Discovery and structure-activity relationships study of positive allosteric modulators of the M3 muscarinic acetylcholine receptor

Article abstract of DOI:10.1016/j.bmc.2020.115531

The M₃ muscarinic acetylcholine receptor (mAChR) is a member of the family of mAChRs, which are associated with a variety of physiological functions including the contraction of various smooth muscle tissues, stimulation of glandular secretion, and regulation of a range of cholinergic processes in the central nerve system. We report here the discovery and a comprehensive structure--activity relationships (SARs) study of novel positive allosteric modulators (PAMs) of the M₃ mAChR through a high throughput screening (HTS) campaign. Compound 9 exhibited potent in vitro PAM activity towards the M₃ mAChR and significant enhancement of muscle contraction in a concentration-dependent manner when applied to isolated smooth muscle strips of rat bladder. Compound 9 also showed excellent subtype selectivity over other subtypes of mAChRs including M₁, M₂, and M₄ mAChRs, and moderate selectivity over the M₅ mAChR, indicating that compound 9 is an M₃-preferring M₃/M₅ dual PAM. Moreover, compound 9 displayed acceptable pharmacokinetics profiles after oral dosing to rats. These results suggest that compound 9 may be a promising chemical probe for the M₃ mAChR for further investigation of its pharmacological function both in vitro and in vivo.

Full text of DOI:10.1016/j.bmc.2020.115531

Journal Pre-proofs
Discovery and Structure-Activity Relationships Study of Positive Allosteric
Modulators of the M3 Muscarinic Acetylcholine Receptor
Hiroaki Tanaka, Kenji Negoro, Takanori Koike, Issei Tsukamoto, Kazuhiro
Yokoyama, Jun Maeda, Yusuke Inagaki, Yukinori Shimoshige, Katsutoshi
Ino, Kenichiro Ishizu, Taisuke Takahashi
PII:
S0968-0896(20)30357-6
https://doi.org/10.1016/j.bmc.2020.115531
BMC 115531
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
5 March 2020
22 April 2020
23 April 2020
Please cite this article as: H. Tanaka, K. Negoro, T. Koike, I. Tsukamoto, K. Yokoyama, J. Maeda, Y. Inagaki, Y.
Shimoshige, K. Ino, K. Ishizu, T. Takahashi, Discovery and Structure-Activity Relationships Study of Positive
Allosteric Modulators of the M3 Muscarinic Acetylcholine Receptor, Bioorganic & Medicinal Chemistry (2020),
doi: https://doi.org/10.1016/j.bmc.2020.115531
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Discovery and Structure-Activity Relationships Study of Positive Allosteric Modulators of the
M3 Muscarinic Acetylcholine Receptor
Hiroaki Tanaka, Kenji Negoro,* Takanori Koike, Issei Tsukamoto, Kazuhiro Yokoyama, Jun Maeda,
Yusuke Inagaki, Yukinori Shimoshige, Katsutoshi Ino, Kenichiro Ishizu, and Taisuke Takahashi
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
*Address correspondence regarding this manuscript to:
Kenji Negoro
Drug Discovery Research
Astellas Pharma Inc.
21 Miyukigaoka, Tsukuba
Ibaraki 305-8585, Japan
Tel: +81-29-829-6631
Fax: +81-29-852-5387
E-mail: kenji.negoro@astellas.com

Abstract
The M3 muscarinic acetylcholine receptor (mAChR) is a member of the family of mAChRs,
which are associated with a variety of physiological functions including the contraction of various
smooth muscle tissues, stimulation of glandular secretion, and regulation of a range of cholinergic
processes in the central nerve system. We report here the discovery and a comprehensive
structure-activity relationships (SARs) study of novel positive allosteric modulators (PAMs) of the
M3 mAChR through a high throughput screening (HTS) campaign. Compound 9 exhibited potent in
vitro PAM activity towards the M3 mAChR and significant enhancement of muscle contraction in a
concentration-dependent manner when applied to isolated smooth muscle strips of rat bladder.
Compound 9 also showed excellent subtype selectivity over other subtypes of mAChRs including
M1, M2, and M4 mAChRs, and moderate selectivity over the M5 mAChR, indicating that compound
9 is an M3-preferring M3/M5 dual PAM. Moreover, compound 9 displayed acceptable
pharmacokinetics profiles after oral dosing to rats. These results suggest that compound 9 may be a
promising chemical probe for the M3 mAChR for further investigation of its pharmacological function
both in vitro and in vivo.

Keywords
M3 muscarinic acetylcholine receptor; positive allosteric modulators; allosteric enhancers.

1. Introduction
Muscarinic acetylcholine receptors (mAChRs) are class A G-protein-coupled receptors
(GPCRs), with five receptor subtypes M1 to M5 having been identified to date. M1, M3 and M5
mAChR subtypes preferentially couple to Gq protein and activate the phospholipase C/inositol
1,4,5-riphosphate (IP3)/Ca2+ signaling pathway, whereas M2 and M4 mAChR subtypes couple to Gi/o
protein and lead to inhibition of adenylyl cyclase activity.1 mAChRs mediate a broad range of actions
throughout the peripheral organs and central nervous system (CNS).
The M3 mAChR is expressed in smooth muscle, glands, and the brain. It plays an essential role
in the contraction of many smooth muscle tissues such as the bladder and gastrointestinal tract, the
stimulation of glandular secretion, and the regulation of a variety of cholinergic processes in the
CNS.2 For example, solifenacin (VESIcare®),3 a competitive M3 mAChR antagonist, blocks the
binding of ACh to its receptors and helps relax the smooth muscle of the bladder, enabling the bladder
to accumulate larger volumes of urine. As a result, it may be effective for controlling and reducing
overactive bladder (OAB) symptoms such as urinary frequency, urinary urgency and incontinence
episodes.4 However, the pharmacological effects of direct agonistic stimulation of M3 mAChRs are
hampered by dose limiting side effects and remain unclear due to the poor subtype selectivity of
available ligands.5 mAChRs are known to have highly homologous sequences, thereby hampering
the identification of subtype selective ligands. Moreover, recent studies have revealed that prolonged
exposure of GPCRs to orthosteric agonists cause a reduction in receptor-mediated signaling via
phosphorylation, G protein uncoupling, internalization and down-regulation.6
Based on these facts, we focused on an alternative approach to further study the
pharmacological functions of the M3 mAChR by targeting the receptor using positive allosteric
modulators (PAMs). A PAM is a compound that binds to an allosteric site other than the orthosteric
binding site, primarily to cause a change in the structure of the receptor, with the effect of increasing
the affinity of an agonist to the receptor and changing the agonist’s activity. In the living body, PAMs
themselves do not exhibit an agonistic effect, but increase the effect of an endogenous agonist. The
general advantages of using a PAM over an agonist include: (1) reduced side effects because PAMs
stimulation-dependently do not primarily induce receptor activation but only enhance the activity of
endogenous agonists; (2) high subtype selectivity because PAMs bind to a site other than the ligand-
binding site; and (3) low chance of desensitization that can be observed with an agonist.7 This strategy
has been applied to M1 and M4 mAChRs, which are considered attractive therapeutic targets for the
treatment of neurodegenerative and neuropsychiatric diseases.8 For example, Beshore et al. recently
reported the discovery of MK-7622, a highly selective PAM of the M1 muscarinic receptor, which is

currently under phase 2 clinical trial for the treatment of Alzheimer’s disease.9 In contrast to emerging
studies on selective PAMs for M1 and M4 mAChRs, to our knowledge, only a few studies have
reported selective PAMs for the M3 mAChR, most of which have relatively low affinity and limited
selectivity.5 Therefore, the identification of potent and selective PAMs of the M3 mAChR are needed
for further investigations into the receptor’s pharmacological functions.
Here, we report the discovery of novel PAMs of the M3 mAChR through a high throughput
screening (HTS) campaign and the subsequent structure-activity relationships (SARs) study.
2. Chemistry
The synthesis of compounds 1–4 were performed as outlined in Scheme 1.10 Commercially
available aryl chlorides were converted to the corresponding aryl piperidines 22, 24, 26, and 28, which
were subjected to condensation with 4-(4-chloro-2-thienyl)-1,3-thiazol-2-amine to yield 29–32. The
benzylamine unit was then attached through a Mannich reaction of (2R)-2-methylpyrrolidine, and
subsequent hydrolysis produced compounds 1–4.
F
F
a, b
HO C
2
Cl
HO C
N
CO Et
2
2
N
N
Cl
21
22
c, d
e
Cl
N
CO Me
2
HO C
2
N
CO Me
2
N
S
N
S
N
N
H
N
H
N
g
h, i
23
24
R CO R'
2
R CO H
2
Cl
Cl
N
O
O
S
S
N
N
HO C
2
Cl
N
HO C
2
N
CO Et
2
29-32
1-4
N
F
25
26
29, 1
30, 2
31, 3
N
N
f
N
HO C
2
N
CO Et
2
HO C
2
Cl
N
N
N
27
28
N
R =
N
N
N
N
N
32, 4
N
Scheme 1. General procedure for preparation of compounds 1–4. Reagents and conditions: (a)
ethyl piperidine-4-carboxylate, DMAc, 80ºC; (b) 10% Pd/C, Et3N, THF-EtOH, rt; (c) tert-butyl
piperidine-4-carboxylate, K2CO3, DMF, 100ºC; (d) 4M HCl/AcOEt, AcOEt, rt, then TFA,
CH2Cl2, rt; (e) ethyl piperidine-4-carboxylate, DIPEA, DMF, 80ºC; (f) ethyl piperidine-4-
carboxylate, DIPEA, DMF, 90ºC; (g) SOCl2, DCE, reflux, then 4-(4-chloro-2-thienyl)-1,3-
thiazol-2-amine, Et3N, 1,4-dioxane, reflux; (h) (2R)-2-methylpyrrolidine L-tartrate, 35%
HCHO aq. AcOH, 80ºC; (i) 1 M NaOH aq. EtOH, 50ºC.

