Discovery of N-(3-(5-((3-acrylamido-4-(morpholine-4-carbonyl)phenyl)amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methylphenyl)-4-(tert-butyl)benzamide (CHMFL-BTK-01) as a highly selective irreversible Bruton's tyrosine kinase (BTK) inhibitor

Article abstract of DOI:10.1016/j.ejmech.2017.03.001

Currently there are several irreversible BTK inhibitors targeting Cys481 residue under preclinical or clinical development. However, most of these inhibitors also targeted other kinases such as BMX, JAK3, and EGFR that bear the highly similar active cysteine residues. Through a structure-based drug design approach, we discovered a highly potent (IC₅₀: 7?nM) irreversible BTK inhibitor compound 9 (CHMFL-BTK-01), which displayed a high selectivity profile in KINOMEscan (S score (35)?=?0.00) among 468 kinases/mutants at the concentration of 1?μM. Compound 9 completely abolished BMX, JAK3 and EGFR's activity. Both X-ray crystal structure and cysteine-serine mutation mediated rescue experiment confirmed 9's irreversible binding mode. 9 also potently inhibited BTK Y223 auto-phosphorylation (EC₅₀: <30?nM), arrested cell cycle in G0/G1 phase and induced apoptosis in U2932 and Pfeiffer cells. We believe these features would make 9 a good pharmacological tool to study the BTK related pathology.

Full text of DOI:10.1016/j.ejmech.2017.03.001

Accepted Manuscript
Discovery of N-(3-(5-((3-acrylamido-4-(morpholine-4-carbonyl)phenyl)amino)-1-
methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methylphenyl)-4-(tert-butyl)benzamide
(CHMFL-BTK-01) as a highly selective irreversible Bruton's tyrosine kinase (BTK)
inhibitor
Qianmao Liang, Yongfei Chen, Kailin Yu, Cheng Chen, Shouxiang Zhang, Aoli Wang,
Wei Wang, Hong Wu, Xiaochuan Liu, Beilei Wang, Li Wang, Zhenquan Hu, Wenchao
Wang, Tao Ren, Shanchun Zhang, Qingsong Liu, Cai-Hong Yun, Jing Liu
PII:
S0223-5234(17)30150-2
10.1016/j.ejmech.2017.03.001
EJMECH 9261
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 January 2017
Revised Date: 27 February 2017
Accepted Date: 1 March 2017
Please cite this article as: Q. Liang, Y. Chen, K. Yu, C. Chen, S. Zhang, A. Wang, W. Wang, H. Wu,
X. Liu, B. Wang, L. Wang, Z. Hu, W. Wang, T. Ren, S. Zhang, Q. Liu, C.-H. Yun, J. Liu, Discovery of
N-(3-(5-((3-acrylamido-4-(morpholine-4-carbonyl)phenyl)amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-
yl)-2-methylphenyl)-4-(tert-butyl)benzamide (CHMFL-BTK-01) as a highly selective irreversible
Bruton's tyrosine kinase (BTK) inhibitor, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.03.001.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of N-(3-(5-((3-acrylamido-4-(morpholine-4-
carbonyl)phenyl)amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-
methylphenyl)-4-(tert-butyl)benzamide (CHMFL-BTK-01) as a highly
selective irreversible Bruton's tyrosine kinase (BTK) inhibitor
1
,2,8
2,3,8
2,4,8
2,3,8
5,8
Qianmao Liang , Yongfei Chen , Kailin Yu , Cheng Chen , Shouxiang Zhang , Aoli
2
,4
2,3
2,4
1,2
2,3
2,3
Wang , Wei Wang , Hong Wu , Xiaochuan Liu , Beilei Wang , Li Wang , Zhenquan
2
,3
2,3
6
3,7
2,3,4,6
5*
Hu , Wenchao Wang , Tao Ren , Shanchun Zhang , Qingsong Liu
, Cai-Hong Yun , Jing
2
,3*
Liu
1
2
3
. Department of Chemistry, University of Science and Technology of China, Hefei, Anhui
30036, P. R. China
2
. High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350
Shushanhu Road, Hefei, Anhui 230031, P. R. China
. CHMFL-HCMTC Target Therapy Joint Laboratory, 350 Shushanhu Road, Hefei Anhui
2
30031, P. R. China
4
5
. University of Science and Technology of China, Hefei, Anhui 230036, P. R. China
. Institute of Systems Biomedicine, Department of Biophysics, Beijing Key Laboratory of
Tumor Systems Biology and Center for Molecular and Translational Medicine, School of
Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, P.R.
China
6
. Precision Targeted Therapy Discovery Center, Institute of Technology Innovation, Hefei
Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230088, P. R.
China
7
8
. Hefei Cosource Medicine Technology Co. LTD., 358 Ganquan Road, Hefei, Anhui 230031,
P. R. China
. These authors contribute equally
AUTHOR INFORMATION

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Corresponding Authors
E-mail address: yunch@hsc.pku.edu.cn (C-H. Yun), E-mail address: jingliu@hmfl.ac.cn (J.
*
Liu).
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. Q.L., Y.C., K.Y., C.C. and S.Z. contributed equally to this
work. Chemical synthesis: Qianmao Liang, Yongfei Chen; Biological evaluation: Kailin Yu,
Cheng Chen, Aoli Wang, Wei Wang, Hong Wu, Xiaochuan Liu, Beilei Wang, Li Wang,
Zhenquan Hu, Wenchao Wang, Tao Ren, Shanchun Zhang; Crystal structure determination:
Shouxiang Zhang, Cai-Hong Yun; Project supervision and manuscript preparation: Qingsong
Liu, Jing Liu
Abstract
Currently there are several irreversible BTK inhibitors targeting Cys481 residue under
preclinical or clinical development. However, most of these inhibitors also targeted other kinases
such as BMX, JAK3, and EGFR that bear the highly similar active cysteine residues. Through a
structure-based drug design approach, we discovered a highly potent (IC : 7 nM) irreversible
5
0
BTK inhibitor compound 9 (CHMFL-BTK-01), which displayed a high selectivity profile in
KINOMEscan (S score (35)=0.00) among 468 kinases/mutants at the concentration of 1 µM.
Compound 9 completely abolished BMX, JAK3 and EGFR’s activity. Both X-ray crystal
structure and cysteine-serine mutation mediated rescue experiment confirmed 9’s irreversible
binding mode. 9 also potently inhibited BTK Y223 auto-phosphorylation (EC50: <30 nM),
arrested cell cycle in G0/G1 phase and induced apoptosis in U2932 and Pfeiffer cells. We believe
these features would make 9 a good pharmacological tool to study the BTK related pathology.
Keywords
BTK; Irreversible inhibitor, Kinase inhibitor; Structure-activity relationship; B-cell lymphoma
Abbreviations used

