Antihyperlipidemic studies of newly synthesized phenolic derivatives: In silico and in vivo approaches

Article abstract of DOI:10.2147/DDDT.S158554

Background: Hyperlipidemia is a worth-mentioning risk factor in quickly expanding cardiovascular diseases, including myocardial infarction and, furthermore, in stroke. Methods: The present work describes the synthesis of phenolic derivatives 4a–e and 6a–c

Full text of DOI:10.2147/DDDT.S158554

Drug Design, Development and erapy
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O r i g i n a l r e s e a r c h
antihyperlipidemic studies of newly synthesized
phenolic derivatives: in silico and in vivo
approaches
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
Muhammad Tahir aqeel1
nisar ur-rahman1
arif-ullah Khan2
Background: Hyperlipidemia is a worth-mentioning risk factor in quickly expanding cardio-
vascular diseases, including myocardial infarction and, furthermore, in stroke.
Methods: The present work describes the synthesis of phenolic derivatives 4a–e and 6a–c with
the aim of developing antihyperlipidemic agents. The structures of the synthesized compounds
were confirmed by spectroscopic data. The in silico docking studies were performed against
human 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase enzyme (PDB ID:
1HWK), and it was observed that compounds 4a and 6a exhibited maximum binding affinity
with target protein having binding energies −8.3 and −7.9 kcal, respectively.
Results: Compound 4a interacts with amino acids Val805 with distance 1.89 Å and Met656,
Thr558, and Glu559 with bonding distances 2.96, 2.70, and 2.20 Å, respectively. The in vivo
antihyperlipidemic activity results revealed that compound 4a indicated minimum weight incre-
ment, ie, 20% compared with 35% weight increment with standard drug atorvastatin during
6 weeks of treatment. Moreover, increment in high-density lipoprotein cholesterol and decrease
in total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels were more
prominent in case of 4a compared to atorvastatin with P,0.05. The synthesized compounds
were nontoxic and well tolerated because none of the mice were found to suffer from any kind
of morbidity and death during 6 weeks of dosing.
Zaman ashraf3
Muhammad latif4
hummera rafique5
Usman rasheed1
1Department of Pharmacy, cOMsaTs
institute of information Technology
abbottabad, abbottabad, Pakistan;
2riphah institute of Pharmaceutical
sciences, riphah international
University, islamabad, Pakistan;
3Department of chemistry, allama
iqbal Open University, islamabad,
Pakistan; 4college of Medicine, centre
for genetics and inherited Diseases
(cgiD),Taibah University, al-Madinah
al-Munawwarah, saudi arabia;
5Department of chemistry, University
of gujrat, gujrat, Pakistan
Conclusion: Based on our pharmacological evaluation, we may propose that compound 4a may
act as a lead structure for the design and development of more potent antihyperlipidemic drugs.
Keywords: phenolic derivatives, synthesis, antihyperlipidemic, in silico docking, HMG CoA
reductase, atorvastatin
Introduction
Human beings are persistently confronting life-threatening morbidities such as
cardiovascular disorders and cancer. Cardiovascular diseases account for ~17 million
deaths annually worldwide. Above all, atherosclerosis is the major factor for the
pathogenesis of myocardial and cerebral infarctions. Increased plasma low-density
lipoproteins and triglycerides along with low levels of high-density lipoproteins are
often the major root cause of atherosclerosis and ultimately myocardial and cerebral
infarctions. Correspondingly, obesity has a direct linkage with atherosclerosis and
myocardial infarction. Exact etiology of obesity is relatively unclear, but still the major
contributors are genetic, environmental, and dietary factors.1
correspondence: Zaman ashraf
Department of chemistry, allama
iqbal Open University, h-8/4,
islamabad 44000, Pakistan
To counter these morbidities, an efficacious and safer pharmacological solution is of
most extreme significance. The process of drug discovery is fastidious and expensive.
The quest for new effective molecules is challenging yet the accessibility of various
Tel +92 32 1519 4461; +92 905 7182
Fax +92 5 1289 1471
email mzchem@yahoo.com
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docking tools and computer-aided methodologies has limited The in silico docking studies against HMG CoA reductase
the gap to a specific level.2,3 Antioxidants have phenolic enzyme (PDB ID: 1HWK) were also performed to compare
flavonoids that scavenge oxygen radicals and are involved the dry laboratory results with wet laboratory findings.
in the inhibition of enzyme activity. Phenolic compounds
are auxiliary metabolites present in fruits and vegetables.4,5
Materials and methods
Drugs and chemicals
They possess diverse pharmacological activities including
cardiovascular,6 antioxidant,5–9 antiatherogenic,10,11 anti-
Atorvastatin was obtained from Global Pharmaceuticals
inflammatory,12–16 and antithrombotic.17–19 Various studies
(Islamabad, Pakistan), and mice were received as gift from
on the experimental animals reported the effect of phenolic
National Institute of Health (Islamabad, Pakistan).
