Design, synthesis and biological evaluation of sulfamoylphenyl-quinazoline derivatives as potential EGFR/CAIX dual inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113300

Multi-target, especially dual-target, drug design has become a popular research field for cancer treatment. Development of small molecule dual-target inhibitors through hybridization strategy can provide highly potent and selective anticancer agents. In this study, three series of quinazoline derivatives bearing a benzene-sulfonamide moiety were designed and synthesized as dual EGFR/CAIX inhibitors. All the synthesized compounds were evaluated against epidermoid carcinoma (A431) and non-small cell lung cancer (A549 and H1975) cell lines, which displayed weak to potent anticancer activity. In particular, compound 8v emerged as the most potent derivative against mutant-type H1975 cells, which exhibited comparable activity to osimertinib. Importantly, 8v exhibited stronger anti-proliferative activity than osimertinib against H1975 cells under hypoxic condition. Kinase inhibition studies indicated that 8v showed excellent inhibitory effect on EGFR^T790M enzyme, which was 41 times more effective than gefitinib and almost equal to osimertinib. Mechanism studies revealed that 8v exhibited remarkable CAIX inhibitory effect comparable to acetazolamide and significantly inhibited the expression of p-EGFR as well as its downstream p-AKT and p-ERK in H1975 cells. Notably, 8v was found to inhibit the expression of CAIX and its upstream HIF-1α in H1975 cells under hypoxic condition. Molecular docking was also performed to gain insights into the ligand-binding interactions of 8v inside EGFR^WT, EGFR^T790M and CAIX binding sites.

Full text of DOI:10.1016/j.ejmech.2021.113300

European Journal of Medicinal Chemistry 216 (2021) 113300
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluation of sulfamoylphenyl-
quinazoline derivatives as potential EGFR/CAIX dual inhibitors
,
b
,
,
b
,
,
,
, b, *
Bin Zhang a 1, Zhikun Liu a 1, Shengjin Xia a b, Qingqing Liu a b, Shaohua Gou a
a Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing, 211189, China
b Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Multi-target, especially dual-target, drug design has become a popular research field for cancer treat-
ment. Development of small molecule dual-target inhibitors through hybridization strategy can provide
highly potent and selective anticancer agents. In this study, three series of quinazoline derivatives
bearing a benzene-sulfonamide moiety were designed and synthesized as dual EGFR/CAIX inhibitors. All
the synthesized compounds were evaluated against epidermoid carcinoma (A431) and non-small cell
lung cancer (A549 and H1975) cell lines, which displayed weak to potent anticancer activity. In particular,
compound 8v emerged as the most potent derivative against mutant-type H1975 cells, which exhibited
comparable activity to osimertinib. Importantly, 8v exhibited stronger anti-proliferative activity than
osimertinib against H1975 cells under hypoxic condition. Kinase inhibition studies indicated that 8v
showed excellent inhibitory effect on EGFRT790M enzyme, which was 41 times more effective than
gefitinib and almost equal to osimertinib. Mechanism studies revealed that 8v exhibited remarkable CAIX
inhibitory effect comparable to acetazolamide and significantly inhibited the expression of p-EGFR as
well as its downstream p-AKT and p-ERK in H1975 cells. Notably, 8v was found to inhibit the expression
Received 16 December 2020
Received in revised form
10 February 2021
Accepted 15 February 2021
Available online 20 February 2021
Keywords:
Dual-target inhibitors
Sulfamoylphenylquinazoline derivatives
EGFR inhibitors
CAIX inhibitors
Anticancer agents
of CAIX and its upstream HIF-1a in H1975 cells under hypoxic condition. Molecular docking was also
performed to gain insights into the ligand-binding interactions of 8v inside EGFRWT, EGFRT790M and CAIX
binding sites.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
combination-based therapies [4]. A rational design to discover
dual-target agents is established based on some pharmacophores
It is currently well-known that most cancers can quickly
develop resistance against any single protein or kinase inhibitor [1].
Despite drug combination therapies are used as an alternative
approach to achieve efficacy, their benefits are often negated by
adverse drug-drug interactions, poor patient compliance, unpre-
dictable pharmacokinetics (PK) and safety profiles [2]. Dual-target
anticancer agents have been developed successfully since the
beginning of modern pharmacology, and their dual-target proper-
ties have contributed to their clinical transformation [3]. Dual-
target drugs help to target different biological receptors with the
expectation of synergistic effects and address the limited efficacy
and resistance or toxicity associated with many single-target or
that are maintained and another is introduced to yield hybrid
molecules [4a].
Epidermal growth factor receptor (EGFR), as a transmembrane-
bound molecule, has important regulatory functions in cell prolif-
eration, apoptosis, migration, differentiation, and survival [5].
Increased EGFR activity resulting from overexpression, mutation, or
amplification of EGFR gene contributes to many human malig-
nancies, including glioblastoma, epithelial cancers of the head and
neck, esophagus cancers, breast cancers, and lung cancers, espe-
cially in non-small-cell lung cancers (NSCLCs) [6]. Accordingly,
interruption of the signaling pathway of EGFR, either extracellularly
by blocking the binding site of EGFR or intracellularly by inhibiting
the tyrosine kinase activity, is important in cancer prevention and
treatment [7]. First-generation (e.g., gefitinib or erlotinib) and
second-generation (e.g., afatinib or dacomitinib) quinazoline-based
EGFR tyrosine kinase inhibitors (TKIs) (Fig. 1) have remarkably
improved survival in advanced EGFR mutant NSCLC patients [1b,8].
However, the emergence of acquired point mutations, particularly
* Corresponding author. Jiangsu Province Hi-Tech Key Laboratory for Biomedical
Research, Southeast University, Nanjing, 211189, China.
E-mail address: 2219265800@qq.com (S. Gou).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113300
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
inhibitors as antitumor therapeutics. Recently, the relationship
between hypoxia-mediated gene transcription (such as HIF-1a and
Abbreviations
CAIX) and EGFR expression in hypoxic cells has been reported [16].
Studies suggested that EGFR signaling enhances the cellular
response to hypoxia and acts as a survival factor [17]. Thus, the
inhibition of hypoxia-mediated gene transcription may provide an
attractive treatment strategy for overcoming the tumor resistance
of EGFR TKI.
In the current medical era, quinazoline is one of the most
important heterocyclic scaffolds in cancer drug discovery and forms
a large array of EGFR inhibitors (Fig. 1) [18]. Besides, sulfonamide is
a privileged scaffold that is well known for its anticancer activity by
many mechanisms such as cyclin-dependent kinase (CDK) inhibi-
tion, matrix metalloproteinase (MMP) inhibition and carbonic
anhydrase (CA) inhibition (Fig. 1) [19].
EGFR
TKIs
CAIX
HIF-1
ERK
Epidermal growth factor receptor
Tyrosine kinase inhibitors
Carbonic anhydrase IX
Hypoxia inducible factor-1a
Extracellular signal-regulated kinase
Protein kinase B
a
AKT
DMF-DMA N,N-Dimethylformamide dimethyl acetal
DMF
IC50
N,N-Dimethylformamide;
Half-maximum inhibitory concentration
T790 M mutation, has weakened their therapeutic efficacy, leading
to drug resistance [9]. Thus, the third-generation EGFR covalent
inhibitors (e.g., osimertinib or rociletinib) targeting T790 M muta-
tion have been developed [10]. Unfortunately, C797S point muta-
tion and/or other mechanisms have been shown to be associated
with the acquired resistance to the third-generation EGFR TKIs [11].
Subsequently, the fourth-generation EGFR TKIs such as EAI045 and
other noncovalent inhibitors are being developed to be a major
breakthrough against these tertiary mutations [12]. Even so, there
is still an unmet medical need to develop novel small molecule
inhibitors or therapeutic approaches to overcome multipoint mu-
tations of EGFR.
Oxygen supply is crucial for the growth of cells and is often
diminished in solid tumors, especially at the center of the tumor
mass, as tumor cells grow faster than the endothelial cells that are
crucial for the formation of blood vessels [13]. Tumor cells within
hypoxic regions contribute to increased invasion, metastatic spread
and therapy resistance [14]. Hypoxia can regulate the expression of
several genes, including that encoding carbonic anhydrase IX
Herein we report the synthesis and cytotoxic evaluation of a
number of new quinazoline derivatives containing substituted
anilide as well as sulfamoylphenyl fragments as EGFR/CAIX dual-
target inhibitors. The cytotoxicity of the target compounds was
evaluated against three cancer cell lines (human epidermoid car-
cinoma (A431) and non-small cell lung cancer cells (A549 and
H1975)) by MTT assay. Besides, the most active members were
examined for their EGFR (EGFRWT and EGFRT790M) and CA (CAII and
CAIX) inhibitory activity. These compounds elicit potent inhibition
of EGFRT790M and CAIX in biochemical settings that translates nicely
into improved activity on pertinent NSCLC cell lines. Molecular
docking of the promising compound was performed inside the
binding site of EGFRWT, EGFRT790M and CAIX to gain insights into
the molecular interactions and possible modes of action.
2. Results and discussion
2.1. Chemistry
(CAIX), through binding of the hypoxia inducible factor HIF-1a to a
hypoxia-responsive element in the gene. Membrane-bound CAIX
contributes to acidification of the tumor environment by efficiently
catalyzing the hydration of carbon dioxide to bicarbonate and
protons, thereby leading to acquisition of metastatic phenotypes
and chemoresistance to weakly basic anticancer drugs [15].
Therefore, many efforts have been made to generate effective CAIX
The structures and preparation of target compounds 8a-w, 15a-n
and 18a-n are described in Schemes 1 and 2. In Scheme 1, compounds
8a-w containing 6-hydroxyl-7-methoxy-4-arylaminoquinazoline
frameworks (4a-w) equipped with specific sulfamoylphenyl war-
heads were obtained by a substitution reaction of the 6-hydroxyl
group with 5-bromo-N-(4-sulfamoylphenyl)pentanamide (7). The
Fig. 1. Chemical structures of selected EGFR inhibitors (I-VII) and CA inhibitors (VIII and IX).
2

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
Scheme 1. General procedures of preparing compounds 8a-8w.11Reagents and conditions: (i) (CH3CO)2O, pyridine, reflux; (ii) SOCl2, DMF, reflux; (iii) substituted aniline, CH3CN,
reflux; (iv) NH3$H2O, CH3OH, reflux; (v) pyridine, DMF, rt; (vi) K2CO3, DMF, KI.
synthesis of the anilinoquinazoline scaffold 4a-w was performed as
previously reported with minor adaptations [20]. Compound 7 was
obtained by means of acylation of benzenesulfonamide with 5-
bromopentanoyl chloride (6) in DMF. In Scheme 2, intermediates
12a-n were synthesized by reacting 5-nitroanthranilonitrile (9) with
DMF-DMA to afford intermediate 10, which cyclized upon reaction
with different anilines in refluxing glacial acetic acid into 6-nitro-4-
anilinoquinazolines 11a-n through Dimroth rearrangement. Reduc-
tion of the latter using Fe/NH4Cl mixture afforded 6-amino-4-
anilinoquinazolines 12a-n, which were subsequently treated with
compounds 14 or 17 in DMF at 60 ꢀC overnight to produce the target
compounds 15a-n and 18a-n. 4-Isothiocyanatobenzenesulfonamide
(14) was prepared by reaction of 4-aminobenzenesulfonamide with
thiophosgene in water containing concentrated hydrochloric acid
[21]. Isothiocyanate 17 was obtained by treated 4-(2-aminoethyl)
benzenesulfonamide with dicyclohexylcarbodiimide and carbon di-
sulfide in THF at room temperature. The purities of all compounds
were evaluated by HPLC (each compound > 95%). Their structures
were characterized by HR-MS and 1H and 13C NMR spectrometry.
available drugs (gefitinib, erlotinib and osimertinib) were used in
this test as positive controls.
As shown in Table 1, the obtained results revealed that most of
the synthesized compounds showed weak to potent anti-
proliferative activities against the tested cell lines. For the activity
against the wild-type non-small cell lung cancer cell line (A549),
compounds 8a, 8b, 8c, 8d, 8e, 8m, 8r, 8t, 8v and 8w were found to
be more potent than gefitinib (IC50 ¼ 15.59
m
M) and erlotinib
(IC50 ¼ 16.43
m
M) with IC50 values ranging from 6.54 to 15.53 m
M,
while the other compounds had equally or less potent activity than
gefitinib and erlotinib. For the wild-type human epidermoid car-
cinoma cell line (A431), compounds 8a, 8b, 8h, 8i, 8l, 8m, 8o, 8t, 8v
and 8w exhibited effective cytotoxic activities against A431 cell line
with IC50 values ranging from 3.91 to 8.02
potent than gefitinib (IC50 ¼ 8.37 M). Notably, compounds 8a, 8b
and 8v showed stronger cytotoxic activity than osimertinib
M). For the activity against the mutant-type non-
mM, which were more
m
(IC50 ¼ 5.32
m
small cell lung cancer cell line (H1975), compounds 8b, 8k, 8m, 8n,
8q, 8t, 8v and 8w showed potent activity against H1975 cell line
with IC50 values ranging from 1.94 to 10.34
active than gefitinib (IC50 ¼ 10.78 M), but all the target compounds
had weaker activity against H1975 cell line than osimertinib
M). Observing the results of antiproliferative tests, we
found that benzene sulfonamide substituted for morpholine could
contribute to increasing inhibitory effects. Moreover, better anti-
tumor activities presented when 3-positon of the aryl group was
occupied with a trifluoromethyl group. More importantly, 8v had a
mM, which were more
m
2.2. Antiproliferative activity assay
(IC50 ¼ 0.98
m
The antiproliferation activity of the target compounds against
human epidermoid carcinoma cells (A431) and non-small cell lung
cancer cells (A549 and H1975) were evaluated by MTT assay. A431
and A549 cells have overexpressed EGFRWT, while H1975 cells
harbor EGFRL858R/T790M mutation [20a,22]. Three commercially
3

