Synthesis and anticancer activity of new 1-[(5 or 6-substituted 2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives

Article abstract of DOI:10.1016/j.bmc.2010.09.028

A series of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives were synthesized and evaluated as an anticancer agent. From screening of quinoxalinyl-piperazine compound li

Full text of DOI:10.1016/j.bmc.2010.09.028

Bioorganic & Medicinal Chemistry 18 (2010) 7966–7974
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anticancer activity of new 1-[(5 or 6-substituted
2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives
Young Bok Lee a, , Young-Dae Gong b, , Heejeong Yoon a, Chang-Ho Ahn a, Moon-Kook Jeon c,
⇑
,
⇑
,
Jae-Yang Kong c
a Rexahn Pharmaceuticals, Inc., Rockville, MD 20850, USA
b Innovative Drug Library Research Center, Department of Chemistry, Dongguk University-Seoul, Pil-dong 3-ga, Jung-gu, Seoul 100-715, Republic of Korea
c Center for Drug Discovery Platform Technology, Korea Research Institute of Chemical Technology, PO Box 107, Sinseongno, Yuseong-gu, Daejeon 305-600, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2010
Revised 9 September 2010
Accepted 14 September 2010
Available online 19 September 2010
A series of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alkoxyquinoxalinyl)amino-
carbonyl]-4-(hetero)arylpiperazine derivatives were synthesized and evaluated as an anticancer agent.
From screening of quinoxalinyl-piperazine compound library, we identified that many compounds inhib-
ited proliferation of various human cancer cells at nanomolar concentrations. Among them, one of the
fluoro quinoxalinyl-piperazine derivatives showed its IC50 values ranging from 11 to 21 nV in the growth
inhibition of cancer cells. This compound also displayed a more potent effect than paclitaxel against pac-
litaxel resistant HCT-15 colorectal carcinoma cells. The potency of this novel compound was further con-
firmed with the synergistic cytotoxic effect with several known cancer drugs such as paclitaxel,
doxorubicin, cisplatin, gemcitabine or 5-fluorouracil in cancer cells. This strong cell killing effect was
derived from the induction of apoptosis. Mechanistic studies have shown that this quinoxalinyl-pipera-
zine compound is a G2/M-specific cell cycle inhibitor and inhibits anti-apoptotic Bcl-2 protein with p21
induction. Thus the results suggest that our compound has potential use in the growth inhibition of drug
resistant cancer cells and the combination therapy with other clinically approved anticancer agents as
well.
Keywords:
G2/M-Specific cell cycle
2-Alkoxyquinoxalin-(hetero)arylpiperazine
Anticancer
Combination
Resistant
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
pyrans,6 oxazoles,7 pyrazoles,8 oxadiazoles,9 thiadiazoles,9 various
thiazoles,10,11 and pyrimidinediones.12 Inhibition of cell prolifera-
The importance of G2/M-specific cell cycle inhibitor in the
mediation of normal and pathological processes has motivated
considerable efforts to identify cell proliferation. Especially com-
pounds to induce G2/M arrest and apoptosis in cancer cells were
well known; for example, paclitaxel and related taxens,1,2
N-methyl-N0-nitro-N-nitrosoguanidine (MNNG), a DNA alkylating
agent,3 benzazepine (BBL22),4 curcumin,5 etc.
To identify small molecule inhibitors of G2/M-specific cell cycle,
we screened 1200 drug-like compounds using a cell-based reporter
assay that measures the transcriptional activity of G2/M-specific
cell cycle. The G2/M-specific cell cycle transcriptional activity
was dependent on cell proliferation and could be used to monitor
the activity of the cancer cell proliferation pathway. The 1200 com-
pounds that were screened represent 20,000 structurally diverse
and druggable, heterocyclic compound libraries, including benzo-
tion of various cancer cells by the 1200 compounds were screened
at a compound concentration of 5 M and compounds that repro-
ducibly inhibited growth by over 50% were selected. In the first
round of screening, quinoxalinyl-piperazine derivatives were
shown to reproducibly have IC50 below 5 lM. Therefore, in connec-
l
tion with a project for exploring the novel potent G2/M-specific
cell cycle inhibitors, we have designed some target compounds
for quinoxalinyl-piperazine-based library for optimization of the
structural features of the primary screening results through high-
throughput screening efforts. Although quinoxalinyl-piperazine
compounds which have an unsubstituted quinoxaline ring were
presented13 and one of the unsubstituted quinoxalinyl-piperazine
compounds was shown to exhibit microtubule-inhibiting activities
as a candidate for anticancer agent with the ability to inhibit the
angiogenesis of endothelial cells and to induce the apoptosis of tu-
mor cells,14 little attention was directed towards substituted qui-
noxalinyl-piperazine compounds at quinoxaline ring as a new
therapeutic molecule for the treatment of disorders such as
tumors.
⇑
Corresponding authors. Tel.: +1 240 328 6905 (Y.B.L.); tel.: +82 2 2260 3206
(Y.-D.G.).
E-mail addresses: leeyb@rexahn.com (Y.B. Lee), ydgong@dongguk.edu
(Y.-D. Gong).
In this Letter, a series of novel 5 or 6-substituted compounds at
quinoxaline ring of 1-[(2-alkoxyquinoxalin-3-yl)aminocarbonyl-4-
These authors contributed equally.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.028

Y. B. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7966–7974
7967
(hetero)arylpiperazine derivatives were made and tested as a
prominent anticancer agent in various types of human cancer cell
lines. The best compound was further characterized through com-
bination assay with several known cancer drugs, drug resistant
cells, and apoptosis assay.
reported related reactions17,18 and was supported by X-ray crystal-
lographic analysis of compound 19b (Fig. 1).
3. Result and discussion
The in vitro cytotoxicity experiments were performed with the
synthesized 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocar-
bonyl]-4-(hetero)arylpiperazine derivatives (Table 1) against hu-
man cancer cell lines using a colorimetric SRB-assay.19 Initially
we tested the compounds against three cancer cell lines such as
2. Chemistry
The compounds 5–8 with fluorine or chlorine atom at 5 position
of quinoxaline ring system were regiospecifically synthesized from
the corresponding 3-chloro-2-(methylthio)quinoxalines 1a and
1b15 as described in Scheme 1. Sequential substitution reactions
by treatment of the starting materials 1a and 1b with ammonia fol-
lowed by methoxide afforded 3-amino-2-methoxy derivatives 3a
and 3b through the formation of 3-amino-2-(methylthio)quinoxa-
line derivatives 2a and 2b. The final quinoxalinyl-piperazine com-
pounds 5–8 were prepared from the reactions of carbamate
derivatives 4a and 4b of the intermediates 3a and 3b with arylpip-
erazines. For the preparation of the quinoxalinyl-piperazine deriv-
atives 13–16 substituted with 5-methyl or 5-methoxy group, the
compounds 11a and 11b were synthesized by the application of
the same reaction sequence as that for the synthesis of intermedi-
ates 3a and 3b from compounds 1a and 1b to the corresponding
2,3-dichloroquinoxaline starting materials 9a and 9b16 (Scheme 2).
