Novel bioactive sulfated alkene and alkanes from the Mediterranean ascidian Halocynthia papillosa

Article abstract of DOI:10.1021/np000281o

Three sulfated alkene and alkanes - (R)-2,6-dimethylheptyl sulfate (1), 6-methylheptyl sulfate (2a), and (E)-5-octenyl sulfate (3a) - with cytotoxic activity in vitro, have been isolated from the Mediterranean ascidian Halocynthia papillosa. The structure

Full text of DOI:10.1021/np000281o

1590
J . Nat. Prod. 2000, 63, 1590-1592
Novel Bioa ctive Su lfa ted Alk en e a n d Alk a n es fr om th e Med iter r a n ea n Ascid ia n
Ha locyn th ia pa pillosa
Anna Aiello,† Sabina Carbonelli,† Giuseppe Esposito,‡ Ernesto Fattorusso,*,† Teresa Iuvone,‡ and
Marialuisa Menna†
Dipartimento di Chimica delle Sostanze Naturali, Universita` degli Studi di Napoli Federico II, via D. Montesano 49,
I-80131, Napoli, Italy, and Dipartimento di Farmacologia Sperimentale, Universita` degli Studi di Napoli Federico II,
via D. Montesano 49, I-80131, Napoli, Italy
Received J une 2, 2000
Three sulfated alkene and alkaness(R)-2,6-dimethylheptyl sulfate (1), 6-methylheptyl sulfate (2a ), and
(E)-5-octenyl sulfate (3a )swith cytotoxic activity in vitro, have been isolated from the Mediterranean
ascidian Halocynthia papillosa. The structures of the new compounds 2a and 3a have been elucidated
by spectroscopic analysis.
Ta ble 1. IC50 (µg/mL) of Compounds 1, 2a , and 3a a
Sulfated alkanes and alkenes have been isolated recently
from several marine sources, most of them exhibiting
Cell line
1
2a
3a
6-MP
pharmacological effects, such as antibacterial, antifungal,
and antiproliferative activity.1-6 Some of these compounds
were shown to be present in relatively remarkable quanti-
ties in ascidians, thus indicating that they could play a
biological role in these organisms.3-6
WEHI
C6
20.9 ( 2
15.0 ( 1
12.2 ( 0.9
1.3 ( 0.03
30.2 ( 1.5
inactive
545.4 ( 7.5
515.2 ( 5.2
a
Results are expressed as mean ( S. E. M. of three experiments
in quadruplicate.
In the course of our search for bioactive metabolites of
Mediterranean ascidians, we found that the MeOH extracts
of the hepatopancreas, gonads, body muscle, and tunic of
the species Halocynthia papillosa Hartmeyer 1904 (family
Pyuridae) were toxic to WEHI 164 (murine fibrosarcoma)
cells. A bioassay-guided fractionation of each extract
revealed that the activity was mainly due to the presence
of a mixture of sulfated alkanes and alkenes; it was
separated and purified, thus affording compounds 1, 2a ,
and 3a ; they showed cytotoxic activity, which has been
estimated on WEHI 164 and C6 (rat glioma) cell lines
(Table 1).
The normonoterpenoid 1 was previously found in the
Mediterranean ascidian Policitor adriaticus;3 it was also
isolated, as a racemate, from another species of Halocyn-
thia, H. roretzi,4 but it was shown to be present only in
the hepatopancreas. Alkyl sulfates 2a and 3a are new
compounds, and their isolation and structure elucidation
are described in this paper.
were dissected from the body muscle. Each tissue, after
homogenization, was separately extracted with methanol;
the concentrated aqueous residues were partitioned be-
tween water and ethyl acetate and, subsequently, the polar
layers were re-extracted with n-BuOH. The n-BuOH
extracts, all showing cytotoxic activity, were subjected to
reversed-phase C18 chromatography, using a linear gradi-
ent of MeOH (from 0 to 100%) in H2O. The bioactive
fractions, eluted with MeOH/H2O (3:7), were further sepa-
rated and purified by RP18 HPLC (MeOH/H2O, 1:1) to give
compounds 1, 2a , and 3a in the pure state.
