Flexible synthesis of poison-frog alkaloids of the 5,8-disubstituted indolizidine-class. II: Synthesis of (-)-209B, (-)-231C, (-)-233D, (-)-235B", (-)-221I, and an epimer of 193E and pharmacological effects at neuronal nicotinic acetylcholine receptors.

Abstract

The 5,8-disubstituted indolizidines constitute the largest class of poison-frog alkaloids. Some alkaloids have been shown to act as noncompetitive blockers at nicotinic acetylcholine receptors but the proposed structures and the biological activities of most of the 5,8-disubstituted indolizidines have not been determined because of limited supplies of the natural products. We have therefore conducted experiments to confirm proposed structures and determine biological activities using synthetic compounds. Recently, we reported that one of this class of alkaloids, (-)-235B', acts as a noncompetitive antagonist for α4β2 nicotinic receptors, and its sensitivity is comparable to that of the classical competitive antagonist for this receptor, dihydro-β-erythroidine.
The enantioselective syntheses of (-)-209B, (-)-231C, (-)-233D, (-)-235B", (-)-221I, and what proved to be an epimer of natural 193E, starting from common chiral lactams have been achieved. When we performed electrophysiological recordings to examine the effects of the synthetic alkaloids on two major subtypes of nicotinic receptors (α4β2 and α7) expressed in Xenopus laevis oocytes, (-)-231C effectively blocked α4β2 receptor responses (IC(50 )value, 1.5 μM) with a 7.0-fold higher potency than for blockade of α7 receptor responses. In contrast, synthetic (-)-221I and (-)-epi-193E were more potent in blocking α7 receptor responses (IC(50 )value, 4.4 μM and 9.1 μM, respectively) than α4β2 receptor responses (5.3-fold and 2.0-fold, respectively).
We achieved the total synthesis of (-)-209B, (-)-231C, (-)-233D, (-)-235B", (-)-221I, and an epimer of 193E starting from common chiral lactams, and the absolute stereochemistry of natural (-)-233D was determined. Furthermore, the relative stereochemistry of (-)-231C and (-)-221I was also determined. The present asymmetric synthesis of the proposed structure for 193E revealed that the C-8 configuration of natural 193E should be revised. The selectivity for α4β2 and α7 nicotinic receptors differed markedly for the 5,8-disubstituted indolizidines tested, and thus it appears that the nature of the side chains in these indolizidines is crucial with regard to subtype-selectivity.

Full text

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Open Access
Beilstein Journal of
Organic Chemistry
Full Research Paper
Flexible synthesis of poison-frog alkaloids of the 5,8-
disubstituted indolizidine-class. II: Synthesis of (-)-209B, (-)-231C,
(-)-233D, (-)-235B", (-)-221I, and an epimer of 193E and
pharmacological effects at neuronal nicotinic acetylcholine
receptors
Soushi Kobayashi1, Naoki Toyooka*1, Dejun Zhou1, Hiroshi Tsuneki1,
Tsutomu Wada1, Toshiyasu Sasaoka1, Hideki Sakai1, Hideo Nemoto1, H
Martin Garraffo2, Thomas F Spande2 and John W Daly2
Address: 1Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Sugitani 2630, Toyama, 930-0194, Japan and
2Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS,
Bethesda, MD 20892, USA
Email: Soushi Kobayashi - d0560009@ems.u-toyama.ac.jp; Naoki Toyooka* - toyooka@pha.u-toyama.ac.jp;
Dejun Zhou - zej4060@yahoo.co.jp; Hiroshi Tsuneki - htsuneki@pha.u-toyama.ac.jp; Tsutomu Wada - twada-tym@umin.ac.jp;
Toshiyasu Sasaoka - tsasaoka@pha.u-toyama.ac.jp; Hideki Sakai - sakaih@pha.u-toyama.ac.jp; Hideo Nemoto - nemotoh@pha.u-toyama.ac.jp;
H Martin Garraffo - garraffo@helix.nih.gov; Thomas F Spande - thomassp@bdg8.niddk.nih.gov; John W Daly - jdaly@nih.gov
* Corresponding author
Abstract
Background: The 5,8-disubstituted indolizidines constitute the largest class of poison-frog alkaloids. Some alkaloids
have been shown to act as noncompetitive blockers at nicotinic acetylcholine receptors but the proposed structures and
the biological activities of most of the 5,8-disubstituted indolizidines have not been determined because of limited
supplies of the natural products. We have therefore conducted experiments to confirm proposed structures and
determine biological activities using synthetic compounds. Recently, we reported that one of this class of alkaloids, (-)-
235B', acts as a noncompetitive antagonist for α4β2 nicotinic receptors, and its sensitivity is comparable to that of the
classical competitive antagonist for this receptor, dihydro-β-erythroidine.
