Synthesis, molecular docking, and apoptogenic efficacy of novel N-heterocycle analogs to target B-cell lymphoma 2/X-linked inhibitors of apoptosis proteins to regress melanoma

Article abstract of DOI:10.1007/s00044-019-02357-x

The novel series of piperidine conjugated benzophenone analogs with amide link 11a–l were synthesized in a multistep process. The structures of these compounds were confirmed by IR, ¹H, ¹³C, NMR, and mass spectra and also by elementa

Full text of DOI:10.1007/s00044-019-02357-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02357-x
ORIGINAL RESEARCH
Synthesis, molecular docking, and apoptogenic efficacy of novel N-
heterocycle analogs to target B-cell lymphoma 2/X-linked inhibitors
of apoptosis proteins to regress melanoma
Zabiulla Zabiulla1 Vikas H. Malojirao Yasser Hussein Eissa Mohammed Prabhu Thirusangu B. T. Prabhakar
2 ●
1 ●
3 ●
2 ●
●
1
Shaukath Ara Khanum
Received: 14 February 2019 / Accepted: 8 May 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The novel series of piperidine conjugated benzophenone analogs with amide link 11a–l were synthesized in a multistep
1
13
process. The structures of these compounds were confirmed by IR, H, C, NMR, and mass spectra and also by elemental
analyses. The newly synthesized molecules were screened for selectivity against cancers of different origin through cell
based assay system using B16F10, A375, A549, HepG2, ACHN, and MCF7 cells. The results postulated that compound 11f
with two bromo groups at the para position in rings A and E and two methyl groups at ortho position in rings B and D
evokes target specific action against melanoma highlighting the importance of substituted groups. Down the line studies
further inferred compound 11f evokes the apoptotic cellular event leading to cell death. Investigation of eventual mechanism
revealed that compound 11f turned out to be a dual inhibitor of B-cell lymphoma-2 and X-linked inhibitors of apoptosis
causing the up regulation of Bax and Bad. Further, the antiproliferative effects were mimicked in murine melanoma with
similar mechanisms. Molecular docking experiments further confirmed that compound 11f possessed a superior affinity for
of B-cell lymphoma-2 and X-linked inhibitors of apoptosis through strong hydrogen bonds. The study implies the
identification of compound 11f with selective target against melanoma by inducing apoptogenic effect, which could be the
future hope for the treatment of skin cancer.
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●
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Keywords Benzophenone Piperidine Melanoma Apoptosis of B-cell lymphoma-2 X-linked inhibitors of apoptosis
Introduction
Skin cancers are by far the most common malignancy of
humans and millions of cases detected each year (Gupta
et al. 2016). The growing incidence of cutaneous malig-
nancies has heralded the need for multiple treatment
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02357-x) contains supplementary
options. Although many therapeutic strategies available in
the modern era, new research and fresh innovation are still
required to reduce morbidity and mortality and urgent
approaches for skin cancer may pass through new com-
pounds. One of the most effective ways to combat skin
cancers is through early diagnosis and administration of
effective treatment with target specific drugs. Further, fol-
low up and efficient monitoring will allow physicians to
detect relapsing diseased condition.
Apoptosis, a normal physiological form of cell death, is
critically involved in the regulation of cellular home-
ostasis. In normal physiological condition, the fate of the
cells depends on the cellular microenvironment. Any
material, which is available to authorized users.
*
*
B. T. Prabhakar
prabhakarbt@gmail.com
Shaukath Ara Khanum
shaukathara@yahoo.co.in
1
2
Department of Chemistry, Yuvaraja’s College (Autonomous),
University of Mysore, Mysore, Karnataka, India
Molecular Biomedicine Laboratory, Postgraduate Department of
Studies and Research in Biotechnology, Sahyadri Science College,
Kuvempu University, Shimoga, Karnataka, India
3
Department of Experimental Pathology and Medicine, Mayo
Clinic, Rochester, MN 55902, USA

Medicinal Chemistry Research
mutation or damage will be detected and repair or elim-
ination event takes place depending upon the damage to
the cells. This event is regulated by different proteins
which will sense and act. Whereas, in the pathogenesis of
melanoma, the neoplastic cells are evolved by developing
escaping strategy leading to programmed cell death,
which is necessary for prognosis of the disease (Elmore
Moreover, benzophenones are a class of compounds
obtained from natural sources (Henry et al. 1999) or by
synthetic methods (Khanum et al. 2005; Mohammed et al.
2018; Ranganatha et al. 2013). They are of enormous sig-
nificance fundamentally due to their diverse biological
properties. Further, benzophenones display significant
antitumor activity both in vitro and in vivo (Al-Ghorbani
et al. 2017; Ranganatha et al. 2013; Zabiulla et al. 2016).
Moreover, synthetic benzophenones, such as benzophenone
appended with oxadiazoles (Puttaswamy et al. 2018), ben-
zimidazole benzophenones (Thirusangu et al. 2017), and
pyridine conjugated benzophenones (Al-Ghorbani et al.
2016) have proven to be anticancer agents. Further, para-
methoxy substituted benzophenones were evaluated as p38a
inhibitors with high selectivity and efficacy (Revesz et al.
2004). Whereas, methoxy and amino substituted benzo-
phenones have been reported to be potent cytotoxic agents
against a panel of human cancer cell lines including multi-
drug-resistant cell lines (Schlitzer et al. 2002). Also, analogs
of benzophenone show a selective toxicity for proliferating
endothelial cells by induction of apoptosis (Iyer et al. 1998),
and polyprenylated benzophenone derivatives are also able
to induce caspase mediated apoptosis (Balasubramanyam
et al. 2004). In addition, piperidine derivatives have been
found to display a wide range of biological and therapeutic
activities in view of their wide range of occurrence and also
due to low toxicity (Khanum et al. 2009; Hu et al. 2014;
Wang et al. 2015; Xin-Hua et al. 2012). Besides, chemical
modification and combination of two or more bioactive
compounds is one of the most efficient approach in drug
development. On this basis, an approach was made to the
synthesis of potential apoptogenic molecules by combining
two bioactive molecules like benzophenone and piperidine
collectively through amide linkage 11a–l to attain phar-
macologically efficacious single molecule as anticancer
agent for both in vitro and in vivo studies. The study was
also supported by molecular docking investigation which
illustrated the interaction of the lead compound as dual
inhibitor of both XIAP and Bcl-2 which is very promising.
2
007). These cancer cells regularly started to over express
array of anti-apoptotic proteins that play an imperative
roles in abiding the revival of apoptotic signaling cascade
(
Frenzel et al. 2009). Many members of the B-cell lym-
phoma 2 (Bcl-2) family of apoptosis-related genes have
been found to be differentially expressed in various skin
malignancies (Eberle and Hossini 2008). Most of the Bcl-
2
family genes have been found to play a central reg-
ulatory role in apoptosis induction, which includes both
pro and anti-apoptotic proteins. Bcl-2 is one of the prime
anti-apoptotic proteins which primarily interact and neu-
tralize the pro-apoptotic protein expression and their by
halting the release of apoptogenic molecules from mito-
chondria (Frenzel et al. 2009). Another group of anti-
apoptotic proteins expressed by the cancerous cells are
inhibitors of apoptosis (IAPs), which interacts and
antagonize the executioner caspases, such as caspase-3
and caspase-8. Modulating these proteins is an effective
treatment strategy, which provides the basis for early
diagnosis, understanding the prognosis, and establishment
of therapeutic intervention that targets these apoptosis
pathways (Parrish et al. 2013).
Targeting and mitigating the expression these anti-
apoptotic proteins by novel molecules and thereby restor-
ing the apoptotic event is considered to be an effective
mode of treating cancer.
Current cancer therapy suffers from the major limitation
of side effect and drug resistance, so continued search for
newer and safer anticancer drugs remains critically impor-
tant with target specificity. Nitrogen containing moieties
occurs widely in synthetic and natural products. Besides,
when one or more nitrogen atoms exist in the chemical
structure, intercalating chromophore possesses a polarized
character and optimal interaction occur (Ozkay et al. 2010).
The nitrogen atom containing heterocycles occurs in phar-
maceuticals, natural products, dyes, organic materials and in
particular, in biologically active compounds (Yang et al.
Materials and methods
Experimental section
2
015). These heterocycles produces anticancer effects in
different types of cancer through inhibiting cell growth and
induction of cell differentiation and apoptosis. However,
despite their wide range of biological activities along with
their anticancer activity still there is a need for the devel-
opment of novel, practical, and efficient methods for the
synthesis of nitrogen containing heterocyclic compounds
which nowadays becomes an important goal in modern
organic synthesis.
The chemicals were purchased from Sigma Aldrich, ana-
lytical thin layer chromatography (TLC) was performed on
0.25 mm silica gel plates (Merck 60 F 254) by using solvent
system ethyl acetate: hexane (2:3). Melting point was
determined on a Chemi Line Micro Controller based melt-
ing point apparatus with a digital thermometer. The IR
spectrum was recorded by the potassium bromide pellet
method on Cary 630 FTIR Agilent spectrophotometer,

Medicinal Chemistry Research
NMR spectrum was recorded on a VNMRS-400 MHz
2,5-Dimethyl phenylbenzoate 3b Yield: 82%. M.P.:
−
1
1
Agilent-NMR spectrophotometer in CDCl or DMSO. Mass
66–68 °C. IR (KBr) νmax (cm ): 1725 (ester, C=O). H
NMR (400 MHz) (CDCl ) δ (ppm): 2.42 (s, 6H, 2CH ),
3
spectrum was obtained with a VG70-70H spectrometer.
Elemental analysis results are within 0.4% of the theoretical
calculated value.
3
3
7.20–8.25 (m, 8H, Ar–H). LC-MS m/z 227 (M + 1). Anal.
Cal. for C H O (227): C, C, 79.62; H, 6.24. Found: C,
1
5
14
2
The human cancer lines, A549 (Human lung adeno-
carcinoma), MCF-7 (Human breast cancer), A375 (Human
melanoma cancer), HepG2 (Human liver cancer), ACHN
79.58; H, 6.13%.
2-Methylphenyl 4-bromobenzoate 3c Yield: 90%. M.P.:
61–63 °C. IR (KBr) νmax (cm ): 1715 (ester, C=O). H
NMR (400 MHz) (CDCl ) δ (ppm): 2.46 (s, 3H, CH ),
7.09–8.18 (m, 8H, Ar-H). LC-MS m/z 291 (M+), 293 (M
+ 2). Anal. Cal. for C H BrO (291): C, 57.76; H, 3.81.
Found: C, 57.70; H, 3.69%.
−
1
1
(
Human renal cancer), B16F10 (Mouse melanoma cancer)
were procured from National Center for Cell Science
NCCS), Pune, India. The protease inhibitor cocktail, anti
3
3
(
mouse/rabbit IgG antibodies from Sigma Aldrich, USA.
Dulbecco’s Modified Eagle Media (DMEM), Antibiotic-
antimycotic solution, trypsin- ethylene diamine tetraacetic
acid (EDTA) solution, fetal bovine serum (FBS) from In-
vitrogen (Gibco), USA. Annexin V–FITC staining kit is
from BD Bioscience, USA. A click-it TUNEL vision kit,
Alexa fluor 488-anti mouse/rabbit IgG from Thermo Fisher
Scientific, Watman, MA, USA. All other chemicals used in
the current study are of molecular and analytical grade. Cell
culture plastic wares were from corning Sigma and
Eppendorf, Germany. All bright field and fluorescence
image were taken in EVOS FL cell imaging, Thermo Sci-
entific, USA and the results were assessed and analyzed by
Image J software. All the gel and blotting images were
documented using Bio-rad Gel Documentation TM XR +
Imaging system and quantified using densitometric analysis.
Experiments were performed at minimum three independent
times and analyzed.
1
4
11
2
2-Chloro-6-fluorophenyl 4-chlorobenzoate 3d Yield: 82%.
−
1
M.P.: 50–52 °C. IR (KBr) ν
(cm ): 1720 (ester, C=O).
max
1
H NMR (400 MHz) (CDCl ) δ (ppm): 7.03–8.15 (m, 7H,
3
Ar-H). LC-MS m/z 285 (M+), 287 (M + 2), 289 (M + 4).
Anal. Cal. for C H Cl FO (285): C, 54.77; H, 2.47.
Found: C, 54.68; H, 2.40%.
1
3
7
2
2
2-Methylphenyl 4-methylbenzoate 3e Yield: 93%. M.P.:
59–61 °C. IR (KBr) νmax(cm ): 1710 (ester, C=O). H
NMR (400 MHz) (CDCl ) δ (ppm): 2.37 (s, 6H, 2CH ),
−
1
1
3
3
7.11–8.28 (m, 8H, Ar–H). LC-MS m/z 227 (M + 1). Anal.
Cal. for C H O (226): C, 79.62; H, 6.24. Found: C,
1
5
14
2
79.51; H, 6.13%.
2-Methylphenyl 2-methylbenzoate 3f Yield: 83%. M.P.:
−
1
1
56–58 °C. IR (KBr) νmax (cm ): 1715 (ester, C=O). H
Chemistry
NMR (400 MHz) (CDCl ) δ (ppm): 2.44 (s, 6H, 2CH ),
3
3
7.12–8.24 (m, 8H, Ar–H). LC-MS m/z 227 (M + 1). Anal.
General procedure for the synthesis of phenyl benzoates
Cal. for C H O (226): C, 79.62; H, 6.24. Found: C,
1
5
14
2
3
a–f
79.53; H, 6.19%.
The starting substituted benzoates 3a–f were synthesized by
benzoylation of substituted phenols 1a–c with substituted
benzoyl chlorides (2a–d, 1:1) in the presence of 10%
sodium hydroxide solution. The reaction mixture was stir-
red for 3–4 h at 0–5 °C. The reaction was monitored by
TLC using 4:1 n-hexane: ethyl acetate solvent mixture.
After completion of the reaction, the oily product was
extracted with ether layer (3 × 25 mL). Ether layer was
washed with 10% sodium hydroxide solution (3 × 30 mL)
followed by water (3 × 25 mL) and then dried over anhy-
drous sodium sulfate and the solvent was evaporated under
reduced pressure to afford compounds 3a–f.
General procedure for the synthesis of (4-hydroxy phenyl)
phenyl methanones 4a–f
Substituted (4-hydroxy phenyl) phenyl methanone com-
monly known as hydroxy benzophenones 4a–f were syn-
thesized by Fries rearrangement. Compounds 3a–f
(0.002 mol) were treated with anhydrous aluminum chloride
(0.005 mol) as a catalyst at 150–170 °C without using sol-
vent condition for about 3–4 h. The reaction mixture was
cooled to room temperature and quenched with 6N hydro-
chloric acid in the presence of ice cold water. The reaction
mixture was stirred for about 2–3 h, filtered the solid then
and recrystallized with methanol to obtain compounds 4a–f.
2
-Methyl phenylbenzoate 3a Yield: 93%. M.P.: 62–64 °C.
−
1
1
IR (KBr) νmax (cm ): 1715 (ester, C=O). H NMR
(4-Hydroxy-3-methyl-phenyl)-phenyl-methanone
−
1
(
(
(
400 MHz) (CDCl ) δ (ppm): 2.45 (s, 3H, CH ), 7.11–8.25
4a Yield: 76%. M.P.: 109–111 °C. IR (KBr) ν
1630 (C=O), 3530–3630 (OH). H NMR (400 MHz)
(CDCl ) δ (ppm): 2.35 (s, 3H, CH ), 6.73–7.68 (m, 8H,
(cm ):
3
3
max
1
m, 9H,). LC-MS m/z 213 (M + 1). Anal. Cal. for C H O
1
4
12
2
212): C, 79.22; H, 5.70. Found: C, 79.20; H, 5.76%.
3
3

