Extensive SAR and computational studies of 3-{4-[(benzylmethylamino)methyl] phenyl}-6,7-dimethoxy-2H-2-chromenone (AP2238) derivatives

Article abstract of DOI:10.1021/jm070100g

AP2238 was the first compound published to bind both anionic sites of the human acetylcholinesterase, allowing the simultaneous inhibition of the catalytic and the amyloid-β pro-aggregating activities of AChE. Here we attempted to derive a comprehensive structure-activity relationship picture for this molecule, affording 28 derivatives for which AChE and BChE inhibitory activities were evaluated. Selected compounds were also tested for their ability to prevent the AChE-induced Aβ-aggregation. Moreover, docking simulations and molecular orbital calculations were performed.

Full text of DOI:10.1021/jm070100g

4250
J. Med. Chem. 2007, 50, 4250-4254
Brief Articles
Extensive SAR and Computational Studies of
3-{4-[(Benzylmethylamino)methyl]phenyl}-6,7-dimethoxy-2H-2-chromenone (AP2238)
Derivatives
Lorna Piazzi,* Andrea Cavalli, Federica Belluti, Alessandra Bisi, Silvia Gobbi, Stefano Rizzo, Manuela Bartolini,
Vincenza Andrisano, Maurizio Recanatini, and Angela Rampa
Department of Pharmaceutical Sciences, UniVersity of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
ReceiVed January 25, 2007
AP2238 was the first compound published to bind both anionic sites of the human acetylcholinesterase,
allowing the simultaneous inhibition of the catalytic and the amyloid-â pro-aggregating activities of AChE.
Here we attempted to derive a comprehensive structure-activity relationship picture for this molecule,
affording 28 derivatives for which AChE and BChE inhibitory activities were evaluated. Selected compounds
were also tested for their ability to prevent the AChE-induced Aâ-aggregation. Moreover, docking simulations
and molecular orbital calculations were performed.
Introduction
and the benzylamino moiety itself. A total of 28 analogues
(Table 1) of 1 were synthesized, and their biological activities
were tested using the hAChE and BChE enzymes. Eight
compounds, properly selected, were also tested for their ability
to prevent the AChE-induced Aâ aggregation. In addition,
docking simulations and molecular orbital calculations were
carried out to study the binding mode of the inhibitors at the
hAChE gorge.
Alzheimer’s disease (ADa),1 the most common form of
dementia, was described for the first time a century ago by Alois
Alzheimer. He described typical clinic characteristics such as
memory disturbance and the neuropathological picture with
miliary bodies and dense bundles of fibrils, which are known
today as plaques and tangles, the hallmarks of the disease. The
cholinergic hypothesis in AD states that degeneration of
cholinergic neurons in the basal forebrain nuclei causes distur-
bances in presynaptic cholinergic terminals in the hippocampus
and neocortex, which could account for memory alterations and
other cognitive symptoms.2 One therapeutic approach to enhance
cholinergic neurotransmission is the increase in acetylcholine
(ACh) availability induced by inhibiting acetylcholinesterase
(AChE). Besides its catalytic function, AChE also acts as a
promoter of Aâ fibril formation, this effect being independent
from its normal hydrolyzing activity and associated with the
peripheral anionic site (PAS) of the enzyme.3 This finding
stimulated a great interest toward the design of hybrid molecules
capable of simultaneously inhibiting AChE and AChE-induced
Aâ aggregation. Our research group has been involved in the
development of AChE inhibitors for several years, and recently,
this dual target approach has been addressed by linking a
benzylamino group and a coumarin heterocycle through a phenyl
ring. The combined molecule, 1 (AP2238), proved to be able
to simultaneously contact both the central and the peripheral
anionic sites of AChE.4 In this paper, we intend to extensively
investigate the structure-activity relationships (SARs) of 1. We
modified the lead compound 1 in all its peculiar parts (Chart
1): the coumarin moiety and its substituents, the position of
the benzylamino group with respect to the coumarin nucleus,
Chemistry
The synthesis of the compounds 1-29 is reported in
Scheme 1. The previously synthesized intermediates 30-44
were brominated using N-bromosuccinimide in CCl4 with a
catalytic amount of (PhCOO)2O to give the bromo-derivatives
45-59. The nucleophilic attack of the selected benzylalkylamine
on the bromo-derivatives afforded the compounds 1-18 and
20-28. Methylation of compound 1 with CH3I gave 29.
