Discovery of novel 2,4‐dianilinopyrimidine derivatives containing 4‐(Morpholinomethyl)phenyl and n‐substituted benzamides as potential fak inhibitors and anticancer agents

Article abstract of DOI:10.3390/molecules26144187

Focal adhesion kinase (FAK) is responsible for the development and progression of various malignancies. With the aim to explore novel FAK inhibitors as anticancer agents, a series of 2,4‐ dianilinopyrimidine derivatives 8a–8i and 9a–9g containing 4‐(morpholinomethyl)phenyl and N‐ substituted benzamides have been designed and synthesized. Among them, compound 8a dis-played potent anti‐FAK activity (IC50 = 0.047 ± 0.006 μM) and selective antiproliferative effects against H1975 (IC50 = 0.044 ± 0.011 μM) and A431 cells (IC50 = 0.119 ± 0.036 μM). Furthermore, compound 8a also induced apoptosis in a dose‐dependent manner, arresting the cells in S/G2 phase and inhibiting the migration of H1975 cells, all of which were superior to those of TAE226. The docking analysis of compound 8a was performed to elucidate its possible binding modes with FAK. These results established 8a as our lead compound to be further investigated as a potential FAK inhibitor and anticancer agent.

Full text of DOI:10.3390/molecules26144187

molecules
Article
Discovery of Novel 2,4-Dianilinopyrimidine Derivatives
Containing 4-(Morpholinomethyl)phenyl and N-Substituted
Benzamides as Potential FAK Inhibitors and Anticancer Agents
Chun Han 1,† , Kemin Shen 2,†, Shijun Wang 1 , Zhijun Wang 1, Feng Su 1, Xi Wu 1, Xiaoqin Hu 1, Mengyao Li 1,
Jing Han 3,* and Lintao Wu 1,
*
1
Department of Chemistry, Changzhi University, Changzhi 046011, China; coldspringfibre@126.com (C.H.);
444795415@nwafu.edu.cn (S.W.); czxywzj@czc.edu.cn (Z.W.); sufeng@czc.edu.cn (F.S.);
wuxi040252768@163.com (X.W.); xiaoqin_hu2021@126.com (X.H.); lmysummer@126.com (M.L.)
Department of Public Health and Preventive Medicine, Changzhi Medical College, Changzhi 046011, China;
shenkm@czmc.edu.cn
School of Chemistry and Materials Science, Jiangsu Normal University, Xuzhou 221116, China
Correspondence: hj1986424@jsnu.edu.cn (J.H.); ltwu@live.cn (L.W.); Tel.: +86-516-8340-3166 (J.H.);
+86-355-217-8113 (L.W.)
2
3
*
†
C.H. and K.S. contributed equally to this work.
Abstract: Focal adhesion kinase (FAK) is responsible for the development and progression of various
malignancies. With the aim to explore novel FAK inhibitors as anticancer agents, a series of 2,4-
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
dianilinopyrimidine derivatives 8a
substituted benzamides have been designed and synthesized. Among them, compound 8a displayed
potent anti-FAK activity (IC50 = 0.047 0.006 M) and selective antiproliferative effects against H1975
(IC50 = 0.044 0.011 M) and A431 cells (IC50 = 0.119 0.036 M). Furthermore, compound 8a also
induced apoptosis in a dose-dependent manner, arresting the cells in S/G2 phase and inhibiting
the migration of H1975 cells, all of which were superior to those of TAE226. The docking analysis
of compound 8a was performed to elucidate its possible binding modes with FAK. These results
established 8a as our lead compound to be further investigated as a potential FAK inhibitor and
anticancer agent.
–8i and 9a–9g containing 4-(morpholinomethyl)phenyl and N-
Citation: Han, C.; Shen, K.; Wang, S.;
Wang, Z.; Su, F.; Wu, X.; Hu, X.; Li, M.;
Han, J.; Wu, L. Discovery of Novel
2,4-Dianilinopyrimidine Derivatives
Containing
±
µ
±
µ
±
µ
4-(Morpholinomethyl)phenyl and
N-Substituted Benzamides as
Potential FAK Inhibitors and
Anticancer Agents. Molecules 2021, 26,
4187. https://doi.org/10.3390/
molecules26144187
Keywords: FAK inhibitor; 2,4-dianilinopyrimidine; 4-(morpholinomethyl)phenyl; N-substituted
benzamides; anticancer; molecular docking
Academic Editor: Gian Cesare Tron
Received: 11 June 2021
Accepted: 6 July 2021
Published: 9 July 2021
1. Introduction
Cancer is one of the most dreaded diseases impacting human health, and is one of
the leading causes of death throughout the world today. In 2020, an estimated 19.3 million
new cancer cases and almost 10.0 million cancer deaths were reported worldwide [1]. For
cancer patients, traditional cytotoxic chemotherapy has been the most common treatment
method, but it suffers from a lack of selectivity for tumor cells and frequently causes drug
resistance during the course of treatment [2,3]. However, in recent years, there has been a
significant emphasis on the development of targeted cancer therapies that are designed to
enhance efficacy and selectivity for the treatment of various cancers by targeting specific
cancer biomarkers [3–5].
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Focal adhesion kinase (FAK), also known as protein tyrosine kinase 2 (PTK2), is
a 125 kDa non-receptor tyrosine kinase protein that is involved in a variety of cellular
processes, such as cross-linked signaling events mediated by integrins, stimulating the
dissociation of G protein-coupled receptor subunits, and regulating the production of
growth factors and neurotransmitters, all of which affect cell adhesion, motility, migration,
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conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
apoptosis, and cell–matrix interactions [6]. FAK plays a crucial role in the development and
progression of cancer, and its upregulation or overexpression has been frequently observed
Molecules 2021, 26, 4187. https://doi.org/10.3390/molecules26144187
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4187
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in a variety of malignancies, including colorectal, breast [7], thyroid [8], prostate [9],
brain [10], ovarian [11], liver [12], stomach [13], and lung cancers [14]. In addition, FAK
has been implicated in the development of chemoresistance. For example, ovarian cancer
cells have been shown to confer resistance to platinum-based chemotherapies by increasing
FAK activity needed to sustain cellular vitality and growth [15]. In addition, targeting FAK
in endothelial cells was found to sensitize tumor cells to DNA-damaging therapies, which
implicated FAK in the regulation of tumor chemosensitivity [16]. Other recent studies
have revealed that FAK mediated immune responses to certain cancers. Inhibition of
FAK also induced T cell-mediated tumor regression in mice, corroborating the potential
to modulate FAK activity as a means to control antitumor immunity [17,18]. However,
low-dose FAK inhibition was reported to increase tumor growth and angiogenesis, which
was contrary to higher FAK inhibition, indicating that FAK can have a nonlinear dose-
dependent function in regulating tumor growth and angiogenesis [19]. Nevertheless, FAK
represents a promising biological target for the development of effective new anticancer
therapies.
The FAK protein comprises a multidomain structure featuring an N-terminal FERM
domain, a kinase domain, and a C-terminal domain. Targeting the ATP binding pocket
in the kinase domain using small molecules has been the most common approach toward
FAK inhibition. Several ATP-competitive FAK inhibitors (Figure 1) have been successfully
developed, with some of them, including TAE226, GSK-2256098, VS-6063 (PF-04554878,
Defactinib), PF-00562271 (VS-6062), VS-4718 (PND-1186), CEP-37440, and BI-853520 (IN-
10018), progressing through preclinical development and even clinical trials [20]. These
compounds displayed potent inhibition against FAK and demonstrated antitumor effects
both in vitro and in vivo in many different malignancy models. To date, GSK-2256098 [21],
PF-00562271 [22], CEP-37440 [23], and BI-853520 [24] have successfully completed their
Phase I clinical trials, and VS-6063 [25] recently completed its Phase II clinical trial. How-
ever, the clinical trial of VS-4718 [26] was terminated or withdrawn, and TAE226 [27] was
abandoned due to the discovery of several off-target effects, though TAE226 is extensively
employed as a positive control to compare to other newly developed FAK inhibitors. Due
to the lack of marketed FAK inhibitors, there is an urgent need to develop novel FAK
inhibitors with effective antitumor activity. The majority of novel FAK inhibitors cur-
rently in development comprise a 2,4-diaminopyrimidine core, which was found to be
an important structural moiety for promoting the binding of the inhibitors in the hinge
region of the ATP binding site of FAK, as the compounds are stabilized through hydrogen
bonds between active site residues and the pyrimidine nitrogen and aniline NH of the
2,4-diaminopyrimidine core [28,29]. Therefore, the 2,4-diaminopyrimidine is an attractive
scaffold from which small-molecule inhibitors against FAK can be designed to improve the
inhibition activity against FAK and other physicochemical properties compared to current
inhibitors in development.

