The first synthesis of 4-phenylbutenone derivative bromophenols including natural products and their inhibition profiles for carbonic anhydrase, acetylcholinesterase and butyrylcholinesterase enzymes

Accepted Manuscript The First Synthesis of 4-Phenylbutenone Derivative Bromophenols Including Natural Products and Their Inhibition Profiles for Carbonic Anhydrase, Acetylcholinesterase and Butyrylcholinesterase Enzymes Çetin Bayrak, Parham Taslimi, İlhami Gülçin, Abdullah Menzek PII: DOI: Reference:

S0045-2068(16)30443-6 http://dx.doi.org/10.1016/j.bioorg.2017.03.001 YBIOO 2027

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

29 December 2016 28 February 2017 1 March 2017

Please cite this article as: C. Bayrak, P. Taslimi, I. Gülçin, A. Menzek, The First Synthesis of 4-Phenylbutenone Derivative Bromophenols Including Natural Products and Their Inhibition Profiles for Carbonic Anhydrase, Acetylcholinesterase and Butyrylcholinesterase Enzymes, Bioorganic Chemistry (2017), doi: http://dx.doi.org/ 10.1016/j.bioorg.2017.03.001

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The First Synthesis of 4-Phenylbutenone Derivative Bromophenols Including Natural Products and Their Inhibition Profiles for Carbonic Anhydrase, Acetylcholinesterase and Butyrylcholinesterase Enzymes

Çetin Bayrak, Parham Taslimi, İlhami Gülçin, Abdullah Menzek*

1

Department of Chemistry, Faculty of Science, Atatürk University, Erzurum-25240, Turkey

ABSTRACT: The first synthesis of (E)-4-(3-bromo-4,5-dihydroxyphenyl)but-3-en-2-one (1), (E)-4-(2bromo-4,5-dihydroxyphenyl)but-3-en-2-one (2), and (E)-4-(2,3-dibromo-4,5-dihydroxyphenyl)but-3-en-2one (3) was realized as natural bromophenols. Derivatives with mono OMe of 2 and 3 were obtained from the reactions of their derivatives with di OMe with AlCl3. These novel 4-phenylbutenone derivatives were effective inhibitors of the cytosolic carbonic anhydrase I and II isoenzymes (hCA I and II), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with Ki values in the range of 158.07– 404.16 pM for hCA I, 107.63–237.40 pM for hCA II, 14.81–33.99 pM for AChE and 5.64–19.30 pM for BChE. The inhibitory effects of the synthesized novel 4-phenylbutenone derivatives were compared to acetazolamide as a clinical hCA I and II isoenzymes inhibitor and tacrine as a clinical AChE and BChE enzymes inhibitor. Keywords:

Bromination;

bromophenols;

Carbonic

anhydrase;

Acetylcholinesterase;

Butyrylcholinesterase; Enzyme inhibition; Phenylbutenone; Natural products.

* Corresponding author: E-mail address: [email protected]; Phone: 90 4422314423; Fax: 90 4422360948.

1. INTRODUCTION There are a significant number of bromophenols in naturally occurring organobromine compounds. They are usually isolated from red algae of the family Rhodomelaceae in marine life [1]. Most bromophenols have important biological activities such as antioxidant [2-4], cytotoxicity [5], and feeding deterrent effects [6].

We investigated the carbonic anhydrase (CA) inhibitory effects of some

bromophenol derivatives and determined their inhibitory effects [7-11]. The rhizome of ginger root (Zingiber officinale Roscoe) having gingerols is widely used in Asia as a medicine and seasoning spice [12].

Figure 1. Some natural phenol compounds It was reported that natural products 1–3 were isolated from the red alga (Rhodomela confervoides), and they exhibited antioxidant activity [4,13]. The product 3 that was also isolated previously [14-15] exhibited moderate activity against human leukaemia cells [15]. Carbonic anhydrases (CA) catalyse the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and a proton (H+). This essential biochemical reaction plays a crucial physiological role in a large scale of biological systems. These enzymes are expressed by the most of living organisms and encoded by seven distinct gene families including α-, β-, γ-, δ-, ζ-, n- and θ-CAs [16-18]. These 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. α-CAs have differences in tissue location and distribution, namely 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. 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

2

limited number of normal tissues, such as gastrointestinal mucosa and related structures [19-21]. The αCAs have been extensively studied due to their role in human physiology and disease pathology. In living organisms, CA isoenzymes play a crucial role in a plenty of biochemical and physiological processes including CO2 and HCO3- transportation between tissues and respiratory surfaces, pH homeostasis, bone resorption, electrolyte transport in various epithelia, calcification and biosynthetic reactions including gluconeogenesis, ureagenesis and lipogenesis. CA inhibitors (CAIs) are a class of chemicals or pharmaceuticals that suppress the CA activity. They are clinically used for treatment some diseases including glaucoma and cancer. Additionally, it has lately demonstrated that they have potential as antiobesity, diuretics and anti-infective drugs [22-24]. Cholinergic

enzymes

including

acetylcholinesterase

(E.C.

3.1.1.7,

AChE)

and

butyrylcholinesterase (E.C. 3.1.1.8, BChE) also play important roles in cholinergic transmission by hydrolyzing the neurotransmitter acetylcholine (ACh) to acetate/butyrate and choline (Ch) [25,26]. AChE is a membrane-bound enzyme and present in the cholinergic neurons, muscles, and brain. It consists of multiple subunits. In the mammalian brain, most of the AChE exists in the membrane-bound G4 form, but its level declines as the neurons degenerate [27, 28]. On the other hand, BChE, which expressed by neuroglia found in the liver, intestine, heart, kidney, serum and lung, plays a crucial role in the ester containing compounds metabolism [29,30]. It also can hydrolyze ACh and its levels do not decline or might even increase in AD [31,32]. Normally in the brain AChE is predominant but BChE activity rises while AChE activity remains unchanged or diminishes in the brain of AD patients [33,34]. Therefore, a drug inhibiting of both cholinergic enzymes might be preferable to selective ChEs inhibitors (ChEIs). During the past few years, synthetic ChEIs, including galantamine, rivastigmine, tacrine and donepezil have been used for clinical treatment of AD. Recently, the usage of these drugs has been limited because of their side effects such as hepatotoxicity and gastrointestinal disturbance [35,36]. Due to the adverse side effects of synthetic ChEIs, the development of nontoxic ChEIs as alternatives to synthetic drugs are of great interest among researchers. For this purpose, many natural ChEIs have been isolated or synthesized from natural bio resources [37-39]. In this study, we report the first synthesis of 4-phenylbutenone derivatives and inhibition effects for acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase enzymes.

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2.

RESULTS AND DISCUSSION

2.1. Chemistry Firstly, condensation of prepared corresponding benzaldehyde derivatives 5–7 with acetone was planned for the synthesis of natural products 1–3 and their derivatives. With this aim, the aldehydes 5–7 were prepared from the starting material vanillin by the established methods [7,8,40-42]. Next, the condensation reactions of the aldehydes 5–7 with acetone yielded to the corresponding products 8–10 with high yields (Scheme 1). Compound 9 is known from the products 8–10 [43].

