Development of Halogenated Pyrazolines as Selective Monoamine Oxidase-B Inhibitors: Deciphering via Molecular Dynamics Approach

Halogens have been reported to play a major role in the inhibition of monoamine oxidase (MAO), relating to diverse cognitive functions of the central nervous system. Pyrazoline/halogenated pyrazolines were investigated for their inhibitory activities against human monoamine oxidase-A and -B. Halogen substitutions on the phenyl ring located at the fifth position of pyrazoline showed potent MAO-B inhibition. Compound 3-(4-ethoxyphenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole (EH7) showed the highest potency against MAO-B with an IC50 value of 0.063 µM. The potencies against MAO-B were increased in the order of –F (in EH7) > –Cl (EH6) > –Br (EH8) > –H (EH1). The residual activities of most compounds for MAO-A were > 50% at 10 µM, except for EH7 and EH8 (IC50 = 8.38 and 4.31 µM, respectively). EH7 showed the highest selectivity index (SI) value of 133.0 for MAO-B, followed by EH6 at > 55.8. EH7 was a reversible and competitive inhibitor of MAO-B in kinetic and reversibility experiments with a Ki value of 0.034 ± 0.0067 µM. The molecular dynamics study documented that EH7 had a good binding affinity and motional movement within the active site with high stability. It was observed by MM-PBSA that the chirality had little effect on the overall binding of EH7 to MAO-B. Thus, EH7 can be employed for the development of lead molecules for the treatment of various neurodegenerative disorders.


Introduction
Monoamine oxidases (MAOs) are the principal metabolizing enzymes responsible for the oxidative degradation of various biogenic amines that are related to diverse cognitive functions of the central nervous system (CNS) [1]. Considering the difference in the structure, substrate specificity, biological functions, and catalytic mechanism of MAOs, two types of isoforms are available, namely, MAO-A and MAO-B [2]. Ammonia, aldehyde, and hydrogen peroxide are the major intermediate products formed during MAO-based catalyzed oxidative deamination [3]. These can produce substantial issues, such as astrocyte swelling, unbalancing of the signaling process in the neurotransmission, neuronal loss, and mitochondrial dysfunctions [4]. Eventually, these toxic by-products can lead to numerous neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD) [5]. Selegiline is a selective/irreversible MAO-B inhibitor used as an adjuvant therapy with L -DOPA, which is considered a safe therapy for PD [6,7]. The latest research has also documented that potent, reversible dual targeted MAO-B/AChE inhibitors can decrease the production of β-amyloid peptide levels in AD-related neurons [8]. In 2017, safinamide, as the first selective and reversible MAO-B inhibitor approved by the USFDA for PD treatment, along with levodopa, has promising neuroprotective effects on MPTP-treated mice [9,10].
In the exploration of novel, selective, and reversible MAO-B inhibitors, a general blueprint of drug design has been widely accepted recently, and it consists of a molecular framework of two hydrophobic rings of phenyl/heteroaryl, which are separated by an electron-rich and flexible short spacer unit ( Figure 1) [11]. Many of the molecules from this diverse class, such as pyrazolines, enamides, carboxamides, and α, β-unsaturated ketones, were identified as potent MAO-B inhibitors [12][13][14][15][16][17][18][19][20][21]. The presence of a rotatable bond and electron-rich Michael acceptor unit in the spacer can efficiently make appropriate orientations and binding affinities in the entrance and substrate cavity of MAO-B. The flexibility of the molecules provides good recognition of aromatic systems of the ligand to the hydrophobic cavity of the inhibitor binding cavity of MAO-B by Pi-Pi stacking or Pi-cation interactions. The binding requirements of the selective MAO-B inhibitors include one or more hydrophobic ring, preferably an aromatic or heteroaromatic nucleus with small lipophilic substituents, such as dimethylamino, ethoxy, methoxy, and halogens. The presence of a hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) linker with optimal rotatable bonds between these hydrophobic units had a pivotal role in the design of potent MAO-B inhibitors [22][23][24][25][26]. Pyrazoline, or dihydropyrazole, is the partially reduced form of pyrazole, and 2pyrazoline is the most stable tautomeric from in this class [27]. The presence of an asymmetric carbon atom at the C5 position of pyrazoline was revealed to be due to two epimers of the R and S forms [28]. The ring closure reaction of α, β-unsaturated ketones with hydrazine derivatives in the presence of basic medium is the commonly used synthetic methodology for 2-pyrazolines [29]. The separation of the R and S forms from the racemic mixture of 2-pyrazoline is a considerable task in the identification of biologically more active isomers [30]. Many studies of the literature have documented that the substitution of the 3rd and 5th position of pyrazolines with aryl/heteroaryl units can favor MAO-B inhibition [31]. At the same time, the length and bulkiness of the groups anchoring from the N1 position of pyrazolines can shift the selectivity from MAO-B to MAO-A. The presence of lipophilic groups, such as halogens, hydroxyl, and methoxy groups, on the phenyl ring of the third and fifth positions of the pyrazoline nucleus, can cause significant MAO-B inhibition [32][33][34][35].
Regarding the role of halogens in MAO inhibition, it is generally known that FDAapproved MAO inhibitors like clorgyline, moclobemide, safinamide, and lazabemide are chlorine-containing drugs [36]. The evidence shows that more than 40% of drugs in FDAapproved or clinical trials are in the halogenated form [37]. Recently, our group emphasized the presence and orientation of various halogens on chemical scaffolds, such as chalcones, chromones, coumarins, hydrazothiazoles, and pyrazolines, thereby providing in-depth knowledge on MAO inhibition and B isoform selectivity. It was noted that this can have a new type of impact on the drug-target interaction of these MAO inhibitors in order to fill the small lipophilic pockets in the inhibitor binding cavity (IBC) of MAOs [38].
The objective of the present study was focused on the in vitro evaluation of MAO-A and MAO-B inhibitions of pyrazoline/halogenated pyrazoline derivatives. The lead molecules were further studied for kinetics, reversibility, and blood-brain barrier (BBB) permeation. Detailed molecular dynamics established precisely the protein-ligand binding interactions of the halogenated molecules in their respective active sites of enzyme targets and the role of chirality towards MAO-B inhibition.

