Design, synthesis, and biological evaluation of novel quinazoline derivatives as anti-inflammatory agents against lipopolysaccharide-induced acute lung injury in rats

Article abstract of DOI:10.1111/cbdd.12454

Quinazoline has been reported to exhibit multiple bioactivities. The aim of this study was to discover new quinazoline derivatives with preventive effect on lipopolysaccharide-induced acute lung injury via anti-inflammatory actions. Thirty-three 4-amino quinazolin derivatives were synthesized and screened for anti-inflammatory activities in lipopolysaccharide-induced macrophages. The most potent four compounds, 6h, 6m, 6p, and 6q, were shown dose-dependent inhibition against lipopolysaccharide-induced TNF-α and IL-6 release. Then, the preliminary structure-activity relationship and quantitative structure-activity relationship analyses were conducted. To further determine the effects of quinazolines on acute lung injury treatment, lipopolysaccharide-induced acute lung injury model was employed. Male Sprague Dawley rats were pretreated with 6m or 6q before instillation of lipopolysaccharide. The results showed that 6m and 6q, especially 6q, obviously alleviated lung histopathological changes, inflammatory cells infiltration, and cytokines mRNA expression initiated by lipopolysaccharide. Taken together, this work suggests that 6m and 6q suppressed the lipopolysaccharide-induced acute lung injury through inhibition of the inflammatory response in vivo and in vitro, indicating that quinazolines might serve as potential agents for the treatment of acute lung injury and deserve the continuing drug development and research.

Full text of DOI:10.1111/cbdd.12454

Chem Biol Drug Des 2014
Research Article
Design, Synthesis, and Biological Evaluation of Novel
Quinazoline Derivatives as Anti-inflammatory Agents
against Lipopolysaccharide-induced Acute Lung Injury
in Rats
Jie Hu1,†, Yali Zhang1,†, Lili Dong2, Zhe Wang1,
Received 10 August 2014, revised 12 October 2014 and
accepted for publication 12 October 2014
Lingfeng Chen1, Dandan Liang1, Dengjian Shi1,
Xiaoou Shan2 and Guang Liang1,*
1Chemical Biology Research Center, School of
Acute lung injury (ALI) is a clinical syndrome characterized
Pharmaceutical Sciences, Wenzhou Medical University,
University Town, Wenzhou, Zhejiang 325035, China
2Department of Pediatric, Second Affiliated Hospital of
Wenzhou Medical University, 109 Xueyuan Road,
Wenzhou, Zhejiang 325027, China
by acute inflammatory process that includes neutrophil
accumulation, interstitial edema, and lung parenchymal
injury (1). It occurs mostly in young and is responsible for
thousands of adult and pediatric deaths annually world-
wide (2). Despite increasing insights into ALI, it still pre-
sents the high mortality rate of 30–40% and there are still
few effective measures and special drugs to treat it (1,3,4).
Therefore, the development of novel therapies for ALI is
urgently needed. Recently, evidence from various clinical
studies indicates that a complex network of inflammatory
cytokines and chemokines plays a major role in lung
injury process (5). Thus, treatments aimed at alleviating
non-specific inflammatory reactions may have potential
therapeutic.
*Corresponding author: Guang Liang,
wzmcliangguang@163.com
†These authors contribute equally to this work.
Quinazoline has been reported to exhibit multiple bio-
activities. The aim of this study was to discover new
quinazoline derivatives with preventive effect on lipo-
polysaccharide-induced acute lung injury via anti-
inflammatory actions. Thirty-three 4-amino quinazolin
derivatives were synthesized and screened for anti-
inflammatory activities in lipopolysaccharide-induced
macrophages. The most potent four compounds, 6h,
6m, 6p, and 6q, were shown dose-dependent inhibition
against lipopolysaccharide-induced TNF-a and IL-6
release. Then, the preliminary structure–activity rela-
tionship and quantitative structure–activity relationship
analyses were conducted. To further determine the
effects of quinazolines on acute lung injury treatment,
lipopolysaccharide-induced acute lung injury model
was employed. Male Sprague Dawley rats were pre-
treated with 6m or 6q before instillation of lipopolysac-
charide. The results showed that 6m and 6q, especially
6q, obviously alleviated lung histopathological
changes, inflammatory cells infiltration, and cytokines
mRNA expression initiated by lipopolysaccharide.
Taken together, this work suggests that 6m and 6q
suppressed the lipopolysaccharide-induced acute lung
injury through inhibition of the inflammatory response
in vivo and in vitro, indicating that quinazolines might
serve as potential agents for the treatment of acute
lung injury and deserve the continuing drug develop-
ment and research.
Numerous inflammatory mediators are involved in ALI.
Among them, IL-6 and TNF-a are considered the most
important ones in innate immune response (6). IL-6 is a
well-recognized inflammatory mediator upregulated in
patients with ALI (7). Upon exposure to lipopolysaccharide
(LPS), the lungs become a major source of systemic IL-6
as they become more permeable, allowing the facile trans-
location of IL-6 into the systemic circulation (8). TNF-a is
the most extensively studied cytokine member of TNF
superfamily. In vivo, excess generation of TNF-a facilitates
pro-inflammatory reactions (9). Increasing evidence has
emerged that excessive TNF-a production contributes to
the deterioration of inflammation in ALI, and elevated levels
of TNF-a has been found in bronchoalveolar lavage fluid
(BALF) from patients with ALI (10). Therefore, the inhibition
of the excessive production of pro-inflammatory cytokines,
especially TNF-a and IL-6, stands as a central therapeutic
strategy for ALI.
Quinazoline represents an interesting group of heterocy-
cles, which exhibit various biological activities including
containment of inflammatory disorders such as osteoarthri-
tis (11), neurodegenerative impairments (12), and inflam-
matory bowel syndrome (13), invoking the interest in
Key words: acute lung injury, anti-inflammation, IL-6, quinaz-
oline derivatives, TNF-a
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12454
1

Hu et al.
synthesis and evaluation of their analogs. Additionally, dif-
ferent known anti-inflammatory drugs such as proquazone,
fluoroquazone, and tryptanthrin are bearing quinazolinone
nucleus (Figure 1). Also, it has been reported that some
quinazoline derivatives possess potent anti-inflammatory
activity, especially inhibit NF-jB transcriptional activation
(14–16). Yet the relationship between anti-inflammatory
activity and structure of quinazoline remains uncertain.
Moreover, although numerous studies have addressed the
cytokine-inhibitory potential of quinazoline derivatives, its
ability to attenuate LPS-induced ALI remains poorly under-
stood. In this study, we synthesized a novel series of qui-
nazolinone derivatives, discussed the structure–activity
relationship, and evaluated the anti-inflammatory activity.
Active compounds were selected for the assessment of
their vivo anti-inflammatory effects on LPS-induced ALI
model.
washed with ethyl ether, and dried to yield 6.0 g product
(89.7%).