For the first synthesis of natural bromophenols 1–3, demethylation reactions of 8–10 with BBr3 were performed in CH2Cl2 at 0-25oC. Firstly, natural bromophenols were synthesized from the reactions shown in (Scheme 1). NMR data of the natural bromophenols 1–3 are in agreement with data of isolated bromophenols reported in the literature [4,13-15].

4

Derivatives without double bonds of natural bromophenols 1–3 might be important for potential biological activities. There are several available methods for reduction of double bonds. Many reducing reagents may also reduce bromides in molecules such as bromophenol derivatives 8–10 because the double bonds are structures of α,β-unsaturated ketones. One of the methods used in the reduction of double bonds of α,β-unsaturated carbonyl compounds is transition metal catalyzed borahydride reduction (NiCl2∙6H2O/NaBH4) [44]. With this aim, reductions of the compounds 8–10 with NiCl2∙6H2O/NaBH 4 gave compounds 11–13, respectively (Scheme 2). The demethylation reaction of 13 from the compounds 11–13 with BBr3 in CH2Cl2 at 0–25oC gave bromophenol 14 (Scheme 3). Good results could not be obtained from the demethylation reactions of 11 and 12 with BBr3 under the same conditions.

The natural bromophenols with mono OMe might be obtained from the compounds 8–10, precursor of the natural bromophenols 1–3, by mono demethylation. One demethylation reagent is AlCl 3 [45]. Reaction of 9 with AlCl3 in CH2Cl2 at room temperature (RT) for 48 h yielded to two isomeric products. According to their1H- and 13C-NMR spectra, they are two isomeric products with mono OMe. In the same way, reaction of 10 with AlCl3 was performed and only one product with mono OMe was obtained from the reaction (Scheme 4). However, it is not easy to establish the position of OH in the benzene rings of these three products. The positions of these OH groups were determined by their HMQC and HMBC spectra. The isomeric products are 15 and 16 while the other, which is formed alone, is 17. These products 15, 16 and 17 are known and their syntheses were performed by condensation reactions [46,47]. However, their properties such as melting points and much NMR data could not be found by us.

5

2.2. Enzyme inhibition studies Recently natural products and their synthesized derivatives have continued to attract attention, as many enzyme inhibitors that bind to an enzyme and decrease its activity, exhibit a wide spectrum of activities. [48-51]. CA isoenzymes have attracted this type of interest for the design of inhibitors or activators with biomedical, pharmaceutical and physiological applications. Additionally, we determined the effects of some novel 4-phenylbutenone derivatives against AChE, BChE, hCA I, and II, enzymes. Esterase activity method was used for determination of the inhibition parameters including IC50 and Ki values inhibition effects of novel 4-phenylbutenone derivatives on both CA isoenzymes [52]. CA activity (%) versus inhibitory concentration and 1/V versus 1/[S] graph were drawn using by the LineweaverBurk. The Lineweaver-Burk coordinates were used to obtain the maximal velocity (Vmax) and the inhibition constants (Ki). Then average Ki values were calculated from these graphs (Table 1). Therefore, we report the inhibition effects of novel 4-phenylbutenone derivatives on the esterase activity of hCA I, hCA II, AChE and BChE enzymes as in vitro. The following results are presented in Table 1. (i)

Cytosolic hCA I, and II are both expressed in erythrocyte, which are necessary for maintaining the physiological pH of the blood through production of HCO3− [53, 54]. Abnormal levels of CA I in the blood are used as a marker for hemolytic anemia. The cytosolic and slow isoform hCA I was inhibited by novel the 4-phenylbutenone derivatives, with Ki values ranging between 158.07±42.20 and 404.16±31.40 pM. Furthermore, 4-(2,3-dibromo-4,5-dimethoxyphenyl)butan-2-one (13), which possessed two bromine and two methoxy groups, demonstrated the most powerful hCA I isoenzyme inhibition properties with a Ki value of 158.07±42.20 pM. It is well known that certain isoforms anions show binding affinities in the low micromolar range [55-57]. Unlike the sulfonamides, the

6

anions may connect to the metal ion in three different coordination geometries: trigonal– bipyramidal, tetrahedral, or distorted tetrahedral geometries. Bromide (Br−) adopts either the trigonal-bipyramidal or distorted tetrahedral geometries [10, 58, 59]. In addition, it is known that the molecules, which had methoxy groups, had inhibition affinity against CA isoenzymes [60-62]. The standard and clinically used drug acetazolamide (AZA) demonstrated a K i value of 439.40±181.0 pM. Thus, the investigated novel 4-phenylbutenone derivatives showed better inhibitory profiles when compared to AZA, a clinically used CA inhibitor. (ii)

In addition to red blood cells, CA II is ubiquitously expressed in other tissues including the kidneys, bones, and ocular tissues. hCA II is the most extensively studied and characterized of the CA isoforms. Interestingly, CA II has been associated with several transporters including the Cl−/HCO3 − exchanger, the Na+/HCO3− cotransporter, and the Na+/H+ exchanger. This suggests that CAII acts as a mediator of certain metabolic pathways by further providing the substrates for these various transporters [53]. Consequently, CA II is often associated with several diseases such as osteoporosis, glaucoma, and renal tubular acidosis [60-62]. It has also been shown to be essential for the proper functioning of the water-transport channel, aquaporin-1 (AQP1). Specifically, the relationship between CA II and AQP1 has been shown to be essential for regulation of AQP1 function, maintaining proper CO2 transport in oocytes, and maintenance of a stable intracellular pH [60,63]. hCA II was also effectively inhibited by the novel 4-phenylbutenone derivatives investigated here. These compounds appeared to strongly inhibit hCA II, with K i values ranging from 107.63±0.72 pM to237.40±110.3 pM. Ki values of newly compounds are better than that of the clinically used drug acetazolamide (Ki: 490.27±15.0 pM). All the investigated 4-phenylbutenone derivatives showed potent inhibition against hCA I, but the compound of (E)-4-(3-bromo-4,5dimethoxyphenyl)but-3-en-2-one (8), which possessed one bromine and two methoxy groups, showed an excellent inhibition profile against hCA II with a Ki value of 107.63±0.72 pM. It was reported that the compounds had halogens group had positive effect for the inhibition effects towards cytosolic hCA I, and II isoforms [16].

