6′-Methylpyrido[3,4-b]norhomotropane: Synthesis and outstanding potency in relation to the α4β2 nicotinic receptor pharmacophore model

Article abstract of DOI:10.1016/j.bmcl.2004.12.069

6′-Methylpyrido[3,4-b]norhomotropane [synthesis as the racemate reported here] is more potent at the α4β2 nicotinic receptor than any previous bridged nicotinoid. The two nitrogens and 6′-methyl substituent are superimposable on the two nitrogens and 6-chloro substituent of epibatidine, with the best fit on comparing the chair conformer of the (1R)- pyridonorhomotropane with natural (1R)-epibatidine. In this pharmacophore model, the 6′-methyl substituent may be equivalent to the acetyl methyl of acetylcholine.

Full text of DOI:10.1016/j.bmcl.2004.12.069

Bioorganic& Medicinal Chemistry Letters 15 (2005) 877–881
60-Methylpyrido[3,4-b]norhomotropane: synthesis
and outstanding potency in relation to the a4b2 nicotinic
receptor pharmacophore model
David B. Kanne,a Motohiro Tomizawa,a Kathleen A. Durkinb and John E. Casidaa,*
aEnvironmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management,
University of California, Berkeley, CA 94720-3112, USA
bMolecular Graphics Facility, College of Chemistry, University of California, Berkeley, CA 94720-1460, USA
Received 29 October 2004; revised 21 December 2004; accepted 22 December 2004
Available online 19 January 2005
Abstract—60-Methylpyrido[3,4-b]norhomotropane [synthesis as the racemate reported here] is more potent at the a4b2 nicotinic
receptor than any previous bridged nicotinoid. The two nitrogens and 60-methyl substituent are superimposable on the two nitro-
gens and 6-chloro substituent of epibatidine, with the best fit on comparing the chair conformer of the (1R)-pyridonorhomotropane
with natural (1R)-epibatidine. In this pharmacophore model, the 60-methyl substituent may be equivalent to the acetyl methyl of
acetylcholine.
Ó 2005 Elsevier Ltd. All rights reserved.
Nicotinic acetylcholine (ACh) receptor (nAChR) li-
gands are of interest in the treatment of cognitive and
attention deficits, Parkinsonꢀs disease, schizophrenia
and depression, and in nicotinically mediated analge-
sia.1,2 The diversity of neuronal nAChRs (e.g., a4b2
and a7) provides the potential for developing subtype-
selective therapeutic agents with reduced adverse side ef-
fect liabilities.3,4 These goals are facilitated by successful
pharmacophore mapping, which requires optimized
probes of high potency and defined or predictable con-
formations. Studies on the a4b2 nicotinic receptor were
based initially on nicotine (1) and nornicotine (2) and
later on the exquisitely potent epibatidine (3),5 all with
two critically positioned nitrogens and free rotation of
the pyridyl substituent (Fig. 1). Conformationally re-
stricted ligands, for example, bridged nicotinoids,6 facil-
itate further differentiation of the stericand electronic
requirements of a receptor target. ( )-Pyrido[3,4-b]nor-
homotropane (4)7,8 was the first bridged nicotinoid with
affinity rivaling or surpassing the conformationally free
analogs 1 and 2, and it is therefore an excellent probe
for further refinement of pharmacophore models.9–11
HN
N
CH3N
HN
1
2
3'
2'
6'
N
N
Cl
(-)-1
(1R)-3
( )-2
6
HN
τ2
5'
6'
1
O
2
N
2'
N
B
O
R
A
C
τ3
τ4
ACh
( )-4 R = H
( )-5 R = CH3
Figure 1. Structures of the non-protonated forms of standard nicoti-
noids [(ꢀ)-1, ( )-2, and (1R)-3] and ( )-pyrido[3,4-b]norhomotropanes
[( )-4 and ( )-5] compared with ACh.
A 6-chloro or 6-methyl substituent is tolerated without
major potency loss or even with a potency increase for
1, 2, and 3.12–14 This approach was therefore used here
for 4 by adding a 60-methyl substituent (5) to facilitate
molecular comparisons between the conformationally
flexible and restricted series (Fig. 1).
Keywords: Nicotinic receptor; Pharmacophore model; Pyridonorho-
motropane; Epibatidine; Bridged nicotinoid.
*
Corresponding author. Tel.: +1 510 642 5424; fax: +1 510 642
6497; e-mail: ectl@nature.berkeley.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.069

