Synthetic Studies on Centromere-Associated Protein-E (CENP-E) Inhibitors: 2. Application of Electrostatic Potential Map (EPM) and Structure-Based Modeling to Imidazo[1,2-a]pyridine Derivatives as Anti-Tumor Agents

Article abstract of DOI:10.1021/acs.jmedchem.5b00836

To develop centromere-associated protein-E (CENP-E) inhibitors for use as anticancer therapeutics, we designed novel imidazo[1,2-a]pyridines, utilizing previously discovered 5-bromo derivative 1a. By site-directed mutagenesis analysis, we confirmed the ligand binding site. A docking model revealed the structurally important molecular features for effective interaction with CENP-E and could explain the superiority of the inhibitor (S)-isomer in CENP-E inhibition vs the (R)-isomer based on the ligand conformation in the L5 loop region. Additionally, electrostatic potential map (EPM) analysis was employed as a ligand-based approach to optimize functional groups on the imidazo[1,2-a]pyridine scaffold. These efforts led to the identification of the 5-methoxy imidazo[1,2-a]pyridine derivative (+)-(S)-12, which showed potent CENP-E inhibition (IC₅₀: 3.6 nM), cellular phosphorylated histone H3 (p-HH3) elevation (EC₅₀: 180 nM), and growth inhibition (GI₅₀: 130 nM) in HeLa cells. Furthermore, (+)-(S)-12 demonstrated antitumor activity (T/C: 40%, at 75 mg/kg) in a human colorectal cancer Colo205 xenograft model in mice.

Full text of DOI:10.1021/acs.jmedchem.5b00836

Article
pubs.acs.org/jmc
Synthetic Studies on Centromere-Associated Protein‑E (CENP-E)
Inhibitors: 2. Application of Electrostatic Potential Map (EPM) and
Structure-Based Modeling to Imidazo[1,2‑a]pyridine Derivatives as
Anti-Tumor Agents
Takaharu Hirayama,* Masanori Okaniwa,* Hiroshi Banno, Hiroyuki Kakei, Akihiro Ohashi, Kenichi Iwai,
Momoko Ohori, Kouji Mori,‡ Mika Gotou, Tomohiro Kawamoto, Akihiro Yokota,
and Tomoyasu Ishikawa
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa
251-8555, Japan
S
* Supporting Information
ABSTRACT: To develop centromere-associated protein-E
(CENP-E) inhibitors for use as anticancer therapeutics, we
designed novel imidazo[1,2-a]pyridines, utilizing previously
discovered 5-bromo derivative 1a. By site-directed mutagenesis
analysis, we confirmed the ligand binding site. A docking
model revealed the structurally important molecular features
for effective interaction with CENP-E and could explain the
superiority of the inhibitor (S)-isomer in CENP-E inhibition vs
the (R)-isomer based on the ligand conformation in the L5
loop region. Additionally, electrostatic potential map (EPM)
analysis was employed as a ligand-based approach to optimize
functional groups on the imidazo[1,2-a]pyridine scaffold. These efforts led to the identification of the 5-methoxy imidazo[1,2-
a]pyridine derivative (+)-(S)-12, which showed potent CENP-E inhibition (IC50: 3.6 nM), cellular phosphorylated histone H3
(p-HH3) elevation (EC50: 180 nM), and growth inhibition (GI50: 130 nM) in HeLa cells. Furthermore, (+)-(S)-12
demonstrated antitumor activity (T/C: 40%, at 75 mg/kg) in a human colorectal cancer Colo205 xenograft model in mice.
INTRODUCTION
■
Centromere-associated protein-E (CENP-E) is a mitotic
spindle motor protein belonging to the kinesin superfamily.1−4
CENP-E controls chromosome alignment onto the equatorial
plane during metaphase by capturing the microtubules with
energy from ATP hydrolysis.5−7 Loss of CENP-E function
causes chromosome misalignment during metaphase, leading to
prolonged mitotic arrest and the consequent apoptosis.
Figure 1. Chemical structures of reported CENP-E inhibitors.
Accordingly, inhibition of CENP-E has been reported to
suppress cellular proliferation in various types of cancer.8−12
GSK923295A13−17 is the first reported selective CENP-E
ligand-based electrostatic potential map (EPM) calculations.
inhibitor with an ATP uncompetitive profile to inhibit CENP-E
kinesin motor ATPase activity (Figure 1).13,14 On the basis of
its ATP uncompetitive nature, GSK923295A exhibits CENP-E
inhibitory activity even at high ATP concentrations, and this
was interpreted to contribute to its potent cellular activity.
Recently, we reported imidazo[1,2-a]pyridine inhibitor 1a,
which showed CENP-E inhibitory activity (IC50 = 50 nM) in an
ATP competitive manner, in contrast to GSK923295A (Figure
1).18 In this report, we describe the design, synthesis, and
structure−activity relationships (SAR) of imidazo[1,2-a]-
pyridine inhibitors based on both CENP-E homology docking
modeling supported by site-directed mutagenesis analysis and
The biological profiles of these compounds are also discussed
in detail.
Design of CENP-E Inhibitors. Because X-ray cocrystal
structures have not been reported for inhibitors bound to
human CENP-E, we built a CENP-E homology model utilizing
reported cocrystal structures of human Eg5, also known as
kinesin spindle protein (KSP),1−4 which has 36% amino acid
sequence identity with human CENP-E in the motor domain.19
The predicted binding mode of our lead compound 1a is
Received: June 1, 2015
© XXXX American Chemical Society
A
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Figure 2. (A) Binding mode of lead compound 1a and target residues in the motor domain of CENP-E. Docking model of 1a was built on the basis
of our previous complex model of CENP-E and an initial lead compound.18 (B) Design of compounds 1b−f (R1), 1g−k (R2), and 11−13 (R3).
Figure 3. (A) Chemical structure of 5-bromoimidazo[1,2-a]pyridine derivative 1a and its EPM surface. The EPM was calculated with MOE (version
2011.10)20 for the simplified dimethyl amide analogue (Supporting Information Figure S2). Positive, negative, and neutral charges are colored in
blue, red, and white, respectively. The pyridine moiety of the bicyclic core scaffold is outlined by the white box. Computation used an MMFF94x
force field, and the result is depicted as a Connolly surface in solid display style. (B) EPMs of the simplified dimethyl amide analogues of
imidazo[1,2-a]pyridine derivatives 1e and 1g−k.
shown in Figure 2A (computational 3D model is depicted in
Supporting Information Figure S1). By constructing the
docking model based on a CENP-E mutagenesis analysis
(vide infra), we confirmed that compound 1a could be
accommodated in a reported cavity known as the L5 binding
site13 that is formed by the L5 loop, α2 helix, and α3 helix. This
model is consistent with the previously reported SAR: for
example, the N,N-dimethylaminoethyl group of 1a is an
important part of the pharmacophore to provide potent
inhibitory activity and is most likely to interact with Glu186
on the α3 helix.18
At the same time, this model suggested three additional
binding pockets in a ligand binding site that were predicted to
be potentially useful in increasing affinity (Figure 2B). Thus,
our efforts in the design of new inhibitors focused on
modifications at the following positions: (1) the R1 group on
B
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a
Scheme 1. Synthesis of Imidazo[1,2-a]pyridine-2-carboxamide Derivatives 1a−k
a
Reagents and conditions: (a) N′-(3,4-dichlorobenzyl)-N,N-dimethylethane-1,2-diamine 3, HATU, base, 71−78% for 1a, 1d, and 16% for 1g′ from
4e; (b) 4 M HCl in EtOAc, 38−83% for 1b, 1c, 1e, 1f, and 1i−k; (c) TFA, rt, 1 h, then, 4 M HCl in EtOAc 71%; (d) NaOMe, DMF, rt, 12 h, 49%.
a
Scheme 2. Synthesis of Imidazo[1,2-a]pyridine-2-carboxylic Acid 2a−g and 2i−k
a
Reagents and conditions: (a) NaOMe or NaOtBu, THF; (b) EtOH, reflux, 4−12 h, 24−53% in 2 steps (9a, 9e from 4a, 4e), 19% (9d from 4d′);
(c) MgBr2, THF, 80 °C, 0.5 h, then 6, EtOH, reflux, overnight, 15% in 3 steps (9b); (d) 2 M LiOH or 2 M NaOH, THF, MeOH or EtOH, 40−65
°C, 0.5−4 h, 69−98% (2a, 2b, 2d, 2e, 2i, and 2k), 13−29% in 3 steps (2c, 2f, and 2j); (e) MeI, NaH, THF, rt, 1 h, 85%; (f) tributyl (ethynyl)
stannane, PdCl2(dppf)·CH2Cl2, DME, H2O, 100 °C, 16 h, 18%; (g) CuCN, Pd2(dba)3, dppf, cyclopentyl methyl ether, reflux, 20 h, 32%.
the p-fluorobenzene (pendant phenyl) ring to interact with a
hydrophobic pocket including Pro107, (2) the R2 group on the
imidazo[1,2-a]pyridine scaffold to fit in the hydrophobic pocket
containing Ile182, and (3) the R3 group of the 3,4-
dichlorobenzyl group directed toward the α2 helix and
hydrophobic L5 loop.
First, we investigated 5-bromoimidazo[1,2-a]pyridine deriv-
atives 1b (2-Cl), 1c (3-Cl), 1d (3-F), 1e (3-CH3), and 1f (3-
OCH3), having small substituents (R1) that protrude into the
hydrophobic groove near Pro107. Next, we utilized EPM
analysis to select potential R2 substituents for the 5-position of
the imidazo[1,2-a]pyridine scaffold. In our previous empirical
studies,18 neutral electron charge (Figure 3A, region within the
white box) on imidazo[1,2-a]pyridine was shown to be a useful
predictor of potent CENP-E inhibitory activity.
We analyzed the EPMs of imidazo[1,2-a]pyridine derivatives
1g−k possessing various 5-substituents (R2) in comparison to
that of 1e (R1 = CH3, R2 = Br) (Figure 3B). Almost all of the
proposed compounds 1g−j showed neutral charge on the fused
pyridine ring, suggesting that these compounds had potential to
exhibit potent CENP-E inhibition based on our previous
empirical knowledge.18 On the other hand, 5-CN derivative 1k
C
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Scheme 3. Synthesis of 5-Methoxyimidazo[1,2-a]pyridine-2-carboxamide Derivatives 11a−d, 12−14 Bearing Various
a
Substituents (R3 and L) at the Benzylic Position
a
Reagents and conditions: (a) racemic diamines 16a−d, 17−19, HATU, base, solvent, (HCl), 45−88%; (b) CaCl2, NaBH4, EtOH, THF, rt, 0.3 h,
then 0 °C, 0.7 h, 12−29%.
showed positive charge on the fused pyridine ring. On the basis
of such analysis, we predicted the potency of CENP-E
inhibition in the following order: methoxy (1h), ethynyl (1i)
> cyclopropyl (1j), methylamino (1g), bromo (1e) > cyano
(1k).
equiv of sodium methoxide (NaOMe) in N,N-dimethylforma-
mide (DMF) to afford the desired compound 1h in 49% yield.
The synthesis of imidazo[1,2-a]pyridine-2-carboxylic acids
2a−g and 2i−k is shown in Scheme 2. The β-chloro-α-
ketoester intermediates 4a′ and 4c′−f′ were prepared by
Darzens reaction21 conditions from the corresponding
benzaldehydes 4a and 4c−f, with methyl dichloroacetate 5 in
the presence of NaOMe or sodium tert-butoxide (NaOtBu).
Subsequent reaction of the β-chloro-α-ketoesters 4a′, 4d′, and
4e′ with commercially available 2-amino-6-bromopyridine 6 in
refluxing EtOH provided imidazo[1,2-a]pyridine-2-carboxy-
lates22 9a and 9e in 53% and 24% yields in two steps from
4a and 4e, respectively, and 9d in 19% yield from 4d′. When 2-
chloro-4-fluorobenzaldehyde 4b was treated with methyl
dichloroacetate 5 under Darzens reaction conditions, methyl
2-chloro-3-(2-chloro-4-fluorophenyl)oxirane-2-carboxylate 4b″
was isolated as a stable intermediate. The obtained 4b″ was
treated with magnesium bromide to afford β-bromo-α-ketoester
4b′. Subsequent ring formation of 4b′ with 6 in refluxing EtOH
gave the desired compound 9b in 15% yield over three steps
from 4b. Because EtOH was used in the ring formation as the
solvent, some of the methyl ester was trans-esterified to give
mixtures of the methyl and ethyl esters 9c/10c, 9f/10f, 9g′/
10g′, and 9j/10j. The mixtures of esters 9c/10c, 9f/10f, and
9j/10j were converted into the desired carboxylic acids 2c, 2f,
and 2j by conventional hydrolysis conditions in 13−29% yields
over three steps. A mixture 9g′/10g′ was treated with methyl
iodide to provide the N-methyl derivatives 9g/10g as a mixture
Finally, the introduction of an R3 substituent (compound 11,
Table 4) or cyclization at the R3 position (compounds 12 and
13, Table 4 and dotted line in Figure 2B) were examined to
determine whether modification of the molecular conformation
could impart a hydrophobic interaction in the L5 loop region
based on modeling predictions.
Chemistry. The synthesis of imidazo[1,2-a]pyridine-2-
carboxamide derivatives 1a−k is outlined in Scheme 1.
Imidazo[1,2-a]pyridine-2-carboxylic acids 2a−g and 2i−k
(prepared as shown in Scheme 2) were reacted with
commercially available N′-(3,4-dichlorobenzyl)-N,N-dimethyl-
ethane-1,2-diamine 3 under condensation conditions using
hexafluorophosphate O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tet-
ramethyluronium (HATU) in the presence of a base such as
N,N-diisopropylethylamine (DIPEA), pyridine, or triethylamine
(Et3N) to give the corresponding 2-carboxamide derivatives
1a−f, 1g′, and 1i−k. Oily compounds were converted to the
corresponding solids (1b, 1c, 1e, 1f, and 1i−k) as HCl salts by
treatment with 4 M HCl in EtOAc. N-tert-Butoxycarbonyl
(Boc) protected methylamino derivative 1g′ was treated with
trifluoroacetic acid (TFA) to provide the desired 5-methyl-
amino derivative 1g in 71% yield. Replacement of a 5-bromo
group (1e) with a methoxy group was accomplished using 2
D
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a
Scheme 4. Synthesis of 3-(4-Fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]pyridine-2-carboxylic Acid 15
a
Reagents and conditions: (a) EtOH, 40 °C, 14 h, 52%; (b) NBS, DMF, rt, 1 h, 96%; (c) PdCl2(dppf)·CH2Cl2, Cs2CO3, DME, H2O, 90 °C, 1.5 h,
89%; (d) 2 M NaOH, THF, MeOH, rt, 8 h, 74%.
(85% yield), followed by hydrolysis of the mixture 9g/10g to
give desired carboxylic acid 2g, which was used in the
subsequent coupling reaction without purification. Hydrolysis
of other ester derivatives 9a, 9b, 9d, and 9e was carried out with
2 M NaOH or 2 M LiOH to yield the corresponding carboxylic
acids 2a, 2b, 2d, and 2e in 71−98% yields.
5-Bromo compound 9e proved to be a useful intermediate
for further chemical modification. The palladium-catalyzed
Stille coupling23 of 9e with tributyl(ethynyl)stannane using
[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)
dichloromethane (PdCl2(dppf)·CH2Cl2) proceeded in dime-
thoxyethane (DME)/water (5:1) to give ethynyl derivative 9i
in 18% yield. Replacement of the 5-bromo group with a cyano
group was achieved using 4 equiv of copper(I) cyanide
(CuCN) in the presence of a catalytic amount of tris-
(dibenzylideneacetone) dipalladium(0) (Pd2(dba)3) and 1,1′-
bis(diphenylphosphino)ferrocene (dppf) in cyclopentyl methyl
ether to afford the desired 5-cyano derivative 9k in 32% yield.
