Homodimeric tacrine congeners as acetylcholinesterase inhibitors

Article abstract of DOI:10.1021/jm010308g

In the search for highly selective and potent derivatives of tacrine (1a), a number of homodimeric tacrine congeners were synthesized and conducted for their effects on rat acetylcholinesterase (ACHE) and human butyrylcholinesterase (BChE) inhibitions. Heptylene-linked bis-(6-chloro)-tacrine (3h) was found up to 3000- and 3-fold more potent in inhibiting rat AChE than tacrine and the unsubstituted bis-tacrine 3b, respectively. Changes in the size of the carbocyclic ring of the dimeric tacrine reduced both the selectivity and the potency of AChE inhibition as compared to 3b. Inserting an aza into the tacrine nucleus as the desired isosteres 3j-m resulted in moderate potency but tended to be detrimental to selectivity. The pronounced enhancement of AChE inhibition potency and AChE/BChE selectivity was achieved with incorporation of a halogen at the 6-position of homodimeric tacrines. The assay results of 3a-m also provided evidence that the 7-methylene tether tended to be optimal to AChE inhibition potency.

Full text of DOI:10.1021/jm010308g

J . Med. Chem. 2002, 45, 2277-2282
2277
Hom od im er ic Ta cr in e Con gen er s a s Acetylch olin ester a se In h ibitor s†
,
‡
‡
§
|
Ming-Kuan Hu,* Li-J u Wu, George Hsiao, and Mao-Hsiung Yen
School of Pharmacy and Department of Pharmacology, National Defense Medical Center, 161 Section 6, Minchuan East Road
Taipei, Taiwan 114, Republic of China, and Department of Pharmacology, School of Medicine, Taipei Medical University,
2
50 Wushin Street Taipei, Taiwan 110, Republic of China
Received J uly 5, 2001
In the search for highly selective and potent derivatives of tacrine (1a ), a number of homodimeric
tacrine congeners were synthesized and conducted for their effects on rat acetylcholinesterase
AChE) and human butyrylcholinesterase (BChE) inhibitions. Heptylene-linked bis-(6-chloro)-
(
tacrine (3h ) was found up to 3000- and 3-fold more potent in inhibiting rat AChE than tacrine
and the unsubstituted bis-tacrine 3b, respectively. Changes in the size of the carbocyclic ring
of the dimeric tacrine reduced both the selectivity and the potency of AChE inhibition as
compared to 3b. Inserting an aza into the tacrine nucleus as the desired isosteres 3j-m resulted
in moderate potency but tended to be detrimental to selectivity. The pronounced enhancement
of AChE inhibition potency and AChE/BChE selectivity was achieved with incorporation of a
halogen at the 6-position of homodimeric tacrines. The assay results of 3a -m also provided
evidence that the 7-methylene tether tended to be optimal to AChE inhibition potency.
In tr od u ction
Ch a r t 1. Tacrine (1a ) and Its Derivatives 1b-e
Alzheimer’s disease (AD) has been recognized as one
of the most severe conditions affecting the aged and is
1
life-threatening for this group of people. The disease
is characterized by neuronal loss, synaptic damage, and
neutritic and vascular plaques. At the cellular level, AD
is associated mainly with reduced levels of synaptic
acetylcholine (ACh) and other related neurotransmit-
2
ters. Tacrine (1a , tetrahydroaminoacridine or THA), a
reversible acetylcholinesterase inhibitor (AChEI), has
electron density on the tacrine aromatic rings could
favor π-interactions with nearby residues in active sites
of the enzyme and strongly increase the inhibitory
potency of tacrine. In addition to the structure-activity
approach toward potent tacrine derivatives, an auto-
mated docking program was applied to simulate the
binding possibilities for tacrine and an extra peripheral
site was identified at the binding pocket of AChE.12 On
the basis of these studies, heptylene-linked bis-tacrine
been one of the major approved drugs for use in AD
,4
(
Chart 1).3 The rationale for its use was related to the
elevation of ACh levels that can compensate for the
cholinergic deficiency associated with the brain lesions
in AD. Nevertheless, the deficiency of tacrine in clinic
has been related mainly to elevated liver transaminase
levels and dose-related, low-selective peripheral cholin-
5
,6
ergic effects.
related new candidates is still of interest to medicinal
The search for tacrine analogues or
(3b, see Table 1) was found to be a potent and selective
7
,8
13-15
chemists involved in AD research.
inhibitor of AChE
and the simultaneous binding to
Recent contributions to the development of tacrine-
the active and peripheral sites of AChE was proposed
to be responsible for the enhanced inhibition potency
of 3b.
related agents disclosed that tacrin-1-ol (1b, velna-
9
16
crine), an active metabolite of tacrine, has been chosen
for clinical trial. 6-Fluoro-tacrin-1-ol (1c) was reported
to be slightly more potent than tacrine, and 6-chloro-
tacrin-1-ol (1d ) was found to be 30 times more potent
All of the above contributions suggest that the selec-
tivity and potency of AChEIs for AD may be improved
with manipulation of tacrine. In the present paper, we
took a further step to integrate these findings on both
monomeric and dimeric tacrine derivatives by carrying
out our work on a series of homodimeric tacrine conge-
ners. These were focused on the 6-position substitution,
changes in carbocyclic ring size, and isosteric modifica-
tion of the tacrine nucleus to optimize AChE inhibition
potency and AChE/butyrylcholinesterase (BChE) selec-
tivity.
1
0
than tacrine. In particular 6-chlorotacrine (1e) has
been found to be more potent than other substituted
analogues.1
1a,b
This could be due to favorable orientation
and electron effects that contribute to more efficient
inhibition. On the basis of theoretical calculations, 1e
was also shown to exhibit improved binding strength
toward AChE.1 These suggested that the reduced
1c
*
To whom correspondence should be addressed. Tel: (886)2 8792-
6
030. Fax: (886)2 8792-3169. E-mail: hmk@ndmctsgh.edu.tw.
Dedicated to Professor Daniel H. Rich on the occasion of his 58th
Ch em istr y
†
birthday.
The synthesis of dimeric tacrines is illustrated in
Schemes 1 and 2. Although recent reports for the
synthesis of bis-tacrines began with tacrine itself, this
‡
School of Pharmacy, National Defense Medical Center.