Reduction of compound 18 with H2 over Pd/CaCO3 gave 19.
Syntheses of heterocyclic intermediates 30-44 are reported
in Schemes 2-5 (see Supporting Information (SI)).
Results and Discussion
Evaluation of Structure-Activity Relationships (SARs)
Related to Catalytic hAChE Activity. The inhibitory potencies,
expressed as IC50 values, of compounds 2-29 on recombinant
hAChE and BChE from human serum in comparison with the
reference 1 are reported in Table 2. The selectivity toward AChE
versus BChE was, for all compounds, of 3 orders of magnitude
or more. In view of a clearer presentation of the SAR of the
series, five structural features of 1 were identified:
(a) Position of the Benzylalkylamino Moiety. Compound
2, in which the N-benzyl-N-methyl group is in the meta-position
with respect to the coumarin moiety, showed a lower AChE
inhibitory potency compared to the lead 1. The same trend was
shown for monomethoxy derivatives, 3-6, in which the meta-
analogues 3 and 5 had a higher IC50 than the para-analogues 4
and 6.
* To whom correspondence should be addressed. Phone: +39 051
2099722. Fax: +39 051 2099734. E-mail: lorna.piazzi@unibo.it.
a Abbreviations: SAR, structure-activity relationships; AChE, acetyl-
cholinesterase; hAChE, human acetylcholinesterase; BChE, butyrylcho-
linesterase; Aâ, beta-amyloid peptide; AD, Alzheimer’s disease; PAS,
peripheral anionic site; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; RMSD, root mean square deviation; DFT, density functional
theory; NMR, nuclear magnetic resonance.
10.1021/jm070100g CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/26/2007

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4251
Chart 1. SAR Studies of 1 (AP2238)
Table 1. Synthesized Compounds
cpd
chain
R1
R2
R3
R4
cpd
chain
R1
R2
R3
CH3
R4
1
2
3
4
5
6
7
8
9
10
11
12
13
para
meta
meta
para
meta
para
para
para
para
para
para
para
para
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
14
15
16
17
18
19
22
23
24
25
26
27
28
para
para
para
para
para
para
para
meta
meta
meta
para
para
para
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
NO2
m-OCH3
p-OCH3
CH3
C2H5
C2H5OH
CH3
CH3
CH3
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
NH2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
o-NO2
m-NO2
p-NO2
o-CH3
m-CH3
p-CH3
o-OCH3
CH3
C2H5
OCH3
OCH3
OCH3
H
OCH3
OCH3
H
OCH3
Scheme 1a
the benzylamino group in para-position and introduced both
electron withdrawing and electron-donor substituents on the
terminal phenyl ring. Ortho-, meta-, and para-nitro (7-9),
-methyl (10-12), and -methoxy (13-15) derivatives were
synthesized. All these compounds, with the exception of 13 (o-
methoxy), showed a reduced anticholinesterase activity, probably
indicating that the unsubstituted N-benzyl group completely
filled the bottom of the AChE gorge and that no increase in
hindrance on this part of the molecule was allowed. Exception-
ally, compound 13 showed an IC50 that is of the same order of
magnitude as the lead compound 1. This could be explained,
as seen for other AChE inhibitors,5-7 considering that the
o-methoxy group is able to stabilize the formal positive charge
of the amino group through mixed inductive and mesomeric
effects,8 which is crucial not only to properly interact with the
central aromatic residue by cation-π interaction, but also to
induce the molecule to penetrate the gorge.9
a Reagents: (a) NBS, (PhCOO)2O; (b)
; (c) CH3I;
(d) H2, Pd/CaCO3. bNumber sequence of compounds is referred to Table 1
and Schemes 2-5 (see SI).
