Synthesis and structure-activity relationships of pyrimidine derivatives as potent and orally active FGFR3 inhibitors with both increased systemic exposure and enhanced in vitro potency

Article abstract of DOI:10.1016/j.bmc.2021.116019

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of patients with bladder cancer harboring genetic alterations in FGFR3. We identified pyrimidine derivative 20b, which induced tumor regression following ora

Full text of DOI:10.1016/j.bmc.2021.116019

Bioorg. Med. Chem. 33 (2021) 116019
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure-activity relationships of pyrimidine derivatives as
potent and orally active FGFR3 inhibitors with both increased systemic
exposure and enhanced in vitro potency
,
,1
Ikumi Kuriwakia *, Minoru Kamedaa, Kazuhiko Iikuboa, Hiroyuki Hisamichia
,
Yuichiro Kawamotoa 2, Shigetoshi Kikuchia 3, Hiroyuki Moritomoa, Yutaka Kondoha
,
,
,
,4
Tadashi Terasakaa 5, Yasushi Amanoa, Yukihiro Tateishia, Yuka Echizena, Yoshinori Iwaib,
,
Atsushi Nodab, Hiroshi Tomiyamab, Taisuke Nakazawaa, Masaaki Hiranoa
a Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Research Laboratories, Kotobuki Pharmaceutical Co., Ltd., 198 Kamigomyou Sakaki-Machi, Hanishina-Gun, Nagano 389-0697, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of patients with
bladder cancer harboring genetic alterations in FGFR3. We identified pyrimidine derivative 20b, which induced
tumor regression following oral administration to a bladder cancer xenograft mouse model. Compound 20b was
discovered by optimizing lead compound 1, which we reported previously. Specifically, reducing the molecular
size of the substituent at the 4-position and replacing the linker of the 5-position in the pyrimidine scaffold
resulted in an increase in systemic exposure. Furthermore, introduction of two fluorine atoms into the 3,5-dime-
thoxyphenyl ring enhanced FGFR3 inhibitory activity. Molecular dynamics (MD) simulation of 20b suggested
that the fluorine atom interacts with the main chain NH moiety of Asp635 via a hydrogen bond.
Fibroblast growth factor receptor 3
Bladder cancer
Antitumor effect
X-ray crystal structure
Molecular dynamics simulation
Structure–activity relationships
1. Introduction
medical needs in molecular targeted therapy.
Fibroblast growth factor receptor (FGFR) is a tyrosine kinase, and the
The recent development of molecular targeted therapeutics, which
target specific cancer cell environment, has reduced the side effects of
anticancer drugs and the burden on patients. For this reason, use of
molecular targeted drugs has increased in clinical practice.1 Tyrosine
kinases are one of the main targets of these drugs, and many inhibitors
have been discovered.1 Representative molecular targeted drugs against
tyrosine kinases include dacomitinib (EGFR/HER inhibitor), laro-
trectinib, entrectinib (NTRK1/2/3 inhibitor), lorlatinib (ALK inhibitor),
pexidartinib (CSF1R inhibitor), and gilteritinib (FLT3 inhibitor), which
have been approved since 2018.1,2 However, while the efficacy of these
agents is attractive for patients with a certain genetic background, they
are not effective in all cancer patients. Therefore, there remain unmet
fibroblast growth factor (FGF)/FGFR signaling pathway is known to play
an important role in the development of various organs, in particular, in
the process of cell proliferation and angiogenesis.3 Abnormal activation
of this pathway disrupts these processes in a subset of cancer patients.
Genetic abnormalities, such as amplification, point mutations, trans-
location, and fusion genes of FGFRs, activate this pathway.4–8 Such
FGFR ablation is observed in lung cancer, breast cancer, multiple
myeloma, uterine cancer, stomach cancer, brain tumor, bile duct cancer,
and urothelial cancer.9
Drug discovery targeting FGFRs has been actively conducted in
recent years, and a number of ongoing clinical trials are being conducted
on drug candidates with selective FGFR inhibitory activity.10,11 Some of
* Corresponding author.
E-mail address: ikumi.kuriwaki@astellas.com (I. Kuriwaki).
1
Industrial Property Cooperation Center, Fukagawa Gatharia W3 Building, 1-2-15 Kiba, Koto-ku, Tokyo 135-0042, Japan.
School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Tsukuba Research Laboratories, Tokuyama Corporation, 40 Wadai, Tsukuba, Ibaraki 300-4247, Japan.
2
3
4
5
Kishida Chemical Co., Ltd., 13-2 Ooaza-hoshinosato, Ami-machi, Inashiki-gun, Ibaraki 300-0326, Japan.
Japan Agency for Medical Research and Development, 1-5-5 Nihonbashi-muromachi, Chuo-ku, Tokyo 103-0022, Japan.
https://doi.org/10.1016/j.bmc.2021.116019
Received 27 November 2020; Received in revised form 8 January 2021; Accepted 11 January 2021
Available online 16 January 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
these include AZD454712, infigratinib (NVP-BGJ398)13, derazantinib
(ARQ087)14, pemigatinib (INCB054828)15, E709016, and Debio1347
(CH5183284)17, which target FGFR1, 2, and 3, and erdafitinib (JNJ-
42756493)18, rogaratinib (BAY1163877)19, PRN137120, and futibatinib
(TAS-120)21, which are pan-FGFR inhibitors (Fig. 1). Among these drug
candidates, erdafitinib has received approval from the U.S. Food and
Drug Administration (FDA) for the treatment of adults with locally
advanced or metastatic urothelial carcinoma harboring FGFR3 point
mutations or fusion genes.22,23 In addition, several compounds, such as
infigratinib, derazantinib, pemigatinib, Debio1347, rogaratinib, and
futibatinib have shown good results in clinical trials for intrahepatic
cholangiocarcinoma (iCCA) patients with FGFR2 gene fusions.24
Therefore, FGFRs are promising targets for treating patients with various
types of cancer.
MW: 760
HBA: 15
1
Fig. 2. Structure of compound 1.25
overcome these problems in 1.
Here, we describe the synthesis and biological evaluation of a novel
series of pyrimidine derivatives and studies on their structure–activity
relationships (SARs) using X-ray crystal structure to acquire potent and
orally active pan-FGFR inhibitors with a different chemical structure
from known FGFR inhibitors. Furthermore, we discuss their SARs based
on molecular dynamics (MD) simulations.
We previously reported that the lead compound 125 (Fig. 2) had
potent pan-FGFR inhibitory activity with excellent selectivity over
VEGFR2 (Table 1). However, 1 showed low in vitro cell growth inhibi-
tory activity against UM-UC-14 human bladder cancer cells endoge-
nously expressing FGFR3 S249C (an activating gene point mutation)
with an IC50 value of 84 nM25 and NIH/3T3 cells exogenously over-
expressing FGFR3 S249C with an IC50 value of 99 nM. In addition,
plasma concentrations of 1 were low (98.2 nM) 6 h after oral adminis-
tration of a 30 mg/kg dose to Institute of Cancer Research (ICR) mice.
Given that the relationship between compound’s plasma concentration
and in vitro cellular activity, it would be necessary to improve both the in
vitro cellular potency and systemic exposure to obtain a potent anti-
tumor effect in vivo in a bladder cancer xenograft mouse model.16,18
Therefore, we conducted a study to optimize pyrimidine derivatives to
2. Design and chemistry
2.1. Design
To begin compound design, we first confirmed the physical proper-
ties and metabolic stability of 1. Compound 1 showed low permeability
as indicated in the parallel artificial membrane permeability assay
(PAMPA), poor aqueous solubility in the Japanese Pharmacopoeia 2nd
fluid (JP2) test, and high in vitro intrinsic clearance in ICR mouse liver
AZD4547
Infigratinib (NVP-BGJ398)
Derazantinib (ARQ087)
Debio1347 (CH5183284)
E7090
Pemigatinib (INCB054828)
Rogaratinib (BAY1163877)
Futibatinib (TAS-120)
Erdafitinib (JNJ-42756493)
PRN1371
Fig. 1. Structure of known FGFR inhibitors examined in clinical studies.10,11
2

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
Table 1
In vitro potency and mouse plasma concentration of 1.
Enzyme IC50 (nM)a
Cellular
Plasma
IC50 (nM)
Conc.
(nM)c
FGFR1
FGFR2
3.1
FGFR3
4.3
FGFR4
74
VEGFR2
UM-UC-14a
NIH/3T3
(FGFR3
S249C)b
2.1
a
>1000
84
99
98.2
Data from reference 25.
b
c
NIH/3T3 cells exogenously overexpressing FGFR3 S249C.
Plasma concentration was measured using LC-MS/MS 6 h after single oral administration of 1 at 30 mg/kg to ICR mice (average of 3 mice).
microsomes (Tables 2 and 5). We judged that improving both perme-
ability and aqueous solubility by adjusting lipophilicity would be diffi-
cult. Therefore, we optimized compounds with lower molecular weight
(MW) and fewer hydrogen bond acceptors (HBAs) by applying the Lip-
inski’s rule of five as a reference for drug-likeness.26,27 An improvement
in metabolic stability was also expected.
Solvent Region
Back Pocket
Met529
Hinge Region
Ala558
Our synthetic strategy was based on the X-ray complex crystal
structure of FGFR3 with 225, which is an analog of 1 (Fig. 3). The crystal
structure indicated that three substructures were maintained: (1) the 2-
aminopyrimidine group (two hydrogen bond interactions with Ala558,
located in the hinge region of the ATP binding site); (2) the dimethox-
yphenyl group (van der Waals (vdW) interaction with Met529 in the
back pocket, which contributes to FGFR selectivity over VEGFR225); (3)
the 4-(4-methylpiperazin-1-yl)piperidin-1-yl-benzene group (available
substructure in the solvent region of some kinase inhibitors such as
gilteritinib28 (FLT3/AXL inhibitor), ASP302629 (EML4-ALK inhibitor),
and naquotinib30 (EGFR T790M inhibitor)). In contrast, the N-ethyl-2-
sulfamoyl-anilino group, which fills the sugar pocket and interacts with
Lys508 via a hydrogen bond, had not been optimized. Therefore, we
planned to replace this unit with smaller substituents to decrease MW
and HBA values (Fig. 3).
Lys508
1: X = C(O), Y= NH
2: X and Y= CH2
Sugar Pocket
Fig. 3. Illustration of representative kinase pockets and amino acid residues
with 1 and 2.
sulfamoyl-anilino unit (shown in red) of 1. Next, we considered opti-
mizing the linker between the 3,5-dimethoxybenzene and pyrimidine
(shown in blue) because the sulfamoyl group was located close to the
linker, and conversion of the sulfamoyl group would affect the position
of the 3,5-dimethoxybenzene. Because 225 had an ethylene linker and
The detailed strategy is shown in Fig. 4. First, compounds were
designed to partially or completely reduce the size of the N-ethyl-2-
Table 2
Results of optimizing a smaller structure for substituent R3.
No
Structure
Linker
X
Enzyme
FGFR3
Cellular
FGFR3
PAMPA
Solubility
Log D7.4
>3.9
MWd
760
HBAd
15
R3
(10ꢀ 6 cm/sec)b
(µM)c
Y
IC50 (nM)
IC50 (nM)a
1
C(O)
C(O)
C(O)
NH
4.3e
99
1.4
<1
7a
7b
NH
NH
467
NT
NT
0.4
36
3.7
1.8
653
683
12
14
>1000
<0.2
≥100
7c
2
H
H
C(O)
CH2
NH
>1000
NT
34
<2.9
≥100
<1
2.8
562
745
11
13
CH2
2.8e
1.8
>4.8
11
CH2
CH2
24
324
15.1
≥100
3.5
547
9
a
NIH/3T3 cells exogenously overexpressing FGFR3 S249C.
b
c
Parallel artificial membrane permeability assay (PAMPA) at pH = 6.5.
Aqueous solubility in the Japanese Pharmacopoeia 2nd fluid (JP2) for disintegration test (pH = 6.8 buffer).
MW and HBA values were calculated using ACD/Percepta (ver. 2018.2.5) software.
Data from reference 25.
d
e
3

