Discovery of a novel orally active TRPV4 inhibitor: Part 1. Optimization from an HTS hit

Full text of DOI:10.1016/j.bmc.2019.05.041

Accepted Manuscript
Discovery of a novel orally active TRPV4 inhibitor: Part 1. Optimization from
an HTS hit
Yuichi Sami, Masataka Morita, Hirokazu Kubota, Ryoji Hirabayashi, Ryushi
Seo, Nobuaki Nakagawa
PII:
S0968-0896(19)30506-1
https://doi.org/10.1016/j.bmc.2019.05.041
BMC 14928
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 April 2019
24 May 2019
27 May 2019
Please cite this article as: Sami, Y., Morita, M., Kubota, H., Hirabayashi, R., Seo, R., Nakagawa, N., Discovery of
a novel orally active TRPV4 inhibitor: Part 1. Optimization from an HTS hit, Bioorganic & Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.bmc.2019.05.041
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Discovery of a novel orally active TRPV4 inhibitor: Part 1.
Optimization from an HTS hit
Yuichi Sami,* Masataka Morita, Hirokazu Kubota, Ryoji Hirabayashi, Ryushi Seo and Nobuaki
Nakagawa
Institute for Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki
305-8585, Japan,
*Corresponding author:
Development Project Management, Development
Astellas Pharmaceuticals, Inc.
2-5-1, Nihonbashi-Honcho, Chuo-ku,
Tokyo 103-8411, Japan
Tel: +81-(0)3-3244-5246
Fax: +81-(0)3-3243-5741
E-mail: yuichi.sami@astellas.com

1. Introduction
Transient receptor potential vanilloid type 4 (TRPV4)1 is a non-selective cation channel
constitutively expressed across species and regulates a multitude of physiological mechanisms. Its
first identified function dictated the early nomenclature, vanilloid receptor-related osmotically
activated channel (VR-OAC)2 or osmosensitive transient receptor potential channel 4 (OTRPC4)2b;
When the osmolarity of the extracellular medium diverts from that of the endothelial cells on which
TRPV4 channels are expressed, the cell volumes are controlled through the channel’s trafficking of
extracellular Ca2+. Taken together with the fact that TRPV4 is expressed in organs such as the lung
and the heart, evidences allow one to hypothesize that its malfunction results in pulmonary3 and
cardiovascular3b diseases. Indeed, it was shown by a Glaxo-Smith-Kline, Inc. group with compounds
1 and 2 (figure 1) that a TRPV4 inhibitor can improve surrogate parameters of pulmonary edema, in
high pulmonary venous pressure (PVP) induced ex vivo rodent and canine models4 or in a TRPV4
agonist-induced rodent in vivo model4b, respectively. The results of a series of clinical studies
involving yet another TRPV4 inhibitor GSK-2798745 for cardiac failure, congestive heart failure
and cough will clarify the validity of this therapeutic approach in these areas.5
However, TRPV4 channels are now known to be expressed in a more diverse list of organs, and
hence to be tasked with a wider variety of essential biological functions. One such aspect that we
became intrigued with was its relationship in nociceptive perceptions as the channel is found in the
peripheral nervous systems, e.g. sensory neurons and DRG neurons. It is believed that TRPV4
channel regulates both the inflammatory6a and mechanical pain6b in pathophysiological conditions.
One example of the value of its modulator has been provided by a Shionogi group, demonstrating
the effectiveness of a TRPV4 inhibitor 4 in Freund’s Complete Adjuvant (FCA) induced hyperalgesia
guinea pig model.7a-c Furthermore, another group from the same institute reported TRPV4 inhibitors
such as 1 and 5 can attenuate a loss of grip strength induced by MIA injection in an acute OA pain
model.7d Exemplified by these reports, the pharmacological implications continue to garner wide
attention.
CN
OH
N
O
NH
N
CN
N
N
O
N
N
O
Cl
Cl
Br
N
N
N
N
N
H
CF3
O
N
3
2
(1)
GSK2193874
O
N
N
F
OH
N
F
Cl
N
F F
O
F
F
N
N
Cl
O
O
S
N
N
S
N
H
N
O O
H
NC
N
S
F
(5)
HC- 067047
6
4
Figure 1. Selected small molecule organic compounds with TRPV4 inhibitory activities8
Intrigued by the pharmaceutical value a TRPV4 inhibitor would have for these reasons, we have

decided to start our own research for a TRPV4 inhibitor that can be orally administered to human
patients, especially in the field of pain management. One of the several hit compounds that were
found from an HTS campaign with an internal small molecule library was compound 7. With a unique
structure compared to the known TRPV4 inhibitors (figure 1, 1-68), its decent in vitro potency for an
HTS hit, and also a low species variance between human and rat,9 we have selected this compound
as the starting point towards an orally active TRPV4 inhibitor with requisite safety profile.
Figure 2. HTS hit compound (7) from Astellas chemical library
2. Results and discussion
2.1. Chemistry
Compound 7 and its derivatives on table 1-3 were synthesized according to schemes 1 and 2. 1-
Methyl piperazine was introduced to commercially available 2-aminothiazoles following thiazole C5
bromination. Condensation with aromatic carboxylic acids or acid chlorides gave the desired
compounds.
Scheme 1. Syntheses of 7, 12a-e, 13a-m. Reagents and conditions: (a) NBS, NMP; (b) N-Methyl piperazine, NMP;
(c) (i) ArCOCl, Et3N, DCM or (ii) ArCOOH, HATU, DIPEA, THF, microwave irradiation; then 4M HCl (dioxane),
solvents.
One aromatic carboxylic acid 16 that was used as an intermediate for 13j in scheme 1 was
synthesized as shown in scheme 2.

Scheme 2. Synthesis of 16. Reagents and conditions: (a) (4,4-difluorocyclohexyl)hydrazine hydrogen chloride,
K2CO3, EtOH, 70 °C, 1 d; (b) TFA, DCM, rt, 1 d.
Compound 21a was synthesized as shown in scheme 3. Reaction of a commercially available
Weinreb amide 17 with a Grignard reagent followed by bromination and thiazole ring formation gave
aminothiazole 19. Amide formation, and then replacement of a Cbz group with a methyl group
produced the desired compound.
Scheme 3. Synthesis of 21a. Reagents and conditions: (a) PhMgBr, THF, rt, 3 hr; (b) Br2, AcOH, rt, 3 hr; thiourea,
EtOH, reflux, 1 hr; (c) 3,5-(MeO)2PhCOCl, Et3N, DCE, 50 °C, 12 hr; (d) H2 (3 atm), Pd/C, AcOH, MeOH, DMF,
rt, 6 hr; 37% aq. formaldehyde, NaBH(OAc)3, AcOH, DMF, rt, 5 hr.
Compounds 21b,c,e,f were synthesized according to scheme 4. Bromination followed by
condensation with thiourea afforded a 2-aminothiazole derivative from commercially available
ketones. Following formation of an amide bond by condensation with 3,5-dimethoxybenzoyl
chloride, the chlorine was substituted with various secondary amines to yield the desired compounds.
Scheme 4. Syntheses of 21b, 21c, 21e, 21f. Reagents and conditions: (a) Br2, HBr in EtOH, MeOH; (b) Thiourea,
EtOH; (c) 3,5-(MeO)2PhCOCl, Et3N, DCE; (d) Amine, K3PO4, KI, DMF; then conc. HCl, EtOH.

Compounds 21d and 28a-c were synthesized as drawn in scheme 5. In these cases, intermediates
24b and 25 were first reacted with morpholine or piperidine, and then with either 3,5-
dimethoxybenzoyl chloride or commercially available carboxylic acids to yield the desired
compounds.
Scheme 5. Synthesis of 21d, 28a-c. Reagents and conditions: (a) morpholine, DMF, 60 °C, 2 d; 15 hr; or piperidine,
DMF, 60 °C, 1 d; (b) 3,5-(MeO)2PhCOCl, Et3N, DCE; or ArCOOH, HATU, DIPEA, NMP; then 4M HCl (dioxane).
Compound 21g was synthesized as drawn in scheme 6. Mannich reaction utilizing piperidine
afforded the desired compound in a single step from a commercially available reagent 29.
Scheme 6. Synthesis of 21g. Reagents and conditions: (a) Piperidine, 36% aq. formaldehyde, AcOH, 80 °C, 18 hr;
then 4M HCl (EtOAc).
2.2. Human TRPV4 inhibition and structure-activity relationship
Our goal of this research was to obtain an orally active TRPV4 inhibitor with a safety profile that
permits use in common diseases, e.g. pain management in osteoarthritis patients. Thus, the initial
aim was set on improvements of not only the in vitro potency, but also of factors that affect
effective oral administration; namely, aqueous solubility and intrinsic metabolic clearance. Though
it was initially believed that the poor solubility is primarily a result of the presence of two benzene
rings (regions A, B), region C was also explored with the same objective.
Figure 3. Regions A, B and C of compound 7.

