Design, synthesis and biological evaluation of novel 7H-pyrrolo[2,3-d]pyrimidine derivatives as potential FAK inhibitors and anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2019.111716

A series of 7H-pyrrolo[2,3-d]pyrimidine derivatives possessing a dimethylphosphine oxide moiety were designed, synthesized and evaluated as novel Focal adhesion kinase (FAK) inhibitors. Most compounds potently suppressed the enzymatic activities of FAK, with IC₅₀ values in the 10^?8–10^?9 M range, and potently inhibited the proliferation of breast (MDA-MB-231) and lung (A549) cancer cell lines. The representative compound 25b exhibited potent enzyme inhibition (IC₅₀ = 5.4 nM) and good selectivity when tested on a panel of 26 kinases. 25b exhibited antiproliferative activity against A549 cells (IC₅₀ = 3.2 μM) and relatively less cytotoxicity to a normal human cell line HK2. Compound 25b also induced apoptosis and suppressed the migration of A549 cells in a concentration-dependent manner. Further profiling of compound 25b revealed it had good metabolic stability in mouse, rat and human liver microsomes in vitro and showed weak inhibitory activity against various subtypes of human cytochrome P450. The docking study of compound 25b was performed to elucidate its possible binding modes and to provide a structural basis for further structure-guided design of FAK inhibitors.

Full text of DOI:10.1016/j.ejmech.2019.111716

European Journal of Medicinal Chemistry 183 (2019) 111716
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of novel 7H-pyrrolo[2,3-d]
pyrimidine derivatives as potential FAK inhibitors and anticancer
agents
,
Ruifeng Wang a, Yixuan Chen a b, Xiangxin Zhao a, Sijia Yu a, Bowen Yang a, Tianxiao Wu a,
,
Jing Guo a, Chenzhou Hao a, Dongmei Zhao a *, Maosheng Cheng a
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China
b The School of Life Science and Biopharmaceutical, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 7H-pyrrolo[2,3-d]pyrimidine derivatives possessing a dimethylphosphine oxide moiety were
designed, synthesized and evaluated as novel Focal adhesion kinase (FAK) inhibitors. Most compounds
potently suppressed the enzymatic activities of FAK, with IC50 values in the 10ꢀ8e10ꢀ9 M range, and
potently inhibited the proliferation of breast (MDA-MB-231) and lung (A549) cancer cell lines. The
representative compound 25b exhibited potent enzyme inhibition (IC50 ¼ 5.4 nM) and good selectivity
when tested on a panel of 26 kinases. 25b exhibited antiproliferative activity against A549 cells
Received 11 August 2019
Received in revised form
17 September 2019
Accepted 17 September 2019
Available online xxx
(IC50 ¼ 3.2
mM) and relatively less cytotoxicity to a normal human cell line HK2. Compound 25b also
Keywords:
induced apoptosis and suppressed the migration of A549 cells in a concentration-dependent manner.
Further profiling of compound 25b revealed it had good metabolic stability in mouse, rat and human
liver microsomes in vitro and showed weak inhibitory activity against various subtypes of human cy-
tochrome P450. The docking study of compound 25b was performed to elucidate its possible binding
modes and to provide a structural basis for further structure-guided design of FAK inhibitors.
© 2019 Elsevier Masson SAS. All rights reserved.
FAK inhibitor
Structure-activity relationship
7H-pyrrolo[2,3-d]pyrimidine
Dimethylphosphine oxide
Anticancer
Molecular docking
1. Introduction
and siRNA to FAK have exemplified the role of FAK in cancer cell
progression and metastasis [9,10]. More importantly, recent
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that
has been identified as a key regulator of growth factor receptor- and
integrin-mediated signals. FAK governs fundamental processes in
cancer and normal cells through its kinase activity and scaffolding
function [1e3]. FAK overexpression has been documented in mul-
tiple tumor types, including prostate, breast, lung, ovarian, neck,
head and colon cancer, and it has been correlated with poor
prognosis of cancer patients [3,4]. FAK promotes malignancy by
regulating tumorigenic and metastatic potential through highly
coordinated signaling networks that orchestrate a diverse range of
cellular processes, such as cell proliferation, survival, invasion,
migration, angiogenesis, epithelialemesenchymal transition and
regulation of cancer stem cell activities [5e8]. Similarly, studies
using dominant negative FAK mutants, antisense oligonucleotides
research has identified FAK as a key mediator of the immune
response in certain cancers and has shown that targeting FAK
renders pancreatic cancers responsive to checkpoint immuno-
therapy [11,12]. Furthermore, FAK is a novel regulator of DNA
damage repair in mutant KRAS NSCLC, and its pharmacologic in-
hibition leads to radiosensitizing effects that could be beneficial in
cancer therapy [13]. Thus, the FAK enzyme has emerged as a
promising cancer therapeutic target.
Several potent and selective FAK inhibitors have been designed
and developed, and some compounds have entered preclinical or
clinical trials (see Fig. 1), including TAE226 (1) [14], VS-4718 (2,
phase I, NCT01849744) [15], CEP-37440 (3, phase I, NCT01922752)
[16], defactinib (4, phase II, NCT02465060) [17], PF-562271 (5,
phase I, NCT00666926) [18], and GSK2256098 (6, phase II,
NCT02428270, NCT02523014) [19]. These compounds are orally
bioavailable, ATP competitive inhibitors of FAK, and they exhibit
highly potent inhibitory activity toward FAK and potent antitumor
* Corresponding author.
E-mail address: Medchemzho@163.com (D. Zhao).
https://doi.org/10.1016/j.ejmech.2019.111716
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
Fig. 1. Structures of potent and selective FAK inhibitors.
activities in a panel of in vitro and in vivo cancer models. Moreover,
the progress in evaluating FAK inhibitors in both preclinical models
and human clinical trials is promising. Therefore, the aim of the
work reported herein was to identify a novel scaffold from which to
develop new FAK inhibitors as anticancer agents.
benzene ring or substituted pyrazole ring at the R2 to improve
potency and adjust drug-like properties (see in Fig. 2). On the basis
of the above strategies, this paper describes the design, synthesis,
biological evaluation, and docking study of 7H-pyrrolo[2,3-d]py-
rimidine derivatives as novel FAK inhibitors.
It was observed that 2,4-diaminopyridine and 2,4-
diaminopyrimidine are the major scaffolds found in compounds
described by pharmaceutical companies and academic research
[20e22]. In this study, we presented additional medicinal chem-
istry efforts performed on this scaffold, focusing on the replace-
ment of the 2,4-diaminopyridine scaffold by the 7H-pyrrolo[2,3-d]
pyrimidine-2,4-diamine scaffold. We assumed it was feasible to
introduce an extra interaction with Glu500 by a simple addition of a
hydrogen donor in this region, which would result in hinge-binding
moiety containing three donoreacceptor interactions instead of
two in the pyrimidine case. In addition, various substituents with
hydrogen bond receptors were introduced at R1 to form hydrogen
bond interactions with Asp564 of the DFG motif. Compounds
containing a dimethylphosphine oxide (DMPO) moiety exhibited
better activity to FAK. Given this result, we retained the ortho-
DMPO-substituted benzene at R1 to further explore SAR at R2.
Further modifications were performed by introducing a substituted
2. Chemistry
Compounds 11a-h as well as intermediates 14 and 17 were
synthesized as depicted in Scheme 1. Commercially available 2,4-
dichloro-7H-pyrrolo[2,3-d]pyrimidine (7) was protected by TsCl
to afford intermediate 8, which was equipped with a variety of
substituted anilines or benzylamines via SNAr reactions under basic
conditions to afford intermediates 9a-d. Palladium-catalyzed
Buchwald cross-coupling with corresponding substituted anilines
afforded intermediates 10a-h [23], and was followed by a depro-
tection step to remove the Ts-protection group to produce target
compounds 11a-h. SNAr reactions between 2-fluorobenzonitrile
(12) and N-methylmethane-sulfonamide were conducted under
basic conditions to afford intermediate 13, which was then sub-
jected to reduction by BH3 in THF to yield intermediate 14. N-
methylation of 3-nitropyridin-2(1H)-one (15) followed by
Fig. 2. Rational design of target compounds.

R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
3
Scheme 1. Synthesis of compounds 11a-h and intermediates 14 and 17. Reagents and conditions: (i) Ts-Cl, NaH, anhydrous THF, 0 ꢁC; (ii) NH2-R1, DIPEA, n-BuOH, 100 ꢁC; (iii) 4-
amino-N-methylbenzamide or 2-methoxy-4-morpholinoaniline, Pd(AcO)2, X-phos, Cs2CO3, dioxane, 90 ꢁC; (iv) NaOH, MeOH/H2O, 40 ꢁC. (v) N-methylmethanesulfonamide, Cs2CO3,
CH3CN, 80 ꢁC; (vi) BH3 (2M in THF), anhydrous THF, 60 ꢁC; (vii) iodomethane, NaH, anhydrous THF, 55 ꢁC; (viii) Pd/C, H2, MeOH, 40 ꢁC.
reduction of nitro (H2, Pd/C) generated intermediate 17. Com-
pounds 20a-l, 22a-g and 25a-h were prepared under reaction
conditions similar to those depicted in Scheme 2. The synthesis of
compounds 25a-h is used as an example for illustration. Buchwald
cross-coupling of 9b with appropriate substituted amines in the
presence of Pd(AcO)2, X-phos and Cs2CO3 or Pd(dba)2, BINAP and
Cs2CO3 gave the intermediates 23a-h. Then, the tosyl group was
subsequently removed under basic conditions (NaOH, MeOH/H2O)
to give intermediates 24a-h. An additional deprotection step
removing the Boc-protection group of the solubilizing tail was
conducted to produce target compounds 25a-h. Intermediates 28a-
f and 31a-h were synthesized as depicted in Scheme 3. The starting
1H-pyrazol-4-amine (26) underwent a Mitsunobu reaction with
corresponding alcohols to afford intermediates 27a-c [24], and it
underwent an electrophilic substitution reaction with corre-
sponding halogenated substance to afford intermediates 27d-f. The
nitro group was then reduced by Pd/C in the presence of H2 to yield
amines 28a-f. Coupling of acids 29a-h with tert-butyl 4-
aminopiperidine-1-carboxylate in the presence of HATU and
DIPEA followed by nitro-reduction provided intermediates 31a-h
[25]. Compounds 34b, 35, 38b and 39 were synthesized as depicted
in Scheme 4. We performed the three-step reaction sequence
mentioned above, consisting of SNAr reaction, Buchwald cross-
coupling and deprotection, to synthesize these compounds.
Scheme 2. Synthesis of compounds 20a-l, 22a-g and 25a-h. Reagents and conditions: (i) corresponding aromatic amines, Pd(AcO)2, X-phos, Cs2CO3, dioxane, 90 ꢁC; (ii) corre-
sponding aromatic amines, Pd(dba)2, BINAP, Cs2CO3, dioxane, 90 ꢁC; (iii) NaOH, MeOH/H2O, 40 ꢁC; (iv) a) NaOH, MeOH/H2O, 40 ꢁC; b) HCl-EA, rt; (v) HCl-EA, rt.

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R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
Scheme 3. Synthesis of intermediates 28a-f and 31a-h. Reagents and conditions: (i) for 27a-c, R4-OH, PPh3, DEAD, THF, rt; (ii) for 27d-f, R4-Br, K2CO3, DMF, 40 ꢁC-80 ꢁC; (iii) Pd/C,
H2, EtOH, 40 ꢁC; (iv) tert-butyl 4-aminopiperidine-1-carboxylate, HATU, DIPEA, CH2Cl2, 25 ꢁC-40 ꢁC.
Scheme 4. Synthesis of compounds 34b, 38b, 35 and 39. Reagents and conditions: (i) (2-aminophenyl) dimethylphosphine oxide, DIPEA, DMF, 110 ꢁC; (ii) R8-NH2, Pd(AcO)2, X-phos,
Cs2CO3, dioxane, 90 ꢁC; (iii) R8-NH2, Pd(dba)2, BINAP, Cs2CO3, dioxane, 90 ꢁC; (iv) HCl-EA, rt.
3. Results and discussion
As the starting point, we introduced the dominant fragments of
the reported inhibitors at the R2 moiety (Table 1). Introduction of the
N-methyl-2-benzamide moiety at R1 led to compounds 11a and 11b,
which retained moderate potency against FAK (IC50 values of 34.5
and 91.8 nM, respectively), indicating that the 7H-pyrrolo[2,3-d]py-
rimidine scaffold was suitable for developing new FAK inhibitors.
The different fragments with hydrogen bond receptors at R1 were
introduced to form hydrogen bond interaction with Asp564 of the
DFG motif. The results indicated that the dimethyl(phenyl)phos-
phine oxide or N-methyl-N-phenylmethanesulfonamide moiety at
3.1. In vitro activity against FAK kinase and SAR analyses
All newly synthesized compounds were evaluated for their ac-
tivity against the FAK enzyme using homogeneous time-resolved
fluorescence (HTRF) assays, and TAE-226 was tested for compari-
son. Under the experimental conditions, TAE-226 displayed strong
inhibition against FAK with an IC50 value of 6.2 nM, which was
similar to the previously reported data [21].