Compound 1, which was the major alkyl sulfate in each
extract, was identified as (R)-2,6-dimethylheptyl sulfate by
comparison of its spectral properties (negative FABMS, 1H
and 13C NMR, [R]25D) with those reported in the literature.
3,7
The FABMS (negative ion mode) of 2a showed a [M]-
ion peak at m/z 209, corresponding to the formula C8H17
-
SO4. The presence of a sulfate group was inferred from the
IR absorption band at 1210 cm-1. The main features of its
1H NMR spectrum were a doublet in the high-field region
at δ 0.92 (6H, J ) 6.5), due to the methyl protons of an
isopropyl group, and a triplet signal in the midfield region
at δ 4.02 (2H, J ) 6.8), attributable to the protons of a
methylene bearing the sulfate group. Solvolysis of com-
pound 2a gave the corresponding alcohol 2b, which dis-
played the predictable upfield shift (δ 3.63) for this latter
signal, further proving the presence in 2a of the sulfate
1
moiety. Interpretation of the H-1H COSY allowed delin-
eation of the whole spin sequence H1-H7/H8 and the
identification of compound 2a as 6-methylheptyl sulfate.
The 13C NMR spectrum and DEPT analysis were consistent
with the proposed structure (Table 2).
The tunics were removed from several specimens of H.
papillosa and, then, the hepatopancreas and the gonads
Compound 3a had the molecular formula C8H15SO4 as
deduced from the [M]- ion peak at m/z 207 in the negative
ion FABMS. Mass data and the IR absorption at 1210 cm-1
1
* To whom correspondence should be addressed. Tel.: 39-081-678503.
Fax: 39-081-678552. E-mail: fattoru@unina.it.
attested to the presence of a sulfate function. The H NMR
spectrum of 3a contained two double triplets at δ 5.33 and
5.40, respectively, corresponding to two olefinic protons,
† Dipartimento di Chimica delle Sostanze Naturali.
‡ Dipartimento di Farmacologia Sperimentale.
10.1021/np000281o CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/21/2000

Notes
J ournal of Natural Products, 2000, Vol. 63, No. 11 1591
Ta ble 2. 1H and 13C NMR Data of Compounds 2a and 3a in
40 m. A voucher specimen is deposited at the Dipartimento di
Chimica delle Sostanze Naturali, Napoli, Italy.
CD3ODa
Extr a ction a n d Isola tion of Com p ou n d s 1-3. The tunic
was removed from the specimens of H. papillosa, and then
hepatopancreas and gonads were dissected from the bodies.
Each tissue (hepatopancreas, 11 g dry wt after extraction;
gonads, 4 g; bodies, 16 g; tunic, 170 g) was homogenized and
extracted at room temperature with MeOH. The solvent was
removed, and the concentrated aqueous residues were parti-
tioned between EtOAc and H2O and, subsequently, between
n-BuOH and H2O. The n-BuOH extracts were fractionated by
RP18 MPLC eluting with a linear gradient of MeOH in H2O (0
f 100%). In each case, bioactive fractions, eluted with H2O/
MeOH (7:3), were further chromatographed by HPLC using a
LUNA 3 µm C18 column (4.60 × 150 mm) with MeOH/H2O (1:
1) as the eluent to give compounds 1 (100 mg), 2a (50 mg),
and 3a (5 mg) in the pure state.
