Discovery of novel N-substituted carbazoles as neuroprotective agents with potent anti-oxidative activity

Article abstract of DOI:10.1016/j.ejmech.2013.07.029

Carbazole moiety is an important scaffold with a variety of biological applications, for example, anti-oxidative stress. Our previous synthesized carbazoles were screened for their neuroprotective properties against two individual oxidative stresses. Some of the new carbazole derivatives were observed with modest to good neuroprotective effects on neuronal cells HT22 against cell injury induced by glutamate or homocysteic acid (HCA). Substituents introduced to the carbazole ring system play crucial roles in their biological activities. In particular, a bulky group favors the neuroprotective activity of the compounds. One of the new compounds, 6, showed the best neuroprotective effects, which might result from its anti-oxidative activity with a GSH-independent mechanism. These findings might provide an alternative strategy for the development of novel carbazole derivatives for the treatment of CNS diseases such as Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2013.07.029

European Journal of Medicinal Chemistry 68 (2013) 81e88
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of novel N-substituted carbazoles as neuroprotective agents
with potent anti-oxidative activity
,
b
,
,
,
Daqian Zhu a 1, Meihui Chen b 1, Min Li a b, Bingling Luo a, Yang Zhao b, Peng Huang a,
Fengtian Xue c, Simona Rapposelli d, Rongbiao Pi b , Shijun Wen
,
a,b,
**
*
a State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou 510060, China
b School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China
c The Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201-1140, United States
d Department of Pharmaceutical Sciences, University of Pisa, Via Bonanno 6, Pisa 56126, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Carbazole moiety is an important scaffold with a variety of biological applications, for example, anti-
oxidative stress. Our previous synthesized carbazoles were screened for their neuroprotective proper-
ties against two individual oxidative stresses. Some of the new carbazole derivatives were observed with
modest to good neuroprotective effects on neuronal cells HT22 against cell injury induced by glutamate
or homocysteic acid (HCA). Substituents introduced to the carbazole ring system play crucial roles in
their biological activities. In particular, a bulky group favors the neuroprotective activity of the com-
pounds. One of the new compounds, 6, showed the best neuroprotective effects, which might result from
its anti-oxidative activity with a GSH-independent mechanism. These findings might provide an alter-
native strategy for the development of novel carbazole derivatives for the treatment of CNS diseases such
as Alzheimer’s disease.
Received 14 April 2013
Received in revised form
20 July 2013
Accepted 22 July 2013
Available online 9 August 2013
Keywords:
Carbazole
Alzheimer’s disease
Neuroprotection
HT22 cell
Ó 2013 Elsevier Masson SAS. All rights reserved.
Antioxidant
1. Introduction
cellular oxidative stress, cellular functions’ alteration, and even
cell death. Central nervous system (CNS) is particularly susceptible
Increased oxidative stress has been recognized as a common
culprit of many neurological disorders including Alzheimer’s dis-
ease (AD), Parkinson’s disease (PD), and stroke [1e3]. Oxidative
stress is often the result of unregulated production of reactive ox-
ygen species (ROS) including hydrogen peroxide, nitric oxide, su-
peroxide, and reactive hydroxyl radicals [2]. Cells have developed a
highly elaborate mechanism to regulate cellular level of oxidant
species including ROS using exogenous and endogenous antioxi-
dants [2]. Endogenous antioxidants such as glutathione peroxidase
and superoxide dismutase are essential to detoxify or scavenge
oxidant species. In some circumstances, however, the production of
oxidant species can exceed the scavenging ability of the endoge-
nous antioxidants. The consequent oxidative imbalance can lead to
to oxidative stress because, comparing to other organs, it is rich
in highly oxygen-consuming polyunsaturated fatty acids, and
possesses a relatively low level of antioxidant and low regenerative
capacity [2,3].
Naturally occurring carbazoles have attracted interests of chem-
ists and biologists due to their structural features and intriguing
pharmacological activities. A large array of natural carbazoles and
synthetic derivatives are endowed with profound biological activ-
ities. These unique compounds include anti-inflammatory caprofen
[4], antitumor ellipticine [5], antibiotic carbazomycin B [6], anti-
oxidative carvedilol [7], neuronal cell-protecting agent carbazoma-
durin A and P7C3 [8], and anti-prion tetrahydrocarbazoles [9] (Fig.1).
Anti-oxidative activity of carbazoles might contribute to their
neuroprotective properties although the exact mechanism still re-
mains to be explored [10]. Substituted carbazoles were often re-
ported to be neuroprotective, for example, carvedilol and P7C3
[7,8]. These findings indicated that a high possibility to move sub-
stitutes around the carbazole core and to change their chemical
properties could still keep their neuroprotective activity. Therefore,
it is of high interest to search for a library of substituted carbazoles
Abbreviations: CNS, central nerve system; AD, Alzheimer’s disease; ROS, reactive
oxygen species; mCPBA, meta-chloroperoxybenzoic acid.
* Corresponding author.
** Corresponding author. State Key Laboratory of Oncology in South China, Sun
Yat-Sen University Cancer Center, Guangzhou 510060, China.
E-mail addresses: pirb@mail.sysu.edu.cn (R. Pi), wenshj@sysucc.org.cn (S. Wen).
1
Both authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.029

82
D. Zhu et al. / European Journal of Medicinal Chemistry 68 (2013) 81e88
Scheme 2. Synthesis of 6. Reaction conditions: (A) Pd(PPh3)4, K3PO4, EtOH, reflux, 6 h,
85%. (B) i. NaNO2, 0 ꢀC THF, 4M HCl, ii. KI, r.t., 81%. (C) mCPBA (85%), TfOH, CH2Cl2, r.t.,
2 h, 100%. (D) p-toluidine, Na2CO3, Cu(OAc)2, iPrOH/(CH2OH)2, 90 ꢀC, 16 h, 71%.
Fig. 1. Chemical structures of known carbazoles.
3. Results and discussions
in order to identify a potent lead compound that will be further
developed into a drug for AD.
The serendipitously obtained carbazoles with a structural di-
versity resemble the neuroprotective agents, and they could un-
expectedly provide a good lead to elaborate drug candidates to
target AD. So the synthetic compounds were taken for a biological
screening against the death of neuronal HT22 cells induced by
neurotoxins including glutamate and HCA. Our results indicated
that several of the new carbazoles were significantly neuro-
2. Chemistry
Recently, we have developed a novel strategy to prepare diver-
sified carbazoles from cyclic dipenyleneiodoniums with a broad
range of amines including aromatic and aliphatic amines and aryl
sulfonamides (Scheme 1) [11]. Specifically, our method has
employed an inexpensive catalyst copper (II) acetate (Cu(OAc)2)
and a base Na2CO3. The reaction of diphenyleniodoniums with a
variety of amines in a refluxing isopropanol/ethylene glycol (9/1)
proceeded smoothly in modest to good yields. In the end, a small
chemical library of structurally diversified carbazoles has been
generated.
protective at the concentration of 30 mM (Fig. 2).