R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
5
Table 1
Table 2
The FAK enzymatic activities of compounds 11a-h.
The FAK enzymatic activities of compounds 20a-l.
Compd.
R3
IC50 (nM)a
15.8
Compd.
R3
IC50 (nM)a
64.4
Compd.
R1
IC50 (nM)a
Compd.
R1
IC50 (nM)a
11d
20g
11a
11b
34.5
91.8
11e
11f
31.9
17.1
20a
20b
20c
85.0
38.7
82.0
20h
20i
20j
9.1
38.2
62.1
11c
11d
51.1
15.5
11g
11h
150.8
126.4
TAE-226
6.2
a
The IC50 values are shown as the mean (nM) values from two separate
20d
20e
6.8
20k
21.9
9.3
experiments.
16.7
17.2
20l
R1 (11c-f) retained potent activity against FAK with IC50 values be-
tween 15.5 and 51.1 nM. Replacement of the R1 group by a pyridone-
containing fragment (11g, 11h) resulted in decreased activity toward
FAK (IC50 > 100 nM). In particular, compound 11d, which contained a
DMPO moiety, exhibited better activity to FAK (IC50 ¼ 15.5 nM).
Although phosphorus is abundant in the human body, it is rarely
found in drug molecules, and phosphine oxide is an overlooked but
novel hydrogen bond acceptor [26]. To further explore the potential
of this functionality, we incorporated DMPO into the design of FAK
inhibitors.
20f
TAE-226
6.2
a
The IC50 values are shown as the mean (nM) values from two separate
experiments.
Table 3
The FAK enzymatic activities of compounds 22a-g.
On the basis of compound 11d, we next focused SAR exploration
on the R3 position. As shown in Table 2, the introduction of amide,
sulfonamide or phosphate-containing fragments as R3 yielded
compounds 20a-c, which exhibited lower potency against FAK with
IC50 values between 38.7 and 85.0 nM. Due to the para-substitution
vector R3 pointing toward the solvent region, the introduction of a
hydrophilic fragment at the R3 position may contribute to activity
enhancement. A class of diverse hydrophilic fragment-containing
derivatives (20d-l) were designed and synthesized, and most
compounds exhibited potent inhibitory activity against FAK.
Among these derivatives, addition of the piperidineyl group to
compound 11d gave compound 20d, which significantly increased
the activity toward FAK (IC50 ¼ 6.8 nM). Substitution of the terminal
piperidineyl in 20d by tetrahydro-pyranyl (20e) or 1-
methylpiperidineyl (20f) led to reduced FAK potency. The R-enan-
tiomer 20g exhibited lower potency against FAK (IC50 ¼ 64.4 nM),
but was less potent compared to the S-enantiomer 20h
(IC50 ¼ 9.1 nM). Morpholine or piperazine ring attached to the C2
phenyl via a carbonyl group yielded compounds 20i-j, which were
less potent than compounds 20d-e. The compound bearing a N-
methylpiperazine (20k) directly attached to C2 phenyl displayed
moderate potency (IC50 ¼ 21.9 nM), and 2-fold higher potency
(IC50 ¼ 9.3 nM) was observed for a piperazine-substituted analogue
20l.
Compd.
R4
IC50 (nM)a
156.1
Compd.
R4
IC50 (nM)a
26.2
22a
CH3
22e
22b
22c
132.6
23.1
64.1
22f
176.6
21.1
6.2
22g
22d
TAE-226
a
The IC50 values are shown as the mean (nM) values from two separate
experiments.
yl-substituted pyrazole 22d exhibited moderate potency against
FAK (IC50 ¼ 64.1 nM), and much higher potency (IC50 ¼ 26.2 nM)
was observed for the piperidin-4-yl-substituted analog 22e. Com-
pound 21f bearing a flexible acetamide moiety showed poor po-
tency (IC50 ¼ 176.2 nM), but much higher potency (IC50 ¼ 21.1 nM)
was observed for the morpholine-substituted analog 22g.
Compounds 22a-g represent a series of analogs bearing a (1-
substituted)-1H-pyrazo-4-yl moiety. As shown in Table 3, the
methyl-substituted compound 22a and 1,1-difluoroethyl-
substituted analog 22b were equally potent (156.1 vs 132.6 nM), but
much higher potency (IC50 ¼ 23.1 nM) was observed for the
methoxyethyl-substituted pyrazole 22c. The tetrahydro-pyran-4-

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R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
Table 4
Because compound 20d exhibited the best activity and moder-
The FAK enzymatic activities of compounds 25a-h.
ate stability in mouse liver microsomes (T1/2 ¼ 92.9 min), we
studied its binding modes with FAK by molecular docking to guide
the next round of structural optimization. As shown in Fig. 3A,
compound 20d binds to FAK in the ATP-binding site by adopting a
U-shaped ligand conformation. Briefly, the main interactions
include molecular interactions of the 7H-pyrrol[2,3-d]pyrimidine
scaffold with the hinge region, molecular interactions of the DMPO
moiety with the Asp564 of the DFG motif, and molecular in-
teractions of the piperidine moiety with Cys427. As shown in
Fig. 3B, it's worth noting that the gatekeeperþ2 residue is a smaller
Leu501 for FAK, but it is a bulky Phe or Tyr for most kinases [27].
Therefore, the substituents on the 2-N-aryl will occupy the lower
hinge area pocket and hydrophobically interact with the side chain
of Leu501, which can potentially improve the selectivity of the ki-
nase family.
Based on the above analysis, we started the optimization study
of compound 20d by varying the substituent on the benzene ring.
As shown in Table 4, introducing F-, MeO- or EtO- as the R5 or Me-
or F- as the R6 yielded compounds 25a-c and 25g-h, which retained
high potency against FAK, with IC50 values between 5.1 and
14.0 nM. Similarly, fluoro group introduction para to the methoxy
substituent on the 2-substituted aniline resulted in a potent in-
hibitor 25f (IC50 ¼ 9.4 nM). Further substituting the R6 moiety to
larger i-PrO (25d) and CF3 (25e) groups resulted in remarkable
activity loss toward FAK (IC50 ¼ 80.8 nM and 347.5 nM,
respectively).
Compd.
R5
R6
R7
IC50 (nM)a
20d
25a
25b
25c
25d
25e
25f
25g
25h
TAE-226
H
F
H
H
H
H
H
H
H
Me
F
H
H
H
H
H
H
F
6.8
14.0
5.4
OMe
OCH2CH3
OCH(CH3)2
CF3
OMe
H
5.8
80.8
347.5
9.4
10.3
5.1
H
H
H
6.2
a
The IC50 values are shown as the mean (nM) values from two separate
experiments.
Table 5
The FAK enzymatic activities of compounds 34b, 35, 38b and 39.
Finally, on the basis of representative compounds 22c and 25b,
bioisostere substitution was performed on the 7H-pyrrolo[2,3-d]
pyrimidine scaffold to obtain compounds 34b, 35, 38b and 39
(Table 5). Compared to compound 22c, the thieno[2,3-d]pyrimidine
analogs 34b and 38b were more than 5-fold less potent against FAK.
Similarly, lower potency was observed for the thieno[2,3-d]py-
rimidine analogs 35 and 39 compared to compound 25b. These
results indicated that hydrogen bonding of the 7H-pyrrolo[2,3-d]
pyrimidine and Glu500 contributes to achieving high potency.
Compd.
R8
IC50 (nM)a
Compd.
R8
IC50 (nM)a
22c
34b
38b
23.1
185.6
154.2
25b
35
39
5.4
23.0
20.6
3.2. Kinase selectivity profile
To obtain detailed information for the selectivity profile of this
series, the potent inhibitor 25b was further subjected to kinase
selectivity profiling with a panel of 26 kinases at a concentration of
TAE-226
6.2
a
0.5 mM, and the percent inhibition values were reported in Table 6.
The IC50 values are shown as the mean (nM) values from two separate
Good kinase selectivity was observed for 25b, with only FAK and
ALK producing greater than 80% inhibition. Three kinases (SYK,
PLK1 and TBK1) were inhibited by 25b to an extent of 20%e80%. For
the other 21 kinases, the inhibition rate was less than 20%.
experiments.
Fig. 3. Molecular docking model of compound 20d in the FAK active site. (A) Detailed interactions with the protein residues. Each dashed red line represents a hydrogen bond. (B)
The red frame represents the lower hinge area pocket. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
7
Table 6
Kinase selectivity profile of compound 25b.a
Kinase
%inhibition at 0.5
mM
kinase
%inhibition at 0.5
mM
kinase
%inhibition at 0.5 mM
ABL1
AKT1
ALK
BRAF
BTK
CDK2
CHK1
CSF1R
DDR2
5
0
98
3
0
1
3
12
1
HER2
FGFR1
FLT3
mTOR
KDR
KIT
LCK
ERK2
cMet
12
6
13
0
1
2
14
9
ꢀ2
PIM2
PLK1
PKA
FAK
SYK
RET
TBK1
ZAP70
ꢀ3
43
2
100
73
10
23
ꢀ3
Profiling Service from Life Technologies. The results represent the mean of three independent experiments performed in triplicate.
a
Selectivity profile of compound 25b measured at a concentration of 0.5
mM in a panel of 26 kinases generated with the SelectScreen®.
3.3. Antiproliferative activity
3.5. Effects of compound 25b on cell migration
To test the anticancer activity of the synthesized compounds, we
evaluated the antiproliferative activities of several compounds
against MDA-MB-231 (human breast cancer cell) and A549 (human
lung cancer cell) cell lines, in which FAK has been found to be
overexpressed [28,29], using the MTT assay. TAE-226 was tested for
comparison. As shown in Table 7, most of the target compounds
possessed significant anticancer activities with IC50 values ranging
To further investigate the effect of 25b on the migration of
A549 cells, transwell assays were performed. A549 cells were
incubated with DMSO and 25b (1, 3 and 9 mM) for 72 h. Under these
conditions, as displayed in Fig. 5A and B, the number of cells that
permeated the membrane were reduced in a concentration-
dependent manner. These results demonstrated that 25b has a
significant anti-metastatic ability against A549 cells.
from 0.5 to 17.7
exhibited less than 15
m
M. As a general trend, most of the compounds
M antiproliferative activities against MDA-
m
3.6. In vitro metabolic stability and CYP450 inhibition of compound
25b
MB-231 cells, and six of the compounds (11d, 20b, 20l, 22b, 22d,
25h) showed antiproliferative activities against MDA-MB-231 cells
similar to those of TAE-226. Three compounds (20b, 20l, 25h)
displayed significant potency to A549 cells with IC50 values of
We investigated the in vitro metabolic stability of compound
25b in mouse, rat and human liver microsomes and we evaluated
its ability to inhibit human cytochrome P450 in vitro. As shown in
Table 8 and Table 9, compound 25b possessed good microsomal
metabolic stability, and it had weak inhibitory effect on CYP1A2,
CYP2C9, CYP2C19, CYP2D6 and CYP3A4 metabolism.
0.5e2.0 mM. In addition, normal human cell line HK2 (normal hu-
man tubular epithelial cell) was also used to test their cytotoxicity.
These compounds also showed low cytotoxicity toward the normal
HK2 cells, indicating that these compounds exert less effect on HK2
cells than the cancer cells.
3.7. Molecular modeling analysis
3.4. Effects of compound 25b on cell apoptosis
To elucidate the interaction mode of compound 25b, a docking
study of this compound in the ATP-binding site of FAK (PDB entry
2JKK) was performed. The predicted binding mode is shown in
Fig. 6. The compound is anchored to the hinge region via the ca-
nonical donor-acceptor-donor hydrogen bonding motif between
the nitrogen molecules on the 7H-pyrrolo[2,3-d]pyrimidine-2,4-
diamine moiety and the backbone of residues Glu500 and
Gly502. Further stabilization may be achieved through the hydro-
phobic interactions of the 7H-pyrrolo[2,3-d]pyrimidine ring with
the hydrophobic side chains of Ala452 and Leu553. The DMPO
moiety interacts with Asp564 of the DFG motif via hydrogen
bonding. The o-methoxy on the benzene ring occupies the lower
hinge area pocket and hydrophobically interacts with the side chain
of Leu501. Furthermore, the piperidine moiety points toward the
solvent region by forming a hydrogen bond with Cys427 located at
the edge of the active pocket.
To investigate the antiproliferative mechanism of 25b on
A549 cells, the effects of 25b on apoptosis in A549 cells were tested
by flow cytometry. A549 cells were incubated with DMSO and 25b
(1, 3 and 9 mM) for 72 h. As shown in Fig. 4. Compound 25b strongly
induced apoptosis of A549 cells in a concentration-dependent
manner with apoptotic rates of 22.89%, 42.39%, and 66.71% at
concentrations of 1, 3 and 9 mM, respectively.
Table 7
Antiproliferative activity of several compounds.
Compd.
MDA-MB-231 (
m
M)a
A549 (
m
M)a
HK2 (m
M)a
11d
20b
20d
20e
20h
20i
4.17 0.18
1.31 0.01
17.76 1.06
6.53 0.51
12.41 0.60
8.80 1.14
14.30 0.43
4.35 0.29
3.43 0.15
8.18 0.47
5.53 0.27
14.11 0.12
17.41 1.53
11.29 0.59
4.17 0.24
4.24 0.034
3.45 0.23
1.02 0.09
2.93 0.17
3.72 0.53
2.48 0.25
9.88 0.06
9.80 0.22
0.53 0.04
7.53 0.22
17.40 1.53
7.01 0.22
14.53 1.27
3.20 0.41
2.24 0.47
1.61 0.16
1.02 0.14
9.16 0.18
4.77 0.38
>25
9.75 0.93
>25
>25
20j
20l
8.89 0.37
8.33 0.18
4.40 0.91
>25
14.42 2.09
>25
>25
>25
21.88 3.13
8.20 1.01
4. Conclusions
22b
22c
22d
22e
25b
25f
In conclusion, based on a structure-based design approach, a
series of 7H-pyrrolo[2,3-d]pyrimidine derivatives possessing a
dimethylphosphine oxide moiety were designed and synthesized.
The structure-activity relationship of these compounds was dis-
cussed from the perspective of enzymatic and cellular activities.
Most compounds potently suppressed the enzymatic activities of
FAK with IC50 values in the 10ꢀ8e10ꢀ9 M range and potently
inhibited the proliferation of breast (MDA-MB-231) and lung
(A549) cancer cell lines. In particular, the representative compound
25h
TAE-226
a
Concentration that inhibits the proliferation of cancer cells by 50%. Cell prolif-
eration was determined by MTT assay after incubation with compounds for 72 h.
The mean values of three independent experiments SE are reported.

8
R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
Fig. 4. Effects of compound 25b on A549 cells apoptosis.
Fig. 5. (A). The effect of compound 25b on the migration of A549 cells were investigated by transwell assays. (B). Quantitative analysis of migration.
Table 8
Liver microsomal stability.
parameters
T1/2 (min)
Remaining (%) T ¼ 1 h
Remaininga (%) T ¼ 1 h
CLint(mic)
(
mL/min/mg)
CLint(liver) (mL/min/kg)
in mouse
in rat
in human
137.6
>145
107.6
75.4
80.7
67.9
78.0
76.3
81.2
10.1
<9.6
12.9
39.9
<17.3
11.6
a
The NADPH regeneration system (replaced with buffer) was not added to the sample during the 1 h incubation.

