Novel antioxidant bromophenols with acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase inhibitory actions

Bioorganic Chemistry 74 (2017) 104–114

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Novel antioxidant bromophenols with acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase inhibitory actions _ Necla Öztaskın, Parham Taslimi, Ahmet Marasß ⇑, Ilhami Gülcin, Süleyman Göksu ⇑ Department of Chemistry, Faculty of Science, Atatürk University, 25240 Erzurum, Turkey

a r t i c l e

i n f o

Article history: Received 10 June 2017 Revised 16 July 2017 Accepted 17 July 2017 Available online 20 July 2017 Keywords: Bromophenols Antioxidant activity Acetylcholinesterase Butyrylcholinesterase Carbonic anhydrase

a b s t r a c t In this study, a series of novel bromophenols were synthesized from benzoic acids and methoxylated bromophenols. The synthesized compounds were evaluated by using different bioanalytical antioxidant assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20 -azino-bis (3-ethylbenzothiazoline-6-sul phonic acid) (ABTS+) radical scavenging assays. Also, reducing power of novel bromophenols were evaluated by Cu2+-Cu+ reducing, Fe3+-Fe2+ reducing and [Fe3+-(TPTZ)2]3+-[Fe2+-(TPTZ)2]2+ reducing and ferrous ions (Fe2+) chelating abilities. The compounds demonstrate powerful antioxidant activities when compared to standard antioxidant molecules of a-tocopherol, trolox, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT). Also in the last part of this studies novel bromophenols were tested against some metabolic enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE) enzymes and carbonic anhydrase I, and II (hCA I and hCA II) isoenzymes. The newly synthesized bromophenols showed Ki values in a range of 6.78 ± 0.68 to 126.07 ± 35.6 nM against hCA I, 4.32 ± 0.23 to 72.25 ± 12.94 nM against hCA II, 4.60 ± 1.15 to 38.13 ± 5.91 nM against AChE and 7.36 ± 1.31 to 29.38 ± 3.68 nM against BChE. Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction Bromophenols are natural organic compounds and they are secondary metabolites of marine organisms [1]. Especially, diverse species algae such as red algae, brown algae, and green algae have a higher content of bromophenols [2]. In the last decades, there have been a large number of studies on the isolation and biological activities of bromophenols [2]. It can be seen from these studies that naturally occurring compound 1 shows feeding deterrent [3], anti-cancer [4], antioxidant [5], antidiabetic [6] properties. Aldose reductase inhibition of 2 and 3 [7], DPPH radical scavenging activity [8], protein tyrosine phosphatase [9] and hCA inhibitory actions [10] of 3 have been reported. In addition, antibacterial activity [11] and hCA inhibition [12] of compounds 4 and 5 have also been evaluated. Not only diarylmethane like natural bromophenols but also their diarylmethanone like synthetic derivatives have beneficial biological activities. In this context, we have reported the synthesis, antioxidant and acetylcholinesterase inhibition properties of compounds 6–10 [13] in a recent paper published in this journal (Fig. 1).

⇑ Corresponding authors. E-mail addresses: [email protected] (A. Marasß), [email protected] (S. Göksu). http://dx.doi.org/10.1016/j.bioorg.2017.07.010 0045-2068/Ó 2017 Elsevier Inc. All rights reserved.

In our earlier studies, we only reported the hCA inhibition [10,12] or antioxidant activity and acetylcholinesterase inhibition [13] of bromophenols. In continuation of our studies, we aimed to synthesize some synthetic bromophenols similar to natural product 1 and 2. However, the compounds reported in the current study contain only one Br group at the aromatic ring while compared with 1 and 2. On the other hand, we also declare the synthesis of some novel compounds similar to 6–10. Investigation of antioxidant, AChE, BChE and hCA inhibition properties of the bromophenols can lead to development of drug candidates. Therefore, here we extend our study on the synthesis, antioxidant activity, AChE, BChE and hCA inhibition of the title compounds. Reactive oxygen species (ROS) is a term used to describe the species that are common outcome of normal aerobic cellular metabolic processes. During daily activities and with advanced age, oxidative substances and free radicals accumulate in cells affecting various organs and systems in our body [14]. A free radical is defined as chemical species capable of independent existence, possessing one or more unpaired electrons. Overproductions of these ROS and free radicals are the results of chronic and other degenerative diseases in human bodies [15]. ROS are continuously produced during normal physiological events and can easily initiate the peroxidation of membrane lipids, leading to the accumulation of lipid peroxides [16]. Uncontrolled production of these free

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Br

Br

Br

Br

Br

HO

OH OH

Br Br Br

HO OH

OH 1

CH 2OR

HO

Br

OH

HO

Br

OH

R1

Br

R2

OH

R3

O

4 R=Me 5 R=Et

Br Br

OH

Br Br 3

2

Br OH

OH

Br

Br

OH OH

R4

R6

6 R 1=R 6=H, R4 =Br, R2 =R 3 =R 5=OH R 5 7 R 3=R 6=H, R 4 =Br, R 1=R 2=R 5=OH 8 R 1=R 2= H, R 5 =Br, R 3=R 4=R 6=OH 9 R 1=R 2=R3 =H, R5 =Br, R 4 =R 6=OH 10 R1 =R2 =H, R6 =Br, R 3=R 4=R 5=OH

Fig. 1. Biologically active natural and synthetic bromophenols.

radicals and ROS leads to attack on various biomolecules including lipids, proteins, enzymes and DNA, cellular machinery, cell membrane, causing oxidative stress and ultimately cell death. All aerobic organisms have antioxidant defenses, including antioxidant constituents and antioxidant enzymes for removing or repairing the damaged biomolecules [17]. This defense system against free radicals and ROS improved the taking sufficient amounts of exogenous antioxidants. An antioxidant compound, is a molecule capable of inhibiting the oxidation of other molecules, usually has the ability to scavenge free radicals and ROS [18]. Also, antioxidant compounds can scavenge hazardous effects of free radicals and increase shelf life by retarding the process of lipid peroxidation, which is one of the major reasons for deterioration of pharmaceutical and food products during processing and storage [19]. They can easily protect the human body from free radicals and ROS effects. A huge variety of biological active phenolic compounds contain one or more aromatic rings. It is well known that phenolic compounds had a large spectrum of biological activity including acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase inhibition effects [20]. Acetylcholinesterase (E.C. 3.1.1.7, AChE) plays an important role in cholinergic transmission by hydrolyzing the neurotransmitter acetylcholine (ACh) to acetate and choline [21]. AChE is a membrane-bound enzyme and present in the cholinergic neurons, brain, and muscles [22]. On the other hand, BChE is expressed in neuroglia and also found in the liver, heart, intestine, serum, kidney, and lung [23]. It plays an important role in the ester containing compounds metabolism. BChE also can hydrolyse ACh, and its levels do not decline, or may even increase in Alzheimer disease (AD) [24]. Normally in the brain AChE is predominant but BChE activity rises while AChE activity remains unchanged or diminishes in the brain of AD patients [25]. Both enzymes are responsible for the termination of cholinergic signalling by hydrolyzing ACh. Therefore, a drug inhibiting both enzymes may be preferable to selective ChE inhibitors (ChEIs). Recently, synthetic ChEIs, including galantamine, tacrine, rivastigmine, and donepezil have been used for clinical treatment of AD. The usage of these drugs has been limited because of their side effects such as gastrointestinal disturbance and hepatotoxicity [26]. Because of the adverse side effects of ChEIs, the development of non-toxic ChEIs as alternatives to synthetic drugs is of great interest among researchers. For this purpose, many novel ChEIs have been isolated or synthesized from natural bio resources [27]. Thus, the search for novel compounds remains important for the treatment of AD [28]. On the other hand, carbonic anhydrases (CAs, E.C. 4.2.1.1) represent a superfamily of metalloenzymes, which catalyse a crucial biochemical reaction, the rapid and reversible hydration of carbon dioxide (CO2) and water (H2O) to bicarbonate (HCO 3 ) and a proton

(H+) to maintain acid-base balance in blood and other tissues [29]. Also, they help transport CO2 out of tissues in living systems. CAs are expressed in most living organisms and are encoded by seven distinct gene families, including a-, b-, c-, d-, f-, n- and h-CAs [30]. All of CA families show changes in their preference for the catalytic metal ions located in the active site and catalyse the same reaction of CO2 hydration. However, each CA family demonstrates proper specific characteristics in the primary amino acid sequence. Also, they are dissimilar to molecular properties, such as oligomeric arrangement, cellular localization, distribution tissue and organs, expression levels, kinetic features, and response to different classes of inhibitors. a-CAs, which have sixteen isoenzymes are expressed predominantly in vertebrates and are the only class observed in humans [31]. Moreover, they have differences in tissue location and distribution; cytosolic (CA I, II, III, VII, and XIII), membrane-bound (CA IV, IX, XII, and XIV), mitochondrial (CA VA and VB), and secreted (CA VI) forms have been described. The CA IX and XII isoenzymes are known as the membrane CAs associated with cancers. The both isoenzymes have also been found in a very limited number of normal tissues, such as gastrointestinal mucosa and related structures [32]. An important role of CA IX and XII as major tumour prosurvival pH-regulating enzymes was suggested in genetic research. CA VIII, X and XI are non-catalytic due to the absence of one or more of the coordinating histidine residues and are termed CA related proteins [33].