(b) Substituents on the Heteroaromatic Nucleus. Consider-
ing the methoxy groups in positions 6 and 7, for both meta-
and para-compounds, the substitution in position 6 seemed to
be more important than the substitution in position 7 to maintain
a submicromolar activity. On the basis of previous results, we
introduced an electron-withdrawing (NO2) and an electron-
donating (NH2) substituent in position 6 of the coumarin moiety
with the N-benzyl-N-methyl group in para-position (compounds
18 and 19, respectively). The IC50 of both compounds was
higher than the IC50 of the lead compound, but similar to the
6-monomethoxy derivative 6.
From the analysis of points (a) and (b) of the SAR study, it
clearly emerged that the N-benzyl-N-methyl group in para-
position and the 6,7-dimethoxy coumarin conferred the best
anticholinesterase activity so far (see Table 2).
(c) Substituents on the Terminal Phenyl Ring. On the basis
of previous results, we maintained the dimethoxycoumarin with
(d) Substituent on the Basic Nitrogen. To confirm the
importance of the positive charge, the methyl ammonium
analogue of 1 (29) was synthesized and showed the lowest IC50
of the series. We also replaced the methyl group of the amino
function of 1 with ethyl and hydroxyethyl groups, affording 16
and 17, respectively. Compound 16, showing an IC50 value of
18.3 nM, is more than 2 times more active than compound 1.
This result was in agreement with data presented by other
authors,5,6 where the introduction of an ethyl substituent
conferred a higher potency. The better activity could be related
to the increased lipophilicity of compound 16 compared to that

4252 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Brief Articles
Table 2. Inhibition of AChE and BChE Activities and AChE-mediated Aâ-aggregation
inhibition
inhibition
of Aâ(1-40)
aggregationb
SEM within 5%
of Aâ(1-40)
IC50 (nM)
IC50 (nM)
aggregationb
IC50 (nM)
AChE ( SEM
IC50 (nM)
BChE ( SEM
cpd
AChE ( SEM
BChE ( SEM
SEM within 5%
cpd
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
44.5 ( 6.5
202 ( 9.0
2290 ( 290
892 ( 54
303 ( 9.0)
163 ( 7.0
987 ( 76
368 ( 23
24 600
366 ( 40
122 ( 40
310 ( 36
62.1 ( 1.9
714 ( 39
2260 ( 60
48.9 × 103
279 × 103
>10-5
35
25
16
17
18
19
20
21
22
23
24
25
26
27
28
29
18.3 ( 3.2
49.3 ( 2.3
148 ( 14
281 ( 13
not solub
30 300
1230 ( 60
2020 ( 180
1590 ( 60
2270 ( 270
3100 ( 390
18 100
118 × 103
35.1 × 103
>10-5
38
6
<5
<5
570 × 103
136 × 103
1300 × 103
>10-5
<5
<5
>10-5
not solub
>10-5
>10-5
>10-5
>10-5
>10-5
>10-5
>10-5
>10-5
>10-5
>10-5
>10-5
88.9 × 103
52.9 × 103
15
80 500
10.5 ( 5.0
>10-5
>10-5
43.8 × 103
>10-5
a Human recombinants AChE and BChE from human serum were used. IC50 values represent the concentration of inhibitor required to decrease enzyme
activity by 50% and are the mean of two independent measurments, each performed in triplicate (SEM ) standard error of the meaning). b Determined at
[inhibitor] ) 100 µM, [Aâ(1-40)] ) 230 µM and [AChE] ) 2.3 µM.
of compound 1. This hypothesis could also explain the inhibitory
potency of compound 17, which is very similar to that of 1:
the positive lipophilic effect due to two carbon units is
counterbalanced by the lower lipophilicity caused by introducing
the OH group.
In Figure 1B, the binding mode of 22 and 26 (i.e., the
isoflavone and flavone para-derivatives) is reported along with
1. In this case, we observed that all inhibitors could simulta-
neously interact with both sites of the enzyme, and that while
22 lost the H-bond with Phe295 backbone, 26 kept such an
interaction. The experimental results reported in Table 2 showed
that 22 and 26 were almost 2 orders of magnitude less potent
than 1, and therefore, we had to conclude that docking outcomes
alone were not able to explain the reduced potencies of 22 and
26 when compared to 1.
