Synthesis and evaluation of the anti-inflammatory activity of quinoline derivatives

Article abstract of DOI:10.1007/s00044-015-1323-y

Three types of quinoline derivatives, quinolin-2-one, 1-oxa-3,5-diaza-anthracen-6-one, and cyclopenta[a]anthracene, were designed and synthesized as potential anti-inflammatory agents. Their anti-inflammatory activities were evaluated using the xylene-ind

Full text of DOI:10.1007/s00044-015-1323-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1323-y
ORIGINAL RESEARCH
Synthesis and evaluation of the anti-inflammatory activity
of quinoline derivatives
•
Xiang Wen Shi-Ben Wang Da-Chuan Liu
•
•
•
Guo-Hua Gong Zhe-Shan Quan
Received: 15 September 2014 / Accepted: 4 January 2015
Ó Springer Science+Business Media New York 2015
Abstract Three types of quinoline derivatives, quinolin-
2-one, 1-oxa-3,5-diaza-anthracen-6-one, and cyclopenta
[a]anthracene, were designed and synthesized as potential
anti-inflammatory agents. Their anti-inflammatory activi-
ties were evaluated using the xylene-induced ear-edema
test in mice. Pharmacological analyses showed that com-
pounds 3-(4-methyl-benzyl)-3,4,7,8-tetrahydro-2H,5H-1-
oxa-3,5-diaza-anthracen-6-one (3g) and 9-(4-fluoro-phenyl)-
5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,11b-tetraaza-cyclopenta[a]
anthracene (6d) exhibited the greatest anti-inflammatory
activity (63.19 and 68.28 % inhibition, respectively, 30 min
after intraperitoneal administration) and were more potent
than the reference drug ibuprofen. The peak activity of 3g and
6d was observed 3 h after oral administration, and the com-
pounds exhibited stronger anti-inflammatory activity than
ibuprofen at 50 mg/kg at this time point. The most effective
compound, 6d (hydrochloride salt), was evaluated in lipo-
polysaccharide (LPS)-stimulated RAW264.7 mouse macro-
phages, in which it significantly inhibited LPS-induced
production of tumor necrosis factor-a and interleukin-6 in a
dose-dependent manner.
Keywords Anti-inflammatory ꢀ IL-6 inhibition ꢀ
Mannich reaction ꢀ Quinoline ꢀ Synthesis ꢀ
TNF-a
Introduction
Inflammation remains a common and life-threatening
problem in cases of allergy, autoimmune diseases, and
rejection of transplanted organs (Huerre and Gounon,
1996). Treatment options for inflammatory diseases pro-
duce adverse effects and show a lack of efficacy. Non-
steroidal anti-inflammatory drugs (NSAIDs) are very use-
ful in the treatment of acute and chronic inflammation (Sng
and Schug, 2009), pain (Zahradnik et al., 2010), and fever
(Eccles, 2006), but long-term use of NSAIDs is associated
with gastrointestinal lesions, bleeding, and nephrotoxicity
(Mukherjee et al., 2001). The discovery of novel and safe
anti-inflammatory drugs remains a challenging goal (Vane
and Botting, 1995). There is an increasing need for novel
structures that may be used in the design of new, potent,
and less toxic anti-inflammatory agents.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1323-y) contains supplementary
material, which is available to authorized users.
X. Wen ꢀ S.-B. Wang ꢀ D.-C. Liu ꢀ Z.-S. Quan (&)
Key Laboratory of Natural Resources and Functional Molecules
of the Changbai Mountain, Affiliated Ministry of Education,
Yanbian University College of Pharmacy, Yanji 133000,
People’s Republic of China
Quinoline derivatives have a wide spectrum of biolog-
ical activities, including anti-microbial (Rajanarendar
et al., 2012), anti-depressant (Sun et al., 2011), anti-con-
vulsant (Guan et al., 2008), anti-malarial (N’Da et al.,
2010), and anti-inflammatory effects (El-Gazzar et al.,
2009). Accordingly, much effort has been spent developing
new and more effective quinoline-based therapeutics. In
our studies, 6-alkyl-3,4-dihydro-1H-quinolin-2-one quino-
line derivatives (2a–2i) were obtained by alkylation of
e-mail: zsquan@ybu.edu.cn
G.-H. Gong (&)
Medicinal Chemistry and Pharmacology Institute, Inner
Mongolia University for the Nationalities, No. 22 Huolin He
Street, Tongliao City 028000, Inner Mongolia, People’s
Republic of China
e-mail: gongguohua0211@163.com
6-hydroxy-3,4-dihydro-1H-quinolin-2-one
and
alkyl
123

Med Chem Res
halides, and some of these derivatives were found to pos-
sess modest anti-inflammatory activity. It has been reported
that small heterocycles such as oxazine (e.g., oxazine
(Barco et al., 2007; Kourounakis et al., 2010; Liu and
Ying, 2010) and triazole (Abdel-Aziz et al., 2013; Abuo-
Rahma Gel et al., 2014) can result in increased biological
activity. Therefore, to further investigate the biological
activity of quinoline derivatives, we introduced an oxazine
ring at the sixth and seventh positions of the quinoline ring
and designed a novel series of 1-oxa-3,5-diaza-anthracen-
6-one (3a–3l) derivatives. Furthermore, we also introduced
a triazole ring at the fifth and sixth positions of the 1-oxa-
3,5-diaza-anthracene ring and designed another novel ser-
ies of cyclopenta[a]anthracene (6a–6l) derivatives (Fig. 1).
We report the synthesis of three types of quinoline
derivatives and the evaluation of their anti-inflammatory
activity in a xylene-induced ear-edema test in vivo. The
inflammatory response after lipopolysaccharide (LPS)
challenge was associated with the release of pro-inflam-
matory cytokines and other inflammatory mediators,
including tumor necrosis factor-a (TNF-a) and interleukin-
6 (IL-6) (Soromou et al., 2012). Macrophages are an
essential interface between innate and adaptive immunity
during the inflammatory response. Herein, we studied
active compounds to investigate their protective effects
against inflammation in LPS-stimulated RAW264.7 mouse
macrophages in vitro.
Results and discussion
Chemistry
Based on previous studies carried out by our research team,
we designed three series of 6-alkyl-3,4-dihydro-1H-quin-
olin-2-one (2a–2j), 1-oxa-3,5-diaza-anthracen-6-one (3a–
3l), and cyclopenta[a]anthracene (6a–6l) quinoline deriv-
atives and synthesized them according to Scheme 1.
Compounds 2a–2j were synthesized according to a method
described previously (Sun et al., 2006; Wei et al., 2010;
Sun et al., 2013). The starting material, compound 1, was
treated with benzyl chloride in a solution of sodium
hydroxide in ethanol to yield 6-alkoxy-3,4-dihydroquino-
lin-2(1H)-one (2a–2i). Next, compounds 3a–3l were syn-
thesized by the Mannich reaction. Subsequently, the
starting material, compound 1, was reacted with phospho-
rous pentasulfide in the presence of triethylamine in ace-
tonitrile to obtain compound 4. A key substrate for the
R'
R
O
R
O
O
Ν
N
N
H
O
N
H
O
N
N
N
n
n
2a-2i
3a-3l
6a-6l
Fig. 1 Target compounds 2a–2i, 3a–3l, and 6a–6l
HO
HO
HO
P2S5
HCONHNH2
N
H
O
Et3N
N
N
H
S
N
N
4
1
5
HCHO
RCl
O
RNH2
RNH2
HCHO
14
4
7
3
15
18
5
8
4
R'
9
8
O
5
6
6
7
10
9
O
10
3
2
15
16
11
14
13
12
2
R
R
Ν
N
16
17
13
N
H
O
1
N
n
N
N
11
n
N
H
O
17
12
1
2a-2i
3a-3l
6a-6l
Scheme 1 Synthesis of compounds 2a–i, 3a–l, and 6a–l. R0: 2a H,
2b p-CH3, 2c p-OCH3, 2d o-F, 2e m-F, 2f p-F, 2g o-Cl, 2h m-Cl, 2i p-
Cl; R: n = 0, 3a–6a H, 3b–6b p-CH3, 3c–6c p-OCH3, 3e–6e p-F;
n = 1, 3f–6f H, 3g–6g p-CH3, 3h–6h p-OCH3, 3i–6i p-F, 3j–6j p-Cl,
3k–6k p-CN; 3e–6e p-F; n = 2, 3l–6l H
123

Med Chem Res
Pharmacology
production of 6a–6l, compound 5, was prepared by an
established procedure involving the reaction of compound
4 with carbohydrazide in boiling butanol (Xie et al., 2005).
