Antiobesity, antioxidant and cytotoxicity activities of newly synthesized chalcone derivatives and their metal complexes Dedicated to Professor R. R. Schmidt on the occasion of his 79th birthday.

European Journal of Medicinal Chemistry 76 (2014) 517e530

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Antiobesity, antioxidant and cytotoxicity activities of newly

synthesized chalcone derivatives and their metal complexes

,

b

,

c,d

Mohamed Ramadan El Sayed Aly^a, Hamadah Hamadah Abd El Razek Fodah

,

*

Sherif Yousef Saleh ^e

^aChemistry Department, Faculty of Science, Taif University, Hawyah-Taif, Kingdom of Saudi Arabia, Saudi Arabia

^bChemistry Department, Faculty of Applied Science, Port Said University, 42522 Port Said, Egypt

^cChemistry Department, Faculty of Science, Taif University, Kingdom of Saudi Arabia, Saudi Arabia

^dChemistry Department, Faculty of Science, Damietta University, New Damietta 34517, Egypt

^eBiochemistry Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt

a r t i c l e i n f o

a b s t r a c t

Article history:

Four sets of rationally designed chalcones were prepared for evaluation of their antiobesity, antioxidant

and cytotoxicity activities. These sets include nine oleoyl chalcones as mimics of oleoyl estrone, three

monohydroxy chalcones (chalcone ligands), Schiff base-derived chalcones and four copper as well as zinc

complexes. Oleoyl chalcones 4d, 4e and particularly 6a as an isosteric isomer of oleoyl estrone, were as

active as Orlistat on weight loss and related metabolic parameters using male SD rats in vivo. Chalcone

ligands 10aec and Schiff base-derived chalcones 11 and 14a,b were weakly antioxidants, while, the

copper and zinc complexes 15aed were good antioxidants with zinc chelates 15b,d being more active

than their copper analogues 15a,c in vitro. Compounds 10c and 14a showed good cytotoxicity activities as

Doxorubicin against PC3 cancer cell line in vitro, while, the copper complex 15c showed promising ac-

Received 29 December 2013

Received in revised form

7 February 2014

Accepted 8 February 2014

Available online 15 February 2014

Dedicated to Professor R. R. Schmidt on the

occasion of his 79th birthday.

Keywords:

Chalcone

Metal complex

Antiobesity

Antioxidant

Cytotoxicity

tivity with IC₅₀value of 5.95 mM. The estimated IC₅₀value for Doxorubicin was 8.7 mM. Chalcones 14a,b

are bifunctional probes for potential investigations in cancer diagnosis and radiotherapy by complexation

with Gd^3þor metal radioisotopes followed by posttranslation of Shiga toxin B-subunits that target

globotriosyl ceramide expressing cancer cells.

1. Introduction

they are precursors for ﬂavones, isoﬂavones, aurones and antho-

cyanins which are regarded as cyclic chalcones and have closely

Chalcones

I

(1,3-diaryl-2-peopen-1-ones) (Scheme 1) are

related physiological and medical relevancies. Of the biological

signiﬁcant aspects of natural as well as synthetic chalcones are

their antiobesity [3,4], antioxidant [5,6] and anticancer activities

[7,8].

structurally divergent natural products in edible and medicinal

plants having beneﬁts for human health from life style to protection

from life threatening diseases [1,2]. Besides being natural products,

Oleoyl estrone (OE) II, is an estrone hormone synthesized in the

white adipose tissue then released into the blood stream in animal

and human as well [9e11]. It is able to produce rapid and sustained

weight loss through induction of body fat loss while preserving

protein stores, a common side effect of antiobesity formulations.

Besides being able to reduce circulating plasma lipids, OE is able to

reduce plasma insulin and improve insulin resistance [12,13]. It

functions through hampering the uptake of substrates essential for

lipogenesis and thus favoring lipolysis indirectly, i.e. imbalance the

lipogenesiselipolysis equilibrium. Its sustained action arises from

its tendency to decrease expression of lipogenic enzyme genes

without affecting expression of lipolytic enzyme genes [14]. Obesity

accounts for approximately 20% of all cancer cases; weight loss was

reported to reduce risk for, at least, breast cancer [15]. The

Abbreviations: OE, oleoyl estrone; ROS, reactive oxygen species; SD, sparguee

dawley; Pgp, P-glycoprotein; BCRP, breast cancer resistant protein; MRP1, multi-

drug resistance-associated protein 1; TGA, thermal gravimetric analysis; DTG, dif-

ferential thermogravimetry; TNF-a, tumor necrosis factor-a; FFAs, free fatty acids;

PTPs, protein tyrosine phosphatase; IRS-1, insulin receptor substrate-1; LDL, low

density lipoprotein; HDL, high density lipoprotein; LFD, low fat diet; HFD, high fat

diet; FI, free insulin; ISI, insulin sensitivity index; TG, triglycerides; FBs, fasting

blood sugar; DPPH, 1,1-diphenyl-2-picryl-hydrazyl; Trolox, 6-hydroxy-2,5,7,8-

tetramethylchroman-2-carboxylic acid; AsAc, ascorbic acid; ELISA, enzyme-linked

immunosorbent assay; ANOVA, analysis of variance.

* Corresponding author. Chemistry Department, Faculty of Applied Science, Port

Said University, 42522 Port Said, Egypt; and Chem. Dep., Faculty of Science, Taif

Uni., Taif, KSA.

E-mail address: mrea34@hotmail.com (M.R. El Sayed Aly).

http://dx.doi.org/10.1016/j.ejmech.2014.02.021

518

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

Scheme 1. Structure of model compounds.

physiologic disorders accompanying obesity include hyper-

glycaemia, insulin resistance and growth factors excretion. All these

factors are involved in a complex physiologic pathways lead to

hormone receptor-positive tumors or the so-called obesity-related

cancer [16].

Putting in mind the afﬁnity of the oleoyl chain to cross cellular

membranes [31], it might be concluded that the oleoyl moiety as

well as the copper ions might act as penetration enhancers of

chalcones into cancer cells. Even if they are non cytotoxic to cancer

cells, they might act as inhibitors of drug efﬂux proteins charac-

teristic to cancer cells and responsible for drug resistance of cancer

cells, for instance, P-glycoproteins (Pgp), breast cancer resistance

protein (BCRP) [25,26] and multidrug resistance-associated protein

1 (MRP1) [26]. Chalcones with basic functionalities on Ring A, for

instance IV [25], were proven to act as Pgp-inhibitors, thus, exerted

synergistic effect with doxorubicin [25,27].

Based on the known beneﬁts of natural isofavones on human

obesity and plasma cholesterol levels, besides, their close similarity

to estrogen ring structure, Xiang et al. [17], reported the synthesis

of fatty acid substituted isoﬂavone derivatives, e.g. III, as potential

antiobesity candidates that might retain the antiobesity effect of OE

and bypass the estrogenic effect of OE if applied as antiobesity

agent. III was able to lower the body weight gain, adiposity,

improved plasma lipid proﬁle and reduced plasma insulin with low

toxicity LD₅₀2.1638 g/kg. Isoﬂavone derivatives with other lipid

chains were not as active as III, thus, reﬂecting the known impor-

tance of the oleoyl moiety for the antiobesity effect [18].

As antioxidants, hydroxylated, methoxylated and prenylated

chalcones are known for their powerful antioxidant activities. They

are able to scavenge reactive oxygen species (ROS), free radicals and

inhibit their implication in damage of cell membranes, DNA, pro-

teins and consequent potential prognosis of ageing, cancer and

atherosclerosis [5].

2. Results and discussion

2.1. Chemistry

To obtain 4⁰-(N-oleoylamido)chalcones 4e6 as oleoyl estrone

analogues for screening mainly as potential antiobesity agents, p-

aminoacetophenone 1 was treated with oleoyl chloride 2 in Et₃N to

afford substrate 3 in nearly quantitative yield (Scheme 2). Claisene

Schmidt condensation of 3 with a set of aromatic aldehydes,

including

basically

2-furancarboxaldehyde

and

2-

Chalcone derivatives of diverse chemical architectures are quite

signiﬁcant in anticancer drug discovery. They are known to induce

apoptosis [19], DNA [20] and mitochondrial damage [21], inhibit

angiogenesis [22], tubulin [23], Kinases [24] and drug efﬂux protein

activities [25e27]. They are implemented in cancer diagnosis too

[28].

In this paper, a series of oleoylamidochalcones were prepared as

easily accessible isosteric isomers of OE and oleoylisoﬂavones and

screened as antiobesity agents on SD rat model. Another set of

monohydroxy chalcones (chalcone ligands), Schiff base-derived

chalcones and their copper and zinc complexes were also pre-

pared and investigated as antioxidants and anticancer agents.

While zinc and chalcones are individually antioxidants we welled

that merging should enhance the overall antioxidant power. On the

other hand, cancer cells are known for their high afﬁnity to copper

ions [29], thus, upon chelation of chalcones with copper they will

be more liable to target cancer cells in micro Dendron architecture.

Copper (II) complexes of Schiff base-derived curcumin ligands are

highly cytotoxic [30].

thiophenecarboxaldehyde, afforded the desired chalcones in very

good yields (63%equ) except in the case of aldehydes having p-

electron withdrawing groups, chalcones 4e,f. Molecular ion peaks

were recognizable with good intensities and were in accordance

with the calculated empirical formulas and the same was for

elemental analyses except for 5 where it was not possible to obtain

complete right analysis for it. In the IR spectra, diagnostic bands for

NH stretching, enone C]O stretching overlapped with Amide I and

ﬁnally Amide II bands were all clearly visual near to 3275, 1655 and

1610 cm^ꢀ1, respectively. In some cases, for instance 4f, the enone

C]O stretching band was shifted a little to a higher frequency

1675 cm^ꢀ1while the NO₂symmetric and asymmetric stretching

vibrations were strongly visible at 1515 and 1340 cm^ꢀ1, respec-

tively. ¹H NMR spectra of compounds 4e6 showed the oleoyl

oleﬁnic protons as multiplet at

the aromatic protons. The H_bof the enone system was more

deshielded than the H_adue to -bond delocalization and both

d 5.43 ppm and well separated from

p

appeared in the aromatic region with large coupling constant J 15.6

Hz characteristic for S-trans conﬁguration of the enone system [32].

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

519

Scheme 2. Reagents and conditions: (a) DCM, Et₃N, 0 ^ꢁCert (99%); (b) Aldehyde, Et₂O/EtOH, NaOH, rt (4a, R⁰¼ H, 82%), (4b, R⁰¼ OCH₃, qu), (4c, R⁰¼ CH₃, 73%), (4d, R⁰¼ NMe₂, 63%),

(4e, R⁰¼ Cl, 31%), (4f, R⁰¼ NO₂, 78%), (5, 78%), (6a, X ¼ O, 90%), (6b, X ¼ S, 89%).

In ¹³C NMR, the carbonyl carbon of the enone system was more

deshielded than the amide C]O carbon, presumably, the tautom-

erism in the amide shields the C]O carbon more effectively than

Schmidt condensation of 8aec with p-hydroxybenzaldehyde 9

afforded chalcone ligands 10aec in low yields. These chalcones

showed overlapped NH/OH stretching vibration bands within the

range 3200e3300 cm^ꢀ1. Enone C]O stretching as well as Amide I

and Amide II bands appeared quite similar to those in chalcones 4e

6. In the ¹H NMR spectra, the deuterium exchangeable phenolic OH

the delocalization of the p-orbitals in the enone moiety.

