Synthesis, biological evaluation, and metabolic stability of acrylamide derivatives as novel CCR3 antagonists

Article abstract of DOI:10.1016/j.bmc.2009.06.066

Our laboratory has identified several acrylamide derivatives with potent CCR3 inhibitory activity. In the present study, we evaluated the in vitro metabolic stability (CL_int; mL/min/kg) of these compounds in human liver microsomes (HLMs), and a

Full text of DOI:10.1016/j.bmc.2009.06.066

Bioorganic & Medicinal Chemistry 17 (2009) 5989–6002
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and metabolic stability of acrylamide
derivatives as novel CCR3 antagonists
a
a
a
a
a
Ippei Sato a, , Koichiro Morihira , Hiroshi Inami , Hirokazu Kubota , Tatsuaki Morokata , Keiko Suzuki ,
Kazuki Ohno a, Yosuke Iura b, Aiko Nitta b, Takayuki Imaoka b, Toshiya Takahashi b, Makoto Takeuchi a,
Mitsuaki Ohta a, Shin-ichi Tsukamoto a
*
a Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
b Pharmaceutical Research Laboratories, Toray Industries, Inc., 6-10-1 Tebiro, Kamakura, Kanagawa 248-0036, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Our laboratory has identified several acrylamide derivatives with potent CCR3 inhibitory activity. In the
present study, we evaluated the in vitro metabolic stability (CLint; mL/min/kg) of these compounds in
human liver microsomes (HLMs), and assessed the relationship between their structures and CLint values.
Among the compounds identified, N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-[1-(2-
hydroxybenzoyl)piperidin-4-ylidene]acetamide (30j) was found to be a potent inhibitor (IC50 = 8.4 nM)
with a high metabolic stability against HLMs.
Received 3 June 2009
Revised 25 June 2009
Accepted 26 June 2009
Available online 3 July 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Allergic diseases
CCR3 antagonists
Acrylamide derivatives
CLint
1. Introduction
In previous papers, we reported a series of 6-fluoro-2-naphthyl
derivatives, 13a and 2,3b as novel types of CCR3 antagonists (Fig. 1).
The chemokines are a family of small (8–12 kDa), heparin-bind-
ing, basic proteins that chemoattract and activate leukocytes such
as monocytes, lymphocytes, eosinophils, and basophils. To date,
over 50 distinct chemokines have been characterized and subse-
quently classified into four sub-families, CC chemokines, CXC che-
mokines, C chemokines, and CX3C chemokines. This classification is
based on the positions of their first two conserved N-terminal cys-
teine residues. The chemokine receptors are members of the se-
ven-transmembrane G-protein coupled receptor (GPCR) family,
which are expressed on various immune and inflammatory cells.
Chemokines bind to and activate these receptors in order to exert
their biological effects.1a–e
CC chemokine receptor 3 (CCR3), which is predominantly ex-
pressed on eosinophils, plays an important role in mediating the
eosinophil chemotaxis induced by eosinophilic chemokines such
as eotaxin-1, -2, -3, MCP-3, -4, and RANTES. The recruitment of
eosinophils to sites of inflammation is thought to be a feature of
various inflammatory diseases, such as allergic asthma and atopic
dermatitis. Thus, using small molecule CCR3 antagonists to inhibit
this recruitment process may lead to significant advances in the
treatment of eosinophil-related allergic diseases.2a,b
However, their ability to inhibit CCR3 (IC50 = 20 and 23 nM, respec-
tively) was slightly weaker than that of other CCR3 inhibitors re-
ported by other groups.4a–c Additionally, compound 1 was found
to have a potent CYP2D6 inhibition, as previously disclosed, and
further examination revealed that compound 2, which had im-
proved CYP2D6 inhibition, exhibited poor permeability in a PAMPA
(pH 6.5: 4.7 ꢀ 10ꢁ6 cm/s) likely due to the low C log D7.4 value
caused by the N-oxide moiety. Therefore, to improve the CCR3
inhibitory activity of compound 1, we attempted the further struc-
tural optimization of 1 in the early stages of this study by adding a
novel backbone. We planned a synthetic strategy based on previ-
ously reported key pharmacophores for CCR3 inhibitory activity.
One pharmacophore is a basic nitrogen atom in the center of the
molecule, and the other pharmacophores are the two aryl rings, a
6-fluoronaphthalene moiety and another aryl ring, at the terminus
(Fig. 2).3a We first modified the linkage between the phenylene ring
and the basic nitrogen atom of 1 and found that (3R)-17, which has
a pyrrolidine ring instead of the tropane ring, showed a moderate
activity. We chose this pyrrolidine derivative as the next lead com-
pound, and its optimization led to the discovery of several acryl-
amide derivatives that were potent CCR3 inhibitors.
In this paper, we detail the results of research on the structure–
activity relationships (SARs) of these acrylamide derivatives as
well as their in vitro metabolic stability against human liver micro-
somes (HLMs).
* Corresponding author. Tel.: +81 29 863 6732; fax: +81 29 852 5387.
E-mail address: ippei.sato@jp.astellas.com (I. Sato).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.066

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I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
and appropriate carboxylic acids via condensation with WSCꢂHCl.
The Boc group of 23 was removed under acidic conditions, and sub-
sequent benzoylation yielded 24. Benzenesulfonyl isocyanate was
reacted with the free amines of (3R)-16 and 183a to afford 22a
and 22b, respectively.
O
N
H
N
Scheme 3 illustrates the synthesis of compounds 26, 29, and
30a –30s. The condensation of (3R)-16 with (diethoxyphospho-
ryl)acetic acid quantitatively yielded Horner–Wadsworth–Em-
mons (HWE) reagent 25. Compounds 26 and 27 were obtained
via the HWE reaction between 25 and appropriate ketones. By
using compound 25 slightly more than sodium hydride in this step,
a good yield of the exo-olefin compound was selectively obtained.
The Boc group of 27 was removed under acidic conditions, and sub-
sequent condensation with the corresponding commercially avail-
able carboxylic acids or acid chlorides yielded 29 and 30a–30s. The
intermediate ketone (34), which corresponded to compound 26,
was prepared via the Mitsunobu reaction between 33 and phenol,
followed by acidic hydrolysis of the ketal group (Scheme 4).
F
1
CCR3: IC50 = 20 nM
N
O
O
N
H
N
N+
-O
F
2
3
CCR3: IC50 = 23 nM
O
N
F
N
3. Results and discussion
H
CCR3: IC50 = 38 nM
Each synthesized compound’s ability to inhibit (IC50) eotaxin-
induced Ca2+ influx was evaluated using CCR3-expressing pre-B
cells. The lead compound 1 demonstrated potent CCR3 inhibitory
activity with an IC50 value of 20 nM.
Figure 1. The structure of our previously reported compounds.3a,b
We previously reported that for optimal CCR3 inhibitory activ-
ity, the distance between the phenylene ring and the basic nitrogen
of 1 was a more important factor than the nature of the linkage ele-
ments and that the piperidine derivative 3 was approximately two-
fold less potent than 1.3a In order to find alternative linkage
structures, we initially reinvestigated the effect of replacing the
tropane ring of 1 with another nitrogen-containing ring system
(Table 1). Homopiperazines (7 and 8) and piperazine (11) deriva-
Key-pharmacophore for CCR3 inhibitory activity
O
N
N
H
tives had no inhibitory effect at 10
length.
lM, irrespective of their linkage
F
However, the activity of 3-aminopiperidine [(rac)-14] and the
3-aminopyrrolidine [(rac)-17] derivatives was moderate. Compar-
ison of the two enantiomers of (rac)-17 revealed that the (3R)-iso-
mer was approximately 20-fold more potent than the (3S)-isomer.
Therefore, the structure–activity relationships (SARs) of the (3R)-3-
aminopyrrolidine derivative [(3R)-17] were further studied.
To simplify the SAR results, the terminal phenyl ring of the
biphenyl moiety was removed, and the amide linkage between
the phenyl ring and pyrrolidine was converted. The results were
compared with those of piperidine derivatives that were already
reported (19b and 21b) (Table 2).3a The results obtained for pyrrol-
idine derivatives were markedly different from those obtained for
piperidine derivatives. That is, while the length of linkage was an
important factor for determining the inhibitory potency of piperi-
dine derivatives, the potency of the pyrrolidine derivatives did not
change after elongation of the linker. Among these compounds, the
acrylamide derivative (21a) showed the most potent activity (Ta-
ble 2). Based on these data, we decided to further elongate the lin-
ker part of 21a by incorporating a rigid structure between the
phenyl ring and the acrylamide double bond. We expected toler-
ance for various structural conversions in the linker part since all
compounds listed out in Table 2 exhibited a moderate activity.
The results are summarized in Table 3. The piperidinecarboxamide
derivatives (30a), in which the acrylamide double bond was exo-
substituted at the 4-position on the piperidine ring, showed extre-
mely potent inhibitory activity (IC50 = 4.4 nM). Compared with
30a, compound 24, which has a linkage one atom longer than that
of 30a, showed a weak activity. Compound 26, the cyclohexyl ana-
logue of 30a, also proved to be less active than 30a, whose potency
could be attributed to a newly introduced amide oxygen atom. We
linker part
1
Figure 2. The key pharmacophore for CCR3 inhibitory activity.3a
2. Chemistry
Scheme 1 summarizes the synthesis of compounds 7, 8, 11,
(rac)-14, (rac)-17, (3S)-17, and (3R)-17. The alkylation of homo-
piperazine (4) with 2-(bromomethyl)-6-fluoronaphthalene (5)5
yielded amine 6. The condensation of 6 with biphenyl-2-carboxylic
acid and biphenyl-2-ylacetic acid using WSCꢂHCl as the coupling
reagent afforded 7 and 8, respectively. The alkylation of tert-butyl
piperazine-1-carboxylate (9) with 5, followed by removal of the
Boc group under acidic condition yielded the amine 10, which
was converted into 11 in the same manner as for 8. Compound
(rac)-13 was synthesized by condensation of the amine (rac)-12
with biphenyl-2-carboxylic acid, followed by deprotection of the
Boc group. The alkylation of (rac)-13 with 5, and subsequent con-
version to its hydrochloride salt via treatment with 4 M HCl (g)/
EtOAc yielded (rac)-14. Compounds (rac)-17, (3S)-17, and (3R)-
17 were synthesized in the same manner as for 11, respectively.
The synthetic routes of compounds 19a, 20a,b, 21a, 22a,b, and
24 are shown in Scheme 2. Compounds 19a and 20a were prepared
from (3R)-16 and appropriate acid chlorides, and for compound
20b, compound 183a was used as a starting material instead of
(3R)-16. Compounds 21a and 23 were prepared from (3R)-16

I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
5991
HCl
O
a
b
Br
NH
N
HN
HN
(
)n N
N
+
+
F
F
F
n = 0 ; 7
n = 1 ; 8
4
5
6
O
b
c
2HCl
Br
NH
BocN
N
N
HN
N
F
F
F
HCl
9
5
10
11
H
H2N
H
e
d
NBoc
N
N
N
NH
O
O
F
HCl
(rac)-12
(rac)-13
(rac)-14
f
oxalate
g
N
NH
Boc
O
H2N
N
H
N
F
2HCl
N
F
H
(rac)-15
(3S)-15
(3R)-15
(rac)-16
(3S)-16
(3R)-16
(rac)-17
(3S)-17
(3R)-17
Scheme 1. Synthesis of compounds 7, 8, 11, 14, and 17. Reagents and conditions: (a) K2CO3, MeCN, rt, 20 h, 77%; (b) ArCOOH, WSCꢂHCl, HOBt, DMF, rt, then 4 M HCl/EtOAc; (c)
K2CO3, MeCN, rt, 4.5 h, then 4 M HCl/EtOAc, EtOAc–MeOH, rt, overnight, 86% for two steps; (d) biphenyl-2-carboxylic acid, WSCꢂHCl, HOBt, THF, rt, 18 h, then 2 M HCl/EtOAc,
rt, 4 h, 21% for two steps; (e) 5, K2CO3, MeCN, rt, 25 h, then 4 M HCl/EtOAc, 20% for two steps; and (f) 5, K2CO3, MeCN, rt, then, 4 M HCl/EtOAc, EtOAc, rt; (g) biphenyl-2-
carboxylic acid, WSCꢂHCl, HOBt, Et3N, DMF, rt, then oxalic acid.
O
O
2HCl
H2N
O
N
( )n
c
S
N
N
( )n
N
H
F
H
F
n = 1 ; 22a
n = 2 ; 22b
n = 1 ; (3R)-16
n = 2 ; 18
O
a or b
O
O
O
*
R2N
*
*
R1 =
*
F
N
( )n
R1
n =1;R2 = Boc ; 23
= Bz ; 24
N
n = 1 ; 20a
n = 2 ; 20b
d
F
n = 1 ; 21a
H
n = 1 ; 19a
Scheme 2. Synthesis of compounds 19–22, and 24. Reagents and conditions: (a) ArCOCl, Et3N, CH2Cl2, rt; (b) ArCOOH, WSCꢂHCl, HOBt, Et3N, DMF, rt; (c) benzenesulfonyl
isocyanate, MeCN, 0 °C; and (d) 4 M HCl/EtOAc, EtOAc, rt, overnight, then BzCl, Et3N, CH2Cl2, rt, 3 h, 41% for two steps.
believed that the rigid structure of the piperidinecarboxamide
moiety enhanced inhibitory activity by locking the terminal ben-
zoyl moiety into an active conformation. In order to confirm the
importance of the terminal phenyl ring, the phenyl moiety of 30a
was replaced with a methyl group. The activity of the acetamide
derivative (29) significantly decreased relative to that of 30a. These
results also suggested that the newly added carbonyl moiety and
terminal phenyl ring would work synergistically to increase the
CCR3 inhibitory activity. Thus, we created a potent inhibitor with
high novelty by modifying the acrylamide derivatives.
vs 4.7 ꢀ 10ꢁ6 cm/s), its metabolic stability against human liver
microsomes (HLMs) in vitro was poor. Its clearance value (CLint va-
lue) was 338 mL/min/kg, which is greater than those of 2 (89 mL/
min/kg) and several commercial drugs.6
With the aim of improving both activity and metabolic stability,
we next investigated the effects of various substituents around the
benzoyl moiety of 30a (Table 4). The introduction of an electron-
withdrawing fluorine atom at the 2-position (30b) on the phenyl
ring of 30a yielded a similar activity, but the same introduction
at the 3- or 4-position led to activity decreases of about threefold
and fivefold, respectively. Introduction of an electron-donating
methoxy moiety, at the 2- or 3-position retained the activity of
However, although compound 30a showed better membrane
permeability than 2, as measured in a PAMPA (19.9 ꢀ 10ꢁ6 cm/s

