Synthesis and antiproliferative activity of benzophenone tagged pyridine analogues towards activation of caspase activated DNase mediated nuclear fragmentation in Dalton's lymphoma

Article abstract of DOI:10.1016/j.bioorg.2016.02.001

A series of benzophenones possessing pyridine nucleus 8a-l were synthesized by multistep reaction sequence and evaluated for antiproliferative activity against DLA cells by in vitro and in vivo studies. The results suggested that, compounds 8b with fluoro group and 8e with chloro substituent at the benzoyl ring of benzophenone scaffold as well as pyridine ring with hydroxy group exhibited significant activity. Further investigation in mouse model suggests that compounds 8b and 8e have the potency to activate caspase activated DNase (endonuclease) which is responsible for DNA fragmentation, a primary hallmark of apoptosis and thereby inhibits the Dalton's lymphoma ascites tumour growth.

Full text of DOI:10.1016/j.bioorg.2016.02.001

Bioorganic Chemistry 65 (2016) 73–81
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and antiproliferative activity of benzophenone tagged pyridine
analogues towards activation of caspase activated DNase mediated
nuclear fragmentation in Dalton’s lymphoma
Mohammed Al-Ghorbani a, Prabhu Thirusangu b, H.D. Gurupadaswamy a, V. Girish a,
H.G. Shamanth Neralagundi b, B.T. Prabhakar b, Shaukath Ara Khanum a,
⇑
a Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore 570005, Karnataka, India
b Molecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College (A), Kuvempu University, Shimoga 577203,
Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzophenones possessing pyridine nucleus 8a–l were synthesized by multistep reaction
sequence and evaluated for antiproliferative activity against DLA cells by in vitro and in vivo studies.
The results suggested that, compounds 8b with fluoro group and 8e with chloro substituent at the ben-
zoyl ring of benzophenone scaffold as well as pyridine ring with hydroxy group exhibited significant
activity. Further investigation in mouse model suggests that compounds 8b and 8e have the potency
to activate caspase activated DNase (endonuclease) which is responsible for DNA fragmentation, a pri-
mary hallmark of apoptosis and thereby inhibits the Dalton’s lymphoma ascites tumour growth.
Ó 2016 Elsevier Inc. All rights reserved.
Received 1 January 2016
Revised 1 February 2016
Accepted 2 February 2016
Available online 2 February 2016
Keywords:
Benzophenone-pyridine
DLA
CAD
Endonuclease
1. Introduction
less toxic candidates, which targets the activation of CAD has
become a key strategy for cancer treatment.
Programmed cell death (PCD) or apoptosis, plays a very crucial
role in the maintenance of tissue homeostasis by regulating cell
death [1]. When there is a failure in the cell death, it leads to the
malignant tumour [2,3]. During apoptosis, cells exhibit specific
morphological changes. These include membrane blebbing, cyto-
plasmic condensation, chromatin condensation, nucleosomal frag-
mentation and apoptotic body formation [4]. The fragmentation of
chromosomal DNA into nucleosomal units, producing small DNA
fragments or DNA ladder, is considered as a prominent biochemical
hallmark of apoptotic cell death [4,5]. The molecular characteriza-
tion of this process identifies a specific DNase called Caspase acti-
vated DNase (CAD) or DNA fragmentation factor (DFF-40) that
cleaves cellular DNA in a caspase-dependent manner that eradi-
cates the cells in an appropriate way [6,7]. Apoptosis can be acti-
vated through awakening CAD by dissociating the inhibitor of
CAD (ICAD) from CAD which normally heterodimerised with ICAD
[8–10]. In this manner, the development of the active, selective and
Pyridine is one of the most prevalent heterocyclic compounds
in nature. For example, it is present in the coenzyme vitamin B6
family and in numerous alkaloids, further it plays a central role
as versatile building block in the synthesis of natural products
as well as biologically active compounds. Further, pyridine bases
are widely used in pharmaceutics as nicotinamides and nicotinic
acid derivatives. The various therapeutic potential of pyridine
derivatives have been reported in the treatment of cancers of
diverse cells, by targeting angiogenesis [11,12], apoptosis
[13,14] and by inhibiting wide range of tumour promoting factors
like, FAK [14], CDK [13,16] and topoisomerase II [17]. Neverthe-
less, benzophenone derivatives are extensively used in medicine
research for their recognized potencies against various
pathological conditions including cancer [18–20]. In recent years,
our group has reported
a number of novel benzophenone
conjugated analogues as potent inhibitors targeting angiogenesis
[21–23] and apoptosis [24,25]. In continuation, this current study
is based on the synthesis of benzophenone bearing pyridine
nucleus and their evaluation for antiproliferative and apoptogenic
properties against Dalton’s lymphoma by both in vitro and in vivo
analysis.
⇑
Corresponding author.
E-mail address: shaukathara@yahoo.co.in (S.A. Khanum).
http://dx.doi.org/10.1016/j.bioorg.2016.02.001
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

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M. Al-Ghorbani et al. / Bioorganic Chemistry 65 (2016) 73–81
[25,26] has a promising pharmacological activity against various
2. Results and discussion
pathological conditions including cancer. While, pyridine or nico-
tinic acid is well known for its biological activity against carcino-
genesis [14–19]. In the present investigation, compounds 8a–l
analogues were synthesized, by tagging the benzophenone with
pyridine ring and evaluated for their antiproliferative and cytotoxic
properties against Dalton’s Lymphoma Ascites (DLA) cells by per-
forming MTT, trypan blue and lactate dehydrogenase (LDH) release
assays [Fig. 1]. In the series of compounds 8a–l, the compounds 8b
with fluoro and 8e with chloro group at the para position in the
benzoyl ring of the benzophenone and also with hydroxy pyridine
moiety exhibited a noticeable cytotoxic effect against DLA cells.