2.2.1. Region A
Our structure-activity relationship (SAR) investigation started with replacement of the phenyl with
all three regioisomers of a pyridine (12a-c, table 1). Unfortunately, these analogs did not retain the
in vitro TRPV4 antagonistic activity.
Next, we turned to a series of nonaromatic substituents (12d-e). It was revealed that cyclobutyl (12e)
is a substituent that brings both comparative potency and solubility compared to 7. It can be
speculated that the protein pocket this region binds to prefers a lipophilic ligand with appropriate
size. Although it was encouraging to see improvements in both aspects, we have turned our attention
to region B with the hope of further optimization.
Table 1. Analogs of 7 with different substructures in region A.
N
N
R
H
N
O
O
S
N
O
Aqueous
Solubility*
(M)
Compound
No.
h TRPV4
Measured
R
IC50 (nM)
logD7.4
7**
12a
12b
12c
12d
144
1800
290
<1
≥100
2.2
>4.6
3.7
4.1
4.1
4.0
N
N
>10000
240
1.7
N
15

12e
84
4.7
4.6
* pH6.8. ** Dihydrochloride salt.
2.2.2. Region B
With the objective of further improving the in vitro potency and solubility by exploration of region
B, we have investigated the SAR of various scaffolds. One such scaffold that we became intrigued
with was a dimethylpyrazole (13a, table 2) because a much higher solubility (≥100 M) was achieved
while the ligand lipophilicity efficiency based on measured LogD values was comparable (LipE=3.0
(13a) vs 2.5 (12e))10. This proved to be a critical choice for optimizing the potency and solubility, as
is discussed later in this paper. Subsequently, a benzene ring was introduced to N1 of this scaffold
with the intention of clarifying the spatial requirement of the binding pocket. Thus, the result that
this additional phenyl significantly improved the potency indicates that there is lipophilic region
within the pocket. However, no other aromatic linker between the amide bond and the terminal
benzene ring showed the same degree of potency (13c-f), which in turn means the linkage between
the core and terminal structures are crucial due to either or both of electronic and steric reasons. The
solubility improvement from 12e to 13b is discussed later.
Table 2. Analogs of 12e with different substructures in region B.
N
N
H
N
S
N
R
O
Aqueous
Solubility*
(M)
h TRPV4
Measured
Compound No.
R
IC50 (nM)
logD7.4
O
12e
84
4.7
4.6
3.1
O
13a***
730
≥100
N
N

13b***
13c
11
240
830
3600
81
≥100
NT**
NT**
<1
4.4
NT**
NT**
4.9
N
N
S
13d
O
13e
N
N
N
O
13f***
<1
5.0
* pH6.8. ** NT is a value that has not been tested. *** Dihydrochloride salt.
Next, having observed the success with a simple phenyl ring on N1, further optimization was
conducted. Introduction of a tertiary alkyl substituent resulted in less potency (13g, table 3), but the
bioactivity was regained with a cyclic substituent (13h). While the potency was lost when a THP
ring was introduced (13i), a difluorocyclohexyl substituent retained the potency, albeit with a loss of
solubility (13j). Addition of a fluorine atom to the original benzene moiety improved the potency
while maintaining the high solubility (13k), somewhat contrasting the SAR of the cyclohexane
analogs (13h, j). Introduction of a polar CN group onto the same position (13l), or an additional
methylene spacer between the two aromatic rings (13m), resulted in not only a decreased potency,
but also a compromised solubility.
Table 3. Analogs of 13b with different N1 substituents.
N
N
H
N
S
N
R
N
N
O
Aqueous
Solubility*
(M)
h TRPV4
Measured
Compound No.
R
IC50 (nM)
logD7.4

13b**
13g***
13h**
13i**
13j**
13k
11
≥100
48
4.4
4.5
5.0
3.1
4.3
4.8
78
11
98
O
880
15
≥100
7.6
F
F
F
1.9
≥100
CN
13l
22
30
<1
4.0
4.8
13m**
2.4
F
* pH6.8. ** Dihydrochloride salt. *** Hydrochloride salt.
2.2.3. Region C
As the final part of the initial round of investigations, the SAR of region C was explored to identify
a substituent with improved in vitro potency and physicochemical property. We were intrigued when
an early exploration quickly revealed that the nitrogen directly attached to the core can be substituted
to a carbon atom without loss of activity in the context of cyclic amines (21a, table 4). This finding
also met our desire to remove one of the two amino groups bound to the thiazole, since we suspected
that there is a potential risk of mutagenicity for a diaminothiazole compounds.11,12 Interestingly, the

bioactivity was mostly retained even when the piperidine was exchanged with a linear amine (21b),
possibly because the original piperidine ring was not forcing the ligand in an optimal conformation
to bind to the target protein pocket.
Table 4. Analogs of 7 with different substructures in region C.
R
H
N
O
O
S
N
O
Aqueous
Solubility*
(M)
Compound
No.
h TRPV4
Measured
R
IC50 (nM)
logD7.4
N
N
7**
144
<1
>4.6
N
21a
84
86
≥100
≥100
3.6
3.5
21b**
N
* pH6.8. ** Dihydrochloride salt.
Taking advantage of these findings, we next turned to a series of structures with linear, carbon based
linkers between cyclic amines and the core. With the exception of piperazine that has an additional
basic amine (21e, table 5), the introduced structures maintained the in vitro potency with a propylene
linker (21c, d). Ethylene linker (21f) was found to be optimal from a survey of shorter linkers (21f,
h).
Table 5. Analogs of 21b with different terminal amine structures.
n
H
N
O
O
S
N
R
O
Aqueous
Solubility*
(M)
Compound
No.
h TRPV4
Measured
n
R
IC50 (nM)
logD7.4

21b**
21c**
21d
3
3
3
3
2
1
86
120
99
≥100
≥100
<1
3.5
4.1
4.7
4.1
4.5
4.9
N
N
O
N
N
21e***
21f**
1700
51
≥100
84
N
N
21g****
310
<1
N
* pH6.8. ** Dihydrochloride salt. *** Trihydrochloride salt. **** Hydrochloride salt.
2.2.4. Combination of the obtained SAR
Based on the findings described above, we have decided to prepare one compound that contains all
the optimized substituents (28a, table 6). While the in vitro potency and solubility was significantly
improved compared to 7, the human microsomal clearance value was found to be higher. In an effort
to increase the metabolic stability, two pyridine analogs were prepared (28b, c), and 28c was found
to possess a satisfactory profile across the aspects of our interest.
Table 6. Overall profile of advanced compounds 28a-c in comparison to 7.
R2
R1
H
N
S
N
Ar
O
Aqueous
Solubility*
(M)
Compound
No.
h TRPV4 r TRPV4
Measured h CLint
r CLint
R1
R2
Ar
IC50 (nM) IC50 (nM)
logD7.4 (ml/min/kg)(ml/min/kg)

O
O
N
7**
Ph
144
0.85
1.2
137
1.1
<1
>4.6
4.1
131
527
292
120
>1000
>1000
688
N
F
F
28a**
28b**
28c**
cBu
cBu
cBu
≥100
≥100
≥100
N
N
N
N
N
0.32
0.73
3.8
N
N
N
F
N
N
3.3
4.5
798
N
* pH6.8. ** Dihydrochloride salt.
2.3. In vivo pharmacology
After confirming the species variance in the IC50 values against rat and human TRPV4 is negligible,
we have decided to confirm if 28c, the most metabolically stable compound in human microsomes
from table 6, is also active in vivo. An in vivo rat plasma extravasation experiment utilizing a TRPV4
activator was built to demonstrate the in vivo TRPV4 blockade by 28c, because TRPV4 activators
are known to elicit vascular permeability13 which can be quantified by the leakage of Evans blue dye.
Thus, 28c was orally dosed at 3 mg/kg to a rat pretreated with a TRPV4 agonist, and was successfully
shown to be orally active (88% inhibition). This finding validates our belief that this series of
compounds merits further optimization efforts towards a clinical candidate after future examination
of toxicological aspects.
2.4. Solubility improvements
Improvement of aqueous solubility is one of the key factors in drug discovery research.14 According
to the formulation proposed by Yalkowski and Valvani, aqueous solubility of organic compounds
should be given by15
log SW ≈ –log P – 0.01 x mp + 1.05
(1)
Thus, the mechanisms underlying the solubility improvement that is achieved through modification
to regions A, B and C can be assessed from the two critical aspects noted in this formula; the
lipophilicity (log P) and the melting point (mp).
In tables 4 and 5, compounds with higher pKb values, e.g. 7 (5.8), 21d (6.1) and 21g (6.2), all have

lower solubility values than the others, such as 21a (4.5), 21b (4.1), 21c (4.2), 21e (4.5) and 21f
(4.7).16 This is most critically a product of higher measured lipophilicity that resulted from lower
basicity values for the former group compared to the latter, because the trends of solubility (log SW)
and lipophilicity (measured logD7.4) are opposite to each other. Similar relationship is observed
among 7 and 12a-c. In this case, subtly greater exposure of the basic nitrogen to the surface might
explain the lipophilicity trend, as the dihedral angle values between the pyridine and thiazole moieties
in their most stable conformations in in silico analyses are 53 degrees for 12a and 39, 40 degrees for
12b, 12c.17
In contrast to these observations is the data obtained in tables 2 and 3. Particularly, the solubility
difference between 12e and 13b (4.7 vs ≥100 M) despite similar measured logD7.4 values (4.6 vs
4.4) is interesting. The other critical component of Yalkowski’s formula is the melting point (mp).
Mp continues to be one of the most difficult physicochemical property to predict,18 but it has been
well established from various studies to possess correlation with the symmetry19 and planarity20 of
the compound.21 The structural difference between 12e and 13b is limited to region B, and 12e has a
C2-symmetrical 3,5-(MeO)2-phenyl that also has low dihedral angles between the thiazole-amide-
benzene system. On the other hand, 13b has an unsymmetrical pyrazole, which induces a non-planar,
bent shape to the entire molecular. Therefore, it is reasonable to understand the solubility difference
between the two compounds are caused by these structural factors, and it also highlights the
importance of selecting the right scaffold upon which a compound is built. Further crystallographic
studies are needed for a decisive analysis.
3. Conclusion
Optimization of the two benzene substituents on the thiazole ring of our HTS hit compound 7
resulted in identifications of a cBu and a pyrazole scaffold in regions A and B that led to both
increased solubility and in vitro potency. Another structural modification that yielded similar
improvements was discovered within region C in the form of a linear 2-piperidinylethyl. We believe
this structure also lowered the intrinsic risk for mutagenicity. Combination of these findings yielded
a small set of TRPV4 inhibitors with vastly improved potency and solubility. Comparison of the
human metabolic stability revealed an interesting pattern contrary to the general trend with
lipophilicity within this set of compounds. Finally, 28c was shown in a rat in vivo model to be an
orally active TRPV4 inhibitor. We aim to further optimize this compound and identify a clinical
candidate in the near future.
4. Experimental section
4.1 Biological assays
4.1.1. In vitro human and rat TRPV4 inhibition assay
4.1.1-1 Ca2+ Influx assay
Human TRPV4 channel-expressing HEK293 cells and rat TRPV4 channel-expressing HEK293 cells

