Design, synthesis and biological evaluation of 7H-pyrrolo[2,3-d]pyrimidin-4-amine derivatives as selective Btk inhibitors with improved pharmacokinetic properties for the treatment of rheumatoid arthritis

Article abstract of DOI:10.1016/j.ejmech.2017.12.079

Bruton's tyrosine kinase (Btk) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) and Fcγ receptor (FcR) signaling pathways, which makes it a uniquely attractive target for the treatment of autoimmune diseases, such as rheumatoid arthritis (RA). We reported a series of compounds bearing 7H-pyrrolo [2,3-d]pyrimidin-4-amine scaffold that potently inhibited Btk in vitro. Analysis of the structure-activity relationships (SAR) and drug-like profiles led to the discovery of the optimal compound B16. B16 preferentially inhibited Btk (IC₅₀ = 21.70 ± 0.82 nM) over closely related kinases with moderate selectivity. Cell-based tests also confirmed that B16 significantly inhibited Btk Y223 auto-phosphorylation and PLCγ2 Y1217 phosphorylation. MTT revealed that B16 displayed weak suppression against normal LO2, HEK293 and THP-1 cell lines with IC₅₀ values over 30 μM. Moreover, B16 showed very weak potential to block the hERG channel (IC₅₀ = 11.10 μM) in comparison to ibrutinib (IC₅₀ = 0.97 μM). Owing to its favorable physicochemical properties (ClogP = 2.53, aqueous solubility ≈ 0.1 mg/mL), pharmacokinetic profiles (F = 49.15%, t_1/2 = 7.02 h) and reasonable CYP450 profile, B16 exhibited potent anti-arthritis activity and similar efficacy to ibrutinib in reducing paw thickness in CIA mice. In conclusion, B16 is a potent, selective and durable inhibitor of Btk and has the potential to a safe and efficacious treatment for arthritis.

Full text of DOI:10.1016/j.ejmech.2017.12.079

Accepted Manuscript
Design, synthesis and biological evaluation of 7H-pyrrolo[2,3-d]pyrimidin-4-amine
derivatives as selective Btk inhibitors with improved pharmacokinetic properties for
the treatment of rheumatoid arthritis
Linhong He, Heyin Pei, Chufeng Zhang, Mingfeng Shao, Dan Li, Mingli Tang, Taijing
Wang, Xiaoxin Chen, Mingli Xiang, Lijuan Chen
PII:
S0223-5234(17)31110-8
10.1016/j.ejmech.2017.12.079
EJMECH 10057
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 August 2017
Revised Date: 22 December 2017
Accepted Date: 22 December 2017
Please cite this article as: L. He, H. Pei, C. Zhang, M. Shao, D. Li, M. Tang, T. Wang, X. Chen,
M. Xiang, L. Chen, Design, synthesis and biological evaluation of 7H-pyrrolo[2,3-d]pyrimidin-4-
amine derivatives as selective Btk inhibitors with improved pharmacokinetic properties for the
treatment of rheumatoid arthritis, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.12.079.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of
7
H-pyrrolo[2,3-d]pyrimidin-4-amine derivatives as selective Btk inhibitors with
improved pharmacokinetic properties for the treatment of rheumatoid arthritis
1
,*
1,*
1,*
1
1
Linhong He , Heyin Pei , Chufeng Zhang , Mingfeng Shao , Dan Li , Mingli
1
1
2
1
1,#
Tang , Taijing Wang , Xiaoxin Chen , Mingli Xiang , Lijuan Chen
1
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University and Collaborative Innovation Center. Chengdu, Sichuan 610041,
PR China.
2
Guangdong Zhongsheng Pharmaceutical Co., Ltd., Dongguan, Guangdong 523325,
China.
*
There authors contributed equally to this work.
#
Corresponding Author
Lijiuan Chen
Tel: +86 28 85164063.
Fax: +86 28 85164060.
E-mail address: chenlijuan125@163.com (L-J. Chen)

ACCEPTED MANUSCRIPT
Abstract: Bruton’s tyrosine kinase (Btk) is a Tec family kinase with a well-defined
role in the B cell receptor (BCR) and Fcγ receptor (FcR) signaling pathways, which
makes it a uniquely attractive target for the treatment of autoimmune diseases, such as
rheumatoid arthritis (RA). We reported a series of compounds bearing
7
H-pyrrolo[2,3-d]pyrimidin-4-amine scaffold that potently inhibited Btk in vitro.
Analysis of the structure-activity relationships (SAR) and drug-like profiles led to the
discovery of the optimal compound B16. B16 preferentially inhibited Btk (IC50
1.70 ± 0.82 nM) over closely related kinases with moderate selectivity. Cell-based
=
2
tests also confirmed that B16 significantly inhibited Btk Y223 auto-phosphorylation
and PLCγ2 Y1217 phosphorylation. MTT revealed that B16 displayed weak
suppression against normal LO2, HEK293 and THP-1 cell lines with IC values over
5
0
3
0 µM. Moreover, B16 showed very weak potential to block the hERG channel (IC50
11.10 µM) in comparison to ibrutinib (IC = 0.97 µM). Owing to its favorable
=
5
0
physicochemical properties (ClogP = 2.53, aqueous solubility ≈ 0.1 mg/mL),
pharmacokinetic profiles (F = 49.15%, t1/2 = 7.02 h) and reasonable CYP450 profile,
B16 exhibited potent anti-arthritis activity and similar efficacy to ibrutinib in reducing
paw thickness in CIA mice. In conclusion, B16 is a potent, selective and durable
inhibitor of Btk and has the potential to a safe and efficacious treatment for arthritis.
Key words: Btk, inhibitor, selectivity, bioavailability, aqueous solubility, rheumatoid
arthritis

