Discovery of thieno[3,2-c]pyridin-4-amines as novel Bruton's tyrosine kinase (BTK) inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.05.043

A novel series of BTK inhibitors bearing thieno[3,2-c]pyridin-4-amine framework as the core scaffold were designed, synthesized and well characterized. In this paper, twenty one compounds displayed variant inhibitory activities against BTK in vitro, and c

Full text of DOI:10.1016/j.bmc.2015.05.043

Accepted Manuscript
Discovery of thieno[3,2-c]pyridin-4-amines as novel Bruton’s tyrosine kinase
(BTK) inhibitors
Xinge Zhao, Minhang Xin, Yazhou Wang, Wei Huang, Qiu Jin, Feng Tang,
Gang Wu, Yong Zhao, Hua Xiang
PII:
S0968-0896(15)00453-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.043
BMC 12339
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 March 2015
21 May 2015
22 May 2015
Please cite this article as: Zhao, X., Xin, M., Wang, Y., Huang, W., Jin, Q., Tang, F., Wu, G., Zhao, Y., Xiang, H.,
Discovery of thieno[3,2-c]pyridin-4-amines as novel Bruton’s tyrosine kinase (BTK) inhibitors, Bioorganic &
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.043
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Discovery of thieno[3,2-c]pyridin-4-amines as novel Bruton’s
tyrosine kinase (BTK) Inhibitors
a, b,
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Xinge Zhao
†, Minhang Xin c, †, Yazhou Wang b, Wei Huang d, Qiu Jin b, Feng Tangb,
Gang Wu b, Yong Zhao b, Hua Xiang a, *
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a Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, No. 24,
Tongjiaxiang, Nanjing 210009, P.R. China
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b Jiangsu Simcere Pharmaceutical Co. Ltd., Jiangsu Key Laboratory of Molecular Targeted Antitumor
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Drug Research, No 699-18, Xuan Wu District, Nanjing 210042, P.R. China
c Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an Jiaotong
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University, No 76, Yanta West Road, Xi’an 710061, P.R.China
d Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan, 430079, P.R.China
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Footnotes
*Address correspondence to this author at the Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, No 24 Tongjiaxiang, Nanjing 210009, P.R. China; Tel:
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+86-25-83271096; E-mail: 1020030692@cpu.edu.cn
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† These authors contributed equally to this work.
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Abstract: A novel series of BTK inhibitors bearing thieno[3,2-c]pyridin-4-amine
framework as the core scaffold were designed, synthesized and well characterized. In this
paper, twenty one compounds displayed variant inhibitory activities against BTK in vitro,
and compound 14g showed the most potent inhibitory activity agianst BTK enzyme, with
the IC50 value of 12.8 nM. Moreover, compounds 14g displayed relatively good kinase
selectivity and was subsequently evaluated in vivo for profiling its PK properties. This
work identified the thieno[3,2-c]pyridin-4-amine derivatives as novel BTK inhibitors and
verified the value of thieno[3,2-c]pyridin-4-amine scaffold in drug design.
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Keywords: BTK inhibitors, thieno[3,2-c]pyridin-4-amine, inhibitory activity, kinase
selectivity.
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1. Introduction
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Bruton's tyrosine kinase (BTK) is a member of Tec family of cytoplasmic mammalian
non-receptor tyrosine kinases. It is a crucial terminal kinase in the B cell receptor (BCR)
signaling pathway and essential for the development and activation of B cells. It has been
shown that aberrant activation of B cells plays a central role in the pathogenesis of B-cell
lymphomas and various autoimmune diseases. Therefore, inhibition of BTK has become
an attractive and potential therapeutic approach for the treatment of human diseases
associated with B cells lymphoproliferative disorders and autoimmune disorders, such as
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hematological malignancies and rheumatoid arthritis (RA) 1-2
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During recent years, a number of research groups are eager to pursue small molecular
BTK inhibitors, and many BTK-targeting agents have progressed to clinical stages.
Among these, ibrutinib (1, PCI-32765, ImbruvicaTM) is the most successful BTK
inhibitor, which was developed by Pharmacyclics and approved by US FDA for the
treatment of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) in
the past 2 years. And now, it is also ongoing in the clinical evaluation for other
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indications . In addition, several BTK inhibitors including spebrutinib (2, CC-292,
AVL-292), ONO-4059 (3) and ACP-196 are currently moved into the phase II clinical
evaluation, and several molecules including MSC-2364447, PRN1008, BGB-3111 and
HM-71224 are quickly advanced into phase I clinical trials, although most of their
structures are still not disclosed 4-6. Except that, there are some agents including
CGI-1746, GDC-0834, RN-486 (4) reported in discovery stage 7-15. Recently, we have
also reported our medicinal chemistry work on the discovery of BTK inhibitors, which
are structurally similar to RN-486, such as compounds 5 and 6 16-17. In general, the
reported small molecular BTK inhibitors can be classified into two classes (irreversible
inhibitors and reversible inhibitors) by their binding modes with the BTK catalytic
domains. Irreversible BTK inhibitors contain a Michael addition receptor moiety in the
structures which can form a covalent bind with the conserved cysteine-481 residue of
BTK enzyme to achieve strong binding 1. The agents including ibrutinib, spebrutinib and
ONO-4059 belong to this class. It is believed that irreversible BTK inhibitors can achieve
significant clinical benefit for treating hematological malignancies. For example,
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ibrutinib shows significant progression free survival benefit and overall survival benefit
in both MCL and CLL clinical trials. Nevertheless, it is thought reversible BTK inhibitors
might be more appropriate for treating RA. RA is different from hematological
malignancies, and is non-life-threatening disease. However, the irreversible inhibitors
might give some unexpected off-target toxicity because of the potential of covalently
binding to non-target proteins and achieving the drug–protein conjugate. Thus, the
development of reversible BTK inhibitors would be more suitable to fulfill the treatment
need for non-life-threatening RA disease 18-19. Among the reported reversible BTK
inhibitors, 5-phenylpyrazinone-based or 5-phenylpyridinone-based inhibitors, such as
GDC-0834 and RN-486, demonstrated high BTK inhibitory potency and excellent
selectivity. However, GDC-0834 was suspended in phase I because of poor
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pharmacokinetic properties, and RN486 was not any progressed in the past 3 years .
Thus, there is still eagerly need to develop new classes of reversible BTK inhibitors.
(Insert Figure 1 and title for Figure 1 here)
Ibrutinib is a selective irreversible BTK inhibitor, showing very high potency against
BTK (IC50 = 0.5 nM). Ibrutinib can irreversibly bind to BTK because of the acrylamide
moiety which can form a covalent bind with the conserved cysteine-481 residue of BTK.
In fact, ibrutinib was structurally optimized from the precursor compound PCI-29732,
which was reported to be another potent BTK inhibitor (IC50 = 8.2 nM). Due to lack of
Michael addition receptor moiety, PCI-29732 is regard to non-covalently bind to BTK
enzyme. PCI-29732 is chemically similar to compound B43. The publicly available
crystal structure of B43 with BTK (PDB 3GEN) disclosed the binding mode of this type
of reversible BTK inhibitors 20-21. Generally, the backbone of 4-amino pyrrolopyrimidine
occupies the ATP binding pocket and makes several important interactions with the hinge.
The 4-amine directly forms two H-bonds with the gatekeeper Thr-474 and the backbone
Glu-475. The N-3 nitrogen of the pyrimidine participates in an H-bond with Met-477,
and the phenoxyphenyl group enters a hydrophobic pocket and forms a face-to-edge
π-stacking interaction with Phe-540. These interactions above are critical for BTK
activity. Bioisosteric replacement is a highly attractive method in the drug design and
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discovery, and we have used this strategy to discover several novel chemical series in
hedgehog inhibitors programs 22. Thieno[3,2-c]pyridines are very valuable heterocyclic
scaffolds which are abundantly described as central structural motifs in the design of
kinase inhibitors 23. Based on the deeply understanding of the binding mode of B43 with
BTK enzyme and the principles of bioisosterism, we designed a novel series of BTK
inhibitors containing thieno[3,2-c]pyridin-4-amine scaffold. In the new designed series,
thieno[3,2-c]pyridin-4-amine group are expected to occupy the ATP binding pocket and
keep the key hydrogen bonds, and the C3-phenoxyphenyl and its bioisosteric groups can
form the π-stacking interaction. Meanwhile, we tried to introduce alkyl and aryl groups
on the C7-position of thieno[3,2-c]pyridines to explore the SAR of the
thieno[3,2-c]pyrid-4-amine scaffold. To the best of our knowledge, there is the first time
to describe the SARs on this position. Herein, we report our recent effort on the synthesis
and SARs of this series of BTK inhibitors.
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(Insert Figure 2 and title for Figure 2 here)
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2. Chemistry
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As summarized in Table 1, the thieno[3,2-c]pyridin-4-amine derivatives (12a–h, 14a-i,
19a-b and 24a-b) were designed and synthesized. The synthetic routes for all target
compounds are illustrated in Schemes 1–2.
Compounds 12a–h and 14a-i were synthesized according to Scheme 1. Commercially
available
3-bromothieno[3,2-c]pyridin-4-amine
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was
treated
with
(4-phenoxyphenyl)boronic acid under palladium catalysis to give the Suzuki coupling
product 10 in 96 % yield. Iodination of 10 using N-iodosuccinimide afforded the key
intermediate 11 in 70 % yield. Then treating 11 with various boronic acid reagents under
the Suzuki coupling condition provided the target compounds 12a-h as well as the
intermediate 13 in 35-90 % yields. Deprotection of 13 with trifluoroacetic acid provided
the target compound 14a in 92 % yield, which was subsequently treated with various
halides under alkaline condition to produce target compound 14b-i in 40-95 % yields.
(Insert Scheme 1 and legend for Scheme 1 here)
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Compounds 19a–b and 24a-b were prepared according to Scheme 2. Condensation of
the commercially available boronobenzoic acid (15a-b) and aniline gave 16a-b, which
were subsequently reacted with 3-bromothieno[3,2-c]pyridin-4-amine 9 using Suzuki
coupling protocols to generate 17a-b. Treating 17a-b with N-iodosuccinimide provided
the iodides 18a-b, followed by Suzuki coupling reaction to afford the target compounds
19a-b. Likewise, the similar synthetic routes were employed to synthesize compounds
24a-b. Starting from the commercially available 4-aminophenylboronic acid pinacol ester
(20) and m-chlorobenzoyl chloride, the desired condensation product 21 was obtained,
which was followed by Suzuki coupling reaction, iodination and again Suzuki coupling
reaction, leading to the target compounds 24a-b.
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(Insert Scheme 2 and legend for Scheme 2 here)
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3. Result and discussion
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3.1. In vitro enzymatic assay of the new compounds against BTK kinase
The newly synthesized thieno[3,2-c]pyridin-4-amine derivatives (12a–h, 14a-i, 19a-b
and 24a-b) were evaluated for their abilities to inhibit the BTK enzymatic activity. The
vitro IC50 values are summarized in Table 1.
(Insert Table 1 and title for Table 1 here)
Among the thieno[3,2-c]pyridin-4-amine series, when the six-membered ring aryl
group were introduced at the C7-position, such as phenyl (12a), 4-pyridyl (12b),
4-morpholinyl substituted phenyl (12c), 4-phenoxy substituted phenyl (12d), and
6-methoxynaphthalenyl (12e), the resulting compounds almost lost inhibitory effect against
