Divergent structure-activity relationships of structurally similar acetylcholinesterase inhibitors

Article abstract of DOI:10.1021/jm400990p

The molecular interactions between the enzyme acetylcholinesterase (AChE) and two compound classes consisting of N-[2-(diethylamino)ethyl] benzenesulfonamides and N-[2-(diethylamino)ethyl]benzenemethanesulfonamides have been investigated using organic synthesis, enzymatic assays, X-ray crystallography, and thermodynamic profiling. The inhibitors' aromatic properties were varied to establish structure-activity relationships (SAR) between the inhibitors and the peripheral anionic site (PAS) of AChE. The two structurally similar compound classes proved to have distinctly divergent SARs in terms of their inhibition capacity of AChE. Eight X-ray structures revealed that the two sets have different conformations in PAS. Furthermore, thermodynamic profiles of the binding between compounds and AChE revealed class-dependent differences of the entropy/enthalpy contributions to the free energy of binding. Further development of the entropy-favored compound class resulted in the synthesis of the most potent inhibitor and an extension beyond the established SARs. The divergent SARs will be utilized to develop reversible inhibitors of AChE into reactivators of nerve agent-inhibited AChE.

Full text of DOI:10.1021/jm400990p

Subscriber access provided by A.A. Lemieux Library | Seattle University
Article
Divergent Structure-Activity Relationships of
Structurally Similar Acetylcholinesterase Inhibitors
C. David Andersson, Nina Forsgren, Christine Akfur, Anders Allgardsson, Lotta
Berg, Cecilia Engdahl, Weixing Qian, Fredrik Johan Ekström, and Anna Linusson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400990p • Publication Date (Web): 28 Aug 2013
Downloaded from http://pubs.acs.org on August 31, 2013
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
Divergent StructureꢀActivity Relationships of
7
8
9
Structurally Similar Acetylcholinesterase Inhibitors
10
11
12
13
14
15
16
17
18
19
C. David Andersson,# Nina Forsgren,¤ Christine Akfur,¤ Anders Allgardsson,¤ Lotta Berg,# Cecilia
Engdahl,#,¤ Weixing Qian,# Fredrik Ekström,¤,* and Anna Linusson.#,*
# Department of Chemistry, Umeå University, SEꢀ901 87 Umeå, Sweden. ¤ Swedish Defense Research
Agency, CBRN Defense and Security, SEꢀ906 21 Umeå, Sweden.
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
KEYWORDS: Acetylcholinesterase, molecular recognition, structureꢀactivity relationship, peripheral
anionic site, aromatic interactions, faceꢀtoꢀface, edgeꢀtoꢀface, isothermal titration calorimetry.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 33
1
2
3
ABSTRACT
4
5
6
7
8
9
The molecular interactions between the enzyme acetylcholinesterase (AChE) and two compound
classes consisting of Nꢀ[2ꢀ(diethylamino)ꢀethyl]ꢀbenzenesulfonamides and Nꢀ[2ꢀ(diethylamino)ꢀethyl]ꢀ
benzenemethanesulfonamides have been investigated using organic synthesis, enzymatic assays, Xꢀray
crystallography, and thermodynamic profiling. The inhibitors’ aromatic properties were varied to
establish structureꢀactivity relationships (SAR) between the inhibitors and the peripheral anionic site
(PAS) of AChE. The two structurally similar compound classes proved to have distinctly divergent
SARs in terms of their inhibition capacity of AChE. Eight Xꢀray structures revealed that the two sets
have different conformations in PAS. Furthermore, thermodynamic profiles of the binding between
compounds and AChE revealed classꢀdependent differences of the entropy/enthalpy contributions to the
free energy of binding. Further development of the entropyꢀfavored compound class resulted in the
synthesis of the most potent inhibitor, and an extension beyond the established SARs. The divergent
SARs will be utilized to develop reversible inhibitors of AChE into reactivators of nerve agent inhibited
AChE.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 3 of 33
Journal of Medicinal Chemistry
1
2
3
INTRODUCTION
4
5
6
7
8
9
The interaction between small organic molecules and proteins plays a key role in most biological
processes. To obtain a better understanding of this event, the fundamental mechanisms governing
molecular recognition need to be further clarified and generalized. In this medicinal chemistry program
we investigated the binding between the enzyme acetylcholinesterase (AChE) and a set of small organic
molecules by using a combination of organic synthesis, enzymatic assays, Xꢀray crystallography, and
thermodynamic profiling. The compounds were specifically designed to probe the nonꢀcovalent
interactions present between the inhibitors and the protein, while aiming to discover and develop
compounds for AChE inhibition as well as compounds acting as reactivators of nerve agent inhibited
AChE.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AChE is an essential enzyme that terminates cholinergic transmission by the hydrolysis of the
neurotransmitter acetylcholine (ACh).1 Reversible AChE inhibitors are in use for symptomatic treatment
of e.g., Huntington’s disease,2 and Alzheimer’s disease (AD),3ꢀ4 where increased ACh levels results in
reduced disease symptoms. AChE inhibitors may also act as toxins, e.g., organophosphorusꢀbased nerve
agents, which block the enzymatic activity by covalently binding to the catalytic serine residue. The
lethal condition caused by these agents may be reversed by treatment with reactivators (antidotes) that
are capable of binding in the active site gorge and cleave the bond between the enzyme and the nerve
agent, thereby restoring the enzymatic activity. Both AChE inhibitors currently used in disease
treatment and reactivators used to treat nerve agent intoxication suffer from limited efficacy due to e.g.,
limited bloodꢀbrain barrier penetration capability,5 and they are commonly associated with adverse
effects6 making the search for new AChE inhibitors warranted. The discovery of compounds binding to
AChE may result in new therapeutics but can also be utilized in the development of new reactivators
that can be synthesized from reversible inhibitors by the introduction of a nucleophilic chemical group.7
Additionally, a more thorough understanding of the mechanisms governing the molecular recognition in
AChE may assist in avoiding offꢀtarget effects of drugs aimed at other targets in the body.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 33
The active site of AChE forms a deep and sterically confined gorge that serves as the binding site for
both natural and synthetic ligands. The gorge structure is conserved among the extensively studied Mus
musculus AChE (mAChE),8 Torpedo californica AChE,9 and the recently solved structure for human
AChE (hAChE).10The binding site is composed of two subꢀsites; the peripheral anionic site (PAS)
corresponds to the entrance of the active site gorge, and the catalytic site (CAS) is situated close to the
base of the active site gorge. Common features of compounds binding to AChE are basicꢀ or
permanently charged nitrogens and aromatic systems that can form interactions to one, or both, of the
subsites. Galanthamine11 (halfꢀmaximal inhibitory concentration (IC50) in hAChE of 2.01 ꢁM)12 and
huperzine13 (IC50 of 0.082 ꢂM in mAChE)14 are both examples of AD drugs that bind to the CAS, while
propidium iodide (IC50 in mAChE of 1.1 ꢂM)15 binds in the PAS region.8 Examples of inhibitors that
bind to PAS and CAS simultaneously are some tetracyclineꢀbased compounds displaying an IC50 in the
nanoꢀmolar region (hAChE), tacrin analogues16ꢀ19 including the structurally symmetrical inhibitor bisꢀ
tacrine16, 20 and the nonꢀsymmetrical tacrineꢀcontaining TZ2PA6 (Kd in mAChE of 0.41 nM).21ꢀ22 The
wellꢀstudied class of benzylamines e.g., donepezil, which is a low nanoꢀmolar binder for AChE also
used in symptomatic treatment of AD (Figure 1), binds with a basic nitrogen in the CAS and an
aromatic system in the PAS.23ꢀ27 HIꢀ6 (Figure 1) is an example of a reactivator that binds to both the
CAS and the PAS with its two positively charged aromatic rings that has an IC50 for hAChE of 66
ꢂM.28,29 This list of examples is by no means comprehensive and we also refer to reviews on AChE
inhibitors.30ꢀ32
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Crystal structures of AChE•inhibitor complexes have shown that AChE inhibitors commonly interact
with the gorge residues via arene–arene interactions. Aromatic ringꢀstacking “faceꢀtoꢀface” interactions
are present in complexes with most tacrine analogues that bind to AChE, as well as in the complexes
with donepezil25, 33 and HIꢀ6.29 “Edgeꢀtoꢀface” interactions are less common but have been observed in,
for example, the PAS region of the complex with the nonꢀsymmetrical compound TZ2PA6.22
Alterations in the aromatic systems of ligands may lead to significant changes in affinity and binding
mode in general,34 and have been shown for compounds binding to AChE.23ꢀ24, 26ꢀ27, 35ꢀ38 Changes can be
ACS Paragon Plus Environment