2. Results and discussion 2.1. Chemistry Benzene derivatives give Friedel-Crafts acylation with benzoic acids [34] or benzylalchols [35] in the presence of polyphosphoric acid (PPA) By a similar approach, the reaction of benzoic acids 11, 12, 16 and 17 with methoxylated bromobenzenes 13 and 18 in the presence of PPA prepared from H3PO4 and P2O5 yielded diarylmethanones 14, 15, 19 and 20 in moderate yields. The synthesis of 19 [36] and 20 [36] has also been reported by a different method. We well know from one of our early study that bromobenzene 13 gives acylation reaction from para position of Br [13]. Further, 1H NMR data of synthesized diaryl methanones are also in accordance with the structures (Scheme 1). On the other hand, benzophenones 21–23 were synthesized as described previously [13]. The reduction of benzylic ketone (CO) group to methylene (CH2) group is one of the crucial steps; because of diaryl methanones described here incorporate Br. The reducing of CO may lead to debromination either. There are two methodologies described in the literature for this purpose. One of them is

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R1

O

R1

OMe

R2

OH

R3

800 C 90 min

Br R4

OMe

R3

Br R4

O

Br

O OH

OMe

14 R 1 =R 4=H, R 2=R 3=OMe 60% 15 R 1=H, R2 =R3 =R4 =OMe 62%

13

11 R 1=R 4=H, R2 =R 3=OMe 12 R 1=H, R2 =R 3 =R 4=OMe

OMe

R2

PPA

+

O

Br

PPA

+

OMe

R 16 R=H 17 R=OMe

800 C 90 min

R

OMe 19 R=H 52% 20 R=OMe 59%

18

Scheme 1. The synthesis of diarylmethanones 14, 15, 19 and 20.

tical, an antioxidant has been defined as any substance that is present in low concentrations compared to the oxidizable substrate, which significantly delays or inhibits the oxidation of that substrate [42]. DPPH is a stable nitrogen-bearing free radical that gives a maximal absorbance at 517 nm, which has been utilized to assess antioxidant capacity in those antioxidant candidates, which scavenge this free radical resulting in the decrease in absorbance [43]. The more popular synthetic antioxidants used are phenolic compounds such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate and tert-butylhydroquinone [44]. These antioxidants are the most commonly used for pharmacological and food applications. However, it was reported that some of them (BHA and BHT) have been restricted by legislative rules due to doubts over their carcinogenic and toxic effects [45]. Therefore, there is a growing interest in safer ones. In the present study we spectrophotometrically determined the antioxidant and antiradical potentials of novel bromophenols using different bioanalytical methods including Fe3+-reducing antioxidant assay, cupric ions (Cu2+)-reducing (Cupric) assay, Fe3+-TPTZ reducing assay, ferric ions (Fe2+) chelating activity, DPPH scavenging activity, and ABTS+-scavenging activity. The antioxidant activity of novel bromophenols was summarized in Tables 1 and 2. As can be seen in Schemes 1 and 2, diarylmethanones 14, 15, 19 and 20, and diarylmethanes 24–27, which do not contain any antioxidant phenolic rings, could not demonstrated effective antioxidant activity. Because of this reason, in this part of the study, we will only argue the antioxidant activity of novel bromophenols 28–35, which contain antioxidant phenolic groups.

based on the reduction of ketone to alcohol with NaBH4 followed by conversion of alcohol to Br with PBr3 and then the reduction of benzylic Br with Bu3SnH in the presence of AIBN [5]. This method includes long reaction sequences, which is not preferred. The second one is based on the direct conversation of CO to CH2 with Et3SiH in the presence of trifluoroacetic acid (CF3CO2H) [37]. Here the second method was chosen for the synthesis of diarylmethanes. The reduction of diarylmethanones 19, 21, 22 and 23 with Et3SiH in CF3CO2H at reflux temperature for 2.5 h gave 24 [38], 25 [39], 26 and 27 in moderate yields (Scheme 2). Demethylation of arylmethyl ethers is an important functional group transformation in synthetic organic chemistry and medicinal chemistry for the synthesis of target phenolic compounds [40]. It can conclude from early publications that BBr3 is one of the most efficient reagents for O-demethylation of arylmethyl ethers [35]. Therefore, diarylmethanones 14, 15, 19, 20 and diaryl methanes 24–27 were converted into their bromophenol derivatives 28–35. The synthesized arylmethyl ethers were reacted with BBr3 in CH2Cl2 firstly at 0 °C and then at 25 °C under N2 for 24 h. Followed by the addition of MeOH for quenching of excess BBr3 furnished bromophenols 28–35 in good yields. All bromophenols are new except 30 [41] (Scheme 3). The structures of all synthesized compounds were characterized by 1H and 13C NMR, elemental analysis and IR spectroscopic techniques. 2.2. Antioxidant activities An antioxidant compound is a molecule capable of inhibiting the oxidation of other molecules. In terms of food and pharmaceu-

R1

O

R5

Et3SiH

R2 R3

R6 R4

TFA reflux, 2.5 h

R7

19 R1 =R 2 = R 3 =R 4=R 7 = H, R 5 =Br, R6 = OMe 21 R1 =R 2 = R 3 =R 4=H, R 5 =R7 =OMe , R 6= Br 22 R1 =R 2 = R 4 =H, R 6 =Br, R3 =R5 =R 7 = OMe 23 R1 =R 4 = R 7 =H, R 5 =Br, R2 =R3 =R 6 = OMe

R1

R5

R4

R7

R2 R3

R6

24 R1 =R 2 = R 3 =R 4=R 7 = H, R 5 =Br, R6 = OMe 25 R1 =R 2 = R 3 =R 4=H, R 5 =R7 =OMe , R 6= Br 26 R1 =R 2 = R 4 =H, R 6 =Br, R3=R5 =R 7 = OMe 27 R1 =R 4 = R 7 =H, R 5 =Br, R2=R3 =R 6 = OMe

Scheme 2. The synthesis of diarylmethanes 24–27.

%74 %75 %77 %70

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R1

O

R1

R5

R2 R3

R6 R4

14 15 19 20

R7

1.BBr 3, CH2 Cl2 0-25 oC, 24 h

R2

2. MeOH 0 oC, 15 min.

R3

R1 =R4 =H,R6 =Br, R2 =R 3 =R 5=R 7=OMe R1 =H, R 6=Br, R 2=R 3=R4 =R5 =R7 =OMe R1 =R 2 =R 3=R 4=R 7=H, R5 =Br, R6 =OMe R1 =R 2 =R 4=R 7=H, R 5=Br, R3 =R6 =OMe R1

00 C-25 0C, 24 h R6 R4

R6 R7

28 R 1=R 4=H,R 6=Br, R 2=R 3 =R5 =R 7 =OH 93% 29 R 1=H, R 6=Br, R2 =R3 =R4 =R 5 =R 7=OH 72% 30 R 1=R 2=R 3=R 4=R7 =H, R5 =Br, R 6=OH 80% 31 R 1=R 2=R 4=R 7=H, R5 =Br, R 3 =R 6=OH 88%

BBr 3/CH2 Cl2

R3

R5

R4

R5

R2

O

R5

R4

R7

R3

R7

24 R1 =R 2 = R 3 =R 4=R 7 = H, R 5 =Br, R6 = OMe 25 R1 =R 2 = R 3 =R 4=H, R 5 =R7 =OMe , R 6= Br 26 R1 =R 2 = R 4 =H, R 6 =Br, R3 =R5 =R 7 = OMe 27 R1 =R 4 = R 7 =H, R 5 =Br, R2 =R3 =R 6 = OMe

R1 R2

R6

32 R1 =R 2 = R 3 =R 4=R 7 = H, R 5 =Br, R6 = OH 94% 33 R1 =R 2 = R 3 =R 4=H, R 5 =R7 =OH , R 6= Br 78% 34 R1 =R 2 = R 4 =H, R 6 =Br, R3 =R5 =R 7 = OH 91% 35 R1 =R 4 = R 7 =H, R 5 =Br, R2 =R3 =R 6 = OH 81%

Scheme 3. The synthesis of bromophenols 28–35.

