Epiboxidine and novel-related analogues: A convenient synthetic approach and estimation of their affinity at neuronal nicotinic acetylcholine receptor subtypes

Article abstract of DOI:10.1016/j.bmcl.2008.07.016

Racemic exo-epiboxidine 3, endo-epiboxidine 6, and the two unsaturated epiboxidine-related derivatives 7 and 8 were efficiently prepared taking advantage of a palladium-catalyzed Stille coupling as the key step in the reaction sequence. The target compounds were assayed for their binding affinity at neuronal α4β2 and α7 nicotinic acetylcholine receptors. Epiboxidine 3 behaved as a high affinity α4β2 ligand (K_i = 0.4 nM) and, interestingly, evidenced a relevant affinity also for the α7 subtype (K_i = 6 nM). Derivative 7, the closest analogue of 3 in this group, bound with lower affinity at both receptor subtypes (K_i = 50 nM for α4β2 and K_i = 1.6 μM for α7) evidenced a gain in the α4β2 versus α7 selectivity when compared with the model compound.

Full text of DOI:10.1016/j.bmcl.2008.07.016

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4651–4654
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Epiboxidine and novel-related analogues: A convenient synthetic approach
and estimation of their affinity at neuronal nicotinic acetylcholine receptor
subtypes
a
a
b
b
Luca Rizzi a, Clelia Dallanoce a, , Carlo Matera , Pietro Magrone , Luca Pucci , Cecilia Gotti ,
*
Francesco Clementi b, Marco De Amici a
a Istituto di Chimica Farmaceutica e Tossicologica ‘‘Pietro Pratesi”, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
b CNR, Istituto di Neuroscienze, Farmacologia Cellulare e Molecolare e Dipartimento Farmacologia, Chemioterapia e Tossicologia Medica, Università degli Studi di Milano,
Via Vanvitelli 32, 20129 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Racemic exo-epiboxidine 3, endo-epiboxidine 6, and the two unsaturated epiboxidine-related derivatives
7 and 8 were efficiently prepared taking advantage of a palladium-catalyzed Stille coupling as the key
step in the reaction sequence. The target compounds were assayed for their binding affinity at neuronal
Received 18 June 2008
Revised 3 July 2008
Accepted 4 July 2008
Available online 10 July 2008
a
4b2 and
(Ki = 0.4 nM) and, interestingly, evidenced a relevant affinity also for the
tive 7, the closest analogue of 3 in this group, bound with lower affinity at both receptor subtypes
(Ki = 50 nM for 4b2 and Ki = 1.6 M for 7) evidenced a gain in the 4b2 versus 7 selectivity when
compared with the model compound.
a
7 nicotinic acetylcholine receptors. Epiboxidine 3 behaved as a high affinity
a4b2 ligand
a
7 subtype (Ki = 6 nM). Deriva-
Keywords:
a
l
a
a
a
Neuronal nicotinic acetylcholine receptors
Epiboxidine
Nicotinic ligands
Ó 2008 Elsevier Ltd. All rights reserved.