A substituent at the N9 position of the carbazole is essential for
neuroprotective ability while unsubstituted carbazole did not pre-
vent HT22 from the death induced by glutamate or HCA at the
concentration of 30 mM (Table 1). This was further confirmed by
structurally similar compounds, for example, dibenzofuran (DBF)
and dibenzothiophene (DBT) that showed no neuroprotective ac-
tivity at the concentration of 30 mM. The spatial properties of the
The general procedure for the preparation of these carbazoles
was exemplified by the synthesis of 6 (Scheme 2). Firstly, Suzuki
reaction of 2-iodoaniline with [1,10-biphenyl]-4-ylboronic acid and
subsequent Sandmeyer reaction afforded iodo-terphenyl 6b. Then,
6b was oxidized and subsequently cyclized to form 6a using mCPBA
(85%) in anhydrous dichloromethane with trifluoromethanesulfonic
acid. Finally, 6 was prepared in a good yield using the method dis-
cussed above.
substituents play important roles in their biological activities. The
neuroprotective activity was significantly increased when a bulky
group, such as methoxylphenyl (3), t-butylphenyl (4), tri-
fluorophenyl (11), and N,N-dimethylphenyl (12) was introduced to
the N9 position of the carbazole. So it is not surprising that com-
pound 6 with methylphenyl and phenyl attached to N9 position and
C2 position had displayed the best neuroprotective ability, resulting
Scheme 1. Synthetic carbazoles from diphenyleneiodoniums.

D. Zhu et al. / European Journal of Medicinal Chemistry 68 (2013) 81e88
83
Fig. 2. The effect of carbazoles on the viability of mice hippocampal cell line HT22 cells. Cells were pretreated ahead with carbazoles alone at 30
viability was tested by MTT assay (n ¼ 6). *P < 0.05 compared with glutamate-treated group.
m
M for 24 h. After that, the cell
from cumulative spatial effects. The electronic property of the
substituents on the introduced benzene ring did not make any
significant difference (11 and 12). Substituents attached to C3 po-
sition of carbazoles play a negative role (5). Carbazoles with an N-
alkyl substituent showed none or low neuroprotective capacity (14
or 15, 16). With N-arylsulfonyl, an electron-rich methoxy group on
the side benzene ring of carbazoles was detrimental (19, 20). With
2-subsituted phenyl and trifluoromethyl, or none substitute, the N-
arylsulfonyl carbazoles showed modest neuroprotective ability (21,
23, 17, 18). In general, carbazole with N-alkyl and benzenesulfonyl
substitutions are less potent compared to the ones with aryl sub-
stitutions. However, one exception is compound 25, which showed
neuroprotection while the counterpart 24 did not (see Table 1). In
addition, the neuroprotective effects of compound 6, in glutamate
and HCA model, are in “bell-shape” manners. Further study
demonstrated that compound 6 inhibits the proliferation but not
concentration as low as 3 mM (see Table 1, Fig. 3 and Supplemental
Figure 1). Both glutamate and HCA were reported to induce ROS
through depleting the intracellular GSH [12e14]. Previous study
demonstrated that glutamate-induced ROS is associated with the
cell damage in HT22 cells [15]. To investigate whether the novel
carbazole 6 could decrease the glutamate-induced ROS, an ROS
predictor DHE was used. We found that the cells changed to red
color under fluorescence microscopy when exposed to glutamate
(see Fig. 4B). Compound 6 significantly inhibited the changes in a
concentration-dependent manner (see Fig. 4CeE), suggesting that
6 decreased glutamate-induced ROS level in a concentration-
dependent manner. However, 6 did not reverse the glutamate-
induced intracellular GSH depletion (see Fig. 5), indicating that it
might quench ROS through a GSH-independent way. Previous
report showed that melatonin, a potent natural antioxidant also did
not restore cytosolic GSH depletion although it potently prevented
mitochondrial ROS production [15]. These data suggest that com-
pound 6 might target mitochondria in a similar way as melatonin.
Further study is under way to uncover the exact mechanism(s) of
neuroprotective effects of 6.
induces cell toxicity at 30 mM using LDH assay (cell toxicity assay)
(Data not shown). Further studies are under way to uncover the
exact mechanism.
It is interesting that compound 6 dramatically prevented HT22
cells from the cell death induced by glutamate or HCA at the
4. Conclusion
Table 1
Neuroprotective effects of the carbazoles on HT22 cells against cell injury induced by
neurotoxins, glutamate and HCA.
In summary, we have screened a series of substituted carbazoles
for their neuroprotective activities. The results of the anti-oxidation
screening have revealed that the compounds 3, 4, 6, 9, 11, 12, 17, and
21 showed moderate to excellent neuroprotective capacity. The
most potent compound, 6 prevented cell death through a GSH-
Chemicals Inhibitory rate of cell injury
induced by glutamate (%)
Inhibitory rate of cell injury
induced by HCA (%)
3
m
M
10
mM
30
mM
3
mM
10
mM
30 mM
1
2
3
4
0
7
0
25
35
0
6
0
59
70
0
0
0
0
32
30
0
5
0
71
63
0
0
3
0
0
0
0
2
0
5
6
7
63
0
69
0
50
0
64
0
77
0
44
0
8
0
0
0
0
0
0
9
0
0
0
20
0
0
3
0
0
0
0.8
0
12
3
0
0
4
0
0
14
40
0
0
8
1
10
3
2
3
19
2
10
0
20
0
68
77
5
0
20
4
47
28
4
5.2
30
7
42
0
0
0
0
0
0
0
7
0
4
0
0
0
11
0
0
0
18
0
0
10
30
5
0
8
1
13
6
0
0
18
0
6
40
0
80
81
12
0
10
12
34
30
0
0
30
0
38
0
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Fig. 3. Compound 6 dramatically attenuates the cell death induced by glutamate in a
dose-dependent manner in mice hippocampal cell line HT-22 cells. Cells were pre-
treated ahead with compound 6 at different concentrations for 30 min and then
exposed to glutamate (2 mM) for 24 h. After that, the cell viability was tested by MTT
assay (n ¼ 6).
0
30
16
26
Bold values highlight that these compounds are effectively neuroprotective.

84
D. Zhu et al. / European Journal of Medicinal Chemistry 68 (2013) 81e88
Fig. 4. Compound 6 dramatically attenuates the ROS induced by glutamate in mice hippocampal cell line HT22 cells. HT22 cells were treated with Compound 6 for 30 min and
further exposed to glutamate (2 mM) for 10 h. DHE staining was applied for ROS detection. (A) Vehicle; (B) Glutamate; (C) Compound 6 (30 M); (D) Compound
m
6
(3
m
M) þ glutamate; (E) Compound 6 (10
m
M) þ glutamate and (F) Compound 6 (30
mM) þ glutamate group, respectively. The data showed here are representative results of three
independent experiments.
independent way. With low toxicity and potent neuroprotective
effects, 6 could serve as a potential lead compound to further
develop into novel agents with potent neuroprotection against
oxidative stress-associated CNS diseases such as AD.
multiplet, the signals are reported as intervals. Signals are abbre-
viated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet. Coupling constants are expressed in Hertz. The progress
of the reactions was monitored by thin-layer chromatography on a
glass plate coated with silica gel with fluorescent indicator (GF254).