(iii)

Recently, some studies have shown that the peripheral anionic site of CA is asserted in promoting aggregation of the β-amyloid peptide, which responsible for the neurodegenerative effects in AD. Moreover, it was reported that ChEIs had healing effect for AD treatment on cognition and on behavioral symptoms by clinical studies [64-66]. Furthermore, the designing of new ChEIs that interact with the peripheral anionic site are extremely important for the AD treatment [67,68]. The inhibition effects of the newly synthesized 4-phenylbutenone derivatives on AChE are shown in Table 1. The AChE inhibition profiles of newly synthesized 4-phenylbutenone derivatives investigated here were quite interesting. Overall, the newly synthesized 4-phenylbutenone

7

derivatives had excellent inhibitory, activity with Ki values ranging from 14.81±1.90 pM to 33.99±11.7 pM. It was reported that galantamine as a standard AChE inhibitor had a Ki value of 0.253±0.21 µM. It was reported that some novel monoterpene indole alkaloids had AChE inhibition, and showed moderate activities (IC50 values of 4.1-10.3 µM) [69]. Also, these compounds effectively inhibit BChE with Ki values in ranging of 5.64±1.8 -19.30±4.29 pM. Furthermore, tacrine, used as a standard ChEIs in this study, demonstrated Ki values of 50.72±7.05 pM toward AChE and 19.16±3.73 pM toward BChE, respectively. As the results show, 4phenylbutenone derivatives had much better AChE inhibition effects than that of standard drugs.

3. CONCLUSION

In conclusion, the first synthesis of natural products 1–3 was successfully realized from the corresponding reactions. The double bonds of α,β-unsaturated carbonyl compounds 8–10, precursor of natural bromophenols

1–3,

were reduced to give the corresponding products

11–13

by

NiCl2∙6H2O/NaBH4. By a different way, known [46,47], derivatives with mono OMe 15–17 of 2 and 3 were obtained from the reactions of their derivatives with di OMe with AlCl 3. Picomolar levels of inhibition were achieved in all 4-phenylbutenone derivatives. All of the synthesized compounds effectively inhibited some metabolic enzymes like CA I and II, AChE, and BChE at the picomolar levels. As we discussed above, 4-phenylbutenone derivatives can be good candidate drugs for treatment of some diseases like glaucoma, epilepsy, mountain sickness, gastric and duodenal ulcers, neurological disorders, or osteoporosis as carbonic anhydrase inhibitors. 4. EXPERIMENTAL SECTION 4.1. General Experimental Procedures. Solvents were purified and dried by known methods. For all compounds, values as well as Mp, IR Spectra, 1H and 13C NMR spectra, chemical shift, elemental analyses, and CA inhibitory properties of samples were performed as explained previously [11,70]. PLC (preparative thick-layer chromatography) was used as 1 mm of silica gel 60 PF (Merck, Darmstadt, Germany) on glass plates. HRMS data were obtained by LC-MS-TOF electrospray ionization technique (1200/6210, Agilent). 4.2. Synthesis:

4.2.1. (E)-4-(3-bromo-4,5-dihydroxyphenyl)but-3-en-2-one (1): Standard procedure for the demethylation reaction with BBr3

8

A solution of compound 8 (0.5 g, 1.76 mmol) in CH2Cl2 (10 mL) under N2 gas was cooled to 0oC in an ice-water bath and BBr3 (1.32 g, 5.28 mmol) was added with the aid of a syringe. The resulting mixture was allowed to stir under N2 for 10 min at 0 °C. Then, the cold bath was removed, and the mixture was stirred for 10 h at room temperature (RT) and cooled again to 0oC. To this reaction mixture was added MeOH (10 mL) dropwise and then the solvent was removed under vacuum. The crude product was extracted with EtOAc (22 × 25 mL). The combined extracts were dried over Na 2SO4 and the solvent was removed under vacuum. The residue was purified by column chromatography on silica gel (10 g) using CH2Cl2/MeOH (98:2) eluent. The obtained natural product 1 was crystalized from EtOAc/hexane as black crystal (277 mg %50). M.p: 147-149 oC (184-185 oC) [13].; 1H-NMR (400 MHz, acetone-d6): 9.03 (bs, OH, 1H), 8.54 (bs, OH, 1H), 7.44 (d, A part of AB system, J = 16.3 Hz, olefinic, 1H), 7.37 (d, J = 2.1 Hz, aromatic, 1H), 7.17 (d, J = 2.1 Hz, aromatic, 1H). 6.58 (d, B part of AB system, J = 16.3 Hz, olefinic, 1H), 2.27 (s, CH3, 3H); 13C-NMR (100 MHz, Acetone-d6): 196.88 (CO), 146.18 (C), 145.54 (C), 141.67 (CH), 127.76 (C), 125.41 (CH), 124.36 (CH), 113.53 (CH), 109.70 (C), 26.39 (CH 3).

4.2.2. (E)-4-(2-bromo-4,5-dihydroxyphenyl)but-3-en-2-one (2)

The standard procedure (4.2.1.) described for the synthesis of 1 was performed for this reaction. The compound 9 (1.0 g 3.52 mmol) was used and natural product 2 (577 mg, 52%) was obtained as a black crystal. M.p: 177-180 oC (145-147oC) [4].; 1H-NMR (400 MHz, acetone-d6): 8.98 (bs, OH, 1H), 8.48 (bs, OH, 1H), 7.77 (d, A part of AB system, J = 16.1 Hz, olefinic, 1H), 7.28 (s, aromatic, 1H), 7.10 (s, aromatic, 1H), 6.58 (d, B part of AB system, J = 16.1 Hz, olefinic, 1H). 2.77 (s, CH3, 3H); 13C-NMR (100 MHz, acetone-d6): 197.02 (CO), 148.48 (C), 145.23 (C), 140.69 (CH), 126.53 (CH), 125.81 (C), 119.44 (CH), 115.64 (C), 113.64 (CH), 26.71 (CH3).

4.2.3. (E)-4-(2,3-dibromo-4,5-dihydroxyphenyl)but-3-en-2-one (3)

The standard procedure (4.2.1.) described for the synthesis of 1 was also performed for this reaction. The compound 10 (0.8 g 2.19 mmol) was used and natural product 3 (433 mg, 50%) was obtained as a black crystal. M.p: 160-162 oC (165-166 °C) [14].; 1H-NMR (400 MHz, acetone-d6): 9.209.00 (m, OH, 2H), 7.86 (d, A part of AB system, J = 16.1 Hz, olefinic, 1H), 7.34 (s, aromatic, 1H), 6.54 (d, B part of AB system J = 16.1 Hz, olefinic, 1H). 2.31 (s, CH3, 3H); 13C-NMR (100 MHz, acetone-d6): 196.58 (CO), 146.65 (C), 145.38 (C), 142.30 (CH), 127.97 (CH), 127.21 (C), 117.74 (C), 113.61 (C), 112.64 (CH), 27.12 (CH3).

