Discovery of 5-phenoxy-2-aminopyridine derivatives as potent and selective irreversible inhibitors of bruton’s tyrosine kinase

Article abstract of DOI:10.3390/ijms21218006

As a member of the tyrosine protein kinase Tec (TEC) family, Bruton’s tyrosine kinase (BTK) is considered a promising therapeutic target due to its crucial roles in the B cell receptor (BCR) signaling pathway. Although many types of BTK inhibitors have been reported, there is an unmet need to achieve selective BTK inhibitors to reduce side effects. To obtain BTK selectivity and efficacy, we designed a novel series of type II BTK inhibitors which can occupy the allosteric pocket induced by the DFG-out conformation and introduced an electrophilic warhead for targeting Cys481. In this article, we have described the structure-activity relationships (SARs) leading to a novel series of potent and selective piperazine and tetrahydroisoquinoline linked 5-phenoxy-2-aminopyridine irreversible inhibitors of BTK. Compound 18g showed good potency and selectivity, and its biological activity was evaluated in hematological tumor cell lines. The in vivo efficacy of 18g was also tested in a Raji xenograft mouse model, and it significantly reduced tumor size, with 46.8% inhibition compared with vehicle. Therefore, we have presented the novel, potent, and selective irreversible inhibitor 18g as a type II BTK inhibitor.

Full text of DOI:10.3390/ijms21218006

International Journal of
Molecular Sciences
Article
Discovery of 5-Phenoxy-2-aminopyridine Derivatives
as Potent and Selective Irreversible Inhibitors of
Bruton’s Tyrosine Kinase
1
1
1
1
2
1
Eun Lee , Hyewon Cho , Da Kyung Lee , JuHyun Ha , Byeong Jo Choi , Ji Hye Jeong ,
Jae-Ha Ryu 1 , Jong Soon Kang 2 and Raok Jeon *
1,
1
College of Pharmacy, Sookmyung Women’s University, Seoul 04310, Korea; eunlee178@gmail.com (E.L.);
hy-0802@sookmyung.ac.kr (H.C.); josephin914@sookmyung.ac.kr (D.K.L.); cu1006@sookmyung.ac.kr (J.H.);
jjh4415@naver.com (J.H.J.); ryuha@sookmyung.ac.kr (J.-H.R.)
Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Ochang,
2
Chungcheongbuk-do 28116, Korea; byung127@kribb.re.kr (B.J.C.); kanjon@kribb.re.kr (J.S.K.)
*
Correspondence: rjeon@sookmyung.ac.kr; Tel.: +82-2-710-9571
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 11 August 2020; Accepted: 27 October 2020; Published: 28 October 2020
Abstract: As a member of the tyrosine protein kinase Tec (TEC) family, Bruton’s tyrosine kinase
(
(
BTK) is considered a promising therapeutic target due to its crucial roles in the B cell receptor
BCR) signaling pathway. Although many types of BTK inhibitors have been reported, there is an
unmet need to achieve selective BTK inhibitors to reduce side effects. To obtain BTK selectivity and
efficacy, we designed a novel series of type II BTK inhibitors which can occupy the allosteric pocket
induced by the DFG-out conformation and introduced an electrophilic warhead for targeting Cys481.
In this article, we have described the structure–activity relationships (SARs) leading to a novel series
of potent and selective piperazine and tetrahydroisoquinoline linked 5-phenoxy-2-aminopyridine
irreversible inhibitors of BTK. Compound 18g showed good potency and selectivity, and its biological
activity was evaluated in hematological tumor cell lines. The in vivo efficacy of 18g was also tested
in a Raji xenograft mouse model, and it significantly reduced tumor size, with 46.8% inhibition
compared with vehicle. Therefore, we have presented the novel, potent, and selective irreversible
inhibitor 18g as a type II BTK inhibitor.
Keywords: Bruton’s tyrosine kinase; irreversible kinase inhibitor; hematological malignancy
1
. Introduction
Bruton’s tyrosine kinase (BTK), a member of the tyrosine protein kinase Tec (TEC) family, is expressed
in B cells, myeloid cells, mast cells, and macrophages [
1
,
2
]. BTK is a crucial regulator of B cell development
and function and is activated downstream of the B cell receptor (BCR) [3]. Abnormal functions of BTK
are critical to the dysregulation of several steps in B cell proliferation, development, differentiation,
and apoptosis, which induce overactive inflammation and hematologic malignancy [4].
Ibrutinib, the first US Food and Drug Administration (FDA)-approved BTK inhibitor
(
Figure 1), is an irreversible covalent inhibitor that targets Cys481, the residue adjacent to the
ATP-binding site. It has been approved for the treatment of chronic lymphocytic leukemia (CLL),
Waldenström macroglobulinemia, mantle cell lymphoma, and chronic graft versus host disease [ ].
Another irreversible BTK inhibitor, acalabrutinib, was approved by the FDA for the treatment of mantle
cell lymphoma, and several other covalent inhibitors are in clinical trials (Figure 1) [ ]. Most of the
adverse effects of ibrutinib are thought to be related to off-target effects arising from inhibition of TEC,
janus kinase 3 (JAK3), and epidermal growth factor receptor kinase (EGFRK) not only the BTK [ ].
Based on the location of the targeted Cys residue near the ATP-binding site, those kinases can be
5
6
7
Int. J. Mol. Sci. 2020, 21, 8006; doi:10.3390/ijms21218006
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 8006
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classified as Group 3F [8]. To overcome the issue of side effects, the discovery of highly selective BTK
inhibitors is desirable.
O
F
H
N
N
N
N
O
N
NH
O
O
N
N
O
N HO
NH2
N
N
N
NH2
NH2
N
RN-486
NH
N
N
N
O
N
N
O
N
H
N
O
N
O
N
H
N
N
O
N
O
O
HN
Ibrutinib
Acalabrutinib
Evobrutinib
O
CHMFL-BTK-01
Figure 1. Examples of FDA-approved and preclinical BTK inhibitors.
Small molecule kinase inhibitors can be classified as reversible or irreversible inhibitors, depending
on how they bind to the kinase [ ]. Many BTK inhibitors, such as ibrutinib, acalabrutinib and evobrutinib,
9
include a Michael acceptor with the aim of irreversible blocking by targeting the Cys residue at the
BTK active site. Meanwhile, reversible inhibitors are generally further classified into four groups;
type I to type IV inhibitors based on the conformation of the kinase ATP-binding pocket and where the
inhibitor binds. Type I inhibitors bind to the kinase in adenosine triphosphate (ATP)-competitive mode
at the ATP binding site. For type II inhibitors, the molecules interact with an inactive form of the target
kinase and occupy both the ATP site and DFG pocket. Type III and type IV inhibitors are allosteric
modulators which bind outside the ATP-binding site. RN-486, a representative reversible BTK inhibitor,
can achieve BTK selectivity by targeting not only the adenine pockets but also a BTK specific allosteric
pockets [10]. Recently, some hybridization strategies were attempted to convert reversible inhibitors to
irreversible inhibitors, which resulted in selective inhibitors, such as CHMFL-BTK-01 [11].
Herein, we designed and synthesized aminopyridine derivatives as selective covalent BTK
inhibitors and evaluated their antiproliferative activities in hematological tumor cell lines and their
in vivo efficacy in a Raji xenograft mouse model. Consequently, we have identified a novel class of
5
2
2
-phenoxy-2-aminopyridine compounds as potent and selective BTK inhibitors.
. Results and Discussion
.1. Drug Design
Ibrutinib contains a phenoxyphenyl moiety, which forms hydrophobic interactions with a
selectivity pocket formed by Met449, Ile472, Leu542, and Phe540. The selectivity pocket is behind the
Thr474 gatekeeper residue and is induced by the C-helix-out conformation [12]. For this lipophilic
α
substituent, acalabrutinib has a pyridylamide group instead of a phenoxy group. This particular
structural change resulted in a greatly improved kinase selectivity profile [13], which showed that
modifying the lipophilic group may be a good approach for targeting BTK selectively. Moreover,
we hypothesized that expanding the lipophilic linker could induce not only the
αC-helix-out
conformation, but also the DFG-out conformation, which allows increased affinity and selectivity of
compounds [14]. As there are currently no validated type II BTK inhibitors, the design of a type II
inhibitor is a good starting point to achieve a novel and selective BTK inhibitor. We analyzed 74 BTK
structures co-crystalized with small molecule inhibitors registered in the Protein Data Bank [15] (PDB
IDs are listed in Supplementary Materials). Among the numerous activation states of BTK, there is a
DFG-out conformation BTK structure complexed with a type II SRC kinase inhibitor (PDB ID: 3PJ3) [16],
which provided the structural perspective for the design of type II BTK inhibitors.
When starting from ibrutinib, we considered it was necessary to revise the lipophilic group to
target the allosteric site. To ensure flexibility for modification of the lipophilic group, the bicyclic