Page 5 of 33
Journal of Medicinal Chemistry
introduced by varying arene–substituents resulting in a different overall electrostatic nature of the
aromatic ring by introducing dipoleꢀmoments as well as by altering the van der Waals surfaces.39ꢀ41
In this study, we have designed a set of compounds aimed at exploring the effects of modifying the
PASꢀbinding moiety of an inhibitor of AChE previously identified by us in a highꢀthroughput screening
(HTS) campaign (compound 1, Figure 1).42 The objective of the study was to investigate the structureꢀ
activity relationships (SAR) and the potential to optimize the PASꢀbinding moiety for increased binding
affinity using extensive biochemical and structural characterizations; with the aim to use the results in
future research for new reactivators of nerve agent inhibited AChE.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cl
H2N
O
O
O
O
N+
S
O
O
NH
O
N+
N
N
N
OH
Donepezil
HI-6
1
Figure 1. The chemical structures of donepezil, HIꢀ6, and the hit identified in the HTS campaign
targeting hAChE (1).
RESULTS AND DISCUSSION
Design of AChE Inhibitors with a focus on PAS binding. Starting from compound 1 (Figure 1),
structurally simplified firstꢀgeneration analogues were designed for evaluation in mAChE using well
established experimental protocols for both kinetics and crystallography. Based on the hypothesis that
the paraꢀchloro benzyl and the tertiary amine of 1 would bind in PAS and CAS, respectively, we
simplified the N,Nꢀdibenzylamine of 1 to a N,Nꢀdiethylamine and the substitution pattern on the
phenylmethanesulfonamide were varied resulting in different overall aromatic ring electrostatic
potential (e.g., electron donating and withdrawing substituents) and van der Waals volume (Table 1).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 33
Seven “benzylic“ Nꢀ[2ꢀ(diethylamino)ꢀethyl]ꢀbenzenemethanesulfonamides and twelve “nonꢀbenzylic”
Nꢀ[2ꢀ(diethylamino)ꢀethyl]ꢀbenzenesulfonamides were synthesized in a oneꢀstep reaction forming the
sulfonamide from the corresponding sulfonyl chloride and N,Nꢀdiethylethyleneꢀdiamine (Scheme 1).
1
2
3
4
5
6
7
8
9
Scheme 1. General synthesis of the AChE inhibitors included in the studya
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
N
a
O
n
O
n
R
Cl
R
S
S
N
O
O
n = 0, 1
n = 0, 1
aReagents and conditions: (a) CH2Cl2, N, Nꢀdiethylethyleneꢀdiamine, 0 °C → rt, 4 h.
The inhibitory effect of the benzene- and benzenemethane sulfonamides on AChE. The inhibitory
effect of the compounds were evaluated on two occasions (two bioreplicates) on independently
expressed and purified mAChE using the Ellman assay,43 and the results are presented in Table 1
(details are given in the Supporting Information). The bioreplicates gave statistically coherent results,
and only the results from bioreplicate B will therefore be discussed henceforth. The inhibitors displayed
a range in potency between an IC50 of 7 ꢁM and 139 ꢁM. This is comparable to the potency of many
currently used reactivators, e.g., HIꢀ6.28 Notably, representatives of both benzylicꢀ and nonꢀbenzylic
compounds were among the most potent inhibitors (cf, 8 and 11). The SAR conclusions, however, are
different for the two compound classes.
Table 1. Inhibition of the enzymatic activity of mAChE for compounds in set 1.
R1
H
O
N
S
N
R2
O
n
R3
Bioreplicate B
ID
2
n
1
1
R1
R2
H
R3 IC50 (ꢁM) 95% CI (ꢁM)a
H
H
H
139
49
122 ꢀ 158
41 ꢀ 58
CH3
H
3
ACS Paragon Plus Environment

Page 7 of 33
Journal of Medicinal Chemistry
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
F
Cl
H
H
H
H
H
H
H
H
F
40
12
12
10
7
34 ꢀ 46
11 ꢀ 13
10ꢀ14
9ꢀ11
4
1
2
5
3
4
6
5
CF3
Br
Cl
H
6
7
8
9
10
11
12
13
H
7
Cl
H
6 ꢀ 8
8
H
31
41
7
27 ꢀ 36
36 ꢀ 46
6 ꢀ 8
9
H
H
10
14
H
OCH3
CF3
Cl
H
H
H
H
H
H
H
H
H
F
11
12
13
14
15
16
17
18
19
20
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
22
18
19 ꢀ 24
15ꢀ20
H
OCH3
CH3
F
119
135
62
97 ꢀ 145
117 ꢀ 156
54 ꢀ 70
H
H
Cl
H
88
74 ꢀ 106
81 ꢀ 111
80 ꢀ 103
40 ꢀ 63
NO2
Cl
H
95
H
91
F
F
H
50
a 95% confidence interval calculated from at least 4 replicates.
SAR analysis of benzylic and non-benzylic inhibitors. The SAR of the benzylic inhibitors (Table 1)
gave a clear indication that a substituent in the paraꢀposition was beneficial for the potency while the
unsubstituted compound 2 was the least potent. Regarding the electronic properties of the substituents
on the aromatic ring, the electronꢀwithdrawing paraꢀchloro or paraꢀ and metaꢀchloro (5 and 8,
respectively) proved to be more beneficial than the weakly electronꢀdonating methylꢀgroup (3). Paraꢀ
trifluoromethyl substituted inhibitor 6 displayed the same potency as the paraꢀchloro compound 5,
indicating that increasing the electronꢀwithdrawing capacity of the substituent might not be more
beneficial for potency. A trend in the paraꢀhalogenated inhibitors could be seen, where bromine (7)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 33
gave the most potent compound followed by chlorine (5) and fluorine (4) in decreasing potency order,
which may be an effect of decreasing the van der Waals volume of the substituent.
1
2
3
4
5
6
7
8
Different SAR conclusions were drawn from the analysis of the nonꢀbenzylic inhibitors (Table 1)
compared to the benzylic compounds. In contrast to the benzylic compounds, the electron donating or
withdrawing capabilities had little influence, although the position of the substituent on the aromatic
ring proved critical for the potency of the compounds. The presence of a metaꢀsubstituent was favorable
and the most potent inhibitor was metaꢀmetoxy substituted compound 11, while a paraꢀsubstituent
proved unfavorable (cf, paraꢀmetoxy in 14 and paraꢀnitro in 18). There was an indication that a smaller
substituent, such as fluorine, was less disadvantageous since inhibitors with this substituent (16 and 20)
had slightly lower IC50 than the other paraꢀsubstituted inhibitors. Orthoꢀfluoro substituted and
unsubstituted compounds were equipotent inhibitors (10 and 9, respectively), and were slightly more
potent than the most potent paraꢀsubstituted inhibitor (20).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Previously, the potency of donepezil26 and donepezilꢀlike compounds in AChE inhibition have been
examined via substituent replacements on the indanone35 or the corresponding PASꢀbinding moieties in
similar molecules.23ꢀ24, 27, 35ꢀ38 These studies showed, in agreement with ours, that altering the presumed
PASꢀbinding moiety has a great effect on the potency, and that electrostatics and van der Waals
surfaces, i.e., the lowest unoccupied molecular orbital energy,38 and LennardꢀJones potentials44 may be
used to explain some of these effects. These conclusions were, however, not supported by Xꢀray
crystallography data.
The thermodynamic binding-profile of the inhibitors. The thermodynamic profiles of the binding
between mAChE and two benzylic (5; paraꢀchloro and 6; paraꢀtrifluoromethyl) and two nonꢀbenzylic
(12; metaꢀtrifluoromethyl, and 13; metaꢀchloro) inhibitors were determined using ITC (Figure 2). These
compounds were selected for further characterization since they have similar substitution patterns (and
thus presumably similar solvation properties) and a comparable inhibition potency for mAChE. ITC
heatꢀdiagrams and titration curveꢀfitting are presented in the Supporting Information.
ACS Paragon Plus Environment

