Design and synthesis of diamide-coupled benzophenones as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2016.03.040

A series of diamide-coupled benzophenone, 2-(4-benzoyl-phenoxy)-N-{2-[2-(4-benzoyl-phenoxy)-acetylamino]-phenyl}-acetamide analogues (9a-l) were synthesized by multistep reactions and all compounds were well characterized. Among the series (9a-l), compound 9k with three methyl groups at ortho position in rings A, B, and D and bromo group at the para position in ring E was selected as a lead compound by screening through multiple cancer cell types by in-vitro cytotoxic and antiproliferative assay systems. Also, the cytotoxic nature of the compound 9k resulted the regression of the tumor growth in-vivo, which could be due to decreased vascularisation in the peritoneum lining of the mice which regress the tumor growth. The results were reconfirmed in-vivo chorioallantoic membrane model which indicates a scope of developing 9k into potent anticancer drug in near future.

Full text of DOI:10.1016/j.ejmech.2016.03.040

Accepted Manuscript
Design and synthesis of diamide-coupled benzophenones as potential anticancer
agents
Zabiulla, H.G. Shamanth Neralagundi, A. Bushra Begum, B.T. Prabhakar, Shaukath
Ara Khanum
PII:
S0223-5234(16)30222-7
10.1016/j.ejmech.2016.03.040
EJMECH 8465
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 January 2016
Revised Date: 15 March 2016
Accepted Date: 16 March 2016
Please cite this article as: Zabiulla, H. Shamanth Neralagundi, A Bushra Begum, B.T Prabhakar, S.A.
Khanum, Design and synthesis of diamide-coupled benzophenones as potential anticancer agents,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.040.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design and synthesis of diamide-coupled benzophenones as potential
anticancer agents
Zabiullaa, Shamanth Neralagundi HGb, Bushra Begum Aa, Prabhakar B.Tb and Shaukath Ara
Khanuma*
aDepartment of Chemistry, Yuvaraja’s College (Autonomous), University of
Mysore,
Karnataka, India.
bMolecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in
Biotechnology, Sahyadri Science College (Autonomous), Kuvempu University, Karnataka,
India.
O
C
O
N
H
D
O
O
B
NH
O
H3C
E
H3C
Compound 9k
Br
CH3
O
A

ACCEPTED MANUSCRIPT
Design and synthesis of diamide-coupled benzophenones as potential
anticancer agents
Zabiullaa, Shamanth Neralagundi HGb, Bushra Begum Aa, Prabhakar B.Tb and Shaukath Ara
Khanuma*
aDepartment of Chemistry, Yuvaraja’s College (Autonomous), University of
Mysore,
Karnataka, India.
bMolecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in
Biotechnology, Sahyadri Science College (Autonomous), Kuvempu University, Karnataka,
India.
*Correspondence Author:
Dr. Shaukath Ara Khanum
Associate Professor, Department of Chemistry
Yuvaraja’s College
University of Mysore, Mysore.
Phone: +91 - 99018 88755Fax: +821- 2419239
Email: shaukathara@yahoo.co.in
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ABSTRACT
A series of diamide-coupled benzophenone, 2-(4-benzoyl-phenoxy)-N-{2-[2-(4-benzoyl-
phenoxy)-acetylamino]-phenyl}-acetamide analogues (9a-l) were synthesized by multistep
reactions and all compounds were well characterized. Among the series (9a-l), compound 9k
with three methyl groups at ortho position in rings A, B, and D and bromo group at the para
position in ring E was selected as a lead compound by screening through multiple cancer cell
types by in-vitro cytotoxic and antiproliferative assay systems. Also, the cytotoxic nature of the
compound 9k resulted the regression of the tumor growth in-vivo, which could be due to
decreased vascularisation in the peritoneum lining of the mice which regress the tumor growth.
The results were reconfirmed in-vivo chorioallantoic membrane model which indicates a scope
of developing 9k into potent anticancer drug in near future.
Key words: Benzophenone-diamide, Antiproliferation, Antiangiogenesis.
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1. Introduction
Cancer is one of the deadliest diseases affecting mankind. Although a number of
chemotherapeutic agents are available, most of them also kill the normal cells that divide rapidly
and cause several side effects such as immune suppression, inflammation of the digestive tract
lining and hair loss [1]. Angiogenesis is a biological process where new blood vessels branch out
from the existing vasculature [2]. This process is tightly controlled for normal physiologies such
as reproduction, development and wound repair. However, it becomes out of control when it is
implicated in diseases such as cancer, age-related or diabetic retinopathy and so on [3]. Tumor
cells, in general, proliferate very fast and the demand for essential nutrients, oxygen, etc. is
always high. The immediate environment of cancers increasing in size, however, often becomes
heterogeneous and some regions of large cancers often possess micro environmental niches,
which exhibit a significant gradient of critical metabolites including oxygen, glucose, other
nutrients, and growth factors [4]. Thus, many cancer cells get the critical metabolites by
randomly recruiting new blood vessels, a phenomenon commonly known as angiogenesis, to
survive under such severe conditions. The literature survey reveals that benzophenone and its
derivatives are an emerging class of molecules with multiple pharmacokinetic properties. New
molecules with benzophenone moiety emerging day by day with potent biological activity in
recent times [5-8]. Several analogues of benzophenone are well known for their antitumor and
antiangiogenic potentials which are under clinical trials [9, 10].
Being very much focused on this aspect by our research group, a series of benzophenone
have been synthesized with special emphasis given to the anticancer activity with the
establishment of the mechanism of action [11-14]. In this connection, several molecules have
been identified with potentiality in inhibiting angiogenesis, which plays a very important role in
tumor establishment, prognosis of cancer[15-17] and identification of its molecular target [18,
19]. Such identification of novel molecular target for cancer therapy has led to a paradigm shift
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in the drug development process which can effectively inhibit the signaling pathways involved in
cancer development and Prognosis [20, 21]. As a continued approach of screening for novel
drug, a series of diamide-coupled benzophenones, 2-(4-benzoyl-phenoxy)-N-{2-[2-(4-benzoyl-
phenoxy)-acetylamino]-phenyl}-acetamides (9a-l) were synthesized. The synthesis of amides is
one of the most fundamental methods in organic chemistry used to obtain potent compounds [22,
23]. Hence we considered it worthwhile to pursue further modifications on the substituted
benzophenone part by appending diamide subunit and screened against multiple cancer cell lines,
such as lung carcinoma (A-549), breast cancer (MCF-7) and lymphoma (DLA) in-vitro. Then,
antiproliferative efficacy was verified in-vivo in the Daltons lymphoma murine model and
mechanism of tumor inhibition.
2. Result and discussion
2.1. Chemistry
The synthesis of the title compounds 9a–l was accomplished by a synthetic procedure as
shown in Scheme 1. All the synthesized compounds were established by IR, proton NMR and
mass spectral data. First, the benzoylated products 3a–f were synthesized by the benzoylation of
substituted phenols 1a–c with substituted benzoyl chlorides 2a-d under low temperature and the
structures were confirmed by the appearance of the carbonyl stretching band for the ester group
in the IR spectra and the disappearance of broad singlet of the OH proton of substituted
phenols 1a–c in proton NMR spectra. Fries rearrangement of compounds 3a–f using anhydrous
aluminium chloride as a catalyst under neat condition afforded hydroxy benzophenones 4a–f,
and these compounds were established by the disappearance of the carbonyl stretching band of
the ester group and appearance of the OH stretching band in IR spectra and also, the appearance
of broad singlet for OH proton and decrease in one aromatic proton in proton NMR spectra.
