Design, synthesis, and pharmacological profile of novel fused pyrazolo[4,3-d]pyridine and pyrazolo[3,4-b][1,8]naphthyridine isosteres: A new class of potent and selective acetylcholinesterase inhibitors

Article abstract of DOI:10.1021/jm020391n

A new family of tacrine (THA) analogues (7-9, 12), containing the azaheterocyclic pyrazolo-[4,3-d]pyridine or pyrazolo[3,4-b] [1,8]naphthyridine systems as isosteres of the quinoline ring of THA, has been synthesized. The compounds were tested in rat brai

Full text of DOI:10.1021/jm020391n

1144
J . Med. Chem. 2003, 46, 1144-1152
Design , Syn th esis, a n d P h a r m a cologica l P r ofile of Novel F u sed
P yr a zolo[4,3-d ]p yr id in e a n d P yr a zolo[3,4-b][1,8]n a p h th yr id in e Isoster es: A New
Cla ss of P oten t a n d Selective Acetylch olin ester a se In h ibitor s
Eliezer J . Barreiro,*,†,‡ Celso A. Camara,‡ Hugo Verli,†,‡ Leonora Brazil-Ma´s,§ Newton G. Castro,§
Wagner M. Cintra,§ Yasco Aracava,§ Carlos R. Rodrigues,† and Carlos A. M. Fraga†,‡
Laborato´rio de Avaliac¸a˜o e S´ıntese de Substaˆncias Bioativas (LASSBio), Faculdade de Farma´cia,
Universidade Federal do Rio de J aneiro, C. P. 68.006, 21944-910, Rio de J aneiro, RJ , Brazil,
Departamento de Quı´mica Orgaˆnica, Instituto de Quı´mica, Universidade Federal do Rio de J aneiro,
Rio de J aneiro, RJ , Brazil, and Departamento de Farmacologia Ba´sica e Cl´ınica, Instituto de Cieˆncias Biome´dicas,
Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de J aneiro, Rio de J aneiro, RJ , Brazil
Received September 10, 2002
A new family of tacrine (THA) analogues (7-9, 12), containing the azaheterocyclic pyrazolo-
[4,3-d]pyridine or pyrazolo[3,4-b][1,8]naphthyridine systems as isosteres of the quinoline ring
of THA, has been synthesized. The compounds were tested in rat brain cholinesterases using
Ellman’s method, and all were fully efficacious in inhibiting the enzymes. Compounds 9 and
12b were the most potent against acetylcholinesterase (AChE), showing IC50 of 6.0 and 6.4
µM, and were less active against rat brain butyrylcholinesterase, showing selectivity indexes
of 5.3 and 20.9, respectively. Compounds 7-9 and 12 were also tested for their acute
neurotoxicity in vitro, using cultured rat cortical cells. Compounds 7 and 8 were not significantly
toxic; 9 was toxic at 500 µM, but not at 100 µM. The naphthyridine derivatives 12a and 12b
showed a significant concentration-dependent neurotoxicity, being able to kill most cells at
500 µM. Molecular dynamic simulation using the X-ray crystal structure of AChE from Torpedo
californica was used to explain the possible binding mode of these new THA isosteres.
In tr od u ction
developing new AChE inhibitors with improved activity
and reduced adverse side effects.
Alzheimer’s disease (AD) is a neurodegenerative
disorder characterized by a progressive impairment of
cognitive function which seems to be associated with
deposition of amyloid protein and neuronal loss, as well
as with altered neurotransmission in the brain.1 Much
of the functional deficit in AD has been correlated with
extensive morphological and functional derangement of
the central cholinergic system. The efficacy of cholin-
ergic therapies in this disease validates and supports
the cholinergic hypothesis of AD.2 Several strategies
have been explored to enhance cholinergic transmis-
sion,3 such as acetylcholinesterase (AChE) inhibitors4
(e.g., galanthamine5 (1)), acetylcholine (ACh) precur-
sors1 (e.g., choline, 2), nicotinic agonists6 (e.g., ABT-418,
3), muscarinic agonists7 (e.g., arecholine, 4) or acetyl-
choline releasers3 such as linopirdine (5). AChE inhibi-
tion has proven to be a successful strategy to ameliorate
cholinergic deficit and to promote symptomatic improve-
ment of AD patients.8 Tacrine (9-amino-1,2,3,4-tetra-
hydroacridine, THA, 6), described in 1953, was the first
AChE inhibitor approved for the palliative treatment
of AD senile dementia.9 Its clinical usefulness is limited,
however, owing to undesirable side effects, specially
hepatotoxicity,10,11 and current research is focused on
In the scope of a research program aiming at the
design, synthesis, and evaluation of new neuroactive
lead-compound candidates with beneficial effects in
treatment of AD as AChE inhibitors, we became inter-
ested in developing new THA analogues. The new com-
pounds 7-9 described in this study were structurally
planned with basis in the bioisosteric relationship
between the quinoline ring, included in the 1,2,3,4-
tetrahydroacridine system of THA, and the azahetero-
cyclic pyrazolo[3,4-b]pyridine system.12 This new iso-
steric relationship was discovered in the study of the
new antimalarial compound 10, an analogue of meflo-
quine (11) (Figure 1).12
* To whom correspondence should be addressed. Fax/Phone: +55-
21-25626644. E-mail: eliezer@ufrj.br. http://www.farmacia.ufrj.br/
lassbio.
† LASSBio, Faculdade de Farma´cia, Universidade Federal do Rio
de J aneiro.
The new THA analogues 7-9 (Figure 1) are positional
isomers, designed in order to investigate the eventual
contribution of different N-phenyl orientations around
the azaheterocyclic system in the AChE activity.
‡ Instituto de Qu´ımica, Universidade Federal do Rio de J aneiro.
§ Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de
J aneiro.
10.1021/jm020391n CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/28/2003

Novel Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1145
nitrile derivative 17, by applying the successive oxy-
mation of the formyl group followed by phosphorus
oxychloride dehydration of the oxyme moiety19 (Scheme
1). On the other hand, key intermediates for the
pyrazolo[4,3-b]pyridine derivatives 8 were constructed
through inverse functionalization of the pyrazolyl chlo-
ride derivative 13, which was initially submitted to a
regioselective nitration of C-4 at the pyrazole nucleus,
by using fuming nitric acid and acetic anhydride under
rigid temperature control.20 Next, nucleophilic hetero-
aromatic substitution of chlorine atom at C-5 of nitro
derivative 18 by cyanide anion, under phase transfer
catalysis,21 followed by chemoselective reduction of the
nitro group,22 furnished the desired o-amino nitrile
derivative 20.
The functionalyzed pyrazolo[3,4-b]pyridine derivative
21, precursor of the compounds of pyrazolo[3,4-b][1,8]-
naphthyridine series 12, was prepared through one-pot
Knovenagel condensation of malononitrile with o-amino-
aldehyde derivative (16) followed by intramolecular
cyclization with the vicinal amino group.21
F igu r e 1. Design concept of new pyrazolopyridine THA
analogues 7-9.