Binding affinity
Neuronal nicotinic acetylcholine receptors (nAChRs) make up a
family of pentameric ligand-gated ion channels, which are formed
by combinations of alpha and beta subunits1,2 or exist as homo-
(À)-epibatidine 2 (Fig. 1), a highly toxic alkaloid identified in the
skin of the Ecuadorian frog Epipedobates tricolor,10 has inspired a
huge amount of research aimed at designing subtype selective nic-
otinic agonists.1,2,9,11,12 Although the pharmacological effects of
(À)-2 are mediated by a variety of nAChRs,13 which preclude any
therapeutic potential for epibatidine, the analgesic potency,
roughly 100 times higher than that of morphine and 30 times high-
er than that of nicotine, has been attributed to its high affinity for
pentamers, in the cases of
a7,
a
8, and
a
9 receptors, which are
a (a2–a10) and three
inhibited by
a
-bungarotoxin.3 To date, nine
b (b2–b4) isoforms have been characterized, though only a rela-
tively small subset of combinations generates functionally and
physiologically relevant channels.4 Nicotinic receptors are widely
distributed in the brain, where they primarily modulate the release
of other neurotransmitters and, to a lesser extent, mediate synaptic
transmission.5 Neuronal nAChRs, selectively activated by (S)-nico-
tine 1 (Fig. 1), are involved in various processes such as cognition,
learning and memory, cerebral blood flow and metabolism, as well
as an array of pathological conditions such as Alzheimer’s and Par-
kinson’s diseases, mild cognitive impairment (MCI), schizophrenia,
epilepsy, Tourette’s syndrome, anxiety, depression, attention-defi-
cit hyperactivity disorder (ADHD), and nicotine addiction.1,2,6,7
the
a
4b2 subtype. Worth mentioning, (À)-2 and (+)-2 are charac-
terized by similar affinities for nAChRs and almost identical ED50
values in the mouse tail-flick antinociception assay.13d,14,15
Among the synthetic epibatidine-related compounds, ( )-epib-
oxidine 3 (Fig. 1), in which the 3-methylisoxazolyl moiety has re-
placed the chloropyridinyl ring of the parent derivative, behaved
as a potent a4b2 nicotinic receptor agonist, 10-fold less potent than
epibatidine as antinociceptive agent but about 20-fold less toxic.16
The presence of the 3-methylisoxazole ring was similarly effective
The heteromeric
a4b2 receptors, the most abundant subtype in
in the structure of (S)-ABT-418 4 (Fig. 1), a nicotine-related a4b2
the mammalian CNS, and the homomeric
a
7 channels are privi-
selective full agonist, which entered Phase I for the treatment of
cognitive dysfunction and was later on discontinued.2,7,17 A further
relevant example of structural analogue of epibatidine is the unsat-
urated derivative ( )-UB-165 5, containing the 9-azabicy-
clo[4.2.1]nonene skeleton, a high affinity nicotinic partial agonist
leged biological targets in the search for selective nAChR ligands
with therapeutic potential.2,8 As far as the number of nicotinic ago-
nists isolated from natural sources is taken into consideration,9
which allowed to clarify the involvement of the
a4b2 subtype in
* Corresponding author. Tel.: +39 02 50319327; fax: +39 02 50319326.
E-mail address: clelia.dallanoce@unimi.it (C. Dallanoce).
modulating dopamine release from rat striatal synaptosomes.18,19
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.016

4652
L. Rizzi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4651–4654
CH
H
CH
3
H
3
Cl
N
N
N
N
N
N
O
O
N
N
CH
3
CH
3
1
(
S
)-Nicotine
3
( )-Epiboxidine
4
(
S)-ABT-418
2
(−)-Epibatidine
H
CH
H
N
R
3
Cl
N
N
N
N
O
O
CH
3
N
5
( )-UB-165
6 ( )-endo-Epiboxidine
( )-7: R=H
( )-8: R=CH
3
Figure 1. Structures of model and target compounds in this study.
In the present study, we aimed at further investigating epibox-
idine, that is, at 7 nAChRs, a subtype on which, to the best of our
Boc
N
a
Br
knowledge, it was never assayed. To this end, we thought about a
flexible synthesis of ( )-3 allowing also the preparation of unsatu-
rated structurally related derivatives. Accordingly, we describe a
novel synthetic route to epiboxidine 3 (exo-isomer), its epimer
endo-epiboxidine 6, and the two unprecedented dehydro-ana-
logues of epiboxidine 7 and 8. The target chiral compounds, whose
structures are reported in Figure 1, were prepared and tested as
racemates.
Br
a
+
N
Boc
CO Me
2
CO Me
2
13
14
15
Boc
N
As illustrated in the upper part of Scheme 1, the known prepa-
O
rations of
3
took advantage of the reaction of 7-azabicy-
b-d
e,f
15
11
clo[2.2.1]heptane-2,7-dicarboxylic acid diesters 9a and 9b with
the dianion of acetone oxime, followed by acidic treatment of
intermediates 10a and 10b.16,20 As an alternative approach, we
planned to make use of a Stille palladium-catalyzed cross-coupling
reaction,21 which involved the coupling of enoltriflate 11 with 3-
methyl-5-tributylstannylisoxazole 12, as depicted in the lower
part of Scheme 1.
