Benzoflavones as cholesterol esterase inhibitors: Synthesis, biological evaluation and docking studies

Article abstract of DOI:10.1016/j.bmcl.2017.01.020

A library of forty 7,8-benzoflavone derivatives was synthesized and evaluated for their inhibitory potential against cholesterol esterase (CEase). Among all the synthesized compounds seven benzoflavone derivatives (A-7, A-8, A-10, A-11, A-12, A-13, A-15) exhibited significant inhibition against CEase in in vitro enzymatic assay. Compound A-12 showed the most promising activity with IC₅₀value of 0.78?nM against cholesterol esterase. Enzyme kinetic studies carried out for A-12, revealed its mixed-type inhibition approach. Molecular protein–ligand docking studies were also performed to figure out the key binding interactions of A-12 with the amino acid residues of the enzyme's active site. The A-12 fits well at the catalytic site and is stabilized by hydrophobic interactions. It completely blocks the catalytic assembly of CEase and prevents it to participate in ester hydrolysis mechanism. The favorable binding conformation of A-12 suggests its prevailing role as CEase inhibitor.

Full text of DOI:10.1016/j.bmcl.2017.01.020

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzoflavones as cholesterol esterase inhibitors: Synthesis, biological
evaluation and docking studies
Harbinder Singh a, Jatinder Vir Singh a, Manish K. Gupta b, Palwinder Singh c, Sahil Sharma a,
,
⇑
Kunal Nepali a, , Preet Mohinder S. Bedi a,
⇑
⇑
a Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab 143005, India
b Lloyd Institute of Management and Technology, Greater Noida, UP, India
c Department of Chemistry, Guru Nanak Dev University, Amritsar, Punjab 143005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of forty 7,8-benzoflavone derivatives was synthesized and evaluated for their inhibitory poten-
tial against cholesterol esterase (CEase). Among all the synthesized compounds seven benzoflavone
derivatives (A-7, A-8, A-10, A-11, A-12, A-13, A-15) exhibited significant inhibition against CEase in
in vitro enzymatic assay. Compound A-12 showed the most promising activity with IC50 value of
0.78 nM against cholesterol esterase. Enzyme kinetic studies carried out for A-12, revealed its mixed-type
inhibition approach. Molecular protein–ligand docking studies were also performed to figure out the key
binding interactions of A-12 with the amino acid residues of the enzyme’s active site. The A-12 fits well at
the catalytic site and is stabilized by hydrophobic interactions. It completely blocks the catalytic assem-
bly of CEase and prevents it to participate in ester hydrolysis mechanism. The favorable binding confor-
mation of A-12 suggests its prevailing role as CEase inhibitor.
Received 9 August 2016
Revised 30 December 2016
Accepted 9 January 2017
Available online xxxx
Keywords:
Benzoflavone
Baker Venkataraman rearrangement
Cholesterol esterase inhibition
Enzyme kinetics
Ó 2017 Elsevier Ltd. All rights reserved.
Docking studies
Cholesterol is a vital component of cell membrane and pos-
sesses many physiological functions. The greatest percentage of
cholesterol is used in cytoplasm for bile acid synthesis.1 Hyperc-
holesterolemia produced either by cholesterol feeding or by
cholesterol-free, purified diets (‘‘endogenous” Hypercholes-
terolemia) results in the accumulation of cholesterol in adipose tis-
sue. As there is a well-established link between plasma cholesterol
level and coronary artery disease, the reduction of cholesterol level
in plasma, particularly in low density lipoprotein (LDL) lowers the
risk of cardiovascular events.2 The contribution of elevated plasma
cholesterol specifically, LDL-cholesterol, to other diseases includ-
ing cancer, obesity, and diabetes has made control of plasma
cholesterol a major health aim.3
treating hypercholesterolemia and associated diseases such as
coronary heart disease.6
From the last decade, there are several classes of potent
CEase inhibitors have been developed,7 so far, including
6-chloro-2-pyrones,8 thieno[1,3]-oxazin-4-ones,9 carbamates,10
aryl phosphates and phosphonates,11 chloroisocoumarins,12
phosphaisocoumarins,13
flavonoids,14
thiazolidinediones,5
phosphorylated
2-(1H-Indol-3-yl)-4-phenylquinolines15 and 3-phenyl substituted
1,3,4-oxadiazol-2(3H)-ones16 (Fig. 1). However, most of these
inhibitors are not highly selective and they could also inhibit other
serine hydrolases, such as acetylcholinesterase (AChE), butyryl-
cholinesterase (BChE), Pseudomonas species lipase (PSL), chy-
motrypsin (CT) and trypsin.14
Pancreatic cholesterol esterase is the member of a/b hydrolase
family of proteins which catalyses the hydrolysis of dietary choles-
terol ester into free cholesterol in the lumen of small intestine.4 It
is also thought that the transport of cholesterol micelles to entero-
cytes is performed by this enzyme.5 As the combined role of CEase
in the absorption and transport of cholesterol, its inhibition is
important by the development of novel moieties which helps in
Flavonoids, including flavones, flavonols, isoflavones and fla-
vanones, are a large class of polyphenolic compounds widely dis-
tributed in herbs and foods of plant origin, and exhibit
diversified biological activities, such as antioxidant, anti-prolifera-
tive, anti-tumor, anti-microbial, estrogenic, acetyl cholinesterase,
anti-inflammatory activities and are also used in cancer, cardiovas-
cular disease, neurodegenerative disorders and enzyme inhibi-
tion.17 Among them, the flavones have been considerably
explored due to their ability to modulate several enzyme systems
involved in a number of diseases.14
⇑
Corresponding authors.