Tracing the fact that hydroxylated chalcones are potential

antioxidant and cytotoxic, besides, the high afﬁnity of cancer cells

to intake copper ions, a series of chalcone ligands having an OH

group as donor group, compounds 10aec as well as Schiff base-

derived chalcones 14a,b were prepared (Scheme 3). Thus, Claisene

signal appeared downﬁeld at

the aromatic amide 10c, 10 ppm, was more deshielded compared

with the aliphatic analogues 10a,b, 1.6e1.2 ppm.

d 10.0e10.5 ppm. The amide proton in

d

Scheme 3. Reagents and conditions: (a) C₁₅H₃₁COCl or CH₃COCl or PhCOCl, Et₂O/DCM, Et₃N (8a, R ¼ C₁₅H₃₁, 43%), (8b, R ¼ CH₃, 91%), (8c, R ¼ Ph, 96%); (b) EtOH, NaOH (10a,

R ¼ C₁₅H₃₁, 66%), (10b, R ¼ CH₃, 21%), (10c, R ¼ Ph, 43%).

520

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

Chalcones 14a,b were prepared from Schiff’s base 13 by Claisene

silica due to acidity, however, elution in the presence of 1% Et₃N or

chromatography on alumina as stationary phase failed to purify the

products.

The ORTEP diagram (Fig. 1) of 14a crystallized in a monoclinic

P2₁/c system illustrates the E-geometry around the double bond of

the enone moiety. This is in good agreement with the observed

high coupling constant, ¹H NMR, of H_aand H_b. The OeH and the

azomethine nitrogen are well oriented for intramolecular hydrogen

bonding and supports planarity of the whole molecule. Bond

lengths are given in (Table 1).

To investigate the effect of merging chalcones with metal ions

on the antioxidant and cytotoxicity of chalcones, chalcones 14a,b

were treated with copper and zinc acetates in reﬂuxing EtOH to

afford 15aed as ML₂chelate complexes, despite, the reaction pro-

portions were 1:1 (Scheme 4). A set of diagnostic variations in

absorption bands could be recognized by comparing the IR spectra

of 15aed and their parent chelators 14a,b. These variations include,

disappearance of the phenolic OH-azomethine intramolecular H-

bond (OHeN) at z2970 cm^ꢀ1, shift of the azomethine (C]N) ab-

sorption to lower frequency (1618 / 1605 cm^ꢀ1), and the phenolic

Schmidt condensation with anisaldehyde and p-tolualdehyde

affording 14a in weak yield (27%) as single medium sized prisms

and 14b (57%) as sun-shaped crystals (Scheme 4). Besides, the

desire to check chalcones having non-chelating Schiff’s base tags

for SAR analysis, compound 11 was prepared in 76% yield in one

step by treatment of 7 with anisaldehyde in the presence of NaOH.

Upon acid hydrolysis of 11 (86%), intermediate amine 12 was

treated with salicylaldehyde affording 14a in much better yield

(75%) but most interestingly is the crystal color and morphology

which was better and enabled obtaining of X-ray ORTEP structure

shown in (Fig. 1). The OH IR-stretching band of 14a,b could not be

seen due to strong intramolecular H-bonding with the azomethine

nitrogen which appears, instead, at about 2970 cm^ꢀ1. For the same

reason it was highly deshielded

The imine hydrogen appeared as singlet at

d

z 13 ppm and D₂O exchangeable.

8.7 ppm. Attempts to

d

prepare more derivatives of the type 14 having different sub-

stituents at Ring B were unsuccessful. Despite, products were

visible on TLC, all trials to purify them even by ﬂash chromatog-

raphy were unsuccessful. It was anticipated that it decomposes by

Scheme 4. Reagents and conditions: (a) Anisaldehyde, NaOH, EtOH (76%); (b) HCl, Acetone (86%); (c) Salicylaldehyde, EtOH, rfx. [Only 14a from 12, 75% (Method A); 68% for 13]; (d)

Anisaldehyde or tolualdehyde, NaOH, EtOH [27% for 14a, R ¼ OMe (Method B) and 57% for 14b, R ¼ Me]; (e) Cu(OAc)₂. 5H₂O or Zn(OAc)₂. 2H₂O, EtOH, rfx. (62% for 15a, R ¼ OMe,

M ¼ Cu^2þ; 61% for 15b, R ¼ OMe, M ¼ Zn^2þ; 66% for 15c, R ¼ Me, M ¼ Cu^2þ; 46% for 15d, R ¼ Me, M ¼ Zn^2þ).

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

521

Fig. 1. Ortep diagram of 14a.

CeO absorption z1290 cm^ꢀ1to higher frequency 1330 cm^ꢀ1due to

enhancement of the mesomeric effect upon complexation. The

formation of complexes 15aed gives rise to extra bands at the low

frequency region, deﬁnitively, around 530 and 450 cm^ꢀ1which

point to formation of MeO and MeN bonds, respectively.

(IRS-1) dephosphorylation and insulin resistance [36]. Proin-

ﬂammatory cytokines released from excess adipose tissues are

responsible for elevation of blood pressure and elevation in both

insulin resistance and glucose. They are major risk factors of car-

diovascular diseases [37]. Elevation of plasma triglycerides

(hypertriglyceridemia), and total cholesterol (hypercholesterole-

mia), i.e. low density lipoprotein-cholesterol (LDL-cholesterol) and

high density lipoprotein-cholesterol (HDL-cholesterol), are related

physiologic disorders to obesity [38].

Nine oleoylamidochalcones 4aef, 5 and 6a,b were prepared as

isosteric isomers of oleoyl estrone for screening their antiobesity

and related metabolic disorders. Choose of the chalcone moiety as

alternative for the estrone ring was based on successful results

exhibited by oleoylisoﬂavones, while, the oleoyl group was retained

as necessary component for antiobesity stimulation where other

saturated and unsaturated alternatives were ineffective. Besides

being easily accessible in more diverse chemical architectures than

isoﬂavones, chalcones have closely related physiological proﬁles as

isoﬂavones which gives better opportunity for potential physiologic

effects. Thus, eight of these chalcones 4aef and 6a,b were screened

as weight loss stimulants and some related metabolic disorders

including levels of triglycerides, free fatty acids, total cholesterol,

fast blood sugar, free insulin and insulin sensitivity index. Experi-

ments were done in vivo using male SD rats. Animals were divided

into eleven groups, one low-fat-diet (LFD) and another high fat diet

(HFD) as negative control. A third group was treated with Orlistat as

positive control while the remaining eight groups were treated

with compounds 4aef and 6a,b individually. Vehicle doses (30 mg/

day/kg of rat weight) were given by intragastric administration

once daily for 30 days.

In body weight experiments (Fig. 8A) feeding with HFD signif-

icantly increases body weight to 49.5% of the normal group, while,

it was attenuated signiﬁcantly by 23.5% upon treatment of HFD

individuals with Orlistat. Variation between Orlistat treated obese

rats and normal one was insigniﬁcant. Chalcones 4d with p-dime-

thylaminophenyl, 4e with p-chlorophenyl and 6a with 2-furanyl

moieties, as Ring B terminals were able to reduce the body

weight signiﬁcantly by 28.0, 22.0 and 36.6%, respectively, compared

with HFD rats without having signiﬁcant variations with normal

rats. Other chalcones varied insigniﬁcantly with HFD rats reﬂecting

their negative effects on body weight loss. Chalcone 4a was

regarded as pseudo active chalcone, where it has insigniﬁcant

variation with both Orlistat and HFD rats.

In case of free fatty acids analysis (Fig. 8B), HFD rats showed a

signiﬁcant increment in FFAs level by 63% compared with normal

rats, while, Orlistat treatment could attenuate signiﬁcantly its level

again by 38.9% and return it to its normal levels. Chalcones 4d, 4e

and 6a varied signiﬁcantly with HFD rats without having signiﬁcant

variations with Orlistat treated and normal rats.

The UV-spectra (Figs. 2 and 3) supports the IR data, thus, the

absorption intensity of 14a at 346 and 14b at 333 nm decreased

from about 3.1 to 2.2 in case of zinc chelates 15b,d and more pro-

nounced to about 0.5 in case of copper complexes 15a,c. These

variations denote to incorporation of the lone-pair of electrons of

the azomethine nitrogen atom in chelation and it is no more

available for efﬁcient transition motions.

In the ¹H NMR spectra of the diamagnetic zinc complexes 15b,d,

the phenolic proton disappeared clearly as a good evidence for

chelation, however, the shift in the imine proton was minute and

negligible. The absence of acetate CH₃signal in DMSO at d 1.91 ppm

[33] supports the proposed structure of ML2 and the spontaneous

deprotonation of phenolic protons under the neutral reaction

conditions. Thus, the complexes are non-electrolytes and no ace-

tates are available outside the coordination sphere, therefore, they

showed very low electrical conductivity, L_Mvalues of 1e

2

U^ꢀ1cm²mol^ꢀ1in DMF, as further conﬁrmation of their neutral

and non-electrolytic nature.

Thermal analysis (TGAeDTG) of complexes 15aed up to 800 ^ꢁC

(Figs. 4e7) showed the thermal stability of zinc complexes 15b,d

compared with the copper chelates 15a,c. Complexes 15b,c are half

hydrated due to weight loss of 1.2% (Calcd. 1.14%) at 547.15 K in the

case of 15b and 1.24% (Calcd. 1.19%) at 537.15 K in the case of 15c.

This water of crystallization was neither visible in the IR spectra nor

effective on the elemental analysis. Copper complexes 15a,c

showed main endothermic decomposition at 618.87 and 604.19 K

due to loss of one chalcone moiety 35.75e39.96% (Calcd. 36.23e

41.46%), while the zinc analogues 15b,d showed their main

decomposition event at 625.38 and 618.97 K due to loss of only

cinnamoyl fragment 19.96e20.92% (Calcd. 20.47e19.45%).

2.2. Biology

2.2.1. Antiobesity screening

Adipocytes in obese individuals function as endocrine gland

releases a variety of molecules including protein hormones such as

leptin, cytokines like TNF-a, free fatty acids (FFAs) and enzymes like

protein tyrosine phosphatases (PTPs). PTPs dephosphorylate mul-

tiple tyrosine residues of the insulin receptor tyrosine kinase, thus,

retarding insulin-stimulated glucose uptake. The pancreas re-

sponds by pumping more insulin to push glucose uptake with

concurrent elevation of both insulin and glucose levels, prognosis of

insulin resistance and Type II diabetes [34,35]. Increased resistance

to insulin mediated inhibition of lipolysis lead to elevation of

plasma free fatty acid levels, blood pressure, impair large artery

endothelial functions and cardiovascular diseases. Theses elevated

plasma fatty acid levels assist also in insulin receptor substrate-1

Free insulin augmentation by HFD feeding (95.5%) was attenu-

ated signiﬁcantly by Orlistat treatment (51.4%) without signiﬁcant

variation with normal rats (Fig. 8C). In this case, only 4d and 6a

522

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

Table 1

5

ꢀ

Bond lengths [A] for compound 14a.

