Synthesis and biological activity of fused furo[2,3-d]pyrimidinone derivatives as analgesic and antitumor agents

Article abstract of DOI:10.1007/s11164-015-2064-8

Tumor growth is usually associated with persistent pain, especially during mid and terminal stages of cancer development. Nonetheless, a medicinal compound that possesses both anticancer and analgesic properties has not been identified. The 2-alkylthio-benzofuro[3,2-d]pyrimidin-4(3H)-ones (Code 5a-d) and 1-aryl-2-alkylthio-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-ones (Code 10a-g) were synthesized by using the bioisostere concept, which were obtained via the aza-Wittig reaction of functionalized iminophosphoranes reacted with carbon disulfide and further reaction of the product with alkyl halides or halogenated aliphatic esters. The analgesic properties of 5a-d and 10a-g were studied using rat chronic constriction injury model and the antitumor properties of these chemicals were assessed using MTS cell proliferation assay. Results showed that 5a-d and 10a-g were found to attenuate thermal and mechanical allodynia induced by neuropathy and inhibited the proliferation of three human cancer cell lines (A459, HepG2, and HeLa). Among these compounds, 10g showed highly positive effects in both assessments, and would be selected for future work.

Full text of DOI:10.1007/s11164-015-2064-8

Res Chem Intermed
DOI 10.1007/s11164-015-2064-8
Synthesis and biological activity of fused
furo[2,3-d]pyrimidinone derivatives as analgesic
and antitumor agents
Qing Li1,2 Yong-Mei Chen3 Yang-Gen Hu1,2
•
•
•
Xin Luo4 Joshua Ka Shun Ko Chi Wai Cheung
5
4
•
•
Received: 22 March 2015 / Accepted: 16 April 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Tumor growth is usually associated with persistent pain, especially
during mid and terminal stages of cancer development. Nonetheless, a medicinal
compound that possesses both anticancer and analgesic properties has not been
identified. The 2-alkylthio-benzofuro[3,2-d]pyrimidin-4(3H)-ones (Code 5a–d)
and 1-aryl-2-alkylthio-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-ones
(Code 10a–g) were synthesized by using the bioisostere concept, which were ob-
tained via the aza-Wittig reaction of functionalized iminophosphoranes reacted with
carbon disulfide and further reaction of the product with alkyl halides or halo-
genated aliphatic esters. The analgesic properties of 5a–d and 10a–g were studied
using rat chronic constriction injury model and the antitumor properties of these
chemicals were assessed using MTS cell proliferation assay. Results showed that
5a–d and 10a–g were found to attenuate thermal and mechanical allodynia induced
by neuropathy and inhibited the proliferation of three human cancer cell lines
&
Yang-Gen Hu
huyg@hbmu.edu.cn
Chi Wai Cheung
cheucw@hku.hk
1
2
3
4
5
Department of Anesthesiology, Institute of Anesthesiology, Taihe Hospital of Hubei University
of Medicine, Shiyan 442000, China
Hubei Key Laboratory of Wudang Local Chinese Medicine Research and Institute of Medicinal
Chemistry, Hubei University of Medicine, Shiyan 442000, China
Department of Medical Laboratory Science, Taihe Hospital of Hubei University of Medicine,
Shiyan 442000, China
Department of Anaesthesiology, Queen Mary Hospital, Li Ka Shing Faculty of Medicine, The
University of Hong Kong, Hong Kong 999077, China
Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist
University, Hong Kong 999077, China
123

Q. Li et al.
(A459, HepG2, and HeLa). Among these compounds, 10g showed highly positive
effects in both assessments, and would be selected for future work.
