2-Fluoro-4-pyridinylmethyl analogues of linopirdine as orally active acetylcholine release-enhancing agents with good efficacy and duration of action

Article abstract of DOI:10.1021/jm9803424

In an effort to improve the pharmacokinetic and pharmacodynamic properties of the cognition-enhancer linopirdine (DuP 996), a number of core structure analogues were prepared in which the 4-pyridyl pendant group was systematically replaced with 2-fluoro-4

Full text of DOI:10.1021/jm9803424

J . Med. Chem. 1998, 41, 4615-4622
4615
2-F lu or o-4-p yr id in ylm eth yl An a logu es of Lin op ir d in e a s Or a lly Active
Acetylch olin e Relea se-En h a n cin g Agen ts w ith Good Effica cy a n d Du r a tion of
Action
Richard A. Earl,*,†,| Robert Zaczek,‡ Christopher A. Teleha,† Barbara N. Fisher,† Carla M. Maciag,‡
Maria E. Marynowski,‡ Andrew R. Logue,‡ S. William Tam,‡,| William J . Tinker,‡ Shiew-Mei Huang,§ and
Robert J . Chorvat*,†
The DuPont Pharmaceuticals Company, Experimental Station, P.O. Box 80500, Wilmington, Delaware 19880-0500
Received J une 3, 1998
In an effort to improve the pharmacokinetic and pharmacodynamic properties of the cognition-
enhancer linopirdine (DuP 996), a number of core structure analogues were prepared in which
the 4-pyridyl pendant group was systematically replaced with 2-fluoro-4-pyridyl. This strategy
resulted in the discovery of several compounds with improved activity in acetylcholine (ACh)
release-enhancing assays, in vitro and in vivo. The most effective compound resulting from
these studies, 10,10-bis[(2-fluoro-4-pyridinyl)methyl]-9(10H)-anthracenone (9), is between 10
and 20 times more potent than linopirdine in increasing extracellular hippocampal ACh levels
in the rat with a minimum effective dose of 1 mg/kg. In addition to superior potency, 9 possesses
an improved pharmacokinetic profile compared to that of linopirdine. The half-life of 9 (2 h)
in rats is 4-fold greater than that of linopirdine (0.5 h), and it showed a 6-fold improvement in
brain-plasma distribution over linopirdine. On the basis of its pharmacologic, pharmacokinetic,
absorption, and distribution properties, 9 (DMP543) has been advanced for clinical evaluation
as a potential palliative therapeutic for treatment of Alzheimer’s disease.
In tr od u ction
various animal models support the role of ACh in
cognition and learning.18-22 The slow nature of the
degenerative process as well as the redundancy of the
connectivity of the brain area provides a window of
opportunity for palliative therapy early in the disease.
The positive effects of the cholinesterase inhibitors
tacrine23 and donepezil24,25 observed in a subpopulation
of AD patients support this concept and the basis that
compounds that do indeed bolster the cholinergic system
should provide theraputic benefit.
Alzheimer’s disease (AD) is a neurodegenerative
disorder that is accompanied in its later stages by a
variety of deficiencies that include deterioration of
memory, loss of cognitive function, and privation of
personality.1 This condition is reflective of the wide-
spread pathology found in the brain of AD patients at
autopsy. Both familial and sporadic forms of the disease
have been identified, and causative roles for abnormal
amyloid deposition,2,3 phosphorylated microtubule pro-
teins,4 and neuroinflammatory mechanisms5-7 have
been put forth.
In addition to the presence of plaques and tangles,
neurochemical alterations are also included as hall-
marks of the disease. In particular, decreases in the
markers of cortical cholinergic innervation are one of
the earliest and most consistently present correlates of
AD. These observations have led to the hypothesis that
associates this type of dementia with cholinergic losses.8
Neurotransmitter receptor sites are also altered in
AD. Both muscarinic M29 and nicotinic10,11 receptors
decrease with the progression of the disease, whereas
the number of muscarinic M1 receptors does not
change.12 Patients with AD show reduced activity of
acetylcholine esterase (AChE), high-affinity choline
uptake (HACU), and choline acetyltransferase (ChAT)
reflective of a damaged cholinergic system and directly
related to their cognitive impairments.8,13-17 Moreover
Over the past several years, we have identified a
series of compounds that possess the unique ability to
enhance the potassium-evoked release of acetylcholine
(ACh).26,27 Since this neurotransmitter is known to be
deficient in AD,16,17 these compounds are potential
therapeutics for the treatment of this disease. Unlike
direct receptor agonists or cholinesterase inhibitors, we
anticipated these agents would not cause receptor down-
regulation due to sustained receptor interaction.28 More-
over, since these agents only affect evoked release, they
should not cause neurotransmitter depletion or overload
toxicity, a problem with agents affecting basal re-
lease.13,29,30 The first member of this series to be tested
in clinical trials was linopirdine (AVIVA, DuP 996), a
compound whose development was largely supported by
behavioral pharmacology.31,32 The results from these
studies, however, were mixed and unconvincing.33 More
recent animal pharmacokinetic studies have suggested
that the absence of a more definitive clinical effect with
linopirdine may have been due to its short and variable
half-life and poor blood-brain barrier (BBB) penetra-
tion.34,35 Therefore, the neurotransmitter release en-
† Chemical and Physical Sciences.
