Synthesis of 6-amino-1,4-dihydropyridines that prevent calcium overload and neuronal death

Article abstract of DOI:10.1016/j.ejmech.2007.06.001

The synthesis and pharmacology of 6-amino-1,4-dihydropyridines, such as ethyl 6-amino-4-aryl-5-cyano-1,4-dihydro-2-methyl-3-pyridinecarboxylic acids (3-16) and 2-amino-4-aryl-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydro-3-quinolinenitriles (17-21) are described. Compounds 18 and 21, at the concentration of 0.3 μM, proved to be the best blockers of the [Ca²⁺] overload induced by depolarization with high [K⁺] of SH-SY5Y neuroblastoma cells, with values of 63.8% and 50.4%, respectively. Most of the compounds induced a remarkable neuroprotective effect against toxicity caused by high [K⁺]-elicited [Ca²⁺] overload, and against H₂O₂-generated free radicals, in SH-SY5Y cells.

Full text of DOI:10.1016/j.ejmech.2007.06.001

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 668e674
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of 6-amino-1,4-dihydropyridines that prevent calcium
overload and neuronal death
a,b a,b
Rafael Leon , Cristobal de los Rıos , Jose Marco-Contelles
a,
*
´
Manuela G. Lopez , Antonio G. Garcıa , Mercedes Villarroya
´
´
´
,
b,**
b
b,c
´
´
a Laboratorio de Radicales Libres (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
b Instituto Teo´filo Hernando, Departamento de Farmacolog´ıa y Terape´utica, Facultad de Medicina,
Universidad Auto´noma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain
c Servicio de Farmacolog´ıa Clinica, Hospital Universitario de la Princesa, C/Diego de Leo´n 62, 28006 Madrid, Spain
Received 10 April 2007; accepted 12 June 2007
Available online 5 July 2007
Abstract
The synthesis and pharmacology of 6-amino-1,4-dihydropyridines, such as ethyl 6-amino-4-aryl-5-cyano-1,4-dihydro-2-methyl-3-pyridine-
carboxylic acids (3e16) and 2-amino-4-aryl-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydro-3-quinolinenitriles (17e21) are described. Compounds
18 and 21, at the concentration of 0.3 mM, proved to be the best blockers of the [Ca2þ] overload induced by depolarization with high [Kþ] of
SH-SY5Y neuroblastoma cells, with values of 63.8% and 50.4%, respectively. Most of the compounds induced a remarkable neuroprotective
effect against toxicity caused by high [Kþ]-elicited [Ca2þ] overload, and against H2O2-generated free radicals, in SH-SY5Y cells.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 6-Amino-1,4-dihydropyridines; Ethyl 6-amino-4-aryl-5-cyano-1,4-dihydro-2-methyl-3-pyridinecarboxylic acids; 2-Amino-4-aryl-7,7-dimethyl-5-oxo-
1,4,5,6,7,8-hexahydro-3-quinolinenitriles; Voltage-dependent calcium channels; Neuroprotection
1. Introduction
and position of C4-aryl ring substituent were determinant of
voltage-dependent calcium channel (VDCC) antagonist activ-
ity. In general, the presence of an aryl group at C4, and esters,
acyl, sulphonyl or nitrile groups at C3 and C5 of the 1,4-DHP
ring has proved to be a fundamental requirement for the phar-
macological activity [6e8]. In spite of the widely developed
chemistry of the 1,4-DHPs, little is known about 1,4-DHPs
bearing substituents other than hydrogen atoms or alkyl groups
at C2 and C6 [9,10]. In this context, it is surprising to recog-
nize the scarce number of studies that report the pharmacology
of 6-amino substituted 1,4-DHPs (I) (Fig. 1). A survey of the
current literature has shown that compounds of type I have
been described in the literature [11,12], and claimed to be
active in the central nervous system, but without reporting
the biological relevant data [13]. Similarly, related 2-amino-
4-aryl-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydro-3-quinoline-
nitriles (II) (Fig. 1) are also well known [14], but to the best of
our knowledge, their pharmacology has never been addressed.
1,4-Dihydropyridine (1,4-DHP) [1] calcium antagonists
that produce vascular smooth muscle relaxation, specially in
arterial beds, such as nifedipine (1) or nimodipine (2)
(Fig. 1), are relevant drugs for the treatment of peripheral
vascular diseases such as angina pectoris and hypertension
[2]. The design of DHPecalcium channel modulators has
prompted studies to investigate the functional and geometrical
requirements at the DHP binding site [3]. Structureeactivity
relationships (SAR) show that the combination of the substit-
uents at the C3, C4 and C5 positions of nifedipine (1) modu-
lates the activity [4] and tissue selectivity [5], while the nature
* Corresponding author. Tel.: þ34 91 5622900; fax: þ34 91 5644853.
** Corresponding author.
E-mail address: iqoc21@iqog.csic.es (J. Marco-Contelles).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.06.001

R. Leo´n et al. / European Journal of Medicinal Chemistry 43 (2008) 668e674
669
MeO
X
X
O2N
MeO2C
O
O
NO2
CO2-iPr
RO2C
Y
CO2Me
CN
NH2
O
N
H
NH2
N
H
(1)
N
H
N
H
(2)
(I) (Y= CN, CO2R´)
(II)
Fig. 1. Nifedipine (1), nimodipine (2), and general structure for the target 1,4-DHP (I, II).
