Pyrrolo[2,3-b]pyridine derivatives as potent Bruton's tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.06.023

A series of pyrrolo[2,3-b]pyridine-based derivatives were designed as potent Bruton's tyrosine kinase (BTK) inhibitors by using a scaffold-hopping strategy. Structure-activity relationship studies identified five compounds (3n, 3p, 3q, 3r, and 3s) with IC

Full text of DOI:10.1016/j.bmc.2015.06.023

Accepted Manuscript
Pyrrolo[2,3-b]pyridine derivatives as potent Bruton’s tyrosine kinase inhibitors
Xinge Zhao, Wei Huang, Yazhou Wang, Minhang Xin, Qiu Jin, Jianfeng Cai,
Feng Tang, Yong Zhao, Hua Xiang
PII:
S0968-0896(15)00517-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.06.023
BMC 12379
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
13 May 2015
9 June 2015
10 June 2015
Please cite this article as: Zhao, X., Huang, W., Wang, Y., Xin, M., Jin, Q., Cai, J., Tang, F., Zhao, Y., Xiang, H.,
Pyrrolo[2,3-b]pyridine derivatives as potent Bruton’s tyrosine kinase inhibitors, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.06.023
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Pyrrolo[2,3-b]pyridine derivatives as potent Bruton’s tyrosine
kinase inhibitors
a, b, †
b, †
, Wei Huang , Yazhou Wang , Minhang Xin , Qiu Jin , Jianfeng Cai ,
b
c
b
b
Xinge Zhao
b
Feng Tang , Yong Zhao , Hua Xiang *
b
a,
a
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, No. 24,
Tongjiaxiang, Nanjing 210009, P.R. China
b
Jiangsu Simcere Pharmaceutical Co. Ltd., Jiangsu Key Laboratory of Molecular Targeted Antitumor
Drug Research, No 699-18, Xuan Wu District, Nanjing 210042, P.R. China
c
Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an Jiaotong
University, No 76, Yanta West Road, Xi’an 710061, P.R.China
Footnotes
*Address correspondence to this author at the Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, No 24 Tongjiaxiang, Nanjing 210009, P.R. China; Tel:
+
86-25-83271096; E-mail: xianghua@cpu.edu.cn
†
These authors contributed equally to this work.
1

Abstract: A series of pyrrolo[2,3-b]pyridine-based derivatives were designed as potent
Bruton's tyrosine kinase (BTK) inhibitors by using a scaffold-hopping strategy.
Structure–activity relationship studies identified five compounds (3n, 3p, 3q, 3r, and 3s)
with IC of less than 10 nM in BTK enzyme assay and five compounds (3m, 3n, 3o, 3p,
50
and 3t) with IC of less than 20 nM in Ramos cell assay. As one of the most potent
5
0
inhibitors, compound 3p exhibited superior activity to that of compound 1 (RN486) and
pyrrolo[2,3-d]pyrimidine derivative 2 in both BTK enzymatic (IC50 = 6.0 nM) and
cellular inhibition (IC50 = 14 nM) assays. In addition, 3p displayed favorable overall
pharmacokinetic profiles compared with 1 and 2.
Keywords: BTK kinase, Inhibitor, Pyrrolo[2,3-b]pyridine, SAR.
2

1
. Introduction
1
,2
Bruton's tyrosine kinase (BTK) belongs to the TEC family of non-receptor kinases
that is involved in multiple signaling pathways downstream of the B cell receptor and Fc
3
receptor. Therefore, targeting BTK is an attractive and potential therapeutic approach
,4
5
–7
for the treatment of several diseases, including cancer and inflammatory diseases.
Many small-molecule inhibitors of BTK show antitumor activity in animal models
5
and clinical studies. The US FDA recently approved Ibrutinib (PCI-32765) for the
,8
9
treatment of mantle cell lymphoma and chronic lymphocytic leukemia. Several BTK
1
0–14
inhibitors also demonstrated efficacy in animal models ofrheumatoid arthritis (RA).
However, only a few of these inhibitors have entered clinical trials for RA.
(
Insert Figure 1 and title for Figure 1 here)
Among the studied inhibitors, compound 1 (RN486) displayed high and selective
BTK potency and appropriate drug-like properties, which useful for developing new BTK
11,12
inhibitors.
The structure of compound 1 can be divided into four parts, namely, blocks
A, B, C, and D.
By analyzing the binding mode of compound 1 with BTK kinase complex (PDB
code, 4OTR), we found that the bidentate hydrogen bonds between block C and Met477
was crucial for maintaining BTK activity. The block C assumed pseudocyclic
conformations, while the block D stacked between residues Gly480 and Leu408. Our
previous studies showed that introducing pyrrolo[2,3-d]pyrimidine to restrain key
pharmacophoric groups conformationally within the molecule lead to synthesis of the
1
potent BTK inhibitor 2. Also, the closely related 7-azaindoles have recently received a
5
1
6-19
lot of attention due to their potential biological activities and drugi-like abilities.
1
9-22
Accordingly, we postulated that scaffold hopping
from pyrrolo[2,3-d]pyrimidine to
pyrrolo[2,3-b]pyridine might be effective in developing new BTK inhibitors as the key
hydrogen bonds and hydrophobic interactions could be retained. In this paper, we report
the synthesis, structure–activity relationship (SAR), and preliminary pharmacokinetic
(
PK) study of pyrrolo[2,3-b]pyridine-based BTK inhibitors 3.
2
2
. Result and discussion
.1. Chemistry
Pyrrolo[2,3-b]pyridine derivatives (3a–3t), imidazo[4,5-b]pyridine derivative (3u),
3

and indole derivative (3v) were designed and synthesized (Table 1). The assembly of
1
halogenated fused heterocycles (block C–D) with boronic ester (block A–B) was
5
2
3
designed to generate target compounds.
(Insert Scheme 1 and legend for Scheme 1 here)
Scheme 1 shows that the synthesis of brominated pyrrolo-pyridines 4b–4t started
from commercially available 4-bromo-1H-pyrrolo[2,3-b]pyridine (4a) as the same key
intermediate. The sulfonylation of 4a produced compound 5 that protects the 7-NH group
and
-Bromo-2-methyl-1H-pyrrolo[2,3-b]pyridine (4b) was prepared using C-2 methylation
LDA, MeI), followed by deprotection of 6 under NaOH. The C-2 bromination (LDA,
,2-dibromo-1,1,2,2-tetrachloroethane) of 5 was followed by selective Suzuki coupling
Pd(dppf)Cl ·CH Cl , Na CO ] with a variety of arylboronic acid, which yielded 2-aryl
improves
the
reactivity
of
C-2
carbon-hydrogen
bond.
4
(
1
[
2
2
2
2
3
substituted 4-bromo-1H-pyrrolo[2,3-b]pyridines 4c–4l after deprotection. The
condensation of 5 with tert-butyl 4-oxopiperidine-1-carboxylate resulted in intermediate
9
. The subsequent one-pot dehydration and deprotection of 9 generated
-bromo-2-(1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine 4m. Thus, 4m was
4
treated with acyl chloride, sulfonyl chloride or oxetan-3-one to produce 4n–4t.
(Insert Scheme 2 and legend for Scheme 2 here)
The nitration-reduction reaction of 4-chloro-2-aminopyridine 10 followed by
cyclization with 4-formyl-N,N-dimethylbenzamide yielded 4-(7-chloro-3H-imidazo-
4,5-b]pyridin-2-yl)-N,N-dimethylbenzamide 4u (Scheme 2). Similar to the synthesis of
[
4
4
p, 4-(4-bromo-1H-indol-2-yl)-N,N-dimethyl-5,6-dihydropyridine-1(2H)-carbox-amide
v was ultimately produced by protection–condensation–deprotection–
dehydration–acylation reactions of 4-bromo-indole 15.
(Insert Scheme 3 and legend for Scheme 3 here)
From 4a–4v, target compounds 3a–3v were synthesized through direct
palladium-catalyzed Suzuki coupling reaction of 4 with boronic ester 20, followed by
15
deprotection.
2
.2. In vitro BTK enzyme and cellular inhibition
The newly synthesized pyrrolo[2,3-b]pyridine derivatives were initially assessed for
their ability to inhibit BTK enzymatic activity using a similar procedure reported in the
4

