Structure-activity relationship investigation for benzonaphthyridinone derivatives as novel potent Bruton's tyrosine kinase (BTK) irreversible inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.06.016

Through a structure-based drug design approach, a tricyclic benzonaphthyridinone pharmacophore was used as a starting point for carrying out detailed medicinal structure-activity relationhip (SAR) studies geared toward characterization of a panel of propo

Full text of DOI:10.1016/j.ejmech.2017.06.016

Accepted Manuscript
Structure-activity relationship investigation for benzonaphthyridinone derivatives as
novel potent Bruton's tyrosine kinase (BTK) irreversible inhibitors
Beilei Wang, Yuanxin Deng, Yongfei Chen, Kailin Yu, Aoli Wang, Qianmao Liang,
Wei Wang, Cheng Chen, Hong Wu, Chen Hu, Weili Miao, Wooyoung Hur, Wenchao
Wang, Zhenquan Hu, Ellen L. Weisberg, Jinhua Wang, Tao Ren, Yinsheng Wang,
Nathanael S. Gray, Qingsong Liu, Jing Liu
PII:
S0223-5234(17)30462-2
10.1016/j.ejmech.2017.06.016
EJMECH 9511
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 March 2017
Revised Date: 6 June 2017
Accepted Date: 7 June 2017
Please cite this article as: B. Wang, Y. Deng, Y. Chen, K. Yu, A. Wang, Q. Liang, W. Wang, C. Chen,
H. Wu, C. Hu, W. Miao, W. Hur, W. Wang, Z. Hu, E.L. Weisberg, J. Wang, T. Ren, Y. Wang, N.S. Gray,
Q. Liu, J. Liu, Structure-activity relationship investigation for benzonaphthyridinone derivatives as novel
potent Bruton's tyrosine kinase (BTK) irreversible inhibitors, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.06.016.
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ACCEPTED MANUSCRIPT
Structure-Activity Relationship Investigation for Benzonaphthyridinone
Derivatives as Novel Potent Bruton's Tyrosine Kinase (BTK) Irreversible
Inhibitors
Beilei Wang1,2,9, Yuanxin Deng1,2,9, Yongfei Chen1,3,9, Kailin Yu1,2,8, Aoli Wang1,2 , Qianmao
Liang1 , Wei Wang1,3, Cheng Chen1,2, Hong Wu1,3, Chen Hu1,2, Weili Miao4, Wooyoung Hur5,
Wenchao Wang1,2, Zhenquan Hu1,3, Ellen L. Weisberg5, Jinhua Wang6, Tao Ren7, Yinsheng
Wang4, Nathanael S Gray6, Qingsong Liu1,2,3,7,8*, Jing Liu,1,3*
1. High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350
Shushanhu Road, Hefei, Anhui 230031, P. R. China
2. University of Science and Technology of China, Hefei, Anhui 230036, P. R. China
3. CHMFL-HCMTC Target Therapy Joint Laboratory, 350 Shushanhu Road, Hefei Anhui
230031, P. R. China
4. Department of Chemistry, University of California-Riverside, 900 University Ave.,
Riverside, CA 92521, USA
5. Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School,
450 Brookline Ave., Boston, MA 02115, USA
6. Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School,
250 Longwood Ave, SGM 628, Boston, MA 02115, USA.
7. Precision Targeted Therapy Discovery Center, Institute of Technology Innovation, Hefei
Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230088, P. R.
China
8. Hefei Science Center, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei, Anhui,
P. R. China, 230031
9. These authors contributed equally to this manuscript
AUTHOR INFORMATION
Corresponding Authors

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*E-mail address: qsliu97@hmfl.ac.cn (Q. Liu), E-mail address: jingliu@hmfl.ac.cn (J. Liu).
AUTHOR CONTRIBUTIONS
All authors listed contributed to this manuscript. All authors have approved the final version of
the manuscript.
ABSTRACT
Through a structure-based drug design approach, a tricyclic benzonaphthyridinone
pharmacophore was used as a starting point for carrying out detailed medicinal structure-activity
relationhip (SAR) studies geared toward characterization of a panel of proposed BTK inhibitors,
including 6 (QL-X-138), 7 (BMX-IN-1) and 8 (QL-47). These studies led to the discovery of the
novel potent irreversible BTK inhibitor, compound 18 (CHMFL-BTK-11). Kinetic analysis of
compound 18 revealed an irreversible binding efficacy (kinact/Ki) of 0.01 µM-1s-1. Compound 18
potently inhibited BTK kinase Y223 auto-phosphorylation (EC50 <100 nM), arrested cell cycle in
G0/G1 phase, and induced apoptosis in Ramos, MOLM13 and Pfeiffer cells. We believe these
features would make 18 a good pharmacological tool for studying BTK-related pathologies.
Keywords
BTK; Kinase inhibitor; Irreversible inhibitor; Structure-activity relationship; B-cell lymphoma
Abbreviations used
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 lymphocytic leukemia;
WM, waldenström's macroglobulinemia; TEK, endothelial tyrosine kinase; ITK, interleukin-2-
inducible T-cell kinase; RLK, LysM domain receptor-like kinase 1; BLK, B lymphocyte kinase;
MAP2K7, dual specificity mitogen-activated protein kinase kinase 7;
PLCγ2, 1-
phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2; PDB, protein data bank;
SAR, structure-activity relationship.
1. Introduction
BTK, also known as agammaglobulinemia tyrosine kinase (ATK) or B-cell progenitor kinase

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(BPK), is a non-receptor tyrosine kinase that was initially identified as the defective protein in
human X-linked agammaglobulinemia (XLA).1,2 The family of Tec tyrosine kinases is the
second largest family of cytoplasmic tyrosine kinases, which consists of five members including
BTK, BMX, TEC, ITK, TXK with tissue-specific expression patterns in normal cells.2 Bruton's
tyrosine kinase (BTK) is predominately expressed in B cells,3-5 monocytes, macrophages and
neutrophils, as well as in B-cell malignancies.6 BTK activation is usually associated with chronic
B-cell receptor (BCR) activation in B-cell-mediated lymphoma (e.g. ABC-DLBCL) and
hematological malignancies (e.g. CLL, AML etc), which make BTK an attractive drug discovery
target.7-9 Currently, there are several kinase inhibitors that inhibit BTK kinase activity through
formation of a covalent bond with the non-catalytic cysteine residue located in the kinase hinge
segment in BTK (i.e., Cys481), a non-conserved amino acid within the active site of the enzyme.
These include 1 (PCI-32765)10, 2 (CC-292)11, 3 (CNX-774)12, 4 (HM71224)13, 514, 6 (QL-X-
138)15, 7 (BMX-IN-1)16 and 8 (QL47)17 (Figure 1). There are 10 other kinases including the TEC
family (TEC, ITK, ETK, RLK), EGFR family (EGFR, Her2, Her4) as well as BLK, JAK3 and
MAP2K7 kinases, which possess similar cysteine residues at the identical positions. Among
them, compound 1 was the first clinically approved BTK kinase inhibitor that was developed
from a pyrzaolopyrimidinamine scaffold. Besides BTK activity, 1 was also potent against other
targets, including EGFR and FLT3-ITD18,19. Compounds 2, 3 and 4 were based on
aminopyrimidine pharmacophores, while 5 was a unique aminopyrimidine-based type II
irreversible inhibitor that binds to the DFG-out inactive conformation of BTK14. We have
previously reported the unique tricyclic fused benzonaphthyridinone pharmacophore BTK/MNK
kinase dual inhibitor 6 and selective BTK kinase inhibitors 7 and 8. Compound 8 was also
reported to bear antiviral activity20,21. Recently, we discovered another tricyclic
benzonaphthyridinone-based BTK inhibitor 18 (CHMFL-BTK-11), which displayed impressive
anti-inflammatory activity in the murine models22. Here, we report detailed structure-activity
relationship (SAR) data for this series of compounds with activity toward BTK as a target. The
starting point for our analyses was an mTOR inhibitor compound 9 (Torin2), and subsequently
included similar characterization of compounds 6, 7, 8, and finally 18. Importantly, the anti-
cancer effects of compound 18 were studied and are presented in this report.