The synthesized o-amino nitrile derivatives 17, 20,
21 and commercial 3-amino-4-cyano-1-phenylpyrazole23
(22) were submitted to the standard Friedlander reac-
tion protocols24 with cyclopentanone and/or cyclohex-
anone, using aluminum chloride as Lewis acid, to
furnish the title compounds 7-9 and 12. All structures
were in agreement with analytical and spectral data.
The crystallographic structure for the AChE enzyme
complexed with THA is available.13 On the basis of these
structural data, we performed a preliminary modeling
study with the new azaheterocyclic compounds 7-9 in
order to verify the main structural features that deter-
mine their binding and to evaluate the interactions
observed in the crystallographic structure of the complex
THA-AChE. Due to the molecular rigidity of these
compounds, only the structure energy of each molecule
was minimized and overlaid on the template (THA)
using the program Search_Compare within Insight-II.14
After performing a preliminary molecular dynamic
study15 with AChE, we were also able to identify
interactions of the azaheterocyclic ring of the new
derivatives 7-9 with Phe330 and Trp84 of AChE, as
found for the quinoline ring of 6 in the THA-AChE
complex. In addition, we observed that for the linear
extended isomer 9 there would be supplementary π-stack-
ing interactions and favorable steric access of the NH2
group to the anionic site of AChE. These results sug-
gested a potential benefit in the overall interactions
involving the extended pyrazolo[4,3-d]pyridine system
of 9 and prompted us to syntesize the new analogues
belonging to the pyrazolo[3,4-b][1,8]naphthyridine class
12 (Figure 2).
P h a r m a cologica l Assa ys. To investigate the poten-
tial effects specifically in the central nervous system,
compounds 7-9 and 12 were evaluated as cholin-
esterase inhibitors in rat brain tissue. The main enzyme
form expressed in the brain of rats and humans is the
tetramer of T-type subunits which is bound to mem-
branes through a hydrophobic tail.25,26 Commercially
available enzyme preparations from muscle, electric
organ, or erythrocytes contain other enzyme forms,
which vary in their sensitivity to inhibitors. Concentra-
tion-response curves were obtained for all seven new
compounds 7-9 and 12, and their mean inhibitory
concentration (IC50) was estimated (Table 1). All com-
pounds were fully efficacious in inhibiting brain cholin-
esterase, as illustrated for compound 12b in Figure 3.
For comparison, THA (6) and galanthamine (GAL, 1)
were tested in the same assay conditions, showing mean
IC50's of 0.16 µM and 3.1 µM, respectively. The new THA
analogues 7-9 and 12 were all less potent than the
parent compound (6) and could be separated in two
groups according to the IC50. Compounds 9 and 12b
showed similar potency, with IC50 of 6.01 and 6.39 µM,
respectively, comparable to that of GAL (1), while
compounds 7-8 and 12a fell into another group, with
IC50's ranging from 23.2 µM to 32.2 µM. The most active
compounds (9, 12b) were also tested in a rat brain
butyrylcholinesterase (BuChE) assay (Table 1). The
maximum rate of butyrylthiocholine hydrolysis in the
absence of inhibitors was approximately 10% of that of
acetylthiocholine hydrolysis when assayed in the same
rat brain samples, showing that the total cholinesterase
IC50 largely reflects AChE inhibition. Compounds 9 and
12b showed significant selectivity toward AChE, with
selectivity indices (SI) of 5.3 and 20.9, respectively,
whereas THA (6) was less selective, with a SI of 1.5. It
must be considered that the substrate concentrations
Resu lts a n d Discu ssion
Ch em istr y. The isomeric series of pyrazolo[3,4-b]-
pyridine derivatives 7 and pyrazolo[4,3-b]pyridine de-
rivatives 8 were prepared by classical synthetic meth-
ods, exploring 5-chloro-3-methyl-1-phenylpyrazole16 (13)
as the common key intermediate, as illustrated in
Scheme 1. Initially, key intermediates for the prepara-
tion of pyrazolo[3,4-b]pyridine derivatives 7 were target.
Pyrazole derivative 13 was regioselectively formylated
at C-4, using Vilsmeier-Haack conditions,16 followed by
aza-functionalization of C-5, exploring the nucleophilic
displacement of chlorine atom by azide anion, catalyzed
by the phase transfer agent tetrabutylammonium bro-
mide (TBAB).17 Chemoselective reduction of the azide
group of compound 15 by treatment with iron powder
in acidic media18 furnished o-amino aldehyde derivative
16, which was converted into the corresponding o-amino

1146 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Barreiro et al.
F igu r e 2. (a) Superimposition of the minimum energy conformers of tacrine (THA, 6), pyrazolo[4,3-b]pyridine derivatives (7b,
8b, 9) and the tetracyclic analogue (12b). (b) Structural design of new pyrazolo[3,4-b][1,8]naphthyridine derivatives (12).
Sch em e 1a
a
Reagents: (a) POCl3, DMF, 70 °C, 7 h, 85%; (b) NaN3, TBAB, DMSO, rt, 24 h, 90%; (c) Fe, NH4Cl, AcOEt/H2O, rt, 4 h, 92%; (d)
NH2OH.HCl, pyridine, EtOH, rt, 2 h, 92%; (e) POCl3, reflux, 30 min, 65%; (f) HNO3, Ac2O, 3-5 °C, 1 h, 64%; (g) NaCN, TBAB, DMSO,
80 °C, 16 h, 73%; (h) Fe, NH4Cl, EtOH/H2O, reflux, 15 min, 75%; (i) NCCH2CN, Et3N, MeOH, reflux, 3 h, 72%; (j) cyclopentanone or
cyclohexanone, AlCl3, ClCH2CH2Cl, reflux, 3 h.
used in our assays were high, which is expected to
underestimate the potency of competitive inhibitors.
Although this had little impact in the IC50 of THA,
whose inhibition is mostly noncompetitive,27 it might
have affected the novel compounds to an unknown
extent. A detailed analysis of mechanism would require
further enzyme kinetics studies which are beyond the
scope of this paper, but the rankings of IC50 and SI
among the compounds were deemed adequate for an
initial discussion of SAR.
These results clearly indicated that the tetracyclic and
the linear extended pyrazolopyridine derivatives 12b

Novel Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1147
Ta ble 1. IC50 of New THA Analogues (7-9, 12), THA (6), and
Galanthamine (1) for Inhibition of Rat Brain Cholinesterases
(ChE) (µM)a
compound
total ChEb
BuChE
SIc
THA (6)
7a
7b
8a
8b
9
12a
12b
GAL (1)
0.16 ( 0.03 (4)
31.2 ( 4.2 (2)
28.5 ( 0.3 (3)
32.2 ( 4.4 (2)
26.7 ( 3.1 (3)
6.01 ( 1.22 (3)
23.2 ( 5.3 (3)
6.39 ( 0.86 (3)
3.10 ( 0.18 (2)
0.24 ( 0.10 (2)
1.5
31.9 ( 10.1 (2)
134 ( 15 (2)
5.3
20.9
a
Values are means ( SEM of the IC50 from the indicated
b
number of animals in parentheses. Total cholinesterase (ChE)
and butyrylcholinesterase (BuChE) activities were determined by
Ellman’s method,28 as described in the Experimental Section. c SI
) selectivity index ) (IC50 BuChE)/(IC50 total ChE) ratio.