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1
. Introduction
Bruton tyrosine kinase (Btk) is a member of Tec family kinases including Tec,
Itk, Txk and Bmx [1-4]. Btk is one of the most important drug target, since it broadly
expresses in all hematopoietic cells except of plasma and T cells, and plays a critical
role in B cell receptor (BCR) and Fcγ receptor (FcγR) signaling pathways, where it
regulates the activation and proliferation of B cells [5-6]. Upon activation by
upstream kinases (such as Lyn and Syk), Btk subsequently phosphorylates and
activates phospholipase-Cγ (PLCγ), leading to calcium ion transportation and
activation of NF-κB and MAP kinase pathways [1-6]. Thus, Btk is involved in
pro-inflammatory signals and regulates the expression of pro-inflammatory cytokines,
chemokines and cell adhesion molecules. Taken together, Btk’s role in these pathways
makes it a uniquely attractive target for the treatment of B cell related diseases, such
as the autoimmune disorder rheumatoid arthritis (RA) [1-6].
RA is an autoimmune disease that involves systemic dysregulation of T and B
lymphocytes with immune responses against self-antigens, and ultimately resulting in
circulating autoantibodies, synovial inflammation, pannus formation, and cartilage
and bone destruction in affected joints [7-8]. B cell function and autoantibody
production have been proven to promote the progression of RA [1,7-8]. The treatment
of RA with anti-CD20 antibody rituximab to deplete mature B cells in clinical studies,
clearly demonstrates that therapies directly or indirectly targeting B cells have the
potential to attenuate disease progression of RA [9-10]. Pharmacological and genetic
studies in animal models also verify Btk inhibitors can be efficacious against RA [3-6].
Moreover, RANK signaling is Btk dependent and controls osteoclastogenesis from
monocytic precursors [5,6]. Therefore, Btk has been identified as a potent target for
the treatment of RA. During recent years, more and more Btk inhibitors have been
developed and demonstrated efficacies in preclinical animal models for RA (ibrutinib,
RN486, CGI1746 and so on ) or entered into clinical trials for RA (CC-292 and
HM71224) (Figure 1) [9-11].

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Figure 1. Btk inhibitors in development for RA
Until now, the most successful Btk inhibitor is ibrutinib, which has been
clinically used for the treatment of mantle cell lymphoma (MCL), chronic lymphatic
lymphoma (CLL), and waldenström's macroglobulinemia (WM) [12-13]. Owing to its
excellent inhibition to Btk, ibrutinib not only efficiently reverses arthritic
inflammation in a therapeutic collagen induced arthritis (CIA) models, but also
prevents clinical arthritis in collagen antibody-induced arthritis (CAIA) models,
indicating its potential use for the treatment of RA [14-15]. However, ibrutinib can
also strongly inhibit numerous kinases with structural homology, such as EGFR,
JAK3, and ErbB2 [12,14,16], which can lead to significant toxicities, including
bleeding, myelosuppression, diarrhea, atrial fibrillation (AF) and renal impairment
[
16-19]. Potent inhibition of EGFR by ibrutinib may explain the observed diarrhea,
which has been observed in clinical practices with EGFR inhibitors [19]. The
moderate inhibition against hERG current (IC = 0.97 µM) by ibrutinib is considered
5
0
as a low-potency blocker and closely associated with AF [18,20]. RA, quite different
from hematological malignancies, is a non-life-threatening disease that calls for safe

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therapies with unexpected off-target toxicities [13]. This has encouraged the
development of more selective Btk inhibitors, including GDC-0834 and RN-486, but
GDC-0834 was suspended in phase I because of its poor pharmacokinetics (PK)
properties [13,21,22]. Hence, developing highly selective Btk inhibitors with
favorable PK properties will be more suitable and efficacious to fulfill the treatment
need for RA disease [13,21,22].
Encouraged by the excellent anti-arthritis activity of ibrutinib, we decided to
optimize the structure of ibrutinib in an attempt to improve its kinase selectivity,
toxicity and PK profiles. Given that the scaffolds of Btk inhibitors are major
representative structures [23,33], and replacing the central core of ibrutinib with
6
-amino-7H-purin-8(9H)-one (ONO-4059) maintains the potency of inhibiting Btk
[
19], we selected 7H-pyrrolo[2,3-d]pyrimidin-4-amine as the new scaffold to replace
the pharmacophore of ibrutinib by employing the theory of bioisostere. As illustrated
in Figure 2, similar to ibrutinib, 7H-pyrrolo[2,3-d]pyrimidin-4-amine scaffold can
also occupy the ATP binding pocket and make several important hydrogen bond
interactions with respective Thr474, Glu475 and Met477 in the hinge.
4
-Piperidinylmethyl was selected as a linker to connect the scaffold and electrophilic
warheads, which allowed acrylamide to form a covalent bond with the conserved
Cys481 residue in Btk domain. As expected, the phenoxyphenyl group of B1 was
overlaid with the same fragment of ibrutinib, which completely entered the
hydrophobic pocket and formed a face-to-edge π-stacking interaction with Phe540. In
our effort to improve the ClogP, aqueous solubility, as well as PK profiles, structural
modifications on C3-position of pyrryl was firstly performed. In the further research
of exploring the structure-activity relationships (SAR) of this series derivatives and
hunting for more potent Btk inhibitors, structural modifications were also conducted
on linker, scaffold and electrophilic warhead region. Herein, we report the synthesis
and pharmacological evaluation of these novel compounds as potent Btk inhibitors, of
which the representative compound B16 exhibits excellent kinase selectivity to Btk,
low toxicity, favorable PK properties and good efficacy for the treatment of RA

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though oral administration.
Figure 2. Design of the 7H-pyrrolo[2,3-d]pyrimidin-4-amines as Btk inhibitors. A
covalent docking model: similar to ibrutinib, compound B1 also can form a covalent
bind with the conserved cys481 residue in Btk domain (ibrutinib: orange; B1: green).
2
. Compounds synthesis
The synthesis route of compounds B1-B24 is described in Scheme 1. By
reference to the preparation of ibrutinib, the starting material 1-1 was directly coupled
with 4 by Mitsunobu reaction in an extremely low yield. In order to improve the
chemical reactivity of 1-1, it was treated with methanesulfonyl chloride in an ice bath
and subsequently with lithium bromide under reflux overnight, giving the important
intermediate 3 that allowed completely reaction with 4 to offer 5 in a good yield. 5
was reacted with ammonia (con.) in an autoclave to give 6, then coupled with a
variety of boronic acid derivatives via Suzuki-Miyaura cross-coupling procedure to
provide 7. Intermediate 8 was obtained by removing the Boc group from 7 in an acid
condition. Finally, the classical electrophilic warhead acryloyl group was introduced
to 8 giving final products B1-B24.
Scheme 1. Preparation of compounds B1-B24.