The compound 10a (Rf = 0.20 for n-hexane/ethyl acetate (4:1) mix-
ture) was isolated from the corresponding major regioisomer 2-
amino-3-chloro-5-methylquinoxaline (Rf = 0.35 for n-hexane/ethyl
acetate (4:1) mixture) by silica gel column chromatography in the
amination step. In the case of 10b, separation from the major 2-
amino-3-chloro regioisomer was difficult and purification by silica
gel column chromatography was performed in the next methoxy-
lation step (for n-hexane/ethyl acetate (1:1) mixture, 11b,
Rf = 0.13; major 2-amino-3-methoxy regioisomer, Rf = 0.20). The
conversion of 11a and 11b to the piperazine products 13–16 were
completed similarly to the examples in Scheme 1. Treatment of
2,3-dichloroquinoxalines 17a–d with ammonia and subsequent
recrystallization of the crude products in appropriate solvents (tet-
rahydrofuran for 18a and dichloromethane for 18c–d) gave the
major regioisomeric products 18a–d in 1H NMR spectroscopically
pure form (Scheme 3). The next steps were performed for the prep-
aration of final compounds 21–40 in analogous way to the cases in
Scheme 2. The regiochemistry of compounds 10 and 18 in Schemes
2 and 3 was determined on the basis of analogy with previously
MDA-MB-231, PANC-1, and UMRC2 at 0.1 and 1
compounds in Table 1, 13 compounds in Table 2 showed more than
50% inhibition at 0.1 M in at least one of these cell lines and they
lM. Among the
l
were further assayed at lower concentrations against more human
cancer cell lines to obtain their IC50 values (Table 2). Despite their
structural similarity to the quinoxalinyl-piperazine core scaffold,
the IC50 values significantly varied; the activity of the compounds
depends on the substitution at quinoxaline ring but also from sub-
stituents at the phenyl ring. Although the fluoro (compounds 5 and
6) or chlorine (compounds 7 and 8) containing compounds at C-5
position of quinoxaline ring might not possess strong anticancer
activity, the introduction of an electron donating group such as
methyl or methoxy at C-5 position of quinoxaline ring resulted in
a significant increase in cytotoxic potency. In particular, com-
pounds 13 and 15 having 3,5-dimethoxyphenyl-piperazine inhib-
ited the cell growth at IC50 values ranged from 29 to 74 nV in
the human cancer cells tested, which are more potent than com-
pounds 14 and 16 having 3,5-dimethylphenyl-piperazine that
might have more than 0.1 lM of IC50 values. Our further efforts
using SAR study to improve the cytotoxic activity of compound
13 or 15 resulted in the introduction of fluorine, chlorine, methyl
or methoxy at C-6 position of quinoxaline ring. Table 2 showed
that among compounds containing F or Cl at C-6 position of quin-
oxaline ring, 3,5-dimethoxyphenyl-piperazine compounds induced
the most cell death. But introduction of methyl or methoxy group
at 2 or 4 position in phenyl reduced the cytotoxic activity of com-
pounds by electronic and steric effects. Interestingly, the introduc-
tion of methyl (compounds 38 and 39) or methoxy (compound 40)
at C-6 position of quinoxaline ring resulted in the increase of IC50
values which is opposite to C-5 substitution of quinoxaline ring.
Compound 30, which showed 19–31 nV of IC50 range and has F-
substitution at C-6, was compared to unsubstituted compound
42 (from U.S. Patent No. 6,683,184) with 20–90 nV of IC50 range.
R1
R1
R1
N
N
NH2
SMe
N
N
Cl
N
NH2
NH3 (g)
NaOMe
DMSO
rt
THF
rt
SMe
N
OMe
3a : R1 = F
1a : R1 = F
1b : R1 = Cl
2a : R1 = F
2b : R1 = Cl
3b : R1 = Cl
Ar
N
O
OEt
NH
OMe
O
N
R1
R1
HN
N Ar
N
N
N
N
NH
OMe
ClCO2Et
pyr
DCM
rt
DBU
THF
60 ºC
4a : R1 = F
5~8 : R1 = F or Cl
4b : R1 = Cl
Scheme 1.

7968
Y. B. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7966–7974
R1
R1
R1
N
N
NH2
Cl
N
N
Cl
Cl
N
N
NH2
NH3 (g)
NaOMe
DMSO
heat
THF
rt
OMe
11a : R1 = Me
9a : R1 = Me
10a : R1 = Me
11b : R1 = OMe
9b : R1 = OMe
10b : R1 = OMe
Ar
N
O
OEt
NH
O
N
R1
R1
HN
N Ar
N
N
N
N
NH
ClCO2Et
pyr
DCM
rt
DBU
OMe
OMe
THF
60 ºC
12a : R1 = Me
13~16 : R1 = Me or MeO
12b : R1 = OMe
Scheme 2.
R2
N
N
NH2
Cl
R2
N
N
Cl
Cl
R2
N
N
NH2
NH3 (g)
NaOMe
DMSO
rt or heat
THF
rt
OMe
19a : R2 = F
19b : R2 = Cl
19c : R2 = Me
17a : R2 = F
18a : R2 = F
18b : R2 = Cl
17b : R2 = Cl
17c : R2 = Me
18c : R2 = Me
19d : R2 = OMe
17d : R2 = OMe
18d : R2 = OMe
Ar
N
O
OEt
NH
O
N
R2
N
N
R2
N
N
NH
HN
N Ar
ClCO2Et
pyr
DBU
THF
OMe
OMe
DCM
rt
60 ºC
21~40 : R2 = F, Cl, Me, or MeO
20a : R2 = F
20b : R2 = Cl
20c : R2 = Me
20d : R2 = OMe
Scheme 3.
This result suggests that introducing fluorine at C-6 in quinoxaline
ring imparted the increased cytotoxic activity. Compound 25
among tested compounds strongly inhibited proliferation of hu-
man cancer cells of the brain, breast, cervix, colon, kidney, liver,
lung, ovary, pancreas, prostate, skin, stomach, and skin (mela-
noma). The growth inhibition by compound 25 appeared to be
dose-dependent and the IC50 for cell growth ranged from 11 to
21 nM in the tested 14 human cancer cells (see Table 2), and we
further characterized this compound.
To evaluate whether any cooperative effect may exist between
cytotoxic drugs and compound 25, we tested the effect of com-
pound 25 in combination with known anticancer drugs of different
classes in the MDA-MB-231 breast cancer cell line. Compound 25
alone at a dose of 20 nM showed an inhibition of MDA-MB-231 cell
growth of approximately 14% (Fig. 2). Therefore, we selected this
low concentration to study whether any cooperative antiprolifera-
tive effect may occur between compound 25 and a series of cyto-
toxic drugs acting by different mechanisms of action. When
MDA-MB-231 cells were treated with compound 25 and a taxane
paclitaxel, a platinum derivative cisplatin, a topoisomerase II-
selective agent doxorubicin and antimetabolites gemcitabine and
fluorouracil, a synergistic growth inhibitory effect was observed
(see Fig. 1). Treatment with 20 nM of compound 25, which inhibits
13.8% of the growth of MDA-MB-231 cells, in combination with 2
or 3 nM paclitaxel, which used alone showed 8.0% and 60.8%
growth inhibition, respectively, caused a 77.4% and 90.6% inhibi-
tion, respectively. In cells treated with 5 or 10 nM of doxorubicin,
which alone cause 4.2% and 59.2% growth inhibition, respectively,
addition of 20 nM of compound 25 showed a growth inhibition of
41.1% and 76.2%, respectively. The growth inhibitory effect by
gemcitabine at 0.1 lM (1.0%) or 0.2 lM (17.7%) was increased to
35.0% and 56.3%, respectively, with the addition of 20 nM of com-
pound 25 to MDA-MB-231 cells. Further study with 5-fluorouracil
or cisplatin occurred also the synergistic inhibition of growth of
MDA-MB-231 cells. In fact, in cells treated with 3 or 5 lM 5-fluo-
rouracil, which alone cause 3.3% and 41.6% growth inhibition,
respectively, addition of 20 nM of compound 25 caused a growth
inhibition of 56.6% and 83.1%, respectively. Cisplatin at 300 lM

Y. B. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7966–7974
7969
Figure 1. The X-ray structure of compound 19b.
