Discovery of 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids as novel PPARα agonists with anti-hyperlipidemic and antioxidant activities: Design, synthesis and in vitro/in vivo biological evaluation

Article abstract of DOI:10.1016/j.bioorg.2021.105170

In the current work, a series of novel 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids (10a-o) and (11a-e) were synthesized and evaluated as new PPARα agonists in order to find new agents with higher activity and fewer side effects. The 2-propanoic acid derivative 10a and the 2-butanoic acid congener 10i showed the best overall PPARα agonistic activity showing E_max% values of 50.80 and 90.55%, respectively, and EC₅₀ values of 8.9 and 25.0 μM, respectively, compared to fenofibric acid with E_max = 100% and EC₅₀ = 23.22 μM, respectively. These two compounds also stimulated carnitine palmitoyltransferase 1A gene transcription in HepG2 cells and PPARα protein expression. Molecular docking simulations were performed for the newly synthesized compounds to study their predicted binding pattern and energies in PPARα active site to rationalize their promising activity. In vivo, compounds 10a and 10i elicited a significant hypolipidemic activity improving the lipid profile in triton WR-1339-induced hyperlipidemic rats, including serum triglycerides, total cholesterol, LDL, HDL and VLDL levels. Compound 10i possessed better anti-hyperlipidemic activity than 10a. At a dose of 200 mg/kg, it demonstrated significantly lower TC, TG, LDL and VLDL levels than that of fenofibrate at the same dose with similar HDL levels. Compounds 10i and 10a possessed atherogenic indices (CRR, AC, AI, CRI-II) like that of fenofibrate. Additionally, a promising antioxidant activity indicated by the increased tissue reduced glutathione and plasma total antioxidant capacity with decreased plasma malondialdehyde levels was demonstrated by compounds 10a and 10i. No histopathological alterations were recorded in the hepatic tissue of compound 10i (200 mg/kg).

Full text of DOI:10.1016/j.bioorg.2021.105170

Bioorganic Chemistry 115 (2021) 105170
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids
as novel PPARα agonists with anti-hyperlipidemic and antioxidant
activities: Design, synthesis and in vitro/in vivo biological evaluation
Rasha M. Hassana, Mona E. Aboutablb, Manuela Bozzic *, Mohammed F. El-Behairye,
,d,
,
,h
Ahmed M. El Kerdawyf g, Beatrice Sampaolesed, Claudia Desideriod, Federica Vincenzonic
,
,
,*
Francesca Sciandrad *, Iman A.Y. Ghannami
a Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618), P.O. 12622,
Dokki, Giza, Egypt
b Medicinal and Pharmaceutical Chemistry Department (Pharmacology Group), Pharmaceutical and Drug Industries Research Division, National Research Centre (ID:
60014618), 33 El Bohouth St., P.O. 12622, Dokki, Giza, Egypt
c
`
Dipartimento Universitario di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Sezione di Biochimica e Biochimica Clinica, Universita
Cattolica del Sacro Cuore di Roma, Largo F. Vito 1, 00168 Roma, Italy
d Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”– SCITEC (CNR) Sede di Roma, Largo F. Vito 1, 00168 Roma, Italy
e Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City, Sadat City 32897, Egypt
f Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
g Department of Pharmaceutical Chemistry, School of Pharmacy, New Giza University, Newgiza, km 22 Cairo-Alexandria Desert Road, Cairo, Egypt
h Fondazione Policlinico Universitario "A. Gemelli", IRCCS, Largo A. Gemelli 8, 00168 Roma, Italy
i Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12622, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the current work, a series of novel 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids (10a-o) and
PPARα agonists
(11a-e) were synthesized and evaluated as new PPARα agonists in order to find new agents with higher activity
Chalcone fibrates
CPT1A
and fewer side effects. The 2-propanoic acid derivative 10a and the 2-butanoic acid congener 10i showed the
best overall PPAR
of 8.9 and 25.0
α
agonistic activity showing Emax% values of 50.80 and 90.55%, respectively, and EC50 values
M, respectively, compared to fenofibric acid with Emax = 100% and EC50 = 23.22 M,
Molecular docking
Anti-hyperlipidemic agents
μ
μ
respectively. These two compounds also stimulated carnitine palmitoyltransferase 1A gene transcription in
HepG2 cells and PPAR
α
protein expression. Molecular docking simulations were performed for the newly syn-
active site to rationalize
thesized compounds to study their predicted binding pattern and energies in PPAR
α
their promising activity. In vivo, compounds 10a and 10i elicited a significant hypolipidemic activity improving
the lipid profile in triton WR-1339-induced hyperlipidemic rats, including serum triglycerides, total cholesterol,
LDL, HDL and VLDL levels. Compound 10i possessed better anti-hyperlipidemic activity than 10a. At a dose of
200 mg/kg, it demonstrated significantly lower TC, TG, LDL and VLDL levels than that of fenofibrate at the same
dose with similar HDL levels. Compounds 10i and 10a possessed atherogenic indices (CRR, AC, AI, CRI-II) like
that of fenofibrate. Additionally, a promising antioxidant activity indicated by the increased tissue reduced
glutathione and plasma total antioxidant capacity with decreased plasma malondialdehyde levels was demon-
strated by compounds 10a and 10i. No histopathological alterations were recorded in the hepatic tissue of
compound 10i (200 mg/kg).
* Corresponding authors at: Dipartimento Universitario di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Sezione di Biochimica e
`
Biochimica Clinica, Universita Cattolica del Sacro Cuore di Roma, Largo F. Vito 1, 00168 Roma, Italy (M. Bozzi); Istituto di Scienze e Tecnologie Chimiche “Giulio
Natta”– SCITEC (CNR) Sede di Roma, Largo F. Vito 1, 00168 Roma, Italy (F. Scaindra); Chemistry of Natural and Microbial Products Department, Pharmaceutical and
Drug Industries Research Division, National Research Centre, Dokki, Cairo 12622, Egypt (I.A.Y. Ghannam).
E-mail addresses: Manuela.Bozzi@unicatt.it (M. Bozzi), francesca.sciandra@cnr.it (F. Sciandra), iman.youssef.ghannam@gmail.com (I.A.Y. Ghannam).
https://doi.org/10.1016/j.bioorg.2021.105170
Received 25 June 2021; Received in revised form 7 July 2021; Accepted 9 July 2021
Available online 16 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
1. Introduction
[15]. Finally, the indole chalcone fibrate hybrid (VIII) displayed an in
vivo anti-hyperlipidemic effect by lowering the levels of cholesterol,
phospholipids, and triglyceride by 31%, 32% and 30%, respectively.
Moreover, the latter compound elicited an antioxidant activity through
the suppression of superoxide ions, hydroxyl radicals and microsomal
lipid peroxidation by 31%, 37% and 35%, respectively [16].
Hypercholesterolemia and hypertriglyceridemia are two main fac-
tors for cardiovascular diseases (CVDs) causing the highest mortality
rate worldwide. According to World Health Organization (WHO), the
death rate due to CVDs is estimated 17.9 million annually [1,2].
Peroxisome proliferator-activated receptors (PPAR) isoforms (
α
, γ, and
These recent findings encouraged us to implement the molecular
hybridization approach, a structural tool commonly used in drug dis-
covery, between the chalcone backbone and the fibrate acidic head to
design chalcone fibrate hybrids. By design, the target hybrids should
β/δ), highly expressed in the liver, heart, kidney, muscles, and adipose
tissues, play a crucial regulatory role in lipid metabolism and fatty acid
oxidation, thus, they are considered main targets in hyperlipidemia
treatment [3,4]. Fibrates, such as fenofibrate (I) and bezafibrate (II)
show comparable binding pattern to that of fibrates in PPARα binding
(Fig. 1), are long standing PPAR
α
agonists that are used clinically to
site. By their carboxylate terminal, they should bind through hydrogen
bonding with the key amino acids His440, Tyr464, Tyr314 and Ser280
and through hydrophobic interactions, they should bind by their 4-
phenyl benzyloxy/(2-phenylethoxy) tail with the hydrophobic region
of the binding site (Fig. 2).
reduce cardiovascular diseases burden via improving the blood lipid
profile [5,6].
Moreover, the fibrates ability to counteract atherosclerosis is not
solely linked to their hypolipidemic effects, but also to their antioxidant
activities [7]. Indeed, initiation and progression of atherosclerosis is
directly connected to the oxidative stress and peroxidative damage of
blood lipoproteins [8,9]. Unfortunately, some fibrates trigger significant
side effects; viz, liver dysfunction and biomarkers alterations of renal
and cardiovascular diseases [4,10,11]. Therefore, the development of
Consequently, a new series of fibrate derivatives were synthesized
and evaluated for their PPARα agonistic activities. Their structure ac-
tivity relationships were characterized and the molecular mechanism
underlying their action was investigated by an in vitro PPAR reporter
activation capability
α
assay which was performed to evaluate the PPAR
α
more effective and safer PPAR
α
agonists is an active topic in medicinal
of the newly synthesized compounds. Molecular docking studies were
also performed to study their predicted binding pattern and energies in
chemistry research. Pemafibrate (III), a selective PPARα modulator, is
one of the best fibrates in terms of efficacy and safety with improved
hepatic and renal functions [12–14].
PPARα binding site to support the in vitro studies. Compounds with the
most promising EC50 values were selected to confirm their ability to
Chalcones represent an important class of compounds displaying
antihyperlipidemic effects [15,16], in addition to a series of pharmaco-
logical potentials including anticancer [17], anti-inflammatory [18],
antibacterial [19], antifungal [20], antiarrhythmic [21], antidiabetic
[22] and antioxidant actions [2]. The chalcone fibrate hybrids IV and V
induce carnitine palmitoyltransferase 1A gene transcription and PPARα
protein expression in HepG2 cells. Finally, their antihyperlipidemic and
antioxidant efficacy was tested in vivo in rats and histopathological ex-
amination of liver tissues was also performed to investigate their safety.
showed a good PPAR
α
agonistic activity (EC50 = 0.06, 64.00 µM,
2. Results and discussion
respectively) [16,23]. The anti-hyperlipidemic coumarin chalcone
fibrate (VI) elicited its anti-hyperlipidemic potential by lowering the total
serum triglyceride level by 25% [2]. The 4-nitrophenylchalcone fibrate
hybrid (VII) showed an anti-hyperlipidemic activity by reducing the total
serum triglyceride and cholesterol levels by 30.4 and 25%, respectively
2.1. Chemistry
The procedure reported in scheme 1 was followed in order to syn-
thesize the different chalcone fibrate hybrids. Hydroxy chalcones 5a-e
were prepared by Claisen–Schmidt condensation of the corresponding
Fig. 1. Chemical structures of some fibrates (I-VIII).
2

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
Fig. 2. Schematic diagram for the design of the chalcone fibrate hybrids.
Scheme 1. Synthesis of chalcone fibrate hybrids 10a-o and 11a-e. Reactions and conditions. i: Na2CO3 anhydrous, DMF, 80 ◦C, overnight, 79–89%; ii: KOH aq.
3
50%, MeOH, reflux, 2–5 h, 65–85%; iii: K2CO3 anhydrous, CH3CN, reflux, 2–4 h, 60–89%; iv: LiOH, THF/MeOH/H2O (3:2:1), rt, overnight, 31–50%.