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
Scheme 2. General procedures of preparing compounds 15a-15n and 18a-18n.11Reagents and conditions: (i) DMF-DMA, reflux; (ii) substituted aniline, glacial acetic acid, reflux; (iii)
Fe, NH4Cl, EtOH/H2O (70%), reflux; (iv) hydrochloric acid, thiophosgen, H2O; (v) DCC, CS2, THF; (vi) 12a-n, DMF.
comparable anti-tumor activity to osimertinib towards H1975 cell
line.
activities against H1975 cells with IC50 values ranging from 4.25 to
10.76 M, more potent than gefitinib. Among this series com-
m
Encouraged by the increased anti-tumor activity of 4-
aminoquinazoline derivatives that contain benzene sulfonamide,
we further explored the anti-proliferation efficacy of more com-
pounds with the same pharmacophores. As shown in Table 2,
compounds 15c, 15d, 15f, 15i, 15l and 15m displayed moderate
activity against A549 cells with IC50 values range from 2.84 to
pounds, compound 18c with a trifluoromethyl group in the 3-
position of the aryl unit also showed the best anti-tumor activity
against H1975 cell line.
The cytotoxic effect of the target compound 8v was also exam-
ined towards normal human liver cells LO2 to investigate the
toxicity of the prepared anticancer agent, using gefitinib and osi-
mertinib as positive controls. As shown in Table S1, gefitinib dis-
25.88 mM. Whereas, these compounds had less activity than gefi-
tinib towards A431 cell line. For the activity against the mutant-
type non-small cell lung cancer cell line (H1975), compound 15c
with a trifluoromethyl group in 3-position of the aryl skeleton
showed the most potent activity against H1975 cell line, whose IC50
value was less than that of gefitinib, but much greater than that of
osimertinib.
played mild cytotoxic effect (IC50 value ¼ 33.17
m
M), while 8v and
osimertinib hardly have cytotoxicity (IC50 value > 40
mM), indi-
cating their relative safety towards normal human liver cells.
2.3. Kinase inhibition study of EGFRWT and EGFRT790M
In order to further explore the effect of increasing carbon chain
length on the antiproliferative activity of the compounds, the
cytotoxicity of 18a-n was tested. As described in Table 3, most of
compounds exhibited stronger antiproliferative activity towards
The inhibitory activity of the selected compounds against
EGFRWT and EGFRT790M were evaluated using a well-established
HTRF KinEASE-TK assay, with gefitinib and osimertinib as positive
controls. As shown in Table 4, most of the tested compounds
exhibited moderate inhibitory activities against EGFRWT (with IC50
13.7e51.2 nM), in which 8b and 8w suppressed EGFRWT more
potently than gefitinib. For mutant EGFR, all the tested compounds
exhibited better inhibitory activities than gefitinib. Notably, 8v
showed excellent inhibitory effect on EGFRT790M, with an IC50 value
of 9.2 nM, which was 41 times as effective as gefitinib. More
importantly, 8v showed comparable EGFRT790M inhibitory activity
A549 than gefitinib, with IC50 values range from 2.77 to 32.36 mM,
which may be attributed to the increase of carbon chain enhancing
the lipid solubility of the compound. In addition, compounds 18h
and 18n displayed effective cytotoxic activities against A431 cell
line with IC50 values 4.21 and 5.77 mM, which were stronger than
gefitinib but almost equal to osimertinib. Moreover, compounds
18c, 18e, 18f, 18l, 18m and 18n possessed effective cytotoxic
4

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
Table 1
Table 2
In vitro antitumor activity of 15a-15n against different cancer cell lines.
In vitro antitumor activity of 8a-8v against different cancer cell lines.
Compounds
R
IC50 (m
M)a
Compounds
R
IC50
A549
15.53 0.82 5.02 0.34
(m
M)a
A549
A431
H1975
A431
H1975
15a
15b
15c
15d
15e
15f
3-Cl, 4-F
3 ꢁ C≡CH
3-CF3
3-CF3, 4-Cl
3-Br, 4-F
3-Br
>40
>40
37.61 1.17 15.75 0.11
15.29 0.21 13.42 0.23
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8 m
8n
8ꢀ
8p
8q
8r
3-Cl, 4-F
3-CF3, 4-Cl
3 ꢁ C≡CH
3-Br
3-Cl
3-F
4-F
4-Cl
4-Br
4-OCH3
35.59 3.45
3.22 0.27
9.27 0.94
7.72 0.25
3.91 0.23
10.14 0.24 21.68 0.16 7.59 0.21
4.50 0.28
>40
17.69 0.94 18.75 1.67
11.37 1.09
11.15 0.88 30.38 2.34 33.18 3.12
18.18 0.32 11.78 0.13
18.21 0.45 9.98 0.26
14.91 0.78 9.75 0.67
25.04 0.65 20.74 0.73 10.96 0.37
>40
>40
>40
>40
>40
29.89 1.89 >40
18.60 1.23 >40
15g
15h
15i
15j
15k
15l
4-CF3
2-F, 3-Cl
3-F
3-Cl
3-CH3
>40
>40
17.96 0.74 17.09 0.25
13.11 0.63 25.14 0.82
7.12 0.27
6.57 0.45
31.78 2.98
23.29 2.19
25.88 1.04 30.08 1.78 9.66 0.15
>40
>40
2.84 0.29
24.64 0.87 38.43 1.85 38.71 0.67
>40 29.51 0.87 10.34 0.17
14.98 0.34 39.79 1.48
23.79 1.26 35.44 0.41
40.02 2.04 39.44 0.41
14.71 0.98 30.19 2.90
35.44 2.56 9.23 0.87
31.57 2.89
6.93 0.67
22.45 1.56 8.77 0.76
3-OCH3, 4-OCH3 >40
3-Br, 4-F
4-CH3
4-CH2CH3
16.02 1.05 8.02 0.47
10.46 0.89 5.56 0.23
>40
16.03 0.97 7.46 0.38
>40 23.41 1.89 19.96 1.89
15 m
15n
2-F, 3-Cl
4-OCF3
3-CH3
3-F, 4-F, 5-F
2-OCH3
4-CF3
32.09 2.79
Gefitinib
Erlotinib
Osimertinib
15.59 1.03 8.37 0.46
16.43 0.96 11.85 0.69 13.12 0.97
5.32 0.43 0.98 0.01
10.78 0.45
24.05 1.93 24.20 2.12 8.03 0.78
14.75 1.08 16.88 1.36 11.98 1.12
e
a
8s
8t
8u
8v
8w
4-CH3
4-CH2CH3
e
>40
35.49 2.67 19.25 1.79
10.34 0.98
25.21 2.12 14.10 1.34
Values are the mean SD of three independent measurements in duplicates.
12.14 0.83 7.22 0.49
>40
6.54 0.59
7.86 0.20
3-CF3
4.04 0.34
7.79 0.45
1.94 0.14
5.67 0.46
Table 3
In vitro antitumor activity of 18a-18n against different cancer cell lines.
Gefitinib
15.59 1.03 8.37 0.46
10.78 0.45
Erlotinib
Osimertinib
16.43 0.96 11.85 0.69 13.12 0.97
5.32 0.43 0.98 0.01
e
a
Values are the mean SD of three independent measurements in duplicates.
Compounds
R
IC50
A549
(m
M)a
to osimertinib, which may be the reason for its potent anti-
proliferative activity against H1975 cell line. These results sug-
gested that substitution of morpholine for a sulfamoylphenyl group
A431
H1975
18a
18b
18c
18d
18e
18f
3-Cl, 4-F
3 ꢁ C≡CH
3-CF3
3-CF3, 4-Cl
3-Br, 4-F
3-Br
4.18 0.12
3.87 0.15
9.05 0.31
30.37 1.32 21.25 1.33 39.52 1.22
5.40 0.54
9.36 0.48
16.72 0.67 11.37 0.16
12.51 0.16 12.67 0.13
16.76 0.13 4.25 0.21
could promote the inhibitory effect on EGFRT790M
.
2.4. CA inhibition study
21.08 1.07 9.43 0.07
17.29 0.75 10.76 0.31
18g
18h
18i
18j
18k
18l
4-CF3
2-F, 3-Cl
3-F
3-Cl
3-CH3
32.36 1.35 26.71 1.21 13.01 0.17
Since sulfonamides and their isosteres were discovered as
potent carbonic anhydrase inhibitors (CAIs), some representative
sulfonamide derivatives were tested to study their inhibition on
cytosolic human carbonic anhydrase isoforms (CAII) and tumor
associated isoforms (CAIX) using acetazolamide as positive control.
The results are given in Table 4. Among the tested derivatives, 8v
exhibited the most potent inhibition on CAIX with an IC50 value of
115.0 nM comparable to that of acetazolamide, and showed the
highest selectivity index (2.4) as well. It was noted that acetazol-
amide had a much stronger inhibitory effect on CAII than 8v with a
small selectivity index of 0.52, hinting its off-target disadvantage.
As for other derivatives, 18c showed stronger CA (CAII and CAIX)
inhibitory activity than 15c, which may be attributed to a longer
carbon chain that enhanced the binding affinity of 18c to CA.
4.53 0.41
5.51 0.36
2.77 0.35
7.05 0.58
4.21 0.07
9.94 0.34
14.84 0.95 11.01 0.29
15.99 0.55 20.82 0.61
13.04 0.62
35.66 0.23
4-CH3
4-CH2CH3
14.66 0.61 13.11 0.76 10.22 0.03
16.62 0.52 39.61 2.07 9.36 0.12
18 m
18n
6.63 0.37
5.77 0.12
5.16 0.05
Gefitinib
Erlotinib
Osimertinib
15.59 1.03 8.37 0.46
10.78 0.45
16.43 0.96 11.85 0.69 13.12 0.97
5.32 0.43 0.98 0.01
e
a
Values are the mean SD of three independent measurements in duplicates.
reference. The results revealed that only a few apoptotic cells were
present in the control group (6.32%) (Fig. 2). Gratifyingly, a dose-
dependent increase in the percentage of apoptotic cells was
observed after being treated with 8v. The apoptosis ratio rose to
2.5. Induced apoptotic cell death of 8v
According to the results of cytotoxicity assay and enzymatic
inhibitory activity, apoptotic analysis of 8v against H1975 cells were
carried out by an Annexin VFITC/PI assay using gefitinib as
29.4% after treatment with 2
mM of 8v for 24 h and further
increased to 40.7% after treatment with 5
mM of 8v, which was
5

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
Table 4
dose-dependent manner (47.44% for 2 mM and 54.85% for 5 mM),
when compared to the untreated control group (14.74%). Also,
gefitinib arrested the cell cycle at the G2/M phase by 33.72% at
In vitro enzymatic inhibitory activity of representative compounds against different
statues of EGFRWT, EGFRT790M, hCAII and hCAIX.
Compounds
IC50 (nM)a
EGFRWT
2
mM. Cell population at the G2/M increased in comparison to
EGFRT790M
hCAII
hCAIX
SIb
control cell. These results revealed that in H1975 cells, compound
8v evidently arrested the G2/M phase of the cell cycle in a dose-
dependent manner.
8a
8b
18.3 5.2
13.7 4.0
61.9 12.0
37.5 8.2
e
e
e
355.8 61.1 224.3 45.5 1.6
8c
8 m
49.2 12.3 48.5 7.4
34.8 11.5 18.0 3.5
e
e
e
e
e
e
8v
8w
27.0 6.8
16.5 3.7
9.2 2.1
22.8 5.0
278.2 31.6 115.0 16.8 2.4
e
e
e
2.7. Inhibition of migration
15c
51.2 10.4 135.0 15.2 526.2 88.3 577.5 94.6 0.9
18c
18n
Gefitinib
Osimertinib
Acetazolamide
42.6 8.5
28.4 4.7
17.1 4.2
58.2 12.6 8.1 2.2
e
93.4 9.8
66.7 5.5
378.4 56.8
241.5 43.7 312.8 55.7 0.8
To study the effect of compound 8v on tumor cell migration, we
performed wound healing assays in H1975 cell line, in which 8v at
e
e
e
e
e
e
e
e
e
0.5 and 1.0
the migration rate decreased significantly with the increase of the
concentration of 8v. After treatment with 1.0 M of 8v, the migra-
mM was used to reduce cell killing. As shown in Fig. 4,
e
45.1 7.4
87.2 9.6
0.5
a
Values are the mean SD of three independent measurements in duplicates.
Selectivity Index (SI) ¼ hCAII IC50 value/hCAIX IC50 value.
m
b
tion rate decreased from 51.2% to 17.9%. The results indicated that
compound 8v effectively inhibited the migration in H1975 cells.
significantly higher than that of treatment with 2 mM of gefitinib
(13.22%). Overall, the results above evidently verified that com-
pound 8v effectively induced apoptosis in H1975 cells.
2.8. Western blot analysis of 8v
2.6. Effect on cell cycle arrest of 8v
The selective inhibition of the representative compound 8v for
the phosphorylation of EGFR and the downstream signaling
transduction (AKT and ERK) was studied by the Western blot assay.
As showed in Fig. 5, the results clearly showed that after 8v treat-
ment, p-EGFR, p-AKT and p-ERK expression levels were decreased
obviously in a dose dependent manner. Interestingly, compound 8v
inhibited the phosphorylation of EGFR and downstream signaling
The cell cycle is controlled by a series of checkpoints, enabling
proliferation only in the presence of a stimulatory signal. The
anticancer agents can arrest the cell cycle at these checkpoints [23].
In order to investigate the effect of compound 8v on cell cycle ar-
rest, the cycle distribution of H1975 cells treated with different
concentrations of 8v after 24 h was analyzed by flow cytometry
with untreated cells as negative control and gefitinib-treated cells
as positive control. The obtained data (Fig. 3) obviously indicated
that compound 8v arrested the cell cycle at the G2/M phase in a
transduction more potently than gefitinib did at 2
mM. These
findings indicated that compound 8v could specifically target EGFR
and inhibit its related downstream phosphorylated protein
expression.
Fig. 2. Induction of apoptosis at 24 h by gefitinib and 8v in H1975 cells. The cells were harvested and labeled with annexin-V-FITC and PI, and analyzed by flow cytometry. Data are
expressed as the mean SEM for three independent experiments.
6