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BTK, Bruton’s tyrosine kinase; BMX, bone marrow tyrosine kinase gene in chromosome X
protein; JAK3, Janus kinase 3; EGFR, epidermal growth factor receptor; BCR, B cell receptor;
RA, rheumatoid arthritis; MCL, mantle cell lymphoma; CLL, chronic lymphatic leukemia; WM,
waldenström's macroglobulinemia; TEK, endothelial tyrosine kinase; ITK, interleukin-2-
inducible T-cell kinase; RLK, LysM domain receptor-like kinase 1; HER2, Tyrosine kinase-type
cell surface receptor HER2; HER4, Tyrosine kinase-type cell surface receptor HER4; BLK, B
lymphocyte kinase; MAP2K7, dual specificity mitogen-activated protein kinase 7; PDB, protein
data bank; SAR, structure activity relationship; PLCε1, 1-phosphatidylinositol 4,5-bisphosphate
phosphodiesterase gamma-1.
1
. Introduction
Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase that is originally identified
in the inherited immunodeficiency disease X-linked agammaglobulinaemia (XLA) [1]. It is in
the downstream of B cell receptor (BCR) mediated signaling transduction pathway in B cells.
Dysregulation of BTK function may result in deficiency of cell division, cell surface markers
activation and high susceptibility to apoptosis [2-7]. BTK is primarily expressed in B cell
leukemias and lymphomas and disruption of its function has been validated for the treatment of a
variety of B cell malignances such as MCL, CLL and AML, etc [8-10]. In the preclinical model
and early clinical trials, BTK inhibitors also demonstrated efficacy for the rheumatoid arthritis
(RA) [11-12]. The critical role of BTK in the pathology has made it an attractive drug discovery
target and to date a number of BTK inhibitors have been developed, either in the clinical use or
in the variety stages of clinical development [10, 13]. PCI-32765 (1) was the first highly potent
irreversible BTK inhibitor and currently was clinically used for the treatment of mantle cell
lymphoma (MCL), chronic lymphatic leukemia (CLL) and waldenström's macroglobulinemia
(
WM) (Figure 1) [14-15]. It formed a covalent bond by its acrylamide moiety with the Cys481
residue located close to the hinge binding area of BTK. A number of irreversible BTK inhibitors
with similar binding mode have been developed such as QL47 (2) [16], CC-292 (3) [17], CNX-
7
74 (4) [18], and compound 5 [19]. The Cys481 residue utilized by these irreversible inhibitors
in BTK was also found in 11 other kinases such as TEK kinase family (BMX, ITK, RLK, TEC),
EGFR family (EGFR, HER2, HER4), as well as BLK, JAK3 and MAP2K7 kinases at the
identical position [19]. Thus most of these irreversible inhibitors may also inhibit these kinases

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by forming the similar covalent bond, e.g., compound 1 and 5 could potently inhibit JAK3,
BMX, EGFR etc [11, 20-21]. CGI1746 (6) was the first highly potent and selective reversible
BTK inhibitor discovered and based on this scaffold, GDC-0834 (7) [22] and RN486 (8) [23]
were developed. Comparably, the reversible inhibitors exhibited better selectivity profile than the
irreversible ones. Given the fact that irreversible inhibitors usually bear longer residence time on
the target and will provide different pharmacological efficacy profiles from the reversible ones
[24], we aimed to seek highly selective irreversible BTK inhibitors which could help to dissect
the pathological and physiological effect of “permanent” block of the BTK mediated signaling
pathway. A structure-based drug design approach staring from the reversible inhibitor 6 like
scaffold led to the discovery of the highly potent and selective irreversible BTK inhibitor
compound 9 (CHMFL-BTK-01) (Figure 2).
Figure 1. Chemical structures of representative BTK inhibitors.
Figure 2. Schematic illustration of discovery of compound 9 (CHMFL-BTK-01).
2
. Results and discussion

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1
. Design and synthesis
Given the high selectivity profile of compound 6 series BTK inhibitors, we decided to apply
the reversible protein kinase inhibitor scaffold based approach to develop the new irreversible
inhibitors [24]. We first analyzed the binding mode of 6 with BTK (PDB ID: 3OCS) (Figure 3
A). The X-ray crystal structure showed that 6 formed two hydrogen bonds with Met477 via its
aminopyrazinone moiety in the hinge binding area. The morpholine moiety directed into the
solvent exposed area adjacent to the hinge binding area. The tert-butyl substituted benzene ring
orientated into the inner hydrophobic pocket. The distances between the R1, R2, and R3 position
of the two benzene rings and the Cys481 are about 7-8 Å, which is feasible for the installment of
the irreversible warhead. Therefore, we hypothesized that introduction of proper electrophile
warhead at R1/R2/R3 positions and modification of the R4/R5/R6 moieties would furnish the
new irreversible inhibitors (Figure 3 B).
Figure 3. Schematic illustration of the structure-activity relationship (SAR) exploration
rationale. (A) Analysis of the BTK/compound 6 binding mode (PDB ID: 3OCS). (B) Illustration
of the SAR exploration sites.
The synthesis of 9-13 started from amide formation between 4-(tert-butyl) benzoic acid and
R3 substituted bromomethylaniline, and then a Suzuki coupling reaction offered pinacol
protected boronic ester fragment (Scheme 1). Amide coupling between substituted benzoic acid
derivatives and morpholine followed by palladium catalyzed amination furnished the other
fragment that bore R1/R2 substitute. Suzuki coupling of the two parts, hydrogenation of the nitro
group and acylation provided the desired compounds.
a
Scheme 1. Synthesis of Compounds 9-13

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a
Reagents and conditions: (a) oxalyl chloride, DMF, DCM, rt, 4 h; (b) ArNH , DIPEA, DCM,
2
rt, 10 min; (c) bis(pinacolato)-diboron, Pd(dppf)Cl , KOAc, dioxane, 100 °C, 8 h; (d)
2
morpholine, HATU, DIPEA, DMF, rt, 8 h; (e) 3,5-dibromo-1-methyl-1,2-dihydropyridin-2-one,
2 3 2 3 3 4 2 3
Pd (dba) , xantphos, Cs CO , dioxane, sealed tube, 110 °C, overnight; (f) Pd(PPh ) , Na CO ,
dioxane, H O, 110 °C, overnight; (g) Pd/C, H , MeOH, rt, 4 h; (h) Acyl chloride, DIPEA, DCM,
2
2
rt, 10 min.
Amidation of R4 substituted benzoyl chlorides with bromomethylaniline followed by Suzuki
coupling offered pinacol boronic ester, which was then coupled with the substituted pyridone
fragment (Scheme 2). Hydrogenation of the nitro group and subsequent acylation with acryloyl
chloride furnished compounds 14-31.
a
Scheme 2. Synthesis of Compounds 14-31

