Discovery of pyrrolo[2,3-d]pyrimidine derivatives as potent Axl inhibitors: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2021.113497

Axl has emerged as an attractive target for cancer therapy due to its strong correlation with tumor growth, metastasis, poor survival, and drug resistance. Herein, we report the design, synthesis and structure-activity relationship (SAR) investigation of a series of pyrrolo[2,3-d]pyrimidine derivatives as new Axl inhibitors. Among them, the most promising compound 13b showed high enzymatic and cellular Axl potencies. Furthermore, 13b possessed preferable pharmacokinetic properties and displayed promising therapeutic effect in BaF3/TEL-Axl xenograft tumor model. Compound 13b may serve as a lead compound for new antitumor drug discovery.

Full text of DOI:10.1016/j.ejmech.2021.113497

European Journal of Medicinal Chemistry 220 (2021) 113497
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of pyrrolo[2,3-d]pyrimidine derivatives as potent Axl
inhibitors: Design, synthesis and biological evaluation
,
c
,
,
c
,
d
,
,
Dandan Xu a 1, Deqiao Sun b
1, Wei Wang a c, Xia Peng b, Zhengsheng Zhan a,
Yinchun Ji b, Yanyan Shen b, Meiyu Geng b d, Jing Ai b
*, Wenhu Duan a
,
c
,
,
c
,
e
,
, c, **
a Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), 555 Zu Chong Zhi Road, Shanghai, 201203,
China
b Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS),
555 Zu Chong Zhi Road, Shanghai, 201203, China
c University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing, 100049, China
d School of Life Science and Technology, Shanghai Tech University, 393 Middle Huaxia Road, Pudong New District, Shanghai, 201210, China
e Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, 1 Xiangshan Branch Lane, Xihu District, Hangzhou, 330106, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Axl has emerged as an attractive target for cancer therapy due to its strong correlation with tumor
growth, metastasis, poor survival, and drug resistance. Herein, we report the design, synthesis and
structure-activity relationship (SAR) investigation of a series of pyrrolo[2,3-d]pyrimidine derivatives as
new Axl inhibitors. Among them, the most promising compound 13b showed high enzymatic and
cellular Axl potencies. Furthermore, 13b possessed preferable pharmacokinetic properties and displayed
promising therapeutic effect in BaF3/TEL-Axl xenograft tumor model. Compound 13b may serve as a lead
compound for new antitumor drug discovery.
Received 27 September 2020
Received in revised form
30 December 2020
Accepted 16 April 2021
Available online 25 April 2021
Keywords:
Axl
© 2021 Elsevier Masson SAS. All rights reserved.
Cancer
Inhibitor
Pyrrolo[2,3-d]pyrimidine
1. Introduction
[17e22], prostate cancer [23], esophageal cancer [24], ovarian cancer
[25], gastric cancer [26], renal cell carcinoma (RCC) [27e30], non-
Axl, which derives from the Greek vocabulary “anexelekto” (un-
controlled for English), serves as a receptor tyrosine kinase belonging
to TAM subfamily which also comprises Tyro3 and Mer kinases [1,2].
Upon binding to the ligand growth arrest-specific 6 (GAS6), Axl
undergoes dimerization and auto-phosphorylation [3,4], resulting in
the activation of its downstream signaling pathways such as PI3K/
AKT, MAPK/ERK, IFNAR/STAT1/SOCS, etc. [1,2,5]. Physiologically,
GAS6/Axl pathway plays a pivotal role in a broad range of cellular
processes [6e10] including cell survival, proliferation, differentia-
tion, migration, invasion, and anti-inflammation. Overexpression of
Axl has been detected in a variety of human malignancies such as
liver cancer [11e13], pancreatic cancer [14e16], breast cancer
small-cell lung cancer (NSCLC) [31e35], leukemia [36,37], etc., and
such overexpression is correlated with cancer recurrence and poor
prognosis [38e42]. Collective evidences have also confirmed that the
aberrant activation of Axl signaling causes epithelial-mesenchymal
transition (EMT) as well as promotion of migration and invasion of
tumor cells, leading to drug resistance to both targeted therapies and
chemotherapy [1,24,43e52]. Moreover, inhibition of the aberrant
Axl/GAS6 axis with genetic tools or small-molecule Axl inhibitors has
proven favorable antitumor efficacy in human cancer cell lines and
animal xenograft tumor models [30,42,53e56]. Therefore, targeting
GAS6/Axl signaling pathway is expected as a potential strategy for
cancer treatment.
* Corresponding author. Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences
(CAS), 555 Zu Chong Zhi Road, Shanghai, 201203, China.
** Corresponding author. Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), 555 Zu Chong Zhi Road, Shanghai,
201203, China
E-mail addresses: jai@simm.ac.cn (J. Ai), whduan@simm.ac.cn (W. Duan).
Dandan Xu and Deqiao Sun contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113497
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
The reported small-molecule Axl inhibitors are ATP-competitive
ones which contain an adenine-mimicking heterocyclic moiety that
anchors the hinge region of ATP-binding site. The inhibitors have
been classified into two types (Type I and Type II) based on the
binding conformation of the inhibitors and Axl kinase domain; type
I inhibitors target the ATP-binding domain adopting a “U shape” to
generate an active conformation; while type II inhibitors adopt a
linear structure to occupy the ATP-binding site as well as an allo-
steric area nearby, forming an inactive conformation [57,58]. Most
of the reported Axl inhibitors always target several other receptor
tyrosine kinases whilst Axl is not the intended primary target, such
as 1 (gilteritinib) [59,60], 2 (crizotinib) [61,62], 3 (foretinib) [63,64],
4 (merestinib) [65], and 5 (NPS-1034) [45] (Fig. 1). Compound 6
(BMS-777607) is a type II Axl inhibitor that potently inhibits c-Met
and TAM kinases; it was initially developed by Bristol Myers-Squibb
as a c-Met inhibitor (IC50 ¼ 3.9 nM), while it has been promoted
phase I clinical trials for its high Axl potency (IC50 ¼ 1.1 nM) [66].
Compound 7 (BPI-9016 M) is a dual Axl/c-Met inhibitor of Betta
Pharma in phase I/II clinical trials for the treatment of NSCLC and
solid tumor [67,68]. Besides, the first selective type I Axl inhibitor 8
(R428, BGB324 or bemcentinib) has been advanced to phase II
clinical trials due to its promising Axl efficacy (IC50 ¼ 14.0 nM)
[69,70]; in 2019, R428 was assigned for the treatment of elderly
patients of relapsed acute myeloid leukemia (AML). Studies have
consolidated that Axl is synergistically collaborated with c-Met and
they both emerge as significant drivers of drug resistance through
EMT process in a variety of human malignancies [45,49,71e73],
which may offer an opportunity for the development of dual Axl/c-
Met inhibitors to overcome drug resistance and improve long-term
survival of cancer patients.
on their binding modes with c-Met due to the structural homology
between c-Met and Axl kinase domain. The reported co-crystal
structure of c-Met kinase and the dual Axl/c-Met inhibitor 6 is
employed to probe the binding mode of type II inhibitors and Axl
kinase. As shown in Fig. 2A, the 2-aminopyridine segment targets
the backbone of Met1160 in the ATP-binding site to form two
hydrogen bonds; the central fluorophenyl ring interacts with the
side chain of Phe1223 through p-p stacking; the 2-pyridone moiety
offers a 1,3-diketone to target the side chain of Lys1110 and the
backbone NH of Asp1222 with two hydrogen bonds; the 4-
fluorophenyl fragment occupies the allosteric pocket by hydro-
phobic interaction. To identify an appropriate chemical scaffold, we
inspected a set of type II Axl inhibitors which share four fragments
in binding with Axl (Fig. 1): 1) an adenine-mimicking heterocyclic
moiety (blue parts in Fig.1) forming hydrogen bonds with Met1160;
2) a central aromatic ring (yellow parts in Fig. 1) offering
p-p
stacking interaction; 3) a 1,3-diketone-skeleton moiety (green parts
in Fig. 1) targeting Lys1110 and Asp1222 by two hydrogen bonds;
and 4) an aromatic moiety (purplish red parts in Fig. 1) occupying
the allosteric pocket [74,75].
Guided by the above-mentioned information, we designed
pyrrolo[2,3-d]pyrimidine derivatives as new Axl inhibitors (Fig. 2B).
Firstly, we employed pyrrolo[2,3-d]pyrimidine to mimic the ATP
adenine ring. Then, a view on the co-crystal structure of c-Met in
complex with 6 demonstrates a large allosteric pocket that can
accommodate diverse chemical groups; and therefore 4-oxo-1,4-
dihydropyridine-3-carboxamide moiety, the bioisostere of 1,3-
diketone moiety in 6, was chosen as the double-hydrogen bond
acceptor, also allowing for the introduction of substituents at its
nitrogen atom. Besides, the central aromatic ring linking the two
fragments above was also required due to its
p-p stacking with
2. Results and discussion
Phe1223. Lastly, it is reasonable to keep the phenyl moiety adjacent
to the 4-pyridone segment which engages the allosteric pocket.
2.1. Molecular design
2.2. SAR exploration
Use of co-crystal structure information is critical for the success
of a structure-based drug design (SBDD) effort. However, no X-ray
structure of Axl in its inactive conformation has been reported.
Therefore, structural features of Axl inhibitors were proposed based
In addition to Axl kinase assessment, we also evaluated the
enzymatic c-Met activities of the new synthesized compounds,
using the well-characterized Axl inhibitor 8 and c-Met inhibitor 2
Fig. 1. Structures of the representative Axl inhibitors.
2

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
Fig. 2. (A) Co-crystal structure of c-Met in complex with compound 6 and main interactions between c-Met and 6 (PDB ID: 3F82). (B) Design of Axl inhibitors based on scaffold
hopping and bioisosteric replacement.
Table 1
Table 2
SAR of N-substituted 4-pyridone segment.
SAR of the phenyl nearby 4-pyridone part.
Compd.
R1
IC50 (nM)a
Axl
c-Met
Compd.
R2
IC50 (nM)a
Axl
9a
9b
9c
CH3
CH(CH3)2
5.0 0.6
0.4 0.0
0.5 0.3
24.5 3.7
18.4 5.1
18.4 5.0
c-Met
9g
4-F
H
4-Cl
4-CF3
3-F
2,4-di-F
e
0.1 0.0
1.0 0.3
0.2 0.1
0.4 0.1
3.0 1.3
0.8 0.1
5.3 0.7
e
7.1 1.7
20.6 1.1
12.3 2.8
33.1 2.7
95.4 16.4
19.8 1.7
e
10a
10b
10c
10d
10e
8
9d
9e
(CH2)3OCH3
>10.0
1.1 0.4
16.7 3.5
13.1 1.5
9f
1.2 0.1
0.1 0.0
3.1 0.8
7.1 1.7
2
e
3.0 0.8
a
9g
Values are the mean SD of two independent assays.
9h
2.3 0.2
4.8 0.1
important to enhance the binding affinity with allosteric pocket.
Then we investigated the substituents of the central benzene
8
2
e
e
5.3 0.7
e
e
ring which may have a critical
p-p stacking interaction with
3.0 0.8
Phe1223 (Table 3). Fluoro substitution at the positions of the ben-
a
Values are the mean SD of two independent assays.
zene ring (11a, 11b, and 11e) was demonstrated similar potencies
as positive controls. To our delight, the first synthesized compound
9a (Table 1) exhibited high potency against Axl kinase as well as
moderate enzymatic c-Met activity, which might offer additional
advantages compared with selective Axl inhibitors. Next, we car-
ried out further structural optimization.
Table 3
SAR of the central phenyl ring.
We first investigated the SAR of the 4-pyridone segment and
introduced a variety of substituents on its 1-position. As shown in
Table 1, most of the derivatives maintained potent activities against
both Axl and c-Met kinases. Incorporation of the bulky (tetrahydro-
2H-pyran-4-yl)methyl group (9g) led to a 50-fold increase in Axl
potency compared with 9a. Of note, replacement of (tetrahydro-
2H-pyran-4-yl)methyl with cyclohexylmethyl (9h) led to a 23-fold
loss in Axl activity while a slight increase in c-Met activity, which
indicated that the oxygen atom of tetrahydro-2H-pyran ring was
essential for Axl activity.
We next examined the impacts of variations on the phenyl next
to 4-pyridone segment (Table 2). Compounds with para-
substitutions (10b and 10c) displayed comparable enzymatic Axl
potencies as the para-fluorophenyl congener 9g. While the com-
pounds with unsubstituted phenyl and meta-fluoro substituted
phenyl (10a and 10d) resulted in significant loss in both kinases,
which suggested that para-substitution on the phenyl was
Compd.
R3
IC50 (nM)a
Axl
c-Met
9g
H
2-F
3-F
2-CH3
2-Cl
2,5-di-F
e
0.1 0.0
0.2 0.1
0.8 0.1
4.0 0.9
3.7 1.7
0.5 0.1
5.3 0.7
e
7.1 1.7
7.3 1.3
2.4 0.4
33.8 1.3
40.5 3.5
3.3 0.5
11a
11b
11c
11d
11e
8
e
2
e
3.0 0.8
a
Values are the mean SD of two independent assays.
3

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
Table 5
Cellular activities of selected compounds.
against Axl/c-Met compared with the unsubstituted congener 9g.
However, larger substituents such as methyl (11c) and chlorine
(11d) had a detrimental effect on potencies.
Compd.
IC50 (nM)a
We also turned our attention to the modifications at pyrrolo[2,3-
d]pyrimidine moiety (Table 4). Similar to the core structure of ATP,
pyrrolo[2,3-d]pyrimidine may interact with hinge region, which
was essential for inhibitory activity. Comparison between 12a and
9g revealed that the attempt to convert R4 from a hydrogen to a
methyl led to significant decreases in potencies against Axl/c-Met,
presumably because the methyl would impact the interaction
with hinge region. Removal of the N-methyl (12b) led to a slight
reduction in Axl potency and a slight increase in c-Met potency
compared with 9g. However, incorporation of larger substituents at
7-position of pyrrolo[2,3-d]pyrimidine (12c-12e) had a detrimental
effect on potencies, indicating that larger substituents might
impact hydrogen bond interaction between inhibitor and the hinge
region of Axl.
BaF3/TEL-Axl cell
MKN-45 cell
9b
9c
9g
<1.0
>100.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
117.2 8.1
e
85.3 21.4
>1000.0
247.9 3.7
317.8 3.7
347.1 34.0
826.3 124.8
500.7 48.3
209.5 34.6
93.7 14.8
53.7 10.4
77.9 0.5
e
10a
10b
10c
10e
11a
11b
11e
12b
8
2
15.0 0.4
a
Values are the mean SD of two independent assays.
Compounds with excellent Axl enzymatic activities (IC50 less
than 1.0 nM) were further profiled in BaF3/TEL-Axl, engineered to
express human TEL-Axl that stably expresses a constitutively active
Axl and features Axl addicted oncogenic phenotypes [76e78], and
MKN-45, MET-amplified human gastric carcinoma cell line [79e81]
for proliferation assays (Table 5). BaF3/TEL-Axl is a classic gain-of-
function model cell line, of which the proliferation is dependent
on AXL fusion; while MKN-45 is MET-amplified cancer cell line;
these two cell lines used here represent Axl- or Met-dependent
scenarios. To our excitement, most of these tested compounds
except 9c demonstrated very potent anti-proliferative effects
against BaF3/TEL-Axl cells with IC50 values less than 1.0 nM, much
better than positive control 8; nevertheless, these potent com-
pounds could be distinguished by potencies in MKN-45 cells and
most of them displayed marginal antiproliferative efficacies. Com-
parison between 9b and 9g suggested that isopropyl was beneficial
to improve activity in MKN-45. Fluoro substitution on 3- or 5-
position of the central phenyl led to higher cellular potencies
(11b and 11e vs than 9g); particularly, 11e displayed a five-fold
increase in potency compared with 9g. The analogue without N-
methyl (12b) also exhibited an increased cellular potency in MKN-
45. Therefore, compounds 9b, 11e, and 12b were chosen for further
optimization.
2.3. Optimization based on cellular potencies
Given the structure characteristics of 9b, 11e, and 12b, we syn-
thesized another two derivatives (13ae13b) using combination
principle (Table 6). As expected, 13a and 13b exerted high potencies
against Axl with IC50 values less than 1 nM in both enzyme and cell
levels. Moreover, 13b exhibited a good antiproliferative effect in
MKN-45 with IC50 value equivalent to positive control 2, suggesting
that the introduction of isopropyl and 2,5-di-F, and the removal of
the substituent at pyrrolo[2,3-d]pyrimidine were beneficial for c-
Met cellular potency.
2.4. Pharmacokinetic profiles of compounds 13a and 13b
The preliminary pharmacokinetic evaluations were conducted
in SpragueꢀDawley (SD) rats. Compound 13a or 13b was orally
administrated by gavage at a dose of 3 mg/kg and pharmacokinetic
parameters were summarized in Table 7. Compound 13a displayed
moderate maximum concentrations and oral exposures, while
compound 13b was distinguished for its preferable pharmacoki-
netic properties. Taken together with the high enzymatic and
cellular Axl/c-Met potencies, we selected 13b for further biological
evaluation.
Table 4
2.5. Kinase selectivity of compound 13b
SAR of the adenine-mimicking heterocyclic moiety.
The kinase selectivity of compound 13b was tested over a panel
of tyrosine kinases. As shown in Table 8, in contrast to its high
potencies against Axl and c-Met, 13b exhibited weak inhibition
against the additional 13 kinases, displaying more than 5000-fold
or 2500-fold selectivity over Axl or c-Met. These data displayed
that 13b possessed high selectivity for Axl and c-Met, especially for
Axl.
2.6. Compound 13b inhibits Axl/c-Met and the downstream
signaling pathways
Compd.
R4
R5
IC50 (nM)a
Axl
c-Met
9g
H
CH3
H
H
H
CH3
CH3
H
(CH2)2CH3
(CH2)2N(CH3)2
0.1 0.0
5.1 0.6
0.2 0.1
1.8 0.1
2.8 0.0
2.7 0.5
7.1 1.7
158.9 28.2
4.4 0.6
5.1 0.1
215.3 16.4
17.0 1.5
To confirm the cellular inhibition of targeting Axl/c-Met
signaling, effects of 13b on the phosphorylation of Axl/c-Met and
their downstream signaling molecules were also investigated in
BaF3/TEL-Axl and MKN-45 cell lines, respectively. As illustrated in
Fig. 3, compound 13b significantly inhibited the phosphorylation of
Axl and its key downstream molecule p-Akt in a dose-dependent
manner. The same results were also found in MKN-45 cells, the
phosphorylation of c-Met as well as downstream signaling activa-
tion was also substantially inhibited. These results revealed that
12a
12b
12c
12d
12e
H
8
2
e
e
e
e
5.3 0.7
e
e
3.0 0.8
a
Values are the mean SD of two independent assays.
4

