Design, synthesis, and structure-activity relationships of a series of 3-[2-(1-benzylpiperidin-4-yl)ethylamino]pyridazine derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1021/jm001088u

Starting from the 3-[2-(1-benzylpiperidin-4-yl)ethylamino]-6-phenylpyridazine 1, we performed the design, the synthesis, and the structure-activity relationships of a series of pyridazine analogues acting as AChE inhibitors. Structural modifications were achieved on four different parts of compound 1 and led to the following observations: (i) introduction of a lipophilic environment in the C-5 position of the pyridazine ring is favorable for the AChE-inhibitory activity and the AChE/BuChE selectivity; (ii) substitution and various replacements of the C-6 phenyl group are possible and led to equivalent or slightly more active derivatives; (iii) isosteric replacements or modifications of the benzylpiperidine moiety are detrimental to the activity. Among all derivatives prepared, the indenopyridazine derivative 4g was found to be the more potent inhibitor with an IC₅₀ of 10 nM on electric eel AChE. Compared to compound 1, this represents a 12-fold increase in potency. Moreover, 3-[2-(1-benzylpiperidin-4-yl)ethylamino]-5-methyl-6-phenylpyridazine 4c, which showed an IC₅₀ of 21 nM, is 100-times more selective for human AChE (human BuChE/AChE ratio of 24) than the reference compound tacrine.

Full text of DOI:10.1021/jm001088u

J . Med. Chem. 2001, 44, 2707-2718
2707
Articles
Design , Syn th esis, a n d Str u ctu r e-Activity Rela tion sh ip s of a Ser ies of
3-[2-(1-Ben zylp ip er id in -4-yl)eth yla m in o]p yr id a zin e Der iva tives a s
Acetylch olin ester a se In h ibitor s
J ean-Marie Contreras,† Isabelle Parrot,† Wolfgang Sippl,‡ Yveline M. Rival,† and Camille G. Wermuth*,†
Laboratoire de Pharmacochimie de la Communication Cellulaire, UMR 7081 du CNRS, Universite´ Louis Pasteur,
Faculte´ de Pharmacie, 74, route du Rhin, 67401 Illkirch Cedex, France, and Institut fu¨r Pharmazeutische Chemie,
Heinrich-Heine-Universita¨t, Du¨sseldorf, Universita¨tsstr. 1, D-40225 Du¨sseldorf, Germany
Received October 2, 2000
Starting from the 3-[2-(1-benzylpiperidin-4-yl)ethylamino]-6-phenylpyridazine 1, we performed
the design, the synthesis, and the structure-activity relationships of a series of pyridazine
analogues acting as AChE inhibitors. Structural modifications were achieved on four different
parts of compound 1 and led to the following observations: (i) introduction of a lipophilic
environment in the C-5 position of the pyridazine ring is favorable for the AChE-inhibitory
activity and the AChE/BuChE selectivity; (ii) substitution and various replacements of the
C-6 phenyl group are possible and led to equivalent or slightly more active derivatives; (iii)
isosteric replacements or modifications of the benzylpiperidine moiety are detrimental to the
activity. Among all derivatives prepared, the indenopyridazine derivative 4g was found to be
the more potent inhibitor with an IC50 of 10 nM on electric eel AChE. Compared to compound
1, this represents a 12-fold increase in potency. Moreover, 3-[2-(1-benzylpiperidin-4-yl)-
ethylamino]-5-methyl-6-phenylpyridazine 4c, which showed an IC50 of 21 nM, is 100-times more
selective for human AChE (human BuChE/AChE ratio of 24) than the reference compound
tacrine.
Ch a r t 1
In tr od u ction
The neurodegenerative Alzheimer’s disease (AD) af-
fecting mainly aging populations in industrialized na-
tions is characterized by three major pathological signs:
â-amyloid plaques, neurofibrillary tangles, and synaptic
loss.1,2 A deficiency in cholinergic neurotransmission is
considered to be one of the major causes of memory
impairments in patients.3,4 A palliative treatment of AD
is possible by the use of agents that restore the level of
acetylcholine.5 Acetylcholinesterase (AChE) inhibitors
such as tacrine,6 donepezil7 (Chart 1), galanthamine,8
and rivastigmine9 are able to enhance memory in AD
patients. Recent studies10,11 have shown that AChE
inhibitors, which interact with both peripheral and
active sites of the enzyme,12,13could in addition to AChE
inhibition act as potential inhibitors of the formation
of âA4-amyloid protein (âAP). Thus, such AChE inhibi-
tors with central bioavailabality represent still a prom-
ising approach to the treatment of AD. In a previous
paper,14 we reported the synthesis and the biochemical
evaluation of a series of 3-aminoalkyl-6-arylpyridazine
derivatives based on the structure of minaprine,15 an
original lead compound with antidepressive properties.
Among all the derivatives investigated, 3-[2-(1-benzylpi-
peridin-4-yl)ethylamino]-6-phenylpyridazine 1 (Chart 1)
was found to be one of the most potent AChE inhibitors
(IC50 ) 120 nM, electric eel) with a selectivity profile
more favorable than tacrine but less than donepezil.
In the present study, we describe the design, the
synthesis (Schemes 1-4), and the structure-activity
relationships (SAR) (Tables 1-5) of a series of 3-[2-(1-
benzylpiperidin-4-yl)ethylamino]pyridazine analogues
prepared with the aim of increasing the potency of
compound 1 and its selectivity toward AChE inhibition
versus BuChE inhibition.
Design
* Address correspondence to Prof. C. G. Wermuth. Tel: +33 388 67
37 22. Fax: +33 388 67 47 94. E-mail: wermuth@pharma.u-strasbg.fr.
† Universite´ Louis Pasteur.
The crystallographic structure of AChE from Torpedo
californica16 has been well-known for 10 years. More-
‡ Heinrich-Heine-Universita¨t.
10.1021/jm001088u CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/20/2001

2708 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Contreras et al.
Ta ble 1. Anticholinesterase Activity of 4- and
5-Substituted-6-aryl-3-benzylpiperidinylethylaminopyridazines
a
b
All melting points refer to hydrochlorides. Electric eel ace-
tylcholinesterase. c Reference 14.
observed in the X-ray structure of AChE complexes.21
Hydrophobic and van der Waals interactions are also
evident for the protein-inhibitor model. Inhibitor 1
takes advantage of the aromatic residues present in the
gorge by making a variety of interactions. van der Waals
interactions of the piperidine ring occur with the phenyl
rings of Phe331 and Tyr334. The benzyl ring of deriva-
tive 1 displays π-π stacking with the aromatic ring of
Trp84. It occupies the binding site for the quaternary
ligands, such as edrophonium or decamethonium.17 The
phenyl-aminopyridazine part of 1 is located at the
entrance of the gorge and interacts with two aromatic
residues (Trp279 and Tyr70) which are involved in the
AChE peripheral site. The phenyl ring, which is not
coplanar with the pyridazine heterocycle, seems to
display a π-π stacking with the aromatic ring of
Trp279. Thus, the aminopyridazine ring occupies the
same region in space as the quaternary trimethylam-
monium group of decamethonium in the corresponding
X-ray structure.17 In general, our inhibitor 1 seems to
interact with both peripheral and active sites of the
enzyme. Huperzine A dimer22 and bis-tetrahydroami-
nacrine23 were found to have the same binding mode.
Although this molecular modeling study provided us
some interesting information for the design of potent
inhibitors, this computational work was performed after
we started a classical structure activity strategy. Thus,
four types of structural modifications were investigated
in order to improve the anti-AChE activity of 1. First,
substitution of pyridazine ring at C-4 and C-5 positions
resulted in a series of 1 analogues (4a -f in Table 1).
Second, introduction of a methylene bridge between the
C-5 pyridazine position and the phenyl group gave
tricyclic analogues of 1 (4g-j in Table 2). Third,
replacement of the phenyl moiety by nonaromatic and
aromatic groups was also investigated (4k , 5, 6a -o, and
7 in Table 3). Finally, benzylpiperidinyl-ethylamine
F igu r e 1. Comparison between the predicted position of
compound 1 (magenta) and the X-ray structure of decametho-
nium (green) are shown. (Nitrogen atoms are colored blue.)
Only the amino acid residues of the binding pocket are
displayed for clarity.
over, X-ray structures of the enzyme complexed with
inhibitors made the binding site characterization
possible.17-20 These studies showed that AChE pos-
sesses two distinct binding sites for the substrate
acetylcholine: an active site (catalytic site) and a
peripheral site (allosteric site). The peripheral site is
localized at the entrance of a deep and narrow channel
(about 20 Å long and as narrow as 4.5 Å). This gorge,
which is lined with aromatic residues (ca. 40% of the
residues present in the binding pocket), leads to the
active site near the bottom. We used the X-ray structure
of AChE to investigate the possible binding modes of
our inhibitors. A preliminary molecular docking study
was carried out (as described in the Computational
Methods section) mainly on the previously described
inhibitor 1. Thus, Figure 1 shows the calculated and
predicted position of compound 1 (magenta) in the
binding pocket of the AChE-decamethonium crystal-
lographic structure17(decamethonium shown in green).
The results of the docking studies allowed the identifi-
cation of some features of the aminopyridazine 1 binding
mode. The positively charged piperidine nitrogen achieves
a cation-π-cloud interaction with Phe330 and Trp84,
residues involved in the “anionic” part of the enzyme
active site. We observed no direct hydrogen bond
between the polar groups of the inhibitor and the
binding site. However, a water-bridged hydrogen bond
may occur between the piperidine nitrogen of the
inhibitor and Tyr121 or Ser122 (as detected in the
AChE-donepezil crystallographic structure20). Other
comparable water-bridged hydrogen bonds have been

Synthesis of Pyridazine Analogues as AChE Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2709
Ta ble 2. Anticholinesterase Activity of
Ta ble 3. Anticholinesterase Activity of
Tricyclic-6-aryl-3-benzylpiperidinylethylaminopyridazines
6-Substituted-3-benzylpiperidinylethylaminopyridazines
a
b
All melting points refer to hydrochlorides. Electric eel ace-
tylcholinesterase. c Reference 14.
chain analogues of compound 1 were also prepared (10,
12, 14a -c, 16a -b, and 18a -b in Table 4).
Ch em istr y
The 3-[2-(1-benzylpiperidin-4-yl)ethylamino]pyridazines
4a -k (Tables 1, 2, and 3) were synthesized according
to Scheme 1. Condensation of the iminochlorides 3 with
the 2-(1-benzylpiperidin-4-yl)ethylamine24 gave the final
pyridazines 4 as described previously.14 Iminochlorides
3 were readily obtained from the corresponding 3(2H)-
pyridazinones 2 by action of phosphorus oxychloride.
