Supplementary Material for Perkin Transactions 1
This journal is © The Royal Society of Chemistry 2001
6-Substituted 2-Azabicyclo[2.2.1]hept-5-enes by Nitrogen-Directed Radical Rearrangement:
Synthesis of an Epibatidine Analogue with High Binding Affinity at the Nicotinic Acetylcholine
Receptor
Electronic supplementary information (ESI): details of biological studies.
David M. Hodgson,*a Christopher R. Maxwell,a Richard Wisedale,a Ian R. Matthews,b Kate J.
Carpenter,c Anthony H. Dickensonc and Susan Wonnacottd
a
Dyson Perrins Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford
OX1 3QY, UK
b
c
Syngenta, Jealott's Hill International Research Centre, Berkshire RG42 6EY, UK
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
d
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
[3H]-Epibatidine binding to rat brain membranes: Competition assays.
P2 Membrane preparation: Crude membrane preparations were prepared from the brains of male SpragueDawley rats, as previously described.45 Briefly, the tissue was homogenised (10% w/v) in Sucrose Buffer
(0.32 M sucrose, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluouride (PMSF), 0.01% (w/v) sodium
azide, pH 7.4) using a glass-teflon homogeniser (8 strokes at 600 rpm), prior to centrifugation (1000 g, 4
°C, 10 min). The pellet was resuspended to 20% (w/v) in Sucrose Buffer and the centrifugation repeated.
The supernatant fractions were combined and the P2 membranes recovered as a pellet by centrifugation
(15,000 g, 4 °C, 30 min). The pellet was washed twice by resuspension in Phosphate Buffer (50 mM
phosphate, 1 mM EDTA, 0.1 mM PMSF, 0.01% (w/v) sodium azide, pH 7.4) and centrifugation (15,000 g,
4 °C, 30 min). The final pellet was resuspended in Phosphate Buffer to a concentration of 2.5 ml/g original
weight of tissue and stored in aliquots at -20 °C.
[3H]-epibatidine binding assay: Membranes were diluted in Assay Buffer (Phosphate Buffer supplemented
with 0.1% (w/v) BSA) to give the desired protein content (typically 0.5 mg) in a final assay volume of 2
ml. Membranes were incubated with 200 pM [3H]-epibatidine, in the presence and absence of 1 mM
nicotine to define total and non-specific binding, or in the presence of serial dilutions of unlabelled
epibatidine 1, and analogues 2 and 35. Samples were incubated for 2 h at room temperature. Incubations
were terminated by dilution with 4 ml phosphate-buffered saline (PBS; 20 mM Na2HPO4, 5 mM KH2PO4,
150 mM NaCl, pH 7.4) at 4 °C and rapid filtration though type A/E glass fibre filters (Gelman Sciences),
presoaked in 0.3% (v/v) polyethyleneimine (PEI) for 3 h, using a Brandel cell harvester. Filters were
1
Supplementary Material for Perkin Transactions 1
This journal is © The Royal Society of Chemistry 2001
washed twice with 4 ml PBS at 4 °C and bound radioactivity determined by liquid scintillation
spectrometry (counting efficiency 45%).
Data analysis: IC50 values (concentration of drug that inhibits 50% of binding) were calculated by nonlinear regression of the specific binding data from competition binding assays, fitting to the Hill Equation,
% bound = 100% / (1+([L]/IC50)n), where n is the Hill number (nH) and [L] is the concentration of
displacing ligand. Ki values were calculated from IC50 values using the Cheng and Prussoff equation,46
assuming a Kd value for [3H]-epibatidine of 40 pM. Curve-fitting was performed using SigmaPlot V2.0
for Windows (Jandel Scientific).
100
H]-epibatidine binding (% maximum)
3[
1
2
35
80
60
40
20
0
-13
-12
-11
-10
-9
-8
-7
-6
-5
Log10 [drug]
Fig. 3. [3H]-epibatidine binding to rat brain P2 membranes, competition assays
The inhibition of binding of 200 pM [3H]-epibatidine to rat brain P2 membranes by serial dilutions of
epibatidine 1, and analogues 2 and 35 is shown. Data points (mean  s.e.m. from three experiments) are
fitted to the Hill equation.