E-mail address: ss.gq2009@gmail.com (S. Sharma).
http://dx.doi.org/10.1016/j.bmcl.2017.01.020
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Singh H., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.01.020

2
H. Singh et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
S
N
NR2
X
OR1
H
N
O
O
O
OR2
P
n
n
R
O
O
O
Cl
O
O
O2N
O2N
n = 0, 1; X = O, CH2
R = Me, Et
thieno[1,3]-oxazin-4-ones
R1,R2 = Alkyl
aryl phosphates
n =0-4
R = Alkyl
6-chloro-2-pyrone
carbamates
Cl
O
O
O
R
O
H or OY
X
H
N
YO
n
m
R
N
H
S
O
X
NH
O
OH
O
N
Ar
m = 0-3; n = 1,2; X = H, NO2
Y = OP(O)(OEt)2
X = S; R = CH2OCH2Ph (SerOBn)
O
R = NO2 ; Ar = Indole-3-yl
phenylquinolines
phosphorylated flavonoids
thiazolidinedione
chloroisocoumarins
O
Y
R2
YO
X
O
O
Cl
O
P
H or OY
O
S
Z
X
O
OR1
O
NH
N
Ar
Y = OP(O)(OR)2
R
N
N
O
X = H, MeO, Cl; Y = H, Cl
R1 = H, Et, Me; R2 = alkyl, aryl
phosphaisocoumarins
Z = H, OH, OP(O)(OR)2
R = Me, Et
O
NH
2-(1H-Indol-3-yl)-4-phenylquinolines
3-phenyl substituted 1,3,
4-oxadiazol-2(3H)-ones
X = O, S
phosphorylated flavonoids
thiazolidinediones
Fig. 1. Reported CEase inhibitors.
In view of various medicinal attributes of flavones, we here
multiplet at 7.26–7.36 ppm). Compound 3 was then cyclized by
treatment with sulphuric acid to yield the desired benzoflavone
(A-1).18 All the reactions proceeded smoothly with diverse ben-
zoylchlorides (Table 1) and products were obtained in good yields.
No Retro-Diels fragmentation was observed for benzoflavones in
the mass spectrum. The structures of the synthesized compounds
were elucidated by 1H NMR, 13C NMR, HRMS and Elemental
Analysis. All spectral data were in accordance with assumed
structures (Supplementary Material).
All the synthesized benzoflavones were evaluated for their inhi-
bitory potential against CEase enzyme using spectrophotometric
assay as described in the literature.15 Compounds with CEase
enzyme inhibition of more than 60% at 50 nM were further evalu-
ated at concentrations of 1, 5, 10 and 25 nM in order to calculate
their IC50 values (Table 1).
report for the first time, benzoflavone derivatives as potent CEase
inhibitors. Thus, in the present study, various benzoflavone analogs
were synthesized and evaluated for their inhibitory potential
against CEase enzyme using spectrophotometric assay. The type
of inhibition and the interactions of the most potent inhibitor with
CEase enzyme had also been figured out.
Benzoflavones were synthesized via Scheme 1. a-Naphthol was
subjected to fries rearrangement and the product (1) was benzoy-
lated using various substituted benzoylchlorides to obtain 2.