The compounds 8b and 8e were found to be exhibiting a promising
antiproliferative effect with IC50 of 8.5 lM and 9.3 lM respectively
in MTT assay [Fig. 1A]. The synchronized results were obtained for
both the compounds 8b and 8e in trypan blue dye exclusion assay
with the cytotoxic effects at IC50 of 8.7 lM and 9.5 lM respectively
[Fig. 1B]. The cellular integrity of the title compounds was evalu-
ated using the LDH release assay [Fig. 1C], which is one of the gold
standard methods to assess the cytotoxic effect of the compounds.
The results obtained from LDH release assay showed that there
was an increase in the release of LDH after the treatment with
2.1. Chemistry
The reaction sequence for various title compounds 8a–l was
outlined in Scheme 1. Substituted phenyl benzoates 3a–d were
synthesized by stirring 2-chloro-6-fluoro phenol 1 with substi-
tuted acid chlorides 2a–d in alkaline medium using triethylamine.
The phenyl benzoates 3a–d were subjected to Fries rearrangement
to afford hydroxy benzophenones 4a–d. Condensation of 4a–d
with ethyl chloroacetate in the presence of anhydrous potassium
carbonate in dry acetone gave ethyl (2-aroyl-4-methylphenoxy)
acetates 5a–d, which on treatment with 99% hydrazine hydrate
in ethanol gave 4-aryloylaryloxyacethydrazides 6a–d. Finally, the
title compounds 8a–l were achieved in excellence yield by cou-
pling 6a–d with substituted nicotinic acids 7a–c in the presence
of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetraflu-
oroborate (TBTU) as coupling agent and 2,6-dimethyl pyridine
(lutidine). The structures of the compounds were elucidated by
IR, 1H NMR and mass spectral studies and also by microanalyses.
2.2. Biology
compounds 8b and 8e, which exhibited IC50 of 8.5
lM and
9.4 M respectively. In the direction of structure and structure
l
2.2.1. In vitro selection of the lead compound and their structure
activity relationships
During the last decade, the researchers focused on benzophe-
none derivative and suggest that the benzophenone conjugated
with various specific bioactive molecules such as benzimidazole
[21], coumarin [22], thiazole [23], oxadiazole [24] and acetamide
activity relationship (SAR), the compounds 8h and 8k with
hydroxy pyridine moiety, but different substituents at the para
position in the benzoyl ring of benzophenone exhibited moderate
antiproliferative activity whereas, compounds 8b and 8e exhibited
noticeable antiproliferative activity. Surprisingly, the compounds
Scheme 1. Synthesis of nicotinic acid N0-[2-(4-benzoyl-phenoxy)-acetyl]-hydrazides 8a–l.

M. Al-Ghorbani et al. / Bioorganic Chemistry 65 (2016) 73–81
75
Results indicate that compounds 8b and 8e have increased the
cell population undergoing apoptosis about ꢀ21% and ꢀ19%
(including early apoptosis) respectively, compared to untreated
[Supplementary 1]. Hence the compounds 8b and 8e were chosen
as lead compounds and investigated further for their in vivo activity.
2.2.2. Compounds 8b and 8e exhibit antioncogenic activity in ascites
lymphoma
The DLA tumour model is frequently adapted to secret ascites
and is a widely used cell line to study the antiproliferative activity
[27–29]. The ascitic tumour implantation induces a local inflam-
matory reaction, with increasing vascular permeability, which
results in cell proliferation, cellular migration and a progressive
ascitic fluid formation. Ascites fluid is the direct nutritional source
for tumour cell growth and a rapid increase in the secretion of this
fluid is an extremely important feature for tumour survival [28–
32]. Therefore repressing the ascites secretion is one of the central
parameters in reticence of the tumour growth. To study the in vivo
antiproliferative effect of compounds 8b and 8e, DLA ascites
tumour models were administered with the concentration of
75 mg/kg body weight intraperitoneally as determined from the
LD50 studies. The results inferred that compounds 8b and 8e signif-
icantly regressed the tumour progression in mice, which is appar-
ent from tumour volume [Fig. 2A]. Administration of compounds
8b and 8e significantly reduced the ascitic fluid secretion about
76.5% and 73.2%, respectively, compared to the control [Fig. 2B]
and diminished the tumour growth by increasing the tumour inhi-
bition at 91.24% and 86.10% respectively, which was clear from
tumour inhibition [Fig. 2C]. An anticancer drug is considered reli-
able if it can prolong the life span of the animal. Interestingly,
treatment of the compounds 8b and 8e have prolonged the life
span of animals from the 10th day to 40th and >40th day, with
three fold increase in Survivalism [Fig. 2D].
Further, the drug induced side effects were evaluated by inject-
ing compounds 8b and 8e (75 mg/kg body weight per mice) con-
secutively for 10 days in normal swiss albino mouse model.
Compounds 8b and 8e treated mice serum containing alkaline
phosphatase, creatinine, urea, RBC and WBC levels reveal that the
compounds 8b and 8e have no or very less adverse effects [Table 1].
These results clearly ascertain that the compounds 8b and 8e
specifically act upon the progressive tumour cells and responsible
for the tumour inhibition without much toxicological effect.
