Development of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and their salts as potent cytotoxic agents and topoisomerase I/IIα inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.09.019

A novel series of 35 angularly fused pentacyclic 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides were designed and synthesized. Their cytotoxic activities were investigated against six human

Full text of DOI:10.1016/j.bmc.2018.09.019

Accepted Manuscript
Development of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and Their
Salts as Potent Cytotoxic Agents and Topoisomerase I/ IIα Inhibitors
Sateesh Kumar Arepalli, Chaerim Lee, Seongrak Sim, Kiho Lee, Hyunji Jo,
Kyu-Yeon Jun, Youngjoo Kwon, Jong-Soon Kang, Jae-Kyung Jung, Heesoon
Lee
PII:
S0968-0896(18)31291-4
https://doi.org/10.1016/j.bmc.2018.09.019
BMC 14544
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 July 2018
27 August 2018
17 September 2018
Please cite this article as: Arepalli, S.K., Lee, C., Sim, S., Lee, K., Jo, H., Jun, K-Y., Kwon, Y., Kang, J-S., Jung,
J-K., Lee, H., Development of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and Their Salts as Potent
Cytotoxic Agents and Topoisomerase I/ IIα Inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.09.019
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Development of 13H-benzo[f]chromeno[4,3-
b][1,7]naphthyridines and Their Salts as Potent Cytotoxic
Agents and Topoisomerase I/ IIα Inhibitors
Sateesh Kumar Arepallia, Chaerim Leea, Seongrak Sima, Kiho Leeb, Hyunji Joc,
Kyu-Yeon Junc, Youngjoo Kwonc, Jong-Soon Kangd, Jae-Kyung Junga and
Heesoon Leea*
aCollege of Pharmacy, Chungbuk National University, Chungbuk 28160, Republic of Korea
bCollege of Pharmacy, Korea University, Sejong 30019, Republic of Korea
cCollege of Pharmacy, Ewha Womans University, Seoul 03760, Republic of Korea
dKorea Research institute of Bioscience and Biotechnology, Chungbuk 28116, Republic of Korea
 Corresponding author. Tel.: +82-43-261-2811; fax: +82-43-268-2732; e-mail: medchem@chungbuk.ac.kr (H. Lee)
1

Abstract
A novel series of 35 angularly fused pentacyclic 13H-benzo[f]chromeno[4,3-
b][1,7]naphthyridines and 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides were
designed and synthesized. Their cytotoxic activities were investigated against six human cancer
cell lines (NCIH23, HCT15, NUGC-3, ACHN, PC-3, and MDA-MB-231). Among all screened
compounds; 28, 30, 34, 35, 46, 48, 52, and 53 compounds exhibited potential cytotoxic activities
against all tested human cancer cell lines. Further, these potent lead cytotoxic agents were
evaluated against human Topoisomerase I and IIα inhibition. Among them, the compound 48
exhibited dual Topoisomerase I and IIα inhibition especially at 20 μM concentrations the
compound 48 exhibited 1.25 times more potent Topoisomerase IIα inhibitory activity (38.3%)
than the reference drug etoposide (30.6 %). The compound 52 also exhibited excellent (88.4 %)
topoisomerase I inhibition than the reference drug camptothecin (66.7 %) at 100 μM
concentrations. Molecular docking studies of the compounds 48 and 52 with topo I discovered
that they both intercalated into the DNA single-strand cleavage site where the compound 48 have
van der Waals interactions with residues Arg364, Pro431, and Asn722 whilst the compound 52
have with Arg364, Thr718, and Asn722 residues. Both the compounds 48 and 52 have π- π
stacking interactions with the stacked DNA bases. The docking studies of the compound 48 with
topo IIα explored that it was bound to the topo IIα DNA cleavage site where etoposide was
situated. The benzo[f]chromeno[4,3-b][1,7]naphthyridine ring of the compound 48 was stacked
between the DNA bases of the cleavage site with π- π stacking interactions and there were no
hydrogen bond interactions with topo IIα.
2

Key words
13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines; Dual human Topoisomerase I and IIα
inhibition; Cytotoxicity; Molecular docking; Imino Diels-Alder reaction
______________________________________________________________________________
1. Introduction
Human topoisomerases are essential nuclear enzymes that play a vital role in DNA
topology by releasing torsional strain induced during replication, transcription, recombination,
mitosis and chromatin remodeling processes.1-5 These enzymes are important target for a number
of clinically used anticancer drugs owing to their over-expression in cancer cells. Two types of
human topoisomerases existed namely, type I topoisomerase (Topo I) and type II topoisomerase
(Topo II). Human Topo I enzyme catalyzes the formation of DNA single-strand breaks in
absence of binding of ATP and Mg (II) and then rejoined. Topo II enzyme acts as homodimer
which engender double strand breaks with binding of ATP and Mg (II).5-6 Further two isoforms
of topo II characterized namely α and β,6-7 but expression of α isoforms is firmly reliant on
cellular proliferation status, though the β isoform is expressed during the cell cycle.8
Camptothecin analogues such as topotecan, irinotecan and belotecan were used topo I as central
9-10
target for treatment of colon cancer.
Additionally topo I expression is discernible in ovarian
cancer, prostate cancer, and malignant lymphoma. Doxorubicin, etoposide, daunorubicin,
teniposide, idarubicin, mitoxantrone and epipodophyllotoxins were used topo II as main target
for treatment of different malignancies11-21 since topo II is also expressed in breast cancer,
ovarian cancer and malignant lymphoma. Most of the reported topo inhibitors displayed selective
3

