Novel 9-(2-(1-arylethylidene)hydrazinyl)acridine derivatives: Target Topoisomerase 1 and growth inhibition of HeLa cancer cells

Article abstract of DOI:10.1016/j.bioorg.2019.102962

A series of 9-(2-(1-arylethylidene)hydrazinyl)acridine and its analogs were designed, synthesized and evaluated for biological activities. Various biochemical assays were performed to determine the free radical scavenging capacity of synthesized compounds (4a–4j). Anticancer activity of these compounds was assessed against two different human cancer cell lines viz cervical cancer cells (HeLa)and liver cancer cells (HepG2)as well as normal human embryonic kidney cell line (HEK 293). Compounds 4b, 4d and 4e showed potential anti-proliferative effects on HeLa cells. Based on results obtained from antioxidant and cytotoxicity studies, 4b, 4d and 4e were further studied in detail for different biological activities. 4b, 4d and 4e reduced the cell growth, inhibited metastatic activity and declined the potential of cell migration in HeLa cell lines. Topoisomerase1 (Top1)treated with compounds 4b, 4d and 4e exhibited inhibition of Top1 and prevented DNA replication. Molecular docking results validate that interaction of compounds 4b, 4d and 4e with Top1-DNA complex, which might be accountable for their inhibitory effects. Further it was concluded that compounds 4b, 4d and 4e arrests the cells at S phase and consequently induces cell death through DNA damage in HeLa cells.

Full text of DOI:10.1016/j.bioorg.2019.102962

Accepted Manuscript
Novel 9-(2-(1-arylethylidene)hydrazinyl)acridine derivatives: Target Topoiso-
merase 1 and growth inhibition of HeLa cancer cells
Md Rafi Haider, Kamal Ahmad, Nadeem Siddiqui, Zulphikar Ali, Md Jawaid
Akhtar, Neeraj Fuloria, Shivkanya Fuloria, Manickam Ravichandran, M. Shahar
Yar
PII:
S0045-2068(19)30372-4
https://doi.org/10.1016/j.bioorg.2019.102962
102962
DOI:
Article Number:
Reference:
YBIOO 102962
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
7 March 2019
21 April 2019
28 April 2019
Please cite this article as: M. Rafi Haider, K. Ahmad, N. Siddiqui, Z. Ali, M. Jawaid Akhtar, N. Fuloria, S. Fuloria,
M. Ravichandran, M. Shahar Yar, Novel 9-(2-(1-arylethylidene)hydrazinyl)acridine derivatives: Target
Topoisomerase 1 and growth inhibition of HeLa cancer cells, Bioorganic Chemistry (2019), doi: https://doi.org/
1
0.1016/j.bioorg.2019.102962
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Novel 9-(2-(1-arylethylidene)hydrazinyl)acridine derivatives: Target
Topoisomerase 1 and growth inhibition of HeLa cancer cells
1
1
1
1
1,4
2
Md Rafi Haider , Kamal Ahmad , Nadeem Siddiqui , Zulphikar Ali , Md Jawaid Akhtar , Neeraj Fuloria , Shivkanya
2
3
1
Fuloria , Manickam Ravichandran , M. Shahar Yar *
1
Department of Pharmaceutical Chemistry, School of Pharmaceutical Education and Research (SPER), Jamia
Hamdard, Hamdard Nagar, New Delhi-110062, (India)
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Semeling Campus, Jalan
Bedong-Semeling, Bedong, Kedah Darul Aman, Malaysia 08100
3
Faculty of Applied Science, AIMST University, Semeling Campus, Jalan Bedong-Semeling, Bedong, Kedah Darul
Aman, Malaysia 08100
4
School of Pharmacy, Bharat Institute of Technology, NH58, Partapur Bypass Meerut-250103
*
Corresponding author.
Dr. M. Shahar Yar
Department of Pharmaceutical Chemistry, School of Pharmaceutical Education and Research (SPER), Jamia
Hamdard, Hamdard Nagar, New Delhi-110062, (India)
Tel.: +91 11 26059681/688x5890, +91 9811811033 (mobile).
E-mail addresses: yarmsy@rediffmail.com
Abstract:
A series of 9-(2-(1-arylethylidene)hydrazinyl)acridine and its analogs were designed, synthesized
and evaluated for biological activities. Various biochemical assays were performed to determine
the free radical scavenging capacity of synthesized compounds (4a-4j). Anticancer activity of
these compounds was assessed against two different human cancer cell lines viz cervical cancer
cells (HeLa) and liver cancer cells (HepG2) as well as normal human embryonic kidney cell line
(
HEK 293). Compounds 4b, 4d and 4e showed potential anti-proliferative effects on HeLa cells.
Based on results obtained from antioxidant and cytotoxicity studies, 4b, 4d and 4e were further

studied in detail for different biological activities. 4b, 4d and 4e reduced the cell growth,
inhibited metastatic activity and declined the potential of cell migration in HeLa cell lines.
Topoisomerase1 (Top1) treated with compounds 4b, 4d and 4e exhibited inhibition of Top1 and
prevented DNA replication. Molecular docking results validate that interaction of compounds
4
b, 4d and 4e with Top1-DNA complex, which might be accountable for their inhibitory effects.
Further it was concluded that compounds 4b, 4d and 4e arrests the cells at S phase and
consequently induces cell death through DNA damage in HeLa cells.
Keywords: Anticancer; Topoisomerase1; Antioxidant; Docking; Acridine
1
. Introduction
Cancer is one of the major health issues globally with several clinical challenges which increase
every year cumulatively [1]. Cancer is characterized by uncontrolled growth of cells which
invades to other parts of the body and considered as most deadly health crisis next to
cardiovascular diseases [2]. Till date, several cytotoxic drugs have already been developed to
fight cancer. However, most of these drugs have lesser therapeutic efficacy and are associated
with many undesirable side effects [3]. Hence, constant search for safer new chemical entities
with substantial anticancer activity, and identification of efficient cellular targets are needed for

