Synthesis and biological activities of the 3,5-disubstituted indolizidine poison frog alkaloid 239Q and its congeners

Article abstract of DOI:10.1002/ejoc.201200974

The first total synthesis of the 3,5-disubstituted indolizidine toad alkaloid 239Q from the known pyrrolidine 1 has been achieved, in seven steps with a 35 % overall yield. The relative stereochemistry of natural 239Q was determined unambiguously by comparison of GC-FTIR spectra of synthetic material with the skin extracts of the toad Melanophryniscus cupreuscapularis. The C7 congeners at the 5-position (12 and 13) showed strong antagonist activities on the α4β2 nicotinic acetylcholine receptors.

Full text of DOI:10.1002/ejoc.201200974

FULL PAPER
DOI: 10.1002/ejoc.201200974
Synthesis and Biological Activities of the 3,5-Disubstituted Indolizidine Poison
Frog Alkaloid 239Q and Its Congeners
Xu Wang,[a] Hiroshi Tsuneki,*[b] Noriko Urata,[a] Yasuhiro Tezuka,[c] Tsutomu Wada,[b]
Toshiyasu Sasaoka,[b] Hideki Sakai,[b] Ralph A. Saporito,[d] and Naoki Toyooka*[a,e]
Keywords: Total synthesis / Medicinal chemistry / Natural products / Alkaloids / Melanophryniscus klappenbachi /
Melanophryniscus cupreuscapularis
The first total synthesis of the 3,5-disubstituted indolizidine
toad alkaloid 239Q from the known pyrrolidine 1 has been
achieved, in seven steps with a 35% overall yield. The rela-
tive stereochemistry of natural 239Q was determined unam-
biguously by comparison of GC-FTIR spectra of synthetic
material with the skin extracts of the toad Melanophryniscus
cupreuscapularis. The C7 congeners at the 5-position (12 and
13) showed strong antagonist activities on the α4β2 nicotinic
acetylcholine receptors.
instead at C-10 (Figure 1), which has also previously been
reported for the 3,5-disubstituted indolizidine 239AB and
the 5,8-disubstituted indolizidine 239D.[2] As part of a pro-
gram directed towards study of the synthesis and biological
activities of poison frog alkaloids,[3] here we report the syn-
thesis of 3,5-disubstituted indolizidine 239Q and its conge-
ners and the determination of their pharmacological activi-
ties on nicotinic acetylcholine receptors.
Introduction
The 3,5-disubstituted indolizidine alkaloid 239Q (3,5-I,
Figure 1) was isolated from methanol extracts of two spe-
cies of toads (Melanophryniscus klappenbachi and Melano-
phryniscus cupreuscapularis) from Argentina as a major
alkaloid, along with the 4,6-disubstituted quinolizidine
275I.[1] The structure and relative stereochemistry of natu-
ral 3,5-I 239Q were determined by mass, FTIR, and NMR
spectral analysis to correspond to (5Z,9Z)-3-(1-hydroxy-
butyl)-5-propylindolizidine, but no total synthesis has been
reported to date. In most cases, the odd-numbered carbon
side chains of indolizidines present in frog skin and/or ar-
thropods are at the C-5 position, but 3,5-I 239Q possesses
an unusual butyl side chain with a hydroxy group. From
the FTIR and 1H NMR spectra, the location of the hydroxy
group in 3,5-I 239Q is anticipated not to be terminal, but
Figure 1. Structure of 239Q.
[a] Graduate School of Science and Technology for Research,
University of Toyama,
Synthesis
3190 Gofuku, Toyama 930–8555, Japan
Homepage: http://pse.eng.u-toyama.ac.jp/life/research.
html#lab7a
The synthesis began with the known pyrrolidine 1
(Scheme 1).[4] Reduction of 1 with Super-Hydride, followed
by oxidation of the resulting alcohol 2 with PCC, gave alde-
hyde 3. Treatment of 3 with nPrMgBr resulted in alcohols
4a and 4b in 1:1 ratio and 85% combined yield. Use of
(nPr)2Zn instead of the Grignard reagent resulted in a 48%
yield of the single alcohol isomer 4a. The stereochemistry
at the newly formed carbinol positions of 4a and 4b was
determined by NOE experiments with the corresponding
oxazolizinone 7.
[b] Graduate School of Medicine and Pharmaceutical Sciences,
University of Toyama,
2630 Sugitani, Toyama 930–0194, Japan
Homepage: http://www.pha.u-toyama.ac.jp/clinphar/
index-j.html
[c] Institute of Natural Medicine, University of Toyama,
2630 Sugitani, Toyama 930–0194, Japan
[d] Department of Biology, John Carroll University, University
Heights,
Ohio 44118, USA
[e] Graduate School of Innovative Life Science, University of
Toyama,
3190 Gofuku, Toyama 930–8555, Japan
Homepage: http://www.ils.u-toyama.ac.jp/index-e.html
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200974
The selectivity of the above addition reaction to 3 was
not quite satisfactory, and so we next examined the re-
duction of ketone 5, derived from the mixture of alcohols
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Poison Frog Alkaloid 239Q and Its Congeners
Scheme 1. Synthesis of alcohols 4a and 4b and determination of the orientations of their OH groups. Reagents and conditions: a) Super-
Hydride, THF, 0 °C (99%); b) PCC, CH2Cl2, 0 °C to room temp. (74%); c) (nPr)2Zn, THF, –78 °C to room temp. (48%); d) NaH, THF/
DMF, 0 °C to room temp. [80% from 4a to 6, from 4a and 4b to 6 (45%) and 7 (43%)]; e) nPrMgBr, THF, 0 °C to room temp. (85%,
dr = 1:1); f) PCC, CH2Cl2, 0 °C to room temp. (91%); g) NaBH4, MeOH, 0 °C to room temp. (94%); h) NaBH4, CeCl3·7H2O, MeOH,
0 °C to room temp. (80%).
Figure 2. Stereoselectivity in the reduction of 5.
Scheme 2. Synthesis of 239Q and 10-epi-239Q. Reagents and conditions: a) Hex-1-en-3-one, second-generation Grubbs catalyst, CH2Cl2,
reflux (94% from 4b to 8, 98% from 4a to 9); b) H2, 20% Pd(OH)2/C, MeOH (83% from 8 to 239Q, 83% from 9 to 10-epi-239Q).
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4a and 4b by PCC oxidation. Reduction of 5 with DIBAL Comparison of Synthetic 3,5-I 239Q and 3,5-I 10-epi-239Q
resulted in an almost 1:1 mixture of 4a and 4b, but under with Natural 3,5-I 239Q
the Luche reduction conditions the alcohol 4b was obtained
The newly synthesized 3,5-I 239Q and 3,5-I 10-epi-239Q
as the major product (5:1, estimated from the 1H NMR
(see Scheme 2) were compared with the naturally occurring
spectrum of crude product) with a 92% combined yield and
3,5-I 239Q present in the skin of the toad Melanophrynsicus
an 80% isolated yield. The selectivity of the reduction of 5
cupreuscapularis by GC–MS and GC-FTIR. The GC–MS
with NaBH4 in MeOH was very high, and we obtained the
retention times and mass spectral properties of the synthetic
alcohol 4a as the sole product in 94% yield. NaBH4 re-
239Q, the 10-epimer 10-epi-239Q, and natural 239Q were
duction of 5 in the CH2Cl2/MeOH (10:1) solvent system,
identical. This was determined by analyzing each alkaloid
however, provided a 1:2.3 mixture of 4a and 4b in 95%
individually, as well as by co-injection of all three alkaloids.
combined yield..
The major GC peak for the co-injection of all three alka-
The stereoselectivity of the reduction of 5 under the two
loids had a retention time of 10.82, an EI base peak at m/z
sets of reaction conditions above was explained by the Fel-
166 accompanied by a peak at m/z 124 (ca. 15%), and a
kin–Anh or chelation-controlled model as shown in Fig-
molecular weight of 239 as determined by CIMS (CH3OH).
ure 2.
GC-FTIR vapor-phase infrared spectra indicated that
After the synthesis of alcohol 4b, our attention was fo-
the synthetic and natural 239Q were indistinguishable from
cused on the conversion of 4b into the indolizidine 239Q.
one another. Co-injection of both compounds resulted in a
A cross-metathesis reaction between 4b and hex-1-en-3-one
very broad absorption at 3528 cm–1, a moderate Bohlmann
in the presence of the second-generation Grubbs catalysis[5]
band at 2798 cm–1, and identical fingerprint regions (Fig-
proceeded smoothly to afford the unsaturated ketone 8
ure 3a). Compound 10-epi-239Q showed
a moderate
(Scheme 2), which was subjected to a catalytic hydrogen-
ation reaction to furnish the indolizidine 239Q as a single
stereoisomer in 85% yield. The stereochemistry of the syn-
thetic indolizidine 239Q was determined by the NOE ex-
periments as shown in Scheme 2. The isomer 10-epi-239Q
was also synthesized from 4a via the corresponding unsatu-
rated ketone 9 in a similar way.
Bohlmann band at 2798 cm–1, but had a different absorp-
tion in the OH region (3649 cm–1) and fingerprint region
(Figure 3b). Therefore, from the retention times and the
mass and FTIR spectral properties, the relative stereochem-
istries of the synthetic and natural 239Q are considered
identical.
Figure 3. a) FTIR spectrum obtained after co-injection of natural and synthetic 3,5-disubstituted indolizidine 239Q. b) FTIR of synthetic
3,5-disubstituted indolizidine 10-epimer 10-epi-239Q.
