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Dispacamide E and other bioactive bromopyrrole alkaloids from two Indonesian marine sponges of the genus Stylissa ab
c
c
Sherif S. Ebada , Mai Hoang Linh , Arlette Longeon , Nicole J. d
e
f
de Voogd , Emilie Durieu , Laurent Meijer , Marie-Lise Bourguetc
b
g
Kondracki , Abdel Nasser B. Singab , Werner E.G. Müller & Peter Proksch
a
a
Institutfür Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine Universität, Geb. 26.23, Universitätsstrasse 1, D-40225 Düsseldorf, Germany b
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street 1, 11566 Cairo, Egypt c
Laboratoire Molécules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Museum National d'Histoire Naturelle, Paris, France d
Naturalis Biodiversity Center, PO Box 9517 2300, RA Leiden, Netherlands e
Centre National de la Recherche Scientifique, Protein Phosphorylation & Human Disease Group, Station Biologique, F-29680 Roscoff, France f
Man Ros Therapeutics, Centre de Perharidy, F-29680 Roscoff, France g
Institute for Physiological Chemistry, University of Medical Center of the Johannes-Gutenberg-University Mainz, Duesbergweg 6, D-55128 Mainz, Germany Published online: 12 Aug 2014.
To cite this article: Sherif S. Ebada, Mai Hoang Linh, Arlette Longeon, Nicole J. de Voogd, Emilie Durieu, Laurent Meijer, Marie-Lise Bourguet-Kondracki, Abdel Nasser B. Singab, Werner E.G. Müller & Peter Proksch (2014): Dispacamide E and other bioactive bromopyrrole alkaloids from two Indonesian marine sponges of the genus Stylissa, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.947496
To link to this article: http://dx.doi.org/10.1080/14786419.2014.947496
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.947496
Dispacamide E and other bioactive bromopyrrole alkaloids from two Indonesian marine sponges of the genus Stylissa Sherif S. Ebadaab*, Mai Hoang Linhc, Arlette Longeonc, Nicole J. de Voogdd, Emilie Durieue, Laurent Meijerf, Marie-Lise Bourguet-Kondrackic, Abdel Nasser B. Singabb, Werner E.G. Mu¨llerg and Peter Prokscha*
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a
Institutfu¨r Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine Universita¨t, Geb. 26.23, Universita¨tsstrasse 1, D-40225 Du¨sseldorf, Germany; bDepartment of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street 1, 11566 Cairo, Egypt; cLaboratoire Mole´cules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Museum National d’Histoire Naturelle, Paris, France; dNaturalis Biodiversity Center, PO Box 9517 2300, RA Leiden, Netherlands; eCentre National de la Recherche Scientifique, Protein Phosphorylation & Human Disease Group, Station Biologique, F-29680 Roscoff, France; fMan Ros Therapeutics, Centre de Perharidy, F-29680 Roscoff, France; gInstitute for Physiological Chemistry, University of Medical Center of the Johannes-Gutenberg-University Mainz, Duesbergweg 6, D-55128 Mainz, Germany (Received 12 June 2014; final version received 19 July 2014) Chemical investigation of methanolic extracts of the two Indonesian marine sponges Stylissa massa and Stylissa flabelliformis yielded 25 bromopyrrole alkaloids including 2 new metabolites. The structures of all isolated compounds were unambiguously elucidated based on extensive 1D and 2D NMR, LR-MS and HR-MS analyses. All isolated compounds were assayed for their antiproliferative and protein kinase inhibitory activities. Several of the tested compounds revealed selective activity(ies) which suggested preliminary SARs of the isolated bromopyrrole alkaloids. Keywords: bromopyrrole alkaloids; antiproliferative; protein kinase; neurodegenerative disorders
1. Introduction Marine natural products can be considered as bioactive chemicals produced by marine organisms to deliver a survival advantage and protection of sessile organisms against their predators (in the case of invertebrates) or their herbivores (in the case of algae). Based on the knowledge of marine natural products chemistry, ecology and biology, researchers were inspired for developing several new pharmaceutical drugs for treatment of cancer or chronic pain (Mayer et al. 2010; Ebada & Proksch, 2012, 2011a, 2011b; Mayer et al. 2013). Bromopyrrole alkaloids constitute a well-known class of marine natural products found majorly in sponges genera belonging to the families Agelasidae, Axinellidae, Dictyonellidae and Halichondridae (Scala et al. 2010; Ebada & Proksch 2012). Oroidin is the parent compound of this class of marine metabolites which was first isolated from the marine sponge Agelasoroides. It is considered as the key precursor since many bromopyrrole alkaloids comprising a pyrrole – imidazole unit can be biosynthetically considered as metabolic derivatives of the C11N5 skeleton of oroidin (Forenza et al. 1971).
*Corresponding authors. Email: [email protected], [email protected] q 2014 Taylor & Francis
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S.S. Ebada et al.
Bromopyrrole alkaloids are also known as a prolific source of compounds with significant pharmacological activities including antiproliferative, antimicrobial and immunosuppressive activities which encouraged natural product chemists to study their total synthesis primarily during the last two decades. These efforts lead to successful total synthesis of many dimeric bromopyrrole –imidazole alkaloids such as sceptrin, oxsceptrin and ageliferin (O’Malley et al. 2007); nagelamides D (Bhandari et al. 2009) and E (O’Malley et al. 2007); and hymenialdisine analogues (Nikoulina et al. 2000). During our ongoing research for bioactive bromopyrrole alkaloids from marine sponges, we have chemically investigated methanolic extracts of two Indonesian marine sponges Stylissa massa and Stylissa flabelliformis implementing HPLC-DAD-MS analysis. In this study, we report the isolation and identification of 25 bromopyrrole alkaloids including 2 new metabolites together with 2 debrominated congeners in addition to their antiproliferative and protein kinase inhibitory activities. For measurement of protein kinase inhibitory activities, a panel of nine different kinases involved in cell proliferation, diabetes and neurodegenerative disorders (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A), CDC-like kinase 1 (CLK-1), casein kinase 1 (CK-1), cyclin-dependent kinase 5 (CDK5), glycogen synthase kinase-3 (GSK-3), cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2/A) and complex of cyclin-dependent kinase 9 and cyclin T (CDK9/cyclin T)) and malaria (PfGSK-3).
