Synthesis and biological evaluation of novel 7-substituted 3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amines as potent Bruton's tyrosine kinase (BTK) inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.08.039

A series of novel 7-substituted 3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amines as potent BTK inhibitors were designed, synthesized and evaluated. These thieno[3,2-c]pyridin-4-amine derivatives displayed variant inhibitory activities against BTK in vitro

Full text of DOI:10.1016/j.bmc.2015.08.039

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel 7-substituted
3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amines
as potent Bruton’s tyrosine kinase (BTK) inhibitors
a,
⇑
,y
b,y
c
b
b
b
b
Minhang Xin
Hua Xiang
a
, Xinge Zhao , Wei Huang , Qiu Jin , Gang Wu , Yazhou Wang , Feng Tang ,
d,
⇑
Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an 710061, PR China
Jiangsu Simcere Pharmaceutical Co. Ltd, Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No. 699-18, Xuan Wu District, Nanjing 210042, PR China
Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, No. 24, Tongjiaxiang, Nanjing 210009, PR China
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 7-substituted 3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amines as potent BTK inhibi-
tors were designed, synthesized and evaluated. These thieno[3,2-c]pyridin-4-amine derivatives displayed
variant inhibitory activities against BTK in vitro. Among these, 7-pyrazol-4-yl substituted 3-(4-phe-
noxyphenyl)thieno[3,2-c]pyridin-4-amine subseries showed high BTK inhibition and several compounds
displayed superior BTK inhibitory activity. Comprehensive SAR was disclosed and compound 13b showed
excellent potency (IC50 = 11.8 nM), outstanding hydrophilicity (AlogP = 3.53), and relatively good kinase
selectivity, being a promising lead for further evaluation.
Received 19 June 2015
Revised 26 August 2015
Accepted 27 August 2015
Available online xxxx
Keywords:
BTK inhibitors
Thieno[3,2-c]pyrid-4-amine
Inhibitory activity
Kinase selectivity
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
Bruton’s tyrosine kinase (BTK) belongs to the Tec family of non-
licensed to Lilly.4 Therefore, BTK inhibitors have now attracted
much attention, and many research groups are eager to develop
BTK inhibitors.
receptor protein tyrosine kinases which is mostly expressed in
hematopoietic cells such as B cells, mast cells and macrophages.
BTK plays a key role in multiple cellular signaling pathways such
as B cell receptor and Fc receptor signaling cascades. Thus, BTK is
regarded as a potential therapeutic target for treating hematologi-
During recent years, many BTK inhibitors have been reported,
and some of them have even advanced to clinical stages, such as
ibrutinib (1), HM-71224 (2), ONO-4059 (3), spebrutinib
5
(4, CC-292, AVL-292) and RN-486 (5). Among these BTK inhibitors,
pyrazolo[3,4-d]pyrimidines, pyrimidines and 5-phenylpyridinones
are the major representative structural scaffolds. Ibrutinib contains
the pyrazolo[3,4-d]pyrimidine scaffold, whereas ONO-4059
(3, phase II) has a nucleus of 7H-purin-8(9H)-one, that is, struc-
turally derivatized from pyrazolo[3,4-d]pyrimidine. Spebrutinib
1,2
cal malignancies and autoimmune diseases.
molecular BTK inhibitor ibrutinib (1, Imbruvica ) was approved
Recently, a small
TM
by FDA for treating mantle cell lymphoma (MCL) and chronic lym-
3
phocytic leukemia (CLL) , which provided the evidence that BTK
inhibitors have become effective therapies for hematological
malignancies in clinic. Meanwhile, BTK inhibitors are still ongoing
in clinical evaluation to identify their use for treating autoimmune
diseases such as rheumatoid arthritis (RA). Recently, a BTK agent
named HM71224 (2) is reported to come through phase I proof-
of-mechanism studies, which is developed by Hanmi Pharmaceuti-
cals with the purpose of autoimmune diseases therapy, and now
has a pyrimidine central core, while HM71224 contains a
thieno[3,2-d]pyrimidine skeleton, that is, considered to be a struc-
tural mimic of pyrimidine. RN-486 possesses a fundamental core of
5-phenylpyridin-2(1H)-one, that was structurally similar to the
6
–16
pharmacophore of CGI-1746 (6) and GDC-0834 (7)
(Fig. 1).
Inspired by the pioneering structural scaffolds, our group
recently reported our medicinal chemistry work on the discovery
1
7–19
of novel BTK inhibitors.
Among these, one series of thieno
[
3,2-c]pyridine analogues were developed using scaffold hopping
In the thieno[3,2-c]pyrid-4-amine series, compound
was found to exhibit the most BTK inhibitory activity,
⇑
Corresponding authors.
E-mail addresses: xmhcpu@163.com (M. Xin), 1020030692@cpu.edu.cn
1
9,20
strategy.
8
(
H. Xiang).
y
with value of 12.8 nM. Considering that the
IC50
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2015.08.039
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Xin, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.039

2
M. Xin et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
N
O
O
O
O
N
HN
HN
NH2
N
NH2
N
N
N
N
N
N
N
N
N
N
O
NH
O
N
F
O
S
O
N
N
N
H
N
H
O
O
spebrutinib
AVL-292, CC-292)
ibrutinib
PCI-32765)
2
3
ONO-4059
1
HM71224
4
(
(
Pharmacyclics/Janssen
Hanmi Phamarceuticals /Lilly Ono Pharmaceutical/Gliead
Avila Therapeutics/Celegene
Phase II)
(
launched)
(Phase II)
(Phase II)
(
N
N
O
N
O
N
N
O
N
O
F
O
N
O
7
N
N
NH
NH
O
N
H
S
N
N
NH
N
H
O
OH
N
O
5
RN-486 (clinic)
Roche
6
CGI-1746
GDC-0834
Genetech
CGI /Genentech
Figure 1. Representative chemical structures of BTK inhibitors.
O
O
NH
2
N
NH
2
S
N
S
O
N
N
R
HN
8
BTK IC50 = 12.8 nM novel 7-substituted 3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amines
Figure 2. Design of novel 7-substituted 3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amines.
thieno[3,2-c]pyrid-4-amines were first reported as potent BTK
inhibitors and the derivatives were relatively limited, we are
presently interested to further explore extensively structural mod-
ification of thieno[3,2-c]pyrid-4-amine skeleton and the compre-
hensively understanding the SAR, thereby designing a series of
novel thieno[3,2-c]pyrid-4-amine derivatives. Herein, in this paper
we report the synthesis and pharmacological evaluation of these
novel thieno[3,2-c]pyrid-4-amine analogues as potent BTK inhibi-
tors, of which the representative compound 13b is also evaluated
about the kinase selectivity in several other kinase assays (Fig. 2).
of commercially available 3-bromothieno[3,2-c]pyridin-4-amine
and (4-phenoxyphenyl)boronic acid gave compound 9, which
was subsequently iodinated by N-iodosuccinimide reagent to
afford compound 10, followed by the Suzuki coupling reaction to
provide compound 11. Then compound 11 was stirred with
trifluoroacetic acid to produce the deprotected intermediate 12.
Compound 12 was then reacted with various halides to afford
the targeted compounds 13a–j (Scheme 1).
