Determination of absolute stereochemistry and an alternative synthesis of homopumiliotoxin 223G: Identification on chiral GC columns with the natural alkaloid

Article abstract of DOI:10.1021/np990641b

An alternative asymmetric synthesis of (+)-(1S,9aS)-homopumiliotoxin 223G (1) was accomplished via (1R,2R,9aS)-1-(benzyloxy)-2-hydroxy-1-methyl-3[(E)-isobutylidene] quinolizidine (4), which was synthesized according to the intramolecular nickel(II)/chromium(II)-mediated cyclization of the N-(iodoalkenyl)aldehyde 2. Compound 4 was converted to the acetate and subjected to reduction with lithium in ammonia, whereupon deprotection of the O-benzyl group and removal of the acetoxyl group occurred in a single operation to afford (+)-homopumiliotoxin 223G. The same sequence using (±)-4 was applied to the synthesis of racemic 223G. Gas chromatography of a sample of racemic 223G showed no separation into enantiomers on four different cyclodextrin-based chiral GC columns. We found, however, that the O-acetates of (±)-223G gave a nearly baseline separation on either a β-cyclodextrin column or a permethylated β-cyclodextrin column. The O-acetate of synthetic (+)-223G was identical on either of these two columns, with the first eluting O-acetate from acetylated (±)-223G and also with the acetylated 223G present in a frog skin extract, thus allowing us to confirm unambiguously the 1S,9aS absolute configurations of natural 223G.

Full text of DOI:10.1021/np990641b

J . Nat. Prod. 2000, 63, 1157-1159
1157
Deter m in a tion of Absolu te Ster eoch em istr y a n d a n Alter n a tive Syn th esis of
Hom op u m iliotoxin 223G: Id en tifica tion on Ch ir a l GC Colu m n s w ith th e Na tu r a l
Alk a loid
Chihiro Kibayashi,*,† Sakae Aoyagi,† Tzu-Chueh Wang,† Kosuke Saito,† J ohn W. Daly,*,‡ and Thomas F. Spande‡
School of Pharmacy, Tokyo University of Pharmacy & Life Science, Horinouchi, Hachioji, Tokyo 192-0392, J apan, and
Laboratory of Bioorganic Chemnistry, National Institutes of Health, Bethesda, Maryland 20892
Received December 29, 1999
An alternative asymmetric synthesis of (+)-(lS,9aS)-homopumiliotoxin 223G (1) was accomplished via
(1R,2R,9aS)-1-(benzyloxy)-2-hydroxy-1-methyl-3[(E)-isobutylidene]quinolizidine (4), which was synthesized
according to the intramolecular nickel(II)/chromium(II)-mediated cyclization of the N-(iodoalkenyl)aldehyde
2. Compound 4 was converted to the acetate and subjected to reduction with lithium in ammonia,
whereupon deprotection of the O-benzyl group and removal of the acetoxyl group occurred in a single
operation to afford (+)-homopumiliotoxin 223G. The same sequence using (()-4 was applied to the
synthesis of racemic 223G. Gas chromatography of a sample of racemic 223G showed no separation into
enantiomers on four different cyclodextrin-based chiral GC columns. We found, however, that the
O-acetates of (()-223G gave a nearly baseline separation on either a â-cyclodextrin column or a
permethylated â-cyclodextrin column. The O-acetate of synthetic (+)-223G was identical on either of
these two columns, with the first eluting O-acetate from acetylated (()-223G and also with the acetylated
223G present in a frog skin extract, thus allowing us to confirm unambiguously the 1S,9aS absolute
configurations of natural 223G.
Neotropical poison-dart frogs of the Dendrobatidae fam-
ily have been a rich source of a remarkable variety of
alkaloids with structurally unique features and biological
significance.1 The pumiliotoxin A class, one of several major
groups of the dendrobatid alkaloids, is characterized by the
6-alkylidene-8-hydroxy-8-methylindolizidine ring system
and has been divided into two subclasses, the pumiliotoxins
and allopumiliotoxins.2 Another bicyclic alkaloid, in which
the indolizidine moiety of the pumiliotoxin subclass of
alkaloids is replaced with a quinolizidine ring, namely,
F igu r e 1. Homopumiliotoxin class alkaloids.
homopumiliotoxin 223G (1), has been isolated in small
quantities from the Panamanian poison frog Dendrobates
as racemic 223G. Comparison on chiral columns of the 1-O-
pumilio and characterized.3 Examination of skin extracts
acetates (6) of synthetic and natural 223G allowed us to
from nondendrobatid amphibians revealed that homo-
confirm unambiguously the 1S,9aS absolute configurations
pumiliotoxin 223G also occurs in the New World genus of
of natural 223G.
