Structure-based drug design of 1,3,5-triazine and pyrimidine derivatives as novel FGFR3 inhibitors with high selectivity over VEGFR2

Article abstract of DOI:10.1016/j.bmc.2020.115453

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of bladder cancer. We identified 1,3,5-triazine derivative 18b and pyrimidine derivative 40a as novel structures with potent and highly selective FGFR3 inhibitory activity over vascular endothelial growth factor receptor 2 (VEGFR2) using a structure-based drug design (SBDD) approach. X-ray crystal structure analysis suggests that interactions between 18b and amino acid residues located in the solvent region (Lys476 and Met488), and between 40a and Met529 located in the back pocket of FGFR3 may underlie the potent FGFR3 inhibitory activity and high kinase selectivity over VEGFR2.

Full text of DOI:10.1016/j.bmc.2020.115453

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-based drug design of 1,3,5-triazine and pyrimidine derivatives as
novel FGFR3 inhibitors with high selectivity over VEGFR2
Ikumi Kuriwakia,⁎, Minoru Kamedaa, Hiroyuki Hisamichia,1, Shigetoshi Kikuchia,2
,
Kazuhiko Iikuboa, Yuichiro Kawamotoa,3, Hiroyuki Moritomoa, Yutaka Kondoha,4
,
Yasushi Amanoa, Yukihiro Tateishia, Yuka Echizena, Yoshinori Iwaib, Atsushi Nodab,
Hiroshi Tomiyamab, Tomoyuki Suzukia, 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 bladder
cancer. We identified 1,3,5-triazine derivative 18b and pyrimidine derivative 40a as novel structures with
potent and highly selective FGFR3 inhibitory activity over vascular endothelial growth factor receptor 2
(VEGFR2) using a structure-based drug design (SBDD) approach. X-ray crystal structure analysis suggests that
interactions between 18b and amino acid residues located in the solvent region (Lys476 and Met488), and
between 40a and Met529 located in the back pocket of FGFR3 may underlie the potent FGFR3 inhibitory activity
and high kinase selectivity over VEGFR2.
Fibroblast growth factor receptor 3
Vascular endothelial growth factor receptor 2
Kinase selectivity
Structure-based drug design
X-ray crystal structure
Structure activity relationships
1. Introduction
have been approved for the treatment of metastatic urothelial carci-
noma. Although several complete response cases have been observed in
Bladder cancer, a type of urothelial cancer, is the most common
malignancy involving the urinary system and the ninth most common
malignancy worldwide.1 Bladder cancer is histologically classified into
three major types: non-muscle invasive, muscle invasive and metastatic
bladder cancer. Although the 5-year survival rate of non-muscle in-
vasive bladder cancer (NMIBC) is over 70%, it recurs frequently or
progresses to muscle invasive bladder cancer. Furthermore, the treat-
ment outcome remains insufficient for muscle invasive and metastatic
bladder cancers, with a 5-year survival rate of less than 50%.2
clinical trials, their objective response rates were only 14–24%.4 Thus,
more effective and safer molecularly targeted therapies for bladder
cancer are warranted.
The signaling pathway activated by fibroblast growth factors (FGFs)
and their receptors, fibroblast growth factor receptors (FGFRs), is one of
the most important in the development from early embryogenesis to the
formation of various organs.5 The mammalian FGF/FGFR family com-
prises 18 ligands and 4 main receptors (FGFR1, 2, 3, and 4). All FGFRs
are single-transmembrane receptors and act as tyrosine kinases.5 FGFs
induce FGFR dimerization, followed by FGFR autophosphorylation and
activation of downstream signaling pathways such as PI3K-AKT and
Ras/Raf/MEK/MAPK.5 In a variety of human cancers, aberrant acti-
vation of FGF/FGFR signaling promotes cellular proliferation, migra-
tion/invasion and angiogenesis.5
Current therapies for NMIBC are transurethral resection of the
bladder tumor and intravesical postoperative Bacille de Calmette et
Guérin (BCG) therapy or chemotherapy, which are insufficiently ef-
fective for relapse prevention.3 The main therapies for muscle invasive
and metastatic bladder cancers are total bladder resection and systemic
administration of chemotherapeutic agents, although these treatments
are associated with low efficacy and side effects.3 Recently, immune
checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1 antibodies,
Recent studies have reported that FGFR3 harbors activating gene
mutations, mainly R248C, S249C, G372C, Y375C, and K652E, observed
in about 50% of bladder cancer cases.6,7 Further, the FGFR gene and
⁎ Corresponding author.
E-mail address: ikumi.kuriwaki@astellas.com (I. Kuriwaki).
1 Present address: Industrial Property Cooperation Center, Fukagawa Gatharia W3 Building, 1-2-15 Kiba, Koto-ku, Tokyo 135-0042, Japan.
2 Present address: Tsukuba Research Laboratories, Tokuyama Corporation, 40 Wadai, Tsukuba, Ibaraki 300-4247, Japan.
3 Present address: School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
4 Present address: Kishida Chemical Co., Ltd., 13-2 Ooaza-hoshinosato, Ami-machi, Inashiki-gun, Ibaraki 300-0326, Japan.
https://doi.org/10.1016/j.bmc.2020.115453
Received 9 February 2020; Received in revised form 18 March 2020; Accepted 20 March 2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:IkumiKuriwaki,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2020.115453

I. Kuriwaki, et al.
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Table 1
In vitro activity of compound 1.
Dovitinib (TKI258, CHIR-258)
Erdafitinib (JNJ-42756493)
Kinase
FGFR1
40
FGFR2
5.1
FGFR3
12
FGFR4
682
VEGFR2
266
Ratioa (fold)
22
IC50 (nM)
a: Ratio of IC50 value of VEGFR2 to FGFR3.
PRN1371
Infigratinib (NVP-BGJ398)
Fig. 1. Structure of erdafitinib (JNJ-42756493),12 dovitinib (TKI258),18 in-
figratinib (NVP-BGJ398),23 and PRN1371.24
transforming acidic coiled-coil (TACC) fusion gene (FGFR3-TACC3 and
FGFR3-BAIAP2L1) are reportedly expressed in a subset of bladder
cancer patients.8,9,10 Therefore, clinical trials of FGFRs inhibitors for
bladder cancer patients harboring FGFR3 point mutations or fusion
genes are currently being conducted.11 The clinical relevance of FGFR3-
targeted therapy has been suggested in reports on erdafitinib (JNJ-
42756493; Figure 1),12,13 which has received accelerated approval
from the U.S. Food and Drug Administration (FDA) for the treatment of
adults with locally advanced or metastatic urothelial carcinoma in
2019.14 Given these findings, FGFR3 is considered an attractive target
for novel therapies for bladder cancer.
N
N
HN
N
N
NH
O
O
S
NH
Me
MeO
In general, acquiring selective ATP-competitive kinase inhibitors is
challenging due to the high conservation of amino acid residues in the
kinase catalytic domain. Furthermore, low kinase selectivity tends to
cause side effects. Interestingly, amino acid sequences in the catalytic
domain of FGFR3 are similar to those of vascular endothelial growth
factor receptor 2 (VEGFR2).15 Therefore, classical FGFR inhibitors have
shown toxicity related to VEGFR2 inhibition.16,17 For example, dovi-
tinib (TKI258, CHIR-258; Fig. 1), a clinical candidate FGFR inhibitor,
inhibits both FGFR3 and VEGFR2 with IC50 values of 9 nM and 13 nM,
respectively (1-fold selectivity).18,19 In clinical trials, dovitinib causes
hypertension as a side effect, which is attributed to its VEGFR2 in-
hibitory activity.20,21 Erdafitinib also shows inhibitory activity against
both FGFR3 and VEGFR2 with IC50 values of 3 nM and 36.8 nM, re-
spectively (12-fold selectivity).22 Further, FGFR-selective inhibitors
such as infigratinib (NVP-BGJ398) and RPN-1371 are currently un-
dergoing clinical trials for the treatment of bladder cancer. These
compounds show 180- and 172-fold higher selectivity for FGFR3 over
VEGFR2 than erdafitinib, respectively.23,24
N
N
compound 1
Me
Fig. 2. X-ray crystal structure of 1 in complex with FGFR2 (PDB code: 6LVL).
as a surrogate. According to the X-ray crystal structure analysis of 1 in
complex with FGFR2, 1 was located in the ATP binding site (Fig. 2).
Among amino acid residues making up the ATP binding site, all re-
sidues were conserved between FGFR2 and FGFR3 except for Ser568
(corresponding to Ala559 in FGFR3), which was located outside the
pocket. Therefore, we predicted the binding modes of compounds with
FGFR3 based on the X-ray crystal structure using FGFR2. The 2-amino-
1,3,5-triazine moiety formed hydrogen bonds with the main chain
atoms of Ala567 (Ala558 in FGFR3) in the hinge region. In addition, the
phenylsulfonamide moiety also formed a hydrogen bond with the side
chain amino group of Lys517 (Lys508 in FGFR3). Based on the char-
acteristic interactions with Ala567 and Lys517, we concluded that
changing the 2-amino-1,3,5-triazine and phenylsulfonamide moieties
would not lead to optimization of the structure of 1. In contrast, the 3-
Therefore, we hypothesized that potent FGFR3 inhibitors without
VEGFR2 inhibitory activity would be promising drug candidates for the
treatment of bladder cancer, and would not cause serious side effects
such as hypertension. In this article, we describe the synthesis and
structure-activity relationships (SARs) of 1,3,5-triazine and pyrimidine
derivatives as potent and highly selective FGFR3 inhibitors.
methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl
moiety
was exposed to the solvent region and formed no interactions. Among
the amino acid residues located adjacent to the solvent region, the side
chain amino group of Lys485 (Lys476 in FGFR3) could act as a hy-
drogen bond donor. Thus, a compound that can interact with Lys476 is
expected to exhibit high inhibitory activity against FGFR3. Further, 1
did not interact with the hydrophobic residues in the back pocket, such
as Met538 (Met529 in FGFR3) and Val562 (Val553 in FGFR3). In
particular, Met529 in FGFR3 is not conserved in VEGFR2 (corre-
sponding to Leu889), suggesting that achievement of an interaction
with Met529 may enhance the inhibitory activity of FGFR3 and im-
prove selectivity over VEGFR2.
2. Design and chemistry
2.1. Structure-based drug design (SBDD)
Compound 1, which was identified as a FGFR3 inhibitor from our
high throughput screening (HTS) library, exhibited moderate inhibitory
activity against FGFR1, 2, and 3 with unsatisfactory selectivity over
VEGFR2 (Table 1). To obtain kinase inhibitors that were highly selec-
tive over VEGFR2, we utilized X-ray crystal structure data to obtain
insight on how to improve kinase selectivity.
We planned two synthetic strategies based on the X-ray crystal
structure analysis findings described above. First, to acquire an
To reveal the binding mode of 1, we used the FGFR2 kinase domain
2