9

4.2.4. Synthesis of (E)-4-(3-bromo-4,5-dimethoxyphenyl)but-3-en-2-one (8): Standard procedure for the condensation reaction with acetone

To a cold (0 oC) solution of aldehyde 5 (1.0 g, 4.1 mmol) in acetone (5 mL) was slowly added the previously prepared solution of NaOH (10%, 0 °C, 10 mL). The resulting mixture was allowed to stir for 30 min at 0°C, and then the cold bath (mixture of ice and water) was removed. The mixture was stirred for 4 h at RT, cooled again to 0 °C, and neutralized with addition of cold HCl solution (1.0 M, 0 °C). The solvent was removed under vacuum, and water (10 mL) was added. The new mixture was extracted with EtOAc (2 × 50 mL), the combined extracts were dried over Na 2SO4 and the solvent was removed under vacuum. The residue was purified by column chromatography on silica gel (20 g) using EtOAc/hexane (1/4) as eluent gave the product 8 (1.044 g, % 90) as a yellow solid. M.p: 75-77 oC; 1H-NMR (400 MHz, CDCl3): 7.33 (d, A part of AB system, J = 16.2 Hz, olefinic, 1H), 7.29 (d, J = 1.9 Hz, aromatic, 1H), 6.98 (d, aromatic, J = 1.9 Hz, 1H), 6.58 (d, B part of AB system, olefinic, J = 16.2 Hz, 1H), 3.86 (s, OCH3, 3H), 3.85 (s, OCH3, 3H), 2.34 (s, CH3, 3H); 13C-NMR (100 MHz, CDCl3)  197.90 (CO), 153.78, 148.28 (C), 141.58 (CH), 131.45 (C), 127.28 (CH), 125.21 (CH), 118.04 (C), 110.60 (CH), 60.71 (OCH3), 56.09 (OCH3), 27.48 (CH3); IR (CH2Cl2) 2938, 1663, 1556, 1452, 1359, 1141, 818 cm-1; Rf = 0.35; EtOAc/hexane (1:4); Elemental Analysis C: 50.55, H: 4.60; calculate: C: 50.35, H: 4.62.

4.2.5. Synthesis of (E)-4-(2-bromo-4,5-dimethoxyphenyl)but-3-en-2-one (9)

The standard procedure (4.2.4.) described for the synthesis of 8 was performed for this reaction. The compound 6 (0.5 g, 2.05 mmol) and acetone (5 mL) were used and the product 9 (0.52 g, 90%) was obtained as a yellow solid. M.p: 105-107 oC; 1H-NMR (400 MHz, CDCl3): 7.81 (d, A part of AB system, J = 16.3 Hz, olefinic, 1H), 7.07 (s, aromatic, 1H), 7.04 (s, aromatic, 1H), 6.52 (d, B part of AB system, J = 16.2 Hz, olefinic, 1H), 3.89 (s, OCH3, 3H), 3.88 (s, OCH3, 3H), 2.40 (s, CH3, 3H); 13C-NMR (100 MHz, CDCl3): 198.63 (CO), 151.73 (C), 149.00 (C), 142.22 (CH), 128.04 (CH), 126.26 (C), 117.61 (C), 115.61 (CH), 109.12 (CH), 56.50 (OCH3), 56.29 (OCH3), 27.15 (CH3); IR (CH2Cl2) 2936, 1715, 1470, 1424, 1378, 1164, 1062, 1007cm-1; Rf = 0.35; EtOAc/hexane (1:4); HRMS: m/z (M+) calcd. For C12H1379 BrO3: 284.0048; found: 284.0044.

4.2.6. Synthesis of (E)-4-(2,3-dibromo-4,5-dimethoxyphenyl)but-3-en-2-one(10).

10

The standard procedure (4.2.4.) described for the synthesis of 8 was performed for this reaction. The compound 7 (1.0 g 3.10 mmol) and acetone (5 mL) were used and the product 10 (1.03 g, %92) was obtained as an orange solid. M.p: 140-143 oC; 1H-NMR (400 MHz, CDCl3): 7.85 (d, A part of AB system, J = 16.2 Hz, olefinic, 1H), 7.09 (s, aromatic, 1H), 6.49 (d, B part of AB system, J = 16.2 Hz, olefinic, 1H), 3.89 (s, OCH3, 3H), 3.87 (s, OCH3, 3H), 2.41 (s, CH3, 3H); ); 13C-NMR (100 MHz, CDCl3): 198.48 (CO), 152.32 (C), 149.24 (C), 142.91 (CH), 131.79 (C), 130.19 (CH), 122.38 (C), 118.81 (C), 109.70 (CH), 60.47 (OCH3), 56.02 (OCH3), 26.40 (CH3); IR (CH2Cl2) 2943, 16713, 1468, 1422, 1262, 1099, 617 cm-1; Rf = 0.32; EtOAc/hexane (1:4); Elemental Analysis C, 39.59, H, 3.32; calculate: 39.42, H, 3.31.

4.2.7. Synthesis of 4-(3-bromo-4,5-dimethoxyphenyl)butan-2-one (11): Standard procedure for the reduction reaction with NiCl2∙6H2O/NaBH4

A solution of the compound 8 (0.7 g 2.46 mmol) in MeOH (10 mL) and THF (10 mL) was cooled at 0 oC in a mixture of ice-water and H2O (2.0 g) and NiCl2∙6H2O (5.8 g, 24.6 mmol) were added to this solution, consecutively. The resulting mixture was allowed to stir for 10 min at 0 °C. Then, NaBH4 (364 mg, 9.84 mmol) was added slowly and carefully, and the mixture was allowed for stir 30 min at 0 °C. Then the cold bath was removed, and the mixture was stirred for 24 h at RT. The reaction mixture was filtered and the solvent of the filtrate was removed under vacuum. The residue was extracted with EtOAc (2 × 25 mL), the combined organic phases were dried over Na 2SO4 and the solvent was removed under vacuum. The residue was purified by column chromatography on silica gel (20 g) using EtOAc/hexane (1/4) as eluent gave reduction product 11 (450 mg, 65%) was obtained as a yellow liquid. 1H-NMR (400 MHz, CDCl3): 6.88 (d, A part of AB system, J = 1.9 Hz, aromatic, 1H), 6.64 (d, B part of AB system, J = 1.9 Hz, aromatic, 1H), 3.79 (s, OCH3, 3H), 3.76 (s, OCH3, 3H), 2.09 (s, CH3, 3H), 2.76 (A2 part of A2B2 system, 2H), 2.69 (B2 part of A2B2 system, 2H);

13

C-NMR (100 MHz, CDCl3):

13

C-NMR (100 MHz,

CDCl3): 207.64 (CO), 153.60 (C), 144.78 (C), 138.58 (C), 124.26 (CH), 117.54 (C), 112.28 (CH), 60.70 (OCH3), 56.22 (OCH3), 45.04, 30.27, 29.31; IR (CH2Cl2) 1715, 1635, 1566, 1490, 1414, 1271, 1141, 1047cm-1; Rf = 0.50; EtOAc/hexane (3:7); Elemental Analysis C: 50.19, H: 5.27; calculate: C: 49.99, H: 5.29.

4.2.8. Synthesis of 4-(2-bromo-4,5-dimethoxyphenyl)butan-2-one (12)

The standard procedure (4.2.7.) described for the synthesis of 11 was performed for this reaction. The compound 9 (1.0 g 3.52 mmol), MeOH (10 mL) and THF (10 mL) were used and the product 12 (695

11

mg, % 70%) was obtained as a yellow liquid. 1H-NMR (400 MHz, CDCl3): 6.95 (s, aromatic, 1H), 6.73 (s, aromatic, 1H), 3.81 (s, OCH3, 3H), 3.80 (s, OCH3, 3H), 2.89 (A2 part of A2B2 system, 2H), 2.71 (B2 part of A2B2 system, 2H), 2.11 (s, CH3, 3H); 13C-NMR (100 MHz, CDCl3): 207.94 (CO), 148.56 (C), 148.24 (C), 132.44 (C), 115.64 (CH), 114.00 (C), 113.53 (CH), 56.36 (OCH3), 56.24 (OCH3), 43.89, 30.24, 30.17; IR (CH2Cl2) 2936, 1714, 1508, 1440, 1384, 1256, 1217, 1164, 1032cm-1; Rf = 0.47; EtOAc/hexane (3:7); Elemental Analysis C: 50.19, H: 5.27; calculate: C: 50.07, H: 5.26.

