Quinoline-azetidinone hybrids: Synthesis and in vitro antiproliferation activity against Hep G2 and Hep 3B human cell lines

Article abstract of DOI:10.1016/j.bmcl.2017.02.043

In search of new heterocyclic anticancer agents, a new quinoline-azetidinone hybrid template have been designed, synthesized and screened for their cytotoxic activity against human cancer cell lines such as Hep G2, and Hep 3B by the MTT assay and results were compared with paclitaxel, 5-fluorouracil and doxorubicin. Interestingly, some of the compounds were found significantly active against both cell lines. The compound 6f (IC₅₀?=?0.04?±?0.01?μM) exhibited potent antiproliferation activity against Hep G2 cell line, and 6j compound (IC₅₀?=?0.66?±?0.01?μM) demonstrated potent antiproliferation activity against Hep 3B cell line and provide to be more potent as cytotoxic agents than standard drugs. Morphological changes suggest the induction of apoptosis and describe the mechanism of action of these hybrid antitumor agents.

Full text of DOI:10.1016/j.bmcl.2017.02.043

Accepted Manuscript
Quinoline-azetidinone hybrids: Synthesis and in vitro antiproliferation activity
against Hep G2 and Hep 3B human cell lines
S.G. Alegaon, P. Parchure, L.D. Araujo, P.S. Salve, K.R. Alagawadi, S.S.
Jalalpure, V.M. Kumbar
PII:
S0960-894X(17)30165-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.043
BMCL 24715
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 October 2016
31 January 2017
15 February 2017
Please cite this article as: Alegaon, S.G., Parchure, P., Araujo, L.D., Salve, P.S., Alagawadi, K.R., Jalalpure, S.S.,
Kumbar, V.M., Quinoline-azetidinone hybrids: Synthesis and in vitro antiproliferation activity against Hep G2 and
Hep 3B human cell lines, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.02.043
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Quinoline-azetidinone hybrids: Synthesis and in vitro antiproliferation activity
against Hep G2 and Hep 3B human cell lines
Authors:
a
a
a
a
a
S.G. Alegaon *, P. Parchure , L. D. Araujo , P. S. Salve , K.R. Alagawadi , S. S.
Jalalpure b,c, V. M. Kumbarc
a
Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy,
Nehrunagar, Belagavi-590 010, Karnataka, India
b Department of Pharmacognosy KLE University’s College of Pharmacy
c BSRC Centre, KLE University, Nehru Nagar, Belagavi 590010, Karnataka, India
Address:
*Corresponding author: Shankar G. Alegaon, M.Pharm, Ph.D. Associate Professor,
Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy,
Nehrunagar, Belagavi-590 010, Karnataka, India. Tel.: +91 8312471399; Fax. +91
8312472387.
E-mail: sgalegaon@klepharm.edu, sgalegaon@gmail.com (S.G.Alegaon)
Abstract
In search of new heterocyclic anticancer agents, a new quinoline-azetidinone
hybrid template have been designed, synthesized and screened for their cytotoxic
activity against human cancer cell lines such as Hep G2, and Hep 3B by the MTT
assay and results were compared with paclitaxel, 5-fluorouracil and doxorubicin.
Interestingly, some of the compounds were found significantly active against both cell
lines. The compound 6f (IC50 = 0.04
0.01 µM) exhibited potent antiproliferation
0.01 µM)
activity against Hep G2 cell line, and 6j compound (IC50 = 0.66
demonstrated potent antiproliferation activity against Hep 3B cell line and provide to
be more potent as cytotoxic agents than standard drugs. Morphological changes
suggest the induction of apoptosis and describe the mechanism of action of these
hybrid antitumor agents.
Keywords: Quinoline, Azetidinone, antiproliferation, Drug likeness.
1