School of Medicine, Taipei Medical University.
§
|
17
Department of Pharmacology, National Defense Medical Center.
1
0.1021/jm010308g CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/26/2002

2
278 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Hu et al.
Ta ble 1. AChE and BChE Inhibition Potency and Selectivity
of Bis-tacrine Congeners
7-9 with a half equivalent of 1,n-diaminoalkanes
1
0a -c in heated phenol provided alkylene-linked ho-
1
9
modimeric tacrine congeners 2-4 (Scheme 2). This
more strategy efficient method could be applied to the
preparation of analogous bis-tacrine derivatives.
Biologica l Resu lts a n d Discu ssion
These homodimeric tacrine congeners were assayed
for AChE (rat cortex) and BChE (human plasma)
2
0
inhibition potency by the Ellman method. According
to the previous suggestions, the optimal methylene
IC50 (nM)
selectivity
18
product
X
Y
m
n
AChEa
BChEb
for AChEc
length bridging tacrine nucleus with simultaneous
binding to the dual sites of AChE is possible with tether
of at least five methylenes. Heptylene-linked bis-tacrine
was found to be a potent and selective inhibitor of AChE
in the series of alkylene-bridged analogues.13 We,
therefore, observed congeners of tacrine-tacrine ho-
modimers spanning with 6-8 methylene units, which
might display the same trends. The biological results
for these congeners are given in Table 1. The IC50 values
against rat brain AChE obtained for tacrine (1, 333 nM)
and reported 3b (0.2 nM) showed somewhat similar
inclination to that observed by Pang (590 and 0.40 nM,
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
a
b
c
a
b
c
d
e
f
H
H
H
H
H
H
F
CH
CH
CH
CH
CH
CH
CH
CH
CH
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
6
7
115 ( 34
75 ( 11
273 ( 38
328 ( 40
165 ( 21
83 ( 19
54 ( 14
53 ( 17
45 ( 21
257 ( 35
164 ( 32
312 ( 72
2.4
4.4
7.5
8
6
7
8
6
7
8
6
7
8
6
7
8
6
7
8
22 ( 4
1.4 ( 0.1
0.2 ( 0.1
1.6 ( 0.7
0.9 ( 0.3
0.6 ( 0.1
0.7 ( 0.2
0.6 ( 0.2
59
221
33.1
50
F
F
428
234
520
371
647
19.4
45.4
12.1
1.2
g
h
i
Cl CH
Cl CH
Cl CH
H
H
H
H
H
H
0.07 ( 0.01 26 ( 1
0.3 ( 0.2
4.8 ( 1.3
1.3 ( 0.2
1.9 ( 0.2
2.5 ( 0.7
2.7 ( 0.4
1.6 ( 0.4
333 ( 39
194 ( 64
j
N
N
N
CH
CH
CH
93 ( 20
59 ( 14
23 ( 4
k
m
a
b
c
1
3
respectively). In comparison with 3a -c (1.6-0.2 nM
for AChE; 83-53 nM for BChE), carbocyclic-shrinked
congeners 2a -c resulted in almost 100-fold (115-22
nM) and 6-fold (328-165 nM) less potency at AChE and
BChE, respectively. In contrast, ring-expanded 4a -c
had moderate potency (2.7-1.6 nM) on both enzymes.
This suggested that the binding pockets of AChE might
3.3 ( 1.4
2.6 ( 1.3
3.8 ( 0.3
89 ( 7
1.6
1.5
0.3
tacrine
a
Assay performed using rat cortex homogenate and ethopro-
pazine as a specific BChE inhibitor. b Assay performed using
human serum and BW284c51 as a specific AChE inhibitor.
1
8
accommodate a little more bulky moiety.
c
Apparent selectivity for AChE is calculated as IC50(BChE)/
IC50(AChE).
It was already known that the chlorine atom at the
6
- or 8-position increases the inhibitory potency of
Sch em e 1a
11
tacrine. Intriguingly, Savini and co-workers recently
reported that 6,8-dichlorotacrine appeared to be more
potent than the monosubstituted 6- and 8-chlorotacrine
2
1
tacrines, while its homodimeric tacrine showed de-
creased AChE inhibition. These data suggested that the
peripheral site of AChE does not tolerate the simulta-
neous substitution at position 6 and 8 of the bis-tacrine
skeleton. In our studies among these homodimeric
congeners, heptylene-linked bis-(6-chloro)tacrine (3h )
showed much improved potency with an IC50 value of
0.07 nM. This is three times as potent as 3b and over
a
Reagents: (a) POCl3, heat, 2 h.
Sch em e 2a
3
000 times more potent than tacrine against rat AChE.
Together, these results indicated that the binding
pockets of AChE might fit to a limited bulky moiety as
1
8
suggested by Carlier et al. Moreover, bis-(6-fluoro)-
tacrines 3d -f exhibited a little amelioration of potency
as compared to 3b,c. Viewing from the data of 3d -i,
the AChE enzyme tends to tolerate and fit a certain
variation at the 6-position of bis-tacrines. The congeners
with an aza inserted in the tacrine nucleus (3j-m ) still
showed moderate AChEI potency (4.8-1.3 nM). These
results might indicate that the AChE enzyme tends to
tolerate certain changes in aromaticity of the tacrine
nucleus. By summarizing the data of 3a -m , the optimal
potency is reserved by a 7-methylene tether among
these homodimeric congeners (Figure 1). This is con-
sistent with the trend on tacrine dimers as recently
a
Reagents: (a) 1,n-Diaminoalkane (10a -c, n ) 6-8), PhOH,
NaI, 180 °C, 2 h.
method proved to be a lot less replicable after numerous
attempts by variation of the base and reaction temper-
ature.18 Therefore, the related chloride 8a and its
congeners 7, 8b-d , and 9 were chosen as critical
intermediates for the synthesis of homodimeric tacrines
Scheme 1). The POCl3-mediated cyclodehydration reac-
tion between a variety of ortho-amino aromatic acids
a -d and cycloketones 6a -c was adapted to efficiently
(
1
3,16
observed by Pang and co-workers.