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
previously synthesized 4-benzyloxy and 4-(2-phenylethoxy)benzalde-
hyde derivatives 3a-e [24,25] with p-hydroxy acetophenone 4 using
reported procedure [15] in the presence of 50% aqueous KOH. The
synthesized chalcones 5a-e were reacted with ethyl 2-bromopropa-
noate/butanoate, ethyl 2-bromo-2-methylpropanoate 6a-c or methyl
4-bromobutanoate 7 in the presence of anhydrous potassium carbonate
[2,26] to yield the new ester compounds 8a-o and 9a-e, respectively.
The ester derivatives 8a-o and 9a-e were then subjected to hydrolysis to
yield the corresponding novel acid derivatives 10a-o and 11a-e. The
ester hydrolysis for compounds 8a-o and 9a-e to produce the corre-
sponding acid derivatives was done in base-catalyzed conditions using
LiOH according to the reported procedure [24]. All chalcones 5a-e, es-
ters 8a-o and 9a-e and acid derivatives 10a-o and 11a-e were in (E)
configuration. This was confirmed by measuring the coupling constant
2.4. In vitro CPT1A gene transcription analysis
The carnitine palmitoyltransferase 1A gene (CPT1A) encodes for an
enzyme which plays a key role in the fatty-acids β-oxidation process, and
it is known to be up-regulated by PPARα through ligand dependent
transcriptional activation [28–32]. In order to further confirm the
PPAR agonistic effect of compounds 10a and 10i, the transcription
α
levels of CPT1A were evaluated in hepatocarcinoma HepG2 cells using
increasing concentrations of 10a, 10i and fenofibrate (FB). In compar-
ison to FB, both 10a and 10i elicited a more pronounced up-regulation
of the CPT1A transcription at all analysed concentrations. In particular,
10a increased the mRNA levels of CPT1A in a dose dependent manner,
whereas the effect elicited by FB and 10i was maximum at 50 µM and
decreased at 100 µM (Fig. 4).
(J) = 15–16 Hz for the protons of the
α
,β-unsaturated ketone in the 1H
NMR spectra and in 2D NOESY spectra (See supporting materials). The
optical activity for the chiral acids 10a-j was measured by a polarimeter
and found as racemic mixtures (±).
2.5. In vitro PPARα protein expression analysis
In HepG2 cells, compounds 10a and 10i induced the expression of
PPAR in a dose-dependent manner as demonstrated by Western blot
analysis. Fig. 5 showed that compounds 10a, 10i and FB stimulated
PPAR expression at a similar extent at 10 µM. At 50 µM, the capability
α
2.2. In vitro PPARα transactivation assay
α
of the compounds to promote PPARα expression is about double for 10a
In order to evaluate PPARα agonistic function, all the newly syn-
and about ten times higher for 10i, compared to FB; at 100 µM the
difference between 10a and 10i becomes even more remarkable, as the
thesized compounds were tested in vitro using a cell-based trans-
activation assay in eukaryotic cells; the reporter system utilizes the
PPARα expression induced by 10a and 10i is about two and fifty times
luciferase reporter gene functionally linked to a PPARα-responsive
higher than that induced by FB, respectively.
2.6. Molecular docking study
promoter. Thus, quantifying changes in luciferase expression provides a
sensitive measure of PPAR activity [27]. The precent of PPAR acti-
vation for all compounds was initially evaluated at 10 M and the results
α
α
μ
were compared with that of 2-[4-(4-chlorobenzoyl) phenoxy]-2-
methylpropanoic acid (fenofibric acid FA), which was used as a refer-
ence standard (Table 1). FA is the active metabolite of fenofibrate (FB).
Compounds showed activation higher than 20% compared to FA were
selected for Emax% and EC50 determination and the results are summa-
rized in Table 2. Among the six compounds selected for Emax% and EC50
determination, the 3-nitro-4-benzyloxy chalcone 2-butanoic acid hybrid
10i showed similar activity to that of FA in terms of Emax% and EC50
(90.55% and 25 µM, respectively vs 100% and 23.22 µM, respectively).
Compounds 10a, 10 k, and 10n showed significantly lower EC50 values
than those of FA (8.9, 8.8 and 2.3 µM, respectively, vs 23.22 µM), as well
as lower Emax% values (50.8, 41.9 and 13.7%, respectively vs 100%).
Molecular docking simulations were performed to investigate the
probable binding pattern of the newly synthesized compounds in PPAR
α
binding site. As planned, the synthesized chalcone fibrate series showed
a common binding pattern with its carboxylate moiety of the head group
forming four hydrogen bonds with the key amino acids Ser280, Tyr314,
Tyr464, and His440 of the PPARα binding site. The alkyl moiety at the
α
-carbon is directed into a hydrophobic pocket bordered by Phe273,
Gln277, Val444, and Leu456, a region at the top end of the so-called
“benzophenone” pocket and this group is responsible for selectivity to-
ward PPARα over PPARγ [33]. The alkyl chain on the α-carbon affects
the binding to the binding site with CH3 < CH2CH3 < gem-dimethyl in
terms of hydrophobic interaction which is reflected in the compounds’
docking score and biological activity (vide infra). The compounds’ distal
phenyl ring is accommodated in the rear sub-pocket interacting through
hydrophobic interaction with the hydrophobic side chains of the lining
amino acids Phe273, Ile339, Leu247, Ile241, Val255, Leu254, Ala333,
Ala250, Val332, Leu344, Met355. Thus, increasing the hydrophobicity
of the distal phenyl substitution increases the hydrophobic interaction
and so the binding affinity and this is reflected in the docking score and
biological activity (Figs. 6-7), provided that it does not sterically clash
with this sub-pocket. Compounds having R and S configurations at the
These results indicate their higher affinity to the target PPARα, but a
lower intrinsic activity, with respect to FA.
2.3. Structure-activity relationship (SAR) of the novel chalcone fibrates
(10a-o) and (11a-e) at the PPAR
In vitro PPAR transactivation assay showed that the PPAR
α
α
α
agonistic activities of the 2-propanoic acid 10a-e, 2-butanoic acid 10f-j
and 2-methyl propanoic acid series 10 k-o were higher than that dis-
played by the 4-butanoic acid series 11a-e except for the 3-chloro-4-phe-
nylethoxy chalcone fibrate 11c. Moreover, the 4-phenylethoxy series
(10e, 10j, 10o and 11e) was found to be the least active series over the
other 4-benzyloxy series. The 3-nitro-4-benzyloxy chalcone fibrates (10i
and 10n) showed a promising activity due to their favored hydrophobic
interactions of the nitro substituent at the distal phenyl ring and the
chiral
α-carbon showed comparable binding pattern and bind scores
indicating that both enantiomers exert the biological activity compa-
rably. The docking scores for the acid compounds are at the range from
ꢀ 14.32 to ꢀ 17.51 kcal/mol (Supporting information Table S1).
2.7. In vivo pharmacological studies
length of the alkyl chain at the
α-carbon. The 3-chloro-4-benzyloxy
chalcone fibrate hybrids (10c, 10 h and 10 m) were found to be less
active compounds, probably due to a steric clash with the rear sub-
pocket (Fig. 3). Among the six selected compounds, the 2-propanoic
acid derivative 10a and the 2-butanoic acid congener 10i showed the
All the synthesized compounds were initially screened for their in
vitro PPARα agonistic activity. Out of the in vitro tested chalcone de-
rivatives, the most active compounds 10a and 10i were further evalu-
ated for their in vivo antihyperlipidemic efficacy in triton-induced
hyperlipidemic rats as well as their antioxidant potential.
best overall PPAR
α
agonistic activity showing Emax% values of 50.80 and
M, respec-
M,
90.55%, respectively, and EC50 values equals 8.9 and 25.0
μ
tively, compared to FA with Emax = 100% and EC50 = 23.22
μ
2.7.1. Acute toxicity
respectively. Hence, these two compounds were selected for further
The acute oral toxicity test of compounds 10a and 10i showed no
signs of toxicity or mortalities for dose up to 2000 mg/kg. Therefore, 1/
investigation through in vitro and in vivo mechanistic studies.
4

Table 1
Fold activation % on PPAR-
α
for compounds (10a-o) and (11a-e) at 10 µM.
Compound
Fold activation
Compound
ID
Fold activation
%
a
R1
R2
R3
n
R1
R2
R3
n
%
ID
10a
10b
10c
10d
10e
10f
10g
10h
10i
26.3
22
22.8
15
10k
10l
H
CH3
Cl
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
C2H5
H
H
H
H
H
H
H
H
H
H
1
1
1
1
2
1
1
1
1
2
H
CH3
Cl
CH3
CH3
1
1
1
1
2
1
1
1
1
2
CH3
CH3
16
19.8
36.2
17
10m
10n
10o
11a
11b
11c
11d
11e
CH3
CH3
14.2
19
NO2
H
NO2
H
CH3
CH3
CH3
CH3
19.3
21.8
11.5
22
11.4
19
H
H
-
-
-
-
-
-
-
-
-
-
CH3
Cl
CH3
Cl
27
15
NO2 C2H5
C2H5
NO2
H
15
12
10j
FA
H
-
Data are represented as the fold induction % for the agonism of the corresponding luciferase reporter cell lines (Indigo Biosciences). Dosing was done in duplicate as a single experiment. aFold activation % of the
synthesized compounds compared to fenofibric acid (FA) at 10 µM.

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
20 and 1/10 of this dose was chosen for the assessment of the in vivo
Table 2
In vitro transactivation PPARα assay.
pharmacological studies.
Compound ID
Emax
%
EC50 (µM)
2.7.2. Lipid lowering profile of compounds 10a and 10i in triton WR-
1339-induced hyperlipidemic rats
10a
10b
10g
10i
10k
10n
FA
50.8
38.47
23.63
90.55
41.9
8.9
17.6
17.5
25.0
8.8
Tyloxapol or Triton WR-1339 is a non-ionic surfactant that has
been widely used as a model for the induction of acute hyperlipidemia
in animals through the inhibition of the enzymatic activity of the
hydrolyzing enzyme; lipoprotein lipase, resulting in the accumulation
of total cholesterol (TC), triglycerides (TG) and low density lipopro-
teins (LDL-c) [34,35].
13.7
2.3
100
23.22
Emax%: Efficacy values were calculated as percentage of the maximum fold in-
duction obtained with FA used as a reference standard.
In the present study, the lipid profile of rats injected with triton
Fig. 3. Structure-activity relationship (SAR) and essential binding features for the synthesized chalcone fibrates.
Fig. 4. Transcription of CPT1A gene. HepG2 cells were treated with increasing
concentrations of FB, 10a and 10i for 24 h. Relative levels of CPT1 mRNA were
evaluated by real-time PCR and standardized to the transcription of GAPDH
housekeeping gene. Data are presented as fold change compared to the samples
from cells treated with vehicle (DMSO 0.3% v/v). Data are expressed as mean
values ± SD obtained by at least three independent experiments. Statistical
analysis was performed using Student’s t-test. *# p < 0.05; **, ## p < 0.01,
***, ### p < 0.001. * 10i vs. FB treated cells; # 10a vs. FB treated cells.
Fig. 5. Densitometry quantification of PPAR
α protein expression. Western blot
analysis of HepG2 cell extracts after 24 h treatment with increasing concen-
trations of FB, 10a and 10i. The PPARα/β-actin values were normalized to that
found with cells treated with vehicle (DMSO 0.3% v/v). Data are expressed as
mean values ± SD obtained by three independent experiments. Statistical
analysis was performed using Student’s t-test. * p < 0.05; * 10i vs. FB
treated cells.
6

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
Fig. 6. 2D diagram (A) and 3D representation (B) showing 10a docking pose interactions with the key amino acids in the PPARα active site.
demonstrated a significant increase in serum TC, TG, LDL and VLDL as
well as cardiac risk ratio, atherogenic coefficient, atherogenic index of
plasma, and castelli’s risk index II followed by a decrease in HDL
compared to control group. Triton WR-1339 injection exhibited high
cholesterol and triglycerides levels in blood, followed by increasing
VLDL-c secretion by the liver [36]. The atherogenic dyslipidemia, which
is one of the main components of metabolic syndrome, is characterized
by elevated blood levels of TG, small dense LDL, and VLDL while
reduced HDLc, thus considered as a relevant risk factor for cardiovas-
cular diseases [37,38].
kg) were used as reference standards [39].
Fenofibrate is a synthetic PPAR agonist lipid lowering drug which
belongs to the fibrate class of drugs [40]. Fibrates exerts its lipid
lowering effect via activation of PPAR which in turn (1) regulates lipid
α
α
and lipoprotein metabolism due to its association with the transcription
of genes that reduces serum TG and increases HDLc levels, (2) increases
the production of lipoprotein lipase and apolipoprotein A-V, and de-
creases apo C-III plasma levels which inhibits LPL activity, thus aug-
menting the catabolism of TG-rich lipoproteins and decreasing serum TG
levels, (3) upregulates the expression of genes implicated in pathways of
β-oxidation which impact fatty acids levels in the liver resulting in
attenuation of hepatic production of VLDL [13]. Fenofibrate acute effect
in reduction of TG and LDL-c levels has been demonstrated 24 h from its
administration [36,41]. Lowered TG and small dense LDL, while
increased HDLc levels, were observed in patients with atherogenic
2.7.2.1. Effect of compounds 10a and 10i on serum total cholesterol
(TC). In the current study, fenofibrate (FB) at doses 100 and 200 mg/
kg and fenofibric acid (FA) (2-[4-(4-chlorobenzoyl) phenoxy]-2-
methylpropanoic acid) which is the active metabolite of FB (100 mg/
7

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
Fig. 7. 2D diagram (A) and 3D representation (B) showing 10i docking pose interactions with the key amino acids in the PPAR
α
active site.
dyslipidemia and treated with fibrates [42].
kg) and compound 10a (200 mg/kg) (Table 3, Figure S74A).
Triton-induced hyperlipidemic rats significantly increased TC level
by 3.66-folds compared to control. Compounds 10a and 10i, FB and FA
at all dose levels demonstrated a significant reduction in TC serum level
compared to triton-induced hyperlipidemic rats (Table 3, Figure S74A).
The synthesized compounds 10a and 10i showed good TC lowering
activity at dose 200 mg/kg compared to dose 100 mg/kg. Treatment
with 10i (200 mg/kg) revealed the highest TC lowering activity through
its significant reduction of TC level by 66, 31.15, 32.64% compared to
triton, FA (100 mg/kg) and FB (200 mg/kg), respectively. Moreover, 10i
(100 mg/kg) demonstrated significant decrease in serum TC level
compared to FB (100 mg/kg) and 10a (100 mg/kg) by 20.11 and
26.33% respectively. Interestingly, 10i at dose 100 mg/kg demonstrated
similar (non-significant) TC reducing effect compared to FB (200 mg/
2.7.2.2. Effect of compounds 10a and 10i on serum triglycerides (TG).
Induction with triton in rats significantly increased serum TG by 5.39-
folds compared to control rats. Treatment with 10i (200 mg/kg)
demonstrated the most significant decrease in serum TG (65.11%) which
was similar to FA (100 mg/kg) (63.94%) used as a reference standard
compared to induction with triton only. Moreover, treatment with 10i
(200 mg/kg) showed significantly lower serum TG level compared to
10a and FB at dose 200 mg/kg by 32.86% and 34.11%, respectively.
Furthermore, treatment with 10i (100 mg/kg) demonstrated a signifi-
cant reduction in mean TG serum level compared to 10a (100 mg/kg)
while no significant difference compared to FB (100 mg/kg) (Table 3,
Figure S74B).
8