Compounds 4a–f on etherification with chloro ethyl acetate using dry acetone as a solvent gave
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substituted (4-benzoyl phenoxy)-acetic acid ethyl esters 5a–f, which were confirmed by the
disappearance of the OH stretching of compounds 4a–f and appearance of carbonyl stretching
band for the ester group in the IR absorption spectra. The proton NMR observations of these
compounds revealed that, broad singlet for the OH proton of compounds 4a–f disappeared and a
triplet and quartet for CH3 and CH2 protons respectively appeared [9]. The compounds 5a–f on
refluxing with aqueous sodium hydroxide in ethanol gave (4-benzoyl-phenoxy)-acetic acids 6a–
f, which was clearly evident with the appearance of carbonyl group stretching band of carboxylic
acid and disappearance of carbonyl stretching of ester group for compounds 5a–f in the IR
spectra. In proton NMR, the appearance of COOH proton and disappearance of triplet and
quartet peaks for CH3 and CH2 protons, respectively has confirmed the formation of the
compounds 6a–f [24]. Compounds 8a-f were obtained by treating compounds 6a-f with 1, 2
diaminobenzene7 in the presence of 2,6 lutidine and O-(benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluroniumtetrafluoroborate (TBTU) as a coupling agent and dichloromethane (DCM)
as a solvent. The structures were confirmed by the disappearance of the carbonyl stretching band
of a carboxylic acid in the IR spectra and in proton NMR, the appearance of NH2 and NH
protons, besides, an increase in four aromatic protons. Finally, all the substituted compounds 6a–
f, on treatment with 8a-f (N-(2-amino-phenyl)-2-(4-benzoyl-phenoxy)-acetamides) using lutidine
and TBTU as a catalyst, afforded the expected title compounds 9a–l in a good yield (70–90%).
This was supported by the disappearance of NH2 and COOH stretching of the compounds 8a–
f and 6a–f respectively in the IR spectra. Also, it is confirmed by the disappearance of COOH
of 6a–f and NH2 of 8a–f proton and appearance of two CONH protons in the NMR spectra.
2.2. Biology
2.2.1. Compound 9k has potent cytotoxic and antiprolifertive effect against multiple cancer types
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In a course of drug development process it has always been suggested to screen against
multiple cancer cell types with different origin for better assessment strategies of the drug. In the
current investigation, the cytotoxic and antiproliferative effects of compounds 9a-l on multiple
cancer cells with different origin A549, MCF-7 and DLA were investigated following 48 h of
exposure using three independent assay systems (trypan blue, MTT and LDH release). These
methods are the preliminary screening methods to eliminate those analogues that do not show
any cytotoxic and antiproliferative effects. The cells were treated with increasing concentrations
of the compounds (0, 10, 20, 50 and 100 ꢀM in DMSO and vehicle alone). The results inferred
that among the series 9a-l, the compound 9k alone has shown significant cytotoxic and
antiproliferative activities against all the three cancer cell lines and it was chosen as a lead
compound (Fig 1A, 1B and 1C). Thus, our studies using three cell lines of different origin
suggest that irrespective of the cancer type, compound 9k could induce cytotoxicity, as shown by
three independent assay study methods and antitumor activity in-vivo was further investigated.
2.2.2. Structure activity Relationship (SAR)
The benzophenone derivatives are known to be pharmacologically active molecules
against various pathological conditions including cancer [25]. The current investigation involves
the multistep synthesis of diamide-coupled benzophenone compounds. Structurally, the title
compounds are having a basic backbone of benzophenone with acetamide link and terminal
bromo group. The IC50 values for compounds as depicted in Fig 1A, 1B and 1C, which suggest
that the compound 9k with three methyl groups at ortho position in rings A, B and D and bromo
group at the para position in ring E showed IC50 value ~20, 23 and 23ꢀM on A549, MCF-7 and
DLA cells respectively as verified by trypan blue, MTT and LDH release assay. The results
illustrated that, the compounds 9a, 9b, 9d, 9e, 9f and 9g do not have bromo group and compound
9c has two bromo groups at the para position in rings A and E. It reveals that the compound 9k is
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important for biological activity. While the other compounds with different substituents have not
shown significant cytotoxicity. In this connection, the compound 9k was selected as a lead
compound based on its significant structure activity relationship compared with other analogues
and it was further evaluated.
2.2.3. Compound 9k regresses tumor growth
It is a rational and a hierarchical approach beginning with toxicology and pharmacology
studies, progressing to primary tumors to identify therapeutic targets and models of metastatic
disease to compare drugs using rigorous, clinically relevant outcome parameters [26, 27]. Ascites
tumor models of mouse origin is an appropriate model system for preliminary pilot screening and
it plays a decisive role in the drug development process [28] because ascites secreting cell
implantation induces typical tumor microenvironment by inducing local inflammation, increased
vascular permeability and an intense edema formation, cellular migration and invasion.
Accumulated fluid serves as a nutritional source for tumor cells and thereby establishing the
tumor growth. Hence, decrease in ascites fluids accounts for the suppression of tumor growth
[29]. In the present study, we have chosen murine DLA in-vivo model system to study the
preliminary antitumor property of the chosen lead compound 9k to evaluate the antitumor
effect.The compound 9k was administered at the concentration of 75 mg/kg body weight i.p.
(intraperitoneally), on every alternative day starting from 4th day of the tumor implantation as
determined by LD50 assay. Results referred that, upon treatment of 9k compound there was a
decrease in tumor growth as assessed by visible tumor morphology (Fig 2A) and dose dependent
decrease in tumor establishment and it accounts for ~80% inhibition after the final dose of
treatment (Fig 2B). The ascites secretion responsible for establishment of tumor was also low
when compared to the untreated mice (Fig 2C) and cell number (Fig. 2D). The regressed tumor
expanded andsurivality of the mouse was increased upto 2.5 fold time where mice survived 37
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days. Treatment was discontinued for test animals after the 10th day to monitor the survivality
(Fig. 2E).
2.2.4. Compound 9k inhibits neovascularisation
Measurement of micro visual density (MVD) is a widely used surrogate measure in
pathological specimens and tumor models to assess disease progression. Extensive
neovascularization has a direct correlation with tumor progression and its inhibition results in
regression of tumor growth [30, 31]. Earlier, our group showed that benzophenone derivatives
conjugated with various substituents are potent antiangiogenic compounds that inhibit
neovascularization and tumor growth in mice [32]. Treatment with compound 9k effectively
reduced the tumor growth and we verified whether the mechanism of inhibition is linked to
neovascularization since angiogenesis is evident in the inner peritoneal lining of EAC-bearing
mice and thus, mouse peritoneum is a reliable model for angiogenesis [33]. Hence, the peritoneal
lining of mice treated with compound 9k was examined to gauge the effects on peritoneal
angiogenesis. Supporting our hypothesis Tumor-bearing mice treated with 9k compound showed
decreased peritoneal angiogenesis compared to the massive angiogenesis in untreated mice
(Fig.3A). Quantification of the neovessel using angioquant software revealed more than 50%
reduction in MVD after treatment.
To reconfirm and authenticate our results we approached non tumorigenic model and
better validative model fertilized egg CAM [34] where we used recombinant vascular endothelial
growth factor 165 (rVEGF165, 10mg/embryo) as a potent angiogenic factor [35] to induce
neovascularization. Simultaneous treatment with 9k induced a vascular zone in the developing
embryos. Notably, newly formed microvessels regressed around the area of the implanted disk
(Fig. 3B). This indicates that the compound 9k primarily acts to inhibit angiogenesis.
