Analogues of the marine alkaloids oroidin, clathrodin, and hymenidin induce apoptosis in human HepG2 and THP-1 cancer cells

Article abstract of DOI:10.1039/c4md00286e

The marine alkaloids clathrodin, oroidin, and hymenidin, which were isolated from Agelas sponges, possess diverse biological activities. Herein, we describe the design of a library of their analogues and the evaluation of their apoptosis-inducing activities against the human hepatocellular carcinoma HepG2 and acute monocytic leukaemia THP-1 cell lines. The screening of the complete library of 96 compounds using the HepG2 cell line allowed us to determine key structural elements and physicochemical properties that are responsible for the apoptosis-inducing activity. The indole-based compounds 24c, 28c, 29c, and 34c were found to be the most potent inducers of apoptosis in HepG2 and THP-1 cell lines with EC₅₀ values in the low micromolar range. Cell cycle analysis assays confirmed that compounds 24c, 28c, 29c, and 34c induce the apoptosis of THP-1 cells at 25 μM, which highlights these oroidin analogues as interesting candidates for further evaluation of their anticancer activity. This journal is

Full text of DOI:10.1039/c4md00286e

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Analogues of the marine alkaloids oroidin,
clathrodin, and hymenidin induce apoptosis in
Cite this: DOI: 10.1039/c4md00286e
human HepG2 and THP-1 cancer cells†
Tihomir Tomasic,a Dominik Nabergoj,ab Sanja Vrbek,ab Nace Zidar,a Zˇiga Jakopin,a
ˇ ˇ
Ales Zˇula,a Zˇiga Hodnik,a Marko Jukic,a Marko Anderluh,a Janez Ilas,a
ˇ
ˇ
ˇ
Marija Sollner Dolenc,a Jean Peluso,bc Genevieve Ubeaud-Sequier,bc
`
a
´
bc
Lucija Peterlin Masic and Danijel Kikelja
ˇ ˇ
*
*
Christian D. Muller,
The marine alkaloids clathrodin, oroidin, and hymenidin, which were isolated from Agelas sponges, possess
diverse biological activities. Herein, we describe the design of a library of their analogues and the evaluation
of their apoptosis-inducing activities against the human hepatocellular carcinoma HepG2 and acute
monocytic leukaemia THP-1 cell lines. The screening of the complete library of 96 compounds using the
HepG2 cell line allowed us to determine key structural elements and physicochemical properties that are
responsible for the apoptosis-inducing activity. The indole-based compounds 24c, 28c, 29c, and 34c
were found to be the most potent inducers of apoptosis in HepG2 and THP-1 cell lines with EC50 values
in the low micromolar range. Cell cycle analysis assays confirmed that compounds 24c, 28c, 29c, and
34c induce the apoptosis of THP-1 cells at 25 mM, which highlights these oroidin analogues as
interesting candidates for further evaluation of their anticancer activity.
Received 1st July 2014
Accepted 25th August 2014
DOI: 10.1039/c4md00286e
www.rsc.org/medchemcomm
clathrodin, hymenidin, and oroidin, and their synthetic
analogues has not yet been evaluated.
Introduction
The oroidin class of alkaloids is structurally relatively simple
(Fig. 1) and possesses drug-like properties according to
Lipinski's rule of ve;9 thus, this class is well suited for use as a
starting point for the design of novel analogues and mimetics
that can be screened for their biological activities. Recently,
inspired by the reported ability of clathrodin to block human
voltage-gated sodium channels,6 we designed and synthesised a
library of clathrodin and oroidin analogues and evaluated some
Marine organisms, particularly sponges, have been recognised
as a rich source of natural products that possess anticancer
activity.1–3 Clathrodin, hymenidin, and oroidin (Fig. 1) are
pyrrole-2-aminoimidazole4 alkaloids that were initially isolated
from the sponges of the genus Agelas. Oroidin acts as a chemical
defence agent against the predatory reef sh and can be
considered a biogenetic precursor of a variety of secondary
metabolites that exhibit great structural complexity and diver-
sity and present a range of biological activities.3,5 The oroidin
class of marine alkaloids has been reported to display modu-
latory activities on voltage-gated sodium6 and calcium7 chan-
nels, and oroidin and its analogues have also been extensively
studied as inhibitors of bacterial biolm formation.8 However,
the apoptosis-inducing activity of the marine alkaloids
a
ˇ
ˇ
University of Ljubljana, Faculty of Pharmacy, Askerceva 7, 1000 Ljubljana, Slovenia.
