Synthesis, nicotinic acetylcholine receptor binding, antinociceptive and seizure properties of methyllycaconitine analogs

Article abstract of DOI:10.1016/j.bmc.2006.10.061

A series of methyllycaconitine (1a, MLA) analogs was synthesized where the (S)-2-methylsuccinimidobenzoyl group in MLA was replaced with a (R)-2-methyl, 2,2-dimethyl-, 2,3-dimethyl, 2-phenyl-, and 2-cyclohexylsuccinimidobenzoyl (1b-f) group. The analogs 1b-f were evaluated for their inhibition of [¹²⁵I]iodo-MLA binding at rat brain α7 nicotinic acetylcholine receptors (nAChR). In order to determine selectivity, MLA and the analogs 1b-f were evaluated for inhibition of binding to rat brain α,β nAChR using [³H]epibatidine. At the α7 nAChR, MLA showed a K_i value of 0.87 nM, analogs 1b-e possessed K_i values of 1.67-2.16 nM, and 1f showed a K_i value of 26.8 nM. Surprisingly, the analog 1e containing the large phenyl substituent (K_i = 1.67 nM) possessed the highest affinity. None of the compounds possessed appreciable affinity for α,β nAChRs. MLA antagonized nicotine-induced seizures with an AD₅₀ = 2 mg/kg. None of the MLA analogs were as potent as MLA in this assay. MLA and all of the MLA analogs, with the exception of 1b, antagonized nicotine's antinociceptive effects in the tail-flick assay. Compound 1c (K_i = 1.78 nM at α7 nAChR) with an AD₅₀ value of 1.8 mg/kg was 6.7 times more potent than MLA (AD₅₀ = 12 mg/kg) in antagonizing nicotine's antinociceptive effects but was 5-fold less potent than MLA in blocking nicotine-induced seizures. Since MLA has been reported to show neuroprotection against β-amyloid_1-42, these new analogs which have high α7 nAChR affinity and good selectivity relative to α,β nAChRs will be useful biological tools for studying the effects of α7 nAChR antagonist and neuroprotection.

Full text of DOI:10.1016/j.bmc.2006.10.061

Bioorganic & Medicinal Chemistry 15 (2007) 678–685
Synthesis, nicotinic acetylcholine receptor binding, antinociceptive
and seizure properties of methyllycaconitine analogs
a
a
a
F. Ivy Carroll,a, Wei Ma, Hernan A. Navarro, Philip Abraham,
*
´
Scott A. Wolckenhauer,a M. I. Damajb and Billy R. Martinb
aChemistry and Life Sciences, Research Triangle Institute, Research Triangle Park, NC 27709, USA
bDepartment of Pharmacology and Toxicology, Medical College of Virginia Commonwealth University, Richmond, VA 23298, USA
Received 26 September 2006; revised 26 October 2006; accepted 28 October 2006
Available online 1 November 2006
Abstract—A series of methyllycaconitine (1a, MLA) analogs was synthesized where the (S)-2-methylsuccinimidobenzoyl group in
MLA was replaced with a (R)-2-methyl, 2,2-dimethyl-, 2,3-dimethyl, 2-phenyl-, and 2-cyclohexylsuccinimidobenzoyl (1b–f) group.
The analogs 1b–f were evaluated for their inhibition of [125I]iodo-MLA binding at rat brain a7 nicotinic acetylcholine receptors
(nAChR). In order to determine selectivity, MLA and the analogs 1b–f were evaluated for inhibition of binding to rat brain a,b
nAChR using [3H]epibatidine. At the a7 nAChR, MLA showed a Ki value of 0.87 nM, analogs 1b–e possessed Ki values of
1.67–2.16 nM, and 1f showed a Ki value of 26.8 nM. Surprisingly, the analog 1e containing the large phenyl substituent
(Ki = 1.67 nM) possessed the highest affinity. None of the compounds possessed appreciable affinity for a,b nAChRs. MLA antag-
onized nicotine-induced seizures with an AD50 = 2 mg/kg. None of the MLA analogs were as potent as MLA in this assay. MLA
and all of the MLA analogs, with the exception of 1b, antagonized nicotine’s antinociceptive effects in the tail-flick assay. Compound
1c (Ki = 1.78 nM at a7 nAChR) with an AD50 value of 1.8 mg/kg was 6.7 times more potent than MLA (AD50 = 12 mg/kg) in antag-
onizing nicotine’s antinociceptive effects but was 5-fold less potent than MLA in blocking nicotine-induced seizures. Since MLA has
been reported to show neuroprotection against b-amyloid1–42, these new analogs which have high a7 nAChR affinity and good selec-
tivity relative to a,b nAChRs will be useful biological tools for studying the effects of a7 nAChR antagonist and neuroprotection.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
protects against the toxicity produced by the Alzheimer’s
disease-related peptide b-amyloid1–42(Ab42).9 The avail-
ability of potent and selective a7 nAChR antagonist will
enhance our ability to study these processes. Although
MLA binds potently to the a7 subtype, recent evidence
suggests that MLA can also interact with a4b2 nAChR
and presynaptic a3/a6 nAChR.10 Clearly, there is a need
for additional and selective a7 nAChR antagonists to fur-
ther characterize the a7 nAChR pharmacophore and to
further study its biological and pharmacological roles.
Nicotinic acetylcholine receptors (nAChRs) are ligand-
gated ion channels formed by the association of five sub-
units, leading to heteromeric and homomeric structures.