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Angiogenesis plays an important role in tumor growth [36, 37] and our compound 9k
inhibits angiogenesis and it also inhibits tumor growth as well as the accumulation of ascites
fluid in mice, which is angiogenesis dependent (Fig. 2). Since the ascites fluid nutritional
requirement for tumor [38] depletion in ascites fluid accounts for suppression of tumor growth.
The results confirm the effect of compound 9k after i.p. injection was a reduction in the
formation of ascites, which directly impacts the growth of the tumor (Fig. 2). This suggests that
the target-specific action of compound 9k corresponds. Hence, novel studies of our compound
9k have antiproliferative effects against multiple cancer cell lines and angioprevention mediated
tumor growth.
3. Conclusion
Summarizing the current investigation, a new series of diamide-coupled benzophenone
molecules (9a-l) was synthesized and evaluated for cytotoxic and antiproliferative activity
against multiple cancer cells with different origin. From the current investigation, structural
activity relationship of these compounds suggests that the position and the type of subsituents on
the aromatic ring in 9a-l are important for the activity. Compound 9k with three methyl groups at
the ortho position in rings A, B and D and bromo group at the para position in ring E has been
selected as lead compound. The cytotoxic nature of compound 9k further verified against tumor
regressing activity and against murine ascities model DLA resulting in antitumor potency. The
mechanism of antitumor activity is a result of the regressed neovessel formation both in
tumorigenic and non tumorigenic model system. This highlights that the compound 9k is a
promising anticancer molecule with target specific action which could be further adopted into the
drug development process.
4. Materials and methods
4.1 Experimental section
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All solvents and reagents were purchased from Sigma Aldrich Chemicals Pvt Ltd. TLC
was performed on aluminum-backed silica plates and visualized by UV-light. Melting points
(M.P) were determined on an electrically heated VMP-III melting point apparatus. The
elemental analysis of the compounds was performed on a Perkin Elmer 2400 elemental
analyzer. The results of elemental analyses were within ±0.4% of the theoretical values. The
FT-IR spectra were recorded using KBr discs and Nujol on FT-IR Jasco 4100 infrared
spectrophotometer. 1H NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer in
CDCl3 and the chemical shifts were recorded in parts per million downfield from
tetramethylsilane. Mass spectra were recorded on LC-MS (API-4000) mass spectrometer. MTT
was purchased from Sigma Aldrich, USA and CD31 antibodies were procured from Santa Cruz,
USA.
4.2. Chemistry
4.2.1. General procedure for the synthesis of phenyl benzoates 3a-f
The starting substituted benzoates 3a-f were synthesized by benzoylation of subsituted
phenols 1a-c with substituted benzoyl chlorides (2a-d, 1:1) in the presence of 10% sodium
o
hydroxide solution. The reaction mass was stirred for 2-3 hours at 0-5 C. The reaction was
monitored by thin layer chromatography (TLC) using 4:1 n-hexane: ethyl acetate solvent
mixture. After completion of the reaction, the oily product was extracted with ether layer (20 mL
× 3). Ether layer was washed with 10% sodium hydroxide solution (40 mL x 3) followed by
water (30 mL × 3) and then dried over anhydrous sodium sulfate and the solvent was evaporated
under reduced pressure to afford compounds 3a-f.
4.2.1.1. Benzoic acid o-tolyl ester 3a. Yield: 93%. M.P.: 52-54 °C. IR (KBr) νmax (cm-1): 1715
(ester, C=O). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.45 (s, 3H, CH3), 7.11-8.25 (m, 9H, Ar-H).
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LC-MS m/z 213 (M+1). Anal. Cal. for C14H12O2 (212): C, 79.22; H, 5.70. Found: C, 79.20; H,
5.76%.
4.2.1.2. Benzoic acid 2,5-dimethyl-phenyl ester 3b. Yield: 80%. M.P.: 65-67 °C. IR (KBr) νmax
1
(cm-1): 1715 (ester, C=O). H NMR (400 MHz) (CDCl3) δ (ppm): 2.41 (s, 6H, CH3), 7.21-8.26
(m, 8H, Ar-H). LC-MS m/z 227 (M+1). Anal. Cal. for C15H14O2 (227): C, C, 79.62; H, 6.24.
Found: C, 79.60; H, 6.19%.
4.2.1.3. 4-Bromo-benzoic acid o-tolyl ester 3c. Yield: 91%. M.P.: 60-62 °C. IR (KBr) νmax (cm-
1
1): 1715 (ester, C=O). H NMR (400 MHz) (CDCl3) δ (ppm): 2.45 (s, 3H, CH3), 7.11-8.25 (m,
8H, Ar-H). LC-MS m/z 292 (M+1). Anal. Cal. for C14H11BrO2 (291): C, 57.76; H, 3.81. Found:
C, 57.71; H, 3.75%.
4.2.1.4. 4-Chloro-benzoic acid 2-chloro-6-fluoro-phenyl ester 3d. Yield: 85%. M.P.: 52-54 °C.
1
IR (KBr) νmax (cm-1): 1715 (ester, C=O). H NMR (400 MHz) (CDCl3) δ (ppm): 2.37 (s, 3H,
CH3), 7.01-8.18 (m, 7H, Ar-H). LC-MS m/z 286 (M+1). Anal. Cal. for C13H7Cl2FO2 (285): C,
54.77; H, 2.47. Found: C, 54.72; H, 2.41%.
4.2.1.5. 4-Methyl-benzoic acid o-tolyl ester 3e. Yield: 90%. M.P.: 54-56 °C. IR (KBr) νmax (cm-
1
1): 1715 (ester, C=O). H NMR (400 MHz) (CDCl3) δ (ppm): 2.35 (s, 6H, CH3), 7.15-8.30 (m,
8H, Ar-H). LC-MS m/z 227 (M+1). Anal. Cal. for C15H14O2 (226): C, 79.62; H, 6.24. Found: C,
79.52; H, 6.15%.
4.2.1.6. 2-Methyl-benzoic acid o-tolyl ester 3f. Yield: 83%. M.P.: 59-61 °C. IR (KBr) νmax (cm-
1
1): 1715 (ester, C=O). H NMR (400 MHz) (CDCl3) δ (ppm): 2.45 (s, 6H, CH3), 7.11-8.25 (m,
8H, Ar-H). LC-MS m/z 227 (M+1). Anal. Cal. for C15H14O2 (226): C, 79.62; H, 6.24. Found: C,
79.58; H, 6.20%.
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4.2.2. General procedure for the synthesis of (4-hydroxy phenyl) phenyl methanones 4a-f
Substituted (4-hydroxy phenyl) phenyl methanone commonly known as hydroxy
benzophenones 4a-f were synthesized by Fries rearrangement. Compounds 3a-f (0.001 mol) and
aluminium chloride (0.002 mol) were blended and the mixture was heated upto 150-170 °C
without using solvent condition for about 2-3 hours. Then the reaction mixture was cooled to
0°C and quenched with 6N hydrochloric acid in the presence of ice water. The reaction mixture
was stirred for about 2-3 hours. The solid was filtered and recrystallized with ethanol to obtain
compounds 4a-f.
4.2.2.1. (4-Hydroxy-3-methyl-phenyl)-phenyl-methanone 4a. Yield: 75%; M.P.: 110-112 °C: IR
1
(KBr) νmax (cm-1): 1640 (C=O), 3510-3600 cm-1 (OH); H NMR (400 MHz) (CDCl3) δ (ppm):
2.35 (s, 3H, CH3), 6.73-7.68 (m, 8H, Ar-H), 12.0 (bs, 1H, OH). LC-MS m/z 213 (M+1). Anal.