Int. J. Mol. Sci. 2020, 21, 8006
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pyrazolopyrimidine ring at the adenine binding pocket was replaced with a monocycle, such as
evobrutinib (Figure 1). We also changed the lipophilic group linker phenyl ring of ibrutinib to
a saturated piperazine ring to reduce structural rigidity (Figure 2). Furthermore, the monocyclic
piperazine linker was also converted to a bicyclic tetrahydroisoquinoline linker to extend hydrophobic
interactions deep into the allosteric pocket. In addition, the aromatic linker between the hinge binder
and covalent warheads (Michael acceptor linker) can be positioned in the H2 pocket between Leu408
and Gly480 [17].
O
NH2
X
NH2
N
N
N
N
N
O
O
N
O
N
O
Figure 2. Design strategy for a novel series of aminopyridine derivatives as inhibitors of
BTK. Red, lipophilic aryl substituent; green, lipophilic group linker; blue, hinge binder; orange,
Michael acceptor linker.
2
.2. Synthesis
Piperazine-linked aminopyridine derivatives were prepared as described in Scheme 1 and
Supplementary Materials. From commercially available 2-amino-3,5-dichloropyridine, the amine
group was converted to a nitro group using potassium persulfate, providing compound
Nucleophilic substitution at the C3 position of pyridine
and deprotection of the Boc group with trifluoroacetic acid (TFA) afforded the 3-piperazinylpyridine
1
.
1
with tert-butyloxycarbonyl (Boc)-piperazine
2
.
N-Alkylation of the piperazine group with the appropriate aryl halide gave the corresponding
nitropyridine Through a nucleophilic substitution reaction with cesium carbonate in
dimethylformamide (DMF), resorcinol was introduced to the pyridine core scaffold and then acrylated
with a Michael acceptor to achieve the intermediate . Since the acryloyl group was removed under the
3.
5
initial reducing conditions that we tested, the nitro group was reduced to the amine using mild acidic
conditions with tin (II) chloride, which afforded the piperazine-linked aminopyridine derivative 6.
R1
NH2
Cl
NO2
NO2
Cl
NH
NO2
Cl
N
Cl
Cl
N
N
N
a
N
b,c
N
d
N
Cl
1
2
3
R1
R1
R1
NO2
N
NO2
N
O
NH2
N
O
N
N
N
e
N
f
N
g
N
O
OH
O
O
O
O
4
5
6
Scheme 1. Synthesis of piperazine-linked aminopyridine derivatives. Reagents and conditions:
◦ ◦
a) K S O , H SO , 90 C, 24 h, 45%; (b) 1-Boc-piperazine, K CO , toluene, 50 C, 15 h, 42%;
2 2 8 2 4 2 3
(
(
(
c) trifluoroacetic acid (TFA), dichloromethane (DCM), rt, 4 h, 79%; (d) R X, K CO , dimethylformamide
1
2
3
◦
DMF), rt, 5 h, 40–93%; (e) resorcinol, Cs CO , DMF, 70 C, 24 h, 20–79%; (f) acryloyl chloride,
2
3
◦
◦
triethylamine (TEA), DCM, 0 C, 3 h, 6–67%; (g) SnCl ·2H O, EtOH, 0 C, 1 h, 4–23%.
2
2

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To develop the bicyclic lipophilic group linker, tetrahydroisoquinoline scaffolds with aryl substituents
weresynthesizedasdepictedinScheme2. Thecommerciallyavailable2-(3-methoxyphenyl)ethylaminewas
treated with ethyl chloroformate and triethylamine (TEA) in dichloromethane (DCM) to form carbamate
7
8
. The subsequent Bischler–Napieralski reaction with polyphosphoric acid (PPA) gave isoquinolinone
. Demethylation with HBr followed by nucleophilic substitution afforded intermediate 10, which was
then further reduced to tetrahydroisoquinoline with LiAlH to obtain lipophilic substituent 11
.
4
The tetrahydroisoquinoline-linked aminopyridine derivatives 17 and 18 were synthesized in a similar
manner to that of the piperazine derivatives (Scheme 3).
O
a
b
O
NH
O
NH2
O
N
H
O
O
7
8
O
O
c
d
e
NH
NH
NH
HO
R O
2
R O
2
9
10
11
Scheme 2. Synthesis of the tetrahydroisoquinoline scaffold for the lipophilic group linker. Reagents and
◦
conditions: (a) ethyl chloroformate, TEA, DCM, rt, 2 h, 100%; (b) polyphosphoric acid (PPA), 120 C, 1.5 h,
6
8%; (c) 48% HBr, reflux, 24 h, 63%; (d) R X, K CO , DMF, rt, 6 h, 27–55%; (e) LiAlH , tetrahydrofuran
2
2
3
4
(THF), reflux, 5 h, 40–95%.
R3
R3
NO2
NO2
a
N
b
N
N
N
1
2
OH
3
Cl
O
1
2
13, 2-OH
14, 3-OH
R3
R3
NO2
NH2
N
N
c
N
N
d
2
2
O
O
O
O
3
3
O
O
1
1
5, 2-acryloyloxy
6, 3-acryloyloxy
17, 2-acryloyloxy
18, 3-acryloyloxy
Scheme 3. Synthesis of tetrahydroisoquinoline-linked aminopyridine derivatives. Reagents and
◦
conditions: (a) 11a–f or 1,2,3,4-tetrahydroisoquinoline, K CO , toluene, 60 C, 10 h, 34–57%;
2
3
◦
b) resorcinol or catechol, K CO , dimethyl sulfoxide (DMSO), 18-crown-6, 100 C, 3 h, 25–74%;
2 3
c) acryloyl chloride, TEA, DCM, rt, 1 h, 30–99%; (d) SnCl ·2H O, THF/EtOH, rt, 1 h, 4–67%.
(
(
2
2
2
.3. Structure–Activity Relationship Analysis
Synthesized aminopyridine derivatives were evaluated for in vitro BTK enzyme inhibitory
activities. In vitro kinase assays were performed using commercially available ADP-Glo (Promega,
Madison, WI, USA) and HotSpot Kinase assays (Reaction Biology Corporation, Malvern, PA, USA) at
an ATP concentration of 10
µM. The SAR study of piperazine-linked aminopyridine
6 was performed
(Table 1). Introduction of either the benzyl (6b
–
d
) or phenylethyl (6e ) group at the nitrogen of the
,f
piperazine led to better activity than the parent compound 6a, suggesting the beneficial interactions of
the substituted lipophilic groups in the lipophilic selectivity pocket. The chlorine substitution (6d) at

