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 Bourguet-

Kondrackib,Abdel Nasser B. Singabf,Werner E. G. Müllergand Peter

Prokscha*

aInstitut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine

Universität, Geb. 26.23, Universitätsstrasse 1, D-40225 Düsseldorf, Germany; bLaboratoire 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

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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

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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 mouse lymphoma cell line

S15

14 Table S6. Protein kinase inhibitory activity assay of isolated compounds

S16

S3

Detailed experimental procedures

General experimental proceduresStylissa 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 C6-

phenyl, 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.

CollectionThe 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 isolationMethanolic 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 C6-

Phenyl) 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 semi-

preparative 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 assayAntiproliferative 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 kinasesAn assay for inhibition of protein kinase was conducted in vitro as previously

described (Xie et al., 2005). In brief, homogenization buffer: 60 mM β-

glycerophosphate, 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 kinase-

3 (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.

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Peak #1 100%

-10,0200 250 300 350 400 450 500 550 595

%

nm

279.7

224.9

559.6

PC 3.3.6.8.4.A 982.86

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)

0,0 5,0 10,0 15,0 20,0 25,0 30,0-40

100

200 SE090514 #5 NdV VLC-16B, IV UV_VIS_1mAU

min

1 - 14,114

WVL:235 nm

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70,0

[M-H]-

[M+H]+

[M+Na]+ [2M+Na]+ [M+H]+

Table S2.1H and 13C NMR data of compound 1

# Ha (mult., J in Hz)Cb,c

(mult.)# Ha (mult., J in Hz)

Cb,c

(mult.)

1 12.69 (1H, br s, NH-pyrrole) 9 2.50 (2H, m) 27.3

2 6.90 (1H, d, 1.3 Hz) 112.6 10 5.96 (1H, t, 7.6 Hz) 116.8

3 97.8 11 129.6

4 104.7 12

5 128.0 13 156.0

6 158.9 14

7 8.28 (1H, t, 6.0 Hz, NH) 15 163.6

8 3.47 (2H, m) 37.4 16 9.38 (2H, s, NH2-16)

aDMSO-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

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Figure S3. 1H–1H COSY and HMBC spectra of 1 in DMSO-d6

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Peak #1 100%

-10,0

70,0

200 250 300 350 400 450 500 550 595

%

nm

278.7

234.7

559.1

4,5-dibromopyrrole-2-carboxylic acid 980.00

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)

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

200

450 SE090409 #10 NdV VLC-3, 2 UV_VIS_1mAU

min

1 - 28,240

2 - 48,082

WVL:240 nm

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[M-H]-[M-H]-

[M-29]-

Table S4.1H and 13C NMR data of compound 2# Ha (mult., J in Hz) Cb,c (mult.)1 9.74 (1H, br s, NH-pyrrole)

2 124.1, C 3 106.7, C 4 100.6, C 5 6.89 (1H, d, 2.8 Hz) 117.8, CH 6 159.7, C 7 4.34 (2H, q, 7.3 Hz) 61.1, CH2 8 1.36 (3H, t, 7.3 Hz) 14.3, CH3

aCDCl3, 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

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Figure S7. 1H–1H COSY and HMBC spectra of 2 in CDCl3

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Table S5. Antiproliferative (MTT) assay of isolated compounds against mouse lymphoma cell line

Compound Tested L5178Y growth in %(@ 10 µg/mL)

