A-366833: A novel nicotinonitrile-substituted 3,6-diazabicyclo[3.2.0]-heptane α4β2 nicotinic acetylcholine receptor selective agonist: Synthesis, analgesic efficacy and tolerability profile in animal models

Article abstract of DOI:10.1016/j.bcp.2007.08.010

5-[(1R,5S)-3,6-Diazabicyclo[3.2.0]heptan-6-yl]nicotinonitrile (A-366833) is a novel nicotinic acetylcholine receptor (nAChR) ligand that binds to the agonist-binding site ([³H]-cytisine) with K_i value of 3.1 nM and exhibits agonist s

Full text of DOI:10.1016/j.bcp.2007.08.010

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journal homepage: www.elsevier.com/locate/biochempharm
A-366833: A novel nicotinonitrile-substituted
3,6-diazabicyclo[3.2.0]-heptane a4b2 nicotinic
acetylcholine receptor selective agonist: Synthesis,
analgesic efficacy and tolerability profile in
animal models
a,
a
a
Jianguo Ji a, , William H. Bunnelle , David J. Anderson , Connie Faltynek ,
Tino Dyhring b, Philip K. Ahring b, Lynne E. Rueter a, Peter Curzon a,
Michael J. Buckley a, Kennan C. Marsh a, Anita Kempf-Grote a,
Michael D. Meyer a
*
*
a Neuroscience Research, Department-R47W, Bldg.-AP9A, Global Pharmaceutical Research and Development, Abbott,
Abbott Park, IL 60064, USA
b NeuroSearch A/S, Ballerup, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
5-[(1R,5S)-3,6-Diazabicyclo[3.2.0]heptan-6-yl]nicotinonitrile (A-366833) is a novel nicotinic
acetylcholine receptor (nAChR) ligand that binds to the agonist-binding site ([3H]-cytisine)
with Ki value of 3.1 nM and exhibits agonist selectivity at a4b2 nAChR relative to the a3b4
nAChR subtype. The analgesic effects of A-366833 were examined across a variety of animal
models including the mouse model of writhing pain (abdominal constriction), the rat
models of acute thermal (hot box), persistent chemical (formalin) and neuropathic (spinal
nerve ligation, SNL) pain. In the abdominal constriction model, A-366833 was effective at
doses ranging from 0.062 to 0.62 mmol/kg (i.p.). In addition, A-366833 demonstrated sig-
nificant effects in acute thermal pain (6.2–19.0 mmol/kg, i.p.), formalin (1.9–19 mmol/kg i.p.)
and SNL (1.9–19 mmol/kg i.p.) models. The systemic effects of A-366833 were attenuated by
pretreatment with mecamylamine (5 mmol/kg i.p.) in both the formalin and SNL models,
suggesting that the analgesic effects of A-366833 in models of persistent nociceptive and
neuropathic pain are mediated by activation of nAChRs. Pharmacokinetic investigations of
A-366833 in rat revealed moderate brain:plasma distribution, half-life of 1.5 h and excellent
oral bioavailability of 73%. Comparison of peak plasma levels at the minimal effective doses
across rat models of acute thermal pain, formalin and SNL with the maximal exposure that
does not evoke emesis in ferret revealed therapeutic margins ranging from 6- to 22-fold.
These studies indicate that compounds like A-366833 with improved agonist selectivity at
a4b2 vs. a3b4 nAChR can elicit a broad spectrum of analgesic efficacy without concurrent
adverse effects.
Received 4 June 2007
Accepted 8 August 2007
Keywords:
nAChR
Nicotine
Epibatidine
A-366833
Pain
Emesis
# 2007 Elsevier Inc. All rights reserved.
* Corresponding authors. Tel.: +1 847 935 3579/1 847 938 9765; fax: +1 847 937 9195.
E-mail addresses: jianguo.ji@abbott.com (J. Ji), william.h.bunnelle@abbott.com (W.H. Bunnelle).
0006-2952/$ – see front matter # 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2007.08.010

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1.
b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 1 2 5 3 – 1 2 6 2
Introduction
Among numerous approaches aimed at satisfying the
significant unmet medical need for the management of
severe pain [1], those targeting nicotinic acetylcholine
receptors (nAChR) with ligands that exhibit superior efficacy
and tolerability profiles continue to receive considerable
attention [2–4]. It is well recognized that nicotine (1), as well
other nAChR agonists including epibatidine (2), demon-
strate significant antinociceptive effects across a variety of
rodent pain models [5–8]. Clinical validation of the nAChR
approach has been achieved, but development of first
generation nAChR agents for the management of moder-
ate-to-severe pain has been limited due to adverse effects
such as emesis and nausea [9–13]. The search for second-
generation nAChR analgesics has been driven by the
hypothesis that side effects are attributable to poor
selectivity among nAChR subtypes [14]. nAChRs are penta-
meric ligand-gated ion channel proteins that are widely
expressed throughout the central and peripheral nervous
systems including in regions associated with nociceptive
transmission. Neuronal nAChRs are derived from multiple a
(a2–a10) and b (b2–b4) subunit genes. Each distinct subunit
gene composition defines a certain nAChR subtype, with
characteristic pharmacological and biophysical properties
[15]. While the nAChR subunit combinations that mediate
analgesic effects remain to be precisely defined, two distinct
mechanisms for nAChR-mediated effects have been identi-
fied. The first, which requires receptor activation by
agonists, can involve peripheral and central sites of action
and appears to be responsible for the analgesic properties of
compounds like epibatidine and A-85380 [6,16]. A growing
body of evidence, largely from gene knockout and antisense
studies, strongly implicates the involvement of the a4b2
nAChR combination in this type of nAChR agonist mediated
antinociception [17–21]. On the other hand, the a3b4 nAChR
combination predominates in the sympathetic ganglia and
is thought to mediate adverse events such as gastrointest-
inal effects [14]. Accordingly, medicinal chemistry efforts
over the past few years have primarily focused on the
identification of selective a4b2 nAChR agonists to test the
hypothesis that robust analgesic efficacy with wider toler-
ability vs. adverse effects can be achieved [2–8]. More
recently, blockade of a9a10 nAChRs has also been shown to
alleviate chronic pain resulting from peripheral nerve injury
or inflammation, possibly via neuro-immunomodulatory
mechanims [22–24].