Cancer is a prevalent and second leading cause of death worldwide.1 It is
responsible for increase in the mortality rate and hence it has become a life
threatening disease affecting to all the people at all ages in both developing and
developed countries.2 Cancer is a disease which is characterized by uncontrolled cell
growth and proliferation;3 which is due to mutation or mis-regulation of cell cycle
regulatory genes and proteins.4 Altered apoptosis is one of the central steps in
cancer development. It has become an attractive approach for anticancer
therapeutics which can overcome the resistance to apoptosis by directly activating
the normal cell death machinery.5 Many efforts have been taken to fight against
cancer which includes successful treatment of certain tumor types and hence it still
continues to be challenge owing to their aggressiveness, the mechanism of
malignant cell metastasis, chemoresistance and lack of selectivity of drugs.
Therefore, the development of novel anticancer agents is required to synthesize
which would be safe and more effective.6
Literature data has been established that nitrogen containing heterocyclic
compounds plays an important role in designing new class of structural entities for
medicinal applications.7 The skeleton of quinoline is one of the key building elements
for a large number of natural and synthetic heterocycles. The derivatives of quinoline
that belong to this class have shown wide applications as drugs and
pharmaceuticals. Quinoline scaffolds have demonstrated that they have high ability
to draw out an anti-proliferative and antitumor activity based upon the reports on
natural, synthetic and semisynthetic biologically active molecules. They act through
different mechanism of action such as growth inhibitors by cell cycle arrest,
apoptosis, inhibition of angiogenesis, and disruption of cell migration and modulation
of nuclear receptor responsiveness. The two examples of cytotoxic quinolines with
antitumor activity are natural alkaloid camptothecin and its semisynthetic
analogtopotecan which act through inhibition of the DNA enzyme topoisomerase-I.
According to literature survey, it has been proven that quinoline derivatives have an
excellent class of broad spectrum anticancer activity against variety of cancer cell
lines such as breast cancer, hepatocellular carcinoma, and acute myelogenous
leukemia.8, 9 Certain drugs based on quinoline moiety such as bosutinib, lenvatinib
and cabozantinib have been established as one of the most effective class of
anticancer agents in clinical use with broad application in the treatment of several
types of cancer.9
2

The derivatives of quinolines also have varieties of biological activities like
anti-microbial,10 antitubercular,11-15 antimalarial,16-18 antibiotic,19 antihypertensive,20
anti-inflammatory,21 tyrokinase PDGF-RTK inhibition22 and anti-HIV properties.23,24
Few examples of quinoline derivatives include Quinine (antipyretic, antimalarial,
analgesic and anti-inflammatory properties), Chloroquine (antimalarial), Amidaquine
(antimalarial and anti-inflammatory properties), Camptothecin (DNA enzyme
topoisomerase II) and Saquinavir (anti-HIV drug), Ciprofloxacin and Moxifloxacin
(anti-TB drugs), which are actively used in pharmacological field.7 Azetidinone and
their derivatives have attracted interest in organic medicinal chemistry, exhibiting
biological and therapeutic properties, since the introduction of various monobactams
into clinical applications. Chemical modifications of these four–membered
heterocycles constantly result in compounds with
a
wide spectrum of
pharmacological activities such as anticancer, anti-microbial, anti-tubercular, anti-
inflammatory, anti-malarial, thrombin inhibitor, human leukocyte elastase inhibitor,
human cytomegalovirus inhibitor, cysteine protease cathepsin K inhibitor,
antidiabetic, and antiparkinsonian agents.25 In addition, recently, Geesala et al. in
2016 have reported azetidinone derivatives are potential anti-cancer agents.26 Based
on the above findings, in an attempt to pursue new potent anticancer agents with
potent inhibitory activity, a series of new quinoline-azetidinone compounds were
synthesized using molecular hybridization technique (Fig.1.)9, 26-28 and their inhibitory
activity against two human cancer cell lines (Hep G2, Hep 3B) were evaluated. In
addition, Molecular parameter and possible drug likeness were also illustrated.
3

Figure 1. Design of quinoline-azetidinone compound using molecular hybridization
technique
The reaction route employed for synthesis of the title compounds (6a-o) are
illustrated in Scheme 1. The two step procedure was adopted for the synthesis of
key intermediate 7-chloro-4-(2-(4-substitutedbenzylidene)hydrazinyl)quinoline (5a-o)
compounds. The 7-chloro-4-hydrazinylquinoline (3) was synthesized by
condensation of the 4, 7 dichloroquinoline with hydrazine hydrate.Reaction of the 7-
4

chloro-4-hydrazinylquinoline (3) with appropriate aryl/hetero aryl aldehydes in
ethanol afforded the corresponding 7-chloro-4-(2-(4-substitutedbenzylidene)
hydrazinyl) quinoline (5a-o) compounds. 29, 30 The prepared Schiff bases (5a-o) were
reacted with chloroacetyl chloride and trimethylamine in dichloromethane and
refluxed for 24 h. The reaction was monitored by TLC for completion. Mixture was
filtered, washed with dichloromethane to obtain 3-chloro-1-((7-chloroquinolin-4-
yl)amino)-4-(4-substituted phenyl)azetidin-2-one (6a-o) analogous.31The synthesized
1
quinoline-azetidinone (6a-o) hybrids were characterized by IR, H NMR, and mass
spectral analysis. The IR spectra of quinoline-azetidinone (6a-o) compounds
displayed absorption band at 1685 - 1710 cm-1 attributed to a carbonyl group of
azetidinone, their proton NMR spectra showed characteristic new singles around δ
4.25 ppm and δ 5.73 ppm attributed to (CH of C3 and C4) azetidinone protons,
which represents the formation of azetidinone ring system. The structures of the
synthesized quinoline-azetidinone (6a-o) hybrids were further confirmed by mass
spectra analysis.
Scheme 1. Reagents and conditions: (a) Reflux 8 h; (b) Dry ethanol, stirred at RT 24
h; (c) Chloroacetyl chloride, Dichloromethane, Triethylamine, reflux 24 h
The cytotoxicity activity of target compounds (6a-o) against Hep-G2 and Hep-
3B (human hepatocellular) carcinoma cell lines was measured by MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.32 Paclitaxel, 5-
fluorouracil and doxorubicin was selected as standard drug. The in vitro structural
activity relationship of the target compounds was obtained from the analysis of
biological data as summarized in Table 1.
Table 1. In vitro anticancer activity (IC50) of quinoline-azetidinone hybrid compounds
(6a-o) expressed in µM
5