24
literature. IC50 values are summarized in Table 1.
(Insert Table 1 and title for Table 1 here)
15
Similar to the SAR of compound 2, removal of block D resulted in a significant
potency loss in 3a (R = H, IC50 = 562.2 nM) and 3b (R = Me, IC50 = 395 nM). Thus, the
hydrophobic interactions of block D with residues Gly480 and Leu408 play critical roles
1
1
in maintaining BTK potency. A partial recovery of the lost potency was noted with the
introduction of 4-hydroxyl (3c), 4-methoxy (3d), 3-amino (3e), or 4-acyl (3f) substituted
phenyl group in block D (IC50 of 21.3–110.3 nM). Replacing 4-acyl group of 3f with
4
-carbamyl group led to compound 3g, which displayed further increased enzymatic
activity with IC50 of 12.1 nM, which is comparable to that of compound 1 (IC50 = 13.2
nM). The introduction of piperidinyl group (3h) with more steric hindrance
(
N,N-dimethyl for 3g) led to a 3.1-fold decrease in BTK potency. Interestingly, the
replacement of the 4-carbamyl group with 4-morpholinomethyl group provided equivalent
potency (3i, IC50 = 18.1 nM). The bioisosteres of 4-acetylphenyl or 4-methoxyphenyl
group in block D with 5-acetylthiophen-3-yl group or 6-methoxypyridin-3-yl group led to
slight decrease in BTK inhibitory activity (2.5–3.7 fold, cf. 3j and 3f; 3k and 3d).
Compound 3l, bearing 1-methyl-pyrazol-3-yl group, also displayed excellent inhibitory
activity (IC50 = 20.6 nM), suggesting that the pyrazol ring was well tolerated in block C.
The phenyl group in block D was further replaced with unsaturated six-membered
heterocyclic rings. Compound 3m (3,6-dihydro-pyridine-4-yl) exhibited comparable
potency to 3c (4-hydroxy-phenyl), indicating that the hydrophobic non-aromatic ring is
also well tolerated in block D. Based on the SAR of the aryl series (3c–3l), the carbonyl or
sulfonyl group bearing potential hydrogen-bond acceptor was further introduced into the
N-1 position of the 3,6-dihydro-pyridine ring. The results showed that compounds 3n–3s
exhibited excellent BTK inhibitory activities (IC of 6.7–10.4 nM) that are superior to
50
that of 1. Compounds 3n, 3p, 3q, and 3r (IC of 6.0–7.3 nM) displayed comparable BTK
5
0
potency to that of pyrrolo[2,3-d]pyrimidine derivative 2 (IC50 = 7 nM).
Subsequently, the replacement of the pyrrolo[2,3-b]pyridine ring in block C with
imidazo[4,5-b]pyridine (3u) resulted in slight potency loss (2.6-fold lower potency
compared with 3g). The bioisosteres of pyrrolo[2,3-b]pyridine with indole eliminated the
hydrogen bonding interaction with Met477, leading to a dramatic loss of potency on BTK
5

(
3v). These results suggest that the bidentate hydrogen bonding interactions with the
backbone of Met477 are crucial for maintaining BTK activity.
The cellular (Ramos cell) inhibitory activity of all of the newly synthesized
2
+
24
compounds were further investigated through a cell-based Ca flux assay. Table 1
shows that the potency in the cellular assay are in agreement with that in BTK enzymatic
assay. Compounds 3l, 3m, 3n, 3o, 3p, and 3t (IC50 of 3–16 nM) displayed cellular BTK
inhibitory activity that is superior to that of compound 1 (IC50 = 27.3 nM) and compound 2
(
IC50 = 21.3 nM). Compounds 3m and 3t displayed the most potent cellular BTK activity
with IC50 values of 3 nM (approximately 7- to 9-fold higher than that of compound 1 and 2).
Only compound 3r demonstrated excellent BTK enzymatic inhibitory activity (IC50 = 7.1
nM) and low potency in the cellular assay (IC50 = 410 nM), probably because of the poor
cellular permeability of the 1-methylsulfonyl-1,2,3,6-tetrahydropyridine-4-yl group.
Overall, the SARs of cell-based and enzyme-based activities are similar.
2.3. Docking study
To better understand SAR in a structural context, docking analysis was performed
using CDOCKER program of the Discovery Studio 2.5 software package. The docking
orientation and interactions of compound 3p with BTK enzyme (PDB code 4OTR) are
shown in Figure 2. Superimposed models of 3p and 1 (RN486) revealed similar docking
poses of the two compounds (Figure 2A). Figure 2B shows that the entire molecule of 3p
was favorably located in the BTK pocket, as expected. The N atom in the 1’-position and
N–H group in the 7’-position of pyrrolo[2,3-b]pyridine led to the conservation of key
1
1,15
bidentate hydrogen bonding interactions with Met477.
This has also been confirmed
by the results of SAR studies that the closely related indole derivative 3v without N atom
in the 1’-position displayed no BTK inhbitory activity (IC50 > 5000 nM). The carbamyl
group in block D formed an additional hydrogen bond with Lys406, whereas 1, 2 and 3m
did not display any similar interaction. This interaction led 3p achieved 4-fold improved
BTK potency compared with 3m. The tetrahydropyridine ring was stacked between
residues Gly480 and Leu408. The hydroxyl group in block B formed hydrogen bonds
with Asp539, Lys430, and Ser538, whereas the isoquinolinone ring in block A of 3p
adopted a conformation that is opposite to that of 1. The H-bond between 1-carbonyl
2
group of isoquinolinone and Lys430 disappeared, but a new H-bond with water (H O-50)
6

formed. Block A still pointed into a selectivity pocket formed by the side chains of
Gln412, Phe413 (both Gly-rich loops), Asp521, Asn526 (both catalytic loops), Asp539
1
DFG), Leu542, Ser543, Val546, and Tyr551 (all activation loops). Overall, 3p should
1
(
demonstrate a higher enzyme potency compared to 1, which is consistent with the SAR
study.
(Insert Figure 2 and title for Figure 2 here)
2.4. In vivo PK study
Compound 3p, which showed excellent potency both in BTK enzyme and cellular
assays in vitro, was selected for preliminary evaluation of PK properties in rats. Table 2
shows that when orally administered at 4 mg/kg dose, the plasma exposure (AUC) of 3p
reached 3423.0 h·ng/mL, which is 1.9–3.2-fold higher than that of 1 (RN486) and
pyrrolo[2,3-d]pyrimidine derivative 2. When orally administered with the same dose, the
maximal plasma concentration (C ) of 3p reached 739.3 ng/mL, which is 1.8–5.5-fold
max
higher than that of 1 (RN486) and pyrrolo[2,3-d]pyrimidine derivative 2. The clearance
(
Cl) of 3p was 1.2 L/h/kg, which is 2.2–2.7-fold lower than that of 1 and 2. These results
suggest that 3p displayed favorable overall PK profiles compared with 1 and 2.
Insert Table 2 and title for Table 2 here)
. Conclusion
In summary, we reported the development of pyrrolo[2,3-b]pyridine derivatives as a
(
3
class of potent BTK inhibitors by utilizing a scaffold-hopping drug-design strategy. SAR
studies were conducted and a number of compounds were identified to display high BTK
inhibitory potency. Among the compounds, 3n, 3p, 3q, 3r, and 3s exhibited IC values of
5
0
less than 10 nM in BTK enzyme assay, whereas compounds 3m, 3n, 3o, 3p, and 3t
showed IC values of less than 20 nM in Ramos cell assay. The results of molecular
5
0
docking indicate similar binding modes of 3p and 1 with BTK kinase. As one of the most
potent inhibitors, compound 3p exhibited superior activity to that of compound 1 (RN486)
and pyrrolo[2,3-d]pyrimidine derivative 2 both in BTK enzymatic (IC50 = 6.0 nM) and
cellular inhibition (IC50 = 14 nM) assays. 3p also displayed favorable overall PK profiles
compared with 1 and 2. Further evaluation of the efficacy of 3p in animal models ofRA
will be conducted.
4
. Experimental
7

4
.1. Chemistry
All chemicals were purchased from commercial sources and used without further
purification unless especially noted. Melting points of compounds were measured on a
1 13
Melt-Temp II apparatus and uncorrected. H NMR spectra (400 MHz) and C NMR (100
MHz) spectra were recorded on a Bruker BioSpin AG (Ultrashield Plus AV 400)
3 6 6
spectrometer as deuterochloroform (CDCl ) or dimethyl sulfoxide-d (DMSO-d )
solutions using tetramethylsilane (TMS) as an internal standard (δ = 0) unless noted
otherwise. MS spectra were obtained on an Agilent technologies 6120 quadrupole LC/MS
(
ESI). High-resolution mass spectra (HRMS) were recorded on a Water Q-Tof micro
mass spectrometer. The purity of the compounds was verified by the HPLC study using a
mixture of solvent methanol/water or acetonitrile/water at the flow rate of 2 mL/min and
2 2
peak detection at 254 nm under UV. The solvents (such as MeOH, EtOAc, EtOH, CH Cl
and others) were C.P. grade purchased from Nanjing Chemical Co. Ltd. and used without
further purification. Column chromatography (CC) was carried out on silica gel (200–300
mesh, Qindao Ocean Chemical Company, China). Thin-layer chromatography (TLC)
analyses were carried out on silica gel GF254 (Qindao Ocean Chemical Company,
China).
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(1H-pyrrolo[2,3-b]-
pyridin-4-yl)benzyl acetate (21a). A solution of 20 (120 mg, 0.31 mmol), 4a (104 mg,
.31 mmol) and Pd(dppf)Cl ·CH Cl (25 mg, 0.031 mmol) in 1,4-dioxane (10 mL) was
0
2
2
2
degassed with nitrogen for 3 min followed by addition Na
2
CO
3
(65 mg, 2M in water)
o
under continuous flow of nitrogen. The reaction mixture was stirred at 100 C for 2 h.
The reaction mixture was cooled, filtered through celite, diluted with water (5 mL), and
extracted with ethyl acetate (3 × 10 mL). The combined organic layers were dried over
sodium sulfate and were concentrated in vacuo. The crude product was used in the next
+
reaction without further purification. MS (ESI) m/z 468.2 [M + H] .
6
-cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(1H-pyrrolo[2,3-b]pyridin-4-yl)-
phenyl)isoquinolin-1(2H)-one (3a). A mixture of the crude product 21a (70.2 mg, 0.15
o
mmol), LiOH (11 mg, 0.45 mmol), i-PrOH (5 ml) and THF (5 ml) was stirred at 50 C for
5
h. The reaction mixture was cooled, diluted with water (15 mL), and extracted with
8