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Fig. 3. (a) Illustration of representative kinase pockets and targeted amino acid residues with compound 1. (b) Structure of PD173074 with the representative kinase
pockets.25
interaction via a hydrogen bond with the Lys476 adjacent to the solvent
region, we introduced a hydrogen bond acceptor into the 3′-position of
the benzene ring at the right side of 1 (blue circle in Fig. 3a). We expect
that the 3′-methoxy moiety can easily rotate to the opposite side of the
benzene ring due to sufficient space around the 3′-position. Therefore,
we designed compounds based on further extension of the methoxy
moiety. Optimization of a linker was also important for adjusting the
distance from the 3′-position to the side chain of Lys476. Second, to
achieve a hydrophobic interaction with Met529 in the back pocket, we
identified the 1-position of 1,3,5-triazine as the most favorable site for
accessing the back pocket. Although the 1-position of 1,3,5-triazine
cannot be substituted, the 1,3,5-triazine scaffold can be replaced with a
pyrimidine because the nitrogen atom at the 1-position did not show
any interactions in the X-ray structural analysis (red circle in Fig. 3a).
To fill the back pocket, we introduced a 3,5-dimethoxyphenyl group, a
substructure of PD173074 (Fig. 3b), to the corresponding position of
the pyrimidine.25 PD173074 is a known classical FGFR inhibitor and is
capable of filling the back pocket according to X-ray structural ana-
lysis.25 In addition, careful optimization of a linker was also necessary
to identify steric differences between the side chain of Met529 in FGFR3
and Leu889 in VEGFR2.
2.2. Chemistry
Synthesis of the target compounds is outlined in Schemes 1–6. The
synthesis of compounds 4, 7, 8, 13a, and 13b is shown in Scheme 1.
Commercially available 2 was reacted with 326 to give 4 via a nu-
cleophilic aromatic substitution (SNAr) reaction under acidic condi-
Scheme 1. Reagents and conditions: (a) 3, metha-
nesulfonic acid, EtOH, 80 °C or 100 °C; (b) H2, Pd/C,
MeOH, room temperature; (c) 10% NaOH aq., EtOH,
80 °C; (d) 1-(tert-butoxycarbonyl)piperazine, K2CO3,
DMF, 80 °C; (e) 3, DIPEA, NMP, microwave (µW),
120 °C; (f) TFA, CH2Cl2, room temperature; (g) 37%
HCHO aq., NaBH(OAc)3, CH2Cl2, room temperature.
Scheme 2. Reagents and conditions: (a) morpholine, K2CO3, KI, MeCN, 60 °C; (b) N-methyl-piperazine, K2CO3, NMP or DMSO, 150 °C; (c) FeCl3 hexahydrate,
hydrazine monohydrate, activated carbon, EtOH/H2O, 80 °C or 100 °C; (d) 3, AcOH, room temperature.
3

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21
c, d
b
e
a
20
22
23
19
24
Scheme 3. Reagents and conditions: (a) 5-bromo-2,4-dichloropyrimidine, NaH, THF, 50 °C; (b) 21, DIPEA, NMP/IPA, µW, 150 °C; (c) 3,5-dimethoxyphenylboronic
acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane/H2O, 110 °C; (d) AcOH/H2O, 100 °C; (e) N-methyl-piperazine, NaBH(OAc)3, Et3N, CH2Cl2, room temperature.
Scheme 4. Reagents and conditions: (a) 26, DIPEA, NMP, 130 °C; (b) 1 M NaOH aq., THF/MeOH, room temperature; (c) N,O-dimethylhydroxylamine hydrochloride,
EDCI hydrochloride, HOBt, Et3N, DMF, room temperature; (d) 3,5-dimethoxyphenylmagnesium bromide, THF, −78 °C to room temperature; (e) m-CPBA, CH2Cl2,
room temperature; (f) 31, IPA, 90 °C; (g) NaBH4, THF/MeOH, room temperature; (h) triethylsilane, TFA, CH2Cl2, room temperature.
Scheme 5. Reagents and conditions: (a) 31 or 2, methanesulfonic acid, IPA, µW, 130°C; (b) 1-ethynyl-3,5-dimethoxybenzene, Pd(PPh3)4, CuI, Et3N, DMF, 120°C; (c)
H2, Pd/C, MeOH/THF, room temperature.
tions. Reduction of nitro compound 527 gave 6, which was converted to
7 by an SNAr reaction with 3. Hydrolysis of ester 7 led to 8. Introduc-
tion of a 1-(tert-butoxycarbonyl)piperazine unit to commercially avail-
able 9a and 9b gave 10a and 10b, which were subsequently trans-
formed to 11a and 11b via hydrogenation of the nitro moiety. SNAr
reaction of 11a and 11b with 3 gave 12a and 12b, which were con-
verted to 13a and 13b by deprotection of the tert-butoxycarbonyl (BOC)
moiety and subsequent methylation.
Scheme 2 shows the synthesis of compounds 18a–c. Compound 15a
was prepared by alkylation of commercially available 14 with
Scheme 6. Reagents and conditions: (a) 2,4-dichloro-5-nitropyrimidine, DMAc, room temperature; (b) 31, AcOH, room temperature; (c) H2, Pd/C, EtOH/THF, room
temperature; (d) 3,5-dimethoxybenzoyl chloride, DIPEA, THF, room temperature; (e) aryl carboxylic acid, HATU, DIPEA, DMF, room temperature.
4