Most of the 3(2H)-pyridazinones 2 were prepared ac-
cording to the literature.25-31 The synthesis of 5-alkyl-
6-phenyl-3(2H)-pyridazinones 2c-e was realized by
using the one-pot procedure described by Coates et al.32
Condensation of the 3-chloro-5,6-dihydrobenzo[h]cinno-
line 3h 33 with the benzylpiperidinyl-ethylamine by
using acid condition (NH4Cl) gave the benzocinnoline
4i (Table 2) in 25% yield. The dihydrobenzocinnoline
derivative 4h was obtained in neutral condition without
solvent (e condition, Scheme 1). Suzuki coupling of
available aryl boronic acids with 6-chloro-3-[2-(1-ben-
zylpiperidin-4-yl)ethylamino]pyridazine 5 described pre-
viously34 was used to prepare 6-aryl-3-[2-(1-benzylpiper-
idin-4-yl)ethylamino]pyridazines 6a -o with good yields
(Scheme 2, Table 3). Nucleophilic displacement of
compound 5 by NaOMe afforded methoxypyridazine 7
in 50% yield (Scheme 2). The preparation of the isosteric
compounds 10 and 12 (Table 4) is shown in Scheme 3.
A slightly modified method described by Dutta et al.35
allowed us to obtain the 1-benzyl-4-[(ethoxycarbonyl)-
methylene]piperidine 8 in 84% yield, which gave com-
pound 9 after reduction with LiAlH4. Condensation of
the sodium alcoolate of 9 with 3-chloro-6-phenylpy-
ridazine36 provided the alkoxypyridazine derivative 10.
Treatment of 9 with thionyl chloride allowed 1-benzyl-
4-(2-chloroethyl)piperidine37 11 to be obtained which is
condensed with 3-mercapto-6-phenylpyridazine38 to give
the alkylthiopyridazine derivative 12. The acetamidopy-
ridazine derivatives 14a -c (Scheme 4, Table 4) were
prepared by coupling the activated carboxylic acids
a
b
All melting points refer to hydrochlorides. Electric eel ace-
tylcholinesterase. c Reference 14. Reference 34.
d
13a -c with the 3-amino-6-phenylpyridazine. The 2-(1-
benzylpiperidin-4-yl)acetic acid 13a was obtained by the
hydrolysis product of 2-(1-benzylpiperidin-4-yl)acetoni-
trile.14 Alkylation of commercial 1-benzylpiperazine and
4-benzylpiperidine by means of ethyl chloroacetate
followed by saponification gave, respectively, the 2-(4-
benzylpiperazin-1-yl)acetic acid 13b and the 2-(4-ben-
zylpiperidin-1-yl)acetic acid 13c. The diamine chains
15a and 15b were synthesized, respectively, by alkyla-
tion with halogenonitrile of the secondary amine (1-
benzylpiperazine and 4-benzylpiperidine, respectively)
followed by reduction. Gabriel synthesis with the com-
mercially available bromoalkylphthalimide can also be
used for the preparation of 15a and 15b (Scheme 4).
The carboxyamine chains 17a and 17b were prepared
starting from the corresponding commercially available
secondary amines by alkylation with chloroacetic chlo-
ride followed by a Gabriel synthesis (Scheme 4). Con-
densation of the 3-chloro-6-phenylpyridazine36 with
primary amines 15a -b and 17a -b yielded, respec-
tively, compounds 16a -b (acid conditions) and com-
pounds 18a -b (basic conditions) (Scheme 4).

2710 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Contreras et al.
Sch em e 2a
Ta ble 4. Anticholinesterase Activity of 6-Phenylpyridazines
with Various Side Chains
a
Reagents and conditions: (a) 2-(1-benzylpiperidin-4-yl)ethyl-
amine, HCl/H2O/acetone, 100 °C; (b) ArB(OH)2, base, solvent, 100
°C; (c) MeONa, DMF, 130 °C.
Sch em e 3a
a
b
All melting points refer to hydrochlorides. Electric eel ace-
tylcholinesterase. c Reference 14.
Sch em e 1a
a
Reagents and conditions: (a) (EtO)2P(O)CH2CO2Et, THF,
K2CO3, reflux; (b) LiAlH4, THF, reflux; (c) Na, THF; (d) 3-chloro-
6-phenylpyridazine, reflux; (e) SOCl2, CH2Cl2, reflux; (f) 6-phenyl-
3-thiolpyridazine, EtONa, EtOH, reflux.
a
Reagents and conditions: (a) R3-CO-CO2H, rt; (b) NH2NH2‚H2O,
led to compounds showing weaker activities (Table 1).
Thus, isopropyl derivative 4b was found to be 3 times
less potent than the lead compound 1. On the other
hand, the presence of an alkyl group in the C-5 position
(4c-e) increased the anti-AChE potency. Compound 4c
reflux; (c) POCl3, 75 °C; (d) 2-(1-benzylpiperidin-4-yl)ethylamine,
NH4Cl, n-BuOH, 130 °C; (e) 2-(1-benzylpiperidin-4-yl)ethylamine,
100 °C.
Resu lts a n d Discu ssion
was the best compound from this series with an IC50
)
The pyridazine derivatives were tested for in vitro
inhibition of acetylcholinesterase on the commercially
available electric eel AChE (Tables 1-4). The results
obtained with this enzyme preparation allowed us to
investigate molecular modeling studies (described in
another paper39) using the published AChE structure
which is also derived from the electric eel enzyme.16 In
addition to electric eel AChE, we used human erythro-
cytes as a source of human AChE and human serum as
a source of human butyrylcholinesterase (BuChE) to
determine the selectivity profile of the most potent
inhibitors (Table 5). The esterase activity was deter-
mined according to the method of Ellman et al.40
We observed that introduction of a lipophilic and
aliphatic group in the C-4 position of the pyridazine ring
21 nM (a 6-fold increase in activity compared to 1).
Slight increases were observed for derivatives with an
ethyl (4d ) or propyl (4e) group. The combined introduc-
tion of an aromatic environment in the C-4 and C-5
position (phthalazine ring) gave a less potent derivative
(4f). In general, it seems that the C-5 substitution with
an aliphatic group is particularly favorable for anti-
AChE activity.
All the constrained tricyclic pyridazines (4g-j) showed
an increased inhibitory activity (Table 2). With an IC50
value of 10 nM, the indenopyridazine derivative 4g was
found to be 12 times more potent than compound 1. The
benzocinnoline compound 4i showed a slight increase
in activity compared to its saturated analogue 4h . The
ring extension (n ) 3) led to the benzocycloheptapy-

Synthesis of Pyridazine Analogues as AChE Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2711
Sch em e 4a
a
Reagents and conditions: (a) NaOH, EtOH, reflux; (b) ClCH2CO2Et, toluene, TEA, reflux; (c) NaOH, MeOH, reflux; (d) (COCl)2, rt;
(e) 3-amino-6-phenylpyridazine, CH2Cl2, TEA, rt; (f) NCCH2Cl, K2CO3, reflux; (g) LiAlH4, THF, rt; (h) N-bromoethylphthalimide, xylene,
130 °C; (i) NH2NH2‚H2O, EtOH, reflux; (j) 3-chloro-6-phenylpyridazine, NH4Cl, n-BuOH, 130 °C; (k) ClCH2COCl, CH2Cl2, reflux; (l)
phthalimide-K, DMF, rt; (m) NH2NH2‚H2O, EtOH, ∆; (n) 3-chloro-6-phenylpyridazine, pyridine, reflux.
Ta ble 5. Comparative Activity on Various Cholinesterases
AChE activity. A hydrophobic and van der Waals
interaction with the enzyme binding site is probably
responsible for the potency increase.
Surprisingly, the deletion of the phenyl moiety (Table
3) induces only a 2-fold decrease in activity (4k , IC50
)
240 nM). This finding could suggest the slight impor-
tance of the phenyl ring in the binding (hydrophobic
interaction) with the peripheral site. The replacement
of the phenyl group by a halogen atom (5) produces a
weak increase of the anti-AChE activity whereas the
replacement by a methoxy (7) leads to a less active
compound (compared to 1). The isosteric substitution
of the C-6 phenyl ring with thiophene (6n ) and pyridine
(6o) gives, respectively, comparable and 2 times more
potent derivatives. Phenyl ring substitution with elec-
trostatic or hydrophobic groups leads, in most of the
cases, to comparable or slightly more active compounds
(6a -6m ) than 1. Derivative 6c showed a very weak
anti-cholinesterase activity (IC50 ) 3 µM). Among the
6-arylpyridazines investigated, 6g and 6i show, respec-
tively, IC50 values of 56 and 54 nM, which represent a
2-fold increase in potency. On the other hand, the
p-dimethylaminophenyl derivative 6m was found to be
2 times less active than 1. The weak modification (2-
fold maximum) produced by the C-6 pyridazine substi-
tution could suggest rather a π-π stacking between the
pyridazine ring (instead of the phenylpyridazine moiety)
and the indole part of Trp279.
a
b
From electric eel. From human erythrocytes. c From human
serum.
ridazine 4j which is comparable in activity to compound
4i (IC50 ) 22 nM on electric eel AChE). The phenyl ring
of compound 4g is coplanar with the pyridazine hetero-
cycle contrary to derivative 4j. It seems that the
correlation between the uncoplanar phenyl ring ob-
served and the π-π stacking with Trp279 is not so
evident. However, the introduction of a flexible (4c-e)
or constraint (4g-j) lipophilic environment in the C-5
pyridazine position seems to be critical for the anti-
The isosteric replacement of the exo nitrogen atom
of compound 1 by an oxygen and sulfur atom yield,
respectively, the ethoxypyridazine 10 and the ethylthi-
opyridazine 12 (Table 4). Derivative 10 was comparable
to 1 whereas 12 showed a 2-fold increase activity with
an IC50 of 63 nM. The 4-benzylpiperazine 16a and

2712 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Contreras et al.
4-benzylpiperidine 16b analogues were, respectively, 10
and 200 times less potent than the 1-benzylpiperidine
derivative 1. The position of the nitrogen atom in the
aliphatic heterocycle appears to be important for the
anti-AChE activity. The introduction of a carbonyl group
in the ethyl chain was realized in order to obtain an
additional interaction with the enzyme. In all cases
(14a -c and 18a -b), a decrease of the activity in
comparison to the non-carbonyl analogues (1, 16a , and
16b) was observed. All of these observations confirm
that the 1-benzylpiperidine linked to a hydrophobic
chain (as shown previously14) is favorable for the
interaction with the enzyme.
The most active and representative compounds, as
well as two reference inhibitors, tacrine and donepezil,
were then compared in order to determine their relative
inhibitory effects (Table 5) toward human acetyl and
butyrylcholinesterase (ratio of IC50 hBuChE/hAChE).
Compound 4g, which was the most active derivative on
electric eel AChE, and compound 4j have comparable
BuChE/AChE ratios remaining, however, inferior to
that of compound 1. The other tricyclic derivatives (4h
and 4i) did not present a selective profile (ratio close to
1). The 5-alkylpyridazines 4c and 4d were found to be,
respectively, 3- and 5-fold more selective for hAChE
than the reference compound 1. Thus, 3-[2-(1-benzylpi-
peridin-4-yl)ethylamino]-5-methyl-6-phenylpyridazine 4c
was the more selective inhibitor with a ratio of 24 (IC50
of 39 nM for hAChE). The O- (10) and S-isosteric
derivatives (12) present a weak selectivity profile de-
spite their satisfactory anti-AChE activity. Compared
to tacrine, compound 4c is about 100 times more
selective but remains less selective than donepezil.
