Discovery of piperazinylimidazo[1,2-a]pyridines as novel S4 binding elements for orally active Factor Xa inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.12.024

We have recently reported the discovery of orally active sulfonylalkylamide Factor Xa (FXa) inhibitors, as typified by compound 1 (FXa IC₅₀ = 0.061 μM). Since the pyridylpiperidine moiety was not investigated in our previous study, we conducted detailed structure-activity relationship studies on this S4 binding element. This investigation led to the discovery of piperazinylimidazo[1,2-a]pyridine 2b as a novel and potent FXa inhibitor (FXa IC₅₀ = 0.021 μM). Further modification resulted in the discovery of 2-hydroxymethylimidazo[1,2-a]pyridine 2e (FXa IC₅₀ = 0.0090 μM), which was found to be a selective and orally bioavailable FXa inhibitor with reduced CYP3A4 inhibition.

Full text of DOI:10.1016/j.bmc.2007.12.024

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3125–3140
Discovery of piperazinylimidazo[1,2-a]pyridines as novel S4
binding elements for orally active Factor Xa inhibitors
Yasuhiro Imaeda,* Tetsuji Kawamoto, Mamoru Tobisu,ꢀ Noriko Konishi,
Katsuhiko Hiroe, Masaki Kawamura, Toshimasa Tanaka and Keiji Kubo
Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
Received 22 November 2007; revised 11 December 2007; accepted 12 December 2007
Available online 27 December 2007
Abstract—We have recently reported the discovery of orally active sulfonylalkylamide Factor Xa (FXa) inhibitors, as typified by
compound 1 (FXa IC50 = 0.061 lM). Since the pyridylpiperidine moiety was not investigated in our previous study, we conducted
detailed structure–activity relationship studies on this S4 binding element. This investigation led to the discovery of piperazinylim-
idazo[1,2-a]pyridine 2b as a novel and potent FXa inhibitor (FXa IC50 = 0.021 lM). Further modification resulted in the discovery
of 2-hydroxymethylimidazo[1,2-a]pyridine 2e (FXa IC50 = 0.0090 lM), which was found to be a selective and orally bioavailable
FXa inhibitor with reduced CYP3A4 inhibition.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
with many drugs and food.1 Despite careful medical
treatment, it is difficult to protect patients from the risk
of bleeding. Therefore, there is a clear need to develop
improved anticoagulants which are orally active,
have long duration of action, and have useful in vivo po-
tency without unpredictable or serious bleeding
complications.
Intravascular clot formation is an essential factor in a
number of cardiovascular diseases such as myocardial
infarction, unstable angina, deep vein thrombosis, pul-
monary embolism, and ischemic stroke. The interrup-
tion of the coagulation cascade is one of the most
effective strategies for inhibition of clot formation,
which is an important approach for the prevention
and treatment of these thrombotic disorders. Warfarin
prevents the synthesis of vitamin K-dependent coagula-
tion factors in the liver and inhibits the activation of
blood coagulation leading to thrombus formation. As
the only oral anticoagulant in a clinical setting, warfarin
is widely used for the prophylaxis and treatment of ve-
nous thromboembolic disease. However, warfarin ther-
apy necessitates a routine blood coagulation test and
dose adjustment for each individual, because it has sub-
stantial drawbacks such as narrow therapeutic dose win-
dow, slow onset and offset of action, and interaction
The trypsin-like serine protease Factor Xa (FXa), con-
verting prothrombin to thrombin, occupies a unique po-
sition at the final convergence point of the intrinsic and
the extrinsic coagulation pathways.2 Since this process
involves signal amplification, with one molecule of
FXa activating many molecules of prothrombin to
thrombin,3 FXa inhibitors are expected to be more effi-
cacious in interrupting the coagulation cascade than di-
rect thrombin inhibitors. Moreover, it was also shown
recently in in vivo studies that FXa inhibitors may have
less bleeding risk than thrombin inhibitors.4 Thus, FXa
has been identified as an attractive target enzyme for
antithrombotic therapy.5
A variety of low-molecular-weight FXa inhibitors
have been described6 and recently many orally active
FXa inhibitors have been extensively explored.5e–h
For example, some non-amidine FXa inhibitors such
as apixaban5f and rivaroxaban7 have been reported
to be orally active FXa inhibitors and are under clin-
ical study.
Keywords: Factor Xa inhibitor; Anticoagulant; Piperazinylimi-
dazo[1,2-a]pyridines; Oral bioavailability.
*
Corresponding author. Tel.: +81 6 6300 6458; fax: +81 6 6300
6306; e-mail: Imaeda_Yasuhiro@takeda.co.jp
Present address: Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka
565-0871, Japan.
ꢀ
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.024

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Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
We have previously reported orally active sulfonylalkyla-
mide FXa inhibitors, as typified by compound 1.8 The
structure–activity relationships (SAR) for this series re-
vealed that the FXa enzyme rigorously recognized the 6-
chloronaphthalene moiety in the S1 site of FXa, whereas
the recognition of the pyridylpiperidine moiety as the S4
binding element appeared to be loose. Our binding model
of compound 1 in FXa8 indicated that the basic pyridine
ring formed no salt bridge with the carboxyl group of any
amino acid residue in the S4 site, however, it did form
hydrophobic contacts with the aromatic rings of Tyr99,
Phe174, and Trp215 in ‘the S4 aryl binding site’.9 These re-
sults prompted us to modify the S4 binding element of
compound 1, because it remained uninvestigated in our
previous work. Zhu and Scarborough suggested that
rather diverse S4 binding elements can be utilized to give
highly potent FXa inhibitors and that diverse S4 binding
elements would provide opportunities to modulate the
physiochemical profiles.6d We conducted preliminary
chemical modification in an attempt to enhance the
hydrophobic contact by extension of the p electron system
(e.g., a 5,6-fused heteroaromatic ring, such as an imi-
dazo[1,2-a]pyridine, an imidazo[1,2-b]pyridazine, and a
benzimidazole ring). As a result of these initial efforts,
we found piperazinylimidazo[1,2-a]pyridine derivative
2a to be a weak FXa inhibitor and 2b, which showed more
potent FXa inhibitory activity in vitro (FXa
IC50 = 0.021 lM) than 1, as shown in Table 1. Therefore,
we decided to conduct detailed SAR studies on the S4
binding element using 2b as a new lead, since there is no
report on FXa inhibitors bearing an imidazo[1,2-a]pyri-
dine ring and the introduction of the varied substituents
on the imidazo[1,2-a]pyridine ring appears to be possible
by use of robust chemistry. In this paper, we describe the
synthesis, SAR, ex vivo anticoagulant activity, selectivity,
and pharmacokinetics of the piperazinylimidazo[1,2-
a]pyridine derivatives.10 It is also worthwhile to report
the synthesis and the properties of imidazo[1,2-a]pyri-
dines bearing an alkylamino group at the 5-, 6-, or 7-posi-
tion, because there are few literature reports on
imidazo[1,2-a]pyridines.11
2. Chemistry
The synthesis of the 5-(1-piperazinyl)imidazo[1,2-a]pyr-
idines 2a–r is shown in Schemes 1–3. 5-Chloroimi-
dazopyridines 412 and 5, which were obtained by the
condensation of 2-amino-6-chloropyridine 312 and
chloroacetaldehyde or bromoacetone, were heated
with piperazine without solvent at 100–150 ꢁC fol-
lowed by tert-butoxycarbonylation to give the 5-(1-
piperazyl)imidazopyridines
6 and 7 as shown in
Scheme 1. Using formaldehyde in ethanol, direct
hydroxymethylation at the 3-position of the imidazo-
pyridine ring of 6 gave alcohol 8.13 The key interme-
diate ester 10 was prepared by condensation of 3 and
ethyl bromopyruvate followed by displacement of the
chlorine atom with piperazine and introduction of a
Boc group. Hydrolysis of 10 followed by reduction
of the carboxylic group with borane–tetrahydrofuran
complex (BH3–THF) afforded alcohol 11. Treatment
of 10 with 3 equivalent of methylmagnesium bromide
gave a mixture of alcohol 12 and ketone 13. Reduc-
tion of the acetyl group of 13 with NaBH4 afforded
alcohol 14. Removal of the Boc group from imidazo-
pyridines 6–8, 11, 12, and 14 followed by coupling
with 3-(6-chloronaphthalen-2-yl)sulfonylpropionic acid
158 using 1-[3-(dimethylamino)propyl]-3-ethylcarbodi-
imide hydrochloride (WSC) and 1-hydroxybenzotria-
zole hydrate (HOBt) gave the desired amides 2b–g.
5-Chloroimidazopyridine 4 was heated with piperidine
16 without solvent at 100–150 ꢁC to afford piperidy-
limidazopyridine 17. Cleavage of the 1,3-dioxolane
ring of 17 with 4 M HCl, followed by reductive ami-
nation with methylamine using sodium triacetoxyboro-
hydride, introduction of
a Boc group for easy
Table 1. In vitro activities of 1, 2a, and 2b
purification by silica gel chromatography, and removal
of the Boc group, gave N-methyl-N-piperidinamine 18.
Amide 2a was prepared by coupling of amine 18 with
carboxylic acid 15.
The requisite ester 21 was prepared by condensation of
2-amino-6-fluoropyridine 1912 and ethyl 4-chloroace-
toacetate followed by displacement of the fluorine atom
with piperazine and introduction of a Boc group
(Scheme 2). The corresponding displacement reaction
using ethyl (5-chloroimidazo[1,2-a]pyridin-2-yl) acetate
instead of 20 did not proceed. Ester 21 was treated with
ammonia solution to give amide 23. Direct amide for-
mation from 21 with dimethylamine or methylamine
using dimethylaluminum chloride afforded dimethyla-
mide 22 and methylamide 24, respectively. Ester 21
was treated with methylmagnesium bromide and
CeCl314 to give alcohol 25. Treatment of dimethylamide
22 with methylmagnesium bromide followed by reduc-
tion with NaBH4 provided alcohol 26. Removal of the
Boc group from imidazopyridines 21–26 followed by
coupling with 15 gave the corresponding desired amides
2h–m.
Compound
X
Human FXa
a
IC50 (lM)
1
0.061
0.84
2a
2b
0.021
a Inhibitory activity against human FXa. IC50 values shown are means
of duplicate measurement.