Cal. data for C14H12O2 (212): C, 79.22; H, 5.70. Found: C, 79.18; H, 5.69%.
4.2.2.2. (4-Hydroxy-2,5-dimethyl-phenyl)-phenyl-methanone 4b. Yield: 82%; M.P.: 120-122 °C:
1
IR (KBr) νmax (cm-1): 1660 (C=O), 3515-3600 cm-1 (OH); H NMR (400 MHz) (CDCl3) δ
(ppm): 2.35 (s, 3H, CH3), 6.61-7.70 (m, 8H, Ar-H), 11.80 (bs, 1H, OH). LC-MS m/z 227 (M+1).
Anal. Cal. data for C15H14O2 (226): C, 79.62; H, 6.24. Found: C, 79.57; H, 6.20%.
4.2.2.3. (4-Bromo-phenyl)-(4-hydroxy-3-methyl-phenyl)-methanone 4c. Yield: 85%; M.P.: 153-
156 °C: IR (KBr) νmax (cm-1): 1645 (C=O), 3510-3600 cm-1 (OH); 1H NMR (400 MHz) (CDCl3)
δ (ppm): 2.35 (s, 3H, CH3), 6.51-7.60 (m, 7H, Ar-H), 12.0 (bs, 1H, OH). LC-MS m/z 292 (M+1).
Anal. Cal. data for C14H11BrO2 (291): C, 57.76; H, 3.81. Found: C, 57.71; H, 3.72%.
4.2.2.4. (3-Chloro-5-fluoro-4-hydroxy-phenyl)-(4-chloro-phenyl)-methanone 4d. Yield: 78%;
1
M.P.: 147-149 °C: IR (KBr) νmax (cm-1): 1640 (C=O), 3510-3615 cm-1 (OH); H NMR (400
MHz) (CDCl3) δ (ppm): 6.71-7.70 (m, 6H, Ar-H), 11.90 (bs, 1H, OH). LC-MS m/z 286 (M+1).
Anal. Cal. data for C13H7Cl2FO2 (285): C, 54.77; H, 2.47. Found: C, 54.67; H, 2.38%.
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4.2.2.5. (4-Hydroxy-3-methyl-phenyl)-p-tolyl-methanone 4e. Yield: 80%; M.P.: 150-153 °C: IR
1
(KBr) νmax (cm-1): 1650 (C=O), 3520-3620 cm-1 (OH); H NMR (400 MHz) (CDCl3) δ (ppm):
2.35 (s, 6H, CH3), 7.10-7.70 (m, 7H, Ar-H), 12.20 (bs, 1H, OH). LC-MS m/z 227 (M+1). Anal.
Cal. data for C15H14O2 (226): C, 79.62; H, 6.24. Found: C, 79.60; H, 6.17%.
4.2.2.6. (4-Hydroxy-3-methyl-phenyl)-o-tolyl-methanone 4f. Yield: 72%; M.P.: 125-128 °C: IR
1
(KBr) νmax (cm-1): 1640 (C=O), 3510-3600 cm-1 (OH); H NMR (400 MHz) (CDCl3) δ (ppm):
2.35 (s, 6H, CH3), 6.73-7.60 (m, 7H, Ar-H), 11.60 (bs, 1H, OH). LC-MS m/z 227 (M+1). Anal.
Cal. data for C15H14O2 (226): C, 79.62; H, 6.24. Found: C, 79.59; H, 6.18%.
4.2.3. General procedure for the synthesis of (4-benzoyl phenoxy)-acetic acid ethyl ester 5a-f
Compounds 5a-f were obtained by refluxing a mixture of compounds 4a-f (0.013 mol)
and ethyl chloroacetate (0.026 mol) in dry acetone (50 mL) and anhydrous potassium carbonate
(0.019 mol) for 8-10 hours. 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 (50 mL × 3). The ether layer was washed with 10% sodium hydroxide
solution (50 mL × 3) followed by water (30 mL × 3) and then dried over anhydrous sodium
sulfate and evaporated to dryness to obtain crude solid, which when recrystallised with ethanol,
afforded compounds 5a-f.
4.2.3.1. (4-Benzoyl-2-methyl-phenoxy)-acetic acid ethyl ester 5a. Yield: 92%. M.P.: 49-51 oC. IR
(KBr) νmax (cm-1): 1664 (C=O), 1760 (ester, C=O). 1H NMR (400 MHz) (CDCl3) δ (ppm): 1.2 (t,
3H, CH3 of ester), 2.3 (s, 3H, CH3), 4.1 (q, 2H, CH2 of ester), 4.5 (s, 2H, OCH2), 7.1-7.7 (m, 8H,
Ar-H). LC-MS m/z 299 (M+1). Anal. Cal. for C18H18O4 (298): C, 72.48; H, 6.04. Found: C,
72.46; H, 6.02%.
4.2.3.2. (4-Benzoyl-2,5-dimethyl-phenoxy)-acetic acid ethyl ester 5b. Yield: 83%. M.P.: 53-55
oC. IR (KBr) νmax (cm-1): 1664 (C=O), 1760 (ester, C=O). 1H NMR (400 MHz) (CDCl3) δ (ppm):
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1.2 (t, 3H, CH3 of ester), 2.3 (s, 6H, CH3), 4.3 (q, 2H, CH2 of ester), 4.5 (s, 2H, OCH2), 6.9-7.8
(m, 7H, Ar-H). LC-MS m/z 313 (M+1). Anal. Cal. for C19H20O4 (312): C, 73.06; H, 6.45. Found:
C, 73.02; H, 6.41%.
4.2.3.3. [4-(4-Bromo-benzoyl)-2-methyl-phenoxy]-acetic acid ethyl ester 5c. Yield: 92%. M.P.:
47-49 oC. IR (KBr) νmax (cm-1): 1664 (C=O), 1760 (ester, C=O). 1H NMR (400 MHz) (CDCl3) δ
(ppm): 1.2 (t, 3H, CH3 of ester), 2.3 (s, 3H, CH3), 4.4 (q, 2H, CH2 of ester), 4.5 (s, 2H, OCH2),
6.7-7.7 (m, 7H, Ar-H). LC-MS m/z 378 (M+1). Anal. Cal. for C18H17BrO4 (377): C, 57.31; H,
4.54. Found: C, 57.27; H, 4.49%.
4.2.3.4. [2-Chloro-4-(4-chloro-benzoyl)-6-fluoro-phenoxy]-acetic acid ethyl ester 5d. Yield:
o
1
90%. M.P.: 55-57 C. IR (KBr) νmax (cm-1): 1664 (C=O), 1760 (ester, C=O). H NMR (400
MHz) (CDCl3) δ (ppm): 1.3 (t, 3H, CH3 of ester), 4.1 (q, 2H, CH2 of ester), 4.8 (s, 2H, OCH2),
7.1-7.7 (m, 6H, Ar-H). LC-MS m/z 372 (M+1). Anal. Cal. for C17H13Cl2FO4 (371): C, 55.01; H,
3.53. Found: C, 54.95; H, 3.48%.
4.2.3.5. [2-Chloro-4-(4-chloro-benzoyl)-6-fluoro-phenoxy]-acetic acid ethyl ester 5e. Yield:
o
1
91%. M.P.: 47-49 C. IR (KBr) νmax (cm-1): 1664 (C=O), 1760 (ester, C=O). H NMR (400
MHz) (CDCl3) δ (ppm): 1.2 (t, 3H, CH3 of ester), 2.4 (s, 6H, CH3), 4.1 (q, 2H, CH2 of ester), 4.5
(s, 2H, OCH2), 7.1-7.7 (m, 7H, Ar-H). LC-MS m/z 313 (M+1). Anal. Cal. for C19H20O4 (312): C,
73.06; H, 6.45. Found: C, 73.01; H, 6.39%.