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
Table 6
Enzymatic and cellular activities of further optimized compounds.
Compd.
R1
R3
R5
H
IC50 (nM)a
Axl
c-Met
BaF3/TEL-Axl cell
MKN-45 cell
29.8 2.9
13a
2,5-di-F
0.4 0.1
1.7 0.1
<1.0
13b
8
2
CH(CH3)2
e
2,5-di-F
e
H
e
e
<0.2
5.3 0.7
e
4.5 0.4
e
<1.0
117.2 8.1
13.2 1.1
e
e
e
3.0 0.8
e
15.0 0.4
a
Values are the mean SD of two independent assays.
Table 7
Pharmacokinetic profiles of compounds 13a and 13ba.
Compd.
Dose (mg/kg)
Tmax (h)
T1/2 (h)
Cmax (ng/mL)
AUC0/∞ (ng$h/mL)
13a
13b
3
3
0.83
1.08
1.37
2.74
330
14116
385
44335
a
Experiments were carried out in male SpragueꢀDawley rats (n ¼ 3). Data are the mean values.
Table 8
consecutive days. Meanwhile, 8 was administrated twice daily for
14 days as positive control at the dose of 75 mg/kg. A week later,
therapeutic effect of the higher dose group was not potent as ex-
pected, so the dosage was increased to 100 mg/kg from the 8th day
to the end. As depicted in Fig. 4, tumors of the lower dose group
were not inhibited remarkably, while the higher dose group were
suppressed with a maximum tumor growth inhibition rate (TGI) of
67.4% at the dose of 50/100 mg/kg in BaF3/TEL-Axl model. During
the treatment, no severe weight loss was observed. These results
demonstrated that 13b was effective in Axl-driven tumors, exhib-
iting the potential of 13b for further drug development.
Kinase selectivity of compound 13b.
Tyrosine kinase
IC50 (nM)a
Tyrosine kinase
IC50 (nM)a
Axl
c-Met
<0.2
EGFR
ErbB2
ErbB4
Src
Abl
EPH-A2
IGF1R
>1000
>1000
>1000
>1000
>1000
>500
4.5 0.4
>1000
>1000
>1000
>1000
>1000
>1000
VEGFR-1
VEGFR-2
PDGFR-
PDGFR-
Kit
a
b
>1000
Flt-3
a
Values are the mean SD of two independent assays.
2.8. Molecular docking study of compound 13b
To uncover the molecular mechanism that accounted for the
enzymatic activities of our new synthesized compounds, the
representative compound 13b was chosen for a docking study. As
lack of the reported cocrystal complex of Axl and inhibitor in the
inactive conformation, compound 13b was docked to the inactive
conformation of c-Met kinase domain (PDB ID: 3F82) based on the
structural homology of the two kinases. As depicted in Fig. 5, the
7H-pyrrolo[2,3-d]pyrimidin-4-amine moiety anchored the hinge
region of c-Met kinase domain via two hydrogen bonds with
Pro1158 and Met1160. In the allosteric area, the 2,5-difluorophenyl
Fig. 3. (A) Compound 13b suppressed the phosphorylation of Axl and downstream
signaling molecules in BaF3/TEL-Axl cells. (B) Compound 13b inhibited the phos-
phorylation of c-Met as well as downstream cascade pathways in MKN45 cells.
fragment interacted with Phe1223 by p-p stacking interaction and
the oxygen atom of pyridin-4-(1H)-one formed a hydrogen bond
with the backbone NH of Asp1222. Moreover, overlapping the
docking result of 13b with the cocrystal structure of 6 (BMS777607)
and c-Met (PDB: 3F82) showed the similar binding mode, which
further demonstrated the rationality of our structure-based drug
design (SBDD).
13b inhibited both cellular Axl and c-Met signaling pathways
effectively.
2.7. In vivo antitumor efficacy of compound 13b
3. Chemistry
We further assessed the in vivo efficacy of 13b against estab-
lished xenograft models derived from BaF3 cells expressing TEL-
Axl. Mice bearing BaF3/TEL-Axl xenograft were treated orally
with 13b at doses of 10 mg/kg or 50 mg/kg once daily for 14
Synthetic routes to pyrrolo[2,3-d]pyrimidine compounds are
summarized in Schemes 1e3. First, preparation of carboxylic acids
19ae19m was shown in Scheme 1. Substituted or unsubstituted
5

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
Fig. 4. (A) Inhibitory effect of 13b on tumor growth in BaF3/TEL-Axl xenograft nude mice model. The relative tumor volumes are expressed as the mean SEM. Significant dif-
ference from the vehicle group was determined using a t-test, ***P < 0.001. (B) Body weight measurements during treatment.
Fig. 5. (A) Proposed binding mode of compound 13b and c-Met (PDB: 3F82). The hydrogen bonds are indicated by yellow dotted lines. (B) Overlapping of the binding modes of 13b
and 6 with c-Met kinase.
Scheme 1. Syntheses of carboxylic acids 19ae19m. Reagents and conditions: (a) (i) oxalyl chloride, DMF, CH2Cl2, 0 ꢁC to rt; (ii) 2,2-dimethyl-1,3-dioxane-4,6-dione, pyridine, CH2Cl2,
0
ꢁC to rt; (iii) EtOH, 80 ꢁC; (b) (i) 1,1-dimethoxy-N,N-dimethylmethanamine, toluene, 100 ꢁC; (ii) NH4OAc, EtOH, 60 ꢁC. for 17; (i) 1,1-dimethoxy-N,N-dimethylmethanamine,
toluene, 100 ꢁC; (ii) R1NH2, EtOH, 60 ꢁC. for 18de18m; (c) R1X (X ¼ Br or I), K2CO3, DMF, 80 ꢁC; (d) NaOH, H2O, MeOH, rt.
Scheme 2. Syntheses of organic amines 25ae25l. Reagents and conditions: (a) MeI, NaH (60% dispersion in mineral oil), DMF, 0 ꢁC to rt. for 21ae21b; SEMCl, K2CO3, DMF, rt. for 21c;
R5OH, PPh3, DIAD, THF, 0 ꢁC to rt. for 21de21f; (b) bis(pinacolato)diboron, AcOK, PdCl2(dppf), 1,4-dioxane, 90 ꢁC; (c) Na2CO3, Pd(PPh3)4, 1,4-dioxane, H2O, 90 ꢁC; (d) ammonia (aq.),
1,4-dioxane, 100 ꢁC.
6

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
chromatography (TLC) on silica gel plates and visualized under
UV light. Flash chromatography was performed using silica gel
(300e400 mesh). 1H NMR and 13C NMR spectra were generated in
CDCl3 or DMSO‑d6 on Varian Mercury 300, 400, or 500 NMR
spectrometers. Low resolution mass spectra (ESI) were conducted
using an Agilent 6110 Quadrupole LC/MS spectrometer. High res-
olution mass (ESI) data were conducted using an Agilent G6520 Q-
TOF LC-MS spectrometer. All tested compounds were purified to
more than 95% purities as determined by Agilent Infinity 1260 high
performance liquid chromatography (HPLC) coupled with a diode
array detector (DAD) and the ZORBAX Eclipse Plus column (C18,
4.6 ꢂ 150 mm, 5
mm).
5.1.1. General procedure A: syntheses of compounds 16ae16f
To a stirred solution of substituted or unsubstituted phenyl-
acetic acid (14ae14f) (5.0 mmol, 1.0 eq) in anhydrous dichloro-
methane (20 mL) was successively added oxalyl chloride (6.0 mmol,
1.2 eq) and DMF (0.5 mmol, 0.1 eq) at 0 ꢁC under argon atmosphere.
The resulting mixture was subsequently warmed up to room
temperature and stirred for 6 h. The reaction mixture was
concentrated in vacuum to afford the corresponding phenylacetyl
chloride. The solution of the corresponding phenylacetyl chloride
in anhydrous dichloromethane (10 mL) was slowly added to a
stirred solution of 2,2-dimethyl-1,3-dioxane-4,6-dione (5.0 mmol,
1.0 eq), pyridine (10.0 mmol, 2.0 eq) in anhydrous dichloromethane
(10 mL) at 0 ꢁC under argon atmosphere. The resulting mixture was
subsequently warmed up to room temperature and stirred for 5 h.
The reaction mixture was concentrated and the resulting residue
was dissolved in anhydrous ethanol (20 mL), and the mixture was
heated to 80 ꢁC for additional 4 h. After cooling, the mixture was
concentrated to dryness and distributed in water and ethyl acetate.
The organic layer was separated and washed with water and brine
successively, dried over anhydrous sodium sulfate and concen-
trated in vacuum. The resulting residue was purified by silica gel
chromatography (petroleum ether/ethyl acetate, v/v, 99:1 to 90:10)
to give the desired product.
Scheme 3. Syntheses of target compounds 9ae9h, 10ae10e, 11ae11e, 12ae12e and
13ae13b. Reagents and conditions: (a) HATU, DIPEA, DMF, rt; (b) TFA, CH2Cl2, rt.
phenylacetic acids 14ae14f reacted with oxalyl chloride, followed
by condensation to give 16ae16f. The ethyl 3-oxo-4-
phenylbutanoates 16ae16f reacted with 1,1-dimethoxy-N,N-
dimethylmethanamine, followed by ammonium acetate or
substituted amines treatment to yield the corresponding pyridone-
3-carboxylates 17 and 18de18m. While the pyridone-3-
carboxylates 18ae18c could be produced by treatment of the in-
termediate 17 with alkyl halide. Then compounds 21ae21f
(Scheme 2) were prepared from commercially available 4-chloro-5-
iodo-7H-pyrrolo[2,3-d]pyrimidines 20ae20b via Mitsunobu or SN2
reactions. Subsequent Suzuki couplings between 21ae21f and
various phenylboronic acid esters 23ae23f yielded 24ae24l, which
further converted to amines 25ae25l after treatment with aqueous
ammonia. Finally, the target compounds (9ae9h, 10ae10e,
11ae11e, 12a, 12ce12e, Scheme 3) and the SEM-containing com-
pounds 26ae26c were obtained by condensation between car-
boxylic acids 19ae19m and organic amines 25ae25l. Then
26ae26c were deprotected to afford target compounds 12b and
13ae13b.
5.1.1.1. Ethyl 4-(4-fluorophenyl)-3-oxobutanoate (16a). The title
compound was prepared from 15a following general procedure A.
Yield: 86%. 1H NMR (300 MHz, DMSO‑d6)
d 7.26e7.09 (m, 4H), 4.08
(q, J ¼ 7.2 Hz, 2H), 3.88 (s, 2H), 3.66 (s, 2H), 1.17 (t, J ¼ 7.2 Hz, 3H).
4. Conclusion
5.1.1.2. Ethyl 3-oxo-4-phenylbutanoate (16b). The title compound
was prepared from 15b following general procedure A. Yield: 69%.
In summary,
a series of pyrrolo[2,3-d]pyrimidines were
1H NMR (300 MHz, DMSO‑d6)
d
7.41e7.23 (m, 3H), 7.19 (d, J ¼ 7.7 Hz,
designed and synthesized as new Axl inhibitors. The privileged
compound 13b exhibited excellent enzymatic potency and great
selectivity for Axl and c-Met kinases. Western blot analysis revealed
that 13b remarkably suppressed Axl/c-Met and the downstream
signaling pathways in a dose-dependent manner. Furthermore, 13b
demonstrated reasonable pharmacokinetic properties and exhibi-
ted promising antitumor effect in BaF3/TEL-Axl xenograft tumor
model. In addition, the inhibitory activity of 13b against c-Met may
offer an additional advantage with the strength of finding that both
Axl and c-Met are key mediators of acquired resistance. Compound
13b may serve as a new lead compound for antitumor drug
discovery.
2H), 4.09 (q, J ¼ 6.9 Hz, 2H), 3.88 (s, 2H), 3.66 (s, 2H), 1.18 (t,
J ¼ 6.9 Hz, 3H).
5.1.1.3. Ethyl 4-(4-chlorophenyl)-3-oxobutanoate (16c). The title
compound was prepared from 15c following general procedure A.
Yield: 60%. 1H NMR (300 MHz, DMSO‑d6)
7.20 (d, J ¼ 8.3 Hz, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H), 3.89 (s, 2H), 3.67 (s,
2H), 1.17 (t, J ¼ 7.1 Hz, 3H).
d
7.37 (d, J ¼ 8.3 Hz, 2H),
5.1.1.4. Ethyl 3-oxo-4-(4-(trifluoromethyl)phenyl)butanoate (16d).
The title compound was prepared from 15d following general
procedure A. Yield: 43%. 1H NMR (300 MHz, DMSO‑d6)
d 7.68 (d,
5. Experimental section
J ¼ 8.0 Hz, 2H), 7.41 (d, J ¼ 8.0 Hz, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H), 4.03
(s, 2H), 3.71 (s, 2H), 1.18 (t, J ¼ 7.1 Hz, 3H).
5.1. Chemistry
5.1.1.5. Ethyl 4-(3-fluorophenyl)-3-oxobutanoate (16e). The title
compound was prepared from 15e following general procedure A.
All starting materials and reagents were purchased from com-
mercial sources or prepared according to reference procedures. All
purchased chemicals and solvents were used as received without
further purification. All reactions were monitored by thin-layer
Yield: 29%. 1H NMR (300 MHz, DMSO‑d6)
d 7.43e7.30 (m, 1H),
7.16e6.97 (m, 3H), 4.11 (q, J ¼ 7.1 Hz, 2H), 3.92 (s, 2H), 3.67 (s, 2H),
1.19 (t, J ¼ 7.1 Hz, 3H).
7

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
5.1.1.6. Ethyl 4-(2,4-difluorophenyl)-3-oxobutanoate (16f). The title
compound was prepared from 15f following general procedure A.
desired product.
Yield: 47%. 1H NMR (300 MHz, DMSO‑d6)
d
7.37e7.25 (m, 1H),
5.1.4.1. Ethyl 5-(4-fluorophenyl)-1-isopropyl-4-oxo-1,4-
dihydropyridine-3-carboxylate (18d). The title compound was pre-
pared from 16a following general procedure C. Yield: 70%. 1H NMR
(400 MHz, DMSO‑d6)
1H), 7.71e7.60 (m, 2H), 7.28e7.16 (m, 2H), 4.50e4.37 (m, 1H), 4.20
(q, J ¼ 7.1 Hz, 2H), 1.44 (d, J ¼ 6.7 Hz, 6H), 1.26 (t, J ¼ 7.1 Hz, 3H).
7.25e7.14 (m, 1H), 7.10e6.99 (m, 1H), 4.09 (q, J ¼ 7.2 Hz, 2H), 3.95 (s,
2H), 3.71 (s, 2H), 1.18 (t, J ¼ 7.2 Hz, 3H).
d
8.31 (d, J ¼ 2.4 Hz, 1H), 8.01 (d, J ¼ 2.4 Hz,
5.1.2. Synthesis of ethyl 5-(4-fluorophenyl)-4-oxo-1,4-
dihydropyridine-3-carboxylate (17)
The ester 16a (2.0 mmol, 1.0 eq) was dissolved in toluene (5 mL).
1,1-dimethoxy-N,N-dimethylmethanamine (4.0 mmol, 2.0 eq) was
added and the solution was heated to 100 ꢁC for 5 h. The reaction
mixture was concentrated after cooling, and dissolved in anhydrous
ethanol (5 mL). Ammonium acetate (10.0 mmol, 5.0 eq) was added
and the mixture was heated to 60 ꢁC for additional 5 h. After
cooling, the precipitate was collected by filtration and washed with
ethanol, water and ethanol successively. The filter cake was dried to
dryness to give the desired product without further purification.
5.1.4.2. Ethyl 5-(4-fluorophenyl)-4-oxo-1-(tetrahydro-2H-pyran-4-
yl)-1,4-dihydropyridine-3-carboxylate (18e). The title compound
was prepared from 16a following general procedure C. Yield: 72%.
1H NMR (300 MHz, DMSO‑d6)
d
8.33 (d, J ¼ 2.1 Hz, 1H), 8.04 (d,
J ¼ 2.1 Hz, 1H), 7.68 (dd, J ¼ 8.6, 5.9 Hz, 2H), 7.28e7.15 (m, 2H),
4.42e4.26 (m, 1H), 4.20 (q, J ¼ 7.0 Hz, 2H), 4.04e3.95 (m, 2H), 3.40
(t, J ¼ 11.3 Hz, 2H), 2.15e1.87 (m, 4H), 1.27 (t, J ¼ 7.0 Hz, 3H).
Yield: 69%. 1H NMR (400 MHz, DMSO‑d6)
d
11.95 (s, 1H), 8.19 (s, 1H),
5.1.4.3. Ethyl
5-(4-fluorophenyl)-4-oxo-1-((tetrahydrofuran-2-yl)
7.85 (s, 1H), 7.69e7.58 (m, 2H), 7.21 (t, J ¼ 8.9 Hz, 2H), 4.19 (q,
methyl)-1,4-dihydropyridine-3-carboxylate (18f). The title com-
pound was prepared from 16a following general procedure C. Yield:
J ¼ 7.3 Hz, 2H), 1.25 (t, J ¼ 7.3 Hz, 3H).
69%. 1H NMR (300 MHz, DMSO‑d6)
d
8.27 (d, J ¼ 2.2 Hz, 1H), 7.95 (d,
5.1.3. General procedure B: syntheses of compounds 18ae18c
Compound 17 (0.5 mmol, 1.0 eq), corresponding halogenated
compound R1X (X ¼ Br or I) (0.6 mmol, 1.2 eq) and potassium
carbonate (1.5 mmol, 3.0 eq) were dissolved in DMF (2.5 mL), and
the mixture was stirred at 80 ꢁC for 3 h. After cooling, the mixture
was distributed in water and ethyl acetate. The organic layer was
separated and washed with water and brine successively, then
dried over anhydrous sodium sulfate and concentrated in vacuum.
The resulting residue was purified by silica gel chromatography
(dichloromethane/methanol, v/v, 98:2) to give the desired product.
J ¼ 2.2 Hz, 1H), 7.68e7.59 (m, 2H), 7.29e7.18 (m, 2H), 4.27e4.08 (m,
4H), 4.07e3.90 (m, 1H), 3.86e3.72 (m, 1H), 3.72e3.60 (m, 1H),
2.07e1.89 (m, 1H), 1.89e1.74 (m, 2H), 1.60e1.46 (m, 1H), 1.26 (t,
J ¼ 7.1 Hz, 3H).
5.1.4.4. Ethyl 5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1,4-dihydropyridine-3-carboxylate (18g). The title com-
pound was prepared from 16a following general procedure C. Yield:
72%. 1H NMR (300 MHz, DMSO‑d6)
d
8.28 (d, J ¼ 1.8 Hz, 1H), 7.97 (d,
J ¼ 1.8 Hz, 1H), 7.65 (dd, J ¼ 8.4, 5.9 Hz, 2H), 7.27e7.17 (m, 2H), 4.20
(q, J ¼ 7.1 Hz, 2H), 3.92 (d, J ¼ 7.3 Hz, 2H), 3.88e3.79 (m, 2H),
3.33e3.20 (m, 2H), 2.14e1.95 (m, 1H), 1.50e1.39 (m, 2H), 1.35e1.19
(m, 5H).
5.1. 3.1. Ethyl 5-(4-fluorophenyl)-1-methyl-4-oxo-1, 4-
dihydropyridine-3-carboxylate (18a). The title compound was pre-
pared from 17 following general procedure B. Yield: 71%. 1H NMR
(300 MHz, DMSO‑d6)
d
8.26 (d, J ¼ 2.4 Hz, 1H), 7.91 (d, J ¼ 2.4 Hz,
5.1.4.5. Ethyl 1-(cyclohexylmethyl)-5-(4-fluorophenyl)-4-oxo-1,4-
dihydropyridine-3-carboxylate (18h). The title compound was pre-
pared from 16a following general procedure C. Yield: 57%. 1H NMR
1H), 7.77e7.58 (m, 2H), 7.28e7.12 (m, 2H), 4.19 (q, J ¼ 7.1 Hz, 2H),
3.75 (s, 3H), 1.26 (t, J ¼ 7.1 Hz, 3H).
(300 MHz, DMSO‑d6)
d
8.26 (d, J ¼ 2.1 Hz, 1H), 7.96 (d, J ¼ 2.1 Hz,
5.1.3.2. Ethyl 1-(2-(dimethylamino)-2-oxoethyl)-5-(4-fluorophenyl)-
4-oxo-1,4-dihydropyridine-3-carboxylate (18b). The title compound
was prepared from 17 following general procedure B. Yield: 44%. 1H
1H), 7.65 (dd, J ¼ 8.7, 5.8 Hz, 2H), 7.27e7.18 (m, 2H), 4.20 (q,
J ¼ 7.1 Hz, 2H), 3.86 (d, J ¼ 7.3 Hz, 2H), 1.87e1.44 (m, 6H), 1.31e0.89
(m, 8H).
NMR (300 MHz, DMSO‑d6)
d
8.19 (d, J ¼ 1.9 Hz, 1H), 7.82 (d,
J ¼ 1.9 Hz, 1H), 7.66e7.56 (m, 2H), 7.22 (t, J ¼ 8.9 Hz, 2H), 5.04 (s,
2H), 4.20 (q, J ¼ 7.2 Hz, 2H), 2.98 (s, 3H), 2.87 (s, 3H), 1.26 (t,
J ¼ 7.2 Hz, 3H).
5.1.4.6. Ethyl
4-oxo-5-phenyl-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,4-dihydropyridine-3-carboxylate (18i). The title com-
pound was prepared from 16b following general procedure C.
Yield: 76%. 1H NMR (300 MHz, DMSO‑d6)
d 8.27 (s, 1H), 7.94 (s, 1H),
5.1.3.3. Ethyl 5-(4-fluorophenyl)-1-(3-methoxypropyl)-4-oxo-1,4-
dihydropyridine-3-carboxylate (18c). The title compound was pre-
pared from 17 following general procedure B. Yield: 90%. 1H NMR
7.59 (d, J ¼ 7.2 Hz, 2H), 7.44e7.28 (m, 3H), 4.20 (q, J ¼ 7.1 Hz, 2H),
3.92 (d, J ¼ 7.0 Hz, 2H), 3.89e3.79 (m, 2H), 3.26 (t, J ¼ 11.3 Hz, 2H),
2.13e1.96 (m, 1H), 1.44 (d, J ¼ 12.8 Hz, 2H), 1.36e1.15 (m, 5H).
(400 MHz, DMSO‑d6)
d
8.26 (d, J ¼ 2.3 Hz, 1H), 7.96 (d, J ¼ 2.3 Hz,
1H), 7.72e7.61 (m, 2H), 7.27e7.17 (m, 2H), 4.20 (q, J ¼ 7.1 Hz, 2H),
4.05 (t, J ¼ 7.2 Hz, 2H), 3.39e3.28 (m, 2H), 3.22 (s, 3H), 2.06e1.94
(m, 2H), 1.26 (t, J ¼ 7.1 Hz, 3H).
5.1.4.7. Ethyl 5-(4-chlorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1,4-dihydropyridine-3-carboxylate (18j). The title com-
pound was prepared from 16c following general procedure C. Yield:
74%. 1H NMR (300 MHz, DMSO‑d6)
d
8.29 (d, J ¼ 2.0 Hz, 1H), 8.01 (d,
5.1.4. General procedure C: syntheses of compounds 18de18m
The ester (16ae16f) (2.0 mmol, 1.0 eq) was dissolved in toluene
(5 mL). 1,1-dimethoxy-N,N-dimethylmethanamine (4.0 mmol, 2.0
eq) was added and the solution was heated to 100 ꢁC for 5 h. The
reaction mixture was concentrated after cooling and dissolved in
anhydrous ethanol (5 mL). The corresponding primary amine
R1NH2 (2.2 mmol, 1.1 eq) was added and the mixture was heated to
60 ꢁC for additional 12 h. After cooling, the reaction solution was
concentrated and the resulting residue was purified by silica gel
chromatography (dichloromethane/methanol, v/v, 98:2) to give the
J ¼ 2.0 Hz, 1H), 7.66 (d, J ¼ 8.5 Hz, 2H), 7.45 (d, J ¼ 8.5 Hz, 2H), 4.20
(q, J ¼ 6.8 Hz, 2H), 3.92 (d, J ¼ 7.2 Hz, 2H), 3.89e3.80 (m, 2H), 3.26 (t,
J ¼ 11.2 Hz, 2H), 2.13e1.97 (m, 1H), 1.43 (d, J ¼ 12.6 Hz, 2H),
1.35e1.15 (m, 5H).
5.1.4.8. Ethyl 4-oxo-1-((tetrahydro-2H-pyran-4-yl)methyl)-5-(4-(tri-
fluoromethyl)phenyl)-1,4-dihydropyridine-3-carboxylate
The title compound was prepared from 16d following general
procedure C. Yield: 56%. 1H NMR (300 MHz, DMSO‑d6)
8.32 (d,
J ¼ 2.3 Hz, 1H), 8.10 (d, J ¼ 2.3 Hz, 1H), 7.85 (d, J ¼ 8.4 Hz, 2H), 7.75
(18k).
d
8

