5,6-Benzoflavones as cholesterol esterase inhibitors: Synthesis, biological evaluation and docking studies

Article abstract of DOI:10.1039/c7md00565b

In a continued effort to develop potent cholesterol esterase (CEase) inhibitors, a series of 5,6-benzoflavone derivatives was rationally designed and synthesized by changing the position of the benzene ring attached to the flavone skeleton in previously reported 7,8-benzoflavones. All the synthesized compounds were checked for their inhibitory potential against cholesterol esterase (CEase) using a spectrophotometric assay. Among the series of forty compounds, seven derivatives (B-10 to B-16) exhibited above 90 percent inhibition against CEase in an in vitro enzymatic assay. Compound B-16 showed the most promising activity with an IC50 value of 0.73 nM against cholesterol esterase. To determine the type of inhibition, enzyme kinetic studies were carried out for B-16, which revealed its mixed-type inhibition approach. Moreover, to figure out the key binding interactions of B-16 with the amino acid residues of the enzyme's active site, molecular protein-ligand docking studies were also performed. B-16 completely blocks the catalytic assembly of CEase and prevents it from participating in the ester hydrolysis mechanism. The favorable binding conformation of B-16 suggests its prevailing role as a CEase inhibitor. Overall, the study showed that the cisorientation of ring A with respect to the carbonyl group of ring C is responsible for the potent CEase inhibitory activity of the newly synthesized compounds.

Full text of DOI:10.1039/c7md00565b

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Page 1 of 35
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DOI: 10.1039/C7MD00565B
5,6ꢀBenzoflavones as cholesterol esterase inhibitors: Synthesis, biological evaluation and
docking studies
Jatinder V. SinghЇ,a, Anumeet KaurЇ,a, Kavita Bhagata, Manish K. Guptab, Manwinder Singhc,
Harbinder Singh*,a, Preet Mohinder S. Bedi*,a
aDepartment of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab
143005, India
bTERI-Deakin Nanobiotechnology Centre, The Energy and Resources Institute, TERI
Gram, Gual Pahari, Gurugram, Haryana 122001, India
cDepartment of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab
144411, India
Corresponding author: 1) Harbinder Singh
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India
Mob no. +919463148367
2) Dr. P.M.S. Bedi
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India
Mob no. +919815698249
Ї These authors contributed equally to this work.
1

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Abstract: In continuous efforts to develop potent cholesterol esterase (CEase) inhibitors, a series
of 5,6ꢀbenzoflavone derivatives was rationally designed and synthesized by changing the
position of benzene ring attached to flavone skeleton in previously reported (7,8ꢀbenzoflavones).
All the synthesized compounds were checked for their inhibitory potential against cholesterol
esterase (CEase) by using spectrophotometric assay. Among the series of forty compounds,
seven derivatives (B-10 to B-16) exhibited above 90 percent inhibition against CEase in in-vitro
enzymatic assay. Compound B-16 showed the most promising activity with IC50 value of 0.73
nM against cholesterol esterase. To determine the type of inhibition, enzyme kinetic studies were
carried out for B-16, which revealed its mixedꢀtype inhibition approach. Moreover, to figure out
the key binding interactions of B-16 with the amino acid residues of the enzyme’s active site,
molecular protein–ligand docking studies were also performed. The B-16 completely blocks the
catalytic assembly of CEase and prevents it to participate in ester hydrolysis mechanism. The
favorable binding conformation of B-16 suggests its prevailing role as CEase inhibitor. The
overall study stated that the cisꢀorientation of Ring A with respect to carbonyl group of Ring C is
responsible for potent CEase inhibitory activity of newly synthesized compounds.
Keywords: 5,6-benzoflavones, Baker Venkataraman rearrangement, cholesterol esterase
inhibition, enzyme kinetics, docking studies.
2

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Introduction:
Cholesterol is a vital component of cell membrane which possesses many physiological
functions. Plasma cholesterol level is linked to many diseases such as coronary artery disease,
cancer, obesity, and diabetes, which is a major health aim worldwide.1 In this respect, control of
the cholesterol level has gained much attention.
Pancreatic cholesterol esterase (CEase) is an important serine hydrolase that plays imperative
role in the absorption of dietary cholesterol. The transport of cholesterol micelles to enterocytes
is also performed by this enzyme.2 As its dual role in absorption and transportation, the
inhibition of CEase is very important and thus a potential approach to treat hypercholesterolemia
and atherosclerosis.3 In the recent past, several classes of potent CEase inhibitors have been
developed including aryl phosphates and phosphonates,4 carbamates,5 chloroisocoumarins,6 6ꢀ
chloroꢀ2ꢀpyrones,7 2ꢀ(1HꢀIndolꢀ3ꢀyl)ꢀ4ꢀphenylquinolines,8 3ꢀphenyl substituted 1,3,4ꢀoxadiazolꢀ
2(3H)ꢀones,9 phosphaisocoumarins,10 phosphorylated flavonoids,11 thiazolidinediones,2 and
thieno[1,3]ꢀoxazinꢀ4ꢀones12 (Figure 1). However, most of the reported inhibitors are not highly
selective for CEase and could also inhibit other serine hydrolases, such as acetylcholinesterase
(AChE), butylcholinesterase (BuChE), Pseudomonas species lipase, chymotrypsin and
trypsin.3,13ꢀ16 One of the major reasons behind their inꢀselectivity is that all serine enzymes share
the similar catalytic triad of SerꢀHisꢀAsp (Glu) and mechanism of acylationeꢀdeacylation.17
Inspired from various biological attributes of flavones, we have recently reported a series of 7,8ꢀ
benzoflavone derivatives as potential CEase inhibitors.18 Among this whole series of
compounds, twenty seven molecules were found to exhibit above 60% inhibition against CEase
enzyme with the IC50 values ranging from 0.78 to 47.80 nM. Compound A was the most
promising molecule among the series of reported molecules (Figure 2). Prompted from these
significant in-vitro results, a new series of the compounds have been designed by simply
changing the orientation of Ring A, from trans- to cis-configuration with respect to carbonyl
group of Ring C (Figure 2). Docking study demonstrated that, Ring A gets positioned in a wellꢀ
defined cavity formed by Gly106, Gly107, GLu193, Ser194 and Ala19 within the active site of
CEase. While, Ring A of 7,8ꢀbenzoflavone derivatives only surrounded by Ile323 and His435
amino acid residues. We concluded from these particular findings that the cisꢀorientation of Ring
A with respect to carbonyl group of Ring C might be responsible for good CEase inhibitory
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activity of newly designed compounds. In present study, the designed compounds were
synthesized in order to evaluate the inhibitory potential against CEase enzyme by using in-vitro
spectrophotometric assay. The type of inhibition and the various types of interactions of the most
potent inhibitor with CEase had also been figured out.
Results and Discussion:
5,6ꢀbenzoflavone derivatives were synthesized via Scheme 1. βꢀnaphthol was subjected to fries
rearrangement and the product (1) was benzoylated using benzoylchloride to obtain 2. Product 2
was then subjected to Baker Venkataraman rearrangement. The Baker Venkataraman rearranged
product (3) existed in enol form (confirmed by the appearance of singlet for two D2O
exchangeable protons at 11.35 ppm along with the vinylic proton to carbonyl which appeared as
a merged signal in a multiplet at 7.26ꢀ7.36 ppm). Compound 3 was then cyclized by treatment
with sulphuric acid to yield the desired 5,6ꢀbenzoflavone (B-1). Some of the synthesized
molecules are previously reported by various research groups (mentioned below) but most of the
molecules reported herein are novel to the best of our knowledge. All the reactions proceeded
smoothly with diverse benzoylchlorides (Table 1) and products were obtained in good yields. No
RetroꢀDiels fragmentation was observed for derivatives in the mass spectrum. The structures of
1
13
the synthesized compounds were elucidated by H NMR, C NMR and MASS spectrum. All
spectral data were in accordance with assumed structures.