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I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
N
H2N
F
2HCl
(3R)-16
a
PhO
b
O
P
O
O
O
O
N
N
N
H
N
F
F
H
26
25
b
O
6
2
5
R1
3
4
d or e
N
N
O
O
R2
N
N
N
H
N
H
F
F
R1 = Boc ; 27
R1 = H ; 28
c
R2 =
H ; 30a
2-OH ; 30j
3-OH ; 30k
4-OH ; 30l
2-OH-3-MeO ; 30m
2-OH-4-MeO ; 30n
2-OH-5-MeO ; 30o
2-OH-6-MeO ; 30p
2-OH-4-F ; 30q
2-F ; 30b
3-F ; 30c
4-F ; 30d
2-MeO ; 30e
3-MeO ; 30f
4-MeO ; 30g
3,4-MeO ; 30h
d
O
N
O
N
N
H
F
2-OH-4-Cl ; 30r
2-OH-4-Me ; 30s
3,4-OCH2O- ; 30i
29
Scheme 3. Synthesis of compounds 26, 29, and 30a–30s. Reagents and conditions: (a) (diethoxyphosphoryl)acetic acid, WSCꢂHCl, HOBt, Et3N, DMF, rt, 3.5 h, quant.; (b) NaH
(60% oil dispersion), DME, rt, 0.5 h, then ketone, DME, rt; (c) TFA, CH2Cl2, rt, 0.5 h, 97%; and (d) ArCOCl, CH2Cl2; (e) ArCOOH, WSCꢂHCl, HOBt, THF, rt.
a, b
HO
O
sulted in larger CLint values than that for 30a. Furthermore, both
3,4-dimethoxy (30h) and the 3,4-methylenedioxy (30i) derivatives
had CLint values smaller than that for 30a. These results suggested
that the 4-position of benzoyl moiety would be the most suscepti-
ble to metabolic reactions induced by HLMs, and that the CLint val-
ues could be improved by blocking there. The phenol derivatives
(30j, 30k, and 30l) yielded interesting results; the CLint value of
the 4-substituted derivative (30l) was smaller than that of 3-
substituted derivative (30k), which is consistent with the results
described above. However, that of the 2-substituted derivative
(30j) was much smaller than that of 30l, which had the smallest
CLint value in this series of compounds. One reason why this oc-
curred could be the intramolecular hydrogen bond (confirmed to
exist in the NMR spectrum of 30j) between the phenolic hydroxyl
moiety and carbonyl oxygen of the amide moiety. In addition, the
geometrically optimized structure of the model compound (N-2-
hydroxybenzoylpiperidine), which was obtained by calculation
on the HF/6-31* level using the GAUSSIAN 98 program9, also sug-
gested this (Fig. 3). We speculated that this carbonyl moiety plays
an effective role in the interaction between CYPs of hepatic micro-
somes and ligands, and that the intramolecular hydrogen bond in
30j decreased their interaction. To confirm this speculation, we
evaluated the ability of the carbonyl moiety to accept the hydrogen
bond by calculating the interaction energies between ligands
(using substituted N-benzoylpiperidine as a model compound)
and water molecule (as a model of hydrogen bond donor in a
receptor protein). The detailed method is described in Section 5.
O
O
O
33
34
Scheme 4. Synthesis of compound 34. Reagents and conditions: (a) phenol, PPh3,
DEAD, THF, rt, 12 h, 88% and (b) 3 M HCl aq, THF, rt, 8 h, 72%.
30a, while introduction at the 4-position (30g) slightly enhanced
the activity (IC50 = 1.8 nM). The activity of the 3,4-dimethoxy
derivative (30h) was similar to that of 30a, and the 3,4-methylene-
dioxy derivative (30i) exhibited
a slightly improved activity
(IC50 = 1.6 nM). Incorporation of the hydroxyl moiety at the 2-posi-
tion (30j) slightly reduced the inhibitory activity, while that at the
3- or 4-position resulted in activities equal to that of 30a. These re-
sults indicated that the CCR3 inhibitory activity was more suscep-
tible to an electron factor than to a steric factor placed near the
terminal benzoyl part, and that the incorporation of electron-
donating groups at the 4-position would improve the activity.
This series of compounds was also evaluated for its in vitro hu-
man liver microsomal (HLM) metabolic stability (CLint; mL/min/
kg). For both the fluorine- and methoxy-substituted derivatives,
the rank order for metabolic stability (CLint) is: 2-substituted > 3-
substituted > 4-substituted. The 4-fluorine (30d) and 4-methoxy
(30g) derivatives generated much smaller CLint values than 30a.
On the other hand, both the 2- and 3-substituted derivatives re-

I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
5993
Table 1
Table 2
CCR3 inhibitory activity of 6-fluoronaphthalene derivatives
CCR3 inhibitory activity of 6-fluoronaphthalene derivatives
R
N
( )n
R
F
N
H
F
Compound
Ra
IC50 (nM)
b
b
Compound
Ra
n
IC50 (nM)
20 3.9
1c
2d
3c
20 3.9
23 4.7
38 5.4
1c
3c
38 5.4
230 72
Ph
O
O
*
N
Me
Me
Me
19a
1
2
7
NE@10
NE@10
NE@10
l
l
l
N
O
*
19bb
640 270
F
20a
20b
21a
21bb
1
2
1
2
170 9.1
300 150
100 21
*
O
N
8
N
N
Ph
Ph
*
O
*
*
N
*
*
11
9700 3700
O
O
O
O
22a
1
2
250 60
S
H
N
N
(rac)-14
(rac)-17
(3S)-17
550 180
H
N
22b
2000 470
Ph
Ph
O
O
a
Structures of 1 and 3 are shown in Figure 1.
b
c
The IC50 values are shown as the means SEM for at least three determinations.
See Ref. 3a.
*
N
320 89
N
H
derivative (30n) was slightly more potent than that of 30j
(IC50 = 3.3 nM). A substitution at the 5-position (30o) resulted in
less activity (IC50 = 14 nM) and those at the 3- (30m) and 6-posi-
tions (30p) resulted in much less activity than that of 30j
(IC50 = 38 and 45 nM, respectively). The effects of introducing other
substituents at the 4-position were then investigated (Table 6).
While both the electron-withdrawing fluorine and chlorine deriv-
atives had an activity equal to that of 30j, the electron-donating
methyl derivative exhibited the most potent activity in this series
of compounds (IC50 = 0.98 nM).
As we expected, all phenolic derivatives listed out in Table 6
had CLint values smaller than those of the corresponding non-phe-
nolic derivatives (30p vs 30e, 30f vs 30m and 30o, 30n vs 30g, and
30d vs 30q). However, all their CLint values were larger than that of
30j, which indicates that the increase in molecular lipophilicity
caused by each additional substituent was responsible for the
microsomal metabolic lability and that additional methoxy and
methyl moieties would be sensitive to be metabolized by HLMs.
For our series of acrylamide derivatives, the structural modification
of the benzoyl moiety produced substantial changes in metabolic
stability against HLMs.
Ph
Ph
O
O
*
*
N
N
2500 1100
N
H
(3R)-17
140 16
N
H
a
Structures of 1, 2, and 3 are shown in Figure 1.
The IC50 values are shown as the means SEM for at least three determinations.
b
c
See Ref. 3a.
See Ref. 3b.
No effect at 10 lM.
d
e
The results showed that the interaction energy between the N-2-
hydroxybenzoylpiperidene, the model compound of 30j, and a
water molecule was much lower than that of the other model li-
gands (Table 5). This result led us to predict that the degree of
interaction energy between 30j and CYPs would be lower than that
of the other compounds because of the intramolecular hydrogen
bond, and that this weaker interaction meant metabolic stability.
These favorable results prompted us to attempt to further im-
prove the CCR3 inhibitory activity and retain the metabolic stabil-
ity of 30j, and we expected to do this by incorporating an
additional substituent at the benzoyl part (Table 6). First, the elec-
tron-donating methoxy moiety that increased the inhibitory activ-
ity in the study of the mono-substituted derivative (described
above) was substituted at the most appropriate position. The re-
sults showed that the CCR3 inhibitory activity of the 4-substituted
Compounds 30j and 30s were identified as having potent CCR3
inhibitory activity, with CLint values smaller than that of 30a, and
also showed potent dissociation constant (Kb) values (1.0 and
0.44 nM, respectively), which were determined using the method
reported by Lazareno and Bridsall (Table 7).7 Further, compounds
30j and 30s proved to be CCR3-selective inhibitors since they
showed no inhibitory effects on Ca2+ influx signals induced by
other chemokines at 10 lM (using RANTES for CCR1 and CCR5,
and MCP-1 for CCR2, respectively). With regard to species cross-
reactivity, compounds 1 and 2 showed markedly weak inhibition
against mouse and monkey CCR3 as shown in Table 7, while 30j
showed more potent mouse and monkey CCR3 inhibitory activity

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I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
Table 3
CCR3 inhibitory activity of 6-fluoronaphthalene derivatives
N
R
N
F
H
a
Compound
R
IC50 (nM)
1
20 3.9
Figure 3. The geometrically optimized structure of model compound of 30j.
O
21a
26
100 21
210 15
*
Table 5
Interaction energy between the ligand (model compound) and the water molecule
O
O
H
O
*
H
2.5 Å
O
O
N
R
4
*
24
900 2.3
N
N
2
3
O
O
R
Interaction energya (kcal molꢁ1
)
H
2-F
ꢁ2.02
ꢁ1.82
ꢁ1.14
ꢁ2.24
ꢁ2.01
ꢁ1.79
ꢁ2.03
0.89
30a
4.4 1.2
O
3-F
4-F
*
2-MeO
3-MeO
4-MeO
2-OH
3-OH
4-OH
O
N
29
O
160 59
ꢁ2.38
ꢁ1.97
*
a
a
The IC50 values are shown as the means SEM for at least three determinations.
The detailed method is described in Section 5.
Table 6
Table 4
CCR3 inhibitory activity of acrylamide derivatives
CCR3 inhibitory activity and CLint value of acrylamide derivatives
O
2
O
2
3
O
N
3
N
R
O
N
N
6
R
N
H
4
F
6
N
H
4
5
F
5
a
Compound
R
IC50 (nM)
CLint (mL/min/kg)
a
Compound
R
IC50 (nM)
CLint (mL/min/kg)
30a
30j
H
4.4 1.2
8.4 2.8
38 10
3.3 1.4
14 3.2
45 8.3
6.0 0.8
7.5 2.6
0.98 0.32
338
<64
162
232
119
192
116
87
30a
30b
30c
30d
30e
30f
30g
30h
30i
H
4.4 1.2
4.8 1.1
12 3.4
22 7.1
5.1 2.3
5.8 3.3
1.8 0.5
3.6 0.3
1.6 0.1
8.4 2.8
5.0b
338
590
459
237
1147
500
252
246
238
<64
335
120
2-OH
2-F
3-F
4-F
30m
30n
30o
30p
30q
30r
30s
2-OH-3-OMe
2-OH-4-OMe
2-OH-5-OMe
2-OH-6-OMe
2-OH-4-F
2-OMe
3-OMe
4-OMe
3,4-OMe
3,4-OCH2O-
2-OH
2-OH-4-Cl
2-OH-4-Me
156
a
The IC50 values are shown as the means SEM for at least three determinations.
30j
30k
30l
3-OH
4-OH
5.1b
a
The IC50 values are shown as the means SEM for at least three determinations.
Mean of two experiments.
With regard to metabolic stability, we selected compound 30j
for evaluating the pharmacokinetic properties by dosing 1 mg/kg
IV and 3 mg/kg PO (Table 8). Compound 30j exhibited an accept-
able half-life (2.9 h), short maximum drug concentration time
(0.8 h), and moderate oral bioavailability (35%). Membrane perme-
ability of 30j was 29.8 ꢀ 10ꢁ6 cm/s (pH 6.5) as measured in a PAM-
PA, and was 4.0 ꢀ 10ꢁ6 cm/s as measured in a Caco-2 cell assay
(Papp) (Table 7). These properties are highly desirable and strongly
support that 30j would exhibit good absorption on oral administra-
b
(IC50 = 200 and 7.2 nM, respectively) than 1 or 2. In addition, com-
pound 30j exhibited weaker CYP2D6 inhibition than
(IC50 = 29 M vs 0.02 M) and no significant CYP inhibition against
other major subtypes such as CYP1A2, 2C9, 2C19, and 3A4
(Table 7).
1
l
l