Scheme 2 depicted the synthesis of compounds 14a–f and
15a–d. Compound 14a was prepared by treating iodide 10 with
N-Boc-1,4,5,6-tetrahydropyridin-3-ylboronic acid using Suzuki
coupling protocols. Deprotection of 14a with trifluoroacetic acid
yielded compound 14b. Treating 14b with N-methyl
chloroacetamide or acryloyl chloride under alkaline condition at
2
. Chemistry
As summarized in Table 1, the novel 7-substituted
ꢀ
10 °C provided compound 14c and 14d, respectively. Hydrogena-
thieno[3,2-c]pyrid-4-amine derivatives (13a–j, 14a–f and 15a–d)
were designed and synthesized. The synthetic routes for these
compounds are depicted in Schemes 1 and 2.
Scheme 1 detailed the synthesis of the desired thieno[3,2-c]
pyrid-4-amines 13a–j. As described in the literatures,19 the key
intermediate 12 was synthesized by four steps. Suzuki coupling
tion reduction of 14b with Pd/C afforded compound 14e, which
was then treated with acryloyl chloride at ꢀ10 °C to provide
compound 14f. Likewise, compounds 15a–d were synthesized
employed the similar synthetic routes. Coupling of 14a and
N-Boc-1,4,5,6-tetrahydropyridin-4-ylboronic acid using Suzuki
Please cite this article in press as: Xin, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.039

M. Xin et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
reaction provided 15a, which was subsequently reacted with
trifluoroacetic acid to generate 15b. Acylation of 15b with acryloyl
chloride afforded 15c. Meanwhile, compound 15d was prepared by
hydrogenating 15b with Pd/C catalyst in ethanol (Scheme 2).
N1-substituted 1H-pyrazol-4-yl analogues, the 2-piperidyl-2-
oxoethyl analogue 13a and the 2-morpholinyl-2-oxoethyl
analogue 13b displayed equivalent potency (13a: IC50 = 12.2 nM,
13b: IC50 = 11.8 nM), while the 2-oxopiperid-3-yl analogue 13c
showed higher inhibitory activity (13c: IC50 = 9.5 nM), compared
to 2-(methylamino)-2-oxoethyl analogue 8. Replacing the amide
of 8 with a ketone or alkynyl group led to compound 13d and
3
3
. Result and discussion
13e showing the highest BTK inhibition, with the IC50 values of
7.8 nM and 7.4 nM, respectively. However, the oxoethyl chain of
8 was extended to oxopropyl group, leading to deceased BTK inhi-
.1. In vitro BTK kinase inhibition studies for the new
compounds
bitory activity. The 3-(methylamino)-3-oxopropyl analogue 13g
showed an IC50 value of 25.5 nM, while replacing the methylamino
group of 13f with 3-((methylsulfonyl)oxy)piperidin-1-yl group
afforded weaker potency (13g: IC50 = 54.9 nM), whereas the car-
boxyl group showed tolerant (13h: IC50 = 21.0 nM). Interestingly,
compounds 14i bearing acrylamide ethyl moiety showed an IC50
value of 9.9 nM, which was almost 2.5 fold more potent than
All the newly synthesized 7-substituted thieno[3,2-c]pyrid-4-
amines (13a–j, 14a–f and 15a–d) were evaluated in vitro for their
capacity to inhibit BTK kinase. The in vitro IC50 values are depicted
in Table 1.
Among the synthesized C7-substituted thieno[3,2-c]pyrid-4-
amines, the initially designed C7-1H-pyrazol-4-yl derivatives
displayed moderate to high potency against BTK. In the
Table 1
In vitro BTK enzymatic inhibition for thieno[3,2-c]pyrid-4-amine derivatives
O
NH2
N
S
R1
Compds
R1
Btk IC50 [nM]a
12.8
AlogPb
Compds
R1
Btk IC50 [nM]a
2867
H
O
N
N
N
8
3.64
4.76
3.53
14a
O
N
O
N
HN
1
1
3a
3b
N
N
12.2
11.8
14b
14c
3601
O
H
N
O
N
N
N
N
455.7
O
O
O
O
HN
1
1
1
3c
3d
3e
9.5
7.8
7.4
4.23
4.16
5.77
14d
14e
14f
N
342.6
>10000
2295
N
N
N
N
HN
O
O
O
N
N
N
O
O
N
N
1
1
1
3f
N
H
25.5
54.9
21.0
3.89
4.03
4.31
15a
15b
15c
N
1659
853.9
183.3
O
O
S
O
3g
3h
N
N
N
HN
O
O
N
HO
N
N
O
H
N
1
1
3i
3j
N
N
9.9
4.47
15d
>10000
4.0
HN
O
O
ü
ü
N
1687
Ibrutinib
N
a
Values are means of three experiments.
AlogP are calculated by Discovery Studio 3.0 using Calculate Molecular Properties Protocol.
b
Please cite this article in press as: Xin, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.039

4
M. Xin et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
O
O
O
NH2
Br
a
NH2
b
c
NH2
I
N
NH2
N
S
N
N
S
S
S
3
-bromothieno[3,2-c]pyridin-4-amine
9
10
N
N
Boc
11
O
O
O
O
O
N
N
3f: R1
1
=
N
1
3a: R1
=
=
O H
d
e
NH2
2 3
OSO CH
NH2
N
O 13g: R1
13h: R1
13i: R1
=
N
O
1
3b: R1
N
N
S
1
=
S
13c: R =
OH
NH
H
N
O
O
=
N
NH
13d: R1
N
=
R1
O
O
12
13e: R1 =
13j: R1
=
1
3a-13j
Scheme 1. Reagents and conditions: (a) 4-Phenoxyphenylboronic acid, Pd(PPh
3 4
) , Na
2 3 2
CO , EtOH, H O, toluene, 90 °C, 3 h, 96%; (b) NIS, DMF, rt, 1 h, 70%; (c) 1-Boc-pyrazole-4-
boronic acid pinacol ester, Pd(dppf)Cl
2
2
ꢁCH Cl
2
, Na
2
CO
3
, 1,4-dioxane, 100 °C, 1–2 h, 55%; (d) CF
3
COOH, DCM, rt, 12 h, 92%; (e) R
1
X, K
2
CO
3
, DMF, 25 °C, 4–12 h, 40–95%.
d
O
O
O
O
O
NH2
NH2
NH2
N
a
NH2
NH2
b
c
N
N
N
N
S
S
S
S
S
I
O
N
O
N
1
0
N
NH
14b
N
H
Boc
O
1
4a
14c
14d
e
f
O
O
O
NH2
d
N
NH2
NH2
NH2
g
S
N
N
N
S
S
S
1
N
i
NH
4e
O
N
H
1
14f
N
Boc
15b
5a
h
O
O
NH2
NH2
N
O
N
S
S
N
N
H
15c
15d
Scheme 2. Reagents and conditions: (a) Pd(dppf)Cl
rt, 12 h, 75%; (c) N-methyl chloroacetamide, TEA, DCM, ꢀ10 °C, 2 h, 53%; (d) TEA, DCM, ꢀ10 °C, 2 h, acryloyl chloride, 67% for 14d; 68% for 14f; (e) H
7%; (f) Pd(dppf)Cl Cl , Na CO , 1,4-dioxane, 100 °C, 1–2 h, N-Boc-1,4,5,6-tetrahydropyridin-4-ylboronic acid, 63%; (g) CF COOH, DCM, rt, 12 h, 75%; (h) acryloyl chloride,
ꢁCH
TEA, DCM, ꢀ10 °C, 2 h, 77%; (i) H , Pd/C, CH OH, rt, 12 h, 58%.
