Synthesis and anticancer activity of some novel 2-phenazinamine derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.07.017

In this study, we report the synthesis and spectral characterization of a novel series of 2-phenazinamine derivatives. In vitro evaluation for their anticancer activity toward cultured K562 (human chronic myelogenous leukemia), HepG2 (human hepatocellular

Full text of DOI:10.1016/j.ejmech.2013.07.017

European Journal of Medicinal Chemistry 69 (2013) 1e9
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activity of some novel 2-phenazinamine
derivatives
,
,
Xiaochun Gao a 1, Yuanyuan Lu a 1, Lei Fang b, Xubin Fang b, Yingying Xing a,
**
Shaohua Gou b , Tao Xi
,
a,
*
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
b Pharmaceutical Research Center, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we report the synthesis and spectral characterization of a novel series of 2-phenazinamine
derivatives. In vitro evaluation for their anticancer activity toward cultured K562 (human chronic mye-
logenous leukemia), HepG2 (human hepatocellular carcinoma), MGC803 (human gastric carcinoma),
HCT116 (human colorectal carcinoma), MCF7 (human breast adenocarcinoma) cell lines, as well as 293T
(epithelial cells from human embryo kidney) non-cancer cell was carried out. The compounds 4, 7, 16 and
19 showed good positive anticancer activity in vitro. In particular, compound 4, 2-chloro-N-(phenazin-2-
yl)benzamide, possessed a potent anticancer effect comparable to cisplatin against both K562 and HepG2
cancer cells but was very low or had no effect against 293T non-cancer cell. Preliminary anticancer
mechanism of 4 was investigated by cell apoptosis assays compared with cisplatin using flow cytometry.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 3 February 2013
Received in revised form
11 April 2013
Accepted 9 July 2013
Available online 9 August 2013
Keywords:
2-Phenazinamine derivatives
Anticancer activity
MTT assay
Apoptosis
1. Introduction
regarded as secondary metabolites from Streptomyces, Pseudo-
monas, and other marine microorganisms [5].
Cancer is now considered as one of the most serious health
problems all over the world and also one of the leading causes of
death [1e3]. Thus, in the past for several decades researchers have
been struggling to find effective clinical approaches for the treat-
ment of cancer and search for novel anticancer agents. Apart from
the utility of surgical operations and irradiation in past years,
chemotherapy still remains an important option for the manage-
ment of cancer in the clinical settings [4].
Recently, our research group has reported a novel phenazine
derivative N-(2-hydroxyphenyl)-2-phenazinamine (Fig. 1), isolated
from a marine actinomycete BM-17, which showed high cancer cell
cytotoxicity against several cancer cells [24]. This finding prompted
us to further explore it as new potential anticancer agents. Thus, we
describe herein, the synthesis and anticancer activity of a novel
series of 2-phenazinamine derivatives.
Natural and synthetic phenazines have attracted considerable
attention because they have shown interesting biological activities
[5], including broad-spectrum antibiotics [6], antimalarial [7e9],
trypanocidal [10], antihepatitis C viral replication activities [11],
antitumor activity in leukemia and solid tumor [12], anti-
carcinomatous activity [13e15], neuroprotective properties [16e
19], antifungus [20], dual inhibitors of topoisomerase I and II [21].
Phenazines have been associated with anticancer activities since
1959 and they are small molecules that can permeate the tissues
and organs easily [22,23]. Molecules of phenazine class have been
2. Results and discussion
2.1. Chemistry
In this work, the synthesis of a series of 2-phenazinamine de-
rivatives (1e21) was carried out according to the steps showed in
Scheme 1. In the initial step, N1-phenylbenzene-1,2,4-triamine A
was synthesized via the reduction reaction of the commercially
available N-(2,4-dinitrophenyl)benzeneamine in the presence of a
5% Pd/C catalyst under hydrogen atmosphere. The yield was high
and without further purification, the ring closure reaction of the
amine (A) with magnesium sulfate in nitrobenzene afforded the
corresponding phenazin-2-amine (B) [25].
* Corresponding author. Tel.: þ86 25 83271022; fax: þ86 25 83271249.
** Corresponding author. Tel./fax: þ86 25 83272381.
The treatment of phenazin-2-amine (B) with acyl chlorides in
the presence of anhydrous pyridine by stirring under 15 ꢀC for 0.5 h
using dry dichloromethane as solvent of reaction gave the target
E-mail addresses: sgou@seu.edu.cn (S. Gou), taoxi18@hotmail.com (T. Xi).
Both authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.017

2
X. Gao et al. / European Journal of Medicinal Chemistry 69 (2013) 1e9
singlet at
core, four doublets and three triplets of the aromatic protons at
7.44e8.06, more downfield than normal aromatic protons due to
d 6.91 indicated the NH2 group at position 2 of phenazine
d
the effect of two nitrogen heteroatoms, further confirmed the
structure of phenazin-2-amine (B). Moreover, the 13C NMR data
clearly showed the presence of the five aromatic quarter carbon
atoms and seven aromatic carbon atoms containing hydrogen
atoms at the appropriate chemical shift values. IR spectrum of
compound B showed the presence of NeH symmetrical stretching
vibration and asymmetrical stretching vibration of the amino group
at 3194 and 3309 cmꢁ1, respectively, and NeH bending vibration at
Fig. 1. Natural product.
compounds (1e19). Similarly, phenazin-2-amine (B) and benze-
nesulfonyl chloride in the presence of anhydrous pyridine by
refluxing for 1 h using dry dichloromethane as solvent of reaction
afforded N-(2-phenazinyl)sulfon-amide (20) [26], while phenazin-
2-amine (B) and dimethylcarbamyl chloride in the presence of
triethylamine by refluxing for 2.5 h at 110 ꢀC using N,N-dimethyl-
formamide (DMF) as solvent of reaction gave 1,1-dimethyl-3-
(phenazin-2-yl)urea (21).
However, it should be noted that many attempts having been
made to synthesize compound 21 with a N,N-dimethyl substituent
at C]O under various conditions failed, for example, phenazin-2-
amine (B) and dimethylcarbamyl chloride in the presence of trie-
thylamine by refluxing for 5 days at 77 ꢀC using ethyl acetate as
solvent of reaction afforded the same final product (16) as phena-
zin-2-amine (B) and ethyl carbonochloridate under the same pro-
cedure above, instead of target compound 21. This showed that the
solvent ethyl acetate participated in the reaction while the reactant
dimethylcarbamyl chloride could work as a promoter [27,28].
The whole procedure was rather tedious, and the total yield was
modest and significant amounts of byproducts under the harsh
reaction conditions. To date, the synthesis yield of phenazines has
been low since one of the first methods for preparation of phena-
zines was reported in 1901 [5,26].
1640 cmꢁ1
.
Owing to high conjugation of phenazine core, its protons dis-
played a very low activity and difficult substitution [5], and the
active protons of amino group were also poorly active and the
nucleophilicity of phenazin-2-amine (B) was very weak. The
nucleophilicity of the amines are key in Ullmann-type CeN bond
formation coupling reaction [28,29]. Thus, many attempts of the
reactions of phenazin-2-amine (B) with some chemical reagents
under various conditions failed, for example, with other many al-
dehydes targeting Schiff bases were even not successful. In this
paper, we selected strong electrophilic acyl chlorides as chemical
reagents in mild conditions to successfully synthesize target com-
pounds 1e21.
