Synthesis and biological evaluation of simplified analogs of lophocladine B as potential antitumor agents

Article abstract of DOI:10.1007/s00044-016-1717-5

In this study, two series of simplified isoquinolines deriving from lophocladine B were synthesized and evaluated for their antitumor activity. Suzuki-Miyaura and Sharpelss-Fokin reactions were employed to synthesize 26 compounds. Two compounds (25 and 27

Full text of DOI:10.1007/s00044-016-1717-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1717-5
ORIGINAL RESEARCH
Synthesis and biological evaluation of simplified analogs of lophocladine
B as potential antitumor agents
Sewan Theeramunkong1 Opa Vajragupta2 Chawannuch Mudjupa3
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Received: 22 April 2016 / Accepted: 17 August 2016
© Springer Science+Business Media New York
Abstract In this study, two series of simplified iso-
quinolines deriving from lophocladine B were synthesized
and evaluated for their antitumor activity. Suzuki-Miyaura
and Sharpelss-Fokin reactions were employed to synthesize
26 compounds. Two compounds (25 and 27) showed to be
cytotoxic with IC50 values of 10 µM on hepatic cancer cells
and 13 µM on cervical cancer cells, respectively. Further
studies on their mechanism of action showed that com-
pound 27 mediated cytotoxicity through apoptosis
induction.
apparatus is perturbed by chemicals, microtubules are
unable to form a fully operational mitotic spindle, and a
cascade of phosphorylation and caspase-dependent events
leading to apoptosis is triggered (Loo et al., 2002). The
main biological target for antimitotic agents is tubulin,
which, after interaction with these compounds, loses its
ability to polymerize or depolymerize. To date, three dis-
tinct binding sites on tubulin have been reported: the col-
chicine, taxol, and vinblastine-binding sites. Drugs acting
on the taxol and vinblastine-binding site are currently in use
in clinical oncology (Kuppens, 2006; Jordan and Wilson,
2004), while, at the moment, no drugs acting on the
colchicines-binding site has reached the market. Over the
last decade, marine natural products experienced a growing
popularity. Indeed, several bioactive molecules from algae,
sponges, shell-less mollusks and corals have displayed to
possess interesting and unique biological properties (Lei
and Zhou, 2002; Faulkner, 2002; Güven et al., 2010;
Schwartsmann et al., 2001; Boopathy and Kathiresan, 2010;
Gamal, 2010; Itharat et al., 2003; Arif et al., 2004). In
Fig. 1, we reported some compounds from marine natural
sources that possess antimitotic and antitubulin activity
(Pettit et al., 1976; Fuller et al., 1992; Luesch et al., 2001;
Gunasekera et al., 1990).
In this communication we focused on structural mod-
ifications of the bioactive marine alkaloid, lophocladine B
(Fig. 1). This alkaloid was isolated from marine red algae,
Lophocladia sp., a member of the Rhodomelaceae (Cer-
amiales, Rhodophyta), which grows in the tropics and
subtropics. Lophocladine B displayed cytotoxic effect
against human lung tumor NCI-H460 and MDA-MB-435
breast cancer cells. The underlying mechanism of cyto-
toxicity by immunofluorescence assay demonstrated that the
molecular mechanism involves the inhibition of micro-
tubules (Haefner, 2003).
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Keywords Isoquinoline 1,2,3-triazole Cytotoxicity
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Cancel cells Lophocladine B Antitumor compounds
Introduction
Among the various classes of antitumor compounds known
to date, the class of antimitotic agents is well recognized for
its potentiality in cancer therapy. Indeed, when the mitotic
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1717-5) contains supplementary material,
which is available to authorized users.
* Sewan Theeramunkong
sewan@tu.ac.th
1
Faculty of Pharmacy, Thammasat University, 99 Moo 18
Phahonyothin Road, Klongluang 12120 Pathumthani, Thailand
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Mahidol University, 447 Sri-Ayuthaya Road, Rajathevi 10400
Bangkok, Thailand
3
Faculty of Pharmacy, Mahasarakham University, Kantarawichai
District 44150 Mahasarakham, Thailand