Inhibitory Activities against MAO Enzymes
At 10 µM, all derivatives in the EH sequence had potent inhibitory action against MAO-B, with residual activities of about 50% (Table 1). Fluorine-containing compound EH7 showed the greatest inhibitory activities against MAO-B with an IC 50 value of 0.063 µM, followed by chlorine-containing EH6 (IC 50   The enzyme inhibition results obtained in the current study revealed that all of the halogenated pyrazoline derivatives showed good selective MAO-B inhibitory activity. The study mainly confirmed the effect of halogens on the para-position of the B ring of ethoxylated pyrazolines. Among the halogens, fluorine had the greatest impact on MAO-B inhibition with a sub-micromolar level range. It is worth noting that a highly electronegative fluorine atom-containing compound is two times more potent than marketed drugs for the inhibition of MAO-B, such as reversible-type lazabemide (IC 50 = 0.11 µM) and irreversible pargyline (IC 50 = 0.14 µM). Recent reports state that the anisotropy of charge circulation around the fluorine atom, which is the reason for the directional and stabilizing property of the active site, proves that halogen enhances the analogy of drugs of new molecules [38].
Recently, we evaluated MAO-B inhibitory activities for ethoxylated head of chalcone derivatives and found that chlorine-, fluorine-, or bromine-containing chalcones had good IC 50 values of 0.57, 0.053, and 2.01 µM, respectively [39]. Cyclization with hydrazine hydrate of the same halogenated molecules retained higher MAO-B inhibitory activities than the unsubstituted one. This finding clearly shows the importance of halogen incorporation both in ethoxylated chalcones and their pyrazolines moieties for MAO-B inhibition. Interestingly, in both series, fluorine-containing compounds showed dominant MAO-B inhibition profiles. For the purpose of the development of compounds with good MAO-B inhibitory activity, the bonding of fluorine to the molecule was an auspicious lead.