N-(3-fluorophenyl)-6-nitroquinazolin-4-amine (5a).
A
mixture of 3 (218.21 mg, 1 mM) and 3-fluoroaniline (4a,
1 mM) was heated and stirred at reflux in acetic acid
(10 mL) for 1 h. The yellow precipitate that formed was fil-
tered hot, washed with hot acetic acid, diethyl ether, and
dried to give the desired nitroquinazoline 5a as light yellow
solid (180 mg, rate 63.2%). m.p: 230.7–232.6 °C (227–
229 °C, lit.(17)). ESI-MS m/z: 284.8 (M)+, 306.8 (M+Na)+.
N-(3-chlorophenyl)-6-nitroquinazolin-4-amine (5b). By
the procedure described above for 5a using
3
(218.21 mg, 1 mM) and 3-chloroaniline (4b, 127.57 mg,
1 mM) to give 233 mg (77.7%) of yellow powder. m.p:
276.0–279.0 °C (278–281 °C, lit.(17,18)). ESI-MS m/z:
300.8 (M)+.
Experimental Section
N-(3-bromophenyl)-6-nitroquinazolin-4-amine (5c). By
the procedure described above for 5a using 3 (1091 mg,
5 mM) and 3-bromoaniline (4c, 851 mg, 5 mM) to give
1470 mg (85.0%) of yellow powder. m.p: 268.4–271.3 °C
(267–270 °C, lit.(18,19)). ESI-MS m/z: 346.8 (M)+.
Chemical Synthesis
Materials and Methods
In general, reagents, solvents, and other chemicals were
used as purchased without further purification. 5-Nitroan-
thranilonitrile (1), dimethylformamide dimethyl acetal (2),
and various substituted aniline (4a-4n) were purchased
from Alfa Aesar or Sigma–Aldrich. Other chemicals were
obtained from local suppliers and were used without fur-
ther purification. All reactions were monitored by thin-
layer chromatography (250 silica gel 60 F254 glass plates).
1H-NMR and 13C-NMR spectra were recorded on Bruker
600 MHz instruments, and the chemical shifts were pre-
sented in terms of parts per million with TMS as the
internal reference. Electron-spray ionization mass spectra
in positive mode (ESI-MS) data were obtained with a
Bruker Esquire 3000+ spectrometer. Chromatographic
purification was carried out on Silica Gel 60 (E. Merck,
70–230 mesh).
6-nitro-N-(3-(trifluoromethyl)phenyl)quinazolin-4-amine
(5d). By the procedure described above for 5a using 3
(218.21 mg, 1 mM) and 3-trifluoromethylaniline (4d,
161.12 mg, 1 mM) to give 193.1 mg (57.8%) of yellow
powder. m.p: 203.8–205.2 °C (203–205 °C, lit.(17)). ESI-
MS m/z: 334.9 (M)+.
6-nitro-N-(3,4,5-trifluorophenyl)quinazolin-4-amine
(5e). By the procedure described above for 5a using 3
(218.21 mg, 1 mM) and 3,4,5-trifluoroaniline (4e, 147 mg,
1 mM) to give 150.7 mg (47.1%) of yellow powder. m.p:
277.4–278.5 °C. 1H-NMR (600 MHz, DMSO-d6) d(p.p.m.):
9.527(s, 1H, Ar-H), 8.766 (s,1H, -N=CH-N-), 8.541 (d,
J = 9.0 Hz, 1H, Ar-H), 7.927 (d, J = 9.0 Hz, 1H, Ar-H),
7.857–7.885(m, 2H, Ar-H).13C-NMR(600 MHz, DMSO-d6)
d(p.p.m.):106.47, 106.64, 114.44, 120.52, 126.73,
129.57, 135.33, 144.66, 148.94, 149.00, 150.58, 152.75,
157.06, 158.29. ESI-MS m/z: 320.8(M)+, 342.8 (M+Na)+.
N-(2-cyano-4-nitrophenyl)-N,N-dimethylformamide
(3). 5-Nitroanthranilonitrile 1 (4.89 g, 30 mM) and dimeth-
ylformamide dimethyl acetal (5.36 g, 45 mM) in toluene
(50 mL) and the mixture was refluxed for 2 h. The resulting
mixture was cooled to room temperature and refrigerated
overnight. The yellow precipitate that formed was filtered,
N-(3-ethynylphenyl)-6-nitroquinazolin-4-amine (5f). By
the procedure described above for 5a using
3
(218.21 mg, 1 mM) and 3-aminophenylacetylene (4f,
Figure 1: Several anti-inflammatory drugs
bearing quinazolinone nucleus.
2
Chem Biol Drug Des 2014

Quinazoline Derivatives Alleviated ALI
117 mg, 1 mM) to give 195 mg (67.2%) of yellow powder.
m.p: 272.2–274.1 °C (271–272 °C, lit.(20)). ESI-MS m/z:
290.8 (M)+.
DMSO-d6) d(p.p.m.): 11.382 (s, 1H, -NH), 9.884 (s, 1H,
Ar-H), 8.873 (s, 1H, N=CH-N), 8.585 (d, J = 9.6 Hz,1H,
Ar-H), 8.423 (d, J = 6.6 Hz, 1H, Ar-H), 8.000 (d,
J = 8.4 Hz, 1H, pyridine-H), 7.939–7.966 (m, 1H, pyri-
dine-H), 7.305 (d, J = 7.2 Hz, 1H, pyridine-H). 13C-NMR
(600 MHz, DMSO-d6) d (p.p.m.):115.31, 119.53, 121.58,
122.99, 124.58, 126.96, 129.63, 130.00, 141.25,
144.25, 147.91, 157.07, 165.78. ESI-MS m/z: 301.8
(M+H)+.
6-nitro-N-(m-tolyl)quinazolin-4-amine (5g). By the
procedure described above for 5a using 3 (218.21 mg,
1 mM) and 3-methylaniline (4g, 107 mg, 1 mM) to give
208.6 mg (74.4%) of yellow powder. m.p: 247.6–249.3 °C
(250–252 or 267–270 °C, lit.(18,21)). ESI-MS m/z: 280.8
(M)+, 302.8 (M+Na)+.
N-(1-methyl-1H-indol-5-yl)-6-nitroquinazolin-4-amine
(5o). By the procedure described above for 5a using 3
(218.21 mg, 1 mM) and 1-methyl-1H-indol-5-amine (4o,
146 mg, 1 mM) to give 179 mg (56.1%) of red powder.
m.p: 241.2–242.8 °C. 1H-NMR (600 MHz, DMSO-d6) d
(p.p.m.): 10.463 (s, 1H, -NH), 9.669 (d, J = 2.4 Hz, 1H,
Ar-H), 8.617 (s, 1H, Ar-H), 8.524 (d, J = 9.0 Hz, 1H, Ar-
H), 7.955 (s, 1H, -N=C-N-), 7.885 (d, J = 9.0 Hz, 1H,
Ar-H), 7.475 (d, J = 8.4 Hz, 1H, Ar-H), 7.452 (d,
J = 9.0 Hz, 1H, Ar-H), 7.358 (d, J = 3.0 Hz,1H, indole-
H), 6.459 (d, J = 3.0 Hz, 1H, indole-H), 3.745(s, 3H,
-CH3). 13C-NMR (600 MHz, DMSO-d6) d (p.p.m.): 32.59,
100.45, 109.43, 114.39, 115.42, 118.27, 120.87,
126.37, 127.76, 129.26, 129.98, 130.39, 134.18,
144.29, 153.17, 158.07, 159.12. ESI-MS m/z: 319.8
(M)+, 341.7 (M+Na)+.