Synthesis of compounds 5–14 started with the corresponding acetophenones 33–35 (Scheme
2). Compounds 33–35 were treated with phenyltrimethylammonium tribromide, followed by
cyclization with thiourea to yield 2-aminothiazoles 36–38. Amide formation using the carboxylic acid
chloride in pyridine gave compounds 39–41. Subsequently, the amine moiety was directly attached
using a Mannich reaction or via a 5-acetoxymethyl thiazole intermediate, and subsequent hydrolysis
gave the desired analogues 5–14.
R2
R1
S
N
S
N
H
N
H
N
S
N
N
N
O
a, b
c
d, e
NH2
N
CO2Et
N
CO2H
R1
Me
R1
O
N
R1
O
N
33-35
36-38
39-41
5-14
8
Cl
Cl
N
33, 36, 39, 5
34, 37, 40, 6
35, 38, 41, 7-14
5-7
9
N
N
CF3
O
R2 =
R1 =
10
13
11
12
N
N
N
CF3
CF3
O
N
14
N
MeO
Scheme 2. General procedure for preparation of compounds 5–14.a Reagents and conditions:
(a) phenyltrimethylammonium tribromide, THF, rt; (b) thiourea, EtOH, 80ºC; (c) 28, POCl3,
pyridine, rt or TBTU, DIPEA, THF, microwave 150ºC; (d) amine, 36% HCHO aq., DIPEA,
AcOH, 90ºC or 36% HCHO aq., Ac2O, AcOH, microwave 170ºC then amine, DIPEA, DMF,
100ºC; (e) 1 M NaOH aq. EtOH, THF, 50ºC.
a
For the synthesis of 6, amine was attached in the presence of DIPEA in THF at 150ºC under
irradiation with microwaves.
Functionalization at the 5-position of the 2-aminothiazole in compound 38 was accomplished
through trifluoroacetyl protection (Scheme 3). Compound 38 was first treated with trifluoroacetic
anhydride (TFAA) to yield the trifluoroacetylated intermediate, which underwent a Mannich reaction
to attach the 3,3-dimethyl piperidine, followed by removal of the trifluoroacetyl group to give
compound 42. Subsequently, amide formation using N-[({[(1Z)-1-cyano-2-ethoxy-2-
oxoethylidene]amino}oxy)(morpholin-4-yl)methylene]-N-methylmethanaminium
hexafluorophosphate (COMU)11 as a condensation reagent gave compound 43. The carboxylic acid

fragments were connected by nucleophilic aromatic substitution, and subsequent hydrolysis produced
the desired analogues 15–17. Alternatively, pyrazinoic acid analogue 18 was attained via thiazole
intermediate 38, and compounds 19–20 were successfully transformed from 42 using the procedure
described above.
H
N
CO2H
15
16
S
N
S
N
S
N
N
N
N
H
N
H
N
NH2
N
N
a, b, c
e, f
d
Cl
R
N
N
O
38
R =
O
N
N
O
N
CO2H
MeO
MeO
MeO
CF3
CF3
CF3
42
43
CO2H 17
15-17
N
g
S
N
S
N
S
N
N
H
H
N
H
N
N
N
h, i
CO2Et
CO2Et
CO2H
j
O
N
O
N
O
N
MeO
MeO
MeO
44
45
18
CF3
CF3
CF3
S
N
H
N
O
19
19 : k, l
CO2H
N
R
42
R =
O
20 : m, n
MeO
O H
20
CF3 19-20
Scheme 3. General procedure for preparation of compounds 15–20. Reagents and conditions:
(a) TFAA, Et3N, CH2Cl2, rt; (b) 3,3-dimethyl piperidine hydrochloride, 36% HCHO aq.,
DIPEA, AcOH, 60ºC; (c) 6 M NaOH aq., EtOH, THF, 80ºC; (d) 5-chloropyrazine-2-carboxylic
acid, COMU, DIPEA, 1,4-dioxane, rt; (e) amine, DIPEA, DMF, 80ºC or alcohol, NaH, NMP,
80ºC; (f) 1 M NaOH aq., EtOH, 60ºC; (g) 29: 5-(ethoxycarbonyl)pyrazine-2-carboxylic acid,
POCl3, pyridine, rt; 30: HATU, DIPEA, DMF, 100ºC; 31–32: TBTU, DIPEA, 1,4-dioxane,
microwave 130ºC; (h) 36% HCHO aq., Ac2O, AcOH, 90ºC; (i) 3,3-dimethyl piperidine, DIPEA,
DMF, 80ºC; (j) 1 M NaOH aq., EtOH, 60ºC; (k) 4-(2-tert-butoxy-2-oxoethoxy)benzoic acid,
COMU, DIPEA, 1,4-dioxane, 80ºC; (l) 1 M NaOH aq. EtOH, THF, 50ºC; (m) 4-acetoxybenzoic
acid, COMU, DIPEA, 1,4-dioxane, 80ºC; (n) 1 M NaOH aq., MeOH, rt.
3. Results and Discussion
All analogues described in this study were screened with a Fluorometric Imaging Plate Reader
(FLIPRTM) assay using CHO-K1 cells stably expressing the human M3 mAChR with carbachol (CCh)
as a ligand. The PAM activity of the M3 mAChR was determined from the shift in the CCh
concentration-response curve (CCh-CRC) toward lower concentrations following exposure to the test
substance. That is, PAM activity was determined by dividing the EC50 value of CCh in the absence

of the test substance by the EC50 value of CCh in the presence of the test substance. For example,
when the EC50 value of CCh in the absence of the test substance was 200 nM and the EC50 value of
CCh in the presence of the test substance was 10 nM, the PAM activity of the test substance showed
a 20-fold shift. In the tables below, values in the presence of the test substances at 10 μM are shown
for human M3 PAMs (-fold shift).
To identify PAMs of M3 mAChR, we performed a HTS campaign of the Astellas internal
chemical library, which culminated in the identification of compound 1 (Figure 1).12 In functional
assays, 1 produced a 132-fold shift in CCh-CRC at 10 μM, and the CCh-CRC shifted toward lower
concentrations at increasing concentrations of the compound (Figure 2). Because analogues of 1
reportedly have agonistic activity for c-myeloproliferative leukemia virus type P (c-Mpl),13 also
known as human thrombopoietin receptor (TPOR), we assessed the c-Mpl agonistic activity of 1
using a human c-Mpl-introduced Ba/F3 cell proliferative assay and found that 1 showed c-Mpl
agonistic activity with an EC30 value of 3.2 nM. Thrombopoietin (Tpo) is the cytokine responsible
for the primary regulation of megakaryocytes and platelet production via activation of the intracellular
JAK-STAT signaling pathway, which is triggered when Tpo binds to c-Mpl.14 Therefore, the
agonistic activity for c-Mpl may potentially hamper clinical studies because it has been shown to
increase platelet production in humans15 and is associated with the risk of adverse side effects such
as thromboembolism.16 To elucidate whether SARs of M3 PAM activity and c-Mpl agonistic activity
are distinct, we next evaluated c-Mpl agonistic activity and the M3 PAM activity of the test
compounds.
F
S
N
N
H
N
CO H
N
2
Cl
O
N
S
1
Figure 1. Structure of HTS hit 1a.
a hydrochloride salt

Figure 2. In vitro human M3 PAM activity of 1.
CCh-CRCs in human M3-expressing cells in the absence (DMSO) or presence of the indicated concentrations of 1.
Initial efforts focused on attempts to replace the pyridyl-piperidine moiety of 1 (Table 1).
Compound 2, in which the positions of pyridine and piperidine were switched, decreased M3 PAM
activity to a 2.8-fold shift. Although exposure to pyrimidine analogue 3 reduced the shift in CCh-
CRC by half compared to 1, pyrazine analogue 4 produced a substantial increase in activity, resulting
in a 186-fold shift. Interestingly, replacement of 4-chlorothiophene at the 4-position of 2-
aminothiazole with substituted benzenes resulted in a significant reduction in c-Mpl agonistic activity
and a slight reduction in M3 PAM activity (compounds 5–7), suggesting that SARs for c-Mpl
agonistic activity and M3 PAM activity were distinct. We noted that 7 caused a drastic reduction in
c-Mpl agonistic activity (EC30 value >10 μM). The result also suggested that c-Mpl differently
recognized this region in accordance with the position (compound 5 and 6), the lipophilicity and the
electrical property (compound 6 and 7) of substituents. Therefore, analogue 7 was adopted as a
template and used to investigate the amine moiety at the 5-position of 2-aminothiazole.
Table 1. In vitro SARs for human M3 PAM activity and human c-Mpl-introduced Ba/F3 cell
proliferative activity for analogues following replacement of the fluoro pyridine core and 4-
chlorothiophene.