Kinetics of MAO Inhibition
Kinetic studies on MAO-A and MAO-B inhibitions by EH6 and EH7 were performed. Lineweaver-Burk plots and secondary plots showed that EH6 was a competitive inhibitor of MAO-A and MAO-B (

Reversibility Study
Reversibility studies on MAO-B inhibition were carried out on EH7. In these experiments, inhibition of MAO-B by EH7 was recovered from 12.5% (value of A U ) to 89.5% (value of A D ). The recovery value was similar to that of the reversible reference lazabemide (from 35.3% to 75.7%); however, it was different from that of the irreversible inhibitor pargyline (from 41.3% to 44.9%), which was not recovered (Figure 4). Similar to the reversible reference level, inhibition of MAO-B by EH7 was reversed in these experiments, indicating that EH7 is a reversible inhibitor of MAO-B.

Blood-Brain Barrier (BBB) Permeation Studies
The CNS bioavailability of the molecules was confirmed by a parallel artificial membrane permeability assay (PAMPA) [40]. From the assay, highly effective permeabilities and high CNS bioavailabilities were observed for the halogenated pyrazolines with Pe ranges of 14.13~14.56 × 10 −6 cm/s ( Table 2). All halogenated substituted derivatives showed higher CNS permeabilities than unsubstituted ones. The root mean square deviation (RMSD) can be used to estimate the conformational stability of a system during a production run [41]. The differential structural perturbatory effect of the ligands on the structure of MAO-B relative to MAO-A was first explored. MAO-B_EH6, MAO-B_EH7, and MAO-B_EH8 attained equilibration at around 20 ns ( Figure 5). However, MAO-B_EH1 achieved convergence earlier in the simulation run but eventually elicited a somewhat high structural movement at around 45 ns. In comparison, EH1, EH6, EH7, and EH8 did not elicit similar stabilizing effects on the MAO-A protein; this could be due to the IC 50 values experimentally reported, suggesting their low inhibitory potential towards MAO-A. The superior stabilizing effect elicited by the ligands against MAO-B when compared to MAO-A binding points to the high selective targeting of the ligands for MAO-B. As indicated by the IC 50 , EH7 had the highest stabilizing impact on MAO-B with an average RMSD value of 1.49 Å, while EH1, EH6, and EH8 had 2.29, 1.61, and 1.79 Å, respectively (Table 3).  The orientation and position of a ligand are considerably indicative of how reactive the ligands can be within the active site. The characteristics of the ligands were therefore investigated. Interestingly, while EH7 elicited some motional movement within the active site, which made it flexible enough to interact with crucial amino acid residues, it also exhibited good stability-just enough to be adequately anchored for overall binding ( Figure 6).

EH7 Exhibited Low Residual Motion and Trajectory Movement upon Binding to MAO-A and MAO-B
Principal component analysis (PCA) is a common molecular dynamic simulation analysis used in the estimation of the essential production run of a system when computed on a low-dimensional free energy space [42]. This estimation is associated with correlated vibrational modes; the translation of the MD trajectory towards the geometric center abolishes the initial rotational and translational movements of the atoms. The decomposition of the trajectory along PC1 and PC2 reveals that upon EH7 binding to MAO-B, the system exhibits very low diagonalization when compared with other systems ( Figure 7A). This further corroborates the results discussed above in the stability section, which attest to the superior activity of EH7 over other ligands in the series. In contrast, due to the low specificity of EH1, EH6, EH7, and EH8 towards MAO-A, less high trajectory motion was observed in the ligand-bound MAO-A ( Figure 7B).
The root mean square fluctuation (RMSF) computes the residual deviation of a protein over a period of time using a reference position, such as the averaged residual position. RMSF estimates the level of fluctuation of residue from its original position [43]. As  Figure 7D).