‡ Central Nervous System Research.
§ Drug Metabolism and Pharmacokinetics Section.
| Current address: NitroMed, Inc., 12 Oak Park Dr., Bedford, MA
01730.
10.1021/jm9803424 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/10/1998

4616 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Earl et al.
Sch em e 1
Ta ble 1. Physicochemical Properties of Linopirdine Analogues
hancement approach to AD may not have been ad-
equately tested with this drug.
pyridinylmethyl pendant groups of linopiridne were
subject to N-oxidation causing this compound to have a
short plasma half-life.34 Metabolic N-oxidation of aro-
matic nitrogens, such as pyridine, is known to be a
cytochrome P-450-dependent process37 and may result
in rapid metabolism or clearance of active substance.
Indeed, the major oxidative metabolites of linopirdine
(DuP 996) were the bis- and mono-N-oxides in rats, dogs,
and humans34 (Scheme 1). It was expected that sub-
stituting a fluorine atom for the hydrogen ortho to the
pyridine nitrogens would reduce the basicity of the
heterocycles as well as their ability to undergo N-
Our requirements, then, for a second-generation
neurotransmitter release-enhancing compound were
greater brain penetration and longer duration of action
than that of linopirdine. To achieve these goals, two
important observations were taken into consideration.
First, other core structures, such as 4-azafluorene and
anthrone, when dialkylated with the 4-pyridinylmethyl
group, were not only more potent than linopirdine but
also more lipophilic, a property that can facilitate BBB
penetration.36 Second, it had been shown that the

ACh Release-Enhancing Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4617
Sch em e 2
Sch em e 3a
oxidation.38 There is no evidence in the literature to
suggest that 2-fluoro-substituted pyridines undergo
metabolic N-oxidation.39
To assess the effects of fluorine substitution into the
2-position of the pyridinyl pendant groups, we system-
atically replaced 4-pyridinylmethyl with 2-fluoro-4-
pyridinylmethyl in three core structure types: oxindole
(1, 4, 7), 4-azafluorene (2, 5, 8), and anthrone (3, 6, 9)
(Table 1). These compounds were then evaluated for
their ability to enhance release of acetylcholine in a
potassium-evoked slice assay and in vivo using micro-
dialysis in conscious animals.
Ch em istr y
Chlorination of commercially available 2-fluoro-4-
methylpyridine (10) with N-chlorosuccinimide under
free radical conditions provided a mixture of 2-fluoro-
4-(chloromethyl)pyridine (11), 2-fluoro-4-(R,R-dichloro-
methyl)pyridine (12), and unreacted starting material
(3:1:1) (Scheme 2). Subsequent treatment of this mix-
ture under Finkelstein conditions gave only one iodin-
ated compound, 2-fluoro-4-(iodomethyl)pyridine (13).
The byproducts from the chlorination reaction are not
reactive and are readily removed by chromatography
after the subsequent alkylation reaction.
The symmetrically dialkylated compounds 2, 7, 8, and
9 were prepared according to Scheme 3, utilizing N-
phenyloxindole,40 4-azafluorene,41,42 and anthrone and
either 4-picolyl chloride, 2-fluoro-4-(chloromethyl)pyri-
dine, or 2-fluoro-4-(iodomethyl)pyridine in the presence
of base (such as sodium hydride). The anthrone product
3 was best prepared from 4-picolyl chloride hydrochlo-
ride and anthrone under phase-transfer catalyzed con-
ditions.43 In the case of 9, the yield using 2-fluoro-4-
(iodomethyl)pyridine as an alkylating agent was usually
greater than 55%, whereas from the chloromethyl
compound it was typically less than 25%.
Unsymmetrically dialkylated compounds (“mixed pen-
dant group” compounds) 4, 5, and 6 were also prepared
to determine if one fluorinated pyridine would be
sufficient to improve pharmacodynamics while main-
taining or increasing potency. Oxindole 4 was prepared
from the well-known 1,3-dihydro-1-phenyl-3-(4-pyridin-
ylmethyl)-2H-indol-2-one44,45 and 2-fluoro-4-(chloro-
methyl)pyridine in the presence of sodium hydride
(Scheme 4). The 4-azafluorene analogue 5 was synthe-
sized via the procedure in Scheme 5, starting from
a
(a) Picolyl chloride (2 equiv), NaH, THF, room temperature;
(b) picolyl chloride hydrochloride (2 equiv), benzyltriethylammo-
nium chloride, toluene, NaOH, 50 °C; (c) 2-fluoro-4-(chlorometh-
yl)pyridine (2 equiv), NaH, THF, room temperature; (d) 2-fluoro-
4-(iodomethyl)pyridine (2 equiv), NaH, THF, room temperature.
