Trimethoxylated Halogenated Chalcones as Dual Inhibitors of MAO-B and BACE-1 for the Treatment of Neurodegenerative Disorders

Six halogenated trimethoxy chalcone derivatives (CH1–CH6) were synthesized and spectrally characterized. The compounds were further evaluated for their inhibitory potential against monoamine oxidases (MAOs) and β-secretase (BACE-1). Six compounds inhibited MAO-B more effectively than MAO-A, and the 2′,3′,4′-methoxy moiety in CH4–CH6 was more effective for MAO-B inhibition than the 2′,4′,6′-methoxy moiety in CH1–CH3. Compound CH5 most potently inhibited MAO-B, with an IC50 value of 0.46 µM, followed by CH4 (IC50 = 0.84 µM). In 2′,3′,4′-methoxy derivatives (CH4-CH6), the order of inhibition was –Br in CH5 > -Cl in CH4 > -F in CH6 at the para-position in ring B of chalcone. CH4 and CH5 were selective for MAO-B, with selectivity index (SI) values of 15.1 and 31.3, respectively, over MAO-A. CH4 and CH5 moderately inhibited BACE-1 with IC50 values of 13.6 and 19.8 µM, respectively. When CH4 and CH5 were assessed for their cell viability studies on the normal African Green Monkey kidney cell line (VERO) using MTT assays, it was noted that both compounds were found to be safe, and only a slightly toxic effect was observed in concentrations above 200 µg/mL. CH4 and CH5 decreased reactive oxygen species (ROS) levels of VERO cells treated with H2O2, indicating both compounds retained protective effects on the cells by antioxidant activities. All compounds showed high blood brain barrier permeabilities analyzed by a parallel artificial membrane permeability assay (PAMPA). Molecular docking and ADME prediction of the lead compounds provided more insights into the rationale behind the binding and the CNS drug likeness. From non-test mutagenicity and cardiotoxicity studies, CH4 and CH5 were non-mutagenic and non-/weak-cardiotoxic. These results suggest that CH4 and CH5 could be considered candidates for the cure of neurological dysfunctions.


MAOs and BACE1 Inhibition Studies
MAO events were assessed with recombinant MAO-A and MAO-B, as well as the substrates kynuramine (0.06 mM) and benzylamine (0.3 mM). The kinetics of MAO-B were analyzed under 2.4 × K m (i.e., 0.051 mM) of benzylamine. BACE-1 was measured using a β-secretase (BACE-1) assay kit at 320 nm for excitation and 405 nm for emission using a fluorescence spectrometer. The inhibitory activities of CH1-CH6 against MAO-A, MAO-B and BACE-1 were observed at 10 µM. IC 50 values of CH4 and CH5, which showed residual activities of <50% for MAO-A and MAO-B, were evaluated, as described previously. The IC 50 value of CH5 for BACE-1 was also analyzed [55,56].

Enzyme Inhibition and Kinetic Studies
The inhibitory actions of the six molecules against MAO-A, MAO-B, and BACE-1 were first tested at 10 µM. Initially, the IC 50 values of the compounds for MAOs were calculated [57]. At three inhibitory concentrations and five substrate concentrations, kinetic tests were conducted on CH4 and CH5, which inhibited MAO-B most potently.

Inhibitor Reversibility Analysis
Dialysis was used to assess the reversibility of MAO-B inhibitions by CH4 and CH5 after preincubating them for 30 min at 0.15 µM with MAO-B. For contrast, MAO-B was preincubated with lazabemide (a reference reversible MAO-B inhibitor) or pargyline (a reference irreversible MAO-B inhibitor) at 0.20 and 0.30 µM. Reversibility patterns were analyzed by comparing the activities of dialyzed (A D ) and undialyzed (A U ) samples [58].

Cytotoxicity and ROS Assay
The lead molecules CH4 and CH5 in the current series were further evaluated for their cytotoxic nature and ROS scavenging ability by the previously reported procedure [59,60] (Supplementary Material).

