Design, synthesis, and biological evaluation of quinazoline derivatives containing piperazine moieties as antitumor agents

Article abstract of DOI:10.1177/1747519820910384

A series of novel quinazoline derivatives containing piperazine analogs are synthesized via substitution reactions with 6,7-disubstituted 4-chloroquinazoline and benzyl piperazine (amido piperazine). Potent antiproliferative activities are observed against A549, HepG2, K562, and PC-3 with N-(3-chlorophenyl)-2-(4-(7-methoxy-6-(3-morpholino-propoxy)quinazoline-4-yl)piperazine-1-yl)acetamidename C9 showing excellent activity. This active derivative was screened for cell migration ability, proliferation effects, and apoptosis against A549 and PC-3 cells, with the result showing biological activity almost equal to that of the control gefitinib.

Full text of DOI:10.1177/1747519820910384

910384CHL0010.1177/1747519820910384Journal of Chemical ResearchLi et al.
research-article2020
Research Paper
Journal of Chemical Research
1–8
© The Author(s) 2020
Article reuse guidelines:
sagepub.com/journals-permissions
Design, synthesis, and biological evaluation of
quinazoline derivatives containing piperazine
moieties as antitumor agents
https://doi.org/10.1177/1747519820910384
DOI: 10.1177/1747519820910384
journals.sagepub.com/home/chl
Wen Li1, Shu-Yi Chen1, Wei-Nan Hu1, Mei Zhu1, Jia-Min Liu1,
Yi-Hong Fu1, Zhen-Chao Wang1,2 and Gui-Ping OuYang1,2,3
Abstract
A series of novel quinazoline derivatives containing piperazine analogs are synthesized via substitution reactions with
6,7-disubstituted 4-chloroquinazoline and benzyl piperazine (amido piperazine). Potent antiproliferative activities are
observed against A549, HepG2, K562, and PC-3 with N-(3-chlorophenyl)-2-(4-(7-methoxy-6-(3-morpholino-propoxy)
quinazoline-4-yl)piperazine-1-yl)acetamidename C9 showing excellent activity. This active derivative was screened for
cell migration ability, proliferation effects, and apoptosis against A549 and PC-3 cells, with the result showing biological
activity almost equal to that of the control gefitinib.
Keywords
quinazoline derivatives, cytotoxicity, migratory capacity, colonization activity, apoptosis
Date received: 1 December 2019; accepted: 10 February 2020
phosphorylation, was approved by the US Food and Drug
Introduction
Administration (FDA) for locally advanced or metastatic
It has been reported that the quinazoline unit is of great
non-small cell lung cancer therapy—is involved in cellu-
value as a chemical skeleton with diverse pharmaceutical
lar signal-transduction pathways that regulate essential
and physiological utility, including antimicrobial,1 antihy-
perlipidemic,2 anticonvulsant,3 antihypertensive,4 and
1College of Pharmacy, Guizhou University, Guiyang, P.R. China
anti-inflammatory5 activities. Therefore, many quinazo-
2State Key Laboratory of Functions and Applications of Medicinal Plants,
lines have contributed to the quest for an ultimate antitu-
Guizhou Medical University, Guiyang, P.R. China
3Drug Synthetic Engineering Laboratory of Guizhou Province, Guiyang,
P.R. China
mor chemotherapeutic agent. Moreover, several small
molecules such as gefitinib (Figure 1), erlotinib, and
lapatinib, which containing quinazoline analogs, were
designed to inhibit epidermal growth factor receptor
(EGFR) kinase activity. Among them, gefitinib—an oral
small molecule agent that inhibits EGFR tyrosine
Corresponding author:
Zhen-Chao Wang, College of Pharmacy, Guizhou University, Guiyang
550025, P.R. China.
Email: wzc.4884@163.com

2
Journal of Chemical Research 00(0)
converted to the quinazoline 7 by reduction with hydrazine
hydrate followed by ring closure using formic acid and chlo-
rination.22 As shown in route 1, intermediate 7 was reacted
with a series of monosubstituted piperazines 8 to give the tar-
get compounds C1–C5. In addition, intermediate 7 was
reacted with a series of amide-containing monosubstituted
piperazines 9 to give the target compounds C6–C10 (route 2).
All the target compounds were characterized, and the struc-
tures were confirmed by 1H NMR, 13C NMR, and mass spec-
trometry. Taking compound C9 as a representative example,
two singlets appeared at 8.41 and 3.91ppm in CD3OD attrib-
uted to the quinazoline –N=CH and –OCH3, respectively. In
the 13C NMR spectrum, a signal due to C=O was observed at
169.5ppm.
