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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
Department of Agrochemicals and Pest Management, Shivaji University, Kolhapur 416 004, India
Department of Chemistry, Shivaji University, Kolhapur 416 004, India
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 the prediction of toxic properties of chemical
bioactive molecules by means of in-silico techniques structures. It has been help to derive predictions for
which are fast and cost efficient in todays medicinal new structures from a database with experimentally
chemistry research1. The prediction of activity spectra determined toxicity data4.
for substances (PASS) is widely used as in-silico Most of the todays drugs in the market are
technique2. It is based on a strong analysis of structure heterocyclic compounds; among these heterocycles,
activity relationships. It provides more accurate quinoline is important one. The quinoline containing
predictions for compounds belonging to new chemical compounds have wide range of applications such as
classes and to extend the predictable area of new antibacterial5, anticancer6, anti-malarial7 and
biological activities3. This approach can be used at 8
antifungal . Due to such enormous importance, it has
earliest stage of investigation, since only the structural become the synthetic target of many organic and
formula of chemical compound is necessary to obtain medicinal chemistry groups9. Recently, it has been
PASS prediction. The results of prediction are observed that certain bacteria have developed
valuable for planning of the experiment. If the value resistance against well-known antibiotics10. This fact
of Pa is more, there is less probability of false decreases the efficiency of todays antibiotics which
positives in the set of compounds selected for inspired us to search for new antibacterial molecules
biological testing. with high efficiency and less toxicity11,12.
Chemical and pharmaceutical industries and
research institutions need techniques that are capable In continuation of our earlier work on synthesis of
of identifying adverse effect at an early stage of tri-substituted quinoline derivatives13 it encouraged us
product development. This information comes to synthesize different derivatives of quinolines based
rationally from in-vivo testing, but due to the public on the results of PASS and check their in-vitro
pressure to reduce animal experiments and the lack of antibacterial activity. So, here in we wish to report the
important toxicity information for many old synthesis, antibacterial activity and their general
compounds has lead to an increased acceptance of toxicity with brine shrimp (Artemia salina) lethality
alternative, in-silico method. PASS is also used for assay.

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

OH ii O CH3 iV NH

3A-D 5A-D
1 2
R : 3A,3D:Cl-; 3B,3C:H- R :3A,3B:C 6H5-: 3C:CH3-; 3D:2Cl-C 6H4-
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
Possibility of active, bPossibility of inactive.

Result and Discussion I). The synthesized dihydroxypyridazinoquinoline

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

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
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
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).

Table III The brine shrimp lethality assay

Compd LC50a LFLb UFLb Regression equation x2 d

4a 402.35 359.93 472.65 Y=3.65x-4.52 2.74
5a 442.45 394.98 530.63 Y=4.29x-6.36 1.14
a b c d
Lethal concentration, Lower fiducidal limit, Upper fiducidal limit, Chi square limit.

