Design, synthesis and evaluation of 3,4-dihydroxybenzoic acid derivatives as antioxidants, bio-metal
chelating agents and acetylcholinesterase inhibitors.
Laura Friggeri a, Daniela De Vita a*, Fabiana Pandolfi a, Silvano Tortorella a, Roberta Costi a, Roberto Di
Santo a and Luigi Scipione a
Complexation studies
pag.2
Enzymatic inhibition studies
pag.8
Molecular docking studies
pag.10
1
Complexation studies
UV spectra and Job’s plot obtained for each compound with metal cations Cu2+, Fe2+, Fe3+ are shown in
figures S1-S12 in the next pages.
Fig- S1 compound 8 in presence of Cu2+
Fig- S2 compound 8 in presence of Fe2+
2
Fig- S3 compound 8 in presence of Fe3+
Fig- S4 compound 9 in presence of Cu2+
3
Fig- S5 compound 9 in presence of Fe2+
Fig- S6 compound 9 in presence of Fe3+
4
Fig- S7 compound 10 in presence of Cu2+
Fig- S8 compound 10 in presence of Fe2+
5
Fig- S9 compound 10 in presence of Fe3+
Fig- S10 compound 11 in presence of Cu2+
6
Fig- S11 compound 11 in presence of Fe2+
Fig- S12 compound 11 in presence of Fe3+
7
Enzymatic inhibition studies
Dixon’s plot obtained for all compounds with Electric eel AChE are presented in the figures S13 – S16 in
the next pages.
0,0070
0,0065
0,0060
0,0055
1/Rate (µmol/min)
0,0050
0,0045
0,0040
0,0035
0,0030
0,0025
0,0020
ASCh = 200 M
ASCh = 300 M
ASCh = 400 M
0,0015
0,0010
0,0005
-6
-5
-4
-3
-2
-1
0
1
2
3
4
[8] (µM)
Fig- S13. Dixon plot of obtained for AChE in presence of increasing concentrations of carbamate 8. Noncompetitive inhibition was observed (Ki = 5.8 ± 0.4 μM).
0,012
0,011
0,010
1/Rate (µmol/min)
0,009
0,008
0,007
0,006
0,005
0,004
ASCh = 200M
ASCh = 300M
ASCh = 400M
0,003
0,002
0,001
-4
-3
-2
-1
0
1
2
3
4
[9] (µM)
Fig- S14.. Dixon plot of obtained for AChE in presence of increasing concentrations of carbamate 9. Mixed
inhibition was observed (Ki = 8.5 ± 5.6 μM).
8
0,012
0,011
0,010
1/Rate (µmol/min)
0,009
0,008
0,007
0,006
0,005
0,004
ASCh = 200M
ASCh = 300M
ASCh = 400M
0,003
0,002
0,001
-6
-5
-4
-3
-2
-1
0
1
2
3
4
[10] (µM)
Fig- S15.. Dixon plot of obtained for AChE in presence of increasing concentrations of carbamate 10. Mixed
inhibition was observed (Ki = 18.9 ± 17.0 μM).
0,010
0,009
0,008
1/Rate (µmol/min)
0,007
0,006
0,005
0,004
ASCh = 400 M
ASCh = 500 M
ASCh = 600 M
0,003
0,002
0,001
-15
-12
-9
-6
-3
0
3
6
9
12
15
[11] (µM)
Fig- S16. Dixon plot of obtained for AChE in presence of increasing concentrations of carbamate 11. Mixed
inhibition was observed (Ki = 1.5 ± 1.1 μM).
9
Molecular docking studies
The best binding pose identified by EADock DSS software vs native binding mode of donepezil in the
crystal structure of TcAChE (1EVE) is reported in figure S17. The best binding poses of carbamates 8-11 in
the same protein are reported in figures S18-S21.
Fig- S17. The superposition of the re-docked Donepezil (orange) and the native binding mode of Donepezil
(pink) into the active site of TcAChE (1EVE).
10
Fig- S18 The best binding pose of the compound 8 (yellow) in the active site of TcAChE (1EVE). The
selected aminoacids are dark cyan colored.
Fig- S19 The best binding pose of the compound 9 (blue) in the active site of TcAChE (1EVE). The selected
aminoacids are dark cyan colored.
11
Fig- S20 The best binding pose of the compound 10 (red) in the active site of TcAChE (1EVE).
The
selected aminoacids are dark cyan colored.
Fig- S21 The best binding pose of the compound 11 (green) in the active site of TcAChE (1EVE).
The
selected aminoacids are dark cyan colored.
12
The best binding poses of carbamates 8-11 identified by EADock DSS software in the crystal structure of
hBChE (1P0I) are reported in figures S22-S25.
Fig- S22 The best binding pose of the compound 8 (magenta) in the active site of hBChE (1P0I). The
selected aminoacids are orange colored.
13
Fig- S23 The best binding pose of the compound 9 (magenta) in the active site of hBChE (1P0I). The
selected aminoacids are orange colored.
14
Fig- S24 The best binding pose of the compound 10 (blue) in the active site of hBChE (1P0I). The selected
aminoacids are orange colored.
15
Fig- S25 The best binding pose of the compound 11 (magenta) in the active site of hBChE (1P0I). The
selected aminoacids are orange colored.
16