F igu r e 2. Comparison of the experimentally determined
AChE-donepezil complex and the predicted AChE-aminopy-
ridazine complex (magenta). The aminopyridazine inhibitor 4j
(magenta) adopts a conformation similar to that of donepezil
(green) in the corresponding complex.
benzylpiperidine derivative donepezil has been made
available.20 Like donepezil, our most potent inhibitors
contain a benzylpiperidine moiety which shows a similar
position and orientation when compared to the reported
X-ray structure (illustrated with compound 4j in Figure
2). Both kind of inhibitors adopt a comparable confor-
mation and position in the narrow binding pocket. The
indanone ring of donepezil stacks against the indole ring
of Trp279, in the peripheral binding site, by a classical
π-π interaction. A similar interaction was found for the
arylpyridazine part of our inhibitors. However, experi-
mental results showed that the pyridazine ring is
probably the part of the molecule which interacts with
Trp279.
At the present time, we consider synthesizing new
pyridazine derivatives according to the SAR observa-
tions and to a complete molecular modeling study.39 The
combination of the C-5 methyl substitution with, for
instance, the appropriate phenyl substitution should
allow us to obtain more potent inhibitors. Unfortunately,
the synthesis of such compounds is not so evident.
Indeed, the use of the Suzuki reaction for the 6-aryl-5-
methyl-3-[2-(1-benzylpiperidin-4-yl)ethylamino]py-
ridazines preparation is impossible. Moreover, the
synthesis of 6-aryl-5-methyl-3-chloropyridazine by the
classical route (Scheme 1) is in most of the cases very
difficult (low yields for some substituents on the phenyl
ring).
Con clu sion
The synthesis and biochemical evaluation of a series
of compound 1 analogues led to the design of potent and
selective AChE inhibitors. The most potent inhibitor,
4g, which presents an IC50 of 10 nM on electric eel
AChE, is 60 000 times more active than our initial lead
compound, minaprine,14 and 12 times more active than
1. Structure-activity relationships on four different
parts of 1 indicated that (i) introduction of a flexible or
constraint lipophilic environment in the C-5 pyridazine
ring causes an increase of the potency and human
selectivity; (ii) replacement or substitution of C-6 phenyl
group seems to be less determining for the anti-
cholinesterase activity; (iii) isosteric replacement or
modification of the 1-benzylpiperidine ethylamine chain
does not improve the inhibition or the selectivity. Among
all derivatives investigated, 3-[2-(1-benzylpiperidin-4-
yl)ethylamino]-5-methyl-6-phenylpyridazine 4c was found
to be one of the most potent and selective AChE
inhibitors: this derivative presents an IC50 of 21 nM
on electric eel AChE and a human selective ratio of 24
(100 more selective than tacrine).
During our preliminary molecular docking study, we
were able to show that our inhibitors interact with both
the cation-π part of the catalytic site and the peripheral
site of the enzyme. After we had performed our modeling
studies and before we had finished our structure-
activity approach, experimental data were published
which supported the applied docking strategy. The
crystal structure of the AChE complexed with the
Exp er im en ta l Section
Ch em istr y. 1H NMR were recorded on a Bruker AC 200
(200 MHz) or a Bruker DPX 300 (300 MHz) spectrophotometer
at room temperature. Chemical shifts are given in ppm (δ)
relative to SiMe3 as internal standard. Coupling constants (J )

Synthesis of Pyridazine Analogues as AChE Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2713
are in hertz (Hz), and signals are designated as follows: s,
singlet; d, doublet; t, triplet; q, quadruplet; quint., quintuplet;
m, multiplet; brs, broad singlet; etc. The mass spectra were
obtained on a Mariner API-TOF. Melting points were deter-
mined with a Mettler FP62 apparatus and are uncorrected.
Elemental analyses were performed by the CNRS department
of microanalysis (CNRS, Vernaison, France) and are indicated
only by the elemental symbols within (0.4% of the theoretical
values unless otherwise noted. All chemicals and solvents were
obtained from commercial suppliers and used without purifica-
tion. THF and ethyl ether (Et2O) were freshly distilled from
sodium benzophenone ketyl. Flash chromatography was car-
ried out on Geduran silica gel Si 60 (40-63 µm, Merck). Thin-
layer chromatography was carried out using plates silica gel
60 F254 (Merck). Spots were visualized either under UV light
(λ ) 254 nm) or by spraying with molybdate reagent (H2O/
concentrated H2SO4/(NH4)6Mo7O24‚4H2O/(NH4)2-Ce(SO4)4‚
2H2O, 90/10/25/1, v/v/w/w) and charring at 140 °C for a few
minutes. All chemical yields are unoptimized and generally
represent the result of a single experiment.
3-Ch lor o-6-p h en yl-5-p r op ylp yr id a zin e (3e). Yield 59%;
1H NMR (300 MHz, CDCl3) δ 0.87 (t, 3H, J ) 7.5 Hz), 1.55
(m, 2H), 2.62 (t, 2H, J ) 7.9 Hz), 7.42 (s, 1H), 7.49 (brs, 5H).
3-Ch lor o-2,5,6,7-tetr a h yd r o-3H-ben zo[6,7]cycloh ep ta -
[1,2-c]p yr id a zin e (3j). Yield 93%; mp 158 °C (lit.31 155-156
°C); 1H NMR (300 MHz, CDCl3) δ 2.27 (quint., 2H, J ) 7.2
Hz), 2.53 (t, 2H, J ) 7.2 Hz), 2.57 (t, 2H, J ) 7.2 Hz), 7.27-
7.30 (m, 1H), 7.39 (s, 1H), 7.43-7.46 (m, 2H), 7.81-7.84 (m,
1H).
(III) P r ep a r a tion of th e Am in es. The 2-(1-benzylpiperi-
din-4-yl)ethylamine24 chain used in this work was obtained
from a commercial supplier as described in our previous
paper.14 The diamine chain 15a was prepared by alkylation
with an halogenonitrile followed by reduction whereas 15b was
obtained by Gabriel synthesis. These two procedures were
reported previously.14 The carboxyamine chains 17a and 17b
were synthesized starting from commercially secondary amines
according to literature procedure.
2-(1-Ben zylp ip er a zin -4-yl)eth yla m in e (15a ). (a ) 2-(1-
Ben zylp ip er a zin -4-yl)a ceton itr ile. Brown oil; yield 96%; mp
(dihydrochloride) 219 °C; 1H NMR (200 MHz, CDCl3) δ 2.49-
2.53 (m, 4H), 2.59-2.65 (m, 4H), 3.49 (s, 2H), 3.51 (s, 2H),
7.24-7.33 (m, 5H).
(I) 3(2H)-P yr id a zin on es. The 3(2H)-pyridazinones 2 nec-
essary for the synthesis of compounds 4a -k are already known
and were prepared according to literature procedures.25-31 The
3(2H)-pyridazinone 2k is commercially available. Pyridazino-
nes 2c-e and 2j were synthesized by using the one-pot
procedure described by Coates et al.32
(b) 2-(1-Ben zylp ip er a zin -4-yl)eth yla m in e46 (15a ). Yel-
low oil; yield 75%; mp (dihydrochloride) 247 °C; 1H NMR (200
MHz, CDCl3) δ 1.38 (brs, 2H), 2.42 (t, 2H, J ) 6.2 Hz), 2.48
(m, 8H), 2.78 (t, 2H, J ) 6.2 Hz), 3.51 (s, 2H), 7.24-7.33 (m,
5H).
5-Met h yl-6-p h en yl-3(2H )-p yr id a zin on e (2c). A stirred
mixture of glyoxylic acid monohydrate (54 mmol) and pro-
piophenone (163 mmol) was heated at 100-105 °C for 2 h and
allowed to cool to 40 °C, and then H2O (20 mL) was added
followed by concentrated aqueous NH4OH (4 mL). The mixture
was extracted with CH2Cl2 (4 × 25 mL). The ammoniac
solution was stirred with hydrazine hydrate (54 mmol) and
heated under reflux for 2 h. After cooling, the precipitate
formed was collected by filtration and washed with H2O to give
2-(4-Ben zylp ip er id in -1-yl)eth yla m in e (15b). Brown oil;
1
yield 85%; H NMR (200 MHz, CDCl3) δ 1.18-1.38 (m, 2H),
1.50-1.68 (m, 3H), 1.81-2.01 (m, 4H), 2.35 (t, 2H, J ) 6.2
Hz), 2.57 (t, 2H, J ) 6.6 Hz), 2.75 (t, 2H, J ) 6.2 Hz), 2.85
(brd, 2H, J ) 11.7 Hz), 7.14-7.30 (m, 5H).
2-Am in o-1-(4-ben zylp ip er a zin -1-yl)eth a n -1-on e (17a ).
(a ) 1-(4-Ben zylp ip er a zin -1-yl)-2-ch lor oeth a n -1-on e. To a
solution of chloroacetic chloride (13.8 mmol) in CH2Cl2 (20 mL)
was added, at 0 °C, 1-benzylpiperazine (11.5 mmol). The
reaction mixture was stirred at room temperature for 30 min.
H2O (100 mL) was added, and the mixture was rendered
alkaline with a 10% NaHCO3 solution and extracted with
EtOAc. The organic layer, dried over Na2SO4, was evaporated
under reduce pressure to give a yellow oil: yield 95%; 1H NMR
(300 MHz, CDCl3) δ 2.47 (quint., 4H, J ) 4.9 Hz), 3.50 (t, 2H,
J ) 4.9 Hz), 3.53 (s, 2H), 3.63 (t, 2H, J ) 4.9 Hz), 4.06 (s, 2H),
7.25-7.32 (m, 5H).
1
2c: yield 70%; mp 210 °C (lit.32 217-219 °C); H NMR (300
MHz, CDCl3) δ 2.19 (s, 3H), 6.89 (s, 1H), 7.44 (m, 5H), 12.57
(brs, 1H).
5-Eth yl-6-p h en yl-3(2H)-p yr id a zin on e (2d ). Yield 35%;
1
mp 181 °C; H NMR (200 MHz, CDCl3) δ 1.09 (t, 3H, J ) 7.6
Hz), 2,48 (q, 2H, J ) 7.4 Hz), 6.89 (s, 1H), 7.38-7.45 (m, 5H).
6-P h en yl-5-p r op yl-3(2H)-p yr id a zin on e (2e). Yield 27%;
1
mp 128 °C; H NMR (300 MHz, CDCl3) δ 0.86 (t, 3H, J ) 7.0
Hz), 1.26 (m, 2H), 3.58 (m, 2H), 7.45-7.48 (m, 3H), 8.67 (s,
1H), 11.22 (brs, 1H).
