Synthesis, anticancer activity and pharmacokinetic analysis of 1-[(substituted 2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives

Article abstract of DOI:10.1016/j.bmc.2011.12.026

Based on the anticancer activity of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocarbonyl]-4-(hetero) arylpiperazine derivatives published in Bioorg. Med. Chem. 2010, 18, 7966, we further explored the synthesis o

Full text of DOI:10.1016/j.bmc.2011.12.026

Bioorganic & Medicinal Chemistry 20 (2012) 1303–1309
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, anticancer activity and pharmacokinetic analysis of 1-[(substituted
2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives
Young Bok Lee a, , Young-Dae Gong b, , Deog Joong Kim a, Chang-Ho Ahn a, Jae-Yang Kong c,
⇑
⇑
Nam-Sook Kang d
a Rexahn Pharmaceuticals, Inc., Rockville, MD 20850, USA
b Center for Innovative Drug-like Library Research, Dongguk University, Pil-dong 3-ga, Jung-gu, Seoul 100-715, Republic of Korea
c College of Pharmacy, Gyeongsang National University, 816-15 Jinju-daero, Jinju 660-751, Republic of Korea
d Graduate School of New Drug Discovery and Development, Chungnam National University, Daehakno 99, Yuseong-gu, Daejeon 305-764, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the anticancer activity of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alk-
oxyquinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives published in Bioorg. Med. Chem.
2010, 18, 7966, we further explored the synthesis of 7 or 8-substituted quinoxalinyl piperazine deriva-
tives. From in vitro studies of the newly synthesized compounds using human cancer cell lines, we iden-
tified some of the 8-substituted compounds, for example 6p, 6q and 6r, which inhibited the proliferation
of various human cancer cells at nanomolar concentrations. Compound 6r, in particular, showed the low-
est IC50 values, ranging from 6.1 to 17 nM, in inhibition of the growth of cancer cells, which is better than
compound 6k (compound 25 in the reference cited above). In order to select and develop a leading com-
pound among the quinoxaline compounds with substitutions on positions 5, 6, 7 or 8, the compounds
comparable to compound 6k in in vitro cancer cell growth inhibition were chosen and their pharmaco-
kinetic properties were evaluated in rats. In these studies, compound 6k showed the highest oral bio-
availability of 83.4%, and compounds 6j and 6q followed, with 77.8% and 57.6%, respectively. From the
results of in vitro growth inhibitory activities and the pharmacokinetic study, compound 6k is suggested
for further development as an orally deliverable anticancer drug.
Received 11 October 2011
Revised 5 December 2011
Accepted 10 December 2011
Available online 27 December 2011
Keywords:
Pharmacokinetics
Bioavailability
Oral
2-Alkoxyquinoxalin-(hetero)arylpiperazine
Anti-cancer
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Recently we reported the anticancer activities of 5 or 6-substi-
tuted quinoxaline compounds containing piperazine.18 Compound
Since AG1295, which has a relatively simple quinoxaline struc-
ture, has been shown to selectively block tyrosine kinases such as
PDGFR,1–3 the quinoxaline pharmacophore has become an attractive
scaffold for thedevelopmentof active chemotherapeuticagents. Asa
result of such efforts, the pharmacological data of several anticancer
drugs containing a quinoxaline ring has been reported in many jour-
nals.4–9 For example, the quinoxaline anticancer drugs XK469 (2-(4-
((7-chloro-2-quinoxalinyl)oxy)phenoxy)propionic acid) and CQS
(chloroquinoxaline sulfonamide) were found to have activity
against solid tumors10–12 and have been studied in clinical tri-
als.13–15
6k, 1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-
3-yl)aminocarbonyl] piperazine, especially showed strong inhibi-
tion of the growth of human cancer cells (IC50, 11–21 nM), and also
induced cell cycle arrest at G2/M and apoptosis in cancer cells by
the downregulation of Bcl-2 protein level.18 This compound also
significantly inhibited the growth of drug-resistant cancer cells,
and has shown potential use in combination therapy with known
anticancer drugs such as palcitaxel, doxorubicin, gemcitabine, 5-
FU and cisplatin.18
The strong anticancer activity shown in our previous study by
compound 6k, which has 6-fluoro on the quinoxaline ring and
3,5-dimethoxyphenyl piperazine, inspired us to investigate other
substituted quinoxaline compounds containing the piperazine
group to find an even stronger compound. In view of the previous
rationale that was more focused on the quinoxaline derivatives
substituted on the 5 or 6 position, and containing a piperazine
group, and in continuation of an ongoing program aiming to find
a new lead with potential anticancer activity, a series of novel 7-
or 8-substituted compounds (substituted at R3 or R4 of compound
6 in Scheme 1) at the quinoxaline ring of 1-[(2-alkoxyquinoxalin-
3-yl)aminocarbonyl-4-(hetero)arylpiperazine derivatives (shown
There is also a report suggesting unsubstituted quinoxaline
compounds containing piperazine as candidates for anticancer
agents; one of the unsubstituted quinoxalinyl-piperazine com-
pounds was shown to exhibit microtubule-inhibiting activity.16,17
⇑
Corresponding authors. Tel.: +1 240 328 6905 (Y.B.L.); +82 2 2260 3206 (Y.-
D.G.).
