Synthesis, biological evaluation and molecular modelling of diversely functionalized heterocyclic derivatives as inhibitors of acetylcholinesterase/ butyrylcholinesterase and modulators of Ca2+ channels and nicotinic receptors

Article abstract of DOI:10.1016/j.bmc.2004.02.017

The synthesis and the biological activity of compounds 5-40 as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as modulators of voltage-dependent Ca²⁺ channels and nicotinic receptors, are described. These molecules are tacrine analogues, which have been prepared from polyfunctionalized 6-amino-5-cyano-4H-pyrans, 6-amino-5-cyano-pyridines and 5-amino-2-aryl-3-cyano-1,3-oxazoles via Friedlaender reaction with selected cycloalkanones. These compounds are moderate acetylcholinesterase and butyrylcholinesterase inhibitors, the BuChE/AChE selectivity of the most active molecules ranges from 10.0 (compound 29) to 76.9 (compound 16). Interestingly, the 'oxazolo-tacrine' derivatives are devoid of any activity. All compounds showed an important inhibitory effect on the nicotinic acetylcholine receptor. Most of them also blocked L-type Ca ²⁺ channels, and three of them, 64, 19 and 67, the non-L type of Ca²⁺ channels. Molecular modelling studies suggest that these compounds might bind at the peripheral binding site of AChE, which opens the possibility to design inhibitors able to bind at both, the catalytic and peripheral binding sites of the enzyme.

Full text of DOI:10.1016/j.bmc.2004.02.017

Bioorganic & Medicinal Chemistry 12 (2004) 2199–2218
Synthesis, biological evaluation and molecular modelling
of diversely functionalized heterocyclic derivatives as inhibitors of
acetylcholinesterase/butyrylcholinesterase and modulators
+
of Ca2 channels and nicotinic receptors
a,
Jose L. Marco, Cristobal de los Rıos, Antonio G. Garcıa, Mercedes Villarroya,
a,b
b
b
*
ꢀ
ꢀ
ꢀ
ꢀ
M. Carmo Carreiras,c, Carla Martins, Ana Eleuterio, Antonio Morreale,
c
c
d
*
ꢀ
M. Orozcoe and F. Javier Luqued,ꢀ
aLaboratorio de Radicales Libres (IQOG, CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain
b
ꢀ
ꢀ
Instituto Teꢀofilo Hernando, Departamento de Farmacologıa y Terapeutica, Facultad de Medicina,
Universidad Autꢀonoma de Madrid, C/ Arzobispo Morcillo, 4, 28029 Madrid, Spain
cCentro de Estudos de Ci^encias Farmac^euticas, Faculdade de Farmꢀacia da Universidade de Lisboa,
Av. das Forßcas Armadas, 1600-083 Lisboa, Portugal
d
ꢀ
ꢁ
Departament de Fisicoquımica, Facultat de Farmacia, Universitat de Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain
eUnitat de Modelitzaciꢀo Molecular i Bioinformꢁatica, Institut de Recerca Biomꢁedica,
Parc Cientꢀıfic de Barcelona, C/ Josep Samitier 1-5, 08028 Barcelona, Spain
Received 12 December 2003; accepted 11 February 2004
Abstract—The synthesis and the biological activity of compounds 5–40 as inhibitors of acetylcholinesterase (AChE) and butyr-
ylcholinesterase (BuChE), as well as modulators of voltage-dependent Ca2þ channels and nicotinic receptors, are described. These
molecules are tacrine analogues, which have been prepared from polyfunctionalized 6-amino-5-cyano-4H-pyrans, 6-amino-5-cyano-
€
pyridines and 5-amino-2-aryl-3-cyano-1,3-oxazoles via Friedlander reaction with selected cycloalkanones. These compounds are
moderate acetylcholinesterase and butyrylcholinesterase inhibitors, the BuChE/AChE selectivity of the most active molecules ranges
from 10.0 (compound 29) to 76.9 (compound 16). Interestingly, the ꢁoxazolo-tacrineꢂ derivatives are devoid of any activity. All
compounds showed an important inhibitory effect on the nicotinic acetylcholine receptor. Most of them also blocked L-type Ca2þ
channels, and three of them, 64, 19 and 67, the non-L type of Ca2þ channels. Molecular modelling studies suggest that these
compounds might bind at the peripheral binding site of AChE, which opens the possibility to design inhibitors able to bind at both,
the catalytic and peripheral binding sites of the enzyme.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
most common type of dementia in western societies,
which has been causing profound economic and social
impact as the ageing population increases.1 Although
many factors have been implicated in AD, its etiology
and pathogenesis remain unclear. The ꢁcholinergic
hypothesisꢂ2 represents one of the most useful
approaches involved in the design of new agents for the
treatment of AD. This strategy is based on the devel-
opment of drugs with an acetylcholinesterase (AChE)
inhibition profile in order to rectify the deficit of cerebral
acetylcholine (ACh).3 AChE inhibitors such as galan-
thamine,4 donepezil,5 rivastigmine6 and huperzine7
increase the brain ACh levels by preventing the degra-
dation of the released neurotransmitter, thereby
Alzheimerꢂs disease (AD) is a neurodegenerative, irre-
versible disorder that is clinically characterized by a
progressive loss of several cognitive abilities. It is the
€
Keywords: Tacrine analogues; Friedlander reaction; AChE/BuChE
inhibitors; Ca2þ channels; Neuronal nicotinic acetylcholine receptors;
Molecular modelling.
* Corresponding authors. Tel.: +34-91-5622900; fax: +34-91-5644853;
e-mail: iqoc21@iqog.csic.es
ꢀ
To whom questions regarding the molecular modelling should be
addressed.
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.017

2200
J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
enhancing neurotransmission at cholinergic synapses.2
Tacrine (THA, Fig. 1) was the first drug approved in
USA for the treatment of AD. However, severe adverse
effects such as hepatotoxicity represent a drawback.
Despite these limitations, THA appears as an important
reference and a great synthetic and pharmacological
research effort was carried on in order to design safer
and more potent tacrine analogues.8–11
with variable selectivity for these enzymes. The best
values were observed for compounds 1 [AChE (IC50)
(M) 8.68 ꢀ 0.42 · 10ꢁ7],15a 2 [AChE (IC50) (M) 8.22 ꢀ
1.57 · 10ꢁ7],15a 3 [AChE (IC50) (M) 3.77 ꢀ 0.18 · 10ꢁ7
;
BuChE (IC50) (M) > 10ꢁ4 15d
and 4 [AChE (IC50) (M)
]
3.18 ꢀ 0.50 · 10ꢁ7
;
BuChE (IC50) (M) 7.72 ꢀ 0.50 ·
10ꢁ7 15d
(Chart 1). Curiously, the thieno derivatives,
with a similar structure to compounds 3 and 4, were
]
devoid of any inhibitory activity.15d
There is also emerging evidence showing that an altered
calcium homeostasis can play a pivotal role in the
pathogenesis of AD.12;13 On the other hand, ligands of
nicotinic receptors may be strongly associated to neu-
roprotectant effects.14 Thus, we have designed new THA
analogues as AChE/BuChE inhibitors and modulators
of voltage-dependent Ca2þ channels and ligands of nic-
otinic receptors.
In relation to these endeavours, Barreiro et al. have
recently reported interesting results on pyrazolo
[4,3-d]pyridine and pyrazolo[3,4-b][1,8]naphthyridine
analogues.16
We have also found that compounds of type B (Chart
1), in addition to its ability of inhibiting AChE, behave
as modulators or blockers of neuronal nicotinic ACh
receptors (nAChRs) as well as neuronal voltage-depen-
dent Ca2þ channels.15c
In this context, in the last years we have reported the
synthesis and the AChE/BuChE inhibitory activity of a
series of THA analogues where the benzene ring has
been substituted by a 4H-pyran or a pyridine moiety,
and the cyclohexyl ring has been contracted or enlarged
to a cyclopentane or a cycloheptane, respectively. As a
result, we have prepared heterocycles such as cyclo-
penta[b]pyrano[3,2-e]pyridine (A, n ¼ 0), 4H-pyrano-
[2,3-b]quinoline (A, n ¼ 1), cyclohepta[b]-pyrano[3,2-
e]pyridine (A, n ¼ 2),15a;b;c 6H-cyclopenta[b][1,8]naph-
thyridine (B, n ¼ 0), benzo[b][1,8]naphthyridine (B, n ¼
1), 6H-cyclohepta[b][1,8]naphthyridine (B, n ¼ 2),15b
furo (and thieno)[2,3-b]quinoline (C, n ¼ 1) and cyclo-
hepta[e]furo (and thieno) [2,3-b]pyridine (C, n ¼ 2),15c;d
(Chart 1). From this work we concluded that all the
compounds were moderate inhibitors of AChE/BuChE,
Prompted by these results and pursuing our project in
this area, we describe here the synthesis and the
AChE/BuChE inhibitory activities, as well as the mod-
ulation of voltage-dependent Ca2þ channels and/or
nAChR of compounds 5–40 (Chart 2). Finally, molec-
ular modelling calculations are carried out in order to
understand the structural basis for the biological activity
of compounds 1 and 2 (Chart 1), which are represen-
tative of the A and B classes, trying to obtain data for
future endeavours and new rationale synthetic
approaches.
2. Results and discussion
2.1. Chemistry
NH2
2.1.1. Pyrano[3,2-e]pyridines, pyrano[2,3-b]quinolines and
[1,8]naphthyridines. The synthesis of these THA ana-
€
logues has been achieved by Friedlander reaction of
the 6-amino-5-cyano-4H-pyran-3-carboxylic acid alkyl
esters18 and 6-amino-5-cyano-pyridine-3-carboxylic acid
N
17
Tacrine (THA)
Figure 1.
X
X
X
H2N
NH2
N
NH2
N
(CH2)n
EtO2C
Me
EtO2C
Me
(CH2)n
(CH2)n
N
X
O
N
X
A (n= 0, 1, 2)15a-c
B (n= 0, 1, 2)15c
C (X= O, S; n= 1, 2)15c,d
[X= H, 4-Me, 4-Cl, 4-CN, 3-NO2]
OMe
[X= H, 4-OMe]
[X= H, 4-Me, 4-OMe]
X
H2N
NH2
NH2
(CH2)n
EtO2C
Me
EtO2C
N
O
N
N
Me
O
N
X
3 X= Me, n= 1
4 X= H, n= 2
2
1
Chart 1.

J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
2201
X
X
Y
Y
NH2
NH2
N
NH2
N
RO2C
Me
N
O
RO2C
Me
(CH2)n
(CH2)n
(CH2)n
X
O
N
N
30 X= Cl, n= 0
5 X= 3-OMe, Y= H, R= Et, n= 0
6 X= 3-OMe, Y= H, R= Et, n= 1
7 X= 3-OMe, Y= H, R= Et, n= 2
8 X= 2-OMe, Y= H, R= Et, n= 0
9 X= 2-OMe, Y= H, R= Et, n= 1
10 X= 2-OMe, Y= H, R= Et, n= 2
11 X, Y= 3,4-di-OMe, R= Et, n= 0
12 X, Y= 3,4-di-OMe, R= Et, n= 1
13 X, Y= 3,4-di-OMe, R= Et, n= 2
14 X, Y= H, R= i-Pr, n= 1
15 X= H, Y= 3-OMe, R= Et, n= 1
16 X, Y= 3,4-di-OMe, R= Et, n= 1
17 X= H, Y= 4-F, R= Et, n= 1
18 X= H, Y= 4-Cl, R= Et, n= 1
19 X= H, Y= 3-NO2, R= Et, n= 1
20 X= H, Y= 4-Me, R= Et, n= 2
21 X= H, Y= 3-OMe, R= Et, n= 2
22 X= H, Y= 2-OMe, R= Et, n= 2
23 X, Y= 3,4-di-OMe, R= Et, n= 2
24 X= H, Y= 4-F, R= Et, n= 2
25 X= H, Y= 4-Cl, R= Et, n= 2
26 X= H, Y= 3-NO2, R= Et, n= 2
27 X, Y= H, R= Et, n= 3
31 X= OMe, n= 0
32 X= Me, n= 0
33 X= Cl, n= 1
34 X= NO2, n= 1
35 X= OMe, n= 1
36 X= Me, n= 1
37 X= Cl, n= 2
38 X= NO2, n= 2
39 X= OMe, n= 2
40 X= Me, n= 2
28 X= 4-OMe, Y= H, R= Et, n= 3
29 X, Y= H, R= i-Pr, n= 1
Chart 2.
alkyl esters19 with selected cycloalkanones such as
cyclopentanone, cyclohexanone, cycloheptanone and
cyclooctanone.
idenemalononitrile20 followed by enol-promoted 5-exo-
dig cyclization into one of the nitrile groups, in 57%
yield (Scheme 1).
For the synthesis of the pyrano[3,2-e]pyridines and
pyrano[2,3-b]quinolines we required the 6-amino-5-cyano-
4H-pyrans 41–44 (Chart 3). Compounds 41, 42, 44 are
known and have been prepared as previously reported.18
6-Amino-5-cyano-4-(3,4-dimethoxyphenyl)-4H-pyran-3-
carboxylic acid ethyl ester 43 (Chart 3) is a new com-
pound that we describe here for the first time. It was
prepared using the standard protocol, by Michael
addition of ethyl acetoacetate to 3,4-dimethoxybenzyl-
Under standard conditions, using dry 1,2-dichloroeth-
ane as solvent and aluminium trichloride (see Experi-
mental part), 6-amino-5-cyano-4H-pyran-3-carboxylic
acid ethyl or i-propyl esters (41–44) afforded the
expected THA analogues (5–14) employing cyclopenta-
none, cylohexanone or cycloheptanone as the ketone
€
partner in a typical Friedlander reaction (Scheme 2).
Compounds 5–10 and 14 are known products, prepared
in our laboratory some years ago, and have been
X
X
Y
Y
CN
N
RO2C
Me
CN
RO2C
Me
CN
NH2
O
O
NH2
N
NH2
X
45 X= H, Y= 3-OMe, R= Et
46 X, Y= 3,4-di-OMe, R= Et
47 X= H, Y= 4-F, R= Et
48 X= H, Y= 4-Cl, R= Et
49 X= H, Y= 3-NO2, R= Et
50 X, Y= H, R= i-Pr
41 X= H, Y= 3-OMe, R= Et
42 X= H, Y= 2-OMe, R= Et
43 X, Y= 3,4-di-OMe, R= Et
44 X, Y= H, R= i-Pr
59 X= Cl
60 X= NO2
61 X= OMe
62 X= Me
55 X= 4-F, Y= H, R= Et
56 X= 4-Cl, Y= H, R= Et
57 X= 3-NO2, Y= H, R= Et
58 X= 4-Me, Y= H, R= Et
51 X= H, Y= 4-Me, R= Et
52 X= H, Y= 2-OMe, R= Et
53 X, Y= H, R= Et
54 X= H, Y= 4-OMe, R= Et
Chart 3.

