Discovery of huperzine A-tacrine hybrids as potent inhibitors of human cholinesterases targeting their midgorge recognition sites

Article abstract of DOI:10.1021/jm060257t

We describe herein the development of novel huperzine A-tacrine hybrids characterized by 3-methylbicyclo-[3.3.1]non-3-ene scaffolds. These compounds were specifically designed to establish tight interactions, through different binding modes, with the midg

Full text of DOI:10.1021/jm060257t

J. Med. Chem. 2006, 49, 3421-3425
3421
Discovery of Huperzine A-Tacrine Hybrids as Potent Inhibitors of Human Cholinesterases
Targeting Their Midgorge Recognition Sites
Sandra Gemma,†,‡ Emanuele Gabellieri,†,‡ Paul Huleatt,† Caterina Fattorusso,‡,§ Marianna Borriello,‡,§ Bruno Catalanotti,‡,§
Stefania Butini,†,‡ Meri De Angelis,†,‡ Ettore Novellino,‡,§ Vito Nacci,†,‡ Tatyana Belinskaya,| Ashima Saxena,| and
Giuseppe Campiani*,†,‡
Dipartimento Farmaco Chimico Tecnologico, Via Aldo Moro, and European Research Centre for Drug DiscoVery & DeVelopment, UniVersita’
di Siena, 53100 Siena, Italy, Dipartimento di Chimica delle Sostanze Naturali and Dipartimento di Chimica Farmaceutica e Tossicologica,
UniVersita’ di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy, and DiVision of Biochemistry, Walter Reed Army Institute of
Research, SilVer Spring, Maryland 20910
ReceiVed March 7, 2006
We describe herein the development of novel huperzine A-tacrine hybrids characterized by 3-methylbicyclo-
[3.3.1]non-3-ene scaffolds. These compounds were specifically designed to establish tight interactions, through
different binding modes, with the midgorge recognition sites of human acetylcholinesterase (hAChE: Y72,
D74) and human butyrylcholinesterase (hBuChE: N68, D70) and their catalytic or peripheral sites.
Compounds 5a-c show a markedly improved biological profile relative to tacrine and huperzine A.
Chart 1. Reference and Title Compounds
Introduction
The human central nervous system (CNS) contains two
different cholinesterases: acetylcholinesterase (hAChE) and
butyrylcholinesterase (hBuChE). Although 50-60% homolo-
gous, these enzymes are encoded by different genes and clearly
differ in substrate specificity and sensitivity to inhibitors, likely
due mainly to structural differences in the active site gorge of
the two enzymes, leading to a lower electrostatic gradient and
a larger void in BuChE.1,2 While AChE inhibitors (AChEIs)
represent a well-established class of drugs for the symptomatic
treatment of Alzheimer’s disease (AD),3 recent findings point
to BuChE inhibition as an additional tool to increase the
cholinergic activity in AD patients affected by severe symp-
toms.4 Although the forebrain cholinergic system is clearly
perturbed during mild to moderate AD, by the time the disease
has progressed to its severe stage, AChE and choline acetyl-
transferase levels are as much as 85-90% lower than normal.