Table 1
bined with known anticancer drugs of low concentration to treat
the cancer patients more effectively by reducing the side effects
of known anticancer drugs that comes from the usage of high
concentration.
Structure of 1-[(5 or 6-substituted 2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(het-
ero)arylpiperazine derivatives
Ar
N
The development of multidrug resistance (MDR) in tumor cells
by known anticancer drugs has become a serious issue and a major
obstacle in clinical oncology. Our further investigation of com-
pound 25 was extended to examine the effect in drug resistant
cancer cells. In experiment using HCT116 and paclitaxel resistant
HCT-15 cancer cells, compound 25 showed the same potent activ-
ity in both cancer cells, even stronger effect in drug resistant HCT-
15 cells (see Table 3) with 0.72 of Resistant Index (RI). But antican-
cer activity of paclitaxel was decreased by 70-fold in paclitaxel
resistant HCT-15 cells. This result implies that compound 25 can
control and overcome drug resistance that has been a major prob-
lem in anticancer therapy.
Many potential chemotherapeutic agents have been shown to
kill tumor cells by inducing apoptosis (programmed cell death),
without eliciting an inflammatory response in the surrounding
normal tissue. For example, gemcitabine used as an anticancer
agent in various carcinomas including pancreatic, non-small cell
lung, bladder, and breast cancers induces apoptosis.20–22 We con-
firmed the occurrence of apoptosis by compound 25 treatment in
cancer cells using APOPercentage™ Apoptosis Assay that detects
the membrane alteration in apoptotic cells (unpublished result).
Compound 25 also induced the apoptotic cell death through DNA
fragmentation and PARP fragmentation (unpublished data) and
this apoptosis induction by compound 25 was associated with an
increase in p21WAF1 protein, an inhibition of cell cycle progression
at G2/M (Fig. 3) and an inhibition of anti-apoptotic Bcl-2 protein
and an increase of pro-apoptotic Bad protein as shown in Figure 4.
O
N
R1
R2
N
N
NH
OMe
Compound
R1
R2
Ar
5
6
7
F
F
H
H
H
H
H
H
H
H
F
F
F
F
F
F
F
F
F
F
F
F
F
3,5-Dimethoxyphenyl
3.5-Dimethylphenyl
3,5-Dimethoxyphenyl
3.5-Dimethylphenyl
3,5-Dimethoxyphenyl
3.5-Dimethylphenyl
3,5-Dimethoxyphenyl
3.5-Dimethylphenyl
Phenyl
2-Methoxyphenyl
3-Methoxyphenyl
4-Methoxyphenyl
3,5-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
2-Methylphenyl
Cl
Cl
Me
Me
MeO
MeO
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8
13
14
15
16
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
42*
3-Methylphenyl
2,6-Dimethylphenyl
3,5-Dimethylphenyl
2-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
F
F
3-Chlorophenyl
4-Chlorophenyl
4. Conclusion
Cl
Cl
Me
Me
OMe
H
3,5-Dimethoxyphenyl
3,5-Dimethylphenyl
3,5-Dimethoxyphenyl
3,5-Dimethylphenyl
3,5-Dimethylphenyl
3,5-Dimethylphenyl
In conclusion, we screened 1200 heterocyclic small molecules,
and identified a novel hit core skeleton of quinoxalinyl-piperazine,
selected as a potential G2/M-specific cell cycle inhibitor and dem-
onstrated its effects in inhibiting various cancer cell line prolifera-
tion, leading to inhibition of G2/M arrest responses in vitro and
pharmacophore analysis. Further characterization of compound
25 showed that this novel piperazine compound specifically in-
duces the apoptosis by the downregulation of Bcl-2 protein level
and the G2/M arrest and inhibits the growth of various types of
*
Compound 42: from U.S. Patent No. 6,683,184.
(5.0%) or 500 lM (31.6%) showed also the 78.0% and 90.2% of inhi-
bition with the addition of 20 nM of compound 25, respectively.
The combination result suggest that compound 25 can be com-

7970
Y. B. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7966–7974
Table 2
Inhibition of cell growth (IC50
,
l
M) by quinoxalinyl-piperazine compounds against human cancer cell lines
13
15
22
23
25
28
30
34
36
37
38
39
40
42*
MDA-MB-231
Caki-1
UMRC2
PANC-1
A549
MKN-45
HepG2
HCT116
HT29
PC-3
U251
HeLa
SK-MEL-28
OVCAR-3
0.060
0.032
0.030
0.054
0.056
0.057
0.063
0.050
0.055
0.057
0.054
0.062
0.064
0.039
0.056
0.033
0.029
0.042
0.064
0.052
0.057
0.060
0.074
0.057
0.058
0.063
0.068
0.044
0.064
0.11
0.10
0.35
0.20
0.093
0.12
0.16
0.15
0.22
0.14
0.15
0.26
0.076
0.063
0.047
0.050
0.062
0.15
0.012
0.011
0.013
0.021
0.021
0.020
0.019
0.020
0.021
0.021
0.015
0.021
0.020
0.012
0.036
0.024
0.032
0.039
0.081
0.023
0.080
0.043
0.050
0.060
0.050
0.056
0.11
0.023
0.019
0.022
0.024
0.023
0.027
0.021
0.025
0.031
0.022
0.019
0.024
0.022
0.025
0.040
0.064
0.077
0.28
0.050
0.053
0.065
0.098
0.073
0.064
0.063
0.068
0.079
0.076
0.056
0.073
0.11
0.037
0.056
0.06
0.032
0.029
0.039
0.070
0.055
0.040
0.045
0.047
0.048
0.063
0.031
0.051
0.047
0.023
0.050
0.071
0.050
0.28
0.097
0.080
0.081
0.10
0.14
0.11
0.085
0.10
0.13
0.057
0.066
0.10
0.33
0.11
0.081
0.081
0.11
0.12
0.14
0.050
0.035
0.027
0.038
0.021
0.021
0.060
0.24
0.10
0.076
0.069
0.068
0.076
0.11
0.079
0.069
0.081
0.11
0.05
0.12
0.076
0.077
0.097
0.11
0.064
0.090
0.070
0.070
0.046
0.068
0.036
0.090
0.020
0.030
0.020
0.026
0.023
0.13
0.080
0.086
0.19
0.075
0.11
0.14
0.024
0.055
0.042
0.056
0.065
0.059
*
Compound 42: from U.S. Patent No. 6,683,184.
Compound 25
Paclitaxel
Gemcitabine
Cisplatin
100
90
80
70
60
50
40
30
20
10
Doxorubicin
5-Fluorouracil
Compound 25 + Cytotoxic drugs
0
a
b
c
d
e
f
g
h
i
j
Figure 2. Combination effect of compound 25 with different cytotoxic drugs on the growth of MDA-MB-231 cancer cells. The drugs were used at the following doses: (a–b): 2
and 3 nM paclitaxel; (c–d): 5 and 10 nM doxorubicin; (e–f): 100 and 200 nM gemcitabine; (g–h) 3 and 5 M fluorouracil; (i–j): 300 and 500 M cisplatin. Data are expressed
l
l
as percentage growth inhibition in reference to the growth of untreated control cells. 0.5% DMSO in media was used as a control for drugs. The open portion of the bars
represents the percentage growth inhibition values for compound 25. The striped or squared portion of the bars represents the percentage growth inhibition values for the
cytotoxic drugs as indicated in the respective legends. The height of the bars on the left represents the sum of the individual agents’ effects and the expected percentage
growth inhibition if drugs are additive when used in combination. The total height of the solid bar indicates the actual observed growth inhibition when drugs were used in
combination. The data represent means and standard errors of triplicate determination of at least two experiments.
