Discovery of indeno[1,2-b]quinoxaline derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2015.11.031

We have synthesized certain indeno[1,2-b]quinoxaline derivatives for antiproliferative evaluation. Among them, 11-{[3-(dimethylamino)propoxy]imino}-N-[3-(dimethylamino) propyl]-11H-indeno[1,2-b]quinoxaline-6-carboxamide (10a) was active against the growth of MDA-MB231, PC-3, and Huh-7 with IC₅₀ values of 0.87 (selectivity index, SI = 36.22), 0.82 (SI = 38.43), and 0.64 μM (SI = 49.23) respectively. Compound 10a was inactive against the growth of normal human fetal lung fibroblast cell line (MRC-5) with an IC₅₀ value of 31.51 μM. Its analogs, 10b and 10c, were also active against the growth of MB231, PC-3, and Huh-7 with IC₅₀ values of <1.0 μM in each case. Our results have also indicated compounds 10a-10c exhibited comparable inhibitory activities against topo I and topo II with the positive compound 2 at a concentration of 10 μM. Mechanism studies indicated that compound 10a induced cell cycle arrest at S phase via activation of caspase-3, -7 and an increase in the protein expression of Bad and Bax but a decrease in expression of Bcl-2 and PARP, which consequently cause cell death. In addition, compound 10a attenuated the levels of phosphorylated Src, Akt-1, and Akt-2 protein levels but did not affect the total protein expression of Akt. We have also implanted human hepatocellular carcinoma cells into the yolk sac of zebrafish larvae and incubated larvae with various concentrations of 10a. Our results of the zebrafish xenograft assay confirmed the anti-tumor effect of 10a in vivo.

Full text of DOI:10.1016/j.ejmech.2015.11.031

Accepted Manuscript
Discovery of Indeno[1,2-b]quinoxaline Derivatives as Potential Anticancer Agents
Chih-Hua Tseng, You-Ren Chen, Cherng-Chyi Tzeng, Wangta Liu, Chon-Kit Chou,
Chien-Chih Chiu, Yeh-Long Chen
PII:
S0223-5234(15)30368-8
10.1016/j.ejmech.2015.11.031
EJMECH 8221
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 May 2015
Revised Date: 6 November 2015
Accepted Date: 19 November 2015
Please cite this article as: C.-H. Tseng, Y.-R. Chen, C.-C. Tzeng, W. Liu, C.-K. Chou, C.-C. Chiu, Y.-
L. Chen, Discovery of Indeno[1,2-b]quinoxaline Derivatives as Potential Anticancer Agents, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.11.031.
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ACCEPTED MANUSCRIPT
Discovery of Indeno[1,2-b]quinoxaline Derivatives as Potential Anticancer
Agents
Chih-Hua Tseng, You-Ren Chen, Cherng-Chyi Tzeng, Wangta Liu, Chon-Kit Chou, Chien-Chih Chiu,
and Yeh-Long Chen
O
N
N
N
NMe2
IC = 0.87 µM (MDA-MB231)
50
0.82 µM (PC-3)
.64 µM (Huh-7)
> 10 µM (MRC-5)
0
Me N
N
O
2
H
10a
Compound 10a induced cell cycle arrest at S phase via activation of caspase-3, -7 and
increase in the expression of Bad and Bax but decrease in Bcl-2 and PARP.

ACCEPTED MANUSCRIPT
Discovery of Indeno[1,2-b]quinoxaline Derivatives as Potential Anticancer Agents
1
,2,3
4
2,3,4
5
5
Chih-Hua Tseng,
You-Ren Chen, Cherng-Chyi Tzeng,
Wangta Liu, Chon-Kit Chou,
5,6*
2,3,4*
Chien-Chih Chiu, and Yeh-Long Chen
1
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City
8
07, Taiwan
2
Research Center for Natural Products & Drug Development, Kaohsiung Medical University,
Kaohsiung City 807, Taiwan
3
Center for Infectious Disease and Cancer Research, Kaohsiung Medical University,
Kaohsiung City 807, Taiwan
4
Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung
City 807, Taiwan
5
Department of Biotechnology, College of Life Science, Kaohsiung Medical University,
Kaohsiung City 807, Taiwan
6
Research Center for Environment Medicine, Kaohsiung Medical University Hospital,
Kaohsiung Medical University, Kaohsiung City 807, Taiwan
*
2
7
To whom correspondence should be addressed. For Y.L. Chen: Tel. (886) 7-3121101 ext
684; Fax. (886) 7-3125339; e-mail: yeloch@kmu.edu.tw For C.C. Chiu: Tel. (886)
.
-3121101 ext 2368; e-mail: cchiu@kmu.edu.tw
.
1

ACCEPTED MANUSCRIPT
Abstract
We have synthesized certain indeno[1,2-b]quinoxaline derivatives for antiproliferative
evaluation. Among them, 11-{[3-(dimethylamino)propoxy]imino}-N-[3-(dimethylamino)
propyl]-11H-indeno[1,2-b]quinoxaline-6-carboxamide (10a) was active against the growth of
MDA-MB231, PC-3, and Huh-7 with IC values of 0.87 (selectivity index, SI = 36.22), 0.82
5
0
(SI = 38.43), and 0.64 µM (SI = 49.23) respectively. Compound 10a was inactive against the
growth of normal human fetal lung fibroblast cell line (MRC-5) with an IC value of 31.51
50
µM. Its analogs, 10b and 10c, were also active against the growth of MB231, PC-3, and
Huh-7 with IC values of < 1.0 µM in each case. Our results have also indicated compounds
5
0
1
0a – 10c exhibited comparable inhibitory activities against topo I and topo II with the
positive compound 2 at a concentration of 10 µM. Mechanism studies indicated that
compound 10a induced cell cycle arrest at S phase via activation of caspase-3, -7 and an
increase in the protein expression of Bad and Bax but a decrease in expression of Bcl-2 and
PARP, which consequently cause cell death. In addition, compound 10a attenuated the levels
of phosphorylated Src, Akt-1, and Akt-2 protein levels but did not affect the total protein
expression of Akt. We have also implanted human hepatocellular carcinoma cells into the
yolk sac of zebrafish larvae and incubated larvae with various concentrations of 10a. Our
results of the zebrafish xenograft assay confirmed the anti-tumor effect of 10a in vivo.
Key words: Indeno[1,2-b]quinoxaline derivatives; Zebrafish xenograft assay, Anticancer
agents; Cell cycle; Apoptosis; Antiproliferative activity.
2

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1
. Introduction
A number of polycyclic nitrogen heterocycles, for example, ellipticine [1-3], batracylin
4,5], and anthracycline antibiotics are of interest as anticancer agents whose action is based
[
mainly on DNA damage such as DNA intercalation and the inhibition of topoisomerase II.
Ellipticine (5, 11-dimethyl-6H-pyrido[4,3-b]carbazole) is a potent antitumor alkaloid bearing
a tetracyclic ring system and was first isolated from the evergreen tree Ochrosia elliptica
Labill. Batracylin (8-aminoisoindolo[1,2-b]quinazolin-12(10H)-one) is a heterocyclic amine
that exhibits antitumor activity in a number of in vivo and in vitro models. However, the most
noticeable examples of DNA-intercalating agents are anthracycline antibiotics such as
doxorubicin and daunorubicin which possess a tetracyclic system where three of the rings are
coplanar.
The versatile quinoxaline ring constitutes a large number of potential bioactive agents
which possess broad spectrum of pharmacological activities such as anti-infective [6-8],
anti-inflammatory [9,10], and anticancer activities [11-13]. Recently, we have synthesized
and evaluated antiproliferative activities of certain DNA intercalating agents with the
tetracyclic indeno[1,2-c]quinoline scaffold [14-20]. Among them, 9-methoxy-
6
-(piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one O-3-aminopropyl oxime (1) and
E)-6-hydroxy-9-methoxy-11H-indeno[1,2-c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl
oxime (2) exhibited IC value of 0.64 and 0.89 µM respectively against the growth A549,
(
5
0
which was more active than camptothecin and topotecan. 9-Methoxy-6-{4-[(oxiran-2-yl)
methyl]piperazin-1-yl}-11H-indeno[1,2-c]quinolin-11-one O-(oxiran-2-yl)methyl oxime (3)
was also found to exhibit strong antiproliferative activities aginst the growth of Hela, SKHep,
MDA-MB-231, and H1299 cells with an IC50 value of 0.54, 0.99, 0.79, and 1.18 µM
respectively [20]. In continuation of our search for potentential anticancer agents, the present
study describes the preparation and antiproliferative activities of certain tetracyclic
indenoquinoxaline derivatives (Figure 1) whose structures can be considered as aza analogs
of indenoquinoline derivatives.
3

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<
Insert Figure 1 here >
2
. Chemistry
Treatment of the known 11H-Indeno[1,2-b]quinoxalin-11-one (4a) [21] with NH OH
2
gave 11H-Indeno[1,2-b]quinoxalin-11-one oxime (5a) in 94% yield. Its aminoalkyl
derivatives 6a, 7a – 7c were obtained as hydrochloride salts by the reaction of 4a with
various aminoalkoxyamines followed by the treatment of 3N HCL as described in Scheme 1.
Preparation of 6-carboxamide derivatives is outlined in Scheme 2. Reaction of
1
1H-Indeno[1,2-b]quinoxalin-11-one-6-carboxylic
acid
(4c)
[21]
and
1
,1'-carbonyldiimidazole gave the imidazole intermediate 8 which was treated with various
amines to afford C-6 amide derivatives 9a - 9d. Treatment of compounds 9a - 9d with
various aminoalkoxyamine gave C-11 aminoalkoxyimino derivatives 10a - 10d in good
overall yields.
<
Insert Scheme 1 and 2 here >
The preparation of di-aminoalkoxyimino derivatives 14 and 15 are described in Scheme 3.
Reaction of 4b with NBS gave 6-(bromomethyl)-11H-indeno[1,2-b]quinoxalin-11-one (11)
which was then oxidized with MnO2 to give 11-oxo-11H-indeno[1,2-b]quinoxaline-
6
-carbaldehyde (12) in a high overall yield. Treatment of 12 with NH OH or
2
aminoalkoxyamines gave di-hydroxyimine 13 or di-aminoalkoxyimino derivatives 14 and 15
respectively. Compound 11 was also treated with various secondary amines to give C-6
aminomethyl substituted intermediates 16a – 16e respectively which was converted to C-11
aminoalkoxy derivatives 17a – 17e in fairly good overall yields as shown in Scheme 4.
<
Insert Scheme 3 and 4 here >
3
3
. Results and Discussion.
.1. Antiproliferative Activity and DNA Unwinding.
All the newly synthesized compounds were evaluated in vitro against four cancer cell
lines (MDA-MB231, H1299, PC-3 and Huh-7) using XTT assay [22]. The normal human
fetal lung fibroblast cell line (MRC-5) was also evaluated since a potential anticancer drug
4