Hydrolysis of ester derivatives 9i and 9k was carried out with 2
M LiOH to provide the corresponding carboxylic acid
derivatives 2i and 2k in 69−94% yields.
yielded the desired hydroxymethyl ( )-11c and hydroxyethyl
( )-11d derivatives in 29% and 12% yields, respectively.
Optical resolution of racemic compounds 11a, 11b, and 12−14
by a chiral HPLC afforded both of the corresponding (+)-and
(−)-enantiomers of 11a, 11b, and 12−14.
The route for the preparation of carboxylic acid 15 is shown
in Scheme 4. Commercially available 6-methoxypyridin-2-
amine 20 was reacted with ethyl 3-bromo-2-oxopropanoate
21 in EtOH at 40 °C to provide the desired imidazo[1,2-
a]pyridine ester 22 in 52% yield. Because performing the
reaction at temperatures higher than 40 °C gave rise to
demethylation of the 5-methoxy group due to hydrogen
bromide generated in situ, it was found to be quite important to
maintain the temperature at 40 °C. Compound 22 was
quantitatively converted to 3-bromo derivative 23 in 96% yield
using N-bromosuccinimide (NBS) in DMF. Pd-catalyzed
Suzuki−Miyaura coupling24 of 23 with 4-fluoro-3-methylphe-
nylboronic acid 24 in the presence of cesium carbonate
(Cs2CO3) in DME/water (5:1) provided ester 25 in 89% yield.
Subsequent hydrolysis of 25 with 2 M NaOH gave the desired
carboxylic acid 15 in 74% yield.
The synthesis of racemic diamine derivatives 16c, 16d, and
17−19 is outlined in Scheme 5. Reductive amination of
commercially available ketones 26c, 26d, 27, and 28 with N,N-
dimethylethane-1,2-diamine 29 or tert-butyl (2-aminoethyl)-
methylcarbamate 30 was carried out using reducing reagents,
such as NaBH4, 2-picoline borane complex, or NaBH3CN, in
the presence of acetic acid (AcOH) to provide racemic
diamines 16c, 16d, and 17−19 in 4−59% yields.
5-Methoxyimidazo[1,2-a]pyridine-2-carboxamide derivatives
(11a−d, 12−14) bearing various substituents at the benzylic
position were synthesized as depicted in Scheme 3.
Condensation of 3-(4-fluoro-3-methylphenyl)-5-
methoxyimidazo[1,2-a]pyridine-2-carboxylic acid 15 (vide
infra) with various diamine intermediates such as commercially
available ( )-N′-[1-(3,4-dichlorophenyl)ethyl]-N,N-dimethyl-
ethane-1,2-diamine (( )-16a),
( )-N′-[1-(3,4-
dichlorophenyl)propyl]-N,N-dimethylethane-1,2-diamine
(( )-16b) or prepared racemic diamines 16c, 16d, and 17−19
(Scheme 5) was carried out by a similar method to that used in
Scheme 1 to provide the racemic 2-carboxamides 11a, 11b,
11c′, 11d′, and 12−14 in 45−88% yields. Subsequent
reduction of compounds ( )-11c′ and ( )-11d′ with sodium
tetrahydroborate (NaBH4) and calcium chloride (CaCl2)
RESULTS AND DISCUSSION
■
Determination of the Plausible Binding Site of Ligand
1a on CENP-E by Site-Directed Point Mutagenesis
Experiments. We hypothesized that imidazo[1,2-a]pyridine
derivatives could be accommodated in the L5 site, which is
known to bind small molecule inhibitors near the L5 loop of
E
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as a negative control because our docking model predicted the
Thr183 side chain to be located outside the plausible binding
pocket. Thus, we prepared two mutants, human CENP-E
Scheme 5. Synthesis of Diamine Derivatives ( )-16c, 16d,
and 17−19
a
I182L
and human CENP-ET183A, for which the indicated amino acid
residues were switched from the human to mouse residues. The
mutants thus prepared were shown to maintain comparable
activity relative to that of the wild type (ATPase activity). The
IC50 values with inhibitor compounds could be measured under
similar ATP concentrations to that used in the CENP-E (WT)
assay, wherein the ATP concentration (24.7 μM) was set near
the Km value.
We evaluated the ATPase inhibitory activities of compound
1a against CENP-EI182L and CENP-ET183A as well as wild-type
(Table 1). Compound 1a exhibited a 12.6 fold reduction in
Table 1. Comparison of Inhibitory Activity of Compound 1a
between Mutant CENP-EsI182L and T183A and Wild-Type
a
reduction ratio of IC50s for ATPase activity based on
CENP-EWT
Reagents and conditions: (a) AcOH, EtOH, rt, 1.5 h, then NaBH4,
MeOH, 0 °C, 1 h then rt, 0.5 h, 5% for 16c, 4% for 16d; (b) AcOH,
THF, MeOH, rt, 1 h then 50 °C, 2 h, then 2-picoline borane complex,
rt, 14 h, 46% for 17, 59% for 18; (c) AcOH, THF, MeOH, NaBH3CN,
50 °C, 18 h, 24% for 19.
compd
CENP-EI182L
CENP-ET183A
a
a
1a
12.6 (150/12)
2.4 (30/12)
a
Values shown as IC50 ratio for mutant and wild-type with IC50 values
given in parentheses (mutant CENP-E/WT CENP-E)
CENP-E motor domain.13 On the basis of this hypothesis, we
built a CENP-E homology model (Figure 4). To validate the
CENP-EI182L inhibition compared to that of CENP-E (WT),
indicating 1a has a strong interaction with the side chain of
Ile182. On the other hand, the IC50 reduction for CENP-ET183A
was only 2.4-fold. These results suggested that imidazo[1,2-
a]pyridine scaffold should be located in a hydrophobic pocket
containing Ile182 in the L5 site, as predicted.
SAR for Substituents (R1) on the Pendant Phenyl Ring.
According to our docking model, the pendant phenyl ring at
the 3-position of imidazo[1,2-a]pyridine scaffold was predicted
to be accommodated in a hydrophobic pocket near Pro107.
With the aim of creating more effective interaction with the
hydrophobic pocket, we designed 5-bromoimidazo[1,2-a]-
pyridine derivatives 1b−f having a various substituents (R1)
that protrude into the hydrophobic groove. The results of their
evaluation for CENP-E ATPase inhibitory activity are shown in
Table 2. Compound 1b (R1 = 2-Cl) exhibited drastically
reduced CENP-E inhibitory activity relative to 1a (R1 = H)
with an IC50 value of 7900 nM. On the other hand, the
introduction of a chloro group at the 3-position for compound
1c (R1 = 3-Cl) enhanced CENP-E inhibitory activity (IC50 = 15
nM) 3-fold over that of 1a. Similarly, 3-F (1d) and 3-CH3 (1e)
derivatives exhibited enhanced CENP-E inhibitory activities
(IC50: 28 and 22 nM, respectively). Compound 1f possessing a
3-OMe group showed the slightly reduced potency (IC50 = 82
nM) compared to other substituents at the 3-position. These
results suggested that the substituents at the 3-position of the
pendant phenyl ring of 1c (3-Cl), 1d (3-F), and 1e (3-Me)
could be properly accommodated in the hydrophobic pocket
including Pro107. On the other hand, the reduced CENP-E
inhibitory activities of 1b (2-Cl) and 1f (3-OMe) could be
caused by a steric clash between substituents on pendant
phenyl ring and protein according to our docking model
(Figure S1A).
Figure 4. Docking model of compound 1a and amino acid residues
bearing species differences in the L5 site of CENP-E (His102, Ser175,
Ile182, Thr183, and Lys184). The docking model is visualized using
PyMOL Molecular Graphics System, version 1.3, Schrodinger, LLC.
̈
CENP-E homology model in L5 site, we conducted site-
directed point mutagenesis analysis in CENP-E motor domain.
Although an alanine scanning method25 is often used for this
purpose, it is not always effective because mutant proteins
sometime lose their enzymatic function through disruption of
their tertiary structures. Here, we hypothesized that the amino
acid residues with species differences would be ideal targets for
point-mutation studies because the mutant proteins would
likely retain their enzymatic activity. On the basis of differences
in the amino acid sequences between human and mouse
CENP-Es,26 five residues in the L5 site were selected: His102
(Cys), Ser175 (Ala), Ile182 (Leu), Thr183 (Ala), and Lys184
(Thr); the amino acid residue in mouse is described in
parentheses, and amino acid alignment between human and
mouse is described in the Supporting Information Table S1.
Among these potential targets, residue Ile182 of the α3 helix
was viewed as the most promising target due to the likelihood
of forming a hydrophobic interaction with compound 1a. On
the other hand, Thr183 (also part of the α3 helix) was viewed
Next, we examined the cellular activities of these compounds,
by measuring the induction of phosphorylated histone H3 (p-
HH3) accumulation as a biomarker for M-phase arrest in HeLa
cells using FACS. The cellular activities are shown as EC50
values wherein the concentration required for 50% response of
F
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Table 2. SAR for Substituents (R1) on the Pendant Phenyl
Ring of 5-Bromoimidazo[1,2-a]pyridine Derivatives 1a−f
Table 3. SAR for 5-Substitutions (R2) on Imidazo[1,2-
a]pyridine 1e and 1g−k
a
b
compd
R2
CENP-E IC50 (nM)
cell (HeLa) GI50 (nM)
a
CENP-E IC50 (nM)
1e
1g
1h
1i
Br
22 (17−29)
9.2 (8.1−10)
1.0 (0.84−1.2)
5.7 (4.5−7.3)
17 (12−25)
2700
810
d
p-HH3
EC50
NHCH3
OCH3
CCH
c-Pr
b
b,c
compd
R1
25 μM ATP
500 μM ATP
(nM)
240
e
460
1a
1b
1c
1d
1e
1f
H
50 (24−110)
7900 (4500−14000)
15 (9.9−23)
1540
>3000
1650
1210
636
f
f
f
f
f
1j
1800
2-Cl
c
1k
CN
83 (58−120)
N.D.
3-Cl
14 (8.3−24)
7.6 (5.0−12)
a
b
3-F
28 (19−41)
22 (17−29)
82 (58−120)
n = 2. 95% Confidence interval (CI) value. Cell proliferation of
HeLa cells was determined by cellular ATP content (n = 1). Cells
collected 3 days after drug treatment. Not determined.
3-CH3
3-OCH3
c
1270
a
b
n = 2. 95% Confidence interval (CI) value. ATP concentration used
in ATPase assay. Prior to catalytic initiation with 500 μM ATP,
compound and enzyme were incubated for 1 h at rt (preincubation).
Percent of phosphorylated histone H3 positive cells was detected
using FACS (n = 1). Cell cycle synchronized cells were used (section
of Cell Cycle Synchronization in the Supporting Information).
c
positive charge in the EPM. Thus, the prediction of CENP-E
inhibitory activity using EPM analysis proved a practical and
effective method to optimize the R2 group and contributed to
the efficient identification of potent substituent of CENP-E
inhibitors such as 1h (R2 = OMe). Reflecting the potent
CENP-E inhibitory activity, 1h exhibited significant growth
suppression of HeLa cells (GI50 = 240 nM).
d
e
f
Asynchronous cells were used.
the maximum p-HH3 positive cell count was obtained (Table 2,
right column). Unexpectedly, among these compounds, only
the 3-CH3 derivative 1e exhibited significant enhancement,
demonstrating 2.6-fold more potent cellular activity than 3-Cl
derivative 1c.
To better understand the difference in cellular activity
between 1e (3-Me) and 1c (3-Cl), we evaluated the ATPase
inhibitory activity of 1e and 1c at high ATP concentration (500
μM) after 1 h preincubation because the cellular ATP
concentration is known to be approximately 1 mM. In
agreement with the above cellular assay results, a lower IC50
value was found for 1e (7.6 nM) than for 1c (14 nM) under
high ATP concentration conditions (Table 2). On the basis of
these results, we concluded that compound 1e has more potent
CENP-E inhibitory activity than 3-Cl derivative 1c under cell-
free and cellular conditions.
Creating Efficient Interaction between the Benzyl
Moiety and L5 Loop of CENP-E. Despite the potent cellular
activity measured in vitro, compound 1h exhibited low potency
in in vivo studies (data not shown). To enhance the potency,
we initiated chemical modifications at the benzylic position,
starting from 1h as our new lead compound. The results from
biological evaluation of 5-methoxyimidazo[1,2-a]pyridines
possessing various substituents (R3 and L) at the benzylic
position are summarized in Table 4. Introduction of a methyl
(( )-11a) or ethyl (( )-11b) group at the benzylic position
resulted in decreased CENP-E inhibition and cellular potency.
It is worth noting that no remarkable differences in CENP-E
inhibitory activities were found between the enantiomers of 11a
or 11b. The introduction of other substituents such as
hydroxymethyl (( )-11c) and hydroxyethyl (( )-11d) groups
also resulted in reduced potency.
SAR for 5-Substituted Imidazo[1,2-a]pyridine Deriva-
tives. Compounds 1g−k were evaluated for their CENP-E
inhibitory activity and cellular growth inhibitory activity (GI50)
in HeLa cells (Table 3). As expected, the relative CENP-E
inhibitory activity for these compounds was largely consistent
with EPM predictions (Figure 3B). Introduction of an electron
donating group such as 5-NHCH3 (1g) or 5-OCH3 (1h) led to
enhanced CENP-E inhibitory activity. In particular, compound
1h exhibited 22-fold more potent inhibitory activity (IC50 = 1.0
nM) along with a favorable neutral charge in the EPM, while
the 5-Br derivative 1e (22 nM) possessed a slightly positive
charge. Upon the introduction of an 5-ethynyl (1i) or 5-
cyclopropyl (1j) group, compound 1i with neutral charge on
the scaffold exhibited more potent CENP-E inhibitory activity
(IC50 = 5.7 nM) than that of 1j (17 nM), bearing slightly more
negative charge. On the other hand, the 5-CN derivative 1k
showed reduced activity (83 nM) as predicted by its strongly
Next, we envisioned that ring formation at the benzyl
position might produce a conformationally rigid bicyclic
construct to efficiently interact with the L5 loop binding
pocket. In accordance with this expectation, the eutomers of
the dihydrobenzofuran derivative ((+)-12: CENP-E IC50 = 3.6
nM, GI50 = 130 nM) and the dihydrobenzopyran derivative
((+)-13: CENP-E IC50 = 9.0 nM, GI50 = 190 nM)
demonstrated potent single-digit nanomolar CENP-E inhib-
itory potency together with slightly increased cellular activity
compared to that of 1h (GI50 = 240 nM). Interestingly, the
distomers of these compounds, (−)-12 and (−)-13, showed
much weaker CENP-E inhibition than observed for the
eutomers of these derivatives. Notably, compound (+)-12
exhibited 640-fold more potent CENP-E inhibitory activity
(IC50 3.6 nM) than (−)-12 (IC50 = 2300 nM), indicating a
highly specific interaction between the structurally constrained
bicyclic moiety in (+)-12 and the hydrophobic groove of the L5
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Table 4. SAR for Various Substituents (R3 and L) at the
Benzylic Position of 5-Methoxyimidazo[1,2-a]pyridines 1h,
11a−d, 12, and 13
because (+)-12 formed only amorphous solids as a salt free
form. In addition, we made efforts to crystallize (+)-12 in salt
form by treating (+)-12 with various acids, such as mandelic
acid, dibenzoyl-L-tartaric acid, p-toluenesulfonic acid, (+)-tar-
tranilic acid, and L-lactic acid, but crystalline salts were still not
obtained. Next, we investigated the crystallization of Boc-
protected derivatives (+)-14 and (−)-14. As a result of solvent
screening, each optical isomer was successfully crystallized from
iPr2O/EtOAc (5/1) to provide single plate crystals. X-ray
crystallographic analysis determined that the absolute config-
urations of (+)-14 and (−)-14 were (S) and (R), respectively
(Supporting Information Figure S3). The obtained compound
(+)-(S)-14 was treated with TFA to remove the Boc-protecting
group, followed by methylation to yield (+)-12. We confirmed
cell
b
that this compound had consistent optical rotation ([α]25
=
(HeLa)
D
GI50
+97.6° (c = 0.169, CHCl3)) with that of an authentic sample of
a
compd
1h
R3
L
CENP-E IC50 (nM)
(nM)
(+)-12 ([α]25 = +101.0° (c = 0.231, CHCl3)) (Figure 5).