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Figure 1. Chemical structures of representative irreversible BTK kinase inhibitors.
Figure 2. Schematic illustration of the discovery of a series of benzonaphthyridinone derivatives.
2. Results and discussion
2.1. Design and synthesis
While searching for a scaffold amenable to the design of irreversible BTK inhibitors, we

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found that the tricyclic quinonline-based pharmacopohre, which was derived from the previously
developed mTOR inhibitor compound 9, exhibited moderate binding activity against BTK in the
KINOMEScanTM profiling23. A molecular modeling study demonstrated that the CF3 group of 9
is approximately 6Å away from the cys481 residue, which could potentially be connected by
attaching an acrylamide to form a covalent bond to achieve the irreversible binding (Figure 3).
Meanwhile, modification of the aminopyridine moiety (R1) and substituents on the benzene ring
(R2, R3) may be necessary to make the molecule more suitable for the new binding mode and
increase the binding affinity and selectivity.
Figure 3. Irreversible inhibitor design rationale. (A) Compound 9 docked into BTK kinase (PDB
ID: 3GEN); (B) Virtual installation of acrylamide to scaffold of 9; (C) Schematic illustration of
SAR modification.
The synthetic route of compounds 18 and 19 is depicted in scheme 1. Nucleophilic
substitution of 1-fluoro-4-nitrobenzene with pyrazole afforded intermediate 31 was then
subjected to reduction to generate the aniline intermediate 32. The aniline then reacted with
diethyl 2-(ethoxymethylene)malonate to give the intermediate 33, which was cyclized and
chlorinated to provide the quinoline intermediate 35. Then, nucleophilic substitution of 35 with
4-methyl-3-nitroaniline and subsequent reduction of the ethyl ester and oxidation of the alcohol
generated the aldehyde intermediate 38. The tricyclic core structure of 39 was built up by a ring
closing Horner-Wadsworth-Emmons olefination reaction. Finally, compounds 18 and 19 were
furnished by reduction of the nitro group followed by acylation with acryloyl chloride or
propionyl chloride.
Scheme 1. Synthesis of compounds 18 and 19.a

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aReagents and conditions: (a) pyrazole, K2CO3, DMF, rt, overnight; (b) SnCl2, EtOH, reflux, 2 h;
o
(c) diethyl 2-(ethoxymethylene)malonate, EtOH, reflux, 12 h; (d) Ph2O, 260 C, 2 h; (e) POCl3,
CH3CN, reflux, 2 h; (f) 4-methyl-3-nitroaniline, 1,4-dioxane, reflux, 2 h; (g) NaBH4, EtOH, rt,
12 h; (h) MnO2, CH2Cl2, rt, 12 h; (i) ethyl 2-(diethoxyphosphoryl)acetate, Cs2CO3, EtOH, reflux,
24 h; (j) SnCl2, EtOH, reflux, 2 h; (k) acryloyl chloride or propionyl chloride, DIPEA, CH2Cl2, rt,
2 h.
2.2 Biological evaluation
2.2.1 SAR investigation
We first chose BTK, BMX, BLK, and JAK3 kinases, which share a similar cysteine residue at
the same position, and used Invitrogen SelectScreen assay for the SAR analysis. We first
replaced the CF3 group in compound 9 with the acrylamide (10), which not only showed strong
BTK activity (IC50=19.1 nM), but also strong to moderate potency against BMX, BLK and JAK3

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kinases. Addition of a methyl group at R2 position (11) did not improve the selectivity among
these four kinases although the potencies were retained. Replacement of the aminopyridine with
the pyridine group at R2 position (12) resulted in significant activity loss against BTK (IC50=115
nM) and did not improve selectivity over other kinases. Interestingly, switching the pyridine to
benzene ring (13) led to increased activity against BTK (IC50=25.3 nM) and increased activity
against BMX kinase (IC50=15.9 nM) and JAK3 (IC50=25.3 nM). Replacement of the benzene
ring with thiophene (14) did not improve the activity and selectivity. A furan substituent at R1
position (15) further increased the activity against BTK (IC50=17.4 nM), BMX (IC50=12.1 nM)
and JAK3 (IC50=17.4 nM) but showed great selectivity over BLK kinase (IC50=2090 nM).
Pyrazole substituent (6) resulted in potency against BTK (IC50=5.34 nM), BMX (IC50=7.36 nM),
BLK (IC50=30.2 nM) and JAK3 (IC50=38.1 nM), with a loss of selectivity of BTK, BMX and
JAK3 over BLK. Removal of the methyl group at R2 position of 6 (16) retained the activity
against BTK (IC50=17.7 nM) and BMX (IC50=10.5 nM) but showed selectivity over BLK
(IC50=293 nM) and JAK3 (IC50=224 nM). Changing the pyrazole (6) to methyl pryazole (17)
further increased the potency against BTK (IC50=13.5 nM) and BMX (IC50=8.16 nM) and
increased the selectivity over BLK (IC50=339 nM) and JAK3 (IC50=343 nM). Interestingly,
switching the connection mode of pyrazole to the quinoline core from C-C to C-N (18) led to a
complete loss of activity against BLK and JAK3 kinases (IC50 >10000 nM) although slightly
decreased the activity against BTK (IC50=75.6 nM) and BMX (IC50=83 nM). In addition,
saturation of the acrylamide of 18 to propionamide (19), which lost the ability to form covalent
bonds, resulted in a significant loss of activity against BTK (IC50=6873 nM), indicating that 18
exerts its inhibitory activity through an irreversible binding mode. A relatively larger substituent
such as styrene (20) decreased BTK kinase activity (IC50=105 nM) but showed higher selectivity
against BMX kinase (IC50=27.8 nM), and retained good selectivity over BLK and JAK3 kinases.
Introduction of the 1-methyl-3-phenylurea substituent at R1 position (21) increased the activity
against JAK3 kinase (IC50=218 nM) although retained the activity against BTK (IC50=79.1 nM)
and BMX (IC50=40.6 nM). Installation of the larger hydrophobic group 1-(benzyloxy)-3-
(trifluoromethyl)benzene (22) led to higher selectivity toward BTK (IC50=37.2 nM) and BMX
(IC50=28.1 nM) and resulted in good selectivity over BLK (IC50=2060 nM). The N-
phenylmethanesulfonamide substituent at R1 position (7) led to increased activity against BTK
(IC50=10.4 nM) and BMX (IC50=7.99 nM) but also led to moderate activity against BLK

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(IC50=377 nM) and JAK3 (IC50=175 nM). Installation of 2,3-dihydrobenzo[b][1,4]dioxine (23)
retained the activity against BTK (IC50=16.3 nM) and BMX (IC50=18.5 nM) but also led to
increased selectivity over BLK (IC50=160 nM) and JAK3 (IC50=192 nM). A Quinoline
substituent (24) showed better selectivity over JAK3 kinase (IC50=983 nM) but still displayed
moderate activity against BLK (IC50=295 nM) and retained potency against BTK (IC50=24.5 nM)
and BMX (IC50=18.5 nM).
Table 1. SAR Investigation through modification of the R1/R2 Positionsa
BTK
BMX
BLK
JAK3
R1
R2
H
Compd
10
(IC50/nM) (IC50/nM) (IC50/nM) (IC50/nM)
19.1
16.4
115
15.7
12.9
66.1
15.9
19.7
12.1
336
177
654
177
CH3
CH3
CH3
CH3
CH3
11
1950
250
1520
25.3
38.6
17.4
12
25.3
38.6
17.4
13
342
14
2090
15

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HN
N
CH3
H
5.34
17.7
13.5
75.6
6873
105
7.36
10.5
8.16
83
30.2
293
38.1
224
6
HN
N
16
17
18
19
20
CH3
CH3
CH3
CH3
339
343
>10000
ND
>10000
>10000
5580
ND
27.8
607
CH3
79.1
40.6
1130
218
21
CH3
CH3
37.2
10.4
28.1
7.99
2060
377
650
175
22
7
CH3
CH3
16.3
24.5
13.3
18.5
160
295
192
983
23
24
aIC50 values were reported as mean values of duplicates.
We then fixed the R3 moiety as pyrrolidine ring that attached the acrylamide and investigated
the SAR of the R1 moiety. With the methyl pyrazole in the R1 position, compound 8 showed

