Design, synthesis and biological evaluation of 1,3-diphenylbenzo[f][1,7]naphthyrdines

Article abstract of DOI:10.1016/j.bmc.2017.08.030

A novel series of twenty 1,3-diphenylbenzo[f][1,7]benzonaphthyrdine derivatives were designed and synthesized through intermolecular imino Diels-Alder reaction. Their in vitro cytotoxic activities were evaluated against six human cancer cell lines (NCIH23

Full text of DOI:10.1016/j.bmc.2017.08.030

Accepted Manuscript
Design, Synthesis and Biological Evaluation of 1,3-diphenylbenzo[f][1,7]naph-
thyrdines
Sateesh Kumar Arepalli, Byeongwoo Park, Kiho Lee, Hyunji Jo, Kyu-Yeon Jun,
Youngjoo Kwon, Jong-Soon Kang, Jae-Kyung Jung, Heesoon Lee
PII:
S0968-0896(17)31321-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.030
BMC 13933
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 June 2017
16 August 2017
17 August 2017
Please cite this article as: Arepalli, S.K., Park, B., Lee, K., Jo, H., Jun, K-Y., Kwon, Y., Kang, J-S., Jung, J-K., Lee,
H., Design, Synthesis and Biological Evaluation of 1,3-diphenylbenzo[f][1,7]naphthyrdines, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.08.030
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Design, Synthesis and Biological Evaluation of
1,3-diphenylbenzo[f][1,7]naphthyrdines
Sateesh Kumar Arepallia, Byeongwoo Parka, 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)
Abstract
A novel series of twenty 1,3-diphenylbenzo[f][1,7]benzonaphthyrdine derivatives were
designed and synthesized through intermolecular imino Diels-Alder reaction. Their in vitro
cytotoxic activities were evaluated against six human cancer cell lines (NCIH23, HCT15,
NUGC-3, ACHN, PC-3, and MDA-MB-231). Majority of synthesized compounds exhibited
significant cytotoxic activities against all tested human cancer cell lines. Among them 4l, 4m,
and 4o derivatives exhibited most promising cytotoxic activities. Furthermore these compounds
were evaluated against human Topoisomerase IIα inhibition. Interestingly, the compound 4l
exhibited 1.3 and 1.2 times more potent human Topoisomerase IIα inhibition than the reference
drug etoposide in both 100 µM and 20 µM concentrations respectively. Molecular docking
studies for the compound 4l have also been executed by Sybyl X-2.1 in which it reveals the
1

binding site of the compound 4l with topo IIα DNA cleavage site where etoposide was situated.
The benzo[f][1,7]naphthyridine ring was stacked between the DNA bases of the cleavage site.
Key words
1,3-diphenylbenzo[f][1,7]benzonaphthyrdines; human Topoisomerase IIα inhibitors; Cytotoxic
agents; molecular docking studies; imino Diels-Alder reaction
______________________________________________________________________________
1. Introduction
In DNA topology, topoisomerases I and II are most important enzymes to maintain and
play vital role in DNA replication, transcription, recombination, mitosis and chromatin
remodeling processes.1-5 Topotecan, irinotecan and belotecan (Figure 1) were only camptothecin
derivatives approved as Topo I inhibitors for clinical use.6-10 However, low water solubility of
camptothecin, hydrolytically unstable lactone ring, and reversibility of camptothecin-DNA-Topo
I cleavage complex were the basis for its disapproval for clinical usage.11-13 Further fused
heterocyclic systems such as indenoquinolines, indenoisoquinolines, and benzonaphthyrdines
have been developed (Figure 1)14-18 as alternatives for camptothecin. Remarkably, some of the
indenoisoquinolines also exhibited potent Topo II inhibition.19-21
Human DNA topoisomerase IIα is a privileged target presently for many drugs engaged
in cancer chemotherapy.22-23 In the literature, a distinctive range of molecules have been reported
which interfering with eukaryotic topoisomerase II activity and also displayed potent anticancer
activities.22, 24-26 Typically the agents which either impede with binding of the enzyme to DNA,
2

inhibit ATP binding or stabilize non-covalent complexes are categorized as Topoisomerase II
catalytic inhibitors.27 Examples of Topo II catalytic inhibitors are suramin,28 aclarubicin,29
merbarone (Figure 2),30 bisdioxopiperazines31, 32and novobiocin.33 Topo II poisons are agents
which can proceed by trapping the enzyme in the divisible complex which results the
accumulation of condensed DNAs in the cell, thus transforming the enzyme into a cellular
poison.23,34 FDA approved Topo II poisons (Figure 2) are doxorubin, daunorubicin, idarubicin;35-
40 acridines,41, 42 ellipticines,43 and epipodophyllotoxins.44
Figure 1. The representative research objective
On account of interest in the design and development of novel heterocyclic motifs with potential
biological applications,9, 45-50 in this report we have designed and synthesized novel 1,3-
3

diphenylbenzo[f][1,7]benzonaphthyrdine derivatives as potent cytotoxic agents and Topo IIα
inhibitors. The 1,3-diphenylbenzo[f][1,7]benzonaphthyrdines were synthesized via one-pot
intermolecular imino Diels-Alder reaction. As we know that hetero Diels-Alder reaction is one of
the most important and powerful method for the synthesis of poly fused heterocyclic systems
with enantio-, regio- and diastereo- selectivity. 51
Figure 2. Marked Topo II catalytic inhibitors and poisons
4

Scheme 1. Synthesis of 1,3-diphenylbenzo[f][1,7]benzonaphthyrdines (4a-s) by Intermolecular
imino Diels-Alder reaction and synthesis of 4aʹ, 4jʹ. Reaction conditions: 1.0 equiv of 1, 0.4
equiv. of substituted aromatic or heteroaromatic aldehydes (2a-k), 0.8 equiv. of 3a/3b, 20 mol %
of Yb(OTf)3, 20 mol % of CuI, and 0.5 equiv. of 4Ao molecular sieves in 5-10 mL of acetonitrile;
(2) 30mg of 4a or 4j in 10 ml of MeOH, to this added 1 ml Conc. HCl at 0oC and then stir at RT
for 2h.
2. Results and Discussion
2.1 Chemistry
Our studies were commenced with the design of novel scaffolds for synthesis and
evaluate their targeted biological applications. As shown in figure 1, indenoquinolines,
indenoisoquinolines, and benzonaphthyrdines were developed as alternatives for camptothecin
5