Estimation of Free Binding Energies of EH1, EH6, EH7, and EH8 on MAO-A and MAO-B
The estimation of free binding energy using an MM/PBSA protocol can be employed to gain insight into the models and further obtain the semiquantitative values of their stability. We therefore used a representative snapshot and active site residue decomposition to further explore the time-wise bond interaction occurring between major residues in the active sites of MAO-A/MAO-B and the ligands. MM/PBSA has found useful application in the drug design space used in the estimation of binding affinity between ligands and biomolecules [44].
As indicated in Table 4, EH7 had higher binding affinity (−36.29 kcal/mol) towards MAO-B than the other ligands, while the binding of EH1, EH6, and EH8 was recorded to be −4.11, −11.5, and −5.48 kcal/mol, respectively. Of note is the surprising high binding affinity (−32.55 kcal/mol) of EH7 for MAO-A. This could be due to the high electronega-tivity potential of fluorine, which is characteristic of compound EH7. In agreement with the IC 50 , the binding affinity of the compounds also followed the same trend. The individual energy contributions of the active sites were determined using the MM/PBSA per residue energy decomposition protocol [45]. The high binding energy exhibited by EH7 was contributed by electrostatic interactions of Arg42, Arg87, Arg98, Lys296, and Lys386, with energy values of −28.4, −12.8, −15.3, −41.9, and −20.6 kcal/mol, respectively ( Figure 8C). In the interaction between compound EH6 and MAO-B, residues Arg42, Gly58, Tyr60, Arg87, Pro98, and Arg100 contributed an electrostatic energy of −2.68, −0.04, −0.12, −15.17, −0.14, and −17.27 kcal/mol, respectively ( Figure 8B). Despite the electrostatic interactions and other favorable bonds elicited by EH1, EH6, and EH8, compound EH7 still had the best binding energy and highest contribution of electrostatic interactions. This behavior may not be unrelated to the impact of fluorine found on compound EH7. Furthermore, the energetically favored disposition assumed by EH7 could be attributed to the hydrogen bond formed between the fluorine of EH7 and hydrogen of Trp386 ( Figure 9C). The ligand interaction profile of the interaction of EH1 (A), EH6 (B), EH7 (C), and EH8 (D) with MAO-B is shown in Figure 9.   Since our compounds contain a pyrazoline moiety, it is assumed that they possess an asymmetric carbon atom and, hence, exist as enantiomers. We therefore generated the R and S enantiomers of EH7 as a representative structure of the compounds, taking into consideration the fact that it has the best IC 50 and total free energy values. We explored the differential binding and estimated the total free binding energy of the R isomeric form of EH7 (MAO-B_EH7R) and its S counterpart (MAO-B_EH7S) using MM/PBSA calculations. Our calculations revealed that MAO-B_EH7R and MAO-B_EH7S possess binding energies of −36.29 and −36.17 kcal/mol (Table 5). This suggests that the chirality does not change considerably in the overall binding of EH7 to MAO-B. However, MAO-B_EH7S has more van der Waals interactions than MAO-B_EH7R. Despite having total free binding energy values that are close, dissimilar residues present in the active sites are responsible for the binding interactions. Chiefly among the residues are Tyr435, Tyr398, and Leu328 (jointly shared by MAO-B_EH7R), which contribute van der Waals binding energies of −2.93, −1.58, and −1.00 kcal/mol, respectively ( Figure 10).

Target Predicition and ADME Analysis
The biological target and ADME predictions of the lead molecule of EH7 were assessed by the online web-based program SwissTarget. The lead molecule showed good prediction results on biological targets of MAO and AChE ( Figure 11A). The bioavailability radar gave saturation, size, flexibility, insolubility, and polarity of the molecule, which provided a quick judgement of the drug-likeness of the molecule. The lead molecule was recognized to be within the pink region (optimal ranges) and could be considered a compound endowed with drug-like characteristics ( Figure 11B). The BOILED-Egg model also predicted the passive human BBB permeation and gastrointestinal absorption (HIA) for the compound ( Figure 11C). The results revealed that EH7 had comparatively higher probability of BBB and HIA.

Synthesis
A series of ethoxylated chalcone (0.0078 M) was refluxed with (0.0629 M) hydrazine hydrate under ethanol medium. After 14-16 h of reflux, the product was cooled, acidified with dil. HCl, washed thoroughly with water, filtered, dried, and recrystallized from ethanol. The progress of the formation of pyrazoline product was monitored by thin-layer chromatography (TLC; ethylacetate-hexane = 1:9). The synthetic route is depicted in Scheme 1.