ACCEPTED MANUSCRIPT
candidate should selectively affect only tumor cells and not somatic cells. The concentration
that inhibited the growth of 50% of cells (IC ) was determined from the linear portion of the
5
0
curve by calculating the concentration of agent that reduced absorbance in treated cells,
compared to control cells, by 50%. The results of IC50 values are summarized in Table 1.
Indeno[1,2-b]quinoxalin-11-one (4a) and its 11-hydroxyimino derivative 5a were inactive
against the growth of all cell lines tested. However, introduction of the aminoalkyl side chain
at C-11 position improved growth inhibitory effect in which compounds 6a and 7a exhibited
moderate antiproliferative activity. Further substitution of methyl group at C-5, compound 4b,
did not improve growth inhibitory effect. Indeno[1,2-b]quinoxaline-11-oxo-5-carboxylic acid
(
4c) was also inactive against the growth of all cell lines tested. Its C-5 amide derivatives
exhibited moderate effect against certain types of tested cells with exception of compounds
d which is inactive. Further introduction of aminoalkoxyimino side chains on C-11 position
of these amide derivatives enhanced antiproliferative activity. Among them,
1-{[3-(dimethylamino)propoxy]imino}-N-[3-(dimethylamino)propyl]-11H-indeno[1,2-b]-
9
1
quinoxaline-6-carboxamide (10a) was active against the growth of MDA-MB231, PC-3, and
Huh-7 with IC values of 0.87 (selectivity index, SI = 36.22), 0.82 (SI = 38.43), and 0.64
5
0
µM (SI = 49.23) respectively. Compound 10a was inactive against the growth of normal
human fetal lung fibroblast cell line (MRC-5) with an IC value of 31.51 µM. Its analogs,
5
0
1
0b and 10c, were also active against the growth of MDA-MB231, PC-3, and Huh-7 with
IC50 values of < 1.0 µM in each case. Compound 10d was selectively active against the
growth of MDA-MB231 with an IC50 value of < 1.0 µM and was found to be less active
against the growth of normal human fetal lung fibroblast cell line (MRC-5) with IC values
5
0
of 11.52 µM. Compound 12 was inactive while its dihydroxyimino derivative 13 was
marginally active against all cell lines tested. Antiproliferative activities were further
enhanced by the introduction of aminoalkyl side chains. The same structure-activity
relationship (SAR) was observed for compounds 16 and 17 in which C-11 oxo derivatives
1
6a - 16e were inactive while their aminoalkoxyimino counterparts 17a – 17c exhibited
5

ACCEPTED MANUSCRIPT
potent inhibitory activities on all the cells tested. However, 17d was only marginally active
while 17e was selectively active against MDA-MB231 and Huh-7 with IC values of < 1.0
5
0
µM. Compound 10a was found to inhibit the growth of MDA-MB231, PC-3 and Huh-7 but
not MRC-5 and therefore was selected for further mechanism studies.
Previous studies [16-20] indicated that most of the indeno[1,2-c]quinolin-11-one
derivatives interact with DNA and therefore, DNA unwinding assay of these newly
synthesized indeno[1,2-b]quinoxaline derivatives was carried out. Results from Table 1
indicated that compounds 10a-d, 14 and 17a-c are capable of intercalating DNA.
<
Insert Table 1 here >
3
.2. Topoisomerase Inhibition.
The topoisomerase I (topo I) and topoisomerase II (topo II) inhibitory activities of these
indeno[1,2-b]quinoxaline derivatives were carried out with the known compound 2 as a
positive control [16]. Results are summarized in Figure 2 and Table 2. All compounds were
evaluated to inhibit topo I at 100 µM (Figure 2A) and 10 µM (Figure 2B) respectively. From
the gels shown in Figure 2A, most of them exhibited moderate inhibition of Topo I at 100
µM. However, only compounds 10a – 10d, 14, 15, 17a – 17c, and 17e exhibited moderate
inhibition of Topo I at 10 µM. Quantitation of Topo II inhibition was also analyzed at 100
µM (Figure 2C) and 10 µM (Figure 2D) respectively. Compounds 6a, 7a, 7b, 9a – 9c
demonstrated strong topo II inhibitory activities at 100 µM (Figure 2C) while compounds
1
0a – 10d, 14, and 15 inhibit topo II with moderate activities. However, only compounds
0a – 10c, 14, and 15 exhibited moderate inhibition of Topo II at 10 µM (Figure 2D).
Our results indicated compounds 10a – 10c, 14, and 15 exhibited comparable inhibitory
1
activities against topo I and topo II with the positive compound 2 at a concentration of 10 µM.
These compounds could be further optimized to discover dual topo I/II inhibitory agents.
<
Insert Figure 2 and Table 2 here >
3
.3. Compound 10a induces growth inhibition and S-phase arrest in Huh-7 cells.
To investigate the mechanisms leading to loss of cell proliferation by 10a, the effects of
6

ACCEPTED MANUSCRIPT
1
0a treatment on cell cycle arrest or apoptosis were examined. As shown in Figure 3 and
Table 3, cells treated with 1-10 µM of 10a for 24h notably increased the percentage of cells
in the S-phase from 11.4% to 38.4%, with a parallel reduction in the percentage of cells in
G0/G1 and G2/M phase, while the hypodiploid (Sub G1 phase) cells increased. These results
suggested that 10a inhibited the cellular proliferation of Huh-7 cells via S-phase arrest of the
cell cycle. Morphological changes of cells treated with 10a can be visually observed with
light microscopy (Figure 4). We found that the Huh-7 cells treated with 10a at 5 µM for 24 h
became shrinked. Such morphological changes were not apparent in the control cells.
<
Insert Figure 3, Table 3, and Figure 4 here >
3
.4. Compound 10a causes a decrease in the expression levels of cyclins D1, A, and B1.
Perturbation in cell cycle progression mediated by alterations in cell cycle-related
proteins plays a vital role in the proliferation of cancer cells. To elucidate the role of these
molecules in the inhibition of cell cycle induced by 10a treatment, protein extract was
prepared from the 10a-treated cells for 24 h. As shown in Figure 5, 10a treatment of 1-10 µM
caused a marked decrease in cyclin A, cyclin B1, and cyclin D1 expression. These results
suggested that 10a induced S-phase arrest, possibly by altering the S-phase cell cycle-related
protein expression in Huh-7 cells.
<
Insert Figure 5 here >
3
.5. Compound 10a-induced apoptosis in Huh-7 cells
To understand the mechanism of 10a-induced apoptosis, we examined the changes of
the intracellular proteins related to apoptosis, such as Bcl-2 family proteins (Bcl-2 and Bax),
caspase-3, -7, and PARP in cells treated with 10a. Bcl-2 is the first identified member of a
large family of apoptosis-regulating proteins, consisting of blockers (such as Bcl-2) and
promoters (such as Bax) of cell death. Caspases play a crucial role in apoptotic cell death.
Caspase-3 and -7 in particular, could be activated by the proteolytic processing of procaspase
3
and -7 in response to exogenous apoptosis inducers [23]. PARP is involved in DNA repair
predominantly in response to environmental stress, and is important for the maintenance of
7

ACCEPTED MANUSCRIPT
cell viability [24]. Our results indicated that caspase-3 and -7 were activated and increased
the protein expression of Bax, but decreased expression of Bcl-2 and PARP after the
treatment of 10a for 24 h in a concentration-dependent manner (Figure 6). Thus, compound
1
0a induced cell cycle arrest at S phase via activation of caspase-3, -7 and an increase in the
protein expression of Bad and Bax but a decrease in expression of Bcl-2 and PARP, which
consequently cause cell death.
<
Insert Figure 6 here >
3
.6. Compound 10a attenuates activation of Src and Akt signaling
The signaling pathways include the Src and Akt, which play a significant role in
mitogenic and cell survival [25]. Akt/protein kinase B (PKB) is the downstream molecules of
PI3K and previous studies have demonstrated that Akt plays a major role in the survival of
cells under a variety of conditions [26]. To examine whether the activities of Src and Akt are
down-regulated by 10a, the phosphorylation of Src, Akt-1 and Akt-2 activation in Huh-7
cells was analyzed. As shown in Figure 7, Compound 10a treatment attenuated the levels of
phosphorylated Src, Akt-1 and Akt-2 protein levels of in 10a-treated cells. However, 10a did
not affect the total protein expression of Akt.
<
Insert Figure 7 here >
3
.7. Antitumor efficacy of 10a in zebrafish xenograft assay
To examine the anti-tumor effect of 10a in vivo, we implanted human hepatocellular
carcinoma cells into the yolk sac of zebrafish larvae and incubated larvae with indicated
concentrations of 10a. The intensity of red fluorescence is proportional to the xenograft
tumor size. Results indicated compound 10a significantly reduced tumor size in a
dose-dependent manner as shown in Figure 8.
<
Insert Figure 8 here >
4
. Conclusion
A number of indeno[1,2-b]quinoxaline derivatives were synthesized and evaluated for
antiproliferative activities. Among them, 11-{[3-(dimethylamino)propoxy]imino}-N-[3-
8

