The critical role of novel benzophenone analogs on tumor growth inhibition targeting angiogenesis and apoptosis

Article abstract of DOI:10.1039/c7md00593h

In modern biology, one of the major topics of importance is progress in anti-cancer drugs with specific targets. The angiopreventive and in vitro tumor inhibition activities of novel synthetic benzophenone analogs have been investigated intensively and explored in a very systematic way. Novel benzophenone analogs (9a-d and 10a-d) substituted with methyl, chloro and fluoro groups at different positions on an identical chemical backbone and incorporating variations in the number of substituents have been synthesized in a multistep process and characterized. In this study, we further evaluate the newly synthesized compounds for their cytotoxic and anti-proliferative effects against A549, HeLa and MCF-7 cells. The potent lead compound was further assessed for anti-angiogenic effects. Through the structure-activity relationship, we found that an increase in the number of methyl, chloro and fluoro groups in a benzophenone ring on compound 9d resulted in higher potency compared to other compounds. Tumor inhibition was notably promoted, and this was reflected in effects on neovessel formation in in vivo systems, such as the CAM. Compound 9d interacts with rVEGF through hydrogen bonds in silico, thereby down-regulating the expression of VEGF in angiogenesis. From our investigation, it is suggested on the basis of clonogenesis and cell migration assays that compound 9d has the potency to exhibit prolonged activity against cancer progression, through cell cycle arrest at the G2/M phase. In addition, compound 9d inhibits A549 cells through caspase-activated DNase-mediated apoptosis.

Full text of DOI:10.1039/c7md00593h

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The critical role of novel benzophenone analogs on tumor growth inhibition
targeting angiogenesis and apoptosis
1
,2
1*
Yasser Hussein Eissa Mohammed and Shaukath Ara Khanum .
1
Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore- 570005
Karnataka, India.
2
Department of Biochemistry, Faculty of Applied Science College, University of Hajjah, Yemen.
*
Correspondence Author:
Dr. Shaukath Ara Khanum
Associate Professor and Head
Department of Chemistry,
Yuvaraja’s College,
University of Mysore, Mysuru.
Phone: +91 ꢀ 99018 88755
Fax: +821ꢀ 2419239
Email: shaukathara@yahoo.co.in
1

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ABSTRACT
In modern biology, one of the major importance is the progress of antiꢀcancer drugs with
specific targets. The angiopreventive and in vitro tumor inhibition activities of novel synthetic
benzophenone analogs were given more attention and explored in a very professional way. The
multistep synthesis of novel benzophenone analogs (9a-d and 10a-d) allowing substitution with
methyl, choloro and flouro groups at different positions on the identical chemical backbone and
the variations in the number of substituents were synthesized and characterized. In this study, we
further evaluated the newly synthesized compounds for cytotoxic and antiꢀproliferative effects
against the A549, HeLa and MCFꢀ7 cells. For antiꢀangiogenic effects, further assessments were
made for the potent lead compound. Through structure activity relationship, we found that an
increase in the number of methyl, chloro and fluoro groups in a ring of benzophenone on
compound 9d resulted in higher potency compared to other compounds. The tumor inhibition
was importantly suppressed and reflected by the neovessel formation in in vivo systems, such as
the CAM. Compound 9d interacts with rVEGF by hydrogen bond inꢀsilico, thereby down
regulates the VEGF expression in angiogenesis. Through the investigation, it is suggested that
the compound 9d on clonogenesis and cell migration assays have the potency to exhibit the
prolonged activity with cell cycle arrest on G2/M phase against cancer progression. In addition
to that, through caspase activated DNase mediated apoptosis the compound 9d inhibit A549
cells.
Keywords: Benzophenone; MCFꢀ7; A549; VEGF; Docking.
2

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1
. Introduction
Angiogenesis, which are considered as one of the most crucial processes in tumor
progression, are known as the development of new blood vessels from the earlier vasculature.
Angiogenesis word which can set up its own particular blood supply is called tumor growth [1].
The activity of many proangiogenic factors that include Vascular endothelial growth factor
(VEGF), fibroblast growth factor 1 and 2, tumor necrosis factor alpha, angiogenin, tumor growth
factor beta, plateletꢀderived growth factor, angiopoietin and pleiotrophin of the few elements
portrayed, intercedes the enlistment of new blood vessel growth by a tumor [2,3]. The vascular
endothelial growth factor (VEGF) is one of the most important motivating factor in charge of
liquid gathering in the ovarian hyper incitement syndrome. It can be found in different tissues
including edema arrangement in the cerebrum, synovial fluid and malignant ascites [4]. In most
human diseases, the major part of the tumor cells mystery VEGF [5] and VEGF mRNA is
severely upregulated [6]. Prolonged intratumoral miniaturized scale vessel thickness in breast
cancer patients is strongly linked up with VEGF expression [7]. It has been obviously defined
that angiogenesis are vital for a tumor to end up prominently expansive and dangerous. A new
healing window to growth treatment may be given when there is an interference of the procedure
of angiogenesis either by concealment of angiogenic progress factors. The ability to increase, as
well as to down regulate cell passing and generate angiogenesis are mainly considered as major
requirements for creating tumor neovasculature. Consequently, researchers have paid more
attention and focused deeply in the study of the expansion of antiꢀangiogenesis and apoptosis as
one of the most dominant concentrations in current tumor treatment. The improvement of such
novel, powerful and less or no toxic compounds with different methods of activity for the
3

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focused growth treatment leads to the existence of a creative method and efforts which are
directed toward finding such antiꢀcancer agents [8ꢀ10].Created by both natural and synthetic
ways, benzophenones are the class of pharmacologically active molecules. Different analogues
of benzophenones which are featured by their high activity as antiꢀtumor and antiꢀangiogenic
possibilities have been found and they are under clinical trials. Benzophenone moiety alone is
not that much potentially active as antiꢀtumor in comparison to benzophenone integrated with
heterocyclic analogues. In this unique circumstance, to get a potential approach towards the
disclosure of antiꢀangiogenic drug the benzophenones are amalgamated with another
arrangement of synthetic gatherings which have a vital part in the field of medicinal chemistry.
Subsequently, it is extremely fundamental to incorporate and create novel benzophenone
analogues with different targets [11, 12]. Several assessments have been made during this study
for the purpose of studying the antiꢀangiogenic and proapoptotic and some benzophenone
analogues have also been synthesized.
2
2
. Results and discussion
.1. Chemistry
Synthesis of the target compounds 2ꢀ(4ꢀbenzoylphenoxy)acetoꢀN(Nꢀmethyl
imidazole)carbonyl hydrazides 9a-d and 2ꢀ(4ꢀbenzoylphenoxy)acetoꢀN(pyrone ꢀ2ꢀone)carbonyl
hydrazides 10a-d were performed according to the reactions illustrated in scheme 1. The key
starting compounds, phenyl benzoates 3a-d, were prepared according to the published
procedures [11], starting from the commercially available substituted phenols 1a-b with
substituted benzoyl chlorides 2a-d. Fries rearrangement of compounds 3a-d with anhydrous
aluminum chloride as a catalyst gave hydroxybenzophenones 4a-d. Furthermore, etherification
of 4a-d with chloro ethyl acetate afforded the substituted ethyl esters 5a-d, which were
4

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converted to the corresponding acetohydrazides 6a-d upon treatment with hydrazine hydrate.
The corresponding final compounds 9a-d and 10a-d were successfully synthesized by coupling
compounds 6a-d with Nꢀmethyl imidazoleꢀ4ꢀcarboxylic acid (7) and 2ꢀoxoꢀ2Hꢀpyrone ꢀ5ꢀ
carboxylic acid (8) respectively using TBTU as a coupling reagent and lutidine as a base. All the
structures of newly synthesized compounds were assigned on the basis of their spectroscopic
data such as, IR, NMR, LCꢀMS and also by C, H, N analysis. The compounds 3a-d, 4a-d and
5
a-d were synthesized and characterized earlier by our research group [15]. The spectra of the
compound 6a was considered as a representative example of the series 6a-d. In IR spectrum, the
ꢀ1
compound 6a showed bands at 1610, 1645 and 3100ꢀ3205 cm corresponding to aromatic
1
carbonyl, amide carbonyl and NHꢀNH stretching frequencies respectively. In H NMR spectrum
2
compound 6a showed three singlets for NH , OCH and NH at δ 4.35, δ 4.89 and δ 9.02
2
2
respectively and also showed multiplet signals in the range δ 7.01ꢀ8.86 for aromatic protons.
