Novel nalidixic acid derivatives targeting topoisomerase II enzyme; Design, synthesis, anticancer activity and effect on cell cycle profile

Article abstract of DOI:10.1016/j.bioorg.2018.10.058

Aim: Design and synthesis of novel nalidixic acid derivatives of potent anticancer and topoisomerase II inhibitory activities were our major aim. Materials & methods: All the newly synthesized nalidixic acid derivatives were submitted to the National Canc

Full text of DOI:10.1016/j.bioorg.2018.10.058

Accepted Manuscript
Novel Nalidixic Acid Derivatives Targeting Topoisomerase II Enzyme; Design,
Synthesis, Anticancer Activity and Effect on Cell Cycle Profile
Omneya M. Khalil, Ehab M. Gedawy, Afaf A. El-Malah, Mina E. Adly
PII:
S0045-2068(18)30418-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.058
YBIOO 2592
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
28 April 2018
22 October 2018
29 October 2018
Please cite this article as: O.M. Khalil, E.M. Gedawy, A.A. El-Malah, M.E. Adly, Novel Nalidixic Acid Derivatives
Targeting Topoisomerase II Enzyme; Design, Synthesis, Anticancer Activity and Effect on Cell Cycle Profile,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.10.058
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Novel Nalidixic Acid Derivatives Targeting Topoisomerase II Enzyme;
Design, Synthesis, Anticancer Activity and Effect on Cell Cycle Profile.
Omneya M. Khalila, Ehab M. Gedawy a,b*, Afaf A. El-Malah a and Mina E. Adly a
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562,
Egypt.
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Badr University In Cairo BUC, Cairo,
Egypt.
Corresponding author;
Ehab M. Gedawy, Ph.D. Pharmaceutical Organic Chemistry, Faculty of
Pharmacy, Cairo University, Cairo 11562, Egypt.
Address: 33 Kasr El-Aini Street, Cairo, Egypt.
Tel: +20 23639307
Fax:+20 23635140
E-mail address: ehab.gedawy@pharma.cu.edu.eg
Abstract
Aim: Design and synthesis of novel nalidixic acid derivatives of potent anticancer
and topoisomerase II inhibitory activities were our major aim. Materials &
methods: All the newly synthesized nalidixic acid derivatives were submitted to
the National Cancer Institute (NCI), Bethesda, USA and were accepted for single
dose screening. Further investigation via IC50 determination of the most potent
compound 6a against K-562 and SR leukemia cell lines. Finally, the
topoisomerase II inhibitory activity, the cell cycle analysis and molecular docking
of 6a were performed in order to identify the possible mechanism of the
anticancer activity. Results: Compound 6a showed interesting selectivity against
leukemia especially K-562 and SR subpanels with IC50 35.29 µM and 13.85 µM
1

respectively. Moreover, compound 6a revealed potent topoisomerase IIα and
topoisomerase IIβ inhibitory activity compared with known topoisomerase
inhibitors such as doxorubicin and topotecan with IC50 1.30 µM and 0.017 µM
respectively. Cell cycle analysis indicated that compound 6a induced cell cycle
arrest at G2-M phase leading to inhibition of cell proliferation and apoptosis.
Molecular modeling demonstrated that the potent topoisomerase inhibitory
activity of 6a was due to the interaction with the topoisomerase II enzyme
through coordinate bonding with the magnesium ion Mg2+, hydrogen bonding
with Asp 545 and arene cation interaction with His 759.
Keywords: Nalidixic acid; Synthesis; Design; Anticancer acyivity;
Topoisomerase II enzyme; Cell cycle analysis; Apoptosis.
1. Introduction
Topoisomerase II enzyme is a nuclear enzyme that plays crucial functions during
DNA processes such as, replication, transcription and repair via catalysis of the
relaxation and unwinding of double-stranded DNA [1–7]. Recent studies showed
that mammalian topoisomerase II is an important target for many anticancer
agents [8–10]. Topoisomerase II inhibitors can be classified into two classes, the
traditional topoisomerase II inhibitors which are called topo II poisons that act
through stabilization of the formed covalent complex between the enzyme and
DNA, accumulation of these complexes finally induces apoptosis [11,12]. Topo
II poisons include, epipodophyllotoxin (e.g. etoposide) that binds to ATP binding
site [13] and anthracyclines (e.g. doxorubicin) which bind to DNA binding site
[14,15]. The recent class of topoisomerase II inhibitors are called the catalytic
inhibitors (e.g. staurosporine and merbarone) [16–19] that block the catalytic
activity of topo II through several mechanisms such as inhibition of
topoisomerase II-mediated DNA cleavage (merbarone) [12] (Figure 1). Nalidixic
acid which is the parent of the quinolone antibiotics class showed moderate
2

selective anticancer activity against leukemia by inhibiting topoisomerase II.
However, it was reported that eukaryotic topoisomerase II less sensitive than
prokaryotic topoisomerase against nalidixic acid [20]. Vosaroxin; a naphthyridine
analog was found to be topoisomerase II inhibitor and is now in phase III clinical
trials for the treatment of acute myeloid leukemia (AML) [21]. Ever since, many
trials were made to synthesize cytotoxic agents from ciprofloxacin [22],
lomefloxacin [23], norfloxacin [24] and ofloxacin [25]. Most studies were focused
on the C7 piperazine ring [26–28] while little research was dedicated to the C3
carboxylic acid group. Based on the abovementioned findings, we herein reported
the design and synthesis of nalidixic acid derivatives as inhibitors of
topoisomerase II enzyme via introduction of various moieties in position 3 such as
different arylidene, pyrrole, pyrrolidine, isoindoline, arylureido and thioureido
groups. At the same time, we kept the two coplanar carbonyl groups to ensure
coordination with Mg2+ ion of topoisomerase enzyme that represents an important
component of the catalytic machinery of the enzyme [29] (Figure 2). The newly
synthesized compounds were evaluated for their in vitro cytotoxic activity
utilizing 60 different cancer cell lines. Also, the topoisomerase II inhibitory
activity was evaluated using human DNA topoisomerase II Elisa Kit. Finally,
molecular docking studies were done to predict the possible mechanism of action
and binding mode for the most potent compound in order to explain its anticancer
activity.
2. Results and discussion:
2.1. Chemistry
An outline for the synthesis of the target compounds is shown in schemes 1-3.
Nalidixic acid hydrazide 2 was prepared by direct hydrazinolysis of nalidixic acid
1. Condensation of hydrazide 2 with the appropriate ketones afforded compounds
3a,b and 4a-d. 1H NMR spectra of 3a,b and 4a-d revealed disappearance of NH2
of compound 2 and appearance of only one NH signal at  12.44-13.19 ppm.
3