bufonid toads Melanophryniscus4 and the Madagascan
genus of mantelline frogs Mantella.5 The former genus
contains further members of the homopumiliotoxin class,
characterized as homopumiliotoxins 319A, 319B, and 321B
(Figure 1).5
Recent studies concerned with the total synthesis of (+)-
We reported7 previously that the N-(iodoalkenyl) alde-
hyde 2, chirally prepared from (S)-N-Boc-2-acetylpiperidine
(3), can be subjected to intramolecular chromium(II)-
mediated coupling to give the trans-quinolizidine 4. We
envisioned that compound 4 could be utilized for an
alternative approach to the asymmetric synthesis of (+)-
homopumiliotoxin 223G have provided confirmation of the
homopumiliotoxin 223G (1) (Scheme 1). An initial attempt
proposed relative stereochemistry of natural homopu-
miliotoxin 223G.6 However, because of the small quantity
of material available for study from the natural source, the
absolute chemistry and optical rotations have not been
at deoxygenation of 4 through the tosylate was unsuccess-
ful, because tosylation of 4 resulted in the formation of a
complex mixture. However, upon treatment with acetic
anhydride and triethylamine at room temperature, 4 could
determined for all these homopumiliotoxins, although they
readily be converted to the acetate 5 in 93% yield. Reduc-
have tentatively been assigned as 1S,9aS based on the
tion with lithium in ammonia at -78 °C was carried out
analogy with the established absolute configurations of the
to simultaneously remove the O-benzyl protecting group
pumiliotoxin class alkaloids, such as 251D, pumiliotoxin
and the acetoxyl group to produce (+)-(lS,9aS)-homopu-
A (307A), and pumiliotoxin B (323A).1b
In the present work, we report an alternative total
synthesis of (+)-(1S,9aS)- homopumiliotoxin 223G as well
miliotoxin 223G (1), [R]28 ) +1.7° (c 1.00, CHCl3). The
D
hydrochloride salt of 1 had mp 183-184 °C and [R]25
)
D
1
+48.0° (c 0.48, MeOH). The H NMR, vapor-phase FTIR,
and mass spectroscopic data of the synthetic substance
were identical with those reported for natural 223G.3,5
The same sequence of reactions starting with racemic
N-Boc 2-acetylpiperidine [(()-3] was also used to synthesize
racemic 223G [(()-1]. A part of the free base was converted
* To whom correspondence should be addressed (C.K.) Tel.: +81(426)-
76-3275. Fax.: +81(426)76-4475. E-mail: kibayasi@ps.toyaku.ac.jp.
(J .W.D.) Tel.: (301) 496- 4024. Fax: (301) 402-0008. E-mail: J ohnD@
bdg8.niddk.nih.gov.
† Tokyo University of Pharmacy & Life Science.
‡ National Institutes of Health.
10.1021/np990641b CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/29/2000

1158 J ournal of Natural Products, 2000, Vol. 63, No. 8
Notes
Sch em e 1
to the corresponding hydrochloride salt, which was recrys-
tallized from chloroform-EtOAc to give colorless pillars, mp
227-229 °C.
On chiral cyclodextrin-based gas chromatographic col-
umns, the synthetic material of the (+)-enantiomer of 223G
was found to have retention times identical with the
natural alkaloid found in frog skin. This strongly suggested
that the natural 223G is also dextrorotatory and that its
absolute configuration is 1S,9aS. However, it remained
possible that both enantiomers of 223G do not separate
on those chiral columns. Accordingly, a sample of the
racemate of 223G was subjected to chiral gas chromatog-
raphy, but unfortunately it showed no separation into
enantiomers on four different cyclodextrin-based chiral GC
columns (see Experimental Section). We found, however,
the O-acetates of (()-223G that is (()-6, gave a nearly
baseline separation on either a â-cyclodextrin column or a
permethylated â-cyclodextrin column (Figure 2). The O-
acetate of synthetic (+)-223G was identical on either of
these two columns with the first eluting O-acetate from
acetylated (()-223G and also with the O-acetate of natural
223G, thus allowing us to unambiguously confirm the 1S,-
9aS absolute configurations of natural 223G.
