Hydrazones of 2-aryl-quinoline-4-carboxylic acid hydrazides: Synthesis and preliminary evaluation as antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2006.08.022

A new series of 2-arylquinoline-4-carboxylic acid hydrazide-hydrazones was synthesized using an appropriate synthetic route. All the target compounds were evaluated for their in vitro antimicrobial activity against Staphylococcus aureus as an example for

Full text of DOI:10.1016/j.bmc.2006.08.022

Bioorganic & Medicinal Chemistry 14 (2006) 8675–8682
Hydrazones of 2-aryl-quinoline-4-carboxylic acid hydrazides:
Synthesis and preliminary evaluation as antimicrobial agents
Kamel A. Metwally,a,* Lobna M. Abdel-Aziz,a El-Sayed M. Lashine,a
Mohamed I. Husseinyb and Rania H. Badawya
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
bDepartment of Microbiology, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
Received 19 April 2006; revised 15 August 2006; accepted 15 August 2006
Available online 1 September 2006
Abstract—A new series of 2-arylquinoline-4-carboxylic acid hydrazide–hydrazones was synthesized using an appropriate synthetic
route. All the target compounds were evaluated for their in vitro antimicrobial activity against Staphylococcus aureus as an example
for Gram-positive bacteria, Escherichia coli as an example for Gram-negative bacteria, and Candida albicans as a representative of
fungi. The minimum inhibitory concentration (MIC) was determined for test compounds as well as for reference standards. Among
the compounds tested, compounds having nitro substituents at the arylidene moiety showed the most potent antifungal as well as
antibacterial activities against E. coli. Compound 23 displayed an antifungal activity comparable to that of nystatin. However, none
of the compounds demonstrated any antibacterial activity against S. aureus. Hydrophobicity of the target compounds correlated
weakly with their antibacterial and antifungal activities. The most potent compounds namely, 7, 18, 19, 22, and 23 were assessed
for hemolytic toxicity and found to be non-hemolytic up to a concentration of 100 lg/mL. In addition, the most potent compound
(23) was evaluated for in vitro cytotoxic activity against various cancer cell lines. This compound was found to display no cytotoxic
activity but rather it induces the proliferation rate of Hep-G2 cells.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Being a structural building block in many natural prod-
ucts, the quinoline ring is one of the most commonly
encountered heterocycles in medicinal chemistry. Litera-
ture survey revealed that substituted quinolines possess
diverse chemotherapeutic activities including antibacte-
rial,6,7 antifungal,8,9 antiamoebic,10,11 antileishma-
nial,12,13 antimalarial,14,15 and antitumor activities.16,17
Meanwhile, hydrazide–hydrazones have been claimed
to exhibit appreciable antimicrobial activity.18–21 On
the basis of these observations, we had the impetus to
synthesize a number of hydrazide–hydrazones of the
synthetically accessible 2-arylquinoline-4-carboxylic
acids and subsequently evaluate their in vitro
antimicrobial activity.
The dramatically rising prevalence of multi-drug resis-
tant microbial infections in the past few decades has be-
come a serious health care problem. In particular, the
emergence of multi-drug resistant strains of Gram-posi-
tive bacterial pathogens such as methicillin-resistant
Staphylococcus aureus and Staphylococcus epidermis
and vancomycin-resistant Enterococcus is a problem of
ever-increasing significance.1–5 One way to counterbal-
ance this challenge is the controlled use of the currently
marketed antibiotics; the other is the development of
novel antimicrobial agents. Consequently, the search
for new antimicrobial agents will always remain an
important and challenging task for medicinal chemists.
2. Chemistry
The synthesis of the target compounds was carried out
as outlined in Scheme 1. The versatile Pfitzinger reac-
tion22 was utilized to synthesize the starting 2-arylquin-
oline-4-carboxylic acids (1a–j) in satisfactory yields
by reacting isatin or substituted isatins with
Keywords: Pfitzinger reaction; 2-Arylquinoline-4-carboxylic acids;
Hydrazones; Antimicrobial activity.
*
Corresponding author. Tel.: +20552308021; fax: +20552303266;
e-mail: kametwally@hotmail.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.08.022

8676
K. A. Metwally et al. / Bioorg. Med. Chem. 14 (2006) 8675–8682
COOH
O
O
X
X
a
C
H3
O
+
N
H
N
R
(1a-j)
R
b
CONHNH2
COCl
X
X
c
N
N
(3a-j)
(2a-j)
R
R
d
R`
CONHN
C
H
X
X = H, Cl
N
R = H, Cl, Br, CH3, OCH3
R` = H, 4-Cl, 4-Br, 4-OCH3, 2-NO2, 4-NO2
(4-23)
R
Scheme 1. Synthetic route for the target compounds. Reagents and conditions: (a) NaOH, aq EtOH; (b) SOCl2, dry benzene; (c) NH2NH2ÆH2O,
EtOH; (d) substituted benzaldehydes, HOAc.
a-methylketones in aqueous ethanol. The acids were
subsequently treated with thionyl chloride in refluxing
benzene to give the corresponding acid chlorides (2a–j)
which were used directly to prepare the hydrazides
(3a–j) through reaction with hydrazine hydrate in reflux-
ing ethanol. The requisite hydrazones (4–23) were
obtained by condensing the acid hydrazides (3a–j) with
the appropriate aldehydes in glacial acetic acid under
reflux conditions.
employed as the reference antifungal agent. The MIC
values for test compounds as well as reference standards
are given in Table 1.