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strong inhibitory activity against BTK (IC50=6.59 nM) and BMX (IC50=6.73 nM). In addition, it
displayed good selectivity over JAK3 kinase (IC50=5180 nM) and retained moderate activity
against BLK (IC50=366 nM). Removal of the methyl group from pyrazole (25) retained the
activity against BTK (IC50=12.3 nM) and BMX (IC50=8.75 nm) but also gained selectivity over
BLK (IC50=246 nM) and JAK3 (IC50= 2150 nM). Switching the methylpyrzole to aminopyridine
(26) led to similar activity and selectivity as was observed for compound 8. A relatively larger
substituent quinoline (27) retained the activity against BTK (IC50=7.15 nM) and BMX
(IC50=5.62 nM) but gained back the activity against BLK (IC50=191 nM) and JAK3 kinase (983
nM). Installation of N-(tert-butyl)benzenesulfonamide (28) at R1 position achieved great
selectivity over JAK3 kinase (IC50 >10000 nM). In addition, 28 not only retained good activity
against BTK and BMX kinases (IC50= 10.7 nM and 18.1 nM), but also increased activity against
BLK kinase significantly (IC50=83.6 nM). Introduction of the 1-methyl-4-phenylpiperazine
resulted in significant loss of activity against BLK kinase (IC50 >10000 nM) as well as loss of
potency against BTK (IC50=155 nM) and BMX (IC50=399 nM).
Table 2. SAR Investigation of the R1 moiety by fixing the R3 moietya
O
N
R3
O
N
R1
N
BTK
(IC50/nM)
BMX
(IC50/nM)
BLK
JAK3
R1
Compds
(IC50/nM) (IC50/nM)
6.59
12.3
6.01
6.73
8.75
13.3
366
246
300
5180
2150
5640
8
HN
N
25
26

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7.15
10.7
5.62
18.1
191
983
27
28
83.6
>10000
155
399
>10000
1560
29
aIC50 values were reported as mean values of duplicates.
2.2.2 Binding mode and kinetics studies for compound 18
A molecular modeling study with the X-ray structure of BTK (PDB: 3GEN) showed that 18
could form a hinge binding hydrogen bond with Met477. In addition, a covalent bond between
Cys481 and the warhead acrylamide of 18 was formed, indicating an irreversible binding mode
(Figure 4A-B). Furthermore, the N atom at the pyrazole ring formed a hydrogen bond with the
N-H of Cys481 residue, which generated a 20-member big ring together with the newly formed
covalent bond that might contribute to its strong binding affinity. In order to further confirm the
irreversible binding mode, we then performed the mass spectrometry study. The result
demonstrated that after incubation with the intact purified BTK kinase protein, 18 indeed
modified the Cys481 residue containing fragment (Figure 4C-D).

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Figure 4. Binding mode exploration of compound 18. (A, B) Putative binding model of 18
within the inactive pocket of BTK. Hydrogen bonds are indicated by red lines to the key amino
acid residues. (C, D) LC-MS/MS in the MRM mode revealed the 18-conjugated Cys481-
containing tryptic peptide derived from recombinant BTK after reaction with 18. Shown in (C)
are the selected-ion chromatograms (SICs) for monitoring the indicated transitions arising from
the fragmentations of the [M+3H]3+ ions of the unmodified (top) and 18-modified (bottom)
Cys481-containing peptides. Displayed in (D) are the MS/MS for the [M+3H]3+ ions of the
unmodified (top) and 18-modified (bottom) cysteine-containing peptides averaged from the peak
at around 74.2 min. From the peak areas found in the SICs, it can be estimated that more than
90% of Cys481 was modified.

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The inactivation of BTK occurs through a two-step process that begins with reversible binding
of the inhibitor to BTK followed by covalent bond formation with the thiol of Cys481 to form a
covalent adduct. Since an inhibitor’s inactivation rate is governed by a pseudo-second-order rate
constant (kinact/Ki), it is highly dependent on the inhibitor concentration and its chemical property.
With this kinetic system, we then evaluated the irreversible binding efficiency of this series of
compounds (Table 3). The results demonstrate that compounds 6 and 7 exhibited the best
reversible binding affinity (Ki= 2.0 and 3.2 nM). Compound 8 was slightly weaker than them (Ki
=14.2 nM) and 18 was much less active (Ki=97 nM). For the covalent bond formation,
compounds 6, 7 and 8 showed similar efficiency (kinact=0.13-0.15 min-1) and 18 was the least
efficient (kinact=0.07 min-1). The order of overall irreversible binding efficiency was 6>7>8>18.
Table 3. Comparison of kinetic parameters Ki, kinact and kinact/Ki determined for compounds 6, 7,
8 and 18.a
Compound
Ki [nM]
2.0±0.4
3.2±0.06
14.2±2.8
97±30
kinact [min-1]
0.14±0.04
0.15±0.01
0.13±0.05
0.07±0.03
kinact/Ki [µM-1s-1]
1.17±0.21
6
7
0.78±0.12
0.15±0.05
8
18
0.01±0.01
a All values were obtained by testing in triplicate. Shown are mean+/-S.D.
2.2.3 Anti-proliferative effects of compounds 1, 18 and 19 on kinase-transformed BaF3
isogenic cells
Since compound 18 achieved the best selectivity over BLK and JAK3 kinases in the
biochemical assays, we then further tested it with the BaF3 isogenic cell-based assays using BTK
irreversible inhibitor 1 as the control compound (Table 4). The data show that 18 had no
apparent inhibitory activity against BLK, JAK1, JAK2, JAK3 and EGFR kinases. In accordance
with the biochemical assay, 18 also strongly inhibited BMX kinase (GI50=0.018 µM), which is
similar to compound 1 (GI50=0.009 µM). In addition, 1 exhibited strong activities against

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EGFR/T790M and EGFR/L858R mutations, which we have previously reported and which has
also been reported by the Fang group24,19. In contrast, 18 did not show activity against these
EGFR mutants. Finally, the reversible version compound 19 did not inhibit these targets.
Table 4. Anti-proliferation effects of compounds 18, 19 and 1 against a panel of BaF3 isogenic
cell lines.a
Compd. 18
(GI50/μM)
Compd. 19
(GI50/μM)
Compd. 1
(GI50/μM)
Cell line
BaF3
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
TEL-EGFR-BaF3
2.657
0.9182
0.292
1.593
0.018
2.67
>10
TEL-EGFR/T790M-BaF3
TEL-EGFR/L858R-BaF3
TEL-EGFR/T790M/L858R-BaF3
TEL-BMX-BaF3
0.069
0.004
2.756
0.009
8.927
9.023
1.046
TEL-JAK1-BaF3
TEL-JAK2-BaF3
2.1
TEL-JAK3-BaF3
9.674
a All GI50 values were obtained by testing in triplicate.
2.2.4 Anti-proliferative effects of compounds 1, 18 and 19 on established cancer cell lines
and primary patient cells
We next evaluated compound 18 against a panel of established cancer cell lines and CLL
primary patient cells using compounds 1 and 19 as controls. Not surprisingly, 18 did not exhibit
anti-proliferation effects against colorectal carcinoma, epithelial carcinoma, lung cancer,
adenocarcinoma, prostate cancer, Burrkit lymphoma, AML, CML and MCL cell lines similar to
what was observed with compound 1 (Table 5). Since BTK kinase inhibitors have been tested
clinically against CLL, and in order to evaluate the potential clinical efficacy of 18, we
compared the activities of 1 and 18 against a panel of CLL primary patient cells. The results

