Synthetic studies of centromere-associated protein-E (CENP-E) inhibitors: 1.Exploration of fused bicyclic core scaffolds using electrostatic potential map

Article abstract of DOI:10.1016/j.bmc.2013.05.067

Centromere-associated protein-E (CENP-E), a mitotic kinesin that plays an important role in mitotic progression, is an attractive target for cancer therapeutic drugs. For the purpose of developing novel CENP-E inhibitors as cancer therapeutics, we investigated a fused bicyclic compound identified by high throughput screening, 4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide 1a. Based on this scaffold, we designed inhibitors for efficient binding at the L5 site in CENP-E utilizing homology modeling as well as electrostatic potential map (EPM) analysis to enhance CENP-E inhibitory activity. This resulted in a new lead, 5-bromoimidazo[1,2-a]pyridine 7, which showed potent CENP-E enzyme inhibition (IC₅₀: 50 nM) and cellular activity with accumulation of phosphorylated histone H3 in HeLa cells. Our homology model and EPM analysis proved to be useful tools for the rational design of CENP-E inhibitors.

Full text of DOI:10.1016/j.bmc.2013.05.067

Accepted Manuscript
Synthetic Studies of Centromere-Associated Protein-E (CENP-E) Inhibitors: 1.
Exploration of Fused Bicyclic Core Scaffolds Using Electrostatic Potential Map
Takaharu Hirayama, Masanori Okaniwa, Takashi Imada, Akihiro Ohashi,
Momoko Ohori, Kenichi Iwai, Kouji Mori, Tomohiro Kawamoto, Akihiro
Yokota, Toshimasa Tanaka, Tomoyasu Ishikawa
PII:
S0968-0896(13)00536-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.05.067
BMC 10898
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
12 April 2013
28 May 2013
29 May 2013
Please cite this article as: Hirayama, T., Okaniwa, M., Imada, T., Ohashi, A., Ohori, M., Iwai, K., Mori, K.,
Kawamoto, T., Yokota, A., Tanaka, T., Ishikawa, T., Synthetic Studies of Centromere-Associated Protein-E (CENP-
E) Inhibitors: 1. Exploration of Fused Bicyclic Core Scaffolds Using Electrostatic Potential Map, Bioorganic &
Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.05.067
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Synthetic Studies of Centromere-Associated Protein-E (CENP-E) Inhibitors:
1
. Exploration of Fused Bicyclic Core Scaffolds Using Electrostatic Potential
Map
1
a
a
a
a
a
Takaharu Hirayama *, Masanori Okaniwa, Takashi Imada, Akihiro Ohashi, Momoko Ohori,
a
a,b
a
a
a
Kenichi Iwai, Kouji Mori, Tomohiro Kawamoto, Akihiro Yokota, Toshimasa Tanaka,
2
a
and Tomoyasu Ishikawa *,
a
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited: 26-1, Muraoka-
Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b
Present address: Chief Medical & Scientific Officer Office, Takeda Pharmaceutical Company
Limited: 1-1, Doshomachi 4-chome, Chuo-ku, Osaka 540-8645, Japan
1
*Corresponding author: Phone: +81-466-32-1160; Fax: +81-466-29-4448; E-mail:
takaharu.hirayama@takeda.com
2
*Corresponding author: Phone: +81-466-32-1155; Fax: +81-466-29-4449; E-mail:
tomoyasu.ishikawa@takeda.com
1

1
Abbreviations: H-NMR, proton nuclear magnetic resonance; CENP-E, centromere-
associated protein-E; EPM, electrostatic potential map; PD, pharmacodynamic; SAR, structure-
activity relationship; DSC, differential scanning calorimetry
Keywords:
CENP-E;
4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide;
imidazo[1,2-a]pyridine; electrostatic potential map (EPM)
2

Abstract: Centromere-associated protein-E (CENP-E), a mitotic kinesin that plays an important
role in mitotic progression, is an attractive target for cancer therapeutic drugs. For the purpose of
developing novel CENP-E inhibitors as cancer therapeutics, we investigated a fused bicyclic
compound identified by high throughput screening, 4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-
carboxamide 1a. Based on this scaffold, we designed inhibitors for efficient binding at the L5 site
in CENP-E utilizing homology modeling as well as electrostatic potential map (EPM) analysis to
enhance CENP-E inhibitory activity. This resulted in a new lead, 5-bromoimidazo[1,2-a]pyridine
7
, which showed potent CENP-E enzyme inhibition (IC : 50 nM) and cellular activity with
5
0
accumulation of phosphorylated histone H3 in HeLa cells. Our homology model and EPM
analysis proved to be useful tools for the rational design of CENP-E inhibitors.
Graphical Abstract
Electrostaticpotential map (EPM)
3

1.
Introduction
Centromere-associated protein-E (CENP-E) is one of the mitotic spindle motor proteins
1
–4
belonging to the kinesin superfamily, and contains an ATPase domain in its motor region.
CENP-E utilizes energy obtained from ATP hydrolysis to control chromosome alignment by
5
–7
capturing microtubules during metaphase. Loss of CENP-E function causes mitotic arrest with
a cellular phenotype that is characterized by misaligned chromosomes during metaphase, and
results in subsequent apoptosis by inspection of spindle assembly checkpoint (SAC) machinery
during the prolonged mitotic arrest. In general, the mitotic proliferation rate of cancer cells in
malignant tumors is explosively faster than that of normal cells, and inhibition of CENP-E is
8
–12
known to induce an anti-tumor effect. Therefore, CENP-E inhibition is expected to be a
1
3–15
promising new approach for cancer therapy. GSK923295A
is a selective CENP-E inhibitor
that has been evaluated in clinical trials (Figure 1). Despite the promising results of
GSK923295A in preclinical studies, sufficient accumulation of phosphorylated histone H3 in
tumors, a pharmacodynamic (PD) marker, was not detected in the clinical trials, while an
1
6
elevation of aspartate transaminase (AST) and fatigue were observed as dose-limiting toxicities.
Thus, further studies of novel CENP-E inhibitors unrelated to GSK923295A are required to
determine whether inhibition of this promising drug target has the potential to become a useful
new approach in cancer therapy.
[Figure 1]
We initiated studies on novel CENP-E inhibitors aimed at demonstrating clinical efficacy.
From a high throughput screening for ATPase inhibition of the CENP-E motor domain, we
1
7
identified 4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide derivative 1a as exhibiting
4

CENP-E inhibitory activity with an IC value of 2.1 µM (Figure 2). Initial structure-activity
5
0
relationship (SAR) studies revealed that substitution of the 3,4-dichloro groups on the benzyl
moiety (1b) slightly enhanced CENP-E inhibitory activity with an IC value of 1.5 µM. Thus, we
5
0
selected 1b as our lead compound.
[Figure 2]
1
8
Few studies on CENP-E binding sites and no X-ray co-crystal structures of CENP-E
with inhibitors have thus far been reported. We hypothesized that CENP-E would have a similar
binding site to that of Eg5 on the basis of the high sequence identity (36%) in their motor
1
8
domains. Therefore, we built a homology model for the rational design of inhibitors based on
the reported co-crystal structures of Eg5. Eg5 is also known as kinesin spindle protein (KSP), and
1
–4
plays a crucial role in the mitotic spindle assembly. Eg5 is also considered an attractive target
1
9,20
21–23
for cancer treatment, and several small molecule inhibitors such as monastrol and ispinesib
have been developed. In addition, X-ray co-crystal structural analysis of the motor domain of Eg5
2
4
with various inhibitors suggested that several different types of Eg5 inhibitors were
accommodated in the same cavity, known as the L5 binding site, which is formed by loop 5, 2
1
9,20
helix, and 3 helix.
Based on this hypothesis, we built a CENP-E homology model using the X-ray co-crystal
2
4
structure of Eg5 with a pyrrolotriazine-4-one-based inhibitor (PDB: 2GM1) (see Experimental
section). The results of a docking study of our lead 1b in this CENP-E homology model are
shown in Figure 3A. In this study, the fused bicyclic 4-oxo-4,5-dihydrothieno[3,4-c]pyridine
scaffold was accommodated in the hydrophobic cavity lined with Ile182, Tyr131, Phe207 and
Met97 in the motor domain of CENP-E (Figure 3B). In addition, the 3,4-dichlorobenzyl group of
5