Blood-Brain Barrier (BBB) Permeability
The CNS bioavailability of the molecules was confirmed by a parallel artificial membrane permeability assay (PAMPA) by Di et al. [61].

Computational Studies
X-ray coordinates of the crystal structures of both the MAO isoforms and BACE-1 were retrieved from the Protein Data Bank with the entries 2Z5X, 2V5Z and 3TPP, respectively [62][63][64]. Protein and ligand structures were treated with the Protein Preparation Wizard and Ligprep Tool, respectively [65][66][67]. These preliminary steps are crucial for further docking analyses, allowing the generation of the correct protonation states at physiological pH or the generation of all allowed tautomers and possible conformations. Enclosing boxes were centered on the center of a mass of cognate ligand, and SP docking protocols were employed using GLIDE software [68]. To corroborate the reliability of docking simulations, redocking analyses were performed on the cognate ligands in their binding sites. Cognate ligands were moved back to the original positions with Root Mean Square Deviations (RMSD), considering all the heavy atoms, equal to 0.155 Å, 0.208 Å, and 0.467 Å for BACE-1, MAO-A, and MAO-B cognate ligands, respectively. Discovery Studio 2.5 software package (Accelrys, San Diego, CA, USA) was used to predict the ADMET parameters of all the molecules. The Vega and PredHerg platforms were employed to assess the mutagenicity and cardiotoxicity potentials [69,70].

Synthesis
The synthesis of two small series of trimethoxylated halogenated chalcones was achieved by the Claisen-Schmidt condensation of 2,4,6-trimethoxy acetophenone/2,3,4trimethoxy acetophenone with various para-substituted halogenated benzaldehydes. 1 H NMR showed a sharp doublet peak for Hα and Hβ at 7.65-7.62 and 7.50-7.47, respectively. A large coupling constant of 15Hz of the double bond of chalcones revealed its trans conformation [71]. The protons of methoxy groups showed a singlet peak at 3.92, while the 13 C NMR showed a peak at 55.95-62.17 for the methoxy carbon and a characteristic peak of carbonyl carbon at 190. 63-193.91. The HRMS analysis showed the molecular weight of the targeted compounds [Supplementary Material].

MAO-A, MAO-B, and BACE1 Inhibition Studies
The six compounds CH1-CH6 inhibited MAO-B more effectively than MAO-A, and CH4 and CH5 revealed the most inhibitory activities against MAO-A and MAO-B at 10 µM with residual activities of <50% (Table 1). Compound CH5 most potently inhibited MAO-A with an IC 50 value of 0.46 µM, followed by CH4 (IC 50 = 0.84 µM). In the compounds, 2 ,3 ,4 -methoxy moiety in CH4-CH6 was more effective for MAO-B inhibition than 2 ,4 ,6methoxy moiety in CH1-CH3. In 2 ,3 ,4 -methoxy derivatives (CH4-CH6), inhibitory activities against MAO-B increased in the order of -Br in CH5 > -Cl in CH4 > -F in CH6 at para-position in ring B. CH5 and CH4 were selective for MAO-B with selectivity index (SI) values of 31.3 and 15.1, respectively, over MAO-A. No compounds effectively inhibited BACE-1, except CH4 and CH5, which moderately inhibited BACE-1 with IC 50 values of 13.6 and 19.8 µM, respectively ( Table 1). The IC 50 value of CH4 for BACE-1 (13.6 µM) was similar to that of reference quercetin (IC 50 = 13.4 µM) Based upon the above small selected library of compounds, it is possible to expand some interesting structure-activity relationships (SARs) on the basis of the orientation pattern of methoxyl groups on the various positions of phenyl ring A and the presence of various halogens on the para-position of ring B of chalcones. The results clearly documented that all the trimethoxylated halogenated derivatives showed moderate to good inhibition of MAO-B as compared to MAO-A. Considering the orientation pattern, it is clearly understood that introduction of methoxyl groups on the 2 , 4 , and 6 positions does not have much influence when compared with the positions of 2 , 3 , and 4 . This suggests that the crowding of methoxyl groups without the separation of a carbon atom on the A ring is crucial for the activity. As regards halogen substitution on the B ring, bromine or chlorine improved MAO-B inhibitory activity when compared to fluorine substitution (the residual activities were 0.93% and 0.38% at 10.0 µM, respectively). The two representative MAO-B inhibitors, CH4 and CH5, showed good BACE-1 inhibition with 54.3% and 64.3% residual activity, respectively, at 10.0 µM. The IC 50 value of the BACE-1 inhibition of CH4 was 13.6 µM, which, interestingly, is very close to the standard value. In BACE-1 inhibition, both chlorine and bromine have a large impact on the fluorine substitution