2.2.3. Compounds 8b and 8e induce the CAD mediated DNA
fragmentation in DLA cells
The intracellular chemical mediators of programmed cell death
have been relatively well characterized [33]. Activation of effective
members of the caspase family of cysteine proteases is the key pro-
cess in apoptotic death signalling [29]. ICAD is the major apoptotic
endonuclease responsible for internucleosomal DNA cleavage and
chromatin condensation, and their activation requires cleavage of
ICAD primarily by caspase-3 through the dissociation of the
CAD–ICAD complex [6,7,30]. This process allows the CAD to enter
the nucleus and degrade chromosomal DNA into small DNA frag-
ments which causes the apoptotic cell death and apoptotic body
formation [5]. Besides, the tumour inhibitory mechanism of com-
pounds 8b and 8e was verified by various assays such as immuno-
blot, endonuclease assay, DNA fragmentation assay and giemsa
stain. Mechanism underlying the compound 8b and 8e exhibiting
apoptosis studied by immunoblots and the results inferred that
compounds 8b and 8e induces the activation of CAD by cleaving
the ICAD/CAD complex through the expression of caspase-3
[Fig. 3A]. Cell fractionation (cytosolic and nuclear proteins) studies
were performed for evaluating the nuclear translocation of active
CAD and is suggested that compounds 8b and 8e promotes the
nuclear localization of CAD where it can degrade the cellular DNAs
Fig. 1. In vitro selection of the active anti-mitogenic molecule. IC50 Value of
cytotoxic compounds 8a–l were determined against DLA cells by performing MTT,
Trypan blue dye exclusion and LDH release assay (A) IC50 values of 8a–l determined
through (A) MTT assay (B) Trypan blue (C) LDH release assay, in DLA cells. In these
series 8a–l, compounds 8b and 8e which showed a minimal inhibitory concentra-
tion (IC50) were chosen as lead compounds.
8a, 8c, 8d, 8f, 8g, 8i and 8j lacking a hydroxy pyridine moiety
showed a very negligible cytotoxicity against DLA cells, even
though fluoro and chloro groups are present in benzophenone.
These results highlight that the presence of hydroxy pyridine moi-
ety is fundamental for the antiproliferative effect of the drugs and
also fluoro and chloro groups in the benzophenone is responsible
for the enhancement of the activity of the compounds which is evi-
dent from cytotoxic studies. Based on the cytotoxic assay results
and SAR, it is clear that compounds 8b and 8e have the potency
to exhibit the antioncogenic effect against DLA cells. For further
validation of cytotoxic effect, DLA cells were treated with and
without compounds 8b and 8e at 10
lM for 48 h and FACS were
carried out for detecting whether it could be due to apoptosis.

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M. Al-Ghorbani et al. / Bioorganic Chemistry 65 (2016) 73–81
Fig. 2. Compound 8b and 8e has potency to show the inhibition in Dalton’s lymphoma ascites (DLA) proliferation in vivo. Ascites tumour was induced in mice by culturing
DLA cells in peritoneum and administered with 75 mg/kg (i.p) of compound 8b and 8e for three doses. Compound 8b and 8e has ability to exhibit (A) the reduction in tumour
volume, (B) the decrease in ascites secretion, (C) the inhibition of tumour growth and (D) the extended survivalism compared to control.
Table 1
degrades the DNA as observed by DNA fragmentation assay, which
Hematological and serum profile of non tumour bearing mice following treatment
is concurrent to immunoblot and endonuclease assay results
with compounds 8b and 8e at day 10. Values are indicated in mean SEM.
[Fig. 3E]. The cell morphology analysed by Giemsa staining further
Hematological and serum
profile parameters
Control mice
8b treated
mice
8e treated
mice
inferred that compounds 8b and 8e treated cells posed cell shrink-
age, membrane blebbing and irregular shape with apoptotic bodies
which are major features of apoptotic hallmarks [Fig. 3F]. These
results confirm that compounds 8b and 8e specifically induce the
CAD mediated apoptotic DNA fragmentation in DLA tumour.
Alkaline phosphatase (IU/L)
Creatinine (mg/dl)
Urea (mg/dl)
129.55 2.95 135.65 5.6 132.05 4.7
0.59 1.05 0.63 0.335
45 2.78
5.28 1.45
3.32 1.6
0.55 0.25
39 2.8
5.67 1.85
3.10 0.56
46 1.9
5.08 1.05
3.21 0.40
RBC (106/
WBC (106/
l
l
l)
l)
3. Conclusion
In summary, a series of benzophenone tagged pyridine analo-
gous 8a–l were synthesized and evaluated for antiproliferative
activity against DLA cells by in vitro and in vivo studies. From the
present investigation, structural activity relationship of these com-
pounds suggests that hydroxy pyridine moiety and fluoro and
chloro group at the benzoyl ring of the benzophenone scaffold as
in compounds 8b and 8e are significant for activity. Further
investigation in mouse model suggests that compounds 8b and
8e have the potency to activate CAD which is responsible for
[Fig. 3B&C]. Endonuclease assay was performed for studying the
activation of CAD, and the results showed that compounds 8b
and 8e have promoted the endonuclease (CAD) activation as visu-
alized by DNA lysis zone. Increased concentration of 8b and 8e
treated cytosolic fractions (400 and 600 lg) exhibit enlarged DNA
lysis zone (8b with 0.4–0.6 cm DNA lysis and 8e with 0.3–0.5 cm
of DNA lysis) compared to control, which is evident for endonucle-
ase (CAD) activation [Fig. 3D]. Eventually the activated CAD

M. Al-Ghorbani et al. / Bioorganic Chemistry 65 (2016) 73–81
77
Fig. 3. Compound 8b and 8e exhibits CAD mediated DNA fragmentation in DLA cells in vivo. (A) Immunoblot photograph shows that compound 8b and 8e have induced the
activation of CAD through cleaved casspase-3 and inhibits the ICAD in vivo. (B) Cytosolic expression of active CAD (C) Compound 8b and 8e promotes the nuclear translocation
of active CAD. (D) The endonuclease assay images illustrates that the DNA degrading potentiality (DNA lysis zone) of compound 8b and 8e through activated endonuclease
(DNase) in concentration dependant manner (400 and 600 lg/well) and respective graphs shows the increased DNase activity in DNA lysis zone. (E) DNA fragmentation
profile from control and compound 8b & 8e treated samples. (F) Giemsa stain photograph depicting that regular shape of the cells, none of apoptotic bodies in control and
irregular shape and membrane blebbing, apoptotic bodies in Compound 8b and 8e treated cells.