2. Results and discussion The methanolic extract of the marine sponge S. massa collected from Papua island (Indonesia) was subjected to HPLC-DAD-MS analysis which revealed a complex pattern of different molecular weights. Implementing different chromatographic separation methods, 20 bromopyrrole alkaloids including 2 new metabolites (1 and 2) together with 2 debrominated derivatives were purified and their structures were unambiguously elucidated based on extensive 1D and 2D NMR and HR-MS analyses and comparison with reported data in the literature. Known metabolites were identified as 4-bromopyrrole-3-carboxamide (3) (Hassan et al. 2007), 3,4-dibromopyrrole-2-carboxamide (4) (Hassan et al. 2007; Supriyono et al. 1995), (2 )-longamide B (5) (Cafieri et al. 1998; Patel et al. 2005), (2 )-longamide B methyl ester (6) (Patel et al. 2005), (2 )-longamide B ethyl ester (hanishin, 7) (Mancini et al. 1997), aldisine (8) (Schmitz et al. 1985), 2,3-dibromoaldisine (9) (Hassan et al. 2007), 2bromoaldisine (10) (Schmitz et al. 1985; Hassan et al. 2007), 3-bromoaldisine (11) (Hassan et al. 2007), (2 )-mukanadin C (12) (Li et al. 1998; Uemoto et al. 1999), (2 )-longamide (13) (Cafieri et al. 1995), latonduine A (14) (Linington et al. 2003), (2 )-dibromophakellin (15) (Sharma & Magdoff-Fairchild 1977), (2 )-monobromoisophakellin (16) (Assmann & Ko¨ck 2002), (2 )-dibromocantherelline (17) (De Nanteuil et al. 1985), (2 )-hymenine (18) (Kobayashi et al. 1986; Xu et al. 1997), spongiacidin B (19) (Inaba et al. 1998), (10Z)debromohymenialdisine (20) (Sharma et al. 1980; Cimino et al. 1982), (10Z)-hymenialdisine (21) (Cimino et al. 1982) and (10Z)-3-bromohymenialdisine (22) (Supriyono et al. 1995). Furthermore, the purification of the methanolic extract of the marine sponge S. flabelliformis collected from south Sulawei (Indonesia) by using different chromatographic separation methods yielded 11 known dibromopyrrole alkaloids: 3,4-dibromopyrrole-2-carboxamide (4), 2-bromoaldisine (10), (2 )-longamide (13), latonduine A (14), (2 )-dibromoisophakellin (17), (10Z)-hymenialdisine (21), (10E)-hymenialdisine (23) (Plisson et al. 2014), latonduine B ethyl ester (24) (Linington et al. 2003), 3-debromolatonduine A (25) (Fouad et al. 2012), stevensine (26) (Albizati & Faulkner 1985) and 12-N-methyl stevensine (27) (Fouad et al. 2012), furnishing five additional bromopyrrole alkaloids for SARs.
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Compound 1 was isolated as a faint yellow amorphous solid. It showed two pseudomolecular ion clusters at m/z 404, 406 and 408 [M þ H]þ; and at m/z 426, 428 and 430 [M þ Na]þ, each at a 1:2:1 ratio in its ESI mass spectrum indicating that it was a dibrominated compound. The 13C NMR spectrum showed 11 signals representing two methylene groups in addition to nine sp2 carbons including two methine groups and seven fully substituted carbons. The molecular þ formula of 1 was C11H79 11Br2N5O2 as indicated by HR-FT-MS (m/z 405.9320 [M þ H] calcd for C11H79 12Br2N5O2, D þ 1.0 ppm). The structure of 1 was further confirmed by interpretation of 1 H – 1H COSY and HMBC spectra (Figure 1). The 1H – 1H COSY spectrum showed a cross peak between NH-pyrrole (dH 12.69, br s) and the aromatic proton H-2 (dH 6.90, d, 1.3 Hz; dC 112.6) in addition to an extended spin system among NH-7 (dH 8.28, t, 6.0 Hz), ¼ CH2-8 (dH 3.47, m; dC 37.4), ¼ CH2-9 (dH 2.50, m; dC 27.3) and H-10 (dH 5.96, t, 7.6 Hz; dC 116.8). The HMBC spectrum (Figure 1) further supported the proposed structure and revealed correlations between H-2 to C-4 (dC 104.7) and C-5 (dC 128.0); H-10 to C-8 (dC 37.4), C-11 (dC 129.6) and C-15 (dC
O
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R'' R''' Br
Br N H
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H N O
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9: R'= R''= Br 10: R'= Br, R''= H
7: R= Et
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O 8: R'= R''= H
5: R= H O 6: R= Me
3: R'= H, R''= CONH2, R'''= Br 4: R'= CONH2, R''=R'''= Br
1
NH
R''
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NH2
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OH
12: R'= H, R''= Br 13: R'= R''= Br
11: R'= H, R''= Br
R
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O
NH
NH2
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16: R'= H, R''= Br
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Br
NH
O 20: R'= R''= H
Br NH N H
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21: R'= Br, R''= H 22: R'= R''= Br
Br
O H N
N H O
Br
1
Br
N N H 1
NH2
H-1H COSY
5
2 6 O N H
N R
HN
O 2
HMBC
Figure 1. Key HMBC and ROESY correlations of compounds 1 and 2.