SId HEP SId HEP
Comp
Ar
4-Cl-C6H4
4-F-C6H4
4-OCH3-C6H4
3,4-di-OCH3-C6H3
HEP G2a
HEP 3Bb
VEROc
G2
52.22
63.43
72.01
100.91
71.24
>100
57.12
54.43
>100
>100
33.67
71.78
>100
70.98
56.14
-
3B
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
0.85 0.01 3.37 0.02 44.39 1.0
0.76 0.01 1.72 0.01 48.21 1.1
0.71 0.01 0.89 0.02 51.13 1.2
0.36 0.01 0.74 0.02 36.33 0.9
13.17
28.02
57.44
49.09
47.28
30.42
35.86
24.35
55.42
82.77
33.67
26.21
35.93
49.14
13.76
-
3,4,5-tri-OCH3-C6H2 0.73 0.02 1.10 0.01 52.01 1.5
4-OH-C6H4
4-NO2-C6H4
2,3-di-Cl-C6H3
2,4-di-Cl-C6H3
3,4-di-Cl-C6H3
2-Cl-C6H4
3-Br-4-F-C6H3
4-N(CH3)2-C6H4
4-CN-C6H4
2-Thiophene
-
0.04 0.01 1.72 0.01 52.33 1.3
0.86 0.01 1.37 0.01 49.13 1.6
0.51 0.01 1.14 0.01 27.76 1.7
0.38 0.01 0.89 0.02 49.33 0.9
0.51 0.01 0.66 0.01 54.63 0.8
0.96 0.02 0.96 0.05 32.33 0.9
0.65 0.01 1.78 0.05 46.66 1.8
0.31 0.01 1.15 0.01 41.33 1.3
0.54 0.01 0.78 0.01 38.33 1.2
0.75 0.01 3.06 0.01 42.11 1.5
6o
Pace
5-FUf
Doxg
0.3 0.03 0.73 0.05
2.10 0.20 2.66 0.01
0.54 0.02 1.15 0.01
-
-
-
-
-
-
-
-
-
b
aHEP G2, human liver carcinoma cell line(Cat No- 11965-092); HEP 3B, Human
hepatoma cell line (Cat No- 11965-092);
d
cVERO, Monkey Kidney cell line ( Cat No- 11095-080); Selectivity indexs (SI) were
calculated by IC50 values in normal cell line devided by IC50 values in cancer cell
line; ePaclitaxel; f5-Fluorouracil; gDoxorubicin
The IC50 values are the average of the triplicate assay analysis. All quinoline-
azetidinone hybrid compounds (6a-o) inhibited cell proliferation with IC50 values in
range of 0.04 to 3.37 µM, As shown in Table 1, most of test compounds exhibited
comparable activity when compared to standard drugs. It was observed that among
the fifteen synthesized compounds, 6f displayed much higher antiproliferation activity
(IC50 = 0.04 µM) than standard paclitaxel (IC50 = 0.30 µM) against Hep-G2 cell line,
compounds like 6m, 6d, and 6i,showed good inhibitory activity against Hep-G2 cell
line with IC50 values of 0.31 µM, 0.36 µM, and 0.38 µM respectively, which are
comparable to paclitaxel and doxorubicin. Compounds 6h, 6j, and 6n revealed
moderate inhibitory activity against Hep-G2 cell line with IC50 values of 0.51 µM, 0.51
µM, and 0.54 µM respectively. Table 1 also revealed that, compound 6j exhibited
better antiproliferation activity (IC50 = 0.66 µM) than standard drugs against Hep-3B
6