Column chromatography was performed on silica gel (200e300
mesh). Purity of all final compounds was determined by Agilent
1100 HPLC system using an Eclipse XDB-C18 column. In HPLC
conditions, flow rate was set at 1 mL/min, and gradient elution was
run for 20 min, from 85% MeOH/H2O to 95% MeOH/H2O in 15 min
and 95% MeOH/H2O in 5 min.
5. Experiments
5.1. Chemistry
5.1.1. General information
All solvents were commercially available and were used without
a further purification unless stated. The chemicals used were either
purchased from commercial sources or prepared according to
literature procedures. The 1H and 13C nuclear magnetic resonance
(NMR) spectra were recorded on a Bruker Avance spectrometer 400
at 400 MHz and 100 MHz respectively. Chemical shifts are given in
5.1.2. Synthesis and characterization of the carbazole derivatives
General procedure for the synthesis of carbazoles: To a stirred
solution of cyclic diphenyliodonium trifluoromethanesulfonate
(100 mg, 234 mmol, 1 eq) in iPrOH (1.8 mL) and ethylene glycol
(0.2 mL), was added amine (4 eq), sodium carbonate (3 eq),
Cu(OAc)2 (0.2 eq). The reaction proceeded at a reflux for 16 h under
argon atmosphere before iPrOH was removed by a rotary evapo-
ration. The remained mixture was partitioned between water and
EtOAc, and the aqueous phase was extracted with EtOAc. The
combined organic phases were washed with H2O and brine, dried
over anhydrous Na2SO4, evaporated in a vacuo. The residue was
purified by column chromatography on a silica gel (PE/EtOAc) to
provide carbazole derivatives.
ppm (d
) referenced to CDCl3 with 7.26 for 1H and 77.10 for 13C, and
to d6-DMSO with 2.50 for 1H and 39.50 for 13C. In the case of
5.1.2.1. 2-Iodo-1,10:40,100-terphenyl (6b). To a stirred solution of [1,10-
biphenyl]-4-ylboronic acid (542.5 mg, 2.74 mmol) in EtOH (10 mL)
was added 2-iodoaniline (500 mg, 2.28 mmol), K3PO4 (1.21 g,
5.71 mmol), Pd(PPh3)4 (131.9 mg, 0.11 mmol). The reaction pro-
ceeded at a reflux for 6 h under argon atmosphere before EtOH was
removed by rotary evaporation. The remained mixture was parti-
tioned between water and EtOAc, and the aqueous phase was
extracted with EtOAc. The combined organic phases were washed
with H2O and brine, dried over anhydrous Na2SO4, and evaporated
in a vacuo. The residue was purified by column chromatography on
a silica gel (PE/EtOAc ¼ 20/1e10/1) to provide coupled amino-
terphenyl (476 mg, 85% yield) as a yellow liquid. To a stirred
Fig. 5. Compound 6 does not reverse the GSH depletion induced by glutamate in mice
hippocampal cell line HT22 cells. HT22 cells were treated with Compound 6 for 30 min
and further exposed to glutamate (2 mM) for 10 h. GSH levels were analyzed by the
GSH detection kit. The data showed here are representative results of three inde-
pendent experiments.

D. Zhu et al. / European Journal of Medicinal Chemistry 68 (2013) 81e88
85
solution of amino-terphenyl (278 mg, 1.13 mmol) in THF (5 mL) was
J ¼ 3.6 Hz, 9H) ppm. 13C NMR (100 MHz, CDCl3)
d
150.5, 141.1, 135.0,
added 4 M aqueous HCl (2.8 mL), and the solution was cooled in an
ice water bath. A solution of NaNO2 (93.8 mg, 1.36 mmol) in H2O
(3 mL) was added dropwise. After 20 min, a solution of KI
(470.3 mg, 2.83 mmol) in H2O (5 mL) was added, and stirred for
10 min in the ice water bath. Then the solution was slowly warmed
up to r.t. and stirred for 1 h before 1 M aqueous Na2S2O3 was added
until the color of the mixture didn’t change. The phases were
separated, and the aqueous phase extracted with EtOAc (15
mL ꢁ 3). The combined organic layers were washed with H2O
(5 mL ꢁ 2) and brine (5 mL ꢁ 1), dried over anhydrous Na2SO4,
concentrated in a vacuo. The residue was purified by column
chromatography on silica gel (PE/EtOAc ¼ 20/1e10/1) to provide 6b
(326 mg, 81% yield) as a yellow liquid.
126.7, 126.6, 125.8, 123.3, 120.2, 119.7, 34.8, 31.4 ppm. IR y 3129,
3048, 2953, 1913, 1781, 1670, 1597, 1515, 1451, 1317, 1230, 1182, 1110,
1013, 920, 834, 742, 625, 551 cmꢂ1. LRMS (ESI, m/z): 300.2 [M þ H]þ.
5.1.2.8. 9-(4-(tert-butyl)phenyl)-3-methoxy-9H-carbazole (5). A white
solid. HPLC tR ¼ 9.517 min, 98.76%. 1H NMR (400 MHz, CDCl3)
d 8.13e
8.05 (m, 1H), 7.65e7.55 (m, 3H), 7.52e7.46 (m, 2H), 7.43e7.31 (m, 3H),
7.25 (s, 1H), 7.07e7.00 (m, 1H), 3.94 (d, J ¼ 5.5 Hz, 3H), 1.42 (d,
J ¼ 5.5 Hz, 9H) ppm. 13C NMR (100 MHz, CDCl3)
d 154.2, 150.2, 141.4,
136.0, 135.2, 126.7, 126.4, 125.8, 123.6, 123.1, 120.2, 119.3, 114.9, 110.7,
110.0, 103.1, 56.1, 34.8, 31.4 ppm. IR
y 3052, 2945, 1598, 1464, 1270,
1187, 1023, 840, 741, 620 cmꢂ1. M.P. 104.9e106.4 ꢀC. LRMS (ESI, m/z):
330.2 [M þ H]þ. HRMS calcd for C23H23NO [M þ H]þ: 330.1852, found:
330.1867.