BTK (IC50 > 1000 nM). However, the five-membered ring aryl substituted compounds 12f
and 14a displayed weak potency against BTK. C7-furan-3-yl alogue 12f showed an IC50
value of 767.5 nM, while C7-1H-pyrazol-4-yl alogue 14a was weaker than 12f, with an
IC50 value of 1052 nM. The introduction of three-membered ring of cyclopropyl (12g) or
linear alkyl of n-butyl (12h) in the C7-position resulted in BTK inhibitory activity lost
(IC50 > 10000 nM, both). This initial SAR trend appeared that five-membered aryl
substituted at C7-position was a preference. Subsequently, a survey of several substituents
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at the N1-position of pyrazolyl was investigated. Interestingly, Compounds 14b bearing
N1-methyl group showed an IC50 value of 107.4 nM, which was 10 fold more potent than
no-substituted pyrazolyl 14a, while N1-methyl was changed into bulky alkyl groups such
as ethyl (14c), n-butyl (14d), benzyl (14e) or methoxypropyl (14f) afforded reduced BTK
inhibitory activity, compared to 14b. However, unexpectedly, compound 14g bearing a
2-(methylamino)-2-oxoethyl at N1-position of pyrazolyl exhibited a higher potency
against BTK, with an IC50 value of 12.8 nM. Compared with these,
2-(methylamino)-2-oxoethyl (14g) produced increased activity likely due to the amide or
the carbonyl group. An attempt of replacement of 2-(methylamino)-2-oxoethyl with
dimethylaminosulfuryl (14h) and acetyl (14i) led to reduced potency, which appeared
that the position of the amide or the carbonyl group was critical for BTK inhibitory activity.
N-(2-(methylamino)-2-oxoethyl)-1H-pyrazol-1-yl moiety at the C7-position may provide
an extra interaction with BTK residue, thereby showing the higher inhibitory activity.
Based on the best two substituents at the C7-position, the C3-position modification was
attempted to carry out. The replacement of the oxygen linker of C3-phenoxyphenyl with
an amide afforded compounds 19a and 19b, as well as 24a and 24b. However,
unfortunately, these compounds showed reduced inhibitory activities. This finding
suggested that the π-electron properties of the subsutituents on the C3-position of
thieno[3,2-c]pyrid-4-amine scaffold significantly affected the BTK inhibitory activity.
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3.2. Kinase selectivity assay
From the BTK inhibition results above, compound 14b and 14g were picked out as the
representative BTK inhibitors among the thieno[3,2-c]pyridin-4-amine series, and they
were further evaluated for their inhibitory activities against 4 kinds of our in-house RTKs.
The data were summarized in Table 2. Compared to BTK enzyme, both compounds only
showed moderate inhibitory activities against KDR kinase, and moreover, compound 14g
displayed 10-fold more selective against BTK than KDR. This result indicated that 14g was
a relatively selective BTK inhibitor, and deserving further identification against other
kinase profiles.
(Insert Table 2 and title for Table 2 here)
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3.3. In vivo pharmacokinetic (PK) profiles
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Compound 14g stood out as the most potent BTK inhibitor in this
thieno[3,2-c]pyridin-4-amine series, with an IC50 value of 12.8 nM. Therefore, it was
further investigated by profiling its pharmacokinetic (PK) properties in vivo. Table 3
illustrated the PK profiles of compound 14g in SD rats, by iv (2.5 mg/kg) and po (10
mg/kg) administration. Following intravenous injection with 2.5 mg/kg in SD rats, 14g
showed a satisfactory exposure (AUC = 1459.1 hr*ng/mL) and volume of distribution
(Vz = 900 mL/kg), but a short half-time (T1/2 = 0.36 h). The short elimination phase half
life was probably due to its large systemic clearance (Cl = 1716.4 mL/hr/kg).
Subsequently, compound 14g was further evaluated by oral administration of 10 mg/kg
dose. It was found the drug was also quickly eliminated, with large systemic clearance of
8670.1 mL/hr/kg and short half time (T1/2 = 1.01 h), although the area-under-curve (AUC
= 1365.7 hr*ng/mL) and the oral bioavailability (F = 23.4 %) are acceptable in this dose.
The PK properties of compound 14g suggested that some groups in this structure maybe
easily be metabolized and eliminated, thereby exhibiting a short half-life. Thus, in the
next structural modification, efforts should be made for reducing the metabolic effect and
optimizing the PK characteristics, meanwhile, increasing the in vitro biological activities.
(Insert Table 3 and title for Table 3 here)
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3.4. Docking study
In an effort to elucidate the binding mode for this kind of
thieno[3,2-c]pyridin-4-amines, We performed the molecular docking of compound 14g
with BTK enzyme (PDB code 3GEN). The CDOCKER program of the Discovery Studio
2.5 software package was used, and the binding model of compound 14g with BTK
enzyme was shown in Figure 3. It was found that the entire structure of 14g was
favorably located in the BTK pocket. The 4-amino group of thieno[3,2-c]pyridine
fragment of 14g formed two hydrogen bonds with Thr-474 and Glu-475, respectively.
Furthermore, the N atom in the 5-position of thieno[3,2-c]pyridine generated another
hydrogen bonding interaction with the backbone of Met-477 in the BTK kinase hinge
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region. These three hydrogen bonds were very critical for maintaining the BTK inhibitory
activity. Meanwhile, the N’ atom in the 2-position of pyrazolyl group substituted at
C7-position of thieno[3,2-c]pyridin-4-amine formed a hydrogen bond with the water-36,
which was similar to B43 with BTK. Interestingly, it was noticeable that the carbonyl of
2-(methylamino)-2-oxoethyl group in 1-position of pyrazolyl could afford a hydrogen
bond interaction with Cys-481, which might contribute to the higher BTK inhibition of
compound 14g in the series. Overall, the docking results afforded some understandings
on the observed BTK inhibitory activities for the thieno[3,2-c]pyridin-4-amines.
(Insert Figure 3 and title for Figure 3 here)
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4. Conclusion
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In this paper, we have developed a novel series of BTK inhibitors bearing
thieno[3,2-c]pyridin-4-amine framework as the core scaffold. Twenty one compounds
were design, synthized, and evaluated. These compounds displayed variant inhibitory
activities against BTK in vitro, and compound 14g gave the highest potency with the IC50
value of 12.8 nM. Moreover, compounds 14g displayed relatively good kinase selectivity.
However, in vivo evaluation showed that compound 14g had poor PK properties. Thus,
further structural modification is deserved to be made for improving the PK
characteristics, as well as increasing the in vitro biological activities.
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5. Experimental
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5.1 Chemistry
All chemical reagents were purchased from commercial vendors and used without
further purification unless noted especially. The melting points for the compounds were
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performed on a Melt-Temp II apparatus and uncorrected. H NMR spectra (400 MHz)
and 13C NMR (100 MHz) spectra data were recorded in CDCl3 or DMSO-d6 on a Bruker
BioSpin AG (Ultrashield Plus AV 400) spectrometer. MS data were recorded at an
Agilent-6120 quadrupole LC/MS (ESI) while HRMS were recorded at a Water Q-Tof
micro mass spectrometer. The HPLC study for the compounds was verified using a
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mixture of solvent (methanol/water or acetonitrile/water) at the flow rate of 2 mL/min
and peak detection at 254 nm under UV. Column chromatography was carried out on
silica gel (200–300 mesh) purchased from Qindao Ocean Chemical Company of China.
Thin-layer chromatography (TLC) analyses were carried out on silica gel GF254.
Ibrutinib was synthesized according to the reference literature.
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3-(4-Phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
(10).
A
solution
of
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3-bromothieno[3,2-c]pyridin-4-amine (3 g, 13 mmol), Pd(PPh3)4 (1.5 g, 1.3 mmol) and
(4-phenoxyphenyl)boronic acid (3.06 g, 14.3 mmol) in toluene (20 mL) was degassed
with nitrogen for 5 min followed by addition of EtOH (4 mL), H2O (2 mL) and Na2CO3
(3.5 g, 32.5 mmol) under continuous flow of nitrogen. The reaction mixture was stirred at
90 oC for 2 h. The reaction mixture was cooled, filtered through celite, diluted with water
(45 mL), and extracted with (3 × 60 mL) ethyl acetate. The combined organic layers were
dried over sodium sulfate and were concentrated in vacuo, the crude product was purified
on a silica gel column using (20-80 % ethyl acetate/hexanes) as eluent to afford 10 (4.0 g,
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96 %) as a white solid. MS m/z 319.1 [M+H]+; H NMR (400 MHz, DMSO-d6) δ ppm
7.84-7.82 (d, J=5.60 Hz, 1H), 7.59 (s, 1H), 7.48-7.43 (m, 4H), 7.27-7.26 (d, J=5.60 Hz,
1H), 7.22-7.18 (t, J=14.4 Hz, 1H), 7.14-7.10 (t, J=15.6 Hz, 4H), 5.43 (brs, 2H).
7-Iodo-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine (11). A solution of
3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
(2.5
g,
7.9
mmol)
and
N-Iodosuccinimide (2.14 g, 9.5 mmol) in DMF (10 mL) was stirred at r t for 2 h. After
completion of reaction, the reaction mixture was diluted with the mixed ice and water, the
precipitated solid was filtered off and the crude product was purified on a silica gel
column using (20-80 % ethyl acetate/ petroleum ether) as eluent to afford 11 (2.4 g,
70.0 %) as a yellow solid. MS m/z 445.0 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ ppm
8.02 (s, 1H), 7.57 (s, 1H), 7.48-7.42 (m, 4H), 7.22-7.18 (t, J=14.8 Hz, 1H), 7.14-7.10 (t,
J=15.2 Hz, 4H), 5.61 (brs, 2H).
3-(4-Phenoxyphenyl)-7-phenylthieno[3,2-c]pyridin-4-amine (12a). A solution of
7-iodo-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine (100 mg, 0.225 mmol),
Pd(PPh3)4 (26 mg, 0.0225 mmol) and phenylboronic acid (33 mg, 0.27 mmol) in
Ethylene glycol dimethyl ether (10 mL) was degassed with nitrogen for 5 min followed
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by addition of H2O (2 mL) and Na2CO3 (72 mg, 0.675 mmol) under continuous flow of
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nitrogen. The reaction mixture was stirred at 80 C for 3 h. The reaction mixture was
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cooled, filtered through celite, diluted with water (45 mL), and extracted with (3 × 20 mL)
ethyl acetate. The combined organic layers were dried over sodium sulfate and were
concentrated in vacuo, the crude product was purified on a silica gel column using
(20-50 % ethyl acetate/ petroleum ether) as eluent to afford 12a (71 mg, 80.1 %) as a
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white solid. Mp:172-173.4 C; MS m/z 395.1 [M+H]+; H NMR (400 MHz, CDCl3) δ
ppm 7.99 (s, 1H), 7.66 (s, 1H), 7.52-7.39 (m, 9H) 7.20-7.18 (t, J=14.8 Hz, 1H), 7.13-7.09
(t, J=15.2 Hz, 4H), 4.86 (brs, 2H); HPLC 95.4 %; HRMS (ESI) m/z calcd for
C25H18N2OS [M+H]+. 395.1140, found 395.1218.
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3-(4-Phenoxyphenyl)-7-(pyridin-4-yl)thieno[3,2-c]pyridin-4-amine (12b). 12b was
synthesized by the general method described above compound 12a (27 mg, 66.3 %). Mp:
230.2-231.8 oC; MS m/z 396.1 [M+H]+; 1H NMR (400 MHz, CDCl3) δ ppm 8.72-8.71 (d,
J=5.56 Hz, 2H), 8.04 (s, 1H), 7.63-7.62 (d, J=5.52 Hz, 2H), 7.45-7.39 (dd, J=8.44 Hz,
J=7.64 Hz, 4H), 7.20-7.19 (t, J=4.44 Hz, 2H), 7.12-7.10 (d, J=8.56 Hz, 4H), 4.87 (brs,
2H); HPLC 99.4 %; HRMS(ESI) m/z calcd for C24H18N3OS [M+H]+. 396.1092, found
396.1172.
7-(4-Morpholinophenyl)-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine (12c).
12c was synthesized by the general method described above compound 12a (28 mg,
o
67.1 %). Mp: 207.7-209.4 C; MS m/z 480.2 [M+H]+;1H NMR (400 MHz, DMSO-d6) δ
ppm 7.85 (s, 1H), 7.62 (s, 1H), 7.53-7.43 (m, 7H), 7.22-7.21 (t, J=8.54 Hz, 1H),
7.15-7.07 (m, 5H), 5.45 (brs, 2H), 3.78-3.76 (t, J=8.00 Hz, 4H), 3.19-3.17 (t, J=8.00 Hz,
4H); HPLC 95.4 %; HRMS (ESI) m/z calcd for C29H25N3O2S [M+H]+. 480.1740, found
480.1756.
3,7-Bis(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine (12d). 12d was synthesized
by the general method described above compound 12a (22 mg, 76.1 %). Mp: 185.5-187.3
oC; MS m/z 487.2 [M+H]+;1H NMR (400 MHz, CDCl3) δ ppm 7.92 (s, 1H), 7.63-7.61 (d,
J=8.04 Hz, 2H), 7.46-7.43 (d, J=8.34 Hz, 2H), 7.42-7.36 (dd, J=8.00 Hz, J=8.00 Hz,4H),
7.20-7.17 (t, J=13.20 Hz, 2H), 7.15-7.10 (m, 8H); HPLC 95.4 %; HRMS (ESI) m/z calcd
for C31H22N2O2S [M+H]+. 487.1478, found 487.1482.
11

