Novel Mannich bases with strong carbonic anhydrases and acetylcholinesterase inhibition effects: 3-(aminomethyl)-6-{3-[4-(trifluoromethyl)phenyl]acryloyl}-2(3H)-benzoxazolones

In this study, a new series of Mannich bases, 3-(aminomethyl)-6-{3-[4-(trifluoromethyl)phenyl]acryloyl}-2( 3H )-benzoxazolones ( 1a–g ), were synthesized by the Mannich reaction. Inhibitory effects of the newly synthesized compounds towards carbonic anhydrases (CAs) and acetylcholinesterase (AChE) enzymes were evaluated to find out new potential drug candidate compounds. According to the inhibitory activity results, Ki values of the compounds 1 and 1a–g were in the range of 12.3 ± 1.2 to 154.0 ± 9.3 nM against hCA I, and they were in the range of 8.6 ± 1.9 to 41.0 ± 5.5 nM against hCA II. Ki values of acetazolamide (AZA) that was used as a reference compound were 84.4 ± 8.4 nM towards hCA I and 59.2 ± 4.8 nM towards hCA II. Ki values of the compounds 1 and 1a–g were in the range of 35.2 ± 2.0 to 158.9 ± 33.5 nM towards AChE. Ki value of Tacrine (TAC), the reference compound, was 68.6 ± 3.8 nM towards AChE. Furthermore, docking studies were done with the most potent compounds 1d , 1g , and 1f (in terms of hCA I, hCA II, and AChE inhibition effects, respectively) to determine the binding profiles of the series with these enzymes. Additionally, the prediction of ADME profiles of the compounds pointed out that the newly synthesized compounds had desirable physicochemical properties as lead compounds for further studies.

As conclusion, we first aimed to synthesize novel Mannich bases of the previously synthesized chalcone compound, 6-[3-(4-trifluoromethylphenyl)-2-propenoyl]-2(3H)-benzoxazolone, via aminomethylation from the 3rd position of the benzoxazolone ring. Carbonic anhydrase (CA) and acetylcholinesterase (AChE) inhibitory activities of the compounds were evaluated to find out new potential drug candidate molecule/s. Furthermore, docking studies were done to confirm and explain the inhibition effects of the newly synthesized compounds.

Chemistry
All chemicals and solvents were Sigma-Aldrich (Germany) and Merck (Germany). Bruker AVANCE III 400 MHz (Bruker, Karlsruhe, Germany) spectrometer [400 Hz ( 1 H) and 100 Hz ( 13 C)] was used to record the nuclear magnetic resonance (NMR) spectra ( 1 H NMR, 13 C NMR). Dimethyl sulfoxide (DMSO)-d6 was used as a solvent. NMR VTU technique was applied to keep the solubility of the compounds stable. The 1 H and 13 C NMR spectra of the compounds were recorded with dynamic NMR method at 328.15 K by using the Topspin 2.1 NMR program. The internal standard was tetramethylsilane. IR spectra of the compounds were recorded with the FTIR-ATR method (400-4000 cm -1 ) on a IRSpirit Fourier transform (FT)-IR spectrophotometer (Shimadzu, Kyoto, Japan). Mass spectra of the samples were recorded using a liquid chromatography ion trap-time of flight tandem mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization (ESI) source, operating in both positive and negative ionization mode. For data analysis, Shimadzu's LCMS Solution program was used. Electrothermal 9100 instrument (IA9100, Bibby Scientific Limited, Staffordshire, UK) was used to determine the melting points of the compounds.

