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Supplementary Information

Steroids from the Brazilian Zoanthids Palythoa caribaeorum

and Palythoa variabilis

Francisco C. L. Pinto,a José Gustavo L. Almeida,

a Edilberto R. Silveira,

a Arinice M. Costa,

b

Larissa A. Guimarães,b Diego V. Wilke,

b Letícia V. Costa-Lotufo,

c Maria da Conceição M.

Torresd and Otília Deusdênia L. Pessoa*

,a

aDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60021-970

Fortaleza-CE, Brazil

bDepartamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, 60430-270

Fortaleza-CE, Brazil

cDepartamento de Farmacologia, Universidade de São Paulo, 05508-900 São Paulo-SP, Brazil

dDepartamento de Química, Universidade Estadual da Paraíba, 58429-500

Campina Grande-PB, Brazil

Experimental details of the isolation of the known compounds

The P. caribaeorum n-hexane extract (21.0 g) was initially fractionated over silica gel CC

(108 g) using as the elution solvent n-hexane, n-hexane/EtOAc 4:1, 3:2, 2:3, 1:4, EtOAc and, finally,

MeOH. The n-hexane/EtOAc 4:1 fraction (3.6 g), a white amorphous precipitate, was purified by addition

of MeOH followed by filtration to yield campesterol1 (3.0 g). The n-hexane/EtOAc 3:2 fraction (0.9 g)

was subjected to silica gel CC by elution with n-hexane/EtOAc gradient (7:3 → 0:10) and then with

MeOH to give 6 fractions (F1-F6) after TLC analysis. F2 (230.8 mg), obtained by elution with

n-hexane/EtOAc 4:1, was rechromatographed over silica gel using an isocratic mixture of CH2Cl2/EtOAc

7:3 to yield 32 subfractions of 8 mL each, from which F2(4-5), a white amorphous powder, was identified

as 5α,8α-epi-dioxycampesterol2 (15.9 mg). The n-hexane/EtOAc 2:3 fraction (1.1 g) was subjected to

silica gel CC by elution with n-hexane/EtOAc gradient (5:5 → 0:10) followed by MeOH to afford 3 main

fractions (F1-F3), after TLC analysis. F2 (676.9 mg), obtained by elution with n-hexane/EtOAc 4:6, was

subjected to silica gel CC using n-hexane/EtOAc gradient (8:2 → 0:10) followed by MeOH to yield

6 subfractions [F2(1) to F2(6)] after TLC analysis. F2(3), a white amorphous powder, was identified as

7α-hydroxycampesterol3 3 (39.1 mg).

_____________________________ *e-mail: otilialoiola@gmail.com

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The P. caribaeorum EtOH extract (61.7 g), suspended in MeOH/H2O 7:3 (250 mL) was

partitioned with CH2Cl2 and then with EtOAc (3 × 250 mL of each solvent). Both CH2Cl2 and EtOAc

fractions were evaporated under reduced pressure to yield 11.6 and 3.6 g, respectively. The CH2Cl2

fraction (11.6 g) was subjected to successive silica gel CC eluted with a gradient of n-hexane/EtOAc

(3:7 0:10) to yield a white amorphous powder identified as 24(R)-ergost-7-en-3β,5α,6β-triol4

(29.9 mg).

The P. variabilis n-hexane extract (19.8 g) was fractionated over a silica gel CC by elution with

n-hexane, n-hexane/CH2Cl2 1:1, CH2Cl2, CH2Cl2/EtOAc 1:1, EtOAc, and, finally, MeOH. The

n-hexane/CH2Cl2 1:1 and CH2Cl2 fractions were combined (9.4 g) and subjected to successive silica gel

CC eluted with n-hexane/EtOAc gradients to yield campesterol1 (6.3 g). The CH2Cl2/EtOAc 1:1 fraction

(4.3 g) was subjected to silica gel CC using a gradient of n-hexane/EtOAc (7:3 → 0:10) followed by

MeOH to yield 6 sub-fractions (F1-F6), after TLC analysis. F2 (723.6) afforded a mixture of

1-hexadecylglicerol and 7α-hydroxycampesterol3 3 (57.4 mg) after washing with n-hexane/EtOAc 1:1. F3

(537.0 mg) was subjected to silica gel CC by elution with n-hexane, CH2Cl2, and finally EtOAc to give

5α,8α-epi-dioxycampesterol2 (21.3 mg) after elution with n-hexane/EtOAc 4:6.

