Synthesis, activity and theoretical study of ABT-418 analogues

Article abstract of DOI:10.1016/S0040-4020(02)00388-5

This report shows the synthesis and biological evaluation of two new conformationally restricted ABT-418 analogues. This restriction is introduced by the incorporation of the 7-azabicyclo[2.2.1]heptane skeleton. Furthermore, we report here a high-level qu

Full text of DOI:10.1016/S0040-4020(02)00388-5

TETRAHEDRON
Pergamon
Tetrahedron 58 ꢀ2002) 4505±4511
Synthesis, activity and theoretical study of ABT-418 analogues
a,p
a
b
c
Alberto Avenoza, Jes u s H. Busto, Carlos Cativiela, Albert Dordal, Jordi Frigola
c
a,p
and Jes u s M. Peregrina
a
Departamento de Qu Âõ mica, Grupo de S Âõ ntesis Qu Âõ mica de La Rioja, Universidad de La Rioja, U.A.-C.S.I.C., 26006 Logro nÄ o, Spain
b
Departamento de Qu Âõ mica Org a nica, Instituto de Ciencia de Materiales de Arag o n,
Universidad de Zaragoza-C.S.I.C., 50009 Zaragoza, Spain
c
Laboratorios Dr. Esteve S.A., 08041 Barcelona, Spain
Received 4 February 2002; revised 25 March 2002; accepted 5 April 2002
AbstractÐThis report shows the synthesis and biological evaluation of two new conformationally restricted ABT-418 analogues. This
restriction is introduced by the incorporation of the 7-azabicyclo[2.2.1]heptane skeleton. Furthermore, we report here a high-level quantum
mechanical study of their conformations in the gas phase. q 2002 Published by Elsevier Science Ltd.
1. Introduction
similar studies directed toward ABT-418 analogues would
also provide useful information.
The alkaloid epibatidine 1 is a very speci®c agonist of the
neuronal nicotinic acetylcholine receptor ꢀnAChR) that
1
ABT-418 was obtained from l-proline 5 with an ef®cient
route that involved the conversion of the ester function into
acts as a powerful analgesic through a non-opioid mecha-
nism. Since the discovery of this alkaloid by Daly and
2
8
the corresponding b-keto oxime ꢀScheme 1). Recently, we
co-workers in 1992, several synthetic approaches have
3
have developed a method to obtain a type of constrained
proline 6 ꢀAhc) and on the basis of our experience in the
9
been reported by numerous research groups. Its therapeutic
3
b,10
potential as a nicotinic receptor agonist is interesting to
treat the pain and other neurological disorders, including
Alzheimer's and Parkinson's diseases.
epibatidine ®eld,
we report here the synthesis and the
biological activity of two new 3-methylisoxazol-5-yl deri-
vatives 7a and 8a, both including a 7-azabicyclo[2.2.1]hep-
tane system. These compounds can be regarded as
conformationally restricted analogues of ABT-418.
4
However, the epibatidine has a high toxicity and is not
suitable for clinical development. In this context, several
groups have begun to study epibatidine analogues by
combining structural features of the known alkaloids. One
of these known alkaloids is the nicotine 2, and since it has
strong undesired effects on cardiovascular and digestive
systems, several nicotine analogues have been synthesized,
2. Results and discussion
2.1. Chemistry
We used the benzamide 9 ꢀprecursor of the amino acid Ahc
particularly, analogues with rigid structures ꢀ7-azabicyclo-
5
[
ꢀ
2.2.1]heptane skeleton) have become interesting targets
3) ꢀFig. 1).
The replacement of the pyridinyl ring in nicotine by a
methylisoxazolyl ring represents a relative alteration. This
compound, ABT-418 4, has been reported to have antinoci-
6
ceptive effects on mice. Since the synthesis and evaluation
of bridged analogues of nicotine have provided an insight
7
into the pharmacophore for the nAChR, we envisioned that
Keywords: amino acids and derivatives; bicyclic heterocyclic com-
pounds; biologically active compounds; isoxazoles; molecular modeling/
mechanics.
p
Corresponding authors. Tel./Fax: 134-941-299-655;
e-mail: alberto.avenoza@dq.unirioja.es
Figure 1. Structures of interesting nicotinic receptor agonists.
0040±4020/02/$ - see front matter q 2002 Published by Elsevier Science Ltd.