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a
Reagents and conditions: (a) oxalyl chloride, DMF, DCM, rt, 4 h; (b) 3-bromo-2-methylaniline,
DIPEA, DCM, rt, 10 min; (c) bis(pinacolato)diboron, Pd(dppf)Cl , KOAc, Ar, dioxane, 100 °C,
2
8
h; (d) S4b, Pd(PPh ) , Na CO , dioxane, H O, 110 °C, overnight; (e) Pd/C, H , MeOH, rt, 4 h;
3 4 2 3 2 2
(f) acryloyl chloride, DIPEA, DCM, rt, 10 min.
R5 substitution was introduced by amide coupling of 4-amino-2-nitrobenzoic acid with variety
of amine derivatives (Scheme 3). Palladium catalyzed amination, Suzuki coupling that connected
R4 substituted fragments, and installation of the acrylamide moiety provided compounds 34 and
3
6-46. To obtain 32-33 and 35, the nitroaniline derivatives were first coupled with
dibromopyridone and then similar approach was applied (Scheme 4).
a
Scheme 3. Synthesis of Compounds 34, 36-46

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a
Reagents and conditions: (a) R -NH , HATU, DIPEA, DMF, rt, 8 h; (b) 3,5-dibromo-1-
5
2
methyl-1,2-dihydropyridin-2-one, Pd (dba) , xantphos, Cs CO , dioxane, sealed tube, 110 °C,
2
3
2
3
overnight; (c) S2a (for S11a-g) or S7f (for S11h-l), Pd(PPh ) , Na CO , dioxane, H O, 110 °C,
3
4
2
3
2
overnight; (d) Pd/C, H , MeOH, rt, 4 h; (e) acryloyl chloride, DIPEA, DCM, rt, 10 min.
2
a
Scheme 4. Synthesis of Compounds 32-33, 35
a
Reagents and conditions: (a) 3,5-dibromo-1-methyl-1,2-dihydropyridin-2-one, Pd (dba) ,
2
3
xantphos, Cs CO , dioxane, sealed tube, 110 °C, overnight; (b) 4-(tert-butyl)-N-(2-methyl-3-
2
3
3 4 2 3
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)benzamide, Pd(PPh ) , Na CO , dioxane,

ACCEPTED MANUSCRIPT
H O, 110 °C, overnight; (c) Pd/C, H , MeOH, rt, 4 h; (d) acryloyl chloride, DIPEA, DCM, rt, 10
2
2
min.
Compounds 47-51 were prepared from 3,5-dibromo-1-methylpyridin-2(1H)-one and
amination yielded R6 substituted intermediates (Scheme 5). Suzuki coupling and TFA
deprotection followed by acylation afforded the target molecules.
a
Scheme 5. Synthesis of Compounds 47-51 .
a
Reagents and conditions: (a) R -NH , Pd (dba) , xantphos, Cs CO , dioxane, sealed tube, 110
6
2
2
3
2
3
°
C, overnight; (b) 4-(dimethylamino)-N-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)benzamide, Pd(PPh , Na CO , dioxane, H O, 110 °C, overnight; (c) TFA, DCM, rt,
h; (d) acryloyl chloride, DIPEA, DCM, rt, 10 min.
3
)
4
2
3
2
1
2
.2 Structure-activity relationship (SAR) exploration.
Considering the synthetic feasibility, we started with a scaffold that bore an aminopyridinone
instead of aminopyrazinone in 6. Introduction of acrylamide at R1 position afforded 9 (IC : 7
5
0
nM) that was slightly more potent than 6 (IC50: 21 nM) (Table 1). Installment of acrylamide at
R2 position resulted in 11-fold potency decrease (10, IC : 231 nM) compared to 6. Compound
5
0
1
1 that bears acrylamide at R3 position lost over 80-fold potency compared to 6. These indicated
that R1 position might be a suitable warhead site. Changing warhead from acrylamide to
chloroacetamide (12) slightly decreased the inhibitory activity about 9-fold compared to 9.
Saturation of the acrylamide to propionamide (13) caused obvious activity loss (over 23-fold to
9
), which indicated that 9 might exert its inhibitory activity through an irreversible binding
mode.

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a
Table 1. SAR Exploration Focused on the R1/R2/R3 Positions
BTK
Compd.
R1
R2
H
R3
H
(
IC : nM)
5
0
6
9
H
21±7
H
H
7±0.6
1
1
1
1
0
1
2
3
H
H
H
231±41
1697±48
63±5
H
H
H
H
H
163±8
a
All IC values were obtained by triple testing.
5
0
We next explored R4 position that would occupy the inner hydrophobic pocket of BTK
Table 2). Removal of the terminal tert-butyl group on the benzene ring (14) significantly
(
decreased the activity compared to 9 (IC50: 3736 nM vs 7 nM). Adding more hydrophobicity by
a methyl group (15, IC : 114 nM), ethyl group (16, IC : 210 nM) or isopropyl group (17, IC :
5
0
50
50
3
2 nM) started to gain back the activity but still weaker than the tert-butyl group. A
trifluoromethyl group (18, IC : 76 nM) exhibited a slightly better activity than the methyl group,
5
0
presumably due to the higher hydrophobicity of the compound. Introduction of the dimethyl
amino group at this position furnished 19 which showed similar potency to 9 (IC : 5.8 nM).
5
0
However, a methoxyl group at the same position (20) resulted in significant activity loss (IC :
5
0
2
610 nM), indicating that it should not be factors such as hydrogen bond that made 19 more

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potent than 17, but probably the higher solubility due to the more nitrogen atoms or the polarity
presented around there. Larger substitutes containing the nitrogen atom such as pyrrolidine (21)
or morpholine (22) led to about 12-40 fold activity loss. Simply switching the CF group from
3
para- to meta- position (23) of the benzene ring completely abolished the activity (IC50 > 10
µM), while the methoxyl group position change (24) brought the potency back to the moderate
level but was still about 170-fold weaker than 9. Interestingly, 25 that bears the para-methyl
3
group and the meta-CF group at the same time exhibited an activity (IC50: 1261 nM) between
the two compounds bearing each single substitution, i.e., 15 and 23. Installment of the methoxyl
group at both para- and meta- position (26) lost about 700-fold activity (IC50: 4996 nM). A five
membered 1,3-dioxolane ring (27, IC : 282 nM), six membered 1,4-dioxane ring (28, IC : 89
5
0
50
nM) or simple six membered cyclohexane ring (29, IC : 57 nM) started to gain back the activity
5
0
compared to 26 bearing two free methoxyl groups but still much weaker than 9. Replacement of
the benzene ring in 29 with a thiophene ring (30) gave an activity (IC : 16.5 nM) better than 31
5
0
but less than 9. Tri-substitution of the methoxyl group at para- and two meta- positions (31)
caused significant activity loss (over 200 fold, IC : 1436 nM). These SAR explorations
5
0
indicated that the hydrophobic substituent at para- position of the benzene ring on the R4
position was critical for the inhibitory activity and the tert-butyl group (9) or the dimethyl amino
group (19) was the best choice.
a
Table 2. SAR Exploration Focused on the R4 Position
BTK
BTK
Compd.
R4
Compd.
23
R4
(
IC : nM)
(IC : nM)
50
50
1
1
4
5
3736±206
114±9
>10000
24
1191±381