Sch em e 4a
a
(a) 2-Fluoro-4-(iodomethyl)pyridine (2 equiv), NaH, THF, room
temperature.
4-azafluorenone.46 Anthrone 6 was produced according
to Scheme 6, by the presence of equivalent amounts of
both alkylating agents. The desired unsymmetrically
appended product 6 was purified from a mixture with
the symmetrically appended compounds 3 and 9. At-
tempts to prepare a monoalkylated anthrone and con-
vert it to 6 were not successful.
Biologica l Activity
Compounds were initially assessed for their ability
to enhance the K+-stimulated release of [3H]acetylcho-
line from rat hippocampal slices preloaded with [3H]-

4618 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Earl et al.
Sch em e 5
/C
Sch em e 6
F igu r e 1. In vitro ACh release-enhancing effects of 9 and
linopirdine: dose response. Assays were performed as de-
scribed previously.47 Each point represents data from at least
three individual determinations. EC50 values calculated using
peak efficacy as 100% activity were found to be 0.83 µM for 9
and 4.5 µM for linopirdine.
experimental procedure, an animal is surgically im-
planted with a guide-cannula into the hippocampus 2-4
days prior to testing. On the day of testing, a microdi-
alysis probe is placed through the guide and is perfused
with artificial CSF. Physostigmine, a cholinesterase
inhibitor, is also perfused in the system to prevent the
degradation of released ACh, which diffuses into the
probe and is collected. The dialysate is injected every
20 min onto an HPLC system which measures ACh.
Test compound or vehicle is administered orally to
animals after the fourth 20-min collection period. Ex-
periments are usually carried out for 14 collection
periods. Results are expressed as percent increase of
ACh over baseline for each collection period. Linopir-
dine showed a rapid onset of action, a short duration,
and inactivity below 10 mg/kg, po (Table 2, Figure 2).
The monofluorinated and bisfluorinated oxindoles 4 and
7 are likewise inactive at doses lower than 10 mg/kg.
The azafluorenes and anthrones show an increased
ability to enhance ACh release. Generally, upon oral
administration, the nonfluorinated compounds 2 and 3
and the monofluorinated compounds 5 and 6 showed a
similar fast onset and short duration of action like
linopirdine. The bisfluorinated compounds 8 and 9,
however, show a slower onset of action, along with a
very extended duration (Figure 2). Compounds 8 and
9 are less water-soluble and more lipophilic than lino-
pirdine by >1 log unit. These properties can influence
absorption and distribution of agents in vivo and most
likely are contributing to the in vivo pharmacological
profile.
choline.47 This assay is a standard slice superfusion
procedure in which release is stimulated for 4 min at
two points, denoted S1 and S2, by increasing the
concentration of potassium in the superfusion medium
to 25 mM. Test compound is introduced after the
control stimulation period (S1) and remains in the
superfusion medium until the end of the second stimu-
lation period (S2). The ratio of S2/S1 is used to measure
the degree of drug-induced release enhancement.
Figure 1 shows dose-response curves for ACh release
enhancement for linopirdine and 9. From these curves,
the EC50 value, which is the concentration that causes
an enhancement that is 50% of the maximal response
of a given compound, was calculated. For 9 and linopir-
dine, these values are 0.83 and 4.5 µM, respectively,
with the maximum response of 9 higher than that
observed for linopirdine. The EC50’s for the other
compounds are shown in Table 2. With the exception
of the oxindoles 4 and 7, within a core series, release
activity follows nonfluoro ≈ monofluoro > difluoro >
linopirdine.
The exact mechanism of ACh release enhancement
by these compounds has yet to be unequivocally proven.
However, the association of the ability of linopirdine and
related compounds to block certain K+ channels at
concentrations comparable to those causing neurotrans-
mitter release enhancement has been reported.48 In
particular, blockade of M-type potassium channels49
currently serves as a possible molecular target respon-
sible for the ability of these agents to cause ACh release
enhancement. Further work is currently underway to
support this proposal.