Initially, we made use of a known procedure to prepare 7-tert-
butoxycarbonyl-7-azabicyclo[2.2.1]heptan-2-one 16, a key inter-
mediate in one of the syntheses of ( )-epibatidine.22 The first step
of the sequence, a [4+2] cycloaddition of N-Boc-pyrrole 13, used in
large excess, to methyl 3-bromopropiolate 14, was performed un-
der microwave heating (Scheme 2).23 These experimental condi-
tions allowed reduction of the reaction time (from 30 to 1.5 h)
and improvement of the yield of the Diels-Alder adduct 15 (from
60% to 85%). Subsequently, we prepared the known trifluorometh-
16
H
Cl
CH
3
g
+
12
N
HO
SnBu
3
17
18
Scheme 2. (a) MW, 90 °C, 1.5 h, neat; (b)–(d) see Ref. 22; (e) (iPr)2NH/n-BuLi,
À78 °C; (f) N-(5-chloro-2-pyridyl)bis(trifluoromethanesulfonimide), À78 °C to rt;
(g) NEt3/THF, rt, 5 h.
anesulfonate 1124 in 62% isolated yield by treatment of ketone 16
with LDA in THF at À78 °C25 followed by reaction with N-(5-
chloro-2-pyridyl)bis(trifluoromethanesulfonimide)26 (Scheme 2).
3-Methyl-5-tributylstannylisoxazole 12 was obtained through the
previously described 1,3-dipolar cycloaddition of tributylethynyl-
tin 17 to acetonitrile oxide.27 As a modification of the known pro-
cedure, we chose to isolate the stable precursor of the 1,3-dipole,
that is, the acetohydroximoyl chloride 18, which in turn was syn-
thesized by reacting acetaldoxime with benzyltrimethylammoni-
um tetrachloroiodate (BTMA ICl4).28,29 In this way, the target
tributylstannylisoxazole 12 was prepared in acceptable yields
(45–50%).
RO C
RO C
2
2
OH
CH
O
N
N
N
a
b
CO Et
2
3
3
9a: R = Et
9b: R = tert-Bu
10a: R = Et
10b: R = tert-Bu
(a) Acetone oxime, nBuLi; (b) conc. HCl, 80ºC.
The Stille cross-coupling reaction of triflate 11 to isoxazole 12,
performed in the presence of the Tris(dibenzylideneacetone)dipal-
ladium(0)-chloroform adduct, triphenylphosphine, and anhydrous
ZnCl2, produced the 7-azabicyclo[2.2.1]hept-2-ene derivative 19 in
90% yield (Scheme 3).30 Removal of the N-Boc protection of 19 with
a 4 N solution of HCl in dioxane afforded the secondary amine 7,
which was converted into the corresponding N-methyl derivative
8 by a standard reaction with formaldehyde followed by reduction
Boc
CH
3
N
OTf
3
+
N
Bu Sn
3
O
11
12
Scheme 1. (A) Key steps of the known synthetic strategy to epiboxidine 3. (B)
Retrosynthesis of 3 based on a Stille coupling reaction.