Results: The enantioselective syntheses of (-)-209B, (-)-231C, (-)-233D, (-)-235B", (-)-221I, and what proved to be
an epimer of natural 193E, starting from common chiral lactams have been achieved. When we performed
electrophysiological recordings to examine the effects of the synthetic alkaloids on two major subtypes of nicotinic
receptors (α4β2 and α7) expressed in Xenopus laevis oocytes, (-)-231C effectively blocked α4β2 receptor responses
(IC50 value, 1.5 μM) with a 7.0-fold higher potency than for blockade of α7 receptor responses. In contrast, synthetic (-
)-221I and (-)-epi-193E were more potent in blocking α7 receptor responses (IC50 value, 4.4 μM and 9.1 μM,
respectively) than α4β2 receptor responses (5.3-fold and 2.0-fold, respectively).
Conclusion: We achieved the total synthesis of (-)-209B, (-)-231C, (-)-233D, (-)-235B", (-)-221I, and an epimer of
193E starting from common chiral lactams, and the absolute stereochemistry of natural (-)-233D was determined.
Furthermore, the relative stereochemistry of (-)-231C and (-)-221I was also determined. The present asymmetric
synthesis of the proposed structure for 193E revealed that the C-8 configuration of natural 193E should be revised.
Published: 28 September 2007
Beilstein Journal of Organic Chemistry 2007, 3:30 doi:10.1186/1860-5397-3-30
Received: 19 July 2007
Accepted: 28 September 2007
This article is available from: http://bjoc.beilstein-journals.org/content/3/1/30
© 2007 Kobayashi et al; licensee Beilstein-Institut
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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The selectivity for α4β2 and α7 nicotinic receptors differed markedly for the 5,8-disubstituted indolizidines tested, and
thus it appears that the nature of the side chains in these indolizidines is crucial with regard to subtype-selectivity.
Introduction
In the preceding paper [1], we have reported the synthesis
of the chiral lactam building blocks (1, 2, Schemes 1, 2)
for the flexible synthesis of poison-frog alkaloids of the
5,8-disubstituted indolizidine class. The utility of these
chiral building blocks was demonstrated by the synthesis
of alkaloids (-)-203A, (-)-205A from 1, and of (-)-219F
from 2. Although the biological activity of most of the 5,8-
disubstituted indolizidines has not been investigated, cer-
tain 5,8-disubstituted indolizidines have been shown to
act as noncompetitive blockers of nicotinic acetylcholine
receptors. [2,3]
Scheme 1: Syntheses of (-)-209B, (-)-231C, (-)-233D, and (-)-
235B".
Scheme 2: Syntheses of (-)-221I and (-)-7 (an epimer of 193E).