2202
J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
X
Y
NC
Y
CN
O
O
X
43 (X,Y= OMe) (57%)
55 (X= F, Y= H) (75%)
EtO
CN
RO2C
Me
piperidine, toluene
Me
CN
OH
Scheme 1. Synthesis of 4H-pyrans 43 and 55.
X
X
X
Y
CN
NH2
Y
Y
RO2C
Me
a
RO2C
Me
CN
RO2C
Me
CN
(CH2)n
(CH2)n
O
O
N
H
O
N
41 X= H, Y= 3-OMe, R= Et
42 X= H, Y= 2-OMe, R= Et
43 X, Y= 3,4-di-OMe, R= Et
44 X, Y= H, R= i-Pr
X
X
Y
N
Y
NH
H2O
NaOH
RO2C
RO2C
Me
5-14
(CH2)n
(CH2)n
Me
O
N
H
O
N
€
Scheme 2. Synthesis of tacrine-analogues 5–14 by Friedlander reaction. Reagents and conditions: (a) cyclopentanone (n ¼ 0), cyclohexanone (n ¼ 1),
cycloheptanone (n ¼ 2), (CH2)2Cl2, AlCl3, reflux.
obtained as previously mentioned.15c Precursor 43
afforded the new THA analogues 11–13 in mild condi-
tions and good chemical yield (Scheme 2).
benzo[b][1,8]naphthyridines (15–19, 29) (Chart 2) have
been prepared successfully from the corresponding
€
aminopyridines 45–50 (Chart 3) in the Friedlander
reaction (Scheme 3).17
The THA analogues exhibiting the cycloannu-
lated[b][1,8]naphthyridine ring system (15–29) (Chart 2)
have also been synthesized from the corresponding
Substituted 2-amino-pyridines 45–47, 49 and 50 (Chart
3) are reported here for the first time. They were pre-
pared from the corresponding 4H-pyran-3-carboxylic
acid ethyl esters 41, 43, 55, 57 and 44 (Chart 3),
respectively, by reaction with ammonium acetate in
glacial acetic acid (Scheme 4).19 Moreover, 4H-pyran 55
(Chart 3) was previously described in a non-easily
available literature report.21 We have synthesized it in
75% yield, starting from known 4-fluorobenzylidene-
6-amino-5-cyanopyridines (45–54)19 (Chart 3) using the
17
€
Friedlander reaction (Scheme 3).
€
Very unusually, the Friedlander reaction with cyclo-
pentanone did not afford the expected 6H-cyclo-
penta[b][1,8]naphthyridines. In general, this reaction
with cyclopentanone has always led to low yields of the
envisaged adducts. The reasons for this disappointing
result are not clear for us. Conversely, the
X
X
Y
Y
X
AcONH4, AcOH,
reflux
Y
CN
NH2
RO2C
Me
CN
NH2
RO2C
Me
RO2C
Me
CN
a
N
O
15-29
N
NH2
45 X= H, Y= 3-OMe, R= Et (41%)
46 X, Y= 3,4-di-OMe, R= Et (52%)
47 X= H, Y= 4-F, R= Et (41%)
49 X= H, Y= 3-NO2, R= Et (53%)
50 X, Y= H, R= i-Pr (40%)
41 X= H, Y= 3-OMe, R= Et
43 X, Y= 3,4-di-OMe, R= Et
55 X= 4-F, R= Et
45 X= H, Y= m-OMe, R= Et
46 X, Y= 3,4-di-OMe, R= Et
47 X= H, Y= 4-F, R= Et
48 X= H, Y= 4-Cl, R= Et
49 X= H, Y= 3-NO2, R= Et
50 X, Y= H, R= i-Pr
57 X= 3-NO2, R= Et
51 X= H, Y= 4-Me, R= Et
52 X= H, Y= 2-OMe, R= Et
53 X, Y= H, R= Et
44 X, Y= H, R= i-Pr
54 X= H, Y= 4-OMe, R= Et
58 X= H, Y= 4-Me, R= Et
42 X= H, Y= 2-OMe, R= Et
51 X= H, Y= 4-Me, R= Et (44%)
52 X= H, Y= 2-OMe, R= Et (31%)
€
Scheme 3. Synthesis of tacrine-analogues 15–29 by Friedlander reac-
tion. Reagents and conditions: (a) cyclohexanone (n ¼ 1) or cyclo-
heptanone (n ¼ 2) or cyclooctanone (n ¼ 3), (CH2)2Cl2, AlCl3, reflux.
Scheme 4. Synthesis of pyridines 45–47, 49 and 50–52.

J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
2203
malononitrile22 after condensation with ethyl acetoace-
(CH2)n
(n= 0, 1, 2)
tate, using piperidine as catalyst18 (Scheme 1) (see
Experimental part).
O
CN
NH2
N
(CH2)2Cl2, reflux/ AlCl3
O
30-40
X
6H-Cyclohepta[b][1,8]naphthyridines (20–26) (Chart 2)
were synthesized from the related aminopyridines 51, 45,
59 X= Cl
60 X= NO2
61 X= OMe
62 X= Me
17
€
52, 46–49 (Chart 3) via Friedlander reaction, using
cycloheptanone (Scheme 3). The 4-methyl and 2-meth-
oxy substituted aminopyridines 51 and 52 (Chart 3) are
new compounds that were prepared as usual from the
known pyrans 5818 and 42,18 respectively (Scheme 4).
€
Scheme 5. Synthesis of tacrine-analogues 30–40 by Friedlander reac-
tion.
ated according to the standard methodology25;26 (see
Experimental part).
Finally, the 6H-cycloocta[b][1,8]naphthyridines 27 and
28 (Chart 2) were prepared in a similar process by the
condensation of aminopyridines 5319 and 5419 (Chart 3)
with cyclooctanone (Scheme 3).
For comparative purposes we have also included the
IC50 (M) for AChE of the formerly evaluated THA
analogue 1 (Chart 1).15 Comparing with THA (IC50
1.8 ꢀ 0.2 · 10ꢁ7), the new synthesized compounds man-
All the new compounds showed analytical and spec-
troscopic data in good agreement with these structures
(see Experimental part) and with the data we formerly
observed and reported in similar compounds.15
ifest lower activity. The most active compound is still 1
(IC50 8.7 ꢀ 0.42 · 10ꢁ7
)
(around 4–5-fold less active).
15b
For the same substituents (3-, 2-methoxy, 3,4-dime-
thoxy) in the aromatic ring, the cyclohexane-fused
compounds (n ¼ 1) showed the best activity (6, 9, 12)
and the cyclopentane (n ¼ 0) compounds manifested the
worst. Considering the same ring size, the 3-methoxy
derivatives presented the lowest activities (5, 6, 7).
Hence, we conclude that the 4-methoxy group leads to
more active analogues than the 2-, 3-methoxy groups,
and the 3,4-dimethoxy substituents do not impart better
selectivity. Analysis of compound 14, with a similar
carboxylic acid ester,15b reveals that simple conversion
of the ethyl into the i-propyl group in the carboxylic acid
moiety introduced a strong negative effect for the
inhibitory activity (from 1.6 ꢀ 0.1 · 10ꢁ6 to >3.0 · 10ꢁ5).
2.1.2. Cyclopenta[b]oxazolo[4,5-e]pyridines, oxazolo[5,4-
b]quinolines and cyclohepta[b]oxazolo[4,5-e]pyridines.
The relevant AChE/BuChE inhibitory activities previ-
ously attained with the furo-THA derivatives (com-
pounds of type C, Chart 1)15d urged us to undertake the
synthesis and biological evaluation of new, related
derivatives. Therefore, we have designed the oxazolo-
THA analogues 30–40 (Chart 2) by removing one of the
aromatic rings of the furo derivatives C15c (Chart 1) and
incorporating a nitrogen atom instead in the heterocy-
cle.
The synthesis of these THA analogues was also
17
€
accomplished by Friedlander reaction of the related 2-
aryl substituted 5-amino-4-cyano-1,3-oxazoles.23 These
compounds are readily prepared from commercially
available aminopropanedinitrile p-toluenesulfonate and
acid chlorides, as reported by Freeman and Kim.24
Following this protocol, and for the preliminary tests,
we have prepared the known 1,3-oxazoles 59–6124 and
compound 62, which had not been reported in the lit-
erature yet (Chart 3).
The AChE inhibitory activity calculated for the
[1,8]naphthyridine series of derivatives is presented in
Table 1. AChE IC50 (M) data for the inhibitory activity of compounds
5–14
X
Y
NH2
NH2
€
RO2C
H3C
The Friedlander reaction of these precursors with cy-
(CH2)n
clopentanone, cyclohexanone and cycloheptanone pro-
ceeded as expected to afford compounds 30–32, 33–36
and 37–40 (Chart 2), respectively, from low to moderate
chemical yield (see Experimental part) (Scheme 5). All
the new compounds showed excellent analytical and
spectroscopic data in good agreement with these struc-
tures.
N
O
N
Tacrine (THA)
1, 5-14
n
X, Y
––
R
IC50 (M)
Tacrine
1
––
1
0
0
0
1
1
1
2
2
2
1
––
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
i-Pr
1.8 ꢀ 0.2 · 10ꢁ7
8.7 ꢀ 0.4 · 10ꢁ7
>10ꢁ4
H, 4-OMe
H, 3-OMe
H, 2-OMe
3,4-Di-OMe
H, 3-OMe
H, 2-OMe
3,4-Di-OMe
H, 3-OMe
H, 2-OMe
3,4-Di-OMe
H, H
5
8
2.4 ꢀ 0.6 · 10ꢁ5
2.7 ꢀ 0.1 · 10ꢁ5
>3.0 · 10ꢁ5
11
6
2.2. Biological activity
9
7.7 ꢀ 0.8 · 10ꢁ6
4.6 ꢀ 0.9 · 10ꢁ6
1.3 ꢀ 0.2 · 10ꢁ5
2.1 ꢀ 0.3 · 10ꢁ5
3.3 ꢀ 0.4 · 10ꢁ5
>3.0 · 10ꢁ5
12
7
2.2.1. Pyrano[3,2-e]pyridines, pyrano[2,3-b]quinolines and
[1,8]naphthyridines
2.2.1.1. Evaluation of AChE and BuChE. The AChE
inhibitory activities in the pyrano[3,2-e]pyridines and
pyrano[2,3-b]quinolines 5–14 (see Table 1) were evalu-
10
13
14

2204
J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
Table 2. For comparative purposes we have also
included the IC50 (M) for AChE of compound 2 (Chart
1).15b
n ¼ 0, 2 or 3. Very interestingly, as in the pyran-like
THA substrates, the isopropyl ester 29 is still less active
than the similar ethyl ester THA derivative.15b
Among these THA analogues, compound 17, with a
4-fluor substituent in the aromatic ring, is found to be
the most active. Actually, it is even more active than the
other pyrano[3,2-e]pyridines, and pyrano[2,3-b]quino-
lines (Table 1). It is noteworthy that the
benzo[b][1,8]naphthyridines (n ¼ 1) are the most active
in this series, regardless of the substituent in the aro-
matic ring, if we compare with the other inhibitors for
The most potent compound of each group was assayed
to determine its activity on BuChE. Data are given in
Table 3. All the compounds inhibited AChE more
strongly than BuChE. Overall, the best BuChE/AChE
selectivity was found in compound 16 (76.9). These data
are very interesting since tacrineꢂs adverse effects are
ascribed to its ability to inhibit BuChE, and suggest
future structural modifications for activity improve-
ment.
2.2.1.2. 45Ca2 uptake into bovine chromaffin cells.
+
Table 2. AChE IC50 (M) data for the inhibitory activity of compounds
15–29
Three different types of experiments were performed in
order to find out whether the new compounds, at the
concentration of 3 lM, had an action on: (i) basal Ca2þ
uptake, (ii) Ca2þ taken up by cells under conditions of
depolarization with high Kþ (70 mM with equimolar
reduction of Naþ); and (iii) Ca2þ uptake by cells stim-
ulated by the nicotinic agonist dimethylphenylpiper-
azinium (DMPP). The results obtained for some of the
compounds with AChE/BuChE inhibitory data reported
here, were published before.15c
X
Y
NH2
NH2
RO2C
(CH2)n
N
Me
N
N
Tacrine (THA)
2, 15-29
n
X, Y
R
IC50 (M)
Tacrine
2
15
––
H, H
H, 3-OMe
––
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
i-Pr
1.8 ꢀ 0.2 · 10ꢁ7
8.2 ꢀ 1.6 · 10ꢁ7
1.2 ꢀ 0.3 · 10ꢁ6
1.3 ꢀ 0.3 · 10ꢁ6
7.1 ꢀ 1.7 · 10ꢁ7
1.3 ꢀ 0.2 · 10ꢁ6
9.3 ꢀ 1.9 · 10ꢁ7
1.3 ꢀ 0.1 · 10ꢁ5
>3.0 · 10ꢁ5
1
1
1
1
1
1
2
2
2
2
2
2
2
3
3
1
In basal conditions, resting cells took up 781 ꢀ 78 cpm
of 45Ca2þ when incubated in the presence of 1 mM
40Ca2þ for 5 min (data from five different batches of
cells). After 5 min incubation, compounds 24 and 25
affected basal Ca2þ uptake (Chart 2) around 20% in-
crease; compounds 20 and 21increased Ca2þ uptake by
37% and 45%, respectively. Other compounds such as 66
(Chart 4),15b 18 and 19 (Chart 2) inhibited Ca2þ uptake
around 20% (Table 4).
16
17
3,4-Di-OMe
H, 4-F
H, 4-Cl
18
19
H, 3-NO2
H, 4-Me
H, 3-OMe
H, 2-OMe
3,4-Di-OMe
H, 4-F
20
21
22
23
2.2 ꢀ 0.5 · 10ꢁ5
6.4 ꢀ 0.7 · 10ꢁ6
5.4 ꢀ 1.5 · 10ꢁ6
1.9 ꢀ 0.3 · 10ꢁ5
1.4 ꢀ 0.3 · 10ꢁ5
1.7 ꢀ 0.4 · 10ꢁ5
1.7 ꢀ 0.6 · 10ꢁ5
1.2 ꢀ 0.2 · 10ꢁ6
24
25
H, 4-Cl
H, 3-NO2
H, H
Looking for a possible modulatory effect on voltage-
dependent Ca2þ channels (VDCCs), we analyzed the
action of the compounds on 45Ca2þ entry into Kþ-
depolarized chromaffin cells. High Kþ stimulation for
5 s induced 45Ca2þ uptake (in the presence of 1 mM
26
27
28
29
H, 4-OMe
H, H
Table 3. IC50 for BuChE inhibition for some selected compounds and the ratio BuChE/AChE
OMe
X
Y
NH2
NH2
N
NH2
N
NH2
N
EtO2C
Me
EtO2C
Me
RO2C
Me
N
N
O
N
Tacrine (THA)
15-19, 29
1
2
X, Y
––
R
AChE (M)
BuChE (M)
BuChE/AChE
Tacrine
1
––
1.8 ꢀ 0.2 · 10ꢁ7
8.7 ꢀ 0.4 · 10ꢁ7
8.2 ꢀ 1.6 · 10ꢁ7
1.2 ꢀ 0.3 · 10ꢁ6
1.3 ꢀ 0.3 · 10ꢁ6
7.1 ꢀ 1.7 · 10ꢁ7
1.3 ꢀ 0.2 · 10ꢁ6
9.3 ꢀ 1.9 · 10ꢁ7
1.2 ꢀ 0.2 · 10ꢁ6
3.6 ꢀ 0.4 · 10ꢁ8
6.2 ꢀ 0.6 · 10ꢁ6
5.0 ꢀ 0.6 · 10ꢁ6
3.1 ꢀ 0.2 · 10ꢁ5
>10ꢁ4
1.2 ꢀ 0.4 · 10ꢁ5
5.2 ꢀ 0.9 · 10ꢁ5
2.1 ꢀ 0.3 · 10ꢁ5
1.2 ꢀ 0.2 · 10ꢁ5
0.2
7.1
2
6.1
15
16
17
18
19
29
H, 3-OMe
3,4-Di-OMe
H, 4-F
Et
Et
Et
Et
Et
i-Pr
25.4
76.9
16.9
40.0
22.6
10.0
H, 4-Cl
H, 3-NO2
H, H