4.2.9. Synthesis of 4-(2,3-dibromo-4,5-dimethoxyphenyl)butan-2-one (13)

The standard procedure (4.2.7.) described for the synthesis of 11 was performed for this reaction. The compound 10 (2.0 g, 5.48 mmol), MeOH (10 mL) and THF (10 mL) were used. The product 13 (1.43 g, 72%) was obtained as a grey liquid. 1H-NMR (400 MHz, CDCl3): 6.77 (s, aromatic, 1H), 3.75 (s, OCH3, 3H), 3.71 (s, OCH3, 3H), 2.91 (A2 part of A2B2 system, 2H), 2.68 (B2 part of A2B2 system, 2H), 2.06 (s, CH3, 3H); 13C-NMR (100 MHz, CDCl3): 207.41 (CO), 152.58 (C), 146.16 (C), 138.04 (C), 121.47 (C), 116.89 (C), 113.67 (CH), 60.59 (OCH3), 55.95 (OCH3), 42.99, 32.01, 30.30.; IR (CH2Cl2) 2936, 1715, 1470, 1424, 1378, 1163, 1062, 1007 cm-1; Rf = 0.50; EtOAc/hexane (3:7); Elemental Analysis C: 39.38, H: 3.86; calculate: C: 39.22, H: 3.88.

4.2.10. Synthesis of 4-(2,3-dibromo-4,5-dihydroxyphenyl)butan-2-one (14)

The standard procedure (4.2.1.) described for the synthesis of 1 was performed for this reaction. The compound 10 (1.25 g, 3.5 mmol) and CH2Cl2 (10 mL) were use and the crude product was submitted to silica gel (10 g) column chromatography using CH2Cl2/MeOH (96:4) eluent. Product 14 (698 mg, 60%) was obtained as a white crystal. M.p: 102-104 oC. 1H-NMR (400 MHz, Aseton-d6): 8.82 (bs, OH 1H), 8.19 (bs, OH 1H), 6.87 (s, aromatic, 1H), 2.89 (A2 part of A2B2 system, 2H), 2.74 (B2 part of A2B2 system, 2H), 2.10 (s, CH3, 3H); 13C-NMR (100 MHz, Aseton-d6):  206.73 (CO), 144.97 (C), 142.86 (C), 133.25 (C), 116.01 (CH), 115.14 (C), 113.31 (C), 43.09, 31.33, 31.19; IR (Acetone) 3426, 1703, 1406, 1273, 1181, 1090 cm-1; Rf = 0.25; MeOH/CH2Cl2 (4:96); Elemental Analysis C: 35.54, H: 2.98;calculate: C: 35.44, H: 3.00.

4.2.11. Reaction of the compound 9 with AlCl3

12

To a solution of the compound 9 (1.0 g, 3.52 mmol) in CH2Cl2 (10 mL) was added AlCl3 (1.40 g, 10.56 mmol) at RT. The resulting the mixture was stirred for 48 h at RT and water (5 mL) was added slowly and carefully. After the mixture was allowed to stir for 5 min at RT, the solvent (CH2Cl2) of the mixture was evaporated and the first residue was extracted with EtOAc with (2 × 15 mL). The combined organic extracts were dried over Na 2SO4 and the solvent was removed under vacuum. The residue was purified on silica gel (15 g) by column chromatography using EtOAc/hexane (3:7) eluent and 15 (480 mg, 50%, yellow crystal) and 16 (380 mg, 40%, green crystal) were obtained, respectively. 4.2.11.1. The compound 15. M.p: 90-92oC; 1H-NMR (400 MHz, CDCl3): 7.83 (d, A part of AB system, J = 16.3 Hz, 1H), 6.50 (d B part of AB system, J = 16.3 Hz, 1H). 7.17 (s, 1H), 7.08 (s, 1H), 6.06 (s, aromatic OH, 1H), 3.90 (s, OCH3, 3H), 2.41 (s, CH3, 3H); 13C-NMR (100 MHz, CDCl3):  198.87 (CO), 148.64 (C), 146.40 (C), 142.31 (CH), 127.60 (CH), 126.10 (C), 118.95 (CH), 117.99 (C), 108.32 (CH), 56.32 (OCH3), 26.12 (CH3); IR (CH2Cl2) 34419, 16249, 15869, 1503, 1270, 1204, 965 cm-1; Rf = 0.21; EtOAc/Hexane (3:7); HRMS: m/z (M+) calcd. For C11H1179 BrO3: 269,9892; found: 269,9899.

4.2.11.2. The compound 16: M.p: 128-130 oC; 1H-NMR (400 MHz, CDCl3): 7.79 (d, A part of AB system, J = 16.2 Hz, 1H), 6.50 (d, B part of AB system, J = 16.2 Hz, 1H). 7.20 (s, 1H), 7.04 (s, 1H), 5.89 (s, aromatic, OH, 1H), 3.91 (s, OCH3, 3H), 2.39 (s, CH3, 3H); 13C-NMR (100 MHz, CDCl3):  198.01 (CO), 149.24 (C), 145.47 (C), 142.03 (CH), 128.31 (CH), 127.46 (C), 116.21 (C), 115.39 (CH), 112.77 (CH), 56.42 (OCH3), 27.71 (CH3); IR (CH2Cl2) 3410, 1599, 1502, 1439, 12619, 1169, 1026, 968 cm-1; Rf = 0.28; EtOAc/Hexane (3:7); HRMS: m/z (M+) calcd. For C11H1179 BrO3: 269,9892; found: 269, 9884. 4.2.12. Reaction of the compound 10 with AlCl3

The standard procedure (4.2.11.) described for the reaction of 9 was performed for this reaction. The compound 10 (1.0 g 2.75 mmol), AlCl3 (1.10 g, 8.24 mmol) and CH2Cl2 (10 mL) were used and the product 17 (0.82 g, 85%, orange solid) was obtained as alone. M.p: 138-140 oC; 1H NMR (400 MHz, CDCl3) 7.89 (d, A part of AB system, J = 16.2 Hz, 1H), 7.07 (s, 1H), 6.48 (d, B part of AB system, J = 16.2 Hz, 1H), 6.48 (s, OH 1H), 3.94 (s, OCH3, 3H), 2.42 (s, CH3, 3H; 13C NMR (100 MHz, CDCl3) δ 198.67 (CO), 146.54 (C), 146.48 (C), 143.51 (CH), 129.04 (CH), 127.71 (C), 120.67 (C), 113.16 (C), 108.20 (CH), 56.75 (OCH3), 27.18 (CH3); IR (CH2Cl2) 3408, 1627, 1590, 1487, 1389, 1269.2, 1269.7, 1180, 967cm-1; Rf = 0.15; EtOAc/Hexane (3:7); HRMS: m/z (M+) calcd. For C11H1079Br2O3: 347,8997; found: 347,8987