E-mail: lucija.peterlin@ffa.uni-lj.si; Fax: +386-1-4258031; Tel: +386-1-4769635
bLaboratoire d'Innovation Therapeutique, UMR 7200, Faculte de Pharmacie,
´
´
´
Universite de Strasbourg, 67401 Illkirch, France. E-mail: cdmuller@unistra.fr; Fax:
+33-368854310; Tel: +33-688285839
cPlateforme eBioCyt, Faculte de Pharmacie
´
´
& Federation Translationnelle de
´
´
Medecine, Universite de Strasbourg, 67401 Illkirch, France
† Electronic supplementary information (ESI) available: Experimental procedures,
compound characterization data, 1H and 13C spectra of the most active
compounds, analysis of molecular descriptors, biological activity data, and
description of biological assays. See DOI: 10.1039/c4md00286e
Fig.
1 Structures of the pyrrole-2-aminoimidazole alkaloids
clathrodin, hymenidin, and oroidin, and their structural modifications
(type A–C analogues).
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of the resulting compounds to determine their modulatory an early indicator of apoptosis.17 Propidium iodide, which is
activity on human voltage-gated sodium channels10,11 and their widely used as a DNA intercalating dye for the evaluation of cell
antibacterial activity.12
viability or DNA content in cell cycle analyses, was used to
The clathrodin, hymenidin, and oroidin molecules possess a determine the membrane integrity.18 Using this double-staining
potentially unstable double bond in the linker between the apoptosis assay, we were able to distinguish between living cells
2-aminoimidazole and pyrrole moieties.13 Therefore, we (annexin V-negative, PI-negative), early apoptotic cells (annexin
designed and synthesised a library of their analogues by V-positive, PI-negative) and late apoptotic/secondary necrotic
modifying the central part of the molecule (Fig. 1, type A to C cells (annexin V-positive, PI-positive).19 All the compounds were
analogues) to obtain more stable and conformationally tested at 50 mM in four independent experiments (Tables S1–S4
restricted compounds as well as the western (Fig. 1, type A–C in the ESI†). The HPLC purity of the tested compounds was
analogues) and eastern parts (type A–C analogues) of the above 95% monitored at 254 nm. Syntheses of the majority of
molecule to enable structure–activity relationship studies. In compounds are reported elsewhere10–12,20,21 and their analytical
the present work, we studied the apoptosis-inducing activity of data (1H- and 13C-NMR, mass spectra) can be found in the ESI.†
clathrodin, oroidin, and hymenidin, and their type A–C Synthesis of the most active compounds 24c, 28c–30c and 34c is
analogues against HepG2 and THP-1 human cancer cell lines to summarised in Scheme 1.
evaluate their potential as anticancer agents against hepato-
cellular carcinoma and acute monocytic leukaemia.
The marine alkaloids clathrodin, oroidin and hymenidin,
which were synthesised in our laboratory,20 and their analogues
Hepatocellular carcinoma is the most common type of liver 1–4 were found to possess only weak apoptosis-inducing activity
cancer, and its high incidence has been attributed to persistent in the HepG2 cell line with 25–38% apoptotic cells at 50 mM
infection with hepatitis B or C virus, contact with hepatocarci- (Table S1†).
nogens (e.g., aatoxins), and cirrhosis. The development of drug
In the series of type A analogues (Fig. 1), conformational
resistance in hepatocellular carcinoma tumour cells aer drug restriction was achieved by replacing the (E)-5-(3-aminoprop-1-
therapy indicates the important need for the discovery of novel enyl)-1H-imidazol-2-amine moiety by the 4,5,6,7-tetrahy-
anticancer agents for the successful treatment of liver cancer.14 drobenzo[d]thiazol-2-amine core in which the imidazole was
Acute monocytic leukaemia,
a
type of acute myeloid isosterically replaced by the thiazole ring.10 However, type A
leukaemia, is a hematopoietic cancer characterised by a analogues 1a–14a containing also various modications in the
disorder of hematopoietic progenitor cells, which lose their central and eastern parts of the parent molecules (Table S2†)
ability for normal differentiation and response to normal did not display improved apoptosis-inducing activity compared
regulators of proliferation. Its incidence increases with age. with oroidin (20–38% apoptotic HepG2 cells at 50 mM).