inhibition toward topo I or topo II but scarce compounds available for dual topo I and topo II
inhibition.22-26 Therefore, an agent exhibits both topo I and topo II inhibition as dual topo
27-28
inhibitor has received greater attention due to broader anticancer spectrum and advantages.
In recent times, researchers focused to design and develop dual top I & II inhibitors with
potential cytotoxicities.29-36 In this regard as part of developing novel potential anticancer agents,
our group also developed novel 1,8-diazaanthraquinone derivatives and 1,3-
diphenylbenzo[f][1,7]naphthyridines as human topoisomerase IIα inhibitors with potential
cytotoxicities against several cancer cell lines.37-38 Besides, the chromene moiety is an important
ubiquitous structural motif in a wide range of biologically active compounds and natural
products.39 Chromene fused heterocycles are also well known for their biological and
pharmacological
properties.
For
instance
6H-chromeno[4,3-b]quinolines
and
chromenopyridinones are act as potent HSP90 inhibitors,40 anti-inflammatory and potent
cytotoxic agents.41-43 Angularly fused heterocycles (Fig. 1) such as chromeno[4,3-b]pyridines,
44,45
indeno[1,2-b]pyridines,46
benzo[a]phenazines,47
benzofuro[3,2-b]pyridines,48,49
dihydrothieno[2,3-h]quinolines,50 dihydro-1,10-phenanthrolines51 are acknowledged for their
non-intercalative topo I and II dual catalytic inhibition properties. The preamble of angular fused
heterocycles with conformational rigidity on topo I/ IIα-targeting compounds fit further
efficiently into the active site of enzymes and work as an intercalator in the enzyme−DNA
complex.52-55 Since rigid molecules are typically assumed to have less conformational entropy
and can be built into the active site of a receptor. On the basis of these considerations, in this
study we have designed and synthesized 35 angularly fused and pentacyclic 13H-
benzo[f]chromeno[4,3-b][1,7]naphthyridines
(21-39)
and
13H-benzo[f]chromeno[4,3-
b][1,7]naphthyridin-5-ium chlorides (40-56) as shown in Scheme 1. The synthesized compounds
4

were evaluated for cytotoxic activities against six human cancer cell lines, topo I and IIα
inhibitory activities and also carried out docking studies for the potent lead compounds.
Fig 1. Representative angularly fused heterocycles as topo I/IIα inhibitors
2. Results and discussion
2.1. Chemistry
We initially envisaged the synthesis of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines
as a part of our continuous interest in the synthesis of medicinally and biologically important
heterocyclic scaffolds56-61 collectively with an urge to develop an operationally simple and best
synthetic route for desired molecules unlike multistep synthesis for angularly fused heterocycles.
5

Scheme 1. General synthetic one-pot method for the synthesis of 13H-benzo[f]chromeno[4,3-
b][1,7]naphthyridines and 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides.
Hence we developed one-pot, atom-economic and regioselective intramolecular imino Diels-
Alder reaction method for 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines 21-39 as shown in
Scheme 1. After screened different reaction parameters, the reaction condition for the synthesis
of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines 21-39 found to involve 20 mol% CuI, 20
mol% Yb(OTf)3, 0.5 equiv. of 4 Å Molecular sieves in 5–10 mL of CH3CN at reflux temperature
afford the desired intramolecular imino Diels-Alder products 21-39 as shown in scheme 1. The
1
structures of all synthesized products were unambiguously confirmed by H and 13C NMR
spectra. Subsequently, the salt formation was accomplished by suspending the corresponding
13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines in methanol and addition of Conc. HCl at 0oC
then stir 1 h and followed by stir at room temperature for overnight. The hydrochloride salts of
13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines 40-56 were obtained in almost quantitative
yields by concentration of reaction mixture under reduced pressure. These compounds structures
6

13
(Scheme 1 and Table 1) were also confirmed by 1H and C NMR spectra. The compounds 40-
1
13
56 were evidently distinguishable by H and C NMR from the compounds 21-39. To explain
1
the characteristic peak difference among them; for instance in the H NMR spectra, the
compound 30 displayed characteristic singlet peaks at δ 9.47 and 5.09 ppm respectively while its
hydrochloride salt compound 48 shown downfield singlet peaks at δ 10.00 and 5.19 ppm
respectively (Table S3). Similarly the compound 34 displayed characteristic singlet peaks at δ
9.46, 9.02 and 5.54 ppm respectively whereas its salt derivative 52 shown downfield singlet
peaks at δ 10.08, 9.32 and 5.69 ppm respectively (Table S4).
2.2.1. Cytotoxic activity
Having 35 novel chemical libraries in hand, we started evaluation against six different
human cancer cell lines. The utilized human cancer cell lines in this study were lung cancer cell
line (NCIH23), colon cancer cell line (HCT15), gastric cancer cell line (NUCG-3), renal cancer
cell line (ACHN), prostate cancer cell line (PC-3), and breast cancer cell line (MDA-MB-231)
with reference drug doxorubicin (ADR) as shown in Table 1 and Fig. 3, S1 & S2. The growth
inhibitory activity (GI50) is expressed as micromolar concentration as shown in Table 1 and these
values are taken as a mean from three experiments and correspond to the agent’s concentration
causing a 50% decrease in net cell growth. Among all tested chemical libraries, 8 compounds 28,
30, 34, 35, 46, 48, 52, and 53 displayed potential cytotoxicities with GI50 values less than 10μM
range in all tested human cancer cell lines as shown in Table 1. Amongst the compounds 28, 35,
46, 48, 52, and 53 exhibited strong inhibition against all human cancer cell lines. This
phenomenon also supported by the analysis of cell viabilities at 10μM range concentrations (Fig.
3) and which is also established to be most consistent with GI50 values estimated in this study
7