The obtained results revealed that the nature of substit-
uents and substitution pattern on the quinoline ring may
have a considerable impact on the antibacterial and
antifungal activities of the target hydrazones. Of partic-
ular importance, a nitro group in the arylidene side
chain generally enhances the antifungal activity and
antibacterial activity against E. coli. In this context, para
substitution appears to be more beneficial for activity
compared to the ortho substitution pattern. In the ab-
sence of nitro substituents in the arylidene moiety, how-
ever, the introduction of a chloro group at C-6 of the
quinoline nucleus imparts appreciable antibacterial
activity against E. coli. On the other hand, the nature
of substituent at the 2-phenyl ring appears to have little
influence on antimicrobial activity. Consistent with
these results, it was not surprising that compound 23
was found to be the most potent among the test hydraz-
ones. Interestingly, all the target compounds were found
to be devoid of antibacterial activity against S. aureus.
Log P values were calculated for the target compounds
using ACD/LogP software and plotted against antibac-
terial and antifungal activities expressed as ꢀlog MIC
values as illustrated in Figures 1 and 2. The hydrophobic
character of the test compounds correlated weakly with
antibacterial (r = 0.38) and antifungal (r = 0.11) activi-
ties with lipophilicity optima of approximately 4.98
and 5.31, respectively.
The new hydrazones were characterized by their physi-
cal, analytical, and spectral data. IR spectra of the target
hydrazones showed NH and C@O stretching bands at
3140–3190 and 1650–1660 cmꢀ1
, respectively. The
absorption bands associated with other functional
groups appeared in the expected regions. In the 1H
NMR spectra of the target hydrazones, the azomethine
proton appeared at 8.3–8.6 ppm as a sharp D2O non-
exchangeable singlet, whereas a broad more downfield
D2O exchangeable singlet at 12.1–12.6 ppm was charac-
teristic of the NH group. The other protons appeared at
the expected chemical shifts and integral values. All the
target hydrazones were microanalyzed satisfactorily for
C, H, and N.
3. Results and discussion
All the newly synthesized hydrazones were assayed in vi-
tro for antibacterial activity against S. aureus (example
for Gram-positive bacteria) and Escherichia coli (exam-
ple for Gram-negative bacteria), and the antifungal
activity was evaluated against Candida albicans. The
MIC values were determined by the twofold serial dilu-
tion technique in Mueller–Hinton broth and Sabouraud
dextrose broth for the antibacterial and antifungal as-
say, respectively. For comparison, ampicillin was used
as the reference antibacterial agent and nystatin was
Compounds that exhibited the most potent antibacte-
rial and antifungal activities namely, 7, 18, 19, 22, and
23, were evaluated for hemolytic toxicity and minimal
hemolytic concentrations (MHCs) were determined.
Up to a concentration of 100 lg/mL, the test com-
pounds showed no hemolytic activity. Even at as high

K. A. Metwally et al. / Bioorg. Med. Chem. 14 (2006) 8675–8682
Table 1. MIC values for the target hydrazones
8677
Compound
log Pa
MIC (lg/mL)
Gram-positive bacteria
Staph. Aureus ATCC 25923
Gram-negative bacteria
E. coli ATCC 10536
Fungi C. albicans
ATCC 10231
4
5.04 0.41
5.97 0.48
6.15 0.54
5.32 0.49
4.29 0.41
5.22 0.48
5.69 0.40
5.12 0.41
5.87 0.42
6.05 0.49
5.58 0.41
6.44 0.43
5.81 0.44
5.87 0.48
5.97 0.51
5.79 0.50
5.69 0.43
5.87 0.50
5.03 0.44
4.85 0.44
—
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
12.5
>200
>200
>200
25–50
>200
>200
>200
50
50–100
50–100
100–200
25–50
100–200
50
5
6
7
8
9
10
50–100
50–100
50–100
25–50
50–100
50–100
50–100
100–200
50–100
50
11
12
50–100
100
50
13
14
15
16
50
50–100
100
17
18
25–50
25–50
50
19
20
50
21
22
50
25–50
25–50
25
25–50
25–50
25
23
Ampicillin
Nystatin
—
25
—
—
—
a log P values were calculated using ACD/LogP software.