Considering the aging population and the fact that acute
The type B analogues 1b–10b were obtained by modication
myeloid leukaemia has the lowest survival rate of all leukae- of the central part through replacement of the 3-aminoprop-1-
mias, new anticancer agents against acute myeloid leukaemia enyl linker between the 2-aminoimidazole and pyrrole moieties
are urgently needed to ght this type of cancer in the future.15 by the less exible N-methylenepiperazine group (Fig. 1).21
Modication of the central part together with variations in the
eastern and western parts of the parent molecules gave only
weakly active compounds 1b–10b (Table S3,† 20–34% apoptotic
Results and discussion
HepG2 cells at 50 mM). In the type C analogues, the 1,3- or 1,4-
To evaluate the potential of the marine alkaloids clathrodin,
disubstituted phenyl ring was incorporated in place of the prop-
oroidin, and hymenidin, and their type A–C analogues (a library
of 96 compounds) to induce apoptosis in an in vitro human liver
1-enyl linker to obtain a conformationally restricted central part
of the molecule without changing the length of the molecule
cancer model, we conducted a primary screening of all these
compounds on the human hepatocellular carcinoma cell line
HepG2 (ATCC® HB-8065™) using the annexin V/propidium
iodide (PI) apoptosis assay with microcapillary ow cytometry
(Guava EasyCyte™, Millipore/Merck, CA, USA) as the readout.16
Annexin V was used to assess the loss of membrane asymmetry,
which is characterised by externalisation of phosphatidylserine,
(Fig. 1).11 The analysis of the apoptosis-inducing activity of the
type C analogues revealed a similar trend to those observed with
the type A and B analogues. If the substituent in the eastern part
of the molecule was a ve-membered pyrrole (1c–3c, 14c, 19c,
20c, 31c, 37c, 38c, and 43c), pyrrolidine (4c, 21c, 40c, 41c, and
44c), furan (15c, 17c, and 32c), or imidazole (39c) ring, the
Scheme 1 Synthesis of the most active compounds 24c, 28c–30c and 34c. Reagents and conditions: (a) corresponding carboxylic acid, TBTU,
NMM, CH2Cl2, 35 ꢀC, 24 h; (b) HCl(g), THF–EtOH, rt, 5 h.
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compounds were only weakly active (Table S4, Fig. S1,† 13–44%
Of the 96 compounds screened, compounds 24c, 28c–30c,
apoptotic HepG2 cells at 50 mM). The only exceptions were the and 34c, which were among the most active type C oroidin
pyrrole-based compounds 35c and 36c, which contained a analogues, were selected for further characterisation. First, the
benzyl moiety on the imidazole ring nitrogen and induced EC50 values for their apoptosis-inducing activity against the
apoptosis in 54% and 86% of HepG2 cells at a concentration of HepG2 cell line were determined using the annexin V/PI
50 mM (Table S4†), respectively. In contrast, a signicant gain in apoptosis assay (Table 1). 6-Fluoroindole 28c was found to be
the apoptosis-inducing activity was observed in the compounds the most active compound with an EC50 value of 13 mM,
containing an indole or substituted indole moiety in place of followed by 6-chloroindole 29c (EC50 ¼ 16 mM), 6-methoxy-
the pyrrole ring of the parent marine alkaloids.
indole 24c (EC50 ¼ 18 mM), indole 34c (EC50 ¼ 20 mM), and
The indole-based compounds 5c–12c, 16c, 18c, and 42c and 4H-thieno[3,2-b]pyrrole 30c (EC50 ¼ 42 mM).