IC50

(µg/mL) (µM)Dispacamide E (1) 77.2Ethyl 3,4-dibromo-1H-pyrrole-2-carboxylate (2) 27.24-Bromopyrrole-3-carboxamide (3) 100.03,4-Dibromopyrrole-2-carboxamide (4) 75.8(-)-Longamide B (5) 100.0(-)-Longamide B methyl ester (6) 70.0(-)-Longamide B ethyl ester, Hanishin (7) 14.1 9.3 24.47Aldisine (8) 100.02,3-Dibromoaldisine (9) 98.82-Bromoaldisine (10) 100.03-Bromaldisine (11) 100.0(-)-Mukanadin C (12) 100.0(-)-Longamide (13) 100.0Latonduine A (14) 18.6 10.0 26.81(-)-Dibromophakellin (15) 1.0 11.0 28.28(-)-Monobromoisophakellin (16) 73.7(-)-Dibromocantharelline (17) 35.3(-)-Hymenine (18) 16.0Spongiacidin B (19) 0.0 2.40 7.41(10Z)-Debromohymenialdisine (20) 0.0 1.55 6.33(10Z)-Hymenialdisine (21) 0.0 2.70 8.33(10Z)-3-Bromohymenialdisine (22) 0.0 3.90 9.68(10E)-Hymenialdisine (23) ndLatonduine B ethyl ester (24) 1.73-Debromolatonduine A (25) 6.6Stevensine (26) 7.512-N-methyl stevensine (27) 86.1 3.5 8.75Kahalalide F (positive control) 6.30 4.30

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Table S6. Protein kinase inhibitory activity assay of isolated compoundsSample tested IC50 (µM)

CLK-1

CDK5

GSK-3

DYRK1A

CK-1

CDK1

Cdk2/A

Cdk9/cyclin

T

PfGSK-

3Dispacamide E (1) 10.

416 2.1 6.2 4.9 nd nd nd 18.8

4-Bromo-1H-pyrrole-3-carboxamide (3)

10.6

>53 >53 12.7 48 nd nd nd >53

3,4-Dibromo-1H-pyrrole-2-carboxamide (4)

7.5 >37.3

>37.3

14.9 18.7

nd nd nd >37.3

(-)-Longamide B (5) 6.5 >28.4

>28.4

15.6 2.1 nd nd nd 25.6

(-)-Longamide B ethyl ester, hanishin (7)

1.7 >26.3

>26.3

>26.3 7.9 nd nd nd >26.3

Aldisine (8) 13.4

>61 >61 50.6 25.6

nd nd nd >61

2,3-Dibromoaldisine (9) 2.0 >31 31 11.2 3.7 nd nd nd >312-Bromoaldisine (10) 2.3 >41.

2>41.

2>41.2 1.6 >10 >10 >10 >41.2

3-Bromoaldisine (11) 1.6 27.6 21 10.3 1.3 nd nd nd 15.6(-)-Mukanadin C (12) 0.5 2.4 2.4 0.6 0.6 nd nd nd 1.3(-)-Longamide (13) 1.9 13.2 7.4 7.1 1.6 >10 >10 >10 16.8Latonduine A (14) 3.2 24 18.8 6.2 6.2 >10 >10 >10 18.8(-)-Dibromophakellin (15) 2.8 3.9 3.1 3.6 6.4 nd nd nd 5.4(-)-Dibromocantharelline (17) 0.3 0.6 0.8 0.3 0.3 3 >10 >10 0.6(-)-Hymenine (18) 0.2 0.4 0.3 0.2 0.4 nd nd nd 0.4Spongiacidin B (19) 0.0

30.09 0.04 0.04 0.0

6nd nd nd 0.04

(10Z)-Debromohymenialdisine (20)

0.01

0.09 0.13 0.02 0.05

nd nd nd 0.16

(10Z)-Hymenialdisine (21) 0.01

0.26 0.29 0.01 0.13

0.079

0.18 0.17 0.2

(10Z)-3-Bromohymenialdisine (22)

0.04

0.07 0.04 0.07 0.27

nd nd nd 0.07

(10E)-Hymenialdisine (23) <0.03

0.12

0.072

<0.03

0.059

0.11

0.27

0.38 nd

Latonduine B ethyl ester (24)

>10

>10 >10 >10 >10

>10

>10 >10 nd

3-Debromolatonduine A (25)

2 >10 0.21

1.7 0.78

>10

>10 >10 nd

Stevensine (26) 0.36

0.78

0.39

0.31 0.9 0.49

2.5 2.2 nd

12-N-methyl stevensine (27)

4.1 6 2.2 3.2 5.8 2.4 >10 1.3 nd

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