Int. J. Mol. Sci. 2020, 21, 8006
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the para position of the phenyl ring led to the highest potency within this series. However, the insertion
of additional carbons (6f) between the piperazine and the terminal phenyl ring reduced the activity,
implying a steric requirement for fitting into the selectivity pocket.
Table 1. Structure–activity relationship of piperazine-linked aminopyridine 6.
Compound
R1
% Inhibition 1
IC50 (µM) 2
ND 3
3.17
ND
1.54
5.01
ND
6
a
H
Bn
p-FBn
p-ClBn
PhEt
14
85
83
92
89
52
77
6
b
6
c
6
d
6
6
e
f
p-ClPhEt
benzyloxycarbonyl
6
g
ND
Ibrutinib
0.0002
1
Relative percentage inhibition at 20
µ
M of compound compared with staurosporine, HotSpot kinase assay
2
3
(
Reaction Biology Corp.); HotSpot kinase assay (Reaction Biology Corp.); ND = not determined.
To identify a suitable linker moiety, the piperazine linker was replaced with tetrahydroisoquinoline.
Eight of nine tetrahydroisoquinoline derivatives showed complete inhibition of enzyme activity in
single dose determination (20 µM), which indicated improved potency compared with the piperazine
derivatives, supporting our hypothesis (Table 2). In the case of tetrahydroisoquinoline derivatives,
additional modifications resulted in direction toward the H2 pocket where the phenoxy linker-bearing
Michael acceptor was positioned. The substitution position of the Michael acceptor significantly
influenced the activity: the potency of the C2-substituted catechol-type compound 17a was higher
than that of C3-substituted resorcinol type 18a; conversely, that of 17d was less than that of 18d.
Table 2. SAR of tetrahydroisoquinoline linked aminopyridines 17 and 18.
Compound
7a
7d
8a
8b
8c
8d
8e
8f
8g
Position
R3
% Inhibition 1
IC50 (µM) 2
1
1
1
1
1
1
1
1
2
2
3
3
3
3
3
3
3
H
OBn
H
98
98
98
99
96
97
53 *
98
0.63
0.46
2.20
0.97
1.20
0.10
8.53
1.00
0.19
n
O Pr
c
OCH Pr
2
OBn
Op-FBn
Op-ClBn
Op-OMeBn
1
92 *
1
Relative percentage inhibition at 20
HotSpot kinase assay (Reaction Biology Corp.); ADP-Glo Assay (Promega). IC50 values were calculated using
µM of compound compared with staurosporine, ADP-Glo Assay (Promega),
2
*
Sigmaplot software ver. 12.0.
In a series of resorcinol-type compounds, the effect of substituents at the C6 position of the
tetrahydroisoquinoline ring was examined. The aromatic ring (18d) was favored, followed by n-propane
(18b), cyclopropane (18c), or no substituent (18a). The benzyloxy-substituted compound 18d was the
most potent, with an IC50 value of 0.1
lowered potency (IC50 values of 8.5
µ
M. Halogen substitution on the terminal phenyl ring (18e and 18f
)
µM and 1.0 M, respectively). Meanwhile, methoxy substitution
µ
(18g) recovered the potency (IC50 = 0.2 µM).
Based on their sub-micromolar IC50 values, compounds 18d and 18g were selected for further
study. The catechol-type compound 17d was also chosen to compare the effects of the position of the
acryloyloxy substitution. The kinase selectivity profiles for these compounds were explored over some
TEC and SRC family kinases (Table 3). Overall, resorcinol-type compounds (18d and 18g) showed better
selectivity profiles than the catechol-type compound (17d) with lower kinase inhibition against EGFRK,

Int. J. Mol. Sci. 2020, 21, 8006
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interleukin-2-inducible T-cell kinase (ITK), TEC, and TXK tyrosine kinase (TXK). Compound 18g was
the most selective over EGFRK with 33.5% inhibition compared with 17d and 18d (87.0% and 60.5%,
respectively). All three compounds significantly inhibited BMX/ETK and ERBB4. This off target activity
could be a safety concern, but also provides the potential for additional applications.
Table 3. Kinase selectivity profiles of 17d, 18d, and 18g.
%
Inhibition 1
18d
Kinase
1
7d
18g
BMX/ETK
EGFRK
ERBB2
ERBB4
ITK
JAK3
TEC
TXK
98.7 ± 0.6
87.0 ± 0.3
43.4 ± 1.0
95.0 ± 0.1
75.1 ± 0.0
64.8 ± 0.3
84.1 ± 0.2
88.8 ± 0.1
95.5 ± 0.1
60.5 ± 4.4
82.3 ± 1.6
96.2 ± 0.7
52.8 ± 1.8
46.2 ± 4.2
71.3 ± 1.0
37.5 ± 3.3
96.0 ± 0.1
33.5 ± 0.5
78.8 ± 0.7
95.0 ± 0.1
46.0 ± 2.3
77.1 ± 3.0
66.4 ± 0.9
49.7 ± 2.5
1
Relative percentage inhibition at 20
µ
M of compound compared with staurosporine, HotSpot kinase assay (Reaction
90% inhibition; medium red, 80–90% inhibition;
light pink, 60–80% inhibition; no color, <60% inhibition. Values are expressed as means standard deviation (SD):
Biology Corp.). Compounds were tested in duplicate. Red,
≥
±
BMX/ETK, bone marrow X-linked kinase/epithelial and endothelial tyrosine kinase; EGFRK, epidermal growth
factor receptor kinase; ERBB, erythroblastic leukemia viral oncogene homolog; ITK, interleukin-2-inducible T-cell
kinase; JAK3, janus kinase 3; TEC, tyrosine protein kinase Tec; TXK, TXK tyrosine kinase.
2
.4. Molecular Docking Analysis
Molecular docking analysis of 18g was performed based on the human BTK structure (PDB ID:
3PJ3), which has an allosteric pocket induced by the DFG-out conformation (Figure 3). The results
suggested that the tetrahydroisoquinoline linker of 18g placed the lipophilic moiety in the allosteric
pocket of the BTK kinase domain as predicted for a type II inhibitor. The aminopyridine hinge
binder forms two hydrogen bonds with the hinge residue Glu475 and the gatekeeper residue
Thr474. The para-methoxybenzyl group is directed into the allosteric hydrophobic pocket formed
by Met449, Leu452, Leu512, and Phe517. The orientation of the lipophilic substituent was stabilized
by the teterahydroisoquinoline ring, which resulted in van der Waals interactions with Lys430.
In addition, the Michael acceptor linker phenyl ring was located in the cleft between Leu408 and
Gly480. This allowed hydrogen bonding between the carbonyl of the acryl group and the backbone
of Cys481.
Figure 3. Docking analysis of 18g (PDB ID: 3PJ3). (a) Predicted binding mode of 18g to the kinase
domain of BTK. Green dashed line, hydrogen bond interaction. (b) Hydrophobicity map of the active
site. Purple dashed line, alkyl-alkyl interaction.

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2
.5. Effects on Experimental Models of Hematological Malignancy
Next, we examined the effects of the most potent compound 18g and the representative catechol
compound 17d on two cell lines representative of hematological malignancy and compared them to
ibrutinib. Both compounds 17d and 18g showed similar GI50 values in Ramos cell lines, and were less
active than ibrutinib. In the Raji cell lines, 17d exhibited a GI50 value lower than 18g with an activity
similar to ibrutinib (Table 4). All three compounds showed selective cytotoxicity in the two tested
cancer cell lines over normal cells (MRC-5 and MCF10A). Among them, 18g showed the most selective
cytotoxicity against the tested cancer cell lines.
Table 4. In vitro cell growth inhibitory activity of 17d and 18g.
GI50 (µM) 1
Compound
Raji 2
Ramos 2
MRC-5 3
MCF10A 3
1
1
7d
8g
Ibrutinib
16.1 ± 0.5
25.4 ± 1.4
9.5 ± 0.2
12.5 ± 0.1
16.2 ± 1.2
6.3 ± 4.8
28.4 ± 4.0
33.4 ± 2.2
>100
>100
40.4 ± 0.9
4.6 ± 1.1
2
1
CellTiter-Glo assay (Promega). Values are expressed as means
8g were tested with four replicates and ibrutinib was tested with six replicates; compounds were tested with
±
standard deviation (SD); compounds 17d and
3
1
four replicates. GI50 values were calculated using GraphPad Prism: Raji and Ramos, human B Burkitt-lymphocyte
lymphoma cell lines; MRC-5, human lung normal cell; MCF10A, non-malignant human breast cell line.
We evaluated the in vivo efficacy of 17d and 18g in a Raji xenograft mouse model. Doxorubicin
hydrochloride (Dox) was used as a positive control, and 17d and 18g were administered at a dose
of 50 mg/kg once daily via i.p. injected for 2 weeks. There were no significant symptoms of toxicity
based on clinical and necropsy findings. The control group showed slight weight loss, but not
statistically significant, but no weight loss was observed in the 17d and 18g groups. Compound 18g
3
significantly reduced tumor size, with inhibition of 46.8% (229.5
±
116.5 mm ) compared with vehicle
3
(431.8 ± 155.2 mm ) (Figure 4).
Figure 4. In vivo activity of 17d and 18g in Raji xenograft model. (a) Body weight change, (b) tumor size.
Compounds were administered to Raji xenograft mice via i.p. (n = 5/group). Values are expressed as
means ± standard deviation (SD): * p < 0.05 (vs. Vehicle); mpk, mg/kg.
3
. Materials and Methods
3
.1. Chemistry
Most reagents and solvents were purchased from commercial sources and used without further
purification. Flash column chromatography was performed using silica gel 60 (Merck Millipore,
Burlington, MA, USA, 230–400 mesh) with the indicated solvents. To monitor the completion of the