Figure 1. The structure of gefitinib.
functions such as proliferation, differentiation, and apop-
tosis.6–14 However, its efficient use is seriously hampered
by EGFR mutation. Even though tumors were sensitive to
gefitinib, tumor regrowth occurs after several months and
all of the mechanisms of the resistance have not been clar- Biological activity
ified. Thus, it is necessary to exploit novel antitumor
The antitumor activities of the synthesized compounds
drugs with high efficiency and low toxicity due to the
against four cell lines were evaluated using gefitinib as the
control. The IC50 values indicated that the compounds
exerted antitumor effects, with varying degrees of antitumor
activity against the four cell lines. As shown in Table 1, the
IC50 values ranged from 35.56±9.39 to 8.24±1.40µM
against A549, from 14.57±5.52 to 5.62±3.23µM against
PC-3, from 56.42±0.14 to 8.63±0.86µM against HepG2,
and from 159.3±14.28 to 15.02±0.88µM against K562,
being typically better than gefitinib (IC50 values were
5.475±1.06µM againstA549, 5.99±1.47µM against PC-3,
31.48±10.23µM against HepG2, and 13.67±1.44µM
against K562). The cytotoxicities of the target compounds
against the normal cell line NRK-52E in vitro are shown in
the Supporting Information Figure S1). The result showed
that the survival rate of the cells at low concentration was
close to 100%, and the cytotoxicity of compound C9 was the
weakest among these compounds.
To further investigate the cell migration ability of com-
pound C9 with the A549 and PC-3 cell lines, wound healing
assays were examined with gefitinib and C9 at concentra-
tions of 5µM for 24h, respectively. Representative micro-
graphs showed that compound C9 efficiently inhibited
A549 and PC-3 migration in comparison with dimethyl sul-
foxide (DMSO) in a time-dependent manner. Furthermore,
the migration ratios of A549 ranged from 13.39% to 53.18%
and from 12.40% to 51.54%, and the migration ratio of
PC-3 from 5.50% to 16.74% and from 3.03% to 12.60%
after being cultured with gefitinib and C9, respectively
(Figure S2).
various side effects of gefitinib.15
Over past decades, a large number of piperazine hetero-
cyclic derivatives have attracted significant attention as
chemotherapeutic drugs. Numerous studies have illustrated
that the introduction of piperazine moieties can influence
the physicochemical properties and enhance the bioactivity
of compounds. It is known that piperazine is versatile and
privileged in drug discovery,16–20 for example, in ciproflox-
acin for the treatment of bacterial infections, trifluopera-
zine for psychosis, cetirizine for the treatment of allergies,
and tanitinib for the treatment of acute myeloid leukemia. A
series of 4-amino-2H-benzo(h)chromen-2-one analogs
containing the piperazine moiety were reported by Chen in
2019, and bioassays showed that these compounds exhib-
ited potent antagonistic potency against androgen receptors
(AR; inhibition >50%), and exhibited potent AR binding
affinities as well as displaying higher activities than finas-
teride toward LNCaP cells (AR-rich) versus PC-3 cells
(AR-deficient).21
Based on the active substructure combination theory, the
piperazine ring was utilized in this research. Thus, two
series of novel 4-piperazine quinazoline derivatives were
designed and synthesized. The preliminary antitumor activ-
ity of these new compounds was also evaluated in vitro. In
addition, the primary apoptotic effect mechanism induced
by a representative compound C9 was also investigated by
flow cytometry.
Results and discussion
To evaluate the cell proliferation effects of C9, we
performed colony formation on the A549 and PC-3 cell
lines. The results showed that the colonies presented sig-
Synthesis
The synthetic route for the preparation of the novel quinazo- nificant corresponding downregulated trends on A549
line derivatives containing a piperazine moiety is summarized and PC-3 cells when stimulated by a range of C9 doses
in Scheme 1. The key intermediate 7 was synthesized in six (1.25, 2.5, 5, 10, and 20 µM). It was obvious that there
steps (a to f). 3-Hydroxy-4-methoxybenzaldehyde (1) was was very little colony at 20 µM, and the effect of C9 was
chosen as the starting material and was reacted with 1-bromo- in a dose-dependent manner. As Figure S3 shows, the
3-chloropropane followed by reaction with NH2OH·HCl, clonogenic ratio of A549 was from 3.87% to 14.53% and
AcOH, and AcONa to form 3-(3-chloropropoxy)-4-methoxy- that of PC-3 was from 3.60% to 46.13% after being cul-
benzonitrile (3). This was subjected to nitration using nitric tured with C9 at different concentrations (Figure S3).
acid and the product 4 reacted with morpholine under potas-
In order to investigate the induction of apoptosis in A549
sium iodide (KI) catalysis to give intermediate 5. This was and PC-3 cells, the Hoechst staining assay was conducted in

Li et al.
3
Scheme 1. Synthetic routes to the target compounds C1–C5 and C6–C10.
Table 1. The IC50 values of compounds C1 to C10 against four cancer cell lines.