the effective cancer treatment [4, 5]. From literature survey, it was evident Topoisomerase 1
Top1) express itself constitutively in several forms of cancer (lymphoma, breast, leukemia,
lung, ovarian and prostate) and have vital roles in cancer cell growth, survival and development
6-9]. Top1 is an enzyme essential for DNA replication and transcription. During cellular
(
[
processes of replication or transcription, double stranded DNA acquires significant degree of
both positive and negative supercoiling [10, 11]. Top1 resolves the topological problems arising
due to supercoiling and allow effective replication and transcription [11]. Top1 imparts its action
with the help of a nucleophilic tyrosine residue that nicks a single strand of the DNA and allows
“controlled rotation” of the DNA around the other strand, thus relax the double helix of DNA
[
10-12]. Since, Top1 is especially expressed in the S-phase of the cell cycle and an increased
level has been proven in several solid human tumors, it has long been acknowledged as an
effective target for the design and development of cancer chemotherapeutics [10, 12]. Use of
some of the established Top1 inhibitors include Camptothecin, Irinotecan, Topotecan, Rubitecan,
indenoisoquinolines depicted in Fig. 1, remains limited due to side effects, high toxicity and
emerging drug resistance [13-16]. Therefore, there is necessity to find a novel drug that can
reveal promising anti-proliferative activity with least toxicity and lower side effects.
In an effort to find active anticancer agents targeting Top1, several constructive works
have been made on the improvement of heterocyclic moieties depending on their structural
framework. Acridine and its analogues have fascinated the synthetic and medicinal chemists
across the globe owing to their various uses [17]. It is among one of the several
pharmacologically active scaffold in medicinal chemistry with biological activities like
antimicrobial [18], anticancer [17, 19, 20], antimalarial [21], antiviral [22], anti-alzheimer [23]
etc. Due to its planar structure, it can easily slid between the bases of double stranded DNA and
interact with its machinery during replication, transcription and chromosome segregation [17].
Considering the promising therapeutic effects of acridine, we synthesized of new series of 9-(2-
(
1-phenylethylidene)hydrazinyl)acridine analogs with an objective to develop effective and safe
anticancer agents against Top1(Fig. 2) [41,42].
. Results and Discussion
The title compounds (4a-4j) were synthesized in three steps as outlined in the synthetic protocol
Scheme 1). 2-chlorobenzoic acid (1) yielded 2-(phenylamino)benzoic acid (2) on condensation
with aniline using copper/copper oxide, anhydrous potassium carbonate in Dimethyl formamide
2
(

(
DMF). Compound (2) on cyclization with phosphorous oxychloride (POCl ) afforded 9-
3
chloroacridine (3) in good yield. A series of different acetophenone hydrazones were condensed
with 9-chloroacridine (3) to yield final compounds (4a-4j). The purity of compounds was
1
determined by thin layer chromatography and melting point measurement. Data from IR, H
1
3
NMR, C NMR spectroscopy and mass spectrometry were used for the structural establishment
of the synthesized compounds.
9
-(2-(1-phenylethylidene)hydrazinyl)acridine derivatives 4a-4j exhibited IR absorption for N-H
-1
1
at 3200-3300 cm . The characteristic H NMR peaks of NH proton, for compounds 4a-4j were
1
3
found around δ 10.7 ppm. In the C NMR spectra of compounds 4a-4j, C=N carbon peaks were
observed near δ 160 ppm. Mass spectra of some of the title compounds exhibited a molecular ion
peak M+ at an m/z equivalent to their molecular formula (see supplementary data).
2
2
.2. In vitro antioxidant activity
.2.1. DPPH free radical scavenging assay
The radical scavenging potential of synthesized compounds was determined by DPPH assay and
compounds (4b and 4e) showed remarkable antioxidant activities (Fig.3a). These compounds
showed notable DPPH radicals inhibition in a dose-dependent manner at different concentrations
(
5 - 400 µM). The free radical-scavenging action of compounds increased steadily from 5 - 100
µM and then become nearly constant. At a concentration 100 µg/ml, the radical inhibition by 4b
and 4e were found to be 97.01 and 97.24 % respectively as compared to L-AA which exhibited
scavenging activity of 96.11 %. The calculated EC50 values of 4b and 4e were 9.55 ± 0.26 µM
and 9.31 ± 0.32 µM (P <0.05) respectively compared to 9.65 ± 0.11 µM for L-AA (Table 1).
2
.2.2. Superoxide radical scavenging assay
The outcomes depicted in Fig. 3b exhibits the superoxide radical-scavenging activity of
synthesized compounds with the standard. The superoxide radical-scavenging activity of 4b
(
92.14 %) and 4e (93.24 %) were more significant (P <0.05) compared to L-AA (89.44 %) at
1
00 μM concentration.
2
.2.3. Nitric oxide radicals
Nitric oxide free radical scavenging potential was noted to be maximum for compounds 4b and
e at 400 µM concentration compared to the standard L-AA (Fig.3c). A significant difference (P
0.003) was also found in the EC50 values of 4b & 4e and the standard (Table 1).
.2.4. Hydrogen peroxide radicals
4
<
2

The free radical scavenging potential of compounds was determined by hydrogen peroxide
) scavenging assay. Compounds 4b and 4e exhibited maximum scavenging effect
(
H
2
O
2
compared to standard as shown in Fig. 3d in concentration dependent manner. The significant
difference (P <0.005) was also observed between EC50 values of 4b & 4e and the standard (Table
1
).
2
.2.5 Total reducing assay
The reducing power of newly synthesized compounds has been shown in Fig.3e. In present
study, free-radical scavenging potential and reducing properties of these molecules by several
free-radical generating systems was performed and the quantitative estimation proved that
compounds 4b and 4e have higher reducing power than the L- ascorbic acid. Our results suggest
that antioxidant capacity of compounds 4b and 4e is significantly large.
2
2
.3. In vitro anticancer activity
.3.1. MTT assay
The in vitro cytotoxic activity of synthesized compounds was evaluated by MTT assay method
MTT (3-(4,5-dimethylthiaazol-2-yl-2,5-diphenyl tetrazolium bromide) is reduced to formazan in
metabolically active cells. The MTT assay reveals that with the increase in absorbance, the
viability of cells also increases and the cell inhibition decreases. The reduction in absorbance
represents the cell inhibition. This is a reliable method to determine cytotoxicity of compounds.
Synthesized compounds (4a-4j) were tested against the human embryonic kidney cells
(
HEK293) for cytotoxicity activity with Camptothecin as standard (Fig. 4A). IC50 values of
compounds (4a-4j) against HEK293 cells were in the range 31.38 µM to 277.13µM respectively
Table 2).The anticancer activity of compounds (4a-4j) against HeLa and HepG2 cell lines with
(
increasing concentration exhibited a significant reduction in cells viability when compared to
Camptothecin as standard. In case of cell proliferation assay on ovarian cancer cell line (HeLa),
with increasing concentrations of compounds (4a-4j), varying degree of % growth inhibition was
observed (Fig. 4B). The IC50 values of compounds were calculated and found to be in the range
of 18.89 - 52.64 µM (Table 2). Compounds 4b (20.97 µM), 4d (18.89 µM) and 4e (20.66 µM)
showed the lower IC50 values than standard (24.00 µM), which suggests that these compounds
are more active against HeLa. Cell proliferation assay against liver cancer cells (HepG2)
exhibited varying degree of % growth inhibition (Fig. 4C). The IC50 values of compounds were
calculated and found to be in the range of 18.7 - 108.34 µM (Table 2). Compounds 4b (95.76

