SUPPLEMENTARY MATERIAL
Dispacamide E and Other Bioactive Bromopyrrole Alkaloids from
Two Indonesian Marine Sponges of the GenusStylissa
Sherif S. Ebadaa,f*,Mai Hoang Linhb,Arlette Longeonb,Nicole J. de
Voogdc,Emilie Durieud,Laurent Meijere,Marie-Lise BourguetKondrackib,Abdel Nasser B. Singabf,Werner E. G. Müllergand Peter
Prokscha*
a
Institut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine
Universität, Geb. 26.23, Universitätsstrasse 1, D-40225 Düsseldorf, Germany;
b
Laboratoire Molécules de Communication et Adaptation des Micro-organismes,
UMR 7245 CNRS, Museum National d’Histoire Naturelle, Paris, France; cNaturalis
Biodiversity Center, PO Box 9517 2300 RA Leiden, Netherlands; dCentre National de
la Recherche Scientifique, Protein Phosphorylation & Human Disease group. Station
Biologique, F-29680 Roscoff, France; eManRos Therapeutics, Centre de Perharidy,
F-29680 Roscoff, France; fDepartment of Pharmacognosy and Phytochemistry,
Faculty of Pharmacy, Ain-Shams University, Organization of African Unity Street 1,
11566 Cairo, Egypt; gInstitute for Physiological Chemistry, University of Medical
Center of the Johannes-Gutenberg-University Mainz, Duesbergweg 6, D-55128
Mainz, Germany
*Corresponding authors. Tel.: +49-211-81-14163; Fax: +49-211-81-11923.
Email addresses: sherif_elsayed@pharma.asu.edu.eg; proksch@uni-duesseldorf.de
S1
Dispacamide E and Other Bioactive Bromopyrrole Alkaloids from
Two Indonesian Marine Sponges of the GenusStylissa
Chemical investigation of methanolic extracts of the two Indondesian marine
spongesStylissa massaand Stylissa flabelliformisyielded twenty seven
bromopyrrole alkaloids including two 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
S2
Contents of Supplementary Materials
No.
Contents
Page
1
Detailed experimental procedures
S4
2
Table S1. HPLC, UV spectrum, LRESIMS and HRFTMS data of1
S9
3
Table S2.1H and 13C NMR data of compound 1
S10
4
Figure S1. 1H NMR spectrum of 1 in DMSO-d6
S10
5
Figure S2. 13C NMR spectrum of 1 in DMSO-d6
S10
6
Figure S3. 1H–1H COSY and HMBC spectra of 1 in DMSO-d6
S11
7
Table S3. HPLC, UV spectrum, LRESIMS and HRESIMS data of2
S12
8
Table S4.1H and 13C NMR data of compound 2
S13
9
Figure S4. 1H NMR spectrum of 2 in CDCl3
S13
10
Figure S5. 13C NMR spectrum of 2 in CDCl3
S13
11
Figure S6. DEPT spectrum of 2 in CDCl3
S13
12
Figure S7. 1H–1H COSY and HMBC spectra of 2 in CDCl3
S14
13
Table S5. Antiproliferative (MTT) assay of isolated compounds against
S15
mouse lymphoma cell line
14
Table S6. Protein kinase inhibitory activity assay of isolated compounds
S3
S16
Detailed experimental procedures
General experimental procedures
Stylissa massa
Optical rotation was recorded using a Perkin-Elmer-241 MC polarimeter
(Perkin-Elmer®, Waltham, MA, USA). ESI-MS were obtained on a
ThermoFinnigan LCQ DECA (ThermoFinnigan ®, Bremen, Germany) mass
spectrometer coupled to an Agilent 1100 HPLC system equipped with a
photodiode array detector. HR-FT-MS was recorded on a LTQ FT-MS-Orbitrap
(ThermoFinnigan®, Bremen, Germany). 1D and 2D NMR spectra were
recorded at 300ºK on either Bruker ARX-400 or Bruker ARX-500 NMR
(Bruker®, Billerica, MA, USA) spectrometer locked to the major deuterium
resonance of the solvent, CD3OD.
