2-(3-Aryl-2-propenoyl)-3-methylquinoxaline-1,4-dioxides: A novel cluster of tumor-specific cytotoxins which reverse multidrug resistance

Article abstract of DOI:10.1016/j.bmc.2009.04.021

A series of 2-(3-aryl-2-propenoyl)-3-methylquinoxaline-1,4-dioxides 3a-l were prepared by condensation of various aryl aldehydes with 2-acetyl-3-methylquinoxaline-1,4-dioxide 2. These compounds inhibit the growth of human Molt 4/C8 and CEM T-lymphocytes a

Full text of DOI:10.1016/j.bmc.2009.04.021

Bioorganic & Medicinal Chemistry 17 (2009) 3909–3915
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-(3-Aryl-2-propenoyl)-3-methylquinoxaline-1,4-dioxides: A novel cluster
of tumor-specific cytotoxins which reverse multidrug resistance
a
a
b
b
c
Umashankar Das a, , Hari N. Pati , Atulya K. Panda , Erik De Clercq , Jan Balzarini , Joseph Molnár ,
*
Zoltán Baráth c, Imre Ocsovszki c, Masami Kawase d, Li Zhou e, Hiroshi Sakagami f, Jonathan R. Dimmock a,
*
a Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5C9
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
c Institute of Medical Microbiology and Immunology, University of Szeged, Szeged, Hungary
d Faculty of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan
e Meikai Pharmaco-Medical Laboratory (MPL), Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Saitama 350-0238, Japan
f Division of Pharmacology, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Saitama 350-0238, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 February 2009
Revised 8 April 2009
Accepted 11 April 2009
Available online 17 April 2009
A series of 2-(3-aryl-2-propenoyl)-3-methylquinoxaline-1,4-dioxides 3a–l were prepared by condensa-
tion of various aryl aldehydes with 2-acetyl-3-methylquinoxaline-1,4-dioxide 2. These compounds inhi-
bit the growth of human Molt 4/C8 and CEM T-lymphocytes and the IC50 values are mainly in the 5–
30 lM range. The quinoxaline 1,4-dioxide 3j inhibited the growth of 58 human tumor cell lines, partic-
ularly leukemic and breast cancer neoplasms. All of the compounds 3a–l reversed the multidrug resis-
tance (MDR) properties of murine L-5178Y leukemic cells which were transfected with the human
MDR1 gene. The MDR-reversing effect may be due to the conjugated p-electron system forming a weak
Keywords:
a,b-Unsaturated ketones
electron charge transfer complex with the P-glycoprotein-mediated efflux pump. The compounds in ser-
ies 2 and 3 were assessed against HL-60, HSC-2, HSC-3 and HSC-4 malignant cells as well as HGF, HPC and
HPLF normal cell lines which revealed that the majority of the compounds displayed a greater toxicity to
neoplastic than normal cells. Various ways in which the project may be expanded are presented.
Ó 2009 Elsevier Ltd. All rights reserved.
Quinoxalines
Cytotoxicity
Multidrug resistance revertants
Structure–activity relationships
QSAR
1. Introduction
other scaffolds may lead to additional series of compounds which
reverse MDR.
A major interest in our laboratory is the design and synthesis of
conjugated arylidene ketones as antineoplastic agents.1,2 This deci-
The aim of the present investigation was to prepare a series of
2-(3-aryl-2-propenoyl)-3-methylquinoxaline 1,4-dioxides 3a–l in
order to explore the hypothesis that such compounds represent a
novel class of cytotoxic agents which reverse MDR. Such com-
pounds may be regarded as dual agents which exert their antineo-
plastic effect and are not effluxed rapidly from malignant cells. The
decision was made to attach the 3-aryl-2-propenoyl group to a
quinoxaline 1,4-dioxide scaffold and the reasons for the choice of
this heterocycle are as follows. (1) A number of neoplasms have
hypoxic regions8 which may permit reduction of the N-oxides to
the corresponding tertiary amines. Various tumors are more acidic
than the corresponding normal cells9 and in such cases there will
be a higher percentage of protonated amines among the tumors.
The quadrivalent nitrogen atoms will exert a strongly electron-
attracting influence on the 3-aryl-2-propenoyl group thereby
increasing the fractional positive charge on the b carbon atom of
the olefinic linkage and thus increasing the thiol-alkylating proper-
ties of the molecules. If this process of reduction followed by
protonation occurs preferentially in tumor cells, then the rate
and extent of interactions with cellular constituents will be greater
in neoplasms and selective toxicity to tumors takes place. (2)
sion is based on the avidity of
a,b-unsaturated ketones for thiols
and not amino or hydroxyl groups.3,4 Hence interactions with
nucleic acids (which contain amino and hydroxyl functionalities,
but not mercapto groups) should be avoided and the problems of
genotoxicity associated with a number of anticancer drugs5 should
be absent. Hence the present study is a continuation of the quest to
find novel antineoplastic agents which are structurally divergent
from drugs used today. Thus the problem of cross-resistance may
be absent and such prototypic molecules may be able to treat
drug-resistant tumors.
In addition, the discovery in our laboratories of the multidrug
resistance (MDR) revertant properties of compounds containing
the 3-aryl-2-propenoyl pharmacophore has recently been
reported.6,7 Hence the attachment of this structural moiety to
* Corresponding authors. Tel.: +1 306 966 6358; fax: +1 306 966 6377 (U.D.), tel.:
+1 306 966 6331; fax: +1 306 966 6377 (J.R.D.).
E-mail addresses: umashankar.das@usask.ca (U. Das), jr.dimmock@usask.ca (J.R.
Dimmock).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.021

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U. Das et al. / Bioorg. Med. Chem. 17 (2009) 3909–3915
Table 1
Studies have revealed that certain malignant cells are more sensi-
tive to sequential chemical attacks than the corresponding normal
cells.10,11 In other words, greater chemosensitization may occur
with neoplasms than non-malignant cells. Hence the successive
reduction of the two N-oxides thereby permitting an alteration in
the rate of thiol alkylation may lead to preferential cytotoxicity
towards neoplasms. In the heterocycle chosen, the two nitrogen
atoms are in different molecular environments and in order to
exacerbate this differential, the electron-releasing methyl group
was placed in the 3 position of the heterocycle.