The fact that these compounds showed good activity
upon oral administration implies that they have a
higher bioavailability, greater brain penetration, longer
half-life (compared to linopirdine), or a combination of
effects. Indeed, the most effective compound resulting
from these studies is 9, being between 10 and 20 times
more potent than linopirdine in increasing extracellular
hippocampal ACh levels in the rat with a minimum
effective dose of 1 mg/kg (Table 2). In addition to
To determine neurotransmitter release-enhancing
activity in vivo, compounds were evaluated in micro-
dialysis assays that measure extracellular brain con-
centrations of ACh in freely moving rats.50-52 In this

ACh Release-Enhancing Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4619
Ta ble 2. Acetylcholine Release Enhancement Data
in vivo microdialysis ACh release (po)
in vitro ACh
release EC50 (µM)
in vitro ACh
compd
release max efficacya
dose, vehicleb
peakc
durationd
1 (linopirdine)
4.5 ( 1.24
337% at 10 µM
20, methocel
10, methocel
5, water
10, methocel
10, water
5, water
10, methocel
10, water
117 ( 36
65 ( 13
50 ( 16
IA
140
80
20
2
3
4.12 ( 2.28
0.45 ( 0.12
725% at 10 µM
600% at 10 µM
127 ( 31
18 ( 15
27 ( 38
52 ( 10
47 ( 32
75 ( 12
44 ( 32
201 ( 68
74 ( 29
115 ( 27
40 ( 21
100
NSP
NSP
>40
NSP
100
NSP
>80
120
4
5
10.0 ( 10.1
1.08 ( 0.2
433% at 10 µM
317% at 10 µM
5, water
6
7
8
0.41 ( 0.23
15.2 ( 6.67
1.60 ( 0.95
452% at 1 µM
326% at 10 µM
440% at 3 µM
5, methocel
10, methocel
5, methocel
1, methocel
1, methocel
0.5, methocel
9
0.83 ( 0.28
413% at 3 µM
>120
NSP
a
b
Percent control, control ) 100%, at the dose specified; determined as described previously.47 Doses in mg/kg; methocel, compound
suspended in 25% methocel in water and bead-milled overnight. c Percent increase over control, control ) 0; IA, inactive. Number of
consecutive minutes at significance; NSP, no significant points.
d
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Electro-
thermal capillary apparatus and are uncorrected. Proton
NMR spectra were obtained using Varian Unity 300 spectrom-
eters (Varian Instruments, Palo Alto, CA); chemical shifts were
recorded in ppm (δ) from an internal TMS standard in
deuteriochloroform or deuteriodimethyl sulfoxide as specified.
Coupling constants were measured in hertz (Hz). Mass
spectra were measured with a Hewlett-Packard 5988A or a
Finnigan MAT 8230 mass spectrometer with a particle beam
interface using NH3 or CH4 for chemical ionization. High-
resolution mass spectra (HRMS) were obtained on a VG 70-
VSE instrument with NH3 as a carrier gas for chemical
ionization. Elemental analyses were performed by Quantita-
tive Technologies, Inc., Whitehouse, NJ , or Micro-Analysis,
Inc., Wilmington, DE. Solvents and reagents were obtained
from commercial vendors and used without further purification
unless otherwise indicated.
2-F lu or o-4-(ch lor om eth yl)p yr id in e (11). To a solution
of 2-fluoro-4-picoline (10; 13.33 g, 120 mmol) and N-chloro-
F igu r e 2. In vivo microdialysis comparison of test compounds
8 and 9 to linopirdine. Values are expressed as percent control.
Drug was administered at time zero.
succinimide (23.98 g, 180 mmol) in 500 mL of carbon tetra-
chloride was added 1.5 g of benzoyl peroxide, and the mixture
was heated at reflux. After 4 h, the mixture was briefly cooled
to room temperature, an additional 0.5 g of benzoyl peroxide
was added, and reflux was continued overnight. After cooling
to room temperature, the reaction mixture was filtered through
Celite, washed with saturated sodium thiosulfate, saturated
sodium bicarbonate, water, and brine, and dried over magne-
sium sulfate. The solvent was removed in vacuo to give a
yellow liquid, 17.845 g, which contained 60 mol % 11, 24 mol
% 12, and 16 mol % starting material (10) by 1H NMR (300
MHz, CDCl3) integration. This product was used directly in
alkylation reactions or converted to the iodide 13.
2-F lu or o-4-(iod om eth yl)p yr id in e (13). To a solution of
sodium iodide (19.79 g, 0.132 mol) in 500 mL of acetone was
added 11 (17.46 g, 0.120 mol; 29.6 g of the chlorination
mixture), and the solution was heated at reflux for 2 h. The
precipitated sodium chloride was filtered off through Celite,
and the solvent was removed in vacuo. The residue was
redissolved in ether, washed with saturated sodium thiosul-
fate, water, and brine, and dried over magnesium sulfate. The
solvent was evaporated to give a yellow oil, 33.29 g. 1H NMR
(300 MHz, CDCl3) showed that the methylene singlet of the
chloride at 4.57 ppm had shifted to 4.34 ppm and the 6-H
pyridine doublet had shifted from 8.23 to 8.16 ppm. The
spectrum showed 100% conversion of 11 to 13 and the other
unchanged side products of the chlorination reaction (12 and
10). This product was used directly in alkylation reactions.