Finally, test derivatives 6a–6l were prepared by the Man-
nich reaction of compound 5 with diverse amines and
formaldehyde in a solution of ethanol. All synthesized
In vivo inhibition of xylene-induced ear edema
In the primary screening, we used dimethyl sulfoxide
(DMSO) as the vehicle and ibuprofen as the reference drug.
In this test, all synthesized compounds were screened in a
xylene-induced ear-edema test in mice, in which anti-
1
compounds were confirmed by H NMR, 13C NMR, IR
spectroscopy, and MS.
Table 1 Anti-inflammatory activity of compounds 2a–i, 3a–l, and 6a–l after i.p. administration
Comp.
R
Dose (mg/kg)
Number of mice
Edema mean SEM (mg)
Inhibition rate (%)
2a
2b
2c
H
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
5.25 1.10**
5.00 1.04*
8.07 0.72
56.18
58.26
32.64
40.73
57.85
60.77
46.74
59.52
28.88
55.51
39.90
36.56
33.22
45.24
43.82
63.19
–
p-CH3
p-OCH3
o-F
2d
2e
7.10 1.47
m-F
5.05 1.36*
4.70 0.26**
6.38 1.17*
4.85 1.02**
8.52 1.81
2f
p-F
2g
2h
2i
o-Cl
m-Cl
p-Cl
H
3a
3b
3c
5.33 1.42**
7.20 1.14*
7.60 0.92*
8.00 0.98*
6.56 1.48*
6.73 0.52**
4.41 1.16**
10.42 0.91
6.37 1.09**
8.35 1.01
p-CH3
p-OCH3
p-F
3d
3e
p-Cl
H
3f
3g
3h
3i
p-CH3
p-OCH3
p-F
46.83
30.30
20.37
26.88
21.70
42.40
–
3j
p-Cl
p-CN
H
3k
3l
9.54 1.38
8.76 1.88
6a
6b
6c
H
9.38 1.07
p-CH3
p-OCH3
p-F
6.90 0.99**
10.75 1.97
3.80 1.11**
6.30 1.46**
5.43 0.89**
4.93 0.82**
4.48 1.22**
7.98 1.21*
9.30 1.06
6d
6e
68.28
47.41
54.67
58.85
62.60
33.39
22.37
49.50
38.98
–
p-Cl
H
6f
6g
6h
6i
p-CH3
p-OCH3
p-F
6j
p-Cl
p-CN
H
6k
6l
6.05 1.06**
7.31 1.54*
11.98 1.16
4.42 0.81**
DMSO
Ibuprofen
–
–
63.11
– no anti-inflammatory activity
* 0.01 \ p \ 0.05; ** p \ 0.01 compared to the vehicle group
123

Med Chem Res
Table 2 Anti-inflammatory activity of compounds 3g and 6d administered orally at different times before xylene application
Time (h)
Dose (mg/kg)
Inhibition (%)
3g
6d
Ibuprofen
1
100
100
100
100
100
100
24.88
34.19
58.13*
30.33*
23.27
–
28.18
40.76*
64.73*
33.53**
31.59*
–
20.53
26.65
41.58
21.17
20.81
–
2
3
4
5
24
– No anti-inflammatory activity
* 0.01 \ p \ 0.05; ** p \ 0.01 compared to ibuprofen at the corresponding time
inflammatory activity was assessed by the ability of each
compound to prevent edema. Table 1 showed that most of
the newly synthesized compounds had modest anti-
inflammatory activity when administered at a dose of
100 mg/kg via the intraperitoneal route. Compounds 3-(4-
methyl-benzyl)-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-diaza-
anthracen-6-one (3g) and 9-(4-fluoro-phenyl)-5,8,9,10-tet-
rahydro-4H-7-oxa-2,3,9,11b-tetraaza-cyclopenta[a]anthra-
cene (6d) inhibited inflammation by 63.19 and 68.28 %,
respectively, and were thus more effective than ibuprofen
(63.11 %). Among the novel compounds, 2a, 2f, 2h, 3a, 3f,
3i, 6b, 6e–h, and 6k possessed anti-inflammatory activity
similar to that of ibuprofen. In addition, 2b, 2e, 2g, 3b–d,
3e, 6i, and 6l revealed modest anti-inflammatory activity in
comparison with the vehicle group. The other compounds
did not exhibit anti-inflammatory activity.
Finally, at a dose of 100 mg/kg administered via the
intraperitoneal route, 6d (hydrochloride salt) continued to
exhibit potent anti-inflammatory activity (Fig. 4). How-
ever, in this test, 3g (hydrochloride salt) showed strong
toxicity: All 10 mice in the group treated with 3g died after
the compound was administered.
In vitro inhibition of cytokine (TNF-a and IL-6) production
To ascertain the toxicity of 6d (hydrochloride salt) and 3g
(hydrochloride salt) to RAW264.7 cells, cell viability was
evaluated by the MTT assay after incubating RAW264.7
cells for 18 h in the absence or presence of LPS. We found
that 6d had no effect on the viability of RAW264.7 cells at
10, 20, or 40 lg/mL. However, concentrations of 6d up to
50 lg/mL reduced cell viability. The hydrochloride salt 3g
was found to reduce cell viability at 2, 10, 20, 40, and
50 lg/mL. These observations showed that the effects of
6d were not due to cytotoxicity.
On the basis of the considerable anti-inflammatory
activity demonstrated by the results listed in Tables 1, two
outstanding quinoline derivatives, 3g and 6d, were chosen
for further screening, whereby 100 mg/kg of each com-
pound was administered via the oral route at multiple time
points (1, 2, 3, 4, and 5 h) prior to xylene application.
Similarities in anti-inflammatory activity were obvious
among 3g, 6d, and ibuprofen (Table 2; Fig. 2). As the
interval lengthened, the anti-inflammatory activities of 3g,
6d, and ibuprofen first increased and then declined. Peak
activity was observed at 3 h, and they did not exhibit anti-
inflammatory activity at 5 h. Compounds 3g and 6d
showed stronger anti-inflammatory activity than ibuprofen
at all intervals (1–5 h), and these differences were espe-
cially significant at the 3 h interval.
LPS is a major pro-inflammatory constituent of the cell
wall of gram-negative bacteria and has a key role in many
inflammatory diseases (Asti et al., 2000). LPS enters the
bloodstream and elicits inflammatory responses that may
lead to shock and death (Hudson et al., 1995). Production
of pro-inflammatory cytokines by macrophages in response
to inflammatory stimuli and microbial products is well
established (Kim et al., 2002). TNF-a and IL-6 are the
primary cytokines involved in inflammatory processes in
LPS-stimulated RAW264.7 cells. TNF-a is the earliest and
primary endogenous mediator involved in inflammation in
RAW264.7 cells (Yang et al., 2004), and it promotes the
aggregation and activation of leukocytes at sites of
inflammation and stimulates the release of various
inflammatory mediators (Jiang et al., 2004). IL-6 is an
important inflammatory mediator released from monocytes
and vascular endothelial cells. In the present study, cells
were treated with LPS alone or LPS with different con-
centrations of the test compounds for 24 h, and levels of
In a separate experiment, the inhibitory effects of
compounds 3g, 6d, and ibuprofen against ear inflammation
were evaluated and compared at lower doses (50 mg/kg)
administered via the oral route 3 h before xylene applica-
tion (Table 3; Fig. 3). Compounds 3g and 6d possessed
more activity than ibuprofen at a dose of 50 mg/kg when
administered 3 h prior to xylene administration.
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Med Chem Res
Fig. 2 Anti-inflammatory
activities of the compounds
were tested in the xylene-
induced ear-edema test in mice.