NH N H
N H
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HN
Br
NH O
15
H2N
NH O Br
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O 14: R= Br, R'= H 24: R= Br, R'= COOEt 25: R= R'= H
NH
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Br
NH2
H2N
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26: R=H 27: R=Me
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163.6); ¼ CH2-8 to C-9 (dC 27.3), C-10 (dC 116.8) and C-6 (dC 158.9). According to the aforementioned data and by comparison with other dispacamide derivatives reported in the literature (Cafieri et al. 1997, 1996), compound 1 was confirmed to be N-((9E)-3-(2-amino-4oxo-1H-imidazol-5(4H)-ylidene) propyl)-3,4-dibromo-1H-pyrrole-2-carboxamide that was given the trivial name, dispacamide E. Compound 2 was isolated as white needle crystals. It showed pseudomolecular ion peaks at m/z 294, 296 and 298 [M 2 H]- at a 1:2:1 ratio in its ESI mass spectrum, indicating that it was a dibrominated compound with a daughter ion peak at m/z 268.1, indicating the loss of 29 amu that can represent an ethyl moiety. The molecular formula C7H79 7 Br2NO2 was indicated by HR-ESIBr NO , D þ 0.3 ppm). This was confirmed based MS (m/z 295.8916 [M þ H]þcalcd for C7H79 8 2 2 on results of 1D and 2D NMR. In this study, 2 was obtained for the first time as a naturally occurring bromopyrrole alkaloid. The 13C NMR spectrum showed 7 signals representing one methyl and one methylene group in addition to five sp2 carbons including one methine group and four fully substituted carbons. The structure of 2 was further confirmed by interpretation of 1 H – 1H COSY and HMBC spectra (Figure 1). The 1H – 1H COSY spectrum showed a cross peak between NH proton (dH 9.74, br s) and the aromatic proton H-5 (dH 6.89, d, 2.8 Hz; dC 117.8). The HMBC spectrum (Figure 1) supported the presence of the aromatic proton at C-5 and also revealed correlations between H-5 to C-2 (dC 124.1) and C-3 (dC 106.7). The presence of an ethyl ester group was deduced from 1H – 1H COSY that revealed a clear correlation between methylene group (dH 4.34, q, 7.3 Hz; dC 61.1) and methyl group (dH 1.36, t, 7.3 Hz; dC 14.3); and HMBC spectra showed a correlation of the methylene protons to the carbonyl group C-6 (dC 159.7). Moreover, the methylene and methyl protons showed a HMQC correlation to C-7 (dC 61.1) and C-8 (dC 14.3), respectively. Therefore, 2 was confirmed to be ethyl 3,4-dibromo-1Hpyrrole-2-carboxylate. This compound was previously only known as a synthetic product (Handy et al. 2004). All isolated bromopyrrole alkaloids from the methanolic extract of S. massa were subjected to a cell proliferation (MTT) assay against mouse lymphoma (L5178Y) cells. Some of the tested compounds (Table 1) were shown to possess significant antiproliferative activities with IC50 values ranging between 6.33 and 28.28 mM compared to kahalalide F (IC50 ¼ 4.30 mM). Among the hymenialdisine analogues, (10Z) debromohymenialdisine (20) was the most potent derivative with an IC50 value of 6.33 mM. Furthermore, all isolated bromopyrrole alkaloids were assayed for their protein kinase inhibitory activities against nine different protein kinases, namely DYRK1A, CDK5, GSK-3, CLK-1, CK-1, CDK1, CDK2/A, CDK9/cyclin T and Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3). Results of the protein kinase inhibitory activity assay of hymenialdisine derivatives (Table 2) uncovered a correspondence with the respective antiproliferative assay results, particularly with regard to the inhibition of CLK-1 and CDK-5 which are both known to play an important role in cell proliferation. It has been repeatedly shown that protein kinase inhibition may be a plausible mechanism underlying the antiproliferative activity of cytotoxic compounds (Bharate et al. 2012). Moreover, dispacamide E (1), the brominated aldisines (9– 11), (2 )-mukanadin C (12) and (2 )-longamide (13), which were inactive in the antiproliferative assay, revealed significant inhibitory activities against GSK-3, DYRK1A and CK-1 with IC50 values ranging between 0.6 and 6.4 mM which may inspire the search for new potent inhibitors of those kinases playing important roles in diabetes and neurodegenerative disorders, e.g. Alzheimer’s disease (Bharate et al. 2012). In addition, (2 )-hymenine (18) and hymenialdisine derivatives (20 – 23) exhibited potent inhibitory activity against PfGSK-3 with IC50 values in the nanomolar range which may strengthen their role as potential antimalarial candidates.
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Table 1. Antiproliferative (MTT) assay of isolated compounds against mouse lymphoma cell line. IC50
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Compounds tested Dispacamide E (1) Ethyl 3,4-dibromo-1H-pyrrole-2-carboxylate (2) 4-Bromopyrrole-3-carboxamide (3) 3,4-Dibromopyrrole-2-carboxamide (4) (2)-Longamide B (5) (2)-Longamide B methyl ester (6) (2)-Longamide B ethyl ester, Hanishin (7) Aldisine (8) 2,3-Dibromoaldisine (9) 2-Bromoaldisine (10) 3-Bromaldisine (11) (2)-Mukanadin C (12) (2)-Longamide (13) Latonduine A (14) (2)-Dibromophakellin (15) (2)-Monobromoisophakellin (16) (2)-Dibromocantharelline (17) (2)-Hymenine (18) Spongiacidin B (19) (10Z)-Debromohymenialdisine (20) (10Z)-Hymenialdisine (21) (10Z)-3-Bromohymenialdisine (22) (10E)-Hymenialdisine (23)a Latonduine B ethyl ester (24)a 3-Debromolatonduine A (25)a Stevensine (26)a 12-N-methyl stevensine (27)a Kahalalide F (positive control) a
L5178Y growth in % (@ 10 mg/mL) 77.2 27.2 100.0 75.8 100.0 70.0 14.1 100.0 98.8 100.0 100.0 100.0 100.0 18.6 1.0 73.7 35.3 16.0 0.0 0.0 0.0 0.0 nd 1.7 6.6 7.5 86.1
(mg/mL)
(mM)
9.3
24.47
10.0 11.0
26.81 28.28
2.40 1.55 2.70 3.90
7.41 6.33 8.33 9.68
3.5 6.30
8.75 4.30
Data previously published (Fouad et al. 2012).
According to the results of the antiproliferative (MTT) and protein kinase inhibitory activity assays, two different structural key elements can be clearly recognised, the pyrroloazepine moiety fused to aminoimidazolone is crucial for antiproliferative and protein kinase inhibitory activities including PfGSK-3. However, the sole existence of pyrroloazepine moiety diminished and/or abolished antiproliferative activity; whereas it retained the protein kinase inhibitory activities against kinases related to diabetes and neurodegenerative disorders. 3. Conclusions In summary, during the course of our chemical investigation of methanolic extracts of the Indonesian marine sponges S. massa and S. flabelliformis (Dictonellyidae), we identified and purified 25 bromopyrrole alkaloids including 2 new derivatives together with 2 debrominated congeners. All isolated compounds were assayed for their antiproliferative and protein kinase inhibitory activities. Some derivatives revealed potent and selective activities towards one or more kinases which may be proposed as potential candidates for development of new therapeutics for treatment of cancer, diabetes, Alzheimer’s disease and malaria.
10.4 10.6 7.5 6.5 1.7 13.4 2.0 2.3 1.6 0.5 1.9 3.2 2.8 0.3 0.2 0.03 0.01 0.01 0.04 ,0.03 . 10 2 0.36 4.1
Dispacamide E (1) 4-Bromo-1H-pyrrole-3-carboxamide (3) 3,4-Dibromo-1H-pyrrole-2-carboxamide (4) (2 )-Longamide B (5) (2 )-Longamide B ethyl ester, hanishin (7) Aldisine (8) 2,3-Dibromoaldisine (9) 2-Bromoaldisine (10) 3-Bromoaldisine (11) (2 )-Mukanadin C (12) (2 )-Longamide (13) Latonduine A (14) (2 )-Dibromophakellin (15) (2 )-Dibromocantharelline (17) (2 )-Hymenine (18) Spongiacidin B (19) (10Z)-Debromohymenialdisine (20) (10Z)-Hymenialdisine (21) (10Z)-3-Bromohymenialdisine (22) (10E)-Hymenialdisine (23) Latonduine B ethyl ester (24) 3-Debromolatonduine A (25) Stevensine (26) 12-N-methyl stevensine (27)
Note: nd – not determined.