The a7 receptor is the only homomeric nAChR widely
distributed in the mammalian central nervous system.
Binding sites in the brain that show high affinity for
[
125I]a-bungarotoxin ([125I]a-BTX) have been correlated
with the a7 nAChR.1–4 Methyllycaconitine (1a, MLA),
an alkaloid isolated from Delphinium brownii,5,6 is report-
ed to be the most potent non-protein competitive a7
nAChR antagonist presently available.7 Recent studies
have suggested that a7 nAChRs may play an important
role in cognitive dysfunction, neurodegenerative diseases,
vestibular function, epilepsy, and possibly smoking cessa-
tion.8 It is particularly interesting to note that MLA (1a)
In this study, we report the syntheses and inhibition of
[
125I]iodo-MLA and [3H]epibatidine binding at the a7
and a,b nAChRs for MLA (1a) and the MLA analogs
1b–f. Unfortunately, there is a lack of selective behavior-
al measure for a7-mediated response. Therefore, we
evaluated the ability of MLA analogs to antagonize nic-
otine-induced seizures and antinociception in mice using
both the tail-flick and hot-plate assays because of
MLA’s unique actions in these three assays. These
assays were chosen because seizures are mediated at
least in part through a7 receptors (sensitive to MLA),
Keywords: Methyllycaconitine; a7 nAChR; Antagonist; MLA analogs.
Corresponding author. Tel.: +1 919 541 6679; fax: +1 919 541
8868; e-mail: fic@rti.org
*
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.061

F. Ivy Carroll et al. / Bioorg. Med. Chem. 15 (2007) 678–685
679
the hot-plate assay is a4b2-receptor-mediated (insensi-
tive to MLA), and the tail-flick assay is mediated
through multiple nAChRs (sensitive to MLA).
1.1. Chemistry
The MLA analogs 1b–f were synthesized as outlined in
Scheme 1. The succinic acids 2b–f were converted to
the corresponding succinic anhydrides 3 by refluxing
the appropriate acid with acetic anhydride for 12 h.
Treatment of each anhydride with anthranilic acid in
chloroform provided the desired substituted methylly-
coctonic acids, which are most likely a mixture of 4b–f
and 5b–f except for the symmetrical analog (4d or
5d).11 The mixture of acids was refluxed under a Dean
Stark tube in toluene containing triethylamine for 12 h
to yield the appropriate 2-substituent or 2,3-disubstitut-
ed succinimidobenzoic acids 6b–f. The acids 6b–f were
coupled to the primary hydroxyl group of lycoctonine
(7) in the presence of p-toluenesulfonyl chloride and pyr-
idine to give the desired MLA analogs 1b–f.12
1.2. Biology
The Ki values for the inhibition of [125I]iodo-MLA and
[3H]epibatidine binding for MLA (1a) and the MLA
analogs 1b–f were determined using previously reported
procedures and are listed in Table 1.13,14 MLA (1a) and
the MLA analogs 1b–f were evaluated for their ability to
antagonize the effects of nicotine in the tail-flick, hot-
plate, and seizure test using previously reported proce-
dures and the AD50 values are listed in Table 2.15
2. Results and discussion
The a7 nAChR subtype is the second most prevalent in
the brain and has been implicated as playing a key role
in conditions such as nicotine addiction, schizophrenia,
Alzheimer’s disease, and epilepsy. Despite recent pro-
gress in the synthesis of competitive agonists selective
for the a7 nAChR subtype, very few antagonists are
known that bind with high affinity and selectivity at this
receptor. These include the peptide toxins a-bungaro-
toxin and the norditerpenoid alkaloid methyllycaconi-
tine (MLA, 1a).
Scheme 1. Reagents and conditions: (a) acetic anhydride, reflux at
12 h; (b) anthranilic acid; (c) toluene, (C2H5)3N reflux at 12 h; (d)
CH3C6H4SO2Cl, pyridine.
Table 1. Radioligand binding data for MLA (1a) and the MLA analogs 1b–fa
Compound
RTI-7527-
R1
R2
R3
a7 nAChR[125I]iodo-MLA Ki, nM (ꢀnH)
a,b nAChR[3H]epibatidine Ki, nM (ꢀnH)
1a
1b
1c
1d
1e
1f
MLA
58
60
CH3
H
H
H
0.87 0.13 (1.09 0.15)
2.12 0.47 (1.2 0.34)
1.78 0.15 (0.92 0.05)
2.46 0.87 (0.98 0.18)
1.67 0.75 (1.44 0.42)
26.8 3.62 (0.88 0.21)
739 43 (0.68 0.02)
352 47 (0.89 0.08)
602 127 (0.93 0.09)
321 33 (0.88 0.08)
219 23 (0.75 0.04)
1103 400 (1.21 0.02)
CH3
CH3
H
H
CH3
CH3
C6H5
C6H11
H
62
59
CH3
H
H
61
H
H
ꢀnH is the Hill coefficient determined from a four-parameter logistic fit to the data.
a Data represent means SE from at least three independent experiments

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F. Ivy Carroll et al. / Bioorg. Med. Chem. 15 (2007) 678–685
Table 2. Pharmacological evaluation of MLA (1a) and MLA analogs
1b–f as nicotinic antagonistsa
fold loss in affinity relative to MLA at the a7 nAChR,
which suggests that the angular-2-methyl-group on the
succinimidobenzoyl group is essential for potent a7
nAChR binding.12,29
Compound Seizures
(AD50, mg/kg)
Tail-flick
(AD50, mg/kg)
Hot-plate
(AD50 mg/kg)
1a
1b
1c
1d
1e
1f
2 (0.5–4)
12 (2–59)
15% at 20
0% at 30
13% at 20
25% at 20
7% at 20
0% at 20
In the present study, we found that replacement of (S)-2-
methylsuccinimidobenzoyl group in MLA (1a,
Ki = 0.87 nM) with the enantiomeric (R)-2-methyl-
succinimidobenzoyl group to give 1b, Ki = 2.12 nM,
resulted in a 2.4-fold loss in affinity for the a7 nAChR.