1
b can approach the 2 helix to coordinate with Loop 5. On the other hand, 1b showed no
interaction with the carboxy group of Glu186 that is directed toward the ligand. To better
understand the vacancy of “Glu186 space (Figure 3B)”, we calculated the distances between the
1
2
carboxy group of Glu186 and either the R position or R position as 4.1 Å and 3.8 Å,
1
respectively (Figure 3C). These results suggested that introduction of a side chain at either R or
2
R would likely to be acceptable and that it would be possible to enhance CENP-E inhibitory
activity by interaction with the carboxy group of Glu186. Thus, we designed and evaluated
1
2
compounds possessing various substituents at R or R (compounds 2 and 3).
[Figure 3]
According to our docking study, the 4-oxo group on the 4-oxo-4,5-dihydrothieno[3,4-
c]pyridine ring of 1b did not appear to make a significant interaction with any residues in the
hydrophobic pocket. We assumed that this scaffold (ring A) could be replaced by indoles (4 and
5
) or thieno[3,2-b]pyrrole 6 (Figure 4A). Based on further docking studies of the thieno[3,2-
3
b]pyrrole scaffold 6, we examined the ability of various 3-substituents (R ) to occupy the
hydrophobic space (approximately 4 Å) between the ligand and the 2 helix of CENP-E (Figure
4
B).
3
To explain the structure-activity relationship (SAR) at R for 6, we visualized the
2
5–29
molecular surface of our scaffolds using an electrostatic potential map (EPM).
The EPM
results correlated well with the observed SAR and resulted in the discovery of our new lead, 5-
substituted imidazo[1,2-a]pyridine 7 (Figure 4A). In this report, we describe the design and
synthesis of novel CENP-E inhibitors bearing various fused bicyclic scaffolds utilizing CENP-E
homology modeling and EPM analysis.
6

[Figure 4]
2
.
Chemistry
The synthesis of 5-methylated (R = Me) 4-oxo-4,5-dihydrothieno[3,4-c]pyridine
1
1
7
derivatives 1b and 3 is shown in Scheme 1. Known ethyl ester 8 was hydrolyzed with 1 M
NaOH, and the resulting carboxylic acid was condensed with commercially available 1-(3,4-
dichlorophenyl)-N-methylmethanamine 9 using hexafluorophosphate O-(7-azabenzotriazol-1-yl)-
N,N,N',N'-tetramethyluronium (HATU) in the presence of triethylamine (Et N) to obtain the
3
desired N-methyl amide derivative 1b in 11% yield over 2 steps. The preparation of 2-(N,N-
dimethylamino)ethylated compound 3 was accomplished using the corresponding commercially
available N'-(3,4-dichlorobenzyl)-N,N-dimethylethane-1,2-diamine 10 in a method similar to that
used for the preparation of 1b (Yield: 18% in 2 steps).
[
Scheme 1]
1
The synthesis of 5-substituted (R ) compounds (2a−c) is shown in Scheme 2. Rh-
1
7
catalyzed heteroatom–hydrogen insertion of reported thiophenecarboxylic acid 11 was
performed using -diazophosphonate 13, which was prepared in situ by a reaction of
commercially available ethyl (diethoxyphosphoryl) acetate 12 with 4-acetamidobenzenesulfonyl
azide in the presence of sodium hydride. Subsequent treatment of the reaction mixture with 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) promoted an intramolecular Horner–Wadsworth–
3
0
Emmons reaction , to afford bicyclic lactone ethyl ester 14 in quantitative yield. Treatment of 14
with three different amines in tetrahydrofuran (THF) resulted in the ring-opening reaction of
7

lactone to obtain the corresponding enol esters 15a and 15b in 80–91% yields. The
intramolecular condensation of 15a using 4-toluenesulfonic acid monohydrate (TsOH·H O)
2
generated tricyclic lactone 16a in 80% yield. Direct amidation of 16a was accomplished by
aluminum amide, which was prepared from 9 and trimethylaluminum in situ to obtain the desired
2
-hydroxyethyl derivative 2a in 60% yield. The intramolecular condensation of 15b with
TsOH·H O was performed to obtain the desired ethyl esters 16b in quantitative yield. Hydrolysis
2
of 16b with 8 M NaOH, followed by condensation with 9 in the presence of HATU, afforded 2-
(
N,N-dimethylamino)ethyl compound 2b in 33% yield over 2 steps from 16b. The desired 3-
N,N-dimethylamino)propyl compound 2c was synthesized from 14 in 23% yield over 4 steps by
(
the similar method to that of 2b.
[
Scheme 2]
The synthesis of indoles 4 and 5, substituted with a 4-fluorophenyl group, is shown in
Scheme 3. The 3-arylindole 4 was prepared from commercially available N-methylated 3-
bromoindole
17.
The
[1,1•-bis(diphenylphosphino)ferrocene]dichloropalladium(II)
3
1,32
dichloromethane (PdCl (dppf)·CH Cl ) catalyzed Suzuki–Miyaura coupling
of 17 with 4-
2
2
2
fluorophenylboronic acid proceeded in the presence of cesium carbonate (Cs CO ) in
2
3
dimethoxyethane (DME) to obtain ester 18 in 80% yield. Hydrolysis of 18 with NaOH solution,
followed by condensation of the obtained carboxylic acid with 10 in the presence of HATU and
Et N produced the desired compound 4 in 38% yield over 2 steps. The Chan–Lam–Evans
3
3
3,34
coupling reaction of commercially available 19 with 4-fluorophenylboronic acid was carried
out using 2 equivalents of Cu(OAc) in the presence of Et N. Optimal conditions were established
2
3
using trifluoromethylbenzene as a solvent as well as adding 4Å molecular sieves (see below).
8

This reaction proceeded regioselectively to afford the N-1 substituted derivative 20 in 78% yield
without formation of the corresponding C-3 adduct. Compound 20 was converted into the desired
3
-methylated compound 21 in 95% yield by Pd-catalyzed coupling with tetramethyltin(IV).
Similar hydrolysis of 21 followed by condensation with amine 10 to that of 4 generated the
desired indole 5 in 84% yield over 2 steps.
[
Scheme 3]
3
5
The synthesis of thieno[3,2-b]pyrroles 6 with various 3-substituents is depicted in
Scheme 4. Commercially available 4-bromo-2-thiophenecarbaldehyde 22 was treated with ethyl
2
-azidoacetate in the presence of sodium ethoxide (NaOEt) in ethanol (EtOH) to afford azide 23
3
6
in 30% yield. Since azides are generally known to be explosive, the thermal stability of the
obtained azide 23 was given a preliminary assessment by differential scanning calorimetry
(
DSC). As a result, 23 exhibited exothermic decomposition in the range of 95–126 ºC with a peak
at 114 ºC releasing 425 J/g of energy (Supplemental data, Figure S2, see Experimental section).
This result indicated that 23 exhibited moderate potential for self-reaction and explosion, and
thus careful handling was required. To control the desired cyclization of 23, a solution of 23 in
xylene was added slowly to boiling xylene, which produced 3-bromothieno[3,2-b]pyrrole 24 in
3
5
33,34
8
5% yield. The subsequent Chan-Lam-Evans coupling of 24 with 4-fluorophenylboronic acid
using 2.5 equiv of Cu(OAc) in the presence of 3Å molecular sieves (MS) generated the desired
2
N-adduct 25a in 85% yield. As previously mentioned, the addition of molecular sieves
significantly accelerated the reaction and improved the yield relative to conditions without MS
(yield <40%). In these experiments, the use of 3Å MS gave higher yields than 4Å MS (85% vs.
7
5% yield). Of the solvents tested for this reaction, trifluoromethylbenzene gave significantly
9