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morpholine. Subsequent introduction of the N-methyl-piperazine unit
to 15a or commercially available 15b and 15c yielded 16a–c, which
was followed by reduction of the nitro moiety using hydrazine mono-
hydrate to give 17a–c. Corresponding aryl amines 17a–c were sub-
stituted with 3 to give 18a–c via an SNAr reaction.
complex between FGFR2 and 18b (Fig. 4). As expected, the oxygen
atom of the morpholine moiety of 18b formed a weak hydrogen bond
(3.1 Å) with the side chain amino group of Lys485 in FGFR2. Therefore,
the C2 linkers such as 7, 8, and 13a were thought to be too short to
reach the side chain amino group of Lys476. In addition, the results of
18a-c suggested that the presence and location of an oxygen atom were
important for the interaction with Lys485.
Compound 24 was synthesized according to Scheme 3. Aryl amine
1928 was reacted with 5-bromo-2,4-dichloropyrimidine to give 20,
which was subsequently converted to 22 by introduction of 21.29 Su-
zuki coupling reaction of 22 with 3,5-dimethoxyphenylboronic acid
gave the intermediate, and subsequent hydrolysis of the ketal group
gave 23. Compound 24 was obtained by reductive amination of 23 with
N-methyl-piperazine.
Unexpectedly, the methylene unit of the morpholine moiety of 18b
formed a hydrophobic interaction with the side chain of Met497 in
FGFR2 (Met488 in FGFR3), which corresponds to Glu848 in VEGFR2. In
the case of VEGFR2, such a hydrophobic interaction with the side chain
of Glu848 might be weaker because Glu848 is a hydrophilic amino acid.
These interactions with Lys485 and Met497 in FGFR2 by the morpho-
line moiety of 18b are expected to contribute to the potent inhibitory
activity against FGFR3 and improved selectivity over VEGFR2.
The synthesis of compound 34 is shown in Scheme 4. SNAr reaction
between commercially available 25 and 26 gave 27, which was sub-
sequently converted to 28 by hydrolysis of the ester group. The car-
boxylic acid moiety of 28 was transformed to a phenyl ketone using
Grignard reagent via Weinreb amide 29.30 The sulfide group of 30 was
oxidized using m-CPBA to give sulfoxide, which was substituted with
commercially available 31 to yield 32. Reduction of the ketone moiety
of 32 using NaBH4 gave 33, which was converted to 34 by deox-
ygenation with triethylsilane.
3.3. Results of targeting the interaction with Met529 in the back pocket
Table 3 shows the FGFR3 and VEGFR2 inhibitory activities of the
pyrimidine derivatives. Direct introduction of a 3,5-dimethoxyphenyl
ring to the 5-position of pyrimidine 24 increased the FGFR3 inhibitory
activity, with an IC50 value of 4.1 nM (ca. 3-fold more potent than 1).
However, the kinase selectivity of 24 did not improve. Next, we eval-
uated compounds with a linker between the 5-position of pyrimidine
and 3,5-dimethoxybenzene. Introduction of a methylene linker (34)
resulted in a reduction in FGFR3 inhibitory activity, whereas an ethy-
lene linker (37) retained potency. In addition, 37 showed improved
selectivity over VEGFR2, with a decrease in VEGFR2 inhibitory activity
at an IC50 value of 292 nM (ca. 8-fold less potent than 24). Furthermore,
replacement of ethylene with an amide linker (40a) resulted in a sig-
nificant reduction in VEGFR2 inhibitory activity (>233-fold se-
lectivity).
Scheme 5 shows the synthesis of compounds 35b and 37. Com-
pound 20 was introduced to 31 or 2 via an SNAr reaction to give 35a
and 35b. Compound 35a was coupled with 1-ethynyl-3,5-dimethox-
ybenzene by a Sonogashira reaction to give 36. The alkyne linkage of
36 was reduced to yield 37.
Compounds 40a–g were synthesized according to Scheme 6. Com-
pound 26 was reacted with 2,4-dichloro-5-nitropyrimidine by an SNAr
reaction to give 38, which was subsequently converted to 39a by in-
troduction of 31 under acidic condition. Reduction of the nitro group of
39a gave 39b, which was treated with 3,5-dimethoxybenzoyl chloride
or condensed with the corresponding aryl carboxylic acid to yield
40a–g.
Finally, we examined the effect of substituting the phenyl moiety.
Conversion of the 3,5-dimethoxyphenyl moiety, such as shifting the
position of the dimethoxy moiety (40b and 40c) and replacing it with
other substituents (40d and 40e), resulted in a reduction in FGFR3
inhibitory activity compared to 40a. In contrast, introduction of pyr-
imidine instead of benzene enhanced VEGFR2 inhibitory activity (40f
and 40g) compared to 40a. The 3,5-dimethoxyphenyl moiety was the
best substituent for obtaining both potency and selectivity among these
compounds.
3. Results and discussion
The synthesized compounds were evaluated using an ADP-Glo lu-
minescent kinase assay with human recombinant FGFR3 and VEGFR2
enzymes.
3.1. Results of targeting the interaction with Lys476 located adjacent to the
solvent region
3.4. Discussion of the adopted strategy based on structural information on
37
Table 2 shows the FGFR3 and VEGFR2 inhibitory activities of 1,3,5-
triazine derivatives. Although the amine moiety at the 4′-position of 4
was different from that of 1, it showed the same tendency for both
potency and selectivity (based on the results for 1 and 4). Therefore, the
N-methyl-piperazine unit was substituted in every evaluated compound
in this table. Introduction of various C2 units, such as an ester (7),
carboxylic acid (8), and ether (13a), did not result in any enhancement
of FGFR3 inhibitory activity compared to that of 4. Next, we evaluated
compounds with longer linker groups attached to a linear or cyclic ether
moiety. Although 13b did not show improved FGFR3 inhibitory ac-
tivity, 18b showed highly potent and specific FGFR3 inhibitory activity
with IC50 values of 4.1 nM (FGFR3) and 570 nM (VEGFR2), respectively
(139-fold selectivity). Interestingly, introduction of an N-methyl-pi-
perazine moiety (18c) instead of morpholine (18b), and propylene
linker (18a) instead of ethylene decreased FGFR3 inhibitory activity.
The X-ray structure of 37 with FGFR3 is shown in Fig. 5a. Similar to
1 and 18b, compound 37 formed hydrogen bonds with Ala558 and
Lys508 in FGFR3. As expected, the 3,5-dimethoxyphenyl moiety occu-
pied the back pocket and formed hydrophobic interactions. Specifically,
this substituent formed van der Waals (vdW) interactions. We predicted
that 24 did not improve the selectivity over VEGFR2 because, while the
3,5-dimethoxybenzene of 24 located in the back pocket, it was unable
to reach the side chain of Met529 in FGFR3. Further, compound 35b (5-
bromopyrimidine derivative, structure shown in Scheme 5) also ex-
hibited low kinase selectivity with IC50 values of 1.8 nM (FGFR3) and
6.6 nM (VEGFR2). In addition, studies have reported that a 5-bromo-
pyrimidine scaffold like that of 35b often shows multi-kinase inhibi-
tion.31,32 Our results suggested that the 3,5-dimethoxybenzene of 24
had a similar effect to that of a bromide moiety.
3.2. Discussion of the adapted strategy based on structural information on
18b
Fig. 5b shows the detailed structure of the back pocket in compar-
ison with VEGFR2 (PDB code 4AG8).31 The 3,5-dimethoxyphenyl
moiety of 37 formed vdW interactions with the Sδ and Cε atoms of
Met529 in FGFR3. In contrast, this substituent formed vdW interactions
with only the Cδ atom of Leu889 in VEGFR2. These interactions in the
back pocket, especially with Met529, are thought to be responsible for
maintaining the inhibitory activity against FGFR3 and improving the
To confirm whether 18b would undergo the expected interactions,
we performed X-ray structural analysis. FGFR2 was again used as a
surrogate. Similar to 1, compound 18b formed hydrogen bonds with
Ala567 and Lys517 in FGFR2, according to the crystal structure of the
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Table 2
SARs of 1,3,5-triazine derivatives.
Compound
R
Enzyme IC50 (nM)
Ratioa (fold)
FGFR3
VEGFR2
N
N
4
-Me
15
62
65
17
21
341
316
120
310
550
23
5
7
–CH2CO2Et
–CH2CO2H
–CH2CH2OMe
HN
N
NH
O
O
S
8
2
NH
Me
13a
13b
18
O
26
N
N
18b
4.1
570
139
23
N
O
Me compound 18b
18cb
15
341
Fig. 4. X-ray crystal structure of 18b in complex with FGFR2 (PDB code: 6LVK).
1 is superimposed and shown as a gray stick model in gray.
18a
63
736
12
Varian 400-MR, JEOL Lambda, or JEOL EX-400, and chemical shifts are
expressed in δ (ppm) values with tetramethylsilane as an internal re-
ference (s = singlet, d = doublet, t = triplet, q = quartet, m = mul-
tiplet, dd = doublet of doublets, qd = quartet of doublets, and
br = broad peak). Mass spectra (MS) were recorded on Thermo
Electron LCQ Advantage, Thermo Electron TSQ 700, Waters ZQ2000,
Waters SQD, or Agilent Quadrupole. Electrospray ionization (ESI) po-
sitive 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 (C, H, N), DIONEX DX-500 (S, halogen), DIONEX
ICS-2000 (S, halogen), or DIONEX ICS-3000 (S, halogen) instruments,
and the results were within 0.3% of theoretical values. The following
abbreviations are used: AcOH, acetic acid; MeCN, acetonitrile; HATU,
a: Ratio of IC50 value of VEGFR2 to FGFR3.
b: Tetrahydrochloride salt.
selectivity over VEGFR2. The kinase selectivity of 40a was greater than
that of 37, suggesting that the amide linker was more rigid and slightly
shorter than the ethylene linker. Such differences were thought to re-
strict 40a from being positioned close to the side chain of Leu889 and
weaken the interaction between the 3,5-dimethoxyphenyl moiety and
Leu889, and contribute to the high selectivity of 40a over VEGFR2.
Subsequently, 40f and 40g showed potent VEFGR2 inhibitory activity
compared to 40a possibly because the two nitrogen atoms enhanced the
vdW interaction between the dimethoxy moiety and Leu889.
3.5. Further pharmacological evaluations of 40a
O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexa-
fluorophosphate; m-CPBA, 3-chloroperoxybenzoic acid; Et2O, diethyl
ether; IPE, diisopropyl ether; DIPEA, N,N-diisopropylethylamine;
DMSO, dimethyl sulfoxide; DMAc, N,N-dimethylacetamide; DMF, N,N-
dimethylformamide; EtOH, ethanol; EtOAc, ethyl acetate; EDCI, 1-
Given the promising features of 40a including potent FGFR3 in-
hibitory activity and selectivity over VEGFR2, we further examined this
compound in a pharmacological study. Compound 40a showed pan-
FGFR inhibitory activity with IC50 values of 2.1 nM (FGFR1), 3.1 nM
(FGFR2), and 74 nM (FGFR4). In addition, this compound suppressed
proliferation of a UM-UC-14 human bladder cancer cell line, which
expresses a FGFR3 S249C point mutation, with an IC50 value of 84 nM.
ethyl-3-(3-dimethylaminopropyl)carbodiimide;
HOBt,
1-hydro-
xybenzotriazole monohydrate; IPA, isopropyl alcohol; MeOH, me-
thanol; NMP, 1-methyl-2-pyrrolidone; NaBH(OAc)3, sodium triacetox-
yborohydride; THF, tetrahydrofuran; Et3N, triethylamine; TFA,
trifluoroacetic acid; PPh3, triphenylphosphine.
4. Conclusion
5.1.1. N-Ethyl-2-({4-[3-methoxy-4-(4-methylpiperazin-1-yl)anilino]-
1,3,5-triazin-2-yl}amino)benzene-1-sulfonamide (4)
We conducted structural optimization of compound 1, identified in
a HTS, to explore novel FGFR3 inhibitors with decreased activity
against VEGFR2 via an SBDD approach. Following X-ray crystal struc-
ture analysis, we performed studies based on the Lys476 in the solvent
region and Met529 in the back pocket of FGFR3. Structural optimiza-
tion led to identification of 1,3,5-triazine derivative 18b and pyr-
imidine derivative 40a, which showed potent FGFR3 inhibitory activity
with high selectivity over VEGFR2. In particular, 40a is more attractive
lead compound because of the significant reduction in VEGFR2 in-
hibitory activity. Further optimization studies of 40a will be reported in
due course.
To a solution of 3-methoxy-4-(4-methylpiperazin-1-yl)aniline (2,
120 mg, 0.54 mmol) in EtOH (3.0 mL) was added methanesulfonic acid
(70 µL, 1.08 mmol). The mixture was stirred at room temperature for
15 min, and 2-[(4-chloro-1,3,5-triazin-2-yl)amino]-N-ethylbenzene-1-
sulfonamide (3, 204 mg, 0.65 mmol) was added. After stirring at 80 °C
for 2 h, the mixture was neutralized with sat. NaHCO3 aq. and then
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 (CHCl3/MeOH/28% NH3
aq. = 100:0:0 to 100:10:1) to give the product (102 mg, 38%) as a
beige solid. 1H NMR (CDCl3): δ 1.04 (3H, t, J = 7.3 Hz), 2.37 (3H, s),
2.54–2.73 (4H, m), 2.99 (2H, q, J = 7.3 Hz), 3.03–3.17 (4H, m), 3.84
(3H, br s), 4.79–4.99 (1H, m), 6.85–6.93 (1H, m), 6.93–7.01 (1H, m),
7.15–7.23 (1H, m), 7.28–7.43 (1H, m), 7.49–7.63 (1H, m), 7.93 (1H,
dd, J = 7.8, 1.5 Hz), 8.37 (1H, br s), 8.45 (1H, d, J = 7.8 Hz),
8.74–9.07 (1H, m); MS (ESI) m/z [M+H]+ 499; Anal. Calcd for
5. Experimental
5.1. Chemistry
1H-NMR spectra were recorded on Varian 400, Varian VNS-400,
6

I. Kuriwaki, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
(a)
Table 3
SARs of pyrimidine derivatives.
Compound
R
Enzyme IC50 (nM)
Ratioa (fold)
9
OMe
FGFR3
4.1
VEGFR2
N
OMe
24
38
HN
N
N
NH
O
O
S
NH
Me
OMe
34
37
>1000
2.8
>1000
292
NCb
104
N
N
compound 37
Me
40a
40b
4.3
54
>1000
435
>233
8
(b)
40c
40d
40
35
461
715
12
20
40e
40f
40g
275
1.8
34
554
131
227
2
73
7
Fig. 5. (a) X-ray crystal structure of 37 in complex with FGFR3 (PDB code:
6LVM). (b) The same as (a) with focus on the back pocket. Superimposed
structure of FGFR3 and VEGFR2 (PDB code: 4AG8).33 Amino acid residues
making up the back pocket are shown using a cyan and orange stick model for
FGFR3 and VEGFR2, respectively. Residue names of VEGFR2 are shown in
parentheses.
a: Ratio of IC50 value of VEGFR2 to FGFR3.
b: Not calculated.
C23H30N8O3S.0.3CHCl3·0.6H2O: C, 51.33; H, 5.82; N, 20.55; S, 5.88; Cl,
5.85. Found: C, 51.37; H, 5.73; N, 20.51; S, 5.82; Cl, 5.62.
After stirring at 100 °C for 3 h, the mixture was neutralized with sat.
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 = 50:1 to 20:1) to give the product (141 mg, 49%) as a yellow
solid. 1H NMR (CDCl3): δ 1.05 (3H, t, J = 7.2 Hz), 1.28 (3H, t,
J = 7.2 Hz), 2.37 (3H, s), 2.50–2.75 (4H, m), 2.98 (2H, q, J = 7.2 Hz),
3.01–3.28 (4H, m), 4.26 (2H, q, J = 7.2 Hz), 4.66 (2H, br s), 5.54 (1H,
br s), 6.88–6.95 (2H, m), 7.17–7.21 (1H, m), 7.26–7.72 (3H, m), 7.94
(1H, dd, J = 8.0, 1.6 Hz), 8.33 (1H, br s), 8.42 (1H, d, J = 8.4 Hz), 8.92
5.1.2. Ethyl [5-amino-2-(4-methylpiperazin-1-yl)phenoxy]acetate (6)
To a solution of ethyl [2-(4-methylpiperazin-1-yl)-5-nitrophenoxy]
acetate (5, 275 mg, 0.85 mmol) in MeOH (3 mL) was added 5% Pd/C
(50% wet, 30 mg) under an argon atmosphere. After stirring at room
temperature under a hydrogen atmosphere for 7 h, the mixture was
passed through a Celite pad and washed with MeOH. The filtrate was
concentrated in vacuo. The residue was purified by column chromato-
graphy on silica gel (CHCl3/MeOH = 25:1 to 10:1) to give the product
(180 mg, 72%) as a brown oil. 1H NMR (CD3OD): δ 1.29 (3H, t,
J = 7.2 Hz), 2.66 (3H, s), 2.92–3.30 (8H, m), 4.24 (2H, q, J = 7.2 Hz),
4.68 (2H, s), 6.32–6.34 (2H, m), 6.80 (1H, d, J = 9.2 Hz); MS (ESI) m/z
[M+H]+ 294.
(1H, br s); HRMS (ESI) m/z Calcd for
C
26H35N8O5S [M+H]+
:
571.2446. Found: 571.2444.
5.1.4. [5-({4-[2-(Ethylsulfamoyl)anilino]-1,3,5-triazin-2-yl}amino)-2-(4-
5.1.3. Ethyl [5-({4-[2-(ethylsulfamoyl)anilino]-1,3,5-triazin-2-yl}amino)-
2-(4-methylpiperazin-1-yl)phenoxy]acetate (7)
methylpiperazin-1-yl)phenoxy]acetic acid (8)
To a solution of 7 (116 mg, 0.20 mmol) in EtOH (1 mL) was added
10% NaOH aq. (0.3 mL). After stirring at 80 °C for 1 h, the mixture was
concentrated in vacuo. The residue was diluted with H2O, and 10% HCl
aq. was added to the mixture. The resulting precipitate was filtered and
To a solution of 6 (180 mg, 0.61 mmol) in EtOH (4 mL) was added
methanesulfonic acid (66 µL, 1.02 mmol). The mixture was stirred at
room temperature for 20 min, and 3 (160 mg, 0.51 mmol) was added.
7