E-mail addresses: leeyb@rexahn.com (Y.B. Lee), ydgong@dongguk.edu (Y.-D.
Gong).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.026

1304
Y. B. Lee et al. / Bioorg. Med. Chem. 20 (2012) 1303–1309
R1
R1
R4
R1
R4
R2
R3
N
NH2
Cl
R2
R3
N
Cl
Cl
R2
R3
N
NH2
NH3 (g)
NaOMe
THF,rt
DMSO
N
N
1
N
OMe
rt or heat
R4
2
3
R5
R5
R6
O
OEt
HN
N
R1
R4
R1
R4
H
5
N
N
R2
R3
N
N
N
R2
R3
N
NH
R6
ClCO2Et, Pyr
DCM,rt
DBU
O
THF, 60 °C
OMe
N
OMe
4
6
Scheme 1.
methoxide afforded 3-amino-2-methoxy derivatives through the
formation of 3-amino-2-chloroquinoxaline derivatives. The final
quinoxalinyl-piperazine compounds were prepared from the reac-
tions of carbamate derivatives of 3-amino-2-methoxy intermedi-
ates with arylpiperazines.
Table 1
Structures of 1-[(substituted 2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(het-
ero)arylpiperazine derivatives
R5
R1
H
R2
R3
N
N
N
N
N
3. Results
O
R6
OMe
3.1. In vitro cell growth inhibition
R4
The in vitro cytotoxicity experiments were performed with
the synthetic 1-[(7 or 8-substituted alkoxyquinoxalinyl)amino-
carbonyl]-4-(hetero)arylpiperazine derivatives (Table 1) in hu-
man cancer cell lines using a colorimetric sulphorhodamine B
(SRB) assay.19 The IC50 values (Table 2) varied significantly and
were dependent on the substitution on the quinoxaline ring
but also on substituents on the phenyl ring. The data from our
previous paper indicated that compounds containing methyl or
methoxy group at the C-5 position of the quinoxaline ring pos-
sessed stronger anticancer activities with 3,5-dimethoxyphenyl-
piperazine group than 3,5-dimethylphenyl-piperazine group.
Compounds with the 3,5-dimethoxyphenyl-piperazine group to-
gether with 6-methyl or 6-F substitution on the quinoxaline ring
showed more potent anticancer activity than those with the 3,5-
dimethylphenyl-piperazine group. This stronger activity of com-
pounds with 3,5-dimethoxyphenyl-piperazine group also applied
to the compounds containing methyl or methoxy at the C-8 po-
sition of the quinoxaline ring (see Table 2). In the case of the
compounds with substitutions at the C-7 position, the compound
6l, which contains a methyl group at the C-7 position of the
quinoxaline ring, displayed stronger activity than the equivalent
C-7 methoxy compound (6m). However, their IC50 values are
higher than those of the 6- or 8-substituted compounds. Among
the compounds tested, compound 6r, which has both a methoxy
group at the C-8 position of the quinoxaline ring and the 3,5-
dimethoxyphenyl-piperazine group, was found to be the most
active compound (IC50 from 6.1 to 17 nM in the cell lines tested),
and followed by compound 6k.