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Reactions and conditions: (a) MsCl, Et N, DCM, 0 ºC, 2 h; (b) LiBr, ACE, reflux,
3
overnight; (c) K CO , DMF, 80 ºC, 3-4 h; (d) NH OH, 1,4-dioxane, 98 ºC, overnight;
2
3
4
(
e) boronic acid (R ), K CO , PdCl (dppf), 1,4-dioxane/H O, 98 ºC, 3-4 h; (f)
1 2 3 2 2
1
,4-dioxane, HCl in 1,4-dioxane, 50 ºC, overnight; (g) acryloyl chloride, DIEA, dry
DMF, r.t., 2 h.
To search the SAR of linker connecting 7H-pyrrolo[2,3-d]pyrimidin-4-amine and
electrophilic warheads, we synthesized another three kinds of analogues containing
favorable side chains of 4-phenoxyphenyl or benzo[d][1,3]dioxol-5-yl on the scaffold.
Target compounds B25-B26 were prepared according to the same procedures in
Scheme
1
from
the
starting
material
of
(R)-tert-butyl-3-(hydroxymethyl)piperidine-1-carboxylate (1-2), while B27-B28 and
B29-B30
were
obtained
from
(S)-tert-butyl-3-
(hydroxymethyl)piperidine-1-carboxylate (1-3) and tert-butyl-4-(2-hydroxyethyl)
piperidine-1-carboxylate (1-4), respectively.
Scheme 2. Preparation of compounds B25-B30.

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On the basis of the potent compounds B1 and B16, analogues B31-B34 also had
been synthesized to evaluate the functions of electrophilic warheads, and their
synthesis routes were shown in Scheme 3. Intermediate 8-1 was treated with
2
-chloroethanesulfonyl chloride in anhydrous dichloromethane giving B31, while B33
was obtained in the same way from 8-16. Another two compounds B32 and B34 were
provided by coupling (E)-4-(dimethylamino)but-2-enoic acid with respective amides
in low yields (< 35%).
Scheme 3. Preparation of compounds B31-B34
Reactions and conditions: (a) B31 and B33, 2-chloroethanesulfonyl chloride, DIEA,
dry DCM, r.t., 1 h; B32 and B34, (E)-4-(dimethylamino)but-2-enoic acid
hydrochloride, DIEA, PyBOP, DCM, r.t., overnight.
Then we focused on the structural modifications on the scaffold region. As

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described in Scheme 4, compounds bearing 1H-pyrazolo[3,4-d]pyrimidin-4-amine,
the scaffold of ibrutinib, were also prepared. Commercially available material
1
H-pyrazolo[3,4-d]pyrimidin-4-amine (9) was reacted with n-iodosuccinimide (NIS)
to offer 10, and the following reactions were similar as in Scheme 1. Eventually, we
got new compounds B35-B38.
Scheme 4. Preparation of compounds B35-B38
Reactions and conditions: (a) NIS, DMF, r.t., 2 h; (b) K CO , DMF, 80 ºC, 3-4 h; (c)
2
3
K CO , PdCl (dppf), 1,4-dioxane/H O, 98 ºC, 3-4 h; (d) 1,4-dioxane, HCl in
2
3
2
2
1
,4-dioxane, 50 ºC, overnight; (e) B35 and B37, acryloyl chloride, DIEA, dry DMF,
r.t., 2 h; B36 and B38, 2-chloroethanesulfonyl chloride, DIEA, dry DCM, r.t., 1 h.
3
. Result and discussion
3
.1 In vitro potency
All the synthesized compounds were assessed the inhibitory activities against
Btk at the concentrations of 1 µM and 0.1 µM, and the results were summarized in
Table 1. As expected, compound B1 with 4-phenoxyphenyl substitution, structurally
similar to ibrutinib, displayed a strong Btk inhibition potency. The potency of B1
slightly decreased by contrast to ibrutinib. (Figure 2). Compounds with
4
-(benzyloxy)phenyl (B2) and 4-(3,4-difluorobenzyloxy)phenyl (B24) substitutions
decreased the potencies of inhibiting Btk, which may due to their large substituent

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groups interfered in forming π-stacking interaction. The 4-(pyridin-2-yloxy)phenyl
(B3) substitution also could not maintain excellent potency that probably attributed to
its lower hydrophobicity than B1. The 4-fluorophenyl analogue (B7) was more potent
than 3-chlorophenyl (B6) substitutions. Nevertheless, the potencies decreased as the
volume of substituent groups increased (B8 and B9). Similar trend was observed on
4
-hydroxyphenyl compound (B12) and its derivatives (B13, B14 and B15).
Surprisingly, compound B16 substituted with benzo[d][1,3]dioxol-5-yl exhibited good
inhibition to Btk, but other fused heterocyclics (B18, B21 and B22) slightly decreased
the efficacies of inhibiting Btk. Compounds with polar groups on phenyl, such as B4,
B11 and B19, dramatically lost in activities might be attributed to the missing
π-stacking and hydrophobic interactions in the hydrophobic pocket. In brief,
compounds B1 and B16 were worth further structural optimizations.
a
Table 1 The structure of B1-B24 and their activities in enzymatic assay
Btk Mean_inh%
µM 0.1 µM
Btk Mean_inh%
Comp.
Ibruti
R1
Comp.
B13
R1
1
1 µM
0.1 µM
-
99.6 ± 0.1 99.3 ± 0.5
97.6 ± 0.0 94.1 ± 0.3
91.0 ± 1.4 54.0 ± 2.6
89.7 ± 0.2
39.5 ± 0.2
-nib
B1
B2
B14
B15
87.4 ± 0.21 35.8 ± 0.52
84.3 ± 0.5
32.6 ± 0.8

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B3
B4
B5
B6
90.0 ± 0.1 45.3 ± 0.5
B16
B17
B18
B19
98.3 ± 1.1
73.5 ± 20.5
88.6 ± 1.5
34.9 ± 3.8
82.4 ± 0.2
0.6 ± 4.4
8.3 ± 0.4
6.5 ± 4.4
43.1 ± 4.6 12.5 ± 6.5
73.3 ± 0.4 6.5 ± 4.4
31.7 ± 1.1
12.4 ± 1.6
B7
B8
92.8 ± 0.9 50.3 ± 1.5
52.8 ± 0.4 9.2 ± 4.1
73.6 ± 1.4 16.0 ± 0.5
94.2 ± 0.5 47.4 ± 6.0
34.0 ± 1.9 14.7 ± 2.0
94.9 ± 0.0 48.5 ± 1.1
B20
B21
B22
B23
B24
73.3 ± 0.9
62.4 ± 4.0
78.2 ± 1.5
16.6 ± 1.6
52.7 ± 5.7
18.4 ± 5.4
14.9 ± 2.6
17.0 ± 2.7
4.7 ± 4.7
B9
B10
B11
B12
10.6 ± 1.2