and its analogues. At this juncture, we also developed a series of novel 1, 8 diaza heterocyclic
systems as potential cytotoxic and Topo inhibitors.45 Michel, S. et al52 have also synthesized and
evaluated a series of benzo[b]chromeno[6,5-g][1,8]naphthyridinones as antitumor agents. After
these reports, we were attracted towards the naphthyridine pharmacophore since it’s also
exhibited broad spectrum of biological applications.53-54 But the construction of this core unit
requires multistep synthesis. As a result in this report, we have described one-pot, regio-selective,
atom economy, multi-component reaction strategy for the synthesis of an array of 1,3-
diphenylbenzo[f][1,7]benzonaphthyrdines. After screening different reaction conditions, we
optimized the reaction condition55 which involve 1.0 equiv of 3-aminoquinoline, 0.4 equiv. of
substituted aromatic or heteroaromatic aldehyde, 0.8 equiv. of 1-ethynyl-4-methoxybenzene, 20
mol % of Yb(OTf)3, 20 mol % of CuI, and 0.5 equiv. of 4Ao molecular sieves in 5-10 mL of
acetonitrile afforded the novel series of 1,3-diphenylbenzo[f][1,7]benzonaphthyrdine (4a-s) as
shown in Scheme 1 & Table 1. All the synthesized compounds were confirmed by 1H, 13C NMR
and mass spectra. Furthermore, we also synthesized 4aʹ, 4jʹ compounds quantitatively by
dissolving the compounds 4a & 4j in 10 ml of MeOH solvent separately and then added 1 ml of
Conc. HCl in each case and stir them at 0oC for 2h. After the reactions progressed, the reaction
mixtures were concentrated under reduced pressure to obtain 4aʹ and 4jʹ compounds as shown
Scheme 1. The two compounds 4aʹ and 4jʹ were also confirmed by 1H and 13C NMR spectra. In
1H NMR spectra of 4aʹ, 4jʹ compounds showed their characteristic downfield singlets at δ 10.05;
8.33 and δ 9.55; 8.39 ppm respectively.
2.2 Biological activity
2.2.1. Cytotoxic activity
6

With the chemical library of twenty one novel series of 1,3-
diphenylbenzo[f][1,7]benzonaphthyrdines and its derivatives in hand, we started cytotoxic
evaluation against six human cancer cell lines including lung (NCIH23), colon (HCT15), gastric
(NUCG-3), renal (ACHN), prostate (PC-3), and breast (MDA-MB-231) cell lines with reference
doxorubicin (ADR) as shown in Figure 3 and Table 1. Among the set, almost half of the tested
compounds (eleven compounds) such as 4a, 4f, 4g, 4i, 4l, 4m, 4n, 4o, 4r, 4a`, and 4j` displayed
significant cytotoxic activities with GI50 values less than 10µM range in all tested human cancer
cell lines (Table 1). Among them three compounds 4l, 4m, and 4o compounds exhibited
maximum growth inhibition against all tested six human cancer cell lines. From the analysis of
cell viabilities of 1,3-diphenylbenzo[f][1,7]naphthyrdines (Figure 3), it was clearly evident that
the compounds 4a, 4f, 4g, 4i, 4l, 4m, 4n, 4o, 4r, 4a`, and 4j`have displayed significant cytotoxic
activities against all tested human cancer cell lines at 10 µM range concentrations.
100
90
80
70
NCIH23
60
HCT15
50
NUGC-3
ACHN
PC-3
MDA-MB-231
40
30
20
10
0
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r 4s 4a` 4j`
1,3-diphenylbenzo[f][1,7]naphthyridines
Figure 3. The cell viabilities of benzo[f][1,7]naphthyrdine analogues (4a-s; 4a`& 4j`) at 10 µM
concentration on all tested human cancer cell lines.
7

The cell viabilities were very useful to comparison and they were found to be most consistent
with GI50 values in cases where they were estimated. To explain the SAR studies of
diphenylbenzo[f][1,7]naphthyrdines, primarily we fixed 4-methoxyphenyl group at 1st position of
benzo[f][1,7]naphthyrdine core motif and tried different substitution pattern at 2nd & 3rd
positions as shown in Table 1. We started with 1-naphthyl ring at 3rd position as 4a; interestingly
it was exhibited cytotoxic activities against all tested human cancer cell lines in the range of GI50
(µM):1.654 – 3.382 as shown in Table 1. As anticipated by this result, we tried 2-naphthyl ring
at 3rd position of benzo[f][1,7] naphthyrdine core motif as 4b; surprisingly it was not exhibited
any significant inhibition in the cancer cell lines. Yet we tried electron donating group (–OMe)
on naphthyl ring and phenyl ring at 3rd position of benzo[f][1,7] naphthyrdine core as 4c & 4d
respectively, they were also not exhibited any significant effect of the growth inhibition of the
tested cancer cell lines. To examine the effect of benzo[f][1,7] naphthyrdine core motif further,
we substituted different heterocyclic units at 3rd position of core as 4f, 4g & 4h albeit 4f and 4g
only exhibited moderate cytotoxic activities against all six human cancer cell lines in the range
of GI50 (µM): 2.001 – 7.694 as shown in Table 1.
Table 1.
Isolated yields of products and in vitro cytotoxic activities of 1,3-
diphenylbenzo[f][1,7]benzonaphthyrdines on different human cancer cell lines
Entry
Products
Human Cancer cell lines GI50 (µM)b
(Yield %)a
MDA-MB-
231
NCIH23 HCT15 NUGC-3 ACHN
PC-3
(Lung)
(Colon) (Gastric) (Renal) (Prostate)
(Breast)
8

1
1.654
2.724
2.156
5.361
3.382
2.809
4a (13%)
4b (33%)
2
3
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
4c (26%)
4d (17%)
4
5
4e (20%)
4f (27%)
6
7
3.296
7.061
2.001
5.652
6.876
7.694
2.068
4.881
3.033
5.613
6.151
3.035
4g (15%)
4h (29%)
8
9
> 10
> 10
> 10
> 10
> 10
> 10
4.016
4.384
5.828
2.133
3.067
7.283
4i (55%)
10
> 10
> 10
> 10
> 10
> 10
> 10
9

4j (31%)
4k (26%)
11
12
> 10
> 10
> 10
> 10
> 10
> 10
1.720
1.698
1.620
1.862
4.817
1.974
1.951
2.170
1.849
1.981
1.688
4.947
2.984
1.531
6.752
2.302
2.935
2.585
4l (15%)
4m (30%)
4n (22%)
13
14
15
1.888
1.786
2.028
3.859
3.147
1.785
4o (14%)
4p (23%)
4q (26%)
16
17
18
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
2.554
2.146
3.001
3.266
4.457
3.319
4r (42%)
4s (40%)
19
> 10
> 10
> 10
> 10
> 10
> 10
10