On the other hand, amlodipine, a 1,4-DHP in clinical use,
and carvedilol prevent cytotoxicity in cortical neurons isolated
from stroke-prone spontaneously hypertensive rats [15]. Pro-
tective effects of a selective L-type voltage-sensitive calcium
channel blocker, S-312-d, have been reported [16]. Similarly,
a 1,4-DHP blocker such as nimodipine (2) (Fig. 1) behaves
as neuroprotectant drug [17]. Thus, it seems reasonable to
study the effects of new synthetic 1,4-DHPs on the increase
of [Ca2þ] elicited by depolarization with high [Kþ] (70 mM)
in SH-SY5Y human neuroblastoma cells, as well as their
possible effect on cell viability.
These facts prompted us to synthesize the readily available
ethyl 6-amino-4-aryl-5-cyano-1,4-dihydro-2-methyl-3-pyridi-
necarboxylic acids (3e16)/2-amino-4-aryl-7,7-dimethyl-5-
oxo-1,4,5,6,7,8-hexahydro-3-quinolinecarbonitriles (17e21)
(Fig. 2), and test them to a pharmacological screening includ-
ing VDCC blockade, and neuroprotection.
[12] starting from the corresponding substituted benzalde-
hydes via the 2-arylmethylene-3-oxo-butanoic acid ethyl
esters 22 [19], 23 [20], 25 [21] and 26 [21] (Scheme 1).
2-[(40-Biphenyl)methylene]-3-oxo-butanoic acid ethyl ester
(24) has been synthesized here for the first time from the bi-
phenyl-4-carboxaldehyde. Treatment of compounds 22e26
with 3,3-diaminoacrylonitrile [12], obtained in situ from ethyl
2-cyanoacetimidate hydrochloride (27) in the presence of
ammonium acetate, gave the 4-amino-1,4-DHPs (5, 13e16)
in good yields (Scheme 1).
In the group of 2-amino-4-aryl-7,7-dimethyl-5-oxo-
1,4,5,6,7,8-hexahydro-3-quinolinenitriles (17 [14], 18 [22],
19 [14], 20 [14]), compound 21 has been prepared here for
the first time by reacting [2-(4-pyridyl)methylene]malononi-
trile (28) [23] with 3-amino-5,5-dimethyl-2-cyclohexenone
(29) [24] in methanol at reflux, in the presence of ammonium
acetate (Scheme 2).
With these compounds in hands, we first carried on the eval-
uation of cytosolic [Ca2þ] concentrations in the presence of the
compounds 3e21. A control with nimodipine (2) (Fig. 1),
a blocker of L-type VDCC, was included in every individual ex-
periment. All compounds were assayed at the concentration of
0.3 mM. The results are shown in Table 1. As can be noticed,
most of the compounds blocked the increase in [Ca2þ] induced
2. Results and discussion
In the family of ethyl 6-amino-4-aryl-5-cyano-1,4-dihydro-
2-methyl-3-pyridinecarboxylic acids (3e16), compounds 3
[12], 4 [18], and 6e12 [18] (Fig. 2) have been reported. The
other analogues (5, 13e16) have been easily synthesized
X
N
Y
5´
3´
2´
5´
3´
X
4´
X
6´
6´
O
O
O
O
2´
3
1´
1´
4a
3 CN
CN
5
CN
CN
6
5
8
4
EtO
4
EtO
7
6
N
H
NH2
2
N
H
NH2
2
N
H
NH2
N
H
NH2
8a
(17) X = H
(18) X = F
(19) X = Me
(20) X = OMe
(15) X = N, Y= CH
(16) X = CH, Y= N
(21)
(3) X = H
(4) X = 4'-F
(5) X = 2'-CF3
(6) X = 2'-NO2
(7) X = 3'-NO2
(8) X = 4'-NO2
(9) X = 4'-Me
(10) X = 2'-OMe
(11) X = 3'-OMe
(12) X = 4'-OMe
(13) X = 3',4'-di-OMe
(14) X = 4'-C6H5
Fig. 2. Target 6-amino-1,4-DHP.

670
R. Leo´n et al. / European Journal of Medicinal Chemistry 43 (2008) 668e674
CN
located at C30; thus, no additivegroup effect was observed in this
case. Regarding the heterocycle containing compounds 15 and
16, the most potent correlated by far to the 40-pyridyl substituted
molecule (16).
H2N
OEt
Cl
Y
W
X
Y
X
(27)
W
To assess a possible neuroprotective effect of these new
compounds against [Ca2þ] overload, the usual protocol was
followed (see Section 4). Nimodipine (2) (Fig. 1) was used
as a reference compound. All the compounds, including nimo-
dipine, were assayed at the concentration of 0.3 mM. The
results are shown in Table 2. Most of the compounds induced
a remarkable neuroprotective effect, with values ranging from
12.9% to 54.7%. Comparing 2-amino-4-aryl-3-cyano-7,7-di-
methyl-5-oxo-1,4,5,6,7,8-hexahydroquinolines (17e21) with
6-amino-4-aryl-5-cyano-1,4-dihydropyridines (3e16), the for-
mer group of molecules showed a higher protection with
values in the range of 39.5e54.7%, the best neuroprotectant
compound being compound 17, with a 54.7% protection;
note also that compounds 19 (40-Me) and 20 (40-MeO) with
electron-donating groups were more potent than those bearing
electron-withdrawing groups (18 and 21); this is just the oppo-
site behavior of that described for the blockade of [Ca2þ] in-
crease in SH-SY5Y cells for the same molecules (see Table 1).