Acetylcholinesterase inhibition assay
Acetylcholinesterase enzyme inhibition assay was performed according to our previous studies [4,41,[51][52][53][54][55]. The inhibition effects of the compounds 1 and 1a-g on AChE enzyme, obtained from electric eel [56], were recorded in accordance with Ellman's method as demonstrated previously in detail [51,52,57]. The substrates of acetylthiocholine iodide (AChI) and 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB) was used for cholinergic enzymatic reaction. For this purpose, 1 mL of Tris / HCl buffer (1.0 M, pH 8.0) and 10 μL of different concentrations of sample solution were dissolved in deionized water. Then, an aliquot of (50 μL) AChE enzyme was transferred and incubated at room temperature for 10 min. After the incubation period, an aliquot of DTNB (0.5 mM, 50 μL) was added. Then, the enzymatic reaction was started by adding 50 μL of AChI (10 mM). The breakdown of these substrates was monitored spectrophotometrically by the yellow color formation of 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine from hydrolysis of AChI with absorption at a wavelength of 412 nm [58,59].

Carbonic anhydrase inhibition assay
CA inhibition assay was recorded according to previous studies [4,10,27,49,[60][61][62][63][64]. The hCA I and II isoenzymes were purified from human red blood cells by sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography [65], which was used as an affinity matrix for selective retention of both hCA isoenzymes [27,66]. The inhibition effects of the compounds 1 and 1a-g on both hCA isoenzymes were spectrophotometrically measured according to the previous method described in detail [49,60,67]. p-Nitrophenylacetate (PNA) was used as a substrate and changed to p-nitrophenolate ions (PNP). One CA isoenzyme unit is accepted as the amount of CA, which had absorbance change at 348 nm of PNA to PNP over a period of 3 min at 25 °C. After sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography, the enzyme quantity was spectrophotometrically measured at 280 nm [68]. Moreover, the protein effluent was spectrophotometrically determined at 595 nm according to the Bradford method. Purity controls of both CA isoenzymes were performed according to the Laemmli procedure as described in previous studies. Both isoenzymes were visualized by two different acrylamide concentrations (10% and 3% acrylamide), containing 0.1% sodium dodecyl sulfate (SDS) [69,70].

Molecular docking studies
The most active compounds 1d (pyrrolidine derivative) in terms of hCA I inhibition effect, 1g (N-methyl piperazine derivative) in terms of hCA II inhibition effect, and 1f (morpholine derivative) in terms of AChE inhibition effect were docked at the binding sites of the mentioned enzymes to describe and confirm the inhibition effects of the newly synthesized compounds. The Protein Data Bank (PDB 1 ) was used to get the structures of hCA I (4WR7), hCA II (3HS4), and AChE (1C2O) enzymes. The pdb files of the enzymes were prepared and transferred to AutoDockTools (ADT ver.1.5.6). Water molecules of the structures were removed and only polar hydrogen and Kollman charges were added to the proteins. Finally, the pdbqt files of the proteins were saved.
AutoDockTools (ADT ver.1.5.6) was used for molecular docking studies. The Lamarckian genetic algorithm with local search (GALS) was used as a search engine, with a total of 10 runs. The active sites of enzymes were defined by a grid box of 70 × 70 × 70 points. Ten conformers of the compound were considered to evaluate the docking results. Finally, the conformer with the lowest binding free energy was evaluated using Python Molecule Viewer (PMV ver.1.5.6) and PyMOL (ver. 2.3.3, Schrodinger, LLC).

Estimation of physicochemical and ADME properties
In silico prediction of the ADME parameters and physicochemical properties the compounds was performed using the SwissADME 3 web tool. The structures of the compounds 1 and 1a-g were drawn and transformed to SMILES (simplified molecular-input line-entry system). Finally, the ADME parameters and physicochemical parameters of the compounds 1 and 1a-g were calculated by running the program.
According to IR spectra of the compounds, the lactam and ketone C=O stretching bands were seen about 1770 cm -1 and 1650 cm -1 . According to 1 H NMR spectra of the synthesized compounds, the methylene protons between two nitrogen atoms of Mannich bases appeared at the area of 4.5-5.5 ppm as expected. On the other hand, 1 H NMR spectra of the compounds showed that all compounds were configured trans, as understood from coupling constant J = 15.2 -16.1 Hz for vinyl protons or from the results of the NOESY NMR of the compounds. The DEPT NMR spectra of the compound 1a were recorded to determine primary, secondary, and tertiary carbon atoms of the series. According to the DEPT NMR results of the compound 1a, carbons of the carbonyl groups were seen about 187 ppm (ketone) and 155 ppm (lactam). Signals of the other quaternary carbons were seen at 142.55, 136.89, 135.18, 132.75, 128.71, and 110.02 ppm. In the aliphatic region, the carbon of methylene group (secondary) was seen as a negative signal while the carbons of methyl groups (primary) were seen as a positive signal. The chemical structures of the newly synthesized compounds 1, 1a-g were confirmed and characterized by IR, 1 H NMR, 13 C NMR, and HRMS (see the experimental part for details).