Medicinal Chemistry Research
Ar–H), 12.01 (bs, 1H, OH). LC-MS m/z 213 (M + 1). Anal.
residual mass was triturated with ice water to remove
potassium carbonate and then extracted with ether (3 ×
40 mL). The ether layer was washed with 10% sodium
hydroxide solution (3 × 25 mL) followed by water (3 ×
25 mL) and then dried over anhydrous sodium sulfate and
evaporated to dryness to obtain crude solid, which when
recrystallized with ethanol, afforded compounds 5a–f.
Cal. data for C H O (212): C, 79.22; H, 5.70. Found: C,
1
4
12
2
7
9.17; H, 5.68%.
(
4
1
4-Hydroxy-2,5-dimethyl-phenyl)-phenyl-methanone
−
1
b Yield: 84%. M.P.: 121–123 °C. IR (KBr) ν
(cm ):
max
1
650 (C=O), 3505–3590 (OH). H NMR (400 MHz)
(
CDCl ) δ (ppm): 2.35 (s, 6H, 2CH ), 6.61–7.70 (m, 7H,
3
3
Ar–H), 11.80 (bs, 1H, OH). LC-MS m/z 227 (M + 1). Anal.
Ethyl 2-(4-benzoyl-2-methylphenoxy)acetate 5a Yield:
−
1
Cal. data for C H O (226): C, 79.62; H, 6.24. Found: C,
93%. M.P.: 50–52 °C. IR (KBr) ν
(cm ): 1665 (C=O),
1
5
14
2
max
1
7
9.54; H, 6.22%.
1755 (ester, C=O). H NMR (400 MHz) (CDCl ) δ (ppm):
3
1
.20 (t, 3H, CH of ester), 2.31 (s, 3H, CH ), 4.13 (q, 2H,
3
3
(
4
1
4-Hydroxy-3-methyl-phenyl)-(4-Bromo-phenyl)-methanone
CH of ester), 4.54 (s, 2H, OCH ), 7.20–7.83 (m, 8H, Ar-
2 2
−
1
c Yield: 85%. M.P.: 153–156 °C. IR (KBr) ν
(cm ):
H). LC-MS m/z 299 (M + 1). Anal. Cal. for C H O4
max
18 18
1
655 (C=O), 3520–3610 (OH). H NMR (400 MHz)
(298): C, 72.48; H, 6.04. Found: C, 72.38; H, 6.06%.
(
CDCl ) δ (ppm): 2.35 (s, 3H, CH ), 6.51–7.60 (m, 7H,
3
3
Ar–H), 12.02 (bs, 1H, OH). LC-MS m/z 291 (M+), 293 (M
2). Anal. Cal. data for C H BrO (291): C, 57.76; H,
Ethyl 2-(4-benzoyl-2,5-dimethylphenoxy)acetate 5b Yield:
−
1
+
92%. M.P.: 52–55 °C. IR (KBr) ν
(cm ): 1650 (C=O),
14
11
2
max
1
3
.81. Found: C, 57.73; H, 3.75%.
1745 (ester, C=O). H NMR (400 MHz) (CDCl ) δ (ppm):
3
1
.20 (t, 3H, CH of ester), 2.34 (s, 6H, 2CH ), 4.31 (q, 2H,
3
3
(
3-Chloro-5-fluoro-4-hydroxy-phenyl)-(4-chloro-phenyl)-
CH of ester), 4.52 (s, 2H, OCH ), 6.93–7.82 (m, 7H, Ar-
2 2
methanone 4d Yield: 79%. M.P.: 146–149 °C. IR (KBr)
H). LC-MS m/z 313 (M + 1). Anal. Cal. for C H O
(312): C, 73.06; H, 6.45. Found: C, 72.97; H, 6.36%.
19
20
4
−
1
1
νmax (cm ): 1660 (C=O), 3530–3625 (OH). H NMR
400 MHz) (CDCl ) δ (ppm): 6.73–7.71 (m, 6H, Ar–H),
(
3
1
2
5
1.90 (bs, 1H, OH). LC-MS m/z 285 (M + 2), 287 (M + 2),
Ethyl 2-(4-(4-bromobenzoyl)-2-methylphenoxy)acetate 5c
−
1
89 (M + 4). Anal. Cal. data for C H Cl FO (285): C,
Yield: 89%. M.P.: 46–48 °C. IR (KBr) ν
(cm ): 1650
max
13
7
2
2
1
4.77; H, 2.47. Found: C, 54.68; H, 2.39%.
(C=O), 1760 (ester, C=O). H NMR (400 MHz) (CDCl ) δ
3
(
ppm): 1.26 (t, 3H, CH of ester), 2.32 (s, 3H, CH ), 4.53 (q,
3
3
(
4
1
4-Hydroxy-3-methyl-phenyl)-4-methylphenyl-methanone
2H, CH of ester), 4.64 (s, 2H, OCH ), 6.92–7.83 (m, 7H,
2 2
Ar-H). LC-MS m/z 377 (M+), 379 (M + 2). Anal. Cal. for
−
1
e Yield: 82%. M.P.: 151–154 °C. IR (KBr) ν
(cm ):
max
1
640 (C=O), 3510–3620 (OH). H NMR (400 MHz)
C H BrO (377): C, 57.31; H, 4.54. Found: C, 57.22;
1
8
17
4
(
CDCl ) δ (ppm): 2.35 (s, 6H, 2CH ), 7.10–7.70 (m, 7H,
H, 4.41%.
3
3
Ar–H), 12.20 (bs, 1H, OH). LC-MS m/z 227 (M + 1). Anal.
Cal. data for C H O (226): C, 79.62; H, 6.24. Found: C,
Ethyl 2-(2-chloro-4-(4-chlorobenzoyl)-6-fluorophenoxy)acet-
1
5
14
2
−
1
7
9.59; H, 6.20%.
ate 5d Yield: 91%. M.P.: 55–57 °C. IR (KBr) ν (cm ):
max
1
1
660 (C=O), 1755 (ester, C=O). H NMR (400 MHz)
(
4
1
4-Hydroxy-3-methyl-phenyl)-2-methylphenyl-methanone
(CDCl ) δ (ppm): 1.35 (t, 3H, CH of ester), 4.16 (q, 2H,
3
3
−
1
f Yield: 72%; M.P.: 124–127 °C: IR (KBr) νmax (cm ):
CH of ester), 4.83 (s, 2H, OCH ), 7.14–7.75 (m, 6H, Ar-
2
2
1
650 (C=O), 3500–3590 (OH). H NMR (400 MHz)
H). LC-MS m/z 371 (M+), 373 (M + 2), 375 (M + 4).
(
CDCl ) δ (ppm): 2.34 (s, 6H, 2CH ), 6.74–7.61 (m, 7H,
Anal. Cal. for C H Cl FO (371): C, 55.01; H, 3.53.
3
3
17 13
2
4
Ar–H), 11.60 (bs, 1H, OH). LC-MS m/z 227 (M + 1). Anal.
Found: C, 54.92; H, 3.46%.
Cal. data for C H O (226): C, 79.62; H, 6.24. Found: C,
1
5
14
2
7
9.60; H, 6.20%.
Ethyl 2-(2-methyl-4-(4-methylbenzoyl)phenoxy)acetate 5e
−
1
Yield: 92%. M.P.: 48–50 °C. IR (KBr) ν
(cm ): 1665
max
1
General procedure for the synthesis of ethyl 2-(4-
(C=O), 1750 (ester, C=O). H NMR (400 MHz) (CDCl ) δ
3
benzoylphenoxy) acetates 5a–f
(ppm): 1.22 (t, 3H, CH of ester), 2.46 (s, 6H, 2CH ), 4.13
3
3
(
q, 2H, CH of ester), 4.55 (s, 2H, OCH ), 7.12–7.74 (m,
2
2
To a solution of compounds 4a–f (0.013 mol) in dry acetone
7H, Ar-H). LC-MS m/z 313 (M + 1). Anal. Cal. for
(
50 mL), anhydrous potassium carbonate (0.019 mol) and
C H O (312): C, 73.06; H, 6.45. Found: C, 73.04;
1
9
20
4
ethyl chloroacetate (0.026 mol) were added and the reaction
mass was heated to 60 °C for 5–6 h. After completion of the
reaction was monitored by TLC, the reaction mixture was
cooled and the solvent was removed by distillation. The
H, 6.41%.
Ethyl 2-(2-methyl-4-(2-methylbenzoyl)phenoxy)acetate 5f
Yield: 90%. M.P.: 50–52 °C. IR (KBr) νmax (cm ): 1660
−
1

Medicinal Chemistry Research
1
(
(
2
C=O), 1760 (ester, C=O). H NMR (400 MHz) (CDCl ) δ
[2-Methyl-4-(4-methyl-benzoyl)-phenoxy]-acetic acid 6e
3
−
1
ppm): 1.32 (t, 3H, CH of ester), 2.33 (s, 6H, 2CH ), 4.25 (q,
H, CH of ester), 4.90 (s, 2H, OCH ), 6.84–7.63 (m, 7H, Ar-
Yield: 93%. M.P.: 118–120 °C. IR (KBr) νmax (cm ): 1680
3
3
1
(C=O), 1750 (acid, C=O), 3455–3570(acid OH). H NMR
2
2
H). LC-MS m/z 313 (M + 1). Anal. Cal. for C H O (312):
(400 MHz) (CDCl ) δ (ppm): 2.32 (s, 6H, 2CH ), 4.39 (s,
19
20
4
3
3
C, 73.06; H, 6.45. Found: C, 73.04; H, 6.55%.
2H, OCH ), 6.85–7.57 (m, 7H, Ar-H), 13.04 (s, 1H,
2
COOH). LC-MS m/z 285 (M + 1). Anal. Cal. for C H O
17
16
4
General procedure for the synthesis of (4-benzoyl
(284): C, 71.82; H, 5.67. Found: C, 71.74; H, 5.63%.
phenoxy)-acetic acid 6a–f
[
2-Methyl-4-(2-methyl-benzoyl)-phenoxy]-acetic acid 6f
−
1
A mixture of compounds 5a–f (6.0 mmol) dissolved in
ethanol (15 mL) and an aqueous solution of sodium
hydroxide (15 mmol) was refluxed for 4–5 h. The reaction
mass was cooled and acidified with 3N hydrochloric acid
and the aqueous layer The precipitate was filtered, washed
with ice water and recrystallized with ethanol to afford
compounds 6a–f in a good yield.
Yield: 84%. M.P.: 126–128 °C. IR (KBr) νmax (cm ): 1620
(C=O), 1765 (acid, C=O), 3475–3570 (acid OH). H NMR
1
(400 MHz) (CDCl ) δ (ppm): 2.55 (s, 6H, 2CH ), 4.43 (s,
3
3
2H, OCH ), 6.95–7.82 (m, 7H, Ar–H), 12.76 (s, 1H,
2
COOH). LC-MS m/z 285 (M + 1). Anal. Cal. for C H O
17
16
4
(284): C, 71.82; H, 5.67. Found: C, 71.73; H, 5.60%.
General procedure for the synthesis of N-(2-amino-phenyl)-
(
4-Benzoyl-2-methylphenoxy)-acetic acid 6a Yield: 86%.
2-(4-benzoyl-phenoxy)-acetamide 8a–f
−
1
M.P.: 131–133 °C. IR (KBr) νmax (cm ): 1660 (C=O),
1
1
720 (acid, C=O), 3420–3520 (acid OH). H NMR
To a solution of compounds 6a–f (0.0037 mol) in dry
dichloromethane (DCM), lutidine (0.0074 mol) was added at
25–30 °C, followed by the addition of o-phenylenediamine
(7, 0.0037 mol). The reaction mixture was stirred for 30 min
at room temperature, then the reaction was cooled to 0–5 °C,
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetra-
fluoroborate (TBTU) (0.0037 mol) was added over a period
of 30 min while maintaining the temperature below 5 °C.
The reaction was allowed to stirred overnight and monitored
by TLC using hexane and ethyl acetate (4:1). The reaction
mixture was diluted with 25 mL of DCM and treated with
1.5 N HCl solution (30 mL). The organic layer was washed
with water (3 × 30 mL) and brine (3 × 30 mL). Finally, the
organic layer was dried over anhydrous sodium sulfate and
concentrated to yield compounds 8a–f.
(
(
400 MHz) (CDCl ) δ (ppm): 2.44 (s, 3H, CH ), 4.81
3
3
s, 2H, OCH ), 6.90–7.83 (m, 8H, Ar-H), 13.13 (s, 1H,
2
COOH). LC-MS m/z 271 (M + 1). Anal. Cal. for
C H O (270): C, 71.10; H, 5.22. Found: C, 71.06; H,
5
1
6
14
4
.14%.
(
4-Benzoyl-2,5-dimethylphenoxy)-acetic acid 6b Yield:
−
1
8
(
7%. M.P.: 152–154 °C. IR (KBr) νmax (cm ): 1655
1
C=O), 1730 (acid, C=O), 3400–3530(acid OH).
H
NMR (400 MHz) (CDCl ) δ (ppm): 2.44 (s, 6H, 2CH ),
4
1
C H O (284): C, 71.82; H, 5.67. Found: C, 71.70; H,
5
3
3
.53 (s, 2H, OCH ), 7.15–7.63 (m, 7H, Ar-H), 12.52 (s,
2
H, COOH). LC-MS m/z 285 (M + 1). Anal. Cal. for
1
7
16
4
.58%.
[
4-(4-Bromo-benzoyl)-2-methylphenoxy]-acetic acid 6c
N-(2-Amino-phenyl)-2-(4-benzoyl-2-methyl-phenoxy)-aceta-
−
1
Yield: 92%. M.P.: 141–143 °C. IR (KBr) νmax (cm ): 1610
(
(
mide 8a Yield: 92%. M.P.: 191–193 °C. IR (KBr) ν
max
1
−1
C=O), 1750 (acid, C=O), 3455–3560(acid OH). H NMR
400 MHz) (CDCl ) δ (ppm): 2.21 (s, 3H, CH ), 4.41 (s,
(cm ): 1665 (C=O), 1735 (amide, C=O), 3110–3230
1
(NH), 3315–3410 (NH ). H NMR (400 MHz) (CDCl ) δ
3
3
2
3
2
H, OCH ), 7.14–7.85 (m, 7H, Ar-H), 12.71 (s, 1H,
(ppm): 2.44 (s, 3H, CH ), 4.73 (s, 2H, NH ), 4.91 (s, 2H,
2
3 2
COOH). LC-MS m/z 349 (M+), 351 (M + 2). Anal. Cal. for
OCH ), 6.71–7.73 (m, 12H, Ar-H), 9.27 (s, 1H, NH). LC-
2
C H BrO (349): C, 55.04; H, 3.75. Found: C, 55.05;
MS m/z 361 (M + 1). Anal. Cal. for C H N O (360): C,
1
6
13
4
22 20
2
3
H, 3.78%.
73.32; H, 5.59; N, 7.77. Found: C, 73.20; H, 5.55;
N, 7.69%.
[
6
2-Chloro-4-(4-chloro-benzoyl)-6-fluorophenoxy]-acetic acid
d Yield: 94%. M.P.: 139–141 °C. IR (KBr) νmax (cm ):
6440 (C=O), 1770 (acid, C=O), 3495–3600 (acid OH).
−
1
N-(2-Amino-phenyl)-2-(4-benzoyl-2,5-dimethyl-phenoxy)-
1
acetamide 8b Yield: 80%. M.P.: 147–150 °C. IR (KBr)
1
−1
H NMR (400 MHz) (CDCl ) δ (ppm): 4.45 (s, 2H, OCH ),
νmax (cm ): 1685 (C=O), 1755 (amide, C=O), 3140–3260
3
2
1
6
.84–7.86 (m, 6H, Ar-H), 12.93 (s, 1H, COOH). LC-MS
(NH), 3320–3420 (NH ). H NMR (400 MHz) (CDCl ) δ
2
3
m/z 343 (M+), 345 (M + 2), 347 (M + 4). Anal. Cal. for
(ppm): 2.35 (s, 6H, 2CH ), 4.67 (s, 2H, NH ), 4.88 (s, 2H,
3 2
C H Cl FO (343): C, 52.50; H, 2.64. Found: C, 52.50;
OCH ), 6.84–7.76 (m, 11H, Ar–H), 9.32 (s, 1H, NH). LC-
1
5
9
2
4
2
H, 2.65%.
MS m/z 375 (M + 1). Anal. Cal. for C H N O (374): C,
2
3
22
2
3