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
3127
Scheme 1. Synthesis of 5-(1-piperazinyl)imidazo [1, 2-a]pyridines. Reagents: (a) ClCH2CHO or BrCH2COMe; (b) piperazine; (c) (Boc)2O; (d) aq
HCHO; (e) BrCH2COCO2Et; (f) NaOH; (g) BH3/THF; (h) MeMgBr; (i) NaBH4; (j) concd HCl; (k) WSC, HOBt, DBU, Et3N; (l) 4; (m) 1—4 M HCl;
2—MeNH2, NaBH(OAc)3, AcOH; 3—(Boc)2O; 4—concd HCl; (n) 15, WSC, HOBt, Et3N, DBU.
3-Aminoimidazo[1,2-a]pyridine 2o was synthesized by
displacement of the chlorine atom in 2715 with 1-Boc-
piperazine, removal of the Boc group, coupling with
15, and subsequent reduction of the nitro group with
iron powder and CaCl2. 3-Aminoimidazo[1,2-a]pyridine
2o was acetylated to give acetamide 2p (Scheme 3).
7-(1-Piperazinyl)imidazo[1,2-a]pyridine 2r was synthe-
sized as shown in Scheme 5. 7-(4-Boc-piperazinyl)imi-
dazo[1,2-a]pyridine 34 was prepared by displacement
of the chlorine atom of 2-amino-4-chloropyridine 3216
with 1-Boc-piperazine followed by cyclization with chlo-
roacetaldehyde. Treatment of 34 with concentrated HCl
and subsequent coupling with carboxylic acid 15 affor-
ded the desired amide 2r.
The synthesis of 6-(1-piperazinyl)imidazo[1,2-a]pyridine
2q is depicted in Scheme 4. Displacement of the bromine
atom of 5-bromo-2-nitropyridine with 1-Boc-piperazine
followed by reduction of nitro group afforded amino-
pyridine 30, which was condensed with chloroacetalde-
hyde to give imidazopyridine 31. Removal of the Boc
group from 31 and subsequent coupling with carboxylic
acid 15 afforded the desired amide 2q.
3. Results and discussion
The compounds thus synthesized were evaluated in vitro
for inhibitory potency against human FXa, expressed as
IC50 values, and their activity in the prolongation of hu-

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Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
Scheme 2. Synthesis of 5-(1-piperazinyl)imidazo[1,2-a]pyridines 2h–m. Reagents: (a) ClCH2COCH2CO2Et; (b) piperazine; (c) (Boc)2O; (d) aq NH3;
(e) MeNH2 or Me2NH, Me2AlCl; (f) MeMgBr, CeCl3; (g) NaBH4; (h) 1—concd HCl; 2—15, WSC, HOBt, DBU, Et3N.
Scheme 3. Synthesis of 5-(1-piperazinyl)imidazo[1,2-a]pyridines 2n–p. Reagents: (a) 1-Boc-piperazine, i-Pr2NEt; (b) concd HCl; (c) 1—15, WSC,
HOBt, DBU, Et3N; 2—4 M HCl; (d) Fe, CaCl2; (e) Ac2O.
Scheme 4. Synthesis of 6-(1-piperazinyl)imidazo[1,2-a]pyridine 2q. Reagents: (a) 1-Boc-piperazine; (b) H2, Pd/C; (c) ClCH2CHO; (d) 1—concd HCl;
2—15, WSC, HOBt, DBU, Et3N.
Scheme 5. Synthesis of 7-(1-piperazinyl)imidazo[1,2-a]pyridine 2r. Reagents: (a) 1-Boc-piperazine; (b) ClCH2CHO, NaHCO3; (c) 1—concd HCl; 2—
15, WSC, HOBt, DBU, Et3N.

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
3129
man prothrombin time (PT), expressed as the concentra-
tion of compound required to double the clotting time
(PT2) in the PT assay. To estimate the oral bioavailabil-
ity of these compounds, the ex vivo PT prolonging activ-
ity was also determined 1 h after oral administration to
mice at a dose of 30 mg/kg and expressed as ratios of the
PT of the compound-treatment animals with that of
control group.
and PT assays are listed in Table 2. 5-(1-Piperazinyl)imi-
dazo[1,2-a]pyridine 2b (FXa IC50 = 0.021 lM) was
found to be the most potent FXa inhibitor among these
regioisomers. This fact suggests that the configuration of
the imidazo[1,2-a]pyridine ring in the S4 site in FXa
and/or the position of the basic nitrogen atom at the
1-position in the imidazopyridine ring is important for
the activity. Additionally, compound 2b showed the
most potent human PT prolonging activity among these
regioisomers.
The results obtained for the imidazo[1,2-a]pyridine
derivatives, which have a piperazine ring at the 5- (2b),
6- (2q), or 7-positions (2r), in in vitro FXa inhibition
The discovery of the 5-(1-piperazinyl)imidazo[1,2-a]pyr-
idine 2b led us to explore the C-2 or C-3 modification on
the imidazopyridine ring in an effort to further improve
potency as shown in Table 3. All compounds bearing
various substituents at the 2-position exhibited FXa
inhibitory activities with IC50 values of 10À8 M order.
2-Methyl derivative 2c slightly improved the FXa inhib-
itory activity compared to 2b. This suggests that the
methyl group could fill the S4 hydrophobic site in FXa
more effectively through van der Waals interactions
leading to an increase in the FXa inhibitory activity.
Among the compounds bearing a hydroxyl group,
hydroxymethyl derivative 2e, 1-hydroxy-1-methylethyl
derivative 2f, 1-hydroxyethyl derivative 2g, 2-hydroxy-
2-methylpropyl derivative 2l, and 2-hydroxypropyl
derivative 2m were equipotent or slightly more potent
with respect to anticoagulant activity compared to 2b.
The anticoagulant activities of ester 2h and amides 2i–
2k were equipotent or slightly reduced compared to
the corresponding alcohols 2e–g, 2l, and 2m. Regarding
the 3-substituted imidazo[1,2-a]pyridines, as shown in
Table 2. In vitro activities of Imidazo[1,2-a]pyridines 2b, 2q, and 2r
Compound
Position
Human FXa
a
IC50 (lM)
Human PT
b
PT2 (lM)
2b
2q
2r
5
6
7
0.021
0.087
0.078
1.4
2.9
2.2
a See corresponding footnotes in Table 1.
b PT (prothrombin time) is defined as the concentration of compound
required to double the time to clot formation in the PT assay. PT2
values shown are means of duplicate measurement.
Table 3. In vitro and ex vivo activities of imidazo[1,2-a]pyridines 2b–p
Compound
R1
R2
In vitro
Ex vivo
Human FXa
a
IC50 (lM)
Human PT
b
PT2 (lM)
CYP3A4
(% inhibition at 10 lM)
Mouse PT
b
PT2 (lM)
Mouse PT
ratioc
2b
2c
2e
2f
H
Me
H
0.021
0.012
0.0090
0.018
0.020
0.093
0.057
0.045
0.041
0.022
0.018
0.029
0.024
0.035
0.024
1.4
0.96
1.4
2.1
1.2
5.1
2.5
2.1
2.0
0.86
1.1
1.7
3.6
0.73
1.8
45
58
1.7
1.3
2.5
2.9
H
CH2OH
H
15
1.4
1.3
C(Me)2OH
CH(Me)OH
CH2CO2Et
CH2CONMe2
CH2CONH2
CH2CONHMe
CH2C(Me)2OH
CH2CH(Me)OH
H
H
NTd
23
NTd
1.8
NTd
2.5
2g
2h
2i
H
H
NTd
NTd
NTd
NTd
26
NTd
NTd
NTd
NTd
1.7
NTd
NTd
NTd
NTd
2.4
H
2j
H
2k
2l
H
H
2m
2d
2n
2o
2p
H
33
1.5
3.2
CH2OH
NO2
NH2
NHAc
NTd
NTd
52
NTd
NTd
1.5
NTd
NTd
2.4
H
H
H
NTd
NTd
NTd
a,b See corresponding footnotes in Tables 1 and 2.
c Ex vivo mouse PT prolonging activity was determined 1 h after oral administration of the compound at a dose of 30 mg/kg and expressed as ratios
of the PT of the compound-treatment mice with that of control group (n = 3).
d NT means ‘not tested’.

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Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
Table 3, amine 2o showed slightly more potent anticoag-
ulant activity compared to hydroxymethyl derivative 2d,
nitro derivative 2n, and acetamino derivative 2p.
Since many trypsin-like proteases have essential physio-
logical functions,20 an important consideration for the
development of FXa inhibitors is the ability of the mol-
ecule to bind selectively. In several enzyme inhibition as-
says, compound 2e displayed >100-fold selectivity for
FXa versus several coagulation (thrombin, kallikrein)
and fibrinolytic enzymes (plasmin, tissue plasminogen
activator (t-PA)) (Table 4). Furthermore, 2e showed
no significant binding to trypsin.
The ex vivo PT prolonging activities of a select set of the
potent inhibitors in mice were determined and are
shown in Table 3. Compounds 2b and 2c significantly
prolonged mouse PT by 2.5- and 2.9-fold, respectively.
These results suggested that 2b and 2c resembled the or-
ally active pyridylpiperidine 1 in showing good pharma-
cokinetics in mice.8 More polar compounds 2e, 2g, 2l,
2m, and 2o significantly prolonged mouse PT by 1.3-,
2.5-, 2.4-, 3.2-, and 2.4-fold, respectively. These results
suggested that the polar substituents such as hydroxyl
or amino group had not significantly affected the oral
bioavailability in mice. The reason why the in vitro
mouse PT of 2e was equipotent to those of other imi-
dazo[1,2-a]pyridines 2b, 2c, 2g, 2l, and 2m but the
ex vivo anticoagulant activity of 2e was lower than those
of them might be attributed to the difference of the plas-
ma concentration of the compounds 1h after oral
administration in mice.
The pharmacokinetic profile of compound 2e was evalu-
ated in cynomolgus monkeys. As illustrated in Table 5,
2e displayed good oral bioavailability (31%) after oral
administration at a dose of 3 mg/kg in fasted monkeys.
To predict the binding mode of these inhibitors, a
molecular modeling study on 2e was carried out using
the program GOLD and the X-ray structure of FXa
complexed with an inhibitor reported by Sanofi-Aven-
tis21 (Fig. 1). In the model, the imidazo[1,2-a]pyridine
ring forms no salt bridge with the carboxyl group of
Asp189 at the bottom of the S1 pocket, while the 6-chlo-
ronaphthyl group is situated in the hydrophobic S1 re-
gion and a close contact is observed between the
chlorine atom and the benzene ring of Tyr228.7,9b,22
Compound 2e represents a parallel example in FXa inhi-
bition wherein a basic group thought to be essential in
forming the salt bridge with Asp1896d,21 can be elimi-
nated and replaced by a neutral group capable of con-
tributing enough favorable interaction to produce
good affinity to the enzyme. The imidazo[1,2-a]pyridine
ring is deeply buried inside the hydrophobic S4 site and
makes hydrophobic contacts with the aromatic rings of
Tyr99, Phe174, and Trp215 without the salt bridge with
any amino acid residue in this site. In particular, the imi-
dazo[1,2-a]pyridine ring extends across the face of the
Phe174 phenyl ring with a favorable p–p stacking inter-
action, and thus appears to make the hydrophobic con-
tact with the S4 site in FXa more favorably than the
pyridine ring of 1 by extension of the p electron system
(Fig. 2). Moreover, a hydrogen bonding interaction be-
tween the oxygen atom of the piperazine amide bond
and the amide linkage of Gly219 is observed.21,22c,e,23
On the contrary, a hydrogen bond is not observed be-
In the course of further evaluation of 2b, it was found
to inhibit the human cytochrome P450 enzyme
CYP3A4 (45% inhibition at 10 lM), which is known
to be a primary factor responsible for the metabolism
of most drugs.17 Drugs that show CYP inhibition
influence the pharmacokinetics of other co-adminis-
tered drugs and can cause undesired effects in pa-
tients. It might be reasoned that the inhibitory
potency toward CYP3A4 is attributed to coordination
of the basic N(1) atom of the imidazopyridine ring
(pKa = 9.9 for 2b)18 to the heme.19 We thought that
the introduction of a substituent at the 2 or 3 posi-
tion of the imidazopyridine ring might prevent coordi-
nation to the heme and reduce the inhibitory potency.
The CYP3A4 inhibition of some hydroxyl-containing
derivatives, such as 1-hydroxymethyl derivative 2e
and 1-hydroxyethyl derivative 2g, was successfully
lowered as shown in Table 3. On the basis of the po-
tency, the ex vivo PT prolongation activity, and the
weakest CYP3A4 inhibitory potency, 2e was selected
for further evaluation.
Table 4. Selectivity profile for compound 2e
Serine protease
FXa
Thrombin
0.96
Kallikrein
14
Trypsin
>60
Plasmin
>60
t-PA
>60
Kia (lM)
0.0080
a Data are expressed as means of three determinations.
Table 5. In vivo cynomolgus monkey pharmacokinetics of compound 2e (n = 3, fasted)a
iv dose
C5 min (lg/mL)
0.54 0.06
AUC (lg h/mL)
0.73 0.25
Tmax (h)
MRT (h)
Vd(ss) (L/kg)
0.81 0.09
MRT (h)
CLtotal (L/h/kg)
0.44 0.12
B.A. (%)
0.3 mg/kg
po dose
3 mg/kg
1.97 0.63
AUC (lg h/mL)
2.26 0.87
Cmax (lg/mL)
0.50 0.17
1.33 0.58
3.60 0.43
30.5 1.5
a Values are expressed as means SD of three determinations.