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
Table 3
Effects of novel compounds 10a and 10i on TC, TG, HDL-c, LDL-c, and VLDL-c in Triton WR-1339-induced hyperlipidemic rats.
Lipid profile
Groups
TC (mg/dL)
%
TG (mg/dL)
%
HDL (mg/dL)
%
LDL (mg/dL)
%
VLDL (mg/dL)
%
Reduction*
Reduction*
Increase*
Reduction*
Reduction*
Control
Triton
FA
88.05 ± 1.51b
322. 67 ± 9.11a
159.32 ± 3.81ab
–
66.56 ± 7.78b
358.78 ± 6.68a
129.37 ± 2.20ab
–
51.61 ± 3.62b
21.31 ± 3.60a
–
–
24.69 ± 1.89b
224.74 ± 7.23a
93.03 ± 5.20ab
–
–
13.31 ± 1.56b
71.76 ± 1.34a
–
–
–
–
50.62
63.94
39.39 ± 2.74b 84.85
31.13 ± 1.24ab 46.11
33.17 ± 3.31ab 55.68
36.86 ± 1.26ab 73
58.6
25.87 ± 0.44ab 63.94
43.47 ± 0.25ab 39.42
57.36 ± 2.28ab 20.06
48.92 ± 1.43ab 31.82
37.29 ± 1.90ab 48.03
38.00 ± 1.11ab 47.05
25.04 ± 1.30ab 65.11
(100 mg/kg)
FB
(100 mg/kg)
10a
100 mg/kg)
10i
(100 mg/kg)
FB
(200 mg/kg)
10a
(200 mg/kg)
10i
(200 mg/kg)
229.93 ± 3.23 ab
249.34 ± 4.53 ab
183.70 ± 2.92 ab
162.85 ± 3.84 ab
173.34 ± 8.51 ab
109.70 ± 1.33 ab
28.74
22.73
43.07
49.53
46.28
66
217.35 ± 1.23ab
286.79 ± 11.42ab
244.62 ± 7.17ab
186.44 ± 9.48ab
189.99 ± 5.56ab
125.18 ± 6.49ab
39.42
20.06
31.82
48.03
47.05
65.11
157.41 ± 4.24ab 29.96
161.11 ± 4.17ab 28.31
98.35 ± 3.31ab
79.55 ± 2.94ab
56.24
64.6
47.25 ± 3.03b 121.73
44.83 ± 3.66b 110.4
49.78 ± 0.71b 133.6
100.96 ± 7.30ab 55.08
35.21 ± 1.71b
84.33
Data represented as means ± SEM. a significantly different from control group p < 0.05. b significantly different from Triton group p < 0.05.
* Represents % reduction or % increase from triton group.
2.7.2.3. Effect of compounds 10a and 10i on serum high-density lipopro-
teins (HDL). Measurement of serum HDL in rats administered with
triton (300 mg/kg) revealed 0.4-fold decrease compared to control
values. Treatment with 10a (200 mg/kg) and 10i (200 mg/kg) signifi-
cantly increased serum HDL level by 110.4% and 133.6%, respectively,
compared to triton group and were not statistically significant form FB
(200 mg/kg) (121.73%) and FA (84.85%). Treatment with 10i (200 mg/
kg) demonstrated a significant increase in serum HDL compared to 10a
(100 mg/kg) and FB (100 mg/kg). Interestingly, both compounds 10a
and 10i at dose 200 mg/kg exhibited similar increase in HDL compared
to their corresponding dose (100 mg/kg) by 26% (Table 3, Figure S74C).
hyperlipidemic rats (Table 3, Figure S74E).
2.7.3. Effect of compounds 10a and 10i on atherogenic indices
Atherogenic indices are considered as a valuable tool for the
assessment of the risk of development of cardiovascular diseases.
Increment of cardiac risk ratio, atherogenic coefficient, atherogenic
index, and castelli’s risk index II indicates a predisposing risk for the
development of cardiovascular diseases.
2.7.3.1. Effect of compounds 10a and 10i on cardiac risk ratio. Cardiac
risk ratio (CRR) is a ratio between TC and HDL cholesterol [43]. Triton
administration significantly increased CRR compared to control value.
Both compounds 10a and 10i at both dose levels significantly reduced
the triton mediated increase in CRR. Interestingly, compounds 10a (200
mg/kg) and 10i (200 mg/kg) as well as reference standards FA (100 mg/
kg) and FB (200 mg/kg) demonstrated a non-significant increase in CRR
compared to the control group. Treatment with 10i (200 mg/kg)
significantly decreased the CRR compared to FB (100 mg/kg) and 10a
(100 mg/kg) (Fig. 8A).
2.7.2.4. Effect of compounds 10a and 10i on serum low density lipopro-
teins (LDL). The mean serum LDL level of rats induced with hyperlip-
idemia using triton demonstrated 9.1-fold increase compared to control
values. All treatments demonstrated a significant decrease in serum LDL
level compared to triton-induced group. Treatment with 10i (200 mg/
kg) significantly decreased serum LDL level compared to FB (200 mg/
kg), FA, 10i (100 mg/kg), 10a (200 mg/kg), FB (100 mg/kg) and
compound 10a (100 mg/kg) by 55.74, 62.15, 64.20, 65.13, 77.63 and
78.15 %, respectively. In addition, 10a (200 mg/kg) significantly
decreased serum LDL level compared to FB (100 mg/kg) and 10a (100
mg/kg) while similar serum LDL levels were observed compared to FA
(100 mg/kg) (Table 3, Figure S74D).
2.7.3.2. Effect of compounds 10a and 10i on atherogenic coefficient.
Atherogenic coefficient (AC) was calculated as (Non-HDLc)/(HDLc)
where non-HDLc=(TC-HDLc)/HDLc. AC is an indicator measure of
cholesterol in LDLc, VLDLc, intermediate-density lipoprotein (IDLc) li-
poprotein fractions with respect to good cholesterol or HDLc. The higher
the value of atherogenic coefficient the higher the possibility of devel-
oping cardiovascular disorders as it reflects atherogenic potential of the
entire spectrum of lipoprotein fractions. Non HDLc is the second target
of therapy after LDLc as per ATPIII guidelines [44] especially in in-
dividuals with hyper-triglyceridemia. Triton-induced hyperlipidemic
rats demonstrated high atherogenic coefficient compared to control and
treated rats. Treatment with the standard drugs and compounds 10a and
10i at both dose levels significantly attenuated the triton mediated in-
crease in AC. Treatment with 10i (200 mg/kg) demonstrated a non-
significant decrease in AC compared to FB (200 mg/kg), 10a (200
mg/kg) and FA (100 mg/kg). Compound 10a (200 mg/kg) and 10i at
2.7.2.5. Effect of compounds 10a and 10i on serum very low-density li-
poproteins (VLDL). Induction of hyperlipidemia with triton resulted in
5.39-fold increase in VLDL level. Treatment with 10i (200 mg/kg)
demonstrated a significant reduction in serum VLDL level compared to
FB and 10a at both dose levels and 10i (100 mg/kg). Interestingly, 10i
(200 mg/kg) exhibited a similar reduction in VLDL level compared to
the reference standard FA (100 mg/kg). Moreover, 10a (200 mg/kg)
and FB (200 mg/kg) demonstrated similar decrease in VLDL level which
reached 47 and 48%, respectively while 10i (100 mg/kg) and FB (100
mg/kg) acted in a similar manner and the percentage reduction reached
31.82 and 39.42%, respectively compared to triton-induced
9

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
Fig. 8. Effect of novel compounds 10a and 10i on atherogenic indices in Triton
WR-1339-induced hyperlipidemic rats. Cardiac risk ratio (A), atherogenic co-
efficient (B), atherogenic index (C), and Castelli’s risk index II (D). Data are
represented as means ± SEM; p < 0.05 was considered statistically significant.
asignificantly different from control group p < 0.05. bsignificantly different
from Triton WR-1339 group p < 0.05. Lines represents significance between
two groups p < 0.05.
18
A
a
16
14
12
10
8
ab
ab
both dose levels demonstrated a significant decrease in AC compared to
6
ab
10a (100 mg/kg) (Fig. 8B).
b
b
b
4
b
b
2.7.3.3. Effect of compounds 10a and 10i on atherogenic index.
Atherogenic index (AI) calculated as log TG/HDLc has been used as a
significant predictor of atherosclerosis and coronary artery disease
[45–48]. Thus, consideration of AI values offers new insights for the
prediction of cardiovascular diseases. Triton induced hyperlipidemic
rats demonstrated significantly higher AI values compared to control
and all standard and treated groups. Compound 10i (200 mg/kg)
showed a significant decrease in AI value compared to FB and 10a at
dose level of 100 and 200 mg/kg as well as 10i (100 mg/kg). Interest-
ingly, the AI of 10a, compound 10i and FB at dose (200 mg/kg) were not
statistically significant compared to FA (100 mg/kg) (Fig. 8C).
2
0
ol
g)
g)
g)
g)
g)
g)
g)
r
on
k
k
k
k
k
k
g/
m
k
g/
m
t
i
r
T
g/
g/
(
g/
g/
g/
(
ont
C
m
m
m
m
m
100
100
100
100
200
200
200
(
(
(
(
(
FA
FB
FB
10i
10i
10a
10a
16
14
12
10
8
B
a
ab
ab
2.7.3.4. Effect of compounds 10a and 10i on Castelli’s risk index II (CRI-
II). Castelli’s risk index II (CRI-II) is ratio between LDL cholesterol and
HDL cholesterol. Triton administration significantly increased CRI-II
compared to all standard and treated groups at both dose levels. Cas-
telli’s risk index II values of FB (200 mg/kg) and 10i (200 mg/kg) were
non-significant from control values. Compound 10i (200 mg/kg)
showed significant decrease in CRI-II compared to FB (100 mg/kg), 10a
(100 and 200 mg/kg) and 10i (100 mg/kg) while non-significant
decrease compared to FA (100 mg/kg) (Fig. 8D).
6
ab
4
b
b
b
2
b
b
0
ol
g)
g)
g)
k
g)
g)
k
g)
k
g/
m
200
(
g)
r
on
t
k
k
k
k
g/
m
i
r
g/
g/
g/
g/
g/
ont
C
T
m
m
m
m
m
100
100
100
100
200
10a
200
(
(
(
(
(
(
FA
FB
FB
10i
10i
10a
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
C
2.7.4. Effect of compounds 10a and 10i on antioxidant activity in triton-
induced hyperlipidemic rats
a
Development of therapeutic agents having both lipid lowering
properties and antioxidant activity are an ultimate choice for the man-
agement of cardiovascular diseases. Progression of vascular damage has
been linked to oxidative stress. Peroxidative damage caused by reactive
oxygen species (ROS) to blood lipoproteins has been linked to the
initiation and progression of atherosclerosis2. Furthermore, FB exhibi-
ted potent antioxidant effect where it increased catalase and superoxide
dismutase levels, while decreased thiobarbituric acid reactive sub-
stances, and myeloperoxidase levels in diabetic rats [49].
ab
ab
ab
ab
ab
ab
ab
b
2.7.4.1. Effect of compounds 10a and 10i on tissue reduced glutathione
(GSH). Triton administration significantly reduced liver GSH level in
rat by 0.49-folds compared to control values. Treatment with com-
pounds 10a (100 and 200 mg/kg) and 10i (100 and 200 mg/kg)
significantly attenuated the triton mediated inhibition in GSH liver tis-
sue level by 38.6, 94.8, 66.3 and 92.9%, respectively. Treatment with
10a and 10i at dose 200 mg/kg demonstrated no significant difference
in GSH liver levels compared to the reference standard FB (200 mg/kg)
and FA (100 mg/kg) while showed significantly higher liver GSH level
compared to 10a (100 mg/kg) (Fig. 9A).
ol
Contr
on
t
kg)
g/
kg)
kg)
g/
kg)
g/
kg)
g/
20
(
a
kg)
kg)
g/
m
i
r
g/
g/
T
m
m
m
m
m
m
0
0
0
0
0
0
0
10
10
10
10
20
20
(
(
(
(
(
(
a
FA
FB
FB
10i
10i
10
10
D
12
10
8
a
6
ab
ab
2.7.4.2. Effect of compounds 10a and 10i on plasma lipid peroxidation
(MDA). Plasma malondialdehyde (MDA) level was significantly elevated
by 4.54-folds in triton induced group compared to control group.
Treatment with compounds 10a (100 and 200 mg/kg) and 10i (100 and
200 mg/kg) significantly attenuated the triton mediated increase in lipid
peroxidation (MDA plasma level) by 37, 48, 54 and 74 %, respectively.
Treatment with 10i (200 mg/kg) significantly decreased the MDA plasma
level by 59.4, 58.9, and 50.5 % compared to 10a (100 and 200 mg/kg) as
well as FB (100 mg/kg), respectively (Fig. 9B).
4
ab
ab
ab
b
2
b
b
0
ol
g)
g)
g)
g)
g)
g)
g)
r
on
t
k
k
k
k
k
k
k
i
r
T
g/
g/
m
g/
g/
g/
m
g/
g/
m
ont
m
m
m
m
C
100
100
10a
100
100
200
10a
200
200
(
(
(
(
(
(
(
FA
FB
FB
10i
10i
(caption on next column)
10

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
2.7.4.3. Effect of compounds 10a and 10i on plasma total antioxidant
capacity (TAC). Total antioxidant capacity (TAC) level measured in
plasma of rats induced with triton demonstrated significant reduction by
(0.5-folds) compared to the control values. Compounds 10a and 10i at
doses (100 and 200 mg/kg) significantly raised the TAC level by 55.5,
106.3, 40.4, and 95.1, respectively compared to triton-induced hyper-
lipidemic rats. Treatment with 10a and 10i (200 mg/kg) demonstrated
similar TAC levels compared to control group and the reference FB (200
mg/kg) and significantly higher TAC levels compared to FA (100 mg/
kg), and compounds 10a and 10i (100 mg/kg) (Fig. 9C).
2.7.5. Histopathological study
The results of the histopathological examination of hepatic tissues
revealed that in control livers, there was no histopathological alteration
and the normal histological structure of the central vein and surround-
ing hepatocytes in the parenchyma were recorded in Fig. 10A.
In triton induced hyperlipidemic rats, vacuolar degeneration was
detected in the cytoplasm of the hepatocytes in diffuse manner all over
the parenchyma (Fig. 10B) associated with inflammatory cells infiltra-
tion in between the newly formed bile ducts and congested portal vein at
the portal area (Fig. 10C). In agreement with our study, Iturriaga and
coworkers [50] reported that rats treated with triton demonstrated
moderate Kupffer cell hyperplagia and light sinusoidal dilatation as well
as the presence of vacuoles in different size. The vacuoles were
numerous at 24 h after triton injection.
Treatment with FA demonstrated vacuolar degeneration in the
cytoplasm of the hepatocytes and decreased inflammation in the portal
area (Fig. 10D). The treatment achieved the reaction in the portal area
with disappearance of the inflammatory reaction portal vein congestion
and prebiliary inflammation reaction.
Diffuse kupffer cells proliferation was detected in between the he-
patocytes in the group treated with FB (100 mg/kg) (Fig. 10E) while no
histopathological alteration was recorded in treatment with FB (200
mg/kg) (Fig. 10F). Similarly, Garg et al., 2018 demonstrated that
treatment with FB (65 mg/g) showed a portal tract area similar to that of
the control group as it reversed the minimal portal inflammation of the
portal tract that was induced by triton for 48 h [51].
Treatment with 10a (100 mg/kg) showed mild vacuolar degenera-
tion in the cytoplasm of the hepatocytes (Fig. 10G). Moreover, diffuse
kupffer cells proliferation was detected in between the hepatocytes of
livers treated with 10a (200 mg/kg) (Fig. 10H).
Furthermore, treatment 10i (100 mg/kg) demonstrated mild
degenerative change that was detected in some of the hepatocytes
(Fig. 10I). In addition, there was no histopathological alteration as
recorded in 10i (200 mg/kg) (Fig. 10J).
3. Conclusion
In the present study, the effect of fibrate chemical modification on
PPARα agonism was studied. Novel chalcone fibrate hybrids (10a-o) and
(11a-e) were designed. Compounds 10a and 10i exhibited good
agonistic activity on PPARα compared to fenofibric acid in terms of EC50
and Emax% values. These two compounds were further tested in vitro
through CPT1A expression and revealed upregulation in CPT1A tran-
scription at 10, 50 and 100 µM compared to fenofibrate. PPAR
α
expression was also performed through Western blot analysis in HepG2
cells, and it was shown that 10i, 10a and fenofibrate stimulated PPAR
expression at 50 µM in a ratio of 10:2:1, respectively.
α
Moreover, in vivo pharmacological assessment of 10a and 10i in
trition-WR1339 induced hyperlipidemia model revealed that both
compounds significantly decreased TC, TG, LDL and VLDL while
increased HDL level compared to triton-induced hyperlipidemic group.
Overall, 10i possessed better anti-hyperlipidemic activity than 10a.
Compound 10i (200 mg/kg) demonstrated significantly lower TC, TG,
LDL and VLDL level while similar HDL level compared to FB (200 mg/
kg). In addition, 10i (200 mg/kg) showed significantly reduced TC and
Fig. 9. Effect of novel compounds 10a and 10i on antioxidant parameters in
Triton WR-1339-induced hyperlipidemic rats. Glutathione reductase (GSH) (A),
lipid peroxidation [MDA] (B), and total antioxidant capacity (C). Data are rep-
resented as means ± SEM; p < 0.05 was considered statistically significant.
asignificantly different from control group p < 0.05. bsignificantly different from
Triton group p < 0.05. Lines represents significance between two groups p < 0.05.
11