F igu r e 4. Neurotoxicity of compounds 7-9 and 12, as
compared to that of THA (6). Rat cortical neurons were exposed
to the compounds for 4 h, and cell damage was evaluated by
the amount of LDH released. Bar heights and error bars are
means and SD from three culture wells. Very similar results
were obtained in other two experiments.
THA (6) affects membrane fluidity of hepatocytes as
early as 30 min after exposure to concentrations above
250 µM, and that this effect might underlie the drug-
induced LDH release.31 It is possible that the distinct
composition of neuronal membranes makes them less
susceptible to the membrane-destabilizing effect of THA
(6). The neurotoxic mechanism of compounds 9, 12a ,
and 12b is unknown at present, and may be unrelated
to the cytotoxic mechanisms of THA (6). Interestingly,
compounds 7-8 were not significantly toxic to neurons
and actually showed a tendency to reduce LDH release
below control levels in a concentration-dependent man-
ner. This possibly neuroprotective effect could be a
desirable additional feature in Alzheimer’s treatment
and is worth further investigation.
Molecu la r Dyn a m ics Stu d ies. The most active
derivatives 9 and 12b were next studied by molecular
dynamics (MD) in order to assess their probable binding
mode in the active site of AChE. The active site of AChE
is defined by a few key residues: Ser200, His440,
Glu327, Phe330, Trp84, Trp432, and Tyr121. The
contact distances between most of these residues and
the THA (6) pharmacophore groups are shorter than 4
Å, involving hydrophobic interactions. Initially, MD
simulations were performed in the template THA-
AChE, for which the key interactions responsible for the
stability of the complex have been determined.15 After
the minimization step, the inhibitor THA (6) remains
stacked with the aromatic residues Phe330 and Trp84
and is directly hydrogen-bonded to the backbone oxygen
atom of His440 through its +NH group (Figure 5a) as
observed in the crystal structure. Moreover, the NH2
group of THA (6) is hydrogen-bonded to two water
molecules (W634 and W643) (Figure 5a). The conforma-
tions and interactions achieved were in good agreement
with the crystallographic structure of the THA-AChE
complex.13 The model was then used to compare the
binding mode of the more active new heterocyclic
isosteric derivatives 9 and 12b with AChE.
F igu r e 3. Cholinesterase inhibition curves for compound 12b.
The best-fitting curves had IC50’s of 6.5 and 123.5 µM for total
cholinesterase and butyrylcholinesterase, respectively, in these
two representative experiments. Data points and error bars
are means and SD from three tissue samples.
and 9, respectively, presented the best inhibitory profile
on AChE of this series, with a selectivity index superior
to that of THA (6). On the other hand, derivatives of
the series 7 and 8 were less active, indicating that the
angular pattern adopted by the N-phenyl group at-
tached to the pyrazolopyridine system in these deriva-
tives introduced severe steric constraints into the
molecular fit with Ile439/His440 and Tyr334/Trp432 on
the active site of AChE. Additionally, the tetracyclic
cyclopentane derivative 12a was four times less potent
(IC50 ) 23.2 µM) than the corresponding cyclohexane
derivative 12b (IC50 ) 6.39 µM), possibly due to entropic
effects related to the more conformationally flexible
cyclopentane ring in this compound.
Compounds 7-9 and 12 were also tested for their
acute neurotoxicity in vitro. Rat cortical neurons in
high-density cultures were exposed to compounds 7-9
and 12 for 4 h, and the cell damage was evaluated by
the release of intracellular lactate dehydrogenase
(LDH).29 In the control conditions, a small amount of
LDH was released due to the stress of washing and
removal of serum from the culture medium, as shown
by the dashed line in Figure 4. Compounds 12a and 12b
showed a significant concentration-dependent neuro-
toxicity, being able to kill most cells at 500 µM.
Compound 9 was toxic at 500 µM, but not at 100 µM.
In contrast, THA (6) (up to 500 µM) was not neurotoxic,
suggesting that the mechanisms underlying its well-
known acute toxicity to other cell types, particularly
hepatocytes,11,30 were not operative in our assay. This
is unexpected, in view of a recent study showing that
The knowledge of the exact binding mode of deriva-
tives 9 and 12b with AChE could be useful for the
optimization of these new lead-compounds. Therefore,

1148 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Barreiro et al.
F igu r e 5. Plots of the main interactions between AChE and (a) THA (6); (b) compound 12b; (c) compound 9. Superimposition
between THA (6), 12b, and 9 positioned in the putative binding mode, (d) frontal view; (e) orthogonal view.
the MD simulation was performed to investigate the
stability of the AChE-12b and AChE-9 complexes
(Figure 5, parts b and c, respectively) and to examine
the occurrence of local structural changes in the residues
forming the active site. Further insight into the suit-
ability of the binding model was gained by comparison
with the results obtained from THA simulations. Analy-
sis of the collected structures allowed us to determine
key interactions between AChE and new THA-isosteres
9 and 12b. As detailed below, the model developed
predicts significant differences between the binding
modes of THA and both derivatives 9 and 12b. Thus,
the largest difference was found for the azaheterocyclic
ring of 9, which is orthogonally oriented relative to the
4-aminoquinoline ring of THA (6) in the active site of
AChE. Therefore, it is likely that the steric demand
imposed by some residues in the active site would
significantly affect the orientation of the N-heterocyclic
ring of 9 and 12b upon binding to AChE. This reorien-
tation changes the pattern of interactions in the binding
pocket for both derivatives, so that, for instance, the
hydrogen bond observed for 6 with His440 is not
observed with either derivative. A T-stacking interaction
was observed between the imidazole group of His440
and the pyridine ring of compound 9, with the imid-
azolyl-NH oriented toward the phenyl ring of Phe330.
