Synthesis, acetylcholinesterase inhibition and neuroprotective activity of new tacrine analogues

Article abstract of DOI:10.1016/j.bmc.2004.11.020

The synthesis and the biological evaluation (acetylcholinesterase inhibition activity and neuroprotection) of the new tacrine analogues 2-14 is described.

Full text of DOI:10.1016/j.bmc.2004.11.020

Bioorganic & Medicinal Chemistry 13 (2005) 1167–1175
Synthesis, acetylcholinesterase inhibition and
neuroprotective activity of new tacrine analogues
a,b
Rafael Leon, Jose Marco-Contelles, Antonio G. Garcıa and Mercedes Villarroya
a,
b
b,
*
*
´
´
´
aLaboratorio de Radicales Libres (IQOG, CSIC), C/ Juan de la Cierva 3, 28006Madrid, Spain
b
´
´
´
Instituto Teofilo Hernando, Departamento de Farmacologıa y Terapeutica, Facultad de Medicina,
´
Universidad Autonoma de Madrid, C/ Arzobispo Morcillo, 4, 28029 Madrid, Spain
Received 19 October 2004; revised 3 November 2004; accepted 9 November 2004
Available online 25 November 2004
Abstract—The synthesis and the biological evaluation (acetylcholinesterase inhibition activity and neuroprotection) of the new
tacrine analogues 2–14 is described.
ꢀ 2004 Published by Elsevier Ltd.
1. Introduction
was the first drug approved in the United States for
the palliative treatment of AD, but the poor selectivity
of this drug for AChE resulted in a number of side ef-
fects, specially hepatotoxicity,6b and current research is
focused on developing new AChE inhibitors with im-
proved activity and reduced adverse side effects.
AlzheimerÕs disease (AD) is the most common type of
dementia in western societies.1 Of the many pathogenic
hypotheses of AD (i.e., b-amyloid deposits, hyper-
phosphorylation of protein s, cholinergic neuron loss
in the basal nucleus of Meynert2) an alteration of cell
Ca2+ homeostasis might be at the centre of the meta-
bolic crossroad leading neurons to apoptotic death.3,4
The neuropsychiatric symptoms associated with the ill-
ness are related to the reduction in the number of func-
tional neuronal nicotinic receptors (nAChR);5 for this
reason the only therapeutical strategy that, up to now,
has proven to have some efficacy in slowing progression
of AD is that which improves cholinergic neurotrans-
mission, counteracting the deficit of cerebral acetylcho-
line.6 Therefore, acetylcholinesterase (AChE) inhibition
is currently the therapeutic strategy most commonly
used to treat Alzheimer patients. On the other hand,
the multifactorial pathogenesis of AD suggests that drug
treatments with two or more mechanisms of action,
acting in a complementary manner, could be more
efficacious to patients suffering the disease. In fact,
galanthamine, a drug currently used to treat AD, is a
mild inhibitor of AChE that sensitizes the nicotinic
receptor to AChE7 and prevents cell death induced by
b-amyloid or thapsigargin.8 On the other hand, tacrine
(1, Chart 1), a potent and reversible AChE inhibitor,
In this context, a few years ago, we started a project
aimed at the synthesis and the pharmacological study
of new tacrine analogues. As a result, we obtained a
number of compounds of class A and B, with a substi-
tuted benzene-moiety at C-4 position, that keep their
AChE inhibitory properties and behave as mild Ca2+
promotors, enhancing the entry of Ca2+ into excitable
(chromaffin) and smooth muscle cells (Chart 1).9
Continuing with this project we now report the synthesis
of compounds 2–14 with a 3- or 4-pyridyl, 2-(N-acetyl)-
pyrrolyl and 2-thienyl groups at position C-4 (Chart 1).
In these tacrine analogues we wanted to evaluate the
biological activity of related substrates with a heterocy-
clic ring moiety at the C4-position. We also describe
some aspects of their biological profile, like the acetyl-
cholinesterase (AChE) inhibition activity and their
properties as neuroprotector agents.
2. Results and discussion
2.1. Chemistry
Keywords: Tacrine analogues; Inhibitors; AChE; Neuroprotection.
*
The synthesis of these new tacrine analogues 2–11 and
12–14 has been achieved by Friedla¨nder reaction10 of
Corresponding authors. Tel.: +34 91 5622900; fax: +34 91 5644853;
e-mail: iqoc21@iqog.csic.es
0968-0896/$ - see front matter ꢀ 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2004.11.020

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R. Leon et al. / Bioorg. Med. Chem. 13 (2005) 1167–1175
1168
X
X
NH2
NH2
N
NH2
N
EtO2C
Me
EtO2C
Me
(CH2)n
4
(CH2)n
4
N
O
N
Tacrine (1)
A (n= 0, 1, 2)
B (n= 0, 1, 2)
X
Y
N
X
NH2
N
NH2
N
NH2
EtO2C
EtO2C
Me
EtO2C
(CH2)n
(CH2)n
(CH2)n
Me
O
O
Me
N
N
8 X= S, n= 0
9 X= S, n= 1
10 X= S, n= 2
2 X= N, Y =CH, n= 0
3 X= N, Y= CH, n= 1
4 X= N, Y= CH, n= 2
5 X= CH, Y =N, n= 0
6 X= CH, Y= N, n= 1
7 X= CH, Y= N, n= 2
12 n= 0
13 n= 1
14 n= 2
11 X= NCOMe, n= 1
Chart 1.
X
N
X
Y
Y
S
X
EtO2C
Me
CN
EtO2C
Me
CN
EtO2C
Me
CN
EtO2C
Me
CN
N
NH2
NH2
O
NH2
O
NH2
26
15 X= N, Y= CH
16 X= CH, Y= N
17 X= S
19 X= N, Y= CH
25 X= CH, Y= N
18 X= NCOCH3
24 X= O
Chart 2.
the corresponding heterocyclic precursors, such as, ethyl
6-amino-5-cyano-4H-pyran-3-carboxylates11
(15–18)
(Chart 2) and ethyl 6-amino-5-cyano-4-pyridine-3-car-
boxylate12 (19), respectively (Chart 2) with selected
ketones (cyclopentanone, cyclohexanone or cyclo-
heptanone).
boxaldehyde (20), via the Knoevenagel condensation
product 3-pyridylmethylidenemalononitrile (21),14 not
isolated, after in situ Michael addition with ethyl aceto-
acetate, catalyzed by piperidine (Scheme 3). Finally, the
Friedla¨nder reaction10 of compound 16 with cyclopenta-
none, cyclohexanone and cycloheptanone gave products
5 (73%), 6 (88%) and 7 (77%), respectively (Scheme 3).
For the pyran-like tacrine derivatives 2–11 (Chart 1),
under the standard conditions,10 using dry 1,2-dichloro-
ethane as solvent and aluminium trichloride as
promotor, known ethyl 6-amino-5-cyano-4-(4-pyridyl)-
4H-pyran-3-carboxylate 1513 (Chart 2) and ethyl 6-ami-
no-5-cyano-4-(2-thienyl)-4H-pyran-3-carboxylate 17,13
(Chart 2) afforded the expected tacrine analogues 2–4
(Scheme 1) and 8–10 (Scheme 2), respectively, after reac-
tion with cyclopentanone, cyclohexanone and cyclohep-
tanone, in mild conditions and moderate chemical
yields.
Similarly, pyran 18 (Chart 2) has been prepared for the
first time by Michael reaction of ethyl acetoacetate and
N-acetyl-2-pyrrolylmethylidenemalononitrile (23), that
we were able to synthesize by simple acetylation of the
N
N
NH2
EtO2C
Me
a
EtO2C
Me
CN
(CH2)n
O
N
O
NH2
Pyran 16 (Chart 2) is available from commercial sources
in small amounts (Akos, Lithuania), but very surpris-
ingly it has never been reported in the literature. We
have prepared it using the standard protocol12 by Ôone-
potÕ reaction from commercially available 3-pyridinecar-
2-4
15
Scheme 1. Reagents and conditions: (a) 1,2-dichloroethane, AlCl3,
cyclopentanone (2, n = 0, 65%); cyclohexanone (3, n = 1, 85%),
cycloheptanone (3, n = 2, 97%).

