Synthesis of Novel Bromophenol with Diaryl Methanes—Determination of Their Inhibition Effects on Carbonic Anhydrase and Acetylcholinesterase

In this work, nine new bromophenol derivatives were designed and synthesized. The alkylation reactions of (2-bromo-4,5-dimethoxyphenyl)methanol (7) with substituted benzenes 8–12 produced new diaryl methanes 13–17. Targeted bromophenol derivatives 18–21 were synthesized via the O-Me demethylation of diaryl methanes with BBr3. Moreover, the synthesized bromophenol compounds were tested with some metabolic enzymes such as acetylcholinesterase (AChE), carbonic anhydrase I (CA I), and II (CA II) isoenzymes. The novel synthesized bromophenol compounds showed Ki values that ranged from 2.53 ± 0.25 to 25.67 ± 4.58 nM against hCA I, from 1.63 ± 0.11 to 15.05 ± 1.07 nM against hCA II, and from 6.54 ± 1.03 to 24.86 ± 5.30 nM against AChE. The studied compounds in this work exhibited effective hCA isoenzyme and AChE enzyme inhibition effects. The results show that they can be used for the treatment of glaucoma, epilepsy, Parkinson’s as well as Alzheimer’s disease (AD) after some imperative pharmacological studies that would reveal their drug potential.

By hydrolyzing the neurotransmitter acetylcholine (ACh), the enzyme acetylcholinesterase (AChE) modulates cholinergic transmission at the synaptic level [42,43]. AChE affects cell adhesion, proliferation, and differentiation; the formation of tumors, apoptosis, and amyloid protein deposition in organs as well as AChE are all important cholinergic functions [44][45][46]. Abnormal levels of AChE are associated widely with neurodegenerative disorders such as myasthenia gravis, Parkinson's disease (PD), and Alzheimer's disease (AD). Currently, oral active AChE inhibitors that only provide palliative, symptomatic relief are the mainstay of treatment for AD [47][48][49].
The construction or extension of chemical libraries is very important for the development of novel lead compounds in the field of drug design and discovery. Therefore, in this study, we synthesized some novel bromophenols and evaluated their hCA I, hCA II, and AChE inhibitory properties.
By hydrolyzing the neurotransmitter acetylcholine (ACh), the enzyme acetylcholinesterase (AChE) modulates cholinergic transmission at the synaptic level [42,43]. AChE affects cell adhesion, proliferation, and differentiation; the formation of tumors, apoptosis, and amyloid protein deposition in organs as well as AChE are all important cholinergic functions [44][45][46]. Abnormal levels of AChE are associated widely with neurodegenerative disorders such as myasthenia gravis, Parkinson's disease (PD), and Alzheimer's disease (AD). Currently, oral active AChE inhibitors that only provide palliative, symptomatic relief are the mainstay of treatment for AD [47][48][49].
The construction or extension of chemical libraries is very important for the development of novel lead compounds in the field of drug design and discovery. Therefore, in this study, we synthesized some novel bromophenols and evaluated their hCA I, hCA II, and AChE inhibitory properties.

Chemistry
In this study, novel bromophenol derivatives 18-21 were synthesized in two steps. To synthesize desired diaryl methane compounds 13-17, compound 7 was first synthesized according to the procedure described by Crombie and Josephs [50]. The alkylation of substituted benzenes is a very important reaction for the synthesis of novel alkyl benzenes. The synthesis of diaryl methanes can be achieved via the reaction of benzylalcohol with substituted benzenes in the presence of AlCl 3 [51]. The application of this methodology to (2-bromo-4,5-dimethoxyphenyl)methanol (7) and benzene derivatives 8-12 in CH 2 Cl 2 (DCM) in the presence of AlCl 3 afforded novel compounds 13-16 and a known compound 17 [52], with good yields (75-92%). The O-Me demethylation of arylmethyl ethers with BBr 3 is an important strategy for the synthesis of bioactive phenols [21]. Therefore, the targeted novel bromophenols 18-21 were synthesized from the demethylation reaction of 13-16 with BBr 3 in DCM, with the yields ranging from 73 to 82% (Scheme 1). The structures of all the compounds described in this paper were characterized by IR, elemental analysis, and the 1 H and 13 C-NMR techniques.
Molecules 2022, 27, x FOR PEER REVIEW 3 of 13 of substituted benzenes is a very important reaction for the synthesis of novel alkyl benzenes. The synthesis of diaryl methanes can be achieved via the reaction of benzylalcohol with substituted benzenes in the presence of AlCl3 [51]. The application of this methodology to (2-bromo-4,5-dimethoxyphenyl)methanol (7) and benzene derivatives 8-12 in CH2Cl2 (DCM) in the presence of AlCl3 afforded novel compounds 13-16 and a known compound 17 [52], with good yields (75-92%). The O-Me demethylation of arylmethyl ethers with BBr3 is an important strategy for the synthesis of bioactive phenols [21]. Therefore, the targeted novel bromophenols 18-21 were synthesized from the demethylation reaction of 13-16 with BBr3 in DCM, with the yields ranging from 73 to 82% (Scheme 1). The structures of all the compounds described in this paper were characterized by IR, elemental analysis, and the 1 H and 13 C-NMR techniques.