Fig. 1 – Chemical structures of nicotine, epibatidine and 3,6-
diazabicyclo[3.2.0]heptane nAChR agonists used in this
study.
2.
Materials and methods
2.1.
Compound synthesis
2.1.1. A-366833, 5-[(1R,5S)-3,6-diazabicyclo[3.2.0]heptan-6-
yl]nicotinonitrile (3)
Under N2, (1S,5S)-benzyl 3,6-diazabicyclo[3.2.0]heptane-3-car-
boxylate [25] (4.64 g, 20.0 mmol) was stirred with 5-bromo-
nicotinonitrile (5.50 g, 30.0 mmol), Pd2(dba)3 (366 mg, 0.4 mmol),
BINAP (0.75 g, 1.2 mmol) and Cs2CO3 (13.12 g, 40.0 mmol) in
anhydrous toluene (200 mL) at 110 8C for 40 h. The reaction
mixture was cooled to ambient temperature and the solid was
removed by filtration. The organic filtrate was concentrated
under reduced pressure, and the residue was purified by
chromatography (SiO2, eluted with EtOAc/hexane = 80/20,
Rf = 0.30) to give (1S,5S)-benzyl 6-(5-cyanopyridin-3-yl)-3,6-
diazabicyclo[3.2.0]heptane-3-carboxylate (6.1 g, 91.3% yield) as
white solid: mp 101.4–102.9 8C; [a]D20 = À277.468 (c = 0.43,
MeOH)]. This was taken in trifluoroacetic acid (30.0 mL) and
stirred at 65 8C. After 1 h, the mixture was concentrated under
reduced pressure. The residue was basified to pH = 8–9 with 1N
NaOH and extracted with CHCl3 (3 Â 50 mL). The combined
extract was concentrated to an oil that was purified by
chromatography (SiO2, eluted with CH2Cl2/MeOH/NH3ÁH2O
= 90/10/2, Rf = 0.15) to provide A-366833 as the free base (mp:
101.4–102.9 8C). This material (1.40 g, 7 mmol) was converted to
bis(tosylate) salt bystirring with p-TsOHÁH2O (2.66 g, 14.0 mmol)
in iPrOH(50 mL) at80 8C for 1 h, then atambient temperaturefor
10 h. The salt was crystallized, collected by filtration and dried
under vacuum (3.20 g, 84.0% yield). mp 240–243 8C; 1H NMR
(MeOH-D4, 400 MHz) d 2.36 (s, 6H), 3.23 (dd, J = 13.0, 3.8 Hz, 1H),
3.34(dd, J = 12.6, 7.1 Hz, 1H), 3.73(d, J = 12.3 Hz, 1H), 3.82–3.98 (m,
3H), 4.18 (t, J = 8.6 Hz, 1H), 5.13 (dd, J = 6.4, 3.7 Hz, 1H), 7.21 (d,
J = 8.0 Hz, 4H), 7.64 (d, J = 8.0 Hz, 4H), 7.83 (dd, J = 2.6, 1.4 Hz, 1H),
8.22 (d, J = 2.5 Hz, 1H), 8.42 (d, J = 0.9 Hz, 1H) ppm; MS (DCI/NH3)
m/z 218 (M + NH4)+, 201 (M + H)+. Anal. Calcd. for
C11H12N4Á2.00C7H8SO3: C, 55.13; H, 5.18; N, 10.29. Found: C,
55.04; H, 4.93; N, 9.96.
Our in-house efforts to discover second generation a4b2
nAChR subtype selective agonists with diminished interac-
tions at the a3b4 nAChRs, led to the discovery of novel
nicotinonitrile-substituted
3,6-diazabicyclo[3.2.0]heptane
nAChR ligands represented by compounds including A-
366833 (3), A-365193 (4), A-424600 (5) and A-369452 (6) Fig. 1
[25]. This study describes the chiral synthesis, binding and
functional evaluation of compounds 3–6. Among the analogs
evaluated, 5-[(1R,5S)-3,6-diazabicyclo[3.2.0]heptan-6-yl]ni-
cotinonitrile (A-366833, 3) emerged as an a4b2 nAChR
subtype selective agonist, and accordingly, was further
characterized in vivo across models of efficacy and toler-
ability.
2.1.2. A-365193, 5-[(1S,5R)-3,6-diazabicyclo[3.2.0]heptan-6-
yl]nicotinonitrile (4)
The free base of A-365193 was prepared according to the
procedure described for A-366833, with the exception that

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(1R,5R)-benzyl 3,6-diazabicyclo[3.2.0]heptane-3-carboxylate
[25] was used as the starting material. The free base was
converted to the bis(fumarate) salt by stirring with two
equivalents of fumaric acid in EtOAc/EtOH (10/1) for 10 h.