Kinetic Study
Kinetic experiments were carried out at five concentrations of the substrates and three inhibitor concentrations. In the kinetic studies of MAO-B binding by CH4 and CH5, Lineweaver-Burk plots showed that CH4 and CH5 were competitive inhibitors of MAO-B (Figure 1a,c), and their secondary plots showed that their K i values were 0.68 ± 0.17 and 0.31 ± 0.014 µM, respectively (Figure 1b,d). These results suggest that CH4 and CH5 compete with the substrate at the active site of MAO-B.

Reversibility Studies
The reversibilities of the inhibitions were analyzed using dialysis after preincubating MAO-B with CH4 or CH5 for 30 min, as previously described. In the experiments, the concentrations used were the following: CH4 at 1.68 µM, CH5 at 0.92 µM, lazabemide (a reference reversible inhibitor) at 0.22 µM, and pargyline (a reference irreversible inhibitor) at 0.28 µM. The relative activities for undialyzed (A U ) and dialyzed (A D ) samples were compared to determine the reversibility patterns. In reversibility experiments using dialysis, the inhibition of MAO-B by CH4 and CH5 was recovered from 30.9% (A U ) to 96.9% (A D ), and from 37.2% to 73.8%, respectively ( Figure 2). The recovery values were indistinguishable to those of the reversible reference lazabemide (from 33.0% to 89.2%), contrary to the inhibition of MAO-B by the irreversible inhibitor pargyline, which was not recovered (i.e., from 37.2% to 38.5%). These experiments indicated that CH4 and CH5 were reversible inhibitors of MAO-B. ysis, the inhibition of MAO-B by CH4 and CH5 was recovered from 30.9% (AU) to 96.9% (AD), and from 37.2% to 73.8%, respectively ( Figure 2). The recovery values were indistinguishable to those of the reversible reference lazabemide (from 33.0% to 89.2%), contrary to the inhibition of MAO-B by the irreversible inhibitor pargyline, which was not recovered (i.e., from 37.2% to 38.5%). These experiments indicated that CH4 and CH5 were reversible inhibitors of MAO-B.

Blood-Brain Barrier (BBB) Permeation Studies
From the assay, highly effective permeabilities and high CNS bioavailabilities were observed for the halogenated chalcones with Pe ranges of 13.22 ~ 15.65 × 10 −6 cm/s ( Table  2). All halogenated substituted derivatives showed higher CNS permeabilities than un-

Blood-Brain Barrier (BBB) Permeation Studies
From the assay, highly effective permeabilities and high CNS bioavailabilities were observed for the halogenated chalcones with Pe ranges of 13.22~15.65 × 10 −6 cm/s (Table 2). All halogenated substituted derivatives showed higher CNS permeabilities than unsubstituted ones.