The mass spectra of compound 6a gave significant stable M+1 peak at m/z 270. The IR spectra
ꢀ1
ꢀ1
of compounds 9a and 10a has shown new absorption bands for amide 1735 cm and 1725 cm
respectively compared to compound 6a also shown disappearance of the NH protons peak of the
2
1
compound 6a. In addition, H NMR spectra of compounds 9d and 10a showed disappearance of
NH protons peak at 4.35 and appearance of new NH proton peak at δ 10.39 and 10.34
2
respectively. Also, there is an increase in aromatic protons in compounds 9d andn10a as
compared with compound 6a which revealed the formation of the title compounds 9a and 10a.
The mass spectra of compounds 9a gave significant stable peak at m/z 379 (M+1) and 10a at m/z
1
3
3
92 (M+). Further, all the target compounds 9a-d and 10a-d were clearly confirmed by C
NMR spectroscopy.
2
.2. Biology
5

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2
.2.1. Evaluation of IC values and in vitro selection of lead compound
50
The benzophenone derivatives are known to be pharmacologically effective molecules
against various pathological conditions including cancer. Earlier, our group has reported
synthesis of benzophenones conjugated with thiazole, benzimidazole, coumarin and oxadiazole
moieties having an antiꢀtumor, antiꢀangiogenic and proapoptotic properties against various cell
lines [11, 14, 15ꢀ18]. ]. On the other hand, as per the literature survey imidazole and pyrone
nucleus derivatives have excellent pharmacological characteristics against the variety of
pathological conditions including cancer of the different cells. In the present investigation, new
potent analogues were synthesized, by integrating imidazole and pyrone nucleus to
benzophenone moiety. Initially, antiꢀproliferative efficacy of benzophenone analogues 9a-d and
1
0a-d bearing imidazole and pyrone nucleus were evaluated against human cancer cells such
as, human lung adenocarcinoma (A549), human cervical adenocarcinoma (HeLa), human breast
adenocarcinoma (MCFꢀ7) and normal fibroblast cells (NIHꢀ3T3) by performing trypan blue, 3ꢀ
(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) and , lactate dehydrogenase
(
LDH) leak assays [table 1.1]. In this series, compound 9d was found to exhibit a promising antiꢀ
neoplastic effect in trypan blue and MTT assay with half maximal inhibitory concentration IC50
values as 9.6±1.7, and 8.8±0.12 ꢁM, in A549 cells, 10.2±0.3 and 9.9±1.5 ꢁM in Hela cells and
9
.5±0.7 and 9.8±1.0 ꢁM in MCFꢀ7 cells respectively. Remarkably compound 9d exhibited very
negligible toxicity 94.2±1.7 ꢁM to NIHꢀ3T3 cells. The evaluation of cellular integrity using an
LDH release assay is one of the incomparable methods to assess the cytotoxic effect of
compounds. In our investigation the results shown concentration dependent increase in the
release of LDH as the cells lose their integrity upon treatment with compound 9d with IC50
values as 9.1±0.5, 10.4±2.1 and 9.4±0.9 ꢁM in A549, HeLa and MCFꢀ7 cell lines respectively.
6

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Altogether these results suggest that, the compound 9d was potent and prone to exhibit a
cytotoxic effect against the cells of different origin. The effect of compound 9d could be due to
the presence of methyl, chloro and fluoro groups at benzophenone moiety and also due to methyl
group at imidazole ring, which is unique from other compounds of the same series [figure 2].
Hence the compound 9d was chosen as a lead compound and investigated further for its in vitro
and in silico activities.
2
.2.2. Structure activity relationship (SAR).
The compounds which contain the benzophenone pharmacophores are known to be
pharmacologically active molecules which are utilized against several pathological conditions
including cancer [16, 19, 20]. The multistep synthesis of benzophenone containing imidazole and
pyrone analogues is involved in our current study. It is apparent that the compound 9d containing
the same backbone structure of benzophenone with aceto moiety and Nꢀmethyl imidazole ring as
in the same series but with methyl group in the benzophenone moiety has shown good activity
compared same series. The IC values as shown in table 1 suggested that the compound 9d with
5
0
a methyl, chloro and fluoro groups at benzophenone moiety and another methyl group at
imidazole ring shown average IC value as ~9.6 ꢁM as verified by trypan blue, MTT and LDH
5
0
release assay. The results illustrated that, the compounds 9a without halo groups, 9b and 9c with
chloro and fluoro groups shown decreased activity as compared to 9d [table 1]. It reveals that the
compound 9d with a methyl, chloro and fluoro groups in benzophenone moiety and a methyl
group in the imidazole ring [figure.2.] is an important compound for biological activity. While
other compounds with different substituents has shown poor cytotoxicity [table 1]. Therefore
compound 9d was selected as a lead compound based on its significant structure activity
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relationship compared with other analogues and further evaluated by in vitro, in vivo and in silico
studies.
2
.2.3. Compound 9d exhibit the prolonged anti-mitogenicity activity.
The colony formation assay is an appropriate method to investigate the long term
antiꢀmitogenicity of cytotoxic molecules in cancer cell proliferation. The reticence in colony
formation considers as a prolonged cytotoxic effect of the active biomolecule [13]. In this
analysis, A549 cells was treated with or without compound 9d for analyzing long term effect.
Results revealed that the compound 9d visibly diminished the clonogenic efficiency of A549
cells. Compound 9d were found to inhibit the colony formation of A549 cells by 25 and 50 µM
compared to the standard cisplatin [figure 5.3. a and c], and density of the colony formation is
remarkably reduced by compound 9d which is apparent from microscopic analysis of the
colonies [figure 5.3 b].
2
.2.4. Counteracts the cancer cell migration by compound 9d.
Most of the cancers, including lung, liver, renal, cervical breast cancer, etc., are
extremely metastatic with increased migration and invasive characteristics [13, 21]. To
understand the role of compound 9d in migration, we performed scratch wound assay with A549
cells which is highly in vitro migratory. Cell migration into the wound was quantified by taking
snapshot pictures with a regular inverted microscope at 0 and 48 h post scratch. The A549 cells
migrated over a period of 48 h to fill the wound, however, compound 9d treated cells failed to
migrate into the wound and depicted a 78.83 % and 15.59 % at 50 µM and 25 µM respectively
[
figure 5.4. aꢀd]. Therefore, compound 9d has the potency to counteract the angiogenesis and
metastasis by targeting cancer cell migration.
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2
.2.5. Compound 9d potentially inhibits the neovascularization in non-tumor model
systems.
To investigate the antiꢀangiogenic activity, compound 9d was tested in different
angiogenesis models induced by rVEGF . Based on SAR, the compound 9d with chloro, flouro
1
65
and methyl groups in benzophenone moiety and methyl group in imidazole moiety was shown
highest IC value in all assay by in vitro studies which lead us to select compound 9d for in vivo
5
0
studies in which it was showed effect on VEGFr in different chorioallontoic membrane (CAM)
assays. Also, decreased in the VEGF protein levels most effectively as measured by the
decreased of blood vessels in CAM. The total sprouting vessels was quantified by considering
rVEGF165 induced control as 100 % in in vivo CAM and ex vivo CAM [2, 14]. The PBS alone
treated in normal developing CAM was found to be 20 % of growing blood vessels in both in
vivo, and ex vivo CAM assay. A clear avascular zone around the implanted disc with compound
9
d was clearly evident for the regression of neovessels in the developing embryos in both in vivo
and ex vivo CAM [figure.5. a and b] with the inhibition of 24 and 26 % respectively. The total
vessel length in compound 9d treated eggs was drastically decreased to ~85 % compared to
untreated eggs in both the assays [figure.5. d and e]. To reꢀevaluate the effect of compound 9d on
neovascularization on angiogenesis dependent tumor progression, CAM xenograft was
performed by using A549 cells with rVEGF . The results show that the compound 9d and
1
65
cisplatin evidently reduced the tumor progression to 1.7 and 4.0 fold respectively. This results is
based on the decrease in rVEGF165 induced micro vessel density (MVD) / HPF of compound 9d
with 3.6 ± 1.6 (86.2 %) and cisplatin with 9.1± 2.5 (52.2 %) compared to untreated with 30.1 ±
1
.5, in a concentration dependent manner [figure.5. c and f].