3g, 6d, and ibuprofen were
administrated p.o. at a dose of
100 mg/kg, and edema was
quantified at different intervals
(1, 2, 3, 4, and 5 h). *p \ 0.05;
**p \ 0.01 versus the control
#
group, and p \ 0.05 versus the
ibuprofen group at the same
time point
Table 3 Anti-inflammatory activity of compounds 3g and 6d administered orally at different doses
Time (h)
Dose (mg/kg)
Inhibition (%)
3g
6d
Ibuprofen
3
3
100
50
56.41*
23.92
60.55*
28.72*
39.84
18.62
– No anti-inflammatory activity
* 0.01 \ p \ 0.05; * p \ 0.01 compared to ibuprofen at the corresponding dose
TNF-a and IL-6 in culture supernatants were detected
using enzyme-linked immunosorbent assays (ELISA)
(Fig. 5a, b). In comparison with the control group, treat-
ment of RAW264.7 cells with LPS alone led to a signifi-
cant increase in cytokine production. However, upon
treatment with LPS and different concentrations of 6d,
levels of TNF-a and IL-6 fell significantly in a dose-
dependent manner, which suggested that the anti-inflam-
matory activity of 6d was due to inhibition of production of
TNF-a and IL-6.
3a–3l. These findings suggest that the introduction of an
oxazine ring to the derivatives conferred anti-inflammatory
activity, and that the triazole ring dramatically improved
this activity.
Next, we assessed the SARs of all compounds sequen-
tially for each series. First, comparing derivatives 2a–2i
with different halogen substituents on the benzene ring, the
order of activity was m-Cl [ p-F [ m-F [ o-Cl [
o-F [ p-Cl. Comparing compounds with different electron-
donating substituents, the order of activity was p-CH3 [
p-OCH3. Electron-donating groups seemed to be a more
beneficial structural feature than electron-withdrawing
groups for anti-inflammatory activity.
Structure–activity relationships
By evaluating the activities of the synthesized test com-
pounds, we obtained structure–activity relationships
(SARs). Compounds 2a–2i, 2f, and 2h showed significant
anti-inflammatory activity with inhibitory rates of 56.18,
60.77, and 59.52 %, respectively. These results showed
that quinolin-2-one possessed potential anti-inflammatory
activity. As mentioned above, compounds 3a–3l and 6a–6l
were prepared to evaluate the influence of the oxazine ring
and the triazole ring, respectively, on anti-inflammatory
activity. Among compounds 3a–3l, 3a, 3f, 3g, and 3i
showed significant anti-inflammatory activity, and the anti-
inflammatory effect of 3g was 63.19 %. Among 6a–6l,
compounds 6b, 6d, 6e, 6f, 6g, 6h, and 6k possessed sig-
nificant anti-inflammatory activity, and the anti-inflamma-
tory effect of 6d was 68.28 %. Compounds 6a–6l
possessed more potential anti-inflammatory activity than
Second, for compounds 3a–3l (with an oxazine ring),
different substitutions at the third position appeared to
greatly influence the activity. Comparing derivatives without
substitutions on the benzene ring, the order of activity was
–C6H5 [ –CH2C6H5 [ –CH2CH2C6H5. For derivatives
with different electron-donating substituents (methyl and
methoxy), the order of activity was –CH3 [ –OCH3.
Moreover, most of the derivatives with electron-withdraw-
ing substituents on the benzene ring showed modest activity,
and the order of activity was F [ Cl [ CN.
Third, the effects of different substitutions at the ninth
position on the activities of compounds 6a–6l (with oxazine
and triazole rings) were observed. Comparing derivatives
without substitutions on the benzene ring, the order of
activity was –CH2C6H5 [ –CH2CH2C6H5 [ –C6H5. Com-
parison of halogen-substituted derivatives indicated that
123

Med Chem Res
Fig. 3 3g, 6d, and ibuprofen were evaluated and compared at lower
doses (50 mg/kg) that were orally administered 3 h before xylene
application. *p \ 0.05; **p \ 0.01 versus the control group
Fig. 5 Cells were treated with LPS alone or LPS plus different
concentrations of the test compounds for 24 h. The values presented
are the mean SEM of three independent experiments. ##p \ 0.01
versus the control group; **p \ 0.01 versus the LPS group
Fig. 4 6d and 6da (hydrochloride salt) were homogenized with
physiological saline at a dose of 100 mg/kg and administered i.p. to
mice. **p \ 0.01, ***p \ 0.001 versus the control group, and
##p \ 0.01 versus the ibuprofen group
showed weak anti-inflammatory activity; most compounds
showed modest anti-inflammatory activity. In parti-
cular,
3-(4-methyl-benzyl)-3,4,7,8-tetrahydro-2H,5H-1-
different halogen atoms contributed to the anti-inflammatory
activity in the order F [ Cl. Comparing derivatives with
different electron-donating substituents (methyl and meth-
oxy), the order of activity was –CH3 [ –OCH3 for the phe-
nyl groups and –CH3 = –OCH3 for the benzyl groups.
Finally, the electron-donating and electron-withdrawing
groups made similar contributions to the anti-inflammatory
activity.
oxa-3,5-diaza-anthracen-6-one (3g) and 9-(4-fluoro-
phenyl)-5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,11b-tetraaza-
cyclopenta[a]anthracene (6d) showed potent anti-inflam-
matory activity at 100 mg/kg (intraperitoneally), with
inflammatory inhibition values of 63.19 and 68.28 %,
respectively, which were greater than that of ibuprofen
(63.11 %). Compounds 3g and 6d showed anti-inflamma-
tory activity upon oral administration at doses of 50 and
100 mg/kg.
SAR analyses suggested that oxazine and triazole rings
could improve the anti-inflammatory therapeutic efficacy
of quinoline derivatives. Active compounds were selected
for further anti-inflammatory investigation in vitro, and 6d
(hydrochloride salt) showed dose-dependent inhibition of
LPS-induced TNF-a and IL-6 expression.
Conclusion
Three categories of derivatives, quinolin-2-one, 1-oxa-3,5-
diaza-anthracen-6-one, and cyclopenta[a]anthracene, were
synthesized, and their anti-inflammatory activities were
evaluated. In the xylene-induced ear-edema test in mice,
only two of the newly synthesized compounds (3h, 6c)
Further studies are necessary to gain insight into the
mechanisms of actions of these quinoline analogs, but these
123

Med Chem Res
results suggest that further development of such com-
pounds may be of therapeutic interest.
C-17), 130.83 (C-9), 148.26 (C-12), 150.09 (C-5), 171.83
(C-1). IR (KBr, cm-1): 3,202, 1,514 (N–H), 1,678 (C=O),
1,230, 1,024 (C–O–C), 1,094 (C–N). ESI-HRMS calcd. for
C17H16N2O2 (M?): 280.1212; found: 280.1206.
?
Experimental protocols
3-p-Tolyl-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-diaza-anthra-
1
cen-6-one (3b) Yield: 24.44 %; m.p.: 230 °C. H NMR
Chemistry
(CDCl3, 300 MHz): d 2.27 (s, 3H, –CH3), 2.60 (t, 2H, J =
7.5 Hz, H-8), 2.89 (t, 2H, J = 7.5 Hz, H-7), 3.52 (s, 2H,
–CH2N), 4.55 (s, 2H, –CH2O–), 6.51 (s, 1H, H-10), 6.62
(s, 1H, H-9), 6.99–7.09 (m, 4H, H–Ar), 9.17 (s, 1H,
–HNCO–). 13C NMR (CDCl3, 75 MHz): d 20.54 (CH3),
25.28 (C-3), 30.65 (C-2), 50.40 (C-11), 80.11 (C-10), 113.
27 (C-4), 116.15 (C-6), 118.70 (C-14, C-16), 119.72 (C-7),
123.73 (C-8), 129.82 (C-13, C-17), 130.79 (C-15), 131.24
(C-9), 145.94 (C-12), 150.07 (C-5), 172.02 (C-1). IR (KBr,
cm-1): 3,203, 1,514 (N–H), 1,672 (C=O), 1,230, 1,028 (C–
Reactions were monitored by thin-layer chromatography
on silica gel plates precoated with F254 gel. Developed
plates were examined under an ultraviolet lamp (254 nm).
