final concentration (weight/vol-
ume) of 0.5%, 1% and 2% and soakedin filter paper discs (What-
mann no. 4, 5 mm diameter). These discs were placed on the
already seeded plates and incubated at 28 ꢁ 2 ꢀC for four
days. To avoid the activity of the solvent that is used in the
test solutions, a solvent only treated platewas maintained, which
showed a 1 mm diameter zone of inhibition. Finally, after four
days, the inhibition zone which appeared around the discs in
each plate was measured and subtracting the diameter of inhibi-
tion zone formed by the solvent only treated plate, the results are
tabulated in Table 4. Carbendazim was used as standard.
The cultures were shaken periodically thrice a day. The
dividing cells were arrested at metaphase, 45 min before harvest
of culture, by adding 0.05 mL of colchicine solution (0.01%).
The contents in the vial were centrifuged at 1000 rpm for
5 min at the end of the colchicine treatment. The supernatant
was discarded and 7 mL of prewarmed hypotonic solution
(0.075 M KCl) was added to the cell button. The cells were
incubated for 7 min, sedimented after centrifugation at
1000 rpm for 5 min and then fixed in freshly prepared fixative
Cl
N
R4
CHO
R3
R2
NH2NH2.H2O
EtOH
+
reflux, 3 h
OH
R1
16
5
R4
Cl
N
R4
Cl
R3
R2
N
CH=N-NH2
OH
R3
R2
NH
N
R1
R1
17
Scheme 4. (5,17)a: R1 ¼ R2 ¼ R3 ¼ R4 ¼ H, 3.5 h, 78%; b: R1 ¼ CH3; R2 ¼ R3 ¼ R4 ¼ H, 3 h, 90%; c: R2 ¼ CH3; R1 ¼ R3 ¼ R4 ¼ H, 2.5 h, 74%; d: R3 ¼ Cl;
R1 ¼ R2 ¼ R4 ¼ H, 1.5 h, 63%; e: R1 ¼ R4 ¼ CH3; R2 ¼ R3 ¼ H, 3.5 h, 82%.

1132
R. Nandhakumar et al. / European Journal of Medicinal Chemistry 42 (2007) 1128e1136
Table 2
Synthesis of diazepino[4,5-b]quinolinesa 17(aee)
activity against S. typhi and A. hydrophila. Introduction of
two methyl groups at C6 and C8 positions increased the activ-
ity against all the pathogens. Replacement of hydrogen atom
at C6 of the quinoline intermediate by a chlorine atom en-
hanced the antibacterial activity against all the species. Never-
theless, the compounds 17(aee) show similar results but has
significant enhancement in their antibacterial activities against
all the pathogens when compared to the intermediate quino-
lines 5(aee). This enhancement may be due the diazepine het-
erocyclic ring system which has additional electron donating
nitrogen atoms. However, according to the observation, the
toxicity increases with the increase in concentration of the
test solution containing the new compounds. Although, all
the compounds are active, they did not reach the effectiveness
of the conventional bacteriostatic streptomycin. Table 3 con-
fines that the diazepines show more prominent results than
the intermediate quinolines. The variation in the effectiveness
of different compounds against different organisms depends
either on the impermeability of the cells of the microbes or
diffusion in ribosomes of microbial cells [44].
Entry Substrates R1
R2
R3
R4
Reaction
Time (h)
Yieldsb (%)
1
2
3
4
5
17a
17b
17c
17d
17e
H
H
H
H
H
Cl
H
H
3.5
3
78
90
74
63
82
CH3
H
H
H
CH3
H
H
2.5
1.5
3.5
H
CH3
H
CH3
H
a
All reactions were carried out at 80 ꢀC.
Isolated yields after column chromatography on silica gel.
b
(methanol:glacial acetic acid, 3:1, v/v). Two or three changes of
the fixative were given. Slides were prepared by placing a drop
of cell suspension on a clean chilled slide and immediately dry-
ing the slide at 40 ꢀC for a few seconds. The slides were rou-
tinely stained in a 4% buffered solution of Giemsa [43]. Three
hundred and fifty well-banded metaphases were analyzed for
each treatment under oil immersion lens.
