Design and synthesis of boron-containing diphenylpyrimidines as potent BTK and JAK3 dual inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.115236

Bruton's tyrosine kinase (BTK) and Janus kinase 3 (JAK3) are very promising targets for hematological malignancies and autoimmune diseases. In recent years, a few compounds have been approved as a marketed medicine, and several are undergoing clinical trials. By recombining the dominant backbone of known active compounds, constructing a foused library, and screening a broad panel of kinases, we found a class of compounds with dual activities of anti-BTK and anti-JAK3. Some of the compounds have shown 10-folds more active in the enzyme and cell-based assays than a known active compound. Furthermore, liver microsome stability experiments show that these compounds have better stability than ibrutinib. These explorations offered new clues to discover benzoxaborole fragment and pyrimidine scaffold as more effective BTK and JAK3 dual inhibitors.

Full text of DOI:10.1016/j.bmc.2019.115236

Journal Pre-proofs
Design and Synthesis of Boron-containing Diphenylpyrimidines as Potent
BTK and JAK3 Dual Inhibitors
Jing Ren, Wei Shi, Damin Zhao, Qinglin Wang, Xiayun Chang, Xiangyi He,
Xiaojin Wang, Yong Gao, Peng Lu, Xiquan Zhang, Hongjiang Xu, Yinsheng
Zhang
PII:
S0968-0896(19)31649-9
https://doi.org/10.1016/j.bmc.2019.115236
BMC 115236
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
23 September 2019
21 November 2019
23 November 2019
Please cite this article as: J. Ren, W. Shi, D. Zhao, Q. Wang, X. Chang, X. He, X. Wang, Y. Gao, P. Lu, X.
Zhang, H. Xu, Y. Zhang, Design and Synthesis of Boron-containing Diphenylpyrimidines as Potent BTK and
JAK3 Dual Inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
2019.115236
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Design and Synthesis of Boron-containing Diphenylpyrimidines as
Potent BTK and JAK3 Dual Inhibitors
Jing Ren, Wei Shi, Damin Zhao, Qinglin Wang, Xiayun Chang, Xiangyi He, Xiaojin
Wang, Yong Gao, Peng Lu, Xiquan Zhang, Hongjiang Xu*, Yinsheng Zhang*
Institute for Innovative Drug Discovery
Chia Tai Tianqing Pharmaceutical Group Co., Ltd
1099 Fuying Road, Jiangning District, Nanjing, Jiangsu Province, China
*Correspondence to: Yinsheng Zhang (zys@cttq.com), Institute for Innovative Drug
Discovery, Hongjing Xu (xuhongjiang@cttq.com), Institute for Pharmacological
Evaluation, Chia Tai Tianqing Pharmaceutical Group Co., Ltd, 1099 Fuying Road,
Jiangning District, Nanjing, Jiangsu Province, China
ABSTRACT:
Bruton's tyrosine kinase (BTK) and Janus kinase 3 (JAK3) are very promising targets
for hematological malignancies and autoimmune diseases. In recent years, a few
compounds have been approved as a marketed medicine, and several are undergoing
clinical trials. By recombining the dominant backbone of known active compounds,
constructing a foused library, and screening a broad panel of kinases, we found a class
of compounds with dual activities of anti-BTK and anti-JAK3. Some of the compounds
have shown 10-folds more active in the enzyme and cell-based assays than a known
active compound. Furthermore, liver microsome stability experiments show that these
compounds have better stability than ibrutinib. These explorations offered new clues to
discover benzoxaborole fragment and pyrimidine scaffold as more effective BTK and
JAK3 dual inhibitors.
Keywords; BTK, JAK3, dual inhibitor, benzoxaborole, pyrimidine, hematological,
autoimmune diseases
1. Introduction
Bruton’s tyrosine kinase (BTK) is a non-receptor Tec family tyrosine kinase that is
broadly expressed in hematopoietic cells, with the exception of T cells. B cells are

essential to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA).
BTK plays a crucial role in signaling through the B-cell antigen receptor (BCR) and the
Fcγ receptor (FcγR) in B cells and myeloid cells, respectively [1,2]. It is necessary for
BCR-dependent proliferation of B cells as well as the production of pro-inflammatory
cytokines and co-stimulatory molecules [3,4,5]. BTK deregulation has been observed
in numerous B-cell-derived malignancies, including acute lymphoblastic leukemia
(ALL), chronic lymphocytic Leukemia (CLL), Non Hodgkin Lymphoma (NHL),
mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia (WM) and multiple
myeloma (MM)[6,7]. Taken together, BTK could be a potential target for the treatment
of autoimmune diseases and hematologic malignancies. A few inhibitors are launched
such as ibrutinb [8,9] and acalabrutinib [10,11] and several are under clinical trials for
the treatment of CLL, MCL, WM, rheumatoid arthritis, etc.
The Janus kinases (JAKs) are a family of non-receptor tyrosine protein kinases crucially
involved in immune signaling [12]. All four JAK family members, namely JAK1, JAK2,
JAK3 and tyrosine kinase (TYK) 2 signal via the JAK/STAT (signal transducers and
activators of transcription pathway). JAK3 was found to play an important role in
cytokine-induced proliferation and survival of normal and leukemic B-cell precursors
[13,14]. JAK3 also regulates the anti-apoptotic PI3K-AKT pathway and their
downstream targets, some of which have been implicated as important oncogenic
proteins [15]. Furthermore, selective JAK3 inhibitors mediate cytokine signaling
through the γ-common chain receptors [16]. As a result, JAK3 is an attractive target for
hematologic malignant and autoimmune disease. Currently, the selective JAK3
inhibitor PF-06651600 is in the clinical phase II study for the treatment of rheumatoid
arthritis (NCT02969044), alopecia (NCT02974868) and ulcerative colitis
(NCT02958865).
The 2,4 di-substituted pyrimidine is a class of dominant scaffold with broad biological
activity in multiple kinase targets, such as EGFR, ALK, CDK, BCR-ABL and so on.
The nitrogen atom on pyrimidine ring as a hydrogen bond acceptor and the common 2-

or 4-substituted amino group in the pyrimidine core as an H bond donor can form strong
hydrogen bonds with ATP pocket hinge region. It is noticed that 22 of the 48 kinase
inhibitors currently on the market owns the pyrimidine core. Some represent
compounds are shown in Figure 1.
O
H
N
O
N
P
H
N
H
N
NH
N
N
O
N
N
O
S
N
O
Cl
N
OH
N
O
N
N
1
Osimertinib, EGFR
2
Brigatinib, ALK
O
N
F
N
N
N
N
N
N
N
H
H
N
N
N
N
N
O
S
N
H
OH
N
O
F
Abemaciclib, CDK4\6
3
4
Imatinib,BCR-ABL, PDGFR, c-KIT
Figure 1. The examples of the marked drugs with a pyrimidine core.
In recent years, the application of boron atoms in drug development has received more
and more attention. The boron atom is located at the 5th position of the periodic table
as a non-metallic element and is a necessary trace element for plants grown in some
natural environments. Boron is in Group 3 of the periodic table, and the outer electrons
have a p-orbital orbital that forms a coordination bond with the lone pair of O and N.
Many boron-containing compounds take advantage of this feature to form covalent
bonds with the amino acid residues of the receptor protein, thereby increasing their
binding activity [17]. There are currently four boron-containing drug molecules on the
market, and many are at different stages of clinical research. Figure 2 shows some
active compounds containing a common boron heterocycle, benzoxaboroles, such as
crisaborole 5 [18], GSK565 6 [19], antimalarial agent 7 [20] and anti-Wolbachia agent
8 [21]. Some studies on benzoxaboroles show they have drugable properties and could
be a class of dominant pharmacophore [22, 23, 24, 25].

HO
O
OH
O
B
O
O
B
NC
HCl
NH2
OH
Cl
crisaborole,PDE-4
5
GSK656,Leucyl-tRNA Synthetase
(Mycobacterium tuberculosis) Inhibitors
6
OH
OH
F
O
HO
HO
O
N
N
O
B
O
B
O
O
N
O
O
antimalarial activity
7
AN11251,anti-Wolbachia activity
8
Figure 2. The boron-containing drugs on the market and in clinical trials.
To create a new chemical entity we combined these two types of dominant
pharmacophores by introducing the benzoxaboroles into the 2- or 4-position of
pyrimidine. A total of 31 compounds were synthesized to construct a small focused
library. Then, we performed a broad kinases screening (23 kinases) of these compounds
and found seventeen hit compounds for eight targets (Table 1), among which the IC50
of compound 9 is 2.97 nM for BTK and 0.50 nM for JAK3 (Figure 3).
Table 1. Number of hits with >50% inhibition rate at 100 nM
Targets
BTK
ALK
EGFR
(L858R\
T790M)
4
JAK1
JAK3
BLK
ABL1 FLT3
Number of hits
Inhibition rate
at 100 nM
1
6
2
1
1
1
1
94%
54-63%
51-98%
66-89%
98%
68%
69%
82%

R
N
N
O
R
N
NH
O
H
4
B
CF3
3 N
2, 4-substituted pyrimidine
HN
N
5
6
OH
O
X
+
B
N
2
N
N
1
H
HO
R
O
N
B
9
H
R
OH
BTK IC50= 2.97 nM
JAK3 IC50=0.50 nM
31 compounds
focused library
benzoxaboroles
Figure 3. Design of a focused library and results of a broad anti-kinases screening.
Inspired by the above results, we then designed and synthesized a series of derivatives
of compound 9 containing an organic boron fragment based on the known structure-
activity relationships (SARs) of effective BTK inhibitors. Interestingly, these newly
designed boron-containing compounds exhibited potent inhibitory activity against both
BTK and JAK3 enzymes. In this paper, we describe these newly discovered BTK and
JAK3 dual inhibitors, including their synthesis, biological evaluation in vitro and SARs.
2. Results and Discussion
2.1. Molecular docking of compound 9 with BTK and JAK3
To provide a direction for subsequent structural optimization we first probed the
binding mode of the compound 9 with BTK using MOE (Figure 4a). As shown in
Figure 4a, besides a covalent bond force between the essential acrylamide functionality
of 9 with the Cys481 residue, compound 9 forms several important hydrogen bonds
with the ATP pocket of BTK kinase domain. The NH at 2-position and the nitrogen
atom at 1-position of the pyrimidine ring make two hydrogen bonds with the backbone
carbonyl and NH of Met477 residue, respectively. Additionally, the hydroxyl group of
boric acid acts as an extra hydrogen bond donor to form another hydrogen bond with
the carbonyl group of Ala478 residue. The phenyl ring attached to the amino group at
2-position of the pyrimidine and the pyrimidine ring itself make a favorable
hydrophobic interaction with Leu408 and Leu528. The binding mode of the known
molecule, CC292, is similar to compound 9 as a whole, but CC292 does not form the
hydrogen bond with Ala478 residue (Figure 4b).