L. Rizzi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4651–4654
4653
CH
Boc
N
3
N
b,c
a
d
O
11
7
8
19
Boc
N
CH
Boc
3
N
N
e
f
b
b
O
3 x HCl
19
O
CH
3
N
20
21
g
g
7
8
20
7 fumarate
8 fumarate
6 x HCl
Scheme 3. (a) [Pd2(dba)3]ÁCHCl3, PPh3, 12, anhydrous ZnCl2/THF, À78 °C to rt; (b) 4 N HCl/dioxane, rt; (c) aq Na2CO3; (d) HCHO (37%, aq), NaBH3CN/CH3CN; (e) H2, 10% Pd/C,
CH3OH; (f) tert-BuOK/tert-BuOH, reflux, 16 h, flash chromatography; (g) C4H4O4, CH3OH.
of the intermediate aldimine with sodium cyanoborohydride. Next,
hydrogenation of the carbon–carbon double bond of 19 over 10%
Pd/C allowed the isolation of N-Boc-endo-epiboxidine 20 as the
sole isomer in 87% yield.31 Epimerization of 20 using potassium
tert-butoxide in refluxing tert-butyl alcohol22,31 provided the de-
sired N-Boc-exo-epiboxidine 21 in 82% yield. The two epiboxidine
epimers endo-6 and exo-3 were isolated as crystalline hydrochlo-
rides following acidic treatment of the corresponding N-Boc pro-
tected endo-20 and exo-21 precursors, respectively (Scheme 3).
Specifically, the physical and spectroscopic features of 3 hydro-
chloride16,32 matched those of the known compound, now com-
mercially available from Sigma. Since the hydrochlorides of 7 and
8 were amorphous salts, the free bases were reacted with a solu-
tion of fumaric acid in methanol to give rise to the corresponding
1:1 fumarates (Scheme 3).
0.035 nM for 2 and 0.4 nM for 3) combined with a lower affinity
at 7 receptors (Ki values 10.2 nM for 2 and 6.0 nM for 3). As a con-
sequence, a meaningful difference in the 4b2 versus 7 selectivity
ratio (290 for 2 and 15 for 3) characterizes the two structural ana-
logues. An intermediate selectivity has been recently found for the
3-methylisoxazolyl nicotine-related derivative ABT-418 (S)-4.36 If
the data on epiboxidine 3 are taken into account, the binding affin-
a
a
a
ity of (S)-4 at the
more than at the
an 4b2 versus
a7 subtype (Ki = 1.2 lM) is negatively affected
a
4b2 receptors (Ki = 20 nM), thus bringing about
a
a
7 selectivity ratio of 60.
In terms of binding affinity, UB-165 5, the semirigid homologue
of epibatidine, showed a considerable degree of discrimination be-
tween the two neuronal nAChRs under study, the 4b2 versus
selectivity being higher than 10,000, due to a partially conserved
affinity at the 4b2 subtype (Ki = 0.27 nM) coupled with a sizeable
loss of affinity at the 7 subtype (Ki = 2.8
M).19 Also derivative 7,
the unsaturated analogue of epiboxidine, was characterized by a
marked drop of the affinity at the 7 subtype (Ki = 1.6 M) when
a
a7
a
The target racemic compounds 3, 6, 7, and 8 were assayed for
a
l
binding affinity at rat
a4b2 and a
7 nAChR subtypes, using [3H]epi-
batidine and [125I]
a
-bungarotoxin as radioligands, respectively.
a
l
The Ki values, calculated from the competition curves of three
separate experiments by means of the LIGAND program,33 are col-
lected in Table 1. The corresponding values reported for ( )-epi-
batidine 2, (S)-4 (ABT-418), and ( )-5 (UB-165) have been
included for comparison.