20
21
22
1.812
1.977
1.680
2.249
1.567
1.506
4a` (quantitative)
2.425
4.369
2.87
3.917
3.586
3.493
4j` (quantitative)
0.090
0.077
0.086
0.080
0.076
0.068
ADR (Reference)
aIsolated Yields; bGI50 values are taken as a mean from three experiments and correspond to the agent’s
concentration causing a 50% decrease in net cell growth.
After examined the biphenyls and heterocyclic systems, we screened phenyl ring at 3rd position
of core motif, interestingly compound 4i retain the growth inhibition activities against all human
cancer cell lines as shown in Table 1. We initiated screening with different groups on phenyl ring
as R1, R2, R3, R4, R5 positions. Among them, electron releasing groups at R3 position exhibited
most significant cytotoxic activities than electron withdrawing groups. For instance the
compounds 4l (–NMe2), 4m (–Cl), 4o (–Br) exhibited most significant cytotoxic activities than
4j (–NO2), 4p (–CF3), and 4q (–CF3). In order to explore the potential cytotoxic activities of
benzo[f][1,7]naphthyrdines alone against tested cell lines (Table 1), for lung cancer cell lines; 4n
(GI50: 1.620 µM) exhibited better cytotoxic activity than remaining derivatives such as 4a (GI50:
1.654 µM), 4m (GI50: 1.698 µM), 4l (GI50: 1.720 µM), 4j` (GI50: 1.812 µM), and 4o (GI50: 1.888
µM). Albeit these derivatives exhibited improved growth inhibition activities with GI50 values
less than 2µM. For Colon (HCT15) cell lines; 4o (GI50: 1.786 µM) exhibited better cytotoxic
activity than 4l (GI50: 1.862 µM), 4n (GI50: 1.974 µM), 4j` (GI50: 1.977 µM), 4r (GI50: 2.146 µM)
and 4a (GI50: 2.724 µM). For gastric (NUGC-3) cell lines; 4j` (GI50: 1.680 µM), 4n (GI50: 1.849
µM), and 4l (GI50: 1.951 µM) exhibited better cytotoxic activities (GI50: <2 µM) than remaining.
11

For renal (ACHN) cell lines; 4m (GI50: 1.688 µM) and 4l (GI50: 1.981 µM) exhibited best
cytotoxic activities (GI50: <2 µM) than other analogues. For prostate (PC-3) cancer cell lines; 4m
(GI50: 1.531 µM) and 4j` (GI50: 1.567 µM) also exhibited better cytotoxic activities (GI50: <2 µM)
than remaining. For breast (MDA-MB-231) cancer cell lines; 4j` (GI50: 1.506 µM) and 4o (GI50:
1.785 µM) candidates exhibited better cytotoxic activities (GI50: <2 µM) than remaining. To sum
up the cytotoxic activities, the compounds 4l, 4m, and 4o exhibited overall strong inhibition
against all tested human cancer cell lines than the remaining tested compounds.
2.2.2. Topoisomerase IIα inhibition:
After evaluation of cytotoxic activities of benzo[f][1,7]naphthyrdines, we only considered lead
compounds 4l, 4m, and 4o for the evaluation of the conversion of super coiled plasmid DNA to
relaxed DNA by topo IIα as shown in Figure4 and Table 2. In this study, etoposide was used as
reference with our compounds which is lead drug in this study.56 From the results of Figure 4a
and Table 2, it is clear that the compound 4l showing excellent catalytic inhibitory action (91.3%)
towards human topoisomerase IIα than the reference drug candidate etoposide (70.7%) at 100µM
concentration. But the compounds 4m, and 4o exhibited no catalytic inhibitory action towards
topoisomerase IIα. However, the compound 4l also exhibited strong potential inhibition (42.4%)
of topoisomerase IIα than the reference compound etoposide (34.7%) at even 20µM
concentration (Figure 4 and Table 2). The compounds 4m and 4o topoisomerase IIα inhibition
indicates the fewer DNA breaks in gel, revealing their potential cytotoxicities in human cancer
12

cell lines which were in agreement with the cell viability and cytotoxicity data (Table 1 and
Figure 3).
Figure 4. (a) Human Topo IIα inhibition by the 1,3-diphenylbenzo[f][1,7]benzonaphthyrdines.
Effects
of
decreasing
concentrations
at
100µM
and
20
µM
of
1,3-
diphenylbenzo[f][1,7]benzonaphthyrdines; Lane D: pBR322 only; Lane T: pBR322 + Topo II;
Lane E: pBR322 + Topo II + Etoposide; (At 100 µM): Lane 4l: pBR322 + Topo II + compound
4l; Lane 4m: pBR322 + Topo II + compound 4m; Lane 4o: pBR322 + Topo II + compound 4o;
(At 20 µM): Lane 4l: pBR322 + Topo II + compound 4l. (b) Graphical representation of % topo
IIα inhibition of compounds with reference drug etoposide at 100µM and 20 µM concentrations
respectively.
13

Table 2. Human Topo IIα inhibitory activities of lead benzo[f][1,7]naphthyridines
Compounds
Human Topo IIα (%inhibition)
100 µM
20 µM
Etoposide (ref)
70.7
34.7
91.3
42.4
3.0
0.2
--
--
14

2.2.3. Molecular docking study
The docking studies were performed to evaluate the binding modes and nature of
interactions for the lead compound 4l with active site residues of human DNA topoisomerase IIα.
The structure used as template for the docking study was prepared by using the X-ray structures
3QX357 for human topo IIα. Currently, the structure of topo IIβ complexed with etoposide and
DNA is the only structure available with drug bound to topo II. Therefore, homology model of
topo IIα was generated by Modeller (ver. 9.13)58 using 3QX3 as the template. The receptor
protein structure was prepared by removing all the water molecules, ligands and adding
hydrogen atoms. The coordinate files for the structure of compound 4l was constructed using
Sybyl X-2.1 and energetically minimized using Tripos force field with Gasteiger-Huckel charges.
15