R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
9
Table 9
5.1.1. Preparation of 2,4-dichloro-7-tosyl-7H-pyrrolo[2,3-d]
pyrimidine (8)
Cytochrome P450 inhibition.a.
NaH (1.1 equiv) was slowly added at 0 ꢁC to a 2,4-dichloro-7H-
pyrrolo[2,3-d]pyrimidine (1 equiv) solution in THF. The reaction
mixture was stirred at 0 ꢁC for 0.5 h. p-Toluensulfonyl chloride (1
equiv) was then added and it was stirred at 0 ꢁC for 4 h. The solvent
was evaporated to dryness, and the crude product was diluted with
H2O and extracted thrice with CH2Cl2. The organic layer was
washed with brine, dried over anhydrous Na2SO4, and filtered. The
filtrate was concentrated to obtain the crude product for the next
step.
isozyme
CYP1A2
1.5
CYP2C9
11.5
CYP2C19
4.9
CYP2D6
10.7
CYP3A4
10.5
%inhibition at 10 mM
a
Assays were performed in pooled human liver microsomes.
5.1.2. General procedure for the synthesis of intermediates 9a-d
To a solution of 8 (1 equiv) in n-BuOH (20 mL), corresponding
amines (1.1 equiv) and N-ethyl-N-isopropylpropan-2-amine (1.2
equiv) were added. The mixture was stirred at 100 ꢁC for 5 h. The
mixture was diluted with EtOAc and washed successively with
water and brine. The organic layer was dried over Na2SO4, filtered,
and evaporated. The resulting intermediate was purified by
chromatography.
5.1.2.1. 2-((2-Chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)-N-methylbenzamide (9a). 1H NMR (600 MHz, DMSO‑d6)
Fig. 6. Predicted binding mode of 25b in the ATP-binding site of FAK. Each dashed red
line represents a hydrogen bond. (For interpretation of the references to colour in this
figure legend, the reader is referred to the Web version of this article.)
d
11.88 (s,1H), 8.73 (d, J ¼ 4.4 Hz,1H), 8.30 (d, J ¼ 8.2 Hz,1H), 7.99 (d,
J ¼ 8.4 Hz, 2H), 7.76e7.72 (m, 2H), 7.57e7.53 (m, 1H), 7.48 (d,
J ¼ 8.3 Hz, 2H), 7.20 (t, J ¼ 7.5 Hz,1H), 6.75 (d, J ¼ 3.9 Hz,1H), 2.76 (d,
J ¼ 4.5 Hz, 3H), 2.37 (s, 3H).
25b exhibited potent enzyme inhibition (IC50 ¼ 5.4 nM) and good
selectivity for a panel of 26 kinases. Furthermore, compound 25b
effectively induced apoptosis and suppressed the migration of
A549 cells in a concentration-dependent manner. Further investi-
gation demonstrated that compound 25b had good metabolic sta-
bility in mouse, rat and human liver microsomes in vitro and
showed weak inhibitory activity against various subtypes of human
cytochrome P450. Finally, molecular docking demonstrated the
binding pose between compound 25b and FAK, thereby explaining
its anti-FAK activity. Taken together, these results validated the 7H-
pyrrolo[2,3-d]pyrimidine scaffold as a kinase inhibitor platform for
designing FAK inhibitors and phosphorus oxide as a hydrogen bond
receptor that can be used in drug design. Further investigations on
these advanced leads are ongoing and will be reported in the
future.
5.1.2.2. (2-((2-Chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)phenyl)dimethylphosphine oxide (9b). 1H NMR (600 MHz,
DMSO‑d6)
d
12.37 (s, 1H), 8.59 (dd, J ¼ 8.3, 3.8 Hz, 1H), 7.99 (d,
J ¼ 8.4 Hz, 2H), 7.75 (d, J ¼ 3.9 Hz, 1H), 7.65 (dd, J ¼ 13.7, 7.6 Hz, 1H),
7.59 (t, J ¼ 7.9 Hz, 1H), 7.47 (d, J ¼ 8.2 Hz, 2H), 7.18 (t, J ¼ 7.3 Hz, 1H),
6.69 (d, J ¼ 3.7 Hz, 1H), 2.37 (s, 3H), 1.82 (d, J ¼ 13.7 Hz, 6H).
5.1.2.3. N-(2-(((2-chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)methyl)phenyl)-N-methylmethanesulfonamide (9c). 1H NMR
(600 MHz, CDCl3)
d
8.06 (d, J ¼ 8.3 Hz, 2H), 7.58 (d, J ¼ 6.5 Hz, 1H),
7.36e7.28 (m, 5H), 6.62 (s, 1H), 6.37 (d, J ¼ 3.7 Hz, 1H), 5.17 (s, 1H),
4.43 (s, 1H), 3.29 (s, 3H), 3.03 (s, 3H), 2.39 (s, 3H).
5.1.2.4. 2-((2-Chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)-1-methylpyridin-2(1H)-one (9d). 1H NMR (600 MHz,
DMSO‑d6)
d
9.35 (s, 1H), 8.07 (dd, J ¼ 7.4, 1.6 Hz, 1H), 7.97 (d,
J ¼ 8.4 Hz, 2H), 7.69 (d, J ¼ 4.0 Hz, 1H), 7.53 (dd, J ¼ 6.8, 1.7 Hz, 1H),
7.47 (d, J ¼ 8.2 Hz, 2H), 7.14 (d, J ¼ 3.8 Hz, 1H), 6.32 (t, J ¼ 7.1 Hz, 1H),
3.52 (s, 3H), 2.36 (s, 3H).
5. Experimental section
5.1. Chemistry
5.1.3. General procedure for the synthesis of compounds 10a-h
To solutions of 9a-d (1 equiv), corresponding substituted ani-
lines (1.1 equiv), X-phos (0.1 equiv) and Cs2CO3 (3.0 equiv) in 1,4-
dioxane, Pd(AcO)2 (0.05 equiv) was added under a nitrogen atmo-
sphere. The mixture was purged with nitrogen for 5 min and then
heated at 90 ꢁC until completion of the reaction. The mixture was
diluted with ethyl acetate, and the organic layer was washed with
brine, dried over anhydrous Na2SO4 and filtered. The filtrate was
concentrated and purified by chromatography to give the coupling
intermediates 10a-h.
Starting materials, reagents and solvents were obtained from
commercial suppliers and used without further purification unless
otherwise indicated. Anhydrous solvents were dried and stored
according to standard procedures. All reactions were monitored by
thin-layer chromatography (TLC) on silica gel plates with fluores-
cence F-254 and visualized with UV light. Column chromatography
was performed on silica gel (200e300 mesh). 1H NMR, 13C NMR and
DEPT spectral data were recorded in DMSO‑d6 or CDCl3 on Bruker
ARX-600 NMR or Bruker ARX-400 NMR spectrometers with TMS as
an internal standard. IR spectra were recorded on a Bruker IFS 55
spectrometer. High-resolution accurate mass spectrometry (HRMS)
determinations for all final target compounds were obtained on a
Bruker micromass time of flight mass spectrometer equipped with
an electrospray ionization (ESI) detector. All melting points were
obtained on a Büchi melting point B-540 apparatus and are
uncorrected.
5.1.4. General procedure for the synthesis of compounds 11a-h
Intermediates 10a-h were dissolved in methanol (5 mL), and a
2 N sodium hydroxide solution (1.0 mL) was added to the mixture.
The mixture was heated to 40 ꢁC and stirred for 10 h. The majority
of the solvent was removed under vacuum, and the residue was
partitioned between ethyl acetate and water. The organic layer was

10
R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
dried over anhydrous Na2SO4 and concentrated. The residue was
purified by column chromatography to afford compounds 11a-h.
1500.7, 1442.0, 1385.3, 1305.7, 1243.7, 866.3, 755.5. HRMS calcd for
C
457.1512; found 457.1508.
22H23N6O2P, [MþH]þ, 435.1693; found 435.1686; [MþNa]þ,
5.1.4.1. 2-((2-((2-Methoxy-4-morpholinophenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzamide
solid; yield: 73%; mp: 233.2e234.0 ꢁC; 1H NMR (600 MHz,
DMSO‑d6)
(11a). White
5.1.4.5. N-(2-(((2-((2-methoxy-4-morpholinophenyl)amino)-7H-pyr-
rolo[2,3-d]pyrimidin-4-yl)amino)methyl)phenyl)-N-methyl-
methanesulfonamide (11e). Grey solid; yield: 82%; mp:
d
11.66 (s, 1H), 11.29 (s, 1H), 8.88 (d, J ¼ 8.3 Hz, 1H), 8.74
(d, J ¼ 4.4 Hz, 1H), 7.96 (d, J ¼ 8.4 Hz, 1H), 7.74 (d, J ¼ 7.8 Hz, 1H),
7.48e7.40 (m, 2H), 7.04 (t, J ¼ 7.5 Hz, 1H), 6.95 (dd, J ¼ 3.1, 2.4 Hz,
1H), 6.66 (d, J ¼ 2.3 Hz, 1H), 6.50 (dd, J ¼ 8.7, 2.3 Hz, 1H), 6.29 (dd,
J ¼ 3.0, 1.8 Hz, 1H), 3.83 (s, 3H), 3.77e3.75 (m, 4H), 3.11e3.09 (m,
153.4e154.6 ꢁC; 1H NMR (600 MHz, DMSO‑d6)
d 11.05 (s, 1H), 8.18
(d, J ¼ 8.5 Hz, 1H), 7.77 (s, 1H), 7.55e7.50 (m, 1H), 7.41 (dd, J ¼ 6.9,
1.8 Hz, 1H), 7.37e7.28 (m, 2H), 6.94 (s, 1H), 6.78 (s, 1H), 6.61 (d,
J ¼ 2.1 Hz, 1H), 6.45 (s, 1H), 6.36 (dd, J ¼ 8.5, 1.6 Hz, 1H), 4.92 (s, 1H),
4.62 (s, 1H), 3.82 (s, 3H), 3.78e3.67 (m, 4H), 3.28 (s, 3H), 3.12 (s, 3H),
4H), 2.83 (d, J ¼ 4.5 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6)
d 169.48,
156.31, 152.90, 152.53, 150.34, 147.19, 141.11, 131.74, 127.89, 122.37,
122.00, 120.44, 120.35, 119.86, 118.93, 106.65, 99.98, 98.74, 97.24,
66.21 (2C), 55.58, 49.47 (2C), 26.30. IR (thin film, cmꢀ1) 3435.8,
3323.6, 2922.0, 2852.4, 1617.7, 1580.8, 1522.6, 1446.6, 1398.6,
1225.0, 739.5. HRMS calcd for C25H27N7O3, [MþH]þ, 474.2248;
found 474.2253.
3.07e2.95 (m, 4H). 13C NMR (150 MHz, DMSO‑d6)
d 156.36, 155.78,
151.58, 148.15, 145.69, 140.45, 139.64, 128.26, 128.17, 127.68, 126.99,
123.28, 118.53, 118.04, 106.86, 99.99, 98.93, 97.31, 66.21 (2C), 55.66,
49.64 (2C), 40.05, 38.69, 36.13. IR (thin film, cmꢀ1) 3415.1, 2923.2,
2852.7, 1591.1, 1497.0, 1396.8, 1331.4, 1252.8, 1141.5, 1116.6, 731.4.
HRMS calcd for C26H31N7O4S, [MþH]þ, 538.2231; found 538.2228.
5.1.4.2. N-methyl-2-((2-((4-(methylcarbamoyl)phenyl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzamide (11b). White solid;
yield: 78%; mp: 267.9e268.8 ꢁC; 1H NMR (600 MHz, DMSO‑d6)
5.1.4.6. N-methyl-4-((4-((2-(N-methylmethylsulfonamido)benzyl)
amino)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzamide
White solid; yield: 85%; mp: 178.0e179.1 ꢁC; 1H NMR (600 MHz,
DMSO‑d6)
(11f).
d
11.75 (s, 1H), 11.48 (s, 1H), 9.31 (s, 1H), 9.03 (d, J ¼ 8.3 Hz, 1H), 8.76
(d, J ¼ 4.5 Hz, 1H), 8.20 (d, J ¼ 4.5 Hz, 1H), 7.91 (d, J ¼ 8.8 Hz, 2H),
7.79e7.74 (m, 3H), 7.53 (t, J ¼ 8.4 Hz, 1H), 7.10e7.05 (m, 2H), 6.35
(dd, J ¼ 3.3, 1.9 Hz, 1H), 2.84 (d, J ¼ 4.5 Hz, 3H), 2.78 (d, J ¼ 4.5 Hz,
d
11.14 (s, 1H), 8.91 (s, 1H), 8.12 (dd, J ¼ 8.8, 4.3 Hz, 1H),
7.79 (d, J ¼ 8.7 Hz, 2H), 7.72 (s, 1H), 7.62 (d, J ¼ 8.8 Hz, 2H), 7.54 (dd,
J ¼ 7.6, 1.3 Hz, 1H), 7.42 (dd, J ¼ 7.3, 1.5 Hz, 1H), 7.37e7.31 (m, 2H),
6.84 (dd, J ¼ 3.0, 2.5 Hz, 1H), 6.48 (s, 1H), 4.91 (s, 1H), 4.83 (s, 1H),
3.24 (s, 3H), 3.13 (s, 3H), 2.75 (d, J ¼ 4.5 Hz, 3H). 13C NMR (150 MHz,
3H). 13C NMR (150 MHz, DMSO‑d6)
d 169.52, 166.45, 154.97, 152.88,
151.91, 144.24, 141.01, 131.82, 127.96, 127.61 (2C), 125.75, 120.65,
120.63 (2C), 119.19, 116.96 (2C), 99.40, 97.39, 26.33, 26.16. IR (thin
film, cmꢀ1) 3417.9, 2922.0, 2852.1, 1611.4, 1539.0, 1583.0, 1517.5,
1446.9, 1384.9, 1329.7, 751.8. HRMS calcd for C22H21N7O2, [MþH]þ,
416.1830; found 416.1830; [MþNa]þ, 438.1649; found 438.1651.
DMSO‑d6)
d 166.48, 156.54, 155.39, 151.38, 144.64, 140.64, 139.74,
128.36, 128.28, 127.81, 127.51 (2C), 127.02, 125.14, 118.59, 116.46
(2C), 99.00, 97.69, 38.79, 36.12, 26.11. IR (thin film, cmꢀ1) 3424.2,
2922.8, 2852.4, 1615.2, 1464.2, 1384.9, 1319.7, 1242.1, 1137.7, 848.9,
729.2. HRMS calcd for C23H25N7O3S, [MþNa]þ, 502.1632; found
502.1631.
5.1.4.3. (2-((2-((2-Methoxy-4-(piperazin-1-yl)phenyl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide
(11c). White solid; yield: 73%; mp: 238.0e239.1 ꢁC; 1H NMR
(600 MHz, CDCl3)
d
11.08 (s, 1H), 9.59 (s, 1H), 8.96 (dd, J ¼ 8.4,
5.1.4.7. 3-((2-((2-Methoxy-4-morpholinophenyl)amino)-7H-pyrrolo
4.3 Hz, 1H), 8.26 (d, J ¼ 8.6 Hz, 1H), 7.50 (t, J ¼ 7.9 Hz, 1H), 7.24 (dd,
J ¼ 14.2, 7.6 Hz, 1H), 7.16 (s, 1H), 7.04 (t, J ¼ 7.4 Hz, 1H), 6.68 (d,
J ¼ 3.4 Hz, 1H), 6.57 (d, J ¼ 2.3 Hz, 1H), 6.53 (dd, J ¼ 8.7, 2.3 Hz, 2H),
[2,3-d]pyrimidin-4-yl)amino)-1-methylpyridin-2(1H)-one
White solid; yield: 72%; mp: 233.9e234.4 ꢁC; 1H NMR (600 MHz,
DMSO‑d6)
(11g).
d
11.29 (s, 1H), 8.53 (d, J ¼ 7.0 Hz, 1H), 8.21 (s, 1H), 7.86 (d,
3.92e3.88 (m, 7H), 3.14e3.11 (m, 4H), 1.84 (d, J ¼ 13.1 Hz, 6H). 13
C
J ¼ 8.6 Hz, 1H), 7.50 (s, 1H), 7.33 (dd, J ¼ 6.8, 1.5 Hz, 1H), 6.93 (dd,
J ¼ 3.2, 2.3 Hz, 1H), 6.66 (d, J ¼ 2.4 Hz, 1H), 6.50 (dd, J ¼ 8.7, 2.4 Hz,
1H), 6.45 (dd, J ¼ 3.3, 1.8 Hz, 1H), 6.24 (t, J ¼ 7.1 Hz, 1H), 3.82 (s, 3H),
3.78e3.73 (m, 4H), 3.56 (s, 3H), 3.12e3.07 (m, 4H). 13C NMR
NMR (150 MHz, DMSO‑d6)
d 156.28, 153.15, 152.31, 150.42, 147.24,
145.06, 132.29, 130.76 (d, J ¼ 11.4 Hz), 122.35, 122.16, 120.76 (d,
J ¼ 12.0 Hz), 120.26 (d, J ¼ 6.3 Hz), 118.18 (d, J ¼ 92.2 Hz), 117.88,
106.65, 99.99, 98.78, 97.90, 66.21 (2C), 55.58, 49.46 (2C), 18.58 (d,
J ¼ 70.8 Hz, 2C). IR (thin film, cmꢀ1) 3437.2, 2921.7, 2851.7, 1605.0,
1576.5, 1524.5, 1442.0, 1385.2, 1302.0, 1121.7, 755.9. HRMS calcd for
(150 MHz, DMSO‑d6)
130.12, 129.34, 122.84, 122.18, 120.60, 119.82, 106.61, 105.57, 100.00,
98.20, 97.40, 66.20 (2C), 55.54, 49.41 (2C), 37.15. IR (thin film, cmꢀ1
d 157.42, 156.44, 152.86, 152.69, 150.91, 147.51,
)
C
25H29N6O3P, [MþH]þ, 493.2112; found 493.2116.
3437.2, 2921.7, 2851.7, 2344.7, 1624.3, 1581.2, 1522.4, 1488.1, 1384.2,
1227.8, 745.5. HRMS calcd for C23H25N7O3, [MþH]þ, 448.2092;
found 448.2097.
5.1.4.4. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-N-methylbenzamide (11d). White
solid; yield: 76%; mp: 187.7e188.6 ꢁC; 1H NMR (600 MHz,
DMSO‑d6)
d
11.69 (s, 1H), 11.44 (s, 1H), 9.32 (s, 1H), 9.11 (dd, J ¼ 7.4,
5.1.4.8. N-methyl-4-((4-((1-methyl-2-oxo-1,2-dihydropyridin-3-yl)
3.1 Hz, 1H), 8.22 (d, J ¼ 4.0 Hz, 1H), 7.92 (d, J ¼ 8.2 Hz, 2H), 7.77 (d,
J ¼ 8.2 Hz, 2H), 7.61e7.53 (m, 2H), 7.10 (t, J ¼ 7.1 Hz, 1H), 7.01 (s, 1H),
6.41 (s, 1H), 2.79 (d, J ¼ 4.1 Hz, 3H), 1.85 (d, J ¼ 13.5 Hz, 6H). 13C NMR
amino)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzamide
White solid; yield: 78%; mp: 283.0e284.5 ꢁC; 1H NMR (600 MHz,
DMSO‑d6)
(11h).
d
11.50 (s, 1H), 9.33 (s, 1H), 8.75 (dd, J ¼ 7.4, 1.4 Hz, 1H),
(150 MHz, DMSO‑d6)
d
166.42, 154.95, 153.12, 151.76, 145.01, 144.26,
8.34 (s, 1H), 8.20 (q, J ¼ 4.1 Hz, 1H), 7.91 (d, J ¼ 8.8 Hz, 2H), 7.76 (d,
J ¼ 8.8 Hz, 2H), 7.38 (dd, J ¼ 6.8, 1.6 Hz, 1H), 7.04 (dd, J ¼ 3.4, 2.3 Hz,
1H), 6.54 (dd, J ¼ 3.4, 1.9 Hz, 1H), 6.33 (t, J ¼ 7.1 Hz, 1H), 3.58 (s, 3H),
132.37, 130.87 (d, J ¼ 11.0 Hz), 127.59 (2C), 125.73, 120.96 (d,
J ¼ 11.8 Hz), 120.40 (d, J ¼ 6.6 Hz), 120.18, 118.35 (d, J ¼ 91.7 Hz),
116.96 (2C), 99.44, 98.02, 26.15, 18.62 (d, J ¼ 70.4 Hz, 2C). Its 13C
NMR and DEPT spectrum displayed 22 carbon resonances for 3
primary carbon (dC 26.15, 18.62 ꢂ 2), 10 tertiary carbon (dC 132.37,
130.87, 127.59 ꢂ 2, 120.96, 120.40, 120.18, 116.96 ꢂ 2, 98.02), 9
quaternary carbon (dC 166.42, 154.95, 153.12, 151.76, 145.01, 144.26,
125.73, 118.35, 99.44). IR (thin film, cmꢀ1) 3425.6, 2921.1, 1608.8,
2.78 (d, J ¼ 4.5 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6)
d 166.44,
157.50, 154.84, 152.87, 151.96, 144.18, 130.47, 129.29, 127.63 (2C),
125.81, 121.07, 120.61, 116.92 (2C), 105.59, 98.93, 97.66, 37.19, 26.16.
IR (thin film, cmꢀ1) 3424.3, 2922.5, 2852.5, 2344.6, 1626.8, 1511.3,
1475.0, 1384.5, 1324.6, 869.4. HRMS calcd for C20H19N7O2, [MþH]þ,
390.1673; found 390.1672.

