In-vitro cytotoxicity, antioxidant, bleomycin-dependent DNA damage and immunomodulatory evaluation of 1-(4-acetylphenyl)-3-aryloxypyrrolidine-2,5-dione based derivatives

Article abstract of DOI:10.1007/s00044-014-0965-5

A one pot, economical, and efficient synthesis of 1-(4-acetylphenyl)-3- aryloxypyrrolidine-2,5-diones-based derivatives 5a-l have been accomplished in single steps and in satisfactory yields from 1-(4-acetylphenyl)-pyrrole-2,5- diones 3 and phenols 4a-l. All the compounds were characterized by physical, spectroscopic, and elemental analysis. The selection of the bioassays was based on proving the drug receptor binding concept. Compounds 5g, 5k, 5h, 5i, 5a, and 5f showed the highest inhibitory antioxidant activity using ABTS methods. Compounds 5k, 5g, 5c, 5h, 5b, 5d, 5f, and 5j manifested the best protective effect against DNA damage induced by bleomycin. Moreover, an in-vitro cytotoxic activity evaluation of all synthesized compounds was against four cancer cell lines using 5-Fluorouracil as a standard anticancer drug. Springer Science+Business Media 2014.

Full text of DOI:10.1007/s00044-014-0965-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:3907–3915
DOI 10.1007/s00044-014-0965-5
ORIGINAL RESEARCH
In-vitro cytotoxicity, antioxidant, bleomycin-dependent DNA
damage and immunomodulatory evaluation of 1-(4-acetylphenyl)-
3-aryloxypyrrolidine-2, 5-dione based derivatives
•
Jigarkumar R. Patel Bhaveshkumar D. Dhorajiya
•
•
Bharatkumar Z. Dholakiya Farid A. Badria
•
Ahmed S. Ibrahim
Received: 2 October 2013 / Accepted: 17 February 2014 / Published online: 6 March 2014
Ó Springer Science+Business Media New York 2014
Abstract A one pot, economical, and efficient synthesis of
1-(4-acetylphenyl)-3-aryloxypyrrolidine-2,5-diones-based
derivatives 5a-l have been accomplished in single steps and
in satisfactory yields from 1-(4-acetylphenyl)-pyrrole-2,5-
diones 3 and phenols 4a–l. All the compounds were char-
acterized by physical, spectroscopic, and elemental analysis.
The selection of the bioassays was based on proving the drug
receptor binding concept. Compounds 5g, 5k, 5h, 5i, 5a, and
5f showed the highest inhibitory antioxidant activity using
ABTS methods. Compounds 5k, 5g, 5c, 5h, 5b, 5d, 5f, and 5j
manifested the best protective effect against DNA damage
induced by bleomycin. Moreover, an in-vitro cytotoxic
activity evaluation of all synthesized compounds was against
four cancer cell lines using 5-Fluorouracil as a standard
anticancer drug.
Keywords Oxa-michael adducts ꢀ Antioxidant ꢀ
Bleomycin-dependent DNA damage ꢀ
Lymphocyte transformation ꢀ Cytotoxicity
Introduction
Every year almost 9 million cancer cases are newly diag-
nosed in developing countries where cancer incidence
continues to increase at alarming rates. According to can-
cer society statistics, at least one third of these individuals
are not expected to survive the disease, making cancer as
the most prevalent cause of death. Systemic chemotherapy
has emerged as a very promising strategy in treating a wide
variety of cancers (Dhorajiya et al., 2014). Thus, the search
for new ones has been prompted as urgency. Cyclic imides
are an important class of molecules known for their diverse
array of bioactivities (Kasimogullari and Cesur, 2004;
Guzel et al., 2006). These biological activities and various
pharmacological uses of cyclic imides have been attributed
to their unique structural features, which encourage us to
look for newer and more advantageous methods for their
efficient synthesis. Reported methods of Michael addition
on maleimides employed either the use of pyridine as base
in ethanol or triethylamine as base in dichloromethane
(Badawy et al., 1988; Pastor et al., 1985; Wabnitz et al.,
2003). These methods suffer from disadvantages such as
poor yield, longer reaction time, use of hazardous chemi-
cals, and halogenated solvent. To nullify such predica-
ments, essential needs to develop reaction condition such
that eco-friendly, highly efficient, better yielding, and take
place in a short time.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-0965-5) contains supplementary
material, which is available to authorized users.
J. R. Patel ꢀ B. D. Dhorajiya ꢀ B. Z. Dholakiya (&)
Department of Chemistry, S.V.National Institute of Technology,
Ichchhanath, Surat 395007, Gujarat, India
e-mail: bharat281173@gmail.com
J. R. Patel
e-mail: pateljigar.svnit@gmail.com
B. D. Dhorajiya
e-mail: bhavesh.orgchem@gmail.com
F. A. Badria
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura
University, Mansoura, Egypt
e-mail: faridbadria@gmail.com
A. S. Ibrahim
In recent years, reaction in water has attained significant
importance because of its advantages over other solvents.