1
Moreover H NMR spectra of compounds 4a-d disclosed appearance of the
additional CH aliphatic protons of the corresponding cycloalkyl moieties.
Compounds 5a-c were prepared by reaction of the nalidixic acid hydrazide with
1
acids anhydrides in glacial acetic acid. H NMR spectrum of compound 5b
showed appearance of absorption band at 7.20 ppm corresponding to the two
olefinic CH protons.
Reaction of compound 2 with isocyanates and isothiocyantes in absolute ethanol
1
yielded compounds 6a,b and 7a-d respectively. H NMR spectra of compounds
6a-d revealed appearance of 3NH at 9-77-12.31 ppm while compounds 7a,b
showed 3NH at 8.54-11.28 ppm.
2.2. Biological Evaluation
2.2.1. In vitro anticancer screening
All synthesized compounds were selected by the National Cancer Institute (NCI),
Bethesda, USA for cytotoxic screening against 60 human cancer cell lines
representing 9 tumor types including leukemia, melanoma, lung, colon, CNS,
ovarian, renal, prostate, and breast cancers. Screening was performed utilizing a
single dose (10-5 molar concentration) and cell were incubated for 48 hours.
sulforhodamine B (SRB) dye was used for determination of end point. Results
were reported as a graph representing growth percent. The percent inhibition was
calculated, and the results were summarized in (Table 1). Compound 6a showed
selective potent activity against leukemia subpanal especially against k-562 and
SR cell lines with growth inhibition percent of 81.11 and 57.8 respectively.
Structure activity relationship of the newly synthesized nalidixic acid derivatives
disclosed that:
- Condensation of the 3-hydrazide group with aryl or cyclic aliphatic ketones
(compounds 3a,b and 4a-d) reduced the anticancer activity. Compound 4c
showed moderate potency against K-562 and MOLT-4 Leukemia cell lines
4

with GI% = 27.99 and 28.99, respectively. In addition, A498 renal cell line
showed significant sensitivity towards compound 4c with GI% = 42.04.
- Presence of bulky cyclic amide moiety substituted in position-3 of 1,8-
naphthyridine ring (compounds 5a-c) abolished the anticancer activity this
may be attributed to the steric hindrance.
- 1,8-naphthyridine-3-carbonylhydrazinecarbothioamide
derivatives, compounds 6a-d) and
(thioureido
1,8-naphthyridine-3-
carbonylhydrazinecarboxamide (ureido derivatives, compounds 7a,b)
showed moderate to weak anticancer activity. Concerning the substitution
position on the aryl ring, meta substitution interestingly increases the
potency (compound 6a revealed potent anticancer activity against leukemia,
colon, non-small cell lung and renal cancer with GI% range from 81.11 to
18.35). on the other hand, ortho and para substitution dramatically decreased
the potency.
2.2.2. In vitro cytotoxicity IC50 determination by MTT colorimetric assay
Since compound 6a showed the highest potency in the single dose in vitro
screening assay, it was subjected to further studies. Five concentrations were
employed for IC50 determination against k-562 and SR leukemia cell lines. MTT
colorimetric assay was used, which determines the effect of the newly synthesized
compound on the overall growth of adherent cell lines. The mitochondrial enzyme
succinate dehydrogenase causes the cleavage of MTT tetrazolium salt into
formazan which is a blue colored product [30]. Doxorubicin and staurosporine
were used as standards. IC50 values, which are the concentrations required to
reduce the cell growth by 50% were determined and compared to the IC50
achieved by doxorubicin and staurosporine. Results were demonstrated in (Figure
3) which revealed that compound 6a showed comparable activity with
staurosporine against K-562 cell line (with IC50 35.29 and 28.74 M
respectively). Moreover, 6a was found to be equipotent with doxorubicin against
5

SR cell lines (with IC50 of 13.85 and 13.83 M respectively) additionally, it was
1.2 folds more potent than staurosporine.
2.2.3. Cell Cycle analysis
Significant alternations in the cell phases were observed when leukemia SR cells were
treated with compound 6a (Figure 4). Results showed a decrease in the percentage of
cells at the G0-G1 and S phases with 22.61% and 33.83%, respectively in comparison
with control SR leukemia cell line which showed 38.31% and 55.44%, respectively.
Such decrease in the percentage of cells at the G0-G1 and S phases were found to be
comparable to that induced by doxorubicin (25.18% and 28.76%). On the other hand,
the percentage of cells at G2-M was 43.56% which was significantly higher than
control 5.25% and comparable to that of doxorubicin (46.56%). Finally, the
percentage of cells at pre-G1 phase increased by 5.25 folds the control while
doxorubicin caused an increase by 8.62 folds. Therefore, it can be assumed that
compound 6a exhibited cell cycle arrest at G2-M phase leading to inhibition of cell
proliferation and induced apoptosis.
2.2.4. Determination of apoptosis using annexin-V
The results showed a significant elevation in the percentage of the early and late
apoptosis when leukemia SR cells were treated with compound 6a (3.49 and
10.92 folds, respectively more than control leukemia SR cells). Furthermore,
doxorubicin revealed an increase in both the early and late apoptosis with 5.76
and 18.22 folds higher than control, respectively. Also, the necrosis percent
induced by compound 6a and doxorubicin was 2.69% and 4.08%, respectively
which were noticeably higher than control that showed only 0.88% necrosis
(Figure 5).
6

2.2.5. Topoisomerase II inhibitory activity
The inhibitory activity of topoisomerase IIα and topoisomerase IIβ was evaluated
for compound 6a by using Topoisomerase Elisa Kit. Five concentrations were
used and IC50 was calculated. Doxorubicin and topotecan, known inhibitors of
topoisomerase enzymes, were used as reference. The results are reported in (
Figure 6). Compound 6a showed potent inhibitory activity against both
topoisomerase II subclasses, alpha and beta with IC50 of 1.30 and 0.017 M
respectively which are comparable to the used standards doxorubicin and
topotecan.
2.2.6. Molecular modeling study
Several crystal structures for topoisomerase enzyme were available in the protein
data bank. In this work we selected (PDB ID: 4FM9) [3] which has topoisomerase
IIα co-crystallized with DNA. DNA chains were removed from the active site.
Molecular docking setup was validated by carrying out the docking of the
inhibitor merbarone in the DNA binding site and was compared to a previously
reported study [31]. The binding mode of merbarone indicated the used setup was
suitable for the docking study. Merbarone demonstrated energy score (S) =
−20.72 kcal/mol and was able to reproduce the coordinate bond interactions with
Mg2+ and arene cation interaction with His 759. Compound 6a was able to interact
in a similar pattern by forming coordinate bond interactions with Mg2+ via the
oxygen atoms of both carbonyl groups at positions 3 and 4, arene cation
interaction with His 759 and an additional hydrogen bond with Asp 545.
Compound 6a yielded energy score (S) = −33.75 kcal/mol (Figure 7).
Concerning compounds 3a&b and 4a-d the steric hindrance caused by the cyclic
or disubstituted hydrazones prohibited the docking poses from establishing the
key interactions accomplished by the ligand, merbarone within the active site.
7

