Intervention in cyclophosphamide induced oxidative stress and DNA damage by a flavonyl-thiazolidinedione based organoselenocyanate and evaluation of its efficacy during adjuvant therapy in tumor bearing mice

Article abstract of DOI:10.1016/j.ejmech.2013.12.015

A novel flavonyl-thiazolidinedione based organoselenocyanate compound was synthesized and established as nontoxic at the doses of 2.5 and 5 mg/kg b.w. in mice. Oral administration of the compound in combination with cyclophosphamide (CP) resulted in an im

Full text of DOI:10.1016/j.ejmech.2013.12.015

European Journal of Medicinal Chemistry 73 (2014) 195e209
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Intervention in cyclophosphamide induced oxidative stress and DNA
damage by a flavonyl-thiazolidinedione based organoselenocyanate
and evaluation of its efficacy during adjuvant therapy in tumor
bearing mice
*
Somnath Singha Roy, Pramita Chakraborty, Sudin Bhattacharya
Chittaranjan National Cancer Institute, Department of Cancer Chemoprevention, 37, S. P. Mukherjee Road, Kolkata 700 026, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel flavonyl-thiazolidinedione based organoselenocyanate compound was synthesized and estab-
lished as nontoxic at the doses of 2.5 and 5 mg/kg b.w. in mice. Oral administration of the compound in
combination with cyclophosphamide (CP) resulted in an improved therapeutic efficacy which was
mostly evidenced in terms of tumor burden and protection of normal cells. The adjuvant therapy was
proved to be immensely significant in increasing the mean survivability of the tumor bearing hosts.
Reduction in the tumor volume was manifested through the induction of apoptosis and generation of
ROS in transformed cells. Moreover, the organoselenium compound could efficiently suppress CP-
induced DNA damage, chromosomal aberration, hepatic damage and enhanced the activities of
various antioxidant enzymes in normal cells.
Received 17 June 2013
Received in revised form
11 December 2013
Accepted 12 December 2013
Available online 25 December 2013
Keywords:
Organoselenium
Cyclophosphamide
Chemoprotection
Chemoenhancement
ROS
Ó 2013 Elsevier Masson SAS. All rights reserved.
Biodistribution
1. Introduction
Organoselenium compounds in recent years have got wide
attention due to their versatile biological activities which may vary
Most of the anticancer drugs currently in use are cytotoxic to
normal cells, leading to unwanted side effects [1,2]. Chemotherapy
associated adverse effects in cancer patients have been linked to
oxidative stress generation in normal tissues [3]. Therefore, drugs
that could reduce these side effects without concomitant protec-
tion of tumor tissue will be of great help in improving cancer
treatment strategies.
enormously depending on the organic scaffold and the substituents
on it. In this present study, flavonyl-thiazolidinedione is used as the
organic scaffold. Thiazolidinediones have been described as health-
promoting, disease-preventing dietary supplements which are
extremely safe and associated with low toxicity [9]. As antioxidants,
these compounds may prevent the progressive impairment of
pancreatic b-cell function due to oxidative stress and thus reducing
As an alkylating agent CP has been used to treat various types of
cancer starting from chronic and acute leukemias, multiple
myeloma, lymphomas, rheumatic arthritis and also in preparation
for bone marrow transplantation [4]. However, the use of CP as an
effective chemotherapeutic agent is often restricted because of its
wide adverse side effects and toxicities that include nausea, vom-
iting, alopecia, hematopoietic suppression, carcinogenicity [5],
nephrotoxicity [6], hepatotoxicity, urotoxicity, cardiotoxicity [7],
immunotoxicity and mutagenicity [8].
the occurrence of type 2 diabetes [10]. On the other hand, flavo-
noids can act as powerful antioxidants for their free radical scav-
enging activity [11]. Sometimes they even showed more activities
than the traditional vitamins [12]. The rationale for introducing
selenium into the organic scaffold molecules like flavones and
thiazolidinediones is that the essential redox active trace element
selenium generally did not interfere with chemotherapy, it actually
enhanced the efficacy of the chemotherapy [13], prevented the
body’s development of chemotherapeutic drug resistance [14] and
decreased the toxicity of chemotherapeutics drugs [15,16].
Hence, the objective of this present study was to synthesize and
characterize a novel flavonyl thiazolidinedione based organo-
selenocyanate compound and evaluate its toxicity in normal Swiss
albino mice. Then a nontoxic dose was applied to the tumor bearing
* Corresponding author. Tel.: þ91 33 24765101x316.
E-mail address: sudinb19572004@yahoo.co.in (S. Bhattacharya).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.015

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S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
Knoevenagel condensation with formyl flavone (V) to yield flavonyl
thiazolidinedione bromopentyl derivative (VI). The precursor
bromo compound was then converted to selenocyanate (VII) by the
reaction with potassium selenocyanate (KSeCN) in acetone at room
temperature. All the compounds were purified through column
chromatography over silica gel and further crystallized from
appropriate solvents. Finally, IR, 1H NMR, 13C NMR, ES-MS of the
compounds further confirmed their structures.
2.2. Study of the toxicity of the compound
Compared to vehicle control group, no significant (p > 0.05)
changes in the body weight were observed in normal Swiss albino
mice treated with 2.5 mg and 5 mg/kg body weight (b.w.) of
organoselenium compound for 30 days. Oral administration of the
compound at different doses (2.5 mg and 5 mg/kg b.w.) did not
change the hemoglobin level significantly (p > 0.05) compared
with the vehicle control group (Table 1). In addition, no alteration in
the ALT, AST, ALKP, BUN and creatinine level was observed when
treated with organoselenium compound at the dose of 2.5 mg and
5 mg/kg b.w. (Table 1). However, all these toxicity markers showed
enhancement when treated with the higher dose (10 mg/kg b.w.) of
the selenium compound. Treatment with 10 mg/kg b.w. also caused
a significant (p < 0.05) decrease in the body weight at the 4th week
of the treatment compared to the vehicle control group (Fig. 1).
Histopathological examination of selected organs (liver and
kidney) from both vehicle control and organoselenium treated
groups (at the dose of 2.5 and 5 mg/kg b.w.) showed normal ar-
chitecture of the hepatocytes and kidney cells. The overall cellular
structure remains unaltered with no necrosis, inflammation or
other significant lesion. Where as treatment with the higher dose
(10 mg/kg b.w.) resulted in infiltration of mononuclear cells in both
liver and kidney tissues (Figs. 2 and 3).
Scheme 1. Synthesis of VII. (a) Pyridine, heat at 60 ꢁC for 30 min (yield: 81%); (b) KOH,
pyridine, heat at 100 ꢁC for 30 min (yield: 75%); (c) conc. H2SO4, stirred at room
temperature for 30 min (yield: 73%); (d) N-bromosuccinimide, hot benzene, benzoyl
peroxide, reflux at 100 ꢁC for 6 h (yield: 67%); (e) hexamethylenetetramine (HMTA),
acetic acid (%50, v/v), refluxed for 15 min again HCl:H2O (1:1) added and refluxed for
another 10 min (yield: 76%); (f) potassium hydroxide, ethanol, stirred at 40 ꢁC for 2 h
(yield: 87%); (g) 1,5-dibromopentane, DMF, stirred at room temperature for 16 h (yield:
71%); (h) TB, piperidine, benzene, reflux for 16 h under nitrogen, (yield: 64%); (i)
KSeCN, Acetone, stirred at room temperature for 48 h (yield: 73%).
Swiss albino mice to see the antitumor efficacy of the compound.
Finally, we applied the compound along with the standard
chemotherapeutic drug CP to investigate the ‘chemoprotective’
(protection against chemotherapeutic drug induced toxicity) and
‘chemoenhancement’ (enhancement of therapeutic efficacy of
conventional chemotherapeutic drugs) efficacy of the compound in
Ehrlich Ascites Carcinoma (EAC) bearing mice.
2.3. Fluorescence property and biodistribution of organoselenium
compound in cancer cell
The absorption maxima and the emission maxima of the com-
pound were observed at 332 nm and 438 nm in 1% dioxaneewater
mixture respectively (Fig. 4).
2. Result
Fluorescence microscopy provides a powerful tool to study the
biodistribution of fluorescent compounds in living cells. Here we
used Ehrlich ascites carcinoma cells, which were exposed to 10ꢀ5 M
concentration of the compound for 2 h, 4 h and 6 h. A significant
cellular accumulation was observed with cells exposed to the
organoselenium compound. As expected, due to the fluorescence
characteristics described above, the cellular uptake of the com-
pound after 2 h, 4 h and 6 h was confirmed clearly by the significant
emission of the compound (Fig. 5B, C and D). However as the time
progresses, the observed cellular intensity was found to increase.
From the microphotographs it was clear that 6 h exposure to the
compound produce highest fluorescence intensity in the cellular
compartments. In contrast to this, no fluorescence intensity was
2.1. Synthesis
The protocol for the synthesis of flavonyl thiazolidinedione
based organoselenocyanate is shown in Scheme 1. The general
method which is known as BakereVenkataraman method was used
to prepare 3-methyl flavone (III). The methyl group of the flavone
was converted to bromomethyl (IV) with N-bromosuccinimide and
a catalytic amount of benzoyl peroxide, and then this group was
subjected to the Sommelet reaction that gave rise to the aldehyde
(V). The N-alkylation of thiazolidinedione with 1,5-dibromo
pentane was carried out via the formation of its potassium salt.
The N-alkylated thiazolidinedione (TB) was then subjected to a
Table 1
Effect of flavonyl-thiazolidinedione based organoselenocyanate on hematological parameters of different groups of mice.
Groups
Hemoglobin
(g/dL)
Alanine transaminase
(IU/ml)
Aspartate transaminase
(IU/ml)
Alkaline
phosphatase
Blood urea nitrogen
(mg/dL)
Creatinine
(mg/dL)
Gr. I
15.08
14.4
15.9
78 ꢂ 5.8
82 ꢂ 7.9
75 ꢂ 5.6
93 ꢂ 9.3
132 ꢂ 12.5
128 ꢂ 18.9
138 ꢂ 7.5
57 ꢂ 2.6
68 ꢂ 4.6
57 ꢂ 2.8
69 ꢂ 5.1*
52 ꢂ 1.5
46 ꢂ 2.8
53 ꢂ 1.7
57 ꢂ 4.1
0.4 ꢂ 0.01
0.3 ꢂ 0.02
0.4 ꢂ 0.02
0.4 ꢂ 0.04
Gr. II
Gr. III
Gr. IV
12.04
160 ꢂ 15.7*
Blood samples were collected 30 days after treatment with organoselenium compound. Results are expressed as means ꢂ SD. n ¼ 6 animals per group.
(*) Denotes significant difference from vehicle control group at p < 0.05 (one-way ANOVA followed by Tukey test).

