Synthesis and biological evaluation of gambierol analogues

Article abstract of DOI:10.1016/S0960-894X(03)00467-0

Gambierol is a polycyclic ether toxin, isolated as a toxic constituent from the marine dinoflagellate Gambierdiscus toxicus. We describe here the synthesis and biological evaluation of structural analogues of gambierol. The present preliminary structure-a

Full text of DOI:10.1016/S0960-894X(03)00467-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2519–2522
Synthesis and Biological Evaluation of Gambierol Analogues
Haruhiko Fuwa,a,y Noriko Kainuma,a Masayuki Satakeb and Makoto Sasakib,*
aGraduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bGraduate School of Life Sciences, Tohoku University, Tsutumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
Received 4 April 2003; accepted 6 May 2003
Abstract—Gambierol is a polycyclic ether toxin, isolated as a toxic constituent from the marine dinoflagellate Gambierdiscus toxicus.
We describe here the synthesis and biological evaluation of structural analogues of gambierol. The present preliminary structure–
activity relationship studies clearly indicate that the H ring functionality and the unsaturated side chain of gambierol are crucial for
its potent toxicity.
# 2003 Elsevier Ltd. All rights reserved.
The polycyclic ether class of marine natural products,
exemplified by brevetoxins, ciguatoxins and maitotoxin,
has received much attention due to the diverse biological
activities with extreme potency and their complex
molecular architecture.1 Among the most prominent
examples are ciguatoxin and its congeners.2,3 These are
the principal toxins for ciguatera fish poisoning that is
prevalent in circumtropical areas with more than 20,000
victims annually. The causative toxins originate in an
epiphytic dinoflagellate, Gambierdiscus toxicus, and are
accumulated in fish through the food chain, thus caus-
ing human intoxication.4 The ciguatoxins are extremely
potent neurotoxins that bind to voltage-sensitive
sodium channels and alter their function.5
completed total synthesis by us10 and the Yamamoto
group.11 The convergent and flexible entry to gambierol
by our total synthesis10 has since allowed the generation
of a series of analogues to elucidate the structure–activity
relationship (SAR). We report here the synthesis of
gambierol analogues with modifications in the H ring
and the side chain and evaluation of their toxicity
against mice. The described preliminary SAR studies,
which were made possible only through chemical synth-
esis, provided the first insight into the critical structural
elements for the potent lethality of gambierol (Fig. 1).
As a first step of the SAR study of gambierol, we
focused on modifications of the right-hand portion of
the molecule, namely, the H ring functionality and the
lipophilic triene side chain. Initially, we prepared com-
pounds 3 and 4 in order to investigate whether the
polyether core of 1 alone is sufficient for exerting toxi-
city. Removal of the benzyl groups of 29k,10 by hydro-
genolysis provided diol 3, which was then deacetylated
to give tetraol 4 (Scheme 1).
Gambierol (1) is another polycyclic ether toxin isolated
with ciguatoxin congeners from the culture cells of G.
toxicus collected at the Rangiroa Peninsula in French
Polynesia.6,7 It exhibits potent lethality against mice at
50 mg/kg (ip), and its neurological symptoms caused in
mice resemble those shown by ciguatoxins, implying the
possibility that gambierol is also responsible for cigu-
atera fish poisoning. However, the extremely limited
availability of this toxin from natural sources has ham-
pered detailed biological studies.8 Therefore, supply of
useful quantities of this natural product by chemical
synthesis is strongly demanded and a number of syn-
thetic efforts have been reported to date,9 culminating in
Next, analogues 9, 13, and 16, lacking the C3012 methyl
group, were synthesized. Enone 510 was subjected to
*Corresponding author. Tel.: +81-22-717-8828; fax: +81-22-717-
8897; e-mail: msasaki@bios.tohoku.ac.jp
yPresent address: Graduate School of Pharmaceutical Sciences, The
University of Tokyo, Japan.
Figure 1. Structure of gambierol (1).
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00467-0

2520
H. Fuwa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2519–2522
Luche reduction13 to give stereoselectively allylic alco-
hol 6 (78% yield), which was then converted to alcohol
7 by a five-step sequence of protective group manipu-
lations (Scheme 2). The strategy used in the total
synthesis of 1 allowed for conversion of 7 into com-
pound 9 via (Z)-vinyl bromide 8.10
Syntheses of compounds 13 and 16 commenced with
replacement of the acetyl groups of 29k,10 with tert-
butyldimethylsilyl (TBS) groups to give bis(silyl) ether
11 in high yield (Scheme 3). Selective liberation of the
C32primary hydroxyl group followed by oxidation and
Wittig reaction of the derived aldehyde led to (Z)-olefin
12. Removal of the TBS ether and hydrogenolysis of the
benzyl ethers with concomitant reduction of the
Scheme 1. Reagents and conditions: (a) H2, Pd(OH)2/C, EtOAc, rt,
94%; (b) K2CO3, MeOH, rt, 84%.