5.1.2.2. 3-phenyldibenzo[b,d]iodol-5-ium trifluoromethanesulfonate
(6a). To a stirred solution of 6b (150 mg, 421.1
CH2Cl2 (5 mL) was added mCPBA (75%, 145.4 mg, 631.7
(111.5 L, 1.26 mmol). The solution was stirred for 1 h at r.t. before
CH2Cl2 was removed by rotary evaporation. Et2O (5 mL) was added
to the remained solid. The mixture was stirred for 20 min, and then
filtered. The obtained solid was washed with Et2O three times,
dried in high vacuo to provide 6a (213 mg, 100% yield) as a black
solid.
m
mol) in anhydrous
5.1.2.9. 9-(4-fluorophenyl)-9H-carbazole (7). A yellow solid. HPLC
mmol), TfOH
tR ¼ 7.206 min, 96.54%. 1H NMR (400 MHz, CDCl3)
d 8.15 (d,
m
J ¼ 7.7 Hz, 2H), 7.56e7.50 (m, 2H), 7.45e7.39 (m, 2H), 7.35e7.28 (m,
6H) ppm. 13C NMR (100 MHz, CDCl3)
d
160.6 (d, 1J ¼ 247.4 Hz),140.0,
132.6, 128.0 (d, 3J ¼ 8.6 Hz), 126.7, 125.0, 123.7 (d, 3J ¼ 8.8 Hz), 122.2,
119.3,118.9,115.8 (d, 2J ¼ 22.6 Hz),115.0 (d, 2J ¼ 22.9 Hz),108.5 ppm.
IR y 3054, 2946, 1897, 1599, 1505, 1448, 1328, 1224, 1093, 1025, 916,
823, 739, 568 cmꢂ1. LRMS (ESI, m/z): 262.2 [M þ H]þ.
5.1.2.3. 2-phenyl-9-(p-tolyl)-9H-carbazole (6). Following the gen-
eral procedure for the synthesis of carbazoles, 6 was obtained as a
yellow solid at 71% yield. HPLC tR ¼ 9.074 min, 95.6%. 1H NMR
5.1.2.10. 9-(3-fluorophenyl)-9H-carbazole (8). A yellow solid. HPLC
tR ¼ 8.188 min, 98.44%. 1H NMR (400 MHz, CDCl3)
d 8.15
(d, J ¼ 7.7 Hz, 2H), 7.58 (q, J ¼ 8.0 Hz, 1H), 7.50e7.36 (m, 5H), 7.35e
(400 MHz, CDCl3)
d
8.00 (d, J ¼ 7.9 Hz, 1H), 7.64e7.56 (m, 2H), 7.50
7.27 (m, 3H), 7.18 (td, J ¼ 8.4, 2.5 Hz, 1H) ppm. 13C NMR
(d, J ¼ 1.5 Hz,1H), 7.47e7.36 (m, 4H), 7.32 (t, J ¼ 7.3 Hz,1H), 7.21e7.12
(m, 2H), 7.07 (m, 5H), 2.30 (s, 3H). 13C NMR (100 MHz, CDCl3)
143.8,
141.9, 139.8, 131.2, 130.0, 129.5, 128.9, 128.8, 127.5, 127.3, 120.5, 118.6,
(100 MHz, CDCl3)
d
140.5, 131.0 (d, 3J ¼ 9.3 Hz), 126.1, 123.5, 122.7
d
(d, 4J ¼ 3.1 Hz), 120.4, 120.3, 114.5 (d, 3J ¼ 6.9 Hz), 114.3 (d,
3J ¼ 8.7 Hz),109.7 ppm. IR
y 3041, 2958,1895,1597, 1500,1450,1319,
114.0, 101.2, 20.9 ppm. IR
y
3048, 2949, 1709, 1589, 1423, 1333, 1252,
1223, 1083, 1015, 913, 822, 746, 570 cmꢂ1. LRMS (ESI, m/z): 262.2
[M þ H]þ.
1120, 1056, 969, 780, 650, 589 cmꢂ1. M.P. 107.2e108.1 ꢀC. LRMS (ESI,
m/z): 334.2 [M þ H]þ. HRMS calcd for C25H20N [M þ H]þ: 334.1596,
found: 334.1602.
5.1.2.11. 9-(4-chlorophenyl)-9H-carbazole (9). A purple solid. HPLC
tR ¼ 10.229 min, 98.45%. 1H NMR (400 MHz, CDCl3)
d 8.14 (d,
5.1.2.4. 9-phenyl-9H-carbazole(1). A white solid. HPLC tR ¼ 8.079 min,
J ¼ 7.6 Hz, 2H), 7.55 (q, J ¼ 8.0 Hz, 4H), 7.46e7.34 (m, 4H), 7.30 (t,
100%. 1H NMR (400 MHz, CDCl3)
d
8.15 (d, J ¼ 7.7 Hz, 2H), 7.65e7.54
J ¼ 6.8 Hz, 2H) ppm. 13C NMR (100 MHz, CDCl3)
d
140.7, 136.3, 133.1,
(m, 4H), 7.47 (t, J ¼ 6.9 Hz, 1H), 7.41 (d, J ¼ 3.7 Hz, 4H), 7.33e7.27 (m,
2H) ppm. 13C NMR (100 MHz, CDCl3)
140.9, 137.8, 129.9, 127.5, 127.2,
125.9, 123.4, 120.3, 119.9, 109.8 ppm. IR 3047, 2926, 1675, 1589, 1446,
130.1, 128.4, 126.1, 123.5, 120.4, 120.2, 109.6 ppm. IR y 3032, 2949,
d
1895, 1569, 1436, 1358, 1238, 1080, 1032, 836, 759, 650, 533 cmꢂ1
.
y
LRMS (ESI, m/z): 278.2 [M þ H]þ.
1321, 1226, 1169, 1011, 922, 746 cmꢂ1. LRMS (ESI, m/z): 244.2 [M þ H]þ.
5.1.2.12. 9-(4-bromophenyl)-9H-carbazole (10). A yellow solid. HPLC
5.1.2.5. 9-(p-tolyl)-9H-carbazole (2). A white solid. HPLC tR ¼ 9.764
tR ¼ 10.997 min, 98.46%. 1H NMR (400 MHz, CDCl3)
d 8.14 (d,
min, 99.21%. 1H NMR (400 MHz, CDCl3)
d
8.14 (d, J ¼ 7.8 Hz, 2H),
J ¼ 7.6 Hz, 2H), 7.83e7.61 (m, 3H), 7.50e7.35 (m, 5H), 7.30
7.47e7.35 (m, 9H), 7.29 (d, J ¼ 1.9 Hz, 1H), 2.49 (s, 3H) ppm. 13C NMR
(t, J ¼ 6.8 Hz, 2H) ppm. 13C NMR (100 MHz, CDCl3)
d 140.6, 138.9,
(100 MHz, CDCl3)
d
141.1, 137.4, 135.0, 130.5, 127.0, 125.8, 123.2,
136.8, 133.1, 132.4, 129.9, 129.4, 128.7, 128.0, 126.1, 124.4, 123.5, 120.9,
120.2, 119.7, 109.8, 21.2 ppm. IR
y
3039, 2933, 1601, 1589, 1456, 1331,
120.4, 120.2, 109.6 ppm. IR y 3052, 2928, 1902, 1576, 1474, 1328, 1227,
1222, 1169, 1011, 931, 735 cmꢂ1. M.P. 100.7e101.0 ꢀC. LRMS (ESI, m/
z): 258.2 [M þ H]þ. HRMS calcd for C19H16N [M þ H]þ: 258.1277,
found: 258.1285.
1063, 1003, 828, 747, 615, 529 cmꢂ1. LRMS (ESI, m/z): 322.2, 324.2
[M þ H]þ.