Figure 5. Binding mode of the compound 4l with topo IIα: 4l and the interacting residues of the
receptor are shown in sticks colored by atom types and orange for carbon atoms of the compound.
The DNA phosphate backbone and bases are shown in cartoons in cyan.
59, 60
The receptor and ligand file were prepared according to the original publication protocols.
Docking were carried out with Sybyl X-2.1 using the default parameters. From figure 5, it is
revealed that the binding site of the compound 4l with topo IIα DNA cleavage site where
16

etoposide was situated. The benzo[f][1,7]naphthyridine ring was stacked between the DNA bases
of the cleavage site and there was no hydrogen bond interactions with topo IIα.
3. Conclusion
In conclusion, a series of novel 1,3-diphenylbenzo[f][1,7]benzonaphthyrdines have been
designed and synthesized through one-pot intermolecular imino Diels-Alder reaction. This
synthetic method has several advantages such as economical, atom-economy, multi-component
reaction, regio-selective, and broad substrate scope. The synthesized twenty one compounds
were evaluated their cytotoxicities against six human cancer cell lines including lung (NCIH23),
colon (HCT15), gastric (NUCG-3), renal (ACHN), prostate (PC-3), and breast (MDA-MB-231)
cell lines. Remarkably, half of the synthesized compounds exhibited promising positive cell
viabilities and cytotoxic activities albeit among them 4l, 4m, and 4o exhibited most potent
cytotoxic activities. We also evaluated the lead compounds’ 4l, 4m, and 4o catalytic inhibitory
action towards topoisomerase IIα. As anticipated the compound 4l displayed excellent catalytic
inhibition (91.3%, 42.4%) than the reference drug candidate etoposide (70.7%, 34.7%) at 100
and 20 µM respectively. For an explanation of the topoisomerase IIα activity of the compound 4l,
molecular docking studies were performed with Sybyl X-2.1 using the default parameters to
provide the reasonable binding site of the compound 4l with topo IIα DNA cleavage site where
etoposide was situated. The core of benzo[f][1,7]naphthyridine ring was mounded between the
DNA bases of the cleavage site and there was no hydrogen bond interactions with topo IIα.
Overall, these novel results provide an insight for future directions of drug design and
development of new topoisomerase IIα inhibitors.
17

4. Experimental Section
All the reactions were carried out in a flame or 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
o
evaporation with a water bath temperature below 40 C. All the products obtained were purified
by column chromatography using silica gel (100-200 mesh). Thin layer chromatography was
performed on E Merck silica gel GF-254 pre-coated plates; identification was performed under
UV illumination. Flash column chromatography was performed on E Merck silica gel (230-400
1
13
mesh). Hexane was used as a co-eluent. H and C NMR were recorded in Bruker DPX 400,
Bruker Avance 500 and 101, 126 MHz, JEOL 400 and 101 MHz spectrometer respectively. The
1
chemical shifts are reported in ppm downfield to TMS (δ = 0) for H NMR and relative to the
central CDCl3 resonance (δ = 77.0) for 13C NMR. Data are reported as follows: chemical shift in
ppm (δ), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant (Hz),
and integration. High resolution mass spectra (HRMS) recorded on LCQ Fleet-Thermo
Scientifics.
4.1. General Procedure for the synthesis of 1,3-diphenylbenzo[f][1,7]naphthyridines (4a-s)
In an ace pressure tube equipped with a magnetic stirring bar, 1.0 equiv of 3-
aminoquinoline, 0.4 equiv. of substituted aromatic or heteroaromatic aldehyde, 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 imine
formation as indicated by the TLC, to the above reaction mixture 0.8 equiv. of 1-ethynyl-4-
methoxybenzene (3a) or 1-methoxy-4-(prop-1-yn-1-yl)benzene (3b) was added and stirred at
18

reflux temperature. After completion of the reaction as indicated by TLC, dichloromethane
(DCM, 10 mL) was added to the crude reaction mass and then filtered through Celite® pad and
the combined organic layer was washed with brine (10 mL) and extracted with DCM (2x10 mL)
dried over MgSO4. After removing of solvent under vacuum, the crude product was purified by
column chromatography eluting with ethyl acetate/hexane afforded the corresponding products
(4a-s).
4.1.1. 1-(4-methoxyphenyl)-3-(naphthalen-2-yl)benzo[f][1,7]naphthyridine (4a)
1
Yield: 13%; Brown solid; Rf : 0.5 (eluent 1:2=Ethylacetate:Hexanes); H NMR (400MHz,
CDCl3) δ 9.69 (s, 1H), 8.71 (d, 1H, J = 1.4 Hz), 8.45 (dd, 1H, J = 8.6, 1.8 Hz), 8.22 (dd, 1H, J =
8.2, 1.3 Hz), 8.15 (s, 1H), 8.06-7.98 (m, 2H), 7.94-7.89 (m, 1H), 7.74 (dd, 1H, J=8.5, 1.1 Hz),
7.66 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.58-7.52 (m, 2H), 7.48-7.43 (m, 2H), 7.29 (ddd, 1H, J = 8.4,
13
7.0, 1.4 Hz), 7.17-7.10 (m, 2H), 3.97 (s, 3H). C NMR (101 MHz, CDCl3) δ 160.13, 156.78,
155.78, 148.77, 145.35, 143.54, 12975, 128.88, 128.64, 127.87, 126.95, 126.63, 114.91, 55.57.
HRMS m/z [M+H]+ calculated for C29H21N2O : 413.1654 ; Found : 413.1652.
4.1.2. 1-(4-methoxyphenyl)-3-(naphthalen-1-yl)benzo[f][1,7]naphthyridine (4b)
1
Brown solid; Yield: 33%; Rf : 0.5 (eluent 1:2=Ethylacetate:Hexanes); H NMR (400MHz,
CDCl3) δ 9.68 (s, 1H), 8.26 (dd, 2H, J = 11.6, 8.8 Hz), 7.97 (dd, 2H, J = 12.8, 7.4 Hz), 7.88 (s,
1H), 7.81 (d, 2H, J = 6.9 Hz), 7.72-7.67 (m, 1H), 7.65-7.60 (m, 1H), 7.57-7.52 (m, 2H), 7.45 (d,
13
2H, J = 8.5 Hz), 7.34-7.29 (m, 1H), 7.09 (d, 2H, J = 8.5 Hz), 3.94 (s, 3H). C NMR (101MHz,
CDCl3) δ 160.16, 159.25, 155.64, 148.41, 145.52, 143.30, 130.49, 129.79, 129.77, 129.76,
128.26, 127.05, 127.03, 126.47, 126.25, 125.47, 125.45, 114.89, 55.53. HRMS m/z [M+H]+
calculated for C29H21N2O : 413.1654 ; Found : 413.1652.
19