The salt was crystallized, collected by filtration and dried
under vacuum. 1H NMR (MeOH-D4, 400 MHz) d 3.20 (dd, J = 12.7,
3.7 Hz, 1H), 3.35–3.42 (m, 1H), 3.48–3.53 (m, 1H), 3.70–3.85 (m,
3H), 4.10 (t, J = 8.1 Hz, 1H), 5.00 (dd, J = 6.5, 3.8 Hz, 1H), 6.70 (s,
5.0H), 7.36 (d, J = 2.7, 1.7 Hz, 1H), 8.10 (d, J = 3.0 Hz, 1H), 8.26 (d,
J = 1.4 Hz, 1H) ppm; MS (DCI/NH3) m/z 218 (M + NH4)+, 201
(M + H)+. Anal. Calcd. for C11H12N4Á2.50C4H4O4Á0.70H2O: C,
50.14; H, 4.69; N, 11.14. Found: C, 49.83; H, 4.30; N, 11.10.
compound were tested in duplicate. Non-specific binding was
determined in the presence of 10 mM of (À)-nicotine. Bound
radioactivity was isolated by vacuum filtration onto pre-
wetted glass fiber filter plates (Millipore, Bedford, MA) using a
96-well filtration apparatus (Packard Instruments, Meriden,
CT) and were then rapidly rinsed with 2 mL of ice-cold BSS
buffer (120 mM NaCl/5 mM KCl/2 mM CaCl2/2 mM MgCl2).
Packard MicroScint-201 scintillation cocktail (40 mL) was
added to each well and radioactivity determined using a
Packard TopCount1 instrument.
2.2.2. Fluorometric imaging plate reader (FLIPR)-based
functional assay
2.1.3. A-424600, 5-[(1S,5S)-3,6-diazabicyclo[3.2.0]heptan-3-
yl]nicotinonitrile (5)
HEK-293 cell lines expressing the recombinant human
nAChRs (a4b2 and a3b4 subunit combinations) were used in
the determination of functional nAChR agonist activity by
measuring intracellular calcium changes using the fluoro-
metric imaging plate reader (FLIPR; Molecular Devices,
Sunnyvale, CA, USA). Cells were plated at densities of
25,000–50,000 cells/well in DMEM (GIBCO) supplemented with
10% FBS (GIBCO) in 96-well clear bottom black walled plates
precoated with poly-D-lysine (Sigma, 75 mL/well of 0.01 g/L
solution ꢀ30 min) and allowed to incubate for 24–48 h at 37 8C
in 5% CO2 in a humidified environment. After aspirating off the
media, the cell lines were incubated in the dark at room
temperature for 45–60 min with 2–4 mM Fluo-4 AM calcium
indicator dye (Molecular Probes, Eugene, OR) dissolved in 0.1 to
0.2% (v/v) of DMSO (Sigma, UK) in NMDG Ringer buffer (in mM:
140 NMDG, 5 KCl, 1 MgCl2, 10 HEPES, 10 CaCl2, pH = 7.4). Cells
were placed in the FLIPR and 50 mL of 3Â stock concentrations
of test compounds or buffer prepared in the same NMDG
ringer buffer were added. Raw fluorescence data were
corrected by subtracting fluorescence values from wells that
received buffer only additions. Peak fluorescent values were
exported to Microsoft Excel, corrected for background signal
and expressed as a percentage of the reference peak response
for the positive control of 100 mM nicotine.
The free base of A-424600 was prepared from (1R,5S)-tert-butyl
3,6-diazabicyclo[3.2.0]heptane-6-carboxylate [25] according to
the procedure described for A-366833 and then converted to
the tosylate salt by stirring with 1.2 equivalents of p-TsOHÁH2O
in EtOAc/EtOH (10/1) for 10 h. The salt was crystallized,
collected by filtration and dried 1H NMR (MeOH-D4,
300 MHz) d 2.30 (s, 4.2H), 3.16 (dd, J = 10.7, 6.3 Hz, 1H), 3.25
(dd, J = 12.8, 7.8 Hz, 1H), 3.47–3.62 (m, 1H), 3.74 (dd, J = 11.2,
5.1 Hz, 1H), 3.98 (d, J = 10.5 Hz, 1H), 4.20 (d, J = 12.5 Hz, 1H), 4.28
(dd, J = 11.0, 8.6 Hz, 1H), 5.08 (dd, J = 6.8, 5.1 Hz, 1H), 7.22 (d,
J = 8.1 Hz, 2.8H), 7.69 (d, J = 8.5 Hz, 2.8H), 7.76 (dd, J = 2.7, 1.7 Hz,
1H), 8.39 (d, J = 1.7 Hz, 1H), 8.47 (d, J = 2.7 Hz, 1H) ppm; MS (DCI/
NH3) m/z 201 (M + H)+; Anal. Calc. for C11H12N4Á1.40C7H8SO3: C,
56.61; H, 5.30; N, 12.70. Found: C, 56.74; H, 5.29; N, 12.49.
2.1.4. A-369452, 5-[(1R,5R)-3,6-diazabicyclo[3.2.0]heptan-3-
yl]nicotinonitrile (6)
The free base of A-369452 was prepared from (1S,5R)-tert-butyl
3,6-diazabicyclo[3.2.0]heptane-6-carboxylate [25] according to
the procedure described for A-366833 and then converted to
the fumarate salt by stirring with 1.2 equivalents of fumaric
acid in EtOAc/EtOH (v. 10/1) for 10 h. The salt was crystallized,
collected by filtration and dried under vacuum. 1H NMR
(MeOH-D4, 300 MHz) d 3.14 (dd, J = 10.6, 6.2 Hz, 1H), 3.24 (dd,
J = 12.8, 5.0 Hz, 1H), 3.55 (m, 1H), 3.75 (dd, J = 11.0, 5.0 Hz, 1H),
3.96 (d, J = 10.6 Hz, 1H), 4.18 (d, J = 12.2 Hz, 1H), 4.28 (dd, J = 10.9,
8.4 Hz, 1H), 5.00 (dd, J = 6.8, 4.8 Hz, 1H), 6.40 (s, 2H), 7.65 (dd,
J = 2.9, 1.2 Hz, 1H), 8.33 (d, J = 1.2 hz, 1H), 8.45 (d, J = 2.8 Hz, 1H)
ppm; MS (DCI/NH3) m/z 201 (M + H)+; Anal. Calc. for
C11H12N4Á1.00C4H4O4Á0.50H2O: C, 55.38; H, 5.27; N, 17.22. Found:
C, 55.00; H, 5.27; N, 17.06.