Table 1 Determination of reducing power of same concentration (20 lg/mL) of novel bromophenols and their methylated precursors using by FRAP, CUPRAC and ferric ions (Fe3+) reducing methods. Antioxidants

BHA BHT a-Tocopherol Trolox 14 15 19 20 24 25 26 27 28 29 30 31 32 33 34 35

Fe3+-Fe2+ reducing

Cu2+-Cu+ reducing 2

k700

R

2.007 ± 0.005 1.551 ± 0.008 1.779 ± 0.009 1.371 ± 0.006 0.238 ± 0.006 0.203 ± 0.005 0.186 ± 0.004 0.196 ± 0.005 0.305 ± 0.005 0.338 ± 0.005 0.265 ± 0.004 0.253 ± 0.009 2.117 ± 0.006 1.984 ± 0.004 1.216 ± 0.003 1.435 ± 0.004 0.507 ± 0.005 1.886 ± 0.008 1.403 ± 0.008 2.069 ± 0.013

0.9828 0.9717 0.9918 0.9682 0.9924 0.9744 0.9688 0.9919 0.9525 0.9911 0.9998 0.9791 0.9740 0.9494 0.9933 0.9889 0.9618 0.9677 0.9783 0.9287

Fe3+-TPTZ reducing 2

k450

R

1.826 ± 0.004 1.709 ± 0.003 1.002 ± 0.004 0.780 ± 0.004 0.166 ± 0.009 0.157 ± 0.004 0.203 ± 0.004 0.195 ± 0.011 0.280 ± 0.002 0.331 ± 0.009 0.298 ± 0.006 0.182 ± 0.006 1.958 ± 0.004 1.775 ± 0.003 1.065 ± 0.004 0.987 ± 0.005 0.501 ± 0.009 1.502 ± 0.009 0.963 ± 0.013 2.397 ± 0.006

0.9913 0.9755 0.9655 0.9883 0.9622 0.9559 0.9938 0.9748 0.9770 0.9378 0.9690 0.9830 0.9619 0.9977 0.9770 0.9930 0.9494 0.9581 0.9174 0.9848

Reducing power of bioactive compounds reflects the electrondonating capacity and is associated with its antioxidant activity. A reducing agent is a substance that donates electrons and, thereby, causes another reactant to be reduced. Reducing ability of novel bromophenols 28–35 was evaluated by different reducing methods including Fe3+-reducing, cupric ions (Cu2+)-reducing (Cupric), and Fe3+-TPTZ reducing assays. First of all, the reducing capacity of novel bromophenols 28–35 was measured by the direct reduction of Fe[(CN)6]3 to Fe[(CN)6]2 [46]. The absorbance values of 20 lg/mL concentration of novel bromophenols 28–35 and standards decreased in the following order: 28 (2.117 ± 0.006, r2: 0.9740) > 35 (2.069 ± 0.013, r2: 0.9287) > BHA (2.007 ± 0.005, r2: 0.9828) > 29 (1.984 ± 0.004, r2: 0.9494) > 33 (1.886 ± 0.008, r2: 0.9677) > a-tocopherol 2 (1.779 ± 0.009, r : 0.9918)  BHT (1.551 ± 0.008, r2: 0.9717) > 31

k593

R2

1.570 ± 0.006 1.392 ± 0.008 1.602 ± 0.012 1.421 ± 0.009 0.382 ± 0.006 0.327 ± 0.007 0.298 ± 0.006 0.404 ± 0.004 0.331 ± 0.008 0.338 ± 0.012 0.344 ± 0.006 0.419 ± 0.008 1.541 ± 0.003 1.639 ± 0.006 0.701 ± 0.003 0.983 ± 0.007 0.716 ± 0.007 1.394 ± 0.009 1.089 ± 0.020 1.839 ± 0.016

0.9424 0.9140 0.9682 0.9911 0.9590 0.9633 0.9494 0.9929 0.9758 0.9484 0.9958 0.9639 0.9887 0.9819 0.9963 0.9742 0.9935 0.9829 0.9270 0.9299

(1.435 ± 0.004, r2:0.9889) > 34 (1.403 ± 0.008, r2: 0.9783) > Trolox (1.371 ± 0.006, r2: 0.9682) > 30 (1.216 ± 0.003, r2: 0.9933) > 32 (0.507 ± 0.005, r2:0.9618). In this method, addition of free Fe3+ to novel bromophenols 28–35 leads to the formation of the intense Perl’s Prussian blue complex, Fe4[Fe(CN)6]3, which has a strong absorbance at 700 nm [47]. Cu2+ reducing ability of antioxidant bromophenols was measured [48]. As can be seen in Table 1, the absorbance values of 20 lg/mL concentration of novel bromophenols 28–35 and standards decreased in the following order: 35 (2.397 ± 0.006, r2: 0.9848) > 28 (1.958 ± 0.004, r2: 0.9619) > BHA (1.826 ± 0.004, r2: 0.9913) > 29 (1.775 ± 0.003, r2: 0.9977) > BHT (1.709 ± 0.003, r2: 0.9755) > 33 (1.502 ± 0.009, r2: 0.9581) > 30 (1.065 ± 0.004, r2: 0.9770) > a-tocopherol (1.002 ± 0.004, r2: 0.9655)  31 (0.987 ± 0.005, r2: 0.9930) > 34 (0.963 ± 0.013, r2: 0.9174) >

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Table 2 Determination of half maximal concentrations (IC50) of novel bromophenols and their methylated precursors and standards for chelating methods. Antioxidant compounds BHA BHT a-Tocopherol Trolox 14 15 19 20 24 25 26 27 28 29 30 31 32 33 34 35 EDTA

scavenging 21.65 32.99 23.01 19.24 230.17 312.16 222.11 195.54 199.18 198.21 192.01 193.01 16.44 14.43 43.31 19.24 96.88 13.32 13.86 15.75 –

r2 0.9727 0.9810 0.9682 0.9902 0.9911 0.9618 0.9791 0.9889 0.9641 0.9719 0.9889 0.9688 0.9584 0.9944 0.9696 0.9770 0.9580 0.9919 0.9694 0.9824 –

Trolox (0.780 ± 0.004, r2: 0.9883) > 32 (0.501 ± 0.009, r2 : 0.9494). As the last reduction method, FRAP reducing power was used for screening of reducing bility of novel antioxidant bromophenols 28–35 [49]. The absorbance values of 20 lg/mL concentration of novel bromophenols 28–35 and standards decreased in the following order: 35 (1.839 ± 0.016, r2: 0.9299) > 29 (1.639 ± 0.006, r2: 0.9819) > atocopherol (1.602 ± 0.012, r2: 0.9682) > BHA (1.570 ± 0.006, r2: 0.9424) > 28 (1.541 ± 0.003, r2: 0.9887) > Trolox (1.421 ± 0.009, r2: 0.9911) > 33 (1.394 ± 0.009, r2: 0.9829) > BHT (1.392 ± 0.008, r2: 0.9140) > 34 (1.089 ± 0.020, r2: 0.9270) > 31 (0.983 ± 0.007, r2: 0.9742) > 32 (0.716 ± 0.007, r2: 0.9935)  30 (0.701 ± 0.003, r2: 0.9963). It was reported that the reducing results obtained from in vitro Cu2+ ion reducing measurements might be more efficiently extended to the possible reactions of antioxidants [49]. The radical scavenging capacity of novel bromophenols was determined and compared to that of BHA, BHT, a-tocopherol and trolox by using the DPPH and ABTS+ radical scavenging methods [50]. The results (Table 2) showed that the novel bromophenols were effective in scavenging the stable radical DPPH radical dot to the yellow colored non-radical form diphenyl picrylhydrazine (DPPH2), indicating the novel bromophenols 28–35 was active in DPPH radical dot radical scavenging. IC50 values of DPPH radical scavenging effect of novel bromophenols 28–35 and standards were found in the following order: 33 (13.32 lg/mL, r2: 0.9919)  34 (13.86 lg/mL, r2: 0.9694)  29 (14.43 lg/mL, r2: 0.9944) < 35 (15.75 lg/mL, r2: 0.9944) < 28 (16.44 lg/mL, r2: 0.9584) < 31 (19.24 lg/mL, r2: 0.9770) = Trolox (19.24 lg/mL, r2: 0.9902) < BHA (21.65 lg/mL, r2: 0.9727) < a-tocopherol (23.01 lg/mL, r2: 0.9692) < BHT (32.99 lg/mL, r2: 0.9810) < 30 (43.31 lg/mL, r2: 0.9696) < 32 (96.88 lg/mL, r2: 0.9580). The reason of lower IC50 values of DPPH radical scavenging effect of bromophenol 32 is due to it monophenol structure when compared to the other polyphenol structures of bromophenols. It is well known that ortho-substitution of -OH group with electrondonating groups can also increase the antioxidant activity [51]. Similarly, novel bromophenols 28–35 had effective ABTS+ radical scavenging effects. ABTS+ has an intrinsic absorbance at 734 nm [52]. The ABTS+ cation radical developed from its reaction with ABTS in water solution of K2S2O8 at room temperature for 12 h. The absorbance at 734 nm was measured for each sample

scavenging 11.36 8.77 7.37 6.72 125.11 130.26 122.68 117.01 69.30 86.62 98.88 77.01 6.55 6.86 26.65 7.35 23.11 6.86 5.08 7.71 –