Furthermore, the energy scores of these compounds were found to be less than
that of compound 6a (Table 6, Figure 8a,b). The cyclic amide structure of
compounds 5a-c seems to prevent the proper binding to the active site (Figure
8c). Compounds 6b-d and 7a&b yielded less energy scores (with range from -
24.3206 to -30.4713 kcal/mol) than the most active compound 6a (-33.6344).
This may be due to the position of the aryl ring substituents. The meta substituent
seems to fit in the active site more properly than the ortho and para positioned
substituents (Figure 8d,e). The above findings could explain the low activity
associated with compounds 3a&b, 4a-d, 5a-c, 6b-d and 7a&b compared with the
potent derivative 6a.
3. Conclusion
A group of new C-3 nalidixic acid derivatives were synthesized and characterized
by their spectral data. Single dose cytotoxic NCI 60 cell line screening revealed
that compound 6a has selective cytotoxic activity against leukemia especially K-
562 and SR cell lines. Five dose MTT assay showed that compound 6a has
comparable anticancer activity to doxorubicin against K-562 leukemia cell line
with IC50 35.29 M and it was worth mentioning that 6a was equipotent to
doxorubicin against SR leukemia cell line. Compound 6a induced cell cycle arrest
at G2-M phase that was demonstrated through accumulation of SR leukemia cells
at G2-M and pre-G1 phases with increase of 8.29 and 5.28 folds respectively,
relative to the control. Moreover, compound 6a showed significant increase in the
percentage of total apoptotic cells (5.28 folds more than control) and in the
necrosis percentage by three folds than control. At the same time, compound 6a
also showed potent topoisomerase IIα and topoisomerase IIβ inhibitory activity
with IC50 1.30 and 0.017 µM respectively. Based on the molecular docking study
the potent activity of 6a can be attributed to the coordinate bonding interaction
with Mg2+, the hydrogen bonding with Asp 545 and the arene cation interaction
8

with His 759. Finally, this study showed that compound 6a represents a promising
lead topoisomerase II inhibitor and a selective cytotoxic agent against leukemia
that requires further investigation.
Acknowledgment
Authors wish to express their deepest gratitude to the Development Therapeutics
Program of the National Cancer Institute, Bethesda, MD, USA, for in vitro
evaluation of anticancer activity. The authors are thankful Dr. Esam Rashwan,
Head of the confirmatory diagnostic unit VACSERA-EGYPT, for carrying out in
vitro topoisomerase II inhibition assays and cell cycle analysis. This work was
funded by the Faculty of Pharmacy, Cairo University, Egypt.
4. Experimental
4.1. Chemistry
Chemicals were purchased from Sigma-Aldrich, USA and Acros Organics,
Belgium. The reactions progress was monitored by TLC using precoated
aluminum sheet silica gel MERCK 60F 254 (Merck, Germany) and was
visualized by UV lamp. The solvent system used was chloroform: benzene:
methanol [9: 5: 1]. Melting points were obtained using Stuart electrothermal
1
melting point apparatus SMP10 and were uncorrected. H NMR spectra were
13
carried out using a Bruker Advance 400 MHz NMR spectrometer. C NMR
spectra were carried out using a Bruker Advance 100 MHz spectrometer.
Chemical shifts were recorded in ppm on  scale using DMSO-d6 as solvent and
coupling constants (J) in Hz.Mass spectra were run at 70 ev on Hewlett Packard
5988 spectrometer, Micro Analytical Center, Cairo University, Egypt. Element
analyses were carried out at the Regional Center for Mycology and
Biotechnology, Faculty of Pharmacy, Al Azhar University, Egypt.
9

4.1.1. Synthesis of 1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carbohydrazide (Nalidixic acid hydrazide) (2)
A mixture of nalidixic acid 1 (0.01 mol, 2.31 g) and hydrazine hydrate (100 %, 25
mL) was heated under reflux for 24 hours. The reaction mixture was allowed to
cool. The formed precipitate was filtered, dried and recrystallized from 70%
ethanol to afford 2; mp: 192-193 oC (as reported)[32]; Yield: 1.67 g, 68%.
4.1.2. General procedure for the synthesis of 1-ethyl-7-methyl-4-oxo-N'-(1-
(aryl)ethylidene)-1,4-dihydro-1,8-naphthyridine-3-carbohydrazide (3a,b).
Nalidixic acid hydrazide 2 (0.25 g, 1 mmol) was dissolved in 10 mL absolute
ethanol containing 0.20 mL glacial acetic acid. Equimolar amount of the
heterocyclic ketone was added and the solution was heated under reflux for 6
hours. The formed precipitate was filtered while hot and recrystallized from
methanol to afford 3a,b.
4.1.2.1. 1-Ethyl-7-methyl-4-oxo-N'-(1-(pyridin-3-yl)ethylidene)-1,4-dihydro-
1,8-naphthyridine-3-carbohydrazide (3a)
Creamy white solid; mp: > 300 oC; yield: 0.32 g, 91.40%; IR (KBr) max: 3421
1
(NH), 1678 (C=O) cm-1; H NMR (400 MHz, DMSO-d6):  1.44 (t, 3H, CH3,
J=7.0 Hz), 2.44 (s, 3H, CH3), 2.71 (s, 3H, CH3), 4.66-4.68 (q, 2H, CH2, J=7.0
Hz), 7.49 (t, 1H, ArH, J=8.0 Hz), 7.56 (d, 1H, ArH, J=8.0 Hz), 8.23 (d, 1H, ArH,
J=8.0 Hz), 8.62-8.64 (m, 1H, ArH), 8.67 (d, 1H, ArH, J=8.0 Hz), 9.03 (s, 1H,
ArH), 9.17 (s, 1H, ArH) and 13.19 (s, 1H, NH, D2O exchangeable) ppm; MS
[m/z, %]: 350 [M+1˥.+, 2.27], 349 [M+, 2.46] and 215 [M - C7H8N3˥· +, 100];
Anal. Calcd for C19H19N5O2 (349.39): C, 65.32; H, 5.48; N, 20.04. Found: C,
65.49; H, 5.70; N, 20.31.
10

4.1.2.2. 1-Ethyl-7-methyl-4-oxo-N'-(1-(thiophen-2-yl)ethylidene)-1,4-dihydro-
1,8-naphthyridine-3-carbohydrazide (3b)
o
Buff solid; mp: > 300 C; yield: 0.30 g, 83.30%; IR (KBr) max: 3340 (NH),
1
1670 (C=O) cm-1; H NMR (400 MHz, DMSO-d6):  1.43 (t, 3H, CH3, J=7.0
Hz), 2.43 (s, 3H, CH3), 2.70 (s, 3H, CH3), 4.63-4.68 (q, 2H, CH2, J=7.0 Hz), 7.11-
7.13 (m, 1H, ArH), 7.54-7.56 (m, 2H, ArH), 7.62 (d, 1H, ArH, J=8.0 Hz), 8.65 (d,
1H, ArH, J=8.0 Hz), 9.13 (s, 1H, ArH) and 13.05 (s, 1H, NH, D2O exchangeable)
13
ppm; C NMR (100 MHz, DMSO-d6):  14.79, 15.53, 25.34, 46.86, 112.07,
119.83, 122.22, 127.97, 128.33, 129.08, 136.44, 143.53, 147.00, 148.46, 148.74,
160.57, 163.96 and 176.51; MS [m/z, %]: 355 [M+1˥·+, 13.37], 354 [M.+, 53.87]
and 215 [M - C6H7N2S˥·+, 100]; Anal. Calcd for C18H18N4O2S (354.43): C, 61.00;
H, 5.12; N, 15.81. Found: C, 60.78; H, 5.24; N, 15.97.
4.1.3. General procedure for the synthesis of compounds 4a-d.
To a solution of nalidixic acid hydrazide 2 (0.25 g, 1 mmol) in 25 mL hot distilled
water the appropriate cyclic ketone (1 mmol) was added, then the mixture was
stirred vigorously for 24 hours. The formed precipitate was filtered and
recrystallized from absolute ethanol to yield compounds 4a-d.
4.1.3.1. N1-Cyclopentylidene-1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carbohydrazide (4a)
o
Buff solid; mp: 238-240 C; yield: 0.19 g, 59.40%; IR (KBr) max: 3143 (NH),
1670 (C=O) cm-1; 1H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H, CH3, J=7.0 Hz),
1.74-1.79 (m, 2H, CH2), 1.84-1.88 (m, 2H, CH2), 2.35-2.43 (m, 4H, 2CH2), 2.68
(s, 3H, CH3), 4.58-4.63 (q, 2H, CH2, J=7.0 Hz), 7.50 (d, 1H, ArH, J=8.2 Hz), 8.58
(d, 1H, ArH, J=8.2 Hz), 9.09 (s, 1H, ArH) and 12.44 (s, 1H, NH, D2O
13
exchangeable) ppm; C NMR (100 MHz, DMSO-d6):  15.47, 24.73, 24.98,
11

