3-Arylidene-1-(4-nitrophenylmethylene)-3,4-dihydro-1H-naphthalen-2-ones and related compounds displaying selective toxicity and reversal of multidrug resistance in neoplastic cells

Article abstract of DOI:10.1016/j.bmcl.2005.01.054

The incorporation of the 1-aryl-5-(4-nitrophenyl)-3-oxo-1,4-pentadienyl group into 3,4-dihydro-1H-naphthalenyl, cyclohexyl, 2,3-dihydro-1H-indenyl and cyclopentyl scaffolds led to the discovery of various compounds with selective toxicity for malignant cells and ability to reverse multidrug resistance.

Full text of DOI:10.1016/j.bmcl.2005.01.054

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1633–1636
3-Arylidene-1-(4-nitrophenylmethylene)-3,4-dihydro-1H-
naphthalen-2-ones and related compounds displaying selective
toxicity and reversal of multidrug resistance in neoplastic cells
Jonathan R. Dimmock,a,* Umashankar Das,a H. Inci Gul,a, Masami Kawase,b
c
Hiroshi Sakagami, Zoltan Barath, Imre Ocsovsky and Joseph Molnar
d
e
d
´
´
´
aCollege of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, SK, Canada S7N 5C9
bFaculty of Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan
cDepartment of Dental Pharmacology, Meikai University School of Dentistry, Saitama 350-0283, Japan
dInstitute of Medical Microbiology and Immunobiology, Albert Szent-Gyo¨rgyi Medical Centre, University of Szeged,
Szeged H-6720, Hungary
eDepartment of Biochemistry, Albert Szent-Gyo¨rgyi Medical Centre, University of Szeged, Szeged H-6720, Hungary
Received 13 December 2004; revised 21 January 2005; accepted 24 January 2005
Abstract—The incorporation of the 1-aryl-5-(4-nitrophenyl)-3-oxo-1,4-pentadienyl group into 3,4-dihydro-1H-naphthalenyl,
cyclohexyl, 2,3-dihydro-1H-indenyl and cyclopentyl scaffolds led to the discovery of various compounds with selective toxicity
for malignant cells and ability to reverse multidrug resistance.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The major emphases in these laboratories are the design,
syntheses and antineoplastic evaluations of various con-
jugated arylidene ketones. These compounds react pref-
erentially with thiols rather than hydroxy and amino
groups.1 Thus these enones may exert their bioactivities
without interactions with the amino and hydroxy por-
tions of nucleic acids thereby avoiding the genotoxic
problems of a number of anticancer drugs.2 In addition,
the theory of sequential cytotoxicity states that succes-
sive chemical attacks by candidate antineoplastic agents
may be more harmful to malignant cells rather than the
corresponding normal tissues.3,4 On the basis of this
hypothesis, a number of 1,3-diarylidene-2-tetralones
were prepared which had different electron densities on
the olefinic carbon atoms which may permit sequential
thiol addition by cellular constituents.5 In general, these
compounds were more than twice as cytotoxic as the
corresponding 1-arylidene-2-tetralones suggesting that
the theory of sequential cytotoxicity was worthy of fur-
ther consideration. In particular, the aryl substituents of
1a were chosen for the following reasons. First, the
Hammett r values of the 4-nitro and 3,4,5-trimethoxy
substituents are 0.78 and À0.03, respectively,6 indicating
that nucleophilic attack on the olefinic carbon atom
adjacent to ring A would be predicted to occur initially
followed by thiolation at the second olefinic group. Sec-
ond, under the hypoxic conditions of certain tumours,
the nitro group could be reduced to one or more toxic
metabolites;7 this process would occur more readily in
such tumours rather than normal cells. Third, the
3,4,5-trimethoxyphenyl group is an integral part of a
number of anticancer agents such as podophyllotoxin8
and various combretastatins.9 These considerations
were validated when 1a displayed excellent cytotoxicity
when evaluated against 56 human tumour cell lines hav-
ing an average IC50 value of 0.59 lM.5 Furthermore,
mice tolerated doses up to and including 300 mg/kg of
this compound5 (see Fig. 1).