N1eC11

N1eC15

C2eC3

C2eC22

C3eC24

C4eC9

C4eC24

O5eC10

C6eC12

C6eC22

C6eC26

C7eC10

C7eC11

C7eC27

O8eC13

O8eC21

1.282 (2)

1.422 (3)

1.319 (3)

1.477 (3)

1.459 (3)

1.391 (3)

1.396 (3)

1.359 (2)

1.390 (3)

1.493 (3)

1.393 (3)

1.396 (3)

1.449 (3)

1.399 (3)

1.365 (2)

1.421 (3)

C9eC13

C10eC14

C12eC15

C13eC15

C14eC17

C15eC19

C16eC20

C17eC23

O18eC22

C19eC25

C20eC24

C23eC27

C25eC26

C2eH2

1.371 (3)

1.392 (3)

1.388 (3)

1.394 (3)

1.367 (3)

1.390 (3)

1.368 (3)

1.392 (3)

1.222 (2)

1.383 (3)

1.389 (3)

1.395 (3)

1.384 (3)

0.960 (2)

O5eH5

C9eH9

0.921 (14)

0.960 (2)

0.960 (3)

0.960 (2)

4

3

2

1

0

14b

C11eH11

C12eH12

C14eH14

C16eH16

C17eH17

C19eH19

C20eH20

C21eH21A

C21eH21B

C21eH21C

C23eH23

C25eH25

C26eH26

C27eH27

λ_max

=

333 nm

15d

15c

300

350

400

450

500

C3eH3

C4eH4

λ

(nm)

Fig. 3. UV spectrum of 14b, 15c,d.

reduced signiﬁcantly this metabolic parameter by 49.2 and 61.6%,

respectively, while, 4e varied insigniﬁcantly with HFD rats.

Regarding insulin sensitivity (Fig. 8D), HFD feeded rats acquired

signiﬁcant depression in insulin sensitivity than normal LFD rats by

21.5%, while, Orlistat treatment augmented this parameter signiﬁ-

cantly by 48.4% compared with HFD group. Chalcones 4d, 4e and 6a

augmented free insulin signiﬁcantly quite as Orlistat.

structure of 6a with oleoyl estrone gives insight to the importance

of the oxygenated terminal ﬁve membered ring in both compounds

besides the oleoyl moiety for the antiobesity activity, whatever, the

oxygen is exocyclic as in OE or endocyclic as in 6a which are the

most similar structural features between both compounds.

HFD individuals exhibited signiﬁcant triglyceride and fast blood

sugar gain, while, total cholesterol varied insigniﬁcantly (Fig. 8E).

Orlistat treated group got signiﬁcant drop in triglyceride level by

42.7% without signiﬁcant variation with normal rats. The drug has

no statistical signiﬁcant effect on total cholesterol or FBs on com-

parison with HFD rats. As observed in last parameters, only com-

pounds 4d, 4e and 6a could attenuate signiﬁcantly TG levels

compared with HFD rats by 39.7, 34.1 and 49.7%, respectively, but

they varies insigniﬁcantly with Orlistat and normal rats.

Despite, Orlistat, 4d and 4e could not depress signiﬁcantly FBs

levels, 6a could do by 41.3% compared with HFD and return it to its

normal levels. Neither Orlistat nor test compounds could affect

total cholesterol signiﬁcantly.

2.2.2. Cytotoxicity assay

Many natural as well as synthetic chalcones are continually

reported for their anticancer activity against various cancer cell

lines with hydroxylated and methoxylated species being in front of

anticancer known chalcones. Nineteen compounds and Doxoru-

bicin as positive control were evaluated for their cytotoxicity

against prostate cancer cell line PC3 in vitro. In terms of these re-

sults (Fig. 9) the cytotoxic activity of compounds with fatty acyla-

mido groups as Ring A 4aef, 5, 6a,b and 10a regardless their Ring B

structure were weakly signiﬁcantly compared with the control.

Other small sized chalcones 10b,c, 11 and 14a,b showed good

cytotoxic signiﬁcant activities with IC₅₀values within the range

15.74e10.91

10c was signiﬁcantly more cytotoxic, IC₅₀10.92

15.74 M, thus, the phenyl group has positive impact on the cyto-

toxicity compared with the aliphatic one, particularly, if it is lipidic

as in 10a. All Schiff-base derived chalcones either nonchelating 11

or chelating 14a,b were signiﬁcantly active within the range 12.52e

m

M, while, it was 8.91

m

M for the control. Compound

In terms of these results, the compounds that showed signiﬁ-

cant activities are arranged in comparison to Orlistat in the

following order 6a > Orlistat, 4d > 4e > 4a. Thus, the p-dimethy-

laminophenyl and p-chlorophenyl moieties are valuable as Ring B

terminals, while, the 2-furanyl is much better for relieving weight

gain and related metabolic disorders. Comparing the inertness of

6b with the activity of 6a reﬂects the preference of the furanyl

oxygen rather than the thiophenyl sulfur, the unique difference, for

the antiobesity effect of 6a. On the other hand, comparing the

m

M, than 10b, IC₅₀

m

10.90 mM. Compounds 10c and 14a were signiﬁcantly as active as

Doxorubicin without any signiﬁcant variations between them.

As it was planned, copper and zinc complexes 15aed of

chelating chalcones 14a,b were screened as well. The cytotoxicity of

the parent chalcones 14a,b was reduced upon complexation

0.4

2.0x10

14a

1.8

0.3

0.0

λ_max

= 346 nm

1.6

15b

15a

-2.0x10

0.2

0.1

0.0

DTG

1.4

-4.0x10

1.2

-6.0x10

1.0

TG

0.8

345.72

300

C

-8.0x10

300

350

400

450

500

0

100

200

400

500

600

700

800

λ

(nm)

Temp [ C]

Fig. 4. TGAeDTG thermogram of 15a.

Fig. 2. UV spectra of 14a, 15a,b.

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

523

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

afﬁnity which is well related to the antioxidant power on quantum

molecular basis from the E_HOMOand E_LUMOvalues [42].

0.00

Thus, the energy of the highest occupied molecular orbital

(E_HOMO) measures the electron-donating character of a compound

(ionization potential), while (E_LUMO) measures its electron-acceptor

character (electron afﬁnity). Compounds of high E_HOMOand low

E_LUMOvalues and, consequently, low energy gab (D_E) are classiﬁed

as good electron-releasing species. As shown in (Table 2), the

powerful antioxidants in this study 15aed exhibited the lowest D_E

(E_LUMOꢀ E_HOMO) values (2.75e7.79) compared with (11.61e11.99)

for the rest of compounds under consideration reﬂecting their high

electron-releasing afﬁnities [43].

Medically, antioxidants are auxiliary drugs co-administered

with various formulations, particularly anticancer drugs, to scav-

enge free radicals and reactive oxygen species stemming from their

oxidative stress. Therefore, compounds combining an antioxidant

and anticancer activities, compound 15c in this consideration, are

desirable in cancer chemotherapy [44].

-2.50x10

-5.00x10

-7.50x10

DTG

TG

352.23

300 400

Temp [ C]

C

0

100

200

500

600

700

800

Fig. 5. TGAeDTG thermogram of 15b.

(15a,b,d) with the exception of the copper chelate 15c whose ac-

tivity was increased two-fold signiﬁcantly compared with 14b and

showed signiﬁcant cytotoxicity than doxorubicin (31.6% more

active), the unique in this investigation. Probably, these complexes

due to their expected high stability did not liberate their parent

ligands whose chelating centres are necessary for cytotoxicity and

that the OH group is necessary for the activity. However, this is

opposed by the high activity of 15c. Eventually, the activity is

attributed to the presence of the p-methylphenyl moiety of Ring B

and the copper ion.

3. Conclusion

In summary, a series of oleoyl chalcones, hydroxy chalcones,

Schiff base-derived chalcones as well as their copper and zinc

complexes were prepared for screening their antiobesity, cytotox-

icity and antioxidant activities. Oleoyl chalcone 6a graphted with

furanyl moiety exhibited interesting antiobesity activity. The

structure similarity between 6a and OE suggest it to mimic OE,

therefore, its least effective dose and mechanism of action, either by

pancreatic lipase inhibition or interference with gene expression of

lipolytic and lepogenic enzymes have to be considered. This work

also showed the importance of hydroxy chalcones modiﬁed with

simple amide and Schiff base moieties as anticancer agents. The

copper complex 15c of a Schiff’ base-derived chalcone 14b showed

preference to Doxorubicin against PC3 cell line. As antioxidants,

complexation of Schiff base-derived ligands 14a,b with copper and

zinc, led to potent antioxidant complexes 15aed. Thus, compound

15c as powerful cytotoxic complex and good antioxidant is another

fruit of this work for further investigations. The toxicity of 6a and

15c will be considered in due course.

2.2.3. Antioxidant activity

Compounds showed potential cytotoxic activity including

chalcone ligands 10a,b, precursor 11, as well as Schiff-base derived

chalcones 14a,b and metal complexes 15aed of the later species

were selected for antioxidant activity estimation using both the

DPPH-radical scavenging and the hydroxyl-radical scavenging as-

says at 30 nM. The DPPH-scavenging assay of test compounds was

recorded spectrophotometrically at 517 nm taking 6-hydroxy-

2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) as positive

control [39,40]. As shown in (Fig. 10), Precursor 11, chalcone ligands

10a,b and chelating chalcones 14a,b showed low signiﬁcant scav-

enging activities within the range 19e25% of Trolox reﬂecting their

weak antioxidant power.

4. Experimental section

However, upon metal complexation of Schiff base-derived

chalcone ligands 14a,b with copper and zinc acetates, signiﬁcant

increment in the antioxidant power was observed. Zinc complexes

15b,c were a little more powerful than the copper analogues 15a,c.

Thus, in comparison with the signiﬁcant differences with Trolox,

the zinc complex 15b which showed the highest activity was less

active by 10.7%. Its analogue 15d was less active by 20.9%, while the

copper complexes 15a and 15c were less active by 27e28% without

signiﬁcant variations between both of them.

4.1. Chemistry

Melting points were determined on Gallenkamp apparatus and

are uncorrected. Flash chromatography was carried out on silica gel

(Baker, 30e60

mm), Type I, or [Lichroprep Si 60 (E. Merck; f 15e

25 m)], Type II. TLC Monitoring tests were carried out using plastic

m

sheets precoated with silica gel 60 F₂₄₅(layer thickness 0.2 mm)

purchased from Merck. Spots were visualized by their ﬂuorescence

under UV-lamp (l 245 and 366 nm) or staining with iodine vapor,

In the second approach, Hydroxyl radical scavenging activity

was measured according to Fenton method [41] (Fig. 11). Results

showed that chalcones 10a,b, 11 and 14a,b were signiﬁcantly

weakly active exactly as in the DPPH^ꢂAssay and increased signiﬁ-

cantly upon metal complexation. Metal complexes of 14b either

copper 15c or zinc 15d were signiﬁcantly more active than those of

14a. 15d was 6.7% signiﬁcantly more active than 15c and variation

between 15a and 15b was insigniﬁcant.