Figure 4. a) Binding mode of compound 9 with BTK kinase domain generated by MOE
(PDB ID: 5P9J). b) The overlay of CC292 (red，co-crystal) and compound 9 (yellow)
in ATP pocket of BTK kinase domain. c) Binding mode of compound 9 with JAK3
kinase domain generated by MOE (PDB ID: 5TOZ). d) The overlay of PF-06651600
(blue，co-crystal) and compound 9 (yellow) in ATP pocket of JAK3 kinase domain.
We also probed the binding mode of the compound 9 with JAK3 using MOE (Figure
4c). As shown in Figure 4c, compound 9 forms several important biding forces with the
ATP pocket of the JAK3 protein. Besides a covalent bond force between the essential
acrylamide functionality of 9 with the Cys356 residue, the NH at 2-position and the
nitrogen atom at 1-position of the pyrimidine ring make two hydrogen bonds with the
backbone carbonyl and NH of Leu323 residue, respectively. Two aromatic ring systems

of 9 are engaged in a hydrophobic association with leu73 and leu497. In addition, the
hydroxyl group of boric acid acts as an extra hydrogen bond donor to form another
hydrogen bond with the carbonyl group of Pro324 residue. The binding mode of the
known compound, PF-06651600, at ATP pocket of JAK3 kinase domainat is not
exactly the same as that of compound 9, but both have a covalent bond with the Cys356
residue and a hydrogen bond with Leu323 residue (Figure 4d).
2.2. Chemistry
Scheme 1. Synthesis of compounds 14a-p
R1
R1
O
R3
NH2
O
R1
X1
Cl
N
X1
13
O
R2
N
H
X1
R2
H
N
R2
N
N
H
b
a
N
R3
Cl
N
N
N
Cl
N
H
R3=
R3=
R3=
O
13a
,
14a-e
14f-n
14o-p
(Table 1)
(Table 2)
(Table 3)
B
10
11
12
HO
1
2
1
12a
12b
12c
12d
12e,
, R =H,R =CF3, X =NH
1
1
2
10a
10b
10c
, X = NH2, R =H
11a
,R =CF3,
1
2
1
O
13b,
13c,
, R =H,R =F, X =NH
1
1
2
, X =NH2, R =OMe
11b
11c
11d
11e
,R =F,
B
1
2
1
, R =H,R =Cl, X =NH
1
1
2
HO
, X =OH, R =H
,R =Cl,
1
2
1
O
, R =H,R =Me, X =NH
3
,
2
14l'
R =
,R =Me,
B
1
2
1
O
R =H,R =OMe, X =NH
2
O
,R =OMe
B
1
2
1
c
c
12f
, R =H,R =OMe, X =O
HO
1
2
1
HO
12g,
R =OMe, R =CF3, X =NH
R3=
B
HO
14l
,
R3=
O
13d,
13e,
13f,
B
O
3
14m',
HO
R =
B
O
3
O
R =
B
HO
R3=
HO
14m,
B
HO
R3=
O
B
OH
F
3
O
13g,
R =
B
HO
O
R3=
R3=
13h
,
B
O
O
13i
,
B
O
NH
R3=
13j,
Reagents and conditions: (a) DIPEA, n-BuOH, rt; (b): (1) TFA, n-BuOH, reflux, for compound 14a-p except for 14n;
(2) Pd2(dba)3, X-PHOS, K2CO3, t- BuOH, MW, 120oC, 30 min, for compound 14n; (c) Isobutylboronic, HCl, MeOH, heptane, rt, 1.5h.
The syntheses of compounds 14a-p were convenient, and the general routes are shown
in Scheme 1. Generally, to synthesize these boron-containing molecules, the key
intermediates, 3- or 4-subtituted N-phenylacrylamide (10) and animo-substituted
benzoxaboroles (13) were first prepared according to the know methods [24, 25, 26,
27] or purchased in case they are commercially available. Substitution of 2,4-dichloro-
pyrimidine (11a-e) with aniline or phenol derivatives (10a-c) at the 2-position in the
presence of DIPEA gave 2-substituted chloro-pyrimidine derivatives (12a-g), which
were further substituted with boron-containing anilines (13a-j) at the 4-position in the

presence of TFA to afford the final compounds (14a-m, 14o-p, Scheme 1). Intermediate
12a reacted with 13j under Buchwald–Hartwig condition to give compound 14n
(Scheme 1).
In some cases, the boron-containing anilines had to selectively substitute 2-chloro
group of 2,4-dichloro-5-(trifluoromethyl)pyrimidine (11a) first using ZnCl2 as a
catalyst. Then, the further substitution of the intermediate (15a-b) with anilines or
phenol (10a, 10c-e) at the 4-position under microwave heating condition furnished the
final compounds (16a-d, Scheme 2).
Scheme 2. Synthesis of compounds 16a-d
R4-X1
10
R4
X1
Cl
Cl
CF3
CF3
CF3
N
R3 X2
N
N
R3
X2
a
R3
X2
b
N
N
Cl
N
13b,k
11a
15a-b
16a
(Table 1)
16b-d
(Table 3)
O
4
4
16c'
, R =
10a
, R =
X1=NH2
O
3
2
3
H2N
N
15a
, R -X =
N
13b
13k
, R =
O
B
O
3
H
B
4
c
HO
HO
H
N
O
X2=NH2
O
4
4
16c
, R =
10c
, R =
3
2
O
N
15b
, R -X =
N
B
H
3
4
X1=OH
H
, R =
O
HO
B
3
HO
X2= OH
10d
, R =
4
Boc
X1=NH2
N
H
4
10e
, R =
N
O
X1=NH2
Reagents and conditions: (a) ZnCl2, Et3N, 1,2-Dichloroethane, t-BuOH, 0oC-rt, 16h; (b) EtOH, MW, 100oC, 30 min;
(c) trans-4-Dimethylaminocrotonic acid hydrochloride, HATU, DIPEA, DMF, rt, 1h.
2.3. Biological activity against BTK
Based on the molecular docking of compound 14a with BTK, We first conducted a
preliminary modification at the three positions of R1, R2, and X2 of structure A (Table
2, structure A). We replaced the trifluoromethyl group (R2=CF3) with other
hydrophobic groups such as F, Cl, and Me. The results showed that the activity of
compound 14a with a trifluoromethyl group was the best, and the activity of other
comppounds (14b, 14c, 14d) with F, Cl and Me group was slightly decreased
(compound 14a→ 14b, 14c, 14d). Our results indicate that the R2 group may be located
in a conservative hydrophobic pocket, which is consistant with the result of the

molecular docking. After replacing the NH at the X2 position of structure A with an
oxygen atom (compound 14a→16a), we found that the activity of compound 16a was
basically lost. This indicates that NH at this position acts as a hydrogen bond donor
required for keeping the activity, which verifies the result of the docking (Figure 4).
Introduction of a methoxy group (R1=OMe) resulted in the loss of activity, probably
because the steric hindrance of methoxy group prevented the compound 14e from
entering the covalent binding capsule of protein, thus losing the covalent binding effect.
Table 2 The inhibitory activities of compound 14a-e and 16a against BTK
R1
O
N
NH
N
H
R2
N
O
X2
B
HO
A
R1
R2
X2
IC50 (nM) a
Compounds
BTK
2.9
14ab
14b
14c
H
H
H
H
CF3
F
NH
NH
NH
NH
8.5
Cl
4.7
14d
Me
6.1
14e
16a
OMe
H
CF3
CF3
NH
O
>1000
>1000
a IC50 stands for 50% inhibition concentration. Dose-response curves were determined at five concentrations.
b14a = 9
Based on the structure-activity relationship (SAR) results from Table 1, we then
fixed R1 group as hydrogen, R2 as trifluoromethyl and X2 as NH, and only examined
the effect of R3 on the activity (Table 3, structure B). We tried many different types of
substituents, including the boron-containing moieties and simple substituted-benzenes.
First of all, the study on compounds 14a, 14f-14h with a boron atom at different
positions of the benzene ring shows that the potency of compound 14h with boron
substitution at 4-position is better than one at 3-position (compound 14a) and

significantly better than those at 2 and 5 positions (14f, 14g). Modifications of the five-
membered ring to the six-membered ring (14i) and dimethyl-substituted five-membered
ring (14j) maintained the activity. However, the introduction of a fluoro group into the
benzoxoborole at 6-position resulted in the significant decrease of the activity. The
boron-containing heterocycle was opened to obtain simple boronic acid derivatives
(compound 14a→14l, compound 14h→14m), and as a result their activity decreased
about 2 times. We further introduced a moiety of phenylhydrazine (14n) as R3 group
instead of the boron-containing moieties, and found that the activity of 14n was
substantially lost (Table 2, case 14n). The activity loss possibly related to a big change
of the electronic property of the compound and lack of a hydrogen bond donor (R2B-
OH).
Table 3 The inhibitory activities of compound 14a and 14f-n against BTK
O
N
NH
N
H
CF3
N
R3
N
H
B
R3
IC50 (nM)
BTK
R3
IC50 (nM) a
BTK
Compounds
Compounds
2.9
2.0
O
B
O
3
14a
14f
14j
HO
B
HO
F
267.8
63.0
14k
156.2
6.5
O
O
B
B
2
HO
HO
HO
14g
14h
14l
B
O
OH
B
5
HO
0.6
14m
1.5
O
HO
B
B
4
HO
OH