Product 2 was then subjected to Baker Venkataraman rearrange-
ment. The Baker Venkataraman rearranged product (3) existed in
enol form (confirmed by the appearance of singlets for two D2O
exchangeable protons at 15.2 and 13.68 ppm along with the vinylic
proton to carbonyl which appeared as a merged signal in a
OH
a
OH
O
CH3
1 (74%)
b
O
O
A-1 (89%)
d
O
O
O
OH
O
O
OH OH
O
c
CH3
C
C
H2
H
2 (80%)
3 (72%)
Scheme 1. Synthesis of benzoflavone derivative. Reagents and conditions: (a) MW, ZnCl2, CH3COOH, 20 min; (b) benzoyl chloride, pyridine, stirring rt, 1 h; (c) KOH, pyridine,
warm, 30 min; and (d) a drop of conc. H2SO4, CH3COOH, reflux, 1 h.
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H. Singh et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 1
Various substituted benzoflavones with their IC50 value against CEase enzyme.
R2
R5
R1
R3
R4
O
O
Code
R1
R2
R3
H
OCH3
OCH3
OCH3
H
OCF3
H
H
F
H
F
H
H
F
H
H
H
H
Br
H
I
H
NO2
H
CH3
CF3
H
H
H
F
CF3
R4
R5
IC50 (nM)
A-1
A-2
A-3
A-4
A-5
A-6
A-7
A-8
H
H
H
OCH3
H
H
F
H
H
F
H
H
OCH3
H
OCF3
H
H
F
H
H
H
F
H
F
Cl
Cl
H
Br
H
H
H
NO2
H
NO2
H
H
CF3
H
CF3
H
H
H
H
F
CF3
H
CH2Cl
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
F
47.80
NA
NA
NA
NA
NA
3.22
5.24
20.10
1.99
1.43
0.78
6.81
14.08
6.99
10.85
11.01
26.09
28.51
16.12
34.15
25.14
37.33
30.04
NA
NA
NA
NA
NA
39.08
38.98
38.89
41.28
42.99
43.03
39.23
40.03
NA
A-9
A-10
A-11
A-12
A-13
A-14
A-15
A-16
A-17
A-18
A-19
A-20
A-21
A-22
A-23
A-24
A-25
A-26
A-27
A-28
A-29
A-30
A-31
A-32
A-33
A-34
A-35
A-36
A-37
A-38
A-39
A-40
F
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
F
H
H
Cl
Br
H
H
I
H
H
H
H
H
H
H
CF3
H
CF3
F
H
H
H
H
H
H
H
H
H
H
NO2
H
H
H
H
CF3
H
H
H
F
F
H
H
H
F
CF3
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
CH2Cl
H
N(CH3)2
H
OCOCH3
H
NA
NA
OCOCH3
Table 1 revealed an interesting structure activity relationship for
benzoflavone derivatives as cholesterol esterase inhibitors. Any
substitution on phenyl ring (at 2nd position of benzo[h]chromen-
4-one nucleus) significantly influences the cholesterol esterase
inhibitory activity. Placement of halogens on this phenyl ring con-
siderably increases the potency against cholesterol esterase enzyme
in the decreasing order with an increase in the size of the halogen.
This pattern of activity is in full agreement with the docking studies
which suggest that compound with difluorophenyl ring fits well at
the catalytic site and is stabilized by H-bonds, polar and van der
Walls interactions and completely blocks the catalytic assembly of
CEase by preventing it to participate in ester hydrolysis mechanism.
Placement of other deactivating groups (nitro, cholomethyl, tri-
floromethyl and acetoxy) on this phenyl ring favors the inhibitory
activity, whereas, substitution of any activating group (dimethy-
lamino, methoxy, methyl and trifloromethoxy) on this phenyl ring
F
R
O
O
Overall preference order of the substituent for the inhibition of cholesterol esterase enzyme:
R = -F > -Cl > -Br > -I > -NO2 > -CH2Cl > -CF3 > -OCOCH3 > -H > -OCF3 > -CH3 > -OCH3 > -N(CH3)2
ACTIVATING GROUPS
F
O
O
BENZOFLAVONE
MOST POTENT COMPOUND
A-12
DEACTIVATING GROUPS
IC50 = 0.78 nM (CEase)
Fig. 2. Structure activity relationship.
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4
H. Singh et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
disfavors the inhibitory potential against the cholesterol esterase
enzyme. Therefore, the overall preference order of the substituent
on phenyl ring at 2nd position of benzo[h]chromen-4-one nucleus
for the inhibition of cholesterol esterase enzyme is as follows:
AF > ACl > ABr > AI > ANO2 > ACH2Cl > ACF3 > AOCOCH3 > AH >
AOCF3 > ACH3 > AOCH3 > AN(CH3)2 (Fig. 2).
