Indian Journal of Chemistry

Vol. 52B, December 2013, pp 1521-1526

In-silico, in-vitro antibacterial activity and toxicity profile of

new quinoline derivatives
P P Sambavekara, M M Aitawadea, D R Patilb, G B Kolekarb, M B Deshmukhb & P V Anbhule*b
a
Department of Agrochemicals and Pest Management, Shivaji University, Kolhapur 416 004, India
b
Department of Chemistry, Shivaji University, Kolhapur 416 004, India
E-mail: pvanbhule2011@gmail.com
Received 15 January 2013; accepted (revised) 29 August 2013

The new substituted quinoline derivatives have been synthesized and characterized by various spectroscopic
techniques. A prediction of activity spectra for substances (PASS) of synthesized quinoline compounds showed their
probabilities of being active for the antibacterial activities. Therefore, all the newly synthesized compounds have been
evaluated for their in-vitro antibacterial activity against Staphylococcus aureus and Escherichia coli. It has been observed
that 6-chloro-4-phenylquinoline-2,3-dicarboxylic acid 4a selectively active against Gram (-)ve E. coli and 8-chloro-10-
phenyl-2,3-dihydropyridazino[4,5-b]quinoline-1,4-dione 5a showed selectivity against Gram (+)ve S. aureus. PASS
prediction study indicates that the compound 5a as a potential candidate with a Pa 0.671, maximum from the series.
According to the results obtained from brine shrimp (Artemia salina) lethality bioassay the compounds prove to be non
toxic. The computer aided results are in good agreement with experimental results.

Keywords: Quinoline derivatives, PASS, Antibacterial activity, Staphylococcus aureus, Escherichia coli, Brine shrimp

A new approach has been used to investigate new
bioactive molecules by means of in-silico techniques
which are fast and cost efficient in todays medicinal
chemistry research1. The prediction of activity spectra
for substances (PASS) is widely used as in-silico
technique2. It is based on a strong analysis of structure
activity relationships. It provides more accurate
predictions for compounds belonging to new chemical
classes and to extend the predictable area of new
biological activities3. This approach can be used at
earliest stage of investigation, since only the structural
formula of chemical compound is necessary to obtain
PASS prediction. The results of prediction are
valuable for planning of the experiment. If the value
of Pa is more, there is less probability of false
positives in the set of compounds selected for
biological testing.
Chemical and pharmaceutical industries and
research institutions need techniques that are capable
of identifying adverse effect at an early stage of
product development. This information comes
rationally from in-vivo testing, but due to the public
pressure to reduce animal experiments and the lack of
important toxicity information for many old
compounds has lead to an increased acceptance of
alternative, in-silico method. PASS is also used for
the prediction of toxic properties of chemical
structures. It has been help to derive predictions for
new structures from a database with experimentally
determined toxicity data4.
Most of the todays drugs in the market are
heterocyclic compounds; among these heterocycles,
quinoline is important one. The quinoline containing
compounds have wide range of applications such as
antibacterial5, anticancer6, anti-malarial7 and
8
antifungal . Due to such enormous importance, it has
become the synthetic target of many organic and
medicinal chemistry groups9. Recently, it has been
observed that certain bacteria have developed
resistance against well-known antibiotics10. This fact
decreases the efficiency of todays antibiotics which
inspired us to search for new antibacterial molecules
with high efficiency and less toxicity11,12.
In continuation of our earlier work on synthesis of
tri-substituted quinoline derivatives13 it encouraged us
to synthesize different derivatives of quinolines based
on the results of PASS and check their in-vitro
antibacterial activity. So, here in we wish to report the
synthesis, antibacterial activity and their general
toxicity with brine shrimp (Artemia salina) lethality
assay.
1522 INDIAN J. CHEM., SEC B, DECEMBER 2013