Melting points were determined in open capillary tubes and
were uncorrected. IR spectra were recorded (in KBr) using
an FTIR1730 Spectrometer (Perkin-Elmer, Waltham, MA,
USA).1H NMR and 13C NMR spectra were recorded using
an AV-300 Spectrometer (Bruker Daltonik, Bremen, Ger-
many), and all chemical shifts were described in parts per
million relative to that of tetramethylsilane. Mass spectra
were measured using an HP1100 LC Spectrometer (Agilent
Technologies, Santa Clara, CA, USA). High-resolution
mass spectra were measured on an MALDI–TOF/TOF
mass spectrometer (Bruker Daltonik). Chemicals were
purchased from Sigma-Aldrich (Saint Louis, MO, USA).
?
O–C), 1,095 (C–N). ESI-HRMS calcd. for C18H18N2O2
(M?): 294.1368; found: 294.1360.
3-(4-Methoxy-phenyl)-3,4,7,8-tetrahydro-2H,5H-1-oxa-
3,5-diaza-anthracen-6-one (3c) Yield: 21.05 %; m.p.:
199 °C. 1H NMR (CDCl3, 300 MHz): d 2.60 (t, 2H, J = 7.
5 Hz, H-8), 2.90 (t, 2H, J = 7.5 Hz, H-7), 3.76 (s, 3H,
–OCH3), 4.51 (s, 2H, –CH2N), 5.27 (s, 2H, –CH2O–), 6.45
(s, 1H, H-10), 6.64 (s, 1H, H-9), 6.81–7.08 (m, 4H, H–Ar),
8.39 (s, 1H, –HNCO–). 13C NMR (CDCl3, 75 MHz): d 25.
29 (C-3), 30.64 (C-2), 50.91 (C-11), 55.51 (OCH3), 80.82
(C-10), 113.19 (C-4), 114.53 (C-14, C-16), 116.17 (C-6),
119.69 (C-7), 120.78 (C-13, C-17), 122.30 (C-8), 123.77
(C-9), 130.73 (C-12), 142.18 (C-15), 150.09 (C-5), 171.84
(C-1). IR (KBr, cm-1): 3,206, 1,512 (N–H), 1,672 (C=O),
1,247, 1,035 (C–O–C), 1,093 (C–N). ESI-HRMS calcd. for
General method for the synthesis of compounds 2a–2i
Compounds 2a–2i were synthesized according to previ-
ously described methods (Sun et al., 2006; Wei et al.,
2010; Sun et al., 2013).
General method for the synthesis of compounds 3a–3l
Compound 1 (10 mmol) was added to a stirred solution of
amine (15 mmol) and methanal (60 mmol) in ethanol
(30 mL), and the reaction mixture was refluxed for 24 h.
The reaction mixture was concentrated under reduced
pressure, diluted with CH2Cl2 (30 mL), washed with
NaOH (1 mol/L, 2 9 30 mL) and H2O (30 mL), and sat-
urated with NaCl (30 mL). The combined organic extracts
were dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure, after which the
obtained crude product was recrystallized in dioxane to
afford white needle-shaped crystal compounds (3a–3l).
?
C18H18N2O3 (M?): 310.1317; found: 310.1312.
3-(4-Fluoro-phenyl)-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-
diaza-anthracen-6-one (3d) Yield: 27.78 %; m.p.: 228 °C.
1H NMR (CDCl3, 300 MHz): d 2.60 (t, 2H, J = 7.5 Hz,
H-8), 2.90 (t, 2H, J = 7.5 Hz, H-7), 4.54 (s, 2H, –CH2N), 5.
28 (s, 2H, –CH2O–), 6.46 (s, 1H, H-10), 6.65 (s, 1H, H-9), 6.
93–7.10 (m, 4H, H–Ar), 8.39 (s, 1H, –HNCO–). 13C NMR
(CDCl3, 75 MHz): d 25.29 (C-3), 30.62 (C-2), 50.88 (C-11),
80.31 (C-10), 113.11 (C-4), 115.65 (C-13, C-17), 115.95 (C-
14, C-16), 116.26 (C-6), 119.38 (C-7), 120.60 (C-8), 123.94
(C-9), 130.92 (C-12), 144.80 (C-15), 149.95 (C-5), 171.77
(C-1). IR (KBr, cm-1): 3,199, 1,510 (N–H), 1,672 (C=O),
1,228, 1,030 (C–O–C), 1,093 (C–N). ESI-HRMS calcd. for
C17H16FN2O2? (M?): 298.1118; found: 298.1117.
3-Phenyl-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-diaza-anthra-
1
cen-6-one (3a) Yield: 29.41 %; m.p.: 197 °C. H NMR
(CDCl3, 300 MHz): d 2.60 (t, 2H, J = 7.5 Hz, H-8), 2.90
(t, 2H, J = 7.5 Hz, H-7), 4.60 (s, 2H, –CH2N), 5.35 (s, 2H,
–CH2O–), 6.50 (s, 1H, H-10), 6.64 (s, 1H, H-9), 6.93–7.31
(m, 5H, H–Ar), 8.84 (s, 1H, –HNCO–). 13C NMR (CDCl3,
75 MHz): d 25.28 (C-3), 30.64 (C-2), 50.22 (C-11), 79.62
(C-10), 113.18 (C-4), 116.26 (C-6), 118.37 (C-14, C-16),
119.69 (C-7), 121.63 (C-15), 123.82 (C-8), 129.30 (C-13,
3-(4-Chloro-phenyl)-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-
diaza-anthracen-6-one (3e) Yield: 16.79 %; m.p.:
223 °C. 1H NMR (CDCl3, 300 MHz): d 2.60 (t, 2H, J = 7.
5 Hz, H-8), 2.90 (t, 2H, J = 7.5 Hz, H-7), 4.56 (s, 2H,
123

Med Chem Res
–CH2N), 5.30 (s, 2H, –CH2O–), 6.44 (s, 1H, H-10), 6.65
(s, 1H, H-9), 7.02–7.24 (m, 4H, H–Ar), 8.09 (s, 1H,
–HNCO–). 13C NMR (CDCl3, 75 MHz): d 25.27 (C-3), 30.
60 (C-2), 50.45 (C-11), 79.51 (C-10), 113.16 (C-4), 116.30
(C-6), 119.30 (C-7), 119.74 (C-13, C-17), 124.00 (C-15),
126.70 (C-8), 129.22 (C-14, C-16), 130.98 (C-9), 146.92
(C-12), 149.88 (C-5), 171.89 (C-1). IR (KBr, cm-1): 3,200,
1,518 (N–H), 1,670 (C=O), 1,228, 1,030 (C–O–C), 1,099
(C-1). IR (KBr, cm-1): 3,200, 1,512 (N–H), 1,672 (C=O),
1,242, 1,029 (C–O–C), 1,097 (C–N). ESI-HRMS calcd. for
C19H21N2O3 ([M ? H]?): 325.1547; found: 325.1550.
?
3-(4-Fluoro-benzyl)-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-diaza-
anthracen-6-one (3i) Yield: 21.55 %; m.p.: 227 °C. 1H
NMR (CDCl3, 300 MHz): d 2.62 (t, 2H, J = 7.5 Hz, H-8),
2.93 (t, 2H, J = 7.5 Hz, H-7), 3.88 (s, 2H, –CH2–Ar), 3.91
(s, 2H, H-4), 4.84 (s, 2H, –CH2O–), 6.36 (s, 1H, H-10), 6.
67 (s, 1H, H-9), 7.02–7.35 (m, 4H, H–Ar), 8.02 (s, 1H,
–HNCO–). 13C NMR (CDCl3, 75 MHz): d 25.32 (C-3), 30.
69 (C-2), 49.30 (CH2), 54.79 (C-11), 82.05 (C-10), 114.06
(C-4), 115.16 (C-14, C-16), 115.44 (C-6), 115.78 (C-13,
C-17), 118.71 (C-7), 123.67 (C-8), 130.41 (C-9), 130.52
(C-12), 133.66 (C-5), 149.80 (C-15), 171.80 (C-1). IR
(KBr, cm-1): 3,199, 1,514 (N–H), 1,685 (C=O), 1,222,
1,018 (C–O–C), 1,095 (C–N). ESI-HRMS calcd. for
(C–N). ESI-HRMS calcd. for C17H16ClN2O2 (M?): 314.