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
Fig. 3. Effects of gefitinib and 8v on cell cycle arrest in H1975 cells. Cells were treated with 2 and 5
content by flow cytometry.
mM of 8v for 24 h. Then the cells were fixed and stained with PI to analyze DNA
Fig. 4. Cell migration inhibition and migration rates of H1975 cells after treated with 0.5 and 1.0 mM of 8v, respectively, for 24 h.
2.9. Cytotoxic activity and Western blot analysis of 8v under
hypoxia
results against H1975 cells under hypoxic and normoxic conditions.
These findings motivated us to further explore the antiproliferative
mechanism of compound 8v under hypoxia.
In order to ascertain the anti-proliferative potential of com-
pound (8v) under hypoxic condition, A549 and H1975 cancer cells
were treated with 8v at the indicated concentrations for 72 h of
incubation. The hypoxic condition was achieved by incubating the
cells under a hypoxic atmosphere (1% O2, 5% CO2, and N2 94%). As
shown in Table 5, 8v caused about 3 times increase in anticancer
activity towards A549 under the hypoxic condition compared with
that under the normoxic condition, and the anticancer activity of 8v
towards H1975 cells was about 2 times better than that of under
normoxic condition. Interestingly, osimertinib presented converse
To further evaluate the effect of 8v on the expression of CAIX, we
performed Western blot analysis of the compound towards
H1975 cells under hypoxic condition using acetazolamide as posi-
tive control. As shown in Fig. 6, acetazolamide successfully inhibi-
ted the expression of CAIX at 2 mM under hypoxic condition. As
expected, 8v also significantly down-regulated the expression level
of CAIX protein in a dose dependent manner. Since CAIX expression
on the cell surface is regulated by HIF-1
expression of HIF-1 was also determined. Interestingly, like CAIX,
8v obviously down-regulated the expression level of HIF-1 protein
a, the effect of 8v on the
a
a
7

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
Fig. 5. Western blot and statistical analysis of the expression level of EGFR, p-EGFR, AKT, p-AKT, ERK and p-ERK in H1975 cells induced by gefitinib and 8v based on three in-
dependent experiments.
docking study by the PYMOL software with EGFRWT protein (PDB
Table 5
code:2ITY) [24] and EGFRT790M mutant protein (PDB code:2JIU)
Cytotoxic effects of 8v against A549 and H1975 cells under hypoxic or normoxic
conditions.
[25]. In the EGFRWT binding model (Fig. 7), the nitrogen atoms in
Compounds
Hypoxia IC50
A549
(m
M)a
H1975
Normoxia IC50
A549
(m
M)a
quinazoline of 8v can bind to Met793 residue by hydrogen bonds,
which is consistent with the binding site of gefitinib in EGFR
domain. Besides, the sulfa warhead of 8v forms hydrogen bonding
with the residue Lys-745 in EGFRWT, due to the U-shaped structure.
In EGFRT790M, 8v maintains the same binding pose as in EGFRWT
(Fig. 7). As expected, the quinazoline core forms the classical
hydrogen bond interactions with Met793. Interestingly, the sulfa
warhead of 8v forms more hydrogen bonds with Arg-841, Asp-855
and Lys-745 in EGFRT790M than those in EGFRWT, which lead to the
binding force of 8v and kinase EGFRT790M more closely and firmly.
This may be the reason that 8v had strong inhibitory activity and
selectivity to EGFRT790M kinase.
H1975
8v
2.21 0.09
e
1.05 0.08
2.08 0.03
6.54 0.59
e
1.94 0.14
0.98 0.01
Osimertinib
a
Values are the mean SD of three independent measurements in duplicates.
in a dose dependent manner. These results indicated that intro-
duction of sulfonamides moieties gave 8v the ability to inhibit the
expression of CAIX and upstream HIF-1a protein.
For the purpose of exploring the binding interaction of com-
pound 8v within CAIX active site, co-crystallization of CAIX protein
with acetazolamide (PDB code:3IAI) was selected. As shown in
Fig. 8, major interactions of 8v with the enzyme active site are
mediated through the deeply buried sulfonamide group, which
make residues locate at the bottom of the active site cavity (Thr199
and Thr200), similar to the binding site of acetazolamide in CAIX
2.10. Molecular docking study of 8v
In order to better illustrate the structural basis of our EGFR and
CAIX dual inhibitors, molecular docking was performed on the
basis of co-crystallization of EGFRWT, EGFRT790M and CAIX.
According to the in vitro antiproliferation activity of the target
compounds, compound 8v was selected to conduct molecular
Fig. 6. Western blot and statistical analysis of the expression level of CAIX and HIF-1
a
in H1975 cells under hypoxic condition. Data represent three individual experiments.
8

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
Fig. 7. 3D model of 8v bound to EGFRWT and EGFRT790M. (A) 3D model of 8v bound to EGFRWT (PDB code:2ITY). (B) 3D model of 8v bound to EGFRT790M (PDB code:2JIU).
Fig. 8. 3D model of 8v bound to CAIX (PDB code:3IAI).
domain. In addition, the oxygen atom of the amide bond forms two
additional hydrogen bonds with residue Asn62 and His64, which
might enhance the binding affinity of 8v to CAIX protein.
These docking results suggest that compound 8v could suc-
cessfully bind to EGFRWT, EGFRT790M and CAIX protein. Besides, the
binding of 8v to EGFRT790M is more favorable than its binding to
9

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
EGFRWT, which is in good agreement with the experimental results.
Upon the molecular docking diagram, we can conclude that the
design idea of EGFR and CAIX dual inhibitors are rational.
The precipitate was purified on silica gel column eluted with DCM/
MeOH (20:1 v/v) to give compound 7 (412 mg, yield 58.6%) as a
white solid. 1H NMR (600 MHz, DMSO‑d6)
d 10.25 (s, 1H), 7.74 (s,
4H), 7.24 (s, 2H), 3.56 (t, J ¼ 6.6 Hz, 2H), 2.39 (t, J ¼ 7.3 Hz, 2H),
3. Conclusions
1.87e1.82 (m, 2H), 1.74e1.69 (m, 2H).
Three different series of quinazoline derivatives bearing
benzene-sulfonamide moiety were prepared and biologically
evaluated as dual EGFR/CAIX inhibitors. All compounds were
examined for their anti-proliferative activity against human
epidermoid carcinoma (A431) and non-small cell lung cancer
(A549 and H1975) cell lines. Among the synthesized compounds,
8v exhibited comparable activity to osimertinib against mutant-
type non-small cell lung cancer cell line H1975. Furthermore, 8v
displayed stronger anti-proliferative activity than osimertinib
against H1975 cells under hypoxic condition. The SAR studies
revealed that introduction of a trifluoromethyl group at 3-positon
of the aryl group and the increase in the length of the carbon
chain between two pharmacophores were beneficial to promote
the antitumor activity of the resulting compound. Several repre-
sentative compounds exhibited good to excellent inhibitory activ-
ities against EGFRWT and EGFRT790M enzymes. It is worth noting
that 8v showed excellent inhibitory effect on EGFRT790M, which was
41 times more effective than gefitinib and almost equal to osi-
mertinib. Mechanism study indicated that 8v exhibited remarkable
CAIX inhibitory effect comparable to acetazolamide, and it could
significantly inhibit the expression of p-EGFR and its downstream
p-AKT and p-ERK in H1975 cells. More importantly, 8v could also
4.2.2. Synthesis of 6-hydroxyl-7-methoxy-4-arylaminoquinazolines
(4a-w)
Compounds 4a-w were prepared according to reported pro-
cedures [20].
4.2.3. Synthesis of 5-((7-methoxy-4-((substituted phenyl)amino)
quinazolin-6-yl)oxy)- N-(4-sulfamoylphenyl)pentanamides (8a-w)
A mixture of intermediates 4a-w (0.25 mmol) and K2CO3
(69 mg, 0.5 mmol) in anhydrous DMF (5 mL) was stirred at room
temperature. Then KI (9 mg, 0.05 mmol) and compound 7 (100 mg,
0.3 mmol) were added to the mixture and stirred at 60 ꢀC over-
night. After completion of reaction, the mixture was treated with
water and extracted with dichloromethane. The combined organic
layers were washed with water and brine, dried over Na2SO4, and
concentrated. The residue was purified on silica gel column eluted
with DCM/MeOH (20:1 v/v) to give the desired products 8a-w.
4.2.3.1.
quinazolin-6-yl)oxy)- N-(4-sulfamoylphenyl)pentanamide (8a). Yel-
low solid (yield: 61.5%). 1H NMR (600 MHz, DMSO‑d6)
10.29 (s, 1H),
5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxy
d
9.52(s,1H), 8.50(s,1H), 8.12 (d, J ¼ 4.8 Hz,1H), 7.80 (s, 2H), 7.76 (s, 4H),
7.44 (t, J ¼ 9.0 Hz,1H), 7.26 (s, 2H), 7.19 (s,1H), 4.23e4.11 (m, 2H), 3.93
(s, 3H), 2.50e2.42 (m, 2H), 1.95e1.77 (m, 4H). 13C NMR (150 MHz,
successfully inhibit the expression of CAIX and its upstream HIF-1
a
DMSO‑d6)
d
171.68, 156.01, 154.51, 153.17 (d, J ¼ 241.5 Hz), 152.63,
in H1975 cells under hypoxic condition. Molecular docking studies
illustrated the binding pattern of 8v with EGFRWT, EGFRT790M and
CAIX proteins, which validated the rational of our design for EGFR
and CAIX dual inhibitors. Therefore, our research offers a promising
way for development of potent multi-target anticancer agents.
148.36, 146.96, 142.20, 138.12, 136.82 (d, J ¼ 2.9 Hz), 126.71, 123.51,
122.34 (d, J ¼ 6.9 Hz), 118.80 (d, J ¼ 18.3 Hz), 118.56, 116.53 (d,
J ¼ 21.5 Hz), 108.78, 107.29, 102.43, 68.52, 55.89, 36.05, 28.20, 21.76.
HRMS (m/z) (ESI): calcd for C26H26ClFN5O5S [MþH]þ: 574.1322;
found: 574.1321.
4.2.3.2. 5-((4-((4-chloro-3-(trifluoromethyl)phenyl)amino)-7-
4. Experimental
methoxyquinazolin
mide (8b). Yellow solid (yield: 68.0%). 1H NMR (600 MHz, DMSO‑d6)
10.31 (s, 1H), 9.72 (s, 1H), 8.54 (s, 1H), 8.38 (s, 1H), 8.28 (d,
-6-yl)oxy)-N-(4-sulfamoylphenyl)pentana-
4.1. Materials and instruments
d
J ¼ 8.0 Hz, 1H), 7.82 (s, 1H), 7.75e7.71 (m, 5H), 7.26e7.21 (m, 3H),
All chemical reagents and solvents used in experiments were
purchased from commercial suppliers and used without further
purification. Thin-layer chromatography (TLC) used to monitor
progress of the reaction and column chromatography was per-
formed using silica gel (200e300 mesh). 1H and 13C NMR spectra
were recorded with a Bruker spectrometer at 600 and 150 MHz,
respectively. Mass spectra were measured by an Agilent 6224 ESI/
TOF MS instrument. The purity of final compounds was analyzed by
RP-HPLC (Waters e2695) using gradient elution with an eluent of
methanol/water (20:80 to 100:0, V/V) (0.1% trifluoroacetic acid in
water). All of the compounds submitted for biological studies were
at least 95%. Besides, all cells used in this article were obtained from
KeyGEN BioTECH Corp. Recombinant human carbonic anhydrase II
and IX were purchased from Sino Biological Inc. Antibodies directed
4.23e4.18 (m, 2H), 3.94 (s, 3H), 2.50e2.47 (m, 2H), 1.94e1.89 (m,
2H), 1.84e1.77 (m, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 171.71,
155.81, 154.69, 152.44, 148.53, 147.16, 142.22, 139.28, 138.13, 131.72,
126.73, 126.54, 126.34, 123.42, 122.96 (d, J ¼ 270.0 Hz), 120.21 (d,
J ¼ 6.0 Hz), 118.57, 108.99, 107.32, 102.40, 68.58, 55.96, 36.06, 28.22,
21.79. HRMS (m/z) (ESI): calcd for C27H26ClF3N5O5S [MþH]þ:
624.1290; found: 624.1281.
4.2.3.3. 5-((4-((3-ethynylphenyl)amino)-7-methoxyquinazolin-
6-yl)oxy)-N-(4- sulfamoylphenyl)pentanamide (8c). Yellow solid
(yield: 67.4%). 1H NMR (600 MHz, DMSO‑d6)
d 10.28 (s, 1H), 9.48 (s,
1H), 8.50 (s, 1H), 7.99 (s, 1H), 7.90 (dd, J ¼ 8.3, 1.2 Hz, 1H), 7.84 (s,
1H), 7.77e7.74 (m, 4H), 7.40 (t, J ¼ 7.9 Hz, 1H), 7.25 (s, 2H), 7.21 (d,
J ¼ 7.6 Hz, 1H), 7.20 (s, 1H), 4.20 (s, 1H), 4.19 (t, J ¼ 6.3 Hz, 2H), 3.93
(s, 3H), 2.48 (t, J ¼ 7.2 Hz, 2H), 1.92e1.88 (m, 2H), 1.86e1.81 (m, 2H).
against p-EGFR, EGFR, p-MEK, MEK, p-ERK, ERK, CAIX and HIF-1
were purchased from Abcam.
a
13C NMR (150 MHz, DMSO‑d6)
d 171.68, 156.12, 154.47, 152.72,
148.33, 147.00, 142.20, 139.83, 138.12, 128.92, 126.71, 126.38, 124.84,
122.66, 121.76, 118.56, 108.92, 107.30, 102.53, 83.53, 80.59, 68.52,
55.89, 36.04, 28.19, 21.76. HRMS (m/z) (ESI): calcd for C28H28N5O5S
[MþH]þ: 546.1806; found: 546.1824.
4.2. General methods for preparation of compounds
4.2.1. Synthesis of 5-bromo-N-(4-sulfamoylphenyl)pentanamide (7)
4.2.3.4.
5-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxy
To a solution of compound 5 (344 mg, 2.0 mmol) in dry DMF
(10 mL), pyridine (316 mg, 4.0 mmol) was added. After stirred at
0
reaction was stirred at room temperature overnight and monitored
by TLC. After completion of reaction, the reaction mixture was
poured into water (250 mL) with stirring to give a white precipitate.
quinazolin-6-yl)oxy) -N-(4-sulfamoylphenyl)pentanamide (8d).
Yellow solid (yield: 65.3%). 1H NMR (600 MHz, DMSO‑d6)
d
10.29 (s,
ꢀC for 5 min, compound 6 (598 mg, 3.0 mmol) was added. The
1H), 9.51 (s, 1H), 8.52 (s, 1H), 8.15 (s, 1H), 7.89 (d, J ¼ 8.1 Hz,1H), 7.84
(s, 1H), 7.77e7.74 (m, 4H), 7.35 (t, J ¼ 8.0 Hz, 1H), 7.28 (d, J ¼ 8.7 Hz,
1H), 7.26 (s, 2H), 7.20 (s, 1H), 4.19 (t, J ¼ 6.1 Hz, 2H), 3.93 (s, 3H), 2.48
(t, J ¼ 7.2 Hz, 2H), 1.92e1.87 (m, 2H), 1.85e1.81 (m, 2H). 13C NMR
10