R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
11
5.1.5. Preparation of N-(2-(aminomethyl)phenyl)-N-
methylmethanesulfonamide (14)
5.1.7.3. Dimethyl(4-((4-((2-(dimethylphosphoryl)phenyl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzyl)phosphonate (20c).
White solid; yield: 77%; mp: 158.3e159.1 ꢁC; 1H NMR (600 MHz,
DMSO‑d6)
To a solution of 2-fluoro-benzonitrile (1 equiv), N-Methyl-
methanesulfonamide (1 equiv), and cesium carbonate (1.2 equiv) in
acetonitrile. The reaction mixture was stirred at 80 ꢁC for 10 h.
Upon completion, the mixture was filtered and then concentrated.
The residue was diluted with water and extracted by CH2Cl2. The
organic phases were combined, washed by brine, dried over
anhydrous Na2SO4 and evaporated to afford crude product 13. In-
termediate 13 (1 equiv) was dissolved in anhydrous tetrahydro-
furan and protected with argon. BH3 (in 2M THF) (1.5 equiv) was
then added, and the mixture was stirred at 70 ꢁC until completion
of the reaction. The reaction was quenched with H2O and extracted
thrice with CH2Cl2, and the organic layer was washed with brine,
dried over anhydrous Na2SO4, and filtered. The filtrate was
concentrated and purified by chromatography to afford interme-
diate 14.
d
11.63 (s, 1H), 11.31 (s, 1H), 9.11 (dd, J ¼ 8.3, 4.0 Hz, 1H),
8.96 (s,1H), 7.74 (d, J ¼ 8.3 Hz, 2H), 7.57 (ddd, J ¼ 14.1, 7.7,1.1 Hz,1H),
7.51 (t, J ¼ 7.9 Hz, 1H), 7.17 (dd, J ¼ 8.6, 2.2 Hz, 2H), 7.07 (t, J ¼ 7.3 Hz,
1H), 6.95 (dd, J ¼ 3.3, 2.3 Hz, 1H), 6.37 (dd, J ¼ 3.4, 2.0 Hz, 1H), 3.61
(s, 3H), 3.60 (s, 3H), 3.21 (s, 1H), 3.17 (s, 1H), 1.83 (d, J ¼ 13.5 Hz, 6H).
13C NMR (150 MHz, DMSO‑d6)
d 155.55, 153.11, 152.08, 145.14,
140.18 (d, J ¼ 3.1 Hz), 132.33, 130.81 (d, J ¼ 11.4 Hz), 129.53 (d,
J ¼ 6.5 Hz, 2C), 123.23 (d, J ¼ 9.0 Hz), 120.78 (d, J ¼ 12.1 Hz), 120.36
(d, J ¼ 6.5 Hz), 119.69, 118.72 (d, J ¼ 3.2 Hz, 2C), 118.13 (d,
J ¼ 91.8 Hz), 99.05, 97.95, 52.30 (d, J ¼ 6.6 Hz, 2C), 30.43 (d,
J ¼ 135.4 Hz), 18.63 (d, J ¼ 70.5 Hz, 2C). IR (thin film, cmꢀ1) 3423.0,
2922.1, 2852.1, 1602.0, 1579.5, 1518.8, 1441.5, 1385.2, 1306.9, 866.2,
757.2. HRMS calcd for C23H27N5O4P2, [MþNa]þ, 522.1430; found
522.1435.
5.1.6. Preparation of 3-amino-1-methylpyridin-2(1H)-one (17)
NaH (1.1 equiv) was slowly added at 0 ꢁC to a 3-nitropyridin-
2(1H)-one (1 equiv) solution in THF. The reaction mixture was
stirred at 0 ꢁC for 0.5 h. Iodomethane (1 equiv) was then added and
the mixture was stirred at 55 ꢁC for 6 h. The reaction was diluted
with H2O and extracted thrice with EA, and the organic layer was
washed with brine, dried over anhydrous Na2SO4, filtered and
concentrated to afford crude product 16. Intermediate 16 (1 equiv)
was dissolved in methanol, and Pd/C (0.1 equiv) was added. The
flask was flushed with H2 and stirred for 5 h at 40 ꢁC. The reaction
mixture was filtered, concentrated and purified by chromatography
to afford intermediate 17.
5.1.7.4. 2-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)benzamide
White solid; yield: 89%; mp: 223.3e224.0 ꢁC; 1H NMR (400 MHz,
DMSO‑d6)
(20d).
d
11.68 (s, 1H), 11.42 (s, 1H), 9.31 (s, 1H), 9.11 (dd, J ¼ 8.3,
4.1 Hz, 1H), 8.15 (d, J ¼ 7.4 Hz, 1H), 7.93 (d, J ¼ 8.7 Hz, 2H), 7.80 (d,
J ¼ 8.7 Hz, 2H), 7.62e7.50 (m, 2H), 7.09 (t, J ¼ 7.1 Hz, 1H), 7.01 (d,
J ¼ 0.9 Hz, 1H), 6.41 (d, J ¼ 1.9 Hz, 1H), 4.01e3.95 (m, 1H), 3.22 (d,
J ¼ 12.5 Hz, 2H), 2.87 (t, J ¼ 11.2 Hz, 2H), 1.91 (d, J ¼ 11.1 Hz, 2H), 1.84
(d, J ¼ 13.5 Hz, 6H), 1.65 (dd, J ¼ 21.4, 11.1 Hz, 2H). 13C NMR
(150 MHz, DMSO‑d6) d 165.56, 154.93, 153.14, 151.72, 145.04, 144.47,
132.40, 130.89 (d, J ¼ 11.2 Hz), 127.99 (2C), 125.59, 120.99 (d,
J ¼ 12.1 Hz), 120.40 (d, J ¼ 6.5 Hz), 120.23, 118.34 (d, J ¼ 91.8 Hz),
116.80 (2C), 99.49, 98.05, 45.18, 43.33 (2C), 29.87 (2C), 18.63 (d,
J ¼ 70.7 Hz, 2C). IR (thin film, cmꢀ1) 3421.0, 2922.3, 2852.1, 1679.5,
1601.8, 1579.2, 1500.7, 1441.1, 1396.1, 1308.8, 867.2, 755.5. HRMS
calcd for C26H30N7O2P, [MþH]þ, 504.2271; found 504.2262.
5.1.7. Preparation of compounds 20a-l, 22a-g and 25a-h
Compounds 20a-l, 22a-g and 25a-h were prepared in a similar
manner as compounds 11a-h.
5.1.7.1. N-(4-((4-((2-(dimethylphosphoryl)phenyl)amino)-7H-pyr-
5.1.7.5. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-N-(tetrahydro-2H-pyran-4-yl)benza-
mide (20e). Grey solid; yield: 78%; mp: 230.8e231.5 ꢁC; 1H NMR
rolo[2,3-d]pyrimidin-2-yl)amino)phenyl)acetamide
White solid; yield: 73%; mp: 217.9e218.5 ꢁC; 1H NMR (600 MHz,
DMSO‑d6)
(20a).
d
11.62 (s, 1H), 11.26 (s, 1H), 9.77 (s, 1H), 9.11 (dd, J ¼ 8.3,
(600 MHz, DMSO‑d6) d 11.69 (s, 1H), 11.42 (s, 1H), 9.32 (s, 1H), 9.12
3.9 Hz, 1H), 8.89 (s, 1H), 7.71 (d, J ¼ 8.8 Hz, 2H), 7.56 (dd, J ¼ 13.7,
7.6 Hz, 1H), 7.51e7.43 (m, 3H), 7.06 (t, J ¼ 7.3 Hz, 1H), 6.94e6.92 (m,
1H), 6.36 (dd, J ¼ 3.1, 1.9 Hz, 1H), 2.02 (s, 3H), 1.83 (d, J ¼ 13.5 Hz,
(dd, J ¼ 8.3, 3.9 Hz, 1H), 8.07 (d, J ¼ 7.7 Hz, 1H), 7.92 (d, J ¼ 8.7 Hz,
2H), 7.79 (d, J ¼ 8.7 Hz, 2H), 7.59 (dd, J ¼ 14.0, 7.7 Hz, 1H), 7.54 (t,
J ¼ 7.8 Hz, 1H), 7.10 (t, J ¼ 7.3 Hz, 1H), 7.02e7.00 (m, 1H), 6.40 (dd,
J ¼ 3.1, 1.9 Hz, 1H), 4.02e3.98 (m, 1H), 3.89 (d, J ¼ 9.3 Hz, 2H),
3.41e3.38 (m, 2H), 1.84 (d, J ¼ 13.5 Hz, 6H), 1.75 (dd, J ¼ 12.6, 2.2 Hz,
6H). 13C NMR (150 MHz, DMSO‑d6)
d 167.63, 155.68, 153.10, 152.16,
145.15, 137.09, 132.44, 132.28, 130.80 (d, J ¼ 11.5 Hz), 120.74 (d,
J ¼ 12.1 Hz), 120.36 (d, J ¼ 6.2 Hz), 119.56, 119.46 (2C), 118.94 (2C),
118.12 (d, J ¼ 91.9 Hz), 98.93, 97.95, 23.83, 18.63 (d, J ¼ 70.7 Hz, 2C).
IR (thin film, cmꢀ1) 3437.1, 2921.6, 2851.9, 1637.0, 1384.5, 1122.4,
573.3. HRMS calcd for C22H23N6O2P, [MþH]þ, 435.1693; found
435.1693; [MþNa]þ, 457.1512; found 457.1515.
2H), 1.61e1.56 (m, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 165.33,
154.91, 153.14, 151.66, 145.05, 144.37, 132.43, 130.93 (d, J ¼ 11.2 Hz),
127.93 (2C), 125.75, 121.00 (d, J ¼ 12.1 Hz), 120.38 (d, J ¼ 6.3 Hz),
120.24, 118.30 (d, J ¼ 91.8 Hz), 116.82 (2C), 99.48, 98.06, 66.29 (2C),
45.59, 32.63 (2C), 18.66 (d, J ¼ 70.7 Hz, 2C). IR (thin film, cmꢀ1
)
3427.1, 2921.9, 2851.9, 2343.4, 1628.1, 1441.7, 1384.4, 1293.4, 1238.0,
867.1. HRMS calcd for C26H29N6O3P, [MþNa]þ, 527.1931; found
527.1927.
5.1.7.2. 4-((4-((2-(dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-N-ethylbenzenesulfonamide
White solid; yield: 83%; mp: 276.1e277.0 ꢁC; 1H NMR (600 MHz,
DMSO‑d6)
(20b).
d
11.71 (s, 1H), 11.46 (s, 1H), 9.51 (s, 1H), 9.07 (dd, J ¼ 8.4,
5.1.7.6. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-N-(1-methylpiperidin-4-yl)benzamide
(20f). White solid; yield: 63%; mp: 230.2e231.0 ꢁC; 1H NMR
4.0 Hz, 1H), 8.01 (d, J ¼ 8.8 Hz, 2H), 7.65 (d, J ¼ 8.8 Hz, 2H), 7.59 (dd,
J ¼ 14.0, 7.6 Hz, 1H), 7.53 (t, J ¼ 7.8 Hz, 1H), 7.30 (t, J ¼ 5.8 Hz, 1H),
7.10 (t, J ¼ 7.2 Hz,1H), 7.04e7.02 (m,1H), 6.41 (dd, J ¼ 3.1, 1.9 Hz,1H),
2.80e2.73 (m, 2H), 1.84 (d, J ¼ 13.5 Hz, 6H), 0.98 (t, J ¼ 7.2 Hz, 3H).
(600 MHz, DMSO‑d6)
d 11.68 (s, 1H), 11.42 (s, 1H), 9.30 (s, 1H), 9.11
(dd, J ¼ 8.3, 3.9 Hz, 1H), 8.01 (d, J ¼ 7.7 Hz, 1H), 7.92 (d, J ¼ 8.7 Hz,
2H), 7.79 (d, J ¼ 8.7 Hz, 2H), 7.58 (dd, J ¼ 14.1, 7.7 Hz, 1H), 7.54 (t,
J ¼ 7.9 Hz, 1H), 7.09 (t, J ¼ 7.3 Hz, 1H), 7.00 (dd, J ¼ 3.1, 2.4 Hz, 1H),
6.41 (dd, J ¼ 3.2, 1.9 Hz, 1H), 3.78e3.71 (m, 1H), 2.81 (d, J ¼ 11.4 Hz,
2H), 2.20 (s, 3H), 2.01 (t, J ¼ 10.6 Hz, 2H), 1.84 (d, J ¼ 13.5 Hz, 6H),
1.77 (d, J ¼ 12.1 Hz, 2H), 1.61 (qd, J ¼ 12.2, 3.5 Hz, 2H). 13C NMR
13C NMR (150 MHz, DMSO‑d6)
d 154.65, 153.15, 151.57, 145.22,
144.90, 132.32, 130.91 (d, J ¼ 11.2 Hz), 130.64, 127.33 (2C), 121.09 (d,
J ¼ 12.0 Hz), 120.49, 120.45, 118.55 (d, J ¼ 91.7 Hz), 117.33 (2C),
99.69, 98.05, 37.48, 18.61 (d, J ¼ 70.8 Hz, 2C), 14.68. IR (thin film,
cmꢀ1) 3425.3, 3332.7, 2922.1, 2852.5, 1631.0, 1579.7, 1579.7, 1479.1,
1439.2, 1302.0, 869.3, 741.9. HRMS calcd for
C
22H25N6O3PS,
(150 MHz, DMSO‑d6) d 165.49, 154.98, 153.17, 151.76, 145.06, 144.37,
[MþNa]þ, 507.1339; found 507.1348.
132.47, 130.94 (d, J ¼ 11.1 Hz), 127.94 (2C), 125.83, 121.04 (d,