25.33, 28.83, 33.00, 46.75, 112.03, 119.79, 122.18, 136.44, 148.44, 148.75,
160.30, 163.93, 166.63 and 176.3 ppm; MS [m/z, %]: 313 [M + 1˥·+, 24.2], 312
[M.+, 100] and 215 [M - C5H9N2˥·+, 95.47]; Anal. Calcd for C17H20N4O2 (312.37):
C, 65.37; H, 6.45; N, 17.94. Found: C, 65.51; H, 6.53; N, 18.12.
4.1.3.2. N1-Cyclohexylidene-1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carbohydrazide (4b)
Buff solid; mp: 252-254 oC; yield: 0.24 g, 72.70%; IR (KBr) max: 3136 (NH),
1
1670 (C=O) cm-1; H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H, CH3, J=7.0
Hz), 1.63-1.68 (m, 6H, 3CH2), 2.32-2.35 (m, 2H, CH2), 2.45-2.48 (m, 2H, CH2),
2.67 (s, 3H, CH3), 4.59-4.64 (q, 2H, CH2, J=7.0 Hz), 7.49 (d, 1H, ArH, J=8 Hz),
8.57 (d, 1H, ArH, J=8 Hz), 9.08 (s, 1H, ArH) and 12.79 (s, 1H, NH, D2O
13
exchangeable) ppm; C NMR (100 MHz, DMSO-d6):  15.52, 25.35, 25.56,
26.02, 27.18, 27.60, 35.20, 46.72, 112.32, 119.83, 122.17, 136.48, 148.48, 148.76,
159.55, 160.60, 163.92 and 176.40 ppm; MS [m/z, %]: 327 [M+1˥·+, 26.1] and
326 [M+, 100]; Anal. Calcd for C18H22N4O2 (326.39): C, 66.24; H, 6.79; N, 17.17.
Found: C, 65.98; H, 6.87; N, 17.41.
4.1.3.3. N1-Cycloheptylidene-1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carbohydrazide (4c)
o
Buff solid; mp: 255-257 C; yield: 0.26 g, 74.10%; IR (KBr) max: 3132 (NH),
1685 (C=O) cm-1; 1H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H, CH3, J=7.0 Hz),
1.58-1.62 (m, 6H, 3CH2), 1.76-1.79 (m, 2H, CH2), 2.48-2.50 (m, 4H, 2CH2), 2.69
(s, 3H, CH3), 4.59-4.65 (q, 2H, CH2, J=7.0 Hz), 7.51 (d, 1H, ArH, J=8.2 Hz), 8.60
(d, 1H, ArH, J=8.2 Hz), 9.10 (s, 1H, ArH) and 12.63 (s, 1H, NH, D2O
13
exchangeable) ppm; C NMR (100 MHz, DMSO-d6):  15.48, 24.34, 25.32,
27.59, 30.12, 30.19, 31.26, 36.77, 46.00, 112.16, 119.77, 122.16, 136.48, 148.42,
148.73, 158.23, 162.07, 163.94 and 171.68 ppm; MS [m/z, %]: 341 [M+1˥ · +,
12

·
22.24],
340
[M+,
81.0]
and
215
[M
-
C7H13N2˥
C19H24N4O2 (340.42): C, 67.04; H, 7.11; N, 16.46. Found: C, 67.23; H, 7.04; N,
16.59.
4.1.3.4. 1-Ethyl-7-methyl-N1-(1-methylpiperidin-4-ylidene)-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carbohydrazide (4d)
o
Buff solid; mp: 238-240 C; yield: 0.27 g, 79.40%; IR (KBr) max: 3132 (NH),
1685 (C=O) cm-1; 1H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H, CH3, J=7.0 Hz),
2.25 (s, 3H, CH3), 2.43-2.45 (m, 2H, CH2), 2.52-2.54 (m, 2H, CH2), 2.67 (s, 3H,
CH3), 3.35-3.50 (m, 4H, 2CH2), 4.58-4.63 (q, 2H, CH2, J=7.0 Hz), 7.48 (d, 1H,
ArH, J=8.2 Hz), 8.56 (d, 1H, ArH, J=8.2 Hz), 9.08 (s, 1H, ArH) and 12.81 (s, 1H,
NH, D2O exchangeable) ppm; 13C NMR (100 MHz, DMSO-d6):  15.48, 25.34,
27.54, 34.35, 45.77, 46.77, 54.33, 55.84, 112.12, 119.76, 122.21, 136.42, 148.43,
148.73, 156.38, 160.69, 163.98 and 176.37 ppm; MS [m/z, %]: 342 [M+1˥ · +,
0.64], 341 [M+, 1.55] and 110 [C6H10N2˥ · +, 100] Anal. Calcd for C18H23N5O2
(341.41): C, 63.32; H, 6.79; N, 20.51. Found: C, 63.50; H, 6.86; N, 20.74.
4.1.4. General procedure for the synthesis of compounds 5a-c.
Equimolar amounts of Nalidixic acid hydrazide 2 (0.25 g, 1 mmol) and the
selected acid anhydride were dissolved in glacial acetic acid and heated under
reflux for 12 hours. The reaction mixture was allowed to cool, filtered, dried and
recrystallized from absolute ethanol to give compounds 5a-c.
4.1.4.1. N-(2,5-Dioxopyrrolidin-1-yl)-1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (5a)
Buff solid; mp: 260-262 oC; yield: 0.27 g, 81%; IR (KBr) max: 3278 (NH), 1732
1
(C=O), 1670 (C=O) cm-1; H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H, CH3,
13