superior potency, 9 possesses an improved pharmaco-
kinetic profile compared to that of linopirdine. The half-
life of 9 (2 h) in rats is 4-fold greater than that of
linopirdine (0.5 h). The oral bioavailability of both 9
and linopirdine is approximately 30%. The levels of
linopirdine were previously evaluated in plasma and
brain upon oral dosing. The brain-to-plasma ratio of
linopirdine was approximately 1:6.53,55 The brain-to-
plasma ratio of 9 observed in parallel studies was
approximately 1:1.56 Thus, this improvement in brain-
plasma distribution of 9 and the in vivo effects of this
compound suggest that it may also possess the better
brain penetration, as compared to linopirdine, that we
sought.
In summary, we have discovered that substitution of
2-fluoro-4-pyridinylmethyl pendant groups for 4-pyrid-
inylmethyl groups, in conjuction with a more potent
tricyclic systems, has led to a compound possessing
superior pharmacolgical and pharmacodynamic proper-
ties relative to linopirdine. On the basis of this phar-
macological and pharmacokinetic profile, 9 (DMP 543)
is undergoing clinical development.54
5,5-B is (4-p y r id in y lm e t h y l)-5H -in d e n o [1,2-b ]p y r i-
d in e, Hem ih yd r a te (2). To a suspension of sodium hydride
(60% in oil, 3.34 g, 83.5 mmol) in 100 mL of THF at room

4620 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Earl et al.
temperature were added a solution of 4-azafluorene (14)41,42
(4.16 g, 25 mmol) and 1 drop of ethanol. A solution of 4-picolyl
chloride in benzene (prepared from 9.79 g, 59.7 mmol, of the
hydrochloride salt, free-based with sodium carbonate, ex-
tracted into benzene, and dried over sodium sulfate) was
subsequently added to the reaction mixture via addition
funnel. After the mixture stirred at room temperature for 3
h, saturated ammonium chloride was added, and the mixture
was extracted with ethyl acetate. The aqueous layer was
washed with more ethyl acetate, and the combined organic
solution was washed with water and brine and dried over
magnesium sulfate. The product was purified via column
chromatography (silica gel, 5% methanol in methylene chlo-
ride) and recrystallized from ethyl acetate/hexane to give 2
as white crystals, 7.09 g, 81% yield: mp 169-171 °C; 1H NMR
(300 MHz, CDCl3) δ 3.40 (s, 4H), 6.51 (d, J ) 6.2 Hz, 4H),
7.18 (dd, J ) 4.8, 7.7 Hz, 1H), 7.37 (dt, J ) 0.7, 7.3 Hz, 1H),
7.47 (dt, J ) 0.7, 7.3 Hz, 1H), 7.59 (d, J ) 7.7 Hz, 1H), 7.67
(dd, J ) 1.5, 7.7 Hz, 1H), 7.74 (d, J ) 7.3 Hz, 1H), 8.15 (dd, J
) 1.5, 4.8 Hz, 4H), 8.45 (dd, J ) 1.5, 5.1 Hz, 1H); MS (CI/
NH3) m/e 350 (M + H). Anal. (C24H19N3‚0.5H2O) C, H, N.
10,10-Bis(4-p yr id in ylm eth yl)-9(10H)-a n th r a cen on e (3).
To a mechanically stirred slurry of 50 g (0.257 mol) of
anthrone, 92.9 g (0.566 mol) of 4-picolyl chloride hydrochloride,
and 5.14 g (0.0226 mol) of benzyltriethylammonium chloride
in 1000 mL of toluene was added 129 mL of 50% NaOH via
addition funnel over the period of 15 min. After addition, the
mixture was slowly heated to 50 °C and maintained at that
temperature for 18 h. The mixture was then diluted with 50
mL of water and cooled to room temperature. The toluene
layer was separated, washed with water and brine, dried over
magnesium sulfate, and evaporated in vacuo to give a dark-
brown solid. The product was dissolved in methylene chloride,
chromatographed on silica gel with 10% ethyl acetate/hexane,
and recrystallized from methylene chloride/hexane to give 3
as white crystals, 32 g, 33% yield: mp 231-2 °C; 1H NMR
(300 MHz, CDCl3) δ 3.73 (s, 4H), 6.19 (dd, J ) 1.5, 4.8 Hz,
4H), 7.54 (dt, J ) 1.1, 7.5 Hz, 2H), 7.79 (dt, J ) 1.5, 7.5 Hz,
2H), 7.98 (d, J ) 8.1 Hz, 2H), 8.02 (dd, J ) 1.5, 4.8 Hz, 4H),
8.12 (dd, J ) 1.5, 7.7 Hz, 2H); IR (KBr) 1656, 1600, 1328, 1316,
694 cm-1; MS (CI/NH3) m/e 377 (M + H). Anal. (C26H20N2O)
C, H, N.