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Poison Frog Alkaloid 239Q and Its Congeners
Our previous results on the pharmacological activities of hibited nicotine-evoked [3H]dopamine release.[7] Alkaloids
synthetic poison frog alkaloids on nicotinic acetylcholine 235BЈ and 237D each possess a C7 side chain at the 5-posi-
receptors showed that 5,8-disubstituted indolizidine (–)- tions in their indolizidine nuclei.
235BЈ (Figure 4) displayed strong and selective antagonist
In view of these results, we planned the synthesis of C7
activity for α4β2 nicotinic acetylcholine receptors,[6] and congeners 12 and 13 (Scheme 3), 19 and 20 (Scheme 4, be-
that the 5,8-disubstituted indolizidine (–)-237D also in- low), and 21 and 22 (Scheme 5, below) of 3,5-I 239Q to test
their biological activities on nicotinic acetylcholine recep-
tors. The alcohols 4a and 4b were converted into the unsat-
urated ketones 10 and 11, which were subjected to hydro-
genation to provide the congeners 12 and 13.
The ketone 14 (Scheme 4), derived from the aldehyde 3,
was transformed into the alcohols 15 and 16, which were
converted into the congeners 19 and 20 (Scheme 4) and 22
and 24 (Scheme 5), via the unsaturated ketones 17, 18, 21,
and 23, by the same procedure.
Figure 4. Structures of (–)-235BЈ and (–)-237D.
Scheme 3. Synthesis of the 239Q analogues 12 and 13. Reagents and conditions: a) Dec-1-en-3-one,[8] second-generation Grubbs catalyst,
CH2Cl2, reflux (80% from 4a to 10, 96% from 4b to 11); b) H2, 20% Pd(OH)2/C, MeOH (79% from 10 to 12, 90% from 11 to 13).
Scheme 4. Synthesis of the 239Q analogues 19 and 20. Reagents and conditions: a) n-HexylMgBr, THF, 0 °C to room temp. (75%);
b) PCC, CH2Cl2, 0 °C to room temp. (89%); c) NaBH4, MeOH, 0 °C to room temp. (90%, dr = 14:1); d) NaBH4, CeCl3·7H2O, MeOH,
0 °C to room temp. (80%, dr = 6:1); e) hex-1-en-3-one, second-generation Grubbs catalyst, CH2Cl2, reflux (94% from 15 to 17, 99% from
16 to 18); f) H2, 20% Pd(OH)2/C, MeOH (71% from 17 to 19, 88% from 18 to 20).
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Scheme 5. Synthesis of the 239Q analogues 22 and 24. Reagents and conditions: a) Dec-1-en-3-one,[8] second-generation Grubbs catalyst,
CH2Cl2, reflux (99% from 15 to 21, 79% from 16 to 23); b) H2, 20% Pd(OH)2/C, MeOH (66% from 21 to 22, 62% from 23 to 24).
CI: 1.3–2.7). In addition, 239Q blocked the currents medi-
ated by the α7 nicotinic receptor in a concentration-depend-
ent manner, with an IC50 value of 5.7 μm (95% CI: 2.7–
11.8), and 13 was 4.1 times more potent (IC50: 1.4 μm, 95%
Pharmacological Activities of Synthesized
Alkaloids
To investigate the pharmacological activities of the syn-
thesized alkaloids on nicotinic acetylcholine receptors, we
CI: 0.8–2.3) than 239Q (data not shown).
conducted electrophysiological recordings from Xenopus la-
evis oocytes ectopically expressing nicotinic receptors by the
methods described previously.[6] In brief, either the mixture
of mouse α4 and β2 subunit cDNA in a ratio of 1:1 or the
mouse α7 subunit cDNA alone was injected into Xenopus
oocytes. The oocytes were then cultured for 4–8 days. An
oocyte was placed in a recording chamber in which Ringer’s
solution [NaCl (82.5 mm), KCl (2.5 mm), CaCl2 (2.5 mm),
MgCl2 (1 mm), and HEPES (5 mm), pH 7.4] containing at-
ropine (1 μm, Sigma, MO) to block endogenous muscarinic
receptors was perfused. Current responses were recorded in
the two-electrode voltage-clamp mode at a holding poten-
tial of –60 mV, with a GeneClamp 500 amplifier and
pClamp7 software (Axon Instruments, CA). After treat-
ment with the synthetic alkaloid for 3 min, acetylcholine
(ACh, Sigma, 1 μm for α4β2 nicotinic receptors, 1 mm for
α7 nicotinic receptors) was applied in combination with the
alkaloid for 5 s through a computer-controlled valve. Fit-
tings of sigmoid concentration/inhibition curves were car-
ried out with the aid of Prism software (GraphPad Soft-
ware, Inc., CA) to calculate the half-maximum inhibitory
concentration (IC50) with 95% confidence interval (95%
CI).
When the oocytes expressing recombinant α4β2 nicotinic
receptors were treated with the alkaloid 239Q, the peak am-
plitudes of the ACh-elicited currents were greatly decreased
(Figure 5, a). The blocking effects of 239Q were concentra- thetic analogues b) 13, and c) 20 on ACh-induced currents in Xen-
opus oocytes expressing α4β2 nicotinic acetylcholine receptors.
tion-dependent, and the IC50 value was 11.0 μm (95% CI:
7.2–16.7). The 239Q analogues 13, possessing a C7 side
chain at the 5-position, and 20, possessing it at the 3-posi-
alkaloids (3 μm). Horizontal bars indicate the period of perfusion
Figure 5. Inhibitory effects a) of the alkaloid 239Q, and of its syn-
Left. Typical traces showing the ACh-elicited (1 μm) currents
through α4β2 nicotinic receptors in the absence or presence of the
tion, also showed concentration-dependent blocking effects
on the α4β2-receptor-mediated currents (Figure 5, b and c).
Analogue 13 was 22.0 times more potent than 239Q in in-
hibiting α4β2 nicotinic receptors, whereas analogue 20 was
5.8 times more potent than 239Q: the IC50 value of 13 was
with ACh for 5 s. Vertical scale bars represent 500 nA. Right. Con-
centration/response curves for the alkaloids on α4β2 nicotinic re-
ceptors. The current responses to ACh in the presence of alkaloids
in each oocyte were normalized to the ACh response in the pres-
ence of vehicle alone (0.1% dimethyl sulfoxide) in the same oocyte.
Values represents the means ϮS.E.M.s for three to six separate ex-
0.5 μm (95% CI: 0.4–0.6) and that of 20 was 1.9 μm (95% periments.
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Poison Frog Alkaloid 239Q and Its Congeners
To investigate the influence of C7 side chain addition to alkaloids tested on α4β2 and α7 nicotinic receptors were
the 5- and/or 3-position(s) of the indolizidine nuclei on reversible, with the ACh responses being recovered 3–6 min
pharmacological activities further, the blocking effects of after removal of the alkaloids (data not shown).
10-epi-239Q, 12, 19, and 22 were compared. The analogue
10-epi-239Q blocked the currents mediated by the α4β2 ni-
cotinic receptor in concentration-dependent manner (Fig-
ure 6, a) with an IC50 value of 2.7 μm (95% CI: 1.9–3.9).
Conclusions
We have achieved the first synthesis of the 3,5-disubsti-
The analogue 12 (Figure 6, b), possessing a C7 side chain
tuted indolizidine poison frog alkaloid 239Q, from the
at the 5-position, was 3.9 times more potent than 10-epi-
known pyrrolidine 1 in seven steps with 35% overall yield.
239Q in blocking the ACh-elicited currents through α4β2
The relative stereochemistry of natural 239Q was deter-
nicotinic receptors, and its IC50 value was 0.7 μm (95% CI:
mined by comparison of the GC-FTIR spectra of synthetic
0.4–1.2). However, the effects of 19 (Figure 6, c), possessing
a C7 side chain at the 3-position, were comparable to those
material with extracts of the skin of the toad Melanophryns-
icus cupreuscapularis. Taken together, we found that 239Q
of 10-epi-239Q (IC50: 2.6 μm, 95% CI: 1.ng C7 side chains
is a natural alkaloid that possesses antagonistic activities on
at both the 5- and the 3-positions, was 2.4 times less potent
α4β2 and α7 nicotinic acetylcholine receptors. In addition,
than 10-epi-239Q in inhibiting α4β2 nicotinic receptors
a single C7 side chain added to the 5-position of 239Q-
(IC50: 6.4 μm, 95% CI: 5.4–7.6). The blocking effects of all
related structure 13, designed from the structure of (–)-
237D, appears to serve as a moiety to enhance the blocking
(antagonistic) activities against nicotinic acetylcholine re-
ceptors.
Experimental Section
General Information: Flash chromatography was performed with
Kanto Kagaku silica gel (60N, 63–210 μm). NMR spectra were re-
corded with a JEOL a-GX 400 or ECP-NMR 600 spectrometer in
the solvents indicated. Chemical shifts (δ) are given in ppm down-
field from TMS and referenced to CHCl3 (δ = 7.26 ppm) as an
internal standard. Peak multiplicities are designated by the follow-
ing abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad and coupling constants (J) are given in Hz.
High-resolution mass spectroscopic data were obtained with a
JEOL MStation JMS-700 instrument. All commercial reagents
were used as received unless otherwise noted.