14i
1.9
14n
>1000
NH
O
B
OH
a IC50 stands for 50% inhibition concentration. Dose-response curves were determined at five concentrations.
According to the result of molecular docking, the X1 moiety (Table 4, structure C ) did
not form a hydrogen bond with the hinge region. Referring to the structure-activity
relationship of the compound (AC0058) currently studied in clinical phase [28], we
replaced NH with an oxygen atom (compound 14h→16b) and found that its activity
decreased about 10 folds (Table 4, structure C). Furthermore, when R2 moiety was
changed from an electron-withdrawing group (CF3) to an electron-donating group
(OMe), the activity of the compound 14o decreased about 10 folds, too. Interestingly,
once both X1 and R2 were simultaneously modified from NH and CF3 to O and OMe,
respectively, the activity of the resulted compound 14p was 4-5 times higher than that
of the single modified compound (16b or 14o), indicating that the two groups had a
certain synergic effect.
With reference to the covalent inhibitors currently reported, such as afatinib and
ibrutinib, a few warheads (Michael addition acceptor) were selected to explore the
effect of R4 moiety (compounds 16c and 16d). It was found that the compounds 16c &
16d displayed much lower activity (11.8 nM and 36.8 nM, respectively) than compound
14h (0.62 nM).
Table 4 The inhibitory activities of compounds 14h, 14o-p and 16b-d against BTK
R4
X1
R2
N
HO
B
O
N
N
H
C
Compounds
R2
R4
X1
IC50 (nM) a
BTK

O
O
O
O
14h
14o
14p
16b
16c
16d
CF3
OMe
OMe
CF3
NH
NH
O
0.6
8.2
N
H
N
H
1.7
N
H
O
6.5
N
H
O
CF3
NH
NH
11.8
36.8
N
N
H
CF3
N
O
a IC50 stands for 50% inhibition concentration. Dose-response curves were determined at five concentrations.
2.4. Biological activity against BTK and JAKs
Similar to the BTK protein, the ATP pocket of the JAK3 protein also has a homologous
cysteine residue that can form irreversible covalent binding to small molecule inhibitors.
In addition to the separate application of BTK inhibitors, JAK3 inhibitors are also used
in the field of immunotherapy. Both inhibitors of the BTK pathway and the JAK3
pathway might have synergistic effects in clinical applications [29, 30]. Therefore, we
selected some compounds with better BTK inhibitory activity and tested their inhibitory
activity against JAK family kinases (Table 5). From the results of the activity tests, the
compounds showed potent inhibitory activity against JAK3 and most of the compounds
had less than 1 nM of IC50, while the inhibitory activity against JAK1, JAK2 and TYK2
was very low. The selectivity of JAK3 over JAK1, JAK2 and TYK2 was great (>1000
folds).
We also compared our compounds with two currently marketed drugs and a testing
drug in clinical studies. In terms of BTK inhibitory activity, our compounds were much

more active than CC292, but comparable to Ibrutinb. In terms of JAK3 inhibitory
activity, our compounds were more active than the three positive control compounds.
Overall, our compounds were a class of very potent BTK/JAK3 dual inhibitors.
Table 5 The inhibitory activitiesa of selected compounds against BTK and JAKs
Compounds
IC50 (nM)
BTK(WT)
IC50 (nM)
JAK1
IC50 (nM)
JAK2
IC50 (nM)
JAK3
IC50 (nM)
TYK2
14a
14h
2.9
0.6
>1000
>1000
971.6
657.9
0.5
0.4
>1000
>1000
14i
1.9
2.0
1.5
>1000
>1000
>1000
>1000
>1000
891.5
1.5
1.0
0.3
>1000
>1000
>1000
14j
14m
14p
Ibrutinb
CC292
1.7
>1000
>1000
>1000
8.3
>1000
>1000
533.7
14.3
0.8
>1000
NT
0.3
20.3
8.5
9.8
NT
Tofacitinib
>100
14.1
235
a IC50 stands for 50% inhibition concentration. Dose-response curves were determined at five concentrations. NT =
not test
2.5. Antiproliferative Activities against several hematopoietic cell lines
To further validate the anti-proliferative activity of these compounds, the molecules
with IC50 values below 5 nM were chosen to examine the effect in cancer cells. Five
typical hematopoietic cell lines associated with the BTK signaling pathway were used
for the cellular inhibitory activity by CCK-8 assay. The results were depicted in Table
6. For comparison, ibrutinib and CC292 were also tested as reference compounds.
Significant cytotoxicity with IC50 in the μM range was determined for most of
hematopoietic cell lines employed in the screening program.
In cell-based assays, both 14a and 14h showed inhibition on the proliferations of Raji,

Ramos and HEL, Jeko-1 and OCI-LY-10 cells at concentrations ranging from 0.5 to 10
μmol/L. Compound 14h with the best kinase-based activity displayed better efficacy
than CC292 and Ibrutinib in the four cells of Raji, Romas, HEL, and Jeko-1.
Table 6 Antiproliferative activitiesa of compound 14a and 14h against hematopoietic
cells
Compounds
IC50 (μM)
IC50 (μM)
IC50 (μM)
IC50 (μM)
IC50 (μM)
Raji
6.8
Romas
2.3
HEL
5.1
Jeko-1
1.2
OCI-LY10
>10
14a
14h
1.7
0.5
1.1
0.5
1-10
CC292
17.6
>20
3.9
2.4
5.3
1.3
3.0
>10
Ibrutinb
>20
1-10
a IC50 stands for 50% inhibition concentration. Dose-response curves were determined at five concentrations.
2.6 Human liver microsome stability
The selected compounds 14a and 14h along with two know drugs, ibrutinb and CC292
were then submitted to metabolic stability studies in human liver microsome. Our tests
(Table 7) showed that the metabolic stability of the compound 14a and 14h in the liver
microsome was very close (T1/2 56 vs. 60 min), however, better than CC292 (T1/2 43
min) and significantly better than ibrutinib (T1/2 5.5 min).
Table 7 Human liver microsomal stability assay of compounds 14a, 14h and the known
drugs
Compounds
T1/2
(min)
55.9
58.8
43.4
5.5
Clint
Remaining
(T=60min)
45.0%
(µL/min/mg)
24.8
14a
14h
23.6
47.2%
CC292
Ibrutinb
31.9
38.8%
254.2
0.04%

3. Conclusion
Both BTK and JAK3 kinase are very popular targets in hematological malignancies and
autoimmune diseases. We recombined two types of dominant scaffold, the 2,4-
disubstituted pyrimidine, and benzoxaboroles, and constructed a small library of 31
compounds. By screening 23 kinases, we found a class of compounds with a potent
BTK inhibitory activity. Through structural optimization, we obtained more active
compounds 14h, 14i, 14m and 14p with IC50 < 2 nM against both BTK and JAK3
kinase. Therefore, a new class of compounds may be considered as a dual inhibitor of
BTK and JAK3. The cell activity screen showed that the compound 14h was more
active than CC292 and Ibrutinib by 10 folds. Metabolic stability experiment in human
liver microsome exhibited that the compounds 14h had better stability than the know
drugs. As a dual-target inhibitor, these compounds are expected to exert better effects
in hematological and immune diseases through synergy. The further pre-clinical
evaluation of the compound 14h is underway.
4. Experimental section
4.1. Chemistry.
All reagents were purchased from commercial sources and were used as received.
Routine monitoring of reactions was performed by thin layer chromatography (TLC)
using pre-coated Haiyang GF254 silica gel TLC plates. NMR spectra were recorded on
a Bruker AVANCE 500 spectrometer at 500 MHz with tetramethylsilane used as an
internal reference. High resolution mass spectra (HRMS) were performed on Agilent
Acrrurate-Mass Q-ToF LC/MS 6520 mass spectrometer with electron spray ionization
(ESI) mode. Microwave reactions were done on a CEM Discover SP.
4.1.1 Intermadiates 10a-10e & 11a-d
Intermediates 10a, 10c-d, 11a-e were purchased from Nanjing Ally Chemical S&T Co.,
Ltd, Shanghai biochempartner Co., Ltd and Sigama-aldrich. Intermediates 10b [27]
and 10e [28] were prepared according to the literatures’ methods.
4.1.2 General procedure A for preparation of intermediates 12a-g