IC50 (µM)a
Compound
A549
PC-3
5.99±1.47
HepG2
K562
Gefitinibb
C1
C2
C3
C4
C5
C6
C7
C8
5.475±1.06
13.37±6.40
24.85±4.06
13.53±2.35
15.34±7.35
13.94±6.38
8.99±6.29
12.96±1.80
22.51±1.24
8.24±1.40
35.56±9.39
31.48±10.23
24.69±2.73
32.23±3.20
43.45±3.85
29.07±5.65
22.29±2.81
51.29±13.08
31.05±5.53
8.63±0.86
13.67±1.44
8.19±1.53
14.57±5.52
8.51±3.20
6.44±2.31
5.62±3.23
15.26±5.12
13.72±4.32
13.42±4.87
7.66±1.27
10.42±1.97
70.69±10.24
60.94±13.41
15.02±0.88
62.96±13.17
45.52±2.55
17.63±8.20
159.3±14.28
50.00±5.13
21.96±3.51
62.56±5.36
C9
C10
38.62±2.41
40.64±13.18
aThe average of three trials.
bCommercial gefitinib was used as a positive control.
nuclear morphology by fluorescent microscopy. As shown in the nucleus, and chromatin condensation after culturing with
Figure S4, it was easy to observe the distinct pattern of mor- gefitinib and C9 at different concentrations (1.25, 2.5, 5, 10,
phological changes, including cell shrinkage, fragmentation of and 20µM) in A549 and PC-3 cell lines for 24h (Figure S4).

4
Journal of Chemical Research 00(0)
Figure 2. C9 and gefitinib increase the apoptosis of A549 and PC-3 cells. (a, b) Annexin/PI statin of A549 and (c, d) Annexin/PI
statin of PC-3.
Figure 3. (a) Compound C9 (colored by atom—carbon: gray, nitrogen: dark blue, oxygen: red, chlorine: green, and hydrogen:
blue) is bound into EGFR. The dotted lines show the hydrogen bonds. (b) The surface model structure of binding of compound C9
with the EGFR complex is shown.
Flow cytometry
Binding mode analysis
Apoptosis assays can provide preliminary information In order to further evaluate these promising compounds and
on the mechanism of growth inhibition of tumor cells. to guide structure–activity relationship (SAR) studies, the
Therefore, the apoptotic effects of C9 against the A549 interaction effect between compound C9 and the EGFR
and PC-3 cell lines were investigated. The results are crystal structure (3W2S.pdb) was investigated. Compound
given in Figure 2. After treatment of A549 and PC-3 cell C9 was inserted into the active site of EGFR kinase for
lines for 24 h with gefitinib and C9 at different concen- molecular docking.All docking runs were applied in Surflex-
trations (5, 10, and 20 µM), the apoptosis ratio of A549 Dock using Sybyl-X 2.0. The binding mode of compound
(including the early and late apoptosis rates) was from C9 and EGFR kinase is depicted in Figure 3. As shown in
41.9% to 57.4% with gefitinib and from 16.4% to 39.8% Figure 3(a), compound C9 was potently bound to the active
with C9. Furthermore, the apoptosis ratios of PC-3 was site of EGFR kinase by hydrophobic interactions, and the
from 35.3% to 43.8% with gefitinib and from 28.7% to binding mode was stabilized by two hydrogen bonds. The
38.7% with C9, being remarkably influenced by gefi- enzyme surface model is shown in Figure 3(b), which
tinib and C9 in a dose-dependent manner.
revealed that the target molecule C9 was well embedded in

Li et al.
5
the active pocket of EGFR kinase. This molecular docking and concentrated to give compound 5 (yield 56%). This
result, along with the biological assay data, suggested that intermediate 5 (12.11g, 0.038mol) and FeCl3 (0.38g,
compound C9 was a potential inhibitor of EGFR kinase.
0.002mol) were dissolved in a mixture of methanol and
water (3:1, 120mL) followed by 80% N2H4·H2O (10mL),
and the mixture was heated under reflux (3h) and then con-
centrated at 40°C. Water (37.5mL), HCl (36%–38%,
45mL), and HCOOH (>88%, 60mL) were added to the
filtrate until the temperature of reaction mixture reached
RT. The mixture was then refluxed at 130°C for 3.5h.
After completion of the reaction, as evidenced by TLC, the
water (75mL) was added to the mixture until the tempera-
ture of the reaction mixture reached RT, after which NaOH
was added to adjust the pH to 7. A yellow and white solid
was obtained by extracting with CHCl3 (3mL×200mL)
and drying the organic layer with anhydrous MgSO4. Ethyl
acetate (20mL) was added into the solid and the solution
heated to boiling. The residue was filtered and dried to
obtain white solid (6) (yield 58%) which could be further
converted into key intermediate 7 by reduction with thionyl
chloride (SOCl2) in DMF (1.0g, yield 94%) at 84°C.
Conclusion
In conclusion, a series of quinazoline derivatives contain-
ing a piperazine moiety have been designed and synthe-
sized. Furthermore, the biological activities of these
compounds were evaluated according to the Thiazolyl Blue
Tetrazolium Bromide (MTT) method, wound healing
assay, clonogenic survival assay, Hoechst staining, and
flow cytometry. Compound C9 with excellent activity may
represent a promising lead candidate for EGFR-inhibiting
therapeutics.