Note once more that nimodipine (2) was significantly less
potent (35.9%) than the best neuroprotectant compound (17)
analyzed in this study. Comparatively, the protection values
recorded for 6-amino-4-aryl-5-cyano-1,4-dihydropyridines
(3e16) (Table 2) were also stronger than those shown in Table
1 for the blockade of [Ca2þ] increase in SH-SY5Y cells for the
same compounds; in this family, products bearing electron-
withdrawing groups at the aromatic ring [(8) 4-NO2
(45.7%), (15, 16) (30-, 40-pyridyl) (43.4%, 43.3%), (6) 20-
NO2 (42.6%)] showed higher protection than those bearing
electron-donating groups. For a definite substituent, the nitro
group for instance, the order of potency was displayed as com-
pound 8 > 6 [ 7, corresponding to the position of the group
at C40, C20 and C30, while for the methoxy group, the order of
potency showed compound 11 > 10 [ 12, referring to the po-
sition of the group at C30, C20 and C40. In general, it is clear
that the more electron-withdrawing the groups are, the more
effective neuroprotection was observed (compare compound
8 with 4), and the less electron-donating the groups are, the
more potent neuroprotection was detected (compare com-
pound 9 with 10e14).
O
AcONH4, MeOH
reflux
O
CN
NH2
EtO
EtO
N
H
O
(22) W,Y = CH,X = 2'-CF3
(23) W,Y = CH,X = 3',4'-di-OMe
(24) W,Y = CH,X = 4'-Ph (76%)
(25) W = N,Y = CH,X = H
(26) W = CH,Y = N,X = H
(5) (43%)
(13) (77%)
(14) (60%)
(15) (66%)
(16) (85%)
Scheme 1. Synthesis of 1,4-DHP (5, 13e16).
by high [Kþ] in a statistically significant manner, the values
ranging between 7.2% and 63.9%. From the SAR analysis,
some conclusions can be drawn. In general, 2-amino-4-aryl-
3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinolines
(17e21) induced higher blockade of [Ca2þ] elevation than
6-amino-4-aryl-5-cyano-1,4-dihydropyridines (3e16); for in-
stance, the most potent derivatives found in this study, the hex-
ahydroquinolines 18 and 21, with a 40-F and a 40-pyridyl
substituent, respectively, showed blockade values of 63.9%
and 50.4%, respectively (compared with the 1,4-DHP analogues
4 and 16, showing a 7.2% and 32.6% blockade). It is also very
clear that in the 2-amino-4-aryl-3-cyano-7,7-dimethyl-5-oxo-
1,4,5,6,7,8-hexahydroquinoline family of compounds (17e
21), the most potent bears electron-withdrawing groups such
as 40-F or 40-pyridyl. On the other hand, compounds that possess
electron-donating groups [19 (40-Me) and 20 (40-MeO)] only
show modest blockade values. Very interestingly, compounds
18 and 21 were more active than nimodipine (2). In the 6-
amino-4-aryl-5-cyano-1,4-dihydropyridine family of com-
pounds (3e16), the most potent molecule corresponded to the
unsubstituted member (3) with a 34.0% blockade, followed by
substituted analogues with electron-withdrawing groups such
as 40-pyridyl (32.6%) (16), 20-CF3 (30.4%) (5), and 30-NO2
(27.8%) (7). Considering the same substituent, for instance a ni-
tro or methoxy group, the most potent compounds were 7 and 11,
bearing these groups at C30. Note also that compound 13, with
two methoxy groups at C30 and C40, is more active (21.8%)
than compound 12 (11.8%) with one methoxy at C40, but some-
what less activethan molecule 11 (25%) with the methoxy group
Furthermore, the effect of our compounds as neuroprotec-
tants towards oxidative stress elicited by free-radical generation
was evaluated. For this purpose, SH-SY5Y cells were exposed
N
N
O
O
CN
NC
+
AcONH4, MeOH
reflux
NH2
CN
N
H
(21)
NH2
71%
(29)
(28)
Scheme 2. Synthesis of the 2-amino-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydro-3-quinolinenitrile (21).