5
Inspection of the BChE data for these dimeric con-
geners indicated no clear trend in inhibition potency
(Figure 2). Interestingly, cycloheptyl-fused congeners
produce in situ the corresponding chlorides 7-9 with
moderate yields (54-94%). Treatment of the chlorides

Homodimeric Tacrine Congeners as Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2279
0.1 mM), 10 µL of homogenate, and followed by 50 µL
(
of acetylthiocholine iodide (4.8 mM) after 2 min. The
reaction was terminated by the addition of 5,5′-dithiobis-
(2-nitrobenzoic acid) (0.2% w/v, 50 µL). Enzyme activity
was determined by measuring the absorbance at 420
nm (Shimadzu UV-160A) after 7 min, relative to the
drug-free control.
Deter m in a tion of BCh E In h ibition . Plasma was
preincubated for 5 min with BW284c51 (2 mM). To a 1
mL UV cell were added 880 µL of Tris buffer (0.1 mM,
pH 7.4), 5 µL of the tested compound (0.1 mM), 10 µL
of plasma, and followed by 50 µL of butyrylthiocholine
iodide (6.4 mM) after 2 min. The reaction was termi-
nated by the addition of 5,5′-dithiobis(2-nitrobenzoic
acid) (0.2% w/v, 50 µL). Enzyme activity was determined
by measuring the absorbance at 420 nm after 7 min,
relative to the drug-free control.
F igu r e 1. AChE inhibition by homodimeric tacrine congeners
as a function of number of methylene units.
Triplicate measurements were performed at typically
a total of six concentrations for all above enzyme
studies. The IC50 values were determined from a plot
of percentage of inhibition vs -log [drug], which was
processed by a software of Sigma Plot 4.0.
Con clu sion s
In these studies, we integrated previous findings to
carry out on the 6-position substitution, changes in
carbocyclic ring size, and isosteric modification toward
enhanced optimization for AChE inhibition potency. We
discovered 3h to be a highly potent and selective
inhibitor of AChE. The data from these studies fit the
putative model of dual site binding on AChE as previ-
ously suggested. Moreover, most homodimeric tacrines
with potent AChE inhibition (e.g., 3a -i, except 3h ) are
still less active toward BChE, consistent with the
tentative suggestion of the absence of a peripheral site
on the enzyme. These studies also provided the evidence
that a tether of 7-methylene units is optimal for AChE
inhibition and AChE/BChE selectivity among these
homodimeric bis-tacrine congeners. The pronounced
enhancement of AChE inhibition potency and AChE/
BChE selectivity was achieved with incorporation of a
halogen at the 6-position of homodimeric tacrines.
F igu r e 2. BChE inhibition by homodimeric tacrine congener
as a function of number of methylene units.
4
a -c showed similar accommodation to both AChE and
BChE with IC50 values in the range of 4-1 nM, while
those (e.g., 2a -c) with cyclopentyl nucleus showed the
least fit to both enzymes (114-22 nM and 328-165 nM).
These results disclosed that BChE seems to be better
able than AChE to accommodate steric bulk around the
catalytic site.18 Most 6-substituted bis-tacrines are
considerably potent in inhibiting AChE and still less
active toward BChE (e.g., 3d -i, except 3h , IC50 values,
3
12-45 nM). These observations come up with retaining
high AChE/BChE selectivity. The marked AChE/BChE
selectivity strongly implied the existence of the periph-
eral site at binding pockets, which was suggested as a
strategy for the development of highly selective AChE
1
3
inhibitors.
Deta iled P h a r m a cologica l P r oced u r e
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. All commercial chemicals
were obtained from Sigma Co. (St. Louis, MO) and were
used without further purification. All of the instruments
Ch em istr y. All ortho-amino aromatic acids and cycloke-
tones were obtained from Aldrich (Milwaukee, WI). H and
C nuclear magnetic resonance (NMR) spectra were recorded
on a Varian Gemini-300 instrument. High-resolution mass
spectra were obtained on a J EOL J . M. S.-300 spectrometer.
Elemental analysis was performed by the Taipei Instrumental
Center, National Science Council (Taipei, Taiwan). Reactions
were followed by thin-layer chromatography (TLC) on Merck
1
1
3
(e.g., scissors, forceps, spoons, clips, and homogenizers)
were kept clean and cool in an ice bath before use.
P r ep a r a tion of Ra t Br a in Hom ogen a te. Rats were
decapitated, and the brain was dissected on ice. Rat
brain homogenate was obtained by centrifugation (2530
rpm, 10 min) of homogenized frontal cortex in 10 mM
Tris buffer (3 mL, pH 7.4) at 4 °C (Eppendorf, Centrifuge
(0.2 mm) aluminum-packed precoated silica gel plates (60 F254
)
that were visualized by phosphomolybdic acid alcoholic solu-
tion under a heating plate.
8
-Ch lor o-2,3-dih ydr o-1H-cyclopen ta[1,2-b]qu in olin e (7).
To a mixture of acid 5a (4.11 g, 30.0 mmol) and ketone 6a
2.65 mL, 30.0 mmol) was carefully added 25 mL of POCl at
5
402). It was kept in several vials at -80 °C and used
as a source of AChE.
(
3
P r ep a r a tion of Hu m a n Ser u m . Human serum was
obtained by centrifugation of 10 mL of heparinized
whole blood (3500 rpm, 10 min) at 4 °C. It was kept in
several vials at -25 °C and was the source of BChE.
Deter m in a tion of ACh E In h ibition . Cortex homo-
genate was preincubated for 5 min with ethopropazine
ice bath. The mixture was heated under reflux for 2 h, then
cooled at room temperature, and concentrated to give a slurry.
The residue was diluted with EtOAc, neutralized with aqueous
K
2
CO
over anhydrous K
a pale brown solid. It was recrystallized from acetone to give
3
, and washed with brine. The organic layer was dried
2
CO and concentrated in vacuo to furnish
3
1
7
)
(5.50 g, 90%); mp 85-87 °C. H NMR (CDCl
3
): δ 8.09 (d, J
(20 mM). To a 1 mL UV cell were added 880 µL of Tris
8.4 Hz, 1H), 7.99 (d, J ) 8.4 Hz, 1H), 7.63 (t, J ) 6.9 Hz,
buffer (0.1 mM, pH 7.4), 5 µL of the tested compound
1H), 7.52 (t, J ) 7.1 Hz, 1H), 3.18 (t, J ) 7.9 Hz, 2H), 3.10 (t,

2
280 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11
Hu et al.