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a
Enzymatic assay is performed in the presence of ATP concentration at K 90 µM.
m
Mean_inh% represents Mean ± SD from at least two independent experiments.
In order to investigate the SAR of linker, we further synthesized
3
-piperidinylmethyl analogues, including R-enantiomers (B25 and B26) and
S-enantiomers (B27 and B28). Unfortunately, neither of them displayed better
potencies of inhibiting Btk than the corresponding 4-piperidinylmethyl analogues
except B27 (Table 2). Unlike the R enantiomer of ibrutinib, B27 is S enantiomer and
slightly decreased the activity in contrast to ibrutinib. Another two 4-piperidinoethyl
derivatives, B29 and B30, were also less potent than the corresponding
4
-piperidinylmethyl ones.
Common electrophilic warheads (E)-2-(4-(dimethylamino)but-2-vinyl and
vinylsulfonyl were respectively introduced to the bottom piperidyl in an attempt to
increase the inhibitory activity to Btk. In comparison to vinyl carbonyl, vinylsulfonyl
substitution (B31 and B33) did some improvement in activity, implying that sulfuryl
can form a stronger covalent bond with cysteine than vinyl carbonyl. Unfortunately,
the modification on vinyl carbonyl (B32 and B34) resulted in different degrees of loss
in activities, and B34 even totally lost the potency of inhibiting Btk, of which
N,N-dimethyl substitution might interfere vinyl carbonyl to form a covalent bond with
cysteine. It could be simply summarized that most compounds with
benzo[d][1,3]dioxol-5-yl substitution on C3-position of pyrryl exhibited less
potentials of inhibiting Btk than the 4-phenoxyphenyl analogues.
a,b,c
Table 2. In vitro potencies of selected compounds
aqueous
aqueous
solubility
Btk
Btk
b
b
Comp.
ClogP
solubility
Comp.
ClogP
a
a
IC /nM
IC /nM
5
0
50
c
c
(
mg/mL)
(mg/mL)
ibrutinib
B1
0.81 ± 0.11
6.09 ± 1.45
77.04 ± 0.73
4.07
4.66
4.25
< 0.01
B28
B29
B30
500.21 ± 1.01
36.83 ± 0.93
597.81 ± 1.24
2.53
5.19
3.06
≈ 0.1
< 0.01
< 0.01
< 0.01
< 0.1
B2

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B16
B25
B26
B27
21.70 ± 0.82
7.34 ± 0.92
83.55 ± 0.91
1.71 ± 1.54
2.53
4.66
2.53
4.66
≈ 0.1
< 0.01
≈ 0.1
B31
B32
B33
B34
4.24 ± 2.05
25.64 ± 1.03
1.32 ± 1.20
4.70
4.98
2.57
2.84
< 0.01
< 0.01
≈ 0.1
< 0.01
2369.28 ± 1.01
< 0.1
a
IC values of Btk are an average of at least three independent dose-response curves
5
0
b
at K ATP (5 nM Btk and 90 µM ATP), mean ± SD. ClogP values are calculated by
m
c
ChemBioDraw software Ultra 12.0. The solubility was measured by a method as
follows: 1.0 mg of the appropriated compound dissolved in 1.0 mL of water at
ambient temperature, continuous another 1.0 mL of water was added until the
compound was completely dissolved.
In order to explain the different enzymatic potencies of those compounds
respectively substituted with benzo[d][1,3]dioxol-5-yl and 4-phenoxyphenyl on the
C3-position of pyrryl and find out their interactions, compounds B1 and B16 were
selected to dock into Btk protein domain using the program Covalent Dock Clound.
As illustrated in Figure 3A, similar to B1 (Figure 2), B16 maintained the potential to
form three key hydrogen bond interactions with respective Thr474, Glu475 and
Met477 in the hinge, and a covalent bond with Cys481. Although the carbony on B16
was clearly observed to form another H-bond with Asn484, this interaction was lower
than the π-stacking interaction with Phe540 in comparison to ibrutinib. The analysis
of hydrophobic interactions between Btk protein and selected compounds explained
that the slight potency decrease of B16 was not only attributed to its weaker
hydrophobicity of benzo[d][1,3]dioxol-5-yl than phenoxyphenyl group on B1 or
ibrutinib, but also not completely filled of the hydrophobic pocket.

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Figure 3. Selected compounds docked into Btk kinase domain. (A): Analysis of
compound B16 and ibrutinib binding into Btk kinase domain (B16: purple; ibrutinib:
orange). (B): The analysis of hydrophobic interaction between Btk protein and
selected compounds B1, B16 and ibrutinib (B1: green; B16: purple; ibrutinib:
orange).
Taken the docking results together, the scaffold of ibrutinib was found to form
one more hydrogen bond interaction with water molecules than the pharmacophore of
B1 and B16. On the basis of this finding, we had synthesized compounds B35-B38
bearing the scaffold of ibrutinib. As expected, compounds B35 and B36 slightly
increased the potencies of inactivating Btk in comparison to the corresponding
compounds B1 and B31. However, on the contrary, benzo[d][1,3]dioxol-5-yl
analogues, B37 and B38, were both weaker to inhibit Btk than B16 and B33,
respectively.
a,b,c
Table 3. In vitro potencies of selected compounds
aqueous
aqueous
solubility
Btk
Btk
b
b
Comp.
ClogP
solubility
Comp.
ClogP
a
a
IC /nM
IC /nM
5
0
50
c
c
(
mg/mL)
(mg/mL)
ibrutinib
B35
0.80 ± 0.07
2.46 ± 1.57
1.79 ± 1.56
4.07
4.08
4.04
< 0.01
B37
B38
70.70 ± 1.14
3.22 ± 1.31
1.91
1.95
≈ 0.1
< 0.01
< 0.01
≈ 0.1
B36