Water is one of the most environmentally benign and
Department of Biochemistry, Faculty of Pharmacy, Mansoura
University, Mansoura, Egypt
e-mail: ahmedsalahibrahim@yahoo.com
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Med Chem Res (2014) 23:3907–3915
inexpensive liquid used in organic synthesis. Moreover, the
reaction in water is greener approach to the synthesis of
molecule of interest. Many organic reactions have been
tried and tested in an aqueous medium with mixed results
(Cheng and Comer, 2002; Zahouily et al., 2003; Beheshtia
et al., 2010). The insolubility of most organic reagents in
water has created a need to improve their compatibility in
water with use of Phase-transfer catalyst, surfactants, or
ionic liquids. Nevertheless, there are inherent difficulties in
handling such reactions. In continuation of our work on the
synthesis of biologically active motifs, we report here the
preparation of a new series of Michael adducts with the
objective of obtaining new biologically active compounds.
(6.00 g). The drying agent is removed by filtration before
the organic layer was concentrated by rotary evaporation
(The bath temperature is increased from 30 to 45 °C, 30–60
mmHg) yielding the crude product as brown solid. To
generalize the reaction, conjugate addition with a different
aromatic phenol were tried as well (Scheme 1). The reac-
tion proceeded well in all cases, and the products were
obtained within 1 h and 30 min with 70–93 % yields as
shown in Table 1. Hydrogen-bond formation between
water and the carbonyl oxygen of maleimide causes elec-
trophilic activation, making the b-position more suscepti-
ble to neucleophilic attack. The oxygen atom of water in
turn forms a hydrogen bond with the phenol hydrogen and
increases the electron density at the oxygen, resulting in
activation of nucleophile, which attacks the b-position of
maleimide. This mechanism illustrated as in (Scheme 2)
can be assumed to take place through the proposed tran-
sition state TS-I. Thus, because of hydrogen bonding
between the carbonyl oxygen and water results in an
increase the electrophilicity at b-carbon, and similarly the
hydrogen bonding between the nucleophile and the water
Result and discussion
With an ever interesting quest for greener solvents and
exploration of new reactions in water, we herein report the
use of water for Michael addition of phenols to maleimide
resulted afford products in good yield, within short reaction
time, and in neutral conditions without use of any kind of
catalyst. Maleimides were prepared by condensation of
maleic anhydride with a different p-amino acetophenone
using DABCO catalyst to give corresponding maleimide in
excellent yield (Khatik et al., 2006). As a model reaction,
250 ml three necked flask equipped with a Teflon-coated
3.2-cm egg-shaped magnetic stirrer bar, internal tempera-
ture probe and a reflux condenser. Charged with distilled
water, 1-(4-acetylphenyl)-pyrrole-2,5-diones (3) (0.21 g, 1
mmol, 1.00 equiv) and p-bromophenol (0.15 g, 1 mmol,
1.00 equiv) were added and the solution was stirred for 30
min. The reaction mixture was heated at 60–65 °C internal
temperature for 1 h. After 1 h, TLC analysis revealed that
the reaction was complete. The reaction flask was allowed
to cool to ambient temperature. The reaction mixture was
transformed to separating funnel and washed with saturated
sodium bicarbonate solution and extracted with ethyl ace-
tate. The combined organic layer was dried over MgSO4
Table 1 Isolated yield of 1-(4-acetylphenyl)-3-aryloxypyrrolidine-
2,5-diones 5a–l
Entry
R0
Isolated yield (%)
5a
5b
5c
5d
5e
5f
a- Napthyl
85
76
70
68
72
91
93
92
94
90
92
90
p-Br-C6H4
m-Cresyl
o-Cresyl
p- Cresyl
p-NO2-C6H4
o-COOH-C6H4
o-CHO-C6H4
2,4,6-[NO2]-C6H4
p-NH2–C6H4
Diphenyl
5g
5h
5i
5j
5k
5l
N-CH3-Quinolin-4-yl
O
O
R'
O
Water
N
R'
OH
+
N
65 °C
O
O
O
O
CH3
CH3
5a-l
4a-l
3
Scheme 1 Synthesis of 1-(4-acetylphenyl)-3-aryloxypyrrolidine-2,5-diones 5a–l
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Med Chem Res (2014) 23:3907–3915
3909
H
H
H
R'
O
O
O
H
H
CH
R'OH
O
O
N
O
O
O
O
N
N
O
CH3
O
CH3
O
CH3
3'
3
TS-I
R'
O
R'
O
O
OH
O
O
N
N
O
CH3
O
CH3
5
5'
Scheme 2 Proposed mechanism for addition of phenols to maleimide
leads to nucleophilic activation. Here, the dual activation
as standard antioxidant (positive control) (Abdel-Wahab
et al., 2009). Blank sample was run without ABTS and using
MeOH/phosphate buffer (1:1) instead of tested compounds.
Negative control was run with ABTS and MeOH/phosphate
buffer (1:1) only. The absorbance A(test) was measured and
the reduction in color intensity was expressed as % inhibition.
The inhibition for each compound was calculated from the
following equation.
role of water can be seen (Blokzij et al., 1991).