Experimental
Materials and methods
All reagents were purchased from commercial sources as
analytical grade and were used without further purification.
All reactions were monitored by thin-layer chromatography
(TLC). 1H NMR and 13C NMR spectra were performed on a
Bruker Ascend 400 NMR (Bruker, Germany) and JEOL-
ECX 500 NMR spectrometers (JEOL, Japan) using metha-
nol (CD3OD) as the solvent and tetramethylsilane as the
internal standard. Chemical shifts are expressed in ppm
(parts per million) and coupling constants are given in Hz
(Hertz). Mass spectra (MS) were recorded on an Agilent
1100 MSD-Trap-VL series with an electron spray ionization
(ESI) source. High-resolution mass spectra (HRMS) were
recorded on Thermo Scientific Q Exactive series with an
ESI source.
General procedure for the preparation of C1–C5.
Taking compound C1 as a representative example,
4-(3-((4-chloro-7-methoxyquinazolin-6-yl)oxy)propyl)
morpholine (7) (0.50g, 1.48mmol), 1-benzylpiperazine
(0.31g, 1.78mmol), and i-PrOH (15mL) were added in turn
to a flask and refluxed for 4h. After completion of the reac-
tion, as indicated by TLC, the solvent was evaporated to
give a yellowish-brown oily substance. This substance was
dissolved in methanol and isolated by column chromatogra-
phy (CHCl3: MeOH=15:1). Pure compound was evapo-
rated to give the yellow oily substance (0.08g, 0.17mmol).
General procedure for the preparation of C6–C10.
Taking compound C6 as a representative example,
4-(3-((4-chloro-7-methoxyquinazolin-6-yl)oxy)propyl)
morpholine (0.50g, 1.48mmol), N-phenyl-2-piperazinyl-
acetamide (0.39g, 1.78mmol), and i-PrOH (15mL) were
added to a flask and refluxed for 4h. After completion of
the reaction, as indicated by TLC, the solvent was evapo-
rated to give a yellowish-brown oily substance. This sub-
stance was dissolved in methanol and isolated by column
chromatography (CHCl3: MeOH=15:1). The pure com-
pound was evaporated to give the brownish black oil sub-
stance (0.10g, 0.19mmol).
The synthesis of intermediate 7. To a solution of 1 in N,
N-dimethylformamide (DMF) (40mL), K2CO3 (15.20g,
0.11mol) and 1-bromo-3-chloropropane (18.89g, 0.12mol)
were added. The mixture was stirred and refluxed at 85°C
for 4h. After completion of the reaction, as monitored by
TLC, the solution was added to cold water and the mixture
filtered and concentrated under reduced pressure to give the
ether 2 (yield 80%). Ether 2 (15.50g, 0.068mol),
NH2OH·HCl (10g, 0.136mol), and AcONa (11.12g,
0.136mol) were dissolved in AcOH (40mL), and the solu-
tion was heated at 105°C for 8h. After completion of the
reaction, as monitored by TLC, the solution was added to
saturate sodium chloride (200mL), and the mixture filtered
and concentrated to give the nitrile 3 (yield 90%). Interme-
diate 3 (17.26g, 0.076mol) was added to a mixture of
AcOH (50mL) and Ac2O (50mL) keeping the temperature
between 0 and 5°C, and then HNO3 (40mL) was added
dropwise to the solution. The mixture was stirred and
refluxed at room temperature (RT) for 72h, and then the
solution was added to cold water. Ammonia was added to
adjust the pH to 7, and then the mixture was filtered and
dried to obtain the nitro compound 4 (yield 68%). To a
solution of 4 (14.16g, 0.063mol) in DMF (40mL), KI
(0.57g, 0.0035mol) and morpholine (10.90g, 0.126mol)
4-(4-Benzylpiperazin-1-yl)-7-methoxy-6-[3-(4-morpholinyl)pro-
poxy]quinazoline (C1). Yield 11%, 0.08g; yellow oily sub-
stance; 1H NMR (CD3OD, 500MHz): δ 8.44 (s, 1H),
7.23–7.35 (m, 5H), 7.12 (s, 2H), 4.12 (t, J=6.0Hz, 2H),
3.94 (s, 3H), 3.68–3.70 (m, 8H), 3.58 (s, 2H), 2.49–2.63
(br, m, 10H), 2.03–2.06 (br, m, 2H); 13C NMR (CD3OD,
125MHz): δ 163.6, 155.5, 152.0, 148.2, 137.1, 129.3,
128.1, 127.2, 110.6, 105.8, 104.5, 66.9, 66.3, 62.6, 55.4,
55.3, 53.5, 52.6, 49.2, 25.6; ESI-MS: m/z 478.2 [M+H]+.