,

radical scavenging and metal

r2

Metal chelating

r2

0.9693 0.9424 0.9595 0.9023 0.9792 0.9824 0.9604 0.9884 0.9868 0.9368 0.9213 0.9399 0.9596 0.9833 0.9930 0.9684 0.9298 0.9128 0.9758 0.9644 –

49.44 76.01 43.31 86.62 98.01 86.62 173.26 99.88 111.41 113.42 86.62 115.50 23.08 30.13 97.18 34.65 109.47 57.75 38.51 30.13 18.73

0.9804 0.9938 0.9107 0.9491 0.9084 0.9749 0.9880 0.9471 0.9555 0.9936 0.9636 0.9889 0.9727 0.9628 0.9591 0.9942 0.9704 0.9390 0.9549 0.9572 0.9424

relative to a blank. A decreased absorbance in a sample correlates to ABTS+ cation radical scavenging activity. IC50 values of ABTS+ scavenging effect of novel bromophenols 28–35 and standards were found in the following order: 34 (5.08 lg/mL, r2: 0.9758) < 28 (6.55 lg/mL, r2: 0.9596)  Trolox (6.72 lg/mL, r2: 0.9023)  29 (6.86 lg/mL, r2: 0.9833) = 33 (6.86 lg/mL, r2: 0.9128)  31 (7.35 lg/mL, r2: 0.9684)  a-tocopherol (7.37 lg/mL, r2: 0.9595)  35 (7.71 lg/mL, r2: 0.9944) < BHT (8.77 lg/mL, r2: 0.9424) < BHA (11.36 lg/mL, r2: 0.9693) < 32 (23.11 lg/mL, r2: 0.9298) < 30 (26.65 lg/mL, r2: 0.9930). Ferrous ion chelating activities of novel bromophenols 28–35, BHA, BHT, a-tocopherol and trolox are shown in Table 2. IC50 values of ferrous ions (Fe2+) chelating effect of novel bromophenols 28–35 and standards were decreased as following order: EDTA (18.73 lg/mL, r2: 0.9424) < 28 (23.08 lg/mL, r2: 0.9727) < 29 (30.13 lg/mL, r2: 0.9628) = 35 (30.13 lg/mL, r2: 0.9572) < 31 (34.65 lg/mL, r2: 0.9942) < 34 (38.51 lg/mL, r2: 0.9549) < atocopherol (43.31 lg/mL, r2: 0.9107) < BHA (49.44 lg/mL, r2: 0.9804) < 33 (57.75 lg/mL, r2: 0.9390) < BHT (76.01 lg/mL, r2: 0.99384) < Trolox (86.62 lg/mL, r2: 0.9491) < 30 (97.18 lg/mL, r2: 0.9591) < 32 (109.47 lg/mL, r2: 0.9704). Minimizing ferrous ions (Fe2+) may afford protection against oxidative damage by inhibiting production of ROS and molecular damage [53]. 2.3. Enzymes inhibition effects The a-CAs have been extensively studied due to their role in human physiology and disease pathology [54]. In living organisms, CA isoenzymes play a crucial role in a lot of physiological and biochemical processes including CO2 and HCO transportation 3 between tissues and respiratory surfaces, bone resorption, pH homeostasis, electrolyte transport in various epithelia, calcification and biosynthetic reactions including gluconeogenesis, ureagenesis and lipogenesis [55]. The CA I, and II isoenzymes are present at higher concentrations in the cytosol of erythrocytes. CA inhibitors (CAIs) are a class of chemicals or pharmaceuticals that suppress the CA activity. They are clinically used for treatment of some diseases including glaucoma and cancer. Additionally, it has lately demonstrated that they have potential as anti-obesity, diuretics and anti-infective drugs [56]. The hCA I isoenzyme is found in many tissues and occurs in high concentrations in the blood and gastrointestinal tract. It is involved

N. Öztaskın et al. / Bioorganic Chemistry 74 (2017) 104–114

in retinal and cerebral edema, and the inhibition of hCA I may be a valuable tool for fighting these conditions [57]. CA inhibitors are clinically used as diuretics, anticonvulsant, antiglaucoma, antiobesity and antitumor agents for decades [58]. For hCA I, the Ki values were found in a range of 6.78 ± 0.68–126.07 ± 35.6 nM. In comparison, the Ki for the broad-spectrum CA inhibitor AZA, an efficient hCA II inhibitor, was 145.96 ± 29.6 nmol/L against hCA I. All novel antioxidant bromophenols had effective inhibition effects than that of AZA. Also, among novel antioxidant bromophenols, (4-bro mo-2,5-dimethoxyphenyl)(3,4-dimethoxyphenyl)methanone (14), which had four methoxy groups (-OMe) and one bromine group (-Br), was the best hCA I inhibitor (Ki 6.78 ± 0.68 nM). It is well known that compounds containing carbonyl (-CO) and halogen groups are effective CA inhibitors [59]. Human CA isoenzyme II (hCA II) is an ubiquitous cytosolic isoform [60]. Its role in diseases such as glaucoma has been well characterized. Indeed, HCO 3 production serves as a mechanism to transport sodium ions (Na+) into the eye along with the influx of water leading to an increase in intraocular pressure [61]. CA II inhibition decreases HCO 3 production and subsequently aqueous humor secretion, which leads to decreased pressure in the eye [62]. The physiologically dominant cytosolic isoform, hCA II, which showed a very interesting inhibition profile with the novel antioxidant bromophenols reported here. The inhibitory profile of novel antioxidant bromophenols against physiologically relevant isoenzymes hCA I, and II isoforms was assessed by esterase activity assay as reported earlier. Novel antioxidant bromophenols synthesized in this study effectively inhibited hCA II with Ki in the low nanomolar range. Ki values were found between 4.32 ± 0.23 and 72.25 ± 12.94 nM. On the other hand, (2-bromo-4-hydroxyphe nyl)(4-hydroxyphenyl)methanone (31) is in fact the best inhibitor in this series, being 21.4 times a better hCA II inhibitor compared to the clinical candidate drug (Ki of AZA: 92.63 ± 10.91 nM). Another interesting case is 14, 15 and 19, which had methoxy (AOMe) and bromine (ABr) groups. These bromophenols are also an effective hCA II inhibitor, being 18 times more effective than AZA in inhibiting hCA II isoenzyme. The acetylcholinesterase inhibitors (AChEIs) and butyrylcholinesterase inhibitors (BChEIs) are used for blocking the cholinergic degradation of acetylcholine (Ach) or butyrylcholine (Bch). Therefore, they are considered to be a promising approach for the treatment of AD. Also, it was known that tacrine, which used for the treatment of AD and various other memory impairments [63], has been shown to lower AChE/BChE inhibition effects (Kis: 45.01 ± 9.50/42.40 ± 4.37 nM). In our study, AChE was also highly inhibited by novel antioxidant bromophenols at the low nanomolar inhibition with Ki values in range of 4.60 ± 1.15–38.13 ± 5.91 nM (Table 3). These results clearly showed that novel synthesized bromophenols had effective AChE inhibition properties. However, the most powerful AChE inhibition was observed by novel bromophenol 31 with Ki value of 4.60 ± 1.15 nM. Also, all the remaining newly synthesized bromophenols reported here were highly efficient inhibition constants against AChE. On the other hand, tacrine (1,2,3,4-tetrahydroacri din-9-amine), which first centrally acting cholinesterase inhibitor approved for the treatment of AD demonstrated Ki value of 45.01 ± 9.50 nM against cholinergic AChE. Tacrine has since been removed from the market due to adverse effects, including hepatotoxicity in a significant percentage of patients [64]. Just like the AChE, BChE has a specific role in cholinergic neurotransmission and it has been associated with AD. Based on this, the strategy of inhibiting the cholinesterase enzymes responsible for ACh degradation, AChE and BChE, are proved more successful and are currently as backbone of AD pharmacotherapy [64]. ChEIs differ from each other with respect to their pharmacologic