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1
1
6
7
210±10
32±6
25
26
1261±39
4996±82
1
8
76±7
27
282±3
1
2
2
9
0
1
5.8±0.9
2610±183
86±4
28
29
30
89±14
57±11
16.5±2.8
2
2
273±45
31
1426±42
a
All IC values were obtained by triple testing.
5
0
We then fixed the R1 substituent and explored the SAR of the R4 and R5 positions (Table
). When R4 position was the para-tert butyl benzene moiety as in 9, simply taking off the acyl
3
morpholine moiety from 9 offered 32 that completely lost the inhibitory activity (IC > 10 µM).
5
0
A smaller hydrophobic methyl group at R5 position (33) gained back the activity to 365 nM but
still much lower than 9 (IC50: 7 nM). Installment of the terminal dimethyl amide moiety at R5
(34) only resulted in near 5 fold activity loss compared to 9, and the methyl ester group (35) was
even much weaker presumably due to its hydrophobic feature (IC50: 197 nM). The five
membered pyrrolidine ring replacement (36) gave 19 nM inhibitory activity, and the
cyclopropanamine replacement (37) caused more activity loss (IC : 41 nM). The N-methyl
5
0
substituted piperazine (38) generated moderate activity (IC : 127 nM), while a bulky Boc
5
0
protection (39) started to gain back the activity to 68 nM. Introduction of more hydrophilic
moiety such as piperidinol (40) further improved the activity (IC50: 21 nM). However,

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decreasing the hydrophilicity by adding methyl ether to the piperidine (41) resulted in slight
activity loss (IC : 65 nM). When R4 position was the para-dimethyl amino benzene moiety as
5
0
in 19, aliphatic PEG-like chains (42 and 43) at R5 led to significant activity loss (IC : 667 nM
5
0
and 3187 nM). Similarly, neither the free piperidinol (44 and 45, IC50: 187 nM and 244 nM) nor
the methyl ether protected form (46, IC : 596 nM) gave a better potency. Hence these SAR
5
0
studies demonstrated that for R5 position the acyl morpholine in 9 was still the best option.
Besides, we also tried to explore if the warhead could be installed on an aliphatic ring instead of
the benzene ring (Table 4). However, all of the six membered ring replacement at the R6
position resulted in significant or complete loss of the activity (47-51, IC50: 2946 nM or > 10
µM).
a
Table 3. SAR Exploration Focused on the R4/R5 Positions
BTK
Compd
R4
R5
(
IC : nM)
50
3
3
3
3
2
3
4
5
H
>10000
365±37
34±2
CH
3
197±12

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3
3
3
3
4
4
4
4
4
4
4
6
7
8
9
0
1
2
3
4
5
6
19±3
41±0.7
127±0.7
68±14
21±5
65±5
667±204
3187±53
187±16
244±35
596±100
a
All IC50 values were obtained by triple testing.
a
Table 4. SAR Exploration Focused on the R6 Position

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BTK
IC50: nM)
Compd
R6
(
4
5
4
7
8
9
>10000
>10000
>10000
5
5
0
1
>10000
2946±164
a
All IC values were obtained by triple testing.
5
0
2
.3 Compound 9’s selectivity profiling
Since compounds 9, 19 and 30 exhibited the best inhibitory activities (IC : 7 nM, 5.8 nM,
5
0
and 16.5 nM respectively), we then tested these compounds to compare with known BTK kinase
inhibitors 1-5 among the kinases which share structurally identical cysteine residues such as
BTK, BMX, EGFR and JAK3 kinases by ADP-Glo assay (Table 5). The results demonstrated
that compound 9 exhibited the best selectivity and completely abolished BMX, EGFR and JAK3
kinase activity. While compound 19 showed good selectivity among these kinases compared to
compounds 1-5, compound 30 started to bring BMX kinase activity (IC : 341 nM) although the
5
0
selectivity over JAK3 and EGFR was remained. Therefore, we then subjected 9 to the kinome
wide selectivity profiling platform KINOMEscan to investigate its selectivity [25]. The results

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demonstrated that at 1 µM concentration, compound 9 only bound to BTK but not any other
kinase in a panel of 468 kinases (Figure 4 and Supplemental Table 1), which is as same as
compound 6’s selectivity [26]. Even the relative percent inhibition threshold (S score) was set as
3
5 (percent control number) (S score (35) =0.00), 9 still only had one target, i.e. BTK.
Table 5. Comparison of compounds 1-5 and 9’s biochemical IC values against BTK, BMX,
5
0
a
JAK3 and EGFR kinases
BTK
BMX
JAK3
EGFR
Compd.
(
IC50: nM)
(IC50: nM)
(IC50: nM)
(IC50: nM)
9
7±1
5.8±0.9
16.5±2.8
12±8
>10000
1359±29
341.3±105.2
8±3
>10000
>10000
5882±714
6863±24
123±5
1
9
0
2014±805
1033±68
146±29
3
1
2
3
4
5
12±7
54±28
3336±210
1753±451
1533±478
1705±156
874±208
442±46
737±149
72±16
9±0.4
2665±494
>10000
24±4
9.5±2.7
25±7
a
All IC50 values were obtained by triple testing.

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Figure 4. Kinome-wide selectivity profiling of compound 9 by DiscoveRx’s KINOMEscan
technology.
2
.4 Exploration of compound 9’s binding mode
To elucidate the detailed binding mode of 9 with BTK, we obtained a high-resolution crystal
structure (1.585Å) of the kinase domain of BTK (spanning residues 382-659) in complex with 9
Figure 5A). The overall structure adopted the DFG-in/C-helix-out inactive conformation that
closely resembled the previously released crystal structure of BTK in complex with 6 (PDB ID:
OCS) except for the clear covalent bond formed between the Cys481 side chain of BTK and the
(
3
acrylamide moiety of 9. The main chain amide and carbonyl of Met477 formed two hydrogen
bonds with 9, tethering the inhibitor to the hinge of the kinase. Interestingly, 9 formed another
hydrogen bond with the Lys430 side-chain, which is a conserved key regulatory residue of
protein kinases that forms a salt-bridge with a glutamine residue from the αC-helix (Glu445 in
BTK) when the kinase is in the active conformation [27].
The complex crystal structure of BTK/compound 9 could well explain the observed SAR
results. For example, SAR data showed that the acrylamide group contributed to the potency of
the compound only when it was attached to the R1 position, which was explained by the
structure that this position is the one closest to the Cys481 side chain among R1, R2 and R3
positions. And the merit of tert-butyl group at R4 could also be clearly explained that the upper
half of BTK residues around this region are hydrophobic, including Leu542, Phe413 and Val546,
which interacted with the tert-butyl moiety of 9 through hydrophobic interactions (Figure 5B).
However, the lower half of residues around this region are uncharged polar residues (Asn526 and

