Synthesis and in vitro anti-bladder cancer activity evaluation of quinazolinyl-arylurea derivatives

Article abstract of DOI:10.1016/j.ejmech.2020.112661

Based on the structural modification of molecular-targeted agent sorafenib, a series of quinazolinyl-arylurea derivatives were synthesized and evaluated for their anti-proliferative activities against six human cancer cell lines. Compared with other cell lines tested, T24 was more sensitive to most compounds. Compound 7j exhibited the best profile with lower IC₅₀ value and favorable selectivity. In this study, we focused on 7j-induced death forms of T24 cells and tried to elucidate the reason for its potent proliferative inhibitory activity. Compound 7j treatment could trigger three different cell death forms including apoptosis, ferroptosis, and autophagy; which form would occur depended on the concentrations and incubation time of 7j: (1) Lower concentrations within the initial 8 h of 7j treatment led to apoptosis-dependent death. (2) Ferroptosis and autophagy occurred in the case of higher concentrations combining with extended incubation time through effectively regulating the Sxc^?/GPx4/ROS and PI3K/Akt/mTOR/ULK1 pathways, respectively. (3) The above death forms were closely associated with intracellular ROS generation and decreased mitochondrial membrane potential induced by 7j. In molecular docking and structure-activity relationship analyses, 7j could bind well to the active site of the corresponding receptor glutathione peroxidase 4 (GPx4). Compound 7j could be a promising lead for molecular-targeted anti-bladder cancer agents’ discovery.

Full text of DOI:10.1016/j.ejmech.2020.112661

Journal Pre-proof
Synthesis and in vitro anti-bladder cancer activity evaluation of quinazolinyl-arylurea
derivatives
Jia-Nian Chen, Ting Li, Li Cheng, Tai-Sheng Qin, Ye-Xiang Sun, Chu-Ting Chen,
Yue-Zhen He, Guang Liu, Di Yao, Ying Wei, Qiu-Yin Li, Guang-Ji Zhang
PII:
S0223-5234(20)30633-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112661
EJMECH 112661
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 April 2020
Revised Date: 1 July 2020
Accepted Date: 12 July 2020
Please cite this article as: J.-N. Chen, T. Li, L. Cheng, T.-S. Qin, Y.-X. Sun, C.-T. Chen, Y.-Z. He, G.
Liu, D. Yao, Y. Wei, Q.-Y. Li, G.-J. Zhang, Synthesis and in vitro anti-bladder cancer activity evaluation
of quinazolinyl-arylurea derivatives, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112661.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Elsevier Masson SAS. All rights reserved.

Graphic abstract
A series of quinazolinyl-arylurea derivatives were synthesized. Compound 7j
exhibited the best profile with lower IC50 value against T24 cells and favorable
selectivity. Compound 7j could induce three different death forms including apoptosis,
ferroptosis, and autophagy.

1
2
Synthesis and in vitro anti-bladder cancer activity evaluation of
quinazolinyl-arylurea derivatives
3
4
Jia-Nian Chen*†, Ting Li†, Li Cheng†, Tai-Sheng Qin†, Ye-Xiang Sun, Chu-Ting Chen,
Yue-Zhen He, Guang Liu, Di Yao, Ying Wei, Qiu-Yin Li, Guang-Ji Zhang
5
6
7
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal
Resources, Guangxi Normal University, Yucai Road 15, Guilin 541004, Guangxi, PR
China
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
† These authors contributed equally to this work.
* Correspondence: cjn288@sina.com (J.-N. Chen). Tel. & Fax: +86-773-2120958.
ORCID: 0000-0002-5551-8240.
Authors’ e-mails: cjn288@sina.com (J.-N. Chen); litingfendou@163.com (T. Li);
missliesel@163.com
wks72018@sina.com
(L.
Cheng);
Sun);
qintaisheng@sina.com
cctichen@sina.com
(T.-S.
(C.-T.
Qin);
(Y.-X.
Chen);
yzhe226@sina.com (Y.-Z. He); 517877698@qq.com (G. Liu); petto19@163.com (D.
Yao); weiying423@sina.com (Y. Wei); lqy061828@sina.com (Q.-Y. Li);
zhanggj914@126.com (G.-J. Zhang).
Running title: quinazolinyl-arylurea, anti-bladder cancer, death form, ferroptosis
- 1 -

1
2
Abstract
Based on the structural modification of molecular-targeted agent sorafenib, a series of
quinazolinyl-arylurea derivatives were synthesized and evaluated for their
anti-proliferative activities against six human cancer cell lines. Compared with other
cell lines tested, T24 was more sensitive to most compounds. Compound 7j exhibited
the best profile with lower IC50 value and favorable selectivity. In this study, we
focused on 7j-induced death forms of T24 cells and tried to elucidate the reason for its
potent proliferative inhibitory activity. Compound 7j treatment could trigger three
different cell death forms including apoptosis, ferroptosis, and autophagy; which form
would occur depended on the concentrations and incubation time of 7j: (1) Lower
concentrations within the initial 8 h of 7j treatment led to apoptosis-dependent death.
(2) Ferroptosis and autophagy occurred in the case of higher concentrations
combining with extended incubation time through effectively regulating the
Sxc-/GPx4/ROS and PI3K/Akt/mTOR/ULK1 pathways, respectively. (3) The above
death forms were closely associated with intracellular ROS generation and decreased
mitochondrial membrane potential induced by 7j. In molecular docking and
structure-activity relationship analyses, 7j could bind well to the active site of the
corresponding receptor glutathione peroxidase 4 (GPx4). Compound 7j could be a
promising lead for molecular-targeted anti-bladder cancer agents’ discovery.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Keywords:
quinazolinyl-arylurea;
apoptosis;
ferroptosis;
autophagy;
structure-activity relationship
- 2 -