ꢀ
ꢀ
.
Scheme 2. Reagents and conditions: (a) NaBH4, CeCl3 7H2O, MeOH/CH2Cl2 (1:1), 0 C, 78%; (b) TBSOTf, Et3N, CH2Cl2, 0 C; (c) LiDBB, THF,
À78!À40 ꢀC, 75% (two steps); (d) TBDPSCl, Et3N, DMAP, CH2Cl2, rt, 85%; (e) TBSOTf, Et3N, CH2Cl2, rt; (f) CSA, MeOH/CH2Cl2 (1:1), 0 ꢀC,
81% (two steps); (g) SO3 pyr, Et3N, DMSO/CH2Cl2 (1:1), 0 ꢀC; (h) CBr4, PPh3, Et3N, CH2Cl2, 0 ꢀC, 47% (two steps); (i) n-Bu3SnH, Pd(PPh3)4,
.
ꢀ
.
benzene, rt; (j) HF pyr, THF, rt; (k) 10, Pd(PPh3)4, CuCl, LiCl, DMSO/THF (1:1), 60 C, 34% (three steps).
Scheme 3. Reagents and conditions: (a) K2CO3, MeOH, rt; (b) TBSOTf, Et3N, DÀMF, 0 ꢀC, 99% (two steps); (c) CSA, MeOH/CH2Cl2 (1:1), 0 ꢀC,
ꢀ
59%; (d) TPAP, NMO, 4 A molecular sieves, CH2Cl2, rt; (e) CH3(CH2)5P+Ph3Br , NaHMDS, THF, 0 C, 45% (two steps); (f) HF pyr, THF, rt,
.
72%; (g) H2, Pd(OH)2/C, MeOH/EtOAc (1:1), rt, quant; (h) LiDBB, THF, À78!À40 ꢀC; (i) TBDPSCl, Et3N, DMAP, CH2Cl2, rt, 79% (two steps);
ꢀ
ꢀ
ꢀ
.
(j) TBSOTf, Et3N, CH2Cl2, 0 C!rt; (k) CSA, MeOH/CH2Cl2 (1:1), 0 C, 80% (two steps); (l) SO3 pyr, Et3N, DMSO/CH2Cl2 (1:1), 0 C; (m) CBr4,
ꢀ
.
PPh3, Et3N, CH2Cl2, 0 C, 92% (two steps); (n) n-Bu3SnH, Pd(PPh3)4, benzene, rt, 47%; (o) HF pyr, THF, rt, 97%; (p) 10, Pd(PPh3)4, CuCl, LiCl,
DMSO/THF (1:1), 60 ꢀC, 68%.

H. Fuwa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2519–2522
2521
Scheme 4. Reagents and conditions: (a) H2, Pd/C, EtOAc, rt, 95%; (b) SO3 pyr, Et3N, DMSO/CH2Cl2 (1:1), 0 ꢀC; (c) CBr4, PPh3, Et3N, CH2Cl2,
.
ꢀ
ꢀ
.
0 C; (d) n-Bu3SnH, Pd(PPh3)4, benzene, rt, 74%ꢀ(three steps); (e) HF pyr, THF, rt, quant; (f) 10, Pd(PPh3)4, CuCl, LiCl, DMSO/THF (1:1), 60 C,
+
À
ꢀ
.
.
64%; (g) SO3 pyr, Et3N, DMSO/CH2Cl2 (1:1), 0 C; (h) CH3(CH2)5 P Ph3Br , NaHMDS, THF, 0 C, 58% (two steps); (i) HF pyr, THF, rt, 81%;
(j) H2, Pd/C, EtOAc, rt, 59%.
C32–C33 double bond then afforded compound 13. On
the other hand, 11 was converted into alcohol 14 by a
four-step sequence of reactions including reductive
debenzylation with lithium di-tert-butylbiphenilide
(LiDBB), two-step protection of the C1 and C6
hydroxyl groups, and selective liberation of the C32
hydroxyl group under acidic conditions. Conversion of
14 into compound 16 via 15 followed the same protocol
as described earlier.
completely inactive, indicating that the octacyclic poly-
ether core alone is not sufficient for exhibiting toxicity.
