Synthesis and evaluation of oxime derivatives as modulators for amyloid beta-induced mitochondrial dysfunction

Article abstract of DOI:10.1016/j.ejmech.2012.12.033

Starting from quinuclidinyl oxime 1 identified by preliminary screening, a series of azacycles-containing oxime derivatives was synthesized. Their mPTP blocking activities were evaluated by a JC-1 assay, measuring the change of mitochondrial membrane potential. The inhibitory activity of nine compounds against amyloid beta-induced mPTP opening was comparable or even superior to that of piracetam. Among them, 12d effectively maintained mitochondrial function and cell viabilities on the ATP assay, the MTT assay, and the ROS assay. In addition, it exhibited favorable in vitro stability and pharmacokinetic characteristics, which hold a promise for further development of AD therapeutics.

Full text of DOI:10.1016/j.ejmech.2012.12.033

European Journal of Medicinal Chemistry 62 (2013) 71e83
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of oxime derivatives as modulators for
amyloid beta-induced mitochondrial dysfunction
,
,
,
Young Seub Kim a b, Sun hwa Jung a c, Beoung-Geon Park a d, Min Kyung Ko a,
Hyun-Seo Jang a, Kihang Choi c, Ja-Hyun Baik d, Jiyoun Lee e, Jae Kyun Lee a,
Ae Nim Pae a b, Yong Seo Cho a , Sun-Joon Min
,
,
b,
a,b,
*
*
a Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu,
Seoul 136-791, South Korea
b Biomolecular Science, University of Science and Technology (UST), 176 Gajung-dong, 217 Gajungro, Yuseong-gu, Daejeon 305-333, South Korea
c Department of Chemistry, Korea University, Seoul 136-701, South Korea
d School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea
e Department of Global Medical Science, Sungshin Women’s University, Seoul 142-732, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from quinuclidinyl oxime 1 identified by preliminary screening, a series of azacycles-containing
oxime derivatives was synthesized. Their mPTP blocking activities were evaluated by a JC-1 assay,
measuring the change of mitochondrial membrane potential. The inhibitory activity of nine compounds
against amyloid beta-induced mPTP opening was comparable or even superior to that of piracetam.
Among them, 12d effectively maintained mitochondrial function and cell viabilities on the ATP assay, the
MTT assay, and the ROS assay. In addition, it exhibited favorable in vitro stability and pharmacokinetic
characteristics, which hold a promise for further development of AD therapeutics.
Received 8 September 2012
Received in revised form
13 December 2012
Accepted 17 December 2012
Available online 26 December 2012
Keywords:
Ó 2012 Elsevier Masson SAS. All rights reserved.
Alzheimer’s disease
Mitochondrial permeability transition pore
(mPTP)
Oxime derivatives
Amyloid beta
Pharmacokinetics
1. Introduction
accumulated in mitochondria of the AD brain, which is responsible
for mitochondrial dysfunction and oxidative damage ultimately
Alzheimer’s disease (AD) is a progressive, neurodegenerative
disorder, characterized by an age-dependent loss of memory and an
impairment of multiple cognitive functions [1]. Although the
mechanism of AD pathogenesis is largely unknown, deposition of
resulting in structural and functional damage of AD neurons [4].
Therefore, modulation of mitochondrial proteins related to amyloid
beta-induced mitochondrial dysfunction might be viable thera-
peutic strategies for the prevention and treatment of AD.
extracellular amyloid beta (A
b
) plaques and formation of intracel-
The effects of amyloid beta on mitochondria have not been fully
elucidated, but recent reports show that mitochondrial perme-
ability transition pore (mPTP) is involved in mitochondrial
dysfunction induced by amyloid beta toxicity [5]. The mPTP is
a multiprotein complex that is formed in the inner membrane of
the mitochondria under certain pathological conditions such as
oxidative stress, ischemia, stroke, and traumatic brain injury [6].
The opening of the mPTP allows uncontrolled transport in and out
of mitochondria for molecules with molecular weight of less than
1.5 kDa. Structurally, it comprises three major components:
a voltage-dependent anion channel (VDAC) in the outer membrane,
the adenine nucleotide translocator (ANT) in the inner membrane,
and cyclophilin D (CypD) in the mitochondrial matrix. In the
lular neurofibrillary tangles (NFT’s) in brain of AD patients sug-
gested that amyloid beta plays a pivotal role in the pathophysiology
of AD [2]. It has been studied that amyloid beta is associated with
a diverse range of neuronal properties including loss of neuron,
synapses and synaptic function, mitochondrial abnormalities and
inflammatory responses [3]. Despite its various impacts on
neurons, increasing evidence indicated that amyloid beta is
* Corresponding authors. Center for Neuro-Medicine, Brain Science Institute,
Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu,
Seoul 136-791, South Korea.
E-mail addresses: ys4049@kist.re.kr (Y.S. Cho), sjmin@kist.re.kr (S.-J. Min).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.033

72
Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
normal state of mPTP, reversible opening and closing of the mPTP is
regulating the membrane potential, which maintains calcium
homeostasis in the cell. However, an accumulation of soluble
amyloid beta in AD neurons induces excessive calcium entry into
cytosol resulting in elevated calcium level of mitochondria. High
localization of calcium in mitochondria promotes the opening of
the mPTP, which causes severe mitochondrial swelling, outer
membrane rupture and incapability of ATP production. Finally,
a release of proapoptotic proteins accompanied by mPTP opening
leads to neuronal cell death [7e9].
Cyclosporin A (CsA), a cyclic nonribosomal peptide clinically
used as an immunosuppressant, specifically inhibits the mPTP by
binding to CypD in the matrix side of the inner mitochondrial
membrane [10,11]. N-Methyl-4-isoleucin cyclosporin A (NIM811),
a non-immunosuppressant analog of CsA, is also reported as
a potent inhibitor of the mPTP in isolated liver mitochondria [12].
Although these compounds are effectively blocking the activity of
the mPTP, they are not useful for therapeutics because the delivery
of peptide to the brain is limited by poor bioavailability and low BBB
(blood brain barrier) penetration. On the other hand, it has been
reported that Dimebon, an antihistamine drug, blocks the mPTP
opening and protects neurons against amyloid beta-induced cell
death although the mechanism of action is not completely under-
stood [13e15]. Currently, however, there are no safe and selective
mPTP blockers available for therapeutic application to AD treat-
ment. Therefore, given the increasing evidence of protective role of
mPTP inhibition in regulating amyloid beta-induced mitochondrial
dysfunction, nonpeptidyl mPTP blockers should be further devel-
oped for their potential as pharmacological therapeutics in clinical
treatment of AD. Herein, we report the discovery of novel oxime
derivatives that inhibit the activity of the mPTP, which is a potential
drug target for treatment of AD.
2. Results and discussion
Initially, we identified quinuclidinyl oxime 1 as an mPTP blocker
by screening the KIST chemical library (Fig. 1). The activity to block
the mPTP opening induced by amyloid beta was evaluated using
a cell-based JC-1 assay by measuring the change of mitochondrial
membrane potential [16,17]. The color shift from green to red as the
membrane potential increases indicates the recovery of mito-
chondrial function responding to applied amyloid beta toxicity. In
fact, compound 1 exhibited 59% of increased green to red ratio,
which means that it was able to reduce 41% of mitochondrial
damage induced by amyloid beta. Along with this good in vitro
inhibitory activity against mPTP opening, the in vitro stability of 1
on plasma and microsomes was examined to show that it has good
plasma and metabolic stability. However, the pharmacokinetic
parameters for compound 1 following intraperitoneal and oral
administration in rat and mice suggested that the level of drug
exposure (AUC) is not appropriate for therapeutic agents although
compound 1 exhibited good permeability to the brain tissue.
On the basis of the preliminary data of the hit compound 1, we
decided to synthesize the analogs of compound 1 to improve
pharmacokinetic profile as well as in vitro efficacy. To verify the
effect of the 2-benzyloxyphenyl moiety on mPTP blocking activity,
quinuclidinyl oxime derivatives containing
a wide range of
substituents on the aromatic region were initially synthesized as
shown in Scheme 1. Quinuclidine hydroxylamine hydrochloride 4
was synthesized by Mitsunobu reaction and hydrazinolysis fol-
lowed by salt formation. Next, we performed condensation reaction
of 4 with either commercially available substituted benzaldehydes
or the substituted benzyloxybenzaldehydes, easily prepared from
O-alkylation of various benzyl halides with salicylaldehyde. The JC-
1 assay data of the synthesized oxime derivatives 5 and 6 were
JC-1 assay: 59% (increased g/r ratio @ 5 µM)
Plasma stability: < 15% for 2 h
HLM stability: 85% (% remaining @ 1 h)
PK parameters
PO (10 mg/kg, ICR mice, n = 3)
IP (3 mg/kg, SD male rats, n = 3)
AUC0– (µg min/ml)
AUClast (µg min/ml)
Terminal half-life (min)
Cmax (µg/ml)
-
59.41 8.19
55.77 7.79
365.8 1.0
0.524 0.036
25(19-31) b
-
31.49
-
0.330
Tmax (min)
5
B/P ratio (%)
273.0 33.4
AUC0 , total area under the plasma concentration–time curve from time zero to time infinity; AUClast, total area
under the plasma concentration–time curve from time zero to last measured time; Cmax, peak plasma
concentration; Tmax, time to reach Cmax: PO, oral administration; IP, intraperitoneal administration; B/P ratio,
brain to plasma ratio
Fig. 1. Structure and pharmacological profile of compound 1.

Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
73
Scheme 1. Synthesis of the quinuclidinyl oxime derivatives 5 and 6.
determined and listed in Table 1. Unfortunately, we observed that
the lowest increased green/red ratio in the JC-1 assay was only 69%
(6j, R1 ¼ 2,4-diCl), indicating the mPTP blocking activities of 5aek
and 6aem are less potent than that of 1. This result suggested that
the aromatic moiety in this series may have a negative effect on the
inhibitory activity against mPTP opening (Table 1).
beta-induced mPTP opening. Among the tested compounds, the
inhibitory activities of nine compounds were comparable or even
superior to that of piracetam. While the compounds having the
simple substituents such as methoxy and trifluoromethyl on the
phenyl ring (10e13a/b series) showed the low inhibitory activities,
the oxime derivatives derived from the substituted benzylox-
ybenzaldehydes (10e13cef series) displayed excellent blocking
activities up to 18% of increased green/red ratio (10f).
To complement the JC-1 assay, we performed a luciferase-based
assay for cellular ATP levels to validate the effect of the selected
compounds on mitochondrial function [20]. Because a main role for
the mitochondria is the production of ATP, a compound which is
able to block mPTP opening damaged by amyloid beta should
eventually repair mitochondrial dysfunction by promoting ATP
generation. As shown in Table 3, all the tested compounds did not
exhibit significant cell toxicity. However, only compound 12d,
bearing two chlorine atoms at the 3,4-position of benzyloxy group,
possessed highest inhibitory activity against amyloid beta-induced
ATP reduction as well as reasonable cell viability, which are
comparable to those of piracetam. Thus, we selected compound
12d as a lead compound for further investigation.
In order to further investigate the structure activity relationship
(SAR), we then decided to modify the other side of compound 1 by
replacing the quinuclidine segment with monocyclic tertiary
amines such as N-alkyl-pyrrolidines or piperidines. It was known
that such a simple azacycle could be a stable precursor for quinu-
clidine synthesis or a common intermediate of quinuclidine
metabolism [18]. Thus, the second class of oxime derivatives 10e13
was synthesized as shown in Scheme 2. Following the similar
procedure as described above, we synthesized various pyrrolidine/
piperidine hydroxyl amines 9aed as hydrogen chloride salt forms,
which were subjected to condensation reaction with substituted
benzaldehydes to afford the compounds 10e13. In this event,
considering the substituent effect of the phenyl ring moiety in 5
and 6 on the mPTP blocking activity, we selected six different
benzaldehydes for this transformation. The result of JC-1 assay of
the second series is summarized in Table 2. In addition to CsA,
piracetam was also used as a control because it has been reported
that it was able to restore mitochondrial dysfunction and neurite
reduction associated with amyloid beta [19]. Compared to the
quinuclidine oxime derivatives 5 and 6, some pyrrolidine/piperi-
dine analogs 10e13 showed high blocking activity against amyloid
In addition to the JC-1 assay and the luciferase-based assay, we
executed the MTT assay for measuring mitochondrial dehydroge-
nase activity and a CM-H2DCFDA-fluorescenet assay for detecting
cellular reactive oxygen species (ROS) resulting from amyloid beta
toxicity [21,22]. In fact, it was found that the viability of cells treated
with amyloid beta was ameliorated by compound 12d. We also
Table 1
In vitro blocking activity of quinuclidinyl oximes 5 and 6 against amyloid beta-induced mPTP opening (JC-1 assay).
Compds
R1
Increased g/r ratio (%)a
Compds
R1
Increased g/r ratio (%)a
Compds
R1
Increased g/r ratio (%)a
5a
5b
5c
5d
5e
5f
5g
5h
CsA
3-PhO
3-F
3-Br
3-MeO
3-PhCH2O
3-CF3
2-PhO
2-F
138
92
95
86
121
74
84
96
46
5i
5j
2-Br
2-MeO
2-CF3
2-Cl
3-Cl
4-Cl
2-F
93
71
86
88
101
85
6f
4-F
93
96
96
77
69
91
90
85
6g
6h
6i
6j
6k
6l
4-MeO
2,6-diCl
3,4-diCl
2,4-diCl
2-CF3
5k
6a
6b
6c
6d
6e
82
75
3-CF3
4-CF3
3-F
6m
a
% Increase of fluorescence-ratio (green/red) after treatment of each compound and amyloid beta with regard to that of amyloid beta alone (100%). See the text for more
detailed information.