compounds such as quercetin and rutinosides on the lipid
metabolism causing a decrease in the serum lipid levels.20,21
synthesis of 2-methoxyphenyl chloroacetate (2)
The 2-methoxyphenol (1) (0.01 mol) was dissolved in
Derivatives of natural poly-phenols have shown remark-
able bioactivity in various disease areas both in prevention
anhydrous dichloromethane (25 mL), and triethylamine
and treatment. Among numerous classes of polyphenols,
(0.01 mol) was added slowly. The resulting solution was
hydroxycinnamic acid derivative is the renowned group of
then cooled in an ice salt mixture to 0 to −5°C. The chlo-
phenols found in cereals, roasted coffee, and green vegetables
roacetyl chloride (0.01 mol) in dry dichloromethane was
and is reported as a potential antioxidant.22–24 Sinapic acid and
added drop wise to the reaction mixture with constant stirring
ferulic acid are the most studied hydroxycinnamic acid deriv-
over a period of 1 h maintaining the temperature constant.
atives and were found to be highly effective as antioxidant,25,26
The reaction mixture was then stirred at room temperature
and in recent data, they are found to be preventive and
for further 5 h and washed with 5% HCl and 5% sodium
therapeutic agents in several other diseases including athero-
hydroxide solution. The organic layer was washed with
sclerosis, inflammatory injury, and cancer.27,28 Caffeic acid
saturated aqueous NaCl, dried over anhydrous magnesium
derivatives are another class of biologically active phenolic
sulfate and filtered, and the solvent was removed under
derivatives as antioxidant29,30 and anticancer.31–33
reduced pressure. The crude product was purified by silica
Recently, few studies have demonstrated polyphenolic
gel column to afford the corresponding 2-methoxyphenyl
moieties as potential antihyperlipidemic agents by inhibit-
chloroacetate (2): melting point, 56°C–58°C; reaction time,
4 h; yield, 78%; Rf, 0.62 (n-hexane:ethyl acetate 3:1); FTIR
ing 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA)
reductase in in silico models.34 Considering such enormous
νmax (1/cm): 2,903 (sp2 C−H), 2,863 (sp3 C−H), 1,719 (C=O
pharmacological potential, derivatives of natural phenols are
exciting prospect for future drug development especially in
antihyperlipidemic and cardiovascular therapeutic domains.
ester), 1,589 (C=C aromatic), 1,164 (C−O ester).
The use of computational analysis to establish the bind- general procedure for the synthesis of title
ing affinity of the drug-like compounds with different compounds 4a–4e and 6a–6c
enzymes, receptors, and proteins is categorized in in silico The substituted benzoic acids (3a–3e) (0.01 mol), triethyl
pharmacology.35 The in silico methods have been frequently amine (0.01 mol), and potassium iodide (0.01 mol) were
used in the discovery and optimization of novel molecules with mixed in dimethylformamide (25 mL) and stirred at room
affinity to a target and in the optimization of pharmacokinetic temperature. The 2-methoxyphenyl chloroacetate inter-
parameters.36,37 Various studies have been reported in which mediate (2) was then added to the reaction mixture slowly
theinsilicoassessmentofthedrug-likecompoundsshowedthe and at the same temperature overnight (Scheme 1). The
binding affinity with enzymes, proteins, transporter proteins, final products (4a–4e) were then extracted with ethyl acetate
and receptors and is very well correlated with their in vitro and (3×25 mL). The combined ethyl acetate layer was then
in vivo pharmacological effects.38 Consequently, the in silico washed with 5% HCl, 5% sodium carbonate, and finally
pharmacology paradigm is ongoing and presents a vast plat- brine solution. The organic layer was dried over anhydrous
form for the discovery of new drug targets as well as screening magnesium sulfate and filtered, and the solvent was removed
of new drug-like compounds that can be considered as drug.
under reduced pressure to afford the crude products (4a–4e).
In this perspective, current study is aimed to develop The title compounds (4a–4e) were purified by silica gel
potent antihyperlipidimic agents. The phenolic derivatives column chromatography (n-hexane:ethyl acetate 3:1).
4a–e and 6a–c have been synthesized and characterized; The same procedure was used for the preparation of final
the in vivo antihyperlipidemic evaluation was carried out. products (6a–6c) (Scheme 2).
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antihyperlipidemic studies of newly synthesized phenolic derivatives
O
(C2H5)3N/CH2CI2
0°C to –5°C
O
+
HO
O
CI
CI
CI
OCH3
OCH3
(1)
(2)
O
O
OH
O
+
(C2H5)3N/KI
DMF
O
O
O
CI
R
R
OCH3
O
OCH3
(2)
(3a–3e)
(4a–4e)
R =
3a = 3-OH
3c = 3,4-di-OH
3e = 3,5-di-OH
3b = 4-OH
3d = 2,4-di-OH
R is same as in (3a–3e)
Scheme 1 synthesis of phenolic derivatives (4a–4e).