Medicinal Chemistry Research
7
3.78; H, 5.92; N, 7.48. Found: C, 73.67; H, 5.80;
5 °C. The reaction allowed to stir overnight and monitored
by TLC using hexane and ethyl acetate (4:1). After com-
pletion of reaction, the reaction mixture was diluted with
30 mL of DCM and treated with 10% sodium bicarbonate
solution (3 × 30 mL). The organic layer was washed with
water (3 × 20 mL), dried over anhydrous sodium sulfate and
concentrated to afford compounds 9a–l.
N, 7.45%.
N-(2-Amino-phenyl)-2-[4-(4-bromo-benzoyl)-2-methyl-phe-
noxy]-acetamide 8c Yield: 94%. M.P.: 186–188 °C. IR
−
1
(
KBr) νmax (cm ): 1680 (C=O), 1750 (amide, C=O),
1
3
150–3265 (NH), 3305–3405 (NH ). H NMR (400 MHz)
2
(
CDCl ) δ (ppm): 2.32 (s, 3H, CH ), 4.75 (s, 2H, NH ), 4.93
3
3
2
(
s, 2H, OCH ), 6.65–7.87 (m, 11H, Ar-H), 9.21 (s, 1H,
2-(4-Benzoyl-2-methyl-phenoxy)-N-{2-[2-(4-benzoyl-2-
methyl-phenoxy)-acetylamino]-phenyl}-acetamide
2
NH). LC-MS m/z 438 (M+), 440 (M + 2). Anal. Cal. for
−
1
C H BrN O (438): C, 60.15; H, 4.36; N, 6.38. Found: C,
9a Yield: 80%. M.P.: 88–90 °C; IR (KBr) νmax (cm ):
2
2
19
2
3
1
6
0.08; H, 4.30; N, 6.27%.
1665 (C=O), 1740 (amide, C=O), 3100–3210 (NH). H
NMR (400 MHz) (CDCl ) δ (ppm): 2.29 (s, 6H, 2CH ),
3
3
N-(2-Amino-phenyl)-2-[2-chloro-4-(4-chloro-benzoyl)-6-
4.76 (s, 4H, 2OCH ), 6.95–7.65 (m, 20H, Ar-H), 9.67 (s,
2
1
3
fluoro-phenoxy]-acetamide 8d Yield: 84%. M.P.:
2H, 2NH). C NMR (DMSO-d ) δ: 194.8 (2C=O), 167.0
6
−
1
1
58–160 °C. IR (KBr) νmax (cm ): 1655 (C=O), 1705
(2CONH), 159.7 (2C, C1, C19), 138.2 (2C, C7, C25),
132.5 (2C, C13, C14), 132.4 (2CH, C10, C28), 130.7 (2CH,
C3, C21), 130.4 (2CH, C8, C26), 130.1 (2CH, C12, C30),
129.6 (2C, C4, C22), 129.6 (2CH, C9, C25), 128.8 (2CH,
C11, C29), 128.7 (2CH, C5, C23), 127.0 (2CH, C16, C17),
126.3 (2C, C2, C20), 126.0 (2CH, C15, C18), 111.2 (2CH,
C6, C24), 67.5 (2OCH ), 16.5 (2CH ). LC-MS m/z 613 (M
1
(
amide, C=O), 3140–3240 (NH), 3310–3410 (NH ). H
2
NMR (400 MHz) (CDCl ) δ (ppm): 4.55 (s, 2H, NH ), 4.84
3
2
(
s, 2H, OCH ), 6.82–7.95 (m, 10H, Ar–H), 9.72 (s, 1H,
2
NH). LC-MS m/z 432 (M+), 434 (M + 2), 436 (M + 4).
Anal. Cal. for C H Cl FN O (432): C, 58.22; H, 3.49; N,
2
1
15
2
2
3
6
.47. Found: C, 58.10; H, 3.45; N, 6.36%.
2
3
+
1). Anal. Cal. for C H N O (612): C, 74.49; H, 5.26;
38 32 2 6
N-(2-Amino-phenyl)-2-[2-methyl-4-(4-methyl-benzoyl)-phe-
N, 4.57. Found: C, 74.37; H, 5.19; N, 4.40%.
noxy]-acetamide 8e Yield: 91%. M.P.: 180–182 °C. IR
−
1
(
3
(
(
KBr) νmax (cm ): 1685 (C=O), 1750 (amide, C=O),
2-(4-Benzoyl-2,5-dimethyl-phenoxy)-N-{2-[2-(4-benzoyl-2,5-
dimethyl-phenoxy)-acetyl amino]-phenyl}-acetamide 9b
1
150–3250 (NH), 3315–3415 (NH ). H NMR (400 MHz)
CDCl ) δ (ppm): 2.66 (s, 6H, 2CH ), 4.32 (s, 2H, NH ), 4.71
s, 2H, OCH ), 6.83–7.86 (m, 11H, Ar-H), 9.62 (s, 1H, NH).
2
−
1
Yield: 75%. M.P.: 92–94 °C. IR (KBr) νmax (cm ): 1685
3
3
2
1
(C=O), 1740 (amide, C=O), 3110–3230 (NH). H NMR
2
LC-MS m/z 375 (M + 1) Anal. Cal. for C H N O (374): C,
(400 MHz) (CDCl ) δ (ppm): 2.45 (s, 12H, 4CH ), 4.78 (s,
23
22
2
3
3
3
73.78; H, 5.92; N, 7.48. Found: C, 73.80; H, 5.95; N, 7.43%.
4H, 2OCH ), 6.58–7.69 (m, 18H, Ar–H), 9.38 (s, 2H,
2
1
3
2
NH). C NMR (DMSO-d ) δ: 194.0 (2C=O), 165.5
6
N-(2-Amino-phenyl)-2-[2-methyl-4-(2-methyl-benzoyl)-phe-
(2CONH), 162.8 (2C, C1, C19), 139.5 (2C, C7, C25), 138.5
(2C, C5, C23), 135.2 (2C, C13, C14), 132.4 (2CH, C10,
C28), 131.7 (2CH, C3, C21), 129.2 (2C, C4, C22), 128.3
(2CH, C8, C26), 127.5 (2CH, C12, C30), 126.0 (4CH, C9,
C11, C27, C29), 124.7 (2CH, C16, C17), 123.5 (2CH, C15,
C18), 122.1 (2C, C2, C20), 117.6 (2CH, C6, C24), 68.7
(2OCH ), 19.0 (2CH ), 16.0 (2CH ). LC-MS m/z 641 (M +
noxy]-acetamide 8f Yield: 92%. M.P.: 190–192 °C. IR
−
1
(
3
KBr) νmax (cm ): 1660 (C=O), 1725 (amide, C=O),
1
115–3225 (NH), 3310–3410 (NH ). H NMR (400 MHz)
2
(
CDCl ) δ (ppm): 2.42 (s, 6H, 2CH ), 4.64 (s, 2H, NH ),
3
3
2
4
.95 (s, 2H, OCH ), 6.67–7.52 (m, 11H, Ar-H), 9.33 (s, 1H,
2
NH). LC-MS m/z 375 (M + 1). Anal. Cal. for C H N O
23
22
2
3
2
3
3
(
374): C, 73.78; H, 5.92; N, 7.48. Found: C, 73.76; H, 5.85;
1). Anal. Cal. for C H N O (640): C, 74.49; H, 5.26; N,
40 36 2 6
N, 7.35%.
4.57. Found: C, 74.40; H, 5.21; N, 4.49%.
General procedure for the synthesis of 2-(4-
benzoylphenoxy)-N-{2-[2-(4-benzoyl-phenoxy)-
acetylamino]-phenyl}-acetamide 9a–l
N-{2-[2-(4-Benzoyl-2-methylphenoxy)-acetylamino]-phe-
nyl}-2-[2-methyl-4-(4-methyl-benzoyl)-phenoxy]-acetamide
−
1
9c Yield: 71%. M.P.: 90–92 °C. IR (KBr) νmax (cm ):
1
1
675 (C=O), 1750 (amide, C=O), 3140–3260 (NH). H
To a solution of compounds 8a–f (0.003 mol) in dry DCM
NMR (400 MHz) (CDCl ) δ (ppm): 2.33 (s, 9H, 3CH ),
3
3
(
20 mL), substituted (4-benzoyl-phenoxy)-acetic acids 6a–f
4.84 (s, 4H, 2OCH ), 6.73–7.87 (m, 19H, Ar–H), 9.77 (s,
2
1
3
were added at 25–30 °C, followed by the addition of luti-
dine (0.003 mol). The reaction mixture was stirred at room
temperature for 30–40 min. The reaction mixture was
cooled to 0–5 °C and TBTU (0.001 mol) was added over a
period of 30 min while maintaining the temperature below
2H, 2NH). C NMR (DMSO-d ) δ: 193.4 (2C=O), 166.3
6
(2CONH), 161.4 (2C, C1, C19), 140.4 (2C, C7, C25),
136.5 (2C, C13, C14), 133.8 (2CH, C8, C26), 133.3 (2CH,
C12, C30), 132.5 (2CH, C3, C21), 131.4 (2CH, C9, C27),
130.8 (2CH, C11, C29), 129.5 (2C, C4, C22), 128.6 (2C,

