Condensed 1,4-dihydropyridines with various esters and their calcium channel antagonist activities

Article abstract of DOI:10.1016/j.ejmech.2008.10.008

New alkyl 2,6,6-(2,7,7)-trimethyl-4-(2-fluoro-3-chloro-5-trifluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylates and 9-(3-chloro-2-fluoro-5-trifluoromethylphenyl)-6,6(7,7)-dimethyl-6,7-dihydrofuro[3,4-b]quinoline-1,8-diones have been sy

Full text of DOI:10.1016/j.ejmech.2008.10.008

European Journal of Medicinal Chemistry 44 (2009) 2052–2058
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Condensed 1,4-dihydropyridines with various esters and their calcium
channel antagonist activities
a
b
b
a
b
¨
Bahadir Bu¨lbu¨l , Go¨kçe Sevim Oztu¨rk , Mert Vural , Rahime S¸ims¸ek , Yusuf Sariog˘lu ,
c
d
a,
*
¨
Anthony Linden , Mert Ulgen , Cihat S¸afak
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey
b Department of Pharmacology, Faculty of Medicine, Gazi University, Ankara, Turkey
c Institute of Organic Chemistry, University of Zurich, Zurich, Switzerland
d
_
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Istanbul, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
New alkyl 2,6,6-(2,7,7)-trimethyl-4-(2-fluoro-3-chloro-5-trifluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hex-
ahydroquinoline-3-carboxylates and 9-(3-chloro-2-fluoro-5-trifluoromethylphenyl)-6,6(7,7)-dimethyl-
6,7-dihydrofuro[3,4-b]quinoline-1,8-diones have been synthesised and their calcium antagonistic
activities on isolated rabbit sigmoid colon have been investigated and compared with Nifedipine. The
investigation examined the influence of ester groups in the 3-position of the HHQ ring and the 2-
methoxyethyl analogs were found to be the most active derivatives.
Received 19 March 2008
Received in revised form
3 July 2008
Accepted 9 October 2008
Available online 1 November 2008
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords:
Hexahydroquinoline
Furoquinoline
Calcium antagonistic activity
Biotransformation
1. Introduction
nondihydropyridines (non-DHP, represented by verapamil and
diltiazem) (Fig. 2).
According to the WHO (World Health Organization) reports, 17.5
million people worldwide died from cardiovascular disease in 2005
in the World. This represents 30% of all global deaths. Of these
deaths, 7.6 million were due to heart attacks and 5.7 million were
due to stroke. Cardiovascular diseases include disorders of the heart
and blood vessels, hypertension, peripheral artery disease, rheu-
matic heart disease, congenital heart disease and heart failure.
Calcium channel antagonists inhibit muscle contraction by block-
ing the influx of Ca2þ through calcium channels and are used as anti-
anginal and antihypertensive drugs [1–11].1,4-Dihydropyridine (DHP)
derivatives are the most studied group and Nifedipine is the prototype
of calcium channel antagonists. Nifedipine (Fig. 1) was synthesised in
1882 and introduced to therapy 100 years later [12,13].
Many active compounds have been synthesised by making
modifications on the Nifedipine molecule. One of these modifica-
tions is to replace the ester function by the various acyl groups such
as amide, nitro, nitrile, ketone. It is well known that, in the
unsymmetrical diacyl series, when one of the acyl groups is
a methyl or ethyl ester, the activity increases due to lipophilic
properties. In addition, in order to fix one carbonyl group in an
antiperiplanar position, the 1,4-DHP structure has been annelated.
Thus condensed 1,4-DHP derivatives have been obtained and these
analogs showed calcium antagonistic activity [14–28].
According to structure–activity relationships of Nifedipine
analogs the C-2 and C-6 substituents and the C-3 and C-5 esters
must be the same. Although asymmetrical derivatives also exert
calcium channel blocker effects, the biological activity depends on
the presence of an axial aromatic ring substituent at the 4-position
of the 1,4-DHP. The C-3 and C-5 substituents restrict the orientation
of the aromatic ring such that its plane bisects the DHP. The DHP
ring also adopts a flattened boat conformation. Calcium channels
Today, calcium entry blockers are divided into two struc-
tural groups the 1,4-DHPs, represented by Nifedipine, and
Abbreviations: WHO, World Health Organization; DHP, dihydropyridine; HHQ,
hexahydroquinoline; eNOS, endothelial nitric oxide synthase; COX, cyclooxygenase;
are like a pore having one a1 primary subunit and a2
-d, b and g
subunits. 1,4-DHP derivatives have their binding location on the a1
subunit. These drugs are used as an antihypertensive and anti-
anginal agents by effecting the transportation of Ca2þ through the L
type calcium channels [10,29].
L
-NAME,
Nu-nitro-L-arginine methyl ester; TEA, tetraethylammonium; DMSO,
dimethylsulfoxide.
* Corresponding author. Tel.: þ90 312 3052341; fax: þ90 312 3051872.
E-mail address: csafak@hacettepe.edu.tr (C. S¸afak).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.008

