Tihomir Tomašič a, Dominik Nabergoj ab, Sanja Vrbek ab, Nace Zidar a, Žiga Jakopin a, Aleš Žula a, Žiga Hodnik a, Marko Jukič a, Marko Anderluh a, Janez Ilaš a, Marija Sollner Dolenc a, Jean Peluso bc, Geneviève Ubeaud-Séquier bc, Christian D. Muller *bc, Lucija Peterlin Mašič *a and Danijel Kikelj a
aUniversity of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia. E-mail: lucija.peterlin@ffa.uni-lj.si; Fax: +386-1-4258031; Tel: +386-1-4769635
bLaboratoire d'Innovation Thérapeutique, UMR 7200, Faculté de Pharmacie, Université de Strasbourg, 67401 Illkirch, France. E-mail: cdmuller@unistra.fr; Fax: +33-368854310; Tel: +33-688285839
cPlateforme eBioCyt, Faculté de Pharmacie & Féderation Translationnelle de Médecine, Université de Strasbourg, 67401 Illkirch, France
First published on 26th August 2014
The marine alkaloids COMPOUND LINKS
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Explore further on Open PHACTSclathrodin, 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 COMPOUND LINKS
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Download mol file of compound34c 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 COMPOUND LINKS
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Download mol file of compound34c 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.
Fig. 1 Structures of the pyrrole-2-aminoimidazole alkaloids COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSclathrodin, hymenidin, and oroidin, and their structural modifications (type A–C analogues). |
The oroidin class of alkaloids is structurally relatively simple (Fig. 1) and possesses drug-like properties according to Lipinski's rule of five;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 COMPOUND LINKS
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Explore further on Open PHACTSclathrodin and oroidin analogues and evaluated some of the resulting compounds to determine their modulatory activity on human voltage-gated sodium channels10,11 and their antibacterial activity.12
The clathrodin, hymenidin, and oroidin molecules possess a potentially unstable double bond in the linker between the 2-aminoimidazole and pyrrole moieties.13 Therefore, we designed and synthesised a library of their analogues by modifying the central part of the molecule (Fig. 1, type A to COMPOUND LINKS
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Download mol file of compoundC analogues) to obtain more stable and conformationally restricted compounds as well as the western (Fig. 1, type A–C analogues) and eastern parts (type A–C analogues) of the molecule to enable structure–activity relationship studies. In the present work, we studied the apoptosis-inducing activity of COMPOUND LINKS
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Explore further on Open PHACTSclathrodin, oroidin, and hymenidin, and their type A–C analogues against HepG2 and THP-1 human cancer cell lines to evaluate their potential as anticancer agents against hepatocellular carcinoma and acute monocytic leukaemia.
Hepatocellular carcinoma is the most common type of liver cancer, and its high incidence has been attributed to persistent infection with hepatitis B or C virus, contact with hepatocarcinogens (e.g., aflatoxins), and cirrhosis. The development of drug resistance in hepatocellular carcinoma tumour cells after drug therapy indicates the important need for the discovery of novel anticancer agents for the successful treatment of liver cancer.14
Acute monocytic leukaemia, a type of acute myeloid leukaemia, is a hematopoietic cancer characterised by a disorder of hematopoietic progenitor cells, which lose their ability for normal differentiation and response to normal regulators of proliferation. Its incidence increases with age. Considering the aging population and the fact that acute myeloid leukaemia has the lowest survival rate of all leukaemias, new anticancer agents against acute myeloid leukaemia are urgently needed to fight this type of cancer in the future.15
Scheme 1 Synthesis of the most active compounds COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound24c, 28c–30c and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound34c. Reagents and conditions: (a) corresponding carboxylic acid, TBTU, NMM, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSCH2Cl2, 35 °C, 24 h; (b) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSHCl(g), COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSTHF–COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSEtOH, rt, 5 h. |
The marine alkaloids COMPOUND LINKS
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Explore further on Open PHACTSclathrodin, oroidin and hymenidin, which were synthesised in our laboratory,20 and their analogues 1–4 were found to possess only weak apoptosis-inducing activity in the HepG2 cell line with 25–38% apoptotic cells at 50 μM (Table S1†).