I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
5995
Table 7
Comparison of pharmacological profiles between 1, 2, 30j, and 30s
5. Experimental
1
2
30j
30s
5.1. Chemistry
Human CCR3, IC50 (nM)
Human Kb, (nM)a
20
3.7
23
1.4
8.4
1.0
0.98
0.44
NEc
NEc
NEc
NTd
NTd
156
27.3
NTd
NTd
In general, reagents and solvents were used as purchased with-
out further purification. Melting points were determined with a
Yanaco MP-500D melting point apparatus and left uncorrected.
1H NMR spectra were recorded on a JEOL JNM-LA300 or a JEOL
JNM-EX400 spectrometer. Chemical shifts were expressed in d
(ppm) values with tetramethylsilane as an internal standard
(NMR descriptions; s = singlet, d = doublet, t = triplet, dt = double
triplet, m = multiplet, and br = broad peak). Mass spectra were re-
corded on a JEOL JMS-LX2000 spectrometer. High resolution (HR)-
mass spectra were recorded using a Waters QTOF Premier spec-
trometer. The elemental analyses were performed with a Yanaco
MT-5 microanalyzer (C, H, and N) and Yokogawa IC-7000S ion
chromatographic analyzer (halogens) and were within 0.4% of
the theoretical values.
Human CCR1, IC50 (nM)
Human CCR2, IC50 (nM)
Human CCR5, IC50 (nM)
Mouse CCR3, IC50 (nM)b
Monkey CCR3, IC50 (nM)b
CLint (mL/min/kg)
NEc
NEc
NEc
NEc
200
NTd
>30
NTd
0.02
NEc
NEc
NEc
1100
613
87
NEc
NEc
NEc
230
7.2
<64
29.8
4.0
PAMPA (ꢀ10ꢁ6 cm/s)
4.7
Caco-2, Papp (ꢀ10ꢁ6 cm/s)
NTd
29
CYP2D6 inhibition, IC50
(lM)
29
a
See Ref. 7.
See Refs. 8a–c.
No effect at 10
Not tested.
b
c
lM.
d
tion to humans. Further biological and pharmacokinetic evalua-
tions of 30j are now carried out, and further structural optimiza-
tion studies are also planned to determine the SAR of our
acrylamide derivatives, and the results of these compounds’ bio-
logical and pharmacokinetic evaluations will be reported in due
course.
5.1.1. 1-[(6-Fluoro-2-naphthyl)methyl]-1,4-diazepane (6)
To a solution of homopiperazine (4) (2.09 g, 20.9 mmol) in
MeCN (50 mL) were added
5 (1.0 g, 4.18 mmol) and K2CO3
(1.73 g, 12.54 mmol), and the mixture was stirred at room temper-
ature for 20 h. This mixture was concentrated in vacuo. The residue
was then partitioned between CHCl3 and H2O, and the organic
layer was washed with brine, dried over MgSO4, filtered, and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (CHCl3/MeOH = 95:5–85:15) to yield 6 (832 mg,
77%) as a pale yellow solid. 1H NMR (300 MHz, CDCl3) 1.72–1.82
(m, 2H), 2.67–2.76 (m, 4H), 2.88–2.94 (m, 2H), 2.95–3.00 (m,
2H), 3.80 (s, 2H), 7.20–7.28 (m, 1H), 7.42 (dd, J = 9.9, 2.6 Hz, 1H),
7.55 (d, J = 8.6 Hz, 1H), 7.71–7.82 (m, 3H); MS (FAB) m/z = 259
[M+H]+.
4. Conclusions
In summary, to improve the CCR3 inhibitory activity of the lead
compound 1, we designed novel compounds based on previously
reported key pharmacophores, which led to the successful estab-
lishment of a novel series of acrylamide derivatives with potent
activity. The SAR studies showed that the CCR3 inhibitory activity
would be susceptible to electrons around the terminal benzoyl
part, and that the incorporation of electron-donating groups at
the 4-position would be critical for potent activity. In addition,
the study of the relationship between structure and metabolic sta-
bility against HLMs (CLint; mL/min/kg) revealed that the structural
modification of the benzoyl moiety gave rise to considerable
changes in the CLint values. The 2-hydroxy derivatives (30j) had
the lowest CLint values in this series of compounds. The intramolec-
ular hydrogen bond between the 2-hydroxy moiety and the car-
bonyl oxygen of the amide moiety effectively improved
microsomal metabolic stability by decreasing the amount of en-
ergy used to interact with CYPs of hepatic microsomes. Finally,
the introduction of an electron-donating methyl moiety at the 4-
position of the benzoyl moiety of 30j led to the identification of
30s, which had the most potent CCR3 inhibitory activity in this ser-
ies of compounds.
5.1.2. 1-(Biphenyl-2-ylcarbonyl)-4-[(6-fluoro-2-naphthyl)-
methyl]-1,4-diazepane hydrochloride (7)
To a solution of 6 (460 mg, 1.78 mmol) in DMF (10 mL) were
added biphenyl-2-carboxylic acid (378 mg, 1.78 mmol), HOBt
(289 mg, 2.14 mmol), and WSCꢂHCl (412 mg, 2.14 mmol), and this
mixture was stirred at room temperature overnight. The mixture
was then partitioned between EtOAc and satd NaHCO3 aq, and
the organic layer was washed with brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. The crude oil was converted to
its hydrochloride salt by treating it with 4 M HCl (g)/EtOAc. The
crude solid was recrystallized from MeOH–EtOAc to yield 7
(224 mg, 26%) as a colorless solid. Mp: 230–232 °C. 1H NMR
(400 MHz, DMSO-d6) d: 1.24–1.28 (m, 1H), 1.62–1.80 (m, 1H),
2.00–2.40 (m, 1H), 2.63–2.89 (m, 1H), 2.95–3.55 (m, 5H), 4.08–
4.35 (m, 2H), 4.36–4.60 (m, 1H), 7.24–7.60 (m, 10H), 7.73–7.94
(m, 2H), 7.94–8.20 (m, 3H), 11.35–11.83 (m, 1H); MS (FAB) m/
z = 439 [M+H]+. Anal. Calcd for C29H27FN2OꢂHClꢂ0.2H2O: C, 72.78;
H, 5.98; N, 5.85; Cl, 7.41; F, 3.97. Found: C, 72.85; H, 5.99; N,
5.77; Cl, 7.39; F, 3.96.
Based on the desirable properties of 30j, including its high met-
abolic stability against HLMs, moderate bioavailability in mice, and
high membrane permeability, we expected 30j to show good phar-
macokinetic properties in humans.
Table 8
5.1.3. 1-(Biphenyl-2-ylacetyl)-4-[(6-fluoro-2-naphthyl)methyl]-
1,4-diazepane hydrochloride (8)
Pharmacokinetic properties of 30j in mice
Compound 8 was prepared from 6 and biphenyl-2-ylacetic acid
in a manner similar to that described for compound 7, in a yield of
33% as a colorless solid. Mp: 212 °C (dec) (MeOH). 1H NMR
(400 MHz, DMSO-d6) d: 1.84–2.07 (m, 1H), 2.24–2.37 (m, 1H),
2.62–3.00 (m, 2H), 3.17–3.71 (m, 7H), 4.05–4.15 (m, 1H), 4.36–
4.52 (m, 2H), 7.17–7.41 (m, 9H), 7.48–7.56 (m, 1H), 7.77–7.88
(m, 2H), 7.98–8.08 (m, 2H), 8.12–8.19 (m, 1H), 11.35 (s, 1H); MS
(FAB) m/z = 453 [M+H]+. Anal. Calcd for C30H29FN2OꢂHCl: C,
73.68; H, 6.18; N, 5.73; Cl, 7.25; F, 3.88. Found: C, 73.79; H, 6.05;
N, 5.69; Cl, 7.25; F, 3.89.
30j
IVa (1 mg/kg)
POa (3 mg/kg)
CLtot (mL/min/kg)
t1/2 (h)
Tmax (h)
88 7.9
2.1 0.5
—
—
2.9 0.3
0.8 0.3
Cmax (h)
—
41
—
3
Vdss (mL/kg)
AUC0-1 (ng h/mL)
F (%)
9877 692
190 17
200 47
35
8
a
The values are shown as the means SEM for at least three determinations.