(b) N-[2-(4-Ben zylp ip er a zin -1-yl)-2-oxoeth yl]p h th a lim -
id e. A mixture of 1-(4-benzylpiperazin-1-yl)-2-chloroethan-1-
one (45.1 mmol) and potassium phthalimide (45.1 mmol) in
DMF (60 mL) was stirred at room temperature for 5 h. A
saturated NH4Cl solution (400 mL) was added, and the
reaction mixture was extracted with EtOAc. The organic layer
was washed with brine solution, dried over Na2SO4, and
evaporated under reduce pressure to give a white solid: yield
2,5,6,7-Tetr a h yd r o-3H-ben zo[6,7]cycloh ep ta [1,2-c]p y-
r id a zin -3-on e (2j). Yield 21%; mp 243 °C (lit.30 235-239 °C);
1H NMR (200 MHz, CDCl3) δ 2.03 (quint., 2H, J ) 7.3 Hz),
2.35 (t, 2H, J ) 6.9 Hz), 2.54 (t, 2H, J ) 6.9 Hz), 6.74 (s, 1H),
7.10-7.32 (m, 3H), 7.42-7.46 (m, 1H), 12.21 (brs, 1H).
(II) Gen er a l P r oced u r e for 3-Ch lor op yr id a zin es. The
appropriate substituted 3-(2H)-pyridazinone 2 was heated at
80 ( 5 °C for 4 h with an excess (10 equiv) of phosphorus
oxychloride (POCl3). The excess of POCl3 was removed by
distillation under reduced pressure, and the residue was
carefully poured onto ice. The water was rendered alkaline
with 20% NaOH solution and extracted with EtOAc. The crude
3-chloropyridazine was purified by recrystallization in EtOH
or i-PrOH or by flash chromatography using a mixture of
hexanes-EtOAc as eluant. The 3-chloropyridazines 3 are
already known and were prepared according to literature
procedures.28,31-33,36,41-43
1
95%; mp 152 °C; H NMR (200 MHz, CDCl3) δ 2.43-2.56 (m,
4H), 3.55 (s, 2H), 3.57-6.65 (m, 4H), 4.49 (s, 2H), 7.29-7.35
(m, 5H), 7.70-7.74 (m, 2H), 7.84-7.89 (m, 2H).
(c) 2-Am in o-1-(4-ben zylpiper azin -1-yl)eth an -1-on e (17a).
A mixture of N-[2-(4-benzylpiperazin-1-yl)-2-oxoethyl]phthal-
imide (8.2 mmol) and hydrazine hydrate (12.4 mmol) in EtOH
(25 mL) was refluxed for 30 min. The precipitate was filtered
off and washed with EtOH, and the filtrate was concentrated
under reduced pressure. Et2O was added to the residue, and
the precipitate was filtered. The filtrate, dried over Na2SO4,
was evaporated to give a colorless oil: yield 98%; 1H NMR (200
MHz, CDCl3) δ 2.30-2.36 (m, 6H), 3.27 (t, 2H, J ) 4.7 Hz),
3.33 (s, 2H), 3.43 (s, 2H), 3.55 (t, 2H, J ) 4.7 Hz), 7.22-7.26
(m, 5H).
3-Ch lor o-4-isop r op yl-6-p h en ylp yr id a zin e (3b). Yield
94%; mp 72 °C (lit.44 71-74 °C); 1H NMR (200 MHz, CDCl3) δ
1.36 (d, J ) 6,9 Hz, 6H), 3.35 (sept., 1H, J ) 6.9 Hz), 7.51-
7.55 (m, 3H), 7.71 (s, 1H), 8.02-8.07 (m, 2H).
3-Ch lor o-5-m eth yl-6-p h en ylp yr id a zin e (3c). Yield 95%;
mp 123 °C (lit.32,45 123-124 °C); 1H NMR (300 MHz, CDCl3) δ
2.37 (s, 3H), 7.42 (s, 1H), 7.48-7.57 (m, 5H).
2-Am in o-1-(4-ben zylp ip er id in -1-yl)eth a n -1-on e (17b).
(a ) 1-(4-Ben zylp ip er id in -1-yl)-2-ch lor oeth a n -1-on e. Or-
1
ange oil; yield 83%; H NMR (200 MHz, CDCl3) δ 1.71-1.90
(m, 5H), 2.51-2.66 (m, 3H), 3.05 (m, 1H), 3.81-3.86 (m, 1H),
4.08 (s, 2H), 4.52-4.62 (m, 1H), 7.13-7.28 (m, 5H).
3-Ch lor o-5-eth yl-6-p h en ylp yr id a zin e (3d ). Yield 89%;
1
mp 64 °C; H NMR (300 MHz, CDCl3) δ 1.18 (t, 3H, J ) 7.5
(b) N-[2-(4-Ben zylp ip er id in -1-yl)-2-oxoeth yl]p h th a lim -
id e. White solid; yield 75%; 1H NMR (200 MHz, CDCl3) δ
Hz), 2.68 (q, 2H, J ) 7.5 Hz), 7.46 (s, 1H), 7.49 (brs, 5H).

2714 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Contreras et al.
1.15-1.37 (m, 2H), 1.38-1.86 (m, 3H), 2.53-5.62 (m, 3H),
3.03-3.12 (m, 1H), 3.83 (brd, 1H, J ) 13.5 Hz), 4.48 (s, 2H),
4.54 (m, 1H), 7.14-7.33 (m, 5H), 7.70-7.74 (m, 2H), 7.84-
7.88 (m, 2H).
(c) 2-Am in o-1-(4-ben zylpiper idin -1-yl)eth an -1-on e (17b).
Yellow oil; yield 91%; 1H NMR (200 MHz, CDCl3) δ 0.96-1.20
(m, 2H), 1.56-1.75 (m, 3H), 1.84 (brs, 2H), 2.38-2.51 (m, 3H),
2.78 (td, 1H, J 1 ) 13.3 Hz, J 2 ) 1.2 Hz), 3.33 (s, 2H), 3.56
(brd, 1H, J ) 13.5 Hz), 4.50 (brd, 1H, J ) 13.2 Hz), 7.02-7.12
(m, 5H).
3-[2-(1-Ben zylp ip er id in -4-yl)eth yla m in o]- 6-p h en yl-5-
p r op yl-p yr id a zin e (4e). The free base (colorless oil) was
purified by flash chromatography (EtOAc and then EtOAc with
2% (v) TEA): yield 10%; 1H NMR (200 MHz, CDCl3) δ 0.87 (t,
3H, J ) 7.3 Hz), 1.29-1.76 (m, 9H), 1.98 (brt, 2H, J ) 10.8
Hz), 2.55 (t, 2H, J ) 7.9 Hz), 2.91 (brd, 2H, J ) 11.4 Hz), 3.40-
3.50 (m, 2H), 3.52 (s, 2H), 5.08 (brs, 1H), 6.53 (s, 1H), 730-
7.36 (m, 5H), 7.43-7.49 (m, 5H).
Dihydrochloride (i-PrOH): mp 177 °C. Anal. (C27H34N4‚
2HCl‚1.5H2O) C, H, N.
3-[2-(1-B e n zy lp ip e r id in -4-y l)e t h y la m in o ]-6-p h e n -
ylp h th a la zin e (4f). The free base (yellow solid) was purified
by flash chromatography (EtOAc-MeOH, 9:1): yield 59%; mp
170 °C; 1H NMR (300 MHz, CDCl3) δ 1.17-1.29 (m, 3H), 1.55-
1.67 (m, 4H), 1.81 (brt, 2H, J ) 10.9 Hz), 2.75 (brd, 2H, J )
10.6 Hz), 3.39 (s, 2H), 3.73 (m, 2H), 6.37 (brs, 1H), 7.19-7.27
(m, 5H), 7.38-7.46 (m, 3H), 7.57-7.69 (m, 4H), 7.84-7.87 (m,
1H), 8.10-8.13 (m, 1H).
(IV) Gen er a l P r oced u r e for th e Su bstitu tion of th e
Im in och lor id es by Dia m in es. All the final 3-aminoalkylpy-
ridazines were prepared by substituting the iminochlorides 3
with the suitable amines. Compounds 4a -g, 4i-k , 16a , and
16b were obtained by using acid conditions (d condition,
Scheme 1) described in our previous paper.14 Compound 4h
was synthesized in neutral conditions (e condition, Scheme 1)
whereas aminoethanone pyridazine derivatives 18a and 18b
were prepared in basic conditions (n condition, Scheme 4).
The corresponding hydrochlorides were prepared by treating
the free base dissolved in Et2O and/or EtOAc with gaseous
hydrogen chloride or with 2.1 equiv of 37% HCl. The collected
solids were crystallized in i-PrOH with Et2O or (i-Pr)2O.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]-4-m et h yl-6-
p h en ylp yr id a zin e (4a ). A mixture of 3-chloro-4-methyl-6-
phenylpyridazine 3a 41(0.63 g, 3.1 mmol), 2-(1-benzylpiperidin-
4-yl)ethylamine14(1.34 g, 6.2 mmol), and ammonium chloride
(0.17 g, 3.1 mmol) in 1-butanol (10 mL) was refluxed for 48 h.
The solvent was removed by evaporation, and the residue was
diluted with 10% K2CO3 (100 mL) and extracted with EtOAc.
The organic layer was washed with a 10% citric acid solution,
and the combined aqueous phases were extracted with EtOAc.
The aqueous layer was rendered alkaline with K2CO3 and then
extracted with EtOAc. After drying over Na2SO4 and removing
the solvent by evaporation, the free base (yellow oil) was
purified by flash chromatography (EtOAc-MeOH, 9:1 with 2%
(v) TEA): yield 19%; 1H NMR (200 MHz, CDCl3) δ 1.21-1.40
(m, 3H), 1.66-1.77 (m, 4H), 1.96 (brt, 2H, J ) 11.3 Hz), 2.17
(s, 3H), 2.88 (brd, 2H, J ) 12.0 Hz), 3.49 (s, 2H), 3.65-3.75
(m, 2H), 4.19 (brt, 1H, J ) 5.1 Hz), 7.31-7.45 (m, 9H), 7.97-
8.01 (m, 2H).
Dihydrochloride (i-PrOH): mp 206 °C. Anal. (C28H30N4‚
2HCl‚3H2O) C, H; N: calcd, 10.19; found, 9.65.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]-5H -in d en o-
[1,2-c]p yr id a zin e (4g). The free base (beige solid) was
purified by flash chromatography (EtOAc-MeOH, 9:1 with 2%
1
(v) TEA): yield 13%; mp 186 °C; H NMR (200 MHz, CDCl3)
δ 1.29-1.39 (m, 3H), 1.57-1.73 (m, 4H), 1.95 (brt, 2H, J )
11.4 Hz), 2.88 (brd, 2H, J ) 11.7 Hz), 3.40-3.47 (m, 2H), 3.49
(s, 2H), 3.82 (s, 2H), 4.83 (brt, 1H, J ) 5.3 Hz), 6.74 (s, 1H),
7.24-7.32 (m, 5H), 7.36-7.50 (m, 3H), 8.11-8.15 (m, 1H).
Dihydrochloride (i-PrOH): mp dec. Anal. (C25H28N4‚2HCl‚
1.5H2O) C, H; N: calcd, 11.57; found, 11.15.