2.3.
In vivo studies
2.3.1. Animals
Adult male mice (Jackson Laboratories, Bar Harbor, ME),
Sprague–Dawley rats (Charles River, Portage, MI) and male
ferrets (Marshall BioResources, North Rose, NY) were used for
the studies. These animals were housed in AAALAC-approved
facilities at Abbott Laboratories in a temperature-regulated
environment with lights on between 7:00 a.m. and 10:00 p.m.
Food and water were available ad libitum at all times except
during testing. Studies were conducted following procedures
outlined in protocols approved by Abbott’s Institutional
Animal Care and Use Committee.
2.2.
In vitro assays
2.2.1. [3H]-Cytisine binding
[3H]-Cytisine binding assay conditions were modified from the
procedures described in Pabreza et al. [26]. Membrane
enriched fractions from rat brain minus cerebellum (ABS
Inc., Wilmington, DE) were slowly thawed at 4 8C, washed and
resuspended in 30 vol of BSS–Tris buffer (120 mM NaCl; 5 mM
KCl; 2 mM CaCl2; 2 mM MgCl2; 50 mM Tris–Cl, pH 7.4, 4 8C).
Samples containing 100–200 mg of protein and 0.75 nM [3H]-
cytisine (30 Ci/mmol; Perkin Elmer/NEN Life Science Products,
Boston, MA) were incubated in a final volume of 500 mL for
75 min at 4 8C. Seven log-dilution concentrations of each
2.3.2. Abdominal construction model
Experiments were performed using mice weighing 20–25 g
(Jackson Laboratories, Bar Harbor, ME) as described previously
[27]. A solution of A-366833 bis(tosylate) was prepared in saline
and injected (i.p.). After 30 min 0.3 mL of 0.6% acetic acid was
dosed (i.p.) to evoke writhing. Animals were placed separately
under clear cylinders for observation and quantification of
abdominal constriction responses. These responses were

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b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 1 2 5 3 – 1 2 6 2
defined as a mild constriction and elongation passing caudally
along the abdominal wall, accompanied by a slight twisting of
the trunk and followed by bilateral extension of the hind
limbs. The total number of abdominal constrictions was
recorded from 5 to 20 min after the acetic acid injection.
Results are presented as percent of responses relative to
vehicle-treated animals.
The wound was cleaned, the membrane sewn with 4.0
dissolvable Vicryl suture and the skin closed with wound
clips. Following nerve ligation surgery, animals were group-
housed. Rats had access to food and water ad libitum. Two
weeks after surgery, rats were acclimated to the testing box
that was constructed of Plexiglas with a wire mesh floor to
allow access to the planter surface of the hind paws. On the
testing days, mechanical allodynia in the affected paw of
animals that had undergone spinal nerve ligation was
evaluated using von Frey filaments. The maximal withdrawal
threshold was 15 g. Using the Dixons up–down method, the
baseline level of allodynia was determined, with allodynia
defined as a withdrawal threshold ꢁ4 g of pressure. Immedi-
ately after baseline testing, a saline solution of A-366833 was
administered intraperitoneally, and subsequent withdrawal
thresholds were determined 15, 30, 60 and 120 min post-
dosing. In experiments designed to assess the role of nAChRs,
mecamylamine was administered (i.p.) after baseline testing,
10 min prior to the administration of A-366833.
2.3.3. Acute thermal pain model (hot box)
For hot-box experiments,
a commercially available paw
thermal stimulator was used as previously described [8]. Rats
were placed in Plexiglas cubicles that were located on a glass
surface of the apparatus. The surface of the glass was
maintained at 30 8C. A thermal stimulus was applied to the
bottom of the rear foot of the rat via a movable focused
projection bulb. The stimulus current was maintained at
4.8 amp. The latency until the animal moved its foot from the
stimulus was recorded automatically by use of photodiode
motion sensors. A 20-s cut-off was used to limit possible tissue
damage after exposure to the stimulus. For any given measure
(e.g., time point), one foot of each of the six animals was tested
and then the process was repeated for the opposite foot. All
2.3.6. Emetic effects in ferrets
Male ferrets weighing 1–1.7 kg were used in all experiments as
described previously [31,32]. Animals were fasted overnight
before emesis testing. Briefly, animals were placed in
individual polycarbonate cages with ventilated tops and
allowed to acclimate to the experimental room for 1 h prior
to commencing study. A-366833 was dissolved in vehicle
(saline) and administered (i.p.). After dosing, the animals were
observed for emetic responses for a period of 90 min. The
percentage of animals that experienced emesis was recorded.
studies began with
a 20-min acclimation period. After
determination of base line responses, compound was admi-
nistered (i.p.) and measures were taken 15, 30 and 45 min post-
treatment.