ethyl acetate (3 × 10 mL), the combined organic layers were dried over sodium sulfate
and were concentrated in vacuo. The crude product was purified on a silica gel column
3
using (1-10% CH OH/ DCM) as eluent to afford 3a (40 mg, 62.8 %) as a white solid. Mp:
o + 1
25-227 C. MS (ESI) m/z 426.2 [M + H] . H NMR(400 MHz, DMSO-d
2
4
2
6
) : 8.29 (d, J =
.80 Hz, 1H), 7.60 (d, J = 8.00 Hz, 1H), 7.56 (d, J = 8.00 Hz, 1H), 7.51 (d, J = 8.00 Hz,
H), 7.41 (d, J = 8.00 Hz, 2H), 7.26 (s, 1H), 7.18 (d, J = 8.00 Hz, 1H), 6.98 (d, J = 16.00
Hz, 1H), 6.60 (d, J = 8.00 Hz, 1H), 6.24 (s, 1H), 4.71 (d, J = 12.00 Hz, 1H), 4.23 (d, J =
1
0
1
6.00 Hz, 1H), 4.18 (d, J = 16.00 Hz, 1H), 2.10-2.08 (m, 1H), 1.11-1.09 (m, 2H),
1
3
.89-0.87 (m, 2H). C NMR (100 MHz, DMSO-d
42.25, 141.71, 140.18, 139.92, 139.64, 136.04, 135.28, 130.19, 128.62, 128.51, 126.46,
18.98, 118.27, 116.14, 110.70, 110.48, 104.05, 99.00, 57.19, 15.38, 10.72, 10.68. HPLC
6
) : 158.29, 151.78, 151.64, 148.49,
1
9
+
.
7.9 %. HRMS(ESI) m/z calcd for C26
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-methyl-1H-pyrrolo-
2,3-b]pyridin-4-yl)benzyl acetate (21b). Using a method similar to that of 21a,
compound 21b was synthesized as a white solid (113 mg, 54 %). MS (ESI) m/z 482.2 [M
3 2
H20FN O [M+H] 426.1539, found 426.1612.
2
[
+
H] . H NMR(400 MHz, DMSO-d
1
+
=
6
) : 11.67 (s, 1H), 8.19 (d, J = 5.20 Hz, 1H), 7.68 (t, J
16.00 Hz, 1H), 7.52 (d, J = 8.00 Hz, 1H), 7.49 (d, J = 8.00 Hz, 1H), 7.44 (d, J = 8.00
Hz, 1H), 7.28 (s, 1H), 7.01 (s, 1H), 6.97 (d, J = 5.20 Hz, 1H), 6.62 (d, J = 8.00 Hz, 1H),
5
2
.93 (s, 1H), 4.82 (d, J = 16.00 Hz, 1H), 4.72 (d, J = 16.00 Hz, 1H), 2.43 (s, 3H),
.10-2.07 (m, 1H), 2.43 (s, 3H), 1.11-1.09 (m, 2H), 0.89-0.87 (m, 2H).
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-methyl-1H-pyrrolo[2,3-b]-
pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3b). Using a method similar to that of 3a,
o
compound 3b was synthesized as a white solid (43 mg, 57 %). Mp: 248-250 C. MS (ESI)
+
1
m/z 440.2 [M + H] . H NMR(400 MHz, DMSO-d
H), 7.58 (t, J = 16.00 Hz, 1H), 7.49 (d, J = 8.00 Hz, 1H), 7.40 (d, J = 8.00 Hz, 2H), 7.27
s, 1H), 7.11 (d, J = 5.20 Hz, 1H), 6.98 (d, J = 12.00 Hz, 1H), 6.60 (d, J = 8.00 Hz, 1H),
.95 (s, 1H), 4.62 (s, 1H), 4.19-4.18 (s, 2H), 2.40 (s, 3H), 2.07-2.05 (m, 1H), 1.12-1.08
6
) : 11.61 (s, 1H), 8.17 (d, J = 5.20 Hz,
1
(
5
1
m, 2H), 0.88-0.85 (m, 2H). C NMR (100 MHz, DMSO-d
3
(
6
) : 160.60, 158.46, 151.87,
1
1
48.71, 141.60, 140.79, 140.17, 140.07, 138.17, 137.36, 135.85, 135.19, 130.19, 128.64,
28.32, 120.14, 118.25, 116.03, 110.73, 110.51, 104.17, 96.84, 57.18, 15.37, 13.48, 10.68,
9

+
.
1
4
0.65. HPLC 97 %. HRMS(ESI) m/z calcd for C27
40.1776.
3 2
H22FN O [M+H] 440.1768, found
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(4-hydroxyphenyl)-1H-
pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21c). Using a method similar to that of 21a,
+
compound 21c was synthesized as a gray solid. MS (ESI) m/z 560.3 [M + H] .
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(4-hydroxyphenyl)-1H-
pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3c). Using a method similar
to that of 3a, compound 3c was synthesized as a white solid (33 mg, 64.1 %). Mp:
o
+ 1
82-184 C. MS (ESI) m/z 518.2 [M + H] . H NMR(400 MHz, DMSO-d
1
1
6
) : 12.13 (s,
H), 9.74 (s, 1H), 8.22 (s, 1H), 7.75 (d, J = 8.00 Hz, 2H), 7.58 (t, J = 12.00 Hz, 2H), 7.43
(
s, 2H), 7.27 (s, 1H), 7.16 (s, 1H), 6.98 (d, J = 12.00 Hz, 1H), 6.83 (d, J = 8.00 Hz, 2H),
.61 (d, J = 8.00 Hz, 1H), 6.52 (s, 1H), 4.77 (s, 1H), 4.22 (s, 2H), 2.08-2.05 (m, 1H),
6
1
1
1
1
C
1
3
6
.12-1.07 (m, 2H), 0.88-0.86 (m, 2H). C NMR (100 MHz, DMSO-d ) : 160.64, 158.36,
57.66, 151.67, 149.74, 141.82, 141.62, 140.21, 140.00, 139.36, 138.54, 135.98, 135.34,
30.28, 128.62, 128.55, 126.94, 122.42, 120.52, 118.28, 116.59, 115.65, 110.71, 110.49,
04.00, 94.20, 57.17, 15.39, 10.68, 10.65. HPLC 98.4 %. HRMS(ESI) m/z calcd for
+
.
[M+H] 518.1874, found 518.1879.
32
H24FN
3
O
3
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(4-methoxyphenyl)-1H-
pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21d). Using a method similar to that of 21a,
+
compound 21d was synthesized as a gray solid. MS (ESI) m/z 574.3 [M + H] .
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(4-methoxyphenyl)-1H-
pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3d). Using a method similar
to that of 3a, compound 3d was synthesized as a white solid (38 mg, 65.7 %). Mp:
o
+ 1
2
1
5
1
1
1
1
6
58-260 C. MS (ESI) m/z 532.3 [M + H] . H NMR(400 MHz, DMSO-d ) : 12.22 (s,
H), 8.24 (d, J = 5.20 Hz, 1H), 7.88 (d, J = 8.00 Hz, 2H), 7.63-7.56 (m, 2H), 7.44 (d, J =
.00 Hz, 2H), 7.27 (s, 1H), 7.18 (d, J = 5.00 Hz, 1H), 7.02 (d, J = 8.00 Hz, 2H), 6.97 (s,
H), 6.61 (d, J = 5.00 Hz, 2H), 4.72 (s, 1H), 4.22 (s, 2H), 3.85 (s, 3H), 2.08-2.05 (m, 1H),
1
3
6
.12-1.07 (m, 2H), 0.88-0.86 (m, 2H). C NMR (100 MHz, DMSO-d ) : 163.23, 160.64,
59.27, 158.37, 158.32, 151.69, 151.59, 149.79, 141.92, 141.83, 140.20, 139.93, 138.83,
35.98, 135.32, 130.30, 128.65, 128.59, 126.84, 123.97, 120.44, 118.28, 116.67, 114.32,
10