For analytical HPLC analysis, samples were injected into a HPLC system
equipped with a photodiode array detector (Dionex, Munich, Germany).
Routine detection was at 235, 254, 280, and 340 nm. The separation column
(125 × 4 mm ID) was prefilled with C-18 Eurosphere, 5 µm (Knauer, Berlin,
Germany). Separation was achieved by applying a linear gradient from 90%
H2O (pH 2.0) to 100% MeOH over 40 min. TLC analysis was carried out using
aluminium sheet precoated with silica gel 60 F254 (Merck, Darmstadt,
Germany).
Preparative HPLC separations were performed on a LaChrom-Merck Hitachi HPLC
system, pump L-7100, UV detector L-7400 using a C-18 column (Knauer, 300 × 8
mm ID, prefilled with C-18 Eurosphere, flow rate 5 mL/min, UV detection at 280
nm); the solvent system consisted of a linear gradient of MeOH and nanopure H2O.
Stylissa flabelliformis
Optical rotations were recorded on a Perkin-Elmer 341 polarimeter (Perkin-Elmer®,
USA). IR spectra were recorded on a FT-IR Shimadzu 8400 S spectrometer. UV
spectra were recorded on a UVIKON 930 (Kontron®, Germany) spectrometer. Mass
spectra were recorded on an API Q-STAR PULSAR I (Evisa®) of Applied Biosystem.
NMR Spectra were obtained on either a Bruker Avance 3HD 400 or 600 (USA)
spectrometer using standard pulse sequences. The acquisition of HMBC spectra were
optimized for either 7 or 8.3 Hz. Flash chromatographies were performed with a
S4
Buchi C615/601 pump system using silica gel cartridges (40-63 µm) (Buchi®,
Switzerland). TLC analysis was carried out using aluminum-backed sheets (Silica gel
60 F254, Merck, Darmstadt, Germany) and visualized under UV (254 nm) and
Lieberman spray reagent. Analytical and semi-preparative reverse-phase (Gemini C6phenyl, 5 µm, Phenomenex, USA, 250x4.6 ID, flow rate 1 mL/min and 250x10 ID,
flow rate 3 mL/min respectively) columns were performed with an Alliance apparatus
(model 2695, Waters, USA), equipped with a photodiode array detector (model 2998,
Waters), an evaporative light-scattering detector (model Sedex 80, Sedere, France),
and the software Empower.
Collection
The sponge identified as Stylissa massa was collected off the shores of Papua island
in 2008 during a scientific excursion to Indonesia. Identification of the sponge was
performed by Dr. N. J. de Voogd and a voucher specimen is kept in ethanol under the
registration number NdV012008 at Naturalis Biodiversity Center, Leiden,
Netherlands.
Specimens of Stylissa flabelliformis were collected off South Sulawesi (Spermonde
Archipelago off Ujung Pandang) in 1998 during a previous scientific excursion to
Indonesia. Identification of the sponge was performed by Dr. R. Van Soest and a
voucher specimen is kept in ethanol under the registration number
98/SS/MAY01/BH/039 at the Zoological Museum of the University of Amsterdam,
Netherlands.
Extraction and isolation
Methanolic extract of the sponge Stylissa massawas evaporated under reduced
pressure to yield 40 g residue. Then, the residue was partitioned between H2O
and EtOAc and afterwards between H2O and BuOH. The EtOAc soluble
fraction was concentrated and then subjected to vacuum liquid chromatography
(VLC) on a silica gel column employing a step gradient of n-hexane-EtOAc
and then CH2Cl2-MeOH yielding 22 fractions each of 750 ml. These fractions
were dried and examined by TLC on premade silica gel plates (Merk,
Darmstadt, Germany) using a dichloromethane-methanol based solvent system.
Further purification of the fractions obtained by preparative reversed phase
HPLC (C18 Eurosphere 100) using the appropriate gradient elution of
MeOH:H2O to afford 4 mg of compounds (1 and 16), 7 mg of compounds
S5
(2and 18), 5 mg of compounds (3, 11 and 15), 6 mg of compounds (4, 10 and
14), 9 mg of 5, 2 mg of compounds (6, 7 and 17), 17 mg of 8, 1 mg of 9, and 3
mg of compounds (12 and 13).