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
3131
Figure 1. Binding model of compound 2e in FXa.
Figure 2. Binding model of compounds 2e (white) and 1 (orange) in FXa.
tween compound 1 and Gly219 (Fig. 2). Recently, other
researchers have reported that the important interaction
for high affinity to FXa is not an ionic interaction with
Asp189, but a hydrogen bonding interaction with the
carbonyl group in the amide linkage of Gly219.9b,21,22c
The reason why imidazo[1,2-a]pyridine 2e is more po-
tent than pyridylpiperidine 1 could be due to the p–p
stacking interaction and the hydrogen bond (Fig. 2).
The sulfone oxygen is involved in a hydrogen bond with
the Gln192 main chain nitrogen.6d
4. Conclusion
In order to clarify SAR of the S4 binding element of our
FXa inhibitor 1, we conducted modification of its S4

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Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
binding element. This investigation led to the discovery
of piperazinylimidazo[1,2-a]pyridine 2b as a potent FXa
inhibitor. Further investigation resulted in discovery of
the 2-hydroxymethylimidazo[1,2-a]pyridine 2e, which is
a potent, selective, and orally bioavailable FXa inhibitor
with reduced CYP3A4 inhibition. Various other optimi-
zation strategies using this imidazo[1,2-a]pyridine series
will be reported in due course.
temperature for 1 h and concentrated in vacuo. The res-
idue was diluted with water (200 mL) and extracted with
EtOAc (200 mL). The extract was washed with brine,
dried over anhydrous MgSO4, and concentrated in va-
cuo. The residue was purified by silica gel chromatogra-
phy (EtOAc/EtOH = 10:1) to give 6 (8.39 g, 93%) as a
1
pale yellow solid. H NMR (200 MHz, CDCl3) d: 1.50
(9H, s), 3.06–3.11 (4H, m), 3.54–3.82 (4H, m), 6.30
(1H, d, J = 7.2 Hz), 7.18 (1H, dd, J = 9.2 and 7.2 Hz),
7.42 (1H, d, J = 8.8 Hz), 7.57 (1H, s), 7.66 (1H, s).
5. Experimental
5.1. Chemistry
5.1.4. tert-Butyl 4-(2-methylimidazo[1,2-a]pyridin-5-
yl)piperazine-1-carboxylate (7). Compound 7 was pre-
pared in a manner similar to that described for 6. H
1
Melting points were determined with a Yanagimoto
melting point apparatus or a Buchi melting point appa-
¨
NMR (200 MHz, CDCl3) d: 1.50 (9H, s), 2.48 (3H, s),
2.97–3.15 (4H, m), 3.58–3.78 (4H, m), 6.23 (1H, d,
J = 8.2 Hz), 7.13 (1H, dd, J = 8.8 and 7.0 Hz), 7.28–
7.35 (2H, m).
ratus B-545 and are uncorrected. 1H NMR spectra were
obtained at 300 or 200 MHz on a Varian Ultra-300 or a
Varian Gemini-200 spectrometer. Chemical shifts are gi-
ven in d values (ppm) using tetramethylsilane as the
internal standard. Reactions were followed by TLC on
Silica gel 60 F 254 precoated TLC plates (E. Merck)
or NH TLC plates (Fuji Silysia Chemical Ltd). Chro-
matographic separations were carried out on silica gel
60 (0.063–0.200 or 0.040–0.063 mm, E. Merck) or basic
silica gel (Chromatorexꢂ NH, 100–200 mesh, Fuji Sily-
sia Chemical Ltd) using the indicated eluents. Yields
are unoptimized. Chemical intermediates were charac-
5.1.5. tert-Butyl 4-[3-(hydroxymethyl)imidazo[1,2-a]pyri-
din-5-yl]piperazine-1-carboxylate (8). To a solution of 6
(5.00 g, 16.5 mmol) in EtOH (20 mL) was added aque-
ous formaldehyde solution (37%; 50.5 mL, 678 mmol)
and the resulting mixture was stirred at 85 ꢁC for 16 h.
The reaction mixture was concentrated in vacuo, and
water (20 mL) was added to the residue. The solution
was basified with 8 M NaOH and extracted with CHCl3
(30 mL). The extract was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The res-
idue was purified by silica gel chromatography (EtOAc/
EtOH = 10:1) to give 8 (3.28 g, 60%) as a colorless solid.
1H NMR (200 MHz, CDCl3) d: 1.50 (9H, s), 2.85–2.97
(2H, m), 3.12–3.38 (4H, m), 3.68 (1H, br s), 4.12–4.34
(2H, m), 4.87 (2H, s), 6.57 (1H, dd, J = 7.2 and
1.2 Hz), 7.20 (1H, dd, J = 8.8 and 1.2 Hz), 7.50 (1H, d,
J = 8.8 Hz), 7.56 (1H, s).
1
terized by H NMR.
5.1.1. Ethyl 5-chloroimidazo[1,2-a]pyridine-2-carboxylate
(9). A mixture of 312 (27.7 g, 216 mmol) and ethyl bro-
mopyruvate (50.5 g, 259 mmol) in EtOH (400 mL) was
refluxed for 15 h. The solvent was evaporated in vacuo
and the residue was partitioned between CHCl3
(250 mL) and aqueous K2CO3 (250 mL) solution. The
organic layer was separated, washed with brine, dried
over anhydrous Na2SO4, and concentrated in vacuo.
The residue was washed with diisopropyl ether (IPE)
5.1.6. tert-Butyl 4-(2-ethoxycarbonylimidazo[1,2-a]pyri-
din-5-yl)piperazine-1-carboxylate (10). A mixture of 9
(12.0 g, 53.4 mmol) and piperazine (46.0 g, 534 mmol)
in acetonitrile (200 mL) was refluxed for 72 h under ar-
gon atmosphere. The reaction mixture was concentrated
in vacuo and the residue was partitioned between CHCl3
(250 mL) and water (250 mL). The organic layer was
separated, washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was dis-
solved in EtOH (150 mL), and (Boc)2O (11.7 g,
53.4 mmol) was added to the solution at room tempera-
ture. The resulting mixture was stirred at room temper-
ature for 1 h and concentrated in vacuo. The residue was
diluted with water (200 mL) and extracted with EtOAc
(200 mL). The extract was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The res-
idue was purified by silica gel chromatography (EtOAc
to 10:1 EtOAc/EtOH) to give 10 (17.2 g, 86%) as a col-
1
to give 9 as a beige powder (41.5 g, 86%). H NMR
(300 MHz, CDCl3) d: 1.46 (3H, t, J = 7.2 Hz), 4.49
(2H, q, J = 7.2 Hz), 6.99 (1H, dd, J = 0.9 and 7.5 Hz),
7.24–7.29 (1H, m), 7.67 (1H, dt, J = 0.9 and 9.0 Hz),
8.39 (1H, d, J = 0.9 Hz).
5.1.2. 5-Chloro-2-methylimidazo[1,2-a]pyridine (5). Com-
pound 5 was prepared in a manner similar to that de-
scribed for 9. 1H NMR (300 MHz, CDCl3) d: 2.49
(3H, d, J = 0.9 Hz), 6.82 (1H, dd, J = 7.5 and 0.9 Hz),
7.11 (1H, dd, J = 7.2 and 9.0 Hz), 7.47 (1H, dt, J = 0.9
and 9.0 Hz), 7.52 (1H, t, J = 0.9 Hz).
5.1.3. tert-Butyl 4-(imidazo[1,2-a]pyridin-5-yl)piperazine-
1-carboxylate (6). A mixture of 412 (4.58 g, 30 mmol)
and piperazine (25.8 g, 300 mmol) was heated at
125 ꢁC for 18 h under argon atmosphere. The reaction
mixture was partitioned between CHCl3 (200 mL) and
water (200 mL). The organic layer was separated,
washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was dissolved in
EtOH (100 mL) and di-tert-butyl dicarbonate ((Boc)2O;
6.55 g, 30.0 mmol) was added to the solution at room
temperature. The resulting mixture was stirred at room
1
orless solid. H NMR (300 MHz, CDCl3) d: 1.43–1.51
(12H, m), 2.98–3.17 (4H, m), 3.58–3.85 (4H, m), 4.47
(2H, q, J = 5.7 Hz), 6.36 (1H, d, J = 7.2 Hz), 7.22–7.28
(1H, m), 7.46 (1H, d, J = 10.8 Hz), 8.16 (1H, s).
5.1.7. tert-Butyl 4-[2-(hydroxymethyl)imidazo[1,2-a]pyri-
din-5-yl]piperazine-1-carboxylate (11). A mixture of 10
(8.26 g, 22.1 mmol) and
8
M
NaOH (5.5 mL,