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generally revealed a stronger effect of compound 18 over compound 1 as a single agent (Table
6). Moreover, compound 19 didn’t show any anti-proliferative activity against CLL primary
patient cells, which suggests that 18 inhibited CLL primary patient cells through targeting BTK
with an irreversible binding mode.
Table 5. Anti-proliferative effects of compounds 1, 18 and 19 against a panel of cancer cell linesa
Compd. 18 Compd. 19 Compd. 1
Cell line
Cell type
(GI50/μM)
(GI50/μM)
(GI50/μM)
HCT116
A-431
colorectal carcinoma
epithelial carcinoma
lung cancer
>10
>10
>10
5.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.2
H460
>10
3.7
lung adenocarcinoma
epithelial
A549
H358
NSCLC
>10
5.57
>10
>10
>10
>10
>10
7.9
H1975
HeLa
NSCLC
adenocarcinoma
prostate cancer
prostate cancer
prostate cancer
ABC-DLBCL
ABC-DLBCL
GCB-DLBCL
Burkitt lymphoma
Burrkit lymphoma
ALL
>10
>10
>10
>10
2.2
DU145
RV-1
C4-2
SU-DHL2
U2932
Pfeiffer
Ramos
NAMALWA
NALM6
U937
4.0
5.8
8.4
3.5
3.9
4.5
6.0
1.17
>10
>10
8.5
AML

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PF138
ALL
8.8
0.59
1.5
1.4
1.8
2.5
3.7
3.8
4.3
4.9
5.6
7.8
>10
1.6
3.4
8.5
>10
8.2
MOLM-13
MOLM-14
MOLM-16
HEL
AML
AML
AML
AML
AML
AML
AML
AML
AML
AML
AML
CML
MCL
MCL
MCL
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.3
1.8
4.4
4.0
SKM-1
NB-4
3.9
7.5
HL-60
3.5
CMK
>10
>10
>10
>10
>10
>10
>10
>10
NOMO-1
OCI-AML-3
OCI-AML-2
K562
REC-1
JVM-2
Z-138
a All GI50 values were obtained by testing in triplicate.
Table 6. Anti-proliferative effects of 1, 18 and 19 against a panel of CLL primary patient cellsa
Compd. 18
GI50(µM)
Compd. 19
GI50(µM)
Compd. 1
GI50(µM)
Patient code
p12
p13
10.3
3.7
>10
>10
>10
>10

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p14
p15
2.4
2.9
>10
>10
3.7
3.8
a All GI50 values were obtained by testing in triplicate.
2.2.5 Effects of compound 18 on BTK-mediated signaling, cell cycle progression, and
apoptosis
We next evaluated the effects of compound 18 on the BTK-mediated signaling pathway in the
ABC-DLBCL cell line, Pfeiffer, the Burkitt lymphoma cell line, Ramos and the AML cell line,
MOLM13, using classical irreversible BTK inhibitor compound 1 as a control (Figure 5). Upon
anti-IgM stimulation, compound 18 potently inhibited BTKY223 auto-phosphorylation in
MOLM13 cells and Ramos cells (EC50<100 nM) and less potently in Pfeiffer cells (EC50 <3000
nM) however did not affect the trans-phosphorylation site Y551 (Syk kinase phosphorylation
site) in both cell lines. It inhibited the BTK downstream mediator PLCγ2 Y1217 phosphorylation
sites in Ramos cell line (EC50<1000 nM), but did not affect PLCγ2 Y1217 phosphorylation sites
in Pfeiffer and MOLM13 cell lines, which exhibited the same trends with compound 1. As the
results show, compound 18 arrests cell cycle progression into G0/G1 phase in Ramos, MOLM13
and Pfeiffer cells (Figure 6). In addition, compound 18 induces apoptosis in these three cell lines.
Figure 5. Compound 18’s effects on the BTK mediated signaling pathways in Pfeiffer, MOLM-
13, and Ramos cells.

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Figure 6. Effects of compound 18 on cell cycle progression and apoptosis in Pfeiffer, MOLM-13
and Ramos cell lines.
3 Conclusions
Starting from a tricyclic quinonline core derived from mTOR inhibitor Torin2, through the
structure facilitated irreversible inhibitor design approach, we discovered a novel irreversible
BTK kinase inhibitor 18, which displays great selectivity over other kinases such as BLK,
EGFR, JAK3 that bear a similar cysteine residue at the same positions. Kinetic studies of
compound 18 show that it displays an irreversible binding efficacy (kinact/Ki) of 0.01 µM-1s-1.
Compound 18 potently inhibited BTK Y223 auto-phosphorylation, arrested cell cycle
progression in the G0/G1 phase and induced apoptosis in MOLM13 and Ramos cells. As a
highly selective BTK irreversible inhibitor, we believe that 18 will be a useful tool for the study
of BTK-associated pathology.
4 Experimental section
4.1 Chemistry.

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1
All reagents and solvents were purchased from commercial sources and used as obtained. H
13
NMR and C NMR spectra were recorded with a Bruker 400 or 500 MHz NMR spectrometer
and referenced to deuterium dimethyl sulfoxide (DMSO-d6). Chemical shifts are expressed in
ppm. In the NMR tabulation, s indicates singlet, d indicates doublet, t indicates triplet, q
indicates quartet, m indicates multiplet and br indicates 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.
4.1.1 Compounds 10-17 and 20-24 were prepared following previous reported synthetic
procedures for compound 7,16 and compounds 25-29 were prepared following previous reported
synthetic procedures for compound 8.17 The characterization of compounds 11, 17, 24 and 28
were reported in a previous study,21 as well as compounds 16 and 23.20
4.1.1.1 N-(3-(9-(6-aminopyridin-3-yl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-
yl)phenyl)acrylamide (10) Yield: 12%. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 9.05 (s,
1H), 8.27 (d, J = 9.4 Hz, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 11.3
Hz, 2H), 7.64 (t, J = 8.0 Hz, 1H), 7.19 (d, J = 14.8 Hz, 3H), 6.90 (d, J = 9.4 Hz, 1H), 6.45 (dd, J
13
= 17.7, 9.2 Hz, 2H), 6.26 (d, J = 16.8 Hz, 3H), 5.77 (d, J = 11.2 Hz, 1H). C NMR (100 MHz,
DMSO-d6) δ 163.92, 163.06, 159.58, 150.76, 148.21, 146.03, 142.12, 141.35, 141.32, 140.50,
135.91, 135.28, 131.99, 131.20, 131.02, 128.19, 127.97, 124.50, 123.07, 122.10, 121.12, 120.39,
119.94, 118.03, 113.80, 108.48. LC/MS (ESI, m/z) [M+H]+: 434.16.
4.1.1.2 N-(2-methyl-5-(2-oxo-9-(pyridin-3-yl)benzo[h][1,6]naphthyridin-1(2H)-
1
yl)phenyl)acrylamide (12) Yield: 15%. H NMR (400 MHz, DMSO-d6) δ 9.75 (s, 1H), 9.13 (s,
1H), 8.54 (d, J = 3.6 Hz, 1H), 8.47 (s, 1H), 8.29 (d, J = 9.4 Hz, 1H), 8.11 (d, J = 8.5 Hz, 1H),
8.05 (d, J = 8.5 Hz, 1H), 7.71 (s, 1H), 7.59 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.40 (d,
J = 4.2 Hz, 1H), 7.22 (d, J = 7.7 Hz, 2H), 6.92 (d, J = 9.4 Hz, 1H), 6.63 – 6.53 (m, 1H), 6.25 –
13
6.13 (m, 1H), 5.73 (d, J = 8.9 Hz, 1H), 2.43 (s, 3H). C NMR (100MHz, DMSO-d6) δ 163.90,
163.06, 151.79, 149.18, 148.96, 148.29, 142.63, 140.45, 138.57, 138.42, 134.99, 134.60, 134.35,
133.35, 132.47, 131.88, 131.51, 128.87, 127.60, 126.00, 124.84, 124.13, 122.28, 118.01, 113.83,
18.27. LC/MS (ESI, m/z) [M+H]+: 433.17.