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a
IC values of Btk are an average of at least three independent dose-response curves
5
0
b
at K ATP (5 nM Btk and 90 µM ATP), mean ± SD. ClogP values are calculated by
m
c
ChemBioDraw software Ultra 12.0. The solubility was measured by a method as
follows: 1.0 mg of the appropriated compound dissolved in 1.0 mL of water at
ambient temperature, continuous another 1.0 mL of water was added until the
compound was completely dissolved.
It is well known that, molecules with suitable CLogP values and aqueous
solubilities will have more chances to own good PK profiles for oral administration
and enhance potencies in vivo [24-25]. Smith C.R. et al also claimed that CLogP
values ranging from 0.5 to 3 were the best for oral administration [26]. Thus, we
calculated ClogP values for selected compounds by ChemBioDraw software Ultra
1
2.0, and determined their solubilities in water at ambient temperature. Similar to
ibrutinib, compounds substituted with 4-phenoxyphenyl (B1, B25, B27, B29, B31,
B32, B35 and B36) or 4-(benzyloxy)phenyl (B2) have high ClogP values up to 4,
along with poor aqueous solubilities less than 0.01 mg/mL, which may result in
unfavorable bioavailability. In our previous studies, candidate compounds with
excellent enzymatic potencies but low aqueous solubilities or poor bioavailabilities
had demonstrated that they could not enter into further in vivo tests or achieve ideal
treatment outcomes in animal models. Satisfying our aim for developing this series
compounds, benzo[d][1,3]dioxol-5-yl derivatives, such as B16, B28, B37 and B38,
exhibited proper ClogP values (1.91~3.06) and aqueous solubilities (≈ 0.1 mg/mL)
meeting with the concept of drug-like physical chemical criteria, which promised
favorable PK properties to enhance the potencies in vivo.
3
.2 Cytotoxicity assay
Cytotoxicity to normal cells is often used to evaluate the preliminary toxicities of
compounds. Since one of the most important side effects of ibrutinib is renal toxicity,
the inhibitory activities against HEK293 cells for selected compounds were assessed

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using MTT assay. As shown in Table 4, compounds with vinylsulfonyl (B31, B33,
B36 and B38) as electrophilic warhead, as well as B25 and B27, exhibited high
toxicities to HEK293 cells with IC values below 3 µM, indicating that they might
5
0
result in renal impairment. Fortunately, compound B16 displayed weaker cytotoxicity
than ibrutinib to HEK293 cells. The similar results were also observed in another two
normal cell lines of LO2 and THP-1. As a whole, B16 showed the minimal
cytotoxicity that nearly two-fold reduced than ibrutinib on normal cell lines.
Table 4. Cytotoxicity for selected compounds
(
IC50
/
µM) a
Comp.
HEK293
LO2
THP-1
Comp.
B31
HEK293
LO2
THP-1
3.3 ± 0.4
3.2 ± 1.1
3.7 ± 0.7
ibrutinib 19.3 ± 1.2 17.4 ± 0.5 20.1 ± 0.7
2.7 ± 0.5
4.8 ± 0.8
2.5 ± 0.4
3.2 ± 0.4
6.6 ± 0.9
3.2 ± 0.4
B1
13.3 ± 0.9 16.0 ± 1.1 16.7 ± 1.0
34.2 ± 1.9 31.3 ± 2.1 38.9 ± 1.5
2.9 ± 0.2 11.7 ± 1.9 8.8 ± 1.0
1.3 ± 0.2 14.2 ± 2.1 10.7 ± 1.5
10.2 ± 1.6 15.2 ± 2.0 18.9 ± 2.8
B32
B16
B25
B27
B29
B33
B35
11.2 ± 1.1 10.9 ± 1.6 15.7 ± 2.2
1.6 ± 0.2 19.1 ± 3.5 18.3 ± 2.7
2.8 ± 0.3 10.1 ± 2.1 14.4 ± 0.9
B36
B38
a
IC = compound concentration required to inhibit cell proliferation by 50%. Data
5
0
are expressed as the mean ± SD from the dose-response curves of at least three
independent experiments.
3
.3 Kinase selectivity profile for compound B16
In consideration of the overall profiles of those compounds, B16 was selected to
further investigation because of its good enzymatic potency, low cytotoxicity and
suitable physical chemical properties. The kinase selectivity profile for B16 was first
detected over the closely related Tec and Src family kinases [27-28], as well as
kinases that carry a thiol in an analogous position to Cys481 of Btk [16]. A summary
of data for kinases screen set was listed in Table 5. Unlike ibrutinib, B16 only
exhibited good inhibition to Btk with an IC value of 21.7 nM, moderately inhibited
5
0
Bmx (IC50 = 61.5 nM) and several other kinases (IC50 > 145.0 nM), and had no

ACCEPTED MANUSCRIPT
inhibition to EGFR, ErbB2, ErbB4, Itk and JAK3 (IC50 > 10000 nM). Since
irreversible inhibitors can bring unexpected off-target toxicities resulting from the
potential of covalently binding to non-target and achieving the drug protein conjugate
[
13]. Thus, irreversible Btk inhibitors with high kinase selectivity will be more
suitable to fulfill the treatment need of non-life-threatening RA disease. Although less
potent than ibrutinib on Btk, B16 was identified as a relatively highly selective Btk
inhibitor that could be safer for RA treatment than ibrutinib.
Table 5. Inhibition efficacy and selectivity among different kinases by B16 (IC /
5
0
nM)
a
31
a
31
kinase
B16
Ibrutinib
0.5
kinase
B16
Ibrutinib
n.f.
*
Btk
21.7
MKK6
>10000
657.4
*
#
Bmx
61.5
0.8
Fgr
2.3
*
#
Tec
210.6
78
Frk
1129.3
1007.8
636.5
29.2
96
*
#
Itk
>10000
330.7
10.7
n.f.
Fyn
*
#
Txk
Lck
33.2
200
*#
#
Blk
EGFR
503.7
0.5
Lyn
259.0
*
*
#
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
5.6
Src
1000.9
145.9
171
#
ErbB2
9.4
Yes
6.5
*
#
ErbB4
n.f.
Hck
1111.6
>10000
>10000
>10000
>10000
>10000
639.8
3.7
*
MKK7β
n.f.
Flt3
73
*
JAK3
16.1
>10000
>10000
n.f.
Fms
5545
n.f.
JAK1
mTOR
p70S6K
PKC
Ret
JAK2
n.f.
CaMKI
n.f.
CaMKII
CaMKIV
CaMKK1
CaMKK2
CSK
n.f.
36.5
>10000
n.f.
n.f.
Rsk1
Rsk2
Rsk3
Rsk4
Tak
>10000
>10000
>10000
>10000
>10000
>10000
n.f.
n.f.
n.f.
2.3
n.f.
GSK3α/β
MEK1
n.f.
n.f.
n.f.
Syk
>10000