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
(d, J ¼ 8.4 Hz, 2H), 4.21 (q, J ¼ 7.1 Hz, 2H), 3.93 (d, J ¼ 7.5 Hz, 2H),
3.89e3.81 (m, 2H), 3.31e3.20 (m, 2H), 2.14e1.96 (m, 1H), 1.44 (d,
J ¼ 12.0 Hz, 2H), 1.36e1.17 (m, 5H).
J ¼ 1.1 Hz, 1H), 7.83e7.72 (m, 2H), 7.30 (t, J ¼ 9.0 Hz, 2H), 4.69e4.51
(m, 1H), 4.10e3.95 (m, 2H), 3.51e3.34 (m, 2H), 2.24e2.06 (m, 2H),
2.06e1.91 (m, 2H). MS (ESIꢀ) m/z: 362.1 [M þ HCOO]-.
5.1.4.9. Ethyl 5-(3-fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1,4-dihydropyridine-3-carboxylate (18l). The title com-
pound was prepared from 16e following general procedure C. Yield:
5.1.5.6. 5-(4-Fluorophenyl)-4-oxo-1-((tetrahydrofuran-2-yl)methyl)-
1,4-dihydropyridine-3-carboxylic acid (19f). The title compound
was prepared from 18f following general procedure D. Yield: 90%.
19%. 1H NMR (300 MHz, DMSO‑d6)
d 8.30 (s, 1H), 8.07 (s, 1H),
1H NMR (300 MHz, DMSO‑d6)
d
8.74 (d, J ¼ 2.1 Hz, 1H), 8.37 (d,
7.58e7.38 (m, 3H), 7.22e7.11 (m,1H), 4.21 (q, J ¼ 7.3 Hz, 2H), 3.92 (d,
J ¼ 6.8 Hz, 2H), 3.89e3.80 (m, 2H), 3.32e3.19 (m, 2H), 2.14e1.96 (m,
1H), 1.43 (d, J ¼ 13.2 Hz, 2H), 1.34e1.13 (m, 5H).
J ¼ 2.1 Hz, 1H), 7.78e7.68 (m, 2H), 7.36e7.26 (m, 2H), 4.44e4.29 (m,
1H), 4.27e4.15 (m, 2H), 3.87e3.74 (m, 1H), 3.72e3.62 (m, 1H),
2.09e1.93 (m, 1H), 1.91e1.75 (m, 2H), 1.65e1.46 (m, 1H). MS (ESIꢀ)
m/z: 362.1 [M þ HCOO]-.
5.1.4.10. Ethyl 5-(2,4-difluorophenyl)-4-oxo-1-((tetrahydro-2H-py-
ran-4-yl)methyl)-1,4-dihydropyridine-3-carboxylate (18m). The title
compound was prepared from 16f following general procedure C.
5.1.5.7. 5-(4-Fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,4-dihydropyridine-3-carboxylic acid (19g). The title com-
pound was prepared from 18g following general procedure D.
Yield: 50%. 1H NMR (300 MHz, DMSO‑d6)
d 8.34 (s, 1H), 7.92 (s, 1H),
7.44 (dd, J ¼ 15.7, 8.1 Hz, 1H), 7.34e7.22 (m, 1H), 7.19e7.07 (m, 1H),
4.20 (q, J ¼ 7.1 Hz, 2H), 3.96e3.80 (m, 4H), 3.31e3.20 (m, 2H),
2.11e1.92 (m, 1H), 1.44 (d, J ¼ 12.8 Hz, 2H), 1.33e1.11 (m, 5H).
Yield: 88%. 1H NMR (300 MHz, DMSO‑d6)
d 8.77 (s, 1H), 8.41 (s, 1H),
7.80e7.70 (m, 2H), 7.31 (t, J ¼ 8.8 Hz, 2H), 4.14 (d, J ¼ 7.0 Hz, 2H),
3.91e3.80 (m, 2H), 3.25 (t, J ¼ 11.4 Hz, 2H), 2.20e2.04 (m, 1H),
1.49e1.20 (m, 4H). MS (ESIꢀ) m/z: 376.1 [M þ HCOO]-.
5.1.5. General procedure D: syntheses of compounds 19ae19m
The ester (18ae18m) (1.0 mmol, 1.0 eq) and NaOH aqueous
solution (1 N, 3.0 mL, 3.0 mmol, 3.0 eq) were dissolved in methanol
(5 mL) and stirred for 1 h at room temperature. The reaction
mixture was concentrated to dryness and the resulting residue was
cooled in ice bath, acidified with hydrochloric acid (1 N) and
extracted with dichloromethane. The combined organic layers
were dried over anhydrous sodium sulfate and concentrated in
vacuum. The resulting residue was purified by silica gel chroma-
tography (dichloromethane/methanol, v/v, 99:1 to 95:5) to give the
desired product.
5.1.5.8. 1-(Cyclohexylmethyl)-5-(4-fluorophenyl)-4-oxo-1,4-
dihydropyridine-3-carboxylic acid (19h). The title compound was
prepared from 18h following general procedure D. Yield: 73%. 1H
NMR (300 MHz, DMSO‑d6) d 8.73 (s, 1H), 8.40 (s, 1H), 7.82e7.66 (m,
2H), 7.30 (t, J ¼ 8.8 Hz, 2H), 4.09 (d, J ¼ 7.1 Hz, 2H), 1.95e1.75 (m,
1H), 1.75e1.46 (m, 5H), 1.30e0.89 (m, 5H). MS (ESIþ) m/z: 330.2
[M þ H]þ.
5.1.5.9. 4-Oxo-5-phenyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1,4-
dihydropyridine-3-carboxylic acid (19i). The title compound was
prepared from 18i following general procedure D. Yield: 88%. 1H
5.1.5.1. 5-(4-Fluorophenyl)-1-methyl-4-oxo-1,4-dihydropyridine-3-
carboxylic acid (19a). The title compound was prepared from 18a
following general procedure D. Yield: 85%. 1H NMR (300 MHz,
NMR (300 MHz, DMSO‑d6)
d 8.77 (s, 1H), 8.39 (s, 1H), 7.68 (d,
J ¼ 7.3 Hz, 2H), 7.51e7.37 (m, 3H), 4.15 (d, J ¼ 7.3 Hz, 2H), 3.93e3.77
(m, 2H), 3.25 (t, J ¼ 11.6 Hz, 2H), 2.22e2.03 (m, 1H), 1.44 (d,
J ¼ 11.6 Hz, 2H), 1.38e1.22 (m, 2H). MS (ESIþ) m/z: 314.2 [M þ H]þ.
DMSO‑d6)
d
8.74 (d, J ¼ 2.1 Hz,1H), 8.35 (d, J ¼ 2.1 Hz,1H), 7.79e7.70
(m, 2H), 7.35e7.26 (m, 2H), 3.95 (s, 3H). MS (ESIþ) m/z: 248.1 [M þ
H]þ.
5.1.5.10. 5-(4-Chlorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,4-dihydropyridine-3-carboxylic acid (19j). The title com-
pound was prepared from 18j following general procedure D. Yield:
5.1.5.2. 1-(2-(Dimethylamino)-2-oxoethyl)-5-(4-fluorophenyl)-4-
oxo-1,4-dihydropyridine-3-carboxylic acid (19b). The title com-
pound was prepared from 18b following general procedure D.
45%. 1H NMR (300 MHz, DMSO‑d6)
d 8.77 (s, 1H), 8.44 (s, 1H), 7.73
Yield: 74%. 1H NMR (300 MHz, DMSO‑d6)
d 8.68 (s, 1H), 8.28 (s, 1H),
(d, J ¼ 8.1 Hz, 2H), 7.54 (d, J ¼ 8.1 Hz, 2H), 4.14 (d, J ¼ 7.0 Hz, 2H),
3.94e3.76 (m, 2H), 3.24 (t, J ¼ 11.4 Hz, 2H), 2.25e1.98 (m, 1H), 1.43
(d, J ¼ 11.6 Hz, 2H), 1.38e1.19 (m, 2H). MS (ESIꢀ) m/z: 392.1
[M þ HCOO]-.
7.75e7.66 (m, 2H), 7.31 (t, J ¼ 8.9 Hz, 2H), 5.27 (s, 2H), 3.00 (s, 3H),
2.89 (s, 3H). MS (ESIþ) m/z: 319.1 [M þ H]þ.
5.1.5.3. 5-(4-Fluorophenyl)-1-(3-methoxypropyl)-4-oxo-1,4-
dihydropyridine-3-carboxylic acid (19c). The title compound was
prepared from 18c following general procedure D. Yield: 82%. 1H
5.1.5.11. 4-Oxo-1-((tetrahydro-2H-pyran-4-yl)methyl)-5-(4-(tri-
fluoromethyl)phenyl)-1,4-dihydropyridine-3-carboxylic acid (19k).
The title compound was prepared from 18k following general
NMR (300 MHz, DMSO‑d6)
d
8.74 (d, J ¼ 2.2 Hz, 1H), 8.40 (d,
J ¼ 2.2 Hz, 1H), 7.80e7.71 (m, 2H), 7.35e7.26 (m, 2H), 4.28 (t,
J ¼ 7.0 Hz, 2H), 3.42e3.28 (m, 2H), 3.20 (s, 3H), 2.20e2.00 (m, 2H).
MS (ESIþ) m/z: 306.2 [M þ H]þ.
procedure D. Yield: 77%.1H NMR (300 MHz, DMSO‑d6)
d 8.81 (s,1H),
8.50 (s, 1H), 7.92 (d, J ¼ 8.2 Hz, 2H), 7.83 (d, J ¼ 8.2 Hz, 2H), 4.15 (d,
J ¼ 7.0 Hz, 2H), 3.93e3.78 (m, 2H), 3.25 (t, J ¼ 11.7 Hz, 2H),
2.22e2.01 (m, 1H), 1.44 (d, J ¼ 12.1 Hz, 2H), 1.39e1.17 (m, 2H). MS
(ESIꢀ) m/z: 426.3 [M þ HCOO]-.
5.1.5.4. 5-(4-Fluorophenyl)-1-isopropyl-4-oxo-1,4-dihydropyridine-
3-carboxylic acid (19d). The title compound was prepared from 18d
following general procedure D. Yield: 87%. 1H NMR (400 MHz,
DMSO‑d6)
d
8.79 (d, J ¼ 2.0 Hz, 1H), 8.45 (d, J ¼ 2.0 Hz, 1H), 7.74 (dd,
5.1.5.12. 5-(3-Fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,4-dihydropyridine-3-carboxylic acid (19l). The title com-
pound was prepared from 18l following general procedure D. Yield:
J ¼ 8.8, 5.7 Hz, 2H), 7.30 (t, J ¼ 8.8 Hz, 2H), 4.76e4.61 (m,1H),1.51 (d,
J ¼ 6.8 Hz, 6H). MS (ESIþ) m/z: 276.3 [M þ H]þ.
65%. 1H NMR (300 MHz, DMSO‑d6)
d 8.79 (s, 1H), 8.48 (s, 1H),
5.1.5.5. 5-(4-Fluorophenyl)-4-oxo-1-(tetrahydro-2H-pyran-4-yl)-
1,4-dihydropyridine-3-carboxylic acid (19e). The title compound
was prepared from 18e following general procedure D. Yield: 79%.
7.62e7.46 (m, 3H), 7.33e7.21 (m, 1H), 4.14 (d, J ¼ 7.2 Hz, 2H),
3.99e3.76 (m, 2H), 3.24 (t, J ¼ 11.6 Hz, 2H), 2.21e2.03 (m, 1H), 1.43
(d, J ¼ 12.1 Hz, 2H), 1.37e1.17 (m, 2H). MS (ESIꢀ) m/z: 376.1
[M þ HCOO]-.
1H NMR (300 MHz, DMSO‑d6)
d
8.78 (d, J ¼ 1.1 Hz, 1H), 8.47 (d,
9