Int. J. Mol. Sci. 2020, 21, 8006
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reaction, thin-layer chromatography (TLC) was performed using Kieselgel 60 F254 plates (Merck Millipore,
Burlington, MA, USA). IR spectra were recorded by a Nicolet iS5 spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA) using the ATR method. NMR spectra were recorded on an Avance III HD 500
1
13
spectrometer (Bruker, Billerica, MA, USA, H 500 MHz, and C 125 MHz). High performance liquid
chromatography (HPLC) was used to evaluate compound purity. A Waters 1525 (Waters Corporation,
Milford, MA, USA) system fitted with a reverse-phase column (Inertsil ODS-2 C18 column, 150 × 4.6 mm
i.d., GS Sciences, Tokyo, Japan) was eluted with 75% methanol 1 mL/min and monitored at 254 nm.
The purity was calculated based on the relative peak areas on the HPLC chromatogram. Mass spectra
(ESI) were obtained using an Agilent single quadrupole LC-MS high-resolution mass spectrometer.
3
.1.1. General Procedures for the Synthesis of Compound 6
◦
5 (1.0 equiv.) in EtOH at 0 C, was added tin (II) chloride dihydrate
To a mixture of compound
5.0 equiv.). After stirring at rt for 2 h, the mixture was basified with NaHCO (aq) and filtered onto a
(
3
celite pad with MeOH. The combined organic layers were concentrated under reduced pressure and
extracted with DCM. The combined organic layers were dried over MgSO , filtered, and concentrated
4
in vacuo. The residue was purified by flash column chromatography (n-hexane/EtOAc) to obtain
compound 6.
3
.1.2. Acrylic Acid 3-(6-Amino-5-piperazinylpyridin-3-yloxy)phenyl Ester (6a)
Brown solid (yield 4%); R = 0.40 (EtOAc 100%); IR (neat, cm 1) 3340, 2923, 2852, 1735, 1640, 1595,
−
f
1
1
7
8
466, 1367, 1335, 1272, 1244, 1176, 1137, 1043, 993, 967; H NMR (500 MHz, CDCl )
δ
7.63 (1H, br s),
3
.14 (1H, t, J = 8.0 Hz), 6.96 (1H, d, J = 2.5 Hz), 6.58 (1H, dd, J = 10.5, 17.0 Hz), 6.55 (1H, dd, J = 2.0,
.0 Hz), 6.49 (1H, dd, J = 2.0, 8.0 Hz), 6.38 (1H, t, J = 2.0 Hz), 6.33 (1H, dd, J = 1.5, 17.0 Hz), 5.75 (1H, dd,
13
J = 1.5, 10.5 Hz), 4.90 (2H, br s), 3.81 (2H, br s), 3.71 (2H, br s), 2.90 (4H, br s); C NMR (125 MHz,
CDCl3)
δ
165.8, 159.7, 157.8, 151.0, 150.2, 134.5, 133.7, 130.7, 128.9, 127.2, 121.1, 110.3, 109.2, 104.4, 51.0,
+
5
0.5, 46.3, 42.5; LCMS (ESI) m/z calcd for C H N O , 340.15; Found 341.5 [M + H] .
18
20
4
3
3
.1.3. Acrylic Acid 3-[6-Amino-5-(4-benzylpiperazinyl)pyridin-3-yloxy]phenyl Ester (6b)
Brown solid (yield 15%); R = 0.44 (n-hexane/EtOAc = 1:5); IR (neat, cm 1) 3058, 2942, 2820, 1664,
−
f
1
1
594, 1462, 1369, 1336, 1265, 1214, 1178, 1134, 996, 737, 700; H NMR (500 MHz, CDCl )
δ
7.69 (1H, d,
3
J = 2.5 Hz), 7.34–7.26 (6H, m), 6.98 (1H, d, J = 2.5 Hz), 6.83–6.80 (2H, m), 6.70 (1H, t, J = 2.0 Hz), 6.58 (1H,
dd, J = 1.0, 17.5 Hz), 6.28 (1H, dd, J = 10.5, 17.5 Hz), 6.00 (1H, dd, J = 1.0, 10.5 Hz), 4.63 (2H, br s),
3
.58 (2H, s), 2.93 (4H, br s), 2.60 (4H, br s); 13C NMR (125 MHz, CDCl3)
δ 164.3, 159.7, 151.6, 151.4,
1
45.1, 138.0, 135.2, 134.3, 132.8, 130.1, 129.3, 128.4, 127.9, 127.3, 120.1, 115.6, 114.2, 110.4, 63.1, 53.5, 50.6;
+
LCMS (ESI) m/z calcd for C H N O , 430.20; Found 431.7 [M + H] .
25
26
4
3
3
.1.4. Acrylic Acid 3-{6-Amino-5-[4-(4-fluorobenzyl)piperazinyl]pyridin-3-yloxy}phenyl Ester (6c)
−
1
Brown solid (yield 12%); R = 0.32 (n-hexane/EtOAc = 1.5:1); IR (neat, cm ) 2939, 2821, 1740,
f
1
1
602, 1507, 1479, 1402, 1366, 1312, 1243, 1220, 1157, 1025, 1002, 841, 796, 736, 700; H NMR (500 MHz,
CDCl3)
.81 (2H, t, J = 8.5 Hz), 6.70 (1H, t, J = 2.5 Hz), 6.58 (1H, dd, J = 1.0, 17.5 Hz), 6.28 (1H, dd, J = 10.5,
7.5 Hz), 6.00 (1H, dd, J = 1.0, 10.5 Hz), 4.71 (2H, br s), 3.53 (2H, s), 2.93 (4H, br s), 2.58 (4H, br s);
δ 7.68 (1H, d, J = 2.5 Hz), 7.31–7.27 (3H, m), 7.01 (2H, t, J = 8.5 Hz), 6.98 (1H, d, J = 2.5 Hz),
6
1
13
C NMR (125 MHz, CDCl3) δ 164.3, 162.2 (d, JC–F = 243.9 Hz), 159.7, 151.7, 151.5, 145.1, 135.3, 134.2,
1
1
33.7 (d, JC–F = 3.1 Hz), 132.9, 130.7 (d, JC–F = 7.9 Hz), 130.2, 127.9, 120.3, 115.6, 115.2 (d, JC–F = 21.1 Hz),
+
14.2, 110.4, 62.3, 53.5, 50.6; LCMS (ESI) m/z calcd for C H FN O , 448.19; Found 449.6 [M + H] .
25
25
4
3
3
.1.5. Acrylic Acid 3-{6-Amino-5-[4-(4-chlorobenzyl)piperazinyl]pyridin-3-yloxy}phenyl Ester (6d)
Brown solid (yield 10%); R = 0.32 (n-hexane/EtOAc = 1.5:1); IR (neat, cm 1) 3048, 2940, 2821, 1742,
−
f
1
1
594, 1488, 1402, 1367, 1311, 1293, 1247, 1212, 1157, 1086, 1002, 795, 736; H NMR (500 MHz, CDCl )
3