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Figure 1. Structures of ATP-competitive FAK inhibitors in preclinical and clinical trials.
With the aim of developing novel FAK-targeted inhibitors, a series of novel 2,4-
dianilinopyrimidine derivatives containing 4-(morpholinomethyl)phenyl and N-substituted
benzamides were designed and synthesized. In our design (Figure 2), we introduced a
4-(morpholinomethyl)phenyl moiety into the N2 position of the 2,4-diaminopyrimidine
scaffold to provide flexibility and conformational freedom in order to better enable interac-
tions with active site residues. In the N4 position of the scaffold, various alkanolamines
were coupled with benzoic acid to form N-substituted benzamides to increase hydrogen
bonding interactions with the target. Using this design approach, we synthesized and
biologically evaluated the target 2,4-dianilinopyrimidines 8a–8i. Moreover, nitric oxide
(NO) plays an important role in cancer biology, and the use of NO donor-based therapies
to fight cancer has generated significant progress [30]. Therefore, (phenylsulfonyl)furoxan
was introduced into the scaffold as a potential NO donor, affording target compounds
9a–9g. Their inhibitory activity against FAK, antiproliferative activity against tumor cells,
cellular selectivity, apoptosis-inducing activities, and effects on cell cycle distribution and
cell migration were thoroughly evaluated. In addition, molecular docking studies were
also performed to elucidate the binding interactions between the inhibitor and FAK.

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Figure 2. Design strategy of the target compounds.
2. Results and Discussion
2.1. Synthetic Pathways
The synthesis of compounds 8a
tuted aniline
and subsequent reduction of the nitro group. Meanwhile, anilinopyrimidine
pared by nucleophilic aromatic substitution of the 4-Cl of 2,4,5-trichloropyrimidine with
the nucleophilic aniline . Then, aniline underwent a regioselective nucleophilic aromatic
substitution with the 2-Cl of to obtain the 2,4-dianilinopyrimidines . After saponifi-
cation, intermediate was converted to the corresponding acid , which was condensed
with various alkanolamines to yield the target compounds 8a 8i. Compounds 8a 8g were
–
8i and 9a
–
9g is illustrated in Scheme 1. The substi-
3
was prepared from 4-nitrobenzyl bromide (
1
) by amination with morpholine
was pre-
5
4
3
5
6
6
7
–
–
then reacted with 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10) to afford the target
compounds 9a–9g.

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Scheme 1. Synthes◦is of 2,4-dianilinopyrimidines derivatives 8a
–
8i and 9a
–
9g. Reagents and conditions: (
a
) morpholine,
K2CO3, DMSO, 90 C, 3 h, 94%. (
b
) 10% Pd/C, EtOH, H2 atm, rt, 8 h, 89%. (
c) 2,4,5-trichloropyrimidine, DIPEA, 2-BuOH,
80 ◦C, 72 h, 76%. (
d
) TFA, 2-BuOH, 90 ◦C, 48 h, 25%. (
e) NaOH, 1,4-dioxane/H2O, rt, 5 h, 95%. (
f
) HROH, EDCI, HOBt,
◦
DMF, rt, 24 h. (g) 3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10), NaH, THF, 0 C, 24 h.
2.2. Kinase Inhibitory Activity and Structure–Activity Relationships
The inhibitory activity of the target compounds 8a–8i and 9a–9g against FAK ki-
nase was evaluated using the ADP-GloTM assay, with TAE226 employed as the positive
control. As provided in Table 1, most of these novel derivatives suppressed FAK ac-
tivity at low-nanomolar concentrations. Compounds 8a
exhibited higher potencies compared to the corresponding furoxan-containing derivatives
9a 9g. In particular, 8a (IC50 = 0.047 0.006 M), 8c (IC50 = 0.030 0.007 M), and 8d
(IC50 = 0.040 ± 0.011 µM) displayed the highest inhibitory activities against FAK compared
to the other compounds tested. In addition, compounds 8c 8d
9c (IC50 = 0.089 ± 0.014 µM),
and 9d
IC50 = 0.066 ± 0.014 µM), which featured methyl substituents at R, had higher in-
hibitory constants against FAK compared to 8i (IC50 = 0.126 0.021 M), which featured
a bulkier group for R. However, the presence of an additional hydroxyl group of R in 8h
(IC50 = 0.072 0.007 M) did not furnish an increase in the inhibition against FAK relative
to 8b 8g featuring R with cyclic or longer
–8g featuring hydroxyl groups
–
±
µ
±
µ
,
,
(
±
µ
±
µ
(
IC50 = 0.061 ± 0.009 µM). Lastly, analogues 8e
–
alkanolamines exhibited lower inhibitory constants against FAK compared to compounds
8a, 8c, and 8d.