Fig. 2. The cell viabilities of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines (2a) and 13H-
benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides (2b) at 10 μM concentration on
human lung cancer (NCIH23), colon cancer (HCT15), gastric cancer (NUCG-3), renal cancer
(ACHN), prostate cancer (PC-3), and breast cancer (MDA-MB-231) cell lines.
(Fig. S1 & S2). After synthesize different substitutions on core motifs of benzo[f]chromeno[4,3-
b][1,7]naphthyridines and benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides, we
noticed the substituents on C and E rings (Scheme 1) were crucial to explain structure activity
relationship (SAR). When methyl substituent on C ring with different substitution like –I and +I
8

groups on E ring (compounds 21-29) did not exhibit considerable cytotoxic activities against all
tested human cancer cell lines except the compound 28 which is having methoxy substituent (+M
group) on E ring. It was exhibited significant cytotoxic activities against all six human cancer
cell lines in the range of GI50: 1.520-2.622 μM as shown in Table 1. Interestingly, when we
screened the scaffolds with no methyl substitution on C ring and –I groups such as –F and –Cl on
E ring exhibited significant cytotoxicities against all human cancer cell lines. For example
compound 34 (GI50: 1.847-3.205 μM) and compound 35 (GI50: 1.185-2.716 μM) were exhibited
significant cytotoxicities against all human cancer cell lines. We also noted that tolyl group on C
ring with no substitution on E ring (compound 30) exhibited considerable cytotoxic activities
against colon (HCT15), gastric (NUCG-3), renal (ACHN), prostate (PC-3), and breast cancer cell
lines in the range of GI50: 2.398-5.024 μM. After screening of 13H-benzo[f]chromeno[4,3-
b][1,7]naphthyridines (21-39), we observed that only four compounds exhibited moderate to
considerable cytotoxicities but remaining compounds did not exhibit even nominal cytotoxicities
against all tested human cancer cell lines due to their partial solubilities in DMSO media. Hence
in order to improve the solubilities of 21-39, 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-
ium chlorides (40-56) were synthesized and evaluated against all six human cancer cell lines as
shown in Table 1. As anticipated the compounds 40-56 have displayed strong cytotoxicities
against all screened human cancer cell lines than the 13H-benzo[f]chromeno[4,3-
b][1,7]naphthyridines (21-39). Among them, the compounds 46, 48, 52 and 53 were exhibited
higher cytotoxicities against all tested human cancer cell lines in the range of GI50: 0.900-2.699
μM. In order to explain the potential cytotoxic activities of all compounds 21-39 and 40-56
toward each human cancer cell lines, for lung cancer cell line (NCIH23) compound 53 exhibited
higher cytotoxicity (GI50: 0.900 μM) than the remaining compounds.
9

Table 1. In vitro cytotoxic activities of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and
13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides on different human cancer cell
lines
Human Cancer cell lines GI50 (µM)a
MDA-
MB-231
NCIH23
(Lung)
HCT15
(Colon)
NUGC-3
(Gastric)
ACHN
(Renal)
PC-3
Compounds
(Prostate)
(Breast)
(21-39)b & (40-56)
0.090
>10
0.077
>10
0.086
>10
0.080
>10
0.076
>10
0.068
>10
ADR (Reference)
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
10

>10
>10
>10
>10
>10
>10
>10
1.557
>10
>10
1.787
>10
>10
2.622
>10
>10
2.191
>10
>10
1.607
>10
>10
1.520
>10
>10
>10
4.230
3.852
2.398
5.024
5.616
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
11

1.867
1.185
>10
2.195
1.567
>10
1.847
2.208
>10
3.205
2.559
>10
2.794
1.496
>10
2.204
2.716
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
12

>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.392
1.289
1.556
2.008
2.059
1.670
>10
>10
>10
>10
>10
>10
2.059
2.715
2.699
2.284
1.528
1.787
13