2-(2-Methoxyphenoxy)-2-oxoethyl 3-hydroxybenzoate (DMSO-d6, δ ppm): 7.90 (d, J=8.8 Hz, 2H, H-2, H-6), 7.26
(4a): solid; melting point, 81°C–83°C; yield, 76%; Rf, 0.56 (ddd, J=6.8, 1.6, 1.4 Hz, 1H, H-4′), 7.11–7.16 (m, 2H, H-3′,
(n-hexane:ethyl acetate 3:1); FTIR νmax (1/cm): 3,248 (O−H), H-6′), 6.97 (ddd, J=6.4, 1.6, 1.21H, H-5′), 6.90 (d, J=8.8 Hz,
3,965 (sp2 C−H), 2,864 (sp3 C−H), 1,723 (C=O ester), 1,595 2H, H-3, H-5), 5.15 (s, 2H, −CH2), 3.78 (s, 3H, −OCH3), 3.33
(C=C aromatic), 1,164 (C−O ester); 1H NMR (CDCl3, δ ppm): (s, 1H, −OH); 13C NMR (DMSO-d6, δ ppm): 166.8 (C=O
7.69 (m, 1H, H-6), 7.58 (dd, J=1.6, 1.6 Hz, 1H, H-2), 7.33 ester), 165.4 (C=O ester), 162.9 (C-4), 151.1 (C-2′), 139.0
(dd, J=4.8, 4.4 Hz, 1H, H-5), 7.24 (m, 1H, H-4), 7.12 (dd, (C-1′), 132.2 (C-2, C−), 127.7 (C-5′), 123.0 (C-1), 121.3
J=6.4, 1.6 Hz, 1H, H-6′), 7.07 (dd, J=3.6, 1.8 Hz, 1H, H-3′), (C-3′), 119.6 (C-4′), 116.0 (C-3, C-5), 113.5 (C-6′),
6.95–6.99 (m, 2H, H-4′, H-5′), 5.65 (s, 1H, −OH), 5.10 60.9 (−CH2), 56.3 (−OCH3); anal calcd for C16H14O6: C,
(s, 2H, −CH2), 3.85 (s, 3H, −OCH3); 13C NMR (CDCl3, δppm); 63.57; H, 4.63; found C, 63.66; H, 4.73.
166.1 (C=O ester), 165.7 (C=O ester), 155.8 (C-3), 150.9
2-(2-Methoxyphenoxy)-2-oxoethyl 3,4-dihydroxy-
(C-2′), 139.0 (C-1′), 130.4 (C-5), 129.7 (C-6), 127.3 (C-2), benzoate (4c): solid; melting point, 154°C–156°C; yield,
122.6 (C-1), 122.3 (C-4), 121.4 (C-4′), 120.8 (C-6′), 116.6 72%; Rf, 0.48 (n-hexane:ethyl acetate 3:1); FTIR νmax
(C-3′), 114.5 (C-5′), 61.0 (−CH2), 55.8 (−OCH3); anal calcd (1/cm): 3,166 (O−H), 2,935 (sp2 C−H), 2,837 (sp3 C−H),
for C16H14O6: C, 63.57; H, 4.63; found C, 63.64; H, 4.71.
1,722 (C=O ester), 1,600 (C=C aromatic), 1,148 (C−O
1
2-(2-Methoxyphenoxy)-2-oxoethyl 4-hydroxybenzoate ester); H NMR (DMSO-d6, δ ppm): 7.43 (d, J=2.0 Hz,
(4b): solid; melting point, 126°C–128°C; yield, 78%; Rf, 1H, H-2), 7.39 (dd, J=6.0, 2.0 Hz, 1H, H-6), 7.25 (ddd,
0.53 (n-hexane:ethyl acetate 3:1); FTIR νmax (1/cm): 3,149 J=3.6, 2.0, 1.6 Hz, 1H, H-4′), 7.12–7.16 (m, 2H, H-3′,
(O−H), 2,932 (sp2 C−H), 2,842 (sp3 C−H), 1,718 (C=O H-6′), 6.97 (ddd, J=4.0, 2.4, 1.6 Hz, 1H, H-5′), 6.84 (d,
ester), 1,593 (C=C aromatic), 1,148 (C−O, ester); 1H NMR J=8.0 Hz, 1H, H-5), 5.13 (s, 2H, −CH2), 3.80 (s, 3H, −OCH3),
2
2+
ꢃ&ꢅ+ꢆꢄꢁ1ꢇ.,
5
ꢀ
2
2
2
5
2
&,
'0)
2
2&+ꢁ
2
2&+ꢁ
ꢀꢄꢂ
ꢀꢁD±ꢁFꢂ
ꢀꢃD±ꢃFꢂ
5ꢂ
ꢁDꢂ ꢂ±+
ꢁFꢂ ꢂ±&,
ꢁEꢂ ꢂ±2+
5ꢂLVꢂVDPHꢂDVꢂLQꢂꢃꢁD±ꢁFꢄ
Scheme 2 synthesis of phenolic derivatives (6a–6c).
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aqeel et al
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3.42 (s, 2H, −OH); 13C NMR (DMSO-d6, δ ppm): 171.4 (C=O (CDCl3, δ ppm); 167.7 (C=O ester), 166.1 (C=O ester), 150.9
ester), 166.8 (C=O ester), 165.5 (C-4), 151.4 (C-3), 151.1 (C-2″), 146.3 (C-2), 139.2 (C-1″), 134.1 (C-5″), 130.0 (C-2′,
(C-2′), 148.1 (C-1′), 139.0 (C-5′), 127.9 (C-6), 123.2 (C-4′), C-6′), 128.9 (C-3′, C-5′), 128.2 (C-4′), 127.2 (C-1′), 122.6
122.7 (C-3′), 121.1 (C-6′), 119.8 (C-2), 116.9 (C-5), 113.5 (C-3″), 120.7 (C-4″), 116.7 (C-6″), 112.5 (C-1), 60.5 (−CH2),
(C-1), 60.9 (−CH2), 56.1 (−OCH3); anal calcd for C16H14O7: 55.9 (−OCH3); anal calcd for C18H16O5: C, 69.23; H, 5.12;
C, 60.37; H, 4.40; found C, 60.44; H, 4.46.
found C, 69.29; H, 5.17.