were plated at a density of 3.5 × 103 cells/well for human TRPV4 and 6.5 × 103 cells/well for rat
TRPV4 into black-wall, clear bottom, Poly-D-lysine coated, 384-well plate and incubated with
Dulbecco’s Modified Eagle Medium alpha (DMEM) containing 10% fetal bovine serum, 50 units /
mL penicillin, and 50 μg/mL streptomycin. At approximately 72 hr after cell plating, the medium
was removed and the cells were loaded with 20 μL/well of Loading solution {1μM Fluo4-AM
(DOJINDO, Kumamoto, Japan) diluted in assay buffer (1xHBSS containing 20mM HEPES and 1%
CHAPS. In case of rat TRPV4 assay, 1.25mM CaCl2 was also added)}. After approximately 1.5-h
incubation at room temperature in shaded condition, 10μL/well of diluted test compounds (final, 0-
10 , vehicle (assay buffer) or Ruthenium Red (final, 3.3  as positive control TRPV4 inhibitor
were added to respective wells. After approximately 30min incubation at room temperature in shaded
condition, 25μl of hypotonic solution (88% H2O in assay buffer for hTRPV4 and 100% H2O for rat
TRPV4) was added to respective wells and change in RFU (excitation wavelength: 475-495 nm,
emission wavelength: 515-575 nm) from immediately before to 3 min after the addition of hypotonic
solution was recorded as an indicator of change in Ca2+ influx. Data in an experiment was obtained
in duplicate.
4.1.1-2 Data Analysis
The response to each concentration of test compound was expressed as a percentage
where average values of the responses to vehicle and Ruthenium Red in each test plate
were defined as 0% and 100% inhibition, respectively. IC50 value was calculated by Sigmoid-Emax
model non-linear regression analysis using the Statistical Analysis System (SAS Institute Japan Ltd.,
Tokyo, Japan). IC50 value was defined as the test compound concentration which produces 50%
inhibition.
4.1.2. In vivo rat TRPV4 inhibition assay
4.1.2-1 Measurement of plasma extravasation
As TRPV4 activator elicits vascular permeability13, the effect of TRPV4 inhibitors were evaluated
in a TRPV4 activator22 induced vascular permeability test. Female Lewis rats (7 weeks of age;
Charles River Laboratories Japan, Yokohama, Japan) were used. All animal experimental procedures
were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc.
All animals were used under fed conditions. The animals’ back fur was shaved under isoflurane
anesthesia. After rats awoke, 0.5% methyl cellulose (MC) as a negative control or a TRPV4 inhibitor
of choice was orally administered (N = 4–5). After 1 hour, 1.5% Evans blue dye (3 mL/kg) was
injected via the tail vein immediately after intradermal application of the TRPV4 activator (10 ,
10 L) and PEG-400 (Vehicle, 10 L) into 4 points symmetrically on the dorsal region under
isoflurane anesthesia. After 10 minutes, the rats were killed by cervical dislocation. The skin area
around the TRPV4 activator and Vehicle application point (8 mm in diameter) was removed, and the
Evans blue dye was extracted by incubating the skin samples in 1 mL of formamide for
approximately 24 hours at room temperature. The absorbance of Evans blue dye extracted in
formamide from individual skin samples was measured by spectrophotometry at 620 nm using a plate

reader (SPECTRAmax PLUS 384; Molecular Devices Japan, Tokyo, Japan). The mean value of the
four skin samples where Vehicle or TRPV4 activator was injected was used as the Vehicle or TRPV4
activator-induced Evans blue extravasation value.
4.1.2-2 Data analysis
The concentration of the Evans blue was calculated from a standard curve, subtracted by the value
corresponding to skin samples when Vehicle was injected. Inhibitory activity (%) was calculated as
follow.
Inhibitory activity (%) = {1-Value(Test compound) / Value(MC)} x 100
4.1.3. In vitro human and rat microsomal clearance
Pooled human and rat liver microsomes (Xenotech LLC.) were diluted in 100 mM
KH2PO4/Na2HPO4 buffer (pH7.4) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). The
incubation mixtures (270 μL total volume), which contained 0.2 mg/mL of microsomal proteins, and
0.2 μM of substrates (0.093 μM in the case of compound 7) were pre-incubated for approximately
about 15 min at 37 °C. Reactions were initiated by the addition of 1 mM NADPH (30 μL). After the
appropriate incubation time (0, 15, 30, and 45 min), 40 μL of incubation mixture was transferred into
80% acetonitrile containing internal standard (50 nM propranolol, 250 μL), stood at 4 °C for over 10
min, and centrifuged for 20 min at 2800 rpm. The supernatant (200 μL) was prepared and analyzed
via LC-MS/MS with UPLC system (Waters) and Xevo TQ (Waters). The in vitro intrinsic clearance
(CLint, vitro) was calculated using Equation 1, which is based on the time course of the residual ratio
of the compounds.
ퟏ
풎품
풎푳
(
)
(
풎품
)
풌품
푲풆 퐦퐢퐧 × 푴푺풄풐풏풕풆풏풕
CLint, vitro (mL/min/kg) =
(2)
(
)
푴푺 푷풓풐풕풆풊풏 푪풐풏풄.
where the Ke is the disappearance rate constant.
4.2. Physicochemical property assay
4.2.1. Aqueous solubility
Solubility in Japanese Pharmacopoeia 2nd fluid for disintegration test (JP2; pH = 6.8 buffer) was
evaluated by following method. 10 mM DMSO stock solution of test compounds were prepared and
added into JP2. Shook by 1000 rpm at 25 °C light protected condition for 20 h. Precipitates were
filtrated through PVDF membrane filter (pore size 0.22 μm, MERCK) and the compound
concentration (μM) of filtrate was assayed by HPLC.
4.2.2. Measured logD7.4
LogD7.4 value was calculated as the natural logarithm of the compound concentration ratio between
1-octanol and Dulbecco’s phosphate buffered salts (PBS) aqueous solution (pH7.4). The

concentration values within the two layers were respectively evaluated by LC-MS after 10 mM
DMSO stock solution of the compound was partitioned at 23 °C.
5. Syntheses
5.1. Materials
The following abbreviations are used: NMP, N-methylpyrrolidone; NBS, N-bromosuccinimide;
TFA, trifluoroacetic acid; DMF, N,N-dimethylformamide; MeOH, methanol; EtOH, ethanol; HATU,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-Oxide
Hexafluorophosphate; DIPEA, diisopropylethylamine; DMSO, N,N-dimethylsulfoxide; EtOAc,
ethyl acetate; IPA, isopropanol; Et2O, diethylether; IPE, diisopropylether.
5.2. Instrumentation
1H NMR spectra were recorded on a Varian VNS-400, JEOL JNM-LA400 or JEOL JNM-AL400
spectrometer. Chemical shifts were expressed in δ values (ppm) using tetramethylsilane as the
internal standard (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = double doublet,
dt = double triplet, br = broad peak, and brs = broad singlet). Mass spectra (MS) were recorded on a
JEOL LX-2000, Waters ZQ-2000, Waters LCT Premier mass spectrometer or Thermo Fisher
Exactive Plus Orbitrap. Elemental analyses were conducted using a Yanaco JM10 (C, H, N),
Elementar Vario EL III (C, H, N), Dionex ICS-3000 (S, halogene), and Dionex DX-5000 (S,
halogene) and were within ±0.4% of theoretical values. All reactions were carried out using
commercially available reagents and solvents without further purification.
5.3. Synthetic procedures
5.3.1. 5-(4-Methylpiperazin-1-yl)-4-phenyl-1,3-thiazol-2-amine (10)
To a mixture of 8 (10.0 g, 57 mmol) and NMP (100 mL) was added NBS (10.6 g, 60 mmol, 1.1
equiv.) portionwise on ice water bath, and the whole was stirred on the same bath for 1 hr, then at rt
for 2 hr. To the reaction mixture was added N-methylpiperazine (28.5 g, 285 mmol, 5.0 equiv.) in
NMP (20 mL) on an ice water bath, and the whole was stirred at 100 °C for 8 hr. To the reaction
mixture was added H2O, and the whole was stirred at room temperature for 10 minutes. The
precipitate was collected by filtration, washed with H2O and dried in vacuo to give the title compound
1
(12.5 g, 45 mmol, 80%) as a pale brown powder. H NMR (400 MHz, CDCl3): δ = 2.35 (3H, s),
2.50–2.63 (4H, m), 2.85–2.97 (4H, m), 4.80 (2H, s), 7.21–7.28 (1H, m, overlapped with CHCl3),
7.32–7.42 (2H, m), 8.04–8.14 (2H, m); MS (ESI) m/z 275 [M+H]+.
5.3.2. 3,5-Dimethoxy-N-[5-(4-methylpiperazin-1-yl)-4-phenyl-1,3-thiazol-2-yl]benzamide
dihydrochloride (7)
To a mixture of 10 (5.0 g, 18 mmol) and CH2Cl2 (60 mL) was added Et3N (5.4 mL, 39 mmol, 2.1

