S6.7 (SI. 59â. 55.3 (s). II. -6.0 (SJ. 76.9 (Si. 76.0 (sj. 76.8 (s). 86.8 (s). 12 ... formamide group appeared at 6~ 5.09 (brs, NH) and 8.04 (brs, H-21) /6, 160.1. while ...
f~~rriihlmi
Lerren.
Vol. 36. Vn 47. pp. 8637-8640,
1995
Elsewer Science Ltd Pinted III Great Botain 0040.4039195
Antifouling
Kalihinenes from the Marine Acanthella
Tatsufumi
$9.50+0.00
Sponge
cavernosa
Okino, Erina Yoshimura, Hiroshi and Nobuhiro Fusetani*
Hirota,
Fuserum Biofouling Project, E.wplortrton. Research for .4dvanced Technology (ER.~POJ. Research Development C‘orporatron oflupan (JRDC), c/o Niigata Engineering Co , Ltd.. Isogo-Xu. Yokohama 2.35, Japan
Abstract: rhree new kalihinenrs here isolated tram the marme sponge ;2ran#1el/n cnvernosn together with kalihmol A and I@-fornlamidokalihlnenr Their structures were elucidated to be 1-3 on the basis of 7D NMR data. These compounds showed potent antifouling activities against the barnacle larvae of Bdanu~ nmphirrite
Marine sezs~le orgamsms. such ah barnacles and rnu~el~. cause serious problems by settling on manmade submersed structures.
Organotln
compounds
have been widely used for control of these fouling
orgamsms until recently. when they were found toxic to marine organisms. I32 Therefore, nontoxic antifouling substances are urgent]) needed. Marine orgamsm\ are believed to possess chemical defenses against fouling orgamsms.
hence then secondary metabolites
hromopyrroles,? methylgraminey
pukalides.J,y
sponge, .-lcnnthelln
renillafoulma.”
were reported as antifouhng
search for antlfoulmg
tmght be potential turospongolide.’
nontoxic
antifouling
ubiquinone
agents.
In fact,
8,* and 2,5,6-tribromo-l-
xubxtances against barnacles from marine organisms. During our
substances from Japanese marine invertebrates, we found that the extract of the marine
caverno.cul().
collected off ‘r’akushlma island, 1000 km southwest of Tokyo, inhibited larval
settlement and metamorphosis of the barnacle Bnkmrrs
trmphitrire.
extract of the sponge yielded three new drterpene tormamldrs,
Bioassay-guided
fractionation of the ethanol
kalihinenes X (l), Y (2), and Z (3) along with
8638
Table 1, 1% NMR (CDCl$
Data for Kahhinene Derivatives
2a
3a
5a.b
&
Carbon 1 2
la 45.9 (d) 19.1 (t)
3 4 i 6 7 8 9 10 II 12 13 l-1 15 16 Ii
10 h It) 179.7 (SJ 178.0 (d) J5.U (d) SO.8 (d) 2.5 (t, J2.X (t, S.T.2 (S) -6.0 (SJ JY.0 (t) 77.5 (t) 6.5.0 (d) -5.7 ISI 77 x tqi 30.5 (qJ
18
71 0 (q)
19.6 (q)
18.4 (q)
19.2 (9)
19.7 (9)
19 30 ?I 22
‘3.2 (q) ‘7 2 (q) 162.X (d)
23.9 (q) 19.0 IqJ 162.7 (dj
23.2 (q) 27.2 (q) 162.X (d)
29.0 (4 20.7 (4 157’ 153 c
23.3 (9) 27.1 (4 162.8 (d) 153.9 c
49.7 (d) 23.3 (t) 30.4 132.6 125.5 39.4 53.6 23.9 42.0 55.3 76.9 38.2 27.7 64.8 75.7 22.8 30.9
(I) (s) (d) (dr (d, (tl it1 (\i (Si ttj (1) (d) (5) (q) (q)
45.8 (d) 19.5 (t)
42.3 (d) 21.6 (t)
30.6 130.3 128.0 35.1 48.6 22.9 33.2 S6.7 76.0 29.2 ‘5.7 64.6 73.8 23.8 29.3
32.6 70.5 63.7 36.0 48.4 21.9 39.7 59” 76.8 38.0 27.4 64.1 76.0
(t, (sj (d) (d) (d) (t) (t) (SI (sj (t) (t) (d) (si (q) (q,
45.8 (d) 19.4 (t)
(t) (s) C (d) (d) (t, (t)
30.7 130.6 126.9 35.4 48.6 24.6 32.6 55.3 86.8 37.3 25.7 82.9 60.5 26.3 25.2
(s) (t) (t) (d) (s) 22.8 (4 30.5 (q)
a: s-tram ~wtner; h. nrse data are not literature ones.~III INIT ~~~ptxmental data Especially
5 was reassigned
(t) (s) (d) (d) (d) (t) (t) (s) (s) (t) (t) (d) C (q) (q)
by HMBC
data;
C: Signal appears as a hrosd triplet.