J=7.0 Hz), 1.80 (s, 4H, 2CH2), 2.69 (s, 3H, CH3), 4.60-4.62 (q, 2H, CH2, J=7.0
Hz), 7.52 (d, 1H, ArH, J=8.0 Hz), 8.59 (d, 1H, ArH, J=8.0 Hz), 9.02 (s, 1H, ArH)
and 11.60 (s, 1H, NH, D2O exchangeable) ppm; 13C NMR (400 MHz, DMSO-d6):
 15.45, 20.83, 25.34, 26.84, 46.80, 110.58, 111.42, 120.01, 122.39, 136.41,
148.49, 149.37, 161.69, 163.42, 166.82 and 175.85 ppm; MS [m/z, %]: 329
[M+1˥·+, 2.61], 328 [M.+, 12.35] and 215 [M - C4H5N2O2˥·+, 100]; Anal. Calcd
for C16H16N4O4 (328.32): C, 58.53; H, 4.91; N, 17.06. Found: C, 58.67; H, 5.08;
N, 17.23.
4.1.4.2. N-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-ethyl-7-methyl-4-oxo-1,4-
dihydro-1,8-naphthyridine-3-carboxamide (5b)
o
Pale yellow solid; mp: 265-267 C; yield: 0.24 g, 72%; IR (KBr) max: 3510
(NH), 1732 (C=O), 1670 (C=O) cm-1; 1H NMR (400 MHz, DMSO-d6):  1.41 (t,
3H, CH3, J=7.0 Hz), 2.69 (s, 3H, CH3), 4.57-4.62 (q, 2H, CH2, J=7.0 Hz), 7.20 (s,
2H, 2CH), 7.53 (d, 1H, ArH, J=8.1 Hz), 8.59 (d, 1H, ArH, J=8.1 Hz), 9.03 (s, 1H,
13
ArH) and 11.35 (s, 1H, NH, D2O exchangeable) ppm; C NMR (100 MHz,
DMSO-d6):  15.43, 25.35, 46.80, 110.43, 120.11, 122.40, 134.20, 136.47,
148.65, 149.56, 164.05, 164.27, 168.41 and 175.94 ppm; MS [m/z, %]: 327
[M+1˥·+, 4.66], 326 [M.+, 22.14] and 215 [M - C4H3N2O2˥·+, 100]; Anal. Calcd
for C16H14N4O4 (326.31): C, 58.89; H, 4.32; N, 17.17. Found: C, 59.12; H, 4.23;
N, 17.03.
4.1.4.3. N-(1,3-Dioxoisoindolin-2-yl)-1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carboxamide (5c)
o
Pale yellow solid; mp: 298-298 C; yield: 0.35 g, 92%; IR (KBr) max: 3522
1
(NH), 1747 (C=O) cm-1; H NMR (400 MHz, DMSO-d6):  1.42 (t, 3H, CH3,
J=7.0 Hz), 2.70 (s, 3H, CH3), 4.58-4.63 (q, 2H, CH2, J=7.0 Hz), 7.54 (d, 1H, ArH,
J=8.0 Hz), 7.94-8.00 (m, 4H, ArH), 8.60 (d, 1H, ArH, J=8.0 Hz), 9.07 (s, 1H,
14

13
ArH) and 11.60 (s, 1H, NH, D2O exchangeable) ppm; C NMR (100 MHz,
DMSO-d6):  15.45, 25.36, 46.84, 110.46, 120.12, 122.43, 124.13, 130.16,
135.61, 136.46, 148.65, 149.59, 164.10, 164.23, 165.55 and 176.02 ppm; MS
[m/z, %]: 377 [M+1˥·+, 25.56], 376 [M.+, 100] and 215 [M - C8H5N2O2˥·+, 100];
Anal. Calcd for C20H16N4O4 (376.37): C, 63.82; H, 4.28; N, 14.89. Found: C,
63.70; H, 4.41; N, 14.95.
4.1.5. General procedure for the synthesis of compounds 6a-d.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbohydrazide
(2)
(0.50 g, 2 mmol) and the appropriate isothiocyanate (2 mmol) were dissolved in
absolute ethanol and heated under reflux for 5 hours. The reaction mixture was
left to cool and the formed precipitate was filtered, dried and recrystallized from
95% ethanol to yield compounds 6a-d.
4.1.5.1. 2-(1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carbonyl)-N-(m-tolyl)hydrazinecarbothioamide (6a)
o
White solid; mp: >300 C; yield: 0.70 g, 87.50%; IR (KBr) max: 3286 (NH),
1643 (C=O) cm-1; 1H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H, CH3, J=7.0 Hz),
2.29 (s, 3H, CH3), 2.69 (s, 3H, CH3), 4.59-4.64 (q, 2H, CH2, J=7.0 Hz), 6.95 (d,
1H, ArH, J=7.6 Hz), 7.2 (t, 1H, ArH, J=7.6 Hz), 7.31-7.36 (m, 2H, ArH), 7.52 (d,
1H, ArH, J=8.0 Hz), 8.60 (d, 1H, ArH, J=8.0 Hz), 9.05 (s, 1H, ArH), 9.79 (s, 2H,
13
2NH, D2O exchangeable) and 10.53 (s, 1H, NH, D2O exchangeable) ppm; C
NMR (100 MHz, DMSO-d6):  15.54, 21.46, 25.36, 46.62, 111.72, 119.98,
122.20, 125.59, 125.78, 128.48, 128.61, 136.51, 137.95, 138.10, 139.59, 148.47,
148.56, 152.07, 163.89 and 175.83 ppm; MS [m/z, %]: 396 [M+1˥·+, 2.46], 395
[M.+, 9.19] and 91 [C7H7˥· +, 100]; Anal. Calcd for C20H21N5O2S (395.48): C,
60.74; H, 5.35; N, 17.71. Found: C, 60.51; H, 5.44; N, 17.92.
15

4.1.5.2. 2-(1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carbonyl)-N-(4-methoxyphenyl)hydrazinecarbothioamide (6b)
White solid; mp: 276-278 oC; yield: 0.76 g, 92.40%; IR (KBr) max: 3286 (NH),
1
1651 (C=O) cm-1; H NMR (400 MHz, DMSO-d6):  .41 (t, 3H, CH3, J=7.0
Hz), 2.69 (s, 3H, CH3), 3.75 (s, 3H, CH3), 4.59-4.65 (q, 2H, CH2, J=7.0 Hz), 6.89
(d, 2H, 2ArH, J=8.8 Hz), 7.35 (d, 2H, 2ArH, J=6.8 Hz), 7.52 (d, 1H, ArH, J=8.0
Hz), 8.61 (d, 1H, ArH, J=8.0 Hz), 9.05 (s, 1H, ArH), 10.07 (s, 1H, NH, D2O
exchangeable), 11.44 (s, 1H, NH, D2O exchangeable) and 12.23 (s, 1H, NH, D2O
13
exchangeable) ppm; C NMR (100 MHz, DMSO-d6):  15.33, 25.23, 46.90,
55.64, 110.33, 113.96, 119.75, 122.36, 127.68, 131.93, 136.31, 144.34, 148.33,
157.25, 162.03, 164.29, 171.26 and 175.95 ppm; MS [m/z, %]: 412 [M+1˥ · +,
0.46], 411 [M.+, 1.68] and 215 [M - C8H10N3OS˥ · +, 100]; Anal. Calcd for
C20H21N5O3S (411.48): C, 58.38; H, 5.14; N, 17.02. Found: C, 58.38; H, 5.27; N,
16.89.
4.1.5.3. 2-(1-Ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carbonyl)-N-(p-tolyl)hydrazinecarbothioamide (6c)
o
White solid; mp: 250-252 C; yield: 0.64 g, 81%; IR (KBr) max: 3286 (NH),
1
1651 (C=O) cm-1; H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H, CH3, J=7.0
Hz), 2.28 (s, 3H, CH3), 2.69 (s, 3H, CH3), 4.59-4.64 (q, 2H, CH2, J=7.0 Hz), 7.13
(d, 2H, 2ArH, J=8.0 Hz), 7.34-7.38 (m, 2H, 2ArH), 7.51 (d, 1H, ArH, J=8.0 Hz),
8.60 (d, 1H, ArH, J=8.0 Hz), 9.04 (s, 1H, ArH), 9.77 (s, 1H, NH, D2O
exchangeable), 10.38 (s, 1H, NH, D2O exchangeable) and 12.31 (s, 1H, NH, D2O
13
exchangeable) ppm; C NMR (100 MHz, DMSO-d6):  15.26, 20.85, 25.18,
46.86, 111.17, 119.56, 122.29, 125.07, 129.26, 133.50, 135.06, 136.10, 136.46,
148.14, 152.20, 164.16, 169.24 and 175.76 ppm; MS [m/z, %]: 396 [M+1˥ · +,
2.46], 395 [M.+, 2.12] and 215 [M - C8H10N3S˥ · +, 100]; Anal. Calcd for
16