R. Leo´n et al. / European Journal of Medicinal Chemistry 43 (2008) 668e674
671
Table 1
Table 2
Effects of 6-amino-4-aryl-5-cyano-1,4-dihydropyridines (3e16) and 2-amino-
4-aryl-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinolines (17e21)
on [Ca2þ] increase elicited by 70 mM [Kþ] in SH-SY5Y cells (% inhibition
with respect to a control without any drug)
Neuroprotective effects of 6-amino-4-aryl-5-cyano-1,4-dihydropyridines (3e
16) and 2-amino-4-aryl-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydro-
quinolines (17e21), expressed as reduction in the increase of LDH released
in the presence of 70 mM [Kþ], in neuroblastoma cells
Compound
Substituent
% Blockade [Ca2þ] uptake
Compound
Substituent
LDHa release
(% control)
% Protection
([Ca2þ] overload)
Nimodipine (2)
3
e
45.5 ꢀ 6.1***
34.03 ꢀ 6.6**
7.2 ꢀ 4.7 ns
30.4 ꢀ 3.5**
16.1 ꢀ 5.02 ns
27.8 ꢀ 3.2**
17.6 ꢀ 3.2 ns
16.9 ꢀ 3.4 ns
24.3 ꢀ 5.6*
H
40-F
Nimodipine (2)
3
e
75.00 ꢀ 2.2***
76.26 ꢀ 2.4***
72.80 ꢀ 2.9***
72.11 ꢀ 2.7***
71.11 ꢀ 3.6***
79.4 ꢀ 3.8*
35.9 ꢀ 2.8
34.1 ꢀ 3.2
39.8 ꢀ 4.03
40.2 ꢀ 3.6
42.6 ꢀ 5.2
30.3 ꢀ 5.5
45.7 ꢀ 2.6
40.8 ꢀ 4.4
31.5 ꢀ 3.7
32.1 ꢀ 3.8
14.4 ꢀ 4.4
31.2 ꢀ 4.9
12.9 ꢀ 1.9
43.4 ꢀ 3.5
43.3 ꢀ 5.7
54.7 ꢀ 2.6
42.3 ꢀ 3.05
44.4 ꢀ 4.01
43.2 ꢀ 2.01
39.5 ꢀ 2.4
4
H
40-F
5
20-CF3
20-NO2
30-NO2
40-NO2
40-Me
20-OMe
30-OMe
40-OMe
30,40-Di-OMe
40-Phenyl
30-Pyridyl
40-Pyridyl
H
4
6
5
20-CF3
20-NO2
30-NO2
40-NO2
40-Me
20-OMe
30-OMe
40-OMe
3,040-Di-OMe
40-Phenyl
30-Pyridyl
40-Pyridyl
H
7
6
8
7
9
8
68.9 ꢀ 2***
10
11
12
13
14
15
16
17
18
19
20
21
9
72.3 ꢀ 3.1***
78.2 ꢀ 2.6**
77.9 ꢀ 2.9**
89.9 ꢀ 3.05 ns
80.5 ꢀ 4.3*
25 ꢀ 3.7**
10
11
12
13
14
15
16
17
18
19
20
21
11.8 ꢀ 6.3 ns
21.8 ꢀ 4.05*
23.9 ꢀ 3.7*
14.1 ꢀ 4.6 ns
32.6 ꢀ 4.3**
20.8 ꢀ 4.3 ns
63.9 ꢀ 3.2***
28.6 ꢀ 3.5**
12.6 ꢀ 3.7 ns
50.4 ꢀ 6.7***
90.9 ꢀ 8.7 ns
70.5 ꢀ 2.6***
70.5 ꢀ 4.01***
61.2 ꢀ 1.8***
69.9 ꢀ 2.2***
68.4 ꢀ 2.8***
69.3 ꢀ 1.5***
71.4 ꢀ 1.8***
40-F
40-Me
40-MeO
40-Pyridyl
40-F
40-Me
40-MeO
40-Pyridyl
Data are expressed as means ꢀ SEM of at least three different cultures in qua-
druplicate.
*p < 0.05, **p < 0.01, ***p < 0.001; ns ¼ not significant. All compounds
were assayed at the concentration of 0.3 mM.
Data are expressed as means ꢀ SEM of at least three different cultures in qua-
druplicate.
*p < 0.05, **p < 0.01, ***p < 0.001; ns ¼ not significant. All compounds
were assayed at the concentration of 0.3 mM.
a
Lactic dehydrogenase activity.
for 24 h to 60 mM H2O2, following the usual protocol (see
Section 4). The results are reported in Table 3. Nimodipine
(2), and catalase as a reference antioxidant compound, were
included. In general, protection afforded by these compounds
was from low to moderate, with most of the values below
30%, but some of them showed more than 40% inhibition.
Comparing 2-amino-4-aryl-3-cyano-7,7-dimethyl-5-oxo-1,4,5,
6,7,8-hexahydroquinolines (17e21) with 6-amino-4-aryl-5-cy-
ano-1,4-dihydropyridines (3e16), the former group of mole-
cules revealed less protection. Accordingly, only compounds
9, 13 and 16 were more effective, with protection values of
42.4%, 40.8% and 46.7%, respectively. These compounds
were more potent than nimodipine (2) (36.0%), but twofold
less active than catalase. No obvious relationship was observed
between the type of substituent and the neuroprotective effect;
however, for a given substituent, regardless of its electronic
nature, being at the C40 position conferred to compounds, the
best neuroprotective profile.
significant manner, and induced a remarkable neuroprotective
effect at the concentration of 0.3 mM, with values ranging
from 12.9% to 54.7% protection against cell death caused by
[Ca2þ] overload, induced upon incubation of SH-SY5Y cells
with 70 mM [Kþ]. The antioxidant effect of the compounds
was less pronounced, with protection values around 30% in
most of the cases. As representative examples, compound 18
showed a 63.9% blockade for the increase in [Ca2þ] induced
by high [Kþ] in SH-SY5Y cells; molecule 17 displayed
a 54.7% protection for cell viability in the presence of
70 mM [Kþ]; finally, product 16 exhibited significant protec-
tion (46.7%) against the free-radical insult, while nimodipine
(2), a well known calcium antagonist used as a reference com-
pound, presented lower values in all these experiments when
compared with the most active compound in each experiment.