J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
2205
OMe
additional blockade. Only 64 (Chart 4),15b 19 (Chart 2)
and 67 (Chart 4)15b presented an additional inhibition of
25%, 19% and 39%, respectively, which could indicate
an effect on the other subtypes of Ca2þ channels (N, P/
Q) (Table 4).
X
X
NH2
N
NH2
N
NH2
N
EtO2C
Me
EtO2C
Me
EtO2C
Me
N
N
N
63 X= H
66 X= H
67 X= 4-OMe
Stimulation with the nicotinic receptor agonist DMPP
(100 lM during 5 s) induced 45Ca2þ uptake (in the
presence of 1 mM 40Ca2þ) of 455 ꢀ 48 cpm per 2 · 105
cells (12 individual wells data from three different bat-
ches of cells). After 15 min incubation all the compounds
blocked Ca2þ uptake induced by nicotinic stimulation
with DMPP, with inhibition values ranging from 50% to
almost 100% (compound 16) (Table 4).
65
64 X= 4-OMe
Chart 4.
Table 4. Effect of the different compounds on 45Ca2þ uptake into
bovine chromaffin cells (% inhibition concerning a control without any
drug); all the compounds were tested at the concentration of 3 lMa
2.2.2. Cyclopenta[b]oxazolo[4,5-e]pyridines, oxazolo[5,4-
b]quinolines and cyclohepta[b]oxazolo[4,5-e]pyridines
2.2.2.1. Evaluation of AChE. We have noted that these
substrates were particularly insoluble in most of the
usual solvents, which was a real problem in order to
carry out biological evaluation (see below). With the
purpose to solve this, we have also prepared the hy-
drochlorides of compounds 33–35 and 37–39, but their
conversion into salts did not improve the solubility
either.
NH2
EtO2C
Me
N
N
2
Compound
Basal
70 mM Kþ
100 lM DMPP
63
64
2
ꢁ0.8 ꢀ 5.7b
ꢁ8.1 ꢀ 5
24.3 ꢀ 6.4
14.9 ꢀ 2.4
19.7 ꢀ 4.6
10.3 ꢀ 5.6
11.7 ꢀ 7.2
39.2 ꢀ 6
51.6 ꢀ 4.8
71.1 ꢀ 3.8
52.2 ꢀ 2.2
76.0 ꢀ 4.1
69.7 ꢀ 3.2
82.3 ꢀ 2.9
49.7 ꢀ 9.1
99.6 ꢀ 0.2
63.0 ꢀ 7.5
83.4 ꢀ 4.6
61.5 ꢀ 8.2
68.1 ꢀ 4
4.3 ꢀ 4.3
65
66
67
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
10.6 ꢀ 6
Due to these solubility difficulties, we only obtained the
following data (% inhibition) for the AChE inhibition at
10 lM concentration for a limited number of substrates
[(39): 14.1 ꢀ 0.2%; (32) 27.5 ꢀ 2.9%; (30): 39.9 ꢀ 4.4%;
(37): 45.6 ꢀ 5.8%]. Then, we tested the 1,3-oxazole pre-
cursors and observed the best value (63.2%) for com-
pound 60 (Chart 3) at 10 lM concentration. These low
results prevented us to investigate at any other different
concentrations.
22.2 ꢀ 4.1
7.1 ꢀ 4.5
5.8 ꢀ 2.1
15.5 ꢀ 3
9.1 ꢀ 9.3
26 ꢀ 0.8
14.2 ꢀ 7.6
20.6 ꢀ 4.2
25.1 ꢀ 8.9
ꢁ36.6 ꢀ 12
ꢁ45.3 ꢀ 11.3
4.5 ꢀ 6.9
38.5 ꢀ 7.9
34.4 ꢀ 3.3
18.4 ꢀ 6.1
23.8 ꢀ 2.8
8.9 ꢀ 3.6
16.9 ꢀ 2.7
9.2 ꢀ 4.8
17.2 ꢀ 4.4
26.9 ꢀ 2.7
12.7 ꢀ 9.4
10.3 ꢀ 1.8
19.6 ꢀ 3.8
9.7 ꢀ 10.9
61.7 ꢀ 7.5
89.5 ꢀ 3.9
96.8 ꢀ 3.6
70.6 ꢀ 3.2
62.9 ꢀ 3.7
74.4 ꢀ 5.5
81.9 ꢀ 4.8
73.3 ꢀ 4.3
81.8 ꢀ 12.1
4.0 ꢀ 6.3
ꢁ16.9 ꢀ 4
ꢁ19.0 ꢀ 7.6
6.1 ꢀ 5.4
2.3. Molecular modelling
9.0 ꢀ 6.2
2.3.1. Pyrano[3,2-e]pyridines, pyrano[2,3-b]quinolines and
[1,8]naphthyridines. Despite the variety of chemical
modifications introduced into the basic core of the
AChE inhibitors examined here and their high structural
similarity with THA, the most potent compounds are
ꢂ4-fold less active than THA. Moreover, it is also
intriguing that the mechanism of AChE inhibition
strikingly varies from competitive to noncompetitive
type due to apparently minor structural modifica-
tions,15b thus suggesting that these compounds cannot
be easily accommodated in the AChE active site.
Therefore, the putative binding mode of compounds 1
and 2, which are representative members of the A- and
B-like ligands (see Chart 1) and are amongst the most
potent AChE inhibitors (see above) was investigated by
means of molecular dynamics simulations. To this end,
compounds 1 and 2 were initially positioned into the
binding site by superposing the N and NH2 groups of
the central ring to those of THA in the X-ray crystal-
lographic structure of the Torpedo californica AChE–
THA complex (PDB entry 1ACJ).27 The aromatic and
alicyclic rings were then placed over those of THA (di-
rect binding mode; d) or by rotating the molecule 180ꢁ
7.5 ꢀ 5.4
13.8 ꢀ 9.6
a Data are expressed as the meansþSEM of at least three different
cultures in quadruplicate.
b Negative values mean an increase in 45Ca2þ uptake.
40Ca2þ) of 401 ꢀ 14 cpm per 2 · 105 cells (16 individual
wells data from three different batches of cells). In basal
conditions for this time of incubation (5 s in Krebs-
HEPES solution containing 5 lCi mLꢁ1 45Ca2þ plus
1 mM 40Ca2þ), cells retained 85 ꢀ 1 cpm (16 individual
wells data from different batches of cells). Thus, the
mean ratio evoked/basal Ca2þ uptake was about 5-fold
for Kþ-depolarization. Most compounds inhibited Ca2þ
entry around 20%, which could indicate a blockade of
the L-type Ca2þ channels, since the proportion of this
subtype of channels is about 20% of the total in bovine
chromaffin cells. When the experiments were repeated in
the presence of 3 lM nifedipine, a specific blocker of L-
type Ca2þ channels, most compounds did not exert an

2206
J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
through the Nꢃ ꢃ ꢃNH2 axis (inverse binding mode; i).
Therefore, four (1R-d, 1R-i, 1S-d and 1S-i) and two (2-d
and 2-i) starting binding modes were examined for
compounds 1 and 2, respectively. For each binding
mode, a molecular dynamics (MD) simulation was
performed to explore the structural and energetic
properties of the drug–enzyme complex using the sim-
ulation protocol adopted in our previous studies11;28 (see
Experimental part).
The root-mean square deviation (rmsd) profiles deter-
mined for the subset of residues that form the wall of the
ꢂ
active site was in all cases larger than 2.4 A, thus indi-
cating the occurrence of relevant local distortions in the
protein as a consequence of the binding of these mole-
cules. As a reference, the corresponding rmsd deter-
mined for the binding of THA and huprine Y, a mixed
tacrine–huperzine
A derivative, was only around
28
ꢂ
0.6 A. For three complexes of compound 1 (1R-d, 1R-i
and 1S-d) and one complex of compound 2 (2-d) the
rmsd increased steadily along the trajectory, it being
Figure 2. Representation of the binding mode of compounds 1 (1S-i;
top) and 2 (2-i; bottom) found from molecular dynamics simulations.
Superposition of the inhibitor with THA (cyano) obtained by fitting
the enzymeꢂs backbone atoms for the AChE-inhibitor complex and the
X-ray crystallographic structure of the AChE–THA complex (1ACJ) is
also displayed. Hydrogens are not shown for the sake of clarity.
ꢂ
larger than 3 A at the end of the simulation. For the two
remaining binding modes (1S-i and 2-i) the rmsd
ꢂ
remained stable at ꢂ2.5 A along the simulation. The
energetic analysis also pointed out that these latter
binding modes were also those leading to the most stable
complexes. The greater stability of the binding modes
1S-i and 2-i is mostly due to the internal energy of the
enzyme (by more than 20 kcal/mol with respect to the
other binding modes). For compound 1 the interaction
energy between inhibitor and enzyme also favours the
binding mode 1S-i. For compound 2, the enzyme–drug
interaction term slightly favoured the binding mode 2-d,
but this was counterbalanced by both the solvation free
energy and the destabilization in the internal energy of
the enzyme, which favour binding mode 2-i.
through a noncompetitive mechanism,15b might exert
their inhibitory activity by interacting at the peripheral
binding site of AChE, which is postulated to be Trp279,
as it has already been noted for propidium,29 which is a
selective ligand for the peripheral site. This finding
might be of particular relevance in view of recent studies
pointing out that inhibitors binding to the peripheral
site might play a role in controlling the formation of
amyloid fibrils.30
The preceding results suggest that the binding site of
AChE is not flexible enough as to easily accommodate
compounds 1 and 2. For compound 1, the methoxy-
benzene unit interferes with the stacking interaction
between the aminoacridine unit of THA and the rings of
Trp84 and Phe330, thus forcing the drug to reorient
inside the binding pocket (see Fig. 2; top). Such a dis-
rupting effect is less marked in compound 2, since the
stacking interaction is roughly preserved along the
simulation (see Fig. 2; bottom). Nevertheless, unfa-
vourable steric contacts due to the ester side chain forces
the displacement of the inhibitor to the position occu-
pied by THA, thus leading to the loss of specific inter-
actions such as the hydrogen bonding between the NH
group of THA and the C@O group of His440. Overall, it
seems that the chemical groups present in 1 and 2 con-
strain these compounds to occupy a large fraction of (or
even to exceed) the allowable molecular size of the
binding pocket. Thus, whereas the cavity at the catalytic
2.3.2. Cyclopenta[b]oxazolo[4,5-e]pyridines, oxazolo[5,4-
b]quinolines and cyclohepta[b]oxazolo[4,5-e]pyridines.
The preceding results show that the different oxazolo-
THA compounds 30–40 are devoid of any AChE
inhibitory activity, a result that is in contrast with the
inhibitory activity exhibited by the furo derivatives 3
and 4 (see Introduction). Such a drastic reduction in the
inhibitory potency must be related to the replacement of
the aromatic ring in the related furo derivatives (C,
Chart 1) by a nitrogen atom in compounds 30–40. Based
on the molecular modelling results presented above for
the 4H-pyrano[2,3-b]quinoline and benzo[b][1,8]naph-
thyridine derivatives, as well as on structure–activity
studies of 11H-indeno[1,2-b]quinolin-10-ylamine ana-
logues of THA,31 we suggest that compounds 3 and 4
might also bind to the AChE peripheral site. Therefore,
it is reasonable to expect that their binding should be
mediated by a cation-p interaction between the pro-
tonated ligand and the ring of Trp279. Thus, the
reduced activity of 30–40 might be due to the increased
acidity of these compounds due to the electron-with-
drawing nature of the oxazole ring.
3
ꢂ
binding site is estimated to be ꢂ340 A , the van der
Waals volumes of compounds 1 and 2 are ꢂ330 and
3
ꢂ
ꢂ300 A , which are noticeably larger than the van der
3
ꢂ
Waals volume of tacrine (ꢂ170 A ). Finally, since
attachment of those bulky substituents might difficult
the pass through the gorge to reach the catalytic binding
site, we suggest that these compounds, which act

J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
2207
NH2
N
tion 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 car-
ried out under vacuum with a rotary evaporator. Flash
column chromatography was performed using silica gel
68 X= NH, Y= CH
69 X= O, Y= CH
Y
X
70 X= O, Y= N
Chart 5.
1
60 (230–400 mesh, Merck). H spectra were recorded
with a Varian VXR-200S spectrometer, using tetra-
methylsilane as internal standard and 13C NMR spectra
were recorded with a Bruker WP-200-SY. Known 4H-
pyrans (41, 42, 44, 56–58),18 pyridines (45–47, 49, 50)19
and oxazoles (59–61)24 have been synthesized as
described.
To explore this possibility, we determined the well depth
of the electrostatic potential32 generated in the vicinity
of the N atom in the central ring for THA and for
compounds 68–70 (Chart 5). The energy of the mini-
mum in THA (ꢁ76.8 kcal/mol) is similar to that found
for compounds 68 (ꢁ75.0 kcal/mol) and 69 (ꢁ75.4 kcal/
mol). However, a lower (in absolute term) value of
ꢁ68.7 kcal/mol is found for the oxazolo compound 70.
Therefore, the reduced activity of compounds 30–40
might be related to their increased acidity, which hinders
the formation of the cation-p interaction with Trp279.
4.2. General method for the synthesis of 6-amino-4-aryl-5-
cyano-4H-pyran-3-carboxylic acid ethyl esters
To a solution of the corresponding aldehyde (1 equiv) in
dry toluene (1 mL/mmol), under argon, malononitrile
(1 equiv) and a catalytic amount of piperidine were ad-
ded. The mixture was stirred at rt for 3–7 h. The solvent
was evaporated and the crude was purified by column
cromathography eluting with mixtures of hexane/ethyl
acetate. Next, to a solution of the arylidenemalononit-
rile in dry toluene, ethyl acetoacetate (1 equiv) and a
catalytic amount of piperidine were added. The mixture
was stirred at rt for 2 h, and the precipitated solid was
isolated by filtration, washed with cold toluene, dried
and recrystallized.
3. Conclusions
In this work we have reported the synthesis, the AChE
and BuChE inhibitory activities, as well as the modu-
latory effect on nAChRs and VDCCs of a series of
tacrine analogues 5–40. These molecules have been
prepared from readily available polyfunctionalized ethyl
[4H-pyrans and 6-amino-5-cyanopyridines]-3-carboxyl-
€
ates via Friedlander condensation with selected ketones.
The most active compounds in these series correspond
to pyrano- or pyridine-like tacrines with saturated
cyclohexane rings. The type of the substituent at the
aromatic ring in C4 does not seem to have a deep
influence on the inhibitory activity. Overall, the substi-
tution of a benzene ring in tacrine by pyran or pyridine
rings generates analogues with strong capacity to inhibit
AChE, with lower values, but very close to those shown
by tacrine (1). With respect to modulation of VDCC and
nAChR in the bovine chromaffin cell, [1,8]naphthyr-
idines seem to have an important inhibitory effect on
nAChR. Furthermore, most of the members of this
family could be blocking L-type Ca2þ channels, and
three of them, 64,15b 6715b and 19, non-L type Ca2þ
channels. Molecular modelling results suggest that the
binding of these compounds to the AChE catalytic site
might be hampered by unfavourable steric interactions.
As a result, there might be a displacement of the drug to
the peripheral binding site, leading then to a noncom-
petitive inhibitory mechanism. This finding could be of
interest to explore new inhibitors able to bind at both
catalytic and peripheral binding sites of AChE.
4.3. General method for the synthesis of 6-amino-4-aryl-5-
cyano-pyridine 3-carboxylic acid ethyl esters
To a solution of ammonium acetate (10 equiv) in acetic
acid (1.25 mL/mmol) the appropriate ethyl 6-amino-5-
cyano-4H-pyran-3-carboxylate (1 equiv) was added. The
mixture was refluxed (116 ꢁC) for 3–7 h. Then, the
reaction was cooled and the solvent was evaporated.
The crude was treated with an aqueous saturated solu-
tion of sodium bicarbonate until pH 7 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 chromatography
eluting with mixtures of dichloromethane/ethyl acetate.
The isolated product was recrystallized from mixtures of
hexane/dichloromethane.
¨
4.4. General method for the Friedlander reaction
Aluminium chloride (1.2–1.7 equiv) was suspended in
dry 1,2-dichloroethane (10 mL/mmol) at rt under argon.
The corresponding ethyl 6-amino-4-aryl-5-cyano-4H-
pyran-3-carboxylic acid derivatives (1 equiv) and the
ketone (cyclopentanone, cyclohexanone and cyclohep-
tanone; 1.2–1.7 equiv) were added. The reaction mixture
was heated under reflux (10–24 h). After the reaction
was complete (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
4. Experimental part
4.1. General methods
Reactions were monitored by TLC, using precoated
silica gel aluminium plates containing a fluorescent
indicator (Merck, 5539). Detection was accomplished by
UV (254 nm) followed by charring with sulfuric–acetic
acid spray, 1% aqueous potassium permanganate solu-