N-(4-morpholinophenyl)-6-nitroquinazolin-4-amine
(5h). By the procedure described above for 5a using
3
(218.21 mg, 1 mM) and 4-morpholinoaniline (4h,
90 mg, 0.5 mM) to give 152 mg (86.6%) of red
powder. m.p: 281.0–282.7 °C (lit. (22)). ESI-MS m/z:
352.0 (M)+.
N-(4-ethoxyphenyl)-6-nitroquinazolin-4-amine (5i). By
the procedure described above for 5a using
3
(218.21 mg, 1 mM) and 4-ethoxyaniline (4i, 137 mg,
1 mM) to give 211.7 mg (68%) of red powder. m.p:
203.0–205.8 °C (lit.(23)). ESI-MS m/z: 310.9 (M)+, 332.9
(M+Na)+.
N-(2,5-dimethoxyphenyl)-6-nitroquinazolin-4-amine
(5j). By the procedure described above for 5a using 3
(218.21 mg, 1 mM) and 2,5-dimethoxyaniline (4j, 153 mg,
1 mM) to give 305 mg (93.6%) of organic powder. m.p:
187.0–189.6 °C (lit.(24)). ESI-MS m/z: 326.9 (M)+, 348.8
(M+Na)+.
6-nitro-N-(1-propyl-1H-indol-5-yl)quinazolin-4-amine
(5p). By the procedure described above for 5a using 3
(436 mg, 2 mM) and 1-propyl-1H-indol-5-amine (4p,
348 mg, 2 mM) to give 316 mg (45.5%) of red powder.
m.p:183.6–185.2 °C. 1H-NMR (600 MHz, DMSO-d6) d
(p.p.m.): 10.459 (s, 1H, -NH), 9.667 (d, J = 2.4 Hz, 1H,
Ar-H), 8.614 (s, 1H, Ar-H), 8.527 (d, J = 9.0 Hz, 1H, Ar-
H), 7.937(s, 1H, -N=CH-N-), 7.887 (d, J = 9.0 Hz, 1H,
Ar-H), 7.517 (d, J = 9.0 Hz, 1H, Ar-H), 7.426 (d,
J = 8.4 Hz, 1H, Ar-H), 7.407 (d, J = 3.0 Hz, 1H, indole-
H), 6.463 (d, J = 3.0 Hz, 1H, indole-H), 4.112–4.166(m,
2H, N-CH2-), 1.783–1.819 (m, 2H, -CH2-), 0.846–0.870
6-nitro-N-(3,4,5-trimethoxyphenyl)quinazolin-4-amine
(5k). By the procedure described above for 5a using 3
(218.21 mg, 1 mM) and 3,4,5-trimethoxyaniline (4k,
183 mg, 1 mM) to give 160 mg (45%) of red powder. m.p:
230.2–231.2 °C (258 °C, lit.(25)). ESI-MS m/z: 356.9 (M)
+, 378.9 (M+Na)+.
N-(3,5-dimethoxyphenyl)-6-nitroquinazolin-4-amine
(5l). By the procedure described above for 5a using 3
(218.21 mg, 1 mM) and 3,5-dimethoxyaniline (4l, 153 mg,
1 mM) to give 301 mg (92.2%) of red powder. m.p: 223.2–
224.8 °C (220 °C, lit.(25)). ESI-MS m/z: 326.9 (M) +, 348.8
(M+Na)+.
(m, 3H, -CH3). 13C-NMR (600 MHz, DMSO-d6)
d
(p.p.m.): 11.18, 23.20, 47.20, 100.52, 109.57, 114.38,
115.55, 118.24, 120.87, 126.38, 127.80, 129.27, 129.39,
129.49, 133.55, 144.30, 153.18, 158.08, 159.15. ESI-MS
m/z: 348.0(M+H)+.
N-(1-allyl-1H-indol-5-yl)-6-nitroquinazolin-4-amine
(5q). By the procedure described above for 5a using 3
(278 mg, 1.28 mM) and 1-allyl-1H-indol-5-amine (4q,
220 mg, 1.28 mM) to give 175 mg (39.6%) of red powder.
m.p: 229.1–230.1 °C. 1H-NMR (600 MHz, DMSO-d6) d
(p.p.m.): 10.463 (s, 1H, -NH), 9.665 (d, J = 2.4 Hz, 1H,
Ar-H), 8.617 (s, 1H, Ar-H), 8.519–8.538 (d, J = 9.0 Hz,
1H, Ar-H), 7.955 (s, 1H, -N=C-N-), 7.467 (d, J = 8.4 Hz,
1H, Ar-H), 7.467 (d, J = 9.0 Hz, 1H, Ar-H), 7.429 (d,
J = 8.4 Hz, 1H, Ar-H), 7.384 (d, J = 3.0 Hz,1H, indole-H),
6.502 (d, J = 3.0 Hz, 1H, indole-H), 6.001–6.064 (m, 1H,
C-CH=C), 5.169 (d, J = 10.2 Hz, 1H, -C=CH2), 5.048
(d, J = 14.8 Hz,1H, -C=CH2), 4.849 (d, J = 5.4 Hz, 2H,
6-nitro-N-(quinolin-6-yl)quinazolin-4-amine (5m). By
the procedure described above for 5a using
3
(436 mg, 2 mM) and 6-aminoquinoline (4m, 288 mg,
2 mM) to give 432 mg (68%) of yellow powder.
m.p: >300 °C (333–335 °C, lit. (26)). ESI-MS m/z: 317.9
(M)+.
N-(6-chloropyridin-2-yl)-6-nitroquinazolin-4-amine
(5n). By the procedure described above for 5a using
3
(436 mg, 2 mM) and 2-amino-6-chloropyridine (4n,
256 mg, 2 mM) to give 328 mg (51.7%) of yellow
powder. m.p: 249.0–251.5 °C. 1H-NMR (600 MHz,
Chem Biol Drug Des 2014
3

Hu et al.
N-CH2-). 13C-NMR (600 MHz, DMSO-d6)
d
(p.p.m.):
N4-(4-ethoxyphenyl)quinazoline-4,6-diamine
48.17, 106.97, 109.80, 114.38, 115.57, 116.66, 118.38,
120.87, 126.39, 127.94, 129.29, 129.51, 130.08, 133.53,
134.46, 144.30, 153.17, 158.10, 159.15. ESI-MS m/z:
346.1(M+H)+.