1
2
Abbreviations
Annex, Annexin V-FITC; BC, bladder cancer; BCA, bicinchoninic acid; Casp-3,
caspase-3; Casp-8, caspase-8; CLSM, confocal laser scanning microscope; CQ,
chloroquine; cyto c, cytochrome c; DCFH-DA, 2',7'-dichlorodihydrofluorescein
diacetate; DMT1, divalent metal transportor 1; DTNB, 5,5'-dithiobis-(2-nitrobenzoic
acid); EB, ethidium bromide; ECL, enhanced chemiluminescence; FDA, U.S. Food
and Drug Administration; Fer-1, ferrostatin-1; FTH1, ferritin heavy chain; FTL,
ferritin light chain; G gemcitabine; Gef, gefitinib; GPx4, glutathione peroxidase 4;
GSH, glutathione; HCC, hepatocellular carcinoma; HO-1, heme oxygenase-1; Hoe,
hoechst 33342; ICP-MS, inductively coupled plasma-mass spectrometry; IGF-1,
insulin-like growth factor 1; IREB2, iron-responsive element binding protein-2; IRP2,
iron regulatory protein 2; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-
carbocyanine iodide; LC-3, microtubule-associated protein 1 light-chain 3; MDC,
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
monodansylcadaverine;
MTA,
molecular-targeted
agent;
MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NADPH, reduced
nicotinamide adenine dinucleotide phosphate; NSCLC, non-small cell lung cancer;
PCD, programmed cell death; PI, propidium iodide; Rap, rapamycin; ROS, reactive
oxygen species; S, sorafenib; SAR, structure-activity relationship; SDS-PAGE,
SDS-polyacrylamide gel electrophoresis; SI, selectivity index; Sxc , system xc-; zVAD,
-
carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone;
ΔΨm,
mitochondrial membrane potential.
- 3 -

1
2
1. Introduction
Cancer is considered a leading cause of death in both developed and developing
countries [1]. The World Health Organization previously reported an average of 15
million newly discovered cancer cases worldwide each year [2]. In spite of the
immense research endeavors and profound advances in cancer detection and treatment,
cancer is still one of the most challenging health problems in the world [3]. Taking BC
as an example, BC is a common urological disease accounting for > 3% of all global
cancer diagnoses with an estimated incidence of 549,000 new cases and 200,000
deaths in 2018 [4]. Traditional chemotherapeutic agents and radiotherapy provide only
temporal therapeutic benefits as well as being limited by a narrow therapeutic index,
remarkable toxicity and often acquired resistance [3]. Therefore, there is a growing
need for further identification and development of new treatment approaches,
especially the discovery of novel chemotherapeutic agents with improved potency and
minimal side effects. Molecular targeted therapy receives increasing attention in the
last two decades, which refers to the use of MTA including drugs or other substances
that target specific molecules (molecular targets) to block the growth and spread of
cancer cells [5]. Based on the molecular biological differences between malignant and
normal cells, molecular targeted therapy precisely and efficiently delivers MTAs to
the cancer site, which provides a critically-needed approach to improve outcomes for
patients [1]. Molecular targeted therapy mainly includes the application of MTA solely,
different MTAs combination, or MTA plus conventional cytotoxic drugs in clinical
practice [5,6]. Compared with conventional cytotoxic chemotherapy, these targeted
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
- 4 -

1
2
agents offer a more tolerable toxicity profile, thereby promising both optimized dose
intensity and better quality of life [7]. A typical example of diarylurea derivative
sorafenib (BAY 43-9006, Nexavar®) was the first oral multikinase inhibitor targeted
Raf and receptor tyrosine kinases [8]. Sorafenib has been approved by the FDA for the
treatment of advanced renal cell carcinoma in December 2005, unresectable HCC in
November 2007, and differentiated thyroid carcinoma in November 2013 [9]. It was
also subjected to clinical trials for the treatment of other types such as metastatic
NSCLC, BC, and colorectal cancers [2,10]. Despite the diverse merits of sorafenib, it
possesses some drawbacks of poor physicochemical properties accompanying toxicity
[3]. Therefore, more and more attention has been paid for further structural
modification/optimization of sorafenib in order to generate new chemical entities with
improved pharmacological and pharmacokinetic properties [11–15].
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
In recent decades, cell death form has been the most widely discussed topic for
pharmacologists and biologists. In particular, in the field of cancer therapy, more and
more researchers have realized that finding the cause of cancer cell death induced by
chemotherapy agents is very important and necessary because it offers unique
opportunities to interfere with processes associated with cell death and survival [16].
In general, cells die in mainly two ways, by accidental death or programmed cell
death (PCD) [17,18]. Dysregulation and/or dysfunction of PCD contribute to the
development of multiple human diseases, such as cancer [19]. Up to date, PCD is
divided into three main categories: Apoptosis, also namely type I PCD, was
discovered by Kerr et al. in 1972 [20]. Apoptosis was once considered to be the sole
- 5 -

1
2
form of PCD. Type II, named autophagy, is characterized by a prominent formation of
autophagic vacuoles. In addition to apoptosis and autophagy, several other types of
PCD have recently been identified to regulate various physiological and pathological
processes [21]. Type III is defined as regulated necrosis [19,22]. Among Type III PCD,
ferroptosis has attracted considerable attention [23–26]. Ferroptosis, discovered in
2012 by Stockwell et al., results from iron-dependent lipid peroxide accumulation
[23]. Up to date, the Sxc-/GPx4/ROS biological axis is considered to be closely
related to ferroptosis, and GPx4 is identified as an essential regulator [27]. Besides
this signaling pathway, a few regulatory factors (e.g., FTH1, FTL, DMT1, IREB2,
HSPB1, and HO-1) associated with iron metabolism were reported in succession [28].
Ferroptosis inducers can be at least categorized into two classes based on whether
these agents inhibit GPx4 directly: Class I, they suppress the activity of Sxc- and are
characterized by an accumulation of ROS accompanying with GSH depletion, then
down-regulate the expression of GPx4 in a cascaded way (e.g., erastin) [29]. Class II,
they directly inhibit the activity of GPx4 (e.g., RSL3) [30].
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
From 2011 to now, our research group has been committed to the structural
modification of the MTA—sorafenib, in order to find excellent candidates with high
efficacy and selectivity [31–33]. It is well-known that 4-aminoquinazoline in drug
molecular design is an important part to obtain tyrosine kinase inhibitors with potent
anti-cancer activity. Since the successful launch of gefitinib in 2003 for the treatment
of NSCLC, 4-aminoquinazoline derivatives have attracted vast interest [14,34]. With
all this in mind, in this study a series of quinazolinyl-arylurea derivatives were
- 6 -