ACCEPTED MANUSCRIPT
(
dimethylamino)propyl]-11H-indeno[1,2-b]quinoxaline-6-carboxamide (10a) was active
against the growth of MB231, PC-3, and Huh-7 with IC values of 0.87, 0.82, and 0.64 µM
5
0
respectively. Compound 10a was inactive against the growth of MRC-5 with an IC value of
5
0
3
1.51 µM. Our results have also indicated compound 10a exhibited comparable inhibitory
activities against topo I and topo II with the positive compound 2 at a concentration of 10 µM.
Mechanism studies indicated that compound 10a induced cell cycle arrest at S phase via
activation of caspase-3, -7 and an increase in the protein expression of Bad and Bax but a
decrease in expression of Bcl-2 and PARP, which consequently cause cell death. We have
also implanted human hepatocellular carcinoma cells into the yolk sac of zebrafish larvae and
incubated larvae with various concentrations of 10a. Our results of the zebrafish xenograft
assay confirmed the anti-tumor effect of 10a in vivo. Further structural optimization of
compound 10a is on-going.
5
5
. Experimental
.1. General.
TLC: precoated (0.2 mm) silica gel 60 F254 plates from EM Laboratories, Inc.; detection by
UV light (254 nm). All chromatographic separations were performed using silica gel (Merck
1
13
6
0 230–400 mesh). M.p.: Yamato MP-21 melting-point apparatus; uncorrected. H and C
NMR spectra: Varian-Unity-400 spectrometer at 400 and 100 MHz, chemical shifts in ppm
with SiMe as an internal standard (= 0 ppm), coupling constants J in Hz. Mass spectra were
4
recorded on Bruker APEX II (ESI) mass spectrometer. Elemental analyses were carried out
on a Heraeus CHN-O-Rapid elemental analyzer, and results were within ± 0.4% of calculated
values. Microwave reactions were conducted using a CEM Discover Synthesis Unit (CEM
Corp., Matthews, NC).
5
.2.1. 11H-Indeno[1,2-b]quinoxalin-11-one (4a)
A mixture of ninhydrin (1.80 g, 10.0 mmol) and 1,2-phenylenediamine (1.14 g, 10.5 mmol)
in EtOH (100 mL) was refluxed for 2 h (TLC monitoring). The mixture was cooled, then the
resulting precipitate was filtered, purified by column chromatography (CH Cl ), and
2
2
9

ACCEPTED MANUSCRIPT
crystallized from EtOH/CH Cl (5/1) to give 4a (2.16 g, 93% yield) as a yellow solid. Mp:
2
2
o
o
2
18-220 C. (lit. mp. 217-219 C) [21]
5
.2.2. 6-Methyl-11H-indeno[1,2-b]quinoxalin-11-one (4b).
Compound 4b was obtained from ninhydrin (1.80 g, 10.0 mmol), 2,3-diaminotoluene (1.22 g,
0.0 mmol) and EtOH (100 mL) as described for the preparation of 2a in 85% yield as a
1
o
o
yellow needle solid. Mp: 227-229 C. (from EtOH), (lit. mp. 222-224 C from acetone) [21]
.2.3. 11-Oxo-11H-indeno[1,2-b]quinoxaline-6-carboxylic acid (4c).
5
A mixture of ninhydrin (1.80 g, 10.1 mmol) and 2,3-diaminobenzoic acid (1.52 g, 10.0 mmol)
in EtOH (100 mL) was refluxed for 2 h (TLC monitoring). The mixture was cooled, then the
resulting precipitate was filtered and washed with EtOH (50 mL) to give 2c (2.65 g, 96%
o
o
yield) as a yellow solid. Mp: 368-370 C. (decomp.), (lit. mp. > 300 C from ethylene glycol)
21]
.3.1. 11H-Indeno[1,2-b]quinoxalin-11-one oxime (5a).
[
5
A mixture of 4a (0.23 g, 1.0 mmol) and hydroxylamine hydrochloride (0.35 g, 5.0 mmol) in
EtOH (5.0 mL) was refluxed for 6 h (TLC monitoring). The mixture was cooled and poured
into water. The resulting precipitate was filtered and crystallized from EtOH to give 5a (0.23
o
1
g, 94% yield) as a white solid. Mp: 274-276 C (from EtOH, decomp.). H NMR (400 MHz,
DMSO-d ): δ 7.67-7.72 (m, 2H, ArH), 7.78-7.85 (m, 2H, ArH), 8.10-8.13 (m, 2H, ArH),
6
1
3
8
.14-8.17 (m, 1H, ArH), 8.50-8.53 (m, 1H, ArH), 13.48 (s, 1H, NOH). C NMR (100 MHz,
DMSO-d ): δ 122.26, 128.76, 129.30, 129.87, 129.99, 130.58, 132.09, 132.56, 133.07,
6
+
1
35.99, 141.56, 141.85, 147.18, 150.81, 152.89. ESIMS [M+H] :248.01. Anal. calc. for
C H N O．0.2 H O: C 71.81, H 3.78, N 16.75; found: C 71.47, H 3.65, N 16.80. [21]
15
9
3
2
5
.3.2.
11H-Indeno[1,2-b]quinoxalin-11-one
O-[2-(pyrrolidin-1-yl)ethyl]
oxime
hydrochloride (6a).
A mixture of 4a (0.23 g, 1.0 mmol) and 2-(pyrrolidino)ethoxyamine hydrochloride (0.61 g) in
EtOH (5.0 mL) was refluxed for 6 h (TLC monitoring). The mixture was cooled and
concentrated, then dissolved in water (20 mL), neutralized by K CO (0.41 g, 3.0 mmol), and
2
3
1
0

ACCEPTED MANUSCRIPT
extracted with CH Cl three times. The organic layer evaporated in vecuo and purified by
2
2
column chromatography (CH Cl /MeOH 10/1). The oil compound was stirred with 3N HCl
2
2
(3.0 mL) in EtOH (2.0 mL) for 0.5 h, then evaporated in vecuo. The residue was dissolved in
EtOH (2.0 mL), then poured into acetone (50 mL). The resulting precipitate was filtered and
o
1
dried to give 6a (0.33 g, 87% yield) as a yellow solid. Mp: 187-189 C (decomp.). H NMR
(
400 MHz, D O): δ 1.87-1.92 (m, 2H, pyrrolidinyl-H), 2.00-2.05 (m, 2H, pyrrolidinyl-H),
2
2
.96-3.03 (m, 2H, pyrrolidinyl-H), 3.42 (br, 2H, OCH CH N), 3.52-3.57 (m, 2H,
2 2
pyrrolindyl-H), 4.22 (br, 2H, OCH CH N), 6.56-6.57 (m, 1H, ArH), 6.74-6.76 (m, 3H, ArH),
2
2
1
3
6
5
1
.90-6.93 (m, 1H, ArH), 7.06-7.09 (m, 3H, ArH). C NMR (100 MHz, D O): δ 22.78(2C),
2
3.71, 55.08 (2C), 71.26, 121.10, 127.32, 128.29 (2C), 130.30, 130.48, 131.19, 132.46 (2C),
+
33.96, 139.22, 139.76, 146.50, 147.53, 150.54. ESIMS [M+H] : 333.11. Anal. calc. for
C H N O．1.0 HCl．1.9 H O: C 60.74, H 6.03, N 13.50; found: C 60.53, H 5.68, N 13.43.
21
20
4
2
5
.3.3.
hydrochloride (7a).
A mixture of 4a (0.23 g, 1.0 mmol) and 3-(dimethylamino)propoxyamine hydrochloride
0.61 g) in EtOH (5.0 mL) was refluxed for 6 h (TLC monitoring). The mixture was cooled
and concentrated, then dissolved in water (20 mL), neutralized by K CO (0.41 g, 3.0 mmol),
11H-Indeno[1,2-b]quinoxalin-11-one
O-[3-(dimethylamino)propyl]
oxime
(
2
3
and extracted with CH Cl three times. The organic layer evaporated in vecuo and purified by
2
2
column chromatography (CH Cl /MeOH 10/1). The oil compound was stirred with 3N HCl
2
2
(3.0 mL) in EtOH (2.0 mL) for 0.5 h, then evaporated in vecuo. The residue crystallized from
o
EtOH to give 7a (0.30 g, 82% yield) as a white solid. Mp: 220-222 C (from EtOH, decomp.).
1
H NMR (400 MHz, D O): 1.85-1.92 (m, 2H, OCH CH CH N), 2.81 (s, 6H, N(CH ) ),
2
2
2
2
3 2
3
.01-3.05 (m, 2H, OCH CH CH N), 3.87 (br, 2H, OCH CH CH N), 6.61 (d, 1H, J = 6.8 Hz,
2 2 2 2 2 2
ArH), 6.67-6.76 (m, 2H, ArH), 6.82 (d, 1H, J = 6.8 Hz, ArH), 6.86-6.88 (m, 1H, ArH),
13
7
.03-7.05 (m, 1H, ArH), 7.15-7.17 (m, 2H, ArH). C NMR (100 MHz, D O): 23.83, 42.91,
2
(2C), 54.92, 72.66, 121.07, 127.40, 127.82, 128.47, 130.08, 130.54, 130.89, 132.01, 132.20,
+
1
33.73, 139.52, 139.77, 145.28, 147.69, 150.44. ESIMS [M+H] : 345.09. Anal. calc. for
1
1

ACCEPTED MANUSCRIPT
C H N O．1.0 HCl．1.2 H O: C 61.50, H 6.05, N 14.35; found: C 61.17, H 5.92, N 14.29.
20
20
4
2
5
.3.4. 6-Methyl-11H-indeno[1,2-b]quinoxalin-11-one O-[3-(dimethylamino)propyl]
oxime hydrochloride (7b).
Compound 7b was
-(dimethylamino)propoxyamine hydrochloride (0.60 g) as described for the preparation of
obtained
from
4b
(0.25
g,
1.0
mmol)
and
3
o
1
7
a in 85% yield as a white solid. Mp: 231-233 C (from EtOH, decomp.). H NMR (400
MHz, D O): δ 1.74 (s, 3H, ArCH ), 1.95-2.02 (m, 2H, OCH CH CH N), 2.81 (s, 6H,
2
3
2
2
2
N(CH ) ), 3.06-3.10 (m, 2H, OCH CH CH N), 4.02 (t, 2H, J = 5.6 Hz, OCH CH CH N),
3
2
2
2
2
2
2
2
1
3
6
.67-6.69 (m, 1H, ArH), 6.76-6.86 (m, 5H, ArH), 7.05 (d, 1H, J = 6.8 Hz, ArH). C NMR
(
100 MHz, D O): δ 17.03, 24.01, 42.89 (2C), 55.03, 72.58, 121.16, 126.23, 127.93, 129.38,
2
1
30.66, 130.97, 131.65, 131.79, 134.75, 136.06, 139.36 (2C), 145.92, 147.14, 150.15.
+
ESIMS [M+H] : 347.11. Anal. calc. for C H N O．1.0 HCl．1.0 H O: C 62.90, H 6.30, N
2
1
22
4
2
1
5
6
3.98; found: C 62.63, H 6.28, N 13.92.
.3.5. 11-{[3-(Dimethylamino)propoxy]imino}-11H-indeno[1,2-b]quinoxaline-
-carboxylic acid hydrochloride (7c).
7c was obtained
-(dimethylamino)propoxyamine hydrochloride (0.60 g) as described for the preparation of
Compound
from
4c
(0.28
g,
1.0
mmol)
and
3
o
1
7
a in 75% yield as a green solid. Mp: 250-252 C (from EtOH, decomp.). H NMR (400
MHz, D O): δ 2.03-2.10 (m, 2H, OCH CH CH N), 2.86 (s, 6H, N(CH ) ), 3.15-3.19 (m, 2H,
2
2
2
2
3 2
OCH CH CH N), 4.12-4.15 (m, 2H, OCH CH CH N), 6.59 (d, 1H, J = 6.8 Hz, ArH), 6.86 (t,
2
2
2
2
2
2
1
H, J = 6.8 Hz, ArH), 6.92 (t, 1H, J = 6.8 Hz, ArH), 7.10 (d, 1H, J = 6.8 Hz, ArH), 7.24-7.32
1
3
(
m, 2H, ArH), 7.79 (dd, 1H, J = 6.8, 1.6 Hz, ArH). C NMR (100 MHz, D O): δ 23.94,
2
4
1
2.92 (2C), 54.79, 73.75, 121.91, 122.47, 128.15, 130.59, 130.98, 131.03, 133.03, 134.61,
+
34.88, 135.73, 136.92, 139.04, 144.22, 148.52, 148.81, 166.97. ESIMS [M+H] : 377.12.
Anal. calc. for C H N O ．1.0 HCl: C 61.08, H 5.14, N 13.57; found: C 61.06, H 5.29, N
2
1
20
4
3
1
3.41.
.4. General procedure for preparation of compounds 9a-9d.
5
1
2