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Table 1. Biological activities of compounds 8a–8i and 9a–9g.
Antiproliferative Activity IC50 (µM) a
FAK, IC50 (µM) a
SI Values b
Compound
R
H1975
A431
HBE
8a
8b
8c
8d
8e
8f
0.047 ± 0.006
0.061 ± 0.009
0.030 ± 0.007
0.040 ± 0.011
0.129 ± 0.037
0.173 ± 0.023
0.198 ± 0.032
0.044 ± 0.011
0.075 ± 0.011
0.119 ± 0.047
0.119 ± 0.036
0.207 ± 0.036
0.158 ± 0.027
1.039 ± 0.266
1.153 ± 0.283
4.007 ± 0.231
24
15
34
13
0.3
0.4
6
0.293 ± 0.050
1.414 ± 0.132
2.221 ± 0.261
0.287 ± 0.061
0.161 ± 0.082
0.238 ± 0.098
0.195 ± 0.097
0.507 ± 0.019
3.825 ± 0.500
0.435 ± 0.045
0.862 ± 0.125
1.645 ± 0.813
8g
8h
8i
0.072 ± 0.007
0.126 ± 0.021
0.219 ± 0.082
0.815 ± 0.061
0.047 ± 0.006
1.245 ± 0.909
0.688 ± 0.121
3
>20
>24
9a
9b
9c
0.149 ± 0.033
0.338 ± 0.038
0.089 ± 0.014
0.047 ± 0.009
0.164 ± 0.051
0.567 ± 0.086
0.182 ± 0.032
0.138 ± 0.031
1.074 ± 0.154
0.916 ± 0.092
2.078 ± 0.366
2.418 ± 0.546
19
13
4
9d
0.066 ± 0.014
1.094 ± 0.137
2.738 ± 0.206
4.552 ± 0.533
4
9e
9f
>1
0.300 ± 0.009
>1
1.724 ± 0.295
1.570 ± 0.265
0.592 ± 0.073
2.414 ± 0.587
1.739 ± 0.305
0.617 ± 0.040
0.763 ± 0.100
3.761 ± 1.604
3.009 ± 0.090
0.4
2
9g
5
TAE226
Osimertinib
0.0034 ± 0.0005
0.141 ± 0.041
0.392 ± 0.053
>20
1.208 ± 0.177
>142
10
ND
0.116 ± 0.032
0.988 ± 0.119
a
b
IC50 data were means ± SD of three independent experiments. Selectivity index (SI) value = IC50 for HBE cell line/IC50 for H1975 cell
line. ND, not determined.
2.3. Antiproliferative Activity against Tumor Cells
We next determined the antiproliferative effects of the 2,4-dianilinopyrimidines deriva-
tives in H1975 non-small cell lung cancer (NSCLC) cells and A431 human epithelial carci-
noma cells using a CCK-8 assay, with TAE226 and osimertinib (AZD9291, a potent EGFR
inhibitor against the H1975 cell line [31]) employed as controls. As shown in Table 1, most
of the target compounds were potent in the inhibition of H1975 and A431 cells. Consistent
with the trends of their kinase inhibitory activities, compounds 8a
inhibitory effects than 9a 9g against the two tumor cell lines. In general, most compounds
exhibited stronger inhibitory activity against H1975 cells than A431 cells. In addition, 8a
(IC50 = 0.044 0.011 M), 8b (IC50 = 0.075 0.011 M), and 9a (IC50 = 0.047 0.009 M)
demonstrated higher antiproliferative activities against H1975 cells, while nine compounds
8a 8f 8h 9a, and 9b) were found to more potently inhibit the proliferation of A431 cells,
with IC50 values of 0.047–0.238 M, than the positive controls. Combining these results,
8b, and 9a potently inhibit the proliferation of both H1975 and A431 cell
–8g displayed better
–
±
µ
±
µ
±
µ
(
–
,
,
µ
compounds 8a
,
lines.

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2.4. Cellular Selectivity Assay
To determine the selectivity of the target compounds against tumor cells, normal
human bronchial epithelial cells (HBE) were exposed to each of the compounds, and the
viability of the cells was assessed using the CCK-8 assay. From these results, the selectivity
index (SI) of the compounds between HBE and H1975 cells was also calculated. As shown
in Table 1, most of these compounds more weakly inhibited the growth of HBE cells
compared to H1975 cells. The SI values of the three most active compounds previously
identified (8a, 8b, and 9a) were 24, 15, and 19, respectively. Since compound 8a (Figure 3)
displayed the best combination between potency and SI of the rest of the compounds
evaluated, it was carried forward for further studies.
Figure 3. Structure of compound 8a.
2.5. Cell Apoptosis Assay
To better elucidate the mechanism by which these compounds inhibit cellular pro-
liferation, AO/EB staining assays and flow cytometry were performed in H1975 cells
to assess the effects of compound 8a on inducing cellular apoptosis, using TAE226 as a
positive control. As illustrated in Figure 4, H1975 cells that were treated with increasing
concentrations (0.1, 0.5, and 1 µM) of 8a exhibited certain morphological phenomena char-
acteristic of apoptosis, including noticeable nuclear condensation, membrane blebbing,
nuclear fragmentation, and the formation of apoptotic bodies. In addition, the results
of flow cytometric analysis with Annexin V-FITC/PI double staining are summarized in
Figures 5 and 6. It was found that 8a substantially increased the extent of apoptosis in
H1975 cells in a dose-dependent manner. Notably, the ability of 8a to induce apoptosis was
significantly stronger compared to the control TAE226.
Figure 4. Apoptotic morphology of H1975 cells was assessed by AO/EB double fluorescent staining (100
tion).
×
, final magnifica-

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Figure 5. Flow cytometry analysis of apoptosis induction in H1975 cells treated with compound 8a and TAE-226 (0.1, 0.5,
M) for 48 h, and untreated cells (control) as a control. The cells were stained with Annexin V-FITC and PI, followed by
flow cytometry analysis.
1
µ
Figure 6. The quantitative data of the apoptosis of compound 8a and TAE226 are expressed as
the mean SD of the percentages of apoptotic cells from three independent experiments. Key:
±
(***) p < 0.001, (****) p < 0.0001 vs. TAE226 group.
2.6. Cell Cycle Analysis
The effects of compound 8a on cell cycle distribution in H1975 cells were analyzed by
flow cytometry, and TAE226 was employed as a reference drug (Figures 7 and 8). When the
H1975 cells were exposed to compound 8a at concentrations of 0.1, 0.5, and 1 µM for 48 h,
the percentages of cells in G0/G1 phase decreased from 80.74% to 39.06%, while those in S
phase and G2/M phase increased from 5.96% to 23.65% and 13.3% to 37.29% respectively,
compared to the control group. Furthermore, treatment of the H1975 cells with compound
8a induced cell cycle arrest in S/G2 phase in a dose-dependent manner, which was more
potent than TAE226.

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Figure 7. Cell cycle analysis of H1975 cells treated with 8a and TAE226 (0.1, 0.5, 1 µM) for 48 h by flow cytometry assay.
Figure 8. The cell cycle percentage of H1975 cells treated with 8a and TAE226.
2.7. Cell Migration Assay
We further assessed whether compound 8a had any influence on inhibiting the mi-
gration of H1975 cells using a scratch assay (Figure 9). H1975 cells were incubated with
different concentrations of compound 8a and TAE226 (0.1, 0.5, and 1 µM) for 24 h. As
expected, H1975 cells in control groups displayed the farthest cell migration, whereas
incubation of the cells with either compound 8a or TAE226 manifested reduced migration
distances of the H1975 cells. Furthermore, compound 8a inhibited the migration of the
H1975 cells slightly more potently than TAE226.

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Figure 9. Effects of compound 8a and TAE226 on the migration of H1975 cells in vitro determined by the scratch assay.
H1975 cells were incubated with different concentrations of compound 8a and TAE226 (0.1, 0.5, and 1 µM) for 24 h.
2.8. Molecular Docking Study
Molecular docking studies were conducted using the CDOCKER module in the Dis-
covery Studio 2017 R2 software to predict the binding affinity of compound 8a in FAK
(PDB: 2JKK [29]), and the docking results are shown in Figure 10. Compound 8a was
bound in the active pocket of FAK, and its binding pose was nearly overlapped with that of
the ligand TAE226 that was co-crystallized with FAK. Two hydrogen bonds were formed
between compound 8a and FAK, one between the N-1 nitrogen of the pyrimidine ring of
8a and the NH of the amino acid residue Cys502, and the other between the oxygen atom
of the amide group and the NH of Asp564, which was also observed with TAE226. As
expected, hydrogen bonds were also formed between the morpholine oxygen of compound
8a and the OH of Ser509, as well as the terminal hydroxyl group and the oxygen atom of
the carbonyl group of Asn551. Moreover, hydrophobic interactions between compound 8a
and the surrounding residues in the binding pocket were observed. These docking results
provided vital structural information for further rational design of FAK inhibitors featuring
this scaffold as well as derivative scaffolds.