Benzyl
(2S,5R)-(–)-5-Allyl-2-(hydroxymethyl)pyrrolidine-1-carb-
oxylate (2): A solution of Super-Hydride (1.02 m in THF, 4.56 mL,
4.65 mmol) was added at 0 °C to a stirred solution of ester 1[4]
(641 mg, 2.11 mmol) in THF (20 mL), and the resulting solution
was stirred at 0 °C for 2 h. The reaction was quenched with ice/
water, and the aqueous mixture was extracted with CH2Cl2 (10 mL
ϫ4). The extracts were combined, dried with Na2SO4, and concen-
trated to afford the residue, which was chromatographed on silica
gel (25 g, hexane/acetone 15:1 to 8:1) to give 2 (574 mg, 99%) as a
1
colorless oil. [α]2D6 = –2.4 (c = 1.10, CHCl3). H NMR (300 MHz,
CDCl3): δ = 1.64–1.70 (m, 1 H), 1.74–1.78 (m, 1 H), 1.86–1.93 (m,
1 H), 1.99–2.09 (m, 1 H), 2.12–2.25 (m, 1 H), 2.30–2.41 (m, 1 H),
3.55–3.62 (m, 1 H), 3.70–3.73 (m, 1 H), 3.91–3.94 (m, 2 H), 4.45–
4.53 (m, 1 H), 5.03 (d-like, J = 10.2 Hz, 2 H), 5.16 (ABq-like, J =
12.2 Hz, 2 H), 5.68–5.81 (m, 1 H), 7.26–7.38 (m, 5 H) ppm. 13C
NMR (100 MHz): δ = 26.61, 28.19, 39.47, 58.66, 61.95, 67.39,
67.71, 117.48, 127.90, 128.09, 128.50, 134.65, 136.30, 157.46 ppm.
Figure 6. Inhibitory effects a) of the alkaloid 10-epi-239Q, and of
its synthetic analogues b) 12, c) 19, and d) 22 on ACh-induced cur-
rents in Xenopus oocytes expressing α4β2 nicotinic acetylcholine
receptors. Left. Typical traces showing the ACh (1 μm) elicited cur-
rents through α4β2 nicotinic receptors in the absence or presence
of the alkaloids (3 μm). Horizontal bars indicate the period of per-
fusion with ACh for 5 s. Vertical scale bars represent 500 nA.
Right. Concentration/response curves for the alkaloids on α4β2
nicotinic receptors. The current responses to ACh in the presence
of alkaloids in each oocyte were normalized to the ACh response
in the presence of vehicle alone (0.1% dimethyl sulfoxide) in the
same oocyte. Values represents the means ϮS.E.M.s for three to
seven separate experiments.
IR (neat): ν = 3427, 2950, 1682, 1412, 1354, 1111 cm–1. MS (EI):
˜
m/z = 234 [M – C3H5]+. HRMS calcd. for C13H16NO3 [M –
C3H5]+ 234.1130; found 234.1125.
Benzyl (2S,5R)-5-Allyl-2-formylpyrrolidine-1-carboxylate (3): PCC
(127 mg, 0.59 mmol) was added at 0 °C to a stirred solution of
alcohol 2 (108 mg, 0.39 mmol) in CH2Cl2 (5 mL). The resulting
suspension was stirred at room temperature for 20 h. The solvent
was evaporated, and the residue was chromatographed on silica gel
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(13 g, hexane/acetone 20:1 to 15:1) to give 3 (79 mg, 74%) as a
–78 °C to a stirred solution of aldehyde 3 (134 mg, 0.49 mmol) in
colorless oil. The aldehyde was used immediately in the next step.
THF (3 mL) and the reaction mixture was stirred at –78 °C to room
1H NMR (400 MHz, CDCl3): δ = 1.73–1.80 (m, 1 H), 1.96–2.07 temperature for 3 h. The reaction was quenched with aq. NH4Cl
(m, 3 H), 2.11–2.25 (m, 1 H), 2.50 and 2.63 (m, 1 H), 4.02 and 4.10
(each br, 1 H), 4.19 and 4.28 (each br, 1 H), 5.01–5.09 (m, 2 H),
5.13–5.21 (m, 2 H), 5.68–5.81 (m, 1 H), 7.31–7.37 (m, 5 H), 9.46
and 9.55 (each s, 1 H) ppm.
and the aqueous mixture was extracted with CH2Cl2 (10 mL ϫ3).
The organic extracts were combined, dried with Na2SO4, and con-
centrated to afford a residue, which was chromatographed on silica
gel (15 g, hexane/acetone 30:1 to 25:1) to give 4a (65 mg, 48%) as
a colorless oil.
Benzyl
(2S,2αS,5R)-(–)-5-Allyl-2-(1-hydroxybutyl)pyrrolidine-1-
carboxylate (4a) and Benzyl (2S,2αR,5R)-(–)-5-Allyl-2-(1-hy-
droxybutyl)pyrrolidine-1-carboxylate (4b): A solution of nPrMgBr
(1.05 m in THF, 1.27 mL, 1.34 mmol) was added at 0 °C to a stirred
solution of aldehyde 3 (73 mg, 0.27 mmol) in THF (5 mL) and the
reaction mixture was stirred at room temperature for 20 h. The
reaction was quenched with satd. NH4Cl soln. and the aqueous
mixture was extracted with CH2Cl2 (10 mL ϫ3). The extracts were
combined, dried with Na2SO4, and concentrated to afford the resi-
due, which was chromatographed on silica gel (20 g, hexane/acet-
one 30:1 to 25:1) to give 4a and 4b (73 mg, 85%) as a 1:1 mixture
of diastereoisomers and as a colorless oil.
Benzyl
(2S,2αR,5R)-(–)-5-Allyl-2-(1-hydroxybutyl)pyrrolidine-1-
carboxylate (4b) from 5: CeCl3·7H2O (295 mg, 0.79 mmol) was
added at 0 °C to a stirred solution of ketone 5 (100 mg, 0.32 mmol)
in MeOH (4 mL), and the resulting mixture was stirred for 10 min.
NaBH4 (29 mg, 0.76 mmol) was added to the mixture at 0 °C and
the reaction mixture was then stirred at room temperature for 17 h.
The reaction was quenched with H2O and the aqueous mixture was
extracted with EtOAc (5 mL ϫ3). The extracts were combined,
dried with Na2SO4, and concentrated to afford a residue, which
was chromatographed on silica gel (15 g, hexane/acetone 30:1 to
25:1) to give 4a (80 mg, 80%) as a colorless oil. [α]1D7 = –5.5 (c =
1.30, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 0.86–0.98 (m, 3
H), 1.26–1.34 (m, 3 H), 1.59–1.60 (m, 1 H), 1.68–1.78 (m, 2 H),
1.79–1.82 (m, 1 H), 1.84–1.89 (m, 1 H), 2.17–2.23 (m, 1 H), 2.43
(br, 1 H), 3.88–3.90 (m, 1 H), 3.96–3.98 (m, 1 H), 4.03–4.04 (m, 1
H), 5.05 (m, 2 H), 5.13 (s, 2 H), 5.63–5.76 (m, 1 H), 7.26–7.38 (m,
5 H) ppm. 13C NMR (100 MHz): δ = 14.10, 19.44, 24.37, 28.54,
34.53, 39.56, 58.63, 60.35, 67.09, 70.75, 117.28, 127.86, 128.04,
Benzyl (2S,5R)-(–)-5-Allyl-2-butyrylpyrrolidine-1-carboxylate (5):
PCC (127 mg, 0.59 mmol) was added to a stirred solution of
alcohols 4a and 4b (125 mg, 0.39 mmol) in CH2Cl2 (5 mL) and the
resulting mixture was stirred at room temperature for 24 h. The
solvent was evaporated and the residue was chromatographed on
silica gel (20 g, hexane/EtOAc 20:1 to 10:1) to give 5 (113 mg, 91%)
as a colorless oil. [α]2D0 = –20.2 (c = 0.95, CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 0.80 and 0.92 (each t, J = 7.4 Hz, 3 H),
1.45–1.52 (m, 1 H), 1.60–1.67 (m, 2 H), 1.76–1.94 (m, 3 H), 2.05–
2.19 (m, 2 H), 2.32–2.48 (m, 2 H), 4.05 and 3.98 (each br, 1 H),
4.33 and 4.42 (each t, J = 7.8 Hz, 1 H), 5.02 (d, J = 9.0 Hz, 2 H),
5.14 (d, J = 10.3 Hz, 2 H), 5.71–5.85 (m, 1 H), 7.27–7.37 (m, 5
H) ppm. 13C NMR (100 MHz): δ = 13.62 and 13.72, 16.61 and
16.68, 27.05 and 28.04, 28.57 and 29.41, 38.46 and 39.10, 40.60
and 41.38, 58.22 and 58.88, 66.10 and 66.24, 67.11, 117.24, 127.75,
128.07, 128.44, 134.91 and 134.99, 136.31 and 136.55, 154.37 and
128.51, 135.40, 136.55, 156.49 ppm. IR (neat): ν = 3421, 2934,
˜
1684, 1412, 1354, 1103 cm–1. MS (EI): m/z = 276 [M – C3H5]+.
HRMS (EI): calcd. for C16H22O3N [M – C3H5]+ 276.1600; found
276.1590.