D
H
1.0 (0.84−1.2)
240
Analysis of Eutomer (+)-12 and Distomer (−)-12 by
Docking Studies. We conducted docking studies of eutomer
(+)-12 and distomer (−)-12 using a homology model CENP-E
to explain the differences in CENP-E inhibitory activities
between (+)-12 and (−)-12. Analysis of these results revealed
that for the (S)-configuration the dichlorobenzene ring is
located near the carboxylic acid moiety of Glu100 in the L5
loop, providing for effective interaction (Figure 6A). On the
( )-11a
(+)-11a
(−)-11a
CH3
10 (8.7−12)
5.7 (3.7−8.8)
9.5 (6.9−13)
820
c
ND
c
ND
c
( )-11b
(+)-11b
(−)-11b
CH2CH3
16 (15−18)
10 (8.7−12)
19 (15−23)
ND
1900
1600
( )-11c
( )-11d
CH2OH
7.1 (4.9−10)
18 (14−22)
810
810
(CH2)2OH
( )-12
(+)-12
(−)-12
−(CH2)−
−(CH2)2−
4.8 (3.1−7.5)
3.6 (3.1−4.2)
2300 (1800−2900)
270
130
>3000
( )-13
(+)-13
(−)-13
14 (12−16)
9.0 (7.7−11)
390
190
Figure 6. Docking models of the eutomer and distomer around the L5
loop. Proteins are rendered as surfaces, and ligands as CPK space-
filling models by MOE (version 2011.10).20 (A) Model of eutomer
(+)-(S)-12. (B) Model of distomer (−)-(R)-12. Steric clash between
the dihydrobenzofuran moiety and L5 loop is indicated by a white
arrow.
71 (43−110)
1400
a
b
n = 2. 95% Confidence interval (CI) value. Cell proliferation of
HeLa cells was determined by cellular ATP amounts (n = 1). Cells
collected at 3 days after drug treatment. Not determined.
c
loop. Among the compounds described above, compound
(+)-12 revealed the most potent combination of antiprolifer-
ative activity against HeLa cells (GI50 = 130 nM) and p-HH3
elevation (EC50 = 180 nM), thus we selected compound (+)-12
for further biological evaluation.
Determination of the Absolute Configuration of
(+)-12. For the purpose of determining the absolute
configuration of the eutomer (+)-12, we attempted to obtain
single crystals of (+)-12. However, no crystals were obtained
other hand, the (R)-configuration causes a steric clash between
the dihydrobenzofuran moiety and L5 loop, preventing close
interaction with the ligand binding site (Figure 6B). Thus,
introduction of a bicyclic substructure through ring formation
at the benzylic position served to provide a rigid molecular
conformation and the two resulting enantiomers are
distinctively recognized by the L5 site of CENP-E.
Biological Evaluation of CENP-E Inhibitor (+)-(S)-12.
We evaluated the enzymatic activity of representative CENP-E
Figure 5. Synthetic route to (+)-12 from (+)-14.
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inhibitor (+)-(S)-12 against other kinesins, Eg5 (KSP) and
kinesin heavy chain (KHC). Compound (+)-(S)-12 exhibited
weak inhibitory activities against Eg5 and KHC (IC50 > 10000
nM). In addition, (+)-(S)-12 did not show any significant
inhibitory activities (IC50 > 1000 nM) against 36 kinases (list of
evaluated kinases is described in the Supporting Information
Table S2), also suggesting that (+)-(S)-12 is likely a selective
inhibitor of CENP-E.
Next, we conducted immunofluorescence analysis to confirm
the cellular phenotype of mitotic arrest induced by (+)-(S)-12.
In concurrence with the potent enzymatic activity measured for
this compound, chromosome misalignment was observed in
HeLa cells in treatment with 300 nM of (+)-(S)-12 (Figure 7),
75 mg/kg (ip, bid) without severe body weight loss in the same
tumor model (Figure 9).
CONCLUSION
■
With the aim of discovering potent CENP-E inhibitors for use
in the treatment of cancer, we designed novel imidazo[1,2-
a]pyridine derivatives using CENP-E homology modeling
validated by site-directed mutagenesis analysis. The docking
model highlighted the structurally important features of our
imidazo[1,2-a]pyridine inhibitors (R1, R2, and R3) in creating
effective interaction with the CENP-E protein (Pro107, Ile182,
and L5 loop, respectively). Introduction of a 3-methyl group
(R1 = 3-Me) on the pendant phenyl ring (1e) provided not
only potent CENP-E inhibitory activity but also potent cellular
activity. Subsequently, the neutral electron charge on the
pyridyl ring of the imidazo[1,2-a]pyridine scaffold by EPM
analysis was shown to be important for affording high affinity
with the hydrophobic pocket containing Ile182, leading us to
compound 1h (R2 = 5-OCH3). Finally, ring formation at the
benzylic (R3) position provided the chiral dihydrobenzofuran
derivative (+)-(S)-12 with a rigid conformation of its bicyclic
substructure, providing effective interaction with the L5 loop.
The optimized compound (+)-(S)-12 showed potent CENP-E
inhibitory activity (CENP-E IC50 = 3.6 nM) as well as
antiproliferative activity (GI50 = 130 nM) due to the induction
of mitotic arrest (p-HH3 EC50 = 180 nM). Compound (+)-(S)-
12 exhibited in vivo PD effects and significant antitumor
activity (40% T/C at 75 mg/kg, bid) in a human colorectal
cancer Colo205 xenograft mouse model. Consequently,
compound (+)-(S)-12 was judged to represent a promising
new lead compound for further studies of CENP-E inhibitor.
Figure 7. Immunofluorescence analysis in treatment with (+)-(S)-12
(300 nM) in HeLa cells. Misaligned chromosomes indicated by
arrows. Blue, green, and pink signals indicate DNA, α-tubulin, and
CENP-B (kinetochore), respectively.
correlating with the increased p-HH3 (EC50 = 180 nM)
expected to be observed during prolonged mitotic arrest and
the consequent antiproliferative effects (GI50 = 130 nM, Table
4) in these cells. Furthermore, we measured p-HH3 levels as an
in vivo PD marker in a tumor xenograft model by Western
blotting in response to (+)-(S)-12 and (−)-(R)-12 treatment.
Upon intraperitoneal (ip) administration with 150 mg/kg of
either (+)-(S)-12 or (−)-(R)-12 twice a day (bid) in a human
colorectal cancer Colo205 xenograft mouse model, only
eutomer (+)-(S)-12, but not (−)-(R)-12, exhibited elevation
of p-HH3 levels in tumors (Figure 8). Moreover, (+)-(S)-12
demonstrated p-HH3 elevation at a dose of 75 mg/kg.
Reflecting the observed in vivo PD effects, (+)-(S)-12 exhibited
significant antitumor activity in a dose-dependent manner with
tumor growth inhibition (T/C) of 55% at 50 mg/kg and 40% at
EXPERIMENTAL SECTION
■
General Chemistry Information. All commercially available
solvents and reagents for reactions were reagent-grade and were
used as purchased. Analytical thin layer chromatography (TLC) was
performed on silica gel 60 F254 plates (Merck) or NH TLC plates (Fuji
Silysia Chemical Ltd.). Flash column chromatography separations
were performed on Purif-Pack (SI or NH, Fuji Silysia Chemical Ltd.).
Melting points (mp), determined on a SRS OptiMelt automated
melting point apparatus, are uncorrected. Proton nuclear magnetic
resonance (1H NMR) spectra were recorded using Bruker AVANCE II
(300 MHz), Bruker AVANCE III (300 MHz, 400 MHz), or Varian
mercury plus (300 MHz) spectrometers with tetramethylsilane (TMS)
as an internal standard. The NMR data are given as follows: chemical
shift (δ) in ppm, multiplicity (where applicable), coupling constants
(J) in Hz (where applicable), and integration (where applicable).
Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q
(quartet), dd (double doublets), dt, (double triplet), dq (double
quartet), br s (broad singlet), or m (multiplet). Mass spectra (MS)
were aquired using an Agilent LC/MS system (Agilent 1200SL/
Agilent 6130MS), Shimadzu LC/MS system (LC-10ADvp high
pressure gradient system/LCMS-2010A) or Shimadzu UFLC/MS
(Prominence UFLC high pressure gradient system/LCMS-2020)
operating in electron spray ionization mode (ESI+). The column used
was an L-column 2 ODS (3.0 mm × 50 mm I.D., 3 μm, CERI, Japan)
with a temperature of 40 °C and a flow rate of 1.2 or 1.5 mL/min.
Mobile phase A was 0.05% TFA in ultrapure water. Mobile phase B
was 0.05% TFA in acetonitrile, which was increased linearly from 5%
to 90% over 2 min, 90% over the next 1.5 min, after which the column
was equilibrated to 5% for 0.5 min. Elemental analyses and high
resonanse mass spectra (HRMS) were carried out by Takeda
Analytical Laboratories, Ltd. Each compound was confirmed to be
≥95% purity by either LC/MS or elemental analysis. Yields were not
optimized.
Figure 8. p-HH3 levels in tumors in a Colo205 xenograft mouse
model. Intraperitoneal administration was performed twice a day (0
and 8 h). Phosphorylated HH3 in tumors was detected by Western
blotting (n = 2−3) 24 h after the first administration of (+)-(S)-12 or
(−)-(R)-12.
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Figure 9. Mean tumor volumes and body weights in a Colo205 xenograft mouse model dosed with compound (+)-(S)-12 or vehicle. n = 5.
Compound (+)-(S)-12 was delivered in 0.1 mol/L of citric acid with 10% DMSO, 9% cremophor EL, and 18% PEG 400 and intraperitoneally
administered (50 and 75 mg/kg) twice a day on each of the second, third, seventh, and eighth days over 10 days. P ≤ 0.05 (*) vs control at day 10 (t-
test).
5-Bromo-N-(3,4-dichlorobenzyl)-N-[2-(dimethylamino)ethyl]-3-
(4-fluorophenyl)imidazo[1,2-a]pyridine-2-carboxamide (1a). To a
solution of 5-bromo-3-(4-fluorophenyl)imidazo[1,2-a]pyridine-2-car-
boxylic acid 2a (451 mg, 1.34 mmol) in DMF (5.0 mL) was added a
solution of N′-(3,4-dichlorobenzyl)-N,N-dimethylethane-1,2-diamine
3 (349 mg, 1.41 mmol), HATU (560 mg, 1.47 mmol), and DIPEA
(433 mg, 3.35 mmol) in DMF (1.0 mL), and the reaction mixture was
stirred at room temperature for 24 h. The reaction mixture was
partitioned between EtOAc (15 mL) and saturated aqueous NaHCO3
solution (15 mL). The aqueous layer was extracted with EtOAc (3 ×
15 mL), and the combined organic layers were washed with water (15
mL) and brine (15 mL), and dried over anhydrous MgSO4. After
concentration in vacuo, the residue was purified by column
chromatography (NH silica gel, eluted with 20−70% EtOAc in n-
hexane) to give 1a (591 mg, 78%) as a colorless amorphous solid. MS
10.66 (1H, m). Anal. Calcd for C25H21BrCl3FN4O·HCl·1.5H2O: C,
45.34; H, 3.81; N, 8.46.Found: C, 45.13; H, 3.78; N, 8.27.
5-Bromo-3-(3-chloro-4-fluorophenyl)-N-(3,4-dichlorobenzyl)-N-
[2-(dimethylamino)ethyl]imidazo[1,2-a]pyridine-2-carboxamide
Dihydrochloride (1c). To a solution of 5-bromo-3-(3-chloro-4-
fluorophenyl)imidazo[1,2-a]pyridine-2-carboxylic acid 2c (150 mg,
0.41 mmol) in THF (3 mL) was added a solution of 3 (100 mg, 0.40
mmol) in THF (1 mL), HATU (228 mg, 0.60 mmol) and Et3N (275
μL, 2.0 mmol). The reaction mixture was stirred at room temperature
for 14 h. It was then diluted with EtOAc (40 mL) and saturated
aqueous NaHCO3 solution (40 mL). The aqueous layer was extracted
with EtOAc (40 mL). The combined organic layers were washed with
brine (40 mL) and dried over anhydrous MgSO4. After concentration
in vacuo, the residue was purified by column chromatography (NH
silica gel, eluted with 5−80% EtOAc in n-hexane) to give the free base
of 1c as oil. The obtained oily material was dissolved in EtOAc (1.0
mL) and treated with 4 M HCl in EtOAc (1.0 mL, 4.0 mmol) at room
temperature. After being stirred for 15 min, the mixture was diluted
with Et2O (5 mL). The resulting precipitate was collected by filtration
and washed with Et2O to give 1c (102 mg, 38%) as a colorless solid.
1
(ESI+): 563.1 [M + H]+. H NMR (300 MHz, CDCl3) δ 2.04−2.20
(6H, m), 2.38−2.48 (2H, m), 3.41 (2H, m), 4.65−4.80 (2H, m),
6.84−6.99 (1H, m), 7.02−7.19 (5H, m), 7.27−7.35 (1H, m), 7.38
(1H, dd, J = 8.5, 5.3 Hz), 7.44−7.51 (1H, m), 7.62−7.72 (1H, m).
Anal. Calcd for C25H22BrCl2FN4O: C, 53.21; H, 3.93; N, 9.93. Found:
C, 53.44; H, 3.96; N, 10.04.
1
MS (ESI+): 597.0 [M + H]+. H NMR (300 MHz, DMSO-d6) δ
2.64−2.88 (6H, m), 3.05−3.24 (1H, m), 3.42−3.72 (3H, m), 4.52−
4.88 (2H, m), 6.82−7.99 (9H, m), 10.06−10.72 (1H, m). Anal. Calcd
for C25H21BrCl3FN4O·2HCl: C, 44.71; H, 3.45; N, 8.34. Found: C,
44.86; H, 3.73; N, 8.32.