Table 3
Comparison of IC50 values (nM)a of compound 25 and paclitaxel in HCT116 and HCT-
15 cells
5. Experimental
5.1. Chemistry
Compound
HCT116
29 1.4
HCT-15
Resistant indicesb
Compound 25
Paclitaxel
21 0.98
140 11
0.72
70
2
0.08
5.1.1. General
a
IC50 values were determined after incubation of compound 25 and paclitaxel at
All chemicals were reagent grade and used as purchased. Reac-
tions were monitored by TLC analysis using Merck silica gel 60 F-
254 thin layer plates. Flash column chromatography was carried
out on Merck silica gel 60 (230–400 mesh). The crude products
were purified by parallel chromatography using Quad3™. 1H
NMR spectra were recorded in d units relative to deuterated sol-
vent as an internal reference using a Bruker 500 MHz NMR instru-
ment. LC–MS analysis was performed on an ESI mass spectrometer
with PDA detection. LC–MS area % purities of all products were
determined by LC peak area analysis (XTerraMS C18 column,
4.6 mm ꢀ 100 mm; PDA detector at 200–400 nm; gradient, 5–
95% CH3CN/H2O).
various concentrations with the HCT116 cell line (paclitaxel sensitive) and the HCT-
15 cell line (paclitaxel resistant) for 96 h. Data are presented as the mean SD.
b
Resistant indices are defined by IC50 (HCT-15)/IC50 (HCT116).
human cancer cell lines. Compound 25 has also significantly
inhibited growth of drug resistant cancer cells, and has potential
use in combination therapy with known cancer drugs that could
improve its therapeutic index and decrease toxicity to cancer
patients although extensive efforts to further characterize this
molecule should be followed.

Y. B. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7966–7974
7971
100
75
50
25
0
A
Control
Compound 25
Go, G1
S
G2/M Go, G1
S
G2/M Go, G1
S
G2/M
3 hr
6 hr
12 hr
100
75
B
G0, G1
S
S
G2/M
G2/M
50
25
0
0 uM
0.02 uM
0.07 uM
0.2 uM
Compound 25
Figure 3. Cell cycle distribution of MDA-MB-231 cells after treatments with compound 25. The MDA-MB-231 cells were seeded in 10 cm dish and then incubated overnight.
Cells were treated with 70 nM of compound 25 for 3, 6, and 12 h (A) or 20 nM, 70 nM and 200 nM of compound 25 for 12 h (B). Compound 25 treated cells were harvested and
subject to cell cycle analyses as described in experimental section. PI stained cells were analyzed by Guava PCA-AFP instrument using its cell cycle software program (Guava
Technologies, Hayward, CA) and expressed as percentage of cells in G0/G1, S, and G2/M phases of the cell cycle.
p21
Bcl-2
Bad
β-actin
Con
Sample Con
24 h
Sample Con
48 h
Sample
72 h
Con
0 h
Figure 4. Effect of compound 25 on the expression of p21WAF1, Bcl-2, and Bad protein in MDA-MB-231 cancer cells. The cells were treated without (Con) or with sample
(compound 25) for 24, 48, and 72 h and p21WAF1, Bcl-2, and Bad protein expression were determined by western blot analysis. b-Actin was determined as a loading control.
Representative data are shown from two independent cell culture experiments.
5.1.2. Compounds synthesis
5.1.2.1. Synthesis of 3-amino-5-fluoro-2-methylthioquinoxaline
(n-hexane/ethyl acetate = 3:1) to yield the desired compound 2a
(50 mg, 58%): 1H NMR (500 MHz, CDCl3) d 2.75 (s, 3H), 5.84 (br s,
2H), 7.20 (m, 1H), 7.29 (m, 1H), 7.61 (m, 1H); MS (ESI) m/z 210
([M+H]+).
(2a).
Method A: To a stirred solution of 3-chloro-5-fluoro-2-
methylthioquinoxaline (1a) (93 mg, 0.41 mmol) in dimethylsulfox-
ide (20 ml) was injected anhydrous ammonia gas at room temper-
ature. Stirring was continued at room temperature for 48 h and
then water was added to the reaction mixture. The mixture was
extracted with ethyl acetate and the organic layer was washed
with water and dried over MgSO4. After removal of solvent in va-
cuo, the residue was purified by SiO2 column chromatography
5.1.2.2. Synthesis of 3-amino-5-fluoro-2-methoxyquinoxaline
(3a).
Method B: To a stirred solution of 3-amino-5-fluoro-2-
methylthioquinoxaline (2a) (47 mg, 0.22 mmol) in tetrahydrofuran
(2 ml) was added 25 wt % sodium methoxide in methanol (475 mg,
2.20 mmol) at room temperature. Stirring was continued at room

7972
Y. B. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7966–7974
temperature for 48 h. The resulting mixture was concentrated in
vacuo to remove the solvent and then water was added thereto.
The mixture was extracted with dichloromethane and the organic
layer was washed with water and dried over MgSO4. After removal
of solvent in vacuo, the residue was purified by SiO2 column chro-
matography (n-hexane/ethyl acetate = 3:1) to yield the desired
compound 3a (30 mg, 71%): 1H NMR (500 MHz, CDCl3) d 4.16 (s,
3H), 6.28 (br s, 2H), 7.17 (m, 1H), 7.27 (m, 1H), 7.50 (m, 1H); MS
(ESI) m/z 194 ([M+H]+).
method A. The compound 10a (Rf = 0.20 for n-hexane/ethyl acetate
(4:1) mixture, 1.07 g, 39%) was purified from the major regioiso-
meric 2-amino-3-chloro-5-methylquinoxaline (Rf = 0.35 for n-hex-
ane/ethyl acetate (4:1) mixture, 1.37 g, 50%): 1H NMR (500 MHz,
CDCl3) d 2.79 (s, 3H), 5.38 (br s, 2H), 7.34 (t, J = 7.9 Hz, 1H), 7.61
(d, J = 7.9 Hz, 1H), 7.78 (d, J = 7.9 Hz, 1H); MS (ESI) m/z 194
([M+H]+).
5.1.2.9. Synthesis of 3-amino-2-methoxy-5-methylquinoxaline
(11a).
Prepared from compound 10a (1.04 g, 5.37 mmol) in
99% yield (1.01 g) using method B: 1H NMR (500 MHz, CDCl3) d
2.63 (s, 3H), 4.13 (s, 3H), 5.19 (br s, 2H), 7.23 (d, J = 7.7 Hz, 1H),
7.32 (t, J = 7.7 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H); MS (ESI) m/z 190
([M+H]+).
5.1.2.3. Synthesis of ethyl N-(5-fluoro-2-methoxyquinoxalin-3-
yl)carbamate (4a).
Method C: To a stirred solution of 3-ami-
no-5-fluoro-2-methoxyquinoxaline (3a) (26 mg, 0.13 mmol) and
ethyl chloroformate (22 mg, 0.20 mmol) in dichloromethane
(6 ml) at room temperature was added pyridine (16 mg,
0.20 mmol). The resulting mixture was stirred at room tempera-
ture for 40 h, concentrated in vacuo to remove the solvent and
purified by SiO2 column chromatography (n-hexane/ethyl ace-
tate = 5:1) to yield the desired compound 4a (26 mg, 75%): 1H
NMR (500 MHz, CDCl3) d 1.37 (t, J = 7.1 Hz, 3H), 4.17 (s, 3H), 4.34
(q, J = 7.1 Hz, 2H), 7.25 (m, 1H), 7.45 (m, 1H), 7.55 (m, 1H), 7.81
(br s, 1H); MS (ESI) m/z 266 ([M+H]+).