(1S,5R,8S)-5-Allyl-1-propyltetrahydropyrrolo[1,2-c]oxazol-3-one (6)
from 4a: NaH (60%, 15 mg, 0.38 mmol) was added at 0 °C to a
stirred solution of alcohol 4a (78 mg, 0.25 mmol) in THF (3 mL)
and DMF (1 mL) and the resulting solution was stirred at room
temperature for 24 h. The reaction was quenched with H2O and
the aqueous mixture was extracted with CH2Cl2 (10 mL ϫ2). The
organic extracts were combined, dried with Na2SO4, and concen-
trated to afford a residue, which was chromatographed on silica gel
(10 g, hexane/acetone 30:1 to 25:1) to give 6 (42 mg, 80%) as a
solid. M.p. 48–50 °C. [α]1D6 = +9.3 (c = 0.75, CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 0.89 (t, J = 7.4 Hz, 3 H), 1.27–1.43 (m, 2
H), 1.47–1.60 (m, 2 H), 1.69–1.78 (m, 1 H), 1.79–1.86 (m, 1 H),
1.93–1.99 (m, 1 H), 2.05–2.15 (m, 1 H), 2.29 (dt, J = 13.8, 8.4 Hz,
1 H), 2.85 (dm, J = 13.8 Hz, 1 H), 3.58 (tt, J = 3.1, 8.4 Hz, 1 H),
3.69 (ddd, J = 6.3, 8.6, 6.8 Hz, 1 H), 4.12 (dt, J = 6.3, 7.6 Hz, 1
H), 5.04 (d, J = 10.2 Hz, 1 H), 5.08 (d, J = 15.9 Hz, 1 H), 5.64–
5.74 (m, 1 H) ppm. 13C NMR (100 MHz): δ = 13.80, 18.14, 28.44,
33.13, 34.91, 36.13, 54.72, 66.75, 82.38, 118.22, 134.161,
155.16, 209.37 and 209.57 ppm. IR (neat): ν = 2956, 1701, 1508,
˜
1458, 1208 cm–1. MS (EI): m/z = 274 [M – C3H5]+. HRMS (EI):
calcd. for C16H20O3N [M – C3H5]+ 274.1444; found 274.1425.
Benzyl
(2S,2αS,5R)-(–)-5-Allyl-2-(1-hydroxybutyl)pyrrolidine-1-
carboxylate (4a) from 5: NaBH4 (15 mg, 0.39 mmol) was added at
0 °C to a stirred solution of ketone 5 (51 mg, 0.16 mmol) in MeOH
(2 mL) and the reaction mixture was stirred at room temperature
for 22 h. The reaction was quenched with HCl (10%) and the aque-
ous mixture was extracted with CH2Cl2 (5 mL ϫ3). The organic
extracts were combined, dried with Na2SO4, and concentrated to
afford a residue, which was chromatographed on silica gel (15 g,
hexane/acetone 30:1 to 25:1) to give 4a (48 mg, 94%) as a colorless
1
oil. [α]1D7 = –23.4 (c = 2.00, CHCl3). H NMR (400 MHz, CDCl3):
155.93 ppm. IR (KBr): ν = 2959, 1740, 1398, 1069 cm–1. MS (EI):
˜
δ = 0.93 (t, J = 7.1 Hz, 3 H), 1.39–1.47 (m, 3 H), 1.50–1.59 (m, 1
H), 1.68–1.75 (m, 2 H), 1.76–1.90 (m, 1 H), 1.93–2.01 (m, 1 H),
2.13–2.20 (m, 1 H), 2.44 (br, 1 H), 3.48 (m, 1 H), 3.87 (m, 1 H),
4.04 (m, 1 H), 5.03 (m, 2 H), 5.12 and 5.19 (ABq, J = 12.2 Hz, 2
H), 5.71 (m, 1 H), 7.29–7.39 (m, 5 H) ppm. 13C NMR (100 MHz):
δ = 14.24, 18.32, 27.20, 28.75, 36.96, 39.53, 58.68, 64.57, 67.54,
76.41, 117.49, 127.93, 128.10, 128.53, 134.66, 136.56, 158.36 ppm.
m/z = 168 [M – C3H5]+. HRMS (EI): calcd. for C9H14O2N [M –
C3H5]+ 168.1024; found 168.1008.
(1S,5R,8S)-5-Allyl-1-propyltetrahydropyrrolo[1,2-c]oxazol-3-one (6)
and (1R,5R,8S)-5-Allyl-1-propyltetrahydropyrrolo[1,2-c]oxazol-3-
one (7) from 4a and 4b: NaH (60%, 10 mg, 0.25 mmol) was added
at 0 °C to a stirred solution of alcohols 4a and 4b (53 mg,
0.17 mmol) in THF (3 mL) and DMF (1 mL) and the resulting
solution was stirred at room temperature for 20 h. The reaction was
quenched with H2O and the aqueous mixture was extracted with
IR (neat): ν = 3442, 2961, 1670, 1508, 1406, 1113 cm–1. MS (EI):
˜
m/z = 276 [M – C3H5]+. HRMS (EI): calcd. for C16H22O3N [M –
C3H5]+ 276.1599; found 276.1577.
Benzyl
(2S,2αS,5R)-(–)-5-Allyl-2-(1-hydroxybutyl)pyrrolidine-1- CH2Cl2 (10 mL ϫ2). The organic extracts were combined, dried
carboxylate (4a) from 3: A solution of (nPr)2Zn [prepared from
ZnCl2 (334 mg, 2.45 mmol) and nPrMgBr (1.05 m in THF,
4.67 mL, 4.90 mmol) at room temperature for 0.5 h] was added at
with Na2SO4, and concentrated to afford a residue, which was chro-
matographed on silica gel (10 g, hexane/acetone 30:1 to 25:1) to
give 6 (16 mg, 45%) and 7 (15 mg, 43%), both as solids.
7088
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 7082–7092

Poison Frog Alkaloid 239Q and Its Congeners
(1R,5R,8S)-5-Allyl-1-propyltetrahydropyrrolo[1,2-c]oxazol-3-one
(7): M.p. 42–44 °C. [α]1D7 = +45.7 (c = 0.40, CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 0.89 (t, J = 7.4 Hz, 3 H), 1.24–1.45 (m, 2
H), 1.47–1.60 (m, 1 H), 1.61–1.71 (m, 3 H), 1.90–1.97 (m, 1 H),
2.03–2.13 (m, 1 H), 2.24 (dt, J = 13.9, 8.4 Hz, 1 H), 2.93 (dm, J =
13.9 Hz, 1 H), 3.55 (tt, J = 3.4, 8.4 Hz, 1 H), 4.13 (ddd, J = 6.0,
8.0, 8.5 Hz, 1 H), 4.44 (dt, J = 4.5, 8.5 Hz, 1 H), 5.04 (d, J =
11.2 Hz, 1 H), 5.08 (d, J = 17.3 Hz, 1 H), 5.65–5.75 (m, 1 H) ppm.
13C NMR (100 MHz): δ = 13.80, 18.74, 23.34, 32.60, 33.23, 34.88,
NMR (100 MHz): δ = 13.60, 14.01, 17.37, 18.21, 27.03, 29.09,
36.75, 37.98, 41.75, 57.92, 64.43, 67.53, 75.87, 127.86, 128.07,
128.43, 132.40, 135.97, 142.25, 156.12, 200.25 ppm. IR (neat): ν =
˜
3466, 2960, 1699, 1670, 1410, 1313, 1113 cm–1. MS (EI): m/z = 387
[M]+. HRMS (EI): calcd. for C23H34NO4 [M + H]+ 388.2488;
found 388.2515.
10-epi-239Q: Pd(OH)2/C (20%, 5 mg) was added to a stirred solu-
tion of 9 (111 mg, 0.29 mmol) in MeOH (5 mL), and the resulting
suspension was stirred under hydrogen at 1 atm for 28 h. The cata-
lyst was removed by filtration and the filtrate was concentrated to
afford a residue, which was chromatographed on silica gel (10 g,
hexane/acetone 25:1 to 15:1) to give 10-epi-239Q (57 mg, 83%) as
a pale yellow oil. [α]2D6 = +84.5 (c = 1.25, CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 0.90 (t, J = 7.1 Hz, 3 H), 0.93 (t, J =
6.8 Hz, 3 H), 1.01–1.10 (m, 1 H), 1.17–1.25 (m, 4 H), 1.28–1.38 (m,
4 H), 1.42–1.47 (m, 2 H), 1.51–1.57 (m, 2 H), 1.64–1.70 (m, 2
H),1.72–1.85 (m, 3 H), 2.28 (t-like, J = 9.7 Hz, 1 H), 2.35 (t-like, J
= 10.2 Hz, 1 H), 2.83 (t, J = 7.3 Hz, 1 H), 2.96 (br, 1 H) ppm. 13C
NMR (100 MHz): δ = 14.29, 14.39, 19.67, 19.80, 25.28, 29.53,
30.60, 31.11, 33.15, 36.91, 38.19, 62.66, 63.78, 67.99, 74.30 ppm.
54.79, 63.60, 76.30, 118.07, 134.41, 155.70 ppm. IR (KBr): ν =
˜
2934, 1747, 1404, 1063 cm–1. MS (EI): m/z = 168 [M – C3H5]+.
HRMS (EI): calcd. for C9H14O2N [M – C3H5]+ 168.1024; found
168.1006.