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
Fig. 4. Synthetic plan.
exhibited similar potency to 1 against FGFR3, we introduced linkers
consisting of various combinations of pairs of atoms including an
ethylene unit. Finally, according to a reported strategy for enhancing
FGFR inhibitory activity13, we introduced various halogen atoms into
3,5-dimethoxybenzene (shown in green).
13b. Compound 13c was synthesized by introducing 3,5-dimethoxyphe-
nol to 12b via Mitsunobu reaction32,33 to yield 13c. Alkylation of the
phenolic hydroxyl group in 12c using 3,5-dimethoxybenzyl bromide
gave 13d. Compounds 13b–d were converted to 14a–c using Buchwald
reaction condition34
.
Synthesis of compounds 20a–d is shown in Scheme 4. Chlorination of
15 using SO2Cl2 gave 16a. Fluorination of 15 using Selectfluor™ gave
16b and 16c. Compound 16c was chlorinated to yield 16d. Compound
17 was reacted with 4 under microwave (µW) irradiation via an SNAr
reaction to give 18, which was alkynylated with 16a–d via Sonogashira
reaction31 to give 19a–d. The alkyne moiety of 19a–d was hydrogenated
by in-situ generation of a diimide35 to yield 20a–d.
2.2. Chemistry
Synthesis of the target compounds is outlined in Schemes 1–4. Syn-
thesis of compounds 7a–d is shown in Scheme 1. Compounds 3a–c were
introduced to commercially available 4 via a nucleophilic aromatic
substitution (SNAr) reaction under acidic conditions to give 5a–c.
Reduction of the nitro groups of 5a–c gave 6a–c, which were treated
with 3,5-dimethoxybenzoyl chloride or condensed with 3,5-dimethoxy-
benzoic acid to give 7a–c. Compound 7d was synthesized from 6c via
reductive amination reaction with 3,5-dimethoxybenzaldehyde.
Scheme 2 shows the synthesis of compound 11. Commercially
available 8 was reacted with 4 via an SNAr reaction to give 9, which was
subsequently coupled with 1-ethynyl-3,5-dimethoxybenzene via Sono-
gashira reaction31 to yield 10. The alkyne linkage of 10 was reduced by
adding hydrogen to give 11.
3. Results and discussion
Synthesized compounds were evaluated using an ADP-Glo lumines-
cent kinase assay with human recombinant FGFR3 enzyme. Some
compounds were assessed using a cell growth assay in NIH/3T3 cells
exogenously overexpressing FGFR3 S249C. In addition, selected com-
pounds were assessed by examining permeability, aqueous solubility,
metabolic stability, plasma concentration, and/or in vivo inhibitory ac-
tivity against phosphorylation of FGFR3.
Table 2 shows the SAR of the substituent R3 of 1 and 2. Compared to
1, 7a–c had over 100-fold lower FGFR3 enzyme inhibitory activity,
Compounds 14a–c were synthesized according to Scheme 3. Com-
pound 12a was reacted with 3,5-dimethoxybenzoyl chloride to give
13a, whose amide moiety was methylated using iodomethane to give
4
a or b
d, e, or f
3a: R3 = NHPh
7a: R3 = NHPh, X = C(O)
5a: R3 = NHPh, R4 = NO2
5b: R3 = NHCH2CH2SO2Me, R4 = NO2
5c: R3 = H, R4 = NO2
3b: R3 = NHCH2CH2SO2Me
7b: R3 = NHCH2CH2SO2Me, X = C(O)
3c: R3 = H
7c: R3 = H, X = C(O)
7d: R3 = H, X = CH2
c
6a: R3 = NHPh, R4 = NH2
6b: R3 = NHCH2CH2SO2Me, R4 = NH2
6c: R3 = H, R4 = NH2
Scheme 1. Reagents and conditions: (a) 4, AcOH, room temperature, 62–84% for 5a and 5b; (b) 4, MsOH, EtOH, 100 ◦C, 63% for 5c; (c) H2, Pd/C, EtOH/THF or
EtOH, room temperature, 71–100%; (d) 3,5-dimethoxybenzoyl chloride, DIPEA, THF, room temperature, 30% for 7a; (e) 3,5-dimethoxybenzoic acid, HATU, DIPEA,
DMF, room temperature, 33–51% for 7b and 7c; (f) (i) 3,5-dimethoxybenzaldehyde, NaBH(OAc)3, CH2Cl2, room temperature, (ii) NaBH4, MeOH, room temperature,
16% (2 steps) for 7d.
4

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
b
a
8
9
10: X-Y=
11: X-Y= -CH2CH2-
c
Scheme 2. Reagents and conditions: (a) 4, MsOH, IPA, 90 ◦C to 110 ◦C, 79%; (b) 1-ethynyl-3,5-dimethoxybenzene, Pd(PPh3)4, CuI, Et3N, DMF, 110 ◦C, 93%; (c) H2
(3 atm), Pd/C, MeOH/THF, room temperature, 54%.
a, b or c
e
12a: R4 = NH2
12b: R4 = CH2OH
12c: R4 = OH
13a: X = C(O), Y= NH
13b: X = C(O), Y= N(Me)
13c: X = O, Y= CH2
14a: X = C(O), Y= N(Me)
14b: X = O, Y= CH2
d
14c: X = CH2, Y= O
13d: X = CH2, Y= O
Scheme 3. Reagents and conditions: (a) 3,5-dimethoxybenzoyl chloride, DIPEA, THF, room temperature, 77% for 13a; (b) 3,5-dimethoxyphenol, ADDP, n-Bu3P,
THF, room temperature, 53% for 13c; (c) 3,5-dimethoxybenzyl bromide, K2CO3, DMF, room temperature, 60% for 13d; (d) iodomethane, NaH, DMF, room tem-
perature, 73%; (e) 4, Pd(OAc)2, rac-BINAP, Cs2CO3, 1,4-dioxane, 100 ◦C, 33–82%.
a or b
d
15
16a: R1 = R2 = Cl
16b: R1 = R2 = F
16c: R1 = F, R2 = H
16d: R1 = F, R2 = Cl
19a: R1 = R2 = Cl
19b: R1 = R2 = F
19c: R1 = F, R2 = H
19d: R1 = F, R2 = Cl
X-Y=
a
e
20a: R1 = R2 = Cl
20b: R1 = R2 = F
20c: R1 = F, R2 = H
20d: R1 = F, R2 = Cl
X-Y= -CH2CH2-
c
17
18
Scheme 4. Reagents and conditions: (a) SO2Cl2, MeCN, room temperature, 47–83% for 16a and 16d; (b) Selectfluor™, MeCN, room temperature, 20–31% for 16b
and 16c; (c) 4, MsOH, IPA, microwave (µW), 130 ◦C, 83%; (d) Pd(PPh3)4, CuI, DIPEA or Et3N, DMF, 80 ◦C or 100 ◦C, 52–66%; (e) p-toluenesulfonyl hydrazide,
NaOAc, DME/water, 110 ◦C, 24–69%.
whereas 11 showed 9-fold lower FGFR3 enzyme inhibitory activity than
that of 2 (IC50 values were 24 nM and 2.8 nM, respectively). Moreover,
the inhibitory activity of 11 against the proliferation of NIH/3T3 cells
exogenously overexpressing FGFR3 S249C was 10-fold lower than that
of 2 (IC50 values were 324 nM and 34 nM, respectively). With regard to
physical properties, although the aqueous JP2 solubility of 7a–c
improved according to Log D7.4 values, these compounds had poor
permeability. In contrast, compared to the poor physical properties of 2,
11 showed improved permeability and JP2 solubility. Considering that
permeability is generally correlated with the lipophilicity, the improved
permeability of 11 in contrast to 2 was possibly due to its lower MW
(547) and HBA (9) among the two compounds.
dimethoxybenzene and pyrimidine of 11. Compared to 11, compounds
14a, 14b, and 7d showed reduced FGFR3 enzyme inhibitory activity,
whereas 14c exhibited comparable activity. As for the physical prop-
erties, all compounds showed favorable JP2 solubility. The permeability
of 7d and 14c was similar to that of 11, whereas that for 14b improved
to over 30 × 10ꢀ 6cm/sec, possibly because of its smaller surface area.
Fig. 5 shows the X-ray crystal structure of 11 in complex with FGFR3.
Similar to the interaction of 2 with FGFR325, the 2-aminopyrimidine
unit of 11 formed hydrogen bonds with the main chain atoms of
Ala558 in the hinge region. In addition, the 3,5-dimethoxyphenyl group
formed hydrophobic and vdW interactions with the amino acid residues
(Met529, Val555 and Ala634) in the back pocket. In addition, the
ethylene linker was located in the hydrophobic region surrounded by the
Table 3 shows the SAR of the linker X and Y between the 3,5-
5

