Bis-(-)-nor-meptazinols as novel nanomolar cholinesterase inhibitors with high inhibitory potency on amyloid-β aggregation

Article abstract of DOI:10.1021/jm070154q

Bis-(-)-nor-meptazinols (bis-(-)-nor-MEPs) 5 were designed and synthesized by connecting two (-)-nor-MEP monomers with alkylene linkers of different lengths via the secondary amino groups. Their acetylcholinesterase (AChE) inhibitory activities were more greatly influenced by the length of the alkylene chain than butyrylcholinesterase (BChE) inhibition. The most potent nonamethylene-tethered dimer 5h exhibited low-nanomolar IC₅₀ values for both ChEs, having a 10 000-fold and 1500-fold increase in inhibition of AChE and BChE compared with (-)-MEP. Molecular docking elucidated that 5h simultaneously bound to the catalytic and peripheral sites in AChE via hydrophobic interactions with Trp86 and Trp286. In comparison, it folded in the large aliphatic cavity of BChE because of the absence of peripheral site and the enlargement of the active site. Furthermore, 5h and 5i markedly prevented the AChE-induced Aβ aggregation with IC₅₀ values of 16.6 and 5.8 μM, similar to that of propidium (IC₅₀ = 12.8 μM), which suggests promising disease-modifying agents for the treatment of AD patients.

Full text of DOI:10.1021/jm070154q

J. Med. Chem. 2008, 51, 2027–2036
2027
Bis-(-)-nor-meptazinols as Novel Nanomolar Cholinesterase Inhibitors with High Inhibitory
Potency on Amyloid-ꢀ Aggregation
Qiong Xie,†,# Hao Wang,‡,# Zheng Xia,‡ Meiyan Lu,† Weiwei Zhang,‡ Xinghai Wang,† Wei Fu,† Yun Tang,§ Wei Sheng,†
Wei Li,† Wei Zhou,‡ Xu Zhu,‡ Zhuibai Qiu,*,† and Hongzhuan Chen*,‡
Department of Medicinal Chemistry, School of Pharmacy, Fudan UniVersity, 138 Yixueyuan Road, Shanghai 200032, P. R. China, Department
of Pharmacology, Institute of Medical Sciences, Shanghai JiaoTong UniVersity School of Medicine, 280 South Chongqing Road,
Shanghai 200025, P. R. China, and School of Pharmacy, East China UniVersity of Science and Technology,
130 Meilong Road, Shanghai 200237, P. R. China
ReceiVed February 9, 2007
Bis-(-)-nor-meptazinols (bis-(-)-nor-MEPs) 5 were designed and synthesized by connecting two (-)-nor-
MEP monomers with alkylene linkers of different lengths via the secondary amino groups. Their
acetylcholinesterase (AChE) inhibitory activities were more greatly influenced by the length of the alkylene
chain than butyrylcholinesterase (BChE) inhibition. The most potent nonamethylene-tethered dimer 5h
exhibited low-nanomolar IC50 values for both ChEs, having a 10 000-fold and 1500-fold increase in inhibition
of AChE and BChE compared with (-)-MEP. Molecular docking elucidated that 5h simultaneously bound
to the catalytic and peripheral sites in AChE via hydrophobic interactions with Trp86 and Trp286. In
comparison, it folded in the large aliphatic cavity of BChE because of the absence of peripheral site and the
enlargement of the active site. Furthermore, 5h and 5i markedly prevented the AChE-induced Aꢀ aggregation
with IC50 values of 16.6 and 5.8 µM, similar to that of propidium (IC50 ) 12.8 µM), which suggests promising
disease-modifying agents for the treatment of AD patients.
Introduction
Alzheimer’s disease (AD), which is characterized by progres-
sive loss of memory and impairment in cognition,1 is becoming
a serious threat to life expectancy for elderly people. The main
pathological changes in the AD brain are the abnormal formation
of extracellular senile plaques consisting of aggregated amyloid-
ꢀ-peptide (Aꢀ) deposits and intracellular neurofibrillary tangles
(NTFs) consisting of abnormally phosphorylated microtubule-
associated protein τ.2
Current clinical therapy for AD patients is mainly palliative
treatment targeting acetylcholinesterase (AChE). On the basis
of the cholinergic hypothesis,3 inhibition of AChE effectively
increases the available acetylcholine (ACh) within cholinergic
synapses, resulting in modest improvement in cognitive symp-
Figure 1. Catalytic and peripheral sites of AChE active site gorge.
toms. Mounting evidence has indicated that AChE may be
involved in several noncholinergic functions.4 AChE colocalizes
cognitive deficit of AD patients by elevating ACh levels but
also act as disease-modifying agents delaying amyloid plaque
with Aꢀ in senile plaques, promoting the assembly of Aꢀ into
fibrils5 and accelerating Aꢀ peptide deposition.6 Structural
models of the interaction between AChE and Aꢀ have recently
been explored.7 It has been speculated that AChE achieves its
aggregation-promoting action through direct binding with Aꢀ
via the specific region of the enzyme that involves a peripheral
binding site.8 Inhibition of the peripheral site might prevent Aꢀ
peptide deposition induced by AChE. This enzyme has a narrow
20 Å deep active site gorge, the bottom and opening regions of
which are known as catalytic and peripheral sites, respectively
(Figure 1). AChE inhibitors simultaneously blocking both the
catalytic and peripheral sites might not only alleviate the
formation.9a,13
Recently, bivalent ligand strategy has been utilized in the
design of dual binding site AChE inhibitors.9–13 Homobivalent
or heterobivalent ligands are obtained by connecting two
identical or distinct moieties through a linker of suitable length
to make contact with both the catalytic and peripheral sites. A
spatial 12 Å distance was determined by X-ray crystal diffraction
from Trp86 (mammalian numbering), the catalytic anionic site
center, to Trp286 (mammalian numbering), core of the periph-
eral site (Figure 1).14 In many cases of homobivalent ligands
(bis-ligands), AChE inhibitory potency and selectivity improved
relative to the monomer and additional inhibition of AChE-
induced Aꢀ aggregation was observed. Bis-tacrine 1 (Figure 2,
n ) 7)9 was the first bivalent AChE inhibitor reported on this
strategy, presenting a more than 1000-fold increase in AChE
inhibiting potency and a 10000-fold increase in AChE/butyryl-
cholinesterase (BChE) selectivity compared with tacrine.
Bis-galanthamine 2 (Figure 2, n ) 8),10 bis-5-amino-5,6,7,8-
tetrahydroquinolinone 3 (Figure 2, n ) 10 or 12),11 and bis-
* To whom correspondence should be addressed. For Z.Q.: phone, 86-
21-54237595; fax, 86-21-54237264; e-mail, zbqiu@shmu.edu.cn. For H.C.:
phone, 86-21-63846590, extension 776450; fax, 86-21-64674721; e-mail,
yaoli@shsmu.edu.cn.
†
Fudan University.
These authors contributed equally to this work.
Shanghai JiaoTong University.
East China University of Science and Technology.
#
‡
§
10.1021/jm070154q CCC: $40.75
2008 American Chemical Society
Published on Web 03/12/2008

2028 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Xie et al.
Figure 2. Structures of reported homobivalent AChE inhibitors and title compounds 5.
Scheme 1. Synthesis of (-)-nor-MEP 8a
a Reagents and conditions: (i) (a) ClCOOEt, KHCO3, CHCl3, reflux, 1 h; (b) K2CO3 (aq), MeOH, N2, room temp, 1 h, 95%; (ii) 50% H2SO4, N2, reflux,
4 h, 54%.
Scheme 2. Synthesis of 5a,b,e-ka
huperzine B 4 (Figure 2, m ) 2, n ) 10)12 (Figure 2) have also
been reported.