Int. J. Mol. Sci. 2020, 21, 8006
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δ
7.69 (1H, d, J = 2.5 Hz), 7.32–7.26 (5H, m), 6.98 (1H, d, J = 2.5 Hz), 6.83–6.80 (2H, m), 6.70 (1H, t,
J = 2.5 Hz), 6.58 (1H, dd, J = 1.0, 17.5 Hz), 6.28 (1H, dd, J = 10.5, 17.5 Hz), 6.00 (1H, dd, J = 1.0, 10.5 Hz),
.63 (2H, br s), 3.53 (2H, s), 2.93 (4H, br s), 2.58 (4H, br s); 13C NMR (125 MHz, CDCl3)
164.4, 159.8,
51.7, 151.5, 145.1, 136.6, 135.2, 134.5, 133.0, 132.9, 130.5, 130.2, 128.6, 127.9, 120.2, 115.6, 114.2, 110.4,
4
1
δ
+
6
2.3, 53.5, 50.6; LCMS (ESI) m/z calcd for C H ClN O , 464.16; Found 465.6 [M + H] .
25 25 4 3
3
.1.6. Acrylic Acid 3-[6-Amino-5-(4-phenethylpiperazinyl)pyridin-3-yloxy]phenyl Ester (6e)
−
1
Brown solid (yield 19%); R = 0.48 (EtOAc 100%); IR (neat, cm ) 3025, 2942, 2819, 1743, 1608,
f
1
1
464, 1402, 1372, 1336, 1247, 1152, 1023, 1001, 775, 735, 700; H NMR (500 MHz, CDCl )
δ
7.70 (1H, d,
3
J = 2.5 Hz), 7.31–7.28 (3H, m), 7.23–7.21 (3H, m), 7.00 (1H, d, J = 2.5 Hz), 6.83–6.81 (2H, m), 6.71 (1H, t,
J = 2.5 Hz), 6.59 (1H, dd, J = 1.0, 17.5 Hz), 6.29 (1H, dd, J = 10.5, 17.5 Hz), 6.01 (1H, dd, J = 1.0, 10.5 Hz),
4
.65 (2H, br s), 2.97 (4H, br s), 2.85–2.82 (2H, m), 2.69–2.66 (6H, m); 13C NMR (125 MHz, CDCl3)
δ 164.4,
1
1
59.7, 151.7, 151.4, 145.1, 140.3, 135.2, 134.4, 132.9, 130.2, 128.8, 128.6, 127.9, 126.3, 120.2, 115.6, 114.2,
+
10.4, 60.6, 53.7, 50.6, 33.8; LCMS (ESI) m/z calcd for C H N O , 444.22; Found 445.6 [M + H] .
26
28
4
3
3
.1.7. Acrylic Acid 3-(6-Amino-5-{4-[2-(4-chlorophenyl)ethyl]piperazinyl}pyridin-3-yloxy)phenyl
Ester (6f)
Brown solid (yield 4%); R = 0.24 (n-hexane/EtOAc = 1:50); IR (neat, cm 1) 3355, 2938, 2821, 1742,
−
f
1
1
608, 1464, 1402, 1336, 1247, 1154, 1002, 776; H NMR (500 MHz, CDCl )
δ
7.69 (1H, d, J = 2.5 Hz),
3
7
.29 (1H, t, J = 8.5 Hz), 7.25 (2H, d, J = 8.5 Hz), 7.14 (2H, d, J = 8.5 Hz), 6.99 (2H, d, J = 2.5 Hz), 6.83–6.80
(
2H, m), 6.71 (1H, t, J = 2.5 Hz), 6.58 (1H, dd, J = 1.0, 17.5 Hz), 6.29 (1H, dd, J = 10.5, 17.5 Hz), 6.01 (1H,
dd, J = 1.0, 10.5 Hz), 4.68 (2H, br s), 2.96 (4H, br s), 2.81–2.78 (2H, m), 2.65–2.62 (6H, m); LCMS (ESI)
+
m/z calcd for C H ClN , 478.18; Found 479.7 [M + H] .
26
27
4
3.1.8. 4-[5-(3-Acryloyloxyphenoxy)-2-aminopyridin-3-yl]piperazine carboxylic Acid Benzyl Ester (6g)
−
1
Brown solid (yield 23%); R = 0.36 (n-hexane/EtOAc = 1:5); IR (neat, cm ) 3351, 3062, 2827, 1742,
f
1
1
688, 1608, 1466, 1403, 1361, 1333, 1284, 1245, 1152, 1087, 1044, 992, 908, 735, 699; H NMR (500 MHz,
CDCl3)
.82 (2H, td, J = 2.0, 8.0 Hz), 6.70 (1H, t, J = 2.5 Hz), 6.58 (1H, dd, J = 1.0, 17.5 Hz), 6.28 (1H, dd,
J = 10.5, 17.5 Hz), 6.01 (1H, dd, J = 1.0, 10.5 Hz), 5.16 (2H, s), 4.65 (2H, br s), 3.65 (4H, br s), 2.88 (4H,
br s); 13C NMR (125 MHz, CDCl3)
164.3, 159.6, 155.4, 151.7, 151.3, 145.2, 136.7, 135.1, 134.6, 133.0,
30.3, 128.7, 128.3, 128.1, 127.9, 120.4, 115.7, 114.3, 110.5, 67.5, 50.5, 44.4; LCMS (ESI) m/z calcd for
δ 7.72 (1H, d, J = 2.5 Hz), 7.39–7.32 (5H, m), 7.29 (1H, t, J = 8.0 Hz), 6.95 (1H, d, J = 2.5 Hz),
6
δ
1
+
C H N O , 474.19; Found 475.7 [M + H] .
26
26
4
5
3
.1.9. General Procedures for the Synthesis of Compounds 17 and 18
To a mixture of compound 15 or 16 (1.0 equiv.) in THF/EtOH (2:1) was added tin (II) chloride
dihydrate (5.0 equiv.). After stirring at rt for 2 h, the mixture was basified with NaHCO (aq) and
3
filtered onto a celite pad with MeOH. The combined organic layers were concentrated under reduced
pressure and extracted with EtOAc. The combined organic layers were dried over MgSO , filtered, and
4
concentrated in vacuo. The residue was purified by flash column chromatography (n-hexane/EtOAc)
to obtain compound 17 or 18.
3
.1.10. Acrylic Acid 2-[6-Amino-5-(3,4-dihydro-1H-isoquinolin-2-yl)pyridin-3-yloxy]phenyl Ester (17a)
Yellow solid (yield 31%); R = 0.60 (n-hexane/EtOAc = 1:1); IR (neat, cm 1) 2980, 2888, 1596, 1460,
−
f
1
1
380, 1315, 1230, 1315, 1238, 1150, 1023, 971; H NMR (500 MHz, CDCl )
δ
7.69 (1H, d, J = 2.5 Hz),
3
7.21–7.14 (5H, m), 7.10–7.06 (2H, m), 7.03 (1H, d, J = 2.5 Hz), 6.89 (1H, dd, J = 1.5, 8.5 Hz), 6.59 (1H, dd,
J = 1.0, 17.5 Hz), 6.32 (1H, dd, J = 10.5, 17.5 Hz), 6.00 (1H, dd, J = 1.0, 10.5 Hz), 4.66 (2H, br s), 4.06 (2H,
s), 3.22 (2H, t, J = 6.0 Hz), 3.01 (2H, t, J = 6.0 Hz); 13C NMR (125 MHz, CDCl3)
δ
164.1, 151.4, 150.2,