Benzyl (2S,2αR,5R)-(+)-2-(1-Hydroxybutyl)-5-[(E)-4-oxohept-2-en-
yl]pyrrolidine-1-carboxylate (8): Hex-1-en-3-one (0.11 mL,
1.00 mmol) and the second-generation Grubbs catalyst (16 mg,
0.02 mmol) were added to a stirred solution of alcohol 4b (62 mg,
0.20 mmol) in CH2Cl2 (3 mL), and the reaction mixture was heated
at reflux for 23 h. After the mixture had cooled, the solvent was
evaporated, and the residue was chromatographed on silica gel
(15 g, hexane/acetone 15:1 to 10:1) to give 8 (71 mg, 94%) as a pale
yellow oil. [α]2D6 = +8.4 (c = 1.35, CHCl3).1H NMR (400 MHz,
CDCl3): δ = 0.91 (t, J = 7.3 Hz, 6 H), 1.26–1.41 (m, 3 H), 1.56–
1.65 (m, 4 H), 1.70 (br, 1 H), 1.87–2.01 (m, 2 H), 2.33 (br, 1 H),
2.40–2.44 (m, 2 H), 2.60–2.68 (br, 1 H), 3.82–3.92 (m, 1 H), 3.97
(br, 1 H), 4.10 (br, 1 H), 5.10 and 5.16 (ABq, J = 12.2 Hz, 2 H),
6.07 (d, J = 15.6 Hz, 1 H), 6.78 (br, 1 H), 7.32–7.35 (m, 5 H) ppm.
13C NMR (100 MHz): δ = 13.69, 14.09, 17.49, 19.31, 23.94, 28.99,
34.85, 38.02, 42.03, 58.02, 64.88, 67.12, 70.43, 127.83, 128.05,
IR (neat): ν = 3433, 2957, 2870, 2761, 1456, 1119 cm–1. MS (EI):
˜
m/z = 239 [M]+. HRMS (EI): calcd. for C15H30NO [M + H]+
240.2328; found 240.2315.
Benzyl (2S,2αS,5R)-2-(1-Hydroxybutyl)-5-[(E)-4-oxoundec-2-enyl]-
pyrrolidine-1-carboxylate (10): Dec-1-en-3-one (95 mg, 0.61 mmol)
and the second-generation Grubbs catalyst (16 mg, 0.02 mmol)
were added to a stirred solution of alcohol 4a (65 mg, 0.21 mmol)
in CH2Cl2 (3 mL) and the reaction mixture was heated at reflux
for 21 h. After the mixture had cooled, the solvent was evaporated
and the residue was chromatographed on silica gel (15 g, hexane/
acetone 15:1 to 10:1) to give 10 (73 mg, 80%) as a pale yellow oil.
128.47, 131.97, 136.32, 143.04, 156.00, 200.39 ppm. IR (neat): ν =
˜
3447, 2959, 1696, 1676, 1409, 1319, 1103 cm–1. MS (EI): m/z =
387 [M]+. HRMS (EI): calcd. for C23H33NO4 [M]+ 387.2409; found
387.2379.
1
[α]2D6 = +6.1 (c = 0.80, CHCl3). H NMR (400 MHz, CDCl3): δ =
0.88 (t, J = 6.8 Hz, 3 H), 0.92 (t, J = 6.8 Hz, 3 H), 1.21–1.32 (m,
9 H), 1.35–1.48 (m, 2 H), 1.53–1.63 (m, 3 H), 1.65–1.72 (m, 2 H),
1.86–2.03 (m, 2 H), 2.36 (br, 1 H), 2.47 (t, J = 7.4 Hz, 2 H), 2.57
(br, 1 H), 3.39–3.49 (m, 1 H), 3.85–3.93 (m, 1 H), 4.06–4.16 (m, 1
H), 5.16 (s, 2 H), 6.07 (d, J = 16.1 Hz, 1 H), 6.66–6.78 (m, 1 H),
7.29–7.39 (m, 5 H) ppm. 13C NMR (100 MHz): δ = 14.00, 14.10,
18.32, 22.52, 22.53, 24.06, 27.16, 29.02, 29.17, 31.62, 36.92, 38.09,
40.09, 58.03, 64.44, 67.65, 76.68, 127.97, 128.18, 128.53, 132.48,
239Q: Pd(OH)2/C (20%, 5 mg) was added to a stirred solution of
8 (132 mg, 0.34 mmol) in MeOH (5 mL), and the resulting suspen-
sion was stirred under hydrogen at 1 atm for 24 h. The catalyst was
removed by filtration and the filtrate was concentrated to afford a
residue, which was chromatographed on silica gel (7 g, hexane/acet-
one 25:1 to 15:1) to give 239Q (67 mg, 83%) as a pale yellow oil.
[α]2D6 = +50.9 (c = 1.35, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
0.88 (t, J = 7.6 Hz, 3 H), 0.92 (t, J = 7.4 Hz, 3 H) 1.02–1.16 (m, 2
H), 1.17–1.42 (m, 7 H), 1.43–1.60 (m, 4 H), 1.63–1.68 (m, 1 H),
1.69–1.84 (m, 4 H), 2.20–2.35 (m, 2 H), 2.85 (d-like, J = 10.0 Hz,
1 H), 3.58 (br, 1 H) ppm. 13C NMR (100 MHz): δ = 14.36, 14.41,
19.03, 19.59, 22.92, 24.85, 31.63, 32.07, 32.16, 35.58, 37.72, 64.25,
136.08, 142.24, 157.98, 200.50 ppm. IR (neat): ν = 3495, 2951,
˜
1697, 1670, 1413, 1356, 1113 cm–1. MS (EI): m/z = 443 [M]+.
HRMS (EI): calcd. for C27H41NO4 [M]+ 443.3036; found 443.3040.
(3S,3αS,5S,9S)-5-Heptyl-3-(1-hydroxybutyl)octahydroindolizine
(12): Pd(OH)2/C (20%, 5 mg) was added to a stirred solution of 10
(120 mg, 0.27 mmol) in MeOH (5 mL), and the resulting suspen-
sion was stirred under hydrogen at 1 atm for 23 h. The catalyst was
removed by filtration and the filtrate was concentrated to give a
residue, which was chromatographed on silica gel (10 g, hexane/
acetone 40:1 to 30:1) to give 12 (63 mg, 79%) as a pale yellow oil.
[α]2D6 = +63.4 (c = 1.10, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
0.88 (t, J = 6.8 Hz, 3 H), 0.94 (t, J = 6.9 Hz, 3 H), 1.01–1.11 (m,
1 H), 1.13–1.19 (m, 2 H), 1.20–1.31 (m, 9 H), 1.32–1.39 (m, 4 H),
1.42–1.47 (m, 2 H), 1.52–1.62 (m, 3 H), 1.64–1.71 (m, 2 H), 1.73–
1.78 (m, 2 H), 1.83–1.87 (m, 1 H), 2.27 (t-like, J = 10.5 Hz, 1 H),
2.35 (t-like, J = 10.5 Hz, 1 H), 2.83 (t-like, J = 7.4 Hz, 1 H), 2.94–
2.99 (m, 1 H) ppm. 13C NMR (100 MHz): δ = 14.04, 14.27, 19.61,
22.61, 25.25, 26.68, 29.33, 29.51, 29.83, 30.60, 31.11, 31.78, 33.16,
64.51, 67.10, 72.44 ppm. IR (neat): ν = 3452, 2957, 2932, 2870,
˜
2795, 1456 cm–1. MS (EI): m/z = 239 [M]+. HRMS (EI): calcd. for
C15H29NO [M]+ 239.2249; found 239.2251.
Benzyl (2S,2αS,5R)-(+)-2-(1-Hydroxybutyl)-5-[(E)-4-oxohept-2-en-
yl]pyrrolidine-1-carboxylate (9): Hex-1-en-3-one (0.09 mL,
0.79 mmol) and the second-generation Grubbs catalyst (13 mg,
0.016 mmol) were added to a stirred solution of alcohol 4a (50 mg,
0.16 mmol) in CH2Cl2 (3 mL), and the reaction mixture was heated
at reflux for 20 h. After the mixture had cooled, the solvent was
evaporated, and the residue was chromatographed on silica gel
(10 g, hexane/acetone 20:1 to 10:1) to give 9 (60 mg, 98%) as a pale
1
yellow oil. [α]2D6 = +16.0 (c = 1.00, CHCl3). H NMR (400 MHz,
34.75, 35.97, 62.68, 64.02, 67.96, 74.29 ppm. IR (neat): ν = 3368,
˜
CDCl3): δ = 0.90 (t, J = 7.3 Hz, 6 H), 1.33–1.44 (m, 3 H), 1.53–
1.60 (m, 3 H), 1.62–1.67 (m, 2 H), 1.91–1.95 (m, 2 H), 2.36 (br, 1
H), 2.43 (t-like, J = 7.2 Hz, 2 H), 2.53 (br, 1 H), 3.41–3.45 (m, 1
2928, 2858, 2793, 1456, 1302, 1109 cm–1. MS (EI): m/z = 295
[M]+. HRMS (EI): calcd. for C19H37NO 295.2875; found 295.2861.
H), 3.86–3.87 (m, 1 H), 4.09 (br, 1 H), 5.12 (s, 2 H), 6.05 (d, J = Benzyl (2S,2αR,5R)-2-(1-Hydroxybutyl)-5-[(E)-4-oxoundec-2-enyl]-
15.6 Hz, 1 H), 6.69–6.72 (m, 1 H), 7.28–7.36 (m, 5 H) ppm. 13C pyrrolidine-1-carboxylate (11): Dec-1-en-3-one (95 mg, 0.61 mmol)
Eur. J. Org. Chem. 2012, 7082–7092
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7089

H. Tsuneki, N. Toyooka, et al.
FULL PAPER
and the second-generation Grubbs catalyst (16 mg, 0.02 mmol)
were added to a stirred solution of alcohol 4b (65 mg, 0.21 mmol)
in CH2Cl2 (4 mL), and the reaction mixture was heated at reflux
for 20 h. After the mixture had cooled, the solvent was evaporated,
and the residue was chromatographed on silica gel (15 g, hexane/
acetone 20:1 to 10:1) to give 11 (87 mg, 96%) as a pale yellow oil.