878
D. B. Kanne et al. / Bioorg. Med. Chem. Lett. 15 (2005) 877–881
BnN
BnN
BnN
O
c
a
b
5
O
O
H
N
H
6
7
8
Scheme 1. Reagents and conditions: (a) Na2PdCl4, 0.5 M HCl, EtOAc, benzoquinone; (b) NH2OHÆHCl, gl HOAc; (c) Pd(OH)2/C, H2.
N-Benzyl-3-allyl-2-formyl-9-azabicyclo[4.2.1]nonane (6),
an intermediate in the synthesis of ( )-4 prepared as re-
ported earlier,7,8 served as the starting material for the
three-step conversion to ( )-5 (Scheme 1). Wacker oxi-
dation (Na2PdCl4, benzoquinone)15 transformed 6 to
N-benzyl-2-formyl-3-(2-oxopropyl)-9-azabicyclo[4.2.1]-
nonane (7). Ring synthesis proceeded from 7 using
NH2OHÆHCl in refluxing acetic acid to generate N-ben-
zyl-60-methylpyrido[3,4-b]norhomotropane (8)16 which
after hydrogenolysis with Pd(OH)2 on carbon (Pearl-
manꢀs catalyst) yielded 5.17 Compound 4 was prepared
as previously described,7,18 and N-methyl-4 was avail-
able from our earlier study8 and subjected to
rechromatography.18
The diminished activity on N-methylation is also seen
in other 9-azabicyclo[4.2.1]nonane systems such as ana-
toxin-a.26 The loss in activity of 20-Me-427 may result
from unfavorable steric interactions with the receptor
rather than from conformational perturbations; this
proposal is based on the observation that each of 20-
Me-4, 4, and 5 favor the chair conformation over the
boat by 4.6, 3.8, and 4.8 kcal/mol, respectively (see
Table 1 and Supplemental Table).28
The high potency of the conformationally restricted,
bridged nicotinoid 5 makes it an ideal model to predict
the bioactive conformations of more flexible ligands
such as 3 and ACh. In considering possible molecular
overlays, the (1R)-enantiomer of 5 was used based on
analogy with the known stereochemistry of the more
potent enantiomer of the related (+)-anatoxin,8,10,29 an
analogy supported by the observation that both 4 and
(+)-anatoxin undergo major potency loss on N-methyla-
tion.26 The natural (1R)-3 (which is equipotent with its
enantiomer)14 was used in all comparisons.
The binding affinities of the standard nicotinoids [(ꢀ)-1,
( )-2, and ( )-3] and pyridonorhomotropanes [( )-4
and ( )-5] were determined to both a4b2 and a7 nAC-
hRs using (ꢀ)-[3H]nicotine and [125I]-a-bungarotoxin,
respectively, as the radioligands (Table 1).19–21 The a7
nAChR is much less sensitive than the a4b2 to all of
the ligands with the highest selectivity factor for ( )-4
and ( )-5. Most importantly, the affinity of ( )-5 for
the a4b2 receptor was 3-fold greater than that of ( )-4
and 36-fold greater than ( )-2, making 5 the most po-
tent bridged nicotinoid reported for the nicotinic recep-
tor. The enhancing effect of the 60-methyl group is in
sharp contrast to the 600- to 700-fold loss in affinity
resulting from a N-methyl or 20-methyl substituent.8,25
Conformational analysis on protonated 3, using a
molecular mechanics calculation followed by further
optimization at B3LYP/6-31G**++,30 revealed minima
with dihedral angles (C1–C2–C30–C20) of roughly
+15°, +90°, and ꢀ90° (Table 2) in accord with a previ-
Table 2. Conformational analysis on protonated 3, chair and boat
conformers of protonated 5, and ACh as distances, dihedral angles and
relative DG for each set of calculated minima (all values as Gibbs free
energies with zero point energy corrections included)
Table 1. Binding affinities of standard nicotinoids and ( )-pyrido[3,4-
b]norhomotropanes to the a4b2 and a7 nicotinic acetylcholine
receptors
Compound
and
conformation (A)
N–N
Dihedral
angle
(°)
Relative
DG
(kcal/mol)
distance
˚
Compound
Ki (nM SD, n = 3)a
3
C1, C2,
C30, C20
84
a4b2
a7
Standard nicotinoids
4.56
5.29
4.63
0
(ꢀ)-1
( )-2
( )-3b
2.5 0.3
14
0.035 0.004
7200 220
138,000 17,000
10
ꢀ89
1.03
0.67
2
14.7
1
5
C1–H, C1,
C30, C20
ꢀ2.1
( )-Pyrido[3,4-b]norhomotropanes
( )-4
( )-5
1.3 0.2
0.39 0.04
>100,000 (31%)c
ꢁ100,000 (52%)c
Chair
Boat
4.69
4.86
0
4.83
ꢀ31
a a4b2 subtype was immunoisolated by monoclonal antibody (mAb)
270 from mouse fibroblast M10 cells and assayed with 7 nM
[3H]nicotine binding. In the same manner, a7 subtype was immuno-
isolated by mAb 306 from human neuroblastoma SH-SY5Y cells and
assayed with 1 nM [125I]a-BGT binding. Ki Values are calculated
with the equation of Cheng and Prusoff,22 Ki = IC50/(1 + [L]/KD),
with KD values of 3.8 and 1.06 nM for the a4b2 and a7 subtypes,
respectively.23,24
a
ACh
N–O
(carbonyl)
s2
s3
s4
˚
distance (A)
5.06
5.14
gauche
trans
65
166
180
80
ꢀ179 0
180 1.49
180
68
other
other
other
4.28
3.88
ꢀ126 0.27
175 0.62
ꢀ158
80
80 ꢀ110
3.59
ꢀ167 0.53
b Data from Zhang et al.21
c Percent inhibition at the indicated concentration.
a s4: C–O-C(O)–C.