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R. Leon et al. / Bioorg. Med. Chem. 13 (2005) 1167–1175
1169
2), but we were unable to perform any successful
Friedla¨nder reaction on this substrate, probably due to
the lability of the furan derivatives under these experi-
mental conditions.
S
S
NH2
N
a
EtO2C
Me
EtO2C
Me
CN
(CH2)n
O
O
NH2
17
8-10
The naphthyridine-like tacrine derivatives 12–14 (Chart
1) have been synthesized from pyridine 19, prepared
from the corresponding pyran 1513 as usual, after reac-
tion with ammonium acetate in acetic acid,12 as shown
in Scheme 5.
Scheme 2. Reagents and conditions: (a) 1,2-dichloroethane, AlCl3,
cyclopentanone (8, n = 0, 29%); cyclohexanone (9, n = 1, 83%),
cycloheptanone (10, n = 2, 61%).
N
The new pyridines 25 and 26 (Chart 2), prepared from
the corresponding 4H-pyrans 16 (Scheme 5) and 17
(Scheme 6), respectively,12 did not afford the expected
tacrine analogues.
N
N
a
EtO2C
Me
CN
b
NC
O
H
CN
O
NH2
20
2.2. Pharmacology
21
16
N
2.2.1. The AChE inhibitory activity. According to the
standard methodology17 we obtained the following data
for the acetylcholinesterase inhibition activity in the pyr-
ano[3,2-e]pyridines, and pyrano[2,3-b]quinolines 2–10
(see Table 1). For comparative purposes we have also in-
cluded in Table 1 the IC50 (lM) for tacrine (1) and the
previously published analogues 27 and 289b (Chart 3).
None of the test compounds showed an improved AChE
inhibition as compared to tacrine (IC50 0.14mM), with
compound 27 (IC50 0.87mM)9b being the most active
in this assay.
NH2
EtO2C
Me
(CH2)n
O
N
5-7
Scheme 3. Reagents and conditions: (a) malononitrile, then ethyl
acetoacetate, piperidine (84%); (b) 1,2-dichloroethane, AlCl3, cyclo-
pentanone (5, n = 0, 73%); cyclohexanone (6, n = 1, 88%), cyclohep-
tanone (7, n = 2, 77%).
previously described 2-pyrrolylmethylidenemalononit-
rile (22)15 (Scheme 4). Finally, the Friedla¨nder reaction
of compound 18 with cyclohexanone gave product 11
(43%) (Scheme 4). The reaction of pyran 18 (Chart 2)
with cyclopentanone or cycloheptanone failed.
Structure–activity relationship analysis regarding the
heterocyclic ring moiety in the C4-position of tacrine
leads to the following conclusions. For the class A (i)
the pyrrolyl containing substrate (11) is the most active
(10-fold drop compared to tacrine), followed by the 4-
pyridyl containing substrates (2–4). The 3-pyridyl—with
the exception of compound 6—and 2-thienyl derivatives
We have also prepared ethyl 6-amino-5-cyano-4-(2-fur-
yl)-2-methyl-4H-pyran-3-carboxylate (24)10a,b,16 (Chart
O
MeC
O
MeC
N
N
XN
NH2
b
EtO2C
CN
c
EtO2C
NC
CN
Me
O
NH2
Me
O
N
22 X= H
11
18
a
23 X= COMe
Scheme 4. Reagents and conditions: (a) Ac2O, py (98%); (b) ethyl acetoacetate, piperidine (99%); (c) 1,2-dichloroethane, AlCl3, cyclohexanone
(43%).
X
X
N
N
N
Y
Y
NH2
N
EtO2C
Me
CN
a
EtO2C
Me
CN
b
EtO2C
Me
(CH2)n
(from 19)
O
NH2
NH2
12-14
15 X= N, Y= CH
16 X= CH, Y= N
19 X= N, Y= CH
25 X= CH, Y= N
Scheme 5. Reagents and conditions: (a) AcOH, AcONH4 (15–19: 57%), (16–25: 15%); (b) 1,2-dichloroethane, AlCl3, cyclopentanone (12, n = 0,
61%); cyclohexanone (13, n = 1, 49%), cycloheptanone (14, n = 2, 92%).

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R. Leon et al. / Bioorg. Med. Chem. 13 (2005) 1167–1175
1170
Regarding the [1,8]naphthyridine derivatives 12–14
S
S
(class B), it can be seen that the replacement of a
benzene substituent, compound 28 (Chart 3),9b for a
4-pyridyl substituent is not favoured. Again, the six-
membered cycloannulated compound shows the stron-
gest inhibitory effect. This compound is in fact the most
potent in the whole series synthesized. Comparing the
two classes, the [1,8]naphthyridine derivatives (class B)
are the more potent one.
EtO2C
Me
CN
a
EtO2C
Me
CN
O
NH2
N
NH2
17
26
Scheme 6. Reagents and conditions: (a) AcOH, AcONH4 (5%).
Table 1. IC50 (lM) values for the AChE inhibition
2.2.2. Neuroprotection. Inhibition of AChE may tempo-
rarily improve AD symptoms such as memory and cog-
nitive impairment,18 but not the ongoing cellular loss.
Drug treatments with two or more mechanisms of ac-
tion, acting in a complementary manner, could be more
efficacious to patients suffering from the disease. Galan-
thamine, which is an allosteric potentiating ligand of the
nAChR,7 also prevents apoptotic cell death induced by
different toxic stimuli.8 We decided to evaluate the po-
tential cytoprotective effect of the new compounds and
several intermediates on bovine chromaffin cells, a type
of cells that possess all the characteristics and types of
Ca2+ channels of sympathetic neurons, exposed during
24h to veratridine 30lM, a toxin, which induces a
Ca2+ overload and a consequent cell death.19
n
Heterocycle
IC50 (lM)
Tacrine (1)
2
—
0.14
6.6
0
0
0
1
1
1
1
1
2
2
2
1
0
1
2
4-Pyridyl
3-Pyridyl
2-Thienyl
4-(OMe)C6H4
4-Pyridyl
3-Pyridyl
2-Thienyl
2-Pyrrolyl
4-Pyridyl
3-Pyridyl
2-Thienyl
C6H5
5
>100
27.8
0.87
3.0
8
279b
3
6
7.11
24.8
1.7
9
11
4
8.6
7
32.3
14.4
0.82
8.2
10
289b
12
13
14
4-Pyridyl
4-Pyridyl
4-Pyridyl
1.4
5.1
Drugs, at the concentration of 3lM, were administered
24h before the incubation of cells with veratridine and
maintained during the whole of the experiment. After
that, release of lactic dehydrogenase (LDH) was mea-
sured as a parameter of cell death, since this enzyme is
released to the extracellular medium as a consequent cell
death.19 Basal release of LDH by the cells was sub-
tracted from the values obtained for all the compounds
or veratridine. Results are shown in Table 2.
OMe
NH2
N
H
NH2
EtO2C
Me
EtO2C
Me
(CH2)n
Only six of the new compounds showed a tendency of
some neuroprotective action, which-at the drug concen-
tration tested-was statistically significant only in the case
of compound 10 and intermediate 16. None of the test
compounds was as effective as the previously synthesized
compound 28,9b shown here for comparison.