DNA fragmentation, a primary hallmark of apoptosis. Elucidation
of compounds 8b and 8e induced upstream signalling is under
investigation for detailed studies.
by UV-light. Melting points were measured on a Thomas Hoover
capillary melting point apparatus with a digital thermometer. IR
spectra were recorded on FT-IR Shimadzu 8300 spectrophotome-
ter, 1H and 13C NMR spectra were recorded on a Bruker 400 MHz
NMR spectrophotometer using TMS as an internal standard and
DMSO-d6 as solvent. Chemical shifts are given in parts per million
downfield from tetramethylsilane. The mass spectra were obtained
with a VG70-70H spectrophotometer and the elemental analysis
(C, H, and N) was performed on Elementar Vario EL III elemental
analyzer. The results of elemental analyses were within 0.4% of
the theoretical values.
4. Material and methods
4.1. Chemistry
Chemicals were procured from Sigma Aldrich Chemical Co. TLC
was performed on aluminium-backed silica plates and visualized

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M. Al-Ghorbani et al. / Bioorganic Chemistry 65 (2016) 73–81
4.1.1. General procedure for the synthesis of aryl benzoates (3a–d)
2-Chloro-6-fluoro phenol (1, 0.20 mol) was dissolved in
dichloro methane (DCM) and triethylamine (TEA, 0.45 mol) was
added to it. Then the reaction mixture was cooled to 0 °C. Further,
a solution of substituted benzoyl chloride 2a–d (0.21 mol) in DCM
was slowly added to the reaction mixture and stirred for 3 h and
the completion of the reaction was monitored by TLC using 4:1
n-hexane: ethyl acetate solvent mixture. Then the reaction mass
was diluted with DCM (100 ml), washed with 10% sodium hydrox-
ide solution (3 ꢁ 40 ml), followed by water (3 ꢁ 30 ml). The
organic layer was dried over sodium sulphate and the solid
obtained after evaporation of the solvent was recrystallized from
ethanol to give compounds 3a–d. [21] Compound (3a) is taken as
a representative example to explain physical and characterization
data.
C17H13ClF2O4: C, 57.56; H, 3.69; Cl, 9.99; F, 10.71. Found: C,
57.41; H, 3.52; Cl, 9.79; F, 10.88%.
4.1.4. General procedure for the synthesis of 4-aryloyl aryloxy
acethydrazides (6a–d)
Hydrazine hydrate (0.018 mol) was added to a solution of com-
pounds 5a–d (0.018 mol) in ethanol (30 ml) and continuously stir-
red for 2 h at room temperature. After the completion of the
reaction, mointered by TLC using 2:1 chloroform: ethyl acetate as
a mobile phase. A white solid was separated out, which was
quenched with water (50 ml), filtered and washed with water
(50 ml). The solid was dried under vacuum and the product was
recrystallized from ethanol to obtain compounds 6a–d with good
yield [35]. Compound (6a) is taken as a representative example
to explain physical and characterization data.
4.1.4.1. [2-Chloro-6-fluoro-4-(4-fluoro-benzoyl)-phenoxy]acetic acid
hydrazide (6a). Yield: 76%; m.p. 107–109 °C; FT-IR (KBr, cmꢂ1):
1610 (C@O), 1645 (amide, C@O), 3100–3205 (NHANH2); 1H NMR
(DMSO-d6): d 4.35 (bs, 2H, NH2), 4.69 (s, 2H, OCH2), 7.20–7.86
(m, 6H, ArAH), 9.32 (bs, 1H, NH); LC–MS m/z 340 (M+) and 342
(M+2). Anal. Calcd. for C15H11ClF2N2O3: C, 52.88; H, 3.25; Cl,
10.41; F, 11.15; N, 8.22. Found: C, 52.75; H, 3.38; Cl, 10.29; F,
11.24; N, 8.11%.
4.1.1.1. 2-Chloro-6-fluorophenyl-4-fluorobenzoate (3a). Yield: 92%;
m.p. 52–54 °C; FT-IR (KBr, cmꢂ1) 1738 (C@O); 1H NMR (DMSO-
d6): d 7.42–8.28 (m, 7H, ArAH); LC–MS m/z 268 (M+) and 270 (M
+2). Anal. Calcd. for C13H7ClF2O2: C, 58.12; H, 2.63; Cl, 13.20; F,
14.14. Found: C, 58.22; H, 2.43; Cl, 13.30; F, 14.29%.
4.1.2. General procedure for the synthesis of (4-hydroxy aryl) aryl
methanones (4a–d)
Substituted 4-hydroxy benzophenones 4a–d were synthesized
by Fries rearrangement. Compounds 3a–d (0.063 mol) was
blended with aluminium chloride (0.126 mol) and the mixture
was heated to 150 °C and this temperature was maintained for
2 h and the completion of the reaction was monitored by TLC using
4:1 n-hexane: ethyl acetate solvent mixture. Then the reaction
mixture was cooled to room temperature and quenched with 6 N
hydrochloric acid and ice water. The reaction mixture was stirred
for about 1 h, the obtained solid was filtered and recrystallized
with methanol to obtain desired compounds 4a–d. [23] Compound
(4a) is taken as a representative example to explain physical and
characterization data.