IC 50
Ki
Ki
[3H]-Epibatidine K i
[3H]-Cytisine Ki ( 42 re ceptors )3b
Epi bati dine
0.155 nM
0.026 nM
Nicotin e
An alog ue 2
1.3 nM
0.22 n M
Epi bati dine
0.042 nM
An alog ue 35
4.2 n M
0.7 nM
ABT-594
0.037 nM
2
1.0 nM
Supplementary Material for Perkin Transactions 1
This journal is © The Royal Society of Chemistry 2001
Electrophysiological studies of (+)-2 and ()-2
The techniques used here have been described more fully elesewhere.40 Male Sprague-Dawley rats (200–
250 g) were anaesthetised with 2-3% halothane (in 66% N2O and 33% O2) and subsequently maintained at
1.8% halothane. Core temperature of the animal was monitored using a rectal thermometer probe coupled
to a heating blanket. At the end of the experiment, animals were terminated with overdose of anaesthetic.
A laminectomy was performed to expose the segments L4–L5 of the spinal cord, and a parylene-coated
tungsten electrode was descended into the dorsal horn using an Epson Stepper device. The depth of the
recording site was noted from the microdrive readings. Extracellular recordings were made from single
dorsal horn neurones receiving C- and A-fibre input from the skin of the hindpaw, identified by their
ability to respond to both noxious and innocuous stimuli (pinch and touch). Neuronal responses were
elicited by transcutaneous electrical stimulation given in the centre of the receptive field of the neurone in
the ipsilateral hindpaw, at 3 times the threshold current required for C-fibre evoked activity. At 10-minute
intervals, tests consisting of a train of 16 stimuli (2msec-wide pulses at 0.5 Hz) were carried out and poststimulus histograms constructed. These evoked response were separated, according to latency, into Afibre evoked activity (0–20 msec post-stimulus); A-fibre evoked activity (20–90 msec); C-fibre evoked
activity (90–300 msec) and post-discharge of the neurone (300–800 msec). The neuronal response evoked
by the first stimulus of the train is referred to as “input” and consists of the number of action potentials
(90–800msec) evoked by this stimulus. Wind-up, a measure of the enhanced neuronal response elicited by
repetitive stimulation, was quantified as the difference between the total number of action potentials
produced by the 16 stimuli (90–800 ms), and the input  16. Thus the measure of wind-up includes both
the enhanced C-fibre evoked responses and the post-discharge generated as wind-up develops.
Tests were performed at 10-minute intervals, until the neuronal responses evoked at each test differed
by less than 10%. The results of the last three tests were then averaged to give control values for each
parameter.
Cumulative doses of each epibatidine analogue [(+)-2 and ()-2] (0.5, 5 and 50g in 50l saline) were
then applied to the exposed spinal cord into a ‘well’ formed by the laminectomy which held the 50l
volume of drug. The electrically-evoked neuronal responses were followed for 60 minutes after each dose,
after which the solution was removed and the next dose applied.
The effect of analogue (+)-2 was tested on 7 individual neurones; analogue ()-2 was tested on 5
individual neurones.
Data below are shown as percentage of pre-drug control values, with a 60 minute time-course for each
dose of the drug. To generate a dose response curve, the maximum effects of each dose on each neurone
were averaged. Data are presented as mean  s.e.m.
The results clearly show a dose-dependent inhibition of wind-up and post discharge, measures of post
synaptic neuronal hyperexcitability in response to C-fibre evoked noxious stimulation and an inhibition of
input onto the neurones, an index of neurotransmitter release. This dual effect was seen for the population
of neurones and is clearly illustrated in the individual example (page 6) where the initial response of the
neurone is reduced as is the enhanced response seen as the stimulation continues. This profile is very
similar to that seen with another epibatidine analogue.3 The spinal route of administration of (+)-2
confirms this area as a key site in the antinociceptive effects of these nicotinic agents, The marked effects
of (+)-2 contrast with those of ()-2. The interpretation that either ()-2 does not bind or it is too weak to
activate and then desensitise the receptor, is supported by the fact that (+)-2 facilitated the neuronal
responses at low doses yet as the dose increased clear inhibitions were produced whereas ()-2 only
enhanced the responses.