CLK-1
Sample tested 16 . 53 . 37.3 . 28.4 . 26.3 . 61 . 31 . 41.2 27.6 2.4 13.2 24 3.9 0.6 0.4 0.09 0.09 0.26 0.07 0.12 . 10 . 10 0.78 6
CDK5
Table 2. Protein kinase inhibitory activity assay of isolated compounds.
2.1 . 53 .37.3 .28.4 .26.3 . 61 31 .41.2 21 2.4 7.4 18.8 3.1 0.8 0.3 0.04 0.13 0.29 0.04 0.072 . 10 0.21 0.39 2.2
GSK-3 6.2 12.7 14.9 15.6 . 26.3 50.6 11.2 . 41.2 10.3 0.6 7.1 6.2 3.6 0.3 0.2 0.04 0.02 0.01 0.07 , 0.03 . 10 1.7 0.31 3.2
DYRK1A 4.9 48 18.7 2.1 7.9 25.6 3.7 1.6 1.3 0.6 1.6 6.2 6.4 0.3 0.4 0.06 0.05 0.13 0.27 0.059 . 10 0.78 0.9 5.8
CK-1 nd nd nd nd nd nd nd . 10 nd nd . 10 . 10 nd 3 nd nd nd 0.079 nd 0.11 . 10 . 10 0.49 2.4
CDK1
IC50 (mM)
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nd nd nd nd nd nd nd .10 nd nd .10 .10 nd .10 nd nd nd 0.18 nd 0.27 .10 .10 2.5 .10
Cdk2/A nd nd nd nd nd nd nd .10 nd nd .10 .10 nd .10 nd nd nd 0.17 nd 0.38 .10 .10 2.2 1.3
Cdk9/cyclin T
18.8 . 53 . 37.3 25.6 . 26.3 . 61 . 31 . 41.2 15.6 1.3 16.8 18.8 5.4 0.6 0.4 0.04 0.16 0.2 0.07 nd nd nd nd nd
PfGSK-3
6 S.S. Ebada et al.
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Supplementary material Experimental details relating to this paper are available online. Acknowledgements We thank Prof. J. C. Braekman (ULB, Belgium) for giving us the sample of S. flabelliformis and Prof. Dr van Soest (University of Amsterdam, the Netherlands) for its identification.
Funding
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S.S.E. acknowledges the Egyptian Government (Ministry of High Education) for a doctoral scholarship and the University of Du¨sseldorf for a postdoctoral fellowship. P.P. acknowledges BMBF for financial support. We acknowledge the support of the European Union 7th Framework Program Knowledge-Based Bio-economy [grant number FP7-KBBE] – BlueGenics 2012 grant, including a postdoctoral fellowship.
References Albizati KF, Faulkner DJ. 1985. Stevensine, a novel alkaloid from an unidentified marine sponge. J Org Chem. 50:4163–4164. Assmann M, Ko¨ck M. 2002. Monobromoisophakellin, a new bromopyrrole alkaloid from the Caribbean sponge Agelas sp. Z Naturforsch. 57c:153–156. Bhandari MR, Sivappa R, Lovely CJ. 2009. Total synthesis of the putative structure of nagelamide D. Org Lett. 11:1535–1538. Bharate SB, Yadav RR, Battula S, Vishwakarma RV. 2012. Meridianins: marine-derived potent kinase inhibitors. MiniRev Med Chem. 12:618– 631. Cafieri F, Fattorusso E, Mangoni A, Taglialatela-Scafati O. 1995. Longamide and 3,7-dimethylisoguanine, two novel alkaloids from the marine sponge Agelaslongissima. Tetrahedron Lett. 36:7893–7896. Cafieri F, Fattorusso E, Mangoni A, Taglialatela-Scafati O. 1996. Dispacamides, anti-histamine alkaloids from Caribbean Agelas sponges. Tetrahedron Lett. 37:3587–3590. Cafieri F, Carnuccio R, Fattorusso E, Taglialatela-Scafati O, Vallefuoco T. 1997. Anti-histaminic activity of bromopyrrole alkaloids isolated from Caribbean Agelas sponges. Bioorg Med Chem Lett. 7:2283–2288. Cafieri F, Fattorusso E, Taglialatela-Scafati O. 1998. Novel bromopyrrole alkaloids from the sponge Agelasdispar. J Nat Prod. 61:122–125. Cimino G, de Rosa S, de Stefano S, Mazzarella L, Puliti R, Sodano G. 1982. Isolation and X-ray crystal structure of a novel bromo-compound from two marine sponges. Tetrahedron Lett. 23:767–768. De Nanteuil G, Ahond A, Guilhem J, Poupat C, Dau ETH, Potier P. 1985. Invertebresmarins du lagon neo-caledonien-V. Isolement et identification des metabolites d’une nouvelle espece de spongiaire, Pseudaxinyssacantharella. Tetrahedron. 41:6019–6033. Ebada SS, Proksch P. 2011a. Marine organisms and their perspective use in therapy of human diseases. In: Melhorn H, editor. Nature helps . . . parasitology research monograph. New York: Springer; p. 153 –189. Ebada SS, Proksch P. 2011b. Chemistry and biology of guanidine alkaloids from marine invertebrates. Mini Rev Med Chem. 11:225–246. Ebada SS, Proksch P. 2012. Chemistry of marine sponges-alkaloids, peptides, and terpenoids. In: Fattorusso E, Gerwick WH, Taglialatela-Scafati O, editors. Mar Nat Prod. New York: Springer; p. 191 –293. Forenza S, Minale L, Riccio R, Fattorusso E. 1971. New bromopyrrole derivatives from the sponge Agelasoroides. J Chem Soc, D Chem Commun. 1129–1130. Fouad MA, Debbab A, Wray V, Mu¨llerm WEG, Proksch P. 2012. New bioactive alkaloids from the marine sponge Stylissa sp. Tetrahedron. 68:10176 –10179. Handy ST, Sabatini JJ, Zhang Y, Vulfova I. 2004. Protection of poorly nucleophilic pyrroles. Tetrahedron Lett. 45:5057–5060. Hassan W, Elkhayat E, Edrada R, Ebel R, Proksch P. 2007. New bromopyrrole alkaloids from the marine sponge Axinelladamicornis and Stylissaflabelliformis. Nat Prod Commun. 2:1–6. Inaba K, Sato H, Tsuda M, Kobayashi J. 1998. Spongiacidins A–D, new bromopyrrole alkaloids from Hymeniacidon sponge. J Nat Prod. 61:693–695. Kobayashi J, Ohizumi Y, Nakamura H, Hirata Y, Wakamatsu K, Miyazawa T. 1986. Hymenine, a novel a-adrenoceptor blocking agent from the Okinawan marine sponge Hymeniacidon sp. Experientia. 42:1064 –1065. Li C-J, Schmitz FJ, Kelly-Borges M. 1998. A new lysine derivative and new 3-bromopyrrole carboxylic acid derivative from two marine sponges. J Nat Prod. 61:387–389.