S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
197
Fig. 1. Changes in body weight in different groups of mice. Results are expressed as means ꢂ SD. n ¼ 6 animals per group. (*) Denotes significantly different from vehicle control
group at p < 0.05 (one-way ANOVA followed by Tukey’s t-test).
visualized in the control cells (incubated without the compound)
(Fig. 5A).
the dose of 4 mg/kg b.w. in CP treated mice leapt the Hb level and
RBC count by 14.4% (ECD), 38.5% (PECD) and 16.6% (ECD), 44.4%
(PECD) respectively. In addition to that, treatment with only orga-
noselenium treatment in pretreatment (PED) and concomitant (ED)
treatment schedule also rose the Hb level and RBC count signifi-
cantly (p < 0.05) compared to that of EAC control group (E). On the
other hand EAC inoculation drastically increased the WBC count
compared to the vehicle control group (VC). However, CP treatment
led to a massive drop in the WBC count. Here also treatment with
the organoselenium compound boosted up the WBC count
compared to the CP control and normalized the condition.
2.4. Organoselenium compound protected hematopoietic system
from CP induced damage in tumor bearing mice
As shown in Table 2, EAC transplanted mice showed significant
(p < 0.05) lowering of hemoglobin (Hb) level and RBC count
compared to the vehicle control group. Whereas significant
(p < 0.05) higher value of WBC count was observed in EAC trans-
planted mice compared to the vehicle control group. Treatment
with CP further decrease the Hb level and RBC count significantly
(p < 0.05) by 15.3% and 21.4% compared to EAC control group.
Treatment with the organoselenium compound (ECD and PECD) at
After EAC inoculation the two hepatotoxicity markers ALT and
AST activity also rose to 175 ꢂ 11.1 and 308 ꢂ 22.5 U/ml in EAC
control group from 77.5 ꢂ 6.4 and 127.5 ꢂ 10.1 U/ml in vehicle
Fig. 2. Histopathology of liver (400ꢃ); (A) vehicle control group; treatment with organoselenium compound at the dose of (B) 2.5 mg/kg b.w. and (C) 5 mg/kg b.w. showed normal
arrangement of hepatocytes with clear nuclei. (D) Treatment with organoselenium compound at the dose of 10 mg/kg b.w.

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Fig. 3. Histopathology of kidney (400ꢃ); (A) vehicle control group; treatment with organoselenium compound at the dose of (B) 2.5 mg/kg b.w. and (C) 5 mg/kg b.w. showed
normal arrangement of the renal cells with clear nuclei. (D) Treatment with organoselenium compound at the dose of 10 mg/kg b.w.
control group respectively (Table 2). During the CP treatment these
values further upregulated to 213.7 ꢂ 14.9 and 303 ꢂ 24.1 U/ml.
Treatment with organoselenium compound brought back these
activities towards normal in EAC bearing mice and also in CP
treated EAC bearing mice.
(E). These enhancements were inhibited during the treatment with
organoselenium compound in concomitant and pretreatment
schedules. It was evidenced that pretreatment showed highest
ability to prevent the lipid peroxidation in hepatic tissues. Pre-
treatment with the organoselenium compound alone and in com-
bination decreased the lipid peroxidation level by 32% (PED) and 46%
(PECD) respectively compared to their respective control groups.
2.5. Organoselenium compound inhibited CP induced lipid
peroxidation level in tumor bearing mice
2.6. Organoselenium compound diminished CP induced hepatic
damage in tumor bearing animals
As shown in Fig. 6, lipid peroxidation level was increased signif-
icantly (p < 0.05) by 52% in EAC bearing group (E) compared to the
vehicle control group (VC). CP treatment (EC) further enhanced the
lipid peroxidation level by 45% compared to the EAC control group
EAC inoculation caused multiple changes in hepatic tissue
which include congestion of blood vessels, formation of pyknotic
Fig. 4. Normalized (A) absorption profile and (B) emission profile of the compound in 1,4-dioxane/water (1:1) mixture.

S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
199
Fig. 5. Fluorescence image of EAC cells (A) without the compound; after incubated with the organoselenium compound for (B) 2 h, (C) 4 h, (D) 6 h (400ꢃ).
nuclei. In addition, marked hepatic injury with lymphocytic and
granulocytic aggregation, necrosis and hypertrophy of liver cells
was observed after ten days of EAC cells inoculation and CP treat-
ment (EC). Concomitant treatment and pretreatment with orga-
noselenium compound alone in EAC bearing mice produced almost
normal architecture of the hepatic tissue with fewer numbers of
pyknotic nuclei in case of concomitant treatment group (ED). Liver
sections from combination treatment with organoselenium com-
pound and CP in concomitant schedule showed mild infiltration of
mononuclear cells and vacuolization of cytoplasms with no ne-
crosis, however, pretreatment (PECD) showed almost normal ar-
chitecture with some hypertrophic cells (Fig. 7).
with CP alone for 10 consecutive days (EC) further depleted the GSH
level by 39% compared to EAC control group (E). Organoselenium
treatment significantly reversed the GSH content. Prior adminis-
tration of the compound showed better result than concomitant
treatment. GSH content upregulated by almost two fold (p < 0.05)
in PED group and by more than fourfold (p < 0.05) in PECD group
compared to their respective control groups (Fig. 8).
2.8. Treatment with organoselenium compound upregulated the
activities of different antioxidant/detoxifying enzyme system in
tumor bearing mice
The activities of various antioxidant and detoxifying enzymes
like SOD, CAT, GST, GPx and also glutathione content were impacted
significantly to a great extent by EAC inoculation and CP treatment
(Fig. 9). EAC inoculation caused a reduction in the activities of all
the antioxidant and detoxifying enzyme systems and these down
regulations were even more accelerated by CP treatment.
2.7. Organoselenium compound enhanced the GSH level in liver
tissues of tumor bearing animals
EAC inoculation decreased the GSH content compared to the
vehicle control animals by 66% (p < 0.05). EAC bearing mice treated
Table 2
Effect of combination treatment of organoselenium compound and CP on hematological parameters in tumor bearing mice.
Groups
Hemoglobin
(g/dL)
Red blood corpuscle
White blood corpuscle
Alanine transaminase
(IU/ml)
Aspartate transaminase
(IU/ml)
(ꢃ106)
(ꢃ103)
VC
E
ED
PED
EC
14.8 ꢂ 0.2
9.8 ꢂ 0.2*
10.3 ꢂ 0.3#
12.4 ꢂ 0.8#
8.3 ꢂ 0.7#
9.5 ꢂ 0.6&
11.5 ꢂ 0.9&
9.4 ꢂ 0.7
6.1 ꢂ 0.5
13.3 ꢂ 0.9*
9.2 ꢂ 0.5#
8.1 ꢂ 0.3#
3.9 ꢂ 0.3#
4.8 ꢂ 0.4&
6.7 ꢂ 0.1&
77.5 ꢂ 6.4
175 ꢂ 11.1*
123 ꢂ 10.3#
70.4 ꢂ 7.9#
213.7 ꢂ 14.9#
140 ꢂ 9.1&
82 ꢂ 5.7&
127.5 ꢂ 10.1
308 ꢂ 22.5*
178 ꢂ 16.7#
142 ꢂ 12.0#
303 ꢂ 24.1#
221.4 ꢂ 11.2&
191 ꢂ 18.5&
6.6 ꢂ 0.4*
7.5 ꢂ 0.7#
8.2 ꢂ 0.2#
5.4 ꢂ 0.2#
6.3 ꢂ 0.4&
7.8 ꢂ 0.4&
ECD
PECD
Results are expressed as means ꢂ SD. n ¼ 6 animals per group.
(*) p < 0.05 statistically compared with Gr. VC (vehicle control).
(#) p < 0.05 statistically compared with Gr. E (EAC control).
(&) p < 0.05 statistically compared with Gr. EC (CP only treated group) as compared to control group (one-way ANOVA/Tukey).