Keywords Fused furo[2,3-d]pyrimidinone derivatives Á Synthesis Á Analgesic and
antitumor
Introduction
At terminal stages of cancer development, patients can experience severe cancer-
associated pain. In normal practice, both chemotherapeutic and analgesic agents are
prescribed simultaneously. However, none of these drugs possess dual activities in a
single remedy. Even if there were drugs that exerted strong action in one area with
some effect on the other, the power would be usually weak. During the course of our
drug discovery works aiming to synthesize new nitrogen heterocyclic compounds, we
revealed that furo[2,3-d]pyrimidines and fused benzofuro[2,3-d]pyrimidinones have
potent antitumor properties [1, 2]. It was reported that 2,3-disubstituted quinazolin-4-
(3H)-ones may exhibit good analgesic effects [3–5]. Bioisosterism is a useful strategy
during the lead optimization process and molecular modification in rational drug
design [6, 7]. On the basis of the bioisostere concept, we postulated that replacement
of the condensed quinazolinone ring system with furopyrimidinone bioisostere group
could generate compounds with improved biological activities (Fig. 1).
In such respect, as a continuation of our ongoing efforts towards the design and
synthesis of nitrogen-containing heterocycle compounds via aza-Wittig reaction
[8–12], we now report a number of tricyclic and tetracyclic fused benzofuro[2,3-
d]pyrimidinone derivatives 5 and 10 based on the development and identification of
lead molecules by the bioisostere concept. In addition, these fused furo[2,3-
d]pyrimidinone derivatives were screened for their analgesic and antitumor activities.
Results and discussion
Chemistry
As described in detail previously [12], the synthesis of compounds 5 and 10 started
from commercially available salicylic nitrile 1, and the reaction of 2, 3, 4, and 5 had
taken place smoothly at room temperature with good yields (Scheme 1).
Ο
Ο
Ο
Ν
Ο
N
Ο
R1
N
R1
SR2
Ν
Ν
Ν
N
Ν
SR2
R1
SR2
5
10
Fig. 1 Bioisostere-based drug structure design
123

Synthesis and biological activity of fused…
N
C S
N
CN
OH
NH2
CO2C2H5
PPh3
CO2C2H5
CO2C2H5
Ο
Ο
2
Ο
3
1
SR2
N R1
SH
N
N
N R1
Ο
Ο
Ο
Ο
5
4
Scheme 1 Preparation of 3-alkyl-2-alkylthio-benzofuro[3,2-d]pyrimidin-4(3H)-one 5
NHR1
PPh3
N C
NR1
N
N
NH2NH2
N
NH2
CO2C2H5
CO2C2H5
Ο
Ο
2
Ο
Ο
7
6
R1
N
R1
NHR1
PPh3
N
N
N
SR2
N
SH
CS2
N
N
N
N
N
N
Ο
Ο
Ο
Ο
Ο
Ο
10
8
9
Scheme 2 Preparation of 1-aryl-2-alkylthio-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one
10
The synthetic strategy for 6–10 is outlined in Scheme 2. The syntheses of the key
intermediates 6–8 have been described earlier [9, 11]. The functionalized
iminophosphoranes 8 reacted with excess carbon disulfide to give 1-aryl-2-thioxo-
benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one 9. S-Alkylation of 9
with alkyl halides or halogenated aliphatic esters in the presence of potassium
carbonate produced 1-aryl-2-alkylthio- benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]pyrim-
idin-5(1H)-one 10 (Scheme 2) in good yield. The results are listed in Table 1. The
1
structures of the compounds 9 and 10 were deduced based on their H NMR, MS,
IR, and elementary analyses. Furthermore, the structure of 10f was confirmed by
X-ray crystallographic analysis [11].
Biological evaluation
The synthesized compounds 5a–5d and 10a–10g were evaluated for analgesic and
antitumor activities. Because of poor water solubility, all the synthesized
compounds were administered in the form of a suspension in 5 % DMSO.