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MEK2
>10000
>10000
>10000
>10000
>10000
n.f.
Trka
Trkb
Trkc
PI3K
>10000
>10000
>10000
>10000
n.f.
n.f.
MEKK2
MEKK3
n.f.
n.f.
MKK4
n.f.
>10000
a
IC values are an average of at least two independent dose-response curves in the
5
0
*
presence of 10 µM ATP. Kinases that contain a cysteine residue aligning with Cys481
#
in Btk. Kinases that belong to Src family. n.f. = not found.
3
.4 Kinetics study for compound B16
Given that the inactivation of Btk by irreversible inhibitor B16 may occur
through a two-step process that begins with reversible binding of B16 to Btk followed
by irreversible covalent bond formation with the thiol of Cys481, we next examined
the overall inactivation constant (K = Kinact / K , second-order rate constant) of B16
on
i
using an activity-based, time-dependent assay, which is highly dependent on the
inhibitor concentration and its chemical property. As shown in Figure 4A, the
conversion
%
curves describing the activity of Btk presented
a
concentration-dependent and time-dependent manner, which were used to determine
Kobs (exponential rate constant) at each inhibitor (B16) concentration. As we known,
a plot is made of Kobs versus inhibitor concentration, if this plot is linear, the slope is
K and the y-intercept is K . As displayed in Figure 4B, B16 was slowly on binding
on
off
-1
-1
-1
-1
to Btk with a K value of 0.000578 nM .min and a K value of 0.03116 nM .min
on
off
(Figure 4).

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Figure 4. Kinetics study for compound B16. A: The conversion % of Btk after
incubated with different concentrations of compound B16. B: The K (Kinact / Ki)
on
value determined for B16, K : reversible binding affinity; Kinact: inactivation rate
i
constant.
3
.5 Inhibition of hERG current assay for compound B16
Some drugs that induce prolongation of the cardiac action potential and manifest
as a prolongation of the QT interval on an electrocardiogram, have been withdrawn
from market because of their association with a potentially fatal cardiac arrhythmia
called Torsades de Pointes [29-30]. During past decades, numerous efforts to reduce
+
the incidence of sudden cardiac death have focused largely on assays using the K
channel, encoded by the human ether-a-go-go-related gene (hERG), as a standard
component of preclinical safety testing [29-30]. The inhibition of hERG potassium
current by B16 was tested using automated QPatch clamp technology in CHO cells.
B16 inhibited hERG current in a concentration dependent manner with an IC value
5
0
of 11.10 µM, which is 11 folds greater than the IC obtained by ibrutinib (0.97 µM)
50
[
26], suggesting that B16 showed very low potential of blocking the hERG channel
and low risk for cardiotoxicity.

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Figure 5. The inhibition of hERG current by B16. (A). Dose-dependent response
curve of B16 inhibiting hERG potassium current. (B) Raw current traces responding
to vehicle and six doses of B16.
3
.6 Western blot analysis for mediated signaling pathway of Btk by B16
B16 was examined in a series of B lymphoma cells including Daudi cells (Btk
overexpression), U2932 and Raji cells to determine its effect on the Btk mediated
cellular signaling pathway by western blot analysis. As illustrated in Figure 6, B16
potently inhibited Btk Y223 auto-phosphorylation in all tested cells in a concentration
dependent manner, but did not affect the trans-phosphorylation site Y551 (Syk
phosphorylation site), which were consistent with the enzymatic results. As expected,
B16 also exhibited inhibition to the Btk downstream mediator PLCγ2 Y1217
phosphorylation in respective cells. Among these cell lines, B16 showed the most
favorable suppression on the Btk mediated signaling pathway in Daudi cells, of which
0
.1 µM began to reduce Btk Y223 auto-phosphorylation and 0.3 µM started to inhibit
PLCγ2 Y1217 phosphorylation, while had moderated inhibition in U2932 cells and
the least inhibition in Raji cells.

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Figure 6. Western blot analysis for compound B16’s effect on the Btk mediated
signaling pathway in Daudi, U2932 and Raji cells.
3
.7 Pharmacokinetic properties of B16
In rat pharmacokinetic study, B16 exhibited reasonable maximum concentration
(Cmax = 257.82 µg/L), acceptable oral clearance rate (Cl = 2.75 L/h/kg), favorable
half-life (t1/2 = 7.04 h) and good bioavailability (F = 49.15%) at an oral dose of 3
mg/kg, which was much higher than the oral bioavailability of ibrutinib ranging from
1
8 to 23% in a variety of rats [18]. This might be attributed to the significant
improvement of its ClogP and aqueous solubility.
Table 6. Pharmacokinetic parameters for compound B16 in SD rat
Intravenous
3
Oral
3
dose (mg/kg)
Cmax (µg/L)
tmax (h)
3132.28
0.11
257.82
0.96
t (h)
5.18
7.04
1
/2
AUC (µg/L*h)
2522.28
1.21 (V)
8.23V(Cl)
1239.63
26.15 (V/F)
2.75 (Cl/F)
49.15
0
-t
V, V/F (L/kg)
Cl, Cl/F (L/h/kg)
F%

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Data were mean concentrations in rat plasma (n = 5).
Owing to covalent features, B16 was found to be stable in liver microsome of
various species (Table 7). Among them, B16 exhibited high stability in human live
microsome with a half-life of 330.0 min and a low clearance rate of 0.0042
-1
-1
mg ·mL·min . In mice liver microsome, B16 was moderately stable (t = 256.7
1
/2
min). By comparison, B16 was relatively rapidly cleared in rat (t = 187.3 min) and
1
/2
monkey (t1/2 = 169.0 min) liver microsome. To further investigate the cytochrome
P450 activities, the potential of B16 to inhibit the formation of metabolites by six
CYP isoforms (CYP1A2, 3A4, 2C9, 2C19, 2D6 and 2E1) was assessed using human
live microsomes and corresponding substrates through metabolism-dependent CYP
inhibition assay [34]. As shown in Table 8, B16 displayed a strong inhibition to
CYP1A2 (IC50 = 0.059±0.01 µM) indicating that it need to be careful if it was
combined use of other CYP1A2 metabolism drugs. The activities of CYP3A4 and
2
C9, were moderately inhibited by B16 with IC values about 3 µM. B16 displayed
50
very weak inhibition against CYP2C19 and 2D6, and at up to 100 µM didn’t inhibit
CYP2E1.
Table 7. In vitro liver microsome stability expressed as half-lives (t ) and clearance
1
/2
rates (Cl ) of B16
int
human
330.0
rat
dog
mice
monkey
169.0
t (min)
187.3
0.0074
315.0
0.0044
256.7
1
/2
-
1
-1
Cl (mg ·mL·min )
0.0042
0.0054
0.0082
int
Table 8. The CYP inhibition of B16.
CYP450
1A2
3A4
2C9
2C19
2D6
2E1
IC (µM)
0.06±0.01 3.19±0.05 3.54±0.04 41.66±0.03 60.1±0.05 1308027.51±0.07
5
0
CYP = cytochrome P450. HLM CYP assay. IC50 values are an average of three
independent dose-response curves.