equiv.) and 3,5-dimethoxybenzoyl chloride (7.3 g, 36 mmol, 2.0 equiv.) on an ice water bath, and
the whole was stirred on the same bath for 10 minutes, then at rt for 1 d. To the reaction mixture was
added EtOAc, and the organic layer was washed with H2O and sat. brine. The organic layer was dried
over anhydrous MgSO4 and concentrated in vacuo, and the residue was purified on a silica gel
column chromatography (CHCl3:MeOH). To the crude product was added EtOH, EtOAc and 4 M
HCl in dioxane, and the whole was stirred at rt overnight. The precipitation was collected by filtration,
washed with EtOAc and dried in vacuo. The obtained pale yellow powder was dissolved into H2O,
and 1 M aq. NaOH was added therein. The suspension was extracted with CHCl3. The combined
organic layers were concentrated in vacuo, and the residue was purified on a silica gel column
chromatography (CHCl3:MeOH) to give a pale yellow powder. To a solution of this product in
MeOH and EtOAc was added 4 M HCl in dioxane, and the mixture was stirred at rt overnight. The
precipitate was collected by filtration, washed with a hot mixture of EtOH and EtOAc, then dried in
1
vacuo to give the title compound (2.8 g, 5.0 mmol, 28%) as a pale yellow powder. H NMR (400
MHz, DMSO-d6): δ = 2.79–2.88 (3H, m), 3.10–3.35 (6H, m), 3.42–3.53 (2H, m), 3.83 (6H, s), 6.75
(1H, t, J = 2.3Hz), 7.27–7.36 (3H, m), 7.42–7.49 (2H, m), 8.10–8.16 (2H, m), 11.07 (1H brs), 12.59
(1H, brs); MS (ESI) m/z 439 [M+H]+; Anal. Calcd. for C23H26N4O3S·2H2O·2HCl: C, 50.46, H, 5.89,
N, 10.23, S, 5.86, Cl, 12.95. Found: C, 50.59, H, 6.12, N, 10.18, S, 5.74, Cl, 12.71.
5.3.3. 5-(4-Methylpiperazin-1-yl)-4-(pyridin-2-yl)-1,3-thiazol-2-amine (11a)
The title compound was obtained in a similar manner to 10 using 9a as a yellow solid in 10% yield.
1H NMR (400 MHz, DMSO-d6): δ = 2.20 (3H, s), 2.37–2.47 (4H, m), 2.81–2.92 (4H, m), 6.70 (2H,
s), 7.17 (1H, ddd, J = 1.1, 4.7, 7.5 Hz), 7.74 (1H, td, J = 2.0, 7.6 Hz), 7.82–7.88 (1H, m), 8.51–8.58
(1H, m); MS (ESI) m/z 276 [M+H]+.
5.3.4. 3,5-Dimethoxy-N-[5-(4-methylpiperazin-1-yl)-4-(pyridin-2-yl)-1,3-thiazol-2-yl]benzamide
(12a)
The title compound was obtained in a similar manner to 7 using 11a as a pale yellow solid in 43%
yield. 1H NMR (400 MHz, DMSO-d6): δ = 2.24 (3H, s), 2.43–2.55 (4H, m, overlapped with DMSO),
3.00–3.08 (4H, m), 3.83 (6H, s), 6.73 (1H, t, J = 2.3 Hz), 7.26 (1H, ddd, J = 1.1, 4.8, 7.4 Hz), 7.31
(2H, d, J = 2.3 Hz), 7.80–7.87 (1H, m), 8.02–8.07 (1H, m), 8.60–8.64 (1H, m), 12.54 (1H, brs); MS
(ESI) m/z 440 [M+H]+.
5.3.5. 5-(4-Methylpiperazin-1-yl)-4-(pyridin-3-yl)-1,3-thiazol-2-amine (11b)
The title compound was obtained in a similar manner to 10 using 9b as a beige solid in 47% yield.
1H NMR (400 MHz, DMSO-d6): δ = 2.23 (3H, s), 2.42–2.50 (4H, m, overlapped with DMSO), 2.73–
2.80 (4H, m), 6.90 (2H, s), 7.39 (1H, ddd, J = 0.78, 4.8, 8.2 Hz), 8.33 (1H, dt, J = 1.9, 8.0 Hz), 8.40
(1H, dd, J = 1.7, 4.7 Hz), 9.17-9.21 (1H, m); MS (ESI) m/z 276 [M+H]+.
5.3.6. 3,5-Dimethoxy-N-[5-(4-methylpiperazin-1-yl)-4-(pyridin-3-yl)-1,3-thiazol-2-yl]benzamide
(12b)

The title compound was obtained in a similar manner to 7 using 11b as a colorless solid in 10%
yield. 1H NMR (400 MHz, DMSO-d6): δ = 2.25 (3H, s), 2.44–2.59 (4H, m, overlapped with DMSO),
2.90–2.96 (4H, m), 3.83 (6H, s), 6.74 (1H, t, J = 2.3 Hz), 7.30 (2H, d, J = 2.4 Hz), 7.47 (1H, ddd, J
= 0.83, 4.8, 8.0 Hz), 8.42 (1H, dt, J = 2.1, 8.0 Hz), 8.48 (1H, dd, J = 1.7, 4.7 Hz), 9.26–9.30 (1H, m),
12.49 (1H, brs); MS (ESI) m/z 440 [M+H]+; Anal. Calcd. for C22H25N5O3S: C, 60.12, H, 5.73, N,
15.93, S, 7.30. Found: C, 59.98, H, 5.72, N, 15.92, S, 7.30.
5.3.7. 5-(4-Methylpiperazin-1-yl)-4-(pyridin-4-yl)-1,3-thiazol-2-amine (11c)
The title compound was obtained in a similar manner to 10 using 9c as a beige solid in 45% yield.
1H NMR (400 MHz, DMSO-d6): δ = 2.24 (3H, s), 2.43–2.52 (4H, m, overlapped with d-DMSO),
2.72–2.81 (4H, m), 6.91 (2H, s), 7.94–7.99 (2H, m), 8.51-8.56 (2H, m); MS (ESI) m/z 276 [M+H]+.
5.3.8. 3,5-Dimethoxy-N-[5-(4-methylpiperazin-1-yl)-4-(pyridin-4-yl)-1,3-thiazol-2-yl]benzamide
(12c)
The title compound was obtained in a similar manner to 7 using 11c as a beige solid in 28% yield.
1H NMR (400 MHz, DMSO-d6): δ = 2.27 (3H, s), 2.47–2.60 (4H, m, overlapped with DMSO), 2.92–
2.99 (4H, m), 3.83 (6H, s), 6.74 (1H, t, J = 2.3 Hz), 7.30 (2H, d, J = 2.4 Hz), 8.02–8.07 (2H, m),
8.61–8.65 (2H, m), 12.57 (1H, brs); MS (ESI) m/z 440 [M+H]+; Anal. Calcd. for C22H25N5O3S: C,
60.12, H, 5.73, N, 15.93, S, 7.30. Found: 59.85, H, 5.81, N, 15.70, S, 7.23.
5.3.9. 4-Cyclopropyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-amine (11d)
The title compound was obtained in a similar manner to 10 using 9d as a brown solid in 51% yield.
1H NMR (400 MHz, CDCl3): δ = 0.73–0.87 (4H, m), 1.95–2.05 (1H, m), 2.33 (3H, s), 2.48–2.61
(4H, m), 2.81–2.93 (4H, m), 4.63 (2H, s); MS (ESI) m/z 239 [M+H]+.
5.3.10. N-[4-Cyclopropyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-3,5-dimethoxybenzamide
(12d)
The title compound was obtained in a similar manner to 7 using 11d as a pale yellow solid in 83%
yield. 1H NMR (400 MHz, DMSO-d6): δ = 0.80–0.89 (4H, m), 2.00–2.09 (1H, m), 2.23 (3H, s), 2.40–
2.55 (4H, m, overlapped with DMSO), 2.82–2.94 (4H, m), 3.81 (6H, s), 6.69 (1H, t, J = 2.3 Hz), 7.24
(2H, d, J = 2.3 Hz), 12.2 (1H, brs); MS (ESI) m/z 403 [M+H]+; Anal. Calcd. for
C20H26N4O3S·0.5H2O: C, 58.37, H, 6.61, N, 13.61, S, 7.79. Found: C, 58.18, H, 6.56, N, 13.44, S,
7.72.
5.3.11. 4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-amine (11e)
The title compound was obtained in similar manner to 10 using 9e as a brown powder in 62% yield.
1H NMR (400 MHz, CDCl3): δ = 1.80–2.10 (2H, m), 2.10–2.20 (2H, m), 2.24–2.40 (5H, m), 2.44–
2.56 (4H, m), 2.70–2.85 (4H, m), 3.55–3.68 (1H, m), 4.77 (2H, s); MS (ESI) m/z 253 [M+H]+.
5.3.12. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-3,5-dimethoxybenzamide

(12e)
A mixture of 11e (400 mg, 1.6 mmol), HATU (540 mg, 1.7 mmol, 1.1 equiv.), 3,5-
dimethoxybenzoic acid (350 mg, 1.9 mmol, 1.2 equiv.), DIPEA (619 mg, 4.8 mmol, 3.0 equiv.) and
THF (4.0 mL) was stirred under irradiation of microwave at 160 °C for 20 minutes. To the reaction
mixture was added EtOAc, and washed with sat. aq. NaHCO3 and sat. brine. The separated organic
layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by a
silica gel column chromatography (CHCl3:MeOH) and recrystallization from a mixture of EtOAc
and hexane to give the title compound (236 mg, 0.57 mmol, 36%) as a pale yellow powder. 1H NMR
(400 MHz, DMSO-d6): δ = 1.76–1.87 (1H, m), 1.90–2.06 (1H, m), 2.12–2.26 (2H, m), 2.22 (3H, s),
2.28–2.40 (2H, m), 2.42–2.48 (4H, m), 2.75–2.83 (4H, m), 3.64–3.75 (1H, m), 3.82 (6H, s), 6.71
(1H, t, J = 2.3 Hz), 7.27 (2H, d, J = 2.3 Hz), 12.35 (1H, s); MS (ESI) m/z 417 [M+H]+; Anal. Calcd.
for C21H28N4O3S: C, 60.55, H, 6.78, N, 13.45, S, 7.70. Found: C, 60.40, H, 6.68, N, 13.25, S, 7.67.
5.3.13. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-1,5-dimethyl-1H-pyrazole-4-
carboxamide dihydrochloride (13a)
To a dioxane (2.5 mL) solution of the free form of the title compound (261 mg), which was obtained
in a similar manner to 12e using 1,5-dimethyl-1H-pyrazole-4-carboxylic acid, was added 4 M HCl
dioxane (1.5 mL) and concentrated in vacuo. The residue was suspended in a hot mixture of IPA,
EtOH and EtOAc, allowed to rt, and then the resulting precipitation was filtered to give the title
compound as a colorless solid in 35% yield. 1H NMR (400 MHz, DMSO-d6): δ = 1.77–1.89 (1H, m),
1.89–2.04 (1H, m), 2.12–2.24 (2H, m), 2.24–2.40 (2H, m), 2.36 (3H, s), 2.81 (3H, d, J = 4.6 Hz),
3.01–3.13 (4H, m), 3.14–3.27 (2H, m), 3.35–3.48 (2H, m), 3.63–3.76 (1H, m), 3.81 (3H, s), 8.52
(1H, s), 11.02 (1H, brs), 11.93 (1H, brs); MS (ESI) m/z 375 [M+H]+; Anal. Calcd. for
C18H26N6OS·2HCl·0.03C3H8O·0.3H2O: C, 47.79, H, 6.39, N, 18.49, S, 7.05, Cl,15.60. Found: C,
47.78, H, 6.39, N, 18.28, S, 7.19, Cl, 15.55.
5.3.14. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-5-methyl-1-phenyl-1H-
pyrazole-4-carboxamide dihydrochloride (13b)
The title compound was obtained in a similar manner to 13a using 5-methyl-1-phenyl-1H-pyrazole-
4-carboxylic acid as a colorless solid in 56% yield. 1H NMR (400 MHz, DMSO-d6): δ = 1.78–1.91
(1H, m), 1.91–2.05 (1H, m), 2.14–2.26 (2H, m), 2.27–2.42 (2H, m), 2.57 (3H, s), 2.81 (3H, d, J =
4.7 Hz), 3.01–3.30 (6H, m), 3.36–3.51 (2H, m), 3.72 (1H, tt, J = 8.6, 8.6 Hz), 7.46–7.65 (5H, m),
8.55 (1H, s), 11.17 (1H, brs), 12.18 (1H, brs); MS (ESI) m/z 437 [M+H]+; Anal. Calcd. for
C23H28N6OS·2HCl·0.9H2O: C, 52.55, H, 6.10, N, 15.99, S, 6.10, Cl, 13.49. Found: C, 52.56, H, 6.17,
N, 16.03, S, 6.08, Cl, 13.37.
5.3.15. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-4-phenylthiophene-2-
carboxamide (13c)
The title compound was obtained in a similar manner to 12e using 4-phenylthiophene-2-carboxylic
acid in 46% yield. MS (ESI) m/z 439 [M+H]+.