12
R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
J ¼ 12.2 Hz), 120.44 (d, J ¼ 6.4 Hz), 120.26, 118.33 (d, J ¼ 91.8 Hz),
116.84 (2C), 99.50, 98.09, 54.52, 46.25, 45.83 (2C), 31.46 (2C), 18.66
(d, J ¼ 70.9 Hz, 2C). IR (thin film, cmꢀ1) 3423.1, 2921.7, 2851.3,
1631.0, 1499.5, 1440.0, 1384.7, 1308.5, 1242.8, 756.4. HRMS calcd for
153.14, 151.77, 145.00, 143.19, 132.36, 130.86 (d, J ¼ 11.1 Hz), 128.05
(2C), 126.37, 120.97 (d, J ¼ 12.1 Hz), 120.42 (d, J ¼ 6.2 Hz), 120.11,
118.37 (d, J ¼ 91.8 Hz), 117.30 (2C), 99.40, 98.01, 66.16 (2C), 18.61 (d,
J ¼ 70.6 Hz, 2C). Its 13C NMR and DEPT spectrum displayed 23 car-
bon resonances for 2 primary carbon (dC 18.61 ꢂ 2), 2 secondary
C
27H32N7O2P, [MþH]þ, 518.2428; found 518.2420.
carbon
(dC 66.16 ꢂ 2), 10 tertiary carbon
(dC 132.36, 130.86,
5.1.7.7. (R)-4-((4-((2-(dimethylphosphoryl)phenyl)amino)-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl)amino)-N-(piperidin-3-yl)benzamide
(20g). White solid; yield: 73%; mp: 273.5e274.2 ꢁC; 1H NMR
128.05 ꢂ 2, 120.97, 120.42, 120.11, 117.30 ꢂ 2, 98.01), 9 quaternary
carbon (dC 169.44, 155.04, 153.14, 151.77, 145.00, 143.19, 126.37,
118.37, 99.40). IR (thin film, cmꢀ1) 3422.3, 2921.8, 2852.7, 1603.6,
1580.0, 1523.8, 1440.5, 1385.4, 1304.4, 867.5, 758.4. HRMS calcd for
(600 MHz, DMSO‑d6)
d 11.68 (s, 1H), 11.45 (s, 1H), 9.30 (s, 1H), 9.12
(d, J ¼ 4.2 Hz, 1H), 8.04e7.83 (m, 3H), 7.78 (d, J ¼ 8.6 Hz, 2H), 7.59
(dd, J ¼ 14.1, 7.7 Hz, 1H), 7.54 (t, J ¼ 7.7 Hz,1H), 7.10 (t, J ¼ 7.2 Hz, 1H),
7.00 (d, J ¼ 3.0 Hz, 1H), 6.40 (d, J ¼ 3.3 Hz, 1H), 3.87e3.61 (m, 2H),
2.99e2.75 (m, 2H), 2.40 (t, J ¼ 10.5 Hz, 1H), 1.84 (d, J ¼ 13.5 Hz, 6H),
1.66e1.38 (m, 3H), 1.28 (dd, J ¼ 13.5, 7.0 Hz, 1H). 13C NMR (150 MHz,
C
25H27N6O3P, [MþH]þ, 491.1954; found 491.1955; [MþNa]þ,
513.1774; found 513.1774.
5.1.7.11. Dimethyl(2-((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-
7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)phosphine
(20k). White solid; yield: 77%; mp: 179.1e179.6 ꢁC; 1H NMR
(400 MHz, CDCl3)
oxide
DMSO‑d6)
d 165.40, 154.96, 153.14, 151.74, 145.05, 144.31, 132.45,
130.92 (d, J ¼ 11.5 Hz), 127.91 (2C), 125.86, 121.00 (d, J ¼ 11.9 Hz),
120.40 (d, J ¼ 6.2 Hz), 120.23, 118.30 (d, J ¼ 91.9 Hz), 116.81 (2C),
99.46, 98.04, 51.45, 46.82, 45.79, 30.90, 25.58, 18.65 (d, J ¼ 71.0 Hz,
2C). IR (thin film, cmꢀ1) 3427.4, 2921.5, 2851.9, 1634.5, 1440.7,
1384.5, 1308.3, 1125.1, 847.6, 758.5. HRMS calcd for C26H30N7O2P,
[MþNa]þ, 526.2091; found 526.2088.
d
11.11 (s,1H), 9.66 (s, 1H), 8.94 (dd, J ¼ 8.3, 4.4 Hz,
1H), 7.52e7.43 (m, 3H), 7.21 (dd, J ¼ 14.2, 7.5 Hz, 1H), 7.01 (t,
J ¼ 6.6 Hz, 1H), 6.90 (d, J ¼ 8.8 Hz, 2H), 6.86 (s, 1H), 6.57 (d,
J ¼ 3.3 Hz, 1H), 6.51 (d, J ¼ 3.2 Hz, 1H), 3.24e3.18 (m, 4H), 2.68 (s,
4H), 2.41 (s, 3H), 1.83 (d, J ¼ 13.1 Hz, 6H). 13C NMR (100 MHz, CDCl3)
d
156.56, 154.06, 152.63, 146.75, 145.64 (d, J ¼ 2.8 Hz), 133.67,
132.93, 129.65 (d, J ¼ 11.2 Hz), 121.99 (2C), 121.70 (d, J ¼ 6.9 Hz),
121.19 (d, J ¼ 12.4 Hz), 119.07, 117.97 (d, J ¼ 89.0 Hz), 117.46 (2C),
99.88, 99.27, 55.01 (2C), 49.80 (2C), 45.81, 18.97 (d, J ¼ 71.6 Hz, 2C).
IR (thin film, cmꢀ1) 3422.5, 2922.4, 2851.5, 1638.7, 1602.4, 1515.3,
1439.5, 1385.4, 1291.0, 1123.8, 755.0. HRMS calcd for C25H30N7OP,
[MþH]þ, 476.2322; found 476.2301.
5.1.7.8. (S)-4-((4-((2-(dimethylphosphoryl)phenyl)amino)-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl)amino)-N-(piperidin-3-yl)benzamide
Hydrochloride(20h). Yellow solid; yield: 78%; mp: 271.3e272.5 ꢁC;
1H NMR (400 MHz, DMSO‑d6)
d 11.88 (s, 1H), 11.79 (s, 1H), 9.87 (s,
1H), 9.47 (d, J ¼ 8.4 Hz, 1H), 9.07 (d, J ¼ 7.1 Hz, 1H), 8.83 (dd, J ¼ 7.8,
3.7 Hz, 1H), 8.54 (d, J ¼ 7.7 Hz, 1H), 7.92 (d, J ¼ 8.8 Hz, 2H), 7.84 (d,
J ¼ 8.8 Hz, 2H), 7.65 (dd, J ¼ 13.7, 7.5 Hz, 1H), 7.58 (t, J ¼ 7.8 Hz, 1H),
7.19 (t, J ¼ 7.2 Hz, 1H), 7.05 (dd, J ¼ 3.1, 2.4 Hz, 1H), 6.45 (s, 1H),
4.27e4.23 (m, 1H), 3.28 (d, J ¼ 10.3 Hz, 1H), 3.13 (d, J ¼ 11.3 Hz, 1H),
2.91 (td, J ¼ 19.0, 10.3 Hz, 2H), 1.93e1.87 (m, 2H), 1.83 (d, J ¼ 13.6 Hz,
5.1.7.12. Dimethyl(2-((2-((4-(piperazin-1-yl)phenyl)amino)-7H-pyr-
rolo[2,3-d]pyrimidin-4-yl)amino)phenyl)phosphine oxide hydrochlo-
ride (20l). White solid; yield: 90%; mp: 284.9e286.0 ꢁC; 1H NMR
(400 MHz, DMSO‑d6) d 12.39 (s, 1H), 12.15 (s, 1H), 10.35 (s, 1H), 9.61
6H), 1.74e1.65 (m, 2H). 13C NMR (150 MHz, DMSO‑d6)
d
172.04,
(s, 2H), 8.49 (d, J ¼ 4.0 Hz, 1H), 7.70 (ddd, J ¼ 13.7, 7.7, 1.1 Hz, 1H),
7.50e7.42 (m, 3H), 7.27 (t, J ¼ 7.2 Hz,1H), 7.10 (d, J ¼ 9.0 Hz, 2H), 7.05
(dd, J ¼ 3.3, 2.3 Hz, 1H), 6.48 (s, 1H), 3.50e3.40 (m, 4H), 3.26 (s, 4H),
165.51, 153.50, 152.74, 143.65, 143.26, 132.57, 131.21 (d, J ¼ 10.8 Hz),
128.33 (2C), 126.39, 122.50 (d, J ¼ 11.6 Hz), 121.70, 120.79, 120.47 (d,
J ¼ 94.9 Hz), 117.89 (2C), 99.35, 98.98, 46.25, 43.18, 42.92, 27.96,
20.19, 18.56 (d, J ¼ 70.8 Hz, 2C). IR (thin film, cmꢀ1) 3424.8, 2922.3,
2852.1, 1630.0, 1547.7, 1507.6, 1462.9, 1384.2, 869.8, 761.4. HRMS
calcd for C26H30N7O2P, [MþH]þ, 504.2271; found 504.2266.
1.82 (d, J ¼ 13.7 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6)
d 154.25,
150.59, 146.65, 142.27, 132.47, 131.37 (d, J ¼ 10.5 Hz), 130.56, 123.95
(d, J ¼ 9.2 Hz), 123.67 (2C), 122.51, 120.87, 116.82 (2C), 100.13, 98.82,
46.08 (2C), 42.37 (2C), 18.47 (d, J ¼ 70.6 Hz, 2C). IR (thin film, cmꢀ1
)
3428.1, 3100.8, 292.4, 2455.7, 1639.9, 1563.5, 1512.6, 1461.9, 1382.8,
5.1.7.9. (4-((4-((2-(dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)phenyl)(piperazin-1-yl)methanone hy-
drochloride (20i). White solid; yield: 91%; mp: 230.2e231.0 ꢁC; 1H
1370.9, 1120.8, 742.9. HRMS calcd for
462.2166; found 462.2155.
C
24H28N7OP, [MþH]þ,
NMR (600 MHz, DMSO‑d6)
d
11.86 (s, 1H), 11.68 (s, 1H), 9.75 (s, 1H),
5.1.7.13. Dimethyl(2-((2-((1-methyl-1H-pyrazol-4-yl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)phosphine oxide (22a).
White solid; yield: 72%; mp: 138.2e138.9 ꢁC; 1H NMR (600 MHz,
9.38 (s, 2H), 8.87 (s, 1H), 7.85 (d, J ¼ 8.3 Hz, 2H), 7.64 (dd, J ¼ 13.7,
7.6 Hz, 1H), 7.54 (t, J ¼ 7.8 Hz, 1H), 7.43 (d, J ¼ 8.6 Hz, 2H), 7.17 (t,
J ¼ 7.3 Hz, 1H), 7.05e7.02 (m, 1H), 6.44 (s, 1H), 3.75 (s, 4H), 3.16 (s,
DMSO‑d6)
d 11.52 (s, 1H), 11.16 (s, 1H), 9.07 (s, 1H), 8.77 (s, 1H), 7.90
4H),1.83 (d, J ¼ 13.5 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6)
d
172.05,
(s, 1H), 7.57 (dd, J ¼ 13.7, 7.4 Hz, 1H), 7.51 (t, J ¼ 7.6 Hz, 2H), 7.07 (t,
J ¼ 7.2 Hz, 1H), 6.89 (dd, J ¼ 3.3, 2.2 Hz, 1H), 6.34 (dd, J ¼ 3.2, 1.9 Hz,
1H), 3.80 (s, 3H), 1.83 (d, J ¼ 13.5 Hz, 6H). 13C NMR (150 MHz,
169.38, 153.73, 152.11, 143.18, 141.46, 132.51, 131.30 (d, J ¼ 10.8 Hz),
128.29 (2C), 127.84, 122.98 (d, J ¼ 11.4 Hz), 122.19, 120.89, 119.05
(2C), 118.04, 99.30, 99.26, 42.50 (2C), 21.12 (2C), 18.52 (d,
J ¼ 70.8 Hz, 2C). IR (thin film, cmꢀ1) 3422.7, 2922.1, 2852.3, 2375.3,
DMSO‑d6)
d 155.70, 153.35, 152.64, 145.23, 132.28, 130.88 (d,
J ¼ 11.4 Hz), 129.83, 124.50, 120.82 (d, J ¼ 11.8 Hz), 120.40, 120.09,
119.08, 118.39 (d, J ¼ 92.6 Hz), 98.24, 97.94, 38.66, 18.62 (d,
J ¼ 70.7 Hz, 2C). IR (thin film, cmꢀ1) 3421.0, 2922.3, 2852.3, 1631.8,
1578.3, 1438.0, 1385.4, 1291.3, 1124.1, 754.4. HRMS calcd for
1628.7, 1510.6, 1384.3, 1104.8. HRMS calcd for
C25H28N7O2P,
[MþH]þ, 490.2115; found 490.2111; [MþNa]þ, 512.1934; found
512.1938.
C
18H20N7OP, [MþH]þ, 382.1540; found 382.1542; [MþNa]þ,
5.1.7.10. (4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)phenyl)(morpholino)methanone (20j).
White solid; yield: 84%; mp: 191.3e192.1 ꢁC; 1H NMR (600 MHz,
404.1359; found 404.1361.
5.1.7.14. (2-((2-((1-(2,2-Difluoroethyl)-1H-pyrazol-4-yl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide
(22b). White solid; yield: 79%; mp: 136.0e136.6 ꢁC; 1H NMR
DMSO‑d6)
d
11.67 (s, 1H), 11.40 (s, 1H), 9.28 (s, 1H), 9.09 (dd, J ¼ 8.3,
3.9 Hz, 1H), 7.92 (d, J ¼ 8.5 Hz, 2H), 7.58 (dd, J ¼ 13.9, 7.7 Hz, 1H),
7.52 (t, J ¼ 7.8 Hz, 1H), 7.35 (d, J ¼ 8.5 Hz, 2H), 7.09 (t, J ¼ 7.3 Hz, 1H),
7.02e6.97 (m, 1H), 6.40 (s, 1H), 3.62 (s, 4H), 3.53 (s, 4H), 1.83 (d,
(600 MHz, DMSO‑d6) d 11.56 (s,1H),11.19 (s,1H), 9.07 (s,1H), 8.87 (s,
1H), 8.00 (s, 1H), 7.67 (s, 1H), 7.57 (dd, J ¼ 13.9, 7.6 Hz, 1H), 7.51 (t,
J ¼ 13.5 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6)
d 169.44, 155.04,
J ¼ 7.7 Hz, 1H), 7.07 (t, J ¼ 7.2 Hz, 1H), 6.90 (s, 1H), 6.45e6.24 (m,