2 2 2
R O R O R O
1 1 1
R R R
OH i O CH3 iii NH

OH ii O CH3 iV NH
N N N

O O O
3A-D 5A-D
4A-D
1 2
R : 3A,3D:Cl-; 3B,3C:H- R :3A,3B:C 6H5-: 3C:CH3-; 3D:2Cl-C 6H4-
0
i) EtOH, aq. KOH, Reflux 2hrs. ii) Water, 1:1 HCl, 0-5 C

iii) EtOH, hydrazine hydrate, Reflux 2hrs. iV) Glacial acetic acid, Reflux 2hrs

Scheme I

Table I PASS prediction for antibacterial activity and toxicity parameters

Compd Antibacterial activity Mutagenic toxicity Carcinogenic toxicity

Paa Pib Pa Pi Pa Pi
4a 0.619 0.049 0.176 0.064 0.000 0.000
4b 0.567 0.096 0.171 0.067 0.184 0.145
4c 0.208 0.091 0.000 0.000 0.000 0.000
4d 0.556 0.108 0.146 0.084 0.272 0.079
5a 0.671 0.044 0.149 0.081 0.329 0.055
5b 0.626 0.092 0.145 0.085 0.238 0.099
5c 0.571 0.092 0.141 0.088 0.227 0.106
5d 0.617 0.051 0.119 0.107 0.342 0.051
a
Possibility of active, bPossibility of inactive.

Result and Discussion I). The synthesized dihydroxypyridazinoquinoline

Chemistry
The synthesis of target compounds involves
preparation of 2,3,4-trisubstituted quinolines 3a-d.
They were synthesized from different aromatic amino
ketones 1a-d and diethyl acetelylenedicarboxylate
(DEAD) 2 in ethanol as reported by Patil et al.14 and
confirmed by spectroscopic methods. The dicarboxy-
quinoline derivatives 4a-d are prepared from the
corresponding quinoline dicarboxylates 3a-d by
alkaline hydrolysis, which was acidified with 1:1 HCl
at low temperature15 (Scheme I). The compounds 4b-
d are characterized by spectroscopic techniques like
IR, 1H NMR, 13C NMR, GCMS and LCMS data. The
data obtained is in good agreement with the proposed
structure. For the synthesis of
dihydroxypyridazinoquinoline derivatives 5a-d
(Scheme I), the reaction of corresponding
dicarboxylicacidesters 3a-d and hydrazine hydrate
was carried out in ethanol as a solvent. The salt
obtained was then treated with glacial acetic acid to
get the desired products. All the products were
obtained in good yield and in high purity16 (Scheme
derivatives 5b-d were confirmed by the spectroscopic
techniques.
Pharmacology
Prediction of biological activity spectra
Pharma Expert is an in-silico tool for predicting
biological activity. The input MDL Mol files (*.mol)
of synthesized compounds were sketched with the
help of ACD/ChemSketch version 12.0 software.
Details of the method used for biological activity
prediction are carried out by the reported methods17,18.
Prediction of antibacterial activity
The prediction of probabilities of being active (Pa)
and inactive (Pi) for the different biological activities
for dicarboxyquinoline and dihydroxypyridazino-
quinoline were studied. The obtained results help to
study antibacterial activity. The selected biological
activities showed higher value of Pa in comparison
with that of Pi values indicates that compounds have
maximum possibilities of activity (Table I).
 SAMBAVEKAR et al.: NEW QUINOLINE DERIVATIVES 1523

Figure I Graphical representation of activity and toxicity

Table II In-vitro antibacterial activity Prediction of mutagenic and carcinogenic toxicity

