Synthesis and evaluation of a new series of Tri-, Di-, and mono- n -alkylcarbamylphloroglucinols as bulky inhibitors of acetylcholinesterase

Article abstract of DOI:10.1021/tx300119a

1,3,5-Tri-N-alkylcarbamylphloroglucinols (1-4) are synthesized as a new series of bulky inhibitors of acetylcholinesterase that may block the catalytic triad, the anionic substrate binding site, and the entrance of the enzyme simultaneously. Among three series of phloroglucinol-derived carbamates, tridentate inhibitors 1,3,5-tri-N-alkylcarbamylphloroglucinols (1-4), bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5-8), and monodentate inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9-12), tridentate inhibitors 1-4 are the most potent inhibitors of mouse acetylcholinesterase. When different n-alkylcarbamyl substituents in tridentate inhibitors 1-4 are compared, n-octylcarbamate 1 is the most potent inhibitor of the enzyme. All inhibitors 1-12 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. Thus, tridentate inhibitors 1-4 are supposed to be hydrolyzed to bidentate inhibitors 5-8 after the enzyme catalysis. Subsequently, bidentate inhibitors 5-8 and monodentate inhibitors 9-12 are supposed to yield monodentate inhibitors 9-12 and phloroglucinol, respectively, after the enzyme catalysis. This means that tridentate inhibitors 1-4 may act as long period inhibitors of the enzyme. Therefore, inhibitors 1-4 may be considered as a new methodology to develop the long-acting drug for Alzheimer's disease. Automated dockings of inhibitor 1 into the X-ray crystal structure of acetylcholinesterase suggest that the most suitable configuration of inhibitor 1 to the enzyme binding is the (1,3,5)- (cis,trans,trans)-tricarbamate rotamer. The cis-carbamyl moiety of this rotamer does not bind into the acetyl group binding site of the enzyme but stretches out itself to the entrance. The other two trans-carbmayl moieties of this rotamer bulkily block the tryptophan 86 residue of the enzyme.

Full text of DOI:10.1021/tx300119a

Article
pubs.acs.org/crt
Synthesis and Evaluation of a New Series of Tri-, Di-, and
Mono-N-alkylcarbamylphloroglucinols as Bulky Inhibitors
of Acetylcholinesterase
Ming-Chen Lin,† Gin-Zen Lin,‡ Yu-Fong Shen,‡,§ Shuo-Yung Jian,‡ Dean-Kuo Hsieh,∥ James Lin,⊥
and Gialih Lin*,‡,∥
†Division of Internal Medicine, Chung Shan Medical University Hospital, School of Medicine, Chung Shan Medical University,
Taichung 402, Taiwan
‡Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan
§Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
∥Department of Applied Chemistry, Chaoyang University of Technology, Taichung 413, Taiwan
⊥Department of Aquaculture, National Taiwan Ocean University, Keelung City 202, Taiwan
ABSTRACT: 1,3,5-Tri-N-alkylcarbamylphloroglucinols (1−4) are synthe-
sized as a new series of bulky inhibitors of acetylcholinesterase that may block
the catalytic triad, the anionic substrate binding site, and the entrance of the
enzyme simultaneously. Among three series of phloroglucinol-derived
carbamates, tridentate inhibitors 1,3,5-tri-N-alkylcarbamylphloroglucinols
(1−4), bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5−8), and
monodentate inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9−12), tridentate
inhibitors 1−4 are the most potent inhibitors of mouse acetylcholinesterase.
When different n-alkylcarbamyl substituents in tridentate inhibitors 1−4 are
compared, n-octylcarbamate 1 is the most potent inhibitor of the enzyme. All
inhibitors 1−12 are characterized as the pseudo substrate inhibitors of
acetylcholinesterase. Thus, tridentate inhibitors 1−4 are supposed to be
hydrolyzed to bidentate inhibitors 5−8 after the enzyme catalysis. Subsequently,
bidentate inhibitors 5−8 and monodentate inhibitors 9−12 are supposed to yield monodentate inhibitors 9−12 and phloroglucinol,
respectively, after the enzyme catalysis. This means that tridentate inhibitors 1−4 may act as long period inhibitors of the enzyme.
Therefore, inhibitors 1−4 may be considered as a new methodology to develop the long-acting drug for Alzheimer's disease.
Automated dockings of inhibitor 1 into the X-ray crystal structure of acetylcholinesterase suggest that the most suitable configuration
of inhibitor 1 to the enzyme binding is the (1,3,5)- (cis,trans,trans)-tricarbamate rotamer. The cis-carbamyl moiety of this rotamer
does not bind into the acetyl group binding site of the enzyme but stretches out itself to the entrance. The other two trans-carbmayl
moieties of this rotamer bulkily block the tryptophan 86 residue of the enzyme.
INTRODUCTION
■
Acetylcholinesterase (AChE, EC 3.1.1.7) belongs to the serine
hydrolase family that catalyzes the hydrolysis of acetylcholine
(ACh), thus regulating cholinergic neurotransmission.1−4 Both
subtype unselective cholinesterase and AChE-selective inhib-
itors have been used in Alzheimer's disease to amplify the
action of ACh at remaining cholinergic synapses within the
Alzheimer's disease brain. The X-ray crystal structures of AChE
have revealed that AChE contains a catalytic triad similar to
that present in other serine hydrolases. It has also revealed that
the catalytic triad Ser-His-Glu is located near the bottom of a
deep and narrow gorge about 20 Å in depth.2−4
Figure 1. Kinetic scheme for the pseudo substrate inhibition of
carbamate inhibitors in the presence of substrate. E, enzyme (AChE);
S, substrate (acetylthiocholine); ES, acetylenzyme intermediate; I,
inhibitors 1−12; EI, enzyme−inhibitor tetrahedral intermediate; EI′,
carbamyl enzyme intermediate; P, product from substrate reaction
(thiocholine); P′, product from pseudo substrate reaction (phenols);
and Q, the second product (carbamic acids).