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properties. Novel antioxidant bromophenols inhibited BChE with Ki values in a range of 7.36 ± 1.31–29.38 ± 3.68 nM. The Ki values of novel antioxidant bromophenols for AChE and BChE were calculated from Lineweaver-Burk plots [65]. On the other hand, tacrine, which was the first cholinesterase inhibitor to be approved for the management of AD symptoms in 1993, had Ki value of 42.40 ± 4.37 nm. Besides that it was reported that donepezil hydrochloride, which is used for the treatment of mild-tomoderate AD and various other memory impairments, had been shown to lower AChE inhibition activity (IC50: 55.0 nM) [28]. The compounds prepared in this study contained carbonyl, methoxy groups as well as bromine atoms, which all have important roles in biological systems. Additionally, these compounds are soluble in polar solvents such as ethanol and methanol. Therefore, these compounds will be easy to prepare and use in clinical applications. These lower values were originated from lower molecular polarity of the compounds, which contains methoxy groups while compared with ketones or phenolic compounds.

3. Conclusion In summary, a series of diarylmethanones including methoxy and bromine groups were obtained from benzoic acids and methoxylated bromobenzenes. Some of these diarylmethanones were converted into their diarylmethane derivatives 24–27. As phenolic compounds are more potent biological active than their methoxylated derivatives, compounds 14, 15, 19, 20 and 24–27 were converted into phenolic derivatives for their biological activities. Also, there seems to be a significant difference between the activity of the ketones (compounds 14, 15, 19, 20, 28–31) and the arylmethanes (compounds 24–27, 32–35). Generally the arylmethanes seem less active. This situation could perhaps be due to some binding interaction of the ketone group, or the greater flexibility of the arylmethanes. All synthesized novel compounds may be important synthons for further synthetic and biological manners. Additionally, the synthesized compounds were evaluated for antioxidant activity and AChE, BChE, hCA I, and hCA II enzymes inhibition effects.

4. Experimental 4.1. General All chemicals and solvents are commercially available and were used without purification or after distillation and treatment with drying agents. Melting points are uncorrected and they were determined on a capillary melting apparatus (Buechi 530). IR spectra were obtained from solutions in 0.1 mm cells with a PerkinElmer spectrophotometer. The 1H and 13C NMR spectra were recorded on a 400 (100)-MHz Varian and 400 (100)-MHz Bruker spectrometer; d in ppm, Me4Si as the internal standard. Elemental analyses were performed on a Leco CHNS-932 apparatus. All column chromatography was performed on silica gel (60-mesh, Merck). PLC is preparative thick-layer chromatography: 1 mm of silica gel 60 PF (Merck) on glass plates. All biologically evaluated compounds were demonstrated to exist in >95%purity by elemental analysis. The 1H NMR and 13C NMR spectra as well as the InChI codes of the investigated compounds are provided as Supporting Information. 4.2. Chemistry The synthesis of compound 13 was performed according to the procedure described by Etienne et al. [66].

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N. Öztaskın et al. / Bioorganic Chemistry 74 (2017) 104–114

Table 3 Human carbonic anhydrase I, and II (hCA I, and II) isoenzymes, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes inhibition effects of novel antioxidant bromophenols. Compounds

14 15 19 20 24 25 26 27 28 29 30 31 32 33 34 35 AZA* TACW *

W

IC50 (nM)

Ki (nM)

hCA I

r2

hCA II

r2

AChE

r2

BChE

r2

hCA I

hCA II

AChE

BChE

5.68 5.77 7.61 7.51 95.36 92.25 80.32 80.40 7.87 8.15 8.45 6.79 72.88 62.65 49.50 57.08 152.61 –

0.9723 0.9692 0.9711 0.9748 0.9612 0.9605 0.9596 0.9497 0.9789 0.9666 0.9905 0.9788 0.9738 0.9764 0.9726 0.9861 0.9893 –

5.29 5.05 5.58 6.07 77.70 72.09 60.84 65.68 9.24 5.97 6.93 6.18 49.04 25.21 27.15 26.03 114.80 –

0.9985 0.9579 0.9846 0.9758 0.9793 0.9753 0.9647 0.9492 0.9694 0.9727 0.9650 0.9979 0.9887 0.9821 0.9784 0.9970 0.9869 –

15.40 8.77 21.63 15.75 27.01 14.65 21.83 15.20 20.93 24.75 10.82 13.07 37.02 53.97 44.16 50.07 – 110.58

0.9774 0.9679 0.9893 0.9740 0.9735 0.9620 0.9766 0.9826 0.9685 0.9919 0.9755 0.9913 0.9952 0.9772 0.9964 0.9823 – 0.9735

24.66 23.89 21.75 26.65 58.87 55.75 52.98 40.64 31.51 21.48 22.35 30.13 41.88 21.48 25.96 27.37 – 101.47

0.9880 0.9947 0.9745 0.9890 0.9856 0.9851 0.9830 0.9894 0.9922 0.9748 0.9940 0.9882 0.9899 0.9769 0.9821 0.9626 – 0.9834

6.78 ± 0.68 7.62 ± 1.50 7.88 ± 1.04 9.02 ± 2.67 87.35 ± 19.94 126.07 ± 35.6 111.01 ± 25.4 117.82 ± 31.8 10.065 ± 3.50 9.28 ± 2.11 8.44 ± 1.38 8.15 ± 3.47 93.48 ± 43.05 78.01 ± 12.32 67.30 ± 32.90 40.92 ± 4.74 145.96 ± 29.6 –

5.23 ± 1.34 5.64 ± 1.61 5.05 ± 0.75 7.01 ± 1.66 70.25 ± 18.26 72.25 ± 12.94 53.48 ± 20.70 53.05 ± 13.42 8.24 ± 1.72 6.15 ± 1.50 6.17 ± 2.34 4.32 ± 0.23 57.30 ± 18.13 18.41 ± 3.22 27.44 ± 4.82 22.84 ± 5.75 92.63 ± 10.91 –

7.02 ± 2.97 5.01 ± 0.64 11.39 ± 1.65 5.16 ± 0.02 16.88 ± 2.12 8.51 ± 2.81 12.43 ± 1.42 12.12 ± 3.80 9.99 ± 1.01 9.31 ± 1.60 7.81 ± 3.54 4.60 ± 1.15 24.48 ± 5.33 23.74 ± 4.04 17.01 ± 4.40 38.13 ± 5.91 – 45.01 ± 9.50

7.36 ± 1.31 10.32 ± 2.84 8.39 ± 1.91 9.87 ± 2.52 23.43 ± 4.57 24.30 ± 5.72 29.38 ± 3.68 15.13 ± 2.16 8.65 ± 0.95 8.79 ± 2.84 7.42 ± 0.90 14.99 ± 1.93 15.53 ± 2.23 11.38 ± 2.26 7.80 ± 2.63 18.28 ± 2.37 – 42.40 ± 4.37

Acetazolamide (AZA) was used as a standard inhibitor for both carbonic anhydrase I, and II (hCA I and II) isoenzymes. Tacrine (TAC) was used as a standard inhibitor for acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes.