a 10 μM of test substance was used, b hydrochloride salt, c racemate, d NT = Not Tested
The next set of investigations involved modifications of the amine moiety (Table 2). Replacing
(2R)-2-methylpyrrolidine (7) with (2S)-2-methylpyrrolidine (8) decreased M3 PAM activity to a 25-
fold shift, demonstrating that the (2R)-substituent was more favorable for activity. (2R)-Alkylated
pyrrolidine analogues such as ethyl (9) and n-propyl (10) led to increased M3 PAM activity compared
with the methyl analogue (7), whereas hydrophilic methoxymethyl (11) retained the same level of
activity as 7. While (2R)-2-methylpiperidin (12) resulted in a slight increase in M3 PAM activity, 3,3-
dimethylpiperidin (13) caused a 238-fold shift in activity. In contrast, replacement with a branched
alkyl chain (14), in which the (2S)-2-methoxymetylpyrrolidin of 11 was cracked into an alkyl chain,
had little impact on M3 PAM activity, suggesting that this benzyl amine region was only capable of
accepting some flexible conformations. We also noted that none of the analogues prepared in Table
2 showed c-Mpl agonistic activity.
Table 2. In vitro SARs for analogues following replacement of amines at the 5-position of the 2-
aminothiazole.

a 10 μM of test substance was used, b hydrochloride salt
With potent amine moieties at hand, we conducted a SARs study around the pyrazyl-piperidine
moiety using 13 as a template scaffold (Table 3). Transformation of the piperidine (13) to N- or O-
linked analogues (15–17) attenuated M3 PAM activity, and the pyrazinoic acid analogue (18)
drastically reduced activity. Despite the fact that an aromatic amine was not necessarily required at
the terminal region (17), a measurable distance between pyrazine and carboxylic acid was thought to
be essential for retaining M3 PAM activity because the directly attached carboxylic acid in 18
negatively affected activity. Interestingly, benzene analogue 19 exhibited almost the same level of
M3 PAM activity as its direct analogue 17. Of note, our attempt to replace the carboxylic acid with
phenol (20) caused a marked decrease in M3 PAM activity, suggesting that the carboxylic acid might
play a crucial role in activity.
Table 3. In vitro SARs for analogues following replacement of the pyrazyl-piperidine moiety.

a 10 μM of test substance was used, b hydrochloride salt, c potassium salt
4. Further evaluation of a selected compound
Among the compounds synthesized in this study, compound 9, which showed the most potent
PAM activity towards the M3 mAChR, was selected for further evaluation.
As shown in Figure 3, compound 9 concentration-dependently shifted CCh-CRC for human M3
mAChR to the left. The selectivity profiles of compound 9 for other subtypes of human and rat
mAChRs were also evaluated. We found that 9 displayed specific modulation of the M3 mAChR over
the other human and rat mAChR subtypes with the exception of the M5 mAChR, for which it
possessed moderately lower PAM activity than for M3 mAChR (Table 4).

Figure 3. In vitro human M3 PAM activity of 9.
CCh-CRCs in human M3-expressing cells in the absence (DMSO) or presence of the indicated concentrations of 9.
Table 4. Selectivity profile of 9 for all subtypes of human and rat mAChRs.
mAChR PAM (-fold shift at 10 µM)
M1
2.3
2.1
M2
1.3
1.6
M3
269
158
M4
1.0
0.7
M5
94
Human
Rat
110
To assess whether compound 9 enhances the physiological functions mediated by the M3 AChR,
we examined the effect of compound 9 on the electrical field stimulation (EFS)-induced contraction
of isolated rat bladder strips (Table 5). Compound 9 at 3 and 10 µmol/L significantly enhanced EFS-
induced bladder contractions in a concentration-dependent manner. These results suggest that through
M3 PAM activity, compound 9 enhanced the action of ACh, which was released from nerves through
EFS, resulting in increasing contraction of isolated rat bladder strips.
Table 5. Effect of 9 on electrical field stimulation-induced contraction of isolated rat bladder strips
Tension of isolated bladder
Concentration of 9 (µM)
contraction (g)
pre
1
2.21 ± 0.26
2.34 ± 0.32
3
2.79 ± 0.41**

10
3.49 ± 0.50**
Data are expressed as the mean ± SEM (n=7).
** P<0.005: significantly different from the pre-value (Williams’ test using within subject error).
The pharmacokinetics profiles of compound 9 in rats are shown in Table 6. After intravenous
administration (0.3 mg/kg), compound 9 showed moderate total clearance (917.7 mL/h/kg), a steady-
state volume of distribution (1.59 L/kg), and a half-life of 2.43 h. Compound 9 was rapidly absorbed
after oral administration at 1 mg/kg and showed acceptable bioavailability (19.0%). Therefore, 9 has
potential as a chemical probe for investigation in vivo.
Table 6. Pharmacokinetics data for compound 9 in rats.a
Cmax
tmax
(h)
AUCinf
t1/2
(h)
CLtot
Vss
F
Dose
(mg/kg)
0.3
Route
(ng/mL)
(ng·h/mL)
(mL/h/kg)
(L/kg)
(%)b
iv
−
−
330.2
208.8
2.43
2.16
917.7
1.59
−
po
1
85.29
0.31
−
−
19.0
− : not applicable
a
Mean values of individual parameters calculated from the plasma concentrations in each animal
(n=4).
b F = Bioavailability.
5. Conclusion
We reported the discovery of the first potent PAM of the M3 mAChR and a comprehensive
SARs study. Compound 1, initially identified by a HTS campaign, caused a 132-fold shift in CCh-
CRC for the human M3 mAChR at 10 µM. Compound 1 was successfully converted to analogue 7,
which diminished human c-Mpl agonistic activity. In the SARs study around the terminal amine and
pyrazyl-piperidine moiety, 9 was identified as a more promising chemical tool, causing a 269-fold
shift in CCh-CRC at 10 µM and contraction of isolated smooth muscle strips of rat bladder. Moreover,
9 exhibited acceptable PK profiles in rats, excellent subtype selectivity over both human and rat M1,
M2, and M4, and moderate selectivity over M5 mAChRs, indicating that compound 9 is an M3-
preferring M3/M5 dual PAM. Therefore, we anticipate that 9, a newly identified PAM of the M3
mAChR, will be useful as a new class of chemical probe for investigating an array of pharmacological

functions for future drug discovery campaigns. To evaluate the utilities of compound 9 as a potential
pharmacological tool, further experiments are needed (manuscripts are under preparation).
Experimental
Biology
Evaluation of mAChR Positive Allosteric Modulator Activity
Human and rat M1, M2, M3, M4, and M5 mAChR genes were inserted into pcDNA3.1TM
expression vectors (Life Technologies) and the mAChR-expressing vectors were transfected into
CHO-K1 cells (ATCC No. CCL-61) using Lipofectoamine 2000 reagent (Life Technologies). The
cells were cultured in alpha Modified Eagle Minimum Essential Medium (α-MEM) containing 2 mM
glutamic acid, 10% fetal bovine serum, and 2.0 mg/mL Geneticin (Life Technologies) for 4 weeks to
acquire drug-resistant clones. To convert the signaling pathway of M2 and M4 mAChR subtypes,
which are coupled to Gi/o proteins, into a Gq signaling pathway, we also transfected a human G16 G
protein α subunit-expressing vector into human and rat M2- and M4-expressing cells as described
above and drug-resistant clones were selected in the presence of 0.5 mg/mL Hygromycin B.
The day before the experiment, M1, M3, and M5 receptor-expressing CHO-K1 cells were
suspended in α-MEM containing 2 mM glutamic acid, 10% fetal bovine serum, and 0.2 mg/mL
Geneticin, dispensed into a 384-well plate (Lot number 355962, BD Biosciences) at 1.2 to 1.5 × 104
cells/well, and cultured at 37°C and 5% CO2 overnight. M2 and M4 receptor-expressing CHO-K1
cells were suspended in α-MEM containing 2 mM glutamic acid, 10% fetal bovine serum, 0.2 mg/mL
Geneticin, and 50 µg/mL Hygromycin B, seeded and cultured as described above.
The culture medium was replaced with loading buffer (Assay Buffer [Hanks Balanced Salt
Solution (HBSS)], 1 g/L BSA, 20 mM HEPES (pH 7.5), and 2.5 mM Probenecid) containing 3.1 μM
Fluo 4-AM (Dojindo Laboratories), and left at room temperature for about 2 hours. Thereafter, the
cells were washed with assay buffer using plate washing equipment (ELx405TM; BIO-TEK
Instruments), and placed in a FLIPR TetraTM, a system that measures the intracellular Ca2+
concentration (Molecular Devices). A test substance (final concentration 10 μM) and CCh (Sigma,
final concentration 0.0024 nM to 100 μM), each of which were dissolved in assay buffer in advance,
were placed in the FLIPR TetraTM. Test substances were added to the cells inside the device, and CCh
was added to the cells about 5 minutes later. The Ca2+ flux induced by CCh was measured at an
excitation wavelength of 470 nm to 495 nm and fluorescence wavelength of 515 nm to 575 nm.
The activity of mAChR PAMs was determined from the shift in the CCh concentration-
response curve toward lower concentrations following exposure to the test substance. That is, the