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
5.1.5.13. 5-(2,4-Difluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1,4-dihydropyridine-3-carboxylic acid (19m). The title
compound was prepared from 18m following general procedure D.
2H), 2.64 (t, J ¼ 5.4 Hz, 2H), 2.14 (s, 6H).
5.1.8.3. 4-(2-(4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)
ethyl)morpholine (21f). The title compound was prepared from 20a
following general procedure F. Yield: 35%. 1H NMR (300 MHz,
Yield: 97%. 1H NMR (300 MHz, DMSO‑d6)
d 8.82 (s, 1H), 8.37 (s, 1H),
7.55 (dd, J ¼ 15.4, 7.9 Hz, 1H), 7.46e7.31 (m, 1H), 7.27e7.15 (m, 1H),
4.12 (d, J ¼ 7.6 Hz, 2H), 3.94e3.78 (m, 2H), 3.25 (t, J ¼ 11.8 Hz, 2H),
2.18e1.99 (m, 1H), 1.44 (d, J ¼ 11.8 Hz, 2H), 1.36e1.19 (m, 2H). MS
(ESIꢀ) m/z: 394.1 [M þ HCOO]-.
DMSO‑d6)
d
8.63 (s, 1H), 8.04 (s, 1H), 4.38 (t, J ¼ 6.2 Hz, 2H),
3.54e3.43 (m, 4H), 2.69 (t, J ¼ 6.2 Hz, 2H), 2.46e2.37 (m, 4H).
5.1.9. General procedure G: syntheses of compounds 23ae23f
The substituted aniline (22ae22f) (10.0 mmol, 1.0 eq), bis(pi-
nacolato)diboron (12.0 mmol, 1.2 eq), potassium acetate
(30.0 mmol, 3.0 eq) and PdCl2(dppf) (1.0 mmol, 0.1 eq) were sus-
pended in 1,4-dioxane (50 mL) under argon atmosphere and the
reaction mixture was heated to 90 ꢁC for 12 h. The crude mixture
was filtered through a thin plug of Celite. The Celite plug was
washed with ethyl acetate and filtrate was concentrated. The
resulting residue was purified by silica gel chromatography (pe-
troleum ether/ethyl acetate, v/v, 98:2) to give the desired product.
5.1.6. General procedure E: syntheses of compounds 21ae21b
To a stirred solution of 20a or 20b (15.0 mmol, 1.0 eq) in anhy-
drous DMF (30 mL) was slowly added NaH (60% dispersion in
mineral oil) (30.0 mmol, 2.0 eq) at 0 ꢁC under argon atmosphere.
After stirring for 20 min, iodomethane (18.0 mmol, 1.2 eq) was
added dropwise. The reaction mixture was allowed to stir at room
temperature for 2 h. The reaction mixture was poured into iced
water and the precipitate was collected by filtration, washing with
water and ethanol successively. The filter cake was dried to dryness
to give the desired product without further purification.
5.1.9.1. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (23a).
The title compound was prepared from 22a following general
5.1.6.1. 4-Chloro-5-iodo-7-methyl-7H-pyrrolo[2,3-d]pyrimidine
(21a). The title compound was prepared from 20a following gen-
procedure G. Yield: 85%. 1H NMR (400 MHz, DMSO‑d6)
d 7.32 (d,
eral procedure E. Yield: 96%. 1H NMR (300 MHz, DMSO‑d6)
d
8.65 (s,
J ¼ 8.4 Hz, 2H), 6.50 (d, J ¼ 8.4 Hz, 2H), 5.47 (s, 2H), 1.23 (s, 12H).
1H), 7.98 (s, 1H), 3.83 (s, 3H).
5.1.9.2. 2-Fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ani-
line (23b). The title compound was prepared from 22b following
general procedure G. Yield: 38%. 1H NMR (300 MHz, DMSO‑d6)
5.1.6.2. 4-Chloro-5-iodo-2,7-dimethyl-7H-pyrrolo[2,3-d]pyrimidine
(21b). The title compound was prepared from 20b following gen-
eral procedure E. Yield: 100%. 1H NMR (300 MHz, DMSO‑d6)
d
7.85
d
7.18 (d, J ¼ 8.4 Hz, 1H), 7.13 (d, J ¼ 12.5 Hz, 1H), 6.72 (t, J ¼ 8.4 Hz,
(s, 1H), 3.77 (s, 3H), 2.64 (s, 3H).
1H), 5.56 (s, 2H), 1.24 (s, 12H).
5.1.7. Synthesis of 4-chloro-5-iodo-7-((2-(trimethylsilyl)ethoxy)
methyl)-7H-pyrrolo[2,3-d]pyrimidine (21c)
5.1.9.3. 3-Fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ani-
line (23c). The title compound was prepared from 22c following
general procedure G. Yield: 77%. 1H NMR (400 MHz, DMSO‑d6)
To a stirred solution of 20a (30.0 mmol, 1.0 eq) in DMF (90 mL)
was added potassium carbonate (90.0 mmol, 3.0 eq) at room
temperature. After stirring for 20 min, 2-(trimethylsilyl)ethox-
ymethyl chloride (45.0 mmol, 1.5 eq) was added dropwise, and the
mixture was stirred for 2 h. The reaction mixture was poured into
iced water and the precipitate was collected by filtration, washing
with water and ethanol successively. The filter cake was dried to
dryness to give the desired product without further purification.
d
7.26 (t, J ¼ 8.0 Hz, 1H), 6.32 (dd, J ¼ 8.0, 1.9 Hz, 1H), 6.18 (dd,
J ¼ 12.3, 1.9 Hz, 1H), 5.83 (s, 2H), 1.23 (s, 12H).
5.1.9.4. 2-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)an-
iline (23d). The title compound was prepared from 22d following
general procedure G. Yield: 40%. 1H NMR (300 MHz, DMSO‑d6)
d
7.28e7.18 (m, 2H), 6.54 (d, J ¼ 7.8 Hz, 1H), 5.23 (s, 2H), 2.02 (s, 3H),
Yield: 90%. 1H NMR (300 MHz, DMSO‑d6)
d
8.69 (s, 1H), 8.14 (s, 1H),
1.23 (s, 12H).
5.60 (s, 2H), 3.57e3.47 (m, 2H), 0.87e0.77 (m, 2H), 0.10 (s, 9H).
5.1.9.5. 2-Chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ani-
line (23e). The title compound was prepared from 22e following
general procedure G. Yield: 74%. 1H NMR (300 MHz, DMSO‑d6)
5.1.8. General procedure F: syntheses of compounds 21de21f
To a stirred solution of compound 20a (1.0 mmol, 1.0 eq), cor-
responding hydroxyl-substituted compound R5OH (1.5 mmol, 1.5
eq) and triphenylphosphine (2.0 mmol, 2.0 eq) in anhydrous THF
(10 mL) was added diisopropyl azodicarboxylate (2.0 mmol, 2.0 eq)
at 0 ꢁC under argon atmosphere. The mixture was warmed up to
room temperature subsequently and stirred for 2 h. The reaction
solution was concentrated and the resulting residue was purified by
silica gel chromatography (petroleum ether/ethyl acetate, v/v, 95:5
for 21d; dichloromethane/methanol, v/v, 99:1 for 21ee21f) to give
the desired product.
d
7.39 (d, J ¼ 1.2 Hz, 1H), 7.29 (dd, J ¼ 8.0, 1.2 Hz, 1H), 6.74 (d,
J ¼ 8.0 Hz, 1H), 5.78 (s, 2H), 1.25 (s, 12H).
5.1.9.6. 2,5-Difluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
aniline (23f). The title compound was prepared from 22f following
general procedure G. Yield: 72%. 1H NMR (300 MHz, DMSO‑d6)
d
7.04 (dd, J ¼ 11.6, 5.4 Hz, 1H), 6.40 (dd, J ¼ 10.8, 7.0 Hz, 1H), 5.94 (s,
2H), 1.24 (s, 12H).
5.1.10. General procedure H: syntheses of compounds 24ae24l
The iodine substituted compound (21ae21f) (1.0 mmol, 1.0 eq),
boric acid ester (23ae23f) (1.2 mmol, 1.2 eq), sodium carbonate
(3.0 mmol, 3.0 eq) and Pd(PPh3)4 (0.1 mmol, 0.1 eq) were sus-
pended in 1,4-dioxane/H2O (40 mL/10 mL, v/v ¼ 4:1) under argon
atmosphere and the reaction mixture was heated to 90 ꢁC for 14 h.
The crude mixture was filtered through a thin plug of Celite. The
Celite plug was washed with ethyl acetate and filtrate was
concentrated. The resulting residue was purified by silica gel
chromatography (petroleum ether/ethyl acetate, v/v, 90:10 to
70:30) to give the desired product.
5.1.8.1. 4-Chloro-5-iodo-7-propyl-7H-pyrrolo[2,3-d]pyrimidine
(21d). The title compound was prepared from 20a following gen-
eral procedure F. Yield: 98%. 1H NMR (300 MHz, DMSO‑d6)
d 8.62 (s,
1H), 8.05 (s, 1H), 4.21 (t, J ¼ 7.1 Hz, 2H), 1.90e1.72 (m, 2H), 0.80 (t,
J ¼ 7.5 Hz, 3H).
5.1.8.2. 2-(4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-N,N-
dimethylethan-1-amine (21e). The title compound was prepared
from 20a following general procedure F. Yield: 96%. 1H NMR
(300 MHz, DMSO‑d6)
d
8.63 (s, 1H), 8.01 (s, 1H), 4.34 (t, J ¼ 5.4 Hz,
10

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
5.1.10.1. 4-(4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ani-
line (24a). The title compound was prepared from 21a and 23a
following general procedure H. Yield: 89%. 1H NMR (300 MHz,
5.1.10.11. 4-(4-Chloro-7-(2-morpholinoethyl)-7H-pyrrolo[2,3-d]pyr-
imidin-5-yl)aniline (24k). The title compound was prepared from
21f and 23a following general procedure H. Yield: 61%. 1H NMR
DMSO‑d6)
d
8.61 (s, 1H), 7.62 (s, 1H), 7.15 (d, J ¼ 8.1 Hz, 2H), 6.61 (d,
(300 MHz, DMSO‑d6)
2H), 6.61 (d, J ¼ 8.4 Hz, 2H), 5.17 (s, 2H), 4.41 (t, J ¼ 6.3 Hz, 2H),
3.57e3.43 (m, 4H), 2.73 (t, J ¼ 6.3 Hz, 2H), 2.48e2.39 (m, 4H).
d
8.60 (s, 1H), 7.69 (s, 1H), 7.16 (d, J ¼ 8.4 Hz,
J ¼ 8.1 Hz, 2H), 5.15 (s, 2H), 3.85 (s, 3H).
5.1.10.2. 4-(4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-
fluoroaniline (24b). The title compound was prepared from 21a and
23b following general procedure H. Yield: 93%. 1H NMR (300 MHz,
5.1.10.12. 4-(4-Chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyr-
rolo[2,3-d]pyrimidin-5-yl)-2,5-difluoroaniline (24l). The title com-
pound was prepared from 21c and 23f following general procedure
DMSO‑d6)
d
8.63 (s, 1H), 7.70 (s, 1H), 7.12 (dd, J ¼ 12.7, 0.9 Hz, 1H),
H. Yield: 80%. 1H NMR (400 MHz, DMSO‑d6)
d 8.69 (s, 1H), 7.88 (s,
7.06e6.96 (m, 1H), 6.86e6.76 (m, 1H), 5.22 (s, 2H), 3.86 (s, 3H).
1H), 7.08 (dd, J ¼ 11.5, 6.9 Hz, 1H), 6.63 (dd, J ¼ 11.3, 7.7 Hz, 1H), 5.67
(s, 2H), 5.61 (s, 2H), 3.63e3.52 (m, 2H), 0.88e0.78 (m, 2H), ꢀ0.09 (s,
9H).
5.1.10.3. 4-(4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-3-
fluoroaniline (24c). The title compound was prepared from 21a and
23c following general procedure H. Yield: 73%. 1H NMR (300 MHz,
5.1.11. General procedure I: syntheses of compounds 25ae25l
In a sealed tube, chlorinated compound (24ae24l) (0.5 mmol,
1.0 eq) was dissolved in aqueous ammonia (0.5 mL) and 1,4-dioxane
(0.5 mL). The mixture was heated at 100 ꢁC for 48 h. After cooling,
the reaction solution was distributed in water and ethyl acetate. The
organic layer was separated and washed with water and brine
successively, dried over anhydrous sodium sulfate and concen-
trated in vacuum. The resulting residue was purified by silica gel
chromatography (dichloromethane/methanol, v/v, 95:5) to give the
desired product.
DMSO‑d6)
d
8.63 (s, 1H), 7.66 (s, 1H), 7.04 (t, J ¼ 8.6 Hz, 1H),
6.49e6.36 (m, 2H), 5.50 (s, 2H), 3.87 (s, 3H).
5.1.10.4. 4-(4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-
methylaniline (24d). The title compound was prepared from 21a
and 23d following general procedure H. Yield: 40%. 1H NMR
(300 MHz, DMSO‑d6)
6.65 (d, J ¼ 7.8 Hz, 1H), 4.92 (s, 2H), 3.85 (s, 3H), 2.10 (s, 3H).
d 8.61 (s, 1H), 7.61 (s, 1H), 7.09e7.00 (m, 2H),
5.1.10.5. 2-Chloro-4-(4-chloro-7-methyl-7H-pyrrolo[2,3-d]pyr-
imidin-5-yl)aniline (24e). The title compound was prepared from
21a and 23e following general procedure H. Yield: 97%. 1H NMR
5.1.11.1. 5-(4-Aminophenyl)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-
4-amine (25a). The title compound was prepared from 24a
following general procedure I. Yield: 80%. 1H NMR (300 MHz,
(300 MHz, DMSO‑d6)
d
8.64 (s, 1H), 7.73 (s, 1H), 7.32 (d, J ¼ 2.0 Hz,
1H), 7.17 (dd, J ¼ 8.2, 2.0 Hz, 1H), 6.84 (d, J ¼ 8.2 Hz,1H), 5.45 (s, 2H),
DMSO‑d6)
d
8.11 (s, 1H), 7.17e7.02 (m, 3H), 6.65 (d, J ¼ 7.7 Hz, 2H),
6.00 (s, 2H), 5.21 (s, 2H), 3.70 (s, 3H). MS (ESIþ) m/z: 240.2 [M þ H]þ.
3.86 (s, 3H).
5.1.11.2. 5-(4-Amino-3-fluorophenyl)-7-methyl-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (25b). The title compound was prepared from
24b following general procedure I. Yield: 89%. 1H NMR (300 MHz,
5.1.10.6. 4-(4-Chloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-
2,5-difluoroaniline (24f). The title compound was prepared from
21a and 23f following general procedure H. Yield: 55%. 1H NMR
DMSO‑d6)
d
8.12 (s, 1H), 7.17 (s, 1H), 7.05 (dd, J ¼ 12.4, 1.5 Hz, 1H),
(300 MHz, DMSO‑d6)
d
8.65 (s, 1H), 7.74 (s, 1H), 7.07 (dd, J ¼ 11.6,
6.99e6.93 (m, 1H), 6.90e6.80 (m, 1H), 6.04 (s, 2H), 5.22 (s, 2H), 3.70
6.8 Hz, 1H), 6.62 (dd, J ¼ 11.3, 7.7 Hz, 1H), 5.59 (s, 2H), 3.87 (s, 3H).
(s, 3H). MS (ESIþ) m/z: 258.2 [M þ H]þ.
5.1.10.7. 4-(4-Chloro-2,7-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
aniline (24g). The title compound was prepared from 21b and 23a
following general procedure H. Yield: 91%. 1H NMR (300 MHz,
5.1.11.3. 5-(4-Amino-2-fluorophenyl)-7-methyl-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (25c). The title compound was prepared from
24c following general procedure I. Yield: 96%. 1H NMR (300 MHz,
DMSO‑d6)
d
7.49 (s, 1H), 7.14 (d, J ¼ 8.3 Hz, 2H), 6.60 (d, J ¼ 8.3 Hz,
DMSO‑d6)
d
8.11 (s,1H), 7.12 (s,1H), 7.02 (t, J ¼ 8.4 Hz,1H), 6.51e6.40
2H), 5.13 (s, 2H), 3.80 (s, 3H), 2.64 (s, 3H).
(m, 2H), 5.88 (s, 2H), 5.52 (s, 2H), 3.71 (s, 3H). MS (ESIþ) m/z: 258.3
[M þ H]þ.
5.1.10.8. 4-(4-Chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyr-
rolo[2,3-d]pyrimidin-5-yl)aniline (24h). The title compound was
prepared from 21c and 23a following general procedure H. Yield:
5.1.11.4. 5-(4-Amino-3-methylphenyl)-7-methyl-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (25d). The title compound was prepared from
24d following general procedure I. Yield: 66%. 1H NMR (300 MHz,
95%. 1H NMR (300 MHz, DMSO‑d6)
d 8.66 (s, 1H), 7.75 (s, 1H), 7.16 (d,
J ¼ 8.4 Hz, 2H), 6.62 (d, J ¼ 8.4 Hz, 2H), 5.65 (s, 2H), 5.19 (s, 2H), 3.56
DMSO‑d6)
d
8.10 (s, 1H), 7.07 (s, 1H), 7.01 (s, 1H), 6.97 (d, J ¼ 8.0 Hz,
(t, J ¼ 8.0 Hz, 2H), 0.84 (t, J ¼ 8.0 Hz, 2H), ꢀ0.09 (s, 9H).
1H), 6.69 (d, J ¼ 8.0 Hz, 1H), 5.98 (s, 2H), 4.94 (s, 2H), 3.70 (s, 3H),
2.10 (s, 3H). MS (ESIþ) m/z: 254.2 [M þ H]þ.
5.1.10.9. 4-(4-Chloro-7-propyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ani-
line (24i). The title compound was prepared from 21d and 23a
following general procedure H. Yield: 96%. 1H NMR (300 MHz,
5.1.11.5. 5-(4-Amino-3-chlorophenyl)-7-methyl-7H-pyrrolo[2,3-d]
pyrimidin-4-amine (25e). The title compound was prepared from
24e following general procedure I. Yield: 54%. 1H NMR (300 MHz,
DMSO‑d6)
d
8.60 (s, 1H), 7.69 (s, 1H), 7.16 (d, J ¼ 8.4 Hz, 2H), 6.61 (d,
J ¼ 8.4 Hz, 2H), 5.15 (s, 2H), 4.25 (t, J ¼ 7.2 Hz, 2H), 1.93e1.77 (m,
DMSO‑d6)
d
8.12 (s, 1H), 7.24 (d, J ¼ 1.8 Hz, 1H), 7.18 (s, 1H), 7.10 (dd,
2H), 0.85 (t, J ¼ 7.2 Hz, 3H).
J ¼ 8.1, 1.8 Hz, 1H), 6.88 (d, J ¼ 8.1 Hz, 1H), 6.03 (s, 2H), 5.44 (s, 2H),
3.70 (s, 3H). MS (ESIþ) m/z: 274.1 [M þ H]þ.
5.1.10.10. 4-(4-Chloro-7-(2-(dimethylamino)ethyl)-7H-pyrrolo[2,3-d]
pyrimidin-5-yl)aniline (24j). The title compound was prepared
from 21e and 23a following general procedure H. Yield: 86%. 1H
5.1.11.6. 5-(4-Amino-2,5-difluorophenyl)-7-methyl-7H-pyrrolo[2,3-
d]pyrimidin-4-amine (25f). The title compound was prepared from
24f following general procedure I. Yield: 51%. 1H NMR (300 MHz,
NMR (300 MHz, DMSO‑d6)
d 8.60 (s, 1H), 7.69 (s, 1H), 7.16 (d,
J ¼ 8.4 Hz, 2H), 6.61 (d, J ¼ 8.4 Hz, 2H), 5.15 (s, 2H), 4.38 (t, J ¼ 6.3 Hz,
DMSO‑d6)
d
8.12 (s, 1H), 7.18 (s, 1H), 6.99 (dd, J ¼ 11.7, 7.2 Hz, 1H),
2H), 2.70 (t, J ¼ 6.3 Hz, 2H), 2.18 (s, 6H).
6.64 (dd, J ¼ 11.4, 7.7 Hz, 1H), 5.99 (s, 2H), 5.55 (s, 2H), 3.71 (s, 3H).
11