5996
I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
5.1.4. 1-[(6-Fluoro-2-naphthyl)methyl]piperazine dihydrochloride
(10)
1.15–1.32 (m, 1H), 1.63–1.75 (m, 1H), 1.77–1.88 (m, 2H), 2.76–
2.95 (m, 1H), 2.95–3.25 (m, 2H), 3.30–3.42 (m, 1H), 4.01–4.15
(m, 1H), 4.23–4.57 (m, 3H), 7.15–7.33 (m, 5H), 7.34–7.44 (m,
3H), 7.46–7.58 (m, 2H), 7.74–7.85 (m, 2H), 7.98–8.18 (m, 3H),
8.28 (d, J = 7.8 Hz, 1H), 10.68–10.95 (m, 1H); MS (FAB) m/z = 439
[M+H]+. Anal. Calcd for C29H27FN2OꢂHClꢂ0.8H2O: C, 71.17; H, 6.10;
N, 5.72; Cl, 7.24; F, 3.88. Found: C, 71.36; H, 5.96; N, 5.72; Cl,
7.34; F, 3.74.
To
a solution of tert-butyl piperazine-1-carboxylate (9)
(770 mg, 4.14 mmol) in MeCN (15 mL) were added 5 (990 mg,
3.76 mmol) and K2CO3 (1.56 g, 11.3 mmol), and the mixture was
stirred at room temperature for 4.5 h. The residue was then parti-
tioned between EtOAc and satd NaHCO3 aq, and the organic layer
was washed with brine, dried over MgSO4, filtered, and concen-
trated in vacuo. To a solution of this crude compound in EtOAc
(20 mL) and MeOH (10 mL) was added 4 M HCl (g)/EtOAc
(30 mL), and the mixture was stirred at room temperature over-
night. The precipitate was collected by filtration, washed with
EtOAc and Et2O, and dried in vacuo to yield 10 (1.02 g, 86% for
two steps) as a colorless solid. 1H NMR (400 MHz, DMSO-d6)
3.08–3.82 (m, 8H), 4.45–4.65 (m, 2H), 7.51 (ddd, J = 8.8, 8.8,
2.5 Hz, 1H), 7.79 (dd, J = 10.3, 2.0 Hz, 1H), 7.80–7.90 (m, 1H),
7.98–8.08 (m, 2H), 8.17–8.20 (m, 1H), 9.76 (s, 1H), 11.00–12.55
(m, 2H); MS (FAB) m/z = 245 [M+H]+.
5.1.8. (3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-amine
dihydrochloride [(3R)-16]
To a solution of tert-butyl (3R)-pyrrolidin-3-ylcarbamate [(3R)-
15] (20.2 g, 108 mmol) in MeCN (300 mL) were added 5 (31.12 g,
130 mmol) and K2CO3 (17.99 g, 130 mmol), and the mixture was
stirred at room temperature for 7 h. The residue was then parti-
tioned between EtOAc and H2O, and the organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(CHCl3/MeOH = 98:2) to yield tert-butyl {(3R)-1-[(6-fluoro-2-naph-
thyl)methyl]pyrrolidin-3-yl}carbamate (28.35 g, 76%). To a solu-
tion of this compound in EtOAc (500 mL) was added 4 M HCl (g)/
EtOAc (200 mL), and the mixture was stirred at room temperature
for 3 h. The mixture was concentrated in vacuo to yield (3R)-16
5.1.5. 1-(Biphenyl-2-ylacetyl)-4-[(6-fluoro-2-naphthyl)methyl]-
piperazine hydrochloride (11)
Compound 11 was prepared from 10 and biphenyl-2-ylacetic
acid in a manner similar to that described for compound 7, in a
yield of 33% as a colorless solid. Mp: 218–220 °C (MeOH). 1H
NMR (400 MHz, DMSO-d6) d: 2.70–2.91 (m, 2H), 3.05–3.15 (m,
1H), 3.23–3.48 (m, 3H), 3.55–3.68 (m, 2H), 3.81–3.92 (m, 1H),
4.34–4.54 (m, 3H), 7.17–7.40 (m, 9H), 7.52 (ddd, J = 8.8, 8.8,
2.4 Hz, 1H), 7.86–7.85 (m, 2H), 7.98–8.08 (m, 2H), 8.12–8.18 (m,
1H), 11.60 (s, 1H); MS (FAB) m/z = 439 [M+H]+. Anal. Calcd for
C29H27FN2OꢂHCl: C, 73.33; H, 5.94; N, 5.90; Cl, 7.46; F, 4.00. Found:
C, 73.37; H, 5.94; N, 5.95; Cl, 7.50; F, 4.11.
(26.32 g, quant.) as
a
pale pink amorphous solid. 1H NMR
(400 MHz, DMSO-d6) d: 2.00–2.60 (m, 2H), 3.18–3.46 (m, 1H),
3.51–3.79 (m, 3H), 3.81–4.15 (m, 1H), 4.54–4.75 (m, 2H), 7.52
(ddd, J = 9.2, 8.8, 2.5 Hz, 1H), 7.75–7.90 (m, 2H), 7.98–8.03 (m,
2H), 8.15–8.23 (m, 1H), 8.50–8.90 (m, 3H), 11.45–12.08 (m, 1H);
MS (FAB) m/z = 245 [M+H]+.
5.1.9. (3S)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-amine
dihydrochloride [(3S)-16]
5.1.6. (rac)-N-Piperidin-3-ylbiphenyl-2-carboxamide [(rac)-13]
To a solution of (rac)-12 (720 mg, 3.59 mmol) in THF (10 mL)
were added biphenyl-2-carboxylic acid (712 mg, 3.59 mmol), HOBt
(583 mg, 4.31 mmol), and WSCꢂHCl (830 mg, 4.31 mmol), and this
mixture was stirred at room temperature for 18 h. The mixture was
then partitioned between EtOAc and satd NaHCO3 aq, and the or-
ganic layer was washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuo. To a solution of this crude compound
in EtOAc (30 mL) was added 4 M HCl (g)/EtOAc (30 mL), and the
mixture was stirred at room temperature for 4 h. The mixture
was concentrated in vacuo. The residue was dissolved in H2O,
and washed with Et2O. The aqueous layer was alkalified with satd
NaHCO3 aq and extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo to yield (rac)-13 (210 mg, 21% for two steps) as a pale yel-
low solid. 1H NMR (400 MHz, DMSO-d6) 1.12–1.33 (m, 2H), 1.38–
1.49 (m, 1H), 1.56–1.65 (m, 1H), 2.13 (dd, J = 11.8, 9.3 Hz, 1H),
2.28–2.38 (m, 1H), 2.64–2.78 (m, 2H), 3.53–3.64 (m, 1H), 7.29–
7.41 (m, 8H), 7.45–7.51 (m, 1H), 7.85 (d, J = 8.4 Hz, 1H); MS (FAB)
m/z = 281 [M+H]+.
Compound (3S)-16 was prepared from (3S)-pyrrolidin-3-ylcar-
bamate and 5 in a manner similar to that described for compound
(3R)-16, in a yield of 53% as a colorless solid. 1H NMR (400 MHz,
DMSO-d6) d: 2.00–2.60 (m, 2H), 3.18–3.46 (m, 1H), 3.51–3.79 (m,
3H), 3.81–4.15 (m, 1H), 4.54–4.75 (m, 2H), 7.52 (ddd, J = 9.2, 8.8,
2.5 Hz, 1H), 7.75–7.90 (m, 2H), 7.98–8.03 (m, 2H), 8.15–8.23 (m,
1H), 8.50–8.90 (m, 3H), 11.45–12.08 (m, 1H); MS (ESI) m/z = 245
[M+H]+.
5.1.10. (rac)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
amine dihydrochloride [(rac)-16]
Compound (rac)-16 was prepared from (rac)-pyrrolidin-3-ylc-
arbamate and 5 in a manner similar to that described for com-
pound (3R)-16, in a yield of 77% as a pale brown amorphous
solid. 1H NMR (400 MHz, DMSO-d6) d: 2.00–2.60 (m, 2H), 3.18–
3.46 (m, 1H), 3.51–3.79 (m, 3H), 3.81–4.15 (m, 1H), 4.54–4.75
(m, 2H), 7.52 (ddd, J = 9.2, 8.8, 2.5 Hz, 1H), 7.75–7.90 (m, 2H),
7.98–8.03 (m, 2H), 8.15–8.23 (m, 1H), 8.50–8.90 (m, 3H), 11.45–
12.08 (m, 1H); MS (FAB) m/z = 245 (M+H)+.
5.1.11. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}biphenyl-2-carboxamide oxalate [(3R)-17]
5.1.7. (rac)-N-{1-[(6-Fluoro-2-naphthyl)methyl]piperidin-3-yl}-
biphenyl-2-carboxamide hydrochloride [(rac)-14]
To a solution of (3R)-16 (250 mg, 0.79 mmol) in DMF (5 mL)
were added biphenyl-2-carboxylic acid (171 mg, 0.87 mmol),
Et3N (0.36 mL, 2.60 mmol), HOBt (117 mg, 0.87 mmol), and
WSCꢂHCl (181 mg, 0.95 mmol), and this mixture was stirred at
room temperature overnight. The mixture was then partitioned be-
tween EtOAc and satd NaHCO3 aq, and the organic layer was
washed with brine, dried over MgSO4, filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (CHCl3/MeOH = 94:6–90:10). The crude oil was converted to
its oxalate by treating it with oxalic acid. This solid was recrystal-
lized from EtOH–EtOAc to yield (3R)-17 (224 mg, 55%) as a color-
To a solution of (rac)-13 (196 mg, 0.70 mmol) in MeCN (5 mL)
were added
5
(167 mg, 0.70 mmol) and K2CO3 (193 mg,
1.40 mmol), and the mixture was stirred at room temperature for
25 h. The residue was then partitioned between EtOAc and satd
NaHCO3 aq, and the organic layer was washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (CHCl3/MeOH =
98:2–95:5). The crude oil was converted to its hydrochloride salt
by treating it with 4 M HCl (g)/EtOAc. The crude solid was recrys-
tallized from MeOH–EtOAc to yield (rac)-14 (70 mg, 20%) as a col-
orless solid. Mp: 138–142 °C. 1H NMR (400 MHz, DMSO-d6) d:
less powder. Mp: 183–185 °C. ½a D25
ꢃ
+1.99 (c 0.20, EtOH). 1H NMR