HRMS (ESI) m/z calcd for C27H35N5O3 [M+H]+ 478.2813,
found: 478.2808.
were added. The mixture was stirred and refluxed at 70°C 4-(4-(2,6-Dichlorobenzyl)piperazin-1-yl)-7-methoxy-6-[3-
for 10h. After the reaction was finished, the mixture was (4-morpholinyl)propoxy]quinazoline (C2). Yield 7%, 0.05g;
1
added to cold water, and the solid precipitate was filtered yellow oily substance; H NMR (CD3OD, 500MHz): δ

6
Journal of Chemical Research 00(0)
8.43 (s, 1H), 7.35 (d, J=8.0Hz, 2H), 7.22 (t, J=8.0Hz, 148.0, 137.9, 128.6, 124.8, 120.0, 110.9, 105.9, 104.3,
1H), 7.11 (s, 1H), 7.10 (s, 1H), 4.12 (t, J=6.0Hz, 2H), 3.94 67.0, 66.3, 61.6, 55.4, 55.3, 53.5, 52.9, 49.3, 25.7; ESI-MS:
(s, 3H), 3.82 (s, 2H), 3.63–3.69 (m, 8H), 2.74 (t, J=4.6Hz, m/z 521.4 [M+H]+. HRMS (ESI) m/z calcd for C28H36N6O4
4H), 2.49–2.57 (m, 6H), 2.03–2.06 (br, m, 2H); 13C NMR [M+H]+ 521.2871, found: 521.2863.
(CD3OD, 125MHz): δ 163.6, 155.5, 152.0, 148.2, 148.1,
136.8, 133.7, 129.4, 128.3, 110.9, 105.8, 104.6, 66.9, 66.3,
56.0, 55.4, 55.3, 53.5, 52.7, 49.4, 25.7; ESI-MS: m/z 546.4
[M+H]+. HRMS (ESI) m/z calcd for C27H33Cl2N5O3
[M+H]+ 546.2033, found: 546.2026.
2-(4-(7-Methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)pipera-
zin-1-yl)-N-(o-methylphenyl)acetamide (C7). Yield 13%, 0.10g;
yellow oily substance; 1H NMR (CD3OD, 500MHz): δ 8.46
(s, 1H), 7.69 (d, J=8.3Hz, 1H), 7.03–7.18 (m, 5H), 4.12 (t,
J=6.2Hz, 2H), 3.94 (s, 3H), 3.67–3.74 (m, 8H), 3.26 (s, 2H),
2.82 (t, J=4.6Hz, 4H), 2.56 (t, J=7.6Hz, 2H), 2.47 (s, 4H),
2.27 (s, 3H), 2.03–2.06 (br, m, 2H); 13C NMR (CD3OD,
125MHz): δ 169.5, 163.6, 155.5, 152.0, 148.3, 148.1, 135.4,
130.2, 130.1, 126.3, 125.3, 123.0, 111.0, 105.9, 104.3, 67.3,
66.3, 61.4, 55.4, 55.3, 53.5, 53.0, 49.6, 25.7, 16.9; ESI-MS:
m/z 535.5 [M+H]+. HRMS (ESI) m/z calcd for C29H38N6O4
[M+H]+ 535.3027, found: 535.3019.
4-(4-(2-Methylbenzyl)piperazin-1-yl)-7-methoxy-6-[3-(4-mor-
pholinyl)propoxy]quinazoline (C3). Yield 17%, 0.12g; yel-
1
low oily substance; H NMR (CD3OD, 500MHz): δ 8.38
(s, 1H), 6.91–7.13 (br, m, 6H), 3.99 (t, J=6.0Hz, 2H), 3.86
(s, 3H), 3.61 (t, J=4.6Hz, 4H), 3.41–3.51 (br, m, 4H), 3.39
(s, 2H), 2.46–2.49 (m, 6H), 2.27–2.39 (br, m, 7H), 1.94–
1.97 (br, m, 2H); 13C NMR (CD3OD, 125MHz): δ 163.5,
163.4, 155.2, 152.1, 147.9, 137.4, 135.6, 130.2, 129.9,
127.2, 125.4, 110.7, 106.0, 104.3, 67.0, 66.4, 60.5, 55.4,
55.3, 53.5, 52.8, 49.4, 25.7, 18.4; ESI-MS: m/z
492.5[M+H]+. HRMS (ESI) m/z calcd for C28H37N5O3
[M+H]+ 492.2969, found: 492.2963.