2a
3a
position
1H (mult, J )
13C
1H (mult, J )
13C
1
2
3
4
5
6
7
8
4.02 (t, 6.8)
1.69 (m)
68.9
30.6
26.9
30.0
40.3
28.8
23.0
23.0
4.01 (t, 6.8)
1.65 (m)
1.45 (m)
68.9
29.6
26.5
1.43 (m)
1.32 (m)
1.23 (m)
1.58 (m)
2.15 (m)
27.0
5.23 (dt, 11, 6.8)
5.40 (dt, 11, 6.8)
2.08 (m)
129.1
132.0
20.9
0.92 (t, 6.5)
0.92 (t, 6.5)
0.99 (t, 7)
14.7
Assignments were based on 1H-1H COSY and HMQC experi-
a
ments.
and two triplets, one at δ 0.99 (3H) due to a methyl group
and one at δ 4.02 (2H) assigned to a sulfated methylene
group; a series of well-separated methylene signals in the
high-field region of the spectrum (δ 1.45-2.15) were also
present. This good dispersion of proton signals allowed us
to easily establish the gross structure of 3a as 5-octenyl
sulfate, from the connectivities observed in the 1H-1H
COSY spectrum. The Z configuration of the double bond
was assigned on the basis of the value of the H-5/H-6
coupling constant (11 Hz) and of the 13C NMR chemical
shifts (Table 1) of the allylic methylenes C-4 and C-7.4,8
6-Meth ylh ep tyl su lfa te (2a ): colorless amorphous solid;
1
IR (KBr) 1242, 1210, and 1110 cm-1; H and 13C NMR data,
see Table 2; negative FABMS m/z 209.
(E)-5-Octen yl su lfa te (3a ): colorless amorphous solid; IR
1
(KBr) 1240, 1210, and 1110 cm-1; H and 13C NMR data, see
Table 2; negative FABMS m/z 207.
Solvolysis of 2a a n d 3a . Compound 2a (10 mg) was
dissolved in a dioxane-pyridine mixture, 1:1 (5 mL) and
heated at 130 °C (3 h). Water (10 mL) was added to the cooled
solution before extraction with CHCl3 (3 × 5 mL). The organic
phase was evaporated in vacuo to give the alcohol 2b (5 mg):
1H NMR (CDCl3) δ 3.63 (2H, t, J ) 6.8 Hz, H-1); EIMS m/z
130, 112. An identical procedure was used for the solvolysis
of 3a (2 mg), which gave the alcohol 3b (0.6 mg): 1H NMR
(CDCl3), δ 3.64 (2H, t, J ) 6 Hz, H-1); EIMS m/z 128, 110.
Cell Cu ltu r es a n d Biologica l Activity. WEHI 164 cells
(murine fibrosarcoma cell line) were maintained in adhesion
on Petri dishes with Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% (v:v) heat-inactivated fetal
bovine serum (FBS), 25 mM HEPES, penicillin (100 U/mL),
and streptomycin (100 µg/mL). C6 (rat glioma cell line) cells
were maintained in adhesion on Petri dishes with DMEM
medium supplemented with 5%FBS, 25 mM HEPES, glutamine
(2 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL).
As hypothesized for a previously isolated sulfated alkene,
whose structure displayed a recognizable isoprenoid por-
tion,6 normonoterpenoid 1 could originate by oxidative
cleavage from a higher mevalonate precursor. An analogous
process, starting from higher acetate-derived precursors
(e.g., fatty acids), could account for the biogenesis of the
co-occurring sulfates 2a and 3a .
The cytotoxic activity of compounds 1, 2a , and 3a was
evaluated at 96 h on WEHI 164 and C6 cell lines in vitro,
and the effect on both cell lines is reported in Table 1 as
IC50 (the concentration that inhibited the cell growth by
50%). These data show that the compounds act selectively
on fibrosarcoma rather than glioma cells, which are
described in the literature as very resistant also to che-
motherapy.9
All reagents for cell culture were from Biowhittaker. MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-phenyl-2H-tetrazolium bro-
mide] and 6-mercaptopurine (6-MP) were from Sigma.