1
2
designed via a hybrid approach through combining the core pharmacophores of
sorafenib (ureido-) and gefitinib (4-anilinoquinazolinyl-) (Scheme 1); their in vitro
anti-proliferative activities against several cancer cell lines were evaluated (Table 1;
Table S1 in Additional file 1). To our surprise, compared with other cancer cell lines,
T24 was more sensitive to most compounds. Based on the above facts, in this study
we turned our attention to the effects of these quinazolinyl-arylurea derivatives on BC
cell line T24 in vitro. Among them, 7j exhibited the best profile against T24 cells and
favorable selectivity. In this paper, we focused on 7j-induced T24 cell death forms and
tried to give a reasonable explanation. It was found that 7j could at least induce
apoptosis, ferroptosis, and autophagy of T24 cells. Which form would be triggered
depended on its concentrations and incubation time.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2. Results and discussion
2.1 Synthesis
General synthetic route to quinazolinyl-arylurea derivatives (7a–q and 8a–q) is
outlined in Scheme 1. Totally 34 quinazolinyl-arylurea derivatives were prepared, and
among them 32 compounds (7a–p and 8a–p) were reported for the first time. It should
be noted that, although the chemical structures of two compounds (7q and 8q) have
been described in our previous study [31], the activity data of all of these compounds
(including 7q and 8q) were covered for the first time. For the key intermediates
N-(3-aminobenzyl)quinazolin-4-amine (4a) and N-(3-aminobenzyl)-6,7-dimethoxy-
quinazolin-4-amine (4b), the separation and purification were not easy because of
- 7 -

1
2
their high polarity. Here we put forward a simple, but practical solution: A
recrystallization procedure could be employed by using mixed solvents before the
purification by column chromatography. Several solvents were tested, and it was
found that the use of mixed solvents petroleum ether and chloroform (v : v = 5 : 1)
were suitable. As for different aromatic isocyanates 6, they had better been
synthesized before use. It was proved in the experiment that most newly prepared
isocyanates were much higher reactive than those ready-made products purchased
from reagent companies, maybe because of their instability in storage or
transportation. Finally, column chromatography was used to purify these
quinazolinyl-arylurea derivatives. The chemical structures of the target compounds
were confirmed through the melting point determination, 1H and 13C NMR
spectroscopies, and HRMS. Original spectra of representative compounds were listed
in Additional file 2 (see Supplementary data).
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2.2 Proliferation inhibitory activity evaluation
Obtained thirty-four target compounds were evaluated for their inhibitory
activities against the following six human cancer cell lines including BEL-7402 (liver),
MGC-803 (stomach), HCT116 (colon), HCC827 (NSCLC), T24 and RT4 (bladder).
Sorafenib, gefitinib, and gemcitabine were selected as the positive reference drugs. It
was found that, compared with other cancer cell lines, T24 was more sensitive to most
of these derivatives as evidenced by the fact that their IC50 values against T24 cells
were less than 10 µM (Table 1; Table S1 in Additional file 1). Among all the
compounds screened, five compounds (7g, 7i–k, 8k) showed pronounced inhibitory
- 8 -

1
2
activities and their IC50 values were below 5 µM in T24 cell model. In particular,
compound 7j was considered highly potent lead candidate (IC50 = 3.97 ± 0.23 μM;
Incubation time: 48 h). The decline in IC50 values with extended incubation time
suggested that 7j concentration- and time-dependently suppressed the proliferation of
T24 cells (Fig. 1A). At the same concentration, the activity of 7j was stronger than
that of the positive reference drugs (sorafenib and gemcitabine) within 72 hours by the
fact that 7j gave the lowest IC50 values. The selectivity index (SI) values, reflected by
the ratio of IC50 values between non-tumoral and tumoral cell lines, were adopted to
evaluate the selectivity of proliferation inhibitory activity [35]. The normal bladder
epithelium cell line HCV29 was used. Two compounds 7j and 7k showed favorable
selectivity to T24 cell line with the SI values of 5.84 and 4.12, respectively (Table 1).
By contrast, sorafenib, gemcitabine, and other compounds exhibited comparably low
selectivity and their SI values were < 4. The inhibitory effect of 7j on the proliferation
of NSCLC cell line HCC827 was also tested and its SI value was also < 4 (Table S1).
Favorable anti-proliferative activity against T24 cells and fairly good selectivity of 7j
drove us to further explore its molecular mechanisms, which would be discussed in
detail in the following sections.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2.3 Induced T24 cell apoptosis by compound 7j at lower concentrations
Potency of 7j to induce apoptosis in T24 cells was analyzed using Annex/PI dual
staining assay. As shown in Fig. 1B, treatment with 7j (≤ 6 µM) for 8 h resulted in an
increase in the percentage of early plus late apoptotic cells (Q2 + Q3); the apoptosis
- 9 -