3
Supplementary Material for Perkin Transactions 1
This journal is © The Royal Society of Chemistry 2001
Electrophysiological data for (+)-2
C-fibres
10'
Day1 83
Day1 107
Day2 101
Day3
day4 102
Day5 86
Day5 76
Mean
93
Post Discharge
10'
Day1 82
Day1 133
Day2 108
Day3
Day4 108
Day5 112
Day5 129
Mean
112
Input
10'
Day1
Day1 187
Day2 88
Day3
Day4 129
Day5
Day6 143
Mean
137
20'
84
96
96
90
103
98
80
92
0.5ug
30'
73
92
95
114
105
104
91
96
20'
69
148
128
187
107
128
91
123
0.5ug
30'
56
139
122
162
100
129
84
113
40'
71
105
102
119
101
106
84
98
40'
49
123
121
146
97
122
80
105
0.5ug
20'
30'
40'
204
138
133
129
116
64
131
XS-Spikes
149
171
121
112
124
99
129
128
157
136
100
142
69
122
50'
66
110
107
117
89
98
50'
40
145
110
130
87
102
50'
60'
10'
20'
50ug
30'
40'
50'
60'
104
73
108
99
73
105
97
80
103
95
92
101
83
98
100
91
91
93
78
93
84
77
67
80
72
53
75
60
60
74
55
61
72
60
57
75
97
71
91
86
69
86.4
109
84
94.6
110
79
95
110
76
93
106
81
92
103
73
86
103
74
80
107
69
75
98
62
71
96
98
71
73
10'
20'
5ug
30'
40'
50'
60'
10'
20'
50ug
30'
40'
50'
60'
132
95
134
111
125
127
115
113
111
98
118
113
63
117
104
92
94
86
69
90
90
58
44
66
47
28
48
39
36
54
15
34
58
33
35
59
113
69
109
96
64
104.6
128
66
106.6
131
83
109
129
75
98
118
91
96
100
90
88
88
61
63
98
71
58
78
66
55
80
86
47
53
40'
50'
60'
10'
20'
50ug
30'
40'
50'
60'
20'
149
133
126
149
148
140
152
119
123
171
109
128
167
117
94
200
93
89
20
117
123
152
95
85
133
91
68
86
74
72
76
51
51
81
47
55
67
51
55
67
53
160
84
135
142
69
124
107
79
117.8
124
79
123
169
69
125
147
74
89
138
89
119
129
69
101
98
64
78
116
64
76
87
54
64
80
80
63
64
60'
57
105
63
10'
20'
5ug
30'
40'
50'
60'
10'
20'
50ug
30'
40'
50'
60'
96
34
99
86
38
118
87
34
51
91
25
145
91
31
27
68
39
93
69
51
78
63
29
71
50
10
116
47
20
141
25
29
124
41
27
124
112
89
85
89
79
79
84
60
77.2
112
76
72
101
119
96
107
85
68
100
78
76
89
108
79
91
83
67
94
86
71
93
91
78
97
93
69
71
60'
63
135
120
10'
20'
5ug
30'
40'
50'
60'
10'
20'
50ug
30'
40'
50'
60'
131
104
148
107
80
157
127
94
138
92
109
142
69
159
126
95
120
114
52
159
101
35
101
84
19
77
68
15
72
75
6
88
71
19
76
88
122
110
79
116
81
106.3
102
115.3
104
112
102
114
96
106
96
102
91
78
96
65
84
62
83
62
88
68
60'
73
88
133
10'
20'
5ug
30'
40'
50'
60'
10'
20'
50ug
30'
40'
50'
60'
79
121
159
85
115
159
79
119
159
80
131
154
90
128
154
81
126
160
68
126
162
84
121
140
87
110
129
69
93
134
75
103
141
69
96
127
100
66
105
89
66
102.8
96
87
108
96
81
108
99
68
108
96
77
108
96
60
102
91
77
103
98
68
98
98
68
92
101
94
105
97
10'
20'
5ug
30'
40'
50'
60'
10'
20'
50ug
30'
40'