?
0822; found: 314.0812.
3-Benzyl-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-diaza-anthra-
1
cen-6-one (3f) Yield: 20.67 %; m.p.: 211 °C. H NMR
(CDCl3, 300 MHz): d 2.61 (t, 2H, J = 7.5 Hz, H-8), 2.92 (t,
2H, J = 7.5 Hz, H-7), 3.90 (s, 4H, –CH2NCH2), 4.84 (s, 2H,
–CH2O–), 6.41 (s, 1H, H-10), 6.65 (s, 1H, H-9), 7.29–7.36
(m, 5H, H–Ar), 9.06 (s, 1H, –HNCO–). 13C NMR (CDCl3,
75 MHz): d 25.33 (C-3), 30.70 (C-2), 49.31 (CH2), 55.51 (C-
11), 82.27 (C-10), 114.11 (C-4), 115.74 (C-6), 118.85 (C-7),
123.58 (C-15), 127.44 (C-8), 128.48 (C-14, C-16), 128.93
(C-13, C-17), 130.71 (C-9), 138.00 (C-12), 149.85 (C-5),
171.88 (C-1). IR (KBr, cm-1): 3,205, 1,514 (N–H), 1,674
(C=O), 1,236, 1,022 (C–O–C), 1,095 (C–N). ESI-HRMS
calcd. for C18H18N2O2? (M?): 294.1368; found: 294.1363.
?
C18H18FN2O2 ([M ? H]?): 313.1347; found: 313.1347.
3-(4-Chloro-benzyl)-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-diaza-
anthracen-6-one (3j) Yield: 20.39 %; m.p.: 214 °C. 1H
NMR (CDCl3, 300 MHz): d 2.62 (t, 2H, J = 7.5 Hz, H-8),
2.93 (t, 2H, J = 7.5 Hz, H-7), 3.88 (s, 2H, –CH2–Ar), 3.91
(s, 2H, H-4), 4.84 (s, 2H, –CH2O–), 6.37 (s, 1H, H-10), 6.
67 (s, 1H, H-9), 7.31–7.32 (m, 4H, H–Ar), 8.23 (s, 1H,
–HNCO–). 13C NMR (CDCl3, 75 MHz): d 25.33 (C-3), 30.
68 (C-2), 49.36 (CH2), 54.80 (C-11), 82.14 (C-10), 114.03
(C-4), 115.80 (C-6), 118.65 (C-7), 123.70 (C-8), 128.62
(C-14, C-16), 130.20 (C-13, C-17), 130.76 (C-9), 133.19
(C-15), 136.52 (C-12), 149.76 (C-5), 171.76 (C-1). IR
(KBr, cm-1): 3,188, 1,510 (N–H), 1,670 (C=O), 1,240,
1,020 (C–O–C), 1,091 (C–N). ESI-HRMS calcd. for
3-(4-Methyl-benzyl)-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-di-
aza-anthracen-6-one (3g) Yield: 22.88 %; m.p.: 202 °C.
1H NMR (CDCl3, 300 MHz): d 2.37 (s, 3H, –CH3), 2.62 (t,
2H, J = 7.5 Hz, H-8), 2.93 (t, 2H, J = 7.5 Hz, H-7), 3.88
(s, 2H, –CH2–Ar), 3.92 (s, 2H, H-4), 4.85 (s, 2H, –CH2O–),
6.38 (s, 1H, H-10), 6.66 (s, 1H, H-9), 7.18–7.26 (m, 4H, H–
Ar), 8.39 (s, 1H, –HNCO–). 13C NMR (CDCl3, 75 MHz): d
21.14 (CH3), 25.34 (C-3), 30.71 (C-2), 49.26 (CH2), 55.22
(C-11), 82.19 (C-10), 114.13 (C-4), 115.71 (C-6), 118.91
(C-7), 123.54 (C-8), 128.93 (C-14, C-16), 129.16 (C-13,
C-17), 130.70 (C-9), 134.91 (C-12), 137.09 (C-15), 149.88
(C-5), 171.91 (C-1). IR (KBr, cm-1): 3,200, 1,514 (N–H),
1,675 (C=O), 1,222, 1,020 (C–O–C), 1,095 (C–N). ESI-
C18H18ClN2O2 ([M ? H]?): 329.1051; found: 329.1048.
?
4-(6-Oxo-5,6,7,8-tetrahydro-4H-1-oxa-3,5-diaza-anthracen-
3-ylmethyl)-benzonitrile (3k) Yield: 20.45 %; m.p.:
242 °C. 1H NMR (CDCl3, 300 MHz): d 2.39 (t, 2H, J = 7.
5 Hz, H-8), 2.79 (t, 2H, J = 7.5 Hz, H-7), 3.82 (s, 2H,
–CH2–Ar), 3.94 (s, 2H, H-4), 4.80 (s, 2H, –CH2O–), 6.44 (s,
1H, H-10), 6.62 (s, 1H, H-9), 7.54–7.83 (m, 4H, H–Ar), 9.87
(s, 1H, –HNCO–). 13C NMR (CDCl3, 75 MHz): d 25.33 (C-
3), 30.70 (C-2), 49.31 (CH2), 55.51 (C-11), 82.27 (C-10),
109.77 (CN), 114.11 (C-4), 115.74 (C-6), 118.85 (C-7), 123.
58 (C-15), 127.44 (C-8), 128.48 (C-14, C-16), 128.93 (C-13,
C-17), 130.71 (C-9), 138.00 (C-12), 149.85 (C-5), 171.88
(C-1). IR (KBr, cm-1): 3,208, 1,519 (N–H), 1,685 (C=O),
1,230, 1,071 (C–O–C), 1,116 (C–N). ESI-HRMS calcd. for
C19H18N3O2? ([M ? H]?): 320.1394; found: 320.1397.
HRMS calcd. for C19H21N2O2 ([M ? H]?): 309.1598;
?
found: 309.1598.
3-(4-Methoxy-benzyl)-3,4,7,8-tetrahydro-2H,5H-1-oxa-
3,5-diaza-anthracen-6-one (3h) Yield: 35.35 %; m.p.:
212 °C. 1H NMR (CDCl3, 300 MHz): d 2.37 (s, 3H,
–CH3), 2.62 (t, 2H, J = 7.5 Hz, H-8), 2.93 (t, 2H, J = 7.
5 Hz, H-7), 3.52 (s, 3H, –OCH3), 3.83 (s, 2H, –CH2–Ar), 3.
85 (s, 2H, H-4), 4.84 (s, 2H, –CH2O–), 6.36 (s, 1H, H-10),
6.67 (s, 1H, H-9), 6.67–6.91 (m, 4H, H–Ar), 7.99 (s, 1H,
–HNCO–). 13C NMR (CDCl3, 75 MHz): d 25.34 (C-3), 30.
71 (C-2), 49.19 (CH2), 54.85 (OCH3), 55.30 (C-11), 82.02
(C-10), 113.86 (C-14, C-16), 114.05 (C-4), 115.71 (C-6),
118.89 (C-7), 123.58 (C-8), 129.98 (C-12), 130.19 (C-13,
C-17), 130.65 (C-9), 149.89 (C-5), 159.01 (C-15), 171.73
3-Phenethyl-3,4,7,8-tetrahydro-2H,5H-1-oxa-3,5-diaza-anthra-
1
cen-6-one (3l) Yield: 49.12 %; m.p.: 180 °C. H NMR
(CDCl3, 300 MHz): d 2.61 (t, 2H, J = 7.5 Hz, H-8), 2.88
(t, 2H, J = 7.5 Hz, –CH2CH2N), 3.03 (t, 2H, J = 7.5 Hz,
123

Med Chem Res
H-7), 3.52 (t, 2H, J = 7.5 Hz, –CH2CH2N), 3.99 (s, 2H,
chromatography over SiO2 (CH2Cl2/MeOH = 80/1, v/v) to
obtain white solid compounds (6a–6l).