4. Results and discussions
4.2. Antifungal studies
4.1. Antibacterial studies
The in vitro antifungal activity of the novel diazepino quin-
olines 17(aee) and the intermediate quinolines 5(aee) were
studied against the fungal strains viz. A. flavus, P. funiculosum,
A. macrospora, F. oxysporum. Carbendazim was used as
a standard drug whose diameter of zone inhibition is furnished
in Table 4.
All the synthesized novel diazepino quinolines 17(aee) and
the intermediate quinolines 5(aee) were tested for their anti-
bacterial activity in vitro against A. hydrophila, E. coli and
S. typhi. Streptomycin was used as a standard drug whose
diameter of zone inhibition is furnished in Table 3.
In general, all the synthesized novel diazepino quinolines
and the intermediate quinolines exerted a wide range of mod-
est in vitro antibacterial activity against all the tested organ-
isms except some compounds against E. coli. The compound
5a without having any substituents at C5eC8 did not exhibit
any antibacterial activity even at the higher concentration
against E. coli. There was no further improvement in the activ-
ity even after the introduction of the methyl groups (5b, 5c) at
positions C7 or C8. But all of these compounds showed good
All the compounds, except compound 5a, exhibited good
activity against all the pathogenic fungi used in this study.
The introduction of substituents to diazepino quinolines and
the intermediate quinolines such as methyl and chloro groups
increased the antifungal activities significantly. The com-
pounds 5(bed) exerted modest activity against all the species
when compared to compound 5a. However, the replacement of
the hydrogen atom by a chlorine moiety at C6 position ex-
hibited significant antifungal activity against all the fungal
Table 3
Antibacterial activity of the compounds 5(aee) and 17(aee)a
Compound
Diameter of inhibition zone in mm
Aeromonas hydrophila
Escherichia coli
Salmonella typhi
0.125%
0.25%
0.5%
0.125%
0.25%
0.5%
0.125%
0.25%
0.5%
5a
5b
4
4
3
5
4
5
4
5
6
4
8
6
8
9
11
10
14
10
12
11
12
16
11
19
e
e
e
1
e
e
e
5
e
e
2
3
4
4
6
5
4
5
4
6
5
8
5
7
9
11
13
17
13
10
12
13
19
14
18
5c
5d
7
8
9
8
11
8
5e
17a
6
3
5
9
8
e
e
e
3
2
4
6
17b
17c
7
2
6
7
9
1
5
9
17d
17e
11
8
7
11
9
12
9
2
5
Streptomycin
12
6
9
15
10
e No inhibition.
a
Values of the mean of three replicates.

R. Nandhakumar et al. / European Journal of Medicinal Chemistry 42 (2007) 1128e1136
1133
Table 4
Antifungal activity of the compounds 5(aee) and 17(aee)a
Compound
Diameter of inhibition zone in mm
Aspergillus flavus Penicillium funiculosum
Alternaria macrospora
Fusarium oxysporum
0.5%
1%
2%
0.5%
1%
2%
0.5%
1%
2%
0.5%
1%
2%
5a
5b
2
1
3
3
4
3
2
4
5
4
6
3
2
8
5
e
e
1
1
2
5
6
e
2
2
3
3
1
3
4
5
7
7
1
5
4
9
e
e
1
e
3
4
7
5c
5d
5
9
3
8
4
7
4
9
6
10
12
9
2
5
10
11
9
7
11
13
7
2
5
11
12
7
5e
17a
7
3
7
8
3
7
5
1
4
3
e
2
3
17b
17c
4
8
e
3
3
10
11
14
17
21
6
10
12
15
17
18
6
10
11
13
15
18
7
11
13
16
21
6
10
9
3
7
17d
17e
8
6
9
4
8
9
14
5
10
16
11
13
4
11
11
Carbendazim
9
6
e No inhibition.
a
Values of the mean of three replicates.
strains and at all concentrations. As expected, the antifungal
activity of the novel diazepino quinolines 17(aee) were
enhanced compared to the intermediate quinolines 5(aee).