(e) Oxygenated Heteroaryl Moiety. We inverted the position
of the coumarin and the central phenyl ring of the lead
compound so that the 1,2-dimethoxyphenyl moiety could interact
with the PAS and the coumarin could act as a spacer;
unfortunately, this compound (20) was insoluble, and no
biological activity could be measured. The substitution of the
coumarin nucleus with other oxygenated heteroaryl nuclei was
performed: the 3-benzyl-6,7-coumarin was substituted with 2,3-
dimethoxyxanthen-9-one leading to compound 21, which is 3
orders of magnitude less active than 1, probably because it is
too short compared to the lead compound. The coumarin moiety
was also substituted with isoflavone and flavone (compounds
22-24 and 25-28, respectively). For all compounds, the
inhibitory activity toward AChE was lower than for the reference
compound 1, and even if the dimethoxy compounds were better
inhibitors than the monomethoxy derivatives, the significant
decrease in their activities could not be explained by means of
classical SARs.
To provide a possible rationalization for the SARs of these
compounds, molecular orbital calculations within the density
functional theory (DFT) framework were performed. The
calculations were carried out on the 5,6-dimethoxycoumarin and
6,7-dimethoxychromone skeletons. In particular, we computed
the energy of the frontier molecular orbital LUMO (i.e., the
lowest unoccupied molecular orbital) of both scaffolds at the
B3LYP/6-31G* level of theory. The results showed that the
LUMO energy of 5,6-dimethoxycoumarin was almost 9 kcal/
mol less than the LUMO energy of the 6,7-dimethoxychromone.
Furthermore, we also carried out single-point calculations by
adding one electron to both scaffolds (see SI). These calculations
showed that the 5,6-dimethoxycoumarin was more prompted
to accept the additional electron when compared to the 6,7-
dimethoxychromone, with the energy difference being 10.3 kcal/
mol in favor of the coumarin compound. These results showed
the ability of the 5,6-dimethoxycoumarin moiety of 1, which
was to better accept electrons when compared to the 6,7-
dimethoxychromone of 22 and 26 and therefore explained the
higher inhibitory potency of 1 relative to 22 and 26 toward the
hAChE enzyme. Actually, it is now widely accepted that Trp286
of the AChE PAS attracts ACh from the solvent bulk by means
of cation-π interaction,9 demonstrating that this amino acid
preferably donates (rather than accepts) electrons in π-π
stacking with ligands.13 The above-reported results agree with
similar computations carried out on a series of xanthostigmine
derivatives,14 demonstrating that the energy of the frontier
orbitals should be taken into account when designing ligands
binding at tryptophan residues.
Docking Simulations and Molecular Orbital Calculations.
To thoroughly investigate the SARs of the present series of
AChE inhibitors, docking simulations were carried out with the
software GOLD10 and outcomes were rationalized by means of
the clustering algorithm AClAP.11,12 Compound 1, previously
docked by means of a different procedure,4 was studied
according to the present computational strategy (see SI).
Notably, the binding mode of 1 at the gorge of hAChE was
very similar to that previously reported,4 with an overall RMSD
for the nonhydrogen atoms less than 1 Å. In Figure 1A, the
binding mode of 1 and 2 (the meta-analogue of 1) at the hAChE
gorge is reported. It can be seen that while 1 could favorably
interact with both the catalytic site and the PAS of the enzyme,4
2 was not able to properly reach Trp286. In particular, while
the benzylamino moieties of both compounds could interact with
Trp86 of the catalytic pocket, only the coumarin moiety of 1
could also establish a favorable π-π stacking with the PAS
residue Trp286. Moreover, 2 also lost the H-bond interaction
with the backbone of Phe295, and this could likely explain its
decreased activity when compared to 1.