Compounds 1c, Ki = 1.78 nM, and 1d, Ki = 2.62 nM,
which possess an additional methyl group at the 2- or
3-position, showed 2.1- and 3-fold loss in affinity,
respectively. Surprisingly, replacement of the 2-methyl
group on the 2-methylsuccinimido group of MLA with
a phenyl ring to give 1e resulted in a Ki value of
1.67 nM. Thus, the MLA pharmacophore will accom-
modate large flat aromatic groups in this part of the
structure. In contrast, replacement of the 2-methyl
group with a cyclohexyl group to give 1f resulted in a
Ki value of 26.8 nM and thus a 31-fold loss of affinity.
This alteration suggests that even though a phenyl ring
is allowed in the 2-position of the 2-methylsuccinimido
group of MLA, larger more bulky groups like cyclohex-
yl are not allowed. The weak ability of MLA and all the
analogs 1b–f to inhibit [3H]epibatidine binding indicates
that these compounds have low affinity for a,b nAChRs.
40% at 50
10 (9–11)
0% at 50
0% at 50
33 (10–113)
10% at 30
1.8 (0.4–9)
7.0 (3–15)
18.3 (10–31)
7.0 (2.5–18)
Results were expressed as AD50 (mg/kg) confidence limits (CL) or %
effect at the highest dose tested.
a Dose–response curves were determined using a minimum of four
different doses of test compound and at least eight mice were used per
dose group.
b-Amyloid1–42 binds to a7 nAChRs with high affinity.16
Functional studies have shown both a7 nAChR activa-
tion17,18 and inhibition19 with this peptide. Many studies
suggest that a7 nAChRs agonist, such as TC-1698 (8),20
shows neuroprotection against b-amyloid1–42 effects on
a7 nAChR function. It is well-known that a7 nAChRs
undergo extensive desensitization 21–24 so long-term ago-
nist treatment may not be desirable. Interestingly, Mar-
tin et al. reported that a7 nAChR antagonist MLA (1a)
has neuroprotective actions against Ab42-induced
neurotoxicity and suggested that a7 nAChR antagonist
might be useful pharmacotherapies in treating neurode-
generative disorders such as Alzheimer’s disease.9
Unfortunately, lack of highly selective agonists and
antagonists has complicated the elucidation of the
physiological function of a7 nAChRs.
MLA analogs 1b–e showed equipotent binding affinities
toward the a7 nAChR. However, their in vivo profile in
the seizures and tail-flick assays was different from each
other. MLA analogs 1d–f were weak antagonists in the
tail-flick assay (AD50 = 7–18.3 mg/kg). Analog 1b,
which differs from MLA in only the stereochemistry of
the methyl group [(R)-methyl] on the suc-
cinimidobenzoyl group, was inactive in this assay. The
most potent compound, 1c, with an AD50 value of
1.8 mg/kg, was 6.7 times more potent than MLA, which
has an AD50 of 12 mg/kg. The nAChR subtype or sub-
types that mediate the tail-flick response are not known,
but the lack of a high correlation between a7 receptor
affinity and antagonistic potency in the tail-flick assay,
particularly the discrepancy with enantioselectivity
found in the 2-methylbenzoyl group, does not support
a role for a7 receptors in mediating antinociception in
this assay. It should be noted that earlier reports failed
to observe a significant blockade of nicotine’s effects in
the tail-flick test by MLA after sc injection in mice
(5 mg/kg)30 and icv administration in rats (10 lg).31
However, the doses of MLA used in the present study
are much higher than the reported ones. This difference
in the effect observed could mean MLA lacks nAChR
selectivity at higher doses or it is operating by some
other mechanism. MLA and all the analogs had poor
affinity for ab nAChRs as determined in the [3H]epibati-
dine binding assay and they were inactive at antagoniz-
ing the nicotinic effects in the hot-plate test. This is
consistent with earlier observations that a4b2 receptors
are required for this effect.32
MLA (1a) is an ester composed of the alcohol lycocto-
nine (7) and the acid, (S)-2-methylsuccinimidobenzoic
acid. A number of truncated analogs of the parent
MLA (1a) have been synthesized.25 None of the com-
pounds showed appreciable activity.25,26 Reports from
the literature have shown that neither the alcohol 7
nor the acid 9 possesses appreciable affinity for the a7
nAChR.12,27 Thus, it appears that the (S)-2-meth-
ylsuccinimidobenzoyl group connected to the C18 oxy-
gen of lycoctonine is essential for potent a7 nAChR
binding. In addition, esters formed from the acid 9
and alcohols possessing parts of the lycoctonine struc-
ture as well as esters formed from lycoctonine with other
acids were reported to possess low affinity for the a7
nAChR.27,28 In addition, the lycoctonine ester (10a), a
nonditerpenoid alkaloid lacking the 2-(S)-methyl group
on the succinimidobenzoyl group of MLA, and the
dehydro analog (10b) are reported to show 5- and 10-