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
11: R3 = H
2: R3
=
Fig. 5. X-ray crystal structure of 11 in complex with FGFR3 (PDB code: 7DHL). 2 is superimposed and shown as a stick model in grey.25
side chains of Val486, Ala506, Val555, Leu624, and Ala634. Our find-
Table 4
ings suggested that the interaction between 11 and these hydrophobic
Results of optimizing substituents R1 and R2 in the 3,5-dimethoxyphenyl unit.
amino acids would cause 11 to exhibit potent kinase inhibitory activity.
Meanwhile, other compounds (7c, 7d, and 14a–c) that contained some
hetero atoms in the linker unit would not show such optimal hydro-
phobic interactions, indicated that they would exhibit lower potency
than 11. Based on both the potency and physical properties, ethylene
was selected as the best linker for further optimization.
No
Structure
Enzyme
FGFR3
IC50
Cellular
FGFR3
IC50
PAMPA
(10ꢀ 6 cm/
sec)b
Solubility
Log
(µM)c
D7.4
Table 4 shows the SAR of the 3,5-dimethoxyphenyl group of 11.
Although compound 20a, which contained dichloro groups, showed 6-
fold greater enzyme inhibitory activity than 11, the increased lip-
ophilicity significantly reduced the JP2 solubility. In contrast, com-
pound 20b, a difluoro derivative, showed very potent enzyme inhibitory
activity with an IC50 value of 1.2 nM and cellular activity with an IC50
value of 13 nM (20 times and 25 times more potent than 11,
R1
R2
(nM)
(nM)a
11
H
Cl
F
H
Cl
F
24
324
113
13
15.1
18.7
18.4
24.4
19.4
≥100
5
3.5
4.4
3.0
3.2
3.5
20a
20b
20c
20d
4.3
1.2
5.2
1.6
79
F
H
Cl
95
≥100
12
F
31
a
b
c
NIH/3T3 cells exogenously overexpressing FGFR3 S249C.
Parallel artificial membrane permeability assay (PAMPA) at pH = 6.5.
Table 3
Aqueous solubility in the Japanese Pharmacopoeia 2nd fluid (JP2) for
Results of changing the linker X and Y between the 3,5-dimethoxybenzene and
pyrimidine.
disintegration test (pH = 6.8 buffer).
respectively). The JP2 solubility of 20b was 79 µM and is expected to
improve systemic exposure following oral dosing. Compound 20d,
which contained both fluorine and chlorine atoms, showed similar
enzyme inhibitory activity to 20b, but poor solubility comparable to
20a. Furthermore, compound 20c, a monofluoro derivative, showed
lower enzyme inhibitory activity than that of 20b, indicating that both
fluoro groups of 20b were important for potent kinase inhibitory ac-
tivity. In addition, all compounds in this table showed good permeability
similar to 11 due to their suitable Log D7.4 values; therefore, cellular
activity was mostly correlated with enzyme inhibitory activity among
the compounds in Table 4.
No
Linker
X
Enzyme
FGFR3
IC50
PAMPA
(10ꢀ 6
Solubility
Log
Surface
areac
(µM)b
D7.4
Y
cm/sec)a
(nM)
11
CH2
C
CH2
N
24
15.1
3.7
≥100
≥100
3.5
1.8
610.2
624.7
14a
>1000
(O)
O
(Me)
CH2
NH
O
14b
7d
380
78
>30.0
18.1
≥100
≥100
≥100
3.4
2.9
3.2
599.6
603.3
602.0
CH2
CH2
With the aim of clarifying the effect of the fluoro atoms in 20b,
molecular dynamics (MD) simulation by GROMACS37 was performed
using the X-ray crystal structure of 11 in complex with FGFR3 as a
template. Energy-minimized structures of 11, 20a and 20b bound to
FGFR3 were generated and used as a starting model. We focused on Cβ of
Lys508, Cγ of Val555, Cβ of Ala634, and the main chain NH moiety of
14c
45
18.5
a
Parallel artificial membrane permeability assay (PAMPA) at pH = 6.5.
b
Aqueous solubility in the Japanese Pharmacopoeia 2nd fluid (JP2) for
disintegration test (pH = 6.8 buffer).
c
Surface area values were calculated using Molecular Operating Environment
(MOE)36 software (average of 3 times).
6

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
Asp635, which were located within 4.5 Å of the fluorine atoms
substituted in the 3,5-dimethoxybenzene of 20b in the model structure
(Fig. 6a).
explore potent and orally active FGFR3 inhibitors with increased sys-
temic exposure and enhanced in vitro potency against FGFR3. Reducing
the molecular size of the substituent at the 4-position and replacing the
linker of the 5-position in the pyrimidine scaffold improved perme-
ability, aqueous solubility and in vitro metabolic stability, and increased
systemic exposure. Furthermore, introducing two fluorine atoms into
the 3,5-dimethoxyphenyl ring enhanced FGFR3 inhibitory activity. MD
simulation of 20b indicated that the fluorine atoms would interact with
Lys508, Val555, Ala634, and Asp635, and that particularly the main
chain NH moiety of Asp635 would interact with the fluorine atom via a
hydrogen bond. The resulting pyrimidine derivative 20b induced tumor
regression following oral administration to a bladder cancer xenograft
nude mouse model. Further optimization studies on novel FGFR3 in-
hibitors derived from 20b, as an orally active lead compound, will be
reported in the future.
The frequency distribution of distances between each amino acid
residue and the R1/R2 substitutions (hydro/chloro/fluoro) in the MD
simulation are displayed in histograms in Fig. 6b. The histograms show
that the distribution of distances interacting with Lys508 or Val555 was
similar among the three compounds. These findings suggested that a
more hydrophobic substituent was favorable for interacting with
Lys508/Val555, explaining why 20a and 20b were more potent than 11.
In contrast, the distribution of distances interacting with Ala634 or
Asp635 were different among compounds. A smaller R1/R2 substituent
resulted in a shorter distance with Ala634, suggesting that Ala634 could
avoid steric repulsion with the R1/R2 substituent. The fluorine substit-
uent formed a shorter distance with Asp635 than hydrogen or chlorine
substituents. These results suggested that the main chain NH moiety of
Asp635 would interact with the fluorine atom via a hydrogen bond38
because a fluorine atom has greater electronegativity than hydrogen or
chlorine atoms39. This may also explain why 20b showed improved
FGFR3 inhibitory activity compared to 11 and 20a. While 20d exhibited
similar potency to 20b, the FGFR3 inhibitory activity of 20c was lower
than that of 20b. These results suggested that the fluoro atom of 20d
would interact with Asp635 in same manner as that of 20b, while a
chloro substituent would additionally interact with Lys508/Val555.
Moreover, it was speculated that the fluoro substituent of 20c would
interact with either Ala634/Asp635 or Lys508/Val555.
5. Experimental
5.1. Chemistry
1H-NMR spectra were recorded on Varian 400, Varian VNS-400, or
Varian 400-MR and chemical shifts are expressed in δ (ppm) values with
tetramethylsilane as an internal reference (s = singlet, d = doublet, t =
triplet, m = multiplet, dd = doublet of doublets, and br = broad peak).
Mass spectra (MS) were recorded on Thermo Electron LCQ Advantage,
Thermo Trace DSQ, Waters SQD, or Agilent Quadrupole. Electrospray
ionization (ESI) positive high resolution mass spectra (HRMS) were
obtained using Thermo Fisher EXACTIVE-Plus. Elemental analyses were
performed with Yanaco MT-6 (C, H, N), Elementar Vario MICRO cube
(C, H, N), Elementar Vario EL III (C, H, N), DIONEX DX-500 (S, halogen),
DIONEX ICS-3000 (S, halogen), or DIONEX ICS-5000 (S, halogen) in-
struments, and the results were within ±0.4% of theoretical values. The
following abbreviations are used: AcOH, acetic acid; MeCN, acetonitrile;
HATU, O-(7-aza-1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate; ADDP, 1,1′-(azodicarbonyl)dipiperidine; BINAP,
2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; IPE, diisopropyl ether;
DIPEA, N,N-diisopropylethylamine; DME, 1,2-dimethoxyethane; DMSO,
dimethyl sulfoxide; DMF, N,N-dimethylformamide; EtOH, ethanol;
EtOAc, ethyl acetate; IPA, isopropyl alcohol; MsOH, methanesulfonic
acid; MeOH, methanol; Pd(OAc)2, palladium(II) acetate; NaOAc, sodium
acetate; NaBH(OAc)3, sodium triacetoxyborohydride; THF, tetrahydro-
furan; n-Bu3P, tri-n-butylphosphine; Et3N, triethylamine; PPh3,
triphenylphosphine.
Table 5 shows the plasma concentration and in vivo inhibitory ac-
tivity against FGFR3 phosphorylation of orally administered test com-
pounds at 30 mg/kg, and in vitro intrinsic clearance in ICR mouse liver
microsomes. Plasma concentrations were measured using LC-MS/MS 6 h
after administration to ICR mice, and in vivo FGFR3 phosphorylation in
tumors was measured using sandwich ELISA 6 h after administration to
nude mice xenografted with NIH/3T3 cells exogenously overexpressing
FGFR3 S249C. Both 11 and 20b exhibited improved systemic exposure
and lower values of in vitro metabolic stability than 1. Moreover, 11 and
20b showed 21% and 85% in vivo inhibitory activity against FGFR3
phosphorylation, respectively.
The higher plasma concentrations of 11 and 20b were attributed to
not only improved physical properties (PAMPA and JP2 solubility) but
also improved in vitro metabolic stability. Moreover, 20b exhibited more
potent in vivo inhibitory activity against FGFR3 phosphorylation than
11, suggesting that potent cellular activity of 20b was necessary for
potent in vivo activity.
Given the promising features of 20b, including its high plasma
concentration and potent in vivo FGFR3 inhibitory activity, further
pharmacological evaluations were conducted (Table 6). Compound 20b
exhibited potent enzyme inhibitory activity against other FGFR subtypes
with similar IC50 values to that for FGFR3. As for the kinase selectivity of
20b, the enzyme inhibitory activity for FGFR3 was 28-fold more potent
than that for VEGFR2 (IC50 value was 34 nM). Moreover, 20b had potent
cell growth inhibitory activity against the UM-UC-14 human bladder
cancer cell line, with an IC50 value of 21 nM.
5.1.1. N2-{3-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]
phenyl}-5-nitro-N4-phenylpyrimidine-2,4-diamine (5a)
A mixture of 2-chloro-5-nitro-N-phenylpyrimidin-4-amine (3a, 260
mg, 1.04 mmol), 3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-
yl]aniline (4, 316 mg, 1.04 mmol), and AcOH (4.00 mL) was stirred at
room temperature overnight. The mixture was quenched with water,
NaOH (1.0 M aqueous solution), and sat. NaHCO3 aq. in an ice-water
bath and extracted with CHCl3. The organic layer was washed with
brine, dried over MgSO4 and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (CHCl3/MeOH/28%
NH3 aq. = 100:0:0 to 90:9:1) to give the product (450 mg, 84%) as a red
solid. 1H NMR (400 MHz, DMSO‑d6) δ ppm 1.46–1.60 (2H, m),
1.72–1.88 (2H, m), 2.16 (3H, s), 2.20–2.58 (11H, m), 3.24–3.40 (2H, m),
3.42–3.56 (3H, m), 6.63–6.73 (1H, m), 7.03–7.17 (2H, m), 7.17–7.29
(1H, m), 7.30–7.44 (2H, m), 7.52–7.66 (2H, m), 9.08 (1H, s),
10.19–10.41 (2H, m); MS (ESI) m/z [M + H]+ 519.
Finally, we evaluated an in vivo antitumor assay using a nude mouse
model xenografted with UM-UC-14 bladder cancer cells (Table 7).
Compound 20b (0.1, 0.3, 1, 3, and 10 mg/kg) was suspended in vehicle
(0.5% methylcellulose solution) and orally administered to mice once
daily for 11 days. Tumor growth inhibition at doses of 0.3 mg/kg (48%
inhibition) and 1 mg/kg (62% inhibition), and tumor regression at doses
of 3 mg/kg (42% regression) and 10 mg/kg (75% regression) were
observed in a dose-dependent manner. These pharmacological results
suggest that 20b is an orally active lead compound for the treatment of
bladder cancer patients.
5.1.2. N4-[2-(Methanesulfonyl)ethyl]-N2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]phenyl}-5-nitropyrimidine-2,4-diamine
(5b)
4. Conclusion
Compound 5b was prepared from 2-chloro-N-[2-(methanesulfonyl)
ethyl]-5-nitropyrimidin-4-amine (3b) and 4 in 62% yield using a similar
We conducted structural optimization of the lead compound 1 to
7