Our group has been interested in the study of meptazinol
(MEP),15 a racemic marketed opioid analgesic with low addic-
tion liability, and its (-)-enantiomer, which has demonstrated
moderate inhibition of AChE.16 We established an approach to
the resolution of MEP in acceptable yields and determined the
absolute configurations of (-)-MEP and (+)-MEP as S and R,
respectively, by X-ray crystal structures.17 Continuing with our
previous research to find new AChE inhibitors through the
molecular modeling of (-)-MEP derivatives,18 we describe here
the design, synthesis, pharmacological evaluation, and molecular
docking of a series of homobivalent (-)-N-demethylmeptazinols
(bis-(-)-nor-MEPs) 5. Two identical (-)-nor-MEP units are
connected by alkylene linkers of different lengths via the
secondary amino groups in compound 5 (Figure 2).
A suitable length of the alkylene linker, together with an
appropriate point of the coupling position, guarantees that bis-
ligand will simultaneously bind to the catalytic and peripheral
sites of the enzyme. According to our predicted binding mode
of (-)-MEP in AChE active site,18 the azepane ring is located
in the middle of the gorge whereas the phenolic group is oriented
down into the catalytic site at the bottom. Therefore, bivalent
(-)-MEP derivatives were designed as bis-(-)-nor-MEPs
linking, via a point in the azepane ring instead of the phenolic
group (Figure 1). As to the chain length (number of methylene
units), the optimal number in the case of tacrine-based bivalent
ligands was 7.9,19 However, that might not be the case in our
study. To find the most potent compound in our series and
discuss the effect of linker length on inhibitory potency,
compounds possessing methylene spacers varying from 2 to 12
were synthesized.
a Reagents and conditions: (i) R,ω-dihaloalkane (0.5 equiv), triethylamine
(2 equiv), acetonitrile, reflux, 2-5 h, 35-83%.
Newly synthesized compounds were tested in vitro for AChE
and BChE inhibitory potency, and their selectivity for AChE
was calculated. A molecular docking study was performed on
mouse AChE (mAChE) and human BChE (hBChE) to il-
luminate the binding modes of the most potent compound 5h
with both enzymes. Because of the unavailability of crystal-
lographic data of mouse BChE, hBChE was used instead
because of a high sequence identity. The ability of compounds
5g, 5h, and 5i to inhibit the AChE-induced Aꢀ aggregation,
compared with propidium iodine and the reference compound
(-)-MEP, was assessed by means of a thioflavin T-based
fluorometric assay.20 Cell viability was tested by MTT assay
for 5h and 5i.
Results and Discussion
Chemistry. The synthetic methodology employed for the
preparation of bis-(-)-nor-MEP derivatives 5 is illustrated in
Schemes 1-3. Key step in this route is the N-demethylation of
(-)-MEP 6 producing (-)-nor-MEP 8. A few methods have

Bis-(-)-nor-meptazinols as Inhibitors
Scheme 3. Synthesis of 5c,da
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2029
a Reagents and conditions: (i) R,ω-alkanediacyl dihalide (0.5 equiv), triethylamine (2 equiv), dry CH2Cl2, 0 °C, 15 min, 38-41%; (ii) lithium aluminum
hydride (LAH), dry THF, reflux, 1 h, 31-36%.
Table 1. Inhibition of AChE and BChE by Bis-(-)-nor-MEPs 5a-k,
been reported for the demethylation of tertiary amines, such as
(-)-MEP, and Rivastigminea
employment of azodicarboxylic acid esters,21 cyanogen bro-
IC50 (nM)
mide,22 or chloroformates.23,24 Ethyl chloroformate was chosen
chain
length (n)
mice brain
AChE
Mice serum selectivity for
as the reagent, taking both the availability and reaction simplicity
into account. Treating (-)-MEP 6 with ethyl chloroformate in
the presence of KHCO3 in boiling CHC13, followed by dealing
the resulting residue with a mild base, afforded a nonbasic
carbamate intermediate, (-)-N-carboethoxy nor-MEP 7. How-
ever, troubles were encountered in the hydrolysis and decar-
boxylation of the resulting carbamate intermediate 7. All
reactions failed under the reported alkaline condition in KOH24
and acidic conditions in hydrobromic acid or 25% sulfuric acid.
Finally this transformation was accomplished in 50% sulfuric
acid under nitrogen for 4 h, and (-)-nor-MEP 8 was obtained
in a 54% yield (Scheme 1).
compd
BChE
AChEb
5a
5b
5c
5d
5e
5f
2
3
4
5
6
43000 ( 20000
42000 ( 14000
21400 ( 7600
4000 ( 1000
1220 ( 20
270 ( 70
125 ( 9
132 ( 51
104 ( 29
192 ( 41
119 ( 20
102 ( 19
63 ( 8
0.0029
0.0031
0.0049
0.048
0.098
0.38
0.80
2.6
1.8
3.1
2.4
0.29
0.37
7
8
5g
79 ( 19
5 h
5i
5j
5k
9
3.9 ( 1.3
10 ( 3
17 ( 6
10
11
12
9.5 ( 4.5
24 ( 8
74 ( 11
100 ( 55
1600 ( 30
42 ( 20
rivastigmine
(-)-MEP
5500 ( 1500
41000 ( 14000 15000 ( 4000
Alkylation of (-)-nor-MEP 8 with R,ω-dihaloalkanes (0.5
equiv) in the presence of triethylamine, followed by chromato-
graphic purification, easily produced the bis-(-)-nor-MEP
compounds 5a,b,e-k in 35-83% yield (Scheme 2). However,
alkylation with 1,4-dichlorobutane or 1,5-dichloropentane failed
to furnish the bivalent compounds 5c,d, since the N-4-chlo-
robutyl (or 5-chloropentyl)-(-)-nor-MEP intermediate was prone
to forming stable intramolecular five-membered or six-
membered ring structures, resulting in failure to link another
(-)-nor-MEP unit and leading to the generation of spiro
quaternary ammoniums. Structures of two quaternary ammo-
niums derived from the R enantiomer were confirmed by X-ray
crystallographic diffraction.25 Eventually, the synthesis of the
bis-ligands 5c,d was accomplished by acylation with R,ω-
alkanediacyl dihalide (0.5 equiv) to form bis-amides intermedi-
ates 9c,d followed by reduction using lithium aluminum hydride
(LAH) in tetrahydrofuran (THF) (Scheme 3).
a Mice brain homogenate prepared in normal saline was used as a source
of AChE. Mice serum was the source of BChE. AChE was assayed
spectrophotometrically with acetylthiocholine as substrate in the presence
of 1024 M ethopropazine as BChE inhibitor. BChE was assayed similarly
with butyrylthiocholine as substrate and 1025 M BW284C51 as AChE
inhibitor. IC50 values were computed by a nonlinear least squares regression
program that also provided an estimate of statistical precision (standard
error of the mean). b Selectivity for AChE: IC50 for BChE divided by IC50
for AChE.
The chemical structures of all target compounds or their
synthesized hydrochloride salts were characterized by specific
1
rotation [R]D, IR, H NMR, and HR-ESI, as reported in the
Experimental Section. The complicated property of NMR data
from bis-(-)-nor-MEP hydrochloride salts in DMSO-d6 re-
sembled the case of (+)-MEP hydrochloride.26 It was reasonably
explained by conformational switch of the azepane ring and
configurational inversion of nitrogen.
AChE Inhibitory Potency and AChE/BChE Selectivity.
Newly synthesized compounds were tested in vitro for potency
and selectivity as cholinesterase (ChE) inhibitors. Extracts from
mice brain and mice serum were used as sources of AChE and
BChE, respectively. The results showed that both (-)-MEP and
its bis-ligand analogues possessed ChE inhibitory activity. The
IC50 value of (-)-MEP was 41 µM, about 10 times higher than
that obtained with AChE from bovine erythrocytes,16 and the
testing data of the reference drug rivastigmine conformed to
the previous report.27 The AChE inhibitory potency within the
series of bis-(-)-nor-MEP derivatives was closely related to
Figure 3. Correlation between ChE inhibitory potency (-log(IC50))
and the alkylene chain length (n) in compounds 5.
the length of the alkylene chain. The optimal chain length
determined experimentally was achieved in compound 5h, with
nine methylene groups between two (-)-nor-MEP units.