5.1.2.13. 9-(4-(trifluoromethyl)phenyl)-9H-carbazole (11). A brown
5.1.2.6. 9-(4-methoxyphenyl)-9H-carbazole (3). A white solid. HPLC
solid. HPLC tR ¼ 8.929 min, 96.83%. 1H NMR (400 MHz, CDCl3)
d 8.15
tR ¼ 7.452 min, 97.99%. 1H NMR (400 MHz, CDCl3)
d
8.15 (d,
(d, J ¼ 7.7 Hz, 2H), 7.88 (d, J ¼ 7.2 Hz, 2H), 7.73 (d, J ¼ 8.1 Hz, 2H),
J ¼ 7.7 Hz, 2H), 7.49e7.37 (m, 4H), 7.36e7.26 (m, 4H), 7.12 (d,
7.47e7.39 (m, 4H), 7.38e7.28 (m, 2H) ppm. 13C NMR (100 MHz,
J ¼ 8.5 Hz, 2H), 3.93 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3)
d
157.8,
CDCl3) d 139.3, 137.9, 137.7, 133.9, 130.1, 129.6, 128.9, 128.3, 128.1,
140.3, 129.3, 127.5, 124.8, 122.1, 119.2, 118.6, 114.0, 108.6, 54.5 ppm.
127.4, 127.0, 126.5, 126.1 (d, 3J ¼ 6.1 Hz), 125.2, 122.7, 119.5, 119.4 (d,
IR
y
3047, 2939, 2837, 1597, 1511, 1453, 1317, 1242, 1172, 1109, 1026,
2J ¼ 31.8 Hz), 113.6, 108.5 ppm. IR
y 3042, 2949, 1623, 1559, 1424,
829, 749, 583 cmꢂ1. LRMS (ESI, m/z): 274.2 [M þ H]þ. HRMS calcd
1376, 1170, 1031, 950, 750 cmꢂ1. LRMS (ESI, m/z): 312.3 [M þ H]þ.
for C19H16NO [M þ H]þ: 274.1226, found: 274.1240.
5.1.2.14. 4-(9H-carbazol-9-yl)-N,N-dimethylaniline (12). A white solid.
1
5.1.2.7. 9-(4-(tert-butyl)phenyl)-9H-carbazole (4). A yellow solid.
HPLC tR ¼ 9.701 min, 96.32%. H NMR (400 MHz, CDCl3)
d 8.14 (d,
HPLC tR ¼ 15.021 min, 99.21%. 1H NMR (400 MHz, CDCl3)
d
8.14 (m,
J ¼ 7.8 Hz, 2H), 7.45e7.30 (m, 7H), 7.29e7.22 (m, 1H), 6.91 (d,
2H), 7.60 (m, 2H), 7.48 (m, 2H), 7.41 (m, 4H), 7.26 (m, 2H), 1.43 (d,
J ¼ 7.2 Hz, 2H), 3.07 (d, J ¼ 1.5 Hz, 6H) ppm. 13C NMR (100 MHz, CDCl3)

86
D. Zhu et al. / European Journal of Medicinal Chemistry 68 (2013) 81e88
d
148.8, 140.6, 127.2, 125.2, 124.6, 121.9, 119.1, 118.3, 112.0, 108.8,
1H), 7.11 (m, 2H), 6.96 (m, 1H), 3.95 (d, J ¼ 5.1 Hz, 3H), 2.27 (d,
39.6 ppm. IR
y
3049, 2931, 1731, 1608, 1526, 1453, 1345, 1237, 1104,
J ¼ 4.6 Hz, 3H) ppm. 13C NMR (100 MHz, CDCl3)
d 159.8, 144.9, 139.8,
944, 811, 741, 565 cmꢂ1. M.P. 210.4e211.5 ꢀC. LRMS (ESI, m/z): 287.2
[M þ H]þ. HRMS calcd for C20H19N2 [M þ H]þ: 287.1543, found:
287.1556.
138.4, 135.0, 129.7, 126.5, 126.0, 123.9, 120.6, 119.1, 115.1, 112.1, 99.9,
55.8, 21.5 ppm. IR
y 3053, 2929, 1724, 1602, 1446, 1361, 1270, 1165,
986, 821, 668, 585 cmꢂ1. M.P. 125.3e126.1 ꢀC. LRMS (ESI, m/z): 352.2
[M þ H]þ. HRMS calcd for C20H16NO3S [M ꢂ H]ꢂ: 350.0851, found
350.0845.
5.1.2.15. 9-(5-methylpyridin-2-yl)-9H-carbazole (13). A yellow solid.
HPLC tR ¼ 4.486 min, 98.46%. 1H NMR (400 MHz, CDCl3)
d 8.55 (s,
1H), 8.12 (d, J ¼ 7.7 Hz, 2H), 7.81e7.70 (m, 3H), 7.53 (d, J ¼ 8.2 Hz,
1H), 7.43 (t, J ¼ 7.7 Hz, 2H), 7.31 (t, J ¼ 7.5 Hz, 2H), 2.46 (s, 3H) ppm.
5.1.2.22. 3-methoxy-9-tosyl-9H-carbazole (20). A white solid. HPLC
tR ¼ 4.803 min, 94.39%. 1H NMR (400 MHz, CDCl3)
d 8.30 (d,
13C NMR (100 MHz, CDCl3)
d
149.8, 149.5, 139.8, 139.0, 131.0, 126.1,
J ¼ 6.3 Hz,1H), 8.22 (d, J ¼ 7.3 Hz,1H), 7.85 (s,1H), 7.64 (d, J ¼ 6.3 Hz,
2H), 7.48 (d, J ¼ 6.1 Hz, 1H), 7.34 (s, 2H), 7.08 (s, 3H), 3.90 (d,
J ¼ 4.2 Hz, 3H), 2.26 (d, J ¼ 4.4 Hz, 3H) ppm. 13C NMR (100 MHz,
124.1, 120.6, 120.2,118.8,111.0, 18.1 ppm. IR y 3046, 2982,1623,1557,
1423, 1386, 1196, 1125, 945, 749, 683 cmꢂ1. M.P. 104.4e105.1 ꢀC.
LRMS (ESI, m/z): 259.2 [M þ H]þ. HRMS calcd for C18H15N2
[M þ H]þ: 259.1230, found: 259.1233.
CDCl3)
d 156.8, 144.7, 139.1, 134.8, 132.7, 129.6, 127.6, 127.4, 126.6,
126.4, 123.8, 119.9, 116.3, 115.5, 115.3, 103.2, 100.0, 55.8, 21.5 ppm. IR
y
2931, 1602, 1451, 1360, 1174, 1029, 974, 812, 741, 675, 570 cmꢂ1
.
5.1.2.16. 9-butyl-9H-carbazole (14). A white solid. HPLC tR ¼ 7.786
M.P. 127.5e128.3 ꢀC. LRMS (ESI, m/z): 352.3 [M þ H]þ. HRMS calcd
min, 98.96%. 1H NMR (400 MHz, CDCl3)
d
8.12 (d, J ¼ 7.8 Hz,
for C20H16NO3S [M ꢂ H]ꢂ: 350.0851, found: 350.0855.