However, analysis of the interaction energy with His440
indicated a favorable contribution particularly for de-
rivative 9 (-6.98 ( 0.9 kcal mol-1). The indole ring of
Trp84 remained stacking with the pyrazolopyridine ring
of compound 9, as with THA (6). This π-stack interaction
was lost in the case of derivative 12b, but was effectively
replaced by a hydrogen bond between the carbonyl
oxygen atom of Trp84 and the N-H group of 12b plus
van der Waals interactions of the indole ring with the
compound’s cyclohexane ring. The energy profile of the
interaction with Trp84 did not differ among the ligands,
i.e., THA (-12.96 ( 1.1 kcal mol-1), 9 (-12.27 ( 1.4
kcal mol-1) and 12b (-11.11 ( 1.3 kcal mol-1). In
addition, the stacking between compound 12b and

Novel Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1149
azole-4-carbaldehyde (14) (3.00 g, 13.61 mmol), sodium azide
(1.10 g, 16.33 mmol), and tetrabutylammonium bromide (0.526
g, 1.63 mmol) dissolved in 12 mL of DMSO was stirred at room
temperature for 24 h. The reaction mixture was poured in ice
and the crude precipitate collected by filtration, washed with
cooled water, dried, and recrystallized with petroleum ether/
dichloromethane mixture to give 2.84 g (92%) of azido com-
pound 15, as a brown solid, mp 42 °C (lit.,21 42-43 °C): 1H
NMR (200 MHz, CDCl3) δ 2.52 (s, 3H, CH3), 7.38-7.51 (m,
3H, Ph-H3′, Ph-H4′ and Ph-H5′), 7.59 (m, 2H, Ph-H2′ and Ph-
H6′), 9.94 (s, 1H, CHO); 13C NMR (50 MHz, CDCl3) δ 12.9
(CH3), 112.4 (C4), 124.4 (Ph-C2′ and Ph-C6′), 128.7 (Ph-C4′),
129.2 (Ph-C3′ and Ph-C5′), 137.1 (Ph-C1′), 140.6 (C5), 152.2
(C3), 183.8 (CHO); IR (KBr) cm-1: 2156 (NdNdN-), 1672
(CdO).
Phe330 occurred with the N-phenyl substituent on the
pyrazole ring, while in the binding of THA with AChE,
this interaction occurs with the quinoline ring. The
binding modes of AChE ligands THA (6), 12b, and 9 is
depicted in Figure 5.
Con clu sion s
The new analogues of THA 7-12 containing the
azaheterocyclic pyrazolo[4,3-d]pyridine and pyrazolo-
[3,4-b][1,8]naphthyridine system have been designed,
synthesized, and subjected to pharmacological evalua-
tion. The biological results identify compounds 9 and
12b as the most active, presenting an IC50 of 6.01 and
6.39 µM, respectively, each being a cyclohexane deriva-
tive from its N-heterocyclic class. In addition, derivative
12b, was ca. 21 times more selective for acetylcholin-
esterase over butyrylcholinesterase, presenting a selec-
tivity index ca. 10-fold superior to that of THA (6).
Compounds 9 and 12b were toxic to rat cortical neurons
only at concentrations 10× above their IC50, and most
of the other derivatives were even less neurotoxic,
indicating that these new compounds represent useful
templates for development of new anti-AD agents.
5-Am in o-3-m et h yl-1-p h en yl-1H -p yr a zole-4-ca r b a ld e-
h yd e (16). To a mixture of 5-azido-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde (15) (2.80 g, 12.33 mmol), iron powder
(2.22 g, 39.6 mmol), and ethyl acetate (10 mL) was added a
solution of ammonium chloride (3.52 g, 65.8 mmol) in water
(10 mL), and the resulting emulsion was stirred at room
temperature for 4 h. The mixture was filtered and the residue
washed with ethyl acetate (10 mL). The organic layers were
dried with anhydrous sodium sulfate and evaporated under
reduced pressure to furnish a crude oily residue. After column
chromatography on silica gel using dichloromethane as eluent,
2.4 g (91%) of amino aldehyde derivative 16 was obtained as
a white solid, mp 96-97 °C (lit.,33 97.5 °C): 1H NMR (200 MHz,
CDCl3) δ 2.32 (s, 3H, CH3), 5.83 (br, 2H, NH2), 7.28-7.37 (m,
1H, Ph-H4′), 7.42 (m, 4H, Ph-H2′, Ph-H3′, Ph-H5′ and Ph-H6′),
9.60 (s, 1H, CHO); 13C NMR (50 MHz, CDCl3) δ 11.8 (CH3),
105.8 (C4), 123.7 (Ph-C2′ and Ph-C6′), 128.2 (Ph-C4′), 129.9
(Ph-C3′ and Ph-C5′), 137.1 (Ph-C1′), 149.3 (C5), 150.8 (C3),
Exp er im en ta l Section
Ch em istr y. Melting points were determined with a Quimis
1
540 apparatus and are uncorrected. H and 13C NMR spectra
were determined in deuteriochloroform containing ca. 1%
tetramethylsilane as an internal standard with a Brucker DRX
200 spectrometer at 200 and 50 MHz, respectively. Splitting
patterns are as follows: s, singlet; d, doublet; br, broad; m,
multiplet. 1H and 13C NMR assignments given for each
compound were confirmed by HMQC and HMBC experiments.
IR spectra were obtained with a Nicolet-550 Magna spectro-
photometer using KBr pellets. The mass spectra (MS) were
obtained on GC/VG Micromass 12 spectrometer at 70 eV.
Ultraviolet (UV) spectra were determined in methanol solution
on a Beckmann DU-70 spectrophotometer. Microanalysis data
were obtained with a Perkin-Elmer 240 analyzer, using a
Perkin-Elmer AD-4 balance. Prior to concentration under
reduced pressure, all organic extracts were dried over anhy-
drous sodium sulfate powder. The progress of all reactions was
monitored by TLC performed on 2.0 × 6.0 cm aluminum sheets
precoated with silica gel 60 (HF-254, Merck) to a thickness of
0.25 mm. The developed chromatograms were viewed under
UV light at 254 nm. For column chromatography Merck silica
gel (70-230 mesh) was used. Solvents used in the reactions
were dried, redistilled prior to use and stored over +3-4 Å
molecular sieves. Reaction mixtures were generally stirred
under a dry nitrogen atmosphere.
5-Ch lor o-3-m et h yl-1-p h en yl-1H -p yr a zole-4-ca r b a ld e-
h yd e (14). The 5-chloro-3-methyl-1-phenyl-1H-pyrazole (13)
(2.21 g, 11.48 mmol) was heated at 70 °C for 7 h with a mixture
of phosphorus oxychloride (3.52 g, 22.98 mmol) and N,N-
dimethylformamide (1.6 g, 22.98 mmol) and allowed to cool to
40 °C. Then, cold water (80 mL) was added, followed by
neutralization with concentrated aqueous sodium carbonate.
The mixture was extracted with dichloromethane (5 × 80 mL),
and then organic layers were dried with anhydrous sodium
sulfate and concentrated under reduced pressure. The crude
solid was purified by recrystallization in ethanol, yielding 2.12
(84%) of the pyrazole-4-carbaldehyde derivative 14, as yellow
needles, mp 131-132 °C (lit.,32 132 °C): 1H NMR (200 MHz,
CDCl3) δ 2.54 (s, 3H, CH3), 7.47-7.56 (m, 5H, Ph-H2′, Ph-
H3′, Ph-H4′, Ph-H5′ and Ph-H6′), 9.97 (s, 1H, CHO); 13C NMR
(50 MHz, CDCl3) δ 14.0 (CH3), 117.5 (C4), 125.3 (Ph-C2′ and
Ph-C6′), 129.3 (Ph-C4′), 129.4 (Ph-C3′ and Ph-C5′), 137.1 (Ph-
C1′), 133.6 (C5), 151.9 (C3), 184.0 (CHO); IR (KBr) cm-1: 1676
(CdO).