74
Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
Scheme 2. Synthesis of pyrrolidinyl/piperidinyl oxime derivatives 10e13.
observed that 12d reduced amyloid beta-induced ROS production
with 82% inhibition value. These data suggested that compound
12d is a potential mPTP blocker maintaining mitochondrial func-
tion and cell viability (Table 4).
a pyrrolidine ring (compound 12d) significantly enhanced the
in vitro affinity while improving the stability and PK profile.
3. Conclusion
Finally, the pharmaceutical properties of compound 12d were
examined as shown in Tables 5 and 6. The stability assays showed
that 12d was not decomposed in rat plasma for 2 h and it remained
up to 48% at 30 min after treatment of 12d in pooled human hepatic
microsomes. This result indicated that compound 12d has good
plasma and microsomal stability. The pharmacokinetic studies of
12d on intravenous and oral administration in rats showed that all
the PK parameters of this compound were highly improved in
comparison of those of compound 1. In particular, compound 12d
showed high level of drug exposure, moderate clearance, and good
oral bioavailability. Furthermore, the high ratio of brain to plasma
concentration at 2 h after iv or oral administration (14.58 and 0.29)
turned out that 12d penetrated into brain tissue effectively.
Therefore, replacement of the quinuclidine ring in compound 1 by
In this study, we identified compound 12d, a potent mPTP
blocker, starting from quinuclidinyl oxime hit compound
1
screened by the JC-1 assay. The replacement of substituents on the
aromatic ring of compound 1 did not improve the inhibitory
activity against amyloid beta-induced mPTP opening. However, the
introduction of monocyclic azacycles such as pyrrolidine and
piperidine as a quinuclidine surrogate served to increase the in vi-
tro efficacy significantly. In particular, compound 12d having N-
benzylpyrrolidine showed good in vitro profiles with regard to the
assays associated with mitochondrial functions and cell viabilities.
Moreover, we found that the in vitro safety and PK properties of
compound 12d were dramatically improved by such a structural
change. Therefore, compound 12d, a potential mPTP blocker, can be
Table 2
The result of the JC-1 assay.
Compds
R1
R2
Increased g/r ratio (%)a
Compds
R1
R2
Increased g/r ratio (%)a
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
11e
11f
Me
Me
Me
Me
Me
Me
iPr
2-MeO
3-CF3
H
3,4-diCl
3-F
2,4-diCl
2-MeO
3-CF3
H
3,4-diCl
3-F
147
108
69
57
42
18
55
54
19
95
39
61
12a
12b
12c
12d
12e
12f
13a
13b
13c
13d
13e
13f
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
PhCH2
Me
Me
Me
Me
Me
2-MeO
3-CF3
H
3,4-diCl
3-F
2,4-diCl
2-MeO
3-CF3
H
3,4-diCl
3-F
81
123
38
41
42
61
257
84
29
62
iPr
iPr
iPr
iPr
32
64
iPr
2,4-diCl
Me
2,4-diCl
Piracetam
40
a
% Increase of fluorescence-ratio (green/red) after treatment of each compound and amyloid beta compared to that treated with amyloid beta alone (100%). See the text for
more detailed information.

Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
75
Table 3
The result of ATP assay of the selected compounds.
Compds
1
10e
10f
11c
11e
12c
12d
12e
13c
13e
Piracetam
% Recoverya (%)
Viability
13
92
20
87
ꢂ9
ꢂ93
ꢂ223
ꢂ137
104
83
ꢂ201
15
112
9
136
127
88
90
67
94
99
74
a
% Recovery of ATP production at 5 mM of each compound against amyloid beta-suppressed mitochondrial ATP reduction.
considered as a promising lead for further development of new AD
therapeutics.
temperature. The precipitate was filtered off through a pad of
CeliteÒ and the filtrate was washed with brine. The organic layer
was treated with 10% HCl solution and concentrated under reduced
pressure. The residual solid was filtered and recrystallized in
ethanol to give O-(quinuclidin-3-yl)hydroxylamine hydrogen
4. Experimental section
chloride 4 (3.86 g, 46%) as a solid. 1H NMR (CD3OD, 400 MHz)
d 4.67
4.1. Chemistry
(m, 1H), 3.78 (m, 1H), 3.30e3.40 (m, 5H), 2.60 (m, 1H), 2.15 (m, 2H),
1.92 (m, 2H).
General: All reactions were carried out under dry nitrogen
unless otherwise indicated. Commercially available reagents were
used without further purification. Solvents and gases were dried
according to standard procedures. Organic solvents were evapo-
rated with reduced pressure using a rotary evaporator. Analytical
thin layer chromatography (TLC) was performed using glass plates
precoated with silica gel (0.25 mm). TLC plates were visualized by
exposure to UV light (UV), and then were visualized with a p-ani-
saldehyde stain followed by brief heating on hot plate. Flash
column chromatography was performed using silica gel 60 (230e
400 mesh, Merck) with the indicated solvents. 1H and 13C spectra
were recorded on Bruker 300, Bruker 400 or Varian 300 NMR
spectrometers. 1H NMR spectra are represented as follows: chem-
ical shift, multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet), integration, and coupling constant (J)
in Hertz (Hz). 1H NMR chemical shifts are reported relative to CDCl3
(7.26 ppm). 13C NMR was recorded relative to the central line of
CDCl3 (77.0 ppm).
4.1.1.3. 2-(1-Methylpyrrolidin-3-yloxy)isoindoline-1,3-dione
(8a).
Following the same procedure used for the synthesis of 3, the
reaction of 1-methyl-3-hydroxypyrrolidine (0.54 mL, 4.94 mmol),
diisopropyl azodicarboxylate (1.95 mL, 9.89 mmol), N-hydroxyph-
thalimide (1.29 g, 7.91 mmol), and triphenylphosphine (2.59 g,
9.89 mmol) in THF (24 mL) at 0 ꢀC gave the title compound 8a
(1.17 g, 97%). 1H NMR (CDCl3, 300 MHz)
d 7.51 (m, 2H), 7.42 (m, 2H),
3.94 (m, 1H), 3.67 (m, 1H), 3.42 (m, 2H), 2.99 (s, 3H), 2.97 (m, 1H),
2.41 (m, 1H).
4.1.1.4. 2-(1-Isopropylpyrrolidin-3-yloxy)isoindoline-1,3-dione (8b).
Following the same procedure used for the synthesis of 3, the
reaction of 1-isopropyl-3-pyrrolidinol (0.52 mL, 3.87 mmol), dii-
sopropyl azodicarboxylate (1.52 mL, 7.74 mmol), N-hydroxyph-
thalimide (1.01 g, 6.19 mmol), and triphenylphosphine (2.03 g,
7.74 mmol) in THF (19 mL) at 0 ꢀC gave the title compound 8b
(990 mg, 93%). 1H NMR (CDCl3, 300 MHz)
d 7.79 (m, 2H), 7.72 (m,
2H), 4.88 (m, 1H), 3.09 (m, 1H), 2.99 (m, 2H), 2.94 (m, 1H), 2.92 (m,
1H), 2.15 (m, 2H), 1.14 (dd, J ¼ 3.2, 6.3 Hz, 6H).
4.1.1. Synthesis of hydroxylamine hydrogen chloride
4.1.1.1. Representative procedure for synthesis of 2-(quinuclidin-3-
yloxy)isoindoline-1,3-dione (3). To a solution of 3-quinuclidinol 2
(1.00 g, 7.86 mmol) in THF (39 mL) cooled to 0 ꢀC were added
diethyl azodicarboxylate (2.47 mL, 15.7 mmol), N-hydroxyph-
thalimide (2.05 g, 12.6 mmol), and triphenylphosphine (4.12 g,
15.7 mmol). After the reaction mixture was stirred for 6 h (moni-
tored by TLC), it was treated with H2O (40 mL) and extracted with
chloroform (3 ꢁ 30 mL). The combined organic layer was washed
with brine, dried over magnesium sulfate, and concentrated under
reduced pressure. The resulting crude oil was purified by column
chromatography on silica gel (ethyl acetate/hexane ¼ 15:1) to yield
2-(quinuclidin-3-yloxy)isoindoline-1,3-dione 3 (1.80 g, 85%) as an
4.1.1.5. 2-(1-Benzylpyrrolidin-3-yloxy)isoindoline-1,3-dione
(8c).
Following the same procedure used for the synthesis of 3, the
reaction of 1-benzyl-3-pyrrolidinol (0.47 mL, 2.82 mmol), diethyl
azodicarboxylate (0.89 mL, 5.64 mmol), N-hydroxyphthalimide
(740 mg, 4.51 mmol), and triphenylphosphine (1.48 g, 5.64 mmol)
in THF (14 mL) at 0 ꢀC gave the title compound 8c (766 mg, 84%). 1H
NMR (CDCl3, 300 MHz)
d 7.78 (m, 2H), 7.70 (m, 2H), 7.18e7.33 (m,
5H), 4.90 (m, 1H), 2.89 (d, J ¼ 4.2 Hz, 2H), 2.84 (m, 1H), 2.58 (m, 1H),
2.14 (m, 2H).
4.1.1.6. 2-((1-Methylpiperidin-3-yl)oxy)isoindoline-1,3-dione
(8d).
oil. 1H NMR (CDCl3, 400 MHz)
d 7.80 (m, 2H), 7.74 (m, 2H), 4.15 (m,
Following the same procedure used for the synthesis of 3, the
reaction of N-methyl-3-piperidinol (0.5 mL, 4.34 mmol), diisopropyl
azodicarboxylate (1.71 mL, 8.68 mmol), N-hydroxyphthalimide
(1.13 g, 6.95 mmol), and triphenylphosphine (2.28 g, 8.68 mmol) in
THF (22 mL) at 0 ꢀC gave the title compound (1.00 g, 88%). 1H NMR
1H), 3.21 (m, 1H), 2.97e3.05 (m, 2H), 2.79e2.85 (m, 2H), 2.66 (m,
1H), 2.11 (m, 1H), 1.41 (m, 1H), 1.23 (m, 3H).
4.1.1.2. Representative procedure for synthesis of O-(quinuclidin-3-yl)
hydroxylamine hydrogen chloride (4). To a solution of 2-(quinucli-
din-3-yloxy)isoindoline-1,3-dione 3 (10.6 g, 39.3 mmol) in ethanol
(131 mL) was added hydrazine monohydrate (3.57 mL, 59.0 mmol).
The reaction mixture was stirred at 80 ꢀC for 3 h and cooled to room
(CDCl3, 300 MHz) d 7.51 (m, 2H), 7.42 (m, 2H), 3.94 (m, 1H), 3.67 (m,
1H), 3.42 (m, 2H), 2.99 (s, 3H), 2.97 (m, 1H), 2.41 (m, 1H).
4.1.1.7. O-(1-Methylpyrrolidin-3-yl)hydroxylamine hydrogen chloride
(9a). Following the same procedure used for the synthesis of 4, the
Table 4
The result of MTT assay and ROS assay of 12d.
MTT
ROS
Table 5
Plasma and microsomal stability of 12d.
% Inhibition (against
Viability
% Inhibition (against
A
b
induced toxicity)
Ab induced ROS)
Plasma stability
(rat, % remaining at 2 h)
Microsomal stability
(HLM,a % remaining at 30 min)
1
12d
Piracetam
0%
84%
129%
132%
64%
82%
129%
149%
29%
12d
111.17% (ꢃ3.0)
47.81% (ꢃ1.92)
a
HLM, human liver microsomes.