WEHI 164 and C6 (3.5 × 103 cells) were plated on 96-well
microliter plates and allowed to adhere at 37 °C in 5% CO2/
95% air for 2 h. Thereafter, 50 µL of 1:4 (v/v) serial dilution of
compounds 1, 2a , and 3a (2.5 mg/mL) were added and the cells
incubated for 96 h. In some experiments 6-MP was added as
standard compound for antiproliferative activity. Cell viability
was assessed through an MTT conversion assay as previously
described.6 The viability of each cell line in response to
treatment with compounds 1, 2a , 3a , and 6-MP was calculated
as % dead cells ) 100 - (OD treated/OD control) × 100. Table
1 shows the results expressed as IC50 (the concentration that
inhibited the cell growth by 50%). Statistical analysis was
made by paired two-tailed Student’s t-test: The level of
statistically significant difference was defined as p<0.01.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. FABMS (CsI ions,
glycerol matrix) and EIMS (40 eV) were performed on a VG
Prospec (FISONS) mass spectrometer. Optical rotation was
measured in MeOH on
a Perkin-Elmer 192 polarimeter
equipped with a sodium lamp (λ ) 589 nm) and a 10-cm
microcell. IR (KBr) spectra were recorded on a Bruker model
IFS-48 spectrophotometer. H (500.14 MHz) and 13C (125.03
1
MHz) NMR spectra were determined on a Bruker AMX-500
spectrometer; chemical shifts were referred to the residual
solvent signal (CD3OD: δH ) 3.34, δC ) 49.0; CDCl3: δH
7.26;). Methyl, methylene, and methine carbons were distin-
)
1
guished by DEPT experiments. Homonuclear H connectivities
Ack n ow led gm en t. This work is the result of research
supported by MURST PRIN “ Chimica dei composti organici
di interesse biologico”, Rome, Italy. We wish to thank Prof.
Angelo Tursi (Istituto di Zoologia ed Anatomia Comparata,
Universita` di Bari, Italy) for providing and identifying the
organism. Mass and NMR experiments were performed at the
“Centro di Ricerca Interdipartimentale di Analisi Strumen-
tale”, Universita` di Napoli “Federico II”.
were determined using COSY experiments. One-bond hetero-
1
nuclear H-13C connectivities were determined with the Bax-
Subramanian6 HMQC pulse sequence using a BIRD pulse 0.50
s before each scan in order to suppress the signals originating
from protons not directly bound to 13C (interpulse delay set
1
for J CH ) 140 Hz). Medium-pressure liquid chromatography
(MPLC) was performed using a Bu¨chi 861 apparatus with an
RP18 Si gel (particle size 40-63 µm) packed column. High
performance liquid chromatography (HPLC) separations were
achieved on a Waters 501 apparatus equipped with an RI
detector and with an RP18 LiChrospher (250 × 4 mm) column.
Refer en ces a n d Notes
(1) Findlay, J . A.; He, Z. Q.; Calhoun, L. A. J . Nat. Prod. 1990, 53, 1015-
1018.
An im a l Ma ter ia l. Halocynthia papillosa was collected in
(2) Findlay, J . A.; Yayli, N.; Calhoun, L. A. J . Nat. Prod. 1991, 54, 302-
the Corigliano Gulf (Ionian Sea, southern Italy) at a depth of
304.

1592 J ournal of Natural Products, 2000, Vol. 63, No. 11
Notes
(3) Crispino, A.; De Giulio, A.; De Rosa, S.; De Stefano, S.; Milone, A.;
Zavodnik, N. J . Nat. Prod. 1994, 57, 1575-1577.
(4) Tsukamoto, S.; Kato, H.; Hirota, H.; Fusetani, N. J . Nat. Prod. 1994,
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(7) De Rosa, S.; Milone, A.; Crispino, A.; J aklin, A.; De Giulio, A. J . Nat.
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(5) Aiello, A.; Fattorusso, E.; Menna, M.; Carnuccio, R.; D’Acquisto, F.
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(6) Aiello, A.; Fattorusso, E.; Menna, M.; Carnuccio, R.; Iuvone, T.
Tetrahedron 1997, 53, 11489-11492.
NP000281O