I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
5997
(400 MHz, DMSO-d6) d: 1.66–1.75 (m, 1H), 2.05–2.20 (m, 1H),
2.58–2.70 (m, 1H), 2.95–3.10 (m, 2H), 3.16–3.28 (m, 1H), 4.14 (d,
J = 12.7 Hz, 1H), 4.23 (d, J = 12.7 Hz, 1H), 4.30–4.39 (m, 1H), 7.18
(t, J = 7.2 Hz, 1H), 7.27 (dd, J = 7.7, 7.3 Hz, 2H), 7.31–7.33 (m, 2H),
7.35–7.44 (m, 3H), 7.46–7.54 (m, 2H), 7.59 (d, J = 9.0 Hz, 1H),
7.76 (dd, J = 10.2, 2.5 Hz, 1H), 7.95–7.98 (m, 2H), 8.04 (dd, J = 9.0,
5.8 Hz, 1H), 8.42 (d, J = 6.8 Hz, 1H); MS (ESI) m/z = 425 [M+H]+.
Anal. Calcd for C28H25FN2OꢂC2H2O4: C, 70.03; H, 5.29; N, 5.44; F,
3.69. Found: C, 70.02; H, 5.28; N, 5.49; F, 3.65.
1.53–1.63 (m, 1H), 2.06–2.16 (m, 1H), 2.31–2.37 (m, 1H), 2.39–
2.49 (m, 1H), 2.63–2.70 (m, 2H), 3.37 (s, 2H), 3.68 (d, J = 13.2 Hz,
1H), 3.74 (d, J = 13.2 Hz, 1H), 4.09–4.19 (m, 1H), 7.16–7.29 (m,
5H), 7.41 (ddd, J = 8.8, 8.8, 2.7 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H),
7.68 (dd, J = 10.5, 2.7 Hz, 1H), 7.82–7.88 (m, 2H), 7.96 (dd, J = 9.2,
5.8 Hz, 1H), 8.23 (d, J = 5.9 Hz, 1H); MS (FAB) m/z = 363 [M+H]+.
Anal. Calcd for C23H23FN2O: C, 76.22; H, 6.40; N, 7.73; F, 5.24.
Found: C, 76.16; H, 6.43; N, 7.71; F, 5.18.
5.1.16. N-{1-[(6-Fluoro-2-naphthyl)methyl]piperidin-4-yl}-2-
phenylacetamide (20b)
5.1.12. N-{(3S)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}biphenyl-2-carboxamide oxalate [(3S)-17]
Compound 20b was prepared from 183a and phenylacetyl chlo-
ride in a manner similar to that described for compound 19a, in a
yield of 46% as a colorless crystal. Mp: 168–171 °C (MeCN). 1H
NMR (400 MHz, CDCl3) d: 1.27–1.39 (m, 2H), 1.80–1.89 (m, 2H),
2.08–2.27 (m, 2H), 2.69–2.79 (m, 2H), 3.55 (s, 2H), 3.58 (s, 2H),
3.76–3.87 (m, 1H), 5.22 (d, J = 7.8 Hz, 1H), 3.74 (d, J = 13.2 Hz,
1H), 7.20–7.38 (m, 6H), 7.41 (dd, J = 10.0, 2.7 Hz, 1H), 7.45 (d,
J = 8.3 Hz, 1H), 7.66–7.74 (m, 2H), 7.77 (dd, J = 9.1, 5.7 Hz, 1H);
MS (FAB) m/z = 377 [M+H]+. Anal. Calcd for C24H25FN2O: C, 76.57;
H, 6.69; N, 7.44; F, 5.05. Found: C, 76.82; H, 6.80; N, 7.44; F, 5.18.
Compound (3S)-17 was prepared from (3S)-16 and biphenyl-2-
carboxylic acid in a manner similar to that described for compound
(3R)-17, in a yield of 58% as a colorless powder. Mp: 183–185 °C
(EtOH–EtOAc). ½a D25
ꢃ
ꢁ1.99 (c 0.20, EtOH). 1H NMR (400 MHz,
DMSO-d6) d: 1.66–1.75 (m, 1H), 2.05–2.20 (m, 1H), 2.58–2.70 (m,
1H), 2.95–3.10 (m, 2H), 3.16–3.28 (m, 1H), 4.14 (d, J = 12.7 Hz,
1H), 4.23 (d, J = 12.7 Hz, 1H), 4.30–4.39 (m, 1H), 7.18 (t,
J = 7.2 Hz, 1H), 7.27 (dd, J = 7.7, 7.3 Hz, 2H), 7.31–7.33 (m, 2H),
7.35–7.44 (m, 3H), 7.46–7.54 (m, 2H), 7.59 (d, J = 9.0 Hz, 1H),
7.76 (dd, J = 10.2, 2.5 Hz, 1H), 7.95–7.98 (m, 2H), 8.04 (dd, J = 9.0,
5.8 Hz, 1H), 8.42 (d, J = 6.8 Hz, 1H); MS (ESI) m/z = 425 [M+H]+.
Anal. Calcd for C28H25FN2OꢂC2H2O4: C, 70.03; H, 5.29; N, 5.44; F,
3.69. Found: C, 70.06; H, 5.30; N, 5.47; F, 3.67.
5.1.17. (2E)-N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-
3-yl}-3-phenylacrylamide (21a)
Compound 21a was prepared from (3R)-16 and (2E)-3-phenyl-
acrylic acid in a manner similar to that described for compound
(3R)-17, in a yield of 37% as a colorless powder. Mp: 172–174 °C
5.1.13. N-{(rac)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}biphenyl-2-carboxamide fumarate [(rac)-17]
(EtOAc). ½a 2D5
ꢃ
ꢁ15.00 (c 0.10, CHCl3). 1H NMR (400 MHz, DMSO-
Compound (rac)-17 was prepared from (rac)-16 and biphenyl-2-
carboxylic acid in a manner similar to that described for compound
(3R)-17, in a yield of 28% as a pale brown powder. Mp: 165–168 °C
(EtOH–EtOAc). 1H NMR (400 MHz, DMSO-d6) d: 1.49–1.62 (m, 1H),
1.95–2.08 (m, 1H), 2.27 (d, J = 9.8, 4.8 Hz, 1H), 2.50–2.65 (m, 2H),
2.74–2.81 (m, 1H), 3.74 (d, J = 12.7 Hz, 1H), 3.81 (d, J = 12.7 Hz,
1H), 4.16–4.28 (m, 1H), 6.61 (s, 2H), 7.18–7.28 (m, 3H), 7.32–7.56
(m, 8H), 7.70 (dd, J = 10.2, 2.5 Hz, 1H), 7.84–7.92 (m, 2H), 8.04 (dd,
J = 9.6, 5.7 Hz, 1H), 8.25 (d, J = 6.8 Hz, 1H); MS (FAB) m/z = 425
(M+H)+. Anal. Calcd for C28H25FN2OꢂC4H4O4: C, 71.10; H, 5.41; N,
5.18; F, 3.51. Found: C, 70.01; H, 5.40; N, 5.18; F, 3.51.
d6) d: 1.57–1.70 (m, 1H), 2.11–2.23 (m, 1H), 2.38–2.50 (m, 2H),
2.65–2.80 (m, 2H), 3.68–3.79 (m, 2H), 4.25–4.36 (m, 1H), 6.64 (d,
J = 15.8 Hz, 1H), 7.33–7.47 (m, 5H), 7.51–7.57 (m, 3H), 7.67 (dd,
J = 10.3, 2.4 Hz, 1H), 7.84–7.89 (m, 2H), 7.97 (dd, J = 9.0, 5.9 Hz,
1H), 8.25 (d, J = 7.0 Hz, 1H); MS (ESI) m/z = 375 [M+H]+. Anal. Calcd
for C24H23FN2O: C, 76.98; H, 6.19; N, 7.49; F, 5.07. Found: C, 77.02;
H, 6.26; N, 7.49; F, 5.15.
5.1.18. tert-Butyl 4-[(1E)-3-({(3R)-1-[(6-fluoro-2-naphthyl)-
methyl]pyrrolidin-3-yl}amino)-3-oxoprop-1-en-1-yl]piper-
idine-1-carboxylate (23)
Compound 23 was prepared from (3R)-16 and (2E)-3-[1-(tert-
butoxycarbonyl)piperidin-4-yl]acrylic acid10 in a manner similar to
that described for compound (3R)-17, in a yield of 96% as a colorless
amorphous solid. 1H NMR (400 MHz, CDCl3) d: 1.24–1.39 (m, 1H),
1.60–1.74 (m, 2H), 2.18–2.39 (m, 3H), 2.60–2.84 (m, 4H), 2.89–2.96
(m, 1H), 3.75 (s, 2H), 4.02–4.20 (m, 2H), 4.50–4.60 (m, 1H), 5.69 (d,
J = 15.3 Hz, 1H), 5.85 (d, J = 7.8 Hz, 1H), 6.76 (dd, J = 15.3, 6.6 Hz, 1H),
7.21–7.30 (m, 1H), 7.43 (dd, J = 9.8, 2.2 Hz, 1H), 7.48 (d, J = 8.4 Hz,
1H), 7.70–7.82 (m, 3H); MS (ESI) m/z = 482 [M+H]+.
5.1.14. 4-Fluoro-N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]-
pyrrolidin-3-yl}benzamide (19a)
To a solution of (3R)-16 (284 mg, 0.90 mmol) in CH2Cl2 (10 mL)
were added Et3N (0.37 mL, 2.69 mmol) and 4-fluorobenzoyl chlo-
ride (149 mg, 0.94 mmol), and the mixture was stirred at room
temperature for 0.5 h. The mixture was then partitioned between
EtOAc and H2O, and the organic layer was washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (CHCl3/
MeOH = 98:2–90:10). The residue was recrystallized from MeCN
to yield 19a (131 mg, 40%) as a colorless crystal. Mp: 157–159 °C.
5.1.19. (2E)-3-(1-Benzoylpiperidin-4-yl)-N-{(3R)-1-[(6-fluoro-2-
naphthyl)methyl]pyrrolidin-3-yl}acrylamide (24)
½
a 2D5
ꢃ
ꢁ4.99 (c 0.10, CHCl3). 1H NMR (400 MHz, DMSO-d6) d: 1.77–
To a solution of 23 (430 mg, 0.89 mmol) in EtOAc (3 mL) was
added 4 M HCl (g)/EtOAc (3 mL), and the mixture was stirred at
room temperature overnight. The mixture was concentrated in va-
cuo. To a solution of this crude compound (405 mg, 0.89 mmol) in
CH2Cl2 (5 mL) were added Et3N (0.42 mL, 3.04 mmol) and BzCl
(147 mg, 1.04 mmol), and the mixture was stirred at room temper-
ature for 3 h. The mixture was then partitioned between EtOAc and
H2O, and the organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (CHCl3/MeOH = 99:1–
92:8). The residue was recrystallized from hexane–EtOAc to yield
24 (172 mg, 41% for two steps) as a colorless powder. Mp: 104–
1.86 (m, 1H), 2.11–2.22 (m, 1H), 2.42–2.48 (m, 1H), 2.52–2.59
(m, 1H), 2.62–2.70 (m, 1H), 2.82–2.89 (m, 2H), 3.75 (s, 2H), 4.33–
4.44 (m, 1H), 7.24–7.31 (m, 2H), 7.40 (ddd, J = 8.8, 8.8, 2.5 Hz,
1H), 7.55 (d, J = 8.8 Hz, 1H), 7.68 (dd, J = 10.3, 2.5 Hz, 1H), 7.84–
8.00 (m, 5H), 8.49 (d, J = 6.9 Hz, 1H); MS (FAB) m/z = 367 [M+H]+.
Anal. Calcd for C22H20F2N2O: C, 72.12; H, 5.50; N, 7.65; F, 10.37.
Found: C, 72.05; H, 5.42; N, 7.67; F, 10.39.
5.1.15. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-phenylacetamide (20a)
Compound 20a was prepared from (3R)-16 and phenylacetyl
chloride in a manner similar to that described for compound 19a,
in a yield of 15% as a colorless crystal. Mp: 114–116 °C (MeCN).
106 °C (hexane–EtOAc). ½a D25
ꢃ
+13.00 (c 0.10, CHCl3). 1H NMR
(400 MHz, DMSO-d6) d: 1.20–1.40 (m, 2H), 1.53–1.86 (m, 3H),
½
a 2D5
ꢃ
+41.12 (c 0.107, MeOH). 1H NMR (400 MHz, DMSO-d6) d:
2.08–2.18 (m, 1H), 2.32–2.50 (m, 3H), 2.61–2.75 (m, 2H), 2.78–

5998
I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
3.18 (m, 2H), 3.45–3.66 (m, 1H), 3.67–3.76 (m, 2H), 4.17–4.28 (m,
1H), 4.34–4.58 (m, 1H), 5.90 (d, J = 15.5 Hz, 1H), 6.56 (d, J = 15.5,
5.4 Hz, 1H), 7.32–7.48 (m, 6H), 7.53 (d, J = 8.4 Hz, 1H), 7.67 (dd,
J = 10.3, 2.4 Hz, 1H), 7.82–7.88 (m, 2H), 7.96 (dd, J = 9.0, 5.8 Hz,
1H), 8.06 (d, J = 7.0 Hz, 1H); MS (ESI) m/z = 486 [M+H]+. Anal. Calcd
for C30H32FN3O2ꢂ0.2H2O: C, 73.66; H, 6.68; N, 8.59; F, 3.88. Found:
C, 73.59; H, 6.66; N, 8.53; F, 3.91.
dropwise over 0.5 h, and this mixture was stirred at room temper-
ature for 0.5 h. To this mixture was added tert-butyl 4-oxopiperi-
dine-1-carboxylate (5.40 g, 27.1 mmol) in DME (50 mL) dropwise,
and this mixture was stirred at room temperature for 3 h. The mix-
ture was then partitioned between CHCl3 and H2O, and the organic
layer was washed with satd NaHCO3 aq, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by silica gel
column
chromatography
(CHCl3/MeOH/NH4OH = 99:1:0.1–
5.1.20. N-({(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}carbamoyl)benzenesulfonamide (22a)
96:4:0.1) to yield compound 27 (13.0 g, quant.) as a colorless
amorphous solid. To a solution of the above-obtained compound
(9.23 g, 19.7 mmol) in CH2Cl2 (50 mL) was added TFA (50 mL),
and the mixture was stirred at room temperature for 0.5 h. The
mixture was then concentrated in vacuo. The residue was dis-
solved in CHCl3, and this organic layer was washed with satd NaH-
CO3 aq, dried over Na2SO4, filtered, and concentrated in vacuo to
yield compound 28 (7.05 g, 97%) as a pale red amorphous solid.
1H NMR (400 MHz, DMSO-d6) d: 1.54–1.63 (m, 1H), 2.04–2.15
(m, 3H), 2.32 (dd, J = 9.5, 5.7 Hz, 1H), 2.42–2.49 (m, 1H), 2.58–
2.83 (m, 8H), 3.69 (d, J = 13.1 Hz, 1H), 3.73 (d, J = 13.1 Hz, 1H),
4.14–4.25 (m, 1H), 5.59 (s, 1H), 7.41 (ddd, J = 8.8, 8.8, 2.4 Hz, 1H),
7.53 (d, J = 8.3 Hz, 1H), 7.68 (dd, J = 10.5, 2.7 Hz, 1H), 7.82–7.88
(m, 2H), 7.92–8.00 (m, 2H); MS (FAB) m/z = 368 [M+H]+.
A solution of (3R)-16 (390 mg, 1.23 mmol) in EtOAc (50 mL)
was washed with 1 M NaOH aq and brine. The organic layer was
dried over Na2SO4, filtered, and concentrated in vacuo. To an ice-
cooled solution of this product in MeCN (50 mL) was added ben-
zenesulfonyl isocyanate (283 mg, 1.55 mmol) in MeCN (3 mL)
dropwise over 10 min, and this mixture was stirred at room tem-
perature for 0.5 h. To this mixture was then added PS-trisamine
(800 mg), and this mixture was stirred slowly at room temperature
for 4 h. The residue was then diluted with MeOH (300 mL), filtered,
and concentrated in vacuo. The crude solid was recrystallized from
MeOH to yield 22a (154 mg, 30%) as a colorless crystal. Mp: 150–
152 °C. ½a 2D5
ꢃ
+27.27 (c 0.11, MeOH). 1H NMR (400 MHz, DMSO-d6)
d: 1.42–1.58 (m, 1H), 2.04–2.16 (m, 1H), 2.36–2.50 (m, 2H), 2.59–
2.70 (m, 1H), 2.72–2.82 (m, 1H), 3.70–3.88 (m, 2H), 3.94–4.02
(m, 1H), 6.54–6.72 (m, 1H), 7.43 (ddd, J = 8.8, 8.8, 2.5 Hz, 1H),
7.50–7.65 (m, 4H), 7.70 (dd, J = 10.2, 2.5 Hz, 1H), 7.83–7.90 (m,
5H), 7.98 (dd, J = 9.2, 5.8 Hz, 1H); MS (FAB) m/z = 428 [M+H]+. Anal.
Calcd for C22H22FN3O3Sꢂ0.5H2O: C, 60.53; H, 5.31; N, 9.63; F, 3.51;
S, 7.35. Found: C, 60.57; H, 5.16; N, 9.82; F, 4.49; S, 7.48.
5.1.24. 4-Phenoxycyclohexanone (34)
To a solution of 33 (4.82 g, 30.5 mmol) in THF (100 mL) were
added phenol (2.73 g, 29.0 mmol), PPh3 (7.60 g, 29.0 mmol), and
DEAD (5.31 g, 30.5 mmol), and this mixture was stirred at room
temperature for 12 h. The mixture was then concentrated in vacuo.
The residue was purified by silica gel column chromatography
(hexane/EtOAc = 5:1) to yield 8-phenoxy-1,4-dioxaspiro[4.5]dec-
ane (5.94 g, 88%) as a colorless oil. To an ice-cooled solution of
the above-obtained compound (5.94 g, 25.4 mmol) in THF
(200 mL) was added 3 M HCl aq (60 mL), and the mixture was stir-
red at room temperature for 8 h. The mixture was neutralized with
NaHCO3 (pH 8), and extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (hexane/EtOAc = 7:1–5:1) to yield 34 (3.78 g, 72%) as a color-
less oil. 1H NMR (400 MHz, CDCl3) d: 2.02–2.12 (m, 2H), 2.25–2.37
(m, 4H), 2.65–2.75 (m, 2H), 4.67–4.73 (m, 1H), 6.94–7.01 (m, 3H),
7.29–7.35 (m, 2H); MS (FAB) m/z = 191 [M+H]+.
5.1.21. N-({1-[(6-Fluoro-2-naphthyl)methyl]piperidin-4-
yl}carbamoyl)benzenesulfonamide hydrochloride (22b)
Compound 22b was prepared from 18 and benzenesulfonyl iso-
cyanate in a manner similar to that described for compound 22a, in
a yield of 13% as a colorless amorphous solid. 1H NMR (400 MHz,
DMSO-d6) d: 1.62–2.01 (m, 4H), 2.91–3.12 (m, 2H), 3.17–3.40 (m,
2H), 3.45–3.58 (m, 1H), 4.39 (s, 2H), 7.08 (d, J = 5.9 Hz, 1H), 7.41
(ddd, J = 8.6, 8.5, 2.4 Hz, 1H), 7.60 (dd, J = 7.5, 7.5 Hz, 2H), 7.65–
7.71 (m, 1H), 7.73–7.83 (m, 2H), 7.90 (d, J = 7.5 Hz, 2H), 7.95–
8.08 (m, 2H), 8.12 (s, 1H), 10.49–10.80 (m, 2H); MS (FAB) m/
z = 442 [M+H]+. Anal. Calcd for C23H24FN3O3SꢂHCl: C, 57.79; H,
5.27; N, 8.79; Cl, 7.42; F, 3.97; S, 6.71. Found: C, 57.86; H, 5.21;
N, 8.79; Cl, 7.45; F, 3.85; S, 6.71.
5.1.25. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-(4-phenoxycyclohexylidene)acetamide (26)
5.1.22. Diethyl [2-({(3E)-1-[(6-fluoro-2-naphthyl)methyl]-
pyrrolidin-3-yl}amino)-2-oxoethyl]phosphonate (25)
Compound 26 was prepared from 25 and 34 in a manner similar
to that described for compound 27, in a yield of 79% as a pale yel-
To a solution of (3R)-16 (10.3 g, 32.5 mmol) in DMF (100 mL)
were added (diethoxyphosphoryl)acetic acid (6.52 g, 34.1 mmol),
HOBt (2.19 g, 16.2 mmol), WSCꢂHCl (6.85 g, 35.7 mmol), and Et3N
(9.05 mL, 64.9 mmol), and this mixture was stirred at room tem-
perature for 3.5 h. The mixture was then partitioned between
CHCl3 and H2O, and the organic layer was washed with satd NaH-
CO3 aq, dried over Na2SO4, filtered, and concentrated in vacuo to
yield compound 25 (15.3 g, quant.) as a brown oil. 1H NMR
(400 MHz, DMSO-d6) d: 1.15–1.21 (m, 6H), 1.52–1.62 (m, 1H),
2.08–2.18 (m, 1H), 2.34–2.44 (m, 2H), 2.63–2.72 (m, 2H), 3.57 (s,
2H), 3.68 (d, J = 13.2 Hz, 1H), 3.75 (d, J = 13.2 Hz, 1H), 3.93–4.04
(m, 4H), 4.10–4.20 (m, 1H), 7.42 (ddd, J = 9.3, 8.8, 2.9 Hz, 1H),
7.54 (d, J = 8.7 Hz, 1H), 7.68 (dd, J = 10.2, 2.5 Hz, 1H), 7.84–7.88
(m, 2H), 7.98 (dd, J = 8.8, 5.9 Hz, 1H), 8.15 (d, J = 7.3 Hz, 1H); MS
(FAB) m/z = 423 [M+H]+.
low amorphous solid. ½a D25
ꢃ
+27.99 (c 0.10, MeOH). 1H NMR
(400 MHz, DMSO-d6) d: 1.51–1.69 (m, 3H), 1.85–2.02 (m, 2H),
2.05–2.22 (m, 2H), 2.24–2.36 (m, 2H), 2.42–2.50 (m, 1H), 2.56–
2.68 (m, 2H), 2.68–2.76 (m, 1H), 3.22–3.29 (m, 1H), 3.66–3.76
(m, 2H), 4.14–4.25 (m, 1H), 4.53–4.59 (m, 1H), 5.66 (s, 1H), 6.88–
6.98 (m, 3H), 7.24–7.31 (m, 2H), 7.40 (ddd, J = 8.8, 8.8, 2.0 Hz,
2H), 7.53 (d, J = 8.3 Hz, 1H), 7.68 (dd, J = 10.3, 2.5 Hz, 1H), 7.83–
7.88 (m, 2H), 7.94–8.00 (m, 2H); MS (FAB) m/z = 459 [M+H]+. Anal.
Calcd for C29H31FN2O2: C, 75.96; H, 6.81; N, 6.11; F, 4.14. Found: C,
75.60; H, 6.88; N, 6.08; F, 4.10.
5.1.26. 2-(1-Benzoylpiperidin-4-ylidene)-N-{(3R)-1-[(6-fluoro-
2-naphthyl)methyl]pyrrolidin-3-yl}acetamide (30a)
To an ice-cooled solution of 28 (275 mg, 0.75 mmol) in CH2Cl2
(5 mL) was added BzCl (110 mg, 0.79 mmol), and the mixture
was stirred at 0 °C for 5.5 h. The mixture was then partitioned be-
tween EtOAc and H2O, and the organic layer was washed with satd
NaHCO3 aq and brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (CHCl3/MeOH = 100:0–97:3) to yield 30a (210 mg, 60%) as a
5.1.23. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-piperidin-4-ylideneacetamide (28)
To a solution of NaH (60% oil dispersion, 1.19 g, 29.8 mmol) in
DME (30 mL) was added 25 (13.7 g, 32.5 mmol) in DME (100 mL)