[α]2D6 = +21.9 (c = 0.60, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
0.88 (t, J = 6.9 Hz, 3 H), 0.92 (br, 3 H), 1.21–1.37 (m, 11 H), 1.51–
1.62 (m, 3 H), 1.70 (br, 1 H), 1.81–1.93 (m, 3 H), 2.34 (br, 1 H),
2.40–2.49 (m, 2 H), 2.60 (br, 1 H), 3.88 (br, 1 H), 3.94–4.01 (m, 1
H), 4.06–4.14 (m, 1 H), 5.10 and 5.17 (ABq, J = 12.3 Hz, 2 H),
6.08 (d, J = 15.9 Hz, 1 H), 6.76 (br, 1 H), 7.31–7.39 (m, 5 H) ppm.
13C NMR (100 MHz): δ = 13.95, 14.05, 19.29, 22.49, 23.83, 24.05,
28.97, 29.12, 31.56, 34.91, 37.96, 40.01, 40.18, 57.99, 64.79, 67.06,
70.38, 127.78, 128.00, 128.43, 131.91, 136.31, 143.04, 155.92,
C3H5]+. HRMS: calcd. for C19H26NO3 [M – C3H5]+ 316.1913;
found 316.1914.
Benzyl (2S,2αS,5R)-5-Allyl-2-(1-hydroxyheptyl)pyrrolidine-1-carb-
oxylate (15): NaBH4 (44 mg, 1.15 mmol) was added at 0 °C to a
stirred solution of ketone 14 (170 mg, 0.48 mmol) in MeOH
(5 mL), and the reaction mixture was stirred at room temperature
for 24 h. The reaction was quenched with HCl (10%) and the aque-
ous mixture was extracted with CH2Cl2 (5 mL ϫ3). The organic
extracts were combined, dried with Na2SO4, and concentrated to
afford a residue, which was chromatographed on silica gel (15 g,
hexane/acetone 30:1 to 25:1) to give 15 (155 mg, 90%) as a colorless
oil. [α]2D2 = –26.4 (c = 0.75, CHCl3). H NMR (400 MHz, CDCl3):
1
δ = 0.88 (t, J = 6.8 Hz, 3 H), 1.22–1.35 (m, 8 H), 1.42–1.54 (m, 2
H), 1.64–1.75 (m, 2 H), 1.80–1.88 (m, 1 H), 1.91–1.99 (m, 1 H),
2.12–2.21 (m, 1 H), 2.44 (br, 1 H), 3.47 (t-like, J = 8.1 Hz, 1 H),
3.84–3.89 (m, 1 H), 4.00–4.07 (m, 1 H), 4.98–5.07 (m, 2 H), 5.12
and 5.19 (ABq, J = 12.3 Hz, 2 H), 5.66–5.78 (m, 1 H), 7.29–7.34
(m, 5 H) ppm. 13C NMR (100 MHz): δ = 14.06, 22.60, 25.03, 27.16,
28.73, 29.46, 31.83, 34.74, 39.49, 58.65, 64.52, 67.49, 76.51, 117.44,
200.47 ppm. IR (neat): ν = 3485, 2930, 1699, 1682, 1409, 1350,
˜
1101 cm–1. MS (EI): m/z = 443 [M]+. HRMS (EI): calcd. for
C27H41NO4 443.3036 [M]+; found 443.3041.
(3S,3αR,5S,9S)-5-Heptyl-3-(1-hydroxybutyl)octahydroindolizine
(13): Pd(OH)2/C (20%, 5 mg) was added to a stirred solution of 11 127.89, 128.06, 128.49, 134.62, 136.31, 158.25 ppm. IR (neat): ν =
˜
(161 mg, 0.36 mmol) in MeOH (5 mL), and the resulting suspen-
sion was stirred under hydrogen at 1 atm for 26 h. The catalyst was
removed by filtration and the filtrate was concentrated to give a
residue, which was chromatographed on silica gel (10 g, hexane/
acetone 30:1 to 20:1) to give 13 (97 mg, 90%) as a pale yellow oil.
[α]2D6 = +48.2 (c = 1.50, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
0.86 (t, J = 6.5 Hz, 3 H), 0.92 (t, J = 7.0 Hz, 3 H), 1.01–1.11 (m,
1 H), 1.13–1.19 (m, 2 H), 1.20–1.38 (m, 13 H), 1.41–1.54 (m, 3 H),
1.57–1.68 (m, 3 H), 1.71–1.82 (m, 4 H), 2.21–2.34 (m, 2 H), 2.84 (d-
like, J = 10.2 Hz, 1 H), 3.58 (br, 1 H) ppm. 13C NMR (100 MHz): δ
= 14.02, 14.33, 19.55, 22.60, 22.90, 24.84, 25.96, 29.26, 29.96, 31.61,
31.78, 32.06, 32.20, 35.51, 35.54, 64.51, 64.52, 67.08, 72.48 ppm.
3437, 2930, 1682, 1418, 1101 cm–1. MS (EI): m/z = 318 [M –
C3H5]+. HRMS: calcd. for C19H28NO3 [M – C3H5]+ 318.2070;
found 318.2061.
Benzyl (2S,2αR,5R)-(+)-5-Allyl-2-(1-hydroxyheptyl)pyrrolidine-1-
carboxylate (16): CeCl3·7H2O (391 mg, 1.05 mmol) was added at
0 °C to a stirred solution of ketone 14 (150 mg, 0.42 mmol) in
MeOH (4 mL) and the resulting mixture was stirred for 10 min.
NaBH4 (38 mg, 1.01 mmol) was added to the mixture at 0 °C, and
the mixture was then stirred at room temperature for 21 h. The
reaction was quenched with H2O, and the aqueous mixture was
extracted with EtOAc (10 mL ϫ3). The extracts were combined,
dried with Na2SO4, and concentrated to afford a residue, which
was chromatographed on silica gel (20 g, hexane/acetone 30:1 to
20:1) to give 16 (120 mg, 80%) as a colorless oil. [α]2D2 = +2.1 (c =
0.75, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 0.86 (t, J = 6.8 Hz,
3 H), 1.20–1.27 (m, 7 H), 1.29–1.35 (m, 2 H), 1.43–1.48 (m, 1 H),
1.66–1.73 (m, 1 H), 1.75–1.87 (m, 3 H), 2.10–2.21 (m, 1 H), 2.41
(br, 1 H), 3.82–3.87 (m, 1 H), 3.89–3.95 (m, 1 H), 3.96–4.05 (m, 1
H), 4.98–5.03 (m, 2 H), 5.12 (s, 2 H), 5.63–5.72 (m, 1 H), 7.27–7.37
(m, 5 H) ppm. 13C NMR (100 MHz): δ = 14.05, 22.56, 24.26, 26.21,
28.52, 29.30, 31.78, 32.50, 39.41, 58.59, 65.07, 67.02, 71.02, 117.20,
IR (neat): ν = 3456, 2930, 2858, 2794, 1456, 1379, 1103 cm–1. MS
˜
(EI): m/z = 295 [M]+. HRMS (EI): calcd. for C19H37NO 295.2875
[M]+; found 295.2849.
Benzyl (2S,5R)-(–)-5-Allyl-2-heptanoylpyrrolidine-1-carboxylate
(14): A solution of n-hexylMgBr (2.0 m in THF, 2.60 mL,
5.20 mmol) was added at 0 °C to a stirred solution of aldehyde 3
(284 mg, 1.04 mmol) in THF (10 mL), and the reaction mixture
was stirred at room temperature for 24 h. The reaction was
quenched with satd. NH4Cl soln. and the aqueous mixture was
extracted with CH2Cl2 (10 mL ϫ3). The extracts were combined,
dried with Na2SO4, and concentrated to afford the residue, which
was chromatographed on silica gel (25 g, hexane/acetone 30:1 to
25:1) to give 10 (280 mg, 75%) as a 1:1 mixture of diastereomers
and as a colorless oil.
127.79, 127.98, 128.45, 135.35, 136.52, 156.33 ppm. IR (neat): ν =
˜
3437, 2930, 1682, 1418, 1107 cm–1. MS (EI): m/z = 318 [M –
C3H5]+. HRMS: calcd. for C19H28NO3 [M – C3H5]+ 318.2070;
found 318.2071.