2
ꢁCH
2
Cl , Na
2
2
CO
3
, 1,4-dioxane, 100 °C, 1–2 h, N-Boc-1,4,5,6-tetrahydropyridin-3-ylboronic acid, 53%; (b) CF
3
COOH, DCM,
2
, Pd/C, CH
3
OH, rt, 12 h,
7
2
2
2
2
3
3
2
3
Please cite this article in press as: Xin, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.039

M. Xin et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 2
Kinase selectivity profiles of compounds 13b
Compds
Structures
IC50 (nM)
JAK3
BTK
11.8
JAK2
6864
KDR
191
c-Met
2427
OPh
S
1
3b
3068
NH
2
O
N
N
N
N
O
compound 13f, likely due to that the acrylamide could provide a
covalently binding with BTK kinase cysteine residue. However,
unfortunately, an attempt of replacing acrylamide ethyl moiety
with short acryloyl group (14h) led to the loss of BTK inhibitory
activity, which appeared that the size of the substituent at
N1-position of 1H-pyrazol-1-yl was critical for BTK inhibitory
activity.
compound 13b only showed moderate inhibitory activities against
KDR kinase, with IC50 value of 191 nM, which was more than
16-fold weaker than BTK inhibition. This result indicated that
13b was a relatively selective BTK inhibitor. In future, it should
be still further identification against other kinase profiles (Table 2).
4
. Conclusion
After various substituents at the N1-position of 1H-pyrazol-4-yl
linked to C7-position of the central thieno[3,2-c]pyrid-4-amine
skeleton, several piperidyl and tetrahydropyridinyl substituents
at the C7-position were attempted to extend the structural
diversity. All the tested compounds 14a–f and 15a–d displayed
remarkably reduced inhibitory activities. Compounds bearing
N-Boc-1,4,5,6-tetrahydropyridin-3-yl or 1,4,5,6-tetrahydropy-
ridin-3-yl group were almost inactive against BTK (14a and 14b:
IC50 > 1000 nM), although the introduction of 2-(methylamino)-
In summary, we have designed a series of novel 7-substituted
-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amines as potent BTK
inhibitors. Twenty thieno[3,2-c]pyridin-4-amine derivatives were
design, synthesized, and evaluated. These compounds displayed
variant inhibitory activities against BTK in vitro. 7-pyrazol-4-yl
3
substituted
3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
subseries showed high BTK inhibition, and compounds 13d and
3e gave the highest potency with the IC50 value of 7.8 nM and
.4 nM, respectively. Compounds 13b displayed favorable BTK
1
7
2
-oxoethyl or acryloyl group led to weak potency (14c:
IC50 = 455.7 nM, 14d: IC50 = 342.6 nM). Moreover, extending the
tetrahydropyridin-3-yl to piperid-3-yl group affording the com-
pounds 14e and 14f, the BTK inhibitory activities were completely
lost (14e and 14f: IC50 > 1000 nM). Migration of the nitrogen atom
from the 3-position of the tetrahydropyridinyl (or piperidyl) to
inhibition with an IC50 value of 11.8 nM, fine hydrophilicity, and
relatively good kinase selectivity, which could be considered as
an ideal lead candidate for further evaluation.
5
5
. Experimental
4
-position afforded several tetrahydropyridin-4-yl (or piperidyl)
analogues 15a–15d. It was found that compounds bearing
tetrahydropyridin-4-yl moiety showed slightly higher potency
than the corresponding tetrahydropyridin-3-yl ones (15a vs 14a,
.1. Chemistry
All chemical reagents were purchased from commercial ven-
dors and used without further purification unless noted especially.
1
5b vs 14b, 15c vs 14c), even though compound 15d with the
piperid-4-yl was inactive for BTK kinase. In this subseries including
compounds 14a–f and 15a–d, Compound 15c bearing an acryloyl
tetrahydropyridin-4-yl showed the best potency, with an IC50
value of 183.3 nM, however, which was still nearly 25-fold weaker
than compound 13e (Table 1).
The melting points (Mp) for the compounds were performed on a
1
Melt-Temp II apparatus and uncorrected.
(
H NMR spectra
13
400 MHz) and C NMR (100 MHz) spectra data were recorded
in CDCl or DMSO-d on a Bruker BioSpin AG (Ultrashield Plus AV
00) spectrometer. MS data were recorded at an Agilent-6120
3
6
4
Furthermore, in order to assess the drug-like possibility of these
compounds with IC50 values below 100 nM, their AlogP values
were calculated by using Calculate Molecular Properties Protocol
of the Discovery Studio 2.5 software package. The results were
illustrated in Table 1. Despite compounds 13d and 13e displayed
higher BTK inhibitory activities in vitro, both showed weaker
hydrophilicity properties (13d: AlogP = 4.16, 13e: AlogP = 5.77),
compared to compound 8 (AlogP = 3.64). It means both com-
pounds might afford inadequate druggability. Among these com-
pounds, 13b bearing 2-morpholinyl-2-oxoethyl hydrophilic
moiety showed an improved hydrophilicity, with AlogP value of
quadrupole LC/MS (ESI) while HRMS were recorded at a Water
Q-Tof micro mass spectrometer. The HPLC study for the com-
pounds was verified using a mixture of solvent (methanol/water
or acetonitrile/water) at the flow rate of 2 mL/min and peak detec-
tion at 254 nm under UV. Column chromatography was carried out
on silica gel (200–300 mesh) purchased from Qindao Ocean Chem-
ical Company of China. Thin-layer chromatography (TLC) analyses
were carried out on silica gel GF254. Ibrutinib was synthesized
8
according to the reference literature.
5
(
.1.1. 7-Iodo-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
10)
It was prepared as we recently reported, yellow solid (2.4 g,
3
.53, moreover, whose BTK inhibition was also favorable. Based
on the drug-like prediction study, compound 13b was considered
as the ideal lead candidate, deserving further evaluation (Table 1).
1
9
1
7
7
2
6
0.0%). H NMR (400 MHz, DMSO-d ) d 8.02 (s, 1H), 7.57 (s, 1H),
.48–7.42 (m, 4H), 7.22–7.18 (m, 1H), 7.14–7.10 (m, 4H), 5.61 (s,
H); MS m/z 445.0 [M+H] .