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Figure 10. The predicted docked pose of compound 8a in the FAK active site (PDB: 2JKK). (a) Image showing the overlap of
compound 8a (blue sticks) and TAE226 (yellow sticks). Hydrogen bonds are shown in green dots. (
b) Interactions between
compound 8a and the key residues in the bonding site, where different interactions are shown in different colors. Hydrogen
bonds: green; hydrophobic interactions: pink.
3. Materials and Methods
3.1. Chemistry
Starting materials, reagents, and solvents were commercially available. Melting points
of individual compounds were measured without correction on a SGWX-4 Micro Melting
1
Point apparatus (Shanghai Precision & Scientific Instrument Co. Ltd., Shanghai, China). H
and 13C Nuclear Magnetic Resonance (NMR) spectral data were recorded in DMSO-d6 on a
0
Bruker 400 54 Ascend spectrometer (Bruker BioSpin AG, Faellanden, Switzerland) at 303 K
with tetramethylsilane (TMS) as an internal standard. High-Resolution Mass Spectrometry
(HRMS) determinations were obtained on a Bruker compact
Daltonik GmbH, Bremen, Germany). Compound was prepared from 4-nitrobenzyl
bromide ( ) by amination and subsequent reduction, as described previously [32]. 3,4-
™ QTOF system (Bruker
3
1
Bis(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10) was prepared via a three-step reaction
sequence using thiophenol as the starting material according to a previously described
methodology [33].
3.1.1. Methyl 2-((2,5-Dichloropyrimidin-4-yl)amino)benzoate (5)
2,4,5-Trichloropyrimidine (14.560 g, 0.0794 mol), methyl anthranilate (4, 10 g, 0.0662 mol),
and N,N-diisopropylethylamine (DIPEA) (1◦0.262 g, 0.0794 mol) were added to 100 mL
of 2-BuOH. The reaction was heated at 80 C for 3 days. After being cooled to room
temperature, the precipitate was filtered out and washed with ethanol and water to obtain
◦
the desired product as a white solid (15 g, 76% yield). mp 230–232 C. 1H NMR (400 MHz,
DMSO-d6)
δ 11.12 (s, 1H), 8.52 (s, 1H), 8.49 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.1 Hz, 1H), 7.72
(t, J = 8.4 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 3.89 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ
168.21, 157.11, 156.94, 156.19, 139.79, 134.66, 131.36, 124.22, 122.09, 118.34, 115.39, 53.15.
ESI-HRMS (m/z): [M + H]+ calcd for C12H10Cl2N3O2 298.0150, obsd 298.0183.
3.1.2. Methyl 2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)
amino)benzoate (6)
A solution composed of intermediate
5 (7.753 g, 0.026 mol), 3 (5 g, 0.026 mol), and
TFA (4.450 g, 0.039 mol) in 70 mL of 2-BuOH was stirred at 90 ◦C for 48 h. After being

Molecules 2021, 26, 4187
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concentrated in vacuo, the residue was dissolved in a small amount of methanol and added
dropwise into a saturated NaHCO3 aqueous solution. A yellow solid was precipitated
and then dried after filtration. The product was purified by chromatography on silica gel,
eluting with 120:1 (v/v) dichloromethane-methanol (3.64 g, 31% yield). mp 223–225 ◦C. 1H
NMR (400 MHz, DMSO-d6)
δ 10.97 (s, 1H), 9.50 (s, 1H), 8.91 (d, J = 8.5 Hz, 1H), 8.27 (s, 1H),
8.03 (d, J = 8.0 Hz, 1H), 7.63–7.57 (m, 3H), 7.30–7.09 (m, 3H), 3.91 (s, 3H), 3.64–3.51 (m, 4H),
3.41 (s, 2H), 2.35 (brs, 4H). 13C NMR (101 MHz, DMSO-d6) δ 168.47, 158.21, 155.40, 141.55,
139.37, 134.48, 131.67, 131.30, 129.54, 122.53, 121.59, 120.26, 115.99, 105.57, 66.66, 62.56, 53.56,
53.06. ESI-HRMS (m/z): [M + H]+ calcd for C23H25ClN5O3 454.1640, obsd 454.1615.
3.1.3. 2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)
benzoic acid (7)
NaOH (1.028 g, 25.7 mmol) was dissolved in 10 mL of water, which was added to
a solution of compound
mixture was stirred at room temperature for 5 h and acidified to subacidity (pH
6
(2.913 g, 6.43 mmol) in 10 mL of 1,4-dioxane. The resulting
6) with
≈
diluted hydrochloric acid. The precipitate was collected as a yellow sticky solid and then
stirred in ethyl acetate to disp◦erse. After filtration, a powdery yellow solid was obtained
δ 12.13 (s, 1H), 9.53 (s,
(2.7 g, 96% yield). mp > 300 C. 1H NMR (400 MHz, DMSO-d6)
1H), 8.91 (d, J = 7.3 Hz, 1H), 8.19 (s, 1H), 8.05 (d, J = 7.5 Hz, 1H), 7.65 (d, J = 8.0 Hz, 2H),
7.49 (t, J = 7.2 Hz, 1H), 7.27 (d, J = 8.1 Hz, 2H), 7.09 (t, J = 7.3 Hz, 1H), 3.69 (t, J = 4.4 Hz,
6H), 2.64 (br s, 4H). 13C NMR (101 MHz, DMSO-d6)
δ 170.60, 158.15, 155.52, 154.98, 141.72,
140.13, 133.29, 131.70, 130.20, 129.26, 122.06, 120.96, 120.03, 118.66, 105.92, 65.96, 61.80, 52.93.
ESI-HRMS (m/z): [M + H]+ calcd for C22H23ClN5O3 440.1484, obsd 440.1468.
3.1.4. General Procedure for the Preparation of 8a–8i
A flask was charged with compound 7 (200 mg, 0.455 mmol), EDCI (174 mg, 0.909 mmol),
HOBt (80 mg, 0.591 mmol), and DMF (2 mL). After stirring for 0.5 h, alkanolamine
(2.27 mmol) was added. The mixture continued to react overnight, and then was diluted
with water (10 mL) and extracted thrice with ethyl acetate. The organic layers were com-
bined, concentrated, and purified using flash chromatography (dichloromethane-methanol
20:1 v/v).
2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)-N-(2-
hydroxyethyl)benzamide (8a)
The title compound was obtained starting from compound
7
and ethanolamine. As
11.55 (s, 1H),
a yellow solid, 18% yield. mp 230–231 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ
9.47 (s, 1H), 8.82 (s, 1H), 8.75 (d, J = 6.7 Hz, 1H), 8.22 (s, 1H), 7.84 (d, J = 7.5 Hz, 1H), 7.62
(d, J = 7.7 Hz, 2H), 7.48 (t, J = 7.4 Hz, 1H), 7.20 (d, J = 8.0 Hz, 2H), 7.15 (t, J = 7.3 Hz, 1H),
4.83 (s, 1H), 3.57 (br s, 6H), 3.40 (s, 2H), 3.37 (t, J = 5.3 Hz, 2H), 2.34 (br s, 4H). 13C NMR
(101 MHz, DMSO-d6) δ 169.10, 158.23, 155.44, 155.06, 139.74, 139.55, 131.86, 131.36, 129.55,
128.72, 122.36, 121.86, 121.41, 120.02, 105.48, 66.66, 62.58, 59.88, 53.57, 42.66. ESI-HRMS
(m/z): [M + H]+ calcd for C24H28ClN6O3 483.1906, obsd 483.1911.
2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)-N-(3-
hydroxypropyl)benzamide (8b)
The title compound was obtained starting from compound
7
and 3-amino-1-propanol.
◦
As a yellow solid, 19% yield. mp 200–202 C. 1H NMR (400 MHz, DMSO-d6)
δ
11.48 (s, 1H),
9.44 (s, 1H), 8.82–8.67 (m, 2H), 8.22 (s, 1H), 7.77 (dd, J = 7.9, 1.3 Hz, 1H), 7.61 (d, J = 8.4 Hz,
2H), 7.47 (t, J = 8.4 Hz, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.16 (t, J = 7.6 Hz, 1H), 4.51 (t, J = 5.0 Hz,
1H), 3.58 (t, 4H, J = 4.4 Hz, 4H), 3.49 (q, J = 6.0 Hz, 2H), 3.41 (s, 2H), 3.38–3.35 (m, 2H),
2.35 (br s, 4H), 1.77–1.65 (quint, J = 6.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 168.92,
158.25, 155.45, 155.09, 139.65, 139.55, 131.80, 131.43, 129.55, 128.56, 122.44, 121.93, 121.66,
120.06, 105.46, 66.68, 62.59, 59.07, 53.58, 37.17, 32.60. ESI-HRMS (m/z): [M + H]+ calcd for
C25H30ClN6O3 497.2062, obsd 497.2062.