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
3133
44.1 mmol) in EtOH (80 mL) was stirred at room tem-
perature for 30 min. The reaction mixture was neutral-
ized with concentrated HCl and EtOH was evaporated
in vacuo. The residue was diluted with water (50 mL),
acidified to pH 3–4 with concentrated HCl, and ex-
tracted with CHCl3 (100 mL). The extract was dried
over anhydrous MgSO4 and concentrated in vacuo. To
a solution of BH3–THF in THF (1 M, 68.2 mL,
68.2 mmol) was added the residue and the resulting
mixture was stirred at room temperature under argon
atmosphere for 1 h. The reaction mixture was poured
into ice-water (300 mL) and acidified to pH 1–2 with
concentrated HCl cautiously. After stirring at room
temperature for 1 h, the mixture was basified to pH
10–11 with 8 M NaOH and extracted with EtOAc
(150 mL). The extract was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The res-
idue was purified by silica gel chromatography (EtOAc
to 5:1 EtOAc/EtOH) to give 11 (4.77 g, 65%) as a color-
5.1.10. 5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propa-
noyl}piperazin-1-yl)imidazo[1,2-a]pyridine hydrochloride
(2b). A solution of 6 (8.39 g, 27.8 mmol) in concentrated
HCl (22.8 mL) was stirred at room temperature for
20 min. The reaction mixture was diluted with EtOH
(85 mL) and concentrated in vacuo. The precipitate
was collected by filtration and washed with EtOH and
Et2O to give 5-(piperazin-1-yl)imidazo[1,2-a]pyridine
dihydrochloride (5.24 g, 69%) as a colorless powder.
To a mixture of 158 (0.75 g, 2.5 mmol) and HOBt
(0.57 g, 3.72 mmol) in MeCN (15 mL) was added WSC
(0.72 g, 3.76 mmol) and the mixture was stirred at room
temperature for 20 min. A solution of the 5-(piperazin-
1-yl)imidazo[1,2-a]pyridine dihydrochloride (0.83 g,
3.02 mmol), Et3N (1.05 mL, 7.5 mmol), and 1,8-diazabi-
cyclo[5.4.0]-7-undecene (DBU; 0.90 mL, 6.03 mmol)
was added and the resulting mixture was stirred at room
temperature for 3 h. The solvent was evaporated in va-
cuo and the residue was partitioned between CHCl3
(50 mL) and water (50 mL). The organic layer was sep-
arated, washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was
purified by silica gel chromatography (EtOAc/
EtOH = 5:1) to give 5-(4-{3-[(6-chloronaphthalen-2-
yl)sulfonyl]propanoyl}piperazin-1-yl)imidazo[1,2-a]pyr-
idine (1.09 g, 91%) as a pale yellow amorphous powder.
1
less solid. H NMR (200 MHz, CDCl3) d: 1.44 (9H, s),
2.94–3.10 (4H, m), 3.51–3.67 (4H, m), 4.61 (2H, d,
J = 5.4 Hz), 5.16 (1H, t, J = 5.4 Hz), 6.41–6.45 (1H,
m), 7.21–7.24 (2H, m), 7.60 (1H, s).
5.1.8.
dazo[1,2-a]pyridin-5-yl]piperazine-1-carboxylate (12) and
tert-butyl 4-(2-acetylimidazo[1,2-a]pyridin-5-yl)pipera-
tert-Butyl
4-[2-(1-hydroxy-1-methylethyl)imi-
zine-1-carboxylate (13). To a solution of 10 (7.17 g,
19.1 mmol) in THF (60 mL) was added a solution of
methylmagnesium bromide in Et2O (1 M, 60.0 mL,
60.0 mmol) dropwise at 0 ꢁC under argon atmosphere
and the mixture was stirred at room temperature for
30 min. The reaction mixture was poured into ice-water
(100 mL) and extracted with EtOAc (100 mL). The ex-
tract was washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was
purified by silica gel chromatography (EtOAc to 10:1
EtOAc/EtOH) to give 12 (more polar; 2.96 g, 41%) as
a pale yellow oil and 13 (less polar; 2.82 g, 45%) as a pale
yellow oil. 12: 1H NMR (200 MHz, CDCl3) d: 1.50 (9H,
s), 1.69 (6H, s), 2.99–3.13 (4H, m), 3.58–3.77 (4H, m),
6.29 (1H, d, J = 7.0 Hz), 7.18 (1H, dd, J = 8.8 and
The product obtained (1.09 g, 2.26 mmol) was dissolved
in EtOH (15 mL) and concentrated HCl (0.44 mL,
5.40 mmol) was added. The resulting mixture was concen-
trated in vacuo and the residue was suspended in EtOH
and Et2O. The precipitate was collected by filtration and
washed with Et2O to give 2b (1.02 g, 73%) as a colorless
1
powder. Mp 137–139 ꢁC. H NMR (200 MHz, DMSO-
d6) d: 2.83 (2H, t, J = 7.4 Hz), 2.94–3.08 (2H, m), 3.08–
3.20 (2H, m), 3.48–3.84 (6H, m), 6.98 (1H, d,
J = 7.4 Hz), 7.68–7.76 (2H, m), 7.90–8.04 (2H, m), 8.18–
8.24 (2H, m), 8.24–8.32 (3H, m), 8.68 (1H, br s). Anal.
Calcd for C24H23ClN4O3SÆHClÆ2.2H2O: C, 51.56; H,
5.12; N, 10.02. Found: C, 51.87; H, 5.52; N, 9.75.
1
7.0 Hz), 7.36 (1H, d, J = 8.8 Hz), 7.44 (1H, s). 13: H
The following compounds 2c, 2d, 2f, and 2g were pre-
pared in a manner similar to that described for 2b.
NMR (200 MHz, CDCl3) d: 1.51 (9H, s), 2.73 (3H, s),
2.98–3.17 (4H, m), 3.58–3.81 (4H, m), 6.37 (1H, d,
J = 6.8 Hz), 7.27 (1H, d, J = 9.2 and 7.2 Hz), 7.45 (1H,
d, J = 9.2 Hz), 8.13 (1H, s).
5.1.11. 5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propa-
noyl}piperazin-1-yl)-2-methylimidazo[1,2-a]pyridine hydro-
chloride (2c). Yield 66%, as a colorless powder. Mp
125–127 ꢁC. 1H NMR (200 MHz, DMSO-d6) d: 2.52
(3H, s), 2.84 (2H, t, J = 7.4 Hz), 2.94–3.08 (2H, m),
3.08–3.23 (2H, m), 3.23–3.54 (2H, m), 3.54–3.78 (4H,
m), 6.93 (1H, d, J = 7.2 Hz), 7.60 (1H, d, J = 8.8 Hz),
7.71–7.76 (1H, m), 7.88 (1H, dd, J = 8.8 and 8.0 Hz),
7.99 (1H, s), 8.03–8.04 (1H, m), 8.18–8.32 (3H, m),
8.68 (1H, br s). Anal. Calcd for C25H25ClN4O3SÆHClÆ3-
H2O: C, 51.11; H, 5.49; N, 9.54. Found: C, 51.01; H,
5.46; N, 9.45.
5.1.9. tert-Butyl 4-[2-(1-hydroxyethyl)imidazo[1,2-a]pyri-
din-5-yl]piperazine-1-carboxylate (14). To a solution of
13 (4.13 g, 12.0 mmol) in EtOH (50 mL) was added
NaBH4 (0.55 g, 14.8 mmol) and the mixture was stirred
at room temperature for 15 min. The reaction mixture
was poured into ice-water (100 mL) and extracted with
EtOAc (100 mL). The extract was washed with brine,
dried over anhydrous MgSO4, and concentrated in va-
cuo. The residue was purified by silica gel chromatogra-
phy (EtOAc/EtOH = 10:1) to give 14 (3.95 g, 95%) as a
colorless amorphous powder. 1H NMR (200 MHz,
DMSO-d6) d: 1.44–1.63 (12H, m), 2.94–3.12 (4H, m),
3.48–3.70 (4H, m), 4.77–4.95 (1H, m), 5.21 (1H, d,
J = 4.8 Hz), 6.41–6.45 (1H, m), 7.17–7.30 (2H, m), 7.54
(1H, s).
5.1.12. [5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propa-
noyl}piperazin-1-yl)imidazo[1,2-a]pyridin-3-yl]methanol
hydrochloride (2d). Yield 47%, colorless powder. Mp
1
192–194 ꢁC. H NMR (200 MHz, DMSO-d6) d: 2.56–
3.01 (5H, m), 3.12–3.33 (2H, m), 3.33–3.84 (3H, m),