O
O
N
2-11
27
Heterocycle
H
N
N
NH2
NH2
EtO2C
Me
EtO2C
Me
3. Conclusions
(CH2)n
N
N
N
To summarize, we have reported the synthesis, acetyl-
cholinesterase (AChE) inhibitory activities and possible
neuroprotective properties of new tacrine analogues (2–
14). With respect to inhibition of AChE, none of the
test compounds showed superior activity to tacrine
(IC50 0.14mM). For the pyrano[3,2-e]pyridines, and
pyrano[2,3-b]quinolines group (class A), test compound
(11) (IC50 1.7mM) and for the [1,8]naphthyridine
derivatives, compound 13 (IC50 1.4mM) were the most
potent. In addition, comparing our current data with
the ones obtained in previous works from these labora-
tories,9 it is clear that the incorporation of heterocyclic
rings systems on these substrates, still gives us com-
pounds with some AChE inhibitory profile, in the
lM range, but significantly less active compared with
the simple benzene-substituted derivatives.9 Regarding
neuroprotective properties of the different tacrine ana-
28
12-14
Chart 3.
are poor AChE inhibitors (IC50 > 10mM). (ii) The six-
membered cycloannulated compounds (n = 1) (3, 6, 9)
were more potent than their five- and seven-membered
analogues. In those cases where it was possible to pre-
pare the three cycloalkyl fused-ring onto the central 4-
aminopyridine nucleus (n = 0, n = 1, n = 2), the most
active compounds were always the 4-pyridyl derivatives
(2–4). This was particularly obvious for compounds 2
(4-pyridine substituent; IC50 6.6mM) and its close ana-
logue compound 5 (3-pyridine substituent; IC50
100mM).
>

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R. Leon et al. / Bioorg. Med. Chem. 13 (2005) 1167–1175
1171
Table 2. Cell viability expressed as increase of LDH released in the
presence of veratridine
tures, and with related molecules, previously synthesized
in our laboratory.9
Compound (3lM)
LDHf (% total)
% Protection
4.2. General method for the synthesis of ethyl 6-amino-4-
aryl-5-cyano-2-methyl-4H-pyran-3-carboxylates
Vehicle
Tacrine
2
31.2 1.1
30.8 2.9
35.1 2.9
30.6 4.7
1.3
0
To a solution of the corresponding aldehyde (1equiv) in
dry toluene (1mL/mmol), under argon, malononitrile
(1equiv) and a catalytic amount of piperidine were
added. The mixture was stirred at rt for 3–7h. The sol-
vent was evaporated and the crude was purified by col-
umn chromatography, eluting with mixtures of hexane/
ethyl acetate. Next, to a solution of this arylidenemalo-
nonitrile in dry toluene, ethyl acetoacetate (1equiv) and a
catalytic amount of piperidine were added. The mixture
was stirred at rt for 2h, and the precipitated solid was
isolated by filtration, washed with cold toluene, dried
and recrystallized.
3
1.9
3.8
0
4
30
4
5
32.2 4.4
32.5 3.2
24 4.5
6
0
7
23.1
2.6
9
40.1***
10.3
0
8
30.4 2.1
28.4 1.7
9
10
11
12
13
14
15
16
17
18
19
25
26
289b
18.7
4
28 1.8
31.6 1.8
30.7 0.8
1.6
9.6
0
28.2
1
31.9 2.1
19.8 1.4
29.8 2.1
30.3 2.5
36.5***
4.5
2.9
1.6
3.2
4.8
44.2***
4.2.1. General method for the synthesis of ethyl 6-amino-
4-aryl-5-cyano-2-methyl-pyridine-3-carboxylates. To a
solution of ammonium acetate (10equiv) in acetic acid
(1.25mL) the appropriate ethyl 6-amino-5-cyano-2-
methyl-4H-pyran-3-carboxylate (1equiv) was added.
The mixture was refluxed (bath temperature at 116ꢁC)
for 3–7h. Then, the reaction was cooled and the solvent
was evaporated. The crude was treated with an aqueous
saturated solution of sodium bicarbonate until pH7 and
extracted with ethyl acetate. The organic phase was
dried with sodium sulfate, filtered and the solvent was
removed. The crude was purified by column chromato-
graphy eluting with mixtures of dichloromethane/ethyl
acetate. The isolated product was recrystallized from
mixtures of hexane/dichloromethane.
30.7
2
30.2 1.7
29.7 1.4
17.4 1.4
*** p 6 0.001.
logues, some showed a clear tendency in reducing the
veratridine-induced LDH release at the concentration
tested. This effect was statistically significant only for
compound 10 and intermediate 16. Unfortunately,
none of the new tacrine analogues showed a profile
in which these two pharmacological parameters were
combined.
¨
4.2.2. General method for the Friedlander reaction. Alu-
minium chloride (1.2–1.7equiv) was suspended in dry
1,2-dichloroethane (10mL) at rt under argon. The corre-
sponding ethyl 6-amino-4-aryl-5-cyano-2-methyl-4H-
pyran 3-carboxylate (1equiv) and the ketone (cyclopen-
tanone, cyclohexanone or cycloheptanone; 1.2–
1.7equiv) were added. The reaction mixture was heated
under reflux (10–24h). When the reaction was over
(TLC analysis), a mixture of THF/H2O(2:1) was added
at rt. An aqueous solution of sodium hydroxide (10%)
was added dropwise to the mixture until the aqueous
solution was basic. After stirring for 30min, the mixture
was extracted three times with dichloromethane. The or-
ganic layer was washed with brine, dried over anhydrous
sodium sulfate, filtered and the solvent was evaporated.
The resultant solid was purified by silica gel flash chro-
matography using methanol/dichloromethane mixtures
as eluent to give pure compounds.
4. Experimental part
4.1. General methods
Reactions were monitored by TLC using precoated sil-
ica gel aluminium plates containing a fluorescent indica-
tor (Merck, 5539). Detection was done by UV (254nm)
followed by charring with sulfuric–acetic acid spray, 1%
aqueous potassium permanganate solution or 0.5%
phosphomolybdic acid in 95% EtOH. Anhydrous
Na2SO4 was used to dry organic solutions during
work-ups and the removal of solvents was carried out
under vacuum with a rotary evaporator. Flash column
chromatography was performed using silica gel 60
(230–400 mesh, Merck). Melting points were determined
on a Kofler block and are uncorrected. IR spectra were
obtained on a Perkin–Elmer Spectrum One spectropho-
1
tometer. H NMR spectra were recorded with a Varian
4.3. Ethyl 6-amino-5-cyano-2-methyl-4-(3-pyridyl)-4H-
pyran-3-carboxylate (16)
VXR-200S spectrometer, using tetramethylsilane as
internal standard and 13C NMR spectra were recorded
with a Bruker WP-200-SY. All the assignments for pro-
tons and carbons were in agreement with 2D COSY,
gHSQC, gHMBC and 1D NOESY spectra. Elemental
analyses were conducted on a Carlo Erba EA 1108
apparatus. All new compounds showed analytical and
spectroscopic data in good agreement with these struc-
Following the General method, 3-pyridinecarboxalde-
hyde (1.40g, 13.0mmol), malononitrile (0.858g,
13.0mmol), ethyl acetoacetate (1.69g, 13.0mmol), piper-
idine (15 drops), ethanol (20mL), after 3.5h, compound
16 (3.0g, 84%) was obtained: mp 180–183ꢁC; IR (KBr) m
3342, 3041, 2196, 1724, 1684, 1614, 1221, 1067,

´
R. Leon et al. / Bioorg. Med. Chem. 13 (2005) 1167–1175
1172
714cmꢀ1
;
1H NMR (DMSO-d6, 200MHz) d 8.04 (d,
[M+Na+], 305. Anal. Calcd for C15H14N4O2: C, 63.82;
H, 5.00; N, 19.85. Found: C, 63.94; H, 4.86; N, 19.95.
