(S)-(-)-5-ethynyl-3-(1-methyl-2-pyrrolidinyl)pyridine maleate (SIB- 1508Y): A novel anti-Parkinsonian agent with selectivity for neuronal nicotinic acetylcholine receptors

Full text of DOI:10.1021/jm960328w

J . Med. Chem. 1996, 39, 3235-3237
3235
rolidinone anion followed by decarboxylation and ring
closure of the intermediate gave the imine 4. Reduction
(NaBH4) and N-methylation under Eschweiler-Clarke
conditions13 afforded 5.14a Palladium-catalyzed cross-
coupling15 of 5 with 2-methyl-3-butyn-2-ol provided the
5-alkynyl derivative 6 which underwent base-catalyzed
deprotection16 to give 2.14b Conversion of 2 to 7 was
achieved by hydrogenation (H2, Pd-C).14b In order to
evaluate the relative contributions of the individual
enantiomers [(S)-2 and (R)-2] to the activity of the
racemate (2), the synthesis was modified to provide the
component isomers separately (Scheme 1). Reduction
of 4 with NaBH4/CBZ-D-proline17,18 followed by N-
methylation provided enantiomerically enriched 5 (30%
ee) which was crystallized as the dibenzoyl-L-tartrate
salt to give, after Pd-mediated alkyne coupling and
deprotection, (S)-2 in >99% ee.14c Similarly, the use of
NaBH4/CBZ-L-proline, followed by crystallization with
di-p-toluoyl-D-tartaric acid, ultimately provided the
antipode (R)-2 (97% ee).14d,19
(S)-(-)-5-Eth yn yl-3-(1-m eth yl-2-p yr r oli-
d in yl)p yr id in e Ma lea te (SIB-1508Y): A
Novel An ti-P a r k in son ia n Agen t w ith
Selectivity for Neu r on a l Nicotin ic
Acetylch olin e Recep tor s
Nicholas D. P. Cosford,* Leo Bleicher,
Audrey Herbaut, J . Stuart McCallum,
J ean-Michel Vernier, Heather Dawson,
J effrey P. Whitten, Pamala Adams,
Laura Chavez-Noriega, Lucia D. Correa,
J ames H. Crona, Lorrence S. Mahaffy,
Frederique Menzaghi, Tadimeti S. Rao, Richard Reid,
Aida I. Sacaan, Emily Santori,
Kenneth A. Stauderman, Kevin Whelan,
G. Kenneth Lloyd, and Ian A. McDonald
SIBIA Neurosciences Inc., 505 Coast Boulevard South,
Suite 300, La J olla, California 92037
Received May 2, 1996
The selective modulation of neuronal receptor sub-
types has considerable potential for the treatment of
disease with minimal adverse side effects. Specifically,
the ideal central nervous system (CNS) drug would
discriminate not only between the target receptor and
those in the periphery but also between receptor sub-
types within the brain.1 The importance of nicotinic
acetylcholine receptors (NAChRs)2,3 in several disease
states, including Alzheimer’s disease (AD)4,5 and Par-
kinson’s disease (PD),6,7 is now well established. Indeed
the NAChR agonist ABT-418 is presently in clinical
trials for the treatment of AD.8 In this communication,
the synthesis and pharmacological evaluation of (S)-
(-)-5-ethynyl-3-(1-methyl-2-pyrrolidinyl)pyridine [(S)-
2, SIB-1508Y], a novel agonist of human neuronal
NAChRs, are reported.9 The characterization of this
compound was accomplished using both classical meth-
ods and functional assays employing mammalian cell
lines expressing NAChRs comprised of recombinant
human NAChR subunits.
The results of the binding and in vitro dopamine (DA)
release experiments with 1, 2, 5, and 7 are presented
in Table 1. The data indicate that all the compounds
tested exhibited a high degree of selectivity for the [3H]-
Nic (nicotine) binding site compared with the [3H]QNB
(muscarinic) binding site in rat cortical membranes.
Differences were observed, however, in the ability of the
compounds to release DA from rat striatal slices.
Whereas 2 tended toward a greater efficacy than 1 in
this bioassay, 7 was significantly less active. Further-
more, the data demonstrate that (S)-2 was the enan-
tiomer exhibiting the highest affinity in the binding
assay and was primarily responsible for the DA release
stimulated by 2, an effect which is blocked by the
NAChR antagonists mecamylamine and dihydro-â-
erythroidine.23 The results illustrate the utility of a
functional assay for the differentiation of two closely
related analogues (2 and 7) and led to the decision to
more fully evaluate 2 and (S)-2.
Electrophysiological recording of current responses in
Xenopus oocytes expressing recombinant human NAChRs
is a sensitive assay for detecting the activity of agonists
or antagonists. Thus voltage clamp recordings of oo-
cytes expressing human R2â2, R2â4, R3â2, R3â4, R4â2,
R4â4, or R7 NAChR subtypes were obtained (Figure 1).24
The inward current elicited by 2 was normalized to the
response produced by a prior application of acetylcholine
(ACh; 10 µM) to the same cell. At a concentration of 10
µM, 2 produced currents that ranged between 20% and
50% of the response elicited by ACh (10 µM) in oocytes
expressing the R2â2, R2â4, R3â2, R4â2, and R4â4
NAChR subtypes. No detectable inward currents were
observed at the human R7 receptor subtype,25 and only
a minimal response at the R3â4 subtype was detected.