4.1.3. 3-(4-methoxynaphthalen-1-yl)-1-(4-methoxyphenyl)benzo[f][1,7]naphthyridine (4c)
1
Yield: 26%; Brown solid; Rf : 0.4 (eluent 1:2=Ethylacetate:Hexanes); H NMR (400MHz,
CDCl3) δ 9.67 (s,1H), 8.36 (dddd, 2H, J = 7.8, 4.5, 1.9, 0.6 Hz), 8.23 (dd, 1H, J = 8.2, 1.3 Hz),
7.85 (s, 1H), 7.81-7.74 (m, 2H), 7.67 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.59-7.51 (m, 2H), 7.47-
7.41 (m, 2H), 7.30 (ddd, 1H, J = 8.5, 7.0, 1.5 Hz), 7.12-7.07 (m, 2H), 6.97(d, 1H, J = 8.1 Hz),
4.09 (s, 3H), 3.94 (s, 3H). 13C NMR (101MHz, CDCl3) δ 156.64, 155.67, 148.21, 143.34, 130.45,
129.85, 129.78, 128.95, 125.61, 127.52, 126.99, 126.40, 126.05, 125.62, 125.16, 122.54, 114.86,
103.57, 55.80, 55.54. HRMS m/z [M+H]+ calculated for C30H23N2O2 : 443.1760 ; Found :
443.1759.
4.1.4. 1,3-bis(4-methoxyphenyl)benzo[f][1,7]naphthyridine (4d)
1
Yield: 17%; Brown solid; Rf : 0.4666 (eluent 1:2=Ethylacetate:Hexanes); H NMR (400MHz,
CDCl3) δ 9.60 (s, 1H), 8.26-8.16 (m, 3H), 7.92 (s, 1H), 7.72-7.66 (m, 1H), 7.62 (ddd, 1H, J = 8.3,
7.1, 1.3 Hz), 7.42-7.36 (m, 2H), 7.26(ddd, 2H, J = 8.5, 7.0, 1.4 Hz), 7.13-7.02 (m, 5H), 3.95 (s,
3H), 3.89 (s, 3H). 13C NMR (101MHz, CDCl3) δ 161.30, 160.45, 156.61, 155.75, 148.55, 145.24,
143.47, 133.69, 130.92, 130.40, 129.70, 128.89, 128.37, 126.82, 126.35, 125.04, 124.88, 123.74,
114.83, 114.46, 55.53. HRMS m/z [M+H]+ calculated for C26H21N2O2 : 393.1603; Found :
393.1605.
4.1.5. 3-(2-bromophenyl)-1-(4-methoxyphenyl)-2-methylbenzo[f][1,7]naphthyridine (4e)
1
Yield: 20%; Brown solid; Rf : 0.4333 (eluent 1:2=Ethylacetate:Hexanes); H NMR (400MHz,
CDCl3) δ 9.56 (s,1H), 8.18 (dd, 1H, J = 8.2,1.3 Hz), 8.09-7.94 (m, 1H), 7.71 (dd, 1H, J = 7.8,0.6
Hz), 7.62 (ddd, 1H, J = 8.2, 7.0, 1.3 Hz), 7.54-7.47 (m, 2H), 7.40 (dt, 1H, J = 8.6, 1.9 Hz), 7.33
(ddt, 2H, J = 8.4, 6.2 ,3.7 Hz), 7.23 (ddd, 1H, J = 8.5, 6.3, 1.5 Hz), 7.20-7.01 (m, 4H), 3.95 (s,
20

3H), 2.06 (s, 3H). 13C NMR (101MHz, CDCl3) δ 160.18, 159.78, 155.47, 147.15, 145.81, 141.64,
140.83, 134.52, 132.92, 132.08, 130.54, 130.35, 130.08, 129.75, 129.38, 128.58, 127.98, 126.93,
126.58, 122.63, 115.53, 55.51, 18.11. HRMS m/z [M+H]+ calculated for C26H19BrN2O :
455.0759; Found : 455.0824.
4.1.6. 1-(4-Methoxyphenyl)-3-(thiophen-2-yl)benzo[f][1,7]naphthyridine (4f)
1
Yield: 27%; Orange solid; Rf: 0.4833 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (500MHz,
CDCl3) δ 9.58 (S, 1H), 8.19 (dd, 1H, J = 8.1, 1.2 Hz), 8.14 (dd, 1H, J = 2.9, 1.2Hz), 7.91 (dd, 1H,
J = 5.1, 1.2Hz), 7.86 (S, 1H), 7.69 (dd, 1H, J = 8.5, 1.0 Hz), 7.64 (ddd, 1H, J = 8.2, 7.1, 1.3 Hz),
13
7.48 (dd, 1H, J = 5.0, 3.0Hz), 7.42-7.37 (m, 2H), 7.27 (dd, 3H, 15.5, 1.4 Hz), 3.96 (S, 3H). C
NMR (101MHz, CDCl3) δ 160.03, 155.56, 153.01, 148.58, 145.24, 143.38, 141.45, 133.41,
130.37, 129.58, 128.42, 126.79, 126.74, 126.57, 126.34, 125.38, 125.24, 123.61, 114.80, 55.47.
HRMS m/z [M+H]+ calculated for C23H17N2OS: 369.1061; Found : 369.1056.
4.1.7. 3-(1-Ethyl-1H-indol-3-yl)-1-(4-methoxyphenyl)benzo[f][1,7]naphthyridine (4g)
1
Yield: 15%; Brown solid; Rf: 0.3167 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (400MHz,
CDCl3) δ 9.63 (S, 1H), 8.74 (dd, 1H, J = 6.5, 2.6 Hz), 8.19 (d, 1H, J = 8.0 Hz), 7.94 (s, 1H), 7.90
(s, 1H), 7.66 (d, 1H, J = 8.4 Hz), 7.61 (t, 1H, J = 7.6 Hz), 7.46-7.40 (m, 3H), 7.37-7.33 (m, 2H),
7.24 (d, 1H, J =7.2 Hz), 7.11 (d, 2H, J = 8.6 Hz), 4.29 (q, 2H, J = 7.3 Hz), 3.96 (s, 3H), 1.57 (t,
3H, J = 7.3 Hz). 13C NMR (101MHz, CDCl3) δ 159.94, 157.08, 155.65, 155.24, 147.68, 143.74,
137.13, 133.94, 130.35, 129.75, 128.61, 127.86, 126.61, 126.51, 126.18, 125.43, 124.05, 122.70,
122.32, 121.35, 115.23, 114.78, 109.83, 55.45, 41.56, 15.42. HRMS m/z [M+H]+calculated for
C29H24N3O: 430.1919; Found: 430.1914.
4.1.8. 3-(1-Hexyl-1H-indol-3-yl)-1-(4-methoxyphenyl)benzo[f][1,7]naphthyridine (4h)
21