C20H21N5O2S (395.48): C, 60.74; H, 5.35; N, 17.71. Found: C, 60.89; H, 5.52; N,
17.54.
4.1.5.4. N-(4-Chlorophenyl)-2-(1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carbonyl)hydrazinecarbothioamide (6d)
o
White solid; mp: 293-295 C; yield: 0.70 g, 84%; IR (KBr) max: 3286 (NH),
1651 (C=O) cm-1; 1H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H, CH3, J=7.0 Hz),
2.69 (s, 3H, CH3), 4.59-4.64 (q, 2H, CH2, J=7.0 Hz), 7.38 (d, 2H, 2ArH, J=8.8
Hz), 7.52 (d, 1H, ArH, J=8.0 Hz), 7.55-7.58 (m, 2H, 2ArH), 8.60 (d, 1H, ArH,
J=8.0 Hz), 9.05 (s, 1H, ArH), 9.91 (s, 1H, NH, D2O exchangeable), 10.70 (s, 1H,
13
NH, D2O exchangeable) and 11.33 (s, 1H, NH, D2O exchangeable) ppm; C
NMR (100 MHz, DMSO-d6):  15.34, 25.24, 46.88, 115.86, 119.74, 122.32,
128.60, 128.96, 136.28, 138.19, 148.31, 164.23, 173.63 and 175.88 ppm; MS
[m/z, %]: 416 [M+1˥·+, 0.09], 415 [M.+, 0.31] and 215 [M - C7H7ClN3S˥·+, 100];
Anal. Calcd for C19H18ClN5O2S (415.90): C, 54.87; H, 4.36; N, 16.84. Found: C,
54.95; H, 4.08; N, 16.95.
4.1.6. General procedure for the synthesis of compounds 7a,b.
A mixture of Nalidixic acid hydrazide 2 (0.25 g, 1 mmol) and the appropriate aryl
isocyanate (1mmol) in absolute ethanol was heated under reflux for 5h. The
reaction mixture was left to cool and the formed precipitate was filtered, dried and
recrystallized from 95% ethanol to afford white solid (7a.b).
4.1.6.1. N-(2,6-Dichlorophenyl)-2-(1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-
naphthyridine-3-carbonyl)hydrazinecarboxamide (7a)
White solid; mp: 308-310 oC; yield: 0.28 g, 64.50%; IR (KBr) max: 3294 (NH),
1
1693 (C=O), 1643 (C=O) cm-1; H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H,
CH3, J=7.0 Hz), 2.69 (s, 3H, CH3), 4.61 (q, 2H, CH2, J=7.0 Hz), 7.30 (t, 1H, ArH,
17

J=8 Hz), 7.50-7.53 (m, 3H, ArH), 8.54 (s, 1H, NH, D2O exchangeable), 8.60 (d,
1H, ArH, J=8.0 Hz), 8.81 (s, 1H, NH, D2O exchangeable), 9.08 (s, 1H, ArH) and
13
11.28 (s, 1H, NH, D2O exchangeable) ppm; C NMR (100 MHz, DMSO-d6):
 15.45, 25.31, 46.54, 111.92, 117.47, 118.62, 119.86, 121.96, 128.47, 128.88,
128.98, 136.38, 141.52, 147.81, 148.43, 148.53, 163.75, 163.84 and 175.89 ppm;
MS [m/z, %]: 435 [M+1˥·+, 0.23], 434 [M.+, 0.09] and 215 [M - C7H6Cl2N3O˥·+,
100]; Anal. Calcd for C19H17Cl2N5O3 (434.28): C, 52.55; H, 3.95; N, 16.13.
Found: C, 52.78; H, 4.07; N, 16.40.
4.1.6.2. N-(2-Chloro-6-methylphenyl)-2-(1-ethyl-7-methyl-4-oxo-1,4-dihydro-
1,8-naphthyridine-3-carbonyl)hydrazinecarboxamide (7b)
White solid; mp: 308-310 oC; yield: 0.29 g, 65.90%; IR (KBr) max: 3309 (NH),
1697 (C=O), 1651 (C=O) cm-1 1H NMR (400 MHz, DMSO-d6):  1.41 (t, 3H,
CH3, J=7.0 Hz), 2.25 (s, 3H, CH3), 2.69 (s, 3H, CH3), 4.59-4.64 (q, 2H, CH2,
J=7.0 Hz), 7.15-7.22 (m, 2H, ArH), 7.32 (d, 1H, ArH, J=6.5 Hz), 7.51 (d, 1H,
ArH, J=8.0 Hz), 8.29 (s, 1H, NH, D2O exchangeable), 8.60 (d, 1H, ArH, J=8.0
Hz), 8.65 (s, 1H, NH, D2O exchangeable), 9.07 (s, 1H, ArH) and 11.23 (s, 1H,
13
NH, D2O exchangeable) ppm; C NMR (100 MHz, DMSO-d6):  15.52, 18.88,
25.34, 49.06, 120.19, 122.20, 123.88, 127.22, 127.79, 129.43, 132.53, 134.35,
136.51, 143.60, 148.57, 151.20, 153.41, 163.64, 163.92 and 176.01 ppm; MS
[m/z, %]: 414 [M+1˥·+, 0.27], 413 [M.+, 0.96] and 215 [M - C8H9ClN3O˥·+, 100];
Anal. Calcd for C20H20ClN5O3 (434.28): C, 58.04; H, 4.87; N, 16.92. Found: C,
58.21; H, 5.11; N, 16.78.
18

4.2. Biological evaluation
4.2.1. In vitro anticancer screening
60 different human cancer cell lines were utilized to screen the newly synthesized
compounds for anticancer activity according to the previously reported standard
procedure [33–35] as follows:
1- RPMI 160 medium containing 2 mM L-glutamine and fetal bovine serum was
used to grow the human cancer cell lines.
2- Cells were inoculated into 96 well microtiter plates into 100 L at optical
density ranging from 4000-5000 cells/well depending on the replication time of
individual cell lines.
o
3- The microtiter plates were incubated at 37 C, 5% CO2, 95% air and 100%
relative humidity for 24 hours.
4- Trichloroacetic acid (TCA) was used to fix two plates of each cell line in situ.
This represented a measurement of cell population at the time of drug addition
(Tz).
5- The synthesized compounds were dissolved in dimethyl sulfoxide (DMSO) at
400 folds the desired final maximum concentration and frozen prior to use.
6- The frozen concentrate was melted at time of drug addition and diluted to twice
the final concentration with complete medium containing 50 g/mL gentamycin.
7- 100 L of the synthesized compounds were added to the appropriate microtiter
wells already containing 100 L yielding the final compound concentration.
o
8- Following the compounds addition, the plates were incubated at 37 C, 5%
CO2, 95% air and 100% relative humidity for 48 hours.
19