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
MS (ESIþ) m/z: 276.3 [M þ H]þ.
carboxamide (9a). The title compound was prepared from 19a and
25a following general procedure J. Yield: 82%. 1H NMR (300 MHz,
5.1.11.7. 5-(4-Aminophenyl)-2,7-dimethyl-7H-pyrrolo[2,3-d]pyr-
imidin-4-amine (25g). The title compound was prepared from 24g
following general procedure I. Yield: 55%. 1H NMR (300 MHz,
DMSO‑d6)
d
13.06 (s, 1H), 8.69 (d, J ¼ 2.2 Hz, 1H), 8.16 (d, J ¼ 2.2 Hz,
1H), 8.15 (s, 1H), 7.81 (d, J ¼ 8.6 Hz, 2H), 7.77e7.67 (m, 2H), 7.43 (d,
J ¼ 8.6 Hz, 2H), 7.37e7.22 (m, 3H), 6.11 (s, 2H), 3.93 (s, 3H), 3.74 (s,
DMSO‑d6)
d
7.08 (d, J ¼ 8.4 Hz, 2H), 7.02 (s, 1H), 6.65 (d, J ¼ 8.4 Hz,
3H). 13C NMR (126 MHz, DMSO‑d6)
d 174.4, 162.2, 161.7, 157.1, 151.5,
2H), 6.02 (s, 2H), 5.50 (s, 2H), 3.67 (s, 3H), 2.38 (s, 3H). MS (ESIþ) m/
z: 254.2 [M þ H]þ.
150.4, 145.2, 141.2, 137.3, 131.0 (2C), 130.3, 129.9, 129.5, 128.9 (2C),
124.2, 120.1 (2C), 118.0, 114.8 (2C), 114.7, 99.8, 44.2, 30.7. HRMS
(ESIþ) m/z C26H22FN6O2: calcd 469.1788, found 469.1793 [M þ H]þ.
5.1.11.8. 5-(4-Aminophenyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine (25h). The title compound
was prepared from 24h following general procedure I. Yield: 57%.
5.1.12.2. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-5-(4-fluorophenyl)-1-isopropyl-4-oxo-1,4-dihydropyridine-
3-carboxamide (9b). The title compound was prepared from 19d
and 25a following general procedure J. Yield: 66%. 1H NMR
1H NMR (300 MHz, DMSO‑d6)
d 8.14 (s, 1H), 7.20 (s, 1H), 7.11 (d,
J ¼ 8.4 Hz, 2H), 6.66 (d, J ¼ 8.4 Hz, 2H), 6.07 (s, 2H), 5.50 (s, 2H), 5.31
(s, 2H), 3.53 (t, J ¼ 8.1 Hz, 2H), 0.83 (t, J ¼ 8.1 Hz, 2H), ꢀ0.08 (s, 9H).
MS (ESIþ) m/z: 356.3 [M þ H]þ.
(400 MHz, DMSO‑d6)
d
13.04 (s, 1H), 8.76 (d, J ¼ 2.0 Hz, 1H), 8.27 (d,
J ¼ 2.0 Hz, 1H), 8.16 (s, 1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.74 (dd, J ¼ 8.5,
5.7 Hz, 2H), 7.44 (d, J ¼ 8.4 Hz, 2H), 7.36e7.24 (m, 3H), 6.13 (s, 2H),
4.70e4.57 (m, 1H), 3.74 (s, 3H), 1.51 (d, J ¼ 6.6 Hz, 6H). 13C NMR
5.1.11.9. 5-(4-Aminophenyl)-7-propyl-7H-pyrrolo[2,3-d]pyrimidin-4-
amine (25i). The title compound was prepared from 24i following
(126 MHz, DMSO‑d6)
d 174.7, 162.2, 161.8, 157.1, 151.5, 150.4, 142.2,
general procedure I. Yield: 74%. 1H NMR (300 MHz, DMSO‑d6)
d 8.09
137.8, 137.3, 131.2 (2C), 130.4, 130.3, 130.0, 128.9 (2C), 124.2, 120.1
(2C), 118.3, 114.7 (2C), 114.7, 99.8, 59.3, 30.7, 22.0 (2C). HRMS (ESIþ)
m/z C28H26FN6O2: calcd 497.2101, found 497.2107 [M þ H]þ.
(s, 1H), 7.14 (s, 1H), 7.10 (d, J ¼ 8.4 Hz, 2H), 6.65 (d, J ¼ 8.4 Hz, 2H),
5.98 (s, 2H), 5.20 (s, 2H), 4.08 (t, J ¼ 7.2 Hz, 2H), 1.90e1.67 (m, 2H),
0.85 (t, J ¼ 7.2 Hz, 3H). MS (ESIþ) m/z: 268.2 [M þ H]þ.
5.1.12.3. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-1-(2-(dimethylamino)-2-oxoethyl)-5-(4-fluorophenyl)-4-
oxo-1,4-dihydropyridine-3-carboxamide (9c). The title compound
was prepared from 19b and 25a following general procedure J.
5.1.11.10. 5-(4-Aminophenyl)-7-(2-(dimethylamino)ethyl)-7H-pyr-
rolo[2,3-d]pyrimidin-4-amine (25j). The title compound was pre-
pared from 24j following general procedure I. Yield: 42%. 1H NMR
(300 MHz, DMSO‑d6)
d
8.11 (s, 1H), 7.17 (s, 1H), 7.10 (d, J ¼ 8.4 Hz,
Yield: 83%. 1H NMR (300 MHz, DMSO‑d6)
d 12.98 (s, 1H), 8.63 (d,
2H), 6.66 (d, J ¼ 8.4 Hz, 2H), 6.01 (s, 2H), 5.20 (s, 2H), 4.29 (t,
J ¼ 6.5 Hz, 2H), 2.93e2.68 (m, 2H), 2.33 (s, 6H). MS (ESIþ) m/z: 297.4
[M þ H]þ.
J ¼ 1.8 Hz,1H), 8.15 (s, 1H), 8.07 (d, J ¼ 1.8 Hz, 1H), 7.81 (d, J ¼ 8.3 Hz,
2H), 7.70 (dd, J ¼ 8.4, 5.5 Hz, 2H), 7.44 (d, J ¼ 8.3 Hz, 2H), 7.35e7.24
(m, 3H), 6.12 (s, 2H), 5.25 (s, 2H), 3.74 (s, 3H), 3.02 (s, 3H), 2.90 (s,
3H). 13C NMR (126 MHz, DMSO‑d6)
d 174.8, 166.2, 162.2, 161.7, 157.1,
5.1.11.11. 5-(4-Aminophenyl)-7-(2-morpholinoethyl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine (25k). The title compound was prepared from
24k following general procedure I. Yield: 98%. 1H NMR (300 MHz,
151.4, 150.4, 146.0, 141.7, 137.2, 130.9 (2C), 130.3, 130.0, 128.9 (3C),
124.2, 120.2 (2C), 117.9, 114.9 (2C), 114.8, 99.8, 57.4, 35.6, 35.3, 30.7.
HRMS (ESIþ) m/z C29H27FN7O3: calcd 540.2159, found 540.2169
[M þ H]þ.
DMSO‑d6)
d
8.10 (s, 1H), 7.17 (s, 1H), 7.10 (d, J ¼ 8.4 Hz, 2H), 6.66 (d,
J ¼ 8.4 Hz, 2H), 6.00 (s, 2H), 5.29 (s, 2H), 4.26 (t, J ¼ 6.7 Hz, 2H),
3.58e3.52 (m, 4H), 2.72 (t, J ¼ 6.7 Hz, 2H), 2.56e2.45 (m, 4H). MS
(ESIþ) m/z: 339.3 [M þ H]þ.
5.1.12.4. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-5-(4-fluorophenyl)-1-(3-methoxypropyl)-4-oxo-1,4-
dihydropyridine-3-carboxamide (9d). The title compound was pre-
pared from 19c and 25a following general procedure J. Yield: 42%.
5.1.11.12. 5-(4-Amino-2,5-difluorophenyl)-7-((2-(trimethylsilyl)
ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (25l). The title
compound was prepared from 24l following general procedure I.
1H NMR (300 MHz, DMSO‑d6)
d
13.02 (s, 1H), 8.69 (d, J ¼ 2.2 Hz,1H),
Yield: 92%. 1H NMR (400 MHz, DMSO‑d6)
d 8.14 (s, 1H), 7.29 (s, 1H),
8.20 (d, J ¼ 2.2 Hz,1H), 8.15 (s,1H), 7.81 (d, J ¼ 8.5 Hz, 2H), 7.78e7.69
(m, 2H), 7.44 (d, J ¼ 8.5 Hz, 2H), 7.36e7.21 (m, 3H), 6.08 (s, 2H), 4.24
(t, J ¼ 7.0 Hz, 2H), 3.74 (s, 3H), 3.36 (t, J ¼ 5.9 Hz, 2H), 3.24 (s, 3H),
7.10e6.90 (m, 1H), 6.78e6.50 (m, 1H), 6.08 (s, 2H), 5.58 (s, 2H), 5.51
(s, 2H), 3.63e3.47 (m, 2H), 0.92e0.75 (m, 2H), ꢀ0.08 (s, 9H). MS
(ESIþ) m/z: 392.3 [M þ H]þ.
2.16e1.99 (m, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 174.6, 162.2,
161.8, 157.1, 151.5, 150.4, 144.5, 140.3, 137.2, 131.1 (2C), 130.3, 130.0,
129.8,128.9 (2C),124.2,120.2 (2C),118.2,114.8 (2C),114.7, 99.8, 68.3,
57.9, 54.5, 30.7, 29.9. HRMS (ESIþ) m/z C29H28FN6O3: calcd 527.2207,
found 527.2194 [M þ H]þ.
5.1.12. General procedure J: syntheses of compounds 9ae9h,
10ae10e, 11ae11e, 12a, 12ce12e and 26ae26c
The carboxylic acid intermediate (19ae19m) (0.2 mmol, 1.0 eq),
organic amine intermediate (25ae25l) (0.2 mmol, 1.0 eq) and 2-(7-
azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexa-
fluorophosphate (0.3 mmol,1.5 eq) were dissolved in DMF (2 mL) at
room temperature. N-ethyl-N-isopropylpropan-2-amine
5.1.12.5. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-5-(4-fluorophenyl)-4-oxo-1-(tetrahydro-2H-pyran-4-yl)-
1,4-dihydropyridine-3-carboxamide (9e). The title compound was
prepared from 19e and 25a following general procedure J. Yield:
(0.6 mmol, 3.0 eq) was added and the mixture was stirred for 3 h.
The reaction mixture was distributed in water and ethyl acetate.
The organic layer was separated and washed with water and brine
successively, dried over anhydrous sodium sulfate and concen-
trated in vacuum. The resulting residue was purified by silica gel
chromatography (dichloromethane/methanol, v/v, 98:2 to 95:5) to
give the desired product.
74%. 1H NMR (300 MHz, DMSO‑d6)
d 12.99 (s, 1H), 8.76 (s, 1H), 8.28
(s, 1H), 8.15 (s, 1H), 7.81 (d, J ¼ 8.2 Hz, 2H), 7.78e7.72 (m, 2H), 7.43
(d, J ¼ 8.2 Hz, 2H), 7.35e7.22 (m, 3H), 6.11 (s, 2H), 4.64e4.46 (m,
1H), 4.11e3.95 (m, 2H), 3.74 (s, 3H), 3.44 (t, J ¼ 11.3 Hz, 2H),
2.22e1.93 (m, 4H). 13C NMR (126 MHz, DMSO‑d6)
d 174.8, 162.1,
161.8, 157.1, 151.4, 150.4, 142.7, 137.9, 137.2, 131.3 (2C), 130.3 (2C),
130.0, 128.9 (2C), 124.2, 120.2 (2C), 118.3, 114.7 (3C), 99.8, 66.2 (2C),
63.5, 32.2 (2C), 30.7. HRMS (ESIþ) m/z C30H28FN6O3: calcd 539.2207,
found 539.2217 [M þ H]þ.
5.1.12.1. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-5-(4-fluorophenyl)-1-methyl-4-oxo-1,4-dihydropyridine-3-
12

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
5.1.12.6. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydrofuran-2-yl)methyl)-
1,4-dihydropyridine-3-carboxamide (9f). The title compound was
prepared from 19f and 25a following general procedure J. Yield:
2H), 7.73 (d, J ¼ 8.4 Hz, 2H), 7.52 (d, J ¼ 8.4 Hz, 2H), 7.44 (d,
J ¼ 8.4 Hz, 2H), 7.30 (s, 1H), 6.12 (s, 2H), 4.11 (d, J ¼ 6.8 Hz, 2H),
3.91e3.81 (m, 2H), 3.74 (s, 3H), 3.35e3.18 (m, 2H), 2.22e2.01 (m,
1H), 1.53e1.40 (m, 2H), 1.40e1.20 (m, 2H). 13C NMR (126 MHz,
67%. 1H NMR (300 MHz, DMSO‑d6)
d
13.00 (s,1H), 8.72 (d, J ¼ 2.1 Hz,
DMSO‑d6) d 174.5, 162.1, 157.1, 151.4, 150.4, 144.6, 140.6, 137.2, 132.8,
1H), 8.19 (d, J ¼ 2.1 Hz, 1H), 8.15 (s, 1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.72
(dd, J ¼ 8.4, 6.1 Hz, 2H), 7.44 (d, J ¼ 8.4 Hz, 2H), 7.35e7.24 (m, 3H),
6.09 (s, 2H), 4.36e4.31 (m, 1H), 4.27e4.11 (m, 2H), 3.90e3.63 (m,
5H), 2.15e1.92 (m, 1H), 1.93e1.74 (m, 2H), 1.65e1.49 (m, 1H). 13C
132.5, 130.8 (2C), 130.0, 129.4, 128.9 (2C), 128.0 (2C), 124.2, 120.2
(2C), 118.2, 114.8, 99.8, 66.4 (2C), 61.7, 35.8, 30.7, 29.3 (2C). HRMS
(ESIþ) m/z C31H30ClN6O3: calcd 569.2068, found 569.2070 [M þ
H]þ.
NMR (126 MHz, DMSO‑d6)
d 174.7, 162.2, 161.8, 157.2, 151.5, 150.4,
145.0, 140.8, 137.2, 131.0 (2C), 130.3, 130.0, 129.4, 128.9 (2C), 124.2,
120.2 (2C), 117.9, 114.9 (2C), 114.7, 99.8, 77.3, 67.5, 59.9, 30.7, 28.1,
25.1. HRMS (ESIþ) m/z C30H28FN6O3: calcd 539.2207, found
539.2206 [M þ H]þ.
5.1.12.11. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)methyl)-5-(4-(tri-
fluoromethyl)phenyl)-1,4-dihydropyridine-3-carboxamide
(10c).
The title compound was prepared from 19k and 25a following
general procedure J. Yield: 63%. 1H NMR (300 MHz, DMSO‑d6)
5.1.12.7. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,4-dihydropyridine-3-carboxamide (9g). The title com-
pound was prepared from 19g and 25a following general procedure
d
12.94 (s, 1H), 8.76 (d, J ¼ 2.0 Hz,1H), 8.33 (d, J ¼ 2.0 Hz,1H), 8.16 (s,
1H), 7.93 (d, J ¼ 8.4 Hz, 2H), 7.86e7.77 (m, 4H), 7.44 (d, J ¼ 8.4 Hz,
2H), 7.31 (s, 1H), 6.13 (s, 2H), 4.13 (d, J ¼ 7.1 Hz, 2H), 3.94e3.80 (m,
2H), 3.74 (s, 3H), 3.33e3.20 (m, 2H), 2.23e2.00 (m, 1H), 1.55e1.42
J. Yield: 51%. 1H NMR (300 MHz, DMSO‑d6)
d
13.01 (s, 1H), 8.72 (d,
(m, 2H), 1.42e1.17 (m, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 174.5,
J ¼ 1.8 Hz,1H), 8.22 (d, J ¼ 1.8 Hz,1H), 8.16 (s, 1H), 7.81 (d, J ¼ 8.4 Hz,
2H), 7.74 (dd, J ¼ 8.4, 5.9 Hz, 2H), 7.44 (d, J ¼ 8.4 Hz, 2H), 7.35e7.24
(m, 3H), 6.10 (s, 2H), 4.11 (d, J ¼ 6.8 Hz, 2H), 3.93e3.80 (m, 2H), 3.74
(s, 3H), 3.34e3.21 (m, 2H), 2.22e2.01 (m, 1H), 1.47 (d, J ¼ 12.1 Hz,
2H), 1.33 (td, J ¼ 12.3, 4.0 Hz, 2H). 13C NMR (126 MHz, DMSO‑d6)
162.0, 157.0, 151.3, 150.4, 144.9, 141.3, 138.2, 137.2, 130.0, 129.7 (2C),
129.2, 128.9 (2C), 128.1, 125.5e123.1 (4C), 120.2 (2C), 118.5, 114.8,
99.8, 66.4 (2C), 61.7, 35.8, 30.7, 29.3 (2C). HRMS (ESIþ) m/z
C
32H30F3N6O3: calcd 603.2331, found 603.2326 [M þ H]þ.
d
174.6, 162.2, 161.8, 157.1, 151.4, 150.4, 144.5, 140.4, 137.2, 131.1 (2C),
5.1.12.12. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)phenyl)-5-(3-fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,4-dihydropyridine-3-carboxamide (10d). The title com-
pound was prepared from 19l and 25a following general procedure
130.2, 130.0, 129.7, 128.9 (2C), 124.2, 120.2 (2C), 118.1, 114.8 (2C),
114.8, 99.8, 66.4 (2C), 61.7, 35.8, 30.7, 29.3 (2C). HRMS (ESIþ) m/z
C
31H30FN6O3: calcd 553.2363, found 553.2367 [M þ H]þ.
J. Yield: 96%. 1H NMR (300 MHz, DMSO‑d6)
d 12.96 (s, 1H), 8.73 (d,
5.1.12.8. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-1-(cyclohexylmethyl)-5-(4-fluorophenyl)-4-oxo-1,4-
dihydropyridine-3-carboxamide (9h). The title compound was pre-
pared from 19h and 25a following general procedure J. Yield: 80%.
J ¼ 2.2 Hz,1H), 8.29 (d, J ¼ 2.2 Hz,1H), 8.16 (s,1H), 7.81 (d, J ¼ 8.6 Hz,
2H), 7.64e7.47 (m, 3H), 7.44 (d, J ¼ 8.6 Hz, 2H), 7.31 (s, 1H),
7.27e7.17 (m, 1H), 6.12 (s, 2H), 4.12 (d, J ¼ 7.4 Hz, 2H), 3.93e3.80 (m,
2H), 3.74 (s, 3H), 3.38e3.12 (m, 2H), 2.18e2.04 (m, 1H), 1.53e1.39
1H NMR (300 MHz, DMSO‑d6)
d
13.03 (s, 1H), 8.68 (d, J ¼ 2.1 Hz, 1H),
(m, 2H), 1.39e1.20 (m, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 174.5,
8.21 (d, J ¼ 2.1 Hz, 1H), 8.15 (s, 1H), 7.80 (d, J ¼ 8.6 Hz, 2H), 7.76e7.68
(m, 2H), 7.44 (d, J ¼ 8.6 Hz, 2H), 7.35e7.23 (m, 3H), 6.09 (s, 2H), 4.06
(d, J ¼ 7.3 Hz, 2H), 3.74 (s, 3H),1.93e1.48 (m, 6H),1.33e0.95 (m, 5H).
162.1, 161.8,157.0,151.3,150.4,144.6,140.9,137.2,136.2,130.0,129.9,
129.2, 128.9 (2C), 125.0, 124.3, 120.2 (2C), 118.4, 115.7, 114.8, 114.5,
99.8, 66.4 (2C), 61.7, 35.8, 30.7, 29.3 (2C). HRMS (ESIþ) m/z
13C NMR (126 MHz, CDCl3)
d
175.5, 162.8, 162.6, 156.9, 151.7, 150.8,
C
31H30FN6O3: calcd 553.2363, found 553.2357 [M þ H]þ.
144.0, 138.4, 137.8, 132.1, 130.7 (2C), 130.2, 129.6, 129.3 (2C), 123.7,
121.1 (2C), 119.4, 116.0, 115.6 (2C), 101.0, 64.6, 39.2, 31.2, 30.3 (2C),
25.9, 25.4 (2C). HRMS (ESIþ) m/z C32H32FN6O2: calcd 551.2571,
found 551.2575 [M þ H]þ.
5.1.12.13. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-5-(2,4-difluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,4-dihydropyridine-3-carboxamide (10e). The title com-
pound was prepared from 19m and 25a following general pro-
5.1.12.9. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-4-oxo-5-phenyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-1,4-
dihydropyridine-3-carboxamide (10a). The title compound was
prepared from 19i and 25a following general procedure J. Yield:
cedure J. Yield: 60%. 1H NMR (300 MHz, DMSO‑d6)
d 12.85 (s, 1H),
8.76 (d, J ¼ 2.1 Hz, 1H), 8.19 (d, J ¼ 2.1 Hz, 1H), 8.15 (s, 1H), 7.79 (d,
J ¼ 8.6 Hz, 2H), 7.61e7.48 (m, 1H), 7.43 (d, J ¼ 8.6 Hz, 2H), 7.40e7.31
(m, 1H), 7.30 (s, 1H), 7.25e7.15 (m, 1H), 6.11 (s, 2H), 4.10 (d,
J ¼ 7.6 Hz, 2H), 3.95e3.79 (m, 2H), 3.74 (s, 3H), 3.35e3.18 (m, 2H),
2.19e1.98 (m, 1H), 1.51e1.42 (m, 2H), 1.40e1.20 (m, 2H). 13C NMR
45%. 1H NMR (300 MHz, DMSO‑d6)
d 13.05 (s, 1H), 8.72 (s, 1H), 8.20
(s, 1H), 8.15 (s, 1H), 7.80 (d, J ¼ 8.3 Hz, 2H), 7.67 (d, J ¼ 8.3 Hz, 2H),
7.54e7.34 (m, 5H), 7.30 (s, 1H), 6.11 (s, 2H), 4.11 (d, J ¼ 6.5 Hz, 2H),
3.94e3.79 (m, 2H), 3.74 (s, 3H), 3.35e3.21 (m, 2H), 2.21e2.01 (m,
1H), 1.56e1.40 (m, 2H), 1.41e1.15 (m, 2H). 13C NMR (126 MHz,
(126 MHz, DMSO‑d6)
d 174.2, 162.2, 162.0, 159.9, 157.0, 151.4, 150.4,
145.1, 141.8, 137.2, 133.3, 130.0, 128.9 (2C), 125.7, 124.3, 120.1 (2C),
118.3, 117.9, 114.8, 111.2, 104.0, 99.8, 66.4 (2C), 61.6, 35.9, 30.7, 29.2
(2C). HRMS (ESIþ) m/z C31H29F2N6O3: calcd 571.2269, found
571.2270 [M þ H]þ.
DMSO‑d6)
d 174.7, 162.3, 156.8, 151.08, 150.3, 144.4, 140.5, 137.3,
134.0, 130.8, 129.9, 129.0 (2C), 128.9 (2C), 128.0 (2C), 127.8, 124.3,
120.2 (2C), 118.1, 114.9, 99.8, 66.4 (2C), 61.6, 35.9, 30.7, 29.3 (2C).
HRMS (ESIþ) m/z C31H31N6O3: calcd 535.2458, found 535.2442 [M þ
H]þ.
5.1.12.14. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)-2-fluorophenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-py-
ran-4-yl)methyl)-1,4-dihydropyridine-3-carboxamide
(11a).
5.1.12.10. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-5-(4-chlorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,4-dihydropyridine-3-carboxamide (10b). The title com-
pound was prepared from 19j and 25a following general procedure
The title compound was prepared from 19g and 25b following
general procedure J. Yield: 90%. 1H NMR (300 MHz, DMSO‑d6)
d
13.23 (s,1H), 8.74 (s,1H), 8.57 (t, J ¼ 8.1 Hz,1H), 8.23 (s,1H), 8.16 (s,
1H), 7.81e7.66 (m, 2H), 7.41e7.21 (m, 5H), 6.21 (s, 2H), 4.11 (d,
J ¼ 6.6 Hz, 2H), 3.93e3.80 (m, 2H), 3.74 (s, 3H), 3.40e3.14 (m, 2H),
2.23e2.03 (m, 1H), 1.54e1.40 (m, 2H), 1.40e1.21 (m, 2H). 13C NMR
J. Yield: 91%. 1H NMR (300 MHz, DMSO‑d6)
d
12.98 (s, 1H), 8.72 (d,
J ¼ 1.8 Hz,1H), 8.25 (d, J ¼ 1.8 Hz,1H), 8.16 (s, 1H), 7.80 (d, J ¼ 8.4 Hz,
13