J ) 7.5 Hz, 2H), 2.19 (qint, J ) 7.4 Hz, 2H). FABMS: m/z [M
465.2. HR-FABMS: exact mass calcd for C31
H
37
N
4
[M + H]+,
+
+
+
H] 204.0. HR-FABMS: exact mass calcd for C12
H
11NCl [M
465.3017; found, 465.3012. Anal. (C31
calcd, 77.13; found, 77.82.
H
36
N
4
2
‚H O) H, N. C:
+
H] , 204.0580; found, 204.0579.
-Ch lor o-1,2,3,4-tetr a h yd r oa cr id in e (8a ).19a Compounds
a (7.4 g, 53.9 mmol) and 7b (5.36 mL, 51.7 mmol) were
N,N′-Bis-(2,3-d ih yd r o-1H-cyclop en ta [1,2-b]qu in olin -8-
yl)-1,8-d ia m in oocta n e (2c). Compounds 7 (0.82 g, 4.00
mmol) and 10c (0.29 g, 2.00 mmol) were combined as above
9
5
condensed as above to afford 8a (11.4 g, 94%); mp 68-70 °C
1
1
to afford 2c (0.30 g, 31%) as a brown solid; mp 50-52 °C. H
(
literature mp 66-68 °C). H NMR (CDCl ): δ 8.13 (d, J )
3
NMR (CDCl ): δ 7.91 (d, J ) 8.7 Hz, 2H), 7.74 (d, J ) 8.4 Hz,
3
7
1
2
2
.5 Hz, 1H), 8.00 (d, J ) 8.3 Hz, 1H), 7.63 (dd, J ) 9.2, 7.5 Hz,
H), 7.51 (dd, J ) 9.2, 8.3 Hz, 1H), 3.10 (t, J ) 6.3 Hz, 2H),
2
H), 7.55 (t, J ) 7.1 Hz, 2H), 7.36 (t, J ) 7.3 Hz, 2H), 4.72 (s
+
br, 2H, 2 NH), 3.57 (q, J ) 6.3 Hz, 2H), 3.19 (t, J ) 7.4 Hz,
4H), 3.05 (t, J ) 7.7 Hz, 4H), 2.14 (quint, J ) 7.4 Hz, 4H),
.97 (t, J ) 4.8 Hz, 2H), 1.91 (s br, 4H). EIMS: 217 (M , 100),
+
19 (M + 2 , 33). HR-EIMS: exact mass calcd for C13
H
12NCl
+
+
1.70-1.55 (m, 4H), 1.50-1.30 (m, 8H). FABMS: m/z [M + H]
[M] , 217.0659; found, 217.0648.
+
4
4
79.2. HR-FABMS: exact mass calcd for C32
H
39
N
4
[M + H] ,
9
-Ch lor o-6-flu or o-1,2,3,4-tetr a h yd r oa cr id in e (8b). Com-
79.3174; found, 479.3175. Anal. (C32
H
38
N
4
‚2H
2
O) C, H, N.
pounds 5b (3.0 g, 19.6 mmol) and 6b (2.05 mL, 19.6 mmol)
Hexylen e-Lin k ed Bis-ta cr in e (3a ). Compounds 8a (0.75
g, 3.5 mmol) and 10a (0.2 g, 1.75 mmol) were combined as
above to afford 3a (0.47 g, 56%) as an amber glass foam; mp
were condensed as above to afford 8b (2.49 g, 54%) as a light
1
brown solid; mp 75-77 °C. H NMR (CDCl
3
): δ 8.11 (dd, J )
9
1
.3, 6.0 Hz, 1H), 7.57 (dd, J ) 9.1, 2.5 Hz, 1H), 7.29-7.26 (m,
H), 3.06 (s br, 2H), 2.96 (s br, 2H), 1.92 (t, J ) 3.3 Hz, 4H).
1
9
(
4-96 °C. H NMR (CDCl
3
): δ 7.94 (d, J ) 4.1 Hz, 2H), 7.91
d, J ) 4.1 Hz, 2H), 7.54 (t, J ) 7.0 Hz, 2H), 7.32 (t, J ) 7.0
+
FABMS: m/z [M + H] 236.0. HR-FABMS: exact mass calcd
Hz, 2H), 4.00 (s br, 2H), 3.47 (t, J ) 7.1 Hz, 4H), 3.06 (s br,
+
for C13
,9-Dich lor o-1,2,3,4-tetr ah ydr oacr idin e (8c). Compounds
c (8.58 g, 50.0 mmol) and 6b (5.18 mL, 50.0 mmol) were
condensed as above to afford 8c (11.67 g, 93%) as a brown solid;
H12NFCl [M + H] , 236.0642; found, 236.0644.
4
1
H), 2.68 (s br, 4H), 1.89 (t, J ) 2.4 Hz, 8H), 1.65 (s br, 4H),
6
+
.41 (s br, 4H). FABMS (NBA as matrix): m/z [M + H] 479.2.
5
+
HR-FABMS: exact mass calcd for C32
H
39
N
4
[M + H] , 479.3173;
found, 479.3187. Anal. (C32
Hep tylen e-Lin k ed Bis-ta cr in e (3b). Compounds 8a
H
38
N
4
‚H
2
O) C, H, N.
1
mp 81-83 °C. H NMR (CDCl
7
2
3
): δ 8.88 (s 1H), 8.27 (dd, J )
17
.08, 1.38 Hz, 1H), 7.74 (dd, J ) 7.10, 1.98 Hz, 1H), 3.64 (s br,
(0.75 g, 3.5 mmol) and 10b (0.23 g, 1.75 mmol) were combined
H), 3.09 (s br, 2H), 2.02 (s br, 4H). EIMS: m/z [M]+ 251
1
as above to afford 3b (0.40 g, 47%) as an amber oil. H NMR
+
(100%). HR-EIMS: exact mass calcd for C13
H
11NCl
2
[M] ,
(
7
3
CDCl ): δ 7.97 (d, J ) 3.2 Hz, 2H), 7.94 (d, J ) 3.3 Hz, 2H),
2
51.0271; found, 251.0277.