Briefly, accurate weighing the synthesized compounds were dissolved in 0.5 ml
100 % DMSO, respectively, and then diluted to 10 ml with normal saline as
suspension in 10 % v/v Tween-80. Final concentrations of DMSO in assay solutions
never exceed 1 %. The animals used in this experiment were provided by the
123

Q. Li et al.
Table 1 Preparation of fused furo[2,3-d]pyrimidinone derivatives (5b and 10) and the intermediates 9
Compound
R1
R2
Condition
Yielda (%)
5a
CH2CH2CH3
CH2CH2CH3
CH2CH2CH2CH3
PhCH2
50 °C/4 h
50 °C/4 h
50 °C/4 h
50 °C/4 h
40–50 °C/24 h
40–50 °C/24 h
50–60 °C/6 h
50–60 °C/6 h
50–60 °C/6 h
50–60 °C/6 h
50–60 °C/6 h
50–60 °C/6 h
50–60 °C/6 h
86
82
85
86
86
80
75
72
78
83
80
72
81
5b
CH2CH2CH3
5c
CH2CH2CH3
5d
CH2CH2CH3
CH2CO2C2H5
H
9a
Ph
9bb
10a
10b
10c
10d
10e
10f#
10g
a
4-F-Ph
Ph
H
CH2CH2CH2CH3
C(CH3)2CO2C2H5
CH2CH2CO2C2H5
CH2CO2C2H5
HC:CCH2
Ph
Ph
Ph
Ph
4-F-Ph
4-F-Ph
CH3CH2CH2CH2CH2CH2
CH2CO2C2H5
Isolated yields were estimated based on those of 4, 8, and 9
b
Known compounds were identified [11, 12]
Experimental Animal Center of Hubei University of Medicine. This study was
carried out in strict accordance with the recommendations in the Guide for the Care
and Use of Laboratory Animals of Hubei University of Medicine. The protocol was
approved by the Committee on the Ethics of Animal Experiments of Hubei Univer-
sity of Medicine. All surgery was performed under sodium pentobarbital anesthesia,
and all efforts were made to minimize suffering. Human A549 (lung carcinoma),
HepG2 (hepatocellular carcinoma), and HeLa (cervical adenocarcinoma) cells
(kindly provided by Institute of Basic Medical Sciences, Hubei Medical University,
Shiyan, China) were cultured in a proper medium supplemented with 10 % fetal
bovine serum under humidified atmosphere of 5 % CO2 at 37 °C.
Analgesic activity
The analgesic activity of the synthesized compounds 5a–5d and 10a–10g was
evaluated using the rat paw withdrawal technique. Namely, test for analgesic
activity was performed by using the Plantar and the electronic von Frey Test
Analgesia Meter for detection of thermal paw hyperalgesia (IITC Life Science,
USA). For the establishment of chronic pain, a constriction injury was applied to the
sciatic nerve, which induced neuropathic pain characterized by hyperalgesia and
allodynia. All injections were given intraperitoneally with the compounds 500 mg/kg
of body weight. Results showed that the replacement of 2,3-disubstituted
quinazolinones by its polysubstitution fused furo[2,3-d]pyrimidinone derivatives
promoted potential analgesic activities (Table 2). The test compounds (10a, 10g)
showed good analgesic activity.
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Synthesis and biological activity of fused…
Table 2 Behavioral studies of paw withdrawal latency (n = 5, xꢀÆ s)
Compound
Concentration
(mg/ml)
Paw withdrawal latency (PWL) (TS(s), MS(g))
Before drug treatment After drug treatment
30 min
60 min
5a
50
TS
8.90 0.72
35.00 2.00
7.09 0.68
36.72 3.50
6.45 1.82
36.38 2.80
9.51 2.80
36.74 2.67
11.44 1.36
35.56 1.27
10.99 2.37
39.02 3.84
12.25 2.78
32.50 3.00
13.80 2.92
38.80 2.46
6.07 1.21
38.4 4.31
8.65 0.84
35.70 4.14
9.63 1.46
32.09 2.68
11.40 2.89
29.50 1.64
11.65 0.61
35.98 3.50
8.40 0.87
10.81 0.70
57.85 4.50*