Interestingly, it has been reported by several authors that this
loss of AChE is accompanied by an increase in glial BuChE
levels by up to 2-fold,3 suggesting that BuChE can partly
compensate for AChE action in severe AD.3-5 Despite this topic
still being a matter of debate,6 clinical studies using rivastigmine,
a potent inhibitor of both BuChE and AChE, proved the
increased therapeutic efficacy of this compound in mild-to-
moderate and moderately severe AD compared to currently used
AChE selective inhibitors, indicating that clinical benefits of
rivastigmine are related to its dual inhibitory action.7 Four
AChEIs have been approved by the European and US regulatory
authorities: tacrine (THA, 1, Chart 1), donepezil, rivastigmine,
and galantamine. These drugs are often associated with adverse
events (e.g., liver damage, nausea, and vomiting) and their
therapeutic potential is limited. Huperzine A (HA, 2a) is an
alkaloid, presently under clinical evaluation.8 As with the
previously mentioned AChEIs, huperzine A interacts with the
catalytic binding site of AChE, which is located at the bottom
of a deep gorge. A second binding site, at the lip of the gorge,
namely the peripheral “anionic” site (PAS), is located around
18 Å away from the active site.9 Through the development of
specific tacrine heterodimers, our group recently targeted a
“midgorge recognition site” in hAChE. In addition, we uncov-
ered the existence of a peripheral binding site and a midgorge
interaction site also in the BuChE family of enzymes.10,11 In
the past, we have reported the synthesis of HA and of a large
number of closely related analogues.12 Others hybridized the
structure of HA to synthesize huprine X (3),13 a potent AChEI,
which incorporates key structural features from both HA and
THA. In addition, HA-THA hybrids (4a and 4b) containing
the bicyclic tetrahydroquinolinone fragment of HA have been
reported.14 Huprine X interacts with the catalytic site of AChE,
displaying a low affinity for BuChE, while the hybrids described
by Carlier can interact with the catalytic site and/or PAS of
AChE, depending on the length of the linker. In particular, 4b
showed low affinity while its superior homologue 4a (character-
ized by a 10-carbon methylene linker) displayed nanomolar
* To whom correspondence should be addressed. Tel: 0039-0577-
234172. Fax: 0039-0577-234333. E-mail: campiani@unisi.it.
† Dipartimento Farmaco Chimico Tecnologico, Universita’ degli Studi
di Siena.
‡ European Research Centre for Drug Discovery and Development,
Universita’ di Siena.
§ Universita’ degli Studi di Napoli “Federico II”.
| Walter Reed Army Institute of Research.
10.1021/jm060257t CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/04/2006

3422 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Brief Articles
Scheme 1a
Scheme 2a
a Conditions: (a) (1) N-(7-aminoheptyl)-1,2,3,4-tetrahydroacridin-9-
amine, AcOH, pH 6; (2) NaBH3CN; (b) (1) Me3SiI; (2) MeOH, reflux.
ester 8 was achieved by a Michael addition-aldol reaction
sequence catalyzed by 1,1,3,3-tetramethylguanidine (TMG) to
yield a bicyclic ketol intermediate.12c The endo-olefin 9 was
finally obtained by dehydration of the alcohol precursor (as a
mixture of stereoisomers) through a two-step process involving
mesylate formation followed by collidine-induced elimination.
Starting from â-keto ester 9 the next step of the synthetic plan
required the installation of the (E)-ethylidene side chain at C9.
A standard Wittig olefination protocol employing n-BuLi and
ethyl(triphenylphosphonium)bromide furnished only trace amount
of the desired olefination product along with a complex mixture
of fragmentation products of the bridged bicyclic skeleton and
of the MEM ether. Investigation of a variety of reaction
conditions led us to obtain a 8:1 mixture of (Z)- and (E)-alkenes
10 in 75% yield when potassium bis(trimethylsilyl)amide and
ethyl(triphenylphosphonium)bromide were used.17 As the reac-
tion product consisted largely of the incorrect isomer, mixture
10 was subjected to an isomerization reaction employing
thiophenol and 2,2′-azobisisobutyronitrile (AIBN), which re-
sulted in an intractable mixture, probably due to the instability
of MEM ether under radical conditions. Alternatively, hydro-
chloric acid-promoted deprotection of the hydroxy group
followed by oxidation of the alcohol using PDC furnished ketone
11 in good overall yield. Radical isomerization of the (Z)-
ethylidene chain of the resulting ketone 11 afforded an 18:1
mixture of (E)- and (Z)-olefins in 95% yield. Next, the ester
was hydrolyzed by 20% sodium hydroxide in a 2:1 tetrahydro-
furan/methanol mixture to furnish the E-acid 12 as a single
geometric isomer (the more hindered Z-isomer failed to undergo
hydrolysis under these conditions). The resulting acid was
treated with diphenyl azidophosphate, and the intermediate
isocyanate was reacted with methanol to provide the key
urethane intermediate 13. Initial attempts to carry out the
reductive amination reaction of ketone 13 with N-(7-amino-
heptyl)-1,2,3,4-tetrahydroacridin-9-amine18 proved to be prob-
lematic. A variety of reaction conditions, using different
dehydrating agents, solvents, and reaction conditions (temper-
ature, sealed tube reactions) failed to afford the desired
compound or the corresponding imine intermediate. Success was
ultimately achieved through a two-step protocol that involved
carbonyl activation by titanium tetraisopropoxide followed by
treatment of the crude reaction mixture with sodium cyanoboro-
hydride in ethanol.19 Trimethylsilyl iodide-promoted deprotec-
tion of the carbamate group finally furnished the desired
heterodimer 5a.