2.3.4. Formalin model
Male Sprague–Dawley rats weighing 200–400 g were used in all
experiments as previously described [28]. Injection of formalin
(50 mL of 5% formalin in saline) into the dorsal aspect of the rat
rear paw produces a transient inflammatory response (Phase
1) [29], and characterized by behaviors including licking, biting
and flinching the affected paw. This acute phase subsides after
about 10 min, with the nocifensive behavior renewed (about
20 min post-dose) in a persistent response (Phase 2) that is
considered to reflect central sensitization. In this study, only
the Phase 2 response was scored. A-366833 was dosed (i.p.)
5 min prior to administration (s.c.) of formalin (50 mL of 5%
formalin in saline) into the dorsal aspect of one of the rear
paws. Animals were then returned to the clear observation
cages suspended above mirror panels. Effects of A-366833
were evaluated in the time interval 30–50 min after the
administration of formalin (Phase 2 response). The number
of flinches was recorded as a measure of persistent pain for 6–8
rats per dose group. In experiments designed to assess the role
of nAChRs, mecamylamine was administered 10 min before
A-366833 treatment.
2.3.7. Data analysis
In radioligand binding experiments, the IC50 values were
determined by nonlinear regression using Microsoft Excel1
software and Ki values were calculated using the Cheng-
Prusoff equation, where Ki = IC50/(1 + [ligand]/KD). For calcium
flux experiments, concentration-response data were fitted
using a single sigmoidal function in GraphPad Prism (San
Diego, CA) for determination of EC50 and maximum response
values. Statistical analysis was performed using either t-test or
one-way ANOVA. P < 0.05 was considered significant. Data are
expressed as means Æ S.E.M.
3.
Results
3.1.
In vitro pharmacology
The predominant receptor with high affinity to (À)-[3H]-
cytisine binding in rodent brain is composed of a4 and b2
subunits [33,34]. The binding affinities of ligands at nAChRs
were determined by assessing their ability to displace (À)-[3H]-
cytisine binding to rat brain membranes [35]. The Ki values of
the compounds for displacing rat brain [3H]-cytisine binding is
shown in Table 1, A-366833 (3) shows binding affinity
(Ki = 3.1 nM), comparable to that of nicotine (1) (Ki = 1.0 nM),
but substantially less potent than epibatidine (2) (Ki = 0.05 nM).
The affinities for A-365193 (4) and A-424600 (5) are slightly
higher. A-369452 (6) demonstrated the highest binding affinity
(Ki = 0.1 nM) among four isomers 3–6 tested.
2.3.5. Spinal nerve (L5-L6) ligation model
Male Sprague–Dawley rats weighing 80–100 g at the time of
surgery were used for all experiments. Prior to surgery,
animals were group-housed and maintained in a tempera-
ture-regulated environment as described above. Under
halothane anesthesia, the L5 and L6 spinal nerves were tightly
ligated in the manner described previously by Chung et al. [30].
An incision was made on the dorsal portion of the hip and the
muscle was blunt dissected to reveal the spinal processes. The
L6 transverse process was removed, and the left L5 and L6
spinal nerves were tightly ligated with 5.0-braided silk suture.

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Table 1 – Comparative in vitro properties of nicotinonitrile-substituted 3,6-diazabicyclo[3.2.0]-heptanes (3–6) and nicotine
(1), as well epibatidine (2) in radioligand binding and functional assays
Compounds
[3H]-cyt.a
Ca2+ flux (FLIPR)
Ki (nM) (pKi)
ha4b2
ha3b4
Max. (%)c
EC50 (mM) (pEC50
)
Max. (%)c
b
b
EC50 (mM) (pEC50
)
A-366833
A-365193
A-424600
A-369452
Nicotine
3.1 (8.50 Æ 0.17)
0.9 (9.07 Æ 0.05)
1.7 (8.77 Æ 0.06)
0.1 (9.92 Æ 0.05)
1.0 (9.03 Æ 0.31)
0.05 (10.33 Æ 0.13)
5.9 (5.29 Æ 0.10)
11.4 (5.04 Æ 0.34)
14.6 (4.88 Æ 0.08)
0.6 (6.38 Æ 0.17)
6.6 (5.18 Æ 0.06)
0.05 (7.27 Æ 0.04)
65 Æ 2
34 Æ 2
N.C.d
16 Æ 2
47 Æ 8
84 Æ 3
102 Æ 3
89 Æ 3
103 Æ 15
13.7 (4.91 Æ 0.15)
7.1 (5.15 Æ 0.03)
0.3 (6.49 Æ 0.02)
8.0 (5.10 Æ 0.04)
0.01 (7.92 Æ 0.07)
102 Æ 6
123 Æ 5
101 Æ 3
133 Æ 13
Epibatidine
a
Displacement studies with [3H]-cytisine using rat brain membranes. The Ki represents mean values obtained from at least three independent
experiments.
b
Values represent mean potencies of compounds, assessed by measuring fluorescence changes using FLIPR technology in HEK 293 cell lines
expressing human a4b2 and a3b4 nAChRs.
c
Max. values represent maximal response of the ligand relative to the peak response for the positive control of 100 mM nicotine.
d
NC, EC50 not reliably calculable for agonists with maximal response below 20%.