4.2.3.6. [2-Methyl-4-(2-methyl-benzoyl)-phenoxy]-acetic acid ethyl ester 5f. Yield: 89%. M.P.:
50-52 oC. IR (KBr) νmax (cm-1): 1664 (C=O), 1760 (ester, C=O). 1H NMR (400 MHz) (CDCl3) δ
(ppm): 1.3 (t, 3H, CH3 of ester), 2.3 (s, 6H, CH3), 4.2 (q, 2H, CH2 of ester), 4.9 (s, 2H, OCH2),
6.8-7.6 (m, 7H, Ar-H). LC-MS m/z 313 (M+1). Anal. Cal. for C19H20O4 (312): C, 73.06; H, 6.45.
Found: C, 73.02; H, 6.52%.
14

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4.2.4. General procedure for the synthesis of (4-benzoyl phenoxy)-acetic acid 6a-f
Compounds 5a-f (6.0 mmol) were dissolved in ethanol (15 mL) and treated with a
solution of sodium hydroxide (15 mmol) in water (5 mL). The reaction mixture was refluxed
for 5-6 h, cooled and acidified with 1N hydrochloric acid. The precipiatae was filtered, washed
with water and recrystallised with methanol to afford compounds 6a-f in a good yield.
4.2.4.1. (4-Benzoyl-2-methyl-phenoxy)-acetic acid 6a. Yield: 82%. M.P.: 130-132 oC. IR (KBr)
νmax (cm-1): 1675 (C=O), 1730 (acid, C=O), 3400-3500(acid OH). 1H NMR (400 MHz) (CDCl3)
δ (ppm): 2.3 (s, 3H, CH3), 4.61 (s, 2H, OCH2), 7.2-7.7 (m, 8H, Ar-H), 9.5 (s, 1H, COOH). LC-
MS m/z 271 (M+1). Anal. Cal. for C16H14O4 (270): C, 71.10; H, 5.22. Found: C, 71.06; H,
5.20%.
o
4.2.4.2. (4-Benzoyl-2,5-dimethyl-phenoxy)-acetic acid 6b. Yield: 88%. M.P.: 153-155 C. IR
1
(KBr) νmax (cm-1): 1660 (C=O), 1738 (acid, C=O), 3410-3540(acid OH). H NMR (400 MHz)
(CDCl3) δ (ppm): 2.4 (s, 6H, CH3), 4.53 (s, 2H, OCH2), 7.1-7.6 (m, 8H, Ar-H), 9.4 (s, 1H,
COOH). LC-MS m/z 285 (M+1). Anal. Cal. for C17H16O4 (284): C, 71.82; H, 5.67. Found: C,
71.78; H, 5.62%.
4.2.4.3. [4-(4-Bromo-benzoyl)-2-methyl-phenoxy]-acetic acid 6c. Yield: 93%. M.P.: 141-143 oC.
IR (KBr) νmax (cm-1): 1615 (C=O), 1760 (acid, C=O), 3465-3575(acid OH). 1H NMR (400 MHz)
(CDCl3) δ (ppm): 2.2 (s, 3H, CH3), 4.41 (s, 2H, OCH2), 7.1-7.8 (m, 7H, Ar-H), 9.7 (s, 1H,
COOH). LC-MS m/z 350 (M+1). Anal. Cal. for C16H13BrO4 (349): C, 55.04; H, 3.75. Found: C,
55.01; H, 3.73%.
4.2.4.4. [2-Chloro-4-(4-chloro-benzoyl)-6-fluoro-phenoxy]-acetic acid 6d. Yield: 89%. M.P.:
o
1
137-139 C. IR (KBr) νmax (cm-1): 1640 (C=O), 1750 (acid, C=O), 3480-3585(acid OH). H
NMR (400 MHz) (CDCl3) δ (ppm): 2.3 (s, 3H, CH3), 4.46 (s, 2H, OCH2), 6.9-7.7 (m, 8H, Ar-H),
15

ACCEPTED MANUSCRIPT
9.5 (s, 1H, COOH). LC-MS m/z 344 (M+1). Anal. Cal. for C16H14Cl2FO4 (343): C, 52.50; H,
2.64. Found: C, 52.47; H, 2.61%.
4.2.4.5. [2-Methyl-4-(4-methyl-benzoyl)-phenoxy]-acetic acid 6e. Yield: 90%. M.P.: 119-121 oC.
IR (KBr) νmax (cm-1): 1665 (C=O), 1735 (acid, C=O), 3470-3580(acid OH). 1H NMR (400 MHz)
(CDCl3) δ (ppm): 2.3 (s, 6H, CH3), 4.39 (s, 2H, OCH2), 6.8-7.5 (m, 7H, Ar-H), 9.7 (s, 1H,
COOH). LC-MS m/z 285 (M+1). Anal. Cal. for C17H16O4 (284): C, 71.82; H, 5.67. Found: C,
71.79; H, 5.64%.
4.2.4.6. [2-Methyl-4-(2-methyl-benzoyl)-phenoxy]-acetic acid 6f. Yield: 85%. M.P.: 124-126 oC.
IR (KBr) νmax (cm-1): 1630 (C=O), 1755 (acid, C=O), 3470-3575(acid OH). 1H NMR (400 MHz)
(CDCl3) δ (ppm): 2.5 (s, 6H, CH3), 4.40 (s, 2H, OCH2), 6.9-7.7 (m, 7H, Ar-H), 9.6 (s, 1H,
COOH). LC-MS m/z 285 (M+1). Anal. Cal. for C17H16O4 (284): C, 71.82; H, 5.67. Found: C,
71.80; H, 5.63%.
4.2.5. General procedure for the synthesis of N-(2-amino-phenyl)-2-(4-benzoyl-phenoxy)-
acetamide 8a-f
To compounds 6a-f (0.0037 mol) in dry dichloromethane (15 mL), lutidine (1.2 vol.) was
added at 25-30oC , followed by the addition of 1,2 diaminobenzene (7, 0.0037 mol). The reaction
o
mixture was stirred at 25-30o C for 30 min. The reaction was cooled to 0-5 C, TBTU (0.0037
mol) was added over a period of 30 min while maintaining the temperature below 5o C. The
reaction was strirred overnight and monitored by TLC using hexane and ethyl acetate (4:1). The
reaction mixture was diluted with 20 mL of dichloromethane and treated with 1.5 N HCl solution
(20 mL). The organic layer was washed with water (25 mL × 3) and brine (25 mL × 3). Finally,
the organic layer was dried over anhydrous sodium sulfate and concentrated to afford
compounds 8a-f.
16

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4.2.5.1. N-(2-amino-phenyl)-2-(4-benzoyl-2-methyl-phenoxy)-acetamide 8a. Yield: 92%. M.P.:
o
190-192 C. IR (KBr) νmax (cm-1): 1675 (C=O), 1730 (amide, C=O), 3120-3220 (amide CO-
1
NH). H NMR (400 MHz) (CDCl3) δ (ppm): 2.2 (s, 3H, CH3), 4.74 (s, 2H, NH2), 4.91 (s, 2H,
OCH2), 6.5-7.6 (m, 12H, Ar-H), 9.2 (s, 1H, NH). LC-MS m/z 361 (M+1). Anal. Cal. for
C22H20N2O3 (360): C, 73.32; H, 5.59. Found: C, 73.26; H, 5.48%.