5.3.16. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-5-phenylfuran-2-carboxamide
(13d)
The title compound was obtained in a similar manner to 12e using 5-phenylfuran-2- carboxylic acid
in 35% yield. MS (ESI) m/z 423 [M+H]+.
5.3.17. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-1-methyl-5-phenyl-1H-
pyrazole-3-carboxamide (13e)
The title compound was prepared in a similar manner to 12e using 1-methyl-5-phenyl-1H-pyrazole-
3-carboxylic acid as a colorless solid in 14% yield. 1H NMR (400 MHz, DMSO-d6): δ = 1.76–1.88
(1H, m), 1.89–2.03 (1H, m), 2.11–2.25 (2H, m), 2.22 (3H, s), 2.25–2.38 (2H, m), 2.40–2.54 (4H, m,
overlapped with DMSO), 2.74–2.87 (4H, m), 3.65 (1H, tt, J = 8.6, 8.6 Hz), 3.96 (3H, s), 7.15 (1H,
s), 7.45–7.64 (5H, m), 11.64 (1H, s); MS (ESI) m/z 437 [M+H]+; Anal. Calcd. for C23H28N6OS: C,
63.28, H, 6.46, N, 19.25, S, 7.34. Found: C, 63.07, H, 6.40, N, 19.23, S, 7.24.
5.3.18. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-4-methyl-2-phenyl-1,3-
oxazole-5-carboxamide dihydrochloride (13f)
The title compound was prepared in a similar manner to 13a using 4-methyl-2-phenyl-1,3-oxazole-
1
5-carboxylic acid as a pale yellow solid in 52% yield. H NMR (400 MHz, DMSO-d6): δ = 1.81–
1.92 (1H, m), 1.92–2.06 (1H, m), 2.16–2.28 (2H, m), 2.29–2.43 (2H, m), 2.86 (3H, s), 2.95–3.07
(2H, m), 3.11–3.20 (2H, m), 3.20–3.31 (2H, m), 3.44–3.51 (2H, m), 3.54 (3H, s, overlapped with
residual H2O), 3.73 (1H, tt, J = 8.6, 8.6 Hz), 7.54–7.69 (3H, m), 8.29–8.38 (2H, m); MS (ESI) m/z
438 [M+H]+; Anal. Calcd. for C23H27N5O2S·2HCl·0.3H2O: C, 53.55, H, 5.78, N, 13.58, S, 6.22, Cl,
13.74. Found: C, 53.65, H, 5.78, N, 13.51, S, 6.28, Cl, 13.87.
5.3.19. 1-tert-Butyl-N-[4-cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-5-methyl-1H-
pyrazole-4-carboxamide hydrogen chloride (13g)
The title compound was prepared in a similar manner to 12e using 1-tert-butyl-5-methyl-1H-
1
pyrazole-4-carboxylic acid as a colorless solid in 33% yield. H NMR (400 MHz, DMSO-d6): δ =
1.61 (9H, s), 1.77–1.88 (1H, m), 1.89–2.03 (1H, m), 2.13–2.23 (2H, m), 2.26–2.39 (2H, m), 2.74
(3H, s), 2.82 (3H, d, J = 4.3Hz), 2.99–3.13 (4H, m), 3.15–3.27 (2H, m), 3.4–3.49 (2H, m), 3.70 (1H,
tt, J = 8.7, 8.7Hz), 8.23 (1H, s), 10.80 (1H, brs), 11.95 (1H, s); MS (ESI) m/z 417 [M+H]+; Anal.
Calcd. for C21H32N6OS·HCl·1.7H2O: C, 52.15, H, 7.59, N, 17.38, S, 6.63, Cl, 7.33. Found: C, 51.92,
H, 7.68, N, 17.32, S, 6.63, Cl, 7.61.
5.3.20. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-1-cyclohexyl-5-methyl-1H-
pyrazole-4-carboxamide dihydrochloride (13h)
The title compound was obtained in a similar manner to 13a using 1-cyclohexyl-5-methyl-1H-
pyrazole-4-carboxylic acid as an off white amorphous semisolid in 20% yield. 1H NMR (400 MHz,
DMSO-d6): δ = 1.11–1.29 (1H, m), 1.33–1.51 (2H, m), 1.60–1.88 (8H, m), 1.89–2.02 (1H, m), 2.10–

2.24 (2H, m), 2.26–2.41 (2H, m), 2.57 (3H, s), 2.81 (3H, d, J = 4.7 Hz), 2.99–3.30 (6H, m), 3.36–
3.54 (2H, m), 3.70 (1H, tt, J = 8.5, 8.5 Hz), 4.11–4.29 (1H, m), 8.32 (1H, s), 10.99 (1H, brs), 11.95
(1H, brs); MS (ESI) m/z 443 [M+H]+; Anal. Calcd. for C23H34N6OS·1.9HCl·1.9H2O: C, 50.58, H,
7.33, N, 15.39, S, 5.87, Cl, 12.33. Found: C, 50.60, H, 7.40, N, 15.27, S, 5.91, Cl, 12.19.
5.3.21. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-5-methyl-1-(oxan-4-yl)-1H-
pyrazole-4-carboxamide dihydrochloride (13i)
The title compound was obtained in a similar manner to 13a using 5-methyl-1-(oxan-4-yl)-1H-
pyrazole-4-carboxylic acid as a pale yellow amorphous semisolid in 40% yield. 1H NMR (400 MHz,
DMSO-d6): δ = 1.74–1.88 (3H, m), 1.89–2.09 (3H, m), 2.13–2.24 (2H, m), 2.27–2.39 (2H, m), 2.60
(3H, s), 2.81 (3H, d, J = 4.8Hz), 2.95–3.14 (4H, m), 3.15–3.28 (2H, m), 3.40–3.55 (4H, m), 3.64-
3.77 (1H, m), 3.96 (2H, dd, J = 4.0, 11.0Hz), 8.35 (1H, s), 10.84 (1H, brs), 11.99 (1H, brs); MS (ESI)
m/z 445 [M+H]+; Anal. Calcd. for C22H32N6O2S·1.7HCl·2H2O: C, 48.70, H, 7.00, N, 15.49, S, 5.91,
Cl, 11.11. Found: C, 48.86, H, 7.24, N, 15.39, S, 5.76, Cl, 10.96.
5.3.22. tert-Butyl 1-(4,4-difluorocyclohexyl)-5-methyl-1H-pyrazole-4-carboxylate (15)
A mixture of (4,4-difluorocyclohexyl)hydrazine hydrochloride (1047 mg, 5.6 mmol), tert-butyl 2-
[(dimethylamino)methylidene]-3-oxobutanoate (14) (1300 mg, 6.1 mmol, 1.1 equiv.), and K2CO3
(620 mg, 6.3 mmol, 1.1 equiv.) in EtOH (14 mL) was stirred at 70 °C for 1 d. To the mixture was
added H2O, and the mixture was extracted with CHCl3. The organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo. The residue was purified on a silica gel column chromatography
1
(hexane:EtOAc) to give the title compound (718 mg, 2.4 mmol, 43%) as a yellow solid. H NMR
(400 MHz, CDCl3): δ = 1.55 (9H, s), 1.80–2.00 (4H, m), 2.25–2.40 (4H, m), 2.54 (3H, s), 4.07–4.21
(1H, m), 7.79 (1H, s); MS (ESI) m/z 301 [M+H]+.
5.3.23. 1-(4,4-Difluorocyclohexyl)-5-methyl-1H-pyrazole-4-carboxylic acid (16)
TFA (4.5 mL, 59 mmol, 25 equiv.) was added to a solution of 15 (718 mg, 2.4 mmol) in CH2Cl2
(4.5 mL) at rt, and the mixture was stirred at rt for 1 d. The reaction mixture was concentrated in
vacuo. Toluene was added to the residue, and the mixture was concentrated in vacuo. EtOAc was
added to the residue, and then the mixture was concentrated in vacuo to give the title compound (570
mg, 2.3 mmol, 98%) as an off white solid. 1H NMR (400 MHz, DMSO-d6): δ = 1.86–2.20 (8H, m),
2.53 (3H, s), 4.38–4.54 (1H, m), 7.75 (1H, s), 12.17 (1H, brs); MS (ESI) m/z 245 [M+H]+.
5.3.24. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-1-(4,4-difluorocyclohexyl)-5-
methyl-1H-pyrazole-4-carboxamide dihydrochloride (13j)
The title compound was obtained in a similar manner to 13a using 16 as a colorless solid in 11%
1
yield. H NMR (400 MHz, DMSO-d6): δ = 1.76–2.25 (12H, m), 2.26–2.40 (2H, m), 2.59 (3H, s),
2.81 (3H, d, J = 4.7 Hz), 3.00–3.13 (4H, m), 3.14–3.30 (2H, m), 3.38–3.50 (2H, m), 3.70 (1H, tt, J =
8.6, 8.6 Hz), 4.44–4.59 (1H, m), 8.35 (1H, brs), 10.95 (1H, brs), 11.99 (1H, brs); MS (ESI) m/z 479
[M+H]+; Anal. Calcd. for C23H32F2N6OS·2HCl·0.5H2O: C, 49.28, H, 6.29, N, 14.99, S, 5.72, Cl,