MLA was also effective in blocking nicotine-induced sei-
zures in an enantioselective manner. The seizures are
mediated by a7 nAChRs and these results are consistent

F. Ivy Carroll et al. / Bioorg. Med. Chem. 15 (2007) 678–685
681
with an a7 mechanism. However, as with the tail-flick
assay, there was a poor correlation between a7 receptor
affinity and antagonistic potency. The results from the
seizure study also support the view that nicotine-induced
seizures and antinociception may be mediated by non-
a4b2 and non-a7 receptor subtypes. Indeed, pharmaco-
logical and genetic approaches initially suggested the
involvement of a7 nicotinic subtypes in nicotine-induced
seizures. However, additional studies on different mouse
inbred strains and transgenic mice have also implicated
a4, a5, a3, and b4.33 The in vivo results show also that
analogs 1b and 1e are much less potent than MLA in
blocking nicotinic effects despite having good affinity
for the a7 nAChR. This suggests that these two analogs
have a different selectivity profile than MLA. The differ-
ence seen between in vitro binding affinity and in vivo
potency in the various behavioral tests suggests that
these MLA analogs have lower selectivity for the a7
receptor subtype. Ultimately, in vitro functional selec-
tivity on various nAChR subtypes in expressed cells
would confirm this in vivo selectivity. While MLA and
its derivatives antagonize both nicotine-induced antino-
ciception and seizures, they apparently do so by acting
at multiple sites or different mechanisms, since there is
little correlation between antagonistic potencies in these
two measures. The fact that MLA does not readily cross
the blood–brain barrier raises the question of whether
pharmacokinetic differences may explain some of these
findings. However, it seems unlikely that the pharmaco-
kinetics would be so different among these analogs to ex-
plain their pharmacological dissimilarities.
cal rotations were determined at the sodium D-line using
a Rudolph Research Autopol III polarimeter (1-dm
cell). H NMR were determined on a Bruker Avance
DPX-300 or Bruker AMX-500 spectrometer using tetra-
methylsilane as an internal standard. Silica gel 60 (230–
400 mesh) was used for all column chromatography. All
reactions were followed by thin-layer chromatography
using Whatman silica gel 60 TLC plates and were visu-
alized by UV or by charring using 5% phosphomolybdic
acid in ethanol. All solvents were reagent grade.
1
MLA was isolated from Delphenium elatum (pacific
giant) seeds (purchased from Flowers of Tomorrow
Inc., Parma, Idaho 83660) and hydrolyzed to lycocto-
nine (7).34,35 [3H]epibatidine (s.a. = 66.6 ci/mmol) was
obtained from DuPont New England Nuclear (Boston,
MA).
4.1. (R)-Methylsuccinamic acids (4b and 5b)
A suspension of (R)-methylsuccinic acid (2b) (0.89 g,
6.75 mmol) in acetic anhydride (10 mL) was heated to re-
flux overnight. After removal of the excess of acetic anhy-
dride by distillation, the residue was triturated and dried
under reduced pressure to give the anhydride 3b that was
dissolved in 10 mL CHCl3 and added to a suspension of
anthranilic acid (0.925 g, 6.75 mmol) in 10 mL CHCl3.
The reaction mixture was heated on a steam bath for
30 min, and the CHCl3 removed under reduced pressure.
The product was purified on silica gel using hexane/ethyl
acetate/methanol/acetic acid (125:45:5:4) as eluent to give
1.56 g (92%) of succinamic acids (4b and 5b): mp 165–
167 ꢁC; 1H NMR (CD3OD) d 1.25–1.36 (2d, 3H),
2.50–2.60 (m, 1H), 2.76–2.99 (m, 2H), 7.12–7.18 (t, 1H,
aromatic), 7.53–7.56 (t, 1H, aromatic), 8.08–8.11 (d,
1H, aromatic), 8.54–8.57 (d, 1H, aromatic).
3. Conclusions
Replacement of the (S)-2-methylsuccinimidobenzoyl
group in MLA (1a) by an (R)-2-methylsuccinimido- ben-
zoyl to give 1b caused only a small reduction in binding at
the a7 nAChR, however, 1b showed no ability to antago-
nize seizures and antinociception induced by nicotine.
Surprisingly, compound 1c which has methyl groups pres-
ent in both the (2S) and (2R)-position of the succinimido
group is 6.7 times more potent than MLA in antagonizing
the antinociception induced by nicotine in the tail-flick
test. However, 1c has essentially the same affinity for the
a7 nAChR as 1b. These analogs have a pharmacological
profile distinct from that of MLA despite relatively mod-
est changes in structure. It would seem that they are acting
at sites other than a7 receptors. Regardless of their mech-
anism of action, the (S)-2-substituted succinimidobenzoyl
group is critical. Further development of MLA analogs
may lead to even better probes for characterizing these
receptor sites and to biological tools for studying the
effects of a7 nAChR antagonist in several neurological
disorders.