Nicotinic receptors are ligand-gated ion channels com-
posed of five subunits. [4] To date, 12 nicotinic receptor
subunits (α2-α10, β2-β4) have been identified. Subtypes
of neuronal nicotinic receptors are constructed from
numerous subunit combinations, which confer varied
functional and pharmacological characteristics. [5] Nico-
tinic receptors have been implicated in a wide range of
neuronal dysfunctions and mental illness, such as epi-
lepsy, Tourette's syndrome, Alzheimer's disease, Parkin-
son's disease, and schizophrenia. [5,6] Since different
subtypes of nicotinic receptors are involved in different
neurological disorders, subtype-selective nicotinic ligands
would be valuable for investigation and potentially for
treatment of cholinergic disorders of the central nervous
system. However, there are only a limited number of com-
pounds that elicit subtype-selective blockade of nicotinic
receptors because of the similarity of receptor-channel
structure among the subtypes. Recently, we have investi-
gated the effect of synthetic (-)-235B', one of the 5,8-dis-
ubstituted indolizidine class of poison-frog alkaloids, on
several subtypes of nicotinic receptors, and found that this
alkaloid exhibits selective and potent blocking effects at
the α4β2 nicotinic receptor. [3] The potency of (-)-235B'
for this receptor is comparable to that of the classical α4β2
competitive antagonist, dihydro-β-erythroidine. In this
study, we have synthesized 5,8-disubstituted indolizi-
dines (-)-209B, (-)-231C, (-)-233D, (-)-235B", (-)-221I,
and an alkaloid that proved to be an epimer of natural
indolizidine 193E. The alkaloids (-)-209B and (-)-235B"
are known to be noncompetitive nicotinic blockers [2],
but effects of the other compounds have not yet been
tested. To explore possible subtype selectivity, we exam-
ined the effects of (-)-231C, (-)-221I and (-)-epi-193E on
α4β2 and α7 nicotinic receptors, the most abundant sub-
types in the mammalian brain. [4]
Results and Discussion
Reduction of the lactam 1 [1] with LiAlH4 followed by
Swern oxidation of the resulting alcohol and Wittig reac-
tion gave the olefin 3 in 78% overall yield (Scheme 1).
Hydrogenation of the double bond in 3 with 10% Pd/C
provided (-)-209B, whose spectral data were identical
with reported values. [7] The lactam 1 was also converted
to the alcohol 4, [1] which was transformed into (-)-
235B" by Swern oxidation followed by Wittig reaction
under high dilution and 'salt free' conditions (Scheme 1).
The spectral data of synthetic (-)-235B" were identical
with reported values. [8,9] Indolizidines (-)-231C [10]
and (-)-233D [10] were synthesized from common inter-
mediate 5 prepared from the alcohol 4. Thus, the Swern
oxidation of 4 and then the Wittig reaction of the resulting
aldehyde under Stork's conditions [11] provided the Z-
iodoolefin 5 in a highly stereoselective manner. The
Sonogashira coupling reaction [12] of 5 with TMS-acety-
lene followed by cleavage of the trimethylsilyl group with
K2CO3 afforded (-)-231C. Although the rotation of the
natural alkaloid is unknown, the relative stereochemistry
N
OH
H
Me
O
H
H
N
H
Me
H
H
OH
N
H
Me
H
H
N
H
Me
H
H
N
H
Me
H
H
I
N
H
Me
H
H
N
H
Me
H
H
N
H
Me
H
H
1
1) LiAlH4, THF, reflux
2) Swern oxidn.
3) n-BuP+Ph3Br-, n-BuLi, THF
0 ˚C to rt (78%)
3
(-)-209B
4
5
(-)-233D
(-)-231C
(-)-235B''
10% Pd/C, H2
EtOAc, 1 atm
(95%)
ref. 1
1) Swern oxidn.
2) ICH2P+Ph3I-, NaHMDS
HMPA, THF, -78 ˚C to rt
(61%)
1) CuI, TMS-acetylene
Pd(Ph3P)4, i-Pr2NH
THF, rt (90%)
2) K2CO3, MeOH, rt
(89%)
NiCl2(dppp), vinylMgBr
Et2O, rt (86%)
1) Swern oxidn.
2) n-PrP+Ph3Br-, NaHMDS
THF, -78 ˚C to rt (66%)
N
OH
H
H
O
H
N
H
H
H
CO2Me
O
N
H
H
H
N
H
H
H
2
1) Swern oxidn.
2) NaClO2, NaH2PO4
t-BuOH/H2O, 0 ˚C to rt
3) ClCO2Et, Et3N, THF
0 ˚C
4) CH2N2, Et2O, rt
5) PhCO2Ag, Et3N, MeOH
rt (81%)
6
(-)-7
(-)-221I
1) LiAlH4, THF, reflux
2) Swern oxidn.
3) MeP+Ph3I-, n-BuLi, THF
0 ˚C to rt (63%)
1) LiAlH4, THF, reflux
2) Swern oxidn.
3) n-PrP+Ph3I-, NaHMDS, THF
-78 ˚C to rt (62%)

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was determined to be 5,8-E and 5,9-Z by GC-MS and GC-
FTIR comparison with natural 231C in extracts from a
Panamanian dendrobatid frog, Dendrolbates pumilio. A
similar, Ni-catalyzed cross coupling [13] reaction of 5
with vinylmagnesium bromide provided the (-)-233D,
whose spectral data were identical with values reported
for the natural alkaloid isolated from the Panamanian
dendrobatid frog. [10] Although differing in magnitude,
the HCl salts of both synthetic (-) 233D and the natural
alkaloid had negative optical rotations.