Carbonic anhydrases inhibitory activities
The CA inhibition effects of the compounds 1a-g were reported for the first time in this study, and the CA inhibition results are shown in Table 1. Acetazolamide (AZA) was used as a reference drug, and its K i values were 84.4 ± 8.4 nM towards hCA I and 59.2 ± 4.8 nM towards hCA II.
The compounds 1 and 1a-g had K i values of in the range of 12.3 ± 1.2 to 154.0 ± 9.3 nM towards hCA I ( Table  1). According to hCA I inhibitory activity results of the compounds, all Mannich bases synthesized (1a-g) had higher inhibitory activities than the reference compound, AZA. On the other hand, compound 1 had both the lowest inhibitory activity in the series and lower inhibitory activity than the reference compound, AZA. As a result, the aminomethylation from the 3rd position of the benzoxazolone ring by Mannich reaction caused a significant increase (in the range of 2.1-12.5 times) in hCA I inhibition effects, as expected. This result suggested that the prevention of tautomerization between the nitrogen atom and carbonyl of the benzoxazolone ring had a crucial role in hCA I inhibitory activity. According to the K i values presented in Table 1, the pyrrolidine derivative compound 1d had the highest inhibition effect in the series with a lower K i value (12.3 ± 1.2 nM) than AZA (K i = 84.4 ± 8.4 nM) towards hCA I. Including the nitrogen atom of Mannich base in a cyclic structure (pyrrolidine derivative compound 1d) led to an approximately three-fold increase in hCA I inhibitory activity comparing to diethylamine derivative compound 1b. On the other hand, replacing the pyrrolidine ring (compound 1d) with a six-membered ring (piperidine derivative compound 1e) led to an approximately two-fold decrease in hCA I inhibitory activity. Comparing the K i values of compounds 1e and 1f pointed out that the replacement of the carbon atom on the piperidine heterocycle with the oxygen atom caused an increase in the hCA I inhibition effect. This may be due to the formation of a hydrogen bond between the oxygen atom and the active site of hCA I isoenzyme.
According to Table 1, K i values of the compounds 1, 1a-g were in the range of 8.6 ± 1.9 to 41.0 ± 5.5 nM towards hCA II isoenzyme. The hCA II inhibitory activity results of the compounds presented in Table 1 showed that all compounds synthesized (1 and 1a-g) showed higher inhibition effects with their lower K i values than the reference compound AZA. Thus, the results presented in Table 1 pointed out that the aminomethylation from the 3rd position of the benzoxazolone ring by Mannich reaction generally resulted in an increase (except compounds 1b and 1f) in hCA II inhibitory activity. The most potent compound in the series was N-methyl piperazine derivative compound 1g with the remarkable K i value of 8.6 ± 1.9 nM, which is approximately 7 times lower than AZA. Thus, compound 1g was the most important compound of the series in terms of hCA II inhibitory activity and can be regarded as a lead molecule for further investigations. According to Table 1, the morpholine derivative compound 1f had the lowest hCA II inhibition effect in the series with the K i value of 41.0 ± 5.5 nM which is 1.4 times lower than AZA. The pyrrolidine derivative compound 1d including the nitrogen atom of Mannich base in a cyclic structure had higher inhibitory activity (K i = 19.01 ± 3.0 nM) about 2 times towards hCA I than diethylamine derivative compound 1b (K i = 37.6 ± 4.3 nM). Comparing the K i values of compounds 1e and 1f towards hCA II showed that the replacement of the carbon atom on the piperidine heterocycle with the oxygen atom led to a decrease about 2 times in the hCA II inhibition effect. On the other hand, the inhibition results in Table 1 suggested that the compounds carrying the nitrogen atom of Mannich base in a cyclic structure (compounds 1d -g) generally had higher inhibitory activities towards hCA II isoenzyme than the compounds carrying the nitrogen atom of Mannich bases on a straight chain (compounds 1a-c).