R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
13
2H), 4.56 (td, J ¼ 14.8, 2.8 Hz, 2H), 1.83 (d, J ¼ 13.5 Hz, 6H). 13C NMR
3H), 2.87 (s, 3H), 1.83 (d, J ¼ 13.5 Hz, 6H). 13C NMR (150 MHz,
(150 MHz, DMSO‑d6)
d
155.58, 153.39, 152.49, 145.23, 132.34,
DMSO‑d6) d 166.94, 155.51, 153.36, 152.38, 145.18, 132.35, 130.87 (d,
131.44, 130.92 (d, J ¼ 11.3 Hz), 125.06, 120.85 (d, J ¼ 11.3 Hz), 120.30,
120.16, 119.22, 118.35 (d, J ¼ 94.3 Hz), 114.31 (t, J ¼ 241.4 Hz), 98.40,
98.00, 53.06 (t, J ¼ 25.9 Hz), 18.64 (d, J ¼ 70.7 Hz, 2C). IR (thin film,
cmꢀ1) 3422.1, 2922.1, 2852.4, 1601.7, 1579.4, 1439.8, 1387.2, 1325.2,
1292.8, 755.6. HRMS calcd for C19H20F2N7OP, [MþH]þ, 432.1508;
found 432.1509. [MþNa]þ, 454.1327; found 454.1322.
J ¼ 11.4 Hz), 130.11, 124.43, 120.80 (d, J ¼ 9.4 Hz), 120.47, 120.32,
119.09,118.32 (d, J ¼ 98.3 Hz), 98.26, 97.99, 53.06, 36.01, 35.21, 18.63
(d, J ¼ 70.9 Hz, 2C). IR (thin film, cmꢀ1) 3424.0, 2922.6, 2852.3,
1639.6, 1579.7, 1475.3, 1438.7, 1385.0, 1123.1, 755.7. HRMS calcd for
C
21H25N8O2P, [MþNa]þ, 475.1730; found 475.1736.
5.1.7.19. 2-(4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-1-
morpholinoethan-1-one (22g). Grey solid; yield: 65%; mp:
5.1.7.15. (2-((2-((1-(2-Methoxyethyl)-1H-pyrazol-4-yl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide
(22c). White solid; yield: 82%; mp: 130.4e131.2 ꢁC; 1H NMR
152.7e153.4 ꢁC; 1H NMR (600 MHz, DMSO‑d6)
d 11.53 (s, 1H), 11.16
(600 MHz, DMSO‑d6)
d
11.53 (s,1H),11.17 (s,1H), 9.08 (s,1H), 8.77 (s,
(s, 1H), 9.07 (s, 1H), 8.81 (s, 1H), 7.90 (s, 1H), 7.59e7.49 (m, 3H), 7.06
(t, J ¼ 7.2 Hz, 1H), 6.88 (dd, J ¼ 3.1, 2.3 Hz, 1H), 6.34 (dd, J ¼ 3.0,
1.9 Hz, 1H), 5.07 (s, 2H), 3.61e3.57 (m, 4H), 3.51 (d, J ¼ 6.5 Hz, 2H),
3.46 (d, J ¼ 4.4 Hz, 2H), 1.82 (d, J ¼ 13.5 Hz, 6H). 13C NMR (150 MHz,
1H), 7.93 (s, 1H), 7.60e7.54 (m, 2H), 7.52 (t, J ¼ 7.9 Hz, 1H), 7.07 (t,
J ¼ 7.3 Hz, 1H), 6.89 (dd, J ¼ 3.3, 2.3 Hz, 1H), 6.35 (dd, J ¼ 3.2, 1.9 Hz,
1H), 4.19 (t, J ¼ 5.3 Hz, 2H), 3.68 (t, J ¼ 5.3 Hz, 2H), 3.24 (s, 3H), 1.83
(d, J ¼ 13.5 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6)
d
155.70, 153.33,
DMSO‑d6) d 165.88, 155.61, 153.34, 152.61, 145.25, 132.35, 130.88 (d,
152.61, 145.24, 132.29, 130.88 (d, J ¼ 11.3 Hz), 130.16, 124.25, 120.77
(d, J ¼ 12.0 Hz), 120.29, 119.73, 119.06, 118.29 (d, J ¼ 91.9 Hz), 98.25,
97.94, 70.85, 58.04, 51.39, 18.63 (d, J ¼ 71.0 Hz, 2C). Its 13C NMR and
DEPT spectrum displayed 20 carbon resonances for 3 primary car-
bon (dC 58.04, 18.63 ꢂ 2), 2 secondary carbon (dC 70.85, 51.39), 8
tertiary carbon (dC 132.29, 130.88, 130.16, 120.77, 120.29, 119.73,
119.06, 97.94), 7 quaternary carbon (dC 155.70, 153.33, 152.61,
145.24, 124.25, 118.29, 98.25). IR (thin film, cmꢀ1) 3424.6, 2922.2,
2852.3, 1828.3, 1439.8, 1384.8, 1291.5, 1118.2, 755.8. HRMS calcd for
J ¼ 11.4 Hz), 130.17, 129.66, 124.61, 120.75 (d, J ¼ 11.5 Hz), 120.37,
119.09, 98.28, 97.95, 66.06, 65.99, 52.93, 44.89, 41.81, 18.63 (d,
J ¼ 70.5 Hz, 2C). IR (thin film, cmꢀ1) 3364.3, 2920.8, 2850.5, 1657.7,
1633.2, 1580.0, 1468.9, 1403.2, 1240.2, 1116.4, 720.4. HRMS calcd for
C
23H27N8O3P, [MþH]þ, 495.2017; found 495.2022; [MþNa]þ,
517.1836; found 517.1847.
5.1.7.20. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-3-fluoro-N-(piperidin-4-yl)benzamide
hydrochloride (25a). White solid; yield: 87%; mp: 253.2e254.0 ꢁC;
C
20H24N7O2P, [MþNa]þ, 448.1621; found 448.1627.
1H NMR (600 MHz, DMSO‑d6)
d 12.16 (s, 2H), 9.73 (s, 1H), 9.11 (s,
5.1.7.16. dimethyl(2-((2-((1-(tetrahydro-2H-pyran-4-yl)-1H-pyr-
azol-4-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)
phosphine oxide (22d). Yellow solid; yield: 63%; mp:
2H), 8.62 (d, J ¼ 6.5 Hz, 1H), 8.45 (s, 1H), 8.10 (s, 1H), 7.88 (d,
J ¼ 11.7 Hz, 1H), 7.76 (d, J ¼ 8.0 Hz, 1H), 7.69 (dd, J ¼ 12.0, 7.6 Hz, 1H),
7.45 (s, 1H), 7.25 (s, 1H), 7.09 (s, 1H), 6.49 (s, 1H), 3.31 (d, J ¼ 10.2 Hz,
2H), 2.99 (s, 2H), 1.97 (d, J ¼ 11.7 Hz, 2H), 1.89e1.80 (m, 8H). 13C
196.4e197.0 ꢁC; 1H NMR (600 MHz, DMSO‑d6)
d 11.51 (s, 1H), 11.16
(s,1H), 9.06 (s,1H), 8.78 (s,1H), 8.00 (s,1H), 7.58 (dd, J ¼ 14.0, 7.6 Hz,
1H), 7.54 (s, 1H), 7.50 (t, J ¼ 7.8 Hz, 1H), 7.08 (t, J ¼ 7.3 Hz, 1H), 6.89
(dd, J ¼ 3.2, 2.3 Hz, 1H), 6.37e6.33 (m, 1H), 4.31 (ddd, J ¼ 15.5, 11.1,
4.3 Hz, 1H), 3.97 (dd, J ¼ 11.1, 3.2 Hz, 2H), 3.48 (td, J ¼ 11.6, 1.9 Hz,
2H), 1.99e1.90 (m, 4H), 1.82 (d, J ¼ 13.5 Hz, 6H). 13C NMR (150 MHz,
NMR (150 MHz, DMSO‑d6)
d
164.18, 153.71, 153.26 (d, J ¼ 245.5 Hz),
151.23, 142.53, 132.46, 131.39 (d, J ¼ 10.5 Hz), 130.03, 129.67, 129.59
(d, J ¼ 10.5 Hz), 123.82, 123.62, 123.01, 122.49, 121.38, 114.64, 114.50,
99.69, 99.40, 44.58, 42.12 (2C), 28.11 (2C), 18.47 (d, J ¼ 70.8 Hz, 2C).
IR (thin film, cmꢀ1) 3424.8, 2924.2, 1639.2, 1618.8, 1541.5, 1501.6,
1463.3, 1383.0, 1339.0, 1311.0, 870.6, 736.0. HRMS calcd for
DMSO‑d6)
d 155.72, 153.38, 152.60, 145.23, 132.32, 130.94 (d,
J ¼ 11.2 Hz), 129.76, 124.17, 120.85 (d, J ¼ 10.1 Hz), 120.39, 119.08,
118.48 (d, J ¼ 88.7 Hz), 117.11, 98.24, 97.97, 66.01 (2C), 57.12, 33.12
(2C), 18.64 (d, J ¼ 70.7 Hz, 2C). IR (thin film, cmꢀ1) 3425.1, 2922.9,
2851.9, 1601.3, 1578.7, 1440.8, 1385.8, 1325.0, 1292.3, 755.4. HRMS
C
26H29FN7O2P, [MþH]þ, 522.2177; found 522.2180.
5.1.7.21. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-3-methoxy-N-(piperidin-4-yl)benza-
mide(25b). White solid; yield: 83%; mp: 183.3e184.1 ꢁC; 1H NMR
calcd for
C
22H26N7O2P, [MþH]þ, 452.1958; found 452.1960;
[MþNa]þ, 474.1778; found 474.1778.
(600 MHz, DMSO‑d6)
d
11.64 (s, 1H), 11.57 (s, 1H), 8.90 (dd, J ¼ 8.1,
3.8 Hz, 1H), 8.57 (d, J ¼ 8.6 Hz, 1H), 8.17 (d, J ¼ 7.6 Hz, 1H), 7.72 (s,
1H), 7.64e7.53 (m, 4H), 7.12 (t, J ¼ 7.2 Hz,1H), 7.04 (d, J ¼ 3.1 Hz,1H),
6.41 (d, J ¼ 3.1 Hz, 1H), 3.98 (s, 3H), 3.86 (s, 1H), 2.98 (d, J ¼ 11.3 Hz,
2H), 2.71e2.52 (m, 2H), 1.84 (d, J ¼ 13.5 Hz, 6H), 1.75 (d, J ¼ 10.7 Hz,
2H), 1.46 (dd, J ¼ 20.1, 11.2 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6)
5.1.7.17. Dimethyl(2-((2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)
amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)phosphine
oxide hydrochloride (22e). White solid; yield: 92%; mp:
247.3e248.2 ꢁC; 1H NMR (600 MHz, DMSO‑d6)
d 12.13 (s, 2H), 10.18
(s, 1H), 9.47 (s, 1H), 9.23 (s, 1H), 8.26 (s, 1H), 7.79 (s, 2H), 7.57 (s, 2H),
7.37 (s, 1H), 7.03 (s, 1H), 6.50 (s, 1H), 4.39 (s, 1H), 3.36 (d, J ¼ 10.1 Hz,
2H), 3.07 (s, 2H), 2.16 (s, 4H), 1.79 (d, J ¼ 12.4 Hz, 6H). 13C NMR
d 165.00, 154.60, 153.27, 151.74, 146.79, 144.75, 132.54, 132.48,
131.01 (d, J ¼ 11.1 Hz), 126.62, 121.29 (d, J ¼ 12.0 Hz), 120.56, 120.52,
120.21, 118.85 (d, J ¼ 91.8 Hz), 116.56, 109.22, 99.67, 98.10, 56.07,
47.49, 45.52 (2C), 33.21 (2C), 18.58 (d, J ¼ 70.7 Hz, 2C). Its 13C NMR
and DEPT spectrum displayed 27 carbon resonances for 3 primary
carbon (dC 56.07, 18.58 ꢂ 2), 4 secondary carbon (dC 45.52 ꢂ 2,
33.21 ꢂ 2), 10 tertiary carbon (dC 132.48, 131.01, 121.29, 120.56,
120.52, 120.21, 116.56, 109.22, 98.10, 47.49), 10 quaternary carbon
(150 MHz, DMSO‑d6) d 155.77, 153.53, 152.74, 145.54, 132.26, 130.94
(d, J ¼ 11.0 Hz), 129.43, 124.15, 120.60, 120.42, 119.07, 116.85, 98.42,
98.02, 59.18, 45.24 (2C), 33.77 (2C), 18.59 (d, J ¼ 70.6 Hz, 2C). IR
(thin film, cmꢀ1) 3426.6, 2922.5, 2852.3, 2343.6, 1638.9, 1546.1,
1463.5, 1384.0, 1124.8, 725.9. HRMS calcd for C22H27N8OP, [MþH]þ,
451.2118; found 451.2123.
(
dC 165.00, 154.60, 153.27, 151.74, 146.79, 144.75, 132.54, 126.62,
118.85, 99.67). IR (thin film, cmꢀ1) 3421.7, 2922.3, 2852.0, 1602.9,
1581.4, 1518.6, 1485.5, 1440.8, 1384.0, 1326.5, 1124.1, 755.8. HRMS
calcd for C27H32N7O3P, [MþNa]þ, 556.2196; found 556.2192.
5.1.7.18. 2-(4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyr-
rolo[2,3-d]pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)-N,N-dimethyla-
cetamide (22f). White solid; yield: 62%; mp: 207.9e208.8 ꢁC; 1H
NMR (600 MHz, DMSO‑d6)
d
11.55 (s, 1H), 11.17 (s, 1H), 9.08 (s, 1H),
5.1.7.22. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-3-ethoxy-N-(piperidin-4-yl)benzamide
(25c). White solid; yield: 89%; mp: 246.1e246.8 ꢁC; 1H NMR
8.83 (s, 1H), 7.89 (s, 1H), 7.54 (ddd, J ¼ 24.5, 14.9, 7.8 Hz, 3H), 7.08 (s,
1H), 6.91e6.87 (m, 1H), 6.35 (d, J ¼ 0.9 Hz, 1H), 5.03 (s, 2H), 3.04 (s,