>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.026
1.564
1.371
1.004
1.273
1.234
0.900
1.089
1.162
1.079
1.671
1.046
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
14

aGI50 values are taken as a mean from three experiments and correspond to the agent’s concentration causing a 50%
decrease in net cell growth. bThese derivatives have partial solubility
For colon cancer cell line (HCT15) compounds 53 and 46 exhibited higher cytotoxicities (GI50:
1.089 & 1.289 μM respectively) than other compounds. For gastric cancer cell line (NUGC-3)
compounds 53 and 52 exhibited higher cytotoxicities (GI50: 1.162 & 1.371 μM respectively). For
renal cancer cell line (ACHN) compounds 52 and 53 exhibited higher cytotoxicities (GI50: 1.004
& 1.079 μM respectively). For prostate cancer cell line (PC-3) compounds 52 and 35 exhibited
higher cytotoxicities (GI50: 1.273 & 1.496 μM respectively). For breast cancer cell line (MDA-
MB-231) compounds 52 and 53 exhibited higher cytotoxicities (GI50: 1.234 & 1.046 μM
respectively). The cytotoxicity of these compounds on each human cancer cell line was also
shown in Fig. S1 and Fig S2. To sum up the cytotoxic activities, 8 compounds such as 28, 30, 34,
35, 46, 48, 52, and 53 demonstrated potential cytotoxic activities against all tested human cancer
cell lines as shown in Table 1, Fig 3, S1 and S2. In particular, these compounds are more
effective toward lung cancer (NCIH23) and prostate cancer (PC-3) cell lines than other tested
human cancer cell lines.
2.2.2. Topoisomerase I & IIα inhibition
Subsequent to the discovery of lead 8 cytotoxic agents (28, 30, 34, 35, 46, 48, 52, and 53),
we targeted to evaluate the effect of these compounds on human DNA topo I and IIα with
relaxing assay using super coiled plasmid DNA. Camptothecin and etoposide are used as
references for topo I and II inhibitors respectively. The inhibitory activities of these compounds
on topo I and IIα are shown in Fig. 3 & 4 and by using densitometry analysis and their quantified
activities are listed in Table 2.
15

Fig. 3. (a) Human DNA topo I inhibitory activity of lead 13H-benzo[f]chromeno[4,3-
b][1,7]naphthyridines and 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides at the
concentration of 100 μM and 20 μM. Land D: pBR322 DNA only; lane T: pBR322 DNA+
Topo I; lane C: pBR322 DNA+ Topo I + Camptothecin; lane 34: pBR322 DNA+ Topo I +
compound 34; lane 30: pBR322 DNA+ Topo I + compound 30; lane 52: pBR322 DNA+ Topo I
+ compound 52; lane 48: pBR322 DNA+ Topo I + compound 48; lane 53: pBR322 DNA+
Topo I + compound 53; lane 28: pBR322 DNA+ Topo I + compound 28; lane 46: pBR322
DNA+ Topo I + compound 46. (b) Graphical representation of Topo I inhibition for the lead
compounds at 100 μM concentrations.
16

Fig. 4. (a) Human DNA topo IIα inhibitory activity of lead 13H-benzo[f]chromeno[4,3-
b][1,7]naphthyridines and 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides at the
concentration of 100 μM and 20 μM. Land D: pBR322 DNA only; lane T: pBR322 DNA+ Topo
II; lane E: pBR322 DNA+ Topo II + Etoposide; lane 34: pBR322 DNA+ Topo II + compound
34; lane 30: pBR322 DNA+ Topo II + compound 30; lane 52: pBR322 DNA+ Topo II +
compound 52; lane 48: pBR322 DNA+ Topo II + compound 48; lane 53: pBR322 DNA+ Topo
II + compound 53; lane 28: pBR322 DNA+ Topo II + compound 28; lane 46: pBR322 DNA+
Topo II + compound 46. (b) Graphical representation of Topo IIα inhibition for the lead
compounds at 100 and 20 μM concentrations.
17

Table 2. Inhibitory activities of lead 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines and 13H-
benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides on human Topo I and IIα
% Inhibition of Topo I
% Inhibition of Topo IIα
100μM
20μM
100μM
20μM
Compounds
Camptothecin
66.7
NTa
14.4
3.0
44.4
NTa
NTa
NTa
NTa
NTa
NTa
11.1
4.2
NTa
70.4
0.0
NTa
30.6
NTa
NTa
NTa
NTa
NTa
38.3
NTa
NTa
Etoposide
28
30
14.9
14.5
0.0
34
7.0
35
1.7
46
10.2
57.2
88.4
12.3
23.5
69.8
12.2
0.0
48
52
53
NTa
aNot tested
When we screened the inhibitory activities of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines
against Topo I and IIα, initially we tested their activities at 100 μM concentration level. If they
exhibited more than 30% inhibitory activities then we further tested at 20 μM concentration
levels. Each experiment was performed with different concentrations with positive controls such
as camptothecin for Topo I and etoposide for Topo IIα. Since the percentage of inhibition of
Topo I and IIα are often dose-independent.48, 62 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines
such as the compounds 28, 30, 34 and 35 exhibited 14.4%, 3.0%, 7.0%, and 1.7% nominal
18