Thus 1a was considered a lead molecule with demon-
strated cytotoxicity without short-term marked toxicity.
The objectives of the present study were to prepare a
small group of prototypic molecules related to 1a with
a view to determining whether selective toxicity to
malignant cells and reversal of multidrug resistance
(MDR) would be demonstrated. If such properties were
noted, an indication of those structural features which
*
Corresponding author. Tel.: +1 306 966 6331; fax: +1 306 966
6377; e-mail: dimmock@skyway.usask.ca
On leave from Ataturk University, Erzurum, Turkey.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.054

1634
J. R. Dimmock et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1633–1636
R2
R2
general, electron-releasing substituents were placed in
R1
R3
R1
R3
ring B. The disparity between the combined Hammett
r values of the aryl substituents in rings A and B in ser-
ies 1–4 is presented in Table 1. These data reveal that
with the exception of 2g, the Dr figures were equal or
greater than 1a. Second, further molecular modifications
of series 1 were undertaken in order to glean some idea
of the contributions to bioactivity of ring C as well as
the size of the alicyclic ring; such considerations led to
the decision to prepare series 2–4.
B
B
B
C
O
O
A
A
A
O2N
O2N
1a-f
R2
2a-g
The synthesis of the compounds in series 1–4 was
accomplished as follows. 1-(4-Nitrophenylmethylene)-
3,4-dihydro-1H-naphthalen-2-one was prepared by a lit-
erature method10 and reacted with various aryl alde-
hydes in acidic media to yield 1a–f. The enones 2a–g
were synthesized in a similar manner from 2-(4-nitro-
phenylmethylene)cyclohexanone.11 Morpholine was re-
acted with either 2,3-dihydro-1H-inden-2-one or
cyclopentanone to produce the corresponding enamines
to which 4-nitrobenzaldehyde was added. After treating
the products with acid, the resultant enones were con-
densed with the appropriate aryl aldehyde under acidic
conditions giving rise to 3a and 4a,b. The purity of com-
R2
R3
R3
R1
B
B
R1
O
O
A
A
O2N
4a-b
O2N
3a
1
pounds was established by elemental analyses and H
Figure 1. The general structures of compounds in series 1–4. The
nature of the aryl substituents are indicated in Table 1.
NMR spectroscopy. This latter determination revealed
the stereohomogeneity of the compounds and the ole-
finic double bonds were assigned the E configuration
since X-ray crystallography had proved that various 1-
arylidene-2-tetralones,5 2-arylidenecyclohexanones10,12
and a 2,6-bis(arylidene)cyclohexanone13 possessed the
E stereochemistry.
contributed to these favourable bioactivities would be
sought.