a Conditions: (a) NaBH4; (b) 1 N HCl; (c) MEMCl, DIPEA; (d) KH,
35% in mineral oil, DMC; (e) (1) methacrolein, TMG; (2) MsCl, Et3N,
DMAP; (3) 2,4,6-collidine; (f) Ph3PEtBr, KHMDS (0.5 M in toluene); (g)
1 N HCl; (h) PDC; (i) PhSH, AIBN; (j) NaOH 20%, MeOH/THF 1:2; (k)
(1) (PhO)2PON3, Et3N; (2) MeOH, reflux; (l) (1) N-(7-aminoheptyl)-1,2,3,4-
tetrahydroacridin-9-amine, Ti(O-i-Pr)4; (2) NaBH3CN; (m) (1) Me3SiI; (2)
MeOH, reflux.
affinity, likely due to the interaction of the second protonatable
nitrogen with rat AChE PAS.14 Interestingly, 4a and 4b were
found to be poorly active on rat BuChE.14 Since much evidence
suggests that BuChE may represent an intriguing target for the
treatment of AD,1-4,7,8 enzyme selectivity may not be a key
issue in drug development for treating neurodegenerative
disorders of the Alzheimer’s type, and specific ligands targeting
both enzymes may represent a new and valuable therapeutic
approach for use in mild, moderate, and severe dementia.
Accordingly, taking into account the information previously
acquired on the existence of multiple interaction sites in either
the AChE or the BuChE family of enzymes (catalytic, midgorge,
and peripheral), we developed the new HA-THA hybrids 5a-
c, shown in Chart 1, with the aim to obtain inhibitors targeting
both enzymes. These compounds are characterized by a nano-
molar inhibitory activity on both hAChE and hBuChE.
Chemistry
Our background in the synthesis of potent structural analogues
of HA (a methyl group at C-10 axial and equatorial positions
provided analogues more potent than HA)12b was exploited. For
the synthesis of compound 5a, our main goal was the setup of
a suitable synthetic strategy to obtain the (()-methyl (9E)-9-
ethylidene-3,6,6-trimethyl-7-oxobicyclo[3.3.1]non-3-en-1-ylcar-
bamate key intermediate 13.
The synthesis begins by reducing the carbonyl group of the
known monoethylene ketal 615 (Scheme 1) using sodium
borohydride followed by deprotection of the ketal function by
exposure to 1 N hydrochloric acid in acetone to obtain ketol 7.
The hydroxy group was then protected as a methoxyethoxy-
methyl ether (MEM ether). The resulting compound was
regioselectively transformed into the â-keto ester 8 by means
of potassium hydride and dimethyl carbonate (DMC). This
reaction, carried out using Mander’s reagent,16 failed. The
introduction of the unsaturated carbon bridge across the â-keto
The synthesis of compounds 5b and 5c is described in Scheme
2. Ketones 15 and 16, readily accessible from commercially
available 1,4-cyclohexanedione monoethylene ketal following
a previously described procedure,12c were reacted with N-(7-

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3423
aminoheptyl)-1,2,3,4-tetrahydroacridin-9-amine to furnish final
compounds 5c and, after carbamate deprotection, 5b.
Results and Discussion
We previously reported10,11 that a protonatable nitrogen at
the tether level of tacrine homo- and heterodimers was a key
determinant for potency improvement toward ChEs. This is due
to interactions with the midgorge recognition site, characterized
by the presence of a conserved Asp residue (hAChE, D74;
hBuChE, D70). Interestingly, at the midgorge level AChE and
BuChE differ in their amino acid composition, since AChEs
present several aromatic residues that are replaced by aliphatic
ones in BuChEs. These structural differences are responsible
for the larger electrostatic gradient and for the narrower void
along the AChE gorge with respect to BuChE, affecting ligand
selectivity.10,11 Flexible docking studies showed that 5a-c can
adopt different binding modes within hAChE and hBuChE
active site gorges, and results are displayed in Figures 1 and 2.