(6i). Celadon powder, 78.3% yield, m.p:160.1–161.2 °C.
1H-NMR (600 MHz, DMSO-d6) d (p.p.m.): 9.291 (s, 1H,
-NH), 8.330 (s, 1H, N=CH-N), 7.669–7.776 (m, 2H, Ar-H),
7.486–7.517 (m, 1H, Ar-H), 7.351 (s, 1H, Ar-H), 7.209–
7.228 (m, 1H, Ar-H), 6.911–6.948 (m, 2H, Ar-H), 5.537 (s,
2H, -NH2), 4.000–4.043 (m, 2H, O-CH2), 1.357–1.319 (m,
3H, -CH3). 13C-NMR (600 MHz, DMSO-d6) d (p.p.m.):
14.71, 63.13, 101.35, 114.12, 114.12, 116.25, 123.67,
124.13, 124.13, 127.04, 132.15, 147.51, 149.29, 154.93,
156.52, 162.67. ESI-MS m/z: 280.8(M)+.
N4-(3-fluorophenyl)quinazoline-4,6-diamine (6a). 6-
Nitroquinazoline 5a (142 mg, 0.5 mM, 1.0 equiv) and iron
(140 mg, 2.5 mM, 5.0 equiv) were suspended in aqueous
ethanol (13 mL, 70% v/v) containing acetic acid
(0.45 mL, 7 mM, 14 equiv) and heated at reflux until dis-
appearance of starting material. The reaction mixture was
cooled to room temperature and alkalinized by addition of
concentrated ammonia or NaOH until the pH is adjusted
to 8–9. Insoluble material was removed by filtration
through celite, and the filtrate was evaporated under
reduced pressure. The water phase was extracted twice
with ethyl acetate, and the organic extracts were washed
with brine, dried over MgSO4, and concentrated under
reduced pressure without heating. Chromatography on
silica gel eluting with a mixture of ethyl acetate/petroleum
ether gave the expected product with 52.2% yield
(66.3 mg). m.p: 128.2–129.5 °C (122–124 °C, lit.(17)).
ESI-MS m/z: 254.7(M)+.
N4-(quinolin-6-yl)quinazoline-4,6-diamine (6m). Celadon
powder, 58.5% yield, m.p: 237.4–239.2 °C. 1H-NMR
(600 MHz, DMSO-d6) d(p.p.m.): 9.658 (s, 1H, -NH),
8.778–8.784 (m, 1H, Ar-H), 8.581 (s, 1H, Ar-H), 8.423 (s,
1H, Ar-H), 8.3015 (d, 1H, J = 7.8 Hz, Ar-H), 8.188 (d, 1H,
J = 9 Hz, Ar-H), 7.997 (d, 1H, J = 9 Hz, Ar-H), 7.571 (d,
1H, J = 9 Hz, Ar-H), 7.473–7.494 (m, 1H, Ar-H), 7.429 (s,
1H, Ar-H), 7.288–7.269 (m, 1H, Ar-H), 5.634 (s, 2H,
-NH2). ESI-MS m/z: 287.9 (M)+.
N4-(6-chloropyridin-2-yl)quinazoline-4,6-diamine
(6n). Celadon powder, 34.2% yield, m.p: 206.3–
209.8 °C. 1H-NMR (600 MHz, DMSO-d6) d(p.p.m.): 9.957
(s, 1H, -NH), 8.463 (s,1H, -N=C-N-), 8.391 (d, J = 8.6 Hz,
1H, Ar-H), 7.844–7.870 (m, 1H, Ar-H),7.584 (d,
J = 9.0 Hz, 1H, Ar-H), 7.481 (s, 1H, Ar-H), 7.285 (d,
J = 9.0 Hz, 1H, Ar-H), 7.157 (d, J = 7.8 Hz, 1H, Ar-H),
5.611 (s, 2H, -NH2). 13C-NMR (600 MHz,DMSO-d6) d
(p.p.m.):101.16, 113.44, 116.95, 117.83, 124.21, 128.66,
140.98, 143.03, 147.62, 147.80, 149.12, 153.07, 155.00.
ESI-MS m/z: 271.8(M)+.
N4-(3-chlorophenyl)quinazoline-4,6-diamine (6b). Cela-
don powder, 51.9% yield. m.p: 162.2–163.2 °C (235–
237 °C or 186–189 °C, lit.(18,27)). ESI-MS m/z: 270.7(M)+.
N4-(3-bromophenyl)quinazoline-4,6-diamine (6c). Celadon
powder, 77.9% yield. m.p: 199.1–200.4 °C (204–206 °C,
lit.(18,19)). ESI-MS m/z: 314.8, 316.7(M)+.
N4-(3-(trifluoromethyl)phenyl)quinazoline-4,6-diamine
(6d). Celadon powder, 37.5% yield. m.p: >250 °C (>250 °C,
lit.(17,27,28)). ESI-MS m/z: 304.9(M)+.
N4-(1-methyl-1H-indol-5-yl)quinazoline-4,6-diamine
(6o). Celadon powder, 41.7% yield. m.p: 217.4–
219.6 °C. 1H-NMR (600 MHz, DMSO-d6) d(p.p.m.): 9.253
(s, 1H, -NH), 8.243 (s, 1H, N=CH-N), 7.976 (s, 1H, Ar-H),
7.490–7.505 (m, 1H, Ar-H), 7.443–7.461 (m, 1H, Ar-H),
7.394–7.408 (m, 2H, Ar-H), 7.300 (s, 1H, Ar-H), 7.226–
7.207 (m, 1H, Ar-H), 6.411–6.415 (m, 1H, Ar-H), 5.488 (s,
2H, -NH2), 3.790 (s, 3H, -CH3). 13C-NMR (600 MHz,
DMSO-d6) d (p.p.m.): 32.60, 100.31, 101.39, 109.24,
114.48, 116.59, 118.19, 123.25, 127.86, 128.51, 130.05,
131.59, 133.64, 142.33, 147.02, 150.39, 156.72. ESI-MS
m/z: 289.8 (M)+.
N4-(3,4,5-trifluorophenyl)quinazoline-4,6-diamine
(6e). Celadon powder, 30.1% yield. m.p:245.1–
247.8 °C. 1H-NMR (600 MHz, DMSO-d6) d(p.p.m.): 9.575
(s, 1H, -NH), 8.418 (s,1H, -N=CH-N-), 7.939 (s, 2H, Ar-H),
7.570(d, J = 7.2 Hz, 1H, Ar-H), 7.284 (s, 2H, Ar-H), 5.683
(s, 2H, -NH2). 13C-NMR (600 MHz, DMSO-d6) d (p.p.m.):
100.44, 104.90, 105.06, 116.62, 124.08, 128.86, 136.56,
136.63, 142.75, 147.57, 149.20, 149.23, 150.60, 155.43.