Indolizidine (-)-7 with the relative stereochemistry pro-
posed for 193E [14] and indolizidine (-)-221I [14] were
synthesized from the lactam 2 [1] via the ester 6 (Scheme
2). The two-step oxidation of 2 followed by Arndt-Eistert
homologation of the resulting carboxylic acid provided
the ester 6. Reduction of both lactam and ester moieties of
6 with LiAlH4 followed by Swern oxidation and Wittig
reaction of the resulting aldehyde furnished the indolizi-
dine (-)-7. Coinjections of synthetic material with an alka-
loid fraction from a Madagascan mantellid frog, Mantella
viridis that contained natural 193E, revealed that the syn-
thetic material had a slightly longer GC retention time
than the natural product. The GC-mass spectra of (-)-7
and natural product were virtually identical and their GC-
FTIR spectra were very similar in the Bohlmann band
region (indicating 5,9-Z configurations in both),
although differing slightly in their fingerprint regions.
These results indicate that the natural 193E is most likely
the 8-epimer of (-)-7 and that the proposed configuration
[14] of the ethyl substituent at C-8 was in error. The
indolizidine (-)-221I was also synthesized from 6 follow-
ing a procedure similar to that used for the synthesis of (-
)-7 as shown in Scheme 2.
The relative stereochemistry of natural 221I was deter-
mined to be the same as that of synthetic (-)-221I by GC-
MS and GC-FTIR comparison with natural 221I, in the
alkaloid fraction from the Madagascan mantellid frog,
Mantella viridis (See Additional File 1 for experimental
details relating to all syntheses).
We then conducted electrophysiological experiments to
examine the effect of three of the synthetic alkaloids on
nicotinic receptors. When Xenopus laevis oocytes express-
ing the α4β2 nicotinic receptor were treated with 3 μM (-
)-231C, the peak amplitude of the acetylcholine (ACh)-
elicited currents was greatly decreased, whereas the
responses elicited in oocytes expressing the α7 nicotinic
receptor were not strongly affected (Figure 1A). When the
concentration-response curves were compared between
these receptor subtypes, (-)-231C blocked the α4β2 recep-
tor-mediated currents [50% inhibitory concentration
(IC50) = 1.5 μM, 95% confidence intervals (CI): 1.1 to 2.1
μM] with 7.0-fold higher sensitivity than blockade of the
Inhibitory effect of (-)-231C on ACh-induced currents in X. laevis oocytes expressing recombinant nicotinic receptors
Figure 1
Inhibitory effect of (-)-231C on ACh-induced currents in X. laevis
oocytes expressing recombinant nicotinic receptors. Currents
were recorded in the voltage-clamp mode at -60 mV. Concentra-
tions of ACh used were 1 μM for the α4β2 receptor and 100 μM
for the α7 receptor. For test responses, oocytes were preincu-
bated with (-)-231C for 3 min and then exposed to ACh with (-)-
231C. A, representative traces showing the ACh-elicited cur-
rents in the absence and presence of (-)-231C (3 μM). Horizontal
bars indicate the period of perfusion with ACh for 5 s. Vertical
scale bars represent 0.5 μA on the α4β2 receptor, and 0.1 μA on
the α7 receptor. B, concentration-response curves for (-)-231C
on recombinant nicotinic receptors. Current responses to ACh
in the presence of (-)-231C in each oocyte were normalized to
the ACh responses (control responses) recorded in the same
oocytes. Values represent the mean ± S.E.M. for five to six sepa-
rate experiments.
?7
?4?2
ACh
ACh
(-)-231C
(-)-231C
A
0
-7-6-5
0.2
0.4
0.6
0.8
Log conc. of (-)-231C
normalized response
?7
?4?2
B 1
-4

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α7 receptor-mediated currents (IC50 = 10.7 μM, 95% CI:
8.6 to 13.3 μM) (Figure 1B). These results indicate that (-
)-231C selectively blocked the responses mediated by the
α4β2 receptor.