The structural assignments of a series of 2-phenazinamine de-
rivatives (1e21) were based on a full characterization by IR, 1H
NMR, 13C NMR, ESI-MS spectra, all target compounds were in
accordance with the proposed structures, with spectral data re-
ported in experimental section of this paper. Yield, melting point,
molecular formula, molecular weight and recrystallization solvent
of the newly synthesized compounds were provided in Table 1.
2.2. In vitro anticancer screening
The purity of compounds was checked by single-spot TLC using
petroleum ether/ethyl acetate (1:1) solvent systems spots located
under UV light. The final products were purified by flash chroma-
tography column (petroleum ether/ethyl acetate gradient, 1:1e1:0)
and recrystallized with ethyl acetate/petroleum ether or ethyl ac-
etate, methanol, trichloromethane/petroleum ether, dichloro-
methane/petroleum ether, acetone/petroleum ether.
The structures of the newly synthesized compounds were
appropriately characterized by spectral data. The 1H NMR spectrum
of compound B showed that the presence of the two active proton
Twenty-one of the newly synthesized compounds were sub-
jected to in vitro cytotoxicity evaluation against K562 (human
chronic myelogenous leukemia), HepG2 (human hepatocellular
carcinoma), MGC803 (human gastric carcinoma), HCT116 (human
colorectal carcinoma), MCF7 (human breast adenocarcinoma) cell
lines, as well as 293T (epithelial cells from human embryo kidney)
non-cancer cell by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) reduction assay, respectively, using
cisplatin as the reference drug [30,31]. Since the tested compounds
showed poor aqueous solubility, they were all dissolved in DMSO
and then diluted in culture medium so that the effective DMSO
concentration did not exceed 0.2%. The percent inhibition of
viability for each concentration of the compounds was calculated
with respect to the control and IC50 values were estimated with the
software SPSS version 16.0 for Windows. Each experiment was
repeated three times and the results were summarized in Table 2.
Blank wells of all agents, containing the same concentrations of test
compounds, but without MTT, had very low absorbance (data not
shown). Therefore, interference of the color of compounds in the
assay seemed unlikely. The response parameter calculated was IC50
value (Table 2), which corresponded to the compound concentra-
tion causing 50% mortality in net cells.
and a broad singlet at
d 6.47 corresponded to an NH2 group, one
Compound 4 showed 1.7-fold more potent against the human
chronic myelogenous leukemia K562 cell line and 1.1-fold more
potent against the human hepatocellular carcinoma HepG2 cell line
than cisplatin in terms of IC50 values. However it illustrated less
potent against the human colorectal carcinoma HCT116 cell line
than the positive controls in terms of IC50 values and poor or no
activity against other cell lines, while it was very low or no effect on
the epithelial cells from human embryo kidney 293T non-cancer
cell (Fig. 2 and Table 2).
Scheme 1. The synthesis of the compounds (1e21).

X. Gao et al. / European Journal of Medicinal Chemistry 69 (2013) 1e9
3
Table 1
Some properties of the compounds (1e21).
H
N
N
N
R
Compd. no.
R
Yield (%)
32
Mp (ꢀC)
Mol. formula
C15H13N3O
Mol. weight
251
Recrystallization solvent
CH3
CH3
1
200e202
Ethyl acetate/petroleum ether
O
2
3
25
34
184e186
183e186
C16H15N3O
C20H15N3O
265
313
Ethyl acetate/petroleum ether
Ethyl acetate/petroleum ether
O
O
O
4
5
28
34
187e189
227e229
C19H12ClN3O
C19H12ClN3O
333
333
Ethyl acetate/petroleum ether
Ethyl acetate
Cl
O
Cl
O
6
81
211e213
C20H15N3O
313
Ethyl acetate/petroleum ether
CH3
O
7
8
35
11
113e116
105e108
C20H15N3O
C20H15N3O
313
313
Methanol
CH3
O
Trichloromethane/petroleum ether
H3C
O
9
15
18
113e116
206e209
C20H15N3O2
329
305
Ethyl acetate/petroleum ether
Ethyl acetate/petroleum ether
OMe
O
10
C19H19N3O
11
12
42
69
183e186
230e233
C20H21N3O
C23H15N3O
319
349
Ethyl acetate/petroleum ether
Ethyl acetate/petroleum ether
O
O
O
13
14
17
43
178e180
170e173
C17H11N3O
289
305
Acetone/petroleum ether
O
O
C17H11N3OS
Dichloromethane/petroleum ether
S
O
S
15
16
44
42
130e133
199e200
C18H13N3OS
C15H13N2O2
319
267
Ethyl acetate/petroleum ether
Ethyl acetate
O
CH3
Cl
O
17
18
69
15
190e192
188e190
C15H12ClN3O
C19H13N3O
285
299
Acetone
O
O
Ethyl acetate/petroleum ether
CH3
19
23
234e236
C14H11N3O
237
Ethyl acetate/petroleum ether
O
(continued on next page)

4
X. Gao et al. / European Journal of Medicinal Chemistry 69 (2013) 1e9
Table 1 (continued )
Compd. no.
R
Yield (%)
23
Mp (ꢀC)
Mol. formula
C18H13N3O2S
Mol. weight
335
Recrystallization solvent
O
S
O
20
21
229e231
Ethyl acetate/petroleum ether
O
13
113e115
C15H14N3O
266
Ethyl acetate/petroleum ether
N
H3C
CH3
Compound 7 exhibited the highest growth inhibitory activity
against the human breast adenocarcinoma MCF7 cell line with IC50
embryo kidney 293T non-cancer cell (Table 2) were used. Results
showed that compounds 8 and 2 had effect on non-cancer cells, the
rest of the compounds generally showed low or no effect. It should
be noted that the standard reference anticancer drug cisplatin
displayed magnificent effect on the normal cells, a phenomenon
that has been reported in other literature [32e34].
As it could be seen in Tables 1 and 2, although the newly syn-
thesized 2-phenazinamine derivatives, including aliphatic 1, 2, 17,
19, aromatic 3e9, 12, 18, aliphatic cyclic 10, 11, heterocyclic 13e15,
ester 16, sulfuryl 20 and ureal 21, showed chemical structural di-
versity with this phenazine-containing structures, it was extremely
difficult to derive a uniform structureeactivity relationship due to
the derivatization of the phenazine core with diverse pendant
functionality [35].
values of 11.63
control cisplatin with IC50 values of 28.03
m
M, which was 2.4-fold more potent than positive
M. However, it was less
m
potent against the human gastric carcinoma MGC803 cell line and
no activity against other cell lines. Compound 13 was close to
cisplatin and compounds 12, 17, 21 showed significant activity
against the MCF7 cell line. Although compound 8 was less potent
against MGC803 cell line, it was 1.1-fold more potent against HepG2
cell line than cisplatin. Meantime, it had effect on the 293T non-
cancer cell.