Page 9 of 33
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Figure 2. Thermodynamic parameters and dissociation constants measured with ITC for two benzylic
(5; paraꢀchloro and 6; paraꢀtrifluoromethyl) and two nonꢀbenzylic (12; metaꢀtrifluoromethyl and 13;
metaꢀchloro) inhibitors of mAChE.
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Inhibitors 5, 6 and 12 bound equally strong to mAChE, both in terms of the change in Gibbs free
energy (ꢃG) and dissociation constant (Kd), while 13 bound somewhat weaker (Figure 2). Benzylic
inhibitors 5 and 6 had a ꢃG of ꢀ6.8 and ꢀ6.9 kcal/mol, respectively, while nonꢀbenzylic 12 and 13 had a
ꢃG of ꢀ6.6 and ꢀ6.0 kcal/mol, respectively. Clear differences between the two inhibitor groups emerged
when the ꢃG was decomposed into enthalpy (ꢃH) and entropy (ꢃS) components. The benzylic
compounds displayed a more favorable enthalpic component while the entropy component was
unfavorable. The nonꢀbenzylic compounds clearly had beneficial entropy components, where a large
proportion of the binding energy came from an entropy gain upon binding. This indicates that the
benzylic compounds have more direct and specific enthalpyꢀdriven interactions with mAChE and may
be less flexible upon binding, while the nonꢀbenzylic inhibitors causes a gain in entropy through
increased flexibility and/or affecting water molecules via the hydrophobic effect.45 The fact that the four
compounds have different entropy/enthalpy contributions but roughly the same ꢃG can be explained by
the entropyꢀenthalpy compensation phenomenon,46 which we have reported before for a pair of
enantiomeric inhibitors of AChE.47
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 33
X-ray crystallography of AChE inhibitors. The bioactive conformations of seven of the inhibitors
(3, 4, 5, 10, 11, 12, and 17) were determined by Xꢀray crystallography at resolutions ranging from 2.1 to
2.8 Å. The crystal structures showed that the inhibitors’ aromatic moieties bound to the PAS region of
mAChE, in accordance with our design hypothesis. Numerous faceꢀtoꢀface and edgeꢀtoꢀface aromatic
interactions were observed between the inhibitors and mAChE. Furthermore, all inhibitors formed an
internal hydrogen bond between one of the sulfonamide oxygens and the tertiary amine (Figure 3a and
b) with an average heavy atom distance of 3.1 Å (SD 0.1 Å)48. The interaction patterns in the CAS were
similar in all seven structures, and consisted of electrostatic interactions, hydrogen bonds and van der
Waal contacts. The inhibitors’ diethylꢀamine moieties presumably interact with Trp86 via hydrogen
bonds of the NC–Hꢄꢄꢄarene type, where the average distance of the closest carbon to the sixꢀmembered
ring centroid of Trp86 was 3.8 Å (SD 0.1 Å).48 Other interactions shared by the majority of the
inhibitors in the CAS included contacts between the inhibitors’ Nꢀethyl groups and Tyr337, Gly120,
Gly121, Glu202 and His477. The following paragraphs will include a brief analysis of the inhibitors’
interactions with the PAS region of AChE.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Crystal structures of mAChE•inhibitor complexes with mAChE residues in gray and (a) the
benzylic inhibitor 3 (purple), and (b) the nonꢀbenzylic inhibitor 11 (green). (c) The PAS binding mode
of the nonꢀbenzylic inhibitors 17 (dark blue), 11 (green), and 10 (pink) and residue Phe338 from the
ACS Paragon Plus Environment

Page 11 of 33
Journal of Medicinal Chemistry
respective complexes in the same coloring. (d) The multiple conformers with similar occupancy of
inhibitor 17 and Phe338 as seen in chain A (inhibitor conformers A and B marked in darkꢀ and light
blue, respectively) and chain B (marked in orange).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Benzylic inhibitors display a conserved binding conformation. The benzylic inhibitors are
coordinated in the PAS in nearly identical conformations, which is exemplified by inhibitor 3 in Figure
3a. The inhibitors’ aromatic rings were located between Tyr341 and Tyr124 allowing parallelꢀdisplaced
faceꢀtoꢀface interactions with Tyr341 with an average phenyl centroid–centroid distance of 4.2 Å (SD
0.1 Å).48 The paraꢀsubstituent and benzyl carbon of the inhibitor and the hydroxyl of Tyr341 were
oriented in a 90º angle with respect to each other, which is a preferred orientation in these stacking
interactions.49 In addition, the benzylic inhibitors interacted with Tyr124 via O–H···arene type bonds
(phenyl centroid–oxygen distance of 3.1 Å (SD 0.1 Å)).48 In comparison with other compounds binding
to AChE, the benzylic inhibitors form similar interactions with Tyr341 as the pyridinium oxime moiety
of HIꢀ6. This interaction is, however, not observed in the complex with donepezil. Unlike our inhibitors,
both donepezil and HIꢀ6 form aromatic interactions with Trp286, a residue in the PAS of AChE that
commonly participates in key interactions to many AChE inhibitors. For further details regarding key
proteinꢀligand interactions, and structural comparisons to HIꢀ6 and donepezil, see the Supporting
Information. The most potent benzylic inhibitors included in this study all contain a paraꢀsubstituent (5,
6, 7 and 8). The increased potency can, on a structural basis, be interpreted as an effect from a
combination of increased van der Waals interactions (halogen–arene interactions),50 and beneficial
dipole–dipole interactions with Tyr341, and in particular with Trp286. A similar case involving paraꢀ
chloroꢀsubstituted ligands of the serine protease factor Xa revealed the same trend as observed here; a
chlorine substituent resulted in more potent inhibitors compared to fluorine and methyl, which was
shown to be due to interactions with a tryptophan.51
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 33
Divergent binding conformations of the non-benzylic inhibitors. The nonꢀbenzylic inhibitors
displayed three different binding modes depending on the arene substitution pattern, and these are
shown in Figures 3b and 3c. The metaꢀsubstituted compounds 11 and 12 (exemplified by 11 in Figure
3b) participated in edgeꢀtoꢀface aromatic interactions with Tyr341 and in faceꢀtoꢀface (parallelꢀ
displaced) interactions with Phe338 (phenyl centroid–centroid distance 4.6 Å, SD 0.3 Å).48 Overall, the
most potent nonꢀbenzylic metaꢀsubstituted inhibitors 11 and 12 had a higher number of favorable
interactions with PAS compared to the paraꢀ and orthoꢀsubstituted ones. In comparison to the binding
mode of donepezil, 11 and 12 have ring conformations in a different plane compared to the dimethoxyꢀ
indanone moiety of donepezil, thus interacting with other aromatic sites in PAS (e.g. Phe338). They also
lack the stacking interaction donepezil has with Trp286.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Interestingly, three different substituentꢀdependent conformations of the PASꢀbinding moieties of the
nonꢀbenzylic inhibitors were observed (Figure 3c). The metaꢀsubstituted 11 and 12 had the same phenyl
conformation, while two alternative conformations were observed for orthoꢀsubstituted 10 and the low
affinity paraꢀsubstituted 17, respectively, where the ring was rotated 90º in the former and highly
mobile in the latter (Figure 3c and 3d). The orthoꢀsubstituted 10 only participated in edgeꢀtoꢀface
aromatic interactions with Tyr341. For compounds 11 and 12, however, the metaꢀsubstituents formed
contacts to Trp286 in addition to the edgeꢀtoꢀface interaction with Trp341. The pyridinium moiety of the
reactivator HIꢀ6 that stacks with Tyr341 has a similar orientation as the orthoꢀsubstituted 10, and
consequently a different orientation compared to the metaꢀ and paraꢀsubstituted nonꢀbenzylic inhibitors.
The deviant binding mode of the orthoꢀsubstituted 10 was most likely a result of the rotational
restriction imposed by the fluorine atom due to its proximity to the sulfonamide oxygens. Notably, the
fluorine in its present position may form a hydrogen bond to the sulfonamide nitrogen with a heavy
atom distance of 2.45 Å. This is most likely the preferred orientation over the significantly less occupied
conformation (visible in chain A in the mAChE•10 complex), where the ring is rotated 180 degrees
around the phenylꢀsulfur bond.
ACS Paragon Plus Environment

Page 13 of 33
Journal of Medicinal Chemistry
Paraꢀchloroꢀsubstituted 17 displayed three different conformations in the PAS (two in chain A and
one in Chain B), and as a consequence Phe338 displayed an assembly of different conformations
(Figure 3d). Modeling of the dominating conformers of 17 and Phe338 suggests that the structural
changes result in a stacking interaction (chain A) or edgeꢀtoꢀface interaction (chain B) between the
inhibitor and mAChE. The conformational changes observed for 17 appear to be energetically
unfavorable, which might explain the lower potency. The loss of potency of paraꢀsubstituted
compounds compared to the metaꢀsubstituted may be because a paraꢀsubstituent, such as chlorine in 17,
would result in a clash with the side chain of Tyr341 if compound 17 would adopt the binding
conformation displayed by the metaꢀsubstituted compounds.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Investigation of a second generation non-benzylic inhibitors with bicyclic aromatic systems. The
binding of the nonꢀbenzylic compounds to AChE were shown to have beneficial entropies and the
compounds displayed a range of binding modes in the PAS region. Increasing the number of interaction
points between the inhibitor and AChE, and especially areneꢀstacking with Trp286, was deemed a
reasonable strategy in the development of new inhibitors and reactivators leads. Hence, a second
generation of nonꢀbenzylic compounds was synthesized to investigate the level of modifications needed
to increase potency and alter the binding mode to AChE, and thereby to go beyond the previously
established SAR. Four new molecules (set 2), all containing a bicyclic aromatic system in the PASꢀ
binding moiety, were therefore synthesized using the same strategy as the previous analogues (Scheme
1), and were evaluated for inhibition of mAChE enzymatic activity (Table 2) on two separate occasions
(see the Supporting Information). The naphthalene compound 21, extended with an additional ring in
positions corresponding to ortho-/meta to the sulfonamide, was clearly more potent than compound 22
with the additional phenyl in positions corresponding to para-/meta. Hence, these two compounds seem
to adhere to the previously established SAR, showing that a paraꢀsubstituent is detrimental to potency.
Extending with paraꢀchloro benzene (23) in the paraꢀposition also diminished the inhibition of AChE,
suggesting that the SAR holds for this class of compounds as well. Interestingly, extending the PASꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 33
binding benzene with an additional phenyl (i.e., 2ꢀchloroꢀ6ꢀnitroꢀbenzene, 24) linked with an etherꢀ
bridge proved to be the most potent compound with an IC50 of 2.5 ꢂM, i.e., three times more potent that
the most potent nonꢀbenzylic compound 11 in set 1. Notably, the paraꢀmetoxy 14 displayed an IC50 of
only 119 ꢂM, indicating that 24 does not seem to adhere to the SAR established for the nonꢀbenzylic
compounds, which might indicate a different binding mode of inhibitor 24.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Inhibition of mAChE hydrolysis activity of compounds in set 2.
O
O
S
N
N
H
R
Bioreplicate C1
ID
R
IC50 (ꢁM) 95% CI (ꢁM)a
44
32ꢀ58
21
>700
>700
ꢀ
ꢀ
22
23
Cl
Cl
2.5
2.3ꢀ2.8
24
O2N
O
a 95% confidence interval calculated from at least four replicates.
Crystallographic investigation of the most potent inhibitor. The crystal structure of mAChE in
complex with 24 was determined at 2.5 Å resolution (for details, see the Supporting Information). The
crystal structure showed that aromatic moieties of 24 bound in the PAS region of AChE but with a
slightly different conformation, and that the diethyl amine bound in the CAS region with similar binding
mode, compared to the previous inhibitors, as exemplified by an overlay with the metaꢀmetoxy
substituted 11 (Figure 4a). Only one conformation was observed for the adjacent amino acids and
inhibitor 24, as opposed to the paraꢀsubstituted nonꢀbenzylic compound 17 which displayed multiple
conformations (Figure 3d). Compared to compound 11, compound 24 also stack with Phe338 (Figure
ACS Paragon Plus Environment