The design of analogues of 1a was based on the follow-
ing reasoning. First, in order to ensure a differential be-
tween the fractional positive charges on CA and CB, the
4-nitrophenylmethylene group was retained while, in
All of the compounds in series 1–4 were evaluated
against four neoplastic cell lines, namely HSC-2, HSC-
3, HSG and HL-60 cells as well as the nonmalignant
Table 1. The aryl substituents and cytotoxic evaluation of the compounds in series 1–4
b
Compound
Aryl substituentsa
CC50 (lM)
HL-60 HGF
0.55
R1
R2
R3
Drc
HSC-2
0.55
HSC-3
HSG
3.0
HPC
HPLF
SId
1a
1b
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OCH3
0.81
0.93
1.03
1.05
1.10
1.15
0.81
0.93
1.03
1.05
1.10
1.15
0.55
0.81
0.81
1.05
—
2.9
4.2
>400
>400
33
>400
>400
83
>400
>400
21
>229
>159
15
0.92
1.45
4.4
5.8
0.56
0.95
4.4
2.2
2.5
1.1
4.3
3.4
1c
1d
3.8
OCH3
11
32
47
5.0
11
8.3
13
>400
>400
85
>400
>400
113
>400
>400
75
>17
>92
12
1e
OCH2O
2.6
5.2
0.76
2.3
2.8
7.6
1f
2a
OH
14
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OCH3
4.7
4.4
336
53
347
82
378
95
95
13
2b
2c
5.2
11
>400
>400
>400
15
165
>400
>400
391
171
>400
330
>44
2d
2e
OCH3
>400
308
8.1
1.5
221
>400
189
13
>400
>400
4.4
4.1
318
>400
>400
>400
191
ꢀ1.0
OCH2O
ꢀ1.2
ꢀ3.8
46
2f
2g
OH
Cl
>400
345
153
>400
332
4.6
3a
4a
OCH3
OCH3
OCH3
OCH3
OCH3
—
OCH3
OCH3
>400
>400
>400
2.9
>400
>400
>400
89
>400
>400
>400
>400
ꢀ0.6
ꢀ1.0
ꢀ1.3
>400
63
>400
>400
>400
>400
233
>400
>400
414
4b
Doxorubicin
—
—
0.68
1.9
0.47
>235
a For clarity, hydrogen atoms are omitted.
b Concentrations of compound to kill 50% of the cells.
c Dr refers to the differences in the r values between the substituents in rings A and B in series 1–4. The sigma values used in these calculations were
taken from Ref. 16.
d The letters SI indicate the selectivity index which was calculated as follows: SI = [CC50(HGF)+CC50(HPC)+CC50(HPLF)]/[CC50(HSC-2)+
CC50(HSC-3)+CC50(HSG)+CC50 (HL-60] · 4/3.

J. R. Dimmock et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1633–1636
1635
HGF, HPC and HPLF cells.14 The potency of the lead
compound 1a towards four cancer cell lines is revealed
in Table 1. The average CC50 figure of 1a to HSC-2,
HSC-3, HSG and HL-60 cells was 1.75 lM or 85% of
the average potency of doxorubicin towards these four
cell lines. The average potencies of the other compounds
prepared in this study were less than 1a revealing that
3,4,5-trimethoxy substitution in aryl ring B was optimal
(1a > 1b–f), the presence of ring C increases potency
(1a > 2a) and a six-membered alicyclic ring was pre-
ferred to a five-membered one (1a > 3a). In order to ob-
tain an answer to the first query as to whether
preferential toxicity for malignant cells was displayed,
a selectivity index (SI) value was calculated for each of
the compounds in series 1–4. These figures are presented
in Table 1 which reveal that within series 1 and 2, many
of the compounds displayed excellent selectivity. In par-
ticular, 1a rivals doxorubicin in this regard. A review of
the SI data revealed the following structure–activity
relationships (SAR). First, marked selective potencies
for malignant cells was noted in series 1 and 2 but not
3 and 4 indicating the importance of a six-membered
rather than a five-membered alicyclic ring. Second, in
general, the presence of ring C in series 1 enhanced selec-
tivity since the SI figures of 1a,b,d–f were greater than
2a,b,d–f, respectively. Third, 1a and 2a possessed the
highest SI figures in series 1 and 2, respectively, indicat-
ing that the 3,4,5-trimethoxy substitution pattern was
optimal. However no correlation exists between the SI
values and the Dr figures revealing that other factors im-
pinge on the selective toxicity for malignant cells such as
the specific locality of the nuclear methoxy groups which
enable good ligand–receptor binding to occur. In the fu-
ture, chemically divergent substituents should be placed
in both rings A and B and comparisons made between
the Dr and SI figures in a further evaluation of the the-
ory of sequential cytotoxicity.