µM), 4d (84.93 µM) and 4e (108.34 µM) showed the lowest IC50 values against HepG2
compared to standard (22.58 µM).
The selectivity index (SI) values for the effectiveness of selected compounds against
cancerous cells were calculated. High SI value (>3) of a compound suggests selective inhibition
towards cancer cells. While the compounds with SI value <3 is considered to be toxic for normal
cells [39]. Compounds 4b, 4d and 4e exhibited better selectivity against HeLa cell lines since
their SI values were more than 3 compared to other compounds of the series (Table 2). The SI
values of compounds 4b, 4d and 4e were found to be less than 3 against HepG2 cell lines. The
result suggests that compounds 4b, 4d and 4e are selective and effective against HeLa cell line.
Besides, the effect of the compounds 4b, 4d and 4e on HeLa cells showed profound anti-
proliferative activity in comparison to standard which was further complemented by cell
migration assay, cell nuclear morphology assay and cell cycle analysis.
2
.3.2. Structure activity relationship of 9-(2-(1-arylethylidene)hydrazinyl)acridine derivatives
On the basis of various free radical scavenging assays performed, it was inferred that compounds
b and 4e exhibit excellent antioxidant potential due to presence of hydroxyl group attached to
4
the aromatic ring linked to acridine. Substitution of aromatic ring with methyl group results in
moderate antioxidant activity while methoxy group caused poor activity. Substitution at ortho
position results in better activity than para substitution. Anticancer activity of synthesized
compounds performed by MTT assay against HeLa cells showed that compounds 4b, 4d and 4e
possessed better activity than standard drug. Substitution in aromatic ring linked to acridine
scaffold at ortho position resulted in better activity than para substitution. Presence of hydroxyl
group at both positions resulted in more or less equal growth inhibition. Substitution with methyl
group at ortho position improves the activity but at para position, there is considerable decrease
in activity. All three compounds inhibited of Top1 enzyme during inhibition assay. Anticancer
potential of all the three compounds has been further supported by molecular docking.
2
.3.3. Cell nuclear morphology assay
Necrotic and apoptotic cells were observed through fluorescence microscopy technique (Nikon
Eclipse, Inc., Japan) on the basis of cell membrane integrity and general cell morphology.
Initially, fluorescence microscopic evaluation showed untreated HeLa cells tainted with a
uniform blue fluorescence and intact shape. 48 h post treatment with compounds 4b, 4d and 4e at
IC50 concentrations, the cells were observed for morphological alterations as shown in Fig. 5A.

Mean and the standard deviations were also calculated based on numbers of apoptotic
cells. An increase was observed in number of apoptotic cells at IC50 concentration of 4b, 4d and
4
e (Fig. 5B). Percentage of apoptotic cells was determined for being statistically significant from
the 4b, 4d and 4e treated HeLa cancer cells. Only a small fraction of apoptotic cells were found
in the control cells.
2
.3.4. Cell migration assay
The wound-healing study to determine cell migration demonstrated considerable decrease in cell
migration on treatment with compounds 4b, 4d and 4e at IC50 concentrations in HeLa cells.
Photomicrographs of HeLa cells post treatment with 4b, 4d and 4e is shown in Fig. 5a. The
wound closure rate images were taken after 48 h treatment. Scale bars were added with the help
of Image J, as depicted in Fig. 5b. As shown in Fig. 5a, wound area significantly increased on
treatment with compounds 4b, 4d and 4e after 48 h. However, in control cells a reduction in area
was observed. Besides, it was observed that 4b, 4d and 4e exhibited better reduction in cell
migration compared to control (Fig. 5a & Fig. 5b).
2
.3.5. Effect on cell cycle regulation
The cell cycle analysis in HeLa cells on treatment with compounds 4b, 4d and 4e was studied by
flow cytometry using propidium iodide staining technique. The percentage of apoptotic cells as
well as cell cycle stage 48 h post treatment with compounds is shown in Fig. 5I & II. Treatment
with compounds 4b, 4d and 4e at IC50 concentrations in HeLa cells induce 12.4%, 2.4%, and
1
1.7% of apoptosis, respectively compared against the untreated control (1.4%) as presented in
Fig. 5I.
Treatment of HeLa cells with compounds 4b, 4d and 4e at IC50 concentration caused an increase
in the percentage of cells in the S phase against control. Compound 4b also resulted in rise of
cells in G0/G1 phase against control. The percentage of cells got reduced in G2/M phase in
comparison to control cells. The flow cytometry analysis suggested that the effect of compounds
4
b, 4d and 4e on the accumulation of cells in the S phases subsequently resulted in cell cycle
arrest (Fig. 5I & II).
.4. Topoisomerase 1(Top1) inhibition assay
2
Top1 is a key cellular enzyme that cuts DNA and makes it to wind and unwind, thereby plays an
important role in transcription, replication and chromosome structure. The strategy to inhibit
Top1 enzyme leads to death of cancer cells, thereby acts as target in human cancer