4.2. (R)-Methyllycactonic acid (6b)
A solution of the above (R)-methylsuccinamic acids (4b
and 5b) (1.5 g, 6 mmol) and triethylamine (3.05 g,
30.1 mmol) in 200 mL of toluene was heated to reflux
overnight. The toluene was removed by simple distilla-
tion, and the residue was purified on silica gel using hex-
ane/ethyl acetate/methanol/acetic acid (125:45:5:4) as
eluent to obtain 0.9 g (64%) of pure (R)-methyllycacton-
ic acid (6b): mp 117–119 ꢁC; 1H NMR (CD3OD) d 1.38–
1.40 (d, 3H), 2.41–2.61 (m, 1H), 3.04–3.15 (m, 2H),
7.28–7.31 (d, 1H, aromatic), 7.56–7.58 (t, 1H, aromatic),
7.65–7.68 (t, 1H, aromatic), 8.11–8.14 (d, 1H, aromatic).
Anal. (C12H11NO4Æ0.5H2O) C, H, N.
4.3. (R)-Methyllycaconitine (1b) citrate
To a stirred solution of imido acid 6b (49.9 mg,
0.214 mmol) in 1 mL of dry pyridine was added
76.4 mg (0.428 mmol) of p-toluenesulfonyl chloride.
The mixture was cooled to 0 ꢁC, 100 mg (0.214 mmol)
of lycoctonine was added, and the solution was stirred
for additional 1 h at 0 ꢁC and then placed in the refrig-
erator for 24 h. The yellow solution was dissolved in
10 mL NH4OH (aq, pH 9) and extracted with 3·
10 mL CHCl3. Organic phases were combined and
4. Experimental
Melting points were determined on a Thomas–Hoover
capillary tube apparatus and are not corrected. Elemen-
tal analyses were obtained by Atlantic Microlabs Inc.,
and are within 0.4% of the calculated values. All opti-

682
F. Ivy Carroll et al. / Bioorg. Med. Chem. 15 (2007) 678–685
washed with 10 mL NH4OH (aq, pH 9) and dried over
anhydrous Na2SO4. After removal of the solvent, the
residue was purified on silica gel using cyclohex-
ane:CHCl3/(Et)3N (6:4:1) as eluent to give 66 mg
(45%) of pure (R)-methyllycaconitine (1b). An analytical
reflux overnight. After removal of the excess of acetic
anhydride by distillation, the residue was triturated
and dried under reduced pressure to give the anhydride
3d which was dissolved in 15 mL CHCl3 and added to a
suspension of anthranilic acid (1.88 g, 13.7 mmol) in
15 mL CHCl3. The reaction mixture was heated on a
steam bath for 30 min and the CHCl3 removed under re-
duced pressure. The product was purified on silica gel
sample was prepared as the citrate salt: mp 99 ꢁC (dec);
20
½aꢁ þ 48:3ꢂ (c, 0.64, EtOH) (free base). Anal.
D
(C43H58N2O17Æ1.5H2O) C, H, N.
using
hexane/ethyl
acetate/methanol/acetic
acid
(125:45:5:4) as eluent to obtain 3 g (83%) of 2,3-dim-
4.4. 2,2-Dimethylsuccinamic acids (4c and 5c)
1
ethylsuccinamic acids 4d and 5d: H NMR (CDCl3) d
2,2-Dimethylsuccinic anhydride (3c) (2.0 g, 15.6 mmol)
was dissolved in 15 mL CHCl3 and added to a suspen-
sion of anthranilic acid (2.14 g, 15.6 mmol) in 15 mL
CHCl3. The reaction mixture was heated on a steam
bath for 30 min and CHCl3 removed under reduced
pressure. The product was purified on silica gel using
hexane/ethyl acetate/methanol/acetic acid (125:45:5:4)
as eluent to obtain 3.5 g (85%) of 2,2-dimethylsuccinam-
130–138 (m, 6H), 2.75–2.85 (m, 2H), 7.07–7.12 (t, 1H,
aromatic), 7.54–7.59 (t, 1H, aromatic), 8.01–8.04 (d,
1H, aromatic), 8.64–8.67 (d, 1H, aromatic).
4.8. 2,3-Dimethyllycactonic acid (6d)
A solution of the above 2,3-dimethylsuccinamic acids 4d
and 5d (3 g, 11.3 mmol) and triethylamine (5.65 g,
56.5 mmol) in 300 mL of toluene was heated to reflux
overnight. The toluene was removed by simple distilla-
tion, and the residue was purified on silica gel using hex-
ane/ethyl acetate/methanol/acetic acid (125:45:5:4) as
eluent to obtain 0.8 g (29%) of pure 2,3-dimethyllycac-
1
ic acids (4c and 5c): mp 138–140 ꢁC; H NMR (CDCl3)
d 1.41 (s, 6H, 2CH3), 2.78 (s, 2H, -CH2), 7.07–7.20 (t,
1H, aromatic), 7.56–7.61 (t, 1H, aromatic), 8.05–8.08
(d, 1H, aromatic), 8.77–8.80 (d, 1H, aromatic). Anal.
(C13H15NO5Æ0.5H2O) C, H, N.
1
tonic acid (6d): mp 163–165 ꢁC; H NMR (CDCl3) d
4.5. 2,2-Dimethyllycactonic acid (6c)
1.32–1.44 (2 d, 6H), 2.62–3.16 (2 m, 2H), 7.26–7.29 (d,
1H, aromatic), 7.51–7.56 (t, 1H, aromatic), 7.67–7.70
(t, 1H, aromatic), 8.17–8.20 (d, 1H, aromatic). Anal.
(C13H13NO4Æ25H2O) C, H, N.