PII: S0040-4020ꢀ02)00388-5

4506
A. Avenoza et al. / Tetrahedron 58 72002) 4505±4511
Scheme 4. ꢀa) LiAlH
MeOH, 16 h, 98%; ꢀc) ꢀi) Jones' reagent, acetone, 4 h; ꢀii) CH
ether, 97%; ꢀd) ꢀi) 2equiv. n-BuLi, acetone oxime, THF, 08C!rt!re¯ux;
ii) HCl conc. 808C, 41% two steps.
4
, THF 16 h, re¯ux, 99%; ꢀb) ꢀBoc)
2
O, PdꢀOH)
2
,
2
N
2
, diethyl
ꢀ
formed into the subsequent hydrochloride salts 7b and 8b
by treatment with 2N HCl ꢀScheme 3).
Scheme 1. Retrosynthetic analysis of ABT-418 from l-proline and of its
analogues 7a and 8a from Ahc.
In order to improve the yield of the target molecules, we
changed the benzamide group of the compound 9 by another
suitable group as Boc. With this aim, compound 9 was
treated with LiAlH and amide and ester functions were
4
transformed into amino and alcohol groups by the corre-
sponding reduction. The hydrogenolysis of 10 in the
6
) as starting material in the synthesis. It was obtained from
9
methyl 2-benzamidoacrylate in ®ve steps. In a ®rst attempt,
we tried the reaction of methyl ester with dianion oxime
using benzamide group as N-protected group. We employed
two methods, one with the isolation of the intermediate
b-keto oxime, and another without isolation, but with
immediately cyclization, dehydration and deprotection,
presence of ꢀBoc) O in MeOH gave the N-Boc alcohol 11
2
in an excellent yield. A subsequent oxidation with Jones'
reagent and methylation with addition of diazomethane
allowed to obtain the necessary compound 12. The intro-
duction of the methylisoxazolyl ring was carried out using
the reaction with the oxime above described in only one step
to give the key compound 7a in a 41% yield ꢀScheme 4).
1
1
1
2
obtaining better yields in this second case. In this way,
compound 7a was obtained from 9 in a 25% yield
ꢀ
Scheme 2).
2.2. Biology
To obtain the other ABT-418 analogue, the amine 7a was
methylated with formic acid±formaldehyde giving a good
4
Af®nity data for both ABT-418 analogues 7b and 8b
towards nicotinic and muscarinic receptors are shown in
Table 1, together with comparison data for the epibatidine,
nicotine, citisine, atropine and pirenzepine. The two ana-
logues do not show af®nity for the muscarine receptor but
differ in af®nity for the nicotinic receptor; while 8b do not
show af®nity for this receptor, the IC -value for compound
yield of 8a. Taking into consideration that some ligands
interact with their corresponding receptor in the protonated
form, both isoxazolyl compounds 7a and 8a were trans-
5
0
7b demonstrates that the af®nity is approximately 100 times
lower than in the case of nicotine.
Even though the two analogues possess structures very simi-
lar to that of ABT-418 but less ¯exible due to the rigidity
introduced by the 7-azabicyclic framework, they did not
show a high af®nity with the nAChR. This information
provides an important insight into the design of new
nAChR ligands.
Scheme 2. ꢀa) 2equiv. n-BuLi, acetone oxime, THF, 08C!rt!re¯ux;
ꢀ
b) HCl conc., 808C, 25% two steps.
Further evaluation of analgesic effects in vivo revealed that
Table 1. Nicotinic and muscarinic receptors af®nities
a
b
Compound
Nicotinic receptor
Muscarinic receptor
2
6
26
Shift ꢀ10 M) IC50 ꢀnM) %Shift ꢀ10 M) IC50 ꢀnM)
%
Epibatidine
Nicotine
Citisine
Atropine
Pirenzepine
±
87.6
[10 M± ]ˆ92
K
K
K
i
i
ˆ0.20
ˆ41.3
±
±
±
±
±
±
27
i
ˆ3.3
27
±
±
[10 M]ˆ95
K
i
ˆ0.4
±
62.4
1.9
78
214.8
23.2
K
i
ˆ24.5
7
8
b
b
K
i
ˆ386
.10,000
.10,000
.10,000
a
3
Inhibition of [ H]-ꢀ^)-epibatidine in rat brain.
b
3
Inhibition of [ H]-QNB in rat cortex.