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4.1.1.3 N-(2-methyl-5-(2-oxo-9-phenylbenzo[h][1,6]naphthyridin-1(2H)-yl)phenyl)acrylamide
1
(13) Yield: 16%. H NMR (400 MHz, DMSO-d6) δ 9.73 (s, 1H), 9.11 (s, 1H), 8.29 (d, J = 9.4
Hz, 1H), 8.08 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.75 (s, 1H), 7.50 (d, J = 7.9 Hz, 1H),
7.37 (d, J = 7.4 Hz, 3H), 7.29 (s, 1H), 7.22 (dd, J = 15.0, 7.4 Hz, 3H), 6.91 (d, J = 9.4 Hz, 1H),
6.59 (d, J = 16.1 Hz, 1H), 6.20 (d, J = 17.1 Hz, 1H), 5.74 (d, J = 13.1 Hz, 2H), 2.43 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 163.83, 163.12, 151.42, 148.76, 142.64, 140.47, 139.42, 138.68,
138.45, 137.33, 132.92, 132.49, 131.92, 131.24, 129.27, 128.99, 128.21, 127.59, 127.35, 125.80,
124.45, 123.70, 122.14, 118.00, 113.78, 18.35. LC/MS (ESI, m/z) [M+H]+: 432.17.
4.1.1.4 N-(2-methyl-5-(2-oxo-9-(thiophen-3-yl)benzo[h][1,6]naphthyridin-1(2H)-
1
yl)phenyl)acrylamide (14) Yield: 15%. H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.09 (s,
1H), 8.29 (d, J = 9.4 Hz, 1H), 8.05 (d, J = 2.1 Hz, 2H), 7.72 (s, 1H), 7.58 (s, 1H), 7.53 (d, J = 9.6
Hz, 2H), 7.26 (s, 1H), 7.20 (d, J = 6.5 Hz, 1H), 6.97 (d, J = 3.8 Hz, 1H), 6.91 (d, J = 9.4 Hz, 1H),
13
6.57 (d, J = 10.2 Hz, 1H), 6.21 (d, J = 16.4 Hz, 1H), 5.74 (d, J = 9.4 Hz, 1H), 2.43 (s, 3H). C
NMR (100 MHz, DMSO-d6) δ 163.87, 163.11, 151.16, 148.59, 142.48, 140.91, 140.49, 138.63,
138.37, 133.18, 132.55, 132.43, 131.90, 131.20, 128.75, 127.68, 127.61, 126.23, 125.86, 124.90,
122.48, 122.37, 122.13, 118.06, 113.83, 18.33. LC/MS (ESI, m/z) [M+H]+: 438.13.
4.1.1.5 N-(5-(9-(furan-3-yl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-yl)-2-
1
methylphenyl)acrylamide (15) Yield: 14%. H NMR (400 MHz, DMSO-d6) δ 9.79 (s, 1H), 9.07
(s, 1H), 8.27 (d, J = 9.4 Hz, 1H), 8.01 (d, J = 8.6 Hz, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.86 (s, 1H),
7.70 (s, 2H), 7.54 (d, J = 7.9 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.08 (s, 1H), 6.90 (d, J = 9.4 Hz,
1H), 6.65 – 6.52 (m, 1H), 6.31 (s, 1H), 6.21 (d, J = 16.6 Hz, 1H), 5.75 (d, J = 9.9 Hz, 1H), 2.43
(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.89, 163.07, 150.97, 148.57, 144.86, 142.26,
140.44, 138.56, 138.31, 133.12, 132.44, 131.88, 131.16, 129.43, 128.31, 127.62, 125.90, 125.73,
125.08, 122.13, 121.60, 118.07, 113.81, 108.50, 18.26. LC/MS (ESI, m/z) [M+H]+: 422.15.
4.1.1.6 (E)-N-(2-methyl-5-(2-oxo-9-styrylbenzo[h][1,6]naphthyridin-1(2H)-
1
yl)phenyl)acrylamide (20) Yield: 11%. H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 9.06 (s,
1H), 8.27 (d, J = 9.3 Hz, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.70 (s, 1H),
7.54 (dd, J = 14.9, 7.7 Hz, 3H), 7.37 (d, J = 7.3 Hz, 2H), 7.30 (d, J = 6.6 Hz, 1H), 7.16 (d, J =
6.9 Hz, 1H), 7.07 (s, 1H), 6.92 (t, J = 13.9 Hz, 2H), 6.75 (d, J = 16.2 Hz, 1H), 6.66 – 6.54 (m,
13
1H), 6.25 (d, J = 17.0 Hz, 1H), 5.77 (d, J = 10.0 Hz, 1H), 2.39 (s, 3H). C NMR (100 MHz,

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DMSO-d6) δ 164.05, 163.07, 151.03, 148.97, 142.48, 140.41, 138.64, 138.24, 137.16, 134.57,
133.46, 132.38, 131.89, 130.85, 130.12, 129.18, 128.97, 128.43, 128.33, 127.56, 126.97, 125.92,
125.68, 123.24, 122.11, 118.04, 113.82, 18.35.LC/MS (ESI, m/z) [M+H]+: 458.19.
4.1.1.7 N-(2-methyl-5-(9-(4-(3-methylureido)phenyl)-2-oxobenzo[h][1,6]naphthyridin-1(2H)-
1
yl)phenyl)acrylamide (21) Yield: H NMR (400 MHz, DMSO-d6) δ 9.77 (s, 1H), 9.08 (s, 1H),
8.69 (s, 1H), 8.28 (d, J = 9.4 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.71 (s,
1H), 7.53 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.23 (s, 2H), 7.10 (d, J = 8.4 Hz, 2H),
6.90 (d, J = 9.3 Hz, 1H), 6.58 (d, J = 9.4 Hz, 1H), 6.20 (d, J = 16.4 Hz, 1H), 6.05 (s, 1H), 5.74
13
(d, J = 9.3 Hz, 1H), 2.67 (d, J = 4.0 Hz, 3H), 2.46 (s, 3H). C NMR (100 MHz, DMSO-d6) δ
163.81, 163.15, 156.17, 150.99, 148.40, 142.46, 141.02, 140.51, 138.68, 138.41, 137.24, 132.96,
132.50, 131.89, 131.74, 131.11, 128.65, 127.62, 127.58, 125.87, 124.48, 122.48, 122.03, 118.18,
118.05, 113.73, 26.73, 18.40.LC/MS (ESI, m/z) [M+H]+: 504.20.
4.1.1.8 N-(2-methyl-5-(2-oxo-9-(4-((3-
(trifluoromethyl)phenoxy)methyl)phenyl)benzo[h][1,6]naphthyridin-1(2H)-yl)phenyl)acrylamide
1
(22) Yield: 12%. H NMR (400 MHz, DMSO-d6) δ 9.76 (s, 1H), 9.11 (s, 1H), 8.28 (d, J = 9.2
Hz, 1H), 8.09 (d, J = 8.3 Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.73 (s, 1H), 7.54 (d, J = 7.4 Hz, 1H),
7.47 (d, J = 6.0 Hz, 3H), 7.34 (d, J = 8.9 Hz, 3H), 7.27 (d, J = 7.9 Hz, 3H), 7.20 (d, J = 6.9 Hz,
1H), 6.91 (d, J = 9.1 Hz, 1H), 6.59 (s, 1H), 6.21 (d, J = 16.7 Hz, 1H), 5.74 (d, J = 13.1 Hz, 1H),
13
5.24 (s, 2H), 2.41 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 163.83, 163.11, 159.05, 151.46,
148.80, 142.62, 140.46, 139.10, 138.65, 138.44, 136.89, 136.51, 133.09, 132.48, 131.90, 131.26,
131.21, 128.95, 128.71, 127.56, 127.44, 125.86, 124.60, 123.69, 122.14, 119.59, 117.99, 117.83,
113.78, 111.82, 69.67, 18.30. LC/MS (ESI, m/z) [M+H]+: 606.20.
4.1.1.9 1-(1-Acryloylindolin-6-yl)-9-(1H-pyrazol-4-yl)benzo[h][1,6]naphthyridin-2(1H)-one (25).
1
Yield: 7%. H NMR (400 MHz, DMSO-d6) δ 13.06 (s, 1H), 9.05 (s, 1H), 8.28 (s, 1H), 8.14 (s,
1H), 7.97 (d, J = 12.0 Hz, 2H), 7.74 (s, 1H), 7.60 (s, 1H), 7.35 (s, 1H), 7.16 (s, 1H), 7.13 (s, 1H),
6.92 (s, 1H), 6.77 (s, 1H), 6.23 (d, J = 16.1 Hz, 1H), 5.83 (s, 1H), 4.41 (s, 2H), 3.42 (s, 2H). 13C
NMR (100 MHz, DMSO-d6) δ 164.17, 163.20, 150.60, 148.09, 144.89, 142.14, 140.52, 139.89,
134.17, 131.19, 130.31, 129.97, 129.62, 128.32, 126.79, 124.81, 122.06, 121.08, 120.61, 118.12,
117.31, 113.78, 48.92, 27.92. LC/MS (ESI, m/z) [M+H]+: 434.16.