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
(150 MHz, DMSO‑d6)
d
171.69, 155.99, 154.53, 152.64,148.39, 147.04,
J ¼ 9.0 Hz, 2H), 7.25 (s, 2H), 7.16 (s, 1H), 6.97 (d, J ¼ 9.0 Hz, 2H), 4.17
(t, J ¼ 6.2 Hz, 2H), 3.92 (s, 3H), 3.77 (s, 3H), 2.50e2.45 (m, 2H),
142.21, 141.31, 138.12, 130.39, 126.72, 125.63, 124.07, 121.21, 120.64,
118.56, 108.95, 107.30, 102.48, 68.54, 55.91, 36.04, 28.20, 21.77.
HRMS (m/z) (ESI): calcd for C26H27BrN5O5S [MþH]þ: 600.0911;
found: 600.0848.
1.90e1.80 (m, 4H). 13C NMR (150 MHz, DMSO‑d6)
d 171.69, 156.70,
155.78, 154.25, 152.92, 148.10, 146.31, 142.21, 138.11, 132.11, 126.70,
124.62, 118.56, 113.67, 108.66, 106.98, 102.77, 68.49, 55.83, 55.24,
36.03, 28.17, 21.76. HRMS (m/z) (ESI): calcd for C27H30N5O6S
[MþH]þ: 552.1911; found: 552.1881.
4.2.3.5. 5-((4-((3-chlorophenyl)amino)-7-methoxyquinazolin-
6-yl)oxy)-N-(4- sulfamoylphenyl)pentanamide (8e). Yellow solid
(yield: 64.5%). 1H NMR (600 MHz, DMSO‑d6)
d
10.30 (s, 1H), 9.54 (s,
4.2.3.11.
methoxyquinazolin-6-yl)oxy)-
mide (8k). Yellow solid (yield: 63.0%). 1H NMR (600 MHz, DMSO‑d6)
10.29 (s, 1H), 9.39 (s, 1H), 8.41 (s, 1H), 7.84 (s, 1H), 7.78e7.73 (m,
5-((4-((3,4-dimethoxyphenyl)amino)-7-
1H), 8.53 (s, 1H), 8.04 (s, 1H), 7.85 (s, 1H), 7.82 (d, J ¼ 8.3 Hz, 1H),
2.78e7.74 (m, 4H), 7.41 (t, J ¼ 8.1 Hz, 1H), 7.26 (s, 2H), 7.21 (s, 1H),
7.15 (d, J ¼ 7.1 Hz, 1H), 4.19 (t, J ¼ 6.0 Hz, 2H), 3.94 (s, 3H), 2.50e2.45
N-(4-sulfamoylphenyl)pentana-
d
(m, 2H), 1.91e1.82 (m, 4H). 13C NMR (150 MHz, DMSO‑d6)
d
171.69,
4H), 7.36 (d, J ¼ 2.3 Hz, 1H), 7.30e7.28 (m, 1H), 7.24 (s, 2H), 7.16 (s,
156.02, 154.54, 152.63, 148.39, 147.01, 142.21, 141.16, 138.12, 132.75,
130.07, 126.71, 122.75, 121.29, 120.24, 118.56, 108.95, 107.28, 102.51,
68.55, 55.91, 36.04, 28.19, 21.77. HRMS (m/z) (ESI): calcd for
1H), 6.98 (d, J ¼ 8.7 Hz, 1H), 4.18 (t, J ¼ 5.8 Hz, 2H), 3.92 (s, 3H), 3.78
(s, 3H), 3.77 (s, 3H), 2.47 (t, J ¼ 6.7 Hz, 2H), 1.91e1.83 (m, 4H). 13
C
NMR (150 MHz, DMSO‑d6)
d 171.66, 156.64, 154.24, 152.93, 148.44,
C
26H27ClN5O5S [MþH]þ: 556.1416; found: 556.1399.
148.09, 146.48, 145.40, 142.18, 138.11, 132.58, 126.67, 118.54, 115.21,
111.80, 108.73, 108.17, 107.09, 102.78, 68.52, 55.81, 55.77, 55.61,
36.02, 28.17, 21.75. HRMS (m/z) (ESI): calcd for C28H32N5O7S
[MþH]þ: 582.2017; found: 582.1990.
4.2.3.6. 5-((4-((3-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(4- sulfamoylphenyl)pentanamide (8f). Yellow solid
(yield: 70.5%). 1H NMR (600 MHz, DMSO‑d6)
d
10.31 (s, 1H), 9.57 (s,
1H), 8.52 (s, 1H), 7.90 (d, J ¼ 11.9 Hz, 1H), 7.86 (s, 1H), 7.78e7.73 (m,
4H), 7.62 (d, J ¼ 7.9 Hz, 1H), 7.43e7.39 (m, 1H), 7.26 (s, 2H), 7.21 (s,
1H), 6.93e6.90 (m, 1H), 4.20 (t, J ¼ 6.0 Hz, 2H), 3.94 (s, 3H),
2.50e2.47 (m, 2H),1.91e1.81 (m, 4H). 13C NMR (150 MHz, DMSO‑d6)
4.2.3.12.
methoxyquinazolin-6-yl)oxy)
mide (8l). Yellow solid (yield: 64.5%). 1H NMR (600 MHz, DMSO‑d6)
5-((4-((3-bromo-4-fluorophenyl)amino)-7-
-N-(4-sulfamoylphenyl)pentana-
d
10.28 (s, 1H), 9.53 (s, 1H), 8.50 (s, 1H), 8.21 (dd, J ¼ 6.4, 2.6 Hz, 1H),
d
171.70, 162.07 (d, J ¼ 238.5 Hz), 156.07, 154.53, 152.64, 148.38,
7.89e7.85 (m, 1H), 7.82 (s, 1H), 7.75 (s, 4H), 7.41 (t, J ¼ 8.8 Hz, 1H),
7.24 (s, 2H), 7.20 (s, 1H), 4.18 (t, J ¼ 5.9 Hz, 2H), 3.93 (s, 3H),
2.50e2.47 (m, 2H), 1.93e1.77 (m, 4H). 13C NMR (150 MHz,
147.05, 142.22, 141.46 (d, J ¼ 10.5 Hz), 138.12, 129.91 (d, J ¼ 9.0 Hz),
126.72, 118.57, 117.53 (d, J ¼ 3.0 Hz), 109.50 (d, J ¼ 21.0 Hz), 108.99,
108.64 (d, J ¼ 25.5 Hz), 107.31, 102.57, 68.57, 55.91, 36.04, 28.20,
21.78. HRMS (m/z) (ESI): calcd for C26H27FN5O5S [MþH]þ: 540.1711;
found: 540.1677.
DMSO‑d6)
d
171.63, 155.42, 155.26 (d, J ¼ 224.4 Hz), 153.02, 152.59,
148.34, 146.89, 142.17, 138.10, 137.03 (d, J ¼ 4.1 Hz), 126.66, 126.21,
123.03 (d, J ¼ 6.8 Hz), 118.53, 116.32 (d, J ¼ 22.8 Hz), 108.75, 107.28,
107.16 (d, J ¼ 27.6 Hz), 102.48, 68.52, 55.87, 36.01, 28.16, 21.72.
HRMS (m/z) (ESI): calcd for C26H26BrFN5O5S [MþH]þ: 618.0817;
found: 618.0824.
4.2.3.7. 5-((4-((4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)oxy)-N-(4- sulfamoylphenyl)pentanamide (8g). Yellow solid
(yield: 65.5%). 1H NMR (600 MHz, DMSO‑d6)
d 10.30 (s, 1H), 9.52 (s,
1H), 8.43 (s, 1H), 7.84 (s, 1H), 7.79e7.74 (m, 6H), 7.26 (s, 2H), 7.23 (t,
J ¼ 8.8 Hz, 2H), 7.18 (s, 1H), 4.18 (t, J ¼ 6.1 Hz, 2H), 3.93 (s, 3H),
2.50e2.46 (m, 2H),1.91e1.81 (m, 4H). 13C NMR (150 MHz, DMSO‑d6)
4.2.3.13.
methoxyquinazolin-6-yl)oxy)
mide (8m). Yellow solid (yield: 67.8%). 1H NMR (600 MHz,
DMSO‑d6) 10.30 (s, 1H), 9.65 (s, 1H), 8.38 (s, 1H), 7.82 (s, 1H),
5-((4-((3-chloro-2-fluorophenyl)amino)-7-
-N-(4-sulfamoylphenyl)pentana-
d
171.70, 159.18, 157.59, 156.45, 153.60 (d, J ¼ 237.0 Hz), 148.24,
d
146.71, 142.22, 138.12, 135.67, 126.72, 124.53 (d, J ¼ 7.5 Hz), 118.57,
115.05 (d, J ¼ 22.5 Hz), 108.74, 107.17, 102.66, 68.52, 55.87, 36.04,
28.19, 21.77. HRMS (m/z) (ESI): calcd for C26H27FN5O5S [MþH]þ:
540.1711; found: 540.1657.
7.77e7.74 (m, 4H), 7.52 (t, J ¼ 7.1 Hz, 1H), 7.48 (t, J ¼ 7.0 Hz, 1H), 7.28
(t, J ¼ 8.1 Hz, 1H), 7.25 (s, 2H), 7.21 (s, 1H), 4.17 (t, J ¼ 6.2 Hz, 2H),
3.94 (s, 3H), 2.48 (t, J ¼ 7.7 Hz, 2H), 1.91e1.87 (m, 2H), 1.84e1.80 (m,
2H). 13C NMR (150 MHz, DMSO‑d6)
d 171.68, 156.90, 153.77 (d,
4.2.3.8. 5-((4-((4-chlorophenyl)amino)-7-methoxyquinazolin-
6-yl)oxy)-N-(4- sulfamoylphenyl)pentanamide (8h). Yellow solid
J ¼ 240.0 Hz), 153.33, 151.68, 148.34, 146.93, 142.21, 138.11, 128.40
(d, J ¼ 11.9 Hz), 127.18, 126.98, 126.71, 124.94 (d, J ¼ 4.4 Hz), 120.18
(d, J ¼ 16.5 Hz), 118.56, 108.66, 107.13, 102.56, 68.41, 55.91, 36.02,
28.14, 21.73. HRMS (m/z) (ESI): calcd for C26H26ClFN5O5S [MþH]þ:
574.1322; found: 574.1343.
(yield: 73.2%). 1H NMR (600 MHz, DMSO‑d6)
d 10.29 (s, 1H), 9.54 (s,
1H), 8.48 (s, 1H), 7.87e7.84 (m, 3H), 7.77e7.74 (m, 4H), 7.45e7.43
(m, 2H), 7.25 (s, 2H), 7.19 (s, 1H), 4.18 (t, J ¼ 6.2 Hz, 2H), 3.93 (s, 3H),
2.48 (t, J ¼ 7.4 Hz, 2H), 1.92e1.80 (m, 4H). 13C NMR (150 MHz,
4.2.3.14.
5-((7-methoxy-4-((4-(trifluoromethoxy)phenyl)
DMSO‑d6)
d
171.68, 156.15, 154.48, 152.67, 148.32, 146.86, 142.20,
amino)quinazolin-6- yl)oxy)-N-(4-sulfamoylphenyl)pentanamide
138.50, 138.12, 128.33, 126.91, 126.71, 123.76, 118.56, 108.90, 107.22,
(8n). Yellow solid (yield: 69.3%). 1H NMR (600 MHz, DMSO‑d6)
102.60, 68.53, 55.90, 36.04, 28.19, 21.76. HRMS (m/z) (ESI): calcd for
C
d
10.28 (s, 1H), 9.59 (s, 1H), 8.48 (s, 1H), 7.92 (d, J ¼ 9.0 Hz, 2H), 7.85
26H27ClN5O5S [MþH]þ: 556.1416; found: 556.1381.
(s, 1H), 7.78e7.73 (m, 4H), 7.39 (d, J ¼ 8.7 Hz, 2H), 7.24 (s, 2H), 7.20
(s, 1H), 4.19 (t, J ¼ 5.7 Hz, 2H), 3.94 (s, 3H), 2.47 (d, J ¼ 6.8 Hz, 2H),
4.2.3.9. 5-((4-((4-bromophenyl)amino)-7-methoxyquinazolin-
6-yl)oxy)-N-(4- sulfamoylphenyl)pentanamide (8i). Yellow solid
1.91e1.83 (m, 4H). 13C NMR (150 MHz, DMSO‑d6)
d 171.64, 156.17,
(yield: 68.3%). 1H NMR (600 MHz, DMSO‑d6)
d
10.29 (s, 1H), 9.53 (s,
154.51, 152.65, 148.32, 146.94, 143.65, 142.17, 138.74, 138.11, 126.67,
123.55, 121.22, 120.21 (d, J ¼ 254.0 Hz), 118.54, 108.86, 107.24,
102.64, 68.54, 55.87, 36.01, 28.16, 21.73. HRMS (m/z) (ESI): calcd for
1H), 8.48 (s, 1H), 7.85 (s, 1H), 7.82 (d, J ¼ 8.7 Hz, 2H), 7.77e7.74 (m,
4H), 7.56 (d, J ¼ 8.7 Hz, 2H), 7.25 (s, 2H), 7.19 (s, 1H), 4.18 (t,
J ¼ 6.0 Hz, 2H), 3.93 (s, 3H), 2.50e2.46 (m, 2H), 1.90e1.80 (m, 4H).
C
27H27F3N5O6S [MþH]þ: 606.1629; found: 606.1607.
13C NMR (150 MHz, DMSO‑d6)
d
171.67, 156.09, 154.48, 152.65,
4.2.3.15. 5-((7-methoxy-4-(m-tolylamino)quinazolin-6-yl)oxy)-
148.32, 146.88, 142.20, 138.95, 138.11, 131.22, 126.70, 124.07, 118.55,
114.92, 108.92, 107.22, 102.60, 68.53, 55.89, 36.03, 28.18, 21.75.
HRMS (m/z) (ESI): calcd for C26H27BrN5O5S [MþH]þ: 600.0911;
found: 600.0835.
N-(4- sulfamoylphenyl)pentanamide (8o). Yellow solid (yield:
67.7%). 1H NMR (600 MHz, DMSO‑d6)
d
10.29 (s, 1H), 9.42 (s, 1H),
8.45 (s, 1H), 7.87 (s, 1H), 7.78e7.73 (m, 4H), 7.63 (d, J ¼ 8.2 Hz, 1H),
7.58 (s, 1H), 7.29e7.24 (m, 3H), 7.18 (s, 1H), 6.94 (d, J ¼ 7.5 Hz, 1H),
4.19 (t, J ¼ 5.8 Hz, 2H), 3.93 (s, 3H), 2.50e2.45 (m, 2H), 2.34 (s, 3H),
4.2.3.10. 5-((7-methoxy-4-((4-methoxyphenyl)amino)quinazo-
lin-6-yl)oxy)-N-(4- sulfamoylphenyl)pentanamide (8j). Yellow
1.91e1.83 (m, 4H). 13C NMR (150 MHz, DMSO‑d6)
d 171.64, 156.42,
solid (yield: 62.7%). 1H NMR (600 MHz, DMSO‑d6)
d
10.30 (s, 1H),
154.35, 152.78, 148.19, 146.55, 142.17, 139.29, 138.10, 137.56, 128.25,
126.66, 124.18, 122.93, 119.69, 118.53, 108.83, 107.06, 102.72, 68.50,
9.44 (s, 1H), 8.39 (s, 1H), 7.85 (s, 1H), 7.77e7.74 (m, 4H), 7.62 (d,
11