Compound
R1
R2
R3
R4
R5 and R6
6a*
6b*
6c*
6d*
6e*
6f*
6g*
6h*
6i*
6j*
6k*
6l
6m
6n
6o
6p
6q
Me
Me
OMe
OMe
F
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
OMe
F
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
OMe
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
OMe
OMe
Me
OMe
Me
OMe
Me
OMe
Me
OMe
Me
Me
OMe
Me
Me
Me
Me
OMe
Me
OMe
H
H
6r
H
*
From Ref. 18.
in Table 1) were synthesized and tested against various types of
human cancer cell lines, as a second optimization process. We se-
lected several active compounds such as 6p, 6q and 6r, and con-
ducted
a
pharmacokinetic study in rats, together with
representative 5 or 6-substituted compounds including compound
6k, to develop a promising and orally deliverable anticancer drug.
2. Chemistry
3.2. The prediction of physicochemical properties
1-[(7 or 8-Substituted alkoxyquinoxalinyl)aminocarbonyl]-4-
(hetero)arylpiperazine derivatives were regiospecifically synthe-
sized from the corresponding 2,3-dichloroquinoxaline starting
materials as described in Scheme 1 by application of the same
reaction sequence as that for the synthesis of 6-substituted alkoxy-
quinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives
described earlier.18
We also checked the physicochemical properties of the com-
pounds listed in Table 1 using, to save time and cost, an in silico
prediction method for pharmacokinetics, as shown in Table 3. As
discussed by Lipinski, molecular properties are closely related to
the oral bioavailability of a drug.20 In particular, membrane perme-
ation is considered as a common requirement for oral bioavailabil-
ity. It is well known that membrane permeability shows a good
correlation with CLogP.21 Hann et al. suggested lead-likeness as
Sequential substitution reactions by treatment of 2,3-dichloro-
quinoxaline starting materials with ammonia followed by

Y. B. Lee et al. / Bioorg. Med. Chem. 20 (2012) 1303–1309
1305
Table 3
The physicochemical properties of quinoxaline-piperazine compounds
Compound
CLogPa
Solubility (S)b
PSAc
BBBd
hERGe
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
4.75
3.78
4.65
3.68
4.43
3.46
4.75
3.78
4.65
4.43
3.46
4.75
4.65
4.43
4.75
3.78
4.65
3.68
ꢀ5.699
ꢀ4.732
ꢀ5.19
59.338
77.198
68.268
86.128
59.338
77.198
59.338
77.198
68.268
59.338
77.198
59.338
68.268
59.338
59.338
77.198
68.268
86.128
0.219
Ok
Ok
Weak
Ok
Ok
Ok
Ok
Ok
Ok
Ok
Ok
Ok
Ok
Ok
Ok
Ok
Weak
Ok
ꢀ0.375
ꢀ0.078
ꢀ0.671
0.132
ꢀ4.207
ꢀ5.409
ꢀ4.442
ꢀ5.684
ꢀ4.716
ꢀ5.179
ꢀ5.447
ꢀ4.479
ꢀ5.676
ꢀ5.162
ꢀ5.439
ꢀ5.691
ꢀ4.723
ꢀ5.173
ꢀ4.188
ꢀ0.461
0.219
ꢀ0.375
ꢀ0.078
0.132
ꢀ0.461
0.219
ꢀ0.078
0.132
0.219
ꢀ0.375
ꢀ0.078
ꢀ0.671
a
These values are calculated using Tripos Sybyl-X1.3.23
Aqueous solubility; S < ꢀ8.0, extremely low; ꢀ8.0 < S < ꢀ6.0, very low, possible;
ꢀ6.0 < S < ꢀ4.0, low; ꢀ4.0 < S < ꢀ2.0, good; ꢀ2.0 < S < 0.0, optimal; S > 0.0, too
soluble, using Accelrys ADMET module.24
Polar surface area for each molecule using a 2D structure.
The predicted blood–brain barrier using Accelrys ADMET module; BBB > 0.0,
high penetrate; ꢀ0.52 < BBB < 0, medium penetrate; BBB < ꢀ0.52, low
penetrate.
b
c
d
e
The hERG inhibition predicted from Ref. 25.
CLogP values less than 4.2, and ꢀ5 < solubility < 0.5.22 In addi-
tion, we calculated BBB (blood–brain barrier) and hERG (human
ether-a-go-go related gene) inhibition. All compounds showed
stable values for BBB and hERG inhibition although 6c and 6q
gave weak inhibition—about 30–40% at 10 lM.