Compounds 16 and 19 indicated 1.2-fold more potent against
the human hepatocellular carcinoma HepG2 cell line and 1.1-fold
more potent against the human chronic myelogenous leukemia
K562 cell line, respectively, while they both were less potent
against the human breast adenocarcinoma MCF7 cell line than
positive control cisplatin in terms of IC50 values. All of the syn-
thesized compounds were less sensitive and less potent against the
human colorectal carcinoma HCT116 cell line than cisplatin
excepted for compound 1, which was close to positive control
cisplatin in terms of IC50 values.
2.3. Apoptosis assay for compound 4 by flow cytometry
In order to investigate whether the compounds exhibited its
anti-proliferative effect on HepG2 cancer cell through induction of
apoptosis, studies of flow cytometry were undertaken on com-
pound 4 and cisplatin [36,37]. These compounds were incubated
for 24 h at a concentration of 50 mM and the results were shown in
Figs. 3 and 4. Four areas in the diagrams stand for necrotic cells (Q1,
low Annexin V-FITC and high PI signal, left square on the top), late
apoptosis or necrosis cells (Q2, high Annexin V-FITC and high PI
signal, right square on the top), live cells (Q3, low Annexin V-FITC
and low PI signal, left square at the bottom), apoptosis cells (Q4,
high Annexin V-FITC and low PI signal, right square at the bottom),
respectively. As it could be seen in Figs. 3 and 4, compound 4
showed a high population of apoptotic cells (56.6%) and 1.7-fold
higher than cisplatin (33.5%) at the same concentration. The re-
sults demonstrated that the newly synthesized compounds could
induce apoptosis of HepG2 cancer cells. But the proapoptotic
property needs further investigations to better understand the
precise mechanism of action of the compounds.
In order to assess the effect of the newly synthesized com-
pounds on non-cancer cells, the epithelial cells from human
Table 2
In vitro cytotoxicity (IC50, m
M)a of the compounds against human tumor cell lines.
Compd. no.
IC50 (mM)
K562b
HepG2c
MGC803d
HCT116e
MCF7f
293Tg
1
2
3
4
5
6
7
8
57.67
>160
>160
33.43
>160
>160
>160
>160
>160
78.85
157.32
61.22
n.d.h
>160
n.d.
>160
>160
>160
16.46
>160
>160
>160
16.74
>160
>160
>160
44.22
52.25
>160
>160
15.21
>160
>160
>160
>160
>160
18.44
63.93
43.47
>160
>160
>160
>160
27.22
89.01
50.06
67.09
122.33
87.86
n.d.
18.94
62.33
>160
91.93
>160
>160
>160
>160
>160
>160
>160
130.6
86.56
68.14
95.57
>160
141.47
28.21
>160
>160
143.88
16.26
40.73
68.82
>160
>160
>160
>160
11.63
>160
>160
136.71
>160
30.09
28.82
>160
>160
46.09
37.61
>160
82.62
>160
35.07
28.03
>160
109.63
>160
>160
>160
>160
>160
56.16
>160
>160
>160
>160
>160
>160
>160
>160
>160
>160
>160
>160
>160
13.77
9
10
11
12
13
14
15
16
17
18
19
20
21
Cisplatin
>160
n.d.
>160
74.46
>160
49.20
>160
n.d.
>160
59.46
>160
93.07
>160
n.d.
56.04
9.51
a
IC50 is the drug concentration effective in inhibiting 50% of the cell growth
measured by the MTT assay after 48 h drug exposure.
b
Human chronic myelogenous leukemia cell line.
Human hepatocellular carcinoma cell line.
Human gastric carcinoma cell line.
Human colorectal carcinoma cell line.
Human breast adenocarcinoma cell line.
Epithelial cells from human embryo kidney.
n.d. ¼ not determined.
c
d
e
f
g
h
Fig. 2. Structures of highly active anticancer of newly synthesized compounds.

X. Gao et al. / European Journal of Medicinal Chemistry 69 (2013) 1e9
5
Fig. 3. Flow cytometric results after the exposure of HepG2 cell to the active compound 4 and cisplatin (50 mM). Four areas in the diagrams represent four different cell states:
necrotic cells (Q1), late apoptotic or necrotic cells (Q2), living cells (Q3) and apoptotic cells (Q4).
3. Conclusion
apparatus. Infrared spectra were measured on KBr pellets on a
Nicolet IR200 FT-IR spectrometer in the range of 4000e400 cmꢁ1.1H
NMR and 13C NMR spectroscopic measurements were performed on
a Bruker AV-500 NMR spectrometer, using TSP and TMS as internal
references at 298 K, respectively. Electro-spray ionization (ESI) mass
spectra were recorded on a Finnigan MAT SSQ 710 mass spectrom-
eter in a scan range of 100e1200 amu. K562 (human chronic mye-
logenous leukemia), HepG2 (human hepatocellular carcinoma),
MGC803 (human gastric carcinoma), HCT116 (human colorectal
carcinoma), MCF7 (human breast adenocarcinoma) cell lines, as well
as 293T (epithelial cells from human embryo kidney) non-cancer
cell were purchased from American type Cell Culture (ATCC,
Shanghai, China) and maintained in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% fetal bovine serum and antibiotics.
In summary, a novel series of 2-phenazinamine derivatives were
synthesized and characterized. The biological activities of all
compounds were examined against five cancer cell lines and one
non-cancer cell line. The results of in vitro anticancer activity
indicated that compounds 4, 7, 16 and 19 were more active than
cisplatin against both the K562 cell line and the HepG2 cell line, the
MCF7 cell line, the HepG2 cell line, and K562 cell line, respectively.
It was noted that the highly active anticancer compounds were no
effect on the epithelial cells from the 293T non-cancer cell while
the positive control cisplatin was very effective on the normal cells.
Moreover, flow cytometry analysis of compound 4 indicated that it
inhibited tumor proliferation by inducing apoptosis mechanism
similar to cisplatin effect. In conclusion, it is worth studying com-
pound 4 further as new potential anticancer agent for the treat-
ment of human cancers.
4.2. Chemistry: general methods
4.2.1. General procedure for the synthesis of intermediates A and B
N-(2,4-Dinitrophenyl)benzeneamine (5.0 g, 19.3 mmol) was
dissolved in ethyl acetate (300 mL), then catalytically reduced in a
Parr apparatus for 24 h using a 5% Pd/C catalyst under hydrogen
atmosphere. After reduction, the reaction mixture was filtered into
ethyl acetate. The solvent was removed in vacuo, giving the desired
N1-phenylbenzene-1,2,4-triamine A (3.76 g,18.9 mmol, 98%), which
was used without further purification. Compound A was immedi-
ately refluxed for 24 h in nitrobenzene (100 mL) containing MgSO4
in a flask. After nitrobenzene removal in vacuo, and the residue was
quenched with water, extracted with dichloromethane and dried
over anhydrous Na2SO4. The solvent was removed under vacuum
and the residue was purified by silica gel column chromatography,
using petroleum ether/ethyl acetate, 3:1e1:1. The product phena-
zin-2-amine (B) was obtained as a red solid (1.11 g, 30%): mp 285e
286 ꢀC (lit. mp 280 ꢀC) [25]. IR (KBr, cmꢁ1): 3309, 3194, 2918, 1640,
1597, 1514, 1476, 1453, 1335, 1232, 1132, 827, 755. 1H NMR (DMSO-
4. Experimental section
4.1. Materials and instruments
All commercially available chemicals and solvents were of
analytical reagent grade and were used without further purification
unless otherwise specified. Column chromatography was carried
out on silica gel (100e300 mesh). TLC was conducted on silica gel
250 micron, GF254 plates with short-wavelength UV light for visu-
alization. Melting points were measured on a SGWX-4 hot stage
d6, 500 MHz)
d
: 8.06e8.04 (d, J ¼ 8.5 Hz, 1H, AreH), 7.99e7.97 (d,
J ¼ 8.5 Hz, 1H, AreH), 7.91e7.89 (d, J ¼ 9.2 Hz, 1H, AreH), 7.78e7.75
(t, J ¼ 7.5 Hz, 1H, AreH), 7.67e7.64 (t, J ¼ 7.5 Hz, 1H, AreH), 7.46e
7.44 (d, J ¼ 9.2 Hz, 1H, AreH), 6.91 (s, 1H, AreH), 6.47 (s, 2H, NH2).