inhibitory activities respectively on topo I at 100μM concentration level where as 13H-
benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides such as 46, 48, 53 and 52compounds
displayed 10.2%, 57.2%, 12.3% and 88.4% nominal to excellent inhibitory activities respectively
on topo I at 100μM concentration level as shown in Table 2 and Fig 3a. Among all tested 8
compounds, the compound 52 exhibited excellent topo I inhibitory effect (88.4%) than the
reference drug camptothecin (66.7 %) which is 1.32 times more strong topo I inhibition than the
positive control camptothecin at 100μM concentration level as shown in Fig. 3 and Table 2. The
compound 48 also exhibited moderate 57.2 % Topo I inhibitory effect at 100μM concentration
level (Fig. 3a and Table 2). The compounds 48 and 52 also displayed 11.1% and 4.2% topo I
inhibitory activities respectively at 20 μM concentration level (Fig. 3b and Table 2). We have
also assessed the effect of 8 compounds on human DNA topo IIα (Table 2 and Fig. 3). The
compounds 30, 34, 46, 48, and 52 exhibited 14.9%, 14.5%, 23.5%, 69.8% and 12.2% topo IIα
inhibitory activities respectively at 100μM concentration. Among them, the compound 48
exhibited almost similar (69.8%) topo IIα inhibitory activity with the reference drug etoposide
(70.4%) at 100 μM concentrations but at 20 μM concentrations, the compound 48 exhibited
excellent topo IIα inhibitory activity (38.3%) than the reference drug etoposide (30.6 %) which is
1.25 times more strong potent topo IIα inhibition than the reference drug etoposide. Topo I and
IIα inhibition of 8 compounds indicated the fewer DNA breaks in gel (Fig 3 & 4) enlightening
their potential cytotoxicities in human cancer cell lines which were in agreement with the cell
viability and cytotoxic data (Table 1, Fig S1 & S2). Fig 3 & 4 and Table 2 summarized the topo I
and IIα inhibitory activities of 28, 30, 34, 35, 46, 48, 52, and 53 compounds; above all the
compound 48 which contain tolyl group on C ring with no substituents on E ring exhibited
excellent dual topo I and topo IIα inhibitory activities at 100 and 20 μM concentrations than the
19

positive controls camptothecin and etoposide respectively. The compound 52 which contain no
substituent on C ring and –F substituent on E ring exhibited strong top I inhibitory than the
positive control camptothecin at 100 μM concentration. The obtained results indicated that the
compounds 48 and 52 were also found to have broad-spectrum cytotoxic activities against tested
six human cancer cell lines (Table 1 and Fig. 2, S1 & S2). Moreover the substituents on C and E
rings of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chloride moieties have a key role
in exhibiting dual topo I / IIα inhibitory activities. However, there was no direct correlation
between the cytotoxic activities of tested compounds and their top I and IIα inhibitory activities.
2.2.3. Molecular docking study
The molecular docking simulations were performed to better understand the potential
interaction modes of the compounds 48 and 52 with topo I and IIα. From the above studies,
compound 48 inhibited both topo I and topo IIα. Fig. 5a shows the binding site of compound 48
with topo I where it intercalates into the single-strand cleavage site. There is no hydrogen
bonding interactions between the compound 48 and topo I. Compound 48 have van der Waals
interactions with residues Arg364, Pro431, and Asn722 and π- π stacking interactions with the
stacked DNA bases. Fig. 6 depicts the binding site of compound 48 with topo IIα where it bound
to the topo IIα DNA cleavage site where etoposide was situated. The benzo[f]chromeno[4,3-
b][1,7]naphthyridine ring was stacked between the DNA bases of the cleavage site with π- π
stacking interactions. Similar to the binding of topo I, there were no hydrogen bond interactions
with topo IIα.
20

Fig 5. (a) Binding mode of the compound 48 with topo I. (b) binding mode of the compound 52
with topo I: Compounds 48, 52 and the interacting residues of the receptor are depicted in
capped sticks colored by atom types and taupe and magenta for carbon atoms of the receptor and
compounds respectively. The DNA phosphate backbone and bases are displayed in cartoons in
cyan and green.
21

Fig 6. Binding mode of compound 48 with topo IIα: Compound 48 and the interacting residues
of the receptor are shown in capped sticks colored by atom types and magenta for carbon atoms
of the compound. The DNA phosphate backbone and bases are shown in cartoons in cyan and
green.
Hence, it was obvious that the compound 48 acted as dual topo I/ IIα inhibitor. From the
biological part, compound 52 inhibited topo I with excellent activity to the reference drug
camptothecin. Fig. 5b reveals the binding site of the compound 52 with topo I where it was
intercalated into the single-strand DNA cleavage site. There was no hydrogen bonding
interaction between the compound 52 and topo I observed. The compound 52 have van der
22