the known kalihinol
.A (3) t t and IO-formamidokahhinene
structure elucidation of these antifouling
t 5). 12 In this paper we describe the isolation and
substance\.
The frozen sponge (0.5 kg) was extracted with EtOH followed by partitioning The ether layer was then partitioned betwen hexane and MeOH& larval settlement and metamorphosis chromatography
(hexane-ether
column (benzene-EtOAc)
in the barnacle Ralanus
and EtOAc).
Kalihinene
was separated by silica gel column
to afford two antifouling fractrons; the benzene-EtOAc
to afford kalihinene
with lo-formamidokahhinene
(9: 1). The hexane layer which inhibited
The active EtOAc eluate was again fractionated
(4. 1.3 mg), while the fraction eluted with benzene-EtOAc (aqMeOH)
amphitrik
between ether and water.
on a silica gel
(8:2) eluate yielded kalihinol A
(73) was repeatedly purified
X (1, 5.4 mg). kalihmene Y (2, 0.7 mg), and kalihinene
by ODS HPLC
Z (3, 1.0 mg) along
(5. 7.4 mg).
X (1 )t? had a molecular formula of C,tH3&IN02
which was established by HRFABMS
(m/z 368.2347 [M+H]+. 3 -0.9 mmu). Most tH and 1% NMR signals of 1 were doubled due to the presence
8639
of a formamide formamide
(5). 12 Signals assignable to the s-cis
group, as is the case of IO-formamidokalihinene
group appeared at 6~ 5.09 (brs, NH) and 8.04 (brs, H-21) /6, 160.1. while those for the s-trans
form at 6 5.62, 8.20 /162.8. which were secured by COSY and HMQC spectra. 1% chemical shifts for C-l C-10 and C-19 - C-21 were identical with those of the decalin ring part in 5 (Table 1). The presence of a tetrahydropyran especially
ring embracing
a chlorine
6 76.0 (C-l 1J and 75.2 (C-15).
from 13C chemical shifts of C-l 1 to C-18,
atom was inferred Therefore,
1 had the carbon framework
decalin ring system as 5, which was connected to a tetrahydropyran confirmed by 2D NMR experiments. decalin was &-fused
derived from a NOESY configuration.
NOESY cross peaks between
and Me-20 was axial. Axial orientation experiment.
The coupling
Relative stereochemistry
composed of the same
ring identical to that of 4. This was also Me-20 and both H- 1 and H-6 indicated that
of H-13 (6 2.08). Me-16, and Me-18 was also
constants of H-14 (5=12.4, 4.3 Hz) indicated
its axial
of C-7 and C-l 1 was assumed to be the same as that of the other
known kalihinane diterpenes whose structures were determined by X-ray diffraction. The molecular formula of kalihinene 368.2356 [M+H]+.