minimum and maximum value of the CCh response from the concentration-response curve of CCh
were set to 0% and 100%, respectively. Using logistic regression, the concentration of CCh showing
a 50% response was calculated as the EC50 value, and the activity was determined by dividing the
EC50 value of CCh in the absence of the test substance by the EC50 value in the presence of the test
substance.
Human c-Mpl-Introduced Ba/F3 Cell Proliferative Assay
A human c-Mpl receptor gene (GenBank Accession No. M90102.1) was inserted into the pEF-
BOS expression vector (Nucleic Acids Res. 18, 5322, 1990), and the human c-Mpl receptor-
expressing vector was transfected into Ba/F3 cells (RIKEN BRC: RCB0805) using electroporation.
pEF-BOS- c-Mpl (10 μg), pSV2bsr (1 μg; Kaken Pharmaceutical Co., Ltd.) and 1 × 107 Ba/F3 cells
were added to a cuvette with a gap width of 0.4 cm, and electroporated at 1.5 kV (25 μF) using a
Gene Pulser (BioRad). The cells were cultured in RPMI-1640 culture medium containing 0.5% WEHI
conditioned media (BD Biosciences) and 10% fetal bovine serum for 3 days, and subsequently
cultured in RPMI-1640 culture medium containing 10 μg/mL blasticidin for 30 days to acquire a
drug-resistant clone.
The obtained cells were cultured in RPMI-1640 culture medium containing 0.5% WEHI
conditioned media and 10% fetal bovine serum. The day before the experiment, test substances (final
concentration 100 nM to 10 μM) that had been dissolved in culture medium for the assay (RPMI-
1640 culture medium containing 10% fetal bovine serum) were added to a 384-well plate (Lot
No.781185, Greiner Bio-One). The cell medium was replaced with culture medium for the assay. The
cells were then dispensed into a 384-well plate containing the test substances at 1 × 104 cells/well,
and cultured at 37°C and 5% CO2 overnight. On the day of the experiment, a solution from the Cell
Counting Kit (Dojindo Laboratories) was added to each well of the 384-well plate and cultured at
37°C and 5% CO2 for about 5 hours. Thereafter, the absorbance (absorption wavelength 450 nm) in
each well was measured using Safire2TM (TECAN) and used as an index for the number of cells. A
well without the test substance was used the negative control. The absorbance for the negative control
was set to 0%, and the absorbance for the positive control, where 1-(5-{[4-(4-chloro-2-thienyl)-5-
{[(2R)-2-methylpyrrolidin-1-yl]methyl}-1,3-thiazol-2-yl]carbamoyl}-3-fluoropyridin-2-
yl)piperidine-4-carboxylic acid hydrochloride was added at a final concentration of 1 μM, was set to
100%. The cell proliferation rate (%) was calculated in wells containing the test substance, and the
test substance concentration showing 30% proliferation was calculated as the EC30 value using
logistic regression.

Effects on EFS-Induced Contraction of Isolated Rat Bladder Strips
Nine- and ten-week-old female Sprague-Dawley rats (SLC Japan Inc., Shizuoka, Japan) were
euthanized by exsanguination under isoflurane anesthesia. The urinary bladder was isolated, and a
longitudinal bladder strip (approximately 10 mm in length and 2 mm in width) was placed between a
pair of electrodes in 10 mL organ baths containing Krebs solution, maintained at 37°C and constantly
aerated with 95% O2 and 5% CO2. Celphines tied with silk ligatures were applied to each end of the
bladder strip. One end was attached to a holder and the other to an isometric tension transducer (TB-
611T, Nihon Kohden, Tokyo, Japan) coupled to a PowerLab system (Power Lab8/30, ADInstruments,
Aichi, Japan) to record contractile responses. The resting tension of the strips was adjusted to
approximately 1 g, and the strips were left to equilibrate for approximately 1 hour. The strips were
pre-contracted with KCl (final concentration 60 mmol/L in the organ bath) and washed out with Krebs
solution. This step was repeated 2 times.
Neurogenic contraction of bladder strips was induced by applying EFS. Approximately 20 V
of EFS (trains of electrical pulses at 8 Hz, 10 s duration, 0.3 ms pulse width) was applied 2 times at
4-minute intervals to observe the maximum response, and the voltage required to elicit approximately
half the maximum response was determined and fixed.
After observing the pre-contractions, potentiating effects of compound 9 (final concentration 1,
3, 10 µM in the organ bath) on EFS (same conditions as above)-induced contraction were tested.
Compound 9 was added cumulatively and EFS was applied 10 times at each concentration.
Bladder contraction induced by EFS was calculated as the difference between the peak
contraction and the basal tone (the average tension for 2 minutes before contraction). The average of
the contractions and the basal tone measured from the last 3 contractions before the application of
compound 9 was defined as the pre-value. The effect of compound 9 (1, 3 µM) was evaluated from
the average of the contractions and the basal tone measured from the last 3 contractions before the
application of the next concentration of compound 9. The effect of compound 9 (10 µM) was
evaluated using the average of the contractions and the basal tone measured from the last 3
contractions before the end of the experiment.
The contractions and basal tone were compared between pre-value and compound 9-treated
values using Williams’ test with within subject error, and a probability value of less than 0.025 was
considered significant. Data are expressed as the mean ± SEM (n=7).
Pharmacokinetics study

Pharmacokinetic characterization of compound 9 was conducted in male SD rats (Sprague-
Dawley strain SPF rats Crl:CD(SD), 8 weeks of age). Compound 9 was administered intravenously
at 0.3 mg/kg and orally at 1 mg/kg in a mixture of DMF/propylene glycol (PG)/water for injection
containing 1 M sodium hydroxide solution (2 equivalents of 9) (1/1/8). The animals were food
deprived. After each administration, blood samples were drawn at assigned time points up to 24 hours,
and the concentrations of compound 9 in plasma were determined using a validated LC-MS/MS
method. Pharmacokinetic parameters were calculated from the plasma concentrations in each animal
by noncompartmental analysis using the pharmacokinetic software Phoenix WinNonlin.
Chemistry
1H-NMR spectra were recorded on JEOL JNM-EX400, Brucker DPX200, Avance400,
AV400M, Avance III HD500, or Varian VNS-400 spectrometer and were referenced against the
internal standard, tetramethylsilane. The abbreviations used for the signal patterns are as follows: s,
singlet; br, broad; brs, broad singlet; d, doublet; t, triplet; q, quartet; dd, double doublet; tt, triple
triplet; m, multiplet. Mass spectra (MS) were recorded on JEOL LX-2000, Agilent 1100 LC/MSD,
Thermo Fisher Scientific LCQ Advantage, or Waters UPLC/SQD mass spectrometer. Elemental
analyses were performed using a Yanako MT-5 microanalyzer (C, H, N), Elementar Vario EL III (C,
H, N), Dionex ICS-3000 (S, halogen), and Yokogawa IC-7000S ion chromatographic analyzer (S,
halogens), where the analytical results are within ±0.4% of the theoretical values. Electrospray
ionization positive high-resolution mass spectrometry (HRMS) was performed using Thermo
Scientific Exactive Plus. Column chromatography was performed using Wakogel C-200 or Merck
silica gel 60 N (Kanto Chemical, 63–210 μm) or HI-FLASH™ Column (Yamazen). All reactions
were conducted using commercially available reagents and solvents without further purification.
6-[4-(Ethoxycarbonyl)piperidin-1-yl]-5-fluoronicotinic acid (22)
To a mixture of 2,6-dichloro-5-fluoronicotinic acid (21, 21.0 g) and N,N-dimethylacetamide
(200 mL) was added ethyl piperidine-4-carboxylate (39.1 mL), followed by stirring at 80C overnight.
The mixture was concentrated under reduced pressure, and ethyl acetate and a 1 M aqueous hydrogen
chloride solution were added thereto. The organic layer was separated, washed with a 1 M aqueous
hydrogen chloride solution and a saturated aqueous sodium chloride solution, and dried over
anhydrous sodium sulfate. The insoluble materials were separated by filtration and the filtrate was
concentrated under reduced pressure to give a brown solid (34.3 g). The obtained solid was washed
with ethyl acetate/diisopropyl ether to obtain a colorless solid (18.3 g).

To the above obtained solid (15.0 g) in ethanol (150 mL) and tetrahydrofuran (150 mL) was
added triethylamine (19.0 mL) and 10% palladium-supported carbon (50% wet, 1.50 g), followed by
stirring at room temperature overnight at 3 atm under a hydrogen atmosphere. The insoluble materials
were separated by filtration, and 1 M aqueous hydrogen chloride solution (500 mL) was added to the
filtrate, followed by extraction with ethyl acetate. The organic layer was washed with a saturated
aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The insoluble materials
were separated by filtration and the filtrate was concentrated under reduced pressure to give 22 (12.2
g, 50%) as a colorless solid: 1H-NMR (CDCl3) δ 1.27 (3H, t, J = 6.8 Hz), 1.77–1.90 (2H, m), 1.98–
2.06 (2H, m), 2.60 (1H, tt, J = 10.8, 4.0 Hz), 3.12–3.22 (2H, m), 4.16 (2H, q, J = 6.8 Hz), 4.33–4.42
(2H, m), 7.75 (1H, dd, J = 14.0, 2.0 Hz), 8.66 (1H, t, J = 2.0 Hz).
1-[5-(Methoxycarbonyl)pyridin-2-yl]piperidine-4-carboxylic acid (24)
Methyl 6-chloronicotinate (23, 5.56 g), tert-butyl piperidine-4-carboxylate (5.50 g),
dipotassium carbonate (11.2 g), and N,N-dimethylformamide (80 mL) were mixed, followed by
stirring at 100C overnight. The mixture was cooled to room temperature and diluted with ethyl
acetate. The insoluble materials were separated by filtration and the filtrate was washed with water
and a saturated aqueous sodium chloride solution, and dried over anhydrous magnesium sulfate. The
insoluble materials were separated by filtration and the filtrate was concentrated under reduced
pressure. The obtained solid was washed with diisopropyl ether and dried to obtain a white solid (7.97
g).
The obtained solid (7.97 g), ethyl acetate (60 mL), and a 4 M hydrogen chloride/ethyl acetate
solution (63 mL) were mixed, followed by stirring at room temperature for 1 hour. The mixture was
concentrated under reduced pressure, and water and a 1M aqueous sodium hydroxide solution were
added thereto, followed by extraction with ethyl acetate. The organic layer was washed with water
and a saturated aqueous sodium chloride solution, and dried over anhydrous magnesium sulfate. The
insoluble materials were separated by filtration and the filtrate was concentrated under reduced
pressure.
The obtained residue was mixed with dichloromethane (60 mL) and trifluoroacetic acid (20
mL), followed by stirring at room temperature overnight. The mixture was concentrated under
reduced pressure, and water and a 1 M aqueous sodium hydroxide solution were added thereto,
followed by extraction with ethyl acetate. The organic layer was dried over anhydrous magnesium
sulfate. The insoluble materials were separated by filtration and the filtrate was concentrated under
reduced pressure. The obtained residue was washed with diisopropyl ether and dried to obtain 24