Enzyme Assays
MAO activities were assayed as described previously, using recombinant human MAO-A and MAO-B and kynuramine (0.06 mM) and benzylamine (0.3 mM) as substrates, respectively; both substrate concentrations were 1.5 × K m (K m = 0.041 and 0.20 mM, respectively). The enzymes and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) [17].

Analysis of Enzyme Inhibitions and Kinetics
The inhibitory activities of the four compounds against MAO-A and MAO-B were first observed at a concentration of 10 µM. IC 50 values for MAO-B by compounds showing residual activities of <50% were determined. Kinetic studies were performed on the most potent inhibitors, i.e., EH6 and EH7 for MAO-A and MAO-B, at five concentrations of the substrates and three inhibitor concentrations, as previously described [18].

Analysis of Inhibitor Reversibilities
Reversibilities of EH7 were analyzed using a dialysis method after preincubating with MAO-B for 30 min, as previously described. The concentrations used were EH7 at 0.15 µM, lazabemide (a reversible MAO-B reference inhibitor) at 0.20 µM, and pargyline (an irreversible MAO-B reference inhibitor) at 0.30 µM. The relative activities for undialyzed (A U ) and dialyzed (A D ) samples were compared to determine the reversibility patterns [19].

Molecular Dynamics Study
The 3D structures of MAO-A (PDB ID: 2Z5X and MAO-B: 2V5Z), hereafter referred to as MAO-A and MAO-B, respectively, were retrieved from the Protein Data Bank (PDB) [46,47]. Molecules that were co-crystallized with MAO-A and MAO-B were deleted with the exception of the co-enzyme FAD. Missing residues were filled with the aid of MODELLER tools found in the graphic user interface of Chimera [48,49]. Marvin Sketch was employed to draw the 2D structures of EH1, EH6, EH7, and EH8; afterwards, geometrical and structural optimizations of these ligands were carried out using B3LYP/6-311 ++G (d,p) and the UFF forcefield of Gaussian 16, respectively [50]. Active site identification was achieved by centering the grid box around a co-crystallized ligand found on the MAO-A and MAO-B proteins downloaded from PDB. Molecular docking was then carried out using AutoDock Vina [51]. The systems were then subjected to a simulation run of 50 ns using Amber18 software (San Francisco, CA, USA) [52]. Eight systems were set up for simulation (MAO-A:EH1, MAO-A:EH6, MAO-A:EH7, MAO-A:EH8, MAO-B:EH1, MAO-B:EH6, MAO-B:EH7, and MAO-B:EH8). The simulation process was carried out in accordance with an in-house protocol. The CPPTRAJ module of Amber18 software was used to analyze the trajectories emanating from the simulation process [53,54].

Target Predicition and ADME Analysis
The biological target and ADME predictions of the lead molecule of EH7 were assessed at http://www.swisstargetprediction.ch (access on 23 May 2021) [55,56].

Conclusions
A series of four halogenated pyrazolines with ethoxylated heads (EH1, EH6, EH7, and EH8) was synthesized from their corresponding chalcones. All four compounds were explored for their MAO-A and MAO-B inhibitory activities. On the basis of previous studies regarding the role of halogens in various scaffolds with MAO inhibition activities, we mainly focused on the effect of various halogens on the pyrazoline core with the ethoxylated head. According to the results of this study, the synthesized halogenated pyrazolines exhibited good selective MAO-B inhibition. Fluorine-containing pyrazoline (EH7) exhibited an improved selectivity profile against the MAO-B isoform. Kinetics and reversibility studies revealed that EH7 is a selective and competitive inhibitor of MAO-B, with a recovery value similar to that of the reversible reference drug lazabemide.
Pyrazolines are said to be optically active compounds, and, from the MM/PBSA values obtained, it can be clearly observed that chirality has minimal influence on the binding of the compound to MAO-B. It may be suggested that the absence of bulky groups on the N1 position of pyrazoline limited the role of chirality in the binding mode of the lead molecule in the current study.