This increased activity in the case of the novel title compounds
may be due to the additional diazepine heterocyclic ring sys-
tem containing two nitrogen atoms when compared to the
intermediate quinolines. Nevertheless, the activities of the fun-
gal strains were similar to the activities of the bacterial strains.
They also gave a clear picture with regard to the inhibition
zone, which increases with the increase in concentration of
the test solution containing the new compounds. Although
all the compounds show good activity at higher concentrations
they did not reach the effectiveness of the standard carbenda-
zim. Table 4 shows that the diazepines show better results than
the intermediate quinolines.
5. Conclusion
We have demonstrated the utility of VilsmeiereHaack reac-
tion on 4-hydroxyquinaldines resulting in some efficient and
potential intermediate towards the synthesis of newer diaze-
pino systems. An examination of the in vitro antibacterial
and antifungal activity profile of a variety of substituted novel
diazepino quinolines and the intermediate quinolines against
the bacterial strains viz., A. hydrophila, E. coli and S. typhi
and the fungal strains viz., A. flavus, P. funiculosum, A. macro-
spora, and F. oxysporum, respectively, provide a structure
activity correlate, which may be summarized as follows. The
compounds with chloro or the dimethyl functional groups in
the heterocyclic moiety play an important role in eliciting bi-
ological response. Nonetheless, the diazepines show signifi-
cant antibacterial and antifungal activities when compared to
the intermediate quinolines. The cytogenetic studies show its
importance, as the compounds do not have any chromosomal
4.3. Cytogenetic analysis
4.3.1. Analysis of chromosomal aberrations (CA)
All the prepared slides were stained with Giemsa, 20 well-
spread metaphase plates were screened for chromosome aber-
rations. The aberrations include chromatid gaps, chromatid
breaks, isochromatic breaks and chromatid interchanges. Poly-
ploid cells were also screened in all the slides. The clastogenic
properties for the compounds 17(aee) were studied by inves-
tigating the effects of the compounds on human chromosomes
in vitro in leukocyte cultures. It was noted that the chromo-
somal aberrations showed a dose dependant increase. Chromo-
somal aberrations like chromatid gaps and chromatid breaks
were observed. Polyploid cells were absent in all examined
slides. There were no chromatid interchanges, isochromatid
breaks and gaps found in all the samples and at all concentra-
tions. However, the compounds 17a and d caused no CA at all
concentrations. But the compounds 17b, c and e are mild
mutagens at concentration not lower than 20 mg/mL. Although
CA was observed in PBLC treated with the compounds 17b, c
and e, it was noted that the compound 17e caused CA even at
48 h of treatment expressing higher genotoxicity. The results
are presented in Table 5.
Table 5
Chromosomal aberrations in vitro following addition of compounds 17b, c and
e (20 mg/mL) at 0, 24 and 48 h after human peripheral blood leukocyte culture
initiation
Experiment
(mg/mL)
Average no. of
metaphases (n ¼ 3)
Mean number of aberrations (%)
Chromatid gaps
Chromatid breaks
Control Ia
Control IIb
350
350
e
e
e
e
0 h
17b
17c
17e
350
350
350
6 (1.7)
8 (2.3)
11 (3.1)
3 (0.9)
3 (0.9)
5 (1.4)
24 h
17b
17c
17e
350
350
350
2 (0.6)
4 (1.1)
7 (2.0)
1 (0.3)
2 (0.6)
3 (0.9)
48 h
17e
a
350
3 (0.9)
1 (0.3)
Control I was given an equal volume of distilled water.
Control II was given an equal volume of 1% acetone.
b

1134
R. Nandhakumar et al. / European Journal of Medicinal Chemistry 42 (2007) 1128e1136
aberrations in the lower doses. Thus, in future this class of
quinoline intermediates and novel diazepino quinolines may
be used as templates to generate better drugs to combat bacte-
rial and fungal infections.
was removed under reduced pressure. It was further recrystal-
lized with chloroform.
Compound 5a: m.p. 155 ꢀC. IR (KBr): 3438, 1664, 1595.