1
2
rates were 5.45% ± 0.29%, 8.08% ± 0.41%, and 32.82% ± 2.67% at concentrations of
0, 3, and 6 μM, respectively (Table S2). Compared with gemcitabine, 7j possessed a
stronger ability to induce apoptosis as evidenced by higher apoptosis rates in the
groups of 7j-treated T24 cells. After this, Hoe/PI double-labeling was used, and the
results suggested 7j induced T24 cell apoptosis in a time-dependent manner (Fig.
S1A).
3
4
5
6
7
Apoptosis is mediated by activation of a cascade of serine proteases, named
caspases [36]. There are two types of apoptotic caspases: initiator caspases and
effector (executioner) caspases. Initiator caspases (e.g., caspase-8 and -9) cleave
inactive pro-forms of effector caspases, thereby activating them. Effector caspases
(e.g., caspase-3, -6, and -7) in turn cleave other protein substrates within the cell, to
trigger the apoptotic process [36,37]. Among them, caspase-8 (casp-8) is a key
initiator; while caspase-3 (casp-3) serves as a key executioner. Reversal of apoptosis
can be achieved using the pan-caspase inhibitor zVAD, which binds to caspase
proteases irreversibly, preventing the initiation of the proteolytic cascade [38]. To
examine that induced apoptosis by 7j was whether or not associated with caspase
activation, T24 cells were pre-incubated with zVAD (20 µM) for 1 hour, and then 7j
was added. As expected, the addition of zVAD led to an obvious decrease in apoptosis
rates, compared with the group in which T24 cells were treated with 7j alone (Fig.
1B). We further examined the role of caspases during apoptosis induced by compound
7j. In a lower concentration range (≤ 5 µM), 7j treatment resulted in the up-regulation
of casp-8 in a concentration-dependent manner; at the concentration of 5 µM, its
- 10 -
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
expression level reached the peak, and then dropped rapidly (Fig. 1C and D). When
the concentration was ≥ 10 µM, its levels in 7j-treated groups were significantly lower
than that of the negative control. Increased casp-3 activity is generally considered to
be a sign of apoptosis and a positive indicator of efficacy in cancer therapy [37].
When T24 cells were co-incubated with 7j, the changing trend of casp-3 was the same
as that of casp-8, but a higher expression level of casp-3 was observed in T24 cells
after exposure to the same concentration of compound 7j (Fig. 1C and D).
Caspase-dependent cell apoptosis occurs principally via two separate yet interlinked
signaling mechanisms: the extrinsic pathway triggered by the activation of death
receptors and intrinsic mitochondria-mediated pathway initiated by the release of
cytochrome c (cyto c) from the mitochondrial matrix and subsequent caspase
activation [39]. Intrinsic pathway is regulated by a group of proteins which belong to
the Bcl-2 family. Bcl-2 and Bcl-xl are anti-apoptotic proteins and their role is to
prevent the release of cyto c and maintain mitochondrial integrity; while Bax and
other pro-apoptotic proteins of Bcl-2 family allow the release of cyto c from the
mitochondrial intermembrane space into the cytosol to promote the induction of
apoptosis [40]. Intrinsic pathway can be triggered by various extra and intra-cellular
stresses, which at least include oxidative stress, irradiation, and treatment with
chemotherapeutic agents [41]. To better understand the mechanisms of 7j-induced cell
apoptosis, several mitochondrial-related apoptosis proteins were evaluated via western
blot analysis. As shown in Figs. 1E and S1B, compound 7j treatment up-regulated the
pro-apoptotic protein Bax and led to the release of cyto c; while the anti-apoptotic
- 11 -
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
proteins Bcl-2 and Bcl-xl were down-regulated. Our results indicated that 7j treatment
for shorter periods (≤ 8 h) with lower concentrations (≤ 6 µM) led to
caspase-dependent apoptosis of T24 cells as evidenced by the activation of casp-3 and
-8, and the release of cyto c.
3
4
5
6
2.4 Compound 7j treatment led to ferroptosis-dependent death of T24 cells in the
case of prolonged incubation time combining with elevated concentrations
It was very interesting that extended incubation time (e.g., 12 h) with elevated
concentrations of 7j treatment led to a significant decrease (but not increase) in
apoptosis rate; more and more cells were floating, and then dead. From the above
results, at least the following two conclusions could be drawn: (1) Compound
7j-induced apoptosis was highly related to its incubation time and concentrations. (2)
There should be other modes of action associated with 7j-induced death of T24 cells.
It was found that 7j-induced cell death at higher concentrations could be partly
attenuated by a specific inhibitor of ferroptosis—Fer-1, so we investigated whether
T24 cell death was mediated via ferroptosis. As shown in Fig. 2A, when T24 cells
were incubated with 7j/erastin (the combination of 7j with erastin), lower survival
rates in trypan blue exclusion assay were detected, compared with the group treated
with 7j alone. By contrast, when 7j/Fer-1 were added to cell culture medium, the
survival rates were increased. Iron element transport is essential for the execution of
ferroptosis; however, how iron promotes ferroptosis remains unclear. Compound 7j
treatment led to a significant increase in iron content (Table 2). Simultaneously, a
- 12 -
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
significant accumulation of ROS and the depletion of the antioxidant GSH were
detected (Fig. 2B). Compound 7j induced ROS generation would be discussed later
(see Figs. 4 and S4). On the contrary, the addition of Fer-1 led to increased survival
rates, decreased ROS, and elevated GSH levels, compared with the group only treated
with 7j. Reasonable explanations were as follows: (1) Compound 7j-induced ROS
generation and GSH depletion triggered ferroptosis-dependent death of T24 cells; (2)
Compound 7j and erastin could synergistically induce ferroptosis. In contrast to this,
the addition of Fer-1 partially offset the effect of 7j, which contributed to improved
survival rates.
3
4
5
6
7
8
9
-
10
11
12
13
14
15
16
17
18
19
20
21
22
The cystine/glutamate antiporter Sxc consists of a light chain subunit
(xCT/SLC7A11) that confers substrate specificity and a glycosylated heavy chain
subunit (4F2hc/SLC3A2) located on the cell surface [42]. Erastin can inhibit the
uptake of cystine by the cystine/glutamate antiporter, creating a void in the
antioxidant defenses of the cell and ultimately leading to ferroptosis [28]. Inhibiting
the activity of GPx4 by genetic or drug approaches can promote ferroptosis through a
lipid ROS-dependent manner [43]. To investigate the mechanism of 7j-induced
ferroptotic cell death, ferroptosis-related proteins were analyzed. As shown in Fig. 2C,
D and Fig. S2A–D, 7j treatment led to a concentration-dependent decrease in the
expression levels of xCT, 4F2hc, GPx4, and FTH1; meanwhile, the expressions of
FTL and DMT1 were up-regulated. Moreover, the combination of different
concentrations of Fer-1 and 7j exerted different effects on the expression of xCT (Figs.
2C and S2A). However, when the concentration was decreased (< 6 μM), the
- 13 -