Page 15 of 33
Journal of Medicinal Chemistry
4b), but is shifted slightly deeper down in the gorge thereby allowing the 2ꢀchloroꢀ6ꢀnitrophenol of 24
to form an additional faceꢀtoꢀface stacking interaction with Trp286 (benzene centroid–centroid distance
4.1 Å,48 Figure 4b) in a similar conformation as the one observed for the dimethoxyꢀindanone moiety of
donepezil. This key interaction with Trp286 most likely explains the higher potency of this inhibitor.
Another potential key interaction is formed between the nitro group of 24 and the backbone nitrogen of
Phe295 (Figure 4b), with a heavy atom distance of 3.3 Å.48 Hydrogen bonds to this residue have
previously been observed in several structures.25, 29, 42, 47
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Crystal structures of (a) the superposed mAChE•inhibitor complexes with the nonꢀbenzylic
inhibitors 11 (green) and 24 (pink), and (b) inhibitor 24 and PAS residues of mAChE
CONCLUSIONS
We have investigated the molecular interactions between AChE and two structurally similar classes of
compounds and we could show that the two classes displayed different SARs, binding modes, and
thermodynamic profiles when binding to AChE. Variations of the compound areneꢀsubstituents affected
the compounds’ steric, electrostatic, and van der Waals properties, which in turn affected the
compounds’ potency, confirming the importance of aromatic interactions in PAS also shown by
others.23ꢀ24, 27, 35ꢀ38 Our data based on both biochemical and structural data revealed that the SARs in the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 33
PAS of AChE are intricate; relatively small changes in the aromatic rings of the inhibitors can have
striking effects on the binding modes, which implies that inhibitor design strategies based solely on
SARꢀconclusions are insufficient. Our results indicate that larger van der Waals contacts have a greater
influence on aromatic interactions than different electronic distributions in the phenyl rings. For the
benzylic compounds, a larger electronꢀwithdrawing substituent in the paraꢀposition of the benzylic
compounds was beneficial. For the nonꢀbenzylic compounds, a metaꢀsubstituent led to increased
potency, a paraꢀsubstituent led to decreased potency, while an orthoꢀsubstituent led to decreased
potency and alternative binding modes. Analysis of ITC data showed that the two structurally similar
series of inhibitors displayed a difference in the thermodynamic profiles, where the binding of the
benzylic inhibitors to AChE was more enthalpyꢀdriven compared to the nonꢀbenzylic inhibitors. The
more entropyꢀdriven nonꢀbenzylic compound class was investigated with a second generation of
compounds. Inhibitor 24 in the second set proved to be the most potent inhibitor, and extended beyond
the previously established SAR for the nonꢀbenzylic compounds. An additional etherꢀlinked aromatic
ring in the paraꢀposition increased the potency, which again is a reminder of the intricacy of SAR in
AChE.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Taken together, we have utilized information from kinetic, crystallographic and thermodynamic data
to establish detailed SARs specific for two structurally similar compound classes. The established SARs
can be utilized in both inhibitor and reactivator design, focusing on fineꢀtuning the aromatic interactions
in the PAS of AChE. We intend to extend the established divergent SARs of the compound classes
investigated here to also include nerve agent inhibited hAChE,28 and use the results to develop new
reactivators. We believe that a good understanding of the intricate SARs of the PASꢀbinding moiety is
critical in order to optimize the efficacy of reactivators while reducing the inhibition of the active
AChE. Our design strategy will be to combine PASꢀbinding moieties with divergent SARs with sets of
flexible CASꢀbinding moiety with reactivating properties to generate a tailored set of compounds for
biochemically and structurally evaluation of free and nerve agent inhibited hAChE.
ACS Paragon Plus Environment

Page 17 of 33
Journal of Medicinal Chemistry
EXPERIMENTAL SECTION
1
2
3
4
5
6
7
8
9
Chemistry. Thin layer chromatography was performed on Merck precoated Silicaꢀgel 60F254 plates
with detection by ultra violet (UV)ꢀlight and staining with ninhydrin (0.75 g ninhydrin, 250 mL 95%
EtOH, 2.5 mL glacial acetic acid) for detection of amines. Flash column chromatography was
performed on silica gel (grade 9385, pore size 60 Å, 230ꢀ400 mesh) and eluent ethyl acetate/methanol
3:1. Preparative reversedꢀphased HPLC was performed on a prepꢀscale HPLC pump (flowrate 20
mL/min), a 151 UV/VIS detector (detection at 214 nm), and a VP 250/21 Nucleodor (C18 HTec, 5 ꢁm)
column using a water/acetonitrile eluent system. Analytical reversedꢀphased HPLC for compounds 2ꢀ20
was performed on a Gilson® 322 HPLC pump (flowrate 1 mL/min), a Gilson® 172 diode array detector
(detection at 210ꢀ360 nm), a Sedere Sedex 85 lowꢀtemperature evaporative lightꢀscattering detector
(ELSD), and an EC 150/4.6 Nucleodor (C18 HTec, 5 ꢁm) column using a water/acetonitrile/formic acid
(0.005%) eluent system. Analytical reversedꢀphased HPLC for 21ꢀ24 was performed on a Shimadzu®
LCꢀ30AD pump (flowrate 1 mL/min) a Shimadzu SPDꢀM20A diode array detector (detection at 210ꢀ
360nm) an EC 150/4.6 Nucleodor (C18 HTec, 5 ꢁm) column using a water/acetonitrile/formic acid
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
13
(0.005%) eluent system. H and C NMR spectra were recorded on a Bruker DRXꢀ400 instrument in
CDCl3 [residual CHCl3 (δH 7.26 ppm, δC 77.16 ppm)], CD3OD [residual CH3OH (δH 3.31 ppm, δC
49.00 ppm)] or (CD3)2SO [residual (CH3)2SO (δH 2.50 ppm, δC 39.52 ppm)]. NMR spectra and HPLC
chromatograms for all compounds are given in the Supporting Information. Purities of the biologically
evaluated compounds were >95% determined by analytical HPLC.
General procedure for the synthesis of N-[2-(diethylamino)-ethyl]-benzenemethanesulfonamides
and N-[2-(diethylamino)-ethyl]-benzenesulfonamides exemplified by N-[2-(diethylamino)ethyl]-3-
methoxy-benzenesulfonamide (11). 3ꢀMetoxybenzenesulfonyl chloride (0.103 g, 0.5 mmol) was
suspended in CH2Cl2 (2.5 mL). The suspension was cooled to 0 °C, N, Nꢀdiethylethylenediamine
(0.07g, 0.6 mmol) was added slowly over 10 min and the mixture was stirred at rt until the starting
material was consumed (4 h). CH2Cl2 (2.5 mL) was added to the reaction flask and the organic phase
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 33
was washed once with NaHCO3 (aq, satd, 2.0 mL). The organic phase was dried over anhydrous
Na2SO4 and removed under reduced pressure. Purified by flash chromatography (ethyl acetate/methanol
3:1) gave the product in 89% yield. HPLC purity: > 95%.
1
2
3
4
5
6
7
8
9
N-[2-(Diethylamino)ethyl]-1-phenyl-methanesulfonamide (2). Yield 55%. Purified by preparative
HPLC. 1H NMR (CD3OD): δ 1.02 (t, 6H, J=7.3 Hz), 2.46ꢀ2.57 (m, 6H), 2.95ꢀ3.01 (m, 2H), 4.34 (s, 2H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
7.30ꢀ7.46 (m, 5H). C NMR (CD3OD): δ 11.54, 41.76, 48.10, 53.97, 59.33, 129.48, 129.62, 131.49,
131.98.
1
N-[2-(Diethylamino)ethyl]-1-(4-methylphenyl)methanesulfonamide (3). Yield 35%. H NMR
(CD3OD): δ 1.02 (t, 6H, J=7.2 Hz), 2.34 (s, 3H), 2.48ꢀ2.57 (m, 6H), 2.93ꢀ2.99 (m, 2H), 4.29 (s, 2H),
13
7.16ꢀ7.22 (m, 2H), 7.27ꢀ7.34 (m, 2H). C NMR (CD3OD): δ 11.50, 21.20, 41.76, 48.13, 53.94, 59.09,
128.35, 130.25, 131.85, 139.53.
N-[2-(Diethylamino)ethyl]-1-(4-fluorophenyl)-methanesulfonamide (4). Yield 69%. Purified by
flash chromatography. 1H NMR (CDCl3): δ 0.92 (t, 6H, J=7.1 Hz), 2.40ꢀ2.49 (m, 6H), 2.93 (t, 2H, J=5.9
13
Hz), 4.20 (s, 2H), 5.01 (br s, 1H), 6.99ꢀ7.06 (m, 2H), 7.33ꢀ7.39 (m, 2H). C NMR (CDCl3): δ 11.59,
40.90, 46.37, 51.98, 57.78, 115.71 (d, 2C, J=21.8 Hz), 125.57 (d, 1C, J=2.9 Hz), 132.38 (d, 2C, J=8.0
Hz), 162.91 (d, 1C, J=248.0 Hz).
1
1-(4-Chlorophenyl)-N-[2-(diethylamino)ethyl]methanesulfonamide (5). Yield 61%. H NMR
(CD3OD): δ 1.03 (t, 6H, J=7.1 Hz), 2.49ꢀ2.58 (m, 6H), 2.99ꢀ3.04 (m, 2H), 4.34 (s, 2H), 7.37ꢀ7.45 (m,
4H). 13C NMR (CD3OD): δ 11.53, 41.75, 48.12, 53.98, 58.46, 129.70, 130.39, 133.53, 135.55.
N-(2-Diethylamino-ethyl)-C-(4-trifluoromethyl-phenyl)-methanesulfonamide (6). Yield 82%.
1
Purified by flash chromatography. H NMR (CDCl3): δ 0.92 (t, 6H, J=7.1 Hz), 7.37ꢀ7.50 (m, 6H), 2.97
13
(t, 2H, J=5.9 Hz), 4.29 (s, 2H), 5.10 (br s, 1H), 7.46ꢀ7.63 (m, 4H). C NMR (CDCl3): δ 11.54, 40.88,
46.41, 51.95, 58.16, 123.96 (q, 1C, J=272.0 Hz), 125.39ꢀ125.76 (m, 2C), 130.70 (q, 1C, J=32.4 Hz),
131.07, 133.77.
C-(4-Bromo-phenyl)-N-(2-diethylamino-ethyl)-methanesulfonamide (7). Yield 94%. Purified by
flash chromatography. 1H NMR (CDCl3): δ 0.97 (t, 6H, J=7.1 Hz), 2.44ꢀ2.53 (m, 6H), 2.98 (t, 2H, J=5.9
ACS Paragon Plus Environment