Scheme 3. ꢀa) H
2
O, HCO
2
H, HCHO, 14 h re¯ux, 70%; ꢀb) 2N HCl, 100%.

A. Avenoza et al. / Tetrahedron 58 72002) 4505±4511
Table 2. Calculated energy, N±N distance and dihedral angle in nicotine and its analogues
4507
Ê
N±N distance ꢀA)
E ꢀhartree)
Dihedral angle ꢀ8)
p
a ꢀHF/3-21G )
7
8
2567.4086424.32
53.9
4.25
4.75
4.31
4.76
4.65
4.49
4.40
4.13
4.13
4.33
4.12
2
567.412828
567.413777
306.0
190.8
51.3
206.7
235.3
266.5
297.6
42.6
2
2606.225556
p
a ꢀHF/3-21G )
2606.226988
2606.226378
2606.226236
2606.226260
p
b ꢀHF/3-21G )
7
8
2567.802556
567.802556
2606.622452
606.626458
2
317.4
53.8
325.7
p
b ꢀHF/3-21G )
2
pp
b ꢀB3LYP/6-31G )
7
8
2574.5731324.17
2613.888463
2997.061430
2496.130949
51.8
pp
b ꢀB3LYP/6-31G )
4.18
4.50
4.65
311.6
85.1
117.1
1
p
a
Epipatidine H ꢀB3LYP/6-31G )
Nicotine H ꢀB3LYP/6-31G )
1
pp
a
a
Aromatic heterocycle ring rotation angle.
8b was ineffective while 7b needs high concentrations to
show effects in the hot-plate assay.
compared with our compounds because its data arise from
semi-empirical calculations.
1
5
In general, the geometry of the different conformers of
compounds 7a and 8a and its protonated forms 7b and 8b
can be de®ned with the parameter corresponding to the
isoxazol ring rotation angle. In this sense, the study of the
conformation pro®le of the rotation of the isoxazol ring is
shown in Fig. 2.
2
.3. Molecular modeling
The rigidity of our ABT-418 analogues allowed us to apply
0
systematic bond rotations around C1±C5 . We achieved
ab initio methods for compounds 7a and 8a and their
protonated species 7b and 8b.
The low-energy conformers of the two non-protonated
compounds 7a and 8a are gathered in Table 2and can be
divided in two groups, which differ in the disposition of the
isoxazol ring that is rotated 137±2528 and in energy 3.22±
0.44 kcal/mol. Similar features have been observed in the
protonated compounds 7b and 8b, with the difference that
the two minima observed in 7b have the same energy due to
the symmetry of the molecule and in this case, the isoxazol
ring is rotated 2758. The two minima observed in 8b differ in
2.51 kcal/mol and in a rotation of isoxazol ring of 2728.
The calculations were carried our with the Gaussian-98
1
3
p
program using HF/3-21G . The step size for the rotation
in 7a, 8a, 7b and 8b was 308. In addition, the structures with
minimum energy were re-optimized with the hybrid
pp
HF-density functional method B3LYP and the 6-31G
basis set.
The total and relative energies of the minima found are
displayed in Table 2, together with the N±N distance and
the dihedral angles, comparing these data with the infor-
mation from the ab initio calculations contributed for
The N±N distance, that has been reported as an important
parameter to predict af®nity for the nAChRs in different
1
4a
14b
epibatidine
and nicotine.
ABT-418 has not been
1
4±16
pharmacophoric models,
protonated compounds ꢀ4.12±4.33 A) and smaller than
shows close values in the
Ê
1
4a
14b
those described for epibatidine or nicotine ꢀTable 2).
We think that this feature is important since, until now, it
has not been described active compound with internitrogen
distances minor than 4.30 A. In fact, the optimal inter-
nitrogen distance in the currently accepted model used
to identify nicotinic geometries ꢀBeers-Reich/Sheridan
nicotinic pharmacophore) is a question that remain
unanswered.
1
6b
Ê
Taking into account the results of the biological assays of 7b
ꢀfew active) and 8b ꢀinactive), the structural similarities of
the protonated nicotine with 8b and the same N±N distance
observed in both compounds ꢀ7b and 8b), it seems that the
N±N-distance is not alone the only parameter to keep in
mind in order to establish the af®nity for the nAChRs. In
fact, the only difference between 7b and 8b is the N-methyl
substituent and it is well-documented the drastic effect that
1
6a
the N-methylation in some cases has on the af®nity.
