Synthesis and bioactivities of 1-(4-hydroxyphenyl)-2-((heteroaryl)thio)ethanones as carbonic anhydrase I, II and acetylcholinesterase inhibitors

The discovery of enzyme targeting inhibitors is a popular area of drug research. Biological activities of the compounds bearing phenol and heteroaryl groups make them popular groups in drug design targeting important enzymes such as acetylcholinesterase (AChE, E.C.3.1.1.7) and carbonic anhydrases (CAs, EC 4.2.1.1). 1-(4-hydroxyphenyl)- 2-((aryl)thio)ethanones as possible AChE and CAs inhibitors were synthesized, and their chemical structures were confirmed by IR, 1H NMR, 13C NMR, and HRMS. The compounds 2 and 4 were found potent AChE inhibitors with the Ki values of 22.13 ±1.96 nM and 23.71 ±2.95 nM, respectively, while the compounds 2 (Ki = 8.61 ±0.90 nM, on hCA I) and 1 (Ki = 8.76 ±0.84 nM, on hCA II) had considerable CAs inhibitory potency. The lead compounds may help the scientists for the rational designing of an innovative class of drug candidates targeting enzyme-based diseases.

Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc bearing metalloenzymes which contribute to the controlling of pH balance by the acceleration of the hydration reaction of carbon dioxide in the living cells [14][15][16].
Regulation of CAs activity by using inhibitors is used in the clinic as antiglaucoma drugs and diuretics [17,18].
Besides, CAIs might have potential as anticancer, antiobesity, and antiinfective agents [19,20]. To date, 15 α -CA isoforms in mammals have been reported. Among them, the cytosolic CA I and CA II isoforms are well-established targets for several diseases. While CA I is responsible for cerebral edema, CA II inhibition has a role for glaucoma, edema, altitude sickness, and epilepsy [21][22][23]. The sulfonamides and their bioisosteres (sulfamates, sulfamides, etc.) are the main pharmacophore groups which interact with the active site of CA isoenzymes [24]. For instance, sulfonamide having acetazolamide, methazolamide, ethoxzolamide ( Figure 1) are widely used systemic antiglaucoma drugs in the clinic and these drugs have heterocyclic structures in their chemical skeleton. After that, the investigation of CA inhibition capacity and the mechanism of action of the phenol ( Figure 1) led to design novel phenolic compounds targeting CA isoenzymes [25][26][27]. Hence, the substitution of phenol pharmacophore can be considered in the design strategy to have potent compounds with enhanced CA activity. Based on the reports, different heterocyclic rings were combined with the phenol functional group since the phenol is one of the popular CAIs group and promising potential pharmacophore for new AChEIs. This study aimed to report CAs and AChE enzyme inhibitory potencies of the 1-(4-hydroxyphenyl)-2-((aryl)thio)ethanones 1-6 to find out CAs and AChE enzyme inhibitors for further studies.  4-methyl-4H -1,2,4-triazole-3-thiol for 5 (1.1 g), 1-methyl-1H -tetrazole-5-thiol for 6 (1.1 g)] was dissolved at room temperature in fresh methanolic NaOH solution (0.4 g, 25 mL methanol) and stirred for 10 min. Then, 2-bromo-4'-hydroxyacetophenone (9.3 mmol, 2 g) was put into the reaction flask. After the final mixture was stirred for several hours at room temperature, it was taken into the water (50 mL). The white solid obtained was filtered, washed with water three times, and then dried. The compounds were purified by crystallization using suitable solvents such as ethanol (1)(2)(3)(4)6 ) and ethanol: DMF (5).

CAs inhibition assay
Human CA isoforms (hCAI and hCAII) were purified by the Sepharose -4B -L -tyrosine -sulfanilamide affinity segregation method as reported [34,35]. Bradford technique was used to measure protein concentrations at 595 nm [36]. Inhibitory effects of the compounds were investigated by measuring the esterase activity according to Verpoorte et al . [37] as described in previous [38][39][40]. The hCA activity was determined by measuring the conversion of the p-nitrophenyl acetate substrate to p -nitro phenolate at 348 nm by the spectrophotometer (UV -VIS Spectrophotometer, UVmini-1240, Shimadzu Corporation, Kyoto, Japan) [41]. Acetazolamide (AZA) was used as a control drug. Lineweaver-Burk plot was used to calculate inhibition constants (Ki) of the compounds [33]. All chemicals were purchased from Sigma-Aldrich Chemie GmbH.