Compared with (-)-MEP (IC50 ) 41 µM) and rivastigmine (IC50
) 5.5 µM), 5h (IC50 ) 3.9 nM) showed a 10000-fold and 1400-
fold increase, respectively, in the inhibition of mice brain AChE
(Table 1). Further decrease or increase of the chain length
weakened the AChE inhibition (Figure 3). For instance, the

2030 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Xie et al.
inhibitory activities of 5a and 5b (43 and 42 µM, respectively)
are similar to that of (-)-MEP. Therefore, the effective alkylene-
bridged bis-(-)-nor-MEP analogues required chains of suitable
length, which was 9 for the best, to bind at both the catalytic
site and the peripheral site of the AChE binding pocket (gorge).
buried within the core of the enzyme binds with the catalytic
site via face-to-face π-stacking interaction with Trp86 (distance
between the two centroids: 4.27 Å). The other nor-MEP moiety
reaches the peripheral site on the surface of the enzyme by
cation-π and hydrophobic interactions between the seven-
membered azepane ring and Trp286 (distance between the two
centroids: 4.09 Å). The spatial distance between the centroids
of the centric phenyl group and the peripheral azepane ring is
13.5 Å, consistent with the reported distance between two
tryptophanes.14 5h forms two hydrogen bonds, both in the
catalytic active site. The hydroxyl is hydrogen-bonded to the
main-chain carbonyl oxygen of His447 (O · · ·O distance: 2.87
Å). Meanwhile, the protonated azepane amino group is hydrogen-
bonded to the hydroxyl oxygen of Tyr124 (N · · ·O distance: 3.22
Å). In addition, other aliphatic and aromatic residues are
involved in hydrophobic interactions, as shown in Figure 4b.
In comparison, the binding mode of 5h with the active site
of hBChE is shown in Figure 4c,d. 5h is folded in the large
cavity along the aliphatic residue-dominated wall, and the
separation of two terminal (-)-nor-MEP units is relatively short.
Three hydrogen bonds are found: (i) between the phenolic
hydroxyl within the core of the enzyme and the carboxylic acid
oxygen of Asp70 (O· · ·O distance, 2.49 Å); (ii) between the
phenolic hydroxyl at the entrance of the enzyme and the
carboxylic acid oxygen of Glu276 (O · · ·O distance, 2.61 Å);
and (iii) between the oxygen on the phenolic hydroxyl at the
entrance and the side chain amide NH of Gln119 (O · · ·N
distance, 3.22 Å). Ala 277 and Ala328 are not so importantly
involved in the hydrophobic interactions with 5h, unlike the
way their counterparts Try286 and Tyr337 in the mAChE
catalytic site behave. On the contrary, some residues unique to
hBChE, such as Gln119, Leu286, and Val288, form hydrophobic
contacts with 5h.
The difference in the binding mode as well as in pharmaco-
logical activities between 5h with AChE and BChE is funda-
mentally caused by conformational differences between the two
enzymes. One of the most important differences is the lack of
peripheral site in BChE. Residues responsible for π-π or
cation-π interactions at AChE peripheral site are replaced by
aliphatic residues in BChE. For example, Trp286, Tyr72, and
Tyr124 in mAChE are the counterparts of Ala277, Asn68, and
Gln119 in hBChE, respectively. In addition, some bulky
aromatic residues in AChE active site have been replaced by
small aliphatic ones in BChE. As a result, the active site of
BChE is wider and able to accommodate bis-ligands with linkers
of wider-ranging lengths. These might be the main reasons that
the BChE inhibition is less sensitive to the linker length.
Compared with the AChE activity, the BChE inhibitory
potency was less impacted by chain length (Figure 3). The
majority of bis-(-)-MEP analogues showed inhibition on BChE
of about 100 nM, although the highest potency (IC50 ) 10 nM)
was achieved in 5h. This isomer was 1500 times and 150 times
more potent than (-)-MEP (IC50 ) 15 µM) and rivastigmine
(IC50 ) 1.6 µM), respectively, but only 10 times lower than
that of the majority compounds (Table 1). The reason that the
BChE inhibition is less sensitive to the linker length than the
AChE inhibition seems to be the enzymic conformational
difference. There is lack of a functional peripheral site in
BChE,28,29 and the BChE active site is wider throughout.
Therefore, there is no restriction of linker length for bivalent
BChE inhibitors.
Most bis-ligand analogues showed greater selectivity for
BChE because of their low affinity for AChE. Only four
compounds, 5h, 5i, 5j, and 5k, demonstrated slightly more
selectivity for AChE. Recent evidence suggests that both AChE
and BChE may play roles in the etiology and progression of
AD beyond regulation of synaptic ACh levels. In the AD brain,
the activity of AChE decreases progressively in certain regions
to reach 10–15% of normal values, whereas the activity of BChE
stays unchanged or is even increased by 20%.30 Thus, it may
not be an advantage for a ChE inhibitor to be considerably more
selective for AChE; on the contrary, a good balance between
AChE and BChE may result in higher efficacy. As a dual
inhibitor of both AChE and BChE, rivastigmine appears to be
beneficial for people with mild to moderate AD, and BChE
inhibition correlates significantly with cognitive improvement
in these patients.31 In our study, although 5h is slightly more
selective for AChE than BChE (2.6), it had the greatest
inhibition at a nanomolar lever on both enzymes. Therefore, it
was suggested that it was a promising drug candidate worthy
of further investigations.
Molecular Docking Studies. Molecular docking study was
performed to ascertain the possibility for the most potent
compound 5h to bind at both the catalytic and peripheral sites
of AChE and to explore the difference in the interactions of 5h
with AChE and BChE. Mammalian enzymes were used in
docking, compatible with the pharmacological test. Because of
the unavailability of crystallographic data of mouse BChE,
hBChE was used instead because there is a high sequence
identity (82%) especially between mouse and human BChE and
the residues in active sites are highly conservative. Here, we
chose recently resolved X-ray crystal structures of the mAChE
complex with succinylcholine32 with a high resolution of 2.05
Å and of native hBChE28 with a 2.0 Å resolution. GOLD33
docking protocol was employed because it has been proved by
our previous study18 to be accurate and reliable for reproducing
the binding modes of seven AChE inhibitors in their X-ray
crystal structures of Torpedo californica AChE (TcAChE)
complexes. An advanced consensus scoring technology was used
to guide the selection of the most reliable conformation from a
set of candidate conformations that GOLD generated.
Inhibition of AChE-Induced Aꢀ Aggregation. Three com-
pounds, 5g, 5h, and 5i, were selected to assess their abilities to
inhibit Aꢀ aggregation induced by AChE using a thioflavin
T-based fluorometric assay,20 compared with the reference
compound propidium iodine (Sigma-Aldrich), a known specific
peripheral site-binding inhibitor, and the monomer (-)-MEP
(Table 2). Results showed that 5h and 5i markedly prevented
the AChE-induced Aꢀ aggregation with IC50 values of 16.6 and
5.8 µM, similar to that of propidium (IC50 ) 12.8 µM). With a
small IC50 value and a higher efficiency of inhibition, 5i and
5h were the wonderful compounds that inhibited the aggregation
of Aꢀ induced by AChE. In contrast, (-)-MEP and 5g showed
fairly low inhibitory activity (Table 2), which indicated their
limited ability to interact with the peripheral site of the enzyme.
Our results show that 5h is able to simultaneously make
contact with both the catalytic and peripheral sites of mAChE,
as illustrated in Figure 4a. The key interactions of these dimeric
inhibitors with the catalytic and peripheral sites are π-stacking
and cation-π interactions. The phenyl group of the nor-MEP
Different behaviors of these four compounds were attributed
to different lengths of linker, demonstrating that a linker no
shorter than 9 was necessary to inhibit AChE-induced Aꢀ

Bis-(-)-nor-meptazinols as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2031
Figure 4. Representation of 5h (C atoms colored green) docked into the binding sites of mAChE (a) and hBChE (c). The binding site surfaces are
colored according to the vacuum electrostatics protein contact potential, calculated by PyMOL 0.99rc2 (DeLano Scientific LLC, San Carlos, CA).