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4.1.1.10
1-(1-acryloylindolin-6-yl)-9-(6-aminopyridin-3-yl)benzo[h][1,6]naphthyridin-
1
2(1H)-one (26). Yield: 6%. H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H), 8.33 (s, 1H), 8.11 (d, J
= 8.5 Hz, 1H), 7.98 (d, J = 9.3 Hz, 1H), 7.80 (s, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.49 (d, J = 7.6
Hz, 1H), 7.35 (d, J = 7.3 Hz, 1H), 7.20 (s, 1H), 7.14 (s, 1H), 6.95 (d, J = 9.4 Hz, 1H), 6.61 – 6.52
(m, 1H), 6.49 (d, J = 8.4 Hz, 1H), 6.42 (d, J = 16.5 Hz, 1H), 5.78 (d, J = 9.9 Hz, 1H), 4.66 (s,
2H), 4.41 – 4.27 (m, 2H), 3.43 (d, J = 41.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 164.15,
163.19, 159.88, 150.85, 148.21, 146.71, 144.85, 142.39, 140.52, 139.90, 135.49, 135.45, 134.41,
131.22, 129.96, 129.62, 128.12, 126.82, 124.78, 123.22, 122.06, 121.47, 118.04, 117.32, 113.77,
108.26, 48.95,27.91. LC/MS (ESI, m/z) [M+H]+: 460.18.
4.1.1.11
1-(1-Acryloylindolin-6-yl)-9-(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one
1
(27). Yield: 7%. H NMR (400 MHz, CDCl3) δ 9.02 (s, 1H), 8.67 (s, 1H), 8.41 (s, 1H), 8.24 (d,
J = 8.6 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 8.05 (d, J = 8.9 Hz, 2H), 8.01 (s, 1H), 7.82 (d, J = 7.7
Hz, 1H), 7.78 – 7.73 (m, 1H), 7.62 (d, J = 6.9 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.44 (s, 1H),
7.16 (d, J = 7.2 Hz, 1H), 6.99 (d, J = 9.4 Hz, 1H), 6.61 – 6.51 (m, 1H), 6.43 (d, J = 16.1 Hz, 1H),
5.79 (d, J = 10.0 Hz, 1H), 4.29 (s, 2H), 3.35 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 164.21,
163.14, 151.90, 149.47, 148.95, 147.26, 142.68, 140.48, 139.83, 134.58, 134.18, 133.57, 132.28,
131.67, 130.37, 129.97, 129.61, 129.24, 129.09, 128.63, 127.77, 126.93, 124.77, 124.37, 122.34,
117.97, 117.46, 113.93, 48.98, 27.91. LC/MS (ESI, m/z) [M+H]+: 495.17.
4.1.1.12
1-(1-Acryloylindolin-6-yl)-9-(4-(4-methylpiperazin-1-
1
yl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (29). Yield: 7%. H NMR (400 MHz, CDCl3) δ
8.92 (s, 1H), 8.31 (s, 1H), 8.09 (d, J = 8.6 Hz, 1H), 7.97 (d, J = 9.4 Hz, 1H), 7.84 (d, J = 8.7 Hz,
1H), 7.46 (d, J = 7.8 Hz, 1H), 7.33 (s, 1H), 7.15 (s, 1H), 7.11 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 9.4
Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H), 6.61 – 6.49 (m, 1H), 6.41 (d, J = 16.4 Hz, 1H), 5.77 (d, J = 9.7
Hz, 1H), 4.35 – 4.23 (m, 2H), 3.33 (d, J = 13.7 Hz, 2H), 3.28 (d, J = 4.4 Hz, 4H), 2.66 (d, J = 4.4
Hz, 4H), 2.41 (s, 3H). LC/MS (ESI, m/z) [M+H]+: 542.26.
4.1.2 Preparation of compounds 18 and 19.
4.1.2.1 1-(4-nitrophenyl)-1H-pyrazole (31). A mixture of pyrazole (12 mmol, 0.82 g), 4-fluoro-1-
nitrobenzene (10 mmol, 1.41 g), potassium carbonate (15 mmol, 2.07 g) in N,N-
dimethylformamide (20 mL) was heated at 110°C for 10 hours and then cooled to room
temperature. The reaction mixture was poured into water (100ml) and filtered. The filter cake

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was washed with water to give 1-(4-nitrophenyl)-1H-pyrazole (31) (1.70 g, 90% yield) as pale
yellow crystals. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.34 (d, J = 6.0 Hz, 2H), 8.12 (s,
2H), 7.88 (s, 1H), 6.65 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 145.21, 144.54, 143.26,
129.31, 125.85, 118.91, 109.84. LC/MS (ESI, m/z) [M+H]+: 190.06
.
4.1.2.2 4-(1H-pyrazol-1-yl)aniline (32). SnCl2.2H2O (13.1g, 50 mmol) was added to a solution of
4-(3-nitrophenyl)-lH-pyrazole (0.95 g, 5 mmol) in EtOH (50 mL). The reaction mixture was
refluxed for 2 hours and then cooled to room temperature. The reaction mixture was neutralized
with 1 N NaOH to pH = 8 and extracted with EtOAc. The combined organic layers were washed
with brine, dried over anhydrous Na2SO4, and concentrated to afford the crude product 32 (0.66
1
g, 83% yield). H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.64 (s, 1H), 7.48 (s, 2H), 6.71 (s,
2H), 6.43 (s, 1H), 5.25 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 147.87, 140.01, 130.43,
127.32, 120.60, 114.57, 107.16. LC/MS (ESI, m/z) [M+H]+: 160.09
.
4.1.2.3 Diethyl 2-(((4-(1H-pyrazol-1-yl)phenyl)amino)methylene)malonate (33). A mixture of 1-
phenyl-5-aminopyrazole (0.64 g, 4 mmol) and diethyl ethoxymethylenemalonate (1.3 g, 6 mmol)
was refluxed in EtOH (15 mL) for 12 hours. After cooling, the reaction mixture was filtered to
afford the desired product 33 (1.20 g, 90% yield) 1H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H),
8.44 (d, J = 12.9 Hz, 2H), 7.85 (s, 2H), 7.73 (s, 1H), 7.49 (s, 2H), 6.52 (s, 1H), 4.18 (s, 4H), 1.26
13
(d, J = 5.0 Hz, 4H). C NMR (100 MHz, DMSO-d6) δ 151.42, 141.30, 137.93, 136.92, 127.99,
119.95, 118.97, 108.21, 100.00, 93.88, 60.02,14.66. LC/MS (ESI, m/z) [M+H]+: 330.15
.
4.1.2.4 Ethyl 4-chloro-6-(1H-pyrazol-1-yl)quinoline-3-carboxylate (35). 33 (1.00 g, 3 mmol) was
added to diphenyl ether (30 mL) and the reaction mixture heated to 260 °C for 2 h. After cooling,
the corresponding solid was isolated as a gray−white solid by filtration and washing with cold
petroleum. The resultant solid as crude 34 (0.50 g, 1.8 mmol) and POCl3 (0.55 g, 3.6 mmol) in
MeCN (2 mL) was refluxed for 2 h. Excess POCl3 was removed under reduced pressure and the
crude residue was neutralized with saturated sodium bicarbonate solution (100 mL) and extracted
with ethyl acetate. The organic layer was dried over Na2SO4, filtered and evaporated under
reduced pressure. The residue was purified by column chromatography to afford the desired
product 35 (0.45 g, 50% yield, two steps). 1H NMR (400 MHz, DMSO-d6) δ 8.96 (s, 1H), 8.68 (s,
1H), 8.51 (s, 1H), 8.38 (s, 1H), 8.10 (s, 1H), 7.82 (s, 1H), 6.60 (s, 1H), 4.40 (s, 2H), 1.37 (d, J =
5.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.05, 149.41, 147.30, 142.54, 141.68, 139.36,