This contrasts with 1 (data not shown) which is a potent
agonist at both the R3â4 and R7 receptor subtypes in
this assay.27
Initial structure-activity studies focused on the
prototypical NAChR agonist nicotine (1).10 Synthetic
targets designed to probe the topology of the binding
site of 1 revealed that substitution at the 5-position led
to derivatives, such as racemic 5-bromo-3-(1-methyl-2-
pyrrolidinyl)pyridine (5),11 that had attractive pharma-
cological profiles. Compound 5 was therefore selected
for further elaboration, and taking advantage of the aryl
bromide functional group, a series of 5-substituted
analogues was prepared. From this series 5-ethynyl-
3-(1-methyl-2-pyrrolidinyl)pyridine fumarate (2, SIB-
1765F) and 5-ethyl-3-(1-methyl-2-pyrrolidinyl)pyridine
(7) were selected for biological evaluation and compari-
son with 1 and 5.
A functional assay employing cell lines stably ex-
pressing human recombinant NAChR28,29 subtypes pro-
vided further data comparing 1, 2 (S)-2, and (R)-2. In
this assay, the activation of recombinant receptors by
agonists stimulates calcium entry into cells. The sub-
sequent elevation of cytosolic Ca2+ concentration ([Ca2+]i)
is determined by measuring the changes in fluorescence
Racemic 2 and 7 were synthesized according to
Scheme 1. The reported synthesis of 511,12 was modified
to provide an efficient route to this key intermediate.
Thus, treatment of readily available 3 with 1-vinylpyr-
S0022-2623(96)00328-7 CCC: $12.00 © 1996 American Chemical Society

3236 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Communications to the Editor
Sch em e 1a
a
(a) LiN(TMS)2, 1-vinylpyrrolidinone, THF, 25 °C (99% yield); (b) 6 M HCl, ∆ (71% yield); (c) NaBH4, MeOH, HOAc (90% yield); (d)
37% HCHO (aq), HCO2H, ∆ (92% yield); (e) 2-methyl-3-butyn-2-ol, 10% Pd-C, CuI, PPh3, K2CO3, DME, H2O, ∆ (92% yield); (f) NaH
(cat.), PhMe, ∆ (97% yield); (g) H2, 10% Pd-C, MeOH (92% yield); (h) CBZ-D-proline, NaBH4, CH2Cl2 (98% yield, 30% ee); (i) dibenzoyl-
L-tartaric acid, EtOH-EtOAc (93% yield, >99% ee).
Ta ble 1. Binding and Release Dataa
Ta ble 2. Profile of NAChR Agonist Functional Potency
EC50 (µM; mean ( SEM) (n)
IC50 (nM; mean ( SEM)
release ((SEM)
compd
[3H]Nicb
[3H]QNBc
[3H]DAd
compd
R2â4a
R3â4a
R4â4a
R4â2a
R1â1γδb
1
5
2
7
4 ( 1.2
19 ( 4
360 000 ( 40 400
16 000 ( 2300
10 100 ( 1200
5800 ( 870
100
1
2
10 ( 2 (3) 51 ( 2 (3) 7.1 ( 1.8 (3)c 2.1 ( 0.6 (5)c 65 ( 18 (3)
69 ( 11
143 ( 11
27.5 (26, 29)e
163 ( 28
55 ( 15
15 ( 3.6 (7) 41 ( 7 (8) 25 ( 7 (6)c
2.6 ( 1 (7)c 52 ( 6 (3)
1.8 ( 0.7 (4) nd
(S)-2 5 ( 1.4 (3) 23 ( 2 (3) 9 ( 4 (3)
(R)-2 29 ( 8 (3) 68 ( 7 (3) 31 ( 6 (3)
4.6 ( 0.9
11 ( 1.7
3 ( 0.5
inactived
nd
(S)-2
(R)-2
8900 ( 980
a
b
Human embryonic kidney (HEK293) cells. Human RD cells.
75 ( 1.7
4500 ( 120
d
c See ref 23. Highest concentration ) 300 µM. Data derived from
measured changes in [Ca2+]i in cell lines stably expressing the
designated human recombinant NAChR subtypes in response to
compounds 1, 2, (S)-2, and (R)-2. n ) number of determinations.
a
The data represent the mean values from three independent
b
experiments (n ) 3). Binding affinities at NAChRs were deter-
mined by measuring the displacement of [3H]nicotine ([3H]Nic)
from a preparation of rat cortical membranes according to the
procedure of Flynn and Mash.20 c Muscarinic receptor binding (rat
cortical membranes) was determined by displacement of [3H]qui-
nuclidinyl benzylate ([3H]QNB) using the method of Yamamura
Ta ble 3. Profile of NAChR Agonist Efficacy
efficacy (mean ( SEM) (n)
and Synder.21 The efficacy of the compounds (300 µM) to
d
compd
R2â4a
R3â4a
R4â4a
R4â2a
R1â1γδb
stimulate [3H]dopamine ([3H]DA) release was determined using
a preparation of superfused rat striatal slices as described by
Sacaan et al.22 The data are expressed as a percentage of the
[3H]DA release elicited by a maximally efficacious dose of nicotine
(10 µM). e Mean value from two independent experiments, raw
data in parentheses.