15% H₂SO₄, KMnO₄, or Ce(IV)SO₄in H₂SO₄. NMR spectra were

recorded on Bruker 600 MHz spectrometer, central laboratory, King

Abd El Aziz, Jeddah, Saudi Arabia. IR-spectra were recorded on ATR-

Alpha FTeIR Spectrophotometer from 400 to 4000 cm^ꢀ1. Mass

spectra were recorded on GCMS-QP 1000Ex Shimadzu spectrom-

eters in the microanalysis unit at Cairo University. UVevis. Mea-

surements were recorded by Perkin-Elmer Lambda 25 UV/vis

double-beam Spectrophotometer. Electrical conductivity mea-

surements were carried out using Equiptronics digital conductivity

meter model JENWAY 4070 at room temperature for

1 ꢃ 10^ꢀ3mol L^ꢀ1solutions. Thermal analysis measurements were

recorded on a Schimadzu model 50 instrument using 20 mg sam-

ples, micro analytical unit of Cairo University. The nitrogen ﬂow

rate and heating were 20 cm³/min and 10 ^ꢁC/min, respectively.

The antioxidant activity of compounds is related with their

ꢂ

electron or hydrogen radical releasing ability to DPPH^ꢂand OH so

that they become stable diamagnetic molecules. Once, phenolic

derivatives 10a,b and 14a,b showed low antioxidant activities,

these derivatives were regarded as weak hydrogen radical releasing

species and attention was turned to estimate the electron releasing

524

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

3.0

2.5

2.0

1.5

1.0

0.5

0.0

4.1.2. General procedure for the synthesis of 4-(arylacryloylphenyl)

oleamides (4e6)

0.00

Ethanolic NaOH (0.7 M, 0.5 ml) was added to a solution of 3

(0.5 g, 1.2 mmol) and the appropriate aldehyde (1.8 mmol) in Et₂O

and the resulting suspension was stirred at ambient temperature

for 24 h. The mixture was neutralized with AcOH (0.5 ml), evapo-

rated in vacuo and the residue was puriﬁed by ﬂash

chromatography.

DTG

-7.50x10

-1.50x10

-2.25x10

TG

4.1.2.1. N-(4-Cinnamoylphenyl)oleamide (4a). Yield: 82% as ﬁne

colorless crystals upon ﬂash chromatography with gradient of

toluene then (toluene:ethyl acetate, 14:1) and crystallization from

Et₂O; R_f0.41 (toluene:ethyl acetate, 9:1); Mp. 104 ^ꢁC; IR (v, cm^ꢀ1):

331.04

C

0

100

200

300

400

500

600

700

800

Temp [ C]

3290 (NH_str), 1650 (C]O_str.

NMR (600 MHz, CDCl₃): 8.04e8.02 (m, 1H, Ar), 8.03 (d, 1H, J 6.6

,

Amide I), 1598 (Amide II); ¹H

Fig. 6. TGAeDTG thermogram of 15c.

d

Hz, Ar), 7.81 (d, 1H, J 15.6 Hz, COCH]CH), 7.68 (d, 2H, J 9.0 Hz, Ar),

7.65 (d, 1H, J 7.8 Hz, Ar), 7.65e7.64 (m, 1H, Ar), 7.54 (d, 1H, J 15.6 Hz,

COCH]CH), 7.43e7.41 (m, 2H, Ar), 7.42 (dd, 1H, J 1.8, 4.8 Hz, Ar),

7.36 (brs, 1H, NH), 5.35e5.34 (m, 2H, CH]CH), 2.40 (t, 2H, J 7.2,

7.8 Hz, COCH₂), 2.02e1.99 (m, 4H, CH₂CH]CHCH₂), 1.74 (quin, 2H, J

7.8 Hz, COCH₂CH₂), 1.40e1.25 (m, 20H, 10 CH₂), 0.88 (t, 3 H, J 6.6 Hz,

Elemental analyses were carried out on PerkinElmer 2400 Series II

CHNS/O System, Chemistry Department, Faculty of Science at Taif

University, Saudi Arabia.

CH₃); ¹³C NMR (150 MHz, CDCl₃):

d 188.94 (COCH]CH), 171.59

(CONH), 144.54, 142.13, 134.95, 133.71, 130.50, 130.06, 130.00,

129.70, 128.96, 128.44, 121.77, 118.89 (12 C_Ar, 2 CH]CH), 37.94,

31.91, 29.77, 29.70, 29.53, 29.33, 29.32, 29.28, 29.22, 29.11, 27.23,

27.16, 25.43, 22.69 (14 CH₂), 14.13 (CH₃). Exact mass calculated for

4.1.1. N-(4-Acetylphenyl)oleamide (3)

A mixture of 1 (2.0 g, 14.8 mmol) and Et₃N (7.5 ml, 54.0 mmol)

in dry DCM (25 ml) was stirred in ice-salt bath, while, oleoyl

chloride 2 (8.0 ml, 20.4 mmol) in dry DCM (10 ml) was added

dropwise. The mixture was allowed to warm up gradually to

ambient temperature with continuous stirring over 1 h then

evaporated in vacuo. The residue was taken in Et₂O (50 ml),

washed with H₂O (20 ml), dried over Na₂SO₄, evaporated in vacuo

and puriﬁed by ﬂash chromatography (toluene:ethyl acetate, 7:1)

to afford 3 (5.86 g, 99%) as colorless tiny star-like crystals. R_f0.35

(toluene:ethyl acetate, 7:1); Mp. 62e64 ^ꢁC. IR (v, cm^ꢀ1): 3332

(NH_str), 1678 (C]O_str), 1656 (Amide I), 1604 (Amide II); ¹H NMR

C

₃₃H₄₅NO₂(487.3450), EI MS, m/z (%), Found: 487.25 (M^þ, 4), 474.25

(10), 433.15 (31), 397.25 (98), 379.20 (31.5). Anal. Calcd. for

₃₃H₄₅NO₂(487.72): C, 81.27; H, 9.30; N, 2.87. Found. C, 80.90; H,

C

9.07; N, 2.57.

4.1.2.2. N-(4-((E)-3-(4-methoxyphenyl)acryloyl)phenyl)oleamide

(4b). Yield: (qu.) as brilliant yellow solid upon ﬂash chromatog-

raphy using solvent gradient of toluene then (toluene:ethyl acetate,

15:1); R_f0.13 (toluene:ethyl acetate, 15:1); Mp. 64 ^ꢁC; IR (v, cm^ꢀ1):

3307 (NH), 1671 (C]O_str), 1658 (Amide I), 1598 (Amide II); ¹H NMR

(600 MHz, CDCl₃): d 7.93 (d, 2H, J 8.4 Hz, H-3_Ar, H-5_Ar), 7.64 (d, 2H, J

8.4 Hz, H-2_Ar, H-6_Ar), 7.57 (brs., 1H, NH), 5.37e5.32 (m, 2H, CH]

CH), 2.57 (s, 3H, COCH₃), 2.39 (t, 2H, J 7.2, 7.8 Hz, COCH₂), 2.04e1.99

(m, 4H CH₂CH]CHCH₂), 1.73 (quin, 2H, J 7.2, 7.8 Hz, COCH₂CH₂),

1.37e1.26 (m, 20H, 10 CH₂), 0.88 (t, 3H, J 6.6, 7.2 Hz, CH₃); ¹³C NMR

(600 MHz, CDCl₃):

d 8.05, 6.94 (2 d, 4H, J 8.4 Hz, Ar), 7.79 (d, 1H, J

15.6 Hz, COCH]CH), 7.60, 7.22 (2 d, 4H, J 9.0 Hz, Ar), 7.32 (s, 1H,

NH), 7.39 (d, 1H, J 15.6 Hz, COCH]CH), 5.36e5.35 (m, 2H, CH]CH),

3.86 (s, 3H, OCH₃), 2.59 (t, 2H, J 7.2, 7.8 Hz, COCH₂), 2.04e2.00 (m,

4H, CH₂CH]CHCH₂), 1.76 (quin, 2H, J 7.8 Hz, COCH₂CH₂), 1.44e1.27

(m, 20H, 10 CH₂), 0.88 (t, 3H, J 7.2 Hz, CH₃); ¹³C NMR (150 MHz,

(150 MHz, CDCl₃):

d 197.03 (CH₃CO), 173.73 (C]O_Amide), 142.40 (C-

1

_Ar), 132.74 (C-4_Ar), 130.06, 129.77 (CH]CH, C-3_Ar, C-5_Ar), 118.79

(C-2_Ar, C-6_Ar), 37.89 (COCH₃), 31.91, 29.77, 29.70, 29.53, 29.33,

29.32, 29.28, 29.22, 29.11, 27.23, 27.16, 26.45, 25.44, 22.70 (13 CH₂,

COCH₂), 14.27 (CH₃). Exact mass calculated for C₂₆H₄₁NO₂(399.31),

EI MS, m/z (%), Found: 399.30 (M^þ, 79), 368 (33), 337 (100). Anal.

Calcd. for C₂₆H₄₁NO₂(399.61): C, 78.15; H, 10.34; N, 3.51. Found. C,

77.68; H, 10.52; N, 3.62.

CDCl₃): d 189.36 (COCH]CH), 171.82 (CONH), 161.75, 154.10, 144.91,

135.97, 130.28, 130.08, 130.00, 129.70, 127.55, 121.76, 119.49, 114.45

(12 C_Ar, 2 CH]CH), 55.44 (OCH₃), 34.42, 31.91, 29.77, 29.69, 29.53,

29.34, 29.33, 29.16, 29.09, 29.08, 27.24, 27.16, 24.85, 22.69 (14 CH₂),

14.13 (CH₃); Exact mass calculated for C₃₄H₄₇NO₃(517.3555), EI MS,

m/z (%), Found: 517.00 (M^þ, 20), 505.00 (31), 423.00 (44), 340.00

(100). Anal. Calcd. for C₃₄H₄₇NO₃(517.74): C, 78.87; H, 9.15; N, 2.71.

Found. C, 79.19; H, 8.74; N, 2.75.

3.0

4.1.2.3. N-(4-((E)-3-p-Tolylacryloyl)phenyl)oleamide (4c). Yield: 73%

As yellowish ﬁne crystals upon ﬂash chromatography using solvent

gradient of toluene then (toluene:ethyl acetate, 11:1) followed by

recrystallization from Et₂O; R_f0.44 (toluene:ethyl acetate, 9:1); Mp.