S. aureus E. coli The mutagenic and carcinogenic effect of the
Compd
50 (g/mL) 100 (g/mL) 50 (g/mL) 100 (g/mL) synthesized compounds were predicted by the use of
4a -- 04a 16 25 PASS toxicity database. From the dicarboxyquinoline
derivatives, 4a and 4c compounds do not show any
4b -- -- 13 22 carcinogenic property. The compound 4c also did not
4c -- -- 11 20 show any mutagenic characters. In the
4d -- 08 15 23 dihydroxypyridazinoquinoline derivatives, all the
compounds showed very less mutagenic and
5a 15 24 -- --
carcinogenic properties. These results proved that the
5b 09 12 -- 05 compounds are comparatively less toxic in nature
5c 12 20 -- 04 (Table I).
5d 14 22 05 12
The trends of toxicity can be clearly seen in
Ampicillin Figure I. Although compound 4c which has no
(10 g/mL) 23 20 chlorine atom do not show any mutagenic and
a
Zone of inhibition values are given in millimeters, carcinogenic characters, has less antibacterial activity
-- no inhibition, NT=Not taken too. The compound 4a do not show any carcinogenic
property and simultaneously less mutagenic in nature
(Figure I).
SAR analysis
In-vitro antibacterial activity
To correlate the PASS prediction with that of
actual results obtained on screening, a SAR study has To check the theoretical prediction with that of
been done. PASS prediction study indicated that the practical results, we have carried out in-vitro testing
compound 5a as a potential candidate with a Pa 0.671, against two bacteria, S. aureus (ATCC 6538) from
maximum from the series. Gram (+)ve and E. coli (ATCC 8739) from Gram
However, the same PASS prediction indicated it to (-) ve category at two different concentrations.
be more carcinogenic toxicity. The general trend The antibacterial screening results showed that all
being that the activity decreases from 4a to 4c has a the compounds except 4c has only moderate activity
slight increase in case of compound 4d, whereas against E. coli from 4a-d series. Whereas the
compounds belonging to pyridazinoquinoline series compounds belonging to pyridazinoquinoline series
expected to be increasing from 5a to 5d (Figure I). has activity shifted to Gram (+)ve bacteria (Table II).
1524 INDIAN J. CHEM., SEC B, DECEMBER 2013
Table III The brine shrimp lethality assay
Compd LC50a LFLb
4a 402.35 359.93
5a 442.45 394.98
a b
Lethal concentration, Lower fiducidal limit, Upper fiducidal limit, Chi square limit.
Brine shrimp lethality assay
The brine shrimp toxicity test and acute oral
toxicity of rodent and human have correlation19.
Therefore, the general toxicity of synthesized
compounds has been studied. The probit analysis was
done on the probit program version 1.5, Ecological
Monitoring Division, Environmental Monitoring
System Laboratory, U.S., Environmental Protection
Agency, Cincinnati, Ohio 45268. The regression
equation was plot from the dose response curve. The
compound 4a has LC50 402.35 g/mL, while 5a has
442.45 g/mL (Table III).
Experimental Section
The melting points are uncorrected and were
determined in an open capillary. Infrared spectra (in
KBr pellets) were measured on a Perkin Elmer 100
spectrophotometer. The 1H and 13C NMR spectra
were recorded on a Bruker Spectrospin Avance II-300
MHz spectrometer using CDCl3 and DMSO-d6
solvents and tetramethylsilane as an internal standard.
Chemical shifts are given in the delta scale (ppm).
Mass spectra were analyzed on a Shimadzu QP 2010
GCM. The LCMSMS used from Applied Bio-system
API-4000 AB-SCIEX. Diethyl acetylenedicarboxylate
was purchased from Aldrich and used as it is. The
homogeneity of the compounds was checked by using
TLC Silica gel 60-F254 plates.
General procedure for synthesis of dicarboxy-
quinoline derivatives, 4a-d
The stirred solution of dicarboxylic acid ester 3a
(0.767 g, 2 mmol) and aq. potassium hydroxide (20