Scheme 1.
The synthesis of novel bromophenol derivatives.

Biochemistry
Since abnormal levels or behaviors of the majority of the sixteen hCA isoenzymes have frequently been linked to several human diseases [53][54][55]. These CA isoforms are intensively found in different tissues and are involved in many important mechanisms such as electrolyte secretion, cell differentiation, bone resorption, calcification, pH and CO2 homeostasis, gluconeogenesis, and neurotransmission in mammals [56][57][58]. Hence, many pharmaceutical uses have notable goals for a variety of CA isoforms, including antiglaucoma drugs, anticonvulsant factors/diagnostic, diuretics, antiobesity, and antitumor tools [59,60]. For instance, inhibitors of the hCAs IX and XII isozymes have been used as antitumor and antimetastatic agents [61,62].
High amounts of the hCA I isoform have been found in the red blood cells and the gastrointestinal tract of mammals. The inhibition of this enzyme can be a key component in the treatment of conditions or diseases, including cerebral and retinal edema [63,64]. The enzyme results are given in Table 1

Biochemistry
Since abnormal levels or behaviors of the majority of the sixteen hCA isoenzymes have frequently been linked to several human diseases [53][54][55]. These CA isoforms are intensively found in different tissues and are involved in many important mechanisms such as electrolyte secretion, cell differentiation, bone resorption, calcification, pH and CO 2 homeostasis, gluconeogenesis, and neurotransmission in mammals [56][57][58]. Hence, many pharmaceutical uses have notable goals for a variety of CA isoforms, including antiglaucoma drugs, anticonvulsant factors/diagnostic, diuretics, antiobesity, and antitumor tools [59,60]. For instance, inhibitors of the hCAs IX and XII isozymes have been used as antitumor and antimetastatic agents [61,62].
High amounts of the hCA I isoform have been found in the red blood cells and the gastrointestinal tract of mammals. The inhibition of this enzyme can be a key component in the treatment of conditions or diseases, including cerebral and retinal edema [63,64]. The enzyme results are given in Table 1 and Figure 2. In the current study, all the novel, synthesized a series of bromophenols (13)(14)(15)(16)(17)(18)(19)(20)(21)      The dominant cytosolic hCA II isoform plays a critical function in disorders such as glaucoma [65]. In fact, the production of HCO 3 − acts as a method to introduce water and Na + ions into the eye, increasing intraocular pressure. As a result, hCA II isozyme inhibition lowers HCO 3 − generation and eye pressure [66,67]. In the current study, bromophenols (13-21) effectively inhibited hCA II with IC 50 s ranging from 7.45 to 27.72 nM and K i s ranging from 1.63 ± 0.11 to 15.05 ± 1.07 nM. Compound 13 demonstrated the best inhibition effects (K i : 1.63 ± 0.11 nM) (Figure 2). When the K i values of the studied compounds (13)(14)(15)(16)(17)(18)(19)(20)(21) were evaluated against hCA II, the following order was found: 13 (1.63 ± 0.11 nM) > 15 (2.62 ± 0.13 nM) > 14 (4.28 ± 0.86 nM) > 21 (4.97 ± 0.59 nM) > 20 (6.21 ± 1.01 nM) > 16 (7.77 ± 0.57 nM) > 18 (9.15 ± 1.36 nM) > 17 (10.33 ± 1.88 nM) > 19 (15.05 ± 1.07 nM). Furthermore, all the novel, synthesized a series of bromophenols (13-21) exhibited competitive inhibition against the physiologically dominant hCA II isoenzyme. The proposed interaction between the most powerful bromophenols (20) and the CA II isoforms is illustrated in Figure 3. Bromophenol (20) has two dihydroxy benzyl rings. A second hydrogen bond was modeled between the oxygen atom, which attached to the -OH group at the phenol moiety in the ortho-position, and the amide NH of Thr199, a universally conserved amino acid residue in CAs. Thus, phenolic compounds and derivatives bind non-classically to CA, providing