compared to the parent compound. However, the presence of the
double bond in 7 caused a reduction of the affinity even at the
a4b2 subtype (Ki = 50 nM). As a consequence, only a minor
improvement of the 4b2 versus 7 selectivity (from 15 for 3 to
a
a
32 for 7) was attained. Worth noting, in terms of affinity profile
at the two studied nAChRs, the results on racemic 7 are quite com-
parable with those obtained in the same experimental conditions
for the pure isomer ABT-418.36
At variance with the secondary amine 7, the corresponding N-
methyl derivative 8 was characterized as a low affinity unselective
The data found for epiboxidine 3 confirm its very high affinity
(Ki = 0.4 nM) for
a4b2 nAChRs, which complements the results
known from the literature (Ki = 0.6 nM,16 Ki = 1.33 nM,34 and
Ki = 0.46 nM35). A comparison between 2 and 3 evidences about a
10-fold higher affinity of 2 at the
a4b2 receptors (Ki values
nicotinic ligand (Ki = 0.51 lM at a4b2 and 0.39 lM at a7). A similar
lack of selectivity was evidenced by endo-epiboxidine 6, which, in
comparison with the exo-isomer 3, retained a non negligible affin-
Table 1
Affinity of ( )-epibatidine 2, ABT-418 (S)-4, UB-165 5, and derivatives 3, 6, 7, and 8 for
native 4b2 and 7 nAChR subtypes present in rat cortical membranes, labeled by
[3H]epibatidine and [125I]
-bungarotoxin
ity at both
subtypes. The same trend was reported at the
a
4b2 (Ki = 70 nM) and
a
7
(Ki = 0.23
lM) nAChR
a
a
a
4b2 subtype for
a
( )-endo-epibatidine,13d which exhibited a drastic reduction of
affinity when compared to ( )-exo-epibatidine (Ki values 7.6 and
0.035 nM, respectively),37 coupled with the loss of the antinocicep-
tive activity in the tail-flick test.14
Entry
a
4b2 [3H]-Epi Ki (nM)
a a-BgTx Ki (nM)
7 [125I]
( )-2
(S)-4
( )-5
( )-3
( )-6
( )-7
( )-8
0.035a
10.2a
20.0b
1200b
2790c
6.0 (47)
0.27c
In summary, we utilized a Pd-catalyzed Stille coupling reaction
as the pivotal step to prepare the two epiboxidine epimers and two
unsaturated epiboxidine-related analogues. We assessed the four
0.40 (57)
70 (49)
50 (23)
514 (34)
230 (51)
1600 (63)
394 (44)
compounds for binding affinity at a4b2 and a7, the two major neu-
ronal nicotinic receptor subtypes. Noteworthy, ( )-exo-epiboxidine
3 displayed a very high affinity at both nAChRs, whereas in its ana-
logue 7 the presence of the double bond caused a loss of affinity at
The numbers in brackets refer to the % coefficient of variation.
a
Ref. 40, [3H]nicotine was used as radioligand for
Ref. 36.
a4b2 receptors.
b
c
Ref. 19, [3H]nicotine was used as radioligand for
a4b2 receptors.
both subtypes together with a higher degree of
a4b2 versus a7

4654
L. Rizzi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4651–4654
4704; (c) Nicolaou, K. C.; Bulger, P. G.; Sarlah, D. Angew. Chem., Int. Ed. 2005, 44,
4442; (d) Yin, L.; Liebscher, J. Chem. Rev. 2007, 107, 133.
22. Zhang, C.; Trudell, M. L. J. Org. Chem. 1996, 61, 7189.
23. For recent reviews on the use of microwaves in organic synthesis, see (a)
Kappe, C. O. Angew. Chem., Int. Ed. 2004, 43, 6250; (b) Kappe, C. O.; Dallinger, D.
Nat. Rev. Drug Discov. 2006, 5, 51.
24. Carroll, F. I.; Liang, F.; Navarro, H. A.; Brieaddy, L. E.; Abraham, P.; Damaj, M. I.;
Martin, B. R. J. Med. Chem. 2001, 44, 2229.
selectivity. We will broaden the study on this set of compounds by
enriching the number of unsaturated analogues and preparing the
individual isomers of ( )-3 and ( )-7. The pharmacological results
will be further analyzed in the light of recently developed molecu-
lar models for the two investigated receptor subtypes.38,39
25. Tessier, P. E.; Nguyen, N.; Clay, M.; Fallis, A. G. Org. Lett. 2005, 7, 767.
26. Choi, Y.; White, J. D. J. Org. Chem. 2004, 69, 3758.
Acknowledgments
27. Lee, J. S.; Cho, Y. S.; Chang, M. H.; Koh, H. Y.; Chung, B. Y.; Pae, A. N. Bioorg. Med.
Chem. Lett. 2003, 13, 4117. (and references cited therein).