5-Bromo-3-(2-chloro-4-fluorophenyl)-N-(3,4-dichlorobenzyl)-N-
[2-(dimethylamino)ethyl]imidazo[1,2-a]pyridine-2-carboxamide
Hydrochloride (1b). To a solution of 5-bromo-3-(2-chloro-4-
fluorophenyl)imidazo[1,2-a]pyridine-2-carboxylic acid 2b (185 mg,
0.50 mmol) and 3 (124 mg, 0.50 mmol) in a mixed solvent of THF (2
mL) and pyridine (1 mL) was added HATU (228 mg, 0.60 mmol),
and the reaction mixture was stirred at 70 °C for 1 h. The reaction
mixture was cooled to room temperature and quenched with saturated
aqueous NaHCO3 solution (20 mL). The resulting mixture was
extracted with EtOAc (2 × 20 mL). The combined organic layers were
washed with brine (20 mL) and dried over anhydrous MgSO4. After
concentration in vacuo, the residue was purified by column
chromatography (NH silica gel, eluted with 30−100% EtOAc in n-
hexane) to give the free base of 1b as oil. The obtained oily material
was dissolved into Et2O (10 mL) and treated with 4 M HCl in EtOAc
(0.2 mL, 0.80 mmol) at room temperature. Then the resulting
precipitate was collected by filtration and washed with Et2O to give 1b
(263 mg, 83%) as a colorless amorphous powder. MS (ESI+): 597.0
5-Bromo-N-(3,4-dichlorobenzyl)-3-(3,4-difluorophenyl)-N-[2-
(dimethylamino)ethyl]imidazo[1,2-a]pyridine-2-carboxamide (1d).
Compound 1d was prepared from 5-bromo-3-(3,4-difluorophenyl)-
imidazo[1,2-a]pyridine-2-carboxylic acid 2d (144 mg, 0.41 mmol)
using 3 (89 mg, 0.36 mmol) by a similar method to that described for
1a. Yield: 71%, colorless amorphous solid (170 mg). MS (ESI+):
1
581.0 [M + H]+. H NMR (300 MHz, DMSO-d6) δ 1.86−2.11 (6H,
m), 2.15−2.42 (2H, m), 3.18−3.63 (2H, m), 4.49−4.71 (2H, m),
7.05−7.89 (9H, m). Anal. Calcd for C25H21BrCl2F2N4O: C, 51.57; H,
3.64; N, 9.62. Found: C, 51.73; H, 3.80; N, 9.40.
5-Bromo-N-(3,4-dichlorobenzyl)-N-[2-(dimethylamino)ethyl]-3-
(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carboxamide
Dihydrochloride (1e). Compound 1e was prepared from 5-bromo-3-
(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carboxylic acid 2e
(300 mg, 0.86 mmol) using 3 (212 mg, 0.86 mmol) by a similar
method to that described for 1b. Yield: 83%, colorless amorphous
powder (437 mg). MS (ESI+): 577.1 [M + H]+. 1H NMR (300 MHz,
1
[M + H]+. H NMR (300 MHz, DMSO-d6) δ 2.65−2.83 (6H, m),
3.09−3.88 (4H, m), 4.54−5.10 (2H, m), 7.08−7.94 (9H, m), 10.27−
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DMSO-d6) δ 2.20−2.28 (3H, m), 2.74 (6H, t, J = 4.2 Hz), 3.03−3.76
(4H, m), 4.48−4.85 (2H, m), 6.88−7.46 (7H, m), 7.46−7.72 (1H, m),
7.69−7.92 (1H, m), 9.74−10.25 (1H, m). Anal. Calcd for
C26H24BrCl2FN4O·2HCl·1H2O: C, 46.66; H, 4.22; N, 8.37. Found:
C, 46.68; H, 4.24; N, 8.49.
2.26−2.38 (3H, m), 2.63−2.79 (3H, m), 4.29 (2H, q, J = 7.1 Hz,
CH2), 6.65−6.74 (1H, m), 6.98−7.11 (1H, m), 7.13−7.35 (3H, m),
7.69−7.75 (1H, m).
To a mixture of 9g and 10g (107 mg) in a mixed solvent of MeOH
(2 mL) and THF (2 mL) was added 1 M NaOH (1 mL), and the
reaction mixture was stirred at 60 °C for 1 h. The reaction mixture was
cooled to room temperature and diluted with water (20 mL). The
aqueous layer was extracted with EtOAc (2 × 30 mL). The combined
organic layers were washed with brine (30 mL) and dried over
anhydrous MgSO4. After concentration in vacuo, the obtained crude
material of 5-[(tert-butoxycarbonyl) (methyl)amino]-3-(4-fluoro-3-
methylphenyl)imidazo[1,2-a]pyridine-2-carboxylic acid 2g was dis-
solved in THF (3 mL). To the solution were successively added 3 (64
mg, 0.26 mmol), HATU (119 mg, 0.31 mmol), and pyridine (0.5 mL).
The reaction mixture was stirred at room temperature overnight. The
reaction mixture was quenched with saturated aqueous NaHCO3
solution (20 mL) and extracted with EtOAc (2 × 30 mL). The
combined organic layers were washed with brine (30 mL) and dried
over anhydrous MgSO4. After concentration in vacuo, the residue was
purified by column chromatography (NH silica gel, eluted with 30−
100% EtOAc in n-hexane) to give 1g′ (117 mg, 16% in 4 steps from
5-Bromo-N-(3,4-dichlorobenzyl)-N-[2-(dimethylamino)ethyl]-3-
(4-fluoro-3-methoxyphenyl)imidazo[1,2-a]pyridine-2-carboxamide
Dihydrochloride (1f). Compound 1f was prepared from 5-bromo-3-
(4-fluoro-3-methoxyphenyl)imidazo[1,2-a]pyridine-2-carboxylic acid
2f (60 mg, 0.20 mmol) using 3 (55 mg, 0.20 mmol) by a similar
method to that described for 1b. Yield: 45%, colorless amorphous
1
powder (53 mg). MS (ESI+): 593.1 [M + H]+. H NMR (300 MHz,
DMSO-d6) δ 2.72 (6H, d, J = 4.5 Hz), 2.94−3.69 (4H, m), 3.77 (3H,
s), 4.57−4.75 (2H, m), 6.89−7.14 (2H, m), 7.20−7.44 (5H, m), 7.46−
7.65 (1H, m), 7.70−7.95 (1H, m), 9.94−10.64 (1H, m). Anal. Calcd
for C26H24BrCl2FN4O2·2HCl·1.5H2O: C; 44.98, H; 4.21, N; 8.07.
Found: C; 44.92, H; 4.34, N; 8.06.
tert-Butyl [2-{(3,4-Dichlorobenzyl)[2-(dimethylamino)ethyl]-
carbamoyl}-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridin-5-
yl]methylcarbamate (1g′). To a solution of 4e (691 mg, 5.0 mmol)
in THF (15 mL) was added 5 (858 g, 6.0 mmol), and the mixture was
cooled to −78 °C. To the mixture was slowly added sodium
methoxide (324 mg, 6.0 mmol) at −78 °C. The reaction mixture was
warmed to room temperature and stirred for 30 min. It was then
concentrated under reduced pressure. The residue was diluted with
water (40 mL) and extracted with EtOAc/Et2O (1:1, 40 mL). The
organic layer was washed with brine (30 mL) and dried over
anhydrous MgSO4. After concentration in vacuo, the oily residue was
dissolved in EtOH (20 mL). To the solution was added tert-butyl (6-
aminopyridin-2-yl)carbamate 7 (1.05 g, 5.0 mmol), and the reaction
mixture was refluxed at 90 °C for 4 h. The reaction mixture was cooled
at room temperature and concentrated under reduced pressure. The
residue was diluted with saturated aqueous NaHCO3 solution (50
mL), and the mixture was extracted with EtOAc (2 × 50 mL). The
combined organic layers were washed with brine (50 mL) and dried
over anhydrous MgSO4. After concentration in vacuo, the residue was
purified by column chromatography (NH silica gel, eluted with 10−
100% EtOAc in n-hexane) to give a mixture of methyl 5-[(tert-
butoxycarbonyl)amino]-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]-
pyridine-2-carboxylate 9g′ and ethyl 5-[(tert-butoxycarbonyl)amino]-
3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carboxylate 10g′
(514 mg) as a yellow amorphous powder. This material was used for
the next reaction without further purification. The ratio of 9g′/10g′
(ca. 7:3) was determined by 1H NMR. For 9g′: MS (ESI+) 400.2 [M
1
4e) as a colorless oil. MS (ESI+): 628.3 [M + H]+. H NMR (300
MHz, CDCl3) δ 1.28−1.42 (9H, m), 1.99−2.72 (14H, m), 3.26−3.63
(2H, m), 4.53−4.87 (2H, m), 6.50−6.78 (1H, m), 6.85−7.48 (7H, m),
7.57−7.66 (1H, m).
N-(3,4-Dichlorobenzyl)-N-[2-(dimethylamino)ethyl]-3-(4-fluoro-
3-methylphenyl)-5-(methylamino)imidazo[1,2-a]pyridine-2-carbox-
amide Dihydrochloride (1g). A solution of 1g′ (117 mg, 0.19 mmol)
in TFA (0.5 mL) was stirred at room temperature for 1 h. The
reaction mixture was quenched with saturated aqueous NaHCO3
solution (20 mL) and extracted with EtOAc (2 × 30 mL). The
combined organic layers were washed with brine (30 mL) and dried
over anhydrous MgSO4. After concentration in vacuo, the residue was
dissolved in a mixed solvent of Et2O (4 mL) and EtOAc (1 mL). To
the solution was added 4 M HCl in EtOAc (0.2 mL, 0.8 mmol). The
reaction mixture was diluted with Et2O (5 mL). The resulting
precipitate was collected by filtration and washed with Et2O to give 1g
(80 mg, 71%) as a colorless powder. MS (ESI+): 528.2 [M + H]+. 1H
NMR (300 MHz, DMSO-d6) δ 2.22−2.30 (3H, m), 2.67−2.87 (9H,
m), 3.10−3.70 (4H, m), 4.16−4.92 (4H, m), 6.04−6.31 (1H, m),
7.01−7.95 (8H, m), 9.57−10.82 (1H, m). Anal. Calcd for
C27H28Cl2FN5O·2HCl·2.8H2O: C, 49.75; H, 5.51; N, 10.74. Found:
C, 49.86; H, 5.31; N, 10.74.
N-(3,4-Dichlorobenzyl)-N-[2-(dimethylamino)ethyl]-3-(4-fluoro-
3-methylphenyl)-5-methoxyimidazo[1,2-a]pyridine-2-carboxamide
(1h). To a solution of 1e (148 mg, 0.26 mmol) in DMF (1.2 mL) was
added sodium methoxide (28 mg, 0.51 mmol). The reaction mixture
was stirred at room temperature for 12 h. The reaction mixture was
partitioned between EtOAc (15 mL) and water (15 mL), and the
aqueous layer was extracted with EtOAc (2 × 10 mL). The combined
organic layers were washed with water (2 × 10 mL) and brine (15
mL) and dried over anhydrous MgSO4. After concentration in vacuo,
the residue was purified by column chromatography (NH silica gel,
eluted with 20−85% EtOAc in n-hexane) to give 1h (68 mg, 49%) as a
purple oil. MS (ESI+): 529.1 [M + H]+. 1H NMR (300 MHz, CDCl3)
δ 2.01−2.19 (6H, m), 2.29 (3H, br s), 2.32−2.50 (2H, m), 3.32−3.49
(2H, m), 3.73 (3H, d, J = 9.6 Hz), 4.66 (2H, d, J = 4.2 Hz), 5.96−6.06
(1H, m), 6.85−7.09 (3H, m), 7.15−7.24 (3H, m), 7.27−7.34 (2H, m).
Anal. Calcd for C27H27Cl2FN4O2·1.5H2O: C, 58.28; H, 5.43; N, 10.07.
Found: C, 58.19; H, 5.31; N, 9.87.
N-(3,4-Dichlorobenzyl)-N-[2-(dimethylamino)ethyl]-5-ethynyl-3-
(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carboxamide
Dihydrochloride (1i). Compound 1i was prepared from 5-ethynyl-3-
(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carboxylic acid 2i
(55.0 mg, 0.187 mmol) and 3 (42 mg, 0.17 mmol) by a similar method
to that described for 1c. Yield: 77%, colorless amorphous solid (78
mg). MS (ESI+): 523.2 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ
2.25 (3H, d, J = 0.8 Hz), 2.67−2.82 (6H, m), 3.10−3.78 (4H, m),
4.55−4.65 (2H, m), 4.74−4.81 (1H, m), 7.06−7.46 (7H, m), 7.50−
7.66 (1H, m), 7.73−7.91 (1H, m), 9.99−10.50 (1H, m). Anal. Calcd
1
+ H]+. H NMR (300 MHz, CDCl3) δ 1.36 (9H, s), 2.37 (3H, d, J =
1.5 Hz), 3.86 (3H, s, CH3), 6.49 (1H, s), 7.14−7.24 (2H, m), 7.28−
7.41 (3H, m), 7.45−7.53 (1H, m). For 10g′ MS (ESI+): 414.2 [M +
1
H]+. H NMR (300 MHz, CDCl3) δ 1.29 (3H, t, J = 7.2 Hz, CH3),
1.36 (9H, s), 2.37 (3H, d, J = 1.5 Hz), 4.31 (2H, q, J = 7.2 Hz, CH2),
6.49 (1H, s), 7.10−7.23 (2H, m), 7.28−7.39 (3H, m), 7.46−7.53 (1H,
m).
To a mixture of 9g′ and 10g′ (121 mg) in DMF (2 mL) were
successively added 60% sodium hydride in mineral oil (14 mg, 0.36
mmol) and iodomethane (37 μL, 0.60 mmol) at room temperature.
The reaction mixture was stirred at room temperature for 2 h. It was
then quenched with water (20 mL) and extracted with EtOAc (2 × 30
mL). The combined organic layers were washed with brine (30 mL)
and dried over anhydrous MgSO4. After concentration in vacuo, the
residue was purified by column chromatography (silica gel, eluted with
10−100% EtOAc in n-hexane) to give a mixture of methyl 5-[(tert-
butoxycarbonyl) (methyl)amino]-3-(4-fluoro-3-methylphenyl)-
imidazo[1,2-a]pyridine-2-carboxylate 9g and ethyl 5-[(tert-butoxycar-
bonyl) (methyl)amino]-3-4-(fluoro-3-methylphenyl)imidazo[1,2-a]-
pyridine-2-carboxylate 10g (107 mg) as a yellow amorphous powder.
1
The ratio of 9g/10g (ca. 7:3) was determined by H NMR. For 9g:
1
MS (ESI+) 414.2 [M + H]+. H NMR (300 MHz, CDCl3) δ 1.28−
1.34 (9H, m), 2.26−2.38 (3H, m), 2.48−2.62 (3H, m), 3.85 (3H, s,
CH3), 6.58 (1H, d, J = 6.6 Hz), 6.98−7.12 (1H, m), 7.13−7.37 (3H,
1
m), 7.65−7.72 (1H, m). 10g: MS (ESI+) 428.2 [M + H]+. H NMR
(300 MHz, CDCl3) δ 1.21−1.29 (3H, m, CH3), 1.28−1.43 (9H, m),
K
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for C28H25Cl2FN4O·2HCl·0.8H2O: C, 55.06; H, 4.72; N, 9.17. Found:
C, 55.11; H, 4.68; N, 9.10.
bromopyridin-2-amine 6 (318 mg, 1.8 mmol), and the reaction
mixture was refluxed at 90 °C for 14 h. It was then cooled to room
temperature and concentrated under reduced pressure. The residue
was suspended in Et2O (5.0 mL). Insoluble product was collected by
filtration and washed with Et2O (5.0 mL) to give a crude mixture of
methyl 5-bromo-3-(3-chloro-4-fluorophenyl)imidazo[1,2-a]pyridine-
2-carboxylate hydrochloride 9c and ethyl 5-bromo-3-(3-chloro-4-
fluorophenyl)imidazo[1,2-a]pyridine-2-carboxylate hydrochloride
10c. For 9c: MS (ESI+) 383.0 [M + H]+. For 10c: MS (ESI+)
397.0 [M + H]+.