A solution of the above 2,2-dimethylsuccinamic acids
(4c and 5c) (3.5 g, 13.2 mmol) and triethylamine (6.6 g,
66 mmol) in 300 mL of toluene was heated to reflux
overnight. The toluene was removed by simple distilla-
tion, and the residue was purified on silica gel using hex-
ane/ethyl acetate/methanol/acetic acid (125:45:5:4) as
eluent to obtain 1.3 g (40%) of pure 2,2-dimethyllycac-
4.9. 2,3-Dimethyllycaconitine (1d) citrate
To a stirred solution of imido acid 6d (105.8 mg,
0.428 mmol) in 2 mL of dry pyridine was added
152.8 mg (0.856 mmol) of p-toluenesulfonyl chloride.
The mixture was cooled to 0 ꢁC and 100 mg
(0.214 mmol) of lycactonine was added, and the solution
was stirred for additional 1 h at 0 ꢁC and was refrigerat-
ed for 18 h. The deep-red solution was taken up in
15 mL NH4OH (aq, pH 9) and extracted with 3·
10 mL CHCl3. Organic phases were combined and
washed with 10 mL NH4OH (aq, pH 9) and dried over
anhydrous Na2SO4. After removal of the solvent, the
residue was purified on silica gel using cyclohexane/
CHCl3/(Et)3N (6:4:1) as eluent to obtain 74.6 mg
(50%) of pure 2,3-dimethyllycaconitine (1d). An analyt-
ical sample was prepared as citrate salt: mp 84 ꢁC (dec).
Anal. (C44H60N2O17Æ0.5H2O) C, H, N.
1
tonic acid (6c): mp 134–136 ꢁC; H NMR (CDCl3) d
1.39 (s, 6H, 2CH3), 2.70 (s, 2H, -CH2), 7.23–7.26 (d,
1H, aromatic), 7.47–7.52 (t, 1H, aromatic), 7.63–7.68
(t, 1H, aromatic), 8.11–8.14 (d, 1H, aromatic). Anal.
(C13H13NO4Æ0.25H2O) C, H, N.
4.6. 2,2-Dimethyllycaconitine (1c) citrate
To a stirred solution of imido acid 6c (105.8 mg,
0.428 mmol) in 2 mL of dry pyridine was added
152.8 mg (0.856 mmol) of p-toluenesulfonyl chloride.
The mixture was cooled to 0 ꢁC, 100 mg (0.214 mmol)
of lycactonine was added, and the solution was stirred
for an additional 1 h at 0 ꢁC and then placed in the
refrigerator for 15 h. The deep-red solution was dis-
solved in 15 mL NH4OH (aq, pH 9) and extracted with
3· 10 mL CHCl3. Organic phases were combined and
washed with 10 mL NH4OH (aq, pH 9) and dried over
anhydrous Na2SO4. After the solvent was removed,
the residue was purified on silica gel using cyclohex-
ane/CHCl3/(Et)3N (6:4:1) as eluent to obtain 117.5 mg
(79%) of the pure 2,2-dimethyllycaconitine (1c). An ana-
lytical sample was prepared as the citrate salt: mp
104 ꢁC (dec). Anal. (C44H60N2O17ÆH2O) C, H, N.
4.10. Phenylsuccinamic acids (4e and 5e)
Phenylsuccinic anhydride (3e) (2.0 g, 11.36 mmol) was
dissolved in 15 mL CHCl3 and was added to a suspen-
sion of anthranilic acid (1.55 g, 11.36 mmol) in 15 mL
CHCl3. The reaction mixture was heated on a steam
bath for 30 min, and then the CHCl3 solvent was evap-
orated to dryness under reduced pressure. The product
was purified on silica gel, eluting with hexane/ethyl ace-
tate/methanol/acetic acid (125:45:5:4) to obtain 3 g
(84%), phenylsuccinamic acids (4e and 5e): mp 169–
4.7. 2,3-Dimethylsuccinamic acids (4d and 5d)
1
171 ꢁC; H NMR (DMSO) d 2.75–2.82 (dd, 1H), 3.11–
A suspension of 2,3-dimethylsuccinic acid (2d) (2.0 g,
13.7 mmol) in acetic anhydride (15 mL) was heated to
3.20 (dd, 1H), 4.02–4.10 (dd, 1H), 7.12–7.17 (m, 1H,
aromatic), 7.26–7.38 (m, 5H, –C6H5), 7.55–7.60 (m,

F. Ivy Carroll et al. / Bioorg. Med. Chem. 15 (2007) 678–685
683
1H, aromatic), 7.93–7.99 (m, 1H, aromatic), 8.44–8.50
(m, 1H, aromatic).