Medicinal Chemistry Research
C10, C28), 127.4 (2CH, C5, C23), 126.4 (2CH, C16, C17),
C29), 128.5 (2C, C13, C14), 128.6 (2CH, C5, C23), 126.8
(2C, C10, C28), 124.5 (2CH, C15, C18), 123.0 (2CH, C16,
C17), 121.8 (2C, C2, C20), 116.8 (2CH, C6, C24), 65.8
(2OCH ), 15.1 (2CH ). LC-MS m/z 770 (M+), 772 (M +
1
23.6 (2C, C2, C20), 122.7 (2CH, C15, C18), 119.8 (2CH,
C6, C24), 69.4 (2OCH ), 16.5 (2CH ). LC-MS m/z 627 (M
2
3
+
1). Anal. Cal. for C H N O (626): C, 74.74; H, 5.47;
39 34 2 6
2
3
N, 4.47. Found: C, 74.71; H, 5.35; N, 4.37%.
2), 774 (M + 4). Anal. Cal. for C H Br N O (770): C,
38 30 2 2 6
59.24; H, 3.92; N, 3.64. Found: C, 59.20; H, 3.81;
N-{2-[2-(4-Benzoyl-2-methylphenoxy)-acetylamino]-phe-
nyl}-2-[2-methyl-4-(2-methyl-benzoyl)-phenoxy]-acetamide
N, 3.53%.
−
1
9
1
d Yield: 90%. M.P.: 94–96 °C. IR (KBr) νmax (cm ):
665 (C=O), 1730 (amide, C=O), 3110–3230 (NH). H
N-{2-[2-(4-Benzoyl-3-methylphenoxy)-acetylamino]-phe-
nyl}-2-[4-(4-bromo-benzoyl)-2-methyl-phenoxy]-acetamide
9g Yield: 73%. M.P.: 84–86 °C; IR (KBr) νmax (cm ):
1685 (C=O), 1740 (amide, C=O), 3130–3240 (NH). H
NMR (400 MHz) (CDCl ) δ (ppm): 2.13 (s, 6H, 2CH ),
1
−
1
NMR (400 MHz) (CDCl ) δ (ppm): 2.35 (s, 9H, 3CH ),
3
3
1
4
.67 (s, 4H, 2OCH ), 6.57–7.76 (m, 19H, Ar–H), 9.76 (s,
2
1
3
2
H, 2NH). C NMR (DMSO-d ) δ: 193.7 (2C=O), 168.5
6
3
3
(
2CONH), 164.3 (C, C1), 163.1 (C, C19), 140.5 (2C, C7,
4.64 (s, 2H, 2OCH ), 6.53–7.62 (m, 19H, Ar–H), 9.59 (s,
2
1
3
C25), 138.5 (C, C23), 137.4 (2C, C13, C14), 135.6 (2CH,
C10, C28), 133.4 (CH, C3), 132.4 (CH, C21), 131.7 (C,
C22), 130.3 (4CH, C8, C12, C26, C30), 129.6 (C, C4),
1
(
1
(
1
2H, 2NH). C NMR (DMSO-d ) δ: 193.3 (2C=O), 167.3
6
(2CONH), 166.3 (2C, C1, C19), 139.5 (C, C10), 138.5 (C,
C25), 136.6 (C, C7), 135.1 (2C, C13, C14), 134.4 (CH,
C28), 133.3 (2CH, C3, C21), 132.3 (2CH, C26, C30), 131.2
(C, CH C4, C12), 130.4 (C, CH, C8, C22), 129.2 (2CH, C9,
C11), 125.3 (2CH, C27, C29), 124.3 (2CH, C5, C23), 123.6
(2CH, C16, C17), 122.1 (2C, C2, C20), 120.6 (2CH, C15,
C18), 119.3 (2CH, C6, C24), 65.2 (2OCH ), 20.5 (CH ),
28.3 (4CH, C9, C11, C27, C29), 127.4 (CH, C5), 127.6
2CH, C6, C17), 127.6 (C, C2), 124.3 (2CH, C15, C18),
22.8 (C, C20), 121.6 (CH, C24), 119.3 (CH, C6), 70.6
2OCH ), 19.7 (CH ), 16.1 (2CH ). LC-MS m/z 627 (M +
2
3
3
). Anal. Cal. for C H N O (626): C, 74.74; H, 5.47; N,
39 34 2 6
2
3
4
.47. Found: C, 74.68; H, 5.41; N, 4.39%.
16.7 (2CH ). LC-MS m/z 692 (M+), 694 (M + 2). Anal.
3
Cal. for C H N BrO (692): C, 66.00; H, 4.52; N, 4.05.
3
8
31
2
6
N-{2-[2-(4-Benzoyl-2,5-dimethylphenoxy)-acetylamino]-
phenyl}-2-[4-(4-bromo-benzoyl)-2-methyl-phenoxy]-aceta-
mide 9e Yield: 83%. M.P.: 91–93 °C. IR (KBr) ν
Found: C, 65.91; H, 4.19; N, 4.02%.
N-{2-[2-(4-Benzoyl-2,5-dimethylphenoxy)-acetylamino]-
phenyl}-2-(4-benzoyl-2-methyl-phenoxy)-acetamide 9h
Yield: 80%. M.P.: 95–97 °C. IR (KBr) νmax cm 1665
max
−
1
(
(
cm ): 1675 (C=O), 1730 (amide, C=O), 3120–3220
1
−1
NH). H NMR (400 MHz) (CDCl ) δ (ppm): 2.75 (s, 9H,
3
1
3
9
1
CH ), 4.72 (s, 4H, 2OCH ), 6.43–7.52 (m, 18H, Ar–H),
(C=O), 1720 (amide, C=O), 3120–3230 (NH). H NMR
3
2
13
.72 (s, 2H, 2NH). C NMR (DMSO-d ) δ: 194.3 (2C=O),
(400 MHz) (CDCl ) δ (ppm): 2.44 (s, 9H, 3CH ), 4.76 (s,
6
3
3
66.2 (CONH), 165.3 (CONH), 164.4 (C, C19), 142.5 (C,
4H, 2OCH ), 6.89–7.66 (m, 19H, Ar–H), 9.67 (s, 2H,
2
1
3
C1), 140.6 (C, C6), 138.7 (C, C25), 137.3 (C, C10), 136.2
C, C7), 133.5 (C, C4), 132.6 (2C, C13, C14), 131.7 (CH,
C21), 130.8 (2CH, C8, C12), 128.9 (2CH, C26, C30), 127.4
C, C22), 126.7 (2CH, C9, C11), 123.5 (2CH, C27, C29),
2NH). C NMR (DMSO-d ) δ: 194.5 (2C=O), 165.4
6
(
(CONH), 162.6 (CONH), 161.7 (2C, C1, C19), 139.8 (C,
C7), 138.9 (C, C25), 136.4 (2C, C13, C14), 135.9 (2CH,
C26, C30), 134.3 (CH, C10), 132.2 (2CH, C3, C21), 131.7
(2CH, C27, C29), 131.2 (2CH, C8, C12), 128.5 (2C, C4,
C22), 126.3 (2CH, C9, C11), 124.3 (2CH, C5, C23), 122.8
(C, C28), 121.9 (2CH, C16, C17), 120.0 (2C, C2, C20),
118.3 (2CH, C15, C18), 114.2 (2CH, C6, C24), 73.2
(2OCH ), 20.6 (CH ), 16.6 (2CH ). LC-MS m/z 627 (M +
(
1
1
1
22.2 (CH, C23), 121.2 (CH, C3), 120.7 (2CH, C16, C17),
20.6 (C, C20), 119.3 (C, C2), 118.5 (2CH, C15, C18),
16.8 (CH, C5), 115.3 (CH, C24), 69.7 (2OCH ), 18.8
2
(
CH ), 15.6 (2CH ). LC-MS m/z 706 (M+), 708 (M + 2).
3 3
Anal. Cal. for C H BrN O (706): C, 66.39; H, 4.71; N,
3
9
33
2
6
2
3
3
3
.97. Found: C, 66.12; H, 4.68; N, 3.82%.
1). Anal. Cal. for C H N O (626): C, 74.74; H, 5.47; N,
39 34 2 6
4.47; O. Found: C, 74.40; H, 5.21; N, 4.49%.
2
-[4-(4-Bromo-benzoyl)-2-methylphenoxy]-N-(2-{2-[4-(4-
bromo-benzoyl)-2-methyl-phenoxy]-acetylamino}-phenyl)-
N-{2-[2-(4-Benzoyl-2-methylphenoxy)-acetylamino]-phe-
acetamide 9f Yield: 79%. M.P.: 87–89 °C. IR (KBr) ν
nyl}-2-[2-chloro-4-(4-chloro-benzoyl)-6-fluoro-phenoxy]-
max
−
1
(
(
cm ): 1685 (C=O), 1770 (amide, C=O), 3160–3240
acetamide 9i Yield: 88%. M.P.: 81–83 °C. IR (KBr) ν
max
1
−1
NH). H NMR (400 MHz) (CDCl ) δ (ppm): 2.54 (s, 6H,
(cm ): 1685 (C=O), 1750 (amide, C=O), 3130–3230
3
1
2
9
1
CH ), 4.76 (s, 4H, 2OCH ), 6.74–7.85 (m, 18H, Ar–H),
(NH). H NMR (400 MHz) (CDCl ) δ (ppm): 2.43 (s, 3H,
3
2
3
13
.58 (s, 2H, 2NH). C NMR (DMSO-d ) δ: 194.7 (2C=O),
CH ), 4.76 (s, 4H, 2OCH ), 6.78–7.76 (m, 18H, Ar–H),
6
3
2
1
3
65.5 (2CONH), 162.4 (2C, C1, C19), 139.7 (2C, C7,
9.86 (s, 2H, 2NH). C NMR (DMSO-d ) δ: 194.7 (2C=O),
6
C25), 134.9 (4CH, C8, C12, C26, C30), 132.8 (2C, C4,
C22), 130.0 (2CH, C3, C21), 129.3 (4CH, C9, C11, C27,
164.5 (2CONH), 163.3 (2C, C1, C19), 152.3 (C, C19),
145.8 (C, C1), 137.5 (C, C7), 136.2 (C, C5), 135.4 (C,

Medicinal Chemistry Research
1
C25), 133.9 (2C, C13, C14), 133.5 (2CH, C26, C30), 132.4
CH, C10), 131.7 (CH, C21), 130.4 (CH, C3), 129.8 (2CH,
(NH). H NMR (400 MHz) (CDCl ) δ (ppm): 2.43 (s, 9H,
3
(
3CH ), 4.81 (s, 4H, 2OCH ), 6.74–7.62 (m, 18H, Ar–H),
9.72 (s, 2H, 2NH). C NMR (DMSO-d ) δ: 194.2 (2C=O),
3
2
1
3
C27, C29), 128.7 (C, C4), 127.9 (2CH, C8, C12), 126.2 (C,
C22), 124.2 (2CH, C9, C11), 123.5 (CH, C23), 122.4 (CH,
C23),122.0 (2CH, C16, C17), 121.2 (C, C20), 120.8 (2CH,
C15, C18), 119.4 (C, C2), 117.6 (CH, C6), 116.4 (CH,
C24), 65.2 (2OCH ), 20.4 (CH ), 15.3 (2CH ). LC-MS m/z
6
166.9 (2CONH), 165.2 (2C, C1, C19), 138.4 (C, C10),
136.4 (C, C25), 134.3 (C, C7), 133.6 (2C, C13, C14), 132.4
(2CH, C26, C30), 131.8 (2CH, C3, C27), 130.0 (2CH, C8,
C12), 129.8 (2C, C4, C22), 128.6 (2CH, C9, C11), 127.1
(2CH, C5, C23), 126.1 (C, C28), 124.1 (2CH, C16, C17),
122.1 (2C, C2, C20), 121.6 (2CH, C15, C18), 116.4 (2CH,
C6, C24), 65.2 (2OCH ), 19.2 (CH ), 15.3 (2CH ). LC-MS
2
3
3
6
86 (M+), 688 (M + 2), 690 (M + 4). Anal. Cal. for
C H Cl FN O (686): C, 68.83; H, 3.97; N, 4.09. Found:
3
7
27
2
2
6
C, 68.75; H, 3.87; N, 4.02%.
2
3
3
m/z 706 (M+), 708 (M + 2). Anal. Cal. for C H BrN O
3
9
33
2
6
N-{2-[2-(4-Benzoyl-2,5-dimethylphenoxy)-acetylamino]-
phenyl}-2-[4-(2-methyl-benzoyl)-2-methyl-phenoxy]-aceta-
mide 9j Yield: 76%. M.P.: 86–88 °C. IR (KBr) ν
(706): C, 66.39; H, 4.71; N, 3.97. Found: C, 66.35; H, 4.68;
N, 3.93%.
max
−
1
(
(
cm ): 1665 (C=O), 1750 (amide, C=O), 3160–3260
General procedure for the synthesis of 2-(4-Benzoyl-
phenoxy)-N-{2-[[2-(4-benzoyl-phenoxy)-acetyl]-(2-piperidin-
1-yl-ethyl)-amino]-phenyl}-acetamide 11a–l
1
NH). H NMR (400 MHz) (CDCl ) δ (ppm): 2.35 (s, 12H,
3
4
9
1
CH ), 4.81 (s, 4H, 2OCH ), 6.83–7.75 (m, 18H, Ar–H),
3 2
13
.86 (s, 2H, 2NH). C NMR (DMSO-d ) δ: 195.2 (2C=O),
6
67.7 (2CONH), 161.6 (C, C19), 154.9 (C, C1), 147.4 (C,
A mixture of compounds 9a–l (0.001 mol) and 1-(2-chlor-
oethyl)piperidine hydrochloride 10 (0.001 mol) in the pre-
sence of potassium tertiary butoxide (0.005 mol) and
DMSO (10 mL) was refluxed for 8–10 h then cooled. The
reaction was monitored on TLC, after the completion of the
reaction the residual mass was triturated with ice cold water
to remove potassium tertiary butoxide and DMSO then, it
was extracted with DCM (30 mL). The organic layer was
washed with a saturated sodium chloride solution (3 ×
20 mL), 10% sodium hydroxide solution (3 × 20 mL) fol-
lowed by distilled water (3 × 30 mL) and then dried over
anhydrous sodium sulfate. Finally the solvent was evapo-
rated to dryness and recrystallized with ethanol to afford the
title compounds 11a–l.
C6), 136.9 (C, C10), 134.0 (C, C25), 133.5 (C, C7), 133.1
C, C4), 132.5 (2C, C13, C14), 132.7 (2CH, C26, C30),
31.8 (CH, C21), 130.5 (2CH, C8, C27), 130.7 (2CH, C12,
(
1
C29), 130.9 (C, C22), 129.0 (2CH, C9, C11), 128.5 (CH,
C23), 127.4 (CH, C3), 127.2 (2CH, C16, C17), 126.3 (C,
C28), 124.2 (C, C20), 118.7 (C, C2), 115.2 (2CH, C15,
C18), 111.6 (CH, C5), 110.5 (CH, C24), 68.3 (OCH ), 67.5
2
(
OCH ), 16.7 (3CH ). LC-MS m/z 641 (M + 1). Anal. Cal.
2 3
for C H N O (640): C, 74.98; H, 5.66; N, 4.37. Found:
4
0
36
2
6
C, 74.88; H, 5.64; N, 4.32%.
N-(2-{2-[4-(4-Bromo-benzoyl)-2-methylphenoxy]-acetyla-
mino}-phenyl)-2-[2-methyl-4-(2-methyl-benzoyl)-phenoxy]-
acetamide 9k Yield: 87%. M.P.: 94–96 °C. IR (KBr) ν
max
−
1
(
(
cm ): 1675 (C=O), 1710 (amide, C=O), 3130–3250
NH). H NMR (400 MHz) (CDCl ) δ (ppm): 2.44 (s, 9H,
2-(4-Benzoyl-2-methylphenoxy)-N-{2-[2-(4-benzoyl-2-
methylphenoxy)-acetylamino]-phenyl}-N-(2-piperidin-1-yl-
ethyl)-acetamide 11a Yield: 89%. M.P.: 102–104 °C; IR
1
3
3
9
1
CH ), 4.69 (s, 4H, 2OCH ), 6.82–7.71 (m, 18H, Ar–H),
3 2
13
−1
.71 (s, 2H, 2NH). C NMR (DMSO-d ) δ: 194.4 (2C=O),
(KBr) νmax (cm ): 1685 (C=O), 1760 (amide, C=O),
6
1
66.3 (2CONH), 164.6 (2C, C1, C19), 155.7 (C, C8), 150.4
3160–3270 (NH). H NMR (400 MHz, DMSO-d ) δ (ppm):
6
(
(
C, C7), 143.5 (C, C25), 138.7 (2C, C13, C14), 136.8
2CH, C26, C30), 134.9 (CH, C10), 133.3 (2CH, C3, C21),
2.21–2.31 (m, 6H, ring-3CH ), 2.37 (t, 4H, ring-2NCH ),
2
2
2.72 (s, 3H, CH ), 3.29 (s, 3H, CH ), 3.58 (t, 2H, NCH ),
3
3
2
1
32.1 (2CH, C27, C29), 131.3 (2C, C4, C22), 130.7 (CH,
4.03 (t, 2H, CONCH ), 4.53 (s, 2H, OCH ), 4.72 (s, 2H,
2 2
13
C12), 129.9 (C, C4), 128.2 (CH, C9), 124.2 (2CH, C5,
C23), 122.7 (C, C28), 121.6 (CH, C11), 120.5 (2CH, C16,
C17), 119.8 (2C, C2, C20), 117.5 (2CH, C15, C18), 116.3
OCH ), 6.71–7.72 (m, 20H, Ar–H), 8.75 (s, 1H, NH).
C
2
NMR (DMSO-d ) δ: 184.8 (2C=O), 167.0 (CONH), 164.9
6
(CO–N), 162.5 (2C, C1, C19), 133.0 (2C, C7, C25), 132.4
(2C, C13, C14), 130.5 (2CH, C10, C28), 130.3 (2CH C3,
C21), 129.8 (4CH, C8, C12, C26, C30), 129.6 (2C, C4,
C22), 128.7 (4CH, C9, C11, C27, C29), 128.4 (2CH, C5,
C23), 128.2 (2CH, C16, C17), 127.0 (2C, C2, C20), 118.2
(
2CH, C6, C24), 67.9 (2OCH ), 20.3 (CH ), 16.6 (2CH ).
2 3 3
LC-MS m/z 706 (M+), 708 (M + 2). Anal. Cal. for
C H BrN O (706): C, 66.39; H, 4.71; N, 3.97. Found: C,
6
3
9
33
2
6
6.32; H, 4.63; N, 3.86%.
(2CH, C15, C18), 111.3 (2CH, C6, C24), 81.4 (CH2,
N-(2-{2-[4-(4-Bromo-benzoyl)-2-methylphenoxy]-acetyla-
mino}-phenyl)-2-[2-methyl-4-(4-methyl-benzoyl)-phenoxy]-
acetamide 9l Yield: 72%. M.P.:102–104 °C. IR (KBr)
OCH ), 78.2 (CH , OCH ), 56.2 (CH , NCH ), 51.7 (CH ,
2
2
2
2
2
2
NCH of morphiline ring), 46.0 (CH , CO-NCH ), 33.1
2
2
2
(2CH , CCH ), 31.3 (CH , CCH ), 15.5 (2CH ). LC-MS m/
2
2
2
2
3
−
1
νmax (cm ): 1635 (C=O), 1750 (amide, C=O), 3160–3250
z 724.12 (M + 1). Anal. Cal. for C H N O (723.33): C,
45 45 3 6