(5.68 g, 72%) as a white solid: 1H-NMR (DMSO-d6) δ 1.44–1.57 (2H, m), 1.84–1.94 (2H, m), 2.57
(1H, tt, J = 10.9, 4.0 Hz), 3.04–3.14 (2H, m), 3.78 (3H, s), 4.26–4.36 (2H, m), 6.89 (1H, d, J = 9.2
Hz) 7.93 (1H, dd, J = 9.2, 2.5 Hz), 8.63 (1H, d, J = 2.5 Hz), 12.24 (1H, brs).
2-[4-(Ethoxycarbonyl)piperidin-1-yl]pyrimidine-5-carboxylic acid (26)
2-Chloropyrimidine-5-carboxylic acid (25, 4.06 g), ethyl piperidine-4-carboxylate (4.74 mL),
N,N-diisopropylethylamine (9.65 mL), and N,N-dimethylformamide (40 mL) were mixed, followed
by stirring at 80C overnight. The mixture was cooled to room temperature and diluted with ethyl
acetate. The organic layer was washed with an aqueous citric acid solution, water, and a saturated
aqueous sodium chloride solution, and dried over anhydrous magnesium sulfate. The insoluble
materials were separated by filtration and the filtrate was concentrated under reduced pressure. The
obtained solid was washed with diisopropyl ether and dried to obtain 26 (5.71 g, 80%) as a beige
solid: 1H-NMR (DMSO-d6) δ 1.19 (3H, t, J = 7.1 Hz), 1.44–1.58 (2H, m), 1.86–1.98 (2H, m), 2.69
(1H, tt, J = 10.9, 3.9 Hz), 3.12–3.24 (2H, m), 4.08 (2H, q, J = 7.1 Hz), 4.54–4.66 (2H, m), 8.75 (2H,
s), 12.79 (1H, brs).
5-[4-(Ethoxycarbonyl)piperidin-1-yl]pyrazine-2-carboxylic acid (28)
5-Chloropyrazine-2-carboxylic acid (27, 7.84 g), N,N-dimethylformamide (120 mL), ethyl
piperidine-4-carboxylate (16.0 mL), and N,N-diisopropylethylamine (25.0 mL) were mixed, followed
by stirring at 90C for 8 hours. The reaction mixture was cooled to room temperature and ethyl acetate
was added. The mixture was washed with a dilute hydrochloric acid solution and dried over
anhydrous sodium sulfate. The insoluble materials were separated by filtration and the filtrate was
concentrated under reduced pressure. The obtained solid was washed with diisopropyl ether and dried
to obtain 28 (6.80 g, 49%) as a milky white solid: 1H-NMR (CDCl3) δ 1.27 (3H, t, J = 7.0 Hz), 1.74–
1.86 (2H, m), 2.02–2.11 (2H, m), 2.66 (1H, tt, J = 10.5, 4.1 Hz), 3.20–3.30 (2H, m), 4.17 (2H, q, J =
7.1 Hz), 4.33–4.42 (2H, m), 8.02 (1H, d, J = 1.3 Hz), 8.86 (1H, d, J = 1.3 Hz); ESI-MS m/z 280
[(M+H)+].
Ethyl 1-(5-{[4-(4-chloro-2-thienyl)-1,3-thiazol-2-yl]carbamoyl}-3-fluoropyridin-2-yl)
piperidine-4-carboxylate (29)
To a mixture of 6-[4-(ethoxycarbonyl)piperidin-1-yl]-5-fluoronicotinic acid (22, 14.0 g) and
1,2-dichloroethane (140 mL) was added thionyl dichloride (6.89 mL), followed by heating to reflux

for 110 minutes. The mixture was cooled to room temperature and concentrated under reduced
pressure, and the residual solvents were chased with toluene.
The resulting residue was mixed with 1,4-dioxane (180 mL), and 4-(4-chloro-2-thienyl)-1,3-
thiazol-2-amine (6.83 g) and triethylamine (17.5 mL) were added thereto, followed by heating to
reflux overnight. The mixture was cooled to room temperature and concentrated under reduced
pressure. The residue was diluted with ethyl acetate, washed with a saturated aqueous sodium
hydrogen carbonate solution twice and a saturated aqueous sodium chloride solution, and then dried
over anhydrous sodium sulfate. The insoluble materials were separated by filtration and the filtrate
was concentrated under reduced pressure to give a brown amorphous solid (21.2 g). The amorphous
solid was recrystallized from ethanol to obtain 29 (14.1 g, 90%) as a brown powder: 1H-NMR (CDCl3)
δ 1.27 (3H, t, J = 6.8 Hz), 1.77–1.90 (2H, m), 1.98–2.08 (2H, m), 2.61 (1H, tt, J = 11.2, 4.0 Hz), 3.12–
3.23 (2H, m), 3.73 (2H, q, J = 6.8 Hz), 4.33–4.42 (2H, m), 7.04 (1H, d, J = 1.6 Hz), 7.07 (1H, s), 7.22
(1H, d, J = 1.6 Hz), 7.73 (1H, dd, J = 14.4, 2.0 Hz), 8.51 (1H, s), 9.79 (1H, brs); FAB-MS m/z 495
[(M+H)+].
Methyl 6-(4-{[4-(4-chloro-2-thienyl)-1,3-thiazol-2-yl]carbamoyl}piperidin-1-yl)nicotinate (30)
4-(4-Chloro-2-thienyl)-1,3-thiazol-2-amine (500 mg), 1-[5-(methoxycarbonyl)pyridin-2-
yl]piperidine-4-carboxylic acid (24, 611 mg), N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-b]pyridin-
3-yloxy)methylene]-N-methylmethanaminium
hexafluorophosphate
(878
mg),
N,N-
diisopropylethylamine (395 μL), and N,N-dimethylformamide (5.0 mL) were mixed, followed by
stirring at 100C for 2 hours. N,N-diisopropylethylamine (395 μL) was added thereto, followed by
stirring at the same temperature for 15 minutes. N-[(dimethylamino)(3H-[1,2,3]triazolo[4,5-
b]pyridin-3-yloxy)methylene]-N-methylmethanaminium hexafluorophosphate (878 mg) was added
thereto, followed by stirring at the same temperature for 15 minutes. 1-[5-(methoxycarbonyl)pyridin-
2-yl]piperidine-4-carboxylic acid (24, 182 mg) was added thereto, followed by stirring at the same
temperature for 1 hour. The mixture was cooled to room temperature, and diluted with ethyl acetate.
The organic layer was washed with water, a 1 M aqueous sodium hydroxide solution, and a saturated
aqueous sodium chloride solution, and then dried over anhydrous magnesium sulfate. The insoluble
materials were separated by filtration and the filtrate was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (hexanes-ethyl acetate) and then washed
with hexanes to obtain 30 (291 mg, 27%) as a beige solid: 1H-NMR (DMSO-d6) δ 1.61 (2H, ddd, J =
24.7, 12.7, 3.9 Hz), 1.87–1.97 (2H, m), 2.86 (1H, tt, J = 11.3, 3.9 Hz), 2.97–3.10 (2H, m), 3.79 (3H,

s), 4.42–4.56 (2H, m), 6.91 (1H, d, J = 9.0 Hz), 7.53 (1H, d, J = 1.5 Hz), 7.55 (1H, d, J = 1.5 Hz),
7.60 (1H, s), 7.94 (1H, dd, J = 9.0, 2.3 Hz), 8.65 (1H, d, J = 2.3 Hz), 12.44 (1H, s).
Ethyl 1-(5-{[4-(4-chloro-2-thienyl)-1,3-thiazol-2-yl]carbamoyl}pyrimidin-2-yl)piperidine-4-
carboxylate (31)
4-(4-Chloro-2-thienyl)-1,3-thiazol-2-amine (300 mg), 2-[4-(ethoxycarbonyl)piperidin-1-
yl]pyrimidine-5-carboxylic
acid
(26,
464
mg),
N-[(1H-benzotriazol-1-
yloxy)(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate (533 mg), N,N-
diisopropylethylamine (592 μL), and 1,4-dioxane (3.0 mL) were mixed, followed by stirring at 130C
for 30 minutes under irradiation with microwaves. The mixture was cooled to room temperature and
concentrated under reduced pressure. The residue was purified by silica gel column chromatography
(hexanes-ethyl acetate) and then washed with hexanes/ethyl acetate to obtain 31 (313 mg, 47%) as a
white powder: 1H-NMR (DMSO-d6) δ 1.19 (3H, t, J = 7.1 Hz), 1.46–1.61 (2H, m), 1.88–1.99 (2H,
m), 2.71 (1H, tt, J = 10.9, 3.9 Hz), 3.15–3.26 (2H, m), 4.09 (2H, q, J = 7.1 Hz), 4.57–4.66 (2H, m),
7.53 (1H, d, J = 1.5 Hz), 7.58 (1H, d, J = 1.5 Hz), 7.66 (1H, s), 9.00 (2H, s), 12.69 (1H, brs); ESI-MS
m/z 478, 480 [(M+H)+].
The following compound (32) was prepared using a procedure similar to that described for 31.
Ethyl 1-(5-{[4-(4-chloro-2-thienyl)-1,3-thiazol-2-yl]carbamoyl}pyrazin-2-yl)piperidine-4-
carboxylate (32)
Pale brown solid (yield 77%): 1H-NMR (DMSO-d6) δ 1.20 (3H, t, J = 7.1 Hz), 1.53–1.66 (2H,
m), 1.88–2.02 (2H, m), 2.73 (1H, tt, J = 10.9, 3.9 Hz), 3.16–3.27 (2H, m), 4.09 (2H, q, J = 7.1 Hz),
4.37–4.49 (2H, m), 7.54 (1H, d, J = 1.5 Hz), 7.57 (1H, d, J = 1.5 Hz), 7.67 (1H, s), 8.39 (1H, d, J =
1.2 Hz), 8.76 (1H, d, J = 1.2 Hz), 11.82 (1H, brs).
1-(5-{[4-(4-Chloro-2-thienyl)-5-{[(2R)-2-methylpyrrolidin-1-yl]methyl}-1,3-thiazol-2-yl]
carbamoyl}-3-fluoropyridin-2-yl)piperidine-4-carboxylic acid dihydrochloride (1)
To
a
mixture of ethyl 1-(5-{[4-(4-chloro-2-thienyl)-1,3-thiazol-2-yl]carbamoyl}-3-
fluoropyridin-2-yl)piperidine-4-carboxylate (29, 10.0 g) and acetic acid (300 mL) was added (2R)-2-
methylpyrrolidine L-tartrate (6.52 g) and 35% formaldehyde aqueous solution (4.39 mL), followed
by stirring at 80°C overnight. The reaction mixture was cooled to room temperature and concentrated
under reduced pressure. The residue was mixed with a saturated aqueous sodium hydrogen carbonate