4.2.1. General procedure for the synthesis of diarylmethanones [Synthesis of (4-bromo-2, 5-dimethoxyphenyl)(3, 4-dimethoxyphenyl) methanone (14)] PPA was prepared by mixing of H3PO4 (6.4 g, 64.77 mmol) and P2O5 (11.5 g, 81.24 mmol) in a vessel (100 mL) at 80 °C for 15 min. Then 2-bromo-1, 4-dimethoxy benzene (13) (1.67 g, 7.68 mmol) and 3,4-dimetoxybenzoic acid (11) (2.0 g, 10.98 mmol) were added to this mixture quickly. The blend was stirred with a glass stick at 80 °C for 1.5 h. After completion of the reaction, H2O (30 mL) was added to the mixture and it was extracted with EtOAc (2  125 mL). The combined organic phases were dried over Na2SO4. After the solvent was evaporated, the residue was crystallized with EtOAc-hexane to give 14 (2.5 g, 60% yield) as cream crystals. Mp: 118–120 °C. 1 H NMR (400 MHz, CDCl3) d 7.53 (d, 1H, J = 2.1 Hz, Ar-H), 7.27 (dd, 1H, J = 8.5, 2.1 Hz, Ar-H), 7.20 (s, 1H, Ar-H), 6.89 (s, 1H, ArH), 6.83 (d, 1H, J = 8.5 Hz, Ar-H), 3.94 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.84 (s, 3H, OCH3) 3.69 (s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) d 194.1(C@O), 153.9(CO), 151.4(CO), 150.30(CO), 149.2 (CO), 130.5(C), 129.0(C), 126.1(CH), 117.6(CH), 114.2(CBr), 112.9 (CH), 111.1(CH), 110.0(CH), 57.1(OCH3), 56.8(OCH3), 56.3(OCH3), 56.3(OCH3). IR (cm1, CH2Cl2): 3003, 2939, 2841, 1711, 1655, 1595, 1513, 1491, 1418, 1268, 1175, 1146, 1024, Anal. Calcd. for C17H17BrO5: C 53.56; H 4.49, Found: C 52.22; H 4.53. 4.2.2. 4-Bromo-2, 5-dimethoxyphenyl)(3,4,5-trimethoxyphenyl) methanone (15) Yield 62% brown solid. Mp: 102–104 °C. 1H NMR (400 MHz, CDCl3): d 7.26 (s, 1H, Ar-H), 7.08 (s, 2H, Ar-H), 6.92 (s, 1H, Ar-H), 3.94 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.85 (s, 6H, OCH3), 3.72 (s, 3H, OCH3) 13C NMR (100 MHz, CDCl3): d 194.0(C@O), 152.9(2CO), 151.3(CO), 150.2(CO), 142.9(CO), 132.4(C), 128.3(C), 117.4(CH), 114.5(CBr), 112.8(CH), 107.4(2CH), 60.9(OCH3), 56.9(OCH3), 56.6 (OCH3), 56.3(2OCH3). IR (cm1, CH2Cl2): 2997, 2936, 2835, 1712, 1664, 1583, 1493, 1460, 1414, 1381, 1329, 1216, 1127. Anal. Calcld. for C18H19BrO6: C 52.57; H 4.66, Found: C 52.65; H 4.69. 4.2.3. (2-Bromo-4-methoxyphenyl)(phenyl)methanone (19) Yield 52%, white crystals; Mp: 85-87 °C. Lit. Mp: 86–88 °C [36]. 1 H NMR and 13C NMR data are in agreement with data given in the literature [36].

4.2.4. (2-Bromo-4-methoxyphenyl)(4-methoxyphenyl)methanone (20) Yield: 59% white solid. Mp: 48–50 °C. Lit. Mp: 46–48 °C [36]. 1H NMR and 13C NMR data are in agreement with data given in the literature [36].

4.2.5. General procedure for the synthesis of diarylmethanes [Synthesis of (1-benzyl-2-bromo-4-methoxybenzene) (24)] Et3SiH (4.39 mL, 27.48 mmol) was added to a solution of 19 (2 g, 6.87 mmol) in TFA (10 mL) and the reaction mixture was refluxed for 2.5 h under N2 atmosphere. After termination of the reaction, TFA was removed by evaporation. Na2CO3 solution was added to the residue to make the ambient basic. This solution was extracted with EtOAc (3  50 mL). Combined organic layers were washed with dilute HCl solution (20 mL) and it was dried over Na2SO4. After evaporation of the solvent, the residue was purified on 20 g silica gel column with 3–5% EtOAc-hexane. 1-benzyl-2-bromo-4methoxybenzene (24) was obtained (1.4 g, 74% yield) as a colorless liquid. 1 H NMR (400 MHz, CD3COCD3): d 7.58–7.46 (2H, m, Ar-H), 7.44–7.34 (m, 4H, Ar-H), 7.25 (dd, 1H, J = 11.3, 8.5 Hz, Ar-H), 6.97 (dd, 1H, J = 11.3, 8.5 Hz, Ar-H), 4.26 (d, 2H, J = 13.9 Hz, CH2), 3.89 (s, 3H, OCH3). 13C NMR (100 MHz, CD3COCD3): d 158.8 (CO), 140.2 (C), 132.6 (C), 131.6 (CH), 128.7 (CH), 128.6 (CH), 126.4 (CH), 125.2 (CBr), 118.2 (CH), 113.9 (CH), 55.6 (OCH3), 41.1 (CH2). IR (cm1, CH2Cl2): 3026, 2958, 2936, 2835, 1605, 1567, 1493, 1453, 1438, 1312, 1329, 1284, 1037. Anal. Calcld. for C14H13BrO: C, 60.67; H, 4.73. Found: C 60.12; H 4.71.

4.2.6. 1-Benzyl-4-bromo-2, 5-dimethoxybenzene (25) Yield: 75% brown crystals. Mp: 61–63 °C. 1H NMR (400 MHz, CDCl3): d 7.32–7.23 (m, 2H, Ar-H), 7.21–7.17 (m, 3H, Ar-H), 7.05 (s, 1H, Ar-H), 6.66 (s, 1H, Ar-H), 3.92 (s, 2H, CH2), 3.76 (s, 6H, OCH3). 13C NMR (100 MHz, CDCl3): d 151.9 (CO), 150.1 (CO), 140.3 (C), 129.9 (C), 128.8 (CH), 128.4 (CH), 126.1 (CH), 116.6 (CH), 114.9 (CH), 109.1 (CBr), 56.9 (OCH3), 56.2 (OCH3), 35.9 (CH2). IR (cm1, CH2Cl2): 3026, 2938, 2840, 1602, 1494, 1463, 1453, 1305, 1214, 1178, 1036. Anal. Calcld. for C15H15BrO2: C 58.65; H 4.92. Found: C 57.95; H 4.88.

N. Öztaskın et al. / Bioorganic Chemistry 74 (2017) 104–114

4.2.7. 1-Bromo-2, 5-dimethoxy-4-(3-methoxybenzyl)benzene (26) Yield: 77% yellow crystals. Mp: 67–69 °C. 1H NMR (400 MHz, CDCl3), d 7.11 (d, 2H, J = 8.4 Hz, Ar-H), 7.05(s, 1H, Ar-H), 6.83(d, 2H, J = 8.4 Hz, Ar-H), 6.66(s, 1H, Ar-H), 3.87 (s, 2H, CH2), 3.78 (3H, s, OCH3), 3.77 (s, 6H, OCH3). 13C NMR (100 MHz, CDCl3) d 158.1 (CO), 152.0 (CO), 150.2 (CO), 132.5 (C), 130.5 (2CH), 129.9 (C), 116.1 (C), 115.0 (CH), 114.0 (2CH), 109.1 (CBr), 57.1 (OCH3), 56.4 (OCH3), 55.4 (OCH3), 35.2 (CH2). IR (cm1, CH2Cl2): 2999, 2935, 2835, 1611, 1583, 1495, 1463, 1440, 1301, 1245, 1176, 1035. Anal. Calcld. for C16H17BrO3: C 56.99; H 5.08. Found: C 56.12; H 5.00.

4.2.8. 2-Bromo-1-(3, 4-dimethoxybenzyl)-4-methoxybenzene (27) Yield: 70% yellow crystals. Mp: 45–47 °C. 1H NMR (400 MHz, CDCl3), d 7.13 (d, 1H, J = 2.6 Hz, Ar-H), 7.02(d, 1H, J = 8.5 Hz, ArH), 6.78(dd, 2H, J = 8.5, 2.6 Hz, Ar-H), 6.75–6.66(m, 2H, Ar-H), 3.98 (s, 2H, CH2), 3.84(s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.78(s, 3H, OCH3). 13C NMR (100 MHz, CDCl3) d 158.7 (CO), 149.1 (CO), 147.6 (CO), 132.9 (C), 132.8 (C), 131.4 (CH), 125.0 (CBr), 121.1 (CH), 118.1 (CH), 113.8 (CH), 112.4 (CH), 111.4 (CH), 56.1 (OCH3), 56.0 (OCH3), 55.4 (OCH3), 40.6 (CH2). IR (cm1, CH2Cl2): 2999, 2936, 2906, 1604, 1566, 1492, 1463, 1439, 1259, 1237, 1183, 1029. Anal. Calcld. for C16H17BrO3: C 56.99; H 5.08. Found: C 56.22; H 4.99.