chemotherapy. In order to study the possible mechanism of cytotoxic potential exhibited by
synthesized compounds 4b, 4d and 4e were subjected to DNA relaxation study. The supercoiled
plasmid PBR322 was incubated with human Top1 in the presence of the test compounds at 2 μM
concentration each. As shown in Fig. 5III, compounds 4b, 4d and 4e showed Top1 inhibitory
activity based on position of supercoiled DNA. Based on the study, it is suggested that
compounds 4b, 4d and 4e exhibit anticancer activity through Top1 inhibition.
2
.5 Molecular Docking Studies
Molecular docking was performed in order to establish the binding ability of the new synthesized
compounds with Top1. The docking scores of all compounds are presented in Table 3. All the
compounds were found to exhibit good interaction with the target protein.
Among all the titled compounds tested for Top1 inhibition, 4b was found to be most
potent and have highest docking score (-6.8 Kcal/mol) as compared to Camptothecin (Table 3).
Compound 4b demonstrated two hydrogen bonds-one each with DNA residue (DA113; 2.3Å)
and Top1 residue (GLU356; 3.4Å) (Fig. 6A(II)). The compound 4b also assumes favorable
orientation within the Top1-DNA complex binding site by interacting with other residues
(
DC112, ASN352, ARG364, LYS374, TRP416, GLU418, LYS425, TYR426 and ILE427) as
against Camptothecin shown in Table 3 and Fig. 6A(I). Compound 4d showed no hydrogen
bonding with Top1-DNA complex, but exhibited other kinds of interactions with various
residues of Top1-DNA complex (DC112, DA113, ASN352, GLU356, PHY361, ARG364,
LYS374, TRP416, GLU418, LYS425, TYR426 and ILE427) presented in Fig. 6B(i) & 6B(ii).
Compound 4e interacted through hydrogen bonds-one each with DNA residue (DA112; 2.9Å)
and Top1 residue (ARG364; 2.0Å) as compared to Camptothecin shown in Table 3. The
compound 4e also interacts with the Top1-DNA complex binding site by interacting with other
residues (DA113, ASN352, GLU356, PHY361, ARG364, LYS374, TRP416, GLU418, LYS425,
TYR426 and ILE427) shown in Fig. 6C(a). In the Table 3, Camptothecin- an inhibitor of Top1
interacted through one hydrogen bond with Top1-DNA complex (ARG364; 2.4Å). It interacts
with the Top1-DNA complex binding site by interacting with other residues (DC112, DA113,
ASN352, GLU356, LYS374, TRP416, GLU418 and LYS425).
Molecular docking study of compounds suggested that several Van der Waals, covalent,
carbon hydrogen, Pi alkyl and electrostatic interactions are the key force for bonding of
compounds 4b, 4d and 4e together with the Top1. Furthermore, compounds 4b, 4d and 4e

interact to the Top1 supported that the antiproliferative activity of synthesized compounds is
caused due to inhibition of Top1 [40, 41]. Compounds 4b, 4d and 4e bind to Top1 with excellent
docking score and it may be considered as an appreciable inhibitor of the Top1.
Schematic model representing inhibition of Top1 by compounds 4b, 4d and 4e is shown
in Fig. 7. Compounds 4b, 4d and 4e bind to Top1 that can block the DNA replication and
ultimately arrest the cell cycle in S phase and subsequent induction of apoptosis reduce the
malignant alteration of cells.
3
. Conclusion
It is evident from the results that the presence of hydroxyl group attached to the aromatic ring
linked to acridine in compounds 4b and 4e might be responsible for its antioxidant behavior.
Cellular experimental results revealed that compounds 4b, 4d and 4e inhibited cell growth and
cell migration predominately in cervical cancer cell. Death of cancer cells by compounds 4b, 4d
and 4e especially followed apoptosis. A possible S phase arrest might be due to the inhibition of
Top1 activity and triggered apoptosis. The mechanism of action of the compounds 4b, 4d and 4e
in cervical cancer cell could possibly be target Top1 and prevents it DNA replication. In silico
experimental results unveiled that the van der Waals, covalent, carbon hydrogen, Pi alkyl and
electrostatic interactions are the key force responsible for better interaction of compounds
present in 4b, 4d and 4e with the Top1. Moreover, our results suggested that compounds 4b, 4d
and 4e decreases the cell growth of cervical cancer cell lines by inhibition of Top1 and could be
effectively used in the cancer chemoprevention and chemotherapy.
4
. Material and methods
4
.1. General information
Melting points of synthesized compounds were determined using open capillary method and are
uncorrected. Progress of the reactions was monitored using self prepared TLC plates (silica gel
‘G’ as stationary phase) and TEF (Toluene: Ethyl acetate: Formic acid; 5:4:1) as mobile phase.
The spots were visualized and marked by exposure to iodine vapors as well as under UV-light.
Shimadzu Infra Red Spectrometer (FTIR-8400S) was used to record IR spectra by KBr pellet
1
13
method. H NMR and C NMR spectra were recorded on Bruker – 300/400 MHz NMR
spectroscope using DMSO-d /CDCl as solvent media. Chemical shift values has been reported
in parts per million (ppm) using TMS (tetramethylsilane) as internal reference. D O exchange
method was used to establish OH and NH protons. LCMS/MS (Perkin-Elmer and LABINDIA,
6
3
2

Applied Biosystem) model no. API 3000 was employed to record mass and presented as m/z. For
elemental analyses, Perkin-Elmer 240 analyzer was used and found for each element analyzed
(
C, H and N) in the range of ±0.4%.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), 1,1-Diphenyl-2-
picrylhydrazyl (DPPH), 2,4,6-tri (2-pyridyl)-s-triazine (TPTZ) and Ascorbic acid were obtained
from Sigma Aldrich (Steinheim, Germany) and DMSO was purchased from Merck. Cell lines
HeLa, HepG2 and HEK293 were procured from National Centre for Cell Sciences (NCCS),
Pune, India. Dulbecco’s Modified Eagle’s Media (DMEM) and Fetal bovine serum were taken
from Gibco-life technologies.
Each of the newly synthesized test compounds 4a to 4j and standard were dissolved in
0
4
4
.1 % dimethyl sulfoxide (DMSO) and diluted to final concentrations of 5 to 80 μM each.
.2. Synthesis of 9-(2-(1-phenylethylidene)hydrazinyl)acridine analogs
.2.1. Synthesis of 2-(phenylamino)benzoic acid (2)
A mixture of 2-chlorobenzoic acid (1) (100 mmol) and aniline (110 mmol) in sufficient quantity
of DMF containing catalytic amount of copper oxide (3.2 g) and potassium carbonate (27.6 g)
was refluxed for 4 hrs. TLC was used at regular intervals to monitor the progress and
completion of reactions. Once the reaction was complete, the reaction mixture was cooled down
to room temperature, poured in to ice-cold water with stirring, filtered and neutralized with conc.
HCl. The precipitate thus obtained was filtered, dried and recrystallized from a mixture of
ethanol-water (Yield 74%) [24].
4
.2.2. Synthesis of 9-chloroacridine (3)
1
0 g of 2-(phenylamino)benzoic acid (2) was dissolved in 16 ml of freshly distilled phosphorous
oxychloride and warmed on a water bath up to 80 ºC for 15 min. and then refluxed for 3.5 hrs on
oil bath. The progress of reaction was monitored constantly using TLC until completion. The
reaction mixture was then allowed to cool down at room temperature. Excess of Phosphorous
oxychloride was removed by distillation under reduced pressure. To this, a mixture of ice,
ammonia and chloroform was added with vigorous stirring. The chloroform layer was separated
and the aqueous layer was further extracted with chloroform. The chloroform extracts were