Medicinal Chemistry Research
1
7
4.67; H, 6.27; N, 5.81. Found: C, 74.68; H, 6.26;
C=O), 3150–3260 (NH). H NMR (400 MHz, DMSO-d ) δ
6
N, 5.78%.
(ppm): 1.56–1.81 (m, 6H, ring-3CH ), 2.18 (t, 4H, ring-
2
2
NCH ), 2.35 (s, 3H, 3CH ), 2.42 (s, 3H, CH ), 2.55 (s, 3H,
2 3 3
2
-(4-Benzoyl-2,5-dimethylphenoxy)-N-{2-[2-(4-benzoyl-2,5-
CH ), 3.21 (t, 2H, NCH ), 3.64 (t, 2H, CONCH ), 4.79 (s,
3 2 2
dimethylphenoxy)-acetylamino]-phenyl}-N-(2-piperidin-1-
yl-ethyl)-acetamide 11b Yield: 90%. M.P.: 97–99 °C. IR
2H, OCH ), 4.90 (s, 2H, OCH ), 6.8–7.6 (m, 19H, Ar-H),
2 2
1
3
9.6 (s, 1H, NH). C NMR (DMSO-d ) δ: 187.2 (2C=O),
6
−
1
(
KBr) νmax (cm ): 1655 (C=O), 1740 (amide, C=O),
167.8 (CONH), 165.6 (CO–N), 139.9 (C, C8), 138.4 (C,
C7), 136.3 (C, C25), 135.7 (2C, C13, C14), 133.8 (C, C28),
132.4 (CH, C10), 131.7 (2CH, C3, C21), 131.3 (2CH, C26,
C30), 130.6 (C, 2CH, C4, C12, C22), 128.8 (CH, C9, 126.7
(2CH, C27, C29), 124.3 (2CH, C5, C23), 123.9 (CH, C11),
122.3 (2CH, C16, C17), 121.6 (2C, C2, C20), 120.7 (2CH,
C15, C18), 114.2 (2CH, C6, C24), 76.9 (CH , OCH ), 73.2
1
3
1
2
2
190–3270 (NH). H NMR (400 MHz, DMSO-d ) δ (ppm):
6
.51–1.83 (m, 6H, ring-3CH ), 2.13 (t, 4H, ring-2NCH ),
2
2
.31 (s, 6H, 2CH ), 2.35 (s, 3H, CH ), 2.49 (s, 3H, CH ),
3
3
3
.73 (t, 2H, NCH ), 3.54 (t, 2H, CONCH ), 4.75 (s, 2H,
2
2
OCH ), 4.89 (s, 2H, OCH ), 6.56–7.62 (m, 18H, Ar-H),
2
2
13
9
1
.37 (s, 1H, NH). C NMR (DMSO-d ) δ: 186.8 (2C=O),
6
2
2
68.6 (CONH), 166.6 (CO-N), 164.8 (2C, C1, C19), 139.7
(CH , OCH ), 55.7 (CH , NCH ), 53.3 (CH , ring-NCH ),
2 2 2 2 2 2
(
1
2C, C7, C25), 138.5 (2C, C5, C23), 136.6 (2C, C13, C14),
35.5 (2CH, C10, C28), 133.5 (2CH, C3, C21), 132.4 (2C,
48.9 (CH , CO-NCH ), 27.6 (2CH , CCH ), 24.3 (CH ,
2 2 2 2 2
CCH ), 16.3 (CH ), 11.0 (2CH ). LC-MS m/z 738.35 (M +
2 3 3
C4, C22), 131.3 (4CH, C8, C12, C26, C30), 129.1 (4CH,
C9, C11, C27, C29), 126.6 (2CH, C15, C18), 124.5 (2C,
C2, C20), 122.2 (2CH, C16, C17), 117.8 (2CH, C6, C24),
1). Anal. Cal. for C H N O (737.35): C, 74.88; H, 6.42;
46 47 3 6
N, 5.69. Found C, 74.79; H, 6.34; N, 5.58%.
7
5
6.5 (CH , OCH ), 74.6 (CH , OCH ), 55.4 (CH , NCH ),
2-(4-Benzoyl-2,5-dimethylphenoxy)-N-(2-{2-[4-(4-bromo-
benzoyl)-2-methylphenoxy]-acetylamino}-phenyl)-N-(2-
piperidin-1-yl-ethyl)-acetamide 11e Yield: 82%. M.P.:
2
2
2
2
2
2
2.6 (CH , ring-NCH ), 48.6 (CH , CO–NCH ), 26.4
2
2
2
2
(
2CH , CCH ), 23.2 (CH , CCH ), 16.2 (2CH ), 12.4
2 2 2 2 3
−
1
(
2CH ). LC-MS m/z 752.36 (M + 1). Anal. Cal. for
90–92 °C. IR (KBr) νmax (cm ): 1665 (C=O), 1725
3
1
C H N O (751.36): C, 75.08; H, 6.57; N, 5.59. Found: C,
(amide, C=O), 3150–3250 (NH). H NMR (400 MHz,
4
7
49
3
6
7
4.98; H, 6.47; N, 5.48%.
DMSO-d ) δ (ppm): 1.61–1.88 (m, 6H, ring-3CH ), 2.15 (t,
6
2
4
H, ring-2NCH ), 2.31 (s, 3H, CH ), 2.49 (s, 3H, CH ),
2 3 3
N-{2-[2-(4-Benzoyl-2-methylphenoxy)-acetylamino]-phe-
nyl}-2-[2-methyl-4-(4-methyl-benzoyl)-phenoxy]-N-(2-piper-
idin-1-yl-ethyl)-acetamide 11c Yield: 78%. M.P.: 92–94 °C.
2.64 (s, 3H, CH ), 3.13 (t, 2H, NCH ), 3.57 (t, 2H,
3 2
CONCH ), 4.80 (s, 2H, OCH ), 4.97 (s, 2H, OCH ),
2
2
2
1
3
6.67–7.74 (m, 18H, Ar-H), 9.56 (s, 1H, NH). C NMR
−
1
IR (KBr) νmax (cm ): 1685 (C=O), 1710 (amide, C=O),
(DMSO-d ) δ: 189.2 (2C=O), 166.4 (CONH), 164.3
6
1
3
1
2
140–3250 (NH). H NMR (400 MHz, DMSO-d ) δ (ppm):
(CO–N), 162.8 (C, C19), 145.6 (C, C1), 139.5 (C, C7),
138.3 (C, C5), 137.6 (C, C25), 136.3 (2C, C13, C14), 134.8
(2CH, C26, C30), 134.4 (CH, C10), 133.4 (CH, C21),
133.6 (CH, C3), 132.7 (2CH, C27, C29), 131.4 (C, C4),
130.4 (2CH, C8, C12), 130.1 (C, C22), 128.5 (2CH, C9,
C11), 127.7 (CH, C23), 126.2 (C, C28), 124.6 (2CH, C16,
C17), 123.5 (C, C20), 121.3 (2CH, C18, C15), 120.2 (C,
C2), 116.3 (CH, C6), 113.4 (CH, C24), 76.2 (CH , OCH ),
6
.60–1.88 (m, 6H, ring-3CH ), 2.16 (t, 4H, ring-2NCH ),
.39 (s, 3H, 3CH ), 2.44 (s, 3H, CH ), 2.62 (s, 3H,
2
2
3
3
CH ),2.75 (t, 2H, NCH ), 3.57 (t, 2H, CONCH ), 4.81 (s,
3
2
2
2
9
1
H, OCH ), 4.87 (s, 2H, OCH ), 6.72–7.82 (m, 18H, Ar-H),
2 2
13
.54 (s, 1H, NH). C NMR (DMSO-d ) δ: 187.3 (2C=O),
6
68.5 (CONH), 166.2 (CO-N), 142.4 (C, C10), 139.8 (C,
C25), 137.5 (C, C7), 135.3 (2C, C13, C14), 134.3 (C, C28),
33.2 (2CH, C3, C21), 132.4 (2CH, C26, C30), 130.6
2
2
1
74.3 (CH , OCH ), 56.5 (CH , NCH ), 53.8 (CH , ring-
2 2 2 2 2
(
(
4CH, C4, C8, C12, C22), 129.5 (2CH, C9, C11), 128.3
2CH, C27, C29), 126.1 (2CH, C5, C23), 125.8 (2CH, C16,
NCH ), 48.1 (CH , CO–NCH ), 27.7 (2CH , CCH ), 25.8
2 2 2 2 2
(CH , CCH ), 15.7 (CH ), 11.4 (2CH ). LC-MS m/z 815.23
2
2
3
3
C17), 124.5 (2CH, C2, C20), 121.1 (2CH, C15, C18), 116.8
(M+), 817.29 (M + 2). Anal. Cal. for C H BrN O
46 46 3 6
(
5
2CH, C6, C24), 76.9 (CH , OCH ), 74.8 (CH , OCH ),
6.4 (CH , NCH ), 54.0 (CH , ring-NCH ), 49.5 (CH ,
2 2 2 2 2
(815.26): C, 67.64; H, 5.68; N, 5.14. Found): C, 67.69; H,
5.61; N, 5.18%.
2
2
2
2
CO–NCH ), 26.4 (2CH , CCH ), 25.3 (CH , CCH ), 21.2
2
2
2
2
2
(
CH ), 12.4 (2CH ). LC-MS m/z 738.35 (M + 1). Anal. Cal.
2-[4-(4-Bromo-benzoyl)-2-methylphenoxy]-N-(2-{2-[4-(4-
bromo-benzoyl)-2-methyl phenoxy]-acetylamino}-phenyl)-
N-(2-piperidin-1-yl-ethyl)-acetamide 11f Yield: 88%. M.
3
3
for C H N O (737.35): C, 74.88; H, 6.42; N, 5.69.
Found: C, 74.79; H, 6.46; N, 5.67%.
4
6
47
3
6
−
1
P.: 95–97 °C. IR (KBr) νmax (cm ): 1695 (2C=O), 1750
1
N-{2-[2-(4-Benzoyl-2-methylphenoxy)-acetylamino]-phe-
nyl}-2-[2-methyl-4-(2-methyl-benzoyl)-phenoxy]-N-(2-piper-
idin-1-yl-ethyl)-acetamide 11d Yield: 89%. M.P.:
(amide, C=O), 3140–3250 (NH). H NMR (400 MHz,
DMSO-d ) δ (ppm): 1.53–1.86 (m, 6H, ring-3CH ), 2.16 (t,
6
2
4H, ring-2NCH ), 2.31 (s, 3H, CH ), 2.42 (s, 3H, CH ),
2
3
3
−
1
9
8–100 °C. IR (KBr) νmax cm 1695 (C=O), 1780 (amide,
2.73 (t, 2H, NCH ), 3.54 (t, 2H, CONCH ), 4.75 (s, 2H,
2 2