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
Fig. 10. Histological examination of hepatic tis-
sue of rats. (A): Microscopic section of the liver of
control group showing normal histological struc-
ture of central vein (CV) and surrounding hepa-
tocytes (h) in the parenchyma (H & E, ×40). (B)
Liver of rat induced with hyperlipidemia using
triton showing intracytoplasmic vacuoles degen-
eration in the hepatocytes all over the paren-
chyma (H & E, ×40). (C) Liver of rat in triton
group showing severe congestion in the portal
vein (PV) with multiple bile ducts formation with
inflammatory cells infiltration (m) in portal area
(H & E, ×40). (D) Liver of rat treated with FA
showing vacuolar degeneration (VD) in the he-
patocytes (H & E, ×40). (E) Liver of rat treated
with FB (100 mg/kg) showing diffuse kupffer cells
(K) proliferation in between the hepatocytes. (F)
Liver of rat treated with FB (200 mg/kg) showing
normal histological structure (h) (H & E, ×40).
(G) Liver of rat treated with compound 10a (100
mg/kg) showing mild vacuolar degeneration (VD)
in some of the hepatocytes (H & E, ×40). (H) Liver
of rat treated with compound 10a (200 mg/kg)
showing diffuse kupffer cells proliferation (K) in
between the hepatocytes (H & E, ×40). (I) Liver of
rat treated with compound 10i (100 mg/kg)
showing mild degenerative change (VD) in some
hepatocytes (H & E, ×80). (J) Liver of rat treated
with compound 10i (200 mg/kg) showing normal
intact histological alteration (H & E, ×40).
LDL while comparable TG, HDL and VLDL levels compared to FA (100
mg/kg). For the assessment of the risk of development of cardiovascular
diseases, 10i and 10a possessed analogous atherogenic indices (CRR,
AC, AI, CRI-II) compared to FB and FA. The antioxidant activity of both
compounds was demonstrated by increased tissue GSH and plasma TAC
while decreased plasma MDA levels. In conclusion, compound 10i
showed better in vitro PPAR
PPAR
α agonistic activity confirmed by CPT1A and
α
gene expression experiments and promising in vivo hypolipi-
demic and antioxidant activities at a dose of 200 mg/kg without histo-
pathological alterations of hepatic tissue.
12

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
4. Experimental section
4.1.7. General procedure for the Synthesis of chalcones (5a-e)
To a stirred solution of 4-(3-substituted benzyloxy)/(2-phenylethoxy)
benzaldehydes (3a-e) (5 mmol) and p-hydroxyacetophenone (4) (5
mmol) in methanol (40 ml) was added 50% aqueous potassium hy-
droxide (15 ml) and reflux with stirring for 2–5 h. After completion of the
reaction as monitored by TLC, the solvent was evaporated, neutralized
with dilute HCl and extracted with ethyl acetate (2 × 50 ml). The com-
bined organic fractions were washed with water, dried over Na2SO4,
evaporated and purified either by crystallization from ethanol or by
column chromatography using silica gel as stationary phase and ethyl
acetate/petroleum ether 60–80 ◦C, (1:1) as mobile phase to obtain the
pure products.
4.1. Chemistry
All chemicals and reagents were purchased from commercial sour-
ces, Sigma Aldrich, ACROS and Alfa Aser. Thin layer chromatography
(TLC) was performed on precoated silica gel 60 F245 aluminium plates
(Merck) with visualization under UV light. All final compounds
exhibited ≥ 95% purity as determined by HPLC. HPLC analysis was
performed using Agilent 1100 series apparatus equipped with a qua-
ternary pump, a vacuum degasser and detection method was using diode
array (DAD) ultraviolet (UV) detector at 254 nm and HP Chemstation
software. Column used was C18 column Zorbax ODS (4.6 × 150 mm i.
d.,5 mm) Agilent Technologies (Palo Alto, CA), solvent system used was
water: acetonitrile (50:50), flow rate = 1 ml/min, temperature 25 ◦C.
Melting points were determined on an Electrothermal Melting Point
Apparatus with open capillary tubes and were uncorrected. The optical
activity for the chiral acid compounds was measured by an AA-65
Automatic Polarimeter (Optical Activity) LTD. Mass and NMR spectra
4.1.8. (E)-3-(4-(benzyloxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one
(5a) [57,58]
Yield: 85%, yellow solid, mp: 184 ◦C, (Lit. [57,58] 180–182 ◦C). ESI-
MS (m/z, %): 330 (M+, 100%); 331 [M + H]+, 18%.
4.1.9. (E)-1-(4-hydroxyphenyl)-3-(4-((3-methylbenzyl)oxy)phenyl)prop-
2-en-1-one (5b)
for chiral compounds were reported for the racemic mixture. 1H- and 13
C
NMR spectra were recorded at 400 (100) MHz Spectrometer. CDCl3 and
DMSO‑d6 were used as NMR solvents. Chemical shifts (δ) were expressed
in parts per million using tetramethylsilane (TMS) as an internal stan-
dard and were given in parts per million (ppm) relative to TMS.
Coupling constants were reported in Hertz (Hz). Mass spectra were
recorded on GC/MS FINNIGAN MAT SSQ 7000 digital DEC 3000 mass
spectrometer. High-resolution mass spectra (HRMS) were obtained by
ESI-Orbitrap Elite (ThermoFisher Scientific) mass spectrometer flow
injection analysis at flow rate of 8 µl/min and with 240 K resolution in
50–600 m/z acquisition range. ESI-MS data were recorded on Advion
compact mass spectrometer (CMS).
Yield: 70%, shiny yellow crystals, mp: 182–183 ◦C. 1H NMR (400
MHz, DMSO‑d6) δ 2.31 (s, 3H), 5.12 (s, 2H), 6.90 (d, J = 8.4 Hz, 2H),
7.07 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 7.2 Hz, 1H), 7.23–7.29 (m, 3H),
7.65 (d, J = 15.6 Hz, 1H), 7.77 (d, J = 16.0 Hz, 1H), 7.81 (d, J = 8.8 Hz,
2H), 8.06 (d, J = 8.4 Hz, 2H), 10.43 (br s, 1H). 13C NMR (100 MHz,
DMSO‑d6) δ 21.1, 69.5, 115.3, 115.5, 119.8, 124.9, 127.8, 128.4, 128.5,
128.7, 129.4, 130.6, 131.1, 136.7, 137.8, 142.7, 160.3, 162.2, 187.2.
ESI-MS (m/z, %): 344 (M+, 100%); 345 [M + H]+, 30%. Anal. Calcd for
C23H20O3: C, 80.21; H, 5.85. Found: C, 80.32; H, 5.90.
4.1.10. (E)-3-(4-((3-chlorobenzyl)oxy)phenyl)-1-(4-hydroxyphenyl)prop-
2-en-1-one (5c)
4.1.1. General procedure for the Synthesis of 4-(3-substitutedbenzyloxy)/
4-(2-phenylethoxy) benzaldehydes (3a-e)
Yield: 76%, yellow solid, mp: 212 ◦C. 1H NMR (400 MHz, DMSO‑d6)
δ 5.18 (s, 2H), 6.91 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H),
7.37–7.42 (m, 3H), 7.53 (s, 1H), 7.66 (d, J = 15.6 Hz, 1H), 7.78 (d, J =
15.6 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 8.07 (d, J = 8.8 Hz, 2H). 13C NMR
(100 MHz, DMSO‑d6) δ 68.4, 115.2, 115.4, 119.9, 126.2, 127.4, 127.9,
127.9, 129.3, 130.4, 130.5, 131.1, 133.2, 139.3, 142.5, 159.9, 162.2,
187.0. ESI-MS (m/z, %): 364 (M+, 42%); 365 [M + H]+, 12%. Anal.
Calcd for C22H17ClO3: C, 72.43; H, 4.70; Cl, 9.72. Found: C, 72.32; H,
4.76; Cl, 9.84.
A reaction mixture of benzyl chloride derivatives or phenethyl bro-
mide (1a-e) (0.136 mol) and p-hydroxybenzaldehyde (2) (15 g, 0.12 mol),
anhydrous Na2CO3 (17 g, 0.16 mol) in 50 ml DMF was heated and stirred
overnight at 80 ◦C. After cooling to room temperature, the reaction
mixture was poured onto 50 ml of water and extracted with ethyl acetate
(3 × 50 ml). The organic phase was washed with 10 % NaOH (3 × 25 ml)
then with distilled water (2 × 25 ml), dried over anhydrous Na2SO4 and
evaporated under reduced pressure to give compounds (3a-e).
4.1.11. (E)-1-(4-hydroxyphenyl)-3-(4-((3-nitrobenzyl)oxy)phenyl)prop-
2-en-1-one (5d)
4.1.2. 4-(Benzyloxy)benzaldehyde (3a) [52,53]
Yield: 89%, off-white solid, mp: 77 ◦C (Lit. [52] 74–76 ◦C).
Yield: 65%, yellow solid, mp: 231–232 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 5.33 (s, 2H), 6.90 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 8.0 Hz,
2H), 7.66 (d, J = 15.2 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 15.6
Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 7.2 Hz, 1H), 8.06 (d, J =
4.1.3. 4-((3-Methylbenzyl)oxy)benzaldehyde (3b) [25,54]
Yield: 85%, buff oil. 1H NMR (400 MHz, DMSO‑d6) δ 2.30 (s, 3H),
5.16 (s, 2H), 7.14–7.15 (m, 1H), 7.18 (d, J = 8.8 Hz, 2H), 7.24–7.30 (m,
3H), 7.86 (d, J = 8.8 Hz, 2H), 9.87 (s, 1H). 13C NMR (100 MHz,
DMSO‑d6) δ 21.0, 69.7, 115.2, 124.9, 128.4, 128.7, 129.8, 131.8, 136.2,
137.7, 163.3, 191.2. EI-MS (m/z, %): 227 [M + H]+, 35%.
8.4 Hz, 2H), 8.19 (d, J = 8.0 Hz, 1H), 8.33 (s, 1H), 10.42 (br s, 1H). 13
C
NMR (100 MHz, DMSO‑d6) δ 68.1, 115.2, 115.4, 119.9, 122.1, 122.8,
128.1, 129.3, 130.1, 130.6, 131.1, 134.1, 139.1, 142.5, 147.8, 159.7,
162.2, 187.0. ESI-MS (m/z, %): 375 (M+, 100%); 376 [M + H]+, 34%.
Anal. Calcd for C22H17NO5: C, 70.39; H, 4.56; N, 3.73. Found: C, 70.55;
H, 4.67; N, 3.84.
4.1.4. 4-((3-Chlorobenzyl)oxy)benzaldehyde (3c) [52]
Yield: 83%, yellow soild, mp: 51 ◦C (Lit.[52] 52 ◦C).
4.1.12. (E)-1-(4-hydroxyphenyl)-3-(4-phenethoxyphenyl)prop-2-en-1-one
(5e)
4.1.5. 4-((3-Nitrobenzyl)oxy)benzaldehyde (3d) [52]
Yield: 79%, yellow solid, mp: 85 ◦C (Lit. [52] 80–82 ◦C).
Yield: 79%, yellow solid, mp: 175–176 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 3.04 (t, J = 6.8 Hz, 2H), 4.23 (t, J = 6.8 Hz, 2H), 6.92 (d, J
= 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 7.19–7.24 (m, 1H), 7.28–7.34
(m, 4H), 7.66 (d, J = 15.2 Hz, 1H), 7.78 (d, J = 12.0 Hz, 1H), 7.80 (d, J
= 8.0 Hz, 2H), 8.07 (d, J = 8.8 Hz, 2H), 10.45 (s, 1H). 13C NMR (100
MHz, DMSO‑d6) δ 34.9, 68.3, 114.9, 115.4, 119.6, 126.3, 127.6, 128.3,
129.0, 129.4, 130.6, 131.1, 138.2, 142.9, 160.3, 162.1, 187.1. ESI-MS
(m/z, %): 344 (M+, 100%); 345 [M + H]+, 18%. Anal. Calcd for
4.1.6. 4-(2-Phenylethoxy)benzaldehyde (3e) [24,55,56]
Yield: 88%, colourless oil. 1H NMR (400 MHz, CDCl3) δ 3.15 (t, J =
6.8 Hz, 2H), 4.28 (t, J = 6.8 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 7.25–7.37
(m, 5H), 7.84 (d, J = 8.8 Hz, 2H), 9.89 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ 35.7, 69.1, 114.9, 126.8, 128.7, 129.1, 130.0, 132.1, 137.8,
163.9, 190.9. EI-MS (m/z, %): 227 [M + H]+, 100%.
C23H20O3: C, 80.21; H, 5.85. Found: C, 80.35; H, 5.93.
13