combined and dried using rotary evaporator to get 9-chloroacridine as greenish residue. It was
obtained as pure crystals using hexane (Yield 67%) [25].
4
.2.3. Synthesis of 9-(2-(1-phenylethylidene)hydrazinyl)acridine and its analogs (4a-4l)
To the ethanolic solution of 9-chloroacridine (2 mmol) and different acetophenone hydrazones (2
mmol) [26], 2-3 drops of conc. HCl were added and the reaction mixture was refluxed for 3-4
hrs. TLC was taken at regular time intervals to monitor the reaction. Once the reaction was
complete, the reaction mixture was allowed cooled to room temperature and filtered to obtain the
crystals of final product.
9
-(2-(1-phenylethylidene)hydrazinyl)acridine (4a)
-1
Yield 72%, m.p. 212-214 °C, IR (KBr; cm ): 3269 (N-H str.), 3117 (Ar.C-H str.), 2878 (Ali. C-
1
H str.); H NMR (400 MHz, DMSO-d
6
, δ ppm): 2.46 (3H, s, CH ), 6.92-6.96 (2H, t, Ar.H), 7.09-
3
7
8
.12 (2H, m, Ar.H), 7.22-7.26 (3H, m, Ar.H), 7.45-7.50 (2H, t, Ar.H), 7.96-7.98 (2H, d, Ar.H),
1
3
.29-8.39 (1H, q, Ar,H), 8.57-8.59 (1H, d, Ar,H ), 10.71 (1H, s, NH); C NMR (100 MHz,
, ppm): 15.0, 76.6, 77.4, 126.6, 128.4, 129.6, 138.5, 157.7; LC-MS (m/z): 312 [M+1];
Elem. Anal. Calc. for C21 : C, 81.00; H, 5.50; N, 13.49. Found: C, 81.03; H, 5.48; N, 13.47.
-(1-(2-(acridin-9-yl)hydrazono)ethyl)phenol (4b)
CDCl
3
H
17
N
3
4
-1
Yield 72%, m.p. 220-222 °C, IR (KBr; cm ): 3249 (N-H str.), 3107 (Ar.C-H str.), 2888 (Ali. C-
1
H str.); H NMR (300 MHz, DMSO-d
6
, δ ppm): 2.51 (3H, s, CH ), 5.35 (1H, s, -OH), 7.21-7.26
3
(
2H, t, Ar.H), 7.50-7.61 (2H, m, Ar.H), 7.69-7.75 (2H, m, Ar.H), 7.85-7.89 (2H, t, Ar.H), 8.14-
1
3
8
.16 (2H, d, Ar.H), 8.22-8.24 (1H, d, Ar,H), 8.42-8.47 (1H, d, Ar,H ), 11.76 (1H, s, NH); C
, ppm): 15.0, 76.6, 77.4, 126.6, 128.4, 129.6, 138.5, 157.7, 162.10; LC-
MS (m/z): 328 [M+1]; Elem. Anal. Calc. for C21 O: C, 77.04; H, 5.23; N, 12.84. Found: C,
NMR (75 MHz, CDCl
3
H
17
N
3
7
7.54; H, 5.18; N, 12.77.
-(2-(1-(4-methoxyphenyl)ethylidene)hydrazinyl)acridine (4c)
9
-1
Yield 62%, m.p. 198-200 °C, IR (KBr; cm ): 3263 (N-H str.), 3121 (Ar.C-H str.), 2858 (Ali. C-
1
H str.); H NMR (300 MHz, DMSO-d
6
, δ ppm): 2.50 (3H, s, CH
3
), 3.79 (3H, s, OCH ), 7.22-
3
7
8
.27 (2H, t, Ar.H), 7.50-7.61 (2H, m, Ar.H), 7.69-7.75 (2H, m, Ar.H), 7.84-7.89 (2H, t, Ar.H),
.14-8.16 (2H, d, Ar.H), 8.22-8.24 (1H, d, Ar,H), 8.42-8.45 (1H, d, Ar,H ), 11.96 (1H, s, NH);
LC-MS (m/z): 342 [M+1]; Elem. Anal. Calc. for C22
C, 79.03; H, 5.64; N, 12.40.
H
19
3
N O: C, 77.40; H, 5.61; N, 12.31. Found:
9
-(2-(1-(p-tolyl)ethylidene)hydrazinyl)acridine (4d)