5
5
4.5
4
23
22
7
21
19
13
4.5
9
20
22
7
19
18
23
4
11
14 10 16
5,18
15
14 20
21
11
15
12
17
16 12
17
8
6
13
4
3.5
3
3.5
4.5
5
5.5
Log P
6
6.5
4
4.5
5
5.5
Log P
6
6.5
7
Figure 1. Plot of hydrophobicity of the test compounds versus
antibacterial activity against E. coli.
Figure 2. Plot of hydrophobicity of the test compounds versus
antifungal activity.
concentration as 1 mg/mL, the tested compounds
exhibited only 5% hemolysis (Fig. 3). Furthermore,
compound 23, being the most potent in the series,
was evaluated for in vitro cytotoxic activity against
Hep-G2, MCF-7, and 1301 cell lines. This compound
led to a significant induction of the proliferation rate
of Hep-G2 cells (P < 0.01) to around 1.6-fold of the
control cells and this maximum induction seems
unchanged with the increasing of the dose. The tested
compound administration to Raw macrophage 264.7
resulted in a significant elevation in growth rate
(P < 0.05), while it exhibited insignificant increment
in the cell growth of lymphoblastic leukemia (1301).
On the other hand, the tested doses have led to static
unchangeable rate of growth in the treated MCF-7
cells (Fig. 4).
7
6
Cpd No. 7
Cpd No. 18
Cpd No. 19
Cpd No. 22
5
4
3
2
1
0
Cpd No. 23
0
1
2
3
4
Log Concentration
Figure 3. Plot of log concentration versus percentage lyses of test
compounds 7, 18, 19, 22, and 23.

8678
K. A. Metwally et al. / Bioorg. Med. Chem. 14 (2006) 8675–8682
200
160
120
80
C16H11Cl2N3O: C, 57.85; H, 3.34; N, 12.65. Found: C,
58.00; H, 3.39; N, 12.54.
4.4. 6-Chloro-2-(4-bromophenyl)quinoline-4-carboxylic
acid hydrazide (3b)
Yield 82%; mp 253–255 ꢁC (EtOH); IR (KBr, cmꢀ1):
3268 (NHs), 3048, 2922, 1640 (C@O), 1586, 1542,
1
1485, 1074, 885, 828, 722. H NMR (DMSO-d6) d: 5.3
(s, 2H, NH2, D2O-exchangeable), 7.4–7.6 (m, 2H, Ar–
H), 7.6–7.7 (m, 1H, Ar–H), 7.8–7.9 (m, 1H, Ar–H),
8.2–8.3 (m, 2H, Ar–H), 8.3 (s, 1H, Ar–H), 9.1–9.2 (d,
1H, Ar–H), 9.8 (s, 1H, CONH, D2O-exchangeable).
Anal. Calcd for C16H11BrClN3O: C, 51.02; H, 2.94; N,
11.16. Found: C, 51.36; H, 3.18; N, 11.24.
40
0
0
25
Concentration (µM)
Hep-G2 RAW 264.7
1301
50
75
100
4.5. 6-Chloro-2-(4-methylphenyl)quinoline-4-carboxylic
acid hydrazide (3c)
Figure 4. Plot of concentration of test compounds versus cell viability.
Yield 69%; mp 249–251 ꢁC (EtOH); IR (KBr, cmꢀ1):
3261 (NHs), 3057, 2926, 1639 (C@O), 1586, 1542,
1489, 1445, 1352, 1036, 885, 754, 704. 1H NMR
(DMSO-d6) d: 2.4 (s, 3H, CH3), 5.1 (s, 2H, NH2), 7.3–
7.4 (d, 2H, Ar–H), 7.8–7.9 (d, 1H, Ar–H), 8.1–8.2 (m,
3H, Ar–H), 8.4 (s, 1H, Ar–H), 8.6 (s, 1H, Ar–H), 9.9
(s, 1H, CONH). Anal. Calcd for C17H14ClN3O: C,
65.49; H, 4.53; N, 13.48. Found: C, 65.50; H, 4.85; N,
13.73.
4. Experimental protocols
4.1. Syntheses
Melting points were determined on a Gallenkamp melt-
ing point apparatus and are uncorrected. IR spectra
were recorded on a Pye Unicam SP 1000 IR spectropho-
1
tometer as KBr pellets. H NMR spectra were recorded
on
a
Varian-Mercury 200 MHz spectrometer in
DMSO-d6. Chemical shifts were expressed in parts per
million (ppm) with tetramethylsilane (TMS) as an inter-
nal standard. MS spectra were measured with an HP
5995 instrument. Elemental analyses (C, H, and N) were
performed at the National Research Centre, Cairo,
Egypt. All compounds were routinely checked by
thin-layer chromatography (TLC) on aluminum-backed
silica gel plates. All solvents were dried by standard
methods. Compounds (1a–j)23–27 and (3e–g)23,24,28–30
were synthesized according to literature procedures.