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Bioorganic Chemistry 115 (2021) 105170
4.1.13. General Procedure for the Synthesis of the esters (8a-o and 9a-e)
To a hot solution of the synthesized chalcones (5a-e) (7 mmol) in dry
acetonitrile (15 ml) was added, anhydrous K2CO3 (35 mmol), DL-ethyl 2-
bromopropionate, DL-ethyl 2-bromobutyrate, ethyl 2-bromoisobutyrate
(6a-c) or methyl 4-bromobutyrate (7) (7.1 mmol) and the mixture was
heated under reflux with stirring for 2–4 h. The reaction mixture was
monitored by TLC. After completion of the reaction, K2CO3 was removed
by filtration; the solvent was evaporated under vacuum. The residue was
purified by column chromatography using silica gel as stationary phase
and a mixture of ethyl acetate and petroleum ether (60–80 ◦C) (1:2) as
mobile phase to afford pure ester compounds.
7.26–7.38 (m, 5H), 7.43 (d, J = 15.2 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H),
7.79 (d, J = 15.6 Hz, 1H), 8.03 (d, J = 9.2 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 14.3, 18.6, 35.8, 61.6, 68.9, 72.7, 114.8, 115.1, 119.6, 126.7,
127.9, 128.7, 129.1, 130.3, 130.8, 132.2, 138.0, 144.1, 160.9, 161.3,
171.7, 188.8. HRMS Calcd for C28H28O5 m/z: 444.1937 [M + H]+, found
444.1930.
4.1.19. Ethyl (E)-2-(4-(3-(4-(benzyloxy)phenyl)acryloyl)phenoxy)
butanoate (8f)
Yield: 65%, light yellow solid, mp: 84–85 ◦C. 1H NMR (400 MHz,
CDCl3) δ 1.13 (t, J = 7.2 Hz, 3H), 1.28 (t, J = 6.8 Hz, 3H), 2.06 (p, J =
7.2 Hz, 2H), 4.26 (q, J = 7.2 Hz, 2H), 4.68 (t, J = 6.4 Hz, 1H), 5.13 (s,
2H), 6.98 (d, J = 9.2 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H),7.35–7.47 (m,
6H), 7.62 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 15.6 Hz, 1H), 8.03 (d, J = 9.2
Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 9.7, 14.3, 26.1, 61.5, 70.2, 77.7,
114.8, 115.4, 119.7, 127.6, 128.1, 128.3, 128.8, 130.2, 130.8, 132.1,
136.5, 144.0, 160.8, 161.7, 171.2, 188.8. HRMS Calcd for C28H28O5 m/
z: 444.1937 [M + H]+, found 444.1921.
4.1.14. Ethyl (E)-2-(4-(3-(4-(benzyloxy)phenyl)acryloyl)phenoxy)
propanoate (8a)
Yield: 69%, yellowish white solid, mp: 82–83 ◦C. 1H NMR (400 MHz,
CDCl3) δ 1.28 (t, J = 6.8 Hz, 3H), 1.69 (d, J = 6.8 Hz, 3H), 4.26 (q, J =
7.2 Hz, 2H), 4.87 (q, J = 6.8 Hz, 1H), 5.14 (s, 2H), 6.97 (d, J = 8.8 Hz,
2H), 7.03 (d, J = 8.4 Hz, 2H), 7.35–7.48 (m, 6H), 7.62 (d, J = 8.4 Hz,
2H), 7.79 (d, J = 15.2 Hz, 1H), 8.03 (d, J = 8.8 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 14.3, 18.6, 61.7, 70.2, 72.7, 114.8, 115.4, 119.7, 127.6,
128.1, 128.3, 128.8, 130.3, 130.8, 132.2, 136.6, 144.1, 160.8, 161.3,
171.7, 188.9. HRMS Calcd for C27H26O5 m/z: 430.1780 [M + H]+, found
430.1772.
4.1.20. Ethyl (E)-2-(4-(3-(4-((3-methylbenzyl)oxy)phenyl)acryloyl)
phenoxy)butanoate (8g)
Yield: 75%, white solid, mp: 71–72 ◦C. 1H NMR (400 MHz, CDCl3) δ
1.13 (t, J = 7.6 Hz, 3H), 1.28 (t, J = 7.2 Hz, 3H), 2.06 (p, J = 7.2 Hz, 2H),
2.41 (s, 3H), 4.26 (q, J = 7.2 Hz, 2H), 4.68 (t, J = 6.0 Hz, 1H), 5.10 (s,
2H), 6.98 (d, J = 8.8 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 7.18 (d, J = 7.2
Hz, 1H), 7.24–7.33 (m, 3H), 7.44 (d, J = 15.6 Hz, 1H), 7.62 (d, J = 8.4
Hz, 2H), 7.80 (d, J = 15.6 Hz, 1H), 8.03 (d, J = 8.8 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 9.6, 14.2, 21.4, 26.1, 61.4, 70.2, 77.6, 114.7, 115.3,
119.6, 124.6, 128.0, 128.3, 128.6, 129.0, 130.2, 130.7, 132.0, 136.3,
138.4, 144.0, 160.8, 161.6, 171.1, 188.8. HRMS Calcd for C29H30O5 m/
z: 458.2093 [M + H]+, found 458.2082.
4.1.15. Ethyl (E)-2-(4-(3-(4-((3-methylbenzyl)oxy)phenyl)acryloyl)
phenoxy)propanoate (8b)
Yield: 63%, white solid, mp: 87–89 ◦C. 1H NMR (400 MHz, CDCl3) δ
1.29 (t, J = 7.2 Hz, 3H), 1.69 (d, J = 6.8 Hz, 3H), 2.40 (s, 3H), 4.26 (q, J
= 7.2 Hz, 2H), 4.87 (q, J = 6.8 Hz, 1H), 5.09 (s, 2H), 6.97 (d, J = 8.8 Hz,
2H), 7.03 (d, J = 8.8 Hz, 2H), 7.18 (d, J = 7.6 Hz, 1H), 7.24–7.33 (m,
3H), 7.44 (d, J = 15.6 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 15.6
Hz, 1H), 8.04 (d, J = 9.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 14.2,
18.5, 21.4, 61.5, 70.2, 72.6, 114.7, 115.3, 119.6, 124.6, 128.0, 128.3,
128.6, 129.0, 130.2, 130.7, 132.1, 136.4, 138.4, 144.0, 160.8, 161.2,
171.6, 188.7. HRMS Calcd for C28H28O5 m/z: 444.1937 [M + H]+, found
444.1928.
4.1.21. Ethyl (E)-2-(4-(3-(4-((3-chlorobenzyl)oxy)phenyl)acryloyl)
phenoxy)butanoate (8h)
Yield: 67%, yellow solid, mp: 82–83 ◦C. 1H NMR (400 MHz, CDCl3) δ
1.10 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.2 Hz, 3H), 2.03 (p, J = 7.6, 6 Hz,
2H), 4.23 (q, J = 7.2 Hz, 2H), 4.66 (t, J = 6.4 Hz, 1H), 5.09 (s, 2H), 6.95
(d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 7.29–7.31 (m, 3H), 7.41 (d,
J = 15.6 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 15.2 Hz, 1H),
8.00 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 9.7, 14.3, 26.2,
61.5, 69.3, 77.7, 114.8, 115.3, 119.9, 125.4, 127.5, 128.3, 128.4, 130.0,
130.3, 130.8, 132.0, 134.7, 138.6, 143.8, 160.4, 161.7, 171.1, 188.7.
HRMS Calcd for C28H27ClO5 m/z: 478.1547 [M + H]+, found 478.1542.
4.1.16. Ethyl (E)-2-(4-(3-(4-((3-chlorobenzyl)oxy)phenyl)acryloyl)
phenoxy)propanoate (8c)
Yield: 68%, yellowish white solid, mp: 80–81 ◦C. 1H NMR (400 MHz,
CDCl3) δ 1.28 (t, J = 7.2 Hz, 3H), 1.69 (d, J = 6.8 Hz, 3H), 4.26 (q, J =
6.8 Hz, 2H), 4.87 (q, J = 6.8 Hz, 1H), 5.11 (s, 2H), 6.97 (dd, J = 8.8 Hz,
2H), 7.01 (dd, J = 8.4 Hz, 2H), 7.29–7.34 (m, 3H), 7.44 (d, J = 15.6 Hz,
1H), 7.46 (d, J = 4.0 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 15.6
Hz, 1H), 8.03 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 14.1,
18.5, 61.5, 69.2, 72.6, 114.7, 115.3, 119.8, 125.4, 127.4, 128.3, 130.0,
130.17, 130.7, 132.0, 134.6, 138.5, 143.8, 160.3, 161.2, 171.6, 188.7.
HRMS Calcd for C27H25ClO5 m/z: 464.1391 [M + H]+, found 464.1384.
4.1.22. Ethyl (E)-2-(4-(3-(4-((3-nitrobenzyl)oxy)phenyl) acryloyl)
phenoxy)butanoate (8i)
Yield: 61%, yellow solid, mp: 71–72 ◦C. 1H NMR (400 MHz, CDCl3) δ
1.10 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 6.8 Hz, 3H), 2.03 (p, J = 7.6 Hz, 2H),
4.23 (q, J = 6.8 Hz, 2H), 4.65 (t, J = 6.4 Hz, 1H), 5.19 (s, 2H), 6.95 (d, J
= 8.8 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 15.6 Hz, 1H), 7.56
(d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.74–7.77 (m, 2H), 8.00 (d,
J = 8.8 Hz, 2H), 8.19 (d, J = 9.6 Hz, 1H), 8.32 (s, 1H). 13C NMR (100
MHz, CDCl3) δ 9.7, 14.3, 26.2, 61.5, 68.8, 77.7, 114.8, 115.3, 120.1,
122.3, 123.2, 128.9, 129.8, 130.3, 130.8, 132.0, 133.2, 138.8, 143.7,
148.6, 160.0, 161.7, 171.1, 188.7. HRMS Calcd for C28H27NO7 m/z:
489.1788 [M + H]+, found 489.1784.
4.1.17. Ethyl (E)-2-(4-(3-(4-((3-nitrobenzyl)oxy)phenyl)acryloyl)
phenoxy)propanoate (8d)
Yield: 60%, white solid, mp: 101–102 ◦C. 1H NMR (400 MHz, CDCl3)
δ 1.28 (t, J = 7.2 Hz, 3H), 1.68 (d, J = 6.4 Hz, 3H), 4.25 (q, J = 7.2 Hz,
2H), 4.86 (q, J = 6.8 Hz, 1H), 5.21 (s, 2H), 6.96 (d, J = 8.8 Hz, 2H), 7.03
(d, J = 8.8 Hz, 2H), 7.44 (d, J = 15.6 Hz, 1H), 7.58–7.64 (m, 3H),
7.76–7.80 (m, 2H), 8.03 (d, J = 8.8 Hz, 2H), 8.21 (d, J = 8.4 Hz, 1H),
8.34–8.35 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 14.2, 18.5, 61.6, 68.7,
72.6, 114.7, 115.2, 120.0, 122.2, 123.1, 128.6, 129.7, 130.2, 130.7,
131.9, 133.1, 138.7, 143.6, 148.5, 159.9, 161.2, 171.6, 188.6. HRMS
Calcd for C27H25NO7 m/z: 475.1631 [M + H]+, found 475.1627.
4.1.23. Ethyl (E)-2-(4-(3-(4-phenethoxyphenyl)acryloyl)phenoxy)
butanoate (8j)
Yield: 70%, yellow oil. 1H NMR (400 MHz, CDCl3) δ 1.10 (t, J = 7.2
Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H), 2.04 (p, J = 6.8 Hz, 2H), 3.12 (t, J =
7.2 Hz, 2H), 4.20–4.26 (m, 4H), 4.66 (t, J = 6.4 Hz, 1H), 6.92 (d, J = 8.8
Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 7.23–7.36 (m, 5H), 7.41 (d, J = 15.2
Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 15.6 Hz, 1H), 8.01 (d, J =
8.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 9.7, 14.3, 26.2, 35.8, 61.5,
68.9, 77.7, 114.8, 115.1, 119.6, 126.7, 127.9, 128.7, 129.1, 130.3,
4.1.18. Ethyl (E)-2-(4-(3-(4-phenethoxyphenyl)acryloyl)phenoxy)
propanoate (8e)
Yield: 70%, white solid, mp: 60–61 ◦C. 1H NMR (400 MHz, CDCl3) δ
1.28 (t, J = 6.8 Hz, 3H), 1.69 (d, J = 6.8 Hz, 3H), 3.14 (t, J = 6.8 Hz, 2H),
4.23–4.29 (m, 4H), 4.87 (q, J = 6.4 Hz, 1H), 6.94–6.98 (m, 4H),
14