Intermediate 10 (1.2 eq), 11 (1.0 eq), n-butanol and DIPEA (1.5 eq) were added to the
reaction flask at room temperature. The resulted mixture was stirred at room
temperature until the TLC plates showed the completion of the reactions. Most of the
reaction solvent was evaporated. Then, ethyl acetate and water were added to the
residue. The organic phase was washed with saturated brine and dried over anhydrous
sodium sulfate, filtered and then evaporated to dryness. Purification by column
chromatography (low, medium and high pressure preparative chromatography) gave
the desired compounds.
N-(3-((2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide (12a)
Yield 61%; white solid; 1H NMR (300 MHz, DMSO-d6) δ [ppm] : 10.22 (s, 1H), 9.57
(s, 1H), 8.58 (s, 1H), 7.79 (s, 1H), 7.49-7.52 (m, 1H), 7.33-7.38 (m, 1H), 7.13-7.16 (m,
13
1H), 6.40-6.49 (m, 1H), 6.23-6.29 (d, 1H), 5.75-5.78 (m, 1H); C NMR (126 MHz,
DMSO-d6) δ [ppm]: 163.7, 162.9, 158.1, 157.1 (q, J = 5.0 Hz), 139.8, 137.6, 132.2,
129.3, 127.5, 123.7 (q, J = 270.9 Hz, CF3), 121.5, 117.6, 117.2, 106.2 (q, J = 32.8 Hz,
CCF3). HRMS (ESI) m/z calculated for C14H11ClF3N4O [M+H]+: 343.0573, found:
343.0566.
N-(3-((2-chloro-5-fluoropyrimidin-4-yl)amino)phenyl)acrylamide(12b)
Yield 93%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.20 (s, 1H), 10.02
(s, 1H), 8.31 (d, J = 3.5 Hz, 1H), 8.01 (s, 1H), 7.44 (s, 1H), 7.42 (s, 1H), 7.33 (t, J = 8.5
Hz, 1H), 6.45-6.51 (m, 1H), 6.28 (dd，J = 17.0, 1.5 Hz, 1H), 5.76 (dd, J = 10.0, 1.5 Hz,
1H); 13C NMR (126 MHz, DMSO-d6) δ 163.7, 153.4 (q, J = 3.2 Hz), 151.7 (q, J = 11.7
Hz), 145.6 (q, J = 259.4 Hz), 142.0 (q, J = 21.0 Hz), 139.8, 138.4, 132.3, 129.3, 127.4,
117.7, 116.1, 113.6. HRMS (ESI) m/z calculated for C13H10ClFN4O [M+H]+: 293.0605,
found: 293.0598.
N-(3-((2,5-dichloropyrimidin-4-yl)amino)phenyl)acrylamide(12c).
Yield 89%; white solid; 1H-NMR (500 MHz, DMSO-d6) δ [ppm] : 10.20 (s, 1H), 9.55
(s, 1H), 8.38 (s, 1H), 7.92 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H),
7.28-7.30 (d, J = 8.0 Hz, 1H), 6.44-6.50 (m, 1H), 6.26-6.29 (dd, J = 17.0 Hz, 1.5 Hz,
13
1H), 5.77 (dd, J = 10.0 Hz, 1.5 Hz, 1H); C NMR (126 MHz, DMSO-d6) δ [ppm]:
163.7, 157.8, 157.4, 155.9, 139.7, 138.1, 132.3, 129.2, 127.4, 119.6, 116.8, 115.4, 114.1.
HRMS (ESI) m/z calculated for C13H11Cl2N4O [M+H]+ : 309.0310, found: 309.0305.
N-(3-((2-chloro-5-methylpyrimidin-4-yl)amino)phenyl)acrylamide(12d).
Yield 57%; white solid; 1H-NMR (500 MHz, DMSO-d6) δ [ppm] : 10.16 (s, 1H), 8.89

(s, 1H), 8.04 (s, 1H), 7.97 (s, 1H), 7.38 (t, J = 7.0 Hz, 2H), 7.32 (t, J = 8.0 Hz, 1H),
6.45-6.51(m, 1H), 6.28 (dd, J = 17.0, 1.5 Hz, 1H), 5.76 (dd，J = 10.0, 1.5 Hz, 1H),
13
2.18 (s, 3H); C NMR (126 MHz, DMSO-d6) δ [ppm] : 163.6, 160.9, 157.3, 156.7,
139.6, 139.3, 132.4, 129.1, 127.3, 118.5, 115.8, 114.9, 114.4, 14.0. HRMS (ESI) m/z
calculated for C14H14ClN4O [M+H]+ : 289.0856, found: 289.0845.
N-(3-((2-chloro-5-methoxypyrimidin-4-yl)amino)phenyl)acrylamide(12e).
Yield 81%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.15 (s, 1H), 9.28
(s, 1H), 8.01 (s, 1H), 7.94 (s, 1H), 7.48 - 7.38 (m, 2H), 7.29 (t, J = 8.1 Hz, 1H), 6.48
(dd, J = 17.0, 10.2 Hz, 1H), 6.27 (dd, J = 17.0, 2.0 Hz, 1H), 5.76 (dd, J = 10.1, 2.0 Hz,
1H), 3.95 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ 163.6, 153.1, 150.0, 140.3, 139.6,
139.1, 136.2, 132.4, 129.1, 127.3, 117.8, 115.7, 113.7, 57.0. ESI-MS: m/z 305.1
[M+H]+.
N-(3-((2-chloro-5-methoxypyrimidin-4-yl)oxy)phenyl)acrylamide (12f).
Yield 64%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.33 (s, 1H), 8.40
(s, 1H), 7.67 (s, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H), 6.96 (ddd, J =
8.1, 2.4, 1.0 Hz, 1H), 6.44 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0, 1.9 Hz, 1H),
5.79 (dd, J = 10.1, 1.9 Hz, 1H), 3.99 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ [ppm]
:163.8, 160.1, 152.3, 148.3, 142.3, 142.2, 140.8, 132.1, 130.5, 127.8, 117.2, 116.9,
112.7, 57.4. ESI-MS: m/z 306.1 [M+H]+.
N-(3-((2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)amino)-4-
methoxyphenyl)acrylamide (12g).
Yield 16%; white solid; 1H-NMR (500 MHz, DMSO-d6) δ [ppm] : 10.14 (s, 1H), 9.15
(s, 1H), 8.56 (s, 1H), 7.78 (s, 1H), 7.56-7.58 (dd, J = 9.0, 2.5 Hz, 1H), 7.11-7.13 (d, J
= 8.5 Hz, 1H), 6.41-6.46 (m, 1H), 6.22-6.26 (dd, J = 17.0, 1.5 Hz, 1H), 5.73-5.75 (dd,
13
J = 10.0, 1.5 Hz, 1H), 3.76 (s, 3H); C NMR (126 MHz, DMSO) δ [ppm] : 163.3,
163.0, 158.4, 156.8 (q, J = 5.0 Hz), 150.4, 132.6, 132.3, 127.0, 125.9, 123.8 (q, J =
270.9 Hz, CF3), 119.3, 119.2, 112.9, 105.9 (q, J = 32.8 Hz, CCF3), 56.5. HRMS (ESI)
m/z calculated for C15H13ClF3N4O2 [M+H]+: 373.0679, found: 373.0743.
4.1.3 Intermadiates 13a-13k.
Intermediates 13a, 13e-j were purchased from Nanjing Ally Chemical S&T Co. Ltd.
Intermediates 13b, 13c and 13d were prepared according to the literatures’ methods [24,
25].

4.1.4 General procedure B for preparation of compound 14a-m, 14o-p
Intermediate 12 (1 eq), substituted aniline intermediate 13 (1.2 eq), n-butanol and
trifluoroacetic acid (3 eq) were placed in a flask. The resulting mixture was heated to
reflux until the TLC plates monitored the completion of the reactions. Most of the
reaction solvent was evaporated under reduced pressure. The pH of remaining residue
was adjusted to 9 with a saturated aqueous solution of sodium bicarbonate. The basic
mixture was extracted with EtOAc. The organic phase was washed with saturated brine,
dried over anhydrous sodium sulfate, filtered, evaporated to dryness. Purification by
column chromatography (low, medium and high pressure preparative chromatography)
gave the desired compounds.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide（14a）
Yield 44%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.24 (s, 1H), 9.75
(brs, 1H), 9.13 (s, 1H), 8.73 (brs, 1H), 8.36 (s, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.54 (d, J
= 8.5 Hz, 2H), 7.38-7.26 (m, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.05 (brs, 1H), 6.45 (dd, J
= 16.9, 10.1 Hz, 1H), 6.24 (dd, J = 16.9, 2.1 Hz, 1H), 5.74 (dd, J = 10.1, 2.1 Hz, 1H),
4.88 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ [ppm] : 163.6, 161.3, 157.8, 156.2 (q,
J = 5.0 Hz), 148.2, 139.6, 138.9, 138.8, 132.3, 129.1, 129.0, 127.3, 125.2 (q, J = 270.9
Hz, CF3), 123.8, 122.2, 121.5, 121.3, 117.2, 116.6, 98.3 (q, J = 32.8 Hz, CCF3), 70.1;
HRMS (ESI) m/z calculated for C21H18BF3N5O3 [M+H]+: 456.1455, found: 456.1452.
N-(3-((5-fluoro-2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide (14b)
Yield 39%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.06 (s , 1H), 9.38
(s , 1H), 9.12 (s, 1H), 9.07 (s, 1H), 8.10 (d, J =3.5 Hz, 1H), 7.94 (s, 1H), 7.87 (s, 1H),
7.83-7.82 (m, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.27 (t, J = 8.0
Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 6.48-6.43 (m, 1H), 6.25 (dd, J = 16.0, 1.5Hz, 1H),
5.75 (dd, J = 10.0, 1.5 Hz, 1H), 4.91 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ [ppm]
: 163.6, 156.1, 150.3, 147.2, 142.1, 141.1, 140.9, 140.1, 139.6, 139.5, 132.4, 129.1,
127.2, 122.9, 121.4, 121.1, 117.5, 115.1, 113.4, 70.1; 19F NMR (471 MHz, DMSO-d6)
δ [ppm] : -164.13; HRMS (ESI) m/z calculated for C20H18BFN5O3 [M+H]+: 406.1487,
found: 406.1494.
N-(3-((5-chloro-2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide (14c)
Yield 51%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.29 (s, 1H), 9.40