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2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)-N-(2-
hydroxypropyl)benzamide (8c)
The title compound was obtained starting from compound
7
and 1-amino-2-propanol.
◦
As a yellow solid, 23% yield. mp 232–233 C. 1H NMR (400 MHz, DMSO-d6)
δ
11.41 (s, 1H),
9.45 (s, 1H), 8.86–8.63 (m, 2H), 8.21 (s, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 8.1 Hz, 2H),
7.47 (t, J = 7.5 Hz, 1H), 7.19 (dd, J = 8.0 Hz, 2H), 7.16 (t, J = 7.2 Hz, 1H), 4.80 (d, J = 4.2 Hz,
1H), 3.81 (m, 1H), 3.57 (br s, 4H), 3.41 (s, 2H), 3.23 (t, J = 5.3 Hz, 2H), 2.35 (br s, 4H), 1.08
(d, J = 6.0 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 169.05, 158.27, 155.47, 155.08, 139.56,
131.78, 131.43, 130.10, 129.55, 128.72, 122.43, 121.94, 121.83, 120.08, 105.44, 66.68, 65.32, 62.57,
53.58, 47.53, 21.64. ESI-HRMS (m/z): [M + H]+ calcd for C25H30ClN6O3 497.2062, obsd
497.2072.
2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)-N-(2-
hydroxyethyl)-N-methylbenzamide (8d)
The title compound was obtained starting from compound
As a yellow solid, 18% yield. mp 103–105 C. H NMR (400 MHz, DMSO-d6) 9.35 (s,
7
and 2-(methylamino)ethanol.
◦
1
δ
1H), 9.05 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.15 (s, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.50–7.38
(m, 2H), 7.24 (t, J = 7.6 Hz, 1H), 7.15–7.02 (m, 2H), 5.12 (br s, 1H), 3.56 (t, J = 4.0 Hz, 4H),
3.49 (q, J = 5.1 Hz, 2H), 3.41 (s, 2H), 3.34–3.28 (m, 2H), 2.97 (s, 3H), 2.32 (br s, 4H). 13C
NMR (101 MHz, DMSO-d6) δ 169.95, 158.21, 156.15, 154.93, 139.64, 136.73, 131.08, 129.46,
128.15, 127.77, 124.74, 124.32, 123.88, 119.52, 104.63, 66.66, 62.56, 57.60, 53.56, 50.08, 38.96.
ESI-HRMS (m/z): [M + H]+ calcd for C25H30ClN6O3 497.2062, obsd 497.2065.
2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)(4-
hydroxypiperidin-1-yl)methanone (8e)
The title compound was obtained starting from compound
As a yellow solid, 20% yield. mp 138–140 C. H NMR (400 MHz, DMSO-d6) 9.36 (s,
7
and 4-hydroxypiperidine.
◦
1
δ
1H), 8.89 (s, 1H), 8.17 (s, 1H), 8.14 (d, J = 8.2 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.47 (t,
J = 7.2 Hz, 1H), 7.37 (d, J = 7.6 Hz, 1H), 7.26 (t, J = 7.4 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H),
4.77 (d, J = 3.4 Hz, 1H), 4.00 (br s, 1H), 3.72–3.62 (m, 1H), 3.56 (t, J = 4.0 Hz, 4H), 3.48 (br s,
1H), 3.37 (s, 2H), 3.23 (br s, 1H), 3.09 (br s, 1H), 2.32 (br s, 4H), 1.76 (br s, 1H), 1.52 (br s,
1H), 1.35 (br s, 1H), 1.15 (br s, 1H). 13C NMR (101 MHz, DMSO-d6)
δ 168.01, 158.20, 156.07,
155.04, 139.60, 136.52, 131.06, 130.17, 129.44, 128.53, 127.83, 125.25, 124.40, 119.57, 104.44,
66.66, 65.69, 62.55, 53.55, 45.23, 34.95. ESI-HRMS (m/z): [M + H]+ calcd for C27H32ClN6O3
523.2219, obsd 523.2213.
2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)(4-
(hydroxymethyl)piperidin-1-yl)methanone (8f)
The title compound was obtained starting from compound
7 and 4-(hydroxymethyl)
piperidine. As a yellow solid, 11% yield. mp 92–94 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ
9.40 (s, 1H), 8.90 (s, 1H), 8.20 (s, 1H), 8.18 (d, J = 8.2 Hz, 1H), 7.62–7.45 (m, 3H), 7.37 (d,
J = 7.6 Hz, 1H), 7.27 (t, J = 7.4 Hz, 1H), 7.12 (d, J = 7.6 Hz, 2H), 4.51 (br s, 2H), 3.58 (brs, 5H),
3.41 (s, 2H), 3.20 (br s, 2H), 2.97(br s, 1H), 2.75(br s, 1H), 2.32 (br s, 4H), 2.12–1.85 (m, 1H),
1.84–1.41(m, 4H). 13C NMR (101 MHz, DMSO-d6)
δ 167.98, 158.20, 155.99, 155.03, 139.60,
136.49, 131.04, 130.13, 129.46, 128.44, 127.80, 125.09, 124.32, 119.56, 104.46, 66.65, 65.91, 62.56,
53.55, 47.80, 41.99, 38.73, 29.51, 28.87. ESI-HRMS (m/z): [M + H]+ calcd for C28H34ClN6O3
537.2375, obsd 537.2362.
2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)-N-(6-
hydroxyhexyl)benzamide (8g)
The title compound was obtained starting from compound
7
and 6-amino-1-hexanol.
As a yellow solid, 17% yield. mp 197–198 C. 1H NMR (400 MHz, DMSO-d6)
11.42 (s, 1H),
9.43 (s, 1H), 8.74 (t, J = 5.5 Hz, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 7.75 (d, J = 7.6 Hz,
◦
δ