In vitro screening:
CEase inhibition assay of all the synthetics was performed by using spectrophotometric assay as
described in the literature8 and the results were compared with potent cholesterol esterase
inhibitor (PF) reported by Wei Y et al.11
In-vitro results showed that among series of forty compounds, nine compounds exhibited
significant percentage inhibition against CEase enzyme (above 90 % inhibition). Compound B-
16 was found to be endowed with most potent percentage inhibition against CEase with 100%
inhibition. Careful examination of Table 1 revealed an interesting structure activity relationship
similar to the reported benzoflavone derivatives (7,8ꢀbenzoflavones) as CEase inhibitors. Any
substitution on Ring D [phenyl (at 2nd position of 5,6ꢀbenzoflavone)] significantly influences the
cholesterol esterase inhibitory activity. Placement of halogen atoms on this phenyl ring
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considerably increases the potency against cholesterol esterase enzyme. It has also been cleared
that as the size of halogen atom increases, inhibitory potency significantly decreases. Ring D
(phenyl) with deactivating groups (nitro, cholomethyl, trifloromethyl and acetoxy) favors the
inhibitory activity whereas, Ring D with activating groups (dimethylamino, methoxy, methyl and
trifloromethoxy), disfavors the activity against CEase inhibition. Thus, the overall preference
order of the substituent on phenyl ring at 2nd position of 5,6ꢀbenzoflavone nucleus for the
inhibition of cholesterol esterase enzyme is as follows: ꢀCl > ꢀF > ꢀBr > ꢀI > ꢀNO2 > ꢀCH2Cl > ꢀ
CF3 > ꢀOCOCH3 > ꢀH > ꢀOCF3 > ꢀCH3 > ꢀOCH3 > ꢀN(CH3)2 (Figure 3). Compounds with
CEase enzyme inhibition of more than 60% at 50 nM were further evaluated at four different
concentrations (1, 5, 10 and 25 nM) in order to calculate their IC50 values. Exceptionally, the IC50
value of unsubstituted compound B-1 was also calculated to better describe the structure activity
relationship. The IC50 value of most potent compound B-16 (0.73 nM) was found comparable to
that of literature value of phosphorylated flavonoid (PF, IC50 = 0.72 nM)11 (Table 1). Most
excitingly, the whole series was found to be more active as compare to the previous series of
compounds (7,8ꢀbenzoflavones) with the IC50 values ranging from 0.72ꢀ31.35. Moreover, the
experimentation for the evaluation to check the specificity of most potent compound towards the
cholesterol esterase enzyme is also under progress.
Enzyme kinetics study:
The most potent compound among the series (B-16) was further investigated for the type of
inhibition by performing enzyme kinetic studies.19 The LineweaverꢀBurk plot (Figure 4)
revealed that the compound B-16 was a mixedꢀtype CEase inhibitor. The pattern of graph shows
that it is a form of mixed inhibition scenario. 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 Figure 4 it was found that intersecting lines on the graph
converge to the left of the yꢀaxis and above the xꢀaxis which indicates that the value of α (a
constant that defines the degree to which inhibitor binding affects the affinity of the enzyme for
substrate) is greater than 1. This confirms that the inhibitor preferentially binds to the free
enzyme and not to the enzyme substrate complex.
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Docking studies:
Various types of the binding interactions of most potent compound B-16 within the active site of
human cholesterolesterase enzyme (hCEase), were also streamlined by using molecular
modeling studies. The active site apparatus of hCEase consist of a catalytic triad and an oxyanion
hole.20 The catalytic triad is made of Ser194, Asp320, and His435 residues and serves as general
acidꢀbase and nucleophilic catalytic entity along with an oxyanion hole consisting of Gly107,
Ala108, and Ala195 residues.20,21 The hydroxyl group of Ser194 acts as nucleophile and is
necessary for the hydrolytic reaction. The serine lipases and serine proteases also possess the
SerꢀAspꢀHis 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.21
The B-16 fits well at the catalytic site and stabilized by Hꢀbonds, polar and van der Walls
interactions (Figure 5 and 6). Interestingly, the Gly107, Ala108, and Ala195 residues of
oxyanion hole were involved in Hꢀbond interactions with the carbonyl oxygen of Ring C (Hꢀ
bond acceptor; d = 2.01 to 2.19 Å). The three Hꢀbonds showed their significance in the tight
binding of B-16 with hCEase. Rings A, B and C were stabilized by van der Waals interaction
with Ser194, His435 and Ala436. In addition to this, the Ring C of B-16 is placed in a wellꢀ
defined cavity formed by Gly106, Gly107, Glu193, Ser194 and Ala195 and suggested to be
stabilized by dispersion interactions. Ring D (dichlorophenyl) gets positioned in a hydrophobic
cavity created by Trp227, Phe324, Leu392 and Phe393 residues and involved in faceꢀtoꢀface paiꢀ
pai stacking interaction with Trp227 and Phe324. The study showed that the B-16 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 B-16 suggests
its prevailing role as hCEase inhibitor.
While comparing the docking conformation of B-16 and A (lead compound), the B-16 showed
more strong binding with hCEase as shown by higher Gold score than A (Gold score = 54.35 and
46.06 for B-16 and A, respectively). Both compounds shared common pharmacophoric features
except the position of Ring A (Figure 2). In contrary to B-16, the Ring A of “compound A” is in
trans conformation to the carbonyl function at Ring C and surrounded by Ile323 and His435
only. Therefore, this can be suggested that cisꢀconformation of Ring A and carbonyl function at
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Ring C is more favorable for activity than transꢀconformation. This pattern of docking is in full
agreement with the in-vitro results. (Figure 7).
In silico studies:
Furthermore, physicoꢀchemical properties like Absorption, Distribution, Metabolism and
Excretion (ADME) of the synthesized compounds were determined in silico by using the webꢀ
based
applications
MarvinSketch
(http://www.chemaxon.com/)
and
PreADMET
(http://preadmet.bmdrc.org/). The Cacoꢀ2 cell, MDCK cell, Blood Brain Barrier (BBB) & Skin
permeabilities, human intestinal absorption and plasma protein binding affinities were predicted
and summarized in Table 2. Results indicated that the compounds are predicted to have lower
blood brain barrier permeation which are less likely to cause neurotoxicity. In other words we
can say that the synthesized compounds cannot alter the normal activity of the neuronal cells.
Basicity and lipophilicity of the synthesized compounds were determined with ChemAxon
software MarvinSketch and the results are shown in Table 3 which describes the compliance of
all the synthesized compounds with the Lipinski rule of five. Tabular values indicated that a) all
the molecules have molecular weight in the range of 286ꢀ408 which lies in the limit of 180ꢀ500
b) compounds have no Hꢀbond donating property c) compounds followed the Hꢀbond acceptor
criteria (< 10) d) molar refractivity found very consistence in the range of 83.64ꢀ98.07 which lies
well in the accepted value range 40ꢀ130 and e) a log P of all the compounds found lower than 5.6
indicating that the compounds are not very lipophilic. These results suggest that all the
compounds follow the Lipinski rule of five and ADME properties which makes them
pharmacologically efficient for clinical use in future.
Conclusion:
A series of 5,6ꢀbenzoflavone derivatives rationally designed, synthesized and characterized by
using 1H NMR, 13C NMR, MASS and Elemental Analysis. All the synthetics were evaluated for
in-vitro cholesterol esterase inhibitory activity. Among all the derivatives, B-16 was found to be
endowed with most potent enzyme inhibition with IC50 value of 0.73 nM. Enzyme kinetic study
confirmed that the inhibitor B-16 preferentially binds to the free enzyme and not to the enzyme
substrate complex (mixed type inhibition). Docking study suggested that the compound B-16 fits
well in the active site of cholesterol esterase enzyme and completely blocks its catalytic
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assembly. The study also concluded that cisꢀconformation of Ring A and carbonyl function at
Ring C is well tolerable for CEase inhibition. In silico parameters revealed that the compounds
with improved CEase inhibitory potential could be act as a hit lead molecules for further
development of pharmacologically active CEase inhibitory framework.
Experimental:
Materials and measurements
The reagents were purchased from Sigma Aldrich, Loba and CDH, India and used without
further purification. The porcine cholesterol esterase enzyme was also procured from Sigma
Aldrich. All yields refer to isolated products after purification. Products were characterized by
1
comparison with authentic samples and by spectroscopic data (1H, 13C NMR and MASS). H
13
NMR and C NMR Spectra were recorded on JEOL AL 300 NMR Spectrometer. The spectra
1
were measured in CDCl3 relative to TMS (0.00 ppm). In H NMR chemical shifts were reported
in δ values using tetramethylsilane as internal standard with number of protons, multiplicities (sꢀ
singlet, dꢀdoublet, tꢀtriplet, qꢀquartet, mꢀmultiplet, ddꢀdouble doublet) and coupling constants (J)
in Hz (Hertz) in the solvent indicated. HRMS was recorded on micrOTOFꢀQII Bruker Daltonik
LC–MS/MS High Resolution Mass Spectrometer. Melting points were determined in open
capillaries and were uncorrected.
