Novel Sunifiram-carbamate hybrids as potential dual acetylcholinesterase inhibitor and NMDAR co-agonist: simulation-guided analogue design and pharmacological screening

Figures & data

Figure 1. Examples of AChEI drugs.

Figure 2. Reported cognitive enhancers.

Scheme 1. Reported method for Sunifiram synthesis.

Figure 3. The rational of novel NMDA receptor modulators and acetylcholinesterase (AChE) inhibitors.

Scheme 2. Synthesis of N-acylbenzotriazoles 1a-k. Reagent condition: DCC (1.4 eq) in CH₂Cl₂, rt, 12 h.

Scheme 3. Synthesis of Sunifiram (3a) and Sunifiram analogues (3b-j). Reagent condition: a) n-butanol, rt, 3h, b) n-butanol, 60 °C, 12 h. For R, see Table 1 and “Experimental” section.

Figure 4. Cytotoxicity IC₅₀ of compounds 3c-j and 3a (Sunifiram) on A549 cell line.

Table 1. Yield % and cytotoxicity IC₅₀ of compounds 3c-j and Sunifiram 3a on A549 cell line.

Compound no.	Structure	Yield %	Cytotoxicity IC50 µM
			A549 cells
3a (Sunifiram) (DM235)		93	85.93 ± 4.43
3b		86	Not applied
3c		91	101.5 ± 5.24
3d		91	49.26 ± 2.54
3e		92	52.77 ± 2.72
3f		93	32.17 ± 1.66
3g		89	37.98 ± 1.96
3h		84	43.49 ± 2.24
3i		87	96.63 ± 4.98
3j		81	57.51 ± 2.97

Figure 5. Acetylcholine released in response to compounds 3c-j and Sunifiram (3a) from A549 cells.

Table 2. Acetylcholine released in response to compounds 3c-j and 3a from A549 cells.

Compound	1/2 IC50 µM	Acetyl choline release (pg/ml)		ACh (pg/ml)
		Choline
		Total	Free
3c	50.8	400.7 ± 4.56	320.6 ± 1.16	80.1 ± 4.7
3d	24.6	377.4 ± 4.84	235.6 ± 1.3	141.8 ± 5.01
3e	26.4	387.4 ± 8.17	307.7 ± 3.44	79.7 ± 8.86
3f	16.1	397.6 ± 8.62	221.5 ± 2	176.1 ± 8.85
3g	19	374.5 ± 6.94	291 ± 2.83	83.5 ± 7.49
3h	21.7	362.9 ± 22	275.1 ± 3.1	87.8 ± 22.21
3i	48.3	351.6 ± 4.24	240.8 ± 1.53	110.8 ± 4.51
3j	28.8	330.7 ± 4.89	240 ± 2.59	90.7 ± 5.53
Sunifiram(3a)	43	402.5 ± 2.58	258.2 ± 1.52	144.3 ± 2.99
Control	–	316.7 ± 1.9	255.7 ± 9.78	61 ± 9.69

Figure 6. SAR of novel AChEI.

Table 3. Anticholinesterase activity of synthesised Sunifiram analogues.

Compound	AChE inhibition (IC50 [µM])	Compound	AChE inhibition (IC50 [µM])
3c	13.9 ± 0.17	3g	22.57 ± 0.29
3d	19 ± 0.00	3i	10.36 ± 0.23
3e	20.1 ± 0.00	Donepezil	0.0299 ± 0.00015 (29.9 ± 0.15 nM)
3f	0.018 ± 0.0002 (18 ± 0.2 nM)	Rivastigmine	0.0163 ± 0.00017 µM (16.3 nM ± 0.17)

Figure 7. AChE level on rat brain after i.p. administration of 3f and tacrine.

Table 4. AChE level on rat brain after i.p. administration of 3f and tacrine.

Group	Dose ug/kg	AChE (ng/mL)
Control	–	2.498 ± 0.303
3f	10	1.266 ± 0.114
Tacrine	10	1.137 ± 0.064

Table 5. Molecular docking scores of Sunifiram and synthesised analogues.

Compound	Docking score (kcal/mol)
Sunifiram (3a)	−4.5
3b	−4.4
3c	−3.8
3d	−3.8
3e	−3.5
3f	−1.7
3g	−0.5
3h	−0.4
3i	−4.5
3j	−2.5

Table 6. Predicted physicochemical properties of synthesised Sunifiram analogues.