1
2
7-(6-Methoxynaphthalen-2-yl)-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
(12e). 12e was synthesized by the general method described above compound 12a (32 mg,
o
1
3
72.1 %). Mp: 205.1-207.2 C; MS m/z 475.2 [M+H]+; H NMR (400 MHz, CDCl3) δ
ppm 8.06-8.05 (d, J=3.4 Hz, 2H), 7.87-7.85 (d, J=8.04 Hz, 1H), 7.82-7.80 (t, J=8.41 Hz,
1H), 7.77-7.75 (d, J=8.00 Hz, 1H), 7.47-7.45 (d, J=8.20 Hz, 2H), 7.42-7.39 (t, J=12.18
Hz, 2H), 7.21-7.16 (dd, J=6.04 Hz, J=4.84 Hz, 4H), 7.12-7.10 (dd, J=2.32 Hz, J=2.46 Hz,
4H), 4.75 (brs, 2H), 3.96 (s, 3H); HPLC 97.7 %; HRMS (ESI) m/z calcd for
C30H22N2O2S [M+H]+. 485.1474, found 485.1485.
4
5
6
7
8
9
7-(Furan-2-yl)-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine (12f). 12f was
synthesized by the general method described above compound 12a (38 mg, 63.0 %). Mp:
163.4-165.1 oC; MS m/z 385.1 [M+H]+; 1H NMR (400 MHz, CDCl3) δ ppm 8.05 (s, 1H),
7.89 (s, 1H), 7.56 (s, 1H), 7.44-7.38 (dd, J=8.04 Hz, J=8.46 Hz, 4H), 7.21-7.18 (t,
J=12.04 Hz, 2H), 7.11-7.08 (dd, J=3.20 Hz, J=4.00 Hz, 4H), 6.83 (s, 1H), 4.74 (brs, 2H);
HPLC 97.5 %; HRMS (ESI) m/z calcd for C23H16N2O2S [M+H]+. 385.1005, found
385.1018.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
7-Cyclopropyl-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine (12g). 12g was
synthesized by the general method described above compound 12a (21 mg, 42.0 %). Mp:
180.5-182.4 oC; MS m/z 359.1 [M+H]+; 1H NMR (400 MHz, CDCl3) δ ppm 7.69 (s, 1H),
7.40 (s, 4H), 7.15 (s, 2H), 7.09 (s, 4H), 4.66 (brs, 2H), 1.97 (s, 1H), 0.97 (s, 2H), 0.75 (s,
2H). HPLC 95.8 %; HRMS (ESI) m/z calcd for C22H18N2OS [M+H]+. 359.1212, found
359.1211.
7-Butyl-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
(12h).
12h
was
synthesized by the general method described above compound 12a (21 mg, 42.0 %). Mp:
147.5-148.6 oC; MS m/z 375.2 [M+H]+; 1H NMR (400 MHz, CDCl3) δ ppm 7.72 (s, 1H),
7.43-7.37 (dd, J=8.04 Hz, J=7.82 Hz, 4H), 719-7.15 (t, J=15.60 Hz, 1H), 7.12 (s, 1H),
711-7.07 (t, J=15.80 Hz, 4H), 4.56(br s, 2H), 719-7.15 (t, J=15.60 Hz, 1H), 2.78-2.75(t,
J=12.00 Hz, 2H), 1.77-1.70 (m, 2H), 1.45-1.38 (m, 2H), 0.98-0.95 (q, J=12.40 Hz, 3H);
HPLC 95.4 %; HRMS (ESI) m/z calcd for C23H22N2OS [M+H]+. 375.1526, found
375.1538.
Tert-butyl 4-(4-amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-7-yl)-1H-pyrazole
12