better results than DMF, DME, acetonitrile, or pyridine (yields <20%). Conventional hydrolysis
of 25a with 2 M NaOH and subsequent condensation with 10 afforded 3-bromo derivative 6a in
8
5% yield over 2 steps. The 3-hydrogenated derivative 6b was prepared by PdCl (dppf)·CH Cl
2
2
2
catalyzed debromination with formic acid in 61% yield. The Pd-catalyzed coupling of 6a with
tetramethyltin(IV) in DME yielded the desired 3-methyl compound 6c in 77% yield.
Replacement of the 3-bromo group (6a) with a chloro group was accomplished using 5 equiv
CuCl under microwave irradiation to obtain the desired compound 6d in 39% yield. Cyanation of
6
a at the 3-position with 3.5 equiv of CuCN under microwave irradiation provided the desired 3-
cyano analog 6f in 49% yield. Trifluoromethylation of 25a with potassium trifluoroacetate in the
presence of 2 equiv of CuI afforded the desired compound 25e in 72% yield. The desired 3-
trifluoromethyl derivative 6e was obtained in 61% yield in 2 steps using a method similar to that
used for 6a.
[
Scheme 4]
3
7
The synthesis of 5-bromoimidazo[1,2-a]pyridine derivative 7 was shown in Scheme 5.
3
8
Darzens reaction of the commercially available 4-fluorobenzaldehyde 26 with methyl
dichloroacetate in the presence of sodium methoxide (NaOMe) produced the -chloro- -ketoester
2
7 in 70% yield. An imidazo[1,2-a]pyridine formation of 27 with 2-amino-6-bromopyridine in a
refluxing EtOH provided 28 as a HCl salt in 53% yield. Following hydrolysis of 28 and
condensation with amine 10, the desired 5-bromoimidazo[1,2-a]pyridine analogue 7 was
obtained in 71% yield over 2 steps.
[
Scheme 5]
1
0

3
3
.
Results and Discussion
Creating an efficient interaction between the R or R side chains and CENP-E
1
2
.1
1
2
The SARs of the N-5-alkyl groups (R ) and C-6-carboxamide N-alkyl groups (R ) on 4-
oxo-4,5-dihydrothieno[3,4-c]pyridine scaffolds are shown in Table 1. The synthesized
compounds 1b, 2a–c and 3 were tested for inhibition of ATPase activity of human CENP-E. The
1
2
-hydroxyethyl derivative 2a as R showed similar CENP-E inhibitory activity to that of methyl
derivative 1b. On the other hand, the 2-(N,N-dimethylamino)ethyl derivative 2b showed 25-fold
more potent inhibitory activity than 2a with an IC value of 72 nM. We predicted that the N,N-
5
0
dimethylamino group of 2b could interact with Glu186 on the 3 helix. Next, we examined
1
elongation of the methylene linker at R , but compound 2c exhibited reduced CENP-E inhibitory
1
activity relative to 2b. We judged that the two-carbon linker in R would be optimal, due to a
hydrogen bonding interaction of the N,N-dimethylamino group with Glu186. Based on these
2
results, we envisioned that the 2-(N,N-dimethylamino)ethyl group could move to the R position
and still interact with Glu186 effectively, by which the synthetic accessibility for scaffold
exploration could be greatly improved. As expected, the newly designed compound 3 showed
comparable CENP-E inhibitory potency to that of 2b. This result motivated the investigation of
other fused bicyclic scaffolds utilizing the key N-(3,4-dichlorobenzyl)-N-2-(dimethylamino)ethyl
group to interact with Glu186.
[Table 1]
3.2
Scaffold exploration
1
1

The results of studies directed at scaffold exploration are summarized in Table 2. The 4-
oxo-4,5-dihydrothieno[3,4-c]pyridine core in compound 3 was replaced with various fused
bicyclic rings A, with a goal of enhancing CENP-E inhibitory activity. Since the 4-oxo group of
the 4-oxo-4,5-dihydrothieno[3,4-c]pyridine ring appears to have no interaction with any residue,
we examined 3-arylindole derivative 4 and 1-arylindole derivative 5, which showed significantly
decreased CENP-E inhibitory activities compared with 3. Next, bioisosteric replacement of the
benzene in 5 with a thiophene ring resulted in the discovery of 4-arylthieno[3,2-b]pyrrole 6b,
which showed comparable CENP-E inhibitory activity to that of 5.
3
We next examined various 3-substituents (R ) to occupy the hydrophobic space
(
approximately 4 Å) between the ligand and the 2 helix of CENP-E (Figure 4B). As predicted by
docking studies, 3-methyl derivative 6c exhibited enhanced CENP-E inhibitory activity (IC : 370
5
0
nM) 5-fold more potent than that of 3-unsubstituted derivative 6b. Furthermore, the 3-bromo
derivative 6a, which was synthesized as an intermediate in the preparation of 6b and 6c, showed
1
0-fold more potent CENP-E inhibitory activity (IC : 32 nM) than that of 6c. The 3-chloro
5
0
derivative 6d also showed comparable CENP-E inhibitory activity (IC : 35 nM) to that of 6a. On
5
0
the other hand, 3-trifluoromethyl 6e and 3-cyano 6f derivatives exhibited reduced CENP-E
inhibitory activities compared to those of 6a and 6d. Because the van der Waals volumes of these
3
39
3
-substituents of 6a, 6c–f are similar (12–16 cm /mol), it is difficult to explain the SAR of the 3-
substituents in terms of steric factors.
[Table 2]
3.3
Electrostatic potential map (EPM) analysis of designed compounds and application
for scaffold hopping
1
2

Recently, several studies have shown that electrostatic potential map (EPM) analysis is a
2
5–29
useful tool to understand and evaluate the binding affinities of small molecules for proteins.
Thus, we examined the effects of 3-substituents on the thieno[3,2-b]pyrrole scaffold by
3
visualizing the relationship between the SAR and the EPM for the R group.
The electrostatic profiles of the 3-substituted thieno[3,2-b]pyrroles were calculated using
4
0
MOE (version 2008.1002). To focus on the EPMs of the scaffolds, N,N-dimethylamide analogs
6
a•, 6c•–f• were used as model molecules corresponding to 6a, 6c–f because the 5-carboxamide
N-alkyl group had negligible influence on the EPMs of the scaffolds. As shown in Figure 5, the
electrostatic potentials on the surfaces of 6a•, 6c•–f• were visualized by color. Positive, negative,
and neutral charges are shown in blue, red, and white, respectively. Potent CENP-E inhibitors, 3-
bromo (6a•) and 3-chloro (6d•) derivatives, exhibited a neutral charge of electrostatic potential
(
white) on the thiophene moiety. In contrast, the 3-methyl derivative (6c•) showed a negative
electrostatic potential (red), while 3-trifluoromethyl (6e•) and 3-cyano (6f•) derivatives exhibited
a positive charge (blue). These changes on the EPM are induced by the electronic characteristics
of the 3-substituents. Based on these results, we proposed that the optimal EPM of the fused
bicyclic scaffolds should be neutral (white) on the left side. This hypothesis was also supported
by docking studies, which predicted ring A to bind in the hydrophobic pocket constructed by
neutral amino acid residues, including Ile182.
[Figure 5]
Despite possessing potent CENP-E inhibitory activity, compound 6a exhibited
insufficient cellular activity (see below). Accordingly, we sought to discover other scaffolds with
potent cellular activity and conducted in silico evaluation of all proposed scaffolds using EPM
1
3

before initiating actual syntheses. For example, bromo- indoles Br-4• and Br-5• were not
prepared because they were predicted to possess negative EPMs (red) based on the extremely
negative nature of the parent scaffold (H-4• and H-5• in Figure 6). Thus, many bicyclic scaffolds
were screened using in silico EPM evaluation, and eventually we identified imidazo[1,2-
a]pyridine scaffold (H-7•) as a promising scaffold for potent CENP-E inhibition. EPM analysis of
H-7• indicated that the N-1-nitrogen on the right ring plays an important role in withdrawing
electron density, shifting it from the left to the right ring. Based on our SAR study of thieno[3,2-
b]pyrroles (Table 2), introduction of an X group at the imidazo[1,2-a]pyridine 5-position was
thought to be a promising approach to enhancing CENP-E inhibitory activity by occupying the
hydrophobic pocket (Figure 4B). Since the combination of the imidazo[1,2-a]pyridine scaffold
and bromine at the 5-position (Br-7•) showed more favorable EPM than the indole derivatives
(Br-4• and Br-5•), we synthesized 5-bromoimidazo[1,2-a]pyridine 7. As predicted by EPM
analysis, compound 7 showed potent CENP-E inhibitory activity with an IC value of 50 nM,
5
0
comparable to that of 6a.
[Figure 6]
3.4
Evaluation of biological and physicochemical properties of compound 6a and 7
The biological and physicochemical properties of 3-bromothieno[3,2-b]pyrrole (6a) and
5-bromoimidazo[1,2-a]pyridine (7) derivatives are summarized in Table 3. We evaluated these
compounds for their ability to induce accumulation of phosphorylated histone H3 (p-HH3), a PD
marker of mitotic arrest, in HeLa cells using FACS. The cellular activities were measured as the
concentration required to induce a response halfway between the baseline and maximum for p-
HH3 positive cell count (%), and are reported as EC values. Both compounds 6a and 7 showed
5
0
1
4