Carbamate inhibitors, such as Alzheimer's disease drug
Rivastigmine (Exelon), and aryl carbamates are characterized
as the pseudo substrate inhibitors of AChE, butyrylcholinester-
ase (BChE), cholesterol esterase, and lipase.3,5−13 In the pres-
ence of substrate, the kinetic scheme for pseudo or alternate
substrate inhibitions of serine hydrolases by carbamate
inhibitors is proposed (Figure 1).5−15 Therefore, values of Ki
and k2 can be calculated from eq 1.5 In eq 1, the kapp values are
Received: March 20, 2012
Published: June 12, 2012
© 2012 American Chemical Society
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of phloroglucinol with 2 equiv of the corresponding n-alkylisocyanate
and 2 equiv of triethylamine in tetrahydrofuran at 25 °C for 5 h.
For example, condensation of phloroglucinol with 2 equiv of
octylisocyanate and 2 equiv of triethylamine in tetrahydrofuran at 25
°C for 5 h yielded 1,3,5-tri-N-n-octylcarbamylphloroglucinol (1)
(26%), 3,5-di-N-n-octylcarbamyloxy- phenol (5) (12%), and 5-N-n-
octylcarbamyloxyresorcinol (9) (19%). These three products were
separated and purified by liquid chromatography (silica gel, hexane-
first-order rate constants that are obtained by Hosie's method.
The bimolecular rate constant, ki = k2/Ki, is related to overall
inhibitory potency.
kapp = k2[I]/{Ki(1 + [S]/Km) + [I]}
(1)
Recently, dual-site inhibitors of AChE, such as tacrine
dimers,16−20 tacrine-sulfoxide tethers,21 huperzine B dimers,22,23
(−)-nor-meptazinol dimers,24 huperine-tacrine heterodimers,25
tacrine-indole heterodimers,26 tacrine-melatonin hybrids,27 qui-
nazolinimine dimers,28 tacrine-gallamine hybrids,29 cymserine,30
and isosorbide-2-benzyl carbamate-5-salicylate,31 which may bind
to both peripheral anionic substrate binding site (PAS) and
anionic substrate binding site (AS) of AChE, have been actively
studied by many groups. We have also designed biphenyl-4-
acyoxylate-4′-N-butylcarbamates,7 benzene-di-N-substituted car-
bamates,8 and 1,n-alkane-di-N-butycarbamates,9 which may bind
to both the PAS and the catalytic triad or esteratic site (ES) of
AChE. In this paper, we further design more bulky tridentate
inhibitors, 1,3,5-tri-N-n-alkylcarbamylphloroglucinols (1−4)
(Figure 2) that may bulkily block all of the active site of the
1
ethyl acetate gradient) and characterized by H, 13C NMR, and high-
resolution mass spectra.
1,3,5-Tri-N-n-octylcarbamylphloroglucinol (1). H NMR (CDCl3,
1
400 MHz, δ): 0.88 (t, J = 7 Hz, 9H, ω-CH3), 1.28−1.35 (m, 30H, γ- to
ω-1-CH2), 1.50 (m, 6H, β-CH2), 3.20 (dt, J = 6.4 and 13.6 Hz, 6H, α-
CH2), 5.29 (t, J = 5.6 Hz, 3H, NH), and 6.82 (s, 3H, benzene-H). 13
C
NMR (CDCl3, 100 MHz, assignment from DEPT experiments, δ):
14.0 (ω-CH3), 22.5, 26.7, 29.1, 29.1, 29.6 (γ- to ω-1-CH2), 31.7 (β-
CH2), 41.2 (α-CH2), 111.8 (C-2, C-4, and C-6 of benzene ring), 151.4
(C-1, C-3, and C-5 of benzene ring), and 153.8 (carbamate CO).
Mass spectra, exact mass: calculated, 591.4247; found, 591.4242.
Elemental analysis: calculated for C33H57N3O6: C, 66.97; H, 9.71; N,
7.10. Found: C, 66.89; H, 9.80; N, 7.05.
1,3,5-Tri-N-n-heptylcarbamylphloroglucinol (2). 1H NMR
(CDCl3, 400 MHz, δ): 0.85 (t, J = 7 Hz, 9H, ω-CH3), 1.28−1.35
(m, 24H, γ- to ω-1-CH2), 1.47 (m, 6H, β-CH2), 3.13 (dt, J = 6.6 and
13.4 Hz, 6H, α-CH2), 5.37 (t, J= 6 Hz, 3H, NH), and 6.77 (s, 3H,
benzene-H). 13C NMR (CDCl3, 100 MHz, δ): 13.9 (ω-CH3), 22.5,
26.6, 28.8, 29.6 (γ- to ω-1-CH2), 31.6 (β-CH2), 41.1 (α-CH2), 111.8
(C-2, C-4, and C-6 of benzene ring), 151.3 (C-1, C-3, and C-5 of
benzene ring), and 153.6 (carbamate CO). Mass spectra, exact
mass: calculated, 549.3778; found, 549.3771. Elemental analysis:
calculated for C30H51N3O6: C, 65.54; H, 9.35; N, 7.64. Found: C,
65.50; H, 9.53; N, 7.52.