ESI-MS m/z: 290.9(M)+.
N4-(3-ethynylphenyl)quinazoline-4,6-diamine
(6f). Celadon powder, 30.8% yield. m.p:250.0–251.2 °C
(lit. (29)). ESI-MS m/z: 260.7(M)+.
N4-(1-propyl-1H-indol-5-yl)quinazoline-4,6-diamine
(6p). Celadon powder, 50.8% yield, mp 201.5–
203.5 °C. 1H-NMR (600 MHz, DMSO-d6) d(p.p.m.): 9.232
(s, 1H, -NH), 8.223 (s, 1H, Ar-H), 7.943 (s, 1H, Ar-H),
7.493–7.350 (m, 4H, Ar-H), 7.214 (d, J = 2.4 Hz, 1H,
indole-H), 7.200 (d, J = 2.4, 1H, indole-H), 6.412 (s, 1H,
Ar-H), 5.477 (s, 2H, -NH2), 4.119–4.142 (m, 2H, -CH2-),
1.761–1. 809 (m, 2H, -CH2-), 0.838–0.863 (m, 3H, -CH3).
13C-NMR (600 MHz, DMSO-d6) d (p.p.m.): 11.18, 23.20,
47.16, 100.28, 101.36, 109.29, 114.52, 116.54, 118.08,
123.16, 127.86, 128.45, 129.06, 131.46, 132.89, 142.31,
N4-(m-tolyl)quinazoline-4,6-diamine
(6g). Celadon
powder, 62.2% yield. m.p:164.5–167.4 °C (245–248 °C or
173–175.5 °C, lit.(18,27)). ESI-MS m/z: 250.7(M)+.
N4-(4-morpholinophenyl)quinazoline-4,6-diamine
(6h). Celadon powder, 18.2% yield. m.p: 230.2–
232.4 °C (lit. (30)). ESI-MS m/z: 321.9(M)+.
4
Chem Biol Drug Des 2014

Quinazoline Derivatives Alleviated ALI
146.93, 150.30, 156.66. ESI-MS m/z: 318.1(M+H)+, 340.0
(M+Na)+.
Medical University (Wenzhou, China). Animals were
housed at a constant room temperature with a 12:12 hour
light–dark cycle and fed with a standard rodent diet and
water. The animals were acclimatized to the laboratory for
at least 7 days before used in experiments. Protocols
involving the use of animals were approved by the Wenz-
hou Medical University Animal Policy and Welfare Commit-
tee (Approval documents: 2013/APWC/0361).
N4-(3-bromophenyl)-N4-(3-methylbut-2-en-1-yl)quinazoline-
4,6-diamine (6q). Celadon powder, 25.5% yield, mp
136.3–138.2 °C. 1H-NMR (600 MHz, DMSO-d6) d(p.p.m.):
9.239 (s, 1H, -NH), 8.227 (s, 1H, Ar-H), 7.965 (s, 1H, Ar-
H), 7.486 (d, J = 9.1 Hz, Ar-H), 7.327–7. 425 (m, 4H, Ar-
H), 7.199–7.217 (m, 1H, Ar-H), 6.450 (s, 1H, Ar-H),
5.999–6.044 (m, 1H, CH2=CH-CH2-), 5.480 (s, 2H, -NH2),
5.168–5.21 (m, 2H, CH2=CH-CH2-), 4.821 (s, J = 5.4 Hz,
2H, CH2=CH-CH2-). 13C-NMR (600 MHz, DMSO-d6) d
(p.p.m.): 48.13, 100.75, 101.35, 109.53, 114.50, 116.55,
116.55, 118.18, 123.18, 127.95, 128.44, 129.09, 131.67,
132.87, 134.55, 142.29, 146.95, 150.27, 156.64. ESI-MS
m/z: 316.0 (M+H)+, 338.1 (M+Na)+.
Cells and regents
Mouse peritoneal macrophages (MPMs) were isolated as
descript in our previous study (31). Briefly, ICR mice were
stimulated by an i.p. injection of 6% thioglycollate solu-
tion (0.3 g beef extract, 1 g tryptone, 0.5 g NaCl dis-
solved in 100 mL ddH2O, and filtrated through 0.22-lm
filter membrane, 1.5 mL per mouse) and kept in a patho-
gen-free condition for 3 days before MPM isolation. Total
MPMs were harvested by washing the peritoneal cavity
with RPMI-1640 medium (8 mL per mouse), centrifuged,
and suspended in RPMI-1640 medium (Gibco/BRL life
Technologies, Eggenstein, Germany) with 10% FBS (Hy-
clone, Logan, UT, USA), 100 U/mL penicillin, and
100 mg/mL streptomycin. Non-adherent cells were
removed by washing with medium 3 h after seeding.
Before treatment, MPMs were cultured in 35-mm plates
and incubated overnight at 37 °C in a 5% CO2-humidi-
fied air.
N-(1-allyl-1H-indol-5-yl)-6-nitroquinazolin-4-amine
(7a). To a stirred solution of N-(3-bromophenyl)-6-nitro-
quinazolin-4-amine (5c, 347 mg, 1 mM) and allyl bromide
(158 mg, 1.2 mM) in acetonitrile (10 mL) was added potas-
sium carbonate (207 mg) and the mixture was refluxed at
80 °C. Stirring was continued for 8 h, and then, the solvent
was evaporated and the crude was dissolved in EtOAc, fol-
lowed by washed with saturated NH4Cl, dried (MgSO4),
and evaporated. The residue was chromatographed on sil-
ica gel to give 300 mg (59%) of 7a as a orange solid, m.p:
172.1–173.8 °C. 1H-NMR (600 MHz, DMSO-d6) d(p.p.m.):
8.928 (d, J = 3.0 Hz, 1H, Ar-H), 8.430 (d, 1H, J = 9.0 Hz,
Ar-H), 8.123(s, 1H, N=CH-N), 7.588 (d, J = 9.6 Hz, 1H, Ar-
H), 7.233–7.279 (m, 2H, Ar-H), 7.179 (d, J = 7.8 Hz, 1H,
Ar-H), 7.036 (d, J = 7.8 Hz, 1H, Ar-H), 5.986–6.041 (m,
1H, C=CH-C), 5.243–5.284 (m, 2H, CH2=C-), 4.773 (d,
J = 4.8 Hz, 2H, -CH2-). 13C-NMR (600 MHz, DMSO-d6) d
(p.p.m.): 50.98, 117.27, 118.13, 120.01, 121.14, 121.82,
121.86, 124.91, 125.12, 126.80, 130.20, 132.16, 142.32,
144.39, 151.48, 151.51, 152.14. ESI-MS m/z: 384.9(M)+.