H-4), 4.87 (s, 2H, –CH2O–), 6.38 (s, 1H, H-10), 6.62 (s, 1H,
H-9), 7.21–7.29 (m, 5H, H–Ar), 7.90 (s, 1H, –HNCO–). 13
C
NMR (CDCl3, 75 MHz): d 25.32 (C-3), 30.71 (C-2), 34.90
(CH2), 50.12 (CH2), 53.13 (C-11), 82.51 (C-10), 113.94 (C-
4), 115.75 (C-6), 119.06 (C-7), 123.58 (C-15), 126.21 (C-8),
128.40 (C-13, C-17), 128.67 (C-14, C-16), 130.58 (C-9),
139.68 (C-12), 149.97 (C-5), 171.75 (C-1). IR (KBr, cm-1):
3,209, 1,514 (N–H), 1,676 (C=O), 1,232, 1,021 (C–O–C),
9-Phenyl-5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,11b-tetraaza-
cyclopenta[a]anthracene (6a) Yield: 20.77 %; m.p.:
171 °C. 1H NMR (CDCl3, 300 MHz): d 2.88 (t, 2H, J = 7.
5 Hz, H-5), 3.19 (t, 2H, J = 7.5 Hz, H-4), 4.65 (s, 2H,
H-10), 5.43 (s, 2H, –CH2O–), 7.02–7.35 (m, 7H, H–Ar), 8.
66 (s, 1H, –N=CH–). 13C NMR (CDCl3, 75 MHz): d 20.60
(C-3), 25.80 (C-4), 50.45 (C-12), 79.91 (C-11), 114.49 (C-
5), 117.74 (C-16), 118.57 (C-14, C-18), 120.34 (C-7), 122.
09 (C-9), 125.62 (C-8), 127.60 (C-10), 129.40 (C-15,
C-17), 137.27 (C-1), 147.96 (C-13), 150.32 (C-2), 153.00
(C-6). IR (KBr, cm-1): 1,539 (C=N), 1,222, 1,030 (C–O–
C), 1,112 (C–N). ESI-HRMS calcd. for C18H17N4O?
([M ? H]?): 305.1397; found: 305.1401.
?
1,091 (C–N). ESI-HRMS calcd. for C19H21N2O2
([M ? H]?): 309.1598; found: 309.1591.
Synthesis of 6-hydroxy-3,4-dihydro-1H-quinoline-2-thione
(4) To a stirring mixture of acetonitrile (120 mL) and
triethylamine (80 mL) in a round-bottomed flask in an ice-
bath, P2S5 (17.70 g, 80 mmol) was added one portion at a
time after the previous portion had dissolved completely.
Next, compound 1 (10.00 g, 60 mmol) was added, and the
solution was refluxed for 3 h. The reaction mixture was
concentrated to dryness under reduced pressure. The
resulting mixture was diluted with methanol and poured
into cool water. The mixture was left to stand for 10 h and
filtered under reduced pressure to obtain a yellow residue
(10.00 g). Yield: 91.07 %; m.p.: 225 °C. 1H NMR (CDCl3,
300 MHz): d 2.79 (t, 2H, J = 6.6 Hz, H-4), 2.95 (t, 2H,
J = 6.6 Hz, H-3,), 6.69–7.04 (m, 3H, H–Ar), 8.32 (s, 1H,
–NHCO–), 10.93 (s, 1H, HO–). MS-EI m/z 179 (M ? H?).
9-p-Tolyl-5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,11b-tetraaza-
cyclopenta[a]anthracene (6b) Yield: 23.42 %; m.p.:
1
184 °C. H NMR (CDCl3, 300 MHz): d 2.27 (s, 3H, –
CH3), 2.94 (t, 2H, J = 7.5 Hz, H-5), 3.14 (t, 2H, J = 7.
5 Hz, H-4), 4.64 (s, 2H, H-10), 5.37 (s, 2H, –CH2O–), 6.
78–7.10 (m, 6H, H–Ar), 8.54 (s, 1H, –N=CH–). 13C NMR
(CDCl3, 75 MHz): d 20.55 (CH3), 20.59 (C-3), 25.78 (C-
4), 50.63 (C-12), 80.36 (C-11), 114.55 (C-5), 117.64 (C-
16), 118.85 (C-14, C-18), 120.37 (C-7), 125.52 (C-9), 127.
52 (C-8), 129.91 (C-10), 131.73 (C-15, C-17), 137.31 (C-
1), 145.62 (C-13), 150.31 (C-2), 152.99 (C-6). IR (KBr,
cm-1): 1,539 (C=N), 1,223, 1,030 (C–O–C), 1,114 (C–N).
ESI-HRMS calcd. for C19H19N4O? ([M ? H]?): 319.
1553; found: 319.1553.
Synthesis of 4,5-dihydro-[1,2,4]triazolo[4,3-a]quinolin-7-
ol (5) A solution of compound 4 (8.00 g, 45 mmol) and
formohydrazide (6.86 g, 58 mmol) in n-butyl alcohol
(80 mL) was heated at reflux for 24 h. Eighty percent of
the n-butyl alcohol was removed under reduced pressure
and cooled to 0 °C. The resulting mixture was filtered
under reduced pressure to yield a yellow powder (7.20 g).
Yield: 86.15 %; m.p.: 235 °C. 1H NMR (DMSO-d6,
300 MHz): d 2.93 (t, 2H, J = 7.7 Hz, H-5), 3.01 (t, 2H,
J = 7.7 Hz, H-4), 6.74–7.56 (m, 3H, H–Ar), 9.11 (s, 1H,
–N=CH–), 9.2 (s, 1H, H–O). MS-EI m/z 187 (M ? H?).
9-(4-Methoxy-phenyl)-5,8,9,10-tetrahydro-4H-7-oxa-
2,3,9,11b-tetraaza-cyclopenta[a]anthracene (6c) Yield:
1
19.14 %; m.p.: 150 °C. H NMR (CDCl3, 300 MHz): d 2.
95 (t, 2H, J = 7.5 Hz, H-5), 3.15 (t, 2H, J = 7.5 Hz, H-4),
3.75 (s, 3H, –OCH3), 4.59 (s, 2H, H-10), 5.32 (s, 2H,
–CH2O–), 6.79–7.09 (m, 6H, H–Ar), 8.53 (s, 1H, –N=CH–).
13C NMR (CDCl3, 75 MHz): d 20.59 (C-3), 25.78 (C-4), 51.
10 (C-12), 55.50 (OCH3), 81.05 (C-11), 114.53 (C-5), 114.58
(C-15, C-17), 117.61 (C-7), 120.34 (C-9), 120.90 (C-14,
C-18), 125.53 (C-8), 127.52 (C-10), 137.30 (C-1), 141.81(C-
13), 150.30 (C-2), 152.99 (C-6), 155.30 (C-16). IR (KBr,
cm-1): 1,539 (C=N), 1,223, 1,031 (C–O–C), 1,114 (C–N).
General method for the synthesis of compounds (6a–6l)
Compound 5 (10 mmol) was added to a stirred solution of
amine (15 mmol) and methanal (60 mmol) in ethanol
(30 mL), and the reaction mixture was refluxed for 24 h.
The reaction mixture was concentrated under reduced
pressure, diluted with CH2Cl2 (30 mL), washed with
NaOH (1 mol/L, 2 9 30 mL) and H2O (30 mL), and sat-
urated with NaCl (30 mL). The combined organic extracts
were dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure, after which the
obtained crude product was purified by column
ESI-HRMS calcd. for C19H19N4O2 ([M ? H]?): 335.
?
1503; found: 335.1504.
9-(4-Fluoro-phenyl)-5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,
11b-tetraaza-cyclopenta[a]anthracene (6d) Yield: 18.
1
81 %; m.p.: 148 °C. H NMR (CDCl3, 300 MHz): d 2.97
(t, 2H, J = 7.5 Hz, H-5), 3.16 (t, 2H, J = 7.5 Hz, H-4), 4.