110.71, 110.49, 104.02, 94.83, 57.17, 55.19, 15.39, 10.70, 10.66. HPLC 96.4 %.
+
.
3 3
HRMS(ESI) m/z calcd for C33H26FN O [M+H] 532.2031, found 532.2025.
2
-(2-(3-Aminophenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-6-(6-cyclopropyl-8-fluoro-1-
oxoisoquinolin-2(1H)-yl)benzyl acetate (21e). Using a method similar to that of 21a,
+
compound 21e was synthesized as a gray solid. MS (ESI) m/z 559.3 [M + H] .
2
-(3-(2-(3-Aminophenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-(hydroxymethyl)-
phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one (3e). Using a method similar to
that of 3a, compound 3e was synthesized as a white solid (57 mg, 60.9 %). Mp: 237-239
o + 1
C. MS (ESI) m/z 517.3 [M + H] . H NMR(400 MHz, DMSO-d
6
) : 12.18 (s, 1H), 7.62
(
d, J = 8.00 Hz, 1H), 7.56 (d, J = 8.00 Hz, 1H), 7.43 (d, J = 8.00 Hz, 2H), 7.27 (s, 1H),
7
1
2
.17 (d, J = 5.00 Hz, 1H), 7.08 (m, 3H), 6.98 (d, J = 12.00 Hz, 1H), 6.64 (d, J = 8.00 Hz,
H), 6.55 (m, J = 4.80 Hz, 1H), 6.55 (s, 1H), 5.16 (br s, 2H), 4.72 (br s, 1H), 4.21 (s, 2H),
1
3
.08-2.05 (m, 1H), 1.12-1.07 (m, 2H), 0.89-0.85 (m, 2H). C NMR (100 MHz,
) : 163.23, 158.37, 151.71, 151.61, 149.66, 148.94, 142.15, 141.2-6.60 (d, 79,
40.20, 139.90, 139.73, 139.00, 135.98, 135.32, 131.88, 130.25, 129.35, 128.63, 128.59,
20.26, 118.29, 116.61, 114.13, 113.24, 110.71, 110.49, 104.02, 95.41, 57.10, 15.40,
DMSO-d
6
1
1
1
+
.
4 2
0.72, 10.68. HPLC 99.2 %. HRMS(ESI) m/z calcd for C32H25FN O [M+H] 517.2034,
found 517.2039.
-(2-(4-Acetylphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-6-(6-cyclopropyl-8-fluoro-1-
2
oxoisoquinolin-2(1H)-yl)benzyl acetate (21f). Using a method similar to that of 3a,
+
compound 3f was synthesized as a gray solid. MS (ESI) m/z 586.2 [M + H] .
2
-(3-(2-(4-Acetylphenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-(hydroxymethyl)-
phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one (3f). Using a method similar to
that of 3a, compound 3f was synthesized as a white solid (45 mg, 67.3 %). Mp: 243-245
o
+ 1
C. MS (ESI) m/z 544.2 [M + H] . H NMR(400 MHz, DMSO-d
d, J = 4.80 Hz, 1H), 8.10 (d, J = 8.00 Hz, 2H), 8.02 (d, J = 8.00 Hz, 2H), 7.65-7.58 (m,
H), 7.44 (t, J = 12.00 Hz, 2H), 7.27 (s, 1H), 7.23 (d, J = 5.00 Hz, 1H), 6.99 (d, J = 8.00
Hz, 1H), 6.92 (s, 1H), 6.62 (d, J = 8.00 Hz, 1H), 4.70 (br s, 1H), 4.21 (s, 2H), 2.58 (s, 3H),
6
) : 12.52 (s, 1H), 8.35
(
2
1
.07-2.05 (m, 1H), 1.10-1.08 (m, 2H), 0.88-0.86 (m, 2H). C NMR (100 MHz,
3
2
DMSO-d ) : 163.22, 160.63, 158.40, 151.75, 151.66, 150.06, 143.51, 141.79, 140.18,
6
11

1
40.00, 139.60, 137.25, 135.97, 135.71, 135.60, 135.24, 130.34, 128.86, 128.75, 125.31,
20.12, 118.29, 117.04, 110.72, 110.50, 104.09, 98.40, 57.14, 26.68, 15.39, 10.72, 10.69.
1
+
.
3 3
HPLC 98.3 %. HRMS(ESI) m/z calcd for C34H26FN O [M+H] 544.2031, found
5
44.2027.
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(4-(dimethylcarba-
2
moyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21g). Using a method
similar to that of 21a, compound 21g was synthesized as a gray solid. MS (ESI) m/z
+
15.3 [M + H] .
6
4
-(4-(3-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-2-(hydroxymethyl)-
phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-N,N-dimethylbenzamide (3g). Using
method similar to that of 3a, compound 3g was synthesized as a white solid (19 mg,
a
o
+
1
5
1.7 %). Mp: 252-254 C. MS (ESI) m/z 573.3 [M + H] . H NMR(400 MHz,
DMSO-d ) : 12.40 (s, 1H), 8.32 (d, J = 4.80 Hz, 1H), 7.99 (d, J = 8.00 Hz, 2H), 7.64-7.57
m, 2H), 7.48 (d, J = 8.00 Hz, 2H), 7.44 (d, J = 8.00 Hz, 2H), 7.26 (s, 1H), 7.22 (d, J =
6
(
5
1
.00 Hz, 1H), 6.99 (d, J = 8.00 Hz, 1H), 6.82 (s, 1H), 6.61 (d, J = 8.00 Hz, 1H), 4.71 (br s,
H), 4.22 (s, 2H), 2.98 (s, 6H), 2.07-2.05 (m, 1H), 1.10-1.08 (m, 2H), 0.88-0.86 (m, 2H).
1
3
6
C NMR (100 MHz, DMSO-d ) : 168.50, 165.90, 163.63, 163.59, 156.95, 156.85,
1
1
6
C
55.19, 148.25, 147.07, 145.46, 144.98, 144.91, 143.06, 141.27, 141.00, 140.55, 137.52,
35.58, 133.94, 132.91, 130.39, 125.44, 123.55, 122.16, 115.99, 115.78, 109.29, 102.43,
2.43, 40.04, 20.65, 15.91, 15.88. HPLC 97.3 %. HRMS(ESI) m/z calcd for
+
[M+H] 573.2296, found 573.2314.
.
35
H29FN
4
O
3
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(4-(piperidine-1-
carbonyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21h). Using a
method similar to that of 3a, compound 3g was synthesized as a gray solid. MS (ESI) m/z
+
54.3 [M + H] .
6
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(4-(piperidine-1-carbonyl)-
phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3h). Using a
method similar to that of 3a, compound 3g was synthesized as a white solid (40 mg,
o
2.7 %). Mp: 243-245 C. MS (ESI) m/z 544.2 [M + H] . H NMR(400 MHz,
+
1
5
DMSO-d ) : 12.38 (s, 1H), 8.31 (d, J = 4.80 Hz, 1H), 7.99 (d, J = 8.00 Hz, 2H), 7.63-7.55
6
12