76
Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
Table 6
concentrated under reduced pressure. The crude oil was purified by
column chromatography on silica gel (CHCl3/MeOH ¼ 10:1) to give
the title compound 1 (924 mg, 92%) as a solid. 1H NMR (CDCl3,
Mean (ꢃSDa) pharmacokinetic parameters after intravenous (n ¼ 3) and oral (n ¼ 4)
administration (10 mg/kg) of 12d to SD male rats.
Plasma
Intravenous
Oral
300 MHz)
d
8.56 (s, 1H), 7.81 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.28e7.44 (m,
AUC0eN
AUClast
(
m
m
g min/mL)
376.55 ꢃ 98.10
353.17 ꢃ 89.08
140.97 ꢃ 41.01
44.53 ꢃ 4.59
e
143.30 ꢃ 36.56
117.33 ꢃ 41.99
175.35 ꢃ 48.99
0.57 ꢃ 0.30
120 ꢃ 0
6H), 6.98 (m, 2H), 5.10 (s, 2H), 4.37 (m, 1H), 3.18 (m, 1H), 2.75e2.93
(m, 6H), 2.23 (dd, J ¼ 6.3, 3.1 Hz, 1H), 1.85 (m, 1H), 1.70 (m, 1H), 1.56
(
g min/mL)
Terminal half-life (min)
Cmax g/mL)
(m, 1H), 1.35 (m, 1H); 13C NMR (CDCl3, 75 MHz)
d 156.6, 144.9, 136.6,
(
m
131.0, 128.6, 128.1, 127.4, 126.5, 121.4, 121.1, 112.5, 78.9, 70.4, 54.4,
47.7, 47.0, 25.3, 24.4, 19.2; LC/MS (ESIþ): m/z: calcd for C21H24N2O2:
336.18, [M þ H]þ; found: 337.49.
Tmax (min)
CL (mL/min/kg)
28.05 ꢃ 8.58
72.13 ꢃ 18.40
e
MRT (min)b
Vss (mL/kg)
Brain to plasma ratio at 2 h
F (%)
2870.90 ꢃ 528.42
14.58
e
0.29
4.1.2.2. 3-Phenoxybenzaldehyde O-quinuclidin-3-yl oxime (5a).
Following the general procedure, the reaction of O-(quinuclidin-3-
yl)hydroxylamine hydrogen chloride 4 (108 mg, 0.50 mmol), 3-
phenoxybenzaldehyde (0.12 mL, 0.75 mmol) and Na2CO3 (80 mg,
0.75 mmol) in methanol (5.0 mL) gave the title compound 5a
38.06%
AUC0eN, total area under the plasma concentrationetime curve from time zero to
time infinity; AUClast, total area under the plasma concentrationetime curve from
time zero to last measured time; Cmax, peak plasma concentration; Tmax, time to
reach Cmax; Cl, time-averaged total body clearance; MRT, mean residence time; Vss
apparent volume of distribution at steady state; F, bioavailability.
,
(97 mg, 60% yield). 1H NMR (CDCl3, 300 MHz)
d 8.09 (s, 1H), 7.27e
a
SD: standard deviations.
Median (range) for Tmax
7.42 (m, 5H), 7.11 (t, J ¼ 7.3 Hz, 1H), 6.97e7.07 (m, 3H), 4.37 (m, 1H),
3.20 (dd, J ¼ 14.0, 9.0 Hz, 1H), 2.71e2.97 (m, 5H), 2.22 (m, 1H), 1.85
(m, 1H), 1.70 (m, 1H), 1.55 (m, 1H), 1.35 (m, 1H); 13C NMR (CDCl3,
b
.
75 MHz)
d 157.5, 157.0, 148.4, 134.2, 130.1, 129.9, 123.5, 122.1, 120.2,
reaction of 2-(1-methylpyrrolidin-3-yloxy)isoindoline-1,3-dione 8a
(1.17 g, 4.75 mmol) and hydrazine monohydrate (0.53 mL,
7.13 mmol) in ethanol (15 mL) at 80 ꢀC followed by treatment of 10%
HCl (2.74 mL, 9.51 mmol) gave the title compound 9a (880 mg,
d 4.67 (m, 1H), 3.67 (m, 1H), 3.42
(m, 2H), 2.99 (s, 3H), 2.97 (m, 1H), 2.41 (m, 1H).
118.9, 117.1, 79.1, 54.3, 47.7, 47.0, 25.3, 24.2, 19.1; LC/MS (ESIþ): m/z:
calcd for C20H22N2O2: 322.17, [M þ H]þ; found: 323.68.
4.1.2.3. 3-Fluorobenzaldehyde O-quinuclidin-3-yl oxime (5b).
Following the general procedure, the reaction of O-(quinuclidin-3-
yl)hydroxylamine hydrogen chloride 4 (100 mg, 0.46 mmol), 3-
97%). 1H NMR (CD3OD, 400 MHz)
fluorobenzaldehyde (74
0.69 mmol) in methanol (4.67 mL) gave the title compound 5b
(99 mg, 85%). 1H NMR (CDCl3, 300 MHz)
8.09 (s,1H), 7.27e7.36 (m,
mL, 0.69 mmol), and Na2CO3 (74 mg,
4.1.1.8. O-(1-Isopropylpyrrolidin-3-yl)hydroxylamine hydrogen chlo-
ride (9b). Following the same procedure used for the synthesis of 4,
the reaction of 2-(1-isopropylpyrrolidin-3-yloxy)isoindoline-1,3-
dione 8b (145 mg, 0.53 mmol), and hydrazine monohydrate
(0.06 mL, 0.79 mmol) in ethanol (1.7 mL) at 80 ꢀC followed by
treatment of 10% HCl (0.30 mL, 1.06 mmol) gave the title compound
d 4.65 (m, 1H), 3.09
(m, 1H), 2.99 (m, 2H), 2.94 (m, 1H), 2.92 (m, 1H), 2.15 (m, 2H), 1.14
(dd, J ¼ 3.2, 6.3 Hz, 6H).
d
3H), 7.01e7.08 (m, 1H), 4.39 (m, 1H), 3.20 (dd, J ¼ 14.0, 9.0 Hz, 1H),
2.77e3.20 (m, 5H), 2.23 (m, 1H), 1.77 (m, 2H), 1.57 (m, 1H), 1.23 (m,
1H); 13C NMR (CDCl3, 75 MHz)
d
162.9 (d, 1J ¼ 244.7 Hz), 147.8 (d,
4J ¼ 2.9 Hz), 134.5 (d, 3J ¼ 8.0 Hz), 130.3 (d, 3J ¼ 8.3 Hz), 123.2 (d,
4J ¼ 4.2 Hz), 116.8 (d, 2J ¼ 21.3 Hz), 113.2 (d, 2J ¼ 22.7 Hz), 79.1, 54.3,
47.6, 46.9, 25.3, 24.1, 19.1; LC/MS (ESIþ): m/z: calcd for C14H17FN2O:
248.13, [M þ H]þ; found: 249.43.
9b (100 mg, 87%). 1H NMR (CD3OD, 400 MHz)
4.1.1.9. O-(1-Benzylpyrrolidin-3-yl)hydroxylamine hydrogen chloride
(9c). Following the same procedure used for the synthesis of 4, the
reaction of 2-(1-benzylpyrrolidin-3-yloxy)isoindoline-1,3-dione 8c
(380 mg, 1.18 mmol) and hydrazine monohydrate (0.08 mL,
1.77 mmol) in ethanol (3.93 mL) at 80 ꢀC followed by treatment of
10% HCl (0.16 mL, 2.36 mmol) gave the compound 9c (386 mg, 62%).
4.1.2.4. 3-Bromobenzaldehyde O-quinuclidin-3-yl oxime (5c).
Following the general procedure, O-(quinuclidin-3-yl)hydroxyl-
amine hydrogen chloride
bromobenzaldehyde (81 L, 0.69 mmol), and Na2CO3 (74 mg,
0.69 mmol) in methanol (4.67 mL) gave the title compound 5c
(126 mg, 88%). 1H NMR (CDCl3, 300 MHz)
8.05 (s, 1H), 7.75 (s, 1H),
4
(100 mg, 0.46 mmol) 3-
m
d
1H NMR (CD3OD, 400 MHz)
d
7.18e7.33 (m, 5H), 4.65 (m, 1H), 2.89
7.44e7.52 (m, 2H), 7.20e7.30 (m,1H), 4.39 (m,1H), 3.20 (dd, J ¼ 14.0,
(d, J ¼ 4.2 Hz, 2H), 2.84 (m, 1H), 2.58 (m, 1H), 2.14 (m, 2H).
9.0 Hz, 1H), 2.79e2.93 (m, 5H), 2.25 (m, 1H), 1.78 (m, 2H), 1.59 (m,
1H), 1.35 (m, 1H); 13C NMR (CDCl3, 75 MHz)
d 147.6, 134.4, 132.7,
4.1.1.10. O-(1-Methylpiperidin-3-yl)hydroxylamine hydrogen chlo-
ride (9d). Following the same procedure used for the synthesis of 4,
the reaction of 2-((1-methylpiperidin-3-yl)oxy)isoindoline-1,3-
dione 8d (1.96 g, 7.57 mmol) and hydrazine monohydrate
(0.85 mL, 11.3 mmol) in ethanol (25 mL) at 80 ꢀC followed by treat-
ment of 10% HCl (4.34 mL, 15.1 mmol) gave the title compound 9d
130.3,129.5,125.9,122.9, 79.1, 54.2, 47.6, 46.9, 25.2, 24.0,19.0; LC/MS
(ESIþ): m/z: calcd for C14H17BrN2O: 308.05, [M þ H]þ; found: 309.38.
4.1.2.5. 3-Methoxybenzaldehyde O-quinuclidin-3-yl oxime (5d).
Following the general procedure, the reaction of O-(quinuclidin-3-
yl)hydroxylamine hydrogen chloride 4 (100 mg, 0.46 mmol), 3-
(960 mg, 63%). 1H NMR (CD3OD, 400 MHz)
d
4.65 (m, 1H), 3.94 (m,
methoxybenzaldehyde (85
0.69 mmol) in methanol (4.6 mL) gave the title compound 5d
(96 mg, 79%). 1H NMR (CDCl3, 300 MHz)
8.11 (s, 1H), 7.28 (m, 1H),
mL, 0.69 mmol), and Na2CO3 (74 mg,
1H), 3.67(m,1H), 3.42 (m, 2H), 2.99 (s, 3H), 2.97 (m,1H), 2.41 (m,1H).
d
4.1.2. Synthesis of quinuclidinyl oxime derivatives 1, 5 and 6
7.14 (m, 2H), 6.91e6.95 (m, 1H), 4.41 (m, 1H), 3.84 (s, 3H), 3.22 (dd,
J ¼ 14.0, 9.0 Hz, 1H), 2.80e2.92 (m, 5H), 2.26 (m, 1H), 1.74 (m, 2H),
4.1.2.1. Representative procedure for synthesis of O-quinuclidin-3-yl
oxime (1). To a solution of O-(quinuclidin-3-yl)hydroxylamine
hydrogen chloride 4 (642 mg, 2.98 mmol) in methanol (30 mL)
were added 2-(benzyloxy)benzaldehyde (949 mg, 4.47 mmol) and
Na2CO3 (474 mg, 4.47 mmol). The reaction mixture was stirred at
room temperature for 3 h, quenched with water (25 mL) and
extracted with chloroform (3 ꢁ 30 mL). The combined organic layer
was washed with brine, dried over magnesium sulfate, and
1.59 (m, 1H), 1.40 (m, 1H); 13C NMR (CDCl3, 75 MHz)
d 159.9, 149.0,
133.8, 129.8, 120.1, 116.2, 111.3, 78.9, 55.4, 54.4, 47.7, 47.0, 25.3, 24.2,
19.2; LC/MS (ESIþ): m/z: calcd for C15H20N2O2: 260.15, [M þ H]þ;
found: 261.39.
4.1.2.6. 3-(Benzyloxy)benzaldehyde O-quinuclidin-3-yl oxime (5e).
Following the general procedure, the reaction of O-(quinuclidin-3-

Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
77
yl)hydroxylamine hydrogen chloride 4 (87 mg, 0.46 mmol), 3-
(benzyloxy)benzaldehyde (129 mg, 0.60 mmol), and Na2CO3
(64 mg, 0.60 mmol) in methanol (4.0 mL) gave the title compound
4.1.2.11. 2-Methoxybenzaldehyde O-quinuclidin-3-yl oxime (5j).
Following the general procedure, the reaction of O-(quinuclidin-3-
yl)hydroxylamine hydrogen chloride (107 mg, 0.50 mmol), 2-
(86 mg, 63%). 1H NMR (CDCl3, 300 MHz)
d
8.11 (s, 1H), 7.36e7.46 (m,
methoxybenzaldehyde (90
0.74 mmol) in methanol (5.0 mL) gave the title compound 5j
(122 mg, 94%). 1H NMR (CDCl3, 300 MHz)
8.52 (s, 1H), 7.77 (d,
mL, 0.74 mmol), and Na2CO3 (79 mg,
7H), 7.29 (dd, J ¼ 14.1, 6.7 Hz, 1H), 7.07 (m, 1H), 5.09 (s, 2H), 4.43 (m,
1H), 3.25 (dd, J ¼ 14.0, 9.0 Hz, 1H), 2.84e2.99 (m, 5H), 2.29 (m, 1H),
1.78 (m, 2H), 1.64 (m, 1H), 1.63 (m, 1H); 13C NMR (CDCl3, 75 MHz)
d
J ¼ 7.7 Hz, 1H), 7.32 (m 1H), 6.87e6.95 (m, 2H), 4.39 (m, 1H), 3.84 (s,
3H), 3.21 (m, 1H), 2.78e2.95 (m, 5H), 2.24 (m, 1H), 1.89 (m, 1H),
1.68e1.77 (m, 1H), 1.59 (m, 1H), 1.38 (m, 1H); 13C NMR (CDCl3,
d
159.1, 149.1, 136.8, 133.7, 129.8, 128.7, 128.1, 127.6, 120.4, 116.9,
112.5, 78.5, 70.2, 54.2, 47.6, 46.9, 25.2, 23.9, 18.9; LC/MS (ESIþ): m/z:
calcd for C21H24N2O2: 336.18, [M þ H]þ; found: 337.79.
75 MHz)
d 157.5, 145.1, 131.0, 126.3, 120.9, 120.7, 111.0, 78.5, 55.5,
54.3, 47.6, 46.9, 25.2, 24.1, 19.1; LC/MS (ESIþ): m/z: calcd for
C15H20N2O2: 260.15, [M þ H]þ; found: 261.58.
4.1.2.7. 3-(Trifluoromethyl)benzaldehyde O-quinuclidin-3-yl oxime
(5f). Following the general procedure, the reaction of O-(quinu-
clidin-3-yl)hydroxylamine hydrogen chloride
0.48 mmol), 3-(trifluoromethyl)benzaldehyde (96
4
(103.2 mg,
4.1.2.12. 2-(Trifluoromethyl)benzaldehyde O-quinuclidin-3-yl oxime
(5k). Following the general procedure, the reaction of O-(quinu-
mL, 0.72 mmol),
and Na2CO3 (76 mg, 0.72 mmol) in methanol (4.8 mL) gave the title
clidin-3-yl)hydroxylamine hydrogen chloride
0.48 mmol) in methanol (4.8 mL), 2-(trifluoromethyl)benzaldehyde
(95 L, 0.72 mmol), and Na2CO3 (76 mg, 0.72 mmol) gave the title
compound 5k (112 mg, 78%). 1H NMR (CDCl3, 300 MHz)
8.49 (d,
4
(103 mg,
compound 5f (95 mg, 66%). 1H NMR (CDCl3, 300 MHz)
d
8.15 (s, 1H),
7.84 (s, 1H), 7.73 (d, J ¼ 7.7 Hz, 1H), 7.61 (d, J ¼ 7.8 Hz, 1H), 7.49 (t,
J ¼ 7.7 Hz,1H), 4.41 (m,1H), 3.22 (dd, J ¼ 14.0, 9.0 Hz,1H), 2.79e2.94
(m, 5H), 2.26 (m, 1H), 1.85 (m, 2H), 1.74 (m, 1H), 1.59 (m, 1H); 13C
m
d
J ¼ 2.1 Hz, 1H), 8.03 (d, J ¼ 7.7 Hz, 1H), 7.64e7.72 (m 1H), 7.42e7.57
(m, 2H), 4.42 (m, 1H), 3.21 (m, 1H), 2.77e2.94 (m, 5H), 2.23 (m, 1H),
1.69e1.87 (m, 2H), 1.56 (m, 1H), 1.38 (m, 1H); 13C NMR (CD3OD,
NMR (CDCl3, 75 MHz)
d
147.6, 133.4, 131.4 (q, 2J ¼ 32.7 Hz), 130.3,
129.3, 126.3 (q, 3J ¼ 3.6 Hz), 124.0 (q, 1J ¼ 270.1 Hz), 123.6 (q,
3J ¼ 3.7 Hz), 122.1, 79.3, 54.3, 47.7, 47.0, 25.4, 24.2, 19.1; LC/MS
(ESIþ): m/z: calcd for C15H17F3N2O: 298.13, [M þ H]þ; found: 299.45.
100 MHz)
d
146.7, 132.2, 130.1, 129.6, 127.8 (q, 2J ¼ 30.6 Hz), 127.2,
126.7 (q, 1J ¼ 271.2 Hz), 125.6 (q, 3J ¼ 5.68 Hz), 75.4, 52.1, 46.5, 45.7,
24.0, 19.7, 16.1, 19.0; LC/MS (ESIþ): m/z: calcd for C15H17F3N2O:
298.13, [M þ H]þ; found: 299.47.
4.1.2.8. 2-Phenoxybenzaldehyde O-quinuclidin-3-yl oxime (5g).
Following the general procedure, the reaction of O-(quinuclidin-3-
yl)hydroxylamine hydrogen chloride 4 (128 mg, 0.59 mmol), 2-
phenoxybenzaldehyde (178 mg, 0.89 mmol), and Na2CO3 (95 mg,
0.89 mmol) in methanol (4.8 mL) gave the title compound 5g
4.1.2.13. 2-(2-Chlorobenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6a). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (68 mg,
0.32 mmol), 2-(2-chlorobenzyloxy)benzaldehyde (118 mg,
0.48 mmol), and Na2CO3 (51 mg, 0.48 mmol) in methanol (3.2 mL)
gave the title compound 6a (117 mg, 99%). 1H NMR (CDCl3,
(130 mg, 68%). 1H NMR (CDCl3, 300 MHz)
d 8.48 (s, 1H), 7.90 (dd,
J ¼ 7.8, 1.3 Hz, 1H), 7.28e7.35 (m, 3H), 7.07e7.14 (m, 2H), 6.87e6.96
(m, 3H), 4.40 (m, 1H), 3.22 (dd, J ¼ 9.4, 5.0 Hz, 1H), 2.79e2.95 (m,
5H), 2.24 (m, 1H), 1.70e1.86 (m, 2H), 1.58 (m, 1H), 1.37 (m, 1H); 13C
300 MHz)
d
8.58 (s, 1H), 7.81 (dd, J ¼ 7.7, 1.5 Hz, 1H), 7.53 (m, 1H),
NMR (CDCl3, 75 MHz)
d
157.4, 154.9, 144.7, 131.2, 129.9, 126.7, 124.0,
7.41 (m, 1H), 7.27e7.35 (m 3H), 6.93e7.04 (m, 2H), 5.19 (s, 2H), 3.21
(m, 1H), 2.78e2.96 (m, 5H), 2.26 (m, 1H), 1.87e1.91 (m, 1H), 1.70e
1.78 (m, 1H), 1.56e1.62 (m, 1H), 1.33e1.40 (m, 1H); 13C NMR
123.9,123.4,119.5,118.3, 78.7, 54.3, 47.6, 46.9, 25.3, 24.1,19.0; LC/MS
(ESIþ): m/z: calcd for C20H22N2O2: 322.17, [M þ H]þ; found: 323.69.
(CDCl3, 75 MHz) d 156.3,145.0,134.3,132.6,131.0,129.4,129.1,128.8,
4.1.2.9. 2-Fluorobenzaldehyde O-quinuclidin-3-yl oxime (5h).
Following the general procedure, the reaction of O-(quinuclidin-3-
yl)hydroxylamine hydrogen chloride 4 (130 mg, 0.60 mmol), 2-
127.0, 126.6, 121.3, 112.6, 78.6, 67.6, 54.2, 47.5, 46.8, 25.2, 24.1, 19.0;
LC/MS (ESIþ): m/z: calcd for C21H23ClN2O2: 370.14, [M þ H]þ; found:
371.59.
fluorobenzaldehyde (95
0.90 mmol) in methanol (6.0 mL) gave the title compound 5h
(113 mg, 75%). 1H NMR (CDCl3, 300 MHz)
8.36 (s, 1H), 7.79 (t,
mL, 0.90 mmol), and Na2CO3 (96 mg,
4.1.2.14. 2-(3-Chlorobenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6b). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (66 mg,
0.31 mmol), 2-(3-chlorobenzyloxy)benzaldehyde (114 mg,
0.46 mmol), and Na2CO3 (49 mg, 0.46 mmol) in methanol (3.1 mL)
gave the title compound 6b (60 mg, 52%). 1H NMR (CDCl3, 300 MHz)
d
J ¼ 7.4 Hz, 1H), 7.32 (q, J ¼ 7.0 Hz, 1H), 7.01e7.17 (m, 2H), 4.40 (m,
1H), 3.22 (dd, J ¼ 14.5, 8.3 Hz, 1H), 2.73e2.98 (m, 5H), 2.23 (m, 1H),
1.69e1.87 (m, 2H), 1.57 (m, 1H), 1.38 (m, 1H); 13C NMR (CDCl3,
75 MHz)
d
160.8 (d, 1J ¼ 250.5 Hz), 142.7 (d, 3J ¼ 4.2 Hz), 131.3 (d,
3J ¼ 8.2 Hz), 126.7 (d, 4J ¼ 2.7 Hz), 124.3 (d, 3J ¼ 3.4 Hz), 120.2 (d,
2J ¼ 10.8 Hz), 115.9 (d, 2J ¼ 20.9 Hz), 79.0, 54.2, 47.6, 46.9, 25.3, 24.1,
19.0; LC/MS (ESIþ): m/z: calcd for C14H17FN2O: 248.13, [M þ H]þ;
found: 249.41.
d
8.54 (s, 1H), 7.80 (dd, J ¼ 7.7, 1.5 Hz, 1H), 7.28e7.42 (m, 5H), 6.89e
6.99 (m, 2H), 5.07 (s, 2H), 4.41 (m, 1H), 3.23 (m, 1H), 2.74e3.00 (m,
5H), 2.26 (m, 1H), 1.89 (m, 1H), 1.74 (m, 1H), 1.59 (m, 1H), 1.38 (m,
1H); 13C NMR (CDCl3, 75 MHz)
d 156.3, 144.9, 138.3, 131.0, 129.9,
128.2, 127.4, 126.7, 125.3, 121.3, 112.4, 78.6, 69.6, 54.3, 47.6, 46.9,
25.2, 24.1, 19.0; LC/MS (ESIþ): m/z: calcd for C21H23ClN2O2: 370.14,
[M þ H]þ; found: 371.46.
4.1.2.10. 2-Bromobenzaldehyde O-quinuclidin-3-yl oxime (5i).
Following the general procedure, the reaction of O-(quinuclidin-3-
yl)hydroxylamine hydrogen chloride 4 (102 mg, 0.47 mmol), 2-
bromobenzaldehyde (83
0.71 mmol) in methanol (4.7 mL) gave the title compound (106 mg,
72%). 1H NMR (CDCl3, 300 MHz)
m
L, 0.71 mmol), and Na2CO3 (75 mg,
4.1.2.15. 2-(4-Chlorobenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6c). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (83 mg,
0.38 mmol), 2-(4-chlorobenzyloxy)benzaldehyde (143 mg,
0.58 mmol), and Na2CO3 (61 mg, 0.58 mmol) in methanol (3.8 mL)
gave the title compound 6c (142 mg, 96%). 1H NMR (CDCl3,
d
8.52 (s, 1H), 7.86 (dd, J ¼ 7.6,
1.6 Hz, 1H), 7.60 (m 1H), 7.19e7.35 (m, 2H), 4.43 (m, 1H), 3.26 (m,
1H), 2.84e2.95 (m, 5H), 2.28 (m, 1H), 1.73e1.91 (m, 2H), 1.61 (m,
1H), 1.41 (m, 1H); 13C NMR (CDCl3, 75 MHz)
d 148.3, 133.1, 131.6,
131.0, 127.4, 123.7, 79.0, 54.1, 47.5, 46.8, 25.2, 24.0, 18.9; LC/MS
(ESIþ): m/z: calcd for C14H17BrN2O: 308.05, [M þ H]þ; found:
309.52.
300 MHz)
d
8.52 (s, 1H), 7.80 (dd, J ¼ 7.7, 1.6 Hz, 1H), 7.27e7.36 (m,
5H), 6.89e6.98 (m, 2H), 5.07 (s, 2H), 4.39 (m, 1H), 3.21 (m, 1H),
2.78e2.95 (m, 5H), 2.24 (m, 1H), 1.88 (m, 1H), 1.72 (m, 1H), 1.58 (m,