1.5, 4.8 Hz, 1H), 8.73 (dd, J ) 1.5, 4.4 Hz, 2H); MS (CI/NH3)
m/e 257 (M + H). Anal. (C18H12N2) C, H, N.
5-(4-P yr id in ylm eth yl)-5H-in d en o[1,2-b]p yr id in e (20).
To a solution of 19 (0.724 g, 2.8 mmol) in ethanol was added
20% Pd(OH)2/C (150 mg), and the mixture was shaken under
50 psi of hydrogen (Parr bottle) for 1 h. The catalyst was
filtered off, the solvent was removed in vacuo, and the residue
was triturated with ether/petroleum ether to give 20 as a white
1
solid, 0.355 g, 49% yield: mp 126-8 °C; H NMR (300 MHz,
CDCl3) δ 3.03 (dd, J ) 8.0, 13.9 Hz, 1H), 3.27 (dd, J ) 6.6,
13.9 Hz, 1H), 4.26 (t, J ) 7.3 Hz, 1H), 7.09 (d, J ) 5.5 Hz,
2H), 7.11 (d, J ) 5.1 Hz, 1H), 7.37 (m, 2H), 7.47 (dt, J ) 1.5,
7.3 Hz, 1H), 8.05 (d, J ) 7.3 Hz, 1H), 8.51 (dd, J ) 1.5, 4.4 Hz,
2H), 8.57 (1.1, J ) 5.1 Hz, 1H); MS (CI/NH3) m/e 259 (M +
H). Anal. (C18H14N2‚0.2H2O) C, H, N.
5-[(2-Flu or o-4-pyr idin yl)m eth yl]-5-(4-pyr idin ylm eth yl)-
5H-in d en o[1,2-b]p yr id in e (5). This compound was prepared
from 20 (180 mg, 0.697 mmol) and 11 (0.794 mmol from the
crude chlorinated mixture) using sodium hydride (60% in oil,
0.997 mmol, 40 mg) in THF in the same manner described for
2. Purification via column chromatography (ethyl acetate) and
recrystallization from methylene chloride/hexane provided 5
as white crystals, 125 mg, 49% yield: mp 164-5 °C; 1H NMR
(300 MHz, CDCl3) δ 3.40 (s, 2H), 3.46 (d, J ) 4 Hz, 2H), 6.13
(s, 1H), 6.34 (dt, J ) 1.5, 5.1 Hz, 1H), 6.53 (d, J ) 6 Hz, 2H),
7.20 (dd, J ) 4.8, 7.7 Hz, 1H), 7.41 (dt, J ) 1.1, 7.3 Hz, 1H),
7.51 (dt, J ) 1.7, 7.3 Hz, 1H), 7.60 (d, J ) 6.8 Hz, 1H), 7.70
(dd, J ) 1.8, 7.7 Hz, 1H), 7.73 (d, J ) 5.1 Hz, 1H), 7.76 (d, J
) 7.3 Hz, 1H), 8.17 (d, J ) 6 Hz, 2H), 8.47 (dd, J ) 1.5, 5.1
Hz, 1H); IR (KBr) 1610, 1568, 1558, 1452, 1412, 824, 750 cm-1
;
MS (CI/NH3) m/e 368 (M + H). Anal. (C24H18FN3‚0.25H2O)
C, H, N.