4.1.5. General procedure for the synthesis of nicotinic acid N0-[2-(4-
benzoyl-phenoxy)-acetyl]-hydrazides (8a–l)
4-Aryloyl aryloxy acethydrazides 6a–d (0.0010 mol) in dry DCM
(20 ml) was stirred at 25–30 °C, and then lutidine (0.0015 mol) was
added, followed by the addition of nicotinic acid derivatives 7a–c
(0.0010 mol). The reaction mixture was stirred at the same tem-
perature for 30 min, then cooled to 0–5 °C and TBTU
(0.0015 mol) was added over a period of 30 min maintaining the
temperature below 5 °C. The reaction mass was stirred overnight
and monitored by TLC using chloroform: methanol (9:1) as the
mobile phase. The solvent was evaporated at reduced pressure,
quenched by the addition of crushed ice and the obtained solid
was filtered, dried and recrystallized from ethanol to afford com-
pounds 8a–l in good yield.
4.1.2.1. (3-Chloro-5-fluoro-4-hydroxyphenyl)-4-fluorophenyl metha-
none (4a). Yield: 70%; m.p. 146–147 °C; FT-IR (KBr, cmꢂ1): 1671
(C@O), 3545–3635 (OH); 1H NMR (DMSO-d6): d 7.36–7.82 (m,
6H, ArAH), 11.64 (bs, 1H, OH); LC–MS m/z 268 (M+) and 270 (M
+2). Anal. Calcd. for C13H7ClF2O2: C, 58.12; H, 2.63; Cl, 13.20; F,
14.14. Found: C, 58.21; H, 2.52; Cl, 13.20; F, 14.25%.
4.1.5.1. 2,6-Dichloro-nicotinic acid N0-{2-[2-chloro-6-fluoro-4-(4-flu-
oro-benzoyl)-phenoxy]-acetyl}-hydrazide (8a). Yield: 85%; m.p.
140–142 °C; FT-IR (KBr, cmꢂ1): 1640 (C@O), 1665 (amide, C@O),
3240–3350 (NHANH); 1H NMR (DMSO-d6): d 4.91 (s, 2H, CH2),
7.35–8.06 (m, 8H, ArAH), 10.79 (bs, 2H, 2NH); 13C NMR (DMSO-
d6): d 175.6, 169.3, 164.2, 162.3, 158.8, 153.4, 147.3, 138.4, 135.1,
132.4, 131.5, 130.3, 129.2, 128.4, 127.5, 115.6, 113.9, 108.5, 68.3;
LC–MS m/z 514 (M+), 516 (M+2) 518 (M+4) and 520 (M+6). Anal.
Calcd. for C21H12Cl3F2N3O4: C, 49.0; H, 2.35; Cl, 20.66; F, 7.38; N,
8.16. Found: C, 49.30; H, 2.16; Cl, 20.52; F, 7.26; N, 8.47%.
4.1.3. General procedure for the synthesis of ethyl 4-
aryloylaryloxyacetates (5a–d)
To a solution of compounds 4a–d (0.038 mol) in dry DMF
(70 ml), potassium carbonate (0.076 mol) and ethyl chloroacetate
(0.057 mol) were added and the reaction mass was heated to
60 °C for 3 h, the progress of the reactions was monitored by TLC
using 2:1 n-hexane: ethyl acetate. The reaction mass was diluted
with ethyl acetate (60 ml), potassium carbonate was filtered off
and the bed was washed with ethyl acetate (40 ml). Finally, the
organic layer was washed with water (3 ꢁ 30 ml), brine
(2 ꢁ 40 ml), dried over sodium sulphate and evaporated to dryness
to obtain crude solid, which, on recrystallization with ethanol
afforded desired compounds 5a–d. [34] Compound (5a) is taken
as a representative example to explain physical and characteriza-
tion data.
4.1.5.2. 6-Hydroxy-nicotinic acid N0-{2-[2-chloro-6-fluoro-4-(4-flu-
oro-benzoyl)-phenoxy]-acetyl}-hydrazide (8b). Yield: 75%; m.p.
132–134 °C; FT-IR (KBr, cmꢂ1): 1640 (C@O), 1670 (amide, C@O),
3270–3370 (NHANH), 3545–3635 (OH); 1H NMR (DMSO-d6): d
4.93 (s, 2H, CH2), 7.28–7.93 (m, 9H, ArAH), 12.4 (s, 1H, OH),
10.47 (bs, 2H, 2NH); 13C NMR (DMSO-d6): d 175.3, 169.3, 164.3,
163.4, 162.3, 158.8, 144.3, 136.4, 134.4, 132.4, 131.5, 130.3,
129.2, 120.4, 115.8, 114.5, 113.9, 108.5, 68.3; LC–MS m/z 462
(M+) and 464 (M+2). Anal. Calcd. for C21H14ClF2N3O5: C, 54.62; H,
3.06; N, 9.10. Found: C, 54.38; H, 3.20; N, 8.96%.