28. Kanemasa, S.; Matsuda, H.; Kamimura, A.; Kakinami, T. Tetrahedron 2000, 56,
1057.
29. Dallanoce, C.; Rizzi, L.; Magrone, P.; Bazza, P.; Grazioso, G.; Riganti, L.; Gotti, C.;
Clementi, F.; Frydenvang, K.; De Amici, M. Chem. Biodivers., in press.
This research was financially supported by the Italian FIRB
Grant # RBNE03FH5Y_002, the PRIN Grant # 2005054943 (F.C.,
C.G., and M.D.A.), and the Fondazione Cariplo Grant # 2006/0882
(F.C. and M.D.A.).
30. 2-(3-Methylisoxazol-5-yl)-7-azabicyclo[2.2.1]hept-2-ene-7-carboxylic
acid
tert-butyl ester (19). To a magnetically stirred solution of enoltriflate 11
(1.10 g, 3.20 mmol) in anhydrous THF (15 mL) kept at À78 °C under argon
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triphenylphosphine (68 mg, 0.26 mmol), then
a solution of isoxazole 12
(1.70 g, 4.57 mmol) in THF (10 mL) and anhydrous ZnCl2 (437 mg,
3.21 mmol). The mixture was quickly warmed at rt and stirred for about 3
days under argon until disappearance of the starting material (TLC, 10%
ethyl acetate/petroleum ether). After addition of water (20 mL), the crude
reaction was repeatedly extracted with ethyl acetate (4 Â 20 mL). The
combined organic extracts were treated with brine (20 mL), then dried
over anhydrous sodium sulfate. After concentration under reduced pressure,
the residue was purified by silica gel column chromatography (10% ethyl
acetate/petroleum ether) to afford 797 mg (90
% yield) of the required
isoxazolyl derivative 19. Colorless prisms (from n-hexane), mp 84–85 °C.
Rf = 0.50 (20% ethyl acetate/petroleum ether). 1H NMR (300 MHz, CDCl3): d
1.24 (m, 2H), 1.41 (s, 9H), 2.00 (m, 2H), 2.30 (s, 3H), 4.82 (br s, 1H), 4.94 (br
s, 1H), 6.10 (s, 1H), 6.68 (s, 1H). 13C NMR (75 MHz, CDCl3): d 11.65, 24.49,
25.43, 28.41, 61.06, 80.65, 102.20, 133.32, 135.08, 155.22, 160.28, 163.95.
C
15H20N2O3 (276.33): calcd. C 65.20, H 7.30, N 10.14; found C 65.37, H 7.22,
N 10.02.
31. Catalytic hydrogenation of the corresponding olefin for the synthesis of ( )-
epibatidine afforded theendo/exo N-Boc-epibatidines in a 4:1 ratio, as reported
by Fletcher, S. R.; Baker, R.; Chambers, M. S.; Herbert, R. H.; Hobbs, S. C.;
Thomas, S. R.; Verrier, H. M.; Watt, A. P.; Ball, G. R. J. Org. Chem. 1994, 59, 1771.
32. ( )-exo-2-(3-Methylisoxazol-5-yl)-7-azabicyclo[2.2.1]heptane hydrochloride
(3ÁHCl). Pale yellow prisms (anhydrous ether washings), mp 200–205 °C
(dec). 1H NMR (300 MHz, D2O): d 2.04 (m, 5H), 2.28 (s, 3H), 2.40 (m, 1H), 3.57
(dd, 1H, J = 5.5 and 9.2 Hz), 4.41 (br s, 1H), 4.53 (d, 1H, J = 2.8 Hz), 6.26 (s, 1H),
13C NMR (75 MHz, D2O): d 10.54, 25.61, 26.54, 33.84, 37.86, 58.97, 62.43,
103.02, 161.91, 171.90. C10H15ClN2O (214.69): calcd. C 55.94, H 7.04, Cl 16.51,
N 13.05; found C 56.12, H 6.91, Cl 16.77, N 13.15.
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