5-Cyclopropyl-N-(3,4-dichlorobenzyl)-N-[2-(dimethylamino)-
ethyl]-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carbox-
amide Dihydrochloride (1j). Compound 1j was prepared from 5-
cyclopropyl-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-car-
boxylic acid 2j (85 mg, 0.275 mmol) using 3 (61.8 mg, 0.25 mmol) by
a similar method to that described for 1c. Yield: 54%, colorless
1
amorphous solid (83 mg). MS (ESI+): 539.2 [M + H]+. H NMR
(300 MHz, DMSO-d6) δ 0.24−0.56 (2H, m), 0.54−0.84 (2H, m),
1.37−1.73 (1H, m), 2.24 (3H, s), 2.68−2.78 (6H, m), 3.06−3.24 (1H,
m), 3.42−3.72 (3H, m), 4.58−4.82 (2H, m), 6.78−7.81 (9H, m),
10.19−10.92 (1H, m). Anal. Calcd for C29H29Cl2FN4O·2HCl·0.5H2O:
C, 56.05; H, 5.19; N, 9.02. Found: C, 55.89; H, 5.41; N, 8.78.
5-Cyano-N-(3,4-dichlorobenzyl)-N-[2-(dimethylamino)ethyl]-3-
(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carboxamide
Dihydrochloride (1k). Compound 1k was prepared from 5-cyano-3-
(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carboxylic acid 2k
(62.0 mg, 0.21 mmol) using 3 (48.0 mg, 0.195 mmol) by a similar
method to that described for 1c. Yield: 71%, colorless amorphous solid
(83 mg). MS (ESI+): 524.1 [M + H]+. 1H NMR (300 MHz, DMSO-
d6) δ 2.27 (3H, d, J = 1.3 Hz), 2.68−2.79 (6H, m), 3.14−3.25 (1H,
m), 3.39−3.74 (3H, m), 4.57−4.86 (2H, m), 7.08−7.68 (7H, m),
7.79−7.95 (1H, m), 8.02−8.24 (1H, m), 9.75−10.48 (1H, m). Anal.
Calcd for C27H24Cl2FN5O·2HCl·0.5H2O: C, 53.48; H, 4.49; N, 11.55.
Found: C, 53.73; H, 4.59; N, 11.29.
5-Bromo-3-(4-fluorophenyl)imidazo[1,2-a]pyridine-2-carboxylic
Acid (2a). To a solution of methyl 5-bromo-3-(4-fluorophenyl)-
imidazo[1,2-a]pyridine-2-carboxylate hydrochloride 9a (1.2 g, 3.44
mmol) in a mixed solvent of EtOH (6.0 mL) and THF (4.0 mL) was
added 2 M NaOH (7.0 mL). The reaction mixture was stirred at 65 °C
for 30 min. It was then cooled to room temperature and concentrated
under reduced pressure. The residue was diluted with water (100 mL)
and 6 M HCl (2.5 mL), and the resulting precipitate was collected by
filtration and washed with water to give 2a (940 mg, 91%) as colorless
solid. MS (ESI+): 335.0 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ
7.18−7.32 (4H, m), 7.44−7.56 (2H, m), 7.70−7.81 (1H, m), 12.63
(1H, br s).
5-Bromo-3-(2-chloro-4-fluorophenyl)imidazo[1,2-a]pyridine-2-
carboxylic Acid (2b). To a solution of methyl 5-bromo-3-(2-chloro-4-
fluorophenyl)imidazo[1,2-a]pyridine-2-carboxylate 9b (483 mg, 1.26
mmol) in a mixed solvent of MeOH (6.0 mL) and THF (6.0 mL) was
added 2 M lithium hydroxide (3.0 mL, 6.0 mmol), and the reaction
mixture was stirred at 40 °C for 1 h. The reaction mixture was cooled
at room temperature and concentrated under reduced pressure. The
residue was treated with 1 M HCl (10 mL) and extracted with EtOAc/
THF (4:1, 2 × 15 mL). The combined organic layers were washed
with brine (20 mL) and dried over anhydrous MgSO4. After
concentration in vacuo, the residue was washed with Et2O to give
2b (456 mg, 98%) as a colorless solid. MS (ESI+): 368.9 [M + H]+.
1H NMR (300 MHz, DMSO-d6) δ 7.26−7.37 (3H, m), 7.58 (1H, dd, J
The obtained mixture was dissolved in a mixed solvent of THF (2.0
mL) and MeOH (2.0 mL), and the mixture was warmed to 60 °C. To
the mixture was added 2 M NaOH (2.0 mL, 4 mmol). The reaction
mixture was stirred at 60 °C for 1 h. It was then cooled to room
temperature and diluted with EtOAc (30 mL) and water (30 mL). The
aqueous layer was washed with EtOAc (2 × 30 mL) and acidified with
2 M HCl (2 mL, 4.0 mmol) to pH 4. The aqueous layer was extracted
with EtOAc (2 × 30 mL). The combined organic layers were washed
with brine (30 mL) and dried over anhydrous MgSO4. The solvent
was removed under reduced pressure to give the desired compound 2c
(284 mg, 13% in 3 steps from 4c) as a yellow solid. MS (ESI+): 369.0
1
[M + H]+. H NMR (300 MHz, DMSO-d6) δ 7.22−7.66 (4H, m),
7.68−7.85 (2H, m), 12.76 (1H, br s).
5-Bromo-3-(3,4-difluorophenyl)imidazo[1,2-a]pyridine-2-carbox-
ylic Acid (2d). Compound 2d was prepared from methyl 5-bromo-3-
(3,4-difluorophenyl)imidazo[1,2-a] pyridine-2-carboxylate 9d (1.41 g,
3.84 mmol) by a similar method to that described for 2b. Yield 92%,
1
colorless oil (1.25 g). MS (ESI+): 353.0 [M + H]+. H NMR (300
MHz, DMSO-d6) δ 7.24−7.38 (3H, m), 7.48 (1H, dt, J = 11.0, 8.5
Hz), 7.67 (1H, ddd, J = 11.5, 7.9, 2.1 Hz), 7.72−7.82 (1H, m), 12.69
(1H, br s).
5-Bromo-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-
carboxylic Acid (2e). Compound 2e was prepared from methyl 5-
bromo-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2- carbox-
ylate 9e (2.0 g, 5.50 mmol) by a similar method to that described for
2b. Yield: 71%, beige crystalline solid (1.46 g); mp 211 °C
(decomposition). MS (ESI+): 349.0 [M + H]+. 1H NMR (300
MHz, DMSO-d6) δ 2.26 (3H, d, J = 1.5 Hz), 6.87−7.59 (5H, m),
7.54−8.01 (1H, m), 12.59 (1H, br s).
5-Bromo-3-(4-fluoro-3-methoxyphenyl)imidazo[1,2-a]pyridine-2-
carboxylic Acid (2f). To a solution of 4-fluoro-3-methoxybenzalde-
hyde 4f (1.5 g, 9.73 mmol) was added 5 (1.46 g, 10.2 mmol). The
reaction mixture was cooled to −30 °C. To the mixture was added
sodium methoxide (525 mg, 9.72 mmol), and the reaction mixture was
stirred at −30 °C for 10 min. The reaction mixture was gradually
warmed to room temperature over 30 min and stirred at the same
temperature for 6 h. The mixture was concentrated under reduced
pressure, and the oily residue was partitioned between EtOAc (100
mL) and water (100 mL). The organic layer was washed with brine
(100 mL) and dried over anhydrous MgSO4. The solvent was removed
under reduced pressure to give methyl 3-chloro-3-(4-fluoro-3-
methoxyphenyl)-2-oxopropanoate 4f′ as yellow oil. The obtained
material 4f′ was dissolved in EtOH (20 mL). To the solution was
added 6 (1.68 g, 9.73 mmol), and the reaction mixture was refluxed at
90 °C for 12 h. The reaction mixture was cooled to room temperature
and concentrated under reduced pressure. The residue was partitioned
between EtOAc (100 mL) and brine (100 mL), and the organic layer
was dried over anhydrous MgSO4 and concentrated in vacuo. The
residue was purified by column chromatography (silica gel, eluted with
33−100% EtOAc in n-hexane) to give a crude mixture of methyl 5-
bromo-3-(4-fluoro-3-methoxyphenyl)imidazo[1,2-a]pyridine-2-carbox-
ylate 9f and ethyl 5-bromo-3-(4-fluoro-3-methoxyphenyl)imidazo[1,2-
a]pyridine-2-carboxylate 10f. For 9f: MS (ESI+) 379.0 [M + H]+. For
10f: MS (ESI+) 393.0 [M + H]+.
= 8.9, 2.6 Hz), 7.65 (1H, dd, J = 8.6, 6.3 Hz), 7.75−7.83 (1H, m),
12.74 (1H, br s).
5-Bromo-3-(3-chloro-4-fluorophenyl)imidazo[1,2-a]pyridine-2-
carboxylic Acid (2c). To a solution of 3-chloro-4-fluorobenzaldehyde
4c (945 mg, 6.0 mmol) in THF (6.0 mL) was added methyl
dichloroacetate 5 (1.07 g, 7.5 mmol). The reaction mixture was cooled
to 0 °C. To the mixture was carefully added sodium methoxide (405
mg, 7.5 mmol) at 0 °C, and the reaction mixture was stirred at 0 °C for
10 min. The reaction mixture was stirred at room temperature for 1 h.
The reaction mixture was warmed to 80 °C and refluxed for 1 h at 80
°C. The reaction mixture was cooled to room temperature and
quenched with water (30 mL). The resulting mixture was extracted
with EtOAc (2 × 40 mL). The combined organic layers were washed
with brine (30 mL) and dried over anhydrous MgSO4. After
concentration in vacuo, the residue was purified by column
chromatography (silica gel, eluted with 0−50% EtOAc in n-hexane)
to give a crude material of methyl 3-chloro-3-(3-chloro-4-fluorophen-
yl)-2-oxopropanoate 4c′. The obtained crude material 4c′ was
dissolved in EtOH (4.5 mL). To the resulting solution was added 6-
The obtained mixture was dissolved in a mixed solvent of THF (15
mL) and MeOH (3.0 mL). To the solution was added 2 M lithium
hydroxide (6.0 mL, 12 mmol). The reaction mixture was stirred at 45
°C for 4 h. The mixture was concentrated under reduced pressure. The
obtained residue was treated with 2 M HCl (7.0 mL, 14 mmol). The
resulting precipitate was collected by filtration and washed with water.
L
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Drying the solid gave 2f (1.02 g, 29% in three steps from 4f) as a
colorless crystalline solid; mp 214−217 °C. MS (ESI+): 365.0 [M +
H]+. 1H NMR (300 MHz, DMSO-d6) δ 3.80 (3H, s), 7.04 (1H, ddd, J
= 8.3, 4.5, 2.1 Hz), 7.13−7.52 (4H, m), 7.65−8.10 (1H, m).
stirred at room temperature for 50 min. The reaction mixture was
warmed up to 85 °C and stirred at the same temperature for 2 h. It was
then cooled to room temperature and partitioned between EtOAc (90
mL) and water (60 mL). The organic layer was washed with brine (30
mL) and dried over anhydrous MgSO4. After concentration in vacuo,
the residue was purified by column chromatography (silica gel, eluted
with 0−60% EtOAc in n-hexane) to give methyl 3-chloro-3-(4-
5-Ethynyl-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-
carboxylic Acid (2i). Compound 2i was prepared from methyl 5-
ethynyl-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carbox-
ylate 9i (58.0 mg, 0.187 mmol) by a similar method to that described
for 2b. Yield: 94%, yellow solid (51.8 mg). MS (ESI+): 295.1 [M +
H]+. 1H NMR (300 MHz, DMSO-d6) δ 2.26 (3H, d, J = 1.3 Hz), 4.50
(1H, s), 7.04−7.42 (5H, m), 7.73−7.81 (1H, m), 12.46 (1H, br s).
5-Cyclopropyl-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]-
pyridine-2-carboxylic Acid (2j). To a solution of 4-fluoro-3-
methylbenzaldehyde 4e (4.00 g, 29.0 mmol) in THF (45 mL) was
added 5 (1.46 g, 10.2 mmol). The mixture was cooled to −78 °C. To
the mixture was added sodium tert-butoxide (3.34 g, 34.8 mmol), and
the mixture was stirred at −78 °C for 20 min. The mixture was stirred
at room temperature for 3 h. It was then concentrated under reduced
pressure, and the residue was poured into water (50 mL). The
resulting mixture was extracted with EtOAc (50 mL). The organic
layer was washed with brine (30 mL) and dried over anhydrous
MgSO4. After concentration in vacuo, the residue was purified by
column chromatography (silica gel, eluted with 0−50% EtOAc in n-
hexane) to give a crude material of methyl 3-chloro-3-(4-fluoro-3-
methylphenyl)-2-oxopropanoate 4e′ as yellow oil. To a solution of 6-
cyclopropylpyridin-2-amine (8, 1.03 g, 7.70 mmol) in EtOH (6.0 mL)
was added a solution of the obtained crude material 4e′ in EtOH (2.0
mL) at room temperature. The reaction mixture was stirred at 80 °C
for 17 h under N2 atmosphere. After removal of solvents under
reduced pressure, the residue was diluted with EtOAc (30 mL) and
saturated aqueous NaHCO3 solution (30 mL) and the aqueous layer
was extracted with EtOAc (3 × 20 mL). The combined organic layers
were washed with brine (30 mL) and dried over anhydrous MgSO4.
After concentration in vacuo, the residue was purified by column
chromatography (silica gel, eluted with 33−100% EtOAc in n-hexane)
and the desired fraction was concentrated in vacuo. The obtained solid
was triturated in n-hexane/EtOAc (10:1). The resulting precipitate
was collected by filtration and washed with n-hexane/EtOAc (10:1) to
give a crude mixture (1.39 g) of methyl 5-cyclopropyl-3-(4-fluoro-3-
methylphenyl)imidazo[1,2-a]pyridine-2-carboxylate 9j and ethyl 5-
cyclopropyl-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-car-
boxylate 10j. For 9j: MS (ESI+) 325.1 [M + H]+. For 10j: MS (ESI+)
339.1 [M + H]+.
The crude mixture was dissolved in a mixed solvent of THF (6.0
mL) and MeOH (6.0 mL), and the solution was warmed to 60 °C. To
the solution was added 2 M NaOH (8 mL, 16.0 mmol), and the
reaction mixture was stirred at 60 °C for 40 min. The reaction mixture
was cooled at room temperature and concentrated under reduced
pressure. The residue was neutralized with 6 M HCl (2 mL, 12 mmol)
and 2 M HCl (2.0 mL, 4.0 mmol) at 0 °C. The resulting precipitate
was collected by filtration and washed with water. Drying the solid
under vacuum at 60 °C gave 2j (1.21 g, 25% yield in 3 steps from 4e)
as a colorless crystalline solid; mp 208−210 °C. MS (ESI+): 311.1 [M
+ H]+. 1H NMR (300 MHz, DMSO-d6) δ 0.30−0.42 (2H, m), 0.60−
0.70 (2H, m), 1.37−1.54 (1H, m), 2.26 (3H, s), 6.71 (1H, d, J = 7.0
Hz), 7.14 (1H, dd, J = 9.8, 8.3 Hz), 7.25 (1H, dd, J = 9.0, 6.9 Hz),
7.34−7.48 (2H, m), 7.55 (1H, d, J = 8.9 Hz).
5-Cyano-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-
carboxylic Acid (2k). Compound 2k was prepared from methyl 5-
cyano-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]pyridine-2-carboxy-
late 9k (96 mg, 0.31 mmol) by a similar method to that described for
2b. Yield: 69%, colorless solid (63.0 mg). MS (ESI+): 296.1 [M + H]+.