AChE inhibitory activities
The AChE inhibitory activities of the newly synthesized compounds 1 and 1a-g were reported for the first time in this study and the results were presented in Table 1. The reference drug used was tacrine (TAC) and its K i value was 68.6 ± 3.8 nM towards AChE enzyme. According to the results presented in Table 1, K i values of the compounds 1 and 1a-g were in the range of 35.2 ± 2.0 to 158.9 ± 33.5 nM towards AChE. The inhibition results showed that compounds 1f and 1g had higher inhibitory effects than the reference drug TAC. The morpholine derivative compound 1f was the most active compound in the series with the K i value as 35.2 ± 2.0 nM. On the other hand, the chalcone compound 1 had the lowest inhibition effect in the series with the K i value as 158.9 ± 33.5 nM. Thus, the inhibition results showed that synthesis of Mannich derivatives from the 3rd position of the benzoxazole ring of the compound 1 was a modification that contributed to the AChE inhibition effect. This may be due to the prevention of tautomerization between the amino and carbonyl groups of the benzoxazolone ring. Besides, the inhibition results presented in Table 1 pointed out that the compounds where the nitrogen atom of Mannich base was included in a cyclic structure (compounds 1d -g) had higher inhibitory effects on AChE enzyme than the compounds carrying the nitrogen atom of Mannich bases on a straight chain (compounds 1a-c). Even, adding one more heteroatom (oxygen or nitrogen) to this heterocyclic structure (compounds 1f and 1g, respectively) resulted in an increase in AChE inhibitory activity comparing to piperidine derivative compound 1e.