Brine shrimp lethality assay Spectroscopic data of dicarboxyquinoline deri-

The brine shrimp toxicity test and acute oral vatives
toxicity of rodent and human have correlation19.
Therefore, the general toxicity of synthesized
acid, 4a: Colourless solid; Weight 0.569 g; Yield
compounds has been studied. The probit analysis was
87%; m.p. 210C; IR (KBr) 3426, 2922, 1923, 1746,
done on the probit program version 1.5, Ecological
1636 cm-1; 1H NMR (300 MHz, CDCl3): 11.23 (s,
Monitoring Division, Environmental Monitoring
1H, Ar-COOH), 11.13 (s, 1H, Ar-COOH), 8.18- 8.15
System Laboratory, U.S., Environmental Protection
(d, 1H, Ar-H), 7.76-7.72 (1H, dd, Ar-H), 7.51-7.49
Agency, Cincinnati, Ohio 45268. The regression
(m, 3H, Ar-H), 7. 41 -7.40 (d, 1H, Ar-H), 7.33-7.32
equation was plot from the dose response curve. The
(m, 2H, Ar-H); 13C NMR (300 MHz, CDCl3):
compound 4a has LC50 402.35 g/mL, while 5a has
167.90, 166.53, 147.34, 145.87, 144.98, 134.54,
442.45 g/mL (Table III).
134.41, 132.09, 131.62, 129.51, 129.05, 128.68,
Experimental Section 128.55, 128.23, 125.15; MS: (m/z-18) 309 (M+)
4-Phenylquinoline-2,3-dicarboxylic acid, 4b:
The melting points are uncorrected and were White solid; Weight 0.498 g; Yield 85%; m.p. 195C;
determined in an open capillary. Infrared spectra (in IR (KBr) 3436, 3080, 2541, 2012, 1740, 1615, 1597
KBr pellets) were measured on a Perkin Elmer 100 cm-1; 1H NMR (300 MHz, CDCl3): 11.32 (s, 1H, Ar-
spectrophotometer. The 1H and 13C NMR spectra COOH), 11.21 (s, 1H, Ar-COOH), 8.19-8.16 (d, 1H,
were recorded on a Bruker Spectrospin Avance II-300 Ar-H), 7.87-7.82 (t, 1H, Ar-H), 7.65-7.60 (m, 2H, Ar-
MHz spectrometer using CDCl3 and DMSO-d6 H), 7.51-7.49 (m, 3H, Ar-H), 7.35-7.33 (m, 2H, Ar-
solvents and tetramethylsilane as an internal standard. H); 13C NMR (300 MHz, CDCl3): 168.16, 167.01,
Chemical shifts are given in the delta scale (ppm). 147.41, 146.74, 146.50, 135.09, 131.20, 130.12,
Mass spectra were analyzed on a Shimadzu QP 2010 129.63, 129.28, 128.90, 128.54, 127.67, 127.24,
GCM. The LCMSMS used from Applied Bio-system 126.61; MS: (m/z-19) 374 (M+)
API-4000 AB-SCIEX. Diethyl acetylenedicarboxylate 4-Methylquinoline-2,3-dicarboxylic acid, 4c:
was purchased from Aldrich and used as it is. The White solid; Weight 0.378 g; Yield 82%; m.p. 190C;
homogeneity of the compounds was checked by using IR (KBr): 3495, 2924, 2499, 1942, 1720, 1719 cm-1;
TLC Silica gel 60-F254 plates. 1
H NMR (300 MHz, CDCl3): 10.40 (s, 1H, Ar-
COOH), 9.96 (s, 1H, Ar-COOH), 8.25-8.23 (d, 1H,
General procedure for synthesis of dicarboxy- Ar-H), 8.12-8.06 (d, 1H, Ar-H), 7.91-7.86 (t, 1H, Ar-
quinoline derivatives, 4a-d H), 7.81 -7.76 (t, 1H, Ar-H), 2.73 (s, 3H, CH3); 13C
The stirred solution of dicarboxylic acid ester 3a NMR (300 MHz, CDCl3): 169.02, 167.42, 148.00,
(0.767 g, 2 mmol) and aq. potassium hydroxide (20 145.86, 143.44, 131.46, 130.36, 129.30, 127.76,
mole% in 0.5 mL water) in 5 mL ethanol, was 125.38, 15.75; MS: (m/z) 232.1 (M+)
refluxed for 2 hr. The reaction mixture was then 6-Chloro-4-(2-chlorophenyl)quinoline-2,3-dicar-
cooled to RT. The precipitated solid material was boxylic acid, 4d: White solid; Weight 0.579 g; Yield
filtered and washed with 10 mL of ethanol. The 80%; m.p. 120C; IR (KBr): 3427, 2923, 2565, 1934,
product was then dissolved in distilled water and 1727, 1559 cm-1; 1H NMR (300 MHz, CDCl3):
acidified with 1:1 aqueous HCl. The precipitate 11.33 (s, 1H, Ar-COOH), 11.01 (s, 1H, Ar-COOH),
formed was filtered and washed with distilled water, 8.21-8.12 (t, 1H, Ar-H), 7.82-7.79 (d, 1H, Ar-H),
recrystalized from ethanol and interpreted by 7.61-7.45 (m, 3H, Ar-H), 7. 33-7.30 (d, 1H, Ar-H),
spectroscopic data. 7.18 (s, 1H, Ar-H); 13C NMR (300 MHz, CDCl3):