1,3,5-Tri-N-n-hexylcarbamylphloroglucinol (3). 1H NMR (CDCl3,
400 MHz, δ): 0.86 (t, J = 7 Hz, 9H, ω-CH3), 1.28−1.34 (m, 18H, γ- to
ω-1-CH2), 1.48 (m, 6H, β-CH2), 3.42 (dt, J = 6.6 and 13.4 Hz, 6H, α-
CH2), 5.30 (t, J= 6 Hz, 3H, NH), and 6.78 (s, 3H, benzene-H). 13C
NMR (CDCl3, 100 MHz, δ): 13.9 (ω-CH3), 22.4, 26.3, 30.0 (γ- to ω-
1-CH2), 31.3 (β-CH2), 41.2 (α-CH2), 111.8 (C-2, C-4, and C-6 of
benzene ring), 151.4 (C-1, C-3, and C-5 of benzene ring), and 153.8
(carbamate CO). Mass spectra, exact mass: calculated, 507.3308;
found, 507.3302. Elemental analysis: calculated for C27H45N3O6: C,
63.88; H,8.93; N, 8.28. Found: C, 63.79; H, 9.01; N, 8.19.
Figure 2. Chemical structures of inhibitors 1−12.
enzyme simultaneously by three carbamyl moieties of the
inhibitors.
Bidentate 3,5-di-N-n-alkylcarbamyloxyphenols (5−8) (Figure 2)
may be produced from the AChE-catalyzed hydrolysis of tridentate
1−4 (Figure 3). Subsequently, monodentate 5-N-n-alkylcarbamy-
1,3,5-Tri-N-n-butylcarbamylphloroglucinol (4). 1H NMR (CDCl3,
400 MHz, δ): 0.90 (t, J = 7 Hz, 9H, ω-CH3), 1.28 (sextet, J = 7 Hz,
6H, γ-CH2), 1.45 (quintet, J = 7 Hz, 6H, β-CH2), 3.18 (dt, J = 6.6 and
13.4 Hz, 6H, α-CH2), 5.19 (t, J = 6 Hz, 3H, NH), and 6.80 (s, 3H,
benzene-H). 13C NMR (CDCl3, 100 MHz, δ): 13.9 (ω-CH3), 20.1
(γ-CH2), 32.0 (β-CH2), 41.1 (α-CH2), 112.1 (C-2, C-4, and C-6 of
benzene ring), 151.7 (C-1, C-3, and C-5 of benzene ring), and 154.1
(carbamate CO). Mass spectra, exact mass: calculated, 423.2369;
found, 424.2364. Elemental analysis: calculated for C21H33N3O6: C,
59.56; H,7.85; N, 9.92. Found: C, 59.51; H, 7.90; N, 9.83.
Figure 3. Postulated pathway of the AChE-catalyzed hydrolyses of
tridentate inhibitors 1−4 followed by hydrolyses of bidentate
inhibitors 5−8; E, enzyme.
1
3,5-Di-N-n-octylcarbamyloxyphenol (5). H NMR (CDCl3, 400
MHz, δ): 0.86 (t, J = 7 Hz, 6H, ω-CH3), 1.28−1.35 (m, 20H, γ- to ω-
1-CH2), 1.50 (m, 4H, β-CH2), 3.18 (dt, J = 6.6 and 13.4 Hz, 4H, α-
CH2), 5.44 (t, J = 6.0 Hz, 2H, NH), 6.26−6.40 (m, 3H, benzene-H),
and 6.42 (s, 1H, 1-OH). 13C NMR (CDCl3, 100 MHz, assignment
from DEPT experiments, δ): 14.0 (ω-CH3), 22.5, 26.7, 28.9, 29.1, 29.6
(γ- to ω-1-CH2), 31.7(β-CH2), 41.1 (α-CH2), 106.5 (C-2 and C-4 of
benzene ring), 106.8 (C-6 of benzene ring), 151.2 (C-3 and C-5 of
benzene ring), 154.8 (C-1 of benzene ring) and 157.7 (carbamate CO).
Mass spectra, exact mass: calculated, 436.2937; found, 436.2932.
Elemental analysis: calculated for C24H40N2O5: C, 66.03; H, 9.23; N,
6.42. Found: C, 65.96; H, 9.57; N, 6.31.
loxyresorcinols (9−12) (Figure 2) may be from the enzyme-
catalyzed hydrolysis of bidentate 5−8 (Figure 3). Thus, tridentate
inhibitors are designed as long-lived inhibitors of AChE. Therefore,
this methodology may be useful to design the long-acting drug for
Alzheimer's disease.
MATERIALS AND METHODS
■
Materials. Phloroglucinol (1,3,5-benzene-triol), n-alkylisocyanate,
triethylamine, and tetrahydrofuran were purchased from Aldrich. Silica
gel used in liquid chromatography and thin-layer chromatography
plates were obtained from Merck.
1
3,5-Di-N-n-heptylcarbamyloxyphenol (6). H NMR (CDCl3, 400
MHz, δ): 0.86 (t, J = 7 Hz, 6H, ω-CH3), 1.28−1.34 (m, 16H, γ- to ω-
1-CH2), 1.50 (m, 4H, β-CH2), 3.18 (dt, J = 6.6 and 13.4 Hz, 4H, α-
CH2), 5.44 (t, J = 6.0 Hz, 2H, NH), 6.22−6.24 (m, 3H, benzene-H),
and 6.34 (s, 1H, 1-OH). 13C NMR (CDCl3, 100 MHz, assignment
from DEPT experiments, δ): 14.0 (ω-CH3), 22.5, 26.7, 28.9, 29.6
Chemistry. 1,3,5-Tri-N-alkylcarbamyl phloroglucinols (1−4), 3,5-
di-N-alkylcarbamyl-oxyphenols (5−8), and 5-N-alkylcarbamyloxyre-
sorcinols (9−12) (Figure 2) were synthesized from the condensation
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(γ- to ω-1-CH2), 31.7 (β-CH2), 41.1 (α-CH2), 106.5 (C-2 and C-4 of
benzene ring), 106.8 (C-6 of benzene ring), 151.2 (C-3 and C-5 of
benzene ring), 154.8 (C-1 of benzene ring), and 157.7 (carbamate CO).