(
m, 2H), 7.44 (d, J = 8.00 Hz, 2H), 7.38 (d, J = 8.00 Hz, 1H), 7.36 (d, J = 8.00 Hz, 1H),
7
.24 (d, J = 4.00 Hz, 1H), 6.90 (s, 1H), 6.80 (s, 1H), 6.75 (s, 1H), 6.49 (d, J = 8.00 Hz,
H), 4.64 (br s, 1H), 4.21 (s, 2H), 3.82 (s, 4H), 2.07-2.05 (m, 1H), 3.82 (s, 4H), 1.63 (s,
H), 1.53 (s, 4H), 1.10-1.08 (m, 2H), 0.88-0.86 (m, 2H). HPLC 95.9 %. HRMS(ESI) m/z
1
2
+
.
4 3
calcd for C38H33FN O [M+H] 613.2609, found 613.2604.
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(4-(morpholinomethyl)
phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21i). Using a method similar to
that of 21a, compound 21i was synthesized as a gray solid. MS (ESI) m/z 643.3 [M +
+
H] .
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(4-(morpholinomethyl)phenyl)
1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3i). Using a method
similar to that of 3a, compound 3i was synthesized as a white solid (35 mg, 56.3 %). Mp:
-
o
+ 1
2
1
8
6
58-260 C. MS (ESI) m/z 601.3 [M + H] . H NMR(400 MHz, DMSO-d ) : 12.31 (s,
H), 8.29 (d, J = 4.80 Hz, 1H), 7.89 (d, J = 8.00 Hz, 2H), 7.64-7.53 (m, 2H), 7.44 (d, J =
.00 Hz, 2H), 7.38 (d, J = 8.00 Hz, 2H), 7.27 (s, 1H), 7.19 (d, J = 4.00 Hz, 1H), 6.99 (d, J
=
8.00 Hz, 1H), 6.71 (s, 1H), 6.61 (d, J = 8.00 Hz, 1H), 4.75 (br s, 1H), 4.10 (s, 2H), 3.60
(
s, 4H), 3.58 (s, 2H), 2.39 (s, 4H), 2.07-2.05 (m, 1H), 1.10-1.08 (m, 2H), 0.86-0.85 (m,
1
3
2
1
1
6
C
6
H). C NMR (100 MHz, DMSO-d ) : 163.23, 160.64, 158.37, 158.32, 151.70, 151.60,
49.82, 142.46, 141.83, 140.20, 139.82, 139.24, 138.63, 137.88, 136.00, 135.31, 130.68,
30.58, 130.30, 130.16, 125.27, 120.27, 118.28, 116.76, 110.71, 110.49, 104.02, 96.04,
6.17, 62.04, 57.16, 53.14, 15.39, 10.71, 10.67. HPLC 96.4 %. HRMS(ESI) m/z calcd for
+
[M+H] 601.2609, found 601.2605.
.
37
H
33FN
4
O
3
2
-(2-(5-Acetylthiophen-3-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-6-(6-cyclopropyl-8-
fluoro-1-oxoisoquinolin-2(1H)-yl)benzyl acetate (21j). Using a method similar to that
+
of 21a, compound 21j was synthesized as a gray solid. MS (ESI) m/z 592.2 [M + H] .
2
-(3-(2-(5-Acetylthiophen-3-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-2-(hydroxymethyl)
phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one (3j). Using a method similar to
that of 3a, compound 3j was synthesized as a white solid (41 mg, 59.3 %). Mp: 232-234
o + 1
C. MS (ESI) m/z 550.2 [M + H] . H NMR(400 MHz, DMSO-d
6
) : 12.16 (s, 1H), 8.36
(
d, J = 5.20 Hz, 1H), 7.97 (d, J = 4.00 Hz, 1H), 7.76 (d, J = 4.00 Hz, 1H), 7.62 (t, J =
13

1
1
2.00 Hz, 1H), 7.56 (d, J = 8.00 Hz, 1H), 7.54 (d, J = 4.00 Hz, 1H), 7.45 (m, J = 8.00 Hz,
H), 7.27 (s, 1H), 7.24 (s, 1H), 6.98 (d, J = 12.00 Hz, 1H), 6.72 (s, 1H), 6.61 (d, J = 8.00
Hz, 1H), 4.74 (s, 1H), 4.19 (s, 2H), 2.56 (s, 3H), 2.08-2.06 (m, 1H), 1.12-1.08 (m, 2H),
1
3
0
1
1
9
C
6
.88-0.85 (m, 2H). C NMR (100 MHz, DMSO-d ) : 163.23, 160.64, 158.36, 151.70,
49.77, 143.92, 142.71, 142.30, 141.79, 140.18, 140.08, 139.39, 135.99, 135.26, 134.91,
32.16, 130.26, 128.83, 128.75, 125.70, 119.99, 118.27, 117.23, 110.72, 110.51, 104.08,
8.72, 57.12, 26.32, 15.40, 10.70, 10.67. HPLC 98.5 %. HRMS(ESI) m/z calcd for
+
S [M+H] 550.1595, found 550.1589.
.
32
H24FN
3
O
3
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(6-methoxypyridin-3-
yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21k). Using a method similar to that
+
of 21a, compound 21 k was synthesized as a gray solid. MS (ESI) m/z 575.2 [M + H] .
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(6-methoxypyridin-3-yl)-1H-
pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3k). Using a method similar
to that of 3a, compound 3k was synthesized as a white solid (38 mg, 52.5 %). Mp:
o + 1
37-239 C. MS (ESI) m/z 533.3 [M + H] . H NMR(400 MHz, DMSO-d
2
1
6
) : 12.36 (s,
H), 8.76 (s, 1H), 8.29 (d, J = 5.20 Hz, 1H), 8.24 (d, J = 8.00 Hz, 1H), 7.63 (d, J = 5.20
Hz, 1H), 7.58 (t, J = 12.00 Hz, 1H), 7.44 (d, J = 8.00 Hz, 2H), 7.27 (s, 1H), 7.21 (s, 1H),
6
1
0
1
1
9
C
.98 (d, J = 12.00 Hz, 1H), 6.92 (d, J = 8.00 Hz, 1H), 6.72 (s, 1H), 6.61 (d, J = 8.00 Hz,
H), 4.73 (s, 1H), 4.22 (s, 2H), 3.90 (s, 3H), 2.08-2.06 (m, 1H), 1.12-1.08 (m, 2H),
1
3
6
.88-0.85 (m, 2H). C NMR (100 MHz, DMSO-d ) : 163.19, 160.63, 158.37, 151.70,
51.60, 149.86, 143.86, 142.43, 141.84, 140.18, 139.81, 139.18, 136.36, 135.97, 135.28,
30.31, 128.68, 121.39, 120.19, 118.25, 116.84, 112.02, 111.96, 110.75, 110.49, 104.04,
5.90, 57.15, 53.36, 15.39, 10.70, 10.67. HPLC 98.2 %. HRMS(ESI) m/z calcd for
+
.
32
H25FN
4
O
3
[M+H] 533.1910, found 533.1983.
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(1-methyl-1H-pyrazol-
-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21l). Using a method similar to
2
3
that of 21a, compound 21l was synthesized as a gray solid. MS (ESI) m/z 547.3 [M +
+
H] .
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(1-methyl-1H-pyrazol-3-yl)-1H
-
pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3l). Using a method similar
14

to that of 3a, compound 3l was synthesized as a white solid (21 mg, 48.3 %). Mp:
o
+ 1
2
1
8
6
62-264 C. MS (ESI) m/z 506.3 [M + H] . H NMR(400 MHz, DMSO-d ) : 12.09 (s,
H), 8.20-8.19 (d, J = 5.20 Hz, 2H), 7.98 (s, 1H), 7.61 (t, J = 12.00 Hz, 1H), 7.55 (d, J =
.00 Hz, 1H), 7.41 (d, J = 8.00 Hz, 2H), 7.27 (s, 1H), 7.15 (d, J = 5.20 Hz, 1H), 6.98 (d, J
=
12.00 Hz, 1H), 6.61 (d, J = 8.00 Hz, 1H), 6.39 (s, 1H), 4.72 (s, 1H), 4.20-4.19 (s, 2H),
1
3
3
.87 (s, 3H), 2.08-2.05 (m, 1H), 1.12-1.07 (m, 2H), 0.88-0.86 (m, 2H). C NMR (100
MHz, DMSO-d ) : 163.22, 160.63, 158.38, 151.71, 149.23, 141.82, 141.25, 140.19,
40.08, 138.18, 136.56, 135.92, 135.29, 132.65, 130.24, 128.61, 128.30, 120.41, 118.28,
16.52, 114.39, 110.70, 110.48, 104.02, 93.94, 57.09, 39.05, 15.39, 10.72, 10.68. HPLC
6
1
1
9
+
.
7.4 %. HRMS(ESI) m/z calcd for C30
5 2
H24FN O [M+H] 506.1986, found 506.1991.
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(1,2,3,6-tetrahydro-
pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21m). Using a method
similar to that of 21a, compound 21m was synthesized as a gray solid. MS (ESI) m/z
+
49.2 [M + H] .
5
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(1,2,3,6-tetrahydropyridin-4-
yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3m). Using
method similar to that of 3a, compound 3m was synthesized as a white solid (87 mg,
a
o
+
1
4
7.2 %). Mp: 245-247 C. MS (ESI) m/z 601.3 [M + H] . H NMR(400 MHz,
DMSO-d ) : 12.58 (s, 1H), 8.61 (d, J = 4.80 Hz, 2H), 8.38 (d, J = 4.80 Hz, 1H), 7.91 (d, J
8.00 Hz, 2H), 7.56 (d, J = 4.80 Hz, 1H), 7.45 (d, J = 8.00 Hz, 2H), 7.26 (s, 1H), 7.02 (s,
6
=
1
3
0
1
1
3
C
H), 6.97 (d, J = 12.00 Hz, 1H), 6.63 (d, J = 4.00 Hz, 1H), 4.70 (br s, 1H), 4.20 (s, 2H),
.85 (m, 2H), 3.35 (m, 2H), 2.74 (m, 2H), 2.07-2.05 (m, 1H), 1.10-1.08 (m, 2H),
1
3
6
.86-0.85 (m, 2H). C NMR (100 MHz, DMSO-d ) : 163.23, 160.64, 158.36, 150.21,
44.07, 141.83, 140.41, 140.19, 139.48, 138.34, 135.99, 135.55, 135.27, 130.34, 128.86,
28.72, 119.85, 119.35, 118.28, 117.16, 110.74, 110.52, 104.06, 99.34, 57.10, 41.30,
8.70, 31.25, 15.39, 10.70, 10.67. HPLC 96.7 %. HRMS(ESI) m/z calcd for
+
.
31
H27FN
4
O
2
[M+H] 507.2024, found 507.2029.
-(2-(1-Acetyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-6-
6-cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)benzyl acetate (21n). Using a
method similar to that of 21a, compound 21n was synthesized as a gray solid. MS (ESI)
2
(
15