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
(126 MHz, DMSO‑d6)
d
174.6, 162.4, 161.8, 157.1, 152.2, 151.5, 150.5,
C
31H28F3N6O3: calcd 589.2175, found 589.2162 [M þ H]þ.
144.7, 140.5, 131.1 (2C), 130.7, 130.2, 129.8, 125.3, 124.8, 124.3, 121.8,
117.9, 115.1e114.5 (3C), 113.8, 99.6, 66.4 (2C), 61.7, 35.8, 30.8, 29.3
(2C). HRMS (ESIþ) m/z C31H29F2N6O3: calcd 571.2269, found
571.2321 [M þ H]þ.
5.1.12.19. N-(4-(4-amino-2,7-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-
5-yl)phenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-
yl)methyl)-1,4-dihydropyridine-3-carboxamide
compound was prepared from 19g and 25g following general
procedure J. Yield: 73%. 1H NMR (300 MHz, DMSO‑d6)
13.01 (s,
1H), 8.72 (d, J ¼ 1.8 Hz, 1H), 8.22 (d, J ¼ 1.8 Hz, 1H), 7.80 (d,
J ¼ 8.4 Hz, 2H), 7.73 (dd, J ¼ 8.7, 5.7 Hz, 2H), 7.42 (d, J ¼ 8.4 Hz, 2H),
7.34e7.25 (m, 2H), 7.23 (s, 1H), 6.13 (s, 2H), 4.11 (d, J ¼ 7.2 Hz, 2H),
3.93e3.79 (m, 2H), 3.70 (s, 3H), 3.32e3.20 (m, 2H), 2.43 (s, 3H),
2.20e2.00 (m, 1H), 1.52e1.40 (m, 2H), 1.40e1.18 (m, 2H). 13C NMR
(12a). The
title
5.1.12.15. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)-3-fluorophenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-py-
ran-4-yl)methyl)-1,4-dihydropyridine-3-carboxamide (11b). The ti-
tle compound was prepared from 19g and 25c following general
d
procedure J. Yield: 81%. 1H NMR (300 MHz, DMSO‑d6)
d 13.16 (s,1H),
8.73 (d, J ¼ 1.8 Hz, 1H), 8.23 (d, J ¼ 1.8 Hz, 1H), 8.15 (s, 1H), 7.92 (d,
J ¼ 13.0 Hz,1H), 7.73 (dd, J ¼ 8.5, 5.7 Hz, 2H), 7.52e7.20 (m, 5H), 6.05
(s, 2H), 4.11 (d, J ¼ 6.3 Hz, 2H), 3.93e3.83 (m, 2H), 3.75 (s, 3H),
3.33e3.20 (m, 2H), 2.20e2.02 (m, 1H), 1.54e1.41 (m, 2H), 1.41e1.21
(126 MHz, DMSO‑d6)
d 174.6, 162.2, 161.8, 159.3, 156.6, 151.2, 144.5,
140.4, 137.2, 131.1 (2C), 130.2, 130.0, 129.7, 128.8 (2C), 123.8, 120.2
(2C),118.1,114.8,114.8 (2C), 97.6, 66.4 (2C), 61.7, 35.8, 30.7, 29.3 (2C),
25.0. HRMS (ESIþ) m/z C32H32FN6O3: calcd 567.2520, found
567.2516 [M þ H]þ.
(m, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 174.6, 162.6, 161.8, 159.2,
157.2, 151.6, 150.2, 144.7, 140.5, 139.0, 131.9, 131.1 (2C), 130.1, 129.8,
125.4, 117.8, 117.0, 115.8, 114.8 (2C), 107.3, 106.9, 100.7, 66.4 (2C),
61.7, 35.8, 30.8, 29.3 (2C). HRMS (ESIþ) m/z C31H29F2N6O3: calcd
571.2269, found 571.2272 [M þ H]þ.
5.1.12.20. N-(4-(4-amino-7-propyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)
phenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)
methyl)-1,4-dihydropyridine-3-carboxamide (12c). The title com-
pound was prepared from 19g and 25i following general procedure
5.1.12.16. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)-2-methylphenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-
J. Yield: 73%. 1H NMR (300 MHz, DMSO‑d6)
d 13.02 (s, 1H), 8.72 (d,
pyran-4-yl)methyl)-1,4-dihydropyridine-3-carboxamide
(11c).
J ¼ 2.1 Hz,1H), 8.22 (d, J ¼ 2.1 Hz,1H), 8.13 (s, 1H), 7.80 (d, J ¼ 8.5 Hz,
2H), 7.77e7.68 (m, 2H), 7.45 (d, J ¼ 8.5 Hz, 2H), 7.35 (s, 1H),
7.33e7.25 (m, 2H), 6.08 (s, 2H), 4.19e4.04 (m, 4H), 3.92e3.80 (m,
2H), 3.30e3.18 (m, 2H), 2.24e2.02 (m, 1H), 1.93e1.69 (m, 2H),
1.52e1.21 (m, 4H), 0.86 (t, J ¼ 7.4 Hz, 3H). 13C NMR (126 MHz,
The title compound was prepared from 19g and 25d following
general procedure J. Yield: 88%. 1H NMR (400 MHz, DMSO‑d6)
d
12.83 (s, 1H), 8.74 (d, J ¼ 2.0 Hz, 1H), 8.45 (d, J ¼ 8.3 Hz, 1H), 8.22
(d, J ¼ 2.0 Hz, 1H), 8.15 (s, 1H), 7.73 (dd, J ¼ 8.6, 5.8 Hz, 2H),
7.40e7.23 (m, 5H), 6.10 (s, 2H), 4.11 (d, J ¼ 7.2 Hz, 2H), 3.91e3.81 (m,
2H), 3.74 (s, 3H), 3.32e3.22 (m, 2H), 2.39 (s, 3H), 2.18e2.03 (m, 1H),
1.52e1.39 (m, 2H),1.38e1.22 (m, 2H). 13C NMR (126 MHz, DMSO‑d6)
DMSO‑d6) d 174.6,162.2,161.8,155.6,154.2,149.5,144.5,140.5,137.4,
131.1 (2C), 130.2, 129.7, 129.4, 129.0 (2C), 124.2, 120.2 (2C), 118.1,
115.6, 114.8 (2C), 99.4, 66.4 (2C), 61.7, 45.6, 35.8, 29.3 (2C), 23.0, 11.1.
HRMS (ESIþ) m/z C33H34FN6O3: calcd 581.2676, found 581.2687
[M þ H]þ.
d
174.7, 162.1, 161.8, 157.1, 151.4, 150.3, 144.5, 140.4, 136.0, 131.1 (2C),
130.3, 130.2, 129.8, 129.7, 127.7, 126.3, 124.1, 121.0, 118.4, 114.9, 114.9
(2C), 99.8, 66.4 (2C), 61.7, 35.8, 30.7, 29.3 (2C), 18.2. HRMS (ESIþ) m/
z C32H32FN6O3: calcd 567.2520, found 567.2527 [M þ H]þ.
5.1.12.21. N-(4-(4-amino-7-(2-(dimethylamino)ethyl)-7H-pyrrolo
[2,3-d]pyrimidin-5-yl)phenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahy-
dro-2H-pyran-4-yl)methyl)-1,4-dihydropyridine-3-carboxamide
(12d). The title compound was prepared from 19g and 25j
following general procedure J. Yield: 79%. 1H NMR (300 MHz,
5.1.12.17. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)-2-chlorophenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-py-
ran-4-yl)methyl)-1,4-dihydropyridine-3-carboxamide
(11d).
The title compound was prepared from 19g and 25e following
DMSO‑d6)
d
13.02 (s, 1H), 8.72 (d, J ¼ 2.1 Hz, 1H), 8.23 (d, J ¼ 2.1 Hz,
general procedure J. Yield: 54%. 1H NMR (300 MHz, DMSO‑d6)
1H), 8.15 (s, 1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.78e7.68 (m, 2H), 7.44 (d,
J ¼ 8.4 Hz, 2H), 7.37 (s, 1H), 7.29 (t, J ¼ 9.0 Hz, 2H), 6.12 (s, 2H), 4.31
(t, J ¼ 6.2 Hz, 2H), 4.11 (d, J ¼ 7.2 Hz, 2H), 3.95e3.79 (m, 2H),
3.34e3.18 (m, 2H), 2.96e2.73 (m, 2H), 2.31 (s, 6H), 2.21e2.02 (m,
1H), 1.55e1.40 (m, 2H), 1.41e1.19 (m, 2H). 13C NMR (126 MHz,
d
13.26 (s,1H), 8.74 (d, J ¼ 2.1 Hz,1H), 8.65 (d, J ¼ 8.6 Hz,1H), 8.21 (d,
J ¼ 2.1 Hz, 1H), 8.16 (s, 1H), 7.72 (dd, J ¼ 8.8, 5.7 Hz, 2H), 7.57 (d,
J ¼ 1.9 Hz, 1H), 7.43 (dd, J ¼ 8.6, 1.9 Hz, 1H), 7.39 (s, 1H), 7.30 (t,
J ¼ 8.8 Hz, 2H), 6.19 (s, 2H), 4.11 (d, J ¼ 7.5 Hz, 2H), 3.93e3.80 (m,
2H), 3.74 (s, 3H), 3.32e3.21 (m, 2H), 2.20e2.03 (m, 1H), 1.54e1.40
DMSO‑d6) d 174.6, 162.2, 161.8, 157.3, 151.5, 150.3, 144.5, 140.4, 137.2,
(m, 2H), 1.40e1.22 (m, 2H). 13C NMR (126 MHz, DMSO‑d6)
d
174.6,
131.1 (2C), 130.2, 130.0, 129.7, 128.9 (2C), 123.4, 120.2 (2C), 118.1,
114.9, 114.8 (2C), 99.8, 66.4 (2C), 61.7, 58.0, 44.7 (2C), 41.1, 35.8, 29.3
(2C). HRMS (ESIþ) m/z C34H37FN7O3: calcd 610.2942, found
610.2940 [M þ H]þ.
162.6, 161.8, 157.1, 151.5, 150.5, 144.8, 140.5, 134.2, 131.1 (2C), 131.1,
130.2, 129.9, 128.7, 127.4, 124.9, 122.9, 122.2, 117.9, 114.9 (2C), 113.5,
99.6, 66.4 (2C), 61.7, 35.8, 30.8, 29.3 (2C). HRMS (ESIþ) m/z
C
31H29ClFN6O3: calcd 587.1974, found 587.1969 [M þ H]þ.
5.1.12.22. N-(4-(4-amino-7-(2-morpholinoethyl)-7H-pyrrolo[2,3-d]
5.1.12.18. N-(4-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
pyrimidin-5-yl)phenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-
yl)-2,5-difluorophenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-
2H-pyran-4-yl)methyl)-1,4-dihydropyridine-3-carboxamide
(12e).
pyran-4-yl)methyl)-1,4-dihydropyridine-3-carboxamide
(11e).
The title compound was prepared from 19g and 25k following
The title compound was prepared from 19g and 25f following
general procedure J. Yield: 67%. 1H NMR (300 MHz, DMSO‑d6)
general procedure J. Yield: 53%. 1H NMR (300 MHz, DMSO‑d6)
d
13.02 (s, 1H), 8.72 (d, J ¼ 1.8 Hz, 1H), 8.22 (d, J ¼ 1.8 Hz, 1H), 8.14 (s,
d
13.44 (s, 1H), 8.76 (d, J ¼ 2.0 Hz, 1H), 8.46 (dd, J ¼ 11.6, 6.7 Hz, 1H),
1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.74 (dd, J ¼ 8.8, 6.1 Hz, 2H), 7.44 (d,
J ¼ 8.4 Hz, 2H), 7.37 (s, 1H), 7.35e7.25 (m, 2H), 6.09 (s, 2H), 4.29 (t,
J ¼ 6.4 Hz, 2H), 4.11 (d, J ¼ 7.1 Hz, 2H), 3.93e3.80 (m, 2H), 3.65e3.46
(m, 4H), 3.34e3.21 (m, 2H), 2.80e2.64 (m, 2H), 2.49e2.41 (m, 4H),
2.19e2.03 (m, 1H), 1.53e1.40 (m, 2H), 1.40e1.20 (m, 2H). 13C NMR
8.25 (d, J ¼ 2.0 Hz,1H), 8.15 (s, 1H), 7.80e7.67 (m, 2H), 7.40e7.25 (m,
4H), 6.22 (s, 2H), 4.12 (d, J ¼ 7.4 Hz, 2H), 3.92e3.81 (m, 2H), 3.74 (s,
3H), 3.32e3.20 (m, 2H), 2.22e2.00 (m, 1H), 1.58e1.40 (m, 2H),
1.41e1.21 (m, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 174.6, 162.8,
161.8, 157.2, 154.9, 151.6, 150.3, 148.2, 144.8, 140.6, 131.1 (2C), 130.1,
129.9, 126.5, 125.9, 117.5, 117.4e117.0 (m, 1C), 116.7, 114.9 (2C), 108.4,
106.3, 100.5, 66.4 (2C), 61.7, 35.8, 30.8, 29.3 (2C). HRMS (ESIþ) m/z
(151 MHz, DMSO‑d6) d 174.6, 162.2, 161.8, 157.2, 151.5, 150.3, 144.5,
140.5, 137.2, 131.1 (2C), 130.3, 130.1, 129.7, 128.9 (2C), 123.6, 120.2
(2C), 118.1, 114.8 (2C), 114.7, 99.8, 66.4 (2C), 66.2 (2C), 61.7, 57.7, 53.2
14

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
(2C), 40.8, 35.8, 29.3 (2C). HRMS (ESIþ) m/z C36H39FN7O4: calcd
652.3048, found 652.3033 [M þ H]þ.
5.1.13.2. N-(4-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2,5-
difluorophenyl)-5-(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-
4-yl)methyl)-1,4-dihydropyridine-3-carboxamide (13a). The title
compound was prepared from 26b following general procedure K.
5.1.12.23. N-(4-(4-amino-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-
pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-5-(4-fluorophenyl)-4-oxo-1-
((tetrahydro-2H-pyran-4-yl)methyl)-1,4-dihydropyridine-3-
carboxamide (26a). The title compound was prepared from 19g and
25h following general procedure J. Yield: 98%. 1H NMR (300 MHz,
Yield: 74%. 1H NMR (300 MHz, DMSO‑d6)
d 13.43 (s, 1H), 11.90 (s,
1H), 8.76 (d, J ¼ 2.1 Hz, 1H), 8.45 (dd, J ¼ 11.8, 6.6 Hz, 1H), 8.24 (d,
J ¼ 2.1 Hz, 1H), 8.10 (s, 1H), 7.73 (dd, J ¼ 8.7, 5.6 Hz, 2H), 7.44e7.22
(m, 4H), 6.13 (s, 2H), 4.12 (d, J ¼ 7.0 Hz, 2H), 3.95e3.77 (m, 2H),
3.33e3.15 (m, 2H), 2.24e2.03 (m, 1H), 1.58e1.40 (m, 2H), 1.40e1.21
DMSO‑d6)
d
13.03 (s, 1H), 8.72 (d, J ¼ 2.1 Hz, 1H), 8.23 (d, J ¼ 2.1 Hz,
1H), 8.17 (s, 1H), 7.82 (d, J ¼ 8.5 Hz, 2H), 7.77e7.68 (m, 2H), 7.45 (d,
J ¼ 8.5 Hz, 2H), 7.41 (s, 1H), 7.34e7.21 (m, 2H), 6.16 (s, 2H), 5.53 (s,
2H), 4.11 (d, J ¼ 7.3 Hz, 2H), 3.93e3.80 (m, 2H), 3.56 (t, J ¼ 7.8 Hz,
2H), 3.34e3.18 (m, 2H), 2.18e2.03 (m, 1H), 1.52e1.21 (m, 4H), 0.85
(t, J ¼ 7.8 Hz, 2H), ꢀ0.07 (s, 9H). MS (ESIþ) m/z: 669.5 [M þ H]þ.
(m, 2H). 13C NMR (151 MHz, DMSO‑d6)
d 174.7, 162.8, 161.9, 157.3,
154.9, 151.8, 151.3, 148.2, 144.9, 140.6, 131.1 (2C), 130.1, 129.9, 126.5,
122.0, 117.5, 117.5e117.1 (m, 2C), 114.9 (2C), 108.4, 107.0, 100.5, 66.4
(2C), 61.7, 35.8, 29.3 (2C). HRMS (ESIþ) m/z C30H26F3N6O3: calcd
575.2018, found 575.2026 [M þ H]þ.
5.1.12.24. N-(4-(4-amino-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-
pyrrolo[2,3-d]pyrimidin-5-yl)-2,5-difluorophenyl)-5-(4-
fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1,4-
dihydropyridine-3-carboxamide (26b). The title compound was
prepared from 19g and 25l following general procedure J. Yield:
5.1.13.3. N-(4-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2,5-
difluorophenyl)-5-(4-fluorophenyl)-1-isopropyl-4-oxo-1,4-
dihydropyridine-3-carboxamide (13b). The title compound was
prepared from 26c following general procedure K. Yield: 76%. 1H
NMR (400 MHz, DMSO‑d6)
d 13.46 (s, 1H), 11.92 (s, 1H), 8.79 (d,
84%. 1H NMR (400 MHz, DMSO‑d6)
d
13.46 (s, 1H), 8.77 (d, J ¼ 2.1 Hz,
J ¼ 2.2 Hz, 1H), 8.46 (dd, J ¼ 11.7, 6.6 Hz, 1H), 8.29 (d, J ¼ 2.2 Hz, 1H),
8.10 (s, 1H), 7.77e7.69 (m, 2H), 7.38e7.25 (m, 4H), 6.16 (s, 2H),
4.76e4.53 (m, 1H), 1.52 (d, J ¼ 6.7 Hz, 6H). 13C NMR (151 MHz,
1H), 8.47 (dd, J ¼ 11.8, 6.9 Hz, 1H), 8.25 (d, J ¼ 2.1 Hz, 1H), 8.17 (s,
1H), 7.78e7.68 (m, 2H), 7.47 (s, 1H), 7.39e7.26 (m, 3H), 6.30 (s, 2H),
5.54 (s, 2H), 4.12 (d, J ¼ 7.6 Hz, 2H), 3.93e3.80 (m, 2H), 3.60e3.52
(m, 2H), 3.27 (t, J ¼ 11.6 Hz, 2H), 2.20e2.04 (m, 1H), 1.53e1.41 (m,
2H), 1.41e1.21 (m, 2H), 0.89e0.77 (m, 2H), ꢀ0.08 (s, 9H). MS (ESIþ)
m/z: 705.5 [M þ H]þ.
DMSO‑d6) d 174.8, 162.8, 161.9, 157.1, 155.0, 151.6, 151.2, 148.3, 142.7,
138.0, 131.3 (2C), 130.6, 130.3, 126.5, 122.1, 117.7, 117.3, 117.1, 114.8
(2C), 108.4, 107.1, 100.5, 59.5, 22.0 (2C). HRMS (ESIþ) m/z
C
27H22F3N6O2: calcd 519.1756, found 519.1745 [M þ H]þ.
5.1.12.25. N-(4-(4-amino-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-
pyrrolo[2,3-d]pyrimidin-5-yl)-2,5-difluorophenyl)-5-(4-
fluorophenyl)-1-isopropyl-4-oxo-1,4-dihydropyridine-3-carboxamide
(26c). The title compound was prepared from 19d and 25l
following general procedure J. Yield: 86%. 1H NMR (400 MHz,
5.2. Elisa kinase assay
The kinases activities were tested using ELISA assay as reported
previously in local. The IC50 values were calculated from the inhi-
bition curves in two separate experiments using a modified 4-
parameter logistic model. If the inhibitory rate is higher than 50%
at the minimum tested drug concentration, the conclusion is that
IC50 is lower than the tested minimum drug concentration, while
the inhibitory rate is less than 50% at the maximum tested drug
concentration, the conclusion is that IC50 is above the tested
maximum drug concentration [82].
DMSO‑d6)
d
13.49 (s, 1H), 8.80 (d, J ¼ 2.4 Hz, 1H), 8.47 (dd, J ¼ 11.8,
6.7 Hz, 1H), 8.29 (d, J ¼ 2.4 Hz, 1H), 8.17 (s, 1H), 7.77e7.68 (m, 2H),
7.47 (s, 1H), 7.36e7.25 (m, 3H), 6.31 (s, 2H), 5.54 (s, 2H), 4.71e4.59
(m, 1H), 3.61e3.52 (m, 2H), 1.52 (d, J ¼ 6.7 Hz, 6H), 0.89e0.78 (m,
2H), ꢀ0.08 (s, 9H). MS (ESIþ) m/z: 649.5 [M þ H]þ.
5.1.13. General procedure K: syntheses of compounds 12b and
13ae13b
5.3. Cell culture
The SEM-protected compound (26ae26c) (0.3 mmol,1.0 eq) and
TFA (3.0 mmol, 10.0 eq) were dissolved in anhydrous dichloro-
methane (3 mL) at room temperature. The mixture was stirred for
1 h and then basified with potassium carbonate at 0 ꢁC. The reac-
tion solution was extracted with dichloromethane; organic layers
were collected, dried over anhydrous sodium sulfate and concen-
trated in vacuum. The resulting residue was purified by silica gel
chromatography (dichloromethane/methanol, v/v, 95:5) to afford
the desired product.
MKN-45 cells were purchased from Japanese Research Re-
sources Bank and BaF3 cells were purchased from Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).
BaF3 cells were engineered to express human wild-type TEL-Axl
that stably expressed a constitutively active Axl. Cells were cultured
in media and serum concentration according to recommendations
of their suppliers.
5.4. Cell proliferation assay
5.1.13.1. N-(4-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-5-
(4-fluorophenyl)-4-oxo-1-((tetrahydro-2H-pyran-4-yl)methyl)-1,4-
dihydropyridine-3-carboxamide (12b). The title compound was
prepared from 26a following general procedure K. Yield: 70%. 1H
Cells were seeded in 96-well plates at a low density in growth
media. The next day, appropriate controls or designated concen-
trations of compounds were added to each well, and the cells were
incubated for 72 h. Finally, cell proliferation was determined using a
sulforhodamine B (SRB) assay (Sigma-Aldrich, USA) or a cell
counting kit (CCK-8) assay (Dojindo, Japan). IC50 values were
calculated by concentration-response curve fitting using a SoftMax
pro-based four-parameter method (SoftMax® Pro Software
(Version 5.4.1).
NMR (300 MHz, DMSO‑d6)
d 13.00 (s, 1H), 11.77 (s, 1H), 8.72 (d,
J ¼ 2.2 Hz,1H), 8.22 (d, J ¼ 2.2 Hz,1H), 8.10 (s,1H), 7.80 (d, J ¼ 8.6 Hz,
2H), 7.76e7.67 (m, 2H), 7.45 (d, J ¼ 8.6 Hz, 2H), 7.36e7.25 (m, 2H),
7.23 (d, J ¼ 2.4 Hz, 1H), 6.02 (s, 2H), 4.11 (d, J ¼ 7.4 Hz, 2H),
3.93e3.79 (m, 2H), 3.37e3.18 (m, 2H), 2.21e2.03 (m, 1H), 1.54e1.41
(m, 2H), 1.41e1.21 (m, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 174.6,
162.2, 161.8, 157.1, 151.5, 151.4, 144.5, 140.4, 137.2, 131.1 (2C), 130.4,
130.3, 129.7, 129.0 (2C), 120.2, 120.1 (2C), 118.1, 115.5, 114.8 (2C),
99.8, 66.4 (2C), 61.7, 35.8, 29.3 (2C). HRMS (ESIþ) m/z C30H28FN6O3:
calcd 539.2207, found 539.2209 [M þ H]þ.
5.5. Pharmacokinetic parameters obtained in rats
Compound 13a or 13b was administrated to male Sprague-
15