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Bioorganic Chemistry 115 (2021) 105170
130.8, 132.1, 138.0, 144.1, 160.9, 161.7, 171.2, 188.9. HRMS Calcd for
6.4 Hz, 2H), 5.19 (s, 2H), 7.07 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.8 Hz,
2H), 7.33–7.37 (m, 1H), 7.40–7.43 (m, 2H), 7.47–7.49 (m, 2H), 7.68 (d,
J = 15.2 Hz, 1H), 7.80–7.86 (m, 3H), 8.15 (d, J = 9.2 Hz, 2H). 13C NMR
(100 MHz, DMSO‑d6) δ 24.1, 29.9, 51.3, 66.9, 69.4, 114.4, 115.2, 119.6,
127.7, 127.8, 127.9, 128.5, 130.7, 130.8, 136.7, 143.0, 160.3, 162.3,
173.0, 187.20. HRMS Calcd for C27H26O5 m/z: 430.1780 [M + H]+,
found 430.1774.
C
29H30O5 m/z: 458.2093 [M + H]+, found 458.2081.
4.1.24. Ethyl (E)-2-(4-(3-(4-(benzyloxy)phenyl)acryloyl)phenoxy)-2-
methylpropanoate (8k)
Yield 78%, whitish yellow solid, mp: 69–70 ◦C. 1H NMR (400 MHz,
CDCl3) δ 1.25 (t, J = 7.2 Hz, 3H), 1.70 (s, 6H), 4.26 (q, J = 7.2 Hz, 2H),
5.12 (s, 2H), 6.91 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H), 7.35–7.47
(m, 6H), 7.61 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 15.6 Hz, 1H), 8.00 (d, J =
9.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 14.1, 25.5, 61.7, 70.1, 79.4,
115.3, 117.5, 119.6, 127.5, 128.0, 128.2, 128.7, 130.2, 130.3, 132.0,
136.5, 143.9, 159.6, 160.8, 173.8, 188.8. HRMS Calcd for C28H28O5 m/
z: 444.1937 [M + H]+, found 444.1922.
4.1.30. Methyl (E)-4-(4-(3-(4-((3-methylbenzyl)oxy)phenyl)acryloyl)
phenoxy)butanoate (9b)
Yield 85%, white crystals, mp: 93–94 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 2.01 (p, J = 6.8 Hz, 2H), 2.32 (s, 3H), 2.47–2.50 (m, 2H),
3.61 (s, 3H), 4.10 (t, J = 6.0 Hz, 2H), 5.13 (s, 2H), 7.07 (t, J = 9.2 Hz,
4H), 7.15 (d, J = 7.2 Hz, 1H), 7.24–7.30 (m, 3H), 7.68 (d, J = 15.6 Hz,
1H), 7.79–7.85 (m, 3H), 8.14 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ 21.0, 24.1, 29.9, 51.4, 66.9, 69.4, 114.4, 115.2, 119.6,
124.9, 127.6, 128.3, 128.4, 128.6, 130.7, 131.8, 136.6, 137.6, 143.1,
160.3, 162.3, 173.0, 187.2. HRMS Calcd for C28H28O5 m/z: 444.1937
[M + H]+, found 444.1932.
4.1.25. Ethyl (E)-2-methyl-2-(4-(3-(4-((3-methylbenzyl)oxy)phenyl)
acryloyl)phenoxy)propanoate (8l)
Yield 85%, yellowish white solid, mp: 67–68 ◦C. 1H NMR (400 MHz,
CDCl3) δ 1.25 (t, J = 6.8 Hz, 3H), 1.70 (s, 6H), 2.40 (s, 3H), 4.27 (q, J =
6.4 Hz, 2H), 5.10 (s, 2H), 6.91 (d, J = 8.8 Hz, 2H), 7.03 (d, J = 8.8 Hz,
2H), 7.18 (d, J = 7.2 Hz, 1H), 7.24–7.33 (m, 3H), 7.44 (d, J = 15.2 Hz,
1H), 7.62 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 15.6 Hz, 1H), 8.00 (d, J = 8.8
Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 14.1, 21.4, 25.4, 61.7, 70.2, 79.4,
115.3, 117.5, 119.6, 124.6, 128.0, 128.3, 128.6, 129.0, 130.2, 130.3,
132.0, 136.3, 138.4, 144.0, 159.5, 160.8, 173.8, 188.8. HRMS Calcd for
4.1.31. Methyl (E)-4-(4-(3-(4-((3-chlorobenzyl)oxy)phenyl)acryloyl)
phenoxy)butanoate (9c)
Yield 82%, white crystals, mp: 103–104 ◦C. 1H NMR (400 MHz,
DMSO‑d6) δ 2.00 (p, J = 6.4 Hz, 2H), 2.47–2.49 (m, 2H), 3.61 (s, 3H),
4.10 (t, J = 6.4 Hz, 2H), 5.20 (s, 2H), 7.05 (d, J = 8.8 Hz, 2H), 7.09 (d, J
= 8.8 Hz, 2H), 7.39–7.44 (m, 3H), 7.53 (s, 1H), 7.67 (d, J = 15.6 Hz,
1H), 7.79–7.85 (m, 3H), 8.13 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ 24.1, 29.9, 51.4, 67.0, 68.5, 114.4, 115.3, 119.8, 126.3,
127.4, 127.9, 130.5, 130.7, 130.7, 130.8, 133.2, 139.4, 143.0, 160.0,
162.3, 173.0, 187.3. HRMS Calcd for C27H25ClO5 m/z: 464.1391 [M +
H]+, found 464.1389.
C
29H30O5 m/z: 458.2093 [M + H]+, found 458.2086.
4.1.26. Ethyl (E)-2-(4-(3-(4-((3-chlorobenzyl)oxy)phenyl)acryloyl)
phenoxy)-2-methylpropanoate (8m)
Yield 65%, yellow solid, mp: 76–77 ◦C. 1H NMR (400 MHz, CDCl3) δ
1.22 (t, J = 7.2 Hz, 3H), 1.69 (s, 6H), 4.23 (q, J = 6.8 Hz, 2H), 5.07 (s,
2H), 6.88 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 7.29–7.31 (m,
3H), 7.41 (d, J = 15.6 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 15.6
Hz, 1H), 7.97 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 14.2,
25.5, 61.8, 69.3, 79.4, 115.3, 117.6, 119.9, 125.5, 127.5, 128.3, 128.4,
130.0, 130.3, 130.4, 132.0, 134.7, 138.6, 143.8, 159.6, 160.4, 173.8,
188.8. HRMS Calcd for C28H27ClO5 m/z: 478.1547 [M + H]+, found
478.1545.
4.1.32. Methyl (E)-4-(4-(3-(4-((3-nitrobenzyl)oxy)phenyl)acryloyl)
phenoxy)butanoate (9d)
Yield 63%, yellowish white solid, mp: 135–136 ◦C. 1H NMR (400
MHz, DMSO‑d6) δ 2.00 (p, J = 6.8 Hz, 2H), 2.48–2.49 (m, 2H), 3.61 (s,
3H), 4.11 (t, J = 6.4 Hz, 2H), 5.35 (s, 2H), 7.06 (d, J = 9.2 Hz, 2H), 7.13
(d, J = 8.8 Hz, 2H), 7.68 (d, J = 15.6 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H),
7.82 (d, J = 15.6 Hz, 1H), 7.87 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 7.6 Hz,
4.1.27. Ethyl (E)-2-methyl-2-(4-(3-(4-((3-nitrobenzyl)oxy)phenyl)
acryloyl)phenoxy)propanoate (8n)
1H), 8.14 (d, J = 9.2 Hz, 2H), 8.19–8.22 (m, 1H), 8.34–8.35 (m, 1H). 13
C
Yield 68%, yellow solid, mp: 83–84 ◦C. 1H NMR (400 MHz, CDCl3) δ
1.15 (t, J = 7.2 Hz, 3H), 1.59 (s, 6H), 4.16 (q, J = 7.2 Hz, 2H), 5.11 (s,
2H), 6.80 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 15.6
Hz, 1H), 7.48–7.53 (m, 3H), 7.66–7.70 (m, 2H), 7.89 (d, J = 8.8 Hz, 2H),
8.11 (d, J = 8.0 Hz, 1H), 8.24 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 14.1,
25.4, 61.7, 68.7, 79.4, 115.2, 117.6, 120.2, 122.2, 123.1, 128.6, 129.7,
130.2, 130.3, 131.8, 133.2, 138.7, 143.6, 148.5, 159.6, 160.0, 173.7,
188.7. HRMS Calcd for C28H27NO7 m/z: 489.1788 [M + H]+, found
489.1782.
NMR (100 MHz, DMSO‑d6) δ 24.1, 29.9, 51.4, 66.9, 68.1, 114.4, 115.2,
119.8, 122.1, 122.8, 128.0, 130.1, 130.6, 130.7, 130.8, 134.1, 139.2,
142.9, 147.9, 159.8, 162.3, 173.0, 187.2. HRMS Calcd for C27H25NO7
m/z: 475.1631 [M + H]+, found 475.1627.
4.1.33. Methyl (E)-4-(4-(3-(4-phenethoxyphenyl)acryloyl)phenoxy)
butanoate (9e)
Yield 68%, yellowish white crystals, mp: 93–94 ◦C. 1H NMR (400
MHz, DMSO‑d6) δ δ 2.00 (p, J = 6.8 Hz, 2H), 2.47–2.49 (m, 2H), 3.04 (t,
J = 6.8 Hz, 2H), 3.60 (s, 3H), 4.09 (t, J = 6.0 Hz, 2H), 4.23 (t, J = 6.8 Hz,
2H), 7.00 (d, J = 8.8 Hz, 2H), 7.05 (d, J = 9.2 Hz, 2H), 7.20–7.24 (m,
1H), 7.29–7.34 (m, 4H), 7.67 (d, J = 16.0 Hz, 1H), 7.79 (d, J = 12.0 Hz,
1H), 7.81 (d, J = 8.0 Hz, 2H), 8.13 (d, J = 8.0 Hz, 2H). 13C NMR (100
MHz, DMSO‑d6) δ 24.6, 30.3, 35.3, 51.8, 67.4, 68.8, 114.8, 115.3, 119.9,
126.8, 127.9, 128.8, 129.4, 131.1, 131.3, 138.7, 143.6, 160.8, 162.7,
173.5, 187.7. HRMS Calcd for C28H28O5 m/z: 444.1937 [M + H]+, found
444.1931.
4.1.28. Ethyl (E)-2-methyl-2-(4-(3-(4-phenethoxyphenyl)acryloyl)
phenoxy)propanoate (8o)
Yield 77%, yellowish white crystals, mp: 77–78 ◦C. 1H NMR (400
MHz, DMSO‑d6) δ 1.15 (t, J = 8.0 Hz, 3H), 1.61 (s, 6H), 3.05 (t, J = 4.0
Hz, 2H), 4.18 (q, J = 8.0 Hz, 2H), 4.25 (t, J = 8.0 Hz, 2H), 6.89 (d, J =
12.0 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 7.20–7.24 (m, 1H), 7.29–7.35 (m,
4H), 7.68 (d, J = 16.0 Hz, 1H), 7.79 (d, J = 12.0 Hz, 1H), 7.81 (d, J = 8.0
Hz, 2H), 8.12 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6) δ 13.8,
25.0, 34.8, 61.3, 68.3, 79.0, 114.9, 117.2, 119.4, 126.3, 127.4, 128.3,
129.0, 130.5, 130.7, 131.3, 138.2, 143.3, 159.1, 160.4, 172.8, 187.2.
HRMS Calcd for C29H30O5 m/z: 458.2093 [M + H]+, found 458.2086.
4.1.34. General procedure for the synthesis of acids (10a-o and 11a-e)
To a stirred solution of the ester derivatives (8a-o and 9a-e) (3
mmol) in THF, MeOH and H2O (3: 2: 1), LiOH (6 mmol) was added and
the mixture was stirred at rt overnight. The reaction mixture was
monitored by TLC. After completion of the reaction, the solvent was
removed under vacuum. The mixture was neutralized with dilute HCl
and extracted with dichloromethane (3 × 20 ml). The combined organic
phases were washed with water, dried over Na2SO4 and evaporated
4.1.29. Methyl (E)-4-(4-(3-(4-(benzyloxy)phenyl)acryloyl)phenoxy)
butanoate (9a)
Yield 89%, white solid, mp: 91–92 ◦C. 1H NMR (400 MHz, DMSO‑d6)
δ δ 2.02 (p, J = 6.8 Hz, 2H), 2.49–2.50 (m, 2H), 3.62 (s, 3H), 4.11 (t, J =
15