solution and ethyl acetate. The organic layer was separated, washed with a saturated aqueous sodium
chloride solution, and dried over anhydrous sodium sulfate. The insoluble materials were separated
by filtration and the filtrate was concentrated under reduced pressure to give a brown amorphous solid
(11.5 g).
Ethyl acetate (550 mL) and a 4 M hydrogen chloride/ethyl acetate solution (4.86 mL) were
added to the resulting amorphous solid, and the mixture was filtered to give a pale brown solid (10.2
g). The obtained solid was mixed with a saturated aqueous sodium hydrogen carbonate solution,
followed by extraction with ethyl acetate twice. The organic layers were combined, washed with a
saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The insoluble
materials were separated by filtration and the filtrate was concentrated under reduced pressure to give
a brown amorphous solid (10.0 g). The amorphous solid was recrystallized from ethanol to obtain a
slightly brown powder (7.70 g).
The obtained powder (7.66 g) and ethanol (115 mL) were mixed, and a 1 M aqueous sodium
hydroxide solution (38.8 mL) was added thereto, followed by stirring at 50C for 1.5 hours. The
reaction mixture was cooled to room temperature and a 1 M aqueous hydrochloric acid solution (38.8
mL) was added. The precipitated solid was collected by filtration to give a pale brown solid (7.58 g).
The obtained solid, acetonitrile (258 mL), and a 1 M aqueous hydrochloric acid solution (28.7
mL) were mixed, followed by stirring at room temperature for 30 minutes. Acetonitrile (10% aqueous,
130 mL) was further added, and the solid was dissolved under heating. After cooling to room
temperature, the precipitated solid was collected by filtration and dried to obtain 1 (3.54 g, 28%) as a
grey solid: 1H-NMR (DMSO-d6) δ 1H-NMR (DMSO-d6) δ 1.47 (3H, d, J = 6.4 Hz), 1.55–1.80 (3H,
m), 1.85–2.03 (4H, m), 2.15–2.27 (1H, m), 2.60 (1H, tt, J = 10.7, 3.9 Hz), 3.10–3.25 (3H, m), 3.40–
3.63 (2H, m), 4.16–4.30 (2H, m), 4.63 (1H, dd, J = 14.9, 7.1 Hz), 4.87 (1H, d, J = 14.9 Hz), 5.50–
7.50 (2H, m), 7.70–7.75 (2H, m), 8.14 (1H, dd, J = 15.4, 1.7 Hz), 8.72–8.77 (1H, m), 11.13 (1H, brs),
12.89 (1H, brs); FAB-MS m/z 564 [(M+H)+]; HRMS (M+H)+ calcd for C25H28O3N5ClFS2 564.1301;
found 564.1303. Anal. calcd for C25H27ClFN5O3S2∙2HCl∙2H2O: C, 44.61; H, 4.94; N, 10.41; S, 9.53;
Cl, 15.80; F, 2.82. Found: C, 44.82; H, 5.07; N, 10.66; S, 9.57; Cl, 15.65; F, 2.82.
The following compounds (2–4) were prepared using a procedure similar to that described for 1.
6-[4-({4-(4-Chloro-2-thienyl)-5-[(2-methylpyrrolidin-1-yl)methyl]-1,3-thiazol-2-yl}
carbamoyl)piperidin-1-yl]nicotinic acid dihydrochloride (2)

White solid (yield 34%): 1H-NMR (DMSO-d6) δ 1H-NMR (DMSO-d6) δ 1.42 (3H, d, J = 6.5
Hz), 1.59–1.72 (3H, m), 1.82–2.02 (4H, m), 2.12–2.26 (1H, m), 2.92 (1H, tt, J = 11.2, 3.8 Hz), 3.05–
3.20 (3H, m), 3.38–3.60 (2H, m), 3.60—5.00 (2H, m), 4.41–4.53 (2H, m), 4.59 (1H, dd, J = 15.0, 7.4
Hz), 4.88 (1H, dd, J = 15.0, 3.1 Hz), 7.06 (1H, d, J = 9.2 Hz), 7.66 (1H, d, J = 1.3 Hz), 7.72 (1H, d,
J = 1.3 Hz), 8.01 (1H, dd, J = 9.2, 2.3 Hz), 8.56 (1H, d, J = 2.3 Hz), 10.51 (1H, brs), 12.65 (1H, s);
ESI-MS m/z 546, 548 [(M+H)+]; HRMS (M+H)+ calcd for C25H29O3N5ClS2 546.1395; found
546.1395.
1-(5-{[4-(4-Chloro-2-thienyl)-5-{[(2R)-2-methylpyrrolidin-1-yl]methyl}-1,3-thiazol-2-yl]
carbamoyl}pyrimidin-2-yl)piperidine-4-carboxylic acid (3)
White powder (yield 63%): 1H-NMR (DMSO-d6) δ 1H-NMR (DMSO-d6) δ 1.16 (3H, d, J = 5.7
Hz), 1.32–1.45 (1H, m), 1.45–1.59 (2H, m), 1.59–1.76 (2H, m), 1.84–2.02 (3H, m), 2.12–2.27 (1H,
m), 2.50–2.66 (2H, m), 2.95–3.09 (1H, m), 3.14–3.26 (2H, m), 3.56 (1H, d, J = 14.7 Hz), 4.19 (1H,
d, J = 14.7 Hz), 4.54–4.66 (2H, m), 7.46 (1H, s), 7.57 (1H, s), 8.98 (2H, s), 12.29 (1H, brs), 12.51
(1H, brs); ESI-MS m/z 547, 549 [(M+H)+]; HRMS (M+H)+ calcd for C24H28O3N6ClS2 547.1347;
found 547.1351.
1-(5-{[4-(4-Chloro-2-thienyl)-5-{[(2R)-2-methylpyrrolidin-1-yl]methyl}-1,3-thiazol-2-yl]
carbamoyl}pyrazin-2-yl)piperidine-4-carboxylic acid dihydrochloride (4)
Pale yellow solid (yield 68%): 1H-NMR (DMSO-d6) δ 1H-NMR (DMSO-d6) δ 1.47 (3H, d, J =
6.4 Hz), 1.51–1.65 (2H, m), 1.65–1.80 (1H, m), 1.86–2.03 (4H, m), 2.14–2.28 (1H, m), 2.65 (1H, tt,
J = 10.8, 4.0 Hz), 3.10–3.30 (3H, m), 3.40–3.63 (2H, m), 4.35–4.50 (2H, m), 4.63 (1H, dd, J = 15.0,
7.2 Hz), 4.87 (1H, dd, J = 15.0, 3.1 Hz), 7.72 (1H, d, J = 1.3 Hz), 7.73 (1H, d, J = 1.3 Hz), 8.40 (1H,
d, J = 1.2 Hz), 8.76 (1H, d, J = 1.2 Hz), 9.89 (3H, brs), 11.22 (1H, brs), 12.09 (1H, brs); ESI-MS m/z
547, 549 [(M+H)+]; HRMS (M+H)+ calcd for C24H28O3N6ClS2 547.1347; found 547.1346.
4-[4-Methoxy-3-(trifluoromethyl)phenyl]-1,3-thiazol-2-amine (38)
1-[4-Methoxy-3-(trifluoromethyl)phenyl]ethanone (35, 15 g) and tetrahydrofuran (270 mL)
were mixed, and phenyltrimethylammonium tribromide (28.42 g) was added thereto, followed by
stirring at room temperature for 30 minutes. The precipitated insoluble materials were separated by
filtration and the filtrate was concentrated under reduced pressure.
The obtained residue and ethanol (260 mL) were mixed, and thiourea (6.81 g) was added thereto,
followed by stirring at 80C for 3 hours. The reaction mixture was cooled to room temperature, and