63 (s, 2H, H-10), 5.34 (s, 2H, –CH2O–), 6.81–7.08 (m, 6H,
123

Med Chem Res
H–Ar), 8.55 (s, 1H, –N=CH–). 13C NMR (CDCl3,
75 MHz): d 20.59 (C-3), 25.81 (C-4), 51.08 (C-12), 80.55
(C-11), 114.49 (C-5), 116.09 (C-14, C-18), 117.74 (C-7),
120.03 (C-15, C-17), 120.78 (C-9), 124.87 (C-8), 125.71
(C-10), 127.71 (C-1), 137.27 (C-13), 144.47 (C-16), 150.31
(C-2), 152.86 (C-6). IR (KBr, cm-1): 1,539 (C=N), 1,222,
1,002 (C–O–C), 1,130 (C–N). ESI-HRMS calcd. for
C18H16FN4O? ([M ? H]?): 323.1303; found: 323.1304.
9-(4-Methoxy-benzyl)-5,8,9,10-tetrahydro-4H-7-oxa-
2,3,9,11b-tetraaza-cyclopenta[a]anthracene (6h) Yield:
1
16.13 %; m.p.: 138 °C. H NMR (CDCl3, 300 MHz): d 3.
01 (t, 2H, J = 7.5 Hz, H-5), 3.21 (t, 2H, J = 7.5 Hz, H-4),
3.84 (s, 3H, –OCH3), 3.87 (s, 2H, –CH2–Ar), 4.00 (s, 2H,
H-10), 4.92 (s, 2H, –CH2O–), 6.83–7.29 (m, 6H, H–Ar), 8.
52 (s, 1H, –N=CH–). 13C NMR (CDCl3, 75 MHz): d 20.67
(C-3), 25.83 (C-4), 49.06 (CH2), 54.90 (OCH3), 55.31 (C-
12), 82.36 (C-11), 113.93 (C-15, C-17), 115.34 (C-5), 117.
19 (C-7), 119.56 (C-9), 125.45 (C-8), 127.32 (C-10), 129.
59 (C-13), 130.15 (C-14, C-18), 137.29 (C-1), 150.35 (C-
2), 152.91 (C-6), 159.13 (C-16). IR (KBr, cm-1): 1,537
(C=N), 1,249, 1,029 (C–O–C), 1,111 (C–N). ESI-HRMS
9-(4-Chloro-phenyl)-5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,11b-
tetraaza-cyclopenta[a]anthracene (6e) Yield: 20.58 %; m.
1
p.: 145 °C. H NMR (CDCl3, 300 MHz): d 2.97 (t, 2H,
J = 7.5 Hz, H-5), 3.17 (t, 2H, J = 7.5 Hz, H-4), 4.65 (s,
2H, H-10), 4.90 (s, 2H, –CH2O–), 6.82–7.25 (m, 6H, H–
Ar), 8.54 (s, 1H, –N=CH–). 13C NMR (CDCl3, 75 MHz): d
20.57 (C-3), 25.80 (C-4), 50.85 (C-12), 79.78 (C-11), 114.
48 (C-5), 116.07 (C-7), 117.81 (C-9), 119.95 (C-14, C-18),
125.75 (C-16), 127.80 (C-8), 129.34 (C-15, C-17), 137.25
(C-10), 143.09 (C-1), 146.62 (C-13), 150.31 (C-2), 152.80
(C-6). IR (KBr, cm-1): 1,539 (C=N), 1,222, 1,002 (C–O–
C), 1,116 (C–N). ESI-HRMS calcd. for C18H16ClN4O?
([M ? H]?): 339.1007; found: 339.1022.
calcd. for C20H21N4O2 ([M ? H]?): 349.1659; found:
?
349.1657.
9-(4-Fluoro-benzyl)-5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,11b-
tetraaza-cyclopenta[a]anthracene (6i) Yield: 15.88 %; m.
1
p.: 146 °C. H NMR (CDCl3, 300 MHz): d 3.01 (t, 2H,
J = 7.5 Hz, H-5), 3.20 (t, 2H, J = 7.5 Hz, H-4), 3.90 (s,
2H, –CH2–Ar), 4.00 (s, 2H, H-10), 4.90 (s, 2H, –CH2O–),
6.83 (s, 1H, H-6), 6.99 (s, 1H, H-11), 7.03–7.36 (m, 4H, H–
Ar), 8.51 (s, 1H, –N=CH–). 13C NMR (CDCl3, 75 MHz): d
20.66 (C-3), 25.84 (C-4), 49.21 (CH2), 54.80 (C-12), 82.37
(C-11), 115.33 (C-5), 115.55 (C-15, C-17), 117.29 (C-7),
119.38 (C-9), 125.55 (C-8), 127.44 (C-10), 130.40 (C-14,
C-18), 130.50 (C-13), 133.31 (C-1), 137.28 (C-2), 150.25
(C-6), 152.80 (C-16). IR (KBr, cm-1): 1,537 (C=N), 1,250,
1,030 (C–O–C), 1,116 (C–N). ESI-HRMS calcd. for
C19H18FN4O? ([M ? H]?): 337.1459; found: 337.1455.
9-Benzyl-5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,11b-tetraaza-
cyclopenta[a]anthracene (6f) Yield: 11.76 %; m.p.:
131 °C. 1H NMR (CDCl3, 300 MHz): d 2.99 (t, 2H, J = 7.
5 Hz, H-5), 3.17 (t, 2H, J = 7.5 Hz, H-4), 3.92 (s, 2H,
–CH2–Ar), 4.00 (s, 2H, H-10), 4.92 (s, 2H, –CH2O–), 6.82
(s, 1H, H-6), 6.99 (s, 1H, H-11), 7.25–7.37 (m, 5H, H–Ar),
8.52 (s, 1H, –N=CH–). 13C NMR (CDCl3, 75 MHz): d 20.
49 (C-3), 25.67 (C-4), 49.14 (CH2), 55.41 (C-12), 82.45
(C-11), 115.64 (C-5), 116.98 (C-7), 119.50 (C-9), 125.36
(C-8), 127.17 (C-16), 127.48 (C-10), 128.46 (C-15, C-17),
128.82 (C-14, C-18), 137.55 (C-13), 137.68 (C-1), 150.15
(C-2), 152.72 (C-6). IR (KBr, cm-1): 1,537 (C=N), 1,250,
1,029 (C–O–C), 1,111 (C–N). ESI-HRMS calcd. for
C19H19N4O? ([M ? H]?): 319.1553; found: 319.1555.
9-(4-Chloro-benzyl)-5,8,9,10-tetrahydro-4H-7-oxa-
2,3,9,11b-tetraaza-cyclopenta[a]anthracene (6j) Yield:
1
10.18 %; m.p.: 130 °C. H NMR (CDCl3, 300 MHz): d 3.
00 (t, 2H, J = 7.5 Hz, H-5), 3.19 (t, 2H, J = 7.5 Hz, H-4),
3.90 (s, 2H, –CH2–Ar), 3.99 (s, 2H, H-10), 4.90 (s, 2H, –
CH2O–), 6.83 (s, 1H, H-6), 6.98 (s, 1H, H-11), 7.28–7.33
(m, 4H, H–Ar), 8.51 (s, 1H, –N=CH–). 13C NMR (CDCl3,
75 MHz): d 20.62 (C-3), 25.80 (C-4), 49.24 (CH2), 54.85
(C-12), 82.45 (C-11), 115.37 (C-5), 117.26 (C-7), 119.33
(C-9), 125.52 (C-8), 127.43 (C-10), 128.71 (C-15, C-17),
130.16 (C-14, C-18), 133.35 (C-16), 136.16 (C-13), 137.31
(C-1), 150.32 (C-2), 152.78 (C-6). IR (KBr, cm-1): 1,537
(C=N), 1,250, 1,030 (C–O–C), 1,114 (C–N). ESI-HRMS
calcd. for C19H18ClN4O? ([M ? H]?): 353.1164; found:
353.1163.
9-(4-Methyl-benzyl)-5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,11b-
tetraaza-cyclopenta[a]anthracene (6g) Yield: 12.38 %; m.
1
p.: 136 °C. H NMR (CDCl3, 300 MHz): d 1.72 (s, 3H,
–CH3), 3.01 (t, 2H, J = 6.0 Hz, H-5), 3.20 (t, 2H, J = 6.
