M. Cieslikiewicz-Bouet, et al.
Biochemical Pharmacology 177 (2020) 114010
added dropwise a solution of 11 (58 mg, 0.28 mmol, 1.2 equiv.) in dry
dichloromethane (1 mL). The reaction mixture was stirred for 16 h at
r.t. The reaction mixture was washed with a saturated aqueous solution
1H), 7.37–7.39 (m, 1H), 7.46–7.49 (m, 2H), 7.72 (m, 2H), 7.82–7.85
(m, 1H), 7.93–8.04 (m, 3H), 8.23 (br s, NH), 8.54 (br s, NH). MS (ESI
+
+): m/z (%): 678 (55), 677 (100) [M + H] . HRMS (ESI+): m/z calcd
35
of NaHCO
3
. The organic phase was separated, dried over MgSO
4
and
for C38
42 8 2 R
H N O Cl 677.3119; found 677.3122. HPLC: t = 20.1 min
concentrated under reduced pressure. The crude product was purified
(purity = 96.2%).
by flash chromatography (cyclohexane/EtOAc, 7/3, v/v) to give
1
MB099 as a yellow oil (70 mg, 78%). HNMR (300 MHz, CDCl
3
)
2.2. Docking studies
δ = 1.62–1.76 (m, 3H), 1.94–1.99 (m, 2H), 2.53 (t, J = 2.4 Hz, 1H)
2
.72–2.96 (m, 6H), 3.01–3.09 (m, 1H), 3.53 (t, J = 7.1 Hz, 1H), 4.65
The crystal structure of the mouse AChE-anti-TZ2-PA6 complex
PDB code 1Q84) was used for molecular docking [40]. Docking cal-
culations were carried out using Autodock Vina [56]. MTDL structures
were created and minimized using the MMFF94 force field implanted in
ChemBio3D Ultra 12.0 (PerkinElmer, Inc. Waltham, MA, USA). MTDL
and mouse AChE were further prepared using Autodock Tools 1.5.6
(
(
3
3
d, J = 2.5 Hz, 2H), 5.84 (m, 1H, NH), 6.91–7.00 (m, 4H), 7.18–7.24
m, 1H), 7.58–7.61 (m, 1H), 8.34–8.48 (m, 2H). MS (ESI+): m/z (%):
(
92 (100) [M + H]+. HRMS (ESI+): m/z calcd for C23
H
26
N
3
O
3
92.1969; found 392.1974.
2
.1.10. ( ± )-4-[(1-{2-[(3-chloro-5,6,7,8-tetrahydroacridin-9-yl)amino]
[
57]. Structural water molecules were conserved in the model in order
to improve docking accuracy. Residues in the active site of AChE (Tyr
2, Trp 86, Tyr 124, Tyr 286, Tyr 337, Tyr 341) were selected as
ethyl]-1,2,3-triazol-4-yl)methoxy]phenyl
azabicyclo[2.2.2]octan-3-yl]carbamate MB105
N-[2-(pyridin-3-ylmethyl)-1-
7
A solution of MB099 (202 mg, 0.52 mmol, 1 equiv.), MB320
flexible. The 3D affinity grid box was designed to include the full active-
site gorge of AChE. Docking calculations were performed using the
default parameter set of AutoDock Vina to generate nine docking poses
per molecule. Vina generated the estimated total Gibbs free energy of
binding in kcal/mol, which could be converted to the apparent con-
(
157 mg, 0.52 mmol, 1 equiv.) and CuI (99 mg, 0.52 mmol, 1 equiv.) in
dry acetonitrile (5.2 mL) was stirred away from the light at r.t. for
days. After removal of solvent, the crude product was purified by flash
chromatography on silica gel (EtOAc/MeOH, 8/2 to 1/1, v/v) to afford
3
1
MB105 (295 mg, 82%). HNMR (300 MHz, CD
1
(
(
3
OD) δ = 1.68 (m, 4H),
stant, K
i
i
, using the relationship ΔG = RT.ln (K ). Docking poses were
.77–1.90 (m, 4H), 1.92 (s, 1H), 2.54 (m, 2H), 2.71 (m, 2H), 2.85–2.98
visualized using the PyMOL molecular graphics system [58].
m br, 6H), 3.46 (m, 1H), 3.59 (m, 1H), 3.96–4.04 (m, 2H), 4.55–4.64
m, 2H), 4.98 (s, 2H), 6.82–6.92 (m, 4H), 7.22 (m, 1H), 7.32–7.36 (m,
2
H), 7.69 (s, 1H), 7.74–7.84 (m, 4H), 8.39 (br s, NH), 8.48 (br s, NH).
2
.3. Evaluation of the inhibitory activity toward human AChE and BChE
1
3
CNMR (75 MHz, CD OD) δ = 23.3, 23.7, 24.9, 25.9, 26.4, 30.4, 34.1,
3
4
1
1
8.4, 48.8, 49.1, 50.1, 51.9, 55.7, 62.2, 73.7, 116.2, 116.8, 117.0,
18.3, 119.6, 123.6, 125.0, 125.6, 125.9, 126.5, 135.5, 136.2, 139.0,
The capacity of tested compounds to inhibit human AChE and BChE
activity was assessed by the Ellman method [59]. The assay was per-
formed at 37 °C in a Jasco V-530 double beam Spectrophotometer
44.8, 146.3, 147.8, 147.9, 150.7, 151.0, 152.1, 156.3, 156.8, 160.5.