13C NMR (DMSO-d6, 125 MHz)
d: 151.0 (AreC), 145.8 (AreC), 143.2
(AreC), 139.7 (AreC), 139.3 (AreC), 130.1 (AreC), 130.0 (AreC),
129.2 (AreC), 128.0 (AreC), 127.0 (AreC), 126.7 (AreC), 101.2 (Are
C). HRMS (ESI): m/z [M þ H]þ calcd for C12H9N3: 196.08747, found:
196.08707.
4.2.2. General procedure for the synthesis of final target compounds
(1e21)
Phenazin-2-amine (B) (195 mg, 1 mmol) was dissolved in the
solution of dry CH2Cl2 (20 mL) and anhydrous pyridine (1 mL). Acyl
Fig. 4. Percentage of apoptotic HepG2 cell in total cells following treatment with
compound 4 at 50 mM compared with cisplatin (50 mM).

6
X. Gao et al. / European Journal of Medicinal Chemistry 69 (2013) 1e9
chlorides (14 mmol) diluted in CH2Cl2 (30 mL) then added drop-
wise to the above solution. The mixture was stirred under 15 ꢀC for
0.5 h, and then 100 mL of CH2Cl2 was added, followed by washing
with dilute 2 N hydrochloric acid, saturated sodium bicarbonate
and water, respectively. Separating the organic layer, the solvent
was removed by distillation. The residue was purified by flash
chromatography on silica gel, using petroleum ether/ethyl acetate,
1:1e1:0, and then recrystallized with ethyl acetate/petroleum
ether or ethyl acetate, methanol, trichloromethane/petroleum
ether, dichloromethane/petroleum ether and acetone/petroleum
ether to give the corresponding 2-phenazinamine derivatives (1e
19), respectively. Compound 20 was prepared as follows: Phenazin-
2-amine (B) (195 mg, 1 mmol) and benzenesulfonyl chloride
(14 mmol) were dissolved in the solution of dry CH2Cl2 (50 mL)
anhydrous pyridine (1 mL). The mixture was refluxed for 1 h and
the other procedures were the same as the preparation of com-
pounds (1e19). Compound 21 was prepared as follows: Phenazin-
2-amine (B) (195 mg, 1 mmol) was dissolved in the solution of N,N-
dimethylformamide (DMF, 20 mL) and triethylamine (3 mL).
Dimethylcarbamyl chloride (14 mmol) was added to the above
solution. The mixture was refluxed under 100 ꢀC for 2.5 h and the
other procedures were similar to the preparation of compounds
(1e19). All the substituents at the phenazin-2-amine were listed in
Scheme 1 and Table 1.
7.60e7.57 (td, J ¼ 7.5, 1.5 Hz, 1H, AreH), 7.55e7.51 (t, J ¼ 7.5 Hz, 1H,
AreH). 13C NMR (DMSO-d6, 125 MHz)
: 165.8 (CO), 143.7 (AreC),
d
143.2 (AreC), 142.0 (AreC), 140.6 (AreC), 140.4 (AreC), 136.4 (Are
C), 131.5 (AreC), 130.9 (AreC), 130.1 (AreC), 130.0 (AreC), 129.9
(AreC), 129.7 (AreC), 129.3 (AreC), 129.1 (AreC), 129.0 (AreC),
127.3 (AreC), 126.3 (AreC), 114.6 (AreC). HRMS (ESI): m/z [M þ H]þ
calcd for: 334.07471, found: 334.07432.
3-Chloro-N-(phenazin-2-yl)benzamide (5): bright yellow solid.
IR (KBr, cmꢁ1): 3377, 3240, 3058, 1686, 1617, 1545, 1487, 1473, 1450,
1257, 1215, 1192, 1143, 758, 741. 1H NMR (DMSO-d6, 500 MHz)
d:
10.99 (s, 1H, NH), 8.88 (s, 1H, AreH), 8.30e8.24 (m, 4H, AreH), 8.13
(s, 1H, AreH), 8.04e8.03 (d, J ¼ 7.0 Hz, 1H, AreH), 7.96e7.92 (t,
J ¼ 9.5 Hz, 2H, AreH), 7.74e7.73 (d, J ¼ 7.0 Hz, 1H, AreH), 7.65e7.62
(t, J ¼ 7.0 Hz,1H, AreH). 13C NMR (DMSO-d6,125 MHz)
d: 165.0 (CO),
143.5 (AreC), 142.9 (AreC), 141.9 (AreC), 140.7 (AreC), 140.6 (Are
C), 136.3 (AreC), 133.2 (AreC), 131.7 (AreC), 130.9 (AreC), 130.4
(AreC), 130.0 (AreC), 129.6 (AreC), 129.2 (AreC), 128.8 (AreC),
127.6 (AreC), 127.0 (AreC), 126.7 (AreC), 115.0 (AreC). HRMS (ESI):
m/z [M þ H]þ calcd for: 334.07471, found: 334.07432.
4-Methyl-N-(phenazin-2-yl)benzamide (6): bright yellow solid.
IR (KBr, cmꢁ1): 3471, 3255, 1652, 1573, 1546, 1505, 1486, 1460, 1438,
1354, 1312, 1279, 1190, 1118, 831, 759, 747. 1H NMR (DMSO-d6,
500 MHz)
d
: 10.74 (s, 1H, NH), 8.86 (d, J ¼ 2.0 Hz, 1H, AreH), 8.29e
8.22 (m, 4H, AreH), 7.99e7.97 (d, J ¼ 8.0 Hz, 2H, AreH), 7.96e7.89
(m, 2H, AreH), 7.41e7.40 (d, J ¼ 8.5 Hz, 2H, AreH), 2.43 (s, 3H, CH3).
N-(Phenazin-2-yl)propionamide (1): bright yellow solid. IR (KBr,
cmꢁ1): 3278, 3066, 2974, 1667, 1571, 1547, 1506, 1482, 1460, 1439,
13C NMR (DMSO-d6, 125 MHz)
d: 166.3 (CO), 143.8 (AreC), 143.1
1361, 1231, 1190, 1125, 829, 757. 1H NMR (DMSO-d6, 500 MHz)
d:
(AreC), 142.1 (AreC), 142.0 (AreC), 141.0 (AreC), 140.5 (AreC),131.6
(AreC), 130.8 (AreC), 129.9 (AreC), 129.5 (AreC), 129.3 (AreC),
129.0 (2ꢂ AreC), 128.9 (AreC), 127.9 (2ꢂ AreC), 127.1 (AreC), 114.8
(AreC), 21.0 (CH3). HRMS (ESI): m/z [M þ H]þ calcd for: 314.12934,
found: 314.12905.