cellular p-HH3 accumulation activity with EC values of 3000 nM and 1540 nM, respectively. It
5
0
was suggested that both compounds demonstrate anti-mitotic activity in HeLa cells along with
CENP-E inhibition. In a parallel artificial membrane permeability (PAMPA) assay system,
compound 7 showed higher cellular permeability (pH 7.4: 353 nm/sec) than that of 6a (41
nm/sec). We assumed that the large improvement in PAMPA value would likely be caused by
lower lipophilicity (CLog P: 5.3 vs 5.7). Another explanation of the improved PAMPA value
could be intramolecular hydrogen bonding between the protonated dimethyl amino group and the
4
1,42
electron lone pair of the imidazo[1,2-a]pyridine nitrogen atom under physiological conditions.
Thus, we judged 5-bromoimidazo[1,2-a]pyridine derivative 7 to represent a novel class of
promising CENP-E inhibitors.
[Table 3]
4.
Conclusion
We designed and synthesized fused bicyclic scaffolds for use in the synthesis of CENP-E
inhibitors. The introduction of a basic 2-(dimethylamino)ethyl sidechain into the 4-oxo-4,5-
dihydrothieno[3,4-c]pyridine scaffold significantly enhanced CENP-E inhibitory activity, which
indicated that the basic substituent could likely interact with Glu186. This result motivated the
investigation of other fused bicyclic scaffolds utilizing the key N-(3,4-dichlorobenzyl)-N-2-
(
dimethylamino)ethyl group to interact with Glu186. SAR studies of various fused scaffolds led
us to the identification of 3-bromothieno[3,2-b]pyrrole 6a, which showed potent CENP-E
inhibitory activity. To better understand the SAR of 3-substituted thieno[3,2-b]pyrrole derivative
6
, EPM analysis was found to be an effective tool for predicting the potency of CENP-E
inhibitors. On the basis of this finding, we designed 5-bromoimidazo[1,2-a]pyridine derivative 7,
1
5

which proved to be a novel and potent CENP-E inhibitor (IC : 50 nM) and exhibited enhanced
5
0
cellular activity (p-HH3 EC : 1540 nM).
5
0
Thus, the use of homology modeling and EPM analysis enabled us to accelerate the
rational drug design of CENP-E inhibitors, with compound 7 being a promising new compound
in this area.
5
.
Experimental Section
Chemistry
5
5
.1
.1.1 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
0 F254 plates (Merck) or NH TLC plates (Fuji Silysia Chemical Ltd.). Flash column
6
chromatography separations were performed on Purif-Pack (SI or NH, Fuji Silysia Chemical
Ltd.). Melting points, determined on a SRS OptiMelt automated melting point apparatus, are
1
uncorrected. The proton nuclear magnetic resonance ( H 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 ( ) 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 (Agilent1200SL/Agilent6130MS), 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
1
6

ionization mode (ESI+). The column used was an L-column 2 ODS (3.0 x 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 minutes, 90% over the next 1.5 minutes, after which
the column was equilibrated to 5% for 0.5 minutes. Elemental analyses (Anal.) 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.
The following abbreviations are used. DME = 1,2-dimethoxyethane, DMF = N,N-
dimethylformamide, DMSO = dimethyl sulfoxide, Et N = triethyl amine, Et O = diethyl ether,
3
2
EtOAc = ethyl acetate, EtOH = ethanol, HATU = O-(7-azabenzotriazol-1-yl)-1,1,3,3-
i
tetramethyluronium hexafluorophosphate, Pr O = diisopropyl ether, NMP = 1-methyl-2-
2
i
pyrrolidone,
Pr EtN
=
N,N-diisopropylethylamine,
PdCl (dppf)·CH Cl
[1,1'-
2
2
2
2
=
bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex, THF =
tetrahydrofuran.
5
.1.2 N-(3,4-Dichlorobenzyl)-7-(4-fluorophenyl)-N,5-dimethyl-4-oxo-4,5-
dihydrothieno[3,4-c]pyridine-6-carboxamide (1b).
To a solution of ethyl 7-(4-fluorophenyl)-5-methyl-4-oxo-4,5-dihydrothieno[3,4-
1
7
c]pyridine-6-carboxylate 8 (290 mg, 0.88 mmol) in THF/EtOH (1:1, 5 mL) was added 1 M
NaOH (3.5 mL, 3.5 mmol), and then the reaction mizture was refluxed for 6 h. After cooling at
room temperature, the solvents were evaporated under reduced pressure. The resulting solid was
dissolved in water (10 mL) and the pH was adjusted to 3 with 1 M HCl. The mixture was
extracted with EtOAc/THF (1:1, 3 × 50 mL), dried over MgSO and concentrated in vacuo to
4
give 7-(4-fluorophenyl)-5-methyl-4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxylic acid (230
1
7

1
mg, 88%) as a light yellow solid. H NMR (300 MHz, DMSO-d ) 3.42 (3H, s), 7.28–7.34 (3H,
6
m), 7.41–7.45 (2H, m), 8.59 (1H, d, J = 3.3 Hz), 14.01 (1H, br s).
To a solution of 7-(4-fluorophenyl)-5-methyl-4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-
carboxylic acid (101 mg, 0.33 mmol) in DMF (2.0 mL) were added successively 1-(3,4-
dichlorophenyl)-N-methylmethanamine 9 (95 mg, 0.50 mmol), HATU (190 mg, 0.50 mmol) and
Et N (101 mg, 1.0 mmol) at room temperature. The resulting mixture was stirred at room
3
temperature for 36 h. The reaction mixture was diluted with EtOAc/water (1:1, 60 mL). The
separated aqueous layer was extracted with EtOAc (2 × 20 mL). The separated organic layers
were combined, and dried over MgSO and concentrated in vacuo. The residue was purified by
4
column chromatography (silica gel, eluted with 0–100% EtOAc in n-hexane) and crystallized
+
from Et O to give 1b (18.6 mg, 12%) as colorless crystals; mp 113–115 °C. MS (ESI+): [M + H]
2
1
4
75.0. H NMR (300 MHz, CDCl ) 2.71 (3H, s), 3.50 (3H, s), 3.93–4.02 (1H, m), 4.85–4.94
3
(1H, m), 6.49–6.55 (1H, m), 7.02–7.25 (5H, m), 7.27–7.55 (2H, m), 8.42–8.45 (1H, m). HRMS
+
(
ESI): calcd for C H Cl FN O S [M + H] 475.0445. Found 475.0406.
2
3
17
2
2
2
5
.1.3 N-(3,4-Dichlorobenzyl)-7-(4-fluorophenyl)-5-(2-hydroxyethyl)-N-methyl-4-oxo-4,5-
dihydrothieno[3,4-c]pyridine-6-carboxamide (2a).
To a solution of 9 (950 mg, 5.0 mmol) in toluene (2.0 mL) was added 1.8 M
trimethylaluminum in toluene (2.8 mL, 5.0 mmol) at room temperature. The resulting mixture
was stirred at room temperature for 0.5 h. To the mixture was added a solution of 10-(4-
fluorophenyl)-3,4-dihydro-1H,6H-thieno[3',4':4,5]pyrido[2,1-c][1,4]oxazine-1,6-dione 16a (155
mg, 0.5 mmol) in toluene (2.0 mL) at room temperature, and the resulting mixture was stirred at
room temperature for 0.5 h and then at 80 °C for 2 h. After cooling at room temperature, the
reaction mixture was quenched with water (20 mL) and diluted with EtOAc (20 mL). After
®
removal of insoluble precipitates by filtration through a pad of Celite , the filtrate was extracted
1
8