184.0 (CHO); IR (KBr) cm-1
(CdO).
: 3341 and 3224 (NH2), 1640
5-Am in o -3-m e t h y l-1-p h e n y l-1H -p y r a zo le -4-c a r b o -
n itr ile (17). A solution of 5-amino-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde (16) (1.0 g, 4.96 mmol) in ethanol (5
mL), maintained at room temperature, was slowly added to a
premixed solution of hydroxylamine hydrochloride (0.68 g, 9.9
mmol) and pyridine (0.79 g, 9.9 mmol) and the resulting
mixture stirred for 2 h. The reaction mixture was cooled, and
the precipitate formed was filtered off and used for the next
step without further purification, yielding 0.98 g (92%) of the
oxyme intermediate, as an amorphous yellow solid, mp 188-
189 °C. Next, 5-amino-3-methyl-1-phenyl-1H-5-pyrazole carb-
aldehyde oxime (0.75 g, 3.46 mmol) was added in small
portions to cooled (0 °C) phosphorus oxychloride (6 mL), and
the resulting mixture was then carefully refluxed for 30 min.
The reaction mixture was cooled, slowly added to ice-water
(ca. 50 g), and neutralized with 10% aq sodium carbonate
solution. The brown precipitate formed was filtered off and
recrystallized from ethanol yielding 0.45 g (65%) of title
compound 17 as yellow needles, mp 96 °C: 1H NMR (200 MHz,
CDCl3) δ 2.30 (s, 3H, CH3), 4.69 (br, 2H, NH2), 7.40-7.54 (m,
5H, Ph-H2′, Ph-H3′, Ph-H4′, Ph-H5′ and Ph-H6′); 13C NMR
(50 MHz, CDCl3) δ 13.0 (CH3), 76.3 (C4), 114.7 (CN), 124.2
(Ph-C2′ and Ph-C6′), 128.7 (Ph-C4′), 130.0 (Ph-C3′ and Ph-
C5′), 137.1 (Ph-C1′), 150.3 (C3), 151.1 (C5); IR (KBr) cm-1
:
3334 and 3226 (NH2), 2218 (CN). Anal. (C11H10N4) C, H, N.
5-Ch lor o-3-m eth yl-4-n itr o-1-p h en yl-1H-p yr a zole (18).
To a solution of 5-chloro-3-methyl-1-phenylpyrazole (13) (30
g, 155.7 mmol) in 150 mL of acetic anhydride at 0 °C was added
dropwise 30 mL of fuming nitric acid in such a manner that
the temperature was maintained at 0-5 °C. After addition of
the acid, the temperature of the reaction mixture was allowed
to gradually reach 25-30 °C and then was maintained at this
temperature for 4 h. At the end of this period the reaction
mixture was poured over crushed ice, and the precipitate was
filtered and washed with water. After drying, the precipitate
was crystallized from ethanol to give 23.8 g (64%) of the
compound 18, as a yellow light crystals, mp 115-116 °C (lit.,20
115-116 °C): 1H NMR (200 MHz, CDCl3) δ 2.62 (s, 3H, CH3),
5-Azid o-3-m et h yl-1-p h en yl-1H -p yr a zole-4-ca r b a ld e-
h yd e (15). A mixture of 5-chloro-3-methyl-1-phenyl-1H-pyr-
7.52 (s, 5H, Ph-H2′, Ph-H3′, Ph-H4′, Ph-H5′ and Ph-H6′); 13
C

1150 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Barreiro et al.
NMR (50 MHz, CDCl3) δ 14.7 (CH3), 125.5 (Ph-C2′ and Ph-
C6′), 128.1 (C5), 129.5 (Ph-C3′ and Ph-C5′), 129.9 (Ph-C4′),
CH3), 2.75 (t, J ) 7.0 Hz, 2H, C5H2), 3.04 (t, J ) 7.0 Hz, 2H,
C7H
2), 4.58 (br, 2H, NH2), 7.21 (t, J ) 7.1 Hz, 1H, Ph-H4′),
136.9 (Ph-C1′), 147.6 (C3); IR (KBr) cm-1
: 1542 and 1354
7.44 (t, J ) 7.1 Hz, 2H, Ph-H3′ and Ph-H5′), 8.19 (d, J ) 7.9
Hz, 2H, Ph-H2′ and Ph-H6′); 13CNMR (50 MHz, CDCl3) δ 15.3
(CH3), 23.6 (C6), 26.4 (C5), 34.7 (C7), 104.8 (C3a), 112.5 (C4a),
121.2 (Ph-C2′ and Ph-C6′), 125.1 (Ph-C4′), 129.0 (Ph-C3′ and
Ph-C5′), 140.3 (Ph-C1′), 140.6 (C3), 144.1 (C4), 153.6 (C8a),
167.8 (C7a); IR (KBr) cm-1: 3482 and 3380 (NH), 2922 and
(NO2).
3-M e t h y l-4-n i t r o -1-p h e n y l-1H -p y r a z o le -5-c a r b o -
n itr ile (19). A mixture of 5-chloro-3-methyl-4-nitro-1-phenyl-
1H-pyrazole (18) (1.18 g, 5.1 mmol), sodium cyanide (0.27 g,
5.5 mmol), and tetrabutylammonium bromide (0.177 g, 0.55
mmol) dissolved in 6 mL of DMSO was stirred at 80 °C for 16
h. The reaction mixture was poured in ice and the solid
collected by vacuum filtration in a Bu¨chner, washed with
cooled water, dried, and used in the next step without further
purification, yielding 0.83 g (73%) of cyanide derivative 19, as
a light brown solid, mp 126-128 °C: 1H NMR (200 MHz,
CDCl3) δ 2.69 (s, 3H, CH3), 7.60 (m, 3H, Ph-H3′, Ph-H4′ and
Ph-H5′), 7.71 (m, 2H, Ph-H2′ and Ph-H6′); 13C NMR (50 MHz,
CDCl3) δ 13.6 (CH3), 108.0 (CN), 113.4 (C5), 118.2 (C3), 123.4
(Ph-C2′ and Ph-C6′), 130.0 (Ph-C3′ and Ph-C5′), 130.6 (Ph-
C4′), 137.3 (Ph-C1′), 147.4 (C4); IR (KBr) cm-1: 2245 (CN),
1502 and 1360 (NO2). Anal. (C11H8N4O2) C, H, N.