The last aspect of the design of series 3 involved the choice of
the substituents in ring A which may enable an understanding of
which physicochemical parameters influence any cytotoxic and
MDR-revertant properties that may be observed. The differing
electronic, hydrophobic and steric effects of aryl substituents
may be measured using the Hammett sigma, Hansch pi and molec-
ular refractivity (MR) constants, respectively. The groups in ring A
Atomic charges on the olefinic atoms of 3a and related compounds
X
CH3
α
Y
β
O
X
Y
Atomic charges (esu)
atom b atom
a
N?O
N
(+) NH
N
N?O
N
N
(+)NH
(+)NH
À0.231
À0.230
À0.284
À0.333
À0.377
À0.024
À0.035
0.046
0.081
0.153
(+)NH
Table 2
Evaluation of 2 and 3a–l against human Molt 4/C8 and CEM T-lymphocytes and as
candidate MDR revertants
have divergent
r and p values and are present in all four quadrants
of the Craig plot.12 The MR figures varied from 2.98 to 23.61.
A preliminary communication revealed that 3a–j,l are cytotoxic
to two murine neoplasms, namely B16 melanoma and L1210
leukemia cell lines.13 This report describes the design and synthe-
ses of 3a–l and their evaluation against human cell lines and also
as candidate MDR revertants.
Compound
Aryl substituents
IC50
Molt 4/C8
(
l
M)
FAR valuesb
a
CEM
20
lM
200 lM
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
—
H
44.6 24.0
6.76 1.5
25.8 5.4
18.5 10.0
5.51 0.7
24.2 0.2
8.40 0.8
7.75 0.1
10.1 3.2
17.1 1.0
6.40 0.4
261 67
87.4 54.6
5.92 0.3
27.6 18.6
19.2 16.5
5.22 0.1
28.1 10.6
22.4 13.7
16.0 12.3
15.4 0.6
11.6 7.5
10.0 2.7
164 12
1.06
10.9
4.25
5.01
8.15
1.58
9.79
2.63
4.42
2.03
4.81
1.28
1.89
—
1.04
36.8
31.7
23
4-OCH3
3,4-(OCH3)2
3,4,5-(OCH3)3
3,4-OCH2O
4-CH3
4-Cl
41.9
3.67
27.2
5.97
11.9
2.81
25.8
2.25
3.75
—
2. Results
The compounds in series 2 and 3 were synthesized using the
procedure outlined in Scheme 1. The electron densities on the ole-
finic carbon atoms of 3a and related compounds were undertaken
and the results are portrayed in Table 1. The N-oxides 2 and 3a–l
were examined for cytostatic properties using human Molt 4/C8
and CEM T-lymphocytes and these data are presented in Table 2.
Correlations were sought between the magnitude of the cytostatic
3,4-Cl2
4-Br
4-F
4-NO2
3-NO2
—
3l
16.3 6.4
3.24 0.8
15.8 6.0
2.47 0.3
Melphalanc
a
The IC50 figures are the concentrations required to inhibit the growth of the
tumor cells by 50%.
and MDR-revertant properties of 3a–l with first, the
r, p and MR
b
The fluorescence activity ratio (FAR) values are the ratios of the fluorescent
constants of the R1–R3 atoms or groups and second, the torsion
angle between the olefinic moiety and the adjacent aryl ring. The
growth-inhibitory properties of three representative compounds,
namely 3b,c,j, were assessed against a panel of 59 human tumor
cell lines from nine different neoplastic conditions using a concen-
intensities of rhodamine 123 in treated and untreated murine L-5178Y tumor cells
which are transfected with the human MDR1 gene. The FAR value of a reference
compound verapamil is 7.86 using a concentration of 5.2 lM.
c
The data were previously reported in Ref. 31 [copyright (2006) by Elsevier].
tration of 10 lM. The greatest potency was displayed by 3j as illus-
lymphoma cells transfected with the human MDR1 gene and the
results are given in Table 2. All of the compounds in series 2 and
3 were assayed against HL-60 promyelocytic leukemic cells and
HSC-2, HSC-3 and HSC-4 squamous cell carcinomas. In addition,
these molecules were evaluated using non-malignant HGF gingival
trated in Figure 1. This compound was evaluated further using
concentrations of 10À4, 10À5, 10À6, 10À7 and 10À8 molar and the
marked inhibitory potency towards some leukemic and breast can-
cer cell proliferation is presented in Figure 2. The MDR-revertant
properties of 2 and 3a–l were examined using murine L-5178
O
O
O
R1
N
CH3
R2
R3
4
N
CH3
CH3
3
N
N
ii
i
α
O
2
N
O
N
O
1
β
O
O
1
3
2
R1
H
H
R2
H
OCH3
R3
H
H
H
R1
H
h: Cl
R2
Cl
Cl
Br
F
R3
H
H
H
H
H
H
a:
b:
g:
c: OCH3 OCH3
d: OCH3 OCH3 OCH3
i:
j:
k:
H
H
H
e:
f:
OCH2O
H
H
NO2
H
H
CH3
l: NO2
Scheme 1. The reagents used in the syntheses of 2 and 3 are as follows, namely (i) CH3COCH2COCH3/N(C2H5)3 and (ii) aryl aldehyde/NaOH.

U. Das et al. / Bioorg. Med. Chem. 17 (2009) 3909–3915
3911
100
80
60
40
20
0
3j
3b
3c
-20
M14
PC-3
A549
A498
U251
K-562
EKVX
HL-60
MCF7
T-47D
UO-31
SW620 SF-295
DU-145
HCT-15
SNB-75
CAKL-1
HOP-92
IGROV1
NCI-H23
RXF 393
HS 578T
HCT-116
LOXIMVI
UACC-62
NCI-H460 NCI-H522
OVCAR-3 OVCAR-4 OVCAR-8
HCC-2998
RPMI-8226
CCRF-CEM
MDA-MB-435
MDA-MB-468
NCI/ADR-RES
Leukemia
Non-small
cell Lung
Melanoma
Renal
Prostate
Breast
Colon
CNS
Ovarian
Figure 1. The growth-inhibiting properties of 10
l
M of 3b,c,j on a number of human tumor cell lines.