Waals interactions with residues Arg364, Thr718, and Asn722. Hence, the compound 52 acted as
topo I inhibitor.
3. Conclusion
Operationally simple, one-pot, regioselective and atom-economic synthetic pathway was
successfully pioneered to the pentacyclic 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines (21-
39) and 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides (40-53). We have
identified certain 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides as a new type
of dual topo I and topo IIα inhibitors with potential cytotoxic activities. In this study, we have
screened novel 35 compounds against six different human cancer cell lines including lung cancer
cell line (NCIH23), colon cancer cell line (HCT15), gastric cancer cell line (NUCG-3), renal
cancer cell line (ACHN), prostate cancer cell line (PC-3), and breast cancer cell line (MDA-MB-
231) with reference drug doxorubicin (ADR). Among all tested compounds, 8 compounds 28, 30,
34, 35, 46, 48, 52, and 53 shown potential cytotoxicities against all tested human cancer cell
lines. We have also evaluated the lead 8 cytotoxic agents against catalytic inhibitory action
toward human topo I and topo IIα. As anticipated the compound 48 displayed dual topo I and
topo IIα inhibition and compound 52 acts as potent topo I inhibitor. In particular at 20 μM
concentrations, the compound 48 exhibited 1.25 times more potent topo IIα inhibitory activity
than the positive control etoposide drug. The compound 52 exhibited 1.32 times more potent
topo I inhibition than the reference drug camptothecin at 100 μM concentrations. The potency of
these compounds dictates the importance of substituents on C and E rings of core motif of 13H-
benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chlorides albeit the combination of tolyl group
23

at C ring with no substituents at E ring and no substituent on C ring and –F substituent on E ring
being the most favorable. We have also carried out molecular docking studies with Sybyl X-2.1
using the default parameters to investigate the binding site of compounds 48 and 52 with human
topo I and IIα respectively. The docking studies of the compounds 48 and 52 with topo I
revealed they intercalated into the single-strand cleavage site where the compound 48 have van
der Waals interactions with residues Arg364, Pro431, and Asn722 whereas the compound 52
with residues Arg364, Thr718, and Asn722. Both the compounds 48 and 52 have π- π stacking
interactions with the stacked DNA bases. The docking studies of the compound 48 with topo IIα
explored that the compound 48 bound to the topo IIα DNA cleavage site where etoposide was
situated. The benzo[f]chromeno[4,3-b][1,7]naphthyridine ring of the compound 48 was stacked
between the DNA bases of the cleavage site with π- π stacking interactions and there were no
hydrogen bond interactions with topo IIα. Conclusively, the results of the present study provide
noticeable rationale to design and synthesis of angularly fused molecules targeting dual topo I
and topo IIα inhibition with potential wide spectrum of cytotoxic activities.
4. Experimental Section
All the reactions were carried out in oven dried glassware with freshly distilled dry
solvents under anhydrous conditions unless otherwise indicated and all commercial chemicals
were used as obtained. Evaporation of organic solutions was achieved by rotary evaporation with
o
a water bath temperature below 40 C. All the products obtained were purified by column
chromatography using activated aluminum oxide basic (Brockmann I, 199443) and silica gel
(100-200 mesh). Thin layer chromatography was performed on E Merck silica gel GF-254 pre-
coated plates and aluminum oxide TLC plates; identification was performed under UV
24

1
13
illumination. Hexane was used as a co-eluent. H and C NMR were recorded in JEOL 400 and
101 MHz spectrometer respectively. The chemical shifts are reported in ppm downfield to TMS
1
13
(δ = 0) for H NMR and relative to the central CDCl3 resonance (δ = 77.0) for C NMR. Data
are reported as follows: chemical shift in ppm (d), multiplicity (s = singlet, d = doublet, t = triplet,
m = multiplet), coupling constant (Hz), and integration. High resolution mass spectra (HRMS,
QTOF) recorded on LCQ Fleet-Thermo Scientifics.
4.1. General Procedure for the synthesis of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines
(21-39)
In an ace pressure tube or a round bottom flask equipped with a magnetic stirring bar,
1.0 equiv of 3-aminoquinoline, 0.5 equiv. of O-propargylated salicylaldehydes, 20 mol % of
Yb(OTf)3, 20 mol % of CuI, 0.5 equiv. of 4Ao molecular sieves and 5-10 mL of acetonitrile was
added. Reaction mixture was stirred at reflux temperature. After completion of the reaction, as
indicated by the Al2O3 TLC, dichloromethane (DCM, 10 mL) was added to the crude reaction
mass and then filtered through Celite® pad and concentrated under the reduced pressure. Product
was purified by column chromatography on activated aluminum oxide basic (Brockmann I,
199443) (eluent: hexanes/ethyl acetate) afforded the corresponding products (21-39).
4.1.1. 9-Bromo-14-methyl-13H-benzo[f]chromeno[4,3-b][1,7]naphthyridine (25)
1
Off-white solid (40.9%); Rf: 0.70 (eluent 2:3 Ethyl acetate: Hexanes); H NMR (400 MHz,
CDCl3) δ 9.49 (s, 1H), 8.64 (dd, J = 13.6, 5.5 Hz, 2H), 8.27 (dd, J = 8.1, 1.4 Hz, 1H), 7.79 (ddd,
J = 8.2, 7.1, 1.3 Hz, 1H), 7.70 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.46 (dd, J = 8.7, 2.4 Hz, 1H), 6.91
13
(d, J = 8.6 Hz, 1H), 5.57 (s, 2H), 2.96 (s, 3H). C NMR (101 MHz, CDCl3) δ 164.31, 155.74,
150.18, 139.67, 134.80, 130.98, 128.77, 128.35, 128.14, 127.05, 126.82, 118.95, 115.34, 114.82,
25