Compound 9 lacking the axial C30 methyl group still
retained a significant level of lethality, being approxi-
mately 5-fold less active than the parent 1. Therefore,
the C30 methyl group is not critical for exerting toxicity,
while possibly important. Compound 19, 34,35,37,38-
tetrahydro derivative of 1, exhibited toxicity to a lower
extent, which is approximately 25-fold lower than that
of 1, whereas compound 18, in which the C28–C29
double bond is removed, suffered a further decrease in
its toxicity (ca. 120-fold less). Mice died within 15–30
min after ip injection of these three active compounds.
In contrast, compounds 13, 16, 20, and 21 showed no
detectable toxicity against mice even at >7.6 mg/kg
dose level.15 These results clearly demonstrate that the
H ring double bond and the side chain containing the
C28–C29 (Z)-double bond are critical structural
requirements for potent lethality of 1. These structural
elements would serve to orientate the side chain to the
proper direction.16 Moreover, the proper length of the
side chain also seems to be necessary for toxicity
because truncating the side chain (compound 21) resul-
ted in loss of activity.
Syntheses of analogues 18–20, having the axial C30
methyl group, are summarized in Scheme 4. Conversion
of 1710 into compound 18 was carried out following the
protocol used for the synthesis of 9 and 16, while com-
pound 19 was prepared from 17 in a similar way used
for the synthesis of 12. Finally, hydorogenation of 19
gave compound 20.
With the gambierol analogues (3, 4, 9, 13, 16, and 18–
20) in hand, the respective toxicities against male mice
(ddY strain, 12–17 g body weight) were determined by
intraperitoneal injection of suspensions in 1% Tween 60
at appropriate dose levels, along with the parent syn-
thetic 1 and analogue 2110 with truncated side chain
(Table 1).14 Not unexpectedly, compounds 3 and 4 were
In summary, the present SAR studies indicate that the
structural elements required for potent toxicity of 1 are
not only the fused polycyclic ether core structure but
also the H ring double bond as well as the side chain
with an appropriate length and a double bond between
C32and C33. Although the analogues we have prepared
are inactive or less potent than 1, these synthetic ana-
logues cannot be prepared by derivatization from nat-
ural 1. On the basis of the present studies, further efforts
aimed at understanding the molecular basis of the
potent lethality of gambierol are currently underway
and will be reported in due course.
Table 1. Minimal lethal dose values (mg/kg) of compounds 1, 3, 4, 9,
13, 16 and 18–21 in mice
Compd
Minimal lethal dose
(mg/kg)
1
3
0.05–0.07
>8.2
4
9
>18.3
0.34
>7.6
>11.9
7.9
1.6
Acknowledgements
12
16
18
19
20
21
We are very grateful to Professor Kazuo Tachibana,
The University of Tokyo, for his helpful discussion.
This work was financially supported by CREST, Japan
Science and Technology Corporation (JST) and Sun-
tory Institute for Bioorganic Research (SUNBOR).
>12.9
>7.6

2522
H. Fuwa et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2519–2522
Lett. 1998, 39, 6369. (c) Kadota, I.; Ohno, A.; Matsukawa, Y.;
References and Notes
Yamamoto, Y. Tetrahedron Lett. 1998, 39, 6373. (d) Kado-
waki, C.; Chan, P. W. H.; Kadota, I.; Yamamoto, Y. Tetra-
hedron Lett. 2000, 41, 5769. (e) Fuwa, H.; Sasaki, M.;
Tachibana, K. Tetrahedron Lett. 2000, 41, 8371. (f) Kadota, I.;
Ohno, A.; Matsuda, K.; Yamamoto, Y. J. Am. Chem. Soc.
2001, 123, 6702. (g) Fuwa, H.; Sasaki, M.; Tachibana, K.
Tetrahedron 2001, 57, 3019. (h) Kadota, I.; Takamura, H.;
Sato, K.; Yamamoto, Y. Tetrahedron Lett. 2001, 42, 4729. (i)
Sakamoto, Y.; Matsuo, G.; Matsukura, H.; Nakata, T. Org.
Lett. 2001, 3, 2749. (j) Cox, J. M.; Rainier, J. D. Org. Lett.
2001, 3, 2919. (k) Fuwa, H.; Sasaki, M.; Tachibana, K. Org.
Lett. 2001, 3, 3549. (l) Kadota, I.; Park, C.-H.; Sato, K.;
Yamamoto, Y. Tetrahedron Lett. 2001, 42, 6195. (m) Kadota,
I.; Kadowaki, C.; Takamura, H.; Yamamoto, Y. Tetrahedron
Lett. 2001, 42, 6199. (n) Kadota, I.; Kadowaki, C.; Park, C.-
H.; Takamura, H.; Sato, K.; Chan, P. W. H.; Thorand, S.;
Yamamoto, Y. Tetrahedron 2002, 58, 1799. (o) Kadota, I.;
Ohno, A.; Matsuda, K.; Yamamoto, Y. J. Am. Chem. Soc.