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1H), 7.60 (d, J = 7.9 Hz, 2H), 7.47 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 8.1 Hz, 2H), 7.16 (t, J = 7.5 Hz,
1H), 4.34 (t, J = 4.4 Hz, 1H), 3.58 (t, J = 4.0 Hz, 4H), 3.44–3.39 (m, 4H), 3.28 (q, J = 5.6 Hz,
2H), 2.36 (br s, 4H), 1.54 (quint, J = 6.0 Hz, 2H), 1.42 (quint, J = 6.0 Hz, 2H), 1.36–1.28 (m,
4H). 13C NMR (101 MHz, DMSO-d6)
δ 168.84, 158.26, 155.49, 155.07, 139.38, 131.73, 131.41,
130.11, 129.59, 128.53, 122.48, 121.95, 120.07, 105.47, 66.64, 62.55, 61.14, 53.55, 32.95, 29.38,
26.86, 25.75. ESI-HRMS (m/z): [M + H]+ calcd for C28H36ClN6O3 539.2532, obsd 539.2521.
2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)-N-(2,3-
dihydroxypropyl)benzamide (8h)
The title compound was obtained starting from compound
7
and 3-amino-1,2-propane-
11.46
diol. As a yellow solid, 15% yield. mp 159–161 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ
(s, 1H), 9.46 (s, 1H), 8.78–8.66 (m, 2H), 8.22 (s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.61 (d, J = 8.4 Hz,
2H), 7.48 (t, J = 7.8 Hz, 1H), 7.20 (t, J = 8.6 Hz, 2H), 7.15 (d, J = 7.5 Hz, 1H), 4.87 (d, J = 4.7 Hz,
1H), 4.62 (t, J = 6.0 Hz, 1H), 3.73–3.65 (m, 1H), 3.57 (t, J = 4.4 Hz, 4H), 3.46–3.42 (m, 1H), 3.41
(s, 2H), 3.40–3.37 (m, 2H), 3.27–3.16 (m, 1H), 2.35 (br s, 4H). 13C NMR (101 MHz, DMSO-d6)
δ 169.14, 158.23, 155.43, 155.09, 139.63, 139.55, 131.84, 131.36, 129.57, 128.75, 122.41, 121.89,
121.61, 120.03, 105.46, 70.48, 66.66, 64.51, 62.57, 53.57, 43.51. ESI-HRMS (m/z): [M + H]+
calcd for C25H30ClN6O4 513.2012, obsd 513.1995.
2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)-N-(1-
hydroxy-3-methylpentan-2-yl)benzamide (8i)
The title compound was obtained starting from compound
7
and L-isoleucinol. As
11.27 (s, 1H),
a yellow solid, 20% yield. mp 264–265 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ
9.46 (s, 1H), 8.67 (d, J = 7.1 Hz, 1H), 8.37 (d, J = 8.6 Hz, 1H), 8.21 (s, 1H), 7.81 (d, J = 7.7 Hz,
1H), 7.61 (d, J = 8.0 Hz, 2H), 7.47 (t, J = 7.6 Hz, 1H), 7.22–7.14 (m, 3H), 4.61 (t, J = 5.1 Hz,
1H), 3.94–3.84 (m, 1H), 3.62–3.49 (m, 6H), 3.41 (s, 2H), 2.35 (br s, 4H), 1.78–1.65 (m, 1H),
1.52–1.42 (m, 1H), 1.19–1.09 (m, 1H), 0.90 (d, J = 6.6 Hz, 3H), 0.85 (t, J = 7.2 Hz, 3H). 13
C
NMR (101 MHz, DMSO-d6) δ 168.87, 158.26, 155.49, 155.06, 139.58, 139.28, 131.57, 131.37,
129.57, 128.90, 122.70, 122.45, 121.96, 120.04, 105.38, 66.65, 62.56, 61.42, 56.05, 53.55, 35.44,
25.49, 16.04, 11.59. ESI-HRMS (m/z): [M + H]+ calcd for C28H36ClN6O3 539.2532, obsd
539.2528.
3.1.5. General Procedure for the Preparation of 9a–9g
To a solution of compounds 8a
–
8g (0.195 mmol) in 2 mL of THF, NaH (14 mg,
◦
0.585 mmol) was slowly added under stirring at 0 C. After stirring for 5 min, 3,4-
bis(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (71.4 mg, 0.195 mmol) was added, and the
obtained mixture was allowed to react overnight at room temperature, then quenched by a
few drops of water and concentrated in vacuo. The crude product was purified by column
chromatography (dichloromethane-methanol 40:1 v/v).
4-(2-(2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-
yl)amino)benzamido)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (9a)
As a yellow solid, 15% yield. mp 174–176 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 11.48
(s, 1H), 9.49 (s, 1H), 9.11 (s, 1H), 8.79 (d, J = 6.0 Hz, 1H), 8.21 (s, 1H), 7.97 (d, J = 6.9 Hz, 2H),
7.86 (d, J = 6.7 Hz, 1H), 7.75 (d, J = 6.5 Hz, 1H), 7.62 (d, J = 6.7 Hz, 2H), 7.60–7.47 (m, 3H),
7.21 (d, J = 6.2 Hz, 3H), 4.61 (br s, 2H), 3.78 (br s, 2H), 3.58 (br s, 4H), 3.44 (s, 2H), 2.37 (br s,
4H). 13C NMR (101 MHz, DMSO-d6)
δ 169.44, 159.34, 158.19, 155.42, 155.07, 139.97, 139.63,
137.68, 136.43, 132.23, 130.29, 130.10, 129.64, 128.75, 122.39, 121.95, 120.85, 120.02, 110.99,
105.56, 69.84, 66.57, 62.47, 53.49, 38.40. ESI-HRMS (m/z): [M + H]+ calcd for C32H32ClN8O7S
707.1798, obsd 707.1798.