-Ch lor o-1,2,3,4-t et r a h yd r o-cycloh exa [1,2,-b]p yr id o-
2,3-b]p yr id in e (8d ). Compounds 5d (4.14 g, 30 mmol) and
b (3.11 mL, 30 mol) were condensed as above to afford 8d
3.92 g, 86%) as a brown solid; mp 146-149 °C. 1H NMR
CDCl ): δ 9.02 (dd, J ) 1.7, 4.2 Hz, 1H), 8.49 (dd, J ) 1.8,
.4 Hz, 1H), 7.46 (dd, J ) 4.2, 8.3 Hz, 1H), 3.19 (s br, 2H),
.00 (t, J ) 6.1 Hz, 2H), 2.03-1.90 (m, 4H). EIMS: m/z 220
.55 (t, J ) 6.8 Hz, 2H), 7.34 (t, J ) 7.1 Hz, 2H), 4.10 (s br,
9
2H), 3.50 (t, J ) 7.4 Hz, 4H), 3.07 (s br, 4H), 2.69 (s br, 4H),
1.90 (s br, 8H), 1.65 (s br, 4H), 1.37 (s br, 6H). FABMS: m/z
[
6
(
(
8
3
+
[M + H] 493.2. HR-FABMS: exact mass calcd for C33
H
41
N
4
+
[M + H] , 493.3330; found, 493.3346. Anal. (C33
H
40
N
4
2
‚1.5H O)
C, H, N.
3
Octylen e-Lin k ed Bis-ta cr in e (3c).17 A mixture of 8a (0.75
g, 3.5 mmol), 10c (0.25 g, 1.75 mmol), phenol (2.0 g), and NaI
+
+
[
M + 2 , 33], 218 [M , 100].
0-Ch lor o-2,3,4,5-tetr ah ydr o-1H-cycloh epta[1,2,-b]qu in -
olin e (9). Compounds 5a (2.05 g, 14.9 mmol) and 6c (1.27 mL,
4.9 mmol) were condensed as above to afford 9 (1.55 g, 45%)
(0.1 g) was treated as above to afford 3c (0.46 g, 51%) as an
1
amber oil. H NMR (CDCl
(
3
): δ 7.97 (d, J ) 8.5 Hz, 2H), 7.92
1
d, J ) 8.5 Hz, 2H), 7.55 (t, J ) 7.1 Hz, 2H), 7.34 (t, J ) 6.0
Hz, 2H), 3.49 (t, J ) 7.4 Hz, 4H), 3.07 (s br, 4H), 2.70 (s br,
4H), 1.91 (s br, 8H), 1.65 (s br, 4H), 1.45-1.20 (m, 8H).
1
1
as a pale brown solid; mp 87-89 °C. H NMR (CDCl
d, J ) 8.4 Hz, 1H), 8.20 (d, J ) 8.2 Hz, 1H), 7.77 (t, J ) 6.7
Hz, 1H), 7.69 (t, J ) 7.6 Hz, 1H), 3.54 (t, J ) 4.5 Hz, 2H),
3
): δ 8.56
+
FABMS: m/z [M + H] 507.2. HR-FABMS: exact mass calcd
(
+
for C34
(C34
H
N
43
N
‚H
4
[M + H] , 507.3487; found, 507.3470. Anal.
+
H
42
4
2
O) C, H, N.
3
2
2
.24-3.16 (m, 2H), 1.90-1.70 (m, 6H). FABMS: m/z [M + H]
32.0. HR-FABMS: exact mass calcd for C14
32.0869; found, 232.0888.
+
Hexylen e-Lin k ed Bis-(6-flu or o)ta cr in e (3d ). Compounds
b (0.5 g, 2.13 mmol) and 10a (0.18 g, 1.07 mmol) were
H15NCl [M + H] ,
8
combined as above to afford 3d (0.31 g, 56%) as brown glass
N,N′-Bis-(2,3-d ih yd r o-1H-cyclop en ta [1,2-b]qu in olin -8-
1
foam; mp 83-85 °C. H NMR (CDCl ): δ 7.95 (dd, J ) 9.3, 6.1
3
yl)-1,6-d ia m in oh exa n e (2a ). A mixture of 7 (0.60 g, 3.00
mmol), 10a (0.18 g, 1.50 mmol), phenol (1.5 g), and NaI (0.07
g) was carefully heated at 180 °C under an inert system for 2
h and then cooled at room temperature. The mixture was
diluted with EtOAc and made basic with 10% KOH solution.
The organic layer was washed with water and brine and dried
Hz, 2H), 7.50 (dd, J ) 10.2, 2.4 Hz, 2H), 7.09 (td, J ) 7.2, 2.4
Hz, 2H), 3.47 (t, J ) 2.6 Hz, 4H), 3.02 (s br, 4H), 2.75-2.65
(
4
C
m, 4H), 1.90-1.80 (m, 8H), 1.78-1.60 (m, 4H), 1.50-1.25 (m,
+
H). FABMS: m/z [M + H] 515.2. HR-FABMS: calcd for
+
32
H
37
N
4
F
2
[M + H] , 515.2985; found, 515.2996. Anal.
‚2H O) C, H. N: calcd, 10.19; found, 9.34.
H ep t ylen e-Lin k ed Bis-(6-flu or o)t a cr in e (3e). Com-
(C
32
H
36
N
4
F
2
2
over anhydrous MgSO
residue was purified on silica gel chromatography (CH
MeOH:NH OH ) 10:1:1) to give 2a (0.23 g, 34%) as amber
glass foam; mp 64-66 °C. H NMR (CDCl
4
. After concentration, the resulting
2
2
Cl :
pounds 8b (0.5 g, 2.13 mmol) and 10b (0.14 g, 1.07 mmol) were
combined as above to afford 3e (0.26 g, 47%) as brown glass
4
1
3
): δ 7.91 (d, J )
.3 Hz, 2H), 7.71 (d, J ) 8.7 Hz, 2H), 7.55 (t, J ) 7.7 Hz, 2H),
.35 (t, J ) 7.7 Hz, 2H), 4.68 (s br, 2H, 2 NH), 3.59 (q, J ) 6.6
1
foam; mp 92-94 °C. H NMR (CDCl
3
): δ 7.99 (dd, J ) 9.6, 6.2
8
7
Hz, 2H), 7.58 (dd, J ) 9.9, 2.2 Hz, 2H), 7.10 (td, J ) 8.0, 2.4
Hz, 2H), 3.53 (t, J ) 7.1 Hz, 4H), 3.05 (s br, 4H), 2.66 (s br,
Hz, 2H), 3.17 (t, J ) 6.9 Hz, 4H), 3.05 (t, J ) 7.6 Hz, 4H),
.20-2.00 (m, 4H), 1.70-1.55 (m, 4H), 1.55-1.40 (m, 4H).