4.2.9. General procedure for the synthesis of bromophenols [Synthesis of (4-bromo-2, 5-dihydroxyphenyl)(3, 4-dihydroxyphenyl)methanone (28)] (4-Bromo-2, 5-dimethoxyphenyl)(3, 4-dimethoxyphenyl) methanone (14) (0.5 g, 1.31 mmol) was dissolved in anhydrous DCM (50 mL) and chilled to 0 °C under nitrogen. BBr3 (2.63 g, 10.49 mmol) was added to this stirred solution drop wise. After it was stirred at this temperature for 30 min, the reaction mixture was allowed to warm to room temperature. The mixture was stirred for 24 h. at rt. The reaction mixture was cooled to 0 °C. Methanol (30 mL) was added to the mixture drop wise. The residue was dissolved in EtOAc (30 mL) and H2O (20 mL) was added to the solution. The organic layer was separatedH2O phase was extracted with EtOAc (2  30mL). The organic layers were combined and dried over Na2SO4. Removal of the solvent gave the crude product 28 (0.4 g, 94% yield), which was crystallized with EtOAc-n-hexane to give brown crystals. Mp: 159–161 °C. 1H NMR (400 MHz, CD3OD) d 7.25 (d, 1H, J = 2.2 Hz, Ar-H), 7.19 (dd, 1H, J = 8.2, 2.2 Hz, Ar-H) 7.16 (s, 1H, Ar-H), 7.12 (s, 1H, Ar-H), 6.88 (d, 1H, J = 8.2 Hz, Ar-H), 4.88 (bs, 4H, OH and overlapping with OH coming from CD3OD). 13C NMR (100 MHz, CD3OD) d 199.5 (C@O), 154.8 (CO), 151.8 (CO), 147.6 (CO), 146.4 (CO), 130.6 (C), 124.5 (CH), 122.5 (CH), 122.4 (C), 118.9 (CH), 118.1 (CBr), 117.6 (CH), 115.8 (CH). IR (cm1, CH2Cl2): 3202, 2934, 1709, 1588, 1440, 1326, 1292, 1123, 1111, 1013, 957 Anal. Calcd. for C13H9BrO5: C 48.03; H 2.79, Found: C 47.85; H 2.76.

4.2.10. (4-Bromo-2, 5-dihydroxyphenyl)(3, 4, 5-trihydroxyphenyl) methanone (29) Yield: 82% brown solid Mp: 265–267 °C. 1H NMR (400 MHz, CD3 COCD3) d 11.13(s, 1H, OH) 8.69 (s, 1H, OH) 8.43 (s, 2H, OH), 8.26 (s, 1H, OH), 7.32 (s, 1H, Ar-H), 7.21 (s, 1H, Ar-H), 6.87–6.86 (m, 2H, Ar-H). 13C NMR (100 MHz, CD3COCD3) d 198.7 (C@O), 155.4 (2CO), 146.2 (CO), 145.5 (CO), 138.1 (CO), 128.6 (C), 121.8 (CH), 121.7 (CH), 119.0 (C), 118.6 (CH), 117.9 (CBr), 109.6 (2CH) IR (cm1, CH2Cl2): 3369, 2993, 1697, 1611, 1586, 1441, 1357, 1190, 1113, 1023, 867 Anal. Calcd. for C13H9BrO6: C 45.77; H 2.66, Found: C 45.32; H 2.61.

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4.2.11. (2-Bromo-4-hydroxyphenyl)(phenyl)methanone (30) Yield: 90% brown solid. Mp: 106–108 °C. 1H NMR (400 MHz, CD3OD) d 7.77 (d, 2H, J = 8.4, Ar-H), 7.66 (t, 1H, J = 8.4 Hz, Ar-H), 7.51 (t, 2H, J = 8.4 Hz, Ar-H), 7.28 (d, 1H, J = 8.4, Ar-H), 7.14 (d, 1H, J = 2.1 Hz, Ar-H), 6.89 (dd, 1H, J = 8.4, 2.1 Hz, Ar-H), 4.88 (bs, 1H, OH overlapping with OH coming from CD3OD), 13C NMR (100 MHz, CD3OD) d 196.6 (C@O), 160.4 (CO), 137.3 (C), 133.4 (CH), 131.3 (C), 131.2 (CH), 129.9 (2CH), 128.5 (2CH), 120.6 (CBr), 119.9 (CH), 114.2 (CH), IR (cm1, CH2Cl2): 3216, 2919, 2852, 1648, 1597, 1579, 1493, 1282, 1036, 933, 871. Anal. Calcd. for C13H9BrO2: C 56.34; H 3.27, Found: C 56.49; H 3.30. 4.2.12. (2-Bromo-4-hydroxyphenyl)(4-hydroxyphenyl)methanone (31) Yield: 88% cream crystals. Mp: 226–228 °C. 1H NMR (400 MHz, CD3OD) d 7.67(d, 2H, J = 8.5 Hz Ar-H), 7.25 (d, 1H, J = 8.5 Hz, Ar-H), 7.18 (d, 1H, J = 2.3 Hz, Ar-H), 6.89–6.85 (m, 3H, Ar-H), 4.88 (bs, 2H, OH overlapping with OH coming from CD3OD). 13C NMR (100 MHz, CD3OD) d 195.6 (C@O), 163.2 (CO), 159.8 (CO), 132.8 (C), 131.8 (2CH), 130.6 (CH), 128.6 (C), 120.2 (CBr), 119.7 (CH), 115.1 (2CH), 114.1 (CH), IR (cm1, CH2Cl2): 3202, 2994, 1636, 1599, 1439, 1315, 1286, 1228, 1032, 934. Anal. Calcld. for C13H9BrO3: C 53.27; H 3.09, Found: C 52.95; H 3.07. 4.2.13. 4-Benzyl-3-bromophenol (32) Yield: 94% yellow oil. 1H NMR (400 MHz, CD3OD), d 8.65 (s, 1H, OH), 7.34–7.27 (m, 2H, Ar-H), 7.22–7.17(m, 3H, Ar-H), 7.13 (d, 1H, J = 2.5 Hz, Ar-H), 7.11 (d, 1H, J = 8.5 Hz, Ar-H), 6.84 (dd, 1H, J = 8.5, 2.5 Hz, Ar-H), 4.88 (bs, 1H, OH overlapping with OH coming from CD3OD), 4.26 (s, 2H, CH2). 13C NMR (100 MHz, CD3OD) d 157.5 (CO), 141.2 (C), 132.7 (C), 131.9 (CH), 129.6 (2CH), 129.2 (2CH), 126.8 (CH), 125.2 (CBr), 120.2 (CH), 115.9 (CH), 41.2 (CH2). IR (cm1, CH3COCH3): 3210, 3025, 2958, 1605, 1585, 1491, 1451, 1434, 1227, 1195, 1073, 1030. Anal. Calcld. for C13H11BrO: C 59.34; H 4.21. Found: C 58.88; H 4.10. 4.2.14. 2-Benzyl-5-bromobenzene-1, 4-diol (33) Yield: 78% brown crystals. Mp: 104–106 °C. 1H NMR (400 MHz, CD3OD), d 7.27 (d, 2H, J = 7.5 Hz, Ar-H), 7.21(d, 2H, J = 7.5 Hz, Ar-H), 7.16(t, 1H, J = 7.5 Hz, Ar-H), 6.91(s, 1H, Ar-H), 6.56(s, 1H, Ar-H), 4.88 (bs, 2H, OH overlapping with OH coming from CD3OD), 3.85 (s, 2H, CH2). 13C NMR (100 MHz, CD3OD) d 149.7 (CO), 148.0 (CO), 142.1 (C), 130.6 (C), 130.0 (2CH), 129.3 (2CH), 126.9 (CH), 119.7 (CH), 118.9 (CH), 107.7 (CBr), 35.9 (CH2). IR (cm1, CH3COCH3): 3340, 3193, 2924, 1600, 1494, 1409, 1314, 1195, 1072, 1032. Anal. Calcld. for C13H11BrO2: C 55.94; H 3.97. Found: C 54.80; H 3.88. 4.2.15. 2-Bromo-5-(4-hydroxybenzyl)benzene-1, 4-diol (34) Yield: 91% brown crystals. Mp: 141–143 °C. 1H NMR (400 MHz, CD3OD), d 7.04 (d, 2H, J = 8.2 Hz, Ar-H), 6.92(s, 1H, Ar-H), 6.72(d, 2H, J = 8.2 Hz, Ar-H), 6.55(s, 1H, Ar-H), 4.88 (bs, 2H, OH overlapping with OH coming from CD3OD), 3.75 (s, 2H, CH2). 13C NMR (100 MHz, CD3OD) d 156.4 (CO), 149.7 (CO), 147.9 (CO), 132.8 (C), 131.6 (2CH), 130.8 (C), 119.8 (C), 118.9 (CH), 116.1 (2CH), 107.5 (CBr), 35.5 (CH2). IR (cm1, CH3COCH3): 3204, 2924, 1608, 1511, 1496, 1413, 1191, 1072, 1030. Anal. Calcld. for C13H11BrO3: C 52.91; H 3.76. Found: C 52.25; H 3.70. 4.2.16. 4-(2-Bromo-4-hydroxybenzyl)benzene-1, 2-diol (35) Yield: 81% dirty white. Mp: 157–159 °C. 1H NMR (400 MHz, CD3COCD3), d 8.39 (s, 1H, OAH), 7.54 (s, 1H, OAH), 7.47 (s, 1H, OAH), 6.98–6.87(m, 2H, Ar-H), 6.59 (d, 1H, J = 8.2 Hz, Ar-H), 6.59 (d, 1H, J = 8.2 Hz, Ar-H), 6.51(s, 1H, Ar-H), 6.40 (d, 1H, J = 8.2 Hz, Ar-H), 3.72(s, 2H, CH2). 13C NMR (100 MHz, CD3COCD3) d 157.4 (CO), 145.8 (CO), 144.2 (CO), 132.8 (C), 136.6 (C), 132.5 (CH),