Int. J. Mol. Sci. 2020, 21, 8006
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1
45.8, 140.8, 135.1, 134.5, 133.9, 132.9, 129.1, 127.6, 127.1, 126.6, 123.7, 123.3, 119.9, 118.0, 53.2, 48.9, 29.8;
+
LCMS (ESI) m/z calcd for C H N O , 387.16; Found 388.16 [M + H] .
23
21
3
3
3
2
.1.11. Acrylic Acid
-[6-Amino-5-(6-benzyloxy-3,4-dihydro-1H-isoquinolin-2-yl)pyridin-3-yloxy]phenyl Ester (17d)
−
1
Ivory solid (yield 23%); R = 0.52 (n-hexane/EtOAc = 1:1); IR (neat, cm ) 2980, 2888, 1735, 1610,
f
1
1
461, 1380, 1239, 1150, 1015, 969; H NMR (500 MHz, CDCl )
δ
7.68 (1H, d, J = 2.5 Hz), 7.42 (2H, d,
3
J = 7.5 Hz), 7.38 (2H, t, J = 7.5 Hz), 7.31 (1H, t, J = 7.5 Hz), 7.15 (2H, t, J = 7.5 Hz), 7.07 (1H, t, J = 7.5
Hz), 7.01 (1H, d, J = 2.5 Hz), 6.97 (1H, d, J = 8.5 Hz), 6.88 (1H, d, J = 8.0 Hz), 6.81–6.77 (2H, m), 6.57 (1H,
dd, J = 1.0, 17.0 Hz), 6.31 (1H, dd, J = 10.5, 17.0 Hz), 5.98 (1H, dd, J = 1.0, 10.5 Hz), 5.04 (2H, s), 4.72 (2H,
br s), 3.98 (2H, s), 3.17 (2H, t, J = 5.5 Hz), 2.95 (2H, t, J = 5.5 Hz); 13C NMR (125 MHz, CDCl3)
δ
164.1,
1
1
59.8, 157.6, 154.5, 151.4, 150.2, 145.7, 140.7, 137.2, 135.1, 135.1, 133.7, 132.9, 130.2, 128.7, 128.1, 127.5,
27.5, 127.1, 126.9, 123.6, 123.2, 119.8, 117.9, 114.7, 113.2, 70.2, 52.7, 48.7, 30.0; LCMS (ESI) m/z calcd for
+
C H N O , 493.20; Found 494.20 [M + H] ; purity 97%.
30
27
3
4
3
.1.12. Acrylic Acid 3-[6-Amino-5-(3,4-dihydro-1H-isoquinolin-2-yl)pyridin-3-yloxy]phenyl Ester (18a)
Yellow solid (yield 30%); R = 0.60 (n-hexane/EtOAc = 1:1); IR (neat, cm 1) 2980, 2888, 1461, 1382,
−
f
1
1
239, 1151, 966; H NMR (500 MHz, CDCl )
δ
7.73 (1H, d, J = 2.5 Hz), 7.30 (1H, t, J = 8.0 Hz), 7.21–7.15
3
(
3H, m), 7.07 (1H, d, J = 7.0 Hz), 7.06 (1H, d, J = 2.5 Hz), 6.83 (1H, dd, J = 2.5, 8.5 Hz), 6.82 (1H, dd,
J = 2.0, 8.5 Hz), 6.73 (1H, dd, J = 2.0, 2.5 Hz), 6.59 (1H, d, J = 1.5, 17.5 Hz), 6.29 (1H, dd, J = 10.5,
7.5 Hz), 6.01 (1H, d, J = 1.5, 10.5 Hz), 4.73 (2H, br s), 4.09 (2H, s), 3.24 (2H, t, J = 6.0 Hz), 3.02 (2H, t,
1
13
J = 6.0 Hz); C NMR (125 MHz, CDCl3)
32.9, 130.2, 129.1, 127.9, 126.7, 126.5, 126.1, 120.9, 115.7, 114.2, 110.4, 53.3, 48.9, 29.6; LCMS (ESI) m/z
calcd for C H N O , 387.16; Found 388.16 [M + H]+.
δ 164.4, 159.7, 151.8, 151.6, 144.9, 135.4, 134.3, 133.8, 133.8,
1
23
21
3
3
3
.1.13. Acrylic Acid 3-[6-Amino-5-(6-propoxy-3,4-dihydro-1H-isoquinolin-2-yl)pyridin-3-yloxy]phenyl
Ester (18b)
−
1
Yellow solid (yield 23%); R = 0.56 (n-hexane/EtOAc = 1:1); IR (neat, cm ) 2980, 2883, 1595,
f
1
1
459, 1399, 1381, 1314, 1237, 1151, 1023, 981; H NMR (500 MHz, CDCl )
δ
7.72 (1H, d, J = 2.5 Hz),
3
7
(
4
.30 (1H, t, J = 8.5 Hz), 7.05 (1H, d, J = 2.5 Hz), 6.98 (1H, d, J = 8.5 Hz), 6.83–6.81 (2H, m), 6.75–6.70
3H, m), 6.58 (1H, dd, J = 1.0, 17.5 Hz), 6.29 (1H, dd, J = 10.5, 17.5 Hz), 6.00 (1H, dd, J = 1.0, 10.5 Hz),
.73 (2H, br s), 4.02 (2H, s), 3.91 (2H, t, J = 7.0 Hz), 3.21 (2H, t, J = 6.0 Hz), 2.97 (2H, t, J = 6.0 Hz), 1.80 (2H,
1
3
sext., J = 7.0 Hz), 1.03 (3H, t, J = 7.0 Hz); C NMR (125 MHz, CDCl3)
δ
164.3, 159.6, 157.8, 151.6, 151.5,
1
5
44.9, 135.3, 134.9, 133.8, 133.2, 132.8, 130.2, 127.8, 127.3, 126.2, 125.4, 120.9, 115.5, 114.3, 114.1, 110.2,
+
2.7, 52.0, 51.5, 48.7, 29.2; LCMS (ESI) m/z calcd for C H N O , 445.20; Found 446.21 [M + H] .
26
27
3
4
3
3
.1.14. Acrylic Acid
-[6-Amino-5-(6-cyclopropylmethoxy-3,4-dihydro-1H-isoquinolin-2-yl)pyridin-3-yloxy]phenyl
Ester (18c)
Yellow solid (yield 67%); R = 0.60 (n-hexane/EtOAc = 1:1); IR (neat, cm 1) 2981, 2884, 1595, 1459,
−
f
1
1
399, 1369, 1311, 1276, 1237, 1153, 1129, 1021; H NMR (500 MHz, CDCl )
δ
7.70 (1H, d, J = 2.5 Hz),
3
7.30 (1H, t, J = 8.0 Hz), 7.05 (1H, d, J = 2.5 Hz), 6.98 (1H, d, J = 8.5 Hz), 6.82 (2H, dd, J = 2.5, 8.5
Hz), 6.76–6.70 (3H, m), 6.59 (1H, dd, J = 1.5, 17.5 Hz), 6.29 (1H, dd, J = 10.5, 17.5 Hz), 6.01 (1H, dd,
J = 1.5, 10.5 Hz), 4.79 (2H, br s), 4.02 (2H, s), 3.79 (2H, d, J = 7.0 Hz), 3.21 (2H, t, J = 6.0 Hz), 2.97 (2H, t,
J = 6.0 Hz), 1.30–1.23 (1H, m), 0.64 (2H, dd, J = 5.0, 12.5 Hz), 0.34 (2H, dd, J = 5.0, 10.0 Hz); 13C NMR
(
125 MHz, CDCl3)
δ 164.3, 159.6, 157.7, 151.6, 151.5, 144.9, 135.3, 134.9, 133.9, 132.8, 130.1, 127.8, 127.7,
1
27.4, 126.4, 120.9, 120.6, 115.5, 114.3, 114.1, 113.0, 110.3, 52.7, 50.7, 48.6, 40.6, 29.8; LCMS (ESI) m/z calcd
+
for C H N O , 457.20; Found 458.22 [M + H] .
27
27
3
4