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4-(3-(2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-
yl)amino)benzamido)propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (9b)
As a yellow solid, 17% yield. mp 144–146 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 11.40
(s, 1H), 9.44 (s, 1H), 8.87 (t, J = 5.7 Hz, 1H), 8.71 (d, J = 8.1 Hz, 1H), 8.19 (s, 1H), 8.06 (dd,
J = 8.4, 1.1 Hz, 2H), 7.90 (t, J = 7.6 Hz, 1H), 7.80 (dd, J = 7.9, 1.3 Hz, 1H), 7.76 (t, J = 7.9 Hz,
2H), 7.61 (d, J = 8.4 Hz, 2H), 7.49 (t, J = 7.8 Hz, 1H), 7.22–7.15 (m, 3H), 4.47 (t, J = 6.0 Hz,
2H), 3.58 (t, J = 4.4 Hz, 4H), 3.47 (q, J = 6.3 Hz, 2H), 3.42 (s, 2H), 2.36 (br s, 4H), 2.12–2.01
(quint, J = 6.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 169.21, 159.40, 158.23, 155.43,
155.06, 139.67, 139.57, 137.65, 136.60, 131.93, 130.53, 129.58, 128.87, 128.63, 122.46, 121.98,
121.54, 120.06, 110.97, 105.45, 69.61, 66.64, 62.54, 53.55, 36.02, 28.61. ESI-HRMS (m/z): [M +
H]+ calcd for C33H34ClN8O7S 721.1954, obsd 721.1952.
4-((1-(2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-
yl)amino)benzamido)propan-2-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (9c)
As a yellow solid, 14% yield. mp 187–189 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 11.29
(s, 1H), 9.44 (s, 1H), 8.93 (t, J = 5.2 Hz, 1H), 8.74 (d, J = 7.2 Hz, 1H), 8.20 (s, 1H), 7.96 (d,
J = 8.1 Hz, 2H), 7.81–7.70 (m, 2H), 7.64–7.54 (m, 4H), 7.51 (t, J = 7.6 Hz, 1H), 7.24–7.15 (m,
3H), 5.22–5.11 (m, 1H), 3.76 (t, J = 4.0 Hz, 1H), 3.72 (t, J = 4.4 Hz, 1H), 3.58 (t, J = 4.0 Hz,
4H), 3.43 (s, 2H), 2.37 (br s, 4H), 1.42 (d, J = 4.0 Hz, 3H). 13C NMR (101 MHz, DMSO-d6)
δ
169.48, 158.91, 158.21, 155.41, 155.07, 139.77, 139.60, 137.63, 136.38, 132.12, 130.22, 129.63,
128.81, 128.69, 122.41, 121.98, 121.19, 120.04, 111.11, 105.51, 78.17, 66.60, 62.49, 53.50, 43.28,
17.69. ESI-HRMS (m/z): [M + H]+ calcd for C33H34ClN8O7S 721.1954, obsd 721.1951.
4-(2-(2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-yl)amino)-N-
methylbenzamido)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (9d)
◦
As a yellow solid, 25% yield. mp 169–171 C. 1H NMR (400 MHz, DMSO-d6)
δ 9.34 (s,
1H), 8.99 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.13 (s, 1H), 7.97 (d, J = 7.6 Hz, 2H), 7.85–7.78 (m,
1H), 7.75–7.63 (m, 2H), 7.54 (t, J = 7.6 Hz, 2H), 7.50 (d, J = 7.6 Hz, 2H), 7.32–7.20 (m, 1H),
7.11 (d, J = 8.1 Hz, 2H), 4.64 (br s, 2H), 3.93 (br s, 2H), 3.57 (t, J = 4.0 Hz, 4H), 3.40 (s, 2H),
3.05 (s, 3H), 2.35 (br s, 4H). 13C NMR (101 MHz, DMSO-d6)
δ 169.80, 159.31, 158.17, 156.07,
154.98, 139.74, 137.58, 136.70, 136.56, 130.43, 129.57, 128.70, 128.39, 128.05, 125.95, 125.22,
124.16, 119.57, 111.07, 104.59, 69.64, 66.54, 62.43, 53.45, 46.28, 39.09. ESI-HRMS (m/z): [M +
H]+ calcd for C33H34ClN8O7S 721.1954, obsd 721.1935.
4-((1-(2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-
yl)amino)benzoyl)piperidin-4-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (9e)
As a yellow solid, 19% yield. mp 130–132 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 9.38
(s, 1H), 9.00 (s, 1H), 8.19 (s, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 7.6 Hz, 2H), 7.86 (t,
J = 7.4 Hz, 1H), 7.72 (t, J = 7.8 Hz, 2H), 7.51 (t, J = 8.0 Hz, 3H), 7.46 (d, J = 7.3 Hz, 1H),
7.30 (t, J = 7.5 Hz, 1H), 7.09 (d, J = 8.3 Hz, 2H), 5.16–5.00 (m, 1H), 3.67 (br s, 2H), 3.62–3.51
(m, 5H), 3.37 (s, 3H), 2.32 (br s, 4H), 2.00 (br s, 1H), 1.84 (br s, 2H), 1.64 (br s, 1H). 13C
NMR (101 MHz, DMSO-d6) δ 168.25, 158.22, 156.23, 155.10, 139.61, 137.71, 136.71, 136.54,
130.99, 130.47, 130.10, 129.45, 128.85, 128.34, 128.06, 125.60, 124.58, 119.58, 110.88, 104.47,
77.51, 66.82, 66.64, 62.53, 53.52, 29.51. ESI-HRMS (m/z): [M + H]+ calcd for C35H36ClN8O7S
747.2111, obsd 747.2092.
4-((1-(2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-
yl)amino)benzoyl)piperidin-4-yl)methoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (9f
)
As a yellow solid, 29% yield. mp 109–111 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 9.36
(s, 1H), 8.99 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.14 (s, 1H), 7.98 (d, J = 7.2 Hz, 2H), 7.84 (t,
J = 7.5 Hz, 1H), 7.70 (t, J = 7.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 8.4 Hz, 2H), 7.40
(d, J = 7.6 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 8.4 Hz, 2H), 4.53 (br s, 1H), 4.21 (d,
J = 5.9 Hz, 2H), 3.70 (br s, 1H), 3.58 (t, J = 4.0 Hz, 4H), 3.38 (s, 2H), 3.03 (br s, 1H), 2.81

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(br s, 1H), 2.33 (br s, 4H), 2.13–1.90 (m, 2H), 1.75 (br s, 1H), 1.60–1.42 (m, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 168.05, 159.25, 158.19, 156.05, 155.01, 139.65, 137.63, 136.74, 136.55,
130.47, 130.32, 129.51, 128.75, 128.20, 127.92, 125.24, 124.33, 119.58, 110.85, 104.56, 75.08,
70.25, 66.59, 62.48, 53.49, 52.77, 35.08, 29.55, 29.21. ESI-HRMS (m/z): [M + H]+ calcd for
C36H38ClN8O7S 761.2267, obsd 761.2235.
4-((6-(2-((5-Chloro-2-((4-(morpholinomethyl)phenyl)amino)pyrimidin-4-
yl)amino)benzamido)hexyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (9g)
As a yellow solid, 30% yield. mp 120–122 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ 11.46
(s, 1H), 9.48 (s, 1H), 8.80 (t, J = 5.4 Hz, 1H), 8.72 (d, J = 7.8 Hz, 1H), 8.20 (s, 1H), 8.02 (d,
J = 7.5 Hz, 2H), 7.88 (t, J = 7.5 Hz, 1H), 7.79–7.73 (m, 3H), 7.62 (d, J = 8.3 Hz, 2H), 7.48 (t,
J = 7.6 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.17 (t, J = 7.2 Hz, 1H), 4.38 (t, J = 6.2 Hz, 2H), 3.59
(t, J = 4.4 Hz, 4H), 3.45 (s, 2H), 3.32 (t, J = 6.2 Hz, 2H), 2.39 (br s, 4H), 1.75 (quint, J = 5.6 Hz,
2H), 1.58 (quint, J = 6.1 Hz, 2H), 1.42–1.34 (m, 4H). 13C NMR (101 MHz, DMSO-d6)
δ 168.89,
159.31, 158.22, 155.45, 155.04, 139.63, 139.58, 137.72, 136.56, 131.76, 130.47, 129.63, 128.74,
128.55, 122.46, 121.99, 121.89, 120.00, 110.87, 105.44, 71.85, 66.58, 62.49, 53.49, 29.24, 28.28,
26.42, 25.25. ESI-HRMS (m/z): [M + H]+ calcd for C36H40ClN8O7S 763.2424, obsd 763.2405.
3.2. In Vitro Kinase Enzymatic Assay
The ADP-Glo™ Kinase Assay Kit and FAK Kinase Enzyme (Catalog. V9301) were
purchased from Promega Corporation (Madison, WI, USA). The experiments were per-
formed according to the detailed and complete protocols of the manufacturer. Briefly, the
concentration of FAK determined by optimization experiments was 0.1 µg/µL. Suitable
concentration gradients from 1.6 to 1000 nM were set for all the tested compounds. A
kinase reaction buffer, a substrate/ATP mixture, and a FAK solution were prepared right
before use. The 5
strate/ATP mixture, and 2
by 3 L of kinase reaction buffer and 2
was prepared using 1 L of kinase reaction buffer instead of a compound solution. The
kinase reactions were added to a 384-well plate and incubated at room temperature for 60
min, then 5 L of ADP-Glo Reagent was added to stop the kinase reaction and deplete
the unconsumed ATP, and incubated at room temperature for 40 min. After adding 10
µ
L kinase reaction consisted of 1
L of FAK solution. The control without enzyme was provided
L of substrate/ATP mixture. The negative control
µL of compound solution, 2 µL of sub-
µ
µ
µ
µ
µ
™
µ
L of Kinase Detection Reagent, the plate was incubated at room temperature for 30 min
and then luminescence was recorded (integration time 0.5–1 s) on a TriStar® LB942 Multi-
mode Microplate Reader (BERTHOLD TECHNOLOGIES GmbH & Co. KG., Bad Wildbad,
Germany). Curve fitting and data presentations were performed using GraphPad Prism
version 8.0.
3.3. Cell Viability Determination
The H1975 human NSCLC cell line was obtained from ATCC. A431 human epithelial
carcinoma and HBE human bronchial epithelial cell lines were kind gifts from Fuheng
Biological Company Ltd. (Shanghai, China) and YuXi Biotech Company (Jiangsu, China),
respectively. The cells were maintained in RPMI 1640 or DMEM (Gibco®, Thermo Fisher
Scientific Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco®) and
1% penicillin-streptomycin (Beyotime Company, Shanghai, China) at 37 ◦C in a 5% CO2
incubator.
A total of 3000 cells of H1975 or HBE and 5000 cells of A431 were seeded for each
well into a 96-well plate and cultured in 10% FBS respective growth medium overnight.
Various concentrations (0–20
µM) of each compound were then added in triplicate to the
cells. After 72 h of incubation, 10
µL of CCK-8 solution (5.0 mg/mL, Biotool Company,
Kirchberg, Switzerland) was added to each well and the cells continued to culture for 4 h.
The optical density (OD) was determined at 450 nm with a microplate reader (Thermo
Fisher Scientific, Waltham, MA, USA). IC50 values were calculated employing GraphPad
Prism version 8.0.