1
2
-1-carboxylate (13). A solution of 11 (150 mg, 0.337 mmol), 1-Boc-pyrazole-4-boronic
acid pinacol ester (100 mg, 0.337 mmol) and Pd(dppf)Cl2·CH2Cl2 (27 mg, 0.0337 mmol)
in 1,4-dioxane (10 mL) was degassed with nitrogen for 5 min followed by addition
Na2CO3 (107 mg, 2M in water) under continuous flow of nitrogen. The reaction mixture
was stirred at 80 oC for 1 h, the catalyst was removed by filtration through Celite and the
filtrate concentrated to a residue. The residue was used in the next reaction without
further purification. MS m/z 485.1 [M+H]+.
3
4
5
6
7
8
3-(4-Phenoxyphenyl)-7-(1H-pyrazol-4-yl)thieno[3,2-c]pyridin-4-amine (14a).
A
9
mixture of the crude product 13 (100 mg, 0.21 mmol) was stirred in CF3COOH (3 mL) at
r t for 2 h, after completion of reaction, the reaction mixture was diluted with the mixed
ice and water, and then 1M NaOH solution was added to alkalify to pH 7-8, the
precipitated solid was filtered, and concentrated under reduced pressure. The crude
product was purified on a silica gel column using (5-10 % CH3OH/ DCM) as eluent to
afford 14a (59 mg, 75 %) as a gray solid. Mp: 232.3-234.2 oC; MS m/z 385.1 [M+H]+.1H
NMR (400 MHz, DMSO-d6) δ ppm 13.09 (brs, 1H), 8.15 (brs, 1H), 8.06 (s, 1H), 7.95 (br
s, 1H), 7.54 (s, 1H), 7.48-7.43 (dd, J=7.20 Hz, J=8.20 Hz, 4H), 7.22-7.18 (t, J=6.80 Hz,
1H), 7.14-7.12 (dd, J=2.80 Hz, J=2.80 Hz, 4H), 5.42 (brs, 2H); HPLC 99.7 %; HRMS
(ESI) m/z calcd for C22H16N4OS [M+H]+.385.1117, found 385.1128.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
7-(1-Methyl-1H-pyrazol-4-yl)-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
(14b). A suspension of 14a (100 mg, 0.26 mmol), CH3I (55 mg, 0.39 mmol) and K2CO3
(71.7 mg, 0.52 mmol) in DMF (5 mL) was stirred for 12 h at rt. After completion of
reaction, the reaction mixture was diluted with the mixed ice and water, the precipitated
solid was filtered off and the crude product was purified on a silica gel column using
(20-50%) ethyl acetate/ petroleum ether) as eluent to afford 14b (19mg, 42.0%) as a
o
1
white solid. Mp: 201-203 C; MS m/z 456.2 [M+H]+; H NMR (400 MHz, DMSO-d6)
δppm 8.17 (s, 1H), 8.07 (br s, 2H), 7.93 (s, 1H), 7.56 (s, 1H), 7.51-7.43 (dd, J=8.00 Hz,
J=8.20 Hz, 4H), 7.22-7.19 (t, J=6.40 Hz, 1H), 7.14-7.12 (dd, J=4.40 Hz, J=4.00 Hz, 4H),
5.44 (brs, 2H), 4.86 (s, 2H), 2.65 (s, 1H); HPLC 98.6%; HRMS(ESI) m/z calcd for
C25H21N5O2S [M+H]+. 456.1488, found 456.1501.
7-(1-Ethyl-1H-pyrazol-4-yl)-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
13