3
.2. Kinase selectivity assay
+
From the BTK inhibition results and the drug-like study above,
compound 13b stood out as the suitable lead candidate. For further
evaluating its kinase selectivity, compound 13b was assessed for
the inhibitory activities against 4 kinds of our in-house RTKs and
the data were summarized in Table 2. In addition to BTK kinase,
5.1.2. 3-(4-Phenoxyphenyl)-7-(1H-pyrazol-4-yl)thieno[3,2-c]pyr-
idin-4-amine (12)
It was synthesized according to our recent route,19 gray solid
1
(59 mg, 75%). Mp: 232.3–234.2 °C; H NMR (400 MHz, DMSO-d
6
)
Please cite this article in press as: Xin, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.039

6
M. Xin et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
d 13.09 (s, 1H), 8.15 (s, 1H), 8.06 (s, 1H), 7.95 (s, 1H), 7.54 (s, 1H),
.48–7.43 (m, 4H), 7.20 (t, J = 6.80 Hz, 1H), 7.14–7.12 (m, 4H), 5.42
5.1.8. 3-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
7-yl)-1H-pyrazol-1-yl)-N-methylpropanamide (13f)
7
+
(
s, 2H); HPLC 99.7%; MS m/z 385.1 [M+H] ; HRMS (ESI) m/z calcd
13f was prepared by the general method as described above for
+
1
for C22
H
16
N
4
OS [M+H] 385.1117, found 385.1128.
preparation of 13a (15 mg, 46.0%). Mp: 140–142 °C; H NMR
(
3
400 MHz, CDCl ) d 7.99 (s, 1H), 7.89 (s, 1H), 7.84 (s, 1H), 7.44–
5
7
.1.3. 2-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
-yl)-1H-pyrazol-1-yl)-1-(piperidin-1-yl)ethanone (13a)
A suspension of 12 (100 mg, 0.26 mmol), 2-chloro-1-(piperidin-
7.38 (m, 4H), 7.19–7.17 (m, 2H), 7.11–7.08 (m, 4H), 5.71 (s, 1H),
4.88 (s, 2H), 4.53–4.48 (m, 2H), 2.80–2.75 (m, 2H), 2.77 (s, 3H);
C NMR (100 MHz, DMSO-d ) d 170.3, 157.5, 156.6, 153.7, 146.7,
6
1
3
1
-yl)ethan-1-one (63 mg, 0.39 mmol) and
K
2
CO
3
(71.7 mg,
140.0, 137.3, 136.8, 131.5, 131.2, 130.7, 127.6, 124.5, 123.7,
0
.52 mmol) in DMF (5 mL) was stirred at rt for 12 h. Then the reac-
119.7, 118.9, 118.7, 118.0, 114.0, 48.3, 36.3, 26.0; HPLC 96.2%;
+
tion mixture was diluted with the mixed ice and water, the precip-
itated solid was filtered off and purified by silica gel column using
MS m/z 470.2 [M+H] ; HRMS (ESI) m/z calcd for C26
H
23
N
5
O
2
S
+
[M+H] 492.1464, found 492.1468.
(
20–50%) ethyl acetate/petroleum ether) to afford 13a as a white
1
solid (30 mg, 53.7%). Mp: 155–157 °C.
H
NMR (400 MHz,
5.1.9. 1-(3-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyr-
idin-7-yl)-1H-pyrazol-1-yl)propanoyl)piperidin-3-yl methane-
sulfonate (13g)
DMSO-d ) d 8.12 (s, 1H), 8.08 (s, 1H), 7.92 (s, 1H), 7.56 (s, 1H),
6
7
5
2
1
1
2
.48–7.43 (m, 4H), 7.20 (t, J = 12.80 Hz, 1H), 7.14–7.11 (m, 4H),
.45 (s, 2H), 5.21 (s, 2H), 3.47 (s, 4H), 1.60–1.55 (m, 4H), 1.29 (s,
13g was prepared by the general method as described above for
1
3
1
H); C NMR (100 MHz, DMSO-d
6
) d 165.4, 157.5, 156.6, 153.6,
preparation of 13a (34 mg, 42.5%). Mp: 155–157 °C; H NMR
46.7, 139.9, 137.3, 136.8, 131.5, 131.2, 130.7, 129.1, 124.5,
23.8, 119.7, 119.0, 118.7, 118.3, 114.0, 53.5, 45.8, 42.9, 26.4,
6
(400 MHz, DMSO-d ) d 8.27 (s, 1H), 8.00 (s, 2H), 7.75 (s, 1H),
7.53–7.48 (m, 4H), 7.23–7.17 (m, 5H), 6.10 (s, 2H), 4.80–4.68 (m,
+
5.7, 24.4; HPLC 95.7%; MS m/z 510.2 [M+H] ; HRMS(ESI) m/z calcd
1H), 4.48–4.42 (m, 2H), 3.72 (s, 3H), 3.23–3.18 (m, 4H), 1.95–
+
13
for C29
H
27
N
5
O
2
S [M+H] 510.1958, found 510.1975.
6
1.90 (m, 2H), 1.50–1.25 (m, 4H); C NMR (100 MHz, DMSO-d ) d
1
69.3, 157.8, 156.5, 137.9, 137.1, 131.5, 130.7, 130.0, 128.7,
5
7
.1.4. 2-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
-yl)-1H-pyrazol-1-yl)-1-morpholinoethanone (13b)
3b was prepared by the general method as described above for
126.2, 124.5, 119.7, 119.4, 118.9, 116.5, 114.5, 76.2, 49.6, 48.3,
+
44.9, 38.3, 29.8, 29.2, 22.3; HPLC 95.0%; MS m/z 618.2 [M+H] ;
+
1
HRMS (ESI) m/z calcd for C31
31
H N
5
O
S
5 2
[M+H] 618.1839, found
1
preparation of 13a (36 mg, 56.4%). Mp: 135–137 °C; H NMR
400 MHz, CDCl 7.96–7.92 (m, 3H), 7.44–7.39 (m, 4H),
.20–7.17 (m, 2H), 7.12–7.07 (m, 4H), 5.30 (s, 2H), 5.09 (s, 2H),
618.1844.
(
3
) d
7
3
5.1.10. 3-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
7-yl)-1H-pyrazol-1-yl)propanoic acid (13h)
+
.67 (s, 8H); HPLC 95.1%; MS m/z 512.2 [M+H] ; HRMS(ESI) m/z
+
calcd for C28
H
25
N
5
O
3
S [M+H] 512.1953, found 510.1972.
13 h was prepared by the general method as described above
1
for preparation of 13a (30 mg, 41.7%). Mp: 245–247 °C; H NMR
5
7
.1.5. 3-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
-yl)-1H-pyrazol-1-yl)piperidin-2-one (13c)
3c was prepared by the general method as described above for
6
(400 MHz, DMSO-d ) d 8.35 (s, 1H), 8.18 (s, 1H), 8.04 (s, 1H), 7.89
(s, 1H), 7.56 (s, 1H), 7.49–7.43 (m, 4H), 7.20 (t, J = 12.40 Hz, 1H),
1
7.14–7.11 (m, 3H), 5.44 (s, 2H), 4.42–4.38 (m, 2H), 2.75–2.70 (m,
1
13
preparation of 13a (21 mg, 38.2%). Mp: 170–172 °C; H NMR
6
2H); C NMR (100 MHz, DMSO-d ) d 166.8, 157.5, 156.5, 153.6,
(
(
(
400 MHz, DMSO-d
s, 1H), 7.52–7.44 (m, 4H), 7.21 (t, J = 12.40 Hz, 1H), 7.16–7.12
m, 4H), 5.46 (s, 2H), 5.07–5.03 (m, 1H), 3.20–3.15 (m, 2H), 2.38–
6
) d 8.19 (s, 1H), 8.09 (s, 1H), 7.93 (s, 1H), 7.91
146.8, 140.0, 137.3, 136.6, 131.5, 131.2, 130.7, 127.7, 124.5,
123.8, 119.7, 118.9, 118.7, 117.9, 114.0, 48.8, 29.4; HPLC 99.1%;
+
MS m/z 457.2 [M+H] ; HRMS (ESI) m/z calcd for C25
H
20
N
4
O
3
S [M
+
2
.35 (m, 1H), 2.32–2.28 (m, 1H), 2.02–1.97 (m, 1H), 1.89–1.85
+H] 457.1328, found 457.1332.