Mass spectra, exact mass: calculated, 408.2624; found, 408.2618. Elemental
analysis: calculated for C22H36N2O5: C, 64.68; H, 8.88; N, 6.86. Found: C,
64.60; H, 9.01; N, 6.53.
3,5-Di-N-n-hexylcarbamyloxyphenol (7). 1H NMR ((CD3)2SO,
400 MHz, δ): 0.86 (t, J = 7 Hz, 6H, ω-CH3), 1.28−1.34 (m, 12H, γ- to
ω-1-CH2), 1.44 (m, 4H, β-CH2), 3.02 (dt, J = 7 and 13 Hz, 4H,
α-CH2), 6.26−6.32 (m, 3H, benzene-H), and 7.68 (t, J = 5.6 Hz, 2H,
NH), and 9.79 (s, 1H, 1-OH). 13C NMR ((CD3)2SO, 100 MHz,
assignment from DEPT experiments, δ): 14.0 (ω-CH3), 22.5, 26.7,
28.9, 29.6 (γ- to ω-1-CH2), 31.7 (β-CH2), 41.1 (α-CH2), 106.5 (C-2
and C-4 of benzene ring), 106.8 (C-6 of benzene ring), 151.2 (C-3 and
C-5 of benzene ring), 154.8 (C-1 of benzene ring), and 157.7
(carbamate CO). Mass spectra, exact mass: calculated, 380.2311;
found, 380.2306. Elemental analysis: calculated for C20H32N2O5: C,
63.13; H, 8.48; N, 7.36. Found: C, 63.02; H, 8.62; N, 7.31.
3,5-Di-N-n-butylcarbamyloxyphenol (8). 1H NMR ((CD3)2SO,
400 MHz, δ): 0.89 (t, J = 7 Hz, 6H, ω-CH3), 1.29 (sextet, J = 7 Hz,
4H, γ-CH2), 1.44 (quintet, J = 7 Hz, 4H, β-CH2), 3.08 (dt, J = 6.6 and
13.2 Hz, 4H, α-CH2), 6.27−6.33 (m, 3H, benzene-H), 7.70 (t, J = 5.6
Hz, 3H, NH), and 9.80 (s, 1H, 1-OH). 13C NMR ((CD3)2SO, 100
MHz, δ): 13.5 (ω-CH3), 19.4 (γ-CH2), 31.3 (β-CH2), 40.1 (α-CH2),
105.4 (C-2 and C-6 of benzene ring), 105.8 (C-4 of benzene ring),
152.1 (C-1 of benzene ring), 153.9 (C-3 and C-5 of benzene ring), and
158.1 (carbamate CO). Mass spectra, exact mass: calculated, 324.1685;
found, 324.1680. Elemental analysis: calculated for C16H24N2O5: C,
59.24; H, 7.46; N, 8.64. Found: C, 59.11; H, 7.58; N, 8.37.
1
5-N-n-Octylcarbamyloxyresorcinol (9). H NMR ((CD3)2SO, 400
MHz, δ): 0.90 (t, J = 7 Hz, 3H, ω-CH3), 1.28−1.35 (m, 10H, γ- to ω-
1-CH2), 1.52 (m, 2H, β-CH2), 3.21 (t, J = 6.8 Hz, 2H, α-CH2), 4.63
(s, 1H, NH), 4.90 (s, 2H, 1-OH), 6.03−6.11 (m, 3H, benzene-H). 13C
NMR ((CD3)2SO, 100 MHz, assignment from DEPT experiments, δ):
14.4 (ω-CH3), 23.6, 27.8, 30.1, 30.7, 31.8 (γ- to ω-1-CH2), 32.9 (β-
CH2), 42.0 (α-CH2), 100.7 (C-2 of benzene ring), 101.6 (C-4 and C-6
of benzene ring), 154.1 (C-5 of benzene ring), and 157.2 (carbamate
CO), and 159.9 (C-3 and C-5 of benzene ring). Mass spectra, exact
mass: calculated, 281.1627; found, 281.1622. Elemental analysis:
calculated for C15H23NO4: C, 64.03; H, 8.24; N, 4.98. Found: C,
63.91; H, 8.37; N, 4.86.
5-N-n-Heptylcarbamyloxyresorcinol (10). 1H NMR ((CD3)2SO,
400 MHz, δ): 0.89 (t, J = 7 Hz, 3H, ω-CH3), 1.29−1.35 (m, 8H, γ- to
ω-1-CH2), 1.52 (m, 2H, β-CH2), 3.20 (t, J = 6.8 Hz, 2H, α-CH2), 4.63
(s, 1H, NH), 4.89 (s, 2H, 1-OH), 6.03−6.11 (m, 3H, benzene-H). 13C
NMR ((CD3)2SO, 100 MHz, assignment from DEPT experiments, δ):
14.4 (ω-CH3), 23.6, 27.8, 30.7, 31.7 (γ- to ω-1-CH2), 32.9 (β-CH2),
42.0 (α-CH2), 100.7 (C-2 of benzene ring), 101.6 (C-4 and C-6
of benzene ring), 154.1 (C-5 of benzene ring), and 157.2 (carbamate
CO), and 159.9 (C-3 and C-5 of benzene ring). Mass spectra, exact
mass: calculated, 267.164; found, 267.158. Elemental analysis: cal-
culated for C14H21NO4: C, 62.90; H, 7.92; N, 5.24. Found: C, 62.78; H,
8.02; N, 5.08.