I. Kuriwaki, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
dried to give the product (46 mg, 42%) as a pale yellow solid. 1H NMR
(CD3OD): δ 1.00 (3H, t, J = 7.2 Hz), 2.89–3.33 (9H, m), 3.51–3.76 (4H,
m), 4.60 (2H, br s), 6.97 (1H, d, J = 8.8 Hz), 7.17–7.25 (2H, m),
7.35–7.45 (1H, m), 7.51–7.63 (1H, m), 7.90 (1H, dd, J = 8.0, 1.6 Hz),
similar approach to that described for 12a. 1H NMR (CDCl3): δ 1.04
(3H, t, J = 7.6 Hz), 1.49 (9H, s), 2.92–3.07 (6H, m), 3.37 (3H, s),
3.51–3.63 (6H, m), 3.69–3.71 (2H, m), 3.83–3.92 (2H, m), 4.07–4.23
(2H, m), 4.97 (1H, br s), 6.83–7.03 (2H, m), 7.17–7.21 (1H, m),
7.49–7.62 (1H, m), 7.93 (1H, dd, J = 8.0, 1.6 Hz), 8.38 (1H, br s), 8.45
(1H, d, J = 8.0 Hz), 8.93 (1H, br s); MS (ESI) m/z [M+H]+ 673.
8.33–8.54 (2H, m); HRMS (ESI) m/z Calcd for C24H31N8O5S [M+H]+
:
543.2133. Found: 543.2138.
5.1.5. tert-Butyl
4-[2-(2-methoxyethoxy)-4-nitrophenyl]piperazine-1-
5.1.11. N-Ethyl-2-({4-[3-(2-methoxyethoxy)-4-(4-methylpiperazin-1-yl)
anilino]-1,3,5-triazin-2-yl}amino)benzene-1-sulfonamide (13a)
carboxylate (10a)
To a solution of 1-chloro-2-(2-methoxyethoxy)-4-nitrobenzene (9a,
4.229 g, 18.26 mmol) in DMF (40 mL) were added 1-(tert-butox-
ycarbonyl)piperazine (3.400 g, 18.26 mmol) and K2CO3 (5.047 g,
36.52 mmol). After stirring at 80 °C for 48 h, the mixture was diluted
with H2O and extracted with EtOAc. The organic layer was washed with
H2O followed by brine, dried over Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography on silica gel (n-
hexane/EtOAc = 9:1 to 5:1) to give the product (1.560 g, 22%) as a
yellow oil. 1H NMR (CDCl3): δ 1.49 (9H, s), 3.15–3.25 (4H, m), 3.45
(3H, s), 3.54–3.64 (4H, m), 3.79–3.81 (2H, m), 4.21–4.23 (2H, m), 6.85
(1H, d, J = 8.8 Hz), 7.71 (1H, d, J = 2.4 Hz), 7.86 (1H, dd, J = 8.8,
2.8 Hz); MS (ESI) m/z [M+H]+ 382.
To a solution of 12a (120 mg, 0.19 mmol) in CH2Cl2 (2 mL) was
added TFA (1 mL). After stirring at room temperature for 1 h, the
mixture was concentrated in vacuo. The residue was diluted with sat.
NaHCO3 aq, and extracted with CHCl3/MeOH. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (CHCl3/40% CH3NH2 MeOH
sol. = 100:0 to 60:1) to give the product (80 mg, 79%) as a gray solid.
To a solution of the obtained intermediate (65 mg, 0.12 mmol) in
CH2Cl2 (1.5 mL) was added 37% HCHO aq. (92 µL, 1.23 mmol). The
mixture was stirred at room temperature for 1 h, and NaBH(OAc)3
(51 mg, 0.24 mmol) was added. After stirring at room temperature for
6 h, the mixture was neutralized with sat. NaHCO3 aq. and extracted
with CH2Cl2. The organic layer was dried over Na2SO4 and con-
centrated in vacuo. The residue was purified by column chromato-
graphy on silica gel (CHCl3/MeOH = 40:1 to 15:1) to give the product
(35 mg, 53%) as a pale yellow solid. 1H NMR (CDCl3): δ 1.03 (3H, t,
J = 7.6 Hz), 2.37 (3H, s), 2.52–2.74 (4H, m), 2.97 (2H, q, J = 7.2 Hz),
3.00–3.22 (4H, m), 3.44 (3H, s), 3.72–3.83 (2H, m), 4.02–4.20 (2H, m),
5.36 (1H, br s), 6.85–7.01 (2H, m), 7.16–7.20 (1H, m), 7.20–7.78 (3H,
m), 7.93 (1H, dd, J = 8.0, 1.2 Hz), 8.33 (1H, br s), 8.44 (1H, d,
J = 8.4 Hz), 8.93 (1H, br s); HRMS (ESI) m/z Calcd for C25H35N8O4S
[M+H]+: 543.2496. Found: 543.2484.
5.1.6. tert-Butyl
4-{2-[2-(2-methoxyethoxy)ethoxy]-4-nitrophenyl}
piperazine-1-carboxylate (10b)
Compound 10b was prepared from 1-chloro-2-[2-(2-methox-
yethoxy)ethoxy]-4-nitrobenzene (9b) in 15% yield using a similar ap-
proach to that described for 10a. 1H NMR (CDCl3): δ 1.49 (9H, s),
3.14–3.28 (4H, m), 3.38 (3H, s), 3.55–3.61 (6H, m), 3.69–3.71 (2H, m),
3.89–3.91 (2H, m), 4.23–4.26 (2H, m), 6.85 (1H, d, J = 8.8 Hz), 7.71
(1H, d, J = 2.8 Hz), 7.85 (1H, dd, J = 8.8, 2.4 Hz); MS (ESI) m/z [M
+H]+ 426.
5.1.7. tert-Butyl
4-[4-amino-2-(2-methoxyethoxy)phenyl]piperazine-1-
5.1.12. N-Ethyl-2-[(4-{3-[2-(2-methoxyethoxy)ethoxy]-4-(4-
methylpiperazin-1-yl)anilino}-1,3,5-triazin-2-yl)amino]benzene-1-
sulfonamide (13b)
carboxylate (11a)
Compound 11a was prepared from 10a in 98% yield using a similar
approach to that described for 6. 1H NMR (CDCl3): δ 1.48 (9H, s), 3.41
(3H, s), 3.53–3.76 (6H, m), 3.94–4.10 (4H, m), 4.14–4.17 (2H, m),
6.28–6.31 (2H, m), 7.94–8.11 (1H, m); MS (ESI) m/z [M+H]+ 352.
Compound 13b was prepared from 12b in 2 steps in 55% yield
using a similar approach to that described for 13a.
1H NMR (CDCl3): δ 1.03 (3H, t, J = 7.2 Hz), 2.37 (3H, s), 2.52–2.72
(4H, m), 2.97 (2H, q, J = 7.2 Hz), 3.00–3.23 (4H, m), 3.36 (3H, s),
3.54–3.56 (2H, m), 3.69–3.72 (2H, m), 3.83–3.92 (2H, m), 4.04–4.21
(2H, m), 5.30 (1H, br s), 6.85–7.02 (2H, m), 7.16–7.20 (1H, m),
7.26–7.70 (3H, m), 7.93 (1H, dd, J = 7.6, 2.0 Hz), 8.35 (1H, br s), 8.43
(1H, d, J = 8.4 Hz), 8.93 (1H, br s); HRMS (ESI) m/z Calcd for
5.1.8. tert-Butyl
4-{4-amino-2-[2-(2-methoxyethoxy)ethoxy]phenyl}
piperazine-1-carboxylate (11b)
Compound 11b was prepared from 10b in 74% yield using a similar
approach to that described for 6. 1H NMR (CDCl3): δ 1.48 (9H, s), 3.36
(3H, s), 3.36–3.80 (8H, m), 3.84–3.86 (2H, m), 3.98–4.15 (4H, m),
4.18–4.21 (2H, m), 6.32–6.34 (2H, m), 7.98–8.21 (1H, m); MS (ESI) m/
z [M+H]+ 396.
C
27H39N8O5S [M+H]+: 587.2759. Found: 587.2754.
5.1.13. 4-[3-(2-Chloro-5-nitrophenoxy)propyl]morpholine (15a)
To a solution of 2-(3-bromopropoxy)-1-chloro-4-nitrobenzene (14,
1.0 g, 3.40 mmol) in MeCN (20 mL) were added morpholine (355 µL,
4.07 mmol), K2CO3 (562 mg, 4.07 mmol) and KI (113 mg, 0.68 mmol).
After stirring at 60 °C for 15 h, the mixture was diluted with H2O 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 = 100:0 to 95:5)
to give the product (646 mg, 63%) as a yellow solid. 1H NMR (CDCl3): δ
2.03–2.11 (2H, m), 2.43–2.52 (4H, m), 2.57 (2H, t, J = 7.0 Hz),
3.66–3.79 (4H, m), 4.21 (2H, t, J = 6.3 Hz), 7.39–7.63 (1H, m),
7.75–7.83 (2H, m); MS (ESI) m/z [M+H]+ 301.
5.1.9. tert-Butyl 4-[4-({4-[2-(ethylsulfamoyl)anilino]-1,3,5-triazin-2-yl}
amino)-2-(2-methoxyethoxy)phenyl]piperazine-1-carboxylate (12a)
To a solution of 11a (187 mg, 0.53 mmol) and 3 (200 mg,
0.64 mmol) in NMP (2 mL) was added DIPEA (180 µL, 1.06 mmol).
After stirring at 120 °C under microwave irradiation for 20 min, the
mixture was diluted with H2O. The resulting precipitate was filtered
and dried. The obtained solid was purified by column chromatography
on silica gel (n-hexane/EtOAc = 2:1 to 1:2) to give the product
(120 mg, 36%) as a brown solid. 1H NMR (CDCl3): δ 1.04 (3H, t,
J = 7.2 Hz), 1.49 (9H, s), 2.89–3.08 (6H, m), 3.44 (3H, s), 3.52–3.66
(4H, m), 3.72–3.82 (2H, m), 4.02–4.26 (2H, m), 5.26 (1H, br s), 6.82
(1H, d, J = 8.0 Hz), 6.83–7.03 (1H, m), 7.17–7.21 (1H, m), 7.26–7.74
(3H, m), 7.93 (1H, dd, J = 7.6, 2.0 Hz), 8.33 (1H, br s), 8.43 (1H, d,
J = 8.4 Hz), 8.93 (1H, br s); MS (ESI) m/z [M+H]+ 629.
5.1.14. 4-{3-[2-(4-Methylpiperazin-1-yl)-5-nitrophenoxy]propyl}
morpholine (16a)
To a solution of 15a (400 mg, 1.33 mmol) in DMSO (3.3 mL) were
added N-methyl-piperazine (293 µL, 2.66 mmol) and K2CO3 (368 mg,
2.66 mmol). After stirring at 150 °C overnight, the mixture was diluted
with H2O and extracted with CHCl3. The organic layer was dried over
MgSO4 and concentrated in vacuo. The residue was diluted with CHCl3.
The organic layer was washed with H2O and concentrated in vacuo to
5.1.10. tert-Butyl 4-{4-({4-[2-(ethylsulfamoyl)anilino]-1,3,5-triazin-2-yl}
amino)-2-[2-(2-methoxyethoxy)ethoxy]phenyl}piperazine-1-carboxylate
(12b)
Compound 12b was prepared from 11b and 3 in 44% yield using a
8