In Vitro Toxicity Evaluation
An in vitro toxicity evaluation of CH4 and CH5 was carried out using an MTT assay. We used the African Green Monkey kidney cell line (Vero) as a valuable in vitro model for basal toxicity studies [72]. To check the toxicity of the tested compounds, the Vero cell line was incubated with different concentrations of CH4 and CH5 (1 to 500 µg/mL), and the relative cell viability was measured after 48 h. Both the compounds induced toxicity at concentrations higher than 200 µg/mL (Figures 3A and 4A). The result of the cell viability assay was represented as a dose-response curve using the GraphPad Prism 6.0 software (USA). The IC 50 value was calculated at a 198.5 µg/mL for CH4 ( Figure 3B) and 126.4 µg/mL for CH5 ( Figure 4B). The reduction in cell number and also the membrane damage were very evident in treatment with CH4 and CH5 at IC 50 and higher concentrations, as analyzed by phase-contrast microscope. These two experiments strongly support that CH4 and CH5 are one hundred times safer than the effective concentration of one micromolar ( Figures 3C and 4C). Both results demonstrate that the undesired changes in membrane integrity and a reduction in cell number are indicative of oxidative stress only at higher concentrations.

ROS Assay
Our next attempt was to study the effect of CH4 and CH5 on inhibiting the excess reactive oxygen species (ROS) involved in the pathogenesis of neurodegenerative diseases. This ROS causes neuronal damage via oxidative damage in the brain during neurodegenerative diseases [73,74]. We examined the intracellular ROS generation on Vero cell cultures after exposure to exogenous H 2 O 2 . During our experiment, it was noted H 2 O 2 -treated Vero cells had a higher ROS generation. When we incubated Vero cells with CH4 and CH5 at IC 50 , we observed a decrease in ROS levels compared with non-treated cells (Figure 5a,b). Based on this observation, it is understood that the disruption of the balance between pro-oxidant and antioxidant levels can be retained by CH4 and CH5 at IC 50 concentrations. CH4 and CH5 are one hundred times safer than the effective concentration of one micromolar ( Figures 3C and 4C). Both results demonstrate that the undesired changes in membrane integrity and a reduction in cell number are indicative of oxidative stress only at higher concentrations.   CH4 and CH5 are one hundred times safer than the effective concentration of one micromolar ( Figures 3C and 4C). Both results demonstrate that the undesired changes in membrane integrity and a reduction in cell number are indicative of oxidative stress only at higher concentrations.   rodegenerative diseases [73,74]. We examined the intracellular ROS generation on Ve cell cultures after exposure to exogenous H2O2. During our experiment, it was noted H2O treated Vero cells had a higher ROS generation. When we incubated Vero cells with CH and CH5 at IC50, we observed a decrease in ROS levels compared with non-treated cel (Figure 5a,b). Based on this observation, it is understood that the disruption of the balan between pro-oxidant and antioxidant levels can be retained by CH4 and CH5 at IC50 co centrations.

Figure 5.
Effects of CH4 (a) and CH5 (b) on Vero cells treated with H2O2 to induce ROS. Vero cells were treated with 100 µg/mL of 30% H2O2, and the production of ROS was evaluated immediately as described in "Experimental Procedures."

Computational Studies
Compounds CH5 and CH4 can bind the MAO-A binding pocket with similar posin (Figure 6). The para-bromine and para-fluorine styrene can establish Pi-Pi interaction with F208, and tri-methoxy phenyl rings can cause Pi-Pi interaction with Y407, facin FAD. Docking score values were equal to −6.416 kcal/mol and −6.465 kcal/mol for CH and CH4, respectively. As far as MAO-B is concerned, the para-bromine and para-fluorin styrene can interact with the MAO-B-selective residue Y326, and trimethoxy phenyl ring face the FAD cofactor, forming Pi-Pi interactions with the side chain of the Y398 aromat residue (Figure 7). Docking score values were equal to −9.824 kcal/mol and −10.25 kcal/mol for CH5 and CH4, respectively. As far as BACE-1 is concerned, compounds CH and CH4 can establish mainly hydrophobic interactions. In particular, the tri-methoxy a omatic rings can face a hydrophobic region made by side chains of F108 and I118, and th halogenated styrene moieties take place towards Y14 side chain (Figure 8). Docking sco values were equal to −6.749 kcal/mol and −6.762 kcal/mol for CH5 and CH4, respectivel Computational analyses were performed in order to shed light on the interaction behind the higher affinity towards MAO-B. The interaction between CH5 and CH4 wi Y326 MAO-B selective residue is crucial for enhancing activity and selectivity toward MAO-B. Furthermore, despite the binding poses of the two compounds being similar b tween MAO binding pockets, the gap between the docking score values towards the tw receptors significantly favors MAO-B. For this reason, a more detailed analysis of th