The stronger electrostatic gradient present along the AChE gorge
drives the diprotonated (see Table 1 of the Supporting Informa-
tion) 3-methylbicyclo[3.3.1]non-3-ene moiety of 5b (KihAChE
)
15.7 nM) in the catalytic site. At this level the protonated
primary amine group of 5b interacts with W86, through a
cation-π interaction, and H447 and G448, through a bridged
water molecule, placing the bridged 3-methyl group and the
ethylidene chain in a similar orientation to HA in complex with
TcAChE (PDB code 1VOT). In addition, the secondary proto-
nated amine of the bicyclic scaffold interacts with D74 and
presents a suitable H-bond distance from the Y124 side chain
oxygen (2.8 Å), also establishing a water-mediated hydrogen
bond with T83. Finally, the THA moiety protrudes to the PAS,
setting up a polarized π-π interaction with W286 (Figure 1A).
On the other hand, the introduction of two methyl substituents
at C-4 (5a; KihAChE ) 16.5 nM) induces a partial rotation of the
HA moiety, which results in a direct interaction between the
5a primary amine group and the catalytic site (G448, H447)
and in the displacement of the water molecule present in 5b
(Figure 1, A vs B). Compound 5a preserves a direct interaction
with D74 through the secondary amino group, but its orientation
promotes an H-bond with the Y337 side chain (Y124 in 5b).
The results of our docking studies suggested an opposite binding
mode for 5a and 5b in hBuChE, with respect to hAChE. This
also reflects the different activities of THA and HA toward both
enzymes (Table 1). Indeed, 5a and 5b place the THA and the
3-methylbicyclo[3.3.1]non-3-ene moieties in the BuChE cata-
lytic and midgorge sites, respectively (Figure 2A,B). In par-
ticular, the bulkier C-4 methyl groups of 5a (KihBuChE ) 20.8
nM; Figure 2B) are perfectly accommodated in the lipophilic
environment at the midgorge level of the enzyme: (i) establish-
ing favorable van der Waals interactions with P285 and I69,
(ii) directly anchoring the protonated primary amine group to
D70 and to the side chain carbonyl oxygen of N68, and (iii)
interacting with D70 (secondary amine group) and Q71 (primary
amine group) through a water-mediated hydrogen-bond network.
This accommodation allows an optimal orientation of the THA
moiety in the catalytic site (Figure 2B). A similar orientation is
observed for compound 5c (KihBuChE ) 19.5 nM), thus allowing
an optimal π-π interaction between W82 and the THA moiety
and also H-bond formation between the exo-cyclic THA
nitrogen and T120 side chain (Figure 2C). On the contrary, for
the less hindered compound 5b (KihBuChE ) 30.8 nM) the
electrostatic interaction with D70 prevails, constraining 5b in
an orientation that weakens interactions with the catalytic site
(Figure 2A). As we have previously observed,10,11 the potency
of multisite inhibitors (i.e., binding more than one site along
Figure 1. Docked complexes of (A) 5b/hAChE (ligand, white; protein,
magenta), (B) 5a/hAChE (ligand, orange; protein, cyan), and (C) 5c/
hAChE (ligand, green; protein, yellow). Heteroatoms and water
molecules are colored by atom type. The van der Waals volume of
W86 is displayed. Hydrogen bonds are highlighted by green dashed
lines. Ligand and protein hydrogens are omitted for clarity, with the
exception of those involved in hydrogen-bond interactions.
the active site gorge of ChEs) is modulated by the mutual
orientation of the binding groups of the ligand.
Compound 5c contains an ester function in place of the
primary amine group on the HA fragment. Consequently, this
substitution induces a change in the binding mode of 5c (KihAChE
) 6.4 nM) to AChE with respect to 5a,b, placing the THA
moiety in the catalytic site similar to that observed in BuChE
with the HA fragment favorably interacting with W286 (PAS)
(Figure 1C). Moreover, 5c preserves the electrostatic interaction
at the midgorge level with D74 and establishes an additional
interaction with Y72 (Figure 1C). An H-bond is formed between
the hydroxyl group of the Y72 side chain and the carbonyl
oxygen of the inhibitor. Interestingly, 5c establishes an analo-
gous H-bond interaction with the side chain of the corresponding
residue in hBuChE (N68; Figure 2C). The introduction of the
3-methylbicyclo[3.3.1]non-3-ene scaffold in THA-HA hybrids
conferred a significant increase in inhibitory potency on AChE

3424 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Brief Articles
hAChE and hBuChE. In particular, in hBuChE we hypothesized
a binding mode involving interactions at the catalytic site (W82
and H438) and at the recently discovered midgorge recognition
site (D70, N68, and Q71). On the other hand, by adopting an
“upside down” binding-mode on hAChE, compounds 5a and
5b showed interactions with the catalytic and the midgorge and
the PAS (W286) recognition sites. Conversely, the potent
inhibitor 5c places the THA moiety in the catalytic site, still
being able to interact with the three AChE binding sites and
establishing an extra interaction with Y72 through the carbonyl
oxygen of its ester group. Compounds 5a-c represent potent
ChE multisite inhibitors of human ChEs, showing comparable
inhibitory activities for both hAChE and hBuChE, thus provid-
ing useful tools for the evaluation of a new therapeutic approach
against the cognitive impairment in moderate and severe AD.