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131.58, 128.95, 126.18, 124.00, 112.08, 109.18, 62.39, 14.42. LC/MS (ESI, m/z) [M+H]+:
302.07
.
4.1.2.5 Ethyl
4-((4-methyl-3-nitrophenyl)amino)-6-(1H-pyrazol-1-yl)quinoline-3-carboxylate
(36). 4-methyl-3-nitroaniline (0.18 g, 1.2 mmol) was added to a solution of 35 (0.30 g, 1 mmol)
in 1,4-dioxane (3 mL) at room temperature in a sealed tube. . The resultant solution was heated
o
to 110 C for 2 h. After the mixture was cooled to room temperature, a solution of NaOH (1N)
was added to neutralize the solution followed by dilution with water and extraction with ethyl
acetate. After the organic extracts were dried over Na2SO4, the solvents were removed and the
residue was purified by flash column chromatography to furnish product 36 (0.37 g, 90% yield).
1H NMR (400 MHz, DMSO-d6) δ 9.76 (s, 1H), 8.87 (s, 1H), 8.65 (s, 1H), 8.55 (s, 1H), 8.33 (s,
1H), 8.09 (s, 1H), 7.79 (s, 1H), 7.69 (s, 1H), 7.36 (d, J = 6.9 Hz, 1H), 7.27 (s, 1H), 6.60 (s, 1H),
3.94 (s, 2H), 2.45 (s, 3H), 1.09 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 166.47, 151.08,
149.49, 148.46, 146.98, 142.83, 142.03, 137.65, 133.80, 131.49, 128.62, 126.51, 123.54, 123.41,
122.07, 114.57, 112.15, 111.56, 108.82, 61.43, 19.40, 14.16. LC/MS (ESI, m/z) [M+H]+: 418.15
.
4.1.2.6 (4-((4-methyl-3-nitrophenyl)amino)-6-(1H-pyrazol-1-yl)quinolin-3-yl)methanol
(37).
NaBH4 (0.6g, 16 mmol) was added to a solution of compound 36 (0.33 g, 0.8 mmol) in
EtOH/THF (1 mL/3 mL) at 0 oC .. The resultant solution was stirred at room temperature for 12
h before being diluted with EtOAc and filtered through Celite. The filtrate was concentrated
under vacuum, and the residue was dissolved in EtOAc and brine. After separation, organic layer
was dried over MgSO4 and concentrated under vacuum. The residue was purified by flash
1
column chromatography to afford product 37 (0.18 g, 60% yield). H NMR (400 MHz, DMSO-
d6) δ 9.02 (s, 1H), 8.90 (s, 1H), 8.58 (s, 1H), 8.46 (s, 1H), 8.27 (d, J = 7.4 Hz, 1H), 8.16 (d, J =
8.5 Hz, 1H), 7.79 (s, 1H), 7.34 (s, 1H), 7.28 (d, J = 7.8 Hz, 1H), 6.93 (s, 1H), 6.59 (s, 1H), 5.45
13
(s, 1H), 4.52 (s, 2H), 2.40 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 151.82, 149.66, 147.09,
144.81, 141.98, 141.59, 137.76, 133.83, 131.46, 128.67, 124.86, 123.17, 121.48, 120.37, 111.62,
110.80, 108.75, 99.99, 58.87, 19.28. LC/MS (ESI, m/z) [M+H]+: 376.14
.
4.1.2.7 1-(4-methyl-3-nitrophenyl)-9-(1H-pyrazol-1-yl)benzo[h][1,6]naphthyridin-2(1H)-one
(39). MnO2 (260 mg, 3 mmol) was added to a solution of compound 37 (187 mg, 0.5 mmol) in
CH2Cl2 (5 mL) at room temperature. . After 12 h, the reaction mixture was filtered through Celite.
The filtrate was concentrated in a sealed tube and the residue as crude 38 was dissolved in dry

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EtOH (5 mL), after which K2CO3 (410 mg, 3 mmol) and triethyl phosphonoacetate (81 mg, 0.36
o
mmol) were added. The resulting mixture was heated to 110 C for 24 h before cooling to room
temperature. Upon removal of the solvents under vacuum, the residue was diluted with water
followed by extraction with EtOAc. The residue was purified by flash column chromatography
1
to provide product 39 (100 mg, 50% yield, two steps H NMR (400 MHz, CDCl3) δ 8.92 (s, 1H),
8.13 (d, J = 9.0 Hz, 1H), 8.08 (s, 1H), 7.98 (d, J = 9.5 Hz, 1H), 7.93 (dd, J = 9.0, 2.0 Hz, 1H),
7.63 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 2.2 Hz, 1H), 7.18 (d,
J = 1.8 Hz, 1H), 6.94 (d, J = 9.4 Hz, 1H), 6.39 (s, 1H), 2.77 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 162.97, 150.16, 150.10, 147.71, 141.93, 141.60, 139.65, 138.51, 137.13, 135.59, 134.91,
133.26, 132.21, 126.06, 125.28, 122.53, 121.55, 117.58, 113.82, 113.77, 108.29, 20.43. LC/MS
(ESI, m/z) [M+H]+: 398.13
.
4.1.2.8 1-(3-amino-4-methylphenyl)-9-(1H-pyrazol-1-yl)benzo[h][1,6]naphthyridin-2 (1H)-one
(40). SnCl2 .2H2O (226 mg, 1 mmol) was added to a solution of compound 39 (40 mg, 0.1 mmol)
in EtOH (2 ml) at room temperature.,. The resultant solution was refluxed for 2 h. After the
mixture was cooled to room temperature, a solution of saturated sodium bicarbonate was added
to quench the reaction followed by dilution with water and extraction with ethyl acetate. After
the organic extracts were dried over Na2SO4, the solvents were removed to afford the crude
1
product 40 (24 mg, 60% yield); H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.26 (s, 1H),
8.18 (s, 1H), 8.11 (s, 1H), 7.73 (s, 1H), 7.45 (s, 1H), 7.19 (s, 2H), 6.91 (s, 1H), 6.65 (s, 1H), 6.53
13
(s, 2H), 5.30 (s, 2H), 2.24 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 162.93, 151.25, 149.18,
147.68, 142.43, 141.89, 140.14, 138.85, 136.73, 132.09, 132.03, 126.86, 123.07, 122.53, 122.29,
118.02, 115.97, 113.95, 113.81, 113.68, 108.70, 17.63. LC/MS (ESI, m/z) [M+H]+: 368.15
.
4.1.2.9 N-(2-methyl-5-(2-oxo-9-(1H-pyrazol-1-yl)benzo[h][1,6]naphthyridin-1(2H)-yl)phenyl)
acrylamide (18). The crude compound 40 (7.3 mg, 0.02 mmol) was dissolved in dry CH2Cl2 (0.4
mL) in an ice bath. Diisopropylethylamine (28 mg, 0.06 mmol) was added. After 5 min, acryloyl
chloride (3 mg, 0.03 mmol) was added, and the reaction mixture was stirred for additional 2 h.
The solvent was evaporated, and the residue was dissolved in ethyl acetate and washed three
times with a NaHCO3 solution (4%). The organic phase was dried over Na2SO4, filtered, and
concentrated. The residue was purified by flash column chromatography to afford product 18
(7.0 mg, 80% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.77 (s, 1H), 9.11 (s, 1H), 8.30 (s,
1H), 8.18 (s, 1H), 8.13 (s, 1H), 7.70 (s, 2H), 7.61 (s, 1H), 7.54 (s, 1H), 7.23 (s, 1H), 7.11 (s,