3-[2-(1-Be n zylp ip e r id in -4-yl)e t h yla m in o]-5,6-d ih y-
d r oben zo[h ]cin n olin e (4h ). The free base (white solid)
obtained after 24 h at 100 °C was purified by flash chroma-
tography (EtOAc with 2% (v) NH4OH): yield 15%; mp 113 °C;
1H NMR (300 MHz, CDCl3) δ 1.26-1.47 (m, 3H), 1.59-1.73
(m, 4H), 2.81-2.93 (m, 6H), 3.42-3.47 (m, 2H), 3.49 (s, 2H),
4.47 (brt, 1H, J ) 5.2 Hz), 6.43 (s, 1H), 7.18-7.38 (m, 8H),
8.40-8.43 (m, 1H).
Dihydrochloride (i-PrOH): mp 270 °C. Anal. (C26H30N4‚
2HCl‚2H2O) C, H; N: calcd, 11.04; found, 11.57.
3-[2-(1-Ben zylp ip er id in -4-yl)eth yla m in o]ben zo[h ]cin -
n olin e (4i). The free base (yellow solid) was purified by flash
chromatography (EtOAc): yield 25%; mp 160 °C; 1H NMR (200
MHz, CDCl3) δ 1.26-1.45 (m, 3H), 1.54-1.76 (m, 4H), 1.98
(brt, 2H, J ) 11.2 Hz), 2.90 (brd, 2H, J ) 12.2 Hz), 3.41-3.49
(m, 2H), 3.50 (s, 2H), 5.08 (brs, 1H), 6.78 (s, 1H), 7.29-7.39
(m, 6H), 7.58-7.81 (m, 4H), 9.33-9.36 (m, 1H).
Dihydrochloride (i-PrOH): mp 246 °C. Anal. (C25H30N4‚
2HCl‚0.5H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)eth yla m in o]-4-isop r op yl-
6-p h en ylp yr id a zin e (4b). The free base (brown oil) was
purified by flash chromatography (EtOAc with 2% (v) TEA):
1
yield 13%; H NMR (200 MHz, CDCl3) δ 1.30-1.45 (m, 3H),
1.32 (d, 6H, J ) 6.9 Hz), 1.59-1.78 (m, 4H), 1.97 (brt, 2H, J )
10.9 Hz), 2.73 (m, 1H), 2.89 (brd, 2H, J ) 10.9 Hz), 3.50 (s,
2H), 3.66-3.76 (m, 2H), 4.30 (brs, 1H), 7.22-7.31 (m, 5H),
7.39-7.41 (m, 4H), 7.97-8.02 (m, 2H).
Dihydrochloride (i-PrOH): mp 103 °C. Anal. (C27H34N4‚
2HCl‚2H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]-5-m et h yl-6-
p h en ylp yr id a zin e (4c). The free base (yellow oil) was puri-
fied by flash chromatography (EtOAc-MeOH, 9:1 and then
EtOAc with 2% (v) TEA): yield 22%; 1H NMR (200 MHz,
CDCl3) δ 1.28-1.43 (m, 3H), 1.56-1.63 (m, 2H), 1.69 (brd, 2H,
J ) 11.7 Hz), 1.93 (brt, 2H, J ) 11.3 Hz), 2.20 (s, 3H), 2.87
(brd, 2H, J ) 11.7 Hz), 3.38-3.45 (m, 2H), 3.48 (s, 2H), 5.15
(brt, 1H, J ) 5.6 Hz), 6.50 (s, 1H), 7.22-7.32 (m, 5H), 7.35-
7.46 (m, 3H), 7.50-7.54 (m, 2H).
Dihydrochloride (i-PrOH): mp 269 °C. Anal. (C26H28N4‚
2HCl‚3.5H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]-2,5,6,7-t et -
r a h yd r o-3H-ben zo[6,7]cycloh ep ta [1,2-c]p yr id a zin e (4j).
The free base (colorless oil) was purified by flash chromatog-
raphy (EtOAc-MeOH, 9:1): yield 30%; 1H NMR (300 MHz,
CDCl3) δ 1.24-1.45 (m, 3H), 1.54-1.69 (m, 4H), 1.91 (brt, 2H,
J ) 11.3 Hz), 2.11 (quint., 2H, J ) 7.1 Hz), 2.36 (t, 2H, J )
7.1 Hz), 2.51 (t, 2H, J ) 7.1 Hz), 2.84 (brd, 2H, J ) 11.6 Hz),
3.40-3.44 (m, 2H), 3.45 (s, 2H), 5.05 (brt, 1H, J ) 5.2 Hz),
7.15-7.36 (m, 8H), 7.73-7.76 (m, 1H).
Dihydrochloride (i-PrOH): mp 160 °C. Anal. (C27H32N4‚
2HCl‚H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]p yr id a zin e
(4k ). The free base (brown oil) was purified by flash chroma-
tography (EtOAc-MeOH, 9:1 with 2% (v) TEA): yield 11%;
1H NMR (200 MHz, CDCl3) δ 1.28-1.46 (m, 3H), 1.61-1.74
(m, 4H), 1.98 (brt, 2H, J ) 11.1 Hz), 2.91 (brd, 2H, J ) 11.8
Hz), 3.37-3.49 (m, 2H), 3.52 (m, 2H), 4.92 (t, 1H, J ) 5.0 Hz),
6.64 (dd, 1H, J 1 ) 9.0 Hz, J 2 ) 1.3 Hz), 7.15 (dd, 1H, J 1 ) 9.0
Hz, J 2 ) 4.5 Hz), 7.26-7.35 (m, 5H), 8.54 (dd, 1H, J 1 ) 4.4
Hz, J 2 ) 1.3 Hz).
Dihydrochloride (i-PrOH): mp 120 °C. Anal. (C18H24N4‚
2HCl‚1.5H2O) C, H; N: calcd, 14.14; found, 13.72.
3-[2-(4-Ben zylp ip er a zin -1-yl)eth yla m in o]-6-p h en ylp y-
r id a zin e (16a ). The free base (white solid) was purified by
flash chromatography (EtOAc-MeOH, 9:1 with 2% (v) TEA):
Dihydrochloride (i-PrOH): mp 135 °C. Anal. (C25H30N4‚2
HCl‚1.2H2O) C, H, N.
3-[2-(1-Ben zylpiper idin -4-yl)eth ylam in o]-5-eth yl-6-ph en -
ylp yr id a zin e (4d ). The free base (yellow oil) was purified by
flash chromatography (EtOAc-MeOH, 9:1): yield 28%; 1H
NMR (300 MHz, CDCl3) δ 1.08 (t, 3H, J ) 7.5 Hz), 1.26-1.47
(m, 3H), 1.57-1.64 (m, 2H), 1.70 (brd, 2H, J ) 11,7 Hz), 1.95
(brt, 2H, J ) 11.7 Hz), 2.55 (q, 2H, J ) 7.2 Hz), 2.88 (brd, 2H,
J ) 11.7 Hz), 3.40-3.47 (m, 2H), 3.49 (s, 2H), 5.08 (brt, 1H, J
) 5.2 Hz), 6.53 (s, 1H), 7.23-7.35 (m, 5H), 7.39-7.45 (m, 5H).
Dihydrochloride (i-PrOH): mp 270 °C. Anal. (C26H32N4‚
2HCl‚H2O) C, H; N: calcd, 11.40; found, 10.93.

Synthesis of Pyridazine Analogues as AChE Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2715
1
yield 22%; mp 140 °C; H NMR (200 MHz, CDCl3) δ 2.52 (m,
water (25 mL, stirred 1 h, and extracted with EtOAc (3 × 25
mL). The organic layer, dried over Na2SO4, was evaporated
under reduce pressure to give the boronic acid, purified by
trituration in a mixture Et2O/hexane: yield 80%; 1H NMR (200
MHz, DMSO-d6) δ 1.15 (t, 3H, J ) 7.5 Hz), 2.68 (q, 2H, J )
7.6 Hz), 7.24 (m, 4H), 7.55 (s, 2H); ES-MS m/z 150 [M + H]+
(100%).
8H), 2.68 (t, 2H, J ) 6.2 Hz), 3.52 (s, 2H), 3.53-3.60 (m, 2H),
5.36 (brs, 1H), 6.73 (d, 1H, J ) 9.1 Hz), 7.28-7.34 (m, 5H),
7.42-7.47 (m, 3H), 7.59 (d, 1H, J ) 9.5 Hz), 7.-95-8.00 (m,
2H).
Dihydrochloride (i-PrOH): mp 263 °C. Anal. (C23H27N5‚
3HCl‚H2O) C, H, N.
(b) 3-[2-(1-Ben zylp ip er id in -4-yl)eth yla m in o]-6-(2-eth -
ylp h en yl)p yr id a zin e (6b). The free base (colorless oil) was
purified by flash chromatography (EtOAc-MeOH, 9:1 with 2%
3-[2-(4-Ben zylp ip er id in -1-yl)eth yla m in o]-6-p h en ylp y-
r id a zin e (16b). The free base (white solid) was purified by
flash chromatography (EtOAc-MeOH, 9:1 with 2% (v) TEA):
yield 22%; mp 135 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.32-
1.36 (m, 2H), 1.65-1.69 (m, 3H), 2.05 (m, 2H), 2.37 (t, 2H, J
) 6.5 Hz), 2.75 (d, 2H, J ) 6.9 Hz), 3.03 (m, 2H), 3.62 (m, 2H),
7.05 (d, 1H, J ) 9.4 Hz), 7.27-7.60 (m, 8H), 7.92 (d, 1H, J )
9.0 Hz), 8.09 (m, 2H).
1
(v) TEA): yield 40%; H NMR (200 MHz, CDCl3) δ 1.22 (m,
6H), 1.66 (m, 4H), 1.99 (brt, 2H, J ) 9.0 Hz), 2.84 (m, 4H),
3.54 (m, 4H), 5.27 (s, 1H), 6.78 (d, 1H, J ) 9.0 Hz), 7.30 (m,
9H), 7.68 (d, 1H, J ) 9.0 Hz).
Dihydrochloride (i-PrOH): mp 115 °C. Anal. (C26H32N4‚2HCl‚
2.5H2O) C, H; N: calcd, 10.81; found, 10.29.
Dihydrochloride (i-PrOH): mp 186 °C. Anal. (C24H28N4‚
2HCl‚1.5H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]-6-(2-ch lor o-
p h en yl)p yr id a zin e (6e). The free base (colorless oil) was
purified by flash chromatography (EtOAc-MeOH, 9:1 with 2%
3-[2-(4-Ben zylp ip er a zin -1-yl)eth a n -1-on e-2-a m in o3]-6-
p h en ylp yr id a zin e (18a ). The free base (white solid) obtained
after 24 h reflux in pyridine was purified by flash chromatog-
raphy (EtOAc and then EtOAc with 2% (v) TEA): yield 10%;
mp dec; 1H NMR (200 MHz, CDCl3) δ 2.42-2.50 (m, 4H), 3.49-
3.53 (m, 2H), 3.54 (s, 2H), 3.71 (t, 2H, J ) 5.1 Hz), 4.39 (d,
2H, J ) 3.6 Hz), 5.84 (brs, 1H), 6.86 (d, 1H, J ) 9.1 Hz), 7.31-
7.33 (m, 5H), 7.39-7.49 (m, 3H), 7.60 (d, 1H, J ) 9.4 Hz),
7.95-7.98 (m, 2H).