5.1.2.10. Synthesis of ethyl N-(2-methoxy-5-methylquinoxalin-
3-yl)carbamate (12a).
By method C from compound 11a
(1.01 g, 5.34 mmol) in 73% yield (1.02 g): 1H NMR (500 MHz,
CDCl3) d 1.37 (t, J = 7.1 Hz, 3H), 2.65 (s, 3H), 4.16 (s, 3H), 4.33 (q,
J = 7.1 Hz, 2H), 7.43–7.38 (m, 2H), 7.76 (br s, 1H), 7.82 (d,
J = 8.0 Hz, 1H); MS (ESI) m/z 262 ([M+H]+).
5.1.2.11. 1-(3,5-Dimethoxyphenyl)-4-[(2-methoxy-5-methylqui-
noxalin-3-yl)aminocarbonyl] piperazine (13).
By method D
5.1.2.4. Synthesis of 1-(3,5-dimethoxyphenyl)-4-[(5-fluoro-2-
methoxyquinoxalin-3-yl)amino-carbonyl]piperazine
from compound 12a (24 mg, 0.092 mmol) in 68% yield (27 mg): 1H
NMR (500 MHz, CDCl3) d 2.62 (s, 3H), 3.25–3.27 (m, 4H), 3.79 (s,
6H), 3.85–3.90 (m, 4H), 4.15 (s, 3H), 6.07 (d, J = 1.7 Hz, 1H), 6.13
(s, 2H), 7.25–7.27 (m, 2H), 7.30–7.33 (m, 2H); MS (ESI) m/z 438
([M+H]+).
(5).
Method D: To a stirred solution of ethyl N-(5-fluoro-2 -
methoxyquinoxalin-3-yl)carbamate (4a) (10 mg, 0.037 mmol)
and 1-(3,5-dimethoxyphenyl) piperazine (12 mg, 0.054 mmol) in
tetrahydrofuran (2 ml) at room temperature was added DBU
(8 mg, 0.05 mmol). The resulting mixture was stirred at 60 °C for
27 h, concentrated in vacuo to remove the solvent and purified
by SiO2 column chromatography (n-hexane/ethyl acetate = 2:1)
to yield the desired compound 5 (9 mg, 55%): 1H NMR (500 MHz,
CDCl3) d 3.25 and 3.35 (2m, 4H), 3.78, 3.85, and 4.14 (2m, 4H),
3.82 (s, 6H), 4.17 and 4.20 (2s, 3H), 6.09 (m, 1H), 6.15 (s, 2H),
7.10–7.20, 7.20–7.27, 7.32, 7.40–7.48, and 7.55–7.60 (5m, 4H);
MS (ESI) m/z 442 ([M+H]+).
5.1.2.12. 1-(3,5-Dimethylphenyl)-4-[(2-methoxy-5-methylqui-
noxalin-3-yl)aminocarbonyl] piperazine (14).
In 86% yield:
1H NMR (500 MHz, CDCl3) d 2.29 (s, 6H), 2.63 (s, 3H), 3.24 (m,
4H), 3.78 (m, 3H), 4.15–4.16 (m, 4H), 6.57–6.59 (m, 3H), 7.27 (br
s, 1H), 7.34–7.35 (m, 2H), 7.48–7.50 (m, 1H); MS (ESI) m/z 406
([M+H]+).
5.1.2.13. 1-(3,5-Dimethoxyphenyl)-4-[(2,5-dimethoxyquinoxa-
lin-3-yl)aminocarbonyl]piperazine (15).
NMR (500 MHz, CDCl3) d 3.21–3.23 (m, 4H), 3.78–3.83 (m, 7H),
4.00 (s, 3H), 4.13 (s, 6H), 6.05 (br s, 1H), 6.12 (d, J = 2.1 Hz, 2H),
6.86–6.89 (m, 1H), 7.22–7.23 (m, 2H), 7.30 (br s, 1H); MS (ESI)
m/z 454 ([M+H]+).
Compounds 6–8 were synthesized by the same way with the
preparation of the compound 5 using methods A, B, C, and D.
In 60% yield: 1H
5.1.2.5. 1-(3,5-Dimethylphenyl)-4-[(5-fluoro-2-methoxyquinox-
alin-3-yl)aminocarbonyl] piperazine (6).
In 66% yield: 1H
NMR (500 MHz, CDCl3) d 2.30 and 2.33 (2s, 6H), 3.23 and 3.33
(2m, 4H), 3.79, 3.86, and 4.14 (3m, 4H), 4.17 and 4.20 (2s, 3H),
6.55–6.65 (m, 3H), 7.15–7.20, 7.20–7.26, 7.31, 7.40–7.48, 7.48–
7.52, and 7.52–7.60 (6m, 4H); MS (ESI) m/z 410 ([M+H]+).
5.1.2.14. 1-[(2,5-Dimethoxyquinoxalin-3-yl)aminocarbonyl]-4-
(3,5-dimethylphenyl)piperazine (16).
In 50% yield: 1H NMR
(500 MHz, CDCl3) d 2.29 (s, 6H), 3.20–3.29 (m, 4H), 3.74–3.83 (m,
3H), 4.00 (s, 3H), 4.13 (m, 4H), 6.56–6.59 (m, 3H), 6.85–6.88 (m,
1H), 7.18–7.26 (m, 2H), 7.40 (br s, 1H); MS (ESI) m/z 422 ([M+H]+).
Compounds 21–40 were synthesized also by the same way with
the preparation of the compound 13 using methods A, B, C, and D.
5.1.2.6. 1-(3,5-Dimethoxyphenyl)-4-[(5-chloro-2-methoxyqui-
noxalin-3-yl)aminocarbonyl] piperazine (7).
In 61% yield:
1H NMR (500 MHz, CDCl3) d 3.25 and 3.33 (2m, 4H), 3.81 (s, 6H),
3.87 and 4.15 (2m, 4H), 4.17 and 4.19 (2s, 3H), 6.08 (s, 1H),
6.10–6.20 (m, 2H), 7.15–7.30, 7.40–7.48, 7.55–7.60, 7.60–7.65,
and 7.70–7.75 (5m, 3H); MS (ESI) m/z 458 ([M+H]+).
5.1.2.15. Synthesis of 3-amino-2-chloro-6-fluoroquinoxaline
(18a).
From compound 17a (21.0 g, 96.8 mmol) in 63% yield
(12.1 g) by method A: 1H NMR (300 MHz, DMSO-d6) d 7.25–7.33
(m, 2H), 7.44 (br, 2H), 7.80 (dd, J = 8.7 and 6.2 Hz, 1H); MS (ESI)
m/z 198 ([M+H]+).
5.1.2.7. 1-(3,5-Dimethylphenyl)-4-[(5-chloro-2-methoxyquinox-
alin-3-yl)aminocarbonyl] piperazine (8).
In 72% yield: 1H
NMR (500 MHz, CDCl3) d 2.32 (s, 6H), 3.24 and 3.31 (2m, 4H),
3.84, 3.88, and 4.15 (3m, 4H), 4.18 and 4.20 (2s, 3H), 7.55–7.65
(m, 3H), 7.25–7.30, 7.40–7.49, 7.55–7.60, 7.60–7.65, and 7.70–
7.75 (5m, 3H); (ESI) m/z 426 ([M+H]+).
Compounds 13–16 in Scheme 2 were prepared using methods
A, B, C, and D.
5.1.2.16. Synthesis of 3-amino-6-fluoro-2-methoxyquinoxaline
(19a).