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3
.8 Anti-arthritic activity evaluation of B16 in CIA model.
With a desirable activity and selectivity profile, a relatively clear liability profile,
low toxicity, as well as favorable pharmacokinetic properties, B16 was further
evaluated for anti-arthritis efficacy in the mice model of CIA. All mice that appeared
serious arthritis and reached a clinical score about 4.0 ± 0.3 were randomly divided
into 4 groups based on the weight. On the basis of enzymatic potencies and
bioavailability of B16, as well as making reference of ibrutinib’s treatment in CIA
mice, treatment groups were orally administrated B16 and ibrutinib at a dosage of 20
mg/kg once a day, respectively. As shown in Figure 7, both treated groups suppressed
arthritis progression without any significant changes of body weight (Figure S1). The
clinical scores in mice treated with B16 were reduced to 2.71 ± 1.38 on day 30, a little
lower than that of ibrutinib (2.87 ± 1.36). The forelimb and hindlimb in model mice
without any treatment were both severely swollen (Figure 7B). Treatment groups
potently inhibited edema formation and the increased paw thickness, suggesting that
B16 could become an efficacious treatment for RA.

ACCEPTED MANUSCRIPT
Figure 7. B16 improves clinical symptoms in developing disease in a mice CIA
model. Arthritis was induced in the mice immunization with type II collagen. The
animals were randomized according to body weight, and oral treatment with B16 (20
mg/kg) or ibrutinib (20 mg/kg) was initiated on day 36; n = 8 for all group. (A)
Clinical arthritic scores in each group, B16 treatment led to a significant lower
arthritis score than recorded for the model group. *P< 0.05; **P < 0.01; ***P< 0.001
versus model control. (B) Forelimbs and hindlimbs of each group mice after treatment
with B16 and ibrutinib.
Subsequently, single oral dose toxicity test of B16 was conducted in BALB/c
mice, which was important in the early stage of drug development. 6 female mice and
6
male mice were oral administration with B16 at a dose of 2.0 g/kg, which was
referenced to ibrutinib [18]. All mice were observed to be survival and healthy with

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free movement, normal behavior, and sensitive to light, sound and other stimulations,
and without any clinical signs observed in the 7 consecutive days. Histopathology
study also demonstrated no histopathological changes in heart, liver, spleen, lung and
kidney (Figure S2), indicating that B16 was safe and suitable for in vivo application.
4
. Conclusion
In summary, we have described the identification of irreversible Btk inhibitors
bearing 7H-pyrrolo[2,3-d]pyrimidin-4-amine core. SAR studies of the different parts
of those inhibitors led to the identification of several lead compounds with excellent
inactivation to Btk, including B1, B16, B25, B27, B29, B31, B32, B33, B35, B36 and
B38. However, most of them revealed good inhibition against HEK293 cells, even
better than ibrutinib, which would lead to severe kidney damage. Among them, B16
not only displayed preference towards Btk over closely related kinases in vitro, but
also low cytotoxicity towards HEK293 cells, as well as LO2 and THP-1 cells.
Moreover, B16 could not potently inhibit hERG current with an IC value up to 11.10
5
0
µM, suggesting that B16 showed little or no potential to block the hERG channel.
Western blot analysis also revealed that the inhibition of B16 to Btk was contributed
to its inhibition to Btk Y223 auto-phosphorylation and PLCγ2 Y1217 phosphorylation.
Combining favorable physicochemical properties, kinase selectivity, pharmacokinetic
profiles and low toxicity, B16 exhibited potent anti-arthritis activity and similar
efficacy to ibrutinib in reducing paw thickness in CIA mice. Therefore, B16 is a
potent, selective, safe and durable inhibitor of Btk and has the potential to an
efficacious treatment for arthritis.
Experimental section.
1
Chemistry. Hydrogen nuclear magnetic resonance( H NMR) spectra were recorded at
1
3
4
00 MHz while carbon nuclear magnetic resonance( C NMR) spectra were recorded
at 101 MHz on a Varian spectrometer (Varian, Palo Alto, CA) model Gemini 400 and
reported in parts per million. Chemical shifts (δ) are quoted in ppm relative to