2H), 7.52e7.38 (m, 4H), 7.26e7.20 (m, 2H), 4.32 (t, J ¼ 7.2 Hz, 2H),
1.95e1.80 (m, 2H), 1.42 (dq, J ¼ 14.8, 7.4 Hz, 2H), 0.96 (t, J ¼ 7.4 Hz,
5.1.2.23. 2-phenyl-9-tosyl-9H-carbazole (21). A yellow solid. HPLC
3H) ppm. 13C NMR (100 MHz, CDCl3)
d
140.4, 125.6, 122.8, 120.3,
tR ¼ 9.577 min, 97.28%. 1H NMR (400 MHz, CDCl3)
d 8.58 (s,1H), 8.34
118.7, 108.6, 42.8, 31.1, 20.6, 13.9 ppm. IR
y
3049, 2938, 1740, 1593,
(d, J ¼ 8.4 Hz, 1H), 7.95 (d, J ¼ 8.1 Hz, 1H), 7.92 (d, J ¼ 7.8 Hz, 1H),
7.76e7.70 (m, 4H), 7.61 (d, J ¼ 8.0 Hz, 1H), 7.50 (q, J ¼ 7.6 Hz, 3H),
7.44e7.35 (m, J ¼ 14.7, 7.1 Hz, 2H), 7.11 (d, J ¼ 8.2 Hz, 2H), 2.27 (s,
1460, 1336, 1224, 1142, 1017, 740 cmꢂ1. LRMS (ESI, m/z): 224.2
[M þ H]þ. HRMS calcd for C16H18N [M þ H]þ: 224.1434, found:
224.1445.
3H) ppm. 13C NMR (100 MHz, CDCl3)
d 144.9, 141.2, 140.8, 139.1,
138.8, 135.0, 129.7, 128.9, 127.6, 127.4, 126.5, 126.2, 125.5, 124.0,
5.1.2.17. 9-cyclohexyl-9H-carbazole (15). A yellow solid. HPLC tR
123.4, 120.2, 120.0, 115.2, 113.6, 21.5 ppm. IR y 3048, 2923, 1728,
¼ 9.948 min, 96.99%. 1H NMR (400 MHz, CDCl3)
d
8.11 (d, J ¼ 7.4 Hz,
1597, 1458, 1367, 1283, 1173, 1093, 974, 755, 674, 578 cmꢂ1. M.P.
107.5e108.8 ꢀC. LRMS (ESI, m/z): 398.3 [M þ H]þ. HRMS calcd for
C25H13NO2SNa [M þ Na]þ: 420.1029, found: 420.1029.
2H), 7.57 (d, J ¼ 8.0 Hz, 2H), 7.43 (t, J ¼ 7.7 Hz, 2H), 7.21 (t, J ¼ 7.4 Hz,
2H), 4.59e4.40 (m, 1H), 2.49e2.32 (m, 2H), 2.00 (t, J ¼ 14.5 Hz, 4H),
1.63e1.48 (m, 4H) ppm. 13C NMR (100 MHz, CDCl3)
d 125.2, 120.3,
118.5, 55.4, 30.7, 26.5, 25.7 ppm. IR
y
3054, 2926, 2855, 1595, 1450,
5.1.2.24. 9-tosyl-9H-carbazole-2-carbonitrile (22). A white solid.
1326, 1215, 1134, 814, 743 cmꢂ1. LRMS (ESI, m/z): 250.2 [M þ H]þ.
HPLC tR ¼ 4.004 min, 98.59%. 1H NMR (400 MHz, CDCl3)
d 8.65 (s,
1H), 8.36 (d, J ¼ 8.4 Hz, 1H), 8.04e7.92 (m, 2H), 7.72 (d, J ¼ 7.5 Hz,
2H), 7.67e7.57 (m, 2H), 7.43 (t, J ¼ 7.5 Hz, 1H), 7.16 (d, J ¼ 8.0 Hz,
5.1.2.18. 4-(9H-carbazol-9-yl)butan-1-ol (16). A white solid. HPLC
tR ¼ 2.966 min, 98.66%. 1H NMR (400 MHz, CDCl3)
d
8.10 (d,
2H), 2.30 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3)
d 145.6, 139.3,
J ¼ 7.7 Hz, 2H), 7.50e7.38 (m, 4H), 7.25e7.20 (m, 2H), 4.45e4.29
137.6, 134.5, 130.0, 129.8, 129.4, 127.2, 126.5, 124.8, 124.5, 120.9,
(m, 2H), 3.72e3.57 (m, 2H), 2.06e1.92 (m, 2H), 1.42 (brs, 2H) ppm.
120.8,119.3,119.0,115.2,110.2, 21.6 ppm. IR y 2948, 2245,1576,1413,
13C NMR (100 MHz, CDCl3)
d
140.4, 125.6, 122.9, 120.4, 118.8, 108.6,
3445, 3036, 2975, 1689, 1573, 1439,
1340, 1155, 979, 815, 728, 670, 591 cmꢂ1. M.P. 174.8e176.1 ꢀC. LRMS
(ESI, m/z): 347.2 [M þ H]þ. HRMS calcd for C20H13N2O2S [M ꢂ H]ꢂ:
345.0698, found: 345.0704.
62.5, 42.8, 30.3, 25.5 ppm. IR
y
1336, 1247, 1156, 1055, 760 cmꢂ1. LRMS (ESI, m/z): 240.2 [M þ H]þ.
5.1.2.19. 9-tosyl-9H-carbazole(17). A white solid. HPLC tR ¼ 5.013 min,
5.1.2.25. 9-tosyl-2-(trifluoromethyl)-9H-carbazole (23). A yellow solid.
100%. 1H NMR (400 MHz, CDCl3)
d
8.33 (d, J ¼ 8.3 Hz, 2H), 7.91 (d,
HPLC tR ¼ 6.427 min, 100%. 1H NMR (400 MHz, CDCl3)
d 8.63 (s, 1H),
J ¼ 7.7 Hz, 2H), 7.70 (d, J ¼ 8.2 Hz, 2H), 7.54e7.45 (m, 2H), 7.36 (t,
8.36 (d, J ¼ 8.4 Hz,1H), 7.98 (dd, J ¼ 16.9, 7.9 Hz, 2H), 7.71 (d, J ¼ 8.3 Hz,
J ¼ 7.5 Hz, 2H), 7.10 (d, J ¼ 8.1 Hz, 2H), 2.26 (s, 3H) ppm. 13C NMR
2H), 7.66e7.54 (m, 2H), 7.41 (t, J ¼ 7.6 Hz, 1H), 7.14 (d, J ¼ 8.4 Hz, 2H),
(100 MHz, CDCl3)
d
144.8, 138.4, 135.0, 129.6, 127.4, 126.5, 126.4, 123.9,
2.29 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3)
d 145.4, 139.2, 137.8,
120.0, 115.2, 21.5 ppm. IR
y
2931, 1691, 1589, 1441, 1357, 1146, 959, 843,
134.7, 129.8, 129.1, 128.7, 126.5, 125.1, 124.3, 120.8e120.6 (m), 120.3,
750, 669, 550 cmꢂ1. LRMS (ESI, m/z): 322.0 [M þ H]þ. HRMS calcd for
115.2, 112.5 (d, 3J ¼ 4.3 Hz) ppm. IR
y 2932, 1596, 1433, 1325, 1169, 983,
C19H15NO2SNa [M þ Na]þ: 334.0716, found: 334.0728.