Antioxidant screening assay (ABTS method)
2,20-Azino-bis-3-ethylbenzthiazol-ine-6-sulfonic acid (ABTS)
was purchased from Wako Co., USA. L-ascorbic acid was
obtained from Sigma and all other chemicals were of the
highest quality available. For each of the investigated com-
pounds (2 ml), ABTS solution (60 lM) was added to 3 ml
MnO2 solution (25 mg/ml) all prepared in (5 ml) aqueous
phosphate buffer solution (pH 7, 0.1 M). The mixture was
shaken, centrifuged, filtered, and the absorbance of the
resulting green–blue solution (ABTS radical solution) at
734 nm was adjusted to approx. 0.5. Then, 50 ll of (2 mM)
solution of the tested compound in spectroscopic grade
MeOH/phosphate buffer (1:1) was added. The absorbance
was measured, and the reduction in color intensity was
expressed as inhibition percentage. L-ascorbic acid was used
% Inhibition ¼ ½AðcontrolÞ ꢁ AðtestÞ=AðcontrolÞꢂ ꢃ 100
Ascorbic acid (vitamin C) was used as standard
antioxidant (positive control). Blank sample was run
without ABTS and using methanol/phosphate buffer (1:1)
instead of sample. Negative control sample was run with
methanol/phosphate buffer (1:1) instead of tested
compound (Morimoto et al., 1995; Lissi et al., 1999).
A variety of N-(4-acetylphenyl) maleic imide-based Oxa
Michael adducts were tested for antioxidant activity as
reflected in the ability to control of ABTS assays. The pro-
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Med Chem Res (2014) 23:3907–3915
oxidant activities of the aforementioned derivates were
assessed by their effects on bleomycin-induced DNA
damage. The N-(4-acetylphenyl) maleic imide-based Oxa
Michael adducts manifested potent antioxidative activity in
the ABTS assay. All compounds have been tested to ble-
omycin-dependant DNA damage. The results indicated that
they may have some protective activity to DNA by certain
mechanism. A series of compounds (5g, 5k, 5h, 5i, 5a, 5f)
exhibited a high antioxidant activity. On the other hand,
compounds (5k, 5g, 5c, 5h, 5b, 5d, 5f, 5j) protect the DNA
from the induced damage by bleomycin (Table 2) (Abdel-
Wahab et al., 2009; El-Gazzar et al., 2009).
indicated compound 5j 67 % lymphocyte transformed at
concentration similar to standard, while 50–55 % lym-
phocyte transformed by synthesized adducts 5k and 5a
(Mikhaeil et al., 2004).
Cytotoxicity and antitumor assays
Cytotoxicity was expressed as the concentration that
caused 50 % loss of the cell monolayer (IC50). The assay
was used to examine the newly synthesized compounds.
5-Fluorouracil as a standard anticancer drug was used for
comparison. The results of our preliminary screening
indicates that compound 5j showed strong cytotoxic
activity, where as compounds 5b and 5l showed moderate
cytotoxic activity. The other compounds 5a and
5c–h showed weak cytotoxicity activity (Table 3).
Lymphocyte transformation assay
Lymphocyte transformation assay involves study of a
specific immune response at 50 lM. The assay investigates
the mitogenic effect of synthesized Michael adducts on
T-Lymphocytes. In this assay, standard extract was
observed 74 % at 50 lM. Data obtained from Table 2
The compounds 5i, 5k, and 5l are strong to moderate
activity against MCF-7 and HEPG-2 cell lines, while weak
cytotoxic activity against WI-38 and VERO cell lines.
Subsequently, we may conclude the following structure
activity relationship’s (SAR’s). (1) The presence of basic
skeleton (maleic imide moiety) is necessary for the broad
spectrum of cytotoxic activity toward different cell lines
(HepG2, WI-38, VERO, and MCF-7). (2) Introducing
amino group (electron-donating group) in position 4 of
phenol to form Michael adduct with maleic imide moiety
strong activity toward all cell lines (compound 5j). (3)
Introducing one bromine atoms (electron-withdrawing
group) in position 4 of phenol to form Michael adduct with
Table 2 Biological activities of compounds 5a–l, toward lymphocyte
transformation, ABTS (free radical scavenging), and bleomycin-
dependent DNA damage activities
Comp. Lymphocyte
no.