I. Kuriwaki, et al.
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7.20–7.31 (1H, m), 7.34–7.51 (1H, m), 7.52–7.69 (1H, m), 7.81 (1H,
dd, J = 7.9, 1.3 Hz), 7.87–8.05 (1H, m), 8.32–8.54 (2H, m), 9.02–9.28
(1H, m), 9.70–9.91 (1H, m); MS (ESI) m/z [M+H]+ 612; HRMS (ESI)
m/z Calcd for C29H42N9O4S [M+H]+: 612.3075. Found: 612.3076.
give the product (200 mg, 41%). MS (ESI) m/z [M+H]+ 365.
5.1.15. 4-{2-[2-(4-Methylpiperazin-1-yl)-5-nitrophenoxy]ethyl}
morpholine (16b)
To a solution of 4-[2-(2-chloro-5-nitrophenoxy)ethyl]morpholine
(15b, 731 mg, 2.42 mmol) in NMP (10 mL) were added N-methyl-pi-
perazine (798 µL, 7.25 mmol) and K2CO3 (1.00 g, 7.25 mmol). After
stirring at 150 °C for 6 h, the mixture was diluted with H2O and ex-
tracted with EtOAc. The organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified by
column chromatography on amino functionalized silica gel (n-hexane/
CHCl3 = 70:30 to 0:100) to give the product (465 mg, 55%) as a yellow
oil. 1H NMR (CDCl3): δ 2.35 (3H, s), 2.45–2.68 (8H, m), 2.80–2.90 (2H,
m), 3.24–3.51 (4H, m), 3.53–3.81 (4H, m), 4.10–4.32 (2H, m), 6.88
(1H, d, J = 8.8 Hz), 7.71 (1H, d, J = 2.4 Hz), 7.86 (1H, dd, J = 8.8,
2.5 Hz); MS (ESI) m/z [M+H]+ 351.
5.1.21. N-Ethyl-2-[(4-{4-(4-methylpiperazin-1-yl)-3-[2-(morpholin-4-yl)
ethoxy]anilino}-1,3,5-triazin-2-yl)amino]benzene-1-sulfonamid (18b)
Compound 18b was prepared from 17b and 3 in 15% yield using a
similar approach to that described for 18a. 1H NMR (DMSO-d6): δ 0.95
(3H, t, J = 7.2 Hz), 2.21 (3H, s), 2.36–2.53 (8H, m), 2.62–2.74 (2H, m),
2.83 (2H, q, J = 7.2 Hz), 2.90–3.03 (4H, m), 3.49–3.60 (4H, m),
3.88–4.06 (2H, m), 6.80 (1H, d, J = 8.6 Hz), 6.94–7.13 (1H, m),
7.19–7.29 (1H, m), 7.29–7.49 (1H, m), 7.50–7.69 (1H, m), 7.81 (1H,
dd, J = 7.9, 1.5 Hz), 7.84–8.06 (1H, m), 8.28–8.70 (2H, m), 9.17 (1H,
br s), 9.82 (1H, br s); MS (ESI) m/z [M+H]+ 598; Anal. Calcd for
C28H39N9O4S.0.2IPE·1.2H2O: C, 54.82; H, 6.96; N, 19.70; S, 5.01.
Found: C, 54.78; H, 6.83; N, 19.73; S, 5.06.
5.1.16. 1-Methyl-4-{2-[2-(4-methylpiperazin-1-yl)ethoxy]-4-nitrophenyl}
piperazine (16c)
5.1.22. N-Ethyl-2-[(4-{4-(4-methylpiperazin-1-yl)-3-[2-(4-
Compound 16c was prepared from 1-[2-(2-chloro-5-nitrophenoxy)
ethyl]-4-methylpiperazine (15c) in 43% yield using a similar approach
to that described for 16b. 1H NMR (CDCl3): δ 2.30 (3H, s), 2.36 (3H, s),
2.39–2.72 (12H, m), 2.86 (2H, t, J = 5.6 Hz), 3.22–3.36 (4H, m), 4.19
(2H, t, J = 5.6 Hz), 6.87 (1H, d, J = 9.0 Hz), 7.70 (1H, d, J = 2.5 Hz),
7.85 (1H, dd, J = 8.9, 2.5 Hz); MS (ESI) m/z [M+H]+ 364.
methylpiperazin-1-yl)ethoxy]anilino}-1,3,5-triazin-2-yl)amino]benzene-1-
sulfonamide tetrahydrochloride (18c)
The free form of 18c was prepared from 17c (157 mg, 0.47 mmol)
and 3 (148 mg, 0.47 mmol) using a similar approach to that described
for 18a. To the free form of 18c was added HCl (4.0 M 1,4-dioxane
solution, 1.5 mL, 6.0 mmol). After stirring at room temperature over-
night, the mixture was concentrated in vacuo. IPE/EtOAc was added to
the residue, and the resulting precipitate was filtered and dried to give
the product (112 mg, 31%) as a yellow solid. 1H NMR (DMSO-d6): δ
0.95 (3H, t, J = 7.2 Hz), 2.76–2.92 (5H, m), 2.97–3.13 (2H, m),
3.18–3.33 (2H, m), 3.34–3.55 (4H, m), 3.56–3.89 (6H, m), 3.96–4.62
(9H, m), 6.88–7.01 (1H, m), 7.18–7.52 (3H, m), 7.61–7.73 (1H, m),
7.84 (1H, dd, J = 8.0, 1.2 Hz), 7.91–8.02 (1H, m), 8.16–8.60 (2H, m),
9.16–9.66 (1H, m), 9.77–10.41 (1H, m), 10.93 (1H, br s), 12.05 (1H, br
5.1.17. 4-(4-Methylpiperazin-1-yl)-3-[3-(morpholin-4-yl)propoxy]aniline
(17a)
To a solution of 16a (200 mg, 0.55 mmol) in EtOH (3.1 mL)/H2O
(0.77 mL) were added FeCl3 hexahydrate (45 mg, 0.16 mmol), acti-
vated carbon (46 mg) and hydrazine monohydrate (266 µL,
5.49 mmol). After stirring at 100 °C for 18 h, the mixture was passed
through a Celite pad and washed with EtOAc. The filtrate was dried
over Na2SO4 and concentrated to give the product (170 mg, 93%) as a
yellow solid. MS (ESI) m/z [M+H]+ 335.
s);
MS
(ESI)
m/z
[M+H]+
611;
Anal.
Calcd
for
C29H42N10O3S.4.3HCl·4.5H2O: C, 41.04; H, 6.57; N, 16.51; S, 3.78; Cl,
17.96. Found: C, 41.20; H, 6.71; N, 16.31; S, 3.60; Cl, 17.86.
5.1.18. 4-(4-Methylpiperazin-1-yl)-3-[2-(morpholin-4-yl)ethoxy]aniline
(17b)
5.1.23. tert-Butyl {2-[(5-bromo-2-chloropyrimidin-4-yl)amino]benzene-1-
sulfonyl}ethylcarbamate (20)
Compound 17b was prepared from 16b in 100% yield using a si-
milar approach to that described for 17a. 1H NMR (CDCl3): δ 2.35 (3H
s), 2.44–2.70 (8H m), 2.77–2.88 (4H, m), 2.95–3.11 (4H, m), 3.66–3.77
(4H, m), 4.03–4.11 (2H, m), 6.18–6.32 (2H, m), 6.75–6.79 (1H, m); MS
(ESI) m/z [M+H]+ 321.
To a solution of tert-butyl (2-aminobenzene-1-sulfonyl)ethylcarba-
mate (19, 15.0 g, 49.9 mmol) in THF (120 mL) was added NaH (13.7 g,
59.9 mmol, 55% oil dispersion) in an ice-water bath. After stirring at
room temperature for 1 h, 5-bromo-2,4-dichloropyrimidine was added
to the mixture. After stirring at 50 °C for 12 h, the mixture was diluted
with H2O and extracted with EtOAc. The organic layer was washed with
brine, dried over Na2SO4 and concentrated in vacuo. The residue was
purified twice by column chromatography on silica gel (n-hexane/
EtOAc = 95:5 to 50:50) to give the product (6.97 g, 28%) as a pale
yellow solid. 1H NMR (CDCl3): δ 1.30–1.40 (12H, m), 3.89 (2H, q,
J = 7.0 Hz), 7.24–7.31 (1H, m), 7.61–7.69 (1H, m), 7.88 (1H, dd,
J = 8.2, 1.6 Hz), 8.38 (1H, s), 8.42 (1H, dd, J = 8.4, 1.0 Hz), 9.59 (1H,
s); MS (ESI) m/z [M+H]+ 491.
5.1.19. 4-(4-Methylpiperazin-1-yl)-3-[2-(4-methylpiperazin-1-yl)ethoxy]
aniline (17c)
Compound 17c was prepared from 16c in 100% yield using a si-
milar approach to that described for 17a with the reaction temperature
changed to 80 °C. 1H NMR (CDCl3): δ 2.32–2.39 (3H, m), 2.47 (3H, s),
2.52–2.88 (14H, m), 3.03–3.19 (4H, m), 3.39–3.58 (2H, m), 4.04–4.11
(2H, m), 6.16–6.32 (2H, m), 6.74–6.80 (1H, m); MS (ESI) m/z [M+H]+
334.
5.1.20. N-Ethyl-2-[(4-{4-(4-methylpiperazin-1-yl)-3-[3-(morpholin-4-yl)
propoxy]anilino}-1,3,5-triazin-2-yl)amino]benzene-1-sulfonamide (18a)
A mixture of 17a (170 mg, 0.51 mmol) and AcOH (8.8 mL) was
stirred at room temperature for 30 min before 3 (159 mg, 0.51 mmol)
was added. After stirring at room temperature overnight, the mixture
was concentrated in vacuo. The residue was diluted with sat. NaHCO3
aq., and the mixture was 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 90:10) to give the product (87 mg, 28%). 1H NMR
(DMSO-d6): δ 0.87–1.03 (3H, m), 1.77–1.96 (2H, m), 2.22 (3H, s),
2.28–2.50 (10H, m), 2.71–2.87 (2H, m), 2.87–3.03 (4H, m), 3.47–3.63
(4H, m), 3.77–4.10 (2H, m), 6.72–6.85 (1H, m), 6.95–7.15 (1H, m),
5.1.24. 2-({5-Bromo-2-[4-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)-3-
methoxyanilino]pyrimidin-4-yl}amino)-N-ethylbenzene-1-sulfonamide (22)
To a solution of 20 (1.45 g, 2.95 mmol) in NMP/IPA (16 mL, 1:1)
were added DIPEA (1.26 mL, 7.38 mmol) and 4-(1,4-dioxa-8-azaspiro
[4.5]decan-8-yl)-3-methoxyaniline (21, 1.01 g, 3.84 mmol). After stir-
ring at 150 °C under microwave irradiation for 14 h, the mixture was
diluted with H2O 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 (n-
hexane/EtOAc = 90:10 to 0:100) to give the product (964 mg, 53%) as
a colorless solid. 1H NMR (CDCl3): δ 0.99–1.09 (3H, m), 1.85–1.99 (4H,
m), 2.95–3.06 (2H, m), 3.06–3.17 (4H, m), 3.73 (3H, s), 3.97–4.05 (4H,
m), 4.48 (1H, t, J = 6.0 Hz), 6.81–6.93 (2H, m), 6.95–7.11 (2H, m),
9

