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

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx

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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 b c

Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab 143005, India Lloyd Institute of Management and Technology, Greater Noida, UP, India Department of Chemistry, Guru Nanak Dev University, Amritsar, Punjab 143005, India

a r t i c l e

i n f o

Article history: 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 Docking studies

a b s t r a c t 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 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 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. Ó 2017 Elsevier Ltd. All rights reserved.

Cholesterol is a vital component of cell membrane and possesses many physiological functions. The greatest percentage of cholesterol is used in cytoplasm for bile acid synthesis.1 Hypercholesterolemia produced either by cholesterol feeding or by cholesterol-free, purified diets (‘‘endogenous” Hypercholesterolemia) results in the accumulation of cholesterol in adipose tissue. 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 including cancer, obesity, and diabetes has made control of plasma cholesterol a major health aim.3 Pancreatic cholesterol esterase is the member of a/b hydrolase family of proteins which catalyses the hydrolysis of dietary cholesterol ester into free cholesterol in the lumen of small intestine.4 It is also thought that the transport of cholesterol micelles to enterocytes 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

⇑ Corresponding authors.

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 thiazolidinediones,5 phosphorylated flavonoids,14 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), butyrylcholinesterase (BChE), Pseudomonas species lipase (PSL), chymotrypsin (CT) and trypsin.14 Flavonoids, including flavones, flavonols, isoflavones and flavanones, are a large class of polyphenolic compounds widely distributed in herbs and foods of plant origin, and exhibit diversified biological activities, such as antioxidant, anti-proliferative, anti-tumor, anti-microbial, estrogenic, acetyl cholinesterase, anti-inflammatory activities and are also used in cancer, cardiovascular disease, neurodegenerative disorders and enzyme inhibition.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

E-mail address: [email protected] (S. Sharma). http://dx.doi.org/10.1016/j.bmcl.2017.01.020 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

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H. Singh et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx

O

NR2 O

n Cl

N

S

X n

O

O

O R

O

O2N

n = 0, 1; X = O, CH2 R = Me, Et thieno[1,3]-oxazin-4-ones

n =0-4 6-chloro-2-pyrone

H N

O

O2N

OR1 P OR2 O

R1,R2 = Alkyl aryl phosphates

R = Alkyl carbamates

Cl O

H or OY

YO OH

R

H N

O

X

N H

O

O

Y = OP(O)(OEt)2 phosphorylated flavonoids

O S

X = S; R = CH2OCH2Ph (SerOBn) thiazolidinedione

R

O

N Ar R = NO2 ; Ar = Indole-3-yl phenylquinolines

m

O

X

NH

n

O m = 0-3; n = 1,2; X = H, NO2 chloroisocoumarins

O Y R2 X

P O

YO X

O

S

OR1

Ar

O

NH

X = H, MeO, Cl; Y = H, Cl O R1 = H, Et, Me; R2 = alkyl, aryl X = O, S phosphaisocoumarins thiazolidinediones

H or OY

Z

Y = OP(O)(OR)2 Z = H, OH, OP(O)(OR)2 R = Me, Et phosphorylated flavonoids

O

Cl

O

O

N N

N

NH 2-(1H-Indol-3-yl)-4-phenylquinolines

O

R

3-phenyl substituted 1,3, 4-oxadiazol-2(3H)-ones

Fig. 1. Reported CEase inhibitors.

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 benzoylchlorides (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 inhibitory 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 evaluated at concentrations of 1, 5, 10 and 25 nM in order to calculate their IC50 values (Table 1).

In view of various medicinal attributes of flavones, we here 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 benzoylated using various substituted benzoylchlorides 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 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 CH 3

1 (74%) O

b

O A-1 (89%)

O

O

O

OH CH 3

2 (80%)

c

O

O C H2

d

OH

OH

O

C H

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 Table 1 Various substituted benzoflavones with their IC50 value against CEase enzyme.

R2 R1

R3

O

R4 R5

O

Code

R1

R2

R3

R4

R5

IC50 (nM)

A-1 A-2 A-3 A-4 A-5 A-6 A-7 A-8 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

H H H OCH3 H H F H H F F H F H H Cl Br H H I H H H H H H H CF3 H CF3 F F CF3 H H H H H H H

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 OCOCH3 H

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 H H H F CH2Cl H N(CH3)2 H OCOCH3

H H H H H H H H H H H F F H H H H H H H H H H NO2 H H H H CF3 H H H F CF3 H H H H H H

H H H H H H H H 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

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 NA NA

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]chromen4-one nucleus) significantly influences the cholesterol esterase inhibitory activity. Placement of halogens on this phenyl ring considerably 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, trifloromethyl and acetoxy) on this phenyl ring favors the inhibitory activity, whereas, substitution of any activating group (dimethylamino, methoxy, methyl and trifloromethoxy) on this phenyl ring F

O

R

O

Overall preference order of the substituent for the inhibition of cholesterol esterase enzyme:

F

R = -F > -Cl > -Br > -I > -NO2 > -CH2Cl > -CF3 > -OCOCH3 > -H > -OCF3 > -CH3 > -OCH3 > -N(CH3)2 O BENZOFLAVONE

O DEACTIVATING GROUPS

ACTIVATING GROUPS

MOST POTENT COMPOUND A-12 IC50 = 0.78 nM (CEase)

Fig. 2. Structure activity relationship.

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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 > ACF 3 > AOCOCH3 > AH > AOCF3 > ACH3 > AOCH3 > AN(CH3)2 (Fig. 2). The most potent compound among the series (A-12) was further 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 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 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).

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H. Singh et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx

This confirms that the inhibitor preferentially interacts to the free enzyme and not to the enzyme substrate complex. In order to look into the interactions between the compound A12 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 Hbonds, polar and van der Walls interactions (Fig. 4a–c). Interestingly, 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 carbonyl carbon atom. The ring A, B and C were stabilized by van der Waals interaction with Ile323, Phe324 and His435.21 The difluorophenyl (ring D) gets positioned in a hydrophobic cavity created by Trp227, Leu282, Val285 and Leu392 residues. The ring D displayed 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. In conclusion, a library of 40 benzoflavone derivatives was successfully synthesized and characterized by using 1H NMR, 13C NMR, HRMS and Elemental Analysis. All the synthesized compounds were tested for their in vitro cholesterol esterase inhibitory 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 complex (mixed type inhibition). Docking study suggested that the compound A-12 fits well in the binding pocket of cholesterol esterase 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