In calcium flux assays, functional interactions were
assessed at ha4b2 and ha3b4 nAChRs [36,37]. At the
recombinant human a4b2 nAChR subtype, A-366833 exhibited
a potency (EC50 = 5.9 mM) similar to nicotine, but A-366833 is
somewhat less efficacious with a maximal response 65%
relative to nicotine. At the a3b4 subtype, A-366833 is much less
active than nicotine, exhibiting only about 16% efficacy at the
highest concentration tested (100 mM, Table 1). Thus, A-366833
is a moderately potent partial agonist at the a4b2 nAChR, with
little or no agonist activity at the a3b4 subtype. In comparison,
epibatidine 2 is approximately 100–150-fold more potent than
A-366833 at ha4b2 (EC50 = 0.05 mM, 133% vs. nicotine) with
even greater activity at the a3b4 subtype (EC50 = 0.01 mM, 103%
vs. nicotine). Among the stereoisomers 3–6, A-365193 (4) and
A-424600 (5) were approximately 2-fold less potent than A-
366833 at the a4b2 nAChR subtype, although the 5 is a full
agonist. Neither 4 nor 5 showed any subtype selectivity,
however, as they activated a3b4 nAChRs with EC50 values of
13.7 mM (max. response 47%) and 7.1 mM (max. response 84%),
respectively. Finally, A-369452 (6) exhibited nearly 10-fold
higher potency (EC50 = 0.6 mM, max. response 123%) than A-
366833 at the recombinant human a4b2 nAChR subtype, and
even greater activity (EC50 = 0.3 mM, max. response 102%) at
the recombinant human a3b4 nAChR subtype. Thus, among
analogs within this group, only A-366833 achieved substantial
a4b2 nAChR subtype selectivity (Table 1).
strictions in
reductions in the number of abdominal constrictions
were noted at dose of 0.019 mmol/kg (i.p.), doses of
a dose-related manner (Fig. 2). While no
a
0.062 mmol/kg (i.p) and 0.19-mmol/kg (i.p.) of A-366833
reduced constrictions by 38% and 77%, respectively. At
the highest dose tested (i.p. 0.62 mmol/kg), animals exhibited
85% diminution of abdominal constrictions. (As a positive
control, epibatidine was fully efficacious in this model at
0.012 mmol/kg.)
3.2.2. Rat acute thermal pain
Intraperitoneal administration of A-366833 increased paw
withdrawal latencies in the rat acute thermal pain model
(Fig. 3). Doses of 0.62 mmol/kg and 1.9 mmol/kg showed no
antinociceptive effects relative to control animals whereas
significant antinociceptive effects of A-366833 were observed
at higher doses (6.2–19 mmol/kg). By comparison, epibatidine is
much more potent in this model (ED50 value of 0.004 mmol/kg,
Curzon et al. [38]). In contrast to epibatidine, however no
apparent behavioral side effects, such as hypoactivity, ataxia,
In order to assess the potential for off-target activity of A-
366833, it was evaluated by radioligand binding against a panel
of 75 receptors, channels and enzyme targets (Cerep). Up to a
concentration of 10 mM, A-366833 showed no significant
inhibition (>70%) of radioligand binding except at the 5HT3
receptor (85%).
3.2.
In vivo efficacy
3.2.1. Mice abdominal constriction
Injection of 0.6% acetic acid into the peritoneal cavity of
mice evoked abdominal constriction responses character-
ized by abdominal stretching combined with an exaggerated
extension of the hind limbs. Systemic administration of
A-366833 decreased acetic acid induced abdominal con-
Fig. 2 – Antinociceptive effects of A-366833 in the acetic
acid-induced abdominal constriction model in mouse.
Results are presented as number of constrictions during
the test period (mean W S.E.M.), n = 6/group, (*) Indicates
significant (P < 0.05) differences vs. saline treated group.

1258
b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 1 2 5 3 – 1 2 6 2
Fig. 3 – Antinociceptive effects of A-366833 in the rat acute
thermal pain assay (hot box). A-366833 produced a
significant dose-dependent antinociceptive effect after
systemic (i.p.) administration. Values represent the mean
latency to paw withdrawal (WS.E.M.); n = 6–9/group. (*)
Significantly (P < 0.05) different from saline treated rats.
Fig. 5 – Effect of mecamylamine pretreatment on the
analgesic effect of A-366833 in phase 2 of rat formalin test.
Mecamylamine HCl (5.0 mmol/kg) was administrated
10 min prior to the injection of A-366833 (n = 6/group). (*)
Indicates significantly (P < 0.05) different from saline/
saline group. (+) Indicates significantly (P < 0.05) different
from saline/A-366833 group.
sedation and respiratory changes [17,18], were observed at
efficacious doses of A-368833.
3.2.3. Formalin model
Next, the effect of systemic A-366833 treatment on phase 2
responses of the formalin model was investigated. A-366833
evoked dose-dependent reductions in the nocifensive
responses after formalin injection to the hind paw (1.9–
19.0 mmol/kg, i.p. Fig. 4). Control animals evoked mean
flinches of 80 Æ 1.8 during the 20-min time-period
measured 30–50 min. after the injection of formalin into
the rear paw. At a dose of 1.9 mmol/kg (i.p.), A-366833
significantly attenuated nocifensive responses, decreasing
flinches by about 50%. At 19 mmol/kg (i.p.), A-366833
demonstrated the most robust analgesic effect (mean
flinches, 19 0 Æ 4.3).
To establish the mechanism by which A-366833 produced
analgesic effects in the formalin model, the effect of
pretreatment with the non-competitive nAChR channel
blocker mecamylamine was examined. Mecamylamine
(5.0 mmol/kg, i.p.) was injected 10 min before the adminis-
tration of saline or A-366833. Mecamylamine alone had no
effect on the number of flinches in phase 2 of the formalin
test. However, as shown in Fig. 5, the analgesic effect of A-
366833 at the dose of 6.2 mmol/kg (i.p.) was blocked by
pretreatment with the nAChR antagonist mecamylamine
(5.0 mmol/kg). The effect of
a higher dose of A-366833
(19 mmol/kg, i.p.) was significantly attenuated by pretreat-
ment with mecamylamine indicating that the analgesic
effects of A-366833 in rat formalin model are mediated by
agonist activity at nAChRs. Furthermore, this result argues
against significant contribution of a9a10 nAChRs in the
effects of A-366833, since a9a10 analgesia is evoked by
antagonists [23].