50'
60'
104
109
178
101
94
163
108
97
154
86
98
164
94
109
136
83
99
140
78
103
111
84
106
102
81
94
70
76
85
80
71
85
89
63
87
104
112
94
119
113
105
115.2
114
107
116
114
97
112
108
110
111
109
94
105
96
88
95
88
91
94
83
99
85
89
102
86
91
84
84
85
89
124
40'
64
136
79
158
105
97
107
50'
69
150
110
157
10'
Day1 85
Day1 88
Day2 102
Day3
Day4 114
Day5 100
Day6 56
Mean
91
20'
89
85
100
130
100
101
48
93
0.5ug
30'
87
71
103
164
104
103
81
102
40'
79
92
114
176
96
104
76
105
50'
82
86
124
181
A-beta-low
0.5ug
20'
30'
40'
94
57
145 135 114
111 108 102
172 198 188
114
93
100
110 113 105
75
94
99
117 124 109
10'
Day1 90
Day1 124
Day2 106
Day3
Day4 78
Day5 121
Day6 91
Mean
102
40'
10'
0.5ug
30'
64
151
88
162
95
95
109
A-beta-high
130
88
102
5ug
30'
60'
20'
84
146
88
141
102
77
106
10'
Day1 94
Day1 155
Day2 94
Day3
Day4 104
Day5 89
Mean
107
60'
37
140
114
20'
50'
0.5ug
30'
40'
61
0
102
98
53
70
169
95
99
98
111 102
66
124
94
84
A-delta
123
77
95
10'
5ug
30'
20'
64
68
80
85
102
111
145
94
10'
Day1 106
Day1 77
Day2 111
Day3
Day4 101
Day5
Day6
Mean
99
60'
62
113
99
50'
0
107
81
96
91
75
122
79
110
50'
80
109
99
189
80
111
120
85
100
60'
67
113
96
115
86
95
4
% Control
% Control
% Control
% Control
% Control
% Control
% Control
Supplementary Material for Perkin Transactions 1
This journal is © The Royal Society of Chemistry 2001
5
Supplementary Material for Perkin Transactions 1
This journal is © The Royal Society of Chemistry 2001
Electrophysiological data for ()-2
C-fibres
10'
107
91
106
110
116
106
20'
105
87
63
99
108
92
0.5ug
30'
114
97
104
111
117
109
40'
118
100
96
112
114
108
50'
123
100
89
114
114
108
60'
118
95
96
122
Post Discharge
10'
Day1 121
Day2 79
Day3 152
Day4 338
Day5 139
Mean
166
20'
146
104
72
433
125
176
0.5ug
30'
162
119
143
343
164
186
40'
156
113
112
287
157
165
50'
188
108
93
206
100
139
60'
168
109
111
251
10'
120
87
100
95
120
104
20'
103
96
100
88
113
100
0.5ug
30'
140
111
113
102
120
117
40'
128
101
107
102
140
116
50'
148
120
127
95
127
123
60'
124
120
107
109
10'
94
101
115
356
118
157
20'
140
114
108
421
105
178
0.5ug
30'
106
102
101
344
129
156
40'
132
119
80
284
89
141
50'
132
73
76
259
89
126
60'
146
67
84
261
10'
125
99
121
121
94
112
20'
88
101
57
121
90
91
0.5ug
30'
88
119
139
131
101
116
40'
194
113
105
128
89
126
50'
150
117
67
97
90
104
60'
113
107
102
123
10
95
89
72
138
98
84
20
104
87
44
57
###
###
0.5ug
30
104
87
76
122
107
88
40
108
83
72
98
106
85
50
108
90
127
114
108
100
60
109
81
65
106
10'
Day1 108
Day2 100
Day3 60
Day4
Day5 94
Mean
91
20'
106
100
56
0.5ug
30'
119
102
52
40'
116
90
53
50'