13

4.3. Biochemical Studies

4.3.1. CA isoenzymes purification and inhibition studies The both cytosolic CA isoenzymes purification methods were previously described [71-73]. CA I, and II isoenzymes were purified by a simple one-step method by a Sepharose-4B-L tyrosinesulphanilamide affinity chromatography [74-76]. The protein quantity in purification step was determined spectrophotometrically at 280 nm as previously reported [77-79]. For visualsing of both CA isoenzymes purity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed [80, 81]. CA activities were determined in accordance with the method of Verpoorte et al [82] as described in previous study [83, 84]. The increase in absorbance of the reaction medium was spectrophotometrically recorded at 348 nm. The protein quantity was recorded at 595 nm according to Bradford’s method [85]. Bovine serum albumin (BSA) was used as standard protein as given previously in detail [86, 87]. 4.3.2. AChE/BChE activity determination

The inhibitory effects of 4-phenylbutenone derivatives on cholinergic enzymes (AChE/BChE) activities were measured according to the spectrophotometric method reported by Ellman et al [88]. Acetylthiocholine iodide and butyrylthiocholine iodide (AChI and BChI) were used as substrates of the reaction. 5,5’-Dithiobis(2-nitro-benzoic)acid (DTNB) was used for the measurement of the AChE/BChE activities. The hydrolysis of both substrates was monitored spectrophotometrically by formation of the yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine (ChI), released by enzymatic hydrolysis of AChI or BChI, with absorption maximum at a wavelength of 412 nm [89].

For the determination of the inhibition profile of each 4-phenylbutenone derivative on both hCA isoenzymes and both cholinergic enzymes, an Activity (%)-[4-Phenylbutenone derivative] graph was drawn. The IC50 values were obtained from activity (%) versus compounds plots [90, 91]. For the calculation of Ki values, three different 4-phenylbutenone derivatives concentrations were used. Finally, the Lineweaver–Burk curves were drawn [52].

Notes The authors declare no competing financial interest.

14

ACKNOWLEDGMENTS This study was financed by TÜBİTAK - The Scientific and Technological Research Council of Turkey (Project No: 113Z702) and realized in Atatürk University, Faculty of Science, Department of Chemistry. We are grateful to both institutes for their supports.

References [1] G.W. Gribble, Chem. Soc. Rev. 28 (1999) 335-346. [2] X.J. Duan, X.M. Li, B.G. Wang, J. Nat. Prod. 70 (2007) 1210-1213. [3] H.T. Balaydın, İ. Gülçin, A. Menzek, S. Göksu, E. Sahin, J. Enzym. Inhib. Med. Chem. 25 (2010) 685-695. [4] K. Li, X.M. Li, J.B. Gloer, B.G. Wang, J Agric. Food. Chem. 2011, 59 (2011) 9916-9921. [5] X.L. Xu, F.H. Song, S.J. Wang, S. Li, F. Xiao, J.L. Zhao, Y.C. Yang, S.Q. Shan, L. Yang, J.G. Shi, J. Nat. Prod. 67 (2004) 1661-1666. [6] K. Kurata, K. Taniguchii, K. Takashima, I.Hayashi, M.Suzuki, Phytochemistry 45 (1997) 485-487. [7] H.T. Balaydın, M. Şentürk, S. Göksu, A. Menzek, Eur. J. Med. Chem. 54 (2012) 423-428. [8] H.T. Balaydın, M. Şentürk, A. Menzek, Bioorg. Med. Chem. Lett. 22 (2012) 1352-1357. [9] Y. Çetinkaya, H. Göçer, İ. Gülçin, A. Menzek, Arch. Pharm. 347 (2014) 354-359. [10] M. Boztaş, Y. Çetinkaya, M. Topal, İ. Gülçin, A. Menzek, E. Şahin, M. Tanç, C.T. Supuran, J. Med. Chem. 58 (2015) 640-650. [11] T. Artunç, Y. Çetinkaya, H. Göçer, İ. Gülçin, A. Menzek, E. Şahin, C.T. Supuran, Chem. Biol. Drug. Des. 87 (2016) 594-607. [12] N. Shoji, A. Iwasa, T. Takemoto, Y. Ishida, Y. Ohizumi, J Pharm Sci-Us, 1982, 71 (1982) 11741175. [13] K. Li, X.M. Li, N.Y. Ji, B.G. Wang, Bioorg. Med. Chem. 15(2007) 6627-6631. [14] J.L. Zhao, X. Fan, S.J. Wang, S.A. Li, S.Q. Shang, Y.C. Yang, N.J. Xu, Y. Lu, J.G. Shi, J. Nat. Prod. 67 (2004) 1032-1035. [15] W.L. Popplewell, P.T. Northcote, A. Colensolide Tetrahedron Lett. 50 (2009) 6814-6817. [16] W.M. Eldehna, G.H. Al-Ansary, S. Bua, A. Nocentini, P. Gratteri, A. Altoukhy, H. Ghabbour , HY. Ahmed, C.T. Supuran. Eur. J. Med. Chem. 127 (2017) 521-530. [17] İ. Gülçin, A. Scozzafava, C.T. Supuran, Z. Koksal, F. Turkan, S. Çetinkaya, Z. Bingöl, Z. Huyut, S.H. Alwasel, J. Enzyme Inhib. Med. Chem. 31 (2016) 1698-1702. [18] B. Özgeriş, S. Göksu, L. Polat Köse, İ. Gülçin, R.E. Salmas, S. Durdagi, F. Tümer, C.T. Supuran, Bioorg. Med. Chem. 24 (2016) 2318-2329.