4.1.3.1. Ethyl [2-(4-fluorobenzoyl)-2-chloro-6-fluorophenoxy]acetate
(5a). Yield: 95%; FT-IR (Nujol, cmꢂ1): 1660 (C@O), 1730 (ester,
C@O); 1H NMR (DMSO-d6): d 1.20 (t, J = 7.0 Hz, 3H, CH3 of ester),
4.17 (q, J = 7.50 Hz, 2H, CH2), 5.02 (s, 2H, OCH2), 7.64–7.86 (m,
6H, ArAH); LC–MS m/z 354 (M+) and 356 (M+2). Anal. Calcd. for
4.1.5.3. 2-Amino-nicotinic acid N0-{2-[2-chloro-6-fluoro-4-(4-fluoro-
benzoyl)-phenoxy]-acetyl}-hydrazide (8c). Yield: 82%; m.p. 127–
129 °C; FT-IR (KBr, cmꢂ1): 1640 (C@O), 1655 (amide, C@O),
3220–3385 (broad NHANH and NH2); 1H NMR (DMSO-d6): d 4.86

M. Al-Ghorbani et al. / Bioorganic Chemistry 65 (2016) 73–81
79
(s, 2H, CH2), 5.73 (s, 2H, NH2), 7.31–8.02 (m, 9H, ArAH), 10.63 (bs,
2H, 2NH); 13C NMR (DMSO-d6): d 175.6, 169.3, 164.2, 162.7, 162.3,
158.8, 147.3, 137.4, 135.8, 133.0, 131.5, 130.3, 129.2, 118.4, 116.4,
114.5, 113.9, 108.5, 68.3; LC–MS m/z 461 (M+) and 463 (M+2). Anal.
Calcd. for C21H15ClF2N4O4: C, 54.73; H, 3.82; N, 12.16. Found: C,
55.08; H, 3.74; N, 12.12%.
(s, 2H, CH2), 5.76 (s, 2H, NH2), 7.21–8.07 (m, 9H, ArAH), 10.63
(bs, 2H, 2NH); 13C NMR (DMSO-d6): d 175.6, 169.3, 164.2, 162.7,
158.8, 147.3, 136.3, 133.0, 132.4, 131.5, 130.3, 129.2, 118.4,
115.9, 114.5, 113.9, 108.5, 102.3, 68.3; LC–MS m/z 569 (M+), 571
(M+2). Anal. Calcd. for C21H15ClFIN4O4: C, 44.35; H, 2.66; N, 9.85.
Found: C, 44.59; H, 2.56; N, 9.65%.
4.1.5.4. 2,6-Dichloro-nicotinic acid N0-{2-[2-chloro-4-(4-chloro-ben-
zoyl)-6-fluoro-phenoxy]-acetyl}-hydrazide (8d). Yield: 87%; m.p.
135–137 °C; FT-IR (KBr, cmꢂ1): 1635 (C@O), 1665 (amide, C@O),
3240–3350 (NHANH); 1H NMR (DMSO-d6): d 4.92 (s, 2H, CH2),
7.26–7.93 (m, 8H, ArAH), 10.75 (bs, 2H, 2NH); 13C NMR (DMSO-
d6): d 175.6, 169.3, 164.2, 158.8, 153.4, 147.3, 138.4, 136.7, 134.4,
132.4, 131.5, 130.3, 129.2, 128.4, 127.5, 114.1, 113.9, 108.5, 68.3;
LC–MS m/z 529 (M+), 531 (M+2), 533 (M+4) and 535 (M+6). Anal.
Calcd. for C21H12Cl4FN3O4: C, 47.49; H, 2.28; N, 7.91. Found: C,
47.29; H, 2.14; N, 7.76%.
4.1.5.10. 2,6-Dichloro-nicotinic acid N0-{2-[2-chloro-6-fluoro-4-(4-
methyl-benzoyl)-phenoxy]-acetyl}-hydrazide (8j). Yield: 88%; m.p.
127–129 °C; FT-IR (KBr, cmꢂ1): 1635 (C@O), 1660 (amide, C@O),
3220–3320 (NHANH); 1H NMR (DMSO-d6): d 2.50 (s, 3H, CH3),
4.91 (s, 2H, CH2), 7.25–8.12 (m, 8H, ArAH), 10.79 (bs, 2H, 2NH);
13C NMR (DMSO-d6): d 165.2, 163.3, 152.2, 151.3, 148.0, 144.1,
143.7, 142.0, 134.0, 133.8, 133.5, 130.3, 129.7, 128.2, 127.4,
124.9, 124.1, 119.5, 110.0, 21.6; LC–MS m/z 511 (M+), 513 (M+2),
515 (M+4) and 517 (M+6) Anal. Calcd. for C22H15Cl3FN3O4: C,
51.74; H, 2.96; N, 8.23. Found: C, 51.39; H, 2.92; N, 8.21%.
4.1.5.5. 6-Hydroxy-nicotinic acid N0-{2-[2-chloro-4-(4-chloro-ben-
zoyl)-6-fluoro-phenoxy]-acetyl}-hydrazide (8e). Yield: 77%; m.p.
