Syntheses of the proposed structures of poison-frog alkaloids 179 and 207E and their inhibitory effects on neuronal nicotinic acetylcholine receptors

Article abstract of DOI:10.1055/s-2007-1000831

Syntheses of the structures postulated for 179 and 207E, members of a proposed new class of poison-frog alkaloids, 6,7-dehydro-5,8-disubstituted indolizidines, are described. The FT-IR spectrum and GC retention time of the synthetic 207E were different from those of the natural product; consequently the original structure of 207E needs to be revised. It is likely that the position of the double bond is instead at the 7,8-position. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2007-1000831

LETTER
61
Syntheses of the Proposed Structures of Poison-Frog Alkaloids 179 and 207E
and Their Inhibitory Effects on Neuronal Nicotinic Acetylcholine Receptors
S
ynthese
s
of the
P
a
roposed Str
o
uctures of Po
k
ison-Frog
A
i
lkaloids Toyooka,*a Dejun Zhou,a Soushi Kobayashi,a Hiroshi Tsuneki,a Tsutomu Wada,a Hideki Sakai,a
Hideo Nemoto,a Toshiyasu Sasaoka,a Yasuhiro Tezuka,b Subehan,b Shigetoshi Kadota,b H. Martin Garraffo,c
Thomas F. Spande,c John W. Dalyc
a
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan
E-mail: toyooka@pha.u-toyama.ac.jp
Institute of Natural Medicine, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
b
c
DHHS, Bethesda, MD 20892, USA
Received 1 October 2007
We used the chiral piperidinol 35 as the starting material,
Abstract: Syntheses of the structures postulated for 179 and 207E,
and masked the hydroxy group as a MOM ether to afford
members of a proposed new class of poison-frog alkaloids, 6,7-de-
hydro-5,8-disubstituted indolizidines, are described. The FT-IR
spectrum and GC retention time of the synthetic 207E were differ-
4. Ether 4 was transformed into the enamino ester 5 using
Rubio’s procedure.6 The key Michael-type conjugate ad-
ent from those of the natural product; consequently the original dition of dimethylcuprate or dipropylcuprate to 5 proceed-
structure of 207E needs to be revised. It is likely that the position of
ed smoothly giving rise to the adducts 6 or 7 in about 80%
yield as a single isomer.7 The stereochemistry of 6 was de-
the double bond is instead at the 7,8-position.
Key words: poison-frog alkaloids 179 and 207E, inhibitory effects
on neuronal nicotinic acetylcholine receptors, dehydro-5,8-di-
substituted indolizidines
termined based upon NOE experiments on 1. In difference
NOE experiments, NOE enhancement (ca. 2.7%) was ob-
served on the C-9 proton upon irradiation of the methyl
protons at C-8, and on irradiation of the C-5 proton a weak
but clear NOE was detected on the same C-9 proton, indi-
cating the 5,9-Z and 8,9-E configurations of 6 as shown in
Scheme 1. The stereoselectivity of this key conjugate ad-
dition reaction can be rationalized as follows: The confor-
mation of 5 should prefer conformer A over that of B
owing to relief of A(1,3) strain8 between the urethane moi-
ety and the allyl substituent at the a-position. The stereo-
electronically preferred a-axial-attack9 will occur on A,
followed by protonation of the resulting enolate to afford
adduct 6 or 7. This selectivity can also be explained by
Cieplak’s hypothesis.10 It is noteworthy that the stereo-
chemical course of the attack of the anion is controlled by
a stereoelectronic effect despite the severe 1,3-diaxial re-
pulsion between the axial alkoxy substituent at the 3-posi-
tion and the incoming anion11 (Scheme 2).
The indolizidine ring is found in a great number of natu-
rally occurring alkaloids, and many of these alkaloids
show intriguing biological activities.1 Many alkaloids
containing a 3,5-disubstituted or 5,8-disubstituted indoliz-
idine ring system have been detected in the skin extracts
of poison frogs from neotropical regions.2,3 Very recently,
a new class of poison-frog alkaloids possessing a 6,7-de-
hydro-5,8-disubstituted indolizidine structure has been
proposed, based upon mass and FT-IR spectral data3
(Figure 1). None of these have been isolated for NMR
spectral analysis so the position of the double bond was
tentative. It now appears that the structures of many of
these dehydro-indolizidines must be re-evaluated with re-
spect to the position of the double bond.
As part of our program directed at studying the synthesis
of biologically active alkaloids,4 we report here the first
chiral synthesis of the structures proposed for 179 (1) and
207E (2), and the evaluations of their inhibitory effects on
neuronal nicotinic acetylcholine receptors.