In the series of type A analogues (Fig. 1), conformational restriction was achieved by replacing the (E)-5-(3-aminoprop-1-enyl)-1H-imidazol-2-amine moiety by the 4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine core in which the imidazole was isosterically replaced by the thiazole ring.10 However, type A analogues 1a–14a containing also various modifications in the central and eastern parts of the parent molecules (Table S2†) did not display improved apoptosis-inducing activity compared with oroidin (20–38% apoptotic HepG2 cells at 50 μM).
The type B analogues 1b–10b were obtained by modification of the central part through replacement of the 3-aminoprop-1-enyl linker between the 2-aminoimidazole and pyrrole moieties by the less flexible N-methylenepiperazine group (Fig. 1).21 Modification 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 HepG2 cells at 50 μM). In the type COMPOUND LINKS
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Download mol file of compoundC analogues, the 1,3- or 1,4-disubstituted phenyl ring was incorporated in place of the prop-1-enyl linker to obtain a conformationally restricted central part of the molecule without changing the length of the molecule (Fig. 1).11 The analysis of the apoptosis-inducing activity of the type COMPOUND LINKS
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Download mol file of compoundC 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 five-membered pyrrole (1c–3c, COMPOUND LINKS
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Download mol file of compound39c) ring, the compounds were only weakly active (Table S4, Fig. S1,† 13–44% apoptotic HepG2 cells at 50 μM). The only exceptions were the pyrrole-based compounds COMPOUND LINKS
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Download mol file of compound36c, which contained a benzyl moiety on the imidazole ring nitrogen and induced apoptosis in 54% and 86% of HepG2 cells at a concentration of 50 μM (Table S4†), respectively. In contrast, a significant gain in the apoptosis-inducing activity was observed in the compounds containing an indole or substituted indole moiety in place of the pyrrole ring of the parent marine alkaloids.
The indole-based compounds 5c–12c, COMPOUND LINKS
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Download mol file of compound42c and 4H-thieno[3,2-b]pyrrole COMPOUND LINKS
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Download mol file of compound13c containing a tert-butyloxycarbonyl (Boc) group on the imidazole N1 showed improved activity with 43–91% apoptotic HepG2 cells at 50 μM. A substitution at position 5 of the indole ring with a methoxy (COMPOUND LINKS
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Download mol file of compound12c) group resulted in improved activity compared to the non-substituted indoles COMPOUND LINKS
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Download mol file of compound6c, whereas a hydroxy (COMPOUND LINKS
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Download mol file of compound10c) substitution decreased the potency. A methyl substituent at the imidazole 2-amino group increased the percentage of apoptotic cells compared with those found with the indole-based compounds (COMPOUND LINKS
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Download mol file of compound18c), whereas a reduction of the imidazole to obtain a 2-aminoimidazoline ring reduced the activity (COMPOUND LINKS
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Download mol file of compound42c with a 1,4-disubstituted phenyl ring in the central part and an indole moiety in the eastern part of the molecule was among the most active apoptosis-inducing compounds and was more active than its 1,3-phenylene counterpart COMPOUND LINKS
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In general, the Boc-deprotected indole-based compounds 22c–29c, COMPOUND LINKS
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Download mol file of compound34c and the 4H-thieno[3,2-b]pyrrole-based compound COMPOUND LINKS
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Download mol file of compound30c retained their apoptosis-inducing activity against the HepG2 cell line. Similar to their Boc-protected analogues, the compounds with the 6-fluoro- (COMPOUND LINKS
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Download mol file of compound30c) moieties were the most active with more than 90% apoptotic HepG2 cells at 50 μM.
We also evaluated the apoptosis-inducing activity of compounds 45c–65c (Table S4†), which were identified in the 3D similarity searching, based on the indole COMPOUND LINKS
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Download mol file of compound22c, in the ZINC database of drug-like compounds.22 Interestingly, most of the indoles (COMPOUND LINKS
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Download mol file of compound64c) induced apoptosis in more than 50% of HepG2 cells at 50 μM, regardless of the ring type in the western part of the molecule, which indicates that the indole moiety is a crucial feature for significant apoptosis-inducing activity in type COMPOUND LINKS
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Download mol file of compoundC oroidin analogues. Among compounds 45c–65c, only compound COMPOUND LINKS
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Download mol file of compound52c showed improved apoptosis-inducing activity (92% apoptotic HepG2 cells at 50 μM) compared with the template compound COMPOUND LINKS
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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 five9 (Fig. S2†). The presented charts show that most of the active compounds (>50% apoptotic HepG2 cells at 50 μM) are more lipophilic (log D values between 3 and 5) and have higher molecular weights (MW between 300 and 500) compared with their inactive 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.