Benzyl (2S,2αS,5R)-2-(1-Hydroxyheptyl)-5-[(E)-4-oxohept-2-enyl]-
pyrrolidine-1-carboxylate (17): Hex-1-en-3-one (0.09 mL,
0.73 mmol) and the second-generation Grubbs catalyst (12 mg,
PCC (50 mg, 0.23 mmol) was added to a stirred solution of the
alcohols obtained above (55 mg, 0.15 mmol) in CH2Cl2 (2 mL), and
the resulting mixture was stirred at room temperature for 23 h. The 0.014 mmol) were added to a stirred solution of alcohol 15 (52 mg,
solvent was evaporated and the residue was chromatographed on
silica gel (13 g, hexane/EtOAc 20:1 to 10:1) to give 14 (48 mg, 89%)
as a colorless oil. [α]2D6 = –22.9 (c = 0.70, CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 0.84 (t, J = 6.5 Hz, 3 H), 1.06–1.32 (m, 7
H), 1.50–1.63 (m, 3 H), 1.69–1.95 (m, 3 H), 2.05–2.22 (m, 2 H),
2.40–2.64 (m, 1 H), 3.96 and 4.02 (each br, 1 H), 4.31 and 4.40
(each t, J = 7.6 Hz, 1 H), 4.97–5.15 (m, 4 H), 5.65–5.85 (m, 1 H),
0.15 mmol) in CH2Cl2 (3 mL), and the reaction mixture was heated
at reflux for 23 h. After the mixture had cooled, the solvent was
evaporated, and the residue was chromatographed on silica gel
(12 g, hexane/acetone 20:1 to 10:1) to give 17 (58 mg, 94%) as a
pale yellow oil. [α]2D6 = +2.0 (c = 0.50, CHCl3). 1H NMR (400 MHz,
CDCl3): δ = 0.88 (t, J = 6.7 Hz, 3 H), 0.91 (t, J = 7.4 Hz, 3 H),
1.22–1.40 (m, 7 H), 1.43–1.54 (m, 2 H), 1.56–1.73 (m, 5 H), 1.88–
7.27–7.33 (m, 5 H) ppm. 13C NMR (100 MHz): δ = 13.99, 22.42, 2.03 (m, 2 H), 2.31–2.40 (m, 1 H), 2.46 (t-like, J = 7.2 Hz, 2 H),
23.14, 27.07 and 28.06, 28.55 and 28.84, 28.79 and 29.64, 31.54,
38.43 and 38.74, 39.07 and 39.46, 58.18 and 58.84, 66.06 and 66.21,
67.07, 117.21, 127.72, 128.03, 128.41, 134.89 and 134.98, 136.26
and 136.52, 154.34 and 155.12, 209.50 and 209.70 ppm. IR (neat):
2.52–2.68 (m, 1 H), 3.38–3.47 (m, 1 H), 3.85–3.93 (m, 1 H), 4.06–
4.14 (m, 1 H), 5.15 (s, 2 H), 6.07 (d, J = 15.6 Hz, 1 H), 6.67–6.77
(m, 1 H), 7.29–7.39 (m, 5 H) ppm. 13C NMR (100 MHz): δ = 13.68,
13.99, 17.45, 22.52, 25.02, 27.12, 29.17, 29.35, 31.74, 34.73, 38.06,
41.85, 58.00, 64.28, 67.62, 76.24, 127.94, 128.16, 128.50, 132.49,
ν = 2930, 1703, 1410, 1352, 1111 cm–1. MS (EI): m/z = 316 [M –
˜
7090
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 7082–7092

Poison Frog Alkaloid 239Q and Its Congeners
136.04, 142.26, 158.02, 200.34 ppm. IR (neat): ν = 3418, 2930,
were added to a stirred solution of alcohol 15 (110 mg, 0.31 mmol)
in CH2Cl2 (5 mL), and the reaction mixture was heated at reflux
for 23 h. After the mixture had cooled, the solvent was evaporated
and the residue was chromatographed on silica gel (15 g, hexane/
acetone 20:1 to 10:1) to give 21 (147 mg, 99%) as a pale yellow oil.
˜
1693, 1666, 1408, 1103 cm–1. MS (EI): m/z = 429 [M]+. HRMS
(EI): calcd. for C26H40NO4 [M + H]+ 430.2957; found 430.2952.
Benzyl (2S,2αR,5R)-2-(1-Hydroxyheptyl)-5-[(E)-4-oxohept-2-enyl]-
pyrrolidine-1-carboxylate (18): Hex-1-en-3-one (0.12 mL,
1.02 mmol) and the second-generation Grubbs catalyst (17 mg,
0.02 mmol) were added to a stirred solution of alcohol 16 (73 mg,
0.20 mmol) in CH2Cl2 (4 mL), and the reaction mixture was heated
at reflux for 27 h. After the mixture had cooled, the solvent was
evaporated and the residue was chromatographed on silica gel
(13 g, hexane/acetone 20:1 to 10:1) to give 18 (86 mg, 99%) as a
pale yellow oil. [α]2D6 = +3.0 (c = 0.55, CHCl3). 1H NMR (400 MHz,
CDCl3): δ = 0.88 (t, J = 6.2 Hz, 3 H), 0.91 (t, J = 7.4 Hz, 3 H),
1.21–1.32 (m, 7 H), 1.33–1.58 (m, 4 H), 1.58–1.64 (m, 2 H), 1.65–
1.74 (m, 1 H), 1.82–1.93 (m, 2 H), 2.26–2.33 (m, 1 H), 2.34–2.48
(m, 2 H), 2.52–2.70 (m, 1 H), 3.80–3.92 (m, 1 H), 3.93–3.99 (m, 1
H), 4.05–4.15 (m, 1 H), 5.10 and 5.16 (ABq, J = 12.3 Hz, 2 H),
6.08 (d, J = 16.1 Hz, 1 H), 6.69–6.84 (m, 1 H), 7.27–7.38 (m, 5
H) ppm. 13C NMR (100 MHz): δ = 13.68, 13.99, 17.48, 22.50,
23.88, 26.12, 29.00, 29.23, 31.72, 32.78, 38.00, 42.02, 58.02, 64.81,
67.10, 70.72, 127.80, 128.03, 128.45, 132.34, 136.33, 143.10, 155.98,
1
[α]2D6 = +4.5 (c = 1.05, CHCl3). H NMR (400 MHz, CDCl3): δ =
0.87 (t, J = 7.0 Hz, 3 H), 0.88 (t, J = 6.8 Hz, 3 H), 1.20–1.34 (m,
16 H), 1.42–1.49 (m, 2 H), 1.53–1.58 (m, 2 H), 1.64–1.71 (m, 2 H),
1.87–2.03 (m, 2 H), 2.35 (br, 1 H), 2.47 (t, J = 7.4 Hz, 2 H), 2.59
(br, 1 H), 3.38–3.46 (m, 1 H), 3.86–3.92 (m, 1 H), 4.06–4.14 (m, 1
H), 5.15 (s, 2 H), 6.07 (d, J = 15.6 Hz, 1 H), 6.67–6.78 (m, 1 H),
7.30–7.39 (m, 5 H) ppm. 13C NMR (100 MHz): δ = 13.90, 13.92,
22.44, 23.96, 25.00, 27.04, 28.94, 29.07, 29.10, 29.29, 31.52, 31.69,
34.62, 37.98, 39.91, 53.30, 57.95, 64.32, 67.51, 76.09, 127.84,
128.06, 128.42, 132.39, 136.01, 142.18, 157.93, 200.35 ppm. IR
(neat): ν = 3456, 2927, 1699, 1674, 1410, 1101 cm–1. MS (EI): m/z
˜
= 485 [M]+. HRMS (EI): calcd. for C30H47NO4 [M]+ 485.3505;
found 485.3498.
(3S,3αS,5S,9S)-5-Heptyl-3-(1-hydroxyheptyl)octahydroindolizine
(22): Pd(OH)2/C (20%, 5 mg) was added to a stirred solution of 21
(146 mg, 0.30 mmol) in MeOH (5 mL), and the resulting suspen-
sion was stirred under hydrogen at 1 atm for 22 h. The catalyst was
removed by filtration and the filtrate was concentrated to give a
residue, which was chromatographed on silica gel (10 g, hexane/
acetone 40:1 to 30:1) to give 22 (67 mg, 66%) as a pale yellow oil.
[α]2D6 = +53.4 (c = 1.50, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
0.86–0.90 (m, 6 H), 1.01–1.10 (m, 1 H), 1.16–1.19 (m, 2 H), 1.20–
1.36 (m, 18 H), 1.39–1.47 (m, 3 H), 1.51–1.73 (m, 5 H), 1.75–1.79
(m, 2 H), 1.82–1.86 (m, 1 H), 2.27 (t-like, J = 9.8 Hz, 1 H), 2.35
(t-like, J = 9.8 Hz, 1 H), 2.84 (t-like, J = 7.3 Hz, 1 H), 2.95 (br, 1
H) ppm. 13C NMR (100 MHz): δ = 14.07, 14.08, 22.63, 22.64,
25.28, 26.44, 26.71, 29.35, 29.55, 29.68, 29.85, 30.62, 31.13, 31.82,
31.90, 33.20, 34.75, 36.00, 62.66, 64.03, 67.78, 74.56 ppm. IR
200.37 ppm. IR (neat): ν = 3452, 2930, 1692, 1678, 1408, 1352,
˜
1105 cm–1. MS (EI): m/z = 429 [M]+. HRMS (EI): calcd. for
C26H40NO4 [M + H]+ 430.2957; found 430.2965.
(3S,3αS,5S,9S)-3-(1-Hydroxyheptyl)-5-propyloctahydroindolizine
(19): Pd(OH)2/C (20%, 5 mg) was added to a stirred solution of 17
(100 mg, 0.23 mmol) in MeOH (5 mL), and the resulting suspen-
sion was stirred under hydrogen at 1 atm for 23 h. The catalyst was
removed by filtration and the filtrate was concentrated to give a
residue, which was chromatographed on silica gel (7 g, hexane/acet-
one 50:1 to 40:1) to give 19 (46 mg, 71%) as a pale yellow oil.