ACCEPTED MANUSCRIPT
Tyr551) or negatively charged acidic residues (Asp539 and Asp521), which would favor a polar,
ideally positively charged group at this position (Figure 5C). This potentially explained the
observation that a dimethylamino substituent (19, IC =5.8 nM) at R4 position was a little bit
5
0
better than the tert-butyl substituent (9, IC50=7 nM).
In order to further confirm that this irreversible binding was biologically relevant, we
transfected the wide type (wt) and the C481S mutant BTK into HEK293T cells and examined the
BTK Y223 phosphorylation. The results demonstrated that 9 inhibited wt BTK Y223
phosphorylation with an EC of 4.7 nM, while the EC for the C481S mutant was over 100 nM
50
50
(
Figure 5D). Similarly, 13 (the reversible version of 9) showed EC50 values over 100 nM for both
wt BTK and BTK C481S. These data proved that 9 inhibited the kinase activity of BTK via an
irreversible binding mode which was biologically relevant.
Figure 5. Binding mode exploration of compound 9 against BTK. (A) X-ray crystal structure of
9
in complex with BTK (PDB ID: 5J87). (B) Illustration of the hydrophobic pocket formed by
Leu542, Phe413 and Val546 to favor the tert-butyl substituent at R4 position. (C) Illustration of
the polar pocket formed by Asn526, Tyr 551, Asp539 and Asp521 to favor the dimethylaminio

ACCEPTED MANUSCRIPT
substituent at R4 position. (D) Compound 9 and 13’s inhibitory activities against Y223 in BTK
wide type and C481S mutant in HEK293T cells.
Since irreversible inhibitor usually takes two steps to achieve the inhibition, i.e., first the
reversible binding then the irreversible covalent bond formation, we next examined the
reversible binding affinity (K ) and the inactivation rate constant (kinact) of 9, 19 as well as 30 to
i
compare them with other reported irreversible BTK inhibitors using an activity-based, time-
dependent assay (Table 6). Compound 9 and 30 exhibited a high binding affinity towards BTK
with a K value in the single-digit nanomolar range (K ＝8.9 nM and 6.0 nM), and compounds 1
i
i
and 2 bore similar high reversible binding affinities against BTK. Compounds 3 and 5 displayed
slightly weaker binding affinities while compound 4 and 19 were much weaker (over 10-fold less
active than others). For the inactivation rate constant, compounds 1-4 and 19 exhibited the best
-1
efficiency (kinact over 0.1 min ) and compound 9 and 30 were slightly less efficient (k
= 0.06
inact
-1
-1
-1
min and 0.05 min ), while compound 5 was the least efficient (kinact =0.01 min ). For the
overall effectiveness of inactivation profile, compounds 30 and 1 exhibited the most favorable
-1 -1
-1 -1
inactivation kinetics (kinact/K = 0.42 µM s and 0.38 µM s ). Compounds 2, 3 and 9 bore
i
-1 -1
moderate favorable profiles (kinact/Ki: 0.11-0.15 µM s ), while compounds 4, 5 and 19 were
-1 -1
much less efficient (kinact/K ≤0.05 µM s ).
i
Table 6. Comparison of kinetic parameters K , k
and kinact/K determined for compounds 1-5
i
i
inact
a
and 9, 19 and 30.
-
1
-1 -1
Compd
K [nM]
kinact [min ]
0.06±0.02
0.33±0.08
0.05±0.01
0.11±0.10
0.13±0.05
kinact/K [µM s ]
i
i
9
8.9±1.2
113±12
6.0±0.53
4.8±1.8
14.2±2.8
0.11±0.03
0.05±0.01
0.42±0.03
0.38±0.08
0.15±0.05
1
9
0
3
1
2

ACCEPTED MANUSCRIPT
3
4
5
20.7±0.1
209.2±0.1
27.2±0.1
0.15±0.12
0.15±0.43
0.01±0.01
0.12±0.04
0.01±0.03
0.01±0.02
a
All data were obtained by triple testing.
.5 Compound 9’s effect on B-cell related cancer cell lines
We then tested compounds 9, 13, 19 and 30 against a panel of B cell related cancer cell lines
2
for the anti-proliferation effect in comparison with compounds 1 and 6 (Table 7). The results
demonstrated that 9, 19 and 30 exhibited moderated anti-proliferative efficacies, and most of cell
lines displayed sub-micromolar GI s. This is consistent with variety of previous reports that
5
0
BTK inhibitors did not exert the anti-cancer roles directly and solely depend on the blockage of
cancer cell proliferation but rather through disruption of BCR signaling, Toll like receptor (TLR)
signaling and affection of B cell adhesion, migration and the cells in the tumor
microenvironment [10]. For example, compound 1’s anti-MCL and anti-CLL efficacies mainly
depended on the expulsion of malignant B cells out of their nursing microenvironment [28]. Not
surprisingly, the reversible version of 9, compound 13 exhibited much lower activities, which
again proved the irreversible binding feature of 9. In general, compound 1 displayed better anti-
proliferative effect than 9, 19 and 30 in these B-cell related cell lines, which again reflected its
multiple targets property (FLT3, BMX, EGFR, JAK, etc) [29]. Furthermore, we also tested these
compounds against normal PBMC for the general toxicity. The results demonstrated that none of
them displayed strong anti-proliferative effect on these cells (Figure 6).
Table 7. Compound 9 and 13’s anti-proliferative effects against a panel of B cell related cancer
a
cell lines
Cell line
Compd.1
Compd. 6
Compd. 9
Compd. 13
Compd. 19
Compd. 30
GI (µM)
5
0
SU-DHL-2
0.64±0.076
>10
3.0±0.21
>10
2.6±0.35
1.8±0.5