15

[19] C.T. Supuran, Nat. Rev. Drug Disc. 7 (2008) 168-181. [20] H. Göksu, M. Topal, A. Keskin, M.S. Gültekin, M. Çelik, İ. Gülçin, M. Tanc, C.T. Supuran, Arch. Pharm. 349 (2016) 466-474. [21] C.T. Supuran, A. Scozzafava, A. Casini, Med. Res. Rev. 23 (2003) 146-189. [22] A. Scozzafava, M. Passaponti, C.T. Supuran, İ. Gülçin, J. Enzyme Inhib. Med. Chem. 30 (2015) 586591. [23] L. Polat Kose, İ. Gülçin, H. Özdemir, A. Atasever, S.H. Alwasel, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31 (2016) 773-778. [24] A. Yıldırım, U. Atmaca, A. Keskin, M. Topal, M.Çelik, İ. Gülçin, C.T. Supuran, Bioorg. Med. Chem. 23 (2015) 2598-2605. [25] J. Massoulie, L. Pezzementi, S. Bom, E. Krejci, F.M. Vallette, Prog. Neurobiol. 4 (1993) 31-91. [26] İ. Gülçin, A. Scozzafava, C.T. Supuran, H. Akıncıoğlu, Z. Koksal, F. Turkan S. Alwasel, J. Enzyme Inhib. Med. Chem. 31 (2016) 1060-1066. [27] M. Weinstock, E. Groner, Chem. Biol. Interact. 175 (2008) 216-221. [28] H. Gocer, F. Topal, M. Topal, M. Küçük, D. Teke, İ. Gülçin, S.H. Alwasel, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31 (2016) 441-447. [29] M.R.C. Schetinger, N.M. Porto, M.B. Moretto, V.M. Morsch, J.B.T. Rocha, V. Vieira, F. Moro, R.T. Neis, S. Bittencourt, Neurochem. Res. 25 (2000) 949-955. [30] M. Topal, H. Gocer, F. Topal, P. Kalin, L.Polat Köse, İ. Gülçin, K.C. Çakmak, M. Küçük, L. Durmaz, A.C. Gören, S.H. Alwasel, J. Enzyme Inhib. Med. Chem. 31 (2016) 266-275. [31] F. Rahim, H. Ullah, M. Taha, A. Wadood, MT. Javed, W. Rehman, M. Nawaz, M. Ashraf, M. Ali, M. Sajid, F. Ali, MN. Khan, KM. Khan, Bioorg Chem. 68 (2016) 30-40. [32] A. Scozzafava, P. Kalın, C.T. Supuran, İ. Gülçin, S. Alwasel, J. Enzyme Inhib. Med. Chem. 30 (2015) 941-946. [33] N.H. Greig, T. Utsuki, Q.S. Yu, X. Zhu, H.W. Holloway, T. Perry, B. Lee, D.K. Ingram, Curr. Med. Res. Opin. 3(2001) 159-165. [34] . Öztaşkın, Y. Çetinkaya, P. Taslimi, S. Göksu, İ. Gülçin, Bioorg. Chem. 60 (2015) 49-57. [35] V. Sculz, Phytomedicine 10 (2003) 74-79. [36] A. Akıncıoğlu, H. Akıncıoğlu, I. Gülçin, S. Durdağı, C.T. Supuran, S. Göksu, Bioorg. Med. Chem. 23 (2015) 3592-3602. [37] S.Y. Kang, K.Y. Lee, S.H. Sung, M.J. Park, Y.C. Kim, J. Nat. Prod. 64 (2001) 683-685. [38] E. Ahmed, S.A. Nawaz, A. Malik, I. Choudhary, Bioorg. Med. Chem. Lett. 16 (2006) 573–580. [39] L. Polat Köse, İ. Gülçin, A.C. Gören, J. Namiesnik, A.L. Martinez-Ayala, S. Gorinstein, Ind. Crops Prod. 74 (2015) 712-721.

16

[40] T. Mori, H. Bando, Y. Kanaiwa, T. Amiya, K. Kurata, Chem. Pharm. Bull. 31 (1983) 1754-1756. [41] P.W. Ford, B.S. Davidson, J. Org. Chem. 58 (1993) 4522-4523. [42] Y. Akbaba, H.T. Balaydin, S. Goksu, E. Sahin, A. Menzek, Helv. Chim. Acta 93 (2010) 1127-1135. [43] Q.H. Zhu, X.Y. Wang, Y.G. Xu, Eur. J. Org. Chem. 6 (2012) 1112-1114. [44] J.M. Khurana, P.B. Sharma, Chem. Soc. Jpn. 77 (2004) 549-552. [45] Z.T. Du, J. Lu, H.R. Yu, Y. Xu, A.P. Li, J. Chem. Res. (2010) 222-227. [46] L.C. Raiford, W.F. Talbot, J. Am. Chem. Soc. 54 (1932) 1092-1077. [47] M. Brink, Tetrahedron 25 (1969) 995-999. [48] M. Şentürk, İ. Gülçin, M. Çiftci, Ö.İ. Küfrevioğlu, Biol. Pharm. Bull. 31 (2008) 2036-2039. [49] İ. Gülçin, Ö.İ. Küfrevioğlu, M. Oktay, J. Enzyme Inhib. Med. Chem. 20 (2005) 297-302. [50] E. Köksal, İ. Gülçin, Protein Peptide Lett. 15 (2008) 320-326. [51] M. Şişecioğlu, M. Çankaya, İ. Gülçin, M. Özdemir, Protein Peptide Lett. 16 (2009) 46-49. [52] H. Lineweaver, D. Burk, J. Am. Chem. Soc. 56 (1934) 658–666. [53] M.A. Pinard, B. Mahon, R. McKenna, BioMed Res. Int. (2015) 453-563. [54] M. Güney, A. Coşkun, F. Topal, A. Daştan, İ. Gülçin, C.T. Supuran, Bioorg. Med. Chem. 22 (2014) 3537-3543. [55] C.T. Supuran, J.Y. Winum, Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease Applications, John Wiley & Sons. (2009). [56] M. Topal, İ. Gülçin, Turk. J. Chem. 38 (2014) 894-902. [57] C.T. Supuran, Exp. Opin. Therap. Pat. 23 (2013) 677-679. [58] P. Taslimi, İ. Gulcin, B. Ozgeris, S. Goksu, F. Tumer, S.H. Alwasel, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31 (2016) 152-157. [59] H. Gocer, A. Aslan, İ. Gülçin, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31(2016) 503–507. [60] W. S. Sly, D. Hewett-Emmett, M. P. Whyte, Y. S. Yu, R.E. Panas. 80 (1983) 2752–2756. [61] P. Taslimi, İ.Gülçin,

. Öztaşkın, Y. Çetinkaya, S. Göksu, S.H. Alwasel, C.T. Supuran, J. Enzyme

Inhib. Med. Chem. 31 (2016) 603-607. [62] B. Arabaci, İ. Gülçin, S. Alwasel, Molecules 19 (2014) 10103-10114. [63] R.P. Henry, E.R. Swenson, Resp. Physiol. 121 (2000) 1-12. [64] G.V. De Ferrari, M.A. Canales, I. Shin, L.M. Weiner, I. Silman, N.C. Inestrosa, Biochemistry 40 (2001) 104-117 [65] H. Göçer, A. Akıncıoğlu, S. Göksu, İ. Gülçin, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 30 (2015) 316-320. [66] L. Polat Köse, İ. Gulcin, A. Yıldırım, U. Atmaca, M. Çelik, S. Alwasel, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31 (2016) 1213-1218.