2-(4-(7-Methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)
piperazin-1-yl)-N-(p-methylphenyl)acetamide (C8). Yield
1
16%, 0.13g; yellow oily substance; H NMR (CD3OD,
500MHz): δ 8.74 (s, 1H), 7.44 (d, J=8.6Hz, 2H), 7.16 (d,
J=11.5Hz, 2H), 7.11 (d, J=8.6Hz, 2H), 4.16 (t, J=6.0Hz,
2H), 3.96 (s, 3H), 3.80 (s, 4H), 3.69 (t, J=4.6Hz, 4H), 3.25
(s, 2H), 2.80 (t, J=4.6Hz, 4H), 2.61 (t, J=7.5Hz, 2H), 2.53
(s, 4H), 2.28 (s, 3H), 2.05–2.08 (br, m, 2H); 13C NMR
(CD3OD, 125MHz): δ 169.3, 163.7, 155.6, 152.0, 148.3,
148.1, 135.2, 134.0, 128.9, 120.2, 110.9, 105.8, 104.5,
66.9, 66.2, 61.5, 55.4, 55.3, 53.4, 52.8, 49.3, 25.6, 19.6;
ESI-MS: m/z 535.5 [M+H]+. HRMS (ESI) m/z calcd for
C29H38N6O4 [M+H]+ 535.3027, found: 530.3018.
4-(4-(3-Methylbenzyl)piperazin-1-yl)-7-methoxy-6-[3-(4-mor-
pholinyl)propoxy]quinazoline (C4). Yield 15 %, 0.11g; yel-
1
low oily substance; H NMR (CD3OD, 500MHz): δ 8.39
(s, 1H), 7.12 (t, J=7.5Hz, 1H), 7.04–7.08 (m, 2H), 6.98–
7.00 (m, 2H), 6.94 (s, 1H), 4.02 (t, J=6.0Hz, 2H), 3.88 (s,
3H), 3.63 (t, J=2.1Hz, 4H), 3.58 (s, 4H), 3.42 (s, 2H),
2.40–2.53 (m, 6H), 2.25–2.40 (br, m, 7H), 1.96–1.99 (br,
m, 2H); 13C NMR (CD3OD, 125MHz): δ 163.4, 155.2,
152.1, 148.1, 148.0, 137.7, 137.1, 129.9, 128.0, 127.9,
126.4, 110.8, 105.9, 104.3, 66.9, 66.4, 62.7, 55.4, 55.3,
53.5, 52.7, 49.2, 25.8, 20.4; ESI-MS: m/z 492.5 [M+H]+.
HRMS (ESI) m/z calcd for C28H37N5O3 [M+H]+ 492.2969,
found: 492.2966.
N-(3-Chlorophenyl)-2-(4-(7-methoxy-6-(3-morpholinopropoxy)
quinazolin-4-yl)piperazin-1-yl)acetamide (C9). Yield 15%,
0.12g; yellow oily substance; 1H NMR (CD3OD, 500MHz):
δ 8.41 (s, 1H), 7.74 (t, J=2.5Hz, 1H), 7.40–7.42 (m, 2H),
7.17–7.21 (m, 1H), 6.97–7.03 (m, 2H), 4.06 (t, J=6.0Hz,
2H), 3.91 (s, 3H), 3.63–3.69 (m, 8H), 3.21 (s, 2H), 2.74 (t,
J=7.4Hz, 4H), 2.43–2.51 (m, 6H), 1.99–2.02 (br, m, 2H);
13C NMR (CD3OD, 125MHz): δ 169.5, 163.5, 155.3,
152.0, 148.1, 148.0, 139.4, 134.0, 129.8, 123.8, 119.7,
117.9, 110.9, 105.9, 104.3, 67.0, 66.3, 61.6, 55.4, 55.3,
53.4, 52.8, 49.3, 25.7; ESI-MS: m/z 555.4 [M+H]+.
HRMS (ESI) m/z calcd for C28H35ClN6O4 [M+H]+
555.2481, found: 555.2474.
4-(4-(4-Methylbenzyl)piperazin-1-yl)-7-methoxy-6-[3-(4-mor-
pholinyl)propoxy]quinazoline (C5). Yield 14%, 0.10g; yel-
1
low oily substance; H NMR (CD3OD, 500MHz): δ 8.59
(s, 1H), 8.42 (s, 1H), 7.85 (t, J=9.9Hz, 2H), 7.45 (t,
J=8.2Hz, 2H), 7.06 (s, 2H), 4.10 (t, J=5.7Hz, 2H), 3.93
(s, 3H), 3.74 (br, s, 6H), 3.65 (t, J=4.6Hz, 4H), 3.29 (s,
3H), 2.80 (t, J=4.6Hz, 4H), 2.54 (t, J=7.3Hz, 2H), 2.46 (s,
3H), 2.01–2.04 (br, m, 2H); 13C NMR (CD3OD, 125MHz):
δ 163.6, 155.4, 152.0, 148.2, 148.0, 136.9, 133.9, 129.3,
128.7, 110.9, 105.9, 104.4, 66.9, 66.4, 62.4, 55.4, 55.3,
53.5, 52.6, 49.2, 25.7, 19.9; ESI-MS: m/z 492.5 [M+H]+.
HRMS (ESI) m/z calcd for C28H37N5O3 [M+H]+ 492.2969,
found: 492.2962.