Crucial catalytic and peripheral site (or the counterpart) residues are colored yellow. Hydrogen bonds and hydrophobic contacts between 5h and the
protein residues of mAChE (b) and hBChE (d) are shown by LigPlot 4.4.2.41
aggregation. These findings agree with the results from enzy-
matic test and molecular docking, which indicated that a linker
of 9 or 10 methylenes would help to reach the peripheral site
of AChE.
Cell Viability. The toxicity of the most potent two bis-(-)-
nor-MEPs was determined in human neuroblastoma cell line
SH-SY5Y. Cell viability was not affected for 5h and 5i at
concentrations of 1–100 µM (higher than the IC50 values of 5h
and 5i against ꢀ-amyloid aggregation inhibition (around 80 µM)
and 10000 times higher than their AChE inhibiting IC50 values).
(-)-nor-MEPs 5. Their AChE inhibitory activities were closely
related to the length of the alkylene chain, whereas BChE
inhibition was less influenced. The optimal chain length for
AChE and BChE inhibition was achieved with 9 in 5h, which
showed a 10000-fold and 1500-fold increase in the inhibition
of mice brain AChE and mice serum BChE, respectively,
compared with (-)-MEP. Molecular docking elucidated that 5h
simultaneously bound to the catalytic and peripheral sites via
hydrophobic interactions with Trp86 and Trp286 in mAChE.
In comparison, it folded in the large aliphatic cavity of hBChE.
The differences were explained by the absence of peripheral
site and the enlargement of the active site gorge in BChE.
Furthermore, 5h and 5i markedly prevented the AChE-induced
Conclusion
We have discovered novel nanomolar ChE inhibitors with
high inhibitory potency on Aꢀ aggregation, i.e., a series of bis-

2032 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Xie et al.
Table 2. Inhibition of AChE-Induced Aꢀ Aggregation by
Bis-(-)-nor-MEPs 5g-i and Reference Compounds
elution with EtOH/CHCl3 (0.8:9.2 to 3:7). The eluent was concen-
trated in vacuo to afford 8 (4.58 g, 54%). Salt forming reaction of
8 (1.07 g) was carried out in dry ether by adding dry HCl-ether
and adjusting the pH to 4, which afforded 8·HCl as a white powder
(0.98 g, 79%): mp 73-75 °C; [R]D -7.10° (c 0.286, MeOH); IR
ν 3257, 2932, 1614, 1588, 1481, 1432, 1321, 1276, 1237, 1211
chain
inhibition (%) at inhibition (%) at
compd
length (n)
100 µM
200 µM
IC50 (µM)
12.8 ( 0.4
propidium
iodine
(-)-MEP
5g
5h
5i
85.6 ( 4.4
98.6 ( 5.9
1
cm-1; H NMR (DMSO-d6) 9.42 (s, H, Ar-OH), 8.82 (br s, 1/2
nda
0
90.8 ( 0.2
95.8 ( 0.5
0
nda
H, NH+), 8.24 (br s, 1/2 H, NH+), 7.16 (t, H, J ) 7.8 Hz, ArH),
6.74–6.65 (m, 3H, ArH), 3.49 (d, H, J ) 14.1 Hz, N-CH2), 3.21
(d, H, J ) 14.5 Hz, N-CH2), 3.08–3.00 (m, 2H, N-CH2), 2.14
(m, H, CH2), 1.77–1.55 (m, 7H, CH2), 0.49 (t, 3H, J ) 7.4 Hz,
CH3); MS (ESI) [M + H]+ 220.1. HPLC: tR ) 1.86 min, 98.5%
purity.
8
9
10
15.2 ( 0.2
99.2 ( 0.1
98.4 ( 0.1
nda
16.6 ( 0.5
5.8 ( 0.3
a nd: not determined.
Aꢀ aggregation with IC50 values of 16.6 and 5.8 µM, compatible
with that of propidium (IC50 ) 12.8 µM), which pointed out a
promising disease-modifying action. Further pharmacological
study is needed to evaluate their abilities to reverse memory
impairment in animal models in order to select ideal candidates
for the treatment of AD patients.
General Procedure for the Synthesis of Bis-(-)-Nor-MEP
Compounds 5a,b,e-k. Triethylamine (2 equiv) and R,ω-dihaloal-
kane (0.5 equiv) were added to a solution of (-)-nor-MEP 8 in
acetonitrile. The reaction mixture was refluxed for 2-5 h. Evapora-
tion of the solvent gave a residue, which was diluted with saturated
K2CO3 solution and extracted with CHCl3. The combined CHCl3
extracts were dried (anhydrous Na2SO4) and evaporated under
reduced pressure. The residue was purified by chromatography on
silica gel. Eluting with petroleum ether/EtOAc (1:2) afforded the
corresponding bis-(-)-nor-MEP compounds 5 as a yellow oil.
Addition of dry HCl-ether to a solution of 5 in dry ether and
adjusting the pH to 3-4 gave the final salt 5·2HCl as powder.
N,N′-(1′,2′-Ethylene)-bis-(-)-nor-MEP Hydrochloride (5a ·
2HCl). (-)-nor-MEP 8 (1.50 g, 6.85 mmol), acetonitrile (15 mL),
triethylamine (1.9 mL, 13.7 mmol), and 1,2-dibromoethane (0.297
mL, 3.43 mmol) were used to produce 5a (0.80 g, 50%). Subsequent
salt formation gave 5a·2HCl (0.80 g, 86%): mp 142-145 °C; [R]D
Experimental Section
Chemistry. Melting points were taken in glass capillary tubes
and were uncorrected. Specific rotation ([R]D) was determined on
a JASCOP-1020 rotatory apparatus. IR data were taken on an
AVATAR 360 FT-IR spectrometer (KBr). NMR data were recorded
with a Mercury Plus 400 instrument. Chemical shifts (δ) are
expressed in parts per million (ppm) relative to tetramethylsilane
(TMS) as an internal standard. All signals of active hydrogen
disappeared after D2O exchange. Mass spectra were measured on
an Agilent 1100 series LC/MSD 1946D spectrometer. HRMS
spectra were recorded with an IonSpec 4.7 T FTMS instrument.
The purity of all target compounds (>95%) was verified via HPLC.
The elution was methanol-0.05 mol/L ammonium acetate solution
(adjustment of pH to 7.4 with aqueous ammonia) (70:30 to 80:20).
The chromatographic condition was a flow rate of 1.0 mL/min with
UV detection at 225 nm on a VP-ODS C18 (150 mm × 4.6 mm,
5 µm) column at a temperature of 50 °C. All reagents were of
commercial quality. Rivastigmine hydrochloride standard was
available from Sunve (Shanghai) Pharmaceutical Co., Ltd.
(-)-N-Carboethoxy-nor-MEP (7). A stirred suspension of (-)-
MEP 6 (9.83 g, 42.2 mmol) and KHCO3 (74 g, 740 mmol) in
boiling CHCl3 (500 mL) was treated with ethyl chloroformate (30.5
mL, 320 mmol) and refluxed for 1 h. H2O (350 mL) was added,
and the CHCl3 phase was separated and concentrated in vacuo. The
residue was dissolved in MeOH (400 mL), treated with an aqueous
solution (400 mL) containing 66 g of K2CO3, and stirred under N2
at room temperature for 1 h. After the MeOH was removed, the
residue was neutralized with 6 M HCl (126 mL) and extracted with
Et2O (150 mL × 3). The combined Et2O extracts were washed
with saturated NaCl solution (150 mL) and dried with anhydrous
Na2SO4. Evaporation of the solvent under reduced pressure gave 7
(11.17 g, 95%) as a yellowish oil. Recrystallization of 7 (1.2 g)
from ethyl acetate (2 mL) afforded 7 as off-white crystals (0.64 g,
53%): mp 79-81 °C; [R]D -61.35° (c 0.11, MeOH); 1H NMR
(CDCl3) 7.18 (t, H, J ) 7.8 Hz, ArH), 6.87–6.81 (m, 2H, ArH),
6.70 (d, H, J ) 8.0 Hz, ArH), 6.00 (br s, 1/2 H, Ar-OH), 5.57 (br
s, 1/2 H, Ar-OH), 4.17–4.03 (m, 2H, -OCH2), 3.94 (d, H, N-CH2,
J ) 14.6 Hz), 3.90–3.85 (m, 1/2 H, N-CH2CH3), 3.79–3.73 (m,
1/2 H, N-CH2), 3.52 (d, 1/2H, J ) 14.4 Hz, N-CH2), 3.34 (d,
1/2 H, J ) 14.9 Hz, N-CH2), 3.06–2.95 (m, H, N-CH2), 2.20–2.05
(m, H, CH2), 1.77–1.58 (m, 7H, CH2), 1.24 (m, 3H, -OCH2CH3),
0.56 (t, 3H, J ) 7.3 Hz, CH3); MS (ESI) [M + H]+ 292.2, [M +
Na]+ 314.2, [M + K]+ 330.2.