with EtOAc (2 × 20 mL). The combined organic layers were dried over MgSO and concentrated
4
in vacuo. The residue was purified by column chromatography (NH silica gel, eluted with 0–
1
00% EtOAc in n-hexane) to give 2a (150 mg, 60%) as a light brown amorphous solid. MS
+
1
(
ESI+): [M + H] 505.1. H NMR (300 MHz, CDCl ) 2.69–2.72 (3H, m), 3.34 (1H, t, J = 5.1
3
Hz), 3.79–4.02 (3H, m), 4.06 (1H, d, J = 14.7 Hz), 4.42–4.52 (1H, m), 4.74 (1H, d, J = 14.5 Hz),
.56–6.61 (1H, m), 7.05–7.14 (4H, m), 7.28–7.50 (3H, m), 8.44–8.48 (1H, m). Anal. Calcd for
C H Cl FN O S·0.2H O: C, 56.63; H, 3.84; N, 5.50. Found: C, 56.68; H, 3.93; N, 5.46.
6
2
4
19
2
2
3
2
5
.1.4 N-(3,4-Dichlorobenzyl)-5-(2-(dimethylamino)ethyl)-7-(4-fluorophenyl)-N-methyl-4-
oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide (2b).
To a solution of ethyl 5-(2-(dimethylamino)ethyl)-7-(4-fluorophenyl)-4-oxo-4,5-
dihydrothieno[3,4-c]pyridine-6-carboxylate 16b (96 mg, 0.25 mmol) in EtOH/THF (1:1, 2.0 mL)
was added 8 M NaOH (1.0 mL, 8.0 mmol), and the resulting mixture was stirred at 80 °C for 6 h.
The reaction mixture was neutralized with 6 M HCl, and the reaction mixture was concentrated in
vacuo. The residue was dissolved in EtOH at 50 °C, and the insoluble precipitate was removed
by filtration. The filtrate was concentrated in vacuo to give 5-(2-(dimethylamino)ethyl)-7-(4-
fluorophenyl)-4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxylic acid (89 mg, quantitative) as
a colorless solid. This material was used in the next reaction without further purification. MS
+
1
(
ESI+): [M + H] 361.1. H NMR (300 MHz, DMSO-d ) 2.18 (6H, s), 2.54–2.61 (2H, m), 4.01–
6
4
3
.11 (2H, m), 7.05 (1H, d, J = 3.2 Hz), 7.10–7.20 (2H, m), 7.48–7.56 (2H, m), 8.39 (1H, d, J =
.2 Hz).
Compound 2b (115 mg) was prepared from 5-(2-(dimethylamino)ethyl)-7-(4-
fluorophenyl)-4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxylic acid (89 mg, 0.25 mmol), 9
70 mg. 0.37 mmol), HATU (141 mg, 0.37 mmol) and Et N (62 mg, 0.62 mmol) by a method
(
3
similar to that described for 1b. Yield: 33% in 2 steps; pale yellow amorphous solid. MS (ESI+):
1
9

+
1
[
M + H] 532.1. H NMR (300 MHz, CDCl ) 2.30 (6H, s), 2.56–2.67 (1H, m), 2.70 (3H, s),
3
2
.73–2.81 (1H, m), 3.67–3.82 (1H, m), 3.89 (1H, d, J = 14.5 Hz), 4.23–4.38 (1H, m), 4.90 (1H, d,
J = 14.5 Hz), 6.53 (1H, dd, J = 8.2, 2.0 Hz), 7.02–7.16 (4H, m), 7.22–7.50 (3H, m), 8.43 (1H, d, J
=
3.2 Hz). Anal. Calcd for C H Cl FN O S: C, 58.65; H, 4.54; N, 7.89. Found: C, 58.38; H, 4.66;
2
6
24
2
3
2
N, 7.74.
5
.1.5 N-(3,4-Dichlorobenzyl)-5-(3-(dimethylamino)propyl)-7-(4-fluorophenyl)-N-methyl-4-
oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide (2c).
To a solution of 14 (160 mg, 0.50 mmol) in THF (2.0 mL) was added N,N-
dimethylpropane-1,3-diamine (102 mg, 1.0 mmol) at room temperature. The resulting mixture
was stirred at room temperature for 2 h. The reaction mixture was purified by column
chromatography (silica gel, eluted with 0–100% EtOAc in n-hexane) to give ethyl 3-(4-((3-
(dimethylamino)propyl)carbamoyl)-3-thienyl)-3-(4-fluorophenyl)-2-hydroxyacrylate 15c (210
+
mg, quantitative) as colorless oil. MS (ESI+): [M + H] 421.1.
To a solution of 15c (210 mg, 0.50 mmol) in toluene (5.0 mL) was added 4-
toluenesulfonic acid monohydrate (190 mg, 1.0 mmol) at room temperature. The resulting
mixture was stirred at 80 °C for 2 h. The reaction mixture was diluted with EtOAc/water (1:1, 40
mL). The aqueous layer was extracted with EtOAc (2 × 20 mL). The combined organic layers
were dried over MgSO and concentrated in vacuo. The residue was purified by column
4
chromatography (silica gel, eluted with 0–100% EtOAc in n-hexane) to give Ethyl 5-(3-
(dimethylamino)propyl)-7-(4-fluorophenyl)-4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-
+
carboxylate 16c (201 mg, quantitative) as pale brown oil. MS (ESI+): [M + H] 403.1.
5-(3-(Dimethylamino)propyl)-7-(4-fluorophenyl)-4-oxo-4,5-dihydrothieno[3,4-
c]pyridine-6-carboxylic acid (187 mg) was prepared from 16c (200 mg, 0.50 mmol) and 8 M
NaOH (1.0 mL. 8.0 mmol) by a method similar to that described for 2b. Yield: quantitative;
2
0

colorless solid. This material was used in the next reaction without further purification. MS
+
(
ESI+): [M + H] 375.1.
Compound 2c (62 mg) was prepared from 5-(3-(dimethylamino)propyl)-7-(4-
fluorophenyl)-4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxylic acid (187 mg, 0.50 mmol), 9
114 mg. 0.60 mmol), HATU (460 mg, 0.60 mmol) and Et N (200 mg, 2.0 mmol) by a method
(
3
similar to that described for 1b. Yield: 23% in 4 steps from 14; pale yellow amorphous solid. MS
+
1
(
ESI+): [M + H] 546.1. H NMR (300 MHz, CDCl ) 1.85–2.11 (2H, m), 2.25 (6H, s), 2.31–
3
2.50 (2H, m), 2.65–2.74 (3H, m), 3.53–3.65 (1H, m), 4.06 (1H, d, J = 14.5 Hz), 4.20–4.35 (1H,
m), 4.72 (1H, d, J = 14.5 Hz), 6.48–6.62 (1H, m), 7.05–7.25 (4H, m), 7.27–7.55 (3H, m), 8.42
(
1H, d, J = 3.2 Hz). Anal. Calcd for C H Cl FN O S: C, 59.34; H, 4.80; N, 7.69. Found: C,
2
7
26
2
3
2
5
5
9.67; H, 5.12; N, 7.67.
.1.6 N-(3,4-Dichlorobenzyl)-N-(2-(dimethylamino)ethyl)-7-(4-fluorophenyl)-5-methyl-4-
oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide (3).
Compound 3 (35 mg) was prepared from 7-(4-fluorophenyl)-5-methyl-4-oxo-4,5-
dihydrothieno[3,4-c]pyridine-6-carboxylic acid (8, 101 mg, 0.33 mmol), N'-(3,4-dichlorobenzyl)-
N,N-dimethylethane-1,2-diamine 10 (124 mg. 0.50 mmol), HATU (190 mg, 0.50 mmol) and Et N
3
(
202 mg, 2.0 mmol) by a method similar to that described for 1b. Yield: 20%; colorless
+
1
amorphous solid. MS (ESI+): [M + H] 532.1. H NMR (300 MHz, CDCl ) 2.03–2.13 (6H, m),
3
2
.23–2.44 (2H, m), 2.64–3.50 (2H, m), 3.54–3.59 (3H, m), 4.01–5.02 (2H, m), 6.49–6.65 (1H,
m), 7.05–7.24 (4H, m), 7.28–7.58 (3H, m), 8.40–8.45 (1H, m). Anal. Calcd for C H Cl FN O S:
2
6
24
2
3
2
C, 58.65; H, 4.54; N, 7.89. Found: C, 58.93; H, 4.85; N, 7.64.
.1.7 N-(3,4-Dichlorobenzyl)-N-(2-(dimethylamino)ethyl)-3-(4-fluorophenyl)-1-methyl-1H-
indole-2-carboxamide (4).
5
2
1