+
m/z 591.3 [M + H] .
-(3-(2-(1-Acetyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-
-(hydroxymethyl)phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(2H)-one (3n). Using
a method similar to that of 3a, compound 3n was synthesized as a white solid (37 mg,
2
2
o
+
1
6
5.8 %). Mp: 198-200 C. MS (ESI) m/z 549.3 [M + H] . H NMR(400 MHz,
DMSO-d ) : 12.01 (s, 1H), 8.26 (d, J = 4.80 Hz, 1H), 7.58 (d, J = 8.00 Hz, 1H), 7.51 (d, J
8.00 Hz, 1H), 7.42 (d, J = 8.00 Hz, 2H), 7.26 (s, 1H), 7.14 (d, J = 4.00 Hz, 1H), 6.98 (d,
J = 12.00 Hz, 1H), 6.60 (d, J = 8.00 Hz, 1H), 6.53 (s, 1H), 6.27 (d, J = 8.00 Hz, 1H), 4.69
6
=
(
(
br s, 1H), 4.18 (s, 2H), 4.13 (s, 2H), 3.61 (m, 2H), 2.52 (m, 2H), 2.09 (s, 3H), 2.09-2.06
13
m, 1H), 1.10-1.07 (m, 2H), 0.87-0.85 (m, 2H). C NMR (100 MHz, DMSO-d
6
) : 163.22,
1
60.63, 158.35, 149.66, 142.59, 141.81, 140.19, 139.76, 139.22, 138.85, 135.96, 135.30,
1
30.22, 128.63, 127.17, 126.89, 121.36, 120.74, 119.69, 118.28, 116.48, 110.70, 110.48,
04.01, 96.33, 57.11, 42.38, 41.21, 37.17, 21.69, 15.39, 10.71, 10.67. HPLC 97.2 %.
1
+
.
4 3
HRMS(ESI) m/z calcd for C33H29FN O [M+H] 549.2296, found 549.2290.
2
-(2-(1-(Cyclopropanecarbonyl)-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]
pyridin-4-yl)-6-(6-cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)benzyl acetate
21o). Using a method similar to that of 21a, compound 21o was synthesized as a gray
(
+
solid. MS (ESI) m/z 617.3 [M + H] .
-(3-(2-(1-(Cyclopropanecarbonyl)-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo-
2,3-b]pyridin-4-yl)-2-(hydroxymethyl)phenyl)-6-cyclopropyl-8-fluoroisoquinolin-1(
H)-one (3o). Using a method similar to that of 3a, compound 3o was synthesized as a
2
[
2
o
+
1
white solid (30 mg, 46.1 %). Mp: 190-192 C. MS (ESI) m/z 575.3 [M + H] . H
NMR(400 MHz, DMSO-d ) : 12.01 (s, 1H), 8.26 (d, J = 4.80 Hz, 1H), 7.58 (d, J = 8.00
Hz, 1H), 7.52 (d, J = 8.00 Hz, 1H), 7.42 (d, J = 8.00 Hz, 2H), 7.26 (s, 1H), 7.15 (d, J =
6
4
1
2
.00 Hz, 1H), 6.98 (d, J = 12.00 Hz, 1H), 6.60 (d, J = 8.00 Hz, 1H), 6.53 (s, 1H), 6.28 (s,
H), 4.69 (br s, 1H), 4.18 (s, 2H), 3.87 (s, 2H), 3.45 (s, 2H), 2.52 (m, 2H), 2.09-2.06 (m,
1
3
H), 1.10-1.07 (m, 2H), 0.87-0.85 (m, 2H), 0.75-0.72 (m, 4H). C NMR (100 MHz,
) : 171.35, 163.22, 160.63, 158.35, 158.30, 151.69, 151.60, 149.65, 142.60,
41.82, 140.19, 139.76, 139.22, 138.83, 135.97, 135.30, 130.23, 128.64, 119.69, 118.28,
16.49, 110.70, 110.48, 104.00, 96.28, 57.11, 15.39, 13.93, 11.22, 8.55. HPLC 96.5 %.
DMSO-d
6
1
1
16

+
.
HRMS(ESI) m/z calcd for C35
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(1-(dimethylcarba-
moyl)-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate
4 3
H31FN O [M+H] 575.2453, found 575.2448.
2
(
21p). Using a method similar to that of 21a, compound 21p was synthesized as a gray
+
solid. MS (ESI) m/z 620.3 [M + H] .
4
-(4-(3-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-2-(hydroxymethyl)-
phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-N,N-dimethyl-5,6-dihydropyridine-1(2H)-
carboxamide (3p). Using a method similar to that of 3a, compound 3p was synthesized
o
+ 1
as a white solid (34 mg, 50.7 %). Mp: 181-183 C. MS (ESI) m/z 578.3 [M + H] . H
NMR(400 MHz, DMSO-d ) : 11.92 (s, 1H), 8.25 (d, J = 4.80 Hz, 1H), 7.59 (d, J = 8.00
Hz, 1H), 7.52 (d, J = 8.00 Hz, 1H), 7.41 (d, J = 8.00 Hz, 2H), 7.26 (s, 1H), 7.15 (d, J =
6
4
1
2
.00 Hz, 1H), 6.98 (d, J = 12.00 Hz, 1H), 6.61 (d, J = 8.00 Hz, 1H), 6.51 (s, 1H), 6.27 (s,
H), 4.70 (br s, 1H), 4.20 (s, 2H), 3.88 (m, 2H), 3.52 (m, 2H), 2.76 (m, 6H), 2.54 (s, 2H),
13
.10-2.08 (m, 1H), 1.10-1.07 (m, 2H), 0.87-0.84 (m, 2H). C NMR (100 MHz,
) : 164.15, 163.72, 161.13, 158.90, 150.07, 143.00, 142.26, 140.69, 140.30,
39.71, 139.61, 136.45, 135.76, 130.73, 129.15, 129.07, 127.59, 122.45, 120.25, 118.79,
16.95, 111.24, 111.03, 104.57, 96.59, 57.65, 46.46, 43.58, 38.49, 25.65, 15.88, 11.14,
DMSO-d
6
1
1
+
.
1
5 3
1.11. HPLC 97.7 %. HRMS(ESI) m/z calcd for C34H32FN O [M+H] 578.2561, found
5
78.2531.
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(1-(morpholine-4-
2
carbonyl)-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl
acetate (21q). Using a method similar to that of 21a, compound 21q was synthesized as a
+
gray solid. MS (ESI) m/z 662.3 [M + H] .
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(1-(morpholine-4-carbonyl)-
,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1-
2H)-one (3q). Using a method similar to that of 3a, compound 3q was synthesized as a
1
(
o
+
1
white solid (28 mg, 50.1 %). Mp: 202-204 C. MS (ESI) m/z 620.3 [M + H] . H
NMR(400 MHz, DMSO-d ) : 11.95 (s, 1H), 8.26 (d, J = 4.80 Hz, 1H), 7.59 (d, J = 8.00
Hz, 1H), 7.52 (d, J = 8.00 Hz, 1H), 7.42 (d, J = 8.00 Hz, 2H), 7.27 (s, 1H), 7.16 (d, J =
.00 Hz, 1H), 6.98 (d, J = 12.00 Hz, 1H), 6.61 (d, J = 8.00 Hz, 1H), 6.52 (s, 1H), 6.27 (s,
6
4
17