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125.0 (CBr), 120.9 (CH), 120.1 (CH), 116.1 (CH), 115.8 (CH), 116.6 (CH), 40.5 (CH2). IR (cm1, CH3COCH3): 3213, 2924, 1605, 1522, 1491, 1440, 1282, 1230, 1109, 1030. Anal. Calcld. for C13H11BrO3: C 52.91; H 3.76. Found: C 51.65; H 3.69. 4.3. Biochemical studies 4.3.1. Antioxidant assays For determination of Fe3+ reducing ability of novel antioxidant bromophenols, Fe3+(CN)6 to Fe2+(CN)6 reduction method was used. Spectrophotometer measurement was realized at 700 nm [67]. Cu2+ reducing power was used as a second reducing ability method for novel antioxidant bromophenols. Cu2+ reducing capability was performed according to the method of Apak et al. [68] using ethanolic neocuproine solution. Absorbance of samples was recorded at 450 nm after 30 min [69]. FRAP assay is based upon the reduction of Fe3+-TPTZ complex under acidic medium and conditions. Increased absorbance of the blue-colored ferrous form (Fe2+-TPTZ complex) is recorded at 593 nm [70]. Metal chelating ability of novel antioxidant bromophenols was predicted according to Dinis et al. [71]. Fe2+-binding capacity of novel antioxidant bromophenols was spectrophotometrically recorded at 562 nm. DPPH scavenging activity of novel antioxidant bromophenols was performed according to the method of Blois [72] as described previously in detail. The absorbance values of samples were recorded at 517 nm in a spectrophotometer [73]. ABTS radical (ABTS+) scavenging activity of novel bromophenols was performed using the spectroscopic method described by Re et al. [74]. The ABTS radical cation (ABTS+) was acquired by reacting 7 mM solution of ABTS with 2.45 mM K2S2O8. The extent of decolorization is calculated as percentage reduction of absorbance [75]. Percentage of metal chelating, and radicals scavenging was computed using the following equation: SE (%) = [1  (As/Ac)]  100. In here, SE is radical scavenging effects, Ac is the absorbance value of control and As is absorbance value of sample [76]. 4.3.2. CA isoenzymes purification assays In this study, the activities of the novel antioxidant bromophenols were tested against two physiologically relevant CAs (hCA I, and II), AChE and BChE enzymes. Both hCA isoenzymes were purified by affinity chromatography with a Sepharose-4B–l-tyrosine– sulfanilamide matrix for selective retention of CAs [77]. For purification, the acidity of the homogenate solution was adjusted with solid Tris, and the supernatant was transferred to the prepared column. The quantity of proteins in the eluates was detected spectrophotometrically at 280 nm [78]. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE, 0.1% SDS) was used to determination of both hCA purities. This biochemical method was established with 10% and 3% acrylamide for the running and stacking gels, respectively. A single band was observed for each CA isoenzymes [79]. The quantity of protein was measured spectrophotometrically at 595 nm during the purification steps according to the Bradford method [80]. 4.3.3. CA isoenzymes inhibition assays The CAs activities were determined according to the method of Verpoorte et al. [81] as described previously [77]. For this purpose, p-NPA hydrolysis was assayed by measuring the change in absorbance at 348 nm over a period of 30 min at 25 °C, using a spectrophotometer (Beckman Coulter, Germany). The reaction mixture contained 400 lL Tris-HCl buffer (pH 7.4), 360 lL PNA (3 mM), 220 lL H2O, and 20 lL purified CA isoenzymes. A control measurement was obtained by preparing the same cuvette without enzyme. Bovine serum albumin was used as the standard protein

as described previously [82]. For determination of the inhibition constant (Ki) of each novel antioxidant bromophenols, three concentrations of each novel antioxidant bromophenols were tested, and the activities (%) were plotted against the concentrations of the novel antioxidant bromophenols in Lineweaver-Burk plots [65] as previously described [82]. 4.3.4. AChE and BChE inhibition assays Inhibition effects of novel antioxidant bromophenols on AChE/ BChE activities were measured by Ellman’s method [83]. AChI/ BChI, and DTNB (Product no: D8130-1G, Sigma-Aldrich) were used for the determination of the AChE/BChE activities. Namely, 100 mL of buffer (Tris/HCl, 1 M, pH 8.0), 10 mL of sample solution dissolved in deionized water at different concentrations. Then, 50 mL AChE/ BChE (5.32103 EU) solution was added and incubated for 10 min at 25 °C. After incubation, a portion of DTNB (50 mL, 0.5 mM) was added. Finally, the reaction was started by the addition of 50 mL of AChI/BChI (10 mM, Product no: 01480-1G, Sigma-Aldrich). The enzymatic hydrolysis of both substrates was determined spectrophotometrically by the formation of yellow 5-thio-2nitrobenzoate anion as the result of the reaction of DTNB with thiocholine at a wavelength of 412 nm. For determination of the effect of novel antioxidant bromophenols on AChE, different concentrations of novel antioxidant bromophenols were added to the reaction mixture. Then, AChE/BChE activities were measured. IC50 values were obtained from activity (%) versus compounds plots [27]. Declaration of interest The authors report no conflicts of interests. Acknowledgments The authors of this work are indebted to Ataturk University for financial support. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bioorg.2017.07. 010. References [1] G.W. Gribble, Chem. Soc. Rev. 28 (1999) 335–346. [2] M. Liu, P.E. Hansen, X. Lin, Mar. Drugs 9 (2011) 1273–1292. [3] K. Kurata, K. Taniguchii, K. Takashima, I. Hayashi, M. Suzuki, Phytochemistry 45 (1997) 485–487. [4] D. Shi, J. Li, S. Guo, H. Su, X. Fan, Chin. J. Ocean Lim. 27 (2009) 277–282. _ Gülçin, A. Menzek, S. Göksu, E.J. Sahin, J. Enzyme Inhib. Med. [5] H.T. Balaydin, I. Chem. 25 (2010) 685–695. [6] D. Shi, J. Li, B. Jiang, S. Guo, H. Su, T. Wang, Bioorg. Med. Chem. Lett. 22 (2012) 2827–2832. [7] W. Wang, Y. Okada, H. Shi, Y. Wang, T. Okuyama, J. Nat. Prod. 68 (2005) 620– 622. [8] X.J. Duan, X.M. Li, B.G. Wang, J. Nat. Prod. 70 (2007) 1210–1213. [9] L. Xu, L. Xiaoming, G. Lixin, C. Chuanming, L. Chunshun, L. Jia, W. Bingui, Chin. J. Ocean. Limn. 29 (2011) 686–690. [10] H.T. Balaydın, H. Soyut, D. Ekinci, S. Göksu, Sß . Beydemir, A. Menzek, E. S ß ahin, J. Enzyme Inhib. Med. Chem. 27 (2012) 43–50. [11] N. Xu, X. Fan, X. Yan, X. Li, R. Niu, C.K. Tseng, Phytochemistry 62 (2003) 1221– 1224. [12] Y. Akbaba, H.T. Balaydın, A. Menzek, S. Göksu, E. Sßahin, D. Ekinci, Arch. Pharm. 346 (2013) 447–454. _ Gulcin, Bioorg. Chem. 60 [13] N. Öztasßkın, Y. Çetinkaya, P. Taslimi, S. Göksu, I. (2015) 49–57. [14] (a) B. Uttara, A.V. Singh, P. Zamboni, R.T. Mahajan, Curr. Neuropharmacol. 7 (2009) 65–74;

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