Moreover, it is not known if the N-methyl group directly
Figure 2. Rotational pro®les of ABT-418 analogues 7a, 8a, 7b and 8b,
determined using HF/3-21G .
p

4508
A. Avenoza et al. / Tetrahedron 58 72002) 4505±4511
pp
Figure 3. Three-dimensional structures of singly protonated nicotine, 7b and 8b optimized at the B3LYP/6-31G level and the total atomic charges of
nitrogens.
participates in a ligand±receptor interaction or whether
its major role is to in¯uence the conformation of the
molecule. On the other hand, it has been described that
features offer information on the investigation of the
structure±activity relationship of simple ligands in their
interaction with nAChR.
1
6b
the replacement of the N-methyl group by hydrogen
ꢀ
we observed the contrary effect in 7b and 8b.
16b
i.e. nornicotine) reduces af®nity for the nAChRs and
4. Experimental
4.1. General
In order to verify if the decrease of biological activity in 8b
in comparison with 7b could be due to a change in the
relative basicity of the nitrogens, we have calculated the
Melting points are uncorrected. All the manipulations with
air-sensitive reagents were carried out under a dry argon
atmosphere using standard Schlenk techniques. Solvents
were puri®ed according to standard procedures. The
chemical reagents were purchased from Aldrich Chemical
Co. Analytical TLC was performed using Polychrom SI F254
plates. Column chromatography was performed using
Kieselgel 60 ꢀ230±400 mesh). Organic solutions were
dried over anhydrous Na SO and, when necessary, concen-
1
3
Mulliken charge distribution, showing similar total atomic
charges for the two nitrogens in both compounds. These
total atomic charges are depicted in cursive in Fig. 3.
The three-dimensional structure of the minima correspond-
ing to the protonated nicotine and the closely related
structures 7b and/or 8b are displayed in Fig. 3 and shows
three important differences: ꢀi) the relative orientation of the
N atom of the heterocycle respect to the methyl substituent
in 8b is contrary to the observed in the nicotine, ꢀii) the N±N
distance is minor in 7b and 8b than in nicotine and ꢀiii)
the number of conformers of low-energy is much smaller
in the case of 7b and 8b that in the case of the nicotine, due
to the presence of the ±CH ±CH ± bridge in 7b and 8b.
2
4
trated under reduced pressure using a rotary evaporator. The
nmax ꢀcm ) of IR spectra are given for the main absorption
2
1
1
bands. NMR spectra were recorded at 300 MHz ꢀ H) and at
1
3
75 MHz ꢀ C) and are reported in ppm down®eld from TMS.
Mass spectra were obtained by electrospray ionization ꢀESI).
2
2
4.1.1. 1-ꢀ3-Methylisoxazol-5-yl)-7-azabicyclo[2.2.1]hep-
tane ꢀ7a). Method A: A solution of acetone oxime ꢀ80 mg,
1.10 mmol) in 5 mL of dry THF at 08C was treated dropwise
with n-BuLi ꢀ1.1 mL, 2M in hexane), and the reaction
mixture was stirred at 08C for 45 min. A solution of 9
ꢀ185 mg, 0.71 mmol) in 10 mL of THF was added dropwise
while the reaction mixture was stirred at 08C. After re¯uxing
for 1 h, the reaction was allowed to occur at room tempera-
ture for another 16 h. The stirring was stopped and 2mL of
2N HCl were added carefully after cooling the reaction
mixture in an ice bath. The mixture was diluted with
water and washed with ethyl acetate ꢀ3£10 mL). The
aqueous layer was basi®ed with solid NaHCO /Na CO
3
3
. Conclusion
Starting from a precursor of the amino acid Ahc, we have
synthesized two analogues of ABT-418, 7a and 8a, in which
the ¯exibility of the pyrrolidine ring is limited by the incor-
poration of the rigid 7-azabicyclo[2.2.1]heptane skeleton.