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J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
30 min, the mixture was extracted three times with di-
chloromethane. The organic 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 chromatography using meth-
anol/dichloromethane mixtures as eluent to provide
pure compounds.
4.7. 6-Amino-5-cyano-4-(3-methoxyphenyl)-2-methyl-3-
pyridinecarboxylic acid ethyl ester (45)
This compound was obtained following the general
method for the synthesis of 6-amino-5-cyano-pyridines.
To a stirred solution of ammonium acetate (1.53 g,
19.60 mmol) in acetic acid (25 mL), pyran 41 (625 mg,
1.99 mmol) was added. After the reaction was complete
and following the work-up, the crude was purified to
produce compound 45 (255 mg, 41%): mp 165–167 ꢁC;
IR (KBr) m 3404, 3315, 3170, 2979, 2218, 1705, 1650,
1602, 1562, 1493, 1469, 1459, 1376, 1286, 1252, 1178,
4.5. 6-Amino-5-cyano-4-(3,4-dimethoxyphenyl)-2-methyl-
4H-pyran-3-carboxylic acid ethyl ester (43)
This compound was prepared following the general
method for the synthesis of 4H-pyrans. To a solution of
m,p-dimethoxybenzylidenemalononitrile20 (1.00 g, 4.67
mmol) in dry toluene (23 mL) at rt under argon, ethyl
acetoacetate (592 lL, 4.67 mmol) and piperidine (six
drops) were added. After the reaction was complete and
following the work-up, purification of the resultant
crude afforded compound 43 (909 mg, 57%): mp 134–
136 ꢁC; IR (KBr) m 3397, 3326, 3223, 2934, 2193, 1693,
1675, 1649, 1606, 1512, 1463, 1448, 1418, 1369, 1334,
1159, 1094, 1079, 1069 cmꢁ1 1H NMR (CDCl3,
;
200 MHz) d 7.38 (dd, J ¼ 7:8, 7.9 Hz, 1H, H50), 7.02–
6.86 (m, 3H, H20, H30, H60), 5.42 (s, 2H, NH2), 3.98 (q,
J ¼ 7:2 Hz, 2H, CO2CH2CH3), 3.82 (s, 3H, OCH3), 2.51
[s, 3H, CH3C(2)], 0.90 (t, 3H, CO2CH2CH3); 13C NMR
(CDCl3, 50.2 MHz) d 167.3 (C@O), 160.6 (C6), 159.5
(C30), 159.0 (C2), 153.7 (C4), 136.7 (C10), 129.7 (C50),
120.2 (C60), 120.0 (C3), 115.9 (C@N), 115.4 (C40), 113.2
(C20), 88.9 (C5), 61.3 (OCH2CH3), 55.3 (OCH3), 23.5
[CH3C(2)], 13.5 (CH3CH2O); MS (APCIþ) m=z 312
[(Mþ1)þ, 100], 284 (25), 266 (14). Anal. Calcd for
C17H17N3O3: C, 65.8S; H, 5.80; N, 13.50. Found: C,
65.37; H, 5.82; N, 13.78.
1
1267, 1063, 1022 cmꢁ1; H NMR (CDCl3, 200 MHz) d
6.82–6.70 (m, 3H, C6H3), 4.44 (s, 2 H, NH2), 4.41 (s, 1H,
H4), 4.05 (q, J ¼ 7:2 Hz, 2H, CO2CH2CH3), 3.87, 3.85
(2s, 6H, 2 · OCH3), 2.36 [s, 3H, CH3C(2)], 1.12 (t, 3H,
CO2CH2CH3); 13C NMR (CDCl3, 75.4 MHz) d 165.9
(C@O), 157.3 (C6), 156.3 (C2), 148.8 (C30), 148.1 (C40),
136.5 (C10), 119.6 (C60), 118.9 (CBN), 111.2, 110.9 (C50,
C20), 108.1 (C3), 62.4 (C5), 60.6 (OCH2CH3), 55.8
(2 · OCH3), 38.3 (C4), 18.3 [CH3C(2)], 13.9
(CH3CH2O); MS (APIESþ) m=z 367 [(MþNa)þ, 100],
345 [(Mþ1)þ, 20]. Anal. Calcd for C18H20N2O5: C,
62.78; H, 5.85; N, 8.13. Found: C, 63.04; H, 6.12; N,
8.40.
4.8. 6-Amino-5-cyano-4-(3,4-dimethoxyphenyl)-2-methyl-
3-pyridinecarboxylic acid ethyl ester (46)
This compound was prepared following the general
method for the synthesis of 6-amino-5-cyano-pyridines.
To a stirred solution of ammonium acetate (980 mg,
12.72 mmol) in acetic acid (16 mL), pyran 43 (438 mg,
1.27 mmol) was added. After the reaction was complete
and following the work-up, the crude was purified to
afford compound 46 (226 g, 52%): mp 171–174 ꢁC; IR
(KBr) m 3417, 3329, 3183, 2978, 2215, 1726, 1657, 1604,
1556, 1516, 1463, 1365, 1325, 1276, 1256, 1238, 1175,
1143 cmꢁ1; 1H NMR (CDCl3, 200 MHz) d 6.96–6.87 (m,
3H, C6H3), 5.43 (s, 2H, NH2), 4.01 (q, J ¼ 7; 1 Hz, 2H,
CO2CH2CH3), 3.92, 3.88 (2s, 6H, 2 · OCH3), 2.49 [s,
3H, CH3C(2)], 0.96 (t, 3H, CO2CH2CH3); 13C NMR
(CDCl3, 50.2 MHz) d 168.2 (C@O), 161.5 (C6), 160.7
(C2), 154.2 (C4), 151.3 (C40), 149.9 (C30), 138.7 (C10),
121.0 (C60), 120.8 (C3), 117.5 (CBN), 111.3, 111.2 (C20,
C50), 89.8 (C5), 61.1 (OCH2CH3), 57.9 (2 · OCH3), 23.5
[CH3C(2)], 18.2 (CH3CH2O); MS (APCIþ) m=z 342
(Mþ, 100), 314 (71), 296 (12), 280 (6), 254 (6). Anal.
Calcd for C18H19N3O4: C, 63.33; H, 5.61; N, 12.31.
Found: C, 63.12, H, 5.68, N, 12.33.
4.6. 6-Amino-5-cyano-4-(4-fluorophenyl)-2-methyl-4H-
pyran-3-carboxylic acid ethyl ester (55)
This compound was prepared following the general
method for the synthesis of 4H-pyrans. To a stirred
solution of p-fluorobenzylydenemalonitrile22 (1.00 g,
5.81 mmol) in dry toluene (25 mL) at rt under argon,
ethyl acetoacetate (737 lL, 5.81 mmol) and piperidine
(seven drops) were added. After the reaction was com-
plete and following the work-up, purification of the
resultant crude rendered compound 5521 (1.31 g, 75%):
mp 154–157 ꢁC; IR (KBr) m 3408, 3341, 2194, 1695, 1679,
1649, 1610, 1508, 1368, 1337, 1267, 1059 cmꢁ1; 1H NMR
(CDCl3, 200 MHz) d 7.28–7.15 (m, 2H, H20, H60), 7.05–
6.95 (m, 2H, H30, H50), 4.49 (s, 2H, NH2), 4.46 (s, 1 H,
H4), 4.06 (q, J ¼ 7:2 Hz, 2H, CO2CH2CH3), 2.39 [s, 3H,
CH3C(2)], 1.12 (t, 3H, CO2CH2CH3); 13C NMR (CDCl3,
4.9. 6-Amino-5-cyano-4-(4-fluorophenyl)-2-methyl-3-
pyridinecarboxylic acid ethyl ester (47)
0
50.2 MHz) d 165.8 (C@O), 164.5, 159.6 (JC4 –F ¼ 246 Hz,
C40), 157.4 (C6), 156.9 (C2), 139.7, 139.6 (JC1 –F ¼ 3 Hz,
0
C10), 129.3, 129.1 (JC2 –F ¼ 8 Hz, 2 · C20), 118.8 (CBN),
This compound was obtained following the general
method for the synthesis of 6-amino-5-cyano-pyridines.
To a stirred solution of ammonium acetate (2.55 g,
33.08 mmol) in acetic acid (30 mL), pyran 55 (1.00 g,
3.31 mmol) was added. After the reaction was complete
and subsequently to the work-up, the crude was purified
to give compound 47 (404 mg, 41%): mp 239–241 ꢁC; IR
0
115.7, 115.3 (JC3 –F ¼ 22 Hz, 2 · C30), 108.0 (C3), 62.5
0
(C5), 60.8 (OCH2CH3), 38.2 (C4), 18.5 [CH3C(2)], 14.0
(CH3CH2O); MS (APCIþ) m=z 303 [(Mþ1)þ, 100], 287
(5), 257 (6), 229 (30), 207 (14). Anal. Calcd for
C16H15FN2O3: C, 63.55; H, 5.00; N, 9.27. Found: C,
63.80; H, 5.30; N, 9.51.

J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
2209
(KBr) m 3384, 3317, 3182, 2222, 1714, 1654, 1606, 1567,
1512, 1283, 1184, 1162 cmꢁ1; 1H [(CD3)2CO, 200 MHz] d
7.61–7.38 (2m, 4H, C6H4), 6.77 (s, 2H, NH2), 4.11 (q,
J ¼ 7:1 Hz, 2H, CO2CH2CH3), 2.58 [s, 3H, CH3C(2)],
1.05 (t, 3H, CO2CH2CH3); 13C NMR (CDCl3,
254 (6). Anal. Calcd for C17H17N3O2: C, 69.14; H, 5.80;
N, 14.23. Found: C, 68.97; H, 6.09; N, 14.17.
4.12. 6-Amino-5-cyano-2-methyl-4-(4-methylphenyl)-3-
pyridinecarboxylic acid ethyl ester (51)
0
75.4 MHz) d 167.7 (C@O), 163.2, 159.9 (JC4 –F ¼ 249 Hz,
C40), 161.3 (C6), 159.4 (C2), 153.3 (C4), 132.5 (C10),
This compound was prepared following the general
method for the synthesis of 6-amino-5-cyano-pyridines.
To a stirred solution of ammonium acetate (2.22 g,
28.86 mmol) in acetic acid (30 mL), pyran 58 (861 mg,
2.89 mmol) was added. After the reaction was complete
and following the work-up, the resultant crude was
purified to afford compound 51 (372 mg, 44%): mp 253–
256 ꢁC; IR (KBr) m 3382, 3347, 3185, 2980, 2222, 1713,
1653, 1561, 1514, 1461, 1370, 1280, 1254, 1184,
130.4, 130.3 (JC2 –F ¼ 8 Hz, 2 · C20), 117.2 (C3), 116.4,
0
116.1 (JC3 –F ¼ 23 Hz, 2 · C30), 116.3 (C@N), 89.8 (C5),
0
61.9 (OCH2CH3), 24.0 [CH3C(2)], 14.0 (CH3CH2O); MS
(APIESþ) m=z 300 [(Mþ1)þ, 50], 322[(MþNa)þ, 100].
Anal. Calcd for C16H15FN2O3: C, 64.21; H, 4.71; N,
14.04. Found: C, 64.02, H, 4.55, N, 13.93.
1
1073 cmꢁ1; H NMR (CDCl3, 200 MHz) d 7.24 (s, 4H,
4.10. 6-Amino-5-cyano-2-methyl-4-(3-nitrophenyl)-3-
pyridinecarboxylic acid ethyl ester (49)
C6H4), 5.35 (s, 2H, NH2), 3.98 (q, J ¼ 7:2 Hz, 2H,
CO2CH2CH3), 2.50 [s, 3H, CH3C(2)], 2.40 [s, 3H,
CH3C(40)], 0.90 (t, 3H, CO2CH2CH3); 13C NMR
(DMSO-d6, 50.2 MHz) d 166.8 (C@O), 159.7/159.5 (2C,
C6, C2), 153.5 (C4), 138.5 (C40), 133.1 (C10), 128.8
(2 · C30), 127.6 (2 · C20), 117.8 (C3), 115.3 (CBN), 79.0
(C5), 60.5 (OCH2CH3), 23.0 [CH3C(2)], 20.7 [CH3C(40)],
13.2 (CH3CH2O); MS (APCIþ) m=z 296 [(Mþ1)þ, 100],
268 (50), 250 (32), 224 (5). Anal. Calcd for C17H17N3O2:
C, 69.14; H, 5.80; N, 14.23. Found: C, 68.95, H, 6.00, N,
14.30.
This compound was prepared following the general
method for the synthesis of 6-amino-5-cyano-pyridines.
To a stirred solution of ammonium acetate (1.08 g,
14.12 mmol) in glacial acetic acid (18 mL), pyran 57
(465 mg, 1.41 mmol) was added. After the reaction was
complete and following the work-up, the resultant crude
was purified to afford compound 49 (245 mg, 53%): mp
152–155 ꢁC; IR (KBr) m 3394, 3327, 3172, 2222, 1716,
1658, 1561, 1532, 1353, 1278, 1178, 1074 cmꢁ1; 1H NMR
(CDCl3, 200 MHz) d 8.36 (m, 1H, H40), 8.22 (m, 1H,
H20), 7.71–7.66 (m, 2H, H50, H60), 5.47 (s, 2H, NH2),
4.01 (q, J ¼ 7:1 Hz, 2H, CO2CH2CH3), 2.55 [s, 3H,
CH3C(2)], 0.95 (t, 3H, CO2CH2CH3); 13C NMR
(CDCl3, 50.2 MHz) d 166.8 (C@O), 161.8 (C6), 158.9
(C2), 151.3 (C4), 148.1 (C30), 137.4 (C10), 134.0 (C60),
129.8 (C50), 124.2, 123.1 (C20, C40), 119.5 (C3), 115.3
(CBN), 88.7 (C5), 61.5 (OCH2CH3), 23.9 [CH3C(2)],
13.6 (CH3CH2O); MS (APIESꢁ) m=z 325 [(Mꢁ1)ꢁ,
100], 253 (12), 237 (52), 205 (23). Anal. Calcd for
C16H14N4O4: C, 58.89; H, 4.32; N, 17.17. Found: C,
59.01, H, 4.60, N, 17.32.
4.13. 6-Amino-5-cyano-4-(2-methoxyphenyl)-2-methyl-3-
pyridinecarboxylic acid ethyl ester (52)
This compound was prepared following the general
method for the synthesis of 6-amino-5-cyano-pyridines.
To a stirred solution of ammonium acetate (12.33 g,
0.16 mol) in acetic acid (90 mL), pyran 42 (3.45 g,
10.98 mmol) was added. After the reaction was complete
and following the work-up, the crude was purified to
give compound 52 (1.05 g, 31%): mp 200–203 ꢁC; IR
(KBr) m 3388, 3321, 3181, 2222, 1711, 1653, 1560, 1496,
1
1461, 1285, 1268, 1248, 1180 cmꢁ1; H NMR (CDCl3,
0
0
0
0
200 MHz) d 7.40 (ddd, J3 –4 ¼ 8:3 Hz, J4 –5 ¼ 7:8 Hz,
J4 –6 ¼ 1:9 Hz, 1H, H40), 7.12 (dd, J5 –6 ¼ 7:5 Hz,
0
0
0
0
4.11. 6-Amino-5-cyano-2-methyl-4-phenyl-3-pyridine-
carboxylic acid i-propyl ester (50)
J4 –6 ¼ 1:9 Hz, 1H, H60), 7.00 (m, 2H, H30, H50), 5.36 (s,
2H, NH2), 3.94 (q, J ¼ 7:2 Hz, 2H, CO2CH2CH3), 3.82
(s, 3H, OCH3), 2.54 [s, 3H, CH3C(2)], 0.85 (t, 3H,
CO2CH2CH3); 13C NMR (CDCl3, 50.2 MHz) d 166.1
(C@O), 161.2 (C6), 158.7 (C2), 156.0 (C20), 151.8 (C4),
130.9, 129.3 (C40, C60), 125.1 (C10), 120.6 (C50), 120.1
(C3), 116.0 (C@N), 111.0 (C30), 90.4 (C5), 61.0
(OCH2CH3), 55.6 (OCH3), 23.9 [CH3C(2)], 13.4
(CH3CH2O); MS (APCIþ) m=z 312 [(Mþ1)þ, 100], 240
(8), 645 [(2 · MþNa)þ, 15]. Anal. Calcd for
C17H17N3O3: C, 65.58; H, 5.50; N, 13.50. Found: C,
65.29, H, 5.72, N, 13.43.
0
0
This compound was prepared following the general
method for the synthesis of 6-amino-5-cyano-pyridines.
To a stirred solution of ammonium acetate (2.58 g,
33.52 mmol) in acetic acid (40 mL), pyran 44 (1.00 g,
3.35 mmol) was added. After the reaction was complete
and following the work-up, the resultant crude was
purified to provide compound 50 (395 mg, 40%): mp
222–224 ꢁC; IR (KBr) m 3393, 3328, 3204, 2978, 2927,
2217, 1713, 1663, 1632, 1561, 1282, 1184, 1110 cmꢁ1; 1H
(CDCl3, 200 MHz) d 7.55–7.42 (m, 3H, 2 · H20, H40),
7.37–7.27 (m, 2H, 2 · H30), 5.39 (s, 2H, NH2), 4.85 [h,
J ¼ 6:2 Hz, 1H, CO2CH(CH3)2], 2.50 [s, 3H, CH3C(2)],
0.91 [d, 6H, CO2CH(CH3)2]; 13C NMR (CDCl3,
50.2 MHz) d 166.8 (C@O), 160.4 (C6), 158.9 (C2), 153.6
(C4), 135.7 (C10), 129.4 (C40), 128.6, 128.0 (2 · C20,
2 · C30), 120.6 (C3), 115.9 (CBN), 89.0 (C5), 69.1
[OCH(CH3)2], 23.4 [CH3C(2)], 21.1 [(CH3)2CHO]; MS
(APIESþ) m=z 296 [(Mþ1)þ, 100], 318 [(MþNa)þ, 15],
4.14. 5-Amino-4-cyano-2-(4-methylphenyl)-1,3-oxazole
(62)
To a solution of aminopropanedinitrile p-toluenesulfo-
nate (AMNT) (4.68 g, 18.5 mmol) in dry 1-methyl-2-
pyrrolidinone (40 mL) at rt p-toluyl chloride (3.74 mL,
28 mmol) was added. The mixture was stirred for 74 h,