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
Fig. 6. Molecular simulations of 11, 20a, and 20b. (a) The complex structure of 20b docked with FGFR3 around the back pocket, and amino acid residues located
within 4.5 Å of the fluorine atoms. (b) Frequency distribution of distances between the amino acid residues and R1/R2 substituents in an MD simulation.
8

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
Table 5
Plasma concentration, in vivo inhibitory activity against FGFR3 phosphorylation, and in vitro metabolic stability.
No
Structure
Plasma
conc.
p-FGFR3
CLint
Cellular
FGFR3
PAMPA
Solubility
(% inh.)b
(mL/min/kg)c
(10ꢀ 6 cm/sec)e
(µM)f
(nM)a
IC50 (nM)d
1
98.2
NTg
>600
99
1.4
<1
11
1120
1220
21
85
227
176
324
13
15.1
18.4
≥100
20b
79
a
Plasma concentrations were measured using LC-MS/MS 6 h after single oral administration of test compounds at 30 mg/kg to ICR mice (average of 3 mice).
b
In vivo inhibition rate of FGFR3 phosphorylation in nude mice xenografted with NIH/3T3 cells exogenously overexpressing FGFR3 S249C 6 h after single oral
administration of test compounds at 30 mg/kg (average of 3 mice).
c
d
In vitro intrinsic clearance in ICR mouse liver microsomes.
NIH/3T3 cells exogenously overexpressing FGFR3 S249C.
e
Parallel artificial membrane permeability assay (PAMPA) at pH = 6.5.
f
g
Aqueous solubility in the Japanese Pharmacopoeia 2nd fluid for disintegration test (JP2: pH = 6.8 buffer).
Not tested.
5.1.4. N2-{3-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]
Table 6
phenyl}-N4-phenylpyrimidine-2,4,5-triamine (6a)
In vitro pharmacological profiles of 20b.
To a mixture of 5a (450 mg, 0.868 mmol) in EtOH (15.0 mL) and THF
(25.0 mL) was added 10% Pd/C (50% wet, 110 mg). After stirring at
room temperature under a hydrogen atmosphere (1.0 atm) for 3 h, the
mixture was passed through a Celite pad. The filtrate was concentrated
in vacuo. To the reaction mixture were added EtOH (12.0 mL), THF
(12.0 mL) and 10% Pd/C (50% wet, 240 mg). After stirring at room
temperature under a hydrogen atmosphere (1.0 atm) for 7 h, the mixture
was passed through a Celite pad. The filtrate was concentrated in vacuo
to give the product (506 mg, quantitative yield) as a grey solid. MS (ESI)
m/z [M + H]+ 489.
Enzyme IC50 (nM)
Cellular IC50 (nM)
FGFR1
FGFR2
FGFR3
1.2
FGFR4
4.2
VEGFR2
34
UM-UC-
14
NIH/3T3
(FGFR3
S249C)
1.7
1.2
21
13
Table 7
In vivo antitumor efficacy of once daily oral administration of 20b for 11 days in
a nude mouse model xenografted with UM-UC-14 bladder cancer cells (average
of 5 mice).
5.1.5. N4-[2-(Methanesulfonyl)ethyl]–N2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidine-2,4,5-triamine (6b)
To a mixture of 5b (710 mg, 1.29 mmol) in EtOH (15.0 mL) and THF
(30.0 mL) was added 10% Pd/C (50% wet, 331 mg). After stirring at
room temperature under a hydrogen atmosphere (1.0 atm) for 7 h, the
mixture was passed through a Celite pad. The filtrate was concentrated
in vacuo. The residue was diluted with CHCl3 and dried over MgSO4 and
concentrated in vacuo. The residue was purified by column chromatog-
raphy on silica gel (CHCl3/MeOH/28% NH3 aq. = 100:0:0 to 90:9:1) to
give the product (710 mg, quantitative yield) as a grey solid. 1H NMR
(399 MHz, DMSO‑d6) δ ppm 1.45–1.60 (2H, m), 1.72–1.85 (2H, m), 2.14
(3H, s), 2.19–2.57 (11H, m), 3.02 (3H, s), 3.22–3.34 (2H, m), 3.45 (2H,
t, J = 6.7 Hz), 3.73 (3H, s), 3.76–3.85 (2H, m), 3.98 (2H, s), 6.62 (1H, t,
J = 5.8 Hz), 6.74 (1H, d, J = 8.6 Hz), 7.24 (1H, dd, J = 8.6, 2.2 Hz), 7.30
(1H, d, J = 2.4 Hz), 7.42 (1H, s), 8.29 (1H, s); MS (ESI) m/z [M + H]+
519.
Dose (mg/kg)
0.1
0.3
1
3
10a
Antitumor effect
a
1.4% inh.
48% inh.
62% inh.
42% reg.
75% reg.
Body weight loss was observed.
approach to that described for 5a. 1H NMR (400 MHz, DMSO‑d6) δ ppm
1.47–1.61 (2H, m), 1.74–1.85 (2H, m), 2.14 (3H, s), 2.20–2.56 (11H, m),
3.00 (3H, br s), 3.28–3.39 (2H, m), 3.45–3.59 (2H, m), 3.77 (3H, s),
3.94–4.06 (2H, m), 6.87 (1H, d, J = 8.6 Hz), 7.27 (1H, d, J = 2.4 Hz),
7.31–7.40 (1H, m), 8.97 (1H, s), 9.05 (1H, br s), 10.27 (1H, br s); MS
(ESI) m/z [M + H]+ 549.
5.1.3. N-{3-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]
phenyl}-5-nitropyrimidin-2-amine (5c)
To a mixture of 4 (973 mg, 3.20 mmol) in EtOH (10.2 mL) was added
MsOH (0.415 mL, 6.39 mmol). After stirring at room temperature for 20
min, 2-chloro-5-nitropyrimidine (3c, 510 mg, 3.20 mmol) was added to
the mixture. After stirring at 100 ◦C for 4 h, the mixture was quenched
with sat. NaHCO3 aq. and extracted with CHCl3. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue was purified
by column chromatography on amino functionalized silica gel (CHCl3/
MeOH = 100:0 to 80:20) to give the product (858 mg, 63%) as a brown
solid. 1H NMR (400 MHz, CDCl3) δ ppm 1.74–1.95 (4H, m), 2.30 (3H, s),
2.36–2.74 (11H, m), 3.46–3.59 (2H, m), 3.88–3.92 (3H, m), 6.91–6.97
(1H, m), 7.04–7.09 (1H, m), 7.23–7.29 (1H, m), 7.62 (1H, br s), 9.17
(2H, br s); MS (APCI/ESI) m/z [M + H]+ 428.
5.1.6. N2-{3-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]
phenyl}pyrimidine-2,5-diamine (6c)
To a mixture of 5c (858 mg, 2.01 mmol) in EtOH (15.0 mL) was
added 10% Pd/C (55% wet, 107 mg). After stirring at room temperature
under a hydrogen atmosphere (1.0 atm) for 12 h, the mixture was passed
through a Celite pad. The filtrate was concentrated in vacuo. The residue
was purified by column chromatography on silica gel (CHCl3/MeOH =
100:0 to 80:20) to give the product (567 mg, 71%) as a brown solid. 1H
NMR (400 MHz, CDCl3) δ ppm 1.73–1.93 (4H, m), 2.29 (3H, s),
2.34–2.74 (11H, m), 3.25 (2H, s), 3.40–3.56 (2H, m), 3.87 (3H, s), 6.72
9