814, 728, 660, 577 cmꢂ1. M.P. 153.1e153.9 ꢀC. LRMS (ESI, m/z): 390.2
[M þ H]þ. HRMS calcd for C20H13F3NO2S [M ꢂ H]ꢂ: 388.0619, found:
388.0620.
5.1.2.20. 9-((4-ethylphenyl)sulfonyl)-9H-carbazole (18). A white
solid. HPLC tR ¼ 6.063 min, 100%. 1H NMR (400 MHz, CDCl3)
d 8.34
(d, J ¼ 8.4 Hz, 2H), 7.91 (d, J ¼ 7.7 Hz, 2H), 7.74 (d, J ¼ 6.6 Hz, 2H),
7.50 (t, J ¼ 7.8 Hz, 2H), 7.36 (t, J ¼ 7.5 Hz, 2H), 7.13 (d, J ¼ 7.0 Hz, 2H),
2.57 (q, J ¼ 7.3 Hz, 2H), 1.13 (td, J ¼ 7.6, 2.2 Hz, 3H) ppm. 13C NMR
5.1.2.26. 11-(p-tolyl)-11H-benzo[a]carbazole (24). A white solid.
HPLC tR ¼ 12.615 min, 98.93%. 1H NMR (400 MHz, CDCl3)
d 8.28e
8.15 (m, 2H), 7.99 (d, J ¼ 8.1 Hz, 1H), 7.72 (d, J ¼ 8.4 Hz, 1H), 7.53e
7.39 (m, 6H), 7.39e7.30 (m, 2H), 7.23 (s, 1H), 7.18 (d, J ¼ 7.8 Hz, 1H),
(100 MHz, CDCl3)
d 150.8, 138.4, 135.3, 128.5, 127.4, 126.6, 126.4,
123.9, 120.0, 115.1, 28.7, 14.7 ppm. IR
y
2933, 1919, 1726, 1595, 1439,
2.57 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3)
d 142.2, 138.8, 137.4,
1358, 1168, 970, 827, 751, 653, 578 cmꢂ1. M.P. 122.1e123.0 ꢀC. LRMS
(ESI, m/z): 336.2 [M þ H]þ. HRMS calcd for C20H16NO2S [M ꢂ H]ꢂ:
334.0902, found: 334.0906.
135.6, 133.5, 130.7, 129.1, 128.8, 124.9, 124.7, 123.3, 122.3, 122.1,
121.0, 120.2, 119.4, 119.3, 119.1, 110.4, 21.4 ppm. IR 3030, 2946,
y
1699, 1601, 1463, 1378, 1220, 1083, 1045, 920, 809, 750, 678,
556 cmꢂ1. LRMS (ESI, m/z): 308.1 [M þ H]þ.
5.1.2.21. 2-methoxy-9-tosyl-9H-carbazole (19). A white solid. HPLC
tR ¼ 5.176 min, 96.66%. 1H NMR (400 MHz, CDCl3)
d
8.26 (m, 1H),
5.1.2.27. 11-tosyl-11H-benzo[a]carbazole (25). A yellow solid. HPLC
7.88 (brs, 1H), 7.84e7.73 (m, 2H), 7.69 (m, 2H), 7.39 (m, 1H), 7.31 (m,
tR ¼ 5.900 min, 95.28%. 1H NMR (400 MHz, CDCl3)
d 9.02 (d,

D. Zhu et al. / European Journal of Medicinal Chemistry 68 (2013) 81e88
87
J ¼ 8.4 Hz, 1H), 8.34 (d, J ¼ 8.0 Hz, 1H), 7.95 (d, J ¼ 8.3 Hz, 1H), 7.84
(d, J ¼ 8.6 Hz, 1H), 7.79e7.75 (m, 1H), 7.73e7.64 (m, 2H), 7.57 (t,
J ¼ 7.5 Hz, 1H), 7.45 (t, J ¼ 7.8 Hz, 1H), 7.35 (t, J ¼ 7.2 Hz, 1H), 6.83 (d,
J ¼ 6.8 Hz, 2H), 6.74 (d, J ¼ 7.5 Hz, 2H), 2.14 (s, 3H) ppm. 13C NMR
5.2.5. Estimation of intracellular glutathione
A glutathione assay kit (Jiancheng Biochemical, Nanjing, China)
was used to measure intracellular glutathione (GSH) concentra-
tions. Cells were collected and sonicated in 50 ml of ice-cold lysis
(100 MHz, CDCl3)
d
144.2, 141.8, 136.7, 133.9, 131.5, 130.1, 128.5,
buffer. After centrifuging at 10,000ꢁ g for 15 min at 4 ꢀC, the su-
128.2, 127.8, 127.6, 127.0, 126.9, 126.6, 126.3, 126.1, 125.9, 125.6,
pernatant was collected. 50 ml of TEAM reagent was added to 1 mL
119.8, 119.4, 117.4, 21.4 ppm. IR
y
3050, 2926, 1725, 1589, 1450, 1360,
supernatant. 50
the kit was applied to each sample wells. 150
m
l of sample or the standard solution provided in
1172, 1092, 1029, 915, 815, 755, 669, 569 cmꢂ1. M.P. 144.9e145.7 ꢀC.
LRMS (ESI, m/z): 372.1 [M þ H]þ.
ml of the freshly
prepared assay mixture was added to each of the wells containing
standards and samples. The plate was incubated in the dark on an
orbital shaker at room temperature. Absorbance was measured at
405 nm using a plate reader (Bio-Tek) after 25 min incubation. GSH
concentrations were calculated according to the instructions pro-
vided by the supplier. All GSH values were normalized to per mg
protein of each sample.
5.2. Biological assay
5.2.1. Materials
Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine
serum (FBS) were purchased from Gibico-BRL (Grand Island, NY,
USA). Dihydroethidium (DHE), 3-(3,4-dimehylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (MTT), and all other chemicals
were purchased from SigmaeAldrich (St. Louis, MO, USA) unless
stated otherwise. Carbazoles were dissolved in dimethyl sulfoxide
(DMSO) and stored at ꢂ20 ꢀC.
5.2.6. Statistical analysis
All quantitative data and experiments described in this study
were repeated at least three times. Data were presented as
mean ꢃ S.D. of multiple independent experiments. Statistics were
analyzed with one-way ANOVA followed by a least significant dif-
ference test (SPSS 17.0 software). Statistical difference was consid-
ered at P < 0.05.