-1.96° (c 0.204, MeOH); IR ν 3176, 2935, 1599, 1447, 1229 cm-1
;
1H NMR (DMSO-d6) 10.84 (br s, 1/2 H, NH+), 10.58 (br s, H,
NH+), 9.51, 9.49, 9.43, 9.34(s, 2H, Ar-OH), 9.09 (br s, 1/2 H,
NH+), 7.22–7.08 (m, 2H, Ar-H), 6.83–6.61 (m, 6H, Ar-H),
3.79–3.71 (m, H, N-CH2), 3.47–3.42 (m, H, N-CH2), 3.14–3.10
(m, 2H, N-CH2), 3.03–2.86 (m, 6H, N-CH2), 2.71–2.69 (m, 2H,
N-CH2), 2.14–2.08 (m, 2H, CH2), 1.84–1.46 (m, 14H, CH2), 0.47
(m, 6H, CH3); MS (ESI) [M + H]+ 465.6. HRMS m/z calcd for
C30H45N2O2 [M + H]+, 465.3476; found, 465.3463. HPLC: tR )
4.48 min, 98.2% purity.
N,N′-(1′,3′-Propylene)-bis-(-)-nor-MEP Hydrochloride (5b ·
2HCl). (-)-nor-MEP 8 (1.54 g, 7.03 mmol), acetonitrile (15 mL),
triethylamine (2.0 mL, 14.4 mmol), and 1,3-dibromopropane (0.357
mL, 3.52 mmol) were used to produce 5b (1.20 g, 71%). Subsequent
salt formation of 5b (0.90 g) gave 5b·2HCl (0.90 g, 87%): mp
165-168 °C; [R]D -48.38° (c 0.228, MeOH); IR ν 3176, 2935,
1
1599, 1447, 1229 cm-1; H NMR (DMSO-d6) 10.18, 9.97 (br s,
6/5 H, NH+), 9.58, 9.52, 9.44, 9.42 (s, 2H, Ar-OH), 8.68, 8.59
(br s, 4/5 H, NH+), 7.20–7.13 (m, 2H, Ar-H), 6.90–6.65 (m, 6H,
Ar-H), 3.92 (d, 4/5 H, J ) 14.5 Hz, N-CH2), 3.56 (m, 6/5 H,
N-CH2), 3.44–3.21 (m, 10H, N-CH2), 2.40 (m, 2H, CH2),
2.10–1.50 (m, 16H, CH2), 0.50 (m, 6H, CH3); MS (ESI) [M +
H]+ 479.4, [M + 2H]2+ 240.2. HRMS m/z calcd for C31H47N2O2
[M + H]+, 479.3632; found, 479.3641. HPLC: tR ) 7.00 min,
99.1% purity.
N,N′-(1′,6′-Hexylene)-bis-(-)-nor-MEP Hydrochloride (5e ·
2HCl). (-)-nor-MEP 8 (1.96 g, 8.95 mmol), acetonitrile (20 mL),
triethylamine (2.5 mL, 18.0 mmol), and 1,6-dibromohexane (0.702
mL, 4.48 mmol) were used to produce 5e (0.96 g, 41%). Subsequent
salt formation gave 5e·2HCl (1.06 g, 97%): mp 135-138 °C; [R]D
-47.8° (c 0.175, MeOH); IR ν 3414, 3176, 2938, 2876, 2732, 1600,
1
1585, 1447, 1269, 1232 cm–1; H NMR (DMSO-d6) 10.05 (br s,
1/2 H, NH+), 9.80 (br s, 3/4 H, NH+), 9.56, 9.53, 9.44, 9.42 (s,
2H, Ar-OH), 8.43, 8.33 (br s, 3/4 H, NH+), 7.22–7.13 (m, 2H,
Ar-H), 6.87–6.76 (m, 4H, Ar-H), 6.71–6.66 (m, 2H, Ar-H), 3.84
(m, 3/4 H, N-CH2), 3.55 (m, 5/4 H, N-CH2), 3.34–3.09 (m, 10H,
N-CH2), 2.38 (m, H, CH2), 2.12–1.73 (m, 15H, CH2), 1.57–1.44
(m, 4H, CH2), 1.35–1.25 (m, 4H, CH2), 0.50 (t, 6H, J ) 7.4 Hz,
CH3); MS (ESI) [M + H]+ 521.7, [M + 2H]2+ 261.4. HRMS m/z
calcd for C34H53N2O2 [M + H]+, 521.4102; found, 521.4086.
HPLC: tR ) 8.10 min, 97.6% purity.
(-)-Nor-MEP Hydrochloride (8 ·HCl). A mixture of 7 (11.17
g, 38.38 mmol) in 50% H2SO4 (120 mL) was refluxed under N2
for 4 h. The solution was treated with aqueous ammonia (180 mL),
adjusted to pH 9, and extracted with CHCl3 (200 mL × 3). The
combined CHCl3 extracts were washed with saturated NaCl solution
(200 mL) and dried with anhydrous Na2SO4. The solvent was
removed in vacuo, and the oily residue underwent chromatography
on a column of 170 g of silica gel (200-300 mesh) and gradient

Bis-(-)-nor-meptazinols as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2033
N,N′-(1′,7′-Heptylene)-bis-(-)-nor-MEP Hydrochloride (5f·
2HCl). (-)-nor-MEP 8 (1.70 g, 7.76 mmol), acetonitrile (20 mL),
triethylamine (2.2 mL, 15.8 mmol), and 1,7-dibromoheptane (0.674
mL, 3.88 mmol) were used to produce 5f (0.73 g, 35%). Subsequent
salt formation gave 5f·2HCl (0.70 g, 84%): mp 125-128 °C; [R]D
-44.29° (c 0.218, MeOH); IR ν 3417, 3186, 2938, 2866, 2740,
Subsequent salt formation gave 5j·2HCl (0.47 g, 84%): mp
108-112 °C; [R]D -38.53° (c 0.29, MeOH); IR ν 3423, 3180, 2927,
1
2856, 1600, 1585, 1446, 1267, 1232 cm-1; H NMR (DMSO-d6)
10.13, 10.02 (br s, 7/5 H, NH+), 9.58, 9.55, 9.47, 9.45 (s, 2H,
Ar-OH), 8.41, 8.37 (br s, 3/5 H, NH+), 7.18–7.10 (m, 2H, Ar-H),
6.84–6.64 (m, 6H, Ar-H), 3.81 (d, 3/5 H, J ) 14.1 Hz, N-CH2),
3.52 (d, 7/5 H, J ) 13.7 Hz, N-CH2), 3.42–3.26 (m, 3H, N-CH2),
3.14–3.03 (m, 7H, N-CH2), 2.37–2.32 (m, H, CH2), 2.10–1.97 (m,
3H, CH2), 1.82–1.70 (m, 12H, CH2), 1.54–1.37 (m, 4H, CH2),
1.27–1.24(m, 14H, CH2), 0.47 (t, 6H, J ) 7.4 Hz, CH3); MS (ESI)
[M + H]+ 591.3. HRMS m/z calcd for C39H63N2O2 [M + H]+,
591.4884; found, 591.4901. HPLC: tR ) 15.30 min, 98.9% purity.