4
(
H), 1.90 (t, J ) 3.1 Hz, 8H), 1.80-1.60 (m, 4H), 1.50-1.20
2
+
m, 6H). FABMS: m/z [M + H] 529.2. HR-FABMS: exact
+
FABMS: m/z [M + H] 451.2. HR-FABMS: exact mass calcd
for C30
+
mass calcd for C33
Anal. (C33
H
39
N
‚2H
4
F
2
[M + H] , 529.3142; found, 529.3161.
+
H
N
35
N
4
[M + H] , 451.2860; found, 451.2860. Anal.
‚1.5H O) C, H, N.
H
38
N
4
F
2
2
O) C, H, N.
(C
30
H
34
4
2
Octylen e-Lin k ed Bis-(6-flu or o)ta cr in e (3f). Compounds
b (0.4 g, 1.70 mmol) and 10c (0.13 g, 0.85 mmol) were
N,N′-Bis-(2,3-d ih yd r o-1H-cyclop en ta [1,2-b]qu in olin -8-
8
yl)-1,7-d ia m in oh ep ta n e (2b). Compounds 7 (0.82 g, 4.00
mmol) and 10b (0.26 g, 2.00 mmol) were combined as above
combined as above to afford 3f (0.24 g, 51%) as brown glass
1
foam; mp 78-80 °C. H NMR (CDCl ): δ 7.97 (dd, J ) 9.1, 6.1
3
1
to afford 2b (0.33 g, 36%) as a brown solid; mp 57-59 °C. H
Hz, 2H), 7.55 (dd, J ) 10.4, 2.5 Hz, 2H), 7.09 (td, J ) 7.3, 2.5
Hz, 2H), 3.50 (t, J ) 6.9 Hz, 4H), 3.02 (s br, 4H), 2.65 (s br,
4H), 1.89 (s br, 8H), 1.65 (t, J ) 6.9 Hz, 4H), 1.50-1.20 (m,
NMR (CDCl ): δ 7.93 (d, J ) 8.5 Hz, 2H), 7.75 (d, J ) 8.3 Hz,
3
2
H), 7.55 (t, J ) 7.9 Hz, 2H), 7.35 (t, J ) 7.2 Hz, 2H), 4.70 (s
+
br, 2H, 2 NH), 3.58 (q, J ) 6.8 Hz, 4H), 3.19 (t, J ) 7.1 Hz,
8H). FABMS: m/z [M + H] 543.2. HR-FABMS: exact mass
+
4
1
H), 3.05 (t, J ) 7.8 Hz, 4H), 2.13 (quint, J ) 7.4 Hz, 4H)+,
.70-1.55 (m, 4H), 1.55-1.40 (m, 6H). FABMS: m/z [M + H]
calcd for C34
Anal. (C32
H
41
N
4
F
2
[M + H] , 543.3298; found, 543. 3310.
H
36
N
4
F
2
2
‚2H O) H, N. C: calcd, 70.55; found, 70.07.

Homodimeric Tacrine Congeners as Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 11 2281
1
Hexylen e-Lin ked Bis-(6-ch lor o)tacr in e (3g). Compounds
c (0.75 g, 3.00 mmol) and 10a (0.17 g, 1.50 mmol) were
combined as above to afford 3g (0.25 g, 31%) as amber glass
to afford 4b (64 mg, 22%) as a glass foam; mp 64-66 °C. H
8
3
NMR (CDCl ): δ 7.91 (d, J ) 8.5 Hz, 2H), 7.85 (d, J ) 8.5 Hz,
2H), 7.51 (t, J ) 7.4 Hz, 2H), 7.36 (t, J ) 7.3 Hz, 2H), 3.88 (s
br, 2H, 2 NH), 3.21 (s br, 4H), 3.15-3.10 (m, 4H), 2.91 (s br,
1
foam; mp 73-75 °C. H NMR (CDCl
3
): δ 7.88 (s, 2H), 7.86 (d,
J ) 9.0 Hz, 2H), 7.25 (d, J ) 9.0 Hz, 2H), 4.00 (s br, 2H), 3.47
4H), 1.95-1.70 (m, 16H), 1.50-1.25 (m, 6H). FABMS: m/z [M
+
(
8
5
t, J ) 7.0 Hz, 4H), 3.01 (s br, 4H), 2.63 (s be, 4H), 1.88 (s br+,
+ H] 521.2. HR-FABMS: exact mass calcd for C35
H
‚2H
45
N
2
4
[M
O) C,
+
H), 1.65 (s br, 4H), 1.41 (s br, 4H). FABMS: m/z [M + H]
+ H] , 521.4644; found, 521.3640. Anal. (C35
H
44
N
4
47.1. HR-FABMS: exact mass calcd for C32
H
37
N
Cl
4
Cl
‚H
2
[M +
O) C,
H, N.
+
H] , 547.2395; found, 547.2365. Anal. (C32
H, N.