clues for the identification of new types of CA inhibitors. Such inhibition mechanisms of phenolic compounds, including bromophenols, are known [68,69]. As shown in Table 1, the attachment of three methoxy groups caused a decrease in the hCA II inhibition value. The methoxy group at positions 2, 3, and 4 may have created a steric hindrance in enzyme inhibition. As in the hCA I isoform, the presence of the methoxy group instead of the hydroxyl group in the compounds was more effective in inhibiting hCA II. When the compounds of 19 and 21 were compared with each other, the presence of the hydroxyl group instead of the methyl group caused a 3.03-fold increase in the inhibition value. A similar situation was observed in hCA I inhibition. This may be because the hydroxyl group is more electronegative than the methyl group.
ACh is used as a neurotransmitter component, and AChE is a crucial enzyme that catalyzes ACh breakdown. This enzyme has been linked to therapeutic targets for AD [70,71]. The hypothesis was put forth to explain AD that synaptic depression is hampered because the cholinergic neuron cells impede ACh hydrolysis [72,73]. ACh hydrolysis is hindered because of AChE inhibition. As a result, the development of AChE enzyme inhibitor drugs and/or modulators is of great interest because it is currently one of the main goals in the fight against AD [74,75]. In the current study, bromophenols (13-21) effectively inhibited AChE with IC 50 s ranging from 8.35 to 21.00 nM and K i s ranging from 6.54 ± 1.03 to 24.86 ± 5.30 nM. The inhibitor effects of the studied compounds (13)(14)(15)(16)(17)(18)(19)(20)(21) against AChE were decreased as follows: 21 (6.54 ± 1.03 nM) > 18 ( (13)(14)(15)(16)(17)(18)(19)(20)(21) showed competitive inhibition against the cholinergic enzyme of AChE. As shown in Table 1, in the methoxy-bonded compound groups, the fact that the methyl group (14) is attached instead of the bromine ion (13) did not cause any change in inhibition. When compounds 15 and 16 are compared, the addition of the bromine group to the 4th position caused a rise in the inhibition value. The presence of the -OCH 3 groups in the middle position without the bromine group was more effective in AChE inhibition (15, K i : 24.86 ± 5.30 nM; 16, K i : 16.27 ± 2.98 nM). As in hCA I and II, the presence of the methoxy group instead of the hydroxyl group in the compounds was more effective in inhibiting AChE. When compounds 19 and 21 were compared with each other, the presence of the hydroxyl group instead of the methyl group caused a 2.67-fold increase in the inhibition value. ACh is used as a neurotransmitter component, and AChE is a crucial enzyme that catalyzes ACh breakdown. This enzyme has been linked to therapeutic targets for AD [70,71]. The hypothesis was put forth to explain AD that synaptic depression is hampered because the cholinergic neuron cells impede ACh hydrolysis [72,73]. ACh hydrolysis is hindered because of AChE inhibition. As a result, the development of AChE enzyme inhibitor drugs and/or modulators is of great interest because it is currently one of the main goals in the fight against AD [74,75]. In the current study, bromophenols (13)(14)(15)(16)(17)(18)(19)(20)(21) (24.86 ± 5.30 nM). In addition, all the novel synthesized a series of bromophenols (13)(14)(15)(16)(17)(18)(19)(20)(21) showed competitive inhibition against the cholinergic enzyme of AChE. As shown in Table 1, in the methoxy-bonded compound groups, the fact that the methyl group (14) is attached instead of the bromine ion (13) did not cause any change in inhibition. When compounds 15 and 16 are compared, the addition of the bromine group to the 4th position caused a rise in the inhibition value. The presence of the -OCH3 groups in the middle position without the bromine group was more effective in AChE inhibition (