water, a 1 M aqueous sodium hydroxide solution, and ethyl acetate were added. The organic layer
was washed with a 1 M aqueous sodium hydroxide solution, water, and a saturated aqueous sodium
chloride solution, and dried over anhydrous magnesium sulfate. The insoluble materials were
separated by filtration and the filtrate was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (hexane-ethyl acetate) to obtain 38 (16.18 g, 86%) as
1
a white solid: H-NMR (DMSO-d6) δ 3.91 (3H, s), 7.04 (1H, s), 7.10 (2H, s), 7.27 (1H, d, J = 8.6
Hz), 8.01–8.05 (1H, m), 8.02 (1H, s); ESI-MS m/z 275 [(M+H)+].
The following compounds (36–37) were prepared using a procedure similar to that described for 38.
4-[3-Chloro-5-(trifluoromethyl)phenyl]-1,3-thiazol-2-amine (36)
Yellow solid (yield 107%): 1H-NMR (DMSO-d6) δ 1H-NMR (DMSO-d6) δ 4.36 (2H, brs), 7.48
(1H, s), 7.75 (1H, brs), 8.10 (1H, brs), 8.17 (1H, brs); ESI-MS m/z 279, 281 [(M+H)+].
4-[4-Chloro-3-(trifluoromethyl)phenyl]-1,3-thiazol-2-amine (37)
Off-white solid (yield 93%): 1H-NMR (DMSO-d6) δ 1H-NMR (CDCl3) δ 5.03 (2H, brs), 6.81
(1H, s), 7.50 (1H, d, J = 8.4 Hz), 7.86 (1H, d, J = 8.4, 2.1 Hz), 8.12 (1H, d, J = 2.1 Hz); ESI-MS m/z
279, 281 [(M+H)+].
Ethyl 1-[5-({4-[3-chloro-5-(trifluoromethyl)phenyl]-1,3-thiazol-2-yl}carbamoyl)pyrazin-2-yl]
piperidine-4-carboxylate (39)
A mixture of 4-[3-chloro-5-(trifluoromethyl)phenyl]-1,3-thiazol-2-amine (36, 1.03 g), 5-[4-
(ethoxycarbonyl)piperidin-1-yl]pyrazine-2-carboxylic acid (28, 1.13 g), [(1H-benzotriazol-1-
yl)oxy](dimethylamino)-N,N-dimethylmethaniminium tetrafluoridoborate (1.30 g), N,N-
diisopropylethylamine (1.6 mL), and THF (10 mL) was stirred at 150C for 30 minutes under
irradiation with microwaves. The mixture was diluted with water and then extracted with chloroform.
The organic layer was washed with a saturated aqueous sodium chloride solution and dried over
anhydrous sodium sulfate. The insoluble materials were separated by filtration and the filtrate was
concentrated under reduced pressure. The resulting residue was purified by silica gel column
chromatography (hexanes-ethyl acetate) and then washed with hexanes/ethyl acetate to obtain 39
(1.07 g, 54%) as a pale yellow solid: 1H-NMR (DMSO-d6) δ 1H-NMR (DMSO-d6) δ 1.20 (3H, t, J =
7.1 Hz), 1.54–1.67 (2H, m), 1.92–2.02 (2H, m), 2.68–2.78 (1H, m), 3.17–3.28 (2H, m), 4.09 (2H, q,

J = 7.1 Hz), 4.39–4.49 (2H, m), 7.81 (1H, brs), 8.11 (1H, s), 8.28 (1H, brs), 8.35 (1H, brs), 8.40 (1H,
d, J = 1.3 Hz), 8.77 (1H, d, J = 1.3 Hz), 11.89 (1H, brs); ESI-MS m/z 540, 542 [(M+H)+].
The following compound (40) was prepared using a procedure similar to that described for 39.
Ethyl 1-[5-({4-[4-chloro-3-(trifluoromethyl)phenyl]-1,3-thiazol-2-yl}carbamoyl)pyrazin-2-yl]
piperidine-4-carboxylate (40)
1
Pale yellow powder (yield 76%): H-NMR (DMSO-d6) δ 1.20 (3H, t, J = 7.0 Hz), 1.53–1.67
(2H, m), 1.92–2.02 (2H, m), 2.73 (1H, tt, J = 10.9, 3.9 Hz), 3.17–3.27 (2H, m), 4.09 (2H, q, J = 7.0
Hz), 4.38–4.50 (2H, m), 7.80 (1H, d, J = 8.5 Hz), 7.99 (1H, s), 8.24 (1H, dd, 8.5, 2.0 Hz), 8.36–8.44
(2H, m), 8.77 (1H, d, J = 1.2 Hz), 11.87 (1H, brs).
Ethyl 1-[5-({4-[4-methoxy-3-(trifluoromethyl)phenyl]-1,3-thiazol-2-yl}carbamoyl)pyrazin
-2-yl]piperidine-4-carboxylate (41)
Phosphorous oxychloride (2.25 mL) was added dropwise to a mixture of 4-[4-methoxy-3-
(trifluoromethyl)phenyl]-1,3-thiazol-2-amine (38, 6.00 g), 5-[4-(ethoxycarbonyl)piperidin-1-
yl]pyrazine-2-carboxylic acid (28, 6.72 g), and pyridine (106 mL) at an internal temperature of -5C
to 5C over 10 minutes, and the mixture was warmed to room temperature and stirred for 1 hour. The
reaction mixture was concentrated under reduced pressure. An aqueous citric acid solution was added
to the residue and the mixture was extracted with ethyl acetate. The organic layer was dried over
anhydrous magnesium sulfate. The insoluble materials were separated by filtration and the filtrate
was concentrated under reduced pressure to about half the volume. Silica gel was added to the residue
and the mixture was stirred. The insoluble materials were separated by filtration, and the filtrate was
concentrated under reduced pressure. Diisopropyl ether was added to the residue, and the mixture
was stirred. The solid was collected by filtration and dried to obtain 41 (7.59 g, 65%) as a yellow
1
brown solid: H-NMR (CDCl3) δ 1.28 (3H, t, J = 7.0 Hz), 1.76–1.89 (2H, m), 2.03–2.13 (2H, m),
2.66 (1H, tt, J = 4.1, 10.5 Hz), 3.20–3.30 (2H, m), 3.95 (3H, s), 4.18 (2H, q, J = 7.0 Hz), 4.40 (2H,
dt, J = 3.9, 13.7 Hz), 7.05 (1H, d, J = 8.7 Hz), 7.11 (1H, s), 7.99 (1H, dd, J = 8.7, 2.2 Hz), 8.07 (1H,
d, J = 1.3 Hz), 8.10 (1H, d, J = 2.2 Hz), 8.94 (1H, d, J = 1.3 Hz), 10.64 (1H, s); ESI-MS m/z 536
[(M+H)+].
1-[5-({5-[(3,3-Dimethylpiperidin-1-yl)methyl]-4-[4-methoxy-3-(trifluoromethyl)phenyl]
-1,3-thiazol-2-yl}carbamoyl)pyrazin-2-yl]piperidine-4-carboxylic acid dihydrochloride (13)

Ethyl 1-[5-({4-[4-methoxy-3-(trifluoromethyl)phenyl]-1,3-thiazol-2-yl}carbamoyl)pyrazin-2-
yl]piperidine-4-carboxylate (41, 200 mg), acetic acid (4.0 mL), a 36% aqueous formaldehyde solution
(145 μL), and 3,3-dimethylpiperidine (215 mg) were mixed and the mixture was stirred at 90C for
1 hour. The reaction mixture was cooled to room temperature and concentrated under reduced
pressure, and the residue was diluted with ethyl acetate. The mixture was washed with an aqueous
sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution, and dried over
anhydrous sodium sulfate. The insoluble materials were separated by filtration and the filtrate was
concentrated under reduced pressure. The residue was purified by basic silica gel column
chromatography (hexanes-ethyl acetate).
The obtained residue was mixed with ethanol (4.0 mL) and tetrahydrofuran (2.0 mL), and a 1
M aqueous sodium hydroxide solution (1.90 mL) was added thereto, followed by stirring at 50C for
20 minutes. The reaction mixture was cooled to room temperature, and water and a 1 M aqueous
hydrochloric acid solution (1.90 mL) were added, followed by extraction with ethyl acetate. The
organic layer was washed with a saturated aqueous sodium chloride solution and dried over
anhydrous magnesium sulfate. The insoluble materials were separated by filtration and the filtrate
was concentrated under reduced pressure.
The obtained solid was mixed with ethyl acetate, and a 4 M hydrogen chloride/ethyl acetate
solution (500 μL) was added, and the mixture was concentrated under reduced pressure and washed
with ethyl acetate to obtain 13 (130 mg, 49%) as a yellow solid: 1H-NMR (DMSO-d6) δ 0.84 (3H, s),
1.06 (3H, s), 1.25 (1H, dt, J = 3.7, 13.4 Hz), 1.33–1.41 (1H, m), 1.51–1.71 (3H, m), 1.77–1.88 (1H,
m), 1.90–2.00 (2H, m), 2.44–2.55 (1H, m), 2.64 (1H, tt, J = 4.0, 10.8 Hz), 2.69–2.80 (1H, m), 2.84
(1H, d, J = 11.7 Hz), 3.16–3.31 (3H, m), 3.97 (3H, s), 4.37–4.48 (3H, m), 4.57 (1H, dd, J = 14.8, 3.1
Hz), 4.60–6.20 (2H, m), 7.39 (1H, d, J = 8.7 Hz), 7.83 (1H, d, J = 2.0 Hz), 7.87 (1H, dd, J = 8.7, 2.0
Hz), 8.40 (1H, d, J = 1.1 Hz), 8.77 (1H, d, J = 1.1 Hz), 9.86 (1H, brs), 12.06 (1H, s); ESI-MS m/z
633 [(M+H)+]; HRMS (M+H)+ calcd for C30H36O4N6F3S 633.2465; found 633.2463.
The following compounds (7–12) were prepared using a procedure similar to that described for 13.
1-{5-[(4-[4-Methoxy-3-(trifluoromethyl)phenyl]-5-{[(2R)-2-methylpyrrolidin-1-yl]methyl}
-1,3-thiazol-2-yl)carbamoyl]pyrazin-2-yl}piperidine-4-carboxylic acid dihydrochloride (7)
Yellow solid (yield 71%): 1H-NMR (DMSO-d6) δ 1.35 (3H, d, J = 6.4 Hz), 1.51–1.70 (3H, m),
1.84–2.00 (4H, m), 2.11–2.22 (1H, m), 2.64 (1H, tt, J = 3.9, 10.7 Hz), 3.06–3.18 (1H, m), 3.17–3.28
(2H, m), 3.36–3.56 (2H, m), 3.60–4.10 (2H, m), 3.97 (3H, s), 4.38–4.52 (3H, m), 4.74 (1H, dd, J =