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
55.83, 36.01, 28.15, 21.73, 21.16. HRMS (m/z) (ESI): calcd for
C
142.17, 139.39, 138.10, 128.40, 126.66, 123.42, 122.50, 118.53, 108.86,
107.15, 102.76, 68.51, 55.83, 36.00, 28.15, 21.73. HRMS (m/z) (ESI):
calcd for C26H28N5O5S [MþH]þ: 522.1806; found: 522.1769.
4.2.3.22. 5-((7-methoxy-4-((3-(trifluoromethyl)phenyl)amino)
quinazolin-6- yl)oxy)-N-(4-sulfamoylphenyl)pentanamide (8v).
27H30N5O5S [MþH]þ: 536.1962; found: 536.1930.
4.2.3.16. 5-((7-methoxy-4-((3,4,5-trifluorophenyl)amino)quina-
zolin-6-yl)oxy)- N-(4-sulfamoylphenyl)pentanamide (8p). Yellow
solid (yield: 67.0%). 1H NMR (600 MHz, DMSO‑d6)
d
10.28 (s, 1H),
9.56 (s, 1H), 8.55 (s, 1H), 7.90e7.85 (m, 2H), 7.77e7.73 (m, 5H), 7.24
(s, 2H), 7.21 (s, 1H), 4.18 (t, J ¼ 5.9 Hz, 2H), 3.94 (s, 3H), 2.50e2.44
Yellow solid (yield: 61.2%). 1H NMR (600 MHz, DMSO‑d6)
d 10.31 (s,
1H), 9.67 (s, 1H), 8.53 (s, 1H), 8.24e8.21 (m, 2H), 7.86 (s, 1H),
7.80e7.76 (m, 4H), 7.62 (t, J ¼ 7.8 Hz, 1H), 7.44 (d, J ¼ 7.4 Hz, 1H),
7.26e7.22 (m, 3H), 4.20e4.11 (m, 2H), 3.94 (s, 3H), 2.50e2.48 (m,
2H), 1.94e1.89 (m, 2H), 1.85e1.75 (m, 2H). 13C NMR (150 MHz,
(m, 2H), 1.92e1.83 (m, 4H). 13C NMR (150 MHz, DMSO‑d6)
d 171.63,
155.72, 154.67, 152.34, 151.0 (dd, J ¼ 14.7 Hz, 8.6 Hz), 148.6 (dd,
J ¼ 15.5 Hz, 8.8 Hz), 148.48, 147.09, 142.16, 138.11, 136.13 (t,
J ¼ 18.2 Hz), 126.66, 118.53, 108.84, 107.32, 105.75, 105.51, 102.32,
68.57, 55.90, 36.01, 28.17, 21.73. HRMS (m/z) (ESI): calcd for
DMSO‑d6)
d 171.70, 156.05, 154.59, 152.60, 148.45, 147.09, 142.22,
140.50, 138.13, 129.62, 129.23 (d, J ¼ 31.5 Hz), 126.73, 125.48, 124.33
(d, J ¼ 270.0 Hz), 119.31 (d, J ¼ 3.0 Hz), 118.56, 117.86 (d, J ¼ 4.5 Hz),
108.96, 107.31, 102.48, 68.56, 55.93, 36.06, 28.21, 21.79. HRMS (m/z)
(ESI): calcd for C27H27F3N5O5S [MþH]þ: 590.1680; found: 590.1629.
C
26H25F3N5O5S [MþH]þ: 576.1523; found: 576.1471.
4.2.3.17. 5-((7-methoxy-4-((2-methoxyphenyl)amino)quinazo-
lin-6-yl)oxy) -N-(4-sulfamoylphenyl)pentanamide (8q). Yellow
solid (yield: 73.1%). 1H NMR (600 MHz, DMSO‑d6)
d
10.31 (s, 1H),
4.2.3.23.
5-((4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)
9.13 (s, 1H), 8.28 (s, 1H), 7.83 (s, 1H), 7.77e7.73 (m, 4H), 7.49 (d,
J ¼ 7.7 Hz,1H), 7.25e7.23 (m, 3H), 7.15 (s,1H), 7.12 (d, J ¼ 8.3 Hz,1H),
6.99 (t, J ¼ 7.6 Hz, 1H), 4.16 (t, J ¼ 6.3 Hz, 2H), 3.92 (s, 3H), 3.77 (s,
3H), 2.48 (t, J ¼ 7.3 Hz, 2H), 1.89e1.80 (m, 4H). 13C NMR (150 MHz,
amino)-7- methoxyquinazolin-6-yl)oxy)-N-(4-sulfamoylphenyl)pen
tanamide (8w). Yellow solid (yield: 69.1%). 1H NMR (600 MHz,
DMSO‑d6)
d
10.29 (s, 1H), 9.43 (s, 1H), 8.45 (s, 1H), 7.95 (d, J ¼ 2.6 Hz,
1H), 7.81 (s, 1H), 7.77e7.74 (m, 4H), 7.70 (dd, J ¼ 8.9, 2.5 Hz, 1H),
7.49e7.45(m,1H), 7.34e7.31(m, 2H),7.27e7.25(m,3H),7.20e7.16(m,
2H), 5.25 (s, 2H), 4.18 (t, J ¼ 6.2 Hz, 2H), 3.93 (s, 3H), 2.48 (t, J ¼ 7.2 Hz,
2H), 1.92e1.87 (m, 2H), 1.85e1.80 (m, 2H). 13C NMR (150 MHz,
DMSO‑d6)
d 171.70, 157.68, 154.19, 153.85, 153.22, 148.00, 146.61,
142.21, 138.11, 127.88, 127.38, 126.70, 126.57, 120.21, 118.55, 111.81,
108.75, 107.08, 102.80, 68.40, 55.81, 55.51, 36.04, 28.16, 21.74. HRMS
(m/z) (ESI): calcd for C27H30N5O6S [MþH]þ: 552.1911; found:
552.1935.
DMSO‑d6)
d
171.66, 162.22 (d, J ¼ 242.3 Hz), 156.21, 154.36, 152.83,
149.42, 148.22, 146.82, 142.19, 139.71 (d, J ¼ 7.4 Hz), 138.11, 133.53,
130.57 (d, J ¼ 8.3 Hz), 126.69, 124.03, 123.36 (d, J ¼ 2.6 Hz), 122.20,
121.04, 118.54, 114.71 (d, J ¼ 20.7 Hz), 114.32, 114.06 (d, J ¼ 21.8 Hz),
108.72, 107.28, 102.52, 69.40, 68.49, 55.85, 36.03, 28.18, 21.75. HRMS
(m/z) (ESI): calcd for C33H32ClFN5O6S [MþH]þ: 680.1740; found:
680.1670.
4.2.3.18. 5-((7-methoxy-4-((4-(trifluoromethyl)phenyl)amino)
quinazolin-6- yl)oxy)-N-(4-sulfamoylphenyl)pentanamide (8r).
Yellow solid (yield: 68.4%). 1H NMR (600 MHz, DMSO‑d6)
d 10.28 (s,
1H), 9.70 (s, 1H), 8.55 (s, 1H), 8.11 (d, J ¼ 8.5 Hz, 2H), 7.88 (s, 1H),
7.78e7.73 (m, 6H), 7.24 (s, 2H), 7.23 (s, 1H), 4.21 (t, J ¼ 5.9 Hz, 2H),
3.94 (s, 3H), 2.50e2.45 (m, 2H), 1.92e1.84 (m, 4H). 13C NMR
(150 MHz, DMSO‑d6) d 171.64, 155.95, 154.65, 152.49, 148.47, 147.18,
143.41, 142.17, 138.11, 126.67, 125.63 (d, J ¼ 3.0 Hz), 124.55 (d,
J ¼ 270.0 Hz), 122.79 (d, J ¼ 32.0 Hz), 121.44, 118.54, 109.09, 107.29,
102.58, 68.57, 55.91, 36.01, 28.17, 21.74. HRMS (m/z) (ESI): calcd for
4.2.4. Synthesis of 6-amino-4-arylaminoquinazolines (12a-n)
Compounds 12a-n were prepared according to reported pro-
cedures [26].
C
27H27F3N5O5S [MþH]þ: 590.1680; found: 590.1642.
4.2.3.19. 5-((7-methoxy-4-(p-tolylamino)quinazolin-6-yl)oxy)-
N-(4- sulfamoylphenyl) pentanamide (8s). Yellow solid (yield:
66.5%). 1H NMR (600 MHz, DMSO‑d6)
d
10.29 (s, 1H), 9.42 (s, 1H),
4.2.5. 4-Isothiocyanatobenzenesulfonamide (14)
8.42 (s, 1H), 7.86 (s, 1H), 7.78e7.73 (m, 4H), 7.65 (d, J ¼ 8.4 Hz, 2H),
7.24 (s, 2H), 7.19 (d, J ¼ 8.3 Hz, 2H), 7.17 (s, 1H), 4.18 (t, J ¼ 5.9 Hz,
2H), 3.93 (s, 3H), 2.50e2.45 (m, 2H), 2.31 (s, 3H), 1.91e1.81 (m, 4H).
To a solution of sulfanilamide (344 mg, 2.0 mmol) in 10 mL of
water, hydrochloric acid (1 mL) was added slowly. After stirred at
0
ꢀC for 5 min, thiophosgen (253 mg, 2.2 mmol) was added. The
13C NMR (150 MHz, DMSO‑d6)
d 171.64, 156.48, 154.28, 152.86,
solution was kept stirring at 0 ꢀC for 2 h and monitored by TLC. After
completion of reaction, the reaction mixture was poured into water
(50 mL) with stirring to give a white precipitate. The precipitate
was purified on silica gel column eluted with DCM/MeOH (50:1 v/v)
to give compound 14 (yield 68.6%) as a white solid. 1H NMR
148.13, 146.59, 142.17, 138.10, 136.76, 132.50, 128.84, 126.66, 122.66,
118.53, 108.79, 107.12, 102.75, 68.49, 55.81, 36.01, 28.15, 21.73,
20.53. HRMS (m/z) (ESI): calcd for C27H30N5O5S [MþH]þ: 536.1962;
found: 536.1925.
4.2.3.20. 5-((4-((4-ethylphenyl)amino)-7-methoxyquinazolin-
6-yl)oxy)-N-(4- sulfamoylphenyl)pentanamide (8t). Yellow solid
(600 MHz, DMSO‑d6)
2H), 7.49 (s, 2H).
d
7.86 (d, J ¼ 8.6 Hz, 2H), 7.61 (d, J ¼ 8.6 Hz,
(yield: 65.3%). 1H NMR (600 MHz, DMSO‑d6)
d 10.29 (s, 1H), 9.46 (s,
1H), 8.42 (s, 1H), 7.87 (s, 1H), 7.76 (s, 4H), 7.66 (d, J ¼ 8.0 Hz, 2H),
7.24e7.17 (m, 5H), 4.19 (s, 2H), 3.93 (s, 3H), 2.64e2.58 (m, 2H), 2.48
(d, J ¼ 6.2 Hz, 2H), 1.95e1.80 (m, 4H), 1.22e1.18 (m, 3H). 13C NMR
4.2.6. 4-(2-isothiocyanatoethyl)benzenesulfonamide (17)
(150 MHz, DMSO‑d6)
d
171.64, 156.51, 154.31, 152.81, 148.14, 146.47,
To a solution of DCC (2.06 g, 10 mmol) in dry THF (30 mL), CS2
(495 mg, 6.5 mmol) and compound 16 (2.0 g, 4.0 mmol) was added.
The reaction was stirred at room temperature overnight and
monitored by TLC. After completion of reaction, the solvent was
removed under reduced pressure, and then DCM (100 mL) was
added, washed with brine three times. The organic phase was dried
over Na2SO4, and concentrated to dryness under reduced pressure.
The residue was subjected to flash column chromatograph over
silica gel with DCM/MeOH (100:1 v/v) to give compound 17 (yield
142.17, 139.01, 138.09, 136.93, 127.63, 126.66, 122.76, 118.53, 108.77,
107.02, 102.79, 68.50, 55.82, 36.00, 28.15, 27.68, 21.73, 15.73. HRMS
(m/z) (ESI): calcd for C28H32N5O5S [MþH]þ: 550.2119; found:
550.2077.
4.2.3.21. 5-((7-methoxy-4-(phenylamino)quinazolin-6-yl)oxy)-
N-(4- sulfamoylphenyl)pentanamide (8u). Yellow solid (yield:
63.4%). 1H NMR (600 MHz, DMSO‑d6)
d 10.29 (s, 1H), 9.48 (s, 1H),
8.45 (s, 1H), 7.87 (s, 1H), 7.80e7.75 (m, 6H), 7.39 (t, J ¼ 7.9 Hz, 2H),
7.24 (s, 2H), 7.18 (s, 1H), 7.12 (t, J ¼ 7.4 Hz,1H), 4.19 (t, J ¼ 5.9 Hz, 2H),
3.93 (s, 3H), 2.48 (t, J ¼ 4.8 Hz, 2H), 1.91e1.81 (m, 4H). 13C NMR
62.0%) as a white solid. 1H NMR (600 MHz, DMSO‑d6)
d 7.79 (d,
J ¼ 8.3 Hz, 2H), 7.48 (d, J ¼ 8.3 Hz, 2H), 7.34 (s, 2H), 3.96 (t, J ¼ 6.6 Hz,
(150 MHz, DMSO‑d6) d 171.64, 156.40, 154.37, 152.79, 148.20, 146.72,
2H), 3.04 (t, J ¼ 6.6 Hz, 2H).
12