-1
Yield 76%, m.p. 206-208 °C, IR (KBr; cm ): 3236 (N-H str.), 3100 (Ar.C-H str.), 2890 (Ali. C-
1
H str.); H NMR (300 MHz, DMSO-d
6
, δ ppm): 2.30 (3H, s, CH
3
), 2.95 (3H, s, CH ), 7.23-7.25
3
(
2H, t, Ar.H), 7.50-7.61 (2H, m, Ar.H), 7.70-7.74 (2H, m, Ar.H), 7.83-7.89 (2H, m, Ar.H), 8.14-
8
.17 (2H, t, Ar.H), 8.23-8.25 (1H, d, Ar.H), 8.40-8.43 (1H, d, Ar,H), 11.74 (1H, s, NH); LC-MS
: C, 81.20; H, 5.89; N, 12.91. Found: C, 81.03;
(
m/z): 326 [M+1]; Elem. Anal. Calc. for C22
H
19
N
3
H, 5.78; N, 13.17.
2
-(1-(2-(acridin-9-yl)hydrazono)ethyl)phenol (4e)
-1
Yield 69%, m.p. 202-204 °C, IR (KBr; cm ): 3252 (N-H str.), 3123 (Ar.C-H str.), 2866 (Ali. C-
1
H str.); H NMR (300 MHz, DMSO-d
6
, δ ppm): 2.40 (3H, s, CH ), 5.34 (1H, s, OH), 7.21-7.26
3
(
2H, t, Ar.H), 7.50-760 (2H, m, Ar.H), 7.70-7.76 (2H, d, Ar.H), 7.84-7.89 (2H, t, Ar.H), 8.14-
8
.17 (2H, d, Ar.H), 8.22-8.25 (1H, d, Ar,H), 8.42-8.47 (1H, d, Ar,H ), 11.76 (1H, s, NH); LC-MS
O: C, 77.04; H, 5.23; N, 12.84. Found: C,
(
m/z): 328 [M+1]; Elem. Anal. Calc. for C21
H N
17 3
7
9
6.94; H, 5.21; N, 12.90.
-(2-(1-(o-tolyl)ethylidene)hydrazinyl)acridine (4f)
-1
Yield 72%, m.p. 192-194 °C, IR (KBr; cm ): 3252 (N-H str.), 3127 (Ar.C-H str.), 2898 (Ali. C-
1
H str.); H NMR (300 MHz, DMSO-d
6
, δ ppm): 2.40 (3H, s, CH
3
), 2.87(3H, s, CH ), 7.23-7.28
3
(
2H, t, Ar.H), 7.52-7.62 (2H, m, Ar.H), 7.70-7.76 (2H, m, Ar.H), 7.83-7.89 (2H, t, Ar.H), 8.13-
8
.15 (2H, d, Ar.H), 8.24-8.27 (1H, d, Ar,H), 8.40-8.43 (1H, d, Ar,H ), 11.74 (1H, s, NH); LC-MS
: C, 81.20; H, 5.89; N, 12.91. Found: C, 81.33;
(
m/z): 326 [M+1]; Elem. Anal. Calc. for C22
H
19
N
3
H, 5.93; N, 13.08.
9
-(2-(1-(2-methoxyphenyl)ethylidene)hydrazinyl)acridine (4g)
-1
Yield 78%, m.p. 188-190 °C, IR (KBr; cm ): 3268 (N-H str.), 3110 (Ar.C-H str.), 2882 (Ali. C-
1
H str.); H NMR (300 MHz, DMSO-d
6
, δ ppm): 2.48 (3H, s, CH
3
), 3.78 (3H, s, OCH ), 7.12-
3
7
8
.17 (2H, t, Ar.H), 7.50-7.62 (2H, m, Ar.H), 7.69-7.74 (2H, m, Ar.H), 7.84-7.89 (2H, t, Ar.H),
.14-8.17 (2H, d, Ar.H), 8.22-8.25 (1H, d, Ar,H), 8.42-8.45 (1H, d, Ar,H ), 11.92 (1H, s, NH);
LC-MS (m/z): 342 [M+1]; Elem. Anal. Calc. for C22
C, 78.41; H, 5.59; N, 12.28.
H
19
3
N O: C, 77.40; H, 5.61; N, 12.31. Found:
9
-(2-(1-(4-fluorophenyl)ethylidene)hydrazinyl)acridine (4h)
-1
Yield 62%, m.p. 210-212 °C, IR (KBr; cm ): 3270 (N-H str.), 3120 (Ar.C-H str.), 2871 (Ali. C-
1
H str.); H NMR (300 MHz, DMSO-d
6
, δ ppm): 2.51 (3H, s, CH ), 7.22-7.27 (2H, t, Ar.H), 7.48-
3
7
.60 (2H, m, Ar.H), 7.68-7.74 (2H, m, Ar.H), 7.84-7.89 (2H, t, Ar.H), 8.14-8.17 (2H, d, Ar.H),

8
.23-8.26 (1H, d, Ar,H), 8.42-8.45 (1H, d, Ar,H ), 11.86 (1H, s, NH); Elem. Anal. Calc. for
: C, 76.58; H, 4.90; N, 12.76. Found: C, 77.02; H, 4.88; N, 12.72.
-(2-(1-(2-chlorophenyl)ethylidene)hydrazinyl)acridine (4i)
C
21
H16FClN
3
9
-1
Yield 77%, m.p. 204-206 °C, IR (KBr; cm ): 3260 (N-H str.), 3110 (Ar.C-H str.), 2880 (Ali. C-
1
H str.); H NMR (300 MHz, DMSO-d
6
, δ ppm): 2.52 (3H, s, CH ), 7.20-7.26 (2H, t, Ar.H), 7.48-
3
7
8
.59 (2H, m, Ar.H), 7.69-7.75 (2H, m, Ar.H), 7.82-7.87 (2H, t, Ar.H), 8.16-8.19 (2H, d, Ar.H),
.24-8.27 (1H, d, Ar,H), 8.44-8.47 (1H, d, Ar,H ), 11.71 (1H, s, NH); Elem. Anal. Calc. for
: C, 72.93; H, 4.66; N, 12.15. Found: C, 73.02; H, 4.68; N, 12.12.
-(2-(1-(3-chlorophenyl)ethylidene)hydrazinyl)acridine (4j)
C
21
H16ClN
3
9
-1
Yield 63%, m.p. 194-196 °C, IR (KBr; cm ): 3274 (N-H str.), 3137 (Ar.C-H str.), 2865 (Ali. C-
1
H str.); H NMR (300 MHz, DMSO-d
6
, δ ppm): 2.53 (3H, s, CH ), 7.21-7.26 (2H, t, Ar.H), 7.49-
3
7
8
.61 (2H, m, Ar.H), 7.68-7.75 (2H, m, Ar.H), 7.82-7.86 (2H, t, Ar.H), 8.15-8.18 (2H, d, Ar.H),
.23-8.26 (1H, d, Ar,H), 8.42-8.44 (1H, d, Ar,H ), 11.74 (1H, s, NH); Elem. Anal. Calc. for
: C, 72.93; H, 4.66; N, 12.15. Found: C, 73.02; H, 4.68; N, 12.12.
.3. Determination of In vitro antioxidant activity
C
21
H16ClN
3
4
The antioxidant potential of synthetic derivatives of 9-chloroacridine was determined by
different free radical-scavenging assays. Experiments were performed in triplicate taking L-
ascorbic acid as standard.
DPPH radical-scavenging assay was carried out as per the standard procedure previously
reported by Miliauskas [27]. Superoxide radical-scavenging potential was determined by the
process developed by Liu with minor changes [28]. Hydrogen peroxide (H O2) assay was done to
2
measure the scavenging capacity using the method reported earlier by Ruch with slight
modification [29]. Griess assay method was employed for Nitric oxide free radical-scavenging
activity [30]. Assay for determination of reducing power was performed as per the procedure
reported by Oyazi with minor changes [31].
4
4
.4. In vitro Study
.4.1. MTT assay
Antiproliferative potentials and cell cytotoxicity of synthesized compounds were determined by
MTT assay as reported earlier [32, 33]. Briefly, viable HepG-2, MCF-7 and HEK293 cells were
3
seeded in a 96-well plate (approximately 5×10 cells/well). Cancer cell lines were treated with
increasing concentrations (5–80 μM) and HEK 293 cells with increasing concentrations from