Page 19 of 33
Journal of Medicinal Chemistry
13
Hz), 4.22 (s, 2H), 5.15 (br s, 1H), 7.27ꢀ7.53 (m, 4H). C NMR (CDCl3): δ 11.57, 40.84, 46.39, 51.95,
1
2
3
57.93, 122.84, 128.66, 131.81, 132.22.
4
5
6
1
1-(3,4-Dichlorophenyl)-N-[2-(diethylamino)ethyl]-methanesulfonamide (8). Yield 59%. H NMR
7
8
(CD3OD): δ 1.04 (t, 6H, J=7.2 Hz), 2.50ꢀ2.60 (m, 6H), 3.02ꢀ3.08 (m, 2H), 4.36 (s, 2H), 7.34ꢀ7.40 (m,
9
13
1H), 7.52ꢀ7.57 (m, 1H), 7.60ꢀ7.65 (m, 1H). C NMR (CD3OD): δ 11.51, 41.76, 48.11, 53.97, 57.90,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
131.65, 131.78, 132.44, 133.36, 133.54, 133.87.
N-[2-(Diethylamino)ethyl]-benzenesulfonamide (9). Yield 95%. Purified by flash chromatography.
1H NMR (CDCl3): δ 0.89 (t, 6H, J=7.1 Hz), 2.35 (q, 4H, J=7.0 Hz), 2.45 (t, 2H, J=5.7 Hz), 2.94 (t, 2H,
J=5.8 Hz), 7.49ꢀ7.60 (m, 3H), 7.85ꢀ7.89 (m, 2H). 13C NMR (CDCl3): δ 11.77, 40.32, 46.34, 51.02,
127.23, 129.15, 132.66, 139.82.
1
N-[2-(Diethylamino)ethyl]-2-fluoranyl-benzenesulfonamide (10). Yield 42%. H NMR (CD3OD):
δ 0.98 (t, 6H, J=7.2 Hz), 2.45ꢀ2.58 (m, 6H), 3.00ꢀ3.06 (m, 2H), 7.28ꢀ7.39 (m, 2H), 7.63ꢀ7.70 (m, 1H),
7.85ꢀ7.91 (m, 1H). 13C NMR (CD3OD): δ 11.52, 41.33, 48.02, 53.04, 118.10 (d, 1C, J=21.7 Hz), 125.70
(d, 1C, J=3.7 Hz), 129.80 (d, 1C, J=13.8 Hz), 131.22, 136.27 (d, 1C, J=8.4 Hz), 160.24 (d, 1C, J=254.0
Hz).
N-[2-(Diethylamino)ethyl]-3-methoxy-benzenesulfonamide (11). Yield 89%. Purified by flash
chromatography. 1H NMR (CDCl3): δ 0.86 (t, 6H, J=7.1 Hz), 2.32 (q, 4H, J=7.1 Hz), 2.42 (t, 2H, J=5.9
13
Hz), 2.91 (t, 2H, J=5.9 Hz), 3.80 (s, 3H), 5.38 (br s, 1H), 7.02ꢀ7.08 (m, 1H), 7.32ꢀ7.43 (m, 3H). C
NMR (CDCl3): δ 11.61, 40.27, 46.26, 50.94, 55.65, 111.84, 118.88, 119.19, 130.09, 140.79, 159.94.
N-[2-(Diethylamino)ethyl]-3-(trifluoromethyl)benzenesulfonamide (12). Yield 96%. Purified by
flash chromatography. 1H NMR (CDCl3): δ 0.86 (t, 6H, J=7.1 Hz), 2.34 (q, 4H, J=7.1 Hz), 2.46 (t, 2H,
J=5.8 Hz), 2.94 (t, 2H, J=5.8 Hz), 5.54 (br s, 1H), 7.65 (t, 1H, J=7.8 Hz), 7.81 (d, 1H, J=7.6 Hz), 8.05
(d, 1H, J=7.8 Hz), 8.11 (s, 1H). 13C NMR (CDCl3): δ 11.57, 40.30, 46.32, 50.93, 123.29 (q, 1C, J=272.4
Hz), 124.12 (d, 1C, J=3.6 Hz), 129.21 (d, 1C, J=3.3 Hz), 129.95, 130.43, 131.77 (q, 1C, J=33.3 Hz),
141.20.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 33
1
3-Chloro-N-(2-diethylamino-ethyl)-benzenesulfonamide (13). Yield 23%. H NMR (CD3OD): δ
1.01 (t, 6H, J=7.0 Hz), 2.51ꢀ2.61 (m, 6H), 2.95ꢀ3.01 (m, 1H), 7.54ꢀ7.61 (m, 1H), 7.62ꢀ7.68 (m, 1H),
7.76ꢀ7.82 (m, 1H), 7.84ꢀ7.88 (m, 1H). 13C NMR (CD3OD): δ 11.31, 41.38, 48.11, 52.95, 126.39,
127.91, 131.96, 133.65, 136.19, 143.85.
1
2
3
4
5
6
7
8
9
N-[2-(Diethylamino)ethyl]-4-methoxy-benzenesulfonamide (14). Yield 51%. Purified by
preparative HPLC. 1H NMR (CD3OD): δ 0.99 (t, 6H, J=7.2 Hz), 2.46ꢀ2.58 (m, 6H), 2.87ꢀ2.97 (m, 2H),
3.87 (s, 3H), 7.08 (d, 2H, J=8.9 Hz), 7.79 (d, 2H, J=8.9 Hz). 13C NMR (CD3OD): δ 11.40, 41.32, 48.08,
52.90, 56.20, 115.34, 130.19, 133.15, 164.48.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
N-[2-(Diethylamino)ethyl]-4-methyl-benzenesulfonamide (15). Yield 80%. H NMR (CD3OD): δ
0.98 (t, 6H, J=7.2 Hz), 2.43 (s, 3H), 2.45ꢀ2.55 (m, 6H), 2.92 (m, 2H), 7.38 (d, 2H, J=7.9 Hz), 7.74 (d,
2H, J=7.9 Hz). 13C NMR (CD3OD): δ 11.49, 21.43, 41.42, 48.03, 52.93, 128.07, 130.74, 138.80,
144.72.
1
N-[2-(Diethylamino)ethyl]-4-fluoranyl-benzenesulfonamide (16). Yield 81%. H NMR (CD3OD):
δ 0.98 (t, 6H, J=7.2 Hz), 2.45ꢀ2.57 (m, 6H), 2.92ꢀ2.98 (m, 2H), 7.28ꢀ7.25 (m, 2H), 7.88ꢀ7.95 (m, 2H).
13C NMR (CD3OD): δ 11.50, 41.49, 48.04, 53.00, 117.23 (d, 2C, J=22.8 Hz), 130.94 (d, 2C, J=9.4 Hz),
138.16 (d, 1C J=3.0 Hz ), 166.38 (d, 1C, J=251.6 Hz).
4-Chloranyl-N-[2-(diethylamino)ethyl]benzenesulfonamide (17). Yield 59%. 1H NMR (CD3OD): δ
0.99 (t, 6H, J=7.2 Hz), 2.46ꢀ2.58 (m, 6H), 2.92ꢀ2.99 (m, 2H), 7.56ꢀ7.61 (m, 2H), 7.80ꢀ7.87 (m, 2H). 13C
NMR (CD3OD): δ 11.46, 41.46, 48.05, 53.01, 129.75, 130.45, 139.82, 140.69.
1
N-[2-(Diethylamino)ethyl]-4-nitro-benzenesulfonamide (18). Yield 80%. H NMR (CD3OD): δ
1.01 (t, 6H, J=7.2 Hz), 2.50ꢀ2.61 (m, 6H), 3.00ꢀ3.06 (m, 2H), 8.08ꢀ8.14 (m, 2H), 8.40ꢀ8.46 (m, 2H). 13C
NMR (CD3OD): δ 11.49, 41.59, 48.04, 53.18, 125.42, 129.41, 147.85, 151.45.
4-Chloranyl-N-[2-(diethylamino)ethyl]-2-fluoranyl-benzenesulfonamide (19). Yield 50%. 1H
NMR (CD3OD): δ 1.02 (t, 6H, J=7.2 Hz), 2.49ꢀ2.63 (m, 6H), 3.03ꢀ3.10 (m, 2H), 7.39ꢀ7.45 (m, 1H),
7.46ꢀ7.52 (m, 1H), 7.88 (t, 1H, J=8.3 Hz). 13C NMR (CD3OD): δ 11.48, 41.33, 48.05, 53.14, 118.86 (d,
ACS Paragon Plus Environment