ACCEPTED MANUSCRIPT
A mixture of 4c (0.55 g, 2.0 mmol) and 1,1'-carbonyldiimidazole (0.97 g, 6.0 mmol) in
1
,4-dioxane (20 mL) was refluxed for 4 h (TLC monitoring). The mixture was cooled,
concentrated, and triturated with water. The resulting precipitate was collected and dried to
give intermediate compound 8 (639.8 mg). The compound 8 was stirred in CH Cl (20 mL)
2
2
at room temperature, then substituted amines (6.0 mmol) was added, and the reaction
mixture was stirred for 10 min (TLC monitoring). The mixture was extracted with water
three times, then organic layer evaporated in vecuo and purified by column chromatography
(
CH Cl /MeOH 10/1) and crystallized from EtOH to give 9a-9d.
2
2
5
.4.1.
N-[3-(Dimethylamino)propyl]-11-oxo-11H-indeno[1,2-b]quinoxaline-
6
-carboxamide hydrochloride (9a).
o
1
yellow solid (0.45g, 63% yield); Mp: 172-174 C (from EtOH). H NMR (400 MHz, CDCl ):
3
δ 1.97 (quin, 2H, J = 6.8 Hz, NHCH CH CH N), 2.31 (s, 6H, N(CH ) ), 2.53 (t, 2H, J = 6.8
2
2
2
3 2
Hz, NHCH CH CH N), 3.70 (t, 2H, J = 6.8 Hz, NHCH CH CH N). 7.67-7.71 (m, 1H, ArH),
2
2
2
2
2
2
7
.82-7.87 (m, 2H, ArH), 7.96-7.99 (m, 1H, ArH), 8.06 (d, 1H, J = 7.6 Hz, ArH), 8.36-8.38
1
3
(
m, 1H, ArH), 8.89-8.91 (m, 1H, ArH), 10.37 (br, 1H, CONH). C-NMR (100 MHz, CDCl ):
3
δ 27.74, 38.18, 45.50 (2C), 57.45, 122.60, 125.12, 130.05, 130.12, 133.42, 135.29, 136.53,
+
1
3
5
5
37.00, 137.03, 140.06, 140.38, 142.67, 148.93, 155.31, 164.49, 188.98. ESIMS [M+H] :
61.10. Anal. calc. for C H N O ．2.0 HCl．3.0 H O: C 53.13, H 6.87, N 14.30; found: C
2
6
32
6
2
2
2.90, H 6.89, N 14.22.
.4.2. N-[2-(Dimethylamino)ethyl]-11-oxo-11H-indeno[1,2-b]quinoxaline-6-carboxamide
o
(
9b). yellow solid (0.51 g, 73% yield); Mp: 222-223 C (from EtOH, decomp.), (lit. mp.
o
+
2
5
20-221 C from EtOH) [27]. ESIMS [M+H] : 347.05.
.4.3. 11-Oxo-N-[2-(pyrrolidin-1-yl)ethyl]-11H-indeno[1,2-b]quinoxaline-6-carboxamide
o
1
(
9c). green solid (0.46 g, 62% yield); Mp: 197-199 C (from EtOH, decomp.). H NMR (400
MHz, CDCl ): δ 1.88 (quin, 4H, J = 7.6 Hz, pyrrolidinyl-H), 2.74 (br, 4H, pyrrolidinyl-H),
3
2
.88 (t, 2H, J = 5.6 Hz, NHCH CH N), 3.80 (q, 2H, J = 5.2 Hz, NHCH CH N), 7.64 (td, 1H,
2 2 2 2
J = 1.2, 7.6 Hz, ArH), 7.76 (td, 1H, J = 1.2, 7.6 Hz, ArH), 7.84 (t, 1H, J = 7.6 Hz, ArH), 7.95
1
3

ACCEPTED MANUSCRIPT
(
d, 1H, J = 7.6 Hz, ArH), 8.22 (d, 1H, J = 7.2 Hz, ArH), 8.35 (dd, 1H, J = 7.6, 1.6 Hz, ArH),
1
3
8
2
1
.92 (d, 1H, J = 1.6, 7.6 Hz, ArH), 10.63 (br, 1H, CONH). C-NMR (100 MHz, CDCl ): δ
3
3.61 (2C), 39.18, 54.26 (2C), 55.34, 122.91, 125.00, 129.94, 130.03, 133.29, 135.24,
36.53, 136.62, 136.99, 140.24, 140.75, 142.62, 148.94, 155.36, 164.52, 189.15. ESIMS
+
[
M+H] : 373.08. Anal. calc. for C H N O ．0.2 H O: C 70.26, H 5.48, N 14.90; found: C
2
2
20
4
2
2
6
5
9.98, H 5.50, N 14.76.
.4.4. N-(2-Morpholinoethyl)-11-oxo-11H-indeno[1,2-b]quinoxaline-6-carboxamide (9d).
o
1
green solid (0.48 g, 60% yield); Mp: 217-219 C (from EtOH, decomp.). H NMR (400 MHz,
CDCl ): δ 2.62 (t, 2H, J = 4.8 Hz, morpholinyl-H), 2.78 (t, 2H, J = 6.0 Hz, NHCH CH N),
3
2
2
3
1
7
.77 (t, 2H, J = 4.8 Hz, morpholinyl-H), 3.80 (q, 2H, J = 6.0 Hz, NHCH CH N), 7.68 (td,
2 2
H, J = 1.2, 7.6 Hz, ArH), 7.78 (td, 1H, J = 7.6, 1.2 Hz, ArH), 7.86 (t, 1H, J = 7.6 Hz, ArH),
.97 (d, 1H, J = 7.6 Hz, ArH), 8.17 (d, 1H, J = 7.2 Hz, ArH), 8.37 (dd, 1H, J = 8.0, 1.6 Hz,
1
3
ArH), 8.91 (d, 1H, J = 1.6, 7.6 Hz, ArH), 10.42 (br, 1H, CONH). C-NMR (100 MHz,
CDCl ): δ 36.86, 53.80 (2C), 57.94, 66.82 (2C), 122.54, 125.20, 129.94, 130.12, 133.48,
3
1
35.36, 136.62, 136.67, 137.03, 140.15, 140.53, 142.70, 148.94, 155.30, 164.61, 189.00.
+
ESIMS [M+H] : 389.10. Anal. calc. for C H N O : C 68.02, H 5.20, N 14.43; found: C
2
2
20
4
3
6
7.74, H 5.25, N 14.38.
.5.1. 11-{[3-(Dimethylamino)propoxy]imino}-N-[3-(dimethylamino)propyl]-
5
11H-indeno[1,2-b]quinoxaline-6-carboxamide hydrochloride (10a).
A mixture of 9a (0.36, 1.0 mmol) and 3-(dimethylamino)propoxyamine hydrochloride (0.60
g) in EtOH (5.0 mL) was refluxed for 6 h (TLC monitoring). The mixture was cooled and
concentrated. The residue was dissolved in water (20 mL), neutralized by K CO (0.41 g, 3.0
2
3
mmol), and extracted with CH Cl three times. The organic layer evaporated in vecuo and
2
2
purified by column chromatography (CH Cl /MeOH/NH OH 100/20/1). The oil compound
2
2
4
was stirred with 3N HCl (3.0 mL) in EtOH (2.0 mL) for 0.5 h, then evaporated in vecuo. The
residue crystallized from EtOH to give 10a (0.45 g, 84% yield) as a white solid. Mp:
o
1
2
28-230 C (from EtOH, decomp.). H NMR (400 MHz, D O): δ 1.84-1.92 (m, 2H,
2
1
4