1
(v) TEA): yield 50%; H NMR (200 MHz, CDCl3) δ 1.31 (m,
3H), 1.62 (m, 4H), 1.94 (brt, 2H, J ) 9.0 Hz), 2.84 (m, 4H),
3.49 (m, 4H), 5.48 (s, 1H), 6.73 (d, 1H, J ) 9.4 Hz), 7.08 (d,
1H, J ) 9.3 Hz), 7.31-7.57 (m, 6H), 7.68 (d, 1H, J ) 9.0 Hz),
7.58 (m, 1H), 7.82 (m, 1H), 7.84 (s, 1H).
Dihydrochloride (i-PrOH): mp 153 °C. Anal. (C24H27ClN4‚
2HCl‚2.5H2O) C, H, N.
6-(3-Acet a m id op h en yl)-3-[2-(1-b en zylp ip er id in -4-yl)-
eth yla m in o]p yr id a zin e (6h ). The free base (brown oil) was
purified by flash chromatography (EtOAc-MeOH, 9:1): yield
Dihydrochloride (i-PrOH): mp 225 °C. Anal. (C23H25N5O‚
2HCl‚2H2O) C, H, N.
3-[2-(4-Ben zylp ip er id in -1-yl)eth a n -1-on e-2-a m in o3]-6-
p h en ylp yr id a zin e (18b). The free base (white solid) obtained
after 24 h reflux in pyridine was purified by flash chromatog-
raphy (EtOAc and then EtOAc with 2% (v) TEA): yield 16%;
1
30%; H NMR (300 MHz, CDCl3) δ 1.24-1.37 (m, 3H), 1.55-
1.68 (m, 4H), 2.05 (brt, 2H, J ) 12.4 Hz), 2.17 (s, 3H), 2.93
(brd, 2H, J ) 11.3 Hz), 3.36-3.42 (m, 2H), 3.58 (s, 2H), 5.15
(brs, 1H), 6.69 (d, 1H, J ) 9.4 Hz), 7.28-7.37 (m, 6H), 7.51 (d,
1H, J ) 9.4 Hz), 7.62 (d, 1H, J ) 7.9 Hz), 7.69 (d, 1H, J ) 7.9
Hz), 8.06 (s, 1H), 8.31 (s, 1H).
Dihydrochloride (i-PrOH): mp 243 °C. Anal. (C26H31N5O‚
2HCl‚1.5H2O) C, H, N.
1
mp 144 °C; H NMR (200 MHz, CDCl3) δ 1.18-1.31 (m, 2H),
1.73-1.88 (m, 3H), 2.58 (d, 2H, J ) 6.7 Hz), 2.63-2.71 (m,
1H), 3.01 (brt, 1H, J ) 12.6 Hz), 3.87 (brd, 1H, J ) 13.5 Hz),
4.41 (t, 2H, J ) 3.3 Hz), 4.64 (brd, 1H, J ) 13.3 Hz), 6.05 (t,
1H, J ) 3.6 Hz), 6.89 (d, 1H, J ) 9.3 Hz), 7.13-7.36 (m, 5H),
7.40-7.45 (m, 3H), 7.59 (d, 1H, J ) 9.3 Hz), 7.96-8.05 (2H,
m).
6-(3-Acetylp h en yl)-3-[2-(1-ben zylp ip er id in -4-yl)eth yl-
a m in o]p yr id a zin e (6i). The free base (white solid) was
purified by flash chromatography (CH2Cl2-MeOH, 9:1): yield
Dihydrochloride (i-PrOH): mp 215 °C. Anal. (C24H26N4O‚
HCl‚0.5H2O) C, H; N: calcd, 12.97; found, 12.52.
1
80%; mp 108 °C; H NMR (300 MHz, CDCl3) δ 1.20-1.45 (m,
3H), 1.59-1.72 (m, 4H), 1.98 (brt, 2H, J ) 10.9 Hz), 2.65 (s,
3H), 2.90 (brd, 2H, J ) 11.6 Hz), 3.45-3.48 (m, 2H), 3.51 (s,
2H), 5.09 (brs, 1H), 6.74 (d, 1H, J ) 9.4 Hz), 7.24-7.31 (m,
5H), 7.53 (t, 1H, J ) 7.9 Hz), 7.63 (d, 1H, J ) 9.4 Hz), 7.96 (d,
1H, J ) 7.9 Hz), 8.17 (d, 1H, J ) 7.9 Hz), 8.55 (s, 1H).
Dihydrochloride (i-PrOH): mp 243 °C. Anal. (C26H30N4O‚
2HCl‚H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)eth yla m in o]-6-(3,4-m eth -
ylen ed ioxyp h en yl)p yr id a zin e (6j). The free base (white
solid) was purified by flash chromatography (EtOAc-MeOH,
9:1): yield 66%; mp 160 °C; 1H NMR (300 MHz, CDCl3) δ 1.26-
1.43 (m, 3H), 1.59-1.73 (m, 4H), 1.96 (brt, 2H, J ) 10.9 Hz),
2.89 (brd, 2H, J ) 11.7 Hz), 3.43-3.48 (m, 2H), 3.50 (s, 2H),
4.71 (brs, 1H), 6.01 (s, 2H), 6.66 (d, 1H, J ) 9.0 Hz), 6.87 (d,
1H, J ) 8.3 Hz), 7.24-7.39 (m, 6H), 7.50 (d,, 1H J ) 9.4 Hz),
7.56 (d, 1H, J ) 1.5 Hz).
(V) Sp ecia l P r oced u r es. The 6-chloro-3-[2-(1-benzylpi-
peridin-4-yl)ethylamino]pyridazine 5 necessary for the syn-
thesis of compounds 6a -o and 7 was prepared according to a
literature procedure.34 Aryl boronic acids were commercially
available except for the 2-ethylphenylboronic acid which was
synthesized. Substituted 3-amino-6-arylpyridazines 6a -o were
synthesized by using the Suzuki procedure described in our
previous paper34 where the 6-arylpyridazines 6a , 6c, 6d , 6f,
and 6g were already prepared.
Gen er a l P r oced u r e for th e P r ep a r a tion of 6-Ar yl-3-
[2-(1-ben zylp ip er id in -4-yl)eth yla m in o]p yr id a zin es. Ar-
gon was passed through a suspension of 5 (3.46 mmol, 1 equiv),
arylboronic acid (3.98 mmol, 1.15 equiv), 2 M sodium carbonate
(3.7 mL, 7.34 mmol, 2.12 equiv), toluene (20 mL), and possibly
EtOH for 30 min. Tetrakis(triphenylphosphine)palladium(0)
(0.10 mmol, 0.031 equiv) was added, and the mixture was
heated at 110 °C for 24 h. The toluene was removed in vacuo.
The residue was diluted with H2O and extracted with EtOAc
(3 × 5 mL). The organic layer was washed with H2O (3 × 5
mL) and concentrated in vacuo. The crude product was purified
by chromatography with a mixture of EtOAc, MeOH, and TEA.
The corresponding hydrochlorides were prepared using the
same procedure described previously.
Compounds obtained by this method are listed below.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]-6-(2-et h yl-
p h en yl)p yr id a zin e (6b). (a ) 2-Eth ylp h en ylbor on ic Acid .
A solution of 2-bromoethyl-benzene (17.1 mmol, 1 equiv) in
THF was added dropwise to a solution of Mg turnings (20.5
mmol, 1.2 equiv) in THF. The mixture was stirred at 20 °C
over 1 h, and then after cooling at -78 °C, triisopropylborate
(51.4 mmol, 3 equiv) was added dropwise. The solution was
stirred at -78 °C for 1 h and allowed to warm to room
temperature over 48 h. Then, the mixture was hydrolyzed with
Dihydrochloride (i-PrOH): mp 279 °C. Anal. (C25H28N4O2‚
2HCl‚H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]-6-(4-cya n o-
p h en yl)p yr id a zin e (6k ). The free base (yellow oil) was
purified by flash chromatography (EtOAc-MeOH, 9:1): yield
50%; 1H NMR (300 MHz, CDCl3) δ 1.28 (m, 3H), 1.72 (m, 4H),
1.98 (t, 2H, J ) 8.0 Hz), 2.91 (d, 2H, J ) 11.0 Hz), 3.49 (m,
4H), 4.32 (s, 1H), 6.69 (d, 1H, J ) 9.0 Hz), 7.25 (m, 5H), 7.91
(m, 4H), 8.16 (d, 1H, J ) 9.0 Hz). ES-MS m/z: 398 [M + H]+
(100%).
Dihydrochloride (i-PrOH): mp 247 °C. Anal. (C25H27N5‚
2HCl‚1.75H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]-6-(4-flu or o-
p h en yl)p yr id a zin e (6l). The free base (yellow oil) was
purified by flash chromatography (EtOAc-MeOH, 9:1): yield
45%; 1H NMR (300 MHz, CDCl3) δ 1.31 (m, 3H), 1.67 (m, 3H),
1.67 (m, 4H), 1.81 (t, 2H, J ) 8.0 Hz), 2.75 (d, 2H, J ) 11.0

2716 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Contreras et al.
Hz), 3.50 (m, 4H), 4.73 (s, 1H), 6.84 (d, 1H, J ) 9.4 Hz), 7.75
(m, 7H), 7.98 (d, 1H, J ) 9.4 Hz), 8.01 (m, 2H). ES-MS m/z:
391 [M + H]+ (100%).
Dihydrochloride (i-PrOH): mp 253 °C. Anal. (C24H27FN4‚
2HCl‚0.5H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)eth yla m in o]-6-(4-d im eth -
yla m in op h en yl)p yr id a zin e (6m ). The free base (yellow oil)
was purified by flash chromatography (EtOAc-MeOH, 9:1):
yield 65%; 1H NMR (300 MHz, CDCl3) δ 1.33 (m, 3H), 1.71
(m, 4H), 1.95 (t, 2H, J ) 8.0 Hz), 2.84 (d, 2H, J ) 11.0 Hz),
3.48 (m, 4H), 4.96 (s, 1H), 6.81 (m, 3H), 7.32 (m, 5H), 7.71 (d,
2H, J ) 8.6 Hz), 7.84 (d, 1H, J ) 9.2 Hz). ES-MS m/z: 416 [M
+ H]+ (100%).
The solution was filtered and washed with H2O and EtOAc.
The filtrate was extracted with EtOAc, and the combined
organic layers, dried over Na2SO4, were concentrated to give
a colorless oil: yield 95%; 1H NMR (200 MHz, CDCl3) δ 1.34-
1.53 (m, 5H), 1.95 (brd, 2H, J ) 11.8 Hz), 1.98 (brt, 2H, J )
11.4 Hz), 2.90 (brd, 2H, J ) 11.7 Hz), 3.52 (s, 2H), 3.70 (t, 2H,
J ) 6.6 Hz), 7.30-7.36 (m, 5H).