4H-thieno[3,2-b]pyrrole 13c containing a tert-butyloxycarbonyl
Considering the noteworthy HepG2 apoptosis-inducing
(Boc) group on the imidazole N1 showed improved activity with activity of compounds 24c, 28c–30c, and 34c, these were further
43–91% apoptotic HepG2 cells at 50 mM. A substitution at evaluated using the human monocytic leukaemia THP-1 cell
position 5 of the indole ring with a methoxy (7c), benzyloxy (8c), line (ATCC® TIB-202™) (Table 1). The screening of these
uoro (11c), or chloro (12c) group resulted in improved activity compounds at 50 mM against the THP-1 cell line showed that
compared to the non-substituted indoles 5c and 6c, whereas a compounds 24c, 28c, 29c, and 34c induce apoptosis (87–97%
hydroxy (9c) or triuoromethyloxy (10c) substitution decreased apoptotic THP-1 cells), whereas compound 30c was found to be
the potency. A methyl substituent at the imidazole 2-amino inactive (12% apoptotic THP-1 cells). The dose–response curves
group increased the percentage of apoptotic cells compared showed that compounds 24c, 28c, 29c, and 34c exhibited
with those found with the indole-based compounds (5c vs. 18c), similar activities against the THP-1 cell line with EC50 values
whereas a reduction of the imidazole to obtain a 2-amino- ranging from 20 mM to 24 mM (Table 1). Because activation of
imidazoline ring reduced the activity (18c vs. 16c). Compound the apoptotic pathways is a key mechanism through which
42c with a 1,4-disubstituted phenyl ring in the central part and anticancer drugs kill tumour cells,23 it was important to conrm
an indole moiety in the eastern part of the molecule was among that compounds 24c, 28c, 29c, and 34c de facto exert their
the most active apoptosis-inducing compounds and was more cytotoxic effect against the THP-1 cell line via the induction of
active than its 1,3-phenylene counterpart 5c.
apoptosis and not by necrosis. Hence, THP-1 cells were sub-
In general, the Boc-deprotected indole-based compounds jected to a cell cycle analysis aer exposure to 24c, 28c, 29c, and
22c–29c, 33c, and 34c and the 4H-thieno[3,2-b]pyrrole-based 34c to determine the incidence of fragmented DNA (sub-G1
compound 30c retained their apoptosis-inducing activity population) by PI staining of the nuclei.24 DMSO (0.25%), which
against the HepG2 cell line. Similar to their Boc-protected was used as a negative control, did not affect the cell cycle in
analogues, the compounds with the 6-uoro- (28c), 6-chlor- THP-1 cells (Fig. 2). The results of the cell cycle analysis aer the
oindole (29c), and 4H-thieno[3,2-b]pyrrole (30c) moieties were incubation of THP-1 cells with compounds 24c, 28c, 29c, or 34c
the most active with more than 90% apoptotic HepG2 cells at at 25 mM for 24 h and 48 h (Fig. 2 and 3 and Table S5†) show the
50 mM.
presence of a sub-G1 cell population, which conrmed the
We also evaluated the apoptosis-inducing activity of presence of programed cell death, i.e. apoptosis. Compounds
compounds 45c–65c (Table S4†), which were identied in the 3D 28c and 29c showed similar potency: 41% and 40% of the cells
similarity searching, based on the indole 22c, in the ZINC data- were found in the sub-G1 peak aer 48 h of treatment,
base of drug-like compounds.22 Interestingly, most of the indoles
(45c, 53c, 55c, 57c, 62c, and 63c but not 58c and 64c) induced
apoptosis in more than 50% of HepG2 cells at 50 mM, regardless
Table
1
Cytomic apoptosis-inducing activities of the type
C
of the ring type in the western part of the molecule, which
indicates that the indole moiety is a crucial feature for signicant
apoptosis-inducing activity in type C oroidin analogues. Among
compounds 45c–65c, only compound 52c showed improved
apoptosis-inducing activity (92% apoptotic HepG2 cells at 50 mM)
compared with the template compound 22c.
analogues 24c, 28c–30c, and 34c
The molecular descriptor analysis of our library of oroidin
analogues showed that most of the compounds possess drug-
like properties according to Lipinski's rule of ve9 (Fig. S2†). The
presented charts show that most of the active compounds
(>50% apoptotic HepG2 cells at 50 mM) are more lipophilic
(log D values between 3 and 5) and have higher molecular
weights (MW between 300 and 500) compared with their inac-
tive counterparts (log D values between ꢁ1 and 5, MW between
200 and 500). In contrast, the number of hydrogen bond donors
and acceptors is similarly distributed between the actives and
inactives.
Compound
R1
R2
HepG2a EC50
THP-1a EC50
24c
28c
29c
30c
34c
OCH3
F
Cl
—
H
H
H
H
—
18 ꢂ 1 mM
13 ꢂ 7 mM
16 ꢂ 6 mM
42 ꢂ 16 mM
20 ꢂ 5 mM
20 ꢂ 2 mM
23 ꢂ 4 mM
24 ꢂ 5 mM
>50 mM
CH3
24 ꢂ 1 mM
a
The values are the mean ꢂ SD of three independent experiments
performed in triplicate.