4. Experimental part
3. Conclusions
4.1. General methods
We have reported the synthesis of a number of 6-amino-1,4-
DHPs (compounds 3e21) and investigated for the first time
their pharmacological activity, including the effect on cytosolic
[Ca2þ] increase elicited by depolarization with high [Kþ] of
SH-SY5Y human neuroblastoma cells, the neuroprotective ef-
fects against [Ca2þ] overload, and their neuroprotective action
on free-radical generation. First of all, most of the compounds
blocked the [Kþ]-induced [Ca2þ] increase in a statistically
Reactions were monitored by TLC using precoated silica
gel aluminium plates containing a fluorescent indicator
(Merck, 5539). Detection was done by UV (254 nm) followed
by charring with sulfuriceacetic acid spray, 1% aqueous po-
tassium permanganate solution or 0.5% phosphomolybdic
acid in 95% EtOH. Anhydrous Na2SO4 was used to dry or-
ganic solutions during work-ups and the removal of solvents

672
R. Leo´n et al. / European Journal of Medicinal Chemistry 43 (2008) 668e674
J ¼ 6.9 Hz, 3H, CO2CH2CH3); 13C NMR (CDCl3, 75 MHz)
(major Z isomer) d 203.3 (C]O), 194.4 (CO2), 140.6
(CH]C), 134.0 (CH]C ), 131.6, 130.2, 130.0, 128.8, 128.7,
128.1, 127.9, 127.4, 126.8 (aromatic), 61.8 (OCH2CH3), 26.5
[CH3(CO)], 13.8 (CH3CH2O); MS (API-ESþ) m/z: [M þ 1]þ
295.2; [M þ Na]þ 317.0; [2M þ Na]þ, 611.2. Anal. Calcd.
for C19H18O3: C, 77.53; H, 6.16. Found: C, 77.32; H, 5.97.
Table 3
Cell viability for 6-amino-4-aryl-5-cyano-1,4-dihydropyridines (3e16) and
2-amino-4-aryl-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinolines
(17e21), expressed as reduction in the increase of LDH released in the pres-
ence of 60 mM H2O2
Compound
Substituent
LDHa release
(% control)
% Protection
(free radicals)
Catalase
Nimodipine (2)
e
18.9 ꢀ 1.8***
75.4 ꢀ 2.1***
86.7 ꢀ 1.7 ns
82.2 ꢀ 2.8*
88.3 ꢀ 2.8
36.03 ꢀ 2.8
17.3 ꢀ 2.1
24.7 ꢀ 3.8
28.2 ꢀ 1.5
1.6 ꢀ 3.1
e
4.3. General method for the synthesis of 6-amino-4-aryl-
5-cyano-1,4-dihydro-2-methyl-3-pyridinecarboxylic acid
ethyl esters (5, 13e16)
3
H
40-F
4
5
20-CF3
20-NO2
30-NO2
40-NO2
40-Me
20-OMe
30-OMe
40-OMe
30,40-Di-OMe
40-Phenyl
30-Pyridyl
40-Pyridyl
H
78.6 ꢀ 1.3**
98.8 ꢀ 2.2 ns
84.5 ꢀ 3.2 ns
83.4 ꢀ 1.8 ns
69.7 ꢀ 2.4***
88.4 ꢀ 2 ns
6
7
21.2 ꢀ 4.3
23.1 ꢀ 2.4
42.4 ꢀ 3.2
15 ꢀ 2.6
A solution of ethyl 2-cyanoacetimidate hydrochloride
(1 equiv) and ammonium acetate (1e5 equiv), in methanol,
was warmed at reflux for 15 min. Then, the corresponding
ethyl ester 2-arylmethylene-3-oxo-butanoic acid (1 equiv)
was added and the mixture was refluxed for 15 min, and
cooled at 5 ꢂC overnight. The solid was separated and recrys-
tallized from methanol.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
86.1 ꢀ 3.9 ns
77.7 ꢀ 1.6***
70.2 ꢀ 3.1 ns
80.3 ꢀ 1.1**
84.5 ꢀ 3.8***
66.7 ꢀ 2.6***
84.2 ꢀ 1.4***
75.4 ꢀ 3.1***
88.6 ꢀ 1.4 ns
77.8 ꢀ 1.7***
77.7 ꢀ 2.5***
27.7 ꢀ 4
31.3 ꢀ 2.2
40.8 ꢀ 3.9
26.2 ꢀ 1.7
21.2 ꢀ 4.8
46.7 ꢀ 3.7
20.6 ꢀ 1.8
32.4 ꢀ 4
4.3.1. 6-Amino-5-cyano-1,4-dihydro-4-(2-trifluoromethyl-
phenyl)-2-methyl-3-pyridinecarboxylic acid ethyl ester (5)
Following the method described in Section 4.3, the reaction
of ethyl 2-cyanoacetimidate hydrochloride (27) (437.4 mg,
2.93 mmol) and ammonium acetate (733.5 mg, 9.51 mmol),
in methanol (15 mL), with compound 22 [19] (700.0 mg,
2.44 mmol), in 30 min, gave product 5 (368.0 mg, 43%): mp
243e245 ꢂC; IR (KBr) n 3401, 3340, 3224, 2992, 2833,
40-F
40-Me
40-MeO
40-Pyridyl
14.9 ꢀ 1.8
29 ꢀ 2.2
29.1 ꢀ 3.1
Data are expressed as means ꢀ SEM of at least three different cultures in qua-
druplicate.