Human lung epithelial BEAS-2B cells were obtained from
the American Type Culture Collection (Manassas, VA,
USA). Cells were incubated in Dulbecco’s modified Eagle’s
medium (Gibcoꢀ; Life Technologies, Carlsbad, CA, USA)
supplemented with 10% fetal bovine serum (Gibcoꢀ; Life
Technologies), 100 U/mL of penicillin, and 100 mg/mL of
streptomycin at 37 °C with 5% CO2. Lipopolysaccharide
was purchased from Sigma (Louis, MO, USA). Mouse
TNF-a and IL-6 enzyme-linked immunosorbent assay
(ELISA) kits were purchased from eBioscience, Inc. (San
Diego, CA, USA). All other chemicals were of reagent
grade.
N-(1-allyl-1H-indol-5-yl)-6-nitroquinazolin-4-amine
(7b). Using the procedure described for the preparation
of 7a, 284 mg (68.8%) of a orange solid was obtained
from 5c (347 mg, 1 mM) and 1-chloro-3-methylbut-2-ene
(208 mg,
2 mM),
m.p: 117.2–118.3 °C. 1H-NMR
(600 MHz, DMSO-d6) d(p.p.m.): 8.920 (d, J = 2.4 Hz, 1H,
Ar-H), 8.465 (d, 1H, J = 9.0 Hz, Ar-H), 8.141(s, 1H,
N=CH-N), 7.513 (d, J = 9.6 Hz, 1H, Ar-H), 7.227–7.267
(m, 2H, Ar-H), 7.016–7.033 (m, 1H, Ar-H), 7.163–7.181
(m, 1H, Ar-H), 5.263–5.284 (m, 1H, -C=CH-C-), 4.715 (d,
J = 6.6 Hz, 2H, -CH2-), 1.823 (s, 3H, -CH3), 1.718 (s, 3H,
-CH3). 13C-NMR (600 MHz, DMSO-d6) d (p.p.m.): 18.09,
25.33, 47.57, 116.96, 118.22, 120.08, 121.13, 121.81,
121.87, 124.91, 125.06, 126.93, 130.19, 137.82, 142.25,
144.37, 151.59, 151.60, 151.83. ESI-MS m/z: 412.9 (M)+.
Enzyme-linked immunosorbent assay
Mouse peritoneal macrophages (5 9 105 per plate) in 35-
mm plates were pretreated with 10 lM quinazoline deriva-
tives for 30 min and stimulated with LPS (0.5 lg/mL) for
24 h. After treatment, the culture media and cells were
collected separately. The levels of TNF-a and IL-6 in the
media were determined by ELISA according to the manu-
facturers’ instructions.
Quantitative structure–activity relationship
analysis
Animals
The methods and software used for the quantitative struc-
ture–activity relationship (SAR) model establishment and
analysis (including descriptor calculation and selection,
ICR mice and Sprague Dawley (SD) rats aged 6 weeks
were purchased from the Animal Center of Wenzhou
Chem Biol Drug Des 2014
5

Hu et al.
multiple linear regression analysis, and related software)
were described in our previous publication (32).
(GraphPad, San Diego, CA, USA). p-Value <0.05
(p < 0.05) were considered as significance. All experiments
were repeated more than three times.
Lipopolysaccharide-induced acute lung injury in
rats
Result and Discussion
The 24 healthy male SD rats were randomly divided into
four groups: control group, LPS group, 6m plus LPS
group, and 6q plus LPS group. Each group contained six
rats. 6m and 6q (20 mg/kg) were given orally for 7 days
prior to administration of LPS. Rats from the control and
LPS groups received an equal volume of 0.5% CMCNa.
After being anesthetized by diethyl ether, mice were intra-
tracheal instillation 50 lL LPS (20 lg/mL) to induce lung
injury. Control rats were given 50 lL physiological saline.
Twenty-four hours after LPS administration, animals were
euthanized to collect BALF and lung tissue samples. Col-
lection of BALF was performed three times through a tra-
cheal cannula with autoclaved physiological saline, instilled
up to a total volume of 3 mL.
Chemistry
A general synthetic approach was adopted for the rapid
generation of C4 aniline quinazoline with nitro group or
amino group at the 6-position of the quinazoline ring fol-
lowing the literature method (33). The synthetic route is
depicted in Schemes 1 and 2. Briefly, 2-cyano-4-nitro-ani-
line (1) was reacted with N, N-dimethylformamide dimethyl
acetal (2) to afford formamidine 3. Cyclization of the inter-
mediate 3 followed by corresponding amines in acetic acid
and gave compounds 5a-5n or 5o-5q. Reduction of 5a-
5j, 5m, 5n, or 5o-5q under the same condition composed
of Fe/EtOH/AcOH provided 6a-6j, 6m, 6n, and 6o-6q,
respectively. To investigate the effect of alkylation substitu-
ent on the R2, compounds 7a and 7b were designed and
prepared. The alkylation of N-(3-bromophenyl)-6-nitroqui-
nazolin-4-amine (5c) with allyl bromide or 1-chloro-3-
methylbut-2-ene in the presence of potassium carbonate
gave compounds 7a or 7b, respectively. In addition,
amines 4a-4n were commercially available, while N-substi-
tuted aminoindoles 4o-4q were prepared as procedure
described in lit (33). The structures of all compounds were
shown in Scheme 1 or 2. The new compounds were char-
acterized by 1H-NMR, 13C-NMR, and electron-spray ioni-
zation mass spectrometry [ESI-MS].
Histopathological evaluation
Pulmonary tissues were harvested 24 h after the injection
of LPS. Then, the samples were fixed with 4% paraformal-
dehyde for 24 h, embedded in paraffin, and sectioned
(5 lm). The tissue-sectioned samples were stained with
H&E and immunohistochemical. Pathological changes and
inflammatory cell infiltration were observed under the light
microscopy.
Bronchoalveolar lavage fluid collection, cell count,
and protein concentration
Anti-inflammatory evaluation of quinazoline
derivatives
Rats were anesthetized 24 h after being treated with LPS.
Bronchoalveolar lavage fluid was performed three times by
lavaged with of 1 mL of physiological saline. The lavage
fluid was centrifuged, and the supernatants were used for
analysis. Cell pellets were resuspended in 0.05 mL of
physiological saline and used for total cell counts on a
hemocytometer. Total protein concentration of BALF was
measured using a bicinchoninic acid (BCA) assay kit
(Pierce; Rockford, IL, USA) according to the manufacturer’s
instructions, with bovine serum albumin (BSA) as the
standard.
To evaluate the anti-inflammatory effect of 33 synthetic
quinazoline derivatives, we firstly monitored TNF-a and IL-
6 secretion induced by LPS in MPMs with or without the
pretreatment of compounds. Briefly, the cells were pre-
treated with target compounds at 10 lM or DMSO control
for 30 min and then activated with LPS (0.5 lg/mL) for
24 h. Culture medium was collected and the amount of
TNF-a and IL-6 release were measured by ELISA. The
preliminary screening results are shown in Figure 2. A
majority of 33 tested compounds exhibited significantly
anti-inflammatory activity against LPS-induced TNF-a and
IL-6 expression in vitro. Among these compounds, 5o, 5q,
6b, 6m, and 6o-6q exhibited strong inhibitory abilities in
IL-6 expression (inhibitory rate > 50%, Figure 2A). Mean-
while, four compounds, including 6h, 6m, 6p, and 6q,
showed the inhibitory effect on TNF-a expression with an
inhibition rate higher than 50% (Figure 2B). It is worth to
note that compounds 6h, 6m, 6p, and 6q exhibited great
inhibition against both TNF-a and IL-6 expression, so they
were chosen for further study.