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Linington RG, Williams DE, Tahir A, van Soest R, Anderson RJ. 2003. Latonduines A and B, new alkaloids isolated from the marine sponge Stylissacarteri: structure elucidation, synthesis, and biogenetic implications. Org Lett. 5:2735–2738. Mancini I, Guella G, Amade P, Roussakis C, Pietra F. 1997. Hanishin, a semiracemic bioactive C9 alkaloid of the Axinellid sponge Acanthellacarterifrom the Hanish Islands. A shunt metabolites? Tetrahedron Lett. 38:6271–6274. Mayer AM, Glaser KB, Cuevas C, Jacobs RS, Kem W, Little RD, McIntosh JM, Newman DJ, Potts BC, Shuster DE. 2010. The odyssey of marine pharmaceuticals: a current pipeline perspective. Trends Pharmacol Sci. 31:255–265. Mayer AM, Rodrı´guez AD, Taglialatela-Scafati O, Fusetani N. 2013. Marine pharmacology in 2009–2011: marine compounds with antibacterial, antidiabetic, antifungal, anti-inflammatory, antiprotozoal, antituberculosis, and antiviral activities; affecting the immune and nervous systems, and other miscellaneous mechanisms of action. Mar Drugs. 11:2510– 2573. Nikoulina SE, Ciaraldi TP, Mudaliar S, Mohideen P, Carter L, Henry RR. 2000. Potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes. Diabetes. 49:263–271. O’Malley DP, Li K, Maue M, Zografos AL, Baran PS. 2007. Total synthesis of dimericbromopyrrole-imidazole alkaloids: sceptrin, ageliferin, nagelamide E, oxysceptrin, nakamuric acid, and the axinellamine carbon skeleton. J Am Chem Soc. 129:4762–4775. Patel J, Pelloux-Le´on N, Minassian F, Valle´e Y. 2005. Syntheses of S-enantiomers of hanishin, longamide B, and longamide B methyl ester from L -asparitc acid b-methyl ester: establishment of absolute stereochemistry. J Org Chem. 70:9081–9084. Plisson F, Prasad P, Xiao X, Piggott AM, Huang X, Khalil Z, Capon RJ. 2014. Callyspongisines A–D: bromopyrrole alkaloids from an Australian marine sponge, Callyspongia sp. Org Biomol Chem. 12:1579 –1584. Scala F, Fattousso E, Menna M, Taglialatela-Scafati O, Tierney M, Kaiser M, Tasdemir D. 2010. Bromopyrrole alkaloids as lead compounds against protozoan parasites. Mar Drugs. 8:2162–2174. Schmitz FJ, Gunasekera SP, Lakshmi V, Tillekeratne LMV. 1985. Marine natural products: pyrrololactams from several sponges. J Nat Prod. 48:47–53. Sharma G, Magdoff-Fairchild B. 1977. Natural products of marine sponges.7. The constitution of weakly basic guanidine compounds, dibromophakellin and monobromophakellin. J Org Chem. 42:4118–4128. Sharma GM, Buyer JS, Pomerantz MW. 1980. Characterization of a yellow compound isolated from the marine sponge Phakelliaflabellata. J Chem Soc, Chem Commun. 435 –436. Supriyono A, Schwarz B, Wray V, Witte L, Mu¨ller WEG, van Soest RWM, Sumaryono W, Proksch P. 1995. Bioactivealkaloidsfromthetropical marine sponge Axinellacarteri. Z Naturforsch, 50c. :669–674. Uemoto H, Tsuda M, Kobayashi J. 1999. Mukanadin A-C, new bromopyrrole alkaloids from marine sponge Agelasnakamurai. J Nat Prod. 62:1581–1583. Xu Y-Z, Yakushijin K, Horne DA. 1997. Synthesis of C11N5 marine sponge alkaloids: (^)-hymenine, stevensine, hymenialdisine, and debromohymenialdisine. J Org Chem. 62:456–464.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Dispacamide E and other bioactive bromopyrrole alkaloids from two Indonesian marine sponges of the genus Stylissa ab
c
c
Sherif S. Ebada , Mai Hoang Linh , Arlette Longeon , Nicole J. d
e
f
de Voogd , Emilie Durieu , Laurent Meijer , Marie-Lise Bourguetc
b
g
Kondracki , Abdel Nasser B. Singab , Werner E.G. Müller & Peter Proksch
a
a
Institutfür Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine Universität, Geb. 26.23, Universitätsstrasse 1, D-40225 Düsseldorf, Germany b
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street 1, 11566 Cairo, Egypt c
Laboratoire Molécules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Museum National d'Histoire Naturelle, Paris, France d
Naturalis Biodiversity Center, PO Box 9517 2300, RA Leiden, Netherlands e
Centre National de la Recherche Scientifique, Protein Phosphorylation & Human Disease Group, Station Biologique, F-29680 Roscoff, France f
Man Ros Therapeutics, Centre de Perharidy, F-29680 Roscoff, France g
Institute for Physiological Chemistry, University of Medical Center of the Johannes-Gutenberg-University Mainz, Duesbergweg 6, D-55128 Mainz, Germany Published online: 12 Aug 2014.