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
4.2.7. Synthesis of 4-(3-(4-((substituted phenyl)amino)quinazolin-
6-yl)thioureido) benzenesulfonamide (15a-n) and 4-(2-(3-(4-
((substituted phenyl)amino)quinazolin -6-yl)thioureido)ethyl)
benzenesulfonamide (18a-n)
To a solution of compound 14 or 17 (0.02 mmol) in anhydrous
DMF (5 mL), compound 18a-n (0.02 mmol) was added and stirred
at 60 ꢀC overnight. After completion of reaction, the solvent was
removed under reduced pressure. The residue was purified on silica
gel column eluted with DCM/MeOH (40:1 v/v) to give the desired
products 15a-n and 18a-n.
2H), 7.74 (d, J ¼ 8.0 Hz,1H), 7.53 (d, J ¼ 7.9 Hz, 1H), 7.46 (t, J ¼ 8.0 Hz,
1H), 7.34 (s, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 180.25, 159.50,
150.34, 142.41, 139.51, 139.38, 138.39, 136.24, 134.79, 130.77, 129.27,
127.22, 126.29, 123.59, 122.31, 121.25, 119.95, 118.71, 113.85. HRMS
(m/z) (ESI): calcd for C21H17BrN6O2S2 [MþH]þ: 529.0111; found:
529.0056.
4.2.7.7. 4-(3-(4-((4-(trifluoromethyl)phenyl)amino)quinazolin-
6-yl)thioureido) benzenesulfonamide (15g). Yellow powder (yield:
75.0%). 1H NMR (600 MHz, DMSO‑d6)
d 10.33 (s, 1H), 10.30 (s, 1H),
10.11 (s, 1H), 8.69 (s, 1H), 8.58 (s, 1H), 8.15 (d, J ¼ 7.2 Hz, 2H), 7.93 (d,
4.2.7.1. 4-(3-(4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-
yl)thioureido) benzenesulfonamide (15a). Yellow powder (yield:
J ¼ 8.4 Hz, 1H), 7.84 (d, J ¼ 8.7 Hz, 1H), 7.80e7.73 (m, 6H), 7.32 (s,
2H). 13C NMR (150 MHz, DMSO‑d6)
d 180.56, 157.31, 153.61, 147.38,
71.3%). 1H NMR (600 MHz, DMSO‑d6)
d
11.65 (s, 1H), 11.28 (s, 1H),
143.01, 142.52, 139.47, 137.36, 132.29, 127.83, 126.17, 125.72 (d,
J ¼ 3.5 Hz), 124.47 (d, J ¼ 270.0 Hz), 123.41, 123.19, 121.63, 118.43,
115.24. HRMS (m/z) (ESI): calcd for C22H18F3N6O2S2 [MþH]þ:
519.0879; found: 519.0811.
11.12 (s, 1H), 8.98 (s, 1H), 8.80 (s, 1H), 8.27 (d, J ¼ 6.1 Hz, 1H), 8.03 (s,
1H), 7.97 (d, J ¼ 7.0 Hz, 1H), 7.86e7.71 (m, 5H), 7.57 (s, 1H), 7.35 (s,
2H). 13C NMR (150 MHz, DMSO‑d6)
d 180.25, 159.60, 155.39 (d,
J ¼ 246.2 Hz), 150.38, 142.42, 139.54, 139.40, 136.19, 134.84, 133.97,
126.86, 126.32, 125.51 (d, J ¼ 6.0 Hz), 122.33, 119.96, 119.35 (d,
J ¼ 18.0 Hz), 118.67, 117.10 (d, J ¼ 22.5 Hz), 113.78. HRMS (m/z) (ESI):
calcd for C21H17ClFN6O2S2 [MþH]þ: 503.0521; found: 503.0466.
4.2.7.2. 4-(3-(4-((3-ethynylphenyl)amino)quinazolin-6-yl)thio-
ureido) benzenesulfonamide (15b). Yellow powder (yield: 68.5%).
4.2.7.8. 4-(3-(4-((3-chloro-2-fluorophenyl)amino)quinazolin-6-
yl)thioureido) benzenesulfonamide (15h). Yellow powder (yield:
73.5%). 1H NMR (600 MHz, DMSO‑d6)
d 10.31 (s, 1H), 10.27 (s, 1H),
9.95 (s, 1H), 8.51 (s, 1H), 8.44 (d, J ¼ 1.7 Hz, 1H), 7.89 (dd, J ¼ 8.9,
2.0 Hz, 1H), 7.72e7.78 (m, 3H), 7.73 (d, J ¼ 8.7 Hz, 2H), 7.53e7.49 (m,
2H), 7.32 (s, 2H), 7.29 (t, J ¼ 8.1 Hz, 1H). 13C NMR (150 MHz,
1H NMR (600 MHz, DMSO‑d6)
d
11.56 (s, 1H), 11.27 (s, 1H), 11.10 (s,
DMSO‑d6)
d
180.46, 158.09, 154.15, 152.50 (d, J ¼ 247.8 Hz), 147.63,
1H), 8.96 (s, 1H), 8.80 (s, 1H), 8.26 (s, 1H), 7.96e7.75 (m, 7H),
142.58, 139.42, 137.16, 131.93, 128.08, 128.07 (d, J ¼ 11.8 Hz), 127.53,
126.98, 126.18, 124.97 (d, J ¼ 4.4 Hz), 123.13, 120.20 (d, J ¼ 16.5 Hz),
7.52e7.35 (m, 4H), 4.32 (s, 1H). 13C NMR (150 MHz, DMSO‑d6)
d
180.27, 159.50, 150.41, 142.44, 139.44, 139.39, 137.12, 136.37,
118.12, 114.79. HRMS (m/z) (ESI): calcd for
[MþH]þ: 503.0521; found: 503.0463.
C21H17ClFN6O2S2
134.75, 129.76,129.39, 127.70, 126.32, 125.33, 122.36, 122.16, 120.03,
118.79, 113.87, 82.89, 81.59. HRMS (m/z) (ESI): calcd for
4.2.7.9. 4-(3-(4-((3-fluorophenyl)amino)quinazolin-6-yl)thiour-
eido) benzenesulfonamide (15i). Yellow powder (yield: 72.0%). 1H
C
23H19N6O2S2 [MþH]þ: 475.1005; found: 475.0947.
4.2.7.3. 4-(3-(4-((3-(trifluoromethyl)phenyl)amino)quinazolin-
NMR (600 MHz, DMSO‑d6) d 10.33 (s, 1H), 10.32 (s, 1H), 9.91 (s, 1H),
6-yl)thioureido) benzenesulfonamide (15c). Yellow powder (yield:
8.66 (s, 1H), 8.54 (s, 1H), 7.95 (d, J ¼ 11.0 Hz, 1H), 7.90 (d, J ¼ 7.5 Hz,
75.2%). 1H NMR (600 MHz, DMSO‑d6)
d
11.62 (s, 1H), 11.28 (s, 1H),
1H), 7.82e7.68 (m, 6H), 7.42 (d, J ¼ 6.9 Hz, 1H), 7.32 (s, 2H), 6.94 (s,
11.12 (s, 1H), 8.98 (s, 1H), 8.82 (s, 1H), 8.26 (d, J ¼ 7.4 Hz, 1H), 8.17 (s,
1H). 13C NMR (150 MHz, DMSO‑d6)
d
180.57,162.00 (d, J ¼ 240.0 Hz),
1H), 8.07 (d, J ¼ 5.9 Hz, 1H), 7.97 (d, J ¼ 7.9 Hz, 1H), 7.88e7.69 (m,
157.24, 153.86, 147.70, 142.56, 141.13 (d, J ¼ 10.7 Hz), 139.45, 137.15,
132.12, 130.00 (d, J ¼ 9.5 Hz), 128.08, 126.18, 123.17, 118.45, 117.50,
115.20, 109.90 (d, J ¼ 20.9 Hz), 108.59 (d, J ¼ 25.8 Hz). HRMS (m/z)
(ESI): calcd for C21H18FN6O2S2 [MþH]þ: 469.0911; found: 469.0857.
4.2.7.10. 4-(3-(4-((3-chlorophenyl)amino)quinazolin-6-yl)thio-
ureido) benzenesulfonamide (15j). Yellow powder (yield: 74.7%). 1H
6H), 7.35 (s, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 180.29, 159.54,
150.63, 142.46, 139.44, 139.39, 137.87, 137.00, 134.75, 130.12, 129.48
(d, J ¼ 31.8 Hz), 128.40, 126.32, 124.02 (d, J ¼ 270.5 Hz), 122.82,
122.35, 121.03, 120.49, 118.71, 114.00. HRMS (m/z) (ESI): calcd for
C
22H18F3N6O2S2 [MþH]þ: 519.0879; found: 519.0813.
4.2.7.4.
4-(3-(4-((4-chloro-3-(trifluoromethyl)phenyl)amino)
NMR (600 MHz, DMSO‑d6) d 11.48 (s, 1H), 11.21 (s, 1H), 11.05 (s, 1H),
quinazolin-6-yl) thioureido)benzenesulfonamide (15d). Yellow
8.96 (s, 1H), 8.80 (s, 1H), 8.25 (d, J ¼ 8.6 Hz, 1H), 7.95 (d, J ¼ 8.9 Hz,
1H), 7.92 (s, 1H), 7.86 (d, J ¼ 8.3 Hz, 2H), 7.80 (d, J ¼ 8.3 Hz, 2H), 7.70
(d, J ¼ 7.8 Hz, 1H), 7.52 (t, J ¼ 8.0 Hz, 1H), 7.39 (d, J ¼ 7.6 Hz, 1H), 7.33
powder (yield: 79.1%). 1H NMR (600 MHz, DMSO‑d6)
d
11.48 (s, 1H),
11.11 (s, 1H), 10.99 (s, 1H), 8.98 (s, 1H), 8.78 (s, 1H), 8.34 (s, 1H), 8.22
(d, J ¼ 7.6 Hz, 1H), 8.14 (d, J ¼ 7.0 Hz, 1H), 7.95 (d, J ¼ 8.1 Hz, 1H),
7.85e7.75 (m, 5H), 7.34 (s, 2H). 13C NMR (150 MHz, DMSO‑d6)
(s, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 180.27, 159.36, 150.54,
142.40, 139.38, 138.41, 136.92, 134.61, 132.90, 130.45, 126.26, 126.17,
124.22, 122.99, 122.35, 120.44, 118.68, 113.91. HRMS (m/z) (ESI):
calcd for C21H18ClN6O2S2 [MþH]þ: 485.0616; found: 485.0558.
d
180.33, 159.07, 151.00, 142.41, 139.41, 139.23, 136.95, 134.41,
132.08, 132.02, 128.99, 126.89, 126.71 (d, J ¼ 30.8 Hz), 126.26,
122.99, 122.67 (d, J ¼ 271.5 Hz), 122.46, 121.62, 118.53, 114.17. HRMS
(m/z) (ESI): calcd for C22H17ClF3N6O2S2 [MþH]þ: 553.0490; found:
553.0430.
4.2.7.11.
4-(3-(4-(m-tolylamino)quinazolin-6-yl)thioureido)
benzenesulfonamide (15k). Yellow powder (yield: 78.6%). 1H NMR
(600 MHz, DMSO‑d6) 10.27 (s, 1H), 10.23 (s, 1H), 9.72 (s, 1H), 8.58
d
4.2.7.5. 4-(3-(4-((3-bromo-4-fluorophenyl)amino)quinazolin-6-
yl)thioureido) benzenesulfonamide (15e). Yellow powder (yield:
(s, 1H), 8.53 (s, 1H), 7.86 (d, J ¼ 8.4 Hz, 1H), 7.80e7.73 (m, 5H), 7.66
(s, 2H), 7.33 (s, 2H), 7.27 (t, J ¼ 7.8 Hz, 1H), 6.95 (d, J ¼ 7.1 Hz, 1H),
80.2%). 1H NMR (600 MHz, DMSO‑d6)
d
11.56 (s, 1H), 11.23 (s, 1H),
2.34 (s, 3H). 13C NMR (150 MHz, DMSO‑d6)
d 180.56, 157.51, 154.18,
11.07 (s, 1H), 8.95 (s, 1H), 8.78 (s, 1H), 8.25 (d, J ¼ 7.6 Hz, 1H), 8.14 (s,
147.69, 142.61, 139.44, 139.11, 137.66, 136.89, 131.85, 128.35, 128.02,
126.17, 124.47, 123.23, 122.83, 119.56, 118.67, 115.22, 21.25. HRMS
(m/z) (ESI): calcd for C22H21N6O2S2 [MþH]þ: 465.1162; found:
465.1104.
1H), 7.95 (d, J ¼ 8.1 Hz, 1H), 7.86e7.76 (m, 5H), 7.53 (s, 1H), 7.34 (s,
2H). 13C NMR (150 MHz, DMSO‑d6)
d 180.27, 159.47, 156.36 (d,
J ¼ 243.3 Hz), 150.54, 142.42, 139.43, 139.39, 136.71, 134.67, 134.32,
129.43, 126.30, 126.0 (d, J ¼ 7.8 Hz),122.36, 120.33,118.65,116.81 (d,
J ¼ 23.6 Hz), 113.83, 107.69 (d, J ¼ 21.9 Hz). HRMS (m/z) (ESI): calcd
for C21H17BrFN6O2S2 [MþH]þ: 547.0016; found: 546.9949.
4.2.7.12. 4-(3-(4-(p-tolylamino)quinazolin-6-yl)thioureido)ben-
zenesulfonamide (15l). Yellow powder (yield: 81.2%). 1H NMR
(600 MHz, DMSO‑d6)
d 10.39 (s, 1H), 10.37 (s, 1H), 9.93 (s, 1H), 8.58
4.2.7.6. 4-(3-(4-((3-bromophenyl)amino)quinazolin-6-yl)thio-
ureido) benzenesulfonamide (15f). Yellow powder (yield: 78.3%).
(s, 1H), 8.54 (s, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.80e7.75 (m, 5H), 7.69
(d, J ¼ 8.0 Hz, 2H), 7.33 (s, 2H), 7.21 (d, J ¼ 8.0 Hz, 2H), 2.31 (s, 3H).
1H NMR (600 MHz, DMSO‑d6)
d
11.59 (s, 1H), 11.28 (s, 1H), 11.11 (s,
13C NMR (150 MHz, DMSO‑d6)
d 180.53, 157.69, 153.75, 146.28,
1H), 8.98 (s, 1H), 8.81 (s, 1H), 8.27 (d, J ¼ 8.9 Hz, 1H), 8.03 (s, 1H),
142.59, 139.40, 137.13, 136.30, 133.21, 132.12, 128.96, 127.02, 126.18,
123.07, 122.70, 118.68, 115.01, 20.58. HRMS (m/z) (ESI): calcd for
7.97 (d, J ¼ 8.9 Hz, 1H), 7.87 (d, J ¼ 8.6 Hz, 2H), 7.80 (d, J ¼ 8.6 Hz,
13