2-(2-Methoxyphenoxy)-2-oxoethyl 2,4-dihydroxybenzo-
2-(2-Methoxyphenoxy)-2-oxoethyl (2E)-3-(4-
ate (4d): solid; melting point, 109°C–111°C; yield, 76%; Rf, hydroxyphenyl)prop-2-enoate (6b): solid; melting point,
0.46 (n-hexane:ethyl acetate 3:1); FTIR νmax (1/cm): 3,263 101°C–103°C; yield, 78%; Rf, 0.47 (n-hexane:ethyl acetate
(O−H), 2,942 (sp2 C−H), 2,864 (sp3 C−H), 1,726 (C=O 3:1); FTIR νmax (1/cm): 3,132 (−OH), 2,931 (sp2 C−H), 2,849
ester), 1,606 (C=C aromatic), 1,162 (C−O ester); 1H NMR (sp3 C−H), 1,728 (C=O), 1,609 (C=C aromatic), 1,148 (C−O
1
(DMSO-d6, δ ppm): 7.73 (d, J=8.8 Hz, 1H, H-6), 7.24–7.29 ester); H NMR (DMSO-d6, δ ppm): 7.70 (d, J=16.0 Hz,
(m, 2H, H-3′, H-6′), 7.15 (ddd, J=6.4, 5.6, 1.6, Hz, 1H, 1H, H-2), 7.62 (d, J=8.8 Hz, 2H, H-2′, 6′), 7.26 (ddd,
H-4′), 6.98 (ddd, J=5.2, 3.6, 2.0, Hz, 1H, H-5′), 6.43 (dd, J=7.6, 6.0, 1.6 Hz, 1H, H-4″), 7.11–7.16 (m, 2H, H-3″,
J=6.4, 2.4 Hz, 1H, H-3), 6.34 (d, J=2.0 Hz, 1H, H-5), 5.20 H-6″), 6.98 (ddd, J=7.6, 6.0, 1.6 Hz, 1H, H-5″), 6.80 (d,
(s, 2H, −CH2), 3.78 (s, 3H, −OCH3), 3.44 (s, 2H, −OH); 13C J=8.8 Hz, 2H, H-3′, H-5′), 6.54 (d, J=16.0 Hz, 1H, H-1), 5.06
NMR (DMSO-d6, δ ppm): 168.3 (C=O ester), 166.5 (C=O (s, 2H, −CH2), 3.78 (s, 3H, −OCH3), 3.33 (s, 1H, −OH); 13C
ester), 165.1 (C-2), 163.2 (C-4), 151.1 (C-2′), 139.0 (C-1′), NMR (DMSO-d6, δ ppm): 166.1 (C=O ester), 165.8 (C=O
132.5 (C-5′), 127.8 (C-6), 123.2 (C-4′), 121.3 (C-3′), 119.6 ester), 150.9 (C-2″), 144.8 (C-2), 139.1 (C-1″), 132.6 (C-4′),
(C-6′), 116.0 (C-3), 109.0 (C-5), 103.1 (C-1), 61.1 (−CH2), 129.5 (C-2′, C-6′), 129.3 (C-3′, C-5′), 127.2 (C-5″), 122.7
56.1 (−OCH3); anal calcd for C16H14O7: C, 60.37; H, 4.40; (C-1′), 117.4 (C-3″), 112.6 (C-4″), 112.5 (C-6″), 60.6 (−CH2),
found C, 60.41; H, 4.48.
55.9 (−OCH3); anal calcd for C18H16O6: C, 65.85; H, 4.87;
2-(2-Methoxyphenoxy)-2-oxoethyl 3,5-dihydroxybenzo- found C, 65.93; H, 4.93.