4-Am in o -3-m e t h y l-1-p h e n y l-1H -p y r a zo le -5-c a r b o -
n it r ile (20). A mixture of 3-methyl-4-nitro-1-phenyl-1H-
pyrazole-5-carbonitrile (19) (1.0 g, 4.38 mmol), iron powder
(0.74 g, 13.15 atg), and ammonium chloride (1.16 g, 21.9 mmol)
in 30 mL of a mixture of ethanol and water (2:1) was refluxed
for 15 min. Then, the reaction mixture was filtered hot, and
the filtrate was evaporated to half of its original volume. After
being cooled with an ice bath for 2 h, the yellow crystals of
amino compound 20 (0.65 g, 75%) were collected by filtration,
mp 112-114 °C. 1H NMR (200 MHz, CDCl3) δ 2.26 (s, 3H,
CH3), 3.64 (br, 2H, NH2), 7.34 (m, 1H, Ph-C4′), 7.46 (m, 2H,
Ph-H3′ and Ph-H5′), 7.46 (m, 2H, Ph-H2′ and Ph-H6′); 13C
NMR (50 MHz, CDCl3) δ 10.7 (CH3), 99.8 (C4), 112.0 (CN),
121.2 (Ph-C2′ and Ph-C6′), 127.4 (Ph-C4′), 129.6 (Ph-C3′ and
Ph-C5′), 138.9 (Ph-C1′), 139.2 (C5); IR (KBr) cm-1: 3357 and
3323 (NH), 2223 (CN). Anal. (C11H10N4) C, H, N.
2853 (CH aliph.), 1619 and 1566 (CdN ring); UV (EtOH) λmax
:
217 (log ꢀ ) 4.37), 255 (log ꢀ ) 4.27), 314 (log ꢀ ) 4.0). Anal.
(C16H16N4) C, H, N.
3-Meth yl-1-p h en yl-5,6,7,8-tetr a h yd r o-1H-p yr a zolo[3,4-
b]qu in olin -4-a m in e (7b): Compound 7b was obtained in 42%
yield, from Friedla¨nder condensation of the 4-amino-3-methyl-
1-phenyl-1H-pyrazole-5-carbonitrile (20) with cyclohexanone,
as a yellow solid, mp 136-8 °C; 1H NMR (200 MHz, CDCl3) δ
1.76-1.79 (m, 4H, C6H2 and C7H2), 2.32-2.35 (m, 2H, C8H2),
2.58 (s, 3H, CH3), 2.84-2.86 (m, 2H, C5H2), 4.45 (br, 2H, NH2),
7.09 (t, J ) 7.3 Hz, 1H, Ph-H4′), 7.35 (t, J ) 7.5 Hz, 2H, Ph-
H3′ and Ph-H5′), 8.20 (d, J ) 7.8 Hz, 2H, Ph-H2′ and Ph-H6′);
13CNMR (50 MHz, CDCl3) δ 15.4 (CH3), 22.8 (C8), 23.0 (C6
and C7), 34.3 (C5), 104.4 (C3a), 107.6 (C4a), 120.6 (Ph-C2′ and
Ph-C6′), 124.8 (Ph-C4′), 128.9 (Ph-C3′ and Ph-C5′), 140.3 (Ph-
C1′), 140.5 (C3), 146.2 (C4), 151.3 (C9a), 158.5 (C8a); IR (KBr)
cm-1: 3446 and 3298 (NH), 2930 (CH aliph.), 1643 and 1598
(CdN ring); UV (EtOH) λmax: 218 (log ꢀ ) 4.33), 254 (log ꢀ )
4.23), 316 (log ꢀ ) 3.9). Anal. (C17H18N4) C, H, N.
3-Meth yl-1-p h en yl-1,5,6,7-tetr a h yd r ocyclop en ta [b]p yr -
a zolo[3,4-e]p yr id in -8-a m in e (8a ). Compound 8a was ob-
tained in 40% yield, from Friedla¨nder condensation of the
5-amino-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile (17) with
cyclopentanone, as a yellow solid, mp 173-5 °C; 1H NMR (200
MHz, CDCl3) δ 2.22 (qt, J ) 7 Hz, 2H, C6H2), 2.6 (s, 3H, CH3),
2.76 (t, J ) 7 Hz, 2H, C7H2), 3.08 (t, J ) 7 Hz, 2H, C5H2),
4.03 (br, 2H, NH2), 7.29-7.41 (m, 1H, Ph-H4′), 7.43-7.50 (m,
4H, Ph-H2′, Ph-H3′, Ph-H5′ and Ph-H6′); 13CNMR (50 MHz,
CDCl3) δ 11.2 (CH3), 23.6 (C6), 27.0 (C7), 34.4 (C5), 117.2 (C7a),
123.1 (C3a), 125.7 (Ph-C2′ and Ph-C6′), 127.9 (Ph-C4′), 129.4
(Ph-C3′ and Ph-C5′), 134.47 (C8), 140.5 (Ph-C1′), 142.8 (C8a),
144.1 (C3), 163.4 (C4a); IR (KBr) cm-1: 3496 and 3409 (NH),
2923 and 2859 (CH aliph.), 1625 and 1592 (CdN ring); UV
(EtOH) λmax: 226 (log ꢀ ) 4.57), 256 ((log ꢀ ) 4.26), 308 (log ꢀ
) 4.14). Anal. (C16H16N4) C, H, N.
3-m eth yl-1-p h en yl-5,6,7,8-tetr a h yd r o-1H-p yr a zolo[4,3-
b]qu in olin -9-a m in e (8b): Compound 8b was obtained in 45%
yield, from Friedla¨nder condensation of the 5-amino-3-methyl-
1-phenyl-1H-pyrazole-4-carbonitrile (17) with cyclohexanone,
as a yellow solid, mp 173-5 °C; 1H NMR (200 MHz, CDCl3) δ
1.87-1.89 (m, 4H, C6H2 and C7H2), 2.48-2.50 (m, 2H, C5H2),
2.64 (s, 3H, CH3), 3.01-3.03 (m, 2H, C8H2), 4.12 (br, 2H, NH2),
7.36-7.41 (m, 1H, Ph-H4′), 7.42-7.51 (m, 4H, Ph-H2′, Ph-H3′,
Ph-H5′ and Ph-H6′); 13CNMR (50 MHz, CDCl3) δ 11.2 (CH3),
22.9 (C7), 23.1 (C6), 23.6 (C8), 33.8 (C5), 111.8 (C8a), 123.0
(C3a), 125.7 (Ph-C2′ and Ph-C6′), 127.9 (Ph-C4′), 129.4 (Ph-
C3′ and Ph-C5′), 136.3 (C9a), 140.3 (Ph-C1′), 140.4 (C9), 144.2
(C3), 154.4 (C4a); IR (KBr) cm-1: 3481 and 3379 (NH), 2948
and 2881 (CH aliph.), 1619 and 1566 (CdN ring); UV (EtOH)
6-Am in o-3-m eth yl-1-ph en yl-1H-pyr azolo[3,4-b]pyr idin e-
5-ca r bon itr ile (21). A solution of 5-amino-3-methyl-1-phenyl-
1H-pyrazole-4-carbaldehyde (16) (0.5 g, 2.5 mmol), malono-
nitrile (0.17 mL, 2.6 mmol), and triethylamine (6 drops) in
methanol (15 mL) was refluxed on steam bath for 2 h. The
white colored product obtained after cooling was filtered off
and recrystallized from methanol, yielding 0.44 g (70%) of the
desired fused heterocycle derivative 21, mp 223-225 °C (lit.,21
1
226 °C); H NMR (200 MHz, CDCl3) δ 2.56 (s, 3H, CH3), 5.72
(br, 2H, NH2), 7.19 (t, J ) 7.4 Hz, 1H, Ph-H4′), 7.39 (t, J ) 8.0
Hz, 2H, Ph-H3′ and Ph-H5′), 8.00 (s, 1H, Pyridine-H4), 8.05
(d, J ) 8.4 Hz, 2H, Ph-H2′ and Ph-H6′); 13C NMR (50 MHz,
CDCl3) δ 12.2 (CH3), 87.6 (C5), 110.2 (C3a), 117.2 (CN), 121.0
(Ph-C2′ and Ph-C6′), 125.8 (Ph-C4′), 128.8 (Ph-C3′ and Ph-
C5′), 136.5 (C4), 138.8 (Ph-C1′), 143.9 (C3), 151.0 (C7a), 158.2
(C6); IR (KBr) cm-1: 3331 and 3219 (NH), 2210 (CN).