Medicinal Chemistry Research
OCH ), 4.89 (s, 2H, OCH ), 6.62–7.83 (m, 18H, Ar–H),
125.6 (4CH, C9, C11, C27, C29), 124.2 (CH, C5), 121.3
(2CH, C16, C17), 120.5 (C, C2), 120.4 (2CH, C15, C18),
119.7 (C, C20), 118.0 (CH, C24), 113.8 (CH, C6), 82.1
(CH , OCH ), 75.5 (CH , OCH ), 58.9 (CH , NCH ), 55.4
2
2
13
9
1
.42 (s, 1H, NH). C NMR (DMSO-d ) δ: 187.8 (2C=O),
68.5 (CONH), 165.7 (CO-N), 162.3 (2C, C1, C19), 138.6
6
(
2C, C7, C25), 137.4 (2C, C13, C14), 135.8 (4CH, C8,
2
2
2
2
2
2
C12, C26, C30), 132.2 (2CH, C3, C21), 131.6 (4CH, C9,
C11, C27, C29), 130.3 (2C, C4, C22), 129.6 (2CH, C5,
C23), 128.3 (2C, C10, C28), 127.6 (2CH, C16, C17), 124.3
(CH , ring-NCH ), 52.3 (CH , CO-NCH ), 28.6 (2CH ,
2 2 2 2 2
CCH ), 26.2 (CH , CCH ), 14.7 (CH ), 12.0 (2CH ). LC-
2
2
2
3
3
MS m/z 738.35 (M + 1). Anal. Cal. for C H N O
4
6
47
3
6
(
2C, C2, C20), 121.5 (2CH, C15, C18), 119.5 (2CH, C16,
(737.35) C, 74.88; H, 6.42; N, 5.69. Found: C, 74.75; H,
6.32; N, 5.51%.
C24), 77.5 (CH , OCH ), 74.8 (CH , OCH ), 54.9 (CH ,
2
2
2
2
2
NCH ), 52.5 (CH , ring-NCH ), 49.5 (CH , CO–NCH ),
2
2
2
2
2
2
6.7 (2CH , CCH ), 24.2 (CH , CCH ), 16.6 (2CH ). LC-
N-{2-[2-(4-Benzoyl-2-methylphenoxy)-acetylamino]-phe-
nyl}-2-[2-chloro-4-(4-chloro-benzoyl)-6-fluorophenoxy]-N-
(2-piperidin-1-yl-ethyl)-acetamide 11i Yield: 76%. M.P.:
2
2
2
2
3
MS m/z 879.15 (M + 1), 881.19 (M + 2), 883.21 (M + 4).
Anal. Cal. for C H Br N O (879.15): C, 61.30; H, 4.92;
N, 4.77. Found C, 61.29; H, 4.86; N, 4.74%.
4
5
43
2
3
6
−
1
123–125 °C. IR (KBr) νmax (cm ): 1670 (C=O), 1720
1
(
amide, C=O), 3110–3210 (NH). H NMR (400 MHz,
2
-(4-Benzoyl-2-methylphenoxy)-N-(2-{2-[4-(4-bromo-ben-
zoyl)-2-methylphenoxy]-acetyl amino}-phenyl)-N-(2-piperi-
din-1-yl-ethyl)-acetamide 11g Yield: 79%. M.P.:
02–104 °C. IR (KBr) νmax (cm ): 1665 (C=O), 1735
DMSO-d ) δ (ppm): 1.39–1.73 (m, 6H, ring-3CH ), 2.18 (t,
6
2
4H, ring-2NCH ), 2.36 (s, 3H, CH ), 2.73 (t, 2H, NCH ),
2
3
2
3.46 (t, 2H, CONCH ), 4.69 (s, 2H, OCH ), 4.73 (s, 2H,
2
2
−
1
13
1
OCH ), 6.42–7.69 (m, 18H, Ar–H), 9.77 (s, 1H, NH).
C
2
1
(
amide, C=O), 3140–3240 (NH). H NMR (400 MHz,
NMR (DMSO-d ) δ: 193.7 (2C=O), 170.2 (CONH), 168.5
6
DMSO-d ) δ (ppm): 1.43–1.79 (m, 6H, ring-3CH ), 2.26 (t,
(CO–N), 157.2 (C, C19), 150.5 (C, C1), 142.7 (C, C6),
140.8 (C, C25), 138.7 (C, C10), 137.7 (C, C7), 136.6 (C,
C4), 133.9 (2C, C13, C14), 132.6 (CH, C28), 130.5 (CH,
C21), 129.6 (2CH, C8, C12), 128.2 (2CH, C26, C30), 127.2
(C, C22), 126.7 (2CH, C11, C9), 125.3 (2CH, C27, C29),
124.2 (CH, C23), 123.2 (CH, C3), 122.1 (2CH, C16, C17),
121.5 (C, C20), 120.7 (C, C2), 118.5 (2CH, C15, C18),
115.6 (CH, C5), 113.9 (CH, C24), 83.5 (CH , OCH ), 81.1
6
2
4
H, ring-2NCH ), 2.38 (s, 3H, CH ), 2.47 (s, 3H, CH ),
2 3 3
2
.85 (t, 2H, NCH ), 3.65 (t, 2H, CONCH ), 4.62 (s, 2H,
2
2
OCH ), 4.83 (s, 2H, OCH ), 6.73–7.79 (m, 19H, Ar–H),
2
2
13
9
1
.83 (s, 1H, NH). C NMR (DMSO-d ) δ: 193.4 (2C=O),
6
68.8 (CONH), 166.4 (CO–N), 164.5 (2C, C1, C19), 139.5
(
(
C, C7), 137.4 (C, C25), 136.4 (2C, C13, C14), 134.8
2CH, C26, C30), 133.2 (CH, C10), 132.1 (2CH, C3, C2),
2
2
1
31.5 (2CH, C27, C29), 130.8 (2CH, C8, C12), 130.9 (2C,
C4, C22), 129.6 (2CH, C11, C9), 128.6 (2CH, C5, C23),
27.5 (C, C28), 123.0 (2CH, C10, C15), 122.7 (2C, C2,
C20), 119.6 (2CH, C15, C18), 113.3 (2CH, C6, C24), 77.5
(CH , OCH ), 69.7 (CH , NCH ), 68.4 (CH , ring-NCH ),
2 2 2 2 2 2
53.3 (CH , CO-NCH ), 27.6 (2CH , CCH ), 25.3 (CH ,
2
2
2
2
2
1
CCH ), 14.6 (2CH ). LC-MS m/z 795.23 (M+), 797.22 (M
2 3
+ 2), 799.28 (M + 4). Anal. Cal. for C H Cl FN O
6
4
4
40
2
3
(
(
CH , OCH ), 75.5 (CH , OCH ), 53.4 (CH , NCH ), 52.8
(795.23): C, 66.33; H, 5.06; N, 5.27. Found: C, 66.22; H,
4.96; N, 5.13%.
2
2
2
2
2
2
CH , ring-NCH ), 49.4 (CH , ring-NCH ), 23.3 (2CH ,
2
2
2
2
2
CCH ), 21.2 (CH , CCH ), 11.4 (2CH ). LC-MS m/z
2
2
2
3
8
01.24 (M+), 803.21 (M + 2). Anal. Cal. for
N-{2-[2-(4-Benzoyl-2,5-dimethylphenoxy)-acetylamino]-
phenyl}-2-[2-methyl-4-(2-methyl-benzoyl)phenoxy]-N-(2-
piperidin-1-yl-ethyl)-acetamide 11j Yield: 89%. M.P.:
C H BrN O (801.24): C, 67.33; H, 5.52; N, 5.23. Found:
C, 67.30; H, 5.44; N, 5.28%.
4
5
44
3
6
−
1
1
12–114 °C. IR (KBr) νmax (cm ): 1675 (C=O), 1710
1
N-{2-[2-(4-Benzoyl-2,5-dimethylphenoxy)-acetylamino]-
phenyl}-2-(4-benzoyl-2-methyl phenoxy)-N-(2-piperidin-1-
yl-ethyl)-acetamide 11h Yield: 82%. M.P.: 107–109 °C;
(amide, C=O), 3220–3320 (NH). H NMR (400 MHz,
DMSO-d ) δ (ppm): 1.49–1.78 (m, 6H, ring-3CH ), 2.23 (t,
6
2
4H, ring-2NCH ), 2.39 (s, 3H, CH ), 2.45 (s, 6H, 2CH ),
2
3
3
−
1
IR (KBr) νmax (cm ): 1670 (C=O), 1720 (amide, C=O),
2.54 (s, 3H, CH ), 3.24 (t, 2H, NCH ), 3.51 (t, 2H,
3 2
1
3
1
2
3
225–3325 (NH). H NMR (400 MHz, DMSO-d ) δ (ppm):
.72–1.83 (m, 6H, ring-3CH ), 2.22 (t, 4H, ring-2NCH ),
CONCH ), 4.71 (s, 2H, OCH ), 4.86 (s, 2H, OCH ),
2 2 2
6
1
3
6.81–7.72 (m, 18H, Ar-H), 9.69 (s, 1H, NH). C NMR
2
2
.45 (s, 3H, CH ), 2.62 (s, 6H, 2CH ), 2.82 (t, 2H, NCH ),
(DMSO-d ) δ: 196.8 (2C=O), 173.1 (CONH), 169.2 (CO-
3
3
2
6
.61 (t, 2H, CONCH ), 4.75 (s, 2H, OCH ), 4.90 (s, 2H,
N), 168.5 (C, C1), 147.5 (C, C19), 144.7 (C, C8), 141.5 (C,
C7), 140.9 (C, C28), 135.4 (C, C23), 133.6 (2C, C14),
132.7 (CH, C10), 131.3 (CH, C3), 130.2 (CH, C21), 130.2
(2CH, C30), 130.1 (C, CH, C4, C12), 129.2 (CH, C9),
129.5 (2CH, C27, C29), 128.4 (CH, C5), 127.5 (CH, C11),
126.3 (2CH, C16, C17), 124.6 (C, C2), 123.8 (2CH, C15,
C18), 122.7 (1C, C20), 120.8 (CH, C24), 119.7 (CH, C6),
2
2
1
3
OCH ), 6.62–7.73 (m, 19H, Ar–H), 9.84 (s, 1H, NH).
NMR (DMSO-d ) δ: 190.4 (2C=O), 171.6 (CONH), 169.3
C
2
6
(
CO–N), 168.4 (C, C1), 143.6 (C, C19), 139.5 (2C, C7,
C25), 138.7 (C, C23), 136.7 (2C, C13, C14), 134.9 (2CH,
C10, C28), 133.8 (CH, C3), 132.5 (CH, C21), 130.4 (C,
C22), 128.8 (4CH, C8, C12, C26, C30), 127.4 (C, C4),

Medicinal Chemistry Research
7
8.73 (CH , OCH ), 76.8 (CH , OCH ), 68.9 (CH , NCH ),
CCH ), 19.8 (CH ), 15.1 (2CH ). LC-MS m/z 815.21
2 3 3
2
2
2
2
2
2
6
7.5 (CH , ring-NCH ), 55.1 (CH , CO-NCH ), 30.5
(M+), 817.24 (M + 2). Anal. Cal. for C H BrN O
2
2
2
2
46 46 3 6
(
(
2CH , CCH ), 28.9 (CH , CCH ), 18.4 (CH ), 17.53
(815.26): C, 67.64; H, 5.68; N, 5.14. Found: C, 66.26; H,
4.58; N, 5.02%.
2
2
2
2
3
CH ), 13.22 (2CH ). LC-MS m/z 752.35 (M + 1). Anal.
3
3
Cal. for C H N O (751.36): C, 75.08; H, 6.57; N, 5.59.
4
7
49
3
6
Found: C, 74.98; H, 6.47; N, 5.44%.
Biology
N-(2-{2-[4-(4-Bromo-benzoyl)-2-methylphenoxy]-acetyla-
mino}-phenyl)-2-[2-methyl-4-(2-methyl-benzoyl)-phenoxy]-
N-(2-piperidin-1-yl-ethyl)-acetamide 11k Yield: 85%. M.
Animal Cell culture and growth inhibition studies
The A549, MCF-7, A375, HepG2, ACHN, B16F10 cells
were cultured in DMEM, enriched with 10% FBS with
penicillin and streptomycin and maintained in a 98%
−
1
P.: 109–111 °C. IR (KBr) νmax (cm ): 1670 (C=O), 1735
1
(
amide, C=O), 3125–3225 (NH). H NMR (400 MHz,
DMSO-d ) δ (ppm): 1.42–1.73 (m, 6H, ring-3CH ), 2.19 (t,
humidified atmosphere containing 5% CO at 37 °C. The
6
2
2
4
H, ring-2NCH ), 2.30 (s, 3H, CH ), 2.35 (s, 3H, CH ),
ability of the series of compounds piperidine conjugated
with benzophenone 11a–l to inhibit cancer cell proliferation
was assessed by tryphan blue dye exclusion, MTT assay
and lactate LDH release assay as reported previously
(Thirusangu et al. 2017) and IC50 values were calculated. In
2
3
3
2
.51 (s, 3H, CH ), 2.80 (t, 2H, NCH ), 3.49 (t, 2H,
3
2
CONCH ), 4.79 (s, 2H, OCH ), 4.85 (s, 2H, OCH ),
2
2
2
13
6
.81–7.79 (m, 18H, Ar–H), 9.63 (s, 1H, NH). C NMR
(
(
(
DMSO-d ) δ: 195.1 (2C=O), 170.3 (CONH), 166.5
CO–N), 164.5 (2C, C1, C19) 145.4 (2C, C1, C19), 141.3
C, C8), 138.4 (C, C7), 135.6 (C, C25), 134.5 (2C, C13,
6
3
brief, cells were seeded at 4 × 10 cells per well into 96-well
plate, cultured overnight and treated with increasing con-
centration of compounds 11a–l (0, 2, 5, 10, 25, and 50 µM
in DMSO) diluted in a medium containing 0.5% FBS for
48 h.
C14), 131.7 (2CH, C26, C30), 132.3 (CH, C10), 131.2
2CH, C3, C21), 130.6 (2CH, C27, C29), 129.5 (2C, CH,
C4, C22, C12), 129.1 (CH, C9), 128.6 (2CH, C5, C23),
(
1
1
27.6 (C, C28), 125.5 (CH, C11), 124.3 (2CH, C16, C17),
22.8 (2C, C2, C20), 121.3 (2CH, C15, C18), 116.6 (2CH,
In vitro treatment of compound 11f
C6, C24), 78.7 (CH , OCH ), 76.5 (CH , OCH ), 60.4
2
2
2
2
(
CH , NCH ), 59.9 (CH , ring-NCH ), 52.2 (CH ,
The B16F10 cells were cultured, serum starved for 2 h and
treated with varied concentration of compound 11f (0, 4,
and 8 μM) for 48 h along with appropriate vehicle control
wherever necessary for further analysis.
2
2
2
2
2
CO–NCH ), 31.7 (2CH , CCH ), 29.1 (CH , CCH ), 19.4
(
+
2
2
2
2
2
CH ), 15.1 (2CH ). LC-MS m/z 815.25 (M+), 817.23 (M
3 3
2). Anal. Cal. for C H BrN O (815.26): C, 67.64; H,
46 46 3 6
5
.68; N, 5.14. Found C, 67.55; H, 5.58; N, 5.03%.
Annexin V staining
N-(2-{2-[4-(4-Bromo-benzoyl)-2-methylphenoxy]-acetyla-
mino}-phenyl)-2-[2-methyl-4-(4-methyl-benzoyl)-phenoxy]-
N-(2-piperidin-1-yl-ethyl)-acetamide 11l Yield: 75%. M.
The cultured B16F10 cells were treated with compound 11f
(0, 4, and 8 µM) for 48 h and stained with Annexin V
staining-FITC Fluorescence Microscopy Kit (Malojirao
et al. 2018) as per manufacturer’s instruction and images
were documented.
−
1
P.:102–104 °C. IR (KBr) νmax (cm ): 1675 (C=O), 1730
1
(
amide, C=O), 3120–3220 (NH). H NMR (400 MHz,
DMSO-d ) δ (ppm): 1.51–1.83 (m, 6H, ring-3CH ), 2.13 (t,
6
2
4
2
H, ring-2NCH ), 2.34 (s, 9H, CH ), 2.44 (s, 3H, CH ),
2 3 3
.65 (s, 3H, CH ), 2.73 (t, 2H, NCH ), 3.54 (t, 2H,
TUNEL assay
3
2
CONCH ), 4.75 (s, 2H, OCH ), 4.89 (s, 2H, OCH ),
2
2
2
13
6
.71–7.62 (m, 18H, Ar–H), 9.73 (s, 1H, NH). C NMR
The B16F10 cells were cultured on poly-L-lysine pre coated
cover slips and exposed with a lead compound 11f (0, 4,
and 8 µM) for 48 h. Cells were fixed with 4% paraf-
ormaldehyde for 15 min and permeabilized by 0.25% triton
X-100 for 20 min and TUNEL was performed as per
manufacturer’s instruction (Malojirao et al. 2018).
(
(
DMSO-d ) δ: 194.8 (2C=O), 166.6 (CONH), 165.3
CO–N), 152.4 (2C, C1, C19), 138.5 (C, C10), 136.2 (C,
6
C25), 134.4 (C, C7), 133.6 (2C, C13, C14), 132.4 (2CH,
C26, C30), 131.4 (2CH, C3, C21), 130.7 (2CH, C27, C29),
1
29.8 (2C, C4, C22), 128.4 (2CH, C8, C12), 127.3 (2CH,
C9, C11), 126.2 (2CH, C5, C23), 124.1 (C, C28), 123.1
2CH, C16, C17), 122.6 (2C, C2, C22), 120.7 (2CH, C15,
C18), 118.5 (2CH, C6, C24), 82.6 (CH , OCH ), 78.5
(
Cell fractionation and preparation of cell lysates
2
2
(
4
CH , OCH ), 61.2 (CH , NCH ), 59.3 (CH , ring-NCH ),
9.5 (CH , CO-NCH ), 27.2 (2CH , CCH ), 24.3 (CH ,
2 2 2 2 2
The whole cell lysate was prepared as mentioned before
(Mohammed et al. 2018). In brief, cell treated with or
2
2
2
2
2
2