These analogues have been tested as agonists of nAChR
and we have also evaluated the analgesic activity of their
protonated species 7b and 8b, which have shown to be few
active and inactive, respectively, moreover, a theoretical ab
initio study of their conformations is reported. These
3
2

A. Avenoza et al. / Tetrahedron 58 72002) 4505±4511
4509
mixture and was extracted with CHCl /2-propanol 4:1
3
in water ꢀ0.5 mL), formic acid ꢀ0.25 mL), and formaldehyde
ꢀ0.25 mL) was heated at re¯ux for 14 h. The solution was
evaporated, the residue was partitioned between ethyl
acetate and 2M K CO , and the aqueous layer was extracted
ꢀ
Na SO , ®ltered and evaporated. The b-keto oxime was
3£20 mL). The combined organic layer was dried over
2
4
dissolved in 5 mL of 6N HCl and heated at re¯ux. After
stirring at the same temperature for 7 h, the reaction mixture
was cooled in an ice bath. It was neutralized with solid
NaHCO /Na CO mixture and extracted with CHCl3/
2
3
with ethyl acetate ꢀ2£20 mL). The combined organic layer
was dried, ®ltered and evaporated and the residue was
chromatographed on silica gel using CH Cl /MeOH
3
2
3
2
2
2
over Na SO , ®ltered and evaporated. The residue was
-propanol 4:1 ꢀ3£20 mL). The organic layers were dried
ꢀ90:10) as eluent to give 8a in 70% yield. Anal. calcd for
C H N O; C, 68.72; H, 8.39; N, 14.57; found C, 68.59; H,
8.48; N, 14.40; IR ꢀCH Cl , cm ): 1735, 1620; H NMR
2 2
2
4
11 16
2
2
1
1
chromatographed on silica gel using CH Cl /MeOH/Et N
2
2
3
ꢀ
89:10:1) as eluent to give 12mg of 7a ꢀ10% yield). Method
B: A solution of acetone oxime ꢀ102mg, 1.4 mmol) in
0 mL of dry THF at 08C was treated dropwise with
ꢀCDCl ): d 1.42±1.52 ꢀm, 2H); 1.70±1.81 ꢀm, 2H); 1.90±
3
2.15 ꢀm, 7H); 2.29 ꢀs, 3H); 3.37±3.44 ꢀm, 1H); 6.05 ꢀs, 1H);
C NMR ꢀCDCl ): d 11.4, 32.2, 35.1, 40.2, 57.9, 62.1,
3
1
3
1
n-BuLi ꢀ1.5 mL, 2M in hexane), and the reaction mixture
was allowed to warm to room temperature over 30 min. A
solution of 6 ꢀ259 mg, 1.0 mmol) in 20 mL of THF was
added dropwise while the reaction mixture was stirred at
room temperature. After the reaction mixture was re¯uxed
for 45 min, the THF was removed under argon atmosphere
to give a crude residue that was dissolved in 8 mL of
concentrated HCl and heated at 808C for 4 h. The mixture
was cooled, diluted with water and washed with ethyl
acetate ꢀ2£10 mL). The aqueous layer was basi®ed with
solid NaHCO /Na CO mixture and was extracted with
102.3, 158.3, 174.2.
4.1.4. Hydrochloride salt of 8a ꢀ8b). The compound 8a
was dissolved in 5 mL of 2N HCl and stirred for 10 min.
The solvent was evaporated and the residue was eluted
through a C18 reverse-phase Sep-pak cartridge which, after
1
the removal of the water, gave the salt 8b in 100% yield. H
NMR ꢀD O): d 1.89±2.08 ꢀm, 2H); 2.18±2.40 ꢀm, 7H);
2.45±2.60 ꢀm, 5H); 4.18±4.22 ꢀm, 1H); 6.63 ꢀs, 1H).
2
4.1.5.