With the key tetrasubstituted piperidines 6 and 7 in hand,
we next turned our attention to their conversions into the
lactams 10 and 11. Reduction with Superhydride followed
by Swern oxidation and the Horner–Emmons reaction
provided the a,b-unsaturated esters 8 and 9. These esters
were hydrogenated over Pearlman’s catalyst with con-
comitant cleavage of the Cbz group. Exposure of the re-
sulting amino esters to Weinreb’s reaction12 formed
lactams 10 and 11. To complete the synthesis of 1 and 2,
the lactams 10, 11 were converted into dehydropipe-
ridines 14 and 15 using an E2-elimination reaction of the
corresponding mesylates 12 and 13 with DBU. Finally, re-
duction of the lactam moiety in 14 and 15 with LiAlH4
furnished 113 and 214 in 91% and 70% yield, respectively
(Scheme 1).
H
7
R
6
8
H
H
9
5
R'
N
179: R = Me, R' = n-Pr (1)
207E: R, R' = n-Pr (2)
R = Me, CH2OH, Et, n-Pr, n-Bu
R' = n-Pr, n-Bu, CH2CH=CHMe, (CH2)2CH=CH2
Figure 1 Proposed structures of 6,7-dehydro-5,8-disubstituted in-
dolizidine poison-frog alkaloids, with representative side chains2
The GC-FTIR spectrum and GC retention time of synthet-
ic 2 proved different from those of natural 207E, detected
in the skin extracts of Oophaga granulifera, although the
SYNLETT 2008, No. 1, pp 0061–0064
Advanced online publication: 11.12.2007
DOI: 10.1055/s-2007-1000831; Art ID: U09407ST
x
x.
x
x.
2
0
0
7
© Georg Thieme Verlag Stuttgart · New York

62
N. Toyooka et al.
LETTER
H
H
H
MOMO
H
MOMO
H
HO
LiHMDS
PhSeCl (2 equiv)
MOMCl, (i-Pr)2EtN
H
H
H
–78 °C to r.t.
THF (79%)
N
CO2Me
N
CO2Me
N
CO2Me
CH2Cl2 (98%)
Cbz
Cbz
Cbz
5
4
3
R2CuX, –78 °C to 0 °C
Et2O for 6, THF for 7
Superhydride
THF, 0 °C
H
H
MOMO
R
H
H
H
MOMO
R
H
H
R = Me (88%)
R = n-Pr (84%)
6: R = Me, X = Li (78%)
7: R = n-Pr, X = MgBr (83%)
H
N
CO2Me
OH
N
Cbz
Cbz
1) Swern oxidation CH2Cl2
2) (EtO)2P(O)CH2CO2Et
NaH
H
H
1) 20% Pd(OH)2, H2
MeOH, 1 atm
2) Et3Al, DCE, reflux
H
H
MOMO
H
R
H
MOMO
H
R
H
THF, 0 °C to r.t.
N
CO2Et
8: R = Me (83%)
9: R = n-Pr (90%)
10: R = Me (86%)
11: R = n-Pr (98%)
N
Cbz
O
1) concd HCl, MeOH, reflux
2) MsCl, Et3N, CH2Cl2, 0 °C
H
H
H
MsO
R
R
H
DBU, toluene, reflux
H
H
H
14: R = Me (62% in 3 steps)
15: R = n-Pr (64% in 3 steps)
12: R = Me
13: R = n-Pr
N
N
O
O
H
7
R
H
LiAlH4, THF
6
NOE (R = Me)
8
H
H
H
1: R = Me (91%)
2: R = n-Pr (70%)
5
9
N
N
NOE (R = Me)
16
Scheme 1 Syntheses of structures proposed for the alkaloids 179 (1) and 207E (2)
mass spectra were similar. In the GC-FTIR spectra, both structure of indolizidine 207E would have a 7,8-double
synthetic 2 and the natural product showed a characteristic bond (16). The synthesis of 16 is in progress.