Page 21 of 33
Journal of Medicinal Chemistry
1C, J=25.0 Hz), 126.13 (d, 1C, J=3.6 Hz), 128.98 (d, 1C, J=14.5 Hz), 132.37, 141.21 (d, 1C, J=9.6 Hz),
160.14 (d, 1C, J=257.8 Hz).
1
2
3
4
5
6
7
N-[2-(Diethylamino)ethyl]-3,4-difluoranyl-benzenesulfonamide (20). Yield 60%. 1H NMR
(CD3OD): δ 1.01 (t, 6H, J=7.2 Hz), 2.50ꢀ2.60 (m, 6H), 2.96ꢀ3.01 (m, 2H), 7.45ꢀ7.54 (m, 1H), 7.67ꢀ7.74
8
9
13
(m, 1H), 7.75ꢀ7.83 (m, 1H). C NMR (CD3OD): δ 11.38, 41.44, 48.06, 53.01, 117.86 (d, 1C, J=20.00
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hz), 119.43 (d, 1C, J=18.80 Hz), 125.40ꢀ125.80 (m, 1C), 139.02ꢀ139.37 (m, 1C), 151.48 (dd, 1C,
J=13.9, 271.90 Hz), 154.00 (dd, 13.9, 276.5 Hz).
Naphtalene-1-sulfonic acid (2-diethylamino-ethyl)-amide (21) Yield 64%. Purified by flash
chromatography. 1H NMR (CDCl3): δ 0.75 (t, 6H, J=7.2 Hz), 2.19 (q, 4H, J=7.1 Hz), 2.34 (t, 2H, J=5.9
Hz), 2.85 (t, 2H, J=5.8 Hz), 7.54 (t, 1H, J=7.7 Hz), 7.57ꢀ7.62 (m, 1H), 7.65ꢀ7.70 (m, 1H), 7.94 (d, 1H,
J=8.4 Hz), 7.94 (d, 1H, J=8.4 Hz), 8.27 (d, 1H, J=7.4 Hz), 8.67 (d, 1H, J=8.6 Hz). 13C NMR (CDCl3): δ
11.34, 40.27, 45.85, 50.88, 124.06, 124.43, 126.80, 128.20, 128.26, 129.03, 129.71, 134.05, 134.22.
Naphtalene-2-sulfonic acid (2-diethylamino-ethyl)-amide (22). Yield 25%. Purified by Flash
chromatography. 1H NMR (CDCl3): δ 0.89 (t, 6H J=7.1 Hz), 2.35 (q, 4H, J=7.1 Hz), 2.47 (t, 2H, J=5.7
Hz), 2.94ꢀ3.00 (m, 2H), 7.58ꢀ7.68 (m, 2H), 7.82ꢀ7.86 (m, 1H), 7.89ꢀ7.93 (m, 1H), 7.94ꢀ7.99 (m, 2H),
13
8.41ꢀ8.46 (m, 1H). C NMR (CDCl3): δ 11.68, 40.26, 46.33, 51.10, 122.53, 127.66, 128.04, 128.63,
128.83, 129.34, 129.54, 132.27, 134.90, 136.56.
4’-Chloro-biphenyl-4-sulfonic acid (2-diethylamino-ethyl)-amide (23) Yield 9%. Purified by flash
chromatography. 1H NMR (CDCl3): δ 1.02 (t, 6H J=7.1 Hz), 2.56 (q, 4H, J=7.2 Hz), 2.63ꢀ2.69 (m, 2H),
3.06ꢀ3.12 (m, 2H), 7.41ꢀ7.48 (m, 2H), 7.50ꢀ7.56 (m, 2H), 7.64ꢀ7.71 (m, 2H), 7.91ꢀ7.98 (m, 2H).13C
NMR (CDCl3): δ 11.06, 39.94, 46.85, 51.74, 127.65, 127.85, 128.67, 129.37, 134.86, 137.88, 138.80,
144.34.
4-(2-Chloro-6-nitro-phenoxy)-N-(2-diethylamino-ethyl)-benzenesulfonamide (24). Yield 87%.
1
Purified by HPLC. H NMR ((CD3)2SO): δ 0.87 (t, 6H, J=7.2 Hz), 2.34ꢀ2.45 (m, 6H), 2.76ꢀ2.86 (m,
2H), 7.06ꢀ7.14 (m, 2H), 7.47 (bs, 1H), 7.64 (t, 1H, J=8.3 Hz), 7.76ꢀ7.82 (m, 2H), 8.05ꢀ8.12 (m, 1H),
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 33
8.15ꢀ8.21 (m, 2H). 13C NMR ((CD3)2SO): δ 11.41, 40.71, 46.49, 51.45, 115.59, 125.05, 127.97, 129.02,
129.13, 135.22, 136.05, 142.23, 144.27, 159.09, 163.33.
1
2
3
4
5
6
7
8
Determinination of the half-maximal inhibitory concentration (IC50). Compound stock solutions
were prepared by dissolving compounds (1ꢀ6 mg) in dimethyl sulfoxide (DMSO) to a final
concentration of 0.1 M, and stocks were prepared fresh prior to each IC50ꢀdetermination. Two batches of
recombinant mAChE were independently expressed and purified as previously described.52 Batch A was
immediately used for determination of IC50ꢀvalues (bioreplicate A), whereas batch B was stored on ice
for three years prior to use (bioreplicate B).53 A third stock of AChE was used for IC50 determinations of
compounds 21-24 termed bioreplicate C. All stock solutions and buffers were independently prepared
for determination of the two bioreplicates. The enzymatic activity was measured using the Ellman
assay43 at a wavelength of 412 nm and a temperature of 30 °C. The IC50ꢀvalues were obtained by
determining the enzymatic activity in the presence of various concentrations of the inhibitors using a
buffer composed of 0.2 mM 5,5’ꢀdithiobisꢀ(2ꢀnitrobenzoic acid), 1 mM acetylthiocholine iodide and 0.1
M phosphate buffer pH 8.0. The final assay volume was 200 ꢁL and the enzymatic reaction was
followed during 120 seconds using a BioTek Power Wave plate reader. At least four replicate IC50
determinations were performed for each experiment. The obtained doseꢀresponse curve was analyzed
using the Enzyme Kinetics module of Graph Pad Prism version 5.03. The doseꢀresponse curves was
fitted using the log(inhibitor) vs. response variable slope (four parameters) equation.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Isothermal titration calorimetry. All buffers were degassed and sterile filtered prior to use.
Compoundꢀ and mAChE concentrations were prepared in phosphate buffer 0.1 M, pH 7.4 as follows: 5
1.60 mM, mAChE 0.12 mM; 6, 2.50 mM, mAChE 0.17 mM; 12, 3.00 mM, mAChE 0.20 mM; and 13,
2.80 mM, mAChE, 0.15 mM. The ITC experiments were performed at 25.0 °C (26 °C for 13) on an
ITC200 instrument (MicroCalTM, GE Healthcare Life Sciences). The proteinꢀ and compound solutions
(concentrations above) were loaded into the reaction cell and syringe, respectively, and 35 automated
injections of 1.11 ꢂL each with a 250 s break in between each injection were made. The stirring speed
was 600 rpm. A reference run was performed for each experiment where compound was injected into
ACS Paragon Plus Environment