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
C22H21N6O2S2 [MþH]þ: 465.1162; found: 465.1099.
HRMS (m/z) (ESI): calcd for C24H21ClF3N6O2S2 [MþH]þ: 581.0803;
found: 581.0745.
4.2.7.13. 4-(3-(4-((4-ethylphenyl)amino)quinazolin-6-yl)thiour-
eido) benzenesulfonamide (15m). Yellow powder (yield: 79.4%). 1H
4.2.7.19. 4-(2-(3-(4-((3-bromo-4-fluorophenyl)amino)quinazo-
lin-6-yl)thioureido) ethyl)benzenesulfonamide (18e). Yellow pow-
NMR (600 MHz, DMSO‑d6) d 10.29 (s, 1H), 10.27 (s, 1H), 9.80 (s, 1H),
8.56 (s, 1H), 8.52 (s, 1H), 7.86 (d, J ¼ 8.4 Hz, 1H), 7.80e7.72 (m, 7H),
der (yield: 83.4%). 1H NMR (600 MHz, DMSO‑d6)
d 9.84 (s, 2H), 8.61
7.32 (s, 2H), 7.23 (d, J ¼ 8.1 Hz, 2H), 2.62e2.59 (m, 2H), 1.20 (t,
(s, 1H), 8.39 (s, 1H), 8.31 (dd, J ¼ 6.4, 2.6 Hz, 1H), 8.00 (s, 1H),
J ¼ 7.5 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6)
d 180.55, 162.31,
157.57, 154.06, 147.22, 142.59, 139.42, 136.92, 136.68, 131.90, 127.73,
7.92e7.89 (m, 1H), 7.78e7.75 (m, 4H), 7.45 (d, J ¼ 7.7 Hz, 2H), 7.42 (t,
J ¼ 8.8 Hz, 1H), 7.32 (s, 2H), 3.77 (s, 2H), 2.98 (t, J ¼ 7.3 Hz, 2H). 13
C
127.67, 126.16, 123.18, 122.62, 118.67, 115.13, 27.72, 15.78. HRMS (m/
NMR (150 MHz, DMSO‑d6)
d
181.10, 157.12, 154.44 (d, J ¼ 221.4 Hz),
z) (ESI): calcd for
479.1256.
C
23H23N6O2S2 [MþH]þ: 479.1318; found:
153.57, 147.44, 143.52, 142.17, 136.82 (d, J ¼ 2.9 Hz), 131.67, 129.14,
128.26, 126.15, 125.81, 125.73, 122.96 (d, J ¼ 6.9 Hz), 117.53, 116.37
(d, J ¼ 22.8 Hz), 115.14, 107.24 (d, J ¼ 21.6 Hz), 45.25, 34.32. HRMS
(m/z) (ESI): calcd for C23H21BrFN6O2S2 [MþH]þ: 575.0329; found:
575.0257.
4.2.7.14.
amino)quinazolin-6- yl)thioureido)benzenesulfonamide (15n).
Yellow powder (yield: 83.5%). 1H NMR (600 MHz, DMSO‑d6)
10.27
4-(3-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)
d
(s, 2H), 9.80 (s, 1H), 8.59 (s, 1H), 8.49 (s, 1H), 8.04 (s, 1H), 7.85e7.64
4.2.7.20. 4-(2-(3-(4-((3-bromophenyl)amino)quinazolin-6-yl)
thioureido)ethyl) benzenesulfonamide (18f). Yellow powder (yield:
(m, 7H), 7.47e7.18 (m, 7H), 5.25 (s, 2H). 13C NMR (150 MHz,
DMSO‑d6)
d
180.56, 162.21 (d, J ¼ 241.8 Hz), 157.32, 154.01, 149.65,
78.5%). 1H NMR (600 MHz, DMSO‑d6)
d 9.84 (s,1H), 9.82 (s,1H), 8.64
147.46, 142.57, 139.67 (d, J ¼ 6.5 Hz), 139.44, 137.00, 133.21, 131.94,
130.57 (d, J ¼ 7.4 Hz), 127.94, 126.17, 123.96, 123.32, 123.19, 122.15,
121.07, 118.51, 115.05, 114.71 (d, J ¼ 21.2 Hz), 114.35, 114.03 (d,
J ¼ 21.6 Hz), 69.40. HRMS (m/z) (ESI): calcd for C28H23ClFN6O3S2
[MþH]þ: 609.0940; found: 609.0873.
(s, 1H), 8.42 (s, 1H), 8.24 (s, 1H), 8.01 (s, 1H), 7.92 (d, J ¼ 8.2 Hz, 1H),
7.80e7.75 (m, 4H), 7.45 (d, J ¼ 7.7 Hz, 2H), 7.36 (t, J ¼ 8.0 Hz, 1H),
7.32e7.29 (m, 3H), 3.77 (s, 2H), 2.98 (t, J ¼ 7.3 Hz, 2H). 13C NMR
(150 MHz, DMSO‑d6)
d 181.07, 157.11, 153.68, 147.49, 143.50, 142.17,
141.04, 136.86, 131.67, 130.42, 129.13, 128.27, 125.92, 125.80, 123.99,
121.21, 120.54, 117.56, 115.28, 45.25, 34.32. HRMS (m/z) (ESI): calcd
for C23H22BrN6O2S2 [MþH]þ: 557.0424; found: 557.0347.
4.2.7.15. 4-(2-(3-(4-((3-chloro-4-fluorophenyl)amino)quinazo-
lin-6-yl)thioureido) ethyl)benzenesulfonamide (18a). Yellow pow-
der (yield: 78.0%). 1H NMR (600 MHz, DMSO‑d6)
d
9.85 (s, 2H), 8.62
4.2.7.21. 4-(2-(3-(4-((4-(trifluoromethyl)phenyl)amino)quina-
zolin-6-yl) thioureido)ethyl)benzenesulfonamide (18g). Yellow
(s, 1H), 8.40 (s, 1H), 8.22 (dd, J ¼ 6.8, 2.6 Hz, 1H), 8.01 (s, 1H),
7.86e7.83 (m, 1H), 7.79e7.76 (m, 4H), 7.46e7.43 (m, 3H), 7.32 (s,
2H), 3.77 (s, 2H), 2.98 (t, J ¼ 7.3 Hz, 2H). 13C NMR (150 MHz,
powder (yield: 76.8%). 1H NMR (600 MHz, DMSO‑d6)
d 10.01 (s, 1H),
9.87 (s, 1H), 8.66 (s, 1H), 8.46 (s, 1H), 8.17 (d, J ¼ 8.4 Hz, 2H), 8.02 (s,
DMSO‑d6)
d
181.11, 157.12, 153.68, 153.29 (d, J ¼ 241.7 Hz), 147.44,
1H), 7.83e7.75 (m, 6H), 7.45 (d, J ¼ 7.4 Hz, 2H), 7.32 (s, 2H), 3.77 (s,
143.51, 142.17, 136.88, 136.58 (d, J ¼ 3.0 Hz), 131.67, 129.14, 128.27,
125.81, 123.38, 122.26 (d, J ¼ 6.8 Hz), 118.82 (d, J ¼ 18.3 Hz), 117.53,
116.61 (d, J ¼ 21.5 Hz), 115.15, 45.24, 34.32. HRMS (m/z) (ESI): calcd
for C23H21ClFN6O2S2 [MþH]þ: 531.0834; found: 531.0778.
2H), 2.98 (t, J ¼ 7.1 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 181.07,
157.15, 153.57, 147.60, 143.51, 143.16, 142.18, 137.00, 131.75, 129.14,
128.30, 125.81, 125.71 (d, J ¼ 3.6 Hz), 124.51 (d, J ¼ 269.7 Hz), 123.12
(d, J ¼ 31.8 Hz), 121.44, 117.52, 115.39, 45.26, 34.32. HRMS (m/z)
(ESI): calcd for C24H22F3N6O2S2 [MþH]þ: 547.1192; found: 547.1116.
4.2.7.22. 4-(2-(3-(4-((3-chloro-2-fluorophenyl)amino)quinazo-
lin-6-yl)thioureido) ethyl)benzenesulfonamide (18h). Yellow
4.2.7.16. 4-(2-(3-(4-((3-ethynylphenyl)amino)quinazolin-6-yl)
thioureido)ethyl) benzenesulfonamide (18b). Yellow powder
(yield: 82.8%). 1H NMR (600 MHz, DMSO‑d6)
d 9.84 (s, 1H), 9.79 (s,
1H), 8.62 (s, 1H), 8.43 (s, 1H), 8.09 (s, 1H), 8.00 (s, 1H), 7.94 (d,
J ¼ 8.2 Hz, 1H), 7.79e7.76 (m, 4H), 7.45 (d, J ¼ 7.6 Hz, 2H), 7.41 (t,
J ¼ 7.9 Hz, 1H), 7.33 (s, 2H), 7.23 (d, J ¼ 7.6 Hz, 1H), 4.20 (s, 1H), 3.77
(s, 2H), 2.98 (t, J ¼ 7.3 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6)
powder (yield: 82.0%). 1H NMR (600 MHz, DMSO‑d6)
d 9.91 (s, 1H),
9.85 (s, 1H), 8.50 (s, 1H), 8.34 (s, 1H), 8.02 (s, 1H), 7.81e7.75 (m, 4H),
7.54e7.45 (m, 4H), 7.32 (s, 2H), 7.29 (t, J ¼ 8.0 Hz, 1H), 3.77 (s, 2H),
2.99 (t, J ¼ 7.3 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 181.12,
d
181.08, 157.24, 153.79, 147.51, 143.53, 142.17, 139.60, 136.79, 131.62,
158.00, 153.95, 152.46 (d, J ¼ 247.9 Hz), 147.38, 143.52, 142.18,
136.99, 131.58, 129.15, 128.11 (d, J ¼ 12.0 Hz), 127.45, 126.91, 125.81,
125.73, 124.95 (d, J ¼ 4.4 Hz), 120.20 (d, J ¼ 16.4 Hz), 117.31, 114.88,
45.20, 34.28. HRMS (m/z) (ESI): calcd for C23H21ClFN6O2S2 [MþH]þ:
531.0834; found: 531.0761.
129.15, 128.96, 128.26, 126.67, 125.82, 124.74, 122.56, 121.78, 117.65,
115.28, 83.53, 80.61, 45.26, 34.33. HRMS (m/z) (ESI): calcd for
C
25H23N6O2S2 [MþH]þ: 503.1318; found: 503.1274.
4.2.7.17. 4-(2-(3-(4-((3-(trifluoromethyl)phenyl)amino)quina-
zolin-6-yl) thioureido)ethyl)benzenesulfonamide (18c). Yellow
4.2.7.23.
thioureido)ethyl) benzenesulfonamide (18i). Yellow powder (yield:
84.5%). 1H NMR (600 MHz, DMSO‑d6)
9.85 (s, 2H), 8.64 (s, 1H),
4-(2-(3-(4-((3-fluorophenyl)amino)quinazolin-6-yl)
powder (yield: 80.5%). 1H NMR (600 MHz, DMSO‑d6)
d
9.97 (s, 1H),
9.86 (s, 1H), 8.65 (s, 1H), 8.45 (s, 1H), 8.34 (s, 1H), 8.26 (d, J ¼ 8.1 Hz,
d
1H), 8.02 (s, 1H), 7.80e7.76 (m, 4H), 7.63 (t, J ¼ 7.9 Hz, 1H), 7.45 (d,
8.43 (s, 1H), 8.00 (s, 1H), 7.96 (d, J ¼ 11.9 Hz, 1H), 7.81e7.75 (m, 4H),
7.69 (d, J ¼ 8.1 Hz, 1H), 7.46e7.40 (m, 3H), 7.32 (s, 2H), 6.94 (t,
J ¼ 8.3 Hz, 1H), 3.77 (s, 2H), 2.98 (t, J ¼ 7.2 Hz, 2H). 13C NMR
J ¼ 7.5 Hz, 3H), 7.33 (s, 2H), 3.78 (s, 2H), 2.98 (t, J ¼ 7.2 Hz, 2H). 13
C
NMR (150 MHz, DMSO‑d6)
d 181.12, 157.19, 153.64, 147.53, 143.52,
142.18, 140.23, 136.96, 131.74, 129.66, 129.35, 129.14, 128.31, 125.81,
125.32, 124.25 (d, J ¼ 270.6 Hz), 119.59 (d, J ¼ 3.6 Hz), 117.80 (d,
J ¼ 3.9 Hz), 117.57, 115.27, 45.25, 34.32. HRMS (m/z) (ESI): calcd for
(150 MHz, DMSO‑d6)
d
181.09, 161.99 (d, J ¼ 239.3 Hz), 157.15,
153.69, 147.52, 143.51, 142.17, 141.19 (d, J ¼ 11.0 Hz), 136.86, 131.67,
129.98 (d, J ¼ 9.6 Hz), 129.13, 128.27, 125.81, 117.58, 117.40 (d,
J ¼ 2.3 Hz), 115.30, 109.76 (d, J ¼ 21.0 Hz), 108.42 (d, J ¼ 26.0 Hz),
45.24, 34.32. HRMS (m/z) (ESI): calcd for C23H22FN6O2S2 [MþH]þ:
497.1224; found: 497.1175.
C
24H22F3N6O2S2 [MþH]þ: 547.1192; found: 547.1122.
4.2.7.18.
4-(2-(3-(4-((4-chloro-3-(trifluoromethyl)phenyl)
amino)quinazolin-6-
yl)thioureido)ethyl)benzenesulfonamide
(18d). Yellow powder (yield: 78.9%). 1H NMR (600 MHz, DMSO‑d6)
4.2.7.24. 4-(2-(3-(4-((3-chlorophenyl)amino)quinazolin-6-yl)
thioureido)ethyl) benzenesulfonamide (18j). Yellow powder (yield:
d
10.03 (s, 1H), 9.87 (s, 1H), 8.67 (s, 1H), 8.48 (d, J ¼ 2.5 Hz, 1H), 8.43
(s, 1H), 8.34 (dd, J ¼ 8.8, 2.1 Hz, 1H), 8.02 (s, 1H), 7.82e7.77 (m, 4H),
85.2%). 1H NMR (600 MHz, DMSO‑d6)
d 9.83 (s, 2H), 8.64 (s, 1H),
7.74 (d, J ¼ 8.8 Hz, 1H), 7.45 (d, J ¼ 7.6 Hz, 2H), 7.32 (s, 2H), 3.77 (s,
8.43 (s, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.86 (d, J ¼ 8.3 Hz, 1H),
7.80e7.75 (m, 4H), 7.45 (d, J ¼ 7.5 Hz, 2H), 7.42 (t, J ¼ 8.1 Hz, 1H),
7.32 (s, 2H), 7.16 (d, J ¼ 7.9 Hz, 1H), 3.77 (s, 2H), 2.98 (t, J ¼ 7.2 Hz,
2H), 2.98 (t, J ¼ 7.3 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 181.13,
156.99, 153.47, 147.52, 143.50, 142.18, 139.00, 137.09, 131.84, 131.76,
129.14, 128.35, 126.57, 126.37, 126.26, 125.81, 122.87 (d,
J ¼ 271.2 Hz), 120.23 (d, J ¼ 5.4 Hz), 117.43, 115.24, 45.23, 34.31.
2H). 13C NMR (150 MHz, DMSO‑d6)
d 181.08, 157.13, 153.69, 147.51,
143.51, 142.17, 140.91, 136.87, 132.76, 131.68, 130.11, 129.13, 128.27,
14