1
3
(
m, 1H); C NMR (100 MHz, DMSO-d
6
) d 167.6, 157.5, 156.6,
1
1
2
53.6, 146.7, 140.0, 137.3, 136.7, 131.5, 131.2, 130.7, 128.6,
5.1.11. N-(2-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyr-
idin-7-yl)-1H-pyrazol-1-yl)ethyl)acrylamide (13i)
24.5, 123.7, 119.7, 119.0, 118.6, 117.6, 114.0, 61.1, 41.8, 28.7,
+
1.4; HPLC 98.2%; MS m/z 482.2 [M+H] ; HRMS (ESI) m/z calcd
13i was prepared by the general method as described above for
+
1
for C27
H
23
N
5
O
2
S [M+H] 482.1645, found 482.1651.
preparation of 13a (36 mg, 75%). Mp: 158–160 °C; H NMR
(
6
400 MHz, DMSO-d ) d 8.34–8.31 (m, 1H), 8.14 (s, 1H), 8.05 (s,
5
7
.1.6. 1-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
-yl)-1H-pyrazol-1-yl)propan-2-one (13d)
3d was prepared by the general method as described above for
1H), 7.94(s, 1H), 7.55(s, 1H), 7.50–7.43 (m, 4H), 7.20 (t,
J = 14.76 Hz, 1H), 7.14–7.11 (m, 4H), 6.25–6.18 (m, 1H), 6.09 (dd,
J = 2.24 Hz, J = 2.24 Hz, 1H), 5.59 (dd, J = 2.24 Hz, J = 2.20 Hz, 1H),
1
1
13
preparation of 13a (23 mg, 54.7%). Mp: 151–153 °C; H NMR
5.43 (s, 2H), 4.34–4.29 (m, 2H), 3.64–3.58 (m, 2H); C NMR
(
(
4
400 MHz, DMSO-d
s, 1H), 7.51–7.44 (m, 4H), 7.21 (t, J = 12.4 Hz, 1H), 7.16–7.12 (m,
H), 5.48 (s, 2H), 5.23 (s, 2H), 2.15 (s, 3H); 13C NMR (100 MHz,
) d 203.1, 157.5, 156.6, 153.7, 146.7, 140.1, 137.3, 131.5,
31.2, 130.7, 128.9, 124.5, 123.7, 119.7, 118.9, 118.7, 118.6,
6
) d 8.14 (s, 1H), 8.09 (s, 1H), 7.97 (s, 1H), 7.57
6
(100 MHz, DMSO-d ) d 165.5, 157.5, 156.6, 153.7, 146.7, 140.1,
137.3, 132.0, 131.2, 130.7, 127.9, 125.9, 124.5, 123.7, 119.7,
118.9, 118.7, 118.2, 114.0, 51.2, 29.5; HPLC 95.7%; MS m/z 482.2
+
+
DMSO-d
1
1
6
[M+H] ; HRMS (ESI) m/z calcd for C27
H
23
N
5
O
2
S [M+H] 482.1645,
found 482.1652.
+
13.8, 61.1, 27.4; HPLC 96.1%; MS m/z 441.2 [M+H] . HRMS (ESI)
+
m/z calcd for C25
H
20
N
4
O
2
S [M+H] 441.1379, found 441.1390.
5.1.12. 1-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
7
-yl)-1H-pyrazol-1-yl)prop-2-en-1-one (13j)
5
4
.1.7. 3-(4-Phenoxyphenyl)-7-(1-(prop-2-yn-1-yl)-1H-pyrazol-
-yl)thieno[3,2-c]pyridin-4-amine (13e)
3e was prepared by the general method as described above for
13j was prepared by the general method as described above for
preparation of 13a (33 mg, 68.1%). Mp: 163–165 °C; H NMR
(400 MHz, DMSO-d ) d 8.17 (s, 1H), 8.10 (s, 2H), 7.95 (s, 1H), 7.70
6
1
1
1
preparation of 13a (31 mg, 78.5%). Mp: 150.3–152.5 °C; H NMR
400 MHz, CDCl ) d 8.01 (s, 1H), 7.99 (s, 1H), 7.87 (s, 1H), 7.44–
.38 (m, 4H), 7.20–7.18 (m, 2H), 7.11–7.08 (m, 4H), 5.18 (s, 2H),
(s, 1H), 7.46–7.43 (m, 4H), 7.19–7.10 (m, 4H), 6.94 (d, J = 8.40 Hz,
2H), 6.50 (d, J = 4.0 Hz, 1H), 6.35–6.29 (m, 1H), 5.79 (d,
(
3
1
3
7
4
6
J = 12.0 Hz, 1H); C NMR (100 MHz, DMSO-d ) d 165.5, 156.5,
+
.83 (s, 2H), 2.83 (s,1H). HPLC 96.6%; MS m/z 424.1 [M+H] ; HRMS
153.7, 148.8, 147.0, 140.2, 138.9, 137.3, 131.6, 131.5, 130.8,
128.0, 124.6, 124.5, 123.7, 121.5, 119.8, 118.9, 118.7, 117.6,
+
(
18 4
ESI) m/z calcd for C25H N OS [M+H] 424.1226, found 424.1243.
Please cite this article in press as: Xin, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.039

M. Xin et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
+
1
14.9, 114.3; HPLC 95.1%; MS m/z 439.1 [M+H] ; HRMS (ESI) m/z
5.1.17. 3-(4-Phenoxyphenyl)-7-(piperidin-3-yl)thieno[3,2-c]pyr-
idin-4-amine (14e)
+
calcd for C25
H
18
N
4
O
2
S [M+H] 439.1223, found 439.1233.
A suspension of 14b (400 mg, 1.0 mmol) in MeOH (20 mL) with
the 40 mg 10% palladium carbon was stirred for 5 h at room
temperature under an atmosphere of hydrogen gas. The reaction
5
.1.13. Tert-butyl-5-(4-amino-3-(4-phenoxyphenyl)thieno[3,2-
c]pyridin-7-yl)-3,4-dihydropyridine-1(2H)-carboxylate (14a)
A solution of 10 (150 mg, 0.337 mmol), N-Boc-1,4,5,6-tetrahy-
mixture was filtered and concentrated in vacuo to afford 11a
1
dropyridin-3-ylboronic acid (77 mg, 0.337 mmol) and Pd(dppf)Cl
CH Cl
degassed with nitrogen for 5 min followed by addition Na
2
-
(230 mg, 57.5%). H NMR (400 MHz, CDCl
3
) d 7.80 (s, 1H),
ꢁ
2
2
(27 mg, 0.0337 mmol) in 1,4-dioxane (10 mL) was
7.42–7.37 (m, 4H), 7.17 (t, J = 14.76 Hz, 1H), 7.13 (s, 1H),
2
CO
3
7.11–7.07 (m, 4H), 4.61 (s, 2H), 3.26–3.23 (m, 2H), 2.85–2.79 (m,
1
3
(
107 mg, 2 M in water) under continuous flow of nitrogen. The
3H), 2.02–1.99 (m, 2H), 1.88–1.81 (m, 2H); C NMR (100 MHz,
DMSO-d ) d 157.5, 156.6, 153.7, 148.8, 139.1, 137.5, 131.4, 131.3,
reaction mixture was stirred at 100 °C for 2 h, AND the catalyst
6
was removed by filtration through Celite. The filtrate was concen-
130.7, 124.5, 123.7, 123.4, 119.0, 118.6, 51.8, 46.1, 40.2, 30.7,
+
trated and purified by column chromatography to afford
26.5; HPLC 97.3%; MS m/z 402.2 [M+H] ; HRMS (ESI) m/z calcd
1
+
compound 14a (89 mg, 53.1%). Mp: 144–146 °C;
H
NMR
for C24
H
23
N
3
OS [M+H] 402.1634, found 402.1639.