cell line. Compounds 6d, 6c, 6i, and 6k showed moderate antiproliferation effects
(IC50 0.74 – 0.96 µM), against Hep-3B cell line.
The significant activity of compound 6f against Hep-G2 cell line can be attributed to
the presence of a strong electron donating hydroxyl group at the para position of the
phenyl ring attached to C-4 of azetidinone structure. Compound 6f also showed
activity against Hep-3B cell lines at IC50 = 1.72 µM. Any further changes in the
substitution on the phenyl ring of the azetidinone skeleton resulted in different values
of the biological activity. When two methoxy groups were attached at the meta and
para position (compound 6d), the activity was decreased to 0.36 µM against Hep-G2
cell line and 0.74 µM against Hep 3B cell line. When the methoxy group was
attached only at the para position of the phenyl ring (compound 6c) the activity
reduced to 0.71 µM against Hep-G2 cell line and 0.89 µM against Hep 3B cell line.
Further, compound with 3,4,5-trimethoxy (6e) substitution on the phenyl ring
displayed lowered activity of 0.73 µM against Hep-G2 cell line and 1.10 µM against
Hep-3B cell line. Replacing the substituents at para position by an electron
withdrawing group has caused decrease in the anticancer activity as compared to
the most potent compound 6f. This can be justified by the fact that compounds
bearing electron withdrawing groups like cyno (CN) (6n), fluoro (F) (6b), chloro (Cl)
(6a) and nitro (NO2) (6g) substituents at the para position of the phenyl ring have
exhibited activity at IC50 values 0.54, 0.76, 0.85 and 0.86 µM respectively against
Hep-G2 cell line and at 0.78, 1.72, 3.37 and 1.37 µM respectively against Hep-3B
cell line. Compounds bearing chloro (Cl) group at different positions on the phenyl
ring (compounds 6h-k) have shown different activities. Compound 6k with chloro (Cl)
at ortho position of the phenyl ring showed activity at IC50 = 0.96 µM against both
Hep-G2 and Hep-3B cell lines. Compound 6h with two chloro (Cl) group attached at
ortho and meta positions on the phenyl ring displayed activity at 0.51 0.01 against
Hep-G2 and 1.14 µM against Hep-3B cell lines. When two chloro (Cl) groups were
further attached at ortho and para positions (compound 6i) the activity decreased to
0.38 and 0.89 µM against Hep-G2 and Hep-3B cell lines respectively. The
subsequent substitution of two chloro (Cl) groups at meta and para positions showed
anticancer activity at 0.51 µM against Hep-G2 and at 0.66 µM against Hep-3B cell
lines. Compound 6l with two electron withdrawing substituents bromo (Br) at meta
position and fluoro (F) at para position exhibited activity at 0.65 µM and 1.78 µM
against Hep-G2 and Hep-3B cell lines respectively. Furthermore, inhibitory activities
7

decreased to 0.75 µM against Hep-G2 and 3.06 µM against Hep-3B cell lines when
five membered heterocyclic ring like 2-thiophene was attached to the azetidinone
structure instead of the phenyl group. Subsequently compounds 6d, 6f, 6i and 6m
have remarkable activity against Hep G2 cell lines and 6c, 6d, 6e, 6i, 6j, 6k and 6m
compounds showed significant activity against Hep 3B cell lines, these were
selected to further investigation regarding their mechanism of action. To investigate
whether or not compounds induce chromatin condensation and fragmentation, which
are both recognized morphological features of apoptosis, Hep G2 cells were treated
with the chosen compounds and stained with acridine orange (AO) and ethidium
bromide (EB). Nuclear morphology and alterations in the size and the shape of cells
were observed after 24 h treatment with IC50 concentrations of the compounds. The
microscopic photographs are shown in Fig. 2.
Figure. 2. Apoptosis in Hep G2 cells treatment with IC50 concentrations of
6d, 6f, 6f and 6m compounds
These results revealed that selected compounds can induce apoptosis in Hep G2
cells. Identification of deregulation of apoptosis is a possibility of all cancer cells and
the molecules that are capable of inducing apoptosis in cancer cells are considered
to be significant in anticancer agents. The selected compounds are taken for further
study to assessment of nuclear morphology by fluorescence microscopy using cell-
permeable nucleic acid stain, such as DAPI.33 Nuclear fragmentation and nuclear
condensation of Hep G2 cells in Fig. 3.
8

Figure. 3. Fluorescent microscopy pictures showing morphological changes in HEP
G2 cells treated with compound. The cells were stained with DAPI and observed
under fluorescent microscope. (A) Nuclear Condensation, (B) Blebbing and (C)
Nuclear Fragmentation
Additionally, the in vitro cytotoxic activity of all tested compounds was evaluated
against normal VERO cell line by MTT colorimetric assay in order to study estimate
the safety of these agents. The cytotoxic activity results revealed that none of the
tested compounds exhibited any significant toxicity effect on normal VERO cells.
Selectivity indexs (SI) values were calculated by IC50 values in normal cell line
divided by IC50 values in cancer cell line. The results revealed that most of the tested
compounds were less toxic on VERO in comparison with tumor cells (Table 1). The
safety of 6d, 6f, 6i and 6m compounds were further evaluated against Chang Liver
cell lines. The cytotoxicity results were summarized in Table 2. The data showed
that selected 6d, 6f, 6i and 6m compounds were non-toxic towards Chang Liver cells.
9