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Conclusions
In conclusion, the marine alkaloids clathrodin, oroidin, and
hymenidin and a library of their synthetic analogues were tested
to determine their apoptosis-inducing activities against the
human hepatocellular carcinoma HepG2 cell line. Although the
three tested marine alkaloids were found to be only weakly
active, the library compounds, particularly their indole-based
type C analogues, displayed promising activities. Compounds
24c, 28c–30c, and 34c, which were the most active compounds
in the library, exhibited EC50 values between 13 mM and 42 mM
against the HepG2 cell line and between 20 mM and 24 mM
against the acute monocytic leukaemia THP-1 cell line. Through
cell cycle analysis, it was conrmed that compounds 24c, 28c,
29c, and 34c induce apoptosis at 25 mM in THP-1 cells. These
results render compounds 24c, 28c, 29c, and 34c interesting
hits for further optimisation towards more potent oroidin,
hymenidin and clathrodin analogues that induce apoptosis of
human hepatic and blood cancer cells. Additional investigation
of their mechanism of induction of apoptosis at the molecular
level will be necessary to assess their potential for development
towards new anticancer agents.
Experimental section
Chemistry
General procedure A
Synthesis of compounds 24c, 28c–30c and 34c. A solution of
Boc-protected starting compound (1 mmol) in a mixture of THF
and EtOH ¼ 1 : 2 (5 mL) was saturated with gaseous HCl and
stirred at room temperature for 5 h. The solvent was removed
under reduced pressure, and the solid was ltered off and
washed with diethyl ether and dichloromethane, to afford the
title compound.
Fig.
2 Cell cycle analysis of THP-1 cells after incubation with
compounds 24c, 28c, 29c, and 34c at 25 mM for 24 h and 48 h. DMSO
(0.25%) in culture medium was used as a negative control. Repre-
sentative histograms of three independent experiments are displayed.
2-Amino-4-(3-(5-methoxy-1H-indole-2-carboxamido)phenyl)-1H-
imidazol-3-ium chloride (24c). Prepared from 7c according to
General procedure A. Yield: 96%; off-white solid; mp 237–241 ꢀC;
IR (KBr) n ¼ 3301 (N–H), 3138 (C–H), 2955 (C–H), 2761 (C–H), 1673
(C]O), 1653, 1625, 1585, 1541, 1452, 1418, 1336, 1281, 1238, 1208,
1177, 1153, 1132, 1116, 1022, 883, 839, 788, 755 cmꢁ1. 1H NMR
3
(DMSO-d6) d 3.79 (s, 3H, OCH3), 6.89 (dd, 1H, J ¼ 9.2 Hz, 4J ¼
2.4 Hz, Ar-H), 7.15 (d, 1H, 4J ¼ 2.4 Hz, Ar-H), 7.33 (s, 1H, Ar-H),
7.37–7.49 (m, 6H, 4 ꢃ Ar-H, NH2), 7.69–7.72 (m, 1H, Ar-H), 8.08
(s, 1H, Ar-H), 10.42 (s, 1H, NH), 11.70 (s, 1H, NH), 12.16 (s, 1H,
NH), 12.85 (s, 1H, NH); 13C NMR (DMSO-d6) d 55.25 (OCH3),
102.02, 104.14, 109.43, 113.23, 115.19, 116.46, 119.74, 120.33,
126.39, 127.26, 128.12, 129.30, 131.54, 132.16, 139.45, 147.82,
153.84, 159.72; MS (ESI) m/z (%) ¼ 348.2 ([M ꢁ Cl]+, 100). HRMS
for C19H18N5O2: calculated, 348.1461; found, 348.1459. HPLC:
Phenomenex Luna 5 mm C18 column (4.6 mm ꢃ 150 mm);
mobile phase: 60–90% of MeOH in TFA (0.1%) in 20 min; ow
rate: 1.0 mL minꢁ1; injection volume: 10 mL; retention time:
3.029 min (98.2% at 254 nm, 98.7% at 280 nm).
Fig. 3 Comparison of the sub-G1 cell cycle population after the
treatment of THP-1 cells with compounds 24c, 28c, 29c, and 34c at
25 mM for 24 h and 48 h (n ¼ 3 independent experiments).
respectively. Compounds 34c and 24c displayed more potent
2-Amino-4-(3-(5-uoro-1H-indole-2-carboxamido)phenyl)-1H-
apoptosis-inducing activity with 49% and 60% of the cells in the imidazol-3-ium chloride (28c). Prepared from 11c according to
sub-G1 population, although the EC50 values of all four General procedure A. Yield: 77%; off-white solid; mp 202–205 ꢀC;
compounds were very similar (20–24 mM).