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S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
Fig. 6. Effect of monotherapy and combination treatment on lipid peroxidation level of
different groups of mice. Results are expressed as means ꢂ SD. n ¼ 6 animals per
group. ‘*’ Denotes p value <0.05 compared with Gr. VC (vehicle control). ‘#’ Denotes p
value <0.05 compared with Gr. E (EAC control). ‘#p’ Denotes p value <0.05 compared to
EC (CP only treated group) (one-way ANOVA/Tukey).
Fig. 8. Effect of monotherapy and combination treatment on GSH content of different
groups of mice. Results are expressed as means ꢂ SD. n ¼ 6 animals per group. ‘*’
Denotes p value <0.05 compared with Gr. VC (vehicle control). ‘#’ Denotes p value
<0.05 compared with Gr. E (EAC control). ‘#p’ Denotes p value <0.05 compared to EC
(CP only treated group) (one-way ANOVA/Tukey’s t-test).
Intervention of the organoselenium compound during the CP
treatment resulted a steady rise in these activities. Moreover, the
selenoenzymes, glutathione peroxidase had most prominent
improvement during the adjuvant therapy (Fig. 9).
Fig. 7. Light microscopy photomicrographs depicting sections from liver of mouse (A) receiving a vehicle control; Normal hepatocellular architecture depicting hepatocytes
radiating from the central vein (B) EAC control, EAC cells inoculation displaying congestion of blood vessels (CNG), and most of hepatocytes with enlarged and deeply stained
pyknotic nuclei (black arrows). (C) Liver of CP-treated mouse showed marked hepatic injury with lymphocytic and granulocytic aggregation (white double arrow), necrosis (elbow
arrow connector) and hypertrophy (white arrow). (D) Concomitant treatment and (E) pretreatment with organoselenium compound revealing nearly the similar histological profile
as in vehicle control group with no marked hepatotoxicity. (F) Concomitant treatment with CP and organoselenium compound, ECD. Only few infiltrations of mononuclear cells
(MC) and pyknotic nuclei were observed. (G) Pretreatment with organoselenium compound in CP treated EAC bearing mice, PECD. Treatment with organoselenium compound
together with CP markedly reduced the severity of the CP and EAC induced hepatic damage (H&E staining 400ꢃ).

S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
201
Fig. 9. Effect of monotherapy and combination treatment on (A) SOD activity (B) CAT activity (C) GPX activity and (D) GST activity of different groups of mice. ‘*’ Denotes p value
<0.05 compared with Gr. VC (vehicle control). ‘#’ Denotes p value <0.05 compared with Gr. E (EAC control). ‘#p’ Denotes p value <0.05 compared to EC (CP only treated group) (one-
way ANOVA/Tukey).
2.9. Inhibition of hepatic ROS production by organoselenium
treatment
2.11. Protection from chromosomal damage by organoselenium
treatment
As showed in Fig. 10, the ROS level in hepatic tissue was
significantly (p < 0.05) higher in EAC bearing mice (E) compared to
the vehicle control group (VC). During CP treatment this
enhancement was even more prominent and an increase of 43%
(p < 0.05) in EC group was observed compared to the EAC control
group (E). Treatment with the organoselenium compound had
profound effect on the hepatic ROS generation. Here also pre-
treatment showed greater effect than concomitant treatment. ROS
level was inhibited by 32% (p < 0.05) in PED group and 68% in PECD
group compared to their respective control groups.
EAC inoculation and CP treatment caused multiple types of
chromosomal aberration (stretching, breaking, ring formation,
fragmentation) in bone marrow cells (Fig. 12A). EAC inoculation
resulted almost fivefold rise in the percentage of aberrant cells
compared to the vehicle control group. In addition to that, treat-
ment with CP resulted further two fold increase (p < 0.05) in this
percentage compared to the EAC control group (E). Treatment with
organoselenium compound in pretreatment and concomitant
treatment schedules was able to minimize these chromosomal
aberrations. Pretreatment with organoselenium compound caused
inhibition of chromosomal aberration significantly (p < 0.05) by
55% in PECD group and 48% in PED group compared to their
respective control groups (Fig. 12B).
2.10. Protection from DNA strand breaks of peripheral blood
lymphocyte by organoselenium compound
Alkaline comet assay (SCGE) due to its versatility and sensitivity
is generally considered as a reliable method of DNA strand breaks
assessment. In this present study, comet assay in peripheral blood
lymphocytes was performed to observe the extent of DNA damage.
Fig. 11 depicted various comet formed in different groups of mice.
EAC inoculation and CP treatment induced extended tail length
where as treatment with the organoselenium compound inhibited
the damages of the lymphocytes and shortened the tail length.
According to Table 3, treatment with CP led to significant increase
in percentage of tail DNA, tail length and olive tail moment
compared to the EAC control group (E). Moreover, EAC inoculation
also enhanced these parameters compared to the vehicle control
group (VC). Treatments with organoselenium compound reduced
all these parameters and prevented DNA damage in lymphocytes.
In PECD group, pretreatment with the organoselenium compound
reduced the percentage of tail DNA by 64%, tail length by 74% and
tail moment by 77% compared to the EC control group and in PED
group the reductions were 67%, 70% and 76% respectively compared
to EAC control group.
Fig. 10. Effect of monotherapy and combination treatment on ROS generation in liver
cells of different groups of mice. Results are expressed as means ꢂ SD. n ¼ 6 animals
per group. ‘*’ Denotes p value <0.05 compared with Gr. E (EAC control). ‘#’ Denotes p
value <0.05 compared with Gr. EC (CP only treated group) (one-way ANOVA/Tukey’s
test).