167.36, 166.76, 144.98, 143.74, 134.81, 133.57, In-vitro antibacterial testing

133.12, 132.32, 132.02, 131.33, 130.98, 129.73,
Antibacterial activity was tested by disc diffusion
128.43, 127.59, 127.39, 124.75; MS: (m/z) 362 (MH+)
method on nutrient agar plates. The organisms used in
the present study were obtained from the laboratory
General procedure for synthesis of dihydroxypyri-
stock. The subculture was prepared in the sterilized
dazinoquinoline derivatives, 5a-d
nutrient broth. In this method, first 15-20 mL of
The stirred solution of dicarboxylic acid ester 3a
molten agar was poured into each sterile plate. The
(0.767 g, 2 mmol) and (0.303 g, 6 mmol) hydrazine
incubated plates were allowed to stand for 15 min.
hydrate in 5 mL ethanol were refluxed for 2 hr. After
before applying paper disc. Using sterile forceps the
that reaction mixture was cooled to RT and the
paper absorbent disc of 6 mm diameter coated with
precipitated solid material was filtered and washed
respective chemical was placed on the surface of
with 10 mL ethanol. The solid obtained was acidified
plate. The plates were incubated at temperature of
by adding excess acetic acid (15 mL) and refluxed for
37C. The solutions were prepared at different
2 hr. After cooling to RT, the crystalline precipitate
concentrations in ppm. DMSO is used as solvent. The
was obtained. It is then filtered and washed with
control was run under similar condition to know
ethanol and air dried.
activity of blank. Ampicillin (10 g/mL) is used as a
Spectroscopic data of dihydroxypyridazinoquino- standard reference compound. Antimicrobial activity
was evaluated by measuring the diameter (mm) of
line derivatives
8-Chloro-10-phenyl-2,3-dihydropyridazino[4,5- zone of inhibition around each disc. The zone of
b]quinoline-1,4-dione, 5a: Yellow solid; Weight inhibition for standard reference compound
Ampicillin (10 g/mL) is 23 mm and 20 mm for S.
0.517 g; Yield 80%; m.p. >320C; IR (KBr): 3216,
aureus and E. coli, respectively. An examination of
3066, 1656, 1603 cm-1; 1H NMR (300 MHz, CDCl3):
the data reveals that most of the compounds showed
8.29-8.26 (d, 1H, Ar-H), 7.78-7.77 (d, 1H, Ar-H),
antibacterial activity against standard reference
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,
Brine shrimp lethality assay
CDCl3): 189.45, 134.96, 134.62, 133.76, 133.08,
131.96, 129.64, 128.51, 128.10, 127.90, 127.08, The bioassay experiment was performed according
126.36, 125.99, 124.52, 122.65, 121.08; MS: (m/z) to the procedure described by Meyer et al.20 The
323 (M+) nauplii were drawn in a glass capillary along with
10-Phenyl-2,3-dihydropyridazino[4,5-b]quino- water, and ten of such shrimps were transferred to
line-1,4-dione, 5b: Yellow solid; Weight 0.450 g; each sample in the stem of capillary against lighted
Yield 78%; m.p. >320C; IR (KBr): 3133, 3058, background. The two compounds, 4a and 5a which
1694, 1645, 1553 cm-1; 1H NMR (300 MHz, CDCl3): have good antibacterial activity are selected for final
8.31-8.28 (d, 1H, Ar-H), 7.96 (t, 1H, Ar-H), 7.63 (t, toxicity study. In each experiment, 0.5 mL of the
1H, Ar-H), 7.48-7.46 (m, 4Ar-H, 1-NH), 7.23 (s, 2H, solution of drug was added to 4.5 mL of brine
Ar-H), 1.88 (s, 1H, -NH); 13C NMR (300 MHz, solution. The above experiment was done in
CDCl3): 172.33, 162.00, 149.50, 137.02, 132.61, triplicates. In control vial added 4.5 mL of brine
130.10, 128.89, 128.75, 127.90, 127.85, 127.53, solution and 0.5 mL artificial sea water with 0.2%
102.00; MS: (m/z) 289 (M+) DMSO. After 24 hr, percent deaths counted and LC50
10-Methyl-2,3-dihydropyridazino[4,5-b]quino- values were calculated by probit analysis described by
line-1,4-dione, 5c: Creamy solid; Weight 0.340 g; Finneys method21.
Yield 75%; m.p. >320C; IR (KBr): 3167, 3029,
2927, 1659, 1602, 1559 cm-1; 1H NMR (300 MHz, Conclusion
CDCl3): 11.55 (br s, 1H, -NH), 10.89 (br s, 1H, Synthesis of targeted quinoline derivatives as
-NH), 8.51-8.48 (d, 1H, Ar-H), 8.23-8.20 (d, 1H, Ar- potential antibacterial agents has been reported. The
H), 8.06-7.79 (m, 1H, Ar-H), 7.59-7.53 (t, 1H, Ar-H), 6-chloro-4-phenylquinoline-2,3-dicarboxylic acid 4a
2.07 (s, 3H, -CH3); 13C NMR (300 MHz, CDCl3): was selectively active against E. Coli while 8-chloro-
192.52, 182.08, 130.49, 128.80, 126.03, 116.53, 10-phenyl-2,3-dihydropyridazino[4,5-b]quinoline-1,4-
15.76; MS: (m/z) 227 (M+) dione 5a was active against S. aureus. The brine

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