From compound 18a (550 mg, 2.78 mmol) in 91% yield
(491 mg) by method B: 1H NMR (300 MHz, DMSO-d6) d 4.02 (s, 3H),
7.09 (br s, 2H), 7.04–7.21 (m, 2H), 7.60 (dd, J = 8.8 and 6.1 Hz, 1H);
MS (ESI) m/z 194 ([M+H]+).
5.1.2.8. Synthesis of 3-amino-2-chloro-5-methylquinoxaline
5.1.2.17. Synthesis of ethyl N-(6-fluoro-2-methoxyquinoxalin-3-
(10a).
Prepared from compound 9a (3.00 g, 14.1 mmol) using
yl)carbamate (20a).
From compound 19a (580 mg,

Y. B. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7966–7974
7973
3.00 mmol) in 93% yield (740 mg) by method C: 1H NMR (300 MHz,
CDCl3) d 1.36 (t, 3H, J = 7.2 Hz) 4.15 (s, 3H) 4.33 (q, 2H, J = 7.2 Hz)
7.28 (td, 1H, J = 8.7 and 2.7 Hz) 7.40 (dd, 1H, J = 9.3 and 2.7 Hz)
7.74 (s, 1H) 7.93 (m, 1H); MS (ESI) m/z 266 ([M+H]+).
5.1.2.26.
1-(2,6-Dimethylphenyl)-4-[(6-fluoro-2-methoxyqui-
noxalin-3-yl)aminocarbonyl] piperazine (29).
In 65% yield:
1H NMR (300 MHz, CDCl3) d 2.26 (s, 3H), 3.20 (s, 4H), 3.71 (s,
3H), 4.12–4.18 (m, 4H), 6.99–7.01 (m, 3H), 7.26–7.32 (m, 2H),
7.53–7.81 (m, 2H); MS (ESI) m/z 410 ([M+H]+).
5.1.2.18. Synthesis of 1-[(6-fluoro-2-methoxyquinoxalin-3-
5.1.2.27.
1-(3,5-Dimethylphenyl)-4-[(6-fluoro-2-methoxyqui-
yl)aminocarbonyl]-4-phenylpiperazine (21).
From com-
noxalin-3-yl)aminocarbonyl] piperazine (30).
In 79% yield:
pound 20a (27 mg, 0.10 mmol) in 88% yield (34 mg) by Method
D: 1H NMR (300 MHz, CDCl3) d 3.29 (s, 4H), 3.77 (s, 3H), 4.14 (s,
4H), 6.89–6.97 (m, 4H), 7.24–7.56 (m, 5H), 7.62–7.71 (m, 1H);
MS (ESI) m/z 382 ([M+H]+).
1H NMR (300 MHz, CDCl3) d 2.29 (s, 6H), 3.27 (s, 4H), 3.88 (s,
3H), 4.14 (s, 4H), 6.59 (s, 3H), 7.01–7.10 (s, 1H), 7.24–7.36 (m,
2H), 7.47–7.71 (m, 2H); MS (ESI) m/z 410 ([M+H]+).
5.1.2.28.
1-[(6-Fluoro-2-methoxyquinoxalin-3-yl)aminocar-
In 91% yield: 1H
5.1.2.19.
1-[(6-Fluoro-2-methoxyquinoxalin-3-yl)aminocar-
bonyl]-4-(2-fluorophenyl)piperazine (31).
bonyl]-4-(2-methoxyphenyl)piperazine (22).
In 84% yield:
NMR (200 MHz, CDCl3) d 3.18 (s, 4H), 3.78 (s, 3H), 4.13 (s, 4H),
6.93–7.10 (m, 5H), 7.20–7.34 (m, 1H), 7.46–7.60 (m, 1H), 7.67–
7.74 (m, 1H); MS (ESI) m/z 400 ([M+H]+).
1H NMR (200 MHz, CDCl3) d 3.15 (s, 4H), 3.79–3.87 (m, 6H), 4.11
(s, 4H), 6.86–7.02 (m, 4H), 7.18–7.22 (m, 1H), 7.39–7.50 (m, 1H),
7.65–7.72 (m, 1H); MS (ESI) m/z 412 ([M+H]+).
5.1.2.29.
1-[(6-Fluoro-2-methoxyquinoxalin-3-yl)aminocar-
In 85% yield: 1H
5.1.2.20.
1-[(6-Fluoro-2-methoxyquinoxalin-3-yl)aminocar-
bonyl]-4-(4-fluorophenyl)piperazine (32).
bonyl]-4-(3-methoxyphenyl)piperazine (23).
In 87% yield:
NMR (300 MHz, CDCl3) d 3.19 (s, 4H), 3.77 (s, 3H), 4.13 (s, 4H),
6.88–7.02 (m, 4H), 7.23–7.27 (m, 1H), 7.45–7.71 (m, 3H); MS
(ESI) m/z 400 ([M+H]+).
1H NMR (300 MHz, CDCl3) d 3.28 (s, 4H), 3.80 (s, 6H), 4.13 (s,
4H), 6.45–6.58 (m, 3H), 7.01 (s, 1H), 7.17–7.23 (m, 2H), 7.37–7.70
(m, 2H); MS (ESI) m/z 412 ([M+H]+).
5.1.2.30. 1-(2-Chlorophenyl)-4-[(6-fluoro-2-methoxyquinoxa-
lin-3-yl)aminocarbonyl]piperazine (33).
In 87% yield: 1H
5.1.2.21.
1-[(6-Fluoro-2-methoxyquinoxalin-3-yl)aminocar-
NMR (200 MHz, CDCl3) d 3.14 (s, 4H), 3.79 (s, 3H), 4.13 (s, 4H),
6.97–7.05 (m, 2H), 7.22–7.28 (m, 2H), 7.33–7.40 (m, 2H), 7.46–
7.51 (d, J = 10.2 Hz, 1H), 7.66–7.73 (m, 1H); MS (ESI) m/z 416
([M+H]+).
bonyl]-4-(4-methoxyphenyl)piperazine (24).
In 80% yield:
1H NMR (200 MHz, CDCl3) d 3.18 (s, 4H), 3.79 (s, 6H), 4.08–4.15
(m, 4H), 6.85–6.98 (m, 4H), 7.22–7.76 (m, 4H); MS (ESI) m/z 412
([M+H]+).
5.1.2.31. 1-(3-Chlorophenyl)-4-[(6-fluoro-2-methoxyquinoxa-
lin-3-yl)aminocarbonyl]piperazine (34).
NMR (200 MHz, CDCl3) d 3.30 (s, 4H), 3.76 (s, 3H), 4.13 (s, 4H),
6.77–6.91 (m, 3H), 7.15–7.33 (m, 3H), 7.44–7.58 (d, J = 10.0 Hz,
1H), 7.58–7.75 (m, 1H); MS (ESI) m/z 416 ([M+H]+).
5.1.2.22. 1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyqui-
In 70% yield: 1H
noxalin-3-yl)aminocarbonyl] piperazine (25).