3.2.4. Spinal nerve ligation model
A-366833 (1.9–19 mmol/kg, i.p.) also induced significant dose-
dependent reversal of mechanical allodynia in rats with
neuropathy secondary to the tight ligation of spinal nerves L5
and L6 (Fig. 6). At the 15-min time point following injection of
A-366833 (19 mmol/kg, i.p.), maximal anti-allodynic responses
averaging 13.90 Æ 0.70 (g) were observed. The anti-allodynic
was maintained, although at reduced levels through the 30-
Fig. 4 – Effect of A-366833 in the formalin model of
persistent pain. Administration of A-366833 after the
injection of formalin produced significant dose-dependent
analgesic effects during phase 2 of the formalin test (n = 5/
group). Values represent number of flinches. (*) Indicates
significantly (P < 0.05) different from saline treated rats.

b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 1 2 5 3 – 1 2 6 2
1259
pharmacokinetic profile was also assessed after i.v. and p.o.
administration of 6.2 mmol/kg dose in rats (n = 3). A-366833
showed plasma clearance (CLp), volumes of distribution (Vb)
and half-life values of 3.02 L/h/kg, 6.6 L/h and 1.5 h, respec-
tively. A-366833 also showed excellent oral bioavailability
(73%). Since emetic liability was routinely tested in ferrets, the
pharmacokinetic profile of A-366833 was assessed in this
species as well. Plasma concentrations of A-366833 following
a 2.5 mmol/kg dose (i.p.) in ferret showed a mean Cmax of
373 ng/mL with a mean Tmax of 0.25 h (n = 3) and a half life of
1.5 h.
3.4.
Emesis profile in ferrets
Nausea and emesis have been identified as dose-limiting
adverse effects for multiple experimental nAChR ligands
[9–13,39]. We have previously described a ferret emesis
model to evaluate the emetic potential of these compounds
[31,32]. Administration of 10 mmol/kg A-366833 (i.p.) did not
produce emesis in ferrets (n = 6). Emesis, however, was
observed at higher doses. We compared the highest dose to
produce no emesis (the ‘‘no-emesis threshold’’) in ferret to
exposures corresponding to the minimal efficacious dose
(MED) in rodent pain models to determine a preclinical
therapeutic index for A-366833. This can be done by
comparing the relevant doses (Table 2), providing thera-
peutic indices of 1.6–5.3, depending on the model. Since,
however, the pharmacokinetics differs between rat and
ferret, the therapeutic index was also calculated using
plasma exposures (based upon Cmax extrapolated from PK
experiments described in the previous section). Specifically,
since plasma exposures for a given dose of A-366833 were
higher in ferret compared to rat, this analysis provides
higher therapeutic indices. Thus, the peak plasma exposure
in ferret at 10 mmol/kg (i.p.) projects to 1492 ng/mL. Conse-
Fig. 6 – Effects of multiple doses of A-366833 on paw
withdrawal threshold (mean W S.E.M.) in a test of
mechanical allodynia in the rat spinal nerve ligation (SNL)
model of neuropathic pain. Paw withdrawal threshold
(gram force) was determined using von Frey filament
stimulation and the Dixon’s up-down method. Measures
were taken before agonist injection (baseline) and at 15,
30, 60 and 120 min after agonist injection. Six rats were
used at each dose. (*) Significant (P < 0.05) differences vs.
saline treated rats at the corresponding time intervals
(n = 6/group).
min time point at the same dose. By the 60-min time point, the
antiallodynic effect of A-366833 had abated. This dose
(19 mmol/kg, i.p.) was well tolerated, and behavioral side
effects, such as prostration and ataxia, typically seen with
non-selective nicotinic agonists, were not observed following
treatment with A-366333. Antiallodynic efficacy was also
noted at lower doses (1.9–6.2 mmol/kg, i.p.), although these
effects were limited in duration to the 15-min time point, with
no significant analgesia by 30 post-dose.
In this model, pretreatment with the nAChR antagonist
mecamylamine (5.0 mmol/kg, i.p., 10 min prior to the injection
of A-366833) completely blocked the anti-allodynic effects of
A-366833 (19 mmol/kg, i.p.) (Fig. 7), confirming that the effects
are nAChR agonist mediated.
3.3.
Brain penetration and pharmacokinetic
properties in the rat
It has been previously shown that the central activity of
nAChR agonists is important for their broad-spectrum
analgesic effects [5–8]. The ability of A-366833 to enter the
brain was assessed by measuring concentrations of A-366833
in brain and blood plasma in rat. A-366833 rapidly distributed
into the brain after the i.p. administration (6.2 mmol/kg) with a
brain: plasma ratio of 1–2:1, indicating efficient CNS penetra-
tion. Since in vivo studies were conducted by i.p. administra-
tion, the pharmacokinetic properties of A-366833 were
assessed after i.p. dosing in rats. Plasma concentrations of
A-366833 following a 3.0 mmol/kg (i.p.) in rats reached Cmax of
108 ng/mL with a mean Tmax of 0.3 h (n = 3). In addition, the
Fig. 7 – Effect of mecamylamine pretreatment on the anti-
allodynic effect of 19 mmol/kg A-366833 (i.p.) in SNL
neuropathic pain model. Mecamylamine HCl (5 mmol/kg,
i.p.) was administered 10 min before A-366833.
Withdrawal thresholds to a mechanical stimulus were
determined prior to mecamylamine administration and
15, 30 and 60 min post A-366833 administration. (*)
Significant (P < 0.05) differences vs. other treatment groups
at the corresponding time intervals (n = 6/group).