2
.2.6. Effect of compound 9d on the morphology of A549 cells.
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The inhibitory effect of compound 9d on A549 cell growth may be due to induction of
apoptosis. The A549 cells were stained with geimsa and the slides were observed under
microscope. In the figure.6 dead cells were lightly stained with irregular cell membrane
compared to the darkly stained and spherical live cells.
2
.2.7. Induction of endonuclease upon the compound 9d treatment.
Endonucleases are essential for DNA fragmentation, which is a prominent feature of the
apoptosis [3, 14]. Endonuclease assay results shown that the compound 9d has promoted the
endonuclease activation as visualized by DNA lysis zone [figure.7. a and b]. Furthermore, a
molecular event of compound 9d was analyzed by immunoblot where in it exhibited tumor
inhibition [figure.7. c]. Additionally, degradation of DNA into multiple intermolecular small
fragments of 180ꢀ200 base pairs is a distinct biochemical trait of apoptosis. Nuclear DNA treated
with or without compound 9d was analyzed by agarose gel electrophoresis [14]. The results
confirmed the typical "ladder" formation of DNA treated with compound 9d whereas the DNA
from untreated cells did not show any changes and observed as a single band on an agarose gel.
This study confirms that antiꢀangiogenesis effects of benzophenone analogues are mediated
through apoptosis leading to DNA degradation [figure.7. d].
2
.2.8. Compound 9d induce apoptosis on A549 cells.
To elucidate the possible mechanism of compound 9d mediated tumor inhibition, the
effect of compound 9d on A549 cells was investigated. The treatment of compound 9d to A549
cells induced a significant proportion of cells to undergo apoptosis, as determined by the flow
cytometric analysis [13]. The flow cytometric analysis of the cells indicates the formation of
apoptotic bodies and condensed camptothecin and control cells, upon receiving compound 9d
treatment some of the A549 cells undergo apoptosis and are detected in the subꢀG area [figure.8.
2
1
0

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a and b]. Furthermore, compound 9d promote the apoptotic cell death by caspaseꢀ3 activation.
A molecular event of compound 9d exhibited tumor regression was analyzed by immunoblot
[
figure 8. c]. These results suggested the broken DNA of A549 cells, resulting in tumor killing.
The obvious ramification is the growth arrest of A549 cells.
2
.2.9. Influence of compound 9d on the A549 cell cycle arrest.
Compound 9d induces cell cycle arrest in A549 cells in vitro. Flow cytometric cell cycle
analysis was performed using propidium iodide (PI) DNA staining [13] of A549 cells following
8 h of treatment with 85 µM compound 9d. In A549 cell cycle analysis the compound 9d
4
showed arrest in G2/M phase at 24 h after treatment. However, at 48 h, the cells arrested in the
G2/M phase underwent cell death as evidences by the increase in subꢀG0 percentage [figure.9. a
and b].
2
.2.10. Docking of inhibitors with the active site of VEGF.
Autodock programming was applied to produce the proteinꢀ9d complex in order to
understand the interaction between VEGF protein and ligand 9d. It is clearly noticeable that
ligand 9d is placed in the center of the active site and by hydrogen bonding interactions it is
stabilized. Together with their distances and angles, the hydrogen bonds exhibited in the VEGFꢀ
9
d complex are documented. By taking into account the interaction energies of the 9d with
residues in the active site of the VEGF, noteworthy binding key residues in the active site of the
model were determined and approved. This noticeable evidence, distinguished and it can clearly
prove the relative significance of each residue in a positive restricting interaction shows the
binding energy, ligand efficiency, inhibition constant, van der Waals force, hydrogen bond,
desolvation energy, bonding residues score, and bond length score for the active site of VEGFꢀ
9
d complex through a definition compare to the compound 9d [table 2]. Likewise it is obvious
1
1

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ꢀ1
that the VEGFꢀ9d complex has substantial perfect with bonding energies ꢀ7.66 kJ mol
[
figure.10]. For the protein complex interaction we have to take into consideration all the
important factors mentioned earlier. It was approved by the interaction analysis that ARG842,
ASN923 and ASP1052, the amino acid residues of the VEGF were significantly attaching to the
compound 9d as the main providers for the inhibition interaction [figure.11.aꢀd]. Through the in
silico study it was approved that ARG842, ASN923 and ASP1052 are the most preferred
residues in the inhibitor binding reaction and this point was supported that the interaction energy
does not include the contribution from the water or the expanded protein structure along with the
list of hydrogen bond interactions between the protein and the active site. The VEGF structure
was further applied for proteinꢀligand modeling studies. Positively connected with receptor
binding, docking in VEGF protein play crucial role in sustaining a functional conformation. The
planned interactions between the VEGF and the inhibitors in this study are of a great use for
understanding and acquiring the possible mechanism of inhibitor binding. For the VEGF and the
structure and function of biological molecules, it is apparent that hydrogen bonds have
significant roles. Also, ARG842, ASN923 and ASP1052 are important for strong hydrogen
bonding interaction with the inhibitors.
It is obvious that, molecule 9d, is the most likely inhibitor and ARG842, ASN923 and
ASP1052 residues are involved in inhibitor binding and forms hydrogen bonding with the
inhibitors. It should be taken into consideration that ARG842, ASN923 and ASP1052 are vital
for receptor binding or preserving the hydrophobicity of the inhibitor binding pocket.
3
. Conclusion
Through the current study, two series of novel benzophenone analogues containing
imidazole (9a-d) and pyrone nucleus (10a-d) were synthesized. It was suggested by the
1
2

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structural activity relationship of these compounds that, the compound 9d with a methyl, chloro
and fluoro groups in benzophenone moiety, shown antiꢀangiogenesis effect against A549, HeLa
and MCFꢀ7 cell lines with IC of 9.1±0.5, 10.4±2.1 and 9.4±0.9 ꢁM respectively and selected as
5
0
the lead compound within this series. The results observed in the CAM models for the inhibition
of neovascularization and tumor growth strongly supported our hypothesis. Compound 9d
interacts with rVEGF by hydrogen bond in silico, thereby down regulates the VEGF expression
in angiogenesis. Further, the compounds 9d have the potency to exhibit the prolonged activity
with cell cycle arrest on G2/M phase against cancer progression this may be by the interaction of
the compound 9d with VEGFꢀA secretion as verified by cell cycle analysis study and
immunoblot. Therefore, in conclusion we can state that the compound 9d plays a crucial role as
angiopreventive effect and it can be applied in various fields related to cancer therapeutics
[
figure.12].
4
4
. Materials and methods
.1. Experimental section
The chemicals required for the synthesis of the title compounds 9a-d and 10a-d were
procured from Sigma Aldrich Chemical Co. The completion of reaction was checked by thin
layer chromatography (TLC) which was performed on aluminum backed silica plates and the
spots were detected by exposure to UVꢀlamp at λ= 254 nm. Melting points and boiling point
were measured on a chemiline, microcontroller based melting point/ boiling pointꢀCl725
apparatus with a digital thermometer. IR spectra were recorded on the Agilent Technologies,
1
13
Cary 630 FTIR spectrometer, H and C NMR spectra were recorded on VNMRSꢀ400 Agilentꢀ
NMR spectrophotometer. The mass spectra were obtained with a VG70ꢀ70H spectrometer and
1
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the elemental analysis (C, H, and N) was performed on ElementarVario EL III elemental
analyzer. The results of elemental analyses are within ±0.4 % of the theoretical values.
Cell lines, like A549, HeLa, MCFꢀ7 and normal NIHꢀ3T3 cells were procured from
NCCS, Pune, India. DMEM, sodium carbonate, fetal bovine serum (FBS), MTT from Sigma
Aldrich, USA. All cell culture wares are from Eppendorf, Germany. Penicillinꢀ Streptomycin
(SigmaꢀAldrich, USA). All photographs were taken using Canon power shot Sx500 IS camera.