H
36
N
4
2
2
N,N′-Bis-(2,3,4,5-tetr a h yd r o-1H-cycloh ep ta [1,2,-b]qu in -
olin -10-yl)-1,8-d ia m in oocta n e (4c). Compounds 9 (0.29 g,
1.25 mmol) and 10c (90 mg, 0.63 mmol) were combined as
H ep t ylen e-Lin k ed Bis-(6-ch lor o)t a cr in e (3h ). Com-
pounds 8c (0.75 g, 3.00 mmol) and 10b (0.19 g, 1.50 mmol)
1
above to afford 4c (94 mg, 28%) as an amber oil. H NMR
were combined as above to afford 3h (0.47 g, 56%) as brown
3
(CDCl ): δ 8.05 (d, J ) 8.4 Hz, 2H), 7.94 (d, J ) 8.5 Hz, 2H),
1
glass foam; mp 67-69 °C. H NMR (CDCl
3
): δ 7.92 (s, 2H),
7.54 (t, J ) 7.3 Hz, 2H), 7.41 (t, J ) 8.8 Hz, 2H), 3.37 (t, J )
4.4 Hz, 2H), 3.20 (s br, 4H), 2.90 (s br, 4H), 1.95-1.65 (m, 16H),
7
7
1
.89 (d, J ) 9.0 Hz, 2H), 7.25 (d, J ) 9.0 Hz, 2H), 3.47 (t, J )
+
.1 Hz, 4H), 3.02 (s br, 4H), 2.64 (s br, 4H), 1.90 (s br, 8H),
1.45-1.20 (m, 8H). FABMS: m/z [M + H] 535.2. HR-
+
+
.64 (s br, 4H), 1.36 (s br, 6H). FABMS: m/z [M + H] 561.2.
FABMS: exact mass calcd for C36
H
47
N
4
[M + H] , 535.3801;
+
HR-FABMS: exact mass calcd for C33
H
39
N
4
Cl
‚H
2
[M + H] ,
found, 535.3777. Anal. (C36
found, 77.79.
H
46
N
4
2
‚2H O) H, N. C: calcd, 78.21;
5
61.2556; found, 561.2541. Anal. (C33
H
38
N
4
Cl
2
2
O) C, H, N.
Octylen e-Lin k ed Bis-(6-ch lor o)ta cr in e (3i). Compounds
c (0.75 g, 3.00 mmol) and 10c (0.22 g, 1.50 mmol) were
combined as above to afford 3i (0.40 g, 46%) as brown glass
En zym e P r epar ation s an d In h ibition Stu dies on ACh E
a n d BCh E. AChE and BChE enzyme preparations were
prepared from cortex of decapitated rats and human plasma,
respectively. Rat brain homogenate was obtained by centrifu-
gation (2530 rpm, 10 min) of homogenized frontal cortex in
Tris buffer (10 mM, pH 7.4) at 4 °C and kept at -80 °C, which
was used as a source of AChE. Human plasma was the source
of BChE and obtained by centrifugation of whole blood (3500
rpm, 10 min) at 4 °C and kept at -25 °C. The cholinesterase
assays were performed using colorimetric method reported by
Ellman.20 For the determination of AChE inhibition, cortex
homogenate was preincubated for 5 min with ethopropazine
(20 mM), a selective inhibitor of BChE. Similarly, for the
determination of BChE inhibition, plasma was preincubated
with BW284c51 (2 mM), a selective inhibitor of AChE. A 1
mL mixture containing acetylthiocholine iodide (4.8 mM) or
butyrylthiocholine iodide (6.4 mM), 880 µL of Tris buffer (0.1
mM, pH 7.4), a solution of the tested compound (0.1 mM), and
homogenate or plasma (10 µL) was incubated at 37 °C for 5
min. The reaction was terminated by the addition of 5,5′-
dithiobis(2-nitrobenzoic acid) (0.2% w/v, 50 µL). Enzyme
activity was determined by measuring the absorbance at 420
nm after 7 min, relative to the drug-free control. Triplicate
measurements were carried out at typically a total of six drug
concentrations. The IC50 values were determined from a plot
of percentage of inhibition vs -log [drug], which was processed
by a software of Sigma Plot 4.0.
8
1
foam; mp 62-63 °C. H NMR (CDCl ): δ 7.91 (s, 2H), 7.88 (d,
3
J ) 8.8 Hz, 2H), 24 (d, J ) 8.8 Hz, 2H), 4.12 (s br, 2H), 3.51
s br, 4H), 3.03 (s br, 4H), 2.65 (s br, 4H), 1.89 (s br, 8H), 1.65
s br, 4H), 1.30 (s br, 8H). FABMS: m/z [M + H] 575.2. HR-
FABMS: exact mass calcd for C34
(
(
+
+
H
41
N
4
Cl
2
2
[M + H] , 575.2713;
found, 575.2691. Anal. (C34
H
40
N
4
Cl
2
‚H
O) C, H, N.
N,N′-Bis-(1,2,3,4-t et r a h yd r o-cycloh exa [1,2,-b]p yr id o-
2,3-b]p yr id -9-yl)-1,6-d ia m in oh exa n e (3j). Compounds 8d
0.43 g, 2.0 mmol) and 10a (0.12 g, 1.0 mmol) were combined
[
(
as above to afford 3j (0.12 g, 25%) as amber glass foam; mp
1
1
8
21-124 °C. H NMR (CDCl
3
): δ 8.86 (d, J ) 2.7 Hz, 2H),
.38 (d, J ) 8.2 Hz, 2H), 7.26-7.21 (m, 2H), 4.50 (s br, 2H, 2
NH), 3.60-3.50 (m, 4H), 3.07 (s br, 4H), 2.70-2.60 (m, 4H),
1
.95-1.80 (m, 8H), 1.70-1.60 (m, 4H), 1.45-1.35 (m, 4H).
+
FABMS: m/z [M + H] 481.4. HR-FABMS: exact mass calcd
+
for C30
H
N
37
N
‚2H
6
[M + H] , 481.3080; found, 481.3057. Anal.
O) H, N. C: calcd, 69.72; found, 69.30.