1H NMR (300 MHz, DMSO-d6) δ 2.28 (3H, s), 7.21−7.31 (1H, m),
1
fluorophenyl)-2-oxopropanoate 4a′ (6.58 g, 70%) as a yellow oil. H
NMR (300 MHz, CDCl3) δ 3.86 (3H, s), 6.14 (1H, s), 7.09 (2H, t, J =
8.6 Hz), 7.36−7.47 (2H, m).
To a solution of 6 (1.0 g, 5.85 mmol) in EtOH (5 mL) was added a
solution of 4a′ (1.95 g, 8.47 mmol) in EtOH (3 mL), and the reaction
mixture was refluxed at 90 °C for 12 h. The reaction mixture was
cooled to room temperature and concentrated under reduced pressure
until volume of the solution became ca. 3.0 mL. The residue was
diluted with EtOAc (4.0 mL). The resulting precipitate was collected
by filtration and washed with EtOAc to give 9a (1.20 g, 53%) as a
colorless crystalline solid; mp 192−194 °C. MS (ESI+): 349.0 [M +
H]+. 1H NMR (300 MHz, DMSO-d6) δ 3.66 (3H, s), 7.23−7.32 (2H,
m), 7.34−7.41 (2H, m), 7.48−7.59 (2H, m), 7.76−7.85 (1H, m).
Methyl 5-Bromo-3-(2-chloro-4-fluorophenyl)imidazo[1,2-a]-
pyridine-2-carboxylate (9b). To a solution of 2-chloro 4-fluoroben-
zaldehyde 4b (1.59 g, 10 mmol) in THF (30 mL) was added 5 (2.86 g,
20 mmol), and the mixture was cooled to −78 °C. To the mixture was
carefully added sodium tert-butoxide (1.92 g, 20 mmol) at −78 °C,
and the reaction mixture was stirred at −78 °C for 30 min. It was then
warmed up to room temperature and stirred at room temperature for 3
h. The mixture was partitioned between EtOAc (50 mL) and water
(50 mL), and the separated aqueous layer was extracted with EtOAc
(50 mL). The combined organic layers were washed with brine (50
mL) and dried over anhydrous MgSO4. After concentration in vacuo,
the residue was purified by column chromatography (silica gel, eluted
with 1−10% EtOAc in n-hexane) to give 2-chloro-3-(2-chloro-4-
fluorophenyl)oxirane-2-carboxylate 4b″ (2.53 g, 96%) as a colorless
1
oil. H NMR (300 MHz, CDCl3) δ 3.94 (3H, s), 4.65 (1H, s), 6.99−
7.10 (1H, m), 7.14−7.21 (1H, m), 7.29−7.39 (1H, m).
To a solution of 4b″ (2.17 g, 8.19 mmol) in THF (20 mL) was
added magnesium bromide at room temperature. The reaction mixture
was stirred at 80 °C for 30 min. It was then cooled to room
temperature, and the insoluble material was removed by filtration. The
filtrate was partitioned between EtOAc (30 mL) and water (30 mL),
and the organic layer was washed with brine (30 mL), dried over
anhydrous MgSO4, and concentrated in vacuo to give methyl 3-
bromo-3-(2-chloro-4-fluorophenyl)-2-oxopropanoate 4b′ as oil. To
the oily residue 4b′ was added a solution of 6 (1.42 g, 8.19 mmol) in
EtOH (20 mL), and the reaction mixture was stirred at 80 °C
overnight. The reaction mixture was cooled to room temperature and
quenched with saturated aqueous NaHCO3 solution (50 mL). The
mixture was extracted with EtOAc (2 × 50 mL). The combined
organic layers were washed with brine (50 mL), dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography (silica gel, eluted with 40−100% EtOAc in n-
hexane) to give 9b (481 mg, 15% in 3 steps from 4b) as a colorless
1
amorphous powder. MS (ESI+): 383.0 [M + H]+. H NMR (300
MHz, CDCl3) δ 3.86 (3H, s), 7.05−7.28 (4H, m), 7.44 (1H, dd, J =
8.6, 5.9 Hz), 7.77 (1H, dd, J = 8.9, 1.1 Hz).
Methyl 5-Bromo-3-(3,4-difluorophenyl)imidazo[1,2-a]pyridine-2-
carboxylate (9d). To a solution of 5 (10 g, 70.3 mmol) in THF (100
mL) was added a solution of 3,4-difluorobenzaldehyde 4d (10.5 g, 73.4
mmol) in THF (20 mL), and the mixture was cooled to 0 °C. To the
mixture was carefully added sodium methoxide (3.8 g, 70.3 mmol) at 0
°C. The reaction mixture was stirred at room temperature for 2 h. It
was then concentrated under reduced pressure. The residue was
partitioned between EtOAc (200 mL) and water (100 mL). The
organic layer was washed with brine (100 mL) and dried over
anhydrous MgSO4. After concentration in vacuo, the residue was
purified by column chromatography (silica gel, eluted with 20−60%
EtOAc in n-hexane) to give a crude material of methyl 3-chloro-3-(3,4-
difluorophenyl)-2-oxopropanoate 4d′. A part of the residue 4d′ (5.0 g,
20 mmol) was dissolved in EtOH (50 mL). To the mixture was added
7.36−7.56 (3H, m), 7.82 (1H, d, J = 7.0 Hz), 8.05−8.15 (1H, m).
Methyl 5-Bromo-3-(4-fluorophenyl)imidazo[1,2-a]pyridine-2-car-
boxylate Hydrochloride (9a). To a solution of 4-fluorobenzaldehyde
4a (5.1 g, 41.1 mmol) in THF (40 mL) was added 5 (7.6 g, 53.2 mol),
and the mixture was cooled to 0 °C. To the mixture was carefully
added sodium methoxide (2.9 g, 53.7 mmol) at 0 °C. The reaction
mixture was stirred at 0 °C for 10 min. The reaction mixture was
M
DOI: 10.1021/acs.jmedchem.5b00836
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
6 (3.5 g, 20 mmol) at room temperature. The reaction mixture was
refluxed at 90 °C for 6 h. It was then cooled at room temperature and
concentrated under reduced pressure. The residue was partitioned
between EtOAc (200 mL) and saturated aqueous NaHCO3 solution
(100 mL). The organic layer was washed with brine (100 mL) and
dried over anhydrous MgSO4. After concentration in vacuo, the
residue was purified by column chromatography (silica gel, eluted with
30−100% EtOAc in n-hexane) to give 9d (1.41 g, 3.84 mmol, 19%
from 4d′) as a colorless amorphous powder. MS (ESI+): 367.0 [M +
H]+. 1H NMR (300 MHz, DMSO-d6) δ 3.67 (3H, s), 7.23−7.42 (3H,
m), 7.50 (1H, dt, J = 10.9, 8.4 Hz), 7.61−7.88 (2H, m).
Hz), 3.74−3.86 (3H, m), 4.86−5.62 (1H, m), 6.51 (1H, d, J = 7.4 Hz),
6.98−8.05 (8H, m), 9.71−10.36 (1H, m).
Optical Resolution of ( )-11a. The racemic compound ( )-11a
(130 mg, 0.224 mmol) was purified by chiral preparative HPLC
(CHIRALCEL OD 50 mm × 500 mm column, n-hexane/EtOH = 6/
4) to give the first eluting enantiomer tR1 (49 mg) and the second
eluting enantiomer tR2 (51 mg). Chiral HPLC analysis (4.6 mm ×
250 mm CHIRALCEL OD column with n-hexane/EtOH = 6/4 at 0.5
mm/min). For tR1; tR = 8.50 min and >99.5% ee, For tR2; tR = 10.30
min and 99.5% ee. Each enantiomer, tR2 and tR1, was dissolved in
Et2O (10 mL) and treated with 4 M HCl in EtOAc (0.5 mL, 2.0
mmol), and the resulting precipitate was collected by filtration to
afford (+)-11a (50.5 mg, 0.082 mmol) as a colorless amorphous
powder and (−)-11a (57.7 mg, 0.094 mmol) as a pale-beige
Methyl 5-Bromo-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]-
pyridine-2-carboxylate (9e). Compound 9e was prepared from 4e
(5.0 g, 36 mmol) using 5 (5.4 g, 37.6 mmol) and 6 (6.23 g, 36 mmol)
by a similar method to that described for 9a. Yield: 24%, colorless
amorphous powder. For (+)-11a: [α]25 = +138.3° (c = 0.667,
D
DMSO). MS (ESI+): 543.2 [M + H]+. 1H NMR (300 MHz, DMSO-
d6) δ 1.18−1.66 (3H, m), 2.15−2.34 (3H, m), 2.55−2.81 (6H, m),
2.85−3.21 (2H, m), 3.46−3.77 (2H, m), 3.74−3.88 (3H, m), 5.05
(1H, q, J = 6.6 Hz), 6.54 (1H, d, J = 7.8 Hz), 7.03−7.47 (6H, m),
7.47−7.74 (2H, m), 10.04−10.83 (1H, m). Anal. Calcd for
C28H29Cl2FN4O2·2HCl·2.5H2O: C; 50.85, H; 5.49, N; 8.47. Found
C: 50.71, H; 5.37, N; 8.43. For (−)-11a: [α]25D = −147.9° (c = 0.667,
DMSO). MS (ESI+): 543.1 [M + H]+. 1H NMR (300 MHz, DMSO-
d6) δ 1.16−1.78 (3H, m), 2.12−2.36 (3H, m), 2.56−2.81 (6H, m),
2.83−3.32 (3H, m), 3.60−3.91 (4H, m), 4.97−5.14 (1H, m), 6.44−
6.67 (1H, m), 7.05−7.47 (6H, m), 7.48−7.69 (2H, m), 9.99−10.85
(1H, m). Anal. Calcd for C28H29Cl2FN4O2·2HCl·3.0H2O: C; 50.16,
H; 5.56, N; 8.36. Found: C; 50.34, H; 5.36, N; 8.37.
1
amorphous solid (3.07 g). MS (ESI+): 363.0 [M + H]+. H NMR
(300 MHz, DMSO-d6) δ 2.27 (3H, d, J = 1.5 Hz), 3.66 (3H, s), 7.07−
7.55 (5H, m), 7.67−7.93 (1H, m).
Methyl 5-Ethynyl-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]-
pyridine-2-carboxylate (9i). To a solution of 9e (184 mg, 0.50
mmol) in a mixture of DME (2.5 mL) and water (0.5 mL) were
successively added PdCl2(dppf)·CH2Cl2 (62 mg, 0.075 mmol) and
tributyl(ethynyl)stannane (0.22 mL, 0.75 mmol) at room temperature
under nitrogen atmosphere. The reaction mixture was stirred at 100
°C for 16 h. It was then cooled to room temperature and quenched
with saturated aqueous NaHCO3 solution (50 mL). The aqueous layer
was extracted with EtOAc (2 × 50 mL), and the combined organic
layers were washed with brine (50 mL) and dried over anhydrous
MgSO4. After concentration in vacuo, the residue was purified by
column chromatography (silica gel, eluted with 30−100% EtOAc in n-
hexane) and preparative HPLC (YMC CombiPrep-ODS-A, eluted
with 10−95% acetonitrile in water containing 0.1% TFA). The desired
fractions were collected and concentrated in vacuo, and the residue
was neutralized with saturated aqueous NaHCO3 solution (100 mL).
The aqueous layer was extracted with EtOAc (2 × 75 mL), and the
combined organic layers were washed with brine (50 mL) and dried
over anhydrous MgSO4. The solvent was removed under reduced
pressure to give the desired compound 9i (27.7 mg, 18%) as a yellow
( )-N-[1-(3,4-Dichlorophenyl)propyl]-N-[2-(dimethylamino)-
ethyl]-3-(4-fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]-
pyridine-2-carboxamide Hydrochloride (( )-11b). Compound
( )-11b was prepared from 15 (60 mg, 0.20 mmol) using ( )-N′-
[1-(3,4-dichlorophenyl)propyl]-N,N-dimethylethane-1,2-diamine
( )-16b (55 mg, 0.20 mmol) by a similar method to that described for
1b. Yield: 45%, colorless amorphous powder (53 mg). MS (ESI+):
1
557.2 [M + H]+. H NMR (300 MHz, DMSO-d6) δ 0.44−0.89 (3H,
m), 1.93−2.15 (1H, m), 2.16−2.38 (3H, m), 2.56−3.63 (11H, m),
3.69−3.88 (3H, m), 4.88 (1H, s), 6.47 (1H, s), 6.97−7.67 (8H, m),
10.00 (1H, br s).
1
amorphous solid. MS (ESI+): 309.1 [M + H]+. H NMR (300 MHz,
Optical Resolution of ( )-11b. The racemic compound ( )-11b
(195 mg, 0.35 mmol) was purified by chiral preparative HPLC
(CHIRALPAK AD, 50 mm × 500 mm column, n-hexane/EtOH = 1/
1) to give the first eluting enantiomer tR1 (93 mg) and the second
eluting enantiomer tR2 (85 mg). Chiral HPLC analysis (4.6 mm ×
250 mm CHIRALPAK AD column with n-hexane/EtOH = 1/1 at 0.5
mm/min). For tR1; tR = 9.08 min and >99.9% ee. For tR2; tR = 12.08
min and 99.9% ee. Each enantiomer, tR1 and tR2, was dissolved in
Et2O (10 mL) and treated with 4 M HCl in EtOAc (0.5 mL, 2,0
mmol), and the resulting precipitate was collected by filtration to
afford (+)-11b (87.0 mg, 0.14 mmol) as a colorless powder and
(−)-11b (95.0 mg, 0.15 mmol) as a colorless powder. For (+)-11b:
DMSO-d6) δ 2.27 (3H, d, J = 1.7 Hz), 3.67 (3H, s), 4.52 (1H, s),
7.09−7.21 (1H, m), 7.21−7.43 (4H, m), 7.67−7.88 (1H, m).
Methyl 5-Cyano-3-(4-fluoro-3-methylphenyl)imidazo[1,2-a]-
pyridine-2-carboxylate (9k). To a solution of 9e (364 mg, 1.0
mmol) in cyclopentyl methyl ether (5 mL) were successively added
copper(I) cyanide (359 mg, 4 mmol), dppf (222 mg, 0.2 mmol), and
Pd2(dba)3 (92 mg, 0.1 mmol) at room temperature. The reaction
mixture was refluxed at 120 °C for 14 h. To the mixture were
successively added additional dppf (111 mg, 0.1 mmol) and Pd2(dba)3
(46 mg, 0.05 mmol). The reaction mixture was refluxed at 120 °C for
additional 6 h. It was then cooled to room temperature and quenched
with saturated aqueous NaHCO3 solution (50 mL). The aqueous layer
was extracted with EtOAc (2 × 50 mL), and the combined organic
layers were washed with brine (50 mL) and dried over anhydrous
MgSO4. After concentration in vacuo, the residue was purified by
column chromatography (silica gel, eluted with 20−100% EtOAc in n-
hexane) to give 9k (97.6 mg, 32%) as a white amorphous solid. MS
(ESI+): 310.1 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ 2.28 (3H,
d, J = 1.5 Hz), 3.69 (3H, s), 7.26 (1H, dd, J = 9.8, 8.5 Hz), 7.38−7.54
(3H, m), 7.84 (1H, dd, J = 7.1, 1.0 Hz), 8.09 (1H, dd, J = 9.2, 1.0 Hz).