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
Dawley (SD) rats (n ¼ 3) orally by gavage as a solution in 5% DMSO/
95% (0.5% CMC-Na) at a 3 mg/kg dose. Blood samples were collected
at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h following oral dosing. The blood
samples were placed on wet ice, and serum was collected after
centrifugation. Serum samples were frozen and stored at ꢀ20 ꢁC.
The serum samples were analyzed utilizing HPLC-coupled tandem
mass spectrometry (LC-MS/MS). All animal experiments were
performed according to the institutional ethical guidelines on an-
imal care and approved by the Institute Animal Care and Use
Committee at Shanghai Institute of Materia Medica.
13b was docked into this protein, then the docked complexes were
clustered and sorted according to binding energy and the binding
models with the lowest binding energy were retained and analyzed
to further clarify the binding mechanism. Fig. 5A and B were
generated with PyMOL version 1.3.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
5.6. Western blot analysis
Acknowledgments
Cells were cultured under regular growth conditions to the
exponential growth phase. Then, the cells were treated with the
indicated dose of 13b for 2 h and lysed in 1 ꢂ sodium dodecyl
sulfate (SDS) sample buffer. Then, the cell lysates were subse-
quently resolved on 10% SDS-PAGE and transferred to nitrocellulose
membranes. Proteins were probed with a specific antibody (against
p-AXL (Y702), AXL, p-Met (Y1234/1235), p-Akt (S473), Akt, p-ERK1/
This work was supported by the funds from National Science &
Technology Major Project “Key New Drug Creation and
Manufacturing Program”, China (No. 2018ZX09711002-011-016),
Science and Technology Commission of Shanghai Municipality (No.
18431907100), the National Natural Science Foundation of China
(Nos. 21702220 and 81773762), Youth Innovation Promotion As-
sociation CAS (No. 2018324), the “Personalized Medicines –Mo-
lecular Signature-based Drug Discovery and Development”
Strategic Priority Research Program of the Chinese Academy of
Sciences (Nos. XDA12020000 and XDA12020103), and the Collab-
orative Innovation Cluster Project of Shanghai Municipal Commis-
sion of Health and Family Planning (2020CXJQ02).
2 (T202/Y204), ERK1/2,
nology, USA), c-Met (Santa Cruz Biotechnology, USA), then subse-
quently with secondary horseradish peroxidase-conjugated
b-actin and GAPDH) (Cell Signaling Tech-
a
antibody. Finally, immunoreactive proteins were detected using an
enhanced chemiluminescence detection reagent (Thermo Fisher
Scientific, USA).
5.7. In vivo antitumor activity assay
Appendix A. Supplementary data
Animal procedures were approved by the Institutional Animal
Care and Use Committee of the Shanghai Institute of Materia
Medica (approval No. 2018-05-DJ-37). Cells at density of 5e10 ꢂ 106
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113497.
in 200 mL firstly implanted subcutaneous into the right flank of each
Abbreviations
nude mice and then allowing to grow to 700e800 mm3, defined as
a well-developed tumor. After that, the well-developed tumors
were cut into 1.5 mm3 fragments and transplanted sc. into the right
flank of nude mice. When the tumor volume reached
100e200 mm3, the mice were randomly assigned into vehicle and
treatment groups (n ¼ 6 in treated group, n ¼ 12 in vehicle group).
Vehicle groups were given vehicle alone, and treatment groups
received compounds as indicated doses via po administration once
daily for indicated days. The sizes of the tumors were measured
twice per week using a microcaliper. Tumor volume (TV) ¼
(length ꢂ width2)/2, and the individual relative tumor volume
(RTV) was calculated as follows: RTV ¼ Vt/V0, where Vt is the vol-
ume on a particular day and V0 is the volume at the beginning of the
treatment. The RTV was shown on indicated days as the median
RTV SEM indicated for groups of mice. Percent (%) Tumor growth
inhibition (TGI) values were measured on the final day of study for
drug-treated compared with vehicle-treated mice and are calcu-
lated as 100 ꢂ {1 e [(VTreated Final day e VTreated Day 0)/(VVehicle Final day
e VVehicle Day 0)]}. Significant differences between the treated versus
the vehicle groups (p ꢃ 0.001) were determined using Student’s t-
test.
GAS6
RCC
growth arrest-specific 6
renal cell carcinoma
NSCLC
EMT
AML
SBDD
SAR
non-small-cell lung cancer
epithelial-mesenchymal transition
acute myeloid leukemia
structure-based drug design
structure-activity relationship
tumor growth inhibition rate
N,N-dimethylformamide
2-(trimethylsilyl)ethoxymethyl chloride
diisopropyl azodicarboxylate
tetrahydrofuran
TGI
DMF
SEMCl
DIAD
THF
HATU
2-(7-azabenzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate
N,N-diisopropylethylamine
trifluoroacetic acid
DIPEA
TFA
SEM
2-(trimethylsilyl)ethoxy)methyl
References
[1] J. Antony, R.Y.-J. Huang, AXL-driven EMT state as a targetable conduit in
cancer, Canc. Res. 77 (2017) 3725e3732.
[2] R.M.A. Linger, A.K. Keating, H.S. Earp, D.K. Graham, TAM receptor tyrosine
kinases: biologic functions, signaling, and potential therapeutic targeting in
human cancer, Adv. Canc. Res. 100 (2008) 35e83.
[3] J. Braunger, L. Schleithoff, A.S. Schulz, H. Kessler, R. Lammers, A. Ullrich,
C.R. Bartram, J.W.G. Janssen, Intracellular signaling of the Ufo/Axl receptor
tyrosine kinase is mediated mainly by a multi-substrate docking-site, Onco-
gene 14 (1997) 2619.
[4] B.C. Varnum, C. Young, G. Elliott, A. Garcia, T.D. Bartley, Y.W. Fridell,
R.W. Hunt, G. Trail, C. Clogston, R.J. Toso, D. Yanagihara, L. Bennett, M. Sylber,
L.A. Merewether, A. Tseng, E. Escobar, E.T. Liu, H.K. Yamane, Axl receptor
tyrosine kinase stimulated by the vitamin K-dependent protein encoded by
growth-arrest-specific gene 6, Nature 373 (1995) 623e626.
5.8. Molecular Modeling
The docking studies were carried out on AutoDock 4.2 using the
X-ray crystal structure of c-Met (PDB ID: 3F82). The enzyme
preparation and the hydrogen atoms adding were performed in the
prepared process. After moving native ligand and water molecular,
the c-Met enzyme was defined as a receptor and the enclosing box
centered in native ligand was set based on the binding site of c-Met.
In the docking protocol, Lamarchian genetic algorithm (GA) and
default parameters of GA were applied. The designed compound
16

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
[5] C.V. Rothlin, S. Ghosh, E.I. Zuniga, M.B. Oldstone, G. Lemke, TAM receptors are
pleiotropic inhibitors of the innate immune response, Cell 131 (2007)
1124e1136.
[6] P. Valverde, M.S. Obin, A. Taylor, Role of Gas6/Axl signaling in lens epithelial
cell proliferation and survival, Exp. Eye Res. 78 (2004) 27e37.
[7] T. Bauer, A. Zagorska, J. Jurkin, N. Yasmin, R. Koffel, S. Richter, B. Gesslbauer,
G. Lemke, H. Strobl, Identification of Axl as a downstream effector of TGF-
beta1 during Langerhans cell differentiation and epidermal homeostasis,
J. Exp. Med. 209 (2012) 2033e2047.
[29] H. Yu, R. Liu, B. Ma, X. Li, H.Y. Yen, Y. Zhou, V. Krasnoperov, Z. Xia, X. Zhang,
A.M. Bove, M. Buscarini, D. Parekh, I.S. Gill, Q. Liao, M. Tretiakova, D. Quinn,
J. Zhao, P.S. Gill, Axl receptor tyrosine kinase is a potential therapeutic target
in renal cell carcinoma, Br. J. Canc. 113 (2015) 616e625.
[30] L.E. Zucca, M.A. Morini Matushita, R.J.d.S. Oliveira, C. Scapulatempo-Neto,
M. Alves de Lima, G.G. Ribeiro, C.R. Viana, F.M. Carcano, R.M. Reis, Expression
of tyrosine kinase receptor AXL is associated with worse outcome of meta-
static renal cell carcinomas treated with sunitinib, Urol. Oncol.: Semin Orig.
Invest. 36 (2018) 11.e13e11.e21.
[8] S.M. Woo, K.J. Min, S. Kim, J.W. Park, D.E. Kim, S.H. Kim, Y.H. Choi, T.K. Kwon,
Axl is a novel target of withaferin A in the induction of apoptosis and the
suppression of invasion, Biochem. Biophys. Res. Commun. 451 (2014)
455e460.
[31] R.A. Okimoto, T.G. Bivona, AXL receptor tyrosine kinase as a therapeutic target
in NSCLC, Lung Cancer Targets Ther. 6 (2015) 27e34.
[32] X. Qu, J. Liu, X. Zhong, X. Li, Q. Zhang, Role of AXL expression in non-small cell
lung cancer, Oncol. Lett. 12 (2016) 5085e5091.
[9] S. Pagani, M. Bellan, D. Mauro, L.M. Castello, G.C. Avanzi, M.J. Lewis,
P.P. Sainaghi, C. Pitzalis, A. Nerviani, New insights into the role of Tyro3, Axl,
and Mer receptors in rheumatoid arthritis, Dis. Markers 2020 (2020) 1614627.
[10] L.A. Law, D.K. Graham, J. Di Paola, B.R. Branchford, GAS6/TAM pathway
signaling in hemostasis and thrombosis, Front. Med. 5 (2018) 137.
[11] Z.-S. Niu, X.-J. Niu, W.-H. Wang, Role of the receptor tyrosine kinase Axl in
hepatocellular carcinoma and its clinical relevance, Future Oncol. 15 (2019)
653e662.
[12] R. Brenig, O.T. Pop, E. Triantafyllou, A. Geng, A. Singanayagam, C. Perez-Shi-
bayama, L. Besse, J. Cupovic, P. Kunzler, T. Boldanova, S. Brand, D. Semela,
F.H. Duong, C.J. Weston, B. Ludewig, M.H. Heim, J. Wendon, C.G. Antoniades,
C. Bernsmeier, Expression of AXL receptor tyrosine kinase relates to monocyte
dysfunction and severity of cirrhosis, Life Sci, Alliance 3 (2020), e201900465.
[13] A. Tutusaus, E. de Gregorio, B. Cucarull, H. Cristobal, C. Areste, I. Graupera,
M. Coll, A. Colell, G. Gausdal, J.B. Lorens, P. Garcia de Frutos, A. Morales,
M. Mari, A functional role of GAS6/TAM in nonalcoholic steatohepatitis pro-
gression implicates AXL as therapeutic target, Cell Mol. Gastroenterol. Hep-
atol. 9 (2020) 349e368.
[14] J.-B.M. Koorstra, C.A. Karikari, G. Feldmann, S. Bisht, P.L. Rojas, G.J.A. Offerhaus,
H. Alvarez, A. Maitra, The Axl receptor tyrosine kinase confers an adverse
prognostic influence in pancreatic cancer and represents a new therapeutic
target, Canc. Biol. Ther. 8 (2009) 618e626.
[15] X. Song, H. Akasaka, H. Wang, R. Abbasgholizadeh, J.H. Shin, F. Zang, J. Chen,
C.D. Logsdon, A. Maitra, A.J. Bean, H. Wang, Hematopoietic progenitor kinase 1
down-regulates the oncogenic receptor tyrosine kinase AXL in pancreatic
cancer, J. Biol. Chem. 295 (2020) 2348e2358.
[33] G. Zhang, M. Wang, H. Zhao, W. Cui, Function of Axl receptor tyrosine kinase
in non-small cell lung cancer (review), Oncol. Lett. 15 (2018) 1e9.
[34] S. Nuzzo, S. Catuogno, M. Capuozzo, A. Fiorelli, P. Swiderski, S. Boccella, F. de
Nigris, C.L. Esposito, Axl-targeted delivery of the oncosuppressor miR-137 in
non-small-cell lung cancer, Mol. Ther. Nucleic Acids 17 (2019) 256e263.
[35] G. Zhang, M. Wang, H. Zhao, W. Cui, Function of Axl receptor tyrosine kinase
in non-small cell lung cancer, Oncol. Lett. 15 (2018) 2726e2734.
[36] I. Ben-Batalla, R. Erdmann, H. Jorgensen, R. Mitchell, T. Ernst, G. von Amsberg,
P. Schafhausen, J.L. Velthaus, S. Rankin, R.E. Clark, S. Koschmieder, A. Schultze,
S. Mitra, P. Vandenberghe, T.H. Brummendorf, P. Carmeliet, A. Hochhaus,
K. Pantel, C. Bokemeyer, G.V. Helgason, T.L. Holyoake, S. Loges, Axl blockade by
BGB324 inhibits BCR-ABL tyrosine kinase inhibitor-sensitive and -resistant
chronic myeloid leukemia, Clin. Canc. Res. 23 (2017) 2289e2300.
[37] S. Yan, N. Vandewalle, N. De Beule, S. Faict, K. Maes, E. De Bruyne, E. Menu,
K. Vanderkerken, K. De Veirman, AXL receptor tyrosine kinase as a therapeutic
target in hematological malignancies: focus on multiple myeloma, Cancers 11
(2019) 1727e1743.
[38] V.A. Korshunov, Axl-dependent signalling: a clinical update, Clin. Sci. (Lond.)
122 (2012) 361e368.
[39] K. Flem-Karlsen, E. McFadden, N. Omar, M.H. Haugen, G.F. Oy, T. Ryder,
H.P. Gullestad, R. Hermann, G.M. Maelandsmo, V.A. Florenes, Targeting AXL
and the DNA damage response pathway as a novel therapeutic strategy in
melanoma, Mol. Canc. Therapeut. 19 (2020) 895e905.
[40] T. Burstyn-Cohen, A. Maimon, TAM receptors, phosphatidylserine, inflam-
mation, and cancer, Cell Commun. Signal. 17 (2019) 156e164.
[41] H. Sadahiro, K.D. Kang, J.T. Gibson, M. Minata, H. Yu, J. Shi, R. Chhipa, Z. Chen,
S. Lu, Y. Simoni, T. Furuta, H. Sabit, S. Zhang, S. Bastola, S. Yamaguchi,
H. Alsheikh, S. Komarova, J. Wang, S.H. Kim, D. Hambardzumyan, X. Lu,
E.W. Newell, B. DasGupta, M. Nakada, L.J. Lee, B. Nabors, L.A. Norian, I. Nakano,
Activation of the receptor tyrosine kinase AXL regulates the immune micro-
environment in glioblastoma, Canc. Res. 78 (2018) 3002e3013.
[42] S. Zhang, X.S. Xu, J.X. Yang, J.H. Guo, T.F. Chao, Y. Tong, The prognostic role of
Gas6/Axl axis in solid malignancies: a meta-analysis and literature review,
OncoTargets Ther. 11 (2018) 509e519.
[16] K.F. Ludwig, W. Du, N.B. Sorrelle, K. Wnuk-Lipinska, M. Topalovski,
J.E. Toombs, V.H. Cruz, S. Yabuuchi, N.V. Rajeshkumar, A. Maitra, J.B. Lorens,
R.A. Brekken, Small-molecule inhibition of Axl targets tumor immune sup-
pression and enhances chemotherapy in pancreatic cancer, Canc. Res. 78
(2018) 246e255.
[17] T.M. D’Alfonso, J. Hannah, Z. Chen, Y. Liu, P. Zhou, S.J. Shin, Axl receptor
tyrosine kinase expression in breast cancer, J. Clin. Pathol. 67 (2014) 690e696.
[18] G. Jin, Z. Wang, J. Wang, L. Zhang, Y. Chen, P. Yuan, D. Liu, Expression of Axl
and its prognostic significance in human breast cancer, Oncol. Lett. 13 (2017)
621e628.
[43] F. Wu, J. Li, C. Jang, J. Wang, J. Xiong, The role of Axl in drug resistance and
epithelial-to-mesenchymal transition of non-small cell lung carcinoma, Int. J.
Clin. Exp. Pathol. 7 (2014) 6653e6661.
[44] X. Ying, J. Chen, X. Huang, P. Huang, S. Yan, Effect of AXL on the epithelial-to-
mesenchymal transition in non-small cell lung cancer, Exp. Ther. Med. 14
(2017) 785e790.
}
[19] Y.-X. Zhang, P.G. Knyazev, Y.V. Cheburkin, K. Sharma, Y.P. Knyazev, L. Orfi,
ꢀ
I. Szabadkai, H. Daub, G. Keri, A. Ullrich, AXL is a potential target for thera-
peutic intervention in breast cancer progression, Canc. Res. 68 (2008)
1905e1915.
[20] C. Gjerdrum, C. Tiron, T. Hoiby, I. Stefansson, H. Haugen, T. Sandal, K. Collett,
S. Li, E. McCormack, B.T. Gjertsen, D.R. Micklem, L.A. Akslen, C. Glackin,
J.B. Lorens, Axl is an essential epithelial-to-mesenchymal transition-induced
regulator of breast cancer metastasis and patient survival, Proc. Natl. Acad. Sci.
U.S.A. 107 (2010) 1124e1129.
[21] O. Zajac, R. Leclere, A. Nicolas, D. Meseure, C. Marchio, A. Vincent-Salomon,
S. Roman-Roman, M. Schoumacher, T. Dubois, AXL controls directed migration
of mesenchymal triple-negative breast cancer cells, Cells 9 (2020) 247e263.
[22] S.A. Colavito, AXL as a target in breast cancer therapy, J. Oncol. 2020 (2020)
5291952.
[23] J.D. Paccez, G.J. Vasques, R.G. Correa, J.F. Vasconcellos, K. Duncan, X. Gu,
M. Bhasin, T.A. Libermann, L.F. Zerbini, The receptor tyrosine kinase Axl is an
essential regulator of prostate cancer proliferation and tumor growth and
represents a new therapeutic target, Oncogene 32 (2012) 689e698.
[24] G. Zhang, X. Kong, M. Wang, H. Zhao, S. Han, R. Hu, J. Huang, W. Cui, AXL is a
marker for epithelial-mesenchymal transition in esophageal squamous cell
carcinoma, Oncol. Lett. 15 (2018) 1e7.
[25] E.B. Rankin, K.C. Fuh, T.E. Taylor, A.J. Krieg, M. Musser, J. Yuan, K. Wei, C.J. Kuo,
T.A. Longacre, A.J. Giaccia, AXL is an essential factor and therapeutic target for
metastatic ovarian cancer, Canc. Res. 70 (2010) 7570e7579.
[45] J.K. Rho, Y.J. Choi, S.Y. Kim, T.W. Kim, E.K. Choi, S.J. Yoon, B.M. Park, E. Park,
J.H. Bae, C.M. Choi, J.C. Lee, MET and AXL inhibitor NPS-1034 exerts efficacy
against lung cancer cells resistant to EGFR kinase inhibitors because of MET or
AXL activation, Canc. Res. 74 (2014) 253e262.
[46] W.M. Abdel-Rahman, N.A. Al-Khayyal, V.A. Nair, S.R. Aravind, M. Saber-Ayad,
Role of AXL in invasion and drug resistance of colon and breast cancer cells
and its association with p53 alterations, World J. Gastroenterol. 23 (2017)
3440e3448.
[47] I.K. Park, B. Mundy-Bosse, S.P. Whitman, X. Zhang, S.L. Warner, D.J. Bearss,
W. Blum, G. Marcucci, M.A. Caligiuri, Receptor tyrosine kinase Axl is required
for resistance of leukemic cells to FLT3-targeted therapy in acute myeloid
leukemia, Leukemia 29 (2015) 2382e2389.
[48] M. Scaltriti, M. Elkabets, J. Baselga, Molecular pathways: AXL, a membrane
receptor mediator of resistance to therapy, Clin. Canc. Res. 22 (2016)
1313e1317.
[49] E.B. Rankin, K.C. Fuh, L. Castellini, K. Viswanathan, E.C. Finger, A.N. Diep,
E.L. LaGory, M.S. Kariolis, A. Chan, D. Lindgren, H. Axelson, Y.R. Miao, A.J. Krieg,
A.J. Giaccia, Direct regulation of GAS6/AXL signaling by HIF promotes renal
metastasis through SRC and MET, Proc. Natl. Acad. Sci. U.S.A. 111 (2014)
13373e13378.
[26] T. Sawabu, H. Seno, T. Kawashima, A. Fukuda, Y. Uenoyama, M. Kawada,
N. Kanda, A. Sekikawa, H. Fukui, M. Yanagita, H. Yoshibayashi, S. Satoh,
Y. Sakai, T. Nakano, T. Chiba, Growth arrest-specific gene 6 and Axl signaling
enhances gastric cancer cell survival via Akt pathway, Mol. Carcinog. 46
(2006) 155e164.
[27] B.I. Chung, S.B. Malkowicz, T.B. Nguyen, J.A. Libertino, T.W. McGarvey,
Expression of the proto-oncogene Axl in renal cell carcinoma, DNA Cell Biol.
22 (2003) 533e540.
[50] M.L. Lotsberg, K. Wnuk-Lipinska, S. Terry, T.Z. Tan, N. Lu, L. Trachsel-Moncho,
G.V. Rosland, M.I. Siraji, M. Hellesoy, A. Rayford, K. Jacobsen, H.J. Ditzel,
O.K. Vintermyr, T.G. Bivona, J. Minna, R.A. Brekken, B. Baguley, D. Micklem,
L.A. Akslen, G. Gausdal, A. Simonsen, J.P. Thiery, S. Chouaib, J.B. Lorens,
A.S.T. Engelsen, AXL targeting abrogates autophagic flux and induces immu-
nogenic cell death in drug-resistant cancer cells, J. Thorac. Oncol. 15 (2020)
973e999.
[51] Y.N. Liu, M.F. Tsai, S.G. Wu, T.H. Chang, T.H. Tsai, C.H. Gow, Y.L. Chang, J.Y. Shih,
Acquired resistance to EGFR tyrosine kinase inhibitors is mediated by the
reactivation of STC2/JUN/AXL signaling in lung cancer, Int. J. Canc. 145 (2019)
1609e1624.
[28] A. Gustafsson, D. Martuszewska, M. Johansson, C. Ekman, S. Hafizi,
€
B. Ljungberg, B. Dahlback, Differential expression of Axl and Gas6 in renal cell
carcinoma reflecting tumor advancement and survival, Clin. Canc. Res. 15
(2009) 4742e4749.
[52] G. Zhang, X. Kong, M. Wang, H. Zhao, S. Han, R. Hu, J. Huang, W. Cui, AXL is a
17