General
Commercially purchased chemicals were used without further purification. Solvents were used after distillation or after drying with various drying agents. The melting points were determined using a capillary melting equipment and were not corrected (Buechi 530). A PerkinElmer spectrophotometer was used to collect IR spectra (Lancashire, Great Britain) from liquids in 0.1 mm cells. On a 400 (100) MHz (Varian, Danbury, CT) and 400 (100) MHz (Bruker, Fallanden, Switzerland) spectrometers, the 1 H and 13 C NMR spectra were collected; d was in ppm, with Me 4 Si as the internal standard. On a Leco CHNS-932 apparatus (St. Joseph, Missouri, USA), elemental analyses were performed. The silica gel was used for column chromatography (60-mesh, Merck, Darmstadt, Germany). PLC stands for preparative thick-layer chromatography, which used 1 mm of silica gel (60 PF, Merck, Darmstadt, Germany) on glass plates. The synthesized compounds' 1 HNMR and 13 C NMR spectra are provided as Supplementary Materials.

General Synthesis Procedure for the Synthesis of Compounds 13-17
The compound 2-Bromo-4,5-dimethoxybenzenemethanol (7) (5 mmol), the corresponding benzene derivatives (8-12) (5 mmol), and AlCl 3 (7 mmol) were dissolved in 30 mL of dry CH 2 Cl 2 . The solution was cooled to 0 • C in an ice bath and stirred for 24 h. The reaction mixture was quenched by ice-cold water (20 mL) to remove unreacted AlCl 3 . The organic phase was separated, and the water phase was extracted with CH 2 Cl 2 (2 × 30 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , and the solvent was evaporated. Then, the crude products were separated on a silica gel column by using hexane/EtOAc to obtain the pure products.  (17) 1-(2-bromo-4,5dimethoxybenzyl)-2,3,4-trimethoxybenzene (17) was synthesized by a different method than that of described previously [52].
Yield Diaryl methane compounds (13)(14)(15)(16)(17) were dissolved in CH 2 Cl 2 . The solutions were cooled to 0 • C. To these solutions, for each methoxy group in the structure of these compounds, 3 equivalents of BBr 3 were added dropwise under N 2 atmosphere. Then, the mixtures were stirred at rt for 24 h. The reaction medium was cooled to 0 • C. Ice (20 g) and CH 2 Cl 2 (50 mL) were added to the reaction medium and the organic phases were separated. Then, the water phase was extracted with ethyl acetate (2 × 50 mL). The organic layers were combined, dried over anhydrous Na 2 SO 4 and the solvents were evaporated. The residue was crystallized from EtOAc/Hexane.  In this work, the in vitro inhibition effects of bromophenols (13)(14)(15)(16)(17)(18)(19)(20)(21) on AChE activity were determined by Ellman's method [76], as previously described [77]. The results were recorded spectrophotometrically at 412 nm. Acetylthiocholine iodide (AChI) was used as substrate, according to a prior study [78]. Both hCA isoforms were purified by using the Sepharose-4B-L-Tyrosine-sulfanilamide affinity technique [79]. Then, the purity of these CA isoenzymes was defined via the SDS-PAGE purity technique [80,81]. Furthermore, the hCA activity was determined using the esterase method at 348 nm, according to the method of Verpoorte et al. [82] and as given in prior studies [83,84].

Statistical Analyses
Statistical analyses were performed via an unpaired Student's t-test with the use of the statistical program IBM SPSS Statistics 20. The results were recorded as means with their standard deviation (SD). p < 0.05 was the minimum significance level.

Conclusions
In the current study, new bromophenols were synthesized, and their hCAs and AChE inhibitory properties were investigated. The presence of different biologically functional groups (-OH, -OCH3, and -Br) in aromatic scaffolds of synthesized compounds influenced the activity of the studied enzymes. Our findings indicate that the investigated compounds 13-21 exhibited efficient hCA I, II, and AChE inhibition effects in the low nanomolar levels. These experimental findings confirm that substituted methoxy (-OCH3) and bromophenols may be used as leads for generating potent CAI and AChE inhibitors associated with some global disorders, including AD, epilepsy, and glaucoma.