J = 4.7Hz, 1H), 8.38 (s, 1H), 7.54 (dt, J = 4.7Hz,
J = 6.7Hz, 1H), 7.35 (t, J = 6.7Hz, 1H), 7.03 (s, 2H),
4.34 (s, 1H), 3.95 (q, J = 6.9Hz, 2H), 2.32 (s, 3H), 1.00
(t, J = 6.6Hz, 3H); 13C NMR (DMSO-d6, 50.3MHz) d
165.9, 159.2, 158.3, 149.3, 148.8, 141.1, 135.5, 124.5,
120.2, 106.9, 60.9, 57.1, 37.2, 19.0, 14.4; MS (ES)
[M+1]+ 316 [M+Na]+ 338.0; [2M+Na]+ 653.3. Anal.
Calcd for C15H15N3O3: C, 63.15; H, 5.30; N, 14.73.
Found: C, 63.26; H, 5.45; N, 14.83.
4.7. Ethyl 6-amino-5-cyano-2-methyl-4-(3-pyridyl)-3-
pyridinecarboxylate (25)
Following the General method, from compound 1613
(3.0g, 10.50mmol), ammonium acetate (8.17g,
105.2mmol), acetic acid (131mL), after 24h, compound
25 (450mg, 15%) was obtained: mp 179–181ꢁC; IR
(KBr) m 3386, 3323, 3176, 2221, 1713, 1653, 1574,
4.4. N-Acetyl-2-pyrrolylmethylidenemalononitrile (23)
1554, 1278, 1184cmꢀ1
;
1H NMR (DMSO-d6,
200MHz) d 8.67 (d, J = 4.6Hz, 1H), 8.49 (s, 1H), 7.78
(d, J = 7.5Hz, 1H), 7.53 (dd, J1 = 4.6Hz, J2 = 7.5Hz,
1H) 7.45 (s, 2H), 3.88 (q, J = 7.2Hz, 2H), 2.42 (s, 3H),
0.77 (t, 3H); 13C NMR (DMSO-d6, 50.3MHz) d 166.4,
161.3, 159.8, 151.7, 150.4, 148.2, 136.0, 134.2, 123.7,
118.6, 116.2, 86.9, 61.1, 23.9, 13.7; MS (APCI+) m/z
[M+1]+ 283, [M+Na+], 305. Anal. Calcd for
C15H15N4O2: C, 63.82; H, 5.00; N, 19.82. Found: C,
63.58; H, 4.93; N, 18.58.
To a solution of 2212 (200mg, 1.40mmol) in pyridine
(1mL), acetic anhydride (1mL) was added. After 9h
the solvent was evaporated and the resultant oil was
purified by silica gel flash chromatography using dichlo-
romethane as eluent to give the pure compound 23
(245mg, 98%) as an amorphous solid: IR (KBr) m
3433, 3142, 2219, 1722, 1576, 1446, 1375, 1280, 1259,
1
1127, 1078, 957cmꢀ1; H NMR (CDCl3, 200MHz) d
8.79 (s, 1H), 7.78 (d, J = 3.7Hz, 1H), 7.49 (d,
J = 3.7Hz, 1H), 6.54 (t, J = 3.7Hz, 1H), 2.68 (s, 3H);
13C NMR (CDCl3, 50.3MHz) d 169.9, 148.4, 128.7,
128.7, 124.3, 114.9, 114.6, 114.0, 78.5, 24.7; EM (ES):
[M+1]+ 186, [M+Na]+ 208, [2M+Na+] 393. Anal. Calcd
for C10H7N3O: C, 64.86; H, 3.81; N, 22.69. Found: C,
64.63; H, 4.01; N, 22.58.
4.7.1. Ethyl 6-amino-5-cyano-2-methyl-4-(2-thienyl)-3-
pyridinecarboxylate (26). Following the General method,
from compound 1713 (2g, 6.86mmol), ammonium
acetate (5.29g, 68.6mmol), acetic acid (85mL), after
7h, product 26 (360mg, 5%) was obtained: mp 213–
216ꢁC; IR (KBr) m 3391, 3323, 3179, 2219, 1713, 1651,
1558, 1277, 1183, 1065cmꢀ1 1H NMR (DMSO-d6,
;
4.5. Ethyl 4-(10-acetyl-10H-pyrrol-2-yl)-6-amino-5-cyano-
2-methyl-4H-pyran-3-carboxylate (18)
200MHz) d 7.84 (d, J = 4.9Hz, 1H), 7.36 (s, 2H), 7.25
(d, J = 3.7Hz, 1H), 7.19 (dd, J1 = 3.7Hz, J2 = 4.9Hz,
1H), 3.99 (q, J = 7.1Hz, 2H), 2.36 (s, 3H), 0.94 (t,
3H); 13C NMR (DMSO-d6, 50.3MHz) d 167.5, 160.6,
160.4, 146.5, 135.8, 129.8, 129.7, 128.3, 118.8, 116.5,
87.7, 61.7, 23.8, 14.1; MS (APCI+) m/z [M+1]+ 288,
[M+Na+] 310. Anal. Calcd for C14H13N3O2S: C,
57.92; H, 4.86; N, 9.65; S, 11.04. Found: C, 58.01; H,
4.59; N, 10.01; S, 11.25.
Following the General method, compound 23 (300mg,
1.62mmol) was treated with ethyl acetoacetate
(211mg, 1.62mmol), triethylamine (18 drops), in dry tol-
uene (3mL) for 72h, to give compound 18 (510mg, 99%)
as an amorphous solid: IR (KBr) m 3408, 3311, 3197,
2192, 1717, 1671, 1637, 1605, 1384, 1369, 1305,
1
1120cmꢀ1; H NMR (DMSO-d6, 200MHz) d 7.30 (d,
J = 3.3Hz, 1H), 6.75 (br s, 2H), 6.14 (dd, J = 3.2Hz,
J = 3.3Hz, 1H), 5.94 (d, J = 3.2Hz, 1H), 5.48 (s, 1H),
3.89 (q, J = 6.7Hz, 2H), 2.56 (s, 3H), 2.28 (s, 3H), 0.98
(t, 3H); 13C NMR (DMSO-d6, 50.3MHz) d 170.5,
166.2, 159.4, 157.0, 140.4, 122.6, 120.1, 113.1, 111.5,
107.2, 60.5, 57.4, 30.5, 24.9, 18.5, 14.3; MS (APCI+)
m/z [M+1]+ 316, [M+Na]+ 338.0, [2M+Na]+ 653.3.
Anal. Calcd for C16H17N3O4: C, 60.94; H, 5.43; N,
13.33. Found: C, 61.06; H, 5.70; N, 13.65.
4.8. Ethyl 5-amino-4,6,7,8-tetrahydro-2-methyl-4-(4-pyr-
idyl)-cyclopeta[b]pyran[3,2-e]pyridine-3-carboxylate (2)
Following the General method, from compound 1513
(219.5mg, 0.77mmol), AlCl3 (148mg, 1.12mmol),
ClCH2CH2Cl
1.12mmol)], after 28h, product 2 (175mg, 65%) was ob-
(7.7mL),
cyclopentanone
(99lL,
tained: mp 191–192ꢁC; IR (KBr) m 3429, 3355, 2973,
1687, 1645, 1594, 1415, 1372, 1256, 1212, 1071cmꢀ1
;
1H NMR (DMSO-d6, 200MHz) d 8.39 (d, J = 5.9Hz,
2H), 7.29 (d, J = 5.9Hz, 2H), 5.93 (s, 2H), 5.03 (s,
1H), 4.05 (q, J = 6.7Hz, 2H), 2.67 (m, 4H), 2.36 (s,
3H), 1.95 (m, 2H), 1.18 (t, 3H); 13C NMR (DMSO-d6,
50.3MHz) d 166.2, 161.3, 160.9, 156.5, 153.4, 149.7
(2C), 149.5, 123.5 (2C), 117.6, 106.4, 97.5, 60.4, 36.1,
33.9, 27.7, 22.1, 19.4, 14.3; MS (APCI+) m/z [M+1]