Y (2)14 was determined to be C:!1H-&lN02
by HRFABMS
A 0.0 mmu), thus suggesting that it was an isomer of 1. The carbon framework
(m/z
of 2 was
readily assignable to be the same as that of 1 by the COSY, HMQC. and HMBC spectra. Especially, chemical shifts of the tetrahydropyran
ring in 2 were almost superimposable
suggesting the same stereochemistry of the tetrahydropyran coupling
13C
on those of 1 (Table l), thereby
ring for both 2 and 1. This was confirmed by the
constants of H- 14 (J= 12.2, 4.2 Hz) and NOESY correlations
of H- 13, Me- 16, and Me- 18. On the
other hand, the H-S proton appeared as a broad singlet at 6 6.32, indicating that the dihedral angle between H-5 and H-6 was about 90”. Thus, the decalin ring system must be rrans-fused as is the case of I-epi-kalihinene.15 This was confirmed by a NOESY correlation between H-6 and Me-20 (no cross peak between H-l and Me-201 and the coupling constants of H-6 (J=12, 12 Hz). Therefore, 2 is a I-epimer of 1. Kalihinene [M+H]+,
Z (3)16 was also an isomer of 1 and 2 as determined
A -2.3 mmu).
The carbon framework
by 2D NMR experiments.
confirming
constants of H-14. a 14-epimer
tetrahydropyran
of 3 was readily deduced to be the same as those of 1 and 2
tetrahydropyran
of 1. This was supported
characterized
or a tetrahydrofuran
hydroxykalihinenesl*
by
the similarity
E,17 a 14-epimer of kalihinol by
a saturated
substituent.
belong to the tetrahydrofuran
(3) are the first examples of kalihinene-type The new kalihinenes (l-3) inhibited amphitrite
difference between 1 and 3 was found in the
The broad signal for H-14 in 3 suggested its equatorial
rings of 3 and kalihinol
The kalihinols,
data (m/z 368.2333
Relative stereochemistry of a decalin ring of 3 was also the same as that of 1, which
was inferred from 13C chemical shifts (Table 1). A significant coupling
by HRFABMS
decalin
of 13C chemical
thereby
shifts for the
A (4).
ring, belong
Interestingly,
orientation,
kalihinenes,
to two types: those with a which are octalins, and 6-
series. However, the new kalihinenes X (l), Y (2), and Z
diterpenes in tetrahydropyran
series.
attachment and metamorphosis
with EC,,‘s of 0.49, 0.45, and 1.1 pglmL,
respectively,
of cyprid larvae of the barnacle B.
while no toxicity
was found at these
8640
concentrations.
Kalihinol
A (4, EC,, 0.087 pg/mL) and lo-formamidokalihinene
showed potent antifouling
activity. more active than CuS04 (EC,, 0.15 lg/mL).
Acknowledgements:
(5, EC50 0.095 pg/mL) also
We thank Prof. P. J. Scheuer of the University of Hawaii for reading this manuscript.
Thanks are also due to Dr. Rob van Soest of the University of Amsterdam for sponge identification K. Okamoto and Dr. N. Sata of the University
of Tokyo for collection
and to Dr.
of barnacles and for measurement of
optical rotations, respectively.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
1 I. 12. 13.
14.