5-N-n-Hexylcarbamyloxyresorcinol (11). 1H NMR ((CD3)2SO,
400 MHz, δ): 0.86 (t, J = 7 Hz, 3H, ω-CH3), 1.29−1.35 (m, 6H, γ- to
ω-1-CH2), 1.43 (m, 2H, β-CH2), 3.00 (dt, J = 5.6 and 6.8 Hz, 2H, α-
CH2), 5.92 (s, 2H, 4,6-benzene-H), 6.03 (s, 1H, 2-benzene-H), 7.56 (t,
J = 5.2 Hz, 1H, NH), and 9.37 (s, 2H, 1,3-OH). 13C NMR
((CD3)2SO, 100 MHz, assignment from DEPT experiments, δ): 14.7
(ω-CH3), 22.7, 26.6, 29.9 (γ- to ω-1-CH2), 31.6 (β-CH2), 41.1
(α-CH2), 99.9 (C-2 of benzene ring), 100.7 (C-4 and C-6 of benzene
ring), 153.3(carbamate CO), 154.8 (C-5 of benzene ring), and
159.2 (C-3 and C-5 of benzene ring). Mass spectra, exact mass: cal-
culated, 253.1314; found, 253.1308. Elemental analysis: calculated for
C13H19NO4: C, 61.64; H, 7.56; N, 5.53. Found: C, 61.53; H, 7.64; N, 5.42.
5-N-n-Butylcarbamyloxyresorcinol (12). 1H NMR ((CD3)2SO,
400 MHz, δ): 0.90 (t, J = 7 Hz, 3H, ω-CH3), 1.29 (sextet, J = 7 Hz,
2H, γ-CH2), 1.44 (quintet, J= 7 Hz, 2H, β-CH2), 3.06 (dt, J = 6.4
and 6.8 Hz, 2H, α-CH2), 5.93 (s, 2H, 4,6-benzene-H), 6.04 (s, 1H,
2-benzene-H), 7.57 (t, J = 5.2 Hz, 1H, NH), and 9.36 (s, 2H, 1,3-OH).
Figure 4. Nonlinear least-squares curve fittings of kapp vs inhibitor
concentration ([I]) plots against eq 1 for the pseudo substrate
inhibition of AChE by 1,3,5-tri-N-n-octylcarbamylphloroglucinol (1)
(A), 3,5-di-N-n-hexylcarbamyloxy-phenol (7) (B), and 5-N-n-heptyl-
carbamyloxyresorcinol (10) (C). For A, k2 = 0.0027 0.0001 s−1 and
Ki = 15 3 nM (R2 = 0.96458). For B, k2 = 0.0016 0.0002 s−1 and
Ki = 110 40 nM (R2 = 0.91316). For C, k2 = 0.0018 0.0002 s−1
and Ki = 60 20 nM (R2 = 0.92569).
13C NMR ((CD3)2SO, 100 MHz, assignment from DEPT experiments, δ):
13.551 (ω-CH3), 19.4 (γ-CH2), 31.3 (β-CH2), 40.3 (α-CH2), 105.0 (C-2 of
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Chemical Research in Toxicology
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Table 1. Pseudo Substrate Inhibition Constants of AChE by
Inhibitors 1−12
inhibitors
k2 (10−3 s−1
)
Ki (nM)
ki (103 M−1 s−1
)
k3 (10−4 s−1
)
a
1
2.7 0.1
2.2 0.1
2.5 0.2
2.3 0.5
2.6 0.1
2.7 0.1
1.6 0.2
2.1 0.2
15
34
3
7
180 40
60 10
1.2 0.2
1.8 0.2
2.7 0.3
3.5 0.3
1.1 0.1
1.8 0.2
2.6 0.3
3.4 0.3
1.1 0.1
1.7 0.2
2.8 0.3
3.3 0.3
2
3
81 15
30 10
31
6
4
80 30
5
100 30
110 40
110 40
100 20
26
25
15
21
9
9
5
5
6
7
8
9
1.54 0.08
1.3 0.1
1.8 0.2
1.3 0.1
31
6
50 10
40 10
30 10
10
34 10
60 20
11
12
80 20
16
4
b
donepezil
35
4
a
The ki values were calculated from k2/Ki and uncertainty in ki values =
b
[(uncertainty of k2)2 + (uncertainty of Ki)2]1/2. Ki = 21 3 nM from
Piazzi et al.39
benzene ring), 106.0 (C-4 and C-6 of benzene ring), 155.6 (carbamate
CO), 156.0 (C-5 of benzene ring), and 162.0 (C-3 and C-5 of benzene
ring). Mass spectra, exact mass: calculated, 225.1001; found, 225.0095.
Elemental analysis: calculated for C11H15NO4: C, 58.66; H, 6.71; N, 6.22.
Found: C,58.58; H, 6.90; N, 6.13.
1
Instrumental Methods. H and 13C NMR spectra were recorded
at 400 and 100 MHz, respectively, on a Varian-Gemini 400 spectrometer
with an internal reference tetramethylsilane at 25 °C. Mass spectra were
recorded at 71 eV in a mass spectrometer (Joel JMS-SX/SX 102A).
Elemental analyses were preformed on a Heraeus instrument. All steady
state kinetic data were obtained from a UV−visible spectrometer
(Agilent 8453) with a cell holder circulated with a water bath.