(c) 3-[2-(1-Ben zylp ip er id in -4-yl)et h oxy]-6-p h en ylp y-
r id a zin e (10). A mixture of sodium (7.2 mmol) and 1-benzyl-
2-(hydroxyethyl)piperidine) 9 (7.2 mmol) in dry THF (10 mL)
was stirred vigorously at 50 °C for 1 h. 3-Chloro-6-phenylpy-
ridazine36 (7.2 mmol), dissolved in dry THF (10 mL), was added
dropwise, and the mixture was refluxed for 1 h. After cooling,
the mixture was hydrolyzed with H2O, and THF was removed
under reduced pressure. The residue was extracted with
EtOAc, and the organic layer, dried over Na2SO4, was evapo-
rated under reduce pressure. The crude product was purified
by flash chromatography (EtOAc) to obtain a white solid: yield
Trihydrochloride (i-PrOH): mp 145 °C. Anal. (C26H32N5‚
3HCl‚3.5H2O) C, H, N.
3-[2-(1-Ben zylp ip er id in -4-yl)eth yla m in o]-6-(th iop h en -
2-yl)p yr id a zin e (6n ). The free base (white solid) was purified
by flash chromatography (CH2Cl2-MeOH, 95:5): yield 22%;
1
1
10%; mp 149 °C; H NMR (200 MHz, CDCl3) δ 1.23-1.52 (m,
mp 164 °C; H NMR (300 MHz, CDCl3) δ 1.55-1.59 (m, 3H),
3H), 1.64-1.86 (m, 4H), 1.90-2.03 (m, 2H), 2.89 (brd, 2H, J
) 11.7 Hz), 3.49 (s, 2H), 4.62 (t, 2H, J ) 6.7 Hz), 7.02 (d, 1H,
J ) 9.2 Hz), 7.28-7.34 (m, 5H), 7.44-7.51 (m, 3H), 7.78 (d,
1H, J ) 9.2 Hz), 7.99-8.03 (m, 2H).
1.75-1.88 (m, 4H), 2.17-2.22 (m, 2H), 3.07 (brd, 2H, J ) 10.9
Hz), 3.60-3.66 (m, 2H), 3.69 (s, 2H), 4.83 (brs, 1H), 6.76 (d,
1H, J ) 9.1 Hz), 7.20-7.23 (m, 1H), 7.39-7.47 (m, 6H), 7.56-
7.57 (m, 1H), 7.62 (d, 1H, J ) 9.3 Hz).
Dihydrochloride (i-PrOH): mp 155 °C. Anal. (C24H27N3O‚
2HCl‚0.5H2O) C, H; N.
Dihydrochloride (i-PrOH): mp 227 °C. Anal. (C22H26N4S‚
2HCl‚2H2O) C, H, N.
3-[2-(1-Be n zylp ip e r id in -4-yl)e t h ylt h io]-6-p h e n ylp y-
r id a zin e (12). (a ) 1-Ben zyl-4-(2-ch lor oeth yl)p ip er id in e37
(11). To a solution of 1-benzyl-4-(2-hydroxyethyl)piperidine 9
(4.3 mmol) in CH2Cl2 (10 mL), was added dropwise SOCl2 (8.6
mmol) with ice cooling. The mixture was refluxed for 2 h and
then evaporated. The residue was rendered alkaline with 10%
K2CO3 solution and extracted with EtOAc. The organic layer,
dried over Na2SO4, was evaporated under reduce pressure to
give a yellow oil: yield 81%; 1H NMR (300 MHz, CDCl3) δ
1.21-1.34 (m, 2H), 1.48-1.52 (m, 1H), 1.65-1.76 (m, 4H), 1.96
(td, 2H, J 1 ) 11.5 Hz, J 2 ) 2.1 Hz), 2.88 (brd, 2H, J ) 11.5
Hz), 3.50 (s, 2H), 3.57 (t, 2H, J ) 7.2 Hz), 7.25-7.32 (m, 5H).
(b) 6-P h en yl-3-th iolp yr id a zin e.38 A mixture of 3-chloro-
6-phenylpyridazine (2.6 mmol) and thiourea (2.6 mmol) in
EtOH (10 mL) was refluxed for 30 min. The mixture was
evaporated, and H2O (20 mL) was added to the residue,
followed by Na2CO3 (1.3 mmol). The precipitate was collected
by filtration and washed with Et2O. A yellow solid was
3-[2-(1-Ben zylp ip er id in -4-yl)eth yla m in o]-6-(p yr id in -3-
yl)p yr id a zin e (6o). The free base (colorless oil) was purified
by flash chromatography (EtOAc and then EtOAc-MeOH,
1
9:1): yield 24%; H NMR (300 MHz, CDCl3) δ 1.37-1.42 (m,
3H), 1.62-1.72 (m, 4H), 1.97-2.05 (m, 2H), 2.93 (brd, 2H, J
) 11.5 Hz), 3.44-3.51 (m, 2H), 3.54 (s, 2H), 5.22 (brs, 1H),
6.75 (d, 1H, J ) 9.0 Hz), 7.27-7.39 (m, 6H), 7.59 (d, 1H, J )
9.3 Hz), 8.32 (dt, 1H, J 1 ) 8.1 Hz, J 2 ) 1.8 Hz), 8.60 (dd, 1H,
J 1 ) 4.6 Hz, J 2 ) 1.6 Hz), 9.10 (d, 1H, J ) 1.8 Hz).
Trihydrochloride (i-PrOH): mp 131 °C. Anal. (C23H27N5‚
3HCl‚3H2O) C, H; N: calcd, 13.04; found, 12.08.
3-[2-(1-Ben zylp ip er id in -4-yl)et h yla m in o]-6-m et h oxy-
p yr id a zin e (7). To a solution of 3-[2-(1-benzylpiperidin-4-yl)-
ethylamino]-6-chloropyridazine 5 34 (1.6 mmol) in DMF (10 mL)
was added at room temperature a solution of MeONa (Na, 3.2
mmol) in MeOH (5 mL). The mixture was heated at 130 °C
for 20 h. After cooling, the reaction mixture was poured onto
H2O (100 mL), rendered alkaline with a 10% K2CO3 solution,
and extracted with EtOAc. The organic layer was washed with
brine, dried over Na2SO4, concentrated under reduce pressure,
and purified by flash chromatography (EtOAc-MeOH, 9:1
with 2% (v) TEA) to give a yellow oil: yield 50%; 1H NMR (200
MHz, acetone-d6) δ 1.70-1.90 (m, 7H), 2.60 (m, 2H), 3.21 (brd,
2H, J ) 10.9 Hz), 3.50-3.55 (m, 2H), 3.98 (s, 2H), 6.05 (s, 1H),
6.83 (d, 1H, J ) 9.1 Hz), 7.05 (d, 1H, J ) 9.1 Hz), 7.41-7.47
(m, 3H), 7.67-7.72 (m, 3H).
1
obtained: yield 82%; mp 167 °C (lit.38 160 °C); H NMR (200
MHz, CDCl3) δ 7.48-7.55 (m, 4H), 7.79-7.86 (m, 3H).
(c) 3-[2-(1-Ben zylp ip er id in -4-yl)eth ylth io]-6-p h en ylp y-
r id a zin e (12). To a solution of 6-phenyl-3-thiolpyridazine (1.6
mmol) and EtONa (1.6 mmol) in EtOH (5 mL) was added at
room temperature 1-benzyl-4-(2-chloroethyl)piperidine 11 (1.6
mmol) dissolved in EtOH (5 mL). The mixture was refluxed
for 4 h. The solvent was evaporated, H2O was added, and the
residue was extracted with EtOAc. The organic layer, dried
over Na2SO4, was evaporated under reduced pressure, and the
crude product was purified by flash chromatography (EtOAc-
hexane, 1:1 and then EtOAc) to give a white solid: yield 20%;
Dihydrochloride (i-PrOH): mp 120 °C. Anal. (C19H26N4O‚
2HCl‚2H2O) C, H, N.
3-[2-(1-B e n zy lp ip e r id in -4-y l)e t h o x y ]-6-p h e n y lp y -
r id a zin e (10). (a ) 1-Ben zyl-4-[(eth oxyca r bon yl)m eth yl-
en e]p ip er id in e35 (8). A mixture of K2CO3 (84.7 mmol) and
triethyl phosphonoacetate (84.7 mmol) in dry THF (40 mL)
was stirred at room temperature for 15 min and then refluxed
for 20 min. After cooling, 1-benzyl-4-piperidone (56.4 mmol)
was added, and the mixture was heated under reflux for 48 h.
After cooling, a 10% K2CO3 solution (100 mL) was added, and
the mixture was extracted with EtOAc. The organic phase was
dried over Na2SO4 and evaporated under reduce pressure. The
crude product was purified by flash chromatography (EtOAc-
hexane, 1:9). A colorless oil was obtained: yield 84%; 1H NMR
(300 MHz, CDCl3) δ 1.27 (t, 3H, J ) 7.2 Hz), 2.31-2.36 (m,
2H), 2.53 (t, 4H, J ) 5.6 Hz), 2.98 (t, 2H, J ) 5.6 Hz), 3.53 (s,
1
mp 152 °C; H NMR (200 MHz, CDCl3) δ 1.18-1.39 (m, 2H),
1.39-1.57 (m, 1H), 1.66-1.77 (m, 4H), 1.93 (brt, 2H, J ) 11.3
Hz), 2.86 (brd, 2H, J ) 11.7 Hz), 3.36-3.44 (m, 2H), 3.46 (s,
2H), 7.23-7.35 (m, 6H), 7.45-7.48 (m, 3H), 7.60 (d, 1H, J )
9.0 Hz), 8.00-8.04 (m, 2H).
Dihydrochloride (i-PrOH): mp 210 °C. Anal. (C24H27N3S‚
2HCl) C, H; N.
3-[2-(1-Ben zylp ip er id in -4-yl)a cet a m id o]-6-p h en ylp y-
r id a zin e (14a ). (a ) 2-(1-Ben zylp ip er id in -4-yl)a cetic Acid
Hyd r och lor id e (13a ). A mixture of 2-(1-benzylpiperidin-4-
yl)acetonitrile14 (15.1 mmol) and a 1 M solution of NaOH in
ethanol (EtOH-H2O, 9:1) (30 mL) was refluxed for 28 h. The
reaction mixture was concentrated by evaporation, acidified
with concentrated HCl, and concentrated again by evaporation.
2-Propanol was added, and the residue was evaporated to give
a beige moss: yield 95%; 1H NMR (300 MHz, CD3OD) δ 1.22-
1.34 (m, 2H), 1.66-1.80 (m, 3H), 2.00 (brt, 2H, J ) 12.0 Hz),
2.10 (d, 2H, J ) 7.1 Hz), 2.87 (brd, 2H, J ) 12.0 Hz), 3.49 (s,
2H), 7.22-7.31 (m, 5H).