GC-MS analysis of the n-hexane fractions from P. caribaeorum and P. variabilis

GC-MS analysis was carried out on a Shimadzu GCMS-QP2010-Plus spectrometer using a

capillary column RTx-5 (30 m × 0.25 mm i.d., 0.25 µm film thickness), He as carrier gas, flow rate of

1.7 mL min-1

and split mode (ratio 1:48). Both injector and detector temperatures were 250 and 280 °C,

respectively. The column temperature was programmed from 40 to 180 °C for 48 min and then from 180

to 250 °C for 20 min, and held isothermally for 7 min.

An aliquot of each sample (200 mg) was submitted to esterification reaction with KOH/EtOH

2N. The unsaponifiable material was extracted with CH2Cl2 and further subjected to CC over silica gel

eluting with n-hexane/EtOAc (9:1 and 7:3), and then with pure EtOAc. The n-hexane/EtOAc 7:3 fraction

rich in palysterols, was subjected to GC-MS analysis (Table S2 and Figures S1-S20).

References

1. Zhang, X.; Cambrail, A.; Miesch, M.; Roussi, S.; Raul, F.; Marchioni, E.; J. Agric. Food Chem.

2006, 54, 1196.

2. Ioannoua, E.; Abdel-Razika, A. F.; Zervouc, M.; Christfidisa, D.; Alexi, X.; Vagiasa, C.; Alxis,

M. N.; Roussis, V.; Steroids 2009, 74, 73.

3. Kobayashi, M.; Krishna M. M.; Haribabub, B. B.; Anjaneyulu, V.; Chem. Pharm. Bull. 1993, 41,

87.

4. Madaio, A.; Piccialli, V.; Sica, D.; J. Nat. Prod. 1989, 52, 952.

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Figure S1. Chemical structures of the steroids isolated from P. caribaeorum and P. variabilis.

Figure S2. 1H NMR spectrum (500 MHz, CDCl3) of compound 1.

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Figure S3. 13

C and DEPT 135° NMR spectra (125 MHz, CDCl3) of compound 1.

Figure S4. gCOSY spectrum of compound 1.

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Figure S5. HSQC spectrum of compound 1.

Figure S6. HMBC spectrum of compound 1.

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Figure S7. NOESY spectrum of compound 1.

Figure S8. HRESIMS spectrum of compound 1.

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Figure S9. FTIR (KBr) spectrum of compound 1.

Figure S10. 13

C NMR spectrum (125 MHz, CDCl3) between compounds 1 and 3.

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Figure S11. 1H NMR spectrum (500 MHz, CDCl3) of compound 2.

Figure S12. 13

C and DEPT 135° NMR spectra (125 MHz, CDCl3) of compound 2.

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Figure S13. gCOSY spectrum of compound 2.

Figure S14. HSQC spectrum of compound 2.

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Figure S15. HMBC spectrum of compound 2.

Figure S16. NOESY spectrum of compound 2.

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Figure S17. HRESIMS spectrum of compound 2.

Figure S18. FTIR (KBr) spectrum of compound 2.

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Figure S19. HPLC analyses of fraction AcOEt from P. caribaeorum and P. variabilis.

Figure S20. GC analyses of steroids from P. caribaeorum and P. variabilis.

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Table S1. 13

C NMR data comparison of 24(R)-7α-hydroperoxy-ergost-5-en-3β-ol (1) with

7α-hydroperoxycholesterol (1a) and 7β-hydroperoxycholesterol (1b), and 6β-carboxyl-24(R)-(8→6)-

abeo-ergostan-3β,5β-diol (2) with B-nor-ergostan-3β,5β-diol-6β-carboxyl acid (2a)