ate (4e): solid; melting point, 173°C–175°C; yield, 84%; Rf,
2-(2-Methoxyphenoxy)-2-oxoethyl (2E)-3-(4-chlorophe-
0.45 (n-hexane:ethyl acetate 3:1); FTIR νmax (1/cm): 3,134 nyl)prop-2-enoate (6c): solid; melting point, 73°C–75°C;
(O−H), 2,919 (sp2 C−H), 2,838 (sp3 C−H), 1,729 (C=O yield, 83%; Rf, 0.55 (n-hexane:ethyl acetate 3:1); FTIR
ester), 1,608 (C=C aliphatic), 1,598 (C=C aromatic), 1,145 νmax (1/cm): 2,919 (sp2 C−H), 2,862 (sp3 C−H), 1,725
(C−O, ester); 1H NMR (DMSO-d6, δ ppm): 7.27 (ddd, J=5.6, (C=O ester), 1,611 (C=C aromatic), 1,156 (C−O ester);
3.6, 1.6 Hz, 1H, H-4′), 7.12–7.15 (m, 2H, H-3′, H-6′), 6.96 1H NMR (CDCl3, δ ppm): 7.79 (d, J=16.0 Hz, 1H, H-2),
(ddd, J=6.4, 6.4, 2.4 Hz, 1H, H-5′), 6.89 (d, J=2.4 Hz, 7.50 (d, J=7.6 Hz, 2H, H-2′, 6′), 7.43 (d, J=7.6 Hz, 2H,
2H, H-2, H-6), 6.48 (dd, J=2.4, 1.6 Hz, 1H, H-4), 5.17 H-3′, H-5′), 7.24 (dd, J=6.0, 1.6 Hz, 1H, H-3″), 7.11
(s, 2H, −CH2), 3.78 (s, 3H, −OCH3), 3.33 (s, 2H, −OH); (dd, J=6.4, 1.6 Hz, 1H, H-6″), 6.95–6.99 (m, 2H, H-4″,
13C NMR (DMSO-d6, δ ppm); 166.6 (C=O ester), 165.6 H-5″), 6.57 (d, J=16.0 Hz, 1H, H-1), 5.06 (s, 2H, −CH2),
(C=O ester), 159.1 (C-3, C-5), 151.1 (C-2′), 139.0 (C-1′), 3.86 (s, 3H, −OCH3); 13C NMR (CDCl3, δ ppm); 166.1
130.7 (C-5′), 127.8 (C-2, C-6), 123.0 (C-4′), 121.1 (C-3′), (C=O ester), 165.8 (C=O ester), 151.0 (C-2″), 144.9 (C-2),
113.5 (C-6′), 108.2 (C-4), 107.8 (C-1), 61.3 (−CH2), 56.3 139.1 (C-1″), 136.5 (C-4′), 132.6 (C-2′, C-6′), 129.5 (C-3′,
(C-1″); anal calcd for C16H14O7: C, 60.37; H, 4.40; found C, C-5′), 127.3 (C-5″), 122.7 (C-1′), 121.6 (C-3″), 120.7
60.43; H, 4.47.
(C-4″), 117.3 (C-6″), 60.6 (−CH2), 56.0 (−OCH3); anal calcd
2-(2-Methoxyphenoxy)-2-oxoethyl (2E)-3-phenylprop-2- for C18H15O5Cl: C, 62.33; H, 4.32; found C, 62.41; H, 4.39.
enoate (6a): solid; melting point, 76°C–78°C; yield, 85%; Rf,
0.56 (n-hexane:ethyl acetate 3:1); FTIR νmax (1/cm): 2,938 antihyperlipidemic studies
(sp2 C−H), 2,835 (sp3 C−H), 1,719 (C=O ester), 1,599 (C=C An aggregate of 66 mice (Balb C, male, 25–30 g, aged
1
aromatic), 1,164 (C−O, ester); H NMR (CDCl3, δ ppm): 4–6 weeks) was obtained, kept in National Institute of
7.85 (d, J=16.0 Hz, 1H, H-2), 7.58 (dd, J=4.4, 1.6 Hz, Health, and left to be acclimatized for 1 week before
2H, H-2′, 6′), 7.41–7.43 (m, 3H, H-3′, H-4′, H-5′), 7.24 starting the investigation. Animal experiments were per-
(dd, J=6.0, 2.4 Hz, 1H, H-3″), 7.13 (dd, J=6.4, 2.4 Hz, 1H, formed in accordance with the Institute of Laboratory
H-4″), 6.96–6.99 (m, 2H, H-4″, H-5″), 6.56 (d, J=16.0 Hz, Animal Resources, Commission on Life Sciences, National
1H, H-1), 5.04 (s, 2H, −CH2), 3.86 (s, 3H, −OCH3); 13C NMR Research Council (1996), approved by Ethical Committee
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antihyperlipidemic studies of newly synthesized phenolic derivatives
of Riphah Institute of Pharmaceutical Sciences (ref no
REC/RIPS/2017/001). Mice were divided into 11 groups,
each having six mice. Mice were kept in normal laboratory
conditions of temperature 23°C±1°C and ambient humidity
55%±5%. Mice were housed in stainless steel cages individu-
ally and kept in an isolated room. Lights were maintained on
an artificial 12 h light–dark cycle. All mice were weighed
weekly during 6 weeks of experimental period. Animal
care was in accordance with the guidelines established and
approved by the National Institute of Health. Mice were fed
with high-fat diet (HFD) mentioned in Table 1 containing
cholesterol, cholic acid, peanut oil, and normal laboratory diet
for 6 weeks. Standard drug (atorvastatin in this study) and
test compounds from 4a–e and 6a–c were mixed with HFD,
and the composition of the test compounds was 10 mg/kg.
Experimental compounds from 4a–e and 6a–c were mixed
with HFD, and the dose of the test compounds was 10 mg/kg.
Group I received normal laboratory diet, Group II received
HFD, Group III received standard drug (atorvastatin) along
with HFD, and Groups IV–XI received test compounds from
4a–e and 6a–c, respectively, along with HFD. Mice had free
access to feed and water throughout the study. The speci-
fied quantity of test compounds and standard was dissolved
in 1 mL of DMSO and mixed in pellet diet. All mice were
weighed weekly during 6 weeks of experimental period.