1
2
expression of xCT was unexpectedly up-regulated. In other words, 7j-induced
ferroptosis occurred only in the case of higher concentrations (≥ 6 µM) combining
with prolonged incubation periods (≥ 10 h). IREB2, also known as IRP2, is a critical
switch for cellular and systemic iron homeostasis and plays a central role in the
regulation of intracellular iron metabolism, but its role has not been fully understood
during the onset and progression of cancer [44]. In our study, 7j treatment led to a
dose-dependent increase in the expression level of IREB2 (Figs. 2D and S2D).
Whether sorafenib could induce ferroptosis? In the published literature [29,30],
obtained results from different research groups were not uniform and conclusions
were even contradictory. Our results suggested that sorafenib also could induce
ferroptosis, but it was required to incubate with T24 cells for a longer time (≥ 12 h)
with very high concentrations (≥ 12 μM) (Fig. 2E and F; Fig. S2E and F). When
incubation time was less than 12 h, sorafenib inhibited ferroptosis as evidenced by the
fact that it up-regulated the expression of xCT and GPx4, which was reported for the
first time in BC cell line. In our opinion, whether sorafenib induced ferroptosis in
human cancer cell lines depended on three factors: cell line type, its concentration and
incubation time. For gemcitabine, in the tested range (2–12 µM), the level of xCT was
concentration-dependently up-regulated; meanwhile, it has little or no effect on the
downstream target GPx4, which likely meant it could not induce ferroptosis. In view
of the above results, we speculated that the mode of action of compound 7j at higher
concentrations probably belonged to a complex type: It inhibited the activity of xCT,
also suppressed the expression of GPx4. Additionally, as shown in Figs. 2G and S2G,
- 14 -
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
the phosphorylation of HSPB1 (p-HSPB1) was partly inhibited when ferroptosis
occurred, which was consistent with the previous report [21]. HO-1 is a cytoprotective
enzyme that induces antioxidant activity against oxidative cellular stress through
catalyzing heme degradation to release ferrous iron, biliverdin, and carbon monoxide
[45]. It was found that the expression of HO-1 was down-regulated during 7j-induced
ferroptosis of T24 cells (Figs. 2H and S2H). Collectively, these findings indicated that
high concentrations (≥ 6 μM) with extended incubation time (≥ 10 h) of 7j could
induce ferroptosis via effectively regulating the Sxc-/GPx4/ROS signaling pathway.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2.5 Compound 7j treatment led to autophagic death of T24 cells
It was found that exposure to compound 7j caused the accumulation of
autophagosomes in the cytoplasms of T24 cells. Few autophagic bodies were detected
in untreated and 7j-treated cells for shorter incubation time, while extended
incubation time contributed to the formation of more autophagosomes. Or rather,
when the incubation time was over 8 h, the percentage of autophagosomes (white
arrow) per cytoplasm area was time-dependently increased (Fig. 3A and B). In the
range from 10 to 20 hours, a large number of autophagic vacuoles were observed by
the fact of strong blue fluorescence in MDC assay. At the 16-h time point
(concentration: 6 μM), fluorescence intensity reached the peak. Once incubation time
was further prolonged, the number of autophagosomes in per cytoplasm area
decreased rapidly. When the time was ≥ 24 h, more and more cells were distinctly
broken and dead, accompanying with evident abnormal changes (blue arrow) in
- 15 -

1
2
morphology under a CLSM (Fig. 3B and C). When 7j combining with an autophagy
activator rapamycin (Rap) was co-incubated with T24 cells, quantitative analysis
results indicated that the percentage of autophagosomes was further increased,
compared with the group treated with 7j alone (Fig. S3A). Moreover, the addition of
Rap led to lower survival rates in trypan blue exclusion assay (Fig. S3B). The LC-3 is
a reliable autophagic membrane marker for the detection of early autophagosome
formation, and tracking the conversion of LC3-I to LC3-II is indicative of autophagic
activity [41]. As shown in Figs. 3D and S3C, 7j treatment resulted in a
dose-dependent increase in the ratios of LC3B-II/I from 0.89 ± 0.05 (control) to 1.41
± 0.07, 2.93 ± 0.24, 6.38 ± 0.36, 7.79 ± 0.49 for 7j at 6, 8, 10, and 12 µM,
respectively; meanwhile, the expression of LC3B-II was dose-dependently
up-regulated. It was found that shorter incubation time (e.g., 8 h) with lower
concentrations (≤ 6 µM) led to a decrease in the ratios of LC3B-II/I; maybe under this
condition, T24 cells exhibited apoptotic (but not autophagic) death, which meant in
our study induced autophagy and apoptosis by 7j could not occur simultaneously.
Therefore, higher concentrations (≥ 6 µM) and extended incubation time (> 8 h) of 7j
induced an accumulation of autophagosomes, which resulted in autophagic death of
T24 cells. For sorafenib, similar results were obtained. Sequestosome 1 (SQSTM1),
also known as ubiquitin-binding protein p62, is an important regulatory protein
involved in cargo delivery to the vacuole mediated by autophagosomes [46]. Our
results suggested that the expression level of p62 was decreased after the treatment
with 7j or the combination (7j/Rap). However, down-regulated expressions of p62
- 16 -
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
induced by 7j could be offset by the addition of an autophagy inhibitor CQ (Fig. 3E).
The expression level of ULK1 was decreased, while the level of phosphorylated
ULK1 (p-ULK1) was concentration-dependently increased after the treatment with 7j
(Figs. 3F and S3D). Therefore, activation of autophagic flux was associated with
7j-induced cell death. Based on the previous study [47], the regulation of
PI3K/Akt/mTOR signaling pathway was involved in the process of autophagy. To
evaluate the relationship between this pathway and induced autophagy,
autophagy-related proteins/proteases were analyzed. As shown in Figs. 3G and S3E,
7j treatment had a significant effect on the expressions of PI3K, Akt, mTOR, and
phosphorylated mTOR (p-mTOR). To further verify the effect of 7j, an activator of
this signaling pathway IGF-1 was used [48]. As shown in Figs. 3H and S3F, the
expression level of phosphorylated Akt (p-Akt) was concentration-dependently
decreased, which could be partly neutralized by the addition of IGF-1. These results
indicated that 7j could induce autophagic death of T24 cells in the case of higher
concentrations (≥ 6 µM) with extended incubation time (> 8 h), and autophagy
activation was associated with effective regulation of the PI3K/Akt/mTOR/ULK1
signaling pathway.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
In our study, it was found that apoptosis and autophagy did not co-occur; while 7j
really could simultaneously induce ferroptosis and autophagy. Accordingly, there must
be a close relation between ferroptosis and autophagy. Jiang et al. [22] believed that
ferroptosis was an autophagic cell death process. However, the key molecular target(s)
which connected ferroptosis with autophagy remained unclear.
- 17 -