δC (1) δC (1a) ∆δC δC (1b) ∆δC δC (2) δC (2a) ∆δC

1 37.0 37.1 0.1 37.8 0.2 28.5 28.6 0.1

2 31.4 31.3 0.1 31.5 0.1 28.2 28.2 0.0

3 71.5 71.4 0.1 71.3 0.2 67.3 67.4 0.1

4 42.3 42.2 0.1 42.8 0.5 43.7 43.8 0.1

5 148.8 148.9 0.1 140.0 8.8 82.9 82.9 0.0

6 120.3 119.9 0.4 121.6 1.3 58.7 58.4 0.3

7 78.6 78.5 0.1 86.5 7.9 178.2 178.0 0.2

8 37.3 37.4 0.1 34.5 2.8 42.7 42.8 0.1

9 43.7 43.5 0.2 56.0 12.3 51.4 51.4 0.0

10 37.6 37.7 0.1 36.4 1.2 45.5 45.5 0.0

11 21.1 20.9 0.2 21.3 0.2 21.6 21.7 0.1

12 39.2 39.0 0.2 39.5 0.3 39.8 39.8 0.0

13 42.5 42.3 0.2 42.8 0.3 44.8 44.8 0.0

14 49.2 49.0 0.2 48.7 0.5 56.4 56.6 0.2

15 24.6 24.4 0.2 25.9 1.3 24.2 24.3 0.1

16 28.4 28.3 0.1 28.3 0.1 28.4 28.4 0.0

17 55.9 55.7 0.2 55.6 0.3 55.6 55.9 0.3

18 11.5 11.3 0.2 11.9 0.4 12.7 12.7 0.0

19 18.4 18.2 0.2 18.8 0.4 19.3 18.9 0.4

20 36.3 35.8 0.5 35.8 0.5 36.2 36.4 0.2

21 19.1 18.8 0.3 18.8 0.3 17.7 17.7 0.0

22 33.9 36.1 2.2 36.2 2.3 33.9 35.8 1.9

23 30.6 23.7 6.9 23.9 6.7 30.7 24.9 5.8

24 39.2 39.5 0.3 39.6 0.4 39.2 39.7 0.5

25 31.7 28.0 3.7 28.0 3.7 31.6 24.0 7.6

26 20.7 22.8 2.1 22.8 2.1 20.7 22.7 2.0

27 17.8 22.6 4.8 22.5 4.7 17.7 23.0 5.3

28 15.6 a

a

15.6 a

aMe-28 does not exist in cholestane sterols.

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Table S2. GC-MS data of identified sterols in the hexane extracts of P. caribaeorum and P. variabilis

Compound Major fragment ions (m/z) PCP / % PVP / %

7-Hydroxycampesterol (5)

C28H48O2

380 [M-2H2O]+, 365 [M-CH3]

+

261 [M-side chain-ring B cleavage]+

1.2 1.1

Cholesta-5,22-dien-3-ol

C27H44O

384 [M]+, 369 [M-CH3]

+

271 [M-side chain-2H]+

255 [M-side chain-H2O]+

213 [M-side chain-ring D cleavage-H2O]+

0.5 0.5

Cholesterol

C27H46O

386 [M]+, 371 [M-CH3]

+, 368 [M-H2O]

+

353 [M-CH3-H2O]+, 301 [M-C6H13]

+

255 [M-side chain-H2O]+

213 [M-side chain-ring D cleavage-H2O]+

6.1 5.4

Crinosterol

C28H46O

398 [M]+, 383 [M-CH3]

+, 365 [M-CH3-H2O]

+

271 [M-side chain-2H]+

255 [M-side chain-H2O]+

213 [M-side chain-ring D cleavage-H2O]+

2.2 1.9

Campesterol (3)

C28H48O

400 [M]+, 385 [M-CH3]

+, 382 [M-H2O]

+

367 [M-CH3-H2O]+, 315 [M-C5H11]

+

273 [M-side chain]+

255 [M-side chain-H2O]+

213 [M-side chain-ring D cleavage-H2O]+

69.0 68.7

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Table S2. GC-MS data of identified sterols in the hexane extracts of P. caribaeorum and P. variabilis

(cont.)

Compound Major fragment ions (m/z) PCP / % PVP / %

Cholesta-3,5-dien-7-one

C28H44O

396 [M]+, 391 [M-CH3]

+, 269 [M-side chain]

+

227 [M-side chain-ring D cleavage-]+

174 [M-side chain-ring B cleavage]+

0.6 0.7

Cholest-4-en-3-one

C28H46O

398 [M]+, 383 [M-CH3]

+, 356 [M-CO2]

+

229 [M-side chain-ring D cleavage-]+

124 [M-side chain-ring B cleavage]+

1.1 0.9

Gorgosterol

C30H50O

426 [M]+, 311 [M-CH3]

+

271 [M-side chain-2H]+

255 [M-side chain-H2O]+

213 [M-side chain-ring D cleavage-H2O]+

14.1 15.6

Total 94.8 94.8