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S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
2.12. Enhancement of the therapeutic efficacy of CP during the
adjuvant therapy
Therapeutic efficacy of the adjuvant therapy as well as the
monotherapy were determined by various parameters like tumor
volume, packed cell volume, tumor cell count, mean survival time
(MST) and increasing life span (Table 4). Treatment with organo-
selenium compound alone in EAC bearing mice was capable of
minimizing the tumor volume, packed cell volume and tumor cell
count compared to the untreated EAC control group (E). Organo-
selenium treatment significantly (p < 0.05) decreased the tumor
volume by 34% and 51%, tumor cell count by 47% and 60%, packed
cell volume by 50% and 56% in concomitant and pretreatment
schedule respectively compared to the EAC control group. On the
other hand, CP treatment caused a drastic reduction (of about 90%)
in these parameters compared to the EAC control group. Further-
more, the adjuvant treatment with CP and organoselenium com-
pound in ECD and PECD group significantly (p < 0.05) decreased
the tumor volume by 51% and 76%, tumor cell count by 55% and
80%, packed cell volume by 59% and 71% respectively compared to
the CP treated control group.
Fig. 11. Photographs showing comet of lymphocytes from different groups of mice.
Lymphocytes form vehicle control group showed intact DNA with no tail. EAC inocu-
lation (E) and CP treatment (EC) showed prominent tail length with less percentage of
DNA in the head and greater in the tail. Treatment with the organoselenium compound
able to inhibit the DNA damage (ED, PED, ECD and PECD). Pretreatment with the
organoselenium compound showed less migrated DNA with no prominent tail length.
Table 3
Effect of combination treatment of organoselenium compound and CP on DNA
damage of lymphocyte of different groups of mice.
Groups Head DNA (%) Tail DNA (%) Tail length (
m
m) Olive tail moment
VC
E
ED
PED
EC
94.8 ꢂ 1.1
56.3 ꢂ 1.9*
69.9 ꢂ 2.1#
85.8 ꢂ 3.5#
41.9 ꢂ 3.9#
70.7 ꢂ 2.6&
79.3 ꢂ 4.3&
4.4 ꢂ 2.2
43.6 ꢂ 1.9*
30.1 ꢂ 2.1#
14.1 ꢂ 3.5#
58.1 ꢂ 3.9#
29.2 ꢂ 2.6&
20.7 ꢂ 4.3&
1.5 ꢂ 0.3
17.9 ꢂ 1.1*
10.0 ꢂ 0.8#
5.3 ꢂ 0.5#
33.9 ꢂ 2.8#
13.9 ꢂ 1.0&
8.2 ꢂ 0.6&
0.5 ꢂ 0.07
3.9 ꢂ 0.5*
1.8 ꢂ 1.9#
0.8 ꢂ 0.1#
6.6 ꢂ 0.3#
3.8 ꢂ 0.2&
1.6 ꢂ 0.2&
Fig. 12. A: Photographs of metaphase chromosomes of bone marrow cells collected
from treated and untreated EAC bearing mice. Arrows indicate chromatid gaps (g),
chromatid terminal deletions (TD), breaks (B), sister-chromatid union (SCU), acentric
chromosome (AC), acentric fragments (AF), ring formation (R), and stretching (STR). B:
Effect of monotherapy and combination treatment on percentage chromosomal aber-
ration of bone marrow cells. Results are expressed as means ꢂ SD. n ¼ 6 animals per
group. ‘*’ Denotes p value <0.05 compared with Gr. VC (vehicle control). ‘#’ Denotes p
value <0.05 compared with Gr. E (EAC control). ‘#p’ Denotes p value <0.05 compared to
EC (CP only treated group) (one-way ANOVA/Tukey).
ECD
PECD
Results are expressed as means ꢂ SD. n ¼ 6 animals per group.
(*) p < 0.05 statistically compared with Gr. VC (vehicle control).
(#) p < 0.05 statistically compared with Gr. E (EAC control).
(&) p < 0.05 statistically compared with Gr. EC (CP only treated group) as compared
to control group (one-way ANOVA followed by Tukey’s t-test).

S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
203
Table 4
Effect of organoselenium compound alone or in combination with CP on tumor growth, mean survival time, % ILS and % apoptosis in tumor bearing mice.
Groups
Tumor volume (ml)
Packed cell volume (ml)
Tumor cell count (ꢃ106)
Mean survivability (days)
Increasing life span (%)
Apoptosis (%)
E
ED
PED
EC
ECD
PECD
4.9 ꢂ 0.4
2.8 ꢂ 0.1
67.8 ꢂ 6.0
35.4 ꢂ 3.0*
27.1 ꢂ 1.7*
7.6 ꢂ 0.5*
2.4 ꢂ 0.2#
1.1 ꢂ 0.15#
22.2 ꢂ 1.3
28.1 ꢂ 2.4*
32.3 ꢂ 1.7*
40.3 ꢂ 3.3*
54.2 ꢂ 2.6#
62.4 ꢂ 2.4#
e
5.8 ꢂ 0.2
18.5 ꢂ 1.7*
26.3 ꢂ 2.0*
35.5 ꢂ 2.7*
46.4 ꢂ 2.5#
57.2 ꢂ 6.0#
3.2 ꢂ 0.2*
2.4 ꢂ 0.16*
0.7 ꢂ 0.06*
0.2 ꢂ 0.04#
0.1 ꢂ 0.01#
1.4 ꢂ 0.29*
1.2 ꢂ 0.15*
0.5 ꢂ 0.02*
0.2 ꢂ 0.01#
0.1 ꢂ 0.01#
26.5
45.4
63.1
81.5
181.0
Results are expressed as means ꢂ SD. n ¼ 6 animals per group.
(*) Denotes p value<0.05 compared with Gr. E (EAC control).
(#) Denotes p value < 0.05 compared with Gr. EC (CP only treated group) (one-way ANOVA followed by Tukey’s t-test.).
Organoselenium compound alone could enhance the mean
survivability of the tumor bearing hosts by 26% and 45% (p < 0.05)
in concomitant and pretreatment schedule respectively compared
to the EAC control (E) group. CP treatment for ten consecutive days
also increased the mean survivability by 84% compared to the EAC
control (E) group. The organoselenium compound alone was also
able to moderately enhance the mean survivability of the tumor
bearing host. However, the combination treatment caused an
enormous increase in the mean survivability of the tumor bearing
host compared to the untreated controls and the mice underwent
monotherapy. The enhancements were 35% and 55% in ECD and
PECD groups compared to their respective controls (Table 4).
To evaluate the mechanism of cell death we have carried out
TUNEL assay in tumor cells. It was evidenced form Table 4 that the
treatment with organoselenium compound in concomitant treat-
ment and pretreatment schedule induce 18.5% and 26.3% apoptosis
in tumor cells which was significantly higher than the untreated
EAC bearing control group. Percentage of apoptotic cells in com-
bination treatment were further enhanced by 30% (p < 0.05) in case
of concomitant (ECD) and 61% (p < 0.05) in case of pretreatment
(PECD) than the CP monotherapy (EC).
dinedione was then condensed with formyl flavone. Finally, the
resulting bromo compound was converted to selenocyanate by the
displacement with potassium selenocyanate at room temperature.
Treatment with organoselenium compound at the dose of 2.5
and 5 mg/kg b.w. for 30 days did not alter the lipid peroxidation
level and other toxicity markers of liver (ALT, AST and ALP) and
kidney (BUN and creatinine) significantly compared to the vehicle
control group. From histopathological examination it was observed
that the overall cellular structures of liver and kidney tissues were
unaltered with no necrosis, inflammation or other significant le-
sions at the dose of 2.5 and 5 mg/kg b.w. for 30 days. All these
proved nontoxic character of the compound at the above doses.
However, the toxicity levels were elevated when treated with the
higher dose of 10 mg/kg b.w. In addition, treatment with the higher
dose resulted in infiltration of mononuclear cells in both liver and
kidney tissues. Therefore, we selected 4 mg/kg body weight of the
compound as the primary screening dose for the study of the
‘chemoprotective’ and ‘chemoenhancement’ property of the
compound.
Chemotherapy inevitably leads to the decrease in bone marrow
function that reflects by a substantial decrease in the mature blood
elements [17]. In the present study hemoglobin (Hb) level, WBC
count and RBC count decreased markedly after CP treatment in
tumor bearing mice. Whereas treatment with the organoselenium
compound increased these levels and justified its potential for the
restoration of hematopoiesis in mice after chemotherapy. In the
current study, liver function was significantly impaired after EAC
inoculation which was further appended by CP treatment.
2.13. Production of ROS in tumor cells during adjuvant therapy as
well as monotherapy
Treatment with the organoselenium compound alone was able
to enhance the ROS level in tumor cells significantly compared to
the EAC control group (E). After CP treatment the ROS of tumor cells
in EAC bearing mice were enhanced significantly (p < 0.05) from
90.4 to 282.5 units of fluorescence intensity/mg protein (Fig. 13).
Furthermore, when the organoselenium compound was used along
with CP treatment, ROS in the tumor cells was upregulated by 53%
and 70% in case of concomitant and pretreatment schedule
respectively compared to the CP control group (EC).
3. Discussion
Primarily, we tried to synthesize the flavonyl thiazolidinedione
based organoselenocyanate by N-alkylation of flavonyl thiazolidi-
nedione with 1,5-dibromopentane. In our first attempt, we carried
out the N-alkylation by two different methods e (i) K2CO3 in DMF
and (ii) sodium methoxide in methanol and DMF. But each time it
did not produce the desired one. The reason for the failure of N-
alkylation of the flavonyl thiazolidinedione may be due to the
presence of the terminal double bond adjoining the thiazolidine-
dione moiety which in turn decreased the acidity of the hydrogen
attached to the nitrogen due to the lower resonance stabilization of
the carbonyl group.
Fig. 13. Effect of monotherapy and combination treatment on the reactive oxygen
species of tumor cells. Results are expressed as means ꢂ SD. n ¼ 6 animals per group.
‘*’ Denotes p value <0.05 compared with Gr. E (EAC control). ‘#’ Denotes p value <0.05
compared with Gr. EC (CP only treated group) (one-way ANOVA followed by Tukey’s t-
test).
In the modified pathway, first we did the N-alkylation of thia-
zolidinedione with 1,5-dibromopentane via the formation of the
potassium salt of thiazolidinedione. The alkylated thiazoli