1
2
2.6 Compound 7j induced ROS generation and decreased mitochondrial
membrane potential
3
4
ROS are by-products of cellular oxygen metabolism and useful in the process of
intracellular signal transduction at low concentrations. However, uncontrolled
increase of intracellular ROS induced by exogenous stimuli (e.g., chemotherapeutic
drugs) can activate several death-related signaling pathways, such as apoptosis,
ferroptosis, and autophagy [49]. Therefore, the detection of ROS in different death
forms is essential. As shown in Fig. 4A and B, after the treatment with 7j,
intracellular DCF fluorescence intensity was evidently enhanced, which indicated an
accumulation of ROS induced by 7j. The fluorescence intensity was then
quantitatively analyzed using a luminescence spectrometer. It was found that within
16 hours, ROS levels were concentration-dependently increased. At the 16-h time
point, ROS levels were elevated up to 164.4% ± 7.3%, 213.3% ± 6.5%, and 238.4% ±
8.9% of negative control in response to 4, 8, and 12 μM of 7j treatment groups,
respectively. In this study, several intriguing results were obtained: (1) ROS
generation and lipid peroxidation were closely associated with the concentration and
incubation time of 7j. When its concentration was ≤ 12 μM and incubation time was ≤
16 h, both were concentration- and time-dependently increased (Fig. 4A–C). (2) In
apoptosis analysis, the addition of zVAD (20 µM) led to a decrease in ROS levels,
compared with the group treated with 7j alone (Fig. S4A). Similarly, in ferroptosis
and autophagy analyses, the addition of Fer-1 (0.2 µM) or CQ (10 µM) resulted in a
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
- 18 -

1
2
decrease in ROS levels (Fig. S4B and C). (3) When the incubation time was over 16
hours and concentration was further increased (> 12 μM), intracellular ROS and lipid
peroxidation levels were rapidly decreased. We hypothesized that under so high
concentrations, the mitochondria and other subcellular structures of T24 cells suffered
irreparable damage, which led to the failure to produce enough ROS to maintain cell
survival; more and more cells were forced to dismember and die. It was found that the
autophagy inhibitor CQ significantly suppressed ferroptosis-associated total ROS
accumulation, which was consistent with the previous report [26]. Moreover,
compared with the group treated with 7j alone, the addition of CQ led to an increase
in the expression levels of xCT and GPx4, indicating that CQ also could inhibit cell
ferroptosis (Fig. S4D and E). That’s to say, when intracellular GSH levels dropped
induced by 7j, ROS levels increased quickly. Besides the above western blotting
analysis, it was found that at high concentrations, 7j was able to decrease GPx4
activity in a concentration-dependent manner; while at lower concentrations (< 6 μM),
7j showed weak activation effect (Table 3 and Fig. S4F). Arising from the above, we
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
-
hypothesized that the inhibition of Sxc and GPx4 by 7j suppressed cystine import
resulting in ROS generation and the depletion of GSH.
In order to further investigate the inhibitory effect of target compound 7j, ΔΨm
changes were detected, and a fibrillation-state-specific fluorescent probe—JC-1 was
used. JC-1 accumulates in mitochondria depending on ΔΨm and is present either as
monomer or J-aggregate. The monomer predominating in depolarized mitochondria
emits green fluorescence (~530 nm), whereas the oligomer (J-aggregates) forming in
- 19 -