Leukemia
Breast Cancer
120
100
80
60
40
120
100
80
60
40
20
0
20
0
-20
-40
-60
-80
-20
-40
-60
-80
-8
-7
-6
-5
-4
-8
-7
-6
-5
-4
Log10 of Sample Concentration (Molar)
Log 10 of Sample Concentration (Molar)
CCRF HL-60 K-562 RPMI-8226
HS 578T
T-47D
MDA-MB-468
MCF-7
Figure 2. Dose response curves of 3j towards some leukemic and breast cancer cell lines.
Table 3
A comparison of the cytotoxic effects of 2 and 3a–l on malignant and normal cells
M)a
Human malignant cells, CC50
(lM)
Ave SI
a
Compound
Human normal cells, CC50
(
l
HGF
HPC
HPLF
>400
35 4.3
93 23
156 7.0
33 1.9
72 3.6
23 1.2
12 0.3
5.7 0.1
26 3.6
20 8.6
91 26
18 1.2
Ave
HL-60
SIb
HSC-2
SIb
HSC-3
SIb
HSC-4
SIb
2
>400
352 14
36 1.8
92 11
142 1.5
31 2.5
79 7.3
40 15
21 0.1
13 3.0
43 3.4
48 6.5
>400
384
26 7.5
4.1 0.5
16 1.7
14 1.5
1.9 0.1
13 1.9
4.1 0.8
11 1.1
27 4.1
23 3.4
4.6 0.2
262 39
62 6.8
14.8
8.4
4.9
9.9
17.4
5.8
7.7
1.8
0.6
1.8
196 4.5
8.4 0.3
20 0.2
25 0.5
7.8 0.8
23 2.8
8.4 0.6
13 0.3
17 4.1
19 1.4
6.2 0.1
55 4.7
34 4.0
2.0
4.1
4.0
5.5
4.2
3.3
3.8
1.5
0.9
2.1
5.3
ꢀ3.3
1.1
146 7.5
9.7 0.3
23 0.1
31 1.5
6.6 0.3
25 6.0
14 1.9
38 8.9
84 9.1
56 16
7.7 0.2
>400
2.6
3.5
3.4
4.5
5.0
3.0
2.3
0.5
0.2
0.7
4.3
ꢀ0.5
0.3
134 36
9.6 1.6
11 4.9
38 1.5
6.5 0.3
28 5.5
11 2.6
19 1.7
15 2.6
36 7.4
8.1 0.1
73 20
54 3.9
2.9
3.6
7.2
3.6
5.1
2.7
2.9
1.1
1.1
1.1
4.1
ꢀ2.5
0.7
5.6
4.9
4.9
5.9
7.9
3.7
4.2
1.2
0.7
1.4
5.2
ꢀ1.8
0.7
3a
3b
3c
3d
3e
3f
3g
3h
3i
32 3.7
52 16
117 5.0
35 1.6
75 5.5
32 2.5
27 2.7
29 13
53 11
31 1.0
57 13
47 14
34.3
79.0
138
33.0
75.3
31.7
20.0
15.9
40.7
33.0
>183
37.0
3j
3k
3l
7.2
ꢀ0.7
46 8.4
0.6
122 14
a
The CC50 figure is the concentration of the compound required to reduce the number of viable cells by 50%. The highest concentration used is 400
The letters SI indicate the selectivity index. This figure is the quotient of the average CC50 value of the three normal cell lines and the CC50 figure of a malignant cell line.
l
M.
b
fibroblasts, HPC pulp cells and HPLF periodontal ligament fibro-
blasts. The biodata generated is portrayed in Table 3.
procedure14, reacted with acetylacetone to yield 2-acetyl-3-meth-
ylquinoxaline-1,4-dioxide 2. A number of aryl aldehydes were
condensed with 2 leading to 3a–l. 1H NMR spectroscopy revealed
that the olefinic double bond in 3a–l possesses the E-configuration.
In order to evaluate the hypothesis that reduction followed by
protonation of the compounds in series 3 would lead to increases
in their avidity for cellular thiols vide supra, the atomic charges
on the olefinic carbon atoms of a representative compound 3a
3. Discussion
The synthesis of the desired compounds 3a–l is presented in
Scheme 1. Benzofurazan-1-oxide 1, which was prepared by
hypochlorite oxidation of 2-nitroaniline according to a literature

3912
U. Das et al. / Bioorg. Med. Chem. 17 (2009) 3909–3915
and related molecules were measured. As may be observed from
Table 1, protonation depletes the electron densities on the b carbon
atoms which are the loci of reactions with cellular thiols. These
observations support the design of the compounds that reduction
and subsequent protonation of the compounds in series 3 may
increase thiol alkylation preferentially in neoplastic cells compared
to the corresponding normal tissues.
All of the compounds in series 3 as well as the analog bereft of
an alkylating function namely 2 were evaluated for their inhibitory
activity against human Molt 4/C8 and CEM T-lymphocyte prolifer-
ation. The data are presented in Table 2. The IC50 values of the
evaluated against 59 human tumor cell lines originating from the
following neoplastic conditions, namely leukemia and melanoma
as well as non-small cell lung, colon, central nervous system, ovar-
ian, renal, prostate and breast cancers.17 A concentration of 10
lM
was employed and the data of the effect of 3b,c,j on a number of
these cell lines in Figure 1 reveals clearly that 3j is much more
inhibitory than 3b and 3c. The greater growth-inhibiting properties
of the 4-fluoro analog 3j than 3b and 3c, which contain 4-methoxy
and 3,4-dimethoxy aryl substituents, respectively, may be due, at
least in part, to the positive
r and p values and lower MR figure
of the aryl substituent in 3j. The quinoxaline 1,4-dioxide 3j was
following compounds are less than 10
lM (percentage of the
examined further using concentrations of 10À4–10À8 molar. The
potency of the reference drug melphalan in parentheses) namely
3a (48), 3d (59), 3f (39), 3g (42) and 3j (51) in the Molt 4/C8 assay
and 3a (42) and 3d (47) in the CEM screen which are clearly lead
average IC50 figure of 3.98
lM towards 58 human cancer cell lines
is seven times lower than the inhibitory value for melphalan.18
A
review of the mean graphs19 revealed that significant potencies
are displayed towards various leukemic and breast cancer cells
lines as illustrated in Figure 2. Another noteworthy feature of 3j
is the observation that the IC50 values vary considerably towards
the different malignant cell lines over a 22-fold range, that is, the
IC50 figure of the most sensitive cell line is 22 times lower than
for the most refractory neoplasm. It is conceivable that this differ-
ence in sensitivity may translate into a preferential toxicity for
tumor cells than the corresponding normal tissues. These data
reinforce the decision to pursue these compounds in further
studies.