ACCEPTED MANUSCRIPT
NHCH CH CH N), 2.14-2.21 (m, 2H, OCH CH CH N), 2.91 (s, 6H, N(CH ) ), 2.92 (s, 6H,
2
2
2
2
2
2
3 2
N(CH ) ), 3.16-3.28 (m, 6H, NHCH CH CH N, OCH CH CH N), 4.21 (t, 2H, J = 6.0 Hz,
3
2
2
2
2
2
2
2
OCH CH CH N), 7.09-7.11 (m, 1H, ArH), 7.33-7.46 (m, 2H, ArH), 7.49-7.61 (m, 3H, ArH),
2
2
2
13
7
.97 (dd, 1H, J = 7.2, 1.2 Hz, ArH). C NMR (100 MHz, D O): δ 24.08, 24.29, 37.00, 42.99
2
(2C), 43.12 (2C), 54.98, 55.49, 73.39, 121.84, 126.92, 128.85, 129.72, 131.78, 132.98,
1
33.57, 133.66, 133.74, 133.84, 137.99, 139.84, 145.53, 148.35, 150.54, 165.80. ESIMS
+
[
M+H] : 461.21. Anal. calc. for C H N O ．2.0 HCl．3.0 H O: C 53.13, H 6.87, N 14.30;
2
6
32
6
2
2
found: C 52.90, H 6.89, N 14.22.
5
.5.2.
1H-indeno[1,2-b]quinoxaline-6-carboxamide hydrochloride (10b).
Compound 10b was obtained from 9b (0.35 g,
-(dimethylamino)propoxyamine hydrochloride (0.61 g) as described for the preparation of
N-[2-(Dimethylamino)ethyl]-11-{[3-(dimethylamino)propoxy]imino}-
1
1.0
mmol)
and
3
o
1
1
0a in 89% yield as a white solid. Mp: 244-245 C (from EtOH, decomp.). H NMR (400
MHz, D O): δ 2.14-2.21 (m, 2H, OCH CH CH N), 2.89 (s, 6H, N(CH ) ), 2.94 (s, 6H,
2
2
2
2
3 2
N(CH ) ), 3.23-3.27 (m, 2H, OCH CH CH N), 3.31 (t, 2H, J = 6.4 Hz, NHCH CH N), 3.60
3
2
2
2
2
2
2
(
t, 2H, J = 6.4 Hz, NHCH CH N), 4.27 (t, 2H, J = 6.4 Hz, OCH CH CH N), 7.37-7.39 (m,
2 2 2 2 2
1
8
4
1
4
5
5
H, ArH), 7.41-7.49 (m, 2H, ArH), 7.53 (t, 1H, J = 7.6 Hz, ArH), 7.63-7.66 (m, 2H, ArH),
1
3
.04 (dd, 1H, J = 7.6, 1.2 Hz, ArH). C NMR (100 MHz, D O): δ 24.14, 35.15, 42.94 (2C),
2
3.44 (2C), 54.95, 56.73, 73.36, 122.35, 126.93, 128.84, 129.68, 131.91, 133.02, 133.66,
+
33.81, 133.91, 134.10, 138.25, 139.88, 145.86, 148.68, 151.06, 166.88. ESIMS [M+H] :
47.09. Anal. calc. for C H N O ．2.0 HCl．2.8 H O: C 52.67, H 6.66, N 14.75; found: C
2
5
30
6
2
2
2.52, H 6.51, N 14.41.
.5.3. 11-{[3-(Dimethylamino)propoxy]imino}-N-[2-(pyrrolidin-1-yl)ethyl]-
1H-indeno[1,2-b]quinoxaline-6-carboxamide hydrochloride (10c).
Compound 10c was obtained from 9c (0.37 g,
-(dimethylamino)propoxyamine hydrochloride (0.60 g) as described for the preparation of
1
1.0
mmol)
and
3
o
1
1
0a in 82% yield as a white solid. Mp: 226-227 C (from EtOH, decomp.). H NMR (400
1
5

ACCEPTED MANUSCRIPT
MHz, D O): δ 2.01 (br, 4H, pyrrolidinyl-H), 2.13-2.20 (m, 2H, OCH CH CH N), 2.86 (s, 6H,
2
2
2
2
N(CH ) ), 3.21-3.25 (m, 2H, OCH CH CH N), 3.33-3.39 (m, 6H, NHCH CH N,
3
2
2
2
2
2
2
pyrrolidinyl-H), 3.56 (t, 2H, J = 6.0 Hz, NHCH CH N), 4.26 (t, 2H, J = 6.0 Hz,
2
2
OCH CH CH N), 7.35 (d, J = 6.8 Hz, ArH), 7.41-7.47 (m, 2H, ArH), 7.51 (t, 1H, J = 8.0 Hz,
2
2
2
ArH), 7.61 (d, 1H, J = 8.0 Hz, ArH), 7.65 (d, 1H, J = 6.4 Hz, ArH), 8.03 (d, 1H, J = 7.2 Hz,
1
3
ArH). C NMR (100 MHz, D O): δ 22.84 (2C), 24.16, 36.24, 42.93 (2C), 53.91, 54.81 (2C),
2
5
1
4.95, 73.37, 122.30, 127.02, 128.86, 129.68, 131.97, 133.01, 133.66, 133.77, 133.88,
+
34.15, 138.28, 139.91, 145.93, 148.73, 151.10, 166.79. ESIMS [M+H] : 473.12. Anal. calc.
for C H N O ．2.0 HCl．2.8 H O: C 54.40, H 6.71, N 14.10; found: C 54.11, H 6.75, N
2
4.03.
.5.4.
7
32
6
2
2
1
5
11-{[3-(Dimethylamino)propoxy]imino}-N-(2-morpholinoethyl)-
1
1H-indeno[1,2-b]quinoxaline-6-carboxamide hydrochloride (10d).
Compound 10d was obtained from 9d (0.39 g,
-(dimethylamino)propoxyamine hydrochloride (0.60 g) as described for the preparation of
1.0
mmol)
and
3
o
1
1
0a in 73% yield as a green solid. Mp: 227-229 C (from EtOH, decomp.). H NMR (400
MHz, D O): δ 2.14-2.20 (m, 2H, OCH CH CH N), 3.21-3.25 (m, 2H, OCH CH CH N),
2
2
2
2
2
2
2
3
3
.33-3.42 (m, 6H, NHCH CH N, morpholinyl-H), 3.64 (t, 2H, J = 6.8 Hz, NHCH CH N),
2 2 2 2
.95 (br, 4H, morpholinyl-H), 4.25 (t, 2H, J = 6.4 Hz, OCH CH CH N), 7.40-7.49 (m, 3H,
2
2
2
ArH), 7.53 (t, 1H, J = 7.6 Hz, ArH), 7.62-7.65 (m, 2H, ArH), 8.05 (dd, 1H, J = 7.6, 1.2 Hz,
1
3
ArH). C NMR (100 MHz, D O): δ 24.15, 34..46, 42.95 (2C), 52.42 (2C), 54.97, 56.20,
2
6
1
3.99 (2C), 73.37, 122.40, 127.06, 128.85, 129.66, 131.96, 133.01, 133.67, 133.87, 133.92,
+
34.19, 138.31, 139.95, 145.88, 148.74, 151.12, 167.03. ESIMS [M+H] : 489.17. Anal. calc.
for C H N O ．2.0 HCl．2.6 H O: C 53.29, H 6.51, N 13.81; found: C 53.01, H 6.60, N
2
7
32
6
3
2
1
3.78.
5
.6. 6-(Bromomethyl)-11H-indeno[1,2-b]quinoxalin-11-one (11).
A mixture of 4b (2.46 g, 10.0 mmol), N-bromosuccinimide (1.96 g, 11.0 mmol) and
dibenzoylperoxide (0.24 g, 1.0 mmol) in CCl (50 mL) was refluxed for 2 h (TLC
4
1
6

ACCEPTED MANUSCRIPT
monitoring). After cooling, EtOH (30 mL) was added and stirred for 10 min. The resulting
precipitate was filtered, washed with EtOH (20 mL), and crystallized from EtOH/CH Cl2
2
o
(
4/1) to give 11 (2.99 g, 92% yield) as a yellow solid. Mp: 258-260 C (from EtOH,
1
decomp.). H NMR (400 MHz, CDCl ): δ 5.24 (s, 2H, ArCH Br), 7.60-7.64 (m, 1H, ArH),
3
2
7
.69-7.73 (m, 1H, ArH), 7.77-7.81 (m, 1H, ArH), 7.92-7.94 (m, 2H, ArH), 8.16 (d, 1H, J =
1
3
7
.6 Hz, ArH), 8.19 (dd, 1H, J = 8.4, 1.2 Hz, ArH). C NMR (100 MHz, CDCl ): δ 27.71,
3
1
22.83, 124.78, 129.99, 132.14, 132.67, 133.44, 136.73, 136.86, 137.15, 140.82, 141.53,
42.69, 149.60, 156.08, 189.81. Anal. calc. for C H N OBr: C 59.10, H 2.80, N 8.62; found:
1
1
6
9
2
C 59.36, H 2.93, N 8.73.
5
.7. 11-Oxo-11H-indeno[1,2-b]quinoxaline-6-carbaldehyde (12).
A mixture of 11 (1.63 g, 5.0 mmol) and calcium carbonate (15.0 g, 150 mmol) in
,4-dioxane/water (4/1, 50 mL) was refluxed for 2 days (TLC monitoring). After cooling,
1
the mixture was filtered and washed with 1,4-dioxane (20 mL). The filtrate was evaporated in
vacuo to give yellow solid, then Manganese(IV) oxide (8.69 g, 100.0 mmol) and CH Cl2
2
(100 mL) was added and stirred at room temperature for 12 h. The mixture was filtered by
celite and washed with CH Cl (50 mL). The filtrate was evaporated in vacuo and purified by
2
2
column chromatography (CH Cl ) and crystallized from EtOH to give 12 (1.20 g, 92% yield)
2
2
o
1
as a yellow solid. Mp: 270-272 C (from EtOH, decomp.). H NMR (400 MHz, CDCl ): δ
3
7
.65 (t, 1H, J = 7.6 Hz, ArH), 7.87 (t, 1H, J = 7.6 Hz, ArH), 7.86 (t, 1H, J = 7.6 Hz, ArH),
.94 (d, 1H, J = 7.6 Hz, ArH), 8.13 (d, 1H, J = 7.6 Hz, ArH), 8.39 (dd, 1H, J = 7.6, 1.2 Hz,
7
1
3
ArH), 8.39 (dd, 1H, J = 8.0, 1.2 Hz, ArH), 11.41 (s, 1H, ArH). C NMR (100 MHz, CDCl ):
3
δ 122.91, 124.92, 129.77, 131.42, 132.23, 133.24, 136.93, 137.08, 137.48, 140.97, 142.11,
+
1
42.84, 149.89, 157.09, 189.22, 190.61. ESIMS [M+H] : 260.92. Anal. calc. for C H N O :
1
6
8
2
2
C 73.83, H 3.10, N 10.77; found: C 73.64, H 3.25, N 10.59.
5
.8.1. 11-(Hydroxyimino)-11H-indeno[1,2-b]quinoxaline-6-carbaldehyde oxime (13).
Compound 13 was obtained from 12 (1.0 mmol) and hydroxylamine hydrochloride (8.0
o
mmol) as described for the preparation of 3 in 87% yield as a white solid. Mp: 290-291 C
17