Medicinal Chemistry Research
without compound 11f (0, 4, and 8 μM), were harvested and
whole cell extract was prepared using Radio immune pre-
cipitation assay buffer (RIPA buffer) (100 mM tris pH-7.5,
1% triton X-100, 0.1% sodium dodecyl sulfate (SDS),
140 mM NaCl, 0.5% sodium deoxycholate, 5 mM EDTA,
0.5 mM phenylmethylsulfonyl fluoride (PMSF), and

Medicinal Chemistry Research
Scheme 1 Synthesis of piperidine appended benzophenone analogs
via amide linkage 2-(4-Benzoyl-phenoxy)-N-{2-[[2-(4-benzoyl-phe-
noxy)-acetyl]-(2-piperidin-1-yl-ethyl)-amino]-phenyl}-acetamide ana-
logs (11a–l). Reaction conditions and yield: (i) Aq. NaOH, stirring
Molecular modeling studies
The molecular operating environment (MOE) 2015 was
used for in silco studies which were performed on windows
2010 version. The protein coding gene (Bcl-2 and XIAP)
which is an apoptosis regulator was imported from the
protein data bank (PDB ID: 1gjh and 2opz) and the protein
was visualized using sequence option and non interacting
water molecules were removed. The partial charge of the
protein was adjusted, using the force field method AMBER
0
2
–5 °C for 2–3 h, yield: 82–93%, (ii) Anhy. AlCl3, 150–170 °C for
–3 h, yield: 72–85%, (iii) ClCH COOC H /Dry Acetone, K CO ,
2
2
5
2
3
Reflux, 60 °C for 8–10 h, yield: 89–93%, (iv) Aq. NaOH/Ethanol,
Reflux, for 5–6 h, yield: 84–94%, (v) TBTU/Lutidine, Dry DCM,
Stirring 0–5 °C for 30 min then, overnight at RT, yield: 80–94%, (vi)
TBTU/Lutidine, Dry DCM, Stirring 0–5 °C for 30 min then, overnight
at RT, yield: 71–90%, (vii) Piperidine hydrochloride/DMSO, KOtBu,
Reflux, 8–10 h, yield: 75–90%
99. Later, the protein was subjected to 3D protonation at cut
off 12.0, and further hydrogen was added according to
standard geometry and the enzyme was energy minimized
using force field MMFF94x at 0.01 KJ per mole gradients.
The ligand preparation was done by drawing the structure of
ligand by using a marvin Sketch, and adjusting the partial
charges using Hamilton MMFF94 force field method and
subsequently 3D protonation and hydrogen addition was
performed according to standard geometry. Ligands were
energy minimized at cut off 12 using MMFF94x force field
at 0.01 KJ per mole gradient. Docking was performed using
the option simulation followed by dock on selected active
site amino acids using sequence option, and further docked
with setting options such as: receptor and solvent, selected
residues, alpha triangle, affinity dG, force field refinement,
and best 10 pose. After obtaining docking results, out of the
protease inhibitor cocktail. All cellular fractioned proteins
were quantified in Nanodrop- biospectrophotometer
(
Eppendorf, Germany).
Immunoblot (IB) analysis
The cell lyastes were separated on a precast SDS poly-
acrylamide gels, transferred to polyvinylidene difluoride
membrane, probed with appropriate antibodies and detected
with the BCIP-NBT chromogen (Thirusangu et al. 2017).
The bands were densitometrically analyzed and
documented.
Animals and ethics
10 best posed resulted for each chemical structure, best pose
The BALB/c mice (27–30 g) were used throughout the
study and maintained as per The Committee for the Purpose
of control and supervision of experiments on animals
guidelines with ethical clearance (NCP/IAEC/CL/101/05/
was retained. The resultant best pose score values in the
series were used for analysis of docking and interaction.
Statistical analysis
2
013-14).
Values were represented as mean ± standard deviation (SD).
Statistical significance was evaluated by one-way analysis
of variance (ANOVA) followed by two-tailed 13 Student’s
t-test. MS excel 8.1 version software was used for data
analysis and statistical significant values were expressed as
*p < 0.05 and **p < 0.01.
Melanoma tumor model development and treatment
The murine melanoma B16F10 cell lines cultured as pro-
cedure depicted earlier (Overwijk and Restifo 2001). The
cultured B16F10 cells were harvested by trypsinization
using 0.5% trypsin. The cell were centrifuged at 3000 rpm
for 5 min and washed with DMEM media thrice. A mela-
noma tumor model was developed by injecting B16F10
Results and discussion
6
cells (1 × 10 cells) into the skin of male mice sub-
cutaneously (s.c.). The tumor growth was analyzed by the
black spot size on the skin of each mices (Overwijk and
Restifo 2001). The animals were segregated into three
groups with n = 6. One group served as control and the
other two groups received compound 11f (10 and 20 mg/kg
body weight) thrice a week into the peripheral sites of
tumors for 3 weeks. At the termination of the experiments,
all animals were sacrificed, tumors were photographed,
excised and weighed, and the volumes [0.5 (length ×
Chemistry
The synthesis of the title compounds 11a–l was accom-
plished by a synthetic procedure as shown in Scheme 1. All
1
the synthesized compounds were established by IR, H
1
3
NMR, C NMR, and mass spectral data. First, the ben-
zoylated products 3a–f were synthesized by the benzoyla-
tion of substituted phenols 1a–c with substituted benzoyl
chlorides 2a–d under low temperature. Fries rearrangement
of compounds 3a–f, using anhydrous aluminum chloride as
a catalyst was then carried out under neat condition, to
2
breadth )] were calculated and a part of tumor tissue was
used for lysate preparation.

Medicinal Chemistry Research

Medicinal Chemistry Research
Fig. 1 Compound 11f induces
apoptotic hallmarks. The
B16F10 cell lines treated with
compound 11f at 0, 3 and 5 µM
for 48 h and used for further
experiments. a Annexin V
immunostaining images of
bright field and fluorescent
images. b TUNEL positive cells
of control, DNase treated
(
1
positive control) and compound
1f treated fluorescence.
Hoechst counter stains and
merged images
afford hydroxy benzophenones 4a–f. The compounds 4a–f
on etherification with ethyl chloroacetate using dry acetone
as a solvent gave substituted ethyl 2-(4-benzoylphenoxy)
acetates 5a–f. Further, the compounds 5a–f on refluxing
with aqueous sodium hydroxide in ethanol gave (4-benzoyl-
phenoxy)-acetic acids 6a–f. The compounds 6a–f were
coupled with o-phenylenediamine 7 in the presence of 2,6
lutidine and TBTU as a coupling agent and dichlor-
omethane (DCM) as a solvent to yield substituted N-(2-
Then, all the substituted compounds 6a–f, on treatment with
the compounds 8a–f using coupling agent TBTU and luti-
dine as a catalyst, afforded the expected products 9a–l in a
good yield. Finally, the condensation of 9a–l with 1-(2-
chloroethyl)piperidine hydrochloride 10 for 6 h in the pre-
sence of potassium tertiary butoxide and dimethyl sulph-
oxide (DMSO), furnished the title compounds 11a–l.
Among 3a–f series the spectrum of compound 3a is taken
as a representative example and the formation of this
compound was confirmed by the appearance of the carbonyl
amino-phenyl)-2-(4-benzoyl-phenoxy)-acetamides
8a–f.

Medicinal Chemistry Research
stretching band for the ester group at 1715 cm− in the IR
spectrum and the appearance of nine aromatic protons
between 7.11 and 8.25 ppm in proton NMR spectrum. The
mass spectrum of compound 3a gave significant stable M +
1
1
peak at m/z 213 which also evident for the formation of
compound 3a. Further, the spectrum of compound 4a, was
considered as a representative example of the series 4a–f.
The IR spectrum of the compound 4a, was established by
the disappearance of the carbonyl stretching band of the
ester group of compound 3a and appearance of the OH
−
1
stretching band at 3530–3630 cm . And also, the appear-
ance of broad singlet for OH proton at δ 12.01 ppm and
decrease in one aromatic proton between 6.73 and 7.68 ppm
in proton NMR spectrum. The mass spectrum of compound
4a offered significant stable M + 1 peak at m/z 213 which is
considered as additional evidence for the formation of this
compound. Besides, the compound 5a was taken as a
representative example for the 5a–f series, which was
confirmed by the disappearance of the OH stretching of
compound 4a and appearance of the carbonyl stretching
band for the ester group at 1755 cm− in the IR absorption
spectrum. The proton NMR observations of compound 5a
revealed the disappearance of broad singlet for the OH
proton of compound 4a and appearance of a triplet and
1
Fig. 2 Compound 11f modulating Bcl-2 gene related proteins in vitro:
The B16F10 cell lines treated with compound 11f at 0, 3, and 5 µM for
4
8 h. Whole cell lysate fractions were used for IB. The differential
expression of Bcl-2, XIAP, Bax, Bad, Caspase-3, and β actin. Note:
Statistically significant values expressed as *p < 0.05 and **p < 0.01
quartet for CH and CH protons at δ 2.31 and 4.13 ppm,
3
2
respectively. Also, mass spectrum gave significant stable M
+
1 peak at m/z 299 which clearly affirmed the formation of
Fig. 3 Compound 11f regresses
murine melanoma by activating
modulating Bcl-2 gene related
proteins in vivo: The B16F10
6
cells (1 × 10 ) were tail vein
injected into the BALB/c mice.
The mice received 0, 10, and
2
0 mg/kg/ b. wt compound 11f
every-other day after 15 days of
induction. a Anatomy of skin
exhibiting tumor colonization
and quantification of the tumor
nodules in the skin. b IB studies
signifying the altered
translational expression of genes
such as Bcl-2, XIAP, Bax, Bad,
caspase-3, and β-actin

Medicinal Chemistry Research
compound 5a. Similarly the formation of compound 6a was
confirmed with the appearance of carbonyl and OH groups
stretching bands of carboxylic acid at 1720 and
420–3520 cm− respectively, and disappearance of car-
1
3
bonyl stretching of ester group of compound 5a in the IR
spectrum. In proton NMR, the appearance of singlet peak of
COOH proton at δ 13.13 ppm and disappearance of triplet
and quartet peaks for CH and CH protons, respectively, of
3
2
compound 5a has confirmed the formation of the compound
a. The mass spectrum of compound 6a gave significant
6
stable M + 1 peak at m/z 271 which also affirmed the for-
mation of the compound 6a. Likewise, the structure of
compound 8a was confirmed by the disappearance of the
carbonyl and OH stretching bands of carboxylic acid group
of compound 6a and appearance of NH and NH stretching
2
−
1
bands in the range 3110–3230 and 3315–3410 cm
respectively in the IR spectrum. Also, in proton NMR, the
appearance of NH and NH protons at δ 4.73 and 9.27 ppm,
2
respectively, and an increase in four aromatic protons
confirmed the formation of the product 8a. The mass
spectrum of compound 8a gave significant stable M + 1
peak at m/z 361 which also proves the formation of the
compound 8a. Moreover, the spectrum of the compound 9a
was considered as a representative example for the series
Fig. 4 Compound 11f interacts strongly with Bcl-2 and XIAP proteins.
a 3D structure of the compound 11f in the active site C-terminal trans
activation Bcl-2 and hydrogen-bond interaction view of the ligand
molecule 11f with AlA43 an Glu42 in Bcl-2 protein. b 3D structure of
the compound 11f in the active site of XIAP and hydrogen-bond
interaction view of the ligand molecule 11f with GLN399 in XIAP
protein
Fig. 5 Interaction of compound 11f with Bcl-2 and XIAP. a The compound 11f at the pocket site and residue amino acid at the pocket site of the
complex Bcl2-11f. b The compound 11f at the pocket site and residue amino acid at the pocket site of the complex XIAP-11f