1H NMR (CDCl3, 400 MHz) d: 7.7 (d, 1H, C8-H,
J ¼ 8.1 Hz), 7.6 (t, 1H, C7-H, J ¼ 8.3 Hz), 7.9 (t, 1H, C6-H,
J ¼ 7.3 Hz), 8.2 (d, 1H, C5-H, J ¼ 8.3 Hz), 9.2 (s, 1H, C3-
CHO), 9.4 and 9.6 (2s, 2H, vinylic protons), 16.5 (bs, vi-
nylic-OH, D2O exchangeable). 13C NMR (CDCl3, 400 MHz)
d: 192.419 (eCHO), 189.332 (eCH]CHe), 189.08 (e
CH]CHe), 146.24, 137.30, 135.90, 133.32, 126.93, 125.21,
122.61, 119.32, 118.99 (aryl carbons). Anal. Calcd. for
C12H8NO2Cl (%): C, 61.69; H, 3.45; N, 5.99. Found: C,
61.62; H, 3.38; N, 5.92. MS (EI, 70 eV) m/z 233 (15.2%)
(Mþ), 235 (4.8%) (M þ 2), 206 (66.2%), 164 (35.9%), 190
(100%).
6. Experimental
Thin layer chromatography was used to access the reactions
and the purity of products. Melting points were determined on
a Boetius Microheating Table and Mettler-FP5 melting appa-
ratus and are uncorrected. IR spectra were recorded in Shi-
madzu-8201FT instrument in KBr disc and only noteworthy
1
absorption levels (reciprocal centimeter) are listed. H NMR
spectra were recorded in a AMX-400 MHz spectrometer in
CDCl3 solution; chemical shifts are expressed in parts per
million (d) relative TMS, coupling constants (J ) in hertz and
signal multiplicities are represented by bs (broad singlet), s
(singlet), d (doublet), t (triplet) and m (multiplet). 13C NMR
were also recorded on the same AMX-400 MHz spectrometer
with tetramethylsilane (TMS) as internal standard. Mass spec-
tra were recorded on a Jeol-D300 mass spectrometer. CHN
analyses were carried out on a Carlo Erba 106 and Perkine
Elmer Model 240 analysers.
Compound 6a: m.p. 140 ꢀC. IR (KBr): 3520, 1695, 1610.
1H NMR (CDCl3, 400 MHz) d: 2.4 (s, 3H, CH3), 7.3 (d, 1H,
C8-H, J ¼ 7.84 Hz), 7.5 (t, 1H, C7-H, J ¼ 7.58 Hz), 7.7 (t,
1H, C6-H, J ¼ 7.6 Hz), 8.2 (d, 1H, C5-H, J ¼ 8.28 Hz), 9.4
(s, 1H, CHO), 14.1 (bs, 1H, OH). Anal. Calcd. for
C11H9NO2 (%): C, 70.58; H, 4.85; N, 7.48. Found: C, 70.54;
H, 4.81; N, 7.43. MS (EI, 70 eV) m/z 187 (Mþ).
Compound 7a: m.p.65 ꢀC. IR (KBr): 1590. 1H NMR
(CDCl3, 400 MHz) d: 2.5 (s, 3H, CH3), 7.3 (s, 1H, C3-H),
8.1 (d, 1H, C5-H, J ¼ 8.2 Hz), 7.7 (t, 1H, C6-H,
J ¼ 7.54 Hz), 7.9 (t, 1H, C7-H, J ¼ 7.82 Hz), 7.5 (d, 1H, C8-
H, J ¼ 8.14 Hz).
6.1. Typical procedures
6.1.1. VilsmeiereHaack reaction on 4-hydroxyquinaldines
The Vilsmeier reagent was prepared by taking N,N-dime-
thylformamide 3.86 mL (0.05 mol) in a round-bottomed flask
in an ice cold condition (0e5 ꢀC) with constant stirring. To
this, phosphorous oxychloride 13.04 mL (0.014 mol) was
added drop by drop for a period of half an hour and the re-
sultant mixture was stirred for a further 1 h. The appropriate
4-hydroxyquinaldines (0.02 mol) 4(aee) were added to the
Vilsmeier reagent and stirred for further half an hour and
the reaction mixture was kept in a water bath at 100 ꢀC for
the stipulated period of time. After the reaction has been
completed as inferred through thin layer chromatographic
studies (complete disappearance of the starting material),
the reaction mixture was poured into 500 g of crushed ice
with constant manual stirring. It was kept aside for overnight.