Molecular docking studies
To demonstrate the binding model of the synthesized compounds 1 and 1a-g with the active sites of hCA I, hCA II, and AChE enzymes, docking studies were performed using AutoDockTools (ADT ver.1.5.6). The X-ray crystallographic structures of hCA I (4WR7), hCA II (3HS4), and AChE (1C2O) were obtained from the Protein Data Bank 4 . The chalcone compound 1 and the most potent compounds 1d (pyrrolidine derivative), 1g (N-methyl piperazine derivative), and 1f (morpholine derivative) in terms of hCA I, hCA II, and AChE inhibitory activities (respectively) were docked at the binding sites of the mentioned enzymes to describe and confirm the inhibition effects of the series. First, the active sites of the hCA I, hCA II, and AChE were defined by using BIOVIA Discovery Studio Visualizer. Then, the reference drugs acetazolamide (AZA) and tacrine (TAC) were docked into the human carbonic anhydrases (hCA I and hCA II) and AChE, respectively. These simulations were done successfully and the binding free energy scores of the reference drugs were found as -6.53 (in 4WR7), -6.77 (in 3HS4), and -7.10 (in 1C2O) kcal / mol. Finally, the binding free energy scores and K i values of the chalcone compound 1 and the most potent compounds 1d, 1g, and 1f in the series were calculated by docking simulations and the results were presented in Table 2.
The energy score of the most active compound 1d (pyrrolidine derivative) towards hCA I was found as -8.18 kcal/mol. Thus, this suggested the strong interactions of the compound with the active site of the 4WR7, as expected. According to Figure 3, the carbonyl moiety of the benzoxazolone ring of the compound 1d realized a hydrogen bonding with the amino groups of the amino acids His200. This showed the importance of the prevention of tautomerization between the nitrogen atom and carbonyl of the benzoxazolone ring by Mannich reaction in designing hCA I inhibitory compounds in terms of the strong interactions with the enzyme. Besides, the pyrrolidine ring realized a hydrophobic interaction with the amino acid Thr199. According to the binding pattern of compound 1d presented in Figure 3, the π-cation interaction was exhibited between the benzoxazolone ring of the compound and the side chain of the amino acid His200. On the other 4 https://www.rcsb.org/ hand, seven hydrophobic interactions were exhibited between the compound 1d and the amino acids Phe91, His119, Leu131, Val143, Leu198, and Trp209. Furthermore, trifluoromethyl moiety of the compound 1d involved in a halogen bond with the amino acid Phe91. This pointed out that the trifluoromethyl group of the compounds designed had an important role in strong interactions with the active site of the hCA I isoenzyme, as planned. Compound 1g, N-methyl piperazine derivative, showed higher inhibitory activity (K i = 8.6 ± 1.9 nM) than AZA towards hCA II and its docking score was -8.05 kcal/mol that is lower than the reference drug's binding free energy (-6.77  kcal/mol), as expected. The binding pattern of compound 1g with the active site of the hCA II enzyme (3HS4) is shown in Figure 4. In Figure 4, it was seen that the carbonyl group of benzoxazolone heterocycle of compound 1g realized a hydrogen bond with the side chain of the amino acid Gln92. This shows how the prevention of tautomerization between amino and carbonyl groups by aminomethylation contributes positively to hCA II inhibitory activity. Furthermore, two hydrogen bonds were observed between the oxygen atom of the benzoxazolone ring and the amino acids Asn62 and Asn67. On the other hand, the carbonyl group of chalcone structure of compound 1g realized a hydrogen bond with the side chain of the amino acid Trp5. According to the binding profile of compound 1g (Figure 4), π-stacking interaction was exhibited between the benzoxazolone ring of the compound and the amino acid His94. Besides, four hydrophobic interactions were seen between the compound 1g and the amino acids Trp5, Phe20, and Pro202.
The morpholine derivative compound 1f had the highest inhibition effect on AChE enzyme in the series and docked in 1C2O to explain its inhibition effect. As expected, compound 1f had lower energy score, -10.17 kcal/mol, in AChE than reference drug TAC (-7.10 kcal/mol), recommending a strong interaction between the compound 1f and the 1C2O. According to Figure 5, the oxygen atom on the first position of the benzoxazolone ring interacted with the enzyme via two hydrogen bonds with the amino groups of the amino acids Phe295 and Arg296. Similarly, the oxygen atom of the morpholine moiety formed a hydrogen bond with the amino group of Ser293. Thus, this suggested that aminomethylation of benzoxazolone ring with morpholine was a useful modification in terms of strong interactions with the active site of AChE. On the other hand, eight hydrophobic interactions were detected between compound 1f and the amino acids Asp74, Trp86, Phe297, Tyr337, Phe338, and Tyr341 ( Figure 5). Additionally, one halogen bond was exhibited between the trifluoromethyl group of the compound 1f and the side chain of the amino acid Thr83. This pointed out the importance of the trifluoromethyl moiety in designing in terms of AChE inhibition effect.