*p < 0.05, **p < 0.01, ***p < 0.001; ns ¼ not significant. Catalase was
assayed at the concentration of 50 units/mL. The rest of the compounds
were assayed at the concentration of 0.3 mM.
2182, 1657, 1624, 1489, 1367, 1255 cmꢁ1
;
1H NMR
a
Lactic dehydrogenase activity.
(DMSO-d6, 300 MHz) d 8.70 (s, 1H, NH), 7.58e7.42 (m,
4H, aromatic), 5.62 (s, 2H, NH2), 4.79 (s, 1H, H4), 3.83 (q,
J ¼ 7.1 Hz, 2H, OCH2CH3), 2.28 [s, 3H, CH3], 0.86
(t, J ¼ 7.1 Hz, 3H, CO2CH2CH3); 13C NMR (DMSO-d6,
was carried out under vacuum with a rotary evaporator. Flash
column chromatography was performed using silica gel 60
(230e400 mesh, Merck). Melting points were determined on
a Kofler block and are uncorrected. IR spectra were obtained
on a PerkineElmer Spectrum One Spectrophotometer. 1H
NMR spectra were recorded with a Varian VXR-200S spec-
trometer, using tetramethylsilane as internal standard and
13C NMR spectra were recorded with a Bruker WP-200-SY.
All the assignments for protons and carbons were in agreement
with 2D COSY, gHSQC, gHMBC, and 1D NOESY spectra.
Values with (*) can be interchanged. Elemental analyses
were carried out on a Carlo Erba EA 1108 apparatus.
75 MHz)
d 166.8 (C]O), 151.8 (C2), 148.2e124.4
(aromatic), 146.6 (C6), 121.0 (CN), 101.4 (C3), 59.2
(OCH2CH3), 58.2 (C5), 35.9 (C4), 19.1 (CH3), 14.1
(CH3CH2O); MS (API-ESþ) m/z: [M þ 1]þ 352.3; [M þ Na]þ
374.2; [2M þ Na]þ 723.5. Anal. Calcd. for C17H16F3N3O2: C,
58.12; H, 4.59; N, 11.96. Found: C, 58.26; H, 4.35; N, 11.74.
4.3.2. 6-Amino-5-cyano-1,4-dihydro-4-(3,4-dimethoxy-
phenyl)-2-methyl-3-pyridinecarboxylic acid ethyl ester (13)
Following the method described in Section 4.3, the reaction
of ethyl 2-cyanoacetimidate hydrochloride (27) (545.0 mg,
3.69 mmol) and ammonium acetate (915.9 mg, 11.8 mmol),
in methanol (15 mL), with compound 23 [20] (850.0 mg,
3.05 mmol), after 35 min, afforded compound 13 (797.0 mg,
77%): mp 166e168 ꢂC; IR (KBr) n 3420, 3348, 3224, 2934,
4.2. Ethyl ester 2-[(4-biphenyl)methylene]-3-oxo-
butanoic acid (24)
To a solution of ethyl acetoacetate (1.42 g, 10.9 mmol) in
dry toluene (30 mL) biphenyl-4-carboxaldehyde (2.0 g,
10.9 mmol) and piperidine (15 drops) were added. After 4 h,
the solvent was removed and the crude mixture was submitted
to chromatography (hexane:AcOEt, 15%) to give compound 24
(2.45 g, 76%), as a mixture of Z/E isomers in a 67:33 ratio: oil;
IR (KBr) n 3072, 2985, 1716, 1662, 1623, 1603, 1487,
1232 cmꢁ1; 1H NMR (DMSO-d6, 300 MHz) (major Z isomer)
d 7.75e7.38 (m, 10H, C6H4eC6H5, HC]C), 4.35 (q, J ¼
6.9 Hz, 2H, OCH2CH3), 2.45 [s, 3H, CH3(CO)], 1.56 (t,
2898, 2833, 2179, 1650, 1513, 1266 cmꢁ1
;
1H NMR
(DMSO-d6, 300 MHz) d 8.90 (s, 1H, NH), 6.86 (d, J ¼
8.3 Hz, 1H), 6.71 (d, J ¼ 2.0 Hz, 1H), 6.64 (dd, J ¼ 8.3,
2.0 Hz, 1H), 5.74 (s, 2H, NH2), 4.31 (s, 1H, H4), 3.96 (q,
J ¼ 7.1 Hz, 2H, OCH2CH3), 3.70 (s, 6H, 2 ꢃ OCH3), 2.25 [s,
3H, CH3], 1.10 (t, J ¼ 7.1 Hz, 3H, CO2CH2CH3); 13C NMR
(DMSO-d6, 75 MHz) d 166.6 (C]O), 150.4 (C2), 148.2e
110.8 (aromatic), 147.2 (C6), 121.8 (CN), 100.9 (C3), 59.0
(OCH2CH3), 57.3 (C5), 55.4, 55.3 (2C, 2 ꢃ OCH3), 39.7

R. Leo´n et al. / European Journal of Medicinal Chemistry 43 (2008) 668e674
673
(C4), 18.5 (CH3), 14.0 (CH3CH2O); MS (API-ESþ) m/z:
[M þ 1]þ 344.0; [M þ Na]þ 382.1; [2M þ 1]þ 687.5;
[2M þ Na]þ 709.3. Anal. Calcd. for C18H21N3O4: C, 62.96;
H, 6.16; N, 12.24. Found: C, 62.69; H, 6.25; N, 12.43.