1
2
(14c). 14c was synthesized by the general method described above compound 14b (90
mg, 87.1 %) as a white solid. Mp: 180.5-182.0 oC; MS m/z 399.2 [M+H]+. 1H NMR (400
MHz, CDCl3) δ ppm 7.91-7.90 (d, J=6.20 Hz, 1H), 7.76 (s, 1H), 7.65 (s, 1H), 7.43-7.38
(dd, J=8.40 Hz, J=7.80 Hz, 4H), 7.21-7.15 (dd, J=6.00 Hz, J=7.60 Hz, 2H), 7.12-7.07 (dd,
J=4.80 Hz, J=5.20 Hz, 4H), 5.20 (brs, 2H), 4.00 (s, 3H); HPLC 95.3 %; HRMS (ESI) m/z
calcd for C23H18N4OS [M+H]+.399.1274, found 399.1276.
3
4
5
6
7
7-(1-Butyl-1H-pyrazol-4-yl)-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
(14d). 14d was synthesized by the general method described above compound 14b (38.5
8
o
1
9
mg, 83.0 %). Mp: 160-162 C; MS m/z 413.2 [M+H]+; H NMR (400 MHz, CDCl3) δ
ppm 8.02 (s, 1H), 7.90 (s, 1H), 7.78 (s, 1H), 7.45-7.38 (dd, J=8.40 Hz, J=8.00 Hz, 4H),
7.18-7.16 (t, J=8.20 Hz, 2H), 7.11-7.08 (dd, J=4.20 Hz, J=4.40 Hz, 4H), 4.68 (brs, 2H),
4.28-4.27 (q, J=4.00 Hz, 2H), 1.60-1.56 (t, J=2.40 Hz, 3H); HPLC 99.0 %; HRMS (ESI)
m/z calcd for C24H20N4OS [M+H]+.413.1430, found 413.1432.
10
11
12
13
14
15
16
17
18
7-(1-Benzyl-1H-pyrazol-4-yl)-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-4-amine
(14e). 14e was synthesized by the general method described above compound 14b (18.3
o
1
mg, 57.2 %). Mp: 165-167 C; MS m/z 441.2 [M+H]+; H NMR (400 MHz, CDCl3) δ
ppm 8.00 (s, 1H), 7.89 (s, 1H), 7.76 (s, 1H), 7.44-7.38 (dd, J=8.40 Hz, J=8.00 Hz, 4H),
7.20-7.18 (t, J=8.20 Hz, 2H), 7.12-7.08 (dd, J=4.20 Hz, J=4.40 Hz, 4H), 4.87 (brs, 2H),
19
4.23-4.219(t, J=14.0 Hz, 2H), 1.93 (m, 2H), 1.41 (m, 2H), 1.00-0.97 (t, J=9.20 Hz, 3H)；
20
21
22
23
24
25
HPLC 95.5 %; HRMS (ESI) m/z calcd for C26H24N4OS [M+H]+.441.1670, found
441.1673.
7-(1-(3-Methoxypropyl)-1H-pyrazol-4-yl)-3-(4-phenoxyphenyl)thieno[3,2-c]pyridi
n-4-amine (14f). 14f was synthesized by the general method described above compound
o
1
14b (21 mg, 86.5 %). Mp: 182-183.5 C; MS m/z 475.2 [M+H]+; H NMR (400 MHz,
CDCl3) δppm 8.02 (s, 1H), 7.95 (s, 1H), 7.77 (s, 1H), 7.44-7.30(m, 9H), 7.19-7.15(m,
26
2H), 7.11-7.08 (dd, J=4.20 Hz, J=4.40 Hz, 4H), 5.41 (s, 2H), 4.67 (s, 2H)；HPLC 95.6 %;
27
28
29
HRMS (ESI) m/z calcd for C29H22N4OS [M+H]+. 475.1587, found 475.1588.
2-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-7-yl)-1H-pyrazol-1-yl)-N-
methylacetamide (14g). 14g was synthesized by the general method described above
14