The most potent compound among the series (A-12) was fur-
ther investigated for the type of inhibition and enzyme kinetic
study was carried out.17 The Lineweaver-Burk plot (Fig. 3) revealed
that the compound A-12 was a mixed-type CEase inhibitor. The
pattern of graph shows that it is a form of mixed inhibition sce-
nario. The Km, Vmax and slope are all affected by the inhibitor.
The inhibitor has increased the Km and slope (Km/Vmax) while
decreasing the Vmax. Moreover carefully observing the Fig. 3 it
was found that intersecting lines on the graph converge to the left
of the y-axis and above the x-axis which indicates that the value of
a
(a constant that defines the degree to which inhibitor binding
affects the affinity of the enzyme for substrate) is greater than 1.
Fig. 3. Lineweaver-Burk plot of A-12.
Fig. 4. (a) Docked conformation of A-12 at the catalytic site of hCEase (A-12: carbon, oxygen and fluorine atoms are shown in green, red and yellow respectively); (b) 2D
depiction of various catalytic site residues surrounding A-12 (figure generated by LIGPLOT21); (c) H-bonds between Gly107, Ala108 and Ala195; polar interaction with Ser194
and face-to-face pai-pai stacking interaction with Trp227 are shown (only hydrogens which are involved in H-bond interactions are shown in white color).
Please cite this article in press as: Singh H., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.01.020

H. Singh et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
This confirms that the inhibitor preferentially interacts to the free
Supplementary material
enzyme and not to the enzyme substrate complex.
In order to look into the interactions between the compound A-
12 and the enzyme, molecular docking of the compound in the
active site of the human CEase (PDB entry: 1F6W; Resolution
2.3 Å) was performed. The active site apparatus of hCEase consist
of a catalytic triad and an oxyanion hole.19 The catalytic triad is
made of Ser194, Asp320, and His435 residues and serves as general
acid-base and nucelophilic catalytic entity along with an oxyanion
hole consisting of Gly107, Ala108, and Ala195 residues.19,20 The
hydroxyl group of Ser194 acts as nucleophile and is necessary for
the hydrolytic reaction. The serine lipases and serine proteases also
possess the Ser-Asp-His catalytic triad and share the catalytic
mechanism with hCEase. In the present docking study, the hCEase
residues within the radius of 10 Å around the hydroxyl function of
Ser194 were defined to form the active site of the enzyme.20
The A-12 fits well at the catalytic site and is stabilized by H-
bonds, polar and van der Walls interactions (Fig. 4a–c). Interest-
ingly, the Gly107, Ala108, and Ala195 residues of oxyanion hole
were involved in H-bond interaction with the carbonyl oxygen of
ring C (H- bond acceptor; d = 1.69 to 2.35 Å). The three H-bonds
showed their significance in the tight binding of A-12 with hCEase.
The nucleophilic hydroxyl group of Ser194 is oriented parallel to
the carbonyl group of inhibitor (Ring C) and involved in polar/ionic
interaction with sp2 carbon atom of carbonyl group (d = 2.62 Å).
Involvement of carbonyl group in resonance with the double bond
of ring C may obstruct the nucleophilic attack of serAOH on car-
bonyl carbon atom. The ring A, B and C were stabilized by van
der Waals interaction with Ile323, Phe324 and His435.21 The diflu-
orophenyl (ring D) gets positioned in a hydrophobic cavity created
by Trp227, Leu282, Val285 and Leu392 residues. The ring D dis-
played face-to-face pai-pai stacking interaction with Trp227
(d = 3.89 Å). The study showed that the A-12 completely blocks
the catalytic assembly of hCEase. Its binding with the oxyanion
hole prevents it to participate in ester hydrolysis mechanism. The
favorable binding conformation of A-12 suggests its prevailing role
as hCEase inhibitor.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.01.
020.
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NMR, HRMS and Elemental Analysis. All the synthesized com-
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activity. Compound A-12 was found to be most potent enzyme
inhibitor among the series with IC50 value of 0.78 nM. Enzyme
kinetic study confirmed that the inhibitor A-12 preferentially
interacts to the free enzyme and not to the enzyme substrate com-
plex (mixed type inhibition). Docking study suggested that the
compound A-12 fits well in the binding pocket of cholesterol ester-
ase enzyme and completely blocks the catalytic assembly of CEase
by preventing it to participate in ester hydrolysis mechanism.
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Conflict of interest
The authors declared that there is no conflict of interest.
Please cite this article in press as: Singh H., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.01.020