137–139 °C; FT-IR (KBr, cmꢂ1): 1635 (C@O), 1670 (amide, C@O),
3270–3370 (NHANH), 3540–3635 (OH); 1H NMR (DMSO-d6): d
4.88 (s, 2H, CH2), 7.27–8.0 (m, 9H, ArAH), 10.67 (bs, 2H, 2NH),
12.6 (s,1H, OH); 13C NMR (DMSO-d6): d 175.3, 169.3, 164.3,
163.4, 158.8, 144.3, 136.5, 134.4, 133.0, 132.4, 131.5, 130.3,
129.2, 120.4, 115.8, 114.5, 113.9, 108.5, 68.3; LC–MS m/z 478
(M+), 480 (M+2) and 482 (M+4). Anal. Calcd. for C21H14Cl2FN3O5:
C, 52.74; H, 2.95; N, 8.79. Found: C, 52.38; H, 3.25; N, 8.76%.
4.1.5.11. 6-Hydroxy-nicotinic acid N0-{2-[2-chloro-6-fluoro-4-(4-
methyl-benzoyl)-phenoxy]-acetyl}-hydrazide (8k). Yield: 80%; m.p.
132–134 °C; FT-IR (KBr, cmꢂ1): 1635 (C@O), 1665 (amide, C@O),
3260–3360 (NHANH), 3545–3630 (OH); 1H NMR (DMSO-d6): d
2.40 (s, 3H, CH3), 4.90 (s, 2H, CH2), 7.17–8.09 (m, 9H, ArAH),
10.44 (bs, 2H, 2NH), 12.4 (s,1H, OH); 13C NMR (DMSO-d6): d
168.2, 163.4, 152.5, 152.3, 148.1, 144.5, 143.4, 142.2, 134.8,
133.6, 132.6, 130.7,130.1, 127.8, 127.2, 125.2, 124.4, 120.3, 109.7,
21.5; LC–MS m/z 458 (M+), 460 (M+2). Anal. Calcd. for C22H17ClFN3-
O5: C, 57.71; H, 3.74; N, 9.12. Found: C, 57.54; H, 3.86; N, 9.09%.
4.1.5.6. 2-Amino-nicotinic acid N0-{2-[2-chloro-4-(4-chloro-benzoyl)-
6-fluoro-phenoxy]-acetyl}-hydrazide (8f). Yield: 80%; m.p. 123–
125 °C; FT-IR (KBr, cmꢂ1): 1635 (C@O), 1660 (amide, C@O),
3235–3385 (broad NHANH and NH2); 1H NMR (DMSO-d6): d 4.86
(s, 2H, CH2), 5.83 (s, 2H, NH2), 7.14–8.08 (m, 9H, ArAH), 10.39
(bs, 2H, 2NH); 13C NMR (DMSO-d6): d 175.6, 169.3, 164.2, 162.7,
158.8, 147.3, 144.3, 137.4, 136.3, 132.4, 131.5, 130.3, 129.2,
118.4, 116.3, 114.5, 113.9, 108.5, 68.3; LC–MS m/z 477 (M+), 479
(M+2) and 481 (M+4). Anal. Calcd. for C21H15Cl2FN4O4: C, 52.85;
H, 3.17; N, 11.74. Found: C, 52.50; H, 3.07; N, 11.70%.
4.1.5.12. 2-Amino-nicotinic acid N0-{2-[2-chloro-6-fluoro-4-(4-
methyl-benzoyl)-phenoxy]-acetyl}-hydrazide (8l). Yield: 84%; m.p.
116–118 °C; FT-IR (KBr, cmꢂ1): 1630 (C@O), 1655 (amide, C@O),
3230–3380 (broad NHANH and NH2); 1H NMR (DMSO-d6): d 2.30
(s, 3H, CH3), 4.86 (s, 2H, CH2), 5.70 (s, 2H, NH2), 7.13–7.96 (m,
9H, ArAH), 10.51 (bs, 2H, 2NH); 13C NMR (DMSO-d6): d 167.6,
163.2, 152.6, 152.3, 148.2, 144.7, 143.7, 141.9, 134.6, 133.5,
133.1, 130.7, 129.5, 127.8, 127.5, 125.3, 123.6, 119.4, 109.1, 22.2;
LC–MS m/z 457 (M+), 459 (M+2). Anal. Calcd. for C22H18ClFN4O4:
C, 57.84; H, 3.97; N, 12.26. Found: C, 57.49; H, 4.11; N, 12.39%.
4.1.5.7. 2,6-Dichloro-nicotinic acid N0-{2-[2-chloro-6-fluoro-4-(4-
iodo-benzoyl)-phenoxy]-acetyl}-hydrazide (8g). Yield: 83%; m.p.
156–158 °C; FT-IR (KBr, cmꢂ1): 1630 (C@O), 1660 (amide, C@O),
3245–3345 (NHANH); 1H NMR (DMSO-d6): d 4.91 (s, 2H, CH2),
7.15–8.00 (m, 8H, ArAH), 10.37 (bs, 2H, 2NH); 13C NMR (DMSO-
d6): d 175.6, 169.3, 164.2, 162.3, 158.8, 153.4, 147.3, 137.8, 133.0,
131.5, 130.3, 129.2, 128.4, 127.5, 115.2, 113.9, 108.5, 102.3, 68.3;
LC–MS m/z 620 (M+), 622 (M+2), 624 (M+4) and 626 (M+6). Anal.
Calcd. for C21H12Cl3FIN3O4: C, 40.51; H, 1.94; N, 6.75. Found: C,
40.16; H, 1.92; N, 6.75%.
4.2. Biology
The Dalton’s lymphoma Ascites (DLA) cells were selected for
assessing the cytotoxicity of newly synthesized compounds by
MTT assay, trypan blue dye exclusion assay, and LDH release assay
and for studying the in vivo antitumour effect of the potent com-
pounds by FACS, Immunoblot, Endonuclease assay, DNA fragmen-
tation assay and Giemsa stain.