1
2
compound 14b (23 mg, 65.2 %). Mp: 145-147 oC; MS m/z 457.2 [M+H]+; 1H NMR (400
MHz, DMSO-d6) δ ppm 8.17 (s, 1H), 8.05 (s, 1H), 7.91 (s, 1H), 7.56 (s, 1H), 7.50-7.43
(dd, J=8.80 Hz, J=8.40 Hz, 4H), 7.22-7.19 (t, J=12.4 Hz, 1H), 7.15-7.11 (dd, J=4.40 Hz,
J=4.20 Hz, 4H), 4.86 (brs, 2H), 4.33 (t, 2H), 3.40 (t, 2H), 3.39 (s, 3H), 2.20 (t, 2H);
HPLC 96.6 %; HRMS (ESI) m/z calcd for C26H24N4O2S [M+H]+. 457.1692, found
457.1700.
3
4
5
6
7
4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-7-yl)-N,N-dimethyl-1H-pyra
zole-1-sulfonamide (14h). 14h was synthesized by the general method described above
compound 14b (18 mg, 88.5 %). Mp: 170-172 oC; MS m/z 492.2 [M+H]+; 1H NMR (400
MHz, DMSO-d6) δ ppm 8.61 (s, 1H), 8.43 (s, 1H), 8.22 (s, 1H), 7.63 (s, 1H), 7.51-7.43
(dd, J=8.80 Hz, J=8.40 Hz, 4H), 7.23-7.19 (t, J=12.4 Hz, 1H), 7.15-7.13 (dd, J=4.40 Hz,
J=4.20 Hz, 4H), 5.81 (brs, 2H), 2.93 (s, 6H); HPLC 98.5 %; HRMS (ESI) m/z calcd for
C24H21N5O3S2 [M+H]+. 492.1158, found 492.1172.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1-(4-(4-Amino-3-(4-phenoxyphenyl)thieno[3,2-c]pyridin-7-yl)-1H-pyrazol-1-yl)eth
anone (14i). 14i was synthesized by the general method described above compound 14b
o
1
(25 mg, 62.5 %). Mp: 169-171 C; MS m/z 427.2 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ ppm 8.70 (s, 1H), 8.48 (s, 1H), 8.28 (s, 1H), 7.61 (s, 1H), 7.52-7.44 (dd,
J=8.40 Hz, J=8.40 Hz, 4H), 7.24-7.20 (t, J=12.4 Hz, 1H), 7.16-7.13 (dd, J=4.40 Hz,
J=4.20 Hz, 4H), 5.66 (brs, 2H), 2.71 (s, 3H); HPLC 98.5 %; HRMS (ESI) m/z calcd for
C24H18N4O2S [M+H]+. 427.1223, found 427.1225.
(3-(Phenylcarbamoyl)phenyl)boronic acid (16a). A suspension of 3-boronobenzoic
acid (1.0 g, 5.9 mmol), aniline (0.65g, 7 mmol), Et3N (1.29 g, 12 mmol) and PyBop (3.6
g, 7mmol) in DMF(10 mL) was stirred for 12 h at r t. After completion of reaction, the
solution was diluted with H2O (15 mL), and then the product was extracted three times
with EtOAc (50 mL). The combined organic layer was dried over Na2SO4, and the
solvent was removed in vacuo, the crude product was purified on a silica gel column
using (1-5 %) CH3OH/ DCM as eluent to afford 16a (1.47 g, 75 %) as a white solid. MS
m/z 241.2 [M+H]+.
(4-(Phenylcarbamoyl)phenyl)boronic acid (16b). 16b was synthesized by the
general method described above compound 16a (1.53 g, 40.8 %). MS m/z 241.2 [M+H]+.
15