I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
5999
colorless amorphous solid. ½a D25
ꢃ
+36.36 (c 0.11, MeOH). 1H NMR
C
29H29F2N3O2ꢂ0.25H2O: C, 70.50; H, 6.02; N, 8.51; F, 7.69. Found:
(400 MHz, DMSO-d6) d: 1.53–1.64 (m, 1H), 2.03–2.36 (m, 4H),
2.40–2.50 (m, 1H), 2.59–2.77 (m, 2H), 2.80–3.05 (m, 2H), 3.28–
3.45 (m, 2H), 3.49–3.80 (m, 4H), 4.13–4.26 (m, 1H), 5.73 (s, 1H),
7.37–7.48 (m, 6H), 7.53 (d, J = 8.3 Hz, 1H), 7.67 (dd, J = 10.2,
2.5 Hz, 1H), 7.82–7.88 (m, 2H), 7.97 (dd, J = 8.8, 5.8 Hz, 1H), 8.05
(d, J = 6.8 Hz, 1H); MS (FAB) m/z = 472 [M+H]+. Anal. Calcd for
C29H30FN3O2ꢂ0.5H2O: C, 72.48; H, 6.50; N, 8.74; F, 3.95. Found: C,
72.80; H, 6.42; N, 8.76; F, 3.88.
C, 70.34; H, 6.00; N, 8.39; F, 7.97.
5.1.31. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(2-methoxybenzoyl)piperidin-4-ylidene]acetamide
(30e)
Compound 30e was prepared from 28 and 2-methoxybenzoyl
chloride in a manner similar to that described for compound 30a,
in a yield of 75% as a colorless amorphous solid. ½a D25
ꢃ
+34.99 (c
0.10, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.51–1.64 (m, 1H),
2.03–2.36 (m, 4H), 2.38–2.48 (m, 1H), 2.58–2.75 (m, 2H), 2.80–
2.98 (m, 2H), 3.05–3.21 (m, 2H), 3.56–3.73 (m, 4H), 3.78 (s, 3H),
4.12–4.25 (m, 1H), 5.69, 5.74 (each s, 1H), 6.96–7.02 (m, 1H),
7.05–7.10 (m, 1H), 7.16–7.21 (m, 1H), 7.35–7.44 (m, 2H), 7.50–
7.56 (m, 1H), 7.64–7.70 (m, 1H), 7.81–7.88 (m, 2H), 7.93–8.00
(m, 1H), 8.02–8.08 (m, 1H); MS (FAB) m/z = 502 [M+H]+. Anal. Calcd
for C30H32FN3O3ꢂ0.25H2O: C, 71.20; H, 6.47; N, 8.30; F, 3.75. Found:
C, 71.11; H, 6.36; N, 8.22; F, 3.72.
5.1.27. 2-(1-Acetylpiperidin-4-ylidene)-N-{(3R)-1-[(6-fluoro-2-
naphthyl)methyl]pyrrolidin-3-yl}acetamide (29)
Compound 29 was prepared from 28 and acetyl chloride in a
manner similar to that described for compound 30a, in a yield of
17% as a pale yellow amorphous solid. ½a D25
ꢁ11.05 (c 0.057,
ꢃ
MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.50–1.66 (m, 1H), 2.01
(s, 3H), 2.04–2.16 (m, 2H), 2.18–2.23 (m, 1H), 2.28–2.35 (m, 1H),
2.43–2.50 (m, 1H), 2.59–2.74 (m, 2H), 2.80–2.85 (m, 1H), 2.89–
2.95 (m, 1H), 3.35–3.50 (m, 4H), 3.66–3.76 (m, 2H), 4.15–4.24
(m, 1H), 5.71 (s, 1H), 7.40 (ddd, J = 9.2, 8.8, 2.5 Hz, 1H), 7.53 (d,
J = 8.3 Hz, 1H), 7.68 (dd, J = 10.3, 2.5 Hz, 1H), 7.82–7.88 (m, 2H),
7.97 (dd, J = 8.3, 5.9 Hz, 1H), 8.01–8.06 (m, 1H); HR-MS calcd for
C24H29FN3O2 m/z = 410.2244 [M+H]+. Found: 410.2231.
5.1.32. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(3-methoxybenzoyl)piperidin-4-ylidene]acetamide (30f)
Compound 30f was prepared from 28 and 3-methoxybenzoyl
chloride in a manner similar to that described for compound 30a,
in a yield of 78% as a colorless amorphous solid. ½a D25
ꢃ
+30.83 (c
5.1.28. 2-[1-(2-Fluorobenzoyl)piperidin-4-ylidene]-N-{(3R)-1-
[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}acetamide (30b)
Compound 30b was prepared from 28 and 2-fluorobenzoyl
chloride in a manner similar to that described for compound 30a,
0.12, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.53–1.63 (m, 1H),
2.03–2.38 (m, 4H), 2.39–2.50 (m, 1H), 2.58–2.75 (m, 2H), 2.84–
3.04 (m, 2H), 3.50–3.75 (m, 6H), 3.78 (s, 3H), 4.13–4.25 (m, 1H),
5.72 (s, 1H), 6.92–6.98 (m, 2H), 6.99–7.04 (m, 1H), 7.35 (dd,
J = 7.8, 7.8 Hz, 1H), 7.37–7.43 (m, 1H), 7.53 (d, J = 8.3 Hz, 1H),
7.67 (dd, J = 10.3, 2.5 Hz, 1H), 7.82–7.88 (m, 2H), 7.96 (dd, J = 9.3,
5.9 Hz, 1H), 8.05 (d, J = 7.3 Hz, 1H); MS (FAB) m/z = 502 [M+H]+.
Anal. Calcd for C30H32FN3O3ꢂ0.5H2O: C, 70.57; H, 6.51; N, 8.23; F,
3.72. Found: C, 70.70; H, 6.41; N, 8.19; F, 3.80.
in a yield of 86% as a colorless amorphous solid. ½a D25
ꢃ
+35.99 (c
0.10, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.54–1.66 (m, 1H),
2.04–2.19 (m, 2H), 2.24–2.36 (m, 2H), 2.40–2.48 (m, 1H), 2.58–
2.75 (m, 2H), 2.80–3.03 (m, 2H), 3.18–3.23 (m, 1H), 3.24–3.30
(m, 1H), 3.58–3.76 (m, 4H), 4.13–4.25 (m, 1H), 5.71, 5.76 (each s,
1H), 7.25–7.33 (m, 2H), 7.36–7.45 (m, 2H), 7.46–7.55 (m, 2H),
7.64–7.70 (m, 1H), 7.84–7.88 (m, 2H), 7.93–7.99 (m, 1H), 8.04–
8.09 (m, 1H); MS (FAB) m/z = 490 [M+H]+. Anal. Calcd for
C29H29F2N3O2ꢂ0.4H2O: C, 70.12; H, 6.05; N, 8.46; F, 7.65. Found:
C, 70.12; H, 5.97; N, 8.36; F, 7.82.
5.1.33. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(4-methoxybenzoyl)piperidin-4-ylidene]acetamide (30g)
To a solution of 28 (300 mg, 0.82 mmol) in THF (6 mL) were
added 4-methoxybenzoic acid (131 mg, 0.86 mmol), HOBt
(55 mg, 0.41 mmol), and WSCꢂHCl (172 mg, 0.90 mmol), and this
mixture was stirred at room temperature overnight. The mixture
was then partitioned between CHCl3 and H2O, and the organic
layer was washed with satd NaHCO3 aq, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by silica
gel column chromatography (CHCl3/MeOH/NH4OH = 99:1:0.1–
98:2:0.1) to yield 30g (150 mg, 36%) as a colorless amorphous so-
5.1.29. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(3-methoxybenzoyl)piperidin-4-ylidene]acetamide (30c)
Compound 30c was prepared from 28 and 3-fluorobenzoyl
chloride in a manner similar to that described for compound 30a,
in a yield of 54% as a pale yellow amorphous solid. ½a D25
ꢃ
+30.90 (c
0.11, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–1.63 (m, 1H),
2.05–2.31 (m, 4H), 2.41–2.50 (m, 1H), 2.59–2.76 (m, 2H), 2.86–
3.05 (m, 2H), 3.50–3.76 (m, 6H), 3.78 (s, 3H), 4.14–4.26 (m, 1H),
5.73 (s, 1H), 7.23–7.33 (m, 3H), 7.40 (ddd, J = 9.2, 8.8, 2.6 Hz, 1H),
7.46–7.55 (m, 2H), 7.68 (dd, J = 10.3, 2.5 Hz, 1H), 7.82–7.88 (m,
2H), 7.97 (dd, J = 8.8, 5.9 Hz, 1H), 8.06 (d, J = 6.8 Hz, 1H); MS
(FAB) m/z = 490 [M+H]+. Anal. Calcd for C29H29F2N3O2ꢂ0.4H2O: C,
70.12; H, 6.05; N, 8.46; F, 7.65. Found: C, 70.01; H, 6.02; N, 8.22;
F, 7.73.
lid. ½a 2D5
ꢃ
+30.99 (c 0.10, MeOH). 1H NMR (400 MHz, DMSO-d6) d:
1.53–1.64 (m, 1H), 2.05–2.16 (m, 1H), 2.18–2.27 (m, 2H), 2.29–
2.35 (m, 1H), 2.40–2.50 (m, 1H), 2.59–2.75 (m, 2H), 2.89–2.97
(m, 2H), 3.35–3.61 (m, 4H), 3.68 (d, J = 13.2 Hz, 1H), 3.73 (d,
J = 13.2 Hz, 1H), 3.80 (s, 3H), 4.14–4.25 (m, 1H), 5.73 (s, 1H), 6.98
(d, J = 8.8 Hz, 2H), 7.35–7.43 (m, 3H), 7.53 (d, J = 8.3 Hz, 1H), 7.68
(dd, J = 10.2, 2.4 Hz, 1H), 7.82–7.88 (m, 2H), 7.97 (dd, J = 8.8,
5.9 Hz, 1H), 8.05 (d, J = 6.9 Hz, 1H); MS (FAB) m/z = 502 [M+H]+.
Anal. Calcd for C30H32FN3O3ꢂ0.3H2O: C, 71.04; H, 6.48; N, 8.29; F,
3.75. Found: C, 71.01; H, 6.46; N, 8.22; F, 3.63.
5.1.30. 2-[1-(4-Fluorobenzoyl)piperidin-4-ylidene]-N-{(3R)-1-
[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}acetamide (30d)
Compound 30d was prepared from 28 and 4-fluorobenzoyl
chloride in a manner similar to that described for compound
5.1.34. 2-[1-(3,4-Dimethoxybenzoyl)piperidin-4-ylidene]-N-{(3R)-
1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}acetamide(30h)
Compound 30h was prepared from 28 and 3,4-dimethoxyben-
zoic acid in a manner similar to that described for compound
30a, in a yield of 83% as a colorless amorphous solid. ½a D25
ꢃ
+29.16 (c 0.12, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–
1.64 (m, 1H), 2.05–2.34 (m, 4H), 2.40–2.50 (m, 1H), 2.59–2.75
(m, 2H), 2.88–3.04 (m, 2H), 3.50–3.77 (m, 6H), 4.13–4.25 (m,
1H), 5.71 (s, 1H), 7.23–7.30 (m, 2H), 7.40 (ddd, J = 8.8, 8.8,
2.5 Hz, 1H), 7.45–7.55 (m, 3H), 7.67 (dd, J = 10.8, 2.7 Hz, 1H),
7.82–7.87 (m, 2H), 7.96 (dd, J = 9.0, 5.6 Hz, 1H), 8.06 (d,
J = 6.8 Hz, 1H); MS (FAB) m/z = 490 [M+H]+. Anal. Calcd for
30g, in a yield of 68% as a slightly brown amorphous solid. ½a D25
ꢃ
+9.06 (c 0.11, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–1.63
(m, 1H), 2.05–2.17 (m, 1H), 2.18–2.27 (m, 2H), 2.29–2.35 (m,
1H), 2.40–2.50 (m, 1H), 2.59–2.74 (m, 2H), 2.89–2.99 (m, 1H),
3.35–3.62 (m, 4H), 3.66–3.76 (m, 2H), 3.77 (s, 3H), 3.79 (s, 3H),
4.14–4.24 (s, 1H), 5.75 (s, 1H), 6.94–7.01 (m, 3H), 7.41 (ddd,