2H), 4.15 (q, 2H, J ) 7.2 Hz), 5.64 (s, 1H), 7.17-7.27 (m, 5H).
35
(b) 1-Ben zyl-4-(2-h yd r oxyeth yl)p ip er id in e
(9). To a
suspension of LiAlH4 ((38.5 mmol) in dry THF (30 mL) was
added, at 0 °C, 1-benzyl-4-[(ethoxycarbonyl)methylene]pi-
peridine 8 (77.1 mmol) in dry THF (10 mL). The mixture was
refluxed for 20 h, and after cooling unreacted LiAlH4 was
quenched by careful addition of 10% NaOH solution (60 mL).

Synthesis of Pyridazine Analogues as AChE Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2717
(b) 3-[2-(1-Ben zylpiper idin -4-yl)acetam ido]-6-ph en ylpy-
r id a zin e (14a ). A mixture of 2-(1-benzylpiperidin-4-yl)acetic
acid hydrochloride 13a (5.2 mmol) and (COCl)2 (14 mL) was
stirred at room temperature for 30 min. The (COCl)2 excess
was evaporated, and a solution of 3-amino-6-phenylpyridazine
(2.6 mmol), TEA (7.8 mmol), and CH2Cl2 (30 mL) was added
to the residue. The reaction mixture was stirred at room
temperature for 2 h, and the CH2Cl2 was removed in vacuo. A
10% citric acid solution was added to the residue. The aqueous
layer was washed with EtOAc, rendered alkaline with K2CO3,
and then extracted with EtOAc. The organic layer, dried over
Na2SO4, was concentrated under reduced pressure and purified
by flash chromatography (EtOAc-MeOH, 9:1) to give a white
solid: yield 15%; mp 293 °C; 1H NMR (300 MHz, CDCl3) δ
1.39-1.51 (m, 2H), 1.78 (brd, 2H, J ) 12.8 Hz), 1.96-2.05 (m,
3H), 2.66 (d, 2H, J ) 6.7 Hz), 2.88 (brd 2H, J ) 10.9 Hz,),
3.49 (s, 2H), 7.07-7.23 (m, 5H), 7.24-7.52 (m, 3H), 7.91 (d,
1H, J ) 9.4 Hz), 8.03-8.06 (m, 2H), 8.65 (d, 1H, J ) 9.4 Hz),
10.12 (s, 1H).
5H), 7.40-7.48 (m, 3H), 7.80 (d, 1H, J ) 9.3 Hz), 7.96-8.00
(m, 2H), 8.48 (d, 1H, J ) 9.3 Hz), 10.12 (brs, 1H).
Dihydrochloride (i-PrOH): mp 234 °C. Anal. (C24H26N4O‚
2HCl‚0.5H2O) C; H; N.
Biologica l Stu d ies. In Vitr o Mea su r em en t of Acetyl-
ch olin est er a se a n d Bu t yr ylch olin est er a se In h ib it ion .
Acetylcholinesterase and butyrylcholinesterase inhibitory ac-
tivity were measured by the spectrophotometric method of
Ellman et al.40 Electric eel AChE (Type III, electric eel, Sigma
Chemical Co.) and human erythrocytes AChE (Type XIII,
human erythrocytes, Sigma Chemical Co.) were used as
sources of AChE, and lyophilized human serum (crude powder,
Sigma Chemical Co.) was used as source of BuChE. AChE
preparations (from electric eel and human erythrocytes) and
BuChE (from human serum) were dissolved in 0.1 M potas-
sium phosphate buffer pH 7.2 such as to have an enzyme
solution stock with 2.5 units/mL AChE activity. Acetylthio-
choline iodide and butyrylthiocholine iodide (Sigma Chemical
Co.) were used as the substrates of the enzymatic reaction and
5,5-dithiobis(2-nitrobenzoic) acid (DTNB) for the measurement
of cholinesterase activity. In this procedure, 940 µL of 0.1 M
potassium buffer pH 8 with 60 mg/500 mL DTNB, 20 µL of
test compound solution, and 20 µL of enzyme stock solution
(electric eel and human erythrocytes AChE and human serum
BuChE) were mixed. After 10 min of preincubation, 20 µL of
10 mM acetylthiocholine/butyrylthiocholine iodide was added
to the assay solution. The final assay volume was 1 mL. The
change in absorbance at 412 nm was recorded (spectropho-
tometer SHIMADZU UV-2401PC) during 1 min at 25 °C. The
reaction rate was calculated. Different concentrations (range
of 10-9 M-10-3 M) of the test compound were assayed
(triplicate), and the percent inhibition due to the presence of
test compound was calculated. IC50 values were determined
graphically from log concentration-inhibition curves.
Dihydrochloride (i-PrOH): mp 117 °C. Anal. (C24H26N4O‚2
HCl‚5H2O) H; N; C: calcd, 52.42; found, 52.88.
3-[2-(4-Ben zylp ip er a zin -1-yl)a cet a m id o]-6-p h en ylp y-
r id a zin e (14b). (a ) Eth yl 2-(4-Ben zylp ip er a zin -1-yl)a ce-
ta te. A mixture of ethyl chloroacetate (43.3 mmol), 1-ben-
zylpiperazine (28.8 mmol), and TEA (28.8 mmol) in toluene
(60 mL) was heated at 80 °C for 7 h. After cooling, the solvent
was removed by evaporation and H2O was added. The reaction
mixture was extracted with EtOAc. The organic layer, dried
over Na2SO4, was concentrated under reduce pressure and
purified by flash chromatography (EtOAc) to give a yellow
1
oil: yield 95%; H NMR (200 MHz, CDCl3) δ 1.28 (t, 3H, J )
7.0 Hz), 2.54-2.62 (m, 8H), 3.21 (s, 2H), 3.53 (s, 2H), 4.20 (q,
2H, J ) 7.0 Hz), 7.27-7.34 (m, 5H).
(b) 2-(4-Ben zylp ip er a zin -1-yl)a cetic Acid Dih yd r och lo-
r id e (13b). A mixture of ethyl 2-(4-benzylpiperazin-1-yl)-
acetate (28.0 mmol) and 2 M NaOH solution (28 mL) in MeOH
(100 mL) was refluxed for 1 h. After cooling, the reaction
mixture was concentrated by evaporation, acidified with
concentrated HCl, and concentrated again by evaporation.
Et2O was added to the residue, and the solvent was evaporated
to give a beige solid: yield 77%; mp 249 °C; 1H NMR (200 MHz,
CD3OD) δ 3.67-3.79 (m, 8H), 4.25 (s, 2H), 4.52 (s, 2H), 7.50-
7.65 (m, 5H).
(c) 3-[2-(4-Ben zylpiper azin -1-yl)acetam ido]-6-ph en ylpy-
r id a zin e (14b). This compound was prepared using the same
procedure described in the preparation of 14a . White solid;
yield 18%; mp 241 °C; 1H NMR (200 MHz, CDCl3) δ 2.62-
2.73 (m, 8H), 3.26 (s, 2H), 3.59 (s, 2H), 7.29-7.38 (m, 5H),
7.53-7.57 (m, 3H), 7.92 (d, 1H, J ) 9.2 Hz), 8.06-8.11 (m,
2H), 8.59 (d, 1H, J ) 9.2 Hz), 10.24 (brs, 1H).
Com p u ta tion a l Meth od s. (a ) In h ibitor Str u ctu r es. The
crystal structure of minaprine retrieved from the Cambridge
Structural Database was used as template to construct the
inhibitors. All molecules were assumed to be monoprotonated
under physiological conditions, and their molecular structures
were generated accordingly using the SYBYL 6.5 software
(Tripos Associates, St. Louis, MO). The geometry of the
inhibitors was optimized using the Tripos force field and the
conjugate gradient method until the energy difference between
successive cycles was below 0.01 kcal mol-1. Partial atomic
charges were calculated using the semiempirical method AM1.
(b) Cr ysta l Str u ctu r es. The crystal structures of AChE
from T. californica complexed with decamethonium, edropho-
nium,huperzine,and tacrine were retrieved from the Brookhaven
Protein Database. For the docking studies of the 3-aminopy-
ridazines, we selected the AChE crystal structure complexed
with decamethonium, since our developed inhibitors show the
closest resemblance with the size and shape of decametho-
nium. The ligand structure of the complexes was deleted,
Dihydrochloride (i-PrOH): mp 241 °C. Anal. (C23H25N5O‚
2HCl‚3H2O) C; H; N.
3-[2-(4-Ben zylp ip er id in -1-yl)a cet a m id o]-6-p h en ylp y-
r id a zin e (14c). (a ) E t h yl 2-(4-b en zylp ip er id in -1-yl)a ce-
ta te. This compound was prepared using the same procedure
described in the preparation of ethyl 2-(4-benzylpiperazin-1-
hydrogen atoms were added, and charges from AMBER 47 were
48
loaded. AM1
charges were calculated for the ligands. All
water molecules observed in the receptor crystal structure
were deleted, and the enzyme structure was subjected to a
minimization using the AMBER force field, keeping all protein
backbone atoms at fixed positions.
1
yl)acetate. Orange oil; yield 92%; H NMR (300 MHz, CDCl3)
δ 1.25 (t, 3H, J ) 7.1 Hz), 1.36-1.45 (m, 2H), 1.46-1.54 (m,
1H), 1.61 (brd, 2H, J ) 13.9 Hz), 2.09 (brt, 2H, J ) 11.3 Hz),
2.53 (d, 2H, J ) 6.7 Hz), 2.90 (brd, 2H, J ) 11.3 Hz), 3.16 (s,
2H), 4.16 (q, 2H, J ) 7.1 Hz), 7.11-7.19 (m, 3H), 7.23-7.28
(m, 2H).
(b) 2-(4-Ben zylp ip er id in -1-yl)a cetic Acid Hyd r och lo-
r id e (13c). This compound was prepared using the same
procedure described in the preparation of 13b. Beige moss;
yield 98%; 1H NMR (300 MHz, CDCl3) δ 1.84 (m, 5H), 2.61 (d
2H, J ) 5.7 Hz), 3.03 (m, 2H), 3.68 (brd, 2H, J ) 11.3 Hz),
3.81 (s, 2H), 7.12-7.30 (m, 5H), 7.47 (m, 1H).
(c) 3-[2-(4-Ben zylpiper idin -1-yl)acetam ido]-6-ph en ylpy-
r id a zin e (14c). This compound was prepared using the same
procedure described in the preparation of 14b. White solid;
yield 20%; mp 160 °C; 1H NMR (200 MHz, CDCl3) δ 1.15-
1.65 (m, 5H), 2.17 (brt, 2H, J ) 10.3 Hz), 2.50 (d, 2H, J ) 6.0
Hz), 2.85 (brd, 2H, J ) 11.8 Hz), 3.10 (s, 2H), 7.06-7.25 (m,
Ack n ow led gm en t. We thank Prof. G. Cignarella
(Faculty of Pharmacy, University of Milano) for his
advice on the synthesis of the tricyclic derivatives.
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