4
.2. Chemistry: Plan of the synthesis
Novel benzophenone analogues 9a-d and 10a-d synthesis was accomplished according
to the steps illustrated in scheme1. First, substituted phenols 1a-b on reaction with substituted
benzoyl chlorides 2a-d in the presence of trimethylamine (TEA) gave substituted phenyl
benzoates 3a-d. Compounds 3a-d on subjection to fries rearrangement afforded substituted
diaryl methanones commonly known as hydroxy benzophenones 4a-d. Then (2ꢀaroylꢀ4ꢀ
methylphenoxy) acetates 5a-d were achieved in excellent yield by reacting compounds 4a-d with
ethyl bromoacetate in the presence of anhydrous potassium carbonate and dry acetone. Further,
compounds 5a-d on treatment with 99 % hydrazine hydrate gave different hydrazides 6a-d.
Finally, compounds 6a-d on coupling with 1ꢀmethylꢀ1Hꢀimidazoleꢀ4ꢀcarboxylic acid (7) and
separately with 2ꢀoxoꢀ2Hꢀpyrone ꢀ5ꢀcarboxylic acid (8) in the presence of oꢀ(benzotriazolꢀ1ꢀyl)ꢀ
N,N,N′,N′ꢀ tetra methyl uranium tetra fluoro borate (TBTU) and lutidine as a coupling agent
furnished the title compounds 9d and 10d in excellent yield.
4
.2.1. General procedure for the synthesis of benzoic acid phenyl ester (3a-d).
Substituted phenyl benzoates 3a-d was synthesized by dissolving substituted phenol 1a-b
and TEA (0.45 mol) in dichloro methane (DCM). Then the reaction mixture was cooled to 0 ºC.
1
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Further, a solution of substituted benzoyl chloride 2a-d (0.23 mol) in DCM was slowly added to
the reaction mixture and stirred for 5 h and the completion of the reaction was monitored by TLC
using 4:1 nꢀhexane: ethyl acetate solvent mixture. Then the reaction mass was diluted with DCM
(100 ml) and washed with 10 % sodium hydroxide solution (3×40 ml), followed by water (3×30
ml). The organic layer was dried over anhydrous sodium sulphate and the solid obtained after
evaporation of the solvent was recrystallized from ethanol to give compounds 3a-d.
4
.2.1.1. Phenyl benzoate (3a).
ꢀ1
Yield: 86 %; M.P. 52ꢀ54 ºC; FTꢀIR (KBr, νmax cm ): 1738 (C=O), 1130 (CꢀO), 722 (Ar
1
13
bending); H NMR (DMSOd ): 7.12ꢀ8.41 (m, 10H, ArꢀH); C NMR (DMSOꢀd ) δ: 121.73,
6
6
1
26.02, 128.55, 129.34, 130.41, 133.92, 149.50, 165.23; LCꢀMS m/z: 198 (M+) peak showed
that m/z is equivalent to molecular weight. Anal. Calcd. For C H O : C, 77.93; H, 5.09. Found:
1
3
10
2
C, 77.90; H, 5.04 %.
4
.2.1.2. 2-Fluoro-6-chloro Phenyl-4-fluoro benzoate (3b).
ꢀ1
Yield: 94 %; M.P. 54ꢀ56 ºC; FTꢀIR (KBr, νmax cm ): 1740 (C=O), 1150 (CꢀO), 722 (Ar
1
13
bending); H NMR (DMSOꢀd ) δ: 6.94ꢀ8.28 (m, 7H, ArꢀH); C NMR (DMSOꢀd ) δ: 114.03,
6
6
1
15.46, 125.92, 127.23, 128.55, 129.34, 131.41, 132.02, 140.53, 161.03, 165.31, 16817; LCꢀMS
m/z 268 (M+), 270 (M+2). Anal. Calcd. For C H ClF O : C, 58.12; H, 2.63. Found: C, 58.22;
1
3
7
2
2
H, 2.43 %.
4
.2.1.3. 2-Fluoro-6-chloro phenyl-4-chloro benzoate (3c).
ꢀ1
Yield: 95 %; M.P. 55ꢀ57 ºC; FTꢀIR (KBr, νmax cm ): 1750 (C=O), 1145 (CꢀO), 722 (Ar
1
13
bending); H NMR (DMSOꢀd ) δ: 6.92ꢀ8.12 (m, 7H, ArꢀH); C NMR (DMSOꢀd ) δ: 114.03,
6
6
1
27.32, 128.13, 129.15, 131.04, 131.71, 139.52, 140.53, 161.03165.23; LCꢀMS m/z 285 (M+),
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2
2
87 (M+2), 289 (M+4). Anal. Calcd. ForC H Cl FO : C, 54.77; H, 2.47. Found: C, 54.57; H,
13 7 2 2
.33 %.
4
.2.1.4. 2-Fluoro-6-chloro phenyl-4-methyl benzoate (3d).
Yield: 96 %; M.P. 62ꢀ63 ºC; FTꢀIR (KBr, νmax cmꢀ1): 1780 (C=O) 1138 (CꢀO), 722 (Ar
1
13
bending); H NMR (DMSOꢀd6): δ 2.3 (s, 3H, CH3), 7.38ꢀ8.07 (m, 7H, ArꢀH); C NMR
DMSOꢀd ) δ: 21.34, 114.05, 127.32, 128.13, 130.24, 131.64, 140.51, 143.62, 161.03165.33;
(
6
LCꢀMS m/z 264 (M+), 266 (M+2). Anal. Calcd. For C H ClFO : C, 63.53; H, 3.81. Found: C,
1
4
10
2
6
3.59; H, 3.79 %.
4
.2.2. 4.2.2. General procedure for the synthesis of 4-hydroxy benzophenones (4a-d).
Substituted 4ꢀhydroxy benzophenones 4a-d were synthesized by Fries rearrangement.
Compounds 3a-d (0.065 mol) were blended with aluminium chloride (0.128 mol) and the
mixture was heated to 150 ºC this temperature was maintained for 3 h and the completion of the
reaction was monitored by TLC using 4:1 nꢀhexane: ethyl acetate solvent mixture. Then the
reaction mixture was cooled to room temperature and quenched with 7 N hydrochloric acid and
ice water. The reaction mixture was stirred for about 2 h, the solid obtained was filtered and
recrystallized with methanol to obtain desired compounds 4a-d.
4
.2.2.1. (4-Hydroxyphenyl)(phenyl)methanone (4a).
ꢀ1
1
Yield: 61 %; M.P. 146ꢀ147 ºC; FTꢀIR (KBr, νmax cm ): 1671 (C=O), 3545ꢀ3635 (OꢀH); H NMR
1
3
(
DMSOꢀd ) δ: 6.81ꢀ7.81 (m, 9H, ArꢀH), 9.68 (s, 1H, OH); C NMR (DMSOꢀd ) δ: 116.11,
6
6
1
28.42, 130.01, 130.02, 131.43, 131.74, 132.51, 138.41, 162.03, 194.53; LCꢀMS m/z 198 (M+).
Anal. Calcd. For C H O : C, 78.72; H, 5.08. Found: C, 78.69; H, 5.06 %.
1
3
10
2
4
.2.2.2. (3-Chloro-5-fluoro-4-hydroxyphenyl)(4-fluorophenyl)methanone (4b).
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ꢀ1
1
Yield: 61 %; M.P. 148ꢀ149 ºC; FTꢀIR (KBr, νmax cm ): 1671 (C=O), 3545ꢀ3635 (ꢀOH); H NMR
1
3
(
DMSOꢀd ) δ: 7.36ꢀ7.82 (m, 6H, ArꢀH), 11.64 (bs, 1H, OH); C NMR (DMSOꢀd ) δ: 114.21,
6
6
1
15.23, 125.42, 127.31, 130.32, 131.43, 134.04, 135.51, 148.41, 154.81, 166.03, 194.33; LCꢀMS
m/z 268 (M+) and 270 (M+2). Anal. Calcd. For C H ClF O : C, 58.12; H, 2.63. Found: C,
1
3
7
2
2
5
8.21; H, 2.52 %.
4
.2.2.3. (3-Chloro-5-fluoro-4-hydroxyphenyl)(4-chlorophenyl)methanone (4c).