6000
I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
J = 8.8, 8.8, 2.5 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.67 (dd, J = 10.2,
2.9 Hz, 1H), 7.85–7.88 (m, 3H), 7.97 (dd, J = 9.3, 5.8 Hz, 1H), 8.05
(d, J = 6.9 Hz, 1H); MS (FAB) m/z = 532 [M+H]+. Anal. Calcd for
C31H34FN3O4ꢂ0.5H2O: C, 68.87; H, 6.53; N, 7.77; F, 3.51. Found: C,
68.70; H, 6.61; N, 7.71; F, 3.48.
1H), 9.80 (br s, 1H); MS (ESI) m/z = 488 [M+H]+. Anal. Calcd for
C29H30FN3O3ꢂ0.75H2Oꢂ0.25CHCl3: C, 66.71; H, 6.03; N, 7.91; Cl,
5.01; F, 3.58. Found: C, 66.31; H, 6.06; N, 7.86; Cl, 4.95; F, 3.55.
5.1.39. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(2-hydroxy-3-methoxybenzoyl)piperidin-4-ylidene]-
acetamide (30m)
5.1.35. 2-[1-(1,3-Benzodioxol-5-ylcarbonyl)piperidin-4-ylidene]-
N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}acet-
amide (30i)
Compound 30m was prepared from 28 and 2-hydroxy-3-meth-
oxybenzoic acid in a manner similar to that described for com-
pound 30g, in a yield of 67% as a pale brown amorphous solid.
Compound 30i was prepared from 28 and 3,4-methylenedioxy-
benzoic acid in a manner similar to that described for compound
½
a 2D5 +31.99 (c 0.10, MeOH). 1H NMR (400 MHz, DMSO-d6) d:
ꢃ
30g, in a yield of 56% as a slightly brown amorphous solid. ½a D25
ꢃ
1.53–1.64 (m, 1H), 2.05–2.26 (m, 3H), 2.29–2.35 (m, 1H), 2.40–
2.50 (m, 1H), 2.59–2.65 (m, 2H), 2.83–3.00 (m, 2H), 3.09–3.30
(m, 2H), 3.50–3.75 (m, 4H), 3.81 (s, 3H), 4.13–4.26 (m, 1H), 5.70
(s, 1H), 6.70 (dd, J = 7.8, 1.5 Hz, 1H), 6.81 (dd, J = 8.8, 8.8 Hz, 1H),
6.98 (dd, J = 8.8, 1.5 Hz, 1H), 7.40 (ddd, J = 8.8, 8.8, 2.5 Hz, 1H),
7.53 (d, J = 8.3 Hz, 1H), 7.68 (dd, J = 10.5, 2.7 Hz, 1H), 7.82–7.88
(m, 3H), 7.97 (dd, J = 8.8, 5.8 Hz, 1H), 8.05 (d, J = 6.8 Hz, 1H), 9.00
(s, 1H); MS (FAB) m/z = 518 [M+H]+. Anal. Calcd for
C30H32FN3O4ꢂ0.5H2O: C, 68.42; H, 6.32; N, 7.98; F, 3.61. Found: C,
68.53; H, 6.21; N, 7.91; F, 3.51.
+26.14 (c 0.13, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–
1.64 (m, 1H), 2.05–2.17 (m, 1H), 2.18–2.25 (m, 2H), 2.29–2.36
(m, 1H), 2.40–2.50 (m, 1H), 2.59–2.75 (m, 2H), 2.90–2.98 (m,
1H), 3.35–3.60 (m, 4H), 3.66–3.78 (m, 2H), 4.14–4.26 (m, 1H),
5.72 (s, 1H), 6.07 (s, 2H), 6.90–6.99 (m, 3H), 7.41 (ddd, J = 8.8,
8.8, 2.5 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.67 (dd, J = 10.2, 2.9 Hz,
1H), 7.82–7.88 (m, 3H), 7.97 (dd, J = 9.1, 5.7 Hz, 1H), 8.05 (d,
J = 6.8 Hz, 1H); MS (FAB) m/z = 516 [M+H]+. Anal. Calcd for
C30H30FN3O4ꢂ0.75H2O: C, 68.10; H, 6.00; N, 7.94; F, 3.59. Found:
C, 68.32; H, 5.87; N, 7.90; F, 3.74.
5.1.40. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(2-hydroxy-4-methoxybenzoyl)piperidin-4-ylidene]-
acetamide (30n)
5.1.36. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(2-hydroxybenzoyl)piperidin-4-ylidene]acetamide (30j)
Compound 30j was prepared from 28 and 2-hydroxybenzoic
acid in a manner similar to that described for compound 30g, in
Compound 30n was prepared from 28 and 2-hydroxy-4-meth-
oxybenzoic acid in a manner similar to that described for com-
a yield of 59% as a colorless amorphous solid. ½a D25
ꢃ
+22.30 (c 0.25,
pound 30g, in a yield of 16% as a colorless amorphous solid. ½a D25
ꢃ
MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.51–1.63 (m, 1H),
2.05–2.27 (m, 3H), 2.28–2.35 (m, 1H), 2.40–2.50 (m, 1H), 2.58–
2.74 (m, 2H), 2.83–2.99 (m, 2H), 3.10–3.40 (m, 2H), 3.50–3.77
(m, 4H), 4.13–4.24 (m, 1H), 5.71 (s, 1H), 6.80–6.89 (m, 2H), 7.12
(dd, J = 7.8, 1.5 Hz, 1H), 7.19–7.24 (m, 1H), 7.41 (ddd, J = 8.8, 8.8,
2.9 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.67 (dd, J = 10.2, 2.4 Hz, 1H),
7.82–7.88 (m, 2H), 7.96 (dd, J = 8.8, 5.8 Hz, 1H), 8.04 (d, J = 7.4 Hz,
1H), 9.76 (s, 1H); MS (FAB) m/z = 488 [M+H]+. Anal. Calcd for
C29H30FN3O3ꢂ0.25H2O: C, 70.78; H, 6.25; N, 8.54; F, 3.86. Found:
C, 70.99; H, 6.10; N, 8.50; F, 3.90.
+30.99 (c 0.10, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–
1.64 (m, 1H), 2.05–2.23 (m, 3H), 2.29–2.34 (m, 1H), 2.42–2.50
(m, 1H), 2.59–2.66 (m, 1H), 2.68–2.74 (m, 1H), 2.88–2.93 (m,
2H), 3.35–3.50 (m, 5H), 3.66–3.76 (m, 2H), 3.72 (s, 3H), 4.14–
4.23 (m, 1H), 5.71 (s, 1H), 6.40–6.45 (m, 2H), 7.40 (ddd, J = 8.8,
7.2, 2.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.67 (dd, J = 10.4, 2.4 Hz,
1H), 7.82–7.87 (m, 2H), 7.97 (dd, J = 9.2, 5.6 Hz, 1H), 8.04 (d,
J = 7.2 Hz, 1H), 9.88 (s, 1H); MS (FAB) m/z = 518 [M+H]+. Anal. Calcd
for C30H32FN3O4ꢂ0.5H2O: C, 68.42; H, 6.32; N, 7.98; F, 3.61. Found:
C, 68.78; H, 6.43; N, 7.96; F, 3.56.
5.1.37. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(3-hydroxybenzoyl)piperidin-4-ylidene]acetamide (30k)
Compound 30k was prepared from 28 and 3-hydroxybenzoic
acid in a manner similar to that described for compound 30g, in
5.1.41. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(2-hydroxy-5-methoxybenzoyl)piperidin-4-ylidene]-
acetamide (30o)
Compound 30o was prepared from 28 and 2-hydroxy-5-meth-
oxybenzoic acid in a manner similar to that described for com-
a yield of 22% as a colorless amorphous solid. ½a D25
ꢃ
+54.00 (c 0.10,
MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–1.63 (m, 1H),
2.04–2.36 (m, 4H), 2.40–2.50 (m, 1H), 2.59–2.78 (m, 2H), 2.82–
3.02 (m, 2H), 3.35–3.78 (m, 6H), 4.15–4.23 (m, 1H), 5.72 (s, 1H),
6.65–6.84 (m, 3H), 7.22 (dd, J = 7.9, 7.8 Hz, 1H), 7.40 (ddd, J = 8.9,
8.9, 2.6 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.67 (dd, J = 10.3, 2.4 Hz,
1H), 7.81–7.89 (m, 3H), 7.96 (dd, J = 9.0, 5.9 Hz, 1H), 8.03 (d,
J = 7.0 Hz, 1H), 9.64 (br s, 1H); MS (ESI) m/z = 488 [M+H]+. HR-MS
calcd for C29H30FN3O3 m/z = 488.2344 [M+H]+. Found: 488.2349.
pound 30g, in a yield of 32% as a colorless amorphous solid. ½a D25
ꢃ
+36.36 (c 0.11, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–
1.64 (m, 1H), 2.05–2.25 (m, 3H), 2.29–2.36 (m, 1H), 2.39–2.50
(m, 1H), 2.58–2.75 (m, 2H), 2.83–3.00 (m, 2H), 3.50–3.67 (m,
4H), 3.68 (s, 3H), 3.69–3.76 (m, 2H), 4.14–4.25 (m, 1H), 5.72 (s,
1H), 6.69 (d, J = 2.9 Hz, 1H), 6.76–6.84 (m, 2H), 7.40 (ddd, J = 8.8,
8.8, 3.0 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.67 (dd, J = 10.2, 2.5 Hz,
1H), 7.82–7.86 (m, 2H), 7.97 (dd, J = 9.2, 5.8 Hz, 1H), 8.04 (d,
J = 6.9 Hz, 1H), 9.27 (s, 1H); MS (FAB) m/z = 518 [M+H]+. Anal. Calcd
for C30H32FN3O4ꢂ0.25H2O: C, 69.01; H, 6.27; N, 8.05; F, 3.64. Found:
C, 68.80; H, 6.35; N, 7.82; F, 3.43.
5.1.38. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(4-hydroxybenzoyl)piperidin-4-ylidene]acetamide (30l)
Compound 30l was prepared from 28 and 4-hydroxybenzoic
acid in a manner similar to that described for compound 30g, in
5.1.42. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(2-hydroxy-6-methoxybenzoyl)piperidin-4-ylidene]-
acetamide (30p)
a yield of 74% as a colorless amorphous solid. ½a D25
ꢃ
+35.99 (c 0.10,
MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–1.63 (m, 1H),
2.04–2.16 (m, 1H), 2.17–2.25 (m, 2H), 2.32 (dd, J = 9.3, 4.8 Hz,
1H), 2.42–2.50 (m, 1H), 2.59–2.67 (m, 1H), 2.69–2.75 (m, 1H),
2.86–2.96 (m, 2H), 3.39–3.59 (m, 4H), 3.69 (d, J = 7.2 Hz, 1H),
3.73 (d, J = 7.2 Hz, 1H), 4.17–4.23 (m, 1H), 5.72 (s, 1H), 6.79 (d,
J = 8.5 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 7.40 (ddd, J = 8.9, 8.9,
2.5 Hz, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.67 (dd, J = 10.3, 2.3 Hz, 1H),
7.82–7.88 (m, 3H), 7.96 (dd, J = 9.0, 5.8 Hz, 1H), 8.03 (d, J = 7.0 Hz,
Compound 30p was prepared from 28 and 2-hydroxy-6-meth-
oxybenzoic acid in a manner similar to that described for com-
pound 30g, in a yield of 49% as a pale pink amorphous solid. ½a D25
ꢃ
+27.18 (c 0.103, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–
1.65 (m, 1H), 2.02–2.25 (m, 4H), 2.29–2.36 (m, 1H), 2.59–2.75
(m, 2H), 2.85–2.95 (m, 2H), 3.09–3.15 (m, 1H), 3.16–3.22 (m,
1H), 3.55–3.78 (m, 7H), 4.13–4.27 (m, 1H), 5.67, 5.73 (each s,