Int. J. Mol. Sci. 2020, 21, 8006
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3
3
.1.15. Acrylic Acid
-[6-Amino-5-(6-benzyloxy-3,4-dihydro-1H-isoquinolin-2-yl)pyridin-3-yloxy]phenyl Ester (18d)
Yellow solid (yield 63%); R = 0.61 (n-hexane/EtOAc = 1:1); IR (neat, cm 1) 2980, 1739, 1607, 1462,
−
f
1
1
401, 1378, 1313, 1237, 1151, 1023, 1001; H NMR (500 MHz, CDCl )
δ
7.72 (1H, d, J = 2.5 Hz), 7.43 (2H,
3
d, J = 7.0 Hz), 7.38 (2H, t, J = 7.5 Hz), 7.33–7.28 (2H, m), 7.04 (1H, d, J = 2.5 Hz), 6.99 (1H, d, J = 8.0 Hz),
6
6
.83–6.78 (4H, m), 6.72 (1H, t, J = 2.5 Hz), 6.58 (1H, dd, J = 1.0, 17.5 Hz), 6.29 (1H, dd, J = 10.5, 17.5 Hz),
.00 (1H, dd, J = 1.5, 10.5 Hz), 5.05 (2H, s), 4.72 (2H, br s), 4.02 (2H, s), 3.21 (2H, t, J = 6.0 Hz), 2.97 (2H, t,
13
J = 6.0 Hz); C NMR (125 MHz, CDCl3)
δ
164.4, 159.8, 157.6, 154.5, 151.7, 151.6, 145.0, 137.2, 135.2,
1
7
35.1, 134.5, 132.9, 130.2, 128.7, 128.1, 127.9, 127.6, 127.5, 126.9, 120.5, 115.6, 114.8, 114.2, 113.3, 110.4,
+
0.2, 52.8, 48.7, 29.9; LCMS (ESI) m/z calcd for C H N O , 493.20; Found 494.21 [M + H] .
30
27
3
4
3
3
.1.16. Acrylic Acid
-{6-Amino-5-[6-(4-fluorobenzyloxy)-3,4-dihydro-1H-isoquinolin-2-yl]pyridin-3-yloxy}phenyl
Ester (18e)
Yellow solid (yield 4%); R = 0.60 (n-hexane/EtOAc = 1:1); IR (neat, cm 1) 3321, 2922, 2850, 1735,
−
f
1
1
603, 1508, 1460, 1400, 1379, 1235, 1151, 1022; H NMR (500 MHz, CDCl )
δ
7.71 (1H, d, J = 2.0 Hz), 7.42
3
(
8
1
2
1
6
2H, m), 7.32 (1H, t, J = 8.0 Hz), 7.09 (3H, m), 7.00 (1H, d, J = 8.5 Hz), 6.84 (2H, m), 6.81 (1H, dd, J = 2.0,
.5 Hz), 6.77 (1H, d, J = 2.0 Hz), 6.73 (1H, d, J = 2.0 Hz), 6.57 (1H, d, J = 17.0 Hz), 6.32 (1H, dd, J = 10.5
7.0 Hz), 6.02 (1H, d, J = 10.5 Hz), 5.01 (2H, s), 4.82 (2H, br s), 4.03 (2H, s), 3.23 (2H, t, J = 6.0 Hz),
.99 (2H, t, J = 6.0 Hz); 13C NMR (125 MHz, CDCl3)
164.4, 159.7, 157.4, 151.8, 151.7, 145.0, 135.4, 132.9,
30.2, 129.4, 129.4, 127.9, 127.6, 127.0, 120.8, 115.9, 115.7, 115.7, 115.5, 115.3, 114.8, 114.2, 113.3, 110.4,
,
δ
+
9.5, 53.6, 52.8, 48.7, 29.9; LCMS (ESI) m/z calcd for C H FN O , 511.19; Found 512.19 [M + H] .
30 25 3 4
3
3
.1.17. Acrylic Acid
-{6-Amino-5-[6-(4-chlorobenzyloxy)-3,4-dihydro-1H-isoquinolin-2-yl]pyridin-3-yloxy}phenyl
Ester (18f)
Yellow solid (yield 55%); R = 0.62 (n-hexane/EtOAc = 1:1); IR (neat, cm 1) 2980, 2952, 2888, 1458,
−
f
1
1
443, 1234, 1148, 1015; H NMR (500 MHz, CDCl )
δ
7.72 (1H, d, J = 2.5 Hz), 7.36 (4H, s), 7.30 (1H, t,
3
J = 8.5 Hz), 7.05 (1H, d, J = 2.5 Hz), 7.00 (1H, d, J = 8.5 Hz), 6.83 (1H, d, J = 8.0 Hz), 6.82 (1H, d, J = 8.0
Hz), 6.79 (1H, dd, J = 2.5, 8.0 Hz), 6.76 (1H, d, J = 2.5 Hz), 6.72 (1H, t, J = 2.5 Hz), 6.59 (1H, dd, J = 1.0,
1
4
7.0 Hz), 6.29 (1H, dd, J = 10.0, 17.0 Hz), 6.01 (1H, dd, J = 1.0, 10.0 Hz), 5.02 (2H, s), 4.72 (2H, br s),
.03 (2H, s), 3.22 (2H, t, J = 5.5 Hz), 2.98 (2H, t, J = 5.5 Hz); 1 C NMR (125 MHz, CDCl3)
3
δ
164.4, 159.8,
1
1
5
57.3, 151.7, 151.6, 145.0, 135.7, 135.2, 134.4, 133.9, 132.9, 130.2, 128.9, 128.8, 128.7, 127.9, 127.6, 126.9,
20.6, 115.6, 114.8, 114.2, 113.2, 110.4, 69.4, 52.8, 48.7, 29.9; LCMS (ESI) m/z calcd for C H ClN O ,
30
25
3
4
+
27.16; Found 528.16 [M + H] .
3
3
.1.18. Acrylic Acid
-{6-Amino-5-[6-(4-methoxybenzyloxy)-3,4-dihydro-1H-isoquinolin-2-yl]pyridin-3-yloxy}phenyl
Ester (18g)
Yellow solid (yield 42%); R = 0.57 (n-hexane/EtOAc = 1:1); IR (neat, cm 1) 3345, 2980, 2970, 2930,
−
f
1
1
740, 1609, 1513, 1462, 1400, 1379, 1304, 1241, 1153, 1025, 1001; H NMR (500 MHz, CDCl )
δ
7.70 (1H,
3
d, J = 2.5 Hz), 7.33 (2H, d, J = 8.5 Hz), 7.28 (1H, t, J = 8.0 Hz), 7.03 (1H, d, J = 2.5 Hz), 6.96 (1H, d,
J = 8.5 Hz), 6.90 (2H, d, J = 8.5 Hz), 6.82–6.77 (3H, m), 6.76 (1H, d, J = 2.0 Hz), 6.73 (1H, t, J = 2.5 Hz),
6
.57 (1H, dd, J = 1.0, 17.5 Hz), 6.27 (1H, dd, J = 10.5, 17.5 Hz), 5.98 (1H, dd, J = 1.0, 10.5 Hz), 4.95 (2H,
13
s), 4.82 (2H, br s), 4.00 (2H, s), 3.78 (3H, s), 3.18 (2H, t, J = 6.0 Hz), 2.95 (2H, t, J = 6.0 Hz); C NMR
125 MHz, CDCl3) 164.4, 159.8, 159.6, 157.7, 151.7, 151.7, 145.0, 135.3, 135.1, 134.4, 132.9, 130.2, 129.3,
29.2, 127.9, 127.5, 126.8, 120.6, 115.6, 114.8, 114.2, 114.2, 113.3, 110.4, 70.0, 55.5, 52.8, 48.8, 29.9; LCMS
(
1
δ
+
(ESI) m/z calcd for C H N O , 523.21; Found 524.22 [M + H] ; purity 95%.
31 29 3 5