4.2.5.2. N-(2-amino-phenyl)-2-(4-benzoyl-2,5-dimethyl-phenoxy)-acetamide 8b. Yield: 73%.
o
M.P.: 148-150 C. IR (KBr) νmax (cm-1): 1675 (C=O), 1730 (amide, C=O), 3120-3220 (amide
1
CO-NH). H NMR (400 MHz) (CDCl3) δ (ppm): 2.35 (s, 6H, CH3), 4.6 (s, 2H, NH2), 4.88 (s,
2H, OCH2), 6.7-7.5 (m, 11H, Ar-H), 9.1 (s, 1H, NH). LC-MS m/z 375(M+1). Anal. Cal. for
C23H22N2O3 (374): C, 73.78; H, 5.92. Found: C, 73.68; H, 5.75%.
4.2.5.3. N-(2-amino-phenyl)-2-[4-(4-bromo-benzoyl)-2-methyl-phenoxy]-acetamide 8c. Yield:
o
95%. M.P.: 185-187 C. IR (KBr) νmax (cm-1): 1675 (C=O), 1730 (amide, C=O), 3120-3220
(amide CO-NH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.3 (s, 3H, CH3), 4.7 (s, 2H, NH2), 4.93
(s, 2H, OCH2), 6.6-7.8 (m, 11H, Ar-H), 9.2 (s, 1H, NH). LC-MS m/z 440 (M+1 Anal. Cal. for
C22H19BrN2O3 (439): C, 60.15; H, 4.36. Found: C, 60.07; H, 4.28%.
4.2.5.4. N-(2-amino-phenyl)-2-[2-chloro-4-(4-chloro-benzoyl)-6-fluoro-phenoxy]-acetamide 8d.
o
Yield: 67%. M.P.: 154-156 C. IR (KBr) νmax (cm-1): 1675 (C=O), 1730 (amide, C=O), 3120-
3220 (amide CO-NH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.4 (s, 3H, CH3), 4.6 (s, 2H, NH2),
4.9 (s, 2H, OCH2), 6.7-7.9 (m, 12H, Ar-H), 9.6 (s, 1H, NH). LC-MS m/z 434 (M+1). Anal. Cal.
for C21H15Cl2FN2O3 (433): C, 58.22; H, 3.49. Found: C, 58.17; H, 3.48%.
4.2.5.5. N-(2-amino-phenyl)-2-[2-methyl-4-(4-methyl-benzoyl)-phenoxy]-acetamide 8e. Yield:
o
89%. M.P.: 180-182 C. IR (KBr) νmax (cm-1): 1675 (C=O), 1730 (amide, C=O), 3120-3220
(amide CO-NH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.5 (s, 6H, CH3), 4.4 (s, 2H, NH2), 4.7
17

ACCEPTED MANUSCRIPT
(s, 2H, OCH2), 6.7-7.8 (m, 11H, Ar-H), 9.5 (s, 1H, NH). LC-MS m/z 375 (M+1) Anal. Cal. for
C23H22N2O3 (374): C, 73.78; H, 5.92. Found: C, 73.76; H, 5.89%.
4.2.5.6. N-(2-amino-phenyl)-2-[2-methyl-4-(2-methyl-benzoyl)-phenoxy]-acetamide 8f. Yield:
o
93%. M.P.: 187-189 C. IR (KBr) νmax (cm-1): 1670 (C=O), 1740 (amide, C=O), 3130-3230
(amide CO-NH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.3 (s, 6H, CH3), 4.5 (s, 2H, NH2), 4.8
(s, 2H, OCH2), 6.6-7.5 (m, 11H, Ar-H), 9.3 (s, 1H, NH). LC-MS m/z 375 (M+1). Anal. Cal. for
C23H22N2O3 (374): C, 73.78; H, 5.92. Found: C, 73.75; H, 5.84%.
4.2.6. General procedure for the synthesis of 2-(4-benzoyl-phenoxy)-N-{2-[2-(4-benzoyl-
phenoxy)-acetylamino]-phenyl}-acetamide 9a-l.
To compounds 8a-f (0.001 mol) in dry dichloromethane (15 mL), substituted (4-benzoyl-
o
phenoxy)-acetic acids 6a-f were added at 25-30 C, followed by the addition of lutidine (0.001
o
mol). The reaction mixture was stirred at 25-30 C for 30 min. The reaction was cooled to 0-5
oC, TBTU (0.001 mol) was added over a period of 30 min while maintaining the temperature
o
below 5 C. The reaction was strirred overnight and monitored by TLC using hexane and ethyl
acetate (4:1). The reaction mixture was diluted with 20 mL of dichloromethane and treated with
10% sodium bicarbonate solution (20 mL × 3). The organic layer was washed with water (25
mL × 3), dried over anhydrous sodium sulfate and concentrated to yield compounds 9a-l.
4.2.6.1. 2-(4-Benzoyl-2-methyl-phenoxy)-N-{2-[2-(4-benzoyl-2-methyl-phenoxy)-acetylamino]-
phenyl}-acetamide 9a. Yield: 82%. M.P.: 90-92o C; IR (KBr) νmax (cm-1): 1675 (C=O), 1730
1
(amide, C=O), 3120-3220 (amide CO-NH). H NMR (400 MHz) (CDCl3) δ (ppm): 2.27 (s, 6H,
CH3), 4.78 (s, 4H, OCH2), 6.9-7.6 (m, 20H, Ar-H), 9.6 (s, 2H, NH). 13C NMR (DMSO-d6) δ:
194.8, 167.0, 159.7, 138.2, 132.5, 132.4, 130.7, 130.4, 130.1, 129.7, 129.6, 128.8, 128.7, 127.0,
126.3, 126.0, 111.2, 67.5, 16.5. LC-MS m/z 613 (M+1). Anal. Cal. for C38H32N2O6 (612): C,
74.49; H, 5.26; N, 4.57. Found: C, 74.37; H, 5.19; N, 4.40%.
18

ACCEPTED MANUSCRIPT
4.2.6.2.
2-(4-Benzoyl-2,5-dimethyl-phenoxy)-N-{2-[2-(4-benzoyl-2,5-dimethyl-phenoxy)-
o
acetylamino]-phenyl}-acetamide 9b. Yield: 56%. M.P.: 93-95 C. IR (KBr) νmax (cm-1): 1675
(C=O), 1730 (amide, C=O), 3120-3220 (amide CO-NH).1H NMR (400 MHz) (CDCl3) δ (ppm):
2.35 (s, 12H, CH3), 4.78 (s, 4H, OCH2), 6.5-7.6 (m, 18H, Ar-H), 9.3 (s, 2H, NH). 13C NMR
(DMSO-d6) δ: 194.6, 165.6, 162.8, 139.5, 138.5, 135.2, 132.4, 131.5, 129.7, 128.6, 124.5, 122.3,
117.8, 68.4, 19.2, 16.6. LC-MS m/z 641 (M+1). Anal. Cal. for C40H36N2O6 (640): C, 74.49; H,
5.26; N, 4.57. Found: C, 74.40; H, 5.21; N, 4.49%.
4.2.6.3.