After neutralizing the above with 4 N sodium hydroxide so-
lution, the precipitate obtained was washed well with water
and extracted using ethyl acetate. The combined organic
layers were then collected and dried over anhydrous sodium
sulfate. The silica gel chromatography of the reaction mix-
ture afforded three products 7, 6 and 5 at pet. ether (100),
pet. ethereethyl acetate (94:6) and pet. ethereethyl acetate
(85:5), respectively. The products were recrystallized with
methanol.
Compound 17a: m.p. 182 ꢀC. IR (KBr): 3350, 1580, 1595.
1H NMR (CDCl3, 400 MHz) d: 5.2 (bs, 1H, eNH), 7.4 (s, 1H,
C1-H), 7.5e8.5 (m, 6H, Ar-H). Anal. Calcd. for C12H8N3Cl
(%): C, 62.76; H, 3.51; N, 18.30. Found: C, 62.69; H, 3.46;
N, 18.41. MS (EI, 70 eV) m/z 230 (Mþ).
Compound 5b: m.p. 132 ꢀC. IR (KBr): 3450, 1660, 1595.
1H NMR (CDCl3, 400 MHz) d: 2.8 (s, 3H, CH3), 7.5 (t, 1H,
C6-H, J ¼ 7.92 Hz), 7.7 (d, 1H, C7-H, J ¼ 7.16 Hz), 8.1 (d,
1H, C5-H, J ¼ 8.24 Hz), 9.2 (s, 1H, C3-CHO), 9.4 and 9.5
(2s, 2H, vinylic protons), 16.5 (bs, vinylic-OH, D2O exchange-
able). 13C NMR (CDCl3, 400 MHz) d: 192.359 (eCHO),
189.451 (eCH]CHe), 189.014 (eCH]CHe), 147.224,
136.948, 133.953, 130.233, 126.611, 126.390, 122.993,
121.964, 118.594 (aryl carbons), 18.459 (eCH3). Anal. Calcd.
for C13H10NO2Cl (%): C, 63.04; H, 4.07; N, 5.66. Found: C,
62.02; H, 3.98; N, 5.63. MS (EI, 70 eV) m/z 247 (16.5%)(Mþ),
(M þ 2) 249 (5.2%), 220 (66.7%), 204 (100%), 178 (36.5%).
Compound 6b: m.p.195 ꢀC. IR (KBr): 3500, 1710, 1590.
1H NMR (CDCl3, 400 MHz) d: 2.6 (s, 6H, 2 ꢂ CH3), 7.4 (t,
1H, C6-H, J ¼ 7.6 Hz), 7.6 (d, 1H, C7-H, J ¼ 7.28 Hz), 8.0
(d, 1H, C5-H, J ¼ 8.24 Hz), 9.3 (s, 1H, CHO), 15.3 (bs, 1H,
OH). Anal. Calcd. for C12H11NO2 (%): C, 71.63; H, 5.51;
N, 6.96; Found: C, 71.58; H, 5.44; N, 6.98. MS (EI, 70 eV)
m/z 201 (Mþ).
6.1.2. Synthesis of diazepino[4,5-b]quinolines
Compound 7b: m.p.47 ꢀC. IR (KBr): 1570. 1H NMR
(CDCl3, 400 MHz) d: 2.7 (s, 3H, C2-CH3), 2.8 (s, 3H, C8-
CH3), 7.4 (s, 1H, C3-H), 8.1 (d, 1H, C5-H, J ¼ 7.92 Hz), 7.4
(t, 1H, C6-H, J ¼ 7.2 Hz), 7.6 (d, 1H, C7-H, J ¼ 6.28 Hz).
Compound 17b: m.p. 110 ꢀC. IR (KBr): 3390, 1585, 1603.