	Sunifiram analogues
Physiochemical properties	3a	3b	3c	3d	3e	3f	3g	3h	3i	3j
Chemical formula	C14H18N2O2	C13H16N2O2	C18H25N3O4	C19H27N3O4	C21H31N3O4	C21H31N3O4S	C25H31N3O4	C18H19N3O2	C18H19N3O2	C17H17N3O2
Molecular weight (g/mol)	246.30	232.28	347.41	361.44	389.49	421.55	437.53	309.36	309.36	295.34
Number of heavy atoms	18	17	25	26	28	29	32	23	23	22
Number of aromatic heavy atoms	6	6	6	6	6	6	12	12	12	12
Number of rotatable bonds	4	3	8	8	9	11	10	4	4	4
Number of H-bond acceptors	2	2	4	4	4	4	4	2	2	3
Number of H-bond donors	0	0	1	1	1	1	1	1	1	0
TPSA	40.62Å2	40.62	78.95	78.95	78.95	104.25	78.95	66.64	66.64	53.51
Molar Refractivity	77.19	72.38	100.93	1.80	115.35	122.94	130.22	96.69	96.69	90.08
LogPO/W	1.45	0.94	1.50	1.8	2.31	2.36	2.91	1.71	1.84	1.54
GI absorption	High	High	High	High	High	High	High	High	High	High

Figure 8. Comparative RMSD plots of C-a atoms of Sunifiram (black), 3i (red), and the unbound Apo (green). B) Comparative RMSF plots of individual residues of Sunifiram (black), 3i (red), and the unbound Apo (green) conformations over the simulation period. C) Comparative RoG plots of C-a atoms of Sunifiram (black), 3i (red), and the unbound Apo (green). D) 3D structural superposition of Sunifiram (black), 3i (red), and the unbound Apo (green) to show structural flexibility.

Figure 9. Comparative RMSD plots of C-a atoms of 3i (red), glycine (magenta), and the unbound Apo (green). B) Comparative RMSF plots of individual residues of 3i (red), glycine (magenta), and the unbound Apo (green) conformations over the simulation period. C) Comparative RoG plots of C-a atoms of 3i (red), glycine (magenta) and the unbound Apo (green). D) 3D structural superposition of 3i (red), glycine (magenta) and the unbound Apo (green) to show structural flexibility.

Table 7. MMGBSA- based binding free energy profile of Sunifiram and compound 3i.

System	Energy components (kcal/mol)
	${\text{ΔE}}_{\mathrm{vdw}}$	${\text{ΔE}}_{\mathrm{ele}}$	${\text{ΔG}}_{\mathrm{gas}}$	${\text{ΔG}}_{\mathrm{sol}}$	${\text{ΔG}}_{\mathrm{bind}}$
Sunifiram	−9.50 ± 7.19	−4.25 ± 6.29	−13.75 ± 11.59	7.32 ± 6.78	−6.43 ± 5.68
3i	−38.21 ± 2.95	−17.58 ± 5.64	−55.80 ± 6.30	25.67 ± 4.38	−30.13 ± 3.09

ΔE_ele: electrostatic energy; ΔE_vdW: van der Waals energy; ΔG_bind: total binding free energy; ΔG_sol: solvation free energy; ΔG: gas phase free energy.

Figure 10. Per-residue energy decomposition of glycine binding site residues and their corresponding energy contributions towards the binding and stability of Sunifiram, 3i A) Per-residue energy plots of binding sites residues towards Sunifiram and the 3D representation of intermolecular interactions exhibited by Sunifiram. B) Per-residue energy plots of binding sites residues towards the binding of compound 3i and the 3D representation of intermolecular interactions exhibited by 3i.

Figure 11. 3D binding of compound 3f in the catalytic and peripheral pocket of AChE.

Table 8. Molecular docking scores of Sunifiram analogues towards AChE.

Compound	Docking score (kcal/mol)
Donepezil	−11.1
Rivastigmine	−7.9
3c	−9.2
3d	−9.3
3e	−9.4
3f	−8.8
3g	−10.5
3i	−9.8

Figure 12. Molecular visualisation of 3f-AChE, donepezil-AChE, and rivastigmine-AChE binding pocket.