molecules. The IC50 figures are less than 30 lM for the remaining
compounds in both tests with the exception of the outlier 3k. A
comparison between the IC50 values of 2 and 3a–j,l revealed that
3a,d,f–j,l (Molt 4/C8 assay) and 3a,d,g–j,l (CEM screen) are more
potent than 2 while the remaining compounds in series 3 are equi-
potent with 2. Hence the attachment of the alkylating group to the
heterocycle led to increases in antiproliferative potencies in gen-
eral while some contribution to the antineoplastic properties dis-
played is made by the scaffold to which the alkylating moiety
has been attached.
In order to obtain guidelines for expansion of this project, a
QSAR study as well as molecular modeling were undertaken. The
variation in cytotoxic potencies in series 3 may be governed by
the electronic, hydrophobic and steric properties of the aryl sub-
stituents. Accordingly linear and semilogarithmic plots were made
between the IC50 values of 3a–l generated in the Molt 4/C8 screen
A major problem in cancer chemotherapy is MDR. This phenom-
enon can arise by different biochemical mechanisms including the
overexpression of P-glycoprotein (P-gp) which acts as a drug efflux
pump. The compounds in series 2 and 3 were assayed for MDR-
revertant properties using murine L-5178 lymphoma cells trans-
fected with the MDRI gene.20 These cells have greater amounts of
P-gp than the parental cells. The concentrations of rhodamine
123 in treated and untreated transfected and parental cells were
measured and the ratios of the fluorescence intensities are referred
to as fluorescence activity ratio (FAR) values. A FAR value of greater
than 1 indicates that reversal of MDR has taken place.
and the Hammett
r values, then the Hansch p figures and finally
the molecular refractivity (MR) constants. Logarithmic plots were
also made between the MR values and the IC50 figures, since all
MR constants (but not all
r and p values) are positive. Then the
same approach was used in evaluating series 3 against CEM cells.
No correlations (p > 0.05) were noted although a trend towards a
Initial experiments were conducted using a concentration of
positive correlation was observed between the
r
values and the
20 lM which, in general, is slightly in excess of the IC50 values of
IC50 figures in the Molt 4/C8 screen (p = 0.09) and the CEM bioassay
(p = 0.12). Thus development of these compounds should consider
introducing electron-releasing substituents in the arylidene aryl
ring in order to increase cytotoxic potencies. The statistical analy-
ses were repeated omitting the data for the outlier 3k but no cor-
relations or trends towards significance were observed (p > 0.2).
A further consideration pertaining to physicochemical parame-
ters which may influence cytotoxic potencies of the test com-
pounds is the torsion angles between the arylidene aryl ring and
the adjacent enone group. A number of studies revealed that the
magnitude of bioactivity is dependent on whether an aryl ring is
coplanar or not with an adjacent unsaturated group.15 In some
instances coplanarity is favored while on other occasions a lack
of coplanarity is required for bioactivity. Hence models of 3a–l
were built and the torsion angle h was determined; specific values
are given in the experimental section. Linear, semilogarithmic and
logarithmic plots were constructed between the h figures and the
IC50 values in the Molt 4/C8 and CEM screens. A positive trend
was observed in the Molt 4/C8 assay (p = 0.11) and the CEM test
(p = 0.10) which suggests that a lowering of the interplanar angle
may lead to compounds with greater cytotoxicity. Thus replace-
ment of one or both of the ortho protons of 3a by fluorine (the
MR value of fluorine is 0.92 compared to 1.03 for hydrogen)16
should be considered. No other correlations or trends towards sig-
nificance were observed (p > 0.2).
the compounds towards Molt 4/C8 and CEM cells. These data are
presented in Table 2. The results indicate that all of the compounds
in series 3 have MDR-revertant properties in contrast to 2. Of par-
ticular note is the efficacy of 3a,c,d,f which possess FAR values
greater than 5. The experiment was repeated using a tenfold
increase in compound concentration for two reasons. First, the data
may identify potent MDR-revertants. Second, the FAR values may
rise as the concentration increases. Alternatively, hormesis may
occur, that is, MDR reversal is greater at the lower concentration;
a situation which has been observed previously.21 The data in Table
1 indicate that 3a–d,f,j are potent MDR-revertants having FAR val-
ues in excess of 20. In the case of the other compounds in series 3,
the FAR values increase as the concentration is raised while even
when 200 lM of 2 was used, no reversal of MDR was observed.
These results afford further evidence that the 3-aryl-2-propenoyl
group is an important pharmacophore in counteracting drug
resistance.
In order to seek correlations between the physicochemical
properties of the aryl substituents and MDR reversal potencies,
linear and semilogarithmic plots were constructed between the
r, p and MR constants of the groups in ring A and the FAR values.
Logarithmic plots were also made when the MR values were used.
In addition, linear, semilogarithmic and logarithmic plots were
constructed between the torsion angles h and the FAR values.
When the data for 20
a negative correlation was noted with the
l
M of 3a–l was considered, a trend towards
values (p = 0.10) as
In order to ascertain further the potential of the 2-(3-aryl-2-
propenoyl)-3-methyl quinoxaline 1,4-dioxides 3 as novel candi-
date cytotoxins, three representative compounds 3b,c,f were
r
well as a negative correlation between the h figures and the FAR
values (p = 0.05). When the concentration was increased to

U. Das et al. / Bioorg. Med. Chem. 17 (2009) 3909–3915
3913
200
and the
l
M, a negative correlation was noted between the FAR figures
constants (p < 0.05) and a negative trend towards signif-
4. Conclusions
r
icance found when the h values were considered (p = 0.07). When
these analyses were repeated using 3a–j,l, that is, omitting the data
for the outlier 3k, neither correlations of p < 0.05 nor trends to sig-
nificance of p < 0.1 were noted. One may conclude therefore that,
in general terms, MDR-revertant properties are increased by
employing electron-releasing aryl substituents and reducing the
torsion angles h.