12.65, F, 6.78. Found: C, 49.25, H, 6.29, N, 14.98, S, 5.65, Cl, 12.18, F, 6.76.
5.3.25. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-1-(4-fluorophenyl)-5-methyl-
1H-pyrazole-4-carboxamide (13k)
The title compound was obtained in a similar manner to 12e using 1-(4-fluorophenyl)-5-methyl-
1H-pyrazole-4-carboxylic acid as a pale brown powder in 21% yield. 1H NMR (400 MHz, DMSO-
d6): δ = 1.77–1.87 (1H, m), 1.90–2.04 (1H, m), 2.12–2.25 (5H, m), 2.27–2.39 (2H, m), 2.40–2.48
(4H, m), 2.53–2.59 (3H, m), 2.76–2.82 (4H, m), 3.61–3.73 (1H, m), 7.36–7.45 (2H, m), 7.57–7.66
(2H, m), 8.53 (1H, s), 12.06 (1H, s); MS (ESI) m/z 455 [M+H]+; Anal. Calcd. for C23H27FN6OS: C,
60.77, H, 5.99, N, 18.49, S, 7.05. Found: C, 60.43, H, 5.98, N, 18.13, S, 7.22.
5.3.26. 1-(4-Cyanophenyl)-N-[4-cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-5-methyl-
1H-pyrazole-4-carboxamide (13l)
The title compound was obtained in a similar manner to 12e using 1-(4-cyanophenyl)-5-methyl-1H-
pyrazole-4-carboxylic acid as a beige solid in 48% yield. 1H NMR (400 MHz, DMSO-d6): δ = 1.75–
1.88 (1H, m), 1.90–2.05 (1H, m), 2.12–2.40 (7H, m), 2.44–2.57 (4H, m, overlapped with DMSO),
2.64 (3H, s), 2.74–2.88 (4H, m), 3.67 (1H, tt, J = 8.6, 8.6 Hz), 7.78–7.86 (2H, m), 8.03–8.10 (2H,
m), 8.60 (1H, s), 12.13 (1H, s); MS (ESI) m/z 462 [M+H]+; Anal. Calcd. for
C24H27N7OS·0.07C4H8O2·0.5H2O: C, 61.17, H, 6.04, N, 20.57, S, 6.73. Found: C, 61.53, H, 5.88, N,
20.64, S, 6.79.
5.3.27. N-[4-Cyclobutyl-5-(4-methylpiperazin-1-yl)-1,3-thiazol-2-yl]-1-[(4-fluorophenyl)methyl]-
5-methyl-1H-pyrazole-4-carboxamide dihydrochloride (13m)
The title compound was prepared in a similar manner to 13a using 1-[(4-fluorophenyl)methyl]-5-
methyl-1H-pyrazole-4-carboxylic acid as a colorless solid in 39% yield. 1H NMR (400 MHz, DMSO-
d6): δ = 1.74–2.06 (2H, m), 2.12–2.25 (2H, m), 2.25–2.40 (2H, m), 2.53 (3H, s), 2.81 (3H, d, J = 4.7
Hz), 2.97–3.31 (6H, m), 3.34–3.51 (2H, m), 3.70 (1H, tt, J = 8.6, 8.6 Hz), 5.36 (2H, s), 7.13–7.31
(4H, m), 8.37 (1H, s), 10.95 (1H, brs), 12.03 (1H, brs); MS (ESI) m/z 469 [M+H]+; Anal. Calcd. for
C24H29FN6OS·2HCl·0.8H2O: C, 51.85, H, 5.91, N, 15.12, S, 5.77, Cl, 12.75, F, 3.42. Found: C, 52.15,
H, 5.95, N, 15.23, S, 5.89, Cl, 12.41, F, 3.48.
5.3.28. Benzyl 4-(2-oxo-2-phenylethyl)piperidine-1-carboxylate (18)
To the stirred solution of benzyl 4-{2-[methoxy(methyl)amino]-2-oxoethyl}piperidine-1-
carboxylate (310 mg, 0.97 mmol) in THF (6.2 mL) was added PhMgBr (2.0 M in THF, 968 L, 1.9
mmol, 2.0 equiv.). The reaction mixture was stirred at rt for 3 hr. The reaction was quenched with
sat. aq. NH4Cl, and the mixture was extracted with EtOAc. The organic layer was washed with
brine and dried over anhydrous MgSO4 and concentrated in vacuo. The residue was purified on a
silica gel column chromatography to give the title compound (175 mg, 0.52 mmol, 54%) as a
colorless oil. 1H NMR (400 MHz, CDCl3): δ = 1.13–1.35 (2H, m), 1.78 (2H, d, J = 12.9 Hz), 2.14–
2.26 (1H, m), 2.77–2.92 (2H, m), 2.89 (2H, d, J = 6.4 Hz), 4.18 (2H, brs), 5.12 (2H, s), 7.20–7.40

(5H, m, overlapped with chloroform), 7.43–7.50 (2H, m), 7.53–7.60 (1H, m), 7.91–7.97 (2H, m);
MS (APCI/ESI) m/z 338 [M+H]+.
5.3.29. Benzyl 4-(2-amino-4-phenyl-1,3-thiazol-5-yl)piperidine-1-carboxylate (19)
To a stirred solution of 18 (1.2 g, 3.6 mmol) in AcOH (20 mL) was added bromine (277 L, 5.4
mmol, 1.5 equiv.) in AcOH (4.2 mL) dropwise at rt. The reaction mixture was stirred for 3 hr at the
same temperature. The solvent was evaporated off, and residue was dissolved into EtOAc and
washed with sat. aq. NaHCO3. The organic layer was dried over anhydrous MgSO4 and
concentrated in vacuo. To a solution of the residue in EtOH (20 mL) was added thiourea (300 mg,
3.9 mmol, 1.1 equiv). The whole mixture was refluxed for 1 hr. After cooling to rt, the reaction
mixture was concentrated in vacuo, and water was added. The mixture was extracted with EtOAc,
and the organic layer was washed with brine, dried over anhydrous MgSO4 and concentrated in
vacuo. The residue was purified on a silica gel column chromatography to give the title compound
(224 mg, 0.57 mmol, 16%) as a colorless powder. 1H NMR (400 MHz, CDCl3): δ = 1.93 (2H, d, J
= 12.2 Hz), 2.79 (2H, t, J = 12.2 Hz), 3.11 (1H, tt, J = 12.2, 3.7 Hz), 4.26 (2H, brs), 4.78 (2H, s),
5.14 (2H, s), 7.28–7.50 (10H, m); MS (APCI/ESI) m/z 394 [M+H]+.
5.3.30. Benzyl 4-[2-(3,5-dimethoxybenzamido)-4-phenyl-1,3-thiazol-5-yl]piperidine-1-carboxylate
(20)
The title compound was obtained in similar manner to 7 using 19 as a pale yellow powder in 77%
yield. 1H NMR (400 MHz, CDCl3): δ = 1.66–1.83 (2H, m), 1.98 (2H, d, J = 12.8 Hz), 2.82 (2H, t, J
= 12.8 Hz), 3.21 (1H, tt, J = 11.3, 3.5 Hz), 3.81 (6H, s), 4.30 (2H, brs), 5.16 (2H, s), 6.62 (1H, t, J
= 2.2 Hz), 6.97 (2H, d, J = 2.2 Hz), 7.29–7.48 (10H, m), 9.85 (1H, brs); MS (APCI/ESI) m/z 558
[M+H]+.
5.3.31. 3,5-Dimethoxy-N-[5-(1-methylpiperidin-4-yl)-4-phenyl-1,3-thiazol-2-yl]benzamide (21a)
To a mixture of 20 (185 mg, 0.33 mmol), MeOH (2.0 mL) and DMF (2.0 mL) were added 10%
Pd/C (50% wet, 400 mg) and AcOH (300 L, 5.2 mmol, 16 equiv.). The whole was stirred under 3
atm of H2 atmosphere at rt for 6 hr. The catalyst was removed by filtration and washed with
MeOH. The filtrate was concentrated in vacuo. To the residue was added sat. aq. NaHCO3, and the
precipitate was collected by filtration, washed with H2O and dried in vacuo.
To a mixture of the obtained product (101 mg, 0.24 mmol) and DMF (2.0 mL) was added 37% aq.
formaldehyde (100 mg, 1.2 mmol, 5.2 equiv.), AcOH (68 L, 1.2 mmol, 5.0 equiv.) and
NaBH(OAc)3 (252 mg, 1.2 mmol, 5.0 equiv.) on an ice water bath, and the whole was stirred at
room temperature for 5 hr. To the reaction mixture was added sat. aq. NaHCO3, and the mixture
was extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4 and
concentrate in vacuo. The residue was purified on a silica gel column chromatography
(CHCl3:MeOH), and recrystallized from a mixture of EtOAc and hexane to give the title compound
(57 mg, 0.13 mmol, 55%) as a colorless powder. 1H NMR (400 MHz, DMSO-d6): δ = 1.62–1.75
(2H, m), 1.85–1.95 (4H, m), 2.18 (3H, s), 2.80–3.01 (3H, m), 3.82 (6H, s), 6.73 (1H, t, J = 2.3 Hz),