2-(4-(7-Methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)pipera-
zin-1-yl)-N-(2-nitrophenyl)acetamide (C10). Yield 8%, 0.07g;
yellow oily substance; 1H NMR (CD3OD, 500MHz): δ 8.66
(d, J=7.3Hz, 1H), 8.44 (s, 1H), 8.14 (d, J=6.6Hz, 1H), 7.66
(t, J=7.1Hz, 1H), 7.22 (t, J=7.8Hz, 1H), 7.11 (d, J=9.6Hz,
2H), 4.13 (t, J=6.2Hz, 2H), 3.94 (s, 3H), 3.84 (m, 4H), 3.67
(t, J=4.6Hz, 4H), 3.29 (s, 2H), 2.82 (t, J=4.6Hz, 4H), 2.57
(t, J=7.6Hz, 2H), 2.48 (s, 4H), 2.03–2.06 (br, m, 2H); 13C
NMR (CD3OD, 125MHz): δ 170.7, 163.6, 155.5, 152.0,
148.3, 148.1, 137.3, 135.3, 125.5, 123.7, 121.9, 110.9, 106.0,
104.6, 67.2, 66.3, 61.7, 61.5, 55.4, 53.4, 52.9, 49.5, 25.7;
ESI-MS: m/z 566.4 [M+H]+. HRMS (ESI) m/z calcd for
C28H35N7O6 [M+H]+ 566.2722, found: 566.2716.
2-(4-(7-Methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)
piperazin-1-yl)-N-phenylacetamide (C6). Yield 13%, 0.10g;
1
yellow oily substance; H NMR (CD3OD, 500MHz): δ
8.43 (s, 1H), 7.57 (d, J=8.0Hz, 2H), 7.26 (d, J=9.2Hz,
2H), 7.06 (s, 1H), 7.04 (s, 2H), 4.09 (t, J=6.0Hz, 2H), 3.92
(s, 3H), 3.72 (s, 4H), 3.64 (t, J=4.9Hz, 4H), 3.23 (s, 2H),
2.76 (t, J=4.5Hz, 4H), 2.54 (t, J=7.5Hz, 2H), 2.45 (s, 4H),
2.45–2.52 (m, 4H), 2.02–2.03 (br, m, 2H); 13C NMR
(CD3OD, 125MHz): δ 169.3, 163.5, 155.4, 152.0, 148.2,

Li et al.
7
nuclei were identified by nuclear fragmentation of con-
densed chromatin.27
Biological activity
The antitumor activities of the products against A549, K562,
HepG2, and PC-3 were tested at five concentrations (0.625,
1.25, 2.5, 5, and 10µM). Each test was done five times and
assessed according to the MTT assay. The cell cultures were
obtained from the Kunming Cell Bank. PC-3 was cultured at
37°C and 5% CO2 in Dulbecco’s modified eagle medium
supplemented with 10% fetal bovine serum and 1% penicillin
streptomycin. A549, K562, and HepG2 cells were cultured in
Roswell Park Memorial Institute (RPMI) 1640 supplemented
with the same mixture. The cells were inoculated in a 96-well
plate in 180µL aliquots at 3×104 cells/mL and cultivated for
1day. The plates were further incubated for 48h, and the
assay was terminated by the addition of 20µL of 5mg/mL
MTT. Incubation with the dye was continued for 4h, after
which time the medium was removed and the dye was
extracted from the cells with 150µL of DMSO. The absorb-
ance was measured with a Microplate Reader (TECAN infi-
nite M200 pro, TECAN, Switzerland) at 490nm wavelength.
The growth percentage was calculated on a plate by plate
basis for test wells relative to control wells. The sensitivity of
the cancer cells to each test compound was expressed in terms
of IC50, indicated as mean values±SD for three independent
experiments. Statistical analysis was performed using
GraphPad Prism (version 7) for the Student’s test. Values of
p<.05 were considered significant.
The wound healing assay was as follows. Cells were
seeded in a six-well plate and grown for 24h to 80% to 90%
confluence. Cells were washed twice with phosphate-
buffered saline (PBS), and the monolayers were scraped
with a micropipette tip to create a uniform scratch. Next, cells
were washed with PBS again to remove the detached cells.
The compounds were added in the RPMI medium at 5µM.
DMSO was used as a control. Digital images of the wounded
monolayers were obtained using a photomicroscope (Ti-S,
Nikon, Japan) at 0, 6, 12, and 24h. The unfilled scratched
zones were quantified by Java’s ImageJ software.23,24
Flow cytometry
To evaluate cell apoptosis, a double staining trial with pro-
pidium iodide (PI) and fluorescein isothiocyanate isomer
(FITC)-conjugated Annexin V (Solabio, Beijing, China) was
performed. Cells were seeded in a six-well plate at
1×106 cells/well and were stimulated by a range of com-
pound doses (5, 10, and 20µM). The original culture medium
was collected and the cells were cultured with trypsin, which
were subsequently washed twice in binding buffer. Next,
5µL of Annexin V-FITC and 5µL of PI were adopted for
dying cells for 15min at RT. The apoptotic A549 and PC-3
cells were observed via flow cytometry (BD Accuri™ C6,
BD Biosciences, USA) equipment.28,29
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
study was supported by the National Natural Science Foundation
of China (No. 21867004); the Project of the State Key Laboratory
of Functions and Applications of Medicinal Plants, Guizhou
MedicalUniversity(No. FAMP2018);andtheScienceTechnology
Program of Guizhou Province (No. 20185781).