The activity was continued for 6 weeks under controlled
laboratory conditions (temperature 23°C±1°C and ambient
humidity 55%±5%).39
histopathological evaluation
Animals were euthanized under ether anesthesia, and the
liver was dissected out immediately and weighed. For
histopathological analysis, the liver and adipose tissues were
fixed in 10% formalin at room temperature. Adipose tissues
used were collected from subcutaneous fat of the abdomen
of mice. The tissue was embedded in paraffin, sectioned into
3–4 µm thickness and mounted on the glass microscope slides
using standard histological techniques. The sections were
stained with hematoxylin–eosin and examined using light
microscopy at 200× magnitudes. These light microscopic
fields were assessed by an image analyzer on each section.40
Docking studies
acquisition and preparation of target protein
Crystal structure of HMG CoA reductase was acquired
from Protein Data Bank as PDB format (PDB ID: 1HWK-
Homosapien) (www.rcsb.org/pdb).41 Crystal structure of
target protein was validated using the MolProbity server.42
Ligands and water molecules were removed from crystal
structure using the Accelrys Discovery Studio, and the energy
of protein was minimized using the UCSF Chimera.43 Active
binding site studies were viewed using the PyMOL (Python
molecular visualization) software.44
Preparation of ligand
Structures of phenolic experimental compounds were drawn
in MarvinSketch tools and were cleaned into 3D using Marvin
Space and were downloaded in the PDB file extension.45
Blood collection and analysis
At the end of the experiment, the animals were anesthetized.
Blood samples were collected from the retro orbital vein. The
serum was separated by centrifugation at 2,500×gfor 15 min at
4°C. The amount of serum triglyceride, total cholesterol, high-
density lipoprotein (HDL) cholesterol, and low-density lipo-
protein (LDL) cholesterol were assayed automatically using an
ADVIA 1650 lipid analyzer (Bayer, Wuppertal, Germany).
Docking run
Docking is a virtual screening tool used to predict in silico
interaction of ligand with enzyme and its possible inhibition.
Both ligand and target protein were converted from PDB to
PDBQT files after the addition of hydrogen and removal of
water molecules. Energies of ligand and enzyme were mini-
mized using the UCSF Chimera. These prepared molecules
were uploaded as ligand, macromolecules in PyRx, a Python-
based AutoDock Vina-based scoring function screening tool,
autogrid dimension were calculated for HMG CoA reductase
(X=147.451, Y=87.546, and Z=3.341), and inhibitor and
target were geometrically optimized and docked.46,47
Table 1 Fat diet composition
Ingredients
Percentage
cholesterol
cholic acid
2
1
Peanut oil
5
Wheat flour
Wheat baran
Dry skimmed milk
Fish meal
common salt
Molasses
27
27
20
15
0.5
2
statistical analysis
For the determination of change in body weight, liver weight,
plasma lipids, and HDL and LDL-cholesterol levels, indi-
vidual mouse was considered as an experimental unit. All
data were analyzed by IBM SPSS 20 package for one-way
Mineral + vitamin mix
0.5
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when fed on similar HFD. In the present study, mice fed
with a HFD demonstrated predictable weight, cholesterol,
triglycerides, and LDL increment with a decrease in HDL
levels after 6 weeks in the hyperlipidemic control group
(HFD group) (Figure 1). After 6 weeks, mice fed with 4a
experimental compound indicated least weight increase, ie,
20% compared with 35% weight increase with atorvastatin.
Even 4e, 6a, and 6b showed comparable weight increment vs
atorvastatin, ie, 36, and 35%, respectively (Figure 2). These
results propose that weight increment in case of 4a was sig-
nificantly (P,0.05) less than reference standard and increase
in mice weight in case of 4e, 6a, and 6b was insignificant
but comparable with reference standard (Figure 2).
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Figure 1 impact of phenolic derivatives on body weight changes on mice supple-
mented with high-fat diet.
Note: Mean weight in each group is given (n=6 for each group).
Abbreviations: nD, normal diet; hFD, high-fat diet; std, reference standard
(atorvastatin).
Plasma lipid profile
As already discussed, an increase in serum cholesterol and
LDL levels may lead to atherosclerosis and ultimately to
myocardial infarction. Raised serum triglycerides (TGs)
have also been reported to have a role in the aggravation of
atherosclerosis.50 Meanwhile various studies have shown
that drugs decreasing serum TG, LDL, and cholesterol levels
reduce the risk of coronary artery diseases.51 Various phenols
including gallic acid and linoleic acid have been found to be
antiatherogenic especially in in vivo green tea extracts reduc-
ing serum TGs and cholesterol as well as stimulating energy
expenditure, fat oxidation, and fecal lipid oxidation.52
In this study, it was observed that all experimental
molecules were able to decrease cholesterol, LDL, and trig-
analysis of variance (ANOVA) testing; post hoc Tukey test
was used with P-value ,0.05 considering as statistically
significant.48
Results and discussion
Body weights
Among dietary factors, an increase in carbohydrate and fats
in diet has been associated with obesity.49 Similarly, few
researchers have reported that the differences among strains
with the ability of HFD to promote obesity have been dem-
onstrated in both rats and mice.32 Some strains of mice such
as A/J and C57BL do develop obesity when fed on HFD. lycerides and increase HDL in hyperlipidemic mice, but in
Meanwhile, SWR mice were resistant to develop obesity particular, 4a showed highly significant reduction in serum
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Figure 2 effect of phenolic derivatives on body weight of mice fed on high-fat diet compared with standard (atorvastatin) and high-fat diet alone.