Table 2. Cytotoxicity of compounds against Chang Liver cell line
Cytotoxicity
Concentration
in µM
Cell Viability %
6d
6f
6i
6m
200
100
50
84.45 7.32 95.00 2.69 88.45 3.72 85.00 1.38
93.90 3.72 96.81 1.92 92.72 3.08 89.27 6.05
97.36 1.67 96.90 2.05 94.45 7.09 91.00 7.61
98.45 1.38 97.18 2.12 96.81 3.72 92.72 6.42
98.45 3.59 98.36 2.82 98.90 4.88 94.18 5.65
98.18 3.85 99.16 1.51 99.18 1.41 95.72 7.12
25
12.5
6.25
Moreover, in this work we also performed a computational (Lipinski’s rule of five)
study for prediction of physical-chemical properties34, 35 of all derivatives the results
are summarized in Table 3. Briefly, this simple rule is based upon the observation
that most of medicinal agents have a molecular weight (MW) of 500 or less, a log p
not more than 5, number of hydrogen bond donor atoms of 5 or less and number of
hydrogen bond accepter atoms of 10 or less (N or O). Interestingly our molecular
parameter results pointed that most of the compounds obey the Lipinski ‘rule of 5’.
Number of rotatable bonds is an important topological parameter for molecular
flexibility and conformational modification for binding to the enzyme or receptors. The
data shown that quinoline-azetidinone hybrid compounds showed a good number of
rotatable (n-rotb) (2-6) demonstrating their conformational plasticity.36 The hydrogen
bonding is important molecular descriptors for describing permeability of drugs.37
Most of the quinoline-azetidinone hybrid compounds displayed several hydrogen
bond acceptors (4 to 7) and moderate hydrogen bond donors as shown in Table. 3.
milogP, the logarithm of the octanol/water partition coefficient, provides a amount of
the lipophilicity of a ligand. Lipophilicity is an important parameter of an active
pharmaceutical ingredient as it influences a number of physiological properties like
transport through cell membranes, rate of metabolism and interaction with receptor
binding sites. The results revealed that most of the compounds exhibited low Log
p,by which these compounds have good bioavailability.38 All synthesized quinoline-
azetidinone hybrid compounds displayed molecular weight below 500 g/mol.
10

Table 3. Molecular parameters of quinoline-azetidinone hybrids (6a-o)
miLogpd
Comp
n-
nONb nOHNHc
MWe Rule of
5
Drug- TPSAg Molecular Absorption
rotba
likeness
(Å2)
volumeh percent. %
violation model
(Å3)
-
ABSi
-
scoref
0.37
Rule
of 5
6a
-
≤10
≤5
≤5
≤500
≤1
-
3
3
4
5
6
3
4
3
3
3
3
3
4
3
3
1
0
5
4
4
5
6
7
5
7
4
4
4
4
4
5
5
4
4
4
12
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
2
2
7
4.72
4.21
4.10
3.69
3.68
3.57
4.00
5.31
5.33
5.33
4.68
4.95
4.15
3.80
3.95
-0.711
-0.59
0.57
392.67
376.21
388.25
418.27
448.30
374.22
403.22
427.11
427.11
427.11
392.67
455.1
401.29
383.23
364.25
123
130.08
543.52
45.23
45.23
54.46
63.70
72.93
65.46
91.05
45.23
45.23
45.23
45.23
45.23
48.47
69.02
45.23
68.87
65.72
206.08
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
0
0
3
0.36
0.48
0.53
0.48
0.18
0.51
-0.12
0.45
0.42
0.42
0.35
0.22
0.24
0.25
0.27
0.66
-1.07
1.02
302.88
294.27
314.89
340.43
356.98
296.36
312.68
316.41
316.41
316.41
302.88
312.16
335.25
306.20
280.05
756.60
96.91
93.39
93.39
90.21
87.02
83.83
86.41
77.58
93.39
93.39
93.39
93.39
93.39
92.27
85.18
93.39
85.23
86.32
37.90
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
Pacj
5-FUk
Doxl
459.18
b
an-rotb, number of rotatable bonds ; nON, number of hydrogen bond acceptors;
d
cnOHNH, number of hydrogen bond donors; miLogP, logarithm of compound
e
f
partition coefficient between n-octanol and water; MW, molecular weight; Drug-
g
likeness model score: Calculated using molsoft (http://molsoft.com/mprop/); TPSA,
topological polar surface area; Molecular volume; Absorption percent (%ABS) was
calculated by using %ABS = 109-(0.345 X TPSA); Paclitaxel; 5-Fluorouracil;
lDoxorubicin
h
i
j
k
Finally, Molecular polar surface area (TPSA), volume and percentage of absorption
(% ABS) for the quinoline-azetidinone hybrid compounds are presented in Table. 3.
In conclusions, new quinoline-azetidinone hybrids were synthesized and
structures of newly synthesized compounds were confirmed by FTIR, NMR and Mass
spectral studies. The majority of the compounds exhibited significant antiproliferation
activity against Hep G2, and Hep 3B human cancer cell lines with IC50 values in the
range 0.04 - 3.37 µM. The compound 6f (IC50 = 0.04 0.01 µM) exhibited potent
antiproliferation activity against Hep G2 cell line, and 6j compound (IC50 = 0.66 0.01
µM) demonstrated potent antiproliferation activity against Hep 3B cell line and
11