ACCEPTED MANUSCRIPT
MV4-11
U2932
0.25±0.085
4.4±1.9
4.7±0.38
2.8±0.15
2.1±1.0
4.8±1.2
1.9±0.11
6.9±0.11
2.2±0.17
2.3±0.17
2.4±0.15
＞10
Romas
OCI-AML3
Pfeiffer
U937
3.4±0.38
9.2±0.1
＞10
＞10
＞10
＞10
＞10
＞10
＞10
3.5±1.1
4.6±0.58
9.5±0.15
＞10
2.1±0.45
2.6±0.15
9.0±0.86
2.6±0.26
1.9±0.51
1.0±0.17
3.2±0.56
1.2±0.78
2.3±1.8
3.8±0.21
4.5±0.40
4.8±0.49
6.6±0.93
7.1±0.35
7.3±0.35
1.6±0.35
2.9±0.1
3.5±0.46
1.6±1.1
＞10
NB4
3.0±0.25
3.6±0.93
0.59±0.21
＞10
2.4±0.52
2.8±1.3
SKM-1
M-07e
＞10
＞10
2.8±0.023
a
All GI values were obtained by triple testing.
5
0
Figure 6. Anti-proliferative effects of compounds 1, 6, 9, 19 and 30 against normal PBMC.
In order to examine compound 9’s effect on the BTK mediated signaling pathway in cells, we
tested it in the U2932 and Pfeiffer cells using 1, 6 and 13 as control. Compound 9 potently
inhibited BTK Y223 auto-phosphorylation (EC50: <30 nM) but did not affect the trans-
phosphorylation site Y551 (Syk kinase phosphorylation site) (Figure 7). It did not affect BTK
downstream mediator PLCγ1 Y783 and PLCγ2 Y759 sites, just like 1 and 6 did. Both 9 and 1
exhibited slightly better inhibitory activities than reversible inhibitor 6 for the inhibition of

ACCEPTED MANUSCRIPT
PLCγ2 Y1217 phosphorylation. In both U2932 and Pfeiffer cells, 9 could arrest cell cycle into
G0/G1 phase and induce apoptosis in a concentration dependent manner (Figure 8). Interestingly,
9
exhibited better cell cycle progression blockage and apoptosis induction effects than 1 and 6.
Not surprisingly, the reversible version compound 13 did not show apparent such effects. In
addition, neither compound 6 nor 13 induced apoptosis.
Figure 7. Compound 9’s effect on the BTK mediated signaling pathway in U2932 and Pfeiffer
cells.

ACCEPTED MANUSCRIPT
Figure 8. Compound 9’s effect on cell cycle progression and apoptosis in U2932 and Pfeiffer
cells.
2
.6 In vivo pharmacokinetic evaluation
We then evaluated the pharmacokinetic profiles of compounds 9, 19 and 30 in rats (Table 8).
As the data showed, compound 9 exhibited poor AUC and short half-life values via intravenous
injection (iv). In addition, it was almost not absorbed through oral administration (po).
Compound 19 displayed a better AUC value than 9 (about 2-fold increase) and similar half-life
value via intravenous injection. For oral administration, 19’s AUC value was about 7-fold better
than 9 though still not acceptable. Interestingly, compound 30 exhibited a good AUC value (over
1
500 ng/mL*h) although the half-life was still short (0.6 h) via intravenous injection. It is
noteworthy that reversible and irreversible inhibitors have different requirements for sustained
target inhibition [30]. For irreversible inhibitors, a short half-life is actually preferred to avoid the
potential off-target effects [19]. In addition, compound 30 was absorbed very well through oral
administration (AUC near 1800 ng/mL*h and bioavailability 11.48 %). The Cmax of 30 reached
over 7600 ng/mL and 1100 ng/mL through intravenous injection and oral administration,

ACCEPTED MANUSCRIPT
respectively. All these data suggested that compound 30 might serve as a good research tool for
in vivo studies.
Table 8. Pharmacokinetic evaluation of compounds 9, 19 and 30
AUC(0-t)
AUC(0-∞)
ng/mL*h
MRT(0-t)
h
T1/2
h
Tmax
h
Cmax
F
ng/mL*h
ng/mL
%
9
iv (1 mg/kg)
mean±SD
181.80±4.88 182.70±4.93
0.26±0.15
1.36±0.99
0.23±0.14
1.33±1.61
0.24±0.057
7.87±4.61
0.017±0 897.39±297.36 NA
9
po (10 mg/kg) 22.03±21.84 34.55±15.83
mean±SD
0.5±0.43
32.82±41.51
/
1
9
iv (1 mg/kg) 316.77±14.28 317.33±14.64 0.16±0.003
mean±SD
0.017±0 2439.7±174.44
NA
1
9
po (10 mg/kg)
mean±SD
150.70±8.0 291.28±11.58 3.389±0.373
0.083±0 143.71±109.33 4.76
7617.755
3
0
1
±
562.339
190.937
1562.983
±190.685
iv (1 mg/kg)
mean±SD
0.442±0.058 0.608±0.083 0.017±0
2.344±0.998 3.87±3.288 1.5±2.165
/
±1359.904
3
0
1
±
792.986
633.268
1896.276
±582.12
1119.036
±818.45
po (10 mg/kg)
mean±SD
11.48
3
. Conclusions
Through a reversible inhibitor scaffold based irreversible inhibitor development approach,
starting from a compound resembling 6, with the help of structure-based drug design technology,
we discovered a highly selective irreversible BTK inhibitor 9, which displayed great selectivity
over other kinases such as BMX, EGFR, JAK3 that bear a structurally identical cysteine residue
in BTK. The X-ray crystal structure confirmed the covalent binding mode and this irreversible
binding was approved to be biologically relevant. In addition, 9 exhibited good enzymatic
kinetics for the irreversible inhibition efficiency. Compound 9 strongly inhibited the kinase
activities of BTK in the B cell related cancer cell lines and displayed moderated anti-proliferative

ACCEPTED MANUSCRIPT
efficacies against these cell lines. Compound 30 displayed a suitable pharmacokinetic profile for
both IV injection and PO application. We believe that the superior selectivity profile of 9 among
all of the known BTK irreversible inhibitors would make it a good in vitro research tool to
precisely dissect the BTK mediated signaling pathways in both the pathological and
physiological context. Although 9 might not be used in vivo via oral administration, compound
3
0 that bears a proper pharmacokinetic profile might be a good pharmacological tool for in vivo
study.
4
4
. Experimental section
.1 Chemistry. All reagents and solvents were purchased from commercial sources and used as
1
13
obtained. H NMR and C NMR spectra were recorded with a Bruker 400 NMR spectrometer
and referenced to deuterium dimethyl sulfoxide (DMSO-d ). Chemical shifts are expressed in
6
ppm. In the NMR tabulation, s indicates singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and
br, broad peak. LC/MS were performed on an Agilent 6224 TOF using an ESI source coupled to
an Agilent 1260 Infinity HPLC system operating in reverse mode with an Agilent Eclipse Plus
C18 1.8 µm 3.0×50 mm column. Flash column chromatography was conducted using silica gel
(
Silicycle 40−64 µm). The purities of all compounds were determined to be above 95% by
HPLC.
General Method A. N-(3-Bromo-2-methylphenyl)-4-(tert-butyl)benzamide (S1a). To a
mixture of 4-(tert-butyl)benzoic acid (1 g, 5.61 mmol) in DCM (30 mL) was added oxalyl
o
chloride (0.57 mL, 6.74 mmol) and DMF (0.05 mL) at 0 C. The reaction mixture was stirred at
room temperature for 4 h. The resulting mixture was then concentrated to afford the crude 4-
(tert-butyl)benzoyl chloride. And then to a mixture of 3-bromo-2-methylaniline (1.11 g, 6 mmol)
in dry DCM (50 mL) was slowly added crude 4-(tert-butyl)benzoyl chloride and DIPEA (1 mL,
o
5
mmol) at 0 C. The resulting mixture was stirred for 10 min at room temperature, and then
quenched by MeOH (5 mL), concentrated and purified by flash column chromatography (0−2%
1
MeOH in DCM) to afford S1a (1.86 g, two steps yield 96%) as a white solid. H NMR (400
MHz, DMSO-d ) δ 10.08 (s, 1H), 7.93 (d, J = 7.9 Hz, 2H), 7.55 (t, J = 7.4 Hz, 3H), 7.34 (d, J =
6
7
.3 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 2.28 (s, 3H), 1.32 (d, J = 9.3 Hz, 9H). LC/MS (ESI,
+
m/z)=346.0859 [M + H] .