11.66 0.92
40.14 1.07
13.07 3.16*
44.52 2.80
6.96 1.67
47.92 1.31*
27.99 4.17*
61.12 5.82*
16.12 3.79
61.70 4.00*
18.81 6.85*
47.88 2.48
15.66 3.77
44.12 4.30
7.89 1.50
48.06 5.70*
11.54 1.48
44.80 4.8*
28.01 2.70*
69.80 2.80*
11.58 2.92
27.73 1.26
MS
TS
5b
50
MS
TS
44.98 1.07*
5c
50
7.67 1.26
MS
TS
36.56 1.80
12.80 2.23
45.42 1.61
32.43 5.67*
57.22 3.24*
16.66 3.09
44.42 1.00
18.51 2.55*
59.54 2.41*
25.23 3.51*
62.40 2.01*
15.66 3.37*
55.14 2.60*
12.18 2.15
49.30 1.60*
17.97 0.94*
52.10 4.0*
8.51 0.82
5d
50
MS
TS
10a
10b
10c
10d
10e
10f
50
MS
TS
50
MS
TS
50
MS
TS
50
MS
TS
50
MS
TS
50
MS
TS
10 g
Control
50
MS
TS
Normal saline
MS
31.20 1.07
TS thermal hyperalgesia, MS mechanical hyperalgesia
* Different from normal saline group (p \ 0.05)
Antitumor activity
For evaluation of antitumor activities, the MTS cell proliferation assay was
performed, which was dependent on the amount of MTS being reduced to an
aqueous, soluble formazan by dehydrogenase enzymes found in metabolically
active cells. The antiproliferative activities of 5a–5d and 10a–10g were investigat-
ed, and the results showed that these compounds exhibited cytotoxicity against
human cancer cell lines: A459 (lung cancer), HepG2 (hepatocellular carcinoma) and
HeLa (cervical cancer) with cisplatin as the reference drug. The results are listed in
Table 3 and expressed as IC50 values. Among these compounds, 10g is the most
active, which showed potent anticancer efficacy against the human lung cancer
A549 cell line with an IC50 value of 14.8 lM.
123

Q. Li et al.
HeLad
Table 3 Cytotoxicity of
compounds 5 and 10 in
representative human cancer cell
lines (IC50, lM)a
Compound
A549b
HepG2c
5a
115.2 0.6
124.5 0.3
156.5 0.5
131.4 0.5
91.6 0.8
97.6 1.1
134.5 1.0
122.7 1.2
156.2 0.4
49.3 1.2
14.8 – 0.6
259.3 1.5
166.6 0.8
262.9 0.3
183.2 1.0
168.1 1.3
163.1 3.4
210.2 2.0
142.8 1.7
185.3 2.0
156.4 1.4
78.8 2.4
–
138.1 1.8
128.6 2.3
129.3 1.7
174.6 2.6
121.1 0.6
115.6 1.8
99.4 0.9
223.6 2.0
206.2 2.2
89.4 1.1
53.5 0.8
–
5b
5c
5d
a
Half maximal inhibitory
10a
10b
10c
10d
10e
10f
concentration (IC50); values are
the mean of three independent
experiments performed in
triplicate determined after 24 h
of treatment
b
Lung cancer
c
Liver hepatocellular
carcinoma
10g
Cisplatin
32.7 – 1.3
d
Cervical cancer
Conclusions
In the present study, making use of the bioisostere concept for replacement of 2,
3-disubstituted quinazolinones by fused furo[2,3-d]pyrimidinone derivatives, we
have developed an efficient synthesis for fused furo[2,3-d]pyrimidinone derivatives
via the functionalized iminophosphoranes. Bioassay of the compounds indicated
that 2-alkylthiobenzo-furo[3,2-d]pyrimidin-4(3H)-ones and 1-aryl-2-alkylthio-ben-
zofuro[3,2-d]-1,2,4-triazolo[1,5-a]pyrimidin-(1H)-ones showed potential analgesic
and antitumor activities, for which these compounds can be established as lead
molecules for developing novel analgesic and antitumor drugs. Upon vigorous
screening, compound 10g was found to provide promising dual analgesic and
antitumor activities as a single compound, which could be further developed into an
effective chemotherapeutic agent in treating patients with cancer pain. Further
bioassay, optimization, and structure–activity relationships of the title compounds
are underway.