10.49 (s, 1H, NH), 8.70 (d, J ¼ 2.0 Hz, 1H, AreH), 8.22e8.18 (m, 3H,
AreH), 8.00e7.97 (dd, J ¼ 9.5, 2.0 Hz, 1H, AreH), 7.94e7.87 (m, 2H,
AreH), 2.51e2.47 (q, J ¼ 7.5, 2H, CH2), 1.18e1.15 (t, J ¼ 7.5, 3H, CH3).
13C NMR (DMSO-d6, 125 MHz)
d: 173.1 (CO), 143.9 (AreC), 143.1
(AreC), 141.7 (AreC), 140.8 (AreC), 140.4 (AreC), 130.8 (AreC),
129.8 (AreC), 129.7 (AreC), 129.3 (AreC), 128.9 (AreC), 126.4 (Are
C), 113.3 (AreC), 29.7 (CH2), 9.4 (CH3). HRMS (ESI): m/z [M þ H]þ
calcd for: 252.11369, found: 252.11337.
2-Methyl-N-(phenazin-2-yl)benzamide (7): bright yellow solid.
IR (KBr, cmꢁ1): 3253, 3059, 1668,1569, 1540, 1499, 1482, 1453, 1432,
1355, 1307, 1265, 1188, 1125, 823, 756, 736. 1H NMR (DMSO-d6,
500 MHz)
d
: 10.93 (s, 1H, NH), 8.85 (d, J ¼ 1.5 Hz, 1H, AreH), 8.25e
N-(Phenazin-2-yl)butyramide (2): bright yellow solid. IR (KBr,
8.21 (m, 3H, AreH), 8.17e8.15 (dd, J ¼ 9.5, 2.5 Hz, 1H, A-H), 7.96e
7.89 (m, 2H, AreH), 7.60e7.59 (d, J ¼ 7.0 Hz,1H, AreH), 7.48e7.46 (t,
J ¼ 7.5 Hz, 1H, AreH), 7.38e7.35 (t, J ¼ 7.5 Hz, 2H, AreH), 2.46 (s, 3H,
cmꢁ1): 3164, 3060, 2961, 2870, 1665, 1571, 1549, 1482, 1460, 1437,
1219, 1197, 1121, 841, 755. 1H NMR (DMSO-d6, 500 MHz)
d: 10.49 (s,
1H, NH), 8.70 (d, J ¼ 2.0 Hz, 1H, AreH), 8.22e8.18 (m, 3H, AreH),
8.00e7.97 (dd, J ¼ 9.5, 2.0 Hz, 1H, AreH), 7.94e7.87 (m, 2H, AreH),
2.46e2.43 (t, J ¼ 7.5, 2H, CH2), 1.72e1.68 (m, J ¼ 7.5, 2H, CH2), 0.99e
CH3). 13C NMR (DMSO-d6, 125 MHz)
d: 168.7 (CO), 143.8 (AreC),
143.2 (AreC), 141.9 (AreC), 140.8 (AreC), 140.6 (AreC), 136.6 (Are
C), 135.5 (AreC), 130.9 (AreC), 130.6 (AreC), 130.0 (AreC), 129.9
(AreC), 129.8 (AreC), 129.3 (AreC), 128.9 (AreC), 127.4 (AreC),
127.6 (AreC), 125.7 (AreC), 114.3 (AreC), 19.3 (CH3). HRMS (ESI): m/
z [M þ H]þ calcd for: 314.12934, found: 314.12871.
0.96 (t, J ¼ 7.5, 3H, CH3). 13C NMR (DMSO-d6,125 MHz)
d: 172.3 (CO),
143.9 (AreC), 143.1 (AreC), 141.7 (AreC), 140.8 (AreC), 140.4 (Are
C), 130.8 (AreC), 129.8 (AreC), 129.7 (AreC), 129.3 (AreC), 128.9
(AreC), 126.4 (AreC), 113.4 (AreC), 38.5 (CH2), 18.4 (CH2), 13.6
(CH3). HRMS (ESI): m/z [M þ H]þ calcd for: 266.12934, found:
266.12769.
3-Methyl-N-(phenazin-2-yl)benzamide (8): bright yellow solid.
IR (KBr, cmꢁ1): 3346, 3324, 1651, 1541, 1507, 1487, 1458, 1439, 1356,
1309, 1279, 1224, 1190, 758, 739. 1H NMR (DMSO-d6, 500 MHz)
d:
N-(Phenazin-2-yl)-2-phenylacetamide (3): yellow solid. IR (KBr,
10.80 (s, 1H, NH), 8.87 (s, 1H, AreH), 8.29e8.22 (m, 4H, AreH),
7.96e7.93 (m, 2H, AreH), 7.91e7.84 (m, 2H, AreH), 7.49e7.46 (m,
cmꢁ1): 3377, 3240, 3050, 1666, 1575, 1545, 1488, 1461, 1442, 1358,
1183,1133, 835, 765, 717, 692. 1H NMR (DMSO-d6, 500 MHz)
d
: 10.78
2H, AreH), 2.45 (s, 3H, CH3). 13C NMR (DMSO-d6, 125 MHz)
d: 166.6
(s, 1H, NH), 8.69 (d, J ¼ 1.5 Hz, 1H, AreH), 8.22e8.18 (m, 3H, AreH),
8.01e7.99 (dd, J ¼ 9.5, 2.0 Hz, 1H, AreH), 7.94e7.87 (m, 2H, AreH),
7.42e7.35 (m, 4H, AreH), 7.30e7.26 (t, J ¼ 7.5, 1H, AreH). 3.81 (s,
(CO), 143.8 (AreC), 143.2 (AreC), 141.9 (AreC), 140.9 (AreC), 140.6
(AreC), 137.9 (AreC), 134.5 (AreC), 132.6 (AreC), 130.8 (AreC),
130.0 (AreC), 129.6 (AreC), 129.3 (AreC), 128.9 (AreC), 128.4 (Are
C), 128.3 (AreC), 127.1 (AreC), 125.0 (AreC), 114.9 (AreC), 20.9
(CH3). HRMS (ESI): m/z [M þ H]þ calcd for: 314.12934, found:
314.12893.
2H, CH2). 13C NMR (DMSO-d6, 125 MHz)
d: 170.2 (CO), 143.8 (AreC),
143.1 (AreC), 141.8 (AreC), 140.7 (AreC), 140.4 (AreC), 135.4 (Are
C), 130.8 (AreC), 129.9 (AreC), 129.8 (AreC), 129.3 (AreC), 129.2
(2ꢂ AreC), 128.9 (AreC), 128.3 (2ꢂ AreC), 126.6 (AreC), 126.3 (Are
C), 113.7 (AreC), 43.4 (CH2). HRMS (ESI): m/z [M þ H]þ calcd for:
314.12934, found: 314.12721.