ACCEPTED MANUSCRIPT
General Method B. 4-(tert-Butyl)-N-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)benzamide (S2a).
butyl)benzamide (1.73 g, 5 mmol) in dioxane (100 mL) was added bis(pinacolato)diboron (2.54
g, 10 mmol), Pd(dppf)Cl (365 mg, 0.5 mmol) and KOAc (1.47g, 15 mmol). The reaction
To a mixture of
N-(3-bromo-2-methylphenyl)-4-(tert-
2
o
mixture was stirred at 100 C for 8 h under argon, and then concentrated to remove the dioxane.
The residue was diluted with water (100 mL) and extracted with DCM (3 × 80 mL). The organic
phase was concentrated and purified by flash column chromatography (0−2% MeOH in DCM) to
1
afford S2a (1.76 g, 90%) as a white solid. H NMR (400 MHz, DMSO-d ) δ 9.83 (s, 1H), 7.93
6
(d, J = 7.9 Hz, 2H), 7.55 (t, J = 7.4 Hz, 3H), 7.40 (d, J = 7.6 Hz, 1H), 7.22 (t, J = 7.4 Hz, 1H),
+
2
.38 (s, 3H), 1.33 (s, 21H). LC/MS (ESI, m/z)=394.2630 [M + H] .
General Method C. (4-Amino-2-nitrophenyl)(morpholino)methanone (S3b).To a mixture of
-amino-2-nitrobenzoic acid (1.0 g, 5.49 mmol) in DMF (100 mL) was added morpholine (2.39
4
mL, 27.5 mmol), HATU (3.13 g, 8.23 mmol) and DIPEA (1.36 mL, 8.23 mmol). The reaction
mixture was stirred at room temperature overnight, then concentrated to remove the DMF. The
residue was diluted with water (100 mL) and extracted with DCM (3 × 80 mL). The organic
phase was concentrated and purified by flash column chromatography (0−5% MeOH in DCM) to
1
afford S3b (1.25 g, 91%) as a solid. H NMR (400 MHz, DMSO-d ) δ 7.25 (d, J = 2.1 Hz, 1H),
6
7
.13 (d, J = 8.3 Hz, 1H), 6.89 (dd, J = 8.3, 2.1 Hz, 1H), 6.07 (s, 2H), 3.74 – 3.45 (m, 6H), 3.23
+
(
s, 2H). LC/MS (ESI, m/z) =252.1087 [M + H] .
General
Method
D.
5-Bromo-1-methyl-3-((4-(morpholine-4-carbonyl)-3-
(S4b). To mixture of (4-amino-2-
nitrophenyl)amino)pyridin-2(1H)-one
a
nitrophenyl)(morpholino)methanone (1.21 g, 4.85 mmol) in dioxane (15 mL) was added 3,5-
dibromo-1-methylpyridin-2(1H)-one (1.29 g, 4.85 mmol), Pd (dba) (444 mg, 0.485 mmol),
2
3
2 3
Xantphos (281 mg, 0.485 mmol) and Cs CO (4.74 g, 14.5 mmol). The reaction mixture was
o
stirred at 110 C overnight under argon, and then concentrated to remove the dioxane. The
residue was diluted with water (100 mL) and extracted with DCM (3 × 80 mL). The organic
phase was concentrated and purified with flash column chromatography (0−5% MeOH in DCM)
1
to afford S4b (1.80 g, 85%) as a solid. H NMR (400 MHz, DMSO-d ) δ 8.69 (s, 1H), 7.99 (s,
6
1
3
H), 7.64 (s, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 3.65 (d, J = 11.6 Hz, 4H), 3.52 (s, 5H),
+
.26 (s, 2H). LC/MS (ESI, m/z)= 439.0449 [M + H] .