Experimental
General
Melting points were rectified. MS were measured by using a Finnigan Trace MS
spectrometer. IR were recorded by a PE-983 infrared spectrometer as KBr pellets
1
with absorption in cm-1. H NMR were recorded in CDCl3 or DMSO-d6 by the
Varian Mercury 400 spectrometer with resonances relative to TMS. Elementary
analyses were performed by a Vario EL III elementary analysis instrument. X-ray
diffraction data were collected by a Bruker SMART AXS CCD diffractometer.
Male albino rats weighing 120–150 g were used throughout this assay. They were
kept in the animal house under standard conditions of light and temperature with
free access to food and water. The animals were randomly divided into groups of
123

Synthesis and biological activity of fused…
five rats each. Test for analgesic activity was performed by using the Plantar Test
Analgesia Meter for detection of thermal paw hyperalgesia (IITC Life Science,
USA).
Synthesis of 1-aryl-2-thioxo-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]
pyrimidin-5(1H)-ones 9a–9b
Excess carbon disulfide (15 ml) was added to a solution of iminophosphorane 8
(20 mmol) in anhydrous methylene dichloride and acetonitrile (20 ml, v/v = 1:1).
After the reaction mixture was refluxed for 24–30 h at 40–50 °C, the solvent was
condensed under reduced pressure, with addition of ether (10 ml) to precipitate
triphenylphosphine sulfide. The precipitate was then removed by filtration, while the
filtrate was evaporated to give 1-aryl-2-thioxo-benzofuro[3,2-d]-1,2,4-triazolo[1,5-
a] pyrimidin-5(1H)-ones 9a–9b.
1-phenyl-2-thioxo-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-ones (9a)
1
White crystals (86 % yield), M.p. [ 300 °C. H NMR (400 MHz, DMSO-d6)d:
7.51–7.27 (m, 9H, Ar–H). MS (70 eV) m/z (%): 334/333 (M?, 55/100), 300 (23),
275 (42), 261 (38), 235 (16), 185 (46), 160 (42), 130 (55), 114 (61), 102 (99), 77
(81). Anal. Calcd for C17H10N4O2S (334.4): C, 61.07; H, 3.01; N, 16.76. Found: C,
61.13; H, 3.18; N, 16.70.
1-(4-flurophenyl)-2-thioxo-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-
ones (9b)
As described in detail previously [11].
Synthesis of 1-aryl-2-alkylthio-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]
pyrimidin-5(1H)-ones 10a–10g
Alkyl halide (3.5 mmol) and solid potassium carbonate (4 mmol) were added to a
solution of 1-aryl-2-thioxo- benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-
ones 9 (3 m mol) in dry DMF (10 ml). The mixture was stirred for 5–6 h at
50–60 °C. The resulting solution was cooled and diluted with ice water (30 ml). The
solid product obtained was filtered and recrystallized from methylene dichloride/
petroleum ether (v/v) to give 1-aryl-2-alkylthio-benzofuro[3,2-d]-1,2,4-triazolo[1,5-
a] pyrimidin-5(1H)-ones 10a–10g.
1-phenyl-2-n-butylthio-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a] pyrimidin-5(1H)-
ones (10a)
White crystals; Mp: 210–212 °C. 1HNMR (400 MHz, CDCl3): d = 0.94 (t,
J = 7.2, 3H, CH3), 1.46–1.52 (m, 2H, CH2), 1.68–1.81 (m, 2H, CH2), 3.45–3.48
(s, 2H, CH2), 7.34–7.99 (m, 9H, Ar–H). IR (KBr) nu = 1700 (C=O), 1513,
1227 cm-1; MS (70 eV) m/z (%): 390 (69, M?), 342 (34), 332 (100), 260 (19),
123

Q. Li et al.
184 (17), 159 (17), 129 (38), 113 (74), 100 (52), 76 (36). Anal. Calcd for
C21H18N4O2S (390.5): C, 64.60; H, 4.65; N, 14.35. Found: C, 64.53; H, 4.77; N,
14.19.