2-[4-(4-Bromo-benzoyl)-2-methyl-phenoxy]-N-(2-{2-[4-(4-bromo-benzoyl)-2-methyl-
o
phenoxy]-acetylamino}-phenyl)-acetamide 9c. Yield: 58%. M.P.: 86-88 C. IR (KBr) νmax (cm-
1): 1675 (C=O), 1730 (amide, C=O), 3120-3220 (amide CO-NH). 1H NMR (400 MHz) (CDCl3)
δ (ppm): 2.32 (s, 6H, CH3), 4.82 (s, 4H, OCH2), 6.7-7.8 (m, 18H, Ar-H), 9.5 (s, 2H, NH). 13C
NMR (DMSO-d6) δ: 194.3, 165.8, 162.5, 139.7, 134.2, 132.3, 130.0, 129.5, 128.3, 128.1, 126.8,
124.5, 121.9, 116.8, 65.8, 15.9. LC-MS m/z 771 (M+1). Anal. Cal. for C38H30Br2N2O6 (770): C,
59.24; H, 3.92; N, 3.64. Found: C, 59.20; H, 3.81; N, 3.53%.
4.2.6.4. N-{2-[2-(4-benzoyl-2,5-dimethyl-phenoxy)-acetylamino]-phenyl}-2-(4-benzoyl-2-methyl-
phenoxy)-acetamide 9d. Yield: 79%. M.P.: 94-96o C. IR (KBr) νmax cm-11675 (C=O), 1730
1
(amide, C=O), 3120-3220 (amide CO-NH). H NMR (400 MHz) (CDCl3) δ (ppm): 2.44 (s, 9H,
CH3), 4.76 (s, 4H, OCH2), 6.8-7.6 (m, 19H, Ar-H), 9.6 (s, 2H, NH). 13C NMR (DMSO-d6) δ:
194.2, 165.3, 162.3, 161.2, 139.5, 138.7, 136.5, 135.2, 134.8, 132.7, 131.9, 131.2, 128.7, 126.5,
124.3, 123.3, 122.8, 121.5, 120.7, 118.6, 114.2, 73.2, 20.1, 16.3. LC-MS m/z 627 (M+1). Anal.
Cal. for C39H34N2O6 (626): C, 74.74; H, 5.47; N, 4.47; O. Found: C, 74.40; H, 5.21; N, 4.49%.
4.2.6.5. N-{2-[2-(4-benzoyl-2-methyl-phenoxy)-acetylamino]-phenyl}-2-[2-chloro-4-(4-chloro-
benzoyl)-6-fluoro-phenoxy]-acetamide 9e. Yield: 68%. M.P.: 83-85o C. IR (KBr) νmax (cm-1):
19

ACCEPTED MANUSCRIPT
1
1675 (C=O), 1730 (amide, C=O), 3120-3220 (amide CO-NH). H NMR (400 MHz) (CDCl3) δ
(ppm): 2.41 (s, 3H, CH3), 4.82 (s, 4H, OCH2), 6.7-7.7 (m, 18H, Ar-H), 9.5 (s, 2H, NH). 13C
NMR (DMSO-d6) δ: 194.7, 164.9, 163.3, 152.6, 145.8, 137.5, 136.2, 135.4, 133.9, 133.5, 132.4,
131.7, 130.4, 129.8, 128.5, 127.7, 126.2, 124.2, 123.5, 122.9, 121.5, 120.2, 119.8, 117.3, 116.4,
65.2, 62.8, 15.9. LC-MS m/z 686 (M+1). Anal. Cal. for C37H27Cl2FN2O6 (685): C, 68.83; H,
3.97; N, 4.09. Found: C, 68.75; H, 3.87; N, 4.02%.
4.2.6.6. N-{2-[2-(4-benzoyl-2-methyl-phenoxy)-acetylamino]-phenyl}-2-[2-methyl-4-(2-methyl-
benzoyl)-phenoxy]-acetamide 9f. Yield: 90%. M.P.: 95-97o C. IR (KBr) νmax (cm-1): 1675
(C=O), 1730 (amide, C=O), 3120-3220 (amide CO-NH). 1H NMR (400 MHz) (CDCl3) δ (ppm):
2.35 (s, 9H, CH3), 4.69 (s, 4H, OCH2), 6.5-7.7 (m, 19H, Ar-H), 9.6 (s, 2H, NH). 13C NMR
(DMSO-d6) δ: 193.7, 168.7, 165.3, 140.5, 138.3, 137.7, 135.8, 133.2, 132.2, 131.8, 130.9, 129.2,
128.5, 127.5, 127.1, 124.3, 122.8, 121.6, 119.5, 70.5, 19.3, 16.1. LC-MS m/z 627 (M+1). Anal.
Cal. for C39H34N2O6 (626): C, 74.74; H, 5.47; N, 4.47. Found: C, 74.68; H, 5.41; N, 4.39%.
4.2.6.7. N-{2-[2-(4-benzoyl-2-methyl-phenoxy)-acetylamino]-phenyl}-2-[2-methyl-4-(4-methyl-
benzoyl)-phenoxy]-acetamide 9g. Yield: 62%. M.P.: 89-90o C. IR (KBr) νmax (cm-1): 1675
(C=O), 1730 (amide, C=O), 3120-3220 (amide CO-NH). 1H NMR (400 MHz) (CDCl3) δ (ppm):
2.33 (s, 9H, CH3), 4.84 (s, 4H, OCH2), 6.7-7.8 (m, 19H, Ar-H), 9.7 (s, 2H, NH). 13C NMR
(DMSO-d6) δ: 193.4, 166.5, 161.8, 140.4, 136.5, 133.8, 133.2, 132.1, 131.4, 130.3, 129.6, 128.6,
127.5, 126.9, 123.6, 122.0, 119.7, 113.3, 69.4, 21.3, 16.3. LC-MS m/z 627 (M+1). Anal. Cal. for
C39H34N2O6 (626): C, 74.74; H, 5.47; N, 4.47. Found: C, 74.71; H, 5.35; N, 4.37%.
4.2.6.8. N-{2-[2-(4-benzoyl-3-methyl-phenoxy)-acetylamino]-phenyl}-2-[4-(4-bromo-benzoyl)-2-
methyl-phenoxy]-acetamide 9h. Yield: 59%. M.P.: 87-89 o C; IR (KBr) νmax (cm-1): 1675 (C=O),
1730 (amide, C=O), 3120-3220 (amide CO-NH). 1H NMR (400 MHz) (CDCl3) δ (ppm): 2.31 (s,
20

ACCEPTED MANUSCRIPT
6H, CH3), 4.61 (s, 2H, OCH2), 6.5-7.6 (m, 19H, Ar-H), 9.5 (s, 2H, NH). 13C NMR (DMSO-d6) δ:
193.5, 167.3, 166.8, 139.2, 138.5, 136.3, 135.1, 134.9, 133.5, 132.8, 131.5, 130.4, 129.7,
128.2,126.5, 125.1, 124.8, 123.6, 122.1, 120.3, 119.7, 65.2, 16.7. LC-MS m/z 692 (M+1). Anal.
Cal. for C38H31N2 BrO6 (691): C, 66.00; H, 4.52; N, 4.05. Found: C, 65.96; H, 4.21; N, 4.02%.
4.2.6.9.