ACCEPTED MANUSCRIPT
General Method E. 4-(tert-Butyl)-N-(2-methyl-3-(1-methyl-5-((4-(morpholine-4-carbonyl)-3-
nitrophenyl)amino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)benzamide (S5a). To a mixture of 5-
bromo-1-methyl-3-((4-(morpholine-4-carbonyl)-3-nitrophenyl)amino)pyridin-2(1H)-one(219
mg, 0.5mmol) in dioxane (50ml) was added 4-(tert-butyl)-N-(2-methyl-3-(4,4,5,5-tetramethyl-
1
,3,2-dioxaborolan-2-yl)phenyl)benzamide (197 mg, 0.5 mmol), Pd(PPh ) (58 mg, 0.05 mmol),
3 4
o
2 3 2
Na CO (106 mg, 1 mmol) and H O (5 mL). The reaction mixture was stirred at 110 C
overnight under argon, and then concentrated to remove the dioxane. The residue was diluted
with water (100 mL) and extracted with DCM (3 × 80 mL). The organic phase was concentrated,
and purified by flash column chromatography (0−7% MeOH in DCM) to afford S5a (193 mg,
1
6
3
3
2%) as a solid. H NMR (400 MHz, DMSO-d ) δ 9.91 (s, 1H), 8.64 (s, 1H), 7.96 – 7.93 (m,
6
H), 7.59-7.55 (m, 3H), 7.40-7.34 (m, 3H), 7.30-7.19 (m, 3H), 3.65-3.62 (m, 7H), 3.51 (s, 2H),
+
.24 (s, 2H), 2.24 (s, 3H), 1.34 (s, 9H). LC/MS (ESI, m/z)=624.2885 [M + H] .
General Method F. N-(3-(5-((3-Acrylamido-4-(morpholine-4-carbonyl)phenyl)amino)-1-
methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methylphenyl)-4-(tert-butyl)benzamide (9). To
a
mixture of 4-(tert-butyl)-N-(2-methyl-3-(1-methyl-5-((4-(morpholine-4-carbonyl)-3-
nitrophenyl)amino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)benzamide (187 mg, 0.3 mmol) in
MeOH was added 10% Pd/C (15 mg). The reaction mixture was stirred at room temperature for
4
h under H , and then filtered through a pad of Celite and washed with methanol. Evaporation
2
of the filtrate provided the crude product as a solid, which was used directly. To a mixture of the
crude intermediate (187 mg, 0.30 mmol) in DCM was added acryloyl chloride (28.5 uL, 0.36
o
mmol) and DIPEA (60 uL, 0.36 mmol) at 0 C. Then the reaction mixture was stirred at room
temperature for 10 min, and then diluted with water (50 mL) and extracted with DCM (3 × 80
mL). The organic phase was concentrated and purified by flash column chromatography (0−7%
1
MeOH in DCM) to afford 9 (106 mg, two steps yield 55%) as a white solid. H NMR (400 MHz,
DMSO-d ) δ 9.87 (s, 2H), 8.10 (s, 1H), 7.99 – 7.86 (m, 2H), 7.64 – 7.52 (m, 3H), 7.35 (d, J = 7.6
6
Hz, 1H), 7.27 (t, J = 7.6 Hz, 2H), 7.24 – 7.18 (m, 2H), 7.16 (s, 1H), 7.10 (d, J = 8.3 Hz, 1H),
6
3
1
1
1
.47 (dd, J = 17.0, 10.2 Hz, 1H), 6.22 (d, J = 16.8 Hz, 1H), 5.70 (d, J = 10.5 Hz, 1H), 3.61 (s,
1
3
H), 3.56 (s, 4H), 3.39 (s, 4H), 2.21 (s, 3H), 1.34 (s, 9H). C NMR (100 MHz, DMSO-d ) δ
6
68.16, 165.73, 163.72, 157.37, 154.89, 143.61, 139.06, 137.61, 136.47, 132.58, 132.25, 132.19,
31.98, 129.56, 128.17, 128.01, 127.95 127.45, 126.70, 126.10, 125.62, 121.06, 119.23, 115.86,
+
14.75, 113.21, 66.40, 37.72, 35.13, 31.42, 16.00. LC/MS (ESI, m/z)=648.3226 [M + H] .

ACCEPTED MANUSCRIPT
N-(3-(5-((2-Acrylamido-4-(morpholine-4-carbonyl)phenyl)amino)-1-methyl-6-oxo-1,6-
1
dihydropyridin-3-yl)-2-methylphenyl)-4-(tert-butyl)benzamide (10). (Method F) Yield: 47%. H
NMR (400 MHz, DMSO) δ 9.97 (s, 1H), 9.85 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.66 (s, 1H),
7
1
.57-7.53 (m, 3H), 7.47 (d, J = 8.3 Hz, 1H), 7.33-7.20 (m, 4H), 7.13 (d, J = 7.3 Hz, 1H), 6.73 (s,
H), 6.51 (dd, J = 17.0, 10.2 Hz, 1H), 6.29 (dd, J = 17.0, 1.7 Hz, 1H), 5.79 (dd, J = 11.9, 10.3
Hz, 1H), 3.60 (s, 7H), 3.52 (s, 4H), 2.14 (s, 3H), 1.33 (s, 9H). LC/MS (ESI, m/z)= 648.3190 [M
+
+
H]
N-(4-Acrylamido-2-methyl-3-(1-methyl-5-((4-(morpholine-4-carbonyl)phenyl)amino)-6-oxo-
1
1
,6-dihydropyridin-3-yl)phenyl)-4-(tert-butyl)benzamide (11). (Method F) Yield ：52%. H
NMR (400 MHz, DMSO-d ) δ 9.89 (s, 1H), 9.21 (s, 1H), 8.06 (s, 1H), 7.93 (d, J = 8.3 Hz, 2H),
.56-7.54 (m, 3H), 7.39 – 7.20 (m, 5H), 7.12 (s, 1H), 6.88 (s, 1H), 6.47 (dd, J = 19.4, 9.4 Hz,
6
7
1
1
H), 6.20 (d, J = 19.1 Hz, 1H), 5.68 (d, J = 8.6 Hz, 1H), 3.58 (m, 7H), 3.49 (s, 4H), 2.09 (s, 3H),
+
.33 (s, 9H). LC/MS (ESI, m/z)=648.3183 [M + H] .
4
-(tert-Butyl)-N-(3-(5-((3-(2-chloroacetamido)-4-(morpholine-4-carbonyl)phenyl)amino)-1-
methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methylphenyl)benzamide (12). (Method F) Yield: 46%.
1
H NMR (400 MHz, DMSO-d ) δ 10.09 (s, 1H), 9.88 (s, 1H), 8.15 (s, 1H), 7.94 (d, J = 8.3 Hz,
6
2
H), 7.69 (s, 1H), 7.55 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 7.5 Hz, 1H), 7.29 (s, 2H), 7.23 (t, J = 7.6
Hz, 2H), 7.15 (s, 1H), 7.10 (d, J = 8.2 Hz, 1H), 4.30 (s, 2H), 3.61 (s, 3H), 3.59 (s, 4H), 3.45 (s,
+
4
H), 2.21 (s, 3H), 1.33 (s, 9H). LC/MS (ESI, m/z)=670.2850 [M + H] .
4
-(tert-Butyl)-N-(2-methyl-3-(1-methyl-5-((4-(morpholine-4-carbonyl)-3-
propionamidophenyl)amino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)benzamide (13). (Method F)
1
Yield: 51%. H NMR (400 MHz, DMSO-d ) δ 9.89 (s, 1H), 9.58 (s, 1H), 8.07 (s, 1H), 7.93 (d, J
6
=
7.1 Hz, 2H), 7.55 (m, 3H), 7.33 (m, 1H), 7.30-7.15 (m, 4H), 7.12 (s, 1H), 7.05 (d, J = 6.8
Hz,1H), 3.60 (s, 3H), 3.58 (s, 4H), 3.41 (s, 4H), 2.27 (m, J = 6.9 Hz, 2H), 2.21 (s, 3H), 1.33 (s,
+
9
H), 1.01 (t, J = 7.4 Hz, 3H). LC/MS (ESI, m/z)=650.3433 [M + H] .
1
N-(3-Bromo-2-methylphenyl)benzamide (S6a). (Method A) Yield: 81%. H NMR (400 MHz,
DMSO) δ 10.16 (s, 1H), 7.99 (s, 2H), 7.61 (s, 1H), 7.54 (s, 3H), 7.36 (s, 1H), 7.19 (s, 1H), 2.29
+
(
s, 3H). LC/MS (ESI, m/z)=290.0359 [M + H] .
N-(2-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)benzamide (S7a). (Method
1
B) Yield: 76%. H NMR (400 MHz, DMSO-d
6
) δ 9.92 (s, 1H), 8.01 (s, 2H), 7.57 (d, J = 8.1 Hz,