(
3
400 MHz, CDCl ) d 7.68 (s, 1H), 7.45–7.40 (m, 4H), 7.20–7.17 (m,
2
2
9
H), 7.13–7.06 (m, 4H), 6.29 (s, 1H), 5.58 (s, 2H), 4.25–4.21 (m,
5.1.18. 1-(3-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
7-yl)piperidin-1-yl)prop-2-en-1-one (14f)
H), 1.98–1.92 (m, 2H),1.61–1.57 (m, 2H), 1.48 (s, 9H); HPLC
+
6.3%; MS m/z 500.2 [M+H] ; HRMS(ESI) m/z calcd for C29
29
H N
3
O
3
S
14f was prepared by the general method as described above for
+
1
[
M+H] 500.2002, found 500.2017.
preparation of 14c (35 mg, 67.0%). Mp: 148–150 °C; H NMR
(
3
400 MHz, CDCl ) d 7.70 (t, J = 16.40 Hz, 1H), 7.41–7.38 (m, 4H),
5
.1.14. 3-(4-Phenoxyphenyl)-7-(1,4,5,6-tetrahydropyridin-3-yl)-
7.20–7.16 (m, 2H), 7.11–7.07 (m, 4H), 6.68–6.61 (m, 1H),
6.38–6.30 (m, 1H), 5.74–5.70 (m, 1H), 5.43–5.41 (m, 2H), 4.90
(dd, J = 12.80 Hz, J = 12.40 Hz, 1H), 4.19 (dd, J = 12.80 Hz,
J = 12.4 Hz, 1H), 3.15–3.11 (m, 1H), 2.91–2.88 (m, 1H), 2.24–2.20
thieno[3,2-c]pyridin-4-amineine (14b)
3
A mixture of the 14a (100 mg, 0.21 mmol) in CF COOH (3 mL)
was stirred at rt for 2 h. Then the reaction mixture was diluted
with the mixed ice and water, and then 1 M NaOH solution was
added to alkalify to pH 7–8, the precipitated solid was filtered,
and concentrated under reduced pressure. The crude product was
13
(m, 1H), 2.05–1.93 (m, 3H), 1.69–1.60 (m, 1H);
C NMR
(100 MHz, DMSO-d ) d 164.8, 157.5, 156.6, 153.9, 148.7, 139.2,
6
137.6, 131.4, 131.3, 130.7, 129.0, 127.7, 124.5, 123.6, 123.5,
122.5, 122.0, 119.7, 119.0, 118.6, 51.2, 47.3, 46.1, 29.5, 25.5; HPLC
purified by columu chromatography to afford 14b as a gray solid
1
+
(
76 mg, 75.1%). Mp: 210–212 °C; H NMR (400 MHz, CDCl
3
) d
98.6%; MS m/z 456.2 [M+H] ; HRMS (ESI) m/z calcd for
+
7
4
2
1
1
.66 (s, 1H), 7.42–7.38 (m, 4H), 7.20–7.17 (m, 2H), 7.11–7.06 (m,
H), 6.36 (s, 1H), 5.53 (s, 2H), 3.78–3.75 (m, 2H), 2.43–2.40 (m,
H), 2.08–2.03 (m, 2H); 13C NMR (100 MHz, DMSO-d
) d 157.5,
56.6, 154.0, 147.2, 139.6, 137.1, 134.6, 131.4, 131.3, 130.7,
24.5, 124.4, 124.1, 121.6, 119.7, 119.1, 118.6, 42.3, 25.6, 21.6;
C
27
25
H N
3
O
2
S [M+H] 456.1740, found 456.1743.
6
5.1.19. Tert-butyl-4-(4-amino-3-(4-phenoxyphenyl)thieno[3,2-
c]pyridin-7-yl)-5,6-dihydropyridine-1(2H)-carboxylate (15a)
15a was prepared by the general method as described above for
+
1
HPLC 95.3%; MS m/z 500.2 [M+H] ; HRMS (ESI) m/z calcd for
C
preparation of 14a (235 mg, 63.1%). Mp: 149–151 °C; H NMR
+
24
H
21
N
3
OS [M+H] 400.1478, found 400.1486.
3
(400 MHz, CDCl ) d 7.83 (s, 1H), 7.46–7.38 (m, 4H), 7.19–7.15 (m,
2
H), 7.11–7.06 (m, 4H), 6.12 (s, 1H), 4.66 (s, 2H), 4.13 (s, 2H),
1
3
5
7
.1.15. 2-(5-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
-yl)-3,4-dihydropyridin-1(2H)-yl)-N-methylacetamide (14c)
A suspension of 14b (104 mg, 0.26 mmol), N-methyl chloroac-
3.70 (s, 2H), 2.61 (s, 2H), 1.43 (s, 9H); C NMR (100 MHz, DMSO-
) d 157.5, 156.6, 154.2, 146.7, 140.0, 137.1, 133.2, 132.0, 131.9,
d
6
131.5, 131.2, 130.7, 129.3, 129.2, 124.5, 124.1, 122.2, 122.0,
etamide (42 mg, 0.39 mmol) and K
2
CO
3
(71.7 mg, 0.52 mmol) in
119.7, 119.0, 118.6, 79.4, 43.5, 41.9, 28.6; HPLC 97.7%; MS m/z
+
S [M+H]+
DMF (5 mL) was stirred at rt for 12 h. After completion of reaction,
the reaction mixture was diluted with the mixed ice and water,
and the precipitated solid was filtered off and purified on a silica
gel column using (20–50%) ethyl acetate/petroleum ether) as
500.2 [M+H] ; HRMS (ESI) m/z calcd for C29
H
29
N
3
O
3
500.2002, found 500.2020.
5.1.20. 3-(4-Phenoxyphenyl)-7-(1,2,3,6-tetrahydropyridin-4-yl)-
thieno[3,2-c]pyridin-4-amine (15b)
eluent to afford 14c as a white solid (51 mg, 42.0%). Mp:
1
1
43–145 °C; H NMR (400 MHz, CDCl
3
) d 7.76 (s, 1H), 7.42–7.38
15b was prepared by the general method as described above for
1
(
m, 4H), 7.30 (s, 1H), 7.19–7.14 (m, 2H), 7.11–7.07 (m, 4H), 6.21
preparation of 14b (76 mg, 75.1%). Mp: 226–228 °C; H NMR
(
2
s, 1H), 4.72 (s, 2H), 3.46–3.42 (m, 2H), 3.20 (s, 2H), 2.87 (s, 3H),
6
(400 MHz, DMSO-d ) d 7.81 (s, 1H), 7.50 (s, 1H), 7.47–7.43 (m,
4H), 7.20 (t, J = 14.76 Hz, 1H), 7.14–7.10 (m, 4H), 6.15 (s, 1H),
13
.78–2.75 (m, 2H), 2.46–2.40 (m, 2H);
) d 170.2, 157.5, 156.6, 154.1, 147.2, 139.6, 137.1, 133.2,
31.4, 131.2, 130.7, 124.5, 124.1, 123.8, 121.2, 119.7, 119.1,
18.6, 61.3, 55.1, 49.8, 26.0, 25.9; HPLC 98.4%; MS m/z 471.1 [M
C NMR (100 MHz,
DMSO-d
1
1
6
5.43 (s, 2H), 3.50 (s, 2H), 3.07–3.04 (m, 2H), 2.53–2.48 (m, 2H);
1
3
6
C NMR (100 MHz, DMSO-d ) d 155.3, 154.3, 150.8, 147.1, 146.6,
145.3, 140.0, 137.1, 133.2, 131.5, 131.4, 131.2, 130.9, 130.7,
124.5, 124.0, 122.3, 122.0, 121.7, 119.7, 119.0, 118.7, 118.6, 43.5,
+
+
+
H] ; HRMS (ESI) m/z calcd for C27
26 4 2
H N O S [M+H] 471.1849,
+
found 471.1853.