204
S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
Hepatopathy could lead to the leakage of marker enzymes like ALT
and AST into the blood in conformity with the extent of liver
damage [18] affecting the integrity of liver cells. Oral administration
of the organoselenium compound was effective in reducing hepa-
totoxicity which was observed by the reduction in the ALT and AST
activities in serum. The hepatoprotective efficacy of the organo
selenium compound was also confirmed by the histopathological
study which indicated that EAC and CP induced hepatic damages
were diminished effectively after organoselenium treatment.
Results from the present work clearly demonstrated the po-
tential of CP in inducing lipid peroxidation to the liver cells. In-
crease in the accumulation of MDA in cells can result into cellular
degradation, some biochemical changes and even cell death [19].
The decrease in MDA content in liver tissue homogenates in orga-
noselenium treated groups implied the free radical scavenging
property of the organoselenium compound.
In this present study we also carried out comet assay under
alkaline (pH > 13) condition to evaluate DNA damage in lympho-
cytes [20]. Phosphoramide mustard, the major antineoplastic
metabolite of CP, induces a variety of changes in DNA [21] through
its ability to form labile covalent DNA adducts and cross linkages
[22]. This DNA damage ultimately leads to the secondary tumors in
humans. In the present study, it was observed that CP adminis-
tration was capable of inducing DNA damage in lymphocytes and
both numerical and structural chromosomal aberrations in bone
marrow cells of tumor bearing mice. Administration of organo-
selenium compound before or during CP treatment ameliorated the
mutagenic markers and was able to reduce the damages induced by
EAC and CP treatment. These results indicated that the organo-
selenium compound could be effective in mobilizing DNA repair
mechanisms which may be in part due to the activation of different
DNA repairing enzymes.
Relationships between tissue injury, genotoxicity, chromo-
somal instability and oxidative stress have been well demon-
strated in many experimental animal models [23]. The
compound considerably inhibited intracellular ROS level in he-
patic tissues. These results suggested that the ROS scavenging
activity of the organoselenium compound was directly associ-
ated with its potential to protect normal tissues from CP induced
oxidative stress. Several reports suggest that increased ROS
generation after CP treatment reduced the GSH level and GPx/
GST activity in several organs including liver [24]. Additionally,
free radicals may inactivate GPx through the modification of a
cysteine like essential residue on GPx [25]. In this present study
we found that EAC inoculation significantly reduced the GPx,
SOD, CAT and GST activity in hepatic cells. These activities were
further suppressed by CP treatment compared to the EAC control
group. The balance between the dismutation of superoxide anion
(by SOD) and its conversion to molecular oxygen and water (by
CAT or GPx) may be critical to maintain cellular antioxidant
defense [26]. The diminished activity of various antioxidants and
detoxifying enzyme systems may lead to a net increase in ROS
and may be related to several disease pathologies [27]. Treat-
ment with the organoselenium compound invariably increased
the GPx, SOD, CAT and GST activity and reduced the CP mediated
stress in liver cells. It is possible that the synthesized compound
reversed the CP mediated ROS induction and there by restoring
the balance between various antioxidants and detoxifying
enzyme systems. Another explanation is that, organoselenium
treatment increased the activity of antioxidant selenoenzyme
GPx which, in turn, upregulated the other antioxidant enzyme
systems. Enhancement of CP induced depleted GSH level after
the organoselenium treatment may be another cause which is
playing an important role in maintaining the homeostasis of
antioxidant enzymes.
It is crucial that cytoprotective and rescue agents do not pro-
tect tumor cells from the toxicity of chemotherapeutic agents. Our
study indicated that the organoselenium compound did not exert
any protective effect on tumor cell growth in vivo rather it has
moderate cytoxicity on tumor cells. When the selenium com-
pound was used alone to treat the EAC tumor bearing mice it
showed moderate antitumor efficacy without inducing any hep-
atotoxicity and oxidative stress on normal tissue like liver. In
contrast, the adjuvant therapy resulted in a substantial improve-
ment of the activity of CP. The efficacy of combination treatment
in terms of tumor volume, packed cell volume and cell count was
significantly much higher compared to the CP monotherapy. The
combination therapy also potentiated the monotherapy in terms
of mean survival time and life span of tumor bearing host. This
may be due to the reduction in CP induced cellular toxicities in
normal cell and sensitization of tumor cells towards the chemo-
therapeutic drug CP. Evasion of apoptosis is one of the hallmarks
of cancer. It is responsible for tumor promotion and progression as
well as for treatment resistance [28]. It is well known that the
most cytotoxic agents used in cancer treatment mediate their
anticarcinogenic effects by induction of apoptosis [29]. In this
present study, it was observed that the percentage of apoptotic
cells increased after the treatment with organoselenium com-
pound alone or in combination with CP. To determine whether the
difference in apoptotic cell death was associated with ROS for-
mation, the levels of intracellular ROS in tumor cells were evalu-
ated by fluorescence microscopy with the oxidant sensitive probe
20,70-dichlorofluorescin diacetate (DCFH-DA). As discussed in the
result section, an increase in 20,70-dichlorofluorescein (DCF) fluo-
rescence was observed in tumor cells when they were exposed to
CP, and this increase in fluorescence intensity was further signif-
icantly enhanced in cells treated with the organoselenium com-
pound in combination with CP. Fluorescence intensity in the
tumor cells was also enhanced when the tumor bearing mice was
treated with organoselenium compound alone. However, the
exact mechanism of cell death has not been determined fully. But
from the above discussion it can be inferred that the organo-
selenium compound can generate ROS in tumor cell which
sensitized them to apoptosis.
The mechanism of such selective protection of normal cells over
the tumor cells by the organoselenium compound against CP
induced toxicity is not known. It is possible that organoselenium
compound selectively increased the GSH levels in normal versus
tumor cells. Selective enhancement in the activities of antioxidant
enzymes in the normal cells by an antioxidant may be related to the
differential regulation of detoxifying/antioxidant enzyme system in
normal versus tumor cells. Genomic instability is a hallmark of
tumor cells, and tumor cells may be under a continued environ-
ment of oxidative stress, where antioxidant may act as a prooxidant
[30]. Schwartz [31] suggested that dietary antioxidants may act as
an antioxidant to normal cells and as a prooxidant to cancer cells.
The prooxidant activity of an antioxidant may be the reason for the
antitumor activity of many antioxidants [32]. Whether a different
cellular microenvironment around tumor cells versus normal cells
is responsible for the differential effect needs further investigation.
Another possibility for the selective protection of organoselenium
compound in normal versus tumor cells may be related to the
difference in the cellular uptake of the organoselenium compound.
Tumor cells generate their own antioxidant enzyme which also
includes the selenoenzymes. Hence, the dietary selenium may end
up being used up for selenoenzyme synthesis. Thus, there was a
possibility of higher selenium level in tumor cell which can act as
prooxidant [33]. However, further research work is needed to
establish the exact underlying mechanism of the differential effect
of the organoselenium compound.