(s, 1H), 9.09 (s, 1H), 8.82 (s, 1H), 8.15 (s, 1H), 8.02 (s, 1H), 7.85 (dd, J = 8.0, 1.5 Hz,
1H), 7.82 (s, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 9.0 Hz, 1H), 7.29 (t, J = 8.0
Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 6.50 (dd, J = 17.0, 10.0 Hz, 1H), 6.25 (dd, J = 17.0,
2.0 Hz, 1H), 5.75 (dd, J = 10.0, 2.0 Hz, 1H), 4.90 (s, 2H); 13C NMR (126 MHz,
DMSO-d6) δ [ppm] : 163.6, 158.3, 156.4, 154.9, 147.4, 139.7, 139.6, 139.4, 132.5,
131.0, 129.0, 127.0, 123.3, 121.5, 121.3, 118.8, 115.5, 114.7, 104.5, 70.1; HRMS
(ESI) m/z calculated for C20H18BClN5O3 [M+H]+: 422.1191, found: 422.1200.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)amino)-5-
methylpyrimidin-4-yl)amino)phenyl)acrylamide (14d).
Yield 32%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.06 (s, 1H), 9.06
(s, 1H), 8.91 (s, 1H), 8.33 (s, 1H), 7.97 (s, 1H), 7.92-7.90 (m, 2H), 7.83 (s, 1H), 7.52
(d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.15 (d, J =8.5
Hz, 1H), 6.45 (dd, J = 17.0, 10.5 Hz, 1H), 6.25 (dd, J = 17.0, 2.0 Hz,1H), 5.75 (m, 1H),
13
4.90 (s, 2H), 2.13 (s, 3H); C NMR (126 MHz, DMSO-d6) δ [ppm] : 163.5, 159.7,
158.7, 156.0, 146.6, 140.6, 140.5, 139.4, 132.5, 130.9, 128.9, 127.1, 122.8, 121.3, 120.8,
118.2, 114.7, 114.1, 106.4, 70.1, 14.0; HRMS (ESI) m/z calculated for C21H21BN5O3
[M+H]+: 402.1737, found: 402.1742.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)-4-methoxyphenyl)acrylamide (14e).
Yield 54%; white solid; 1H NMR(500 MHz, DMSO-d6) δ [ppm] : 10.06 (s, 1H), 9.75
(s, 1H), 9.10 (s, 1H), 8.36 (s, 1H), 8.18 (brs, 1H), 7.98 (brs, 1H), 7.80-7.82 (dd, J =
8.5, 1.5 Hz, 1H), 7.61-7.63 (d, J = 8.5 Hz, 1H), 7.10-7.12 (d, J = 8.5 Hz, 1H), 7.05 (s,
1H), 6.40-6.45 (m, 1H), 6.20-6.24 (dd，J = 17.0, 1.5 Hz, 1H), 5.71-5.74 (dd, J =
10.0, 1.5 Hz, 1H), 4.88 (s, 2H), 3.78 (s, 3H), 3.39-3.40 (m, 1H); 13C NMR (126 MHz,
DMSO-d6) δ [ppm] : 163.2, 161.3, 157.9, 155.9 (q, J = 5.0 Hz), 149.6, 148.0, 139.0,
132.4, 132.3, 131.0, 127.2, 126.8, 125.4 (q, J = 270.9 Hz, CF3), 123.3, 121.9, 121.3,
119.1, 118.1, 112.2, 98.1 (q, J = 32.8 Hz, CCF3), 70.1, 56.4; HRMS (ESI) m/z
calculated for C22H20BF3N5O4 [M+H]+ : 486.1560, found: 486.1568.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-7-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide (14f).
Yield 30%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.91 (s, 1H),
10.36 (s, 1H), 9.47 (s, 1H), 8.68 (s, 1H), 7.86 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.42 (t,
J = 8.0 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.01-6.98 (m, 2H), 6.70 (d, J = 6.5 Hz, 1H),
6.51 (dd, J = 16.5, 10.0 Hz, 1H), 6.28 (d, J = 17.0 Hz, 1H), 5.78 (d, J = 10.5 Hz, 1H),

4.43 (d, J = 13.5 Hz, 1H), 4.10 (d, J = 13.5 Hz, 1H); 13C NMR (126 MHz, DMSO-d6)
δ [ppm] : 163.8, 156.3, 153.3, 146.8, 146.3 (q, J = 4.9 Hz), 140.0, 139.8, 138.0, 133.3,
132.4, 129.4, 127.4, 126.9, 126.2, 123.7 (q, J = 270.9 Hz, CF3), 121.2, 117.5, 117.1,
114.3, 99.7 (q, J = 34.0 Hz, CCF3), 64.8; 19F NMR (471 MHz, DMSO-d6) δ [ppm] :
-60.15; HRMS (ESI) m/z calculated for C21H18BF3N5O3 [M+H]+ : 456.1455, found:
456.1485.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-4-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide (14g).
Yield 30%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.20 (s, 1H), 9.18
(s, 1H), 9.09 (s, 1H), 8.65 (s, 1H), 8.34 (s, 1H), 7.82 (s, 1H), 7.64 (d, J = 7.5 Hz, 1H),
7.48 (d, J = 7.5 Hz, 1H),7.40 (d, J = 6.5 Hz, 1H), 7.24-7.21 (m, 1H), 7.14-7.13 (m, 1H),
6.50-6.44 (m, 1H), 6.25(d, J = 17.0 Hz,1H), 5.75 (d, J = 10.0 Hz,1H), 4.98 (s, 2H); 13C
NMR (126 MHz, DMSO-d6) δ [ppm] : 163.6, 161.5, 157.7, 156.3 (q, J = 5.0 Hz), 146.7,
139.6, 138.9, 133.3, 132.4, 131.7, 128.9, 127.6, 127.2, 126.4, 125.9, 125.2 (q, J = 270.9
Hz, CF3), 120.8, 116.8, 116.3, 98.3 (q, J = 32.7 Hz, CCF3), 69.5; 19F NMR (471 MHz,
DMSO-d6) δ [ppm] : -59.22; HRMS (ESI) m/z calculated for C21H18BF3N5O3 [M+H]+ :
456.1455, found: 456.1465.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide (14h).
Yield 19%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.22 (s, 1H), 9.87
(s, 1H), 8.90 (s, 1H), 8.39 (s, 1H), 7.78 (s, 1H), 7.67 (s, 1H), 7.62 (d, J = 7.0 Hz, 1H),
7.46-7.39 (m, 2H),7.33 (m, 1H),7.13 (d, J = 6.5 Hz, 1H), 6.45-6.40 (m,1H), 6.26 (d, J
= 16.5 Hz, 1H), 5.75(d, J = 10.0 Hz, 1H), 4.66 (s, 2H); 13C NMR (126 MHz, DMSO-
d6) δ [ppm] : 163.7, 160.9, 158.3, 156.1 (q, J = 5.0 Hz), 155.4, 142.6, 139.9, 139.1,
132.2, 130.9, 129.4, 129.3, 127.5, 125.2 (q, J = 270.9 Hz, CF3), 122.5, 118.8, 118.0,
117.0, 111.3, 98.0 (q, J = 34.1 Hz, CCF3), 70.2; 19F NMR (471 MHz, DMSO-d6) δ
[ppm] : -59.58; HRMS (ESI) m/z calculated for C21H18BF3N5O3 [M+H]+ : 456.1455,
found: 456.1470.
N-(3-((2-((1-hydroxy-3,4-dihydro-1H-benzo[c][1,2]oxaborinin-7-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide (14i).
Yield 86%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.14 (s, 1H), 9.51
(s, 1H), 8.67 (s, 1H), 8.33-8.29 (m, 2H), 7.76-7.74 (m, 2H), 7.58-7.53 (m, 2H), 7.31 (m,
1H), 7.20 (m, 1H), 6.86 (s, 1H), 6.44 (dd, J = 17.0, 10.5 Hz, 1H), 6.26 (dd, J = 17.0,
2.0 Hz, 1H), 5.76-5.74 (m ,1H), 4.02 (t, J = 5.9 Hz, 2H), 2.74 (t, J = 5.9 Hz, 2H); 13C