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1H), 6.94 (s, 1H), 6.56 (s, 1H), 6.49 (s, 1H), 6.20 (d, J = 17.3 Hz, 1H), 5.75 (s, 1H), 2.41 (s,
13
3H). C NMR (100 MHz, DMSO-d6) δ 163.99, 162.99, 151.21, 147.69, 142.38, 141.85,
140.37, 138.32, 138.11, 136.84, 133.55, 132.65, 132.17, 131.84, 127.61, 127.42, 125.94,
125.12, 122.54, 122.22, 117.92, 113.95, 113.72, 108.62, 18.21. LC/MS (ESI, m/z) [M+H]+,
422.16
.
4.1.2.10
Compound 19 was prepared following the synthetic procedure of compound 18.
N-(2-methyl-5-(2-oxo-9-(1H-pyrazol-1-yl)benzo[h][1,6]naphthyridin-1(2H)-
1
yl)phenyl)propionamide (19). Yield: 78% (last step). H NMR (400 MHz, DMSO-d6) δ 9.52 (s,
1H), 9.11 (s, 1H), 8.28 (s, 1H), 8.18 (s, 1H), 8.13 (s, 1H), 7.70 (s, 1H), 7.59 (s, 2H), 7.49 (s, 1H),
13
7.17 (s, 1H), 7.08 (s, 1H), 6.92 (s, 1H), 6.48 (s, 1H), 2.39 (s, 5H), 1.22 (s, 3H). C NMR (100
MHz, DMSO-d6) δ 172.78, 163.00, 151.22, 147.69, 142.40, 141.88, 140.36, 138.74, 138.10,
136.86, 133.52, 132.48, 132.20, 127.53, 125.59, 125.28, 122.55, 122.33, 117.97, 113.99, 113.77,
108.64, 29.37, 18.16, 10.28. LC/MS (ESI, m/z) [M+H]+, 424.18.
4.2 Biology
4.2.1 Cell lines and cell culture
The human cancer cell lines, HCT116, A-431, H460, A549, H358, H1975, HeLa, DU145,
Ramos, U2932, SU-DHL-2, NAMALWA, HEL, SKM-1, CMK, K562, REC-1, JVM-2, and Z-
138, were purchased from the American Type Culture Collection (ATCC) (Manassas, VA,
USA). The human AML cell lines MOLM-13, MOLM14, HEL and the human ALL cell line
PF382 were provided by Dr. Scott Armstrong, Dana-Farber Cancer Institute (DFCI), Boston,
MA. The NALM6 (pre-B) was generous gift from Dr. David Weinstock. The NOMO-1, OCI-
AML-3, OCI-AML-2 and NB4 cell lines were obtained from Dr. Gary Gilliland. U937, HL-60
and MOLM-16 were purchased from Cobioer Biosciences CO., LTD (Nanjing, China). Pfeiffer
was purchased from Beijing BeiNaChuangLian Biotechnology Research Institute (Beijing,
China). H460, H358, H1975, C4-2, Ramos, NAMALWA, NALM6, PF138, MOLM13,
MOLM14, HEL, SKM-1, CMK, NOMO-1, K562, REC-1, JVM-2, U937, HL-60, Pfeiffer and Z-
138 were cultured in RPMI1640 media (Corning, USA) with 10% FBS and supplemented with
2% L-glutamine and 1% pen/strep. MOLM-16 was cultured in RPMI1640 media (Corning,
USA) with 20% FBS and supplemented with 2% L-glutamine and 1% pen/strep. HCT116, A-
431, A549, HeLa, DU145 and RV-1 were cultured in DMEM media (Corning, USA) with 10%

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FBS and supplemented with 2% L-glutamine and 1% pen/strep. OCI-AML-3 was cultured in α-
MEM media (Corning, USA) with 10% FBS and supplemented with 2% L-glutamine and 1%
pen/strep. OCI-AML-2 was cultured in α-MEM media (Corning, USA) with 20% FBS and
supplemented with 2% L-glutamine and 1% pen/strep. All cell lines were maintained in culture
media at 37 ºC with 5% CO2.
4.2.2 Cell culture and anti-proliferation assay
Cells were grown in 96-well culture plates (2500–3000/well) for 12 h before compounds of
various concentrations were added. Cell proliferation was determined after treatment with
compounds for 72 h. CellTiter-Glo assays were performed according to the manufacturer’s
instructions; and luminescence was measured in a multilabel reader (Envision, PerkinElmer,
Hopkinton, MA, USA). Data were normalized to control groups (dimethyl sulfoxide) and
represented by the mean of three independent measurements with standard error <20%. GI50
values were calculated using Prism 5.0 (GraphPad Software, San Diego, CA, USA).
4.2.3 Activity-based assay for IC50 determination and kinetic characterization
The biochemical test of BLK was provided by Invitrogen (Carlsbad, CA, USA). The ADP-glo
kinase assay (Promega, Madison, WI) was used to screen BTK inhibitors. The Kinase enzymes
in each reaction well were set to BTK (4 ng/µL), BMX (4 ng/µL) or JAK3 (3 ng/µL),
respectively. The peptide substrate poly(4:1 Glu, Tyr) and ATP in the kinase reaction system
were set to 0.2 µg/µL and 50 µM, respectively. The kinase enzymes and the 3-fold series dilution
inhibitors were pre-incubated at room temperature. for 1h before the reactions were started by
adding peptide substrate/ATP mixture. The kinase reactions were carried out for 1 h at 37 °C and
then cooled for 5 min at room temperature. 5 µL of solvent reactions were carried out in a 384-
well plate and the kinase reactions were stopped by adding 5 µL of ADP-Glo reagent to consume
the remaining ATP within 40 min. Then, 10 µL of kinase detection reagent was added into the
well and incubated for 30 min to produce a luminescence signal. The luminescence signal was
measured using an automated plate reader (Envision, USA) and the dose-response curve was
fitted using Prism 5.0 (GraphPad Software Inc., San Diego, CA) to determine the IC50 values.
For kinetic characterization (kinact/Ki) of compounds 6, 7, 8 and 18, the respective compounds
were incubated with BTK over different periods (2-60 min) before initiating the kinase reactions.
The durations of enzymatic, ATP deletion and signal generation reactions were kept constant as

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stated above. Calculated IC50 values were plotted versus incubation time and data was fit using
XLfit (Version 5.5.0.5, IDBS, Munich, Germany) as described previously.25,26
4.2.4 Molecular modeling study
The binding mode of compound 9 to the BTK kinase was obtained by interactive docking
using software YetiX 8.27 The kinase domain of chain A was used for docking (PDB ID: 3GEN).
The receptor and ligand coordinates were prepared with BioX 4.6,28 the atomic AM1-BCC partial
charges were calculated by antechamber.29 Covalent docking of compound 18 to the BTK kinase
was performed by AutoDock4.2 with the flexible side chain method.30 The ligand coordinates
tethered with an S atom were generated and optimized with BioX 4.6. The S atom coordinates of
Cys481 were used to set up the covalent map. The grid map was adjusted as a dimension of
100x100x100 points with a spacing of 0.2 Å. The default parameters were used and a total of 50
runs were performed with a Lamarckian genetic algorithm. The docked models were then
clustered and sorted by binding energy.
4.2.5 Mass Spectrometry Experiments
Recombinant BTK protein (at a final concentration of 10 µM) was incubated with 18 (at a
final concentration of 100 µM) in a buffer containing 50 mM HEPES (pH 7.4), 75 mM NaCl,
and 5% glycerol at room temperature with gentle shaking for 1 h. After the reaction, the
remaining inhibitors were removed by exchange into a 50 mM NH4HCO3 (pH 8.5) buffer with
the use of Microcon centrifugal filters (30,000 NMWL, Millipore). To the protein samples were
subsequently added 8 M urea for protein denaturation, dithiothreitol and iodoacetamide for
cysteine reduction and alkylation, respectively. The proteins were digested with modified MS-
grade trypsin (Thermo Pierce) at an enzyme/substrate ratio of 1:100 in 50 mM NH4HCO3 (pH
8.5) at 37°C for overnight. The peptide mixture was subsequently dried in a Speed-vac, desalted
by employing OMIX C18 pipet tips (Agilent Technologies, Santa Clara, CA), and then subjected
to LC-MS/MS analyses.
The LC-MS/MS experiments were first conducted on a QE Plus Quadrupole-Orbitrap mass
spectrometer. The samples were automatically loaded from a 48-well microplate autosampler
using an EASY-nLC 1200 system (Thermo Fisher Scientific) at 3 µL/min onto a biphasic
precolumn (150 µm i.d.) comprised of a 3.5-cm column packed with 5 µm C18 120 Å reversed-
phase material (ReproSil-Pur 120 C18-AQ, Dr. Maisch). The biphasic trapping column was