Page 23 of 33
Journal of Medicinal Chemistry
pure buffer under the same conditions as for the main experiment. The injection heat peaks were
automatically integrated, the heat peaks from the reference experiment were subtracted, and the data
was analyzed using the singleꢀsite binding model, with 300 iterations of the ChiꢀSqr fitting routine in
Origin SR4 software.54
1
2
3
4
5
6
7
8
9
Crystallographic data collection and structure refinement. mAChE was crystallized as previously
described.55 In short, crystals were grown at a protein concentration of 10 mg/mL with well solutions
composed of 27–31 % (w/v) polyethylene glycol monoꢀmethyl ether 750 and 100 mM HEPES, pH 7.0–
7.1. Drop sizes were 2 ꢂL at a ratio of 1:1 of the protein and the mother liquid. The compounds
presented in this study were soaked into mAChE crystals prior to flashꢀfreezing in liquid nitrogen for
data collection as previously described.42 Xꢀray diffraction data were collected at the MAXꢀlab
synchrotron (Lund, Sweden), on beamlines I911ꢀ2 and I911ꢀ3 equipped with MAR Research CCD
detectors. The images were collected at an oscillation range of 1° per exposure. The intensity data were
indexed and integrated using XDS56 and scaled using Scala.57 The structures were determined by rigidꢀ
body refinement with a modified apo structure of mAChE (PDB entry code 1J06)8 as a starting model.
Further crystallographic refinement was performed using the Phenix software suite.58 Several rounds of
refinement were performed, alternating with manual rebuilding in COOT.59 The quality of the final
models were validated using WHATCHECK60 and the figures were constructed using PyMol.61 The
crystallographic statistics are listed in the Supporting Information. The coordinates and structure factors
of each mAChE•ligand complex structure have been deposited in the RCSB Protein Data Bank
(www.pdb.org) with accession codes 4B7Z, 4B80, 4B81, 4B82, 4B83, 4B84, 4B85, and 4BTL for
mAChE•3, mAChE•4, mAChE•5, mAChE•10, mAChE•11, mAChE•12 and mAChE•17, and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mAChE•24, respectively.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 33
ASSOCIATED CONTENT
1
2
3
4
5
Supporting Information
6
7
8
9
Bioassay results of compound sets 1 and 2; ITC heatꢀdiagrams and titration curveꢀfitting;
crystallographic data collection and structure refinement; general description of the crystal structures
and key proteinꢀligand interactions; structural comparisons of benzylic and nonꢀbenzylic inhibitors with
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
13
donepezil and HIꢀ6; H and C NMRꢀspectra; analytical HPLC spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
PDB accession codes
4B7Z, 4B80, 4B81, 4B82, 4B83, 4B84, 4B85, 4BTL.
ACKNOWLEDGMENTS
AL thanks the Swedish Research Council and Umeå University for financial support and the authors
thank the personnel at the MAXlab beamꢀlines for their excellent technical support.
AUTHOR INFORMATION
Corresponding Authors
FE: Phone: +46ꢀ90ꢀ106815; Eꢀmail: fredrik.ekstrom@foi.se. AL: Phone: +46ꢀ90ꢀ7866890; Eꢀmail:
anna.linusson@chem.umu.se.
Notes
The authors declare no competing financial interest
ABBREVIATIONS USED
AChE, acetylcholinesterase; AD, Alzheimer’s disease, mAChE, mus musculus acetylcholinesterase;
PAS, peripheral anionic site; CAS, catalytic site; SAR, structureꢀactivity relationship; ITC, isothermal
titration calorimetry; IC50, halfꢀmaximal inhibitory concentration; Kd dissociation constant; HTS, highꢀ
ACS Paragon Plus Environment

Page 25 of 33
Journal of Medicinal Chemistry
throughput screening; ꢃG, change in Gibbs free energy; ꢃH, change in enthalpy; ꢃS, change in entropy;
Kd, dissociation constant; SD, standard deviation.
1
2
3
4
5
6
7
REFERENCES
8
9
(1)
(2)
(3)
(4)
(5)
(6)
(7)
Taylor, J. L.; Mayer, R. T.; Himel, C. M., Conformers of acetylcholinesterase ꢀ a mechanism of
allosteric control. Mol. Pharm. 1994, 45, 74ꢀ83.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Holzgrabe, U.; Kapkova, P.; Alptuzun, V.; Scheiber, J.; Kugelmann, E., Targeting
acetylcholinesterase to treat neurodegeneration. Expert Opin. Ther. Tar. 2007, 11, 161ꢀ179.
Perry, E. K.; Perry, R. H.; Blessed, G.; Tomlinson, B. E., Changes in brain cholinesterases in
senile dementia of Alzheimer type. Neuropath. Appl. Neuro. 1978, 4, 273ꢀ277.
Talesa, V. N., Acetylcholinesterase in Alzheimer's disease. Mech. Ageing Dev. 2001, 122, 1961ꢀ
1969.
Johannsen, P., Longꢀterm cholinesterase inhibitor treatment of Alzheimer's disease. CNS Drugs
2004, 18, 757ꢀ768.
Jackson, S.; Ham, R. J.; Wilkinson, D., The safety and tolerability of donepezil in patients with
Alzheimer's disease. Brit. J. Clin. Pharmacol. 2004, 58, 1ꢀ8.
de Koning, M. C.; Joosen, M. J. A.; Noort, D.; van Zuylen, A.; Tromp, M. C., Peripheral site
ligandꢀoxime conjugates: a novel concept towards reactivation of nerve agentꢀinhibited human
acetylcholinesterase. Bioorg. Med. Chem. 2011, 19, 588ꢀ594.
(8)
(9)
Bourne, Y.; Taylor, P.; Radić, Z.; Marchot, P., Structural insights into ligand interactions at the
acetylcholinesterase peripheral anionic site. Embo J. 2003, 22, 1ꢀ12.
Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I., Atomicꢀ
structure of acetylcholinesterase from Torpedoꢀcalifornica ꢀ a prototypic acetylcholineꢀbinding
protein. Science 1991, 253, 872ꢀ879.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 33
(10) Cheung, J.; Rudolph, M. J.; Burshteyn, F.; Cassidy, M. S.; Gary, E. N.; Love, J.; Franklin, M.
C.; Height, J. J., Structures of human Acetylcholinesterase in complex with pharmacologically
important ligands. J. Med. Chem. 2012, 55, 10282ꢀ10286.
1
2
3
4
5
6
7
8
9
(11) Eskander, M. F.; Nagykery, N. G.; Leung, E. Y.; Khelghati, B.; Geula, C., Rivastigmine is a
potent inhibitor of acetylꢀ and butyrylcholinesterase in Alzheimer's plaques and tangles. Brain
Res. 2005, 1060, 144ꢀ152.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(12) Galdeano, C.; Viayna, E.; Arroyo, P.; BidonꢀChanal, A.; Blas, J. R.; MunozꢀTorrero, D.; Luque,
F. J., Structural determinants of the multifunctional profile of dual binding site
acetylcholinesterase inhibitors as antiꢀAlzheimer agents. Curr. Pharm. Des. 2010, 16, 2818ꢀ
2836.
(13) Zhang, R. W.; Tang, X. C.; Han, Y. Y.; Sang, G. W.; Zhang, Y. D.; Ma, Y. X.; Zhang, C. L.;
Yang, R. M., Drugꢀevaluation of huperzineꢀa in the treatment of senile memory disorders. Acta
Pharm. Sin. 1991, 12, 250ꢀ252.
(14) Wang, R.; Yan, H.; Tang, X. C., Progress in studies of huperzine A, a natural cholinesterase
inhibitor from Chinese herbal medicine. Acta Pharmacol. Sin. 2006, 27, 1ꢀ26.
(15) Radić, Z.; Taylor, P., Interaction kinetics of reversible inhibitors and substrates with
acetylcholinesterase and its fasciculin 2 complex. J. Biol. Chem. 2001, 276, 4622ꢀ4633.
(16) Carlier, P. R.; Han, Y. F.; Chow, E. S. H.; Li, C. P. L.; Wang, H.; Lieu, T. X.; Wong, H. S.;
Pang, Y. P., Evaluation of shortꢀtether bisꢀTHA AChE inhibitors. A further test of the dual
binding site hypothesis. Bioorg. Med. Chem. 1999, 7, 351ꢀ357.
(17) Savini, L.; Campiani, G.; Gaeta, A.; Pellerano, C.; Fattorusso, C.; Chiasserini, L.; Fedorko, J.
M.; Saxena, A., Novel and potent tacrineꢀrelated heteroꢀ and homobivalent ligands for
acetylcholinesterase and butyrylcholinesterase. Bioorg. Med. Chem. Lett. 2001, 11, 1779ꢀ1782.
(18) Savini, L.; Gaeta, A.; Fattorusso, C.; Catalanotti, B.; Campiani, G.; Chiasserini, L.; Pellerano,
C.; Novellino, E.; McKissic, D.; Saxena, A., Specific targeting of acetylcholinesterase and
ACS Paragon Plus Environment