5
µM to 200µM of synthesized compounds on following day. After 48h of incubation, mixtures
of medium and synthesized compounds were removed and the cells were rinsed twice with
freshly prepared phosphate buffer saline (PBS) followed by treatment with 20 µl MTT (from 5
mg/ml stock). 100 µl DMEM was added into each well and the plate was further incubated for at
3
7 ºC for next 4-5 h in a CO incubator. At last, the residual MTT and medium were removed
2
and the formazan crystals were dissolved in 100µl DMSO. The plates were shaken for 15-20 min
further. Absorbance was recorded at 570 nm using titerplate reader (BioRad). The absorbance
values thus measured were converted to percentage viability as compared to the control cells.
Camptothecin was taken as positive control for anticancer activities. Selectivity index (SI) values
were calculated as described previously [33].
4
.4.2. Cell nuclear morphology assay
For cell nuclear morphology study, DAPI (4',6-diamidino-2-phenylindole) staining technique
was performed by following the standard protocol reported previously [33]. In brief, HeLa cells
treated with synthesized compounds (4b, 4d and 4e) at their IC50 concentrations were incubated
for for 48 h at 37 ºC while control cells were contained only media. After incubation, the cells
were rinsed with buffer (PBS), fixed using 70% ethanol and suspended again in 40 µL of DAPI
followed by incubation at 37 ºC for next 25 min covered. The cells were viewed under
fluorescence microscope.
4
.4.3. Cell migration assay
As per the standard reported procedure in literature, cell migration assay was carried out [33].
HeLa cells were seeded in a 12-well plate, incubated at 37 ºC for 24 h, treated with synthesized
compounds (4b, 4d and 4e) at IC50 concentrations and incubated again for 48 h at 37 ºC. Control
cells were treated with the media only. After incubation, a wound was made by scratching with
pipette tip and placed again in CO
and snapped post 48 h incubation. Image J tool was used to add scale bars to images.
.4.4. Cell cycle analysis
2
incubator. The percentage of healing of wound was calculated
4
For the study of cell cycle arrest and regulation, flow cytometry was performed according to the
standard procedures reported in literature [34, 35]. Effects on cell cycle of HeLa cell lines at IC50
concentrations of synthesized compounds (4b, 4d and 4e) were analyzed by flow cytometry. On
completion of 80% culture area, cells were starved overnight and treated with synthesized
compounds (4b, 4d and 4e) for 48 h in complete medium. Later, cells were trypsinised, washed

with chilled buffer (PBS) and centrifuged for next 5 min. Further, 800 μL chilled 70% ethanol
was added drop wise to the cell pellet with slow mixing followed by incubation at 4 ºC for 2 h. It
was treated with 20mg/mL of RNase successively, and the mixture was again incubated at 37 ºC
for 45 min. The cells were resuspended in 500 µL PBS, Propidium iodide (50 mg/mL) was
added and further incubated for 20 min. Flow cytometry was then carried out for cell cycle phase
distribution analysis using FACS (Becton Dickinson FACSCaliber System).
4
.5. Top1 Inhibition assay
The inhibition of potential of synthesized test compounds to Top1 was determined by assessing
the relaxation of supercoiled pBR322 plasmid DNA [36]. The assay was carried out in a final
volume of 20 μL reaction volume containing 2 μl of 10× Top1 reaction buffer, 2 μl supercoiled
plasmid DNA pBR322 (Takara Biotechnology, Japan), 10 μl of Top1, 6 μl water, 2 μl test
compound, 2 μl DNA loading dye. After incubation of reaction mixtures at 37 °C for 30 minutes,
electrophoresis was done on a 0.8% agarose gel at a potential 90 V for 55 min using 1X TAE
buffer. The 5 wells of agarose gel were loaded with supercoiled DNA plasmid, Plasmid and
Top1 enzyme, Camptothecin and test compounds (4b, 4d and 4e) respectively. After
electrophoresis, the gel was stained with ethidium bromide for 10 min and washed with water for
5
min. The DNA bands were visualized using BioRAD illuminator.
4
.6. Molecular Docking Study
Molecular docking study was carried out to establish different interactions between the test
compounds and the target protein [37]. Molecular docking study was performed using the 3D
structure of Top1 protein on Maestro 10.5 program (Schrodinger Inc. USA) by 64 bit operating
system [Intel (R) Core (TM) i5-2400 CPU @ 2.40 GHz, 6 GB RAM]. The X-ray crystal
structure of Human DNA Top1 enzyme forming complex with the known inhibitor
Camptothecin (PDB ID: 1T8I) [38] was retrieved from the Protein Data Bank for the present
study. The protein obtained was first prepared using protein preparation wizard module. Water
molecules and all other undesirable residues were removed other than unique ligand after
preprocessing. It was then subjected to hydrogen bond optimization followed by energy
minimization. The active site was generated as grid box using Glide. Structure of ligand
molecules were drawn as mol file using ChemDraw 12.0 software and their energy was
minimized using LigPrep module of Maestro. All possible ionization states at pH 7.0 ± 2.0 were
generated and minimized. Ligand molecules prepared were docked into the active site in extra