352.3. Anal. Calcd for C20H21N3O3: C, 68.36; H, 6.02;
11.96. Found: C, 68.25; H, 5.95; N, 11.64.
4.6. Ethyl 6-amino-5-cyano-2-methyl-4-(4-pyridyl)-3-
pyridinecarboxylate (19)
Following the General method, from compound 15
(1.00g, 3.5mmol), ammonium acetate (2.70g,
33.06mmol), acetic acid (44mL), after 21h, product 19
(203.9mg, 57%) was obtained: mp 229–231ꢁC; IR (KBr)
m 3390, 3320, 3175, 2221, 1711, 1651, 1554, 1377, 1282,
1
1189cmꢀ1; H NMR (DMSO-d6, 200MHz) d 8.80 (d,
J = 5.5Hz, 2H), 7.60 (s, 2H), 7.46 (d, J = 5.4Hz, 2H),
3.98 (q, J = 7.0Hz, 2H), 2.54 (s, 3H), 0.86 (t, 3H); 13C
NMR (DMSO-d6, 50.3MHz) d 164.8, 159.8, 158.4,
150.4, 148.5 (2C), 143.1, 121.3 (2 · C), 114.9, 114.1,
85.4, 59.5, 22.3, 11.8; MS (APCI+) m/z [M+1]+ 283,
4.9. Ethyl 5-amino-6,7,8,9-tetrahydro-2-methyl-4-(4-pyr-
idyl)-4H-pyran[2,3-b]quinoline-3-carboxylate (3)
Following the General method, from compound 1513
(100mg, 0.35mmol), AlCl3 (70.67mg, 0.52mmol),

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R. Leon et al. / Bioorg. Med. Chem. 13 (2005) 1167–1175
1173
ClCH2CH2Cl (5mL), cyclohexanone (50mg, 0.52mmol),
after 24h, product 3 (109.2mg, 85%) was obtained: mp
222–223ꢁC; IR (KBr) m 3438, 3358, 3240, 2930, 1689,
ClCH2CH2Cl
(6mL),
cyclohexanone
(137mg,
1.4mmol)], after 36h, product 6 (225mg, 88%) was
obtained: mp 176–178ꢁC; IR (KBr) m 3436, 2932,
1685, 1643, 1572, 1455, 1302, 1571, 1227, 1209,
1644, 1601, 1571, 1454, 1301, 1207, 1061cmꢀ1 1H
;
NMR (DMSO-d6, 200MHz) d 8.39 (d, J = 5.8Hz, 2H),
7.30 (d, J = 5.8Hz, 2H), 5.77 (s, 2H), 5.05 (s, 1H), 4.05
(q, J = 7.0Hz, 2H), 2.37 (s, 3H), 2.25 (m, 2H), 2.18 (m,
2H), 1.66 (m, 4H), 1.14 (t, 3H); 13C NMR (DMSO-d6,
50.3MHz) d 166.6, 161.3, 154.6, 153.8, 153.3, 151.7,
150.1 (2C), 123.9 (2C), 113.8, 106.5, 97.9, 60.8, 36.5,
32.7, 23.7, 22.9, 22.7, 19.8, 14.7; MS (APCI+) m/z
[M+1]+ 366.1; [M+Na]+ 388.1; [2M+Na]+ 754.1. Anal.
Calcd for C21H23N3O3: C, 69.01; H, 6.35; N, 11.50.
Found: C, 68.92; H, 6.30; N, 11.35.
1099, 1063cmꢀ1 1H NMR (DMSO-d6, 200MHz) d
;
8.63 (d, J = 1.8Hz, 1H), 8.33 (d, J = 4.6Hz, 1H),
7.59 (d, J = 3.6Hz, 1H), 7.25 (dd, J = 3.6Hz,
J = 4.7Hz, 1H), 5.77 (s, 2H), 5.05 (s, 1H), 4.07 (q,
J = 7.0Hz, 2H), 2.39 (s, 3H), 2.31 (m, 2H), 2.19 (m,
2H), 1.68 (m, 4H), 1.15 (t, 3H); 13C NMR (DMSO-
d6, 50.3MHz) d 166.6, 160.6, 154.2, 152.8, 151.2,
149.6, 147.8, 140.4, 135.5, 123.8, 113.4, 106.6, 98.0,
60.8, 34.3, 32.3, 23.3, 22.5, 22.3, 19.4, 14.7; MS (APCI+)
m/z [M+1]+ 366.0, [M+Na]+ 388.1, [2M+Na]+
753.1. Anal. Calcd for C21H23N3O3: C, 69.01; H,
6.35; N, 11.50. Found: C, 68.80; H, 6.50; N,
11.27.
4.10. Ethyl 5-amino-4,6,7,8,9,10-hexahydro-2-methyl-4-
(4-pyridyl)-cyclohepta[b]pyran[3,2-e]pyridine-3-carboxyl-
ate (4)
4.13. Ethyl 5-amino-4,6,7,8,9,10-hexahydro-2-methyl-4-
(3-pyridyl)-cyclohepta[b]pyran[3,2-e]pyridine-3-carboxyl-
ate (7)
Following the General method, from compound 1513
(100mg, 0.35mmol), AlCl3 (70.67mg, 0.52mmol),
ClCH2CH2Cl (4mL), cycloheptanone (138.62mg,
0.35mmol), after 26h, compound 4 (120mg, 97%) wasd
obtained: mp 165–167ꢁC; IR (KBr) m 3401, 3238, 2923,
2851, 1692, 1645, 1597, 1567, 1430, 1260, 1233,
Following the General method, from compound 16
(100mg, 0.35mmol), AlCl3 (70.61mg, 0.52mmol),
ClCH2CH2Cl (4mL), cycloheptanone (58.32lL, 0.52
mmol), after 24h, product 7 (102mg, 77%) was ob-
tained: mp 180–182ꢁC; IR (KBr) m 3459, 3372, 2922,
1
1218cmꢀ1; H NMR (DMSO-d6, 200MHz) d 8.39 (d,
J = 5.7Hz, 2H), 7.28 (d, J = 5.7Hz, 2H), 5.75 (s, 2H),
5.02 (s, 1H), 4.05 (q, J = 7.0Hz, 2H), 2.63 (m, 4H),
2.36 (s, 3H), 1.53 (m, 6H), 1.17 (t, 3H); 13C NMR
(DMSO-d6, 50.3MHz) d 166.2, 160.7, 159.9, 153.9,
153.3, 150.7, 149.7 (2C), 123.5 (2C), 118.6, 106.2, 98.4,
60.3, 38.3, 36.5, 31.9, 27.1, 26.2, 25.1, 19.3, 14.3; MS
(APCI+) m/z [M+1] 380.3, [2M+Na] 782.3. Anal. Calcd
for C22H25N3O3: C, 69.62; H, 6.64; N, 11.08. Found: C,
69.68; H, 6.65; N, 11.02.
2849, 1689, 1640, 1565, 1428, 1260, 1214cmꢀ1 1H
;
NMR (DMSO-d6, 200MHz) d 8.63 (s, 1H), 8.31 (d,
J = 3.8Hz, 1H), 7.56 (d, J = Hz, 1H), 7.23 [dd,
J = 3.8Hz, J = 7.8Hz, 1H), 5.77 (s, 2H), 5.00 (s, 1H),
4.05 (q, J = 6.9Hz, 2H), 2.72 (m, 4H), 2.36 (s, 3H),
1.72 (m, 6H), 1.17 (t, 3H); 13C NMR (DMSO-d6,
50.3MHz) d 166.2 (C@O), 160.4 (C11a), 159.8 (C2),
153.8 (C10a), 150.5, 149.7, 147.8, 140.4, 135.6, 123.8,
118.6, 106.5, 98.8, 60.3, 38.2, 34.5, 32.0, 27.2, 26.2,
25.0, 19.3, 14.3; MS (APCI+) m/z [M+1] 380.0,
[M+Na]+ 402.0, [2M+Na] 781.0. Anal. Calcd for
C22H25N3O3: C, 69.62; H, 6.64; N, 11.08. Found: C,
69.54; H, 6.81; N, 11.13.