( )-N-[1-(3,4-Dichlorophenyl)ethyl]-N-[2-(dimethylamino)ethyl]-
3-(4-fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]pyridine-2-
carboxamide Dihydrochloride (( )-11a). Compound ( )-11a was
prepared from 3-(4-fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]-
pyridine-2-carboxylic acid 15 (120 mg, 0.4 mmol) using ( )-N′-[1-
(3,4-dichlorophenyl)ethyl]-N,N-dimethylethane-1,2-diamine
(( )-16a, 125 mg, 0.4 mmol) by a similar method to that described
for 1b. Yield: 88%, colorless amorphous powder (205 mg). MS (ESI
[α]25 = +144.0° (c = 0.667, DMSO). MS (ESI+): 557.2 [M + H]+.
D
1H NMR (300 MHz, DMSO-d6) δ 0.42−0.93 (3H, m), 1.02−2.16
(2H, m), 2.14−2.37 (3H, m), 2.53−2.81 (6H, m), 2.81−3.72 (4H, m),
3.70−3.94 (3H, m), 4.75−5.41 (1H, m), 6.43−6.86 (1H, m), 6.94−
7.23 (1H, m), 7.23−7.47 (4H, m), 7.47−7.67 (3H, m), 10.06−11.38
(1H, m). Anal. Calcd for C29H31Cl2FN4O2·2HCl·1.5H2O: C; 52.98,
H; 5.52, N; 8.52. Found C; 52.70, H; 5.51, N; 8.57. For (−)-11b:
[α]25 = −145.6° (c = 0.667, DMSO). MS (ESI+): 557.2 [M + H]+.
D
1H NMR (300 MHz, DMSO-d6) δ 0.41−0.91 (3H, m), 1.02−2.14
(2H, m), 2.14−2.35 (3H, m), 2.53−2.82 (6H, m), 2.81−3.71 (4H, m),
3.71−4.04 (3H, m), 4.76−5.24 (1H, m), 6.08−6.69 (1H, m), 6.76−
7.83 (8H, m), 10.40−11.16 (1H, m). Anal. Calcd for
C29H31Cl2FN4O2·2HCl·1.5H2O: C; 52.98, H; 5.52, N; 8.52. Found:
C; 52.67, H; 5.57, N; 8.52.
( )-Ethyl (3,4-Dichlorophenyl)([2-(dimethylamino)ethyl]{[3-(4-
fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]pyridin-2-yl]-
carbonyl}amino)acetate (( )-11c′). Compound ( )-11c′ was
prepared from 15 (238 mg, 0.79 mmol) using ( )-ethyl (3,4-
dichlorophenyl){[2-(dimethylamino)ethyl]amino}acetate ( )-16c
1
+): 543.2 [M + H]+. H NMR (300 MHz, DMSO-d6) δ 1.20−1.76
(3H, m), 2.13−2.35 (3H, m), 2.55−3.20 (9H, m), 3.69 (1H, t, J = 10.9
N
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Journal of Medicinal Chemistry
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(278 mg, 0.87 mmol) by a similar method to that described for 1a.
Yield: 58%, colorless amorphous solid (274 mg). MS (ESI+): 601.2
[M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.19−1.29 (3H, m), 1.94−
2.07 (6H, m), 2.07−2.14 (1H, m), 2.31 (3H, s), 2.40−2.59 (1H, m),
3.06−3.72 (2H, m), 3.76 (3H, s), 4.13−4.22 (2H, m), 5.69 (1H, s),
6.00−6.08 (1H, m), 6.95−7.05 (1H, m), 7.13−7.25 (3H, m), 7.27−
7.45 (4H, m).
(3H, m), 2.05−2.14 (4H, m), 2.33 (3H, s), 2.44−2.62 (1H, m), 3.04−
3.54 (2H, m), 3.77 (3H, s), 4.38−4.89 (2H, m), 5.70−6.27 (2H, m),
6.81−7.12 (3H, m), 7.18−7.26 (2H, m), 7.27−7.37 (2H, m). Anal.
Calcd for C28H27Cl2FN4O3·0.3H2O: C, 59.75; H, 4.94; N, 9.95.
Found: C, 59.78; H, 5.02; N, 9.72. For (−)-12: [α]25 = −97.4° (c =
D
1
0.231, CHCl3). MS (ESI+): 557.0 [M + H]+. H NMR (300 MHz,
CDCl3) δ 1.75−1.99 (3H, m), 2.03−2.14 (4H, m), 2.33 (3H, s),
2.46−2.61 (1H, m), 3.01−3.54 (2H, m), 3.78 (3H, s), 4.40−4.89 (2H,
m), 5.71−6.25 (2H, m), 6.81−7.11 (3H, m), 7.17−7.26 (2H, m),
7.28−7.36 (2H, m). Anal. Calcd for C28H27Cl2FN4O3·0.3H2O: C,
59.75; H, 4.94; N, 9.95. Found: C, 59.74; H, 5.02;N, 9.77. Chiral
HPLC analysis (4.6 mm × 250 mm Chiralpak AD column with n-
hexane/EtOH = 1/1 at 0.5 mm/min). For (+)-12; tR = 14.20 min and
>99.9% ee. For (−)-12: tR = 18.82 min and >99.9% ee.
(
)-N-[1-(3,4-Dichlorophenyl)-2-hydroxyethyl]-N-[2-
(dimethylamino)ethyl]-3-(4-fluoro-3-methylphenyl)-5-
methoxyimidazo[1,2-a]pyridine-2-carboxamide (( )-11c). To a
solution of ( )-11c′ (153 mg, 0.26 mmol) in EtOH/THF (2:1, 4
mL) was added calcium chloride (63 mg, 0.51 mmol) at room
temperature, and the mixture was cooled to 0 °C. To the mixture was
slowly added sodium tetrahydroborate (39 mg, 1.0 mmol), and the
mixture was stirred at room temperature for 20 min. The reaction
mixture was stirred at 0 °C for 40 min and partitioned between EtOAc
(15 mL) and water (15 mL). The aqueous layer was extracted with
EtOAc (3 × 15 mL). The combined organic layers were washed with
brine (15 mL) and dried over anhydrous MgSO4. After concentration
in vacuo, the residue was purified by silica gel column chromatography
(NH silica gel, 40%−100% EtOAc in n-hexane) and the subsequent
preparative HPLC (YMC CombiPrep-ODS-A, eluted with 20−65%
acetonitrile in water containing 0.1% TFA) to give ( )-11c (42 mg,
(
)-N-(7,8-Dichloro-3,4-dihydro-2H-chromen-4-yl)-N-[2-
(dimethylamino)ethyl]-3-(4-fluoro-3-methylphenyl)-5-
methoxyimidazo[1,2-a]pyridine-2-carboxamide (( )-13). Com-
pound ( )-13 was prepared from 15 (148 mg, 0.51 mmol) using
( )-N′-(7,8-dichloro-3,4-dihydro-2H-chromen-4-yl)-N,N-dimethyl-
ethane-1,2-diamine ( )-18 (150 mg, 0.52 mmol) by a similar method
to that described for 1a. Yield: 68%, colorless amorphous solid (192
1
mg). MS (ESI+): 571.2 [M + H]+. H NMR (300 MHz, CDCl3) δ
1.85−2.43 (12H, m), 2.52−3.02 (2H, m), 3.20−3.66 (1H, m), 3.73−
3.81 (3H, m), 3.92−4.33 (1H, m), 4.36−4.52 (1H, m), 5.05−5.91
(1H, m), 6.00−6.08 (1H, m), 6.24−6.62 (1H, m), 6.71−6.89 (1H, m),
6.99−7.12 (1H, m), 7.18−7.26 (2H, m), 7.27−7.40 (2H, m).
1
29%) as a colorless oil. MS (ESI+): 559.2 [M + H]+. H NMR (300
MHz, CDCl3) δ 2.23 (6H, s), 2.27 (3H, s), 2.81−3.20 (2H, m), 3.71
(3H, s), 3.72−3.80 (2H, m), 3.81−4.02 (2H, m), 5.43 (1H, dd, J = 8.9,
4.3 Hz), 6.00 (1H, dd, J = 7.2, 0.9 Hz), 6.92−7.09 (3H, m), 7.16−7.24
(3H, m), 7.28−7.36 (2H, m). HRMS (ESI): calcd for
C28H30Cl2FN4O3 [M + H]+, 559.1674; found, 559.1643.
Optical Resolution of ( )-13. Racemic compound ( )-13 (168
mg, 0.29 mmol) was purified by chiral preparative HPLC
(CHIRALCEL OD, 50 mm × 500 mm column, n-hexane/EtOH =
4/1) to afford the first eluting enantiomer (−)-13 (82 mg) as a
colorless solid and the second eluting enantiomer (+)-13 (81 mg) as a
( )-Ethyl 3-(3,4-Dichlorophenyl)-3-([2-(dimethylamino)ethyl]{[3-
(4-fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]pyridin-2-yl]-
carbonyl}amino)propanoate (( )-11d′). Compound ( )-11d′ was
prepared from 15 (90 mg, 0.27 mmol) using ( )-ethyl 3-(3,4-
dichlorophenyl)-3-{[2-(dimethylamino)ethyl]amino}propanoate
( )-16d (81 mg, 0.27 mmol) by a similar method to that described for
colorless solid. For (+)-13: [α]25 = +98.5° (c = 0.130, CHCl3). MS
D
1
(ESI+): 571.5 [M + H]+. H NMR (300 MHz, CDCl3) δ 1.85−2.43
(12H, m), 2.52−3.02 (2H, m), 3.20−3.66 (1H, m), 3.73−3.81 (3H,
m), 3.92−4.33 (1H, m), 4.36−4.52 (1H, m), 5.05−5.91 (1H, m),
6.00−6.08 (1H, m), 6.24−6.62 (1H, m), 6.71−6.89 (1H, m), 6.99−
7.12 (1H, m), 7.18−7.26 (2H, m), 7.27−7.40 (2H, m). Anal. Calcd for
C29H29Cl2FN4O3·0.5H2O: C, 60.00; H, 5.21; N, 9.65. Found: C,
1
1a. Yield: 55%, yellow oil (92 mg). MS (ESI+): 615.2 [M + H]+. H
NMR (300 MHz, CDCl3) δ 1.08−1.22 (3H, m), 1.91−2.05 (3H, m),
2.11−2.31 (6H, m), 2.31−2.52 (2H, m), 2.89−3.50 (4H, m), 3.75−
3.77 (3H, m), 3.94−4.15 (2H, m), 5.47−5.70 (1H, m), 5.99−6.09
(1H, m), 7.00 (1H, s), 7.13 (1H, d, J = 8.1 Hz), 7.17−7.25 (2H, m),
7.27−7.42 (4H, m).
60.23; H, 5.33; N, 9.40. For (−)-13: [α]25 = −126.0° (c = 0.150,
D
CHCl3). MS (ESI+): 571.5 [M + H]+. 1H NMR (300 MHz, CDCl3) δ
1.87−2.34 (12H, m), 2.56−3.02 (2H, m), 3.25−3.66 (1H, m), 3.74−
3.79 (3H, m), 3.93−4.31 (1H, m), 4.35−4.55 (1H, m), 5.04−5.90
(1H, m), 5.99−6.10 (1H, m), 6.23−6.62 (1H, m), 6.71−6.89 (1H, m),
6.99−7.11 (1H, m), 7.16−7.25 (2H, m), 7.27−7.40 (2H, m). Anal.
Calcd for C29H29Cl2FN4O3·0.5H2O: C, 60.00; H, 5.21; N, 9.65.
Found: C, 59.86; H, 5.26; N, 9.47. Chiral HPLC analysis (4.6 mm ×
250 mm CHIRALCEL OD column with n-hexane/EtOH = 4/1 at 1.0
mm/min). For (−)-13: tR = 6.46 min and 99.9% ee. For (+)-13: tR =
13.76 min and 99.3% ee.
(
)-N-[1-(3,4-Dichlorophenyl)-3-hydroxypropyl]-N-[2-
(dimethylamino)ethyl]-3-(4-fluoro-3-methylphenyl)-5-
methoxyimidazo[1,2-a]pyridine-2-carboxamide (( )-11d). Com-
pound ( )-11d was prepared from ( )-11d′ (88 mg, 0.14 mmol)
by a similar method to that described for ( )-11c. Yield: 12%, pale-
1
orange amorphous solid (10 mg). MS (ESI+): 573.2 [M + H]+. H
NMR (300 MHz, CDCl3) δ 2.03−2.25 (8H, m), 2.32 (3H, d, J = 1.5
Hz), 2.34−2.44 (2H, m), 2.72−2.88 (1H, m), 3.33−3.47 (1H, m),
3.72−3.74 (3H, m), 3.80−4.15 (2H, m), 5.54 (1H, dd, J = 11.1, 3.0
Hz), 6.03−6.10 (1H, m), 6.90 (1H, dd, J = 8.4, 1.6 Hz), 6.96−7.14
(3H, m), 7.19−7.26 (2H, m), 7.28−7.38 (2H, m). HRMS (ESI): calcd
for C29H32Cl2FN4O3 [M + H]+, 573.1830; found, 573.1815.
( )-tert-Butyl {2-[(6,7-Dichloro-2,3-dihydro-1-benzofuran-3-yl)-
{[3-(4-fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]pyridin-2-
yl]carbonyl}amino]ethyl}methylcarbamate (( )-14). Compound
( )-14 was prepared from 15 (111 mg) using tert-butyl {2-[(6,7-
dichloro-2,3-dihydro-1-benzofuran-3-yl)amino]ethyl}methylcarbamate
( )-19 (159 mg, 0.44 mmol) by a similar method to that described for
1a. Yield: 61%, colorless amorphous solid (145 mg). MS (ESI+):
643.3 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.14−1.42 (9H, m),
2.30−2.79 (6H, m), 3.00−3.43 (4H, m), 3.74−3.79 (3H, m), 4.26−
4.86 (2H, m), 5.75−6.45 (2H, m), 6.81−7.11 (3H, m), 7.17−7.26
(2H, m), 7.27−7.34 (2H, m).
( )-N-(6,7-Dichloro-2,3-dihydro-1-benzofuran-3-yl)-N-[2-
(dimethylamino)ethyl]-3-(4-fluoro-3-methylphenyl)-5-
methoxyimidazo[1,2-a]pyridine-2-carboxamide (( )-12). Com-
pound ( )-12 was prepared from 15 (452 mg, 1.51 mmol) using
( )-N′-(6,7-dichloro-2,3-dihydro-1-benzofuran-3-yl)-N,N-dimethyl-
ethane-1,2-diamine ( )-17 (456 mg, 1.66 mmol) by a similar method
to that described for 1a. Yield: 80%, colorless amorphous solid (675
1
mg). MS (ESI+): 557.2 [M + H]+. H NMR (300 MHz, CDCl3) δ
Optical Resolution of ( )-14. Racemic compound ( )-14 (140
mg, 0.217 mmol) was purified by chiral preparative HPLC (Chiralpak
IC, 50 mm × 500 mm column, n-hexane/EtOH = 4/6) to afford the
first eluting enantiomer (−)-14 (76 mg) as a colorless solid and the
second eluting enantiomer (+)-14 (71 mg) as a colorless solid. For
(+)-14: [α]25D = +87.2° (c = 0.155, CHCl3). MS (ESI+): 643.0 [M +
1.78−1.88 (3H, m), 2.04−2.12 (4H, m), 2.33 (3H, s), 2.40−2.63 (1H,
m), 3.02−3.46 (2H, m), 3.77 (3H, s), 4.41−4.87 (2H, m), 5.71−6.25
(2H, m), 6.82−7.09 (3H, m), 7.19−7.26 (2H, m), 7.27−7.35 (2H, m).