S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
205
4. Conclusion
obtained was purified by column chromatography using 10e15%
ethyl acetate/petroleum ether. Yield: 76%; amount of product iso-
lated: 0.607 g; m.p.: 187 ꢁC (lit. m.p. 184 ꢁC [34])
In summary, we found that the organoselenium compound
exerts a definite cytoprotective effect on normal cell and inhibited
the CP induced cellular toxicity in mice. At the same time increase
the therapeutic efficacy of CP in tumor cells. Considering the
nontoxic nature of the organoselenium compound, the potential
use of this agent for the population undergoing cancer treatments
with the drug cyclophosphamide should be considered.
5.1.2. Synthesis of 3-(5-bromopentyl)thiazolidine-2,4-dione (TB)
2.1 g of potassium hydroxide and 10 ml distilled ethanol was
mixed and kept for overnight. 6 ml of this solution was taken and
mixed with 1.6 g (13.6 mmol) of 2,4-thizolidinedione and 4 ml
ethanol. The resulting mixture was heated without cooling at 40 ꢁC
for 2 h. The reaction mixture was then cooled. Soon after the
cooling a white crystal was obtained, which was filtered off and
dried in vacuo. Yield: 87%; amount isolated: 1.83 g; m.p.: 240e
242 ꢁC. 0.92 g (5.93 mmol) K-salt of thiazolidinedione was mixed
with 2.8 ml 1,5-dibrompentane in 10 ml DMF. The reaction mixture
was stirred for 16 h at room temperature. After completion of 16 h
the reaction mixture was poured into ice cold water and extracted
with chloroform. Low melting oil was appeared. The crude product
was then purified by column chromatography using 10e12% chlo-
roform and petroleum ether mixture to get a viscous liquid. Yield:
71%; amount of product isolated: 1.12 g. The compound was liquid
at room temperature. 1H NMR (CDCl3, 300 MHz): 1.46 (m, 2H, e
CH2), 1.63 (m, 2H, eCH2), 1.89 (m, 2H, eCH2), 3.40 (t, J ¼ 6.9, 2H, e
CH2), 3.64 (t, J ¼ 7.2, 2H, eCH2), 3.95 (s, 1H, eCH). 13C NMR (CDCl3,
300 MHz): 25.00, 26.48, 31.69, 33.21, 33.63, 41.50, 171.28, 171.61.
ESI-MS m/z: 266.27 and 268.27 [M þ 1]þ.
5. Experimental procedure
5.1. Chemistry
5.1.1. Synthesis of 6-formylflavone
6-Formyl flavone was prepared following the literature method
[34].
5.1.1.1. Synthesis of 2-benzoyloxy-5-methylacetophenone. A mixture
of benzoyl chloride (1 ml, 8.6 mmol) and 2-hydroxy-5-methyl
acetophenone (1 g, 6.6 mmol) was heated in 25 ml pyridine at
60 ꢁC for 30 min. The reaction mixture was then poured in ice cold
acidified water with constant stirring.
A precipitate of 2-
benzoyloxy-5-methylacetophenone (I) was formed. The product
was purified by column chromatography using 10% chloroform and
petroleum ether mixture. Yield: 81%; amount of product isolated:
1.35 g; m.p.: 63 ꢁC (lit. m.p. 63 ꢁC [34]).
5.1.3. Synthesis of flavonyl thiazolidinedione based organoseleno
cyanate: 5-((4-oxo-2-phenyl-4H-chromen-6-yl)methylene)-3-(5-
selenocyanatopentyl)thiazolidine-2,4-dione (VII)
A mixture of formyl flavone (V) (200 mg, 0.8 mmol), 3-(5-
bromopentyl)thiazolidine-2,4-dione (TB) (280 mg, 0.9 mmol) and
5.1.1.2. Synthesis of 1-(2-hydroxy-5-methylphenyl)-3-phenyl
propane-1,3-dione. 2-Benzoyloxy-5-methylacetophenone (I) (5 g,
19.6 mmol) was treated with 15 g pulverized hot KOH in 25 ml
pyridine and heated at 100 ꢁC for 30 min. The reaction mixture was
poured in acidified (6 N HCl) ice cold water. A yellow precipitate
appeared was then filtered off. The residue was dried in vacuum.
The crude product, 1-(2-hydroxy-5-methylphenyl)-3-phenyl
propane-1,3-dione (II) was then recrystallized from petroleum
ether as yellow shining plates. Yield: 75%; amount of product iso-
lated: 3.73 g; m.p.: 95 ꢁC (lit. m.p. 94 ꢁC [34]).
piperidine (20 ml) was refluxed in 15 ml benzene for 16 h under
nitrogen. A brown yellow precipitate was formed. The reaction
mixture was then cooled down and the solvent benzene was
evaporated. The crude product, flavonyl thiazolidinedione bromo-
pentyl derivative (VI) thus obtained was purified by column chro-
matography using 10e15% mixture of petroleum ether and
chloroform over silica gel (80e120 mesh). M.p.: 153e155 ꢁC; yield:
64%; amount isolated: 256 mg. 1H NMR: (CDCl3, 300 MHz): 3.80 (t,
2H, J ¼ 7.2 Hz, CH2); 3.06 (t, 2H, J ¼ 7.2, CH2); 1.98 (m, 2H, CH2); 1.52
(m, 2H, CH2); 1.76 (m, 2H, CH2); 6.85 (s, 1H, ]CH); 7.54e7.56 (m,
3H); 7.68 (d, 1H, J ¼ 8.7 Hz); 7.82 (dd, 1H, J ¼ 8.7 Hz, 2.1 Hz); 7.92e
7.95 (m, 2H). 7.97 (s, 1H); 8.37 (d, 1H, J ¼ 2.4 Hz). 13C NMR (CDCl3,
300 MHz): 25.14, 26.63, 31.99, 33.71, 41.66, 108.03, 119.53, 124.08,
126.37, 129.19, 130.62, 131.97, 133.64, 159.52, 163.90, 177.51, 190.34.
500 mg (1.0 mmol) of compound VI was mixed with 150 mg
(1.04 mmol) of potassium selenocyanate in 2 ml acetone. The
mixture was stirred at room temperature for 24 h. After that, on the
next day, 75 mg (0.52 mmol) of potassium selenocyanate and 1 ml
acetone was again added to the reaction mixture and further stirred
for 24 h. After completion of 48 h the reaction mixture was dec-
anted and acetone was evaporated in rotary evaporator. The
resulting crude product was then purified by column chromatog-
raphy using mixture of 20e25% chloroform and petroleum ether, to
get pure compound VII. M.p.: 170e172 ꢁC, yield: 73%; amount
5.1.1.3. Synthesis
1-(2-Hydroxy-5-methylphenyl)-3-phenylpropane-1,3-dione
of
6-methylflavone
(III).
(II)
(2.5 g, 9.8 mmol) was stirred in 12.5 ml conc. H2SO4 at room tem-
perature for 30 min. A white precipitate obtained was then filtered
off. The residue was dried in vacuum. The product, 6-methylflavone
(III) obtained was purified by column chromatography using 5e10%
chloroform and petroleum ether mixture. Yield: 73%, Amount of
product isolated: 1.70 g; m.p: 121 ꢁC (lit. mp. 118e120 ꢁC [34]).
5.1.1.4. Synthesis of 6-bromomethylflavone (IV). A mixture of N-
bromosuccinimide (III) (1.2 g, 6.72 mmol) and 6-methylflavone (1 g,
4.2 mmol) was dissolved in 40 ml hot benzene (35 ml) and benzoyl
peroxide (0.1 g) was added. The reaction mixture was refluxed for
6 h. It was then cooled. White crystal of succinimide was appeared.
The whole solution was then decanted and evaporated in the rotary
evaporator. The crude product, 6-bromomethylflavone was purified
by column chromatography using 15e20% ethyl acetate/petro-
leum-ether. Yield: 67%; amount of product isolated: 0.892 g; m.p.:
176 ꢁC (lit. m.p. 173 ꢁC [34]).
isolated: 200 mg. IR (cmꢀ1): 1655 (
g-pyrone CO), 1681 and 1738
(thiazolidine ring CO), 2149 (CN stretching). 1H NMR (CDCl3,
300 MHz): 3.80 (t, 2H, J ¼ 6.9 Hz, CH2); 1.98 (m, 2H, CH2); 1.54 (m,
2H, CH2); 1.76 (m, 2H, CH2); 3.06 (t, 2H, J ¼ 7.2, CH2); 7.98 (s, 1H, ]
CH); 8.37 (d, 1H, J ¼ 2.1 Hz); 7.83 (dd, 1H, J ¼ 9 Hz and 2.1 Hz); 7.68
(d, 1H, J ¼ 9 Hz); 6.88 (s, 1H); 7.58e7.53 (m, 3H) 7.93e7.96 (m, 2H).
ESI-MS m/z: 525.13 [Mþ þ H]. 13C NMR (CDCl3, 300 MHz): 23.04,
27.28, 27.94, 41.78, 63.53, 107.78, 119.36, 122.89, 124.29, 126.28,
127.77, 129.11, 130.42, 131,16, 131.64, 131.93, 134.56, 156.58, 161.05,
163.59, 166.01, 167.31, 177.27.
5.1.1.5. Synthesis of 6-formylflavone (V). A mixture of 6-
bromomethylflavone (IV) (1 g, 3.17 mmol) and hexamethylenetet-
ramine (4 g, 28.0 mmol) in 20 ml acetic acid (%50 v/v) was refluxed
for 15 min. After that HCl:H2O (1:1, 10 ml) was added and refluxed
for another 10 min. The product was separated on dilution of the
reaction mixture with water. The product, 6-formylflavone thus