Procedure for Synthesis of 1-(2-hydroxynaphthalen-1-yl)ethanone (1)
βꢀnaphthol (1mmol) was treated with glacial acetic acid (1.2 mmol) in the presence of Zncl2
(0.41 mmol) under microwave irradiation for 20 mins at 200°C. The crude mixture was
dissolved in methanol and adsorbed on silica (60ꢀ120 #). The desired product was purified by
column chromatography with increasing percentage of ethyl acetate in hexane as eluting solvent.
The characterization data for the 1ꢀ(2ꢀhydroxynaphthalenꢀ1ꢀyl)ethanone is as follows:
1
Yield: 60 %, mp: 62ꢀ68ºC. H NMR (CDCl3, 300 MHz, δ, TMS = 0): 13.48 (1H, s, OH), 8.09
(1H, d, J = 8.4 Hz, ArH ), 7.89 (1H, d, J = 9.0 Hz, ArH), 7.78 (1H, d, J = 8.1 Hz, ArH), 7.55ꢀ
7.60 (1H, m, ArH), 7.37ꢀ7.42 (1H, m, ArH), 7.14 (1H, d, J = 9.0 Hz, ArH), 2.87 (3H, s, COCH3).
13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 35.22, 117.55, 122.59, 126.49, 127.06, 130.85, 131.28,
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132.31, 134.63, 140.25, 166.81, 207.44. Anal. Calcd. For C12H10O2: C, 77.40; H, 5.41; O, 17.18;
Found: C, 77.32; H, 5.55.
Procedure for Synthesis of 1-acetylnaphthalen-2-yl benzoate (2)
To a solution of 1ꢀ(2ꢀhydroxynaphthalenꢀ1ꢀyl)ethanone (0.01 mol) in pyridine (5 ml),
benzoylchloride (1 eq) was added and stirred for 1 hr at room temperature. The mixture was
poured on ice and precipitated solid was collected and dried.
1
Yield: 84 %, mp: 49ꢀ53ºC. H NMR (CDCl3, 300 MHz, δ, TMS = 0): 8.19ꢀ8.21 (2H, m, ArH),
7.81ꢀ7.96 (3H, m, ArH), 7.64ꢀ7.67 (1H, m, ArH), 7.53ꢀ7.55 (4H, m, ArH), 7.36ꢀ7.40 (1H, m,
13
ArH), 2.62 (3H, s, COCH3); C NMR (CDCl3, 75 MHz, δ, TMS = 0): 35.01, 124.08, 127.11,
128.84, 130.34, 131.05, 131.33, 131.44, 132.44, 132.95, 133.65, 134.25, 136.76, 147.57, 167.52,
205.70. Anal. Calcd. for C19H14O3: C, 78.61; H, 4.86; Found: C, 78.93; H, 4.61.
Procedure for Synthesis of 1-(2-hydroxynaphthalen-1-yl)-3-phenylpropane-1,3-dione (3)
The mixture of 1ꢀacetylnaphthalenꢀ2ꢀyl benzoate (0.01 mmol), KOH (1 mmol) and pyridine (2
ml) was warmed on a water bath for 15 mins. Acetic acid solution (10%, 1.3 ml) was added to
the cooled mixture. The crude mixture was dissolved in ethyl acetate and adsorbed on silica (60ꢀ
120 #). The desired product was purified by column chromatography with increasing percentage
of ethyl acetate in hexane as eluting solvent. The characterization data for the 1ꢀ(2ꢀ
hydroxynaphthalenꢀ1ꢀyl)ꢀ3ꢀphenylpropaneꢀ1,3ꢀdione is as follows:
Yield: 65%; 1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 11.35 (1H, s, D2O exchangeable proton),
8.27ꢀ8.30 (1H, d, J = 8.4 Hz, ArH), 7.90 (1H, s, ArH), 7.67ꢀ7.70 (1H, m, ArH), 7.39ꢀ7.42 (1H,
m, ArH), 7.15ꢀ7.26 (5H, m, ArH), 6.66 (1H, s, ArH), 1.29 (2H, s, ꢀCH2ꢀ). Anal. Calcd. for
C19H14O3: C, 78.61; H, 4.86; O, 16.53; Found: C, 78.72; H, 4.73.
Procedure for Synthesis of 5,6-benzoflavones (B-1)
To a solution of the 1ꢀ(2ꢀhydroxynaphthalenꢀ1ꢀyl)ꢀ3ꢀphenylpropaneꢀ1,3ꢀdione (1 mmol) in acetic
acid (5 ml) was added a drop of concentrated sulfuric acid and the mixture was refluxed for 1
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hour. The cooled mixture was poured into ice and the product was collected by simple filtration.
The characterization data for the synthesized derivatives is given below:
1
3ꢀphenylꢀ1Hꢀbenzo[f]chromenꢀ1ꢀone (B-1)22ꢀ24: Yield 91%, mp 157ꢀ162°C. H NMR (CDCl3, 300
MHz, δ, TMS = 0): 9.78 (1H, d, J = 9.0 Hz, ArH), 7.77ꢀ7.79 (1H, m, ArH), 7.73ꢀ7.74 (1H, m, ArH),
13
7.56ꢀ7.61 (3H, m, ArH), 7.36ꢀ7.47 (5H, m, ArH), 6.93 (1H, s, ꢀCHꢀ). C NMR (CDCl3, 75 MHz, δ,
TMS = 0): 108.64, 119.15, 120.43, 122.56, 124.45, 125.32, 126.45, 127.54, 128.32, 129.34, 129.21,
131,65, 131.34, 136.65, 153.34, 162.23, 178.34. MS: m/z: 273 (M++1). Anal. Calcd for C19H12O2: C,
83.81; H, 4.44; Found: C, 83.59; H, 4.54.
All the 5,6ꢀbenzoflavone derivatives were synthesized with above given procedure and their
characterization data is given below:
1
3-(4-methoxyphenyl)-1H-benzo[f]chromen-1-one (B-2)22,25: Yield 88%, mp 72ꢀ78°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.11 (1H, m, ArH), 8.13ꢀ8.17 (3H, m, ArH), 8.07 (1H,
m, ArH), 7.97 (1H, m, ArH), 7.41 (2H, d, J = 8.5 Hz, ArH), 7.01ꢀ7.03 (2H, m, ArH), 6.95 (1H,
13
s, ꢀCHꢀ), 3.96 (3H, s, OCH3). C NMR (CDCl3, 75 MHz, δ, TMS = 0): 56.93, 108.27, 114.16,
118.45, 119.54, 121.66, 123.45, 123.99, 126.75, 127.16, 128.34, 130.12, 130.67, 135.76, 153.34,
162.56, 177.76. MS: m/z: 303 (M++1). Anal. Calcd for C20H14O3: C, 79.46; H, 4.67; Found: C,
79.57; H, 4.42.
1
3-(3,4-dimethoxyphenyl)-1H-benzo[f]chromen-1-one (B-3)22: Yield 84%, mp 99ꢀ105°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.07 (1H, d, J = 8.9 Hz, ArH), 8.12ꢀ8.19 (3H, m, ArH),
7.93ꢀ7.99 (2H, m, ArH), 7.72ꢀ7.78 (3H, m, ArH), 6.94 (1H, s, ꢀCHꢀ), 3.92 (3H, s, OCH3), 3.96
(3H, s, OCH3). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 55.83, 56.35, 101.43, 102.99, 1105.54,
107.34, 110.76, 119.45, 122.76, 123.34, 123.76, 123.45, 128.76, 128.34, 129.65, 130.56, 135.34,
148.76, 153.34, 162.54, 177.34. MS: m/z: 322 (M+ + 1). Anal. Calcd for C21H16O4: C, 75.89; H,
4.85; Found: C, 75.91; H, 4.71.