IR (KBr) n ¼ 3443 (N–H), 3275 (N–H), 3145 (C–H), 2764 (C–H),
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1662 (C]O), 1628, 1607, 1544, 1486, 1449, 1411, 1327, 1287, (KBr) n ¼ 3269, 3182, 3049, 2934, 2865, 2746, 1685, 1635, 1602,
1
1244, 1231, 1204, 1145, 1103, 954, 840, 780, 752, 727 cmꢁ1. H 1541, 1494, 1414, 1368, 1334, 1312, 1248, 1190, 1145, 1115,
3
4
NMR (DMSO-d6) d 7.11 (dt, 1H, J ¼ 9.2 Hz, J ¼ 2.0 Hz, Ar-H), 1060, 1018, 989, 919, 971, 819, 796, 775, 750 cmꢁ1
.
1H NMR
7.33 (s, 1H, Ar-H), 7.40–7.50 (m, 7H, 5 ꢃ Ar-H, NH2), 7.69–7.71 (DMSO-d6) d 2.95 (d, 3H, 3J ¼ 4.8 Hz, CH3), 7.09 (dt, 1H, 3J ¼ 6.8
(m, 1H, Ar-H), 8.08 (t, 1H, 4J ¼ 1.6 Hz, Ar-H), 10.49 (s, 1H, NH), Hz, 4J ¼ 1.2 Hz, indole-H), 7.24 (dt, 1H, 3J ¼ 6.8 Hz, 4J ¼ 1.2 Hz,
11.95 (s, 1H, NH), 12.14 (s, 1H, NH), 12.83 (s, 1H, NH); 13C NMR indole-H), 7.43–7.50 (m, 5H, 5 ꢃ Ar-H), 7.68–7.72 (m, 2H, 2 ꢃ
(DMSO-d6) d 104.34 (d, 1C, 4JC–F ¼ 5 Hz), 105.89 (d, 1C, 2JC–F ¼ 23 Ar-H), 7.84 (q, 1H, 3J ¼ 4.8 Hz, NH), 8.11 (t, 1H, 4J ¼ 2.0 Hz, Ar-H-
Hz), 109.51, 112.65 (d, 1C, 2JC–F ¼ 26 Hz), 113.63 (d, 1C, 3JC–F ¼ 9 20), 10.44 (s, 1H, NH), 11.82 (s, 1H, NH), 12.40 (br s, 1H, NH),
Hz), 116.52, 119.93, 120.39, 126.37, 127.00 (d, 1C, 3JC–F ¼ 9 Hz), 12.62 (br s, 1H, NH); 13C NMR (DMSO-d6) d 30.07 (CH3), 105.00,
128.17, 129.35, 132.96, 133.57, 139.28, 147.81, 157.20 (d, 1C, 1JC–F
¼
110.20, 112.88, 117.28, 120.42, 120.67, 120.87, 122.25, 124.35,
231 Hz), 159.47; 19F NMR (DMSO-d6) d ꢁ123.59 (s, 1F); MS (ESI) m/z 127.43, 127.45, 128.69, 129.72, 131.80, 137.33, 139.87, 149.08,
(%) ¼ 336.1 ([M ꢁ Cl]+, 100). HRMS for C18H15N5OF: calculated, 160.25; MS (ESI) m/z (%) ¼ 332 ([M ꢁ Cl]+, 100). HRMS for
336.1261; found, 336.1264. HPLC: Phenomenex Luna 5 mm C18
C19H18N5O: calculated, 332.1511; found, 332.1499. HPLC: Phe-
column (4.6 mm ꢃ 150 mm); mobile phase: 60–90% of MeOH nomenex Luna 5 mm C18 column (4.6 mm ꢃ 150 mm); mobile
in TFA (0.1%) in 20 min; ow rate: 1.0 mL minꢁ1; injection phase: 10–90% of MeOH in TFA (0.1%) in 20 min; ow rate 1.0
volume: 10 mL; retention time: 3.585 min (99.4% at 254 nm, mL minꢁ1; injection volume: 20 mL; retention time: 17.146 min
99.1% at 280 nm).
(99.2% at 254 nm, 99.2% at 280 nm).