116 ^ꢁC; IR (v, cm^ꢀ1): 3274 (NH), 1655 (C]O_str, Amide I), 1601 (Amide

0.0

2.5

DTG

2.0

1.5

1.0

0.5

0.0

-4.0x10

-8.0x10

-1.2x10

-1.6x10

II); ¹H NMR (600 MHz, CDCl₃):

d 8.03, 7.67 (2d, 4H, J 8.4 Hz, Ar), 7.79

(d, 1H, J 15.6 Hz, COCH]CH), 7.67 (d, 2H, J 8.4 Hz, Ar), 7.55, 7.23 (2d,

4H, J 7.8 Hz, Ar), 7.50 (d,1H, J 15.6 Hz, COCH]CH), 7.31 (brs,1H, NH),

5.35e5.34 ( m, 2H, CH₂CH]CHCH₂), 2.40 (t, 2H, J 7.2, 7.8 Hz,

COCH₂), 2.39 (s, 3H, CH_3Tol), 2.02e2.00 (m, 4H, CH₂CH]CHCH₂),

1.74 (quin, 2H, J 7.2 Hz, COCH₂CH₂), 1.35e1.25 (m, 20H, 10 CH₂), 0.88

TG

345.82

C

0

100 200 300 400 500 600 700 800

(t, 3H, J 6.6, 7.2 Hz, CH₃); ¹³C NMR (150 MHz, DMSO):

d 189.02

Temp [ C]

(COCH]CH), 171.55 (CONH), 144.63, 142.00, 141.03, 132.21, 130.52,

129.95, 129.70, 129.49, 128.98, 128.48, 120.76, 118.86 (12 C_Ar, 2 CH]

Fig. 7. TGAeDTG thermogram of 15d.

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

525

Fig. 8. Effects of treatment of HFD fed male SD rats with compounds 4aef and 6a,b compared with Orlistat treated rats and normal control. (A) for body weight in grams; (B) for

FFAs in

mol/L; (C) for FI in ng/ml; (D) for ISI and (E) for TG, TC and FBs expressed as mg/dL. Different letters on the column for each parameter varied signiﬁcantly at p ꢄ 0.05.

m

CH), 37.95, 29.77, 29.70, 29.53, 29.33, 29.32, 29.28, 29.22, 29.11,

27.23, 27.16, 25.43, 22.69, 21.55, (14 eCH₂e, CH_3Tol), 14.13 (CH₃);

Exact mass calculated for C₃₄H₄₇NO₂(501.3606), EI MS, m/z (%),

Found: 501.00 (M^þ,14), 484.00 (10), 399.00 (26),120.00 (100). Anal.

Calcd. for C₃₄H₄₇NO₂(501.74): C, 81.39; H, 9.44; N, 2.79. Found. C,

80.99; H, 9.15; N, 2.92.

¹H NMR (600 MHz, CDCl₃):

d 8.00 (d, 1H, J 8.4 Hz, Ar), 7.73 (d, 2H, J

7.8 Hz, Ar), 7.79 (d, 1H, J 15.0 Hz, COCH]CH), 7.66e7.60 (m, 3H, J

8.4 Hz, Ar), 7.54 (d, 1H, J 7.8 Hz, Ar), 7.50 (brs, 1H, NH), 7.34 (d, 1H, J

15.0 Hz, COCH]CH), 6.69 (d, 1H, J 9.0 Hz, Ar), 5.35e5.33 (m, 2H,

CH₂CH]CHCH₂), 3.05 (s, 6H, NMe₂), 2.39 (t, 2H, J 7.8 Hz, COCH₂),

2.02 (m, 4H, CH₂CH]CHCH₂),175e1.71 (m, 2H, J 7.2 Hz, COCH₂CH₂),

1.31e1.26 (m, 20H, 10 CH₂), 0.88 (t, 3H, J 6.6, 7.2 Hz, CH₃); ¹³C NMR

4.1.2.4. N-(4-((E)-3-(4-Dimethylaminophenyl)acryloyl)phenyl)ole-

amide (4d). Yield: 63% As orange ﬁne solid upon ﬂash chroma-

tography using solvent gradient of toluene then (toluene:ethyl

acetate, 9:1); R_f0.24 (toluene:ethyl acetate, 9:1); Mp. 54e6 ^ꢁC. IR (v,

cm^ꢀ1): 3331 (NH_str), 1678 (C]O_str), 1656 (Amide I), 1591 (Amide II);

(150 MHz, CDCl₃):

142.39, 130.43, 130.05, 129.76, 129.71, 129.70, 118.78, 111.83 (12 C_Ar

2 CH]CH), 40.14 (NMe₂), 37.89 (COCH₂), 31.91, 29.77, 29.70, 29.53,

29.33, 29.27, 29.22, 29.23, 27.11, 27.16, 26.45, 25.43, 22.70 (13 CH₂),

14.13 (CH₃); Exact mass calculated for C₃₅H₅₀N₂O₂(530.3872), EI

d 196.99 (COCH]CH), 171.68 (CONH), 152.04,

,

526

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

Fig. 9. Cytotoxicity results. Different letters on the column varied signiﬁcantly at p ꢄ 0.05.

MS, m/z (%), Found: 530.50 (M^þ, 12), 526.00 (42), 514.00 (38), 502

(56), 444 (60), 313 (100). Anal. Calcd. for C₃₅H₅₀N₂O₂(530.78): C,

79.18; H, 9.49; N, 5.28. Found. C, 78.75; H, 9.96; N, 4.81.

for C₃₃H₄₄ClNO₂(522.16): C, 75.91; H, 8.49; N, 2.68. Found. C, 75.66;

H, 8.43; N, 2.97.

4.1.2.5. N-4-((E)-3(4-Chlorophenylacryloyl)phenyl)oleamide

(4e).

4.1.2.6. N-(4-((E)-3-(4-Nitrophenyl)acryloyl)phenyl)oleamide

(4f).

Yield: 31% As faint creamy powder upon twice ﬂash chromatog-

raphy on Type I silica followed by Type II using solvent gradient of

toluene then (toluene:ethyl acetate, 9:1) and ﬁnally recrystalliza-

tion from toluene; R_f0.28 (toluene:ethyl acetate, 9:1); Mp. 134e

135 ^ꢁC; IR (v, cm^ꢀ1): 3276 (NH_str), 1655 (C]O_str, Amide I), 1609

Yield: 52% As faint yellow powder upon ﬂash chromatography on

Type I silica followed by Type II using solvent gradient of toluene

then (toluene:ethyl acetate 8:1); R_f0.3 (toluene:ethyl acetate, 8:1);

Mp. 128e130 ^ꢁC; IR (v, cm^ꢀ1): 3326 (NH_str), 1675 (C]O_str), 1660

(Amide I), 1594 (Amide II), 1515 (NO_2asy.str.), 1340 (NO_2sym.str); ¹H

(Amide II); ¹H NMR (600 MHz, CDCl₃):

d 8.01, 7.68, 7.57, 7.39 (4d, 8H,

NMR (600 MHz, CDCl₃):

d 8.28, 7.79 (2d, 4H, J 8.4 Hz, Ar), 8.04,

J 8.4 Hz, Ar), 7.74 (d, 1H, J 15.6 Hz, COCH]CH), 7.51 (d, 1H, J 15.0 Hz,

COCH]CH), 7.49 (brs, 1H, NH), 5.36e5.33 (m, 2H, CH₂CH]CHCH₂),

2.40 (t, 2H, J 7.2, 7.8 Hz, COCH₂), 2.04e1.99 (m, 4H, CH₂CH]CHCH₂),

1.74 (quin, 2H, J 7.2, 7.8 Hz, COCH₂CH₂), 1.38e1.25 (m, 20H, 10 CH₂),

7.70 (2d, 4H, J 9.0 Hz, Ar), 7.82 (d, 1H, J 15.6 Hz, COCH]CH), 7.64 (d,

1H, J 15.6 Hz, COCH]CH), 7.40 (brs, 1H, NH), 5.35e5.34 (m, 2H,

CH₂CH]CHCH₂), 2.41 (t, 2H, J 7.8 Hz, COCH₂), 2.02e2.00 (m, 4H,

CH₂CH]CHCH₂), 1.78e1.73 (quin, 2H, J 7.2, 7.8 Hz, COCH₂CH₂),

1.38e1.25 (m, 20H, 10 CH₂), 0.88 (t, 3H, J 6.6, 7.2 Hz, CH₃); ¹³C NMR

0.88 (t, 3H, J 7.2 Hz, CH₃); ¹³C NMR (150 MHz, CDCl₃):

d 188.63

(COCH]CH), 171.70 (CONH), 143.02, 142.32, 136.38, 133.42, 130.06,

130.00, 129.69, 129.59, 129.24, 122.14, 118.94 (12 C_Ar, 2 CH]CH),

37.92 (COCH₂), 31.91, 29.77, 29.70, 29.53, 29.33, 29.32, 29.28, 29.23,

27.11, 27.23, 26.16, 25.43, 22.69 (13 CH₂), 14.13 (CH₃); Exact mass

calculated for C₃₃H₄₄ClNO₂(521.3060), EI MS, m/z (%), Found:

521.45 (M^þ, 12), 518.30 (18), 368.30 (28), 368.30 (100). Anal. Calcd.

(150 MHz, CDCl₃):

d 187.97 (COCH]CH), 171.66 (CONH), 148.52,

142.64, 141.21, 141.14, 136.25, 133.01, 130.54, 130.15, 130.08, 129.87,

129.68, 128.93, 125.47, 124.23, 123.53, 118.99 (12 C_Ar, 2 CH]CH),

37.93 (COCH₂), 29.76, 29.70, 29.53, 29.32, 29.27, 29.21, 29.11, 27.23,

27.16, 25.40, 22.69 (13 CH₂), 14.13 (CH₃); Exact mass calculated for

C

₃₃H₄₄N₂O₄(532.3301), EI MS, m/z (%), Found: 532.50 (M^þ, 52),

Fig. 10. DPPH Radical scavenging assay. Different letters on the column varied

signiﬁcantly at p ꢄ 0.05.

Fig. 11. OH^ꢂScavenging assay. Different letters on the column varied signiﬁcantly at

p ꢄ 0.05.

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

527

Table 2

3

_Thiop), 7.33 (d, 1H, J 15.6 Hz, COCH]CH), 7.32 (brs, 1H, NH), 7.09

Energy calculations using ZNDO1 semi empirical method built in HyperChem Mo-

lecular package.

(dd, 1H, J 3.6, 4.8 Hz, H-4_Thiop), 5.37e5.33 (m, 2H, CH₂CH]CHCH₂),

2.40 (t, 2H, J 7.2, 7.8 Hz, COCH₂), 2.01e1.99 (m, 4H, CH₂CH]CHCH₂),

1.75 (quin, 2H, J 7.2, 6.6 Hz, COCH₂CH₂), 1.39e1.26 (m, 20H, 10 CH₂),

0.88 (t, 3H, J 6.6, 7.2 Hz, CH₃); ¹³C NMR (150 MHz, CDCl₃):

E_TOTAL(kcal/mol) H_FORMATION(kcal/mol) E_HOMO(eV) E_LUMO(eV) D_E(eV)

10b ꢀ114130

10c ꢀ135289

11 ꢀ145161

14a ꢀ140241

14b ꢀ129582

15a ꢀ312831

15b ꢀ281254

15c ꢀ291443

15d ꢀ259938

ꢀ7748.8

ꢀ9681.0

ꢀ6.970

ꢀ6.960

ꢀ6.749

ꢀ6.850

ꢀ6.860

ꢀ1.275

ꢀ3.285

ꢀ2.352

ꢀ3.273

4.977

4.980

4.860

4.890

5.132

1.478

4.507

0.7083

4.448

11.947

11.940

11.609

11.740

11.992

2.753

7.792

3.06

7.721

d

188.30 (COCH]CH), 171.55 (CONH), 1420.10, 140.47, 136.94,

ꢀ10732.0

ꢀ10222.0

ꢀ10093.0

ꢀ20379.0

ꢀ21270.9

ꢀ20050.7

ꢀ21014.3

133.63, 132.00, 131.06, 19.90, 129.70, 128.72, 128.35, 120.51, 118.88

(10 C_Ar, 2 CH]CH), 37.94 (COCH₂), 31.91, 29.76, 29.70, 29.53, 29.33,

29.32, 29.27, 29.22, 29.11, 27.23, 27.16, 25.43, 22.69 (13 CH₂), 14.13

(CH₃); Exact mass calculated for C₃₁H₄₃NO₂S (493.3014), EI MS, m/z

(%), Found: 493.50 (M^þ, 4.7), 483.30 (8), 463.30 (13), 437.40 (15).