Computational Studies
Compounds CH5 and CH4 can bind the MAO-A binding pocket with similar posing (Figure 6). The para-bromine and para-fluorine styrene can establish Pi-Pi interactions with F208, and tri-methoxy phenyl rings can cause Pi-Pi interaction with Y407, facing FAD. Docking score values were equal to −6.416 kcal/mol and −6.465 kcal/mol for CH5 and CH4, respectively. As far as MAO-B is concerned, the para-bromine and parafluorine styrene can interact with the MAO-B-selective residue Y326, and trimethoxy phenyl rings face the FAD cofactor, forming Pi-Pi interactions with the side chain of the Y398 aromatic residue (Figure 7). Docking score values were equal to −9.824 kcal/mol and −10.251 kcal/mol for CH5 and CH4, respectively. As far as BACE-1 is concerned, compounds CH5 and CH4 can establish mainly hydrophobic interactions. In particular, the tri-methoxy aromatic rings can face a hydrophobic region made by side chains of F108 and I118, and the halogenated styrene moieties take place towards Y14 side chain (Figure 8). Docking score values were equal to −6.749 kcal/mol and −6.762 kcal/mol for CH5 and CH4, respectively. On the basis of ADME prediction, all the compounds in the present study show good absorption and BBB permeability, at 99% and 95%, respectively (Figure 9). A dr target matching analysis was also performed by using our predictive platform MuS (Multifingerprint Similarity Search aLgorithm) [75,76]. It is notable that MAO-A a MAO-B were predicted as reliable targets for CH4 and CH5 [Supplementary Material] Finally, we predicted the mutagenicity and cardiotoxicity potentials for CH4 a CH5 [77]. These are two relevant toxicity endpoints of the utmost importance to priorit compounds in early stages of drug discovery and are often behind the attrition of precl ical and clinical studies [78,79]. Regarding the mutagenicity, four non-test predictive mo els (i.e., CAESAR model version 2.1.13; SARpy/IRFMN model version 1.0.7; ISS mo version 1.0.2; KNN/read-across model version 1.0.0) were employed in consensus to p vide a qualitative assessment of the mutagenic potentials of CH4 and CH5 [80]. Intere ingly, both the compounds were found to be non-mutagenic [Supplementary Materia Predictive QSAR models of hERG blockage were employed for cardiotoxicity analy [81]. In the results, CH4 was returned to be non-cardiotoxic, while CH5 was flagged potentially weak cardiotoxic (Supplementary Materials).