ABTS (Percentage Bleomycin-
of scavenging
transformation
assay at 50 lMa,b,g inhibition)c,d,e,f,g
dependent DNA
damage assayg,h
5a
5b
5c
5d
5e
5f
5g
5h
5i
10
50
35
35
25
20
35
45
25
67
55
39
34.3 0.10
37.5 0.05
42.7 1.15
36.8 0.13
41.1 2.38
35.4 0.24
25.8 1.30
33.2 0.70
33.3 0.14
69.5 0.04
30.7 1.16
46.8 0.15
0.210 1.12
0.017 0.15
0.015 0.25
0.017 0.04
0.080 0.20
0.017 0.05
0.014 1.31
0.016 0.27
0.110 1.13
0.017 0.03
0.011 0.27
0.023 0.03
Table 3 In-vitro cytotoxic activities of compounds against four
human cell lines
Compounds IC50 (lM)a
WI-38
VERO
MCF-7
HEPG-2
5a
5b
5c
5d
5e
5f
98 0.08 84 0.14 74 1.34b 69 0.29
35 0.17 28 0.12 31 1.32b 30 0.29
62 0.05 59 0.13 55 1.30b 67 0.15
88 0.12 84 0.15 86 1.74b 66 0.23
97 0.13 90 0.12 60 1.32b 56 0.21
82 0.12 70 0.15 64 1.25b 70 0.23
95 0.19 88 0.15 78 1.35b 63 0.21
90 0.14 89 0.18 78 1.24b 72 0.21
5j
5k
5l
a
Concentration showing 50 % lymphocyte transformation
b
The positive control was Echinacea purpurea (Ech) extract at
concentration 50 gave 74 % lymphocyte transformation
5g
5h
5i
c
ABTS? Scavenging activity (%) = [Ac - As/Ac] 9 100; where
AC is the absorbance value of the control and AS is the absorbance
value of the added samples test solution
110 0.05 95 0.05 30 0.05
15 0.34 25 0.11 20 0.35
105 0.12 85 0.04 30 0.01
50 0.25 40 0.22 35 0.22
10 0.05 08 1.02 10 0.48
25 0.32
15 0.35
25 0.05
32 3.01
05 1.36
5j
d
The concentration of the pure compounds was 2 mM
5k
5l
e
The concentration showing 50 % inhibition is expressed in mM
f
The positive control was vitamin C, and showed 80.0 1.04 %
5-FU
g
Values are means of 3 replicates SD, and significant difference
at P \ 0.05 by Student’s test
a
(IC50, lM): 1–10 (very strong), 11–25 (strong), 26–50 (moderate),
51–100 (weak), 100–200 (very weak), Above 200 (non-cytotoxic)
h
The positive control was vitamin C, (0.24 mM)and showed absor-
bance 0.0038 0.01 at the same concentration of the tested
compounds
b
Values are means of 3 replicates SD, and significant difference
at P \ 0.05 by Student’s test
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Med Chem Res (2014) 23:3907–3915
3911
maleic imide moiety decreases the activity from strong to
moderate against all cell lines (compound 5b). (4) According
to the above findings, the presence of one –NO2 electron-
withdrawing group in position 4 of phenol showed the weak
cytotoxicity activity toward all cell lines (compound 5f),
while increases number of –NO2 electron-withdrawing
group in position 2, 4, 6 of phenol enhanced the cytotoxicity
activity toward MCF-7 and HEPG-2 (compound 5i). (5) In
compound 5k, the presence of phenyl group in position 4 of
phenol to form Michael adduct with maleic imide moiety
acts as electron-donating group showed nearly strong to
moderate activity against HepG2, MCF-7, and weak activity
against WI- 38, VERO. (6) Introducing heterocyclic phenol
(N-methyl-4-hydroxy Quinoline) to form Michael adduct
with maleic imide moiety decreases showed moderate
cytotoxicity activity toward all cell lines (compounds 5l). (7)
In compounds 5c–d, –CH3 group at respective o, m, and p-
position of phenol showed weak cytotoxicity activity toward
all cell lines. Incorporation of weak electron-donating group
irrespective of their position on phenol was not affect cyto-
toxically on all four cell lines (Fadda et al., 2012; Gutterdge
et al., 1981).
apparatus and are uncorrected. To monitor the reactions, as
well as, to establish the identity and purity of reactants and
products, thin layer chromatography was performed on
microscopic glass slides (2 9 7.5 cm2) coated with silica
gel-G, using toluene–acetone and chloroform–methanol, as
the solvent systems, and spots were visualized under UV
radiation. Elemental analysis (C, H, N) was performed
using a PerkinElmer, USA 2400-II CHN analyzer. FTIR
spectra (4,000–400 cm-1) were recorded on Simadzu
8400-S spectrophotometer using KBr disk. Nuclear mag-
netic resonance spectra were recorded on Varian 400 MHz
model spectrometer using DMSO and or DMF as a solvent
and TMS as internal reference (Chemical shifts in d ppm).
Biological evaluation of the isolated compounds
DNA (Calf Thymus type1), bleomycin sulfate, butylated
hydroxyanisole, thiobarbituric acid (TBA), ethylenediami-
netetraacetic acid (EDTA), and ascorbic acid were obtained
from sigma. 2,20-azo-bis-(2-amidinopropane) dihydrochlo-
ride and ABTS were purchased from Wako Co., USA.
Bleomycin-dependent DNA damage assay
Conclusion
The reaction mixture contained DNA (0.5 mg/ml), bleo-
mycin sulfate (0.05 mg/ml), MgCl2 (5 mM), FeCl3
(50 mM), and samples to be tested in a conc. of 0.1 mg/ml.
L-ascorbic acid was used as positive control. The mixture
was incubated at 370 °C for 1 h. The reaction was termi-
nated by addition of 0.05 ml EDTA (0.1 M). The color was
developed by adding 0.5 ml TBA (1 % w/v) and 0.5 ml
HCl (25 % v/v), followed by heating at 80 °C for 10 min.
After centrifugation, the extent of DNA damage was
measured by increase in absorbance at 532 nm (Abdel-
Wahab et al., 2009; Gutterdge et al., 1981).
We have demonstrated that the reaction of 1-(4-acetyl-
phenyl)-pyrrole-2,5-diones with phenols via Michael
addition reaction resulting 1-(4-acetylphenyl)-3-aryloxy-
pyrrolidine-2,5-diones, enables the efficient synthesis of
Michael adduct in single step in satisfactory overall yields.
The products of this reaction are of potential medicinal
interest. The present approach offers significant advantages
over the previously described methods because of the ready
availability of starting materials and simple operations.