Experimental Section
(9E)-N1-(7-(1,2,3,4-Tetrahydroacridin-9-ylamino)heptyl)-9-
ethylidene-4,4,7-trimethylbicyclo[3.3.1]non-6-ene-1,3-diamine (5a).
A mixture of 13 (30.0 mg, 0.108 mmol), N-(7-aminoheptyl)-1,2,3,4-
tetrahydroacridin-9-amine18 (118.4 mg, 0.46 mmol), and titanium
tetraisopropoxide (1.48 mmol, 0.4 mL) was stirred at room
temperature for 48 h. Then NaBH3CN (44.0 mg, 0.70 mmol) and
ethanol (2.0 mL) were added, and stirring was continued for 120
h. Water was added, and the solid was removed by vacuum filtration
through a bed of silica gel, which was subsequently washed with
ethanol. The filtered solution was concentrated and excess NaBH3-
CN was destroyed by addition of aqueous hydrochloric acid and
stirring for 1 h. The mixture was made alkaline with 1 N aqueous
sodium hydroxide and extracted with dichloromethane. The organic
extracts were dried (Na2SO4), and the solvent was removed. The
residue was purified by flash chromatography eluting with a 10:
1:0.5 mixture of EtOAc, MeOH, and triethylamine to afford a
carbamate intermediate (16.7 mg, 30%) as colorless oil: 1H NMR
(CDCl3) δ 7.98-7.96 (m, 2H), 7.58-7.54 (m, 1H), 7.37-7.33 (m,
1H), 5.42 (d, J ) 5.4 Hz, 1H), 5.22 (q, J ) 6.6 Hz, 1H), 4.77 (bs,
1H), 3.63 (s, 3H), 3.53 (t, J ) 6.8 Hz, 2H), 3.11-3.09 (m, 2H),
2.88 (d, J ) 5.4 Hz, 1H), 2.73-2.63 (m, 5H), 2.57 (dd, J ) 4.8,
12.0 Hz, 1H), 2.49 (d, J ) 8.5 Hz, 1H), 2.44-2.34 (m, 1H), 2.25-
2.19 (m, 1H), 1.95-1.91 (m, 4H), 1.64-1.57 (m, 6H), 1.38-1.18
(m, 10H), 0.91 (s, 3H), 0.86 (s, 3H); ES/MS m/z 573 (M+ + H).
To a solution of the above compound (10 mg, 0.019 mmol) in dry
chloroform (0.5 mL) was added iodotrimethylsilane (15.6 µL, 0.11
mmol), and the resulting mixture was heated under reflux for 5 h.
After cooling to room temperature, the solvent was removed in
vacuo, the residue was dissolved in dry methanol (1 mL), and the
solution was heated under reflux. After 18 h, the solvent was
removed and the residue was purified by flash chromatography
eluting with a 20:1:1 mixture of ethyl acetate, methanol, and
ammonium hydroxide (30%) to obtain 1.1 mg (12%) of 5a as a
colorless oil: 1H NMR (CDCl3 + D2O) δ 7.96-7.91 (m, 2H),
7.56-7.52 (m, 1H), 7.35-7.31 (m, 1H), 5.43-5.38 (m, 2H), 3.48
(t, J ) 7.2 Hz, 2H), 3.07-3.06 (m, 2H), 2.88 (d, J ) 6.2 Hz, 1H),
2.70-2.66 (m, 5H), 2.57 (dd, J ) 4.9, 12.0 Hz, 1H), 2.44-2.38
(m, 2H), 2.19-2.14 (m, 1H), 1.98-1.90 (m, 4H), 1.67-1.59 (m,
6H), 1.47-1.24 (m, 10H), 0.96 (s, 3H), 0.76 (s, 3H); ESI-MS m/z
515 (M + H)+. Anal. (C34H50N4) C, H, N.