D. B. Kanne et al. / Bioorg. Med. Chem. Lett. 15 (2005) 877–881
879
ous report.31 The barrier to rotation is small and 3 exists
as a mixture of the minima with significant contribu-
tions from other conformers. Similar calculations on
the two possible conformations of protonated 5 estab-
lish that the chair is more stable than the boat by
ꢁ5 kcal (Table 2). A systematicsearch for the optimal
superpositioning of conformationally mobile (1R)-3
and chair (1R)-5 focused on four key overlay elements:
(1) position and directionality (as indicated by projec-
tion of NH2+) of the charged ammoniums; (2) posi-
tion and lone pair electron directionality of the sp2
nitrogens; (3) position of 60-substituents; and (4) posi-
tion of carbon skeletons. An excellent overlay (Fig. 2)
is obtained when 3 adopts a dihedral angle of ꢀ12°
(i.e., close to one of the conformational minima) with
the pyridine rings very close to coplanar.32 The N–N
˚
distance for (1R)-3 in this conformation is 4.50 A
˚
compared to 4.69 A for chair (1R)-5.
In a similar fashion, chair (1R)-5 gives an excellent fit
with the conformationally mobile ACh in a ꢁtransꢀ
arrangement (see Fig. 3a and Table 2). This superposi-
tion allows overlap of the charged ammoniums, the H-
bond acceptor, and the corresponding methyl groups.
In this view the 60-methyl group of 5 is equivalent to
the acetyl methyl of ACh. The superposition with
gauche ACh also provides a good fit with the ACh
methyl positioned mid-way between C-50 and C-60 of 5
(Fig. 3b and Table 2).
Figure 3. Superposition of the putative pharmacophoric elements of
chair (1R)-5 with the trans (A) and gauche (B) conformations of ACh.
Three other low energy conformers of ACh (Table 2) provide less
optimal or poor overlays with (1R)-5. Hydrogens are not shown on sp3
nitrogen of 5.
An electrostatic potential map of chair (1R)-5 is shown
in Figure 4 with three designated regions (A, B, and
C) for comparison with Figure 1. The dramatically en-
hanced affinity of 60-Me-4 (i.e., compound 5) compared
to 20-methyl-4 suggests that close approach to the recep-
tor is required at C-20 (region A) in order to achieve the
H-bond receptor contact by the adjacent sp2 nitrogen
(shown in blue) (region B). In this model a C-20 methyl
prevents high affinity binding via an unfavorable steric
interaction with the receptor while the C-60 methyl re-
sides in a region where such groups are well accommo-
dated (region C). The onium site (protonated sp3
nitrogen shown in red) of the ligand will interact with
the p-electron-rich subsite formed by aromatic amino
acids (Trp, Tyr) via a cation-p contact or through
hydrogen bonding with a carboxyl oxygen of aspartic
or glutamic acid as a complementary contributor.33–35
Figure 4. Electrostatic potential map of chair (1R)-5. Key regions of
interaction with the receptor are discussed in the text. Blue (sp2
pyridine nitrogen) is more negative and red (sp3 protonated nitrogen)
is more positive. This figure was generated with gOpenMol
(www.csc.fi/gopenmol) from a Jaguar calculation.30
The bicyclic skeletons of 3 and 5 are accommodated
by a lipophilic pocket of the receptor.
The combination of subnanomolar affinity and confor-
mational rigidity makes 5 an excellent reference tem-
plate for the pharmacophoric elements of other more
flexible ligands. This exceptionally potent bridged
nicotinoid establishes an important role of the 60-methyl
substituent in the pyrido[3,4-b]norhomotropane series,
and serves as a useful model in the design of therapeutic
agents targeted at the a4b2 nAChR.
Figure 2. Superposition of the putative pharmacophoric elements of
chair (1R)-5 and (1R)-3 having dihedral angle (C1, C2, C30, C20) of
˚
˚
ꢀ12°. The N–N interatomicdistances are 4.69 A for (1R)-5 and 4.50 A
˚
(close to 4.63 A from conformational analysis) for (1R)-3 (Table 2).
Hydrogens are shown on sp3 nitrogens of 3 and 5.