mole% in 0.5 mL water) in 5 mL ethanol, was
refluxed for 2 hr. The reaction mixture was then
cooled to RT. The precipitated solid material was
filtered and washed with 10 mL of ethanol. The
product was then dissolved in distilled water and
acidified with 1:1 aqueous HCl. The precipitate
formed was filtered and washed with distilled water,
recrystalized from ethanol and interpreted by
spectroscopic data.
UFLb Regression equation x2 d
472.65 Y=3.65x-4.52 2.74
530.63 Y=4.29x-6.36 1.14
c d
Spectroscopic data of dicarboxyquinoline deri-
vatives
6-Chloro-4-phenylquinoline-2,3-dicarboxylic
acid, 4a: Colourless solid; Weight 0.569 g; Yield
87%; m.p. 210C; IR (KBr) 3426, 2922, 1923, 1746,
1636 cm-1; 1H NMR (300 MHz, CDCl3): 11.23 (s,
1H, Ar-COOH), 11.13 (s, 1H, Ar-COOH), 8.18- 8.15
(d, 1H, Ar-H), 7.76-7.72 (1H, dd, Ar-H), 7.51-7.49
(m, 3H, Ar-H), 7. 41 -7.40 (d, 1H, Ar-H), 7.33-7.32
(m, 2H, Ar-H); 13C NMR (300 MHz, CDCl3):
167.90, 166.53, 147.34, 145.87, 144.98, 134.54,
134.41, 132.09, 131.62, 129.51, 129.05, 128.68,
128.55, 128.23, 125.15; MS: (m/z-18) 309 (M+)
4-Phenylquinoline-2,3-dicarboxylic acid, 4b:
White solid; Weight 0.498 g; Yield 85%; m.p. 195C;
IR (KBr) 3436, 3080, 2541, 2012, 1740, 1615, 1597
cm-1; 1H NMR (300 MHz, CDCl3): 11.32 (s, 1H, Ar-
COOH), 11.21 (s, 1H, Ar-COOH), 8.19-8.16 (d, 1H,
Ar-H), 7.87-7.82 (t, 1H, Ar-H), 7.65-7.60 (m, 2H, Ar-
H), 7.51-7.49 (m, 3H, Ar-H), 7.35-7.33 (m, 2H, Ar-
H); 13C NMR (300 MHz, CDCl3): 168.16, 167.01,
147.41, 146.74, 146.50, 135.09, 131.20, 130.12,
129.63, 129.28, 128.90, 128.54, 127.67, 127.24,
126.61; MS: (m/z-19) 374 (M+)
4-Methylquinoline-2,3-dicarboxylic acid, 4c:
White solid; Weight 0.378 g; Yield 82%; m.p. 190C;
IR (KBr): 3495, 2924, 2499, 1942, 1720, 1719 cm-1;
1
H NMR (300 MHz, CDCl3): 10.40 (s, 1H, Ar-
COOH), 9.96 (s, 1H, Ar-COOH), 8.25-8.23 (d, 1H,
Ar-H), 8.12-8.06 (d, 1H, Ar-H), 7.91-7.86 (t, 1H, Ar-
H), 7.81 -7.76 (t, 1H, Ar-H), 2.73 (s, 3H, CH3); 13C
NMR (300 MHz, CDCl3): 169.02, 167.42, 148.00,
145.86, 143.44, 131.46, 130.36, 129.30, 127.76,
125.38, 15.75; MS: (m/z) 232.1 (M+)
6-Chloro-4-(2-chlorophenyl)quinoline-2,3-dicar-
boxylic acid, 4d: White solid; Weight 0.579 g; Yield
80%; m.p. 120C; IR (KBr): 3427, 2923, 2565, 1934,
1727, 1559 cm-1; 1H NMR (300 MHz, CDCl3):
11.33 (s, 1H, Ar-COOH), 11.01 (s, 1H, Ar-COOH),
8.21-8.12 (t, 1H, Ar-H), 7.82-7.79 (d, 1H, Ar-H),
7.61-7.45 (m, 3H, Ar-H), 7. 33-7.30 (d, 1H, Ar-H),
7.18 (s, 1H, Ar-H); 13C NMR (300 MHz, CDCl3):
 SAMBAVEKAR et al.: NEW QUINOLINE DERIVATIVES
167.36, 166.76, 144.98, 143.74, 134.81, 133.57,
133.12, 132.32, 132.02, 131.33, 130.98, 129.73,
128.43, 127.59, 127.39, 124.75; MS: (m/z) 362 (MH+)
General procedure for synthesis of dihydroxypyri-
dazinoquinoline derivatives, 5a-d
The stirred solution of dicarboxylic acid ester 3a
(0.767 g, 2 mmol) and (0.303 g, 6 mmol) hydrazine
hydrate in 5 mL ethanol were refluxed for 2 hr. After
that reaction mixture was cooled to RT and the
precipitated solid material was filtered and washed
with 10 mL ethanol. The solid obtained was acidified
by adding excess acetic acid (15 mL) and refluxed for
2 hr. After cooling to RT, the crystalline precipitate
was obtained. It is then filtered and washed with
ethanol and air dried.
Spectroscopic data of dihydroxypyridazinoquino-
line derivatives
8-Chloro-10-phenyl-2,3-dihydropyridazino[4,5-
b]quinoline-1,4-dione, 5a: Yellow solid; Weight
0.517 g; Yield 80%; m.p. >320C; IR (KBr): 3216,
3066, 1656, 1603 cm-1; 1H NMR (300 MHz, CDCl3):
8.29-8.26 (d, 1H, Ar-H), 7.78-7.77 (d, 1H, Ar-H),
7.75-7.74 (d, 1H, Ar-H), 7.71-7.69 (d, 1H, -NH),
7.45-7.43 (m, 3H, Ar-H), 7.39-7.38 (d, 1H, -NH),
7.19-7.16 (m, 2H, Ar-H); 13C NMR (300 MHz,
CDCl3): 189.45, 134.96, 134.62, 133.76, 133.08,
131.96, 129.64, 128.51, 128.10, 127.90, 127.08,