206
S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
5.2. Biology
inoculation from day 1 to day 10. Only organoselenium pretreated
group (PED): animals were pretreated with the organoselenium
compound orally at a dose of 4 mg/kg b.w. 15days prior to tumor
inoculation and the treatment was continued 24 h after tumor
inoculation from day 1 to day 10. (The day of EAC cell inoculation
was count as day zero.) Only CP treated group (EC): animals were
received CP at a dose of 25 mg/kg b.w. in water by intraperitoneal
administration and 5.5% propylene glycol in water by oral gavages
from day 1 to day 10. Concomitant treatment with CP and organo-
selenium (ECD): organoselenium compound (4 mg/kg b.w.) along
with CP (25 mg/kg b.w.) was administered from day 1 to day 10.
Pretreatment with CP and organoselenium (PECD): animals were
given organoselenium compound orally at a dose of 4 mg/kg b.w.
15days prior to tumor inoculation and the treatment was continued
from day 1 to day 10 starting from 24 h after tumor inoculation
along with CP (25 mg/kg b.w.).
5.2.1. Cell culture for biodistribution of the organoselenium
compound and its interaction with the DNA from tumor cells
Fluorescence microscopy study: stock cells of EAC were cultured
in RPMI-1640 and supplemented with 10% FBS, penicillin
(100 IU mLꢀ1) and streptomycin (100
m
g mLꢀ1) in a humidified
atmosphere of 5% CO2 at 37 ꢁC. At this condition the cells were
seeded for 24 h in 6 well plates (2 ꢃ 105 cells in each well). The cell
culture medium was then replaced with fresh medium containing
the synthetic organoselenium compound in a concentration of
10ꢀ3 M (0.1% V/V DMSO) and incubated for 2 h, 4 h and 6 h at 37 ꢁC
in a 5% CO2/95% air atmosphere. Fluorescence images were taken at
400ꢃ magnifications under a fluorescence microscope (Model:
Leica DM 4000B) with imaging system and an Ex/Em 350/422 nm
filter.
5.2.2. Experimental animals
5.2.6. Estimation of protein content in the tissue homogenate [35]
Total protein content in tissue homogenate during biochemical
analysis assay was measured through Lowry method using Folin-
Adult (5e6 weeks) Swiss albino female mice (23 ꢂ 2 g), bred in
the animal colony of Chittaranjan National Cancer Institute (CNCI),
Kolkata, were used for this study. Animals were maintained at
controlled temperature under alternating light and dark conditions.
Standard food pellets (devoid of supplemented selenium) from
Lipton India Ltd. and drinking water was provided ad libitum. All
the ethical issues as proposed by the IAEC (Institutional Animal
Ethics Committee) of CNCI were strictly followed during the
maintenance of the animals as well as during their sacrifice.
Phenol reagent. To 10
NaOH, 10 l of 1% CuSO4, 10
and 980 l of 2% Na2CO3 were added and kept for 15 min. 300
m
l unknown tissue sample, 100
l of 2% Sodiumepotassium tartarate
l of
ml 1 N
m
m
m
m
Folin-Phenol reagent (v/v; 1:2 with water) was then added and
kept for 15 min in the dark at room temperature. The absorbance of
the color developed was measured against the blank sample
(without homogenate) at 660 nm using the Shimadzu 160A UV
Spectrophotometer. A standard curve was prepared by measuring
the absorbance of different known concentrations of BSA and the
total protein concentration of the unknown sample was deter-
mined from the standard curve.
5.2.3. Experimental design for the toxicity study of the compound
The organoselenium compound was used as a suspension in
5.5% propylene glycol in water. Animals were divided into four
groups containing six animals (n ¼ 6) in each group. Vehicle treated
group (Gr. I): Each animal received 5.5% propylene glycol in water
orally for 30 days.
Drug (2.5 mg) treated group (Gr. II): organoselenium compound
was given as suspension in aqueous solution of 5.5% propylene
glycol at the dose of 2.5 mg/kg body weight for 30 days.
Drug (5 mg) treated group (Gr. III): organoselenium compound
was given as suspension in aqueous solution of 5.5% propylene
glycol at the dose of 5 mg/kg body weight for 30 days.
Drug (10 mg) treated group (Gr. IV): organoselenium compound
was given as suspension in aqueous solution of 5.5% propylene
glycol at the dose of 10 mg/kg body weight for 30 days.
5.2.7. Quantitative estimation of the level of lipid peroxidation [36]
Spectrophotometric method was applied to estimate the level of
lipid peroxidation in liver microsomes by measuring the formation
of lipid peroxides using thiobarbituric acid (TBA). Microsomal
fraction was isolated from tissue homogenate (in KCl) by centrifu-
gation. To this sample 200 ml SDS, 1.5 ml acetic acid and 1.5 ml
TBA were added. The reaction mixture was then heated at 95 ꢁC in
water bath for 1 h. The reaction results in pink colored complex
(TBAeMDA complex) which was extracted by butanol and pyridine
(15:1; v/v). Absorbance was measured at 532 nm against the
colorless blank sample. The level of lipid peroxidation was
expressed as Thiobarbituric Acid Reactive Substances (TBARS)
formed per milligram protein using extinction co-efficient of
5.2.4. Tumor cells
EAC cells was maintained in Swiss mice by weekly intraperito-
neal (i.p.) transplantation of 1 ꢃ106 viable tumor cells suspended in
phosphate buffer saline (PBS).
1.56 ꢃ 10ꢀ5
M
ꢀ1 cm.
5.2.8. Estimation of reduced glutathione (GSH) level [37,38]
GSH level was estimated in liver cytosol spectrophotometrically
by determination of dithiobis(2-nitro)-benzoic acid (DTNB)
reduced by eSH groups by measuring the absorbance. Cytosolic
fraction was isolated from tissue homogenate. To 0.1 ml of super-
natant, 2.4 ml of 0.02 M EDTA solution was added and kept on ice
bath for 10 min. Then 2 ml of distilled water and 0.5 ml of 50% TCA
were added. This mixture was kept on ice for 10e15 min and then
centrifuged at 3000 g for 15 min. 1 ml of supernatant was taken and
2.0 ml of Tris buffer was added. Then 0.05 ml of DTNB solution
(Ellman’s reagent) was added and vortexed thoroughly. Optical
density (OD) was read (within 2e3 min after the addition of DTNB)
at 412 against a reagent blank. Standards were run simultaneously.
The level of GSH was expressed as nmol/mg of protein.
5.2.5. Experimental groups for the study of chemoprotection and
chemoenhancement
Animals were distributed into seven groups. Gr. VC contained
six (6) animals and other groups contained twelve animals each. Six
animals from these groups were taken for the study of biochemical,
hematological parameters and antitumor activity; whereas the rest
of the animals were kept to check the survivability of each group.
Animals of each group (except VC) were injected with EAC cells
(1 ꢃ 106 cells/mouse) intraperitoneally. The day of EAC cell inocu-
lation was count as day ‘zero’. No treatment was given on the day of
EAC cell inoculation. The groups were: Vehicle control (VC): animals
were given 5.5% propylene glycol in water by oral gavages from day
1to day 10. EAC control (E): animals were given 5.5% propylene
glycol in water by oral gavages from day 1to day 10. Only organo-
selenium concomitant treated group (ED): animals were treated only
with organoselenium compound (4 mg/kg b.w.) 24 h after tumor
5.2.9. Estimation of glutathione-S-transferase (GST) activity [37,39]
Spectrophotometric method was adopted to evaluate the ac-
tivity of GST in liver tissue. GST activity was measured in tissue