+
(
ESI+): m/z (%): 694 (30), 693 (50) [M
+
H]
,
347 (100)
(
Jasco Europe). A stock solution of AChE was prepared by dissolving
human recombinant AChE (Sigma Aldrich, Italy) lyophilized powder in
.1 M phosphate buffer (pH = 8.0) containing Triton X-100 0.1%. A
2
+
35
[
M + 2H] . HRMS (ESI+): m/z calcd for C38
= 20.5 min (unprotonated form), 21.5 min
protonated form) (purity = 95%).
42 8 3
H N O Cl 693.3068;
found 693.3072. HPLC: t
(
R
0
stock solution of BChE from human serum (Sigma Aldrich, Italy) was
prepared by dissolving the lyophilized powder in an aqueous solution of
gelatine 0.1%. Stock solutions of inhibitors (1 or 2 mM) were prepared
in methanol. Five increasing concentrations of the inhibitor were used,
yielding 20–80% inhibition of the enzymatic activity. The assay solu-
tion consisted of 0.1 M phosphate, pH 8.0, with the addition of 340 μM
DTNB, 0.02 unit/mL of human recombinant AChE, or human serum
BChE, and 550 µM substrate, ATCh or BTCh, respectively. 50 µL ali-
quots of increasing concentrations of the tested compound were added
to the assay solution and preincubated for 20 min at 37 °C with the
enzyme, followed by addition of substrate, after which the increase in
absorbance at 412 nm was monitored for 3 min. Assays were carried out
against a blank containing all components except AChE or BChE, in
order to correct for non-enzymatic substrate hydrolysis. The reaction
rates were compared and the percent inhibition due to the presence of
tested inhibitor at increasing concentration was calculated. Each con-
centration was analyzed in triplicate, and IC50 values were determined
graphically from log concentration–inhibition curves (GraphPad Prism
2
.1.11. ( ± )-4-(Prop-2-yn-1-yloxy)-N-[2-(pyridin-3-ylmethyl)-1-
azabicyclo[2.2.2]octan-3-yl]benzamide MB114
To a cooled (0 °C) solution of amine 9 (40 mg, 0.18 mmol, 1 equiv.),
3
NEt (50 µL, 0.36 mmol, 2 equiv.) in dry THF (1 mL) was added
dropwise a solution of benzoyl chloride 10 (35 mg, 0.18 mmol, 1
equiv.) in dry THF (1 mL). The reaction mixture was stirred for 30 min
before removing the solvent under reduced pressure. The crude product
was purified by RP-HPLC to give MB114 as a white solid (41 mg, 61%).
1
HNMR (300 MHz, CD
H), 3.34–3.69 (m, 6H), 3.87–3.94 (dd, J = 5.0 Hz, 7.5 Hz, 1H), 4.36
t, J = 7.1 Hz, 1H), 4.78 (d, J = 2.3 Hz, 2H), 6.97 (m, 2H), 7.50 (m,
3
OD) δ = 1.88–2.30 (m, 5H), 2.99 (t, J = 2.4 Hz,
1
(
2
1
H), 7.71–7.75 (m, 1H), 8.18 (d, J = 7.6 Hz, 1H), 8.35 (d, J = 7.9 Hz,
1
3
3
H), 8.52 (br s, 1H), 8.85 (br s, 1H, NH). CNMR (75 MHz, CD OD)
δ = 18.5, 23.2, 28.8, 34.5, 42.9, 52.1, 56.9, 63.9, 76.3, 77.4, 79.3,
1
MS (ESI+): m/z (%): 376 (100) [M + H] . HRMS (ESI+): m/z calcd
for C23 376.2020; found 376.2014.
15.8, 115.9, 127.3, 130.3, 130.8, 143.8, 145.4, 146.9, 162.2, 168.9.
+
26 3 2
H N O
4.03 software, GraphPad Software Inc.). Each IC50 value was de-
termined from at least two independent experiments each performed in
triplicate.
2
.1.12. ( ± )-4-[(1-{2-[(3-chloro-5,6,7,8-tetrahydroacridin-9-yl)amino]
ethyl}-1,2,3-triazol-4-yl)methoxy]-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo
[
2.2.2]octan-3-yl]benzamide MB118
A solution of MB114 (41 mg, 0.11 mmol, 1 equiv.), MB320 (33 mg,
2.4. Evaluation of the time-dependent carbamoylation of hAChE by MB105
0.11 mmol, 1 equiv.) and CuI (21 mg, 0.11 mmol, 1 equiv.) in dry
acetonitrile (2 mL) was stirred away from the light at r.t. for 36 h. After
removal of solvent, the crude product was purified by flash chromato-
The stopped time assay was performed, in which hAChE o hBChE
and MB105 at a concentration of 0.2 µM, a value close to its IC50 value,
were mixed in the assay buffer at pH 8.0. After 2, 10, 20, and 40 min
incubation at 37 °C, the determination of residual activity of the AChE-
catalyzed hydrolysis of the substrate was carried out by Ellman’s
method [59]. A parallel control (i.e., no inhibitors in the mixture) al-
lowed to adjust activities measured at the same incubation times.
graphy on silica gel (EtOAc/MeOH, 4/1 to 1/1, v/v) to afford MB118
1
(
58 mg, 79%). HNMR (300 MHz, CD
3
OD) δ = 1.66 (t, J = 3.0 Hz, 1H),
1
3
2
.84–1.89 (m, 4H), 1.91–2.01 (m, 4H), 2.60 (m, 2H), 2.94 (m, 2H),
.00–3.16 (m, 6H), 3.49 (m, 1H), 4.06 (m, 1H), 4.22 (t, J = 5.0 Hz,
H,), 4.72 (t, J = 5.0 Hz, 2H), 5.06 (s, 2H), 6.92–6.95 (m, 2H), 7.27 (m,
4