17

[67] A. Kamal, A.B. Shaik, G.N. Reddy, C.G. Kumar, J. Joseph, G.B. Kumar, U. Purushotham, G.N. Sastry, Med. Chem. Res. 23 (2014) 20-80. [68] S. Yılmaz, Y. Akbaba, B. Özgeriş, L.Polat Köse, S. Göksu, İ. Gülçin, S.H. Alwasel, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31 (2016) 1484-1491. [69] X.F. Cao, J.S. Wang, X.B. Wang, J. Luo, H.Y. Wang, L.Y. Kong, Phytochemistry 96 (2013) 38-49. [70] H. Şenol, Ç. Bayrak, A. Menzek, E. Şahin, M. Karakuş, Tetrahedron. 72 (2016) 2587-2592. [71] S.B. Özturk Sarıkaya, F. Topal, M. Şentürk, İ. Gülçin, C.T. Supuran, Bioorg. Med. Chem. Lett. 21 (2011) 4259-4262. [72] M. ar, Y. Çetinkaya, İ. Gülçin, A. Menzek, J. Enzyme Inhib. Med. Chem. 28 (2013) 402-406. [73] A. Akıncıoğlu, Y. Akbaba, H. Göçer, S. Göksu, İ. Gülçin, C.T. Supuran, Bioorg. Med. Chem. 21 (2013) 1379-1385. [74] A. Atasever, H. Özdemir, İ. Gülçin, Ö.İ. Küfrevioğlu, Food Chem. 136 (2013) 864-870. [75] İ. Gülçin, J. Med. Food. 14 (2011) 975-985. [76] E. Köksal, E. Bursal, E. Dikici, F. Tozoğlu, İ. Gülçin, J. Med. Plants Res. 5 (2011) 217-222. [77] K. Aksu, M.

ar, M. Tanç, D.Vullo, İ. Gülçin, S. Göksu, F. Tümer, C.T. Supuran, Bioorg. Med.

Chem. 21 (2013) 2925-2931. [78] B. Aydin, I. Gülcin, S.H. Alwasel, Int. J. Food Propert. 18 (2015) 2735-2745. [79] Z. Koksal, R. Kalın, İ. Gülçin, H. Özdemir, A. Atasever, Int. J. Food Propert. 19 (2016) 1207-1216. [80] M. Küçük, İ. Gulcin, Environ. Toxicol. Pharmacol. 44 (2016) 134-139. [81] M. Şentürk, İ. Gülçin, A. Daştan, Ö.İ. Küfrevioğlu, C.T. Supuran, Bioorg. Med. Chem. 17 (2009) 3207-3211. [82] J.A. Verpoorte, S. Mehta, J.T. Edsall, J. Biol. Chem. 242 (1967) 4221-4229. [83] O. Hisar, Ş. Beydemir, İ. Gülçin, Ş. ArasHisar, T. Yanık, Ö.İ. Küfrevioğlu, Turk. J. Vet. Anim. Sci. 29 (2005) 841-845. [84]

O. Hisar, Ş. Beydemir, İ. Gülçin, Ö.İ. Küfrevioğlu, C.T. Supuran, J. Enzyme Inhib. Med. Chem.

20 (2005) 35-39. [85]

M. Bradford, Anal. Biochem. 72 (1976) 248-254.

[86]

Ş. Beydemir, İ. Gülçin, J. Enzyme Inhib. Med. Chem. 19 (2004) 193-197.

[87]

Ş. A. Hisar, O. Hisar, Ş. Beydemir, İ. Gülçin, T. Yanık, Acta Vet. Hung. 52 (2004) 413-422.

[88]

G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherston, Biochem. Pharmacol. 7 (1961) 88-95.

[89]

D.O. Ozgun, C. Yamali, H.İ. Gül, P. Taslimi, İ. Gülçin, T. Yanik, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 31 (2016) 1498-1501.

[90]

Y. Camadan, H. Özdemir, İ. Gulcin, J. Enzyme Inhib. Med. Chem. 31 (2016) 1335-1341.

[91]

Z. Huyut, S. Beydemir, İ. Gulcin, J. Enzyme Inhib. Med. Chem. 31 (2016) 1199-1205.

18

Table 1. Human carbonic anhydrase I and II isoenzymes (hCA I and II), acetylcholinesterase (AChE) and butyrylcholinesterase (AChE)

enzymes

inhibition parameters

of newly synthesized 4-

phenylbutenone derivatives IC50 (pM)

KI (pM)

No hCA I

r2

hCA II

r2

AChE

r2

BChE

r2

hCA I

hCA II

AChE

BChE

1

352.49

0.9989

199.54

0,9933

38.31

0.9928

18.75

0.9857

359.72±47.24

174.90±17.54

32.38±8.01

8.013±3.06

2

361.31

0.9935

215.68

0,9930

40.22

0.9879

35.96

0.9908

404.16±31.40

237.40±110.3

24.38±2.73

13.28±0.07

3

286.24

0.9824

159.20

0,9868

30.14

0.9442

18.93

0.9909

199.11±47.83

116.16±2.86

19.02±6.15

11.84±3.47

8

318.91

0.9886

196.31

0,9909

48.03

0.9822

22.29

0.9820

245.71±57.62

107.63±0.72

31.75±8.96

9.05±2.43

9

277.97

0.9799

178.56

0,9832

41.05

0.9777

29.96

0.9966

240.16±44.25

144.18±19.45

23.98±3.83

19.30±4.29

10

236.76

0.9660

156.71

0,9927

39.53

0.9632

16.81

0.9864

200.46±34.83

136.45±6.38

22.21±5.71

5.64±1.88

11

302.75

0.9925

237.41

0,9905

45.65

0.9745

13.87

0.9952

251.05±40.08

135.34±11.05

32.75±15.3

7.53±1.62

12

294.39

0.9846

240.87

0,9957

37.30

0.9672

28.99

0.9848

245.97±25.70

148.47±34.06

21.97±4.20

9.17±0.75

13

247.85

0.9898

220.84

0,9904

33.46

0.9806

18.89

0.9915

158.07±42.20

142.82±16.06

14.81±1.90

8.11±1.52

14

243.75

0.9693

236.52

0,9802

34.41

0.9701

20.72

0.9889

182.35±24.37

111.38±15.67

27.09±13.3

13.85±1.82

15

348.59

0.9700

282.85

0,9989

45.14

0.9822

22.74

0.9837

203.06±72.88

207.39±14.80

26.80±10.6

16.02±1.61

16

350.17

0.9951

248.38

0,9944

44.68

0.9908

25.27

0.9943

217.48±68.27

182.91±7.52

33.99±11.7

17.47±4.05

17

246.26

0.9977

193.57

0,9906

30.26

0.9829

18.02

0.9767

171.22±10.51

123.18±5.92

16.77±4.92

9.39±1.18

AZA

638.12

0.9710

594.85

0,9799

-

-

439.40±181.0

490.27±15.0

-

-

TAC

-

-

-

-

73.72

0.9848

-

-

50.72±7.05

19.16±3.73

37.87

0.9888

*Acetazolamide (AZA) was used as a standard inhibitor for both hCA I, and II. **Tacrine (TAC) was used as a standard inhibitor for AChE and BChE enzymes.

19

GRAPHICAL ABSTRACT

20

Highlights

   

The first synthesis of natural bromophenols 1- 3 was realized The synthesis of derivatives 5-17 natural bromophenols 1-3 was also realized The compounds effectively inhibit hCA I and II isoenzymes The compounds were powerful inhibitor against cholinergic AChE and BChE enzymes

21