Molecules 2021, 26, 4187
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3.4. Acridine Orange/Ethidium Bromide (AO/EB) Staining Assay
H1975 cells at a density of 1
105 cells/well in 6-well plates were incubated for 24 h.
×
After discarding the original culture medium, different concentrations of inhibitors were
used. The cells were incubated for 48 h and then washed with phosphate-buffered saline
(PBS) three times. Cells were subsequently incubated in the dye mix containing 1.0
µg/mL
of AO and 1.0
µ
g/mL of EB in PBS at 37 ◦C for 5 min, and then washed with PBS. The
apoptotic, necrotic, and live cells were examined under the fluorescent inverted microscope
(OLYMPUS, Tokyo, Japan).
3.5. Flow Cytometric Analysis
H1975 cells were seeded at a density of 1
and then exposed to different concentrations of inhibitors for 48 h. The cells were harvested.
(1) The cells were stained with Annexin V-FITC (5 L)/propidium iodide (5 L), and the
×
105 cells/well into 6-well plates for 24 h
µ
µ
samples were detected and flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA)
was performed to quantify the number of apoptotic cells. (2) The cells were stained with
propidium iodide (PI) in the dark for 30 min, and then analyzed for cell cycle distribution
by the flow cytometry assay.
3.6. Scratch Assay
H1975 cells were seeded in 6-well plates and incubated for 12 h. When cells were
grown to 90–100% confluency in a monolayer, wounds were generated using a pipette
tip. The cells were washed three times with PBS, treated with different concentrations of
compound 8a or TAE226, and incubated for 24 h. The images were captured to record the
status of cells near the scratch lines at the beginning and after 12 and 24 h.
3.7. Molecular Docking Study
Molecular docking simulations were carried out on the Discovery Studio 2017 R2
software (Dassault Systèmes BIOVIA, San Diego, CA, USA). The crystal structure of the
FAK (PDB: 2JKK) in complex with TAE226 was used and the corresponding PDB files were
directly loaded into the Discovery Studio 2017 R2 software. The FAK kinase was prepared
using the “Prepare Protein” plugin, while the structure of ligand 8a was optimized using
the “Prepare Ligands” plugin. The binding site in FAK was defined from the current
selection when TAE226 was selected. Docking was performed using CDOCKER, while the
parameter for “Pose Cluster Radius” was set as 0.5 and the remaining parameters were at
default settings. The best predicted hit with the highest docking score was used for the
visualization with 2JKK.
3.8. Statistical Analysis
All statistical analyses were performed using the GraphPad Prism software version
8.0 (GraphPad Software Inc., La Jolla, CA, USA). Statistically significant differences were
analyzed using the unpaired t tests, and p-values of less than 0.05 were considered to
indicate statistical significance.
4. Conclusions
In summary, a series of 2,4-dianilinopyrimidine derivatives 8a–8i and 9a–9g bearing 4-
(morpholinomethyl)phenyl and N-substituted benzamides were designed and synthesized.
Many of these derivatives inhibited FAK at low-nanomolar concentrations. In particular,
compounds 8a
,
8c, and 8d exhibited the highest inhibition against FAK compared to other
8b, and 9a against H1975 and
compounds. The antiproliferative effects of compounds 8a
,
A431 cells were stronger than both TAE226 and osimertinib; however, most of the target
compounds weakly inhibited the growth of HBE cells compared to H1975 cells. Compound
8a demonstrated the best combination of potency and selectivity against tumor cells
compared to the other compounds. Unexpectedly, compounds 8a
–8g featuring hydroxyl
groups displayed higher potencies against FAK and antiproliferative activities compared

Molecules 2021, 26, 4187
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to compounds 9a
–
9g bearing furoxan moieties. Therefore, the introduction of furoxan did
not improve the inhibitory activity of the scaffold against FAK or the growth of the cancer
cells. In the further studies of compound 8a using H1975 cells, 8a induced cell apoptosis in
a dose-dependent manner, causing S/G2 arrest and inhibiting cell migration, all of which
were superior to TAE226. Molecular docking studies demonstrated that 8a was bound
in the active pocket of FAK in a conformationally similar manner compared to TAE226,
but two additional hydrogen bonds were formed between 8a and the active site residues.
Overall, these results suggested that compound 8a might be a potential inhibitor of FAK
for treatment of various cancers, although further investigation is required to evaluate the
efficacy in vivo.
Author Contributions: C.H. and K.S. contributed equally to this work. Conceptualization, C.H.
and L.W.; methodology, J.H.; software, K.S.; validation, S.W., Z.W., and F.S.; formal analysis, X.W.;
investigation, X.H.; resources, L.W.; data curation, M.L.; writing—original draft preparation, C.H. and
K.S.; writing—review and editing, L.W.; visualization, K.S.; supervision, J.H.; project administration,
J.H.; funding acquisition, C.H. and L.W. All authors have read and agreed to the published version
of the manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (award
Nos. 21402012 and 21907010), and the Fundamental Research Program of Shanxi Province (award
No. 201901D211465), Shanxi “1331 Project” Key Innovative Research Team.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
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