provide to be more potent as cytotoxic agents than standard drugs. Morphological
changes suggest the induction of apoptosis and describe the probable mechanism of
action of these hybrid antitumor agents. The reported quinoline-azetidinone hybrids
showed better safer profiles against normal cell lines. Synthesized analogues
displayed suitable drug like properties and are expected to extant good bioavailability
property. Altogether, these results suggest that some of these hybrid-compounds
would serve as potential anticancer agents with modifications on pharmacophore,
especially; 6f hybrid-compound was promising subjects for further studies regarding
cancer chemotherapy.
Acknowledgements
Authors are grateful to Principal, KLE University’s College of Pharmacy,
Belagavi, for providing necessary facilities for the research. Authors are also grateful
to NMR Research center, IISC, Bangalore, India, for providing the spectral data.
12

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Experimental: General procedure for synthesis of Synthesis of 7-chloro-4-
hydrazinylquinoline (3): The mixture of 4, 7 dichloroquinoline (25 mmol)
and hydrazine hydrate (50 mL, excess amount) was refluxed for 8h. The
reaction was monitored by TLC for completion. The reaction mixture was left
°
to stand at 5 C overnight. The resulted solid reaction mixture was then
treated with dichloromethane (30 mL), filtered, washed with dichloromethane,
dried and recrystallized from ethanol to yield 7-chloro-4-hydrazinylquinoline
°
(3). Yield 83%; mp: 220-222 C; FTIR (KBr, cm-1): 3336 (NH2), 1612 (C=N);
14

1H-NMR (DMSO-d6, 400 MHz) δ, ppm: 5.7 (bs, 3H, NH & NH2), 6.86 (s, 1H,
Ar-H), 7.38 (s, 1H, Ar-H), 7.74 (s, 1H, Ar-H), 8.16 (s, 1H, Ar-H), 8.36 (s, 1H,
Ar-H). General procedure for synthesis of 7-chloro-4-(2-(4-
substituted benzylidene) hydrazinyl)quinolone (5): A mixture of 7-chloro-4-
hydrazinylquinoline (3) (15 mmol) and appropriate aryl or heteroaryl
aldehydes (4) (18 mmol) in dry ethanol (100 mL) was stirred at room
temperature for 24 h. The reaction was monitored by TLC for completion. The
precipitate formed was filtered, washed with cold ether and dried to get 7-
chloro-4-(2-(4-substituted benzylidene) hydrazinyl)quinolone.7-chloro-4-(2-(4-
chlorobenzylidene)hydrazinyl)quinoline (5a): Yellow powder; Yield 74%; mp:
224°C; FTIR (KBr, cm-1): 3238 (NH), 3172 (Ar-H), 1583 (C=N), 1575 (C=C);
1H-NMR (DMSO-d6, 400 MHz) δ, ppm: 7.3 (s, 1H, CH=N), 7.51 (d, 2H, J=8.4
Hz, Ar-H), 7.55 (s, 1H, Ar-H), 7.80 (d, 2H, J=8.4 Hz, Ar-H), 7.82 (s, 1H, Ar-H),
8.35 (s, 1H, Ar-H), 8.38 (m, 2H, Ar-H), 11.37 (s, 1H, NH). 7-chloro-4-(2-(4-
hydroxybenzylidene)hydrazinyl)quinoline (5f): Yellow powder; Yield 57%; mp:
1
280°C; FTIR (KBr, cm-1): 3647(OH), 3211(NH), 3120 (Ar-H), 1568 (C=N); H-
NMR (DMSO-d6, 400 MHz) δ, ppm: 6.83 (d, 2H, J=8.8 Hz, Ar-H), 7.25 (s, 1H,
Ar-H), 7.49 (s, 1H, Ar-H), 7.62 (d, 2H, J=8.4 Hz, Ar-H), 7.8 (s, 1H,-CH=N),
8.38-8.30 (m, 3H, Ar-H), 9.83 (s, 1H, OH), 11.09 (s, 1H, NH). 7-chloro-4-(2-(4-
nitrobenzylidene)hydrazinyl)quinoline (5g): Yellow powder; Yield 57%; mp:
230°C; FTIR (KBr, cm-1): 3591(NH), 3068 (Ar-H), 1566.20 (C=N), 1585(NO2);
1H-NMR (DMSO-d6, 400 MHz) δ, ppm: 7.84 (s, 1H, -CH=N), 8.0 (d, 2H, J=8.4
Hz, Ar-H), 8.47-8.26 (m, 7H, Ar-H), 11.6 (s, 1H, NH).
31.
General procedure for synthesis of 3-chloro-1-((7-chloroquinolin-4-yl)amino)-
4-(4-substituted phenyl)azetidin-2-one (6). To a stirred solution of 7-chloro-4-
(2-(4-substitutedbenzylidene)hydrazinyl)quinoline (5) (3.7 mmol) and
triethylamine (9 mmol) in dichloromethane (25 mL), chloroacetyl chloride (9
mmol) in dichloromethane (25 mL) was added drowise at 0-5 °C. The reaction
mixture was refluxed for 24 h. The reaction was monitored by TLC for
completion. The precipitate formed was filtered, washed with dichloromethane
and dried to get quinoline-azetidinone (6). 3-chloro-1-((7-chloroquinolin-4-
yl)amino)-4-(4-chlorophenyl)azetidin-2-one (6a): Lime-yellow powder; Yield
60%; mp: 328-330°C; FTIR (KBr, cm-1): 3238 (NH), 3084 (Ar-H), 1705 (C=O);
15