Conclusions
In summary, two sets of trimethoxylated halogenated chalcones were synthesize characterized, and evaluated for their MAO and BACE-1 inhibitory activities. CH4 an CH5 were revealed as competitive and reversible inhibitors of MAO-B, with Ki values 0.68 ± 0.17 and 0.31 ± 0.014 µM, respectively. On the other hand, CH4 and CH5 showe good BACE-1 inhibition with IC50 values of 13.6 and 19.8 µM, respectively. Additionall CH4 and CH5 were found to be safe as analyzed by an in-vitro toxicity study. Additio ally, the pro-oxidant and antioxidant levels can be retained by CH4 and CH5. These r sults suggest that CH4 and CH5 could be considered as a potential multi-targeted ligan for the treatment of neurological disorders.
Supplementary Materials: Available online at www.mdpi.com/xxx/s1, spectral interpretation; c totoxicity studies; ROS Assay; MUSSEL Prediction; Toxicity Predicition. Computational analyses were performed in order to shed light on the interactions behind the higher affinity towards MAO-B. The interaction between CH5 and CH4 with Y326 MAO-B selective residue is crucial for enhancing activity and selectivity towards MAO-B. Furthermore, despite the binding poses of the two compounds being similar between MAO binding pockets, the gap between the docking score values towards the two receptors significantly favors MAO-B. For this reason, a more detailed analysis of the terms that constitute the GLIDE scoring function was performed, revealing that the Glide energy term values, which are a sum of van der Waals and Coulomb energy terms, were equal to −50.263 kcal/mol and −46.128 kcal/mol for CH4 and CH5 bonded to MAO-B, respectively, and −21.341 kcal/mol and −23.449 kcal/mol for CH5 and CH4 bonded to MAO-A, respectively, thus considerably favoring the binding to MAO-B. Regarding computational simulations of BACE-1, the binding was mainly due to hydrophobic interactions and the Glide energy term values were equal to −46.583 kcal/mol and −45.809 kcal/mol, thus comparable to those of MAO-B.
On the basis of ADME prediction, all the compounds in the present study showed good absorption and BBB permeability, at 99% and 95%, respectively (Figure 9). A drug target matching analysis was also performed by using our predictive platform MuSSel (Multifingerprint Similarity Search aLgorithm) [75,76]. It is notable that MAO-A and MAO-B were predicted as reliable targets for CH4 and CH5 [Supplementary Material].
Finally, we predicted the mutagenicity and cardiotoxicity potentials for CH4 and CH5 [77]. These are two relevant toxicity endpoints of the utmost importance to prioritize compounds in early stages of drug discovery and are often behind the attrition of preclinical and clinical studies [78,79]. Regarding the mutagenicity, four non-test predictive models (i.e., CAESAR model version 2.1.13; SARpy/IRFMN model version 1.0.7; ISS model version 1.0.2; KNN/read-across model version 1.0.0) were employed in consensus to provide a qualitative assessment of the mutagenic potentials of CH4 and CH5 [80]. Interestingly, both the compounds were found to be non-mutagenic [Supplementary Materials]. Predictive QSAR models of hERG blockage were employed for cardiotoxicity analysis [81]. In the results, CH4 was returned to be non-cardiotoxic, while CH5 was flagged as potentially weak cardiotoxic (Supplementary Materials).

Conclusions
In summary, two sets of trimethoxylated halogenated chalcones were synthesized, characterized, and evaluated for their MAO and BACE-1 inhibitory activities. CH4 and CH5 were revealed as competitive and reversible inhibitors of MAO-B, with Ki values of 0.68 ± 0.17 and 0.31 ± 0.014 µM, respectively. On the other hand, CH4 and CH5 showed good BACE-1 inhibition with IC50 values of 13.6 and 19.8 µM, respectively. Additionally, CH4 and CH5 were found to be safe as analyzed by an in-vitro toxicity study. Additionally, the pro-oxidant and antioxidant levels can be retained by CH4 and CH5. These results suggest that CH4 and CH5 could be considered as a potential multi-targeted ligand for the treatment of neurological disorders.

Conclusions
In summary, two sets of trimethoxylated halogenated chalcones were synthesized, characterized, and evaluated for their MAO and BACE-1 inhibitory activities. CH4 and CH5 were revealed as competitive and reversible inhibitors of MAO-B, with K i values of 0.68 ± 0.17 and 0.31 ± 0.014 µM, respectively. On the other hand, CH4 and CH5 showed good BACE-1 inhibition with IC 50 values of 13.6 and 19.8 µM, respectively. Additionally, CH4 and CH5 were found to be safe as analyzed by an in-vitro toxicity study. Additionally, the pro-oxidant and antioxidant levels can be retained by CH4 and CH5. These results suggest that CH4 and CH5 could be considered as a potential multi-targeted ligand for the treatment of neurological disorders.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study have been included in this article.