Moreover, we have shown from the biological evaluation
part that most of the 1-(4-acetylphenyl)-3-aryloxypyrroli-
dine-2,5-dione derivatives have an excellent lymphocyte
transformation, ABTS (free radical scavenging), bleomy-
cine-dependent DNA damage properties, and in-vitro
cytotoxic activities against four cell lines.
Antioxidant activity screening assay 2,2‘-azino-bis-3-
ethylbenzthiazoline-6-sulfonic acid method
For each of the investigated compounds, 2 ml of
ABTS solution (60 lM) was added to 3 ml MnO2 solution
(25 mg/ml), all prepared in 5 ml aqueous phosphate buffer
solution (pH, 7; 0.1 M). The mixture was shaken, centri-
fuged, filtered, and the absorbance of the resulting green–
blue solution (ABTS radical solution) at k 734 nm was
adjusted to approximately ca. 0.5. Then, 50 ll of (2 mM)
solution of the tested compound in spectroscopic grade
MeOH/phosphate buffer (1:1) was added. The absorbance
was measured and the reduction in color intensity was
expressed as inhibition percentage. L-ascorbic acid was
used as standard antioxidant (positive control). Blank
sample was run without ABTS and using MeOH/phosphate
Experimental
Materials and instrumentation
All reagents were of analytical reagent grade and were used
without further purification. Solvents employed were
purified by standard procedure before using. Maleic
anhydride, p-aminoacetophenone, and phenols were pur-
chased from Aldrich.12 Melting points were determined in
open capillary on Veego (Model: VMP-D) electronic
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Med Chem Res (2014) 23:3907–3915
buffer (1:1) instead of tested compounds (El-Gazzar et al.,
by titered virus in 100 ml medium containing 10 % (v/v)
calf serum in each well. In each tray, the last row of wells
was reserved for controls that were not treated with com-
pounds. Trays were cultured for 96 h. Trays were inverted
onto a paper towel pad, the remaining cells were rinsed
carefully with medium, and fixed with 3.7 % (v/v) form-
aldehyde in saline for at least 20 min. The fixed cells were
rinsed with water, and examined visually. The cytotoxic
activity is identified as confluent, relatively unaltered
monolayers of stained cells treated with the investigated
compounds. Cytotoxicity was estimated as the concentra-
tion that caused approximately 50 % loss of the monolayer.
5-fluorouracil was used as a positive control.
2009).
Lymphocyte transformation assay
The viable lymphocytes were adjusted to a concentration of
2 9 106 cells/ml in RPMI-1640 medium supplemented
with 600 ll penicillin, 0.1 ml streptomycin, 1 % glutamine,
25 % HEPES (N-2-hydroxyethylpiperazine-N-D2-ethane-
sulfonic acid)-buffer, and 20 % fetal calf serum (FCS). The
lymphocytes were plated into 96-well tissue culture plates
(or Ependorf tubes). 100 ll of the volatile oil solution in
DMF (100 ll/ml) and 20 lg of the mitogen (PHA) were
added to each well. Cell cultures were incubated at 37 °C in
5 % CO2 atmosphere for 72 h, during which the mitogen
produces its maximal effect on DNA synthesis. After cul-
ture, B. R. Mikhaeil et al. Chemistry and Immunomodula-
tory Activity of Frankincense Oil 237 cell films were
stained by Giemsa stain and average count of percentage of
transformed (proliferated) blasts was determined. Aqueous
Echinaceae purpurea extract (Immulone) and levamisole
(Ketrax) were used as positive control (standard immuno-
stimulant) 100 lg/ml of each drug in DMSO.
Synthesis of 40-amino maleimide 3
Mixer of maleic anhydride (1) and p-amino acetophenone
(2) (1:1.1) in diethyl ether, catalyzed by DABCO (1,4-
Diazabicyclo [2.2.2] octane) (0.5 mol) in short reaction
time and high yield was performed to give 40-amino
maleimide (3) (Scheme 1) (Khatik et al., 2006).
General Procedure for the Preparation of 1-(4-
acetylphenyl)-3 aryloxypyrrolidine-2,5-dione (5a–l)
Cytotoxicity and antitumor assay
In a typical experiment, 1-(4-acetylphenyl)-pyrrole-2,5-di-
ones (3) (0.21 g, 1 mmol, 1.00 equiv) was taken in a round-
bottomed flask containing mixture (1:1) of demineralized
water, and 4-bromophenol (4d) (0.15 g, 1 mmol) was added.
The Reaction vessel was subjected to heat for 1 h at tem-
perature 60–65 °C, after that the reaction mixture was
washed with saturated sodium bicarbonate solution and
extracted with ethyl acetate. The solvent was evaporated
under reduced pressure to obtain the product 1-(4-acetyl-
phenyl)-3-(4-bromophenoxy) pyrrolidine-2,5-diones, which
was washed with hexane and dried under vacuum.