The n-BuOH soluble fraction was evaporated under reduced pressure and then
subjected to CC using Diaion HP20 stationary phase and a step gradient from H2O to
MeOH. Further purification was achieved via preparative reversed phase HPLC (C18
Eurosphere 100) using the appropriate gradient elution of MeOH:H2O yielding 4 mg
of 19, 24 mg of 20, 5 mg of 21, and 1 mg of 22.
The fresh sponge Stylissa flabelliformis was subsequently extracted by MeOH. The
crude extract, concentrated in vacuo, was then partitioned between H2O and CH2Cl2,
EtOAc and afterwards n-BuOH to obtain the CH2Cl2 (extract A, 4.2g), the EtOAc
(extract B, 2.16 g) and the n-BuOH extract (extract C, 5.43 g). Each extract was
subsequently subjected to flash Silica gel cartridges chromatography using a step
gradient of CH2Cl2-MeOH yielding 11 fractions.
The recrystallization of the fractions A-4 and B-4 in CH2Cl2:MeOH (2:8, V/V) gave
140 mg of 24. The fraction A-2 was finally purified by semi-preparative (Gemini C6Phenyl) HPLC with appropriate gradient elution of ACN:H2O:0.1% CH3COOH to
afford the compounds 25 (6.1 mg) and 26 (3.2 mg).
The fractions 5-6 of the extracts B and C were purified by recrystallizing in MeOH to
afford 810 mg of 27. Further purification of the fractions C-4 was achieved by semipreparative reversed phase HPLC (Gemini C6-Phenyl) using the appropriate gradient
elution of MeOH:H2O:0.1% CH3COOH to afford 28 (4.5 mg) and 23 (1.3 mg).
Dispacamide E (1)
Faint yellow amorphous solid. UV (MeOH) λmax 225, 280 nm. 1H and 13C NMR (500
MHz, DMSO-d6): 12.69 (1H, brs, NH-pyrrole), 9.38 (2H, s, NH2-16), 8.28 (1H, t, 6.0
Hz, NH-7), 6.90 (1H, d, 1.3 Hz, H-2), 5.96 (1H, t, 7.6 Hz, H-10), 3.47 (2H, m), and
2.50 (2H, m); 13C NMR: 163.6 (C-15), 158.9 (C-6), 156.0 (C-13), 129.6 (C-11), 128.0
(C-5), 116.8 (C-10), 112.6 (C-2), 104.7 (C-4), 97.8 (C-3), 37.4 (C-8), and 27.3 (C-9);
HRFTMS m/z 405.9320 [M+H]+ (calcd for C11H1279Br2N5O2, 405.9332).
Ethyl-3,4-dibromopyrrole-2-carboxylate (2)
White needles. UV (MeOH) λmax 235, 279 nm; NMR (500 MHz, CDCl3); 1H and 13C
NMR (500 MHz, CDCl3-d): 9.74 (1H, brs, NH-pyrrole), 6.89 (1H, d, 2.8 Hz, H-5),
4.34 (2H, q, 7.3 Hz, CH2-7), 1.36 (3H, t, 7.3 Hz, CH3-8); 13C NMR: 159.7 (C-6),
S6
124.1 (C-2), 117.8 (C-5), 106.7 (C-3), 100.6 (C-4), 61.1 (C-7), and 14.3 (C-8);
LRESIMS m/z 296.4 [M-H]-; HRESIMS m/z 295.8916 [M+H]+ (calcd for
C7H879Br2NO2, 295.8922).
Cell proliferation assay
Antiproliferative activity was tested in vitro against mouse lymphoma (L5178Y) cell
line using a microplate based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay and compared to that of untreated controls as previously
described (Ashour et al., 2006). All experiments were carried out in triplicate. The
depsipeptide kahalalide F was used as a positive control.