Anal. Calcd. for C₃₁H₄₃NO₂S (493.74): C, 75.41; H, 8.78; N, 2.84.

Found. C, 74.93; H, 8.34; N, 3.35.

522.50 (43), 509.0 (36), 495.0 (21). Anal. Calcd. for C₃₃H₄₄N₂O₄

(532.71): C, 74.40; H, 8.33; N, 5.26. Found. C, 73.97; H, 8.28; N, 5.15.

4.1.3. N-(3-Acetylphenyl)palmitamide (8a)

Compound 7 (1.0 g, 7.4 mmol) was treated with palmitoyl

chloride (2.5 g, 9.1 mmol) as described for 3. Recrystallization of the

residue from EtOH affords 8a as ﬁne creamy crystals (1.5 g, 54%);

Mp. 95e96 ^ꢁC; R_f0.25 (toluene:ethyl acetate, 6:1); IR (v, cm^ꢀ1):

3288 (NH_str),1672 (C]O_str),1657 (Amide I),1590 (Amide II); ¹H NMR

4.1.2.7. N-(4-((2E,4E)-5-phenylpenta-2,4-dienoyl)phenyl)oleamide

(5). Yield: 78% As brilliant yellow amorphous mass upon ﬂash

chromatography using solvent gradient of toluene then (tolue-

ne:ethyl acetate, 14:1); R_f0.20 (toluene:ethyl acetate, 14:1); IR (v,

cm^ꢀ1): 3339 (NH_str), 1672 (C]O_str), 1650 (Amide I), 1605 (Amide II);

(600 MHz, DMSO-d₆):

d 8.04 (s, 1H, H-2_Ar), 7.91 (d, 1H, J 7.8 Hz, H-

6

_Ar), 7.68 (d, 1H, J 7.8 Hz, H-4_Ar), 7.43 (t, 1H, J_4,5¼ J_5,67.8 Hz, H-5_Ar),

¹H NMR (600 MHz, CDCl₃):

d 7.98, 7.65, 7.50, 7.03 (4d, 8H, J 8.4 Hz,

2.61 (s, 3H, COCH₃), 2.39 (t, 2H, J 7.8 Hz, COCH₂), 1.74 (m, 2H,

COCH₂CH₂), 1.63 (br.s, 1H, NeH), 1.38e1.25 [m, 24H, (CH₂)₁₂], 0.88

(dd, 3H, J 6.6, 7.2 Hz, CH₂CH₃); Anal. Calcd for C₂₄H₃₉NO₂. 0.5H₂O

(382.59) C: 75.34, H: 10.56, N: 3.66. Found: C: 75.95, H: 10.56, N:

4.04.

Ar), 7.60 (dddd, 1H, J 1.2, 1.8, 2.4, 8.4 Hz, H_Oleﬁnic), 7.39e7.32 (m, 4H,

1 Ar, 2 H_Oleﬁnic, NH), 7.10 (d, 1H, J 15.0 Hz, H_Oleﬁnic), 5.35e5.34 (m,

2H, CH₂CH]CHCH₂), 2.40 (t, 2H, J 7.2, 7.8 Hz, COCH₂), 2.02e1.99 (m,

4H, CH₂CH]CHCH₂), 1.74 (quin, 2H, J 7.2, 7.8 Hz, COCH₂CH₂), 1.37e

1.25 (m, 20H, 10 CH₂), 0.88 (t, 3H, J 7.2 Hz, CH₃); ¹³C NMR (150 MHz,

CDCl₃): d 188.90 (COCH]CH), 171.62 (CONH), 144.55, 141.99, 141.80,

4.1.4. General procedure for the synthesis of (10aec)

A mixture of 8aec (0.4 g, 1.1 mmol), 9 (0.6 g, 5.0 mmol) and

NaOH (0.5 g, 12.5 mmol) in EtOH (12 ml) was stirred at room

temperature for 48 h then neutralized with AcOH and evaporated

in vacuo.

136.15, 130.06, 129.87, 129.70, 129.20, 128.86, 128.57, 127.29, 126.99,

124.14, 118.86 (12 C_Ar, 3 CH]CH), 37.94 (COCH₂), 31.91, 29.77, 29.70,

29.53, 29.33, 29.28, 29.22, 29.11, 27.23, 27.16, 25.44, 22.69 (13 CH₂),

14.13 (CH₃); Exact mass calculated for C₃₅H₄₇NO₂(513.3606), EI MS,

m/z (%), Found: 513.40 (M^þ, 60), 495.40 (1), 422.32 (9), 249.10 (100).

Anal. Calcd. for C₃₅H₄₇NO₂(513.75): N, 2.73. Found. N, 2.26.

4.1.4.1. (E)-N-(3-(3-(4-hydroxyphenyl)acryloyl)phenyl)palmitamide

(10a). The residue was puriﬁed by ﬂash chromatography using

solvent gradient of (toluene:ethyl acetate, 10:1 / 8:1) to afford 10a

(0.33 g, 66%) as orange sun-like crystals; Mp. 106 ^ꢁC; R_f0.34 (tol-

uene:ethyl acetate, 4:1); IR (v, cm^ꢀ1): 3197e3171 (OeH_str, NeH_str),

1671 (C]O_str, Amide I), 1601 (Amide II); ¹H NMR (600 MHz, CDCl₃):

4.1.2.8. N-(4-((E)-3-(Furan-2-yl)acryloyl)phenyl)oleamide

(6a).

Yield: 90% As tiny star-like yellow crystals upon ﬂash chromatog-

raphy using solvent gradient of toluene then (toluene:ethyl acetate

8:1); R_f0.28 (toluene:ethyl acetate, 8:1); Mp. 67 ^ꢁC; IR (v, cm^ꢀ1):

3353 (NH_str),1677 (C]O_str),1647 (Amide I),1595 (Amide II); ¹H NMR

d

9.86 (s, 1H, OH), 7.83e7.81 (m, 5H, Ar, COCH]CH), 6.99e6.98 (m,

(600 MHz, CDCl₃):

d 8.03, 7.67 (2d, 4H, J 8.4 Hz, Ar), 7.59 (d, 1H, J

5H, Ar, COCH]CH), 2.37 (t, 2H, J 7.8 Hz, COCH₂), 1.83 (br.s, 1H, NH),

1.64 (m, 2H, COCH₂CH₂), 1.35e1.25 [m, 24H, (CH₂)₁₂], 0.88 (t, 3H, J

7.2 Hz, CH₂CH₃); Exact mass calculated for C₃₁H₄₃NO₂(477.32), EI

MS, m/z (%), Found: 479.00 (17, Mþ2), 423 (17), 383 (46), 381 (18),

369 (8). Anal. Calcd. for C₃₁H₄₃NO₂(477.69): N: 2.95. Found: N 2.48.

15.6 Hz, COCH]CH), 7.53 (d, 1H, J 1.2 Hz, H-5_Fur), 7.46 (d, 1H, J

15.6 Hz, COCH]CH), 7.45 (brs, 1H, NH), 6.71 (d, 1H, J 3.6 Hz, H-3_Fur),

6.51 (d, 1H, J 1.2 Hz, H-4_Fur), 5.37e5.31 (m, 2H, CH]CH), 2.39 (t, 2H,

J 7.2, 7.8 Hz, COCH₂), 2.04e1.99 (m, 4H, CH₂CH]CHCH₂), 1.74 (quin,

2H, J 7.2 Hz, COCH₂CH₂), 1.38e1.17 (m, 20H, 10 CH₂), 0.88 (t, 3H, J

6.6, 7.6 Hz, CH₃); ¹³C NMR (150 MHz, CDCl₃):

d 188.35 (COCH]CH),

4.1.4.2. (E)-N-(3-(3-(4-hydroxyphenyl)acryloyl)phenyl)acetamide

(10b). The residue was puriﬁed by ﬂash chromatography (tolue-

ne:ethyl acetate, 1:1) to afford 10b (50 mg, 21%) as colorless pow-

der; Mp. 228e30 ^ꢁC; R_f0.2 (toluene:ethyl acetate, 1:1); IR (v, cm^ꢀ1):

3339 (OeH_str, NeH_str), 1670 (C]O_str.), 1640 (Amide I), 1601 (Amide

171.80 (CONH),151.73, 144.88,130.45, 130.06,129.92, 129.71, 119.06,

118.89, 116.16, 112.67 (10 C_Ar, 2 CH]CH), 37.93 (COCH₂), 31.91,

29.77, 29.70, 29.53, 29.33, 29.32, 29.28, 29.22, 29.11, 27.23, 27.16,

25.44, 22.69 (13 CH₂), 14.13 (CH₃); Exact mass calculated for

₃₁H₄₃NO₃(477.3242), EI MS, m/z (%), Found: 477.40 (M^þ, 1), 466.45

II); ¹H NMR (600 MHz, DMSO-d₆):

d

10.16 (br.s, 1H, OH), 8.17 (s, 1H,

H-2_Anilide), 7.87 (d, 1H, J_5,67.8 Hz, H-6_Anilide), 7.82 (d, 1H, J_7.8Hz, H-

_Anilide), 7.71 (d, 2H, J 8.4 Hz, H-2_Phenol, H-6_Phenol), 7.67 (d, 1H, J

C

(7), 452.0 (14), 434.0 (38), 399.0 (36), 368 (100). Anal. Calcd. for

₃₁H₄₃NO₃(477.68): C, 77.95; H, 9.07; N, 2.93. Found. C, 77.79; H,

C

4

8.88; N, 2.77.

15.6 Hz, COCH]CH), 7.61 (d, 1H, J 15.6 Hz, COCH]CH), 7.47 (t, 1H,

J_4,5¼ J_5,67.8 Hz, H-5_Anilide), 6.82 (d, 2H, J 8.4 Hz, H-3_Phenol, H-5_Phenol),

2.54 (s, 1H, NH), 2.06 (s, 3H, COCH₃); Exact mass calculated for

4.1.2.9. N-(4-((E)-3-(Thiophen-2-yl)acryloyl)phenyl)oleamide (6b).

Yield: 89% As tiny yellow crystals upon ﬂash chromatography using

solvent gradient of toluene then (toluene:ethyl acetate, 9:1) fol-

lowed by recrystallization from toluene or Et₂O; R_f0.32 (tolue-

ne:ethyl acetate, 8:1); Mp. 62 ^ꢁC; IR (v, cm^ꢀ1): 3342 (NH_str), 1671

(C]O_str), 1652 (Amide I), 1589 (Amide II); ¹H NMR (600 MHz,

C

₁₇H₁₅NO₃(281.11), EI MS, m/z (%), Found: 281 (100, M^þ), 264 (6),

238 (45), 222 (16), 210 (24). Anal. Calcd. for C₁₇H₁₅NO₃. 0.25H₂O

(285.83) C: 71.42, H: 5.24. Found: C: 71.42, H: 5.47.