43.4 mmol) in 300 mL of toluene was heated to reflux
overnight. The toluene solvent was removed by simple
distillation. The residue was purified on silica gel, eluting
4.11. Phenyllycactonic acid (6e)
with
(125:45:5:4) to obtain 1.1 g (42%) of pure cyclohexylly-
cactonic acid (6f): mp 68–70 ꢁC; H NMR (CDCl3) d
1.12–1.35 (m, 5H), 1.69–1.81 (m, 5H), 2.06–2.11 (m,
1H), 2.71–3.10 (m, 3H), 7.22–7.26 (d, 1H, aromatic),
7.51–7.56 (t, 1H, aromatic), 7.67–7.72 (t, 1H, aromatic),
8.16–8.19 (d, 1H, aromatic). Anal. (C17H19NO4Æ0.33-
H2O) C, H, N.
hexane/ethyl
acetate/methanol/acetic
acid
1
A solution of the above phenylsuccinamic acids (4e and
5e) (3 g, 9.58 mmol) and triethylamine (4.79 g,
47.9 mmol) in 250 mL of toluene was heated to reflux
overnight. The toluene solvent was removed by simple
distillation, and the residue was purified on silica gel,
eluting with hexane/ethyl acetate/methanol/acetic acid
(125:45:5:4) to obtain 1.1 g (39%) of pure phenyllycac-
tonic acid (6e): mp 80–82 ꢁC; 1H NMR (CDCl3) d
2.98–3.11 (dd, 1H), 3.36–3.45 (dd, 1H), 4.21–4.26 (dd,
1H), 7.33–7.40 (m, 6H, aromatic), 7.56–7.61 (m, 1H,
aromatic), 7.72–7.76 (m, 1H, aromatic), 8.22–8.25 (m,
1H, aromatic). Anal. (C17H13NO4Æ0.6H2O) C, H, N.
4.15. Cyclohexyllycaconitine (1f) citrate
To a stirred solution of the imido acid (6f) (128.98 mg,
0.428 mmol) in 2 mL of dry pyridine was added
152.8 mg (0.856 mmol) of p-toluenesulfonyl chloride.
The mixture was cooled to 0 ꢁC, and 100 mg
(0.214 mmol) of lycactonine was added. The solution
was stirred for additional 1 h at 0 ꢁC and was then
placed in the refrigerator for 18 h. The deep-red solution
was taken up in 15 mL NH4OH aqueous solution (pH
ꢃ9) and extracted with 3· 10 mL CHCl3. Organic phas-
es were combined and washed with 10 mL NH4OH
aqueous solution (pH ꢃ9) and dried over anhydrous
Na2SO4. After the solvent was removed, the residue
was purified on silica gel, eluting with cyclohexane/
CHCl3/(Et)3N (6:4:1) to obtain 105.7 mg (66%) of the
pure cyclohexyllycaconitine (1f). An analytical sample
was prepared as citrate salt: mp 72 ꢁC (dec). Anal.
(C48H66N2O17Æ1.5H2O) C, H, N.
4.12. Phenyllycaconitine (1e) citrate
To a stirred solution of imido acid 6e (126.3 mg,
0.428 mmol) in 2 mL of dry pyridine was added
152.8 mg (0.856 mmol) of p-toluenesulfonyl chloride.
The mixture was cooled to 0 ꢁC, 100 mg (0.214 mmol)
of lycactonine was added, and the solution was stirred
for additional 1 h at 0 ꢁC and was then placed in the
refrigerator for 18 h. The deep-red solution was taken
up in 15 mL NH4OH aqueous solution (pH ꢃ9) and
extracted with 3· 10 mL CHCl3. Organic phases were
combined and washed with 10 mL NH4OH aqueous
solution (pH ꢃ9) and dried over anhydrous Na2SO4.
After the solvent was removed, the residue was purified
on silica gel, eluted with cyclohexane/CHCl3/(Et)3N
(6:4:1) to obtain 100 mg (63%) of the pure phenyllyca-
conitine (1e). An analytical sample was prepared as the
citrate salt: mp 105 ꢁC (dec). Anal. (C48H60N2O17Æ0.5-
H2O) C, H, N.
4.16. [125I]Iodo-MLA binding assay
Frozen male rat cerebral cortex (includes hippocampus;
Pel-Freez Biologicals, Rogers, AK) was homogenized
(polytron) in 39 volumes of ice-cold 50 mM Tris buffer
(assay buffer; pH 7.4 at 4 ꢁC) containing 120 mM NaCl,
5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2. The homog-
enate was centrifuged at 35,000g for 10 min at 4 ꢁC and
the supernatant discarded. The pellet was washed twice
more with the original volume of buffer. After the last
centrifugation, the pellet was resuspended in 1/10 the ori-
ginal volume of buffer and stored at ꢀ80 ꢁC until needed.
The competition binding experiments were carried out in
1.4 mL polypropylene tubes (Matrix Technologies Cor-
poration, Hudson, NH) in a 96-well array. In a final vol-
ume of 0.5 mL triplicate samples contained
approximately 3 mg of tissue (wet weight; added last),
40–50 pM [125I]iodo-MLA, and 10–12 different concen-
trations of the test compounds. The dilutions of the test
compounds were made using assay buffer containing
10% DMSO (1% final concentration). The total binding
and nonspecific binding (300 lM nicotine) samples also
contained a final concentration of 1% DMSO. The test
compounds were pipetted using a MultiProbe IIEX
(Packard Instruments, Meriden, CT) robotic liquid han-
dling system. The samples were incubated on ice for 2 h.