I. Kuriwaki, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
7.16–7.25 (1H, m), 7.48–7.56 (1H, m), 7.96 (1H, d, J = 8.0 Hz),
8.14–8.27 (1H, m), 8.41 (1H, d, J = 8.4 Hz), 8.97 (1H, s); MS (ESI) m/z
[M+H]+ 619, 621.
extracted with CHCl3. The organic layer was washed with brine, dried
over Na2SO4 and concentrated in vacuo. Et2O was added to the residue,
and the resulting precipitate was filtered and dried to give the product
(1.50 g, 81%) as a colorless solid. 1H NMR (DMSO-d6): δ 0.84 (3H, t,
J = 7.2 Hz), 2.36 (3H, s), 2.75–2.85 (2H, m), 7.31–7.39 (1H, m),
7.59–7.68 (2H, m), 7.85 (1H, dd, J = 7.8, 1.2 Hz), 8.02 (1H, d,
J = 8.2 Hz), 8.74 (1H, s), 10.72 (1H, s), 13.54 (1H, br s); MS (ESI) m/z
[M+H]+ 369.
5.1.25. 2-({5-(3,5-Dimethoxyphenyl)-2-[3-methoxy-4-(4-oxopiperidin-1-
yl)anilino]pyrimidin-4-yl}amino)-N-ethylbenzene-1-sulfonamide (23)
To a solution of 22 (188 mg, 0.30 mmol) in 1,4-dioxane (3.8 mL)
were added Pd(PPh3)4 (35 mg, 0.030 mmol), 3,5-dimethoxyphenyl-
bronic acid (110 mg, 0.61 mmol), and 2 M Na2CO3 aq. (379 µL,
0.76 mmol) under an argon atmosphere. After stirring at 110 °C for 2 h,
the mixture was diluted with EtOAc and passed through a Celite pad.
The filtrate was concentrated in vacuo. The residue was purified by
column chromatography on silica gel (n-hexane/EtOAc = 95:5 to
0:100) to give a pale yellow solid. To this product were added AcOH
(4.0 mL, 48 mmol) and H2O (8.0 mL). The mixture was stirred at 100 °C
for 12 h and then concentrated in vacuo. The residue was neutralized
with Na2CO3 aq. and extracted with CHCl3. 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 = 99:1 to 80:20) to give 23 (190 mg, 100%) as a brown solid.
MS (ESI) m/z [M+H]+ 633.
5.1.29. 4-[2-(Ethylsulfamoyl)anilino]-N-methoxy-N-methyl-2-
(methylsulfanyl)pyrimidine-5-carboxamide (29)
To a solution of 28 (400 mg, 1.09 mmol) in DMF (8.00 mL) were
added
N,O-dimethylhydroxyamine
hydrochloride
(159
mg,
1.63 mmol), EDCI hydrochloride (312 mg, 1.63 mmol), HOBt (220 mg,
1.63 mmol) and Et3N (303 µL, 2.17 mmol). After stirring at room
temperature for 12 h, the mixture was diluted with H2O. The resulting
precipitate was filtered and dried to give the product (429 mg, 96%) as
a colorless solid. 1H NMR (CDCl3): δ 0.96 (3H, t, J = 7.2 Hz), 2.51 (3H,
s), 2.87–3.01 (2H, m), 3.44 (3H, s), 3.66 (3H, s), 5.85–5.98 (1H, m),
7.19–7.31 (1H, m), 7.53–7.62 (1H, m), 8.04 (1H, d, J = 8.0 Hz), 8.39
(1H, d, J = 8.2 Hz), 8.58 (1H, s), 9.53 (1H, s); MS (ESI) m/z [M+H]+
412.
5.1.26. 2-{[5-(3,5-Dimethoxyphenyl)-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-4-yl]amino}-N-
ethylbenzene-1-sulfonamide (24)
5.1.30. 2-{[5-(3,5-Dimethoxybenzoyl)-2-(methylsulfanyl)pyrimidin-4-yl]
amino}-N-ethylbenzene-1-sulfonamide (30)
To a solution of 23 (190 mg, 0.30 mmol) in CH2Cl2 (4.0 mL) was
added N-methyl-piperazine (165 µL, 1.5 mmol). After stirring at room
temperature for 1 h, NaBH(OAc)3 (80 mg, 0.38 mmol) was added. After
stirring at room temperature for 1 h, NaBH(OAc)3 (80 mg, 0.38 mmol)
was added. After further stirring at room temperature for 12 h, the
mixture was diluted with NaHCO3 aq. and extracted with EtOAc. The
organic layer was washed with brine, dried over Na2SO4 and con-
centrated in vacuo. The residue was purified by column chromato-
graphy on amino functionalized silica gel (EtOAc/MeOH = 100:0 to
80:20). IPE/EtOAc was added to the residue, and the resulting pre-
cipitate was filtered and dried to give the product (76 mg, 35%) as a
colorless solid. 1H NMR (DMSO-d6): δ 0.91 (3H, t, J = 7.2 Hz),
1.45–1.60 (2H, m), 1.73–1.86 (2H, m), 2.15 (3H, s), 2.20–2.57 (11H,
m), 2.68–2.78 (2H, m), 3.25–3.36 (2H, m), 3.51–3.65 (3H, m), 3.79
(6H, s), 6.51 (1H, dd, J = 2.3, 2.2 Hz), 6.66 (2H, d, J = 2.4 Hz),
6.72–6.80 (1H, m), 7.15–7.24 (2H, m), 7.33 (1H, d, J = 2.2 Hz),
7.48–7.57 (1H, m), 7.67 (1H, t, J = 5.7 Hz), 7.74 (1H, dd, J = 7.8,
1.6 Hz), 8.10 (1H, s), 8.42 (1H, d, J = 8.2 Hz), 8.78 (1H, s), 9.22 (1H,
s); MS (ESI) m/z [M+H]+ 717; HRMS (ESI) m/z Calcd for C37H49N8O5S
[M+H]+: 717.3541. Found: 717.3541.
To a solution of 29 (200 mg, 0.49 mmol) in THF (6.0 mL) was added
3,5-dimethoxyphenylmagnesium bromide (1.0 M THF solution, 1.7 mL,
1.7 mmol) at –78 °C. After stirring the mixture at –78 °C for 2 h and
warming
to
room
temperature,
additional
3,5-dimethox-
yphenylmagnesium bromide (1.0 M THF solution, 0.5 mL, 0.5 mmol)
was added to the mixture at –78 °C. This sequence of steps was repeated
twice with two further additions of 3,5-dimethoxyphenylmagnesium
bromide (1.0 M THF solution, 1.0 mL, 1.0 mmol then 1.0 M THF so-
lution, 1.9 mL, 1.9 mmol). After stirring at –78 °C for 2 h, the mixture
was quenched with sat. NH4Cl aq. and warmed to room temperature.
The mixture was 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 amino functionalized silica
gel (CHCl3) to give the product (150 mg, 63%) as a colorless solid. 1H
NMR (CDCl3): δ 1.05 (3H, t, J = 7.2 Hz), 2.49 (3H, s), 3.00–3.11 (2H,
m), 3.84 (6H, s), 5.17 (1H, t, J = 5.9 Hz), 6.68 (1H, dd, J = 2.4,
2.2 Hz), 6.83 (2H, d, J = 2.4 Hz), 7.26–7.34 (1H, m), 7.56–7.65 (1H,
m), 8.06 (1H, dd, J = 7.9, 1.5 Hz), 8.31 (1H, dd, J = 8.2, 1.0 Hz), 8.58
(1H, s), 11.17 (1H, s); MS (ESI) m/z [M+H]+ 489.
5.1.31. 2-{[5-(3,5-Dimethoxybenzoyl)-2-{3-methoxy-4-[4-(4-
5.1.27. Ethyl 4-[2-(ethylsulfamoyl)anilino]-2-(methylsulfanyl)pyrimidine-
5-carboxylate (27)
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-4-yl]amino}-N-
ethylbenzene-1-sulfonamide (32)
To a solution of ethyl 4-chloro-2-(methylsulfanyl)pyrimidine-5-
carboxylate (25, 4.65 g, 20 mmol) in toluene (58 mL) were added
DIPEA (3.77 mL, 22 mmol) and 2-amino-N-ethylbenzene-1-sulfonamide
(26, 4.01 g, 20 mmol). After stirring at 110 °C for 20 h, the solvent was
replaced with NMP. After stirring at 130 °C for 6 h, the mixture was
poured into H2O. The resulting precipitate was filtered, washed with
Et2O and dried to give the product (5.61 g, 71%) as a colorless solid. 1H
NMR (CDCl3): δ 1.02 (3H, t, J = 7.2 Hz), 1.41 (3H, t, J = 7.1 Hz), 2.46
(3H, s), 2.94–3.08 (2H, m), 4.44 (2H, q, J = 7.0 Hz), 4.89 (1H, br s),
7.23–7.32 (1H, m), 7.54–7.60 (1H, m), 8.03 (1H, d, J = 7.8 Hz), 8.24
(1H, d, J = 8.4 Hz), 8.84 (1H, s), 10.67 (1H, s); MS (ESI) m/z [M+H]+
397.
To a solution of 30 (150 mg, 0.31 mmol) in CH2Cl2 (4.0 mL) was
added m-CPBA (75% wet, 78 mg, 0.34 mmol) in an ice-water bath.
After stirring in an ice-water bath for 2 h, 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 to give
2-{[5-(3,5-dimethoxybenzoyl)-2-(methanesulfinyl)pyrimidin-4-yl]
amino}-N-ethylbenzene-1-sulfonamide (147 mg, 95%) as a brown solid,
which was used in the next reaction without further purification. To a
solution of 2-{[5-(3,5-dimethoxybenzoyl)-2-(methanesulfinyl)pyr-
imidin-4-yl]amino}-N-ethylbenzene-1-sulfonamide
(147
mg,
0.29 mmol) in IPA (4.0 mL) was added 3-methoxy-4-[4-(4-methylpi-
perazin-1-yl)piperidin-1-yl]aniline (31, 98 mg, 0.32 mmol). After stir-
ring at 90 °C for 12 h, the mixture 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 amino functionalized silica gel (EtOAc/
MeOH = 100:0 to 80:20) and then purified by column chromatography
on silica gel (CHCl3/MeOH = 100:0 to 80:20). EtOAc was added to the
5.1.28. 4-[2-(Ethylsulfamoyl)anilino]-2-(methylsulfanyl)pyrimidine-5-
carboxylic acid (28)
To a solution of 27 (1.98 g, 5.0 mmol) in MeOH/THF (20 mL, 1:1)
was added 1 M NaOH aq. (20 mL, 20 mmol). After stirring at room
temperature for 3 h, the mixture was neutralized with 1 M HCl aq. and
10