4.6. 6-Chloro-2-(4-methoxyphenyl)quinoline-4-carboxylic
acid hydrazide (3d)
Yield 68%; mp 233–236 ꢁC (EtOH); IR (KBr, cmꢀ1):
3282 (NHs), 3034, 2962, 1634 (C@O), 1584, 1542,
1517, 1350, 1250, 1174, 1033, 835, 732. 1H NMR
(DMSO-d6) d: 3.9 (s, 3H, CH3), 4.7 (s, 2H, NH2, D2O
exchangeable), 7.1–7.2 (d, 2H, Ar–H), 7.7–7.8 (d, 1H,
Ar–H), 8.0–8.1 (m, 2H, Ar–H), 8.2–8.3 (m, 3H, Ar–
H), 10.1 (s, 1H, CONH, D2O exchangeable). Anal.
Calcd for C17H14ClN3O2: C, 62.30; H, 4.31; N, 12.82.
Found: C, 62.01; H, 4.70; N, 13.13.
4.2. General method for the synthesis of 6-substituted-2-
arylquinoline-4-carboxylic acid hydrazides (3a–d)
4.7. General method for the synthesis of 6-substituted-2-
arylquinoline-4-carboxylic acid hydrazide–hydrazones (4–23)
A mixture of 6-substituted-2-arylquinoline-4-carboxylic
acids (1a–d; 10 mmol), thionyl chloride (2.4 g, 20 mmol)
in dry benzene (50 mL) was heated at reflux for 4 h. Ben-
zene was distilled under reduced pressure and the resi-
due was dissolved in ethanol (50 mL), hydrazine
hydrate (85%; 3 mL) was added, and the mixture was
heated at reflux for 4 h. The reaction mixture was evap-
orated under reduced pressure and the crude product
was purified by recrystallization from ethanol.
A mixture of 6-substituted-2-arylquinoline-4-carboxylic
acid hydrazides (3a–j; 10 mmol) and the appropriate
aromatic aldehyde (10 mmol) in glacial acetic acid
(50 mL) was heated at reflux for 4–8 h. The reaction
mixture was concentrated under reduced pressure,
cooled, and the obtained solid was filtered, washed with
water and cold ethanol. The crude products were puri-
fied by recrystallization from the appropriate solvent.
4.3. 6-Chloro-2-(4-chlorophenyl)quinoline-4-carboxylic
acid hydrazide (3a)
4.8. 2-(4-Chlorophenyl)quinoline-4-carboxylic acid
benzylidene-hydrazide (4)
Yield 77%; mp 256–258 ꢁC (EtOH); IR (KBr, cmꢀ1):
3265 (NHs), 3057, 2924, 1642 (C@O), 1586, 1540,
1
Yield 65%; mp 239–241 ꢁC (EtOH); H NMR (DMSO-
1
1488, 1090, 886, 830, 723. H NMR (DMSO-d6) d: 4.9
d6) d: 7.1–7.2 (m, 1H, Ar–H), 7.4–7.5 (m, 2H, Ar–H),
7.6–7.9 (complex m, 6H, Ar–H), 8.1–8.3 (m, 2H, Ar–
H), 8.3–8.5 (m, 4H, Ar–H), 12.2 (s, 1H, CONH). Anal.
Calcd for C23H16ClN3O: C, 71.60; H, 4.18; N, 10.89.
Found: C, 71.31; H, 4.16; N, 10.72.
(s, 2H, NH2, D2O-exchangeable), 7.5–7.7 (m, 2H, Ar–
H), 7.8–7.9 (m, 1H, Ar–H), 8.1–8.2 (m, 2H, Ar–H),
8.3–8.4 (d, 2H, Ar–H), 8.8–8.9 (d, 1H, Ar–H), 9.9 (s,
1H, CONH, D2O-exchangeable). Anal. Calcd for

K. A. Metwally et al. / Bioorg. Med. Chem. 14 (2006) 8675–8682
8679
4.9. 2-(4-Bromophenyl)quinoline-4-carboxylic acid
(4-chlorobenzylidene)-hydrazide (5)
Anal. Calcd for C24H18ClN3O: C, 72.09; H, 4.54; N,
10.51. Found: C, 71.84; H, 4.35; N, 10.32.
1
Yield 73%; mp 269–271 ꢁC (EtOH); H NMR (DMSO-
4.15. 6-Chloro-2-phenylquinoline-4-carboxylic acid
benzylidene-hydrazide (11)
d6) d: 7.1–7.3 (dd, 1H, Ar–H), 7.5–7.6 (d, 1H, Ar–H),
7.6–7.9 (m, 5H, Ar–H), 8.1–8.3 (m, 3H, azomethine
CH, Ar–H), 8.3–8.5 (m, 4H, Ar–H), 12.3 (s, 1H,
CONH). MS (m/z): 465 (M+, 14.1%), 326 (54.7%), 312
(42.9%), 284 (53.2%), 203 (base peak), 101 (43.6%), 75
(64.0%). Anal. Calcd for C23H15BrClN3O: C, 59.44; H,
3.25; N, 9.04. Found: C, 59.29; H, 3.07; N, 8.94.