880
D. B. Kanne et al. / Bioorg. Med. Chem. Lett. 15 (2005) 877–881
11. Meyer, M. D.; Decker, M. W.; Rueter, L. E.; Anderson,
Acknowledgements
D. J.; Dart, M. J.; Kim, K. H.; Sullivan, J. P.; Williams,
M. Eur. J. Pharmacol. 2000, 393, 171.
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This paper is dedicated to the memory of Professor
Henry Rapoport. This study was supported by Grant
R01 ES08424 from the National Institute of Environ-
mental Health Sciences (NIEHS), NIH, and its contents
are solely the responsibility of the authors and do not
necessarily represent the official views of the NIEHS,
NIH.
16. Compond 8: The starting material was compound 6.7 1H
NMR (400 MHz, CDCl3, 7.26 ppm): d 9.3 (s, 1H), 7.1–7.7
(m, 5H), 5.3–5.8 (m, 1H), 4.9–5.3 (m, 2H), 3.5–3.7 (m,
4H), 1.2–2.5 (m, 12H). HRMS: calculated for C16H20NO
(M+ꢀC3H5): 242.1545. Observed: 242.1526. To 6 (87 mg,
0.307 mmol) dissolved in 0.5 M HCl (0.76 mL) was added
ethyl acetate (0.42 mL), Na2PdCl4 (22 mg, 0.0748 mmol),
and benzoquinone (11.4 mg, 0.105 mmol). The reaction
(monitored by GC/MS) was complete after 24 h at 20 °C.
Aqueous HCl (6 M, 2 mL) was added to the cooled (5 °C)
reaction mixture, which was stirred for 5 min, and then
partitioned between ether and an aqueous (pH = 13)
solution saturated with NaCl. The organiclayers from
three extractions were dried (Na2SO4), and evaporated in
vacuo to yield 58 mg of the crude product. To the 1,5-
dicarbonyl compound 7 (without purification) was added
a suspension of NH2OHÆHCl (60 mg, 0.86 mmol) in
glacial acetic acid (1.0 mL), and the mixture was heated
at reflux for 1.5 h. Workup by ether extraction and solvent
evaporation as above gave an amber oil (42 mg). This
material was chromatographed on silica with CH2Cl2/
MeOH (95:5) as eluent to yield 20 mg of 8 as a colorless
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2004.12.069.
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