Int. J. Mol. Sci. 2020, 21, 8006
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3
.2. BTK Kinase Assay
3
.2.1. ADP-Glo Assay
To examine the enzyme activity, a kinase assay was performed using the ADP-Glo kinase assay
kit (Promega, Madison, WI, USA) and the BTK enzyme system (Promega, Madison, WI, USA).
Kinase reactions were performed using 10 M ATP. The amount of the enzyme was determined as the
µ
concentration giving a signal-to-background ratio of 10 by titrating before using each enzyme batch.
For 30 min, the inhibitor and enzyme in the kinase buffer were pre-incubated at room temperature. The
ATP/substrate mixture was added and incubated for 30 min in a total kinase reaction volume of 10
at room temperature. The kinase reaction solution and ADP-Glo reagent were mixed in a 1:1 ratio (total
L). After incubation for 30 min at room temperature, a kinase detection reagent (20 L) was added
for another 30 min. The luminescence intensity was read on a GloMax Discover (Promega, Madison,
WI, USA). For the single-dose screening enzyme inhibition assays, compounds were tested at 20 M.
Since the staurosporine completely inhibits BTK at 20 M, it was used as the positive control and
the relative percentage inhibition value was calculated compared to staurosporine as 100%. The IC50
values were measured for a 7-dose, 10-fold serial dilution starting at 100 M, and then calculated using
four parameter logistic standard curve fitting using Sigmaplot software ver. 12.0.
µL
2
0
µ
µ
®
µ
µ
µ
3
.2.2. HotSpot Kinase Assay
In vitro profiling of BTK, BMX, EGFRK, ERBB2, ERBB4, ITK, JAK3, TEC, and TXK kinases was
performed at Reaction Biology Corporation (Malvern, PA, USA) [18]. The kinase and substrate pairs
were prepared in reaction buffer: 20 mM HEPES at pH 7.5, 10 mM MgCl , 1 mM EGTA, 0.02% Brij
2
3
5, 0.02 mg/mL BSA, 0.1 mM Na VO , 2 mM DTT, and 1% DMSO. Compounds were added into the
3 4
33
kinase reaction mixture and incubated for 20 min. Then, P-ATP was delivered to the reaction mixture
to initiate the reaction which was carried out at 10 M total ATP. After 2 h at room temperature, the
µ
reaction mixture was spotted onto P81 ion exchange paper, and then kinase activity was detected by the
filter-binding method. Compounds were tested for single-dose enzyme inhibition at a concentration of
2
0
µ
M. For the control compound, staurosporine was tested in a 10-dose IC50 mode. To determine
their IC50 values, the compounds were tested at 10-concentrations with 3-fold serial dilution starting at
00 µM.
1
3
.3. Molecular Docking Analysis
Molecular docking was performed using CDOCKER, Discovery Studio (DS) 2020 (Dassault
Systèmes, BIOVIA, San Diego, CA, USA). The crystal structure of BTK (PDB ID: 3PJ3) [15] was obtained
from the protein data bank. The docking parameters were set to default. The ligands and protein were
prepared using the CHARMm forcefield. Water molecules were removed and hydrogen atoms were
added during the preparation process. The active site was identified from the original ligand of 3PJ3
using the Define and Edit Binding Site tool. The missing loops of the 3PJ3 protein were not added
back, and we set the CDOCKER parameters to give 10 diverse poses, and the pose cluster radius as 0.1
for precise docking results. The docking results were analyzed and visualized using Discovery Studio.
3
.4. In Vitro Growth Inhibition
Cellular toxicity of the compounds was measured using the CellTiter-Glo luminescent cell viability
assay (Promega, Madison, WI, USA). Raji (ATCC CCL-86) and Ramos (ATCC CRL-1596) cells were
4
seeded on an opaque-walled 96-well plate (1
of 17d and 18g. After 48 h, 100
×
10 cells/well) and treated with various concentrations
µ
L of CellTiter-Glo Reagent (Promega, Madison, WI, USA) was added
to each well and the plate was incubated at room temperature for 10 min. The luminescence signal
was measured using a GloMax-Multi Detection System (Promega, Madison, WI, USA) and GI50 values
were calculated using relative IC50 model in GraphPad Prism (GraphPad, San Diego, CA, USA).

Int. J. Mol. Sci. 2020, 21, 8006
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3
.5. In Vivo Xenograft Model
The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of
Korea Research Institute of Bioscience and Biotechnology on 28 January 2020 and the approval number was
6
registered as KRIBB-AEC-19277. Raji cells (9
×
10 cells/mouse) were inoculated subcutaneously in the right
flank of 6-week-old female BALB/c nu/nu mice (Orient Bio, Seongnam, Gyeonggi-do, Korea). After 1 week,
the mice were randomly distributed between the control and treatment groups, and treated five times per
week with vehicle (5% DMSO and 5% Tween 80 in corn oil), 2 mg/kg of doxorubicin hydrochloride, or
50 mg/kg of compound 17d and 18g. During treatment, tumor volumes were measured with vernier calipers
and estimated by the formula: length (mm) width (mm) height (mm)/2, and body weight was monitored
×
×
three times a week. On day 14, the mice were sacrificed and tumor weights were measured. Statistical
analyses were carried out using GraphPad Prism (GraphPad, San Diego, CA, USA). Changes in body
weight and tumor volume were analyzed using two-way ANOVA and Bonferroni multiple comparison test,
and p values of <0.05 were considered statistically significant.
4
. Conclusions
In summary, 5-phenoxy-2-aminopyridine derivatives were designed and synthesized as covalent BTK
inhibitors and their in vitro and in vivo effects in experimental models of hematological malignancy were
tested. Of the tested compounds, 18g showed the highest in vitro enzyme inhibitory activity and best
selectivity profile. As shown by molecular docking, introducing a tetrahydroisoquinoline linker directed
the lipophilic moiety to occupy the allosteric pocket that was generated by the DFG-out conformation,
which can provide novel perspectives for future structure-based drug discovery of a BTK inhibitors
with a unique BTK binding mode. Further efforts will include corroboration of irreversible binding of
the Michael acceptor-containing compounds by comparing their inhibitory activity against wild-type
and mutant (C481S) BTK. Compound 18g also showed good antiproliferative activity in both Raji and
Ramos cell lines in vitro and the Raji xenograft mouse model in vivo. Therefore, we propose the novel
5-phenoxy-2-aminopyridine derivatives as potent and selective BTK inhibitors with the potential for further
development as chemotherapeutic and anti-inflammatory agents.
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/21/
8
006/s1.
Author Contributions: Conceptualization, E.L. and R.J.; chemical synthesis, data preparation, and investigation,
E.L., H.C., D.K.L., J.H. and J.H.J.; data analysis, E.L. and J.-H.R.; in vitro and in vivo data curation, B.J.C. and J.S.K.;
writing—original draft preparation, E.L.; writing—review and editing, supervision, and project administration,
R.J. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Research Foundation (NRF) of Korea, supported by the
Korean government (MSIP), grant number 2019R1F1A1057386 and 2014R1A2A1A11052761.
Conflicts of Interest: The authors declare no conflict of interest.

Int. J. Mol. Sci. 2020, 21, 8006
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Abbreviations
FDA
JAK3
EGFRK
ATP
SRC
Boc
TFA
DCM
DMF
TEA
PPA
THF
DMSO
ADP
IC50
Bn
Food and Drug Administration
Janus kinase 3
Epidermal growth factor receptor kinase
Adenosine triphosphate
Proto-oncogene tyrosine-protein kinase Src
tert-Butyloxycarbonyl protecting group
Trifluoroacetic acid
Dichloromethane
Dimethylformamide
Triethylamine
Polyphosphoric acid
Tetrahydrofuran
Dimethyl sulfoxide
Adenosine diphosphate
The half maximal inhibitory concentration
Benzyl
Me
Methyl
Et
Ethyl
Pr
Propyl
Ac
Acetyl
GI50
The half maximal growth inhibitory concentration
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