Lung wet-to-dry weight (W/D) ratio
After rats were killed, the inferior lobe of right lungs was
excised, blotted dry, weighed to obtain the ‘wet’ weight
and then placed in an oven at 60 °C for 48 h to obtain
the ‘dry’ weight. To quantify edema, the ratio of wet lung
to dry lung was calculated.
Statistical analysis
Data are presented as mean Æ SEM. Statistics were ana-
lyzed using Student’s t-test mode in GRAPHPAD PRISM 5.0
We draw a preliminary SAR conclusion from the result
of anti-inflammatory screening. It is observed that
6
Chem Biol Drug Des 2014

Quinazoline Derivatives Alleviated ALI
Scheme 1: Synthetic and chemical
structure of quinazoline derivatives 5a-5l,
6a-6i, and 7a, 7b. Reagents and conditions:
(a) toluene, reflux, 2 h, 90%; (b) ArNH2,
AcOH, reflux, 3 h, 80–95%; (c) Fe,
CH3COOH, C2H5OH, 78 °C, reflux, 1 h, 50–
85%; (d) K2CO3, CH3CN, 80 °C, reflux, 8 h.
Scheme 2: Synthetic and chemical
structure of quinazoline derivatives 5m-5q
and 6m-6q. Reagents and conditions: (a)
ArNH2, AcOH, reflux, 3 h, 80–95%; (b) Fe,
CH3COOH, C2H5OH, 78 °C, reflux, 1 h, 50–
85%.
compounds in series 6, which has an amino group at
the 6-position of the quinazoline ring, inhibited IL-6 and
TNF-a release slightly stronger than those nitro
derivatives (series 5). The introduction of allyl or isopen-
tene group at R2 position (compounds 7a and 7b) could
slightly increase the cytokine-inhibitory ability. On the
Chem Biol Drug Des 2014
7

Hu et al.
A
Figure 2: Quinazoline analogs inhibited
lipopolysaccharide (LPS)-induced IL-6 (A)
and TNF-a (B) secretion in mouse peritoneal
macrophages. Macrophages were plated at
a density of 5.0 9 105/plate at 37 °C, and
5% CO2 overnight. Cells were pretreated
with quinazoline analogs (10 lM) for 30 min,
then treated with LPS (0.5 lg/mL) for 24 h.
TNF-a and IL-6 levels in the culture media
were measured by ELISA and were
B
normalized by the total protein. The results
were expressed as the percent of LPS
control. Each bar represents mean Æ SEM
of 3 independent experiments. Statistical
significance relative to LPS group was
indicated, *p < 0.05, **p < 0.01.
other hand, the electronic properties of the substituents
at R1 site seem to have little influence on bioactivity. For
example, compounds containing either electron-withdraw-
ing group (6b and 6c) or electron-donor moiety (6h)
showed strong anti-inflammatory activity. It is noteworthy
that the introduction of heterocycles or heterocyclic
groups at 4-amino-phenyl position could enhance the
anti-inflammatory activity, such as active compounds 5o-
5q, 6m, and 6o-6q.
mental values is illustrated in Figure 3. The statistically
significant models, Eq1 and Eq2, were obtained for anti-
TNF-a and anti-IL-6 activities of compounds, with rela-
tively high regression coefficients (R2) of 0.71 and 0.74,
respectively. The variables in the Eq1 model contained
the geometrical molecular descriptor WHIM (Weighted
Holistic Invariant Molecular Descriptor), 3D-MoRSE (3D
Molecule Representation of Structures based on Electron
diffraction Descriptor), and GETAWAY (GEometry, Topol-
ogy, and Atom-Weights AssemblY Descriptor), while in
the Eq2, the variables contained the geometrical molecu-
lar descriptors SAS (solvent-accessible surface) and GET-
AWAY and quantum chemistry descriptor EEVA
Quantitative structure–activity relationship
To further explore and investigate the SAR of these
compounds in the inhibition of TNF-a and IL-6 expres-
sion, a quantitative structure–activity relationship (QSAR)
was calculated for 33 quinazoline derivatives. During a
QSAR model study, the structure properties of com-
pounds are often represented by their molecular descrip-
tors. Statistically significant models with three variables
were derived for anti-TNF-a and IL-6 activity, respec-
tively. The scatter plot of predicted values versus experi-
(Electronic Eigenvalue Descriptor).
A
more detailed
description of these molecular descriptors is provided in
the experimental section. The QSAR results indicate that
molecular electronegativity and molecular surface may
play a pivotal role in the anti-inflammatory activity of
these compounds. The result of QASR study can guide
us to further design analogs with better bio-activity in
the future study.
A
B
Figure 3: Plots of predicted activity against
the corresponding experimental activity on
TNF-a (A) and IL-6 (B) inhibition. N, the
number of compounds taken into account in
the regression; R2, the multiple correlation
coefficient; Radj2, adjusted multiple
correlation coefficient; s, residual standard
error; and the F value is related to the
F-statistic analysis (Fisher’s test). The
numbers in parentheses mean the standard
deviation of the coefficients.
8
Chem Biol Drug Des 2014

Quinazoline Derivatives Alleviated ALI
A
Figure 4: Four active compounds inhibited
lipopolysaccharide (LPS)-induced IL-6 (A)
and TNF-a (B) release in a dose-dependent
manner. Mouse peritoneal macrophages
were plated at a density of 5.0 9 105/plate
at 37 °C, and 5% CO2 overnight. Cells were
pretreated with compounds at indicated
concentrations (2.5, 5, 10, and 20 lM) for
30 min, then treated with LPS (0.5 lg/ml) for
24 h. IL-6 and TNF-a levels in the culture
media were measured by ELISA and were
normalized by the total protein. The results
were expressed as the percent of LPS
control. Each bar represents mean Æ SEM
of three independent experiments. Statistical
significance relative to LPS group was
indicated, *p < 0.05, **p < 0.01.