1
Yield 68%; mp 244–246 ꢁC (EtOH); H NMR (DMSO-
d6) d: 7.1–7.3 (m, 2H, Ar–H), 7.4–7.6 (m, 3H, Ar–H),
7.6–7.8 (m, 5H, Ar–H), 8.1-8.4 (m, 5H, azomethine
CH, Ar–H), 12.2 (s, 1H, NH, D2O-exchangeable). Anal.
Calcd for C23H16ClN3O: C, 71.60; H, 4.18; N, 10.89.
Found: C, 71.64; H, 4.19; N, 10.82.
4.10. 2-(4-Bromophenyl)quinoline-4-carboxylic acid
(4-bromobenzylidene)-hydrazide (6)
4.16. 6-Chloro-2-phenylquinoline-4-carboxylic acid
(4-chlorobenzylidene)-hydrazide (12)
Yield 80%; mp 264–266 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 7.1–7.2 (d, 1H, Ar–H), 7.2–7.3 (d, 1H,
Ar–H), 7.5–7.7 (m, 4H, Ar–H), 7.8–8.0 (m, 3H, Ar–
H), 8.1–8.3 (m, 2H, Ar–H), 8.3–8.5 (m, 3H, azomethine
CH, Ar–H), 12.3 (s, 1H, CONH). Anal. Calcd for
C23H15Br2N3O: C, 54.25; H, 2.97; N, 8.25. Found: C,
54.15; H, 3.06; N, 8.16.
Yield 71%; mp 276–277 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 7.1–7.3 (dd, 1H, Ar–H), 7.5–7.7 (m, 4H,
Ar–H), 7.7–7.9 (m, 3H, Ar–H), 8.1–8.3 (m, 3H, azome-
thine CH, Ar–H), 8.3–8.5 (m, 3H, Ar–H), 12.3 (s, 1H,
NH). Anal. Calcd for C23H15Cl2N3O: C, 65.73; H,
3.60; N, 10.00. Found: C, 65.65; H, 3.69; N, 9.99.
4.11. 2-(4-Bromophenyl)quinoline-4-carboxylic acid
(2-nitrobenzylidene)-hydrazide (7)
4.17. 6-Chloro-2-phenylquinoline-4-carboxylic acid
(4-bromobenzylidene)-hydrazide (13)
1
Yield 66%; mp 259–261 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 7.1–7.2 (d, 1H, Ar–H), 7.4–7.5 (m, 1H,
Ar–H), 7.6–7.8 (m, 3H, Ar–H), 7.8–7.9 (m, 2H, Ar–
H), 8.1–8.3 (m, 3H, Ar–H), 8.2–8.3 (m, 2H, Ar–H),
8.4 (s, 1H, azomethine CH), 8.8 (s, 1H, Ar–H), 12.5 (s,
1H, CONH). Anal. Calcd for C23H15BrN4O3: C,
58.12; H, 3.18; N, 11.79. Found: C, 58.00; H, 2.92; N,
11.58.
Yield 63%; mp 262–264 ꢁC (EtOH); H NMR (DMSO-
d6) d: 7.1–7.3 (dd, 1H, Ar–H), 7.5–7.7 (m, 5H, Ar–H),
7.8–8.0 (m, 3H, Ar–H), 8.1–8.3 (m, 2H, Ar–H), 8.3–
8.5 (m, 3H, Ar–H), 12.3 (s, 1H, NH). Anal. Calcd for
C23H15BrClN3O: C, 59.44; H, 3.25; N, 9.04. Found: C,
59.73; H, 3.04; N, 8.92.
4.18. 6-Chloro-2-(4-methylphenyl)quinoline-4-carboxylic
acid benzylidene-hydrazide (14)
4.12. 2-(4-methoxyphenyl)quinoline-4-carboxylic acid
benzylidene-hydrazide (8)
1
Yield 70%; mp 291–294 ꢁC (EtOH); H NMR (DMSO-
d6) d: 2.1 (s, 3H, CH3), 7.2–7.3 (m, 2H, Ar–H), 7.4–7.5
(m, 2H, Ar–H), 7.6–7.9 (m, 5H, Ar–H), 8.1–8.3 (m,
2H, Ar–H), 8.3-8.4 (m, 2H, Ar–H), 8.4 (s, 1H, azome-
thine CH), 12.2 (s, 1H, NH). Anal. Calcd for
C24H18ClN3O: C, 72.09; H, 4.54; N, 10.51. Found: C,
71.76; H, 4.16; N, 10.91.