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
125.81, 123.02, 121.17, 120.13, 117.58, 115.28, 45.25, 34.32. HRMS (m/
z) (ESI): calcd for C23H22ClN6O2S2 [MþH]þ: 513.0929; found:
513.0861.
150
m
L of DMSO. The O.D. value was read at 570/630 nm enzyme
labeling instrument. The IC50 values were calculated by SPSS soft-
ware after three parallel experiments.
4.2.7.25. 4-(2-(3-(4-(m-tolylamino)quinazolin-6-yl)thioureido)
ethyl) benzenesulfonamide (18k). Yellow powder (yield: 80.7%). 1H
4.4. EGFR kinase inhibition assay
NMR (600 MHz, DMSO‑d6)
d 9.82 (s, 1H), 9.68 (s, 1H), 8.57 (s, 1H),
8.43 (s, 1H), 7.99 (s, 1H), 7.79e7.74 (m, 4H), 7.67 (s, 2H), 7.46 (d,
J ¼ 6.7 Hz, 2H), 7.34 (s, 2H), 7.27 (t, J ¼ 7.6 Hz,1H), 6.95 (d, J ¼ 7.2 Hz,
1H), 3.78 (s, 2H), 2.99 (s, 2H), 2.34 (s, 3H). 13C NMR (150 MHz,
The EGFR proteins (EGFRWT and EGFRT790M) were purchased
from Carna Bioscience. The HTRF KinEASE-TK kit (Cat#62TK0PEB)
was from Cisbio. All the other chemicals were from Sigma. The
EGFR kinase assays were performed in 384-well plate (Corning
3676, low volume, black, NBS), using Cisbio HTRF KinEASE-TK kit.
The assay buffer contained 50 mM HEPES (Ph 7.0), 0.02% NaN3,
0.01% BSA, 0.1 mM Orthovanadate, 10 mM MgCl2, 2.5 mM DTT and
6.25 nM SEB. EGFR kinases was first incubated with compounds for
2 h respectively, then ATP/Peptide substrate mixture was added to
initiate the reaction. After 30 min reaction at room temperature,
the detection reagents were added. The TR-FRET signal was
measured on PerkinElmer Envision using excitation 320 nm and
emission 615 nm/665 nm. The data was analyzed using Graph Pad
Prism (Version 6.0) software after three parallel experiments.
DMSO‑d6)
d 181.08, 157.43, 154.02, 147.49, 143.56, 142.18, 139.17,
137.64, 136.59, 131.48, 129.17, 128.34, 128.19, 125.84, 124.40, 122.74,
119.48, 117.86, 115.34, 45.29, 34.37, 21.26. HRMS (m/z) (ESI): calcd
for C24H25N6O2S2 [MþH]þ: 493.1475; found: 493.1408.
4.2.7.26. 4-(2-(3-(4-(p-tolylamino)quinazolin-6-yl)thioureido)
ethyl) benzenesulfonamide (18l). Yellow powder (yield: 79.5%). 1H
NMR (600 MHz, DMSO‑d6)
d 9.81 (s, 1H), 9.68 (s, 1H), 8.53 (s, 1H),
8.40 (s, 1H), 7.97 (s, 1H), 7.79e7.71 (m, 6H), 7.45 (d, J ¼ 6.7 Hz, 2H),
7.33 (s, 2H), 7.19 (d, J ¼ 7.9 Hz, 2H), 3.76 (s, 2H), 2.98 (s, 2H), 2.30 (s,
3H). 13C NMR (150 MHz, DMSO‑d6)
d 181.07, 157.41, 154.06, 147.47,
143.55, 142.17, 136.64, 136.50, 132.73, 131.42, 129.15, 128.90, 128.17,
125.82, 122.36, 117.87, 115.29, 45.28, 34.36, 20.56. HRMS (m/z) (ESI):
calcd for C24H25N6O2S2 [MþH]þ: 493.1475; found: 493.1411.
4.2.7.27. 4-(2-(3-(4-((4-ethylphenyl)amino)quinazolin-6-yl)thi-
oureido)ethyl) benzenesulfonamide (18m). Yellow powder (yield:
4.5. CA inhibitory assay
Enzyme inhibition assays of CA (CAII and CAIX) were carried out
as per previous reported method [27]. This assay spectrophoto-
metrically measured the p-nitrophenol, a yellow colored product
which is formed by the hydrolysis of p-nitrophenyl acetate (4-PNA)
catalyzed by CA. Absorbance was measured at 400 nm with the
help of UV/visible spectrophotometer (Shimadzu UV2600) equip-
ped with temperature regulator. The absorption data was analyzed
with the help of Graph Pad Prism (Version 6.0) and the IC50 values
for tested compounds were determined.
83.6%). 1H NMR (600 MHz, DMSO‑d6)
d 9.81 (s,1H), 9.69 (s,1H), 8.53
(s, 1H), 8.40 (s, 1H), 7.97 (s, 1H), 7.78 (d, J ¼ 8.2 Hz, 2H), 7.75e7.72
(m, 4H), 7.45 (d, J ¼ 7.5 Hz, 2H), 7.32 (s, 2H), 7.22 (d, J ¼ 8.3 Hz, 2H),
3.76 (s, 2H), 2.98 (t, J ¼ 7.2 Hz, 2H), 2.62e2.58 (m, 2H), 1.20 (t,
J ¼ 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6)
d 181.07, 157.43,
154.06, 147.48, 143.54, 142.17, 139.20, 136.82, 136.51, 131.42, 129.14,
128.16, 127.70, 125.81, 122.45, 117.87, 115.29, 45.28, 34.35, 27.71,
15.79. HRMS (m/z) (ESI): calcd for C25H27N6O2S2 [MþH]þ: 507.1631;
found: 507.1563.
4.2.7.28. 4-(2-(3-(4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)
4.6. Apoptosis assay
amino)quinazolin
(18n). Yellow powder (yield: 82.2%). 1H NMR (600 MHz, DMSO‑d6)
9.82 (s, 1H), 9.73 (s, 1H), 8.57 (s, 1H), 8.37 (s, 1H), 8.05 (s, 1H), 7.98
-6-yl)thioureido)ethyl)benzenesulfonamide
Apoptosis was detected by flow cytometry with annexin V/PI
staining. H1975 cells were grown in each well of six-well plates at
the density of 5 ꢂ 104 cells per mL in the RPMI 1640 medium with
10% FBS to the final volume of 2 mL. The plates were incubated
overnight and treated with test compounds at the indicated con-
centration for 24 h. Then, cells were harvested and washed twice
with ice-cold PBS, and then suspended in the annexin-binding
buffer at a concentration of 5 ꢂ 105 cells per mL. Cells were then
incubated with 5 mL of annexin V-FITC and 5 mL of PI for 30 min at
room temperature in the dark. The samples were analyzed for
fluorescence with a flow cytometer (FACS Calibur, BD Biosciences).
d
(s, 1H), 7.78e7.70 (m, 5H), 7.47e7.39 (m, 3H), 7.38e7.32 (m, 4H),
7.27 (d, J ¼ 8.8 Hz, 1H), 7.18 (t, J ¼ 7.3 Hz, 1H), 5.25 (s, 2H), 3.77 (s,
2H), 2.98 (s, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 181.09, 162.21 (d,
J ¼ 242.1 Hz),157.20,153.89,149.56,147.39,143.52,142.17,139.68 (d,
J ¼ 7.4 Hz), 136.68, 133.32, 131.51, 130.58 (d, J ¼ 8.3 Hz), 129.14,
128.19, 125.81, 123.80, 123.33 (d, J ¼ 2.3 Hz), 122.01, 121.04, 117.67,
115.15, 114.71 (d, J ¼ 20.7 Hz), 114.36, 114.03 (d, J ¼ 21.8 Hz), 69.40,
45.25, 34.33. HRMS (m/z) (ESI): calcd for C30H27ClFN6O3S2 [MþH]þ:
637.1253; found: 637.1208.
4.3. Cell lines and cell growth inhibition assay
4.7. Cell cycle measurement
The NSCLC cell lines A549, H1975, human epidermoid carci-
noma cell line A431 and normal human liver cells LO2 in this study
were purchased from China Life Science College (Shanghai, PRC).
Roswell Park Memorial Institute (RPMI) 1640 medium, Culture
medium Dulbecco’s modified Eagle medium (DMEM), fetal bovine
serum (FBS), phosphate buffered saline (PBS, pH 7.2), and
Antibiotic-Antimycotic came from KeyGen Biotech Company
(China). Cells were grown in the medium supplemented with 10%
FBS, 100 units per mL penicillin and 100 g/mL streptomycin in a
humidified atmosphere of normoxia (20% O2, 75% N2 and 5% CO2) or
hypoxia (1% O2, 94% N2 and 5% CO2) at 37 ꢀC. Then, the compounds
were dissolved in DMSO and diluted with medium to various
concentrations (the final concentration of DMSO was less than
0.4%). After being incubated at 37 ꢀC for 72 h, cells were stained
H1975 cells with good vitality were transferred into six-well
plates and cultured overnight at 37 ꢀC. Then, compounds at the
indicated concentration were incubated with cells for 24 h. All
adherent and floating cells were collected and washed twice with
PBS. Then, the cells were fixed with 70% EtOH at 4 ꢀC for 24 h. After
that, fixed cells were washed with PBS. After being centrifuged,
cells were stained with 50
taining 100
mg/mL propidium iodide solution con-
m
g/mL RNase at 37 ꢀC for 0.5 h. The samples (at least
1 ꢂ 104 cells) were measured by flow cytometry (FAC Scan, Becton
Dickenson) using Cell Quest software and recording propidium
iodide (PI) in the FL2 channel.
4.8. Wound healing assays
with
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium
H1975 cells were seeded in each well of 6-well plates and
cultured. After the cells were attached to the wall, drew a straight
bromide (MTT, 5 mg/mL) for another 4 h, and then dissolved with
15

B. Zhang, Z. Liu, S. Xia et al.
European Journal of Medicinal Chemistry 216 (2021) 113300
line along the ruler with a yellow pipette tip and washed twice with
PBS and the serum-free medium was added. Then, cells were
Appendix A. Supplementary data
incubated with 0.5 and 1.0
m
M of 8v, respectively. A negative control
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113300.
was treated only with culture medium. After 24 h of incubation, the
scratches were visualized and photographed using an inverted
microscope to observe cellular migration through the wounds. The
area of the wound was precisely circled along the edge of the cell
scratch for quantitative analysis. The migration rate ¼ (0 h wound
area e 48 h wound area)/0 h wound area.
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