2210
J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
the solvent evaporated and the crude was dissolved in
ethyl acetate, washed with an 10% aqueous solution of
NaHCO3, dried with anhydrous Na2SO4 and concen-
trated in vacuum. The resultant crude was submitted to
chromatography with dichloromethane/ethyl acetate (8:
2) to give compound 62 (2.332 g, 67%): mp 255–260 ꢁC;
IR (KBr) m 3300, 3140, 2200, 1640, 1595, 1485,
pension of AlCl3 (198 mg, 1.48 mmol) in dry 1,2-
dichloroethane (9 mL) at rt under argon, pyran 43
(300 mg, 0.87 mmol) and cyclohexanone (154 lL,
1.48 mmol) were added. After the reaction was complete
and following the work-up, the crude was purified to
afford product 12 (319 mg, 86%): mp 77–79 ꢁC; IR (BrK)
m 3420, 3393, 2933, 1706, 1642, 1573, 1513, 1449, 1377,
1
1420 cmꢁ1; H NMR (DMSO-d6 300 MHz, 75 ꢁC) d 7.8
1266, 1224, 1141, 1095, 1060, 1027 cmꢁ1 1H NMR
;
(s, 2H, NH2), 7.6 (d, J ¼ 8:1 Hz, 2H), 7.3 (d, J ¼ 8:1 Hz,
2H), 2.3 (s, 3H, CH3); MS (70 eV) m=z (70 eV) 199 [M]þ
(100), 171 [MꢁCO]þ (39), 156 (58), 155 [Mꢁ(NH2CO)]þ
(7), 119 (71), 118 (79), 117 [p-CH3PhCN]þ (9), 103 (17),
91 [p-CH3Ph]þ (27), 77 (17), 65 (17), 54 [(MꢁCO)-p-
CH3PhCN]þ (1), 44 [NH2CO]þ (10). Anal. Calcd for
C11H9N3O: C, 66.32; H, 4.55; N, 21.09. Found: C, 66.60;
H, 4.82; N, 21.37.
(CDCl3, 200 MHz) d 6.86–6.71 (m, 3H, C6H3), 4.80 (s,
1H, H4), 4.17 (q, J ¼ 7:2 Hz, 2H, CO2CH2CH3), 4.07 (s,
2H, NH2), 3.80, 3.78 (2s, 6H, 2 · OCH3), 2.73 (m, 2H,
H9), 2.43 [s, 3H, CH3C(2)], 2.26 (m, 2H, H6), 1.79 (m,
4H, H7, H8), 1.26 (t, 3H, CO2CH2CH3); 13C NMR
(CDCl3, 50.2 MHz) d 167.1 (C@O), 159.3 (C9a), 154.4
(C2), 154.0 (C10a), 150.6 (C5), 149.3, 148.2 (C30, C40),
136.6 (C10), 120.8 (C60), 113.7 (C5a), 111.7 (C50), 111.1
(C20), 106.9 (C3), 99.8 (C4a), 60.4 (OCH2CH3), 56.0,
55.9 (2 · OCH3), 38.3 (C4), 32.6 (C9), 23.0, 22.7, 22.5
(C6, C7, C8), 19.8 [CH3C(2)], 14.4 (CH3CH2O); MS
(APIESþ) m=z 425 [(Mþ1)þ, 100], 871 [(2 · MþNa)þ,
30]. Anal. Calcd for C24H28N2O5: C, 67.91; H, 6.65; N,
6.60. Found: C, 67.75; H, 6.94; N, 6.90.
OMe
4'
OMe
NH2
3'
2'
1'
6
3
4a
5a
RO2C
H3C
4
11a
10a
10a
10
5
8
7
N
N
9a
9
O
N
8a
9
2
9a
8
11-13
4.17. 5-Amino-4,6,7,8,9,10-hexahydro-4-(3,4-dimethoxy-
phenyl)-2-methyl-cyclohepta[b]pyrano[3,2-e]pyridine-3-
carboxylic acid ethyl ester (13)
4.15. 5-Amino-4,6,7,8-tetrahydro-4-(3,4-dimethoxyphen-
yl)-2-methyl-cyclopenta[b]pyrano[3,2-e]pyridine-3-carb-
oxylic acid ethyl ester (11)
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (139 mg, 1.05 mmol) in dry 1,2-
dichloroethane (9 mL) at rt under argon, pyran 43
(300 mg, 0.87 mmol) and cycloheptanone (123 lL,
1.05 mmol) were added. After the reaction was complete
and following the work-up, the resultant crude was
purified to give compound 13 (205 mg, 54%): mp 180–
183 ꢁC; IR (BrK) m 3440, 3401, 2924, 1710, 1643, 1568,
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (263 mg, 1.97 mmol) in dry 1,2-
dichloroethane (12 mL) at rt under argon, pyran 43
(400 mg, 1.16 mmol) and cyclopentanone (175 lL,
1,97 mmol) were added. After the reaction was complete
and following the work-up, the resultant crude was
purified to produce compound 11 (285 mg, 64%): mp
72–73 ꢁC; IR (BrK) m 3467, 1685, 1644, 1513, 1282, 1274,
1513, 1455, 1263, 1229, 1213, 1076 cmꢁ1 1H NMR
;
(CDCl3, 200 MHz) d 6.89–6.72 (m, 3H, C6H3), 4.81 (s,
1H, H4), 4.14 (q, J ¼ 7:2 Hz, 2H, CO2CH2CH3), 4.09 (s,
2H, NH2), 3.82, 3.78 (2s, 6H, 2 · OCH3), 2.89 (m, 2H,
H10), 2.46 [s, 3H, CH3C(2)], 2.44 (m, 2H, H6), 1.80 (m,
2H, H9), 1.67 (m, 4H, H7, H8), 1.28 (t, 3H,
CO2CH2CH3); 13C NMR (CDCl3, 50.2 MHz) d 167.1
(C@O), 160.6 (C10a), 159.2 (C2), 153.9 (C11a), 149.7
(C5), 149.3, 148.2 (C30, C40), 136.5 (C10), 120.8 (C60),
119.1 (C5a), 111.6 (C50), 111.0 (C20), 106.9 (C3), 100.5
(C4a), 60.4 (OCH2CH3), 56.0, 55.9 (2 · OCH3), 38.7,
38.4 (C10, C4), 32.2 (C9), 26.9 (C7), 26.2 (C8), 25.8 (C6),
19.7 [CH3C(2)], 14.4 (CH3CH2O); MS (APIESþ) m=z
439 [(Mþ1)þ, 100], 301 (5). Anal. Calcd for
C25H30N2O5ÆH2O: C, 65,77; H, 7,06; N, 6.14. Found: C,
66.01; H, 7,04; N, 6.47.
1
1212, 1072 cmꢁ1; H NMR (CDCl3, 200 MHz) d 6.88–
6.71 (m, 3H, C6H3), 4.83 (s, 1H, H4), 4.14 (q,
J ¼ 7:2 Hz, 2H, CO2CH2CH3), 4.01 (s, 2H, NH2), 3.81,
3.78 (2s, 6H, 2 · OCH3), 2.88 (m, 2H, H8), 2.59 (m, 2H,
H6), 2.43 [s, 3H, CH3C(2)], 2.11 (m, 2H, H7), 1.27 (t,
3H, CO2CH2CH3); 13C NMR (CDCl3, 50.2 MHz) d
167.0 (C@O), 162.2 (C8a), 159.2 (C9a), 156.6 (C2), 149.4
(C5), 148.6, 148.3 (C30, C40), 136.7 (C10), 120.7 (C60),
118.2 (C5a), 111.9 (C50), 111.2 (C20), 106.9 (C3), 100.0
(C4a), 60.2 (OCH2CH3), 56.0, 55.9 (2 · OCH3), 38.2
(C4), 34.2 (C8), 27.0 (C6), 22.3 (C7), 19.6 [CH3C(2)],
14.3 (CH3CH2O); MS (APIESþ) m=z 411 [(Mþ1)þ,
100], 433 [(MþNa)þ, 10], 843 [(2 · MþNa)þ, 20]. Anal.
Calcd for C23H26N2O5: C, 67.30; H, 6.38; N, 6.82.
Found: C, 67.04; H, 6.60; N, 6.75.
4'
X
3'
2'
4.16. 5-Amino-6,7,8,9-tetrahydro-4-(3,4-dimethoxyphen-
yl)-2-methyl-4H-pyrano[2,3-b]quinolꢀꢃne-3-carboxylic acid
ethyl ester (12)
Y
NH2
1'
6
3
4a
5a
RO2C
H3C
7
8
12a
11a
11
4
11a
10a
10
8
9
5
9
N
N
10
N
N
9a
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
2
10a
9
15-29

J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
2211
4.18. 5-Amino-6,7,8,9-tetrahydro-4-(3-methoxyphenyl)-2-
methyl-benzo[b][1,8]naphthyridine-3-carboxylic acid
ethyl ester (15)
pension of AlCl3 (167 mg, 1.25 mmol) in dry 1,2-
dichloroethane (5 mL) at rt under argon, pyridine 47
(150 mg, 0.50 mmol) and cyclohexanone (130 lL,
1.25 mmol) were added. After the reaction was complete
and following the work-up, the resultant crude was
purified to afford compound 17 (52 mg, 28%): mp 110–
112 ꢁC; IR (KBr) m 3414, 2927, 1725, 1626, 1570, 1544,
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (340 mg, 2.55 mmol) in dry 1,2-
dichloroethane (9 mL) at rt under argon, pyridine 45
(265 mg, 0.85 mmol) and cyclohexanone (221 lL,
2.13 mmol) were added. After the reaction was complete
and following the work-up, the crude was purified to
give compound 15 (241 mg, 73%): mp 152–154 ꢁC; IR
(KBr) m 3446, 2934, 1725, 1637, 1570, 1546, 1432, 1288,
1
1509, 1223 cmꢁ1; H NMR (CDCl3, 200 MHz) d 7.42–
7.13 (2m, 4H, C6H4), 4.21 (s, 2H, NH2), 4.01 (q,
J ¼ 7:1 Hz, 2H, CO2CH2CH3), 3.04 (m, 2H, H9), 2.68
[s, 3H, CH3C(2)], 2.35 (m, 2H, H6), 1.89 (m, 4H, H7,
H8), 1.01 (s, 3H, CO2CH2CH3); 13C NMR (CDCl3,
0
50.2 MHz) d 168.1 (C@O), 165.6, 160.6 (JC4 –F ¼ 251 Hz,
1248 cmꢁ1
;
1H NMR (CDCl3, 200 MHz) d 7.41 (dd,
C40), 162.4 (C9a), 156.3 (C2), 154.8 (C10a), 148.9 (C5),
J ¼ 7:6 Hz, 7.9 Hz, 1H, H50), 7.04–6.90 (m, 3H, H20,
H30, H60), 4.61 (s, 2H, NH2), 4.02 (q, J ¼ 7:5 Hz, 2H,
CO2CH2CH3), 3.82 (s, 3H, OCH3), 3.10 (m, 2H, H9),
2.69 [s, 3H, CH3C(2)], 2.38 (m, 2H, H6), 1.89 (m, 4H,
H7, H8), 0.99 (s, 3H, CO2CH2CH3); 13C NMR (CDCl3,
50.2 MHz) d 168.0 (C@O), 161.6 (C9a), 159.5 (C30),
156.4 (C2), 154.0 (C10a), 149.5 (C5), 144.4 (C4), 138.2
(C10), 129.9 (C50), 126.8 (C3), 121.0 (C60), 115.0 (C40),
114.1 (C20), 111.5 (C5a), 106.1 (C4a), 61.2 (OCH2CH3),
55.3 (OCH3), 33.7 (C9), 23.5, 23.4 [C6, CH3C(2)], 22.5,
22.3 (C7, C8), 13.7 (CH3CH2O); MS (APIESþ) m=z 392
[(Mþ1)þ, 100], 805 [(2 · MþNa)þ, 30]. Anal. Calcd for
C23H25N3O3: C, 70.57; H, 6.44; N, 10.73. Found: C,
70.82; H, 6.64; N, 10.72.
143.4 (C4), 133.3 (C10), 131.1, 130.9 (JC2 –F ¼ 8 Hz,
0
2 · C20), 127.4 (C3), 116.1, 115.6 (JC3 –F ¼ 22 Hz,
0
2 · C30), 111.8 (C5a), 106.5 (C4a), 61.3 (OCH2CH3),
34.2 (C9), 23.7 (C6), 23.5 [CH3C(2)], 22.7, 22.5 (C7, C8),
13.8 (CH3CH2O); MS (APIESþ) m=z 380 [(Mþ1)þ,
100], 781 [(2 · MþNa)þ, 30]. Anal. Calcd for
C18H20N2O5Æ2H2O: C, 63.60; H, 6.31; N, 10.12. Found:
C, 63.34; H, 6.66; N, 10.51.
4.21. 5-Amino-4-(4-chlorophenyl)-6,7,8,9-tetrahydro-2-
methyl-benzo[b][1,8]naphthyridine-3-carboxylic acid
ethyl ester (18)
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
4.19. 5-Amino-6,7,8,9-tetrahydro-4-(3,4-dimethoxyphen-
yl)-2-methyl-benzo[b][1,8]naphthyridine-3-carboxylic
acid ethyl ester (16)
pension of AlCl3 (116 mg, 0.87 mmol) in dry 1,2-
dichloroethane (4 mL) at rt under argon, pyridine 48
(110 mg, 0.35 mmol) and cyclohexanone (90 lL,
0.87 mmol) were added. After the reaction was complete
and following the work-up, the resultant crude was
purified to produce compound 18 (122 mg, 88%): mp
119–122 ꢁC; IR (KBr) m 3482, 3413, 2937, 2862, 1727,
1632, 1571, 1540, 1493, 1314, 1269, 1219 cmꢁ1; 1H NMR
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (113 mg, 0.85 mmol) in dry 1,2-dichlo-
roethane (4 mL) at rt under argon, pyridine 46 (116 mg,
0.34 mmol) and cyclohexanone (93 lL, 1.00 mmol) were
added. After the reaction was complete and following the
work-up, the crude was purified to give product 16
(39 mg, 33%): mp 107–110 ꢁC; IR (KBr) m 3460, 3413,
2933, 1726, 1617, 1569, 1544, 1514, 1462, 1313,
0
0
(CDCl3, 200 MHz) d 7.47 (d, J2 –3 ¼ 8:6 Hz, 2H,
2 · H20), 7.33 (d, J2 –3 ¼ 8:6 Hz, 2H, 2 · H30), 4.22 (s, 2H,
NH2), 4.01 (q, J ¼ 7:1 Hz, 2H, CO2CH2CH3), 3.03 (m,
2H, H9), 2.68 [s, 3H, CH3C(2)], 2.35 (m, 2H, H6), 1.88
(m, 4H, H7, H8), 1.00 (s, 3H, CO2CH2CH3); 13C NMR
(CDCl3, 50.2 MHz) d 168.0 (C@O), 162.5 (C9a), 156.3
(C2), 154.6 (C10a), 148.8 (C5), 143.2 (C4), 135.8, 135.5
(C10, C40), 130.4 (2 · C20), 128.9 (2 · C30), 127.1 (C3),
111.8 (C5a), 106.3 (C4a), 61.4 (OCH2CH3), 34.2 (C9),
23.7 (C6), 23.5 [CH3C(2)], 22.7, 22.5 (C7, C8), 13.8
(CH3CH2O); MS (APIESþ) m=z 396 [(Mþ1)þ, 100], 813
[(2 · MþNa)þ, 15]. Anal. Calcd for C22H22ClN3O2: C,
66.81; H, 5.61; N, 10.63. Found: C, 66.58; H, 5.90; N,
10.35.
0
0
1
1252 cmꢁ1; H (CDCl3, 200 MHz) d 6.95–6.87 (m, 3H,
C6H3), 4.61 (s, 2H, NH2), 4.04 (q, J ¼ 7:1 Hz, 2H,
CO2CH2CH3), 3.95, 3.87 (2s, 6H, 2 · OCH3), 3.10 (m,
2H, H9), 2.69 [s, 3H, CH3C(2)], 2.39 (m, 2H, H6), 1.90
(m, 4H, H7, H8), 1.03 (s, 3H, CO2CH2CH3); 13C NMR
(CDCl3, 50.2 MHz) 168.4 (C@O), 161.9 (C9a), 156.3
(C2), 154.4 (C10a), 149.6, 149.3, 149.0 (C5, C40, C30),
144.3 (C4), 129.2 (C10), 127.4 (C3), 121.3 (C60), 112.1
(C50), 111.4 (C5a), 111.0 (C20), 106.6 (C4a), 61.3
(OCH2CH3), 56.1, 56.0 (2 · OCH3), 34.0 (C9), 23.6 (C6),
23.5 [CH3C(2)], 22.6, 22.5 (C8, C7), 13.9 (CH3CH2O);
MS (APIESþ) m=z 422 [(Mþ1)þ, 100], 865
[(2 · MþNa)þ, 20]. Anal. Calcd for C24H27N3O4ÆH2O: C,
65.59; H, 6.65; N, 9.56. Found: C, 65.54; H, 6.79; N, 9.49.
4.22. 5-Amino-6,7,8,9-tetrahydro-2-methyl-4-(3-nitrophen-
yl)-benzo[b][1,8]naphthyridine-3-carboxylic acid ethyl
ester (19)
This compound was obtained following the general
€
4.20. 5-Amino-4-(4-fluorophenyl)-6,7,8,9-tetrahydro-2-
methyl-benzo[b][1,8]naphthyridine-3-carboxylic acid
ethyl ester (17)
method for the Friedlander reaction. To a stirred solu-
tion of AlCl3 (116 mg, 0.87 mmol) in dry 1,2-dichloro-
ethane (3 mL) at rt under argon, pyridine 49 (81 mg,
0.25 mmol) and cyclohexanone (90 lL, 0.87 mmol) were
added. After the reaction was complete and following
the work-up, the resultant crude was purified to afford
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-