Note: Mean weight ± sD (n=6 in each group).
Abbreviations: nD, normal diet; hFD, high-fat diet; sTD, reference standard (atorvastatin).
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antihyperlipidemic studies of newly synthesized phenolic derivatives
Table 2 Effect of phenolic derivatives on lipid lowering efficiency in mice (n=6, mean ± sD) compared with normal control, hyperlipidemic
control, and standard drug (atorvastatin)
Group
Dose
(mg/kg)
Cholesterol
(mg/dL)
TG
(mg/dL)
LDL
(mg/dL)
HDL
(mg/dL)
normal control
–
260±7.45
386±12.93
165±7.54
150±4.79*
171±4.47
176±6.41
179±7.49
236±6.80
148±4.38*
150±3.60**
156±3.65
127±5.10
132±4.60
90±4.09
84±4.09
86±5.10
86±4.43
88±5.68
95±4.16
80±2.48**
85±3.68
81±3.31**
128±6.90
317±7.09
89±4.73
64±4.69**
54±4.56**
57±4.35
61±3.89
88±4.02
63±4.00
56±4.85
58±2.09
82±4.14
66±3.52
84±2.85
96±4.35*
71±5.49
69±4.64
70±2.82
67±4.16
68±8.37**
77±4.04**
80±3.46**
hyperlipidemic control
–
atorvastatin
10
10
10
10
10
10
10
10
10
4a
4b
4c
4d
4e
6a
6b
6c
Notes: *P,0.01 vs standard drug (atorvastatin). **P,0.05 vs standard drug (atorvastatin).
Abbreviations: Tg, triglyceride; lDl, low-density lipoprotein; hDl, high-density lipoprotein.
cholesterol compared to standard with P,0.01, highly sig- Fatty liver initially starts with simple steatosis, which later
nificant increase in HDL compared to standard with P,0.01, may develop into advanced fibrosis and cryptogenic cir-
and significant reduction in LDL compared to standard rhosis via steatohepatitis and ultimately to hepatocellular
with P,0.05 (Table 2). In order to unveil dose-dependent carcinoma.53 Obesity is the single main causative factor in
effect of 4a, it was evaluated in same hyperlipidemic mice the development of fatty liver both in children and adults.54
model with three different doses, ie, high dose (10 mg/kg), In our current investigation, 4a fed group of mice showed
medium dose (5 mg/kg) and low dose (2.5 mg/kg). Effect of significant histopathological changes in liver sections under
4a reduced with decrease in dose when compared with high microscopic evaluation (Figure 3). A small loss of nucleus
dose 10 mg/kg, and this decrease was statistically significant and membrane disruption was observed when compared with
(Table 3). In vivo outcomes clearly showing the superiority the normal diet (ND) group, but no fat droplet was observed
of antihyperlipidemic potential of 4a compared with other in case of 4a group when compared with the HFD group.
phenolic derivatives and even compared to standard ator- Histology of adipocytes showed that difference in size was
vastatin. Furthermore, after 42 days of treatment none of evidently less in case of 4a group compared with the HFD
the mice showed any kind of morbidity or mortality, which group (Figure 3).
further advocates that all experimental phenolic compounds
were well tolerated and were fairly safe during the whole Docking studies
experiment. These astounding outcomes establish the com- The in silico docking studies were performed to determine
mendable and plausible antihyperlipidemic potential of our the binding affinity of the synthesized compounds with target
experimental natural phenolic derivatives.
protein. It has been reported that docking is an effective pre-
dictive tool regarding possible interactions of ligands with
target protein.55 It further enables illustration of the possible
histopathological examination
Fatty liver is gaining importance in investigations and now atomic interactions between the particular functional group
is known to be most prevalent cause of abnormal liver. of a ligand in the active binding site of protein. To date, the
Table 3 Dose-dependent antihyperlipidemic effect of 4a (n=6, mean ± sD)
Group
Dose (mg/kg)
Cholesterol
(mg/dL)
TG
(mg/dL)
LDL
(mg/dL)
HDL
(mg/dL)
normal control
–
266±9.90
121±4.12
129±4.60
94±4.09
90±4.13*
87±3.89*
124±3.79
314±7.09
71±4.73*
68±3.35
82±3.66
68±3.52
60±2.85
76±3.64*
96±4.12**
hyperlipidemic control
–
380±10.84
191±7.54*
171±5.41**
148±4.21**
4a
4a
4a
2.5 (low dose)
5.0 (medium dose)
10 (high dose)
61±3.61**
Notes: *P,0.05 vs hyperlipidemic group. **P,0.01 vs hyperlipidemic group.