1
Yield 67%; mp 257–260 ꢁC (EtOH); H NMR (DMSO-
d6) d: 3.9 (s, 3H, OCH3), 7.0–7.1 (d, 2H, Ar–H), 7.6–7.7
(d, 2H, Ar–H), 7.8–7.9 (d, 2H, Ar–H), 8.2–8.3 (d, 2H,
Ar–H), 8.3–8.5 (m, 5H, azomethine CH, Ar–H), 12.1 (s,
1H, CONH). Anal. Calcd for C24H19N3O2: C, 75.57; H,
5.02; N, 11.02. Found: C, 75.23; H, 5.37; N, 10.86.
4.19. 6-Chloro-2-(4-chlorophenyl)quinoline-4-carboxylic
acid (4-chlorobenzylidene)-hydrazide (15)
4.13. 2-(4-Methoxyphenyl)quinoline-4-carboxylic acid
(4-bromobenzylidene)-hydrazide (9)
Yield 79%; mp 287–289 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 7.2–7.3 (dd, 1H, Ar–H), 7.4–7.5 (m, 2H,
Ar–H), 7.6–7.7 (m, 2H, Ar–H), 7.8–7.9 (m, 2H, Ar–
H), 8.1–8.2 (m, 1H, Ar–H), 8.3 (s, 1H, azomethine
CH), 8.3–8.4 (m, 3H, Ar–H), 8.5 (s, 1H, Ar–H), 12.3
(s, 1H, NH). Anal. Calcd for C23H14Cl3N3O: C, 60.75;
H, 3.10; N, 9.24. Found: C, 60.37; H, 3.04; N, 8.92.
1
Yield 75%; mp 278–280 ꢁC (EtOH); H NMR (DMSO-
d6) d: 3.8 (s, 3H, CH3), 7.1–7.2 (m, 3H, Ar–H), 7.4–7.5
(d, 1H, Ar–H), 7.5–7.8 (complex m, 5H, Ar–H), 8.1–8.2
(m, 2H, Ar–H), 8.3–8.4 (m, 3H, Ar–H), 12.2 (s, 1H,
CONH). Anal. Calcd for C24H18BrN3O2: C, 62.62; H,
3.94; N, 9.13. Found: C, 62.27; H, 3.72; N, 8.78.
4.14. 2-(4-Methylphenyl)quinoline-4-carboxylic acid
(4-chlorobenzylidene)-hydrazide (10)
4.20. 6-Chloro-2-(4-chlorophenyl)quinoline-4-carboxylic
acid (4-methoxybenzylidene)-hydrazide (16)
Yield 74%; mp 241–243 ꢁC (EtOH/H2O); 1H NMR
(DMSO-d6) d: 1.8 (s, 3H, CH3), 7.1–7.3 (d, 2H,
Ar–H), 7.3–7.6 (m, 4H, Ar–H), 7.7–7.8 (d, 1H, Ar–H),
7.8–7.9 (d, 1H, Ar–H), 7.9–8.3 (complex m, 5H, Ar–
H), 8.4 (s, 1H, azomethine CH), 12.3 (s, 1H, CONH).
Yield 72%; mp 274–276 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 3.9 (s, 3H, OCH3), 7.1–7.2 (m, 2H,
Ar–H), 7.5–7.6 (m, 1H, Ar–H), 7.8–7.9 (m, 2H, Ar–
H), 8.1–8.2 (m, 3H, Ar–H), 8.2–8.4 (m, 4H, Ar–H),
8.4 (s, 1H, azomethine CH), 12.4 (s, 1H, NH). Anal.

8680
K. A. Metwally et al. / Bioorg. Med. Chem. 14 (2006) 8675–8682
Calcd for C24H17Cl2N3O2: C, 64.01; H, 3.81; N, 9.33.
Found: C, 63.97; H, 4.07; N, 9.38.
8.1–8.2 (m, 2H, Ar–H), 8.2–8.3 (m, 2H, Ar–H), 8.3–
8.4 (m, 2H, Ar–H), 8.4 (s, 1H, azomethine CH), 8.8 (s,
1H, Ar–H), 12.6 (s, 1H, NH). Anal. Calcd for
C24H17ClN4O4: C, 62.55; H, 3.72; N, 12.16. Found: C,
62.19; H, 3.47; N, 11.99.
4.21. 6-Chloro-2-(4-bromophenyl)quinoline-4-carboxylic
acid benzylidene-hydrazide (17)
1
Yield 69%; mp 277–279 ꢁC (EtOH); H NMR (DMSO-
4.27. 6-Chloro-2-(4-methoxyphenyl)quinoline-4-carboxyl-
ic acid (4-nitrobenzylidene)-hydrazide (23)
d6) d: 7.1–7.3 (dd, 1H, Ar–H), 7.6–7.8 (m, 5H, Ar–H),
7.8–8.0 (m, 2H, Ar–H), 8.1–8.3 (m, 2H, Ar–H), 8.3–
8.5 (m, 4H, Ar–H), 12.3 (s, 1H, NH). Anal. Calcd for
C23H15BrClN3O: C, 59.44; H, 3.25; N, 9.04. Found: C,
59.75; H, 3.60; N, 8.66.