To cite this article: Sherif S. Ebada, Mai Hoang Linh, Arlette Longeon, Nicole J. de Voogd, Emilie Durieu, Laurent Meijer, Marie-Lise Bourguet-Kondracki, Abdel Nasser B. Singab, Werner E.G. Müller & Peter Proksch (2014): Dispacamide E and other bioactive bromopyrrole alkaloids from two Indonesian marine sponges of the genus Stylissa, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.947496
To link to this article: http://dx.doi.org/10.1080/14786419.2014.947496
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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.947496
Dispacamide E and other bioactive bromopyrrole alkaloids from two Indonesian marine sponges of the genus Stylissa Sherif S. Ebadaab*, Mai Hoang Linhc, Arlette Longeonc, Nicole J. de Voogdd, Emilie Durieue, Laurent Meijerf, Marie-Lise Bourguet-Kondrackic, Abdel Nasser B. Singabb, Werner E.G. Mu¨llerg and Peter Prokscha*
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a
Institutfu¨r Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine Universita¨t, Geb. 26.23, Universita¨tsstrasse 1, D-40225 Du¨sseldorf, Germany; bDepartment of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street 1, 11566 Cairo, Egypt; cLaboratoire Mole´cules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Museum National d’Histoire Naturelle, Paris, France; dNaturalis Biodiversity Center, PO Box 9517 2300, RA Leiden, Netherlands; eCentre National de la Recherche Scientifique, Protein Phosphorylation & Human Disease Group, Station Biologique, F-29680 Roscoff, France; fMan Ros Therapeutics, Centre de Perharidy, F-29680 Roscoff, France; gInstitute for Physiological Chemistry, University of Medical Center of the Johannes-Gutenberg-University Mainz, Duesbergweg 6, D-55128 Mainz, Germany (Received 12 June 2014; final version received 19 July 2014) Chemical investigation of methanolic extracts of the two Indonesian marine sponges Stylissa massa and Stylissa flabelliformis yielded 25 bromopyrrole alkaloids including 2 new metabolites. The structures of all isolated compounds were unambiguously elucidated based on extensive 1D and 2D NMR, LR-MS and HR-MS analyses. All isolated compounds were assayed for their antiproliferative and protein kinase inhibitory activities. Several of the tested compounds revealed selective activity(ies) which suggested preliminary SARs of the isolated bromopyrrole alkaloids. Keywords: bromopyrrole alkaloids; antiproliferative; protein kinase; neurodegenerative disorders
1. Introduction Marine natural products can be considered as bioactive chemicals produced by marine organisms to deliver a survival advantage and protection of sessile organisms against their predators (in the case of invertebrates) or their herbivores (in the case of algae). Based on the knowledge of marine natural products chemistry, ecology and biology, researchers were inspired for developing several new pharmaceutical drugs for treatment of cancer or chronic pain (Mayer et al. 2010; Ebada & Proksch, 2012, 2011a, 2011b; Mayer et al. 2013). Bromopyrrole alkaloids constitute a well-known class of marine natural products found majorly in sponges genera belonging to the families Agelasidae, Axinellidae, Dictyonellidae and Halichondridae (Scala et al. 2010; Ebada & Proksch 2012). Oroidin is the parent compound of this class of marine metabolites which was first isolated from the marine sponge Agelasoroides. It is considered as the key precursor since many bromopyrrole alkaloids comprising a pyrrole – imidazole unit can be biosynthetically considered as metabolic derivatives of the C11N5 skeleton of oroidin (Forenza et al. 1971).
*Corresponding authors. Email: [email protected], [email protected] q 2014 Taylor & Francis
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Bromopyrrole alkaloids are also known as a prolific source of compounds with significant pharmacological activities including antiproliferative, antimicrobial and immunosuppressive activities which encouraged natural product chemists to study their total synthesis primarily during the last two decades. These efforts lead to successful total synthesis of many dimeric bromopyrrole –imidazole alkaloids such as sceptrin, oxsceptrin and ageliferin (O’Malley et al. 2007); nagelamides D (Bhandari et al. 2009) and E (O’Malley et al. 2007); and hymenialdisine analogues (Nikoulina et al. 2000). During our ongoing research for bioactive bromopyrrole alkaloids from marine sponges, we have chemically investigated methanolic extracts of two Indonesian marine sponges Stylissa massa and Stylissa flabelliformis implementing HPLC-DAD-MS analysis. In this study, we report the isolation and identification of 25 bromopyrrole alkaloids including 2 new metabolites together with 2 debrominated congeners in addition to their antiproliferative and protein kinase inhibitory activities. For measurement of protein kinase inhibitory activities, a panel of nine different kinases involved in cell proliferation, diabetes and neurodegenerative disorders (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A), CDC-like kinase 1 (CLK-1), casein kinase 1 (CK-1), cyclin-dependent kinase 5 (CDK5), glycogen synthase kinase-3 (GSK-3), cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2/A) and complex of cyclin-dependent kinase 9 and cyclin T (CDK9/cyclin T)) and malaria (PfGSK-3).
2. Results and discussion The methanolic extract of the marine sponge S. massa collected from Papua island (Indonesia) was subjected to HPLC-DAD-MS analysis which revealed a complex pattern of different molecular weights. Implementing different chromatographic separation methods, 20 bromopyrrole alkaloids including 2 new metabolites (1 and 2) together with 2 debrominated derivatives were purified and their structures were unambiguously elucidated based on extensive 1D and 2D NMR and HR-MS analyses and comparison with reported data in the literature. Known metabolites were identified as 4-bromopyrrole-3-carboxamide (3) (Hassan et al. 2007), 3,4-dibromopyrrole-2-carboxamide (4) (Hassan et al. 2007; Supriyono et al. 1995), (2 )-longamide B (5) (Cafieri et al. 1998; Patel et al. 2005), (2 )-longamide B methyl ester (6) (Patel et al. 2005), (2 )-longamide B ethyl ester (hanishin, 7) (Mancini et al. 1997), aldisine (8) (Schmitz et al. 1985), 2,3-dibromoaldisine (9) (Hassan et al. 2007), 2bromoaldisine (10) (Schmitz et al. 1985; Hassan et al. 2007), 3-bromoaldisine (11) (Hassan et al. 2007), (2 )-mukanadin C (12) (Li et al. 1998; Uemoto et al. 1999), (2 )-longamide (13) (Cafieri et al. 1995), latonduine A (14) (Linington et al. 2003), (2 )-dibromophakellin (15) (Sharma & Magdoff-Fairchild 1977), (2 )-monobromoisophakellin (16) (Assmann & Ko¨ck 2002), (2 )-dibromocantherelline (17) (De Nanteuil et al. 1985), (2 )-hymenine (18) (Kobayashi et al. 1986; Xu et al. 1997), spongiacidin B (19) (Inaba et al. 1998), (10Z)debromohymenialdisine (20) (Sharma et al. 1980; Cimino et al. 1982), (10Z)-hymenialdisine (21) (Cimino et al. 1982) and (10Z)-3-bromohymenialdisine (22) (Supriyono et al. 1995). Furthermore, the purification of the methanolic extract of the marine sponge S. flabelliformis collected from south Sulawei (Indonesia) by using different chromatographic separation methods yielded 11 known dibromopyrrole alkaloids: 3,4-dibromopyrrole-2-carboxamide (4), 2-bromoaldisine (10), (2 )-longamide (13), latonduine A (14), (2 )-dibromoisophakellin (17), (10Z)-hymenialdisine (21), (10E)-hymenialdisine (23) (Plisson et al. 2014), latonduine B ethyl ester (24) (Linington et al. 2003), 3-debromolatonduine A (25) (Fouad et al. 2012), stevensine (26) (Albizati & Faulkner 1985) and 12-N-methyl stevensine (27) (Fouad et al. 2012), furnishing five additional bromopyrrole alkaloids for SARs.