S.S. Roy et al. / European Journal of Medicinal Chemistry 73 (2014) 195e209
207
cytosolic fractions by determining the increase in absorbance at
340 nm with 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate
and the specific activity of the enzyme was expressed as formation
of CDNBeGSH conjugate per minute per milligram of protein.
dichlorodihydrofluorescein diacetate (DCFH-DA) to make a final
volume of 3 ml and incubated for 45 min. ROS levels were
measured using spectrofluorimeter (Varian Cary Eclipse, with an
excitation set at 485 nm and emission at 530 nm) as a change in
fluorescence intensity because of the conversion of nonfluorescent
DCFH-DA to the highly fluorescent compound 20,70-dichloro
fluorescein.
5.2.10. Estimation of GPx activity [40]
GPx activity was measured by NADPH oxidation using a coupled
reaction system consisting of reduced glutathione, glutathione
reductase and hydrogen peroxide. The tissue homogenate was
centrifuged at 13,000 r.p.m. for 10 min. The supernatant was
5.2.15. Detection of DNA damage by alkaline single cell gel
electrophoresis (comet assay) [20]
considered as the sample for experiment. In a tube 200
sium phosphate buffer, 100 l glutathione reductase, 100
and 100 l NADPH was added. 100 l supernatant was added to the
tube. 300 l distilled water H2O was added to make the volume
900
l. Incubation was done at 37 ꢁC for 10 min. The reaction was
started by adding 100 l 12 mM H2O2. O.D. was measured at 340 nm
m
l potas-
DNA damage was measured by single cell gel electrophoresis or
comet assay. Briefly, lymphocytes were separated from heparinized
peripheral blood by Histopaque. The isolated lymphocytes were
suspended in 0.5% (w/v) low melting agarose. Subsequently, cells
were layered over a frosted microscopic slide previously coated
with a layer of 1.0% normal melting agarose. The slides were then
immersed in a lysis buffer of pH 10 and left overnight. Slides were
transferred into a horizontal electrophoresis chamber containing
alkaline solution (300 mM NaOH, 1 mM Na2EDTA; pH 13.0). A
presoaking of 20 min was done in order to unwind DNA. Electro-
phoresis was then carried out for 20 min (300 mA, 20 V). Slides
were then washed thrice with neutralizing buffer (Tris buffer 0.4 M,
pH 7.5) followed by staining with ethidium bromide (final con-
m
ml GSH
m
m
m
m
m
at 30 s interval for 3 min. Total protein concentration of the un-
known sample was determined from the standard curve prepared
by measuring the absorbance of different known concentrations of
BSA. The enzyme activity was expressed as micromol NADPH uti-
lised/min/mg of protein using molar extinction coefficient of
NADPH at 340 nm as 6200 Mꢀ1 cmꢀ1
.
5.2.11. Catalase activity (CAT) [41]
centration 20 mg/ml). Finally, slides were examined under a Leica
100 mg liver tissue was homogenized in 500 ml of ice-cold M/
fluorescence microscope and subjected to image analysis using
comet assay software programme Komet 5.5. To avoid any selection
bias, at least 100 cells from each sample were counted and the
results were expressed as:
150 phosphate buffer, using a Teflon Homogenizer. The homoge-
nate was centrifuged at 2000 g for 10 min at 4 ꢁC. The supernatant
was taken for the assay of catalase activity. 10 ml sample was taken
in tube containing 3.0 ml of H2O2 phosphate buffer. Time required
for 0.05 OD change was measured at 240 nm against a blank con-
taining the enzyme source in H2O2 free phosphate buffer.
ꢄ Percentage of head DNA and tail DNA.
ꢄ Average tail length due to DNA migration in each group.
ꢄ Olive tail moment.
5.2.12. Superoxide dismutase activity (SOD) [42,43]
5.2.16. Histopathological studies
Liver tissues were fixed overnight in 10% buffered neutral
formalin, processed to paraffin wax, sectioned at 5 mm, and stained
SOD was determined by means of inhibition of pyrogallol
autooxidation by the enzyme. Tissue homogenate was centrifuged
at 10,000 g for 30 min at 4 ꢁC. The supernatant was taken and
with hematoxylineeosin for examination by light microscopy.
125 ml of ethanol and 75 ml of chloroform was added to the su-
pernatant. Then it was vortexed for 5e7 min and centrifuged at
13,000 g for 15 min and the supernatant was used as the enzyme
source. The reaction mixture for autooxidation consisted of 2 ml of
the buffer, 0.4 ml of 2 mM pyrogallol solution and 1.6 ml distilled
water. Auto-oxidation of pyrogallol was measured by the increase
in absorbance at 420 nm (0.02 minꢀ1 and reading taken at 30 s
interval for 3 min). A blank sample was prepared as above using
distilled water instead of tissue sample. The reaction mixture for
5.2.17. Chromosomal aberration form bone marrow cells
Cells arrested in metaphase were examined microscopically for
structural chromosome aberrations. Mice were injected (ip) with
0.03% colchicine @1 ml/100 g body weight and were kept for 1 h
15 min prior to sacrifice. Bone marrow cells were be collected
from the femur by flushing in warm (37 ꢁC), sodium citrate (1%)
solution with a hypodermic syringe aspirate and incubate at 37 ꢁC
for 10 min. The material was then centrifuged and fixed in acetic
acid/methanol. Finally, before preparation of slides, fix material
was again centrifuged and resuspended in a small volume of
fixative by gentle flushing until a cloudy suspension result. Two to
three drops of this suspension was dropped on a clean slide which
was chilled previously in 50% ethanol then the slides were burnt
on a flame, air dry and stain with Giemsa and mount in DPX. Total
of 300 bone marrow cells were be observed (50 from each of 6
mice/set).
SOD estimation containing 10 ml cytosolic extraction and decrease
in auto-oxidation at 420 nm was followed for 3 min at 30 s in-
terval. SOD activity was determined by means of inhibition of
pyrogallol auto-oxidation by the enzyme present in the cytosolic
fraction at 420 nm.
5.2.13. Hepatic marker enzymes alanine and aspartate
transaminase (ALT & AST) assay [44]
ALT and AST activity in serum sample was estimated following
2,4-dinitrophenyl hydrazine (2,4-DNPH) color method principles as
supplied with the kit (Span Diagnostics, India) which follow the
method of Reitman and Frankel.
5.2.18. Hematopathological studies
Hemoglobin (Hb) content of blood sample was measured
following Sahli’s method [46]. Red blood cell (RBC) [47] and white
blood cell (WBC) counts were made following a literature proce-
dure [48]. Differential WBC count [49] was carried out from
Leishman stained blood smears.
5.2.14. Determination of hepatic ROS production [45]
Measurement of hepatic ROS generation was done by employing
the method of Driver et al. with slight modifications. Liver tissues
were homogenized in Locke’s buffer (pH 7.4 containing 140 mM
NaCl, 5 mM KCl, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, and
5.2.19. Tumor growth response [50,51]
10 mM glucose) to yield a 10% w/v homogenate. A total of 250
m
l of
The antitumor effect of the organoselenium compound on
therapeutic efficacy of CP was assessed by measuring the changes
tissue homogenate was taken and loaded with 10
m
M 20,70-

208
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quently, the cells were collected, washed one time with PBS. ROS
levels were measured using spectrofluorimeter (Varian Cary
Eclipse, with an excitation set at 485 nm and emission at 530 nm)
as a change in fluorescence intensity because of the conversion of
nonfluorescent DCFH-DA to the highly fluorescent compound 20,70-
dichlorofluorescein.
5.3. Statistical analysis
Results were shown as mean ꢂ SD for each group. Statistical
analysis was performed using SPSS (Version 11.0) statistical soft-
ware. For multiple comparisons, one-way analysis of variance
(ANOVA) was used. In cases where ANOVA showed significant
differences, post-hoc analysis was performed with Tukey’s t-test.
p < 0.05 was considered as statistically significant.
Acknowledgment
Author Somnath Singha Roy gratefully acknowledges CSIR for
the senior research fellowship (09/30 (0060)/2011. EMR-I). One of
the authors, P. Chakraborty acknowledges ICMR for providing RA.
The authors wish to thank the Director, CNCI for the support in this
study.
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