Data Reduction. Origin (version 6.0) was used for the linear and
nonlinear least-squares curve fittings.
AChE Inhibition. The inhibition reactions of AChE were
determined by the Ellman assay.32 The AChE-catalyzed hydrolysis of
substrate acetylthiocholine (0.1 mM) in the presence of 5,5′-dithio-bis-
2-nitrobenzoate (0.1 mM) and inhibitors 1−12 were followed
continuously at 410 nm on a UV−visible spectrometer at 25 °C, pH
7.1. The temperature was maintained at 25.0 °C by a refrigerated
circulating water bath. All reactions were preformed in sodium
phosphate buffer (1 mL, 0.1 M, pH 7.1) containing NaCl (0.1 M),
CH3CN (2% by volume), detergent triton-X 100 (TX) (0.5% by
weight), substrate acetylthiocholine (Km = 0.10 0.01 mM), and a
varying concentration of the inhibitors. Requisite volumes of stock
solution of substrate acetylthiocholine and the inhibitor in acetonitrile
were injected into reaction buffer via a pipet. The reaction was
followed until 85% of substrate consumption was completed. The Ki
and k2 values were obtained from the nonlinear least-squares of curve
fittings of the kapp values versus inhibition concentration ([I]) plot
against eq 15 (Figure 4 and Table 1). The carbamylation stage is rapid
as compared to subsequent decarbamylation (k2 ≫ k3); thus, the two
stages are easily resolved kinetically. Values of decarbamylation
constants (k3 values) were determined from the first order rate
constants for the percent activity versus time plots (Figure 5).33 The
percent activity was defined as Vi/V0, where Vi is the initial rate for an
inhibitor incubated with AChE for a period of time (T) and V0 is the
initial rate of AChE at time T without an inhibitor. Duplicate sets of
data were collected for each inhibitor concentration.
Figure 5. Decarbamylation constant (k3). The first order rate process
for the time course of percent activity for the pseudo substrate
inhibition33 of PSL by 1,3,5-tri-N-n-octylcarbamylphloroglucinol (1)
(A), 1,3,5-tri-N-n-hexylcarbamyl- phloroglucinol (3) (B), and 1,3,5-tri-
N-n-butylcarbamylphloroglucinol (4) (C). For A, k3 = (1.2 0.2) ×
10−4 s−1 (R2 = 0.99037). For B, k3 = (2.7 0.3) × 10−4 s−1 (R2 =
0.98855). For C, k3 = (3.5 0.3) × 10−4 s−1 (R2 = 0.99232).
AChE Catalyzed the Hydrolysis of Tridentate 1. Powder of
AChE from Electrophorus electricus (500 units) was added to a 1 mL of
phosphate buffer solution (0.1 M, pH 7.1) of 1 (100 mM) at 37 °C.
The progress of the reaction was monitored by TLC (the Rf values of
tridentate 1, didentate 5, monodentate 9, and phloroglucinol were
0.65, 0.25, 0.1, and 0, respectively, developed in hexane−ethyl acetate
(5/1, v/v)). After 30 min, [tridentate 1]:[didentate 5]:[monodentate 9]:
[phloroglucinol] is about 1:6:2:1. After 5 h, the ratio became 1:2:4:3.
Therefore, these results suggested that AChE catalyzed the hydrolyses of
tridentate 1 to didentate 5, didentate 5 to monodentate 9, and
monodentate 9 to phloroglucinol (Figure 3).
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Molecular Modeling of Inhibitors and Automated Docking
Inhibitors into AChE. Molecular structures of (1,3,5)-(cis,tran-
s,trans)- and (trans,trans,trans)-tricarbamate conformation of inhibitor
1 were depicted from the molecular structures after MM-2 energy
minimization (minimum root-mean-square gradient was set to be 0.01)
by CS Chem 3D (version 6.0). Out of all of the entries for AChE from
RCSB protein data bank, mouse AChE (2HA3)4 and recombinant human
AChE (3lii)45 were taken for docking analysis. Protein-inhibitor docking
studies were performed to evaluate the algorithm and scoring function
efficiency between a standalone AutoDock 4.234−37 and experimental
activities. Automated docking was used to locate the appropriate binding
orientations and conformations of various inhibitors in the enzyme
binding pocket. All water molecules were removed from the original
Protein Data Bank file. Polar hydrogen atoms were added, and Kollman
charge, atomic solvation parameters, and fragmental volumes were
assigned to the protein using AutoDock Tools (ADT). For docking
calculations, Gasteiger partial charges were assigned to the tested
derivatives, and nonpolar hydrogen atoms were merged. The torsions
of C−O and carbamate CN partial double bonds were allowed (Figure 8)
or restricted (Figure 9) to rotate during docking. The program AutoGrid
used to generate the grid maps. Each grid was centered at the crystal
structure of the corresponding enzyme. The grid dimensions were 40 ×
40 × 40 Å3 with points separated by 0.375 Å. For all inhibitors, random
starting positions, random orientations, and torsions were used. The
translation, quaternion, and torsion steps were taken from default values in
AutoDock. The Lamarckian genetic algorithm and the pseudo-Solids and
Wets methods were applied for minimization using default parameters. The
standard docking protocol for rigid and flexible inhibitor docking consisted
of 50 independent runs per inhibitor, using an initial population of 150
randomly placed individuals, with 2.5 × 106 energy evaluations, a maximum
number of 27000 iterations, a mutation rate of 0.02, a crossover rate of 0.80,
and an elitism value of 1. The probability of performing a local search on an
individual in the population was 0.06, using a maximum of 300 iterations
per local search. After docking, the 10 solutions were clustered into groups
with rms deviations lower than 1.0 Å. The clusters were ranked by the
lowest energy representative of each cluster. The interactive visualization
and analysis of molecular structures and hydrogen bonds between protein
and inhibitor were preformed by UCSF Chimera.38
RESULTS
■
1,3,5-Tri-N-n-alkylcarbamylphloroglucinols (1−4), 3,5-di-N-n-
alkylcarbamyloxy-phenols (5−8), and 5-N-n-alkylcarbamylox-
yresorcinols (9−12) (Figure 2) were synthesized from one pot
reaction of phloroglucinol with 2 equiv of the corresponding
n-alkylisocyanate and 2 equiv of triethylamine in tetrahydrofur-
an at 25 °C for 5 h. Three products were easily separated by
liquid chromatography. Under this optimal condition, the
product ratio for 1,3,5-tri-N-n-alkylcarbamylphloroglucinols
(1−4):3,5-di-N-n-alkylcarbamyloxyphenols (5−8):5-N-n-alkyl-
carbamyloxyresorcinols (9−12) was about 2:1:1.5. When 3 equiv
Figure 6. Comparisons of the ki values of pseudo substrate inhibitions
of AChE by inhibitors 1−12.