1
3-(2,4-dimethoxyphenyl)-1H-benzo[f]chromen-1-one (B-4)25: Yield 89%, mp 105ꢀ113°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.11 (1H, m, ArH), 8.12ꢀ8.14 (3H, m, ArH), 8.06ꢀ8.09
(2H, m, ArH), 7.76 (2H, m, ArH), 7.22ꢀ7.24 (1H, m, ArH), 7.18 (1H, s, ꢀCHꢀ), 3.94 (3H, s,
OCH3), 3.98 (3H, s, OCH3). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 55.95, 56.40, 101.34,
102.35, 105.43, 107.22, 119.97, 122.60, 123.74, 123.91, 128.10, 128.44, 128.76, 129.45, 130.66,
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DOI: 10.1039/C7MD00565B
135.88, 153.68, 158.75, 160.88, 162.20, 177.33. MS: m/z: 322 (M+ + 1). Anal. Calcd for
C21H16O4: C, 75.89; H, 4.85; Found: C, 75.66; H, 4.91.
3-(3-trifluoromethoxyphenyl)-1H-benzo[f]chromen-1-one (B-5): Yield 83%, mp: 96ꢀ101°C,
1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.08 (1H, d, J = 8.5 Hz, ArH), 8.14ꢀ8.18 (3H, m,
ArH), 8.02ꢀ8.04 (2H, m, ArH), 7.21ꢀ7.26 (2H, m, ArH), 7.09ꢀ7.16 (2H, m, ArH), 6.95 (1H, s, ꢀ
CHꢀ): 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 110.55, 111.42, 113.67, 118.34, 120.21, 120.65,
122.76, 123.45, 123.65, 128.67, 128.99, 129.54, 129.99, 130.43, 131.78, 135.45, 153.76, 159.34,
162.55, 177.23. MS: m/z: 357 (M+ + 1). Anal. Calcd for C20H11F3O3: C, 67.42; H, 3.11; F, 16.00;
Found C, 67.55; H, 2.99; F, 16.09.
3-(4-trifluoromethoxyphenyl)-1H-benzo[f]chromen-1-one (B-6): Yield 87%, mp: 115ꢀ120°C.
1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.09 (1H, d, J = 8.6 Hz, ArH), 8.14ꢀ8.19 (3H, m,
ArH), 8.06ꢀ8.09 (2H, m, ArH), 7.88ꢀ7.91 (2H, m, ArH), 7.23 (2H, d, J = 8.5 Hz, ArH), 6.98 (1H,
s, ꢀCHꢀ). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 110.76, 115.34, 118.95, 120.76, 120.34,
122.23, 123.21, 123.56, 125.11, 128.17, 128.34, 129.76, 130.45, 135.34, 153.87, 162.45, 177.45.
MS: m/z: 357 (M+ + 1). Anal. Calcd for C20H11F3O3; C, 67.42; H, 3.11; F, 16.00; Found: C,
67.36; H, 3.24; F, 16.01.
1
3-(2-fluorophenyl)-1H-benzo[f]chromen-1-one (B-7)23,24: Yield 76%, mp 105ꢀ109°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.06 (1H, d, J = 8.4 Hz, ArH), 8.13 (1H, d, J = 9.0 Hz,
ArH), 7.91ꢀ7.99 (2H, m, ArH), 7.75ꢀ7.81 (1H, m, ArH), 7.35ꢀ7.66 (5H, m, ArH), 7.13 (1H, s, ꢀ
CHꢀ). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 115.16, 115.26, 116.90, 117.08, 117.52, 123.78,
124.36, 124.66, 126.71, 127.20, 128.17, 128.90, 129.33, 129.56, 130.41, 130.62, 132.70, 132.77,
135.65, 137.47, 156.40, 157.53, 180.28. MS: m/z: 291 (M+ + 1). Anal. calcd. for C19H11FO2: C,
78.61; H, 3.82; F, 6.54; Found: C, 78.55; H, 3.91; F, 6.44.
1
3-(3-fluorophenyl)-1H-benzo[f]chromen-1-one (B-8): Yield 78%, mp 123ꢀ128°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.03 (1H, d, J = 8.6 Hz, ArH), 8.12ꢀ8.14 (1H, m, ArH), 7.92ꢀ
7.95 (2H, m, ArH), 7.75ꢀ7.79 (1H, m, ArH), 7.62ꢀ7.66 (3H, m, ArH), 7.34ꢀ7.36 (2H, m, ArH),
13
7.14 (1H, s, ꢀCHꢀ). C NMR (CDCl3, 75 MHz, δ, TMS = 0): 114.13, 115.45, 116.96, 117.56,
117.56, 123.74, 124.33, 124.68, 126.75, 127.27, 128.13, 128.96, 129.34, 129.87, 130.45, 130.34,
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132.77, 132.45, 135.67, 137.44, 156.45, 157.56, 180.45. MS: m/z: 291 (M+ + 1). Anal. calcd. for
C19H11FO2: C, 78.61; H, 3.82; F, 6.54; Found: C, 78.74; H, 3.75; F, 6.55.
1
3-(4-fluorophenyl)-1H-benzo[f]chromen-1-one (B-9): Yield 77%, mp 130ꢀ136°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.04 (1H, d, J = 8.5 Hz, ArH), 8.13ꢀ8.15 (1H, m, ArH), 7.91ꢀ
7.94 (2H, m, ArH), 7.75ꢀ7.79 (2H, m, ArH), 7.62ꢀ7.66 (2H, m, ArH), 7.22 (2H, d, J = 8.9 Hz,
ArH), 7.13 (1H, s, ꢀCHꢀ). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 114.16, 115.43, 116.94,
117.57, 117.54, 123.75, 124.38, 124.64, 126.76, 127.23, 128.16, 128.94, 129.33, 129.84, 130.47,
130.33, 132.72, 132.44, 135.63, 137.45, 156.42, 157.53, 180.42. MS: m/z: 291 (M+ + 1). Anal.
calcd. for C19H11FO2: C, 78.61; H, 3.82; F, 6.54; Found: C, 78.72; H, 3.64; F, 6.59.
1
3-(2,6-difluorophenyl)-1H-benzo[f]chromen-1-one (B-10)23,24: Yield 73%, mp 89ꢀ93°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.05 (1H, d, J = 8.6 Hz, ArH), 8.13ꢀ8.16 (3H, m, ArH),
8.05ꢀ8.09 (2H, m, ArH), 7.55ꢀ7.58 (1H, m, ArH), 7.23ꢀ7.29 (2H, m, ArH), 6.93 (1H, s, ꢀCHꢀ).
13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 114.22, 115.46, 116.93, 117.57, 117.53, 123.74,
128.12, 128.64, 129.96, 130.46, 135.34, 153.05, 158.44, 162.26, 179.99. MS: m/z: 309 (M+ + 1).
Anal. calcd. for C19H10F2O2: C, 74.03; H, 3.27; F, 12.33; Found: C, 74.22; H, 3.17; F, 12.44.
1
3-(2,4-difluorophenyl)-1H-benzo[f]chromen-1-one (B-11): Yield 76%, mp 103ꢀ108°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.07 (1H, d, J = 8.4 Hz, ArH), 8.10ꢀ8.15 (3H, m, ArH),
7.99ꢀ8.03 (2H, m, ArH), 7.64ꢀ7.69 (1H, m, ArH), 7.21ꢀ7.25 (2H, m, ArH), 6.91 (1H, s, ꢀCHꢀ).
13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 115.21, 115.88, 116.97, 117.53, 117.56, 123.73,
128.17, 128.63, 129.93, 130.47, 135.39, 153.03, 158.47, 162.24, 180.34. MS: m/z: 309 (M+ + 1).
Anal. calcd. for C19H10F2O2: C, 74.03; H, 3.27; F, 12.33; Found: C, 74.22; H, 3.36; F, 12.23.
1
3-(3,5-difluorophenyl)-1H-benzo[f]chromen-1-one (B-12): Yield 78%, mp 124ꢀ139°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 9.80 (1H, d, J = 8.4 Hz, ArH), 7.97 (1H, d, J = 9.0 Hz,
ArH), 7.75 (1H, d, J = 8.1 Hz, ArH), 7.42ꢀ7.58 (4H, m, ArH), 7.29ꢀ7.33 (2H, m, ArH), 6.76ꢀ6.85
(1H, m, ꢀCHꢀ). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 114.56, 115.41, 116.93, 117.54,
117.56, 123.77, 128.18, 128.69, 129.94, 130.43, 135.32, 153.01, 158.42, 162.23, 179.94. MS:
m/z: 309 (M+ + 1). Anal. calcd. for C19H10F2O2: C, 74.03; H, 3.27; F, 12.33; Found: C, 73.99; H,
3.34; F, 12.15.