In 72% yield;
mp 149 °C; 1H NMR (200 MHz, CDCl3) d 3.18–3.35 (m, 4H), 3.76
(s, 3H), 3.79 (s, 6H), 4.08–4.15 (m, 4H), 6.07 (s, 1H), 6.11 (s, 2H),
6.83–7.73 (8m, total 4H); MS (ESI) m/z 442 ([M+H]+); MS/MS
(amu) 442.152; 13H NMR (125 MHz, CDCl3) d 45.22 (2 CH2),
49.42 (2 CH2), 54.55 (CH3), 55.43 (2 CH3), 92.37 (aromatic carbon
between 2 OCH3), 95.75 (two aromatic carbon of o-position
OCH3), 111.69 (aromatic carbon of o-position F), 111.88 (aromatic
carbon of o-position F), 116.29 (aromatic carbon of m-position F),
128.02 (aromatic carbon of quinoxaline), 128.10 (aromatic carbon
of quinoxaline), 153.01 (N-attached aromatic carbon), 153.19 (N
attached carbon of quinoxaline), 160.44 (F attached aromatic car-
bon), 161.71 (O attached two aromatic carbons), 162.39 (O at-
tached quinoxaline carbon).
5.1.2.32. 1-(4-Chlorophenyl)-4-[(6-fluoro-2-methoxyquinoxa-
lin-3-yl)aminocarbonyl]piperazine (35).
In 95% yield: 1H
NMR (300 MHz, CDCl3) d 3.25 (s, 4H), 3.78 (s, 3H), 4.13 (s, 4H),
6.86 (d, J = 8.4 Hz, 2H), 7.22–7.26 (m, 3H), 7.44 (m, 1H), 7.70 (m,
1H); MS (ESI) m/z 416 ([M+H]+).
5.1.2.33.
1-[(6-Chloro-2-methoxyquinoxalin-3-yl)aminocar-
bonyl]-4-(3,5-dimethoxyphenyl) piperazine (36).
In 81%
yield: 1H NMR (300 MHz, CDCl3) d 3.21 and 3.29 (2m, 4H), 3.75
(s, 3H), 3.79 (s, 6H), 4.11 and 4.14 (2m, 4H), 6.07 (s, 1H), 6.11 (s,
2H), 7.20–7.97 (8m, 4H); MS (ESI) m/z 458 ([M+H]+).
5.1.2.23.
1-[(6-Fluoro-2-methoxyquinoxalin-3-yl)aminocar-
5.1.2.34.
1-[(6-Chloro-2-methoxyquinoxalin-3-yl)aminocar-
bonyl]-4-(3,4,5-trimethoxyphenyl) piperazine (26).
In 76%
bonyl]-4-(3,5-dimethylphenyl) piperazine (37).
In 79%
yield: 1H NMR (200 MHz, CDCl3) d 3.22 (s, 4H), 3.79–3.85 (m,
12H), 4.13 (s, 4H), 6.19 (s, 2H), 7.20–7.34 (m, 1H), 7.35–7.36 (m,
1H), 7.44 (s, 1H), 7.67–7.70 (m, 1H); MS (ESI) m/z 472 ([M+H]+).
yield: 1H NMR (300 MHz, CDCl3) d 2.29 (s, 6H), 3.19–3.29 (m,
4H), 3.73–3.89 (m, 3H), 4.11–4.14 (m, 4H), 6.58 (s, 3H), 7.19–
7.25 (m, 1H), 7.36–7.64 (m, 2H), 7.67–7.81 (m, 1H); MS (ESI) m/z
426 ([M+H]+).
5.1.2.24.
1-[(6-Fluoro-2-methoxyquinoxalin-3-yl)aminocar-
bonyl]-4-(2-methylphenyl)piperazine (27).
In 73% yield:
5.1.2.35. 1-(3,5-Dimethoxyphenyl)-4-[(2-methoxy-6-methylqui-
1H NMR (200 MHz, CDCl3) d 2.35 (s, 3H), 2.98–3.03 (m, 4H),
3.73–3.78 (m, 3H), 4.10–4.14 (m, 4H), 7.02–7.17 (m, 2H), 7.19–
7.29 (m, 2H), 7.36 (s, 1H), 7.48–7.60 (m, 1H), 7.67–7.74 (m, 1H);
MS (ESI) m/z 396 ([M+H]+).
noxalin-3-yl)aminocarbonyl] piperazine (38).
In 94% yield:
1H NMR (300 MHz, CDCl3) d 2.43 and 2.05 (2s, 3H), 3.21 and 3.30
(2m, 4H), 3.78 (s, 3H), 3.79 (s, 6H), 4.12 (m, 4H), 6.06 (s, 1H),
6.12 (s, 2H), 6.84–7.77 (8m, 4H); MS (ESI) m/z 438 ([M+H]+).
5.1.2.25.
1-[(6-Fluoro-2-methoxyquinoxalin-3-yl)aminocar-
5.1.2.36. 1-(3,5-Dimethylphenyl)-4-[(2-methoxy-6-methylqui-
bonyl]-4-(3-methylphenyl)piperazine (28).
In 90% yield:
noxalin-3-yl)aminocarbonyl] piperazine (39).
In 87% yield:
1H NMR (200 MHz, CDCl3) d 2.33 (s, 3H), 3.26–3.30 (m, 4H),
3.74–3.78 (m, 3H), 4.13 (s, 4H), 6.75–6.78 (m, 3H), 7.14–7.28 (m,
2H), 7.36 (s, 1H), 7.44–7.51 (dd, J = 9.8 and 2.4 Hz, 1H), 7.67–7.74
(m, 1H); MS (ESI) m/z 396 ([M+H]+).
1H NMR (300 MHz, CDCl3) d 2.29 (s, 6H), 2.42–2.48 (m, 3H),
3.20–3.28 (m, 4H), 3.75–3.80 (m, 3H), 4.10–4.13 (m, 4H), 6.59 (s,
3H), 7.00–7.12 (m, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.48–7.65 (m,
2H); MS (ESI) m/z 406 ([M+H]+).

7974
Y. B. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7966–7974
5.1.2.37. 1-[(2,6-Dimethoxyquinoxalin-3-yl)aminocarbonyl]-4-
(3,5-dimethylphenyl)piperazine (40).
In 65% yield: 1H NMR
(300 MHz, CDCl3) d 2.28 (s, 6H), 3.26 (s, 4H), 3.76–3.87 (m, 6H),
4.11 (s, 4H), 6.59 (s, 2H), 6.90 (m, 1H), 7.14 (d, J = 8.7 Hz, 1H),
7.32 (s, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H); MS
(ESI) m/z 422 ([M+H]+).
PBS with 0.1% glucose and 2% FBS. After removal of the superna-
tant, cells were resuspended in 0.5 ml of 70 M propidium iodide
(PI) solution containing 0.1% Triton X-100, 40 mM sodium citrate,
pH 7.4. RNase was added at 50 g/ml final concentration and cells
were incubated at 37 °C for 30 min. PI stained cells were analyzed
by Guava PCA-AFP instrument using its cell cycle software pro-
gram (Guava Technologies, Hayward, CA) and expressed as per-
centage of cells in G0/G1, S, and G2/M phases of the cell cycle.
l
l
5.2. Biology
5.2.1. Growth of cancer cells
5.2.5. Western blotting analysis
Human cancer cell lines were obtained from the following
sources: Caki-1, OVCAR-3, MDA-MB-231, HeLa, PC3, HepG2,
A549, PANC-1, SK-MEL-28, HT29, HCT-15, and HCT116 from the
American Type Culture Collection (Manassas, VA); U251 from
Riken Institutes, Marunouchi, Tokyo, Japan; MKN-45 from DSMZ
(German Collection of Microorganisms and Cell Cultures), Braun-
schweig, Germany; UMRC2 (kidney) from the U.S. National Cancer
Institute (Bethesda, MD). All cell lines except MDA-MB-231,
HCT116, Caki-1, UMRC2, and PANC-1 were grown in RPMI1640
medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal
bovine serum (FBS), 1 mM sodium pyruvate, 10 mM HEPES, and
MDA-MB-231 cells were incubated with compound 25 at 70 nM
for 24, 48 or 72 h. After the incubation, cells were washed once
with PBS and resuspended with the lysis buffer containing
25 mM Tris–HCl, pH 7.5, 300 mM NaCl, 1% Triton X-100 and prote-
ase inhibitors. Total protein concentration was determined by BCA
protein assay according to manufacturer’s manual (Pierce Biotech-
nology, Rockford, IL). Protein was separated on SDS–PAGE gel and
transferred to nitrocellulose membranes. The blots were probed
against anti-p21WAF1 (Santa Cruz, CA), anti-Bcl-2 (Santa Cruz, CA),
and anti-Bad antibody (Santa Cruz, CA). Anti-b-actin antibody
(Santa Cruz, CA) was used as an internal control.