To a solution of ethyl 3-(4-fluorophenyl)-1-methyl-1H-indole-2-carboxylate 18 (412 mg,
1
.4 mmol) in THF/EtOH (1:2, 9 mL) were successively added 8 M NaOH (3.0 mL, 24 mmol)
and 2 M NaOH (5.0 mL, 10 mmol) at 75 °C. The resulting mixture was stirred at 75 °C for 1 h.
After removal of solvent under reduced pressure, the residue was diluted with water (20 mL).
The mixture was acidified to pH 2 with 6 M HCl (6 mL) and the resulting precipitate was
collected by filtration and washed with water to give 3-(4-fluorophenyl)-1-methyl-1H-indole-2-
carboxylic acid (331 mg, 89%) as a colorless solid. This material was used in the next reaction
+
1
without further purification. MS (ESI+): [M + H] 269.9. H NMR (300 MHz, DMSO-d ) 4.02
6
(
3H, s), 7.09–7.17 (1H, m), 7.21–7.31 (2H, m), 7.33–7.48 (4H, m), 7.62 (1H, d, J = 8.4 Hz),
2.98 (1H, br s).
To a solution of 3-(4-fluorophenyl)-1-methyl-1H-indole-2-carboxylic acid (134 mg, 0.50
1
mmol) in DMF (2.5 mL) were added successively 10 (130 mg, 0.60 mmol), HATU (200 mg, 0.60
mmol) and Et N (200 mg, 2.0 mmol) at room temperature. The resulting mixture was stirred at
3
room temperature for 18 h. The reaction mixture was diluted with EtOAc/water (1:1, 40 mL).
The aqueous layer was extracted with EtOAc (2 × 20 mL). The combined organic layers were
dried over MgSO and concentrated in vacuo. The residue was purified by column
4
chromatography (NH silica gel, eluted with 0–100% EtOAc in n-hexane) to give 4 (107 mg,
+
1
4
3%) as colorless oil. MS (ESI+): [M + H] 498.0. H NMR (300 MHz, CDCl ) 2.07–2.29 (6H,
3
m), 2.38–2.72 (2H, m), 3.36–3.72 (2H, m), 3.76–3.89 (3H, m), 4.02–5.05 (2H, m), 6.51–6.79 (1H,
m), 7.07–7.24 (4H, m), 7.31–7.58 (5H, m), 7.69–7.77 (1H, m). HRMS (ESI): calcd for
+
C H Cl FN O [M + H] 498.1510. Found 498.1467.
2
7
26
2
3
5
.1.8 N-(3,4-Dichlorobenzyl)-N-(2-(dimethylamino)ethyl)-1-(4-fluorophenyl)-3-methyl-1H-
indole-2-carboxamide (5).
2
2

To a solution of ethyl 1-(4-fluorophenyl)-3-methyl-1H-indole-2-carboxylate 21 (1.0 g, 3.4
mmol) in THF/EtOH (1:1, 10 mL) was added 2 M NaOH (8.0 mL, 16 mmol). The resulting
mixture was stirred at 65 °C for 1.5 h. To the reaction mixture was added 8 M NaOH (1.0 mL,
8
.0 mmol), and the resulting mixture was stirred at 75 °C for 1 h. After removal of solvent under
reduced pressure, the residue was diluted with water (20 mL). The mixture was acidified to pH 2
with 6 M HCl (4.5 mL), and the resulting precipitate was collected by filtration and washed with
water to give 1-(4-fluorophenyl)-3-methyl-1H-indole-2-carboxylic acid (865 mg, 95%) as a
1
colorless solid. This material was used in the next reaction without further purification. H NMR
(
300 MHz, DMSO-d ) 2.59 (3H, s), 6.99 (1H, d, J = 8.3 Hz), 7.13–7.22 (1H, m), 7.25–7.43 (5H,
6
m), 7.76 (1H, d, J = 7.9 Hz), 12.82 (1H, br s).
To a solution of 1-(4-fluorophenyl)-3-methyl-1H-indole-2-carboxylic acid (192 mg, 0.71
mmol) in DMF (5.0 mL) were successively added 10 (160 mg, 0.65 mmol), HATU (295 mg, 0.78
i
mmol) and Pr EtN (251 mg, 1.9 mmol) at room temperature. The resulting mixture was stirred at
2
room temperature for 30 h. The reaction mixture was partitioned between EtOAc (20 mL) and
water (20 mL). The aqueous layer was extracted with EtOAc (3 × 20 mL). The combined organic
layers were washed with water (2 × 20 mL) and brine (20 mL), dried over Na SO and
2
4
concentrated in vacuo. The residue was purified by column chromatography (NH silica gel,
eluted with 0–30% EtOAc in n-hexane) to give 5 (287 mg, 89%) as pale yellow oil. MS (ESI+):
+
1
[
(
M + H] 498.0. H NMR (300 MHz, CDCl ) 1.96–2.15 (6H, m), 2.19–2.38 (2H, m), 2.39–2.43
3
3H, m), 2.93–3.46 (2H, m), 4.21–5.11 (2H, m), 6.70–6.77 (1H, m), 6.90–7.25 (6H, m), 7.27–
7
.49 (3H, m), 7.59–7.70 (1H, m). Anal. Calcd for C H Cl FN O: C, 65.06; H, 5.26; N, 8.43.
2
7
26
2
3
Found: C, 64.92; H, 5.34; N, 8.41.
5
.1.9 3-Bromo-N-(3,4-dichlorobenzyl)-N-(2-(dimethylamino)ethyl)-4-(4-fluorophenyl)-4H-
thieno[3,2-b]pyrrole-5-carboxamide (6a).
2
3

3-Bromo-4-(4-fluorophenyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (681 mg) was
prepared from ethyl 3-bromo-4-(4-fluorophenyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate 25a (814
mg, 2.2 mmol) and 2 M NaOH (6 mL, 12 mmol) by a method similar to that described for 5.
1
Yield: 90%; colorless solid. H NMR (300 MHz, DMSO-d ) 7.23–7.32 (2H, m), 7.32 (1H, s),
6
7
.39–7.48 (2H, m), 7.65 (1H, d, J = 0.6 Hz), 12.72 (1H, br s).
Compound 6a (502 mg) was prepared from 3-bromo-4-(4-fluorophenyl)-4H-thieno[3,2-
b]pyrrole-5-carboxylic acid (316 mg, 0.93 mmol), 10 (275 mg, 1.1 mmol), HATU (456 mg, 1.2
i
mmol) and Pr EtN (300 mg, 2.3 mmol) by a method similar to that described for 5. Yield: 95%;
2
+
1
pale yellow oil. MS (ESI+): [M + H] 568.0. H NMR (300 MHz, CDCl ) 2.13 (6H, d, J = 3.4
3
Hz), 2.30–2.44 (2H, m), 3.39 (2H, dt, J = 6.3, 3.2 Hz), 4.67 (2H, br s), 6.67 (1H, br s), 6.86 (1H,
br s), 7.07–7.20 (4H, m), 7.31–7.48 (3H, m). Anal. Calcd for C H BrCl FN OS: C, 50.63; H,
2
4
21
2
3
3
.72; N, 7.38. Found: C, 50.81; H, 3.86; N, 7.37.
.1.10 N-(3,4-Dichlorobenzyl)-N-(2-(dimethylamino)ethyl)-4-(4-fluorophenyl)-4H-
5
thieno[3,2-b]pyrrole-5-carboxamide (6b).
To a solution of 6a (214 mg, 0.38 mmol) in DME (1.5 mL) were added PdCl (dppf)
2
·
CH Cl (31 mg, 0.038 mmol), Et N (114 mg, 1.1 mmol) and formic acid (28 µL, 0.75 mmol) at
2
2
3
room temperature. The resulting mixture was stirred at 85 °C under nitrogen atmosphere for 8 h.
The reaction mixture was partitioned between EtOAc (15 mL) and saturated aqueous NaHCO3
(
15 mL). The aqueous layer was extracted with EtOAc (3 × 15 mL). The combined organic layers
were washed with brine(15 mL), dried over MgSO and concentrated in vacuo. The residue was
4
purified by column chromatography (NH silica gel, eluted with 10–40% EtOAc in n-hexane) to
+
1
give 6b (113 mg, 61%) as pale yellow oil. MS (ESI+): [M + H] 490.1. H NMR (300 MHz,
CDCl ) 2.13 (6H, s), 2.39 (2H, t, J = 6.4 Hz), 3.49 (2H, t, J = 6.2 Hz), 4.70 (2H, s), 6.74 (1H, br
3
2
4