ꢀ1
1
Yield: 68 %; M.P. 167ꢀ169 ºC; FTꢀIR (KBr, νmax cm ): 1660 (C=O), 3525ꢀ3625 (ꢀOH); H NMR
1
3
(
DMSOꢀd ) δ: 7.12ꢀ7.74 (m, 6H, ArꢀH), 11.60 (s, 1H, OH); ); C NMR (DMSOꢀd ) δ: 114.21,
6
6
1
25.42, 127.41, 128.62, 131.72, 135.33, 136.54, 138.01, 148.41, 154.81, 166.03, 194.33; LCꢀMS
m/z 285 (M+), 287 (M+2) and 289 (M+4). Anal. Calcd. For C H Cl FO : C, 54.77; H, 2.47.
1
3
7
2
2
Found: C, 54.65; H, 2.32 %.
4
.2.2.4. (3-Chloro-5-fluoro-4-hydroxyphenyl)(4-methylphenyl)methanone (4d).
ꢀ1
1
Yield: 65 %; M.P. 182ꢀ183 ºC; FTꢀIR (KBr, νmax cm ): 1635 (C=O), 3430ꢀ3590 (OH); H NMR
1
3
(
DMSOꢀd6) δ: 2.73 (s, 3H, CH ), 7.15ꢀ7.68 (m, 6H, ArꢀH), 9.46 (s, 1H, OH); ); C NMR
3
(
DMSOꢀd ) δ: 21.13, 114.21, 125.52, 127.41, 128.62, 130.32, 142.23, 148.41, 154.81, 194.33;
6
LCꢀMS m/z 265 (M+) and 267 (M+2). Anal. Calcd. For C H ClFO : C, 63.53; H, 3.81; Cl,
1
4
10
2
1
3.39; F, 7.18. Found: C, 63.55; H, 3.83; Cl, 13.41; F, 7.21 %.
4
.2.3. General procedure for the synthesis of (4-benzoyl-phenoxy)- acetic acid ethyl esters
(5a-d).
To a solution of compounds 4a-d (0.036 mol) in dry acetone (70 ml), potassium
carbonate (0.078 mol) and ethyl bromoroacetate (0.057 mol) were added and the reaction mass
was heated to 60 ºC for 4ꢀ6 h, The progress of the reactions was monitored by TLC using 2:1 nꢀ
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hexane: ethyl acetate solvent mixture. The reaction mixture was cooled and the solvent was
removed by distillation. The residual mass was triturated with cold water to remove potassium
carbonate, and extracted with ether (3 × 50 ml). The ether layer was washed with 10 % sodium
hydroxide solution (3 × 50 ml) followed by water (3 × 30 ml), dried over anhydrous sodium
sulphate and evaporated to dryness to obtain crude solid, which, on recrystallization with ethanol
afforded desired compounds 5a-d.
4
.2.3.1. (4-Benzoylphenoxy)acetic acid ethyl ester (5a).
ꢀ1
Yield: 90 %; M.P. 47ꢀ49 ºC; FTꢀIR (KBr, νmax cm ): 1660 (C=O), 1730 (ester, C=O), 1130 (Cꢀ
1
O); H NMR (DMSOꢀd ) δ: 1.28 (t, 3H, CH of ester), 4.21 (q, J=6 Hz, 2H, CH of ester), 5.03
6
3
2
1
3
(
s, 2H, CH ), 6.92 ꢀ7.83 (m, 9H, ArꢀH); C NMR (DMSOꢀd ) δ: 61.33, 65.31, 114.03, 128.43,
2
6
1
30.02, 130.72.41, 131.37, 138.52, 161.93, 169.41, 194.43; LCꢀMS: m/z: 285 (M+). Anal.
Calcd. For C H O : C, 71.82; H, 5.67. Found: C, 71.85; H, 5.69 %.
1
7
16
4
4
.2.3.2. (4-Fluorobenzoyl-2-chloro-6-fluorophenoxy)acetic acid ethyl ester (5b).
ꢀ1
Yield: 85 %; M.P. 50ꢀ52 ºC; FTꢀIR (KBr, νmax cm ): 1660 (C=O), 1730 (ester, C=O), 1135 (Cꢀ
1
O); H NMR (DMSOꢀd ) δ: 1.27 (t, 3H, CH of ester), 4.21 (q, J=6 Hz, 2H, CH of ester), 5.03
6
3
2
1
3
(
s, 2H, CH ), 7.58ꢀ7.77 (m, 6H, ArꢀH); C NMR (DMSOꢀd ) δ: 61.33, 64.95, 114.03, 115.24,
2
6
1
23.94, 126.93, 133.52, 134.12.41, 135.37, 153.52, 166.93, 169.41, 194.43; LCꢀMS: m/z: 354
(
M+) and 356 (M+2). Anal. Calcd. For C H ClF O : C, 57.56; H, 3.69. Found: C, 57.41; H,
17 13 2 4
3
.52 %
4
.2.3.3. [2-Chloro-4-(4-chloro-benzoyl)-6-fluoro-phenoxy]-acetic acid ethyl ester (5c).
ꢀ1
Yield: 83 %; M.P. 54ꢀ56 ºC; FTꢀIR (KBr, νmax cm ): 1650 (C=O), 1740 (ester, C=O), 1135 (Cꢀ
1
O); H NMR (DMSOꢀd ) δ: 1.27 (t, J=7 Hz, 3H, CH of ester), 4.12 (q, J=6 Hz, 2H, CH of
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1
3
ester), 4.92 (s, 2H, CH ), 7.59ꢀ7.75 (m, 6H, ArꢀH); C NMR (DMSOꢀd ) δ: 61.33, 64.95,
2
6
1
14.03, 115.24, 123.94, 126.93, 133.52, 134.12.41, 135.37, 153.52, 166.93, 169.41, 194.43; LCꢀ
MS: m/z: 371 (M+), 373 (M+2) and 375 (M+4). Anal. Calcd. For C H Cl FO : C, 55.01; H,
1
7
13
2
4
3
.53. Found: C, 55.19; H, 3.41 %.
4
.2.3.4. (4-Methylbenzoyl-2-chloro-6-fluorophenoxy)acetic acid ethyl ester (5d).
ꢀ1
Yield: 83 %; M.P. 62ꢀ63 ºC;FTꢀIR (KBr, νmax cm ): 1650 (C=O), 1740 (ester, C=O), 1125 (Cꢀ
1
O); H NMR (DMSOꢀd ) δ: 1.25 (t, J=7 Hz, 3H, CH of ester), 2.32 (s, 3H, CH ), 4.19 (q, J=6
6
3
3
1
3
Hz, 2H, CH of ester) 5.02 (s, 2H, CH ), 7.24ꢀ7.66 (m, 6H, ArꢀH); C NMR (DMSOꢀd ) δ:
2
2
6
2
1.31, 61.33, 64.95, 114.03, 123.94, 126.21, 127.75, 130.25, 135.37, 142.11, 153.32, 169.41,
94.43; LCꢀMS: m/z: 351 (M+), 352 (M+2). Anal. Calcd. For C H ClFO : C, 61.64; H, 4.60;
1
1
8
16
4
Cl, 10.11; F, 5.42. Found: C, 61.66; H, 4.63; Cl, 10.13; F, 5.44 %.
4
.2.4. General procedure for the synthesis of 2-(4-benzoyl-phenoxy)acetohydrazides (6a-d).
Hydrazine hydrate (0.018 mol) was added to a solution of compounds 5a-d (0.018 mol)
in ethanol (30 ml) and continuously stirred for 3 h at room temperature. The completion of the
reaction was monitored by TLC using 2:1 chloroform: ethyl acetate as a mobile phase. After the
separation of a white solid, it 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 in good yield.
4
.2.4.1. 2-(4-Benzoylphenoxy)acetohydrazide (6a).
ꢀ1
Yield: 76 %; M.P. 107ꢀ109 ºC; FTꢀIR (KBr, νmax cm ): 1610 (C=O), 1645 (amide, C=O),
1
3
100ꢀ3205 (NHꢀNH ); H NMR (DMSOꢀd ) δ: 4.35 (s, 2H, NH ), 4.89 (s, 2H, OCH ), 7.01ꢀ8.86
2
6
2
2
1
3
(
m, 9H, ArꢀH), 9.02 (s, 1H, NH); C NMR (DMSOꢀd ) δ: 66.25, 114.11, 128.44, 130.21,
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1
31.05, 132.45, 138.37, 161.41, 166.31, 194.43; LCꢀMS m/z 270 (M+). Anal. Calcd. For
C H N O : C, 66.66; H, 5.22; N, 10.36. Found: C, 66.68; H, 5.24; N, 8.13 %.