Abbreviations: Tg, triglyceride; lDl, low-density lipoprotein; hDl, high-density lipoprotein.
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aqeel et al
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Figure 3 histopathological evaluation of liver section slides: (A) 4a group; (B) 6a group; (C) 6b group; (D) standard drug group; and (E) hyperlipidemic control.
most successful strategy to control hyperlipidemic disor- of HMG CoA reductase (Figure 7). In contrast, 6a interacts
with Asn658 via hydrogen bond at a distance of 3.06 Å and
with aspartate 767 at a distance of 4.33 Å (Figure 8). These
interaction patterns give a strong impression that synthesized
phenolic derivatives possess good inhibitory potential against
HMG CoA reductase.
ders has been to inhibit HMG CoA reductase. This enzyme
catalyzes the conversion of HMG CoA to mevalonic acid,
ultimately producing cholesterol.56 HMG CoA reductase
model characterized with X-ray diffraction at a resolution of
2.22 Å was downloaded from Protein Data Bank (Figure 4).
The structure was validated by using the Ramachandran plot,
which demonstrated that .95.7% residues were lying in
favorable region and 99.4% residues were lying in allowed
region of plot (Figure 5). The experimental compounds
(phenolic derivatives) tightly entrapped in active bind-
ing sites of HMG CoA reductase enzyme were visualized
through Discovery Studio 4.1 (Figure 6). In the present
study, we compared docking outcomes of our experimental
phenolic compounds with atorvastatin (most widely used
US Food and Drug Administration-approved statin), 4a and
6a showed even better binding affinity (−8.3 and −7.9 kcal/
mol, respectively) energy values compared with atorvastatin
(−7.8 kcal/mol) (Table 4). Analysis of residual interaction
between inhibitor and target enzyme in active binding site
revealed that the compound 4a interacted with Val805 (at
a distance of 1.89 Å), Met656 (at a distance of 2.96 Å),
Thr558 (at a distance of 2.70 Å), and Glu559 (at a distance
of 2.20 Å) via hydrogen bonding in the active binding site
Conclusion
The synthesis of some phenolic derivatives 4a–e and 6a–c
was successfully accomplished starting with simplest pre-
cursors. The results of in silico docking studies showed that
Figure 4 Three dimensional structure of homosapien hMg coa reductase down-
loaded from Protein Data Bank with PDB iD 1hWK bound to ligands.
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antihyperlipidemic studies of newly synthesized phenolic derivatives
Table4Bindingaffinity/energyvaluesagainstHMGCoAreductase
(PDB iD: 1hWK)
Ligand
Binding affinity
RMSD/UB
RMSD/LB
4a
−8.3
−7.1
−7.1
−6.5
−6.4
−7.9
−7.4
−6.3
−7.8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ꢀD
ꢁD
4b
4c
4d
ꢁE
4e
6a
6b
6c
Figure 5 The phenolic derivatives 4a, 6a, and 6b tightly entrapped in active binding
sites of hMg cOa reductase enzyme visualized through Discovery studio 4.1.
atorvastatin
Note: Lesser binding affinity energy means less free energy of the interaction
complex, ultimately illuminating that ligand–target complex is more stable.
Abbreviations: hMg coa, 3-hydroxy-3-methylglutaryl-coenzyme a; rMsD, root
mean square deviation; UB, upper bound; lB, lower bound.
compounds 4a and 6a exhibited maximum binding affinity
with target protein having binding energies −8.3 and −7.9 kcal,
respectively. Compound 4a interacts with amino acids Val805 activity with minimum weight increment (20%) compared to
having distance 1.89 Å and Met656, Thr558, and Glu559 hav- standard atorvastatin (35% weight increment). The synthe-
ing bonding distances 2.96, 2.70, and 2.20 Å, respectively. sized compounds were nontoxic and well tolerated because
Compound 4a exhibited excellent in vivo antihyperlipidemic none of the mice were found to suffer from any kind of
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Figure 6 residual interaction of 4a in binding pocket of hMg cO a reductase.
Notes: (A) shows three dimensional docking in active binding site. (B) shows two
dimensional interaction patterns. The legend inset represents the type of interaction
between the ligand atoms and the amino acid residues of the protein. compound 4a
interacts via hydrogen bonding with Val 805 (at a distance of 1.89a°), Met 656 (at
a distance of 2.96a°), Thr 558 (at a distance of 2.70a°), and glu 559 (at a distance
of 2.20a°) amino acids.
Figure 7 residual interaction of 6a in binding pocket of hMg cO a reductase.
Notes: (A) shows three dimensional docking in active binding site. (B) shows two
dimensional interaction patterns. The legend inset represents the type of interaction
between the ligand atoms and the amino acid residues of the protein. compound 6a
interacts via hydrogen bonding with asn 658 (at a distance of 3.06a°) and with asP
767 (at a distance of 4.33a°) amino acids.
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aqeel et al
180
135
90
Dovepress
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–45
–90
–135
–180
–180 –135 –90 –45
0
45
90
135 180
Phi
Figure 8 ramachandran plot of protein structure of target protein (hMg cOa
reductase).
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4a may act as a lead structure for the design and development
of more potent antihyperlipidemic drugs.
Disclosure
The authors report no conflicts of interest in this work.
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