4.1.4.3. (E)-N-(3-(3-(4-hydroxyphenyl)acryloyl)phenyl)benzamide

(10c). The residue was puriﬁed by ﬂash chromatography with

solvent gradient (toluene then toluene:ethyl acetate, 8:1 / 5:1) to

CDCl₃):

d 8.02, 7.67 (2d, 4H, J 8.4 Hz, Ar), 7.94 (d, 1H, J 15.6 Hz,

COCH]CH), 7.42 (d, 1H, J 4.8 Hz, H-5_Thiop), 7.36 (d, 1H, J 3.6 Hz, H-

528

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

afford 10c (0.37 g, 44%) as brilliant yellow crystals; Mp. 154e156 ^ꢁC;

R_f0.15 (toluene:ethyl acetate, 6.5:1); IR (v, cm^ꢀ1): 3298 (OeH_str, Ne

H

1H, J 7.8 Hz, Ar), 7.90 (t, 1H, J 1.8 Hz, Ar), 7.83 (d, 1H, J 15.6 Hz,

COCH]CH), 7.63 (d, 2H, J 8.4 Hz, Ar), 7.55 (t, 1H, J 7.2, 7.8 Hz, Ar),

7.48 (m, 1H, Ar), 7.44 (d,1H, J 15.6 Hz, COCH]CH), 7.44e7.42 (m,1H,

Ar), 7.41e7.39 (m, 1H, Ar), 7.04 (d, 1H, J 7.8 Hz, Ar), 6.97 (dd, 1H, J 1.2,

7.2 Hz, Ar), 6.95 (d, 1H, J 7.2 Hz, Ar), 6.94 (m, 1H, Ar), 3.86 (s,

_str), 1661 (C]O_str., Amide I), 1611 (Amide II); ¹H NMR (600 MHz,

DMSO-d₆):

d

10.47, 10.15 (2s, 2H, NH_{D O}

,

Exchangeable

2

OH_{D O}_Exchangeable), 8.41 (s,1H, H-2_Anilide), 8.10, 7.89 (2d, 2H, J 7.8 Hz,

2

Ar), 7.99 (d, 2H, J 8.4 Hz, H-2_Phenol, H-6_Phenol), 7.72 (d, 2H, J 9.0 Hz,

Ar), 7.68 (d, 1H, J 14.4 Hz, COCH]CH), 7.60 (d, 1H, J 7.2 Hz, Ar), 7.54

(m, 3H, COCH]CH, Ar), 7.16 (d, 1H, J 7.2 Hz, Ar), 6.83 (d, 2H, J 8.4 Hz,

3H, OCH₃); ¹³C NMR (150 MHz, DMSO-d₆):

d 189.85 (C]O), 163.69

(C]N), 161.80, 161.11, 148.91, 145.26, 139.73, 133.53, 132.56, 130.40,

129.64, 127.39, 126.69, 125.66, 120.64, 119.27, 119.24, 119.00, 117.29,

114.42 (18 C_Ar, COCH]CH), 55.42 (OCH₃); Exact mass calculated for

H-3_Phenol, H-5_Phenol); ¹³C NMR (150 MHz, DMSO-d₆):

d 188.90

(COCH]CH), 165.76 (CONH), 160.27, 144.75, 139.66, 138.47, 137.41,

134.66, 131.86, 131.09, 129.16, 128.97, 128.52, 128.28, 127.77, 125.77,

125.39, 124.60, 123.88, 119.99, 118.49, 115.91 (18C_Ar, COCH]CH);

Exact mass calculated for C₂₂H₁₇NO₃(343.12), EI MS, m/z (%),

Found: 343.00 (28, M^þ), 326 (0.7), 315 (0.6), 284 (0.3), 254 (0.3),

238 (7). Anal. Calcd. for C₂₂H₁₇NO₃(343.38) C: 76.95, H: 4.99.

Found: C: 76.80, H: 5.08.

C

₂₃H₁₉NO₃(357.14), EI MS, m/z (%), Found: 357.00 (100, M^þ), 355

(0.8), 342 (9), 314 (4), 298 (1.5), 284 (0.9). Anal. Calcd. for C₂₃H₁₉NO₃

(357.40) C: 77.29, H: 5.36. Found: C: 77.22, H: 5.40.

4.1.7. (E)-1-(3-((E)-2-hydroxybenzylideneamino)phenyl)-3-p-

tolylprop-2-en-1-one (14b)

Compound 13 (0.8 g, 3.3 mmol) was treated with p-tolualde-

hyde (0.5 ml, 4.2 mmol) and NaOH (0.2 g, 5.0 mmol) in EtOH (10 ml)

then worked up as described for 14a in Method B. The crude pre-

cipitate formed was ﬁltered at the pump then recrystallized from

MeOH to afford 14b (0.62 g, 54%) as buff colored crystals; Mp.

118 ^ꢁC; R_f0.25 (Toluene); IR (v, cm^ꢀ1): 2947 (OHeN), 1662 (C]O_str.),

4.1.5. (E)-1-(3-((E)-4-methoxybenzylideneamino)phenyl)-3-(4-

methoxyphenyl)prop-2-en-1-one (11)

A mixture of 7 (1.0 g, 7.4 mmol), anizaldehyde (2.2 ml,

18.1 mmol) and NaOH (0.2 g, 5.0 mmol) in EtOHeDMF (2:5, 7 ml)

was stirred overnight, ﬁltered at the pump, washed with little H₂O

then EtOH and dried well. Recrystallization from EtOH affords

11(2.1 g, 76%) as ﬁne yellow crystals; Mp. 132 ^ꢁC; R_f0.5 (tolue-

ne:acetone:Et₃N, 9 : 0.2 : 0.1); IR (v, cm^ꢀ1): 1661 (C]O_str.), 1621

1618 (C]N_str.), 1283 (CeO_str.); ¹H NMR (600 MHz, CDCl₃):

d 13.09 (s,

1H, OH), 8.71 (s, 1H, N]CH), 9.43 (d, 1H, J 7.8 Hz, Ar), 7.92 (t, 1H, J

1.8 Hz, Ar), 7.84 (d, 1H, J 15.6 Hz, COCH]CH), 7.597.56 (m, 3H, Ar),

7.52 (d, 1H, J 15.6 Hz, COCH]CH), 7.51e7.50 (m, 1H, Ar), 7.44e7.40

(m, 2H, Ar), 7.25e7.24 (m, 2H, Ar), 7.05 (d, 1H, J 8.4 Hz, Ar), 6.98 (t,

1H, J 7.8, 7.2 Hz, Ar), 1.62 (s, 3H, CH₃); ¹³C NMR (150 MHz, CDCl₃):

(C]N_str.); ¹H NMR (600 MHz, DMSO-d₆):

d 8.46 (s, 1H, N]CH),

7.86e7.89 (m, 2H, Ar), 7.88 (d, 1H, J 9.0 Hz, Ar), 7.86 (t, 1H, J 1.8,

3.6 Hz, Ar), 7.64e7.61 (m, 1H, Ar), 7.62 (d, 1H, J 8.4 Hz, Ar), 7.52 (t,

1H, J 7.8 Hz, Ar), 7.46, 7.83 (2 d, 2H, J_vic15.6 Hz, COCH]CH), 7.41,

7.42 (2 dd, 1H, J 1.2, 1.8, Ar), 7.02e7.00 (m, 1 H, Ar), 7.02 (d, 1H, J

9.0 Hz, Ar), 6.96e6.93 (m,1H, Ar), 6.95 (d,1H, J 8.4 Hz, Ar), 3.87, 3.89

(2s, 6H, 2 OCH₃); Exact mass calculated for C₂₄H₂₁NO₃(371.15), EI

MS, m/z (%), Found: 371 (100, M^þ), 340 (22), 262 (23), 328 (13), 237

(36), 297 (10). Anal. Calcd. for C₂₄H₂₁NO₃(371.43) C: 77.61, H: 5.70,

N: 3.77. Found: C: 77.89, H: 5.72, N: 4.18.

d

189.99 (C]O), 163.76 (C]N), 161.12, 148.99, 145.55, 141.39,

139.60, 133.57, 132.57, 131.93, 129.75, 129.69, 128.62, 126.75, 125.80,

120.68, 120.64, 119.26, 119.00, 117.31 (18 C_Ar, COCH]CH), 21.60

(CH₃); Exact mass calculated for C₂₃H₁₉NO₂(341.14), EI MS, m/z (%),

Found: 343 (Mþ2, 9), 342 (Mþ1, 7), 341 (M, 43), 328 (11), 327 (12),

293 (17), 149 (100). Anal. Calcd. for C₂₃H₁₉NO₂(341.40) C: 80.92, H:

5.61, N: 4.10. Found: C: 80.51, H: 5.37, N: 3.61.

4.1.8. General procedure for the synthesis of metal complexes (15ae

d)

4.1.6. (E)-1-(3-((E)-2-hydroxybenzylideneamino)phenyl)-3-(4-

methoxyphenyl)prop-2-en-1-one (14a)

Metal acetate (0.42 mmol) in EtOH (15 ml) was added dropwise

to a reﬂuxing solution of the ligand 14a or 14b (0.42 mmol) in EtOH

(15 ml). Reﬂux was continued for further 10 min after complete

addition of the salt. The precipitate formed was ﬁltered, washed

with EtOH (3 ml) and dried well.

4.1.6.1. Method A. A mixture of 11 (0.2 g, 0.5 mmol) and conc. HCl

(0.3 ml) in dry acetone (10 ml) was heated under reﬂux for 2 h then

coevaporated with toluene in vacuo. The residue was taken in ethyl

acetate (30 ml), washed with saturated K₂CO₃solution (10 ml),

dried over MgSO₄and evaporated in vacuo. The residue was puri-

ﬁed by ﬂash chromatography (toluene:ethyl acetate, 5:1) to afford

12 (0.11 g, 86%). R_f0.3 (toluene:ethyl acetate, 5:1). Compound 12

(0.11 g, 0.4 mmol) was taken in EtOH (4 ml) treated with salicy-

laldehyde (0.2 ml, 1.9 mmol) and heated under reﬂux for 1 h and

left to be crystallized overnight to afford 14a (0.12 g, 75%) as single

yellow crystals.

4.1.8.1. Bis(2-((E)-(3-((E)-3-(4-methoxyphenyl)acryloyl)phenyl-

imino)methyl)phenoxy)copper (15a). Brown precipitate formed

immediately during addition of the salt (0.1 g, 62%); IR (v, cm^ꢀ1):

1650 (C]O_str), 1604 (C]N_str). Anal. Calcd. For C₄₆H₃₆CuN₂O₆

(776.33): C, 71.17; H, 4.67; N, 3.61. Found: C, 70.67; H, 4.46; N, 3.51.