NMR (126 MHz, DMSO-d6) δ [ppm] : 163.6, 161.2, 157.8, 156.2 (q, J = 5.0 Hz), 140.0,
139.6, 139.0, 137.9, 132.4, 129.1, 129.0, 127.3, 126.8, 125.3 (q, J = 269.6 Hz, CF3),
125.1, 123.4, 121.4, 117.2, 116.5, 98.0 (q, J = 34.5 Hz, CCF3), 63.8, 31.8; HRMS (ESI)
m/z calculated for C22H20BF3N5O3 [M+H]+: 470.1611, found: 470.1608.
N-(3-((2-((1-hydroxy-3,3-dimethyl-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide (14j).
Yield 49%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.25 (s, 1H), 9.68
(s, 1H), 8.92 (s, 1H), 8.68 (s, 1H), 8.35 (s, 1H), 7.80 (d, J = 7.0 Hz, 2H), 7.58 (d, J =
8.0 Hz, 2H), 7.32 (t, J = 8.0 Hz,1H), 7.20 (d, J = 7.5 Hz, 1H), 7.05 (s, 1H), 6.47 (dd, J
= 17.0, 10.0 Hz, 1H), 6.26 (dd, J = 17.0, 2.0 Hz, 1H), 5.74 (dd, J = 10.0, 2.0 Hz, 1H),
13
1.39 (s, 6H); C NMR (126 MHz, DMSO-d6) δ [ppm] : 163.5, 161.2, 157.6, 156.3,
155.5 (q, J = 5.0 Hz), 139.4, 139.2, 132.4, 128.9, 127.1, 125.2 (q, J = 270.9 Hz, CF3),
123.6, 121.8, 120.8, 120.7, 120.5, 116.8, 115.8, 115.4, 98.9 (q, J = 32.0 Hz, CCF3),
82.8, 29.9 (2C); HRMS (ESI) m/z calculated for C23H22BF3N5O3 [M+H]+: 484.1768,
found: 484.1768.
N-(3-((2-((5-fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide (14k).
Yield 31%; white solid; 1H NMR (500MHz, DMSO-d6) δ [ppm] : 10.03 (s, 1H), 9.24
(s, 1H), 9.16 (s, 1H), 8.51 (s, 1H), 8.31 (s, 1H), 7.73 - 7.63 (m, 2H), 7.36 (d, J = 6.1 Hz,
1H), 7.23 (t, J = 9.3 Hz, 2H), 7.18 - 7.09 (m, 1H), 6.43 (dd, J = 17.0, 10.1 Hz, 1H), 6.25
(dd, J = 17.0, 1.9 Hz, 1H), 5.74 (dd, J = 10.1, 1.9 Hz, 1H), 4.95 (s, 2H); 13C NMR (126
MHz, DMSO-d6) δ [ppm] : 163.5, 162.3, 160.1, 158.1, 157.4, 156.4 (q, J = 5.5 Hz),
152.9, 139.3, 132.3, 129.8, 128.7, 127.2, 126.3, 126.3, 126.2, 125.2 (q, J = 270.9 Hz,
CF3), 120.3, 116.1, 109.2, 98.6 (q, J = 31.5 Hz, CCF3), 69.9; HRMS (ESI) m/z
calculated for C21H17BF4N5O3 [M+H]+ : 474.1361, found: 474.1348.
(3-((4-((3-acrylamidophenyl)amino)-5-(trifluoromethyl)pyrimidin-2-
yl)amino)phenyl)boronic acid (14l)
Step 1. The general procedure B was used to prepare N-(3-((2-((3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)amino)-5-(trifluoromethyl)pyrimidin-4-
yl)amino)phenyl)acrylamide (14l’).
Yield 39%; yellow solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 9.60 (s, 1H), 9.65
(s, 1H), 8.74 (s, 1H), 8.37 (s, 1 H), 7.92-7.90 (m, 1H), 7.80-7.81 (m, 1H), 7.63 (s, 1H),
7.56-7.54 (m, 1H), 7.34-7.31 (m, 1H), 7.22-7.20 (m, 2H), 7.03-7.02 (m, 1H), 6.46-6.41
13
(m, 1H), 6.27-6.24 (m, 1 H), 5.76-5.74 (m, 1H), 1.27 (s, 12H); C NMR (126 MHz,

DMSO-d6) δ [ppm] :163.6, 161.1, 157.9, 156.2 (q, J = 4.8 Hz), 139.6, 139.0, 132.3,
129.0, 128.5, 128.1, 127.3, 126.3, 126.2, 125.2 (q, J = 270.9 Hz, CF3), 124.1, 123.3,
121.5, 117.3, 116.6, 98.3 (q, J = 30.4 Hz, CCF3), 84.0 (2C), 25.1 (4C). MS (ESI): m/z
526.4 [M+H]+.
Step 2. To a solution of compound 14l’ (1000 mg, 1.9 mmol) in a mixed solvent of
methanol (4.75 mL) and n-heptane (4.75 mL), were added isobutylboronic acid (776
mg ) and 1 M hydrogen chloride solution (2.5 mL). After the reaction mixture was
stirred at room temperature for 1.5 h, a large amount of white solid precipitated from
the mixture. The n-heptane layer was discarded, and the pH of methanol layer was
adjusted to 7 to obtain a suspension. The solid was collected by filtration and then
triturated with methanol to obtain compound 14l as a yellow solid (200 mg, yield
24%). 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.15 (s, 1H), 9.55 (s, 1H), 8.69 (s,
1H), 8.36 (s, 1 H), 7.9 (s, 1H), 7.78-7.75 (m, 2H), 7.66 (s, 1H), 7.55-7.54 (m, 1H),
7.36-7.29 (m, 1 H), 7.24 (s, 1H), 7.24 (s, 1H), 7.01 (brs, 1H), 6.47-6.42 (m, 1H), 6.28-
6.25 (m, 1H), 5.77-5.75 (m, 1H); 13C NMR (126 MHz, DMSO-d6) δ [ppm] : 163.6,
161.2, 157.9, 156.2 (q, J = 4.8 Hz), 139.6, 139.2, 139.1, 139.0, 132.3, 129.0, 128.5,
127.6, 127.3, 126.6, 125.2 (q, J = 252 Hz, CF3), 122.5, 121.5, 117.2, 116.5, 98.1 (q, J
= 30.4 Hz, CCF3); HRMS (ESI) m/z calculated for C20H18BF3N5O3 [M+H]+ :
444.1455, found: 444.1457.
(4-((4-((3-acrylamidophenyl)amino)-5-(trifluoromethyl)pyrimidin-2-
yl)amino)phenyl)boronic acid (14m).
Step 1. The general procedure B was used to prepare N-(3-((2-((4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)amino)-5-(trifluoromethyl)pyrimidin-4-
yl)amino)phenyl)acrylamide (14m’). Yield 49%; yellow solid; 1H NMR (500 MHz,
DMSO-d6) δ [ppm] : 10.19-10.17 (m, 1H), 9.81 (s, 1H), 8.85-8.36 (m, 1 H), 7.82-7.79
(m, 1H), 7.64-7.52 (m, 3H), 7.40-7.35 (m, 2H), 7.18-7.17 (m, 1H), 7.18-7.06 (m, 1H),
6.47-6.41 (m, 1H), 6.28-6.27 (m, 1H), 6.24-6.23 (m, 1H), 5.76-5.72 (m, 1H), 1.26 (s,
12H); 13C NMR (126 MHz, DMSO-d6) δ 163.7, 160.9, 158.1, 156.1 (q, J = 5.0 Hz),
143.1, 139.8, 138.9, 135.3 (2C), 132.3, 129.1 (2*C), 128.6, 127.2, 125.1 (q, J = 270.9
Hz, CF3), 121.9, 118.5, 117.6, 116.7, 98.4 (q, J = 30.1 Hz, CCF3), 83.7 (2C), 25.1
(4C). MS (ESI): m/z 526.3 [M+H]+.
Step 2. Compound 14m was synthesized starting from 14m’ according to the method
to prepare compound 14l. Yield 53%; yellow solid; 1H NMR (500 MHz, DMSO-d6) δ
10.19-10.18 (m, 1H), 9.69 (s, 1H), 8.82 (s, 1H), 8.39 (s, 1H), 7.82-7.75 (m, 1H), 7.70-
7.60 (m, 1H), 7.59-7.53 (m, 5H), 7.40-7.37 (m, 1H), 7.23-7.22 (m, 1H), 6.47-6.42 (m,
13
1H), 6.28-6.24(m, 1H), 5.75-5.73(m, 1H); C NMR (126 MHz, DMSO-d6) δ 163.7,