1H NMR (CDCl3, 400 MHz) d: 2.3 (s, 3H, CH3), 5.0 (bs, 1H,
Hydrazine hydrate (0.002 mol) 16 and 4-chloro-3-formyl-
2-(2-hydroxy-ethene-1-yl)quinoline (0.002 mol) (5) were dis-
solved in 50 mL of absolute ethanol. They were kept at the
refluxing temperature for 3 h. After the completion of the
reaction inferred through the TLC monitoring, the ethanol

R. Nandhakumar et al. / European Journal of Medicinal Chemistry 42 (2007) 1128e1136
1135
eNH), 7.0 (s, 1H, C1-H), 7.29 (t, 1H, C9-H, J ¼ 7.72 Hz), 7.45
(d, 1H, C4-H, J ¼ 7.56 Hz), 7.58 (d, 1H, C5-H, J ¼ 7.00 Hz),
7.86 (d, 1H, C8-H, J ¼ 8.44 Hz), 8.02 (d, 1H, C10-H,
J ¼ 8.28 Hz). Anal. Calcd. for C13H10N3Cl (%): C, 64.07; H,
4.14; N, 17.24. Found: C, 64.15; H, 4.01; N, 17.11. MS (EI,
70 eV) m/z 244 (Mþ).
Compound 5e: m.p. 170 ꢀC. IR (KBr): 3525, 1680, 1595.
1H NMR (CDCl3, 400 MHz) d: 2.6 (s, 6H, 2 ꢂ CH3), 7.6 (d,
1H, C6-H, J ¼ 7.46 Hz), 7.9 (d, 1H, C7-H, J ¼ 7.96 Hz), 9.3
(s, 1H, eCHO), 9.4 and 9.5 (2s, 2H, vinylic protons), 16.5
(bs, vinylic-OH, D2O exchangeable). 13C NMR (CDCl3,
400 MHz) d: 192.34 (eCHO), 189.38 (eCH]CHe), 189.07
(eCH]CHe), 144.34, 136.38, 133.86, 130.95, 127.54,
126.01, 123.11, 122.45, 118.60 (aryl carbons), 19.80 (C5-
CH3), 18.95 (C8-CH3). Anal. Calcd. for C14H12NO2Cl (%):
C, 64.25; H, 4.62; N, 5.35. Found: C, 64.21; H, 4.57;
N, 5.28. MS (EI, 70 eV) m/z (Mþ) 261, (M þ 2) 263.
Compound 6e: m.p. 155 ꢀC. IR (KBr): 3510, 1702, 1610.
1H NMR (CDCl3, 400 MHz) d: 2.7 (s, 9H, 3 ꢂ CH3), 7.5 (d,
1H, C6-H, J ¼ 7.84 Hz), 7.7 (d, 1H, C7-H, J ¼ 7.68 Hz), 9.2
(s, 1H, CHO), 14.5 (bs, 1H, OH); Anal. Calcd. for
C13H13NO2 (%): C, 72.54; H, 6.09; N, 6.51. Found: C,
72.47; H, 6.01; N, 6.42. MS (EI, 70 eV) m/z (Mþ) 215.
Compound 7e: m.p. 80 ꢀC. IR (KBr): 1580. 1H NMR
(CDCl3, 400 MHz) d: 2.6 (s, 9H, 3 ꢂ CH3), 7.4 (s, 1H, C3-
H), 7.6e7.8 (m, 2H, Ar-H).
Compound 5c: m.p. 148 ꢀC. IR (KBr): 3480, 1670, 1590.
1H NMR (CDCl3, 400 MHz) d: 2.6 (s, 3H, CH3), 7.6 (d, 1H,
C6-H, J ¼ 7.56 Hz), 7.8 (s, 1H, C8-H), 8.1 (d, 1H, C5-H,
J ¼ 8.16 Hz), 9.2 (s, 1H, eCHO), 9.3 and 9.5 (2s, 2H, vinylic
protons), 16.5 (bs, vinylic-OH, D2O exchangeable). 13C NMR
(CDCl3, 400 MHz) d: 192.28 (eCHO), 189.41 (eCH]CHe),
189.09 (eCH]CHe), 145.26, 135.68, 134.06, 131.22,
126.28, 126.05, 122.75, 122.34, 119.70 (aryl carbons), 19.52
(eCH3). Anal. Calcd. for C13H10NO2Cl (%): C, 63.04; H,
4.07; N, 5.66. Found: C, 62.98; H, 4.01; N, 5.59. MS (EI,
70 eV) m/z (Mþ) 247, (M þ 2) 249.