7.31 (2H, d, J = 2.3 Hz), 7.35–7.42 (1H, m), 7.44–7.52 (2H, m), 7.54–7.60 (2H, m), 12.61 (1H,
brs); MS (ESI) m/z 438 [M+H]+.
5.3.32. 5-(3-Chloropropyl)-4-phenyl-1,3-thiazol-2-amine hydrogen bromide (24b)
To a mixture of 5-chloro-1-phenylpentan-1-one (22b) (700 mg, 3.6 mmol), hydrobromic acid (1
drop), and AcOH (3.5 mL) was added Br2 (185 L, 3.6 mmol, 1.0 equiv.) over 5 minutes, and the
mixture was stirred at rt for 1.5 hr. After the mixture was concentrated in vacuo, EtOH (3.0 mL) and
thiourea (271 mg, 3.6 mmol, 1.0 equiv.) were added, and the mixture was stirred at reflux for 5 hr.
After cooling to rt, the mixture was concentrated in vacuo. The residue was washed with EtOAc to
give the title compound (1.1 g, 3.3 mmol, 93%) as a pale brown solid. MS (ESI) m/z 253 [M+H]+.
5.3.33. N-[5-(3-Chloropropyl)-4-phenyl-1,3-thiazol-2-yl]-3,5-dimethoxybenzamide (26b)
The title compound was obtained in a similar manner to 7 using 24b as a pale yellow solid in 60%
yield. 1H NMR (400 MHz, CDCl3): δ = 2.14–2.21 (2H, m), 3.07–3.13 (2H, m), 3.59 (2H, t, J = 6.4
Hz), 3.84 (6H, s), 6.65 (1H, t, J = 2.3 Hz), 7.01 (2H, d, J = 2.3 Hz), 7.32–7.37 (1H, m), 7.40–7.45
(2H, m), 7.52–7.56 (2H, m), 9.62 (1H, brs); MS (ESI) m/z 417 [M+H]+.
5.3.34. N-{5-[3-(Dimethylamino)propyl]-4-phenyl-1,3-thiazol-2-yl}-3,5-dimethoxybenzamide
dihydrochloride (21b)
A mixture of 26b (140 mg, 0.34 mmol), dimethylamine (106 L, 1.0 mmol, 3.0 equiv.), K3PO4 (71
mg, 0.34 mmol, 1.0 equiv.), KI (56 mg, 0.34 mmol, 1.0 equiv.) in DMF (2.0 mL) was stirred at 60 °C
for 1 d. The mixture was concentrated in vacuo. The residue was purified on a silica gel column
chromatography (NH2 silica gel, hexane:EtOAc). To a solution of the obtained free product in EtOH
was added conc. HCl (60 L), and the mixture was allowed to stand at rt for 14 hr. The precipitate
was collected by filtration, washed with EtOH, dried in vacuo to give the title compound (96 mg,
0.19 mmol, 57%) as a colorless solid. 1H NMR (400 MHz, DMSO-d6): δ = 2.01–2.11 (2H, m), 2.72
(6H, d, J = 4.9 Hz), 2.96 (2H, t, J = 7.6 Hz), 3.04–3.13 (2H, m), 3.83 (6H, s), 6.75 (1H, t, J = 2.4
Hz), 7.31 (2H, d, J = 2.4 Hz), 7.37–7.42 (1H, m), 7.45–7.52 (2H, m), 7.61–7.66 (2H, m), 10.45 (1H,
brs), 12.68 (1H, brs); MS (ESI) m/z 426 [M+H]+; Anal. Calcd. for
C23H27N3O3S·2HCl·0.3C2H6O·H2O: C, 53.45, H, 6.23, N, 7.92, S, 6.05, Cl, 13.37. Found: C, 53.67,
H, 6.20, N, 7.88, S, 6.04, Cl, 13.20.
5.3.35. 3,5-Dimethoxy-N-{4-phenyl-5-[3-(piperidin-1-yl)propyl]-1,3-thiazol-2-yl}benzamide
dihydrochloride (21c)
The title compound was obtained in a similar manner to 21b using piperidine as a colorless solid
in 72% yield. 1H NMR (400 MHz, DMSO-d6): δ = 1.29–1.44 (1H, m), 1.64–1.84 (5H, m), 2.05–
2.19 (2H, m), 2.75–2.89 (2H, m), 2.95 (2H, t, J = 7.6 Hz), 3.00–3.10 (2H, m), 3.39 (2H, d, J = 12
Hz), 3.83 (6H, s), 6.75 (1H, t, J = 2.4 Hz), 7.31 (2H, d, J = 2.4 Hz), 7.36–7.42 (1H, m), 7.44–7.52
(2H, m), 7.61–7.67 (2H, m), 10.33 (1H, brs), 12.68 (1H, brs); MS (ESI) m/z 466 [M+H]+.

5.3.36. 5-[3-(Morpholin-4-yl)propyl]-4-phenyl-1,3-thiazol-2-amine (26d)
A mixture of 5-(3-chloropropyl)-4-phenyl-1,3-thiazol-2-amine (242 mg, 0.96 mmol), morpholine
(0.25 mL, 2.9 mmol, 3.0 equiv.) and DMF (3.5 mL) was stirred at 60 °C for 1 d. To the mixture
was added morpholine (0.10 mL, 1.1 mmol, 1.2 equiv.), and the mixture was stirred on the same
bath for 15 hr. After allowing to rt, to the mixture were added H2O and sat. aq. NaHCO3, and the
mixture was extracted with EtOAc. The combined organic layers were washed with H2O, sat. brine,
dried over anhydrous Na2SO4, then concentrated in vacuo. The residue was washed with Et2O to
give the title compound (203 mg, 0.67 mmol, 70%) as a pale yellow solid. 1H NMR (400 MHz,
DMSO-d6): δ = 1.68 (2H, tt, J = 7.4, 7.4 Hz), 2.18–2.34 (6H, m), 2.75 (2H, t, J = 7.4 Hz), 3.47–
3.57 (4H, m), 6.75 (2H, s), 7.25–7.30 (1H, m), 7.34–7.42 (2H, m), 7.48–7.56 (2H, m); MS (ESI)
m/z 304 [M+H]+.
5.3.37. 3,5-Dimethoxy-N-{5-[3-(morpholin-4-yl)propyl]-4-phenyl-1,3-thiazol-2-yl}benzamide
(21d)
The title compound was obtained in a similar manner to 7 using 26d as a colorless solid in 26%
1
yield. H NMR (400 MHz, DMSO-d6): δ = 1.79 (2H, tt, J = 7.2, 7.2 Hz), 2.22–2.35 (6H, m), 2.94
(2H, t, J = 7.5 Hz), 3.48–3.58 (4H, m), 3.83 (6H, s), 6.73 (1H, t, J = 2.3 Hz), 7.31 (2H, d, J = 2.2
Hz), 7.34–7.40 (1H, m), 7.41–7.51 (2H, m), 7.61–7.69 (2H, m), 12.61 (1H, s); MS (ESI) m/z 468
[M+H]+; Anal. Calcd. for C25H29N3O4S·0.5H2O: C, 63.00, H, 6.34, N, 8.82, S, 6.73. Found: C, 62.84,
H, 6.12, N, 8.68, S, 6.68.
5.3.38. 3,5-Dimethoxy-N-{5-[3-(4-methylpiperazin-1-yl)propyl]-4-phenyl-1,3-thiazol-2-
yl}benzamide trihydrochloride (21e)
The title compound was obtained in a similar manner to 21b using N-methylpiperazine as a colorless
solid in 53% yield. 1H NMR (400 MHz, DMSO-d6): δ = 2.09–2.24 (2H, m), 2.82 (3H, s), 2.93–3.04
(2H, m), 3.14–3.93 (10H, m), 3.83 (6H, s), 6.74 (1H, t, J = 2.3 Hz), 7.32 (2H, d, J = 2.2 Hz), 7.36–
7.42 (1H, m), 7.44–7.52 (2H, m), 7.59–7.68 (2H, m), 11.74–12.97 (2H, m); MS (ESI) m/z 481
[M+H]+; Anal. Calcd. for C26H32N4O3S·2.9HCl·2H2O: C, 50.17, H, 6.30, N, 9.00, S, 5.15, Cl, 16.52.
Found: C, 50.13, H, 6.44, N, 8.84, S, 5.13, Cl, 16.42.
5.3.39. 3,5-Dimethoxy-N-{4-phenyl-5-[2-(piperidin-1-yl)ethyl]-1,3-thiazol-2-yl}benzamide
dihydrochloride (21f)
The title compound was obtained in a similar manner to 7 and then 21b, whereas excessive
piperidine was used without any other co-solvent, using 5-(2-chloroethyl)-4-phenyl-1,3-thiazol-2-
amine hydrobromide (24f) in 37% total yield over 2 steps. 1H NMR (400 MHz, DMSO-d6): δ = 1.30–
1.44 (1H, m), 1.65–1.85 (5H, m), 2.85–2.97 (2H, m), 3.29–3.42 (4H, m), 3.45–3.53 (2H, m), 3.83
(6H, s), 6.75 (1H, t, J = 2.3 Hz), 7.31 (2H, d, J = 2.3 Hz), 7.39–7.44 (1H, m), 7.47–7.53 (2H, m),
7.65–7.70 (2H, m), 10.15 (1H, brs), 12.75 (1H, brs); MS (ESI) m/z 452 [M+H]+; Anal. Calcd. for
C25H29N3O3S·1.95HCl·0.1C2H6O·2.3H2O: C, 53.22, H, 6.41, N, 7.39, S, 5.64, Cl, 12.16. Found: C,
53.14, H, 6.33, N, 7.37, S, 5.65, Cl, 12.08.