ACCEPTED MANUSCRIPT
1
(
from EtOH, decomp.). H NMR (400 MHz, DMSO-d ): δ 7.71-7.76 (m, 2H, ArH), 7.82 (t,
6
1
8
H, J = 8.0 Hz, ArH), 8.15 (dd, 1H, J = 8.0, 1.2 Hz, ArH), 8.21-8.25 (m, 2H, ArH),
1
3
.53-8.55 (m, 1H, ArH), 9.28 (s, 1H, CHN), 11.68 (s, 1H, NOH), 13.43 (s, 1H, NOH). C
NMR (100 MHz, DMSO-d ): δ 122.22, 125.51, 128.54, 129.40, 130.65, 130.69, 131.84,
6
+
1
2
1
5
6
32.50, 133.13, 135.69, 139.10, 141.30, 144.14, 146.91, 150.98, 152.09. ESIMS [M+H] :
90.93. Anal. calc. for C H N O : C 66.19, H 3.48, N 19.30; found: C 66.23, H 3.52, N
16
10
4
2
9.20.
.8.2.
11-{[2-(Pyrrolidin-1-yl)ethoxy]imino}-11H-indeno[1,2-b]quinoxaline-
-carbaldehyde O-[2-(pyrrolidin-1-yl)ethyl] oxime hydrochloride (14).
A mixture of 12 (0.26 g, 1.0 mmol) and 2-(pyrrolidino)propoxyamine hydrochloride (1.20 g)
in EtOH (5.0 mL) was refluxed for 6 h. The mixture was cooled and concentrated, then
dissolved in water (20 mL), neutralized by K CO (0.83 g, 6.0 mmol), and extracted with
2
3
CH Cl three times. The organic layer evaporated in vecuo and purified by column
2
2
chromatography (CH Cl /MeOH/NH OH 100/20/1). The oil compound was stirred with 3N
2
2
4
HCl (3.0 mL) in EtOH (2.0 mL) for 0.5 h, then evaporated in vecuo. The residue was
dissolved in EtOH (2.0 mL), then poured into acetone (50 mL). The resulting precipitate was
o
filtered and dried to give 14 (0.38 g, 68% yield) as a green solid. Mp: 217-218 C (decomp.).
1
H NMR (400 MHz, D O): δ 1.96-2.20 (m, 8H, pyrrolindinyl-H), 3.13-3.23 (m, 4H,
2
pyrrolindinyl-H), 3.62-3.76 (m, 8H, (OCH CH N) , pyrrolindinyl-H), 4.50-4.62 (m, 4H,
2
2
2
(
OCH CH N) ), 7.28-7.40 (m, 3H, ArH), 7.45-7.55 (m, 2H, ArH), 7.73-7.82 (m, 2H, ArH),
2 2 2
1
3
8
5
.47 (s, 1H, CHN). C NMR (100 MHz, D O): δ 19.98 (4C), 50.97, 51.05, 52.07 (2C),
2
2.31 (2C), 66.34, 68.82, 119.24, 124.83, 125.43, 126.18, 127.15, 128.25, 128.87, 130.25
+
(
2C), 132.52, 136.24, 136.75, 144.38, 144.63, 146.11, 149.17. ESIMS [M+H] : 485.18.Anal.
calc. for C H N O ．2.0 HCl．2.8 H O: C 55.30, H 6.58, N 13.82; found: C 54.91, H 6.58,
2
8
32
6
2
2
N 13.61.
5
.8.3.
11-{[3-(Dimethylamino)propoxy]imino}-11H-indeno[1,2-b]quinoxaline-
6
-carbaldehyde O-[3-(dimethylamino)propyl] oxime hydrochloride (15).
1
8

ACCEPTED MANUSCRIPT
Compound
15
was
obtained
from
12
(0.26
g,
1.0
mmol)
and
3
1
2
3
1
1
7
1
-(dimethylamino)propoxyamine hydrochloride (1.20 g) as described for the preparation of
o
1
4 in 60% yield as a white solid. Mp: 267-269 C (decomp.). H NMR (400 MHz, D O): δ
2
.11-2.16 (m, 4H, (OCH CH CH N) ), 2.86 (s, 6H, N(CH ) ), 2.89 (s, 6H, N(CH ) ),
2
2
2
2
3 2
3 2
.18-3.29 (m, 4H, (OCH CH CH N) ), 4.17-4.23 (m, 4H, (OCH CH CH N) ), 7.02-7.08 (m,
2
2
2
2
2
2
2
2
H, ArH), 7.14-7.20 (m, 2H, ArH), 7.29-7.38 (m, 2H, ArH), 7.43-7.50 (m, 2H, ArH), 8.06 (s,
1
3
H, CHN). C NMR (100 MHz, D O): δ 24.20 (2C), 42.95 (2C), 43.02 (2C), 55.00, 55.23,
2
0.85, 73.02, 121.75, 126.92, 128.03, 128.49, 129.62, 130.91, 131.50, 132.50, 132.75,
+
34.72, 138.60, 139.41, 146.05, 146.19, 148.65, 151.38. ESIMS [M+H] : 461.15. Anal. calc.
for C H N O ．3.0 HCl．0.7 H O: C 53.58, H 6.31, N 14.42; found: C 53.31, H 6.35, N
2
6
32
6
2
2
1
4.37.
.9.1. 6-[(Dimethylamino)methyl]-11H-indeno[1,2-b]quinoxalin-11-one (16a).
5
Compound 11 (0.98 g, 3.0 mmol) was stirred in THF (20 mL) at room temperature, then
dimethylamine (40% in water, 1.0 mL, 9.0 mmol) was added, and the reaction mixture was
stirred for 10 min (TLC monitoring). The solvent evaporated in vecuo, purified by column
chromatography (CH Cl /MeOH 10/1) and crystallized from EtOH to give 16a (0.62 g, 71%
2
2
o
1
yield) as a yellow solid. Mp: 171-173 C (from EtOH, decomp.). H NMR (400 MHz,
CDCl ): δ 2.50 (s, 6H, N(CH ) ), 4.33 (s, 2H, ArCH N), 7.58 (td, 1H, J = 0.8, 7.6 Hz, ArH),
3
3 2
2
7
.73-7.79 (m, 2H, ArH), 7.90 (d, 1H, J = 7.2 Hz, ArH), 7.98 (d, 1H, J = 7.2 Hz, ArH), 8.11
1
3
(
d, 1H, J = 7.6 Hz, ArH), 8.17 (dd, 1H, J = 7.6, 1.2 Hz, ArH). C NMR (100 MHz, CDCl ):
3
δ 45.11 (2C), 57.09, 122.57, 124.68, 129.89, 131.25, 132.47, 133.71, 136.07, 136.04, 136.67,
+
1
41.63, 141.85, 142.65, 149.07, 155.91, 189.95. ESIMS [M+H] : 290.05. Anal. calc. for
C H N O．0.6 H O: C 72.02, H 5.45, N 14.00; found: C 71.98, H 5.18, N 13.88.
18
15
3
2
5
.9.2. 6-(Pyrrolidin-1-ylmethyl)-11H-indeno[1,2-b]quinoxalin-11-one hydrobromide
16b).
Compound 11 (0.98 g, 3.0 mmol) was stirred in CH Cl (20 mL) at room temperature, then
(
2
2
pyrrolidine (0.43 g, 6.0 mmol) was added, and the reaction mixture was stirred for 10 min
1
9

ACCEPTED MANUSCRIPT
(
TLC monitoring). The mixture was extracted with water three times. The organic layer
evaporated in vecuo and purified by column chromatography (CH Cl /MeOH 10/1) to give
2
2
o
1
1
6b (0.69 g, 58% yield) as a green solid. Mp: 268-270 C (decomp.). H NMR (400 MHz,
CDCl ): δ 2.15 (br, 2H, pyrrolidinyl-H), 2.29 (br, 2H, pyrrolidinyl-H), 3.12 (br, 2H,
3
pyrrolidinyl-H), 3.78 (br, 2H, pyrrolidinyl-H), 5.08 (s, 2H, ArCH N), 7.65 (t, 1H, J = 7.2 Hz,
2
ArH), 7.80 (td, 1H, J = 7.6, 1.2 Hz, ArH), 7.85 (t, 1H, J = 7.6 Hz, ArH), 7.94 (d, 1H, J = 7.2
Hz, ArH), 8.15 (d, 1H, J = 7.6 Hz, ArH), 8.30 (dd, 1H, J = 8.0, 0.8 Hz, ArH), 8.62 (d, 1H, J
1
3
=
7.2 Hz, ArH). C NMR (100 MHz, CDCl ): δ 23.13 (2C), 51.04, 53.32 (2C), 122.79,
3
1
1
6
5
25.00, 128.54, 130.49, 133.22, 133.70, 136.76, 136.87, 136.99, 140.82, 141.34, 142.59,
+
49.82, 156.51, 189.08. ESIMS [M+H] : 316.03. Anal. calc. for C H N O．0.9 HBr: C
2
0
17
3
0.60, H 4.56, N 10.60; found: C 60.56, H 4.61, N 10.40.
.9.3. 6-(Piperidin-1-ylmethyl)-11H-indeno[1,2-b]quinoxalin-11-one (16c).
Compound 16c was obtained from 11 (0.98 g, 3.0 mmol) and piperidine (0.51 g, 6.0 mmol)
as described for the preparation of 16b. Compound was crystallized from EtOH in 62%
o
1
yield as a yellow solid. Mp: 175-176 C (from EtOH, decomp.). H NMR (400 MHz, CDCl ):
3
δ 1.44 (quin, 2H, J = 5.6 Hz, piperidinyl-H), 1.61-1.67 (quin, 4H, J = 5.6 Hz, piperidinyl-H),
2
7
8
5
1
.56 (br, 4H, piperidinyl-H), 4.25 (s, 2H, ArCH N), 7.56 (td, 1H, J = 7.6, 0.8 Hz, ArH),
2
.70-7.77 (m, 2H, ArH), 7.89-7.92 (m, 1H, ArH), 7.95 (dd, 1H, J = 7.2, 0.8 Hz, ArH),
13
.10-8.13 (m, 2H, ArH). C NMR (100 MHz, CDCl ): δ 24.29, 26.09 (2C), 54.80 (2C),
3
7.00, 122.44, 124.63, 129.89, 130.19, 132.24, 132.65, 136.00 (2C), 138.09, 141.89 (2C),
+
42.56, 148.75, 155.50, 190.27. ESIMS [M+H] : 330.09. Anal. calc. for C H N O．0.3
21
19
3
H O: C 75.32, H 5.91, N 12.55; found: C 75.13, H 5.91, N 12.41.
2
5
.9.4. 6-(Morpholinomethyl)-11H-indeno[1,2-b]quinoxalin-11-one (16d).
Compound 16d was obtained from 11 (0.98 g, 3.0 mmol) and morpholine (0.52 g, 6.0 mmol)
o
as described for the preparation of 16b in 72% yield as a yellow solid. Mp: 237-238 C
1
(
from EtOH, decomp.). H NMR (400 MHz, CDCl ): δ 2.64 (t, 4H, J = 4.8 Hz,
3
N(CH CH ) O), 3.76 (t, 4H, J = 4.8 Hz, N(CH CH ) O), 4.29 (s, 2H, ArCH N), 7.58 (td, 1H,
2
2 2
2
2 2
2
2
0