1
2
mitochondria with higher potentials emits red fluorescence (~590 nm) [50]. Relative
green and red fluorescence distinguish between low and high ΔΨm, and also can
reflect ΔΨm changes. As shown in Fig. 4D, JC-1 emitted strong red fluorescence,
meanwhile weak green fluorescent image points were observed in the control group,
which indicated a high ΔΨm because in this case J-aggregates were formed. After
T24 cells were exposed to 7j, JC-1 formed monomers and emitted strong green
fluorescence corresponding to a low ΔΨm. As a result, CLSM images showed a
marked decline in red fluorescence and increase in green fluorescence, suggesting a
loss of ΔΨm upon 7j treatment. After T24 cells were treated with sorafenib, similar
results were obtained. However, at lower concentrations (≤ 6 µM) of gemcitabine,
weak green fluorescence in T24 cells was observed. This result verified lower
cytotoxicity of gemcitabine against T24 cells [51]. The changes from red to green
fluorescence were also determined using flow cytometry and quantitatively analyzed
using a fluorescence spectrophotometer. As shown in Figs. 4E and S4G, strong red
fluorescence (p1 region) and weak green fluorescence (p2 region) were detected in
control cells (high ΔΨm). The ratio of green/red fluorescence intensity was 0.29 ± 0.04,
0.69 ± 0.07, 21.03 ± 1.47, and 29.39 ± 2.32 times after 7j treatment at the
concentrations of 0, 4, 8, and 12 µM, respectively. These results revealed that 7j
treatment resulted in a concentration-dependent decrease in ΔΨm. In our opinion,
intracellular ROS accumulation induced by 7j destroyed structural integrity of the
mitochondrial membranes and decreased ΔΨm. Our results suggested that the two
factors (ROS production and mitochondrial dysfunction) were interdependent to each
- 20 -
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
other, which was closely related to 7j-induced apoptotic/ferroptotic/autophagic cell
death.
3
4
2.7 Molecular docking and SAR analyses
5
Molecular docking is an essential tool in structural biology and computer-assisted
drug design. As described above, GPx4 activity inhibition by exogenous stimuli
would directly trigger ferroptosis [27,29]. Moreover, in our study high concentrations
(≥ 6 μM) of 7j could significantly down-regulate the expression of GPx4. Based on
the above facts, we tried to find the probable binding mode of 7j with the receptor
GPx4. Molecular docking analyses were performed using the AutoDock program.
Three ligands including 7j, 8j, and sorafenib were successfully docked into GPx4
binding pockets. GPx4 consisted of one chain (green), and there were at least three
H-bonds when 7j was interacted with GPx4: The first H-bond was formed between
one nitrogen atom in quinazolinyl unit and hydrogen atom of residue LYS152 with a
distance of 2.7 Å (Fig. 5A and B). Another two hydrogen bonds (H···O···H) were
formed between two hydrogen atoms in the urea group and an oxygen atom of residue
ARG179 with distances of 2.2 and 1.8 Å, respectively. There was an active pocket
(marked as "p1"), and the ligand 7j was almost perfectly embedded into this pocket
(Fig. 5C and D). Very different to 7j, only one H-bond with a distance of 1.9 Å was
formed between a hydrogen atom in the urea group of 8j and residue LYS126 (H···O)
(Fig. S5A). In docking model, 7j was surrounded by a thick "fog" formed by the
chain (Fig. 5C). It should be noted that a proper volume of the ligand was very
- 21 -
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
important, because the pocket was neither too big nor too small, which just held those
ligands whose size was equivalent to 7j. That's to say, if the molecular volume of one
ligand was obviously increased or decreased, the binding force between this ligand
and GPx4 was reduced significantly. In our study, because the smaller pocket "p1"
could not hold larger molecule 8j, which resulted in that 8j had to get into another
bigger binding site "p2" (Fig. 5E; Fig. S5B and C). In fact, "p2" located on the left
side was not a very satisfactory binding pocket, which was a long narrow and shallow
ditch. The interaction between 8j and GPx4 was not strong, therefore calculated
binding energy (-3.1 kcal/mol) was obviously lower than that of 7j (-7.2 kcal/mol).
For sorafenib, three H-bonds were formed (Fig. S5D). Due to smaller molecular size,
sorafenib also could be docked into the pocket "p1", but the binding energy (-5.1
kcal/mol) was weaker (Fig. S5E and F). In our opinion, compared with sorafenib,
besides formed H-bonds, more suitable molecular volume of 7j resulted in higher
affinity with GPx4. That’s to say, compared with sorafenib, 7j had a strong interaction
with the receptor GPx4, and therefore exhibited strong proliferation inhibitory activity
against T24 cells. Even so, we were aware that the above binding model could not
give a convincing explanation as to why low concentrations of 7j could not inhibit the
activity of GPx4. We speculated that produced different pharmacological effects of 7j
on the receptor GPx4 at low concentration (weak activation) and high concentration
(inhibition) may be due to the different binding sites. However, the above only
described the binding mode at higher concentration. It has been reported a systemic
knockout of the entire GPx4 gene is lethal because it plays an important role in
- 22 -
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
embryonic development [52]. In another study [53], the inactivation of GPx4 triggers
acute renal failure in mice. Therefore, the function of human GPx4 and associated
molecular targets in ferroptosis needs to be fully elucidated in the future.
3
4
From Table 1, it was found that the right-most aromatic rings and different
substituent groups in molecular structures of the synthesized compounds displayed
differential anti-proliferative activities against T24 cells. The SARs from the
important results were summarized as follows: (1) The inhibitory activity of two
intermediates (4a and 4b) was very low (IC50 > 30 μM); only the final
products—quinazolinyl-arylurea derivatives exhibited moderate to favorable
anti-proliferative activity, which suggested that the pharmacophore—urea moiety
which linked the left and right aromatic rings was essential. It directly determined
whether these derivatives produced the inhibitory effects on T24 cells. Based on the
above molecular docking results, we speculated that in the interaction key H-bonds
between the urea moiety and corresponding residues of the receptor were probably
formed. (2) As far as the distal aromatic rings were concerned, phenyl was preferred.
When phenyl was substituted by others, such as isoxazolyl or naphthyl, the activity
decreased significantly. For example, the inhibitory activity of the six compounds
(7n–p and 8n–p) was obviously weaker than that of 7j. (3) Phenyl must be directly
linked to the urea moiety. If there was one or multiple methylene (-CH2-) groups
between phenyl and urea moiety, the activity was significantly reduced (e.g., 7m and
8m). (4) There must be one or multiple substituent(s) on the distal benzene ring. If no
substituent was connected, the activity was poor, maybe due to the lack of strong
- 23 -
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
hydrophobic interaction with the receptor. (5) The strength of inhibitory activity was
closely associated with the type of substituents on the distal benzene ring. a.
Replacement of one or multiple hydrogen atoms by chlorine atom (Cl-) or CF3- (7g–k)
significantly enhanced the activity. Compound 7j bearing two substituent groups
(3,4-dichloro) exhibited the best profile. b. The introduction of two different or
identical electron-donating groups such as methyl and methoxyl led to an increase of
the activity, but the effect produced was obviously weaker than that of halogen atom
substitution. c. The introduction of the nitro group was undesirable. (6) As mentioned
above, the size/volume of the target compound was of great importance. The addition
of two methoxyl groups on the far left directly increased molecular volume, which
made derivatives 8a–l not fit well with the receptor GPx4. Therefore, in the case that
substituents on the distal benzene ring were the same, derivatives 8a–l exhibited
relatively weaker inhibitory effects, compared with 7a–l. (7) It was very likely that
hydrophobic interaction between those derivatives and the receptor was enhanced by
the introduction of halogen atoms on the distal benzene ring, which was beneficial for
improved anti-proliferative activity. Moreover, the introduction of quinazolinyl group
on the left side in molecular structure also enhanced hydrophobic interaction.
Finally, the molecular mechanisms underlying the inhibitory effects of 7j on the
proliferation of T24 cells and associated death forms were outlined in Fig. 6. Lower
concentrations (≤ 6 μM) of 7j induced T24 cell apoptosis through a
caspase-dependent pathway within the initial 8 h; 7j treatment led to the upregulation
of the expression of Bax, the release of cyto c, and a decrease in the levels of Bcl-xl
- 24 -
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

1
2
and Bcl-2 (pathway I). In the case of high concentrations combining with prolonged
incubation time, ferroptotic and autophagic cell death occurred by effectively
intervening with the Sxc-/GPx4/ROS (II) and PI3K/Akt/mTOR/ULK1 (III) pathways,
respectively. However, an important issue is yet to be solved: What are driving factors
or key targets during the transformation of the above three death forms? We speculate
that there are several key targets which play an important role in initiating the
switchover from one form to another. Fortunately, at least our study proved that GPx4
was one of the key targets in regulating ferroptosis and autophagy. Moreover, it was
likely that ferroptosis and autophagy exerted overlapping functions, and related
signaling pathways shared one to more regulatory factors. However, exact functions
of GPx4 and other targets such as xCT are unclear, which needs an in-depth study.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
3. Conclusions
In summary, we described a series of novel quinazolinyl-arylurea derivatives as
sorafenib analogues through a hybrid-design approach based upon two privileged
pharmacophores, namely ureido and 4-anilinoquinazolinyl groups. Five compounds
(7g, 7i–k, 8k) showed pronounced proliferative inhibitory activities and their IC50
values were below 5 µM against T24 cell line. Compound 7j was found to be the most
potent and efficacious derivative with IC50 value of 3.97 ± 0.23 μM and favorable
selectivity, which was obviously superior to that of the positive reference drug
sorafenib. Base on the above results, we explored T24 cell death forms induced by
this compound and tried to clarify the underlying molecular mechanisms. The
- 25 -