Figure 2. Docked complexes of (A) 5b/hBuChE (ligand, white;
protein, magenta), (B) 5a/hBuChE (ligand, orange; protein, cyan), and
(C) 5c/hBuChE (ligand, green; protein, yellow). Heteroatoms and water
molecules are colored by atom type. The van der Waals volume of
W86 is displayed. Hydrogen bonds are highlighted by green dashed
lines. Ligand and protein hydrogens are omitted for clarity, with the
exception of those involved in hydrogen-bond interactions.
Table 1. Dissociation Constants for the Inhibition of hAChE and
hBuChE by Compounds 5a-c
Ki (nM) ( SDa
compd
hAChE
hBuChE
5a
5b
5c
16.5 ( 0.3
15.7 ( 0.9
6.4 ( 0.8
137.0
20.8 ( 2.6
30.8 ( 2.0
19.5 ( 1.9
7.0
(9E)-N1-(7-(1,2,3,4-Tetrahydroacridin-9-ylamino)heptyl)-9-
ethylidene-7-methylbicyclo[3.3.1]non-6-ene-1,3-diamine (5b). To
a solution of N-(7-aminoheptyl)-1,2,3,4-tetrahydroacridin-9-amine18
(49.0 mg, 0.16 mmol) in dry tetrahydrofuran and methanol (1:1, 2
mL), maintained at pH 6 by addition of glacial acetic acid, was
added 16 (18.0 mg, 0.07 mmol). The mixture was stirred at room
temperature for 1 h and then NaBH3CN (22.0 mg, 0.36 mmol) was
added, and the reaction mixture was heated to 50 °C. After 72 h
the reaction mixture was cooled to room temperature, water was
added, the solvent was removed in vacuo, and the aqueous phase
was extracted with chloroform. The organic extracts were dried
(Na2SO4) and the solvent was removed. The residue was purified
THA (1)
(()-HA (2a)
47.0
0.026
>1000
120
huprine X (3)b
a Ki is the mean of at least three determinations. b Reference 13.
with respect to 4b. In addition, a significant potency against
BuChE was also observed.
Conclusions
In summary, we designed and synthesized novel and potent
HA/THA hybrids targeting the midgorge recognition site of both

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3425
(7) (a) Lane, R. M.; Potkin, S. G.; Enz, A. Targeting acetylcholinesterase
and butyrylcholinesterase in dementia. Int J. Neuropsychopharmacol.
2006, 1, 101-124. (b) Bartorelli, L.; Giraldi, C.; Saccardo, M.;
Cammarata, S.; Bottini, G.; Fasanaro, A. M.; Trequattrini, A. Effects
of switching from an AChE inhibitor to a dual AchE-BuChE
inhibitor in patients with Alzheimer’s disease. Curr. Med. Res. Opin.
2005, 11, 1809-1818.
(8) Jiang, H.; Luo, X.; Bai, D. Progress in clinical, pharmacological,
chemical and structural biological studies of huperzine A: A drug
of traditional Chinese medicine origin for the treatment of Alzhe-
imer’s disease. Curr. Med. Chem. 2003, 10, 2231-2252.
(9) Pang, Y.-P.; Quiram, P.; Jelacic, T.; Hong, F. Highly potent, selective,
and low cost bis-tetrahydroaminacrine inhibitors of acetylcholinest-
erase. Steps toward novel drugs for treating Alzheimer’s disease. J.
Biol. Chem. 1996, 271, 23646-23649.
(10) Savini, L.; Gaeta, L.; Fattorusso, C.; Catalanotti, B.; Campiani, G.;
Chiasserini, L.; Pellerano, C.; Novellino, E.; McKissic, D.; Saxena,
A. Specific targeting of acetylcholinesterase and butyrylcholinesterase
recognition sites. Rational design of novel, selective, and highly potent
cholinesterase inhibitors. J. Med. Chem. 2003, 46, 1-4.