A number of 2-(2-aryl-2-propenyl)-3-methylquinoxaline-1,4-
dioxides 3a–lhave been prepared. The atomic chargeson the olefinic
carbon atoms of 3a and related compounds support the hypothesis
that a reduction–protonation process in tumors may lead to com-
pounds demonstrating preferential toxicity to malignant cells. The
compounds inhibit the growth of human Molt 4/C8 and CEM cells
in the low micromolar range and in particular the potencies
displayed by 3a,d,j establish them to be lead molecules. One of these
three compounds, namely 3j, displays excellent growth-inhibitory
properties towards a range of human neoplasms, especially leuke-
mic and breast cancer cell lines. All of the molecules in series 3
reverse MDR in murine L-5178Y cells and this observation provides
further evidence that the 3-aryl-2-propenoyl group is a MDR-revert-
antpharmacophore. ThehugeFARvaluesof 3a,b,dofover30are par-
ticularly impressive. The physicochemical properties which
enhance the magnitude of the cytotoxic and MDR-revertant proper-
ties are the insertion of electron-releasing substituents in the arylid-
enearyl ring and theloweringof thetorsionangle h. These guidelines
should be useful in the development of this novel cluster of cytotox-
ins which reverse MDR. Finally the demonstration that the majority
of the compounds in series 2 and 3 display greater toxicity to
neoplasms than normalcells provides further evidence of the impor-
tance of developing these novel cytotoxins.
The statistical analyses suggest that both the cytotoxic poten-
cies and MDR-revertant properties of 3a–l are influenced by the
r
and h figures but not by the p and MR physicochemical parame-
ters. The possibility exists therefore that the IC50 and FAR values
are negatively correlated. In order to examine this hypothesis,
linear, semilogarithmic and logarithmic plots were constructed
between the IC50 values in the Molt 4/C8 and CEM screens with
the FAR figures generated when concentrations of 20 and 200 lM
were used. Negative correlations (p < 0.05) were noted between
the IC50 figures of 3a–l in the Molt 4/C8 bioassay and the FAR val-
ues using 20 and 200
when 20 M of compound was used. A trend towards significance
(p = 0.06) was observed when plots were made between the IC50
figures in the CEM screen and the FAR values when 200 M of
lM concentrations and in the CEM screen
l
l
3a–l was employed. The experiments were repeated omitting the
outlier 3k which revealed a negative correlation between the Molt
4/C8 biodata and the FAR values using 20 lM of the compounds
(p < 0.05). No other correlations of p < 0.05 nor trends to a correla-
tion of p < 0.1 were noted. The fact that there is a correlation
between cytotoxic and MDR-revertant properties suggests that
the increased intracellular retention of candidate cytotoxins con-
tributes to a rise in cytotoxic potencies. This observation enhances
the potential for development of these prototypic molecules in ser-
ies 3 insofar as by exploiting the correlations noted, further com-
pounds may be designed with the likelihood of increased
antineoplastic potencies and MDR-revertant properties.
The data presented indicate that the 2-(3-aryl-2-propenoyl)-3-
methylquinoxaline 1,4-dioxides 3 are a novel group of cytotoxins
and possess MDR-revertant properties which may contribute to
the potencies observed. The crucial issue to be addressed is
whether these compounds display greater cytotoxicity for neo-
plasms than normal cells as suggested by the reduction–proton-
ation hypothesis; a theory supported by the atomic charges
portrayed in Table 1. Accordingly all of the compounds in series
2 and 3 were evaluated against malignant HL-60, HSC-2, HSC-3
and HSC-4 cell lines as well as HGF, HPC and HPLF normal cells.
These data are presented in Table 3.
5. Experimental
5.1. Chemistry
Melting points are in degrees Celsius and were recorded using a
Gallenkamp apparatus and are uncorrected. The melting points of
3a,b,f,g,i,k,l recorded in the literature are of non-hydrated materi-
als in contrast to the compounds prepared in this study. 1H NMR
spectra were determined on a Bruker AMX 500 FT machine while
combustion analyses were obtained using an Elementer analyzer.
5.1.1. 2-Acetyl-3-methylquinoxaline-1,4-dioxide 2
A mixture of benzofurazan-1-oxide 1 (0.05 mol), acetylacetone
(0.55 mol), triethylamine (5 mL) and ethanol (20 mL) was stirred
at room temperature for 24 h. The resultant precipitate was col-
lected by filtration, dried and recrystallized from ethanol to give
2, mp 154 °C (lit.22 mp 152 °C) in 78% yield. 1H NMR (CDCl3): d
2.56 (s, 3H, CH3), 2.75 (s, 3H, COCH3), 7.89(m, 2H, Ar-H), 8.58(dd,
1H, Ar-H), 8.65(dd, 1H, Ar-H).
In vivo, the malignant cells of a neoplastic condition will be sur-
rounded by different types of normal cells. Hence in order to assess
whether the quinoxaline N-oxides 2 and 3 have greater toxicity for
malignancies, the CC50 values of each compound towards a cancer
cell line were compared to the average CC50 figure of the three nor-
mal cells. These calculations led to the selectivity index (SI) values
presented in Table 3. The data reveal that in general the com-
pounds display greater toxicity to the cancers than the normal
cells. Thus 81% of the SI figures are greater than 1. This selectivity
is dependent on the cell line since the average SI values for series 3
towards HL-60, HSC-2, HSC-3 and HSC-4 cells are 5.6, 3.3, ꢀ2.4 and
3.0, respectively. In order to identify lead molecules, an average SI
figure of 4 was chosen arbitrarily as an indication of noteworthy
selective toxicity to neoplasms and this criterion was achieved
by 2,3a–d,f,g. In terms of cytotoxicity, the low CC50 values of
3a,d,f,g to the neoplastic cell lines are apparent. Once again the
useful properties of 3j are noted; in this case in terms of both cyto-
toxic potencies and selective toxicity which confirms it to be a pro-
totypic molecule for analog development. As noted previously, 2
and 3k have low cytotoxic potencies.