4.11. Ethyl 5-amino-4,6,7,8-tetrahydro-2-methyl-4-(3-pyrid-
yl)-cyclopenta[b]pyran[3,2-e]pyridine-3-carboxylate (5)
Following the General method, from compound 16
(219.5mg, 0.77mmol), AlCl3 (148mg, 1.12mmol),
ClCH2CH2Cl (7mL), cyclopentanone (99lL, 1.12
mmol), after 96h, product 5 (203mg, 73%) was ob-
tained: mp 213–215ꢁC; IR (KBr) m 3469, 2186, 2961,
4.14. Ethyl 5-amino-4,6,7,8,9,10-hexahydro-2-methyl-4-
(2-thienyl)cyclohepta[b]pyran[3,2-e]pyridine-3-carboxyl-
ate (8)
1709, 1684, 1644, 1578, 1375, 1261, 1204cmꢀ1 1H
;
NMR (DMSO-d6, 200MHz) d 8.62 (d, J = 1.7Hz,
1H), 8.33 (dd, J = 1.7Hz, J = 4.7Hz, 1H), 7.58 (dd,
J = 1.8Hz, J = 8.2Hz, 1H), 7.24 (dd, J = 1.7Hz, J =
8.2Hz, 1H), 5.96 (s, 2H), 5.03 (s, 1H), 4.07 (q,
J = 6.7Hz, 2H), 2.63 (m, 4H), 2.39 (s, 3H), 1.97 (m,
2H), 1.19 (t, 3H); 13C NMR (DMSO-d6, 50.3MHz) d
166.5, 161.3, 160.9, 156.7, 149.8, 149.5, 148.1, 140.7,
135.7, 124.0, 118.0, 106.8, 98.5, 60.6, 34.5, 34.2, 27.9,
22.3, 19.6, 14.5; MS (APCI+) m/z [M+1]+ 352.0,
[M+Na]+ 374.0, [2M+Na]+ 725.3. Anal. Calcd for
C20H21N3O3: C, 68.36; H, 6.02; N, 11.96. Found: C,
68.12; H, 5.94; N, 11.75.
Following the General method, from compound
1713 (200mg, 0.69mmol), AlCl3 (138mg, 1.03mmol),
ClCH2CH2Cl
(7mL),
cyclopentanone
(247lL,
2.89mmol), after 96h, product 8 (70mg, 29%) was
obtained: mp 178–179ꢁC; IR (KBr) m 3413, 3350,
3205, 2925, 2851, 1691, 1652, 1371, 1253, 1210cmꢀ1
;
1H NMR (DMSO-d6, 200MHz)
d 7.24 (d, J =
4.3Hz, 1H), 6.96 (d, J = 4.0Hz, 1H), 6.85 (dd,
J = 4.3Hz, J = 4.0Hz, 1H), 5.99 (s, 2H), 5.39 (s, 1H),
4.15 (q, J = 7.6Hz, 2H), 2.65 (m, 2H), 2.51 (s, 3H),
1.99 (m, 2H), 1.34 (m, 2H), 1.28 (t, 3H); 13C NMR
(DMSO-d6, 50.3MHz) d 166.0, 160.6, 159.9, 156.2,
149.1, 148.7, 126.2, 124.6, 124.2, 117.1, 107.0, 98.3,
60.1, 33.6, 31.5, 27.4, 21.8, 19.0, 14.1; MS (APCI+)
m/z [M+1]+ 357, [M+Na]+ 379.2, [2M+Na]+ 735.3.
Anal. Calcd for C19H20N2O3S: C, 64.02; H, 5.66; N,
7.86; S, 8.98. Found: C, 63.89; H, 5.80; N, 7.77; S,
8.98.
4.12. Ethyl 5-amino-6,7,8,9-tetrahydro-2-methyl-4-(3-
pyridyl)-4H-pyran[2,3-b]quinoline-3-carboxylate (6)
Following the General method, from compound
16 (200mg, 0.7mmol), AlCl3 (148mg, 1.4mmol),

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R. Leon et al. / Bioorg. Med. Chem. 13 (2005) 1167–1175
1174
4.15. Ethyl 5-amino-6,7,8,9-tetrahydro-2-methyl-4-(2-thi-
enyl)-4H-pyran[2,3-b] quinoline-3-carboxylate (9)
66.82; H, 6.37; N, 10.63. Found: C, 67.00; H, 6.60; N,
10.61.
Following the General method, from compound 1713
(200mg, 0.69mmol), AlCl3 (138mg, 1.04mmol),
4.18. Ethyl 5-amino-7,8-dihydro-2-methyl-4-(4-pyridyl)-
6H-cyclopenta[b][1,8]naphthyridine-3-carboxylate (12)
ClCH2CH2Cl
(7mL),
cyclohexanone
(292lL,
2.89mmol), after 1.5h, product 9 (210mg, 83%) was ob-
tained: mp 204–205ꢁC; IR (KBr) m 3440, 2933, 1691,
1645, 1603, 1572, 1455, 1302, 1229, 1206, 1100,
Following the General method, from compound 19
(150mg, 0.53mmol), AlCl3 (105.3mg, 0.79mmol),
ClCH2CH2Cl
(5mL),
cyclopentanone
(72lL,
1
1061cmꢀ1; H NMR (DMSO-d6, 200MHz) d 7.24 (d,
0.79mmol), after 18h, product 12 (118mg, 61%) was ob-
tained: mp 245–247ꢁC; IR (KBr) m 3492, 3435, 3195,
2956, 1724, 1630, 1593, 1550, 141, 1371, 1271, 1087,
J = 5.0Hz, 1H), 6.97 (d, J = 4.7Hz, 1H), 6.84 (dd,
J = 5.0Hz, J = 4.7Hz, 1H), 5.83 (s, 2H), 5.40 (s, 1H),
4.15 (q, J = 7.0Hz, 2H), 2.55 (m, 2H), 2.35 (s, 3H),
2.26 (m, 2H), 1.71 (m, 4H), 1.24 (t, 3H); 13C NMR
(DMSO-d6, 50.3MHz) d 166.8, 160.5, 154.6, 153.1,
151.8, 149.3, 126.9, 125.5, 125.0, 113.6, 107.7, 98.8,
60.8, 32.7, 32.3, 23.7, 23.0, 22.8, 19.8, 14.8; MS (APCI+)
m/z [M+1]+ 371, [2M+Na]+ 763.3. Anal. Calcd for
C20H22N2O3S: C, 64.84; H, 5.99; N, 7.56; S, 8.66.
Found: C, 64.65; H, 6.10; N, 7.58; S, 8.90.
592cmꢀ1 1H NMR (DMSO-d6, 200MHz) d 8.64 (d,
;
J = 4.3Hz, 2H), 7.35 (d, J = 4.3Hz, 2H), 4.96 (s, 2H),
3.84 (q, J = 7.1Hz, 2H), 2.85 (m, 2H), 2.59 (m, 2H),
2.46 (s, 3H), 1.96 (m, 2H), 0.77 (t, 3H); 13C NMR
(DMSO-d6, 50.3MHz) d 170.6, 167.4, 156.7, 154.7,
150.1 (2C), 147.5, 145.4, 142.7, 125.4, 124.1 (2C),
116.8, 106.1, 61.5, 35.1, 28.1, 23.4, 22.0, 13.6; MS
(APCI+) m/z [M+1]+ 349.2, [2M+Na]+ 719.5. Anal.