7-Benzyl-1-hydroxymethyl-7-azabicyclo[2.2.1]-
3
2
3
CHCl /2-propanol 4:1 ꢀ3£20 mL). The combined organic
heptane ꢀ10). To a solution at 08C containing LiAlH4
ꢀ351 mg, 9.25 mmol) in THF ꢀ35 mL), methyl ester 9
ꢀ600 mg, 2.3 mmol) was added as a solution in THF
ꢀ10 mL). The suspension was re¯uxed overnight under an
inert atmosphere. The suspension was cooled and water
ꢀ10 mL), NaOH 10% ꢀ10 mL) and again water ꢀ10 mL)
were added. This mixture was stirred for 30 min and then
extracted with ethyl acetate ꢀ30 mL). The aqueous layer was
washed with ethyl acetate ꢀ2£20 mL). The combined
organic layers were dried, ®ltered and evaporated to give
494 mg of the pure compound 10 ꢀ99%). Anal. calcd for
C H NO; C, 77.38; H, 8.81; N, 6.45; found C, 77.45; H,
3
layer was dried over Na SO , ®ltered and evaporated. The
4
2
residue was chromatographed on silica gel using CH Cl /
2
2
MeOH/Et N ꢀ89:10:1) as eluent to give 45 mg of 7a ꢀ25%
3
yield). Method C: A solution of acetone oxime ꢀ102mg,
1.4 mmol) in 10 mL of dry THF at 08C was treated dropwise
with n-BuLi ꢀ1.5 mL, 2M in hexane), and the reaction
mixture was allowed to warm to room temperature over
3
0 min. A solution of 11 ꢀ259 mg, 1.0 mmol) in 20 mL of
THF was added dropwise while the reaction mixture was
stirred at room temperature. After the reaction mixture was
re¯uxed for 45 min, the THF was removed under an argon
atmosphere to give a crude residue that was dissolved in
8 mL of concentrated HCl and heated at 808C for 4 h. The
mixture was cooled, diluted with water and washed with
1
4
19
2
1
8.71; N, 6.61; IR ꢀCH Cl , cm ): 3610, 2960, 1605, 1453,
2
2
1
1024; H NMR ꢀCDCl ): d 1.24±1.31 ꢀm, 4H); 1.69±1.75
3
ꢀm, 4H); 3.08±3.13 ꢀm, 1H); 3.37 ꢀbr s, 2H); 3.65 ꢀbr s, 2H);
7.12±7.33 ꢀm, 5H); C NMR ꢀCDCl ): d 27.9, 30.3, 49.0,
59.8, 62.1, 69.4, 126.7, 128.2, 128.5, 140.0.
1
3
ethyl acetate ꢀ2£10 mL). The aqueous layer was basi®ed
3
with saturated NaHCO solution and was extracted with
3
CHCl /2-propanol 4:1 ꢀ3£20 mL). The combined organic
3
layer was dried over Na SO , ®ltered and evaporated. The
2
4.1.6. 7-ꢀtert-Butoxycarbonyl)-1-hydroxymethyl-7-aza-
bicyclo[2.2.1]heptane ꢀ11). A solution of 10 ꢀ492mg,
2.26 mmol) in degassed MeOH ꢀ10 mL) was added over a
4
residue was chromatographed on silica gel using CH Cl /
2
2
MeOH/Et N ꢀ89:10:1) as eluent to give 73 mg of 7a ꢀ41%
3
yield). Anal. calcd for C H N O; C, 67.39; H, 7.92; N,
14
suspension of ꢀBoc) O ꢀ494 mg, 2.26 mmol) and 10%
2
1
0
2
1
cm ): 3625, 2956, 2877, 1609, 1270, 1018; H NMR
5.72; found C, 67.48; H, 7.81; N, 15.90; IR ꢀCH Cl ,
PdꢀOH) /C ꢀ321 mg) in degassed MeOH ꢀ20 mL). The reac-
2
2
2
2
1
1
tion was hydrogenated at atmospheric pressure overnight.
The reaction mixture was ®ltered, over Celite and evapo-
rated to obtain 503 mg of a clean oil, corresponding to
compound 11 ꢀ98%). Anal. calcd for C H NO ; C,
ꢀ
CDCl ): d 1.54±1.62 ꢀm, 2H); 1.76±1.93 ꢀm, 6H); 2.23
3
ꢀ
br s, 1H); 2.28 ꢀs, 3H); 3.76±3.83 ꢀm, 1H); 6.05 ꢀs, 1H);
C NMR ꢀCDCl ): d 11.4, 31.2, 35.1, 57.8, 63.4, 101.4,
1
3
3
12 21
3
1
59.6, 173.5.
64.41; H, 9.31; N, 6.16; found C, 64.53; H, 9.23; N, 6.30;
IR ꢀCH Cl , cm ): 3180±3520 ꢀbr), 1648; H NMR
2
1
1
2
2
4
.1.2. Hydrochloride salt of 7a ꢀ7b). The compound 7a
ꢀCDCl ): d 1.34±1.52ꢀm, 13H); 1.73±1.87 ꢀm, 4H);
3
13
was dissolved in 5 mL of 2N HCl and stirred for 10 min.