F igu r e 2. Chiral gas chromatograms of (a) (()-223G heated with Ac2O
for 6 days at 70 °C; leading peak is unacetylated racemate, and the
pair of peaks at longer retention time are the (+)- and (-)-O-acetate
of 223G as labeled; (b) (()-223G heated with Ac2O for 6 days at 70
°C, coninjected with synthetic (+)-223G treated the same way; and
(c) (()-223G heated with Ac2O for 6 days at 70 °C, coinjected with a
sample of the alkaloid fraction from the Madagascan frog M. viridis,
which had been acetylated for 4 days at 70 °C. In both (b) and (c), the
first of the two 223G O-acetate peaks is enhanced. The column used
(B) and temperature program are described in the Experimental
Section. Imp (impurity) in (c) refers to other alkaloids.
Exp er im en ta l Section
Melting points are uncorrected. 1H and 13C NMR spectra
were taken at 400 MHz. Residual chloroform (7.26 ppm) was
used as the internal reference for 1H NMR spectra measured
in CDCl3. 13C chemical shifts were reported on the δ scale
relative to CDCl3 as an internal reference (77.1 ppm). The
EIMS and vapor-phase FTIR spectrum of 223G-OAc were
obtained with a Hewlett-Packard model 5890 gas chromato-
graph having a 25 m × 0.32 mm i.d. HP-5 fused silica-bonded
capillary column programmed from 100 to 280 °C at 10 °C/
min interfaced with a Hewlett-Packard model 5971 mass
selective detector and a Hewlett-Packard model 5965B IR
instrument with a narrow band (4000-750 cm-1) detector and
a Hewlett-Packard ChemStation (DOS based). Merck Si gel
60 (230-400 mesh) was used for column chromatography.
(1R,2R,3E,9a S)-1-(Ben zyloxy)-l-m et h yl-3-m et h ylp r o-
p ylid en e)octa h yd r o-2H-qu in olizin -2-yl Aceta te (5). To a
stirred mixture of 4 (16 mg, 0.049 mmol), which was obtained
according to the previously described method,7b and triethyl-
amine (15 mg, 0.15 mmol) in CH2Cl2 (0.5 mL) was added acetic
anhydride (10 mg, 0.098 mmol) at room temperature. After
stirring at room temperature for 20 h, the mixture was diluted
with CH2Cl2 (10 mL), washed with brine and saturated
aqueous NaHCO3, and dried over MgSO4. The solvent was
removed in vacuo, and the residual oil was purified by
chromatography on a Si gel column eluting with 500:90:1
CHCl3-MeOH-concd NH4OH to give 5 (17 mg, 93%) as a
colorless oil: [R]27D +2.33° (c 0.77, CHCl3); IR (neat) 2865, 2836,
2758, 1736, 1669 cm-1; 1H NMR (CDCl3) δ 0.91, (3 H, s), 1.00
(3 H, d, J ) 6.5 Hz), 1.15 (3 H, s), 1.21-1.92 (6 H, m), 2.06, (3
H, s), 2.07-2.22 (2 H, m), 2.63 (1 H, dt, J ) 6.7, 2.4 Hz), 2.68
(1 H, d, J ) 13.8 Hz), 2.95 (1 H, br d, J ) 10.3 Hz), 3.48 (1 H,
d, J ) 12.5 Hz), 4.56 (1 H, d, J ) 12.4 Hz), 4.59 (1 H, d, J )
12.4 Hz), 5.40 (1 H, s), 5.48 (1 H, dd, J ) 9.1, 1.1 Hz), 7.18-
7.34 (5 H); 13C NMR (CDCl3) δ 19.2, 21.4, 6 22.6, 22.8, 23.2,

Notes
J ournal of Natural Products, 2000, Vol. 63, No. 8 1159
24.1, 24.8, 25.2, 26.5, 52.0, 57.3, 64.3, 66.1, 76.6, 77.6, 127.1,
127.3, 128.1, 134.7, 139.6, 144.6, 172.5; HREIMS m/z 371.2488
(calcd for C23H33NO3, 371.2460).
(26), 107 (11), 97 (14), 84 (92), 69 (81), 55 (32); vapor-phase
FTIR 2966, 2945, 2874, 2742, 1749, 1450, 1371, 1238 cm-1
.
An aliquot (150 mL) of the alkaloid fraction isolated from
five skins (total weight of skins, 0.93 g in 0.93 mL of methanol)
of the Madagascan frog Mantella viridis, collected 13 km south
of Antsirana in J anuary 1994, was evaporated to dryness,
dissolved in 50 µL of acetic anhydride, and heated at 70 °C
for 4 days. It was then worked up as described above to provide
a methanolic solution containing the O-acetylated natural
223G.