Assay for inhibition of protein kinases
An assay for inhibition of protein kinase was conducted in vitro as previously
described (Xie et al., 2005). In brief, homogenization buffer: 60 mM βglycerophosphate, 15 mM p-nitrophenylphosphate, 25 mM Mops (pH 7.2), 15 mM
EGTA, 15 mM MgCl2, 1 mM dithiothreitol, 1 mM sodium vanadate, 1mM NaF, 1
mM phenylphosphate, 10 µg leupeptin ml-1, 10 µg aprotinin ml-1, 10 µg soyabean
trypsin inhibitor ml-1, and 100 µg benzamidine. Buffer A: 10 mM MgCl2, 1mM
EGTA, 1 mM dithiothreitol, 25 mM Tris-HCl pH 7.5, 50 µg heparin ml-1. Buffer C:
homogenization buffer but 5 mM EGTA, no NaF and no protease inhibitors. Kinase
activities were assayed in duplicates in buffer A or C at 30°C, at a final ATP
concentration of 15 µM. The order of mixing the reagents was: buffers, substrate,
enzyme, and inhibitor. Isolated compounds were tested against a panel of six kinases;
namely, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A
(DYRK1A), cylcin-dependent kinase (CDK5), glycogen synthase kinase-3 (GSK-3),
CDC-like kinase 1 (CLK-1), casein kinase 1 (CK1), cyclin-dependent kinase 1
(CDK1), cyclin-dependent kinase 2 (CDK2/A), complex of cyclin-dependent kinase 9
and cyclin T (CDK9/cyclin T) and Plasmodium falciparum glycogen synthase kinase3 (PfGSK-3).
S7
References
Ashour, M., Edrada-Ebel, R.,Ebel, R., Wray, V., Wätjen, W., Padmakumar, K.,
Müller, W.E.G., Lin,W.H.,& Proksch, P.(2006). Kahalalide derivatives from the
Indian sacoglossan mollusk Elysia grandifolia. Journalof NaturalProducts, 69,
1547-1553.
Xie, X., Lemcke, T., Gussio, R., Zaharevitz, D.W., Leost, M., Meijer, L., & Kunick,
C. (2005). Epoxide-containing side chains enhance antiproliferaitve activity of
paullones. European Journal of Medicinal Chemistry, 40, 655-661.
S8
Table S1. HPLC, UV spectrum, LRESIMS and HRFTMS data of1
Compound 1 (Dispacamide E)
Biological source
Stylissa massa
Sample amount
4.0 mg
Physical description
White amorphous solid
Molecular formula
C11H1179Br2N5O2
Molecular weight
405 g/mol
Retention time (HPLC)
14.11 min (half-time gradient)
200 SE090514 #5
mAU
NdV VLC-16B, IV
UV_VIS_1
WVL:235 nm
70,0
Peak #1 100%
%
1 - 14,114
PC 3.3.6.8.4.A 982.86
279.7
100
224.9
559.6
-40
0,0
min
5,0
10,0
[M+Na]+
15,0
20,0
25,0
30,0
-10,0
200
nm
250
300
350
400
450
[M+H]+
[2M+Na]+
[M+H]+
[M-H]-
S9
500
550
595
Table S2.1H and 13C NMR data of compound 1
#
Ha (mult., J in Hz)
1
12.69 (1H, br s, NH-pyrrole)
2
6.90 (1H, d, 1.3 Hz)
Cb,c (mult.)
#
Ha (mult., J in Hz)
Cb,c (mult.)
9
2.50 (2H, m)
27.3
112.6
10
5.96 (1H, t, 7.6 Hz)
116.8
3
97.8
11
4
104.7
12
5
128.0
13
6
158.9
14
7
8.28 (1H, t, 6.0 Hz, NH)
8
3.47 (2H, m)
a
129.6
156.0
15
37.4
16
163.6
9.38 (2H, s, NH2-16)
DMSO-d6, 500 MHz, bDMSO-d6, 125 MHz, cAssignments are based on extensive 1D
and 2D NMR measurements (1H–1H COSY and HMBC).