Figure 7. Conformational changes between (1,3,5)-(trans,trans,trans)- and (1,3,5)-(cis,trans,trans)-tricarbamate rotamers of inhibitor 1.
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of n-alkylisocyanate and 3 equiv of triethylamine were used, the
reactions only yielded 1,3,5-tri-N-n-alkylcarbamylphloroglucinols
(1−4). When 1 equiv of n-alkylisocyanate and 1 equiv of tri-
ethylamine were used, the reaction yield is too low.
Carbamate inhibitors 1−12 were all characterized as the
pseudo substrate inhibitors of AChE (Figures 1, 4, and 5 and
Table 1). It is assumed that AChE catalyzes the hydrolysis of
one n-alkylcarbamyl group of inhibitors 1−12 (Figure 3). In
other words, inhibitors 1−12 may react with the enzyme
through the nucleophilic attack of the catalytic serine of the
enzyme to one carbamyl carbon of inhibitors 1−12.
For AChE inhibition, the inhibitory potencies are as follows:
tridentate inhibitors 1,3,5-tri-N-n-alkylcarbamylphloroglucinols
(1−4) > monodentate inhibitors 5-N-n-alkylcarbamyloxyresor-
cinols (9−12) > bidentate inhibitors 3,5-di-N-n-alkylcarbamy-
loxyphenols (5−8) (Figure 6 and Table 1). Tridentate inhibitors
1,3,5-tri-N-n-alkylcarbamylphloroglucinols (1−4) were the most
potent inhibitors (Figure 6). Comparison of different alkyl
substituents of inhibitors 1−4 indicates that n-octylcarbamyl
inhibitor 1 is the most potent inhibitors (Figure 6 and Table 1).
The Ki value of inhibitor 1 is about the same with that of
donepezil.39
The (1,3,5)-(trans,trans,trans)-tricarbamate rotamer of 1 can
better bind to AChE when the configuration is changed to
(1,3,5)-(cis,trans,trans) rotamer. Molecular docking of the most
stable (1,3,5)-(trans,trans,trans)-tricarbamate rotamer of inhib-
itor 1 (Figure 7) into the X-ray crystal structure of mouse
AChE results that (1,3,5)-(cis,trans,trans)-tricarbamate rotamer
is the most optimal configuration for the enzyme binding
(Figure 8). Separate docking of (1,3,5)-(trans,trans,trans)- and
(1,3,5)-(cis,trans,trans)-tricarbamate rotamers (Figure 7) into
the enzyme by restricted rotation of the CN partial double
Figure 8. Molecular docking of tridentate inhibitor 1, with the mode
of free rotation around the carbamyl CN partial double bond, into the
active sites of X-ray crystal structure of mouse AChE (2HA3):4 (A)
the active site view and (B) the view from the entrance (mouth) of the
enzyme. The configuration of the inhibitor after docked is the (1,3,5)-
(cis,trans,trans)-tricarbamate rotamer. The carbamyl carbonyl carbon
atom of the inhibitor is close to S203 of the catalytic triad, and the
carbamyl ester oxygen atom of the inhibitor is closed to H447 of the
catalytic triad. Two octylcarbamyl groups of the inhibitor shield W86
of the enzyme.
Figure 9. Molecular docking of (1,3,5)-(trans,trans,trans)-tricarbamate
rotamer of inhibitor 1, with restricted rotations of the carbamyl CN
partial double bonds, into the active sites of X-ray crystal structure of
mouse AChE (2HA3)4 results in two unlikely binding modes. (A) The
carbamyl ester oxygen atom of the inhibitor is too far away from H447
of the catalytic triad. Therefore, this rotamer is not a pseudo substrate
inhibitor of the enzyme. (B) An octylcarbamy group of this rotamer
blocks the hydrogen bond formation between the carbamyl ester
oxygen atom of the inhibitor and the H447 of the catalytic triad.
Therefore, this rotamer can not react with the enzyme.
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bonds of inhibitors suggests that the former rotamer is less
likely to bind into the enzyme (Figure 9), but the latter rotamer
binds well into the enzyme (Figure 8).
DISCUSSION
■
Inhibitory Potencies for Mono-, Bi-, and Tridentate-n-
alkylcarbamyl-phloroglucinols. For AChE inhibition, the
inhibitory potencies are as follows: tridentate inhibitors 1,3,5-
tri-N-n-alkylcarbamylphloroglucinols (1−4) > monodentate
inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9−12) >
bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5−8)
(Table 1 and Figure 6). For tridentate inhibitors 1−4, one
carbamyl group extends to the entrance, and the other two
carbamyl groups bind to W86 of AS of the enzyme (Figure 8).