10-[(2-Flu or o-4-pyr idin yl)m eth yl]-10-(4-pyr idin ylm eth -
yl)-9(10H)-a n th r a cen on e (6). To a mechanically stirred
slurry of 1.94 g (10 mmol) of anthrone, 1.64 g (10 mmol) of
4-picolyl chloride hydrochloride, 1.46 g (10 mmol) of 2-fluoro-
4-(chloromethyl)pyridine, and 0.2 g (0.87 mmol) of benzyltri-
ethylammonium chloride in 50 mL of toluene was added 5 mL
of 50% NaOH via addition funnel over a period of 15 min. After
addition, the mixture was stirred at room temperature for 3
days. The mixture was then diluted with 50 mL of water and
diluted with ethyl acetate. The organic layer was washed with
water and brine, dried over magnesium sulfate, and evapo-
rated in vacuo to give a dark-brown oil. The crude product
was dissolved in methylene chloride, chromatographed on
silica gel with 1:1 ethyl acetate/hexane, and recrystallized from
methylene chloride/hexane to give 6 as beige crystals, 468 mg,
12% yield: mp 199-201 °C; 1H NMR (300 MHz, CDCl3) δ 3.76
(s, 2H), 3.81 (s, 2H), 5.89 (s, 1H), 6.11 (d, J ) 5 Hz, 1H), 6.22
(d, J ) 5 Hz, 2H), 7.48 (t, J ) 7 Hz, 2H), 7.64 (d, J ) 5 Hz,
1H), 7.85 (m, 2H), 8.05 (s, 3H), 8.07 (s, 1H), 8.17 (d, J ) 9 Hz,
2H); IR (KBr) 3030, 2950, 1654, 1600, 1558, 1460, 1412, 1322,
3-[(2-Flu or o-4-pyr idin yl)m eth yl]-3-(4-pyr idin ylm eth yl)-
1,3-d ih yd r o-1-p h en yl-2H-in d ol-2-on e (4). This compound
was prepared from 1744,45 (710 mg, 2.36 mmol) and 11 (2.69
mmol from the crude chlorinated mixture) using sodium
hydride (60% in oil, 3.37 mmol, 135 mg) in THF in the same
manner described for 2. Purification via column chromatog-
raphy (2:1 hexane/ethyl acetate) and recrystallization from
methylene chloride/hexane provided 4 as white crystals, 318
1
mg, 33% yield: mp 174-6 °C; H NMR (300 MHz, CDCl3) δ
3.22 (d, J ) 12 Hz, 1H), 3.26 (d, J ) 12 Hz, 1H), 3.46 (d, J )
13 Hz, 1H), 3.52 (d, J ) 13 Hz, 1H), 6.29 (d, J ) 8 Hz, 1H),
6.52 (s, 1H), 6.63 (d, J ) 10 Hz, 2H), 6.80 (d, J ) 5 Hz, 1H),
6.85 (d, J ) 6 Hz, 2H), 7.10 (m, 2H), 7.34 (m, 2H), 7.43 (d, J
) 8 Hz, 2H), 7.93 (d, J ) 5 Hz, 1H), 8.30 (d, J ) 6 Hz, 2H); IR
(KBr) 3394, 3070, 2928, 1708, 1612, 1564, 1502, 1466, 1412,
1382, 1282, 1270, 1238, 1220, 768 cm-1; MS (CI/NH3) m/e 410
(M + H). Anal. (C26H20FN3O) C, H, N.
1152, 816 cm-1; MS (CI/NH3) m/e 395 (M + H). Anal. (C26H19
FN2O) C, H, N.
-
3,3-Bis[(2-flu or o-4-p yr id in yl)m et h yl]-1,3-d ih yd r o-1-
p h en yl-2H-in d ol-2-on e (7). This compound was prepared
from 1640 (2.09 g, 10 mmol) and 11 (22 mmol from the crude
chlorinated mixture) using sodium hydride (60% in oil, 40
mmol, 960 mg) in THF in the same manner described for 2.
Purification via column chromatography (2% methanol in
chloroform) and recrystallization from ethyl acetate/hexane
provided 7 as pale-yellow crystals, 1.77 g, 42% yield: mp
143-5 °C; 1H NMR (300 MHz, CDCl3) δ 3.25 (d, J ) 12.8 Hz,
2H), 3.53 (d, J ) 12.8 Hz, 2H), 6.34 (d, J ) 7.3 Hz, 1H), 6.50
(s, 2H), 6.65 (m, 2H), 6.80 (d, J ) 5.1 Hz, 2H), 7.17 (m, 2H),
7.37 (m, 4H), 7.95 (d, J ) 5.1 Hz, 2H); MS (CI/NH3) m/e 428
(M + H). Anal. (C26H19F2N3O) C, H, N.
5,5-Bis[(2-flu or o-4-p yr id in yl)m eth yl]-5H-in d en o[1,2-b]-
p yr id in e (8). This compound was prepared from 1441,42 (1.22
g, 7.3 mmol) and 13 (16 mmol from the iodination mixture)
using sodium hydride (60% in oil, 25 mmol, 1.0 g) in THF in
the same manner described for 2. Purification via column
chromatography (1.5% methanol/chloroform) and recrystalli-
zation from ether/petroleum ether provided 8 as white crystals,
5-(4-P yr idin ylm eth ylen e)-5H-in den o[1,2-b]pyr idin e (19).