4-Methoxy-N-(phenazin-2-yl)benzamide (9): bright yellow
solid. IR (KBr, cmꢁ1): 3422, 1651, 1601, 1537, 1502, 1457, 1312, 1255,
1193, 1177, 1022, 840, 758. 1H NMR (DMSO-d6, 500 MHz)
d: 10.68 (s,
2-Chloro-N-(phenazin-2-yl)benzamide (4): bright yellow solid.
1H, NH), 8.85 (d, J ¼ 2.2 Hz, 1H, AreH), 8.29e8.26 (m, 1H, AreH),
8.25e8.21 (m, 3H, AreH), 8.08e8.04 (m, 2H, AreH), 7.95e7.88 (m,
2H, AreH), 7.14e7.10 (m, 2H, AreH), 3.87 (s, 3H, OCH3). 13C NMR
IR (KBr, cmꢁ1): 3316, 3263, 3058, 1661, 1572, 1547, 1484, 1459, 1437,
1355, 1292, 1190, 1118, 751. 1H NMR (DMSO-d6, 500 MHz)
d: 11.15 (s,
1H, NH), 8.83 (s, 1H, AreH), 8.27e8.22 (m, 3H, AreH), 8.13e8.11
(dd, J ¼ 9.5, 2.0 Hz, 1H, AreH), 7.97e7.91 (m, 2H, AreH), 7.73e7.72
(dd, J ¼ 7.5, 1.5 Hz, 1H, AreH), 7.65e7.63 (d, J ¼ 8.0 Hz, 1H, AreH),
(DMSO-d6,125 MHz) d: 165.8 (CO),162.3 (AreC),143.8 (AreC),143.1
(AreC), 141.9 (AreC), 141.1 (AreC),140.5 (AreC), 130.8 (AreC), 129.9
(2ꢂ AreC), 129.8 (AreC), 129.5 (AreC), 129.3 (AreC), 128.9 (AreC),

X. Gao et al. / European Journal of Medicinal Chemistry 69 (2013) 1e9
7
127.2 (AreC), 126.4 (AreC), 114.6 (AreC), 113.7 (2ꢂ AreC), 55.5
13C NMR (DMSO-d6, 125 MHz)
d
: 160.7 (CO), 143.7 (AreC), 143.2
(OCH3). HRMS (ESI): m/z [M þ H]þ calcd for: 330.12425, found:
330.12676.
(AreC), 142.0 (AreC), 140.6 (AreC), 140.5 (thiophenyl-C), 132.8
(thiophenyl-C), 130.9 (thiophenyl-C), 130.0 (2ꢂ AreC), 129.7 (Are
C), 129.3 (AreC), 129.0 (AreC), 128.2 (AreC), 128.1 (AreC), 126.9
(thiophenyl-C), 115.0 (AreC). HRMS (ESI): m/z [M þ H]þ calcd for:
306.07011, found: 306.07194.
N-(Phenaazin-2-yl)cyclohexanecarboxamide (10): yellow solid.
IR (KBr, cmꢁ1): 3435, 3255, 3042, 2932, 2853, 1670, 1573, 1548,
1512, 1486, 1461, 1441, 1250, 1204, 833, 759. 1H NMR (DMSO-d6,
500 MHz)
d
: 10.43 (s, 1H, NH), 8.71 (d, J ¼ 2.0 Hz, 1H, AreH), 8.22e
N-(Phenazin-2-yl)-2-(thiophen-2-yl)acetamide (15): deep yel-
low solid. IR (KBr, cmꢁ1): 3408, 3256, 3163, 3052, 1670, 1576, 1551,
1487,1460, 1439, 1354, 1312,1245, 1182, 1121, 827, 764, 690. 1H NMR
8.17 (m, 3H, AreH), 8.00e7.99 (dd, J ¼ 9.0, 2.0 Hz, 1H, AreH), 7.94e
7.90 (m, 1H, AreH), 7.89e7.86 (m, 1H, AreH), 2.49e2.44 (m, 1H,
cyclohexyl-CH), 1.91e1.89 (m, 2H, cyclohexyl-CH2), 1.81e1.79 (m,
2H, cyclohexyl-CH2), 1.70e1.67 (d, J ¼ 12.0 Hz, 1H, cyclohexyl-CH2),
1.53e1.45 (m, 2H, cyclohexyl-CH2), 1.36e1.27 (m, 2H, cyclohexyl-
CH2), 1.26e1.19 (m, 1H, cyclohexyl-CH2). 13C NMR (DMSO-d6,
(DMSO-d6, 500 MHz)
d
: 10.84 (s,1H, NH), 8.69 (d, J ¼ 2.5 Hz,1H, Are
H), 8.22e8.19 (m, 3H, AreH), 8.00e7.98 (dd, J ¼ 9.5, 2.5 Hz, 1H, Are
H), 7.94e7.91 (m, 1H, AreH), 7.90e7.87 (m, 1H, AreH), 7.44e7.43
(dd, J ¼ 5.5, 1.0 Hz, 1H, thiophenyl-H), 7.07e7.06 (dd, J ¼ 5.5, 1.0 Hz,
1H, thiophenyl-H), 7.02e7.01 (q, J ¼ 5.5 Hz, 1H, thiophenyl-H), 4.04
125 MHz) d: 175.4 (CO), 144.0 (AreC), 143.2 (AreC), 141.8 (AreC),
141.0 (AreC), 140.0 (AreC), 130.8 (AreC), 129.8 (2ꢂ AreC), 129.3
(AreC), 128.9 (AreC), 126.5 (AreC), 113.4 (AreC), 45.1 (cyclohexyl-
CH), 29.1 (2ꢂ cyclohexyl-CH2), 25.4 (cyclohexyl-CH2), 25.2 (2ꢂ
cyclohexyl-CH2). HRMS (ESI): m/z [M þ H]þ calcd for: 306.16064,
found: 306.16178.
3-Cyclopentyl-N-(phenazin-2-yl)propanamide (11): yellow
solid. IR (KBr, cmꢁ1): 3430, 3256, 3050, 2947, 2864, 1670, 1647, 1571,
1544, 1508, 1480, 1440, 1358, 1203, 832, 759. 1H NMR (DMSO-d6,
(s, 2H, CH2). 13C NMR (DMSO-d6, 125 MHz)
d: 169.1 (CO), 143.8 (Are
C), 143.1 (AreC), 141.9 (AreC), 140.5 (AreC), 140.4 (thiophenyl-C),
136.5 (AreC), 130.9 (AreC), 130.0 (AreC), 129.9 (AreC), 129.3 (Are
C), 128.9 (AreC), 126.7 (AreC), 126.6 (thiophenyl-C), 126.2 (thio-
phenyl-C), 125.2 (thiophenyl-C), 113.8 (AreC), 37.6 (CH2). HRMS
(ESI): m/z [M þ H]þ calcd for: 320.08576, found: 320.09090.