Yield 71%; mp 287–290 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 3.9 (s, 3H, OCH3), 7.0–7.2 (m, 3H, Ar–
H), 7.4–7.5 (d, 2H, Ar–H), 7.8–7.9 (m, 2H, Ar–H), 8.4
(s, 1H, azomethine CH), 8.1–8.6 (complex m, 5H, Ar–
H), 12.6 (s, 1H, NH). Anal. Calcd for C24H17ClN4O4:
C, 62.55; H, 3.72; N, 12.16. Found: C, 62.56; H, 4.04;
N, 12.14.
4.22. 6-Chloro-2-(4-bromophenyl)quinoline-4-carboxylic
acid (2-nitrobenzylidene)-hydrazide (18)
Yield 62%; mp 284–286 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 7.2–7.3 (d, 1H, Ar–H), 7.5–7.6 (m, 1H,
Ar–H), 7.7–8.0 (m, 4H, Ar–H), 8.1–8.3 (m, 3H, Ar–
H), 8.3–8.4 (m, 2H, Ar–H), 8.5 (s, 1H, azomethine
CH), 8.8 (s, 1H, Ar–H), 12.6 (s, 1H, NH). Anal. Calcd
for C23H14ClBrClN4O3: C, 54.19; H, 2.77; N, 10.99.
Found: C, 54.00; H, 2.64; N, 10.71.
4.28. Biology
4.28.1. Antimicrobial activity. All the test compounds
were assayed in vitro for antibacterial activity against
S. aureus ATCC 25923 (representative for Gram-posi-
tive bacteria) and E. coli ATCC 10536 (representative
for Gram-negative bacteria), and the antifungal activity
was evaluated against C. albicans ATCC 10231. The
strains used in this study were maintained at the Micro-
biology Department, Faculty of Pharmacy, Zagazig
University. The MIC was determined by using twofold
serial dilution method.31–33 Ampicillin and Nystatin
were used as reference standards to compare the anti-
bacterial and antifungal activities, respectively. For
determining both antibacterial and antifungal activities,
the synthesized compounds and the control drugs were
dissolved in a mixture of sterile water and dimethylform-
amide (8:2, the stock solution 5 mg/mL). Further dilu-
tions were prepared at the required quantities of 400,
200, 100, 50, 25, 12.5, 6.25, and 3.125 lg/mL concentra-
tions. In order to ensure that the solvent had no effect on
bacterial growth, a control test was also performed con-
taining broth supplemented with only DMF at the same
dilution used in our experiment. The MIC values were
obtained from the lowest concentration of the test com-
pound where the tubes remain clear, indicating that the
bacterial growth was completely inhibited at this con-
centration. The MIC values were expressed in lg/mL
and the results are shown in Table 1.
4.23. 6-Chloro-2-(4-bromophenyl)quinoline-4-carboxylic
acid (4-nitrobenzylidene)-hydrazide (19)
1
Yield 74%; mp 298–300 ꢁC (EtOH); H NMR (DMSO-
d6) d: 7.4–7.5 (m, 1H, Ar–H), 7.6–7.8 (m, 3H, Ar–H),
7.8–7.9 (m, 2H, Ar–H), 8.1–8.3 (m, 3H, Ar–H), 8.3–
8.4 (m, 2H, Ar–H), 8.5 (s, 1H, azomethine CH), 8.8 (s,
1H, Ar–H), 12.5 (s, 1H, NH). Anal. Calcd for
C23H14BrClN4O3: C, 54.19; H, 2.77; N, 10.99. Found:
C, 53.65; H, 2.77; N, 10.99.
4.24. 6-Chloro-2-(4-methoxyphenyl)quinoline-4-carboxyl-
ic acid (4-chlorobenzylidene)-hydrazide (20)
Yield 72%; mp 295–297 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 3.9 (s, 3H, OCH3), 7.01–7.2 (d, 2H, Ar–
H), 7.4–7.5 (complex m, 5H, Ar–H), 7.5–7.9 (m, 2H,
Ar–H), 8.1–8.2 (m, 2H, Ar–H), 8.3–8.4 (m, 3H, azome-
thine CH, Ar–H), 12.1 (s, 1H, NH). Anal. Calcd for
C24H17Cl2N3O2: C, 64.01; H, 3.81; N, 9.33. Found: C,
63.62; H, 3.73; N, 9.48.