I. Kuriwaki, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
residue, and the resulting precipitate was filtered and dried to give the
product (89 mg, 41%) as a pale yellow solid. 1H NMR (DMSO-d6): δ
0.83–0.97 (3H, m), 1.44–1.58 (2H, m), 1.74–1.84 (2H, m), 2.15 (3H, s),
2.20–2.57 (11H, m), 2.79–2.90 (2H, m), 3.24–3.44 (5H, m), 3.82 (6H,
s), 6.58–6.80 (4H, m), 7.02–7.16 (1H, m), 7.16–7.29 (1H, m),
7.32–7.43 (1H, m), 7.53–7.70 (2H, m), 7.87 (1H, dd, J = 8.0, 1.6 Hz),
7.95–8.09 (1H, m), 8.43 (1H, s), 9.96 (1H, br s), 11.35 (1H, br s); MS
(ESI) m/z [M+H]+ 745.
2.30 (3H, s), 2.34–2.75 (11H, m), 2.95–3.06 (2H, m), 3.43–3.55 (2H,
m), 3.72 (3H, s), 4.44 (1H, t, J = 6.1 Hz), 6.80–6.88 (2H, m), 6.98 (1H,
dd, J = 8.5, 2.5 Hz), 7.06 (1H, d, J = 2.2 Hz), 7.18–7.24 (1H, m),
7.47–7.55 (1H, m), 7.96 (1H, dd, J = 7.9, 1.5 Hz), 8.22 (1H, s),
8.39–8.44 (1H, m), 8.97 (1H, s); MS (ESI) m/z [M − H]− 657, 659.
5.1.35. 2-({5-Bromo-2-[3-methoxy-4-(4-methylpiperazin-1-yl)anilino]
pyrimidin-4-yl}amino)-N-ethylbenzene-1-sulfonamide (35b)
Compound 35b was prepared from 20 and 2 in 73% yield using a
similar approach to that described for 35a. 1H NMR (DMSO-d6): δ 0.96
(3H, t, J = 7.2 Hz), 2.21 (3H, s), 2.37–2.53 (4H, m), 2.79–2.97 (6H, m),
3.59 (3H, s), 6.76 (1H, d, J = 8.4 Hz), 7.16 (1H, d, J = 8.4 Hz),
7.19–7.24 (1H, m), 7.24–7.32 (1H, m), 7.53–7.61 (1H, m), 7.82 (1H,
dd, J = 7.8, 1.6 Hz), 7.85–7.93 (1H, m), 8.32 (1H, s), 8.39–8.52 (1H,
m), 9.15 (1H, s), 9.31 (1H, s); MS (ESI) m/z [M+H]+ 576, 578; HRMS
5.1.32. 2-[(5-[(3,5-Dimethoxyphenyl)(hydroxy)methyl]-2-{3-methoxy-4-
[4-(4-methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-4-yl)amino]-
N-ethylbenzene-1-sulfonamide (33)
To a solution 32 (110 mg, 0.15 mmol) in THF/MeOH (6.0 mL, 2:1)
was added NaBH4 (10 mg, 0.26 mmol) in an ice-water bath. After
stirring at room temperature for 1 h, NaBH4 (10 mg, 0.26 mmol) was
added to the mixture in an ice-water bath. After stirring at room tem-
perature for 2 h, the mixture was concentrated in vacuo. The residue
was purified by column chromatography on silica gel (CHCl3/
MeOH = 100:0 to 80:20). IPE/EtOAc was added to the residue, and the
resulting precipitate was filtered and dried to give the product (71 mg,
64%) as a pale yellow solid. 1H NMR (DMSO-d6): δ 0.91 (3H, t,
J = 7.1 Hz), 1.43–1.59 (2H, m), 1.72–1.85 (2H, m), 2.16 (3H, s),
2.20–2.55 (11H, m), 2.78–2.90 (2H, m), 3.19–3.40 (2H, m), 3.46–3.55
(3H, m), 3.69 (6H, s), 5.69 (1H, d, J = 3.7 Hz), 6.33 (1H, d,
J = 3.9 Hz), 6.37 (1H, dd, J = 2.4, 2.2 Hz), 6.64 (2H, d, J = 2.4 Hz),
6.69 (1H, d, J = 8.6 Hz), 7.13 (1H, d, J = 8.0 Hz), 7.16–7.24 (1H, m),
7.28 (1H, d, J = 2.0 Hz), 7.41 (1H, t, J = 5.7 Hz), 7.46–7.54 (1H, m),
7.80 (1H, dd, J = 8.0, 1.6 Hz), 7.92 (1H, s), 8.15 (1H, d, J = 7.8 Hz),
9.05 (1H, s), 9.18 (1H, s); MS (ESI) m/z [M+H]+ 747.
(ESI) m/z Calcd for
C
24H31BrN7O3S [M+H]+
: 576.1387. Found:
576.1381.
5.1.36. 2-[(5-[(3,5-Dimethoxyphenyl)ethynyl]-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-4-yl)amino]-N-
ethylbenzene-1-sulfonamide (36)
To a solution of 35a (900 mg, 1.4 mmol) in DMF (18 mL) were
added Pd(PPh3)4 (315 mg, 0.27 mmol), 1-ethynyl-3,5-dimethox-
ybenzene (0.22 g, 1.4 mmol), and Et3N (951 µL, 6.8 mmol) under an
argon atmosphere. After stirring at 120 °C for 0.5 h, additional 1-
ethynyl-3,5-dimethoxybenzene (2.0 g, 12 mmol) in DMF (18 mL) was
added dropwise to the mixture over 4 h. After stirring at 120 °C for 2 h,
the mixture was diluted with EtOAc and 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)
and then purified by column chromatography on amino functionalized
silica gel (EtOAc/MeOH = 100:0 to 80:20). The residue was diluted
with EtOAc and stirred under reflux conditions. After cooling to room
temperature, the resulting precipitate was filtered and dried to give the
product (771 mg, 76%) as a pale yellow solid. 1H NMR (DMSO-d6): δ
0.95 (3H, t, J = 7.2 Hz), 1.46–1.60 (2H, m), 1.75–1.85 (2H, m), 2.14
(3H, s), 2.20–2.56 (11H, m), 2.83–2.94 (2H, m), 3.26–3.38 (2H, m),
3.62 (3H, s), 3.77 (6H, s), 6.53 (1H, dd, J = 2.4, 2.4 Hz), 6.77–6.86
(3H, m), 7.16–7.30 (3H, m), 7.57 (1H, t, J = 7.4 Hz), 7.82 (1H, dd,
J = 8.0, 1.6 Hz), 7.93–8.02 (1H, m), 8.40 (1H, s), 8.55–8.75 (1H, m),
9.52 (2H, br s); MS (ESI) m/z [M+H]+ 741; Anal. Calcd for
5.1.33. 2-[(5-[(3,5-Dimethoxyphenyl)methyl]-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-4-yl)amino]-N-
ethylbenzene-1-sulfonamide (34)
To a solution of 33 (53 mg, 71 µmol) in CH2Cl2 (2.0 mL) were added
triethylsilane (113 µL, 0.71 mmol) and TFA (272 µL, 3.5 mmol) in an
ice-water bath under an argon atmosphere. After stirring at room
temperature for 18 h, additional triethylsilane (113 µL, 0.71 mmol) and
TFA (1.0 mL, 13 mmol) were added to the mixture in an ice-water bath.
After stirring at room temperature for 6 h, the mixture was con-
centrated in vacuo. The residue was purified by column chromato-
graphy on amino functionalized silica gel (EtOAc/MeOH = 100:0 to
80:20) and then purified by column chromatography on silica gel
(CHCl3/MeOH = 100:0 to 80:20). IPE/EtOAc was added to the residue,
and the resulting precipitate was filtered and dried to give the product
(16 mg, 31%) as a colorless solid. 1H NMR (DMSO-d6): δ 0.91 (3H, t,
J = 7.2 Hz), 1.45–1.60 (2H, m), 1.74–1.85 (2H, m), 2.18 (3H, s),
2.22–2.57 (11H, m), 2.69–2.81 (2H, m), 3.25–3.36 (2H, m), 3.60 (3H,
s), 3.69 (6H, s), 3.78 (2H, s), 6.34 (1H, dd, J = 2.2, 2.2 Hz), 6.43 (2H,
d, J = 2.4 Hz), 6.75 (1H, d, J = 8.6 Hz), 7.15–7.23 (2H, m), 7.28 (1H,
d, J = 2.0 Hz), 7.49–7.57 (1H, m), 7.78 (1H, dd, J = 7.8, 1.6 Hz), 7.88
(1H, t, J = 5.7 Hz), 8.01 (1H, s), 8.56 (1H, d, J = 8.6 Hz), 8.80 (1H, s),
9.04 (1H, s); MS (ESI) m/z [M+H]+ 731; HRMS (ESI) m/z Calcd for
C38H51N8O5S [M+H]+: 731.3698. Found: 731.3688.
C
39H48N8O5S. 0.3EtOAc.0.04CHCl3: C, 62.59; H, 6.58; N, 14.51; S,
4.15; Cl, 0.55. Found: C, 62.64; H, 6.61; N, 14.51; S, 4.08; Cl, 0.50.
5.1.37. 2-[(5-[2-(3,5-Dimethoxyphenyl)ethyl]-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-4-yl)amino]-N-
ethylbenzene-1-sulfonamide (37)
To a solution of 36 (200 mg, 0.27 mmol) in THF/MeOH (8.0 mL,
1:1) was added 10% Pd/C (14 mg, 13 µmol) under an argon atmo-
sphere. After stirring at room temperature under a hydrogen atmo-
sphere (1.0 atm) for 6 h, the mixture was passed through a Celite pad.
The filtrate was concentrated in vacuo. To the residue in THF/MeOH
(8.0 mL, 1:1) was added 10% Pd/C (14 mg, 13 µmol) under an argon
atmosphere. After stirring at room temperature under a hydrogen at-
mosphere (2.5 atm) for 4 h, the mixture was passed through a Celite
pad. The filtrate was concentrated in vacuo. The residue was diluted
with EtOAc and stirred under reflux conditions. After cooling to room
temperature, the resulting precipitate was filtered and dried to give the
product (102 mg, 51%) as a pale yellow solid. 1H NMR (DMSO-d6): δ
0.92–1.00 (3H, m), 1.45–1.60 (2H, m), 1.74–1.85 (2H, m), 2.14 (3H, s),
2.19–2.56 (11H, m), 2.69–2.90 (6H, m), 3.26–3.36 (2H, m), 3.61 (3H,
s), 3.71 (6H, s), 6.30 (1H, dd, J = 2.4, 2.2 Hz), 6.50 (2H, d,
J = 2.4 Hz), 6.76 (1H, d, J = 8.8 Hz), 7.15–7.24 (2H, m), 7.29 (1H, d,
J = 2.2 Hz), 7.52–7.59 (1H, m), 7.80 (1H, dd, J = 7.8, 1.6 Hz), 7.95
(1H, t, J = 5.5 Hz), 8.02 (1H, s), 8.69 (1H, d, J = 8.4 Hz), 8.98 (1H, s),
9.01 (1H, s); MS (ESI) m/z [M+H]+ 745; HRMS (ESI) m/z Calcd for
5.1.34. 2-[(5-Bromo-2-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)
piperidin-1-yl]anilino}pyrimidin-4-yl)amino]-N-ethylbenzene-1-
sulfonamide (35a)
To a solution of 20 (1.0 g, 2.0 mmol) in IPA (10 mL) were added 31
(650 mg, 2.1 mmol) and methanesulfonic acid (0.40 mL, 6.1 mmol).
After stirring at 130 °C under microwave irradiation for 1 h, 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 diluted with EtOAc and stirred under reflux
conditions. After cooling to room temperature, the resulting precipitate
was filtered and dried to give the product (1.0 g, 77%) as a colorless
solid. 1H NMR (CDCl3): δ 1.04 (3H, t, J = 7.2 Hz), 1.73–1.96 (4H, m),
11