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1
3-(2,5-difluorophenyl)-1H-benzo[f]chromen-1-one (B-13): Yield 81%, mp 131ꢀ136°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.09 (1H, m, ArH), 8.15ꢀ8.19 (3H, m, ArH), 8.02ꢀ8.05
13
(2H, m, ArH), 7.33ꢀ7.42 (3H, m, ArH), 6.85 (1H, s, ꢀCHꢀ). C NMR (CDCl3, 75 MHz, δ, TMS
= 0): 106.64, 109.09, 109.31, 111.43, 117.36, 126.90, 127.11, 128.24, 129.49, 130.33, 130.71,
135.96, 137.01, 157.27, 162.43, 164.31, 179.84. MS: m/z: 309 (M+ + 1). Anal. calcd. for
C19H10F2O2: C, 74.03; H, 3.27; F, 12.33; Found: C, 74.21; H, 3.08; F, 12.66.
1
3-(3,4-difluorophenyl)-1H-benzo[f]chromen-1-one (B-14): Yield 79%, mp 104ꢀ107°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.06 (1H, m, ArH), 8.12ꢀ8.17 (3H, m, ArH), 8.02ꢀ8.05
(2H, m, ArH), 7.42ꢀ7.45 (1H, m, ArH), 7.38 (1H, s, ArH), 7.29ꢀ7.32 (1H, m, ArH), 6.94 (1H, s, ꢀ
CHꢀ). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 115.23, 115.65, 116.96, 117.53, 117.55, 123.71,
128.16, 128.67, 129.93, 130.42, 135.37, 153.08, 158.41, 162.23, 179.95. MS: m/z: 309 (M+ + 1).
Anal. calcd. for C19H10F2O2: C, 74.03; H, 3.27; F, 12.33; Found: C, 74.32; H, 3.45; F, 12.10.
1
3-(3-chlorophenyl)-1H-benzo[f]chromen-1-one (B-15): Yield 78%, mp 160ꢀ166°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.07 (1H, m, ArH), 8.11ꢀ8.17 (3H, m, ArH), 8.00ꢀ8.04 (2H, m,
ArH), 7.66 (1H, s, ArH), 7.23ꢀ7.30 (3H, m, ArH), 6.96 (1H, s, ꢀCHꢀ). 13C NMR (CDCl3, 75
MHz, δ, TMS = 0): 115.45, 117.45, 117.65, 126.26, 127.64, 128.36, 129.08, 130.33, 131.64,
132.37, 133.35, 134.34, 135.43, 136.55, 153.54, 161.18, 180.05. MS: m/z: 307 (M+ + 1). Anal.
calcd. for C19H11ClO2: C, 74.40; H, 3.61; Cl, 11.56; Found: C, 74.50; H, 3.59; Cl, 11.60.
1
3-(2,3-dichlorophenyl)-1H-benzo[f]chromen-1-one (B-16): Yield 84%, mp 160ꢀ165°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.08 (1H, m, ArH), 8.15ꢀ8.19 (3H, m, ArH), 7.98ꢀ8.03
13
(2H, m, ArH), 7.32ꢀ7.36 (3H, m, ArH), 6.81 (1H, s, ꢀCHꢀ). C NMR (CDCl3, 75 MHz, δ, TMS
= 0): 115.97, 117.26, 117.55, 126.80, 127.16, 127.64, 128.21, 129.02, 129.40, 130.41, 130.69,
131.67, 132.51, 133.71, 134.61, 135.83, 157.79, 159.70, 179.79. MS: m/z: 341 (M+ + 1). Anal.
calcd. for C19H10Cl2O2: C, 66.89; H, 2.95; Cl, 20.78; Found: C, 66.99; H, 2.85; Cl, 20.91.
1
3-(2-bromophenyl)-1H-benzo[f]chromen-1-one (B-17): Yield 75%, mp 153ꢀ159°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.08 (1H, d, J = 8.7 Hz, ArH), 8.13 (1H, d, J = 9.0 Hz, ArH),
7.94 (1H, d, J = 7.5 Hz, ArH), 7.40ꢀ7.80 (7H, m, ArH), 6.78 (1H, s, ꢀCHꢀ). 13C NMR (CDCl3, 75
MHz, δ, TMS = 0): 115.12, 116.89, 117.07, 117.11, 117.51, 119.90, 124.64, 124.66, 126.71,
127.19, 128.18, 128.90, 129.33, 130.61, 132.71, 132.79, 135.68, 157.54, 180.32. MS: m/z: 350
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(M+ + 1). Anal. calcd. for C19H11BrO2: C, 64.98; H, 3.16; Br, 22.75; Found: C, 65.05; H, 3.03;
Br, 22.88.
1
3-(3-bromophenyl)-1H-benzo[f]chromen-1-one (B-18): Yield 78%, mp 168ꢀ171°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.04 (1H, d), 8.15 (1H, d, J = 9.0 Hz, ArH), 7.95 (1H, m,
ArH), 7.77ꢀ7.83 (3H, m, ArH), 7.63 (1H, s, ArH), 7.23ꢀ7.33 (3H, m ArH), 6.98 (1H, s, ꢀCHꢀ).
13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 110.53, 117.23, 117.43, 126.08, 126.79, 127.18,
127.47, 128.16, 129.35, 130.34, 130.42, 130.67, 132.35, 135.66, 157.33, 159.72, 179.97. MS:
m/z: 350 (M+ + 1). Anal. calcd. for C19H11BrO2: C, 64.98; H, 3.16; Br, 22.75; Found: C, 65.02;
H, 3.09; Br, 22.79.
1
3-(4-bromophenyl)-1H-benzo[f]chromen-1-one (B-19): Yield 80%, mp 220ꢀ225°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.06 (1H, d, J = 8.7 Hz, ArH), 8.14 (1H, d, J = 9.0 Hz, ArH),
7.93 (1H, d, J = 8.1 Hz, ArH), 7.78ꢀ7.85 (3H, m, ArH), 7.61ꢀ7.70 (4H, m, ArH), 6.97 (1H, s, ꢀ
CHꢀ). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 110.57, 117.28, 117.46, 126.05, 126.74, 127.16,
127.49, 128.18, 129.34, 130.34, 130.43, 130.65, 132.37, 135.65, 157.32, 159.76, 180.07. MS:
m/z: 350 (M++1). Anal. calcd. for C19H11BrO2: C, 64.98; H, 3.16; Br, 22.75; Found: C, 64.78; H,
3.00; Br, 22.65.
1
3-(2-iodophenyl)-1H-benzo[f]chromen-1-one (B-20): Yield 69%, mp 141ꢀ146°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.05 (1H, m, ArH), 8.19ꢀ8.25 (3H, m, ArH), 8.11ꢀ8.13 (2H, m,
13
ArH), 7.82ꢀ7.84 (1H, m, ArH), 7.31ꢀ7.36 (3H, m, ArH), 6.96 (1H, s, ꢀCHꢀ). C NMR (CDCl3,
75 MHz, δ, TMS = 0): 110.32, 113.65, 119.25, 120.65, 122.82, 124.16, 125.62, 127.26, 128.12,
128.56, 129.47, 130.81, 131.94, 136.03, 138.07, 140.53, 154.02, 165.25, 180.12. MS: m/z: 398
(M+ + 1). Anal. calcd. for C19H11IO2: C, 57.31; H, 2.78; I, 31.87; Found: C, 57.42; H, 2.88; I,
31.93.
1
3-(4-iodophenyl)-1H-benzo[f]chromen-1-one (B-21): Yield 72%, mp 133ꢀ139°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.08 (1H, m, ArH), 8.19ꢀ8.25 (3H, m, ArH), 8.11ꢀ8.13 (2H, m,
ArH), 7.88 (2H, d, J = 8.9 Hz, ArH), 7.25ꢀ7.29 (2H, m, ArH), 6.95 (1H, s, ꢀCHꢀ). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 110.55, 113.69, 119.28, 120.62, 122.87, 124.13, 125.67, 127.22,
128.16, 128.53, 129.42, 130.87, 131.92, 136.34, 138.09, 140.51, 154.07, 165.29, 180.18. MS:
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m/z: 398 (M+ + 1). Anal. calcd. for C19H11IO2: C, 57.31; H, 2.78; I, 31.87; Found: C, 57.28; H,
2.79; I, 31.77.