3134
Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
3.84–4.02 (1H, m), 4.17–4.36 (1H, m), 5.01 (2H, d,
J = 6.2 Hz), 7.21–7.28 (1H, m), 7.72–7.86 (2H, m),
7.86–8.11 (2H, m), 8.11–8.37 (4H, m), 8.68 (1H, s). Anal.
Calcd for C25H25ClN4O3SÆHClÆH2OÆ0.5Et2O: C, 53.64;
H, 5.50; N, 9.27. Found: C, 53.76; H, 5.71; N, 9.49.
5.1.16. 8-(Imidazo[1,2-a]pyridin-5-yl)-1,4-dioxa-8-azaspi-
ro[4.5]decane (17). Compound 17 was prepared in 85%
yield by the same method as described above for the syn-
1
thesis of 10 as a pale yellow solid. H NMR (200 MHz,
CDCl3) d: 1.96 (4H, t, J = 6.0 Hz), 3.22 (4H, t,
J = 4.5 Hz), 4.04 (4H, s), 6.32 (1H, d, J = 7.5 Hz), 7.18
(1H, dd, J = 9.3 and 7.2 Hz), 7.40 (1H, d, J = 8.4 Hz),
7.54 (1H, s), 7.65 (1H, s).
5.1.13. 2-[5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]pro-
panoyl}piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]propan-
2-ol hydrochloride (2f). Yield 65%, colorless amorphous
powder. Mp > 132 ꢁC (dec). 1H NMR (200 MHz,
DMSO-d6) d: 1.63 (6H, s), 2.82 (2H, t, J = 7.2 Hz),
2.92–3.06 (2H, m), 3.06–3.20 (2H, m), 3.44–3.82 (6H,
m), 6.97 (1H, d, J = 7.4 Hz), 7.59 (1H, d, J = 8.8 Hz),
7.74 (1H, dd, J = 8.8 and 2.2 Hz), 7.83–8.04 (3H, m),
8.18–8.32 (3H, m), 8.67 (1H, br s). Anal. Calcd for
C27H29ClN4O3SÆHClÆH2OÆ0.4Et2O: C, 54.94; H, 5.80;
N, 8.96. Found: C, 55.03; H, 6.12; N, 8.73.
5.1.17. 1-(Imidazo[1,2-a]pyridin-5-yl)-N-methylpiperidin-
4-amine hydrochloride (18). A solution of 17 (6.60 g,
25.4 mmol) in 4 M HCl (14 mL) and acetone (25 mL)
was stirred at 50 ꢁC for 6 h, and the reaction mixture
was concentrated in vacuo. The concentrated solution
was basified with 1 M NaOH to pH 11, saturated with
NaCl, and extracted with CHCl3 (100 mL). The extract
was washed with brine (10 mL), dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was dis-
solved in acetic acid (50 mL) and a solution of methyl-
amine (40%, 25 mL) in MeOH was added dropwise
over 30 min at 0 ꢁC. After the mixture was stirred at
room temperature for 30 min, sodium triacetoxyborohy-
dride (6.30 g, 29.7 mmol) was added and then the result-
ing mixture was stirred at room temperature for 2 h. The
reaction mixture was concentrated in vacuo and the con-
centrated solution was basified with 1 M NaOH to pH
11, saturated with NaCl, and extracted with CHCl3
(100 mL). The extract was washed with brine (10 mL),
dried over anhydrous MgSO4, and concentrated in va-
cuo. The residue was dissolved in EtOH (100 mL) and
di-tert-butyl dicarbonate (5.55 g, 25.4 mmol) was added
dropwise at room temperature. After the mixture was
stirred at room temperature for 1 h, the reaction mixture
was concentrated in vacuo and the residue was purified
by silica gel chromatography (EtOAc/EtOH = 5:1). The
obtained product was dissolved in EtOH (10 mL) and
concentrated HCl (21 mL) was added. The resulting
mixture was stirred at room temperature for 1 h and
concentrated in vacuo. The residue was crystallized from
EtOH to give 18 (3.07 g, 38%) as a colorless powder. 1H
NMR (200 MHz, CDCl3) d: 1.91–2.07 (2H, m), 2.34–
2.39 (2H, m), 2.83 (3H, s), 2.98–3.11 (2H, m), 3.39–
3.51 (1H, m), 3.63–3.70 (2H, m), 7.00 (1H, d,
J = 7.6 Hz), 7.58 (1H, d, J = 8.8 Hz), 7.83–7.93 (3H, m).
5.1.14. 1-[5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]pro-
panoyl}piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]ethanol
hydrochloride (2g). Yield 56%, colorless amorphous
powder. Mp 191–193 ꢁC. 1H NMR (200 MHz,
DMSO-d6) d: 1.56 (3H, d, J = 6.2 Hz), 2.73–2.90 (2H,
t, J = 7.6 Hz), 2.90–3.08 (2H, m), 3.08–3.22 (2H, m),
3.22–3.56 (2H, m), 3.56–3.83 (4H, m), 5.07 (1H, q,
J = 6.2 Hz), 6.11 (1H, br s), 6.96 (1H, d, J = 7.8 Hz),
7.60 (1H, d, J = 8.8 Hz), 7.71–7.76 (1H, m), 7.86–8.04
(3H, m), 8.18–8.32 (3H, m), 8.68 (1H, br s). Anal. Calcd
for C26H27ClN4O4SÆHClÆH2OÆ0.4Et2O: C, 54.24; H,
5.61; N, 9.17. Found: C, 54.44; H, 5.77; N, 9.04.
5.1.15. [5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propa-
noyl}piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]methanol
(2e). A solution of 11 (3.99 g, 12.0 mmol) in concentrated
HCl (20 mL) was stirred at room temperature for 20 min,
and the reaction mixture was diluted with EtOH (50 mL)
and 2-PrOH (50 mL). The precipitate was collected by
filtration and washed with 2-PrOH and Et2O to give
[5-(piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]methanol
dihydrochloride (3.66 g, quant.) as a colorless powder.
To a mixture of 15 (0.75 g, 2.51 mmol) and HOBt
(0.57 g, 3.72 mmol) in MeCN (15 mL) was added WSC
(0.72 g, 3.76 mmol) and the mixture was stirred at room
temperature for 20 min. A solution of [5-(piperazin-1-
yl)imidazo[1,2-a]pyridin-2-yl]methanol dihydrochloride
(0.92 g, 3.01 mmol), Et3N (1.05 mL, 7.53 mmol), and
DBU (0.90 mL, 6.03 mmol) was added and the resulting
mixture was stirred at room temperature for 3 h. The
solvent was evaporated in vacuo and the residue was
partitioned between CHCl3 (50 mL) and water
(50 mL). The organic layer was separated, washed with
brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was purified by silica gel chroma-
tography (EtOAc/EtOH = 5:1) and recrystallized from
acetone–EtOH to give 2e (0.77 g, 60%) as a colorless
5.1.18. 3-[(6-Chloronaphthalen-2-yl)sulfonyl]-N-[1-(imi-
dazo[1,2-a]pyridin-5-yl)piperidin-4-yl]-N-methylpropana-
mide (2a). Compound 2a was prepared in a manner
similar to that described for 2b in 37% yield as a color-
less powder. Mp 190 ꢁC. 1H NMR (200 MHz, CDCl3) d:
1.69–1.73 (2H, m), 1.85–1.93 (2H, m), 2.77–3.08 (7H,
m), 3.48–3.52 (2H, m), 3.57–3.62 (2H, m), 3.77–3.95
(0.3H, m), 4.53–4.68 (0.7H, m), 6.27–6.34 (1H, m),
7.14–7.24 (1H, m), 7.38–7.45 (1H, m), 7.50–7.52 (1H,
m), 7.60 (1H, dd, J = 9.0 and 1.8 Hz), 7.64–7.67 (1H,
m), 7.92–7.97 (4H, m), 8.50 (1H, s). Anal. Calcd for
C26H27ClN4O3S: C, 61.11; H, 5.33; N, 10.96. Found:
C, 61.03; H, 5.37; N, 11.21.
1
powder. Mp 194–195 ꢁC. H NMR (200 MHz, CDCl3)
d: 2.88–3.18 (6H, m), 3.56–3.92 (6H, m), 4.88 (2H, s),
6.27 (1H, d, J = 7.4 Hz), 7.19 (1H, dd, J = 9.2 and
7.4 Hz), 7.37 (1H, d, J = 8.8 Hz), 7.52 (1H, s), 7.57–
7.63 (1H, m), 7.94–7.98 (4H, m), 8.49 (1H, br s). Anal.
Calcd for C25H25ClN4O4S: C, 58.53; H, 4.91; N, 10.92.
Found: C, 58.40; H, 4.84; N, 10.78.
5.1.19. Ethyl (5-fluoroimidazo[1,2-a]pyridin-2-yl)acetate
(20). A solution of 1912 (5.00 g, 44.6 mmol) and ethyl 4-
chloroacetoacetate (7.20 mL, 53.3 mmol) in EtOH
(120 mL) was refluxed for 12 h and then concentrated

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
3135
in vacuo. The residue was diluted with saturated aque-
ous NaHCO3 (100 mL) and extracted with EtOAc
(100 mL ·2). The extract was washed with brine, dried
over anhydrous MgSO4, and concentrated in vacuo.
The residue was purified by silica gel chromatography
(2:1 EtOAc/hexane to 20:1 EtOAc/EtOH) to give 20
(7.00 g, 71%) as a brown oil. 1H NMR (200 MHz,
CDCl3) d: 1.30 (3H, t, J = 7.0 Hz), 3.89 (2H, s), 4.22
(2H, q, J = 7.0 Hz), 6.42–6.48 (1H, m), 7.14–7.26 (1H,
m), 7.40 (1H, dd, J = 9.2 and 0.8 Hz), 7.67 (1H, s).
EtOH = 2:1) to give 23 (1.95 g, 84%) as a colorless solid.
1H NMR (200 MHz, CDCl3) d: 1.51 (9H, s), 3.08 (4H, t,
J = 4.8 Hz), 3.68 (4H, br s), 3.76 (2H, s), 5.31 (1H, s),
5.48 (1H, br s), 6.33 (1H, dd, J = 7.0 and 1.0 Hz), 7.23
(1H, dd, J = 8.8 and 7.0 Hz), 7.34 (1H, d, J = 8.8 Hz),
7.46 (1H, s).
5.1.23. tert-Butyl 4-{2-[(N-methylcarbamoyl)methyl]imi-
dazo[1,2-a]pyridin-5-yl}piperazine-1-carboxylate (24).
Compound 24 was prepared in a manner similar to that
1
described for 22. H NMR (300 MHz, CDCl3) d: 1.51
5.1.20. tert-Butyl 4-(2-ethoxycarbonylmethylimidazo[1,2-
a]pyridin-5-yl)piperazine-1-carboxylate (21). A mixture
of 20 (6.40 g, 28.8 mmol) and piperazine (10.0 g,
116 mmol) was stirred at 100 ꢁC for 1 h. After cooling
to room temperature, the reaction mixture was diluted
with CHCl3 (200 mL) and water (100 mL). The organic
layer was separated, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was dissolved in
EtOH (70 mL), and (Boc)2O (6.33 g, 29.0 mmol) was
added dropwise to the solution at room temperature.
The resulting mixture was stirred at room temperature
for 30 min and concentrated in vacuo. The residue was
diluted with water (100 mL) and extracted with EtOAc
(100 mL). The extract was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The res-
idue was purified by silica gel chromatography (EtOAc
to 10:1 EtOAc/EtOH) to give 21 (5.50 g, 49%) as a pale
yellow solid. 1H NMR (200 MHz, CDCl3) d: 1.30 (3H, t,
J = 7.2 Hz), 1.34 (9H, s), 3.08 (4H, t, J = 4.6 Hz), 3.68
(4H, br s), 3.88 (2H, s), 4.22 (2H, q, J = 7.2 Hz), 6.28
(1H, dd, J = 7.4 and 1.0 Hz), 7.17 (1H, dd, J = 9.2 and
7.4 Hz), 7.34 (1H, d, J = 9.2 Hz), 7.55 (1H, s).
(9H, s), 2.82 (3H, s), 3.07 (4H, br s), 3.68 (4H, br s),
3.75 (2H, s), 6.33 (1H, dd, J = 7.4 and 1.2 Hz), 7.19–
7.44 (3H, m).
5.1.24. tert-Butyl 4-[2-(2-hydroxy-2-methylpropyl)imi-
dazo[1,2-a]pyridin-5-yl]piperazine-1-carboxylate (25). A
suspension of CeCl3 (5.72 g, 23.2 mmol) in THF
(30 mL) was vigorously stirred at room temperature for
12 h. To the suspension was added a solution of methyl-
magnesium bromide in THF (1 M, 22.0 mL, 22.0 mmol)
dropwise at 0 ꢁC and the mixture was stirred at that tem-
perature for 2.5 h. A solution of 21 (1.60 g, 4.11 mmol) in
THF (100 mL) was added to the suspension at 0 ꢁC and
then the resulting mixture was stirred at 0 ꢁC for 2 h.
The reaction mixture was poured into 5% aqueous acetic
acid at 0 ꢁC and extracted with EtOAc (50 mL ·3). The
extract was washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was puri-
fied by silica gel chromatography (EtOAc to 2:1 EtOAc/
EtOH). The product obtained was treated with CeCl3
(6.64 g, 26.9 mmol) and methylmagnesium bromide in
THF (1 M, 44.0 mL, 44 mmol) by the same method as de-
scribed above to give 25 (0.22 g, 14%) as a pale yellow so-
lid. 1H NMR (200 MHz, CDCl3) d: 1.26 (6H, s), 1.51 (9H,
s), 2.92 (2H, s), 3.08 (4H, t, J = 5.0 Hz), 3.69 (4H, br s),
6.30 (1H, dd, J = 7.4 and 0.9 Hz), 7.15–7.23 (2H, m),
7.31–7.34 (2H, m).
5.1.21. tert-Butyl 4-[2-(N,N-dimethylcarbamoylmeth-
yl)imidazo[1,2-a]pyridin-5-yl]piperazine-1-carboxylate (22).
To a solution of dimethylamine (2 M in THF; 22.5 mL,
129 mmol)
and
diisopropylethylamine
(22.5 mL,
129 mmol) in CH2Cl2 (100 mL) was added dimethylalumi-
numchloride(1 Minhexane;100 mL, 100 mmol)dropwise
at 0 ꢁC. After stirring at 0 ꢁC for 30 min, a solution of 21
(5.00 g, 12.9 mmol) in CH2Cl2 (50 mL) was added drop-
wise. Afterstirring at room temperaturefor 12 h, the result-
ing mixture was poured into saturated aqueous NaHCO3
solution at 0 ꢁC and extracted with CHCl3 (100 mL ·2).
The extract was dried over anhydrous MgSO4 and concen-
trated in vacuo. The residue was purified by silica gel chro-
matography (EtOAc/EtOH = 5:1 to 2:1) to give 22 (3.45 g,
69%) as a pale yellow solid. 1H NMR (300 MHz, CDCl3) d:
1.50 (9H, s), 2.99 (3H, s), 3.09 (4H, br s), 3.18 (3H, s), 3.66
(4H, br s), 3.93 (2H, s), 6.27 (1H, d, J = 7.0 Hz), 7.16 (1H,
dd, J = 9.0 and 7.0 Hz), 7.32 (1H, d, J = 9.0 Hz), 7.57
(1H, s).
5.1.25. tert-Butyl 4-[2-(2-hydroxypropyl)imidazo[1,2-
a]pyridin-5-yl]piperazine-1-carboxylate (26). A suspen-
sion of CeCl3 (5.72 g, 23.2 mmol) in THF (30 mL) was
vigorously stirred at room temperature for 12 h. To
the suspension was added a solution of 22 (3.00 g,
7.74 mmol) and the mixture was stirred at room temper-
ature for 1 h. A solution of methylmagnesium bromide
in THF (1 M, 24.0 mL, 24.0 mmol) was added dropwise
to the suspension at 0 ꢁC and the resulting mixture was
stirred at that temperature for 1 h. The reaction mixture
was poured into 5% aqueous acetic acid at 0 ꢁC and ex-
tracted with EtOAc (50 mL ·3). The extract was washed
with brine, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel
chromatography (EtOAc to 2:1 EtOAc/EtOH) to give
5.1.22. tert-Butyl 4-(2-carbamoylmethylimidazo[1,2-
a]pyridin-5-yl)piperazine-1-carboxylate (23). To a solu-
tion of 21 (2.50 g, 6.44 mmol) in MeCN (100 mL) was
added 25% aqueous ammonia solution (100 mL) and
the resulting mixture was stirred at 50 ꢁC for 24 h.
MeCN was evaporated in vacuo and extracted with
CHCl3 (50 mL ·2). The extract was dried over anhy-
drous MgSO4 and concentrated in vacuo. The residue
was purified by silica gel chromatography (EtOAc/
tert-butyl 4-[2-(2-oxopropyl)imidazo[1,2-a]pyridin-5-
yl]piperazine-1-carboxylate (1.90 g, 69%) as a yellow oil.
To a solution of the product obtained in EtOH (20 mL)
was added NaBH4 (0.35 g, 9.40 mmol) at 0 ꢁC and the
resulting mixture was stirred at room temperature for
1 h. Water (5 mL) was added to the reaction mixture
and EtOH was evaporated in vacuo. The residue was
partitioned between EtOAc (50 mL) and water