AChE-Induced Aâ Aggregation Inhibition. Compound 1
proved to be able to simultaneously contact both the central
and the peripheral anionic sites of hAChE4 and cause inhibition
of 35% in Aâ (1-40) aggregation. Inhibitors of Aâ oligomer-

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4253
Figure 1. (A) Docking model of 1 (carbon atoms in green) and 2 (carbon atoms in orange) at the hAChE gorge. The H-bond between the carbonyl
of the coumarin moiety of 1 and the hydrogen (cyan) of the Phe295 backbone NH is shown as a dark-red dotted line. It can be seen that while 1
simultaneously contacts both sites of the enzyme gorge, 2 is not properly able to reach Trp286 of the PAS. (B) Docking model of 1 (carbon atoms
in green), 22 (carbon atoms in yellow), and 26 (carbon atoms in magenta) at the hAChE gorge. The same H-bond as reported in (A) is shown. It
can be seen that all derivatives are able to simultaneously contact both sites of the hAChE gorge. The activity difference should, therefore, be
looked for at the electronic rather than at the geometrical level.
ization have become increasingly popular as promising phar-
macotherapy for AD. However, assaying many compounds for
inhibition of Aâ aggregation can be a very expensive endeavor
in light of the large quantities of enzyme and peptide needed
for screening.15 Consequently, for this assay we were forced to
choose a limited number of compounds on the basis of their
inhibitory potency. Thus, because the coumarin nucleus is
supposed to interact with the PAS, some coumarins endowed
with different substituents on the heterocyclic nucleus were
selected. In particular, compounds 4 and 6, with one methoxy
group in positions 6 and 7, respectively, and compounds 18
and 19, with an electron-withdrawing (NO2) and an electron-
donating (NH2) substituent in position 6 of the coumarin moiety,
respectively, were chosen. We also tested compound 2, carrying
the benzylmethylamino group in the meta-position, to evaluate
the effect of this modification on Aâ aggregation. Moreover,
compounds 13, 16, and 17, being the best AChE inhibitors of
the series, were also evaluated. The percentage of inhibition of
AChE-induced Aâ aggregation at 100 µM is reported in Table
2. It may be noticed that the inhibitor concentration in the
aggregation assay is much higher than the IC50 values of the
same compounds. Nevertheless, as was pointed out elsewhere,16
if “normalized” on the basis of the different amount of enzyme
used in the Ellman’s and aggregation assays, the ratio [inhibitor]/
[AChE] is of the same magnitude in both assays. Consequentely,
it seems plausible that similar amounts of inhibitor can
simultaneously carry out both the anti-cholinesterase and anti-
aggregating actions. Most of the tested compounds did not cause
any significant inhibition of Aâ aggregation, apart from 16,
where the part of the molecule interacting with the PAS is
unaffected, which showed inhibition similar to that of 1 (38
and 35%, respectively). Surprisingly, compound 17, which bears
an ethanol substituent on the nitrogen interacting with the
internal anionic site residues, showed a remarkably low inhibi-
tory activity toward the AChE-induced Aâ aggregation. A
possible explanation could be that the presence of a hydroxy
group drives the ligand to interact by means of H-bonding with
tyrosines of the AChE gorge, providing a different binding
mode, which is likely unable to inhibit the pro-aggregating effect
ofAChE.
Conclusions
Starting from the dual AChE inhibitor 1, we evaluated the
SAR of 28 analogues in which the modifications performed
involved the substituents on the coumarin nucleus, the coumarin
nucleus itself, the relative position of the benzylmethylamino
group, the alkyl function on the amino group, and the substit-
uents on the terminal phenyl ring. The few compounds showing
an IC50 of the same order of magnitude of 1 were 13, 16, 17,
and 29, while 16 (AP2243) was twice as potent as the lead
compound. All these derivatives maintained the peculiar features
of 1, namely, the dimethoxy coumarin nucleus and the benzy-
lalkylamino group in the para-position. Thus, the structure of
1 seems to be crucial for optimal activity, because only the
modification performed on compound 16, an ethyl instead of a
methyl group on the basic nitrogen, led to an improvement in
both catalytic and AChE-induced Aâ aggregation activities.