ACCEPTED MANUSCRIPT
J = 7.6, 0.8 Hz, ArH), 7.71-7.79 (m, 2H, ArH), 7.91 (d, 1H, J = 7.6 Hz, ArH), 7.96 (dd, 1H,
J = 7.2, 0.8 Hz, ArH), 8.10 (d, 1H, J = 7.2 Hz, ArH), 8.13 (dd, 1H, J = 7.6, 1.2 Hz, ArH).
1
3
C NMR (100 MHz, CDCl ): δ 53.80 (2C), 56.62, 67.06 (2C), 122.47, 124.69, 129.86,
3
1
30.54, 132.37, 132.68, 136.62, 136.68, 137.13, 141.75, 141.85, 142.59, 148.91, 155.62,
+
1
90.14. ESIMS [M+H] : 332.04. Anal. calc. for C H N O : C 72.48, H 5.18, N 12.68;
20
17
3
2
found: C 72.23, H 5.28, N 12.69.
5
.9.5. 6-[(1,4'-Bipiperidin)-1'-ylmethyl]-11H-indeno[1,2-b]quinoxalin-11-one (16e).
Compound 16e was obtained from 11 (0.98 g, 3.0 mmol) and N-(4-piperidino)piperidine
(1.01 g, 6.0 mmol) as described for the preparation of 16b in 60% yield as a green solid. Mp:
o
1
1
98-200 C (from EtOH, decomp.). H NMR (400 MHz, CDCl ): δ 1.45-1.47 (m, 2H,
3
piperidinyl-H), 1.61-1.87 (m, 8H, piperidinyl-H), 2.17-2.23 (m, 2H, piperidinyl-H),
.36-2.42 (m, 1H, piperidinyl-H), 2.52 (br, 4H, piperidinyl-H), 3.09-3.12 (m, 2H,
piperidinyl-H), 4.26 (s, 2H, ArCH N), 7.57 (td, 1H, J = 7.6, 1.2 Hz, ArH), 7.70-7.78 (m, 2H,
2
2
1
3
ArH), 7.90-7.95 (m, 2H, ArH), 8.09-8.14 (m, 2H, ArH). C NMR (100 MHz, CDCl ): δ
3
2
1
4.54, 25.96 (2C), 27.74 (2C), 50.10 (2C), 53.61 (2C), 56.15, 62.82, 122.44, 124.64, 129.90,
30.29, 132.28, 132.55, 136.62 (2C), 137.98, 141.81 (2C), 142.57, 148.81, 155.53, 190.20.
+
ESIMS [M+H] : 413.11. Anal. calc. for C H N O．0.2 H O: C 75.03, H 6.89, N 13.47;
2
6
28
4
2
found: C 74.95, H 6.85, N 13.40.
.10.1. 6-[(Dimethylamino)methyl]-11H-indeno[1,2-b]quinoxalin-11-one
5
O-[3-(dimethylamino)propyl] oxime hydrochloride (17a).
A mixture of 16a (0.29 g, 1.0 mmol) and 3-(dimethylamino)propoxyamine hydrochloride
(0.61 g) in EtOH (5.0 mL) was refluxed for 6 h (TLC monitoring). The mixture was cooled
and concentrated, then dissolved in water (20 mL), neutralized by K CO (0.41 g, 3.0 mmol),
2
3
and extracted with CH Cl three times. The organic layer evaporated in vecuo and purified
2
2
by column chromatography (CH Cl /MeOH/NH OH 100/20/1). The oil compound was
2
2
4
stirred with 3N HCl (3.0 mL) in EtOH (2.0 mL) for 0.5 h, then evaporated in vecuo. The
o
residue was dried to give 17a (0.31 g, 57% yield) as a green solid. Mp: 234-236 C
2
1

ACCEPTED MANUSCRIPT
1
(
decomp.). H NMR (400 MHz, D O): δ 2.21-2.25 (m, 2H, OCH CH CH N), 2.82 (s, 6H,
2 2 2 2
N(CH ) ), 2.85 (s, 6H, N(CH ) ), 3.24-3.28 (m, 2H, OCH CH CH N), 4.41 (t, 2H, J = 5.6
3
2
3 2
2
2
2
Hz, OCH CH CH N), 4.51 (s, 2H, ArCH N), 7.45-7.53 (m, 2H, ArH), 7.63 (t, 1H, J = 7.6
2
2
2
2
1
3
Hz, ArH), 7.73-7.80 (m, 3H, ArH), 7.88 (d, 1H, J = 7.2 Hz, ArH). C NMR (100 MHz,
D O): δ 24.32, 42.90 (2C), 43.01 (2C), 54.99, 57.27, 73.23, 122.45, 127.58, 128.93, 130.13,
2
1
31.26, 132.10, 132.95, 133.25, 134.24, 135.44, 140.25, 140.31, 146.98, 149.92, 152.68.
+
ESIMS [M+H] : 390.10. Anal. calc. for C H N O．4.0 HCl: C 51.58, H 5.85, N 13.08;
2
3
27
5
found: C 51.55, H 5.84, N 13.20.
.10.2. 6-(Pyrrolidin-1-ylmethyl)-11H-indeno[1,2-b]quinoxalin-11-one
5
O-[3-(dimethylamino)propyl] oxime hydrochloride (17b).
A mixture of 16b (0.32 g, 1.0 mmol) and 3-(dimethylamino)propoxyamine hydrochloride
(0.60 g) in EtOH (5.0 mL) was refluxed for 6 h. The mixture was cooled and concentrated,
then dissolved in water (20 mL), neutralized by K CO (0.41 g, 3.0 mmol), and extracted
2
3
with CH Cl three times. The organic layer evaporated in vecuo and purified by column
2
2
chromatography (CH Cl /MeOH/NH OH 100/20/1). The oil compound was stirred with 3N
2
2
4
HCl (3.0 mL) in EtOH (2.0 mL) for 0.5 h, then evaporated in vecuo. The residue was
dissolved in EtOH (2 mL), then poured into acetone (50 mL). The resulting precipitate was
o
1
filtered and dried to give 17b (0.32 g, 66% yield) as a green solid. Mp: 113-114 C. H NMR
(
400 MHz, D O): δ 1.89-1.95 (m, 2H, pyrrolidinyl-H), 2.08-2.14 (m, 2H, pyrrolidinyl-H),
2
2
.18-2.25 (m, 2H, OCH CH CH N), 2.87 (s, 6H, N(CH ) ), 3.18-3.28 (m, 4H,
2 2 2 3 2
OCH CH CH N, pyrrolidinyl-H), 3.39-3.45 (m, 2H, pyrrolidinyl-H), 4.36 (t, 2H, J = 6.0 Hz,
2
2
2
OCH CH CH N), 4.54 (s, 2H, ArCH N), 7.40-7.48 (m, 2H, ArH), 7.57-7.61 (m, 1H, ArH),
2
2
2
2
1
3
7
2
1
1
6
.66-7.73 (m, 3H, ArH), 7.80 (dd, 1H, J = 6.4, 1.2 Hz, ArH). C NMR (100 MHz, D O): δ
2
2.70 (2C), 24.30, 42.94 (2C), 53.56, 54.47 (2C), 55.02, 73.20, 122.32, 128.53, 128.89,
30.08, 131.00, 132.02, 132.86, 133.13, 133.77, 135.46, 140.13, 140.21, 146.88, 149.72,
+
52.60. ESIMS [M+H] : 416.14. Anal. calc. for C H N O．2.0 HCl．3.3 H O: C 54.78, H
2
5
29
5
2
.93, N 12.78; found: C 54.47, H 6.98, N 12.69.
2
2

ACCEPTED MANUSCRIPT
.10.3. 6-(Piperidin-1-ylmethyl)-11H-indeno[1,2-b]quinoxalin-11-one O-[3-(dimethyl-
5
amino)propyl] oxime (17c).
Compound 17c was obtained from 16c (0.33 g, 1.0 mmol) and
3
1
1
-(dimethylamino)propoxyamine hydrochloride (0.60 g) as described for the preparation of
o
1
7b in 74% yield as a green solid. Mp: 218-220 C (decomp.). H NMR (400 MHz, D O): δ
2
.39-1.69 (m, 4H, piperidinyl-H), 1.82-1.85 (m, 2H, piperidinyl-H), 2.17-2.24 (m, 2H,
OCH CH CH N), 2.86 (s, 6H, N(CH ) ), 2.95-3.02 (m, 2H, piperidinyl-H), 3.22-3.27 (m,
2
2
2
3 2
2
H, OCH CH CH N), 3.35-3.39 (m, 2H, piperidinyl-H), 4.35 (t, 2H, J = 6.4 Hz,
2 2 2
OCH CH CH N), 4.46 (s, 2H, ArCH N), 7.41-7.50 (m, 2H, ArH), 7.57-7.61 (m, 1H, ArH),
2
2
2
2
1
3
7
.65-7.67 (m, 2H, ArH), 7.70 (dd, 1H, J = 6.8, 1.2 Hz, ArH), 7.81-7.83 (m, 1H, ArH). C
NMR (100 MHz, D O): δ 21.14, 22.62 (2C), 24.31, 42.95 (2C), 53.48 (2C), 55.04 (2C),
2
7
1
3.21, 122.25, 127.34, 128.92, 130.00, 131.17, 132.09, 132.92, 133.17, 134.47, 135.53,
+
40.27, 140.58, 146.94, 149.69, 152.62. ESIMS [M+H] : 430.13. Anal. calc. for
C H N O．2.0 HCl．2.9 H O: C 56.28, H 7.06, N 12.62; found: C 55.91, H 7.04, N 12.54.
26
31
5
2
5
.10.4. 6-(Morpholinomethyl)-11H-indeno[1,2-b]quinoxalin-11-one O-[3-(dimethyl-
amino)propyl] oxime hydrochloride (17d).
Compound 17d was obtained from 16d (0.33 g, 1.0 mmol) and
3
-(dimethylamino)propoxyamine hydrochloride (0.61 g) as described for the preparation of
o
1
1
7b in 61% yield as a green solid. Mp: 224-226 C (decomp.). H NMR (400 MHz, CDCl ):
3
δ 2.19-2.26 (m, 2H, OCH CH CH N), 2.88 (s, 6H, N(CH ) ), 3.25-3.29 (m, 4H,
2
2
2
3 2
OCH CH CH N, morpholinyl-H), 3.38-3.41 (m, 2H, morpholinyl-H), 3.66-3.72 (m, 2H,
2
2
2
morpholinyl-H), 3.99-4.03 (m, 2H, morpholinyl-H), 4.36 (t, 2H, J = 6.0 Hz,
OCH CH CH N), 4.60 (s, 2H, ArCH N), 7.43-7.52 (m, 2H, ArH), 7.60-7.65 (m, 2H, ArH),
2
2
2
2
1
3
7
2
1
1
.70 (d, 1H, J = 6.8 Hz, ArH), 7.76-7.80 (m, 2H, ArH). C NMR (100 MHz, CDCl ): δ
3
4.28, 42.95 (2C), 51.92 (2C), 55.03, 55.12, 63.71 (2C), 73.19, 122.32, 126.47, 128.92,
30.04, 131.45, 132.05, 132.91, 133.20, 134.82, 135.47, 140.24, 140.57, 146.87, 149.68,
+
52.69. ESIMS [M+H] : 432.15. Anal. calc. for C H N O ．2.0 HCl．4.6 H O: C 51.11, H
2
5
29
5
2
2
2
3