113.54, 66.56, 19.42. HRMS m/z [M+H]+ calculated for C20H14N2OBr: 377.0289; Found:
377.0270.
4.1.2 9,11-Dichloro-14-methyl-13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines (26)
1
Pale orange color solid; 57.2 % Yield; Rf: 0.50 (eluent 2:3 Ethyl acetate: Hexanes); H NMR
(500 MHz, CDCl3) δ 9.50 (s, 1H), 8.69 (d, J = 7.8 Hz, 1H), 8.44 (d, J = 2.5 Hz, 1H), 8.30 (dd, J
= 8.1, 1.4 Hz, 1H), 7.85 – 7.80 (m, 1H), 7.76 – 7.70 (m, 1H), 7.46 (d, J = 2.5 Hz, 1H), 5.70 (s,
2H), 2.99 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 155.59, 150.92, 146.41, 145.56, 142.37,
139.92, 131.76, 130.98, 128.93, 128.14, 127.72, 127.59, 127.11, 126.80, 124.67, 123.99, 122.87,
67.05, 19.40. HRMS m/z [M+H]+calculated for C20H13N2OCl2: 367.0404; Found: 367.0399.
4.1.3. 8-Bromo-11-methoxy-14-methyl-13H-benzo[f]chromeno[4,3-b][1,7]naphthyridine (27)
1
Off-white solid (41.5%); Rf: 0.45 (eluent 2:3 Ethyl acetate: Hexanes); H NMR (400 MHz,
CDCl3) δ 9.56 (d, J = 22.9 Hz, 1H), 8.67 (d, J = 8.5 Hz, 1H), 8.27 (dt, J = 8.1, 1.6 Hz, 1H), 7.78
(ddd, J = 9.9, 5.6, 2.0 Hz, 1H), 7.69 (ddd, J = 8.5, 7.1, 1.5 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H), 5.50
(d, J = 3.3 Hz, 2H), 3.92 (d, J = 1.7 Hz, 3H), 3.00 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 155.81,
155.50, 149.47, 148.43, 147.75, 145.70, 141.84, 139.12, 136.14, 130.94, 29.67, 128.92, 128.79,
127.00, 122.97, 114.78, 114.04, 110.78, 66.96, 56.41, 19.57. HRMS m/z [M+H]+ calculated for
C21H16BrN2O2: 407.0395; Found: 407.0385.
4.1.4. 14-(p-Tolyl)-13H-benzo[f]chromeno[4,3-b][1,7]naphthyridine (30)
1
Off-white solid (53.3%); Rf: 0.60 (eluent 2:3 Ethyl acetate: Hexanes); H NMR (400 MHz,
CDCl3) δ 9.58 (s, 1H), 8.54 (dd, J = 1.6Hz, 1H), 8.17 (dd, J = 1.2, 1.3 Hz, 1H), 7.62-7.598 (m,
1H), 7.45 (d, J = 7.7 Hz, 2H), 7.41-7.33 (m, 2H), 7.22-7.18 (m, 4H), 6.99 (dd, J = 0.8 Hz, 1H)
26

13
5.08 (s, 2H); C NMR (101 MHz, CDCl3): δ 156.9, 155.5, 149.0, 145.3, 143.7, 142.5, 139.0,
134.5, 132.3, 130.8, 130.5, 128.3, 127.7, 126.6, 126.4, 125.8, 125.6, 123.9, 122.6, 117.0, 66.5,
21.5. HRMS m/z [M+H]+calculated for C26H19N2O: 375.1497; Found: 375.1490.
4.1.5. 9-Chloro-14-methyl-13H-benzo[f]chromeno[4,3-b][1,7]naphthyridine (31)
White solid (47.2%); Rf: 0.70 (eluent 1:4 Ethyl acetate: Hexanes); 1H NMR (400 MHz, CDCl3) δ
9.56 (s, 1H), 8.53 – 8.44 (m, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.60 (dd, J = 8.0, 7.1 Hz, 1H), 7.45 (d,
J = 7.6 Hz, 2H), 7.37 (d, J = 8.6 Hz, 1H), 7.33 – 7.29 (m, 1H), 7.19 (t, J = 6.2 Hz, 3H), 6.95 –
6.87 (m, 1H), 5.06 (d, J = 0.8 Hz, 2H), 2.56 (s, 3H). 13C NMR (101 MHz, CDCl3) 155.46, 155.40,
147.86, 145.53, 144.02, 142.57, 139.20, 134.41, 132.05, 130.96, 130.66, 128.66, 128.05, 127.97,
127.76, 126.75, 126.51, 126.17, 125.34, 123.91, 123.84, 118.64, 66.71, 21.59. HRMS m/z
[M+H]+calculated for C26H18ClN2O: 409.1107; Found: 409.1099 (M+H+).
4.2. General Procedure for the synthesis of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-
ium chlorides (40-56)
20 mg of 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridines dissolved in 10 ml of MeOH at 0°C,
to this reaction mixture added 1 ml of 37% HCl and stirred for 1 hour at 0°C. Then the reaction
mixture continued to stir at room temperature for overnight. The reaction progress was
monitored by TLC, and the hydrochloride salts of 13H-benzo[f]chromeno[4,3-
b][1,7]naphthyridines 40-56 were obtained in almost quantitative yields by concentrating the
reaction mixtures under reduced pressure.
4.2.1. 14-(p-tolyl)-13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chloride (48)
27