3.3. Pharmacokinetic analysis
Shown in Figure 1 are the serum concentration versus time
curves of the selected compounds in Sprague–Dawley male rats
following a single dose given iv or po. The compounds were mea-
sured in the plasma for 24 h post-dosing. Table 4 also shows that
there were marked differences among the tested compounds in
some pharmacokinetic parameters, including the maximal serum
concentrations (Cmax), the half-lives (T1/2), the systemic clearance
(CL), the area under the curves (AUC) and the oral bioavailability
(F). Interestingly, among the six compounds, compound 6r, which
has the strongest anticancer activity in inhibition of cell growth,
showed a relatively low level of oral bioavailability (32.4% F).
Compounds 6b and 6p also showed a low level of oral bioavail-
ability. On the other hand, compounds 6j and 6k displayed a high
level of oral bioavailability, 77.8% and 83.4%, respectively. Com-
pound 6k showed the second best anti-cancer activities against
the tested cancer cell lines but showed the highest oral bioavail-
ability (F), indicating satisfactory plasma levels after oral admin-
istration. The systemic clearance (CL) of compound 6k was 0.61 l/
hr/kg in male rats, which corresponds to only about 18.9% of the
hepatic blood flow of the rat. The apparent volume of distribution
at a steady state (Vss) in male rats was estimated to be 4.5 l/kg,
indicating that compound 6k is likely to be readily distributed
out of the vascular space. The terminal half-life (T1/2) in male rats
was 6.5 and 7.9 h after iv or po administration, respectively.
4. Discussion
We reported previously that compound 6k was characterized
as a G2/M-specific cell cycle inhibitor, with strong inhibition of

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Y. B. Lee et al. / Bioorg. Med. Chem. 20 (2012) 1303–1309
6j
6b
10.000
1.000
0.100
0.010
0.001
i.v. (5 mg/kg)
i.v. (5 mg/kg)
p.o.(5 mg/kg)
p.o.(10mg/kg)
1.000
0.100
0.010
0.001
0
4
8
12 16 20 24 28
Time (hr)
0
4
8
12 16 20 24 28
Time (hr)
6k
6p
1.00
0.10
0.01
i.v. (1 mg/kg)
i.v. (10 mg/kg)
p.o.(10 mg/kg)
10.00
1.00
0.10
0.01
p.o.(10mg/kg)
0
4
8
12
16
20
24
28
0
4
8
12 16 20 24 28
Time (hr)
Time (hr)
6q
6r
i.v. (2.5 mg/kg)
p.o.(10mg/kg)
1.0000
0.1000
0.0100
0.0010
0.0001
i.v. (2 mg/kg)
p.o.(5 mg/kg)
10.00
1.00
0.10
0.01
0
4
8
12 16 20 24 28
Time (hr)
0
4
8
12
16
20
24
28
Time (hr)
Figure 1. Plasma concentration versus time plots for compounds 6b, 6j, 6k, 6p, 6q and 6r after a single iv (s) or oral (d) administration to male Sprague–Dawley rats.
Table 4
Pharmacokinetic parameters of compounds 6b, 6j, 6k, 6p, 6q and 6r after an iv and oral administration to SD male rats (n = 3).
Parameters
6b
6j
6k
6p
6q
6r
iv
Cmax
(
lg/mL)
—
—
—
—
—
—
Tmax (h)
—
—
—
—
—
—
T1/2 (h)
AUC0–24h (ug h/mL)
AUC0–1 (lg h/mL)
Cl (L/kg h)
Vss (L/Kg)
MRT (h)
po
8.1 0.93
2.2 0.29
2.5 0.29
2.0 0.25
22.8 3.08
7.8 0.31
5.6 1.37
1.4 0.23
1.5 0.29
3.4 0.61
18.8 2.53
4.7 0.97
6.5 2.37
19.2 10.98
20.6 11.48
0.61 0.365
4.5 1.88
6.0 0.99
17.8 4.39
0.78 0.285
1.24 0.391
0.86 0.262
22.2 11.24
9.4 0.13
2.7 0.99
1.31 0.335
1.36 0.304
1.89 0.395
6.23 1.759
2.9 1.01
7.4 1.47
3.22 0.173
3.76 0.689
0.54 0.090
4.9 1.71
6.2 2.83
Cmax
Tmax (h)
T1/2 (h)
AUC0–24h
AUC0–1 (lg h/mL)
Cl (L/kg h)
Vss (L/kg)
MRT (h)
F (%)
(
l
g/mL)
0.09 0.024
6.7 1.16
10.7 0.84
1.2 0.45
1.6 0.64
—
0.09 0.098
6.0 2.0
6.7 1.37
1.12 0.872
1.27 1.044
—
1.2 0.37
8.7 1.16
7.9 1.72 (n = 2)
15.9 4.15
17.5 4.85 (n = 2)
—
0.14 0.037
7.3 2.31
24.1 9.63 (n = 2)
1.9 0.45
4.5 0.001 (n = 2)
—
0.21 0.064
9.3 1.16
6.5 (n = 1)
3.01 1.028
4.07 (n = 1)
—
0.17 0.034
4.7 2.31
10.9 2.85 (n = 2)
2.61 1.008
2.72 0.106 (n = 2)
—
(l
g h/mL)
—
—
—
—
—
—
9.6 0.50
27.3
8.6 0.75
77.8
9.6 0.18
83.4
11.2 0.95
25.6
8.9 0.47
57.6
10.8 1.92
32.4
Cmax: maximum serum concentration, Tmax: time to maximum concentration, T1/2: half-life, AUC: area under the curve, CL: systemic clearance, Vss: volume of distribution at a
steady state, MRT: mean residence time, F: bioavailability.