1
2
3-(4-Aminothieno[3,2-c]pyridin-3-yl)-N-phenylbenzamide
(17a).
17a
was
synthesized by the general method described above compound 12a (0.89 g, 78.3 %). MS
3
m/z 345.2 [M+H]+.
4
4-(4-Aminothieno[3,2-c]pyridin-3-yl)-N-phenylbenzamide
(17b).
17b
was
5
synthesized by the general method described above compound 12a (1.21 g, 83.1 %). MS
m/z 345.2 [M+H]+.
6
7
3-(4-Amino-7-iodothieno[3,2-c]pyridin-3-yl)-N-phenylbenzamide (18a). 18a was
synthesized by the general method described above compound 11 (0.41 g, 47.1 %). MS
m/z 470.2 [M+H]+.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
4-(4-Amino-7-iodothieno[3,2-c]pyridin-3-yl)-N-phenylbenzamide (18b). 18b was
synthesized by the general method described above compound 11 (0.56 g, 50.3 %). MS
m/z 470.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.41 (s, 1H), 8.10-8.08 (d, J
= 4.80 Hz, 2H), 8.06 (s, 1H), 7.82-7.80 (d, J = 8.00 Hz, 2H), 7.69 (s, 1H), 7.65-7.63 (d, J
= 8.00 Hz, 2H), 7.39-7.35 (t, J = 16.00 Hz, 2H), 7.14-7.12 (d, J = 8.00 Hz, 1H), 5.62 (brs,
2H).
3-(4-Amino-7-(1-(2-(methylamino)-2-oxoethyl)-1H-pyrazol-4-yl)thieno[3,2-c]pyrid
in-3-yl)-N-phenylbenzamide (19a). 19a was synthesized by the general method
described above compound 13 (21 mg, 45.1 %). Mp: 171-173 oC; MS m/z 483.2 [M+H]+;
1H NMR (400 MHz, DMSO-d6) δ ppm 10.35 (s, 1H), 8.21 (s, 1H), 8.13-8.08 (m, 4H),
7.97 (s, 1H), 7.79-7.77 (d, J = 8.00 Hz, 2H), 7.75-7.68 (q, 3H), 7.38-7.34 (d, J = 16.00 Hz,
2H), 7.13-7.11 (d, J = 8.00 Hz, 1H), 5.41 (s, 2H), 4.88 (s, 2H), 2.65 (s, 3H). HPLC
96.1 %; HRMS (ESI) m/z calcd for C26H22N6O2S [M+H]+. 483.1597, found 483.1601.
4-(4-Amino-7-(1-(2-(methylamino)-2-oxoethyl)-1H-pyrazol-4-yl)thieno[3,2-c]pyrid
in-3-yl)-N-phenylbenzamide (19b). 19b was synthesized by the general method
described above compound 13 (27 mg, 49.8 %). Mp: 178-180 oC; MS m/z 483.2 [M+H]+;
1H NMR (400 MHz, DMSO-d6) δ ppm 10.42 (s, 1H), 8.21 (s, 1H), 8.13-8.10 (m, 4H),
7.97 (s, 1H), 7.83-7.81 (d, J = 8.00 Hz, 2H), 7.68-7.66 (d, 3H), 7.40-7.36 (d, J = 16.00 Hz,
2H), 7.14-7.12 (d, J = 8.00 Hz, 1H), 5.47 (s, 2H), 4.88 (s, 2H), 2.65 (s, 3H). HPLC 98.2%;
HRMS (ESI) m/z calcd for C26H22N6O2S [M+H]+. 483.1598, found 483.1603.
3-Chloro-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)benzamide
16

1
2
(21). 3-chlorobenzoyl chloride (175 mg, 1 mmol) in anhydrous THF (2 mL) was added
dropwise to a solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (219 mg,
1 mmol) and Et3N (152 g, 1.5 mmol) in 10 mL anhydrous THF of cooled to 0°C, and the
reaction mixture was stirred at 0 °C for 1 h and at room temperature for 2 h. The solution
was diluted with H2O (5 mL), and then the product was extracted three times with EtOAc
(20 mL). The combined organic layer was dried over Na2SO4, and the solvent was
removed in vacuo, the residue was used in the next reaction without further purification.
MS m/z 357.2 [M+H]+.
3
4
5
6
7
8
9
N-(4-(4-Aminothieno[3,2-c]pyridin-3-yl)phenyl)-3-chlorobenzamide (22). 22 was
synthesized by the general method described above compound 10 (151 mg, 53.7 %). MS
m/z 379.1 [M+H]+.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
N-(4-(4-Amino-7-iodothieno[3,2-c]pyridin-3-yl)phenyl)-3-chlorobenzamide (23).
13 was synthesized by the general method described above compound 3 (102 mg,
43.2 %). MS m/z 505.1 [M+H]+.
N-(4-(4-Amino-7-(1-methyl-1H-pyrazol-4-yl)thieno[3,2-c]pyridin-3-yl)phenyl)-3-c
hlorobenzamide (24a). 24a was synthesized by the general method described above
compound 12a (30 mg, 38.5 %). Mp: 221-223 oC; MS m/z 460.1 [M+H]+; 1H NMR (400
MHz, CDCl3) δ ppm 8.01 (s, 1H), 7.96 (s, 1H), 7.89-7.88 (m, 2H), 7.78-7.75 (t, J = 12.40
Hz, 4H), 7.57-7.44 (m, 4H), 4.73(s, 2H), 4.01 (s, 3H). HPLC 99.0 %; HRMS (ESI) m/z
calcd for C24H18ClN5OS [M+H]+. 460.0993, found 460.1002.
N-(4-(4-Amino-7-(1-(2-(methylamino)-2-oxoethyl)-1H-pyrazol-4-yl)thieno[3,2-c]p
yridin-3-yl)phenyl)-3-chlorobenzamide (24b). 24b was synthesized by the general
method described above compound 12a (38 mg, 35.8 %). Mp: 220-222 oC; MS m/z 517.1
1
[M+H]+; H NMR (400 MHz, CDCl3) δ ppm 8.01-8.00 (m, 3H), 7.96-7.86 (m, 2H),
7.80-7.88 (d, J = 12.40 Hz, 3H), 7.55-7.47 (m, 4H), 7.21 (s, 1H), 6.45 (s, 1H), 5.01 (s,
2H), 4.90 (s, 2H), 2.85 (s, 3H). HPLC 99.0 %; HRMS (ESI) m/z calcd for
C26H21ClN6O2S [M+H]+. 517.1208, found 517.1225.
5.2 BTK enzymatic assay
The HTRF kinase assay (components supplied as kit by Cisbio) was chosen for BTK
17