Samples were prepared for assay by dissolving in 50 ml of
DMSO (Dimethyl Sulfoxide), and diluting aliquots into
sterile culture medium at 0.4 mg/ml. These solutions were
sub-diluted to 0.02 mg/ml in sterile medium and the two
solutions were used as stocks to test samples at 100, 50, 20,
10, 5, 2, and 1 mg/ml in triplicate in the wells of microtiter
plates. The compounds were assayed in triplicate on
monolayers grown in Dulbecco’s modified Eagle’s medium
supplemented with 10 % (v/v) calf serum (HyClone
Laboratories, Ogden, UT), 60 mg/ml Penicillin G, and
100 mg/ml streptomycin sulfate maintained at 37 °C in a
humidified atmosphere containing about 15 % (v/v) CO2 in
air. All medium components were obtained from Sigma
Chemical Co., St. Louis, MO, unless otherwise indicated.
Cell stocks were maintained at 34 °C in culture flasks filled
with medium supplemented with 1 % (v/v) calf serum.
Subcultures of cells for screening were grown in the wells
of microtiter trays (Falcon Microtest III 96-wells trays,
Becton–Dickinson Labware, Lincolin Park, NJ) by sus-
pending cells in medium following trypsin–EDTA treat-
ment, counting the suspension with a hemocytometer,
diluting in medium containing 10 % calf serum to 2 9 104
cells per 200 ml culture, aliquoting into each well of a tray,
and culturing until confluent. Microtiter trays with con-
fluent monolayer cultures of cells were inverted, the
medium shaken out, and replaced with serial dilutions of
sterile compounds in triplicate in 100 l medium followed
18
19
14
17
16
O
7'
5'
9
4'
13
15
O
6'
N
8
3
12
2
10
O
7'
11
5'
CH3
1
4
O
1-(4-Acetylphenyl)-3-(1-napthyloxy)-pyrrolidine-2,5-dione
(5a)
Brown solid. Yield 85 %; M.p. 145 °C (hexane/MeOH).
FTIR (KBr): 1724, 1599, 1520, 1344, 1H NMR (500 MHz,
DMSO-d6); 3.45 (DMSO solvent); 2.55 (s, 3H); 3.11
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Med Chem Res (2014) 23:3907–3915
3913
(s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.64–8.17 (m, 7H), 7.32
(dd, J = 15, 1H), 7.34 (dd, J = 15, 2H).13C NMR
(500 MHz, DMSO-d6); 22 (C-1), 32 (C-9), 80.8 (C-8), 103
(C-5, C-50), 120 (C-12, C-16), 120.1 (C-17), 125 (C-4,
C-40), 126 (C-11, C-14, C-18, C-19), 127 (C-3), 129
(C-13), 133 (C-6), 145 (C-10), 170.9(C-7, C-70), 191 (C-2)
d ppm; ESIMS m/z 359(M?) Anal. Calc. for C22H17NO4
(359.37): C, 73.53; H, 4.77; N, 3.90 Found: C, 73.51; H,
4.75; N, 3.88.
(C-4, C-40), 130.22 (C-12), 133 (C-3), 138 (C-6), 163
(C-10), 167.78 (C-7, C-70), 171 (C-2) d ppm; ESIMS m/z
324 (M?) Anal. Calc. for C19H17NO4 (323.34): C, 70.58;
H, 5.38; N, 4.33 Found: C, 70.56; H, 5. 34; N, 4.31.
1-(4-Acetylphenyl)-3-(4-methylphenyloxy)-pyrrolidine-2,5-
dione (5e)
Orange brown solid. Yield 72 %; M.p. 152 °C (hexane/
MeOH). FTIR (KBr): 1724, 1599, 1520, 1344, H NMR
1
1-(4-Acetylphenyl)-3-(4-Bromophenyloxy)-pyrrolidine-2,5-
(500 MHz, DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H);
2.5 (s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10,
1H), 6.55 (dd, J = 10, 1H), 7.32 (dd, J = 10, 1H), 7.34
(dd, J = 10, 2H). 13C NMR (500 MHz, DMSO-d6); 11.2
(C-of p-Ar-CH3), 23 (C-1), 31 (C-9), 83 (C-8), 114 (C-11,
C-15), 120 (C-5, C-50), 130.22 (C-12, C-14), 133 (C-13),
138 (C-6), 163 (C-10), 167.78 (C-7, C-70), 171 (C-2) d
ppm; ESIMS m/z 324 (M?) Anal. Calc. for C19H17NO4
(323.34): C, 70.58; H, 5.38; N, 4.33 Found: C, 70.58; H, 5.
33; N, 4.33.
dione (5b)
Brown solid. Yield 76 %; M.p. 166 °C (hexane/MeOH).
FTIR (KBr): 1724, 1599, 1520, 1344, 1H NMR (500 MHz,
DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H); 2.5
(s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10, 1H),
6.55 (dd, J = 10, 1H), 7.32 (dd, J = 10, 1H), 7.34 (dd,
J = 10, 2H).13C NMR (500 MHz, DMSO-d6); 22.8 (C-1),
32.2 (C-9), 83.2 (C-8), 115.4 (C-13), (C-5, C-50), 116.3
(C-11, C-15), 120.3 (C-17), 128 (C-4, C-40),132.4 (C-3),
133 (C12, C-14), 145 (C-6), 159 (C-10), 161(C-7, C-70),
195 (C-2) d ppm; ESIMS m/z 387 (M-1); Anal. Calc. for
C18H14BrNO4 (388.21): C, 55.69; H, 3.63; N, 3.61 Found:
C, 55.63; H, 3.62; N, 3.63.
