Discovery of novel 2-(4-(benzyloxy)-5-(hydroxyl) phenyl) benzothiazole derivatives as multifunctional MAO-B inhibitors for the treatment of Parkinson’s disease

Figures & data

Figure 1. Design strategies of 2-(4-(benzyloxy)-5-(hydroxyl) phenyl) benzothiazole derivatives.

Scheme 1. Synthesis of 2-(4-(benzyloxy)-5-(hydroxyl) phenyl) benzothiazole derivatives (3a–o). Reagents and conditions: (i) 2,4-dihydroxybenzaldehyde, Na₂S₂O₅, DMF; (ii) corresponding substituted benzyl chlorides or benzyl bromides, NaHCO₃, KI, CH₃CN, at 60 °C for 30 h.

Table 1. Inhibitory effects on MAOs and antioxidative activities of 2-(4-(benzyloxy)-5-(hydroxyl) phenyl) benzothiazole derivatives and reference compounds in vitro.

Compounds	R	%Inhibition or IC50 (μM)^a		ORAC^b
		MAO-A	MAO-B
3a	H	24.83 ± 0.11%	3.933 ± 0.259	2.13 ± 0.05
3b	2-F	14.25 ± 0.07%	1.674 ± 0.146	2.32 ± 0.02
3c	2-Cl	19.58 ± 0.06%	2.895 ± 0.031	2.26 ± 0.09
3d	3-F	18.41 ± 0.05%	0.526 ± 0.024	2.24 ± 0.10
3e	3-Cl	31.60 ± 0.28%	1.049 ± 0.083	2.31 ± 0.07
3f	3-CF3	n.a.	0.951 ± 0.062	2.25 ± 0.07
3g	3-OCH3	n.a.	1.523 ± 0.097	2.30 ± 0.04
3h	4-F	39.73 ± 0.54%	0.062 ± 0.005	2.27 ± 0.08
3i	4-Cl	47.14 ± 0.21%	0.314 ± 0.010	2.23 ± 0.06
3j	4-CH3	34.32 ± 0.09%	1.942 ± 0.078	2.39 ± 0.14
3k	4-tBu	n.a.	3.407 ± 0.032	2.48 ± 0.13
3l	4-NO2	25.16 ± 0.13%	0.784 ± 0.028	2.14 ± 0.06
3m	3,4-2F	47.52 ± 0.36%	0.423 ± 0.009	2.24 ± 0.11
3n	3,4-2Cl	9.80 ± 0.19	0.970 ± 0.011	2.21 ± 0.09
3o	3-Cl-4-F	49.91 ± 0.53%	0.521 ± 0.007	2.27 ± 0.05
Safinamide	–	24.9 ± 0.04%	0.0572 ± 0.002	1.12 ± 0.01
Rasagiline	–	1.29 ± 0.06	0.0953 ± 0.001	NT
Iproniazid	–	1.87 ± 0.02	1.23 ± 0.037	NT
Clorgyline	–	0.0025 ± 0.0003	3.66 ± 0.09	NT

n.a.: no active, meaning the percent inhibition is <5.0%; NT: not tested.

^aPercent inhibition was determined at the concentration of 10.0 μM and the IC₅₀ value is the concentration when the compound inhibit enzyme activity by 50%.

^bData are expressed as Trolox equivalents.

Figure 2. Kinetic study of MAO-B inhibitory mechanism by compound 3h. (A) Lineweaver-Burk plots of MAO-B activities in the absence and presence of various concentrations of 3h (0.031, 0.062, and 0.124 μM), (B) the slopes of the Lineweaver-Burk plots vs. the 3h concentrations.

Figure 3. Reversibility of the MAO-B inhibition by 3h, safinamide, and rasagiline.

Figure 4. (A) The docking mode of compound 3h with hMAO-A (PDB ID: 2Z5X), (B) The docking mode of compound 3h with hMAO-B (PDB ID: 2V5Z), 3h and amino acid residues that participate in the interactions were displayed as sticks and coloured by the element, hydrogen bonds are showed as green dashed lines.

Figure 5. (A) UV spectra of compound 3h (75.0 μM in methanol) with or without FeSO₄, ZnCl₂, CuCl₂, FeCl₃, or AlCl₃ (75.0 μM in methanol); (B) UV spectra of compound 3h (75.0 μM in methanol) with FeSO₄ (0, 7.5, 15.0, 30.0, 45.0, 60.0, 75.0, 112.5, 150.0, and 187.5 μM); (C) Determination of the stoichiometry of 3h-Fe²⁺ complex by using the molar ratio method of titrating the methanol solution of compounds 3h with ascending amounts of FeSO₄.

Table 2. Permeability results P_e (×10⁻⁶cm/s) for 3h and its predicted penetration into CNS.

Compound^a	Pe (×10^−6 cm/s)^b	Prediction
3h	4.03 ± 0.14	CNS+

^a3h were dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). The final concentration of each compound was 100 μg/mL.

^bData are the mean ± SD of three independent experiments.

Figure 6. (A) Cytotoxicity of 3h towards PC-12 cells. (B) Neuroprotective effects of 3h and resveratrol. Three independent experiments were performed in triplicate. Data were expressed as mean ± SD. ^##p < 0.01 vs. control; **p < 0.01 vs. model group.

Figure 7. The effects of resveratrol and 3h on the cell viability of BV-2 cells (A) without LPS and (B) in the presence of LPS (1.0 μg/mL). Each assay was carried out at least three times and data are expressed as mean ± SD.

Figure 8. The inhibitory effects of resveratrol and 3h on NO release (A) and TNF-α production (B) in LPS-activated BV-2 microglia cells. The data are expressed as the mean ± SD from three independent experiments. ^#p < 0.05 vs. control group; *p < 0.05 vs. model group.

Figure 9. The inhibitory effects of resveratrol and 3h on LPS-induced ROS release in BV-2 microglia cells. (A) representative images, (B) relative fluorescence values obtained by microplate reader. The data are expressed as the mean ± SD from three independent experiments. ^##p < 0.01 vs. control; *p < 0.05 vs. model group; **p < 0.01 vs. model group.