14
R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
(600 MHz, DMSO‑d6)
d
11.63 (s,1H),11.52 (s,1H), 8.90e8.84 (m,1H),
(150 MHz, DMSO‑d6)
d
163.05, 153.96, 153.42 (d, J ¼ 240.7 Hz),
8.58 (d, J ¼ 8.2 Hz, 1H), 8.27 (d, J ¼ 7.3 Hz, 1H), 7.69 (s, 1H), 7.62 (dd,
J ¼ 13.8, 7.6 Hz, 1H), 7.59e7.53 (m, 3H), 7.13 (t, J ¼ 7.1 Hz, 1H), 7.05 (s,
1H), 6.40 (s, 1H), 4.24 (dd, J ¼ 13.4, 6.6 Hz, 2H), 3.98e3.95 (m, 1H),
3.16 (d, J ¼ 11.8 Hz, 2H), 2.77 (t, J ¼ 11.8 Hz, 2H), 1.90e1.81 (m, 8H),
1.66 (dd, J ¼ 21.6, 10.6 Hz, 2H), 1.47 (t, J ¼ 6.7 Hz, 3H). 13C NMR
150.08, 144.42, 141.81, 132.70, 131.62 (d, J ¼ 10.1 Hz), 130.64 (d,
J ¼ 12.3 Hz), 124.45, 123.52, 121.60, 116.54 (d, J ¼ 11.5 Hz), 111.05,
111.03, 106.94 (d, J ¼ 32.5 Hz), 100.06, 99.27, 56.57, 44.41, 41.90 (2C),
27.99 (2C), 18.39 (d, J ¼ 70.7 Hz, 2C). IR (thin film, cmꢀ1) 3424.7,
2924.0, 2852.4, 1616.9, 1537.4, 1498.1, 1463.0, 1387.0, 1300.3, 1121.7,
873.3, 756.1. HRMS calcd for C27H31FN7O3P, [MþH]þ, 552.2283;
found 552.2287.
(150 MHz, DMSO‑d6) d 165.26, 154.49, 153.26, 151.83,145.83, 144.67,
132.80, 132.34, 131.04 (d, J ¼ 11.2 Hz), 126.22, 121.40 (d, J ¼ 12.0 Hz),
120.63, 120.58, 120.35, 119.09 (d, J ¼ 91.8 Hz), 116.29, 110.22, 99.66,
98.12, 64.32, 45.99, 43.87 (2C), 30.74 (2C), 18.55 (d, J ¼ 70.7 Hz, 2C),
14.70. IR (thin film, cmꢀ1) 3423.5, 2921.8, 2851.8, 1630.6, 1518.8,
1440.1, 1384.1, 1305.0, 1206.3, 1121.6, 865.7, 754.6. HRMS calcd for
5.1.7.26. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-2-methyl-N-(piperidin-4-yl)benza-
mide (25g). White solid; yield: 81%; mp: 243.7e244.2 ꢁC; 1H NMR
C
28H34N7O3P, [MþH]þ, 548.2534; found 548.2544.
(400 MHz, DMSO‑d6)
d
11.66 (s, 1H), 11.37 (s, 1H), 9.11 (dd, J ¼ 8.1,
3.9 Hz, 1H), 9.07 (s, 1H), 7.93 (d, J ¼ 7.9 Hz, 1H), 7.69 (d, J ¼ 7.4 Hz,
2H), 7.61e7.50 (m, 2H), 7.23 (d, J ¼ 8.2 Hz,1H), 7.08 (t, J ¼ 7.1 Hz,1H),
6.97 (d, J ¼ 2.8 Hz, 1H), 6.39 (d, J ¼ 2.9 Hz, 1H), 3.81e3.71 (m, 1H),
2.94 (d, J ¼ 11.9 Hz, 2H), 2.35 (s, 3H), 1.84 (d, J ¼ 13.5 Hz, 6H), 1.74 (d,
J ¼ 11.6 Hz, 2H), 1.42e1.32 (m, 2H). 13C NMR (150 MHz, DMSO‑d6)
5.1.7.23. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-3-isopropoxy-N-(piperidin-4-yl)ben-
zamide (25d). Yellow solid; yield: 82%; mp: 172.4e174.6 ꢁC; 1H
NMR (600 MHz, DMSO‑d6)
d 11.62 (s, 1H), 11.53 (s, 1H), 8.84 (dd,
J ¼ 8.2, 3.8 Hz, 1H), 8.57 (d, J ¼ 8.4 Hz, 1H), 8.07 (d, J ¼ 7.8 Hz, 1H),
7.65e7.59 (m, 2H), 7.56 (t, J ¼ 7.8 Hz, 1H), 7.53 (s, 1H), 7.51 (d,
J ¼ 8.5 Hz, 1H), 7.12 (t, J ¼ 7.2 Hz, 1H), 7.04 (d, J ¼ 3.3 Hz, 1H), 6.38 (d,
J ¼ 3.3 Hz, 1H), 4.80e4.73 (m, 1H), 3.85e3.80 (m, 1H), 2.96 (d,
J ¼ 12.1 Hz, 2H), 1.83 (d, J ¼ 13.5 Hz, 6H), 1.74 (d, J ¼ 10.1 Hz, 2H),
d 168.16,155.19,153.08,151.89,145.10,142.46,135.79,132.45,130.90
(d, J ¼ 11.2 Hz), 129.03, 127.79, 120.87 (d, J ¼ 11.9 Hz), 120.23 (d,
J ¼ 6.3 Hz), 120.03, 119.44, 118.18 (d, J ¼ 91.9 Hz), 114.70, 99.28,
97.99, 47.05, 45.30, 33.09 (2C), 20.18 (2C), 18.66 (d, J ¼ 70.8 Hz, 2C).
IR (thin film, cmꢀ1) 3423.5, 2921.6, 2851.5, 1630.4, 1543.2, 1439.4,
1384.4, 1304.4, 1241.5, 867.9, 754.5. HRMS calcd for C27H32N7O2P,
[MþH]þ, 518.2428; found 518.2432; [MþNa]þ, 540.2247; found
540.2257.
1.46e1.36 (m, 8H). 13C NMR (150 MHz, DMSO‑d6)
d 164.93, 154.42,
153.23, 151.86, 144.67, 144.49, 133.75, 132.26, 131.02 (d, J ¼ 11.1 Hz),
126.43, 121.39 (d, J ¼ 12.0 Hz), 120.66, 120.56 (d, J ¼ 6.6 Hz), 120.41,
119.13 (d, J ¼ 91.8 Hz),116.35,112.18, 99.62, 98.06, 71.37, 47.46, 45.49
(2C), 33.21 (2C), 21.96 (2C), 18.53 (d, J ¼ 70.4 Hz, 2C). Its 13C NMR
and DEPT spectrum displayed 29 carbon resonances for 4 primary
carbon (dC 21.96 ꢂ 2, 18.53 ꢂ 2), 4 secondary carbon (dC 45.49 ꢂ 2,
33.21 ꢂ 2), 11 tertiary carbon (dC 132.26, 131.02, 121.39, 120.66,
120.56, 120.41, 116.35, 112.18, 98.06, 71.37, 47.46), 10 quaternary
carbon (dC 164.93, 154.42, 153.23, 151.86, 144.67, 144.49, 133.75,
126.43, 119.13, 99.62). IR (thin film, cmꢀ1) 3422.2, 2922.7, 2852.4,
1630.8, 1517.3, 1481.2, 1441.4, 1384.1, 1326.8, 1123.3, 755.8. HRMS
calcd for C29H36N7O3P, [MþH]þ, 562.2690; found 562.2702.
5.1.7.27. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-2-fluoro-N-(piperidin-4-yl)benzamide
(25h). White solid; yield: 88%; mp: 277.4e278.5 ꢁC; 1H NMR
(400 MHz, DMSO‑d6)
d 11.70 (s, 1H), 11.56 (s, 1H), 9.51 (s, 1H), 9.07
(dd, J ¼ 8.2, 4.0 Hz, 1H), 8.08 (d, J ¼ 14.2 Hz, 1H), 7.76 (dd, J ¼ 7.5,
3.3 Hz, 1H), 7.62e7.51 (m, 4H), 7.11 (t, J ¼ 7.2 Hz, 1H), 7.04 (d,
J ¼ 3.3 Hz, 1H), 6.42 (d, J ¼ 3.3 Hz, 1H), 3.86e3.77 (m, 1H), 2.94 (d,
J ¼ 12.2 Hz, 2H), 2.53e2.48 (m, 2H), 1.84 (d, J ¼ 13.5 Hz, 6H), 1.76 (d,
J ¼ 10.6 Hz, 2H), 1.44e1.34 (m, 2H). 13C NMR (100 MHz, DMSO‑d6)
d
162.75, 160.01 (d, J ¼ 244.9 Hz), 154.58, 153.14, 151.50, 145.37 (d,
5.1.7.24. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)-3-(trifluoromethyl)
benzamide (25e). Grey solid; yield: 91%; mp: 183.8e184.6 ꢁC; 1H
J ¼ 12.4 Hz), 144.90, 132.44, 130.92 (d, J ¼ 11.3 Hz), 130.19 (d,
J ¼ 4.6 Hz), 121.11 (d, J ¼ 12.0 Hz), 120.51, 120.41 (d, J ¼ 7.0 Hz),
118.53 (d, J ¼ 91.8 Hz), 114.58 (d, J ¼ 14.1 Hz), 113.21, 103.98 (d,
J ¼ 29.1 Hz), 99.68, 98.05, 47.27, 45.18 (2C), 32.98 (2C), 18.61 (d,
J ¼ 70.7 Hz, 2C). IR (thin film, cmꢀ1) 3413.8, 2921.5, 2851.7, 1627.3,
1523.3, 1384.4, 1242.2, 866.6, 754.4. HRMS calcd for C26H29FN7O2P,
[MþH]þ, 522.2177; found 552.2183.
NMR (600 MHz, DMSO‑d6)
d
11.73 (s, 1H), 8.76 (d, J ¼ 4.3 Hz, 1H),
8.46 (d, J ¼ 7.7 Hz, 1H), 8.27 (d, J ¼ 8.5 Hz, 1H), 8.22 (d, J ¼ 1.5 Hz,
1H), 8.19e8.13 (m, 1H), 8.09e7.87 (m, 1H), 7.60e7.56 (m, 1H), 7.39
(t, J ¼ 7.8 Hz, 1H), 7.07 (t, J ¼ 7.3 Hz, 1H), 7.03 (d, J ¼ 3.3 Hz, 1H), 6.39
(d, J ¼ 3.4 Hz, 1H), 3.90e3.84 (m, 1H), 2.98 (d, J ¼ 12.2 Hz, 2H), 2.51
(dd, J ¼ 3.5, 1.7 Hz, 2H), 1.83 (d, J ¼ 13.5 Hz, 6H), 1.77 (dd, J ¼ 11.7,
2.1 Hz, 2H), 1.47e1.43 (m, 2H). 13C NMR (150 MHz, DMSO‑d6)
5.1.8. General procedure for the synthesis of intermediates 28a-f
4-nitro-1H-pyrazole (1 equiv), corresponding alcohols (1 equiv)
and triphenylphosphine (1.5 equiv) were dissolved in THF, and di-
tert-butyl azodicarboxylate (1.5 equiv) was added over approxi-
mately 10 min. The resulting mixture was stirred at room temper-
ature for 5 h and concentrated to dryness. The residue was purified
by column chromatography to give intermediates 27a-c. A solution
of 4-nitro-1H-pyrazole (1 equiv), potassium carbonate (1 equiv)
and the alkylating reagent (1.1 eq) in acetonitrile was heated at
40e80 ꢁC for 8 h. The mixture was diluted with ethyl acetate, and
the organic layer was washed with brine, dried over anhydrous
Na2SO4 and filtered. The filtrate was concentrated and purified by
chromatography to give intermediates 27b-f. Intermediates 27a-f
(1 equiv) were dissolved in ethanol, and Pd/C (0.1 equiv) was added.
The flask was flushed with H2 and stirred for 3 h at 40 ꢁC. The re-
action mixture was filtered through a Celite pad and the filtrate was
concentrated to dryness, yielding intermediates 28a-f.
d
163.72,155.42, 153.16,151.86, 144.80,141.20, 132.20, 131.74, 130.92
(d, J ¼ 11.3 Hz), 128.73, 124.17 (q, J ¼ 273.4 Hz), 125.48 (d, J ¼ 5.0 Hz),
125.16, 121.09 (d, J ¼ 11.9 Hz), 120.62, 120.17 (d, J ¼ 6.4 Hz), 119.64
(q, J ¼ 29.0 Hz), 118.50 (d, J ¼ 91.8 Hz), 99.86, 98.02, 47.69, 45.42
(2C), 33.09 (2C), 18.59 (d, J ¼ 70.4 Hz, 2C). IR (thin film, cmꢀ1
)
3447.5, 2922.3, 2852.3, 1637.1, 1546.0, 1512.5, 1442.5, 1384.1, 1304.5,
866.2, 754.4. HRMS calcd for C27H29F3N7O2P, [MþNa]þ, 594.1965;
found 594.1963.
5.1.7.25. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)-2-fluoro-5-methoxy-N-(piperidin-4-yl)
benzamide hydrochloride (25f). White solid; yield: 86%; mp:
253.5e254.2 ꢁC; 1H NMR (600 MHz, DMSO‑d6)
d 12.45 (s, 1H), 12.02
(s, 1H), 9.16 (s, 3H), 8.35 (d, J ¼ 7.3 Hz, 2H), 8.16 (d, J ¼ 12.2 Hz, 1H),
7.76 (dd, J ¼ 13.1, 7.7 Hz,1H), 7.61 (t, J ¼ 7.7 Hz,1H), 7.34 (t, J ¼ 7.2 Hz,
1H), 7.21 (d, J ¼ 6.5 Hz,1H), 7.14e7.11 (m,1H), 6.54 (s,1H), 4.07e4.02
(m, 1H), 3.91 (s, 3H), 3.28 (d, J ¼ 12.5 Hz, 2H), 2.99 (dd, J ¼ 21.1,
11.7 Hz, 2H), 1.99 (d, J ¼ 10.7 Hz, 2H), 1.84e1.75 (m, 8H). 13C NMR
5.1.9. General procedure for the synthesis of compounds 31a-h
To a solution of tert-butyl 4-aminopiperidine-1-carboxylate (1