Medicinal Chemistry Research
Fig. 6 a The 3D interactions of the compound 11f with hydrophobic
amino acids, which represented by yellow color in Bcl-2 protein. b
The 3D interactions of the compound 11f with hydrophobic amino
acids by red colour in XIAP protein. c The 2D interactions analysis of
the compound 11f with Bcl-2 protein. d The 2D interactions analysis
of the compound 11f with XIAP protein (color figure online)
9
a–l. This was supported by the disappearance of COOH
confirmed the formation of title product. The mass spectrum
of compound 11a exhibited M + 1 peak at m/z 724 which
also revealed the formation of the title compound 11a.
and NH stretching of the compound 6a and 8a respec-
tively, and the appearance of only NH stretching between
3
2
−
1
100 and 3210 cm in the IR spectrum. Interestingly,
compound 9a is a symmetrical molecule, and it was also
Pharmacology
proved by the NMR spectrum by the disappearance of
COOH proton of 6a and NH proton of 8a and appearance
Compound 11f is a lead compound and its structure
activity relationship (SAR)
2
of singlet of NH proton at δ 9.67 ppm. Besides, there is an
increase in aromatic protons in range δ 6.95–7.65 ppm and
mass spectrum of compound 9a gave significant stable M +
In the current decade, it’s been constantly observed that the
increased occurrence of cancer worldwide and the
immediate necessity of novel anti-neoplastic compounds
target specific action. The piperidine derivatives are found
to possess diverse pharmacological activities and form an
essential part of the molecular structure of important drugs
(Khanum et al. 2009; Hu et al. 2014; Wang et al. 2015; Xin-
Hua et al. 2012). Similarly benzophenone analogs are also
great importance fundamentally due to their significant
biological activity both in vitro and in vivo (Al-Ghorbani
1
peak at m/z 613 which clearly affirmed the formation of
product 9a. Finally, in the title compounds series 11a–l,
compound 11a is taken as a representative example to
explain characterizations. The structure was confirmed by
the appearance of 14 CH protons in upfield region and two
2
types of methyl protons at δ 2.72 and 3.29 ppm and OCH2
protons at δ 4.53 and 4.72 ppm and the multiplet signal in
the range δ 6.71–7.72 ppm for aromatic protons as well as a
singlet at 8.75 for NH proton in proton NMR spectrum

Medicinal Chemistry Research
Fig. 7 The sequences of complex Bcl-2-11f and XIAP-11f are arranged to their target sites
et al. 2017; Khanum et al. 2005; Puttaswamy et al. 2018;
Ranganatha et al. 2013; Revesz et al. 2004; Zabiulla et al.
with three independent assay types such as 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
lactate dehydrogenase (LDH) and trypan blue assay. The
growth inhibition value inferred increasing cytotoxicity
against B16F10 > A375 > A549 > ACHN > HepG2 > MCF7
cells (Table 1). The melanoma cell lines of both murine and
human origin showed maximum sensitivity at 8 µM more or
less in all three independent experiments.
2
016). In the event of exploring the new set of anticancer
molecules here we conjugated the two pharmacologically
active backbones namely, benzophenone and piperdine,
appended with amide bond linkage by substituting with
various halogens and methyl groups at different position of
the rings. The antiproliferative studies included the cancers
of different origin viz, melanoma, lung adenocarcinoma,
renal cell carcinoma, hepatocellular, and breast carcinoma
Detailed SAR revealed that, the compound 11f with two
bromo groups at the para position in rings A and E and two

Medicinal Chemistry Research
Fig. 8 Mapping molecular
electrostatic potentials of
compound 11f with Bcl-2 and
XIAP: a Separated conformers
and molecular surfaces using
ribbon models and conformer
explorer of the compound 11f
with amino acid residue in Bcl-
2
. b Separated conformers and
molecular surfaces using ribbon
models and conformer explorer
of the compound 11f with amino
acid residue in XIAP
Fig. 9 a Pose organizer viewer
of the compound 11f with
residue amino acid. b Displays
electrostatic interactions of Bcl-
2
proteins with the compound
1
1f. c Preparation view changed
hydrogen view. d Displays
hydrophobic groups: green is
hydrophobic, orange is
hydrophilic of Bcl-2 protein
with the compound 11f (color
figure online)
methyl groups at ortho position in rings B and D showed a
maximum inhibitory effect of 8 and 10 µM on B16F10 and
A375 cells respectively. The results were not significant
against A549, MCF-7, ACHN, and HepG2 cell lines. The
compounds 11e, 11g, 11k, and 11l with a single bromo
group at the para position of ring E and more number of
CH3 groups at different position did not show anti-
proliferative effect, but indicated the role of bromo and
methyl groups in the biological activity, showing slight
cytotoxicity (Table 1). Whereas the compound 11i with the
other halogens like chloro, fluro and with methyl substituent
has no activity. Further, compounds 11a, 11b, 11c, 11d,
11h, and 11j with only methyl substituent has also did not
displayed activity. The overall inference is the compound
11f with two bromo and two methyl groups at different
position in the rings is responsible for the biological activity
and hence chosen as a lead compound for elucidation of
molecular mechanisms in detail (Table 1).

Medicinal Chemistry Research
Fig. 10 a Pose organizer viewer
of the compound 11f with
residue amino acid. b Displays
electrostatic interactions of the
XIAP protein with the
compound 11f. c Preparation
view changed hydrogen view. d
Displays hydrophobic groups:
green is hydrophobic, orange is
hydrophilic of XIAP protein
with the compound 11f (color
figure online)
Compound 11f triggers cellular apoptotic hallmarks
fluorescence microscopy (Fig. 1a). Framing to more sup-
portive evidence for apoptotic cell damage, DNA damage
was accomplished by TUNEL assay, which detects specific
DNA breaks by end labeling. Upon 11f (0, 4, and 8 µM)
treatment to B16F10 cells, control cells did not show any
significant TUNEL positive nuclei, whereas the percentage
of the same was 31.3% and 46.2% respectively, at two
different concentrations (Fig. 1b). The efficacy was com-
pared with positive control DNAse treatment and counter-
stained with Hoechst stain for specific nuclear localization.
Understanding of cellular process that contribute to cancer,
has increased substantially since Hanahan and Weinberg
detailed the hallmark of cancer, including emerging ideas
and failure to activate the same results in anticancer
response (Hanahan and Weinberg 2011). Thus, one can
foresee a future paradigm where activation of specific
pathways of cell death is still intact and predict the response
to therapies (Ricci and Zong 2006; Plati et al. 2011). Rea-
lizing this point, herewith we have provided an experi-
mental evidence of novel pharmacophore compound 11f
which can target melanoma through the activation of cell
death pathways in a systemic investigation. The mechanism
of cytotoxicity was explored for cellular and molecular
changes in B16F10 cells treated with or without compound
Compound 11f induces double inhibition of X-linked IAP
(XIAP) and Bcl-2 and elevates pro-apoptotic gene
expression
Apoptosis is mainly regulated in two predominant path-
ways; intrinsic and extrinsic apoptotic pathway. Which
includes a series of biochemical changes such as disruption
of the mitochondrial membrane and release of apoptogenic
molecules to cytosol, activation of caspase cascade, induc-
tion of DNA fragmentation. The members of the Bcl-2
family mainly regulate the intrinsic apoptotic signaling
pathway, which comprises of both pro-apoptotic (Bax and
Bad) and anti-apoptotic (Bcl-2) proteins (Thomadaki and
Scorilas 2006). Balance between these two groups decides
the fate of the cells. On the induction of apoptotic stimuli,
1
1f (0, 4, and 8 µM) by annexin V staining and terminal
deoxynucleotidyl transferase dUTP nick end labeling
TUNEL) assay. Translocation of phosphatidyl serine from
(
inner to outer leaflet of the cell membrane is typical hall-
marks of apoptotic event which can be measured by
Annexin V-FITC staining. Flip-flop of B16F10 cell mem-
brane was evident in a concentration dependent manner
which absolutely absent in untreated cells. The intensity of
the fluorescence is a direct measure of the exposed annexin
molecules in 11f treated cells, which was observed under

Medicinal Chemistry Research
Fig. 11 a Ramachandran plot showing the phi/psi values of the
modeled Bcl2 protein. Green color- most favorable regions, red color
region- allowed region, pale yellow- generously allowed region and
white color- disallowed regions. b Graph presenting the rotamer
energy plots of the Bcl-2 protein. c Residue clashing profile of amino
acid residues between this system in production stage run. d The
−1
energy score (kcal mol ) of the complex Bcl2-11f with lowest
−
1
binding energy −8.9 kcal mol (color figure online)
the balance shifts towards the pro-apoptoic proteins and
facilitates the release of apoptogenic molecules. This results
in proteolytic cleavage and maturation of caspases (Cas-
pase-3) to execute cellular fatality. On the other hand, the
majority of the tumors tend to express the higher basal level
of active caspase-3 without apoptotic induction. The
intrinsic drive to apoptosis is restrained by concomitant
upregulation of IAP (XIAP) which counteracts the caspase
activity in tumors (Yang and Cao 2003). Considering the
facts, targeting anti-apoptotic (Bcl-2 and XIAP) in cancer
cells might increase the therapeutic effect by shifting the
balance in favor of pro-apoptotic molecules.
protein expression. In addition to measuring expression
levels of members of the Bcl-2 family, the changes in
expression of IAP family proteins were also investigated.
The results show that the expression of two IAP family
proteins, namely XIAP, were reduced following treatment
with higher doses of compound 11f, as shown in Fig. 2.
This eventually leads to the increased expression of
caspase-3 in a concentration dependent manner. Based on
the results, the conclusion drawn is, compound 11f induces
dual inhibition of XIAP and Bcl-2 and eventually upregu-
lates pro-apoptotic gene expression such as Bax, Bad, and
Caspase-3.
To confirm that the mitochondrial pathway is involved in
compound 11f apoptosis, the protein expression of pro-
apoptotic Bax and Bad and anti-apoptotic Bcl-2 protein was
examined in in vitro system. As shown in Fig. 2, treatment
of B16F10 cells with compound 11f for 48 h resulted in
increased levels of Bax and Bad, counteracting Bcl-2
Compound 11f regresses murine melanoma by dual
inhibition of XIAP and Bcl-2 in vivo
Malignant melanoma is a highly aggressive cancer and most
consistent model for metastasis studies (Miller and Mihm

Medicinal Chemistry Research
Fig. 12 a Ramachandran plot showing the phi/psi values of the
modeled XIAP protein. Green color- most favorable regions, red color
region- allowed region, pale yellow- generously allowed region and
white color- disallowed regions. b Graph presenting the rotamer
energy plots of the XIAP protein. c Residue clashing profile of amino
acid residues between this system in production stage run. d The
−1
energy score (kcal mol ) of the complex XIAP-11f with lowest
−1
binding energy −7.48 kcal mol (color figure online)
2
006; Madan et al. 2010). In the present investigation
assay in vitro (Fig. 1b). Taken together 11f is promising
molecule against the skin cancer and which can be devel-
oped as an anticancer drug for skin cancer in near future.
B16F10 melanoma cells were used to develop a melanoma
tumor model in mice. After the onset of tumor development,
six doses of compound 11f (0, 10, and 20 mg/kg.b.w) given
on every alternate day, resulting in reduction in tumor
growth in a dose dependant manner, along with a notable
reduction in the physical morphology of tumor bearing
animals. The dissected tumor size depicted the tumor
regression with 2.2 to 4.6 folds (Fig. 3a). To explore the
molecular mechanism of compound 11f in vivo, whole cell
lysate of tumor tissue was subjected to immunoblot analy-
sis. The results inferred that compound 11f modulates the
Bcl-2 related proteins by specifically down regulating the
Bcl-2 and XIAP protein. As a consequence proapototic
protein such as Bax and Bad got upregulated leading to
activation of apoptotic inducing protease casapse-3 which
eventually damages the DNA as observed in the TUNEL
Compound 11f interacts strongly with Bcl-2 in docking
studies
The molecular docking was performed as per the standard
procedure (Mohammed et al. 2017; Mohammed and Kha-
num 2018) and the analysis revealed that, the compound 11f
interacts strongly with Bcl-2 and XIAP proteins and 3D
structure shows the interaction compound 11f in the active
site C-terminal trans activation in Bcl-2 and in the active
site of XIAP. The hydrogen-bond interaction view of the
ligand molecule 11f with AlA43 and Glu42 in Bcl-2 protein
with GLN399 in XIAP protein as shown in Fig. 4. Further,
the interaction of compound 11f at the pocket site and

Medicinal Chemistry Research
Fig. 13 Graph representing the bond angles of the complex Bcl2-11f and XIAP-11f

Medicinal Chemistry Research
Table 2 Docking energy scores (kcal/mol) derived from the MOE for the complex Bcl2–11f
Table 3 Docking energy scores (kcal/mol) derived from the MOE for the complex XIAP-11f

Medicinal Chemistry Research
Fig. 14 Graph representing the bond lengths of the complex Bcl2-11f and XIAP-11f

Medicinal Chemistry Research
Fig. 15 Graph representing the dihedral angle of the complex Bcl2-11f and XIAP-11f
residue amino acid of the complex Bcl2-11f and complex
XIAP-11f is predicted (Fig. 5) with also the 3D interactions
of 11f with hydrophobic amino acids, which represented by
yellow and red color in Bcl-2 and XIAP protein respectively
2015 program (Fig. 7). The molecular mapping is the 3D
plot of the electrostatic potentials relating to the electron
density mapped onto the isoelectronic surface. It is helpful
to understand the sites intended for the electrophillic and
nucleophiliic reaction for the hydrogen bond. As well as,
the size, shape and electrostatic potential value. The
(
Fig. 6). The sequences of complex Bcl-2-11f and XIAP-
1f are arranged to their target sites is detected using MOE
1

Medicinal Chemistry Research
mapping, molecular electrostatic potentials using ribbon
models, and conformer explorer of compound 11f with
amino acid residue of Bcl-2 and XIAP proteins has pre-
dicted (Fig. 8). Similarly, we have performed, pose orga-
nizer viewer of the compound 11f with residue amino acid
in Bcl-2 and XIAP and it was displays electrostatic inter-
actions of Bcl-2 and XIAP proteins with the compound 11f
with hydrogen view. The compound 11f displays hydro-
phobic and hydrophilic groups as green and orange respec-
tively in both Bcl-2 and XIAP proteins (Figs 9 and 10).
Ramachandran plot showing the phi/psi values of the
modeled Bcl2 and XIAP proteins. Green color is the most
favorable regions, red color region is allowed region, pale
yellow is generously allowed region and white color is
disallowed regions. The Graph presenting the rotamer
energy plots, residue clashing and contact energy profile of
amino acid residues the Bcl-2 and XIAP proteins as shown
in Figs 11 and 12. Further, we have predicted the bond
angle, bond length and dihedral angle of complex Bcl2-11f
and XIAP-11f (Figs 13–15), the docking domains indicated
a strong binding at the catalytic sites of proteins. The
docking studies showed that the compound 11f can be taken
for further study. Moreover, the possible binding con-
formation and orientations were analyzed by clustering
methods, embedded in MOE 2015. From the Tables 2 and 3,
the compound 11f showed good binding energies at
Acknowledgements Zabiulla gratefully acknowledges the financial
support provided by the Department of Science and Technology, New
Delhi, Under INSPIRE-Fellowship scheme [IF140407]. YHEM is
thankful to the University of Hajja, Yemen, for providing financial
support. SAK thankfully acknowledges the financial support provided
by VGST, Bangalore, under CISEE Programme [Project sanction
order: No. VGST/ CISEE /282]. BTP gratefully acknowledges the
grant extended by DBT (6242-P37/RGCB/PMD/DBT/PBKR/2015)
and VGST (VGST/GRD231/CISEE/2013-14).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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