0 Hz, H-4), 3.89 (s, 2H, –CH2–Ar), 4.00 (s, 2H, H-10), 4.
93 (s, 2H, –CH2O–), 6.83 (s, 1H, H-6), 6.97 (s, 1H, H-11),
7.20–7.27 (m, 4H, H–Ar), 8.51 (s, 1H, –N=CH–). 13C
NMR (CDCl3, 75 MHz): d 20.63 (CH3), 21.14 (C-3), 25.80
(C-4), 49.11 (CH2), 55.25 (C-12), 82.53 (C-11), 115.40 (C-
5), 111.91 (C-7), 119.58 (C-9), 125.40 (C-8), 127.30 (C-
10), 128.48 (C-13), 128.88 (C-14, C-18), 129.24 (C-15,
C-17), 134,53 (C-16), 137.32 (C-1), 150.31 (C-2), 152.91
(C-6). IR (KBr, cm-1): 1,537 (C=N), 1,250, 1,029 (C–O–
C), 1,111 (C–N). ESI-HRMS calcd. for C20H21N4O?
([M ? H]?): 333.1710; found: 333.1700.
4-(5,10-Dihydro-4H-7-oxa-2,3,9,11b-tetraaza-cyclopenta[a]
anthracen-9-ylmethyl)-benzonitrile (6k) Yield: 11.26 %;
1
m.p.: 152 °C. H NMR (CDCl3, 300 MHz): d 3.00 (t, 2H,
J = 6.0 Hz, H-5), 3.17 (t, 2H, J = 6.0 Hz, H-4), 3.99 (s,
2H, –CH2–Ar), 4.00 (s, 2H, H-10), 4.90 (s, 2H, –CH2O–),
6.84 (s, 1H, H-6), 6.94 (s, 1H, H-11), 7.49–7.62 (m, 4H,
123

Med Chem Res
H–Ar), 8.51 (s, 1H, –N=CH–). 13C NMR (CDCl3,
75 MHz): d 20.60 (C-3), 25.80 (C-4), 49.54 (CH2), 55.22
(C-12), 82.60 (C-11), 111.49 (C-16), 115.37 (C-5), 117.41
(C-7), 118.73 (CN), 119.09 (C-9), 125.26 (C-8), 127.61 (C-
10), 128.95 (C-13), 129.28 (C-14, C-18), 132.40 (C-15,
C-17), 143.36 (C-1), 150.15 (C-2), 152.69 (C-6). IR (KBr,
cm-1): 1,539 (C=N), 1,250, 1,039 (C–O–C), 1,140 (C–N).
ESI-HRMS calcd. for C20H18N5O? ([M ? H]?): 344.
1506; found: 344.1499.
tissue plug (7 mm diameter) was excised from the ears of
treated and untreated mice. Edema was quantified by
measuring the difference in weight between the plugs from
the treated and untreated mice. Anti-inflammatory activity
was expressed as the percent reduction in edema in com-
parison with the DMSO-treated control group. The NSAID
ibuprofen was tested in parallel as a reference drug.
Xylene-induced ear-edema model with orally administered
compounds
9-Phenethyl-5,8,9,10-tetrahydro-4H-7-oxa-2,3,9,11b-tetra-
aza-cyclopenta[a]anthracene (6l) Yield: 22.03 %; m.p.:
154 °C. 1H NMR (CDCl3, 300 MHz): d 2.90 (t, 2H, J = 7.
5 Hz, H-5), 2.96–3.07 (m, 4H, –(CH2)2N), 3.17 (t, 2H,
J = 7.5 Hz, H-4), 4.08 (s, 2H, H-10), 4.93 (s, 2H, –CH2O–),
6.78 (s, 1H, H-6), 7.00 (s, 1H, H-11), 7.21–7.32 (m, 5H, H–
Ar), 8.53 (s, 1H, –N=CH–). 13C NMR (CDCl3, 75 MHz): d
20.67 (C-3), 25.81 (C-4), 34.88 (CH2), 50.01 (CH2), 53.14
(C-12), 82.86 (C-11), 115.23 (C-5), 117.21 (C-9), 119.73
(C-7), 125.38 (C-8), 126.31 (C-16), 127.34 (C-10), 128.45
(C-14, C-18), 128.65 (C-15, C-17), 137.29 (C-13), 139.47
(C-1), 150.63 (C-2), 152.97 (C-6). IR (KBr, cm-1): 1539
(C=N), 1,222, 1,032 (C–O–C), 1,111 (C–N). ESI-HRMS
calcd. for C20H21N4O? ([M ? H]?): 333.1710; found:
333.1712.
Test compounds (3g, 6d, and ibuprofen) were homoge-
nized with 0.5 % sodium carboxymethylcellulose (CMC-
Na) and administered via the oral route to mice at 100 mg/
kg (0.4 mL/20 g body weight). Control mice received
0.5 % CMC-Na (0.4 mL/20 g body weight). To explore the
peak activity of the test compounds, edema was quantified
at different intervals (1, 2, 3, 4, and 5 h). In a separate
experiment, compounds 3g, 6d, and ibuprofen were
homogenized with 0.5 % CMC-Na and administered orally
to mice (at a lower dose of 50 mg/kg; 0.4 mL/20 g body
weight). Control mice received 0.5 % CMC-Na (0.4 mL/
20 g body weight) and edema was quantified at the peak
activity time point of 3 h.
Assay of hydrochloride salts in the xylene-induced ear-
edema model via the intraperitoneal route
Pharmacology
In vivo study
Compounds 3g (hydrochloride salt) and 6d (hydrochloride
salt) were homogenized with physiological (0.9 %) saline
and administered to mice via the intraperitoneal route at a
dose of 100 mg/kg. Control mice received an equal volume
of physiological saline via the intraperitoneal route.
Kunming mice (mean body weight, 20–25 g; 10 animals
per group) were purchased from the Laboratory of Animal
Research, College of Pharmacy, Yanbian University
(Yanji, China). Animals were housed at a constant room
temperature with a 12–12 h light–dark cycle and fed a
standard rodent diet and water. Mice were acclimatized to
the laboratory for C7 days before experimentation.
In vitro study
DMSO, LPS (Escherichia coli lipopolysaccharide;
055:B5), and 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl-
tetrazolium bromide (MTT) were purchased from Sigma-
Aldrich. Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum (FBS), penicillin, and streptomycin for
cell culture were obtained from Invitrogen–Gibco (Grand
Island, NY, USA). Mouse TNF-a and IL-6 ELISA kits
were purchased from Biolegend (San Diego, CA, USA).
Xylene-induced ear-edema model with intraperitoneally
administered compounds
In this screening, all novel compounds were evaluated with
regard to anti-inflammatory activity via an in vivo inhibi-
tion assay of xylene-induced ear edema (Sowemimo et al.,
2013) in mice. All freshly prepared test compounds and
ibuprofen (dissolved in DMSO) were administered to mice
via the intraperitoneal route at a dose of 100 mg/kg
(0.05 mL/20 g body weight). Control mice received
DMSO (0.05 mL/20 g body weight) only. Thirty minutes
after administration, mice were used in the xylene-induced
ear-edema test (20 L xylene was daubed by a micropipette
on the surface of the right ear of each mouse). After pre-
venting the mice from struggling for 30 min, a cylindrical
Culture and treatment of cells
The RAW264.7 mouse macrophage cell line was pur-
chased from China Cell Line Bank (Beijing, China). Cells
were cultured in DMEM (Invitrogen–Gibco) supplemented
with 10 % heat-inactivated FBS (Invitrogen–Gibco), at
37 °C under a humidified atmosphere of 5 % CO2 in air. In
all experiments, cells were incubated in the presence or
123

Med Chem Res
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Statistical analyses
Data are represented as the mean SEM. Statistical
analyses for comparisons of the control and experimental
groups were performed using one-way analysis of variance
(ANOVA) followed by the Dunnett’s test using GraphPad
Prism version 5 (GraphPad, San Diego, CA, USA). Results
of P \ 0.05 were considered to be statistically significant.
Acknowledgments This work was supported by the National Nat-
ural Science Foundation of China (Nos. 81160382 and 81360468) and
the National Science and Technology Major Project of China (No.
2012ZX09103-101-044).
Conflict of interest The authors declare that they have no conflict
of interest with respect to this study.
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