4.2.1. Cell Culture and in vitro treatment
The DLA cells were grown in DMEM medium (Gibco-Invitrogen,
USA), supplemented with 10% FBS (In vitrogen, USA), Penicillin–
Streptomycin (Sigma–aldrich, USA) and NaHCO3 (0.37%) in a
humidified CO2 incubator at 37 °C with 5% CO2. The cells were trea-
ted with six different concentrations of compounds 8a–j (0, 5, 10,
25, 50, 100 lM in DMSO) and reincubated at 37 °C for 45 h. MTT
assay, trypan blue dye exclusion assay, and LDH release assays
were performed as reported earlier for cytotoxicity analysis [21].
4.1.5.8. 6-Hydroxy-nicotinic acid N0-{2-[2-chloro-6-fluoro-4-(4-iodo-
benzoyl)-phenoxy]-acetyl}-hydrazide (8h). Yield: 81%; m.p. 106–
108 °C; FT-IR (KBr, cmꢂ1): 1630 (C@O), 1665 (amide, C@O),
3265–3375 (NHANH), 3535–3630 (OH); 1H NMR (DMSO-d6): d
4.90 (s, 2H, CH2), 7.42–7.73 (m, 9H, ArAH), 10.67 (bs, 2H, 2NH),
12.3 (s, 1H, OH); 13C NMR (DMSO-d6): d 175.6, 169.3, 164.2,
163.4, 162.3, 158.8, 144.3, 136.5, 132.4, 131.5, 130.3, 129.2,
120.4, 115.6, 114.5, 113.9, 108.5, 102.3, 68.3; LC–MS m/z 570
(M+), 572 (M+2). Anal. Calcd. for C21H14ClFIN3O5: C, 44.27; H,
2.48; N, 7.38. Found: C, 44.64; H, 2.55; N, 7.34%.
4.2.2. Animal ethics and determination of LD50 value
Swiss female albino mice weighing between 28–30 g were used
throughout the study. The mice were grouped and housed in poly-
acrylic cages with not more than ten animals per cage with ade-
quate food and water supply. All procedures described were
reviewed and approved by the National College of Pharmacy Ethi-
cal Committee, Shimoga, India, in accordance with the CPCSEA
4.1.5.9. 2-Amino-nicotinic acid N0-{2-[2-chloro-6-fluoro-4-(4-iodo-
benzoyl)-phenoxy]-acetyl}-hydrazide (8i). Yield: 78%; m.p. 108–
110 °C; FT-IR (KBr, cmꢂ1): 1630 (C@O), 1665 (amide, C@O),
3235–3395 (broad NHANH and NH2); 1H NMR (DMSO-d6): d 4.94

80
M. Al-Ghorbani et al. / Bioorganic Chemistry 65 (2016) 73–81
guidelines for laboratory animal facility (NCP/IAEC/CL/101/05/
2012-13). LD50 of the compounds 8b and 8e were evaluated as
described earlier and their adverse effects were studied by
injecting 75 mg/kg body weight, intraperitoneally (i.p) to healthy
Swiss albino mice continuously for 10 days [22]. The serum profile
such as alkaline phosphatase (ALP), creatinine, urea, number of
RBC and WBC from the blood of both treated and untreated
animals were evaluated.
4.2.8. Statistical analysis
Values were expressed as mean standard error (SEM). Statisti-
cal significance (5%) was evaluated by one-way analysis of variance
(ANOVA) followed by Student’s t-test (⁄ p < 0.05) and (⁄⁄ p < 0.01).
Acknowledgements
Shaukath Ara Khanum express their sincere gratitude to the
Government of Karnataka, Vision Group on Science and Technol-
ogy, Bangalore for the financial assistance and support [VGST/
CISEE/ 2012-13/282] dated 16th March 2013 and UGC, New Delhi
under the Major Research Project Scheme [F.39/737/2010 (SR)].
Prabhu Thirusangu and Shamanth Neralagundi HG acknowledge
the financial support provided by Lady Tata Memorial Trust, Mum-
bai. B.T. Prabhakar gratefully acknowledge their indebtedness for
the grant extended by UGC (F.No.41-507/2012(SR), SERB-DST
(SR/FT/LS-25/2011), and VGST (VGST/ CISEE/2013-14/ 231), DBT
(6242-P37/RGCB/PMD/DBT/PBKR/2015). Our sincere thanks to Dr.
I.J. Kuppast, Principal, National College of Pharmacy, Shimoga,
India for ethical clearance certificate for animal experiments.
4.2.3. Animal tumour models and treatment
DLA tumours were maintained as ascites by intraperitoneal
serial transplantation in mice. The tumour cells were aspirated
from the tumour bearing mice aseptically and washed thrice in
phosphate-buffered saline. A DLA tumour model was developed
by injecting 5 ꢁ 106 cells/ mouse intraperitoneally. After the onset
of the tumour development (4th day), the mice bearing DLA were
administered with or without compounds 8b and 8e (75 mg/kg
body weight i.p) 3 doses on every alternate day. On day 10, tumour
parameters such as tumour volume, ascites secretion, tumour inhi-
bition and survivality were evaluated.
Appendix A. Supplementary material
4.2.4. Cell Sorting (FACS)
DLA cells were cultured in vitro and after 24 h, the cells were
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.02.
001.
treated with compounds 8b and 8e at 10 lM for 48 h. The har-
vested cells were stained with propidium iodide for determining
the cell death [25]. Compounds 8b and 8e induced cell death were
analysed by WinMDI 2.9 software and documented.
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