2002, 124, 3562. (p) Kadota, I.; Takamura, H.; Sato, K.;
Yamamoto, Y. J. Org. Chem. 2002, 67, 3494. (q) Majumder,
U.; Cox, J. M.; Rainier, J. D. Org. Lett. 2003, 5, 913.
10. (a) Fuwa, H.; Sasaki, M.; Satake, M.; Tachibana, K. Org.
Lett. 2002, 4, 2981. (b) Fuwa, H.; Kainuma, N.; Tachibana,
K.; Sasaki, M. J. Am. Chem. Soc. 2002, 124, 14983.
11. Kadota, I.; Takamura, H.; Sato, K.; Ohno, A.; Matsuda,
K.; Yamamoto, Y. J. Am. Chem. Soc. 2003, 125, 46.
12. The numbering of carbon atoms of all compounds in this
paper corresponds to that of gambierol.
1. For reviews on marine polycyclic ether toxins, see (a)
Yasumoto, T.; Murata, M. Chem. Rev. 1993, 93, 1897. (b)
Scheuer, P. J. Tetrahedron 1994, 50, 3. (c) Murata, M.; Yasu-
moto, T. Nat. Prod. Rep. 2000, 17, 293. (d) Yasumoto, T.
Chem. Rec. 2001, 1, 228.
2. (a) Murata, M.; Legrand, A.-M.; Ishibashi, Y.; Yasumoto,
T. J. Am. Chem. Soc. 1990, 111, 8929. (b) Murata, M.;
Legrand, A.-M.; Scheuer, P. J.; Yasumoto, T. Tetrahedron
Lett. 1992, 33, 525. (c) Satake, M.; Morohashi, A.; Oguri, H.;
Oishi, T.; Hirama, M.; Harada, N.; Yasumoto, T. J. Am.
Chem. Soc. 1997, 119, 11325. (d) Yasumoto, T.; Igarashi, T.;
Legrand, A.-M.; Cruchet, P.; Chinain, M.; Fujita, T.; Naoki,
H. J. Am. Chem. Soc. 2000, 122, 4988 and references cited
therein.
3. (a) Recently, total synthesis of ciguatoxin CTX3C was
reported: Hirama, M.; Oishi, T.; Uehara, H.; Inoue, M.;
Maruyama, M.; Oguri, H.; Satake, M. Science 2001, 294,
1904. (b) Inoue, M.; Uehara, H.; Maruyama, M.; Hirama, M.
Org. Lett. 2002, 4, 4551.
4. Bagnis, R.; Chanteau, S.; Chungue, E.; Hurtel, J. M.;
Yasumoto, T.; Inoue, A. Toxicon 1980, 18, 199.
5. (a) Bidard, J.-N.; Vijverberg, H. P. M.; Freiln, C.; Chun-
gue, E.; Legrand, A.-M.; Bagnis, R.; Lazdunski, M. J. Biol.
Chem. 1984, 259, 8353. (b) Lambet, A.; Bidard, J.-N.; Laz-
dunski, M. FEBS Lett. 1987, 219, 355.
6. Satake, M.; Murata, M.; Yasumoto, T. J. Am. Chem. Soc.
1993, 115, 361.
7. Morohashi, A.; Satake, M.; Yasumoto, T. Tetrahedron
Lett. 1999, 40, 97.
13. Luche, J. L. J. Am. Chem. Soc. 1978, 100, 2226.
14. All samples used in the assay were purified by HPLC
(Asahipak ODP-506D, f 4.6ꢁ150 mm) using CH3CN/H2O as
an eluent.
15. For compounds 18 and 19, upon ip injection at lower dose
levels typical neurological symptoms were observed in mice,
whereas such changes were not observed for other inactive
compounds.
8. Very recently, Inoue et al. reported that gambierol inhibits the
binding of dihydrobrevetoxin B (PbTx-3) to voltage-sensitive
sodium channels, though affinity is much lower than that of
ciguatoxins: Inoue, M.; Hirama, M.; Satake, M.; Sugiyama,
K.; Yasumoto, T. Toxicon 2003, 41, 469.
9. (a) Kadota, I.; Park, C.-H.; Ohtaka, M.; Oguro, N.;
Yamamoto, Y. Tetrahedron Lett. 1998, 39, 6365. (b) Kadota,
I.; Kadowaki, C.; Yoshida, N.; Yamamoto, Y. Tetrahedron
16. Details of conformational analysis of analogues will be
reported in a full account.