D. Xu, D. Sun, W. Wang et al.
European Journal of Medicinal Chemistry 220 (2021) 113497
Chem. 52 (2009) 1251e1254.
[67] X. Cui, X. Zheng, J. Jiang, F. Tan, L. Ding, P. Hu, Simultaneous determination of a
novel c-Met/AXL dual-target small-molecule inhibitor BPI-9016M and its
metabolites in human plasma by liquid chromatography-tandem mass spec-
trometry: application in a pharmacokinetic study in Chinese advanced solid
tumor patients, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1068e1069
(2017) 33e40.
[68] X. Hu, X. Zheng, S. Yang, L. Wang, X. Hao, X. Cui, L. Ding, L. Mao, P. Hu, Y. Shi,
First-in-human phase I study of BPI-9016M, a dual MET/Axl inhibitor, in pa-
tients with non-small cell lung cancer, J. Hematol. Oncol. 13 (2020) 6e15.
[69] S.J. Holland, A. Pan, C. Franci, Y. Hu, B. Chang, W. Li, M. Duan, A. Torneros, J. Yu,
T.J. Heckrodt, J. Zhang, P. Ding, A. Apatira, J. Chua, R. Brandt, P. Pine, D. Goff,
R. Singh, D.G. Payan, Y. Hitoshi, R428, a selective small molecule inhibitor of
Axl kinase, blocks tumor spread and prolongs survival in models of metastatic
breast cancer, Canc. Res. 70 (2010) 1544e1554.
[70] S.M. Woo, K.J. Min, S.U. Seo, S. Kim, P. Kubatka, J.W. Park, T.K. Kwon, Axl in-
hibitor R428 enhances TRAIL-mediated apoptosis through downregulation of
c-FLIP and survivin expression in renal carcinoma, Int. J. Mol. Sci. 20 (2019)
3253e3264.
[71] T. Hara, A. Kimura, T. Miyazaki, H. Tanaka, M. Morimoto, K. Nakai, J. Soeda,
Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced
by growth-arrest-specific 6 and hepatocyte growth factor, Biochem. Biophys.
Rep. 21 (2020) 100726.
[72] S. Salian-Mehta, M. Xu, M.E. Wierman, AXL and MET crosstalk to promote
gonadotropin releasing hormone (GnRH) neuronal cell migration and sur-
vival, Mol. Cell. Endocrinol. 374 (2013) 92e100.
[73] C. Clemenson, C. Chargari, W. Liu, M. Mondini, C. Ferte, M.F. Burbridge,
V. Cattan, A. Jacquet-Bescond, E. Deutsch, The MET/AXL/FGFR inhibitor
S49076 impairs Aurora B activity and improves the antitumor efficacy of
radiotherapy, Mol. Canc. Therapeut. 16 (2017) 2107e2119.
[74] L. Tan, Z. Zhang, D. Gao, J. Luo, Z.C. Tu, Z. Li, L. Peng, X. Ren, K. Ding, 4-Oxo-1,4-
dihydroquinoline-3-carboxamide derivatives as new Axl kinase inhibitors,
J. Med. Chem. 59 (2016) 6807e6825.
marker for epithelial-mesenchymal transition in esophageal squamous cell
carcinoma, Oncol. Lett. 15 (2018) 1900e1906.
[53] J. Liu, K. Wang, Z. Yan, Y. Xia, J. Li, L. Shi, Q. Zou, X. Wan, B. Jiao, H. Wang,
M. Wu, Y. Zhang, F. Shen, Axl expression stratifies patients with poor prog-
nosis after hepatectomy for hepatocellular carcinoma, PloS One 11 (2016)
e0154767/0154761ee0154767/0154713.
[54] L. Lozneanu, P. Pinciroli, D.A. Ciobanu, M.L. Carcangiu, S. Canevari,
A. Tomassetti, I.-D. Caruntu, Computational and immunohistochemical ana-
lyses highlight AXL as a potential prognostic marker for ovarian cancer pa-
tients, Anticancer Res. 36 (2016) 4155e4164.
[55] M. Seike, C.H. Kim, F. Zou, R. Noro, M. Chiba, A. Ishikawa, S. Kappaunugi,
K. Kubota, A. Gemma, AXL and GAS6 co-expression in lung adenocarcinoma as
a prognostic classifier, Oncol. Rep. 37 (2017) 3261e3269.
[56] X. Wu, W. Ma, Q. Zhou, H. Yan, Z.F. Lim, M. Huang, C. Deng, X. Yu, H. Su,
S. Komo, H. Yang, X. Zhang, S. Wen, Z. Zhang, P.C. Ma, AXL-GAS6 expression
can predict for adverse prognosis in non-small cell lung cancer with brain
metastases, J. Canc. Res. Clin. Oncol. 143 (2017) 1947e1957.
[57] S.H. Myers, V.G. Brunton, A. Unciti-Broceta, AXL inhibitors in cancer: a me-
dicinal chemistry perspective, J. Med. Chem. 59 (2016) 3593e3608.
[58] Y. Shen, X. Chen, J. He, D. Liao, X. Zu, Axl inhibitors as novel cancer therapeutic
agents, Life Sci. 198 (2018) 99e111.
[59] M. Mori, N. Kaneko, Y. Ueno, M. Yamada, R. Tanaka, R. Saito, I. Shimada,
K. Mori, S. Kuromitsu, Gilteritinib, a FLT3/AXL inhibitor, shows antileukemic
activity in mouse models of FLT3 mutated acute myeloid leukemia, Invest. N.
Drugs 35 (2017) 556e565.
[60] C.M. Gorcea, J. Burthem, E. Tholouli, ASP2215 in the treatment of relapsed/
refractory acute myeloid leukemia with FLT3 mutation: background and
design of the ADMIRAL trial, Future Oncol. 14 (2018) 1995e2004.
[61] H.Y. Zou, Q. Li, J.H. Lee, M.E. Arango, S.R. McDonnell, S. Yamazaki,
T.B. Koudriakova, G. Alton, J.J. Cui, P.P. Kung, M.D. Nambu, G. Los, S.L. Bender,
B. Mroczkowski, J.G. Christensen, An orally available small-molecule inhibitor
of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through
antiproliferative and antiangiogenic mechanisms, Canc. Res. 67 (2007)
4408e4417.
[75] C. Feneyrolles, A. Spenlinhauer, L. Guiet, B. Fauvel, B. Dayde-Cazals,
P. Warnault, G. Cheve, A. Yasri, Axl kinase as a key target for oncology: focus
on small molecule inhibitors, Mol. Canc. Therapeut. 13 (2014) 2141e2148.
[76] M. Sattler, Y.B. Pride, P. Ma, J.L. Gramlich, S.C. Chu, L.A. Quinnan, S. Shirazian,
C. Liang, K. Podar, J.G. Christensen, R. Salgia, A novel small molecule met in-
hibitor induces apoptosis in cells transformed by the oncogenic TPR-MET
tyrosine kinase, Canc. Res. 63 (2003) 5462e5469.
ꢀ
[62] J.J. Cui, M. Tran-Dube, H. Shen, M. Nambu, P.-P. Kung, M. Pairish, L. Jia, J. Meng,
L. Funk, I. Botrous, M. McTigue, N. Grodsky, K. Ryan, E. Padrique, G. Alton,
S. Timofeevski, S. Yamazaki, Q. Li, H. Zou, J. Christensen, B. Mroczkowski,
S. Bender, R.S. Kania, M.P. Edwards, Structure based drug design of Crizotinib
(PF-02341066),
a
potent
and
selective
dual
inhibitor
of
mesenchymaleepithelial transition factor (c-MET) kinase and anaplastic
lymphoma kinase (ALK), J. Med. Chem. 54 (2011) 6342e6363.
ꢀ
[77] M. Warmuth, S. Kim, X.J. Gu, G. Xia, F. Adrian, Ba/F3 cells and their use in
[63] S.K. Chia, S.L. Ellard, M. Mates, S. Welch, C. Mihalcioiu, W.H. Miller Jr.,
K. Gelmon, C. Lohrisch, V. Kumar, S. Taylor, L. Hagerman, R. Goodwin, T. Wang,
S. Sakashita, M.S. Tsao, E. Eisenhauer, P. Bradbury, A phase-I study of lapatinib
in combination with foretinib, a c-MET, AXL and vascular endothelial growth
factor receptor inhibitor, in human epidermal growth factor receptor 2 (HER-
2)-positive metastatic breast cancer, Breast Cancer Res. 19 (2017) 54e61.
[64] J.P. Eder, G.I. Shapiro, L.J. Appleman, A.X. Zhu, D. Miles, H. Keer, B. Cancilla,
F. Chu, S. Hitchcock-Bryan, L. Sherman, S. McCallum, E.I. Heath, S.A. Boerner,
P.M. LoRusso, A phase I study of foretinib, a multi-targeted inhibitor of c-Met
and vascular endothelial growth factor receptor 2, Clin. Canc. Res. 16 (2010)
3507e3516.
[65] S.B. Yan, V.L. Peek, R. Ajamie, S.G. Buchanan, J.R. Graff, S.A. Heidler, Y.H. Hui,
K.L. Huss, B.W. Konicek, J.R. Manro, C. Shih, J.A. Stewart, T.R. Stewart, S.L. Stout,
M.T. Uhlik, S.L. Um, Y. Wang, W. Wu, L. Yan, W.J. Yang, B. Zhong, R.A. Walgren,
LY2801653 is an orally bioavailable multi-kinase inhibitor with potent activity
against MET, MST1R, and other oncoproteins, and displays anti-tumor activ-
ities in mouse xenograft models, Invest. N. Drugs 31 (2013) 833e844.
[66] G.M. Schroeder, Y. An, Z.W. Cai, X.T. Chen, C. Clark, L.A. Cornelius, J. Dai,
J. Gullo-Brown, A. Gupta, B. Henley, J.T. Hunt, R. Jeyaseelan, A. Kamath, K. Kim,
J. Lippy, L.J. Lombardo, V. Manne, S. Oppenheimer, J.S. Sack, R.J. Schmidt,
G. Shen, K. Stefanski, J.S. Tokarski, G.L. Trainor, B.S. Wautlet, D. Wei,
D.K. Williams, Y. Zhang, Y. Zhang, J. Fargnoli, R.M. Borzilleri, Discovery of N-(4-
(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-
kinase drug discovery, Curr. Opin. Oncol. 19 (2007) 55e60.
[78] A. Drilon, S.I. Ou, B.C. Cho, D.W. Kim, J. Lee, J.J. Lin, V.W. Zhu, M.J. Ahn,
D.R. Camidge, J. Nguyen, D. Zhai, W. Deng, Z. Huang, E. Rogers, J. Liu,
J. Whitten, J.K. Lim, S. Stopatschinskaja, D.M. Hyman, R.C. Doebele, J.J. Cui,
A.T. Shaw, Repotrectinib (TPX-0005) is a next-generation ROS1/TRK/ALK in-
hibitor that potently inhibits ROS1/TRK/ALK solvent-front mutations, Canc.
Discov. 8 (2018) 1227e1236.
[79] Y.W. Zhang, B. Staal, C. Essenburg, Y. Su, L. Kang, R. West, D. Kaufman,
T. Dekoning, B. Eagleson, S.G. Buchanan, G.F. Vande Woude, MET kinase in-
hibitor SGX523 synergizes with epidermal growth factor receptor inhibitor
erlotinib in a hepatocyte growth factor-dependent fashion to suppress car-
cinoma growth, Canc. Res. 70 (2010) 6880e6890.
[80] T. Bachleitner-Hofmann, M.Y. Sun, C.T. Chen, D. Liska, Z. Zeng, A. Viale,
A.B. Olshen, M. Mittlboeck, J.G. Christensen, N. Rosen, D.B. Solit, M.R. Weiser,
Antitumor activity of SNX-2112, a synthetic heat shock protein-90 inhibitor,
in MET-amplified tumor cells with or without resistance to selective MET
Inhibition, Clin. Canc. Res. 17 (2011) 122e133.
[81] Y. Funakoshi, T. Mukohara, R.C. Ekyalongo, H. Tomioka, Y. Kataoka,
Y. Shimono, N. Chayahara, M. Toyoda, N. Kiyota, Y. Fujiwara, H. Minami,
Regulation of MET kinase inhibitor resistance by copy number of MET in
gastric carcinoma cells, Oncol. Res. 21 (2013) 287e293.
[82] Z. Zhan, X. Peng, Q. Liu, F. Chen, Y. Ji, S. Yao, Y. Xi, Y. Lin, T. Chen, Y. Xu, J. Ai,
M. Geng, W. Duan, Discovery of 6-(difluoro(6-(4-fluorophenyl)-[1,2,4]triazolo
[4,3-b][1,2,4]triazin-3-yl)methyl)q uinoline as a highly potent and selective c-
Met inhibitor, Eur. J. Med. Chem. 116 (2016) 239e251.
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607),
a
selective and orally efficacious inhibitor of the Met kinase superfamily, J. Med.
18