the growth of human cancer cells and also induction of apoptosis
by the downregulation of Bcl-2 protein level.18 Although
compound 6k was the strongest inhibitor in the previous study,
it belongs to a small group of 5 or 6-substituted quinoxaline com-

Y. B. Lee et al. / Bioorg. Med. Chem. 20 (2012) 1303–1309
1307
pounds and thus it led us to explore other substituted quinoxaline
compounds containing the piperazine group. In the current study,
we designed and synthesized several compounds substituted at
the 7- or 8-position of the quinoxaline ring (shown in Table 1),
and tested them against human cancer cells. In an in vitro cell
study (Table 2), we found that compounds with a 3,5-dimethoxy-
phenyl-piperazine group as well as 8-methyl (6p) or 8-methoxy
(6r) substitution on the quinoxaline ring displayed similar or bet-
ter anti-cancer activity as compound 6k. Compound 6r especially
showed the possibility of being a new leading compound, as it
had the lowest IC50 range. In order to select the most reliable can-
didate compounds in terms of drug development, we are required
to select some representative compounds from each of the substi-
tution groups, that is, C-5 or C-6 or C-8 substituted compounds.
Compound 6b with C-5 substitution, compounds 6j and 6k with
C-6 substitution and compounds 6p, 6q and 6r with C-8 substitu-
tion were selected based on their both in vitro activities in inhibi-
tion of cell growth and the predicted physicochemical properties
and the pharmacokinetic analyses performed in rats. Compounds
substituted at the C-7 position were excluded from the pharmaco-
kinetic study because they had relatively less activity in cell
growth inhibition. From the PK data (Table 4), it is obvious that
substitution on the phenyl-piperazine group as well as at the quin-
oxaline ring has a direct impact on T1/2, F and AUC of the com-
pounds. Among the six compounds for which there is
pharmacokinetic data, the lead compound 6k exhibited good prop-
erties suitable for a drug, such as oral bioavailability (F; 83%), half-
life (T1/2; 7.9 h in oral), and plasma levels. CLogP of compound 6k is
about 3.46 and water solubility is low (Table 3), which will require
a formulation study to achieve efficient oral delivery of the com-
pound. Oral administration of lipophilic drugs has been challenged
during the past 20 years and the development of orally available
anticancer drugs has made great progress using several platform
technologies such as nanosize formulation,26–28 lipid based deliv-
ery systems29,30 or polymeric micelles.31 More than forty five oral
agents have been approved for the treatment of cancer in US32
and it is expected that more novel oral anticancer drugs will be
in the market in the near future. So from the viewpoints of a potent
anticancer activity and good pharmacokinetic properties, com-
pound 6k could be a promising orally available anticancer drug
and guarantees further ADME and toxicological studies.
60 F-254 thin layer plates. Flash column chromatography was car-
ried out on Merck Silica Gel 60 (230–400 mesh). The crude prod-
ucts 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 300 or 500 MHz
NMR instrument. 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
C
18 column, 4.6 mm ꢁ 100 mm; PDA detector at 200–400 nm; gra-
dient, 5–95% CH3CN/H2O).