For bidentate inhibitors 3,5-di-N-alkylcarbamyloxyphenols
(5−8), one carbamyl group extends to the entrance, and the
other one may bind to W86 of AS of the enzyme. In the
meanwhile, the hydroxyl groups of inhibitors 5−8 are also close
to W86. This encounter (hydrophilic group and tryptophan π
orbital) may weaken the binding between the inhibitor and the
W86 of AS of the enzyme and may cause the reason why
inhibitors 5−8 become the worst inhibitors of AChE among
inhibitors 1−12.
Inhibitory Potencies for Varied the Chain Lengths of
n-Alkylcarbamyl Moieties of Inhibitors 1−12. Compar-
ison of different alkyl substituents of inhibitors 1−4 indicates
that n-octylcarbamyl inhibitor 1 is the most potent inhi-
bitor (Figure 6 and Table 1). One of three n-octylcarbamyl
moieties of inhibitor 1 may extend itself to the entrance or PAS
of the enzyme (Figure 8). Therefore, the longer the carbamy
moieties of the inhibitors carry, the more potent of these
inhibitors are.
The k2 values of AChE inhibition by tridentate inhibitors 1−
12 retain constant when varied with the carbamyl chain length
(Table 1). The optimal substrate chain for acetyl group binding
site (ABS) for AChE is the acetyl group as in ACh. Therefore,
most parts of the alkylcarbamyl moieties of monodentate
inhibitors 1−12 in the AChE-inhibitor tetrahedral intermediate
may not bind inside the acetyl binding site (ABS) but outside ABS.
The alkylcarbamyl moieties of inhibitors 1−12 repulse with the
leaving group parts of the inhibitors in these tetrahedral
intermediates. These repulsions make all inhibitors 1−12 have
similar k2 values (1.3−2.7 × 10−3 s−1). For log ki and pKi values of
inhibitors 1−12, only those of monodentate inhibitors exist in
linear relationships with the alkyl group chain length of the alkyl
chain of the inhibitors (n) or the hydrophobicity constant (π),
which is defined as n/2 (Figure 10).40,41 Thus, the n-alkylcarbamyl
groups of monodentate inhibitors 9−12 may bind to the enzyme
through a common mechanism (or at the same binding sites). The
longer carbamyl groups of monodentate inhibitors 9−12 may bind
better to the enzyme.9,10 On the other hand, tridentate inhibitors
1−4 and bidentate inhibitors 5−8 may bind to the enzyme with
different mechanisms or at different binding sites.
Long-Lived Tridentate Inhibitors. Tridentate inhibitors
1−4 act as the pseudo substrate inhibitors of AChE (Figure 1
and Table 1).5−15 The pseudo substrate inhibition reactions of
tridentate inhibitors 1−4 yield bidentate inhibitors 5−8 that are
also characterized as the pseudo substrate inhibitors of the enzyme
(Figure 3 and Table 1). The pseudo substrate inhibition reactions
of bidentate inhibitors 5−8 yield monodentate inhibitors 9−12
that are also the pseudo substrate inhibitors of the enzyme (Figure
3 and Table 1). Accordingly, tridentate inhibitors 1−4 may act as
long-lived inhibitors of AChE.
Figure 10. pKi (A) and log ki values vs carbamyl carbon numbers (n)
of inhibitors 1−12 for the pseudo substrate inhibition of AChE. (A)
For monodentate inhibitors 9−12, a linear correlation between pKi
and n is observed [pKi = 6.7 0.1 + (0.10 0.02)n; R = 0.97439].
(B) For monodentate inhibitors 9−12, a linear correlation between
log ki and n is observed [log ki = 3.71 0.04 + (0.124 0.006)n;
R = 0.99786].
Docking of Inhibitor 1 into the X-ray Crystal Structure
of AChE. Conformational analysis of inhibitor 1 by MM-2
suggests that the most two stable rotamers are (1,3,5)-
(trans,trans,trans)- and (1,3,5)-(cis,trans,trans)-tricarbamate
forms (Figure 7). The energy barrier from the all-trans- to
(cis,trans,trans)-1 is calculated to be 38 kJ/mol by variations of
both bond angles and bond lengths from semiempirical method
(unpublished result). This activation energy is relative;
therefore, these two rotamers can easily convert to each other
even at −10 °C.42
The result of molecular docking suggests that AChE
selectively binds to the (1,3,5)-(cis,trans,trans)-tricarbamate
form among many conformations of inhibitor 1 (Figure 7).
In this docking, the cis carbamy group of the (cis,trans,trans)-
rotamer of 1 does not bind into the ABS but reaches out itself
into the entrance as the X-ray crystal structure of the long chain
carbamylated AChE.43 Both trans carbamyl moieties of
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Figure 11. Molecular docking of (1,3,5)-(trans,trans,trans)-tricarbamate rotamer of inhibitor 1 into the active sites of X-ray crystal structure of
recombinant human AChE (3lii).45 The carbamyl carbonyl carbon atom of the inhibitor is close to S203 of the catalytic triad, and the carbamyl ester
oxygen atom of the inhibitor is closed to H447 of the catalytic triad. One octylcarbamyl group of the inhibitor binds to W86 of the enzyme.
(cis,trans,trans)-rotamer of 1 bind very well into W86 of the AS.
From another side view, this form completely blocks the
entrance of the enzyme (Figure 8B).
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: gilin@dragon.nchu.edu.tw.
Funding
This work was supported by National Science Council of
Taiwan (NSC 100-2113-M-005-003).
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■
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