1
3-(3-nitrophenyl)-1H-benzo[f]chromen-1-one (B-22): Yield 79%, mp 139ꢀ141°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.15 (1H, m, ArH), 8.42 (1H, m, ArH), 8.11ꢀ8.14 (3H, m,
13
ArH), 7.78ꢀ7.85 (2H, m, ArH), 7.62ꢀ7.65 (2H, m, ArH), 7.07 (1H, s, ꢀCHꢀ). C NMR (CDCl3,
75 MHz, δ, TMS = 0): 112.48, 115.93, 117.35, 124.25, 124.30, 126.77, 126.99, 127.14, 128.20,
128.31, 129.31, 129.62, 136.14, 136.16, 137.45, 157.45, 158.11, 179.74. MS: m/z: 318 (M+ + 1).
Anal. calcd. for C19H11NO4: C, 71.92; H, 3.49; N, 4.41; Found: C, 72.00; H, 3.33; N, 4.34.
1
3-(4-nitrophenyl)-1H-benzo[f]chromen-1-one (B-23): Yield 76%, mp: 102ꢀ106°C. H NMR
(CDCl3, 300 MHz, δ, TMS = 0): 10.14 (1H, d, J = 8.4 Hz, ArH), 8.41 (1H, d, J = 8.7 Hz, ArH),
8.14ꢀ8.19 (3H, m, ArH), 7.95 (1H, d, J = 8.4 Hz, ArH), 7.78ꢀ7.80 (1H, m, ArH), 7.65ꢀ7.68 (2H,
13
m, ArH), 7.08 (1H, s, ꢀCHꢀ). C NMR (CDCl3, 75 MHz, δ, TMS = 0): 112.48, 115.93, 117.35,
124.25, 124.30, 126.77, 126.99, 127.14, 128.20, 128.31, 129.31, 129.62, 136.14, 136.16, 137.45,
157.45, 158.11, 179.74. MS: m/z: 318 (M+ + 1). Anal. calcd. for C19H11NO4: C, 71.92; H, 3.49;
N, 4.41; Found: C, 71.85; H, 3.35; N, 4.65.
1
3-(3,5-dinitrophenyl)-1H-benzo[f]chromen-1-one (B-24): Yield 81%, mp 101ꢀ106°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.15 (1H, s, ArH), 9.08 (1H, s, ArH), 8.65ꢀ8.70 (2H, m,
ArH), 8.11 (1H, m, ArH), 7.97ꢀ7.99 (1H, m, ArH), 7.77ꢀ7.84 (3H, m, ArH), 7.07 (1H, s, ꢀCHꢀ).
13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 112.43, 115.97, 117.39, 124.22, 124.36, 126.73,
126.93, 127.17, 128.24, 128.36, 129.37, 129.67, 136.18, 136.13, 137.46, 157.47, 158.18, 180.75
MS: m/z: 363 (M++1). Anal. calcd. for C19H10N2O6: C, 62.76; H, 2.93; N, 7.35; Found: C, 62.66;
H, 3.03; N, 7.30.
1
3-p-tolyl-1H-benzo[f]chromen-1-one (B-25)24: Yield 85%, mp: 96ꢀ102°C. H NMR (CDCl3,
300 MHz, δ, TMS = 0): 10.04 (1H, m, ArH), 8.12ꢀ8.18 (3H, m, ArH), 8.01ꢀ8.06 (2H, m, ArH),
7.33 (2H, d, J = 8.4 Hz, ArH), 7.11ꢀ7.15 (2H, m, ArH), 7.01 (1H, s, ꢀCHꢀ), 2.45 (3H, s, ꢀCH3).
13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 26.45, 110.26, 119.93, 122.65, 123.77, 123.95, 126.33,
127.46, 128.18, 128.63, 129.07, 129.93, 130.45, 135.32, 138.55, 153.04, 162.25, 180.24. MS:
m/z: 287 (M++1). Anal. calcd. for C20H14O2: C, 83.90; H, 4.93; Found: C, 83.80; H, 4.98.
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3-(4-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-26): Yield 85%, mp: 115ꢀ
119°C, 1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.04 (1H, m, ArH), 8.14ꢀ8.19 (3H, m, ArH),
8.01ꢀ8.05 (2H, m, ArH), 7.88 (2H, d, J = 8.9 Hz, ArH), 7.41ꢀ7.45 (2H, m, ArH) 6.97 (1H, s, ꢀ
CHꢀ). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 110.221, 119.91, 122.62, 123.73, 123.95,
126.52, 128.12, 128.64, 129.93, 130.25, 130.46, 133.57, 135.31, 153.03, 162.22, 177.25. MS:
m/z: 341 (M++1). Anal. calcd. for C20H11F3O2: C, 70.59; H, 3.26; F, 16.75; Found: C, 70.68; H,
3.06; F, 16.82.
3-(3-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-27): Yield 74%, mp 106ꢀ110°C.
1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.07 (1H, m, ArH), 8.13ꢀ8.17 (3H, m ArH), 8.00ꢀ
8.04 (2H, m, ArH), 7.88 (1H, s, ArH), 7.21ꢀ7.33 (3H, m, ArH), 6.97 (1H, s, ꢀCHꢀ). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 111.45, 119.95, 122.66, 122.83, 123.76, 123.93, 124.05, 124.67,
128.17, 128.67, 129.74, 129.93, 130.45, 130.76, 130.92, 135.33, 153.05, 162.26, 180.28. MS:
m/z: 341 (M++1). Anal. calcd. for C20H11F3O2: C, 70.59; H, 3.26; F, 16.75; Found: C, 70.49; H,
3.35; F, 16.65.
3-(2-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-28): Yield: 81%; mp 105ꢀ
108°C. 1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.06 (1H, m, ArH), 8.11ꢀ8.14 (3H, m, ArH),
8.00ꢀ8.04 (2H, m, ArH), 7.88ꢀ7.91 (1H, m, ArH), 7.21ꢀ7.33 (3H, m, ArH), 6.97 (1H, s, ꢀCHꢀ).
13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 110.45, 119.94, 122.63, 122.85, 123.76, 123.96,
124.05, 124.64, 128.13, 128.62, 129.71, 129.92, 130.43, 130.74, 130.95, 135.36, 153.07, 162.28,
180.29. MS: m/z: 341 (M++1). Anal. calcd. for C20H11F3O2: C, 70.59; H, 3.26; F, 16.75; Found:
C, 70.48; H, 3.35; F, 16.38.
3-(3-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-29): Yield 80%, mp 96ꢀ101°C.
1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.09 (1H, m, ArH), 8.13ꢀ8.18 (3H, m, ArH), 7.97ꢀ
8.02 (2H, m, ArH), 7.84 (1H, s, ArH), 7.41ꢀ7.53 (3H, m, ArH), 6.93 (1H, s, ꢀCHꢀ). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 110.48, 119.98, 122.67, 122.86, 123.75, 123.94, 124.03, 124.62,
128.12, 128.61, 129.74, 129.95, 130.46, 130.77, 130.94, 135.33, 153.06, 162.23, 180.25. MS:
m/z: 409 (M++1). Anal. calcd. for C21H10F6O2: C, 61.78; H, 2.47; F, 27.92; Found: C, 61.68; H,
2.55; F, 27.83.
16

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3-(4-fluoro-2-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-30): Yield 81%, mp
155ꢀ159°C. 1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.06 (1H, d, J = 8.6 Hz, ArH), 8.13ꢀ8.16
(3H, m, ArH), 8.06 (1H, m, ArH), 8.01 (1H, m, ArH), 7.64ꢀ7.66 (1H, m, ArH), 7.44ꢀ7.47 (1H,
m, ArH), 7.23 (1H, m, ArH), 6.86 (1H, s, ꢀCHꢀ). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0):
110.65, 110.99, 114.75, 117.47, 119.99, 121.45, 122.69, 123.74, 123.96, 125.94, 126.63, 128.17,
128.67, 129.94, 130.47, 135.34, 153.05, 162.25, 164.55, 180.42. MS: m/z: 359 (M++1). Anal.
calcd. for C20H10F4O2: C, 67.05; H, 2.81; F, 21.21; Found: C, 67.11; H, 2.79; F, 21.33.