sharp and intense Bohlmann band near 2790 cm–1 indicat-
Electrophysiological experiments were used to examine
ing the 5,9-Z configuration in both compounds. Synthetic
the effect of the synthetic materials 1 and 2 on nicotinic re-
2 had an absorption band at 3030 cm–1 indicating the vinyl
C–H stretching. However, natural alkaloid 207E showed
only weak vinyl C–H stretching and also no enamine ab-
sorption. These results strongly indicate that the revised
ceptors expressed in Xenopus laevis oocytes using the
same method as described previously.15 After pre-incuba-
tion with the synthesized indolizidine for 3 minutes, cur-
rent was elicited by 5 seconds application of acetylcholine
(100 mM for a7, 1 mM for a4b2) in combination with the
CO2Me
OBn
MOMO
OBn
N
N
α-axial attack
O
O
B
A
CO2Me
β-axial attack
MOMO
stereoelectronically
favored α-axial attack
R
CO2Me
OBn
N
N Cbz
N
O
Cbz
OMe
R'
–O
A(1,3) strain
–O OMe
MOMO
R
R' = OMOM, R = Me or n-Pr
H
OBn
N
MeO2C
protonation
O
Nu
CO2Me
N
R'
R
MOMO
Cbz
Cieplak's hypothesis
Scheme 2 Stereochemical course of the Michael-type conjugate addition reaction of enamino ester 5
Synlett 2008, No. 1, 61–64 © Thieme Stuttgart · New York

LETTER
Syntheses of the Proposed Structures of Poison-Frog Alkaloids
63
indolizidines. Indolizidine 1 (30 mM) reduced the peak over a4b2 receptors. These results suggest that the struc-
amplitude of the a7-nicotinic-receptor-mediated current tures of 6,7-dehydro-5,8-disubstituted indolizidines have
more effectively than the a4b2-receptor-mediated current some determinant moiety for selective interaction with
(Figure 2, A). When the concentration–inhibition curves a7-nicotinic receptors. Further studies with other ana-
were compared between these receptor subtypes, indoliz- logues are required to identify the moiety.
idine 1 blocked the a7-nicotinic-receptor-mediated cur-
In conclusion, we achieved the first synthesis of the pro-
rent [50% inhibitory concentration (IC50) = 6.9 mM, 95%
posed structure for dehydro-indolizidines 179 and 207E.
confidence intervals (CI): 5.6–8.5 mM] with 4.0-fold high-
Comparison of synthetic 2 and natural 207E on GC and
er sensitivity than the blockade of a4b2-receptor-mediat-
GC-FTIR revealed that the proposed structure for 207E
ed current (IC50 = 26.6 mM, 95% CI: 15.8–44.8 mM;
should be revised. The dehydro-indolizidine 179 could no
Figure 2, B). The indolizidine (2) blocked a7-nicotinic-
longer be found in skin extracts and an IR had not been
receptor responses (IC50 = 6.5 mM, 95% CI: 4.2–9.9 mM)
obtained, so no comparisons with synthetic 1 were possi-
with >5.0-fold higher sensitivity than the blockade of the
ble. Furthermore, we conducted for the first time a study
a4b2-receptor responses (IC50 >30.0 mM; Figure 2, C and
of the pharmacological effects of the 6,7-dehydro-5,8-dis-
ubstituted type of indolizidines on nicotinic receptors, and
these indolizidines showed selective inhibition of a7-nic-
otinic receptors over a4b2 receptors. For the confirmation
D). The blocking effects of 1 and 2 at 30 mM on a7- and
a4b2-nicotinic receptors recovered within 10 minutes af-
ter removal of the alkaloids (data not shown).
In this study, we investigated for the first time the pharma- of the structure of natural 207E, the synthesis of the 7,8-
cological effects of 6,7-dehydro-5,8-disubstituted indoliz- dehydro isomer 16 of 2 is now under investigation, and
idines on nicotinic receptors. The compounds 1 and 2 will be reported.
produced a selective inhibition of a7-nicotinic receptors
Figure 2 Inhibitory effects of 6,7-dehydro-5,8-disubstituted indolizidines on acetylcholine (ACh)-induced current in Xenopus laevis oocytes
expressing a7- or a4b2-nicotinic receptors. Current was recorded in voltage-clamp mode at –60 mV. A and C, typical traces showing inhibition
by (A) indolizidine 1 or (C) indolizidine 2 at 30 mM of a7 current elicited by 100 mM ACh and a4b2 current elicited by 1 mM ACh. Horizontal
bars indicate the period of perfusion with ACh for 5 s. Vertical scale bars represent 200 nA. B and D, concentration–inhibition curves for (B)
indolizidine 1 and (D) indolizidine 2 on a7- ({) and a4b2-nicotinic receptors (z). Peak current responses to ACh in the presence of the alkaloid
in each oocyte were normalized to the ACh responses (control responses) recorded in the same oocytes. Values represent the mean SEM of
five separate experiments.
Synlett 2008, No. 1, 61–64 © Thieme Stuttgart · New York

64
N. Toyooka et al.
LETTER
(12) Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett.