Med Chem Res
Fig. 1 Some molecules with
antitubulin activity
The general synthesis of lophocladine B has been
described (Lotter et al., 2006; Disadee et al., 2011) not-
withstanding, it is still unclear which functional group of
lophocladine B is directly involved in the antitumor activity.
Hence, we focused our research on structural simplification
of the 2,7-naphthyridin-1-amine ring of lophocladine B
followed by the replacement of the benzene ring with a
1,2,3-triazole ring in order to explore a different chemical
space.
Results and discussion
Design and synthesis
Scheme 1 Molecular design and synthesis concept of novel iso-
quinoline derivatives
In this study, we simplified the 2,7-naphthyridin-1-amine
scaffold of lophocladine B using 4-phenylisoquinoline. The
Scheme 2 Synthesis of
isoquinoline-benzene analogs by
Suzuki-Miyaura reaction

Med Chem Res
Table 1 Percent yield of the isoquinoline-benzene analogs in series I
structure was expanded by the substitution (R) on the aro-
matic ring. In addition, the phenyl ring was replaced with a
1,2,3-triazole ring as the triazole ring is considered as a
privileged structural motif in medicinal chemistry, both in
term of synthesis (click chemistry) and in its binding mode.
The synthesis of the compounds was accomplished using
two strategies: a Suzuki-Miyaura coupling reaction for
isoquinoline-benzene derivatives (series I) and the Fokin-
Sharpless reaction for isoquinoline-triazole derivatives
(series II) as shown in Scheme 1.
Entry R1
Yield (%) Entry R1
Yield (%)
62.7
3
80.5
9
Starting from the commercial 4-bromoisoquinoline, the
Suzuki-Miyaura reaction was accomplished using 12 dif-
ferent boronic acids (Scheme 2). The reaction was per-
formed under the following conditions: isoquinoline:
boronic acid 1.2:1; aqueous sodium carbonate, dimethyl-
formamide, 80 °C.
This reaction enabled the formation of compounds 3–14
bearing various substituents (R1) in satisfying yield (approx.
70 %) (Table 1). All purified compounds were submitted to
nuclear magnetic resonance (NMR) analysis to verify the
presence of the desired products. Only the most active
compounds were further analyzed by mass spectrometry
(MS).
For series II, isoquinoline-triazole derivatives, the
replacement of benzene by triazole ring was conducted by
using click chemistry to generate a triazole ring as linker
between isoquinoline and the extended R2. The alkyne 15
(prepared according to a published procedure; Dutta et al.,
2016) was submitted to a copper (I) catalyzed [3 + 2]
cycloaddition with the corresponding azides to afford 12
triazole derivatives 16–27 (Scheme 3).
4
20.1
10
90.5
5
6
72.8
92.4
11
12
35.7
61.4
7
8
78.9
84.2
13
14
88.3
77.6
Scheme 3 Copper catalyzed
azide–alkyne cycloaddition