1
Yellow color solid (Quantitative Yield); H NMR (400 MHz, CD3OD) δ 10.00 (s, 1H), 8.50 (dd,
J = 7.9, 1.6 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.94 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.66 – 7.43 (m,
5H), 7.36 – 7.29 (m, 2H), 7.26 – 7.16 (m, 1H), 7.00 (dd, J = 8.2, 0.7 Hz, 1H), 5.19 (s, 2H), 2.56
(s, 3H). 13C NMR (101 MHz, CD3OD) δ 157.68, 152.04, 151.41, 146.11, 144.39, 141.93, 140.93,
140.36, 138.94, 134.27, 133.74, 132.84, 131.24, 131.03, 129.58, 127.65, 127.18, 125.72, 125.21,
122.64, 121.99, 121.12, 117.63, 117.17, 66.33, 20.18.
4.2.2. 9-chloro-14-(p-tolyl)-13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chloride (49)
1
Pale yellow color solid (Quantitative Yield); H NMR (400 MHz, CD3OD) δ 9.91 (s, 1H), 8.43
(d, J = 2.6 Hz, 1H), 8.21 (dd, J = 8.3, 1.0 Hz, 1H), 7.88 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.60 –
7.52 (m, 3H), 7.51 – 7.45 (m, 1H), 7.41 (dd, J = 8.7, 2.6 Hz, 1H), 7.32 – 7.27 (m, 2H), 6.98 (d, J
= 8.7 Hz, 1H), 5.18 (s, 2H), 2.54 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 156.10, 151.88,
141.07, 140.98, 140.36, 133.25, 133.11, 132.85, 131.09, 131.02, 129.30, 127.76, 127.60, 127.54,
127.02, 125.21, 124.96, 123.03, 122.95, 122.53, 119.08, 118.98, 118.07, 117.88, 66.51, 20.18.
4.2.3. 9-fluoro-13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chloride (52)
Beige color Solid (Quantitative Yield); 1H NMR (400 MHz, CD3OD) δ 9.99 (s, 1H), 9.28 (s, 1H),
9.08 – 8.98 (m, 1H), 8.38 – 8.29 (m, 1H), 8.22 – 8.09 (m, 3H), 7.27 (td, J = 8.4, 3.1 Hz, 1H),
7.12 (dd, J = 9.0, 4.5 Hz, 1H), 5.65 (d, J = 0.9 Hz, 2H). 13C NMR (101 MHz, CD3OD) δ 159.64,
157.27, 150.87, 148.68, 145.11, 143.92, 139.94, 135.32, 133.66, 132.34, 130.91, 127.67, 127.10,
124.13, 121.78, 120.16, 119.23, 110.74, 68.05.
4.2.4. 9-chloro-13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chloride (53)
28

1
Acid green color Solid (Quantitative Yield); H NMR (400 MHz, CD3OD) δ 9.98 (s, 1H), 9.29
(s, 1H), 9.04 (dd, J = 7.7, 1.9 Hz, 1H), 8.48 (t, J = 2.9 Hz, 1H), 8.36 (dd, J = 7.5, 2.1 Hz, 1H),
8.18 – 8.14 (m, 1H), 7.50 (ddd, J = 15.2, 8.8, 2.5 Hz, 1H), 7.36 – 7.31 (m, 1H), 7.16 – 7.12 (m,
1H), 5.71 (d, J = 1.1 Hz, 2H). 13C NMR (101 MHz, CD3OD) δ 156.66, 151.19, 148.62, 145.59,
144.59, 133.19, 132.10, 130.66, 128.93, 127.82, 127.20, 126.92, 124.77, 124.08, 123.99, 122.49,
119.31, 114.17, 68.04.
4.2.5. 13H-benzo[f]chromeno[4,3-b][1,7]naphthyridin-5-ium chloride (55)
1H NMR (400 MHz, CD3OD) δ 9.98 (s, 1H), 9.27 (s, 1H), 9.14 – 8.95 (m, 1H), 8.49 (dd, J = 7.8,
1.6 Hz, 1H), 8.38 – 8.32 (m, 1H), 8.19 – 8.13 (m, 2H), 7.52 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.26
– 7.16 (m, 1H), 7.10 (d, J = 8.1 Hz, 1H), 5.66 (d, J = 1.2 Hz, 2H). 13C NMR (101 MHz, CD3OD )
δ 158.14, 152.77, 150.77, 135.36, 133.72, 132.63, 132.04, 130.71, 129.05, 128.40, 126.85,
125.48, 125.09, 124.05, 122.65, 122.06, 121.49, 117.46, 67.92, 48.29, 48.08, 47.87, 47.65, 47.44,
47.23, 47.01.
5. Materials and methods
5.1. Pharmacology
5.1.1. In vitro cytotoxicity assay
In vitro cytotoxicity assay was performed using the number of cells measured indirectly
by the sulforhodamine B (SRB) method according to the NCI (USA) protocol. Cells were plated
into a 96 well plate at a density of 2 103 cells per well. On the next day (day 0), the compounds
of interest dissolved in DMSO/media were added in quadruplicate. The final concentration of
29