106
99
44
60'
102
91
49
112
94
76
87
88
87
87
84
81
Day1
Day2
Day3
Day4
Day5
Mean
Input
Day1
Day2
Day3
Day4
Day5
Mean
XS-Spikes
Day1
Day2
Day3
Day4
Day5
Mean
A-delta
Day1
Day2
Day3
Day4
Day5
Mean
A-beta-high
Day1
Day2
Day3
Day4
Day5
Mean
A-beta-low
108
160
115
140
111
84
20'
120
96
96
109
105
105
5ug
30'
124
96
94
112
104
106
40'
130
96
92
123
107
110
50'
133
94
90
109
95
104
60'
128
99
81
105
10'
118
100
97
90
20'
133
101
99
81
103
101
104
20'
208
143
158
398
220
225
5ug
30'
274
124
180
263
191
206
40'
288
111
173
223
177
194
50'
274
108
149
101
114
149
60'
243
105
98
99
10'
182
110
139
51
20'
244
106
162
43
136
121
139
119
20'
148
118
127
110
120
125
5ug
30'
136
134
147
102
120
128
40'
169
118
127
150
120
137
50'
173
115
120
88
107
121
60'
140
120
100
88
10'
136
120
133
68
20'
165
115
153
61
112
114
124
20'
139
131
94
347
126
167
5ug
30'
220
64
90
277
116
153
40'
184
76
97
91
112
112
50'
174
75
87
181
81
120
60'
194
69
67
162
10'
137
77
81
140
20'
166
82
83
140
123
109
118
20'
156
127
134
100
79
119
5ug
30'
150
114
114
108
89
115
40'
206
116
116
105
76
124
50'
181
113
120
82
76
114
60'
113
107
75
77
10'
125
114
134
59
20'
169
109
145
33
93
108
114
20
107
81
63
57
97
71
5ug
30
112
77
59
89
94
77
40
116
80
57
122
102
86
50
115
80
56
130
60
82
60
118
82
53
130
10
108
83
59
122
20
118
75
53
65
89
76
66
49
20'
127
72
54
5ug
30'
133
76
66
40'
135
82
52
50'
129
75
52
60'
146
79
50
10'
117
85
51
20'
121
87
50
94
86.7
120
93
100
94
92
90
112
92
92
84
86
10'
120
99
104
109
108
10'
174
123
283
141
180
10'
136
153
95
114
125
10'
134
70
346
107
164
10'
113
116
92
85
102
10
105
65
114
106
80
10'
117
6
50ug
30'
40'
121 123
98
93
102 103
74
36
50'
122
95
101
71
60'
124
100
104
55
89
97
96
50ug
30'
40'
187 161
106 98
154 151
30
9
50'
132
73
149
15
60'
135
93
146
4
105
92
94
50ug
30'
153
115
173
61
40'
144
115
147
41
50'
124
87
127
48
60'
120
94
127
34
126
112
97
94
50ug
30'
40'
121 117
77
58
71
85
82 101
50'
143
77
93
111
60'
157
102
93
91
90
106
111
50ug
30'
40'
113 63
109 107
143 136
26
28
50'
63
103
141
21
60'
56
100
139
21
84
82
79
50ug
30
40
109 109
88
80
52
56
41
33
50
110
87
58
49
60
110
82
65
8
64
71
65
50ug
30'
40'
117 110
80
87
51
54
50'
114
81
53
60'
111
77
52
83
80
99
88
98
64
83
84
% Control
% Control
% Control
% Control
% Control
% Control
% Control
Supplementary Material for Perkin Transactions 1
This journal is © The Royal Society of Chemistry 2001
References
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Wonnacott, Neuropharmacol., 1999, 38, 679.
46 Y.-C. Cheng and W. H. Prussof, Biochem. Pharmacol.,1973, 22, 3099.
7