Calcd for C20H20N4O2: C, 68.95; H, 5.79; N, 16.08.
Found: C, 69.00; H, 5.93; N, 16.34.
4.16. Ethyl 5-amino-4,6,7,8,9,10-hexahydro-2-methyl-4-
(2-thienyl)cyclohepta[b]pyran[3,2-e]pyridine-3-carboxyl-
ate (10)
4.19. Ethyl 5-amino-6,7,8,9-tetrahydro-2-methyl-4-(4-
pyridyl)-benzo[b][1–8]naphthyridine-3-carboxylate (13)
Following the General method, from compound 1713
(200mg, 0.69mmol), AlCl3 (138mg, 1.03mmol),
Following the General method, from compound 19
(120mg, 0.42mmol), AlCl3 (84mg, 0.63mmol),
ClCH2CH2Cl
(7mL),
cycloheptanone
(350lL,
2.90mmol), after 4h, product 10 (160mg, 61%) was ob-
tained: mp 155–157ꢁC; IR (KBr) m 3384, 2919, 2849,
ClCH2CH2Cl
0.63mmol), after 40h, product l3 (75mg, 49%) was ob-
(4mL),
cyclohexanone
(66lL,
1688, 1624, 1564, 1371, 1262, 1208cmꢀ1 1H NMR
;
tained: mp 208–210ꢁC; IR (KBr) m 3498, 3334, 3230,
(DMSO-d6, 200MHz) d 7.25 (d, J = 5.1Hz, 1H), 6.95
(d, J = 4.1Hz, 1H), 6.84 (dd, J = 5.1Hz, J = 4.1Hz,
1H), 5.82 (s, 2H), 5.37 (s, 1H), 4.15 (q, J = 7.0Hz,
2H), 2.52 (m, 4H), 2.24 (s, 3H), 1.46 (m, 6H), 1.26 (t,
3H); 13C NMR (DMSO-d6, 50.3MHz) d 166.4, 160.1,
153.8, 150.7, 148.9, 147.8, 127.3, 126.6, 125.8, 116.1,
107.2, 99.2, 60.3, 38.1, 32.1, 27.3, 26.3, 25.1, 19.4, 18.3,
14.4; MS (APCI+) m/z [M+1]+ 385.1, [2M+Na]+
791.5. Anal. Calcd for C21H24N2O3S: C, 65.60; H,
6.29; N, 7.29; S, 8.34. Found: C, 65.39; H, 6.50; N,
7.45; S, 8.60.
2931, 1725, 1629, 1571, 1540, 1445, 1269, 1223cmꢀ1
;
1H NMR (DMSO-d6, 200MHz) d 8.76 (d, J = 5.9Hz,
2H), 7.48 (d, J = 5.9Hz, 2H), 5.28 (s, 2H), 3.96 (q,
J = 7.0Hz, 2H), 2.87 (m, 2H), 2.52 (s, 3H), 2.36 (m,
2H), 1.81 (m, 4H), 0.88 (t, 3H); 13C NMR (DMSO-d6,
50.3MHz) d 167.4, 161.1, 155.8, 153.7, 150.6, 150.4
(2C), 145.4, 142.7, 126.0, 124.2 (2C), 112.2, 105.6,
61.5, 33.5, 23.9, 23.6, 22.4 (2C), 13.8; MS (APCI+) m/
z [M+1]+ 363.0; [M+Na]+ 385.0; [2M+Na]+ 749.0. Anal.
Calcd for C21H23N4O2: C, 69.59; H, 6.12; N, 15.46.
Found: C, 69.31; H, 6.30; N, 15.28.
4.17. Ethyl 4-(1-acetyl-1H-pyrrol-2-yl)-5-amino-6,7,8,9-
tetrahydro-2-methyl-4H-pyran[2,3-b]quinoline-3-carbox-
ylate (11)
4.20. Ethyl 5-amino-7,8,9,10-tetrahydro-2-methyl-4-(4-
pyridyl)-6H-cyclohepta[b][1,8]naphthyridine-3-carboxyl-
ate (14)
Following the General method, from compound 18
(200mg, 0.64mmol), AlCl3 (127.68mg, 0.96mmol),
Following the General method, from compound 19
(150mg, 0.53mmol), AlCl3 (106mg, 0.80mmol),
ClCH2CH2Cl (5mL), cycloheptanone (95lL, 0.8mmol),
after 4h, product 14 (184mg, 92%) was obtained: mp
210–212ꢁC; IR (KBr) m 3477, 3366, 3032, 2925, 2856,
ClCH2CH2Cl
(6mL),
cyclohexanone
(133lL,
1.28mmol), after 3h, product 11 (110mg, 43%) was ob-
tained: mp 172–174ꢁC; IR (KBr) m 3458, 3347, 3246,
2958, 2930, 1710, 1635, 1572, 1455, 1367, 1302, 1218,
1731, 1638, 1568, 1544, 1272, 1224, 1084cmꢀ1 1H
;
1061, 947, 726, 641cmꢀ1
200MHz)
;
7.16 (d, J = 3.2Hz, 1H), 6.05 (t,
1H NMR (DMSO-d6,
NMR (DMSO-d6, 200MHz) d 8.63 (d, J = 5.7Hz,
2H), 7.37 (d, J = 5.7Hz, 2H), 5.19 (s, 2H), 3.85 (q,
J = 7.0Hz, 2H), 2.54 (m, 4H), 2.45 (s, 3H), 1.62 (m,
6H), 0.77 (t, 3H); 13C NMR (DMSO-d6, 50.3MHz) d
167.6, 167.3, 155.2, 153.0, 150.1 (2C), 149.2, 145.3,
142.6, 126.2, 124.1 (2C), 117.2, 106.5, 61.6, 39.0, 31.6,
27.3, 26.5, 25.3, 23.3, 13.6; MS (APCI+) m/z [M+1]+
377.2, [M+Na]+ 399.22, [2M+Na]+ 775.7. Anal. Calcd
for C22H24N4O2: C, 70.19; H, 6.43; N, 14.88. Found:
C, 70.35; H, 6.76; N, 15.16.
d
J = 3.4Hz, 1H), 5.85 (dd, J = 3.2Hz, J = 3.4Hz, 1H),
5.75 (s, 2H), 5.55 (s, 1H), 3.88 (q, J = 7.0Hz, 2H), 2.57
(s, 3H), 2.22 (s, 3H), 2.16 (m, 2H), 1.60 (m, 6H), 0.98
(t, 3H); 13C NMR (DMSO-d6, 50.3MHz) d 173.4,
166.4, 159.4, 153.9, 152.4, 151.3, 140.9, 121.7, 115.3,
112.9, 111.9, 107.8, 98.7, 60.1, 32.2, 29.2, 25.0, 23.3,
22.6, 22.4, 19.4, 14.3; MS (APCI+) m/z [M+1]+ 396.3;
[2M+Na]+ 813.5. Anal. Calcd for C22H25N3O4: C,

´
R. Leon et al. / Bioorg. Med. Chem. 13 (2005) 1167–1175
1175
4.21. Biological studies
References and notes
1. Guttman, R.; Altman, R. D.; Nielsen, N. H. Arch. Fam.
Med. 1999, 8, 347.
2. Selkoe, D. J. Annu. Rev. Neurosci. 1989, 12, 463.
3. Mattson, M.; Pedersen, W. A. Int. J. Dev. Neurosci. 1998,
16, 737.
4. Mattson, M. P.; Laferla, F. M.; Chan, S. L.; Leissring, M.
A.; Shepel, P. N.; Geiger, J. D. Trends Neurosci. 2000, 23,
222.
