Design, synthesis and biological evaluation of tricyclic pyrazolo[1,5-c][1,3]benzoxazin-5(5H)-one scaffolds as selective BuChE inhibitors

Figures & data

Figure 1. The rational design in this study.

Scheme 1. Synthesis of compounds 3a–3f. Reagents and conditions: (i) 40% NaOH solution, EtOH, 60 °C; (ii) N₂H₄·H₂O, EtOH, reflux; (iii) N,N′-carbonyldiimidazole or 1,1′-thiocarbonyldiimidazole, CH₂Cl₂, r.t.

Scheme 2. Synthesis of compounds 6a–6q. Reagents and conditions: (iv) 40% NaOH solution, 60 °C; (v) N₂H₄·H₂O, EtOH, reflux; (vi) N,N′-carbonyldiimidazole or 1,1′-thiocarbonyldiimidazole, CH₂Cl₂, r.t.

Table 1. Chemical structures of compounds 3a–3f and 6a–6q and their inhibitory activities against EeAChE and eqBuChE^a

Compound	X	R1	R2	R3	IC50, µM (or inhibition % at 20 µM)
					AChE^b	BuChE^c
3a	O	Ph	H	H	^d	9.47 ± 0.23
3b	S	Ph	H	H	^d	9 ± 3%
3c	O	4-F[sbond]Ph	H	H	15 ± 3%	15 ± 2%
3d	O	4-Cl[sbond]Ph	H	Cl	39 ± 1%	41 ± 6%
3e	O	4-CH3[sbond]Ph	H	Cl	32 ± 2%	32 ± 5%
3f	O	4-CH3[sbond]Ph	H	H	48 ± 3%	32 ± 2%
6a	O	CH3	Cl	Cl	39 ± 3%	1.06 ± 0.11
6b	S	CH3	Cl	Cl	25 ± 1%	^d
6c	O	CH3	Br	Br	10.71 ± 0.64	1.63 ± 0.42
6d	S	CH3	Br	Br	26 ± 3%	^d
6e	O	CH3	H	Cl	16 ± 1%	6.29 ± 0.65
6f	S	CH3	H	Cl	18 ± 2%	^d
6g	O	CH3	H	Br	26 ± 1%	1.63 ± 0.34
6h	S	CH3	H	Br	27 ± 2%	^d
6i	O	CH3	Br	Cl	13.03 ± 0.32	2.44 ± 0.44
6j	S	CH3	Br	Cl	14 ± 2%	^d
6k	O	CH3	OCH3	H	35 ± 3%	14 ± 3%
6l	S	CH3	OCH3	H	30 ± 2%	2 ± 2%
6m	O	CH3	H	CH3	^d	14 ± 1%
6n	S	CH3	H	CH3	15 ± 2%	^d
6o	O	CH3	H	H	^d	50.08 ± 4.3%
6p	S	CH3	H	H	^d	^d
6q	O	CH3	Br	H	^d	3.22 ± 1.06
donepezil					0.014 ± 0.001	10.38 ± 0.20

^aEach IC₅₀ value is the mean ± SEM from three experiments (n = 3).

^bAChE from electric eel.

^cBuChE from horse serum.

^dNo inhibitory activity (%) against either EeAChE or eqBuChE at 20 µM.

Figure 2. Lineweaver − Burk plots of esBChE inhibition kinetics of compounds 6a (A), 6g (B). The Lineweaver–Burk secondary plots of compounds 6a (C) and 6g (D). Reciprocals of enzyme activity (esBuChE) vs. reciprocals of substrate (butyrylthiocholine iodide) concentration in the presence of different concentrations (0−20 µM) of inhibitor. Inset: Concentrations used for inhibitor are coded with different graphic symbols.

Figure 3. Cytotoxicity of compounds 6a, 6g and donepezil tested at concentrations in the range 1–50 µM in PC12 cell lines for 24 h. Untreated cells were used as control. Results are expressed as percentage of cell survival vs. untreated cell (control) and shown as mean ± SD (n = 3).

Figure 4. Neuroprotective effect on PC12 cell lines of compounds 6a and 6g. After 24 h incubation at different concentration (25 and 50 µM) with H₂O₂ (300 µM). Untreated cells were used as control. Results represent mean ± SD (n = 3). Statistical significance was calculated using one-way ANOVA and Bonferroni post hoc tests. ###p < .001 compared with the control group; *p < .05 compared with H₂O₂ group.

Table 2. -CDOCKER_INTERACTION_ENERGY of title compounds 6a–6q and 1P0I

Compound	-CDOCKER_INTERACTION_ENERGY ΔG (kcal/mol)
6a	34.3548
6b	28.6536
6c	33.1657
6e	30.9456
6g	32.8973
6i	32.2189
6o	27.7660
6q	31.5867

Figure 5. (A,C) 3D mode of interaction of compounds 6a and 6b and BuChE (PDB code: 1P0I) analysed by Discovery Studio 2017R2. Conventional hydrogen bond and carbon hydrogen bond and alkyl as well as Pi–alkyl are shown by green, light green and pink, respectively. (B,D) Two dimensional mode of interaction of compounds 6a and 6b and BuChE (PDB code: 1P0I) analysed by Discovery Studio 2017R2.

Figure 6. The H-Bonds surface of compound 6a and BuChE (PDB code: 1P0I) analysed by Discovery Studio 2017R2.