4.1.8.2. Bis(2-((E)-(3-((E)-3-(4-methoxyphenyl)acryloyl)phenyl-

imino)methyl)phenoxy)zinc (15b). Yellow precipitate formed

upon concentration of the reaction mixture to half of its volume

and cooling to ambient temperature (0.1 g, 61%); IR (v, cm^ꢀ1): 1651

4.1.6.2. Method B. Compound 7 was treated with salicylaldehyde as

described for the synthesis of 14a in Method A to afford 13 (68%)

upon washing with Et₂O as yellow crystals and was pure enough to

be used in the next step without further puriﬁcation; R_f0.3 (tol-

(C]O_str), 1603 (C]N_str); ¹H NMR (600 MHz, DMSO-d₆):

d 8.71 (s,

uene:ethyl acetate, 5:1); Mp. 90e92 ^ꢁC; IR (v, cm^ꢀ1): 3329 (OeH_str

,

2H, 2 CH]N), 8.02 (s, 2H, Ar), 7.94 (d, 2H, J 7.2 Hz, Ar), 7.72 (d, 4H, J

9.0 Hz, Ar), 7.66 (d, 2H, J 15.6 Hz, 2 CH]CHCO), 7.61 (d, 2H, J 15.6 Hz,

2 CH]CHCO), 7.55e7.54 (m, 2H, Ar), 7.51 (t, 2H, J 7.2, 7.8 Hz, Ar),

7.45 (dd, 2H, J 1.8, 7.8 Hz, Ar), 7.27 (m, 2H, J 1.2, 1.8 Hz, Ar), 6.97 (d,

4H, J 9.0 Hz, Ar), 6.73 (d, 2H, J 8.4 Hz, Ar), 6.60 (t, 2H, J 7.2 Hz, Ar),

3.80 (s, 6H, 2 OCH₃). Anal. Calcd. For C₄₆H₃₆N₂O₆Zn. 0.5H₂O

(778.21): C, 70.18; H, 4.74. Found: C, 70.76; H, 4.46.

very weak), 3060 (CeH_{Ar str}), 2993 (CeH_{Alip. str}), 1671 (C]O_str.),

1614 (C]N_str.).

A mixture of 13 (0.45 g, 1.9 mmol), anisaldehyde (0.3 ml,

2.5 mmol) and NaOH (0.2 g, 5.0 mmol) in EtOH (5 ml) was stirred at

ambient temperature for 48 h then acidiﬁed with AcOH. The pre-

cipitate formed was ﬁltered at the pump, washed with MeOHeEt₂O

and dried well. The residue was recrystallized from EtOH to afford

14a (0.18 g, 27%); R_f0.3 (toluene); Mp.129e30 ^ꢁC. IR (v, cm^ꢀ1): 2947

(OHeN), 1668 (C]O_str), 1616 (C]N_str), 1294 (CeO_str). ¹H NMR

4.1.8.3. Bis(2-((E)-(3-((E)-3-p-tolylacryloyl)phenylimino)methyl)

phenoxy)copper (15c). Brown precipitate formed upon concentra-

tion of the reaction mixture to half of its volume and cooling to

(600 MHz, CDCl₃):

d 13.10 (s, 1H, OH), 8.70 (s, 1H, CH]N), 7.92 (d,

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

529

ambient temperature (0.11 g, 66%); IR (v, cm^ꢀ1): 1655 (C]O_str),

1605 (C]N_str). Anal. Calcd. For C₄₆H₃₆CuN₂O₄. 0.5H₂O (753.35): C,

73.33; H, 4.96; N, 3.71. Found: C, 72.68; H, 4.68; N, 3.41.

4.3.2. Antioxidant activity

4.3.2.1. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH^ꢂ) radical scavenging

activity assay. Each test compound (10 L, 0.03 mM in DMSO) was

added to DPPH (990 L, 0.1 mM in MeOH) and shacked thoroughly

m

4.1.8.4. Bis(2-((E)-(3-((E)-3-p-tolylacryloyl)phenylimino)methyl)

reaching a ﬁnal concentration of (30 nM). Absorbance at 517 nm

was determined spectrophotometrically over a period of 2 h.

Changes in absorbance were minimal within 30 min. 6-hydroxy-

2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used as

positive antioxidant control under the same conditions and all

measurements were undertaken on three replicates. The activity of

the test compounds to scavenge DPPH was calculated as percentage

inhibition from the relation:

phenoxy)zinc

(15d). Yellow

precipitate

formed

upon

concentration of the reaction mixture to half of its volume and

cooling to ambient temperature (0.075 g, 46%); IR (v, cm^ꢀ1): 1655

(C]O_str.), 1607 (C]N_str.); ¹H NMR (600 MHz, DMSO-d₆):

d 8.70 (s,

2 H, 2 CH]N), 8.01 (s, 2 H, Ar), 7.95 (d, 2 H, J 7.8 Hz, Ar), 7.71 (d, 2 H, J

15.6 Hz, 2 CH]CHCO), 7.66 (d, 2 H, J 15.6 Hz, 2 CH]CHCO), 7.65 (d,

4 H, J 7.8 Hz, Ar), 7.55e7.54 (m, 2 H, Ar), 7.51 (t, 2 H, J 7.2, 7.8 Hz, Ar),

7.45 (dd, 2 H, J 1.8, 7.8 Hz, Ar), 7.26 (m, 2 H, J 1.8, 8.4 Hz, Ar), 7.23 (d,

4 H, J 7.8 Hz, Ar), 6.72 (d, 2 H, J 8.4 Hz, Ar), 6.59 (t, 2 H, J 7.2 Hz, Ar),

2.33 (s, 6 H, 2 CH₃). Anal. Calcd. For C₄₆H₃₆N₂O₄Zn (746.20): C,

74.04; H, 4.86; N, 3.75. Found: C, 74.18; H, 4.72; N, 3.73.

Inhibition % ¼ [(A_Bꢀ A_A)/A_B] 100

where A_Ais the absorbance of the test compound or control at

t ¼ 30 min, while, A_Bis that for the blank at t ¼ 0 min.

4.2. Crystallographic structure determination

4.3.2.2. Hydroxyl radical (HO^ꢂ) scavenging assay. Samples (10

ml) of

selected tested compounds and standard compound (Ascorbic acid)

at 30 nM were dissolved in DMSO, then incubated with 9.0 mM

FeSO₄(1.0 ml), 0.3% H₂O₂(1.0 ml) in 0.5 ml salicylic acideEtOH

solution (9.0 mM) for 30 min at 37 ^ꢁC. Hydroxyl radicals were

detected by monitoring absorbance at 510 nm. The total volume of

the mixture in each tube was made up to 3 ml by adding the

required amount of distilled water. Inhibition (I) percentage of

deoxyribose degradation in percent was calculated according

following equation: I (%) ¼ [(A₀ꢀ A₁/A₀)] 100. A₀is the absorbance

of the control reaction and A₁is the absorbance of the test com-

pound. Measurements were done in the Chemistry Department,

Faculty of science at Taif University.

Single crystal X-ray diffraction data were collected using the Mo

ꢀ

K

a

radiation (

l

¼ 0.71073 A) on maXus (Bruker Nonius, Delft &

MacScience, Japan) at 298 K. Structure was solved by direct

methods using SIR97 [45] and reﬁned by full-matrix least-squares

on all F²using maXus [46]. Cell reﬁnement according to HKL Sca-

lepack and data reduction using Denzo and Scalepak [47]. Molec-

ular graphics according to Johnson et al. [48]. Crystallographic data

of 14a was deposited with the Cambridge Crystallographic Data

Centre

(http://www.ccdc.cam.ac.uk/services/structure_deposit),

deposition number: CCDC 960986 (E-mail: deposit@ccdc.cam.ac.

uk).

4.3. Biology

4.4. Cytotoxicity screening

4.3.1. Antiobesity

In vitro anticancer-drug discovery screening of newly synthe-

sized compounds was performed using the colorimetric cytotox-

icity assay for anticancer-drug screening using prostate cancer cell

line PC3 at the National cancer Institute, Cairo University. Cells were

plated in 96-multiwell plates (10⁴cells per well) for 24 h to allow

attachment of cells to the walls. Monolayer cells were incubated

with test compounds individually at concentrations of 5.0, 10.0,

4.3.1.1. Animal experiments. Body weight loss and hypolipidemic

activities in SD Male rats (Purchased from Experimental Animal

Center of Suez Canal University, Ismailia, Egypt) were divided into

eleven groups each group contain 10 rats: rats fed with a low fat

diet (normal), rats treated with high-fat diet-fed (obese), high-fat

diet fed rats treated with Tetrahydrolipstatin (Orlistat) (30 mg/day/

kg of rat weight) as positive control, high-fat diet-fed rats treated

with compound (4aef and 6a,b) at a dose of 30 mg/kg. Low fat diet

was composed of protein 20%, carbohydrate 70%, and fat 10%,

whereas high fat diet was composed of protein 20%, carbohydrate

35%, and fat 45%. They were allowed free access to tap water and

food. Every group of rats, the normal control rats were exception,

were fed with HFD for two months. Then every group of rats were

given vehicle or compounds by intragastric administration

respectively, once daily for one month. After this feeding period, the

rats were sacriﬁced.

20.0, 40.0 and 80.0 m

M at 37 ^ꢁC under 5% CO₂atmosphere. After

48 h, cells were ﬁxed, washed then stained with sulfo-rhodamine-B

stain. Excess stain was washed with acetic acid and attached stain

was recovered with triseEDTA buffer. Color intensity was measured

in an ELISA reader. The relation between surviving fraction and

drug concentration was plotted to get the survival curve for each

compound [49].

4.5. Statistical analysis

All results were subjected to one-way ANOVA and the means

were compared according to the StudenteNewmaneKeuls (SNK)

multiple range test (p ꢄ 0.05).

4.3.1.2. Sample collection. Blood was taken in the morning, after

12e14 h fasting. Sera were obtained after at least 30 min clotting by

centrifugation at 2000 rpm for 15 min. Sera were removed and

frozen at ꢀ20 ^ꢁC.

Acknowledgments

4.3.1.3. Measurements of metabolic parameters. Serum levels of

glucose, TC, TG and FFAs were determined by enzymatic tests from

Roche Diagnostic using the cobase 311 autoanalyser. Insulin levels

were determined by an enzyme-immunoassay which does not

cross-react with proinsulin. These hormone measurements were

done in duplicate and results were accepted if the coefﬁcients of

variation were less than 10%. Inter assay coefﬁcients of variation

were approximately 10%. Insulin sensitivity index was calculated as

FBs/Free insulin level.

The ﬁnancial support of Port Said University, Suez Canal Uni-

versity and Taif University, Grant Number 1-433-1828 are gratefully

acknowledged.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2014.02.021.

530

M.R. El Sayed Aly et al. / European Journal of Medicinal Chemistry 76 (2014) 517e530

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Article Doi

Antiobesity, antioxidant and cytotoxicity activities of newly synthesized chalcone derivatives and their metal complexes Dedicated to Professor R. R. Schmidt on the occasion of his 79th birthday.

DOI: 10.1016/j.ejmech.2014.02.021

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