1-(4-Acetylphenyl)-3-(4-nitrophenyloxy)-pyrrolidine-2,5-
dione (5f)
Brownish black solid. Yield 91 %; M.p. 98 °C (hexane/
MeOH). FTIR (KBr): 1724, 1599, 1520, 1344, H NMR
1
1-(4-Acetylphenyl)-3-(3-methylphenyloxy)-pyrrolidine-2,5-
(500 MHz, DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H);
2.5 (s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10,
1H), 6.55 (dd, J = 10, 1H), 8.32 (dd, J = 15, 1H), 8.34
(dd, J = 15, 2H).13C NMR (500 MHz, DMSO-d6); 22.8
(C-1), 31 (C-9), 81.7 (C-8), 114 (C-11, C-15), 120 (C5,
C-50), 126.9 (C-12, C-14), 129 (C-4, C-40), 133 (C-6),
135.9 (C-3), 138 (C-13), 163 (C-10), 167.78 (C-7, C-70),
171 (C-2) d ppm; ESIMS m/z 354 (M?) Anal. Calc. for
C18H14N2O6 (354.31): C, 61.02; H, 3.98; N, 7.91 Found: C,
59.99; H, 4.01; N, 7.89.
dione (5c)
Brown solid. Yield 70 %; M.p. 149 °C (hexane/MeOH).
FTIR (KBr): 1724, 1599, 1520, 1344, 1H NMR (500 MHz,
DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H); 2.5
(s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10, 1H),
6.55 (dd, J = 10, 1H), 7.32 (dd, J = 10, 1H), 7.34 (dd,
J = 10, 2H).13C NMR (500 MHz, DMSO-d6); 11 (C- of
m-Ar-CH3), 22 (C-1), 31 (C-9), 80 (C-8),114 (C-15), 120
(C-11), 126.9 (C-13), 127.85 (C-5, C-50), 129 (C-4, C-40),
130.22 (C-14), 133 (C-3), 137 (C-12), 138 (C-6), 163
(C-10), 167.78 (C-7,C-70), 171 (C-2) d ppm; ESIMS m/z
324 (M?) Anal. Calc. for C19H17NO4 (323.34): C, 70.58;
H, 5.38; N, 4.33 Found: C, 70.58; H, 5. 36; N, 4.32.
2-((1-(4-Acetylphenyl)-2,5-dioxopyrrolidin-3-
yl)oxy)benzoic acid (5g)
Light brown solid. Yield 93 %; M.p. 115 °C (hexane/
MeOH). FTIR (KBr): 1724, 1599, 1520, 1344, H NMR
1
1-(4-Acetylphenyl)-3-(2-methylphenyloxy)-pyrrolidine-2,5-
dione (5d)
(500 MHz, DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H);
2.5 (s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10,
1H), 6.55 (dd, J = 10, 1H), 7.34 (m, 4H), 10.2 (s, 1H).13C
NMR (500 MHz, DMSO-d6); 22.8 (C-1), 31 (C-9), 80.7
(C-8), 114 (C-15), 120 (C-13), 126.9 (C-5, C-50), 127.85
(C-4, C-40), 130.9 (C-12), 129 (C-6), 133 (C-14), 135.9
(C-6), 138 (C-3), 163 (C-10), 167.78 (C of o-Ar-COOH),
Brown solid. Yield 68 %; M.p. 105 °C (hexane/MeOH).
FTIR (KBr): 1724, 1599, 1520, 1344, 1H NMR (500 MHz,
DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H); 2.5
(s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10, 1H),
6.55 (dd, J = 10, 1H), 7.32 (dd, J = 10, 1H), 7.34 (dd,
J = 10, 2H). 13C NMR (500 MHz, DMSO-d6); 11 (C- of
o-Ar-CH3), 22.3 (C-1), 31 (C-9), 80.7 (C-8),114 (C-15), 120
(C-13), 126.9 (C-5, C-50), 127.85 (C14), 128 (C-11), 129
171 (C-7, C-70), 189 (C-2) d ppm; ESIMS m/z 355 (M?2
)
Anal. Calc. for C19H15NO6 (353.32): C, 64.59; H, 4. 28; N,
3.96 Found: C, 64.57; H, 4.29; N, 4.0.
123

3914
Med Chem Res (2014) 23:3907–3915
2-((1-(4-Acetylphenyl)-2,5-dioxopyrrolidin-3-
DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H); 2.5
(s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10, 1H),
6.55 (dd, J = 10, 1H), 8.32 (dd, J = 15, 1H), 8.34 (dd,
J = 15, 2H).13C NMR (500 MHz, DMSO-d6); 22.8 (C-1),
31 (C-9), 81.7 (C-8), 114 (C-11), 120 (C-13), 126.9 (C-11,
C-110), 127.85 (C-130), 128 (C-12), 129 (C–C-4, C-40,
C-120), 130.22 (C-14), 133 (C-15), 135.9 (C-6), 137 (C-3),
138 (C-100), 163 (C-10), 167.78 (C-7, C-70), 171 (C-2) d
ppm; ESIMS m/z 354 (M?) Anal. Calc. for C18H14N2O6
(354.31): C, 61.02; H, 3.98; N, 7.91 Found: C, 59.99; H,
4.01; N, 7.89.