1-phenyl-2-(1,1-dimethyl-1-ethoxycarbonyl-methylthio)-benzofuro[3,2-d]-1,2,4-
triazolo[1,5-a]-pyrimidin- 5(1H)-ones (10b)
White crystals; Mp: 204–206 °C; 1H NMR (400 MHz, CDCl3): d = 1.29 (t,
J = 7.2, 3H, CH3), 1.78 (s, 6H, 2 9 CH3), 4.22 (q, J = 7.2, 2H, OCH2),
7.35–7.98 (m, 9H, Ar–H). 13C NMR (100 MHz, CDCl3): d = 13.9, 26.5, 55.2,
62.4, 112.7, 121.8, 122.4, 123.3, 127.4, 129.6, 129.8, 130.2, 131.2, 134.0, 144.2,
147.1, 147.4, 150.8, 157.4, 172.2. MS (70 eV) m/z (%): 448 (35, M?), 333 (100),
276 (33), 260 (40), 185 (17), 129 (31), 113 (35), 101 (24), 76 (18). Analytical
calculation for C23H20N4O4S (448.5): C, 61.59; H, 4.49; N, 12.49. Found: C, 61.53;
H, 4.58; N, 12.35.
1-phenyl-2-(2-ethoxycarbonyl-ethylthio)-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]-
pyrimidin-5(1H)-ones (10c)
White crystals; Mp:223–225 °C. 1H NMR (400 MHz, CDCl3): d = 1.22 (t,
J = 7.2, 3H, CH3), 2.96–2.98 (m, 2H, CH2), 3.65–23.71 (m, 2H, CH2), 4.14 (q,
J = 7.2, 2H, OCH2), 7.34–7.98 (m, 9H, Ar–H).MS (70 eV) m/z (%): 434 (M?, 54),
389 (7), 333 (100), 261 (7), 185 (3), 130 (7), 114 (9), 77 (7). Analytical calculation
for C22H18N4O4S (434.5): C, 60.82; H, 4.18; N, 12.90. Found: C, 60.76; H, 4.25; N,
12.82.
1-phenyl-2-(1-ethoxycarbonyl-methylthio)-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]-
pyrimidin-5(1H)- ones (10d)
White crystals; Mp: 252–254 °C; 1H NMR (400 MHz, CDCl3): d = 1.31 (t,
J = 7.2, 3H, CH3), 4.24–4.30 (m, 4H, 2 9 CH2), 7.35–7.98 (m, 9H, Ar–H). IR
(KBr) nu = 1713 (C=O), 1540, 1345, 1105 cm-1. MS (70 eV) m/z (%): 420 (M?,
100), 333 (79), 260 (7), 185 (3), 129 (3), 113 (3). Analytical calculation for
C21H16N4O4S (420.4): C, 59.99; H, 3.84; N, 13.33. Found: C, 59.93; H, 3.80; N,
13.20.
1-phenyl-2-propargylthio-benzofuro[3,2-d]-1,2,4-triazolo[1,5-a]-pyrimidin-5(1H)-
ones (10e)
1
White crystals; Mp: 275–277 °C; H NMR (400 MHz, CDCl3): d = 2.33 (s, H,
CH), 4.22 (s, 2H, CH2), 7.34–7.99 (m, 9H, Ar–H). MS (70 eV) m/z (%): 372 (M?,
89), 333 (73), 275 (95), 260 (100), 185 (29), 169 (60), 129 (82), 113 (97),
102 (76), 76 (58). Analytical calculation for C20H12N4O2S (372.4): C, 64.50; H,
3.25; N, 15.04. Found: C, 64.39; H, 3.19; N, 14.96.