A mixture of 4-azafluorenone (18) (15.21 g, 84 mmol), 4-pico-
line (88 mmol, 8.21 g, 8.6 mL), and acetic acid (8.4 mL) in 100
mL of acetic anhydride was heated at 130 °C for 24 h. Excess
acetic anhydride was removed in vacuo, and the residue was
partitioned between methylene chloride and saturated sodium
carbonate. The organic layer was washed with water and
brine, and dried over magnesium sulfate. The solvent was
removed under reduced pressure, and the residue was purified
via column chromatography (silica gel, 10% THF in methylene
chloride) and recrystallized from methylene chloride/hexane
1
to give 19 as pink plates, 6.0 g, 28% yield: mp 194-6 °C; H
NMR (300 MHz, DMSO-d6) δ 7.18 (dd, J ) 4.8, 8.1 Hz, 1H),
7.54 (m, 2H), 7.60 (d, J ) 5.5 Hz, 2H), 7.69 (dd, J ) 1.5, 7.7
Hz, 1H), 7.94 (m, 1H), 8.04 (s, 1H), 8.12 (m, 1H), 8.53 (dd, J )

ACh Release-Enhancing Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4621
1.60 g, 57% yield: mp 137-41 °C; 1H NMR (300 MHz, CDCl3)
δ 3.43 (d, J ) 13 Hz, 2H), 3.49 (d, J ) 13 Hz, 2H), 6.14 (s,
2H), 6.35 (dt, J ) 1.5, 3.3 Hz, 2H), 7.22 (dd, J ) 5.1, 7.7 Hz,
2H), 7.43 (dt, J ) 1.1, 7.3 Hz, 1H), 7.53 (dt, J ) 1.1, 7.3 Hz,
1H), 7.60 (d, J ) 7.3 Hz, 1H), 7.75 (m, 4H), 8.48 (dd, J ) 1.5
Hz, 4.8H); MS (CI/NH3) m/e 386 (M + H). Anal. (C24H17N3F2)
C, H, N.
10,10-Bis[(2-flu or o-4-p yr id in yl)m eth yl]-9(10H)-a n th r a -
cen on e (9). The title compound was prepared from anthrone
(5.0 g, 26 mmol) and 13 (57 mmol from the iodination mixture)
using sodium hydride (60% in oil, 57 mmol, 2.28 g) in THF in
the same manner described for 2. Purification via column
chromatography (1:1 ethyl acetate/hexane) and recrystalliza-
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tion from ether/methylene chloride provided
9 as white
crystals, 6.17 g, 58% yield: mp 159-61 °C; 1H NMR (300 MHz,
CDCl3) δ 3.76 (s, 4H), 5.85 (s, 2H), 6.04 (t, J ) 3 Hz, 2H), 7.50
(t, J ) 8 Hz, 2H), 7.63 (d, J ) 5 Hz, 2H), 7.83 (t, J ) 8 Hz,
2H), 7.97 (d, J ) 8 Hz, 2H), 8.16 (d, J ) 8 Hz, 2H); IR (KBr)
3068, 3032, 2940, 1660, 1604, 1558, 1460, 1410, 1326, 1272,
936, 702 cm-1; MS (CI/NH3) m/e 413 (M + H). Anal.
(C26H18F2N2O) C, H, N.
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Acetylch olin e Relea se Assa y. These experiments were
performed as described previously.47
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Wuonola, M. A.; Boswell, G. A.; Wilkerson, W. W.; Nickolson,
V. J .; Tam, S. W.; Brittelli, D. R.; Chorvat, R. J .; Zaczek, R.;
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In Vivo Micr od ia lysis Assa y. Compounds were evaluated
for their ability to enhance the release of acetylcholine in vivo
as previously described.55
P h a r m a cok in etic Stu d ies. Pharmacokinetic parameters
were determined in male CD rats that received a 20-h or 7-day
dosing of 9 at 1 or 4 mg/kg/day orally in 0.25% methylcellulose
suspension or 4 mg/kg/day iv. For the iv study, 9 was dissolved
in propylene glycol and administered through ALZET pumps
implanted in a jugular cannula at an infusion rate of 8 or 10
µL/h. At 0.5, 1, 2, 4, 8, and 16 h after dosing, rats were
sacrificed and blood samples were collected into tubes contain-
ing EDTA. Blood and brain tissue were collected at the end
of the infusion. Plasma was separated by centrifugation and
stored frozen until analysis. Compounds were extracted from
plasma by simple liquid-liquid extraction. LC/MS/MS analy-
sis was performed on a Sciex (Thornhill, Ontario) model APIIII
triple quadrapole mass spectrometer interfaced with a turbo
ion spray ionization source. The liquid chromatography
consisted of a Perkin-Elmer series 200 solvent delivery system
(Norwalk, CT), a Perkin-Elmer ISS 200 autoinjector, and a
Waters Symmetry octyl minibore column (2.1 × 50 mm).
Ack n ow led gm en t. We wish to thank Melvyn J .
Myers, Spencer Drummond, J r., Constantine Demopou-
los, J ason S. Xiang, Shyam N. Hirwe, Philip L. Saxton,
and Cecelia L. Chi for technical assistance and Lauren
E. Richards and Wendell W. Wilkerson for pertinent
discussions.
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(56) Unpublished results.
J M9803424