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Compound 1 was isolated as a faint yellow amorphous solid. It showed two pseudomolecular ion clusters at m/z 404, 406 and 408 [M þ H]þ; and at m/z 426, 428 and 430 [M þ Na]þ, each at a 1:2:1 ratio in its ESI mass spectrum indicating that it was a dibrominated compound. The 13C NMR spectrum showed 11 signals representing two methylene groups in addition to nine sp2 carbons including two methine groups and seven fully substituted carbons. The molecular þ formula of 1 was C11H79 11Br2N5O2 as indicated by HR-FT-MS (m/z 405.9320 [M þ H] calcd for C11H79 12Br2N5O2, D þ 1.0 ppm). The structure of 1 was further confirmed by interpretation of 1 H – 1H COSY and HMBC spectra (Figure 1). The 1H – 1H COSY spectrum showed a cross peak between NH-pyrrole (dH 12.69, br s) and the aromatic proton H-2 (dH 6.90, d, 1.3 Hz; dC 112.6) in addition to an extended spin system among NH-7 (dH 8.28, t, 6.0 Hz), ¼ CH2-8 (dH 3.47, m; dC 37.4), ¼ CH2-9 (dH 2.50, m; dC 27.3) and H-10 (dH 5.96, t, 7.6 Hz; dC 116.8). The HMBC spectrum (Figure 1) further supported the proposed structure and revealed correlations between H-2 to C-4 (dC 104.7) and C-5 (dC 128.0); H-10 to C-8 (dC 37.4), C-11 (dC 129.6) and C-15 (dC
O
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Figure 1. Key HMBC and ROESY correlations of compounds 1 and 2.
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163.6); ¼ CH2-8 to C-9 (dC 27.3), C-10 (dC 116.8) and C-6 (dC 158.9). According to the aforementioned data and by comparison with other dispacamide derivatives reported in the literature (Cafieri et al. 1997, 1996), compound 1 was confirmed to be N-((9E)-3-(2-amino-4oxo-1H-imidazol-5(4H)-ylidene) propyl)-3,4-dibromo-1H-pyrrole-2-carboxamide that was given the trivial name, dispacamide E. Compound 2 was isolated as white needle crystals. It showed pseudomolecular ion peaks at m/z 294, 296 and 298 [M 2 H]- at a 1:2:1 ratio in its ESI mass spectrum, indicating that it was a dibrominated compound with a daughter ion peak at m/z 268.1, indicating the loss of 29 amu that can represent an ethyl moiety. The molecular formula C7H79 7 Br2NO2 was indicated by HR-ESIBr NO , D þ 0.3 ppm). This was confirmed based MS (m/z 295.8916 [M þ H]þcalcd for C7H79 8 2 2 on results of 1D and 2D NMR. In this study, 2 was obtained for the first time as a naturally occurring bromopyrrole alkaloid. The 13C NMR spectrum showed 7 signals representing one methyl and one methylene group in addition to five sp2 carbons including one methine group and four fully substituted carbons. The structure of 2 was further confirmed by interpretation of 1 H – 1H COSY and HMBC spectra (Figure 1). The 1H – 1H COSY spectrum showed a cross peak between NH proton (dH 9.74, br s) and the aromatic proton H-5 (dH 6.89, d, 2.8 Hz; dC 117.8). The HMBC spectrum (Figure 1) supported the presence of the aromatic proton at C-5 and also revealed correlations between H-5 to C-2 (dC 124.1) and C-3 (dC 106.7). The presence of an ethyl ester group was deduced from 1H – 1H COSY that revealed a clear correlation between methylene group (dH 4.34, q, 7.3 Hz; dC 61.1) and methyl group (dH 1.36, t, 7.3 Hz; dC 14.3); and HMBC spectra showed a correlation of the methylene protons to the carbonyl group C-6 (dC 159.7). Moreover, the methylene and methyl protons showed a HMQC correlation to C-7 (dC 61.1) and C-8 (dC 14.3), respectively. Therefore, 2 was confirmed to be ethyl 3,4-dibromo-1Hpyrrole-2-carboxylate. This compound was previously only known as a synthetic product (Handy et al. 2004). All isolated bromopyrrole alkaloids from the methanolic extract of S. massa were subjected to a cell proliferation (MTT) assay against mouse lymphoma (L5178Y) cells. Some of the tested compounds (Table 1) were shown to possess significant antiproliferative activities with IC50 values ranging between 6.33 and 28.28 mM compared to kahalalide F (IC50 ¼ 4.30 mM). Among the hymenialdisine analogues, (10Z) debromohymenialdisine (20) was the most potent derivative with an IC50 value of 6.33 mM. Furthermore, all isolated bromopyrrole alkaloids were assayed for their protein kinase inhibitory activities against nine different protein kinases, namely DYRK1A, CDK5, GSK-3, CLK-1, CK-1, CDK1, CDK2/A, CDK9/cyclin T and Plasmodium falciparum glycogen synthase kinase-3 (PfGSK-3). Results of the protein kinase inhibitory activity assay of hymenialdisine derivatives (Table 2) uncovered a correspondence with the respective antiproliferative assay results, particularly with regard to the inhibition of CLK-1 and CDK-5 which are both known to play an important role in cell proliferation. It has been repeatedly shown that protein kinase inhibition may be a plausible mechanism underlying the antiproliferative activity of cytotoxic compounds (Bharate et al. 2012). Moreover, dispacamide E (1), the brominated aldisines (9– 11), (2 )-mukanadin C (12) and (2 )-longamide (13), which were inactive in the antiproliferative assay, revealed significant inhibitory activities against GSK-3, DYRK1A and CK-1 with IC50 values ranging between 0.6 and 6.4 mM which may inspire the search for new potent inhibitors of those kinases playing important roles in diabetes and neurodegenerative disorders, e.g. Alzheimer’s disease (Bharate et al. 2012). In addition, (2 )-hymenine (18) and hymenialdisine derivatives (20 – 23) exhibited potent inhibitory activity against PfGSK-3 with IC50 values in the nanomolar range which may strengthen their role as potential antimalarial candidates.