15
14
2
3
4
.2.4.2. [2-4(4-Fluoro)benzoyl-2-chloro-6-fluorophenoxy]aceto hydrazide (6b).
ꢀ1
Yield: 79 %; M.P. 110ꢀ113 ºC; FTꢀIR (KBr, νmax cm ): 1620 (C=O), 1645 (amide, C=O),
1
3
100ꢀ3205 (NHꢀNH ); H NMR (DMSOꢀd ) δ: 4.35 (s, 2H, NH ), 4.69 (s, 2H, OCH ), 7.20ꢀ7.86
2
6
2
2
1
3
(
m, 6H, ArꢀH), 9.32 (s, 1H, NH); C NMR (DMSOꢀd ) δ: 65.95, 114.31, 123.85, 126.93,
6
1
33.45, 134.01, 135.04, 153.41, 166.61, 194.43; LCꢀMS m/z: 340 (M+) and 342 (M+2). Anal.
Calcd. For C H ClF N O : C, 52.88; H, 3.25; Cl, 10.41; F, 11.15; N, 8.22. Found: C, 52.75; H,
1
5
11
2
2
3
3
.38 %.
.2.4.3. 2-(2-Chloro-4-(4-chlorobenzoyl)-6-fluorophenoxy)acetohydrazide (6c).
4
ꢀ1
Yield: 79 %; M.P. 112ꢀ116 ºC; FTꢀIR (KBr, νmax cm ): 1610 (C=O), 1645 (amide, C=O),
1
3
100ꢀ3205 (NHꢀNH ); H NMR (DMSOꢀd ) δ: 4.35 (s, 2H, NH ), 4.83 (s, 2H, OCH ), 7.20ꢀ7.76
2
6
2
2
1
3
(
m, 6H, ArꢀH), 9.32 (s, 1H, NH); C NMR (DMSOꢀd ) δ: 65.95, 114.31, 115.22, 123.85,
6
1
26.93, 133.45, 134.01, 135.04, 153.41, 166.61, 194.43; LCꢀMS m/z 357 (M+) and 359 (M+2).
Anal. Calcd. For C H Cl FN O : C, 50.44; H, 3.10; Cl, 19.85; F, 5.32; N, 7.84. Found: C,
1
5
11
2
2
3
5
0.46; H, 3.13 %.
4
.2.4.4. [2--4(4-Methyl)benzoyl-2-chloro-6-fluorophenoxy]aceto hydrazide (6d).
ꢀ1
Yield: 79 %; M.P. 117ꢀ123 ºC; FTꢀIR (KBr, νmax cm ): 1610 (C=O), 1645 (amide, C=O),
1
3
100ꢀ3205 (NHꢀNH ); H NMR (DMSOꢀd ) δ: 2.40 (s, 2H, CH ), 4.31 (s, 2H, NH ), 4.79 (s, 2H,
2
6
3
2
1
3
OCH ), 7.20ꢀ7.86 (m, 6H, ArꢀH), 9.32 (s, 1H, NH); ); C NMR (DMSOꢀd ) δ: 21.34, 65.95,
2
6
1
14.31, 123.85, 126.93, 128.73, 130.22, 135.45, 142.21.04, 153.31, 166.61, 194.43; LC–MS m/z
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3
36 (M+) and 338 (M+2). Anal. Calcd. For C H ClFN O : C, 57.07; H, 4.19; Cl, 10.53; F,
16 14 2 3
5
.64; N, 8.32. Found: C, 57.09; H, 4.22 %.
4
.2.5. General procedure for the synthesis of 2-(4-benzoylphenoxy)aceto-N (N-methyl
imidazole)carbonyl hydrazides (9a-d).
ꢀ(4ꢀBenzoylphenoxy)acetohydrazides 6a-d (0.0010 mol) in dry DCM (20 ml) was
2
stirred at 25ꢀ30 ºC, and then lutidine (0.0015 mol) was added, followed by the addition of Nꢀ
methyl imidazoleꢀ4ꢀcarboxylic acid (7, 0.0010 mol). The reaction mixture was stirred at the same
temperature for 30 minutes, then cooled to 0ꢀ5 ºC and TBTU (0.0015 mol) was added and the
temperature was maintained to 5 ºC over a period of 30 minutes. The reaction mass was stirred
overnight and monitored by TLC using chloroform: methanol (9:1) as the mobile phase. Then
the solvent was evaporated at reduced pressure, quenched by the addition of crushed ice and the
solid obtained was filtered, dried and recrystallized from ethanol to afford compounds 9a-d in
good yield.
4
.2.5.1. 2-(4-benzoylphenoxy)aceto-N(N-methyl imidazole)carbonyl hydrazide (9a).
ꢀ1
Yield: 93 %; Purity ˃ 96 % HPLC; M.P. 103ꢀ105 ºC; FTꢀIR (KBr, νmax cm ):1650 (C=O),
1
1
678 (ꢀCH ꢀC=O), 1735 (C=O), 3275ꢀ3360 (NHꢀNH); H NMR (DMSOꢀd ) δ: 3.71 (s, 3H,
2
6
1
3
CH ), 4.86 (s, 2H, OCH ), 6.91ꢀ8.08 (m, 11H, ArꢀH), 9.89 (s,1H, NH), 10.39 (s, 1H, NH), ; C
3
2
NMR (DMSOꢀd ) δ: 32.16, 72.77, 114.09,123.03, 126.04, 128.73, 130.11, 130.59, 131.65,
6
1
32.18, 137.80, 140.21, 165.70, 167.34, 175.45, 187.38; LCꢀMS m/z 379 (M+1) and 380
(
M+2). Anal. Calcd. For C H N O : C, 63.48; H, 4.79; N, 14.81, Found: C, 63.46; H, 4.78; N,
20
18
4
4
1
4.82 %.
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4
.2.5.2. [2-4(4-Fluoro) benzoyl-2-chloro-6-fluorophenoxy]aceto-N (N-methyl imidazole)
carbonyl hydrazide (9b).
ꢀ1
Yield: 96 %; Purity ˃ 87 % HPLC; M.P. 107ꢀ109 ºC; FTꢀIR (KBr, νmax cm ): 1675 (C=O),
1
1
676 (ꢀCH ꢀC=O), 1755 (C=O), 3275ꢀ3360 (NHꢀNH); H NMR (DMSOꢀd ) δ: 3.71 (s, 3H,
2
6
1
3
CH ), 4.86 (s, 2H, OCH ), 6.91ꢀ8.05 (m, 8H, ArꢀH), 9.87 (s, 1H, NH), 10.89 (s, 1H, NH); C
3
2
NMR (DMSOꢀd ) δ: 29.16, 77.07,115.21, 116.03, 120.66,120.72, 123.00, 126.46, 127.54,
6
1
4
8
31.29, 133.65, 133.18, 140.03, 148.44, 155.51, 168.41, 167.04, 173.95, 189.78; LCꢀMS m/z
48 (M+) and 450 (M+2). Anal. Calcd. For C H ClF N O : C, 53.52; H, 3.37; Cl, 7.90; F,
2
0
15
2
4
4
.47; N, 12.48, Found: C, 53.54; H, 3.35; N, 12.46 %.
4
.2.5.3. [2-4(4-Chloro) benzoyl-2-chloro-6-fluorophenoxy]aceto-N (N-methyl imidazole)
carbonyl hydrazide (9c).
ꢀ1
Yield: 85 %; Purity ˃ 85 % HPLC; M.P. 106ꢀ108 ºC; FTꢀIR (KBr, νmax cm ): 1680 (C=O),
1
1
679 (ꢀCH ꢀC=O), 1730 (C=O), 3275ꢀ3360 (NHꢀNH); H NMR (DMSOꢀd ) δ: 3.69 (s, 3H,
2
6
1
3
CH ), 4.86 (s, 2H, OCH ), 6.91ꢀ7.85 (m, 8H, ArꢀH), 9.88 (s, 1H, NH), 10.89 (s, 1H, NH); C
3
2
NMR (DMSOꢀd ) δ: 28.16, 77.27, 116.79, 120.7, 123.66, 126.54, 128.51, 131.29, 133.65,
6
1
36.21, 137.50, 140.18, 148.44, 153.41, 167.01, 175.45, 194.78; LCꢀMS m/z 464 (M+ ) and 466
(
M+2). Anal. Calcd. For C H Cl FN O : C, 51.63; H, 3.25; Cl, 15.24; F, 4.08; N, 12.04,
20
15
2
4
4
Found: C, 51.62; H, 3.24; N, 12.02 %.