(+)-Hom op u m iliotoxin 223G (1). A solution of 5 (36 mg,
0.097 mmol) in THF (4 mL) was added to liquid ammonia (10
mL) at -78 °C. To this mixture was added lithium (14 mg,
2.0 mmol) in several portions with stirring at -78 °C and
stirring was continued for 1 h. The reaction was quenched by
adding saturated aqueous NH4Cl, and the mixture was
extracted with dichloromethane (3 × 20 mL). The combined
extracts were washed with saturated aqueous NaHCO4, dried
over MgSO4, and concentrated under reduced pressure. The
residual oil was chromatographed on Si gel eluting with
chloroform-methanol-concd NH4OH (200:90:1) to give (+)-1
Deter m in a tion of Absolu te Ster eoch em istr y of Hom o-
p u m iliotoxin 223G Usin g Ch ir a l GC Colu m n s. A Hewlett-
Packard model 5890 gas chromatograph with a 3390A inte-
grator-recorder and flame ionization detector was used for all
analyses. A temperature program of 100-165 °C at 1.5 °C/
min was used. Helium was used as the carrier gas at 20 psi.
The following chiral cyclodextrin-based columns (Supelco Inc.,
Bellefonte, PA) were employed: (R-Dex-120 (column A); â-Dex-
120 (B), γ-Dex-120 (C), and, from SGE, Inc. (Phenomenex,
Torrance, CA), a permethylated-â-cyclodextrin column (D)
(25QC2/CYDEX-B) was obtained. Columns A, B, and C were
30 m × 0.25 mm i.d., 25-µm film thickness, while column D
was 25 m × 0.22 mm i.d., 0.25-µm film thickness. Columns
A-D failed to resolve (()-1. Columns B and D did, however,
provide nearly baseline separation of homopumiliotoxin 223G
O-acetates [(+)-6 and (-)-6]. Coinjections were performed by
mixing 10- or 20-µL amounts of the acetylated racemic or (+)-
reference sample of 223G (1) and 10 or 20 µL of the acetylated
frog skin extract, then injecting 1 or 2 µL of the resultant
solutions on the GC columns below.
(9.4 mg, 44%) as a pale yellow oil: [R]28 ) +1.7° (c 1.00,
D
CHCl3); IR (neat) 3527, 2954, 2934, 2865, 2802, 2751, 1733,
1674, 1465, 1390, 1323, 1270, 1126, 1062 cm-1 1H NMR
;
(CDCl3) δ 0.91, (3 H, d, J ) 6.7 Hz), 0.98 (3 H, d, J ) 6.6 Hz),
1.10 (3 H, s), 1.22 (1 H, tt, J ) 13.0, 3.8 Hz), 1.39-1.53 (2 H,
m), 1.56-1.63 (1 H, m), 2.05-2.15 (3 H, m), 2.37 (1H, d, J )
12.2 Hz), 2.53-2.65 (2 H, m), 2.87 (1 H, d, J ) 11.3 Hz), 3.49
(1 H, dd, J ) 12.1, 1.0 Hz), 5.07 (1 H, d, J ) 9.2 Hz); 13C NMR
(CDCl3) δ 23.5 (CH3), 23.6 (CH3), 24.2 (CH2), 24.3 (CH3), 24.7
(CH2), 25.6 (CH2), 26.6 (CH), 49.6 (CH2), 56.7 (CH2), 57.0 (CH2),
69.5 (CH), 69.7 (C), 128.8 (C), 135.2 (CH); EIMS m/ z (rel int)
223 (M+, 12), 208 (5), 190 (5), 180 (43), 178 (12), 162 (11), 136
(7), 126 (11), 107 (4), 98 (26), 84 (100); HREIMS m/ z 223.1939,
(calcd for C14H25NO, 223.1936).
The free base was treated with methanolic HCl and con-
centrated in vacuo to give a light brown solid, which was
dissolved in MeOH and treated with activated carbon at room
temperature. After filtration, the filtrate was concentrated in
vacuo, and the residual solid was recrystallized from chloro-
form-EtOAc to afford the hydrochloride salt of (+)-1 as
The retention times of either (()-1 or (+)-1 and (+)-6 and
(-)-6 on columns B and D follows: Chiral column B: (()-1 or
(+)-1 ) 38.20 min; (+)-6 ) 47.86 min, (-)-6 ) 48.17 min.