123

Synthesis and biological activity of fused…
1-(4-flurophenyl)-2-(1-ethoxycarbonyl-methylthio)-benzofuro[3,2-d]-1,2,4-
triazolo[1,5-a]-pyrimidin-5(1H)- ones (10g)
White crystals; Mp: 198–200 °C; 1H NMR (400 MHz, CDCl3): d = 1.32 (t,
J = 7.2, 3H, CH3), 4.25–4.28 (m, 4H, 2 9 CH2), 7.34–7.95 (m, 8H, Ar–H). IR
(KBr) nu = 1724 (C = O), 1580, 1513, 1307, 1183 cm-1. MS (70 eV) m/z (%):
438 (M?, 7), 436 (100), 353 (44), 350 (84), 279 (27), 240 (4), 114 (14), 102 (9),
95 (6). Analytical calculation for C21H15FN4O4S (438.4): C, 57.53; H, 3.45; N,
12.78; Found: C, 57.43; H, 3.53; N, 12.61.
Pharmacological studies
Anticancer activity
Cancer cells (9 9 104 cells/well) were seeded into each well of a 96-well
microplate, supplemented with 10 % fetal bovine serum (PBS) in each well of
96-well microculture plates, and incubated for 24 h at 37 °C in a CO2 incubator.
Compounds, diluted to the desired concentrations in culture medium, were added to
the wells with respective vehicle control. After 24 h of incubation, MT stetrazolium
salt (3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium) (MTS) (10 ll, 5 mg/ml) was added to each well, and the plates
were further incubated for 4 h. Then the supernatant from each well was carefully
removed, formazan crystals were dissolved in DMSO (100 ll), and absorbance at
570 nm wavelength was recorded. Three independent experiments were performed
in triplicate. An equal amount of DMSO was added to the cells as negative control.
All experiments were performed in triplicate.
Analgesic activity
CCI surgery A neuropathic pain model of sciatic nerve chronic constriction injury
(CCI) was employed to induce sciatic nerve injury [13]. Briefly, rats were
anesthetized with pentobarbital sodium (60 mg/kg, intraperitoneal); following this,
the left sciatic nerve was exposed at mid-thigh level. The distance between two
adjacent ligatures was 1 mm. The wound was irrigated with normal saline (0.9 %)
and closed in two layers with 4–0 silk sutures and surgical skin staples. To confirm
the consequence of nerve injury, a sham operation was performed with exposure of
the left sciatic nerve without ligation. The CCI-treated rats were tested 6 days after
the operation.
Von Frey filament test To evaluate the mechanical sensitivity, a Von Frey filament
method was used. For the test, each rat was placed on a mesh floor inside a
transparent plastic cage. The rats were kept in a silent environment at room
temperature for 20 min, and then force (in grams) was applied to the rats through
the plantar hind paw up-down method. The minimum paw withdrawal threshold was
defined as the minimum gram strength producing two sequential responses at 3-min
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Q. Li et al.
intervals (withdrawal from pressure). The upper pressure limit was 100 g, and the
point at which each rat pulled its paw back was recorded.
Plantar test To evaluate the magnitude of thermal hyperalgesia, plantar test was
used. For this method, each rat was placed in an acrylic compartment on a clear
glass surface. A moveable heat source was then put under the plantar surface of the
hind paw and activated with a light beam. The intensity of the light beam was
adjusted such that the withdrawal latencies in sham-operated rats were around
8–10 s. The built-in digital timer automatically recorded the response latency for
paw withdrawal to the nearest 0.1 s. A cutoff time of 20 s was used to prevent tissue
damage when a rat failed to respond after 20 s.
Statistical analysis
Statistical analysis of the biological activities caused by the synthesized compounds
in animals was evaluated using one-way analysis of variance (ANOVA). In all
cases, post hoc comparisons of the means of individual groups were performed
using t test. A significance level of p \ 0.05 denoted significance. All values are
expressed as mean standard deviations (SD).
Acknowledgments We gratefully acknowledge financial support of this work by the National Natural
Science Foundation of Hubei Provincial (No.2011CDC006) and the fund of Hubei 2011 Cooperative
Innovation Center (No. 2011JH-2014CXTT07), Key Project of Project of Hubei University of Medicine
(No. 2014XKJSXJ06, 2014XKJSSJ04, 2011CXX03 and 2014CXZ-05) and Natural Science Foundation
of Hubei Province of China (No. 2012FFC060); Natural Science Foundation of Hubei University of
Medicine (No. 2011QDZR-2).
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