Optical Resolution of ( )-12. Racemic compound ( )-12 (670
mg, 1.20 mmol) was purified by chiral preparative HPLC
(CHIRALPAK AD, 50 mm × 500 mm column, n-hexane/EtOH =
1/1) to afford the first eluting enantiomer (+)-12 (316 mg) as a
colorless solid and the second eluting enantiomer (−)-12 (319 mg) as
a colorless solid. For (+)-12: [α]25D = +101.0° (c = 0.231, CHCl3). MS
1
H]+. H NMR (300 MHz, CDCl3) δ 1.25−1.41 (9H, m), 2.24−2.83
(6H, m), 3.00−3.50 (4H, m), 3.77 (3H, s), 4.26−4.88 (2H, m), 5.76−
6.41 (2H, m), 6.81−7.12 (3H, m), 7.17−7.26 (2H, m), 7.27−7.35
(2H, m). HRMS (ESI): calcd for C32H34Cl2FN4O5 [M + H]+,
1
(ESI+): 557.0 [M + H]+. H NMR (300 MHz, CDCl3) δ 1.74−1.87
O
DOI: 10.1021/acs.jmedchem.5b00836
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
643.1885; found, 643.1886. For (−)-14: [α]25 = −87.0° (c = 0.195,
(2H, m), 4.62−4.74 (1H, m), 7.00 (1H, d, J = 7.9 Hz), 7.15 (1H, d, J =
7.9 Hz).
D
CHCl3). MS (ESI+): 643.0 [M + H]+. 1H NMR (300 MHz, CDCl3) δ
1.25−1.40 (9H, m), 2.29−2.81 (6H, m), 3.04−3.49 (4H, m), 3.77
(3H, s), 4.27−4.86 (2H, m), 5.77−6.42 (2H, m), 6.83−7.11 (3H, m),
7.18−7.26 (2H, m), 7.27−7.37 (2H, m). HRMS (ESI): calcd for
C32H34Cl2FN4O5 [M + H]+, 643.1885; found, 643.1861. Chiral HPLC
analysis (4.6 mm × 250 mm Chiralpak IC column with n-hexane/
EtOH = 4/6 at 1.0 mm/min). (−)-14: tR = 13.03 min and >99.9% ee.
(+)-14: tR = 16.31 min and 98.6% ee.
( )-N′-(7,8-Dichloro-3,4-dihydro-2H-chromen-4-yl)-N,N-dime-
thylethane-1,2-diamine (( )-18). Compound ( )-18 was prepared
from 7,8-dichloro-2,3-dihydro-4H-chromen-4-one 28 (501 mg, 2.31
mmol) using 29 (0.303 mL, 2.77 mmol) by a similar method to that
described for ( )-17. Yield: 59%, yellow oil (394 mg). MS (ESI+):
289.1 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.97−2.04 (2H, m),
2.23 (6H, s), 2.40−2.47 (2H, m), 2.66−2.84 (2H, m), 3.77 (1H, t, J =
4.3 Hz), 4.31−4.51 (2H, m), 6.95−7.00 (1H, m), 7.06−7.11 (1H, m).
( )-tert-Butyl {2-[(6,7-Dichloro-2,3-dihydro-1-benzofuran-3-yl)-
amino]ethyl}methylcarbamate (( )-19). To a solution of tert-butyl
(2-aminoethyl)methylcarbamate 30 (0.287 mL, 1.61 mmol) in THF
(2 mL) was added 27 (150 mg, 0.74 mmol), and the resulting brown
solution was diluted with MeOH (2 mL) and AcOH (0.5 mL) and to
the solution was added NaBH3CN (228 mg, 3.63 mmol) at room
temperature. The reaction mixture was stirred at 50 °C for 18 h. It was
then quenched with 2 M HCl (2 mL, 4.0 mmol). The mixture was
stirred at room temperature for 10 min and made alkaline with 2 M
NaOH (10 mL, 20 mmol). The mixture was extracted with EtOAc (3
× 20 mL). The combined organic layers were washed with brine (30
mL) and dried over anhydrous MgSO4. After concentration in vacuo,
the residue was purified by column chromatography (silica gel, eluted
with 5−80% EtOAc in n-hexane) to yield ( )-19 (62.8 mg, 24%) as a
3-(4-Fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]pyridine-
2-carboxylic Acid (15). Compound 15 was prepared from ethyl 3-(4-
fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]pyridine-2-carboxy-
late (25, 5.71 g, 17.4 mmol) by a similar method to that described for
2a. Yield: 74%, pale-beige crystalline solid (3.87 g); mp 166−168 °C.
MS (ESI+): 301.1 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ 2.26
(3H, d, J = 1.3 Hz), 3.66 (3H, s), 6.35 (1H, d, J = 7.6 Hz), 7.02−7.19
(1H, m), 7.20−7.36 (3H, m), 7.36−7.49 (1H, m).
( )-Ethyl (3,4-Dichlorophenyl){[2-(dimethylamino)ethyl]amino}-
acetate (( )-16c). To a solution of ethyl (3,4-dichlorophenyl)
(oxo)acetate 26c (3.01 g, 12.2 mmol) in EtOH (20 mL) were
added AcOH (1.40 mL, 24.4 mmol) and N,N-dimethylethane-1,2-
diamine 29 (1.53 mL, 14 mmol), successively. The reaction mixture
was stirred at room temperature for 1.5 h. It was then cooled to 0 °C.
To the mixture were added MeOH (15 mL) and sodium
tetrahydroborate (691 mg, 18.3 mmol), successively. The reaction
mixture was stirred at 0 °C for 1 h. It was then stirred at room
temperature for 30 min, quenched with water (10 mL), and
concentrated under reduced pressure. The residue was partitioned
between n-hexane/EtOAc (1:1, 30 mL) and 2 M HCl (30 mL, 60
mmol). The aqueous layer was washed with n-hexane/EtOAc (1:4, 50
mL). The aqueous layer was made alkaline with 2 M NaOH (30 mL,
60 mmol) and saturated aqueous NaHCO3 (10 mL) to pH 9 and was
extracted with EtOAc (3 × 10 mL). The combined organic layers were
washed with brine (30 mL) and dried over anhydrous MgSO4. The
solvent was removed under reduced pressure to give a suspension.
After decantation of the suspension at −20 °C for 3 days, insoluble
material was separated out. Removal of the insoluble material gave the
desired compound ( )-16c (206 mg, 5%) as a yellow oil. MS (ESI+):
319.1 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.19−1.26 (3H, m),
2.27 (6H, s), 2.45−2.64 (4H, m), 4.10−4.25 (2H, m), 4.33 (1H, s),
7.24 (1H, d, J = 2.1 Hz), 7.39−7.43 (1H, m), 7.52 (1H, d, J = 1.9 Hz).
( )-Ethyl 3-(3,4-Dichlorophenyl)-3-{[2-(dimethylamino)ethyl]-
amino}propanoate (( )-16d). Compound ( )-16d was prepared
from ethyl 3-(3,4-dichlorophenyl)-3-oxopropanoate 26d (1.69 mg, 6.5
mmol) and 29 (0.740 mL, 13 mmol) by a similar method to that
described for ( )-16c. Yield: 4%, yellow oil (84 mg). MS (ESI+):
1
pale-yellow oil. MS (ESI+): 361.1 [M + H]+. H NMR (300 MHz,
CDCl3) δ 1.44 (9H, s), 2.59−2.96 (5H, m), 3.16−3.46 (2H, m),
4.40−4.52 (1H, m), 4.51−4.61 (1H, m), 4.60−4.76 (1H, m), 6.98
(1H, d, J = 7.9 Hz), 7.14 (1H, d, J = 7.9 Hz).
5-Methoxyimidazo[1,2-a]pyridine-2-carboxylate (22). To a sol-
ution of 6-methoxypyridin-2-amine 20 (10 g, 80.6 mmol) in EtOH
(150 mL) was added dropwise ethyl 3-bromo-2-oxopropanoate 21 (21
g, 96.9 mmol) at 40 °C over 30 min. The reaction mixture was stirred
at 40 °C for 14 h. The mixture was diluted with EtOAc/n-hexane (2:1,
450 mL) and washed with saturated aqueous NaHCO3 solution (150
mL). The mixture was filtered, and the separated aqueous layer of the
fitrate was extracted with EtOAc (150 mL). The combined organic
layers were washed with brine (150 mL) and filtered through a pad of
NH silica gel (150 g) using EtOAc (150 mL) as eluting solvent. The
filtrate was evaporated in vacuo. The oily residue (19 g) was dissolved
in EtOAc (40 mL), and the solution was filtered through a pad of NH
silica gel (120 g) using EtOAc/n-hexane (1:2, 600 mL) and EtOAc/n-
hexane (2:1, 900 mL), successively. The filtrate was concentrated in
vacuo until the solvent volume became about 100 mL. The resulting
slurry was diluted with n-hexane (500 mL). The mixture was triturated
at room temperature for 30 min, and the resulting precipitate was
collected by filtration. Drying the solid gave 22 (9.30 g, 52%) as a pale-
1
333.1 [M + H]+. H NMR (300 MHz, CDCl3) δ 1.20 (3H, t, J = 7.2
1
yellow powder. MS (ESI+): 221.0 [M + H]+. H NMR (300 MHz,
Hz), 2.20 (6H, s), 2.39−2.75 (6H, m), 4.02 (1H, dd, J = 7.7, 6.0 Hz),
4.10 (2H, q, J = 7.2 Hz), 7.20 (1H, dd, J = 8.2, 2.0 Hz), 7.39 (1H, d, J
= 8.3 Hz), 7.47 (1H, d, J = 1.9 Hz).
DMSO-d6) δ 1.32 (3H, t, J = 7.1 Hz), 4.11 (3H, s), 4.31 (2H, q, J = 7.2
Hz), 6.45 (1H, d, J = 7.0 Hz), 7.26 (1H, d, J = 9.1 Hz), 7.41 (1H, dd, J
= 9.1, 7.6 Hz), 8.22 (1H, s).
( )-N′-(6,7-Dichloro-2,3-dihydro-1-benzofuran-3-yl)-N,N-dime-
thylethane-1,2-diamine (( )-17). To a solution of 6,7-dichloroben-
zofuran-3(2H)-one 27 (1.0 g, 4.95 mmol) in a mixed solvent of THF
(10 mL), MeOH (18.2 mL), and AcOH (1.8 mL) was added 29
(0.703 mL, 6.44 mmol) at room temperature. The reaction mixture
was stirred at room temperature for 1 h and then stirred at 45−50 °C
for 2 h. To the mixture was added 2-picoline borane complex (695 mg,
6.50 mmol) at room temperature. The mixture was stirred at room
temperature for 14 h. After concentration under reduced pressure, the
residue was diluted with 2 M HCl (40 mL, 80 mmol) and the mixture
was stirred at room temperature for 20 min. Insoluble was filtered off,
and the filtrate was diluted with EtOAc/n-hexane (1:1, 40 mL). The
aqueous layer of the filtrate was made alkaline with 8 M NaOH (10
mL, 80 mmol) and 2 M NaOH (2.0 mL, 4.0 mmol). The resulting
alkaline aqueous solution was extracted with EtOAc (3 × 50 mL). The
combined organic layer was washed with brine (100 mL) and dried
over anhydrous MgSO4. The solvent was removed under reduced
pressure to give ( )-17 (626 mg, 46%) as a brown oil. MS (ESI+):
Ethyl 3-Bromo-5-methoxyimidazo[1,2-a]pyridine-2-carboxylate
(23). To a solution of 22 (5.0 g, 22.7 mmol) in DMF (150 mL)
was added portionwise NBS (4.04 g, 22.7 mmol) at 4 °C. The reaction
mixture was stirred at room temperature for 1 h. The resulting mixture
was concentrated in vacuo, and the oily residue was poured into water
(100 mL). The mixture was stirred at room temperature for 1 h. The
resulting precipitate was collected by filtration, washed with water, and
dried under vacuum to give 23 (6.52 g, 21.80 mmol, 96%) as a
1
colorless amorphous powder. MS (ESI+): 299.0 [M + H]+. H NMR
(300 MHz, DMSO-d6) δ 1.32 (3H, t, J = 7.1 Hz), 4.04 (3H, s), 4.31
(2H, q, J = 7.1 Hz), 6.42 (1H, d, J = 6.8 Hz), 7.10−7.30 (1H, m),
7.30−7.54 (1H, m).
Ethyl 3-(4-Fluoro-3-methylphenyl)-5-methoxyimidazo[1,2-a]-
pyridine-2-carboxylate (25). To a mixture of 23 (6.0 g, 20.1 mmol)
and 4-fluoro-3-methylphenylboronic acid 24 (4.63 g, 30.1 mmol) in
DME (150 mL) were added a solution of Cs2CO3 (19.6 g, 60.2 mmol)
in water (30 mL) and PdCl2(dppf)·CH2Cl2 (1.64 g, 2.01 mmol),
successively, at room temperature under nitrogen atmosphere. The
reaction mixture was stirred at 90 °C for 1.5 h. It was then cooled at
room temperature and partitioned between EtOAc (300 mL) and
1
275.1 [M + H]+. H NMR (300 MHz, CDCl3) δ 2.18 (6H, s), 2.35−
2.44 (2H, m), 2.53−2.64 (2H, m), 2.68−2.80 (1H, m), 4.48−4.59
P
DOI: 10.1021/acs.jmedchem.5b00836
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
brine (150 mL). After a removal of insoluble by filtration, the
separated aqueous layer was extracted with EtOAc (100 mL). The
combined organic layers were washed with brine (100 mL) and dried
over anhydrous MgSO4. After concentration in vacuo, the residue was
purified by silica gel column chromatography (NH silica gel, 10%−
70% EtOAc in n-hexane). The desired fractions were combined and
concentrated in vacuo. The resulting slurry was triturated with EtOAc
(20 mL) and n-hexane (100 mL). The resulting precipitate was
collected by filtration and dried under vacuum to give 25 (5.83 g, 17.8
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00836.
Information methods used in kinesin enzyme assays,
cellular assays, in vivo studies, computational studies, and
single-crystal X-ray structure analysis (PDF)
AUTHOR INFORMATION
(14) Qian, X.; McDonald, A.; Zhou, H.-J.; Adams, N. D.; Parrish, C.
A.; Duffy, K. J.; Fitch, D. M.; Tedesco, R.; Ashcraft, L. W.; Yao, B.;
Jiang, H.; Huang, J. K.; Marin, M. V.; Aroyan, C. E.; Wang, J.; Ahmed,
S.; Burgess, J. L.; Chaudhari, A. M.; Donatelli, C. A.; Darcy, M. G.;
■
Corresponding Authors
*For T.H.: phone, +81-466-32-1160; fax, +81-466-29-4448; E-
mail, takaharu.hirayama@takeda.com.
*For M.O., phone, +81-466-32-1158; E-mail, masanori.
okaniwa@takeda.com.
́
Ridgers, L. H.; Newlander, K. A.; Schmidt, S. J.; Chai, D.; Colon, M.;
Zimmerman, M. N.; Lad, L.; Sakowicz, R.; Schauer, S.; Belmont, L.;
Baliga, R.; Pierce, D. W.; Finer, J. T.; Wang, Z.; Morgan, B. P.;
Morgans, D. J., Jr.; Auger, K. R.; Sung, C.-M.; Carson, J. D.; Luo, L.;
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Present Address
‡For K.M.: Chief Medical and Scientific Officer Office, Takeda
Pharmaceutical Company Limited: 1−1, Doshomachi 4-chome,
Chuo-ku, Osaka 540−8645, Japan
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Takashi Imada and Yuri Suzuki for their assistance in
intermediate synthesis, Toshimasa Tanaka for his supports in
computational chemistry, and Mitsuyoshi Nishitani for his
assistance in crystallographic data deposition to the Cambridge
Crystallographic Data Centre.
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