Compound 6c: m.p. 215 ꢀC. IR (KBr): 3350, 1700, 1600.
1H NMR (CDCl3, 400 MHz) d: 2.5 (s, 6H, 2 ꢂ CH3), 7.6 (d,
1H, C6-H, J ¼ 7.14 Hz), 7.8 (s, 1H, C8-H), 8.0 (d, 1H, C5-H,
J ¼ 8.20 Hz), 9.4 (s, 1H, CHO), 15.1 (bs, 1H, OH). Anal.
Calcd. for C12H11NO2 (%): C, 71.63; H, 5.51; N, 6.96. Found:
C, 71.61; H, 5.46; N, 6.94. MS (EI, 70 eV) m/z 201 (Mþ).
Compound 7c: m.p. 92 ꢀC. IR (KBr): 1585. 1H NMR
(CDCl3, 400 MHz) d: 2.6 (s, 6H, 2 ꢂ CH3), 7.3 (s, 1H, C3-
H), 7.5 (s, 1H, C8-H), 7.8 (d, 1H, C6-H, J ¼ 7.24 Hz), 8.1
(d, 1H, C5-H, J ¼ 8.06 Hz).
Compound 17e: m.p. 190 ꢀC. IR (KBr): 3375, 1578, 1595.
1H NMR (CDCl3, 400 MHz) d: 2.6 (s, 6H, 2 ꢂ CH3), 5.0 (bs,
1H, eNH), 7.3 (s, 1H, C1-H), 7.5e8.4 (m, 4H, Ar-H). Anal.
Calcd. for C14H12N3Cl (%): C, 65.25; H, 4.69; N, 16.30.
Found: C, 65.19; H, 4.58; N, 16.19. MS (EI, 70 eV) m/z
(Mþ) 258.
Compound 17c: m.p. 128 ꢀC. IR (KBr): 3400, 1578, 1597.
1H NMR (CDCl3, 400 MHz) d: 2.5 (s, 3H, CH3), 5.3 (bs, 1H,
eNH), 7.2 (s, 1H, C1-H), 7.4e8.3 (m, 5H, Ar-H). Anal. Calcd.
for C13H10N3Cl (%): C, 64.07; H, 4.14; N, 17.24. Found: C,
64.01; H, 4.19; N, 17.21. MS (EI, 70 eV) m/z 244 (Mþ).
Compound 5d: m.p. 180 ꢀC. IR (KBr): 3470, 1670, 1590.
1H NMR (CDCl3, 400 MHz) d: 7.3 (d, 1H, C7-H,
J ¼ 7.68 Hz), 7.5 (d, 1H, C8-H, J ¼ 7.96 Hz), 8.1 (s, 1H, C5-
H), 9.2 (s, 1H, C3-CHO), 9.3 and 9.5 (2s, 2H, vinylic protons),
16.5 (bs, vinylic-OH, D2O exchangeable). 13C NMR (CDCl3,
400 MHz) d: 192.27 (eCHO), 189.26 (eCH]CHe), 189.08
(eCH]CHe), 133.90, 133.21, 129.80, 129.04, 124.41,
121.13, 120.86, 119.87, 118.34 (aryl carbons). Anal. Calcd.
for C12H7NO2Cl2 (%): C, 53.76; H, 2.63; N, 5.26. Found: C,
53.71; H, 2.65; N, 5.22. MS (EI, 70 eV) m/z (Mþ) 267,
(M þ 2) 269, (M þ 4) 271.
Acknowledgements
TS thank CSIR, New Delhi, India for the award of Senior
Research Fellowship. SIF, Indian Institute of Science, Banga-
lore and Central Drug and Research Institute, Lucknow sup-
ported the spectral details.
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