Of the 96 compounds screened, compounds COMPOUND LINKS
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Download mol file of compoundC oroidin analogues, were selected for further characterisation. First, the EC50 values for their apoptosis-inducing activity against the HepG2 cell line were determined using the annexin V/PI apoptosis assay (Table 1). 6-Fluoroindole COMPOUND LINKS
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Download mol file of compound28c was found to be the most active compound with an EC50 value of 13 μM, followed by 6-chloroindole COMPOUND LINKS
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Download mol file of compound29c (EC50 = 16 μM), 6-methoxyindole COMPOUND LINKS
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Compound | R1 | R2 | HepG2a EC50 | THP-1a EC50 |
---|---|---|---|---|
a The values are the mean ± SD of three independent experiments performed in triplicate. | ||||
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound24c | OCH3 | H | 18 ± 1 μM | 20 ± 2 μM |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound28c | F | H | 13 ± 7 μM | 23 ± 4 μM |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound29c | Cl | H | 16 ± 6 μM | 24 ± 5 μM |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound30c | — | — | 42 ± 16 μM | >50 μM |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound34c | H | CH3 | 20 ± 5 μM | 24 ± 1 μM |
Considering the noteworthy HepG2 apoptosis-inducing activity of compounds COMPOUND LINKS
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Download mol file of compound34c, these were further evaluated using the human monocytic leukaemia THP-1 cell line (ATCC® TIB-202™) (Table 1). The screening of these compounds at 50 μM against the THP-1 cell line showed that compounds COMPOUND LINKS
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Download mol file of compound34c induce apoptosis (87–97% apoptotic THP-1 cells), whereas compound COMPOUND LINKS
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Download mol file of compound30c was found to be inactive (12% apoptotic THP-1 cells). The dose–response curves showed that compounds COMPOUND LINKS
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Download mol file of compound34c exhibited similar activities against the THP-1 cell line with EC50 values ranging from 20 μM to 24 μM (Table 1). Because activation of the apoptotic pathways is a key mechanism through which anticancer drugs kill tumour cells,23 it was important to confirm that compounds COMPOUND LINKS
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Download mol file of compound34cde facto exert their cytotoxic effect against the THP-1 cell line via the induction of apoptosis and not by necrosis. Hence, THP-1 cells were subjected to a cell cycle analysis after exposure to COMPOUND LINKS
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Download mol file of compound34c to determine the incidence of fragmented DNA (sub-G1 population) by PI staining of the nuclei.24 DMSO (0.25%), which was used as a negative control, did not affect the cell cycle in THP-1 cells (Fig. 2). The results of the cell cycle analysis after the incubation of THP-1 cells with compounds COMPOUND LINKS
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Download mol file of compound34c at 25 μM for 24 h and 48 h (Fig. 2 and 3 and Table S5†) show the presence of a sub-G1 cell population, which confirmed the presence of programed cell death, i.e. apoptosis. Compounds COMPOUND LINKS
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Download mol file of compound29c showed similar potency: 41% and 40% of the cells were found in the sub-G1 peak after 48 h of treatment, respectively. Compounds COMPOUND LINKS
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Download mol file of compound24c displayed more potent apoptosis-inducing activity with 49% and 60% of the cells in the sub-G1 population, although the EC50 values of all four compounds were very similar (20–24 μM).
Fig. 2 Cell cycle analysis of THP-1 cells after incubation with compounds COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound24c, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound28c, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound29c, and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound34c at 25 μM for 24 h and 48 h. COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound Explore further on Open PHACTSDMSO (0.25%) in culture medium was used as a negative control. Representative histograms of three independent experiments are displayed. |
Fig. 3 Comparison of the sub-G1 cell cycle population after the treatment of THP-1 cells with compounds COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound24c, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound28c, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound29c, and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound34c at 25 μM for 24 h and 48 h (n = 3 independent experiments). |
Footnote |
† 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 |
This journal is © The Royal Society of Chemistry 2015 |