B
Active compounds inhibit TNF-a and IL-6 release in
a dose-dependent manner
Among active compounds above, four compounds, 6h,
6m, 6p, and 6q, demonstrated the highest anti-inflamma-
tory activities in MPMs. Thus, they were chosen for further
evaluation of their dose-dependent inhibitory effects
against LPS-induced TNF-a and IL-6 release. Macrophag-
es were pretreated with 6h, 6m, 6p, and 6q in a series of
C
A
B
D
Figure 5: Effects of 6m and 6q on lipopolysaccharide (LPS)-mediated lung injury. Effects of 6m and 6q on the total protein concentration
(A), the relative amount of cells in bronchoalveolar lavage fluid (B), and the lung W/D ratio (C) of LPS-induced ALI rats. (D) Effects of 6m
and 6q on histopathological changes in lung tissues in ALI model induced by LPS. Rats were given an intragastric administration of 6m,
6q (20 mg/kg/day), or CMCNa 7 days prior to an intranasal administration of LPS. Then, rats were anesthetized and lung tissue samples
were collected at 24 h after LPS challenge for histological evaluation. These representative histological changes of the lung were obtained
from rats of different groups (hematoxylin and eosin staining, original magnification 200 9 ). *p < 0.05.
Chem Biol Drug Des 2014
9

Hu et al.
A
B
C
D
E
Figure 6: Effects of 6m and 6q on lipopolysaccharide (LPS)-mediated lung inflammation. (A) The changes of macrophage infiltration in
lung tissues after LPS treatment and 6m or 6q pretreatment. The inhibition of 6m and 6q on the expression of inflammatory genes on
lung tissue analyzed by mRNA assay. (B) TNF-a, (C) IL-6, (D) IL-1b, and (E) COX-2. Statistical significance relative to LPS group was
indicated, *p < 0.05, **p < 0.01.
concentrations (2.5, 5, 10, and 20 lM) for 30 min and sub-
sequently incubated with LPS (0.5 lg/mL) for 24 h. As
shown in Figure 4, pretreatment with 6h, 6m, 6p, and 6q
exhibited a dose-dependent inhibition on both TNF-a and
IL-6 release induced by LPS. This further validates that
these compounds have the potential to be used as anti-
inflammatory agents.
experimental section. Lipopolysaccharide has been widely
used to induce the animal models of ALI, which resemble
many characteristics of human ALI (34). Twenty-four
hours after LPS administration, the rats were euthanized
by chloral hydrate and the lung tissues and BALF were
collected. Then, the lung wet/dry ratio, total protein con-
centration, and relative amount of cells in the BALF were
evaluated. The results in Figure 5 indicated that LPS instil-
lation significantly increased total protein concentration
and relative amount of cells in BALF (Figure 5A,B). How-
ever, pretreatment with 6m and 6q obviously decreased
the total protein concentration compared with LPS group.
Meanwhile, 6m and 6q slightly inhibited relative amount
of cells in the BALF (Figure 5B) and lung W/D ratio
(Figure 5C).
Effects of 6 m and 6q on lipopolysaccharide-
mediated lung pathophysiologic changes
Two derivatives 6m and 6q showing the highest anti-
inflammatory activity were chosen for in vivo evaluation in
rats with ALI induced by intratracheal instillation of LPS.
The details of animal experiments were described in
A
B
C
D
Figure 7: Effects of 6m and 6q on the expression of inflammatory genes in the human lung epithelial cells. The levels of TNF-a (A), IL-6
(B), IL-1b (C), and COX-2 (D) were measured by mRNA analysis. The results were expressed as the percent of lipopolysaccharide (LPS)
control. Each bar represents mean Æ SEM of 3 independent experiments. Statistical significance relative to LPS group was indicated,
*p < 0.05, **p < 0.01.
10
Chem Biol Drug Des 2014

Quinazoline Derivatives Alleviated ALI
To evaluate the histological changes in LPS-treated rats,
lung tissues were subjected to H&E staining. Lung tissues
from the control group showed a normal structure and no
histopathological change under a light microscope (Fig-
ure 5D). Lipopolysaccharide instillation resulted in a signifi-
cant inflammatory cells infiltration, thickening of the
alveolar wall, and pulmonary congestion. However, LPS-
induced pathological changes were significantly attenuated
by either 6m or 6q treatment (Figure 5D). These results
indicated that 6m and 6q had protective effect against
LPS-induced ALI by reducing total protein concentration
and attenuating histopathological changes.
expression of these pro-inflammatory cytokines. These
results suggested that the protective effect of 6m and 6q
on LPS-induced ALI may be mainly attributed to their inhi-
bition of inflammatory response.
Conclusion
In summary, 33 quinazoline derivatives were synthesized
and their anti-inflammatory activities were evaluated in
LPS-induced MPMs. A majority of these synthetic com-
pounds exhibited significant anti-inflammatory activities,
and the most potent ones, 6h, 6m, 6p, and 6q, inhibit both
TNF-a and IL-6 release in a dose-dependent manner.
Discussion and conclusions were made regarding struc-
ture–activity relationships. Further, in vivo experiment
reveals that 6m and 6q, especially 6q, could attenuate
LPS-induced ALI in rats, via decreasing inflammatory cyto-
kine production, protein concentration in the BALF, patho-
logical changes, and macrophage infiltration. These
present the possibility that quinazolines might serve as
potential agents for the treatment of ALI. Although the anti-
inflammatory mechanism and underlying targets are still
unknown, the beneficial effects of these compounds on
LPS-induced inflammation make quinazoline one of impor-
tant leads in the continuing drug development and
research.
6m and 6q reduce the expression of inflammatory
genes in vivo and in vitro
To evaluate the effect of 6m and 6q on the LPS-induced
inflammation in lung tissue, we examined the inflammatory
cells infiltration and cytokines expression. Macrophages
infiltration (Figure 6A) was studied by examining the CD68
immunostaining of lung tissue. Lipopolysaccharide treat-
ment aggravated lung macrophages infiltration, which was
significantly attenuated by pretreatment with 6m or 6q.
Inflammatory cytokines mRNA levels in lung tissues were
measured by real-time qPCR. The data showed that the
expression of TNF-a, IL-6, IL-1b, and COX-2 was
increased in the LPS alone group. However pretreatment
with 6m or 6q inhibited the expression of these inflamma-
tory genes (Figure 6B–E). Using the Beas-2B human lung
epithelial cell line, we further confirmed the anti-inflamma-
tory effects of 6m and 6q. Figure 7 showed that LPS
markedly increased the inflammatory genes expression
and preincubated 6m (10 lM) markedly reduced the LPS-
induced production of TNF-a, IL-1b, and COX-2
(p < 0.01), while 6q (10 lM) obviously decreased the LPS-
induced release of all four inflammatory genes (p < 0.05 or
p < 0.01). There is no conclusion to account for the differ-
ence between 6m and 6q on LPS-induced mRNA expres-
sion, maybe the introduction of allyl group increase
solubility behavior of 6q leading to improved inhibition on
the inflammatory genes expression. Taken together, 6m
and 6q, especially 6q, may protect against LPS-induced
ALI by attenuating inflammatory response.
Acknowledgments
Financial support was provided by the National Natural
Science Funding of China (81302642 and 21272179), Zhe-
jiang Provincial Natural Science Funding (LQ14H310003),
High-level Innovative Talent Funding of Zhejiang Department
of Health (G. L.), National ‘863’ project (2011AA02A113).
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