1260
b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 1 2 5 3 – 1 2 6 2
Table 2 – Therapeutic Index of A-366833
Model
Minimal efficacious
dose (MED) (mmol/kg)
Peak plasma concentration
(PPC) (ng/mL)
Therapeutic index
MEDa
PPCb
Hot-box
Formalin
SNL
6.2
1.9
1.9
223
68
1.6
5.3
5.3
6.7
21.9
21.9
68
a
Therapeutic index was calculated by dividing no emesis threshold dose in ferret (10 mmol/kg) by corresponding minimal efficacious dose in
the respective pain model assessed in the rat.
b
Therapeutic index was calculated by dividing the peak plasma concentrations (1492 ng/mL, no emesis threshold dose in ferret) by Cmax level
corresponding to the minimal efficacious dose in the respective pain model assessed in the rat.
quently, the therapeutic index was calculated by dividing
the peak plasma concentrations (1492 ng/mL, no emesis
threshold dose in ferret) by the peak plasma levels
corresponding to the minimal efficacious dose in the
respective pain models assessed in the rat. As shown in
Table 2, A-366833 produced a favorable therapeutic index vs.
no emesis threshold of 6.7, 21.9 and 21.9 in models of acute
thermal pain, formalin and SNL, respectively.
In three other models in rat – hot box for acute thermal pain,
formalin model of persistent pain and spinal ligation model
for neuropathic pain, A-366833 was very efficacious at doses
in the range of 1.9–6.2 mmol/kg.
Pretreatment with mecamylamine (5.0 mmol/kg) signifi-
cantly attenuated the analgesic effect of A-366833 assessed
in the phase 2 of the formalin model and completely blocked
the anti-allodynic effects evoked by A-366833 (19 mmol/kg,
i.p.) in the SNL model. These observations suggest that the
analgesic effects of A-366833 in models of persistent
nociceptive and neuropathic pain are nAChR mediated. As
noted in the Introduction, it has recently been shown that
antagonism of peripherally disposed a9a10 nAChRs also
leads to an analgesic response, and is especially effective in
models of neuropathic pain [22,23]. It is not trivial to
positively define the analgesic mechanism for a nAChR
ligand, in part because relatively few nAChR ligands have
been evaluated for activity at a9a10. Moreover, some
agonists at the a4b2 nAChR, including nicotine, act as
antagonists of the a9a10 receptor [24]. The Abbott com-
pounds described herein have not been evaluated for activity
at the a9a10 nAChR, and contribution of this receptor to the
analgesic activity cannot be ruled out entirely. Nevertheless,
the observation that analgesic responses are blocked by the
(non-competitive) nAChR antagonist mecamylamine is more
consistent with those effects arising from agonist activation
of a4b2 nAChRs.
3.5.
Discussion and summary
Nicotine and other nAChR agonists, such as epibatidine, have
previously demonstrated analgesic activity in a wide variety of
preclinical pain models [5–8]. For example, although the mild
analgesic effect of nicotine (1) in a cat model of visceral pain
was reported decades ago [40], its limited efficacy of nicotine in
eliciting analgesia, as well as accompanying adverse side
effects (convulsions, hypothermia, loss of motor coordination,
hypothermia and emesis) precludes its clinical use as an
analgesic agent [41–45]. Epibatidine was reported to evoke a
robust analgesic response superior to morphine, but at
efficacious doses, this compound exhibited severe adverse
side effects and toxicity [46–48], indicating lack of therapeutic
index between analgesia and nAChR-mediated side effects.
Although therapeutic indices of newer agents including ABT-
594 are somewhat improved relative to epibatidine [4],
opportunities exist to further separate the analgesic activity
from side effects.
Since improved tolerability of nAChR agents remains a
major goal in the development of nAChR-based therapeu-
tics, side effect profiles such as emesis [9–13,39], were
evaluated to determine therapeutic margins. As demon-
strated in Table 2, A-366833 showed substantial separation
of analgesic vs. emetic effects. Although emesis was
observed at higher doses, the favorable therapeutic index
of A-366833 supports a profile adequate for clinical advance-
ment. The separation of analgesic effects from gastrointest-
inal tolerability demonstrates that agonists with improved
a4b2 (vs. a3b4) agonist selectivity can achieve broad-
Although A-366833 exhibited relatively lower a4b2 binding
affinity compared to compounds like epibatidine and ABT-
594, its binding affinity (Ki = 3 nM) is comparable to that of
nicotine (Ki = 1 nM). Compared to the non-selective nAChR
agonist epibatidine, A-366833 showed approximately 60-fold
weaker binding affinity and 110-fold weaker functional
potency for activation of the a4b2 nAChR subtype. Interest-
ingly, A-366833 exhibited superior selectivity towards the
a4b2 nAChR subtype compared to activation of a3b4 nAChR
subtype. Since A-366833 demonstrated improved a4b2 vs.
a3b4 nAChR subtype selectivity, the compound was profiled
in a variety of rodent pain models indicating multiple pain
states, such as models of writhing pain (mice ACA), acute
nociceptive pain (rat acute thermal pain), persistent noci-
ceptive pain (rat formalin) and neuropathic pain (rat SNL). A-
366833 exhibited significant analgesic effects in a variety of
rodent pain models. For example, A-366833 exhibited excel-
lent potency (0.062–0.62 mmol/kg) and robust efficacy in
mouse abdominal construction assay, model of visceral pain.
spectrum efficacy in
a variety of animal pain models,
without concurrent adverse effects.
Acknowledgements
The authors would like to thank Drs. Murali Gopalakrishnan
and Lance Lee for assistance with the preparation of this
manuscript.

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