4
.2.5.4. [2--4(4-Methyl) benzoyl-2-chloro-6-fluorophenoxy]aceto-N (N-methyl imidazole)
carbonyl hydrazide (9d).
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ꢀ1
Yield: 89 %; Purity ˃ 85% HPLC; M.P. 114ꢀ117 ºC; FTꢀIR (KBr, νmax cm ): 1645 (C=O),
1
1
682 (ꢀCH ꢀC=O), 1740 (C=O), 3275ꢀ3360 (NHꢀNH); H NMR (DMSOꢀd ) δ: 2.41 (s, 3H, Arꢀ
2
6
CH ), 3.71 (s, 3H, CH ), 4.86 (s, 2H, OCH ), 6.91ꢀ7.85 (m, 8H, ArꢀH), 9.89 (1H, NH), 10.89 (s,
3
3
2
1
3
1
1
4
H, NH); C NMR (DMSOꢀd ) δ: 26.55, 25.55, 77.93, 116.26, 120.08, 123.84, 126.61, 127.13,
6
28.35, 130.00, 133.12, 134.82, 141.43, 148.81, 153.13, 167.02, 170.22, 187.01; LCꢀMS m/z
44 (M+) and 446 (M+2). Anal. Calcd. For C H ClFN O : C, 56.70; H, 4.08; Cl, 7.97; F, 4.27;
2
1
18
4
4
N, 12.59, Found: C, 56.68; H, 4.07; N, 12.58 %.
4
.2.6. General procedure for the synthesis of 2-(4-benzoylphenoxy)aceto-N(pyrone -2-
one)carbonyl hydrazides (10a-d).
Compounds 6a-d (0.0010 mol) in dry DCM (20 ml) was stirred at 25ꢀ30 ºC, and then
lutidine (0.0015 mol) was added dropwise, followed by the addition of 2ꢀoxoꢀ2Hꢀpyrone ꢀ5ꢀ
carboxylic acid (8, 0.0010 mol). The reaction mixture was stirred at the same temperature for 30
minutes, then cooled to 0ꢀ5 ºC and TBTU (0.0015 mol) was added by maintaining the
temperature below 5 ºC over a period of 30 minutes. 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 compounds 10a-d in good yield.
4
.2.6.1. 2-(4-Benzoylphenoxy)aceto-N(pyrone -2-one)carbonyl hydrazide (10a).
ꢀ1
Yield: 81 %; Purity ˃ 85% HPLC;M.P. 112ꢀ115 ºC; FTꢀIR (KBr, νmax cm ):1650 (C=O),
1
1
678 (ꢀCH ꢀC=O), 1725 (C=O of pyrone e), 3275ꢀ3360 (NHꢀNH); H NMR (DMSOꢀd ) δ: 4.86
2
6
1
3
(
s, 2H, OCH ), 6.31ꢀ8.65 (m, 12H, ArꢀH), 9.79 (s, 1H, NH), 10.34 (s, 1H, NH); C NMR
2
(
DMSOꢀd ) δ: 78.47, 105.29, 113.76, 120.93, 128.26, 130.10, 130.19, 131.20, 131.45, 138.48,
6
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1
46.21, 165.71, 161.83, 165.98, 170.45, 187.38; LCꢀMS m/z 392 (M+ ). Anal. Calcd. For
C H N O : C, 64.28; H, 4.11; N, 7.14, Found: C, 64.26; H, 4.09; N, 7.12 %.
21
16
2
6
4
.2.6.2. [2--4(4-Fluoro) benzoyl-2-chloro-6-fluorophenoxy]aceto-N(pyrone -2-one)carbonyl
hydrazide (10b).
ꢀ1
Yield: 72 %; Purity ˃ 81% HPLC; M.P. 116ꢀ119 ºC; FTꢀIR (KBr, νmax cm ):1660 (C=O),
1
1
4
682 (amide, C=O), 1732 (C=O of pyrone e), 3275ꢀ3360 (NHꢀNH); H NMR (DMSOꢀd ) δ:
6
1
3
.86 (s, 2H, OCH ), 6.31ꢀ8.65 (m, 9H, ArꢀH), 9.69 (s, 1H, NH) 10.24 (s. 1H, NH); C NMR
2
(
DMSOꢀd ) δ: 77.95, 106.73, 115.21, 116.29, 120.76, 120.91, 126.91, 131.20, 131.72, 133.11,
6
1
33.37, 146.51, 148.21, 153.12, 161.03, 165.81, 165.93, 170.38, 187.42; LCꢀMS m/z 462 (M+)
and 464 (M+2). Anal. Calcd. For C H ClF N O : C, 54.50; H, 2.83; N, 6.05, Found: C, 54.48;
2
1
13
2
2
6
H, 2.81; N, 6.04 %.
4
.2.6.3. [2--4(4-Chloro)benzoyl-2-chloro-6-fluorophenoxy]aceto-N(pyrone -2-one)carbonyl
hydrazide (10c).
ꢀ1
Yield: 67 %; Purity ˃ 85% HPLC; M.P. 117ꢀ119 ºC; FTꢀIR (KBr, νmax cm ): 1648 (C=O),
1
1
679 (ꢀCH ꢀC=O), 1728 (C=O of pyrone e), 3275ꢀ3360 (NHꢀNH); H NMR (DMSOꢀd ) δ: 4.86
2
6
1
3
(
s, 2H, OCH ), 6.31ꢀ8.65 (m, 9H, ArꢀH), 9.09 (s, 1H, NH), 10.03 (s, 1H, NH); C NMR
2
(
DMSOꢀd ) δ: 77.97, 114.21, 116.22, 120.72, 120.93, 127.12, 128.69, 131.22, 131.50, 133.11,
6
1
35.91, 137.51, 146.72, 148.82, 153.11, 161.01, 164.07, 170.32, 187.38; LCꢀMS m/z 478 (M+)
and 450 (M+2). Anal. Calcd. For C H Cl FN O : C, 52.63; H, 2.73; N, 5.85, Found: C, 52.62;
2
1
13
2
2
6
H, 2.71; N, 5.83 %.
4
.2.6.4. [2-4(4-Methyl)benzoyl-2-chloro-6-fluorophenoxy]aceto-N(pyrone -2-one)carbonyl
hydrazide (10d).
2
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ꢀ1
Yield: 88 %; Purity ˃ 78 % HPLC; M.P.118ꢀ120 ºC; FTꢀIR (KBr, νmax cm ):1650 (C=O),
1
1
678 (ꢀCH ꢀC=O), 1728 (C=O of pyrone e), 3275ꢀ3360 (NHꢀNH); H NMR (DMSOꢀd ) δ: 2.40
2
6
(
s, 3H, CH ), 4.86 (s, 2H, OCH ), 6.31ꢀ8.65 (m, 9H, ArꢀH), 9.23 (s, 1H, NH), 10.23 (s, 1H, NH);
3 2
1
3
C NMR (DMSOꢀd ) δ: 21.32, 77.92, 109.21, 117.22, 122.71, 126.92, 129.12, 130.69, 131.24,
6
1
31.80, 134.11, 135.81, 138.51, 146.73, 149.83, 153.18, 161.04, 164.06, 171.32, 187.58; LCꢀ
MS m/z 458 (M+) and 460 (M+2). Anal. Calcd. For C H ClFN O : C, 57.59; H, 3.51; N, 6.11,
2
2
16
2
6
Found: C, 57.56; H, 3.50; N, 6.10 %.
4
4
.3. Biological study
.3.1. Cell culture and in vitro treatment.