6.1.2. Compound synthesis
The analytical data of compounds 6a–6k and all of the synthetic
procedure of the intermediate compounds 1–4 can be found in our
previous paper.18 Compounds 6m–6r were synthesized by the
same way with the preparation of the compound 6l.
6.1.2.1. Representative synthetic procedure of 1-(3,5-dimethyl-
phenyl)-4-[(3-methoxy-6-methylquinoxalin-2-yl)amino
car-
bonyl] piperazine (6l).
To a stirred solution of ethyl-(3-
methoxy-6methylquinoxalin-2-yl)carbamate
(4l)
(9.7 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 ace-
tate = 2:1) to yield the desired compound 6l (14.2 mg, 95%): mp
132 °C; 1H NMR (300 MHz, CDCl3): d 2.29 (s, 6H), 2.50 (s, 3H),
3.18–3.29 (m, 4H), 3.73–3.80 (m, 4H), 4.12 (s, 3H), 6.58 (s, 3H),
7.31 (d, J = 8.4 Hz, 1H), 7.43 (s, 1H), 7.55 (s, 1H), 7.72 (d,
J = 8.4 Hz, 1H); 13C NMR (125 MHz, CDCl3); d 153.6, 151.6, 151.3,
148.8, 139.0, 137.4, 137.0, 135.9, 128.9, 127.0, 126.1, 122.6,
114.8, 55.4, 49.6, 45.3, 21.8, 21.6; LC-MS (ESI) m/z 406.20
([M+H]+); MS–MS (EI) m/z M+ for C23H27N5O2 calcd 405.220, found
406.059 (M+1).
6.1.2.2. 1-[(3,6-Dimethoxyquinoxalin-2-yl)aminocarbonyl]-4-
(3,5-dimethylphenyl)piperazine (6m).
In 90% yield; mp
142–145 °C; 1H NMR (300 MHz, CDCl3): d 2.29 (s, 6H), 3.26 (s,
4H), 3.75 (s, 4H), 3.90 (s, 3H), 4.14 (s, 3H), 6.59 (s, 3H), 6.99–7.20
(m, 3H), 7.73–7.75 (m, 1H); LC-MS (ESI) m/z 422.20 ([M+H]+).
6.1.2.3.
1-[(6-Fluoro-3-methoxyquinoxalin-2-yl)aminocar-
5. Conclusion
bonyl]-4-(3,5-dimethylphenyl)piperazine
(6n).
In
61%
yield; mp 148 °C; 1H NMR (500 MHz, CDCl3) d 2.29 (s, 6H), 3.24
(m, 4H), 3.74 (t, J = 8 Hz, 4H), 4.15 (s, 3H), 6.56(d, J = 19 Hz, 3H),
7.24 (m, 1H), 7.40 (dd, J = 4.5, 11 Hz, 1H), 7.80 (dd, J = 9.6, 5.5 Hz,
1H), 14,47 (br s, 1H); LC–MS (ESI) m/z 410.20 ([M+H]+).
Inconclusion, we synthesizeda series of5-, 6-, 7- or 8-substituted
quinoxalinyl-piperazine compounds in the previous and present
studiesandtestedthemagainsthumancancercelllinesfortheselec-
tion of a final lead compound(s). Actually, we found that compounds
6p or 6r displayed similar or better anticancer activity compared to
compound 6k in inhibition of cell growth in vitro. However these
second tier compounds (6p and 6r) do not have better pharmacoki-
netic properties compared to the primary compound 6k. Finally,
among representative compounds that were substituted at the C-
5-, C-6-, C-7-, or C-8 position on the quinoxaline phenyl ring, our
in vitro cell line and pharmacokinetic studies suggest that com-
pound 6k has a strong anti-cancer efficacy and desirable pharmaco-
kinetic properties. These data support further development of
compound 6k to achieve an orally available anticancer drug.
6.1.2.4.
1-(3,5-Dimethylphenyl)-4-[(3-methoxy-5-methylqui-
noxalin-2-yl)aminocarbonyl] piperazine (6o).
In 81% yield;
mp 201 °C; 1H NMR (500 MHz, CDCl3) d 2.29 (s, 6H), 2.56 (s, 3H),
3.21–3.23 (m, 4H), 3.74–4.05 (m, 3H), 4.15 (s, 4H), 6.57–6.59 (m,
3H), 7.25–7.30 (m, 3H), 7.54 (br s, 1H); LC–MS (ESI) m/z 406.00
([M+H]+).