N,N′-(1′,12′-Dodecydene)-bis-(-)-nor-MEP Hydrochloride
(5k·2HCl). (-)-nor-MEP 8 (0.69 g, 3.15 mmol), acetonitrile (10
mL), triethylamine (0.9 mL, 6.47 mmol), and 1,12-dibromodode-
cane (0.53 mL, 1.58 mmol) were used to produce 5k (0.34 g, 36%).
Salt formation of 5k (0.58 g) gave 5k·2HCl (0.49 g, 80%): mp
100-105 °C; [R]D -35.77° (c 0.30, MeOH); IR ν 3419, 2927, 2855,
1600, 1585, 1446, 1269; 1H NMR (DMSO-d6) 10.00 (br s, 4/3 H,
NH+), 9.49, 9.48, 9.40 (s, 2H, Ar-OH), 8.23 (br s, 2/3 H, NH+),
7.19–7.11 (m, 2H, Ar-H), 6.85–6.64 (m, 6H, Ar-H), 3.82 (d, 4/5
H, J ) 13.7 Hz, N-CH2), 3.53 (d, 6/5 H, J ) 13.3 Hz, N-CH2),
3.37–3.30 (m, 3H, N-CH2), 3.15–3.05 (m, 7H, N-CH2), 2.36–2.31
(m, H, CH2), 2.10 (m, H, CH2), 1.81–1.70 (m, 14H, CH2), 1.52–1.42
(m, 4H, CH2), 1.27–1.24 (m, 16H, CH2), 0.48 (m, 6H, CH3); MS
(ESI) [M + H]+ 605.3. HRMS m/z calcd for C40H65N2O2 [M +
H]+, 605.5041; found, 605.5054. HPLC: tR ) 19.73 min, 96.1%
purity.
General Procedure for the Synthesis of Intermediate Am-
ide Compounds 9c,d. Triethylamine (2 equiv) was added to a
solution of (-)-nor-MEP 8 in dry CH2Cl2. Then R,ω-alkanediacyl
dihalide (0.5 equiv) in dry CH2Cl2 was added dropwise at 0 °C.
The mixture was stirred for 15 min at 0 °C. The mixture was washed
with H2O (5 mL), 2 M HCl (8 mL), and then H2O (5 mL). The
combined water layers were back-extracted with CH2Cl2 (10 mL
× 3). All the CH2Cl2 layers were combined and dried with
anhydrous Na2SO4. Evaporation of the solvent under reduced
pressure gave a yellowish foam. Purification by chromatography
on silica gel eluted with petroleum ether/EtOAc (1:3) afforded the
amide intermediates 9c,d as a white foam.
1
1600, 1585, 1447, 1268, 1232 cm-1; H NMR (DMSO-d6) 9.70
(br s, 2/3 H, NH+), 9.52, 9.50, 9.42, 9.41 (s, 2H, Ar-OH), 8.25
(br s, 4/3 H, NH+), 7.22–7.14 (m, 2H, Ar-H), 6.87–6.66 (m, 6H,
Ar-H), 3.85 (d, 4/5 H, J ) 14.1 Hz, N-CH2), 3.56 (d, 6/5 H, J )
14.1 Hz, N-CH2), 3.39–3.01 (m, 10H, N-CH2), 2.40 (m, 6/5 H,
CH2), 2.13 (m, 4/5 H, CH2), 2.00–1.73 (m, 14H, CH2), 1.53–1.45
(m, 4H, CH2), 1.30 (m, 6H, CH2), 0.50 (m, 6H, CH3); MS (ESI)
[M + H]+ 535.8, [M + 2H]2+ 268.4. HRMS m/z calcd for
C35H55N2O2 [M + H]+, 535.4258; found, 535.4258. HPLC: tR )
9.67 min, 99.4% purity.
N,N′-(1′,8′-Octylene)-bis-(-)-nor-MEP Hydrochloride (5g ·
2HCl). (-)-nor-MEP 8 (0.94 g, 4.29 mmol), acetonitrile (10 mL),
triethylamine (1.2 mL, 8.63 mmol), and 1,8-dibromooctane (0.406
mL, 2.15 mmol) were used to produce 5g (0.43 g, 37%). Subsequent
salt formation of 5g (0.31 g) gave 5g·2HCl (0.29 g, 83%): mp
101-106 °C; [R]D -37.8 ° (c 0.099, MeOH); IR ν 3420, 2936,
1
1600, 1447, 1269 cm-1; H NMR (DMSO-d6) 10.11, 9.95 (br s,
4/3 H, NH+), 9.57, 9.54, 9.45, 9.43 (s, 2H, Ar-OH), 8.43, 8.35
(br s, 2/3 H, NH+), 7.19–7.11 (m, 2H, Ar-H), 6.85–6.64 (m, 6H,
Ar-H), 3.81 (d, 2/3 H, J ) 14.1 Hz, N-CH2), 3.53 (d, 4/3 H, J )
13.7 Hz, N-CH2), 3.35–3.28 (m, 3H, N-CH2), 3.15–3.05 (m, 7H,
N-CH2), 2.38–2.33 (m, H, CH2), 2.11–1.96 (m, 3H, CH2),
1.80–1.70 (m, 12H, CH2), 1.51–1.41 (m, 4H, CH2), 1.28–1.24 (m,
8H, CH2), 0.47 (t, 6H, J ) 7.4 Hz, CH3); MS (ESI) [M + H]+
549.4, [M + 2H]2+ 275.2. HRMS m/z calcd for C36H57N2O2 [M +
H]+, 549.4415; found, 549.4428. HPLC: tR ) 12.25 min, 97.9%
purity.
N,N′-(1′,9′-Nonylene)-bis-(-)-nor-MEP Hydrochloride (5h ·
2HCl). (-)-nor-MEP 8 (0.89 g, 4.06 mmol), acetonitrile (10 mL),
triethylamine (1.2 mL, 8.62 mmol), and 1,9-dibromononane (0.423
mL, 2.03 mmol) were used to produce 5h (0.71 g, 62%). Subsequent
salt formation of 5h (0.67 g) gave 5h·2HCl (0.62 g, 82%): mp
118-124 °C; [R]D -39.13° (c 0.32, MeOH); IR ν 3423, 2934, 1600,
1
1585, 1446, 1268 cm-1; H NMR (DMSO-d6) 10.10, 9.95 (br s,
4/3 H, NH+), 9.56, 9.54, 9.44 (s, 2H, Ar-OH), 8.41, 8.34 (br s,
2/3 H, NH+), 7.19–7.11 (m, 2H, Ar-H), 6.84–6.64 (m, 6H, Ar-H),
3.82 (d, 2/3 H, J ) 14.1 Hz, N-CH2), 3.53 (d, 4/3 H, J ) 13.7
Hz, N-CH2), 3.38–3.27 (m, 3H, N-CH2), 3.15–3.04 (m, 7H,
N-CH2), 2.38–2.32 (m, H, CH2), 2.10–2.01 (m, 3H, CH2),
1.79–1.70 (m, 12H, CH2), 1.54–1.41 (m, 4H, CH2), 1.29–1.27 (m,
10H, CH2), 0.47 (t, 6H, J ) 7.4 Hz, CH3); MS (ESI) [M + H]+
563.5, [M + 2H]2+ 282.3. HRMS m/z calcd for C37H59N2O2 [M +
H]+, 563.4571; found, 563.4553. HPLC: tR ) 15.98 min, 98.4%
purity.