R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
15
equiv) and appropriate acids (1 equiv) in CH2Cl2 at rt, HATU (1.2
equiv) and DIEA (1.5 equiv) were added. The resulting mixture was
warmed to 25 ꢁC-40 ꢁC and stirred until completion of the reaction.
The mixture was diluted with ethyl acetate, and the organic layer
was washed with brine, dried over anhydrous Na2SO4 and filtered.
The filtrate was concentrated and purified by chromatography to
yield the intermediates 30a-h. Intermediates 30a-h (1 equiv) were
dissolved in ethanol, and Pd/C (0.1 equiv) was added. The flask was
flushed with H2 and stirred for 3 h at 40 ꢁC. The reaction mixture
was filtered through a Celite pad and the filtrate was concentrated
to dryness, yielding intermediates 31a-h.
(400 MHz, DMSO‑d6)
d
8.72 (dd, J ¼ 7.5, 3.5 Hz, 1H), 8.46 (d,
J ¼ 8.8 Hz, 1H), 8.11 (t, J ¼ 6.3 Hz, 2H), 7.93 (s, 1H), 7.65 (dd, J ¼ 13.8,
7.7 Hz, 1H), 7.60e7.50 (m, 3H), 7.29 (d, J ¼ 5.3 Hz, 1H), 7.19 (t,
J ¼ 7.2 Hz, 1H), 3.96 (s, 3H), 3.90e3.81 (m, 1H), 2.98 (d, J ¼ 11.4 Hz,
2H), 2.55 (s, 2H), 1.83 (d, J ¼ 13.6 Hz, 6H), 1.76 (d, J ¼ 11.5 Hz, 2H),
1.49e1.39 (m, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 164.96, 161.34,
157.18, 154.78, 147.53, 144.11, 134.14, 132.34, 132.05, 131.08 (d,
J ¼ 10.8 Hz), 127.55, 123.80, 122.25, 121.92 (d, J ¼ 6.5 Hz), 120.39 (d,
J ¼ 94.5 Hz), 120.08, 117.93, 109.94, 109.35, 56.01, 47.45, 45.46 (2C),
33.16 (2C), 18.42 (d, J ¼ 70.6 Hz, 2C). IR (thin film, cmꢀ1) 3426.5,
2922.3, 2852.1, 1636.1, 1547.0, 1510.8, 1438.5, 1384.3, 1109.2, 868.0,
755.8. HRMS calcd for C27H31N6O3PS, [MþH]þ, 551.1989; found
551.1985.
5.1.10. Preparation of compounds 34b, 35, 38b and 39
Compounds 34b, 35, 38b and 39 were prepared in a similar
manner as compounds 11a-h.
5.2. Pharmacological assay
5.1.10.1. (2-((2-((1-(2-Methoxyethyl)-1H-pyrazol-4-yl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide
(34b). White solid; yield: 47%; mp: 128.7e129.2 ꢁC; 1H NMR
5.2.1. FAK HTRF assay
The FAK kinase assay was performed using the HTRF® KinE-
ASE™-TK kit (Cisbio Bioassays, France) in white 384-well small
(400 MHz, CDCl3)
d
11.48 (s, 1H), 8.92 (s, 1H), 7.96 (s, 1H), 7.58e7.42
volume plates with a total working volume of 20 mL. Purified FAK
(m, 3H), 7.25e7.17 (m, 1H), 7.05 (s, 1H), 7.00e6.89 (m, 2H), 4.26 (s,
enzyme was purchased from Carna Biosciences (Japan). Com-
pounds were diluted step-by-step from a concentrated stock of
8 mM in 100% DMSO and with serial kinase reaction buffer di-
lutions. The IC50 measurements were performed in replicates. For
2H), 3.77 (s, 2H), 3.33 (s, 3H), 1.84 (d, J ¼ 13.0 Hz, 6H). 13C NMR
(100 MHz, CDCl3)
d
169.14, 156.88, 155.07, 145.08 (d, J ¼ 2.7 Hz),
132.99, 132.99 (d, J ¼ 1.9 Hz), 129.78 (d, J ¼ 11.2 Hz), 123.08, 121.93,
121.8, 121.77, 118.69, 118.20 (d, J ¼ 98.6 Hz), 118.18, 112.52, 71.43,
59.10, 52.47, 19.04 (d, J ¼ 71.7 Hz, 2C). IR (thin film, cmꢀ1) 3438.0,
2922.3, 2852.2, 1628.1, 1384.2, 1106.1, 580.1. HRMS calcd for
each assay,
(ATP þ Substrate TK) and 2
the assay wells. The assay plates were incubated at 30 ꢁC for 50 min
and reactions were terminated by adding 10 L of mix 2 (Sa-
4
mL
of dispensed compounds,
4
m
L
of mix
1
mL of kinase (0.111 ng/mL) were added to
C
20H23N6O2PS, [MþNa]þ, 465.1233; found 465.1238.
m
XL665þTK-Antibody-Cryptate). After a final incubation (60 min at
room temperature), HTRF signals were obtained by reading plates
using an Infinite® F500 microplate reader (Tecan, Switzerland). The
fluorescence was measured at 620 nM (Cryptate) and 665 nM
(XL665). A ratio was calculated (665/620) for each well. For IC50
measurements, values were normalized and fitted with Prism
(GraphPad software).
5.1.10.2. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)thieno[2,3-d]
pyrimidin-2-yl)amino)-3-methoxy-N-(piperidin-4-yl)benzamide hy-
drochloride (35). Yellow solid; yield: 89%; mp: 223.7e224.4 ꢁC; 1H
NMR (600 MHz, DMSO‑d6)
d 12.44 (s, 1H), 9.20e9.03 (m, 3H), 8.72
(s, 1H), 8.57 (d, J ¼ 7.3 Hz, 1H), 8.02 (d, J ¼ 8.0 Hz, 1H), 7.69 (dd,
J ¼ 13.1, 7.6 Hz, 1H), 7.63 (d, J ¼ 1.5 Hz, 1H), 7.57 (dd, J ¼ 8.3, 1.6 Hz,
1H), 7.50 (dd, J ¼ 17.1, 6.9 Hz, 2H), 7.45 (d, J ¼ 5.9 Hz, 1H), 7.23 (t,
J ¼ 7.3 Hz, 1H), 4.11e4.06 (m, 1H), 3.93 (s, 3H), 3.31 (d, J ¼ 12.4 Hz,
2H), 3.00 (dd, J ¼ 22.1,12.0 Hz, 2H), 2.01e1.96 (m, 2H),1.89e1.83 (m,
5.2.2. Cell proliferation assay
The antiproliferative activities of the new synthesized com-
pounds were evaluated against A549, MDA-MB-231 and HK2 cell
lines by the standard MTT assay in vitro [30]. TAE-226 was
employed as the positive control. The cell lines were suspended in
medium to adjust their concentration to approximate 5 ꢂ 104 cells/
8H). 13C NMR (150 MHz, DMSO‑d6)
d 172.01, 165.30, 154.55, 154.40,
149.89, 143.14, 132.51, 131.25 (d, J ¼ 11.2 Hz), 130.01, 123.14 (d,
J ¼ 11.7 Hz), 121.56 (d, J ¼ 6.2 Hz), 121.46, 120.80, 120.38 (d,
J ¼ 90.6 Hz), 120.16, 118.58, 112.91, 110.30, 56.10, 44.64, 42.19 (2C),
28.23 (2C), 18.51 (d, J ¼ 70.8 Hz, 2C). IR (thin film, cmꢀ1) 3450.9,
2923.3, 2852.6, 1627.0, 1546.9, 1526.1, 1441.6, 1384.4, 1340.0, 1109.2,
873.5, 754.5. HRMS calcd for C27H31N6O3PS, [MþH]þ, 551.1989;
found 551.1995.
mL. The suspension was dispensed at 100 mL/well to a 96-well plate
and at 37 ꢁC in a humidified atmosphere with 5% CO2 for 24 h. The
tested compounds at the indicated final concentrations were added
to the culture medium and incubated for 72 h. Fresh MTT was
added to each well at the terminal concentration of 5 mg/mL in PBS,
and incubated with cells at 37 ꢁC for 4 h. The formazan crystals in
each well were dissolved in 150 mL DMSO, and the absorbency of
each test well was performed at l490nm by a Thermo reader
(Mulitiskan go).
5.1.10.3. (2-((2-((1-(2-Methoxyethyl)-1H-pyrazol-4-yl)amino)thieno
[3,2-d]pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (38b).
White solid; yield: 38%; mp: 96.5e97.6 ꢁC; 1H NMR (400 MHz,
CDCl3)
d
11.30 (s, 1H), 8.82 (dd, J ¼ 8.1, 4.2 Hz, 1H), 7.94 (s, 1H), 7.67
(d, J ¼ 5.2 Hz, 1H), 7.53 (dd, J ¼ 16.2, 8.3 Hz, 2H), 7.29e7.19 (m, 2H),
7.08 (t, J ¼ 6.7 Hz, 1H), 7.01 (s, 1H), 4.26 (t, J ¼ 5.3 Hz, 2H), 3.77 (t,
J ¼ 5.3 Hz, 2H), 3.33 (s, 3H), 1.85 (d, J ¼ 13.2 Hz, 6H). 13C NMR
5.2.3. Analysis of cell apoptosis
The ability of 25b to induce apoptosis of A549 cells was quan-
tified by annexin V and PI staining and flow cytometry. The Annexin
V-FITC Apoptosis Kit was purchased from US Everbright® Inc.
Briefly, after treatment with 25b for 72 h, cells were harvested,
washed with PBS twice, and subjected to annexin V and propidium
iodide staining using the annexin V FITC apoptosis kit following the
step-by-step protocol provided by the manufacturer. After staining,
flow cytometry was performed for the quantification of apoptotic
cells.
(100 MHz, CDCl3)
d
161.70, 158.10, 155.34, 144.91 (d, J ¼ 2.5 Hz),
132.91 (d, J ¼ 2.0 Hz), 132.58, 131.93, 129.78 (d, J ¼ 11.2 Hz), 123.86,
123.32, 122.37 (d, J ¼ 7.2 Hz), 122.17 (d, J ¼ 12.4 Hz), 121.83, 118.60
(d, J ¼ 95.1 Hz), 109.87, 71.46, 59.12, 52.48, 19.03 (d, J ¼ 71.4 Hz, 2C).
IR (thin film, cmꢀ1) 3441.2, 2923.0, 2852.6, 1633.2, 1384.1, 1116.9,
867.2, 469.8. HRMS calcd for C20H23N6O2PS, [MþH]þ, 443.1414;
found 443.1420.
5.1.10.4. 4-((4-((2-(Dimethylphosphoryl)phenyl)amino)thieno[3,2-d]
pyrimidin-2-yl)amino)-3-methoxy-N-(piperidin-4-yl)benzamide
(39). White solid; yield: 83%; mp: 193.5e194.2 ꢁC; 1H NMR
5.2.4. Cell migration assay
A
transwell chamber assay was conducted in Transwell

16
R. Wang et al. / European Journal of Medicinal Chemistry 183 (2019) 111716
chambers (Corning Incorporated). The cells were trypsinized and
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The liver microsomal stability assay was performed by incu-
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5.2.6. Cytochrome P450 inhibition assay
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were then centrifuged, and the supernatants were analyzed by LC/
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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