Table 2. Effect of 1a–f, 2a–g and 4a,b on multidrug resistance in
murine L-5178 cells and human colon cancer Colo320 neoplasms
Compound
Fluorescence activity ratios at different
concentrationsa
L-5178
Colo320
4 lg/mL
134
44.1
51.8
4.36
3.27
135
15.6
20.4
1.50
40 lg/mL
4 lg/mL
10.9
7.19
40 lg/mL
1a
1b
1c
1d
1e
1f
120
55.9
123
13.0
11.4
6.20
4.30
2.27
11.7
4.38
4.62
1.13
0.58
0.59
0.60
0.53
1.28
0.37
3.38
2.22
1.95
1.99
7.43
6.12
1.98
0.50
0.67
1.15
0.60
1.96
0.63
7.68
3.87
39.3
75.1
47.3
0.83
0.82
1.23
0.97
0.94
6.62
0.69
2a
2b
2c
2d
2e
2f
0.76
0.91
0.94
1.16
3.06
0.59
2g
4a
4b
a The fluorescence activity ratio (FAR) values are the ratios of the
fluorescence intensities of the treated cells/untreated cells. The FAR
value of a reference compound verapamil was 38.2 and 8.05 using L-
5178 and Colo320 cells, respectively, when a concentration of 10 lg/
mL of verapamil was used (limitations of solubility precluded a
higher concentration of verapamil being utilized).
f > 2a–f), a six-membered alicyclic ring was preferable
to the 2,5-disubstituted cyclopentanones (2a,d > 4a,b)
and in series 1 and 2, maximum inhibition of MDR
was found in 1a and 2a having the 3,4,5-trimethoxy
groups in ring B.
In summary, this study of several series of compounds
containing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharma-
cophore has led to novel, potent cytotoxins, some of
which demonstrated remarkable selective toxicity to
malignant cells and the ability to reverse MDR. The rea-
sons for these important findings may have included the
fact that the theory of sequential cytotoxicity was veri-
fied and/or preferential reduction of the nitro group
took place in those tumours in which greater hypoxia
exists.
In order to address the second question as to the efficacy
or otherwise of these novel cytotoxins to MDR, murine
lymphoma L-5178 cells transfected with the human
MDR1 gene as well as human colon cancer Colo320
neoplasms were treated with two concentrations of the
compounds in series 1, 2 and 4.15 The accumulation of
rhodamine 123 was measured in the treated and un-
treated cells. The data are summarized in Table 2. In
those cases where the fluorescence activity ratio (FAR)
values are greater than 1, reversal of MDR has taken
place. MDR is due, inter alia, to an increase in the efflux
of a compound from the cell. Consequently a reversal of
MDR means that the exodus of rhodamine 123 from the
cells will be either the same or lower than in the parental
cells. The data in Table 2 reveal that 1a–c,f, 2a,b dis-
played a remarkable inhibition of the MDR of human
MDR1 gene-transmitted mouse lymphoma cells. These
cells overexpress the P-gp 170 protein responsible for
drug efflux. The same compounds were also the most
effective on the elevated drug accumulation of human
colon cancer Colo320 cells but in each case the FAR
values were lower than for the murine lymphoma cells.
In general, the SAR of the compounds inhibiting
MDR were the same as for the SI values generated,
namely the presence of ring C was beneficial (1a–
Acknowledgements
The authors thank the Canadian Institutes of Health
Research for an operating grant to J. R. Dimmock.
H. I. Gul was supported by a visiting scholar grant
(NATO-B2) distributed by the Scientific and Technical
Research Council of Turkey (TUBITAK). This study
was supported in part by the grant of Szeged Founda-
tion of Cancer Research and a Grant-in-Aid from the
Ministry of Education, Science, Sports and Culture of
Japan (Sakagami, No. 14370607).
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human normal oral cells (gingival fibroblast HGF, pulp
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determinations.
1
represents the mean from duplicate
15. The compounds were added to mouse lymphoma L-5178
cells or human colon cancer Colo320/MDR and incubated
for 10 min. Rhodamine 123 was added to the media and
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