2212
J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
compound 19 (55 mg, 54%): mp 214–217 ꢁC; IR (KBr) m
3460, 3420, 2924, 2852, 1724, 1630, 1570, 1531, 1462,
H20, H30, H60, H20), 7.05–6.90 (m, 3H, C6H3), 4.53 (s,
2H, NH2), 4.01 (q, J ¼ 7:2 Hz, 2H, CO2CH2CH3), 3.82
(s, 3H, OCH3), 3.18 (m, 2H, H10), 2.69 [s, 3H,
CH3C(2)], 2.60 (m, 2H, H6), 1.81 (m, 4H, H7, H9), 1.60
(m, 2H, H8), 0.98 (t, 3H, CO2CH2CH3); 13C NMR
(CDCl3, 50.2 MHz) d 168.2 (C@O), 167.9 (C10a), 159.6
(C30), 156.4 (C2), 154.3 (C11a), 148.4 (C5), 144.6 (C4),
138.4 (C10), 130.0 (C50), 127.6 (C3), 121.3 (C60), 117.1
(C5a), 115.2 (C40), 114.4 (C20), 107.1 (C4a), 61.4
(OCH2CH3), 55.5 (OCH3) 39.5 (C10), 32.0 (C9), 27.1
(C7), 26.5, 26.1 (C6, C8), 23.6 [CH3C(2)], 13.9
(CH3CH2O); MS (APCIþ) m=z 406 [(Mþ1)þ, 100], 390
(6), 378 (85), 362 (25). Anal. Calcd for
C24H27N3O3ÆH2O: C, 68.07; H, 6.90; N, 9.92. Found: C,
68.27; H, 6.60; N, 9.68.
1351, 1315, 1269, 1222 cmꢁ1
;
1H NMR (CDCl3,
200 MHz) d (CDCl3, 300 MHz) d 8.40–8.30 (m, 2H,
H20, H40), 7.80–7.68 (m, 2H, H50, H60), 4.06 (s, 2H,
NH2), 4.02 (q, J ¼ 7:1 Hz, 2H, CO2CH2CH3), 3.08 (m,
2H, H9), 2.72 [s, 3H, CH3C(2)], 2.38 (m, 2H, H6), 1.91
(m, 4H, H7, H8), 1.11 (t, 3H, CO2CH2CH3); 13C
NMR (CDCl3, 50.2 MHz) d 169.0 (C@O), 162.8 (C9a),
156.5 (C2), 154.8 (C10a), 150.2 (C5), 147.8 (C30), 142.7
(C4), 139.0 (C10), 134.5 (C60), 129.3 (C50), 123.6 (C20,
C40), 127.1 (C3), 111.8 (C5a), 106.3 (C4a), 61.2
(OCH2CH3), 33.7 (C9), 23.3, 23.2 [CH3C(2), C6)],
22.1 (C7, C8), 13.4 (CH3CH2O); MS (APIESþ) m=z 407
[(Mþ1)þ, 100]. Anal. Calcd for C22H22N4O4: C,
65.01;H, 5.46; N, 13.78. Found: C, 64.73; H, 5.62; N,
13.58.
4.25. 5-Amino-7,8,9,10-tetrahydro-4-(2-methoxyphenyl)-
2-methyl-6H-cyclohepta[b][1,8]naphthyridine-3-carbox-
ylic acid ethyl ester (22)
4.23. 5-Amino-7,8,9,10-tetrahydro-2-methyl-4-(4-methyl-
phenyl)-6H-cyclohepta[b][1,8]naphthyridine-3-carboxylic
acid ethyl ester (20)
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-
This compound was prepared following the general
€
pension of AlCl3 (320 mg, 2.40 mmol) in dry 1,2-
dichloroethane (10 mL) at rt under argon, pyridine 52
(300 mg, 0.96 mmol) and cycloheptanone (283 lL,
2.40 mmol) were added. After the reaction was complete
and following the work-up, the resultant crude was
purified to give compound 22 (248 mg, 64%): mp 87–
90 ꢁC; IR (KBr) m 3478, 3400, 2923, 1724, 1630, 1567,
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (91 mg, 0.68 mmol) in dry 1,2-dichloro-
ethane (4 mL) at rt under argon, pyridine 51 (100 mg,
0.34 mmol) and cycloheptanone (80 lL, 0.68 mmol) were
added. After the reaction was complete and following
the work-up, the resultant crude was purified to produce
compound 20 (102 mg, 77%): mp 158–161 ꢁC; IR (KBr)
m 3469, 3420, 2924, 1725, 1631, 1570, 1543, 1226,
1543, 1495, 1224, 1086 cmꢁ1
;
1H NMR (CDCl3,
200 MHz) d 7.51 (ddd, J4 –6 ¼ 1:8 Hz, 1H, H40), 7.23
0
0
1
1092 cmꢁ1; H NMR (CDCl3, 200 MHz) d 7.28 (s, 4H,
(dd, J5 –6 ¼ 7:5 Hz, J4 –6 ¼ 1:8 Hz, 1H, H60), 7.03 (m,
2H, H30, H50), 4.53 (s, 2H, NH2), 3.96 (q, J ¼ 7:2 Hz,
2H, CO2CH2CH3), 3.73 (s, 3H, OCH3), 3.20 (m, 2H,
H10), 2.70 [(t, J ¼ 7:2 Hz, 3H, CH3C(2)], 2.63 (m, 2H,
H6), 1.82 (m, 4H, H7, H9), 1.60 (m, 2H, H8), 1.00 (t,
3H, CO2CH2CH3); 13C NMR (CDCl3, 50.2 MHz) d
168.3 (C@O), 167.8 (C10a), 157.1 (C20), 156.5 (C2),
154.5 (C11a), 148.5 (C5), 142.2 (C4), 131.1, 130.2 (C40,
C60), 127.6 (C3), 126.4 (C10), 120.8 (C50), 116.8 (C5a),
111.5 (C30), 108.2 (C4a), 61.2 (OCH2CH3), 56.0 (OCH3)
39.7 (C10), 32.0 (C9), 27.1 (C7), 26.5 (C8), 26.1 (C6),
23.7 [CH3C(2)], 13.9 (CH3CH2O); MS (APCIþ) m=z
406 [(Mþ1)þ, 100], 334 (2). Anal. Calcd for
C24H27N3O2Æ2H2O: C, 68.07; H, 6.90; N, 9.92. Found:
C, 68.28; H, 6.75; N, 9.83.
0
0
0
0
C6H4), 4.40 (s, 2H, NH2), 3.99 (q, J ¼ 7:2 Hz, 2H,
CO2CH2CH3), 3.18 (m, 2H, H10), 2.69 [s, 3H,
CH3C(2)], 2.58 (m, 2H, H6), 2.43 [s, 3H, CH3C(40)], 1.81
(m, 4H, H7, H9), 1.60 (m, 2H, H8), 0.98 (t, 3H,
CO2CH2CH3); 13C NMR (CDCl3, 50.2 MHz) d 168.3
(C@O), 168.0 (C10a), 156.0 (C2), 154.5 (C11a), 148.0
(C5), 144.8 (C4), 139.1 (C40), 134.2 (C10), 129.3 (2 · C30),
128.8 (2 · C20), 127.7 (C3), 116.9 (C5a), 107.4 (C4a), 61.2
(OCH2CH3), 39.7 (C10), 31.8 (C9), 27.0, 26.4, 26.0 (C7,
C8, C9), 23.4 [CH3C(2)], 21.3 [CH3C(40)], 13.7
(CH3CH2O); MS (APCIþ) m=z 390 [(Mþ1)þ, 100], 362
(2). Anal. Calcd for C24H27N3O2Æ3H2O: C, 64.99; H,
7.50; N, 9.47. Found: C, 65.16; H, 7.76; N, 9.56.
4.24. 5-Amino-7,8,9,10-tetrahydro-4-(3-methoxyphenyl)-
2-methyl-6H-cyclohepta[b][1,8]naphthyridine-3-carbox-
ylic acid ethyl ester (21)
4.26. 5-Amino-7,8,9,10-tetrahydro-4-(3,4-dimethoxyphen-
yl)-2-methyl-6H-cyclohepta[b][1,8]naphthyridine-3-car-
boxylic acid ethyl ester (23)
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (160 mg, 1.20 mmol) in dry 1,2-
dichloroethane (5 mL) at rt under argon, pyridine 45
(150 mg, 0.48 mmol) and cycloheptanone (142 lL,
1.20 mmol) were added. After the reaction was complete
and following the work-up, the crude was purified to
afford compound 21 (103 mg, 53%): mp 125–128 ꢁC; IR
(KBr) m 3436, 2924, 1724, 1631, 1568, 1543, 1286, 1224,
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (66 mg, 0.49 mmol) in dry 1,2-dichloro-
ethane (3 mL) at rt under argon, pyridine 46 (100 mg,
0.29 mmol) and cycloheptanone (59 lL, 0.50 mmol) were
added. After the reaction was complete and following
the work-up, the resultant crude was purified to produce
compound 23 (96 mg, 76%): mp 105–108 ꢁC; IR (KBr) m
3476, 3375, 2923, 1724, 1628, 1567, 1513, 1253, 1234,
1093 cmꢁ1 1H NMR (CDCl3, 200 MHz) d 7.43–7.38
;
(dd, J ¼ 7:8 Hz, 7.9 Hz, 1H, H50), 7.02–6.86 (m, 3H,

J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
2213
1140 cmꢁ1; 1H NMR (CDCl3, 200 MHz) d 6.95–6.89 (m,
3H, C6H3), 4.58 (s, 2H, NH2), 4,03 (q, J ¼ 7:1 Hz, 2H,
CO2CH2CH3), 3.95/3.82 (2s, 6H, 2 · OCH3), 3.20 (m,
2H, H10), 2.68 [s, 3H, CH3C(2)], 2.61 (m, 2H, H6), 1.82
(m, 4H, H7, H9), 1.61 (m, 2H, H8), 1.03 (t, 3H,
CO2CH2CH3); 13C NMR (CDCl3, 75.4 MHz) d 168.2
(C@O), 167.8 (C10a) 155.8 (C2), 154.3 (C11a), 149.4,
148.7, 147.9 (C40, C30, C5), 144.3 (C4), 129.0 (C10), 128.8
(C3), 121.2 (C60), 116.7 (C5a), 112.0 (C50), 110.7 (C20),
107.2 (C4a), 61.1 (OCH2CH3), 55.9, 55.8 (2 · OCH3),
39.5 (C10), 31.7 (C9), 26.8 (C7), 26.2 (C8), 25.8 (C6),
23.2 [CH3C(2)], 13.7 (CH3CH2O); MS (APIESþ) m=z
436 [(Mþ1)þ, 100]. Anal. Calcd for C25H29N3O4Æ2H2O:
C, 63.68; H, 7.05; N, 8.91. Found: C, 63.90; H, 6.96; N,
8.71.
J2 –3 ¼ 8:6 Hz, 2H, 2 · H30), 4.30 (s, 2H, NH2), 4.01 (q,
J ¼ 7:2 Hz, 2H, CO2CH2CH3), 3.16 (m, 2H, H10), 2.68
[s, 3H, CH3C(2)], 2.58 (m, 2H, H6), 1.80 (m, 4H, H7,
H9), 1.59 (m, 2H, H8), 1.01 (t, 3H, CO2CH2CH3);
13C NMR (CDCl3, 50.2 MHz) d 168.4 (C@O), 168.0
(C10a), 156.1 (C2), 154.4 (C11a), 147.6 (C5), 143.2
(C4), 135.6, 135.4 (C10, C40), 130.4 (2 · C20), 128.8
(2 · C30), 127.6 (C3), 117.2 (C5a), 107.1 (C4a), 61.4
(OCH2CH3), 39.7 (C10), 31.8 (C9), 27.0 (C7), 26.3 (C8),
26.0 (C6), 23.4 [CH3C(2)], 13.7 (CH3CH2O); MS
(APIESþ) m=z 410 [(Mþ1)þ, 100], 432 [(MþNa)þ,
60], 382 (90). Anal. Calcd for C23H24ClN3O2Æ2H2O: C,
61.95; H, 6.33; N, 9.42. Found: C, 62.05; H, 6.34; N,
9.18.
0
0
4.29. 5-Amino-7,8,9,10-tetrahydro-2-methyl-4-(3-nitro-
phenyl)-6H-cyclohepta[b][1,8]naphthyridine-3-carboxylic
acid ethyl ester (26)
4.27. 5-Amino-4-(4-fluorophenyl)-7,8,9,10-tetrahydro-2-
methyl-6H-cyclohepta[b][1,8]naphthyridine-3-carboxylic
acid ethyl ester (24)
This compound was prepared following the general
€
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (57 mg, 0.43 mmol) in dry 1,2-dichlo-
roethane (3 mL) at rt under argon, pyridine 49 (70 mg,
0.21 mmol) and cycloheptanone (51 lL, 0.43 mmol) were
added. After the reaction was complete and following
the work-up, the resultant crude was purified to afford
compound 26 (78 mg, 86%): mp 182–185 ꢁC; IR (KBr) m
3519, 3420, 2924, 2853, 1724, 1623, 1566, 1531, 1349,
1269 cmꢁ1; 1H NMR (CDCl3, 200 MHz) d 8.38–8.28 (m,
2H, H20, H40), 7.80–7.64 (m, 2H, H50, H60), 4.07 (s, 2H,
NH2), 4.00 (q, J ¼ 7:1 Hz, 2H, CO2CH2CH3), 3.18 (m,
2H, H10), 2.70 [s, 3H, CH3C(2)], 2.57 (m, 2H, H6), 1.81
(m, 4H, H7, H9), 1.60 (m, 2H, H8), 0.99 (t, 3H,
CO2CH2CH3); 13C NMR (CDCl3, 50.2 MHz) d 169.0
(C@O), 167.7 (C10a), 156.2 (C2), 154.5 (C11a), 147.9
(C30), 146.9 (C5), 141.7 (C4), 139.2 (C10), 134.9 (C60),
129.6 (C50), 127.5 (C3), 124.0, 123.9 (C20, C40), 117.8
(C5a), 106.9 (C4a), 61.6 (OCH2CH3), 39.9 (C10), 31.8
(C9), 27.0 (C7), 26.3 (C8), 26.0 (C6), 23.6 [CH3C(2)],
13.8 (CH3CH2O); MS (APCIþ) m=z 421 [(Mþ1)þ, 100].
Anal. Calcd for C23H24N4O4: C, 65.70; H, 5.75; N,
13.32. Found: C, 65.45; H, 6.02; N, 13.34.
pension of AlCl3 (133 mg, 1.00 mmol) in dry 1,2-
dichloroethane (5 mL) at rt under argon, pyridine 47
(150 mg, 0.50 mmol) and cycloheptanone (118 lL,
1.00 mmol) were added. After the reaction was complete
and following the work-up, the resultant crude was
purified to afford compound 24 (138 mg, 70%): mp 165–
168 ꢁC; IR (KBr) m 3487, 3417, 2924, 1725, 1629, 1567,
1
1509, 1224 cmꢁ1; H NMR (CDCl3, 200 MHz) d 7.41–
7.14 (m, 4H, C6H4), 4.32 (s, 2H, NH2), 4.02 (q,
J ¼ 7:0 Hz, 2H, CO2CH2CH3), 3.17 (m, 2H, H10), 2.69
[s, 3H, CH3C(2)], 2.60 (m, 2H, H6), 1.81 (m, 4H, H7,
H9), 1.61 (m, 2H, H8), 1.02 (t, 3H, CO2CH2CH3); 13C
NMR (CDCl3, 50.2 MHz) d 168.4 (C@O), 168.2 (C10a),
165.6, 160.6 (JC4 –F ¼ 251 Hz, C40), 156.1 (C2), 154.6
0
(C11a), 147.8 (C5), 143.6 (C4), 133.3, 133.2
0
(JC1 –F ¼ 4 Hz, C10), 131.1, 131.0 (JC2 –F ¼ 8 Hz, 2 · C20),
0
0
128.0 (C3), 117.3 (C5a), 116.1, 115.6 (JC3 –F ¼ 22 Hz,
2 · C30), 107.4 (C4a), 61.5 (OCH2CH3), 39.8 (C10),
31.9 (C9), 27.1 (C7), 26.5 (C8), 26.1 (C6), 23.5
[CH3C(2)], 13.9 (CH3CH2O); MS (APCIþ) m=z 394
[(Mþ1)þ, 100], 366 (2). Anal. Calcd for
C23H24FN3O2ÆH2O: C, 67.12; H, 6.37; N, 10.22. Found:
C, 66.85; H, 6.21; N, 10.24.
4.30. 5-Amino-6,7,8,9,10,11-hexahydro-2-methyl-4-phenyl-
cycloocta[b][1,8]naphthyridine-3-carboxylic acid ethyl
ester (27)
4.28. 5-Amino-4-(4-chlorophenyl)-7,8,9,10-tetrahydro-2-
methyl-6H-cyclohepta[b][1,8]naphthyridine-3-carboxylic
acid ethyl ester (25)
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred
suspension of AlCl3 (96 mg, 0.72 mmol) in dry
1,2-dichloroethane (4 mL) at rt under argon, pyridine 53
(100 mg, 0.36 mmol) and cyclooctanone (91 mg,
0.72 mmol) were added. After the reaction was complete
and following work-up, the resultant crude was purified
to give product 27 (111 mg, 79%): mp 174–176 ꢁC; IR
(KBr) m 3485, 3414, 2923, 1724, 1629, 1566, 1542,
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred
suspension of AlCl3 (85 mg, 0.64 mmol) in dry 1,2-
dichloroethane (3 mL) at rt under argon, pyridine 48
(100 mg, 0.32 mmol) and cycloheptanone (74 lL,
0.64 mmol) were added. After the reaction was complete
and following the work-up, the resultant crude was
purified to afford compound 25 (97 mg, 75%): mp
96–99 ꢁC; IR (KBr) m 3496, 3401, 3242, 2890, 1718, 1651,
1442, 1325, 1274, 1078 cmꢁ1
;
1H NMR (CDCl3,
200 MHz) d 7.48–7.44 (m, 3H, H40, 2 · H20), 7.41–7.33
(m, 2H, 2 · H30), 4.43 (s, 2H, NH2), 3.95 (q,
J ¼ 7:2 Hz, 2H, CO2CH2CH3), 3.11 (m, 2H, H11), 2.72
(m, 2H, H6), 2.68 [s, 3H, CH3C(2)], 1.89 (m, 2H, H10),
1
1573, 1546, 1494, 1281, 1229 cmꢁ1; H NMR (CDCl3,
200 MHz) d 7.46 (d, J2 –3 ¼ 8:6 Hz, 2H, 2 · H20), 7.34 (d,
0
0