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
under vacuum to obtain the corresponding acids. The residue was pu-
rified by column chromatography using silica gel as stationary phase
and a mixture of dichloromethane (DCM) and MeOH (9:1) as mobile
phase to obtain pure target compounds.
4.1.40. (±)(E)-2-(4-(3-(4-(benzyloxy)phenyl)acryloyl)phenoxy)butanoic
acid (10f)
Yield: 50%, yellow solid, mp: 212 ◦C. HPLC: purity 97%, RT = 1.71
min. 1H NMR (400 MHz, DMSO‑d6) δ 1.03 (t, J = 7.2 Hz, 3H), 1.84–2.00
(m, 2H), 4.48 (t, J = 6.0 Hz, 1H), 5.20 (s, 2H), 7.01 (d, J = 8.4 Hz, 2H),
7.12 (d, J = 8.4 Hz, 2H), 7.36–7.40 (m, 1H), 7.44 (t, J = 7.6 Hz, 2H),
7.50 (d, J = 7.2 Hz, 2H), 7.70 (d, J = 15.6 Hz, 1H), 7.80–7.87 (m, 3H),
8.12 (d, J = 8.4 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6) δ 10.7, 26.2,
69.8, 80.2, 115.3, 115.7, 120.1, 128.1, 128.2, 128.4, 128.9, 130.4,
130.9, 131.0, 137.2, 143.3, 160.7, 163.4, 174.5, 187.7. HRMS Calcd for
4.1.35. (±)(E)-2-(4-(3-(4-(benzyloxy)phenyl)acryloyl)phenoxy)
propanoic acid (10a)
Yield: 45%, yellow solid, mp: 237–238 ◦C. HPLC: purity 97%, RT
=
1.98 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.44 (d, J = 4.4 Hz, 3H), 4.47
(q, J = 5.2 Hz, 1H), 5.19 (s, 2H), 6.92 (d, J = 6.8 Hz, 2H), 7.10 (d, J =
6.4 Hz, 2H), 7.37–7.47 (m, 5H), 7.66 (d, J = 15.6 Hz, 1H), 7.77–7.85 (m,
3H), 8.07 (d, J = 6.8 Hz, 2H), 9.98 (br. s, 1H). 13C NMR (100 MHz,
DMSO‑d6) δ 19.4, 69.9, 75.8, 115.2, 115.7, 120.2, 128.3, 128.4, 128.9,
130.1, 130.9, 131.0, 137.2, 143.1, 160.7, 163.4, 173.3, 187.6. HRMS
Calcd for C25H22O5 m/z: 402.1467 [M + H]+, found 402.1456.
C
26H24O5 m/z: 416.1624 [M + H]+, found 416.1605.
4.1.41. (±)(E)-2-(4-(3-(4-((3-methylbenzyl)oxy)phenyl)acryloyl)
phenoxy)butanoic acid (10g)
Yield: 38%, yellow solid, mp: 114–115 ◦C. HPLC: purity 96%, RT
=
2.30 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.02 (t, J = 7.2 Hz, 3H),
1.87–1.98 (m, 2H), 2.32 (s, 3H), 4.86 (t, J = 6.4 Hz, 1H), 5.13 (s, 2H),
7.02 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 7.2 Hz,
1H), 7.24–7.30 (m, 3H), 7.68 (d, J = 15.6 Hz, 1H), 7.78–7.85 (m, 3H),
8.13 (d, J = 8.8 Hz, 2H), 13.21 (s, 1H). 13C NMR (100 MHz, DMSO‑d6) δ
9.9, 21.4, 25.8, 69.9, 76.9, 115.2, 115.7, 120.0, 125.3, 128.1, 128.8,
128.8, 129.1, 131.1, 131.2, 131.5, 137.1, 138.1, 143.6, 160.8, 162.0,
172.4, 187.8. HRMS Calcd for C27H26O5 m/z: 430.1780 [M + H]+, found
430.1767.
4.1.36. (±)(E)-2-(4-(3-(4-((3-methylbenzyl)oxy)phenyl)acryloyl)
phenoxy)propanoic acid (10b)
Yield: 46%, yellow solid, mp: 141–142 ◦C. HPLC: purity 96%, RT
=
1.60 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.47 (d, J = 6.8 Hz, 3H), 2.32
(s, 3H), 4.57 (q, J = 6.4 Hz, 1H), 5.12 (s, 2H), 6.95 (d, J = 8.4 Hz, 2H),
7.07 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 7.2 Hz, 1H), 7.23–7.30 (m, 3H),
7.65 (d, J = 15.2 Hz, 1H), 7.76 (d, J = 15.6 Hz, 1H), 7.81 (d, J = 8.4 Hz,
2H), 8.06 (d, J = 8.4 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6) δ 19.3,
21.4, 69.9, 75.3, 115.2, 115.7, 120.1, 125.3, 128.1, 128.8, 128.8, 129.0,
130.3, 130.9, 131.0, 137.1, 138.1, 143.2, 160.7, 163.2, 175.5, 187.7.
HRMS Calcd for C26H24O5 m/z: 416.1624 [M + H]+, found 416.1614.
4.1.42. (±)(E)-2-(4-(3-(4-((3-chlorobenzyl)oxy)phenyl)acryloyl)
phenoxy)butanoic acid (10h)
Yield: 48%, yellow solid, mp: 120–121 ◦C. HPLC: purity 98%, RT
=
4.1.37. (±)(E)-2-(4-(3-(4-((3-chlorobenzyl)oxy)phenyl)acryloyl)
phenoxy)propanoic acid (10c)
1.98 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.00 (t, J = 7.2 Hz, 3H),
1.85–1.95 (m, 2H), 4.46 (t, J = 6.4 Hz, 1H), 5.18 (s, 2H), 6.99 (d, J = 8.4
Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 7.41–7.42 (m, 3H), 7.53 (s, 1H), 7.66
(d, J = 15.6 Hz, 1H), 7.75–7.82 (m, 3H), 8.08 (d, J = 8.4 Hz, 2H) ppm.
13C NMR (100 MHz, DMSO‑d6) δ 10.8, 26.2, 68.9, 80.1, 115.3, 115.7,
120.2, 126.7, 127.8, 128.3, 128.3, 130.4, 130.9, 130.9, 131.0, 133.6,
139.8, 143.2, 160.4, 163.4, 175.0, 187.6. HRMS Calcd for C26H23ClO5
m/z: 450.1234 [M + H]+, found 450.1224.
Yield: 40%, beige solid, mp: 140–141 ◦C. HPLC: purity 97%, RT
=
2.96 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.56 (d, J = 6.8 Hz, 3H), 5.03
(q, J = 6.4 Hz, 1H), 5.22 (s, 2H), 7.02 (d, J = 8.8 Hz, 2H), 7.11 (d, J =
8.4 Hz, 2H), 7.41–7.45 (m, 3H), 7.55 (s, 1H), 7.68 (d, J = 15.2 Hz, 1H),
7.80–7.87 (m, 3H), 8.14 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ 18.6, 68.9, 72.1, 115.1, 115.7, 120.2, 126.7, 127.8, 128.3,
128.3, 130.9, 131.1, 131.2, 131.4, 133.6, 139.8, 143.5, 160.5, 161.7,
173.1, 187.8. HRMS Calcd for C25H21ClO5 m/z: 436.1078 [M + H]+,
found 436.1065.
4.1.43. (±)(E)-2-(4-(3-(4-((3-nitrobenzyl)oxy)phenyl)acryloyl)phenoxy)
butanoic acid (10i)
Yield: 31%, Yellow solid, mp: 232 ◦C. HPLC: purity 96%, RT = 2.00
min. 1H NMR (400 MHz, DMSO‑d6) δ 1.00 (t, J = 7.2 Hz, 3H), 1.83–1.92
(m, 2H), 4.36–4.37 (m, 1H), 5.35 (s, 2H), 6.96 (d, J = 8.4 Hz, 2H), 7.12
(d, J = 8.8 Hz, 2H), 7.66 (d, J = 15.6 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H),
7.78 (d, J = 15.6 Hz, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.94 (d, J = 7.6 Hz,
4.1.38. (±)(E)-2-(4-(3-(4-((3-nitrobenzyl)oxy)phenyl)acryloyl)phenoxy)
propanoic acid (10d)
Yield: 32%, white solid, mp: 171–173 ◦C. HPLC: purity 96%, RT
=
2.45 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.54 (d, J = 6.8 Hz, 3H), 5.02
(q, J = 6.8 Hz, 1H), 5.36 (s, 2H), 7.01 (d, J = 8.8 Hz, 2H), 7.13 (d, J =
8.4 Hz, 2H), 7.68 (d, J = 16.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.82 (d,
J = 15.6 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.94 (d, J = 7.6 Hz, 1H), 8.13
(d, J = 8.8 Hz, 2H), 8.21 (d, J = 8.4 Hz, 1H), 8.34 (s, 1H), 13.21 (s, 1H).
13C NMR (100 MHz, DMSO‑d6) δ 18.2, 68.1, 71.6, 114.7, 115.3, 119.8,
122.1, 122.8, 128.0, 130.1, 130.7, 130.7, 130.9, 134.2, 139.2, 143.0,
147.9, 159.8, 161.3, 172.6, 187.2. HRMS Calcd for C25H21NO7 m/z:
447.1318 [M + H]+, found 447.1309.
1H), 8.07 (d, J = 8.4 Hz, 2H), 8.21 (d, J = 8.0 Hz, 1H), 8.34 (s, 1H). 13
C
NMR (100 MHz, DMSO‑d6) δ 10.9, 26.3, 68.5, 80.8, 115.3, 115.7, 120.3,
122.5, 123.3, 128.5, 130.2, 130.6, 130.9, 131.0, 134.5, 139.6, 143.1,
148.3, 160.2, 163.6, 174.7, 187.7. HRMS Calcd for C26H23NO7 m/z:
461.1475 [M + H]+, found 461.1456.
4.1.44. (±)(E)-2-(4-(3-(4-phenethoxyphenyl)acryloyl)phenoxy)butanoic
acid (10j)
Yield: 40%, light yellow solid, mp: 110–112 ◦C. HPLC: purity 96%,
RT = 1.80 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.02 (t, J = 7.2 Hz, 3H),
1.87–2.03 (m, 2H), 3.06 (t, J = 6.8 Hz, 2H), 4.27 (t, J = 6.8 Hz, 2H), 4.85
(t, J = 6.8 Hz, 1H), 7.01 (d, J = 8.8 Hz, 4H), 7.21–7.25 (m, 1H), 7.31 (d,
J = 8.0 Hz, 1H), 7.33–7.35 (m, 3H), 7.67 (d, J = 15.6 Hz, 1H), 7.77–7.83
(m, 3H), 8.13 (d, J = 8.8 Hz, 2H), 13.19 (br s, 1H). 13C NMR (100 MHz,
DMSO‑d6) δ 9.5, 25.3, 34.8, 68.3, 76.4, 114.7, 114.9, 119.5, 126.3,
127.5, 128.3, 129.0, 130.7, 130.7, 131.0, 138.2, 143.2, 160.4, 161.6,
172.0, 187.2. HRMS Calcd for C27H26O5 m/z: 430.1780 [M + H]+, found
430.1769.
4.1.39. (±)(E)-2-(4-(3-(4-phenethoxyphenyl)acryloyl)phenoxy)
propanoic acid (10e)
Yield: 49%, yellowish white solid, mp: 137–139 ◦C. HPLC: purity
96%, RT = 1.74 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.54 (d, J = 6.4 Hz,
3H), 3.06 (t, J = 6.8 Hz, 2H), 4.26 (t, J = 6.8 Hz, 2H), 4.99 (q, J = 6.4 Hz,
1H), 6.99–7.02 (m, 4H), 7.21–7.25 (m, 1H), 7.30–7.35 (m, 4H), 7.67 (d,
J = 15.2 Hz, 1H), 7.77–7.83 (m, 3H), 8.12 (d, J = 8.8 Hz, 2H) ppm. 13
C
NMR (100 MHz, DMSO‑d6) δ 18.2, 34.8, 68.3, 71.8, 114.6, 114.9, 119.5,
126.3, 127.5, 128.3, 129.0, 130.7, 130.9, 138.2, 143.1, 160.4, 161.3,
172.7, 187.2. HRMS Calcd for C26H24O5 m/z: 416.1624 [M + H]+, found
416.1613.
4.1.45. (E)-2-(4-(3-(4-(benzyloxy)phenyl)acryloyl)phenoxy)-2-
methylpropanoic acid (10k)
Yield: 43%, white solid, mp: 170–172 ◦C. HPLC: purity 98%, RT
=
16

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
1.82 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.49 (s, 6H), 5.18 (s, 2H),
6.93 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 7.33–7.36 (m, 1H),
7.41 (t, J = 7.6 Hz, 2H), 7.47 (d, J = 7.6 Hz, 2H), 7.65 (d, J = 15.6 Hz,
1H), 7.77 (d, J = 15.6 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.8
Hz, 2H). 13C NMR (100 MHz, DMSO‑d6) δ 26.5, 69.8, 81.5, 115.7, 117.5,
120.2, 128.2, 128.2, 128.4, 128.9, 129.8, 130.4, 131.0, 137.2, 143.1,
160.6, 161.7, 176.6, 187.7. HRMS Calcd for C26H24O5 m/z: 416.1624
[M + H]+, found 416.1614.
4.1.51. (E)-4-(4-(3-(4-((3-methylbenzyl)oxy)phenyl)acryloyl)phenoxy)
butanoic acid (11b)
Yield: 41%, yellowish white solid, mp: 145–146 ◦C. HPLC: purity
98%, RT = 5.20 min. 1H NMR (400 MHz, CDCl3) δ 2.20 (p, J = 6.4 Hz,
2H), 2.41 (s, 3H), 2.64 (t, J = 7.2 Hz, 2H), 4.14 (t, J = 6.0 Hz, 2H), 5.10
(s, 2H), 6.99 (d, J = 8.8 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 7.18 (d, J =
7.2 Hz, 1H), 7.25–7.33 (m, 3H), 7.45 (d, J = 15.6 Hz, 1H), 7.62 (d, J =
8.4 Hz, 2H), 7.80 (d, J = 15.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 21.4, 24.3, 30.3, 66.8, 70.2, 114.3, 115.3, 119.6,
124.6, 128.0, 128.3, 128.6, 129.0, 130.1, 130.8, 131.4, 136.3 138.4,
143.9, 160.8, 162.5, 177.8, 188.9. HRMS Calcd for C27H26O5 m/z:
430.1780 ([M + H]+, found 430.1771.
4.1.46. (E)-2-methyl-2-(4-(3-(4-((3-methylbenzyl)oxy)phenyl)acryloyl)
phenoxy)propanoic acid (10l)
Yield: 42%, shiny yellow solid, mp: 124–126 ◦C. HPLC: purity 96%,
RT = 2.08 min. 1H NMR (400 MHz, CDCl3) δ 1.63 (s, 6H), 2.30 (s, 3H),
4.98 (s, 2H), 6.90–6.91 (m, 4H), 7.07–7.28 (m, 5H), 7.33 (d, J = 15.6 Hz,
1H), 7.50–7.52 (m, 2H), 7.71 (d, J = 15.2 Hz, 1H), 7.90–7.92 (m, 2H).
13C NMR (100 MHz, CDCl3) δ 21.4, 25.3, 70.2, 79.4, 115.3, 118.3, 119.5,
124.6, 127.8, 128.3, 128.6 129.0, 130.3, 130.5, 132.4, 136.3, 138.4,
144.7, 159.1, 160.9, 177.2, 189.5. HRMS Calcd for C27H26O5 m/z:
430.1780 [M + H]+, found 430.1769.
4.1.52. (E)-4-(4-(3-(4-((3-chlorobenzyl)oxy)phenyl)acryloyl)phenoxy)
butanoic acid (11c)
Yield: 37%, white solid, mp: 127–128 ◦C. HPLC: purity 98%, RT
=
1.54 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.99 (p, J = 6.4 Hz, 2H), 2.41
(t, J = 7.2 Hz, 2H), 4.11 (t, J = 6.4 Hz, 2H), 5.21 (s, 2H), 7.09 (d, J = 8.8
Hz, 4H), 7.43–7.45 (m, 3H), 7.55 (s, 1H), 7.69 (d, J = 15.6 Hz, 1H),
7.80–7.87 (m, 3H), 8.15 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ 24.6, 30.6, 67.5, 68.9, 114.9, 115.7, 120.2, 126.7, 127.8,
128.3, 130.9, 131.1, 131.1, 131.3, 133.6, 139.8, 143.4, 160.4, 162.9,
174.6, 187.7. HRMS Calcd for C26H23ClO5 m/z: 450.1234 [M + H]+,
found 450.1222.
4.1.47. (E)-2-(4-(3-(4-((3-chlorobenzyl)oxy)phenyl)acryloyl)phenoxy)-
2-methylpropanoic acid (10m)
Yield: 38%, yellow solid, mp: 150–151 ◦C. HPLC: purity 95%, RT
=
2.31 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.47 (s, 6H), 5.20 (s, 2H), 6.94
(d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 7.40–7.44 (m, 3H), 7.54 (s,
1H), 7.65 (d, J = 15.6 Hz, 1H), 7.77 (d, J = 15.6 Hz, 1H), 7.82 (d, J = 8.4
Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6) δ 26.4,
68.9, 81.5, 115.7, 117.5, 120.3, 126.7, 127.8, 128.3, 128.4, 129.8, 130.4,
130.9, 131.0, 133.6, 139.8, 143.0, 160.4, 161.7, 176.8, 187.7. HRMS
Calcd for C26H23ClO5 m/z: 450.1234 [M + H]+, found 450.1223.
4.1.53. (E)-4-(4-(3-(4-((3-nitrobenzyl)oxy)phenyl)acryloyl)phenoxy)
butanoic acid (11d)
Yield: 45%, yellow solid, mp: 172–173 ◦C. HPLC: purity 98%, RT
=
6.46 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.94 (p, J = 6.4 Hz, 2H), 2.24
(t, J = 6.8 Hz, 2H), 4.10 (t, J = 6.4 Hz, 2H), 5.36 (s, 2H), 7.07 (t, J = 8.8
Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 15.6 Hz, 1H), 7.72 (t, J =
7.6 Hz, 1H), 7.82 (d, J = 15.6 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.94 (d,
J = 7.6 Hz, 1H), 8.13 (d, J = 8.8 Hz, 2H), 8.22 (d, J = 8.0 Hz, 1H), 8.35
(s, 1H). 13C NMR (100 MHz, DMSO‑d6) δ 25.3, 32.1, 68.0, 68.5, 114.9,
115.7, 120.3, 122.5, 123.3, 128.5, 130.6, 130.9, 131.1, 131.3, 134.6,
139.6, 143.3, 148.3, 160.3, 163.0, 175.9, 187.7. HRMS Calcd for
4.1.48. (E)-2-methyl-2-(4-(3-(4-((3-nitrobenzyl)oxy)phenyl)acryloyl)
phenoxy)propanoic acid (10n)
Yield: 46%, yellow solid, mp: 131–133 ◦C. HPLC: purity 97%, RT
=
1.73 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.52 (s, 6H), 5.38 (s, 2H),
6.97 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 15.6 Hz,
1H), 7.78 (t, J = 8.0 Hz, 1H), 7.82 (d, J = 15.6 Hz, 1H), 7.87 (d, J = 8.4
Hz, 2H), 7.97 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 8.8 Hz, 2H), 8.24 (d, J =
8.0 Hz, 1H), 8.37 (s, 1H). 13C NMR (100 MHz, DMSO‑d6) δ 26.5, 68.5,
81.6, 115.7, 117.5, 120.4, 122.5, 123.3, 128.5, 129.7, 130.4, 130.6,
131.0, 134.6, 139.6, 143.0, 148.3, 160.2, 161.8, 176.4, 187.7. HRMS
Calcd for C26H23NO7 m/z: 461.1475 [M + H]+, found 461.1461.
C
26H23NO7 m/z: 461.1475 [M + H]+, found 461.1456.
4.1.54. (E)-4-(4-(3-(4-phenethoxyphenyl)acryloyl)phenoxy)butanoic acid
(11e)
Yield: 34%, white solid, mp: 125 ◦C. HPLC: purity 96%, RT = 4.98
min. 1H NMR (400 MHz, CDCl3) δ 2.09 (p, J = 6.8 Hz, 2H), 2.54 (t, J =
7.2 Hz, 2H), 3.05 (t, J = 7.2 Hz, 2H), 4.04 (t, J = 6.0 Hz, 2H), 4.16 (t, J =
7.2 Hz, 2H), 6.87 (ddd, J = 8.4 Hz, 4H), 7.16–7.28 (m, 5H,), 7.34 (d, J =
15.6 Hz, 1H), 7.51 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 15.6 Hz, 1H), 7.94 (d,
J = 8.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 24.3, 30.3, 35.7, 66.8,
68.8, 114.3, 115.0, 119.5, 126.6, 127.8, 128.6, 129.0, 130.2, 130.8,
131.4, 137.9, 144.0, 160.8, 162.5, 178.4, 188.9. HRMS Calcd for
4.1.49. (E)-2-methyl-2-(4-(3-(4-phenethoxyphenyl)acryloyl)phenoxy)
propanoic acid (10o)
Yield: 39%, yellow solid, mp: 160–161 ◦C. HPLC: purity 95%, RT
=
3.48 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.52 (s, 6H), 3.05 (t, J = 6.4
Hz, 2H), 4.25 (t, J = 6.8 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 7.00 (d, J =
8.4 Hz, 2H), 7.21–7.25 (m, 1H), 7.30–7.33 (m, 4H), 7.64 (d, J = 15.6 Hz,
1H), 7.73–7.79 (m, 3H), 8.02 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ 26.3, 35.3, 68.8, 81.2, 115.4, 117.6, 120.0, 126.8, 128.0,
128.8, 128.4, 129.4, 130.0, 130.4, 131.0, 138.7, 143.2, 160.8, 161.5,
177.4, 187.6. HRMS Calcd for C27H26O5 m/z: 430.1780 [M + H]+, found
430.1764.
C
27H26O5 m/z: 430.1780 [M + H]+, found 430.1771.
4.2. Biology - in vitro studies
4.2.1. In vitro PPAR- reporter assay
α
The transcriptional activity of human PPAR
α was determined by
measuring luciferase activity in PPAR reporter assays (Indigo Bio-
α
4.1.50. (E)-4-(4-(3-(4-(benzyloxy)phenyl)acryloyl)phenoxy)butanoic
sciences, cat. IB00111) according to the manufacturer’s instructions.
Briefly, reporter cells were dispensed into 96 wells microplates and
treated for 16 h with different concentrations of the compounds. Control
cells were treated with 0.1% (v/v) DMSO. At the end of the treatment,
the medium was discarded and the luciferase detection reagent was
added. The luminescence intensity of light emission from the luciferase
acid (11a)
Yield: 35%, orange solid, mp: 144–145 ◦C. HPLC: purity 98%, RT
=
1.56 min. 1H NMR (400 MHz, DMSO‑d6) δ 1.99 (p, J = 6.8 Hz, 2H), 2.42
(t, J = 7.2 Hz, 2H), 4.11 (t, J = 6.4 Hz, 2H), 5.19 (s, 2H), 7.09 (t, J = 8.8
Hz, 4H), 7.35 (d, J = 7.2 Hz, 1H), 7.41 (t, J = 7.2 Hz, 2H), 7.48 (d, J =
6.8 Hz, 2H), 7.68 (d, J = 15.2 Hz, 1H), 7.79–7.86 (m, 3H), 8.15 (d, J =
8.8 Hz, 2H), 12.17 (br s, 1H). 13C NMR (100 MHz, DMSO‑d6) δ 24.6,
30.5, 67.5, 69.9, 114.9, 115.7, 120.1, 128.1, 128.3, 128.4, 128.9, 131.1,
131.3, 137.2, 143.5, 160.7, 162.8, 174.5, 187.7. HRMS Calcd for
reaction, that is directly proportional to the relative level of PPAR
α
activation in the reporter cells, was measured using a microplate
luminometer (Victor 3, PerkinElmer). The half effective concentration
(EC50) on luciferase activity was analyzed using GraphPad Prism 6.0
(GraphPad Software Inc., La Jolla, CA).
C
26H24O5 m/z: 416.1624 [M + H]+, found 416.1614.
17