100 units/ml
penicillin–streptomycin
(P/S).
MDA-MB-231,
Acknowledgments
HCT116, Caki-1, UMRC2, and PANC-1 cells were maintained in Dul-
becco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad,
CA) supplemented with 10% FBS, 10 mM HEPES, 100 units/ml P/S,
This research was supported by Rexahn Pharmaceuticals, Inc.,
and Basic Science Research Program through the Korea Research
Institute of Chemical Technology and the National Research Foun-
dation of Korea (NRF) funded by the Ministry of Education, Science
and Technology (2010-0004128).
and 2 mM -glutamine. All cells were incubated at 37 °C under
L
5% CO2 and humidified air.
5.2.2. Cell growth inhibition assay
The growth inhibition by the synthesized quinoxalinyl-pipera-
zine compounds against human cancer cells was assessed by the
sulforhodamine B (SRB) assay,15 along with DMSO as a control. Dif-
ferent human cancer cell lines were treated with compounds at
various concentrations. After 96 h incubation, surviving cells were
fixed with trichloroacetic acid, washed and stained with sulforho-
damine B. For the study of combination effect of compound 25
with cytotoxic drugs, MDA-MB-231 cells were seeded in 96 multi-
well plate and treated with compound 25 in the presence of differ-
ent concentrations of the indicated cytotoxic drug for 96 h.
Absorbance was measured at 530 nm using Benchmark Plus Micro-
plate reader (Bio-Rad Laboratories, Hercules, CA). The drug concen-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.09.028.
References and notes
1. Gallagher, B. M., Jr. Curr. Med. Chem. 2007, 14, 2959.
2. Carlson, R. O. Expert Opin. Invest. Drugs 2008, 17, 707.
3. Park, C.; Choi, B. T.; Cheong, J. H.; Moon, S.-K.; Kim, C.-H.; Lee, W. H.; Choi, Y. H.
Mutat. Res. 2004, 563, 139.
4. Xia, W.; Spector, S.; Hardy, L.; Zhao, S.; Saluk, A.; Alemane, L.; Spector, N. L. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 7494.
5. Weir, N. M.; Selvendiran, K.; Kutala, V. K.; Tong, L.; Vishwanath, S.; Rajaram, M.;
Tridandapani, S.; Anant, S.; Kuppusamy, P. Cancer Biol. Ther. 2007, 6, 178.
6. Hwang, J. Y.; Choi, H.-S.; Seo, J.-S.; La, H.-J.; Yoo, S.-E.; Gong, Y.-D. J. Comb. Chem.
2006, 8, 897.
tration which inhibited the cell growth by 50% (IC50
) was
calculated using Kaledia Graph software program (Synergy soft-
ware, Reading, PA).
7. Hwang, J. Y.; Gong, Y.-D. J. Comb. Chem. 2006, 8, 297.
8. Hwang, J. Y.; Choi, H. S.; Lee, D. H.; Yoo, S.-E.; Gong, Y.-D. J. Comb. Chem. 2005, 7,
136.
9. Hwang, J. Y.; Choi, H.-S.; Lee, D. H.; Gong, Y.-D. J. Comb. Chem. 2005, 7, 816.
10. Lee, T.; Park, J.-H.; Lee, D.-H.; Gong, Y.-D. J. Comb. Chem. 2009, 11, 495.
11. Lee, T.; Park, J.-H.; Jeon, M.-K.; Gong, Y.-D. J. Comb. Chem. 2009, 11, 288.
12. Lee, I. Y.; Kim, S. Y.; Lee, J. Y.; Yu, C. M.; Lee, D. H.; Gong, Y.-D. Tetrahedron Lett.
2004, 45, 9319.
13. Cho, E.-H.; Chung, S.-G.; Lee, S.-H.; Kwon, H.-S.; Kang, D.-W.; Joo, J.-H.; Lee, Y.-
H. U.S. Patent 6,683,184, 2004.
14. Yi, E.-Y.; Jeong, E.-J.; Song, H. S.; Lee, M.-S.; Kang, D.-W.; Joo, J.-H.; Kwon, H.-S.;
Lee, S.-H.; Park, S.-K.; Chung, S.-G.; Cho, E.-H.; Kim, Y.-J. Int. J. Oncol. 2004, 25,
365.
5.2.3. Growth inhibition of drug resistant cancer cells by
compound 25
The growth inhibition by compound 25 and paclitaxel (TaxolÒ)
against HCT116 and paclitaxel resistant HCT-15 colon cancer cells
was assessed as described in ‘cell growth inhibition assay’. Resis-
tance index (RI) was calculated by dividing the IC50 value of the
resistant cell line by the IC50 value of the nonresistant cell line
and represents the drug’s efficacy against drug resistant cell lines
versus the corresponding cancer cell lines (nonresistant).
15. Venkatesh, C.; Singh, B.; Mahata, P. K.; Ila, H.; Junjappa, H. Org. Lett. 2005, 7,
2169.
5.2.4. Cell cycle arrest
16. Gong, Y.-D.; Cho, H.; Jeon, M.-K.; Lee, T.; Choi, G.; Kong, J.-Y.; Park, W.-K.;
Hwang, S.-H.; Kim, J. J.; Lee, C.-H.; Ko, J.; Noh, M.; Han, E. S.; Kim, H.; Yun, J.-W.;
Moh, J. H.; Kim, D. H. PCT Int. Appl. WO 2009041789, 2009.
17. Sarges, R.; Howard, H. R.; Browne, R. G.; Lebel, L. A.; Seymour, P. A.; Koe, B. K. J.
Med. Chem. 1990, 33, 2240.
18. Ceccarelli, S.; D’Alessandro, A.; Prinzivalli, M.; Zanarella, S. Eur. J. Med. Chem.
1998, 33, 943.
19. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
MDA-MB-231 cells were plated at 50–70% saturation in a 10 cm
dish in DMEM medium with 10% FBS and then incubated overnight
in a humidified 37 °C incubator with 5% CO2. The cells were ex-
posed to compound 25 at various concentrations or for various
time intervals. No compound control treatment was also included
(0.25% DMSO). Cells were then trypsinized and harvested by cen-
trifugation at 7000 rpm for 5 min. Cell pellets were resuspended
in 0.2 ml of PBS containing 0.1% glucose and 2% FBS. Subsequently,
5 ml ice-cold 70% ethanol was added dropwise with shaking and
the treated cells were stored at ꢁ20 °C at least 30 min. Cells were
centrifuged at 2000 rpm for 5 min and washed once with 1 ml
20. Plunkett, W.; Huang, P.; Xu, Y. Z.; Heinemann, V.; Grunewald, R.; Gandhi, V.
Semin. Oncol. 1995, 22, 3.
21. Li, Y. W.; Singh, B.; Ali, N.; Sarkar, F. H. Int. J. Mol. Med. 1999, 3, 647.
22. Rundall, B. K.; Denlinger, C. E.; Jones, D. R. Surgery 2005, 138, 360.