5. Schroder, H.; Giacobini, E.; Struble, R. G.; Zilles, K.;
Maelicke, A. Neurobiol. Aging 1991, 12, 259.
6. (a) Standaert, D. G.; Young, A. B. Goodman and GilmanÕs
The Pharmacological Basis of Therapeutics, 9th ed.;
4.21.1. AChE activity. To measure the AChE inhibitory
activity of the compounds, we followed the spectromet-
ric method of Rappaport et al.17 using AChE from
electric eel (Torpedo californica) and acetylcholine chlo-
ride (29.5mM) as a substrate. The reaction took place
in a final volume of 2.5mL of an aqueous solution con-
taining 0.78U of AChE and 1.9mM m-nitrophenol to
produce a yellow colour, which is lost as a function
of the enzymatic activity. Inhibition curves were ob-
tained by incubating with the different compounds
for 30min; a control was always present to determine
the 100% value for the enzymatic activity. After the
30min incubation, the consumption of m-nitrophenol
was determined by measuring absorbance at 405nm
in a spectrophotometric plate reader (iEMS Reader
MF, Labsystems). The concentration of compound,
which produced 50% inhibition of the AChE activity
(IC50) was calculated by transforming the values of
absorbance to Rappaport enzymatic activity units,
extrapolating from a calibration curve obtained previ-
ously. Data are means s.e.m of at least three different
experiments in triplicate.
´
McGraw-Hill: Mexico, 1996; pp 39–556; (b) Relman, A.
S. New. Engl. J. Med. 1990, 324, 349.
7. Maelicke, A.; Schrattenholz, A.; Samochocki, M.; Radina,
M.; Albuquerque, E. Behav. Brain Res. 2000, 113, 199.
´
8. Arias, E.; Ales, E.; Gabilan, N. H.; Cano-Abad, M. F.;
Villarroya, M.; Garcıa, A. G.; Lopez, M. G. Neurophar-
´
´
´
macology 2004, 46, 103.
´
9. (a) Marco, J. L.; Martınez-Grau, A. Bioorg. Med. Chem.
Lett. 1997, 7, 3165; (b) Marco, J. L.; de los Rıos, C.;
´
´
Carreiras, M. C.; Ban˜os, J. E.; Badıa, A.; Vivas, N. M.
Bioorg. Med. Chem. 2001, 9, 727; (c) de los Rıos, C.;
´
´
Marco, J. L.; Carreiras, M. C.; Chinchon, P. M.; Garcıa,
A. G.; Villarroya, M. Bioorg. Med. Chem. 2002, 10, 2077;
´
´
(d) Marco, J. L.; de los Rıos, C.; Carreiras, M. C.; Ban˜os,
J. E.; Badıa, A.; Vivas, N. M. Arch. Pharm. 2002, 7, 347;
4.22. Chromaffin cells
´
´
(e) Marco, J. L.; de los Rıos, C.; Garcıa, A. G.; Villarroya,
M.; Carreiras, M. C.; Martins, C.; Eleuterio, A.; Morreale,
´
´
Bovine adrenomedullary chromaffin cells were isolated
following standard methods20 with some modifica-
tions.21 Cells were suspended in DulbeccoÕs modified Ea-
gleÕs medium (DMEM) supplemented with 5% foetal
calf serum, 10lM cytosine arabinoside, 10lM fluorode-
oxyuridine, 50IUmLꢀ1 penicillin and 50lgmLꢀ1 strep-
tomycin. Cells were plated in 24-multiwell Costar plates
and were used 1–5days after plating. Medium was re-
placed after 24h and again after 2–3days.
A.; Orozco, M.; Luque, F. J. Bioorg. Med. Chem. 2004, 12,
2199.
10. (a) Cheng, C. C.; Yan, S. J. Org. React. 1982, 28, 37, For
´
an early similar approach, see: (b) Aguado, F.; Badıa, A.;
Ban˜os, J. E.; Bosch, F.; Bozzo, C.; Camps, P.; Contreras,
J.; Dierssen, M.; Escolano, C.; Go¨rbig, D. M.; Mun˜oz-
´
Torrero, D.; Pujol, M. D.; Simon, M.; Vazquez, M. T.;
Vivas, N. M. Eur. J. Med. Chem. 1994, 29, 205; (c) Badıa,
´
´
A.; Ban˜os, J. E.; Camps, P.; Contreras, J.; Go¨rbig, D. M.;
´
Mun˜oz-Torrero, D.; Pujol, M. D.; Simon, M.; Vivas, N.
M. Bioorg. Med. Chem. 1998, 6, 427.
Lactic dehydrogenase (LDH) activity. To study the
cytoprotective action of the new compounds against
veratridine-induced cell death, drugs were given at time
zero (t = 0) and maintained for 48h; at t = 24h veratri-
11. Kuthan, J. Adv. Heterocycl. Chem. 1995, 62, 20.
12. (a) Seoane, C.; Soto, J. L.; Zamorano, P.; Quinteiro, M. J.
´
Heterocycl. Chem. 1981, 18, 309; (b) Marugan, M.;
Martın, N.; Seoane, C.; Soto, J. L. Liebigs Ann. Chem.
´
´
dine was added to the medium in the presence of the
1989, 145; (c) Zayed, S. E.; Elmaged, E. I. A.; Metwally, S.
A.; Elnagdi, M. H. Collect. Czech. Chem. Commun. 1991,
56, 2175.
drugs; then, at t = 48h, cell viability was assessed
measuring lactic dehydrogenase (LDH) activity. Extra-
cellular and intracellular LDH activity was spectro-
photometrically measured using a Cytotoxicity Cell
Death kit (Roche-Boehringer Mannheim), according
to manufacturerÕs instructions. Total LDH activity was
defined as the sum of intracellular and extracellular
LDH activity; released LDH was defined as the percent-
age of extracellular activity compared to total LDH
activity.
13. (a) Shestopalov, A. M.; Niazimbetova, Z. I.; Evans, D. H.;
Niyazymbetov, M. Heterocycles 1999, 51, 101; (b) Abdel-
Latif, F. F.; Shaker, R. M. Indian J. Chem., Sect. (B)
1990, 29B, 322; (c) Elgemeie, G. E. H.; Gohar, A.-El.-K.
M.; Regalia, H. A.; Elfahhram, H. A. Arch. Pharm. 1988,
321, 131.
14. Elgemie, G. H.; El-Enany, M. M.; Ismail, M. M.; Ahmed,
E. K. Nucleos. Nucleot. Nucl. 2002, 21, 477.
15. Bergauer, M.; Gmeiner, P. Synthesis 2001, 2281.
16. Ibrahim, N. S. Heterocycles 1986, 24, 935.
17. Rappaport, F.; Fischl, J.; Pinto, N. Clin. Chim. Acta 1959,
4, 227.
Acknowledgements
18. Bonner, L. T.; Peskind, E. R. Med. Clin. N. Am. 2002, 86,
657.
19. Maroto, R.; De la Fuente, M. T.; Artalejo, A. R.; Abad,
R.L. thanks MEC for a fellowship (P20020576). The
´
present work was supported by Fundacion Teofilo Her-
nando, FMUAM Red CIEN (Instituto de Salud Carlos
´
´
F.; Lopez, M. G.; Garcıa-Sancho, J.; Garcıa, A. G. Eur. J.
Pharmacol. 1994, 270, 331.
´
´
´
III), Ministerio de Ciencia y Tecnologıa (Grant no
BFI2003-02722), Laboratorios Ferrer and Fundacion
La Caixa (Barcelona, Spain).
20. Livett, B. G. Physiol. Rev. 1984, 64, 1103.
´
´
Garcıa, A. G. Anal. Biochem. 1990, 185, 243.
´
´
21. Moro, M. A.; Lopez, M. G.; Gandıa, L.; Michelena, P.;