126.36, 125.99, 124.52, 122.65, 121.08; MS: (m/z)
323 (M+)
10-Phenyl-2,3-dihydropyridazino[4,5-b]quino-
line-1,4-dione, 5b: Yellow solid; Weight 0.450 g;
Yield 78%; m.p. >320C; IR (KBr): 3133, 3058,
1694, 1645, 1553 cm-1; 1H NMR (300 MHz, CDCl3):
8.31-8.28 (d, 1H, Ar-H), 7.96 (t, 1H, Ar-H), 7.63 (t,
1H, Ar-H), 7.48-7.46 (m, 4Ar-H, 1-NH), 7.23 (s, 2H,
Ar-H), 1.88 (s, 1H, -NH); 13C NMR (300 MHz,
CDCl3): 172.33, 162.00, 149.50, 137.02, 132.61,
130.10, 128.89, 128.75, 127.90, 127.85, 127.53,
102.00; MS: (m/z) 289 (M+)
10-Methyl-2,3-dihydropyridazino[4,5-b]quino-
line-1,4-dione, 5c: Creamy solid; Weight 0.340 g;
Yield 75%; m.p. >320C; IR (KBr): 3167, 3029,
2927, 1659, 1602, 1559 cm-1; 1H NMR (300 MHz,
CDCl3): 11.55 (br s, 1H, -NH), 10.89 (br s, 1H,
-NH), 8.51-8.48 (d, 1H, Ar-H), 8.23-8.20 (d, 1H, Ar-
H), 8.06-7.79 (m, 1H, Ar-H), 7.59-7.53 (t, 1H, Ar-H),
2.07 (s, 3H, -CH3); 13C NMR (300 MHz, CDCl3):
192.52, 182.08, 130.49, 128.80, 126.03, 116.53,
15.76; MS: (m/z) 227 (M+)
1525
In-vitro antibacterial testing
Antibacterial activity was tested by disc diffusion
method on nutrient agar plates. The organisms used in
the present study were obtained from the laboratory
stock. The subculture was prepared in the sterilized
nutrient broth. In this method, first 15-20 mL of
molten agar was poured into each sterile plate. The
incubated plates were allowed to stand for 15 min.
before applying paper disc. Using sterile forceps the
paper absorbent disc of 6 mm diameter coated with
respective chemical was placed on the surface of
plate. The plates were incubated at temperature of
37C. The solutions were prepared at different
concentrations in ppm. DMSO is used as solvent. The
control was run under similar condition to know
activity of blank. Ampicillin (10 g/mL) is used as a
standard reference compound. Antimicrobial activity
was evaluated by measuring the diameter (mm) of
zone of inhibition around each disc. The zone of
inhibition for standard reference compound
Ampicillin (10 g/mL) is 23 mm and 20 mm for S.
aureus and E. coli, respectively. An examination of
the data reveals that most of the compounds showed
antibacterial activity against standard reference
Ampicillin.
Brine shrimp lethality assay
The bioassay experiment was performed according
to the procedure described by Meyer et al.20 The
nauplii were drawn in a glass capillary along with
water, and ten of such shrimps were transferred to
each sample in the stem of capillary against lighted
background. The two compounds, 4a and 5a which
have good antibacterial activity are selected for final
toxicity study. In each experiment, 0.5 mL of the
solution of drug was added to 4.5 mL of brine
solution. The above experiment was done in
triplicates. In control vial added 4.5 mL of brine
solution and 0.5 mL artificial sea water with 0.2%
DMSO. After 24 hr, percent deaths counted and LC50
values were calculated by probit analysis described by
Finneys method21.
Conclusion
Synthesis of targeted quinoline derivatives as
potential antibacterial agents has been reported. The
6-chloro-4-phenylquinoline-2,3-dicarboxylic acid 4a
was selectively active against E. Coli while 8-chloro-
10-phenyl-2,3-dihydropyridazino[4,5-b]quinoline-1,4-
dione 5a was active against S. aureus. The brine
1526 INDIAN J. CHEM., SEC B, DECEMBER 2013
shrimp general toxicity assay shown that compounds
4a and 5a are very less toxic having LC50
402.35 g/mL and 442.45 g/mL respectively which
is the main outcome of this research finding. The
positive PASS results of the said compounds are good
agreement with the experimental observations.
Acknowledgement
One of author (PVA) is highly thankful to
University Grant Commission, New Delhi for
financial assistance through major research project.
[F. NO. 39-786/2010 (SR)]
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