6.1.2.5. 1-(3,5-Dimethoxyphenyl)-4-[(3-methoxy-5-methylqui-
noxalin-2-yl)aminocarbonyl] piperazine (6p).
In 61% yield;
mp 172 °C; 1H NMR (500 MHz, CDCl3) d 2.56 (s, 3H), 3.23–3.25
(m, 4H), 3.79 (s, 6H), 3.92–3.93 (m, 4H), 4.14 (s, 3H), 6.05–6.06
(m, 1H), 6.12 (d, J = 1.8 Hz, 2H), 7.26–7.28 (m, 3H), 7.52–7.54 (m,
1H); LC–MS (ESI) m/z 437.97 ([M+H]+).
6. Experimental
6.1. Chemistry
6.1.1. General
6.1.2.6. 1-[(3,5-Dimethoxyquinoxalin-2-yl)aminocarbonyl]-4-
All chemicals were reagent grade and used as purchased.
Reactions were monitored by TLC analysis using Merck Silica Gel
(3,5-dimethylphenyl)piperazine (6q).
In 66% yield; mp
130 °C; 1H NMR (300 MHz, CDCl3) d 2.26–2.29 (m, 6H), 3.18–3.30

1308
Y. B. Lee et al. / Bioorg. Med. Chem. 20 (2012) 1303–1309
(m, 4H), 3.74–3.80 (m, 3H), 4.01–4.04 (m, 3H), 4.10–4.22 (m, 7H),
6.59 (s, 2H), 6.78–6.84 (m, 1H), 6.93–6.96 (m, 1H), 7.26–7.45 (m,
3H); LC–MS (ESI) m/z 421.99 ([M+H]+).
6.4. Statistical analysis
Analytical data were processed using Analyst software Version
1.4.1. (Applied Biosystems, Concord, Canada). Pharmacokinetic
parameters were obtained by standard non-compartmental analy-
sis of the plasma concentration-time profiles using PK Solutions 2.0
(Summit Research Services, Montrose, CO, USA).
6.1.2.7. 1-(3,5-Dimethoxyphenyl)-4-[(3,5-dimethoxyquinoxalin-
2-yl)aminocarbonyl]piperazine (6r).
In 81% yield; mp
102 °C; 1H NMR (300 MHz, CDCl3) d 3.20–3.32 (m, 4H), 3.74–3.81
(m, 9H), 4.01–4.04 (m, 3H), 4.12 (br s, 1H), 4.19–4.22 (m, 3H),
6.06–6.07 (m, 1H), 6.11–6.12 (m, 2H), 6.79–6.84 (m, 1H), 6.93–
6.96 (m, 1H), 7.26–7.29 (m, 1H), 7.41–7.44 (m, 1H); LC–MS (ESI)
m/z 453.96 ([M+H]+).
Acknowledgments
This research was supported by Rexahn Pharmaceuticals, Inc. in
USA, and the National R&D Program for Cancer Control (Grant no.
1020050), and the National Research Foundation of Korea (2010-
0004128) funded by the Ministry of Education, Science and Tech-
nology, and by the Ministry of Knowledge Economy, Korea. The
authors extend their appreciation to Dr. Edith C. Wolff for critical
review of the manuscript.
6.2. Biology
6.2.1. Growth of cancer cells
Human cancer cell lines were obtained from the following
sources: OVCAR-3, Hs578T, HeLa, PC3, HepG2, A549, PANC-1, SK-
MEL-28 and HCT116 from the American Type Culture Collection
(Manassas, VA); MKN-45 from DSMZ (German Collection of Micro-
organisms and Cell Cultures), Braunschweig, Germany; UMRC2
(kidney) from the US National Cancer Institute (Bethesda, MD,
USA). All cell lines, except Hs578T, HCT116, UMRC-2 and PANC-1,
were grown in RPMI1640 medium (Invitrogen, Carlsbad, CA) sup-
plemented with 10% fetal bovine serum (FBS), 1 mM sodium pyru-
vate, 10 mM HEPES, and 100 units/mL penicillin–streptomycin (P/
S). Hs578T, HCT116, UMRC-2 and PANC-1 cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carls-
bad, CA) supplemented with 10% FBS, 10 mM HEPES, 100 units/
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.026.
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