5.1.2. 2-(3-Aryl-2-propenyl)-3-methylquinoxaline-1,4-dioxides
3a–l
A
mixture of 2-acetyl-3-methylquinoxaline-1,4-dioxide 2
(0.003 mol) and an aryl aldehyde (0.0035 mol) in methanolic so-
dium hydroxide solution (5% w/v, 10 mL) was stirred at 5–10 °C
for 5–10 min. The solid which deposited was collected, washed
with water and recrystallized from chloroform–ethanol to provide
the compounds in series 3.
5.1.2.1. 3-Methyl-2-(3-phenyl-2-propenoyl)-quinoxaline-1,4-
dioxide (3a). Yield: 62%, mp 172–173 °C (lit.22 mp 154 °C). 1H NMR
(CDCl3): d 2.65 (s, 3H, CH3), 7.16 (d, 1H, @CH, J = 16.15 Hz), 7.45 (m,
3H, Ar-H), 7.59 (m, 3H, @CH and Ar-H), 7.90 (m, 2H, Ar-H), 8.60 (d,
1H, Ar-H, J = 8.60 Hz), 8.70 (d, 1H, Ar-H, J = 8.4 Hz). Anal. Calcd for
C18H14N2O3.0.5ÁH2O: C, 68.55; H, 4.47; N, 8.88. Found: C, 68.56; H,
4.58; N, 9.10.
5.1.2.2. 2-[3-(4-Methoxyphenyl)-2-propenoyl]-3-methyl-quin-
oxaline-1,4-dioxide (3b). Yield: 71%, mp 169–170 °C (lit.23 mp

3914
U. Das et al. / Bioorg. Med. Chem. 17 (2009) 3909–3915
186–187 °C). 1H NMR(CDCl3): d 2.59 (s, 3H, CH3), 3.86 (s, 3H,
OCH3), 6.93 (d, 2H, Ar-H, J = 8.74 Hz), 7.02 (d, 1H, @CH,
J = 16.08 Hz), 7.53 (t, 3H, @CH and Ar-H), 7.90 (m, 2H, Ar-H), 8.62
(d, 1H, Ar-H, J = 8.36 Hz), 8.70 (d, 1H, Ar-H, J = 8.44 Hz). Anal. Calcd
for C19H16N2O4Á0.25H2O: C, 66.99; H, 4.73; N, 8.22. Found: C, 66.65;
H, 4.76; N, 8.20.
J = 8.05 Hz). Anal. Calcd for C18H13Br N2O3Á0.25H2O: C, 55.47; H,
3.36; N, 7.18. Found: C, 55.26; H, 3.33; N, 7.10.
5.1.2.10. 2-[3-(4-Fluorophenyl)-2-propenoyl]-3-methylquinox-
aline-1,4-dioxide (3j). Yield: 66%, mp 195–197 °C. 1H NMR (CDCl3):
d 2.59 (s, 3H, CH3), 7.11 (m, 3H, @CH and Ar-H), 7.57 (m, 3H, @CH and
Ar-H), 7.89 (m, 2H, Ar-H), 8.61 (d, 1H, Ar-H, J = 8.23 Hz), 8.69 (d, 1H,
Ar-H, J = 8.60 Hz). Anal. Calcd for C18H13FN2O3Á0.25H2O: C, 65.74; H,
3.98; N, 8.51. Found: C, 65.58; H, 4.02; N, 8.47.
5.1.2.3. 2-[3-(3,4-Dimethoxyphenyl)-2-propenoyl]-3-methyl-
quinoxaline-1,4-dioxide (3c). Yield: 68%, mp 179–180 °C. 1H NMR
(CDCl3): d 2.63 (s, 3H, CH3), 3.96 (s, 3H, OCH3), 3.99 (s, 3H, OCH3),
6.89 (d, 1H, Ar-H, J = 8.37 Hz), 7.05 (d, 1H, @CH, J = 16.10 Hz), 7.10
(s,1H, Ar-H), 7.18 (dd, 1H, Ar-H), 7.51 (d, 1H, @CH, J = 16.07 Hz),
7.90 (m, 2H, Ar-H), 8.63 (d, 1H, Ar-H, J = 8.36 Hz), 8.69 (d, 1H, Ar-
H, J = 8.60 Hz). Anal. Calcd for C20H18N2O5Á0.25H2O: C, 64.76; H,
4.89; N, 7.55. Found: C, 64.37; H, 4.91; N, 7.50.
5.1.2.11. 3-Methyl-2-[4-nitrophenyl)-2-propenoyl]quinoxaline-
1,4-dioxide (3k). Yield: 42%, mp 222–224 °C (lit.22 mp 232 °C). 1H
NMR (CDCl3): d 2.50 (s, 3H, CH3), 7.43 (d, 1H, @CH, J = 16.52 Hz),
7.97 (m, 5H, @CH and Ar-H), 8.27 (d, 2H, Ar-H, J = 8.59 Hz), 8.44
(d, 1H, Ar-H, J = 8.25 Hz), 8.55 (d, 1H, Ar-H, J = 8.48 Hz). Anal. Calcd
for C18H13N3O5Á0.25H2O: C, 60.75; H, 3.68; N, 11.80. Found: C,
60.54; H, 3.52; N, 11.75.
5.1.2.4. 3-Methyl-2-[3-(3,4,5-trimethoxyphenyl)-2-propenoyl]-
quinoxaline-1,4-dioxide (3d). Yield: 67%, mp 180–182 °C. 1H NMR
(CDCl3): d 2.59 (s, 3H, CH3), 3.88 (s, 6H, 2 Â OCH3), 3.91 (s, 3H, OCH3),
6.81 (s, 2H, Ar-H), 7.04 (d, 1H, @CH, J = 16.10 Hz), 7.48 (d, 1H, @CH,
J = 15.98 Hz), 7.93 (m, 2H, Ar-H), 8.62 (d, 1H, Ar-H, J = 7.73 Hz),
8.70 (d, 1H, Ar-H, J = 8.01 Hz). Anal. Calcd for C21H20N2O6Á0.25H2O:
C, 62.91; H, 5.02; N, 6.98. Found: C, 62.62; H, 5.04; N, 7.03.