A MultiMate harvester (Packard) was used to separate
bound radioligand from free by rapid vacuum filtration
onto GF/B filters presoaked for at least 30 min in assay
buffer containing 0.15% bovine serum albumin. The fil-
ters were washed with approximately 4 mL of ice cold
4.13. Cyclohexylsuccinamic acids (4f and 5f)
A suspension of cyclohexylsuccinic acid (2f) (2.0 g,
10 mmol) in acetic anhydride (15 mL) was heated to re-
flux overnight. The excess of acetic anhydride was re-
moved by sample distillation, the residue (3f) was
triturated and dried under reduced pressure. The anhy-
dride (3f) (1.78 g, 9.78 mmol) was dissolved in 15 mL
CHCl3 and was added to a suspension of anthranilic
acid (1.34 g, 9.78 mmol) in 15 mL CHCl3. The reaction
mixture was heated on a steam bath for 30 min, and
the CHCl3 solvent was evaporated to dryness under re-
duced pressure. The product was purified on silica gel,
eluting with hexane/ethyl acetate/methanol/acetic acid
(125:45:5:4) to obtain 2.9 g (92%) of cyclohexylsucci-
namic acids (4f and 5f): mp 171–173 ꢁC, 1H NMR
(CDCl3) d 1.05–2.04 (m, 11H, -C6H11), 2.61–2.68 (m,
1H), 2.76–2.88 (m, 2H), 7.08–7.13 (t, 1H, aromatic),
7.56–7.61 (t, 1H, aromatic), 8.06–8.08 (d, 1H, aromatic),
8.70–8.73 (d, 1H, aromatic).
4.14. Cyclohexyllycactonic acid (6f)
A solution of the above cyclohexylsuccinamic acids (4f
and 5f) (2.77 g, 8.68 mmol) and triethylamine (4.34 g,

684
F. Ivy Carroll et al. / Bioorg. Med. Chem. 15 (2007) 678–685
10 mM Tris buffer (pH 7.4 at 4 ꢁC) without salts and
dried prior to the addition of 35 lL of Microscint 20
scintillant (Packard). The amount of radioligand remain-
ing on each filter was determined using a Packard Top-
Count microplate scintillation counter (70% efficiency).
and a minimum of four doses were performed for
dose–response curve determination. Antagonism studies
were carried out by pretreating the mice sc with either
saline MLA (1a) or MLA analogs 1b–f at different times
before nicotine. The animals were tested 5 min after
administration of nicotine.
4.17. [3H]Epibatidine binding assay
4.20. Hot-plate test
Adult male rat cerebral cortices (Pelfreeze Biological,
Rogers, AK) were homogenized in 39 volumes of ice-
cold 50 mM Tris buffer (pH 7.4 at 4 ꢁC) containing
120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM
MgCl2, and centrifuged at 37,000g for 10 min at 4 ꢁC.
The supernatant was discarded, the pellet resuspended
in the original volume of buffer, and the wash procedure
repeated twice more. After the last centrifugation, the
pellet was resuspended in 1/10 its original homogeniza-
tion volume and stored at ꢀ80 ꢁC until needed. In a final
volume of 0.5 mL, each assay tube contained 3 mg wet
weight of male rat cerebral cortex homogenate (added
last), 0.5 nM [3H]epibatidine (NEN Life Science Prod-
ucts, Wilmington, DE), and one of 10–12 different con-
centrations of test compound dissolved in buffer (pH 7.4
at room temperature) containing 10% DMSO resulting
in a final DMSO concentration of 1%. Total and non-
specific bindings were determined in the presence of
vehicle and 300 lM (ꢀ)-nicotine, respectively. After 4-
h incubation at room temperature, the samples were
vacuum-filtered over GF/B filter papers presoaked in
0.03% polyethylenimine using a Brandel 48-well harvest-
er and washed with 6 mL of ice-cold buffer. The amount
of radioactivity trapped on the filter was determined by
standard liquid scintillation techniques in a TriCarb
2200 scintillation counter (Packard Instruments, Meri-
den, CT) at approximately 50% efficiency.
Mice were placed into a 10-cm wide glass cylinder on a
hot plate (Thermojust Apparatus) maintained at
55.0 ꢁC. Two control latencies at least 10 min apart were
determined for each mouse. The normal latency (reac-
tion time) was 8–12 s. Antinociceptive response was cal-
culated as percent maximum possible effect (% MPE),
where %MPE = [(test ꢀ control)/40 ꢀ control) · 100].
The reaction time was scored when the animal jumped
or licked its paws. Eight mice per dose were injected sc
with epibatidine analogs and tested 5 min thereafter in
order to establish a dose–response curve.
4.21. Nicotine-induced seizures
Male ICR (20–25 g) obtained from Harlan Laboratories
(Indianapolis, IN) were used throughout the study. Fol-
lowing injection of nicotine, each animal was placed in
a 30 · 30 cm2 Plexiglas cage and observed for 3 min.
Whether a clonic seizure occurred within a 3-min time
period was noted for each animal after sc administration
of nicotine. This time was chosen because seizures occur
very quickly after nicotine administration. Antagonism
studies were carried out by pretreating the mice sc with
either saline or different MLA analogs 10 min before nic-
otine. The percentage of animals exhibiting a seizure was
calculated and dose–response curves were constructed
and AD50 value determined for each of the MLA analogs.
4.18. Data handling
The specific binding data were fit using the nonlinear
regression equations in Prism (GraphPad Prism v. 3.0;
GraphPad Software, San Diego, CA). The Cheng–Prus-
off equation36 [Ki = IC50/(1 + ([L]/Kd)] was used to cal-
culate the Ki from the IC50. The data are reported as
means SEM from at least three independent experi-
ments. Kd values for [125I]iodo-MLA and [3H]epibati-
dine were 1.8 and 0.02 nM, respectively. These Kd
values were determined under conditions identical to
their respective assays.
Acknowledgment
This research was supported by the National Institute
on Drug Abuse, Grant DA12001.
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