Med Chem Res
The azide compounds were prepared according to a
reported procedure (Utsintong et al., 2013). The yields of
these click reactions varied from 19 to 74 % (Table 2).
Then, all purified compounds were submitted to NMR
analysis to verify the presence of the desired triazoles. Only
the most active compounds were further analyzed by MS.
diphenyltetrazoliumbromide) assay) colorimetric method
using the following cancer cell lines: non-small lung cancer
(H460), hepatic cancer (HepG2), breast cancer (MCF-7),
and cervical cancer (HeLa) cell lines. The compounds were
initially screened at 100 μM, and only for those compounds
with an inhibition greater than 50 % IC50 was evaluated
(Table 3).
As shown in Table 3, the results of the present study
indicate that compounds 25 and 27 (Fig. 2) were the most
active, and a full concentration–response curves on HepG2
and HeLa cell line were determined (Fig. 3). Compound 25
displayed an apparent IC50 of 10.14 1.25 µM on HepG2
cell, while compound 27 displayed an apparent IC50 of
12.76 0.69 µM on HeLa cell. Both compounds sharing a
common feature that is a methylene between the triazole
and a para-substituted benzene.
Cytotoxicity testing
All synthesized compounds were then evaluated for cyto-
toxicity by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-
Table 2 Percent yield of the of isoquinoline-triazole analogs in series
II
The mechanism of the cytotoxic activity for compound
27 was then investigated by FACS analysis. The HeLa cells
were treated with compound 27 and the harvested cellular
Table 3 Cytotoxicity of the active compounds in series I and II
Entry R2
Yield (%) Entry R2
Yield (%)
36.3
Entry
IC50 (μM, mean SD)
H460 HepG2
MCF-7
HeLa
16
32.0
22
Series I—Isoquinoline-benzene derivatives
5
102.26 12.14
72.34 0.10
89.04 1.88
>100
>100
>100
57.74 2.94
62.38 6.92
88.02 2.35
>100
6
88.89 9.96
84.73 1.53
74.94 2.58
>100
84.52 2.03
61.82 7.18
>100
7
11
12
17
44.9
23
73.6
27.58 12.66
39.41 14.92
>100
Series II—Isoquinoline-triazole derivatives
19
>100
76.65 4.42
51.77 8.16
10.14 1.25
>100
>100
>100
23
>100
>100
36.49 7.52
>100
25
>100
>100
26
>100
>100
84.26 8.14
12.76 0.69
1.21 0.23
>100
18
19
73.2
25.0
24
25
31.5
23.6
27
>100
0.09 0.008a
23.13 8.95
0.31 0.01
89.52 10.55
>100
Doxorubicin
Lophocladine B
0.62 0.19
>100
>100
IC50 values represent the calculated IC50 values for those compounds
in which a full concentration–response curve was performed. Values
are mean SD of three determinations from three separate
experiments
a
Results were from Mohareb et al. (2014)
20
21
19.2
42.8
26
27
41.1
37.9
Fig. 2 The two most active compounds in isoquinoline-triazole series

Med Chem Res
DNA were stained with FITC-Annexin/PI. From FACS
analysis, the dual parametric dot plots of compound
27-treated cells displayed the late apoptotic cells in the
upper right quadrant (UR, Annexin V-positive/PI-positive)
and some population in the early apoptotic cells in the lower
right quadrant (LR, Annexin V-positive/PI-negative),
whereas the control showed the viable cell population in the
lower left quadrant (Annexin V-negative/PI-negative).
These data show that compound 27 induced apoptosis in
tumor cells (Fig. 4).
a)
HeLa cell line
100
90
80
70
60
50
40
30
20
10
0
Compound 27: (12.76 0.69 uM)
Doxorubicin: (1.21 0.23 uM)
-10
0.1
1
10
100
1000
concentration (uM)
HepG2 cell line
b)
110
100
90
80
70
60
50
40
30
20
10
0
Compound 27: (23.13 8.95 uM)
Compound 25: (10.14 1.25 uM)
doxorubicin: (0.31 0.01 uM)
Conclusions
In conclusion, in this study, we designed and synthesized
simplified analogs of the natural product lophocladine B.
The good cytotoxic activity of two compounds (25 and 27)
against cancer cells and the apoptosis-induction mechanism
of compound 27 confirms the validity of this molecular
scaffold as novel core structure for cancer drug development
alone or as part, for the design of colchicine-based hybrid
anticancer drugs (Punganuru et al., 2016; Wang et al., 2016).
0.0001
0.01
1
100
10000
concentration (uM)
Fig. 3 Concentration–response curve of the most potent on cancer cell
line a compound 27 in HeLa cell line, b compounds 25 and 27 in
HepG2 cell line. Values are mean SD of three determinations from
three separate experiments
N egative Control
b) Doxorubicin
c) C ompound 27
3.86
2.90
6.76
10.97
22.06
33.03
9.51
%LR
10.12
19.63
%UR
%LR+%UR
Fig. 4 Flow cytometric analysis (FACS) of the distribution of HeLa
cells treated with the a negative control, b doxorubicin, c compound
27, Annexin V-PI analysis in HeLa cells, following 16 h of compound
treatment by Annexin-V-FITC method at IC50 concentrations, which
are 1.21 and 12.76 µM, respectively. Results are representative of one
of three independent experiments

Med Chem Res
Acknowledgments This research was financially supported from the
Office of the High Education Commission and Mahidol University
under the National Research Universities and the Commission on
Higher Education (grant number CHE-RG-2551-53). We would like to
thank Dr. Supachoke Mangmool (Department of Pharmacology) and
Dr. Jiraphun Jittikoon (Department of Biochemistry) from Mahidol
University for their biological suggestion, facility support, and
assistance.
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