3136
Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
(50 mL). The organic layer was separated, washed with
brine (50 mL), dried over anhydrous MgSO4, and con-
centrated in vacuo. The residue was purified by silica
gel chromatography (EtOAc/EtOH = 5:1) to give 26
(0.88 g, 79%) as a yellow oil. 1H NMR (200 MHz,
CDCl3) d: 1.30 (3H, d, J = 6.2 Hz), 1.51 (9H, s), 2.79
(1H, dd, J = 14.6 and 8.4 Hz), 2.97 (1H, dd, J = 14.6
and 3.4 Hz), 3.05–3.10 (4H, m), 3.67–3.78 (5H, m),
4.14–4.24 (1H, m), 6.29 (1H, d, J = 7.0 Hz), 7.18 (1H,
dd, J = 8.8 and 7.0 Hz), 7.28–7.35 (2H, m).
CDCl3) d: 2.96 (2H, t, J = 7.5 Hz), 3.03 (3H, s), 3.13
(2H, br s), 3.24–3.26 (5H, m), 3.60 (2H, t, J = 7.5 Hz),
3.80 (4H, br s), 4.26 (2H, s), 6.74 (1H, d, J = 7.4 Hz),
7.59–7.66 (1H, m), 7.73 (1H, d, J = 7.4 Hz), 7.85 (1H,
d, J = 8.8 Hz), 7.93–8.00 (5H, m), 8.50 (1H, s). Anal.
Calcd for C28H30ClN5O4SÆHClÆ0.5H2O: C, 54.81; H,
5.26; N, 11.41. Found: C, 54.67; H, 5.18; N, 11.35.
5.1.28. 2-[5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]pro-
panoyl}piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]acetam-
ide (2j). Yield 62%, colorless powder. Mp 159–161 ꢁC.
1H NMR (200 MHz, CDCl3) d: 2.93–3.12 (6H, m),
3.60 (2H, t, J = 7.4 Hz), 3.77–3.81 (6H, m), 5.47 (2H,
br s), 6.31 (1H, dd, J = 7.0 and 1.2 Hz), 7.25 (1H, dd,
J = 8.8 and 7.0 Hz), 7.37 (1H, d, J = 8.8 Hz), 7.45 (1H,
s), 7.61 (1H, dd, J = 8.8 and 2.2 Hz), 7.94–8.00 (4H,
m), 8.50 (1H, s). Anal. Calcd for C26H26ClN5O4SÆH2O:
C, 55.96; H, 5.06; N, 12.55. Found: C, 56.09; H, 4.90;
N, 12.71.
5.1.26. Ethyl [5-(4-{3-[(6-chloronaphthalen-2-yl)sulfonyl]-
propanoyl}piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]acetate
hydrochloride (2h). A solution of 21 (1.50 g, 3.86 mmol)
in concentrated HCl (10 mL) was stirred at room tem-
perature for 5 min. The reaction mixture was diluted
with EtOH (50 mL) and concentrated in vacuo to give
ethyl [5-(piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]ace-
tate dihydrochloride (1.40 g, quant.) as a pale yellow
amorphous powder.
5.1.29. 2-[5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]pro-
panoyl}piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]-N-methy-
lacetamide (2k). Yield 74%, colorless powder. Mp 186–
188 ꢁC. H NMR (200 MHz, CDCl3) d: 2.80–2.82 (3H,
m), 2.93–3.15 (8H, m), 3.60 (2H, t, J = 6.4 Hz), 3.75–
3.82 (4H, m), 6.42 (1H, dd, J = 7.0 and 1.2 Hz), 7.32–
7.64 (5H, m), 7.64–8.00 (4H, m), 8.50 (1H, s). Anal. Calcd
for C27H28ClN5O4SÆH2O: C, 56.69; H, 5.29; N, 12.24.
Found: C, 56.59; H, 5.18; N, 12.11.
To a mixture of 15 (0.96 g, 3.21 mmol) and HOBt
(0.74 g, 4.83 mmol) in MeCN (20 mL) was added WSC
(0.92 g, 4.8 mmol) and the mixture was stirred at room
temperature for 20 min. A solution of the ethyl [5-(pip-
erazin-1-yl)imidazo[1,2-a]pyridin-2-yl]acetate dihydro-
chloride (1.40 g, 3.86 mmol), Et3N (1.35 mL,
9.69 mmol), and DBU (1.20 mL, 8.04 mmol) was added
and the resulting mixture was stirred at room tempera-
ture for 3 h. The solvent was evaporated in vacuo and
the residue was partitioned between CHCl3 (50 mL)
and water (50 mL). The organic layer was separated,
washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was purified by silica
gel chromatography (EtOAc/EtOH = 5:1) to give ethyl
[5-(4-{3-[(6-chloronaphthalen-2-yl)sulfonyl]propanoyl}-
piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]acetate (1.20 g,
66%) as a colorless oil.
1
5.1.30. 1-[5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]pro-
panoyl}piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]-2-meth-
ylpropan-2-ol hydrochloride (2l). Yield 50%, colorless
amorphous powder. 1H NMR (200 MHz, DMSO-d6)
d: 1.20 (6H, s), 2.82 (2H, t, J = 7.3 Hz), 2.94 (2H, s),
3.01 (2H, br s), 3.12 (2H, br s), 3.64–3.68 (6H, m),
6.95 (1H, d, J = 7.6 Hz), 7.63 (1H, d, J = 8.8 Hz), 7.74
(1H, dd, J = 8.4 and 2.2 Hz), 7.88 (1H, dd, J = 8.8 and
7.6 Hz), 7.96 (1H, s), 8.02 (1H, dd, J = 8.4 and
1.4 Hz), 8.21 (1H, d, J = 8.4 Hz), 8.28–8.32 (2H, m),
8.69 (1H, s). Anal. Calcd for C28H31ClN4O4SÆHClÆ2-
H2O: C, 53.59; H, 5.78; N, 8.93. Found: C, 53.72; H,
5.80; N, 8.72.
The product (1.20 g, 2.11 mmol) was dissolved in EtOAc
(30 mL) and 4 M HCl in EtOAc (1.00 mL, 4.00 mmol)
was added and the resulting mixture was stirred at room
temperature for 10 min. The precipitate was collected by
filtration and washed with EtOAc and Et2O to give 2h
(0.68 g, 53%) as a colorless amorphous powder. 1H
NMR (300 MHz, DMSO-d6) d: 1.24 (3H, t,
J = 7.2 Hz), 2.83 (2H, t, J = 7.2 Hz), 3.00 (2H, br s),
3.12 (2H, br s), 3.61–3.70 (6H, m), 4.12 (2H, s), 4.17
(2H, q, J = 7.2 Hz), 6.96 (1H, d, J = 7.5 Hz), 7.65 (1H,
d, J = 9.0 Hz), 7.73 (1H, dd, J = 8.7 and 2.1 Hz), 7.90
(1H, dd, J = 8.7 and 7.8 Hz), 8.00 (1H, dd, J = 8.4 and
1.8 Hz), 8.11 (1H, s), 8.19 (1H, d, J = 8.4 Hz), 8.26–
8.30 (2H, m), 8.67 (1H, d, J = 1.8 Hz). Anal. Calcd for
C29H29ClN4O5SÆHClÆ1.5H2O: C, 54.04; H, 5.16; N,
8.69. Found: C, 54.24; H, 5.35; N, 8.31.
5.1.31. 1-[5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]pro-
panoyl}piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]propan-
2-ol (2m). Yield 73%, colorless powder. Mp 108–109 ꢁC.
1H NMR (300 MHz, CDCl3) d: 1.30 (3H, d, J = 6.3 Hz),
2.80 (1H, dd, J = 14.7 and 8.7 Hz), 2.94–2.99 (3H, m),
3.08 (4H, d, J = 26.1 Hz), 3.60 (2H, t, J = 7.2 Hz), 3.73
(4H, br s), 4.17–4.23 (1H, m), 6.26 (1H, d, J = 7.2 Hz),
7.18 (1H, dd, J = 8.7 and 7.2 Hz), 7.33–7.35 (2H, m),
7.59 (1H, dd, J = 8.7 and 2.0 Hz), 7.90–7.96 (4H, m),
8.49 (1H, s). Anal. Calcd for C27H29ClN4O4SÆ0.5H2O:
C, 58.95; H, 5.50; N, 10.19. Found: C, 59.25; H, 5.78;
N, 9.83.
The following compounds 2i–m were prepared in a man-
ner similar to that described for 2h.
5.1.32.
tert-Butyl
4-(3-nitroimidazo[1,2-a]pyridin-5-
yl)piperazine-1-carboxylate (28). A mixture of 27 (9.88 g,
50.0 mmol), 1-Boc-piperazine (14.0 g, 75.2 mmol), and
N,N-diisopropylethylamine (34.8 mL, 200 mmol) in
2-PrOH (300 mL) was refluxed for 4 h. The reaction mix-
ture was concentrated in vacuo. The residue was crystal-
5.1.27. 2-[5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]pro-
panoyl}piperazin-1-yl)imidazo[1,2-a]pyridin-2-yl]-N,N-
dimethylacetamide hydrochloride (2i). Yield 67%, color-
less powder. Mp 150–152 ꢁC. 1H NMR (200 MHz,