5.1.2.12. 3-Methyl-2-[3-(3-nitrophenyl)-2-propenoyl]quinoxa-
line-1,4-dioxide (3l). Yield: 58%, mp 206–208 °C (lit.24 mp 220–
222 °C). 1H NMR (CDCl3): d 2.70 (s, 3H, CH3), 7.30 (d, 1H, @CH,
J = 16.18 Hz), 7.63 (t, 1H, Ar-H), 7.51 (d, 1H, @CH, J = 16.09 Hz),
7.94 (m, 3H, Ar-H), 8.30 (d, 1H, Ar-H, J = 8.18 Hz), 8.46 (s, 1H, Ar-
H), 8.61 (d, 1H, Ar-H, J = 7.63 Hz), 8.71 (d, 1H, Ar-H, J = 7.79 Hz).
Anal. Calcd for C18H13N3O5Á0.25H2O: C, 60.75; H, 3.68; N, 11.80.
Found: C, 60.45; H, 3.74; N, 11.69.
5.1.2.5. 2-[3-(3,4-Methylenedioxyphenyl)-2-propenoyl]-3-
methyl-quinoxaline-1,4-dioxide (3e). Yield: 72%, mp 185–
186 °C. 1H NMR (CDCl3): d 2.63 (s, 3H, CH3), 6.05 (s, 2H, O–
CH2–O), 6.83 (d, 1H, Ar-H, J = 8.05 Hz), 6.97 (d, 1H, @CH,
J = 16.05 Hz), 7.07 (dd, 1H, Ar-H), 7.11 (d, 1H, Ar-H, J = 1.47 Hz),
7.50 (d, 1H, @CH, J = 16.04 Hz), 7.89 (m, 2H, Ar-H), 8.61 (d, 1H,
Ar-H, J = 8.37 Hz), 8.68 (d, 1H, Ar-H, J = 8.33 Hz). Anal. Calcd for
C19H14N2O5Á0.25H2O: C, 64.31; H, 3.97; N, 7.89. Found: C,
63.86; H, 4.03; N, 7.97.
5.1.3. Statistical analyses
The Hammett r, Hansch p and MR values were taken from the
literature25 with the exception of the 3,4-methylenedioxy sigma
value which was obtained from another source.26 The MR value
of hydrogen is 1.03 not 0.00. Hence the figures of 1.03, 2.06
(2 Â 1.03) and 3.09 (3 Â 1.03) were added to the MR figures of
the aryl substituents in the case of the disubstituted, monosubsti-
tuted and unsubstituted analogs, respectively. The linear, semilog-
arithmic and logarithmic plots were made using a commercial
software package.27 The following correlations or trends to signif-
icance were noted pertaining to the biodata for 3a–l using linear
(l), semilogarithmic (sl) and logarithmic (log) plots viz. IC50 (Molt
5.1.2.6. 3-Methyl-2-[3-(4-methylphenyl)-2-propenoyl]-quinox-
aline-1,4-dioxide (3f). Yield: 46%, mp 189–191 °C (lit.24 mp 174–
178 °C). 1H NMR (CDCl3): d 2.43 (s, 3H, CH3), 2.59(s, 3H, CH3),
7.11 (d, 1H, @CH, J = 16.15 Hz), 7.23 (d, 2H, Ar-H, J = 7.97 Hz),
7.48 (d, 2H, Ar-H, J = 8.09 Hz), 7.56 (d, 1H, @CH, J = 16.16 Hz),
7.90 (m, 2H, Ar-H), 8.62 (d, 1H, Ar-H, J = 8.32 Hz), 8.70 (d, 1H, Ar-
H, J = 8.32 Hz). Anal. Calcd for C19H16N2O3Á0.25H2O: C, 70.25; H,
4.96; N, 8.62. Found: C, 70.13; H, 5.04; N, 8.31.
4/C8) versus
(Molt 4/C8) versus h: p = 0.11 (l); IC50 (CEM) versus h: p = 0.10
(l); FAR (20 M) versus : p = 0.10 (sl); FAR (20 M) versus h:
p = 0.05 (sl) and p = 0.06 (log); FAR (200 M) versus : p = 0.05
(l) and p = 0.04 (sl); FAR (200 M) versus h: p = 0.11 (l) and
p = 0.07 (sl) and p = 0.09 (log); FAR (20 M) versus IC50 (Molt 4/
r: p = 0.09 (l); IC50 (CEM) versus r: p = 0.12 (l); IC50
l
r
l
l
r
5.1.2.7. 2-[3-(4-Chlorophenyl)-2-propenoyl]-3-methyl-quinox-
aline-1,4-dioxide (3g). Yield: 72%, mp 196–197 °C (lit.22 mp
190 °C). 1H NMR (CDCl3): d 2.59 (s, 3H, CH3), 7.13 (d, 1H, @CH,
J = 16.10 Hz), 7.40 (d, 2H, Ar-H, J = 8.51 Hz), 7.53 (d, 2H, Ar-H,
J = 8.50 Hz), 7.58 (d, 1H, @CH, J = 16.15 Hz), 7.92 (m, 2H, Ar-H),
8.61 (dd, 1H, Ar-H), 8.70 (dd, 1H, Ar-H). Anal. Calcd for
C18H13ClN2O3Á0.25H2O: C, 62.61; H, 3.79; N, 8.11. Found: C,
62.31; H, 3.86; N, 8.08.
l
l
8): p = 0.05 (sl) and p = 0.01 (log); FAR (20
lM) versus IC50
(CEM): p = 0.07 (sl) and p = 0.03 (log); FAR (200
l
M) versus Molt
4/C8: p = 0.09 (sl) and p = 0.04 (log); FAR (200
lM) versus CEM:
p = 0.07 (sl) and p = 0.06 (log). When plots were created using
3a–j, l, the following trend towards significance was observed,
namely FAR (200
lM) versus r: p = 0.12 (l).