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Ta cr in e
An a logu es 7-9 a n d 12. A mixture of 0.5 mmol of the
corresponding o-amino-heteroaryl nitrile derivative 17, 20, 21,
or 22 and 1.1 equiv of cyclohexanone or cyclopentanone was
added to a suspension of anhydrous aluminum chloride (0.5
mmol) in 15 mL of dry 1,2-dichloroethane, under inert atmo-
sphere, and the reaction mixture was heated at reflux for 3 h.
Then, a mixture of THF (3 mL) and water (1 mL) was added,
and the reaction mixture was refluxed for additional 10 min
and neutralized with 10% aq NaOH solution. After cooling,
the organic components were extracted with chloroform and
the organic layers were combined, dried, and evaporated at
reduced pressure to give the desired azaheterocyclic product.
The products were purified by SiO2 flash chromatography
using as eluent a mixture of MeOH/EtOAc/CHCl3/Et3N (1:2:
7:1).
λ
max: 223 (log ꢀ ) 4.52), 255 (log ꢀ ) 4.21), 306 (log ꢀ ) 4.10).
Anal. (C17H18N4) C, H, N.
2-P h en yl-5,6,7,8-tetr a h yd r o-2H-p yr a zolo[3,4-b]qu in o-
lin -4-a m in e (9): Compound 9 was obtained in 45% yield, from
Friedla¨nder condensation of the 3-amino-1-phenyl-1H-pyr-
azole-4-carbonitrile22 (22) with cyclohexanone, as a yellow
1
solid, mp 215-6 °C; H NMR (200 MHz, CDCl3) δ 1.74-1,77
(m, 4H, C6H2 and C7H2), 2.49-2.51 (m, 2H, C5H), 2.75-2.78
(m, 2H, C8H2), 6.52 (br, 2H, NH2), 7.40 (t, J ) 7.2, 1H, Ph-
H4′), 7.57 (t, J ) 7.6 Hz, 2H, Ph-H3′ and Ph-H5′), 7.90 (d, J )
7.7 Hz, 2H, Ph-H2′ and Ph-H6′), 8.94 (s, 1H, C4H); 13CNMR
(50 MHz, CDCl3) δ 23.3 (C6), 23.4 (C7), 23.7 (C5), 34.7 (C8),
104.5 (C4a), 108.0 (C3a), 120.0 (Ph-C2′ and Ph-C6′), 120.0 (C3),
127.9 (Ph-C4′), 130.3 (Ph-C3′ and Ph-C5′), 140.6 (Ph-C1′), 146.8
3-Meth yl-1-p h en yl-1,5,6,7-tetr a h yd r ocyclop en ta [b]p yr -
a zolo[4,3-e]p yr id in -4-a m in e (7a ). Compound 7a was ob-
tained in 38% yield, from Friedla¨nder condensation of the
4-amino-3-methyl-1-phenyl-1H-pyrazole-5-carbonitrile (20) with
cyclopentanone, as a yellow solid, mp 135-6 °C; 1H NMR (200
MHz, CDCl3) δ 2.11 (qt, J ) 6.9 Hz, 2H, C6H2), 2.67 (s, 3H,
(C4), 159.0 (C9a), 152.2 (C10a), 161.2 (C8a); IR (KBr) cm-1
3419 and 3306 (NH), 2927 and 2859 (CH aliph.), 1639 and
:

Novel Acetylcholinesterase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1151
1552 (CdN ring); UV (EtOH) λmax: 208 (log ꢀ ) 4.52), 240 (log
ꢀ ) 4.40), 303 (log ꢀ ) 4.37), 342 (log ꢀ ) 4.00). Anal. (C16H16N4)
C, H, N.
distance from a water oxygen atom to the sphere center
exceeded 16 Å. The minimization and MD simulation steps
were accomplished using the CVFF force field within the
Discover program of the InsightII environment. All calcula-
3-Meth yl-1-p h en yl-1,6,7,8-tetr a h yd r ocyclop en ta [b]p yr -
a zolo[4,3-g][1,8]n a p h th yr id in -5-a m in e (12a ): Compound
12a was obtained in 45% yield, from Friedla¨nder condensation
of the 6-amino-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-
5-carbonitrile (21) with cyclopentanone, as a yellow solid, mp
tions were performed on
a Silicon Graphics O2 R10000
workstation under Irix operational system.
P h a r m a cology. Ch olin ester a se Activity Assa ys. Ell-
man’s colorimetric method28 was adapted for determination
of total cholinesterase and butyrylcholinesterase activities in
rat brain homogenates. Brain tissue from adult Wistar rats
was homogenized at 2% w/v (or 10%, for butyrylcholinesterase)
in 0.1 M sodium phosphate buffer, pH 7.4, with added NaCl
58.5 g/L and Triton X-100 0.05% v/v. Aliquots of homogenate
(20 µL) were incubated with anticholinesterase compounds for
10 min in phosphate buffer pH 8.0 before addition of 5,5′-
dithiobis(2-nitrobenzoic) acid and either 1 mM acetylthio-
choline iodide or 10 mM butyrylthiocholine iodide (Sigma, St.
Louis, MO). The reaction was run at room temperature (22-
25 °C) in a final volume of 235 µL in 96-well microplates and
was followed at 412 nm for 5 min with a plate reader
(SpectraMAX 250, Molecular Devices, Sunnyvale, CA). In
every experiment, cholinesterase-independent (nonspecific)
substrate hydrolysis was determined by including one experi-
mental group treated with tacrine 20 µM; appropriate tissue
and reagent blanks were also included. Reaction velocities
were determined in three or four replicates per condition; these
were averaged and expressed as percent activity relative to
control (solvent), after subtracting the rate of nonspecific
hydrolysis. All compounds were tested in at least five concen-
trations that covered the range producing less than 20% and
greater than 80% inhibition, but limited to 500 µM. The IC50
based on a single-site model was determined by nonlinear
regression. Results are reported as mean ( SEM of IC50
obtained independently from two to four animals.