1H-NMR (DMSO-d6, 400 MHz) δ, ppm: 4.25 (s, 1H, CH), 5.74 (s, 1H, CH),
7.57 (d, 2H, J=8.4 Hz, Ar-H), 7.67 (s, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 7.91(d,
2H, J=8.4 Hz, Ar-H), 8.10 (s, 1H, Ar-H), 8.68 (s, 1H, Ar-H), 8.80 (s, 1H, Ar-H),
13 (s, 1H, NH); LCMS m/z = 393 (M + 1). 3-chloro-1-((7-chloroquinolin-4-
yl)amino)-4-(4-fluorophenyl)azetidin-2-one (6b): Yellow powder; Yield 75%;
mp: 320-322°C; FTIR (KBr, cm-1): 4.25 (s, 1H, CH), 5.73 (s, 1H, CH), 6.78 (d,
2H, J=8.8 Hz, Ar-H), 7.51 (s, 1H, Ar-H), 7.67(d, 2H, J=8.8 Hz, Ar-H), 7.82 (s,
1H, Ar-H), 8.02 (s, 1H, Ar-H), 8.56 (s, 1H, Ar-H), 8.64 (s, 1H, Ar-H), 12.6 (s,
1H, NH); LCMS m/z = 375 (M + 1). 3-chloro-1-((7-chloroquinolin-4-yl)amino)-
4-(3,4-dimethoxyphenyl)azetidin-2-one (6d): Yellow powder; Yield 72%; mp:
302-304°C; FTIR (KBr, cm-1): 3169 (NH), 3081 (Ar-H), 1708 (C=O), 1315
(OCH3); 1H-NMR (DMSO-d6, 400 MHz) δ, ppm: 3.83 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 4.25 (s, 1H, CH), 5.73 (s, 1H, CH), 7.07 (s, 1H, Ar-H), 7.38-7.36
(m, 1H, Ar-H), 7.49 (s, 1H, Ar-H), 7.61 (s, 1H, Ar-H), 7.88-7.85 (m, 1H, Ar-H),
8.05 (s, 1H, Ar-H), 8.64-8.62 (m, 1H, Ar-H), 8.70 (s, 1H, Ar-H), 12.8 (s, 1H,
NH); LCMS m/z = 419 (M + 1); 3-chloro-1-((7-chloroquinolin-4-yl)amino)-4-
(3,4,5-trimethoxyphenyl)azetidin-2-one (6e): Yellow powder; Yield 70%; mp:
328-330°C; FTIR (KBr, cm-1): 3210 (NH), 3084 (Ar-H), 1705 (C=O), 1334
(OCH3); 1H-NMR (DMSO-d6, 400 MHz) δ, ppm: 3.73 (s, 3H, OCH3), 3.88 (s,
6H, OCH3), 4.25 (s, 1H, CH), 5.7 (s, 1H, CH), 7.1(s, 2H, Ar-H), 7.65 (s, 1H,
Ar-H), 7.86 (s, 1H, Ar-H), 8.09 (s, 1H, Ar-H), 8.64 (s, 1H, Ar-H), 8.75 (s, 1H,
Ar-H), 10.93 (s, 1H, NH); LCMS m/z = 550 (M + 2). 3-chloro-1-((7-
chloroquinolin-4-yl)amino)-4-(4-hydroxyphenyl)azetidin-2-one (6f): Yellow
powder; Yield 65%; mp: 340-342 °C; 3647 (OH), 3278 (NH), 3086 (Ar-H),
1703 (C=O); 1H-NMR (DMSO-d6, 400 MHz) δ, ppm: 4.25 (s, 1H, CH), 5.73 (s,
1H, CH), 6.89 (d, 2H, J=8.8 Hz, Ar-H), 7.32 (s, 1H, Ar-H), 7.32 (s, 1H, Ar-H),
7.50 (s, 1H, Ar-H), 7.68 (d, 2H, J=8.8 Hz, Ar-H), 7.81 (m, 1H, Ar-H), 8.09 (s,
1H, Ar-H), 10.21 (s, 1H, OH), 12.9 (s, 1H, NH); LCMS m/z = 375 (M + 1). 3-
chloro-1-((7-chloroquinolin-4-yl)amino)-4-(4-nitrophenyl)azetidin-2-one (6g):
Yellow powder; Yield 40%; mp: 320-322 °C; FTIR (KBr, cm-1): 3157 (NH),
3078 (Ar-H), 1697 (C=O), 1585 (NO2); 1H-NMR (DMSO-d6, 400 MHz) δ, ppm:
4.25 (s, 1H, CH), 5.74 (s, 1H, CH), 7.32 (s, 1H, Ar-H), 7.90 (s, 1H, Ar-H), 8.10
(s, 1H, Ar-H), 8.16 (d, 2H, J=8.8 Hz, Ar-H), 8.33 (d, 2H, J=8.8 Hz, Ar-H), 7.75
(s, 1H, Ar-H), 8.57 (s, 1H, Ar-H), 13.02 (s, 1H, NH); LCMS m/z = 404 (M + 1).
16