161.0, 158.1, 156.1 (q, J = 5.7 Hz), 141.8, 139.8, 139.0, 134.9 (2C), 134.8, 132.3, 129.1
(2C), 127.3, 125.2 (q, J = 269.6 Hz, CF3), 122.0, 118.5, 117.5, 116.8, 98.2 (q, J = 31.1
Hz, CCF3); HRMS (ESI) m/z calculated forC20H18BF3N5O3 [M+H]+ : 444.1455, found:
444.1465.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)amino)-5-
methoxypyrimidin-4-yl)amino)phenyl)acrylamide (14o).
Yield 46%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.09 (s, 1H), 9.08
(s, 1H), 8.82-8.80 (m, 2H), 7.96-7.89 (m, 3H), 7.53-7.45 (m, 4H), 7.31-7.28 (m, 1H),
6.48-6.42 (m, 1H), 6.27-6.23 (m, 1H), 5.76-5.73 (m, 1H), 4.81 (s, 2H), 3.88 (s, 3H);
13C NMR (126 MHz, DMSO-d6) δ [ppm] : 163.6, 155.6, 153.9, 152.3, 144.3, 140.0,
139.6, 137.1, 135.3, 132.4, 130.9, 129.1 (2C), 127.2, 118.2, 117.6, 114.9, 114.1, 109.7,
70.2, 57.3; HRMS (ESI) m/z calculated for C21H21BN5O4 [M+H]+: 418.1687, found:
418.1711.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)amino)-5-
methoxypyrimidin-4-yl)oxy)phenyl)acrylamide (14p).
Yield 48%; brown solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.36 (s, 1H), 9.54
(s, 1H), 8.84 (s, 1H), 8.26 (s, 1H), 7.69 (s, 1H), 7.60-7.58 (m, 1H), 7.52-7.40 (m, 3H),
7.22-7.20 (m, 1H), 6.99-6.97 (m, 1H), 6.46-6.40 (m, 1H), 6.28-6.24 (m, 1H), 5.78-5.75
13
(m, 1H),4.65 (s, 2H), 3.91 (s, 3H); C NMR (126 MHz, DMSO-d6) δ [ppm] : 163.8,
159.9, 155.5, 153.5, 153.3, 144.0, 143.6, 140.8, 135.7, 132.1, 130.9, 130.4, 127.8, 117.6,
117.5, 113.3, 112.8, 109.3, 100.0, 70.2, 57.9; HRMS (ESI) m/z calculated for
C21H20BN4O5 [M+H]+ : 419.1527, found: 419.1528.
N-(3-((2-(2-phenylhydrazinyl)-5-(trifluoromethyl)pyrimidin-4-
yl)amino)phenyl)acrylamide (14n).
A mixture of compound 12a (171 mg, 0.5 mmol), phenylhydrazine (65 mg, 0.6 mmol)
and potassium carbonate (276 mg, 2.0 mmol) in tert-butanol (10 mL) were placed in a
microwave tube. After nitrogen gas was bubbled through the mixture, 2-
dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (12 mg, 0.025 mmol) and
tris(dibenzylideneacetone)dipalladium (23 mg, 0.025 mmol ) were added to the mixture.
Again, after nitrogen gas was bubbled through the mixture, the reaction was carried out
o
at 120 C for 30 minutes in a microwave reactor. The reaction solution was filtered
through a bed of celite, and the filter cake was washed with a small amount of ethyl
acetate. After the filtrate was evaporated to dryness, the residue was purified by medium
pressure preparative chromatography to give compound 27 as a white solid (80 mg,

yield 38%). 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.06 (s, 1H), 9.20 (d, J = 17.4
Hz, 1H), 8.53-8.18 (m, 2H), 7.90 (s, 1H), 7.75-7.15 (m, 3H), 7.09 (t, J = 7.2 Hz, 2H),
6.97 (d, J = 3.7 Hz, 1H), 6.65 (s, 3H), 6.46 (dd, J = 16.8, 10.2 Hz, 1H), 6.28 (d, J = 16.9
Hz, 1H), 5.77 (d, J = 10.2 Hz, 1H); 13C NMR (126 MHz, DMSO-d6) δ [ppm] : 164.7,
163.6, 157.1, 156.6 (q, J = 5.0 Hz), 150.0, 138.9, 132.4, 129.1 (2C), 128.8, 127.3, 125.2
(q, J = 269.6 Hz, CF3), 123.7, 119.2, 118.7, 115.3, 114.6, 112.5 (2C), 97.7 (q, J = 34.0
Hz, CCF3); HRMS (ESI) m/z calculated for C20H18F3N6O [M+H]+ : 415.1494, found:
415.1507.
4.1.5 Preparation of Intermediates.
5-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzo[c][1,2]oxaborol-1(3H)-
ol (15b).
A solution of zinc dichloride (1M, 76 mL, 76 mmol) in diethyl ether was added to a
mixture of 2,4-dichloro-5-trifluoromethylpyrimidine (5.5 g, 25.4 mmol) in 1,2-
dichloroethane and tert-butanol (1:1, 300 ml). The resulting mixture was stirred at 0 °
C for 20 min. Intermediate 13b (4.2 g, 27.9 mmol) in a mixed solution of 1,2-
dichloroethane and tert-butanol (1:1, 50 ml) was slowly added to the reaction mixture.
After stirring for 10 minutes, triethylamine (2.8 g, 27.9 mmol) was then slowly added
dropwise. After the completion of the addition, the mixture was stirred at room
temperature for 16 hours, and then poured into ethyl acetate. The organic layer was
washed with water and brine, dried over anhydrous sodium sulfate, and filtered. After
the filtrate was evaporated to dryness, the residue was purified by medium pressure
preparative chromatography to give compound 15b as a brown solid (3.5 g, yield 42%).
1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.83 (s, 1H), 9.07 (s, 1H), 8.84 (s, 1H),
7.84 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 4.99 (s, 2H); 13C NMR
(126 MHz, DMSO-d6) δ 160.9, 158.6 (q, J = 4.8 Hz), 158.1, 155.5, 141.2, 131.4,
125.7, 123.4 (q, J = 270.9 Hz, CF3), 119.7, 112.8, 112.1 (q, J = 33.4 Hz, CCF3), 70.3.
MS (ESI): m/z 330.2 [M+H]+.
4.1.6 General procedure C for preparation of compounds 16a-d.
A mixture of compound 10 (1.1 eq) and 15 (1.0 eq) in ethanol was placed in a
microwave tube. The reaction was carried out at 100 oC for 30 minutes in a microwave
reactor. After the reaction solution was evaporated to dryness, the residue was purified
by medium pressure preparative chromatography to give the desired compounds.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide (16a).

Step 1. 6-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)oxy)benzo[c][1,2]oxaborol-
1(3H)-ol (15a).
Compound 15a was synthesized starting from intermediate 13k according to the
method to prepare compound 15b. A crude product was obtained and used in the next
step without further purification.
Step 2. The general procedure C was used to prepare compound 16a as a yellow solid
(140 mg, yield 9.4 % for two steps). 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.12
(s, 1H)，9.19 (s, 1H), 9.15 (s, 1H), 8.51 (s, 1H), 7.73 (s, 1H), 7.42-7.37 (m, 3H),
7.27(d, J = 2.5 Hz, 1H), 7.13-7.07 (m, 2H), 6.47-6.42 (m, 1H), 6.29-6.25(m, 1H), 5.76
(dd, J = 10.0, 2.0 Hz, 1H), 5.00 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ [ppm] :
166.6, 163.6, 158.8, 157.8 (q, J = 5.0 Hz), 152.0, 151.1, 139.4, 138.0, 132.4, 132.3,
128.7, 127.4, 125.1, 124.3 (q, J = 270.9 Hz, CF3), 123.4, 122.9, 120.9, 116.8, 116.6,
102.8 (q, J = 32.8 Hz, CCF3), 70.2; 19F NMR (471 MHz, DMSO-d6) δ [ppm] : -60.49;
HRMS (ESI) m/z calculated for C21H17BF3N4O4 [M+H]+ : 457.1295, found: 457.1314.
N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)oxy)phenyl)acrylamide (16b).
Yield 85%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.44 (br, 1H), 10.40
(s, 1H), 8.97 (br, 1H), 8.74 (s, 1H), 7.76 (s, 1H), 7.62-7.60 (d, J = 8.0 Hz, 1H), 7.53-
7.48 (m, 3H), 7.27 (s, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.46-6.41 (dd, J = 17.0, 10.0 Hz,
13
1H), 6.28-6.25 (d, J = 17.0 Hz, 1H), 5.79-5.77 (d, J = 10.0 Hz, 1H); C NMR (126
MHz, DMSO-d6) δ [ppm] : 166.8, 163.9, 162.8, 161.3, 158.3 (q, J = 5.0 Hz), 155.3,
152.5, 141.7, 140.9, 132.0, 131.0, 130.6, 127.9, 124.5, 124.0 (q, J = 269.6 Hz, CF3),
119.0, 117.6, 117.4, 113.2, 101.0 (q, J = 32.8 Hz, CCF3), 70.2; HRMS (ESI) m/z
calculated for C21H17BF3N4O4 [M+H]+ : 457.1295, found: 457.1327.
(E)-4-(dimethylamino)-N-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-
yl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)but-2-enamide (16c).
Step 1: The general procedure C was used to prepare 5-((4-((3-aminophenyl)amino)-5-
(trifluoromethyl)pyrimidin-2-yl)amino)benzo[c][1,2]oxaborol-1(3H)-ol (16c’).
Yield 70%; grey solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.01 (s, 1H), 8.94
(s, 1H), 8.43 (s, 1H), 7.78-7.65 (m, 1H), 7.54-7.48 (m, 1H), 7.48-7.26 (m, 3H), 7.10 (s,
1H), 4.77 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ [ppm] : 167.0, 160.6, 157.8, 156.0
(q, J = 5.0 Hz), 155.5, 147.6, 142.5, 139.9, 135.6, 131.0, 130.0, 125.1 (q, J = 269.6 Hz,
CF3), 118.8, 112.4, 111.4, 100.0, 97.3 (q, J = 32.0 Hz, CCF3), 70.3. MS (ESI): m/z
402.2 [M+H]+.
Step 2: A mixture of compound 16c' (324 mg, 0.81 mmol) and trans-4-dimethylamino