Chemistry
This study reported the synthesis and bioactivities of the compounds having 1-(4-hydroxyphenyl)-2-((aryl)thio) ethanones chemical formula. 2-Bromo-4'-hydroxyacetophenone was attached to the heterocyclic rings via thioether functional group by conducting a single step reaction. The compounds 1, 2, 3, 5, and 6 were found as registered compounds at the Sci Finder database without any article and experimental data, while compound 4 [42] was reported as an intermediate. Therefore, the current study is the first study for the synthesis and bioactivities of 1, 2, 3, 5, and 6.
As a spectral evaluation, in 1 H NMR, methylene protons were seen in the range of 5.07-4.49 ppm as expected. In some cases, the phenolic proton was not seen since it is an exchangeable proton. Signal of methyl substituent for the methyl-substituted compounds was seen in the range of 3.97-2.63 ppm. The carbonyl peak of the compounds in 13 C NMR was seen in the range of 191.0-192.6 ppm. The carbon peak of the methylene bridge was seen at 49.1-41.1 ppm, while the carbon signal of methyl was in the range of 34.1-15.6 ppm. Further, calculated and measured m/z values of the compounds were also found compatible in HRMS analysis. In the IR spectra, C =O stretching band was recorded in the range of 1655-1672 cm −1 .

Acetylcholinesterase inhibitory effects
In this study, the compounds 1-6 were screened against the AChE enzyme due to significant reports on AD of The compounds 1 and 2 having a bicyclic ring showed considerable AChE inhibitory effects with low Ki values. When these two compounds compared with each other, it shows that oxygen atom was more favorable than the sulfur atom. Diazole (3) and tetrazole (6) derivatives were found more effective than triazole (5) derivative against the AChE enzyme. The compound 4 having 5-methyl-1,3,4-thiadiazol ring showed favorable enzyme inhibitory potency with Ki value of 23.71 ±2.95 nM in contrast to other five-membered compounds 3-6.
The compounds reported might be potential candidates for designing novel and more powerful AChE inhibitors for future studies. AD drugs such as donepezil, rivastigmine, galantamine have amine moiety in their chemical structure. So, the compounds having nitrogen atom/s may show favorable enzyme-ligand interactions [43]. Also, for the future concept, the most potent amine bearing phenolic compounds can be synthesized with Mannich reaction by the reaction of amine, formaldehyde, and phenolic compound under suitable reaction conditions as mono or bis Mannich bases against AChE based on our previous work regarding Mannich bases as promising AChE inhibitors [30].

Carbonic anhydrase inhibitory effects
Since the phenol group is an important zinc-binding group, the compounds 1-6 were evaluated towards CAs isoenzymes to show their CA inhibitory potency. The Among the compounds having the most common bicyclic rings, the compound 2 (Ki = 8.61 ±0.90 nM) was 2.5 fold more potent inhibitor against hCAI while the compound 1 (Ki = 8.76 ±0.84 nM) was 2.0 fold more potent against hCAII isoenzyme than reference AZA. On the other hand, among the compounds carrying five-membered rings 3-6, the compound 4 (Ki = 13.81 ±2.47 nM) for hCAI, and the compound 5 (Ki = 14.32 ±5.10 nM) for hCAII were potent CA inhibitors. When diazole (3), triazole (5) and tetrazole (6) derivatives were compared, the following results can be made. The tetrazole derivative 6 had good inhibitory potency against hCAI while triazole derivative 5 was effective inhibitor against hCAII isoenzyme. In series, benzothiazole and benzoxazole bearing compounds 1 and 2 were found the most potent CA inhibitor against the hCAs with the lowest Ki values. Also, it can be stated here that linking bicyclic ring with phenol function was found more rewarding modification than five-membered rings. Additional aromatic hydrophobic interactions with the active site residues of the enzyme may result in increasing enzyme inhibitory potency of these compounds. For future studies, these phenolic compounds can be used to synthesize novel CAIs by the reaction of phenol group with suitable reagents to obtain new sulfamate-based CAIs to see how this modification affects the CAs inhibition activity.

Conclusion
In this study, different heteroaryl mercapto compounds were combined with the phenolic group.