ACCEPTED MANUSCRIPT
6
.91, N 11.92; found: C 50.82, H 6.98, N 11.84.
5
.10.5. 6-[(1,4'-Bipiperidin)-1'-ylmethyl]-11H-indeno[1,2-b]quinoxalin-11-one
O-[3-(dimethylamino)propyl] oxime hydrochloride (17e).
Compound 17e was obtained from 16e (0.41 g, 1.0 mmol) and
3
-(dimethylamino)propoxyamine hydrochloride (0.60 g mmol) as described for the
o
1
preparation of 17b in 72% yield as a white solid. Mp: 225-227 C (decomp.). H NMR (400
MHz, D O): δ 1.37-1.89 (m, 7H, piperidinyl-H), 2.22-2.29 (m, 4H, OCH CH CH N,
2
2
2
2
piperidinyl-H), 2.85 (s, 6H, N(CH ) ), 2.90-3.00 (m, 2H, piperidinyl-H), 3.14-3.30 (m, 5H,
3
2
OCH CH CH N, piperidinyl-H), 3.40-3.63 (m, 5H, piperidinyl-H), 4.46 (t, 2H, J = 6.0 Hz,
2
2
2
OCH CH CH N), 4.61 (s, 2H, ArCH N), 7.50-7.60 (m, 2H, ArH), 7.69-7.85 (m, 3H, ArH),
2
2
2
2
1
3
7
.88 (dd, 1H, J = 8.4, 1.6 Hz, ArH), 7.97 (d, 1H, J = 7.2 Hz, ArH). C NMR (100 MHz,
D O): δ 21.27, 23.09 (2C), 24.40, 42.95 (2C), 50.59 (2C), 51.09, 55.06 (2C), 55.19, 60.01,
2
7
1
3.30, 122.52, 126.98, 129.04, 130.07, 131.60, 132.30, 133.08, 133.27, 134.70, 135.77,
+
40.46, 140.72, 147.24, 150.06, 153.02. ESIMS [M+H] : 513.27. Anal. calc. for
C H N O．3.0 HCl．4.0 H O: C 53.62, H 7.42, N 12.11; found: C 53.29, H 7.45, N 11.97.
31
40
6
2
5
5
.11. Pharmacological methods
.11.1. Antiproliferative Assay.
Cancer cells (MDA-MB231, H1299, PC-3, Huh-7) and normal mammary lung cell
(
MRC-5) were purchased from Bioresources Collection and Research Center, Taiwan. Cell
lines were maintained in the same standard medium and grown as a monolayer in DMEM
Gibco,USA) and supplemented with 10% fetal bovine serum (FBS) and antibiotics i.e. 100
(
IU/mL penicillin, 0.1 mg/mL streptomycin and 0.25 ꢀg/mL amphotercin. Culture was
maintained at 37°C with 5% CO in a humidified atmosphere.
2
3
Cells (5 x 10 cells/well) were treated as indicated for 72 h in medium containing 10%
FBS. Cell viability was quantitated with the use of sodium 3’-[1-(phenylamino-carbonyl)-
3
,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) colorimetric
assay (Biological Industries, Beit-Haemek, Israel). XTT labeling reagent (1 mg/mL) was
2
4

ACCEPTED MANUSCRIPT
mixed with electron-coupling reagent, following the manufacturer's instructions, and 50 ꢀL of
the mixture was added directly to the cells. The plates were further incubated at 37°C for 4 h.
Color was measured spectrophotometrically in a microtiter plate reader at 492 nm and used as
a relative measure of viable cell number. The number of viable cells following treatment was
compared to solvent and untreated control cells and used to determine the percent of control
growth as (Abtreated/ Abcontrol) x100, where Ab represents the mean absorbance (n = 3). The
concentration that killed 50% of cells (GI ) was determined from the linear portion of the
5
0
curve by calculating the concentration of agent that reduced absorbance in treated cells,
compared to control cells, by 50% [22].
5
.11.2. DNA mobility assay (DNA unwinding).
Negative supercoiled pBR322 (400 ng) was incubated in TE buffer, pH = 8.0, with 10
µM of different indeno[1,2-b] quinoxaline derivatives for 12 h at room temperature.
Following the addition of 2 µL of loading buffer (5% sarkosyl, 0.0025% bromophenol blue,
-1
2
5% glycerol), the samples were loaded onto a 1% agarose gel. The gel was run at 6 V cm
for 2.5 h in TAE buffer (40 mM Tris, 20 mM sodium acetate, 1 mM EDTA-Na , pH = 8.5)
2
and stained with ethidium bromide and photographed under UV illumination using a Polaroid
instant film [16].
5
.11.3. Topoisomerases (topo I and topo II) Inhibitory Assay
Type I or type II DNA topoisomerase was assayed by measuring the decreased mobility
of the relaxed isomers of supercoiled pBR322 DNA in an agarose gel after it had been treated
with human topo I or II. The standard topoisomerase assay mixture (20 µl) contained: 40 mM
of Tris-HCl (pH 7.5), 100 mM of KCl, 10 mM of DTT, 0.5 mM of EDTA, 10 mM of MgCl2,
3
0 µg/ml of BSA, 0.2 µg of pBR322 DNA, and two units of enzyme (one unit is defined as
the amount of enzyme required to convert 0.2 µg of supercoiled DNA substrate to the relaxed
form under standard assay conditions). Reactions were done at 37°C for 30 minutes, and then
terminated by adding 0.5% SDS, 0.25 µg/ml of bromophenol blue, and 15% glycerol. The
samples were electrophoresed in a horizontal 1% agarose gel in Tris-acetate/EDTA buffer (40
2
5

ACCEPTED MANUSCRIPT
mM Tris-acetate, 2 mM EDTA [pH 8]) at 1.5 V/cm for 2-3 h at room temperature. DMSO
concentrations in each reaction were maintained at 0.5% by adding serially diluted drug
stocks so as not to produce solvent-mediated inhibition of topo I or II activity. The gels were
stained with ethidium bromide (5 µg/ml), de-stained in water, and photographed under UV
light. The relaxation percentage was measured using a laser microdensitometer (2202
Ultrascan; LKB, Bromma, Stockholm, Sweden) to analyze negative photographs of
supercoiled monomer DNA band fluorescence after ethidium bromide staining, and the area
under the peak was calculated [28, 29].
5
.11.4. Cell cycle analysis.
Huh-7 cells were treated with DMSO, 10a at different concentrations (1.0, 5.0, 10.0 ꢀM)
for 24 h. Cells were harvested, rinsed in PBS, resuspended, fixed in 70% ethanol, and stored
at -20°C in fixation buffer until ready for analysis. The pellets were suspended in 1 mL of
propidium iodide (PI) solution containing 20 µg/ꢀL of PI, 0.2mg/mL RNase, and 0.1% (v /v)
Triton X-100. Cell samples were incubated at room temperature in the dark for at least 30 min
and analyzed by a flow cytometer (Coulter Epics). Data recording was made using Epics
software and cell cycle data were analyzed using Multicycle software (coulter).
5
.11.5. Immunoblot analysis.
After treatment of compound 10a, cells were collected and washed twice with cold
PBS and then lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet
P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO , 10 mM NaF, 1 mM DTT, 1 mM PMSF, 25
3
µg/mL aprotinin, and 25 µg/mL leupeptin) and kept on ice for 30 min. The lysates were
o
o
centrifuged at 12,000g at 4 C for 20 min and the supernatants were stored at -70 C. The
protein concentration was determined by the Bradford method. 20 ꢀg protein were separated
by 10% SDS-PAGE and transferred onto a PVDF membrane using a glycine transfer buffer
(
192 mM glycine, 25 mM Tris-HCl, pH 8.8, and 20% methanol [v/v]). After blocking with
% non-fat dried milk, the membrane was incubated for 2 h with primary antibodies,
followed by 30 min with secondary antibodies in milk containing Tris-buffered saline (TBS)
5
2
6

ACCEPTED MANUSCRIPT
and 0.5% Tween. The membrane was then exposed to X-ray film. Protein bands were
detected using the enhanced chemiluminescence blotting detection system (Amersham,
USA).
5
.11.6. Zebrafish xenograft assay
The zebrafish xenograft assay was used for validating the in vivo anti-liver cancer
effect of 10a. The procedure was performed according to a previous study with minor
modifications [30]. In brief, the 48 hour post-fertilization (hpf) zebrafish embryos were
anesthetized with 0.01%. About 100 cells of Huh-7 were transplanted in to the yolk sac of
embryo. Afterwards, embryos were incubated in water with different concentrations of 10a
for 24 and 48 hour post-injection (hpi) respectively. The images of embryos were captured by
the fluorescence microscope (Nikon Eclipse TE2000-U, Tokyo, Japan).
Acknowledgements
Financial support of this work by grants from the Ministry of Science and Technology, the
Republic of China (NSC 101-2320-B-037-039-MY3; NSC 101-2320-B-037-046-MY3; NSC
1
02-2320-B-037-005-MY2; MOST103-2320-B-037-011-MY3) and Kaohsiung Medical
University (KMU-TP103H06; KMU-TP103H07; KMU-TP103H08; KMU-TP103A17;
KMU-TP104H07; KMU-TP104H08; KMU-TP104E16; KMU-TP104E42; KMU-Q104016)
are gratefully acknowledged. We also thank the National Center for High-Performance
Computing for providing computer resources and chemical database services.
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