2-Amino-4-(3-(5-chloro-1H-indole-2-carboxamido)phenyl)-1H-
imidazol-3-ium chloride (29c). Prepared from 12c according to
General procedure A. Yield: 71%; white solid; mp 201–204 ꢀC; IR
(KBr) n ¼ 3410 (N–H), 3260 (N–H), 3145 (C–H), 3032 (C–H), 2761
Acknowledgements
(C–H), 1693 (C]O), 1667, 1610, 1542, 1485, 1442, 1412, 1326, This work was supported by the Slovenian Research Agency
1301, 1275, 1245, 1224, 1190, 1124, 1056, 914, 854, 798, 782, (Grant no. P1-0208 and Grant no. Z1-5458), by the European
754, 725 cmꢁ1. 1H NMR (DMSO-d6) d 7.25 (dd, 1H, 3J ¼ 8.8 Hz, 4J Union FP7 Integrated Project MAREX: Exploring Marine
¼ 2.0 Hz, Ar-H), 7.33 (s, 1H, Ar-H), 7.40–7.51 (m, 6H, 4 ꢃ Ar-H, Resources for Bioactive Compounds: From Discovery to
4
NH2), 7.69–7.71 (m, 1H, Ar-H), 7.79 (d, 1H, J ¼ 2.0 Hz, Ar-H), Sustainable Production and Industrial Applications (Project no.
8.07 (s, 1H, Ar-H), 10.53 (s, 1H, NH), 12.05 (s, 1H, NH), 12.14 (s, FP7-KBBE-2009-3-245137) and by the COST CM1106 (Chemical
1H, NH), 12.84 (s, 1H, NH); 13C NMR (DMSO-d6) d 103.87, Approaches to Targeting Drug Resistance in Cancer Stem Cells).
109.52, 114.01, 116.53, 119.98, 120.40, 120.82, 123.98, 124.42,
126.37, 127.98, 128.18, 129.36, 132.75, 135.21, 139.23, 147.80,
159.41; MS (ESI) m/z (%) ¼ 352.1 ([M ꢁ Cl]+, 100). HRMS for
Notes and references
C
18H15N5OCl: calculated, 352.0965; found, 352.0959. HPLC:
Phenomenex Luna 5 mm C18 column (4.6 mm ꢃ 150 mm);
mobile phase: 60–90% of MeOH in TFA (0.1%) in 20 min; ow
rate: 1.0 mL minꢁ1; injection volume: 10 mL; retention time:
5.338 min (98.4% at 254 nm, 98.8% at 280 nm).
1 D. Sipkema, M. C. R. Franssen, R. Osinga, J. Tramper and
R. H. Wijffels, Mar. Biotechnol., 2005, 7, 142.
2 R. Singh, M. Sharma, P. Joshi and D. S. Rawat, Anti-Cancer
Agents Med. Chem., 2008, 8, 603.
4-(3-(4H-Thieno[3,2-b]pyrrole-5-carboxamido)phenyl)-2-amino-
1H-imidazol-3-ium chloride (30c). Prepared from 13c according
to General procedure A. Yield: 78%; off-white solid; mp 198–202
ꢀC; IR (KBr) n ¼ 3241 (N–H), 3135 (C–H), 3047 (C–H), 2763
(C–H), 1677 (C]O), 1625, 1541, 1488, 1460, 1385, 1348, 1308,
3 A. Al-Mourabit, M. A. Zancanella, S. Tilvi and D. Romo, Nat.
Prod. Rep., 2011, 28, 1229.
ˇ
ˇ
4 A. Zula, D. Kikelj and J. Ilas, Mini-Rev. Med. Chem., 2013, 13,
1921.
5 B. Forte, B. Malgesini, C. Piutti, F. Quartieri, A. Scolaro and
G. Papeo, Mar. Drugs, 2009, 7, 705.
6 A. L. Rentas, R. Rosa, A. D. Rodriguez and G. E. De Motta,
Toxicon, 1995, 33, 491.
7 U. Bickmeyer, C. Drechsler, M. Kock and M. Assmann,
Toxicon, 2004, 44, 45.
8 J. J. Richards, T. E. Ballard and C. Melander, Org. Biomol.
Chem., 2008, 6, 1356.