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
3137
lized from EtOH to give 28 (17.4 g, 67%) as a yellow pow-
der. H NMR (200 MHz, CDCl3) d: 1.47 (9H, s), 2.50–
3.40 (6H, m), 3.54–4.25 (2H, m), 6.67 (1H, d,
J = 7.6 Hz), 7.50 (1H, d, J = 8.3 Hz), 7.59 (1H, dd,
J = 8.3 and 7.6 Hz), 8.49 (1H, s).
J = 9.0 Hz), 7.61 (1H, dd, J = 9.0 and 7.0 Hz), 7.75
(1H, dd, J = 8.8 and 2.2 Hz), 8.01 (1H, dd, J = 8.8 and
2.0 Hz), 8.20 (1H, d, J = 8.8 Hz), 8.28–8.34 (2H, m),
8.68 (1H, s), 13.95 (1H, br s). Anal. Calcd for
C24H24ClN5O3SÆ2HClÆ0.3EtOAc: C, 50.67; H, 4.79; N,
11.72. Found: C, 50.95; H, 4.55; N, 11.75.
1
5.1.33. 5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propa-
noyl}piperazin-1-yl)-3-nitroimidazo[1,2-a]pyridine (2n).
To a solution of 28 (17.4 g, 50.0 mmol) in MeOH
(300 mL) was added 4 M HCl in EtOAc (80 mL) and
the mixture was stirred at room temperature overnight.
The reaction mixture was concentrated in vacuo and the
residue was crystallized from EtOH to give 3-nitro-5-
(piperazin-1-yl)imidazo[1,2-a]pyridine dihydrochloride
(16.0 g, quant.) as a yellow powder.
5.1.35. N-[5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]pro-
panoyl}piperazin-1-yl)imidazo[1,2-a]pyridin-3-yl]acetam-
ide (2p). A mixture of 2o (0.29 g, 0.50 mmol) and acetic
anhydride (90 mg, 0.88 mmol) in pyridine (5 mL) was
stirred at room temperature for 5 h. The reaction mix-
ture was concentrated in vacuo and the residue was par-
titioned between CH2Cl2 (20 mL) and 10% aqueous
Na2CO3 solution (20 mL). The organic layer was sepa-
rated, washed with brine, dried over anhydrous MgSO4,
and concentrated in vacuo. The residue was purified by
silica gel chromatography (CH2Cl2/MeOH/25%
NH4OH = 200:10:1). The product was crystallized from
EtOAc and Et2O to give 2p (0.20 g, 76%) as a colorless
A solution of the product (9.61 g, 30.0 mmol) and DBU
(8.95 mL, 60.0 mmol) in MeCN (300 mL) was stirred at
room temperature for 10 min. To the solution were
added 15 (8.96 g, 95.6 mmol) and HOBt (5.51 g,
36.0 mmol), and the mixture was cooled to 0 ꢁC. Next
Et3N (8.36 mL, 60.0 mmol) and WSC (6.90 g,
36.0 mmol) were added and the resulting mixture was
stirred at room temperature for 2 days. The reaction
mixture was concentrated in vacuo and the residue
was partitioned between CH2Cl2 (100 mL) and 10%
aqueous Na2CO3 solution (100 mL). The organic layer
was separated, washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was
purified by silica gel chromatography (hexane/
EtOAc = 1:3) and recrystallized from EtOAc to give 2n
(9.95 g, 63%) as a pale yellow powder. Mp 213–
1
powder. Mp 146–148 ꢁC. H NMR (200 MHz, CDCl3)
d: 2.26 (3H, s), 2.38–2.88 (10H, m), 3.82–4.08 (1H, m),
4.54–4.76 (1H, m), 6.40–6.49 (1H, m), 7.08–7.22 (1H,
m), 7.43–7.62 (2H, m), 7.91–8.03 (5H, m), 8.50 (1H, s),
10.19 (1H, s). Anal. Calcd for C26H26ClN5O4SÆ0.6H2O:
C, 56.69; H, 4.98; N, 12.71. Found: C, 56.60; H, 4.89;
N, 12.45.
5.1.36. tert-Butyl 4-(6-nitropyridin-3-yl)piperazine-1-car-
boxylate (29). A solution of 1-Boc-piperazine (2.79 g,
15.0 mmol) and 5-bromo-2-nitropyridine (1.02 g,
5.00 mmol) in N-methylpyrrolidone (15 mL) was stirred
at 120 ꢁC for 3 h. The reaction mixture was diluted
with water and the precipitate was collected by
filtration to give 29 (1.27 g, 82%) as a colorless powder.
1H NMR (200 MHz, CDCl3) d: 1.49 (9H, s), 3.43–3.52
(4H, m), 3.62–3.67 (4H, m), 7.21 (1H, dd, J = 3.0
and 9.0 Hz), 8.14 (1H, d, J = 3.0 Hz), 8.19 (1H, d,
J = 9.2 Hz).
1
215 ꢁC. H NMR (200 MHz, CDCl3) d: 2.60–3.10 (5H,
m), 3.10–3.95 (6H, m), 4.35–4.65 (1H, br s), 6.66 (1H,
dd, J = 7.4 and 1.6 Hz), 7.51–7.66 (3H, m), 7.89–7.99
(4H, m), 8.48 (1H, s), 8.51 (1H, s). Anal. Calcd for
C24H22ClN5O5SÆ0.1H2O: C, 54.41; H, 4.22; N, 13.22.
Found: C, 54.43; H, 4.47; N, 12.94.
5.1.34. 5-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propa-
noyl}piperazin-1-yl)imidazo[1,2-a]pyridin-3-amine dihy-
drochloride (2o). A mixture of 2n (8.24 g, 15.6 mmol),
iron powder (4.36 g, 78.1 mmol), and CaCl2 (3.55 g,
32.0 mmol) in 80% EtOH (600 mL) and DMF (60 mL)
was refluxed overnight. After cooling to room tempera-
ture, iron powder (4.36 g, 78.1 mmol) was added to the
mixture. The resulting mixture was refluxed for 5 h
and filtered through a Celite pad. The filtrate was con-
centrated in vacuo and the residue was partitioned be-
tween CH2Cl2 (100 mL) and 10% aqueous Na2CO3
solution (100 mL). The organic layer was separated,
washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was purified by basic
silica gel chromatography (EtOAc/MeOH = 20:1). The
product was dissolved in EtOAc (20 mL) and EtOH
(10 mL), and then 4 M HCl in EtOAc (5 mL) was
added. After stirring at 0 ꢁC for 30 min, the precipitate
was collected by filtration and washed with EtOAc to
give 2o (1.02 g, 11%) as a colorless powder. Mp 144–
5.1.37. tert-Butyl 4-(6-aminopyridin-3-yl)piperazine-1-
carboxylate (30). A suspension of 29 (0.31 g, 1.01 mmol)
and 10% palladium on carbon (50% water, 80 mg) in
EtOH (20 mL) was hydrogenated for 2 h. The reaction
mixture was filtered and concentrated in vacuo to
give 30 (0.28 g, quant.) as a brown oil. 1H NMR
(300 MHz, CDCl3) d: 1.48 (9H, s), 2.95 (4H, t, J =
4.5 Hz), 3.55–3.58 (4H, m), 6.51 (1H, dd, J = 0.9 and
9.0 Hz), 7.19 (1H, dd, J = 3.0 and 8.7 Hz), 7.73 (1H, d,
J = 2.1 Hz).
5.1.38. tert-Butyl 4-(imidazo[1,2-a]pyridin-6-yl)pipera-
zine-1-carboxylate (31). A solution of 30 (0.24 g,
0.86 mmol) and aqueous chloroacetaldehyde (40%,
0.34 g, 1.70 mmol) in EtOH (20 mL) was refluxed for
15 h. The reaction mixture was concentrated in vacuo
and the residue was partitioned between aqueous NaH-
CO3 solution (10 mL) and EtOAc (10 mL). The organic
layer was separated, dried over anhydrous Na2SO4, and
concentrated in vacuo to give 31 (0.28 g, quant.) as a
1
146 ꢁC. H NMR (200 MHz, DMSO-d6) d: 2.70–3.10
(4H, m), 3.20–3.36 (2H, m), 3.42–3.72 (3H, m), 3.82–
3.94 (1H, m), 4.18–4.30 (1H, m), 5.00–6.20 (4H, br s),
6.81 (1H, d, J = 7.0 Hz), 7.14 (1H, s), 7.48 (1H, d,
1
brown oil. H NMR (200 MHz, CDCl3) d: 1.49 (9H,
s), 3.00 (4H, t, J = 5.2 Hz), 3.58–3.68 (4H, m), 7.05
(1H, dd, J = 2.2 and 9.8 Hz), 7.51–7.57 (4H, m).