All the experimental manipulations were carried out strictly under aseptic conditions in
class II biosafety cabinet (Thermo scientific, USA). The floor of the biosafety cabinet was
thoroughly sprayed and swabbed with 70 % ethanol. The surface of all the vessels and other
instruments used for the study were also cleaned with ethanol. The chamber was then sterilized
within built UV rays continuously for 1h before inoculation. Hands and arms inside the
inoculation chamber were swabbed with ethanol before inoculation. For the present investigation
Dulbecco Modified Eagle Medium (DMEM) supplemented with 10 % heat inactivated fetal
bovine serum (FBS), PenicillinꢀStreptomycin and 0.37 % sodium bicarbonate was used. The
powdered DMEM was prepared with sterilized double distilled water as per the manufacturer’s
recommendation. The prepared media, then subjected to filtering sterilization using 0.2 ꢁ
Whatman filter paper [13]. Further, for the present study three different cell lines of varying
origin were selected. The A549, HeLa and MCFꢀ7 cells were used for determining the IC value
5
0
of newly synthesized series 9a-d and 10a-d by MTT, trypan blue LDH leak and colonogenic
assays. The cells were treated using increasing concentrations of compounds 9a-d and 10a-d (0,
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5
, 10, 25, 50 and 100 ꢁM in DMSO) at various time intervals (0ꢀ48 h) and further used for
experiments. Appropriate vehicle control and cisplatin as positive control were used and each
experiment was repeated for a minimum of three independent times.
4
.3.2. MTT assay.
For the evaluation of the effect of compounds 9a-d and 10a-d on cell proliferation of
A549, HeLa and MCFꢀ7 cells, the MTT assay was performed as described earlier [13]. Cells,
growing in exponential phase were seeded into 96ꢀwell plates in triplicates at an initial density of
4
3
×10 cells per well in 100 µl of complete medium. Following 16 h incubation, compounds 9a-d
and 10a-d were added to the culture medium at 0, 5, 10, 25, 50 and 100 ꢁM concentrations with
appropriate controls and blanks. Cells were incubated for 48 h. Later, 10 µl MTT (5 mg/ml)
o
reagent was added and incubated for 4 h at 37 C. For solubilization of the resultant 5ꢀ(4,5ꢀ
dimethyl thiazolꢀ2ꢀyl)ꢀ1,3ꢀdiphenyl blue (formazan) product which obtained by reduction of
MTT after the incubation period, 100 µl of DMSO was added. The absorbance was measured
using a multimode reader at a test wavelength of 570 nm and a reference wavelength of 630 nm.
Data obtained are expressed as the percentage of control mean ± standard error of the mean
(SEM) of triplicate values.
4
.3.3. Trypan blue dye exclusion assay.
The effects of the compounds 9a-d and 10a-d on A549, HeLa and MCFꢀ7cells were
determined by trypan blue dye exclusion assay [13, 14]. Then the A549, HeLa and MCFꢀ7 cells
were cultured and treated with or without compounds were collected after 48 h. The viable cells
were counted by resuspending the cells in 0.4 % trypan blue and the IC values were estimated.
5
0
4
.3.4. LDH release assay.
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The LDH assay was performed to assess the LDH release by treating with compounds
9
a-d and 10a-d (0, 5, 10, 25, 50 and 100 ꢁM in DMSO) on both A549, HeLa and MCFꢀ7 cells
after 48 h of incubation as described earlier [14]. The cells were lysed using 0.1 % “Tritonꢀ×100”
in PBS. The amount of LDH released in both culture media and cell lysate was measured at 490
nm using an ELISA reader. The percentage of LDH release was calculated as LDH release in the
media (LDH release in media + intracellular LDH release) × 100.
4
.3.5. Colony formation assay.
The colony formation assay has been the gold standard for determining the antiꢀ
neoplastic effects of cytotoxic compounds on cancer cell proliferation in vitro and it was
performed as described earlier for the potent compound 9d against A549 cells [22ꢀ24]. In brief,
the cells were cultured in Tꢀ25 tissue culture flasks for 24 h and exposed to test compounds for
9
d at 10 µM and 20 µM concentrations. The cells were then trypsinized, counted and seeded into
a six well plate at appropriate densities and cultured for 8 days. Colonies were fixed with
methanol, stained with crystal violet (0.4 g/l), photographed, analyzed and counted using ImageJ
(
v1.48) software.
.3.6. Migration assay.
A549 cells were cultured in six well plate a until they reached a confluence of 60ꢀ70 %.
4
A scratch was made to form a wound using micropipette and the monolayer was washed with
growth medium. Then 2 ml medium containing different concentrations of test drugs was added
to the respective wells, incubated for 48 h, fixed with chilled 70 % ethanol and stained with
crystal violet (0.4 g/l). Images were taken from random fields at regular time intervals using an
inverted microscope (Bresser, Biolux). Finally, percentage of cell migration was calculated by
comparing the final gap width to initial gap width [13, 25, 26].
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4
.3.7. In vivo rVEGF165 induced chorioallontoic membrane (CAM) assay.
The In vivo CAM model was performed to evaluate antiꢀangiogenesis activity with slight
modifications as described previously. In brief, fertilized eggs were incubated, windows were
rd
made on the 3 day, and filter paper discs loaded with test compound 9d (10 ꢁM) was placed on
CAMs. Then the eggs were reincubated after proper sealing for 3ꢀ4 days. Finally, the eggs were
opened and changes in the MVD were photographed using Sony steady shot DSCꢀW610 camera.
Based on the changes in the MVD, the inhibition of angiogenesis was evaluated [19, 21, 27, 28].
4
.3.8. In vivo / ex vivo rVEGF165 induced CAM assay.
To confirm the angioprevention effect of compound 9d, the in vitro / ex vivo shell less
CAM assay was performed. In brief, the two days old fertilized incubated eggs were cracked out
and the contents were poured onto a sterilized condiment cup covered with saran wrap. The egg
preparations were covered with a sterilized petri dish and reꢀincubated at the humidified
o
th
condition at 37 C. On day 4 the egg preparations were impregnated with sterile filter discs
containing rVEGF165 followed by the treatment with compound. After 72 h of incubation the
change in the vascularization in both treated and untreated egg preparations was photographed
using Sony steady shot DSCꢀW610 camera and the blood vessels were quantified [20].
4
.3.9. In vivo CAM xenograft assay.
The in vivo CAM xenograft model to assess angiogenesis was developed with slight
modification as described previously [21, 29]. In brief, the cell line used for the present assay is
human lung adenocarcinoma (A549), which was cultured as described previously. The fertilized
Giriraja breed hen’s eggs were procured from local market. The eggs were wiped with 70 %
o
alcohol thoroughly and maintained at the 37 C under the humidified condition for the
development of the embryo. Under aseptic condition 50 ꢁl of extracellular matrix (ECM) gel was
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coated on the bottom of plastic ring of dimension 4mm × 4mm (height × diameter). A small
th
window was made in the fertilized eggs on the 6 day of fertilization and about 1ml of albumin
was drawn carefully without damaging the CAM, using sterilized syringe. The plastic rings with
ECM gel were placed on the growing CAM under sterilized condition. The rings were then
6
adsorbed with 5×10 A549 cell suspension along with rVEGF1 (10 ng/ml). The window was
65
th
sealed using a sterile serine wrap and again kept for incubation. The eggs were opened on the12
day and tumor was separated from CAM, photographed and histological sections were processed
for MVD count.
4
.3.10. Changes in the morphology of cell line by Giemsa staining.
The untreated and treated cells with the compound 9d were trypsinized and stained with
0X Giemsa stock solution at a stain to cell suspension ratio of 1:10. Morphology and apoptotic
2
body formations were evaluated by control versus compound 9d treated cells. Then the cells
were viewed under the 40X objective of the microscope (Biolux, Bresser Germany) mounted
with MagCam, Magnus Analytics and finally the images were analyzed with ImageJ software
[
14, 19, 30].
4
.3.11. Endonuclease assay.
The endonuclease assay was performed to assess the compounds induced DNase
activation by the methods described earlier [20]. Briefly, one portion of cytosolic extracts of
A549 cells prepared in lysis buffer (10 mM trisꢀhydrochloric acid, pH 8.0, 0.1 mM EGTA and
0
.1 %
βꢀmercaptoethanol and protease inhibitors) was treated with camptothecin,
second potion with compound 9d and the third potion with untreated. Then 1 % agarose which
contains 10 µg/ml ethidium bromide and 50 µg/ml of hot denatured salmonꢀsperm DNA was
plated on glass petri dishes and allowed to solidify at room temperature. Wells were prepared in
2
9