I. Sato et al. / Bioorg. Med. Chem. 17 (2009) 5989–6002
6001
1H), 6.46–6.54 (m, 2H), 7.09–7.16 (m, 1H), 7.37–7.45 (m, 1H),
7.50–7.56 (m, 1H), 7.64–7.71 (m, 1H), 7.82–7.90 (m, 2H), 7.94–
8.00 (m, 1H), 8.01–8.09 (m, 1H), 9.60, 9.61 (each s, 1H); MS (FAB)
m/z = 518 [M+H]+. Anal. Calcd for C30H32FN3O4: C, 68.66; H, 6.30;
N, 8.01; F, 3.62. Found: C, 68.42; H, 6.15; N, 7.87; F, 3.61.
5.4 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, and 5.5 mM glucose.
The cell suspension (490 L) was transferred into cuvettes and
l
placed under constant agitation. Changes in fluorescence were
monitored at 25 °C using a spectrophotometer at excitation wave-
lengths of 340 nm and 380 nm and an emission wavelength of
510 nm. Calculation of Ca2+ concentration was performed using
the Kd for the Ca2+ binding at 224 nm. The antagonist was dis-
5.1.43. 2-[1-(4-Fluoro-2-hydroxybenzoyl)piperidin-4-ylidene]-
N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}acet-
amide (30q)
Compound 30q was prepared from 28 and 4-fluoro-2-hydroxy-
benzoic acid in a manner similar to that described for compound
solved in 100% DMSO solution (1 lL) and added to the cuvette
1 min prior to the addition of eotaxin (final concentration of
50 ng/mL). Linear regression analysis using EXSAS-STAT was used
to calculate the IC50 values. Values are reported as means SEM
of triplicate experiments.
30g, in a yield of 31% as a colorless amorphous solid. ½a D25
ꢃ
+24.99
(c 0.10, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.53–1.64 (m,
1H), 2.05–2.23 (m, 3H), 2.29–2.34 (m, 1H), 2.40–2.50 (m, 1H),
2.59–2.66 (m, 1H), 2.67–2.74 (m, 1H), 2.85–2.99 (m, 2H), 3.35–
3.76 (m, 4H), 4.12–4.25 (m, 1H), 5.71 (s, 1H), 6.60–6.70 (m, 2H),
7.18 (dd, J = 8.3, 6.9 Hz, 1H), 7.40 (ddd, J = 8.8, 8.8, 2.5 Hz, 1H),
7.53 (d, J = 8.3 Hz, 1H), 7.68 (dd, J = 10.2, 2.4 Hz, 1H), 7.82–7.88
(m, 2H), 7.97 (dd, J = 8.8, 5.8 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H),
10.33 (s, 1H); MS (FAB) m/z = 506 [M+H]+. Anal. Calcd for
C29H29F2N3O3ꢂ0.2H2O: C, 68.41; H, 5.82; N, 8.25; F, 7.46. Found:
C, 68.31; H, 5.83; N, 8.18; F, 7.47.
5.2.2. In vitro human liver microsomal (HLM) metabolic
stability
Pooled human liver microsomes (Cat No. H161, lot 14, Gentest
Corporation) were diluted in 0.1 M KH2PO4/K2HPO4 buffer, pH
7.4, containing 8 mM MgCl2, 10 mM G6P, and 2 units/mL G6PDH.
The incubation mixtures (1.0 mL total volume), which contained
0.1 mg/mL of microsomal proteins and 0.5
were pre-incubated for 5 min at 37 °C. The reactions were initiated
by the addition of NADPH (50 mM, 10 L) and stopped at the
appropriate time points (0, 10, 20, and 30 min) by the addition of
100 L of 20% TCA aq. The incubation mixtures were then added
to an internal standard (5 mol/L in 10% DMSO, 100 L) and centri-
lmol/mL of substrates,
l
5.1.44. 2-[1-(4-Chloro-2-hydroxybenzoyl)piperidin-4-ylidene]-
N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}acet-
amide (30r)
Compound 30r was prepared from 28 and 4-chloro-2-hydroxy-
benzoic acid in a manner similar to that described for compound
l
l
l
fuged for 5 min at 3000 rpm. One milliliter of the supernatant was
purified by solid phase extraction column (OASIS HLB, 30 mg/1 cc,
Waters) and was then analyzed using LC–MS/MS.
30g, in a yield of 63% as a colorless amorphous solid. ½a D25
ꢃ
+32.06
(c 0.103, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.53–1.64 (m,
1H), 2.03–2.27 (m, 3H), 2.29–2.35 (m, 1H), 2.39–2.50 (m, 1H),
2.57–2.75 (m, 2H), 2.82–3.01 (m, 2H), 3.10–3.28 (m, 2H), 3.49–
3.75 (m, 4H), 4.12–4.25 (m, 1H), 5.71 (s, 1H), 6.88–6.93 (m, 2H),
7.14–7.18 (m, 1H), 7.40 (ddd, J = 8.8, 8.8, 3.0 Hz, 2H), 7.53 (d,
J = 8.8 Hz, 1H), 7.68 (dd, J = 10.3, 2.5 Hz, 1H), 7.82–7.88 (m, 2H),
7.97 (dd, J = 9.3, 5.9 Hz, 1H), 8.05 (d, J = 7.3 Hz, 1H), 10.33 (s, 1H);
MS (FAB) m/z = 522 [M+H]+. Anal. Calcd for C29H29ClFN3O3ꢂ0.5H2O:
C, 65.59; H, 5.69; N, 7.91;Cl, 6.68; F, 3.58. Found: C, 65.74; H, 5.67;
N, 8.05; Cl, 6.45; F, 3.49.
5.3. Interaction energy calculation method
To evaluate the ability to accept hydrogen bonds, the interac-
tion energies between ligands and water molecule (as a model
hydrogen bond donor in a protein) were obtained using ab initio
quantum mechanical calculations. In this study, the interaction en-
ergy is defined as
E ðinteractionÞ ¼ E ðcomplexÞ ꢁ E ðligandÞ ꢁ E ðwaterÞ
where E (interaction) is the interaction energy. E (complex), E (li-
gand), and E (water) are the energy of the ligand/water complex,
that of the ligand, and that of the water molecule, respectively,
which were calculated as described below. Initial coordinates were
built and optimized at the molecular mechanics level using Molec-
ular Operating Environment software (MOE, Chemical Computing
Group, Inc.). The distance between the carboxyl group of the ligand
and the oxygen atom of the water molecule was set at 2.5 Å. After
geometrical optimizations were performed on the HF/6-31* level
using the GAUSSIAN 98 program,9 the single point energy for the com-
plex, ligand, and water molecule was calculated on the B3LYP/6-
311+G** level.
5.1.45. N-{(3R)-1-[(6-Fluoro-2-naphthyl)methyl]pyrrolidin-3-
yl}-2-[1-(2-hydroxy-4-methylbenzoyl)piperidin-4-ylidene]-
acetamide (30s)
Compound 30s was prepared from 28 and 2-hydroxy-4-methyl-
benzoic acid in a manner similar to that described for compound
30g, in a yield of 27% as a pale yellow amorphous solid. ½a D25
ꢃ
+31.99 (c 0.10, MeOH). 1H NMR (400 MHz, DMSO-d6) d: 1.52–
1.63 (m, 1H), 2.05–2.22 (m, 3H), 2.24 (s, 3H), 2.29–2.35 (m, 1H),
2.40–2.50 (m, 1H), 2.59–2.73 (m, 2H), 2.87–2.97 (m, 2H), 3.10–
3.65 (m, 4H), 3.68 (d, J = 12.7 Hz, 1H), 3.73 (d, J = 12.7 Hz, 1H),
4.13–4.25 (m, 1H), 5.71 (s, 1H), 6.63–6.68 (m, 2H), 7.05 (d,
J = 7.8 Hz, 1H), 7.40 (ddd, J = 9.2, 8.8, 2.6 Hz, 2H), 7.53 (d,
J = 8.3 Hz, 1H), 7.68 (dd, J = 10.3, 2.5 Hz, 1H), 7.82–7.88 (m, 2H),
7.97 (dd, J = 9.3, 5.9 Hz, 1H), 8.04 (d, J = 7.3 Hz, 1H), 9.65 (s, 1H);
MS (FAB) m/z = 502 [M+H]+. Anal. Calcd for C30H32FN3O3ꢂ0.5H2O:
C, 70.57; H, 6.51; N, 8.23; F, 3.72. Found: C, 70.72; H, 6.53; N,
8.15; F, 3.60.
Acknowledgments
The authors wish to thank the staff of the Division of Analysis &
Pharmacokinetics Research Laboratories for their help with the
evaluation of in vitro human liver microsomal (HLM) metabolic
stability (CLint) as well as with the elemental analysis and spectral
measurements.
5.2. Biology
References and notes
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CCR3-transfected B300-19 cells11 were loaded with 5
lM Fura-
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a
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