(
C
30
H
36
6
2
N,N′-Bis-(1,2,3,4-t et r a h yd r o-cycloh exa [1,2,-b]p yr id o-
2,3-b]p yr id -9-yl)-1,7-d i-a m in oh ep ta n e (3k ). Compounds
d (0.46 g, 2.1 mmol) and 10b (0.14 g, 1.1 mmol) were
combined as above to afford 3k (0.18 g, 36%) as amber glass
[
8
1
foam; mp 109-111 °C. H NMR (CDCl
Hz, 2), 8.41 (d, J ) 8.4 Hz, 2H), 7.22 (t, J ) 4.0 Hz, 2H), 4.70
s br, 2H, 2 NH), 3.53 (s br, 4H), 3.08 (s br, 4H), 2.64 (s br,
3
): δ 8.84 (d, J ) 2.5
(
4
6
H), 1.86 (s br, 8H), 1.80-1.50 (m, 4H, 4H), 0.150-1.20 (m,
+
H). FABMS: m/z [M + H] 495.4. HR-FABMS: exact mass
Ack n ow led gm en t. This study was in part sup-
ported by a grant from the National Science Council,
Taiwan (NSC 89-2320-B-016-101). We thank National
Taiwan University and National Tsing Hua University
for fast atom bombardment mass spectra (FAB-MS) and
high-resolution mass spectra (HR-MS) analyses.
+
calcd for C31
H
39
N
6
[M + H] , 495.3236; found, 495.3232. Anal.
(C
31
H
38
N
6
‚2H
2
O) C, H, N.
N,N′-Bis-(1,2,3,4-t et r a h yd r o-cycloh exa [1,2,-b]p yr id o-
[
(
2,3-b]p yr id -9-yl)-1,8-d i-a m in oocta n e (3m ). Compounds 8d
0.57 g, 2.6 mmol) and 10c (0.19 g, 1.3 mmol) were combined
as above to afford 3m (0.15 g, 23%) as an amber solid; mp 89-
1
9
)
(
1 °C. H NMR (CDCl
8.3 Hz, 2H), 7.26-7.16 (m, 2H), 4.44 (s br, 2H, 2 NH), 3.50
s br, 4H), 3.06 (s br, 4H), 2.62 (s br, 4H), 1.85 (s br, 8H), 1.70-
3
): δ 8.84 (d, J ) 4.0 Hz, 2H), 8.37 (d, J
Refer en ces
(
1) St. George-Hyslop, P. H. Piecing Together Alzheimer’s. Sci. Am.
+
1
.50 (m, 4H), 1.45-1.20 (m, 8H). FABMS: m/z [M + H] 509.3.
2
000, 76-83.
+
HR-FABMS: exact mass calcd for C32
found, 509.3392. Anal. (C32 ‚2H
N,N′-Bis-(2,3,4,5-tetr a h yd r o-1H-cycloh ep ta [1,2,-b]qu in -
olin -10-yl)-1,6-d ia m in oh exa n e (4a ). Compounds 9 (0.25 g,
1
H
2
41
N
6
[M + H] , 509.3393;
(2) Nordberg, A. Biological Markers and the Cholinergic Hypothesis
H
40
N
6
O) C, H, N.
in Alzheimer’s Disease. Acta Neurol. Scand. 1992, Suppl. 85,
5
4-58.
(
3) Knapp, M. J .; Knopman, D. S.; Soloman, P. R.; Pendlebury, W.
W.; Davis, C. S.; Gracon, S. I. A 30-week Randomized Controlled
Trial of High-dose Tacrine in Patients with Alzheimer’s Disease.
J . Am. Med. Assoc. 1994, 271, 985-991.
.08 mmol) and 10a (63 mg, 0.54 mmol) were combined as
above to afford 4a (75 mg, 22%) as an amber glass form; mp
1
9
5-97 °C. H NMR (CDCl
d, J ) 7.9 Hz, 2H), 7.55 (t, J ) 6.9 Hz, 2H), 7.41 (t, J ) 7.0
Hz, 2H), 3.30 (t, J ) 4.6 Hz, 2H), 3.20-3.10 (m, 4H), 2.90-
.80 (m, 4H), 1.95-1.60 (m, 16H), 1.50-1.30 (m, 4H).
3
): δ 7.98 (d, J ) 7.8 Hz, 2H), 7.88
(4) Crismon, M. L. Tacrine: First Drug Approved for Alzheimer’s
Disease. Ann. Pharmacotherapy 1994, 28, 744-751.
(
(
5) Watkins, P. B.; Zimmerman, H. J .; Knapp, M. J .; Gracon, S. I.;
Lewis, K. W.; Hepatotoxic Effects of Tacrine Administration
Inpatients with Alzheimer’s Disease. J . Am. Med. Assoc. 1994,
271, 992-998.
2
+
FABMS: m/z [M + H] 507.2. HR-FABMS: exact mass calcd
for C34
+
H
N
43
N
‚2H
4
[M + H] , 507.3488; found, 507.3483. Anal.
(6) Ames, D. J .; Bhathal, P. S.; Davies, B. M.; Fraser, J . R. E.;
Gibson, P. R.; Roberts, S. Heterogenecity of Adverse Hepatic
Reactions to Tetrahydroaminoacridine. N. Z. J . Med. 1990, 20,
(C
34
H
42
4
2
O) C, H, N.
N,N′-Bis-(2,3,4,5-tetr a h yd r o-1H-cycloh ep ta [1,2,-b]qu in -
olin -10-yl)-1,7-d ia m in oh ep ta n e (4b). Compounds 9 (0.26 g,
.12 mmol) and 10b (73 mg, 0.56 mmol) were combined above
1
93-195.
(7) Brufani, M.; Filocamo, L.; Lappa, S.; Maggi, A. New Acetylcho-
1
linesterase Inhibitors. Drugs Future 1997, 22, 397-410.

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8) Siddiqui, M. F.; Levey, A.-I. Cholinergic Therapies in Alzheimer’s
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789-793.
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in Primary Cultured Astrocytes of Mouse Cerebral Cortex by
Bis(7)-tacrine, a Novel Anti-Alzheimer’s Agent. Neurosci. Lett.
(
9) Antuono, P. G. Effectiveness and Safety of Velnacrine for the
Treatment of Alzheimer’s Disease. A double-blind, placebo-
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1
55, 1766-1772.
(
10) Shutske, G. M.; Pierrat, F. A.; Kapples, K. J .; Cornfeld, M. L.;
Szewczak, Huger, F. P.; Bores, G. M.; Haroutunian, V.; Davis,
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11) (a) Gregor, V. E.; Emmerling, M. R.; Lee, C.; Moore, C. J . The
Synthesis and In Vitro Acetylcholinesterase and Butyrylcho-
linesterase Inhibitory Activity of Tacrine (Cognex) Derivatives.
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