(DMSO-d6, 300 MHz) d 8.77 (s, 1H, NH), 8.47 (dd, J ¼ 4.5,
1.5 Hz, 2H), 7.12 (dd, J ¼ 4.5, 1.5 Hz, 2H), 5.81 (s, 2H,
NH2), 4.36 (s, 1H, H4), 3.93 (q, J ¼ 7.1 Hz, 2H, OCH2CH3),
2.29 [s, 3H, CH3], 1.04 (t, J ¼ 7.1 Hz, 3H, CO2CH2CH3);
13C NMR (DMSO-d6, 75 MHz) d 166.5 (C]O), 155.9 (C2),
151.0 (C6), 150.0e122.2 (aromatic), 121.6 (CN), 99.6 (C3),
59.5 (OCH2CH3), 56.4 (C5), 40.4 (C4), 19.0 (CH3), 14.3
(CH3CH2O); MS (API-ESþ) m/z: [M þ 1]þ 285.2; [M þ Na]þ
307.3; [2M þ 1]þ 569.5; [2M þ Na]þ 591.5. Anal. Calcd. for
C15H16N4O2: C, 63.37; H, 5.67; N, 19.71. Found: C, 63.24;
H, 5.86; N, 19.70.
4.3.3. 6-Amino-5-cyano-1,4-dihydro-2-methyl-4-
(4-biphenyl)-3-pyridinecarboxylic acid ethyl ester (14)
Following the method described in Section 4.3, the reac-
tion of ethyl 2-cyanoacetimidate hydrochloride (27)
(303.89 mg,
2.03 mmol)
and
ammonium
acetate
(507.83 mg, 6.59 mmol), in methanol (15 mL), with com-
pound 24 (500 mg, 1.69 mmol), after 35 min, gave com-
pound 14 (363 mg, 60%): mp 217e219 ꢂC; IR (KBr) n
3405, 3346, 3225, 2876, 2179, 1630, 1662, 1494, 1369,
4.3.6. 2-Amino-7,7-dimethyl-5-oxo-4-(4-pyridyl)-
1,4,5,6,7,8-hexahydro-3-quinolinecarbonitriles (21)
1270, 1221 cmꢁ1
;
1H NMR (DMSO-d6, 300 MHz) d 8.08
A solution of 3-amino-5,5-dimethyl-2-cyclohexenone (29)
[24] (899.0 mg, 6.44 mmol) and ammonium acetate (1.48 g,
19.33 mmol), in methanol (20 mL), was warmed at reflux,
for 15 min. Then, [2-(4-pyridyl)methylene]malononitrile (28)
[23] (1.0 g, 6.44 mmol) was added, and the mixture was re-
fluxed for 3 h 30 min. The mixture was cooled at 5 ꢂC over-
night, the solid was filtered, washed with cold methanol and
recrystallized from methanol to yield compound 21 (1.35 g,
71%): mp 253e255 ꢂC; IR (KBr) n 3385, 3329, 3220, 2963,
(s, 1H, NH), 7.60 (d, J ¼ 8.8 Hz, 2H), 7.58 (d, J ¼ 8.8 Hz,
2H), 7.43 (t, J ¼ 7.5 Hz, 2H), 7.32 (t, J ¼ 7.5 Hz, 1H),
7.20 (d, J ¼ 7.5 Hz, 2H), 5.74 (s, 2H, NH2), 4.39 (s, 1H,
H4), 3.94 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 2.27 [s, 3H,
CH3], 1.08 (t, J ¼ 7.1 Hz, 3H, CO2CH2CH3); 13C NMR
(DMSO-d6, 75 MHz) d 166.5 (C]O), 150.4 (C2), 146.9e
128.8 (aromatic), 145.2 (C6), 121.7 (CN), 100.6 (C3),
59.1 (OCH2CH3), 57.2 (C5), 39.8 (C4), 18.7 (CH3), 14.0
(CH3CH2O); EM (API-ESþ) m/z: [M þ 1]þ 360.1;
[M þ Na]þ 382.1; [2M þ 1]þ 719.2; [2M þ Na]þ 741.2.