The solvent was evaporated and the residue was eluted
through a C18 reverse-phase Sep-pak cartridge which, after
the removal of the water, gave the salt 7b in 100% yield. H
NMR ꢀD O): d 1.86±1.99 ꢀm, 2H); 2.10±2.35 ꢀm, 9H);
3.87±3.90 ꢀm, 2H); 4.20±4.25 ꢀm, 1H); C NMR
ꢀCDCl ): d 28.3, 29.2, 31.7, 58.3, 61.9, 69.0, 80.1, 155.1.
3
1
4.1.7. Methyl 7-ꢀtert-butoxycarbonyl)-7-azabicyclo [2.2.1]-
heptane-1-carboxylate ꢀ12). A 1.5-fold excess of Jones
reagent was dropwise added to a solution of 11 ꢀ200 mg,
2
4
.34±4.41 ꢀm, 1H); 6.52ꢀs, 1H).
0.98 mmol) in acetone ꢀ10 mL) at 08C over 5 min. The
mixture was stirred at 08C for 4 h. The excess of Jones
reagent was destroyed with 2-propanol. The mixture was
4
[
.1.3. 7-Methyl-1-ꢀ3-methylisoxazol-5-yl)-7-azabicyclo-
2.2.1]heptane ꢀ8a). A mixture of 7a ꢀ61 mg, 0.34 mmol)

4510
A. Avenoza et al. / Tetrahedron 58 72002) 4505±4511
then diluted with water ꢀ10 mL) and extracted with ethyl
acetate ꢀ4£20 mL). The combined organic extracts were
dried and concentrated. The residual white solid ꢀ187 mg)
Pannell, L.; Daly, J. W. J. Am. Chem. Soc. 1992, 114, 3475±
3478.
3. ꢀa) Chen, Z. A.; Trudell, M. L. Chem. Rev. 1996, 96, 1179±
1193 and references cited therein. ꢀb) Avenoza, A.; Busto,
J. H.; Cativiela, C.; Peregrina, J. M. Synthesis 1998, 1335±
1338. ꢀc) Aoyagi, S.; Tanaka, R.; Naruse, M.; Kibayashi, C.
Tetrahedron Lett. 1998, 39, 4513±4516. ꢀd) Pandey, G.;
Bagul, T. D.; Sahoo, A. K. J. Org. Chem. 1998, 63, 760±
768. ꢀe) Sirisoma, N. S.; Johnson, C. R. Tetrahedron Lett.
1998, 39, 2059±2062. ꢀf) Aoyagi, S.; Tanaka, R.; Naruse,
M.; Kibayashi, C. J. Org. Chem. 1998, 63, 8397±8406.
2
1
was identi®ed by NMR. IR ꢀCH Cl , cm ): 3679, 3500,
2
2
1
981, 1691; H NMR ꢀCDCl ): d 1.34±1.50 ꢀm, 11H);
2
1
1
8
3
.72±1.86 ꢀm, 4H); 2.05±2.13 ꢀm, 2H); 4.26±4.30 ꢀm,
H); C NMR ꢀCDCl ): d 27.9, 29.0, 33.8, 59.7, 69.1,
1.6, 156.9, 178.4. The residue was dissolved in diethyl
1
3
3
ether ꢀ50 mL) and esteri®ed with an excess of diazomethane
in diethyl ether for 30 min. The solvent was removed under
vacuum and the required compound was used without
further puri®cation.
4
. Bannon, A. W.; Decker, M. W.; Holladay, M. W.; Curzon, P.;
Donnelly-Roberts, D.; Puttfarcken, P. S.; Bitner, R. S.; Diaz,
A.; Dickenson, A. H.; Porsolt, R. D.; Williams, M.; Arneric,
S. P. Science 1998, 2ꢀ9, 77±81.
4
4
.2. Biological assays
.2.1. Muscarinic binding assays. The method used was
5. Xu, Y.-Z.; Choi, J.; Calaza, M. I.; Turner, S.; Rapoport, H.
J. Org. Chem. 1999, 64, 4069±4078.
1
7
similar to the previously described. Membranes were
prepared from cerebral cortices of male Wistar rats. The
tissues were homogenized at 48C with an Ultra-Turrax
6. Garvey, D. S.; Wasicak, J. T.; Decker, M. W.; Brioni, J. D.;
Buckley, M. J.; Sullivan, J. P.; Carrera, G. M.; Holladay,
M. W.; Arneric, S. P.; Williams, M. J. Med. Chem. 1994,
ꢀ
13,500 rpm) for 15 s in 50 mM sodium potassium phos-
phate buffer, pH 7.4 ꢀ10 vols. w/v). The homogenate were
centrifuged for 10 min at 1000g at 48C, the resulting super-
natant recentrifuged twice for 10 min at 48,000g and the
pellet resuspended in the homogenizing buffer to a dilution
3
ꢀ, 1055±1059.