Figure S1. 1H NMR spectrum of 1 in DMSO-d6
Figure S2. 13C NMR spectrum of 1 in DMSO-d6
S10
Figure S3. 1H–1H COSY and HMBC spectra of 1 in DMSO-d6
S11
Table S3. HPLC, UV spectrum, and LRESIMS data of2
Compound 2 (Ethyl 3,4-dibromo-1H-pyrrole-2-carboxylate)
Biological source
Stylissa massa
Sample amount
7.0 mg
Physical description
White needle crystals
Molecular formula
C7H779Br2NO2
Molecular weight
297 g/mol
Retention time (HPLC)
28.24 min (standard gradient)
450 SE090409 #10
mAU
NdV VLC-3, 2
UV_VIS_1
WVL:240 nm
Peak #1 100%
70,0
%
4,5-dibromopyrrole-2-carboxylic acid 980.00
1 - 28,240
278.7
200
234.7
559.1
2 - 48,082
-50
0,0
min
10,0
20,0
30,0
40,0
50,0
60,0
-10,0
200
[M-H]-
nm
250
300
350
400
450
500
550
[M-H][M-29]-
S12
595
Table S4.1H and 13C NMR data of compound 2
#
Ha (mult., J in Hz)
1
9.74 (1H, br s, NH-pyrrole)
Cb,c (mult.)
2
124.1, C
3
106.7, C
4
100.6, C
5
6.89 (1H, d, 2.8 Hz)
117.8, CH
7
4.34 (2H, q, 7.3 Hz)
159.7, C
61.1, CH2
8
1.36 (3H, t, 7.3 Hz)
14.3, CH3
6
a
CDCl3, 500 MHz, bCDCl3, 125 MHz, cAssignments are based on extensive 1D and
2D NMR measurements (1H–1H COSY and HMBC).
Figure S4. 1H NMR spectrum of 2 in CDCl3
Figure S5. 13C NMR spectrum of 2 in CDCl3
Figure S6. DEPT spectrum of 2 in CDCl3
S13
Figure S7. 1H–1H COSY and HMBC spectra of 2 in CDCl3
S14
Table S5. Antiproliferative (MTT) assay of isolated compounds against mouse
lymphoma cell line
Compound Tested
Dispacamide E (1)
Ethyl 3,4-dibromo-1H-pyrrole-2-carboxylate (2)
4-Bromopyrrole-3-carboxamide (3)
3,4-Dibromopyrrole-2-carboxamide (4)
(-)-Longamide B (5)
(-)-Longamide B methyl ester (6)
(-)-Longamide B ethyl ester, Hanishin (7)
Aldisine (8)
2,3-Dibromoaldisine (9)
2-Bromoaldisine (10)
3-Bromaldisine (11)
(-)-Mukanadin C (12)
(-)-Longamide (13)
Latonduine A (14)
(-)-Dibromophakellin (15)
(-)-Monobromoisophakellin (16)
(-)-Dibromocantharelline (17)
(-)-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)
Kahalalide F (positive control)
L5178Y growth in %
(@ 10 µg/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
S15
IC50
(µg/mL)
(µM)
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
Table S6. Protein kinase inhibitory activity assay of isolated compounds
Sample tested
Dispacamide E (1)
4-Bromo-1H-pyrrole-3-carboxamide (3)
3,4-Dibromo-1H-pyrrole-2-carboxamide (4)
(-)-Longamide B (5)
(-)-Longamide B ethyl ester, hanishin (7)
Aldisine (8)
2,3-Dibromoaldisine (9)
2-Bromoaldisine (10)
3-Bromoaldisine (11)
(-)-Mukanadin C (12)
(-)-Longamide (13)
Latonduine A (14)
(-)-Dibromophakellin (15)
(-)-Dibromocantharelline (17)
(-)-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)
CLK1
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
CDK5
GSK-3
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
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
S16
DYRK1
A
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
IC50 (µM)
CK-1 CDK
1
4.9
nd
48
nd
18.7
nd
2.1
nd
7.9
nd
25.6
nd
3.7
nd
1.6
>10
1.3
nd
0.6
nd
1.6
>10
6.2
>10
6.4
nd
0.3
3
0.4
nd
0.06
nd
0.05
nd
0.13
0.079
0.27
nd
0.059
0.11
>10
>10
0.78
>10
0.9
0.49
5.8
2.4
Cdk2/
A
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
Cdk9/
cyclin T
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
PfGSK-3
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