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Table 1. Antiproliferative (MTT) assay of isolated compounds against mouse lymphoma cell line. IC50
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Compounds tested Dispacamide E (1) Ethyl 3,4-dibromo-1H-pyrrole-2-carboxylate (2) 4-Bromopyrrole-3-carboxamide (3) 3,4-Dibromopyrrole-2-carboxamide (4) (2)-Longamide B (5) (2)-Longamide B methyl ester (6) (2)-Longamide B ethyl ester, Hanishin (7) Aldisine (8) 2,3-Dibromoaldisine (9) 2-Bromoaldisine (10) 3-Bromaldisine (11) (2)-Mukanadin C (12) (2)-Longamide (13) Latonduine A (14) (2)-Dibromophakellin (15) (2)-Monobromoisophakellin (16) (2)-Dibromocantharelline (17) (2)-Hymenine (18) Spongiacidin B (19) (10Z)-Debromohymenialdisine (20) (10Z)-Hymenialdisine (21) (10Z)-3-Bromohymenialdisine (22) (10E)-Hymenialdisine (23)a Latonduine B ethyl ester (24)a 3-Debromolatonduine A (25)a Stevensine (26)a 12-N-methyl stevensine (27)a Kahalalide F (positive control) a
L5178Y growth in % (@ 10 mg/mL) 77.2 27.2 100.0 75.8 100.0 70.0 14.1 100.0 98.8 100.0 100.0 100.0 100.0 18.6 1.0 73.7 35.3 16.0 0.0 0.0 0.0 0.0 nd 1.7 6.6 7.5 86.1
(mg/mL)
(mM)
9.3
24.47
10.0 11.0
26.81 28.28
2.40 1.55 2.70 3.90
7.41 6.33 8.33 9.68
3.5 6.30
8.75 4.30
Data previously published (Fouad et al. 2012).
According to the results of the antiproliferative (MTT) and protein kinase inhibitory activity assays, two different structural key elements can be clearly recognised, the pyrroloazepine moiety fused to aminoimidazolone is crucial for antiproliferative and protein kinase inhibitory activities including PfGSK-3. However, the sole existence of pyrroloazepine moiety diminished and/or abolished antiproliferative activity; whereas it retained the protein kinase inhibitory activities against kinases related to diabetes and neurodegenerative disorders. 3. Conclusions In summary, during the course of our chemical investigation of methanolic extracts of the Indonesian marine sponges S. massa and S. flabelliformis (Dictonellyidae), we identified and purified 25 bromopyrrole alkaloids including 2 new derivatives together with 2 debrominated congeners. All isolated compounds were assayed for their antiproliferative and protein kinase inhibitory activities. Some derivatives revealed potent and selective activities towards one or more kinases which may be proposed as potential candidates for development of new therapeutics for treatment of cancer, diabetes, Alzheimer’s disease and malaria.
10.4 10.6 7.5 6.5 1.7 13.4 2.0 2.3 1.6 0.5 1.9 3.2 2.8 0.3 0.2 0.03 0.01 0.01 0.04 ,0.03 . 10 2 0.36 4.1
Dispacamide E (1) 4-Bromo-1H-pyrrole-3-carboxamide (3) 3,4-Dibromo-1H-pyrrole-2-carboxamide (4) (2 )-Longamide B (5) (2 )-Longamide B ethyl ester, hanishin (7) Aldisine (8) 2,3-Dibromoaldisine (9) 2-Bromoaldisine (10) 3-Bromoaldisine (11) (2 )-Mukanadin C (12) (2 )-Longamide (13) Latonduine A (14) (2 )-Dibromophakellin (15) (2 )-Dibromocantharelline (17) (2 )-Hymenine (18) Spongiacidin B (19) (10Z)-Debromohymenialdisine (20) (10Z)-Hymenialdisine (21) (10Z)-3-Bromohymenialdisine (22) (10E)-Hymenialdisine (23) Latonduine B ethyl ester (24) 3-Debromolatonduine A (25) Stevensine (26) 12-N-methyl stevensine (27)
Note: nd – not determined.
CLK-1
Sample tested 16 . 53 . 37.3 . 28.4 . 26.3 . 61 . 31 . 41.2 27.6 2.4 13.2 24 3.9 0.6 0.4 0.09 0.09 0.26 0.07 0.12 . 10 . 10 0.78 6
CDK5
Table 2. Protein kinase inhibitory activity assay of isolated compounds.
2.1 . 53 .37.3 .28.4 .26.3 . 61 31 .41.2 21 2.4 7.4 18.8 3.1 0.8 0.3 0.04 0.13 0.29 0.04 0.072 . 10 0.21 0.39 2.2
GSK-3 6.2 12.7 14.9 15.6 . 26.3 50.6 11.2 . 41.2 10.3 0.6 7.1 6.2 3.6 0.3 0.2 0.04 0.02 0.01 0.07 , 0.03 . 10 1.7 0.31 3.2
DYRK1A 4.9 48 18.7 2.1 7.9 25.6 3.7 1.6 1.3 0.6 1.6 6.2 6.4 0.3 0.4 0.06 0.05 0.13 0.27 0.059 . 10 0.78 0.9 5.8
CK-1 nd nd nd nd nd nd nd . 10 nd nd . 10 . 10 nd 3 nd nd nd 0.079 nd 0.11 . 10 . 10 0.49 2.4
CDK1
IC50 (mM)
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nd nd nd nd nd nd nd .10 nd nd .10 .10 nd .10 nd nd nd 0.18 nd 0.27 .10 .10 2.5 .10
Cdk2/A nd nd nd nd nd nd nd .10 nd nd .10 .10 nd .10 nd nd nd 0.17 nd 0.38 .10 .10 2.2 1.3
Cdk9/cyclin T
18.8 . 53 . 37.3 25.6 . 26.3 . 61 . 31 . 41.2 15.6 1.3 16.8 18.8 5.4 0.6 0.4 0.04 0.16 0.2 0.07 nd nd nd nd nd
PfGSK-3
6 S.S. Ebada et al.
Natural Product Research
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Supplementary material Experimental details relating to this paper are available online. Acknowledgements We thank Prof. J. C. Braekman (ULB, Belgium) for giving us the sample of S. flabelliformis and Prof. Dr van Soest (University of Amsterdam, the Netherlands) for its identification.
Funding
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S.S.E. acknowledges the Egyptian Government (Ministry of High Education) for a doctoral scholarship and the University of Du¨sseldorf for a postdoctoral fellowship. P.P. acknowledges BMBF for financial support. We acknowledge the support of the European Union 7th Framework Program Knowledge-Based Bio-economy [grant number FP7-KBBE] – BlueGenics 2012 grant, including a postdoctoral fellowship.
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