yl)oxy)benzaldehyde (5h)
Light orange solid. Yield 92 %; M.p. 128 °C (hexane/
MeOH). FTIR (KBr): 1724, 1599, 1520, 1344, H NMR
1
(500 MHz, DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H);
2.5 (s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10,
1H), 6.55 (dd, J = 10, 1H), 7.32 (m, 4H), 7.34 (dd, J = 10,
2H), 8.7 (s, 1H).13C NMR (500 MHz, DMSO-d6); 22.8
(C-1), 31 (C-9), 80.7 (C-8), 114 (C-15), 120 (C-13), 121
(C-5, C-50), 126.9 (C-11), 127.85 (C-12), 129.0 (C-4, C-40),
135.9 (C-14), 137 (C-6), 138 (C-3), 163 (C-10), 168 (C-7,
C-70), 174 (C-2) d ppm; ESIMS m/z 337 (M?) Anal. Calc.
for C19H15NO5 (337.32): C, 67.65; H, 4. 48; N, 4.15
Found: C, 67.63; H, 4.46; N, 4.11.
1-(4-Acetylphenyl)-3-(N-methyl-4-quinolinyloxy)-
pyrrolidine-2,5-dione (5l)
Dark orange solid. Yield 90 %; M.p. 98 °C (hexane/
MeOH). FTIR (KBr): 1724, 1599, 1520, 1344, H NMR
1
1-(4-Acetylphenyl)-3-(2,4,6-trinitrophenoxy)pyrrolidine-
2,5-dione (5i)
(500 MHz, DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H);
2.5 (s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10,
1H), 6.55 (dd, J = 10, 1H), 8.32 (dd, J = 15, 1H), 8.34
(dd, J = 15, 2H).13C NMR (500 MHz, DMSO-d6); 22.8
(C-1), 31 (C-9), 45.3 (C –of N-CH3), 81.7 (C-8), 104.9
(C-11), 108.8 (C-18), 114 (C-16), 120 (C-14), 126.9 (C-5,
C-50), 127.85 (C-15), 128 (C-17), 129 (C-4, C-40), 130.22
(C-12), 133 (C-6), 135.9 (C-3), 138 (C-13), 167.78 (C-7,
C-70), 171 (C-2) d ppm; ESIMS m/z 354 (M?) Anal. Calc.
for C18H14N2O6 (354.31): C, 61.02; H, 3.98; N, 7.91
Found: C, 59.99; H, 4.01; N, 7.89.
Yellow solid. Yield 94 %; M.p. 98 °C (hexane/MeOH).
FTIR (KBr): 1724, 1599, 1520, 1344, 1H NMR (500 MHz,
DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H); 2.5
(s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10, 1H),
6.55 (dd, J = 10, 1H), 8.32 (dd, J = 15, 1H), 8.34 (dd,
J = 15, 2H).13C NMR (500 MHz, DMSO-d6); 22.8 (C-1),
31 (C-9), 81.7 (C-8), 120 (C-5, C-50), 126.9 (C-12, C-14),
127.85 (C-4, C-40), 133 (C-6), 135.9 (C-7), 137 (C-13), 138
(C-11, C-15), 163 (C-10), 167.78 (C-7, C-70), 171 (C-2) d
ppm; ESIMS m/z 354 (M?) Anal. Calc. for C18H14N2O6
(354.31): C, 61.02; H, 3.98; N, 7.91 Found: C, 59.99; H,
4.01; N, 7.89.
Acknowledgments The authors thank the S.V.National Institute of
Technology, Surat, for the financial support, and the members of the
analytical group of the Central Salt & Marine Chemicals Research
Institute, Bhavnagar, and Oxygen Healthcare Private Ltd. Ahmeda-
bad, Gujarat, India, for spectral measurements. The biological part of
this work was supported by the Department of Pharmacognosy,
Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. We
thank Dr. Farid A Badria and his research team for excellent technical
assistance.
1-(4-Acetylphenyl)-3-(4-aminophenyloxy)-pyrrolidine-2,5-
dione (5j)
Dark brown solid. Yield 90 %; M.p. 98 °C (hexane/
MeOH). FTIR (KBr): 1724, 1599, 1520, 1344, H NMR
1
(500 MHz, DMSO-d6); 3.45 (DMSO solvent); 2.04 (s, 3H);
2.5 (s, J = 5, 1H); 5.3 (s, J = 10, 1H), 6.52 (dd, J = 10,
1H), 6.55 (dd, J = 10, 1H), 8.32 (dd, J = 15, 1H), 8.34
(dd, J = 15, 2H).13C NMR (500 MHz, DMSO-d6); 22.8
(C-1), 31 (C-9), 81.7 (C-8), 114 (C-11, C-15), 120 (C-12,
C-14), 126.9 (C-5, C-50), 127.85 (C-4, C-40), 133 (C-6),
135.9 (C-3), 138 (C-13), 163 (C-10), 167.78 (C-7, C-70),
171 (C-2) d ppm; ESIMS m/z 354 (M?) Anal. Calc. for
C18H14N2O6 (354.31): C, 61.02; H, 3.98; N, 7.91 Found: C,
59.99; H, 4.01; N, 7.89.
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