1
2
H), 4.67 (br s, 1H), 4.19 (s, 2H), 3.95 (m, 2H), 3.59 (m, 4H), 3.38 (m, 2H), 3.16 (m, 4H),
1
3
.52 (m, 2H), 2.10-2.08 (m, 1H), 1.10-1.07 (m, 2H), 0.87-0.84 (m, 2H). C NMR (100
) : 163.24, 162.85, 158.37, 151.68, 151.59, 149.61, 142.54, 141.80,
40.20, 139.80, 139.22, 139.03, 135.99, 135.28, 130.22, 128.61, 127.13, 121.72, 119.73,
18.30, 116.46, 110.74, 110.52, 104.02, 96.16, 65.90, 57.15, 46.85, 45.93, 42.91, 38.88,
MHz, DMSO-d
6
1
1
1
6
+
.
5.39, 10.63, 10.60. HPLC 98.7 %. HRMS(ESI) m/z calcd for C36
20.2667, found 620.2669.
5 4
H34FN O [M+H]
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(4-(morpholinomethyl)
phenyl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21r). Using a method similar to
that of 21a, compound 21r was synthesized as a gray solid. MS (ESI) m/z 643.3 [M +
+
H] .
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(4-(morpholinomethyl)phenyl)
1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3r). Using a method
similar to that of 3a, compound 3r was synthesized as a white solid (35 mg, 56.3 %). Mp:
-
o
+ 1
2
1
8
6
58-260 C. MS (ESI) m/z 601.3 [M + H] . H NMR(400 MHz, DMSO-d ) : 12.31 (s,
H), 8.29 (d, J = 4.80 Hz, 1H), 7.89 (d, J = 8.00 Hz, 2H), 7.64-7.53 (m, 2H), 7.44 (d, J =
.00 Hz, 2H), 7.38 (d, J = 8.00 Hz, 2H), 7.27 (s, 1H), 7.20 (d, J = 4.00 Hz, 1H), 6.99 (d, J
=
8.00 Hz, 1H), 6.71 (s, 1H), 6.61 (d, J = 8.00 Hz, 1H), 4.75 (br s, 1H), 4.10 (s, 2H), 3.60
(
s, 4H), 3.58 (s, 2H), 2.39 (s, 4H), 2.07-2.05 (m, 1H), 1.10-1.08 (m, 2H), 0.86-0.85 (m,
1
3
2
1
1
6
C
6
H). C NMR (100 MHz, DMSO-d ) : 163.23, 160.64, 158.37, 158.32, 151.70, 151.60,
49.82, 142.46, 141.83, 140.20, 139.82, 139.24, 138.63, 137.88, 136.00, 135.31, 130.68,
30.58, 130.30, 130.16, 125.27, 120.27, 118.28, 116.76, 110.71, 110.49, 104.02, 96.04,
6.17, 62.04, 57.16, 53.14, 15.39, 10.71, 10.67. HPLC 96.4 %. HRMS(ESI) m/z calcd for
+
[M+H] 601.2609, found 601.2605.
.
37
H
33FN
4
O
3
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(1-(N,N-dimethyl-
sulfamoyl)-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl-
acetate (21s). Using a method similar to that of 21a, compound 21s was synthesized as a
+
gray solid. MS (ESI) m/z 656.3 [M + H] .
4
-(4-(3-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-2-(hydroxymethyl)-
phenyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-N,N-dimethyl-5,6-dihydropyridine-1(2H)-
18

sulfonamide (3s). Using a method similar to that of 3a, compound 3s was synthesized as
o
+
1
a white solid (31 mg, 47.9 %). Mp: 187-189 C. MS (ESI) m/z 614.3 [M + H] . H
NMR(400 MHz, DMSO-d ) : 11.00 (s, 1H), 8.26 (d, J = 4.80 Hz, 1H), 7.58 (d, J = 8.00
Hz, 1H), 7.52 (d, J = 8.00 Hz, 1H), 7.41 (d, J = 8.00 Hz, 2H), 7.26 (s, 1H), 7.16 (d, J =
6
4
1
2
.00 Hz, 1H), 6.98 (d, J = 12.00 Hz, 1H), 6.60 (d, J = 8.00 Hz, 1H), 6.53 (s, 1H), 6.28 (s,
H), 4.70 (br s, 1H), 4.17 (s, 2H), 3.92 (m, 2H), 3.43 (m, 2H), 2.77 (s, 6H), 2.55 (m, 2H),
13
.10-2.06 (m, 1H), 1.10-1.07 (m, 2H), 0.87-0.85 (m, 2H). C NMR (100 MHz,
) : 163.20, 160.63, 158.35, 151.70, 149.62, 142.69, 141.81, 140.19, 139.74,
39.30, 138.64, 135.96, 135.29, 130.22, 128.64, 126.74, 120.27, 119.65, 118.28, 116.51,
DMSO-d
6
1
110.70, 110.49, 104.01, 96.45, 57.11, 45.03, 42.69, 37.79, 25.47, 15.11, 10.71, 10.67.
+
.
5 4
HPLC 96.8 %. HRMS(ESI) m/z calcd for C33H32FN O S [M+H] 614.2231, found
6
14.2231.
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(1-(oxetan-3-yl)-1,2,3,6
tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)benzyl acetate (21t). Using a
2
-
method similar to that of 21a, compound 21t was synthesized as a gray solid. MS (ESI)
+
m/z 605.3 [M + H] .
6
-Cyclopropyl-8-fluoro-2-(2-(hydroxymethyl)-3-(2-(1-(oxetan-3-yl)-1,2,3,6-tetra-
hydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl)isoquinolin-1(2H)-one (3t).
Using a method similar to that of 3a, compound 3t was synthesized as a white solid (37
o
+
1
mg, 56.8 %). Mp: 245-247 C. MS (ESI) m/z 563.3 [M + H] . H NMR(400 MHz,
DMSO-d ) : 11.90 (s, 1H), 8.25 (d, J = 4.80 Hz, 1H), 7.59 (d, J = 8.00 Hz, 1H), 7.52 (d, J
8.00 Hz, 1H), 7.42 (d, J = 8.00 Hz, 2H), 7.27 (s, 1H), 7.15 (d, J = 4.00 Hz, 1H), 6.98 (d,
J = 12.00 Hz, 1H), 6.61 (d, J = 8.00 Hz, 1H), 6.51 (s, 1H), 6.23 (s, 1H), 4.69 (br s, 1H),
6
=
4
3
.60-4.57 (m, 2H), 4.52-4.49 (m, 2H), 4.19 (m, 2H), 3.58-3.55 (m, 1H), 3.45 (m, 4H),
13
.04 (m, 2H), 2.11-2.08 (m, 2H), 1.10-1.07 (m, 2H), 0.87-0.85 (m, 2H). C NMR (100
) : 163.24, 160.64, 158.32, 149.60, 142.37, 141.80, 140.21, 139.84,
39.31, 139.07, 135.99, 135.30, 130.22, 128.63, 128.57, 126.65, 121.87, 119.79, 118.30,
16.40, 110.73, 110.52, 104.02, 95.80, 74.53, 58.24, 57.15, 48.77, 45.53, 38.86, 15.39,
MHz, DMSO-d
6
1
1
1
+
.
4 3
0.65, 10.62. HPLC 96.4 %. HRMS(ESI) m/z calcd for C34H31FN O [M+H] 563.2453,
found 563.2448.
19

2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(4-(dimethylcarba-
moyl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)benzyl acetate (21u). Using a method
similar to that of 21a, compound 21u was synthesized as a gray solid. MS (ESI) m/z
+
16.3 [M + H] .
6
4
-(7-(3-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-2-(hydroxymethyl)-
phenyl)-3H-imidazo[4,5-b]pyridin-2-yl)-N,N-dimethylbenzamide (3u). Using
a
method similar to that of 3a, compound 3u was synthesized as a white solid (42 mg,
o
+
1
5
5.6 %). Mp: 219-221 C. MS (ESI) m/z 574.3 [M + H] . H NMR(400 MHz,
DMSO-d ) : 14.10 (s, 1H), 8.47 (d, J = 4.80 Hz, 1H), 8.21 (d, J = 8.00 Hz, 1H), 7.67 (m,
H), 7.41 (d, J = 4.80 Hz, 1H), 7.28 (s, 1H), 7.00 (d, J = 12.00 Hz, 1H), 6.64 (d, J = 8.00
Hz, 1H), 5.56 (br s, 1H), 4.24-4.16 (m, 2H), 3.01 (s, 3H), 2.93 (s, 3H), 2.08 (m, 1H),
6
7
1
3
1
1
1
6
.10-1.07 (m, 2H), 0.87-0.85 (m, 2H). C NMR (100 MHz, DMSO-d ) : 169.30, 163.60,
60.61, 158.38, 154.76, 151.79, 149.27, 141.61, 140.18, 138.44, 138.17, 137.67, 136.57,
35.40, 131.59, 130.68, 129.77, 129.32, 128.89, 128.53, 128.41, 127.70, 126.62, 119.44,
118.34, 110.75, 104.00, 57.38, 34.72, 15.40, 10.71, 10.67. HPLC 95.2 %. HRMS(ESI)
+
.
5 3
m/z calcd for C34H28FN O [M+H] 574.2248, found 574.2245.
2
-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-6-(2-(1-(dimethylcarba-
moyl)-1,2,3,6-tetrahydropyridin-4-yl)-1H-indol-4-yl)benzyl acetate (21v). Using a
method similar to that of 21a, compound 21v was synthesized as a gray solid. MS (ESI)
+
m/z 618.3 [M + H] .
4
-(4-(3-(6-Cyclopropyl-8-fluoro-1-oxoisoquinolin-2(1H)-yl)-2-(hydroxymethyl)-
phenyl)-1H-indol-2-yl)-N,N-dimethyl-5,6-dihydropyridine-1(2H)-carboxamide (3v).
Using a method similar to that of 21a, compound 21v was synthesized as a white solid
o
+ 1
(
18 mg, 42.6 %). Mp: 238-240 C. MS (ESI) m/z 577.3 [M + H] . H NMR(400 MHz,
DMSO-d ) : 11.34 (s, 1H), 7.54 (t, J = 16.00 Hz, 1H), 7.46 (m, 2H), 7.35 (m, 2H), 7.26 (s,
H), 7.17 (t, J = 16.00 Hz, 1H), 7.09 (s, 1H), 6.98 (d, J = 12.00 Hz, 1H), 6.59 (d, J = 8.00
Hz, 1H), 6.34 (s, 1H), 6.21 (s, 1H), 4.60 (br s, 1H), 4.18 (m, 2H), 3.88 (m, 2H), 3.30 (m,
6
1
2
H), 2.77 (s, 6H), 2.48 (m, 2H), 2.09-2.07 (m, 1H), 1.10-1.07 (m, 2H), 0.87-0.85 (m, 2H).
+
.
4 3
HPLC 95.7 %. HRMS(ESI) m/z calcd for C35H33FN O [M+H] 577.2535, found
5
77.2539.
20