15 16
17
REFERENCES AND NOTES Evans, L. V.; Clarkson, N. J. ilppl. Bacterial. 1993, 74, 119s.124s. Glare. A. S.; Rittschof, D.; Gerhart, D. J.: Maki, 3. S. lrwert. Reprod. Develop. 1992,22, 67-76. Kelfer, P. A.: Schwartz, R. E.; Koker. M. E. S.: Hughes Jr., R. G.; Rittschof, D.; Rinehart, K. L. J. Org. Chem. 1991, .i6, 2965-2975. Gerhart. D. J.; Rittschof, D.; Mayo, S. W. J. (‘hem. Ecol. 1988,14, 19051917. Mizobuchi, S.; Kon-ya, K.; Adachi, K.: Sakai, M.; Miki, W. Fisheries Sci. 1994,60, 345-346. Keifer, P. A.; Rinehart Jr., K. L.; Hooper. I. R. /. Org. Chem. 1986, SI, 4450-4454. Goto, R.; Kado. R.; Muramoto, K.; Kamiya, H. Nippon Suisan Gakknishi 1993,59, 1953. Kon-ya, K.; Shimidzu, N.; Otaki, N.; Yokoyama, A.; Adachi. K.; Miki. W. Experientia 1995.51, 153-155. Ken-ya, K.; Shlmid,w. N.; Adachi. K.; Mlkl. W. Fisheries Sri. 1994, 60, 773-775. The sponge was identified as Acanthella c‘awrnosa Dendy, 1922 (Class Demospongiae, Order Halichondrida, famdy Dictyonellidae) by Dr. Rob van Soest. A voucher specimen (ZMA POR. 11018) wa:, deposited at the Institute for Systematics and Population Biology, University of Amsterdam. Chang, C. W. J.; Patra. A.: Roll, D. M.; Scheuer. P. J.; Matsumoto, G. K.; Clardy, J. J. Am. Chem. Sot- 1984, 106, 4644-4646. Rodriguez, J.; Nieto. R. M.; Hunter. L. M.. Diaz. M. C’.: Crews, P.; Lobkovsky, E.; Clardy, J. Tetrahedron 1994, SO. I 1079. I IOYO. Kalihinene X (1): [n]; +26.7” (c 0.3 I, CHC13): IR ( KBr) 3282, 1677 cm-t; tH NMR of s-trans isomer (CDCI?) 8.20 (IH, dJ=12.5 Hz, H-21). 5.62 (1H. NH). 5.62 (IH, H-S), 3.67 (1H. dd 12.4, 4.3, H141, 2.14 (lH, H-6). 1.96 (2H. H-13), I.95 (3H. H-3), 1.69 (ZH, H-2). 1.62 (2H, H-9), 1.60 (IH, HI). 1.58 (3H, s. H-19). I.58 (lH, H-S), 1.54 r2H. H-12), 1.46 (IH, H-7), 1.40 (3H. s, H-20). 1.33 (3H. s, H-17). 1.24 (3H. s. H-18). 1.20 (3H, s, H-16). 1.13 (IH, H-S). Kahhinene Y (2): [a]: +I 1.0’ (c 0.01, CHCI); IR (KBr) 1677 cm-t; ‘H NMR of s-frans isomer (CDCI,) 8.25 (1H. dJ=11.4 Hz, H-21). 6.32 ;lH, brs. H-S), 5.51 (lH, d 11.1, NH), 3.70 (IH, dd I? 3, 4.3, H-14), 2.09 (IH, H-13). 2.06 (IH. H-6). 1.98 (lH, H-13), 1.97 (IH, H-3). I.89 (lH, H3), 1.86 (lH, H-Q), I.81 (IH, H-2). 1.63 (IH, H-X), 1.63 (3H. s, H-19), 1.59 (lH, H-12). 1.55 (lH, H-9). 1.48 (1H. H-12), 1.38 (3H. s, H-17). 1.29 (?H, s, H-16), 1.27 (lH, H-l), 1.25 (IH, H-7), 1.24 (lH, H-2), 1.23 (3H, s, H-18), 1.19 (3H, s, H-20), 1.07 (lH, H-S). Trimurtulu, G.; Faulkner, D. J. J. Nut. Prod. 1994, 57, 501-506. Kalihinene Z (3): [a]? +I 1.7” (c 0.035. CHClj); IR (KBr) 3282, 1677 cm-t; ‘H NMR of s-truns isomer tCDC13) 8.21 (1H. dJ=12.S Hz, H-21), 5.6.5 (IH. NH), 5.64 (lH, H-S), 3.92 (lH, brs. H-14). 2.30 (lH, H-6), 2.25 (lH, H-13), 1.97 (?H, H-12). 1.94 (2H, H-3), 1.92 (lH, H-13), 1.71 (IH, H-7), 1.69 (2H, H-S), 1.64 (IH. H-l). 1.60 tlH, s. I