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
(1H, s), 6.88 (1H, d, J = 8.4 Hz), 6.99 (1H, dd, J = 8.4, 2.2 Hz), 7.20 (1H,
d, J = 2.2 Hz), 8.01 (2H, s); MS (APCI/ESI) m/z [M + H]+ 398.
twice by column chromatography on silica gel (CHCl3/MeOH/28% NH3
aq. = 100:0:0 to 90:9:1) to give the mixture.
To the mixture in MeOH (5.00 mL) was added NaBH4 (57.1 mg, 1.51
mmol). After stirring at room temperature for 6 h, the mixture was
quenched with water and sat. NaHCO3 aq. and extracted with CHCl3.
The organic layer was washed with brine, dried over MgSO4 and
concentrated in vacuo. The residue was purified by column chromatog-
raphy on silica gel (CHCl3/MeOH/28% NH3 aq. = 100:0:0 to 90:9:1).
EtOAc/IPE was added to the residue, and the resulting precipitate was
filtered and dried to give the product (33 mg, 16%) as an yellow solid.
1H NMR (399 MHz, DMSO‑d6) δ ppm 1.43–1.61 (2H, m), 1.69–1.86 (2H,
m), 2.14 (3H, s), 2.18–2.57 (11H, m), 3.21–3.36 (2H, m), 3.68–3.75 (9H,
m), 4.19 (2H, d, J = 6.3 Hz), 5.89 (1H, t, J = 6.3 Hz), 6.36 (1H, t, J = 2.2
Hz), 6.55 (2H, d, J = 2.4 Hz), 6.74 (1H, d, J = 8.6 Hz), 7.18 (1H, dd, J =
8.6, 2.4 Hz), 7.29 (1H, d, J = 2.4 Hz), 7.92 (2H, s), 8.74 (1H, s); MS (ESI)
m/z [M + H]+ 548.
5.1.7. N-(4-Anilino-2-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-
1-yl]anilino}pyrimidin-5-yl)-3,5-dimethoxybenzamide (7a)
To a mixture of 6a (254 mg, 0.520 mmol) in THF (10.0 mL) were
added DIPEA (98.3 µL, 0.574 mmol) and 3,5-dimethoxybenzoyl chloride
(120 mg, 0.598 mmol). After stirring at room temperature for 3 h, the
mixture was quenched with sat. NaHCO3 aq. and extracted with CHCl3.
The organic layer was washed with brine, dried over MgSO4 and
concentrated in vacuo. The residue was purified by column chromatog-
raphy on silica gel (CHCl3/MeOH/28% NH3 aq. = 100:0:0 to 90:9:1).
EtOAc was added to the residue, and the resulting precipitate was
filtered and dried to give the product (103 mg, 30%) as a grey solid. 1H
NMR (400 MHz, DMSO‑d6) δ ppm 1.48–1.59 (2H, m), 1.76–1.83 (2H,
m), 2.15 (3H, s), 2.20–2.58 (11H, m), 3.26–3.37 (2H, m), 3.59 (3H, s),
3.82 (6H, s), 6.71–6.77 (2H, m), 7.02–7.07 (1H, m), 7.20–7.32 (6H, m),
7.63–7.72 (2H, m), 7.97 (1H, s), 8.59 (1H, s), 9.01 (1H, s), 9.57 (1H, s);
MS (ESI) m/z [M + H]+ 653; HRMS (ESI) m/z Calcd for C36H45N8O4 [M
HRMS (ESI) m/z Calcd for C30H42N7O3 [M + H]+: 548.3344. Found:
548.3341; Anal. Calcd for C30H41N7O3.0.1EtOAc: C, 65.6; H, 7.6; N,
17.6. Found: C, 65.5; H, 7.6; N, 17.5.
+
H]+:
653.3558.
Found:
653.3548;
Anal.
Calcd
for
C
36H44N8O4.0.6H2O: C, 65.16; H, 6.87; N, 16.89. Found: C, 65.11; H,
5.1.11. 5-Bromo-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-
yl]phenyl}pyrimidin-2-amine (9)
6.94; N, 16.87.
To a mixture of 5-bromo-2-chloropyrimidine (8, 3.00 g, 15.5 mmol)
and 4 (3.15 g, 10.3 mmol) in IPA (31.5 mL) was added MsOH (2.01 mL,
31.0 mmol). After stirring at 90 ◦C for 12 h and then 110 ◦C for 6 h, the
mixture was quenched with NaHCO3 aq. and extracted with CHCl3. The
organic layer was dried over Na2SO4 and concentrated in vacuo. The
residue was purified by column chromatography on silica gel (CHCl3/
MeOH = 99:1 to 80:20) to give the product (3.76 g, 79%) as a pale
yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 1.72–1.93 (4H, m), 2.29
(3H, s), 2.36–2.73 (11H, m), 3.45–3.56 (2H, m), 3.87 (3H, s), 6.90 (1H,
d, J = 8.4 Hz), 7.00 (1H, dd, J = 8.6, 2.4 Hz), 7.09 (1H, s), 7.19 (1H, d, J
= 2.4 Hz), 8.39 (2H, s); MS (APCI/ESI) m/z [M + H]+ 461, 463.
5.1.8. N-(4-{[2-(Methanesulfonyl)ethyl]amino}-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-5-yl)-3,5-
dimethoxybenzamide (7b)
A mixture of 6b (200 mg, 0.386 mmol), 3,5-dimethoxybenzoic acid
(84.3 mg, 0.463 mmol), DIPEA (132 μL, 0.771 mmol), and HATU (182
mg, 0.479 mmol) in DMF (4.00 mL) was stirred at room temperature
overnight. The mixture was quenched with water and sat. NaHCO3 aq.
and extracted with EtOAc. The organic layer was washed with brine,
dried over MgSO4 and concentrated in vacuo. The residue was purified
twice by column chromatography on silica gel (CHCl3/MeOH/28% NH3
aq. = 100:0:0 to 90:9:1). EtOAc/IPE was added to the residue, and the
resulting precipitate was filtered and dried to give the product (86 mg,
33%) as a colorless solid. 1H NMR (400 MHz, DMSO‑d6) δ ppm
1.45–1.62 (2H, m), 1.75–1.84 (2H, m), 2.14 (3H, s), 2.20–2.57 (11H, m),
3.01 (3H, s), 3.26–3.35 (2H, m), 3.42 (2H, t, J = 6.8 Hz), 3.74–3.84
(11H, m), 6.70 (1H, t, J = 2.2 Hz), 6.77–6.83 (1H, m), 6.93–7.02 (1H,
m), 7.15 (2H, d, J = 2.4 Hz), 7.30–7.35 (2H, m), 7.77 (1H, s), 8.91 (1H,
s), 9.47 (1H, s); MS (ESI) m/z [M + H]+ 683; HRMS (ESI) m/z Calcd for
5.1.12. 5-[(3,5-Dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (10)
To a mixture of 9 (300 mg, 0.650 mmol), Pd(PPh3)4 (75.1 mg, 65.0
µmol), CuI (6.19 mg, 32.5 µmol), 1-ethynyl-3,5-dimethoxybenzene (316
mg, 1.95 mmol) in DMF (6.00 mL) was added Et3N (223 µL, 1.30 mmol)
under an argon atmosphere. After stirring at 110 ◦C for 4 h, the mixture
was diluted with EtOAc and passed through a Celite pad. To the filtrate
was added NaHCO3 aq. and the mixture was extracted with EtOAc. The
organic layer was washed with brine, dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by column chromatography on
silica gel (CHCl3/MeOH = 100:0 to 80:20) to give crude material, which
was purified again by column chromatography on amino functionalized
silica gel (EtOAc/MeOH = 100:0 to 80:20) to give the product (328 mg,
93%) as an yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 1.73–1.96
(4H, m), 2.30 (3H, s), 2.36–2.74 (11H, m), 3.45–3.58 (2H, m), 3.81 (6H,
s), 3.89 (3H, s), 6.47 (1H, t, J = 2.3 Hz), 6.67 (2H, d, J = 2.3 Hz), 6.92
(1H, d, J = 8.6 Hz), 7.04 (1H, dd, J = 8.4, 2.3 Hz), 7.09 (1H, br s),
7.23–7.28 (1H, m), 8.52 (2H, s); MS (APCI/ESI) m/z [M + H]+ 543.
C
C
33H47N8O6S [M + H]+: 683.3334. Found: 683.3324; Anal. Calcd for
33H46N8O6S.0.08IPE.1.7H2O: C, 55.72; H, 7.06; N, 15.53; S, 4.44.
Found: C, 55.79; H, 6.91; N, 15.34; S, 4.39.
5.1.9. 3,5-Dimethoxy-N-(2-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)
piperidin-1-yl]anilino}pyrimidin-5-yl)benzamide (7c)
Compound 7c was prepared from 6c in 51% yield using a similar
approach to that described for 7b. 1H NMR (400 MHz, DMSO‑d6) δ ppm
1.48–1.61 (2H, m), 1.75–1.85 (2H, m), 2.14–2.71 (14H, m), 3.25–3.37
(2H, m), 3.77 (3H, s), 3.83 (6H, s), 6.73 (1H, t, J = 2.2 Hz), 6.82 (1H, d,
J = 8.6 Hz), 7.12 (2H, d, J = 2.2 Hz), 7.28 (1H, dd, J = 8.5, 2.3 Hz), 7.36
(1H, d, J = 2.4 Hz), 8.73 (2H, s), 9.38 (1H, s), 10.21 (1H, s); MS (ESI) m/
z [M + H]+ 562; HRMS (ESI) m/z Calcd for C30H40N7O4 [M + H]+:
5.1.13. 5-[2-(3,5-Dimethoxyphenyl)ethyl]-N-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (11)
To a mixture of 10 (328 mg, 0.604 mmol) in MeOH (10.0 mL) and
THF (10.0 mL) was added 10% Pd/C (50% wet, 64.0 mg) under an argon
atmosphere. After stirring at room temperature under a hydrogen at-
mosphere (3.0 atm) for 3 h, 10% Pd/C (50% wet, 64.0 mg) was added to
the mixture. After stirring at room temperature under a hydrogen at-
mosphere (3.0 atm) for 5 h, the mixture was passed through a Celite pad.
The filtrate was concentrated in vacuo. The residue was purified by
column chromatography on silica gel (CHCl3/MeOH = 100:0 to 80:20),
diluted with EtOAc and stirred under heating condition. After cooling to
room temperature, the resulting precipitate was filtered and dried to
562.3136. Found: 562.3134; Anal. Calcd for
C30H39N7O4.0.14I-
PE.1.2H2O: C, 61.98; H, 7.31; N, 16.41. Found: C, 62.01; H, 7.45; N,
16.23.
5.1.10. N5-[(3,5-Dimethoxyphenyl)methyl]-N2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidine-2,5-diamine (7d)
To a mixture of 6c (150 mg, 0.377 mmol) in CH2Cl2 (6.00 mL) were
added 3,5-dimethoxybenzaldehyde (56.4 mg, 0.340 mmol) and NaBH
(OAc)3 (120 mg, 0.566 mmol). After stirring at room temperature
overnight, the mixture was quenched with water and sat. NaHCO3 aq.
and extracted with CHCl3. The organic layer was washed with brine,
dried over MgSO4 and concentrated in vacuo. The residue was purified
1
give the product (180 mg, 54%) as a pale yellow solid. H NMR (400
10