crotonate (174 mg, 1.1 mmol) in DMF (5 mL), DIPEA (423 ul, 2.4 mmol) and HATU
(461 mg, 1.2 mmol) were stirred for 1 hour at room temperature. The reaction mixture
was diluted with ethyl acetate (20 mL). The organic phase was washed with water,
saturated brine, dried over anhydrous sodium sulfate, and filtered. After the filtrate
was evaporated to dryness, the residue was purified by medium pressure preparative
chromatography to give the desired compound 16c as a white solid (140 mg, yield
34%). 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 10.27 (s, 1H), 9.90 (s, 1H), 8.91-8.87
(m, 2H), 8.39 (s, 1H), 7.83 (s, 1H), 7.68-7.62 (m, 2H), 7.47-7.45 (m, 1H), 7.40-7.35
(m, 2H), 7.12-7.10 (m, 1H), 6.75-6.69 (m, 1H), 6.34-6.30 (m, 1H), 4.67 (s, 2H), 3.17
(s, 2H) , 2.25 (s, 6H); 13C NMR (126 MHz, DMSO-d6) δ [ppm] : 163.6, 160.9, 158.2,
156.0 (q, J = 5.0 Hz), 155.3, 142.6, 140.6, 140.1, 139.1, 130.9, 130.1, 129.2, 127.3,
125.2 (q, J = 269.6 Hz, CF3), 123.5, 122.0, 118.8, 116.8, 111.3, 98.3 (q, J = 32.0 Hz,
CCF3), 70.2, 59.8, 45.1 (2*C); HRMS (ESI) m/z calculated for C24H25BF3N6O3
[M+H]+ : 513.2033, found: 513.2044.
1-(3-((2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-5-yl)amino)-5-
(trifluoromethyl)pyrimidin-4-yl)amino)piperidin-1-yl)prop-2-en-1-one (16d).
Yield 35%; white solid; 1H NMR (500 MHz, DMSO-d6) δ [ppm] : 9.85 (s, 1H), 8.97-
8.94 (m, 1H), 8.27 (s, 1H), 7.93-7.85 (m, 1H), 7.59-7.52 (m, 2H), 6.89-6.63 (m, 2H),
6.16-5.97 (m, 1H), 5.74-5.53 (m, 1H), 4.86 (s, 2H), 4.32-4.26 (m, 2H), 3.97 (d, J
=11.0 Hz, 1H), 3.25-2.74 (m, 2H), 1.94 (s, 1H), 1.85-1.76 (m, 2H), 1.44 (d, J = 9.0
Hz, 1H); 13C NMR (126 MHz, DMSO-d6) δ [ppm] : 165.2, 161.2, 158.0, 155.4 (q, J =
5.0 Hz), 142.7, 131.1, 128.9, 128.0, 127.5, 125.3 (q, J = 269.6 Hz, CF3), 119.1, 111.8,
111.7, 97.9 (q, J = 32.0 Hz, CCF3), 70.4, 49.5, 47.5, 46.0, 25.4, 24.3; HRMS (ESI)
m/z calculated for C20H22BF3N5O3 [M+H]+ : 448.1768, found: 448.1801.
4.2 Biological studies
4.2.1. Kinase enzymatic assay.
Recombinant histidine-tagged human BTK enzymes was obtained from Drug
Discovery Support Business Division of ThermoFisher Scientific, Inc.
4.2.2.Measurment of BTK inhibitory activity.
Measurement of BTK activity was carried out using LANCE Ultra KinaSelectTM
Kit (LANCE Ultra KinaSelectTM Kit, PerkinElmer, Inc.). Firstly, we optimized the
LanthaScreen™kinase assay for BTK (PerkinElmer, USA) according to the
manufacture's specifications. TR-FRET assays were performed by incubating a dilution

series of compound concentrations with ATP (Sigma), LANCE Ultra ULight-Poly GT
substrate (PerkinElmer) and BTK Kinase (ThermoFisher Scientific) in kinase reaction
buffer (PerkinElmer).The kinase reaction buffer consisted of 50 mM HEPES pH 7.5,
10 mM MgCl2, 2mM DTT, 1 mM EGTA and 0.01% Tween 20. The kinase reaction
mixtures were incubated at room temperature (23±2℃) for 2h before stopping the
kinase reaction by the addition of 10 mM EDTA . The phosphorylation of the substrate
by BTK was detected using Eu-PT66 antibody (PerkinElmer) in TR-FRET LANCETM
Detection Buffer at pH 7.5 (PerkinElmer) and then incubated at room temperature
(23±2℃) for 1h, finally measured by determining the emission ratio of 665/615 nm on
a microplate reader (EnVison, PerkinElmer). IC50 was estimated using the log(inhibitor)
vs. response non-linear fit (GraphPad Prism 6.0). Additional assays were similarly
carried out to determine selectivity over JAK1/2/3 and TYK2 (Carna Biosciences, Inc).
4.2.3. Cell viability assay
4.2.3.1 Effect of compound on Raji cell viability
The Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability after
treatment with different doses of compound. Cells were collected and counted with Cell
Counter, and then they were seeded in 96-well plates at 8 x 104cells/well and allowed
to incubate overnight. Then the compounds were added with Nanoliter pipetting
instrument. After another 72 hours, ten microliters of CCK-8 solution were added to
each culture well and incubated for 2 hours. The absorbance (OD) of each group was
measured with a multifunctional microplate reader (Envision) at a wavelength of 450
nm. The proliferation of Raji cells was observed. Cell proliferation inhibition rate (%)
= (control group OD value - experimental group OD value)/(control group OD value -
blank group OD value) × 100% and calculated IC50 with Graphpad Prism.
4.2.3.2 Effect of compound on Ramos cell viability
The Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability after
treatment with different doses of compound. Cells were collected and counted with Cell
Counter, and then they were seeded in 96-well plates at 5 x104cells/well and allowed to
incubate overnight. Then the compounds were added with Nanoliter pipetting
instrument. After another 72 hours, ten microliters of CCK-8 reagents was added to

each culture well and incubated for 2 hours. The absorbance (OD) of each group was
measured with a multifunctional microplate reader (Envision) at a wavelength of 450
nm. The proliferation of Ramos cells was observed. Cell proliferation inhibition rate
(%) = (control group OD value - experimental group OD value)/(control group OD
value - blank group OD value) × 100% and calculated IC50 with Graphpad Prism.
4.2.3.3 Effect of compound on HEL cell viability
The Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability after
treatment with different doses of compound. Cells were collected and counted with Cell
Counter, and then they were seeded in 96-well plates at 7.5 × 104cells/well and allowed
to incubate overnight. Then the compounds were added with Nanoliter pipetting
instrument. After another 72 hours, ten microliters of CCK-8 reagents were added to
each culture well and incubated for 2 hours. The absorbance (OD) of each group was
measured with a multifunctional microplate reader (Envision) at a wavelength of 450
nm. The proliferation of HEL cells was observed. Cell proliferation inhibition rate (%)
= (control group OD value - experimental group OD value)/(control group OD value -
blank group OD value) × 100% and calculated IC50 with Graphpad Prism.
4.2.3.4 Effect of compound on Jeko-1 cell viability
The Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability after
treatment with different doses of compound. Cells were collected and counted with Cell
Counter, and then they were seeded in 96-well plates at 7.5 × 104cells/well and allowed
to incubate overnight. Then the compounds were added with Nanoliter pipetting
instrument. After another 72 hours, ten microliters of CCK-8 solution was added to
each culture well and incubated for 2 hours. The absorbance (OD) of each group was
measured with a multifunctional microplate reader (Envision) at a wavelength of 450
nm. The proliferation of Jeko-1 cells was observed. Cell proliferation inhibition rate (%)
= (control group OD value - experimental group OD value)/(control group OD value -
blank group OD value) × 100% and calculated IC50 with Graphpad Prism.
4.2.3.5 Effect of compound on OCI-LY10 cell viability
Diffuse large B-cell lymphoma cells OCI-LY10 was cultured and stored in liquid
nitrogen Department of Cancer and Endocrinology, College of Pharmacy, Zhejiang

University. Cells were collected and counted with Cell Counter, and then they were
seeded in 96-well plates and allowed to incubate overnight. Then the compounds were
treated with a dose range from 0.0001 μM - 10μM. After another 72 hours, the
proliferation of OCI-LY10 in vitro was measured by Celltiter. The absorbance (OD) of
each group was measured with a multifunctional microplate reader (Envision) at a
wavelength of 450 nm. Cell proliferation inhibition rate (%) = (control group OD value
- experimental group OD value)/(control group OD value - blank group OD value) ×100%
and calculated IC50 with Graphpad Prism.
4.2.4. Human liver microsome stability test
Human liver microsomes were obtained from BD Gentest. A typical standard
reaction mixture 300 µL consisted of the pooled liver microsomes 0.2 mg/mL, 1 mM
NADPH, 5 mM MgCl2, 100 mM potassium phosphate buffer (pH 7.4) and 0.2 µM of
test compounds. After a 5-min pre-incubation at 37 °C, the reactions were initiated by
addition of NADPH and incubation proceeded for 5, 15, 30, 60 min at 37 °C in a
shaking metal bath. The reaction was stopped by transferring 60 µL aliquots to the tubes
on ice and adding 120 µL amounts of ice-cold acetonitrile containing internal standards.
Concentration of the test compounds was measured by UPLC-MS/MS.
4.3 Molecular docking
Molecular docking study was performed by using Molecular Operating
Environment (MOE v2018.01). Receptors [BTK (PDB ID: 5P9J) and JAK3 (PDB ID:
5TOZ)] were prepared through QuickPrep protocol in MOE. Ligands were protonated
at pH 7 followed by conformation searching. The lowest energy conformation of each
Ligand was maintained and covalently docked into the ATP binding site of receptor
through specified Cysteine (Cys481 for BTK and Cys356 for JAK3). The GBVI/WSA
dG score was selected to evaluate docking poses. The docking models were analyzed
and generated by MOE.
Acknowledgements
The authors would like to thank Jie Wang, Yuhan Li and Jianghao Ma for their
generous help on Mass and NMR analysis.
Conflict of interest
None of the authors of the above manuscript has declared any conflict of interest