N-{2-[2-(4-benzoyl-2,5-dimethyl-phenoxy)-acetylamino]-phenyl}-2-[4-(4-bromo-
benzoyl)-2-methyl-phenoxy]-acetamide 9i. Yield: 83%. M.P.: 91-93o C. IR (KBr) νmax (cm-1):
1
1675 (C=O), 1730 (amide, C=O), 3120-3220 (amide CO-NH). H NMR (400 MHz) (CDCl3) δ
(ppm): 2.45 (s, 9H, CH3), 4.72 (s, 4H, OCH2), 6.4-7.5 (m, 18H, Ar-H), 9.7 (s, 2H, NH). 13C
NMR (DMSO-d6) δ: 194.3, 166.2, 165.2, 164.7, 142.9, 140.3, 138.5, 137.3, 136.2, 133.8, 132.1,
131.7, 130.5, 128.2, 127.3, 126.7, 124.2, 123.8, 122.1, 121.5, 120.7, 119.2, 118.5, 115.3, 69.7,
18.2, 15.6. LC-MS m/z 706 (M+1). Anal. Cal. for C39H33BrN2O6 (705): C, 66.39; H, 4.71; N,
3.97. Found: C, 66.12; H, 4.68; N, 3.82%.
4.2.6.10. N-(2-{2-[4-(4-bromo-benzoyl)-2-methyl-phenoxy]-acetylamino}-phenyl)-2-[2-chloro-4-
o
(4-chloro-benzoyl)-6-fluoro-phenoxy]-acetamide 9j. Yield: 76%. M.P.: 80-82 C. IR (KBr) νmax
1
(cm-1): 1675 (C=O), 1730 (amide, C=O), 3120-3220 (amide CO-NH). H NMR (400 MHz)
(CDCl3) δ (ppm): 2.31 (s, 3H, CH3), 4.81 (s, 2H, OCH2), 6.8-7.7 (m, 17H, Ar-H), 9.6 (s, 2H,
NH). 13C NMR (DMSO-d6) δ: 194.8, 168.3, 161.2, 154.2, 147.6, 136.8, 134.2, 133.9, 133.1,
132.5, 132.3, 131.5, 130.7, 130.2, 130.1, 129.6, 128.8, 127.9, 127.0, 126.3, 124.0, 118.7, 115.2,
111.2, 110.5, 68.3, 67.5, 16.5. LC-MS m/z 765(M+1). Anal. Cal. for C37H26BrCl2FN2O6 (764):
C, 58.14; H, 3.43; N, 3.66. Found: C, 58.08; H, 3.41; N, 3.63%.
4.2.6.11. N-(2-{2-[4-(4-bromo-benzoyl)-2-methyl-phenoxy]-acetylamino}-phenyl)-2-[2-methyl-4-
o
(2-methyl-benzoyl)-phenoxy]-acetamide 9k. Yield: 85%. M.P.: 94-96 C. IR (KBr) νmax (cm-1):
1
1675 (C=O), 1730 (amide, C=O), 3120-3220 (amide CO-NH). H NMR (400 MHz) (CDCl3) δ
(ppm): 2.44 (s, 9H, CH3), 4.79 (s, 4H, OCH2), 6.8-7.7 (m, 18H, Ar-H), 9.7 (s, 2H, NH). 13C
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NMR (DMSO-d6) δ: 194.6, 166.3, 164.2, 155.6, 150.3, 143.5, 138.4, 136.9, 134.2, 133.5, 132.1,
131.3, 130.7, 129.9, 128.2, 128.6, 125.3, 124.2, 122.7, 121.6, 120.5, 119.8, 117.5, 116.3, 67.9,
66.3, 16.3. LC-MS m/z 706 (M+1). Anal. Cal. for C39H33BrN2O6 (705): C, 66.39; H, 4.71; N,
3.97. Found: C, 66.32; H, 4.63; N, 3.86%.
4.2.6.12. N-(2-{2-[4-(4-bromo-benzoyl)-2-methyl-phenoxy]-acetylamino}-phenyl)-2-[2-methyl-4-
(4-methyl-benzoyl)-phenoxy]-acetamide 9l. Yield: 56%. M.P.:102-104 oC. IR (KBr) νmax (cm-1):
1
1675 (C=O), 1730 (amide, C=O), 3120-3220 (amide CO-NH). H NMR (400 MHz) (CDCl3) δ
(ppm): 2.42 (s, 9H, CH3), 4.81 (s, 4H, OCH2), 6.7-7.6 (m, 18H, Ar-H), 9.7 (s, 2H, NH). 13C
NMR (DMSO-d6) δ: 194.2, 166.9, 165.3, 138.5, 136.2, 134.4, 133.6, 132.5, 131.4, 130.4, 129.8,
128.5, 127.7, 126.2, 124.1, 122.7, 121.9, 120.2, , 118.3, 116.4, 65.2, 19.5, 15.3.LC-MS m/z 706
(M+1). Anal. Cal. for C39H33BrN2O6 (705): C, 66.39; H, 4.71; N, 3.97. Found: C, 66.35; H, 4.68;
N, 3.93%.
4.3. Biology
4.3.1. Cell culture and in-vitro compound treatment
Three cell lines from different origin, namely, human lung epithelial carcinoma (A549),
human epithelial adenocarcinoma (MCF-7) and Murine Daltons lymphoma (DLA) cells were
cultured in DMEM (Gibco-Invitrogen, USA), with 10% FBS (In vitrogen, USA), with
necessary antibiotic and antimicotic (Sigma-Aldrich, USA) agents, were maintained and
subjected to treatment with varying concentrations of compounds 9a–l (0, 10, 20,50, and 100
µM in DMSO) for various time intervals for 48h for MTT, trypan blue and LDH release assay
as reported earlier [12]. An appropriate vehicle and positive control 5-flurouracil all
determinations were carried out in triplicate for a minimum of three times independently as
described previously.
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4.3.2. Animal models and ethics
Swiss albino male mice weighing 25g-28g were housed under standard laboratory
conditions with food and water. All procedures for animal experimentation used were approved
by the Institutional Animal Ethics Committee, National College of Pharmacy, Shimoga, India, in
accordance
with
the
CPCSEA
guidelines
for
laboratory
animal
facility
(NCP/IAEC/CL/101/05/2012-13).
4.3.3. Determining the LD50 compound 9k
Non-tumor bearing normal swiss albino mice divided into 5 groups (n = 6) were
subjected to the acute toxicity studies. The LD50 of compound 9k was determined by
intraperitoneal administration of 9k as per the standard CPCSEA guidelines [11].
4.3.4. Animal tumor models and treatment
DLA cells were cultured in-vivo and after the optimal growth of tumor, the mice were
divided into group A and group B. The compound 9k was dissolved in DMSO and three doses
on alternative days were injected i.p.to group A mice at a concentration of 75 mg/kg bodyweight
using a 26 gauge needle. Group B was maintained as an appropriate vehicle (DMSO) control.
The survivability of the animals (n = 10 each) was also determined.
4.3.5. Chorioallontoic membrane (CAM) assay
The in-vivo anti-angiogenic effect induced by rVEGF165 was analyzed following
treatment with 9k in 12 days, fertilized egg CAM as described earlier [11] and changes in the
MVD was analyzed and photographed using Sony steady shot DSC-W610camera.
Acknowledgements
Zabiulla gratefully acknowledges the financial support provided by the Department of
Science and Technology, New Delhi, Under INSPIRE-Fellowship scheme [IF140407]. Shaukath
Ara Khanum thankfully acknowledges the financial support provided by VGST, Bangalore,
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under CISEE Programme [Project sanction order: No. VGST/ CISEE /282]. Shamanth
Neralagundi H.G. acknowledges thankfully for Lady Tata Memorial Trust (LTMT) Mumbai for
the JRF award for the year 2015-16. B.T. Prabhakar thankfully acknowledges the financial
support provided by VGST, Bangalore, under CISEE Programme [Project sanction order: No.
VGST/ CISEE /231].
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