1
2
highlight of this paper was that we found 7j could induce different death forms
including apoptosis, ferroptosis, and autophagy; which form would be triggered
depended on its concentrations and incubation time. The analysis of SARs indicated
that ureido group was a key pharmacophore. One or more electron-withdrawing
groups such as halogen (Cl-, CF3-) substitution on the terminal benzene ring were
beneficial for the activity. Molecular volume played a crucial role in the binding with
the receptor GPx4. Compound 7j possessed the urea moiety, two substituent groups
(3,4-dichloro), and appropriate molecular size; therefore it exhibited potent
proliferation inhibition activity against T24 cell line.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Platinum-based chemotherapy regimens (e.g., gemcitabine
+
cisplatin,
methotrexate + vinblastine + doxorubicin + cisplatin) remain the standard of care for
advanced BC [54,55]. Even so, the response rates of the above regimens are only 40%
to 50% and median overall survival is in the range of 14 to 15 months. Additionally,
non-specific targeting associated with annoying side effects is still an issue [54].
Luckily, erdafitinib, the first MTA for the treatment of patients with advanced
urothelial cancer, was approved by the FDA in April 2019 [56]. However, its efficacy
and side effects need to be further tested in clinical practice. In our study, BC cell line
T24 was found to be sensitive to these quinazolinyl-arylurea derivatives, though this
result has yet to be verified by in vivo experiments. In some ways, our study maybe
provides new ideas for the treatment of bladder cancer. Taken together, compound 7j
may serve as a promising candidate for further development of potent
molecular-targeted anti-bladder cancer agents. The optimization of 7j to improve its
- 26 -

1
2
potency and pharmacokinetic/pharmacodynamic properties for in vivo study is in
progress.
3
4
4. Experimental section
5
4.1. Chemistry
6
4.1.1. General conditions
7
Unless otherwise indicated, all solvents and organic reagents were obtained from
commercially available sources and used without purification. Visualization was
accomplished with UV light (254 nm). Melting points (m.p.) were determined using a
hot-stage microscope (X-4, Beijing Taike Ltd, China) and are uncorrected. 1H NMR
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
13
and C NMR spectra were recorded in DMSO-d6 on a Bruker Avance 500 or
400 MHz NMR spectrometer, respectively; chemical shifts (δ) were reported in
parts per million (ppm) with tetramethylsilane as an internal standard. HRMS
spectra were recorded on an Agilent 1290 HPLC-6224 time-of-fight mass
spectrometer. Column chromatography was performed using silica gel (300–400 mesh,
Qingdao Haiyang Chemical Co., Ltd., PR China).
4.1.2 General synthetic procedures for quinazolinyl-arylurea derivatives
The method used for the preparation of quinazolinyl-arylurea derivatives (7a–q
and 8a–q) was outlined in Scheme 1, which has been described in our previous report
[31]. In brief, 4-chloroquinazoline 3a (or 4-chloro-6,7-dimethoxyquinazoline, 3b) was
reacted with 3-aminobenzylamine in dry CH2Cl2 to afford the key intermediate
- 27 -

1
2
N-(3-aminobenzyl)quinazolin-4-amine 4a (or N-(3-aminobenzyl)-6,7-dimethoxy-
quinazolin-4-amine, 4b), then 4a and 4b were reacted with different aromatic
isocyanates 6 to give the desired target compounds 7a–q and 8a–q, respectively. The
crude products were purified on silica gel column chromatography.
3
4
5
6
4.1.3 Characterization of the target compounds
7
4.1.3.1
N-(2,4-dimethylphenyl)-N'-(3-((quinazolin-4-ylamino)methyl)phenyl)urea
8
(7a)
9
White solid, yield: 65.49%, m.p. 249.7–252.1 °C. 1H NMR (500 MHz, DMSO-d6)
δ (ppm): 8.94 (s, 1H), 8.87 (t, J = 5.8 Hz, 1H), 8.47 (s, 1H), 8.34 (d, J = 7.7 Hz, 1H),
7.82–7.77 (m, 2H), 7.72 (d, J = 7.5 Hz, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.55 (s, 1H),
7.47–7.43 (m, 1H), 7.35 (s, 1H), 7.23 (t, J = 7.9 Hz, 1H), 6.98 (d, J = 6.6 Hz, 2H),
6.94 (d, J = 8.2 Hz, 1H), 4.79 (d, J = 5.9 Hz, 2H), 2.22 (s, 3H), 2.17 (s, 3H). 13C NMR
(125 MHz, DMSO-d6) δ (ppm): 159.87, 155.60, 153.18, 149.70, 140.61, 140.55,
135.22, 133.09, 132.04, 131.16, 129.24, 128.20, 128.04, 127.02, 126.20, 123.14,
121.84, 120.93, 116.84, 116.80, 115.40, 43.98, 20.79, 18.24. ESI-HRMS (m/z): calcd.
for C24H23N5O [M + H]+: 398.19809; found: 398.19698.
10
11
12
13
14
15
16
17
18
19
20
21
22
4.1.3.2
N-(2,5-dimethylphenyl)-N'-(3-((quinazolin-4-ylamino)methyl)phenyl)urea
(7b)
White solid, yield: 67.51%, m.p. 246.3–248.1 °C. 1H NMR (500 MHz, DMSO-d6)
δ (ppm): 8.91 (s, 1H), 8.85 (s, 1H), 8.42 (s, 1H), 8.34 (d, J = 7.6 Hz, 1H), 7.80 (t, J =
- 28 -