The 5,8-disubstituted indolizidine (-)-231C is an ana-
logue of (-)-235B', both of which have a seven-carbon
unsaturated side-chain at C-5 and a methyl at C-8. Both
synthetic compounds have the same absolute stereochem-
istry (5R, 8R, 9S). Our previous [3] and present data dem-
onstrate that both (-)-235B' and (-)-231C produce potent
blockade of the α4β2 nicotinic receptor with a similar
selectivity of 6- to 7-fold over blockade of the α7 receptor.
However, the potency of (-)-235B' in blocking the α4β2
receptor is approximately 20-fold greater than that of (-)-
231C. These results suggest that either flexibility or degree
of unsaturation of the seven-carbon side-chain at C-5 in
these 5,8-disubstituted indolizidines is crucial for potent
interaction with the α4β2 receptor.
The synthetic (-)-221I and (-)-epi-193E are 5,8-disubsti-
tuted indolizidines with an ethyl rather than a methyl at
C-8 and a five-carbon or three-carbon side-chain, respec-
tively, at C-5. The alkaloid (-)-221I blocked α7 receptor
responses (IC50 = 4.4 μM, 95% CI: 3.1 to 6.1 μM) with 5.3-
fold higher potency than for blockade of the α4β2 recep-
tor responses (IC50 = 23.1 μM, 95% CI: 18.5 to 28.9 μM)
(Figure 2). Synthetic (-)-epi-193E was more potent in
blocking the α7 receptor response (IC50 = 9.1 μM, 95% CI:
7.5 to 11.1 μM) compared to blockade of the α4β2 recep-
tor (IC50 = 18.0 μM, 95% CI: 12.2 to 26.7 μM) (Figure 3).
Previously, we examined the effects of three synthetic 5,8-
disubstituted indolizidines with an n-butyl group at C-8
and an n-propyl group at C-5 in blocking different sub-
types of nicotinic receptors. [3] Two of these compounds,
namely (+)-8,9-diepi-223V and (-)-9-epi-223V were 6.7-
fold and 11.2-fold more potent in blocking α7 receptor
compared to blockade of α4β2 receptor, while the third,
(-) 223V, was only slightly more potent at blocking the
responses mediated by the α7 receptor. [3,15] These
results suggest that the α4β2 receptor does not interact
well with indolizidines having substituents larger than
methyl at C-8, while the α7 receptor is more accepting of
larger side-chains at C-8. Further analogous synthetic
alkaloids need to be tested. Overall, the side chains of 5,8-
disubstituted indolizidines appear to be of critical impor-
tance in determining selectivity and potency in blocking
responses mediated by subtypes of neuronal nicotinic
receptors. Further study of structure-activity relationships
of synthetic 5,8-disubstituted indolizidines at nicotinic
subtypes could lead to even more subtype-selective lig-
ands as research probes and as potentially useful drugs.
Neuronal nicotinic receptors have been implicated in the
physiological processes of reward, cognition, learning and
Inhibitory effect of (-)-221I on ACh-induced currents in X. laevis oocytes expressing recombinant nicotinic receptors
Figure 2
Inhibitory effect of (-)-221I on ACh-induced currents in X. laevis
oocytes expressing recombinant nicotinic receptors. Currents
were recorded in the voltage-clamp mode at -60 mV. Concentra-
tions of ACh used were 1 μM for the α4β2 receptor and 100 μM
for the α7 receptor. For test responses, oocytes were preincu-
bated with (-)-221I for 3 min and then exposed to ACh with (-)-
221I. A, representative traces showing the ACh-elicited currents
in the absence and presence of (-)-221I (3 μM). Horizontal bars
indicate the period of perfusion with ACh for 5 s. Vertical scale
bars represent 0.5 μA on the α4β2 receptor, and 0.1 μA on the
α7 receptor. B, concentration-response curves for (-)-221I on
recombinant nicotinic receptors. Current responses to ACh in
the presence of (-)-221I in each oocyte were normalized to the
ACh responses (control responses) recorded in the same
oocytes. Values represent the mean ± S.E.M. for five separate
experiments.