Chiral column D: (()-1 or (+)-1 ) 35.60 min; (+)-6 ) 44.11
min, (-)-6 ) 44.41 min
colorless prisms: mp 183-184 °C; [R]25 ) +48.0° (c 0.48,
D
MeOH); 1H NMR (CDCl3) δ 0.92, (3 H, d, J ) 6.7 Hz), 1.16 (3
H, d, J ) 6.5 Hz), 1.24 (3 H, s), 1.48 (1 H, qt, J ) 13.5, 3.8
Hz), 1.80 (1 H, br d, J ) 17.1 Hz), 2.01 (2 H, br t, J ) 15.8
Hz), 2.26-2.33 (2 H, m), 2.35 (2 H, s), 2.64-2.84 (3 H, m),
3.06 (1 H, dd. J ) 11.8, 9.9 Hz), 3.50 (1 H, br dd, J ) 11.8, 1.5
Hz), 4.12 (1 H, d, J ) 12.8 Hz), 4.15 (1 H, s), 5.44 (1 H, d, J )
9.8 Hz), 11.9 (1 H, br s); 13C NMR (CDCl3) δ 15.66 (CH2), 15.75
(CH3), 15.86 (CH2), 15.91 (CH2), 16.2 (CH3), 18.8 (CH3), 20.4
(CH), 41.0 (CH2), 48.00 (CH2), 49.3 (CH2), 63.1 (C), 64.7 (CH),
112.8 (C), 135.9 (CH); FABMS mlz 224 [MH]+; anal. C 64.35%,
H 9.94%, N 5.47%, calcd for C14H25NO‚HCl, C 64.72%, H
10.09%, N 5.39%.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C NMR spectra
of compounds (+)-1, (+)-1‚HCI, and 5. This material is available free
of charge via the Internet at http://pubs.acs.org.
Refer en ces a n d Notes
(1) For recent reviews in this area, see: (a) Daly, J . W.; Garraffo, H. M.;
Spande, T. F. In The Alkaloids; Cordell, G. A., Ed.; Academic: San
Diego, 1993; Vol. 43, Chapter 3, pp 185-288. (b) Daly, J . W.; Garraffo,
H. M.; Spande, T. F. In Alkaloids: Chemical and Biological Perspec-
tives; Pelletier, S. W., Ed.; Pergamon: New York, 1999; Vol. 13,
Chapter 1, pp 1-161.
(2) For a recent review of total syntheses of these subclasses of alkaloids,
see: Franklin, A. S.; Overman, L. E. Chem. Rev. 1996, 96, 505-522.
Also see: Kibayashi, C.; Aoyagi, S. In Studies in Natural Products
Chemistry; Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, 1997; Vol.
19, pp 52-65. Takahata, H.; Momose, T. In The Alkaloids; Cordell,
G. A., Ed.; Academic: San Diego, 1993; Vol. 44, Chapter 3, pp 189-
256.
O-Acetyla tion of Syn th etic (+)-1 a n d (+)-1 a n d Na tu r a l
Hom op u m iliotoxin 223G. Approximately 0.1-0.2 mg of
either synthetic material of homopumiliotoxin 223G, (+)-1, or
(()-1, was heated for 6 days at 70 °C in small sealed tubes
[Wheaton LVI vial (Alltech Assocs., Deerfield, IL)] with teflon-
lined caps containing 200 µL of acetic anhydride. The contents
were cooled and the reagent removed with a stream of N2 to
near dryness, then MeOH added and the tubes shaken and
that solvent removed also. Then 50 µL of MeOH was added,
and 1 or 2 µL injections were made on the gas chromatograph
as described below, or coinjection mixtures were prepared as
indicated below. GC flame ionization detection indicated 30%
conversion to the O-acetate of homopumiliotoxin 223G [(+)-6
or (()-6], under these conditions with 70% recovered starting
material: EIMS m/ z (rel int) 265 [M]+ (3), 222 (43), 205 (49),
190 (100), 176 (19), 162 (31), 148 (9), 134 (13), 131 (14), 121
(3) Tokuyama, T.; Nishimori, N.; Shimada, A.; Edwards, M. W.; Daly, J .
W. Tetrahedron 1987, 43, 643-652.
(4) Garraffo, H. M.; Spande, T. F.; Daly, J . W.; Baldessari, A.; Gros, E.
G. J . Nat. Prod. 1993, 56, 357-373.
(5) Garraffo, H. M.; Caceres, J .; Daly, J . W.; Spande, T. F.; Andriama-
haravo, N. R.; Andriantsiferana, M. J . Nat. Prod. 1993, 56, 1016-
1938.
(6) Aoyagi, S.; Hasegawa, Y.; Hirashima, S.; Kibayashi, C. Tetrahedron
Lett. 1998, 39, 2149-2152.
(7) (a) Aoyagi, S.; Wang, T.-C.; Kibayashi, C. J . Am. Chem. Soc. 1992,
114, 10653-11654. (b) Aoyagi, S.; Wang, T.-C.; Kibayashi, C. J . Am.
Chem. Soc. 1993, 115, 11393-11409.
NP990641B