(11) Campiani, G.; Fattorusso, C.; Butini, S.; Gaeta, A.; Agnusdei, M.;
Gemma, S.; Persico, P.; Catalanotti, B.; Savini, L.; Nacci, V.;
Novellino, E.; Holloway, W.; Greig, N. H.; Belinskaya, T.; Fedorko,
J. M.; Saxena, A. Development of molecular probes for the
identification of extra interaction sites in the mid-gorge and peripheral
sites of butyrylcholinesterase (BuChE). Rational design of novel,
selective, and highly potent BuChE inhibitors. J. Med. Chem. 2005,
48, 1919-1929.
(12) (a) Campiani, G.; Sun, L. Q.; Kozikowski, A. P.; Aagaard, P.;
McKinney, M. A palladium-catalyzed route to huperzine A and its
analogues and their anticholinesterase activity. J. Org. Chem. 1993,
58, 7660-7669. (b) Kozikowski, A. P.; Campiani, G.; Sun, L.-Q.;
Wang, S.; Saxena, A.; Doctor, B. P. Identification of a more potent
analogue of the naturally occurring alkaloid huperzine A. Predictive
molecular modeling of its interaction with AChE. J. Am. Chem. Soc.
1996, 118, 11357-11362. (c) Kozikowski, A. P.; Campiani, G.;
Tu¨ckmantel, W. An approach to open chain modified heterocyclic
analogues of the acetylcholinesterase inhibitor, Huperzine A, through
a bicyclo[3.3.1]nonane intermediate. Heterocycles 1994, 39, 101-
116. (d) Gemma, S.; Butini, S.; Fattorusso, C.; Fiorini, I.; Nacci, V.;
McKissic, D.; Saxena, A.; Campiani, G. A palladium-catalyzed
synthetic approach to new huperzine A analogues modified at the
pyridone ring. Tetrahedron 2003, 59, 87-93.
(13) Camps, P.; Cusack, B.; Mallender, W. D.; El Achab, R.; Morral, J.;
Munoz, D. T.; Rosenberry, T. L. Huprine X is a novel high-affinity
inhibitor of acetylcholinesterase that is of interest for treatment of
Alzheimer’s disease. Mol. Pharmacol. 2000, 57, 409-417.
(14) Carlier, P. R.; Du, D.-M.; Han, Y.; Liu, J.; Pang, Y.-P. Potent, easily
synthesized huperzine A-tacrine hybrid acetylcholinesterase inhibi-
tors. Bioorg. Med. Chem. Lett. 1999, 9, 2335-2338.
(15) Nelson, L. A. K.; Shaw, A. C.; Abrams, S. R. Synthesis of (+)-,
(-)-, and (()-7′-hydroxyabscisic acid. Tetrahedron 1991, 47, 3259-
3270.
(16) Mander, L. N.; Sethi, S. P. Regioselective synthesis of â-ketoesters
from lithium enolates and methyl cyanoformate. Tetrahedron Lett.
1983, 5425-5428.
(17) Sreeknmar, C.; Darst, K. P.; Still, W. C. A direct synthesis of
Z-trisubstituted allylic alcohols via the Wittig reaction. J. Org. Chem.
1980, 45, 4260-4262.
(18) Carlier, P. R.; Chow, E. S.-H.; Han, Y.; Liu, J.; Yazal, J.; Pang,
Y.-P. Heterodimeric tacrine-based acetylcholinesterase inhibitors:
Investigating ligand-peripheral site interactions. J. Med. Chem. 1999,
42, 4225-4231.
(19) Wagermann, R.; Vilsmaier, E.; Maas, G. Spatially fixed oligoamines
IV. 1 flexibility and protonation of meander-type octamines. Tetra-
hedron 1995, 8815-8826.
by flash chromatography eluting with a 20:1:1 mixture of ethyl
acetate, methanol, and ammonium hydroxide (30%) to afford 10.0
mg (26%) of a carbamate intermediate as a colorless oil: ESI-MS
m/z 545 [M + H]+, 513 (100), 470, 338, 312, 295, 199. To a
solution of the above carbamate (10 mg, 0.018 mmol) in dry
chloroform (0.5 mL) was added iodotrimethylsilane (15.6 µL, 0.11
mmol), and the resulting mixture was heated under reflux for 5 h.