2214
J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
1.58 (m, 2H, H7), 1.47 (m, 2H, H9), 1.33 (m, 2H, H8),
0.92 (t, 3H, CO2CH2CH3); 13C NMR (CDCl3,
50.2 MHz) d 168.1 (C@O), 166.1 (C11a), 156.3 (C2),
154.7 (C12a), 148.7 (C5), 144.6 (C4), 137.1 (C10), 129.1
(C40), 128.9, 128.6 (2 · C20, 2 · C30), 127.4 (C3),
114.3 (C5a), 106.7 (C4a), 61.2 (OCH2CH3), 36.8 (C11),
30.9 (C10), 27.2 (C7), 26.5 (C9), 26.2 (C8), 24.6 (C6),
23.5 [CH3C(2)], 13.7 (CH3CH2O); MS (APIESþ) m=z
390 [(Mþ1)þ, 100]. Anal. Calcd for C24H27N3O2Æ2H2O:
C, 70.72; H, 7.18; N, 10.32. Found: C, 70.47; H, 7.05; N,
10.48.
(CDCl3, 50.2 MHz) d 167.7 (C@O), 162.1 (C9a), 156.2
(C2), 154.6 (C10a), 149.1 (C5), 144.2 (C4), 137.4 (C10),
129.1, 128.7 (2 · C20, 2 · C30, C40), 127.3 (C3), 111.5
(C5a), 106.5 (C4a), 69.0 [CO2CH(CH3)2], 34.2 (C9), 23.6
(C6), 23.4 [CH3C(2)], 22.7, 22.6 (C7, C8), 21.3
[CO2CH(CH3)2]; MS (APIESþ) m=z 376 [(Mþ1)þ, 100],
773 [(2 · MþNa)þ, 35]. Anal. Calcd for C23H25N3O2: C,
73.58; H, 6.71; N, 11.19. Found: C, 73.71; H, 7.00; N,
11.18.
NH2
3a 4a
4
5
7
N
O
9a
10a
6
9a
9
X
7
N
N
2
8a
4.31. 5-Amino-6,7,8,9,10,11-hexahydro-4-(4-methoxy-
phenyl)-2-methyl-cycloocta[b][1,8]naphthyridine-3-car-
boxylic acid ethyl ester (28)
8
N
7a
8a
8
30-40
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
4.33. 4-Amino-2-(4-chlorophenyl)-6,7-dihydro-5H-cyclo-
penta[b]oxazolo[4,5-e]pyridine (30)
pension of AlCl3 (171 mg, 1.28 mmol) in dry 1,2-
dichloroethane (7 mL) at rt under argon, pyridine 54
(200 mg, 0.64 mmol) and cyclooctanone (162 mg,
1.28 mmol) were added. After the reaction was complete
and following the work-up, the resultant crude was
purified to afford product 28 (191 mg, 71%): mp 169–
172 ꢁC; IR (KBr) m 3481, 3412, 2926, 1722, 1620, 1567,
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred
suspension of AlCl3 (0.18 g, 1.36 mmol) in dry chloro-
benzene (20 mL) at rt under argon, 1,3-oxazole 59
(0.20 g, 0.92 mmol) and cyclopentanone (122 lL,
1.37 mmol) were added. The reaction was complete
9 days later. After the work-up, the resultant crude was
purified to produce compound 30 (49.4 mg, 19%): mp
308–315 ꢁC; IR (KBr) m 3360, 3310, 3200, 2890,
1
1513, 1251, 1223 cmꢁ1; H NMR(CDCl3, 200 MHz) d
7.32 (d, J ¼ 8:9 Hz, 2H, 2 · H20), 6.99 (d, 2H, 2 · H30),
4.54 (s, 2H, NH2), 4.00 (q, J ¼ 7:2 Hz, 2H,
CO2CH2CH3), 3.85 (s, 3H, OCH3), 3.12 (m, 2H, H11),
2.73 (m, 2H, H6), 2.67 [s, 3H, CH3C(2)], 1.90 (m, 2H,
H10), 1.59 (m, 2H, H7), 1.47 (m, 2H, H9), 1.33 (m, 2H,
H8), 1.00 (t, 3H, CO2CH2CH3); 13C NMR (CDCl3,
50.2 MHz) d 168.4 (C@O), 166.5 (C11a), 160.0 (C40),
156.0 (C2), 155.1 (C12a), 148.4 (C5), 144.4 (C4), 130.2
(2 · C20), 129.0 (C10), 127.7 (C3), 114.1 (C5a), 114.0
(2 · C30), 107.2 (C4a), 61.2 (OCH2CH3), 55.3 (OCH3),
37.0 (C11), 30.9 (C10), 27.2 (C7), 26.5 (C9), 26.1 (C8),
24.6 (C6), 23.5 [CH3C(2)], 13.8 (CH3CH2O); MS
(APIESþ) m=z 420 [(Mþ1)þ, 100], 392 (6). Anal. Calcd
for C25H29N3O3: C, 71.58; H, 6.97; N, 10.02. Found: C,
71.31; H, 7.20; N, 10.15.
2840, 1630, 1610, 1470, 1330, 1240 cmꢁ1 1H NMR
;
(DMSO-d6 300 MHz) d 8.07 (d, J ¼ 8:7 Hz, 2H, H20,
H60), 7.64 (d, 2H, H30, H50), 6.60 (br s, 2H,
NH2), 2.83 (m, 2H, H7), 2.72 (m, 2H, H5), 2.05 (m,
2H, H6); MS (70 eV) m=z 287 [Mþ2]þ (40), 285 [M]þ
(100), 139 (15), 119 (31), 113 [p-Cl37Ph]þ (5), 111
[p-Cl35Ph]þ (15). Anal. Calcd for C15H12ClN3O: C,
63.05; H, 4.23; N, 14.71. Found: C, 62.95; H, 4.25; N,
14.58.
4.34. 4-Amino-6,7-dihydro-2-(4-methoxyphenyl)-5H-
cyclopenta[e]oxazolo[4,5-b]pyridine (31)
4.32. 5-Amino-6,7,8,9,10,11-hexahydro-2-methyl-4-phenyl-
cycloocta[b][1,8]naphthyridine-3-carboxylic acid i-propyl
ester (29)
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (0.37 g, 2.79 mmol) in dry 1,2-dichlo-
roethane (20 mL) at rt under argon, 1,3-oxazole 61
(0.30 g, 1.39 mmol) and cyclopentanone (247 lL,
2.79 mmol) were added. The reaction was complete
13 days later. After the work-up, the resultant crude was
purified to give compound 31 (160 mg, 35%): mp
277–285 ꢁC (dec); IR (KBr) m 3390, 3340, 3190, 2940,
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (226 mg, 1.69 mmol) in dry 1,2-
dichloroethane (7 mL) at rt under argon, pyridine 50
(200 mg, 0.68 mmol) and cyclohexanone (175 lL,
1.69 mmol) were added. After the reaction was complete
and following the work-up, the resultant crude was
purified to give product 29 (121 mg, 48%): mp 200–
201 ꢁC; IR (KBr) m 3480, 3436, 2978, 2933, 2862, 1720,
1628, 1568, 1543, 1496, 1442, 1372, 1314, 1274, 1225,
2860, 1650, 1620, 1500, 1260, 1180 cmꢁ1 1H NMR
;
(DMSO-d6, 60 ꢁC, 300 MHz) d 8.03 (d, J ¼ 8:6 Hz, 2H,
H20, H60), 7.13 (d, 2H, H30, H50), 6.26 (br s, 2H, NH2),
3.86 (s, 3H, OCH3), 2.85 (m, 2H, H7), 2.75 (m, 2H,
H5), 2.11 (m, 2H, H6); MS (70 eV) m=z 281 [M]þ (100),
280 (45), 266 (27), 238 (7), 140 (6), 119 (11), 92 (8), 77
(4), 65 (6). Anal. Calcd for C16H15N3O2Æ1H2O: C,
66.19; H, 5.56; N, 14.47. Found: C, 66.38; H2, 5.12; N,
14.69.
1
1103, 1076 cmꢁ1; H NMR (CDCl3, 200 MHz) d 7.51–
7.35 (m, 5H, C6H5), 4.87 [h, 1H, J ¼ 6:2 Hz,
CO2CH(CH3)2], 4.23 (s, 2H, NH2), 3.02 (m, 2H, H9),
2.68 [s, 3H, CH3C(2)], 2.33 (m, 2H, H6), 1.87 (m, 4H,
H7, H8), 0.96 [d, 6H, CO2CH(CH3)2]; 13C NMR

J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
2215
4.35. 4-Amino-6,7-dihydro-2-(4-methylphenyl)-5H-cyclo-
penta[e]oxazolo[5,4-b]pyridine (32)
was purified to give 34 (94.7 mg, 35%): mp 319–
326 ꢁC; IR (KBr) m 3400, 3320, 3205, 2920, 2850,
1650, 1610, 1590, 1515, 1340 cmꢁ1; MS (70 eV) m=z 310
[M]þ (100), 295 (20), 282 (60), 263 (23), 236 (18), 133 (6)
104 (6). Anal. Calcd for C16H14N4O3: C, 61.93; H,
4.55; N, 18.05. Found: C 62.15; H, 4.45; N, 17.97.
34ÆHCl: mp 304–305 ꢁC (MeOH; dec); IR (KBr) m
3410, 2944, 1659, 1601, 1520, 1446, 1345 cmꢁ1; MS
(70 eV) m=z 310 [MþꢁHCl] (100), 295 (8), 282 (31), 263
(11), 252 (3), 236 (9), 133 (7), 104 (8), 79 (9), 65
(7). Anal. Calcd for C16H14N4O3ÆHClÆ112H2O: C,
51.41; H, 4.85; N, 14.98. Found: C, 51.56; H, 4.78; N,
14.71.
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (0.53 g, 4 mmol) in dry 1,2-dichloro-
ethane (20 mL) at rt under argon, 1,3-oxazole 62 (0.40 g,
2 mmol) and cyclopentanone (355 lL, 4 mmol) were
added. The reaction was refluxed during 286 h. After the
work-up, the resultant crude was purified to afford
compound 32 (46.7 mg, 9%): mp 278–288 ꢁC; IR (KBr) m
3410, 3360, 3200, 2950, 2880, 1670, 1635, 1520, 1480,
1
1355, 1305 cmꢁ1; H NMR (DMSO-d6 60 ꢁC, 300 MHz)
d 7.98 (d, J ¼ 8:4 Hz, 2H, H20, H60), 7.38 (d, 2H, H30,
H50), 6.30 (s, 2H, NH2), 2.85 (m, 2H, H7), 2.76 (m, 2H,
H5), 2.40 (s, 3H, CH3), 2.08 (m, 2H, H6); MS (70 eV)
m=z 265 [M]þ (100), 147 (4), 132 (8), 119 (28), 91 [p-
CH3Ph]þ (15), 77 (6), 65 (18). Anal. Calcd for
C16H15N3O: C, 72.43; H, 5.70; N, 15.84. Found: C,
72.70; H, 6.00; N, 15.61.
4.38. 4-Amino-5,6,7,8-tetrahydro-2-(4-methoxyphenyl)-
oxazolo[5,4-b]quinoline (35)
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (0.19 g, 1.39 mmol) in dry 1,2-dichlo-
roethane (14 mL) at rt under argon, 1,3-oxazole 61
(0.20 g, 0.93 mmol) and cyclohexanone (145 lL,
1,4 mmol) were added. After 216 h the reaction was
complete. Following the work-up, the resultant crude
was purified to afford product 35 (316.7 mg, 52%): mp
270–276 ꢁC; IR (KBr) m 3370, 3300, 3190, 2900, 2860,
4.36. 4-Amino-2-(4-chlorophenyl)-5,6,7,8-tetrahydro-
oxazolo[5,4-b]quinoline (33)
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (0.18 g, 1.38 mmol) in dry chloroben-
zene (17 mL) at rt under argon, 1,3-oxazole 59 (0.20 g,
0.91 mmol) and cyclohexanone (142 lL, 1.37 mmol)
were added. After 24 h the reaction was complete. Fol-
lowing the work-up, the resultant crude was purified to
give compound 33 (111.6 mg, 41%): 305–313 ꢁC; IR
(KBr) m 3380, 3300, 3200, 2920, 2850, 1630, 1605, 1460,
1635, 1610, 1490, 1460, 1430, 1250 cmꢁ1 1H NMR
;
(CDCl3, 300 MHz) d 8.12 (d, J ¼ 8:9 Hz, 2H, H20, H60),
7.00 (d, 2H, H30, H50), 4.72 (br s, 2H, NH2), 2.92 (m,
2H, H8), 2.53 (m, 2H, H5), 1.91 (m, 4H, H6, H7); MS
(70 eV) m=z 295 [M]þ (100), 280 (17), 267 (26), 147 (7),
134 (13), 106 (9), 77 (9). Anal. Calcd for C17H17N3O2: C,
65.78; H, 6.33; N, 10.93. Found: C, 65.64; H, 6.40; N,
10.83. 35ÆHCl: mp 262–263 ꢁC (MeOH); IR (KBr) m
3455, 3286, 3193, 2945, 1659, 1623, 1603, 1583, 1555,
1499, 1449, 1424, 1247, 1178 cmꢁ1. Anal. Calcd for
C17H17N3O2ÆHClÆ2H2O: C, 55.51; H, 6.03; N, 11.42.
Found: C, 55.51; H, 6.25; N, 11.34.
1
1430, 1320, 1215 cmꢁ1; H NMR (CDCl3, 300 MHz) d
8.11 (d, J ¼ 8:5 Hz, 2H, H20, H60), 7.47 (d, 2H, H30,
H50), 4.75 (br s, 2H, NH2), 2.93 (m, 2H, H8), 2.52 (m,
2H, H5), 1.90 (m, 4H, H6, H7); MS (70 eV) m=z 301
[Mþ2] (36), 299 [M]þ (100), 284 (12), 271 (32), 139 (7),
113 [p-Cl37Ph]þ (2), 111 [p-Cl35Ph]þ (6), 106 (7), 79 (6).
Anal. Calcd for C16H14ClN3O: C, 64.11; H, 4.71; N,
14.02. Found: C, 64.40; H, 4.90; N, 14.31. 33ÆHCl: mp
287–288 ꢁC; IR (KBr) m 3294, 3131, 2934, 1658, 1628,
1484, 1447, 1427, 1089 cmꢁ1; MS (70 eV) m=z 301
[Mþ2ꢁHCl]þ (47), 299 [MꢁHCl]þ (100), 284 (19), 271
(48), 139 (7), 137 (4), 113 [p-Cl37Ph]þ (2), 111 [p-Cl35Ph]þ
(6), 106 (7), 79 (6), 66 (3). Anal. Calcd for
C16H14ClN3O.HCl: C, 57.15; H, 4.49; N, 12.49. Found:
C, 56.88; H, 4.58; N, 12.33.
4.39. 4-Amino-5,6,7,8-tetrahydro-2-(4-methylphenyl)-
oxazolo[5,4-b]quinoline (36)
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (0.54 g, 4 mmol) in dry 1,2-dichloro-
ethane (35 mL) at rt under argon, 1,3-oxazole 62 (0.40 g,
2 mmol) and cyclohexanone (416 lL, 4 mmol) were ad-
ded. After 192 h the reaction was complete. Following
the work-up, the resultant crude was purified to give
compound 36 (452.6 mg, 81%): 267–273 ꢁC; IR (KBr) m
3384, 3333, 3213, 2939, 2854, 1649, 1624, 1499, 1474,
4.37. 4-Amino-5,6,7,8-tetrahydro-2-(4-nitrophenyl)-oxaz-
olo[5,4-b]quinoline (34)
1
1444, 1429 cmꢁ1; H NMR (DMSO-d6 300 MHz) d 7.99
This compound was obtained following the general
€
(d, J ¼ 7:9 Hz, 2H, H20, H60), 7.39 (d, 2H, H30, H50),
6.37 (br s, 2H, NH2), 2.74 (m, 2H, H8), 2.48 (m, 2H,
H5), 2.39 (s, 3H, CH3), 1.77 (m, 4H, H6, H7); MS
(70 eV) m=z 279 [M]þ (100), 264 (19), 251 (44), 133 (6),
118 (7), 106 (6), 91 (8), 79 (6), 65 (5). Anal. Calcd for
C17H17N3O: C, 73.10; H, 6.13; N, 15.04. Found: C,
73.25; H, 6.40; N, 14.99.
method for the Friedlander reaction. To a stirred
suspension of AlCl3 (0.18 g, 1.32 mmol) in dry
1,2-dichloroethane (17 mL) at rt under argon, 1,3-oxa-
zole 60 (0.2 g, 0.88 mmol) and cyclohexanone (136 lL,
1.31 mmol) were added. After 98 h the reaction was
complete. Following the work-up, the resultant crude