Ethyl phenazin-2-ylcarbamate (16): yellow solid. IR (KBr, cmꢁ1):
3217, 3058, 2974, 2921, 1738, 1636, 1608, 1502, 1448, 1445, 1313,
1236, 1202, 1133, 1060, 851, 833, 756. 1H NMR (DMSO-d6, 500 MHz)
500 MHz)
d: 10.49 (s, 1H, NH), 8.70 (d, J ¼ 2.0 Hz, 1H, AreH), 8.21e
8.17 (m, 3H, AreH), 7.98e7.96 (dd, J ¼ 9.0, 2.0 Hz, 1H, AreH), 7.93e
7.90 (m, 1H, AreH), 7.89e7.86 (m, 1H, AreH), 2.48e2.45 (m, 2H,
COCH2), 1.83e1.76 (m, 3H, CH2 and cyclopentyl-CH), 1.71e1.66 (m,
2H, cyclopentyl-CH2), 1.61e1.57 (m, 2H, cyclopentyl-CH2), 1.54e
1.45 (m, 2H, cyclopentyl-CH2), 1.16e1.12 (m, 2H, cyclopentyl-CH2).
d
: 10.33 (s, 1H, NH), 8.40 (d, J ¼ 2.0 Hz, 1H, AreH), 8.20e8.16 (m, 3H,
AreH), 7.97e7.95 (dd, J ¼ 9.5, 2.0 Hz, 1H, AreH), 7.92e7.85 (m, 2H,
AreH), 4.26e4.22 (q, J ¼ 7.1, 2H, CH2), 1.33e1.30 (t, J ¼ 7.1, 3H, CH3).
13C NMR (DMSO-d6, 125 MHz)
d: 153.5 (CO), 143.9 (AreC), 143.1
(AreC),141.6 (AreC), 141.1 (AreC), 140.2 (AreC), 130.8 (AreC),129.9
(AreC), 129.6 (AreC), 129.2 (AreC), 128.8 (AreC), 125.8 (AreC),
111.8 (AreC), 60.8 (CH2), 14.4 (CH3). HRMS (ESI): m/z [M þ H]þ calcd
for: 268.10860, found: 268.10704.
13C NMR (DMSO-d6, 125 MHz)
d: 172.4 (CO), 143.9 (AreC), 143.1
(AreC), 141.7 (AreC), 140.8 (AreC), 140.3 (AreC), 130.8 (AreC),
129.8 (AreC), 129.7 (AreC), 129.2 (AreC), 128.8 (AreC), 126.3 (Are
C), 113.4 (AreC), 39.2 (cyclopentyl-CH), 36.0 (CH2), 32.0 (2ꢂ
cyclohexyl-CH2), 31.1 (cyclohexyl-CH2), 24.7 (2ꢂ cyclohexyl-CH2).
HRMS (ESI): m/z [M þ H]þ calcd for: 320.17629, found: 320.17682.
N-(Phenazin-2-yl)-1-naphthamide (12): yellow solid. IR (KBr,
cmꢁ1): 3430, 3278, 3050, 1649, 1560, 1537, 1509, 1485, 1459, 1430,
1287, 1252, 1194, 1126, 1053, 962, 781, 755. 1H NMR (DMSO-d6,
N-(Phenazin-2-yl)propanamide (17): black solid. IR (KBr, cmꢁ1):
3432, 1697, 1632, 1616, 1557, 1518, 1473, 1455, 1358, 1194, 1138, 756.
1H NMR (DMSO-d6, 500 MHz)
d
: 10.97 (s,1H, NH), 8.74 (d, J ¼ 1.5 Hz,
1H, AreH), 8.23e8.20 (m, 3H, AreH), 8.07e8.05 (dd, J ¼ 9.5, 2.0 Hz,
1H, AreH), 7.96e7.89 (m, 2H, AreH), 3.98e3.95 (t, J ¼ 7.5, 6.0 Hz,
2H, CH2), 3.03e3.00 (t, J ¼ 7.5, 6.0 Hz, 2H, CH2). 13C NMR (DMSO-d6,
500 MHz)
d: 11.22 (s,1H, NH), 8.96 (d, J ¼ 2 Hz,1H, AreH), 8.29e8.20
125 MHz) d: 169.3 (CO), 143.7 (AreC), 143.0 (AreC), 141.8 (AreC),
(m, 4H, AreH), 8.17e8.15 (m, 2H, AreH), 8.08e8.06 (m, 1H, AreH),
7.98e7.96 (m, 1H, AreH), 7.95e7.90 (m, 2H, AreH), 7.70e7.67 (m,
1H, AreH), 7.66e7.63 (m, 2H, AreH). 13C NMR (DMSO-d6, 125 MHz)
140.7 (AreC), 140.5 (AreC), 131.0 (AreC), 130.0 (AreC), 129.9 (Are
C), 129.3 (AreC), 128.8 (AreC), 126.3 (AreC), 113.6 (AreC), 40.5
(CH2), 39.5 (CH2). HRMS (ESI): m/z [M þ H]þ calcd for: 286.07471,
found: 286.07370.
d: 168.1 (CO),143.8 (AreC), 143.2 (AreC),142.0 (AreC), 140.9 (AreC),
140.6 (AreC),134.0 (AreC),133.2 (AreC),130.9 (AreC),130.6 (AreC),
130.0 (AreC), 129.8 (AreC), 129.6 (AreC), 129.3 (AreC), 129.0 (Are
C), 128.4 (AreC), 127.2 (AreC), 126.7 (AreC), 126.5 (AreC), 125.9
(AreC), 125.0 (AreC), 124.9 (AreC), 114.6 (AreC). HRMS (ESI): m/z
[M þ H]þ calcd for: 350.12934, found: 350.13123.
N-(Phenazin-2-yl)benzamide (18): yellow solid. IR (KBr, cmꢁ1):
3469, 3225, 3060, 1649, 1566, 1547, 1483, 1461, 1439, 1358, 1310,
1286,1190,1126, 837, 800, 760, 707. 1H NMR (DMSO-d6, 500 MHz)
d:
10.83 (s,1H, NH), 8.87 (s,1H, AreH), 8.29e8.22 (m, 4H, AreH), 8.07e
8.05 (d, 2H, AreH), 7.96e7.90 (m, 2H, AreH), 7.78e7.65 (t, J ¼ 7.5 Hz,
1H, AreH), 7.62e7.59 (t, J ¼ 7.5 Hz, 2H, AreH). 13C NMR (DMSO-d6,
N-(Phenazin-2-yl)furan-2-carboxamide (13): yellow solid. IR
(KBr, cmꢁ1): 3385, 3264, 3125, 1680, 1587, 1544, 1461, 1439, 1354,
1305,1281,1213,1195,1123,1013, 755.1H NMR (DMSO-d6, 500 MHz)
125 MHz) d: 166.5 (CO), 143.7 (AreC), 143.2 (AreC), 142.0 (AreC),
140.9 (AreC),140.6 (AreC),134.5 (AreC),132.0 (AreC),130.9 (AreC),
130.0 (AreC), 129.6 (AreC), 129.3 (AreC), 129.0 (AreC), 128.5 (2ꢂ
AreC), 127.9 (2ꢂ AreC), 127.1 (AreC), 114.9 (AreC). HRMS (ESI): m/z
[M þ H]þ calcd for: 300.11369, found: 300.11211.
d
: 10.78 (s,1H, NH), 8.82 (d, J ¼ 2.0,1H, AreH), 8.30e8.21 (m, 4H, Are
H), 7.96e7.89 (m, 3H, 2ꢂ AreH and furaneCH), 7.49e7.46 (m, 1H,
furaneH), 6.79e6.78 (m, 1H, furaneH). 13C NMR (DMSO-d6,
125 MHz)
d
: 156.8 (CO),146.3 (furaneC),140.4 (furaneC),130.9 (Are
N-(Phenazin-2-yl)acetamide (19): bright yellow solid. IR (KBr,
cmꢁ1): 3490, 3258, 3158, 3046, 1675, 1574, 1550, 1461, 1441, 1378,
C),130.7 (AreC),130.0 (AreC),129.6 (AreC),129.3 (AreC),129.2 (Are
C),128.9 (AreC),126.9 (AreC),122.4 (AreC),116.0 (AreC),115.8 (Are
C), 115.0 (furaneC), 112.5 (AreC), 112.4 (furaneC). HRMS (ESI): m/z
[M þ H]þ calcd for: 290.09295, found: 290.11491.