After cooling to room temperature, the solvent was removed in
vacuo, the residue was dissolved in dry methanol (1 mL) and the
solution was heated under reflux. After 18 h the solvent was
removed and the residue was purified by flash chromatography
eluting with a 20:1:1 mixture of ethyl acetate, methanol, and
ammonium hydroxide (30%) to obtain 1.1 mg (12%) of 5b as a
colorless oil: 1H NMR (CDCl3 + D2O) δ 7.93-7.91 (m, 2H),
7.55-7.53 (m, 1H), 7.30-7.27 (m, 1H), 5.36-5.28 (m, 2H), 3.68-
3.60 (m, 3H), 3.51 (t, J ) 8.0 Hz, 2H), 3.15-2.87 (m, 3H), 2.75-
2.51 (m, 6H), 2.31-2.22 (m, 2H), 2.03-1.75 (m, 4H), 1.60-1.52
(m, 6H), 1.51-1.22 (m, 10H); ESI-MS m/z 487 (M + H)+. Anal.
(C32H46N4) C, H, N.
(9E)-7-(7-(1,2,3,4-Tetrahydroacridin-9-ylamino)heptylamino)-
9-ethylidene-3-methylbicyclo[3.3.1]non-3-ene-1-carboxylic Acid
Methyl Ester (5c). Reductive amination of ketone 15 and N-(7-
aminoheptyl)-1,2,3,4-tetrahydroacridin-9-amine18 was performed as
described for the synthesis of 5b to afford the title compound 5c
in 23% yield as a colorless oil: 1H NMR (CDCl3 + D2O) δ 7.96-
7.89 (m, 2H), 7.57-7.50 (m, 1H), 7.37-7.29 (m, 1H), 5.47-5.37
(m, 1H), 4.91-4.87 (m, 1H), 3.73 (s, 3H), 3.47 (t, J ) 6.9 Hz,
2H), 3.28-3.15 (m, 3H), 3.05-2.97 (m, 2H), 2.77-2.55 (m, 4H),
2.50-2.47 (m, 3H), 2.34-2.20 (m, 2H), 1.97-1.90 (m, 4H), 1.65-
1.57 (m, 6H), 1.41-1.21 (m, 10H); ESI-MS m/z 530 (M + H)+.
Anal. (C34H47N3O2) C, H, N.
Acknowledgment. Authors thank MIUR (Roma) for finan-
cial support. The opinions or assertions contained herein are
the private views of the authors and are not to be construed as
official or as reflecting the views of the United States Army or
Department of Defense.
Supporting Information Available: Experimental details for
13 and molecular modeling and pharmacology for 5a-c. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Gentry, M. K.; Doctor, B. P. In Cholinesterases: Structure, function,
mechanism, genetics, and cell biology; Massoulie, J., Bacou, F.,
Barnard, E. A., Chatonnet, A., Doctor, B. P., Eds.; American
Chemical Society: Washington, DC, 1991; pp 394-398.
(2) Saxena, A.; Redman, A. M. G.; Jiang, X.; Lockridge, O.; Doctor, B.
P. Differences in active site gorge dimension of cholinesterases
revealed by binding of inhibitors to human butyrylcholinesterase.
Biochemistry 1997, 36, 14642-14651.
(3) Giacobini, E. Cholinergic functions in Alzheimer’s disease. Int. J.
Geriatr. Psychiatry 2003, 18, S1-S5, and references therein.
(4) Greig, N. H.; Utsuki, T.; Yu, Q.-S.; Zhu, X.; Holloway, H. W.; Perry,
T.; Lee, B.; Ingram, D. K.; Lahiri, D. K. A new therapeutic target in
Alzheimer’s disease treatment: Attention to butyrylcholinesterase.
Curr. Med. Res. Opin. 2001, 17, 1-6.
(5) Mesulam, M. M.; Guillozet, A.; Shaw, P.; Levey, A.; Duysen, E.
G.; Lockridge, O. Acetylcholinesterase knockouts establish central
cholinergic pathways and can use butyrylcholinesterase to hydrolyse
acetylcholine. Neuroscience 2002, 110, 627-639.
(6) Kuhl, D. E.; Koeppe, R. A.; Snyder, S. E.; Minoshima, S.; Frey, K.
A.; Kilbourn, M. R. In vivo butyrylcholinesterase activity is not
increased in Alzheimer’s disease synapses. Ann. Neurol. 2006, 59,
13-20.
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