1
Yield: 29%; Brown solid; Rf: 0.60 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (400MHz,
CDCl3) δ 9.61 (s, 1H), 8.82-8.64 (m, 1H), 8.17 (dd, , 1H , J = 8.2, 1.3 Hz), 7.88 (d, 2H, J = 1.9
Hz), 7.68 – 7.63 (m, 1H), 7.59 (ddd, 1H, J = 8.2, 7.1, 1.3 Hz), 7.45 – 7.39 (m, 3H), 7.34 – 7.31
(m, 2H), 7.24-7.21 (m, 1H), 7.13-7.07 (m, 2H), 2.00-1.81 (m, 2H), 1.44-1.19 (m, 8H), 0.86 (t, 4H,
J = 7.1 Hz). 13C NMR (101 MHz, CDCl3) data of δ 159.94, 155.65, 155.29, 147.62, 143.73,
137.40, 130.33, 129.75, 129.31, 127.84, 126.61, 126.17, 125.39, 122.68, 122.39, 121.30, 114.77,
109.96, 55.54, 46.99, 31.50, 30.19, 26.75, 22.59, 14.11. HRMS m/z [M+H]+calculated for
C33H32N3O: 486.2545; Found: 486.2540.
4.1.9. 1-(4-Methoxyphenyl)-3-phenylbenzo[f][1,7]naphthyridine (4i)
1
Yield: 55%; Yellow solid; Rf: 0.4167 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (400MHz,
CDCl3) δ 9.64 (S, 1H), 8.27-8.23 (m, 2H), 8.21 (dd, 1H, J = 8.1, 1.1 Hz), 8.00 (s, 1H), 7.72 (d,
1H, J = 8.5 Hz), 7.68-7.63 (m, 1H), 7.59-7.50 (m, 3H), 7.43-7.39 (m, 2H), 7.29 (dd, 1H, J = 8.5,
1.3 Hz), 7.13 – 7.08 (m, 2H), 3.96 (s, 3H). 13C NMR (101 MHz, CDCl3) data of δ 16.06, 156.91,
155.74, 148.67, 145.33, 143.41, 138.33, 133.52, 130.38, 129.86, 129.62, 129.00, 128.50, 127.43,
126.84, 126.32, 125.34, 114.82, 55.47. HRMS m/z [M+H]+calculated for C25H19N2O: 363.1497;
Found: 363.1492.
4.1.10. 1-(4-Methoxyphenyl)-3-(4-nitrophenyl)benzo[f][1,7]naphthyridine (4j)
1
Yield: 31%; Brown solid; Rf: 0.4667 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (500MHz,
CDCl3) δ 9.64 (S, 1H), 8.48-8.44 (m, 2H), 8.42-8.39 (m, 2H), 8.23 (dd, 1H, J = 8.1, 0.9 Hz), 8.05
(s, 1H), 7.75 (dd, 1H, J = 8.5, 0.9 Hz), 7.69 (ddd, 1H, J =8.2, 7.0, 1.2 Hz), 7.44-7.39 (m, 2H),
7.31 (ddd, 1H, J = 8.5, 7.0, 1.4 Hz), 7.14-7.10 (m, 2H), 3.96 (s, 3H). 13C NMR (101MHz,
CDCl3) δ 160.33, 155.58, 154.11, 149.46, 148.73, 145.62, 144.17, 143.49, 133.07, 130.58,
22

129.63, 129.20, 128.30, 127.07, 126.73, 126.26, 125.45, 124.26, 123.32, 115.04, 55.48. HRMS
m/z [M+H]+ calculated for C25H18N3O3: 408.1348; Found: 408.1343.
4.1.11. 1-(4-Methoxyphenyl)-3-(2-nitrophenyl)benzo[f][1,7]naphthyridine (4k)
1
Yield: 26%; Brown solid; Rf: 0.4167 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (500MHz,
CDCl3) δ 9.54 (S, 1H), 8.22 (dd, 1H, J = 8.2, 1.2 Hz), 8.04 (dd, 1H, J = 8.1, 1.1 Hz), 7.84(dd, 1H,
J = 7.4, 1.4 Hz), 7.79-7.75 (m, 2H), 7.70-7.76 (m, 3H), 7.63 (dd, 1H, J = 7.7, 1.2 Hz), 7.42-7.38
(m, 2H), 7.30 (ddd, 1H, J = 8.5, 7.1, 1.4 Hz), 7.11-7.06 (m,2H), 3.94(S, 3H). 13C NMR (101
MHz, CDCl3) δ 160.18, 155.29, 155.20, 148.79, 145.54, 143.06, 134.66, 132.90, 132.82, 131.73,
130.45, 129.89, 129.67, 128.98, 127.20, 127.01, 126.48, 124.78, 114.85, 55.47. HRMS m/z
[M+H]+ calculated for C25H18N3O3: 408.1348; Found: 408.1343.
4.1.12. 4-(1-(4-Methoxyphenyl)benzo[f][1,7]naphthyridin-3-yl)-N,N-dimethylaniline (4l)
1
Yield: 15%; Brown solid; Rf: 0.3548 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (500MHz,
CDCl3) δ 9.58 (S, 1H), 8.18 (dd, 3H, J = 7.9, 4.3 Hz), 7.91 (S, 1H), 7.66 (d, 1H, J = 8.6 Hz),
7.62-7.58 (m, 1H), 7.42-7.37 (m, 2H), 7.23(dd, 1H, J = 5.7, 4.3 Hz), 7.13-7.07 (m, 2H), 6.84 (d,
2H, J = 9.0Hz), 3.95 (s, 3H), 3.07(s, 6H). 13C NMR (101MHz, CDCl3) δ 159.90, 157.05, 155.76,
151.66, 148.01, 145.04, 143.53, 133.93, 130.28, 129.66, 128.39, 127.91, 126.62, 126.10, 125.82,
124.45, 124.34, 123.86, 114.70, 112.19, 55.45, 40.29. HRMS m/z [M+H]+ calculated for
C27H24N3O: 406.1919; Found: 406.1914.
4.1.13. 3-(4-Chlorophenyl)-1-(4-methoxyphenyl)benzo[f][1,7]naphthyridine (4m)
1
Yield: 30%; Brown solid; Rf: 0.5322 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (500MHz,
CDCl3) δ 9.61 (S, 1H), 8.23-8.17 (m, 3H), 7.94 (S, 1H), 7.71 (dd, 1H, J = 8.5, 0.9 Hz), 7.65 (ddd,
1H, J = 8.2, 7.0, 1.3 Hz), 7.56-7.48 (m, 2H), 7.43-7.36 (m, 2H), 7.31-7.24 (m, 2H), 7.14-7.06 (m,
23