s), 6.87 (1H, d, J = 5.3 Hz), 6.98 (1H, dd, J = 8.2, 1.8 Hz), 7.13–7.25 (4H, m), 7.35–7.46 (3H, m).
+
HRMS (ESI): calcd for C H Cl FN OS [M + H] 490.0917. Found 490.0877.
2
4
22
2
3
5
.1.11 N-(3,4-Dichlorobenzyl)-N-(2-(dimethylamino)ethyl)-4-(4-fluorophenyl)-3-methyl-4H-
thieno[3,2-b]pyrrole-5-carboxamide (6c).
To a solution of 6a (106 mg, 0.19 mmol) in DME (2 mL) were added tetramethyltin(IV)
(
165 mg, 0.93 mmol) and PdCl (dppf)·CH Cl (30 mg, 0.037 mmol) at room temperature. The
2
2
2
resulting mixture was stirred at 90 °C under nitrogen atmosphere for 1 h. After removal of
®
insoluble precipitate by filtration through a pad of Celite , the filtrate was concentrated in vacuo.
The residue was purified by column chromatography (NH silica gel, eluted with 5–35% EtOAc
+
1
in n-hexane) to give 6c (72 mg, 77%) as pale yellow oil. MS (ESI+): [M + H] 504.1. H NMR
(
300 MHz, CDCl ) 1.83 (3H, s), 2.14 (6H, s), 2.38 (2H, t, J = 6.4 Hz), 3.44 (2H, t, J = 6.4 Hz),
3
4
.64–4.77 (2H, m), 6.66 (1H, s), 6.78 (1H, s), 6.91 (1H, br s), 7.10–7.20 (3H, m), 7.31–7.43 (3H,
+
m). HRMS (ESI): calcd for C H Cl FN OS [M + H] 504.1074. Found 504.1065.
2
5
24
2
3
5
.1.12 3-Chloro-N-(3,4-dichlorobenzyl)-N-(2-(dimethylamino)ethyl)-4-(4-fluorophenyl)-4H-
thieno[3,2-b]pyrrole-5-carboxamide (6d).
To a solution of 6a (102 mg, 0.18 mmol) in DMF (0.5 mL) was added copper(I) chloride
(
88 mg, 0.89 mmol) at room temperature. The resulting mixture was stirred under microwave
irradiation at 220 °C for 45 min. The reaction mixture was partitioned between EtOAc (20 mL)
and saturated aqueous NaHCO (20 mL). The aqueous layer was extracted with EtOAc (2 × 20
3
mL). The combined organic layers were washed with water(2 × 20 mL) and brine(20 mL), dried
over MgSO and concentrated in vacuo. The residue was purified by preparative HPLC (YMC
4
CombiPrep-ODS-A, eluted with 40–60% acetonitrile in water containing 0.1% TFA). The
desired fractions were combined and concentrated in vacuo. The residue was neutralized with
saturated aqueous NaHCO (50 mL). The mixture was extracted with EtOAc (2 × 50 mL) and the
3
2
5

combined extracts were washed with brine (50 mL), dried over MgSO and concentrated in vacuo
4
+
1
to give 6d (36 mg, 39%) as pale yellow oil. MS (ESI+): [M + H] 524.1. H NMR (300 MHz,
CDCl ) 2.13 (6H, s), 2.36 (2H, t, J = 6.4 Hz), 3.40 (2H, t, J = 6.4 Hz), 4.67 (2H, br s), 6.67 (1H,
3
br s), 6.78–6.92 (1H, m), 6.98 (1H, d, J = 1.1 Hz), 7.09–7.20 (3H, m), 7.30–7.44 (3H, m). HRMS
+
(
ESI): calcd for C H Cl FN OS [M + H] 524.0528. Found 524.0543.
2
4
21
3
3
5
.1.13 N-(3,4-Dichlorobenzyl)-N-(2-(dimethylamino)ethyl)-4-(4-fluorophenyl)-3-
(trifluoromethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide (6e).
4-(4-Fluorophenyl)-3-(trifluoromethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (310
mg) was prepared from ethyl 4-(4-fluorophenyl)-3-(trifluoromethyl)-4H-thieno[3,2-b]pyrrole-5-
carboxylate 25e (430 mg, 1.2 mmol) and 2 M NaOH (3 mL, 6.0 mmol) by a method similar to
1
that described for 5. Yield: 78%; colorless solid. H NMR (300 MHz, DMSO-d ) 7.23–7.33 (2H,
6
m), 7.39–7.46 (3H, m), 8.27 (1H, s), 12.65 (1H, br s).
Compound 6e (133 mg) was prepared from 4-(4-fluorophenyl)-3-(trifluoromethyl)-4H-
thieno[3,2-b]pyrrole-5-carboxylic acid (101 mg, 0.31 mmol), 10 (91 mg, 0.37 mmol), HATU
i
(
140 mg, 0.37 mmol) and Pr EtN (100 mg, 0.77 mmol) by a method similar to that described for
2
+
1
5
. Yield: 78%; pale yellow oil. MS (ESI+): [M + H] 558.1. H NMR (300 MHz, CDCl ) 2.12
3
(
6H, s), 2.25–2.44 (2H, m), 3.37 (2H, t, J = 6.4 Hz), 4.66 (2H, br s), 6.70 (2H, br s), 7.06–7.20
(
3H, m), 7.38 (3H, dd, J = 8.7, 4.7 Hz), 7.59 (1H, s). Anal. Calcd for C H Cl F N OS: C, 53.77;
2
5
21
2
4
3
H, 3.79; N, 7.52. Found: C, 54.01; H, 3.63; N, 7.40.
5.1.14 3-Cyano-N-(3,4-dichlorobenzyl)-N-(2-(dimethylamino)ethyl)-4-(4-fluorophenyl)-4H-
thieno[3,2-b]pyrrole-5-carboxamide (6f).
To a solution of 6a (100 mg, 0.18 mmol) in NMP (0.5 mL) was added copper(I) cyanide
(
55 mg, 0.62 mmol) at room temperature. The resulting mixture was stirred under microwave
irradiation at 220 °C for 45 min. The reaction mixture was partitioned between EtOAc (15 mL)
2
6