Estimation of physicochemical properties
Both the physicochemical and the ADME properties of a compound are very crucial in oral drug-candidate designing. 'Drug-likeness' is a term that describes the potentiality for a compound to be an oral drug in terms of bioavailability [72]. Lipinski's 5 rules are used to evaluate this potentiality of a molecule [4,73]. In this study, both the physicochemical and the pharmacokinetic properties of the synthesized compounds 1 and 1a-g were estimated using the SwissADME 5 web tool [4,72]. For this purpose, molecular weights, the numbers of hydrogen bond acceptors and donors, the topological surface 5 http://www.swissadme.ch/index.php areas (TPSA; a sum of polar atoms' surfaces), lipophilicities, water solubilities, and bioavailability of the synthesized compounds 1 and 1a-g are presented in Table 3.
The results in Table 3 showed that all of the synthesized compounds were in agreement with Lipinski's rule of 5. The compounds' ClogP values ranged between 3.70 and 5.29 (˂6.5), molecular weight range of 333.26-446.26 (˂500), HBA range of 6-8 (≤ 10), and HBD values 0-1 (˂5), suggesting that compounds 1 and 1a-g have desired drug-likeness properties. On the other hand, all of the compounds had desired logP values and were estimated to have high gastrointestinal absorption and to penetrate the blood-brain barrier. These are advantages for the oral use of the synthesized compounds as AChE inhibitors in AD.
The radar charts of the most potent compounds 1d, 1g, and 1f towards hCA I, hCA II, and AChE, respectively, are shown in Figure 6. The pink-colored areas in these charts demonstrate the ideal ranges for each physicochemical properties; LIPO (lipophilicity, XLogP: between -0.7 and +5.0), size (MW: between 150 and 500 g/mol), POLAR (polarity, TPSA: between 20 and 130 Å 2 ), INSOLU (solubility, log S: not higher than 6), INSATU (saturation; the fraction of carbons in the sp 3 hybridization not less than 0.25), FLEX (flexibility, no more than 9 rotatable bonds) [72]. The radar charts presented in Figure 6 pointed out that the physicochemical properties of the most potent compounds 1d, 1g, and 1f were fully located in the pink-colored area. As a result, the leading compounds 1d, 1g, and 1f of the series were predicted as oral bioavailable drug candidates due to having promising pharmacokinetic properties.

Conclusion
The designed compounds 1a-g, 3-(aminomethyl)-6-{3-[4-(trifluoromethyl)phenyl]acryloyl}-2(3H)-benzoxazolones, were synthesized and purified successfully. Their inhibition effects on hCA I, hCA II, and AChE enzymes were investigated for the first time. The inhibition results of the newly synthesized compounds showed that all Mannich bases had higher inhibitory activities against carbonic anhydrases (hCA I and hCA II) than the reference compound AZA. The pyrrolidine derivative compound 1d had the highest inhibition effect in the series on hCA I with a lower K i value (12.3 ± 1.2 nM) than AZA (K i = 84.4 ± 8.4 nM). Besides, the most potent compound in the series was the N-methyl piperazine derivative compound 1g having K i value as 8.55 ± 1.90 nM towards hCA II. According to the hCA inhibition results, compounds 1d and 1g were the most expressive lead compounds of the study with remarkable K i values (12.3 nM and 8.55 nM, respectively) which are approximately 7 times lower than AZA. According to the AChE inhibitory activity results, the morpholine derivative compound 1f was the most active compound in the series with the K i value as 35.2 ± 2.0 nM, which is about two-fold lower than the reference drug, TAC. On the other hand, the most active compounds 1d (pyrrolidine derivative) towards hCA I, 1g (N-methyl piperazine derivative) towards hCA II, and 1f (morpholine derivative) towards AChE were docked at the binding sites of the mentioned enzymes to explain the inhibitory activities of the compounds. The docking results showed that the compounds 1d, 1g, and 1f had strong interactions with the active sites of hCA I, hCA II, and AChE, as expected. ADME prediction studies of the compounds 1 and 1a-g pointed out that the newly synthesized Mannich bases were not only potent AChE and CAs inhibitory compounds but also had promising physicochemical  (20-130 Å 2 ), e Octanol/water partition coefficient (Consensus logP value, recommended range: −2.0 to 6.5), f Aqueous solubility prediction (not higher than 6), g Abbott bioavailability score (probability at least 10% oral bioavailability in rat).
and ADME properties for further investigations. As a conclusion, in vitro inhibition results and in silico studies of the synthesized compounds showed that the aminomethylation from the 3rd position of the benzoxazolone ring of the chalcone compound 1 by Mannich reaction is a useful modification in terms of carbonic anhydrases and AChE inhibitory activities as well as optimization of physicochemical and pharmacokinetic properties.