3-(2-fluoro-4-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-31): Yield 82%, mp
151ꢀ155°C. 1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.08 (1H, d, J = 8.6 Hz, ArH), 8.14ꢀ8.17
(3H, m, ArH), 8.03ꢀ8.06 (2H, m, ArH), 7.22ꢀ7.43 (3H, m, ArH), 6.85 (1H, s, ꢀCHꢀ). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 110.23, 110.67, 114.74, 117.45, 119.67, 121.65, 122.76, 123.34,
123.78, 125.45, 126.67, 128.45, 128.89, 129.45, 130.67, 135.67, 153.65, 162.67, 164.56, 180.56.
MS: m/z: 359 (M+ +1). Anal. calcd. for C20H10F4O2: C, 67.05; H, 2.81; F, 21.21; Found: C,
67.12; H, 2.76; F, 21.34.
3-(2-fluoro-6-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-32): Yield 73%, mp
122ꢀ127°C. 1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.07 (1H, d, J = 8.6 Hz, ArH), 8.12ꢀ8.14
(3H, m, ArH), 8.02ꢀ8.06 (2H, m, ArH), 7.19ꢀ7.28 (3H, m, ArH), 6.86 (1H, s, ꢀCHꢀ). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 110.64, 110.93, 114.74, 117.45, 119.93, 121.45, 122.66, 123.74,
123.93, 125.92, 126.65, 128.14, 128.64, 129.93, 130.44, 135.36, 153.04, 162.27, 164.54, 180.47.
MS: m/z: 359 (M++1). Anal. calcd. for C20H10F4O2: C, 67.05; H, 2.81; F, 21.21; Found: C, 67.22;
H, 2.76; F, 21.32.
3-(5-fluoro-2-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-33): Yield 74%, mp
1
144ꢀ147°C. H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.05 (1H, m, ArH), 8.10ꢀ8.13 (3H, m,
ArH), 8.02ꢀ8.05 (2H, m, ArH), 7.74ꢀ7.78 (1H, m, ArH), 7.11ꢀ7.23 (2H, m, ArH), 6.85 (1H, s, ꢀ
CHꢀ). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 110.62, 110.92, 114.73, 117.49, 119.98, 121.48,
122.69, 123.78, 123.97, 125.96, 126.68, 128.16, 128.67, 129.95, 130.46, 135.34, 153.07, 162.25,
164.56, 180.43. MS: m/z: 359 (M++1). Anal. calcd. for C20H10F4O2: C, 67.05; H, 2.81; F, 21.21;
Found: C, 67.25; H, 2.65; F, 21.54.
17

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3-(3-fluoro-5-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-34): Yield 69%, mp
1
110ꢀ115°C. H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.11 (1H, m, ArH), 8.14ꢀ8.19 (3H, m,
ArH), 8.02ꢀ8.00 (2H, m, ArH), 7.66 (1H, s, ArH), 7.21 (2H, m, ArH), 6.94 (1H, s, ꢀCHꢀ). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 110.67, 110.95, 114.45, 117.78, 119.45, 121.98, 122.45,
123.67, 123.76, 125.54, 126.67, 128.54, 128.89, 129.43, 130.54, 135.55, 153.43, 162.54, 164.34,
180.23. MS: m/z: 359 (M++1). Anal. calcd. for C20H10F4O2: C, 67.05; H, 2.81; F, 21.21; Found:
C, 67.14; H, 2.74; F, 21.45.
3-(4-fluoro-3-(trifluoromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-35): Yield 81%, mp
1
158ꢀ164°C. H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.10 (1H, m, ArH), 8.16ꢀ8.20 (3H, m,
ArH), 8.04ꢀ8.08 (2H, m, ArH), 7.69 (1H, s, ArH), 7.44 (1H, d, J = 8.9 Hz, ArH), 7.23ꢀ7.25 (1H,
13
m, ArH), 6.96 (1H, s, ꢀCHꢀ). C NMR (CDCl3, 75 MHz, δ, TMS = 0): 110.64, 110.93, 114.74,
117.45, 119.93, 121.45, 122.66, 123.74, 123.93, 125.92, 126.65, 128.14, 128.64, 129.93, 130.44,
135.36, 153.04, 162.27, 164.54, 180.47. MS: m/z: 359 (M++1). Anal. calcd. for C20H10F4O2: C,
67.05; H, 2.81; F, 21.21; Found: C, 67.21; H, 2.66; F, 21.29.
3-(4-(chloromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-36): Yield 74%, mp 116ꢀ120°C.
1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.05 (1H, m, ArH), 8.11ꢀ8.15 (3H, m, ArH), 8.05ꢀ
8.07 (2H, m, ArH), 7.66 (2H, d, J = 8.6 Hz, ArH), 7.29 (2H, d, J = 8.6 Hz, ArH), 6.98 (1H, s, ꢀ
CHꢀ), 4.64 (2H, s, CH2Cl). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 48.54, 110.56, 119.87,
122.45, 123.34, 123.78, 125.56, 128.45, 128.76, 128.99, 129.45, 130.43, 130.55, 135.67, 137.34,
153.56, 162.56, 180.34. MS: m/z: 321 (M++1). Anal. calcd. for C20H13ClO2: C, 74.89; H, 4.08;
Cl, 11.05; Found: C, 74.79; H, 4.18; Cl, 11.25.
3-(3-(chloromethyl)phenyl)-1H-benzo[f]chromen-1-one (B-37): Yield 77%, mp 101ꢀ105°C.
1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.11 (1H, m, ArH), 8.14ꢀ8.17 (3H, m, ArH), 8.06ꢀ
8.09 (2H, m, ArH), 7.33ꢀ7.49 (3H, m, ArH), 6.98 (1H, s, ꢀCHꢀ), 4.69 (2H, s, CH2Cl). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 48.23, 110.23, 119.45, 122.45, 123.65, 123.45, 125.65, 128.54,
128.34, 128.34, 129.45, 130.56, 130.45, 135.23, 137.56, 153.34, 162.23, 180.45. MS: m/z: 321
(M++1). Anal. calcd. for C20H13ClO2: C, 74.89; H, 4.08; Cl, 11.05; Found: C, 74.78; H, 4.24; Cl,
10.96.
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3-(4-(dimethylamino)phenyl)-1H-benzo[f]chromen-1-one (B-38): Yield 74%, mp 100ꢀ105°C.
1H NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.04 (1H, m, ArH), 8.14ꢀ8.17 (3H, m, ArH), 8.05ꢀ
8.07 (2H, m, ArH), 7.41 (2H, d, J = 8.7 Hz, ArH), 7.22ꢀ7.25 (2H, m, ArH), 6.99 (1H, s, ꢀCHꢀ),
13
2.89 (3H, s, N(CH3)2), 2.83 (3H, s, N(CH3)2). C NMR (CDCl3, 75 MHz, δ, TMS = 0): 42.56,
42.78, 110.56, 114.34, 119.54, 119.89, 122.65, 123.45, 123.67, 127.45, 128.34, 128.69, 129.56,
130.45, 135.67, 147.54, 153.56, 162.24, 180.56. MS: m/z: 316 (M++1). Anal. calcd. for
C21H17NO2: C, 79.98; H, 5.43; N, 4.44; Found: C, 79.86; H, 5.66; N, 4.34.
1
3-(3-(acetoxy)phenyl)-1H-benzo[f]chromen-1-one (B-39): Yield 78%, mp 160ꢀ165°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.11 (1H, m, ArH), 8.14ꢀ8.18 (3H, m, ArH), 8.03ꢀ8.05
(2H, m, ArH), 7.77ꢀ7.82 (2H, m, ArH), 7.33ꢀ7.35 (2H, m, ArH), 6.94 (1H, s, ꢀCHꢀ), 2.16 (3H, s,
OCOCH3). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 23.34, 110.65, 119.45, 121.46, 121.84,
122.65, 123.74, 123.92, 126.76, 127.56, 128.16, 128.66, 129.97, 130.47, 135.37, 153.08, 162.28,
168.75, 180.32. MS: m/z: 331 (M++1). Anal. calcd. for C21H14O4: C, 76.35; H, 4.27; Found: C,
76.29; H, 4.34.
1
3-(4-(acetoxy)phenyl)-1H-benzo[f]chromen-1-one (B-40): Yield 81%, mp 159ꢀ163°C. H
NMR (CDCl3, 300 MHz, δ, TMS = 0): 10.10 (1H, m, ArH), 8.14ꢀ8.18 (3H, m, ArH), 8.02ꢀ8.04
(2H, m, ArH), 7.66 (2H, d, J = 8.5 Hz, ArH), 7.13ꢀ7.15 (2H, m, ArH), 6.96 (1H, s, ꢀCHꢀ), 2.14
(3H, s, OCOCH3). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 23.54, 110.25, 119.96, 121.48,
121.86, 122.67, 123.74, 123.95, 126.73, 127.66, 128.14, 128.62, 129.93, 130.42, 135.35, 153.02,
162.22, 168.75, 179.99. MS: m/z: 331 (M++1). Anal. calcd. for C21H14O4: C, 76.35; H, 4.27;
Found: C, 76.42 H, 4.18.