2216
J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
4.40. 4-Amino-2-(4-chlorophenyl)-6,7,8,9-tetrahydro-5H-
cyclohepta[e] oxazolo[4,5-b]pyridine (37)
dichloroethane (20 mL) at rt under argon, 1,3-oxazole
61 (0.20 g, 0.93 mmol) and cycloheptanone (149 lL,
1.39 mmol) were added. After 189 h the reaction was
complete. Following the work-up, the resultant crude
was purified to give compound 39 (144.9 mg, 50%):
mp 257–262 ꢁC; IR (KBr) m 3420, 3350, 3220, 3030,
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (0.18 g, 1.38 mmol) in dry chloroben-
zene (20 mL) at rt under argon, 1,3-oxazole 59 (0.20 g,
0.92 mmol) and cycloheptanone (161 lL, 1.36 mmol)
were added. After 96 h the reaction was complete. Fol-
lowing the work-up, the resultant crude was purified to
give compound 37 (111.2 mg, 39%): mp 301–310 ꢁC; IR
(KBr) m 3360, 3320, 3200, 2900, 2820, 1640, 1610, 1460,
2940, 2870, 1650, 1630, 1505, 1470 cmꢁ1 1H NMR
;
(CDCl3, 300 MHz) d 8.10 (d, J ¼ 9 Hz, 2H, H20, H60),
7.00 (d, 2H, H30, H50), 4.74 (br s, 2H, NH2), 3.86 (s, 3H,
OCH3), 3.01 (m, 2H, H9), 2.66 (m, 2H, H5), 1.87 (m,
2H, H7), 1.70 (m, 2H, H8), 1.63 (m, 2H, H6); MS
(70 eV) m=z 309 [M]þ (100), 294 (20), 280 (36), 268 (7),
255 (5), 135 (7), 119 (6), 92 (5), 77 (5), 65 (3). Anal.
Calcd for C18H19N3O2: C, 69.88; H, 6.19; N, 13.58.
Found: C, 69.71; H, 5.98; N, 13.57. 39ÆHCl: mp 248–
249 ꢁC; MS (70 eV) m=z 309 [MþꢁHCl]þ (100), 294 (32),
280 (50), 268 (11), 255 (9), 135 (8), 119 (7), 92 (5), 77 (5),
65 (4).
1
1320, 1080 cmꢁ1; H NMR (DMSO-d6 300 MHz) d 8.09
(d, J ¼ 8:5 Hz, 2H, H20, H60), 7.66 (d, 2H, H30, H50),
6.42 (br s, 2H, NH2), 2.88 (m, 2H, H9), 2.74 (m, 2H,
H5), 1.80 (m, 2H, H7), 1.58 (m, 2H, H6), 1.51 (m, 2H,
H8); MS (70 eV) m=z 315 [Mþ2]þ (34), 313 [M]þ (100),
298 (26), 284 (48), 272 (9), 259 (11), 139 (17), 113 [p-
Cl37Ph]þ (5), 111 [p-Cl35Ph]þ (15). Anal. Calcd for
C17H16ClN3O: C, 65.07; H, 5.14; N, 13.39. Found: C,
64.85; H, 5.12; N, 13.10. 37ÆHCl: mp 301–302 ꢁC
(MeOH); IR (KBr) m 3435, 2924, 2852, 1661, 1623,
1484 cmꢁ1; MS (70 eV) m=z 315 [(Mþ2)ꢁHClþ] (34), 313
[MꢁHCl]þ (100), 298 (24), 284 (52), 272 (4), 259 (11),
119 (14), 113 [p-Cl37Ph]þ (6), 111 [p-Cl35Ph]þ (16), 102
(10), 92 (11), 75 (11), 65 (17). Anal. Calcd for
C17H16ClN3OÆHClÆH2O: C, 55.45; H, 5.20; N, 11.41.
Found: C, 55.78; H, 5.51; N, 11.34.
4.43. 4-Amino-6,7,8,9-tetrahydro-2-(4-methylphenyl)-5H-
cyclohepta[e]oxazolo[4,5-b]pyridine (40)
This compound was obtained following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (0.54 g, 4 mmol) in dry 1,2-dichloro-
ethane (20 mL) at rt under argon, 1,3-oxazole 62 (0.40 g,
2 mmol) and cycloheptanone (574 lL, 4 mmol) were
added. After 190 h the reaction was complete. Following
the work-up, the resultant crude was purified to give
product 40 (418.9 mg, 71%): mp 284–290 ꢁC; IR (KBr) m
3378, 3340, 3212, 2915, 2848, 1654, 1622, 1499, 1481,
1331 cmꢁ1; 1H NMR (DMSO-d6 70 ꢁC, 300 MHz) d 7.99
(d, J ¼ 8:0 Hz, 2H, H20, H60), 7.39 (d, 2H, H30, H50),
6.17 (br s, 2H, NH2), 2.91 (m, 2H, H9), 2.74 (m, 2H,
H5), 1.80 (m, 2H, H7), 1.57 (m, 4H, H6, H8); MS
(70 eV) m=z 293 [M]þ (100), 278 (28), 264 (46), 252 (9),
239 (9), 119 (11), 91 (9), 65 (6). Anal. Calcd for
C18H19N3O: C, 73.70; H, 6.53; N, 14.32. Found: C,
73.48; H, 6.86; N, 14.33.
4.41. 4-Amino-6,7,8,9-tetrahydro-2-(4-nitrophenyl)-5H-
cyclohepta[e]oxazolo[4,5-b]pyridine (38)
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred sus-
pension of AlCl3 (0.46 g, 3.5 mmol) in dry 1,2-dichlo-
roethane (30 mL) at rt under argon, 1,3-oxazole 60
(0.40 g, 1.74 mmol) and cycloheptanone (410 lL,
3.5 mmol) were added. The reaction was complete
14 days later. After the work-up, the resultant crude was
purified to give compound 38 (327 mg, 58%): mp 310–
319 ꢁC; IR (KBr) m 3460, 3400, 3280, 2960, 2890, 1665,
1635, 1600, 1350 cmꢁ1; MS (70 eV) m=z 324 [M]þ (100),
309 (27), 295 (46), 277 (16), 249 (20), 237 (7), 119 (8), 104
(8), 92 (7), 76 (9), 65 (7). Anal. Calcd for C17H16N4O3:
C, 62.95; H, 4,97; N, 17.27. Found: C, 63.10; H, 5.12; N,
16.99. 38ÆHCl: mp 289–290 ꢁC (MeOH; dec); IR (KBr) m
3395, 3337, 3219, 2924, 2851, 1653, 1623, 1590, 1520,
1477, 1342 cmꢁ1; MS (70 eV) m=z 324 [MþꢁHCl] (100),
309 (17), 296 (29), 295 (38), 277 (10), 249 (16), 237 (6),
119 (11), 104 (10), 91 (9), 80 (6), 76 (12), 65 (11). Anal.
Calcd for C17H16N4O3ÆHCl: C, 56.59; H, 4.74; N, 15.58.
Found: C, 56.84; H, 4.98; N, 15.78.
4.44. Pharmacological studies
4.44.1. Evaluation of AChE activity. The assessment of
the compounds AChE inhibitory activity was performed
following the spectrometric method of Rappaport
et al.25 using AChE from electric eel (Torpedo califor-
nica) and acetylcholine chloride (29.5 mM) as a sub-
strate. The reaction took place in a final volume of
2.5 mL of an aqueous solution containing 0.78 U of
AChE and 1.9 mM m-nitrophenol to produce a yellow
colour, which fades out as a function of the enzymatic
activity. Inhibition curves were plotted from the incu-
bating data with the different compounds for 30 min; a
sample without any compound was always present to
determine the 100% of enzymatic activity. After 30 min
incubation, the disappearance of yellow colour by m-
nitrophenol was evaluated by measuring the absorbance
at 405 nm in a spectrophometric plate reader (iEMS
Reader MF, Labsystems). The concentration of com-
4.42. 4-Amino-6,7,8,9-tetrahydro-2-(4-methoxyphenyl)-
5H-cyclohepta[e]oxazolo[4,5-b]pyridin (39)
This compound was prepared following the general
€
method for the Friedlander reaction. To a stirred
suspension of AlCl3 (0.19 g, 1.39 mmol) in dry 1,2-

J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
2217
pound able to produce 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 previously ob-
tained. Data are means ꢀ SE of three different experi-
ments in triplicate at least.
chromaffin cells, the cells were scraped with a plastic
pipette tip while adding 0.1 mL of 10% trichloroacetic
acid. Scintillation fluid (3.5 mL; Optiphase Hisafe II,
EGG Instruments) was added and the samples counted
in a Packard beta counter. Results are expressed as % of
Ca2þ taken up by control cells.
4.45. Molecular modelling
4.44.2. Evaluation of BuChE activity. The assessment of
the compounds BuChE inhibitory activity was deter-
mined following the method of Ellman et al.26 using
BuChE from human serum and butyryl thiocholine
chloride (5 mM) as a substrate. The reaction took place
in a final volume of 1 mL of a phosphate buffer solution
at pH 7.2 containing 0.035 U of BuChE and 0.25 mM
5,50-dithiobis-2-nitrobenzoic acid (DTNB), which pro-
duces the yellow anion 5-thio-2-nitrobenzoic acid.
Inhibition curves were plotted from the incubating data
with the different compounds for 15 min; a sample
without any compound was always present to determine
the 100% of enzymatic activity. After 15 min incubation,
the production of colour as an indication of enzymatic
activity was evaluated by measuring the absorbance at
412 nm in a spectrophometric plate reader (iEMS
Reader MF, Labsystems).
The simulation system used in MD simulations con-
sisted of the enzyme, the drug and a cap (with a radius
36
ꢂ
of 25 A) of TIP3P water molecules centred on the
ligand. The enzyme was modelled in its physiologically
active form with neutral His440 and deprotonated
Glu327, which form together with Ser200 the catalytic
triad. The standard ionization state at neutral pH was
considered for the ionizable residues, but for Asp392
and Glu443, which were neutral, and His471, which was
protonated, according to previous numerical titration
studies.37 The AMBER (amber99 parm file) all-atom
force field38 was used for the enzyme. For the drug, HF/
6-31G(d) RESP charges were used, and the van der
Waals parameters were taken from those defined for
related atoms in the force field.39 After minimization,
the whole system was partitioned into a mobile and a
rigid region. The former consisted of the ligand, all the
residues having at least an atom at a distance less than
4.44.3. Cell isolation and culture of bovine chromaffin
cells. Bovine adrenomedullary chromaffin cells were
isolated following standard methods33 after accom-
plishing some modifications.34 Cells were suspended in
Dulbeccoꢂs modified Eagleꢂs medium (DMEM) supple-
mented with 5% fetal calf serum, 10 lM cytosine ara-
binoside, 10 lM fluorodeoxyuridine, 50 IU mLꢁ1
penicillin and 50 lg mLꢁ1 streptomycin. Cells were pla-
ted at a density of 2 · 105 cells/well in 96-multiwell
Costar plates and were used 1–5 days after plating.
Medium was replaced after 24 h and then after 2–3 days.
ꢂ
14 A from the ligand and all the water molecules. After
equilibration (0.3 ns), a 2 ns MD simulation (at 298 K)
ꢂ
was carried out. A cutoff of 11 A for nonbonded inter-
actions, together with SHAKE and an integration time
step of 2 fs, were used.
The structural stability of the complexes was examined
from the rmsd plots determined for the subset of resi-
dues that form the walls of the binding pocket with
regard to the crystallographic structure. To examine the
energetic stability of the complexes, a MM/PB-SA
analysis was performed by using 100 snapshots collected
regularly along the last 500 ps of the MD simulation.
Accordingly, the stability of each complex was estimated
from the addition of the internal energy (including the
contributions due to both inhibitor and enzyme and
their interaction energy) and the solvation free energy.
The former term was determined by averaging the cor-
responding energy values as determined with the
AMBER force field. The second was estimated by using
a Poisson–Boltzmann calculation for the electrostatic
component and a surface-area dependent term for the
nonelectrostatic contribution. The solute was assigned a
dielectric of 1, the solvent dielectric was set to 78.4, and
the dielectric boundary was defined using a 1.4 probe
sphere and van der Waals radii taken from the AMBER
force field. A focusing approach (final grid spacing of
4.44.4. Evaluation of 45Ca2 uptake. 45Ca2þ uptake
+
studies were carried out in cells after 2–3 days in culture
following standard methods.35 Before the experiment,
cells were washed twice with 0.1 mL Krebs-HEPES
solution with the following composition (in mM): NaCl
140, KCl 5.9, MgCl2 1.2, CaCl2 1, glucose 11, HEPES
10, at pH 7.2 and 37 ꢁC. 45Ca2þ uptake in chromaffin
cells was studied by incubating the cells at 37 ꢁC with
45CaCl2 at a final concentration of 5 lCi mLꢁ1 in Krebs-
HEPES (basal uptake), high Kþ concentration solution
(Krebs-HEPES containing 70 mM KCl with isosmotic
reduction of NaCl) or 100 lM dimethylphenylpipera-
zinium (DMPP) in Krebs-HEPES. This incubation was
carried out during 5 min for basal uptake or 5 s for high
Kþ or DMPP, in the absence (control) or in the presence
of the drugs. At the end of the incubation period the test
medium was rapidly aspirated and the uptake reaction
was ended by adding 0.1 mL of a cold Ca2þ-free Krebs-
HEPES containing 10 mM LaCl3. Finally, cells were
washed again five times with 0.1 mL of Ca2þ-free Krebs-
HEPES containing 10 mM LaCl3 and 2 mM EGTA, at
15 s intervals. To measure radioactivity retained by
ꢂ
0.5 point/A) was employed. The nonelectrostatic term
was determined using a surface tension for all the atoms
ꢁ2 40
Solvation calculations were per-
ꢂ
equal to 5.4 cal A
.
formed with the CMIP program.41
The molecular electrostatic potentials of compounds 63–
65 were determined at the HF/6-31G(d) level of theory
by using the MOPETE program.42

2218
J. L. Marco et al. / Bioorg. Med. Chem. 12 (2004) 2199–2218
€
J.; Gorbig, D. M.; Munoz-Torrero, D.; Pujol, M. D.;
Simo, M.; Vivas, N. M. Bioorg. Med. Chem. 1998, 6, 427.
~
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~
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~