ACh
(-)-221I
ACh
(-)-221I
?4?2
A
?7
0
-7-6 -5
0.2
0.4
0.6
0.8
1
Log conc. of (-)-221I
normalized response
B
?7
?4?2
-4

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memory. [5,6] Some ligand-binding and autoradiography
studies with postmortem human brain suggest that loss of
neuronal nicotinic receptors is related to central choliner-
gic disorders such as Alzheimer's disease, Parkinson's dis-
ease and schizophrenia. [4,6] For instance, in
schizophrenic patients, decrease in binding of α-bungaro-
toxin (α-Bgt), a major specific ligand for α7 nicotinic
receptors, has been detected in hippocampus, thalamus
and frontal cortex [16,17]. Therefore, loss of α7 nicotinic
ligand-binding appears to be an early presymptomatic
diagnostic marker for schizophrenia. For in vivo mapping
of brain receptors, positron emission tomography (PET)
and single photon emission computed tomography
(SPECT) using specific ligands are powerful, non-invasive
techniques. Although 125I-methyllycaconitine has been
used for α7-selective binding in rat brain, [18] neither PET
nor SPECT ligand of α7 nicotinic receptors has been avail-
able so far. Radiolabeled α-Bgt could not be used for in
vivo mapping because of the large molecular weight, high
toxicity and poor blood-brain barrier permeability. [19]
Indolizidines are low molecular weight, lipophilic com-
pounds that should penetrate well into brain and, as
shown in our research, some exhibit high affinity and
selectivity for either α4β2 or α7 nicotinic receptors. Fur-
ther structure-activity relationship studies of synthetic
indolizidines may lead to the development of radioactive
α4β2-selective or α7-selective ligands useful for in vivo
mapping of these important central nicotinic receptors.
Additional material
Acknowledgements
We are grateful to Dr. John A. Dani (Baylor College of Medicine, Houston,
TX, USA) for his support with electrophysiological data acquisition, and to
Dr. Jerry A. Stitzel (University of Colorado) for providing us with plasmid
DNA. This work was supported in part by a grant-in-aid for Scientific
Research (C, No. 17590004, and No. 16590435) by the Japan Society for
the Promotion of Science (JSPS). Work at NIH was supported by the intra-
mural research program of NIDDK.
References
1. Toyooka N, Zhou D, Nemoto H, Garraffo HM, Spande TF, Daly JW:
Beilstein J Org Chem 2007, 3:30.
2. Daly JW, Nishizawa Y, Padgett WL, Tokuyama T, Smith AL, Holmes
AB, Kibayashi C, Aronstam RS: Neurochem Res 1991, 16:1213-1218.
Additional file 1
Experimental details for the synthesis of (-)-209B, (-)-231C, (-)-233D,
(-)-235B", (-)-221I, and an epimer of 193E and pharmacological effects
at neuronal nicotinic acetylcholine receptors. Experimental data which
includes experimental details on the spectral instruments, elemental ana-
lyzer.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1860-
5397-3-30-S1.doc]
Inhibitory effect of (-)-epi-193E on ACh-induced currents in
X. laevis oocytes expressing recombinant nicotinic receptors
Figure 3
Inhibitory effect of (-)-epi-193E on ACh-induced currents in X.
laevis oocytes expressing recombinant nicotinic receptors. Cur-
rents were recorded in the voltage-clamp mode at -60 mV. Con-
centrations of ACh used were 1 μM for the α4β2 receptor and
100 μM for the α7 receptor. For test responses, oocytes were
preincubated with (-)-epi-193E for 3 min and then exposed to
ACh with (-)-epi-193E. A, representative traces showing the
ACh-elicited currents in the absence and presence of (-)-epi-
193E (3 μM). Horizontal bars indicate the period of perfusion
with ACh for 5 s. Vertical scale bars represent 0.5 μA on the
α4β2 receptor, and 0.1 μA on the α7 receptor. B, concentration-
response curves for (-)-epi-193E on recombinant nicotinic recep-
tors. Current responses to ACh in the presence of (-)-epi-193E in
each oocyte were normalized to the ACh responses (control
responses) recorded in the same oocytes. Values represent the
mean ± S.E.M. for five separate experiments.
?7
?4?2
ACh
ACh
(-)-epi-193E
(-)-epi-193E
A
0
-7-6-5
0.2
0.4
0.6
0.8
1
Log conc. of (-)-epi-193E
normalized response
B1.2
?7
?4?2
-4