3-chloro-1-((7-chloroquinolin-4-yl)amino)-4-(3,4-dichlorophenyl)azetidin-2-one
(6j): Light yellow powder; Yield 81%; mp: 340-342 °C; FTIR (KBr, cm-1):3170
(NH), 3054 (Ar-H), 1695 (C=O);1H-NMR (DMSO-d6, 400 MHz) δ, ppm: 4.26
(s, 1H, CH), 5.73 (s, 1H, CH), 7.80-7.71 (m, 3H, Ar-H), 8.10 (s, 1H, Ar-H),
8.17 (s, 1H, Ar-H), 8.68 (s, 1H, Ar-H), 8.75 (s, 1H, Ar-H), 8.80 (s, 1H, Ar-
H),12.98 (s, 1H, NH); LCMS m/z = 427. 3-chloro-1-((7-chloroquinolin-4-
yl)amino)-4-(2-chlorophenyl)azetidin-2-one (6k): Yellow powder; Yield 50%;
mp: 336-338 °C; FTIR (KBr, cm-1): 3169 (NH), 3082 (Ar-H), 1710 (C=O); 1H-
NMR (DMSO-d6, 400 MHz) δ, ppm: 4.25 (s, 1H, CH), 5.74 (s, 1H, CH), 8.1-
7.68-7.46 (m, 3H, Ar-H), 7.87 (s, 1H, Ar-H), 8.22-8.14 (m, 2H, Ar-H), 8.70 (s,
1H, Ar-H), 8.85 (s, 1H, Ar-H), 9.23 (s, 1H, Ar-H), 13.1 (s, 1H, NH); LCMS m/z
= 393 (M + 1).
32.
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http://www.molinspiration.com/services/properties.html.
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http://www.asteris-app.com/technical-info/core-properties/logp.htm (2016)
17

O
HO
Cl
N
N
HN
N
N
H
HN
N
Structural Modification
Cl
OH
H
IC50 = 52.5 µM against HEP G2
IC50 = 0.04 µM against HEP G2
In search of new heterocyclic anticancer agents, a new quinoline-azetidinone hybrid
template have been designed, synthesized and evaluated in vitro anticancer against
Hep G2 and Hep 3B human cancer cell lines.
18

Highlights:
ꢀ Quinoline-azetidinone hybrids were synthesized
ꢀ Cytotoxic activity against Hep G2, and Hep 3B was reported
ꢀ Compound 6f exhibited significant cytotoxic activity (IC = 0.04 µM) against
50
Hep G2
ꢀ Compound 6j exhibited better cytotoxic activity (IC = 0.66 µM) against Hep
50
3B
19