I. Kuriwaki, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
C39H53N8O5S [M+H]+: 745.3854. Found: 745.3850; Anal. Calcd for
5.1.42. N-(4-[2-(Ethylsulfamoyl)anilino]-2-{3-methoxy-4-[4-(4-
C
39H52N8O5S.0.2EtOAc·1.7H2O: C, 60.3; H, 7.2; N, 14.1; S, 4.0. Found:
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-5-yl)-2,5-
dimethoxybenzamide (40b)
C, 60.3; H, 7.0; N, 14.0; S, 3.8.
Compound 40b was prepared from 39b and 2,5-dimethoxybenzoic
acid using a similar approach to that described for 40f. MS (ESI) m/z [M
+H]+ 760.
5.1.38. 2-[(2-Chloro-5-nitropyrimidin-4-yl)amino]-N-ethylbenzene-1-
sulfonamide (38)
A mixture of 26 (510 mg, 2.6 mmol) and 2,4-dichloro-5-nitropyr-
imidine (530 mg, 2.6 mmol) in DMAc (5.0 mL) was stirred at room
temperature for 3 h. The mixture was diluted with 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 = 100:10 to
75:25) to give the product (720 mg, 77%) as a yellow solid. 1H NMR
(DMSO-d6): δ 0.85 (3H, t, J = 7.2 Hz), 2.78 (2H, qd, J = 7.2, 5.8 Hz),
7.46–7.53 (1H, m), 7.71–7.80 (2H, m), 7.89–7.98 (2H, m), 9.31 (1H, s),
10.78 (1H, s); MS (ESI) m/z [M+H]+ 358.
5.1.43. N-(4-[2-(Ethylsulfamoyl)anilino]-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-5-yl)-2,3-
dimethoxybenzamide (40c)
Compound 40c was prepared from 39b and 2,3-dimethoxybenzoic
acid using a similar approach to that described for 40f. MS (ESI) m/z [M
+H]+ 760.
5.1.44. N-(4-[2-(Ethylsulfamoyl)anilino]-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-5-yl)-3,5-
dihydroxybenzamide (40d)
5.1.39. N-Ethyl-2-[(2-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-
1-yl]anilino}-5-nitropyrimidin-4-yl)amino]benzene-1-sulfonamide (39a)
Compound 39a was prepared from 31 and 38 in 87% yield using a
similar approach to that described for 18a. 1H NMR (DMSO-d6): δ
0.77–0.88 (3H, m), 1.43–1.58 (2H, m), 1.74–1.84 (2H, m), 2.14 (3H, s),
2.18–2.57 (11H, m), 2.71–2.82 (2H, m), 3.26–3.35 (2H, m), 3.39 (3H,
br s), 6.64 (1H, d, J = 8.2 Hz), 7.05 (1H, d, J = 8.0 Hz), 7.14 (1H, br s),
7.36–7.47 (1H, m), 7.53–7.66 (1H, m), 7.73–7.83 (1H, m), 7.88 (1H, d,
J = 7.0 Hz), 7.94–8.07 (1H, m), 9.13 (1H, s), 10.37 (1H, br s), 10.82
(1H, br s); MS (ESI) m/z [M+H]+ 626.
Compound 40d was prepared from 39b and 3,5-dihydroxybenzoic
acid using a similar approach to that described for 40f. MS (ESI) m/z [M
+H]+ 732.
5.1.45. N-(4-[2-(Ethylsulfamoyl)anilino]-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-5-yl)-3,5-
dimethylbenzamide (40e)
Compound 40e was prepared from 39b and 3,5-dimethylbenzoic
acid using a similar approach to that described for 40f. MS (ESI) m/z [M
+H]+ 728.
5.1.40. 2-[(5-Amino-2-{3-methoxy-4-[4-(4-methylpiperazin-1-yl)
piperidin-1-yl]anilino}pyrimidin-4-yl)amino]-N-ethylbenzene-1-
sulfonamide (39b)
5.1.46. N-(4-[2-(Ethylsulfamoyl)anilino]-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-5-yl)-4,6-
dimethoxypyrimidine-2-carboxamide (40f)
To a solution of 39a (2.2 g, 3.5 mmol) in EtOH/THF (70 mL, 4:3)
was added 10% Pd/C (50% wet, 1.6 g, 0.74 mmol) under an argon
atmosphere. After stirring at room temperature under a hydrogen at-
mosphere (1.0 atm) for 6 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/28% NH3
aq. = 100:0:0 to 90:9:1) to give the product (2.1 g, 99%) as a brown
solid. 1H NMR (DMSO-d6): δ 0.93 (3H, t, J = 7.2 Hz), 1.45–1.59 (2H,
m), 1.73–1.85 (2H, m), 2.14 (3H, s), 2.17–2.54 (11H, m), 2.83 (2H, q,
J = 7.0 Hz), 3.24–3.34 (2H, m), 3.62 (3H, s), 4.06 (2H, s), 6.74 (1H, d,
J = 8.6 Hz), 7.12–7.22 (2H, m), 7.27 (1H, d, J = 2.4 Hz), 7.52–7.60
(1H, m), 7.75–7.83 (3H, m), 8.67 (1H, s), 8.71 (1H, d, J = 7.8 Hz), 9.01
(1H, br s); MS (ESI) m/z [M+H]+ 596.
To a solution of 39b (278 mg, 0.47 mmol) in DMF (5.0 mL) were
added
4,6-dimethoxypyrimidine-2-carboxylic
acid
(103
mg,
0.56 mmol), DIPEA (160 μL, 0.93 mmol) and HATU (236 mg,
0.62 mmol) in an ice-water bath. After stirring at room temperature
overnight, the mixture was quenched with 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
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 pre-
cipitate was filtered and dried. To the precipitate was added EtOH/
EtOAc/MeCN, and the mixture was heated to 100 °C. After cooling to
room temperature, the resulting precipitate was filtered and dried to
give the product (96 mg, 27%) as a colorless solid. 1H NMR (DMSO-d6):
δ 0.90 (3H, t, J = 7.2 Hz), 1.46–1.61 (2H, m), 1.74–1.86 (2H, m), 2.14
(3H, s), 2.20–2.58 (11H, m), 2.74–2.85 (2H, m), 3.25–3.39 (2H, m),
3.67 (3H, s), 4.03 (6H, s), 6.48 (1H, s), 6.82 (1H, d, J = 8.4 Hz),
7.16–7.29 (3H, m), 7.51–7.62 (2H, m), 7.77 (1H, dd, J = 7.9, 1.5 Hz),
8.13 (1H, s), 8.74–8.87 (1H, m), 9.14 (1H, br s), 9.27 (1H, s), 10.14
(1H, br s); MS (ESI) m/z [M+H]+ 762; Anal. Calcd for
C36H47N11O6S·1.7H2O: C, 54.56; H, 6.41; N, 19.44; S, 4.05. Found: C,
54.55; H, 6.38; N, 19.45; S, 4.02.
5.1.41. N-(4-[2-(Ethylsulfamoyl)anilino]-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-5-yl)-3,5-
dimethoxybenzamide (40a)
To a solution of 39b (150 mg, 0.25 mmol) in THF (5.0 mL) were
added DIPEA (50 µL, 0.29 mmol) and 3,5-dimethoxybenzoyl chloride
(60 mg, 0.30 mmol). After stirring at room temperature for 1 h, the
mixture was quenched with sat. NaHCO3 aq. and extracted with CHCl3/
IPA (3:1). The organic layer was washed with brine, dried over Na2SO4
and concentrated in vacuo. The residue was purified by column chro-
matography on silica gel (CHCl3/MeOH = 98:2 to 85:15). Et2O was
added to the residue, and the resulting precipitate was filtered and
dried to give the product (93 mg, 49%) as a colorless solid. 1H NMR
(DMSO-d6): δ 0.90 (3H, t, J = 7.2 Hz), 1.46–1.61 (2H, m), 1.75–1.86
(2H, m), 2.17 (3H, s), 2.22–2.59 (11H, m), 2.75–2.85 (2H, m),
3.26–3.38 (2H, m), 3.66 (3H, s), 3.83 (6H, s), 6.73 (1H, dd, J = 2.4,
2.2 Hz), 6.81 (1H, d, J = 8.6 Hz), 7.14–7.29 (5H, m), 7.52–7.63 (2H,
m), 7.79 (1H, dd, J = 8.0, 1.6 Hz), 8.10 (1H, s), 8.78 (1H, d,
J = 8.0 Hz), 9.20 (1H, s), 9.25 (1H, s), 9.88 (1H, s); MS (ESI) m/z [M
+H]+ 760; Anal. Calcd for C38H49N9O6S.0.05CHCl3·1.2H2O: C, 58.03;
H, 6.59; N, 16.01; S, 4.07; Cl, 0.68. Found: C, 57.98; H, 6.50; N, 15.86;
S, 4.03; Cl, 0.60.
5.1.47. N-(4-[2-(Ethylsulfamoyl)anilino]-2-{3-methoxy-4-[4-(4-
methylpiperazin-1-yl)piperidin-1-yl]anilino}pyrimidin-5-yl)-2,6-
dimethoxypyrimidine-4-carboxamide (40g)
Compound 40g was prepared from 39b and 2,6-dimethoxypyr-
imidine-4-carboxylic acid using a similar approach to that described for
40f. MS (ESI) m/z [M+H]+ 762.
5.2. Biology
5.2.1. 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,
12

I. Kuriwaki, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Japan) and test compounds were incubated for 30 or 120 min at room
temperature (RT). After the incubation, substrate and adenosine tri-
phosphate (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% inhibi-
tion). The IC50 value of each experiment was calculated using Sigmoid-
Emax non-linear regression analysis.
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5.2.2. In vitro cell growth inhibitory assay
UM-UC-14 was purchased from ECACC (Salisbury, UK). The cell line
was cultured according to instructions from the supplier. The cells were
seeded in 384-well plates at 900 cells per well and incubated overnight.
On the following day, the cells were exposed to compound 40a 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.
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Funding
18. Renhowe PA, Pecchi S, Shafer CM, et al. Design, structure-activity relationships and
in vivo characterization of 4-amino-3-benzimidazol-2-ylhydroquinolin-2-ones: A
novel class of receptor tyrosine kinase inhibitors. J Med Chem. 2009;52:278–292.
19. Trudel S, Li ZH, Wei E, et al. CHIR-258, a novel, multitargeted tyrosine kinase in-
hibitor for the potential treatment of t(4;14) multiple myeloma. Blood.
2005;105:2941–2948.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
20. Angevin E, Lin C, Pande AU, Lopez, J. A., Gschwend, J., Harzstark, A. L., Shi, M.,
Anak, O., Escudier, B. J.. A phase I/II study of dovitinib (TKI258), a FGFR and VEGFR
inhibitor, in patients (pts) with advanced or metastatic renal cell cancer: Phase I
results. J Clin Oncol. 2010;28:3057.
This research is a collaboration between Astellas Pharma Inc. and
Kotobuki Pharmaceutical Co., Ltd.
21. Roodhart JM, Langenberg MH, Witteveen E, Voest EE. The molecular basis of class
side effects due to treatment with inhibitors of the VEGF/VEGFR pathway. Curr Clin
Pharmacol. 2008;3:132–143.
Acknowledgements
22. Perera TPS, Jovcheva E, Mevellec L, et al. Discovery and pharmacological char-
acterization of JNJ-42756493 (erdafitinib), a functionally selective small-molecule
FGFR family inhibitor. Mol Cancer Ther. 2017;16:1010–1020.
The authors thank Dr. Susumu Watanuki for his helpful support in
the preparation of this manuscript. The authors thank Dr. Aya Kikuchi,
Ms. Ayako Nakayama, Mr. Taisuke Nakazawa, and Dr. Masateru Iizuka
for their contributions to the biological studies. The authors thank the
staff of the Division of Analytical Science Laboratories and Astellas
Research Technologies Co., Ltd. for conducting spectral measurements
and elemental analysis.
23. Guagnano V, Furet P, Spanka C, et al. Discovery of 3-(2,6-dichloro-3,5-dimethoxy-
phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-
urea (NVP-BGJ398), a potent and selective inhibitor of the fibroblast growth factor
receptor family of receptor tyrosine kinase. J Med Chem. 2011;54:7066–7083.
24. Brameld KA, Owens TD, Verner E, et al. Discovery of the irreversible covalent FGFR
inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethox-
yphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the
treatment of solid tumors. J Med Chem. 2017;60:6516–6527.
25. Mohammadi M, Froum S, Hamby JM, et al. Crystal structure of an angiogenesis in-
hibitor bound to the FGF receptor tyrosine kinase domain. EMBO J.
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