Acknowledgment
1977, 4171.
We are grateful to Dr. Jerry A. Stitzel (University of Colorado) for
providing us with plasmid DNA. We also thank Dr. John A. Dani
(Baylor College of Medicine, Houston, TX) for support on electro-
physiological data acquisition. This work was supported in part by
a Grant-in-Aid for Scientific Research from the Japan Society for
the Promotion of Science (to N.T. and H.T.), and Tamura Founda-
tion for Promotion of Science and Technology (to N.T.). Work at
NIH was supported by the intramural research program of NIDDK.
(13) The spectral and analytical data of synthetic 1 are as follows:
IR (neat): 3025, 2957, 2871, 2781, 1456, 1377, 1326, 1287,
1179, 913, 798, 715 cm–1. 1H NMR (500 MHz, CDCl3): d =
0.93 (3 H, t, J = 7.5 Hz), 0.94 (3 H, d, J = 6.8 Hz), 1.25–1.34
(1 H, m), 1.35–1.41 (1 H, m), 1.42–1.52 (2 H, m), 1.61–1.67
(1 H, m), 1.68–1.75 (1 H, m), 1.76–1.83 (1 H, m), 1.84 (1 H,
td, J = 9.1, 6.9 Hz), 1.99–2.07 (2 H, m), 2.08–2.14 (1 H, m),
2.64 (1 H, dtd, J = 11.0, 3.6, 1.9 Hz), 3.35 (1 H, td, J = 8.4,
1.9 Hz), 5.50 (1 H, dt, J = 10.2, 1.9 Hz), 5.57 (1 H, dt, J =
10.2, 1.9 Hz). 13C NMR (75 MHz, CDCl3): d = 14.39 (q),
18.14 (q), 18.64 (t), 20.77 (t), 29.38 (t), 36.04 (t), 37.61 (d),
52.79 (t), 63.00 (d), 67.83 (d), 128.55 (d), 131.53 (d).
HRMS: m/z calcd for C12H21N: 179.1674; found: 179.1686.
[a]D26 +129.3 (c 2.74, CHCl3).
(14) The spectral and analytical data of synthetic 2 are as follows:
IR (neat): 3030, 2957, 2929, 2871, 2781, 1458, 1378, 1329,
1260, 1177, 1105, 929, 803, 713 cm–1. 1H NMR (500 MHz,
CDCl3): d = 0.89 (3 H, t, J = 6.8 Hz), 0.91 (3 H, t, J = 7.3
Hz), 1.12–1.21 (1 H, m), 1.23–1.34 (2 H, m), 1.35–1.40 (2
H, m), 1.43–1.53 (3 H, m), 1.59–1.68 (1 H, m), 1.69–1.74 (1
H, m), 1.76–1.82 (1 H, m), 1.93 (1 H, td, J = 9.4, 6.8 Hz),
1.99–2.06 (3 H, m), 2.63 (1 H, dtd, J = 11.0, 3.4, 1.7 Hz),
3.34 (1 H, td, J = 8.5, 2.1 Hz), 5.60 (1 H, dt, J = 9.8, 1.7 Hz),
5.63 (1 H, dt, J = 9.8, 1.7 Hz). 13C NMR (75 MHz, CDCl3):
d = 14.43 (2 × q), 18.64 (t), 19.67 (t), 20.92 (t), 29.69 (t),
34.91 (t), 36.11 (t), 42.54 (d), 52.76 (t), 62.97 (d), 66.13 (d),
129.01 (d), 129.44 (d). HRMS: m/z calcd for C14H25N
207.1986; found: 207.1973. [a]D26 +109.1 (c 0.32, CHCl3).
The GC-MS instrument is a Finnigan-Thermoquest Polaris
Q with a Restek-RTX-5MS column (30 m × 0.25 mm i.d.)
and the program was 100–280 °C at 10 °C/min with a final
hold time of 10 min; tR = 7.69 min.; natural product from
Oophaga granulifera: tR = 8.41 min. MS (EI): m/z (%) = 208
(12), 207 (4), 206 (8), 164 (100), 162 (13), 136 (13), 134
(13), 132 (8), 120 (52), 106 (8), 93 (22), 92 (11), 91 (10), 79
(13), 77 (16), 70 (16), 67 (24), 65 (14). MS (EI) of natural
product from Dendrobates granuliferous: m/z (%) = 207 (2),
206 (4), 17 (10), 164 (100), 162 (78), 134 (18), 120 (28), 91
(12), 79 (14), 77 (20), 65 (11).
References and Notes
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