Neu r otoxicity. Cortical neurons from Wistar rat fetuses
at 18-20 days of gestation were isolated by trypsinization and
trituration. Cells were seeded onto poly-L-lysine-coated 24-well
plates at a density of four cerebral hemispheres per plate and
were maintained in culture in serum-supplemented minimum
essential medium (MEM, Gibco, Rockville, MD). Glial cell
proliferation was halted by addition of 5-fluoro-2′-deoxyuridine
on the day after plating, and neurons were allowed to mature
for 14-18 days before the experiments. Cells were washed
with serum-free medium, the test compounds were added, and
the plates returned to the incubator at 35 °C for 4 h. At the
end of this incubation period, neurotoxicity was measured by
the amount of LDH released in the culture supernatant.29 The
enzyme’s Vmax was determined kinetically at 37 °C using
reagents from a commercial LDH assay kit (Doles Reagentes,
Brazil) based on the method of Whitaker.34 The reaction was
run in a final volume of 100 µL in 96-well plates and was
followed at 510 nm for 5 min. Results are reported as mean (
SEM of the Vmax from three culture wells treated in parallel.
1
220-2 °C; H NMR (200 MHz, CDCl3) δ 1.95 (qt, J ) 7.1 Hz,
2H, C7H2), 2.50 (s, 3H, CH3), 2.70 (t, J ) 7 Hz, 2H, C6H2),
2.82 (t, J ) 7.5 Hz, 2H, C8H2), 6.93 (br, 2H, NH2), 7.12 (t, J )
7.3, 1H, Ph-H4′), 7.40 (t, J ) 7.6 Hz, 2H, Ph-H3′ and Ph-H5′),
8.32 (d, J ) 7.3 Hz, 2H, Ph-H2′ and Ph-H6′), 9.11 (s, 1H, C4H);
13CNMR (50 MHz, CDCl3) δ 12.3 (CH3), 21.7 (C7), 27.3 (C6),
34.9 (C8), 108.3 (C4a), 111.5 (C5a), 115.1 (C3a), 118.8 (Ph-C2′
and Ph-C6′), 124.3 (Ph-C4′), 126.6 (C4), 128.8 (Ph-C3′ and Ph-
C5′), 139.5 (Ph-C1′), 143.8 (C3), 148.7 (C5), 150.5 (C10a), 155.5
(C9a), 171.6 (C8a); IR (KBr) cm-1: 3360 and 3224 (NH), 2958
and 2923 (CH aliph.), 1644 and 1598 (CdN ring); UV (EtOH)
λ
max: 218 (log ꢀ ) 4.16), 276 (log ꢀ ) 4.55), 344 (log ꢀ ) 3.75).
Anal. (C19H17N5) C, H, N.
3-Meth yl-1-p h en yl-6,7,8,9-tetr a h yd r o-1H-ben zo[b]p yr -
a zolo[4,3-g][1,8]n a p h th yr id in -5-a m in e (12b): Compound
12b was obtained in 52% yield, from Friedla¨nder condensation
of the 6-amino-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-
5-carbonitrile (21) with cyclohexanone, as a yellow solid, mp
215-6 °C; 1H NMR (200 MHz, CDCl3) δ 1.82-1,87 (m, 4H,
C7H2 and C8H2), 2.52-2.56 (m, 2H, C6H2), 2.66 (s, 3H, CH3),
2.89-2.91 (m, 2H, C9H2), 7.14 (br, 2H, NH2), 7.27 (t, J ) 7.3,
1H, Ph-H4′), 7.54 (t, J ) 7.7 Hz, 2H, Ph-H3′ and Ph-H5′), 8.45
(d, J ) 7.9 Hz, 2H, Ph-H2′ and Ph-H6′), 9.33 (s, 1H, C4H);
13CNMR (50 MHz, CDCl3) δ 12.8 (CH3), 22.6 (C7 and C8), 23.7
(C6), 33.7 (C9), 107.8 (C5a), 108.3 (C4a), 116.1 (C3a), 119.4
(Ph-C2′ and Ph-C6′), 124.9 (Ph-C4′), 127.5 (C4), 129.4 (Ph-C3′
and Ph-C5′), 140.0 (Ph-C1′), 144.6 (C3), 151.3 (C11a), 152.2
(C10a), 153.2 (C5), 162.4 (C9a); IR (KBr) cm-1: 3359 and 3221
(NH), 2956 and 2923 (CH aliph.), 1630 and 1600 (CdN ring);
UV (EtOH) λmax: 218 (log ꢀ ) 4.16), 277 (log ꢀ ) 4.52), 349
(log ꢀ ) 3.86). Anal. (C20H19N5) C, H, N.
Molecu la r Mod elin g. Molecu la r Dyn a m ics. We selected
the crystal monomeric structure of the AChE-THA complex,
including crystal waters, from Protein Data Bank (PDB code:
1ACJ ). The hydrogen atoms were added in this structure and
energy minimized with 2500 steps of steepest descent followed
by 5000 steps of conjugate gradient until a gradient norm of
0.01 kcal mol-1 Å-1, keeping all heavy atoms fixed. All residues
and crystal waters beyond a region of 16 Å centered on the
nitrogen of the pyridine ring were discarded. The missing side
chains of some residues inside this region were reconstructed.
Acetyl and N-methylamide blocking groups were used to cap
the truncation points. We assumed that the THA molecule and
its derivatives were all protonated. These molecules, ab initio
optimized with the 6-31G** basis set, were placed in the active
site of the enzyme trough the superimposition of their struc-
tures with the THA crystal coordinates. Charges for THA and
its derivatives used in MD simulations were fitted to the
6-31G** eletrostatic potential using the CHELPG methodology
within PC-SPARTAN-PRO 1.0.5, Wavefunction. To relax
some poor interatomic contacts, all complexes formed were
energy minimized with 5000 steps of steepest descent followed
by 10000 steps of conjugate gradient until a gradient norm of
Ack n ow led gm en t. We gratefully acknowledge fi-
nancial support and fellowships from the PRONEX
(Grant #0888, Brazil), the CNPq (Grants 460.200/00-3
and 50.0033/95-0, Brazil), the FAPERJ (Grant #E-26/
170.399/2000, Brazil), the CAPES (Brazil) and the
FUJ B (Brazil). We also thank the NPPN Analytical
Center of Universidade Federal do Rio de J aneiro
(Brazil) for spectroscopic facilities.
0.01 kcal mol-1 Å-1
.
These systems were equilibrated at 300 K for 300 ps using
the velocity scaling method to control the temperature. For
the production phase the temperature was controlled using
the Berendsen method during 300 ps. We used the time step
of 1 fs for the dynamics and a nonbonded interaction cutoff
radius of 11 Å; the trajectory was sampled every 1 ps. The
leapfrog algorithm was used for integrating the equations of
motion. During the MD simulation the methyl carbon atoms
of acetyl and N-methylamide blocking groups were kept fixed
in order to avoid anomalous behavior due the absence of the
discarded residues. To prevent water evaporation, we applied
a half-harmonic potential (k ) 0.5 kcal mol-1 Å-2) if the
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