41.9, 26.9; HPLC 96.3%; MS m/z 500.2 [M+H] ; HRMS (ESI) m/z
+
calcd for C24
H
21
N
3
OS [M+H] 400.1478, found 400.1476.
5
7
.1.16. 1-(5-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
-yl)-3,4-dihydropyridin-1(2H)-yl)prop-2-en-1-one (14d)
5.1.21. 1-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-
7-yl)-3,4-dihydropyridin-1(2H)-yl)prop-2-en-1-one (15c)
15c was prepared by the general method as described above for
preparation of 14c (85 mg, 77.4%). Mp: 160.9–161.9 °C; H NMR
3
(400 MHz, CDCl ) d 7.75 (s, 1H), 7.42–7.37 (m, 4H), 7.17 (t,
1
4d was prepared by the general method as described above for
1
preparation of 14c (32 mg, 67.8%). Mp: 131–133 °C; H NMR
400 MHz, CDCl 7.83–7.75 (m, 1H), 7.42–7.38 (m, 4H),
.20–7.16 (m, 2H), 7.11–7.07 (m, 4H), 6.66–6.61 (m, 1H),
.37–6.28 (m, 2H), 5.76–5.74 (m, 1H), 4.99 (s, 2H), 4.54 (s, 1H),
.40 (s, 1H), 3.88–3.76 (m, 2H), 2.46–2.43 (m, 2H); HPLC 98.6%;
1
(
3
)
d
7
6
4
J = 14.76 Hz, 1H), 7.13 (s, 1H), 7.11–7.07 (m, 4H), 4.61 (s, 2H),
3.52–3.47 (m, 1H), 3.37–3.34 (m, 1H), 2.87–2.72 (m, 3H), 2.17–
+
13
MS m/z 454.2 [M+H] ; HRMS (ESI) m/z calcd for C27
M+H] 454.1583, found 454.1584.
H
23
N
3
O
2
S
6
2.14 (m, 1H), 1.88–1.80 (m, 4H); C NMR (100 MHz, DMSO-d ) d
+
[
157.5, 156.5, 154.2, 146.7, 140.0, 137.1, 131.5, 131.2, 130.7,
Please cite this article in press as: Xin, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.039

8
M. Xin et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
1
9
24.5, 124.1, 122.0, 119.7, 119.0, 118.6, 43.5, 41.9, 28.7; HPLC
for another 30 min. The TMB reaction was quenched by addition of
0.05 mL of 2 M H SO . The optical density was measured at 450 nm
by an ELISA reader. The IC50 values were calculated for test com-
pounds by using a regression analysis of the concentration/inhibi-
tion data.
+
8.3%; MS m/z 402.2 [M+H] ; HRMS (ESI) m/z calcd for C24
H
23
N
3
OS
2
4
+
[
M+H] 402.1634, found 402.1637.
5
.1.22. 3-(4-Phenoxyphenyl)-7-(piperidin-4-yl)thieno[3,2-c]pyri-
din-4-amine (15d)
5d was prepared by the general method as described above for
1
References and notes
1
preparation of 14e (230 mg, 57.5%). H NMR (400 MHz, CDCl
3
) d
1.
Xu, D.; Kim, Y.; Postelnek, J.; Vu, M. D.; Hu, D. Q.; Liao, C.; Bradshaw, M.; Hsu, J.;
Zhang, J.; Pashine, A.; Srinivasan, D.; Woods, J.; Levin, A.; O’Mahony, A.; Owens,
T. D.; Lou, Y.; Hill, R. J.; Narula, S.; DeMartino, J.; Fine, J. S. J. Pharmacol. Exp. Ther.
2012, 341, 90.
7
1
2
9
.80 (s, 1H), 7.42–7.37 (m, 4H), 7.17 (t, J = 14.76 Hz, 1H), 7.13 (s,
H), 7.11–7.07 (m, 4H), 4.61 (s, 2H), 3.26–3.23 (m, 2H),
.85–2.79 (m, 3H), 2.02–1.99 (m, 2H), 1.88–1.81 (m, 2H); HPLC
+
2. Lou, Y.; Owens, T. D.; Kuglstatter, A.; Kondru, R. K.; Goldstein, D. M. J. Med.
Chem. 2012, 55, 4539.
7.3%; MS m/z 402.2 [M+H] ; HRMS (ESI) m/z calcd for C24
H
23
N
3
OS
+
[
M+H] 402.1634, found 402.1639.
.2. BTK enzymatic assay
The HTRF kinase assay (components supplied as kit by Cisbio)
3.
4.
5.
6.
7.
Davids, M. S.; Brown, J. R. Future Oncol. 2014, 10, 957.
http://www.medkoo.com/Anticancer-trials/HM71224.htm.
https://www.thomson-pharma.com/.
Robak, T.; Robak, E. Expert Opin. Investig. Drugs 2012, 21, 921.
Hendriks, R. W. Nat. Chem. Biol. 2011, 7, 4.
5
8. Pan, Z.; Scheerens, H.; Li, S.; Schultz, B. E.; Sprengeler, P. A.; Burrill, L. C.;
Mendonca, R. V.; Sweeney, M. D.; Scott, K. C. K.; Grothaus, P. G.; Jeffery, D. A.;
Spoerke, J. M.; Honigberg, L. A.; Young, P. R.; Dalrymple, S. A.; Palmer, J. T.
ChemMedChem 2007, 2, 58.
was chosen for BTK enzyme assays. It uses time resolved fluores-
cence resonance energy transfer (TR-FRET) to detect production
of a phosphorylated substrate. A peptide substrate is labeled with
a biotin that can bind to XL665 labeled streptavidin, and the
9.
Kim, K. H.; Maderna, A.; Schnute, M. E.; Hegen, M.; Mohan, S.; Miyashiro, J.; Lin,
L.; Li, E.; Keegan, S.; Lussier, J.; Wrocklage, C.; Nicherson-Nutter, C. L.; Wittwer,
A. J.; Soutter, H.; Caspers, N.; Han, S.; Kurumbail, R.; Dunussi-Joannopoulos, K.;
Douhan, J.; Wissner, A. Bioorg. Med. Chem. Lett. 2011, 21, 6258.