N,N′-(1′,10′-Decylene)-bis-(-)-nor-MEP Hydrochloride (5i ·
2HCl). (-)-nor-MEP 8 (0.83 g, 3.78 mmol), acetonitrile (10 mL),
triethylamine (1.1 mL, 7.91 mmol), and 1,10-dibromodecane (0.438
mL, 1.89 mmol) were used to produce 5i (0.51 g, 47%). Subsequent
salt formation of 5i (0.47 g) gave 5i·2HCl (0.45 g, 85%): mp
104-108 °C; [R]D -38.43° (c 0.27, MeOH); IR ν 3417, 3176, 2932,
2856, 2733, 1600, 1585, 1447, 1269, 1230 cm-1; 1H NMR (DMSO-
d6) 10.09, 9.94 (br s, 4/3 H, NH+), 9.57, 9.54, 9.46, 9.45 (s, 2H,
Ar-OH), 8.41, 8.34 (br s, 2/3H, NH+), 7.18–7.11 (m, 2H, Ar-H),
6.84–6.64 (m, 6H, Ar-H), 3.82 (d, 2/3 H, J ) 14.5 Hz, N-CH2),
3.52 (d, 4/3 H, J ) 14.1 Hz, N-CH2), 3.36–3.27 (m, 3H, N-CH2),
3.14–3.03 (m, 7H, N-CH2), 2.38–2.32 (m, H, CH2), 2.13–1.95 (m,
3H, CH2), 1.79–1.70 (m, 12H, CH2), 1.54–1.41 (m, 4H, CH2),
1.28–1.25 (m, 12H, CH2), 0.47 (t, 6H, J ) 7.0 Hz, CH3); MS (ESI)
[M + H]+ 577.5, [M + 2H]2+ 289.3. HRMS m/z calcd for
C38H61N2O2 [M + H]+, 577.4728; found, 577.4747. HPLC: tR )
22.13 min, 98.9% purity.
N,N′-(1′,4′-Succinyl)-bis-(-)-nor-MEP (9c). (-)-nor-MEP 8
(1.45 g, 6.63 mmol), CH2Cl2 (35 mL), triethylamine (1.84 mL, 12.2
mmol), and succinyl chloride (0.382 mL, 3.30 mmol) were used to
produce 9c (0.73 g, 41%). IR ν 3170, 2966, 2936, 1737, 1614,
1
1596, 1455, 1427, 1266, 1238, 1202 cm-1; H NMR (DMSO-d6)
8.79 (s, 2H, Ar-OH), 7.19 (t, 2H, Ar-H), 6.78–6.70 (m, 6H,
Ar-H), 4.88 (d, 2H, J ) 14.7 Hz, N-CH2), 3.59 (dd, 2H, J1 )
11.7 Hz, J2 ) 7.0 Hz, N-CH2), 3.08 (d, 2H, J ) 15.0 Hz, N-CH2),
2.91 (t, 2H, J ) 11.7 Hz, CH2), 2.83 (d, 2H, J ) 13.6 Hz, N-CH2),
2.39 (dm, 2H, J ) 7.7 Hz, N-CH2), 2.33 (d, 2H, J ) 13.2 Hz,
N-CH2), 1.82–1.48 (m, 14H, CH2), 0.68 (t, 6H, J ) 7.3 Hz, CH3);
MS (ESI) [M + H]+ 521.3.
N,N′-(1′,5′-Glutaryl)-bis-(-)-nor-MEP (9d). (-)-nor-MEP 8
(1.59 g, 7.26 mmol), CH2Cl2 (35 mL), triethylamine (2.02 mL, 14.4
mmol), and glutaryl chloride (0.481 mL, 3.63 mmol) were used to
produce 9d (0.74 g, 38%). IR ν 3383, 2933, 2877, 1616, 1583,
1443, 1264 cm-1; 1H NMR (CDCl3) 8.54, 8.10, 7.77, 7.52 (brs ×
4, 2H, Ar-OH), 7.17 (t, 2H, Ar-H), 7.05–6.93 (m, 2H, Ar-H),
6.82–6.70 (m, 4H, Ar-H), 4.53 (d, H, J ) 14.9 Hz, N-CH2),
3.75–3.49 (m, 3H, N-CH2), 3.41–3.18 (m, 2H, N-CH2), 2.99–2.94
(m, H, N-CH2), 2.54–2.46 (m, H, N-CH2), 2.34–2.17 (m, 4H,
CO-CH2), 1.93–1.50 (m, 18H, CH2), 0.62–0.51 (m, 6H, CH3); MS
(ESI) [M + H]+ 535.3.
General Procedure for the Synthesis of Bis-(-)-nor-MEP
Compounds 5c,d. A solution of 9c,d in dry THF was added
dropwise to lithium aluminum hydride (5 equiv) in dry THF at
room temperature. The mixture was refluxed for 1 h, and then H2O,
15% NaOH, and H2O were added. The mixture was stirred for 15
min at room temperature. The mixture was filtered, and the solid
material was washed with THF. The combined THF solution was
evaporated to remove solvents. The residue was treated with H2O
N,N′-(1′,11′-Undecydene)-bis-(-)-nor-MEP Hydrochloride
(5j·2HCl). (-)-nor-MEP 8 (0.78 g, 3.56 mmol), acetonitrile (10
mL), triethylamine (1.0 mL, 7.20 mmol), and 1,11-dibromounde-
cane (0.418 mL, 1.78 mmol) were used to produce 5j (0.50 g, 48%).

2034 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Xie et al.
(15 mL), drops of aqueous ammonia were added to adjust the pH
to 9, and the residue was extracted with CHCl3 (20 mL × 3).
The combined CHCl3 was dried with anhydrous Na2SO4 and
concentrated in vacuo to give a residue, which was chromato-
graphed on silica gel and eluted with MeOH/CHCl3 (0.5:9.5) to
provide 5c,d as an oil. Addition of dry HCl-ether to the solution
of 5c,d in dry ether, with pH adjusted to 3-4, gave the final
salt 5c,d · 2HCl as a powder.
ligand. The final conformation of the ligand was obtained after
1000 steps of energy minimization using the Tripos force field.
Molecular docking was carried out using GOLD 3.0 (CCDC,
Cambridge, U.K., 2005)33 to generate an ensemble of docked
conformations for the ligand. The active site was defined as all
atoms within a radius of 25 Å around some specific residue atoms:
OH of Tyr337 for mAChE and Nꢀ2 of His438 for hBChE. We chose
more enlarged binding pockets here, concerned that smaller sites
might neither accommodate a large bis-ligand nor include both the
catalytic and peripheral sites of AChE. The 600 genetic algorithm
(GA) runs instead of the default 10 were performed owing to the
high flexibility of the ligand possessing a great many rotatable
bonds. For each GA run, the default GA settings were used except
for the prohibited early termination and the permitted pyramidal
nitrogen inversion.
N,N′-(1′,4′-Butylene)-bis-(-)-nor-MEP Hydrochloride (5c ·
2HCl). Compound 9c (0.56 g, 1.08 mmol), lithium aluminum
hydride (0.20 g, 5.26 mmol), THF (30 mL), H2O (0.20 mL), 15%
NaOH (0.20 mL), and H2O (0.60 mL) were used to produce 5c
(0.19 g, 36%). Subsequent salt formation gave 5c · 2HCl (0.12 g,
55%): mp 110-115 °C; [R]D -51.96° (c 0.092, MeOH); IR ν
1
3254, 2936, 1600, 1586, 1448, 1268 cm-1; H NMR (DMSO-
d6) 9.98, 9.77 (br s, 4/3 H, NH+), 9.56, 9.54, 9.43, 9.42 (s, 2H,
Ar-OH), 8.46 (br s, 2/3 H, NH+), 7.21–7.13 (m, 2H, Ar-H),
6.85–6.65 (m, 6H, Ar-H), 3.83 (m, ∼2/3 H, N-CH2), 3.52 (m,
∼4/3 H, N-CH2), 3.36–3.15 (m, 10H, N-CH2), 2.38 (m, H,
CH2), 2.10–1.46 (m, 19H, CH2), 0.49 (t, 6H, J ) 7.0 Hz, CH3);
MS (ESI) [M + H]+ 493.3, [M + 2H]2+ 247.2. HRMS m/z calcd
for C32H49N2O2 [M + H]+, 493.3791; found, 493.3789. HPLC:
tR ) 7.45 min, 98.0% purity.