78
Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
1H), 1.37 (m, 1H); 13C NMR (CDCl3, 75 MHz)
d
156.3, 144.8, 135.0,
55.3, 54.3, 47.6, 46.9, 25.2, 24.1, 19.0; LC/MS (ESIþ): m/z: calcd for
C22H26N2O3: 366.19, [M þ H]þ; found: 367.2.
133.9, 131.0, 128.8, 128.7, 126.6, 121.3, 121.2, 112.5, 78.7, 69.6, 54.3,
47.6, 46.9, 25.2, 24.1, 19.0; LC/MS (ESIþ): m/z: calcd for
C21H23ClN2O2: 370.14, [M þ H]þ; found: 371.53.
4.1.2.20. 2-(2,6-Dichlorobenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6h). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (72 mg,
0.33 mmol), 2-(2,6-dichlorobenzyloxy)benzaldehyde (141 mg,
0.50 mmol), and Na2CO3 (53 mg, 0.50 mmol) in methanol (3.3 mL)
gave the title compound 6h (135 mg, 98%). 1H NMR (CDCl3,
4.1.2.16. 2-(2-Fluorobenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6d). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (65 mg,
0.30 mmol), 2-(2-fluorobenzyloxy)benzaldehyde (105 mg,
0.45 mmol), and Na2CO3 (48 mg, 0.45 mmol) in methanol (3.0 mL)
gave the title compound 6d (107 mg, 99%). 1H NMR (CDCl3,
300 MHz)
d
8.45 (s, 1H), 7.83 (d, J ¼ 7.7 Hz, 1H), 7.38 (t, J ¼ 6.5 Hz,
3H), 7.28 (t, J ¼ 8.0 Hz, 1H), 7.12 (d, J ¼ 8.3 Hz, 1H), 7.01 (t, J ¼ 7.5 Hz,
1H), 5.31 (s, 2H), 4.36 (m, 1H), 3.20 (m, 1H), 2.72e2.97 (m, 5H), 2.23
(m, 1H), 1.85 (m, 1H), 1.72 (m, 1H), 1.57 (m, 1H), 1.36 (m, 1H); 13C
400 MHz)
d
8.54 (s, 1H), 7.81 (dd, J ¼ 5.7, 1.2 Hz, 1H), 7.47e7.51 (m,
1H), 7.30e7.35 (m, 2H), 7.18 (td, J ¼ 5.6, 0.7 Hz, 1H), 7.10 (td, J ¼ 6.9,
0.8 Hz, 1H), 6.95e6.99 (m, 2H), 5.17 (s, 2H), 4.38 (m, 1H), 3.20 (m,
1H), 2.77e2.94 (m, 5H), 2.24 (m, 1H), 1.86 (m, 1H), 1.72 (m, 1H), 1.57
NMR (CDCl3, 75 MHz) d 156.7, 144.9, 137.0, 131.8, 130.9, 130.5, 128.5,
126.3, 121.8, 121.5, 113.0, 78.7, 65.8, 54.3, 47.6, 46.9, 25.2, 24.2, 19.0;
LC/MS (ESIþ): m/z: calcd for C21H22Cl2N2O2: 404.11, [M þ H]þ;
found: 405.1.
(m, 1H), 1.36 (m, 1H); 13C NMR (CD3OD, 75 MHz)
d 160.8 (d,
1J ¼ 244.5 Hz), 156.7, 146.1, 131.6, 130.2 (d, 3J ¼ 3.7 Hz), 126.2, 124.1
(d, 3J ¼ 3.7 Hz), 123.5 (d, 2J ¼ 14.2 Hz), 121.0, 120.4, 115.0 (d,
2J ¼ 21.0 Hz), 112.8, 74.6, 129.8, 129.7, 126.5, 124.3, 121.4, 121.2, 115.4
(d, 2J ¼ 21.0 Hz), 112.4, 74.6, 64.4, 52.3, 46.5, 45.7, 23.9, 19.7, 16.5; LC/
MS (ESIþ): m/z: calcd for C21H23FN2O2: 354.17, [M þ H]þ; found:
353.77.
4.1.2.21. 2-(3,4-Dichlorobenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6i). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (71 mg,
0.32 mmol), 2-(3,4-dichlorobenzyloxy)benzaldehyde (140 mg,
0.49 mmol), and Na2CO3 (53 mg, 0.49 mmol) in methanol (3.2 mL)
gave the title compound 6i (121 mg, 90%). 1H NMR (CDCl3,
4.1.2.17. 2-(3-Fluorobenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6e). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (72 mg,
0.33 mmol), 2-(3-fluorobenzyloxy)benzaldehyde (115 mg,
0.49 mmol), and Na2CO3 (53 mg, 0.49 mmol) in methanol (3.3 mL)
gave the title compound 6e (117 mg, 97%). 1H NMR (CDCl3,
300 MHz)
d
8.52 (s, 1H), 7.80 (d, J ¼ 7.7 Hz, 1H), 7.48 (m, 2H), 7.25e
7.34 (m, 2H), 6.98 (t, J ¼ 7.5 Hz, 1H), 6.89 (d, J ¼ 8.4 Hz, 1H), 5.04 (s,
2H), 4.40 (m,1H), 3.22 (m, 1H), 2.73e2.99 (m, 5H), 2.24 (m,1H), 1.89
(m, 1H), 1.73 (m, 1H), 1.58 (m, 1H), 1.37 (m, 1H); 13C NMR (CDCl3,
75 MHz) d 156.0, 144.7, 136.8, 131.0, 130.6, 129.2, 126.8, 126.5, 121.5,
300 MHz)
d
8.55 (s, 1H), 7.81 (d, J ¼ 7.7 Hz, 1H), 7.28e7.39 (m, 2H),
112.4, 78.8, 69.0, 54.3, 47.6, 46.9, 25.2, 24.2, 19.1; LC/MS (ESIþ): m/z:
calcd for C21H22Cl2N2O2: 404.11, [M þ H]þ; found: 405.1.
7.18 (t, J ¼ 9.7 Hz, 2H), 6.90e7.06 (m, 3H), 5.10 (s, 2H), 4.40 (m, 1H),
3.22 (m, 1H), 2.75e3.00 (m, 5H), 2.25 (m, 1H), 1.88 (m, 1H), 1.73 (m,
1H), 1.58 (m, 1H), 1.35 (m, 1H); 13C NMR (CD3OD, 100 MHz)
d
162.9
4.1.2.22. 2-(2,4-Dichlorobenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6j). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (76 mg,
0.35 mmol), 2-(2,4-dichlorobenzyloxy)benzaldehyde (148 mg,
0.52 mmol), and Na2CO3 (56 mg, 0.52 mmol) in methanol (3.5 mL)
gave the title compound 4 (137 mg, 97%). 1H NMR (CDCl3, 300 MHz)
(d, 1J ¼ 243.1 Hz), 156.6, 146.2 (d, 3J ¼ 18.5 Hz), 139.6 (d, 4J ¼ 7.3 Hz),
131.5 (d, 3J ¼ 14.8 Hz), 130.1, 126.2, 122.8, 120.9, 120.3, 114.4 (d,
2J ¼ 20.6 Hz), 113.8 (d, 2J ¼ 22.5 Hz),112.7, 74.6, 69.3, 52.3, 46.5, 45.7,
24.0, 19.7, 16.2; LC/MS (ESIþ): m/z: calcd for C21H23FN2O2: 354.17,
[M þ H]þ; found: 355.2.
d
8.54 (s, 1H), 7.81 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.45 (m, 2H), 7.27e7.34
4.1.2.18. 2-(4-Fluorobenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6f). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (83 mg,
0.38 mmol), 2-(4-fluorobenzyloxy)benzaldehyde (134 mg,
0.58 mmol), and Na2CO3 (62 mg, 0.58 mmol) in methanol (3.8 mL)
gave the title compound 6f (137 mg, 98%).1H NMR (CDCl3, 300 MHz)
(m, 2H), 6.98 (t, J ¼ 7.5 Hz, 1H), 6.92 (d, J ¼ 8.3 Hz, 1H), 5.14 (s,
2H), 4.38 (m, 1H), 3.19 (m, 1H), 2.74e2.92 (m, 5H), 2.24 (m, 1H), 1.85
(m, 1H), 1.72 (m, 1H), 1.57 (m, 1H), 1.36 (m, 1H); 13C NMR (CDCl3,
75 MHz) d 156.0, 144.7, 134.3, 133.2, 132.9, 131.0, 129.6, 129.3, 127.3,
126.8, 121.5, 121.4, 112.5, 78.8, 67.1, 54.3, 47.6, 46.9, 25.3, 24.2, 19.1;
LC/MS (ESIþ): m/z: calcd for C21H22Cl2N2O2: 404.11, [M þ H]þ;
found: 405.1.
d
8.52 (s,1H), 7.80 (d, J ¼ 7.6 Hz,1H), 7.39 (dd, J ¼ 8.2, 5.6 Hz, 2H), 7.31
(t, J ¼ 7.3 Hz, 1H), 7.08 (t, J ¼ 8.6 Hz, 2H), 6.91e7.10 (m, 2H), 5.05 (s,
2H), 4.39 (m,1H), 3.22 (m,1H), 2.75e3.00 (m, 5H), 2.25 (m,1H),1.88
(m, 1H), 1.74 (m, 1H), 1.59 (m, 1H), 1.38 (m, 1H); 13C NMR (CDCl3,
4.1.2.23. 2-(2-(Trifluoromethyl)benzyloxy)benzaldehyde O-quinucli-
din-3-yl oxime (6k). Following the general procedure, the reaction
of O-(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (89 mg,
75 MHz)
d
162.5 (d, 1J ¼ 245.1 Hz),156.4, 144.9,132.3, 131.0, 129.2 (d,
3J ¼ 8.0 Hz), 126.5, 121.2 (d, 3J ¼ 8.5 Hz), 115.5 (d, 2J ¼ 21.4 Hz), 112.5,
78.5, 69.8, 54.2, 47.5, 46.8, 25.2, 24.0, 18.9; LC/MS (ESIþ): m/z: calcd
for C21H23FN2O2: 354.17, [M þ H]þ; found: 355.2.
0.41
mmol),
2-(2-(trifluoromethyl)benzyloxy)benzaldehyde
(175 mg, 0.62 mmol), and Na2CO3 (66 mg, 0.62 mmol) in methanol
(4.1 mL) gave the title compound 6k (167 mg, 99%). 1H NMR (CDCl3,
300 MHz)
d
8.58 (s, 1H), 7.82 (dd, J ¼ 7.7, 1.6 Hz, 1H), 7.71 (m, 2H),
4.1.2.19. 2-(4-Methoxybenzyloxy)benzaldehyde O-quinuclidin-3-yl
oxime (6g). Following the general procedure, the reaction of O-
(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (67 mg,
0.31 mmol), 2-(4-methoxybenzyloxy)benzaldehyde (113 mg,
0.46 mmol), and Na2CO3 (49 mg, 0.46 mmol) in methanol (3.1 mL)
gave the title compound 6g (104 mg, 92%). 1H NMR (CDCl3,
7.58 (t, J ¼ 7.5 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz, 1H), 7.30 (m, 1H), 6.98 (t,
J ¼ 7.6 Hz,1H), 6.90 (d, J ¼ 8.3 Hz,1H), 5.30 (s, 2H), 4.40 (m,1H), 3.21
(ddd, J ¼ 8.5, 14.4, 1.8 Hz, 1H) 2.77e2.95 (m, 5H), 2.25 (m, 1H), 1.87
(m, 1H), 1.72 (m, 1H), 1.58 (m, 1H), 1.36 (m, 1H); 13C NMR (CD3OD,
75 MHz)
d 156.4, 146.1, 134.6, 132.3, 131.6, 129.8, 128.4, 127.5 (q,
2J ¼ 30.7 Hz), 126.3, 125.9 (q, 3J ¼ 6.0 Hz), 124.4 (q, 1J ¼ 271.5 Hz),
121.1, 120.3, 112.4, 74.7, 66.9, 52.3, 46.5, 45.7, 24.0, 19.7, 16.2; LC/MS
(ESIþ): m/z: calcd for C22H23F3N2O2: 404.17, [M þ H]þ; found: 405.2.
300 MHz)
d
8.53 (s, 1H), 7.80 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.28e7.36 (m,
3H), 6.89e6.97 (m, 4H), 5.02 (s, 2H), 4.38 (m, 1H), 3.82 (s, 3H), 3.19
(m, 1H), 2.77e2.89 (m, 5H), 2.23 (m, 1H), 1.88 (m, 1H), 1.72 (m, 1H),
1.58 (m, 1H), 1.38 (m, 1H); 13C NMR (CDCl3, 75 MHz)
d
159.5, 156.7,
4.1.2.24. 2-(3-(Trifluoromethyl)benzyloxy)benzaldehyde O-quinucli-
din-3-yl oxime (6l). Following the general procedure, the reaction of
145.0, 131.0, 129.2, 128.6, 126.4, 121.2, 120.9, 114.0, 112.5, 78.6, 70.2,

Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
79
O-(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (75 mg,
0.34 mmol), 2-(3-(trifluoromethyl)benzyloxy)benzaldehyde
13C NMR (CDCl3, 75 MHz)
127.4, 126.5, 121.2, 121.0, 112.4, 82.8, 70.3, 61.5, 55.0, 42.1, 31.6; LC/
MS (ESIþ): m/z: calcd for C19H22N2O2: 310.17, [M þ H]þ; found:
311.2.
d
156.7, 145.3, 136.5, 131.0, 128.6, 128.0,
(147 mg, 0.52 mmol), and Na2CO3 (55 mg, 0.52 mmol) in methanol
(3.4 mL) gave the title compound 6l (136 mg, 96%). 1H NMR (CDCl3,
300 MHz)
d
8.54 (s, 1H), 7.82 (d, J ¼ 7.7 Hz, 1H), 7.50e7.67 (m, 4H),
7.32 (m, 1H), 6.96 (m, 2H), 5.14 (s, 2H), 4.39 (m, 1H), 3.21 (m, 1H),
4.1.3.4. 2-((3,4-Dichlorobenzyl)oxy)benzaldehyde O-(1-methylpyrro-
lidin-3-yl) oxime (10d). Following the general procedure, the
reaction of O-(1-methylpyrrolidin-3-yl)hydroxylamine hydrogen
chloride 9a (75 mg, 0.39 mmol), 2-((3,4-dichlorobenzyl)oxy)benz-
aldehyde (160 mg, 0.59 mmol), and Na2CO3 (63 mg, 0.59 mmol) in
methanol (4 mL) gave the title compound 10d (21 mg,14%). 1H NMR
2.80e2.99 (m, 5H), 2.24 (m, 1H), 1.86 (m, 1H), 1.73 (m, 1H), 1.58 (m,
1H), 1.37 (m, 1H); 13C NMR (CD3OD, 100 MHz)
d 156.5, 146.1, 138.2,
131.6,130.9,130.4 (q, 2J ¼ 31.9 Hz),129.1,126.2, 124.4 (q, 3J ¼ 3.7 Hz),
124.2 (q, 1J ¼ 269.7 Hz),123.7 (q, 3J ¼ 3.7 Hz),121.0,120.3,112.8, 74.7,
69.3, 52.3, 46.5, 45.7, 24.0, 19.7, 16.1; LC/MS (ESIþ): m/z: calcd for
C22H23F3N2O2: 404.17, [M þ H]þ; found: 405.2.
(CDCl3, 300 MHz)
d
8.49 (s, 1H), 7.80 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.48 (m,
2H), 7.23e7.33 (m, 2H), 6.98 (t, J ¼ 7.5 Hz, 1H), 6.88 (d, J ¼ 8.3 Hz,
1H), 5.02 (s, 2H), 4.88 (m, 1H), 2.84e2.94 (m, 2H), 2.65 (dd, J ¼ 10.8,
5.6 Hz, 1H), 2.38 (s, 3H), 2.36 (m, 1H), 2.22 (m, 1H), 2.04 (m, 1H); 13C
4.1.2.25. 2-(4-(Trifluoromethyl)benzyloxy)benzaldehyde O-quinucli-
din-3-yl oxime (6m). Following the general procedure, the reaction
of O-(quinuclidin-3-yl)hydroxylamine hydrogen chloride 4 (87 mg,
NMR (CDCl3, 75 MHz) d 156.1, 144.8, 136.8, 132.7, 132.1, 130.9, 130.6,
0.40
mmol),
2-(4-(trifluoromethyl)benzyloxy)benzaldehyde
129.2, 126.8, 126.5, 121.4, 112.3, 83.1, 69.0, 61.7, 55.0, 42.1, 31.6; LC/
MS (ESIþ): m/z: calcd for C19H20Cl2N2O2: 378.09, [M þ H]þ; found:
379.1.
(170 mg, 0.60 mmol), and Na2CO3 (64 mg, 0.60 mmol) in methanol
(4.0 mL) gave the title compound 6m (146 mg, 90%). 1H NMR
(CDCl3, 300 MHz)
d
8.55 (s, 1H), 7.82 (dd, J ¼ 7.7, 1.6 Hz, 1H), 7.66 (d,
J ¼ 8.2 Hz, 2H), 7.55 (d, J ¼ 8.1 Hz, 2H), 7.31 (m, 1H), 6.97 (t,
J ¼ 3.8 Hz,1H), 6.91 (d, J ¼ 8.3 Hz,1H), 5.16 (s, 2H), 4.40 (m, 1H), 3.21
(m, 1H), 2.78e2.95 (m, 5H), 2.25 (m, 1H), 1.86 (m, 1H), 1.73 (m, 1H),
4.1.3.5. 2-((3-Fluorobenzyl)oxy)benzaldehyde O-(1-methylpyrro-
lidin-3-yl) oxime (10e). Following the general procedure, the reac-
tion of O-(1-methylpyrrolidin-3-yl)hydroxylamine hydrogen chlo-
ride 9a (76 mg, 0.40 mmol), 2-((3-fluorobenzyl)oxy)benzaldehyde
(138 mg, 0.60 mmol), and Na2CO3 (64 mg, 0.60 mmol) in methanol
(4 mL) gave the title compound 10e (19 mg, 14% yield). 1H NMR
1.58 (m, 1H),1.37 (m, 1H); 13C NMR (CD3OD,100 MHz)
d 156.5, 146.1,
141.3, 131.6, 129.6 (q, 2J ¼ 32.1 Hz), 127.5, 126.2, 125.1 (q, 3J ¼ 3.6 Hz),
124.6 (q, 1J ¼ 269.6 Hz), 121.0, 120.3, 112.6, 74.7, 69.2, 52.3, 46.5,
45.7, 24.0, 19.7, 16.2; LC/MS (ESIþ): m/z: calcd for C22H23F3N2O2:
404.17, [M þ H]þ; found: 405.2.
(CDCl3, 300 MHz)
d
8.52 (s, 1H), 7.81 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.27e
7.38 (m, 2H), 7.15 (m, 2H), 6.89e7.02 (m, 3H), 5.06 (s, 2H), 4.88 (m,
1H), 2.84e2.93 (m, 2H), 2.66 (dd, J ¼ 10.8, 5.6 Hz, 1H), 2.38 (s, 3H),
2.36 (m, 1H), 2.23 (m, 1H), 2.03 (m, 1H); 13C NMR (CDCl3, 75 MHz)
4.1.3. Synthesis of pyrrolidinyl/piperidinyl oxime derivatives 10e13
4.1.3.1. 2-Methoxybenzaldehyde O-(1-methylpyrrolidin-3-yl) oxime
(10a). Following the general procedure, the reaction of O-(1-
methylpyrrolidin-3-yl)hydroxylamine hydrogen chloride 9a
d
162.9 (d, 1J ¼ 244.8 Hz), 156.3, 145.0, 139.1 (d, 3J ¼ 7.3 Hz), 130.9,
130.1 (d, 3J ¼ 8.1 Hz), 126.7, 122.7 (d, 4J ¼ 3.0 Hz), 121.4, 121.2, 114.9
(d, 2J ¼ 21.0 Hz), 114.2 (d, 2J ¼ 21.9 Hz), 112.3, 83.1, 69.5, 61.7, 55.0,
42.1, 31.6; LC/MS (ESIþ): m/z: calcd for C19H21FN2O2: 328.16,
[M þ H]þ; found: 329.2.
(74 mg, 0.39 mmol), O-anisaldehyde (71
Na2CO3 (62 mg, 0.58 mmol) in methanol (3.9 mL) gave the title
compound 10a (13 mg, 14%). 1H NMR (CDCl3, 300 MHz)
8.48 (s,
mL, 0.58 mmol), and
d
1H), 7.76 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.32 (m, 1H), 6.93 (t, J ¼ 7.5 Hz, 1H),
6.88 (d, J ¼ 8.4 Hz, 1H), 4.88 (m, 1H), 3.82 (s, 3H), 2.73e2.94 (m, 3H),
2.47 (m, 1H), 2.42 (s, 3H), 2.23 (m, 1H), 2.05 (m, 1H); 13C NMR
4.1.3.6. 2-((2,4-Dichlorobenzyl)oxy)benzaldehyde O-(1-methylpyrro-
lidin-3-yl) oxime (10f). Following the general procedure, the reac-
tion of O-(1-methylpyrrolidin-3-yl)hydroxylamine hydrogen chlo-
ride 9a (75 mg, 0.39 mmol), 2-((2,4-dichlorobenzyl)oxy)
benzaldehyde (160 mg, 0.59 mmol), and Na2CO3 (63 mg,
0.59 mmol)in methanol (3.9 mL) gave the title compound 10f
(CDCl3, 75 MHz) d 157.6, 145.4, 131.0, 126.3, 120.9, 120.7, 111.0, 82.8,
61.6, 55.5, 55.0, 42.1, 31.6; LC/MS (ESIþ): m/z: calcd for C13H18N2O2:
234.17, [M þ H]þ; found: 235.2.
(17 mg, 12%). 1H NMR (CDCl3, 300 MHz)
d 8.52 (s, 1H), 7.80 (dd,
4.1.3.2. 3-(Trifluoromethyl)benzaldehyde O-(1-methylpyrrolidin-3-
yl) oxime (10b). Following the general procedure, the reaction of
O-(1-methylpyrrolidin-3-yl)hydroxylamine hydrogen chloride 9a
J ¼ 7.7, 1.7 Hz, 1H), 7.48 (d, J ¼ 8.3 Hz, 1H), 7.42 (d, J ¼ 2.1 Hz, 1H),
7.27e7.35 (m, 2H), 6.98 (t, J ¼ 7.6 Hz, 1H), 6.92 (d, J ¼ 8.3 Hz, 1H),
5.13 (s, 2H), 4.89 (m, 1H), 2.86e2.97 (m, 2H), 2.69 (dd, J ¼ 10.9,
5.6 Hz, 1H), 2.41 (s, 3H), 2.40 (m, 1H), 2.24 (m, 1H), 2.06 (m, 1H); 13C
(64 mg, 0.38 mmol), 3-(trifluoromethyl)benzaldehyde (68
mL,
0.50 mmol), and Na2CO3 (54 mg, 0.50 mmol) in methanol (3.4 mL)
NMR (CDCl3, 75 MHz) d 156.1, 144.9, 134.3, 133.1, 132.9, 131.0, 129.6,
gave the title compound 10b (42 mg, 45%). 1H NMR (CDCl3,
129.2, 127.4, 126.8, 121.49, 121.41, 112.4, 83.0, 67.0, 61.7, 55.0, 42.1,
31.6; LC/MS (ESIþ): m/z: calcd for C19H20Cl2N2O2: 378.09, [M þ H]þ;
found: 379.1.
300 MHz)
d
8.11 (s, 1H), 7.84 (bs, 1H), 7.73 (d, J ¼ 7.7 Hz, 1H), 7.61 (d,
J ¼ 7.9 Hz,1H), 7.49 (t, J ¼ 7.8 Hz,1H), 4.91 (m,1H), 2.93 (m, 2H), 2.79
(m, 1H), 2.50 (m, 1H), 2.45 (s, 3H), 2.25 (m, 1H), 2.05 (m, 1H); 13C
NMR (CDCl3, 75 MHz)
d
147.7, 133.1, 130.2, 129.1, 126.2, 123.5, 83.3,
4.1.3.7. 2-Methoxybenzaldehyde O-(1-isopropylpyrrolidin-3-yl) oxime
(11a). Following the general procedure, the reaction of O-(1-
isopropylpyrrolidin-3-yl)hydroxylamine hydrogen chloride 9b
61.4, 54.9, 42.0, 31.5; LC/MS (ESIþ): m/z: calcd for C13H15F3N2O:
272.11, [M þ H]þ; found: 273.2.
(48 mg, 0.22 mmol), O-anisaldehyde (40
(35 mg, 0.33 mmol) in methanol (2.2 mL) gave the title compound
11a (33 mg, 57%). 1H NMR (CDCl3, 300 MHz)
8.47 (s, 1H), 7.76 (dd,
mL, 0.33 mmol), and Na2CO3
4.1.3.3. 2-(Benzyloxy)benzaldehyde O-(1-methylpyrrolidin-3-yl) oxime
(10c). Following the general procedure, the reaction of O-(1-
methylpyrrolidin-3-yl)hydroxylamine hydrogen chloride 9a
(66 mg, 0.35 mmol), 2-(benzyloxy)benzaldehyde (100 mg,
0.52 mmol), and Na2CO3 (55 mg, 0.52 mmol) in methanol
(3.2 mL) gave the title compound 10c (15 mg, 14%). 1H NMR
d
J ¼ 7.7, 1.6 Hz, 1H), 7.32 (m, 1H), 6.93 (t, J ¼ 7.5 Hz, 1H), 6.88 (d,
J ¼ 8.4 Hz, 1H), 4.87 (m, 1H), 3.81 (s, 3H), 2.92 (d, J ¼ 4.3 Hz, 1H),
2.81e2.93 (m, 2H), 2.60 (m, 1H), 2.45 (m, 1H), 2.22 (m, 1H), 2.04 (m,
1H), 1.14 (dd, J ¼ 3.2, 6.3 Hz, 6H); 13C NMR (CDCl3, 75 MHz)
d 157.5,
(CDCl3, 300 MHz)
7.42 (m, 6H), 6.95 (m, 2H), 5.08 (s, 2H), 4.88 (m, 1H), 2.77e2.96
d
8.53 (s, 1H), 7.81 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.28e
145.3, 131.0, 126.3, 120.9, 120.7, 111.0, 82.1, 57.5, 55.5, 55.1, 50.6, 31.1,
21.3; LC/MS (ESIþ): m/z: calcd for C15H22N2O2: 262.17, [M þ H]þ;
found: 263.2.
(m, 3H), 2.49 (m, 1H), 2.44 (s, 3H), 2.25 (m, 1H), 2.06 (m, 1H);