+
anti-phosphoresidue antibody is labeled with Eu . Upon phospho-
rylation of the substrate, the antibody binds to phosphorylated
substrate that enables TR-FRET detection in homogenous assay for-
mat. All the reagents used for the BTK kinase assays including their
resources are BTK kinase (Invitrogen), HTRF kinEASE-TK kit (Cisbio
1
0. Di Paolo, J. A.; Huang, T.; Balazs, M.; Barbosa, J.; Barck, K. H.; Bravo, B. J.; Carano,
R. A.; Darrow, J.; Davies, D. R.; DeForge, L. E.; Diehl, L.; Ferrando, R.; Gallion, S.
L.; Giannetti, A. M.; Gribling, P.; Hurez, V.; Hymowitz, S. G.; Jones, R.; Kropf, J.
E.; Lee, W. P.; Maciejewski, P. M.; Mitchell, S. A.; Rong, H.; Staker, B. L.;
Whitney, J. A.; Yeh, S.; Young, W. B.; Yu, C.; Zhang, J.; Reif, K.; Currie, K. S. Nat.
Chem. Biol. 2011, 7, 41.
Bioassays), ATP(Sigma), DTT(Sunshine), MgCl
The assay buffer was composed of 50 mM HEPES (pH 7.0), 5 mM
MgCl , 5 mM DTT, 0.1% NaN , 0.1% BSA and 0.1 mM orthovanadate.
2 2
and MnCl (Sigma).
1
1. Young, W. B.; Barbosa, J.; Blomgren, P.; Bremer, M. C.; Crawford, J. J.; Dambach,
D.; Gallion, S.; Hymowitz, S. G.; Kropf, J. E.; Lee, S. H.; Liu, L.; Lubach, J. W.;
Macaluso, J.; Maciejewski, P.; Maurer, B.; Mitchell, S. A.; Ortwine, D. F.; Paolo, J.
D.; Reif, K.; Scheerens, H.; Schmitt, A.; Sowell, C. G.; Wang, X.; Wong, H.; Xiong,
J. M.; Xu, J.; Zhao, Z.; Currie, K. S. J. Med. Chem. 2014, 86, 664.
2
3
The HTRF assays were preformed according to the manual in the
kit. All reagents were dispensed into each well plate according to
12. Puig de la Bellacasa, R.; Roue, G.; Balsas, P.; Perez-Galan, P.; Teixido, J.;
Colomer, D.; Borrel, J. I. Bioorg. Med. Chem. Lett. 2014, 24, 2206.
the orders as follow: (1) BTK enzyme: 0.5 ng/
pound as well as control: 0.008–50 mM; (3) reagent: 22.4
and 0.15 M substrate, 2 l; (4) inculation: Ambient, at 25 °C,
min; (5) reagent: antibody and XL-665, 8 l. Then following 1 h
ll, 4
ll; (2) each com-
l
M ATP
13. Shi, Q.; Tebben, A.; Dyckman, A. J.; Li, H.; Liu, C.; Lin, J.; Spergel, S.; Burke, J. R.;
McIntyre, K. W.; Olini, G. C.; Strnad, J.; Surti, N.; Muckelbauer, J. K.; Chang, C.;
An, Y.; Cheng, L.; Ruan, Q.; Leftheris, K.; Carter, P. H.; Tino, J.; De Lucca, G. V.
Bioorg. Med. Chem. Lett. 2014, 24, 2206.
l
l
5
l
incubation at room temperature fluorescence was measured on
the PHERAStar FS microplate reader (BMG Lab Technologies). Sig-
nal was expressed in terms of HTRF ratio (fluorescence intensity
at 665 nm/fluorescence intensity at 620 nm).
1
4. Lou, Y.; Han, X.; Kuglstatter, A.; Kondru, R. K.; Sweeney, Z. K.; Soth, M.;
McIntosh, J.; Litman, R.; Suh, J.; Kocer, B.; Davis, D.; Park, J.; Frauchiger, S.;
Dewdney, N.; Zecic, H.; Taygerly, J. P.; Sarma, K.; Hong, J.; Hill, R. J.; Gabriel, T.;
Goldstein, D. M.; Owens, T. D. J. Med. Chem. 2015, 58, 512.
5. Lou, Y.; Sweeney, Z. K.; Kuglstatter, A.; Davis, D.; Goldstein, D. M.; Han, X.;
Hong, J.; Kocer, B.; Kondru, R. K.; Litman, R.; McIntosh, J.; Sarma, K.; Suh, J.;
Taygerly, J.; Owens, T. D. Bioorg. Med. Chem. Lett. 2015, 25, 367.
1
5
.3. ELISA-based kinase selectivity assay
1
6. Wan, H. L.; Wang, Z. R.; Li, L. L.; Cheng, C.; Ji, P.; Liu, J. J.; Zhang, H.; Zou, J.; Yang,
S. Y. Chem. Biol. Drug Des. 2012, 80, 366.
In vitro kinase inhibition assays were carried out as described
elsewhere. Briefly, 96-well plates were pre-coated with 0.2 mg/mL
poly (Glu-Tyr, 4:1) (Sigma) overnight at 37 °C. 0.05 mL aliquot of
17. Zhao, X.; Xin, M.; Huang, W.; Ren, Y.; Jin, Q.; Tang, F.; Jiang, H.; Wang, Y.; Yang,
J.; Mo, S.; Xiang, H. Bioorg. Med. Chem. 2015, 23, 348.
18. Zhao, X.; Huang, W.; Wang, Y.; Xin, M.; Jin, Q.; Cai, J.; Tang, F.; Zhao, Y.; Xiang,
H. Bioorg. Med. Chem. 2015, 23, 891.
0
.01 mmol/L ATP diluted in kinases and reaction medium were
added. Test compounds at various concentrations diluted in
.01 mL of 1% DMSO (V/V) were added. The reaction mixtures were
19. Zhao, X.; Xin, M.; Wang, Y.; Huang, W.; Jin, Q.; Tang, F.; Wu, G.; Zhao, Y.; Xiang,
H. Bioorg. Med. Chem. 2015, 23, 6059.
20. (a) Xin, M.; Wen, J.; Tang, F.; Tu, C.; Shen, H.; Zhao, X. Bioorg. Med. Chem. Lett.
0
2013, 23, 6777; (b) Xin, M.; Wen, J.; Tang, F.; Tu, C.; Huang, W.; Shen, H.; Zhao,
incubated for 60 min at 37 °C. The wells were washed with PBS
containing 0.1% T-PBS for three times. The 0.1 mL Phosphorylated
tyrosine substrate was added. The kinase reaction was incubated
for 60 min at 37 °C, and then the wells were washed three times
and then anti-mouse IgG (ZSGB-BIO; ZB-2305; 0.1 mL/well) cou-
pled with horseradish peroxidase (HRP) was added and incubated
X.; Cheng, L.; Wang, M.; Zhang, L. Bioorg. Med. Chem. Lett. 2014, 24, 983; (c) Xin,
M.; Zhang, L.; Tang, F.; Tu, C.; Wen, J.; Zhao, X.; Liu, Z.; Cheng, L.; Shen, H.
Bioorg. Med. Chem. 2014, 22, 1429; (d) Xin, M.; Zhang, L.; Shen, H.; Wen, J.; Tu,
C.; Liu, Z.; Cheng, L.; Zhao, X. Med. Chem. Res. 2014, 23, 3784; (e) Zhang, L.; Xin,
M.; Wen, J.; Tang, F.; Tu, C.; Shen, H.; Wei, P. Chin. J. Org. Chem. 2014, 34, 1407.
Please cite this article in press as: Xin, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.039