N,N′-(1′,5′-Pentylene)-bis-(-)-nor-MEP Hydrochloride (5d·
2HCl). Compound 9d (0.55 g, 1.03 mmol), lithium aluminum
hydride (0.20 g, 5.26 mmol), THF (30 mL), H2O (0.20 mL), 15%
NaOH (0.20 mL), and H2O (0.60 mL) were used to produce 5d
(0.16 g, 31%). Subsequent salt formation of 5d (0.10 g) gave
5d·2HCl (0.06 g, 52%): mp 80-82 °C; [R]D -48.10° (c 0.084,
MeOH); IR ν 3417, 2937, 1600, 1585, 1447, 1268 cm-1; 1H NMR
(DMSO-d6) 9.96, 9.75 (br s, 5/4 H, NH+), 9.57, 9.52, 9.44, 9.42
(s, 2H, Ar-OH), 8.34 (br s, 3/4 H, NH+), 7.19–7.12 (m, 2H,
Ar-H), 6.83–6.64 (m, 6H, Ar-H), 3.82 (m, 3/4 H, N-CH2), 3.52
(m, 5/4 H, N-CH2), 3.34–3.06 (m, 10H, N-CH2), 2.37 (m, H,
CH2), 2.10–1.21 (m, 21H, CH2), 0.48 (m, 6H, CH3); MS (ESI) [M
+ H]+ 507.3, [M + 2H]2+ 254.2. HRMS m/z calcd for C33H51N2O2
[M + H]+, 507.3945; found, 507.3961. HPLC: tR ) 7.77 min,
96.1% purity.
In Vitro AChE/BChE Inhibition Assay. Inhibitory activity
against AChE was evaluated by a modified Ellman’s method.34
Mice brain homogenate prepared in saline was used as a source of
AChE; mice serum was the source of BChE. The AChE activity
was determined in a reaction mixture containing 200 µL of a
solution of AChE (0.415 U/mL in 0.1 M phosphate buffer, pH 8.0),
300 µL of a solution of 5,5′-dithio-bis(2-nitrobenzoic) acid (3.3
mM DTNB in 0.1 M phosphate buffered solution, pH 7.0,
containing NaHCO3 6 mM), and 30 µL of a solution of the inhibitor
(six to seven concentrations). After incubation for 20 min at 37
°C, acetylthiocholine iodide (300 µL of 0.05 mM water solution)
was added as the substrate, and AChE activity was determined by
UV spectrophotometry from the absorbance changes at 412 nm for
3 min at 25 °C. The concentration of compound that produced 50%
inhibition of the AChE activity (IC50) was calculated by nonlinear
regression of the response-concentration (log) curve. BChE
inhibitory activity determinations were similarly carried out using
butyrylthiocholine iodide (0.05 mM) as the substrate. Results are
reported as the mean ( SEM of IC50 obtained from at least three
independent measures.
Molecular Docking. Molecular simulations were performed on
an R14000 SGI Fuel workstation with software package SYBYL
6.9 (Tripos Inc., St. Louis, MO). Standard parameters were used
unless otherwise indicated. The X-ray crystallographic structures
of the mAChE complex with succinylcholine (PDB code 2HA2)32
and the native hBChE (1P0I)28were retrieved from PDB. Het-
eroatoms and water molecules in the PDB files were removed,
and all hydrogen atoms were subsequently added to the protein.
3D coordinates of the ligand were generated by CORINA 3.0
(Molecular Networks GmbH, Erlangen, Germany, 2004).35 Two
sp3 N atoms were both protonated, and the Gasteiger-Huckel
partial charges36,37 were assigned to each atom of the resultant
Advanced combination approach of clustering and consensus
scoring was used to guide the selection of the most reliable
conformation from among a set of candidate conformations that
GOLD generated. All conformations were evaluated with four
available scoring functions, including three scoring functions
(G_Score, D_Score, and ChemScore) from the CSCORE38 module
in SYBYL and another stand-alone scoring function, X-SCORE
1.2.1.39 The “rank-by-rank” strategy reported by Wang et al.40 was
adopted to make consensus scoring. The final rank of a certain
conformation was its average rank received from all four scoring
functions. GOLD generated clusters based on different rmsd (root
mean square deviation) criteria. A proper rmsd standard of clustering
was important to the selection of representative clusters. Herein,
rmsd values of 2.55 and 2.40 Å were chosen as the criteria to cluster
the docked conformations with mAChE and hBChE, respectively.
Among the top 10 clusters for each ChE, the one with members
possessing the minimum average “rerank” was identified as the
representative cluster. The top “reranked” conformation in the
representative cluster was selected as the representative binding
mode for the ligand.
Inhibition of AChE-Induced Aꢀ Aggregation. Aliquots of 2
µL Aꢀ (1–40) peptide (Biosource), lyophilized from 2 mg/mL HFIP
solution and dissolved in DMSO, were incubated for 48 h at room
temperature in 0.215 M sodium phosphate buffer (pH 8.0) at a final
concentration of 230 µM. For coincubation experiments, aliquots
(16 µL) of human recombinant AChE (Sigma-Aldrich) (final
concentration of 2.3 µM, Aꢀ/AChE molar ratio of 100:1) and AChE
in the presence of 2 µL of the tested inhibitors were added. Blanks
containing Aꢀ, AChE, and Aꢀ plus inhibitors at various concentra-
tions in 0.215 M sodium phosphate buffer (pH 8.0) were prepared.
The final volume of each vial was 20 µL. Each assay was run in
duplicate. Inhibitor stock solutions were prepared (c ) 10 mM)
and diluted in 0.215 M sodium phosphate buffer (pH 8.0) when
used. To quantify amyloid fibril formation, the thioflavin T
fluorescence method was then applied.20
After incubation, the samples containing Aꢀ, Aꢀ plus AChE, or
Aꢀ plus AChE in the presence of inhibitors were diluted with 50
mM glycine-NaOH buffer (pH 8.5) containing 1.5 µM thioflavin
T (Sigma-Aldrich) to a final volume of 2.0 mL. Fluorescence was
monitored by PE LS45, with excitation at 446 nm and emission at
490 nm. A time scan of fluorescence was performed, and the
intensity values reached at the plateau (around 300 s) were averaged
after subtracting the background fluorescence from 1.5 µM thiofla-
vin T and AChE. The percent inhibition of the AChE-induced
aggregation due to the presence of increasing concentrations of the
inhibitor was calculated by the following expression: 100 - (IFi/
IFo × 100), where IFi and IFo were the fluorescence intensities
obtained for Aꢀ plus AChE in the presence and in the absence of
inhibitor, respectively, after subtracting the fluorescence of respec-
tive blanks. Inhibition curves and linear regression parameters were
obtained for each compound, and the IC50 was extrapolated, when
possible.
MTT Assay of Cell Viability. The human neuroblastoma cell
line SH-SY5Y (American Type Culture Collection) cells were
cultured in MEM/F-12 (1:1) medium (Invitrogen, Grand Island,
NY) supplemented with 10% fetal calf serum (FCS, Invitrogen),

Bis-(-)-nor-meptazinols as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2035
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100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified
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and grow. When cells reached the required confluence, they were
placed into serum-free medium and treated with the synthesized
compounds 5h and 5i. Twenty-four hours later the survival of cells
was determined by MTT assay. Briefly, after incubation with 20
µL of MTT (5 mg/mL; Sigma, St. Louis, MO) at 37 °C for 3 h,
living cells containing MTT formazon crystals were solubilized
in 200 µL of dimethyl sulfoxide (DMSO, Sigma). The absor-
bance of each well was measured using a microculture plate
reader with a test wavelength of 570 nm and a reference
wavelength of 655 nm.
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Acknowledgment. We thank the National Natural Science
Foundation of China (Grants 30472088, 30772553, and
30371731), the Program of Shanghai Subject Chief Scientist
(Grant 06XD14011), and the Major Basic Research Project of
Shanghai Municipal Science and Technology Commission
(Grant 07DJ14005) for financial support. We also gratefully
thank Dr. Manuela Bartolini (University of Bologna, Italy) and
Dr. Margarita Dinamarca (Pontificia Universidad Católica,
Chile) for their valuable suggestions in the experiments on
AChE-induced Aꢀ aggregation.
Note Added after ASAP Publication. This manuscript was
released ASAP on March 12, 2008 with errors in the Experimental
and Acknowledgment Sections. The correct version posted on
March 15, 2008.
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aggregation induced by human acetylcholinesterase: inhibition studies.
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