ACCEPTED MANUSCRIPT
tetramethylsilane (TMS) as an internal standard, where (δ) TMS = 0.00 ppm. The
multiplicity of the signal is indicated as s, singlet; brs, broad singlet; d, doublet; t,
triplet; q, quartet; m, multiplet, defined as all multi peak signals where overlap or
complex coupling of signals makes definitive descriptions of peaks difficult. Mass
spectra (MS) were measured by MALDI Q-TOF Priemier mass spectrometer
(Micromass, Manchester, UK). Room temperature is within the range 20-25 °C. The
purity was analyzed by HPLC system (Waters 2695, separations module) with a
photodiode array detector (Waters 2996, Milford, MA, U.S.), and the
chromatographic column was a reversed phase C18 column (Waters, 150 mm × 4.6
mm, i.d. 5 µm). All compounds were supplied in HPLC degree methanol with 10 µL,
which was injected on a partial loop, with gradient elution with methanol/H O (60/40,
2
v/v) to methanol/H O (95/5, v/v) at a flow rate of 1 mL/min. The purity of all tested
2
compounds was ≥ 95% according to our analytical HPLC method.
General procedure for the preparation of compound 2. Methanesulfonyl chloride
(1.15eq, 6.07 g, 52.9 mmol) was added dropwise to a solution of 1-1 (1eq, 10.0 g,
4
6.4 mmol) and triethylamine (1.5eq, 7.11 g, 70.3 mmol) in methylene chloride (120
mL) in an ice bath. After 1 h, the mixture was evaporated. The residue was diluted
with EtOAc and water, extracted with EtOAc, washed with water, brine, then dried
over Na SO , filtered, and finally evaporated to give the crude product 2 as pale
2
4
yellow solid (14.84 g, 95.81% yield).
General procedure for the preparation of compound 3. A mixture of 2 (1eq, 11.72 g,
4
0 mmol) and lithium bromide (5eq, 17.20 g, 200 mmol) in acetone (80 mL) was
heated to reflux overnight. The mixture was evaporated, then the residue was
partitioned between EtOAc and water. The organic layer was washed with s brine,
dried over Na SO , filtered, and evaporated to offer the title product 3 as pale yellow
2
4
oil (9.46 g, 85.40% yield).
General procedure for the preparation of compound 5 and 11. K CO (1.5eq, 5.69 g,
2
3
4
1.25 mmol) was added to a solution of 4 (1eq, 6.98 g, 25 mmol) and 3 (1.1eq, 7.62 g,
2
7.5 mmol) in DMF (25 mL) at r.t.. The resulting mixture was heated to 80 °C and

ACCEPTED MANUSCRIPT
stirred for another 3 h. Then the mixture was cooled to r.t. extracted with EtOAc,
washed with brine, and dried over anhydrous Na SO . The organic layer was
2
4
evaporated and the residue was purified by a flash column to give the product 5 as
white solid (6.02 g, 64.20% yield). Intermediate 11 was synthesized following the
procedure of preparation 5.
General procedure for the preparation of compound 6. A solution of 5 (1eq, 4.58 g, 10
mmol) and NH OH (8 mL) in 1.4-dioxane (30 mL) was stirred in an autoclave at
4
1
00 °C overnight. The mixture was allowed to cool to r.t. and concentrated in vacuo,
then water (30 mL) was added. The mixture was subjected to sonication for 10 min.
The resulting solid was collected by filtration and dried in vacuo to afford the desired
product 6 as off-white solid (~75% yield).
General procedure of Suzuki coupled reaction for the preparation of compounds 7 and
1
2. To a mixture of halides (1eq, 5 mmol), boric acid derivatives (1.1eq, 5.5 mmol),
PdCl (dppf) (0.1eq, 365.85 mg, 0.5 mmol) and K CO (3eq, 2.07 g, 15 mmol) in a
2
2
3
1
00 mL reaction vial under vacuum, 75 mL of H O/1,4-dioxane (1/8, v/v) was added
2
via a syringe. The mixture was re-fed with N and heated to 100 °C for 3-4 h. Then
2
the solvent was evaporated. The residue was dissolved in EtOAc/H O, extracted with
2
EtOAc, washed with water, brine, dried over Na SO , filtered, and evaporated. The
2
4
residue was purified with flash column chromatography to afford coupled product 7
as yellow solids (~50% yield). Intermediate 12 was synthesized according to the
procedure for the preparation of 7.
General procedure for the preparation of compounds 8 and 13. 4M HCl in 1,4-dioxane
(3eq) was added to a stirred solution of 7 (1eq) in 1,4-dioxane at r.t.. The resulting
solution was heated to 55 °C under stirring overnight. The corresponding
hydrochlorides 8 were obtained by filtration as light yellow solid (~60% yield).
Intermediate 13 was prepared according to the procedure of preparation 8.
General procedure for the preparation of compounds 10. NIS (1.1eq, 4.95 g, 22 mmol)
was slowly added into the mixture of 9 (1eq, 2.70 g, 20 mmol) and DMF (50 mL) at
r.t.. The reaction mixture was continued stirred at r.t. overnight. The solvent was

ACCEPTED MANUSCRIPT
removed in vacuo and water was added. The mixture was subjected to sonication for
1
0 min. The precipitate was collected by filtration, rinsed with water, and dried. The
solid 10 was used for the next step without further purification (5.01 g, 96% yield).
General procedure for the preparation of compounds B1-B30, B35 and B37. A
mixture of piperidine hydrochloride (1eq, 0.3 mmol) and DIEA (5eq, 193.5 mg, 1.5
mmol) in dry DMF (2 mL) was added acryloyl chloride (1.3eq, 35.3 mg, 0.39 mmol)
in an ice bath. The reaction mixture was stirred at r.t. for another 1 h and quenched
with cold water. The mixture was extracted with DCM, washed with brine, dried over
anhydrous Na SO , and concentrated under reduced pressure. Purification the residue
2
4
by a flash column gave target compounds as white solids (~37% yield).
General procedure for the preparation of compounds B31, B33, B36 and B38. A
mixture of piperidine hydrochloride (1eq, 0.3 mmol) and DIEA (5eq, 193.5 mg, 1.5
mmol) in dry DCM (6 mL) was added 2-chloroethanesulfonyl chloride (1.3eq, 63.6
mg, 0.39 mmol) in an ice bath. The reaction mixture was stirred at r.t. for another 1 h
and quenched with cold water. The mixture was extracted with DCM, washed with
brine, dried over anhydrous Na SO , and concentrated under reduced pressure.
2
4
Purification the residue using a flash column gave target compounds as white solids
~30% yield).
(
General procedure for the preparation of compounds B32 and B34. The solution of
trans-4-dimethylaminocrotonic acid hydrochloride (1.5eq, 74.5 mg; 0.45 mmol) and
DIEA (1.5eq, 0.07 mL; 0.45 mmol) in DCM (5 mL) was stirred in an ice bath for
about 10 min, then slowly added mixture of piperidine hydrochloride (1eq, 0.3 mmol)
and DIEA (5eq, 193.5 mg, 1.5 mmol) in DCM (6 mL). The reaction mixture was
stirred at r.t. overnight. The solvent was removed by under reduced pressure, and the
residue was purified using a flash column to give target compounds as white solids
(~30% yield).
1
-(4-((4-Amino-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)pi
1
peridin-1-yl)prop-2-en-1-one (B1). Purity: HPLC 95.9%. H NMR (400 MHz,
DMSO-d ): δ 8.14 (d, J = 5.5 Hz, 1H), 7.47 (d, J = 8.6 Hz, 2H), 7.42 (t, J = 7.9 Hz,
6