9- 50 L of cold 50% (w/v) TCA was gently added to fix the cells and the plates
o
were incubated for 60 minutes at 4 C. The supernatant was discarded and the
plates are washed 5 times with tap water and air dried.
10- To each well, sulphorhodamine B (SRB) solution (100 L) at 0.4% (w/v) in
1% acetic acid was added and plates were incubated for 10 minutes at room
temperature.
11- Unbound dye was removed by washing five times with 1% acetic acid and
plates were air dried.
12- 10 m trizma base was used to dissolve the bound stain and the absorbance
was read at 415 nm wavelength using an automated plate reader.
13- The percentage growth inhibition was calculated as:
Where Time zero is Tz, Control growth is C, Test growth in the presence of drug
is Ti.
4.2.2. MTT colorimetric assay
Determination of IC50 using MTT colorimetric assay was done according to the
previously reported standard procedure[36–41] as follows:
1. Leukemia K-562 and SR cells were obtained from the American Type Culture
Collection. K-562 and SR cells were grown in 100 L DMEM media in a U-
bottomed 96-well microtiter tray, supplemented with 50 g/mL penicillin and 25
o
g/mL streptomycin and were incubated at 37 C till the concentration of the
suspended cells in the media was 5x104 cells/mL.
20

2. The tested compound and standards were dissolved in DMEM media at 1
mg/mL concentration. 10 folds serial dilutions were made directly in the 96 well
microtiter tray to obtain the final desired concentrations (100, 10, 1, 0.1, 0.01
g/mL) and were incubated for 24 hours at 37 oC.
3. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was
dissolved in in phosphate buffered saline at concentration 5 mg/mL. 10 L of
MTT solution was added to all wells in the microtiter plate. Incubate the cultures
at 37 oC for 2 hours.
4. 100 L of isopropanol acidified with HCl (2 mL concentrated HCl added to
500 ml isopropanol) were added to each well to dissolve the formed formazan
crystals. Gentle agitation was applied to ensure complete dissolution of the
formazan crystals.
5. The absorbance was measured spectrophotometrically at two different
wavelengths (540 nm and 690 nm) and the results were summarized in (Table 2)
4.2.3. Cell Cycle analysis
The effect of compound 6a on cell cycle progression was analyzed by flow
cytometric analysis according to the following procedure[42]:
1. Leukemia SR cells were treated with compound 6a at concentration of IC50
(13.85 uM) and incubated for 24 hours.
2. Ice-cold phosphate buffer saline (PBS) was used to wash the cells twice.
3. Cells were collected by centrifugation, fixed in 70% (v/v) ethanol, washed
with PBS and re-suspended with 0.1 mg/mL RNase
4. Cells then were stained with 40 mg/mL propidium iodide (PI) and evaluated
by flow cytometry using FACSCalibur (Becton Dickinson).
5. Cell Quest software (Becton Dickinson) was used to calculate cell cycle
distributions and the results were summarized in (Table 3)
21

4.2.4. Determination of apoptosis using Annexin-V
Evaluation of the pro-apoptotic effect of compound 6a was done using Annexin-
V-FITC and propidium iodide using the following procedure[42]:
1. Leukemia SR cells were incubated for 24 hours after treatment with compound
6a at its IC50 (13.85 uM).
2. The cells were then collected by trypsinization and 0.5 x 106 cells were
washed twice with PBS and stained with 5 mL Annexin-V-FITC and 5 mL PI in 1
x binding buffer for 15 minutes at room temperature in the dark.
3. Analysis were performed using FACS Calibur flow cytometer (BD
Biosciences, San Jose, CA) and the results were reported in (Table 4)
4.2.5. Topoisomerase II inhibitory activity
Compound 6a was evaluated for topoisomerase II and topoisomerase
II inhibitory activity utilizing the human DNA topoisomerase Elisa kit and
following the provided procedure:
1. Standards and the tested compound were dissolved in sample diluent and 2
folds serial dilution was achieved.
2. Biotin-conjugated antibody and avidin conjugated Horseradish Peroxidase
(HRP-avidin) were diluted to 10 folds.
3. 100 L of each concentration of standard or test compound were added to each
well. Incubate at 37 oC for 1 hour.
4. The liquid in each well was removed.
5. 100 L of Biotin-conjugated antibody solution was added to each well
followed by incubation for 1 hour at 37 oC.
6. The microtiter plate was allowed to aspirate and washed 3 times.
7. 100 L of avidin conjugated Horseradish Peroxidase (HRP-avidin) solution
was added to each well and the plate was incubated for 1 hour at 37 oC.
22

8. The plate was aspirated and washed 5 times.
9. 90 L of TMB substrate were added to each well and the plate was incubated
for 30 minutes at 37 oC and protected from light.
10. 50 L stop solution was added. The absorbance was measured
spectrophotometrically within 5 minutes at 450 nm and the results were
summarized in (Table 5).
4.2.6. Molecular modeling study
Molecular modeling studies were carried out using Molecular Operating
Environment (MOE, 10.2008) software. All minimizations were carried out with
MOE with MMFF94x force field and the partial and formal charges were
automatically calculated. The x-ray crystallographic structures of topoisomerase
IIα co-crystallized with DNA [3] were downloaded from the protein data bank
[43]. Enzyme was prepared for docking study by removal of DNA chains, water
molecules. Protonate 3D protocol in MOE with default options was used to
prepare the topoisomerase enzyme. For the docking protocol triangle matcher
placement method and London dG scoring function were used. Validation of the
docking setup was done by docking of the reference topoisomerase inhibitor
merbarone and comparing the results with a previously reported study [31]. The
MOE validated setup was used to predict the binding interactions and affinity of
the synthesized compounds at the active site. Reference compound merbarone
was used to compare its binding score and binding interactions with the nalidixic
acid derivatives. Energy scores of all the synthesized compounds are shown in
(Table 6).
23

O
O
N
O
N
Ar
Ar
N
H
N
3a,b
O
O
O
N
O
NH2
H2N-NH2
OH
N
H
N
N
N
O
O
O
1
2
N
X
N
X
H
N
N
4a-d
3b
: Ar =
3a
: Ar =
,
S
N
CH2
4a
: X =
4b
4c
4d
CH -CH -CH ,
: X =
2
CH
CH -CH
,
: X =
2, : X =
N CH3
2
2
2
2
Scheme 1: Synthesis of compounds 3a,b and 4a-d.
24

O
O
O
O
N
O
N
N
O
H
O
N
5a
O
O
N
O
O
NH2
O
O
N
O
N
H
N
N
H
N
O
O
N
2
O
5b
O
O
O
N
O
N
O
N
H
O
N
5c
Scheme 2: Synthesis of compounds 5a-c.
25

O
N
O
H
N
H
N
Ar-NCS
N
H
Ar
S
N
6a-d
O
N
O
NH2
N
H
N
O
N
O
H
N
H
N
2
Ar-NCO
N
H
Ar
O
N
7a,b
O
Cl
CH3
CH3
6b
,
: Ar =
6a
6d
: Ar =
: Ar =
,
6c
, : Ar =
CH3
H3C
Cl
7a
7b
: Ar =
: Ar =
,
Cl
Cl
Scheme 3: Synthesis of compounds 6a-d, 7a,b.
26