1
2
enzyme assays. It uses time resolved fluorescence 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 anti-phosphoresidue antibody
is labeled with Eu+. Upon phosphorylation of the substrate, the antibody binds to
phosphorylated substrate that enables TR-FRET detection in homogenous assay format.
All the reagents used for the BTK kinase assays including their resources are BTK kinase
(Invitrogen), HTRF kinEASE-TK kit (Cisbio Bioassays), ATP(Sigma), DTT(Sunshine),
MgCl2 and MnCl2 (Sigma). The assay buffer was composed of 50 mM HEPES (pH 7.0),
5 mM MgCl2, 5mM DTT, 0.1 % NaN3, 0.1 % BSA and 0.1 mM orthovanadate. The
HTRF assays were preformed according to the manual in the kit. All reagents were
dispensed into each well plate according to the orders as follow: (1) BTK enzyme:
0.5ng/µl, 4 µl; (2) Each compound as well as control: 0.008-50 mM; (3) Reagent: 22.4
3
4
5
6
7
8
9
10
11
12
13
µM ATP and 0.15 µM substrate, 2 µl; (4) Inculation: Ambient, at 25℃, 5 min; (5)
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Reagent: antibody and XL-665, 8 µl. Then following 1 hour incubation at room
temperature fluorescence was measured on the PHERAStar FS microplate reader (BMG
Lab Technologies). Signal was expressed in terms of HTRF ratio (fluorescence intensity
at 665 nm/fluorescence intensity at 620 nm).
5.3 ELISA-based kinase selectivity assay.
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 0.01 mmol/L ATP diluted in kinases and reaction medium were
added. Test compounds at various concentrations diluted in 0.01mL of 1 % DMSO (V/V)
were added. The reaction mixtures were 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 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) coupled with horseradish peroxidase (HRP) was added and
incubated for another 30 min. The TMB reaction was quenched by addition of 0.05mL of
2 M H2SO4. The optical density was measured at 450 nm by an ELISA reader. The IC50
18

1
2
values were calculated for test compounds by using a regression analysis of the
concentration/inhibition data.
3
4
5.4 Pharmacokinetic Profiles of compound 14b in SD rats
5
Compound 14g were administered to 3 male SD rats (weight ranging from 180 g to
240 g) at doses of 2.5 mg/kg for iv administration as well as doses of 10 mg/kg for po
administration for another 3 male SD rats. The dosing volume was 5 mL/kg. After
administration, blood samples were collected at the point including 2 min, 5 min, 15 min,
30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h for analyses, the collected blood samples
6
7
8
9
o
were centrifuged at 4000 rpm for 5 min at 4 C, and then analyzed after protein
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
precipitation. LC/MS/MS analysis of compound 14g was performed under optimized
conditions to obtain the best sensitivity and selectivity of the analyte in selected reaction
monitoring mode (SRM) containing an internal standard. Plasma concentration-time data
were measured by a noncompartmental approach using the software WinNonlin
Enterprise, version 5.2 (Pharsight Co.,Mountain View, CA).
5.5 Molecular docking
The molecular docking procedure was referred to CDOCKER protocol within
Discovery Studio 2.5. The protein-ligand complex crystal structure of B43 bound to Btk
(PDB code 3GEN) was chosen as the template. The initial 3D conformation of compound
14g was optimized in the ChemBio3D Ultra using MM2 energy minimization method.
The whole Btk enzyme was typed with CHARMm force field and the hydrogen atoms
were added. The water molecules were deleted except those occupying at the ligand
pocket. Binding site was defined as a sphere. Compound 14g was docked in the defined
active site. A set of 10 starting random conformations was generated. The top scoring
pose of compound 14g was retained and selected for analyzing.
References and Notes
29
30
31
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Figure 1. Representative structures of BTK inhibitors.
4
5
22

1
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Figure 2. Design of the thieno[3,2-c]pyrid-4-amines as BTK inhibitors
3
4
23

1
2
3
Figure 3. Docking mode of compound 14g with BTK enzyme (PDB code: 3GEN). Selected residues
Thr-474, Glu-475, Met-477, H2O-36 and Cys-481 are shown. Hydrogen bonds are shown as green
dashed lines.
4
5
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7
24

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3
4
5
6
7
Scheme 1. Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, EtOH, H2O, toluene, 90 oC, 3 h, 96 %; (b) NIS, DMF, r t,
o
1 h, 70 %; (c) R1B(OH)2, Pd(PPh3)4, Na2CO3, DME, H2O, 80 C, 2-3 h, 35-90 %; (d) 1-Bocpyrazole-4-boronic acid
pinacol ester, Pd(dppf)Cl2CH2Cl2, Na2CO3, 1,4-dioxane, 100 oC,1-2 h, 55 %; (e) CF3COOH, DCM, rt, 12 h, 92 %; (f)
R2X, K2CO3, DMF, 25 oC, 4-12 h, 40-95 %.
25

1
2
3
4
5
6
7
8
9
Scheme 2. Reagents and conditions: (a) aniline, PyBop, Et3N, DMF, r t, 12 h; (b) compound 9, Pd(PPh3)4,
Na2CO3, DME, H2O, 80 oC, 2-3 h; (c) NIS, DMF, r t, 1h, 50-55 %; (d) Pd(dppf)Cl2·CH2Cl2, Na2CO3,
1,4-dioxane, 100 oC, 2 h; (e) m-Chlorobenzoyl chloride, Et3N, DCM, 0-25 oC, 2 h, 85%; (f) Pd(PPh3)4, Na2CO3,
o
DME, H2O, 80 C, 2-3 h, 80.2 %; (g) NIS, DMF, rt, 2 h, 57 %; (h) 1-Methylpyrazole-4-boronic acid pinacol
ester for 24a , N-methyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)acetamide for 24b,
Pd(dppf)Cl2·CH2Cl2, Na2CO3, 1,4-ioxane, 100 oC, 2 h.
26

1
2
Table 1. In vitro BTK enzymatic inhibition for thieno[3,2-c]pyrid-4-amine derivatives
3
Btk
IC50[nM] a
Btk
IC50[nM] a
Compds
12a
R1
R2
Compds
14d
R1
R2
>10000
387.1
699.2
279.7
12.8
1954
2295
12b
14e
12c
14f
>10000
>10000
12d
14g
O
O
N
S
N
N
527.6
12e
14h
767.5
454.2
291.5
12f
14i
>10000
12g
19a
>10000
1052
137
12h
14a
19b
24a
420.9
107.4
176.2
408.7
4.0
14b
14c
24b
ibrutinib
—
—
4
a Values are means of three experiments
5
6
27

1
2
Table 2. Kinase selectivity profiles of compounds 14b and 14g
IC50 (nM)
JAK3 KDR c-Met
Compds
structures
BTK
JAK2
107.4 >10000 >10000 343 >10000
14b
12.8 >10000 >10000 284 >10000
14g
3
4
5
28

1
Table 3. PK properties study for compound 14g in SD rats a
Dose
Cmax
AUC0-t
Vz
Cl
MRT(0-t) T1/2 Tmax
F
compds
(mg/kg) (ng/mL) (hr*ng/mL) (mL/kg) (mL/h/kg)
(h)
(h)
0.36 0.11
1.01 0.67 23.4
(h)
(%)
2.5 (iv)a 2547.3
10 (po)b
855.5
1459.1
1365.7
900.0
1716.4
8670.1
0.43
14g
13320.6
1.48
2
3
4
5
a Compound 14g was formulated using 5 % DMA + 10 % solutol.
b Compound 14g was formulated using 0.5 % HMPC + 0.5 % Twen-80.
29