ORCID iD
Zhen-Chao Wang
https://orcid.org/0000-0003-1859-0128
Supplemental material
Supplemental material for this article is available online.
Antiproliferative activity was measured by the clono-
genic survival assay after treating with the test compound.
Briefly, cells were seeded in a six-well plate at 3×105 cells/
well and were stimulated by a range of compound doses
(1.25, 2.5, 5, 10, and 20µM). The plates were further incu-
bated for 24h and the cells were plated in six wells at 1000
cells per well cultured for 12days, and the number of sur-
viving colonies (defined as a colony with >50 cells) was
counted. The survival fraction was calculated as the num-
ber of colonies divided by the number of cells seeded times
plating efficiency. Three independent experiments were
performed.25,26
Hoechst staining of the cultured cells was used to
observe apoptotic nuclei by evaluation of nuclear morphol-
ogy using a fluorescence inverted microscope. Cells were
fixed in 4% paraformaldehyde solution for 10min at RT.
Then the cells were incubated with Hoechst 33258 dye for
20min. Next, the cells were rinsed with precooling PBS
and were observed under a fluorescence inverted micro-
scope with 20µL of antifluorescent quencher after air-
drying. Typical nuclei showed non-condensed chromatin
distributed over the whole nucleus. In contrast, apoptotic
References
1. Zhou Y, Murphy DE, Sun Z, et al. Tetrahedron Lett 2004;
45: 8049.
2. Habib NS, Ismail KA and Abdel-Aziem T. Pharmazie 2000;
55: 495.
3. Georgey HH, Abdel-Gawad N and Abbas S. Molecules
2008; 13: 2557.
4. Xue S, McKenna J and Shieh WC. J Org Chem 2004, 69:
6474.
5. Jessy EM, Sambanthan AT and Alex J. Indian J Pharm Sci
2007; 69: 476.
6. Wu X, Li M and Tang W. Chem Biol Drug Des 2011; 78:
932.
7. Thomas ES and Mark AS. The Oncologist 2008; 13: 933.
8. Sharma MJ, Kumar MS and Murahari M. Arch Pharm 2019;
352: 1800381.
9. Friedmann B, Caplin M and Hartley JA. Clin Cancer Res
2004; 10: 6476.
10. Wu X, Li M and Qu Y. Bioorg Med Chem 2010; 18: 3812.
11. Tomizawa Y, Fujita Y and Tamura A. Lung Cancer 2010;
68: 269.
12. Dominguez-Escrig JL, Kelly JD and Neal DE. Clin Cancer
Res 2004; 10: 4874.

8
Journal of Chemical Research 00(0)
13. Zeng YD, Zhang L and Liao H. Asian Pac J Cancer P 2012; 22. Chandregowda V, Venkateshwara RG and Chandrasekhara
13: 909.
RG. Org Process Res Dev 2007; 11: 813.
14. Zheng Y, Li MD and Zhang SN. J Chem Res 2009; 2009: 388. 23. Chandregowda V, Rao VG and Reddy GC. Heterocycles
15. Yun CH, Boggon TJ and Li Y. Cancer Cell 2007; 11: 217.
2007; 71: 39.
16. Yang XB, Qin XL and Wang Q. Heterocycl Commun 2015; 24. Hu S, Li L and Huang W. Cancer Manag Res 2018; 10:
21: 233.
4603.
17. Mao Z, Zheng X and Qi Y. RSC Adv 2016; 6: 7723.
18. Xu Y, Liang P and Ur Rashid H. Med Chem Res 2019; 28: 1618.
25. Teixeira RR, Silva AMD and Siqueira RP. J Brazil Chem
Soc 2019; 30: 541.
19. Yamamoto Y, Tanabe K and Okonogi T. Chem Lett 1999; 26. Feng XP, Yi H and Li MY. Cancer Res 2010; 70: 3450.
28: 103.
27. Li WQ, Yu HY and Zhong NZ. In J Oncol 2015; 46: 1601.
20. Solomon VR, Hu C and Lee H. Bioorg Med Chem 2010; 18: 28. Kolomeichuk SN, Nizhnik YP and Makhova NN. Chem
1563.
Heterocycl Compd 2018; 54: 70.
21. Chen H, Zhang J and Hu P. Bioorg Med Chem 2019; 27: 29. Lee CH, Lee KH and Jang AH. Tuberculosis Respiratory
115081.
Diseases 2017; 80: 83.