2H), 3.95(s, 3H). 13C NMR (101MHz, CDCl3) δ 160.17, 155.63, 155.55, 148.94, 145.37, 136.72,
136.18, 133.38, 130.66, 130.44, 129.66, 129.24, 128.71, 126.92, 126.49, 125.57, 124.98, 114.91,
113.75, 55.54. HRMS m/z [M+H]+ calculated for C25H18ClN2O : 397.1107; Found : 397.1102.
4.1.14. 3-(4-Bromophenyl)-1-(4-methoxyphenyl)benzo[f][1,7]naphthyridine (4n)
1
Yield: 22%; Brown solid; Rf: 0.55 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (500MHz,
CDCl3) δ 9.60 (S, 1H), 8.19 (dd, 1H, J = 8.1, 1.0 Hz), 8.15-8.11 (m, 2H), 7.94 (s, 1H), 7.73-7.69
(m, 1H), 7.69-7.62 (m, 3H), 7.41-7.37 (m, 2H), 7.29-7.25 (m, 2H), 3.95 (s, 3H). 13C NMR
(400MHz, CDCl3) data of δ 160.17, 155.67, 148.99, 145.40, 143.42, 137.22, 133.39, 132.24,
130.48, 129.66, 129.00, 128.75, 126.93, 126.52, 125.65, 124.98, 124.61, 123.54, 114.92, 55.57.
HRMS m/z [M+H]+calculated for C25H18BrN2O: 441.0602; Found: 441.0597.
4.1.15. 3-(1-(4-Methoxyphenyl)benzo[f][1,7]naphthyridin-3-yl)benzonitrile (4o)
1
Yield: 14%; Orange solid; Rf: 0.3710 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (500MHz,
CDCl3) δ 9.63 (S, 1H), 8.58 (t, 1H, J = 1.4Hz), 8.54-8.48 (m, 1H), 8.22 (dd, 1H, J = 8.1, 1.1Hz),
7.97 (S, 1H), 7.81-7.72 (m, 2H), 7.71-7.64 (m, 2H), 7.44-7.38 (m, 2H), 7.30 (ddd, 1H, 8.4, 7.1,
1.4Hz), 7.12 (d, 2H, J =8.7Hz), 3.96 (S, 3H). 13C NMR (101MHz, CDCl3) data of δ 160.30,
155.53, 154.24, 149.46, 139.52, 133.12, 133.05, 131.56, 131.15, 130.55, 129.91, 129.63, 129.05,
127.00, 126.66, 124.93, 123.37, 118.67, 115.02, 113.41, 102.37, 55.58. HRMS m/z [M+H]+
calculated for C26H18N3O: 388.1449; Found: 388.1445.
4.1.16. 1-(4-Methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)benzo[f][1,7]naphthyridine (4p)
1
Yield: 23%; Yellow solid; Rf: 0.5322 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (500MHz,
CDCl3) δ 9.63 (S, 1H), 8.37 (d, 2H, J = 8.1 Hz), 8.21 (dd, 1H, J = 8.1, 1.0 Hz), 8.01 (S, 1H), 7.80
(d, 2H, J = 8.2 Hz), 7.73 (dd, 1H, J = 8.5, 0.8 Hz), 7.63 (ddd, 1H, J =8.2, 7.2, 1.2 Hz), 7.45-7.37
24

(m, 2H), 7.29 (ddd, 1H, J = 8.4, 7.1, 1.3), 7.15-7.08 (m, 2H), 3.96 (S, 3H). 13C NMR (101MHz,
CDCl3) data of δ 162.94, 160.24, 155.67, 155.24, 149.19, 145.51, 143.46.141.66, 133.27, 130.42,
129.66, 128.94, 127.79, 127.00, 126.59, 126.01, 125.97, 125.36, 123.45, 114.97, 55.57. HRMS
m/z [M+H]+calculated for C26H18F3N2O: 431.1371; Found : 431.1366.
4.1.17. 3-(2,4-Bis(trifluoromethyl)phenyl)-1-(4-methoxyphenyl)benzo[f][1,7]naphthyridine
(4q)
1
Yield: 26%; Yellow solid; Rf: 0.50 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (500MHz,
CDCl3) data of δ 9.59 (S, 1H), 8.23 (dd, 1H, J = 8.19, 1.22 Hz), 8.10 (S, 1H), 7.98 (d, 1H, J =
7.94 Hz), 7.91 (d, 1H, J = 8.09 Hz), 7.80 (dd, 1H, J = 8.53, 0.90 Hz), 7.73-7.67 (m, 2H), 7.42-
13
7.38 (m, 2H), 7.35-7.29 (m, 1H), 7.12-7.05 (m, 2H), 3.94 (S, 3H). C NMR (400MHz, CDCl3)
data of δ 160.32, 156.05, 155.28, 148.48, 145.66, 142.91, 142.66, 132.74,131.71, 131.37, 130.55,
129.82, 129.71, 129.51, 129.22, 128.81, 128.19, 127.15, 126.65, 124.74, 123.99, 123.26, 121.99,
55.53. HRMS m/z [M+H]+calculated for C27H17F6N2O : 499.1245; Found : 499.1240.
4.1.18. 3-(2-Chlorophenyl)-1-(4-methoxyphenyl)benzo[f][1,7]naphthyridine (4r)
1
Yield: 42%; Yellow solid; Rf: 0.4833 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (400MHz,
CDCl3) δ 9.63 (S, 1H), 8.22 (d, 1H, 8.2 Hz), 7.94 (S, 1H), 7.86 (dd, 1H, J = 7.4, 1.9 Hz), 7.79 (d,
1H J = 8.5 Hz), 7.68 (t, 1H, J = 7.6 Hz), 7.54 (dd, 1H, J = 7.7, 1.2 Hz), 7.47-7.40 (m, 4H), 7.31
(d, 1H, J = 8.3 Hz), 7.09 (d, 2H, J = 8.6 Hz), 3.94 (S, 3H). 13C NMR (101MHz, CDCl3) δ 160.06,
156.79, 155.40, 147.60, 145.46, 143.21, 138.29, 133.10, 131.80, 130.35, 130.30, 129.69, 129.45,
128.78, 127.37, 127.02, 126.35, 114.79, 55.44. HRMS m/z [M+H]+calculated for C25H18ClN2O:
397.1107; Found: 397.1102.
4.1.19. 3-(2-Bromophenyl)-1-(4-methoxyphenyl)benzo[f][1,7]naphthyridine (4s)
25