80
Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
4.1.3.8. 3-(Trifluoromethyl)benzaldehyde O-(1-isopropylpyrrolidin-
3-yl) oxime (11b). Following the general procedure, the reaction of
O-(1-isopropylpyrrolidin-3-yl)hydroxylamine hydrogen chloride
(CDCl3, 300 MHz)
d
8.51 (s, 1H), 7.81 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.48 (d,
J ¼ 8.3 Hz, 1H), 7.42 (d, J ¼ 2.1 Hz, 1H), 7.27e7.35 (m, 2H), 6.98 (t,
J ¼ 7.6 Hz,1H), 6.92 (d, J ¼ 8.3 Hz,1H), 5.13 (s, 2H), 4.89 (m,1H), 2.85e
3.00 (m, 3H), 2.58 (m, 1H), 2.47 (quin, J ¼ 6.3 Hz, 1H), 2.25 (m, 1H),
2.05 (m, 1H), 1.15 (dd, J ¼ 4.1, 6.3 Hz, 6H); 13C NMR (CDCl3, 75 MHz)
9b (49 mg, 0.23 mmol), 3-(trifluoromethyl)benzaldehyde (45 mL,
0.33 mmol), and Na2CO3 (35 mg, 0.33 mmol) in methanol (2.3 mL)
gave the title compound 11b (35 mg, 51%). 1H NMR (CDCl3,
d 156.1, 145.0, 134.3, 133.2, 132.9, 131.0, 129.6, 129.2, 127.4, 126.8,
300 MHz)
d
8.09 (s, 1H), 7.83 (s, 1H), 7.72 (d, J ¼ 7.7 Hz, 1H), 7.60 (d,
121.5,121.4,112.4, 82.3, 67.0, 57.6, 55.1, 50.6, 31.0, 21.3; LC/MS (ESIþ):
J ¼ 7.8 Hz, 1H), 7.48 (t, J ¼ 7.7 Hz, 1H), 4.89 (m, 1H), 2.81e2.98 (m,
m/z: calcd for C21H24Cl2N2O2: 406.12, [M þ H]þ; found: 407.1.
3H), 2.39e2.57 (m, 2H), 2.23 (m, 1H), 2.02 (m, 1H), 1.13 (dd, J ¼ 4.2,
6.3 Hz, 6H); 13C NMR (CDCl3, 75 MHz)
d
147.5, 133.2, 131.2 (q,
4.1.3.13. 2-Methoxybenzaldehyde O-(1-benzylpyrrolidin-3-yl) oxime
(12a). Following the general procedure, the reaction of O-(1-
benzylpyrrolidin-3-yl)hydroxylamine hydrogen chloride 9c
(45 mg, 0.17 mmol), O-anisaldehyde (31
Na2CO3 (27 mg, 0.25 mmol) in methanol (1.7 mL) gave the title
compound 12a (23 mg, 44%). 1H NMR (CDCl3, 300 MHz)
8.47 (s,
2J ¼ 32.4 Hz), 130.1, 129.1, 126.1 (q, 3J ¼ 3.6 Hz), 123.8 (q,
1J ¼ 270.7 Hz), 123.5 (q, 3J ¼ 3.8 Hz), 82.9, 57.6, 55.0, 50.6, 31.0, 21.3;
LC/MS (ESIþ): m/z: calcd for C15H19F3N2O: 300.14, [M þ H]þ; found:
301.2.
mL, 0.25 mmol), and
d
4.1.3.9. 2-(Benzyloxy)benzaldehyde O-(1-isopropylpyrrolidin-3-yl)
oxime (11c). Following the general procedure, the reaction of O-(1-
isopropylpyrrolidin-3-yl)hydroxylamine hydrogen chloride 9b
(43 mg, 0.19 mmol), 2-(benzyloxy)benzaldehyde (63 mg,
0.29 mmol), and Na2CO3 (31 mg, 0.29 mmol) in methanol (1.9 mL)
gave the title compound 11c (25 mg, 37%). 1H NMR (CDCl3,
1H), 7.75 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.23e7.36 (m, 6H), 6.93 (t,
J ¼ 7.6 Hz,1H), 6.88 (d, J ¼ 8.4 Hz,1H), 4.89 (m,1H), 3.83 (s, 3H), 3.66
(d, J ¼ 4.7 Hz, 2H), 2.79 (m, 3H), 2.50 (m, 1H), 2.21 (m, 1H), 2.00 (m,
1H); 13C NMR (CDCl3, 75 MHz)
d 157.5, 145.1, 130.9, 128.9, 128.2,
126.9, 126.3, 120.7, 111.0, 82.4, 60.4, 59.6, 55.5, 52.8, 31.2; LC/MS
(ESIþ): m/z: calcd for C19H22N2O2: 310.17, [M þ H]þ; found: 311.2.
300 MHz)
d
8.53 (s, 1H), 7.81 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.28e7.42 (m,
6H), 6.95 (m, 2H), 5.08 (s, 2H), 4.88 (m, 1H), 2.84e2.95 (m, 3H), 2.62
(m, 1H), 2.49 (quin, J ¼ 6.3 Hz, 1H), 2.23 (m, 1H), 2.05 (m, 1H), 1.15
4.1.3.14. 3-(Trifluoromethyl)benzaldehyde O-(1-benzylpyrrolidin-3-
yl) oxime (12b). Following the general procedure, the reaction of
O-(1-benzylpyrrolidin-3-yl)hydroxylamine hydrogen chloride 9c
(dd, J ¼ 3.5, 6.3 Hz, 6H); 13C NMR (CDCl3, 75 MHz)
d 156.7, 145.2,
136.5, 131.0, 128.6, 128.0, 127.4, 126.5, 121.3, 121.0, 112.4, 82.2, 70.3,
57.5, 55.2, 50.6, 31.0, 21.2; LC/MS (ESIþ): m/z: calcd for C21H26N2O2:
338.2, [M þ H]þ; found: 339.2.
(41 mg, 0.15 mmol), 3-(trifluoromethyl)benzaldehyde (31 mL,
0.23 mmol), and Na2CO3 (25 mg, 0.23 mmol)in methanol (1.5 mL)
gavethe title compound 12b (26 mg, 49%).1H NMR (CDCl3, 300 MHz)
d
8.11 (s,1H), 7.84 (s,1H), 7.72 (d, J ¼ 7.7 Hz,1H), 7.60 (d, J ¼ 7.8 Hz,1H),
4.1.3.10. 2-((3,4-Dichlorobenzyl)oxy)benzaldehyde O-(1-isopropyl-
pyrrolidin-3-yl) oxime (11d). Following the general procedure, the
reaction of O-(1-isopropylpyrrolidin-3-yl)hydroxylamine hydrogen
chloride 9b (43 mg, 0.19 mmol), 2-((3,4-dichlorobenzyl)oxy)benz-
aldehyde (83 mg, 0.29 mmol), and Na2CO3 (31 mg, 0.29 mmol) in
methanol (1.9 mL) gave the title compound 11d (39 mg, 48%). 1H
7.49 (t, J ¼ 7.7 Hz, 1H), 7.23e7.36 (m, 5H), 4.93 (m, 1H), 3.69 (q,
J ¼ 11.6 Hz, 2H) 2.83 (m, 3H), 2.51 (m,1H), 2.25 (m,1H), 2.03 (m,1H);
13C NMR (CDCl3, 75 MHz)
d 147.3, 138.6, 133.3, 130.1, 129.1, 128.9,
128.2, 127.0,126.1, 123.5, 83.1, 60.3, 59.5, 52.7, 31.1; LC/MS (ESIþ): m/
z: calcd for C19H19F3N2O: 348.14, [M þ H]þ; found: 349.1.
NMR (CDCl3, 300 MHz)
d
8.48 (s, 1H), 7.80 (dd, J ¼ 7.7, 1.7 Hz, 1H),
4.1.3.15. 2-(Benzyloxy)benzaldehyde O-(1-benzylpyrrolidin-3-yl) oxime
(12c). Following the general procedure, the reaction of O-(1-
benzylpyrrolidin-3-yl)hydroxylamine hydrogen chloride 9c (46 mg,
0.17 mmol), 2-(benzyloxy)benzaldehyde (55 mg, 0.26 mmol), and
Na2CO3 (28 mg, 0.26 mmol) in methanol (1.7 mL) gave the title
7.47 (m, 2H), 7.23e7.34 (m, 2H), 6.98 (t, J ¼ 7.5 Hz, 1H), 6.88 (d,
J ¼ 8.3 Hz, 1H), 5.02 (s, 2H), 4.89 (m, 1H), 2.85e2.99 (m, 3H), 2.60
(m, 1H) 2.47 (m, 1H), 2.23 (m, 1H), 2.03 (m, 1H), 1.15 (dd, J ¼ 3.8,
6.3 Hz, 6H); 13C NMR (CDCl3, 75 MHz)
d 156.1, 144.9, 136.8, 132.7,
132.1, 131.0, 130.6, 129.2, 126.8, 126.5, 121.5, 121.4, 112.3, 82.3, 69.0,
57.5, 55.2, 50.6, 31.0, 21.2; LC/MS (ESIþ): m/z: calcd for
C21H24Cl2N2O2: 406.12, [M þ H]þ; found: 407.1.
compound 12c (17 mg, 26%). 1H NMR (CDCl3, 300 MHz)
d 8.53 (s,1H),
7.80 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.22e7.41 (m, 11H), 6.95 (m, 2H), 5.08 (s,
2H), 4.90 (m, 1H), 3.67 (q, J ¼ 6.3 Hz, 2H), 2.79 (m, 3H), 2.21 (m, 1H),
2.50 (m, 1H), 2.05 (m, 1H); 13C NMR (CDCl3, 75 MHz)
d 156.6, 144.9,
4.1.3.11. 2-((3-Fluorobenzyl)oxy)benzaldehyde O-(1-isopropyl-
pyrrolidin-3-yl) oxime (11e). Following the general procedure, the
reaction of O-(1-isopropylpyrrolidin-3-yl)hydroxylamine hydrogen
chloride 9b (45 mg, 0.20 mmol), 2-((3-fluorobenzyl)oxy)benzalde-
hyde (72 mg, 0.31 mmol), Na2CO3 (33 mg, 0.31 mmol) in methanol
(2 mL) gave the title compound 11e (27 mg, 36%). 1H NMR (CDCl3,
138.7, 136.5, 130.9, 128.9, 128.6, 128.2, 128.0, 127.4, 126.9, 126.5, 121.4,
121.0, 112.4, 82.5, 70.3, 60.4, 59.6, 52.8, 31.2; LC/MS (ESIþ): m/z: calcd
for C25H26N2O2: 386.2, [M þ H]þ; found: 387.2.
4.1.3.16. 2-((3,4-Dichlorobenzyl)oxy)benzaldehyde O-(1-benzylpy-
rrolidin-3-yl) oxime (12d). Following the general procedure, the
reaction of O-(1-benzylpyrrolidin-3-yl)hydroxylamine hydrogen
chloride 9c (52 mg, 0.19 mmol), 2-((3,4-dichlorobenzyl)oxy)benz-
aldehyde (68 mg, 0.29 mmol), and Na2CO3 (31 mg, 0.29 mmol)in
methanol (1.9 mL) gave the title compound 12d (24 mg, 22%). 1H
300 MHz)
d
8.52 (s, 1H), 7.81 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.28e7.38 (m,
2H), 7.15 (m, 2H), 6.89e7.05 (m, 3H), 5.07 (s, 2H), 4.89 (m,1H), 2.85e
2.96 (m, 3H), 2.63 (m,1H), 2.49 (m,1H), 2.24 (m,1H), 2.0 (m,1H),1.16
(dd, J ¼ 3.6, 6.3 Hz, 6H); 13C NMR (CDCl3, 75 MHz)
d 162.9 (d,
1J ¼ 244.5 Hz), 156.4, 145.1, 139.2, 139.0, 131.1, 130.2 (d, 3J ¼ 8.1 Hz),
126.7, 122.7 (d, 4J ¼ 2.7 Hz), 121.3, 114.9 (d, 2J ¼ 21.0 Hz), 114.1 (d,
2J ¼ 22.0 Hz), 112.3, 82.2, 69.5, 57.4, 55.2, 50.6, 31.0, 21.2; LC/MS
(ESIþ): m/z: calcd for C21H25FN2O2: 356.19, [M þ H]þ; found: 357.2.
NMR (CDCl3, 300 MHz)
d
8.48 (s,1H), 7.79 (dd, J ¼ 7.7,1.7 Hz,1H), 7.49
(m, 2H), 7.21e7.36 (m, 7H), 6.97 (t, J ¼ 7.5 Hz,1H), 6.88 (d, J ¼ 8.3 Hz,
1H), 5.02 (s, 2H), 4.89 (m,1H), 3.66 (q, J ¼ 11.0 Hz, 2H), 2.75e2.85 (m,
3H), 2.48 (m,1H) 2.23 (m,1H), 2.01 (m,1H);13C NMR(CDCl3, 75 MHz)
d
156.1, 144.7, 138.7, 136.8, 130.9, 130.6, 129.2, 128.9, 128.2, 126.9,
4.1.3.12. 2-((2,4-Dichlorobenzyl)oxy)benzaldehyde O-(1-isopropyl-
pyrrolidin-3-yl) oxime (11f). Following the general procedure, the
reaction of O-(1-isopropylpyrrolidin-3-yl)hydroxylamine hydrogen
chloride 9b (47 mg, 0.21 mmol), 2-((2,4-dichlorobenzyl)oxy)benz-
aldehyde (91 mg, 0.32 mmol), and Na2CO3 (35 mg, 0.32 mmol) in
methanol (2.1 mL) gave the title compound 11f (28 mg, 31%).1H NMR
126.8, 126.5, 121.4, 112.3, 82.6, 69.0, 60.4, 59.6, 52.8, 31.2; LC/MS
(ESIþ): m/z: calcd for C25H24Cl2N2O2: 454.12, [M þ H]þ; found: 455.1.
4.1.3.17. 2-((3-Fluorobenzyl)oxy)benzaldehyde O-(1-benzylpyr-
rolidin-3-yl) oxime (12e). Following the general procedure, the
reaction of O-(1-benzylpyrrolidin-3-yl)hydroxylamine hydrogen

Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
81
chloride 9c (39 mg, 0.14 mmol), 2-((3-fluorobenzyl)oxy)benzalde-
hyde (51 mg, 0.22 mmol), and Na2CO3 (24 mg, 0.22 mmol) in
methanol (1.4 mL) gave the title compound (19 mg, 32%). 1H NMR
64.5, 59.5, 57.4, 41.2, 28.4, 22.6; LC/MS (ESIþ): m/z: calcd for
C20H24N2O2: 324.18, [M þ H]þ; found: 325.2.
(CDCl3, 300 MHz)
d
8.52 (s, 1H), 7.81 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.28e
4.1.3.22. 2-((3,4-Dichlorobenzyl)oxy)benzaldehyde O-(1-methylpipe-
ridin-3-yl) oxime (13d). Following the general procedure, the reac-
tion of O-(1-methylpiperidin-3-yl)hydroxylamine hydrogen chlo-
ride 9d (69 mg, 0.34 mmol), 2-((3,4-dichlorobenzyl)oxy)
benzaldehyde (143 mg, 0.51 mmol), and Na2CO3 (54 mg, 0.51 mmol)
in methanol (3.4 mL) gave the title compound 13d (39 mg, 48%). 1H
7.38 (m, 7H), 7.15 (m, 2H), 6.89e7.05 (m, 3H), 5.12 (s, 2H), 4.89 (m,
1H), 3.67 (q, J ¼ 11.3 Hz, 2H), 2.85e2.96 (m, 3H), 2.49 (m, 1H), 2.24
(m, 1H), 2.0 (m, 1H); 13C NMR (CD3OD, 75 MHz)
d 163.0 (d,
1J ¼ 243.8 Hz), 156.7, 146.6, 139.6 (d, 3J ¼ 7.5 Hz), 131.6, 130.2, 130.0
(d, 3J ¼ 8.2 Hz), 126.2, 122.8 (d, 4J ¼ 3.0 Hz), 120.8, 114.3 (d,
2J ¼ 21.0 Hz), 113.8 (d, 2J ¼ 22.5 Hz), 112.6, 80.0, 69.2, 67.6, 38.7, 30.2,
28.7; LC/MS (ESIþ): m/z: calcd for C25H25FN2O2: 404.19, [M þ H]þ;
found: 405.2.
NMR (CDCl3, 300 MHz)
7.53 (m, 4H), 6.98 (t, J ¼ 7.3 Hz, 1H), 6.88 (d, J ¼ 8.3 Hz, 1H), 5.03 (d,
J ¼ 4.3 Hz, 2H), 4.31 (m, 1H), 4.16 (m, 1H), 3.15 (m, 1H), 2.69 (m, 1H),
d
8.51 (d, J ¼ 6.4 Hz, 1H), 7.82 (m, 1H), 7.23e
2.30 (s, 3H), 1.60e1.98 (m, 5H); 13C NMR (CDCl3, 75 MHz)
d 156.1,
4.1.3.18. 2-((2,4-Dichlorobenzyl)oxy)benzaldehyde O-(1-benzylpyr-
rolidin-3-yl) oxime (12f). Following the general procedure, the
reaction of O-(1-benzylpyrrolidin-3-yl)hydroxylamine hydrogen
chloride 9c (42 mg, 0.16 mmol), 2-((2,4-dichlorobenzyl)oxy)benz-
aldehyde (67 mg, 0.24 mmol), and Na2CO3 (25 mg, 0.24 mmol) in
methanol (1.6 mL) gave the title compound 12f (20 mg, 29%). 1H
144.5,144.2,136.8,131.0,130.6,129.2,126.8,126.7,126.6,126.5,121.5,
112.4, 69.0, 64.5, 59.5, 57.5, 41.3, 28.3, 22.7; LC/MS (ESIþ): m/z: calcd
for C20H22Cl2N2O2: 392.11, [M þ H]þ; found: 393.1.
4.1.3.23. 2-((3-Fluorobenzyl)oxy)benzaldehyde O-(1-methylpiperi-
din-3-yl) oxime (13e). Following the general procedure, the reac-
tion of O-(1-methylpiperidin-3-yl)hydroxylamine hydrogen chlo-
ride 9d (76 mg, 0.37 mmol), 2-((3-fluorobenzyl)oxy)benzaldehyde
(129 mg, 0.56 mmol), and Na2CO3 (59 mg, 0.56 mmol) in methanol
(3.7 mL) gave the title compound 13e (27 mg, 38%). 1H NMR (CDCl3,
NMR (CDCl3, 300 MHz)
d
8.51 (s, 1H), 7.79 (dd, J ¼ 7.7, 1.7 Hz, 1H),
7.48 (d, J ¼ 8.3 Hz, 1H), 7.42 (d, J ¼ 2.1 Hz, 1H), 7.24e7.43 (m, 7H),
6.98 (t, J ¼ 7.6 Hz, 1H), 6.92 (d, J ¼ 8.3 Hz, 1H), 5.13 (s, 2H), 4.90 (m,
1H), 3.67 (q, J ¼ 11.3 Hz, 2H), 2.80 (m, 3H), 2.49 (m, 1H), 2.24 (m,
1H), 2.03 (m, 1H); 13C NMR (CDCl3, 75 MHz)
d
156.1, 144.7, 138.7,
300 MHz)
d
8.55 (d, J ¼ 6.6 Hz, 1H), 7.82 (m, 1H), 7.11e7.38 (m, 4H),
134.3, 133.1, 132.9, 131.0, 129.6, 129.2, 128.9, 128.2, 127.4, 126.9,
126.8, 121.4, 112.4, 82.6, 67.0, 60.4, 59.6, 52.8, 31.2; LC/MS (ESIþ): m/
z: calcd for C25H24Cl2N2O2: 454.12, [M þ H]þ; found: 455.1.
6.99 (m, 3H), 5.07 (d, J ¼ 4.3 Hz, 2H), 4.27 (m, 1H), 4.14 (m, 1H), 3.13
(m, 1H), 2.72 (m, 1H), 2.31 (s, 3H), 1.60e1.86 (m, 5H); 13C NMR
(CDCl3, 75 MHz)
d
163.8 (d, 1J ¼ 231.7 Hz), 144.5, 144.3, 130.8 (d,
3J ¼ 9.3 Hz), 130.1 (d, 3J ¼ 8.0 Hz), 126.6 (d, 4J ¼ 4.7 Hz), 122.7, 121.2,
114.7, 114.2 (d, 2J ¼ 21.8 Hz), 114.1 (d, 2J ¼ 21.8 Hz), 112.4, 112.3, 69.5,
64.4, 59.6, 57.6, 41.4, 28.5, 22.6,; LC/MS (ESIþ): m/z: calcd for
C20H23FN2O2: 342.17, [M þ H]þ; found: 343.2.
4.1.3.19. 2-Methoxybenzaldehyde O-(1-methylpiperidin-3-yl) oxime
(13a). Following the general procedure, the reaction of O-(1-
methylpiperidin-3-yl)hydroxylamine hydrogen chloride 9d
(70 mg, 0.34 mmol), O-anisaldehyde (62
Na2CO3 (54 mg, 0.52 mmol) in methanol (3.4 mL) gave the title
compound 13a (33 mg, 57%). 1H NMR (CDCl3, 300 MHz)
8.50 (d,
mL, 0.52 mmol), and
4.1.3.24. 2-((2,4-Dichlorobenzyl)oxy)benzaldehyde O-(1-methylpipe-
ridin-3-yl) oxime (13f). Following the general procedure, the reac-
tion of O-(1-methylpiperidin-3-yl)hydroxylamine hydrogen chlo-
ride 9d (67 mg, 0.37 mmol), 2-((2,4-dichlorobenzyl)oxy)
benzaldehyde (139 mg, 0.49 mmol), Na2CO3 (53 mg, 0.49 mmol) in
methanol (3.3 mL) gave the title compound 13f (28 mg, 31%). 1H
d
J ¼ 7.8 Hz, 1H), 7.70 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.31 (m, 1H), 6.89 (m,
2H), 4.30 (m, 1H), 4.17 (dd, J ¼ 11.0, 5.2 Hz, 1H), 3.83 (d, J ¼ 3.5 Hz,
3H), 3.17 (m, 1H), 2.71 (m, 1H), 2.30 (s, 3H), 1.60e2.01 (m, 5H); 13C
NMR (CDCl3, 75 MHz)
d 157.5, 144.8, 130.9, 126.3, 120.7, 111.0, 64.5,
59.6, 57.5, 41.3, 28.4, 22.6; LC/MS (ESIþ): m/z: calcd for C14H20N2O2:
248.15, [M þ H]þ; found: 249.2.
NMR (CDCl3, 300 MHz)
d
8.55 (d, J ¼ 6.3 Hz, 1H), 7.82 (m, 1H), 7.48
(dd, J ¼ 8.3, 5.8 Hz, 1H), 7.43 (d, J ¼ 2.0 Hz, 1H), 7.25e7.35 (m, 2H),
6.99 (t, J ¼ 7.5 Hz,1H), 6.92 (d, J ¼ 8.2 Hz,1H), 5.14 (d, J ¼ 3.3 Hz, 2H),
4.33 (m, 1H), 4.23 (m, 1H), 3.14 (m, 1H), 2.66 (m, 1H), 2.31 (s, 3H),
4.1.3.20. 3-(Trifluoromethyl)benzaldehyde O-(1-methylpiperidin-3-
yl) oxime (13b). Following the general procedure, the reaction of
O-(1-methylpiperidin-3-yl)hydroxylamine hydrogen chloride 9d
1.61e1.99 (m, 5H); 13C NMR (CDCl3, 75 MHz)
d 144.5, 144.3, 134.3,
133.2, 132.9, 131.0, 130.9, 129.7, 129.3, 127.4, 126.7, 121.5, 112.5, 67.0,
64.5, 59.5, 57.6, 41.4, 28.4, 22.6; LC/MS (ESIþ): m/z: calcd for
C20H22Cl2N2O2: 392.11, [M þ H]þ; found: 393.1.
(74 mg, 0.36 mmol), 3-(trifluoromethyl)benzaldehyde (73
mL,
0.54 mmol), and Na2CO3 (58 mg, 0.54 mmol) in methanol (3.6 mL)
gave the title compound 13b (35 mg, 51%). 1H NMR (CDCl3,
300 MHz)
d
8.13 (d, J ¼ 5.1 Hz, 1H), 7.83 (s, 1H), 7.73 (d, J ¼ 7.6 Hz,
4.2. Biological evaluation
1H), 7.59 (m,1H), 7.49 (m,1H), 4.35 (m,1H), 4.17 (dd, J ¼ 11.0, 5.2 Hz,
1H), 3.14 (m, 1H), 2.66 (m, 1H), 2.30 (s, 3H), 1.61e2.03 (m, 5H); 13C
4.2.1. Cell culture
NMR (CD3OD, 75 MHz)
d
149.5, 132.9, 132.7, 130.5, 129.4 (q,
HT-22 (mouse hippocampal cells) cells were grown in Dulbec-
co’s Modified Eagle’s Medium (DMEM, GIBCO) supplemented with
3J ¼ 3.75 Hz), 126.4 (q, 3J ¼ 6.0 Hz), 123.9 (q, 1J ¼ 270.0 Hz), 123.2 (q,
3J ¼ 4.5 Hz), 77.8, 74.4, 55.6, 43.0, 26.1, 21.8; LC/MS (ESIþ): m/z: calcd
for C14H17F3N2O: 286.13, [M þ H]þ; found: 287.2.
10% (vol/vol) FBS and antibiotics (100
mg/mL penicillin/strepto-
mycin mix) in a humidified atmosphere at 37 ꢀC with 5% CO2.
4.1.3.21. 2-(Benzyloxy)benzaldehyde O-(1-methylpiperidin-3-yl) oxime
(13c). Following the general procedure, the reaction of O-(1-
methylpiperidin-3-yl)hydroxylamine hydrogen chloride 9d (70 mg,
0.34 mmol), 2-(benzyloxy)benzaldehyde (110 mg, 0.52 mmol),
4.2.2. JC-1 mitochondrial membrane potential assay
HT-22 cells (30,000 per well) were seeded into a clear 96-well
plate (FALCON) at 200 mL per well one day prior to assay. 750 mM
of JC-1 (Stratagene) in DMSO stock solution was dissolved into
Na2CO3 (55 mg, 0.52 mmol) in methanol (3.4 mL) gave the title
phenol red-free Opti-MEM (GIBCO) medium to make final
1
compound 13c (25 mg, 37%). H NMR (CDCl3, 300 MHz)
d
8.56 (d,
concentration of 7.5
the plate, and 100
m
M JC-1 per well. Medium was removed from
J ¼ 5.8 Hz, 1H), 7.82 (m, 1H), 7.25e7.42 (m, 6H), 6.96 (m, 2H), 5.09 (d,
mL per well of JC-1 was added. Plates were
J ¼ 4.4 Hz, 2H), 4.31 (m, 1H), 4.18 (m, 1H), 3.14 (m, 1H), 2.66 (m, 1H),
incubated for 1 h and 15 min at 37 ꢀC and washed twice with 100
mL
2.32 (s, 3H), 1.61e1.78 (m, 5H); 13C NMR (CDCl3, 75 MHz)
d
156.6,
per well PBS. Subsequently, cells were treated with 25
mL solution
144.8, 144.5, 136.5, 130.9, 128.6, 128.0, 127.4, 126.5, 121.0, 112.5, 70.3,
of each compound at 10
m
M in Opti-MEM and incubated at 37 ꢀC for

82
Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
10 min followed by addition of 25
mL of amyloid Beta (American
containing carbamazepine (internal standard) was added to the
peptide, 1e42) solution at 10
mM. Fluorescence was measured at
plasma sample. The plasma samples were then centrifuged and the
every 1 h for 3 h at ex/em 530 nm/580 nm (‘red’) and ex/em
485 nm/530 nm (‘green’). The ratio of green to red fluorescence was
recorded and the percent changes in ratio from each compound
were calculated and normalized using vehicle control as 100%.
supernatants were analyzed by LCeMS/MS.
4.3.2. HLM (human liver microsomes) stability assay
To determine the microsomal stability of the selected
compounds, pooled human liver microsomes (HLM’s) containing
20 mg/mL of total protein were used and purchased from BD
Biosciences (Bedford, MA). A compound was incubated with HLM
at 37 ꢀC, in the presence of an NADPH, in a final incubation volume
4.2.3. Assay for cellular ATP levels
10,000 HT-22 cells per well were seeded into a clear 96-well
plate (FALCON) at 200
Medium was removed from the plate, and cells were treated with
25 L solution of each compound at 10
M and incubated at 37 ꢀ
for 10 min followed by addition of 25 L of amyloid Beta (American
peptide, 1e42) solution at 10
M. Cells were incubated at 37 ꢀC for
mL per well one day prior to assay.
of 200
mL. The final incubation solutions contain 1 mM of the
m
m
C
compound, 1.2 mM NADPH, 0.5 mg/mL (total protein) microsomes,
100 mM phosphate buffer (pH 7.4). The incubation tubes were then
incubated in a water-bath shaker at 37 ꢀC for 30 min. At the time
point, two incubation tubes were removed from the water-bath for
m
m
7 h and washed twice with PBS. Cells were lysed by using 1% Triton-
X 100 in TBST buffer solution and protein concentrations of each
well were determined via BCA protein determination kit (Thermo
scientific). Equal amount of cell lysates from each well were plated
into a white 96-well plate (NUNC) and the amount of ATP levels in
each sample was determined by using ATP determination kit
(Invitrogen). The ATP levels of each sample were subtracted with
vehicle control and percent inhibition were calculated based on the
ATP levels of the vehicle control treated with amyloid Beta. Cell
viability was also calculated based on the ATP levels of each sample
without the treatment of amyloid Beta solution.
duplicate and the reaction was terminated immediately. The 400
mL
of ice-cold acetonitrile containing 0.1 g/mL of internal standard
m
was added to microsomal incubate to terminate the reaction and
precipitate the proteins. The incubation solutions were then
centrifuged and the supernatants were analyzed by LCeMS/MS.
4.3.3. Pharmacokinetic studies
Each compound at a dose of 10 mg/kg was administered intra-
venously or orally to male Sprague Dawley rats. Blood samples were
collected via carotid artery at 0 (to serve as a control),10 (IV only), 50,
150, 0.5, 1, 2, 4, 6, and 8 h after administration of each compound.
4.2.4. MTT assay
After centrifugation at 3000 rpm for 10 min, a 50-mL aliquot of
5000 HT-22 cells per well were seeded and treated as above
plasma samples were stored at ꢂ70 ꢀC until analysis. Pharmacoki-
netic parameters were determined by a non-compartmental anal-
ysis using WinNonlinÒ (Pharsight Corporation, Mountain View, CA)
program. The total area under the plasma concentrationetime curve
from time zero to last measured time (AUClast) was calculated by the
trapezoidal ruleeextrapolation method. Standard methods were
used to calculate the following pharmacokinetic parameters [23];
the time-averaged total body clearance (CL), total area under the
first moment of plasma concentrationetime curve from time zero to
time infinity (AUC0eN), terminal half-life, mean residence time
(MRT), apparent volume of distribution at steady state (Vss). Animal
experimentation was performed according to Guidelines for
Accommodation and Care of Animals.
described method. Cells were incubated at 37 ꢀC for 24 h 10
mL of
MTT solution (Thiazolyl blue tetrazolium bromide, Sigma) was
added directly to each well and incubated at 37 ꢀC for 2 h. After
confirming the formation of blue formazan precipitates under
microscope, 140 mL of solubilizing solution (10% Triton-X 100 in
Isopropanol with 0.1 M HCl) was added to each well and incubate
for another hour at room temperature. Absorbance at 570 nM was
measured and OD values from each well were subtracted with
vehicle control and percent inhibition and cell viability were
calculated by using the same method described for the ATP assay.
4.2.5. ROS assay
10,000 HT-22 cells per well were seeded in black/clear bottom
plate and treated with each compound and amyloid Beta in Opti-
4.3.4. Quantitative analysis
MEM for 6 h. Cells were washed once with HBSS. 100
H2DCFDA (Invitrogen C6827) solution, prepared from dilution of
L of the 1 mM stock solution into 2 mL media (HBSS), was added
into each well. Cells were incubated at 37 ꢀC for 30 min and washed
twice with HBSS. 100 L of nuclear staining (Hoechst), prepared
from dilution of 1 L of nuclear staining into 1 mL HBSS, were added
into each well. ROS production was measured by capturing fluo-
rescent images from each well, then calculated the total signal
intensity from each well by using Operetta high content screening
system (Perkin Elmer). Percent inhibition against amyloid beta-
induced ROS production was determined by calculating percent
ratio of the increased total signal intensity from compounds treated
m
L of CM-
Concentrations of each compound in the above samples were
analyzed using LC-MS/MS. To a 50
a 100 L aliquot of acetonitrile containing 0.1
standard was added. After vortex mixing and centrifugation at
10,000 rpm for 10 min, a 5 L of supernatant was injected onto LCe
mL aliquot of plasma sample,
2
m
m
mg/mL of internal
m
m
m
MS/MS system. The LCeMS/MS system consisted of a HP1100Ò
HPLC system (Agilent, Santa Clara, CA) and API3200Ò triple-
quadrupole mass spectrometer (Applied Biosystems-SCIEX,
Concord, Canada). The HPLC mobile phases consisted of 0.1% for-
mic acid (A) and 0.1% formic acid in 90% acetonitrile (B). Chro-
matographic separation was achieved on a reversed-phase XterraÒ
C18 column (50 ꢁ 2.1 mm, 3.5
mm, Waters Corporation, Milford, MA)
cells to untreated cells in the presence of Ab1e42
4.3. Evaluation of stability and pharmacokinetics
4.3.1. Plasma stability
.
using gradient elution at a flow rate of 0.3 mL/min. The lower limit
of quantitation of each compound in rat plasma was 5 ng/mL. The
values of coefficients of correlation (R) were more than 0.997.
To determine the stability of the selected compounds in plasma,
a stock solution of each compound in acetonitrile (50 g/mL) was
spiked into polyethylene tubes containing drug free rat plasma to
Acknowledgments
m
This research was supported by the Korea Institute of Science and
Technology (KIST) and the Basic Science Research Program through
the National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (NRF2009-0081670).
produce a concentration of 0.5 mg/mL. Each tube was incubated in
a water-bath shaker kept at 37 ꢀC for 0, 15, 30, 60, and 120 min,
respectively. At each time point, 2ꢁ volume of acetonitrile

Y.S. Kim et al. / European Journal of Medicinal Chemistry 62 (2013) 71e83
83
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