4.25. 6-Chloro-2-(4-methoxyphenyl)quinoline-4-carboxyl-
ic acid (4-bromobenzylidene)-hydrazide (21)
4.28.1.1. Antibacterial assay. The cultures were ob-
tained in Mueller–Hinton broth for all the bacteria after
24 h of incubation at 37 ꢁC. Testing was carried out on
Mueller–Hinton broth at pH 7.4 and the twofold serial
dilution technique was applied. The final inoculum size
was 106 CFU/mL. A set of tubes containing only inocu-
lated broth was kept as control. After incubation for
24 h at 37 ꢁC, the last tube with no growth of microor-
ganism was recorded to represent MIC expressed in
lg/mL. Every experiment in the antibacterial assay
was replicated twice in order to define the MIC values.
Yield 69%; mp >300 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 3.9 (s, 3H, OCH3), 7.0–7.2 (m, 3H, Ar–
H), 7.4–7.5 (d, 1H, Ar–H), 7.6–7.8 (m, 3H, Ar–H),
7.8–7.9 (m, 1H, Ar–H), 8.1–8.2 (d, 1H, Ar–H), 8.2 (s,
1H, azomethine CH), 8.3–8.4 (m, 3H, Ar–H), 12.3 (s,
1H, NH). Anal. Calcd for C24H17BrClN3O2: C, 58.26;
H, 3.46; N, 8.49. Found: C, 57.89; H, 3.37; N, 8.65.
4.26. 6-Chloro-2-(4-methoxyphenyl)quinoline-4-carboxyl-
ic acid (2-nitrobenzylidene)-hydrazide (22)
4.28.1.2. Antifungal assay. The yeast C. albicans was
maintained in Sabouraud dextrose broth after incuba-
tion for 24 h at 35 ꢁC. Testing was performed in Sabou-
raud dextrose broth at pH 7.4 and the twofold serial
Yield 62%; mp 258–260 ꢁC (DMF/EtOH); 1H NMR
(DMSO-d6) d: 3.9 (s, 3H, OCH3), 7.1–7.2 (d, 2H, Ar–
H), 7.7–7.8 (m, 1H, Ar–H), 7.8–8.0 (m, 2H, Ar–H),

K. A. Metwally et al. / Bioorg. Med. Chem. 14 (2006) 8675–8682
8681
dilution technique was applied. The final inoculum size
was 105 CFU/mL. A set of tubes containing only inocu-
lated broth was kept as control. After incubation for
48 h at 35 ꢁC, the last tube with no growth of yeast
was recorded to represent MIC expressed in lg/mL.
Every experiment in the antifungal assay was replicated
twice in order to define the MIC values.
dimethylsulfoxide (DMSO, 99.9%, HPLC grade) and
diluted 1000-fold in the assay. The experiment was
repeated four times, and the data were represented as
(means SD). Results were compared with those of
DMSO-treated cells. All culture material was obtained
from Cambrex BioScience (Copenhagen, Denmark),
and all chemicals were from Sigma (USA).
4.28.2. Hemolytic assay. Fresh human blood (5 mL) was
centrifuged at 700g (3500 rpm) for 10 min and the eryth-
rocytes (RBCs) were collected. The erythrocytes were
washed and centrifuged three times with phosphate-buf-
fered saline (PBS, pH 7.4, 50 mL) and the supernatant
was carefully decanted. The erythrocyte suspension
was diluted twice with saline solution and total erythro-
cytes were counted using hemocytometer. Finally neces-
sary dilution was carried out with saline solution to get
total count to 6.4 · 108 cells/mL. The stock solution of
the test compounds was made at an initial concentration
of 4 mg/mL using the same saline solution. Assay was
carried out in 1 mL Eppendorf tubes. In an Eppendorf,
varying volumes (900–650 lL) of the saline solution
were added and subsequently varying volumes of the
stock solution of test compounds (1–250 lL) were added
followed by 100 lL RBC suspension. Plain saline solu-
tion was used as a negative control, whereas Triton X-
100 (10 lL of 20% w/v) was used as a positive control
that produces total cell lyses. The Eppendorf tubes were
then incubated at 37 ꢁC for 30 min, centrifuged at
4000 rpm for 10 min, and the supernatant was carefully
transferred to fresh Eppendorf tubes. The absorbance of
the samples was read at 404 nm with LKB-Biochrom
Novaspec-1049 spectrophotometer. The percentage
lyses was determined according to the following
equation:
4.28.3.2. MTT assay. Antiproliferative activity
against various tumor cell lines was estimated by the
3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoli-
um bromide (MTT) assay, which is based on the cleav-
age of the tetrazolium salt by mitochondrial
dehydrogenases in viable cells.35 Cells (5 · 104 cells/well)
of different cell lines were incubated with various con-
centrations of the tested compound at 37 ꢁC in a ser-
um-free medium, before submitted to MTT assay. The
relative cell viability was expressed as the mean percent-
age of viable cells compared with DMSO-treated cells
(Fig. 4).
Acknowledgment
We thank Dr. Amira Gamal-Eldeen, Cancer Biology
Laboratory, Department of Biochemistry, National
Research Center, Cairo, Egypt, for performing the
MTT assay.
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