. Turner, S. C.; Hongbin, Z.; Rapoport, H. J. Org. Chem. 2000,
5, 861±870.
7
8
6
. ꢀa) Synthesis of ABT-418 from l-proline: Elliott, R. L.;
Kopecka, H.; Lin, N.-H.; He, Y.; Garvey, D. S. Synthesis
of 250 times. The homogenates were incubated with 0.2 nM
3
[
by ®ltration. Non-speci®c binding was de®ned as that in the
H]-QNB for 90 min at 258C and the reactions terminated
1
994, 722±774. ꢀb) Other synthesis of ABT-418: Giard, T.;
Lasne, M.-C.; Plaquevent, J.-C. Tetrahedron Lett. 1999, 40,
5495±5497.
. Avenoza, A.; Busto, J. H.; Cativiela, C.; Fern a ndez-Recio,
presence of 1 mM atropine. IC50 values of displacement
were determined 8by computer-assisted curve ®tting
9
1
EBDA/LIGAND). K values were calculated from IC
ꢀ
values by the equation derived by Cheng and Prusoff.
i
50
M. A.; Peregrina, J. M.; Rodrõguez, F. Tetrahedron 2001,
Â
1
9
5
0. Avenoza, A.; Cativiela, C.; Fern a ndez-Recio, M. A.;
ꢀ, 545±548.
1
4
.2.2. Nicotinic binding assays. The method used was
Peregrina, J. M. Tetrahedron: Asymmetry 1999, 10, 3999±
4
2
0
similar to the previously described. Brain homogenate
was prepared from male Wistar rats. The tissues were homo-
genized at 48C with a Polytron ꢀ14,500 rpm) for 30 s in
007.
1
1
1
1. Singh, S.; Avor, K. S.; Pouw, B.; Sfale, T. W.; Basmadjian,
G. P. Chem. Pharm. Bull. 1999, 4ꢀ, 1501±1505.
50 mM Tris±HCl buffer, pH 7.4 ꢀ10 vols. w/v). The homo-
genate was centrifuged twice for 10 min at 48,000g and the
2. Pandey, G.; Sahoo, A. K.; Grade, S. R.; Trusar, D. B.;
Phalgune, U. D. J. Org. Chem. 1999, 64, 4990±4994.
3. ꢀa) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G.
E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.;
Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.;
Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.;
Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.;
Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford,
S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.;
Morokuma, K.; Salvador, P.; Dannenberg, J. J.; Malick, D.
K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.;
Cioslowski, J.; Ortiz, J. V.; Baboul, A. G.; Stefanov, B. B.;
Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts,
R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M.
W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.;
Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J.
A. Gaussian 98, Revision A.11; Gaussian, Inc.: Pittsburgh
PA, 2001.
pellet resuspended in the same buffer to a dilution of 150
times. The homogenates were incubated with 0.1 nM
3
[
H]-Epibatidine for 180 min at 248C and the reactions
terminated by ®ltration. Non-speci®c binding was de®ned
as that in the presence of 300 mM nicotine. IC50 values of
displacement were determined by computer-assisted curve
1
8
®
IC values by the equation derived by Cheng and Prusoff.
tting ꢀEBDA/LIGAND). K values were calculated from
i
1
9
5
0
Acknowledgements
We thank the Comisi o n Interministerial de Ciencia y
Tecnolog Âõ a ꢀCICYT) and the Comisi o n Europea ꢀproject
2
FD97-1530), the Gobierno de La Rioja ꢀANGI-2001) and
the Universidad de La Rioja ꢀproject API-01/B02) for
support of this research.
1
4. ꢀa) Campillo, N.; Paez, J. A.; Alkorta, I.; Goya, P. J. Chem.
Soc., Perkin Trans. 2 1998, 2665±2669. ꢀb) Elmore, D. E.;
Dougherty, D. A. J. Org. Chem. 2000, 65, 742±747.
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1