4
.2. BTK enzymatic assay
The HTRF kinase assay (components supplied as kit by Cisbio) was chosen for BTK
enzyme assays. It uses time resolved fluorescence resonance energy transfer (TR-FRET)
to detect production of a phosphorylated substrate. A peptide substrate is labeled with a
biotin that can bind to XL665 labeled streptavidin, and the anti-phosphoresidue antibody
+
is labeled with Eu . Upon phosphorylation of the substrate, the antibody binds to
phosphorylated substrate that enables TR-FRET detection in homogenous assay format.
All the reagents used for the BTK kinase assays including their resources are BTK kinase
(
Invitrogen), HTRF kinEASE-TK kit (Cisbio Bioassays), ATP(Sigma), DTT(Sunshine),
MgCl and MnCl (Sigma). The assay buffer was composed of 50 mM HEPES (pH 7.0),
mM MgCl , 5 mM DTT, 0.1 % NaN , 0.1 % BSA and 0.1 mM orthovanadate. The
2
2
5
2
3
HTRF assays were preformed according to the manual in the kit. All reagents were
dispensed into each well plate according to the orders as follow: (1) BTK enzyme: 0.5
ng/µL, 4 µL; (2) Each compound as well as control: 0.008-50 mM; (3) Reagent: 22.4 µM
o
ATP and 0.15 µM substrate, 2 µL; (4) Incubation: Ambient, at 25 C, 5 min; (5) Reagent:
antibody and XL-665, 8 µL. Then following 1 hour incubation at room temperature
fluorescence was measured on the PHERAStar FS microplate reader (BMG Lab
Technologies). Signal was expressed in terms of HTRF ratio (fluorescence intensity at
6
65 nm/fluorescence intensity at 620 nm ).
2
.3. Cellular (Ramos cell) activity in a Ca flux assay.
+
4
Ramos cells (CRL-1596; American Type Culture Collection, Manassas, VA) were
seeded in an assay plate, 20,000 cells per well, in phenol red-free RPMI 1640 medium
Gibco, Life Technologies) containing 2 % heatinactivated fetal bovine serum (Gibco,
(
Life Technologies), and loaded with a calcium dye in Fluo-4 Direct™ Calcium Assay
Kits for 40 min (Invitrogen). Next, cells were treated with either compounds or vehicle
for 20 min in the dark before the plate was transferred to a PHERStar FS (BMG,
Germany). Immediately after the transfer and a 10s recording of baseline fluorescence,
cells were stimulated with a purified goat anti-human IgM antibody (10 µg/mL)
(
SouthernBiotech, USA) for 8 min, during which fluorescence signal was monitored and
recorded per 10 s. Difference between the signal and that at baseline, designated adjusted
relative fluorescence unit, was fitted with GraphPad Prism version 5.00 (GraphPad
21

Software Inc., San Diego, CA) to determine IgM-induced calcium influx and the IC50
values.
4
.4. Molecular modeling
The protein-ligand complex crystal structure of ethyl analog of RN486 bound to BTK
(
PDB code 4OTR) was chosen as the template. The molecular docking procedure was
referred to CDOCKER protocol within Discovery Studio 2.5. The enzyme preparation
and the hydrogen atoms adding was performed in the prepared process. The whole BTK
enzyme was defined as a receptor and the site sphere was selected on the basis of the
binding location of 1 (RN486). 1 and the irrelevant water molecule were moved and
compound 3p was placed. After completion of docking, 10 poses were scored and
selected on the basis of the calculated CDOCKER energy.
4
.5 Pharmacokinetic Profiles of compound 3p in SD rats
Compound 3p were administered to 3 male SD rats (weight ranging from 180 g to 240
g) at doses of 4 mg/kg for po administration. The dosing volume was 5 mL/kg. After
administration, blood samples were collected at the point including 2 min, 5 min, 15 min,
3
0 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h for analyses, the collected blood samples
o
were centrifuged at 4000 rpm for 5 min at 4 C, and then analyzed after protein
precipitation. LC/MS/MS analysis of compound 3p was performed under optimized
conditions to obtain the best sensitivity and selectivity of the analyte in selected reaction
monitoring mode (SRM) containing an internal standard. Plasma concentration-time data
were measured by a noncompartmental approach using the software WinNonlin
Enterprise, version 5.2 (Pharsight Co.,Mountain View, CA).
Acknowledgments
This work was supported by the National Major Science and Technology Project of
China (Innovation and Development of New Drugs, No. 2011ZX09401-008-07).
References and Notes
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[
1]Vetrie, D.; Vorechovsky, I.; Sideras, P.; Holland, J.; Davies, A.; Flinter, F.; Hammarstrom, L.;
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[
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[
7]Honigberg, L. A.; Smith, A. M.; Sirisawad, M.; Verner, E.; Loury, D.; Chang, B.; Li, S.; Pan, Z.;
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[
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24

Figure 1. Design of the target compounds (3)

Figure 2. Docking mode of compound 3p with BTK. (a) Superposed docking poses of 3p (cyan) and 1 (ice
blue). (b) Docking poses of 3p (cyan) with BTK pocket, PDB ID: 4OTR.

Scheme 1. Reagents and conditions: (a) benzenesulfonyl chloride, (CH
3
)
3
COK, THF, rt, 4 h; (b) CH
3
I, LDA,
o
anhydrous THF, -78 C, 3 h; (c) NaOH, CH
3
OH, THF, rt, 2 h; (d) 1,2-dibromo-1,1,2,2-tetrachloroethane,
o
o
LDA, anhydrous THF, -78 C, 3 h; (e) Pd(dppf) Cl
2
·CH
2
Cl
2
, Na
2
CO
3
, 1,4-dioxane, 80 C, 2-3 h; (f) tert-butyl
COOH, con H SO , rt, 12 h; (h)
o
4
-oxopiperidine-1-carboxylate, LDA, anhydrous THF, -78 C, 3 h; (g) CF
3
2
4
acetyl chloride for 4n, cyclopropanecarbonyl chloride for 4o, dimethylcarbamic chloride for 4p,
morpholine-4-carbonyl chloride for 4q, methanesulfonyl chloride for 4r, dimethylsulfamoyl chloride for
o
o
4
3 2
s, DIPEA, anhydrous DMSO, 0 C to rt, 2-12 h, oxetan-3-one for 4t, NaBH CN, ZnCl , MeOH, 50 C, 3 h.

o
Scheme 2. Reagents and conditions: (a) Fuming nitric acid, conc H
2
SO
4
, 0 C to rt, 3 h; (b) SnCl
2
·2H
2
O,
o
o
HCl, CH
3
CH
2
OH, 50 C, 3 h; (c) SOCl
2
, dimethylamine hydrochloride, DIPEA, THF, 0 C to rt, 5 h; (d)
o
nitrobenzene, 150 C, 12 h; (e) benzenesulfonyl chloride, (CH
3
)
3
COK, THF, rt, 4 h; (f) tert-butyl 4-
OH, THF, rt, 2 h; (h) 37%
o
oxopiperidine-1-carboxylate, LDA, anhydrous THF, -78 C, 3 h; (g) NaOH, CH
3
o
HCl, rt, 12 h; (i) dimethylcarbamic chloride, DIPEA, anhydrous DMSO, 0 C to rt, 3 h.

o
Scheme 3. Reagents and conditions: (a) Pd(dppf) Cl
o
2
·CH
2
Cl
2
, Na
2
CO
3
, 1,4-dioxane, 80 C, 2-3 h; (b) LiOH,
THF, i-PrOH, 50 C, 3-7 h.