14.9, 2.4 Hz), 7.40 (1H, d, J = 8.8 Hz), 7.93 (1H, d, J = 2.0 Hz), 7.99 (1H, dd, J = 8.8, 2.0 Hz), 8.40
(1H, d, J = 1.2 Hz), 8.77 (1H, d, J = 1.2 Hz), 10.52 (1H, brs), 12.10 (1H, s); ESI-MS m/z 605
[(M+H)+]; HRMS (M+H)+ calcd for C28H32O4N6F3S 605.2152; found 605.2153.
1-{5-[(4-[4-Methoxy-3-(trifluoromethyl)phenyl]-5-{[(2S)-2-methylpyrrolidin-1-yl]methyl}
-1,3-thiazol-2-yl)carbamoyl]pyrazin-2-yl}piperidine-4-carboxylic acid dihydrochloride (8)
Pale yellow solid (yield 55%): 1H-NMR (DMSO-d6) δ 1.34 (3H, d, J = 6.5 Hz), 1.51–1.68 (3H,
m), 1.83–1.99 (4H, m), 2.12–2.22 (1H, m), 2.59–2.68 (1H, m), 3.07–3.27 (3H, m), 3.37–3.56 (2H,
m), 3.62–3.92 (2H, m), 3.97 (3H, s), 4.38–4.52 (3H, m), 4.72–4.79 (1H, m), 7.40 (1H, d, J = 8.8 Hz),
7.92 (1H, d, J = 2.0 Hz), 7.95–7.99 (1H, m), 8.37–8.42 (1H, m), 8.77 (1H, d, J = 1.2 Hz), 10.25 (1H,
brs), 12.10 (1H, s); ESI-MS m/z 605 [(M+H)+]; HRMS (M+H)+ calcd for C28H32O4N6F3S 605.2152;
found 605.2162.
1-{5-[(5-{[(2R)-2-Ethylpyrrolidin-1-yl]methyl}-4-[4-methoxy-3-(trifluoromethyl)phenyl]
-1,3-thiazol-2-yl)carbamoyl]pyrazin-2-yl}piperidine-4-carboxylic acid dihydrochloride (9)
Yellow solid (yield 55%): 1H-NMR (DMSO-d6) δ 0.80 (3H, t, J = 7.3 Hz), 1.45–2.01 (9H, m),
2.04–2.20 (1H, m), 2.56–2.70 (1H, m), 3.05–3.28 (4H, m), 3.44–3.56 (1H, m), 3.97 (3H, s), 4.07–
4.77 (6H, m), 7.40 (1H, d, J = 8.8 Hz), 7.93 (1H, d, J = 2.0 Hz), 7.99 (1H, dd, J = 8.7, 2.0 Hz), 8.40
(1H, d, J = 1.2 Hz), 8.77 (1H, d, J = 1.2 Hz), 10.62 (1H, brs), 12.10 (1H, s); ESI-MS m/z 619
[(M+H)+]; HRMS (M+H)+ calcd for C29H34O4N6F3S 619.2309; found 619.2306.
1-{5-[(4-[4-Methoxy-3-(trifluoromethyl)phenyl]-5-{[(2R)-2-propylpyrrolidin-1-yl]methyl}
-1,3-thiazol-2-yl)carbamoyl]pyrazin-2-yl}piperidine-4-carboxylic acid dihydrochloride (10)
Yellow solid (yield 57%): 1H-NMR (DMSO-d6) δ 0.81 (3H, t, J = 7.2 Hz), 0.97–1.11 (1H, m),
1.19–1.32 (1H, m), 1.50–1.66 (5H, m), 1.82–2.00 (4H, m), 2.06–2.20 (1H, m), 2.58–2.69 (1H, m),
3.08–3.28 (4H, m), 3.46–3.57 (1H, m), 3.97 (3H, s), 4.03–4.86 (6H, m), 7.40 (1H, d, J = 8.7 Hz), 7.93
(1H, d, J = 2.0 Hz), 7.98 (1H, dd, J = 8.6, 2.0 Hz), 8.40 (1H, d, J = 1.1 Hz), 8.77 (1H, d, J = 1.2 Hz),
10.55 (1H, brs), 12.11 (1H, s); ESI-MS m/z 633 [(M+H)+]; HRMS (M+H)+ calcd for C30H36O4N6F3S
633.2465; found 633.2467.
1-{5-[(5-{[(2S)-2-(Methoxymethyl)pyrrolidin-1-yl]methyl}-4-[4-methoxy-3-(trifluoromethyl)
phenyl]-1,3-thiazol-2-yl)carbamoyl]pyrazin-2-yl}piperidine-4-carboxylic acid dihydrochloride
(11)

Pale yellow solid (yield 30%): 1H-NMR (DMSO-d6) δ 1.52–1.69 (3H, m), 1.80–2.01 (4H, m),
2.06–2.16 (1H, m), 2.58–2.68 (1H, m), 3.16–3.28 (5H, m), 3.48–3.78 (4H, m), 3.94–4.01 (4H, m),
4.31–4.99 (6H, m), 7.39 (1H, d, J = 8.8 Hz), 7.92 (1H, d, J = 2.0 Hz), 8.03 (1H, dd, J = 8.6, 2.0 Hz),
8.39—8.40 (1H, m), 8.77 (1H, d, J = 1.2 Hz), 10.62 (1H, brs), 12.09 (1H, s); ESI-MS m/z 635
[(M+H)+]; HRMS (M+H)+ calcd for C29H34O5N6F3S 635.2258; found 635.2259.
1-{5-[(4-[4-Methoxy-3-(trifluoromethyl)phenyl]-5-{[(2R)-2-methylpiperidin-1-yl]methyl}
-1,3-thiazol-2-yl)carbamoyl]pyrazin-2-yl}piperidine-4-carboxylic acid dihydrochloride (12)
Yellow solid (yield 19%): 1H-NMR (DMSO-d6) δ 1.34 (3H, d, J = 6.3 Hz), 1.31–1.74 (7H, m),
1.76–1.85 (1H, m), 1.90–2.00 (2H, m), 2.58–2.74 (2H, m), 3.09–3.30 (4H, m), 3.60–4.40 (2H, m),
3.97 (3H, s), 4.37–4.48 (3H, m), 4.79 (1H, dd, J = 14.9, 1.7 Hz), 7.39 (1H, d, J = 8.7 Hz), 7.88 (1H,
d, J = 2.0 Hz), 7.93 (1H, dd, J = 8.7, 2.0 Hz), 8.40 (1H, d, J = 1.0 Hz), 8.77 (1H, d, J = 1.0 Hz), 10.34
(1H, brs), 12.12 (1H, s); ESI-MS m/z 619 [(M+H)+]; HRMS (M+H)+ calcd for C29H34O4N6F3S
619.2309; found 619.2318.
1-{5-[(5-{[Isobutyl(2-methoxyethyl)amino]methyl}-4-[4-methoxy-3-(trifluoromethyl)
phenyl]-1,3-thiazol-2-yl)carbamoyl]pyrazin-2-yl}piperidine-4-carboxylic acid (14)
Ethyl 1-[5-({4-[4-methoxy-3-(trifluoromethyl)phenyl]-1,3-thiazol-2-yl}carbamoyl)pyrazin-2-
yl]piperidine-4-carboxylate (41, 1.45 g), acetic acid (10 mL), a 36% aqueous formaldehyde solution
(1.50 mL), and acetic anhydride (1.8 mL) were mixed, followed by stirring at 170C for 30 minutes
under irradiation with microwaves. The reaction mixture was concentrated under reduced pressure,
and water and a saturated aqueous sodium hydrogen carbonate solution were added to the residue,
followed by extraction with ethyl acetate. The organic layer was washed with water and a saturated
aqueous sodium chloride solution, and dried over anhydrous magnesium sulfate. The insoluble
materials were separated by filtration and the filtrate was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (chloroform-ethyl acetate).
The obtained residue was mixed with N,N-dimethylformamide (15 mL), N-(2-methoxyethyl)-
2-methylpropane-1-amine hydrochloride (685 mg), and N,N-diisopropylethylamine (1.4 mL), and the
mixture was stirred at 100C for 1 hour. The reaction mixture was cooled to room temperature and
ethyl acetate was added. The mixture was washed with water and a saturated aqueous sodium chloride
solution, and dried over anhydrous magnesium sulfate. The insoluble materials were separated by
filtration and the filtrate was concentrated under reduced pressure. The residue was purified by silica