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
MHz, DMSO‑d6) δ ppm 1.46–1.59 (2H, m), 1.74–1.83 (2H, m), 2.14 (3H,
s), 2.19–2.54 (11H, m), 2.71–2.82 (4H, m), 3.26–3.36 (2H, m), 3.70 (6H,
s), 3.74 (3H, s), 6.30 (1H, t, J = 2.3 Hz), 6.38 (2H, d, J = 2.3 Hz), 6.79
(1H, d, J = 8.6 Hz), 7.24 (1H, dd, J = 8.6, 2.3 Hz), 7.35 (1H, d, J = 2.3
Hz), 8.26 (2H, s), 9.22 (1H, s); MS (ESI) m/z [M + H]+ 547; HRMS (ESI)
m/z Calcd for C31H43N6O3 [M + H]+: 547.3391. Found: 547.3383; Anal.
Calcd for C31H42N6O3.0.4H2O: C, 67.22; H, 7.79; N, 15.17. Found: C,
67.40; H, 7.77; N, 15.00.
42%) as a pale yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ ppm
1.45–1.60 (2H, m), 1.72–1.84 (2H, m), 2.14 (3H, s), 2.20–2.59 (11H, m),
3.22–3.39 (5H, m), 3.57–3.69 (6H, m), 3.73 (3H, s), 6.32–6.52 (3H, m),
6.79 (1H, d, J = 8.6 Hz), 7.16–7.29 (2H, m), 8.32 (2H, br s), 9.47 (1H, s);
MS (ESI) m/z [M + H]+ 576; HRMS (ESI) m/z Calcd for C31H42N7O4 [M
+ H]+: 576.3293. Found: 576.3288; Anal. Calcd for C31H41N7O4.0.3I-
PE.1.2H2O: C, 62.73; H, 7.64; N, 15.61. Found: C, 62.64; H, 7.40; N,
15.62.
5.1.14. N-(2-Chloropyrimidin-5-yl)-3,5-dimethoxybenzamide (13a)
Compound 13a was prepared from 2-chloropyrimidin-5-amine (12a)
in 77% yield using a similar approach to that described for 7a. 1H NMR
(400 MHz, DMSO‑d6) δ ppm 3.83 (6H, s), 6.78 (1H, t, J = 2.3 Hz), 7.14
(2H, d, J = 2.3 Hz), 9.13 (2H, s), 10.70 (1H, s); MS (ESI) m/z [M + H]+
294.
5.1.19. 5-[(3,5-Dimethoxyphenoxy)methyl]-N-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (14b)
Compound 14b was prepared from 13c and 4 in 82% yield using a
similar approach to that described for 14a. 1H NMR (400 MHz,
DMSO‑d6) δ ppm 1.47–1.60 (2H, m), 1.76–1.83 (2H, m), 2.14 (3H, s),
2.19–2.58 (11H, m), 3.26–3.36 (2H, m), 3.71 (6H, s), 3.76 (3H, s), 4.94
(2H, s), 6.12 (1H, t, J = 2.2 Hz), 6.20 (2H, d, J = 2.2 Hz), 6.81 (1H, d, J
= 8.6 Hz), 7.27 (1H, dd, J = 8.6, 2.4 Hz), 7.36 (1H, d, J = 2.4 Hz), 8.52
(2H, s), 9.50 (1H, s); MS (ESI) m/z [M + H]+ 549; HRMS (ESI) m/z Calcd
for C30H41N6O4 [M + H]+: 549.3184. Found: 549.3182; Anal. Calcd for
5.1.15. N-(2-Chloropyrimidin-5-yl)-3,5-dimethoxy-N-methylbenzamide
(13b)
To a mixture of 13a (690 mg, 2.35 mmol) in DMF (10.0 mL) was
added NaH (113 mg, 2.58 mmol, 55% oil dispersion) in an ice-water
bath. After stirring in an ice-water bath for 15 min, iodomethane
(220 µL, 3.53 mmol) was added to the mixture. After stirring at room
temperature for 4 h, the mixture was quenched with water in an ice-
water bath and extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (n-hexane/EtOAc
= 100:0 to 50:50) to give the product (530 mg, 73%) as an yellow solid.
1H NMR (400 MHz, DMSO‑d6) δ ppm 3.38 (3H, s), 3.68 (6H, s),
6.48–6.53 (3H, m), 8.73 (2H, s); MS (APCI/ESI) m/z [M + H]+ 308.
C30H40N6O4.0.05EtOAc.0.04H2O: C, 65.50; H, 7.37; N, 15.18. Found: C,
65.49; H, 7.43; N, 15.05.
5.1.20. 5-[(3,5-Dimethoxyphenyl)methoxy]-N-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (14c)
Compound 14c was prepared from 13d and 4 in 33% yield using a
similar approach to that described for 14a. 1H NMR (399 MHz,
DMSO‑d6) δ ppm 1.43–1.62 (2H, m), 1.68–1.87 (2H, m), 2.14 (3H, s),
2.17–2.70 (11H, m), 3.23–3.35 (2H, m), 3.74 (6H, s), 3.74 (3H, s), 5.07
(2H, s), 6.46 (1H, t, J = 2.4 Hz), 6.60 (2H, d, J = 2.4 Hz), 6.78 (1H, d, J
= 8.6 Hz), 7.23 (1H, dd, J = 8.6, 2.2 Hz), 7.32 (1H, d, J = 2.2 Hz), 8.29
(2H, s), 9.15 (1H, s); MS (ESI) m/z [M + H]+ 549; HRMS (ESI) m/z Calcd
for C30H41N6O4 [M + H]+: 549.3184. Found: 549.3179; Anal. Calcd for
5.1.16. 2-Chloro-5-[(3,5-dimethoxyphenoxy)methyl]pyrimidine (13c)
To a mixture of (2-chloropyrimidin-5-yl)methanol (12b, 120 mg,
0.805 mmol) in THF (2.40 mL) were added 3,5-dimethoxyphenol (186
mg, 1.21 mmol), ADDP (305 mg, 1.21 mmol), and n-Bu3P (244 mg, 1.21
mmol) in an ice-water bath. After stirring at room temperature for 12 h,
the insoluble material was removed by filtration. The filtrate was
concentrated in vacuo to give a residue, which was purified by column
chromatography on silica gel (n-hexane/EtOAc = 90:10 to 0:100) to give
the product (119 mg, 53%) as a colorless solid. 1H NMR (400 MHz,
CDCl3) δ ppm 3.78 (6H, s), 5.03 (2H, s), 6.11–6.16 (3H, m), 8.70 (2H, s);
MS (APCI/ESI) m/z [M + H]+ 281.
C
30H40N6O4.0.01IPE.0.03H2O: C, 65.6; H, 7.4; N, 15.3. Found: C, 65.9;
H, 7.4; N, 15.0.
5.1.21. 2,4-Dichloro-3-ethynyl-1,5-dimethoxybenzene (16a)
To a mixture of 1-ethynyl-3,5-dimethoxybenzene (15, 500 mg, 3.08
mmol) in MeCN (10.0 mL) was added SO2Cl2 (524 µL, 6.47 mmol) in an
ice-water bath under an argon atmosphere. After stirring at room tem-
perature for 12 h, the mixture was concentrated in vacuo to give a res-
idue, which was purified by column chromatography on silica gel (n-
hexane/EtOAc = 98:2 to 60:40) to give the product (335 mg, 47%) as a
colorless solid. 1H NMR (400 MHz, CDCl3) δ ppm 3.68 (1H, s), 3.92 (6H,
s), 6.58 (1H, s); MS (APCI/ESI) m/z [M + H]+ 231.
5.1.17. 2-Chloro-5-[(3,5-dimethoxyphenyl)methoxy]pyrimidine (13d)
To a mixture of 2-chloropyrimidin-5-ol (12c, 278 mg, 2.13 mmol) in
DMF (3.00 mL) were added K2CO3 (453 mg, 3.28 mmol) and 3,5-dime-
thoxybenzyl bromide (541 mg, 2.34 mmol) in an ice-water bath. After
stirring at room temperature for 7 h, the mixture was quenched with
water and sat. NaHCO3 aq. and extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4 and concentrated in vacuo.
The residue was purified by column chromatography on silica gel (n-
hexane/EtOAc = 100:0 to 75:25) to give the product (360 mg, 60%) as a
colorless solid. 1H NMR (400 MHz, DMSO‑d6) δ ppm 3.75 (6H, s), 5.20
(2H, s), 6.48 (1H, t, J = 2.3 Hz), 6.63 (2H, d, J = 2.4 Hz), 8.60 (2H, s);
MS (APCI/ESI) m/z [M + H]+ 281.
5.1.22. 3-Ethynyl-2,4-difluoro-1,5-dimethoxybenzene (16b)
.
5.1.23. 1-Ethynyl-2-fluoro-3,5-dimethoxybenzene (16c)
To a mixture of 15 (5.00 g, 30.8 mmol) in MeCN (100 mL) was added
Selectfluor™ (24.3 g, 61.7 mmol) in an ice-water bath. The mixture was
allowed to gradually warm to room temperature and stirred for 4 days.
The mixture was quenched with NaHCO3 aq. and extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4 and
concentrated in vacuo. The residue was purified by column chromatog-
raphy on silica gel (n-hexane/EtOAc = 95:5 to 70:30) to give 16c (1.74
g, 31%) as a pale yellow oil and crude material for 16b, which was
purified twice by column chromatography on silica gel (n-hexane/
CHCl3 = 50:50 to 0:100) to give 16b (1.21 g, 20%) as a colorless solid.
1H NMR (400 MHz, CDCl3) δ ppm 3.52 (1H, s), 3.88 (6H, s), 6.66 (1H, t,
J = 8.0 Hz); MS (EI) m/z [M]+ 198 (for 16b); 1H NMR (400 MHz, CDCl3)
δ ppm 3.29 (1H, s), 3.77 (3H, s), 3.86 (3H, s), 6.49 (1H, dd, J = 4.5, 2.9
Hz), 6.54 (1H, dd, J = 7.0, 2.9 Hz); MS (APCI/ESI) m/z [M + H]+ 181
(for 16c).
5.1.18. 3,5-Dimethoxy-N-(2-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)
piperidin-1-yl]anilino}pyrimidin-5-yl)-N-methylbenzamide (14a)
To a mixture of 13b (195 mg, 0.634 mmol), 4 (295 mg, 0.969 mmol),
rac-BINAP (119 mg, 0.191 mmol), and Cs2CO3 (619 mg, 1.90 mmol) in
1,4-dioxane (8.00 mL) was added Pd(OAc)2 (39.0 mg, 0.174 mmol).
After stirring at 100 ◦C for 3 h, the mixture was quenched with water and
extracted with EtOAc. The organic layer was washed with brine, dried
over MgSO4 and concentrated in vacuo. The residue was purified twice
by column chromatography on silica gel (CHCl3/MeOH/28% NH3 aq. =
100:0:0 to 90:9:1). EtOAc/IPE was added to the residue, and the
resulting precipitate was filtered and dried to give the product (153 mg,
11