5.1.2.8. 2-[3-(3,4-Dichlorophenyl)-2-propenoyl]-3-methylqui-
noxaline-1,4-dioxide (3h). Yield: 69%, mp 211–212 °C. 1H NMR
(CDCl3): d 2.59 (s, 3H, CH3), 7.15 (d, 1H, @CH, J = 16.08 Hz), 7.44
(dd, 1H, Ar-H), 7.51 (d, 1H, Ar-H, J = 8.33 Hz), 7.57 (d, 1H, @CH,
J = 16.12 Hz), 7.69 (d, 1H, Ar-H, J = 1.88 Hz), 7.93 (m, 2H, Ar-H),
8.60 (d, 1H, J = 8.31 Hz), 8.70 (d, 1H, Ar-H, J = 8.22 Hz). Anal. Calcd
for C18H12Cl2N2O3Á0.25H2O: C, 56.93; H, 3.18; N, 7.37. Found: C,
56.74; H, 3.23; N, 7.25.
5.1.4. Molecular modeling
Models of 3a–l were built using BioMedCache 6.1 for Windows28
and the lowest energy conformations were found using optimized
geometry calculations in MOPAC and AM1 parameters. The torsion
angles h determined for the compounds in series 3 are as follows,
namely 3a: 14.0; 3b: 13.0; 3c: 14.9; 3d: 16.4; 3e: 22.1; 3f: 15.0;
3g: 13.5; 3h: 17.1; 3i: 14.3; 3j: 17.4; 3k: 20.9; 3l: 18.9.
5.2. Cytostatic and cytotoxic assays
5.1.2.9. 2-[3-(4-Bromophenyl)-2-propenoyl]-3-methylquinoxa-
line-1,4-dioxide (3i). Yield: 54%, mp 209–210 °C (lit.22 mp 212 °C).
1H NMR (CDCl3): d 2.59 (s, 3H, CH3), 7.15 (d, 1H, @CH, J = 16.10 Hz),
7.46 (d, 2H, Ar-H, J = 8.47 Hz), 7.57 (m, 3H, @CH and Ar-H), 7.92 (m,
2H, Ar-H), 8.61 (d, 1H, Ar-H, J = 8.35 Hz), 8.70 (d, 1H, Ar-H,
The methodology for evaluating the inhibitory activity of 2 and
3a–l towards Molt 4/C8 and CEM cell proliferation has been
described previously29 and was based on the determination of the
tumor cell number by a Coulter counter after an incubation period

U. Das et al. / Bioorg. Med. Chem. 17 (2009) 3909–3915
3915
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absence of serial dilutions of the test compound. The assessment of
3b,c,j towards human tumors followed a literature procedure.17
The evaluation of the cytotoxicity of 2 and 3a–l against near
confluent HL-60, HSC-2, HSC-3, HSC-4, HGF, HPC and HPLF cells
utilized a method setting the incubation time of 48 h which has
been described previously.2,30 Human oral normal cells (HGF,
HPC, HPLF) were prepared from the periodontal tissues, according
to the guideline of the Intramural Board of Ethic Committee (No.
A0808), after obtaining the informed consents from the patients.
The biodata for a reference compound melphalan has been deter-
6. Das, U.; Kawase, M.; Sakagami, H.; Ideo, A.; Shimada, J.; Molnár, J.; Baráth, Z.;
Bata, Z.; Dimmock, J. R. Bioorg. Med. Chem. 2007, 15, 3373.
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lM in
parentheses), namely HGF (>200), HPC (>200), HPLF (>200), HL-
60 (6.0), HSC-2 (35) and HSC-4 (81). 2 The CC50 figure of melphalan
towards HSC-3 cells is 115 lM.
16. Hansch, C.; Leo, A. J. Substituent Constants for Correlation Analysis in Chemistry
and Biology; John Wiley and Sons: New York, 1979. p. 49.
5.3. Evaluation of 2 and 3a–l as MDR revertants
17. Boyd, M. R.; Paull, K. D. Drug Dev. Res. 1995, 34, 91.
18. Pati, H. N.; Das, U.; Kawase, M.; Sakagami, H.; Balzarini, J.; De Clercq, E.;
The assay utilized has been described previously20 while a brief
summary of this methodology has been published very recently.21
Dimmock, J. R. Bioorg. Med. Chem. 2008, 16, 5747.
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G.; Vigyikanne, A. V.; Molnár, A.; Molnár, J. In Vivo 2005, 19, 705.
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Acknowledgments
The authors thank the Canadian Institutes of Health Research
and the Szeged Foundation of Cancer Research, Hungary for oper-
ating grants to J. R. Dimmock and J. Molnár, respectively. Appreci-
ation is extended to the Geconcerteerde Onderzoeksacties(GOA 05/
19) who provided funds to J. Balzarini enabling Mrs. L. van Berckel-
aer to undertake the Molt 4/C8 and CEM assays. The National Can-
cer Institute, USA kindly evaluated 3b,c,j against a panel of human
tumor cell lines. The Ministry of Education, Science, Sports and Cul-
ture kindly provided a Grant-in-Aid (No. 19592156) to H. Saka-
gami. The authors thank Beryl McCullough and Sandy Knowles
for typing the manuscript and Vigyikanne Váradi Aniko is grate-
fully acknowledged for technical assistance in flow cytometry.
22. El-Halim, M. S. A.; El-Ahl, A. S.; Etman, H. A.; Ali, M. M.; Fouda, A.; Fadda, A. A.
Monatsh. Fur Chem. 1995, 126, 1217.
23. Benko, P.; Bozsing, D.; Magyar, J. K. US Patent 4,373,101, 1983, February 8.
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25. Hansch, C.; Leo, A. J. Substituent Constants for Correlation Analysis in Chemistry
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Bases; Chapman and Hall: London, 1981. p. 112.
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30. Motohashi, N.; Wakabayashi, H.; Kurihara, T.; Fukushima, H.; Yamada, T.;
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References and notes
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Bioorg. Med. Chem. 2008, 16, 6261.
2. Pati, H. N.; Das, U.; Quail, J. W.; Kawase, M.; Sakagami, H.; Dimmock, J. R. Eur. J.
Med. Chem. 2008, 43, 1.