Page 27 of 33
Journal of Medicinal Chemistry
butyrylcholinesterase recognition sites. Rational design of novel, selective, and highly potent
cholinesterase inhibitors. J. Med. Chem. 2003, 46, 1ꢀ4.
1
2
3
4
5
6
7
8
(19) Campiani, G.; Fattorusso, C.; Butini, S.; Gaeta, A.; Agnusdei, M.; Gemma, S.; Persico, M.;
Catalanotti, B.; Savini, L.; Nacci, V.; Novellino, E.; Holloway, H. W.; Greig, N. H.; Belinskaya,
T.; Fedorko, J. M.; Saxena, A., Development of molecular probes for the identification of extra
interaction sites in the midꢀgorge and peripheral sites of butyrylcholinesterase (BuChE). Rational
design of novel, selective, and highly potent BuChE inhibitors. J. Med Chem. 2005, 48, 1919ꢀ
1929.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(20) Rydberg, E. H.; Brumshtein, B.; Greenblatt, H. M.; Wong, D. M.; Shaya, D.; Williams, L. D.;
Carlier, P. R.; Pang, Y. P.; Silman, I.; Sussman, J. L., Complexes of alkyleneꢀlinked tacrine
dimers with Torpedo californica acetylcholinesterase: binding of bis(5)ꢀtacrine produces a
dramatic rearrangement in the activeꢀsite gorge. J. Med. Chem. 2006, 49, 5491ꢀ5500.
(21) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radić, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.;
Sharpless, K. B., Click chemistry in situ: acetylcholinesterase as a reaction vessel for the
selective assembly of a femtomolar inhibitor from an array of building blocks. Angew. Chem.
Int. Ed. 2002, 41, 1053ꢀ1057.
(22) Bourne, Y.; Kolb, H. C.; Radić, Z.; Sharpless, K. B.; Taylor, P.; Marchot, P., Freezeꢀframe
inhibitor captures acetylcholinesterase in a unique conformation. P. Natl. Acad. Sci. USA 2004,
101, 1449ꢀ1454.
(23) Villalobos, A.; Blake, J. F.; Biggers, C. K.; Butler, T. W.; Chapin, D. S.; Chen, Y. P. L.; Ives, J.
L.; Jones, S. B.; Liston, D. R.; Nagel, A. A.; Nason, D. M.; Nielsen, J. A.; Shalaby, I. A.; White,
W. F., Novel benzisoxazole derivatives as potent and selective inhibitors of acetylcholinesterase.
J. Med. Chem. 1994, 37, 2721ꢀ2734.
(24) Nagel, A. A.; Liston, D. R.; Jung, S.; Mahar, M.; Vincent, L. A.; Chapin, D.; Chen, Y. L.;
Hubbard, S.; Ives, J. L.; Jones, S. B.; Nielsen, J. A.; Ramirez, A.; Shalaby, I. A.; Villalobos, A.;
White, W. F., Design and synthesis of 1ꢀHeteroarylꢀ3ꢀ(1ꢀbenzylꢀ4ꢀpiperidinyl)propanꢀ1ꢀone
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 33
derivatives as potent, selective acetylcholinesterase inhibitors. J. Med. Chem. 1995, 38, 1084ꢀ
1089.
1
2
3
4
5
6
7
8
(25) Kryger, G.; Silman, I.; Sussman, J. L., Structure of acetylcholinesterase complexed with E2020
(Aricept (R)): implications for the design of new antiꢀAlzheimer drugs. Structure 1999, 7, 297ꢀ
307.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(26) Sugimoto, H.; Yamanishi, Y.; Iimura, Y.; Kawakami, Y., Donepezil hydrochloride (E2020) and
other acetylcholinesterase inhibitors. Curr. Med. Chem. 2000, 7, 303ꢀ339.
(27) Ishichi, Y.; Sasaki, M.; Setoh, M.; Tsukamoto, T.; Miwatashi, S.; Nagabukuro, H.; Okanishi, S.;
Imai, S.; Saikawa, R.; Doi, T.; Ishihara, Y., Novel acetylcholinesterase inhibitor as increasing
agent on rhythmic bladder contractions: SAR of 8ꢀ{3ꢀ[1ꢀ(3ꢀfluorobenzyl)piperidinꢀ4ꢀ
yl]propanoyl}ꢀ1,2,5,6ꢀtetrahydroꢀ4Hꢀpyrrolo[3,2,1ꢀij]quinolinꢀ4ꢀone (TAKꢀ802) and related
compounds. Bioorg. Med. Chem. 2005, 13, 1901ꢀ1911.
(28) Artursson, E.; Andersson, P. O.; Akfur, C.; Linusson, A.; Börjegren, S.; Ekström, F., Catalyticꢀ
site conformational equilibrium in nerveꢀagent adducts of acetylcholinesterase: Possible
implications for the HIꢀ6 antidote substrate specificity. Biochem. Pharmacol. 2013, 85, 1389ꢀ
1397.
(29) Ekström, F.; Hörnberg, A.; Artursson, E.; Hammarström, L. G.; Schneider, G.; Pang, Y. P.,
Structure of HIꢀ6 center dot sarinꢀAcetylcholinesterase determined by XꢀRay crystallography
and molecular dynamics simulation: reactivator mechanism and design. PLoS One 2009, 4,
e5957.
(30) Kaur, J.; Zhang, M. Q., Molecular modelling and QSAR of reversible acetylcholinesterase
inhibitors. Curr. Med. Chem. 2000, 7, 273ꢀ294.
(31) Bolognesi, M. L.; Minarini, A.; Tumiatti, V.; Melchiorre, C., Progress in acetylcholinesterase
inhibitors for Alzheimer's disease. Expert Opin. Ther. Pat. 2006, 16, 811ꢀ823.
ACS Paragon Plus Environment

Page 29 of 33
Journal of Medicinal Chemistry
(32) Tumiattit, V.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Milelli, A.; Matera, R.; Melchiorre,
C., Progress in acetylcholinesterase inhibitors for Alzheimer's disease: an update. Expert Opin.
Ther. Pat. 2008, 18, 387ꢀ401.
1
2
3
4
5
6
7
8
9
(33) Saxena, A.; Fedorko, J. M.; Vinayaka, C. R.; Medhekar, R.; Radić, Z.; Taylor, P.; Lockridge, O.;
Doctor, B. P., Aromatic aminoꢀacid residues at the active and peripheral anionic sites control the
binding of E2020 (Aricept (R)) to cholinesterases. Eur. J. Biochem. 2003, 270, 4447ꢀ4458.
(34) Lee, E. C.; Kim, D.; Jurecka, P.; Tarakeshwar, P.; Hobza, P.; Kim, K. S., Understanding of
assembly phenomena by aromaticꢀaromatic interactions: benzene dimer and the substituted
systems. J. Phys. Chem. A 2007, 111, 3446ꢀ3457.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(35) Rizzo, S.; Bartolini, M.; Ceccarini, L.; Piazzi, L.; Gobbi, S.; Cavalli, A.; Recanatini, M.;
Andrisano, V.; Rampa, A., Targeting Alzheimer's disease: novel indanone hybrids bearing a
pharmacophoric fragment of AP2238. Bioorg. Med. Chem. 2010, 18, 1749ꢀ1760.
(36) Sugimoto, H.; Tsuchiya, Y.; Sugumi, H.; Higurashi, K.; Karibe, N.; Iimura, Y.; Sasaki, A.;
Kawakami, Y.; Nakamura, T.; Araki, S.; Yamanishi, Y.; Yamatsu, K., Novel piperidineꢀ
derivatives
ꢀ
Synthesis and antiacetylcholinesterase activity of 1ꢀBenzylꢀ4ꢀ[2ꢀ(Nꢀ
benzoylamino)ethyl]piperidine derivatives. J. Med. Chem. 1990, 33, 1880ꢀ1887.
(37) Kwon, Y. E.; Park, J. Y.; No, K. T.; Shin, J. H.; Lee, S. K.; Eun, J. S.; Yang, J. H.; Shin, T. Y.;
Kim, D. K.; Chae, B. S.; Leem, J. Y.; Kim, K. H., Synthesis, in vitro assay, and molecular
modeling of new piperidine derivatives having dual inhibitory potency against
acetylcholinesterase and A beta(1ꢀ42) aggregation for Alzheimer's disease therapeutics. Bioorg.
Med. Chem. 2007, 15, 6596ꢀ6607.
(38) Piazzi, L.; Cavalli, A.; Belluti, F.; Bisi, A.; Gobbi, S.; Rizzo, S.; Bartolini, M.; Andrisano, V.;
Recanatini, M.; Rampa, A., Extensive SAR and computational studies of 3ꢀ{4ꢀ
[(benzylmethylamino)methyl]phenyl}ꢀ6,7ꢀdimethoxyꢀ2Hꢀ2ꢀchromenone (AP2238) derivatives.
J. Med. Chem. 2007, 50, 4250ꢀ4254.
ACS Paragon Plus Environment