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
5.1.24. 2-Chloro-3-ethynyl-4-fluoro-1,5-dimethoxybenzene (16d)
Compound 16d was prepared from 16c in 83% yield using a similar
approach to that described for 16a. 1H NMR (400 MHz, CDCl3) δ ppm
3.60 (1H, s), 3.89 (3H, s), 3.91 (3H, s), 6.61 (1H, d, J = 7.5 Hz); MS (CI)
m/z [M]+ 214.
6.65 (1H, dd, J = 4.7, 2.9 Hz), 6.79 (1H, dd, J = 7.0, 2.9 Hz), 6.84 (1H, d,
J = 8.6 Hz), 7.27 (1H, dd, J = 8.6, 2.4 Hz), 7.32 (1H, d, J = 2.4 Hz), 8.62
(2H, s), 9.84 (1H, s); MS (ESI) m/z [M + H]+ 561.
5.1.29. 5-[(2-Chloro-6-fluoro-3,5-dimethoxyphenyl)ethynyl]-N-{3-
methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-
amine (19d)
5.1.25. 5-Iodo-N-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]
phenyl}pyrimidin-2-amine (18)
Compound 19d was prepared from 18 and 16d in 66% yield using a
similar approach to that described for 19a. 1H NMR (399 MHz,
DMSO‑d6) δ ppm 1.46–1.62 (2H, m), 1.71–1.88 (2H, m), 2.14 (3H, s),
2.20–2.58 (11H, m), 3.28–3.38 (2H, m), 3.77 (3H, s), 3.89–3.97 (6H, m),
6.84 (1H, d, J = 8.6 Hz), 7.04 (1H, d, J = 8.0 Hz), 7.26 (1H, dd, J = 8.6,
2.2 Hz), 7.35 (1H, d, J = 2.4 Hz), 8.64 (2H, s), 9.91 (1H, s); MS (ESI) m/z
[M + H]+ 595.
To a mixture of 2-chloro-5-iodopyrimidine (17, 1.00 g, 4.16 mmol)
and 4 (1.27 g, 4.16 mmol) in IPA (20.0 mL) was added MsOH (809 µL,
12.5 mmol). After stirring at 130 ◦C for 3 h under µW irradiation, the
mixture was quenched with NaHCO3 aq. and extracted with CHCl3. The
organic layer was dried over Na2SO4 and concentrated in vacuo. The
residue was purified by column chromatography on silica gel (CHCl3/
MeOH = 100:0 to 80:20) to give the crude product, which was purified
again by column chromatography on amino functionalized silica gel
(EtOAc/MeOH = 100:0 to 80:20). EtOAc was added to the residue, and
the resulting precipitate was filtered and dried to give the product (1.76
g, 83%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm
1.74–1.98 (4H, m), 2.25–2.34 (3H, m), 2.36–2.72 (11H, m), 3.46–3.55
(2H, m), 3.85–3.89 (3H, m), 6.86–6.94 (1H, m), 6.94–7.01 (2H, m),
7.16–7.21 (1H, m), 8.49 (2H, s); MS (APCI/ESI) m/z [M + H]+ 509.
5.1.30. 5-[2-(2,6-Dichloro-3,5-dimethoxyphenyl)ethyl]-N-{3-methoxy-4-
[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (20a)
A mixture of 19a (126 mg, 0.206 mmol) and p-toluenesulfonyl hy-
drazide (1.92 g, 10.3 mmol) in DME (2.52 mL) was stirred at 110 ◦C. To
the mixture was added a solution of NaOAc (845 mg, 10.3 mmol) in
◦
water (2.52 mL) over 2 h. After stirring at 110 C for 2 h, p-toluene-
sulfonyl hydrazide (1.92 g, 10.3 mmol) and NaOAc (845 mg, 10.3 mmol)
were added over 2 h. After stirring at 110 ◦C for 2 h, the mixture was
neutralized with NaHCO3 aq. and extracted with CHCl3. The organic
layer was dried over Na2SO4 and concentrated in vacuo. A mixture of the
residue and p-toluenesulfonyl hydrazide (1.92 g, 10.3 mmol) in DME
(2.52 mL) was stirred at 110 ◦C. To the mixture was added a solution of
NaOAc (845 mg, 10.3 mmol) in water (2.52 mL) over 2 h. After stirring
5.1.26. 5-[(2,6-Dichloro-3,5-dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-
[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (19a)
To a mixture of 18 (200 mg, 0.393 mmol), 16a (100 mg, 0.433
mmol), Pd(PPh3)4 (22.7 mg, 19.7 µmol), CuI (3.75 mg, 19.7 µmol) in
DMF (4.00 mL) was added DIPEA (135 µL, 0.787 mmol) under an argon
atmosphere. After stirring at 80 ◦C for 1 h, the mixture was diluted with
EtOAc and water and passed through a Celite pad. The organic layer was
separated and washed with brine, dried over Na2SO4 and concentrated
in vacuo. The residue was purified by column chromatography on silica
gel (CHCl3/MeOH = 100:0 to 80:20) to give the product (126 mg, 52%)
as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 1.74–1.94 (4H,
m), 2.30 (3H, s), 2.37–2.75 (11H, m), 3.47–3.56 (2H, m), 3.89 (3H, s),
3.94 (6H, s), 6.57 (1H, s), 6.91 (1H, d, J = 8.6 Hz), 6.99–7.05 (1H, m),
7.16 (1H, s), 7.31 (1H, d, J = 2.4 Hz), 8.60 (2H, s); MS (APCI/ESI) m/z
[M + H]+ 611, 613.
◦
at 110 C for 2 h, the mixture was neutralized with NaHCO3 aq. and
extracted with CHCl3. The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified by column chromatog-
raphy on silica gel (CHCl3/MeOH/28% NH3 aq. = 100:0:0 to 80:18:2).
EtOAc was added to the residue, and the resulting precipitate was
filtered, washed with IPE, and dried to give the product (53.0 mg, 42%)
as a pale yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ ppm 1.46–1.60
(2H, m), 1.74–1.83 (2H, m), 2.14 (3H, s), 2.20–2.56 (11H, m), 2.65–2.72
(2H, m), 3.08–3.17 (2H, m), 3.26–3.35 (2H, m), 3.75 (3H, s), 3.90 (6H,
s), 6.79 (1H, d, J = 8.8 Hz), 6.83 (1H, s), 7.24 (1H, dd, J = 8.6, 2.4 Hz),
7.37 (1H, d, J = 2.4 Hz), 8.23 (2H, s), 9.28 (1H, s); MS (ESI) m/z [M +
H]+ 615, 617; HRMS (ESI) m/z Calcd for C31H41Cl2N6O3 [M + H]+:
615.2612. Found: 615.2608; Anal. Calcd for C31H40Cl2N6O3.0.2E-
tOAc.0.2H2O: C, 59.98; H, 6.65; N, 13.20; Cl, 11.13. Found: C, 59.95; H,
6.52; N, 13.04; Cl, 11.27.
5.1.27. 5-[(2,6-Difluoro-3,5-dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-
[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (19b)
To a mixture of 18 (182 mg, 0.358 mmol), 16b (85.1 mg, 0.430
mmol), Pd(PPh3)4 (41.4 mg, 35.8 µmol), CuI (3.41 mg, 17.9 µmol) in
DMF (3.64 mL) was added Et3N (123 µL, 0.716 mmol) under an argon
atmosphere. After stirring at 100 ◦C for 4 h, the mixture was diluted with
EtOAc and passed through a Celite pad. The filtrate was diluted with
NaHCO3 aq. and extracted with EtOAc. The organic layer was washed
with brine, dried over Na2SO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (CHCl3/MeOH =
100:0 to 80:20) to give crude material, which was purified again by
column chromatography on amino functionalized silica gel (EtOAc/
MeOH = 100:0 to 80:20). The residue was added EtOAc and stirred
under heating condition. After cooling to room temperature, the
resulting precipitate was filtered and dried to give the product (120 mg,
58%) as an yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ ppm 1.44–1.68
(2H, m), 1.71–1.91 (2H, m), 2.17–2.75 (14H, m), 3.24–3.41 (2H, m),
3.77 (3H, s), 3.90 (6H, s), 6.84 (1H, d, J = 8.6 Hz), 7.10 (1H, t, J = 8.4
Hz), 7.27 (1H, dd, J = 8.6, 2.4 Hz), 7.33 (1H, d, J = 2.4 Hz), 8.64 (2H, s),
9.91 (1H, s); MS (ESI) m/z [M + H]+ 579.
5.1.31. 5-[2-(2,6-Difluoro-3,5-dimethoxyphenyl)ethyl]-N-{3-methoxy-4-
[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (20b)
A mixture of 19b (7.50 g, 13.0 mmol) and p-toluenesulfonyl hydra-
zide (24.2 g, 130 mmol) in DME (75.0 mL) was stirred at 110 ◦C. To the
mixture was added a solution of NaOAc (10.6 g, 130 mmol) in water
◦
(15.0 mL) over 2 h. After stirring at 110 C for 2 h, p-toluenesulfonyl
hydrazide (12.1 g, 64.8 mmol) and NaOAc (5.32 g, 64.8 mmol) were
◦
added over 2 h. After stirring at 110 C for 2 h, p-toluenesulfonyl hy-
drazide (12.1 g, 64.8 mmol) and NaOAc (5.32 g, 64.8 mmol) were added
◦
over 2 h. The mixture was stirred at 110 C for 2 h. The mixture was
neutralized with NaHCO3 aq. and extracted with CHCl3. The organic
layer was dried over MgSO4 and concentrated in vacuo. A mixture of the
residue and p-toluenesulfonyl hydrazide (12.1 g, 64.8 mmol) in DME
(75.0 mL) was stirred at 110 ◦C. To the mixture was added a solution of
NaOAc (5.32 g, 64.8 mmol) in water (15.0 mL) over 2 h. After stirring at
◦
110 C for 2 h, p-toluenesulfonyl hydrazide (12.1 g, 64.8 mmol) and
5.1.28. 5-[(2-Fluoro-3,5-dimethoxyphenyl)ethynyl]-N-{3-methoxy-4-[4-
(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (19c)
Compound 19c was prepared from 18 and 16c in 55% yield using a
similar approach to that described for 19a. 1H NMR (399 MHz,
DMSO‑d6) δ ppm 1.43–1.64 (2H, m), 1.71–1.88 (2H, m), 2.14 (3H, s),
2.20–2.59 (11H, m), 3.26–3.39 (2H, m), 3.74–3.80 (6H, m), 3.85 (3H, s),
NaOAc (5.32 g, 64.8 mmol) were added over 2 h. After stirring at 110 ◦C
for 2 h, the mixture was neutralized with NaHCO3 aq. and extracted with
CHCl3. The organic layer was dried over MgSO4 and concentrated in
vacuo. The residue was purified by column chromatography on silica gel
(CHCl3/MeOH/28% NH3 aq. = 100:0:0 to 80:18:2). EtOAc (140 mL) and
EtOH (10 mL) were added to the residue, and the resulting precipitate
12