1
1231, 1191, 1115, 1084, 963, 877, 827, 748, 711 cmꢁ1. H NMR
3
4
(DMSO-d6) d 7.03 (dd, 1H, J ¼ 5.2 Hz, J ¼ 0.8 Hz, Ar-H), 7.31
(s, 1H, Ar-H), 7.36–7.49 (m, 6H, 4 ꢃ Ar-H, NH2), 7.66–7.69 (m,
1H, Ar-H), 8.06 (t, 1H, 4J ¼ 1.6 Hz, Ar-H), 10.24 (s, 1H, NH), 11.99
(s, 1H, NH), 12.14 (s, 1H, NH), 12.82 (s, 1H, NH); 13C NMR
(DMSO-d6) d 103.91, 109.43, 111.90, 116.27, 119.48, 120.15,
122.94, 126.45, 128.08, 128.28, 129.26, 130.49, 139.64, 141.32,
147.77, 159.41; MS (ESI) m/z (%) ¼ 324.1 ([M ꢁ Cl]+, 100). HRMS
for C16H14N5OS: calculated, 324.0919; found, 324.0911. HPLC:
9 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney,
Adv. Drug Delivery Rev., 2001, 46, 3.
ˇ
ˇ ´
ˇ ˇ
Phenomenex Luna 5 mm C18 column (4.6 mm ꢃ 150 mm); 10 Z. Hodnik, T. Tomasic, L. Peterlin Masic, F. Chan,
mobile phase: 60–90% of MeOH in TFA (0.1%) in 20 min; ow
rate: 1.0 mL minꢁ1; injection volume: 10 mL; retention time:
2.790 min (96.5% at 254 nm, 97.0% at 280 nm).
R. W. Kirby, D. J. Madge and D. Kikelj, Eur. J. Med. Chem.,
2013, 70, 154.
11 N. Zidar, Z. Jakopin, D. J. Madge, F. Chan, J. Tytgat,
ˇ
ˇ ´
ˇ
4-(3-(1H-Indole-2-carboxamido)phenyl)-2-(methylamino)-1H-
imidazol-3-ium chloride (34c). Prepared from 18c according to
General procedure A. Yield: 95%; white solid; mp 190–194 ꢀC; IR
S. Peigneur, M. Sollner Dolenc, T. Tomasic, J. Ilas,
ˇ ˇ
L. Peterlin Masic and D. Kikelj, Eur. J. Med. Chem., 2014,
74, 23.
This journal is © The Royal Society of Chemistry 2014
Med. Chem. Commun.

View Article Online
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Concise Article
ˇ
ˇ
˜
12 N. Zidar, S. Montalvao, Z. Hodnik, D. A. Nawrot, A. Zula, 18 H. Lecoeur, Exp. Cell Res., 2002, 277, 1.
ˇ
ˇ ˇ
J. Ilas, D. Kikelj, P. Tammela and L. Peterlin Masic, Mar. 19 T. Vanden Berghe, S. Grootjans, V. Goossens,
Drugs, 2014, 12, 940.
Y. Dondelinger, D. V. Krysko, N. Takahashi and
13 T. Lindel, G. Breckle, M. Hochgurtel, C. Volk, A. Grube and
M. Kock, Tetrahedron Lett., 2004, 45, 8149.
P. Vandenabeele, Methods, 2013, 61, 117.
ˇ
ˇ
20 A. Zula, D. Kikelj and J. Ilas, Tetrahedron Lett., 2014, 55, 3999.
ˇ
14 S. H. Henry, F. X. Bosch and J. C. Bowers, Adv. Exp. Med. Biol., 21 M. Jukic, R. Frlan, F. Chan, R. W. Kirby, D. J. Madge,
2002, 504, 229.
M. Anderluh and D. Kikelj, Med. Chem. Res., 2014, accepted.
ˇ ´
15 E. Estey and H. Dohner, Lancet, 2006, 368, 1894.
16 M. van Engeland, L. J. Nieland, F. C. Ramaekers, B. Schutte
and C. P. Reutelingsperger, Cytometry, 1998, 31, 1.
22 T. Tomasic, B. Hartzoulakis, N. Zidar, F. Chan, R. W. Kirby,
D. J. Madge, S. Peigneur, J. Tytgat and D. Kikelj, J. Chem. Inf.
Model., 2013, 53, 3223.
17 S. J. Martin, C. P. Reutelingsperger, A. J. McGahon, 23 S. H. Kaufmann and W. C. Earnshaw, Exp. Cell Res., 2000,
J. A. Rader, R. C. van Schie, D. M. LaFace and D. R. Green,
256, 42.
J. Exp. Med., 1995, 182, 1545.
24 C. Riccardi and I. Nicoletti, Nat. Protoc., 2006, 1, 1458.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2014