Table 1: Percent inhibition for synthesized compounds at 10-5 Molar concentration.
Percent inhibition for synthesized compounds at 10-5 Molar concentratio
Cell line
3a
3b
4a
4b
4c
4d
5a
5b
5c
6a
6b
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
8.44
6.63 -10.16
2.84
10.97
10.71
9.02
1.72
10.26
1.2
6.87
-0.06
7.34
0.79
5.57
-0.84
12.85
-3.65
2.52
8.28
19.8
27.99
28.99
11.86
14.18
-0.67
-2.52
12.35
2.87
-1.71
-0.12
4.45
6.35
0.07
0.25
-2.93 -5.36
-6.44 1.32
7.83
1.56
5.67
0.93
7.69
50.68 15.
-2.54 24.19 -0.9
8.69
-5.3
-5.61
-10.8
5.78
5.88
7.75
6.5
81.11 5.3
39.99 3.2
26.8 2.2
57.8 10.
-3.26
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
11.52 11.92
9.68
11.17
6.7
9.36
17.14
1.06
17.61
7.12
11.19
7.8
1.12
0.46
13.11
10.59
6.07
7.1
17.41
6.79
5.23
19.31
18.02
10.51
11.34
7.6
7.68
-3.38
3.41
4.94
4.78
-0.13
8.51
0.91
-2.46 -4.74
-3.39 -0.08
-1.6
1.64
6.19
3.98
29.6 6.9
-2.98
14.33
12.08
8.48
2.21
3.39
6.87
11.73
-0.69
9.53
6.19
8.29
-8.08 32.65 5.2
-8.68 40.94 6.1
HOP-62
HOP-92
-1.09
-1.62
6.05
-6.9
3.46
0.96
0.6
37.62 17.
28.39 13.
18.98 3.1
27.79 19
44.33 3.
49.02 9.
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
1.29
-4.22 -7.15
-0.01 16.25
13.16
10.78
13.44
3.71
4.39
5.06 -11.17 -4.73
5.48
24.74
5.58
9.43
0.43
Colon Cancer
COLO 205
HCC-2998
-4.72
-8.11
-3.66 -16.56
2.24
0.92
-6.49
-17.26 -5.61
-8.25
-105
5.07
-12.76 35.38 -4.6
-1.62 18.35 11.
2.66 -20.27 10.39
-4.73
26

HCT-116
HCT-15
HT29
KM12
SW-620
15.11 13.23
10.88
3.45
3.14
5.32
7.04
8.48
7.11
-1.37
4.09
8.38
22.28
13.85
0.35
12.37
8.81
10.65
-1.5
-6.63
7.24
-0.94
-2.23 -0.39
2.9
2.09
-2.15 44.26 2.8
0.11 66.95 16.
-4.39 32.28 4.2
11.02
4.81
5.91
-0.29
10.38
9.02
9.29
13.27 -2.26 -1.69
12.1 -1.35 0.2
CNS Cancer
3.91
1.92
32.93 7.5
34.73 11.
11.65
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
4.55
14.45
2.88
11
12.95 11.38
4.71
11.55
7.27
3.64
5.51
6.63
7.23
3.77
3.42
13.92
3.45
4.75
5.33
-0.12
6.53
13.54
3.52
15.99
22.3
0.01
11.53
23.76
5.41
8.99
3.76
-4.68
1.64
21.05 -1.44
5.4
-3.16 -0.07
-0.85 -6.81
-2.3
-5
33.98 7.3
39.6 6.9
-1.37
-0.65
-4.2
3.25
2.53
0.99
-6.39 12.43 0.1
0.2 32.73 6.5
-7.18 31.58 20.
0.58
4.48
2.87
42.92 3.0
Melanoma
LOX IMVI
MALME-3M
M14
2.51
3.15 -14.32
-3.23 -2.36
-4.49
1.9
8.18
-2.18
13.07
3.52
9.08
7.18
9.94
21.4
3.13
9.65
-3.88
12.64
-2.29
7.15
2.26
6.29
13.06
5.04
2.87
-3.48
16.08
-4.88
6.19
12.89
12.16
26.77
-0.01
-5.09
-2.15
2.27
-3.55
3.87
3.78
2.51
1.57
20.06 3.2
15.48 -1.
-0.64 -9.82 -13.03 31.68 -3.0
MDA-MB-435 14.34 11.49
12.14 -10.43 -4.5
-0.5 -9.92 -5.46
6.46 -16.32 -5.19
-1.89
7.19
-2.49 21.77 4.2
-5.79 18.18 1.5
-4.55 15.06 -0.
0.59
5.24
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
4.59
2.86
11.07 -1.01
9.28 7.64
13.79 18.26
-1.87
-0.5
1.9
5.97
2.4
0.12
9.23
7.83
23.02 3.1
25.9 6.
16.17
11.66 37.25 11.
Ovarian Cancer
8.18 -8.5 3.89
IGROV1
2.7
3.63
15.01
6.24
18.46
-8.78 28.95 7.6
27

OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
5.01
5.99
11.02
6.8
6.37
6.34
6.61
-0.67
5.64
3.96
0.55
3.18
5.74
16.34
5.96
10.03
10.19
8.82
8.77
13.08
2.38
2.83
3.59
9.39
13.75
20.36
7.42
7.47
10.6
1.82
4.2
7.11
4.05
0.47
-8.53
3.21
5.17
-3.97 -5.59
-5.27 -4.22
-4.82
-6.15 -8.72
-0.4 1.91
-4.73
0.7
32.58 7.1
-1.05 44.58 11.
-8.08 30.44 6.5
4.77
-4.61
4.92
2.78
33.02 5.9
38.6 4.8
9.03 10.
Renal Cancer
3.57 -6.97 -4.21
19.43 11.86 21.53
0.05 -12.43 -2.43
786-0
A498
8.4
9.91
6.94
33.25
13.41
15.8
-3.04
18.95
0.04
9.36
22.45
8.82
12.42
-5.66
14.85
-4.75
14.74
12.77
42.04
15.32
20.1
-0.14
14.53
4.33
-3.18 19.76 7.3
11.38 17.82
16.29
1.7
1.19
50.6 25.
36.94 6.
44.28 16.
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
7.79
15.29
-7.98
22.62 17.74
-6.43
17.1
3.26
8.32
-0.9
18.04
6.31
7.96
2.38 -10.26 -1.67
-3.87 22.51 11.
13.66 22.99 8.9
-1.71 26.63 -3.2
13.05 46.07 24.
16
-1.49
5.16
-8.49
12.8
-6.35
-5.14 -8.18
18.69
24.84
10.45 11.77 14.73
Prostate Cancer
PC-3
DU-145
12.02
-0.65
8.94
-0.1
14.36
11.22
17.31
12.27
-2.13
8.05
-8.17 -9.11
7.04
6.94
42.04 10.
-9.32 -10.81 10.79
-5.17 25.62 -4.3
Breast Cancer
MCF7
MDA-MB-
231/ATCC
HS 578T
BT-549
20.06 12.39
12.34 16.82
16.35 23.93
16.61
6.68
10.83
5.61
22.84
12.86
10.09
8.18
-5.63 -4.16
0.54 -2.67
-2.09 -2.32
6.74
5.62
0.53
8.74
45.93 16.
38.09 3.0
53.54 7.3
-4.52
11.18
16.54
18.13
8.05
19.88
17.03
17.24
2.89
3.34
2.09
-4.95 27.26 7.2
28