and saturated aqueous NaHCO (15 mL). The aqueous layer was extracted with EtOAc (3 × 15
3
mL). The combined organic layers were washed with saturated aqueous NaHCO (15 mL), water
3
(
15 mL) and brine (15 mL), dried over MgSO and concentrated in vacuo. The residue was
4
purified by column chromatography (NH silica gel, eluted with 10–45% EtOAc in n-hexane) to
+
1
give 6f (44 mg, 49%) as yellow oil. MS (ESI+): [M + H] 515.1. H NMR (300 MHz, CDCl )
3
2
.11 (6H, s), 2.35 (2H, d, J = 3.8 Hz), 3.31–3.46 (2H, m), 4.66 (2H, br s), 6.74 (1H, br s), 6.90
(
1H, br s), 7.13–7.24 (3H, m), 7.29–7.38 (1H, m), 7.40–7.51 (2H, m), 7.81 (1H, d, J = 4.3 Hz).
+
HRMS (ESI): calcd for C H Cl FN OS [M + H] 515.0870. Found 515.0836.
2
5
21
2
4
5
.1.15 5-Bromo-N-(3,4-dichlorobenzyl)-N-[2-(dimethylamino)ethyl]-3-(4-
fluorophenyl)imidazo[1,2-a]pyridine-2-carboxamide (7).
To a solution of methyl 5-bromo-3-(4-fluorophenyl)imidazo[1,2-a]pyridine-2-carboxylate
hydrochloride 28 (1.2 g, 3.4 mmol) in EtOH/THF (3:2, 10 mL) was added 2 M NaOH (7.0 mL),
and the resulting mixture was stirred at 65 °C for 0.5 h. The reaction mixture was cooled at room
temperature and concentrated under reduced pressure. The residue was diluted with water (100
mL) and 6 M HCl (2.5 mL) at 0 °C, and the resulting precipitate was collected by filtration and
washed with water (2 × 10 mL) to give 5-bromo-3-(4-fluorophenyl)imidazo[1,2-a]pyridine-2-
carboxylic acid (940 mg, 91%) as a colorless solid. This material was used in the next reaction
+
1
without further purification. MS (ESI+): [M + H] 335.0. H NMR (300 MHz, DMSO-d ) 7.18–
6
7
.32 (4H, m), 7.44–7.56 (2H, m), 7.70–7.81 (1H, m), 12.63 (1H, br s).
To a solution of 5-bromo-3-(4-fluorophenyl)imidazo[1,2-a]pyridine-2-carboxylic acid
(
(
451 mg, 1.3 mmol) in DMF (6.0 mL) were successively added 10 (349 mg, 1.4 mmol), HATU
i
560 mg, 1.5 mmol) and Pr EtN (433 mg, 3.4 mmol), and the resulting mixture was stirred at
2
room temperature for 24 h. The reaction mixture was partitioned between EtOAc (15 mL) and
saturated aqueous NaHCO (15 mL). The aqueous layer was extracted with EtOAc (3 × 15 mL).
3
2
7

The combined organic layers were washed with water (2 × 15 mL) and brine(15 mL), dried over
MgSO and concentrated in vacuo. The residue was purified by column chromatography (NH
4
silica gel, eluted with 20–70% EtOAc in n-hexane) to give 7 (591 mg, 78%) as colorless crystals;
+
1
mp 114–116 °C. MS (ESI+): [M + H] 563.1. H NMR (300 MHz, CDCl ) 2.04–2.20 (6H, m),
3
2
.38–2.48 (2H, m), 3.38–3.45 (2H, m), 4.65–4.80 (2H, m), 6.84–6.99 (1H, m), 7.02–7.19 (5H, m),
.27–7.35 (1H, m), 7.36–7.41 (1H, m), 7.44–7.51 (1H, m), 7.62–7.72 (1H, m). Anal. Calcd for
7
C H BrCl FN O: C, 53.21; H, 3.93; N, 9.93. Found: C, 53.44; H, 3.96; N, 10.04.
2
5
22
2
4
5.1.16 Ethyl diazo(diethoxyphosphoryl)acetate (13).
To a suspension of 60% sodium hydride in mineral oil (0.6 g, 15 mmol) in THF (100 mL)
was added dropwise ethyl (diethoxyphosphoryl)acetate 12 (2.2 g, 10 mmol) at 0 °C, and the
resulting mixture was stirred at 0 °C for 0.5 h. To the reaction mixture was added 4-
acetamidobenzenesulfonyl azide (2.5 g, 10.5 mmol) at 0 °C, and the mixture was stirred at room
temperature for 1 h. The reaction mixture was diluted with EtOAc/water (1:1, 200 mL). After
acidified to pH 2 with 1 M HCl, the aqueous layer was extracted with EtOAc (2 × 50 mL). The
combined organic layers were dried over MgSO and concentrated in vacuo. The residue was
4
purified by column chromatography (silica gel, eluted with 0–100% EtOAc in n-hexane) to give
+
1
1
3 (2.5 g, quantitative) as colorless oil. MS (ESI+): [M + H] 251.0. H NMR (300 MHz, CDCl )
3
1.24–1.41 (9H, m), 4.10–4.32 (6H, m).
5
.1.17 Ethyl 7-(4-fluorophenyl)-4-oxo-4H-thieno[3,4-c]pyran-6-carboxylate (14).
The mixture of 4-(4-fluorobenzoyl)thiophene-3-carboxylic acid 11 (1.0 g, 4.0 mmol), 13
(
1.5 g, 6.0 mmol) and dirhodium(II) tetraacetate (18 mg, 0.04 mmol) in toluene (20 mL) was
stirred at 80 °C for 3 h. After cooling, 1,8-diazabicyclo[5.4.0]undec-7-ene was added to the
reaction mixture at room temperature, and the mixture was stirred at room temperature overnight.
The reaction mixture was diluted with EtOAc/water (1:1, 100 mL). After acidified to pH 1 with 1
2
8

M HCl, the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers
were dried over MgSO and concentrated in vacuo. The residue was purified by column
4
chromatography (silica gel, eluted with 0–100% EtOAc in n-hexane) and solidified from Et O to
2
+
1
give 14 (1.3 g, quantitative) as a colorless amorphous solid. MS (ESI+): [M + H] 319.0. H NMR
(
300 MHz, CDCl ) 1.14 (3H, t, J = 7.2 Hz), 4.17 (2H, q, J = 7.0 Hz), 7.12 (1H, d, J = 3.2 Hz),
3
7
.14–7.21 (2H, m), 7.28–7.35 (2H, m), 8.55 (1H, d, J = 3.2 Hz).
5.1.18 Ethyl 3-(4-fluorophenyl)-2-hydroxy-3-(4-((2-hydroxyethyl)carbamoyl)-3-
thienyl)acrylate (15a).
To a solution of 14 (160 mg, 0.50 mmol) in THF (2.0 mL) was added 2-aminoethanol (60
mg, 1.0 mmol) at room temperature. The resulting mixture was stirred at room temperature for 18
h. The mixture was diluted with EtOAc/water (1:1, 40 mL). The aqueous layer was extracted
with EtOAc (2 × 20 mL). The combined organic layers were dried over MgSO and concentrated
4
in vacuo. The residue was purified by column chromatography (silica gel, eluted with 0–100%
EtOAc in n-hexane) to give 15a (150 mg, 80%) as a colorless amorphous solid. MS (ESI+): [M +
+
1
H] 380.1. H NMR (300 MHz, CDCl ) 1.24 (3H, t, J = 7.2 Hz), 2.33 (1H, t, J = 4.6 Hz), 3.22–
3
3.34 (1H, m), 3.68–3.89 (2H, m), 4.04–4.12 (1H, m), 4.20 (2H, q, J = 7.0 Hz), 4.83 (1H, s), 5.27
(
1H, s), 6.75 (1H, dd, J = 3.0, 1.5 Hz), 6.98–7.08 (2H, m), 7.30–7.37 (2H, m), 8.17 (1H, d, J =
3
.0 Hz).
5.1.19 Ethyl
3-(4-((2-(dimethylamino)ethyl)carbamoyl)-3-thienyl)-3-(4-fluorophenyl)-2-
hydroxyacrylate (15b).
Compound 15b (370 mg) was prepared from 14 (320 mg, 1.0 mmol) and N,N-
dimethylethane-1,2-diamine (180 mg. 2.0 mmol) by a method similar to that described for 15a.
+
1
Yield: 91%; colorless amorphous solid. MS (ESI+): [M + H] 407.1. H NMR (300 MHz, CDCl )
3
1
.16 (3H, t, J = 7.2 Hz), 2.01 (6H, s), 2.15–2.22 (2H, m), 2.48–2.61 (1H, m), 2.89–3.02 (1H, m),
2
9