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
was filtered to give the crude product. EtOH (50 mL) was added to the
crude product, and the resulting precipitate was filtered and dried to
give the product (1.78 g, 24%) as a pale yellow solid. 1H NMR (399 MHz,
DMSO‑d6) δ ppm 1.46–1.59 (2H, m), 1.74–1.82 (2H, m), 2.14 (3H, s),
2.18–2.58 (11H, m), 2.65–2.75 (2H, m), 2.84–2.93 (2H, m), 3.24–3.38
(2H, m), 3.74 (3H, s), 3.81 (6H, s), 6.79 (1H, d, J = 8.8 Hz), 6.85 (1H, t,
J = 8.4 Hz), 7.23 (1H, dd, J = 8.8, 2.4 Hz), 7.34 (1H, d, J = 2.4 Hz), 8.17
(2H, s), 9.25 (1H, s); MS (ESI) m/z [M + H]+ 583; HRMS (ESI) m/z Calcd
for C31H41F2N6O3 [M + H]+: 583.3203. Found: 583.3195; Anal. Calcd
for C31H40F2N6O3.0.1H2O: C, 63.7; H, 6.9; N, 14.4; F, 6.5. Found: C,
63.8; H, 7.1; N, 14.2; F, 6.1.
MOE36 using the crystal structure of FGFR3 in complex with 11 (PDB
code: 7DHL) as a template. Molecular dynamics simulations for the
complexes were performed using GROMACS37 ver. 2016.1 with the
AMBER 99SB-ILDN force field for proteins and the general amber force
field (GAFF) for ligands.
5.3. In vitro kinase inhibitory assay
Inhibitory activity of compounds against FGFR1, 2, 3, and 4, and
VEGFR2 were evaluated using an off-chip mobility shift assay. FGFR1, 2,
3, and 4, and VEGFR2 kinase (Carna Bioscience, Kobe, Japan) and test
compounds were incubated for 30 or 120 min at room temperature (RT).
After the incubation, substrate and adenosine triphosphate (ATP) at 75
µmol/L for VEGFR2, FGFR2, and FGFR3, 125 µmol/L for FGFR1, and
300 µmol/L for FGFR4 were added and the reactions were incubated for
30 min at RT. The kinase reaction was stopped by the addition of
termination buffer. The reaction mixtures were measured using an EZ
Reader II (Perkin Elmer). Wells without ATP were measured as positive
control (100% inhibition), and wells treated with DMSO were measured
as a negative control (0% inhibition). The IC50 value of each experiment
was calculated using Sigmoid-Emax non-linear regression analysis.
5.1.32. 5-[2-(2-Fluoro-3,5-dimethoxyphenyl)ethyl]-N-{3-methoxy-4-[4-
(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-amine (20c)
A mixture of 19c (122 mg, 0.218 mmol) and p-toluenesulfonyl hy-
drazide (2.03 g, 10.9 mmol) in DME (2.44 mL) was stirred at 110 ◦C. To
the mixture was added a solution of NaOAc (893 mg, 10.9 mmol) in
◦
water (1.22 mL) over 2 h. After stirring at 110 C for 2 h, p-toluene-
sulfonyl hydrazide (1.01 g, 5.45 mmol) and NaOAc (447 mg, 5.45 mmol)
were added over 2 h. After stirring at 110 ◦C for 2 h, the mixture was
neutralized with NaHCO3 aq. and extracted with CHCl3. The organic
layer was dried over MgSO4 and concentrated in vacuo. The residue was
purified by column chromatography on amino functionalized silica gel
(EtOAc/MeOH/28% NH3 aq. = 100:0:0 to 80:18:2). EtOAc/IPE was
added to the residue, and the resulting precipitate was filtered and dried
to give the product (63.0 mg, 51%) as a pale yellow solid. 1H NMR (399
MHz, DMSO‑d6) δ ppm 1.45–1.61 (2H, m), 1.73–1.83 (2H, m), 2.14 (3H,
s), 2.19–2.56 (11H, m), 2.70–2.77 (2H, m), 2.79–2.87 (2H, m),
3.25–3.34 (2H, m), 3.70 (3H, s), 3.74 (3H, s), 3.78 (3H, s), 6.37 (1H, dd,
J = 5.1, 2.9 Hz), 6.55 (1H, dd, J = 7.0, 2.9 Hz), 6.79 (1H, d, J = 8.6 Hz),
7.24 (1H, dd, J = 8.6, 2.4 Hz), 7.35 (1H, d, J = 2.2 Hz), 8.23 (2H, s), 9.24
(1H, s); MS (ESI) m/z [M + H]+ 565; HRMS (ESI) m/z Calcd for
5.4. In vitro cell growth inhibition assay
NIH/3T3 cells exogenously overexpressing FGFR3 S249C were
seeded in 96-well plates at 3000 cells per well and incubated overnight.
The following day, the cells were exposed to test compounds for 5 days.
Cell viability was measured using CellTiter-Glo (Promega, Madison, WI,
USA). Data are presented as mean values from a single experiment
performed in duplicate.
UM-UC-14 cells were purchased from ECACC (Salisbury, UK). The
cells were cultured according to instructions from the supplier. The cells
were seeded in 384-well plates at 900 cells per well and incubated
overnight. The following day, the cells were exposed to 20b for 5 days.
Cell viability was measured using CellTiter-Glo (Promega, Madison, WI,
USA). Data are presented as mean values from a single experiment
performed in duplicate.
C
C
31H42FN6O3 [M + H]+: 565.3297. Found: 565.3287; Anal. Calcd for
31H41FN6O3.0.4H2O: C, 65.10; H, 7.37; N, 14.70; F, 3.32. Found: C,
65.18; H, 7.24; N, 14.55; F, 3.33.
5.1.33. 5-[2-(2-Chloro-6-fluoro-3,5-dimethoxyphenyl)ethyl]-N-{3-
methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidin-2-
amine (20d)
5.5. In vitro metabolic stability
A mixture of 19d (126 mg, 0.212 mmol) and p-toluenesulfonyl hy-
drazide (1.97 g, 10.6 mmol) in DME (2.52 mL) was stirred at 110 ◦C. To
the mixture was added a solution of NaOAc (870 mg, 10.6 mmol) in
To estimate the intrinsic clearance of compounds against mouse
hepatic CYPs, test compounds (0.2 µM) were incubated with pooled
mouse liver microsomes (0.2 mg protein/mL), NADPH (1 mM) and
EDTA (0.1 mM) in pH 7.4 Na+-K+ phosphate buffer (100 mM) at 37 ◦C.
Incubations were conducted for 0, 15, 30, and 45 min. The peak area of
the compounds and internal standard was measured using liquid
chromatography-tandem mass spectrometry (LC-MS/MS) and analyzed
to calculate CLint (mL/min/kg). Values are shown as an average of
duplicate experiments.
◦
water (2.52 mL) over 2 h. After stirring at 110 C for 2 h, p-toluene-
sulfonyl hydrazide (1.97 g, 10.6 mmol) and NaOAc (870 mg, 10.6 mmol)
were added over 2 h. After stirring at 110 ◦C for 2 h, the mixture was
neutralized with NaHCO3 aq. and extracted with CHCl3. The organic
layer was dried over Na2SO4 and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (CHCl3/MeOH/28%
NH3 aq. = 100:0:0 to 80:18:2) to give crude material, which was purified
again by column chromatography on amino functionalized silica gel
(EtOAc/MeOH/28% NH3 aq. = 100:0:0 to 80:18:2). EtOAc/IPE was
added to the residue, and the resulting precipitate was filtered and dried
to give the product (88.0 mg, 69%) as a pale yellow solid. 1H NMR (400
MHz, DMSO‑d6) δ ppm 1.47–1.61 (2H, m), 1.73–1.85 (2H, m), 2.17 (3H,
s), 2.21–2.60 (11H, m), 2.65–2.75 (2H, m), 2.93–3.03 (2H, m),
3.24–3.39 (2H, m), 3.74 (3H, s), 3.84–3.88 (6H, m), 6.79 (1H, d, J = 8.6
Hz), 6.84 (1H, d, J = 7.8 Hz), 7.23 (1H, dd, J = 8.6, 2.4 Hz), 7.35 (1H, d,
J = 2.4 Hz), 8.18 (2H, s), 9.26 (1H, s); MS (ESI) m/z [M + H]+ 599;
HRMS (ESI) m/z Calcd for C31H41ClFN6O3 [M + H]+: 599.2907. Found:
599.2897; Anal. Calcd for C31H40ClFN6O3.0.4H2O: C, 61.41; H, 6.78; N,
13.86; Cl, 5.85; F, 3.13. Found: C, 61.39; H, 6.78; N, 13.70; Cl, 5.91; F,
3.10.
5.6. Permeability
Parallel artificial membrane permeability assay (PAMPA) was con-
ducted using STARlet 8ch (Hamilton Robotics, Reno, NV). In this assay,
a ‘sandwich’ is formed by a 96-well microtiter plate (pION Inc., Billerica,
MA) and a 96-well filter plate (pION Inc.) such that each composite well
is divided into two chambers, with the donor at the bottom and acceptor
at the top, separated by a lipid (pION Inc.)-coated microfilter disc.
DMSO stock solutions (10 mM) of the test compounds were added to a
mixture of aqueous buffer (pH 6.5) and DMSO (9:1). The drug solutions
were filtered through a 96-well filter plate (PVDF, Corning Inc., Corning,
NY) and added to the donor compartments. The plates were sandwiched
together and acceptor buffer (pH 7.4, pION Inc.) was added to the
acceptor compartment. The sandwiched plates were incubated at room
temperature for 2 h under saturated water vapor conditions. After in-
cubation, the amount of test compounds in the donor and acceptor
5.2. Molecular modeling
Docking models of 11, 20a, and 20b with FGFR3 were prepared by
13

I. Kuriwaki et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116019
compartments was assayed using high performance liquid chromatog-
raphy (HPLC). Permeability was calculated using PAMPA Evolution
software (pION Inc.).
Acknowledgements
The authors thank Dr. Susumu Watanuki for his valuable advice in
the preparation of this manuscript; Drs. Tomoyuki Suzuki, Aya Kikuchi,
Masateru Iizuka, and Ms. Ayako Nakayama for their contributions to the
biological studies; Drs. Akihiro Yamada, Yoichi Naritomi, Toshiko
Yahata, Yoshiteru Kamiyama, and Fumihiko Ushigome for their con-
tributions to the pharmacokinetic studies; Mr. Noritaka Hamada and Dr.
Kouji Yamazaki for their contribution to physicochemical studies; Drs.
Kazuki Ono and Yohsuke Hagiwara for their contributions to molecular
modeling; and the staff of the Division of Analytical Science Laboratories
and Astellas Research Technologies Co., Ltd. for conducting screening
studies for metabolic clearance, permeability, solubility, partition co-
efficient, elemental analysis, and spectral measurements.
5.7. Aqueous solubility
Solubility of the test compounds was evaluated using the Japanese
Pharmacopoeia 2nd fluid for disintegration test (JP2; pH = 6.8 buffer).
DMSO stock solutions (10 mM) of the test compounds were prepared
and added to JP2. The solutions were shaken at 1000 rpm at 25 ◦C under
light-protected conditions for 20 h. Precipitates were filtered through a
PVDF membrane filter (pore size 0.22
μm, MERCK) and the concentra-
tion ( M) of the compound in the filtrate was assayed using HPLC.
μ
Funding
5.8. Animal experiments
This study was funded by Astellas Pharma Inc. and Kotobuki Phar-
maceutical Co., Ltd.
All animal experimental procedures were approved by the Institu-
tional Animal Care and Use Committee of Astellas Pharma Inc.
Furthermore, Astellas Pharma Inc. Tsukuba Research Center was
awarded Accreditation Status by the AAALAC International. All efforts
were made to minimize the number of animals used and to avoid
suffering and distress.
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Declaration of Competing Interest
The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
[Ikumi Kuriwaki, Minoru Kameda, Kazuhiko Iikubo, Hiroyuki Hisa-
michi, Yuichiro Kawamoto, Hiroyuki Moritomo, Yoshinori Iwai, Atsushi
Noda, Hiroshi Tomiyama are inventors of the following patent: WO
2013/129369 (A1). This research is a collaboration between Astellas
Pharma Inc. and Kotobuki Pharmaceutical Co., Ltd. Ikumi Kuriwaki,
Minoru Kameda, Kazuhiko Iikubo, Hiroyuki Moritomo, Yasushi Amano,
Yukihiro Tateishi, Yuka Echizen, Taisuke Nakazawa, Masaaki Hirano
are employees of Astellas Pharma Inc.; Yoshinori Iwai and Atsushi Noda
are employees of Kotobuki Pharmaceutical Co., Ltd.; and Hiroshi
Tomiyama is President and CEO of Kotobuki Pharmaceutical Co., Ltd., as
of the date of manuscript submission].
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