1
Yield: 40%; Yellow solid; Rf: 0.40 (eluent 1:2 Ethyl acetate: Hexanes); H NMR (400MHz,
CDCl3) data of δ 9.63 (S, 1H), 8.22 (d, 1H, J = 8.15 Hz), 7.91 (S, 1H), 7.85-7.77 (m, 2H), 7.73
(d, 1H, J = 8.0Hz,), 7.67 (t, 1H, J = 7.6 Hz), 7.50 (t, 1H, J = 7.6 Hz), 7.46-7.40 (m, 2H), 7.37-
7.27 (m, 2H), 7.09 (d, 2H, J = 8.5 Hz), 3.94 (S, 3H). 13C NMR (101MHz, CDCl3) δ 160.07,
158.12, 155.38, 147.52, 145.49, 143.12, 140.28, 133.50, 133.11, 131.72, 130.46, 130.39, 129.70,
129.46, 128.80, 127.91, 127.03, 126.36, 123.46, 121.94, 114.80, 55.45. HRMS m/z
[M+H]+calculated for C25H18BrN2O: 441.0602; Found : 441.0597.
4.2. General procedure for the synthesis of 4a` and 4j` compounds:
30 mg of 1-(4-methoxyphenyl)-3-(naphthalen-1-yl)benzo[f][1,7]naphthyridine (4a) or 1-(4-
Methoxyphenyl)-3-(4-nitrophenyl)benzo[f][1,7]naphthyridine (4j) was dissolved in 10 ml of
MeOH at 0°C, to this reaction mixture added 1 ml of 37% HCl and stirred for 2 hours. Then the
reaction mixture was concentrated under reduced pressure to obtain 4a` or 4j` in quantitative
yield.
4.2. 1. 1-(4-Methoxyphenyl)-3-(naphthalen-1-yl)benzo[f][1,7]naphthyridin-4-ium (4a`)
Yield: Quantitative; Orange yellow solid; 1H NMR (400 MHz, CD3OD) δ 10.05 (s, 1H), 8.42 –
8.33 (m, 3H), 8.16 (d, 1H, J = 8.2 Hz), 8.12 – 8.03 (m, 3H), 7.98 (td, 1H, J = 6.9, 1.8 Hz), 7.78 –
7.69 (m, 2H), 7.68 – 7.58 (m, 4H), 7.28 – 7.23 (m, 2H), 3.98 (d, 3H, J = 1.9 Hz); 13C NMR (101
MHz, CD3OD) δ 160.38, 156.24, 155.39, 154.89, 154.20, 151.78, 149.65, 149.51, 148.85,
147.09, 143.84, 143.66, 132.94, 132.12, 130.56, 130.49, 130.31, 130.12, 129.82, 129.70, 129.16,
128.37, 127.27, 127.02, 126.72, 126.38, 126.29, 126.07, 125.86, 124.61, 124.19, 123.36, 115.40,
55.93.
4.2. 2. 1-(4-methoxyphenyl)-3-(4-nitrophenyl)benzo[f][1,7]naphthyridin-4-ium (4j`)
26

Yield: Quantitative; Orange yellow solid; 1H NMR (400 MHz, (CD3)2SO) δ 9.55 (s, 1H), 8.71 –
8.60 (m, 2H), 8.38 (dd, J = 10.1, 3.1 Hz, 3H), 8.14 (d, J = 7.9 Hz, 1H), 7.77 – 7.62 (m, 2H), 7.49
13
(dd, J = 6.7, 4.8 Hz, 2H), 7.36 (t, J = 7.8 Hz, 1H), 7.23 – 7.08 (m, 2H), 3.87 (s, 3H). C NMR
(101 MHz, (CD3)2SO) δ 160.38, 156.24, 155.39, 154.89, 154.20, 151.78, 149.65, 149.51,
148.85, 147.09, 143.84, 143.66, 132.94, 132.12, 130.56, 130.49, 130.31, 130.12, 129.82, 129.70,
129.16, 128.37, 127.27, 127.02, 126.72, 126.38, 126.29, 126.07, 125.86, 124.61, 124.19, 123.36,
115.40, 55.93.
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
each compound ranged from 1 nM to 10 µM and the final concentration of DMSO was <0.1%.
Seventy-two hours later, the cells were fixed with 10% trichloroacetic acid (TCA) overnight at
4°C, washed extensively with distilled water and dried in air. SRB solution (0.4% in 1% acetic
acid) was then added to each well at room temperature for 1 h. The wells were washed with 1%
acetic acid and bound dye was dissolved in 10 mM Tris. The absorbance was measured at 690
nm using a micro plate reader. The absorbance of the day 0 sample was subtracted from the
absorbance of the day 3 sample.
27

5.1.2. In vitro DNA topoisomerase-IIα inhibition assay
The mixture of 200 ng of super coiled pBR322 plasmid DNA and 4 units of human DNA
topoisomerase-IIα (Sigma, USA) was incubated with the compounds [4l, 4m, 4o and etoposide
(Eto)] in the assay buffer (10 mM Tris–HCl (pH 7.9) containing 50 mM NaCl, 5 mM MgCl2, 1
mM EDTA, 1 mM ATP, and 15 µg/mL bovine serum albumin) for 30 min at 30 oC. The reaction
in a final volume of 20 µL was terminated by the addition of 3 µL of 7 mM EDTA. Reaction
products were analyzed on a 1% Agarose gel at 60 V for 1 h with a running buffer of TAE. Gels
were stained for 30 min in an aqueous solution of ethidium bromide (0.5 µg/mL). DNA bands
were visualized by transillumination with UV light.
Acknowledgments
This research was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-
2015R1D1A1A01058142), Korea Research Fellowship Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-
2016H1D3A1937140) and Medical Research Center Program (2017R1A5A2015541).
Supplementary data
1
13
For all synthesized molecules H NMR, C NMR and MS spectra are available free of
charge. Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.
28

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