3138
Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
5.1.39. 6-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propa-
noyl}piperazin-1-yl)imidazo[1,2-a]pyridine (2q). A solu-
tion of 31 (0.26 g, 0.86 mmol) in concentrated HCl
(2 mL) and EtOH (2 mL) was stirred at room tempera-
ture for 1 h and the reaction mixture was concentrated
in vacuo. The water was azeotroped with EtOH to give
6-(piperazin-1-yl)imidazo[1,2-a]pyridine dihydrochlo-
ride (0.25 g, quant.) as a brown amorphous powder.
5.1.42. 7-(4-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propa-
noyl}piperazin-1-yl)imidazo[1,2-a]pyridine (2r). Com-
pound 2r was prepared in a manner similar to that
described for 2q in 24% yield as a colorless powder.
1
Mp 200–202 ꢁC. H NMR (300 MHz, CDCl3) d: 2.91–
2.96 (2H, m), 3.13 (2H, t, J = 5.1 Hz), 3.21 (2H, t,
J = 5.1 Hz), 3.56–3.61 (2H, m), 3.65 (2H, t,
J = 5.1 Hz), 3.71 (2H, t, J = 5.1 Hz), 6.55 (1H, dd,
J = 2.4 and 7.5 Hz), 6.80 (1H, d, J = 2.1 Hz), 7.38 (1H,
t, J = 0.6 Hz), 7.48 (1H, d, J = 1.5 Hz), 7.57 (1H, dd,
J = 1.8 and 8.7 Hz), 7.89–7.96 (5H, m), 8.48 (1H, d,
J = 1.5 Hz). Anal. Calcd for C24H23ClN4O3SÆ0.2H2O:
C, 59.24; H, 4.85; N, 11.51. Found: C, 59.00; H, 4.69;
N, 11.32.
To a mixture of 15 (0.26 g, 0.86 mmol) and HOBt
(0.20 g, 1.31 mmol) in MeCN (25 mL) was added WSC
(0.25 g, 1.30 mmol) and the mixture was stirred at room
temperature for 20 min. To the mixture was added a
solution of 6-(piperazin-1-yl)imidazo[1,2-a]pyridine
dihydrochloride (0.25 g, 0.86 mmol), Et3N (0.26 g,
2.6 mmol), and DBU (0.25 mL, 1.71 mmol), and the
resulting mixture was stirred at room temperature for
15 h. The reaction mixture was concentrated in vacuo
and the residue was diluted with aqueous NaHCO3 solu-
tion (20 mL), THF (20 mL), and EtOAc (20 mL). The
organic layer was separated, dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was
purified by basic silica gel chromatography (EtOAc) to
give 2q (0.13 g, 31%) as a green amorphous powder:
1H NMR (300 MHz, CDCl3) d: 2.91–2.97 (4H, m),
3.06 (2H, t, J = 5.1 Hz), 3.56–3.61 (2H, m), 3.66 (2H,
t, J = 5.1 Hz), 3.72 (2H, t, J = 5.1 Hz), 7.01 (1H, dd,
J = 2.1 and 9.9 Hz), 7.51–7.60 (5H, m), 7.89–7.97 (4H,
m), 8.48 (1H, s). Anal. Calcd for C24H23ClN4O3SÆ0.5-
H2OÆ0.4EtOAc: C, 58.32; H, 5.20; N, 10.63. Found: C,
58.05; H, 5.41; N, 10.76.
5.2. Biology
5.2.1. In vitro assays for the inhibition of human FXa. Hu-
man Factor Xa (0.3 U/mL) was obtained from Roche
Diagnostics. Chromogenic substrate, S-2765 (Chromog-
enix-Instrumentation Laboratory), was used for the
measurement of the inhibition of FXa. Anti-FXa activ-
ity was assayed in a buffer containing 50 mM Tris–HCl,
145 mM NaCl, and 2 mM CaCl2 at pH 8.3. Enzyme as-
say was carried out in 96-well microtiter plates. Test
compounds were diluted in DMSO. Compound dilu-
tions and 10 lL of enzyme solution were added to the
well containing buffer, and preincubated. The enzymatic
reactions were initiated with the addition of 10 lL of
3 mM substrate and the mixture was incubated for
10 min at 37 ꢁC. The reaction was terminated with the
addition of 25 lL of 50% acetic acid. The color develop-
ment from the release of p-nitroanilide from each chro-
mogenic substrate was measured at 405 nm on a
microtiter plate reader (Multiscan Ascent, Dainippon
Sumitomo Pharmaceutical Co.). Each absorbance [T]
was calculated by subtracting the absorbance measured
without the substrate. The control [C] was performed
using DMSO solution in place of test compound. Inhib-
itory effect (%) was calculated according to the equation
(1 À [T]/[C]) · 100. IC50 values were calculated from the
regression line based on the method of least squares be-
tween inhibitory effect and concentration. Data for new
compounds were compared to positive control DX-
9065a6c (FXa IC50 = 0.13 lM). The 95% confidence
interval for the IC50 of DX-9065a was 0.12–0.15 lM
(duplicate).
5.1.40. tert-Butyl 4-(2-aminopyridin-4-yl)piperazine-1-
carboxylate (33).
A solution of 1-Boc-piperazine
(13.0 g, 69.8 mmol) and 3216 (6.00 g, 46.7 mmol) in
EtOH (50 mL) was refluxed for 7 h. The reaction mix-
ture was concentrated in vacuo and the residue was par-
titioned between CHCl3 (100 mL) and aqueous K2CO3
solution (100 mL). The organic layer was separated,
dried over anhydrous Na2SO4, and concentrated in va-
cuo. The residue was washed with IPE to give 33
(10.8 g, 83%) as
a
colorless powder. 1H NMR
(300 MHz, CDCl3) d: 1.48 (9H, s), 3.23–3.28 (4H, m),
3.52–3.57 (4H, m), 4.25 (2H, br s), 5.85 (1H, d,
J = 3.3 Hz), 6.18 (1H, dd, J = 3.9 and 9.3 Hz), 7.83
(1H, d, J = 9.2 Hz).
5.1.41. tert-Butyl 4-(imidazo[1,2-a]pyridin-7-yl)pipera-
zine-1-carboxylate (34). A solution of 33 (1.39 g, 4.99
mol), NaHCO3 (0.42 g, 5.00 mmol), and aqueous chlo-
roacetaldehyde (40%, 1.18 g, 6.00 mmol) in EtOH
(20 mL) was refluxed for 3 h. The reaction mixture
was concentrated in vacuo and the residue was parti-
tioned between aqueous K2CO3 solution (30 mL) and
CHCl3 (30 mL). The organic layer was separated, dried
over anhydrous Na2SO4, and concentrated in vacuo.
The residue was purified by basic silica gel chromatogra-
phy (EtOAc) to give 34 (1.26 g, 83%) as a brown pow-
5.2.2. In vitro PT assays. The assay of plasma clotting
time was performed using an automatic coagulometer
(STA Compact, Diagnostica Stago). PT was measured
with STA PT reagents (Roche Diagnostics). 1.5 lL of
compound dilutions in DMSO was added to 48.5 lL
of human normal plasma (fresh human plasma: FFP,
Sekisui Chemical Co.), and the mixture was preincu-
bated at 37 ꢁC for 4 min. Coagulation was initiated with
the addition of 100 lL of thromboplastin, and coagula-
tion time was measured. Coagulation time prolonging
ratio (%) was calculated based on coagulation time
when DMSO was added instead of test compound.
The plasma clotting time doubling concentration (PT2)
was calculated from the regression line based on the
method of least squares. Data for new compounds were
1
der. H NMR (300 MHz, CDCl3) d: 1.49 (9H, s), 3.17
(4H, t, J = 5.2 Hz), 3.60 (4H, t, J = 5.2 Hz), 6.59 (1H,
dd, J = 2.4 and 7.6 Hz), 6.81 (1H, d, J = 2.6 Hz), 7.38
(1H, t, J = 0.6 Hz), 7.47 (1H, d, J = 1.0 Hz), 7.94 (1H,
d, J = 7.8 Hz).

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 3125–3140
3139
compared to positive control DX-9065a6c (PT2 = 0.81
0.029 lM (mean SEM, n = 3)).
Lineweaver–Burk plot, when the optical densities were
measured with different concentrations of substrates.
5.2.3. Measurement of CYP inhibition activity. Inhibition
activity of test compounds of CYP3A4 was evaluated by
incubating 100 lM testosterone with 10 nM CYP3A4
derived from CYP3A4-expressing human B-lympho-
blastoid cells (BD Biosciences) in the presence of
10 lM test compound. The incubation mixture was al-
lowed to stand for 30 min at 37 ꢁC. The concentration
of 6b-hydroxytestosterone was measured by HPLC sys-
tem equipped with a UV detector.
5.2.6. Pharmacokinetic analysis in cynomolgus monkeys.
Test compound was administered to fasted cynomolgus
monkeys (male, n = 3) intravenously (1 mg/kg, DMA/
PEG400) and orally (1 mg/kg, 0.5% methylcellulose sus-
pension). Before and 5, 10, 15, 30 min, 1, 2, 4, 8, 24 h
after intravenous administration, or before and 15,
30 min, 1, 2, 4, 8, 24 h after oral administration, blood
samples were collected from femoral vein. The blood
samples were centrifuged to obtain the plasma fraction.
The plasma samples were deproteinized with acetoni-
trile. After centrifugation, the supernatant obtained
was diluted with the same volume of 0.01 mol/L
HCO2NH4 (adjusted to pH 3.0 with HCO2H) and cen-
trifuged again. The compound concentration in the
supernatant was measured by LC/MS/MS with an
API3000 triple quadrupole mass spectrometer (Perkin-
Elmer Sciex). The mass spectrometer was equipped with
a turbo ionspray source and operated in positive ion
mode. The HPLC conditions were as follows: column,
an L-column ODS (2.1 · 50 mm); mobile phase,
0.01 mol/L HCO2NH4 (adjusted to pH 3.0 with
HCO2H)/acetonitrile = 6:4; flow rate, 0.2 mL/min; col-
umn temperature, 40 ꢁC.
5.2.4. Measurement of ex vivo PT in mouse after oral
administration. Male ICR mice (30–35 g, Clea Japan
Inc.) fasting for more than 12 h were employed. Test
compounds were orally administered to these animals.
An hour after administration, 800 lL blood was col-
lected from artery abdominal aorta using 3.8% sodium
citrate (whole blood: sodium citrate solution = 9:1, v/
v), under anesthesia with pentobarbital (50 mg/kg, ip).
The citrated blood was centrifuged at 1000g for 10 min
to obtain platelet poor plasma (PPP). PT was measured
with an automatic coagulometer. Fifty microliters of
PPP was preincubated at 37 ꢁC for 4 min. The PPP
was mixed with 100 lL of thromboplastin solution,
and then its coagulation time was measured. Test com-
pounds were suspended in 0.5% methyl cellulose (Meto-
lose cp100, Shin-Etsu Chemical), and as control, 0.5%
methylcellulose was administered instead of test com-
pounds. Activity of each test compound was shown in
ratio (%) determined by comparing the clotting time of
compound-treatment mice with that of control group.
We have validated the ex vivo PT assay for the evalua-
tion of oral bioavailability in mice by use of the positive
control compound DX-9065a.6c DX-9065a prolonged
mouse PT by 1.1-fold at a dose of 30 mg/kg and by
1.4-fold at a dose of 100 mg/kg.
Acknowledgments
The authors thank Mr. Terufumi Takagi for his docking
model study of compound 2e and helpful discussions,
and the DMPK group at Takeda Pharmaceutical Com-
pany for carrying out CYP3A4 inhibition measurements
for the compounds highlighted in Table 3 and obtaining
monkey pharmacokinetic data for compound 2e in Ta-
ble 5.
5.2.5. Enzyme inhibition assays. Enzyme assays using
chromogenic substrates were performed as follows. Hu-
man Factor Xa was obtained from Roche diagnostics.
Human thrombin was obtained from Sigma Chemical
Co. Human trypsin was obtained from Atheus Research
and Technology, Inc. Human kallikrein and plasmin
were purchased from BioPur AG. Human t-PA was
obtained from Kyowa Hakko Kogyo Co. The chromo-
genic substrates used were S-2222, S-2366, S-2222, S-
2366, S-2302, and S-2288 for FXa, thrombin, trypsin,
plasmin, kallikrein, and t-PA, respectively, and obtained
from Chromogenix-Instrumentation Laboratory. All
enzyme assays were performed at 37 ꢁC in 96-well
microtiter plates. The final enzyme concentrations were
0.024 U/mL, 0.080 U/mL, 0.040 lg/mL, 0.040 U/mL,
0.020 U/mL, and 4000 U/mL for FXa, thrombin, tryp-
sin, plasmin, kallikrein, and t-PA, respectively. Com-
pound dilutions were added to the wells containing
buffer and enzyme, and incubated for 30 min. The
enzyme reactions were initiated by the addition of sub-
strate and the color developed from the release of
p-nitroanilide from each chromogenic substrate was
monitored continuously for 5 min at 405 nm on a micro-
titer plate reader. The Ki values were determined from a
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