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
4.2.2. Cell culture
the compound of interest. Fenofibric acid (FA) was docked as reference.
Human HepG2 hepatocellular carcinoma cells were grown in DMEM,
supplemented with 10% Fetal Bovine Serum, 10 mg/ml streptomycin/
penicillin and 1x MEM non-Essential Amino Acids (Gibco-Life Tech-
nologies), at 37 ◦C in humidified air with 5% CO2.
4.4. In vivo pharmacological studies
4.4.1. Animals
Adult male Albino rats weighing 180–200 g were purchased from the
Animals House Colony of the National Research Centre, Giza, Egypt and
were housed in polypropylene cages under the under conditions of
controlled light (12 h light/dark cycle) and temperature (23 ± 2 ◦C). The
animals had free access to water and standard chow ad libitum. Rats were
allowed to acclimatize to laboratory conditions before the start of the
experiment. All tests were performed between 09:00 a.m. and 04:00p.m.
All experimental procedures involving animals were performed ac-
cording to the guidelines of the ethical committee of the National
Research Centre for Experimental Animal Use [Ethical Approval Num-
ber: 20046].
4.2.3. Gene Expression. RNA isolation and RT-PCR
Total RNA of HepG2 cells was isolated using the RNeasy Mini Kit
(Qiagen, Hilden, Germany) according to the manufacturer’s in-
structions. For removing residual DNA amounts an additional on-
column DNase treatment was performed. RNA was reverse transcribed
from 1 µg total RNA in a 20 µl reaction mixture using the Enhanced avian
RT-PCR kit (Sigma-Aldrich) following the manufacturer’s instructions.
Quantitative RT-PCR was performed in triplicate using a standard
TaqMan® PCR protocol on a StepOne Real time PCR System (Applied
Biosystems, Foster City, CA, USA). The 25 µl PCR mix included 1,5 µl of
cDNA, 1 × TaqMan® Universal PCR Master Mix (Applied Biosystems)
and 1.25 µl TaqMan® assay including probes specific for human CPT1A
and GAPDH genes. The reactions were incubated at 50 ◦C for 2 min and
95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 s and 60 ◦C for 1
min. All reactions were run in triplicate. The human GAPDH house-
keeping gene was used as reference. Briefly, average GAPDH CT values
were subtracted from average CPT1A CT values to obtain ΔCT values.
ΔΔCT values were then obtained by subtracting ΔCT values of treated
samples from ΔCT values of samples treated with vehicle (DMSO).
4.4.2. Acute toxicity study
An acute toxicity test was conducted according to the procedure of
Organization of Economic Co-operation and Development (OECD)
guideline 425 for testing of chemicals [62]. The compounds (10a and
10i) were suspended in 1% Tween 80 in distilled water and adminis-
tered orally at an initial single dose of 2000 mg/kg to 10 weeks old rats.
Rats were observed individually for signs of toxicity during the first 30
min after dosing, then periodically during the first 24 h, with special
attention during the first 4 h, and then daily for a total period of 14 days.
Relative gene expression levels were then calculated as 2ꢀ ΔΔCT
.
4.2.4. Western blot
4.4.3. Experimental induction of hyperlipidemia
HepG2 cells for western blot were scraped in cold PBS buffer, cen-
trifugated at 1000 rpm for 5 min and resuspended in lysis buffer (150
mM NaCl, 1% Triton X-100, 50 mM Tris HCl pH 8.0) containing protease
and phosphatase inhibitors (Roche, Basel, Switzerland). Protein con-
centrations were measured by the Bio-Rad protein assay (Bio-Rad Lab-
oratories, Hercules, CA). Western blot experiments were carried out as
previously described [59]. Briefly, samples were denaturated in
SDS–PAGE sample buffer for 5 min at 99 ◦C, loaded onto a 10% SDS
PAGE gel (Biorad) and transferred to a nitrocellulose membrane. After
transfer, the membrane was blocked for 2 h at room temperature with
3% nonfat dried milk (AppliChem GmbH, Germany) in TBS containing
0.01% Tween 20 (Sigma–Aldrich). Subsequently, the membrane was
washed and incubated overnight at 4 ◦C with the polyclonal antibody
Hyperlipidemia was induced by a single intraperitoneal (i.p.) injec-
tion of tyloxapol (triton WR-1339) (Acros organicsTM) at dose of 300
mg/kg [63,64] dissolved in 0.89% saline, to rats of all groups except the
control group. Standard drugs: fenofibrate (Mina pharm, Egypt) and
fenofibric acid (Sigma-Aldrich, St. Louis, MO, USA) as well as com-
pounds 10a and 10i were suspended in 1% Tween 80 and administered
by oral gavage (p.o.).
4.4.4. Experimental design
The rats were randomly divided into nine groups each comprising six
rats. Group 1 received the vehicle (1% Tween 80) and served as control
group, Groups 2–9 received triton and 1 h later was administered with
the vehicle or standard or treatment via oral gavage [65]. Group 2
received the vehicle and served as positive control [hyperlipidemic or
triton-induced group]. Group 3 received fenofibric acid (FA) [standard
drug] (100 mg/kg) [66]. Group 4 and 5 received fenofibrate (FB)
[standard drug] at a dose of 100 and 200 mg/kg, respectively [67,68].
Group 6 and 7 received compound 10a at dose of 100 and 200 mg/kg,
respectively. Group 8 and 9 received compound 10i at dose of 100 and
200 mg/kg, respectively. After 24 h, blood samples were collected
through the retro-orbital puncture. Serum was separated by centrifuga-
tion at 3000 rpm for 15 min at 4 ◦C for the assessment of lipid markers.
For plasma separation, blood was collected in heparin coated collecting
tubes then centrifuged at 3,000 rpm for 10 min at 4 ◦C. The plasma was
stored at ꢀ 80 ◦C until analysis of plasma lipid peroxidation [Malon-
dialdehyde: MDA] and total anti-oxidant capacity. Then, rats were
sacrificed and the liver tissue was excised for homogenization for the
determination of tissue reduced glutathione and histopathological
examination.
against PPAR
α (1:000; Invitrogen, Carlsbad, CA, USA) or with the
monoclonal antibody against β-actin (1:5000, Abcam, Cambridge, UK),
used as loading control protein.
4.3. Molecular docking study
All the molecular modeling studies were carried out using Molecular
Operating Environment (MOE, 2020.0901) software. All minimizations
were performed with MOE until an RMSD gradient of 0.05 kcal∙molꢀ 1Åꢀ 1
with MMFF94x force field and the partial charges were automatically
calculated. The X-ray crystallographic structure of PPARα co-crystallized
with GW735 [EC50 = 20 nM] (PDB ID: 2P54) was downloaded from the
protein data bank [60,61]. Water molecules and peptide chain that are not
involved in binding were removed. Then, the protein was prepared for
docking study using Protonate 3D protocol in MOE with default options.
The co-crystalized ligand (GW735) was used to define the binding site for
docking. Triangle Matcher placement method and London dG scoring
function were used for docking. Docking setup was first validated by self-
docking of the co-crystallized ligand (GW735) in a close proximity to the
binding site of the protein with energy score (S) = ꢀ 15.77 kcal/mol and
RMSD of 0.630 Å and by the ability of the docking pose to reproduce all
the key interactions accomplished by the co-crystallized ligand with the
binding site hot spots (Ser280, Tyr314, His440 and Asp464) (See sup-
porting information, Figures S51 and S52). The validated setup was then
used in predicting the ligand-receptor interactions at the binding site for
4.4.5. Measurement of serum lipid profile
Total cholesterol (TC), triglycerides (TG), high-density lipoprotein
cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c)
were measured spectrophotometrically using commercially available
kits (MG Science & Technology Center “STC”, Giza, Egypt and Bio-
diagnostic, Giza, Egypt). The result of very low-density lipoprotein-
cholesterol (VLDL-c) was estimated by the equation: VLDL-c = TG/5
[69]. The cardiac risk ratio, atherogenic coefficient, atherogenic index,
18

R.M. Hassan et al.
Bioorganic Chemistry 115 (2021) 105170
and Castelli’s risk index II were calculated by the following equations
[43,70,71].
Acknowledgment
Cardiac risk ratio (CRR) = [TC]/[HDL-c]
The authors would like to thank the National Research Centre (NRC)
- Egypt and Consiglio Nazionale delle Ricerche (CNR) - Italy for funding
this research through the bilateral project of NRC - Egypt and CNR -
Italy; Project ID: IT II 020702.
Atherogenic coefficient (AC) = [Non-HDL-c]/[HDL-c]
Atherogenic index (AI) = log ([TG] / [HDL-c])
Castelli’s risk index II (CRI-II) = [LDL-c]/[HDL-c].
The authors would like to thank Prof. Dr. Adel Bakeer (Pathology
Department, Faculty of Veterinary Medicine, Cairo University, Egypt) for
his assistance in the histopathological examinations and Prof. Andrea
Urbani (Dipartimento Universitario di Scienze Biotecnologiche di Base,
4.4.6. Measurement of antioxidant parameters
4.4.6.1. Determination of reduced glutathione (GSH). Liver reduced GSH
contents was estimated using Biodiagnostic kit (Biodiagnostic, Giza,
Egypt), according to the method described by [72] and expressed as mg/
g wet tissue. Briefly, liver tissue was excised, weighted and minced/
grinded on ice. The tissue was homogenized in homogenization buffer
[50 mM potassium phosphate buffer in 1 mM EDTA pH 7.5] to prepare
10% homogenate (w/v). The homogenate was centrifuged at 4000 rpm
for 15 min at 4 ◦C. The supernatant was collected and stored at ꢀ 80 ◦C.
`
Cliniche Intensivologiche e Perioperatorie, Universita Cattolica del Sacro
Cuore di Roma, Italy) for high-resolution mass-spectrometry experiments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105170.
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