2
39 ( 3 (7) 34 ( 4.2 (8) 23 ( 4 (6)
47 ( 10 (7) 53 ( 17 (3)
(S)-2 49 ( 5.2 (3) 52 ( 7.5 (3) 26 ( 10.4 (3) 49 ( 8.5 (4) nd
(R)-2 9 ( 1.7 (3) 7 ( 2.3 (3) 8 ( 4 (3)
inactivec
nd
a
b
Human embryonic kidney (HEK293) cells. Human RD cells.
c Highest concentration ) 300 µM. Data derived from measured
changes in [Ca2+]i in cell lines stably expressing the designated
human recombinant NAChR subtypes in response to compounds
2, (S)-2, and (R)-2. The data are expressed as a percentage of the
value for the maximally effective concentration of nicotine (1) in
each cell line. n ) number of determinations.
the NAChR antagonists d-tubocurarine and mecamyl-
amine (data not shown). While (S)-2 showed its greatest
potency at the R4â2 NAChR subtype (Table 2), (R)-2
exhibited low activity in the â4-containing cell lines and
was inactive in the cell line expressing the R4â2
subtype. Furthermore, at a maximally efficacious con-
centration, (S)-2 was 26-52% as efficacious as 1 in each
cell line, whereas (R)-2 displayed weak efficacy (Table
3). The data establish that (S)-2 is the more active
enantiomer which is in agreement with the binding and
DA release results (Table 1).
F igu r e 1. Voltage clamp recordings in Xenopus oocytes, the
current evoked by 2 (10 µM) in Xenopus oocytes expressing
the designated recombinant human NAChR subtypes. The
ordinate shows the fractional response relative to 10 µM ACh.
Since 2 stimulated the release of DA from rat striatal
slices, it was evaluated in an animal model of Parkin-
son’s disease. The effect of 2 on ipsilateral rotations of
rats with unilateral nigrostriatal 6-hydroxydopamine (6-
OHDA) lesions32 (rat-turning model) is shown in Table
4. At a dose of 25 mg/kg given subcutaneously (sc), 2
produced a significant increase in ipsilateral rotations
compared with saline-treated animals, an effect which
is blocked by mecamylamine. Futhermore, 2 was more
efficacious than 1 at the maximum effective dose with
of the Ca2+-sensitive dye 1-[2-amino-5-(2,7-dichloro-6-
hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-(2-amino-5-me-
thylphenoxy)ethane-N,N,N′,N′-tetraacetic acid (fluo-3).30
The compounds were tested in cell lines expressing
recombinant human R2â4, R3â4, R4â4, and R4â2 NAChR
subtypes and a cell line expressing the fetal form
(R1â1γδ) of human neuromuscular NAChRs (Tables 2
and 3).31 In each cell line 1, 2, and (S)-2 elicited
increases in [Ca2+]i, and these effects were blocked by

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3237
Novel Isoxazoles and Isothiazoles as Bioisosteric Replacements
for the Pyridine Ring in Nicotine. J . Med. Chem. 1994, 37,
4455-4463.
Ta ble 4. Data for the Rat-Turning Model
compd
dosemax
rotations ((SEM)
% rotationsb
a
(14) (a) Converted to the dihydrobromide salt for biological evalua-
tion. (b) Converted to the fumarate salt for biological evaluation.
(c) Converted to the maleate salt for biological evaluation. (d)
Converted to the di-p-toluoyl-D-tartrate salt for biological evalu-
ation.
1
2
0.4
25
62 ( 21
100
263
163 ( 44c
a
Maximally effective dose (mg/kg free amine, sc). Intolerable
side effects were observed for nicotine (1) at a dose of 1 mg/kg.
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b
Ipsilateral rotations measured over a period of 120 min, ex-
pressed as a percentage of the value for 1. c p
t-test). Each group contained eight animals.
0.05 (Student’s
minimal adverse side effects. These studies led to the
preclinical development of (S)-2 for the treatment of PD.
In conclusion, the novel NAChR agonist (S)-2 has
been synthesized and evaluated in a range of in vitro
and in vivo assays. Significantly, the differentiation of
(S)-2 from structurally related agonists with quite
similar binding affinities for endogenous NAChRs was
accomplished on the basis of results derived from
functional assays, including a novel functional assay
employing cell lines stably expressing recombinant
human NAChR subtypes. This assay provides a power-
ful method for the discovery of subtype selective NAChR
agonists and antagonists, acting either at the ACh
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mm) at 125 °C with a flow rate of 3.0 mL/min. The absolute
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