In vitro cholesterol esterase assay
Porcine cholesterol esterase inhibition was assayed spectrophotometrically at 405 nm at 25˚C.
Assay buffer was 100 mM sodium phosphate, 100 mM NaCl, pH 7.0. A stock solution of CEase
(200 µg/mL) was prepared in 100 mM sodium phosphate buffer, pH 7.0 and kept at 0˚C. A 1:200
dilution was done with the same buffer immediately before starting the measurement. Sodium
taurocholate (12 mM) was dissolved in assay buffer and kept at 25˚C. A stock solution of paraꢀ
nitrophenyl butyrate (20 mM) was prepared in acetonitrile. The final concentration of
acetonitrile was 3%, of the substrate paraꢀnitrophenyl butyrate 20 µM, and of sodium
taurocholate 6 mM. Assays were performed with a final concentration of 10 ng/mL of CEase.
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Into a cuvette containing 430 ꢁL assay buffer, 500 ꢁL of the sodium taurocholate solution, 20 ꢁL
acetonitrile, 10 ꢁL of the paraꢀnitrophenyl butyrate solution, and 30 ꢁL of an inhibitor solution
in DMSO were added and thoroughly mixed. After incubation for 5 min at 25˚C, the reaction
was initiated by adding 10 ꢁL of the enzyme solution 1µg/mL. All the experiments were
performed in triplicate and values were expressed as means of three experiments.8
Enzyme kinetics study
Synthesized compounds were further investigated for the type of inhibition and enzyme kinetics
study were carried out. The LineweaverꢀBurk plot will be established from which we can
calculate the Km, Vmax of the slope of inhibitor and the value of α (a constant that defines the
degree to which inhibitor binding affects the affinity of the enzyme for substrate).19
Docking study
To study the binding mode of the synthesized compounds, we have docked the most potent
compound B-16 at the catalytic site of lipase. The Xꢀray coordinates of catalytic domain of
human bile salt activated lipase (hBAL, hCEase) was obtained from protein data bank (PDB
entry: 1F6W; Resolution 2.3Å).20 The docking study was carried out using GOLD v5.3
software.26 Gold performs genetic algorithm based ligand docking to optimize the conformation
of ligand at the receptor binding site. The Goldscore fitness function was used to evaluate the
various conformations of ligand at the binding site. Goldscore comprises of four components:
protein–ligand hydrogen bond energy, protein–ligand van der Waals (vdw) energy, ligand
internal vdw energy and ligand torsional strain energy.26 B-16 was docked ten times and
conformation associated with highest scoring value was considered to analyze various drugꢀ
receptor interactions. The Structure of B-16 was drawn in ChemDraw Ultra (2010) and subjected
to energy minimization using the MM2 force field as implemented in Chem 3D Ultra software.
Conflict of interest
The authors declare no competing interests.
20

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Acknowledgement
Authors are grateful to University Grants Commission for providing funds under university with
potential for excellence (UPE) Scheme and Rajiv Gandhi National Fellowship (RGNF) to carry
out the research work. The authors are also thankful to Dr. Sahil Sharma, Research Fellow,
Memorial Sloan Kettering Cancer Center, New York, USA for his valuable support and
guidance.
Captions
Figure 1. Reported CEase inhibitors18
Scheme 1. Synthesis of 5,6ꢀbenzoflavone. Reagents and conditions: (a) MW, ZnCl2, CH3COOH,
20 min; (b) benzoyl chloride, pyridine, stirring rt, 1 h; (c) KOH, pyridine, warm, 15 min; (d) a
drop of conc. H2SO4, CH3COOH, reflux, 30 min.
Figure 2. Design strategy for target compounds
Figure 3. Structure Activity Relationship
Figure 4. LineweaverꢀBurk plot of B-16
Figure 5. Schematic 2D representations of hCeaseꢀB-16 complex showing Hꢀbond and van der
Walls interactions (Figure generated by LIGPLOT27)
Figure 6. Docked conformation of B-16 at the catalytic site of hCEase (B-16: carbon atoms are
shown in green; only hydrogens which are involved in Hꢀbond interactions are shown in white
color)
Figure 7. Docked conformation of B-16 and A (lead compound) at the catalytic site of hCEase
(B-16: shown in green; A: shown in purple)
Table 1. Various substituted 5,6ꢀBenzoflavone derivatives and their CEase inhibitory activity
Table 2. In silico ADME properties of 5,6ꢀBenzoflavone derivatives
Table 3. Physicochemical parameters of 5,6ꢀBenzoflavone derivatives
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Cl
O
OR1
OR2
H
n
O
O
N
m
n
P
R
O
X
O
O
Cl
O
O
O2N
O2N
O
R1,R2 = Alkyl
aryl phosphates
m = 0ꢀ3; n = 1,2; X = H, NO2
n =0ꢀ4
6-chloro-2-pyrone
R = Alkyl
carbamates
chloroisocoumarins
Y
R2
O
O
H or OY
YO
O
O
OR1
O
X
P
R
N
N
O
R
OH
O
O
X = H, MeO, Cl; Y = H, Cl
R1 = H, Et, Me; R2 = alkyl, aryl
phosphaisocoumarins
N
Ar
Y = OP(O)(OEt)2
phosphorylated flavonoids
3-phenyl substituted 1,3,
4-oxadiazol-2(3H)-ones
R = NO2 ; Ar = Indoleꢀ3ꢀyl
phenylquinolines
S
N
O
NR2
X
X
R
O
O
X
NH
H
Cl
S
N
n
NH
N
S
Ar
H
N
O
n = 0, 1; X = O, CH2
R = Me, Et
thieno[1,3]-oxazin-4-ones
O
NH
2-(1H-Indol-3-yl)-4-phenylquinolines
X = O, S
thiazolidinediones
X = S; R = CH2OCH2Ph (SerOBn)
O
thiazolidinedione
Figure 1. Reported CEase inhibitors18
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O
CH3
O
CH3
OH
OH
O
a
b
O
1; yield = 60%
2; yield = 84%
c
O
O
HO
O
OH
OH
d
3; yield = 65%
O
O
B-1; yield = 91%
Scheme 1. Synthesis of 5,6ꢀbenzoflavones. Reagents and conditions: (a) MW, ZnCl2,
CH3COOH, 20 min; (b) benzoyl chloride, pyridine, stirring rt, 1 h; (c) KOH, pyridine, warm, 15
min; (d) a drop of conc. H2SO4, CH3COOH, reflux, 30 min.
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Ring gets positioned in a wellꢀdefined cavity formed
by Gly106, Gly107, GLu193, Ser194 and Ala19
Only surrounded by Ile323 and His435 amino acid residues
D
R
8
B
5
O1
C
A
D
8
R
1
7
6
2
3
O
Position of Ring A changed
2
3
7
C
B
4
6
A
4
O
5
O
7,8ꢀbenzoflavones
5,6ꢀbenzoflavones
Target compounds
Most potent (A); R = 3,5ꢀdifluoro
IC50 = 0.78 nM
Figure 2. Design strategy for target compounds
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Cl
Cl
R
D
O
C
O
O
Overall preference order of the substituent for the inhibition of cholesterol esterase enzyme:
B
R = ꢀCl > ꢀF > ꢀBr > ꢀI > ꢀNO2 > ꢀCH2Cl > ꢀCF3 > ꢀOCOCH3 > ꢀH > ꢀOCF3 > ꢀCH3 > ꢀOCH3 > ꢀN(CH3)2
A
O
ACTIVATING GROUPS
MOST POTENT COMPOUND
B-16
5,6-benzoflavones
DEACTIVATING GROUPS
IC50 = 0.73 nM (CEase)
Figure 3. Structure Activity Relationship
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Figure 4. LineweaverꢀBurk plot of B-16
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Figure 5. Schematic 2D representations of hCEaseꢀB-16 complex showing Hꢀbond and van der
Waals interactions (Figure generated by LIGPLOT27)
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