[α]2D6 = +72.9 (c = 0.70, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
0.88 (t, J = 7.1 Hz, 3 H), 0.91 (t, J = 7.2 Hz, 3 H), 1.00–1.12 (m,
1 H), 1.14–1.27 (m, 4 H), 1.28–1.42 (m, 9 H), 1.44–1.47 (m, 2 H),
1.48–1.58 (m, 1 H), 1.59–1.62 (m, 2 H), 1.63–1.72 (m, 2 H), 1.73–
1.81 (m, 2 H), 1.82–1.86 (m, 1 H),2.25–2.39 (m, 2 H), 2.84 (t-like,
J = 7.3 Hz, 1 H), 2.93–2.97 (m, 1 H) ppm. 13C NMR (100 MHz):
δ = 14.07, 14.37, 19.79, 22.61, 25.26, 26.45, 29.52, 29.45, 30.60,
31.11, 31.89, 33.13, 34.68, 38.19, 62.69, 63.79, 67.98, 74.53 ppm.
(neat): ν = 3408, 2926, 2856, 2797, 1456, 1379, 1142 cm–1. MS (EI):
˜
m/z = 337 [M]+. HRMS (EI): calcd. for C22H43NO [M]+ 337.3344;
found 337.3319.
Benzyl (2S,2αS,5R)-2-(1-Hydroxyheptyl)-5-[(E)-4-oxoundec-2-enyl]-
pyrrolidine-1-carboxylate (23): Dec-1-en-3-one (134 mg, 0.87 mmol)
and the second-generation Grubbs catalyst (25 mg, 0.029 mmol)
were added to a stirred solution of alcohol 16 (104 mg, 0.29 mmol)
in CH2Cl2 (5 mL) and the reaction mixture was heated at reflux
for 22 h. After the mixture had cooled, the solvent was evaporated,
and the residue was chromatographed on silica gel (20 g, hexane/
acetone 20:1 to 10:1) to give 23 (110 mg, 79%) as a pale yellow oil.
IR (neat): ν = 3375, 2930, 2858, 2793, 1468 cm–1. MS (EI): m/z =
˜
281 [M]+. HRMS (EI): calcd. for C18H35NO [M]+ 281.2719; found
281.2710.
(3S,3αR,5S,9S)-3-(1-Hydroxyheptyl)-5-propyloctahydroindolizine
(20): Pd(OH)2/C (20%, 5 mg) was added to a stirred solution of 18
(145 mg, 0.34 mmol) in MeOH (5 mL), and the resulting suspen-
sion was stirred under hydrogen at 1 atm for 25 h. The catalyst was
removed by filtration and the filtrate was concentrated to give a
residue, which was chromatographed on silica gel (10 g, hexane/
acetone 50:1 to 40:1) to give 20 (84 mg, 88%) as a pale yellow oil.
[α]2D6 = +51.8 (c = 0.80, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
0.88 (t, J = 7.0 Hz, 3 H), 0.91 (t, J = 7.2 Hz, 3 H), 1.02–1.12 (m,
1 H), 1.13–1.17 (m, 1 H), 1.18–1.23 (m, 2 H), 1.24–1.34 (m, 9 H),
1.37–1.54 (m, 4 H), 1.55–1.62 (m, 1 H), 1.63–1.71 (m, 2 H), 1.72–
1.74 (m, 1 H), 1.75–1.84 (m, 3 H),2.23–2.37 (m, 2 H), 2.83–2.89
(m, 1 H), 4.54–4.60 (m, 1 H) ppm. 13C NMR (100 MHz): δ = 14.05,
14.42, 19.03, 22.60, 22.90, 24.84, 26.30, 29.58, 31.62, 31.79, 32.06,
1
[α]2D6 = +4.5 (c = 1.05, CHCl3). H NMR (400 MHz, CDCl3): δ =
0.87 (t, J = 7.0 Hz, 3 H), 0.88 (t, J = 6.8 Hz, 3 H), 1.20–1.34 (m,
16 H), 1.42–1.49 (m, 2 H), 1.53–1.58 (m, 2 H), 1.64–1.71 (m, 2 H),
1.87–2.03 (m, 2 H), 2.35 (br, 1 H), 2.47 (t, J = 7.4 Hz, 2 H), 2.59
(br, 1 H), 3.38–3.46 (m, 1 H), 3.86–3.92 (m, 1 H), 4.06–4.14 (m, 1
H), 5.15 (s, 2 H), 6.07 (d, J = 15.6 Hz, 1 H), 6.67–6.78 (m, 1 H),
7.30–7.39 (m, 5 H) ppm. 13C NMR (100 MHz): δ = 13.90, 13.92,
22.44, 23.96, 25.00, 27.04, 28.94, 29.07, 29.10, 29.29, 31.52, 31.69,
34.62, 37.98, 39.91, 53.30, 57.95, 64.32, 67.51, 76.09, 127.84,
128.06, 128.42, 132.39, 136.01, 142.18, 157.93, 200.35 ppm. IR
(neat): ν = 3456, 2927, 1699, 1674, 1410, 1101 cm–1. MS (EI): m/z
˜
= 485 [M]+. HRMS (EI): calcd. for C30H47NO4 [M + H]+ 486.3583;
found 486.3592.
32.15, 33.37, 37.72, 64.26, 64.40, 67.11, 72.64 ppm. IR (neat): ν =
˜
3452, 2930, 2858, 2795, 1456 cm–1. MS (EI): m/z = 281 [M]+.
(3S,3αR,5S,9S)-5-Heptyl-3-(1-hydroxyheptyl)octahydroindolizine
(24): Pd(OH)2/C (20%, 5 mg) was added to a stirred solution of 23
(110 mg, 0.23 mmol) in MeOH (5 mL), and the resulting suspen-
HRMS: calcd. for C18H35NO [M]+ 281.2719; found 281.2698.
Benzyl (2S,2αS,5R)-2-(1-Hydroxyheptyl)-5-[(E)-4-oxoundec-2-enyl]-
pyrrolidine-1-carboxylate (21): Dec-1-en-3-one (141 mg, 0.92 mmol) sion was stirred under hydrogen at 1 atm for 24 h. The catalyst was
and the second-generation Grubbs catalyst (26 mg, 0.03 mmol) removed by filtration and the filtrate was concentrated to give a
Eur. J. Org. Chem. 2012, 7082–7092
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7091

H. Tsuneki, N. Toyooka, et al.
FULL PAPER
residue, which was chromatographed on silica gel (10 g, hexane/
acetone 40:1 to 30:1) to give 24 (47 mg, 62%) as a pale yellow oil.
[α]2D6 = +32.3 (c = 1.10, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
0.88 (t, J = 6.8 Hz, 6 H), 1.02–1.07 (m, 1 H), 1.08–1.14 (m, 1 H),
1.15–1.20 (m, 1 H), 1.23–1.36 (m, 18 H), 1.40–1.46 (m, 1 H), 1.47–
1.53 (m, 1 H), 1.54–1.63 (m, 5 H), 1.64–1.71 (m, 1 H), 1.72–1.78
(m, 2 H),1.79–1.84 (m, 1 H), 2.22–2.34 (m, 2 H), 2.83–3.61 (m, 1
H), 3.54–3.61 (m, 1 H) ppm. 13C NMR (100 MHz): δ = 14.06,
14.40, 22.62, 22.91, 24.86, 25.99, 26.29, 29.19, 29.28, 29.58, 29.99,
31.64, 31.81, 31.91, 32.09, 32.24, 33.36, 35.53, 64.50, 64.55, 67.13,
Michigan Medical Center) for providing us with plasmid DNA. We
also thank Dr. J. A. Dani (Baylor College of Medicine, TX), Dr. S.
Kobayashi, K. Yamaguchi, and M. Fujita (University of Toyama,
Japan) for their technical assistance in the pharmacological assay.
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72.69 ppm. IR (neat): ν = 3447, 2928, 2856, 2795, 1458 cm–1. MS
˜
(EI): m/z = 337 [M]+. HRMS (EI): calcd. for C22H43NO
[M + H]+ 338.3423; found 338.3416.
Spectral Analyses: GC–MS was performed with a Varian Sa-
turn 2100 T ion trap MS instrument coupled to a Varian 3900 GC
with a 30 mϫ0.25 mm i.d. Varian Factor Four VF-5ms fused silica
column. GC separation of alkaloids was achieved by use of a tem-
perature program from 100 to 280 °C at a rate of 10 °C per minute
with He as the carrier gas (1 mLmin–1). Alkaloids were analyzed
both by electron impact MS and by chemical ionization MS with
MeOH as the reagent.
Vapor-phase FTIR spectroscopic data (GC-FTIR) were obtained
with a Hewlett–Packard model 5890 gas chromatograph and a Phe-
nomenex Zebron ZB-5 capillary column (30 m, 0.32 mm i.d.,
0.25 μm), with use of the same temperature program as above, cou-
pled with a model 5965B (IRD) narrow band (4000–750 cm–1) in-
frared detector. A Hewlett–Packard ChemStation was used to gen-
erate FTIR spectra.
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Supporting Information (see footnote on the first page of this arti-
1
cle): H NMR and 13C NMR spectra of compounds 2, 4a, 4b, 5–
1
8, 239Q, 9, 10-epi-239Q, and 10–24, H NMR spectrum of com-
pound 3, and NOE data for compounds 7 and 239Q.
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Acknowledgments
We are grateful to Drs. H. Martin Garraffo and Thomas F. Spande
(NIH) for providing us with the skin extracts of the toad Melano-
phryniscus cupreuscapularis and to Dr. J. A. Stitzel (University of
Received: July 24, 2012
Published Online: November 7, 2012
7092
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 7082–7092