Anal. Calcd. for C22H21N3O2: C, 73.52; H, 5.89; N,
11.69. Found: C, 73.27; H, 5.90; N, 11.83.
1
2884, 2183, 1657, 1598, 1477, 1370, 1270 cmꢁ1; H NMR
(DMSO-d6, 300 MHz) d 8.97 (s, 1H, NH), 8.43 (d, J ¼
6.0 Hz, 2H), 7.11 (d, J ¼ 6.0 Hz, 2H), 5.86 (s, 2H, NH2),
4.32 (s, 1H, H4), 2.45e2.29 (m, 2H, 2H6), 2.20e1.97 (m,
2H, 2H8), 1.05, 0.90 (s, s, 3H, 3H, 2 ꢃ CH3); 13C NMR
(DMSO-d6, 75 MHz) d 194.3 (C5, C]O), 155.4 (C2), 150.9
(C8a), 150.7e122.5 (aromatic), 121.5 (CN), 107.8 (C4a),
57.5 (C3), 50.3 (C6), 40.1 (C8), 37.2 (C4), 32.4 (C7), 29.1,
27.0 (2C, 2 ꢃ CH3); MS (API-ESþ) m/z: [M þ 1]þ 295.1;
[M þ Na]þ 317.1; [2M þ Na]þ 611.3. Anal. Calcd. for
C17H18N4O: C, 69.37; H, 6.16; N, 19.03. Found: C, 69.50;
H, 6.21; N, 18.91.
4.3.4. 6-Amino-5-cyano-1,4-dihydro-2-methyl-4-(3-pyridyl)-
3-pyridinecarboxylic acid ethyl ester (15)
Following the method described in Section 4.3, the reaction
of ethyl 2-cyanoacetimidate hydrochloride (27) (883.2 mg,
5.9 mmol) and ammonium acetate (1.37 g, 17.9 mmol), in
methanol (15 mL), with compound 25 [21] (1 g, 4.56 mmol),
after 25 min, gave product 15 (855 mg, 66%): mp 226e
228 ꢂC; IR (KBr) n 3401, 2971, 2920, 2182, 1661, 1491,
1
1327 cmꢁ1; H NMR (DMSO-d6, 300 MHz) d 8.74 (s, 1H,
4.4. Pharmacological protocols
NH), 8.38e7.30 (m, 4H, aromatic), 5.77 (s, 2H, NH2), 4.40
(s, 1H, H4), 3.94 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 2.28 [s,
3H, CH3], 1.04 (t, J ¼ 7.1 Hz, 3H, CO2CH2CH3); 13C NMR
(DMSO-d6, 75 MHz) d 166.5 (C]O), 150.8 (C2), 148.4e
124.0 (aromatic), 146.2 (C6), 121.7 (CN), 100.2 (C3), 59.5
(OCH2CH3), 57.1 (C5), 37.1 (C4), 18.7 (CH3), 14.3
(CH3CH2O); MS (API-ESþ) m/z: [M þ 1]þ 285.2; [M þ Na]þ
307.3; [2M þ 1]þ 569.5; [2M þ Na]þ 591.5. Anal. Calcd. for
C15H16N4O2: C, 63.37; H, 5.67; N, 19.71. Found: C, 63.07;
H, 5.90; N, 20.02.
4.4.1. Culture of SH-SY5Y cells
SH-SY5Y cells, at passages between 3 and 16 after de-
freezing, were maintained in a Dulbecco’s modified Eagle’s
medium (DMEM) containing 15 non-essential amino-acids
(NEAAs) and supplemented with 10% fetal calf serum
(FCS), 1 mM glutamine, 50 units/mL penicillin and 50 mg/
mL streptomycin (reagents from Gibco, Madrid, Spain).
Cultures were seeded into flasks containing supplemented
medium and maintained at 37 ꢂC in 5% CO2/humidified air.
Stock cultures were passaged 1:4 twice weekly. For assays,
SH-SY5Y cells were sub-cultured in 24-well plates at a seed-
ing density of 2 ꢃ 105 cells per well, or in 96-well plates at
a seeding density of 8 ꢃ 104 cells per well. For the cytotoxicity
experiments cells were treated with drugs before confluence,
in DMEM free of serum.
4.3.5. 6-Amino-5-cyano-1,4-dihydro-2-methyl-4-(4-pyridyl)-
3-pyridinecarboxylic acid ethyl ester (16)
Following the method described in Section 4.3, the reaction
of ethyl 2-cyanoacetimidate hydrochloride (27) (816.0 mg,
5.47 mmol) and ammonium acetate (1.37 g, 17.9 mmol), in
methanol (15 mL), with compound 26 [21] (1 g, 4.56 mmol),
after 30 min, gave product 16 (855 mg, 66%): mp 252e
254 ꢂC; IR (KBr) n 3425, 3347, 3228, 2981, 2172, 1642,
4.4.2. Measurement of lactic dehydrogenase (LDH) activity
Extracellular and intracellular LDH activity was spectro-
photometrically measured using a Cytotoxicity Cell Death
;
1592, 1485, 1370, 1328, 1264, 1217 cmꢁ1 1H NMR

674
R. Leo´n et al. / European Journal of Medicinal Chemistry 43 (2008) 668e674
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