1356,1292,1122, 838, 765. 1H NMR (DMSO-d6, 500 MHz)
d: 10.55 (s,
1H, NH), 8.68 (d, J ¼ 2.2 Hz, 1H, AreH), 8.22e8.18 (m, 3H, AreH),
7.97e7.87 (m, 3H, AreH), 2.20 (s, 3H, CH3). 13C NMR (DMSO-d6,
N-(Phenazin-2-yl)thiophene-2-carboxamide (14): yellow solid.
IR (KBr, cmꢁ1): 3316, 3088, 1663, 1653, 1573, 1550, 1522, 1508, 1414,
125 MHz) d: 169.4 (CO), 143.9 (AreC), 143.1 (AreC), 141.8 (AreC),
140.8 (AreC),140.4 (AreC),130.8 (AreC),129.8 (AreC),129.7 (AreC),
129.3 (AreC), 128.9 (AreC), 126.3 (AreC), 113.4 (AreC), 24.3 (CH3).
HRMS (ESI): m/z [M þ H]þ calcd for: 238.09804, found: 238.09638.
N-(Phenazin-2-yl)benzenesulfonamide (20): red solid. IR (KBr,
cmꢁ1): 3223, 3060, 1638, 1601, 1519, 1483, 1453, 1362, 1330, 1156,
1360, 1274, 1190, 758, 729. 1H NMR (DMSO-d6, 500 MHz)
d: 10.79 (s,
1H, NH), 8.80 (d, J ¼ 0.5 Hz, 1H, AreH), 8.25e8.22 (m, 4H, AreH),
8.17e8.16 (dd, J ¼ 4.0, 1.0 Hz, 1H, thiophenyl-H), 7.97e7.96 (dd,
J ¼ 5.0, 1.0 Hz, 1H, thiophenyl-H), 7.95e7.92 (m, 1H, AreH), 7.91e
7.88 (m, 1H, AreH), 7.32e7.30 (q, J ¼ 5.0, 4.0 Hz, 1H, thiophenyl-H).
1094, 922, 906, 832, 748. 1H NMR (DMSO-d6, 500 MHz)
d: 11.26 (s,

8
X. Gao et al. / European Journal of Medicinal Chemistry 69 (2013) 1e9
1H, NH), 8.18e8.15 (m, 3H, AreH), 7.97e7.96 (d, J ¼ 7.2 Hz, 2H, Are
H), 7.93e7.87 (m, 2H, AreH), 7.80e7.75 (td, J ¼ 10.2, 2.3 Hz, 2H, Are
were added into each well and incubated with cells for 24 h at 37 ꢀC
in 5% CO2. Cisplatin was used as positive control and 10% CS was
used as a negative control for HepG2. Cell apoptosis assays were
performed by Flow cytometry using an Annexin V-FITC Apoptosis
Detection Kit (Biouniquer, Nanjing, China) according to the manu-
facturer’s instructions. Cells were harvested and washed twice with
cold PBS, then collected by centrifugation for 5 min and stained
H), 7.64e7.59 (m, 3H, AreH). 13C NMR (DMSO-d6, 125 MHz)
d: 143.2
(AreC),143.1 (AreC),141.9 (AreC),140.2 (AreC),139.8 (AreC),139.1
(AreC),133.4 (AreC),131.1 (AreC),130.8 (AreC),130.1 (AreC),129.5
(2ꢂ AreC), 129.3 (AreC), 128.8 (AreC), 126.7 (2ꢂ AreC), 125.5 (Are
C), 112.5 (AreC). HRMS (ESI): m/z [M þ H]þ calcd for: 336.08067,
found: 336.07862.
with annexin V-FITC (100 ng/mL) and propidium iodide (2 mg/mL)
1,1-Dimethyl-3-(phenazin-2-yl)urea (21): brown solid. IR (KBr,
in annexin-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM
CaCl2, pH 7.4). After 15 min incubation at room temperature, the
fluorescence of cells was measured using a flow cytometer (FAC
Scan, Becton Dickenson, USA) in FL1 and FL2 channel, respectively.
The results were obtained by using FCSExpress software and rep-
resented as percentage of normal and apoptotic cells.
cmꢁ1): 3380, 3047, 2915, 1636, 1617, 1582, 1476, 1449, 1396, 1360,
1099, 832, 771, 756. 1H NMR (DMSO-d6, 500 MHz)
d: 8.18e8.16 (d,
J ¼ 7.5 Hz, 1H, AreH), 8.16 (s, 1H, AreH), 8.13e8.12 (d, J ¼ 8.5 Hz, 1H,
AreH), 8.07e8.05 (d, J ¼ 9.5 Hz, 1H, AreH), 7.89e7.86 (m, 1H, Are
H), 7.83e7.80 (m, 1H, AreH), 7.70e7.69 (d, J ¼ 9.0 Hz, 1H, AreH),
7.51 (s, 1H, NH), 3.14 (s, 3H, CH3), 3.06 (s, 3H, CH3). 13C NMR (DMSO-
d6, 125 MHz)
d
: 155.1 (2ꢂ C, CO and AreC), 154.6 (AreC), 144.8 (Are
Acknowledgments
C), 142.9 (AreC), 141.0 (AreC), 131.4 (AreC), 130.3 (AreC), 129.3
(AreC), 129.2 (AreC), 128.8 (AreC), 128.6 (AreC), 112.4 (AreC), 39.7
(CH3), 34.2 (CH3). HRMS (ESI): m/z [M þ H]þ calcd for C15H14N4O:
267.12459, found: 267.12463.
This study was supported by the Project Program of National
Nature Science Foundation of China for Young Scientists (No.
81001395), State Key Laboratory of Natural Medicines, China
Pharmaceutical University (No. JKGQ201102), and Qing Lan Project.
4.3. In vitro anticancer screening
4.3.1. Cell culture
Appendix A. Supplementary data
All adherent cell lines including K562 (human chronic myelog-
enous leukemia), HepG2 (human hepatocellular carcinoma),
MGC803 (human gastric carcinoma), HCT116 (human colorectal
carcinoma), MCF7 (human breast adenocarcinoma), as well as 293T
(epithelial cells from human embryo kidney) non-cancer cell
cultured in a humidified, 5% CO2 atmosphere at 37 ꢀC, and main-
tained in monolayer culture in RPMI-1640 medium supplemented
with 10% fetal bovine serum (FBS), 100 mg/mL of streptomycin and
100 mg/mL of penicillin.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.07.017.
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