Experimental Section
Chemistry. 3-{4-[(Benzylmethylamino)methyl]phenyl}-6,7-
dimethoxychromen-2-one (1). A mixture of 45 (1.32 g, 3.52 mmol)
and benzylmethylamine (0.92 mL, 7.04 mmol) in toluene (125 mL)
was stirred under reflux for 20 h. The solution was washed with
water and extracted with 6 N HCl; the aqueous layer was basified
with Na2CO3, and extracted with CH2Cl2. The combined organic
layers were dried and concentrated under reduced pressure to
1
give an oil. H NMR (CDCl3): δ 2.42 (s, 3H), 3.68 (s, 2H), 3.69
(s, 2H), 3.93 (s, 3H), 3.95 (s, 3H), 6.84 (d, 2H), 7.19-7.48 (m,
7H), 7.63 (d, 2H), 7.77 (s, 1H). The product was then converted in
the hydrochloride salt affording 1‚HCl (90 mg, 6% yield): mp 230-
232 °C (MeOH). ES-MS m/z: 452 (M + 1). Anal. (C26H26ClNO4)
C, H, N.
6-Amino-3-{4-[(benzylmethylamino)methyl]phenyl}-chromen-
2-one (19). A solution of 18 (2.62 g, 6.55 mmol) in THF (250
mL) was hydrogenated in the presence of Pd/CaCO3 at atmospheric
pressure and room temperature. The mixture was filtered and the

4254 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Brief Articles
(6) Tumiatti, V.; Rosini, M.; Bartolini, M.; Cavalli, A.; Marucci, G.;
Andrisano, V.; Angeli, P.; Banzi, R.; Minarini, A.; Recanatini, M.;
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noncovalent inhibitors based on a polyamine backbone. Role of the
substituents on the phenyl ring and nitrogen atoms of caproctamine.
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Andrisano, V.; Recanatini, M.; Rampa, A. Cholinesterase inhibi-
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docking. J. Mol. Biol. 1997, 267, 727.
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filtrate was evaporated to give a residue that was purified by
gradient flash chromatography. Eluting (from CH2Cl2 100% to
CH2Cl2/EtOH 98:2) afforded 19 (1.7 g, 70% yield) as a dark yellow
1
solid: mp 166-167 °C. H NMR (CDCl3): δ 2.22 (s, 3H), 3.57
(s, 2H), 3.58 (s, 2H), 6.73 (d, 1H), 6.84 (dd, 1H), 7.14-7.32 (m,
8H), 764-7.70 (m, 3H). ES-MS m/z: 371 (M + 1). Anal.
(C24H22N2O2) C, H, N.
Benzyl-[4-(6,7-dimethoxy-2-oxo-2H-chromen-3-yl)benzyl]dim-
ethyl Ammonium Iodide (29). A solution of CH3I (54 mg, 0.38
mmol) and 1‚HCl (85 mg, 0.19 mmol) in MeOH (25 mL) was
stirred overnight and then poured into diethyl ether. The filtered
solid afforded 29 (51 mg, 24% yield) as a light beige solid: mp
145-147 °C. 1H NMR (CD3COCD3): δ 3.26 (s, 6H), 3.88 (s, 3H),
3.98 (s, 3H), 5.14 (s, 2H), 5.17 (s, 2H), 7.02 (s, 1H), 7.28 (s, 1H),
7.23-7.62 (m, 3H), 7.75-7.78 (m, 4H), 7.91-7.98 (m, 2H), 8.22
(s, 1H). ES-MS m/z: 558 (M + 1). Anal. (C27H28INO4) C, H, N,
I.
Acknowledgment. This work was supported by MIUR,
Rome (Grant No. FIRB RBNE03FH5Y). We thank Ms. Luisa
Ceccarini for her technical assistance.
Supporting Information Available: Schemes 2-5; experi-
mental details for intermediates 30-70 and final compounds 2-18
and 20-28; computational chemistry and biological evaluation
methods; and elemental analyses and 13C NMR of final compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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