5.2.2. Cell culture and cell treatment
HT22 murine hippocampal neuronal cells (a gift from Prof. Jun
Liu, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, China)
[16] were maintained in DMEM supplemented with 10% (v/v) FBS
and incubated at 37 ꢀC under 5% CO2. To study the neuroprotective
effects of carbazoles, cells were seeded in 96-well plates at a den-
sity of 4000 cells per well. The solution of carbazoles (30 mM in
DMSO) was diluted in DMEM supplemented with 10% (v/v) FBS
before addition to each well at the desired final concentrations.
After 30 min, cells were exposed to toxins, glutamate (2 mM) or
Conflict of interest statement
The authors declare that there is no conflict of interest.
Acknowledgments
This study was supported by a start-up grant from Sun Yat-sen
University and Research Fund for the Doctoral Program of Higher
Education of China (No.20110171120098) to S Wen and in part by
Fundamental Research Funds for the Central Universities
(No.10ykpy23), Guangdong Provincial International Cooperation
Project of Science & Technology (No. 2012B050300015), and Ex-
ecutive Program of Scientific and Technological Cooperation be-
tween the Government of the Italian Republic and the Government
of the People’s Republic of China for the years 2013e2015 (MAE-
M00705/CN13MO9) to R Pi.
HCA (500 mM). Cells in the control group were treated with vehicle
alone. Cell morphology could be observed by an inverse phase
contrast microscopy (Olympus, Japan).
5.2.3. MTT assay
For determining cell viability, the MTT assay was used. MTT
(10
concentration of 500
1 h at 37 ꢀC; and the liquid in the wells was removed thereafter.
DMSO (150 l) was added to each well, and the absorbance was
ml, at 5 mg/mL) was added to each well in 96-well plate at a final
mg/mL; the mixture was further incubated for
m
Appendix A. Supplementary data
read with microplate reader (Bio-Tek) at 570 nm. Results were
expressed as the percentage of MTT reduction and the absorbance
of control cells was set as 100%.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.07.029.
5.2.4. Measurement of intracellular ROS accumulation
The intracellular reactive oxygen species (ROS) level was
measured using fluorescent dyes, Dihydroethidium (DHE). Once
internalized, the DHE reacts with superoxide production [16,17]
and is dehydrogenated to ethidium, a red fluorescent product,
which then intercalates into DNA [18]. A working stock of 10 mM
DHE in anhydrous DMSO was freshly prepared immediately before
use. In the presence of an oxidant, DHE, a dye, combines with ROS,
especially hydrogen peroxide or peroxyl radicals to produce red
fluorescence [16,17]. For assay, HT22 cells were cultured for over-
night and then treated with glutamate in the presence or absence of
carbazole for 12 h. Cells were washed twice with PBS (pH 7.4) and
References
[1] C. Sánchez-Moreno, J.F. Dashe, T. Scott, D. Thaler, M.F. Folstein, A. Martin,
Stroke 35 (2004) 163e168.
[2] D. Praticò, Trends Pharmacol. Sci. 29 (2008) 609e615.
[3] K.J. Barnham, C.L. Masters, A.I. Bush, Nat. Rev. Drug Discov. 3 (2004) 205e214.
[4] (a) B.P. Bandgar, L.K. Adsul, H.V. Chavan, S.S. Jalde, S.N. Shringare, V. Masand,
Bioorg. Med. Chem. Lett. 22 (2012) 5839e5844;
(b) A. Zall, D. Kieser, N. Höttecke, E.C. Naumann, B. Thomaszewski, B. Schmidt,
Bioorg. Med. Chem. 19 (2011) 4903e4909.
[5] (a) D. Mousset, R. Rabot, P. Bouyssou, G. Coudert, I. Gillaizeau, Tetrahedron
Lett. 51 (2010) 3987e3990;
ꢀ
(b) M. Stiborová, J. Sejbal, L. Borek-Dohalská, D. Aimová, E. Frei, Cancer Res. 64
(2004) 8374e8380.
[6] (a) D. Crich, S. Rumthao, Tetrahedron 60 (2004) 1513e1516;
(b) K.-I. Sakano, K. Ishimaru, S. Nakamura, J. Antibiot. 33 (1980) 683e689.
[7] S. Butler, R. Wang, S.L. Wunder, H.-Y. Cheng, C.S. Randall, Biophys. Chem. 119
(2006) 307e315.
[8] (a) Y. Hieda, T. Choshi, S. Kishida, H. Fujioka, S. Hibino, Tetrahedron Lett. 51
(2010) 3593e3596;
(b) A.A. Pieper, S. Xie, E. Capota, S.J. Estill, et al., Cell 142 (2010) 39e51.
[9] T. Kimura, J. Hosokawa-Muto, K. Asami, T. Murai, K. Kuwata, Eur. J. Med. Chem.
46 (2011) 5675e5679.
incubated with DHE (10 mM) in serum-free DMEM for 30 min. After
washed twice with PBS, multiple viewing fields were photographed
under a fluorescence microscope (IX71; Olympus, Tokyo, Japan)
and the fluorescence intensity of DHE was measured by a fluori-
metric plate reader Flex Station 3 (Molecular Devices, USA) at an
excitation wavelength of 485 nm and an emission wavelength of
595 nm.

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D. Zhu et al. / European Journal of Medicinal Chemistry 68 (2013) 81e88
[10] (a) H.-J. Knölker, K.R. Reddy, Chem. Rev. 102 (2002) 4303e4427;
(b) H.-J. Knölker, Top. Curr. Chem. 244 (2005) 115e148;
[14] L. Wang, E. Hinoi, A. Takemori, N. Nakamichi, Y. Yoneda, J. Biol. Chem. 281
(2006) 24553e24565.
(c) H.-J. Knölker, Chem. Lett. 38 (2009) 8e13.
[15] F. Herrera, V. Martin, G. García-Santos, J. Rodriguez-Blanco, I. Antolín,
C. Rodriguez, J. Neurochem. 100 (2007) 736e746.
[16] Z. Zhao, R. Lu, B. Zhang, J. Shen, L. Yang, S. Xiao, J. Liu, W.Z. Suo, Neurol. Res. 34
(2012) 38e43.
[17] N. Li, K. Ragheb, G. Lawler, J. Sturgis, B. Rajwa, J.A. Melendez, J.P. Robinson,
J. Biol. Chem. 278 (2003) 8516e8525.
[18] S.L. Budd, R.F. Castilho, D.G. Nicholls, FEBS Lett. 415 (1997) 21e24.
[11] D. Zhu, Q. Liu, B. Luo, M. Chen, R. Pi, P. Huang, S. Wen, Synthesis of carbazoles via
one-pot copper-catalyzed amine insertion into cyclic diphenyleneiodoniums as
a strategy to generate a drug-like chemical library, Adv. Synth. Catal. 355 (2013)
(Accepted).
[12] S. Tan, Y. Sagara, Y. Liu, P. Maher, D. Schubert, J. Cell Biol. 141 (1998) 1423e1432.
[13] Y. Li, P. Maher, D. Schubert, Neuron 19 (1997) 453e463.