precision mode (XP) using Glide. Docking of compound 4b, 4d and 4e into the active site of
Human Top1 exhibited several molecular interactions (hydrogen bond, pi-pi interaction and
hydrophobic interaction) and were considered to be responsible for the observed activity of the
compounds.
4
.7. Statistical Analysis
Effects were presented as mean value ± SD (n = 3). Regression evaluation was performed to find
the dose response relation among the synthesized compounds (4b, 4d and 4e). Linear regression
was performed to get correlation coefficient. Student’s t-test was used to determine Significance
and a value of p < 0.05 was considered to be significant.
Acknowledgments
One of the Research Scholar (RH) is thankful to Ministry of Minority Affairs, Government of
India, for providing timely Maulana Azad National Fellowship financial assistance [F1-
1
7.1/2014-15/MANF-2014-15-MUS-BIH-38609] and the other author (KA) is thankful to Indian
council of medical research (ICMR), India, for award of Research Associateship [No-
/2/3/16/2018/Online Onco Fship/NCDIII].
3
Conflict of Interests
The authors confirm that this article contents have no conflicts of interest.
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Figure Captions:
Fig. 1: Some established Topoisomerase I inhibitors as anticancer drugs.
Fig. 2: Rationally designed acridine derivatives bearing hydrazinyl linker.
Fig. 3: Free radical-scavenging activity of compounds 4a-4j: (a) DPPH radicals, (b)
superoxide radicals, (c) hydrogen peroxide radicals and (d) nitric oxide radicals. (e) Reducing
potential. L-ascorbic acid (L-AA) were considered as standard antioxidants. The values are
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Fig. 4: Cytotoxicity assay: (A). Compounds on HEK293 cells. (B) Compounds on Hela cells
and (C) Compounds on HepG-2 cells. Data represent three independent tests of experiments and
results are shown as the mean ± SD. Camptothecin is used as a positive control. p < 0.05
compared with the control.
Fig. 5: Effect of compounds on HeLa cells: Cell nuclear morphology: A) without treatment
compound (Control), treated with compounds 4b, 4d and 4e. B) Percentage of apoptotic cells
against their IC50 concentrations of compounds 4b, 4d and 4e. Cell migration: a) Width of

wound increases effectively after the treatment of compounds 4b, 4d and 4e in HeLa cell lines as
compared without treatment compound (Control). b) The percentage of wound against their IC50
concentrations of compounds 4b, 4d and 4e as compared without treatment of compound
(
4
Control). Cell cycle regulation: I) Cell cycle phase distribution with treatment of compounds
b, 4d and 4e as compared without treatment of compound (Control). Compounds 4b arrests
HeLa cell cycle in G0/G1 and S phase while 4d and 4e arrests HeLa cell cycle in S phase as
determined by flow cytometry. II) Bar graph indicating cell distribution percentage against IC50
concentrations of compounds 4b, 4d and 4e as compared without treatment of compound
(
Control).III) Top1 inhibition assay of compounds 4b, 4d and 4e depicting the relaxation of
supercoiled pBR322 plasmid DNA as compared to Camptothecin. Data represent three
independent tests of experiments and results are shown as the mean ± SD. p < 0.05 compared
with the control. Representative images (40X magnification) by fluorescence microscope.
Fig. 6: Molecular docking of compounds with Top1: A) (I) 2D schematic diagram showing
interactions of compound 4b. (II) Cartoon view of Top1 protein with compound 4b. B) (i) 2D
schematic diagram showing interactions of compound 4d. (ii) Cartoon view of Top1 protein with
compound 4d. C) (a) 2D schematic diagram showing interactions of compound 4e. (b) Cartoon
view of Top1 protein with compound 4e. D) (I) 2D schematic diagram showing interactions of
m-Amsacrine. (II) Cartoon view of Top1 protein with m-Amsacrine. Residues involved in
hydrogen bonding, van der Waals interactions, carbon hydrogen and Pi-alkyl are represented in
different color indicated in inset.
Fig. 7: Proposed schematic model for the inhibition of Top1 by compounds 4b, 4d and 4e.

Fig. 1: Some established Topoisomerase I inhibitors as anticancer drugs

Fig. 2: Rationally designed acridine derivatives bearing hydrazinyl linker

Fig. 3: Free radical-scavenging activity of compounds 4a-4j: (a) DPPH radicals, (b)
superoxide radicals, (c) hydrogen peroxide radicals and (d) nitric oxide radicals. (e) Reducing
potential. L-ascorbic acid (L-AA) were considered as standard antioxidants. The values are
expressed as mean ± standard deviation (n = 3) and p < 0.05 compared with the control.

Fig. 4: Cytotoxicity assay: (A). Compounds on HEK293 cells. (B) Compounds on Hela cells
and (C). Compounds on HepG-2 cells. Data represent three independent tests of experiments and
results are shown as the mean ± SD. Camptothecin is used as a positive control. p < 0.05
compared with the control.

Fig. 5: Effect of compounds on HeLa cells: Cell nuclear morphology: A) without treatment
compound (Control), treated with compounds 4b, 4d and 4e. B) Percentage of apoptotic cells
against their IC50 concentrations of compounds 4b, 4d and 4e. Cell migration: a) Width of
wound increases effectively after the treatment of compounds 4b, 4d and 4e in HeLa cell lines as
compared without treatment compound (Control). b) The percentage of wound against their IC50
concentrations of compounds 4b, 4d and 4e as compared without treatment of compound

(
4
Control). Cell cycle regulation: I) Cell cycle phase distribution with treatment of compounds
b, 4d and 4e as compared without treatment of compound (Control). Compounds 4b arrests
HeLa cell cycle in G0/G1 and S phase while 4d and 4e arrests HeLa cell cycle in S phase as
determined by flow cytometry. II) Bar graph indicating cell distribution percentage against IC50
concentrations of compounds 4b, 4d and 4e as compared without treatment of compound
(
Control).III) Top1 inhibition assay of compounds 4b, 4d and 4e depicting the relaxation of
supercoiled pBR322 plasmid DNA as compared to Camptothecin. Data represent three
independent tests of experiments and results are shown as the mean ± SD.p < 0.05 compared
with the control. Representative images (40X magnification) by fluorescence microscope.

Fig. 6: Molecular docking of compounds with Top1 (PDB ID: 1T8I): A) (I) 2D schematic
diagram showing interactions of compound 4b. (II) Cartoon view of Top1 protein with
compound 4b. B) (i) 2D schematic diagram showing interactions of compound 4d. (ii) Cartoon
view of Top1 protein with compound 4d. C) (a) 2D schematic diagram showing interactions of
compound 4e. (b) Cartoon view of Top1 protein with compound 4e. D) (I) 2D schematic
diagram showing interactions of m-Amsacrine. (II) Cartoon view of Top1 protein with m-
Amsacrine.Residues involved in hydrogen bonding, van der Waals interactions, carbon hydrogen
and Pi-alkyl are represented in different color indicated in inset.