5.3.40. 3,5-Dimethoxy-N-{4-phenyl-5-[(piperidin-1-yl)methyl]-1,3-thiazol-2-yl}benzamide
hydrogen chloride (21g)
A mixture of 3,5-dimethoxy-N-(4-phenyl-1,3-thiazol-2-yl)benzamide (29) (120 mg, 0.35 mmol),
piperidine (73 L, 0.74 mmol, 2.1 equiv.), 36% aq. formaldehyde (42 L, 0.56 mmol, 1.6 equiv.)
and AcOH (2.0 mL) was stirred at 80 °C for 18 hr. The reaction mixture was concentrated in vacuo,
and sat. aq. NaHCO3 was added. The mixture was extracted with CHCl3. The organic layer was dried
over anhydrous MgSO4, then concentrated in vacuo. The residue was purified on a silica gel column
chromatography (CHCl3:MeOH). The obtained crude free product was washed with EtOH. After 4
M HCl in EtOAc (0.15 mL) was added to the suspension of the resultant solid, the mixture was
concentrate in vacuo. The residue was recrystallized from EtOH to give the title compound (116 mg,
0.24 mmol, 69%) as a colorless solid. 1H NMR (400 MHz, DMSO-d6): δ = 1.21-1.42 (1H, m), 1.53–
1.78 (5H, m), 2.69–2.84 (2H, m), 3.19–3.31 (2H, m), 3.83 (6H, s), 4.56 (2H, d, J = 4.7 Hz), 6.77 (1H,
t, J = 2.2 Hz), 7.32 (2H, d, J = 2.3 Hz), 7.43–7.57 (3H, m), 7.60–7.67 (2H, m), 10.01 (1H, brs), 12.97
(1H, s); MS (ESI) m/z 438 [M+H]+; Anal. Calcd. for C24H27N3O3S·HCl·0.3H2O: C, 60.13, H, 6.01,
N, 8.76, S, 6.69, Cl, 7.39. Found: C, 60.19, H, 5.91, N, 8.83, S, 6.64, Cl, 7.44.
5.3.41. 4-Cyclobutyl-5-[2-(piperidin-1-yl)ethyl]-1,3-thiazol-2-amine (27)
In a 1000ml three neck flask which was fitted with a thermometer, a dropping funnel and a reflux
condenser, to a mixture of 4-chloro-1-cyclobutylbutan-1-one (23) (36 g, 222 mmol), 20% HBr in
EtOH (9.0 mL) and MeOH (131 mL) was added bromine (11.4 mL, 222 mmol, 1.0 equiv.) on an
ice-salt water bath over 5 minutes. It was stirred for 1 hr at 0 °C, then it was allowed to rt and
stirred for 1.5 hr. H2O was added and stirred for 1hr. It was diluted with H2O, and then extracted
with Et2O. The combined organic layers were washed with 10% aq. K2CO3, sat. brine, dried over
anhydrous MgSO4 and concentrated in vacuo. To the residue, thiourea (18 g, 233 mmol, 1.1 equiv.)
and EtOH (130 mL) were added, and the mixture was refluxed for 12 hr under Ar. The reaction
mixture was cooled to rt, passed through a Kiriyama filter, and then was concentrated in vacuo. The
residue was diluted with EtOAc, and the organic layer was washed with sat. aq. NaHCO3. The two
phases were separated, and the aq. phase was extracted with EtOAc. The combined organic layers
were washed with brine, dried over anhydrous Na2SO4, and then concentrated in vacuo. The
residue was purified on a silica gel column chromatography (hexane:EtOAc) to give the
intermediate (25) (14 g, 65 mmol).
A mixture of the obtained intermediate (25) (5 g, 23 mmol), piperidine (9.8 g, 115 mmol, 5.0
equiv.) and DMF (60 mL) was stirred at 60 °C for 1 d. H2O was added to the mixture, and the
whole was stirred at rt for 14 hr. The precipitation was collect by filtration, and was washed with
IPE to give the title compound (3.3 g, 12 mmol, 16%) as a pale yellow solid. 1H NMR (400 MHz,
DMSO-d6): δ = 1.28–1.53 (6H, m), 1.68–1.95 (2H, m), 2.00–2.12 (2H, m), 2.15–2.39 (8H, m), 2.60
(2H, t, J = 7.2 Hz), 3.38 (1H, tt, J = 8.6, 8.6 Hz), 6.57 (2H, s); MS (ESI) m/z 266 [M+H]+.
5.3.42. N-{4-Cyclobutyl-5-[2-(piperidin-1-yl)ethyl]-1,3-thiazol-2-yl}-1-(4-fluorophenyl)-5-methyl-

1H-pyrazole-4-carboxamide dihydrochloride (28a)
To a mixture of 27 (330 mg, 1.2 mmol), 1-(4-fluorophenyl)-5-methyl-1H-pyrazole-4-carboxylic
acid (329 mg, 1.5 mmol, 1.2 equiv.) and NMP (6.6 mL) was added DIPEA (482 mg, 3.7 mmol, 3.0
equiv.) and HATU (567 mg, 1.5 mmol, 1.2 equiv.), and the whole was stirred at 70 °C for 1 d. The
reaction was quenched with sat. aq. NaHCO3, and the mixture was extracted with EtOAc. The
organic layer was concentrated in vacuo. The resulting crude product were purified on a silica gel
column chromatography. The obtained residue was dissolved in 4 M HCl dioxane, and then was
concentrated in vacuo. The residue was recrystallized from IPA to give the title compound (297
mg, 0.55 mmol, 44%) as a colorless solid. 1H NMR (400 MHz, DMSO-d6): δ = 1.29–1.45 (1H, m),
1.67–1.92 (6H, m), 1.92–2.08 (1H, m), 2.16–2.30 (2H, m), 2.30–2.43 (2H, m), 2.55 (3H, s), 2.79–
2.99 (2H, m), 3.04–3.29 (4H, m), 3.43–3.57 (2H, m), 3.71 (1H, tt, J = 8.6, 8.6 Hz), 7.37–7.47 (2H,
m), 7.57–7.66 (2H, m), 8.56 (1H, s), 10.56 (1H, brs), 12.29 (1H, brs); 13C NMR (126 MHz,
DMSO-d6): δ = 11.59, 17.81, 19.59, 21.38, 22.23, 25.39, 28.45, 32.94, 51.68, 56.19, 61.90, 113.31,
116.11 (d, J = 23.2 Hz), 117.72, 127.62 (d, J = 9.1 Hz), 134.73 (d, J = 2.7 Hz), 139.65, 143.81,
149.93, 155.56, 160.56, 160.71, 162.51; MS (ESI) m/z 468 [M+H]+; Anal. Calcd. for
C25H30FN5OS·2HCl·0.5H2O: C, 54.64, H, 6.05, N, 12.74, S, 5.83, Cl, 12.90, F, 3.46. Found: C,
54.28, H, 6.12, N, 12.46, S, 5.75, Cl, 12.82, F, 3.38.
5.3.43. N-{4-Cyclobutyl-5-[2-(piperidin-1-yl)ethyl]-1,3-thiazol-2-yl}-1-(5-fluoropyridin-2-yl)-5-
methyl-1H-pyrazole-4-carboxamide dihydrochloride (28b)
The title compound was obtained in a similar manner to 28a using 1-(5-fluoropyridin-2-yl)-5-
methyl-1H-pyrazole-4-carboxylic acid as a colorless solid in 60% yield. 1H NMR (400 MHz, DMSO-
d6): δ = 1.31–1.46 (1H, m), 1.67–1.90 (6H, m), 1.92–2.07 (1H, m), 2.19–2.29 (2H, m), 2.30–2.42
(2H, m), 2.81 (3H, s), 2.85–2.95 (2H, m), 3.07–3.15 (2H, m), 3.16–3.24 (2H, m), 3.42–3.59 (2H, m),
3.66–3.74 (1H, m), 7.89 (1H, dd, J = 4.1, 9.0 Hz), 8.03 (1H, td, J = 3.0, 8.5 Hz), 8.58 (1H, s), 8.60
(1H, d, J = 3.0 Hz), 10.68 (1H, brs), 12.31 (1H, brs); ; 13C NMR (126 MHz, DMSO-d6): δ = 12.44,
17.80, 19.60, 21.38, 22.24, 25.39, 28.45, 32.95, 51.70, 56.20, 61.90, 114.46, 117.81, 119.78 (d, J =
5.5 Hz), 126.64 (d, J = 20.5 Hz), 135.65 (d, J = 26.4 Hz), 140.29, 144.47, 148.16 (d, J = 3.0 Hz),
150.02, 155.46, 157.12, 159.14, 160.61; MS (ESI) m/z 469 [M+H]+; Anal. Calcd. for
C24H29FN6OS·2HCl·0.5H2O·0.6C3H7O: C, 52.88, H, 6.23, N, 14.34, S, 5.47, Cl, 12.10, F, 3.24.
Found: C, 52.47, H, 6.34, N, 14.06, S, 5.35, Cl, 11.89, F, 3.21.
5.3.44. N-{4-Cyclobutyl-5-[2-(piperidin-1-yl)ethyl]-1,3-thiazol-2-yl}-1-(5-fluoropyridin-2-yl)-3-
methyl-1H-pyrazole-4-carboxamide dihydrochloride (28c)
The title compound was obtained in a similar manner to 28a using 1-(5-fluoropyridin-2-yl)-3-
methyl-1H-pyrazole-4-carboxylic acid as a colorless solid in 44% yield. 1H NMR (400 MHz, DMSO-
d6): δ = 1.31–1.46 (1H, m), 1.66–1.92 (6H, m), 1.93–2.07 (1H, m), 2.19–2.42 (4H, m), 2.83–2.97
(2H, m), 3.07–3.25 (4H, m), 3.45–3.57 (2H, m), 3.70 (1H, tt, J = 8.6, 8.6 Hz), 7.95–8.00 (2H, m),
8.55–8.59 (1H, m), 9.61 (1H, s), 10.47 (1H, brs), 12.40 (1H, brs); 13C NMR (126 MHz, DMSO-d6):
δ = 13.67, 17.84, 19.62, 21.38, 22.24, 28.46, 32.97, 51.71, 56.21, 113.75 (d, J = 5.0 Hz), 114.90,

117.75, 126.81 (d, J = 20.4 Hz), 129.59, 136.16 (d, J = 26.4 Hz), 146.44 (d, J = 2.5 Hz), 150.09,
152.70, 155.38, 156.89, 158.88, 160.35; MS (ESI) m/z 469 [M+H]+; Anal. Calcd. for
C24H29FN6OS·1.9HCl·2.1H2O: C, 50.07, H, 6.15, N, 14.60, S, 5.57, Cl, 11.70, F, 3.30. Found: C,
49.84, H, 6.12, N, 14.44, S, 5.59, Cl, 11.76, F, 3.31.
6. Acknowledgements
The corresponding author would like to thank all the staff members of Astellas Research
Technologies Co., Ltd. for generating the data that supported this research. Special gratitude is due
towards Dr. Susumu Watanuki, Mr. Hiroyuki Kaizawa, Dr. Makoto Oku and Dr. Ayako Moritomo
for their invaluable advices on the manuscript preparation.
7. Declarations of interest
The authors declare no conflict of interest.

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12 In a separate study, we were not able to substitute the amino group on thiazole C2 and maintain
the in vitro potency.
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14 See the Biopharmaceutics Classification System (BCS) guideline issued by the FDA.
15 Yalkowski, S. H.; Valvani, S. C. J. Pharm. Sci. 1980, 69, 912–922.
16 pKb values were obtained by calculation on Molecular Operating Environment (MOE), 2016.08;
Chemical Computing Group Inc., 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada,
H3A 2R7, 2016.
17 The conformations with the minimum energy potentials were calculated on MOE as follows;
Potential conformations were generated with LowMode function, and the energy potentials were
evaluated with PM6 semiempirical parameterization under the Amber10:EHT forcefield,
Generalized Born implicit solvent model. All of these functions are implemented in the MOE
software bundle, or its software package MOPAC (version 2016).