B. Bu¨lbu¨l et al. / European Journal of Medicinal Chemistry 44 (2009) 2052–2058
2053
seen. In the 1H NMR spectra, 6,6-dimethyl- or 7,7-dimethyl protons
of the HHQ and furoquinoline ring were seen at 0.80–0.90 ppm as
separate singlets. The chemical shifts of the aromatic, 2-methyl,
methylene, methine, and ester protons of the compounds have
expected values. The N–H signals were seen at 9.30–10.00 ppm.
The 13C NMR spectra of the compounds displayed the appropriate
number of resonances that exactly fitted the number of carbon
atoms. The DEPT spectra of one compound are in accordance with
its structure. The DEPT spectra of compound 4m, displayed three
methine, two methylene and six methyl peaks. In the COSY spec-
trum of the compound 4m, 2,3,5-trisubstituted aryl ring protons
resonate at 7.41–7.42 and 7.76–7.77 ppm.
The mass spectra of the compounds were recorded using the
electrospray ionisation technique. Molecular ion peaks were seen
in the spectra of all compounds. The base peak forms by cleavage of
the aryl ring from the parent molecule. In further fragmentation,
the ion is formed by the cleavage of the ester group. Aromatisation
of the DHP ring to the pyridine analogue was also realised. These
findings are in accordance with the literature [22–24]. The
elemental analysis results are also consistent with the postulated
structures. The X-ray crystal structure analysis1 of compound 4f
confirmed the structure of this compound (Fig. 3).
NO2
H3COOC
H3C
COOCH3
N
H
CH3
Fig. 1. Nifedipine.
Voltage-gated calcium channels are integral membrane proteins
that open via electrical depolarization of the plasma membrane and
mediate the entry of calcium ions into the cell. Calcium channels
are found in every excitable cell, including neurons, myocytes and
pancreatic
b cells and form the most efficient molecular link
between membrane depolarization and intracellular biochemical
signaling. Ca2þ entering the cell through voltage-gated Ca2þ
channels serves as the second messenger of electrical signaling,
initiating intracellular events such as contraction, secretion,
synaptic transmission and gene expression [1].
The cytosolic calcium concentration regulates many cellular
functions. The influx of extracellular Ca2þ is a prerequisite for many
cellular functions including cell proliferation and motility. In
gastrointestinal smooth muscle, the upstroke of action potential is
principally mediated by Ca2þ influx through voltage-dependent L-
type Ca2þ channels and is responsible for initiation of contraction.
Calcium influx through voltage-gated channels contributes to this
rise in cytosolic calcium. Because of their central role in the exci-
tation–contraction coupling for cardiac and smooth muscle,
voltage-sensitive calcium channels have become a target for the
drug therapy of cardiovascular and gastrointestinal diseases [6,30].
The aim of this work was to synthesise the compounds having
a 1,4-DHP ring in a condensed system with different ester moieties
in the C-3 position and also including furoquinoline derivatives as
a metabolite of this group of compounds and to screen calcium
channel blocker activity. In this paper the influence of the different
ester groups in the C-3 position of the condensed 1,4-DHP ring was
evaluated. Synthesised compounds were tested as a racemate. The
cardiovascular activities of the synthesised compounds were
assayed on isolated rabbit sigmoid colon by using Nifedipine as
standard.
In this structure, intermolecular hydrogen bonds between the
amine group and the oxo O atom of a neighbouring molecule link
the molecules into infinite one-dimensional chains.
2.2. Pharmacology
The maximum relaxant effects (Emax) and pD2 values of the
compounds and Nifedipine on isolated strips of rabbit sigmoid
colon circular smooth muscle are given in Table 1. Nifedipine and
several compounds produced concentration-dependent relaxation
in rabbit sigmoid colon circular smooth muscle strips, such that
Nifedipine>. 4k ꢀ 4d ꢀ 4h > 4b ¼ 4i ꢀ 4a. Compounds 4c, 4e–g,j,l,n,
5a,b displayed inhibitory responses, which were not statistically
significant from control inhibition produced by DMSO.
The main site of action of the Ca2þ channel blockers is believed
to be the voltage-dependent channels, where inhibition of the
influx of extracellular Ca2þ results in uncoupling of excitation and
contraction. Calcium channel blockers may not only directly
decrease the concentration of cytoplasmic Ca2þ but also cause
a decrease in Ca2þ release from intracellular stores [32].
The Ca2þ channel types described for smooth muscle consist of
the L- and T-types, with slow and fast inactivation characteristics,
respectively [31]. L-type Ca2þ channels are inhibited by Mg2þ and
by DHPs such as Nifedipine, nimodipine and isradipine [32]. The
function of L-type Ca2þ channels can be regulated both by guano-
sine 50-triphosphate(GTP)-binding proteins and by phosphoryla-
tion, the latter often having a negative effect on smooth muscle
[32–35].
Given the overall powerful selectivity of action of the clinically
available 1,4-DHPs for the L-type channel, some evidence suggests
that a component of their vascular selective and vasodilating
properties may arise from an ability to stimulate nitric oxide release
from vascular endothelial cells [36]. Such an action would add to
the vasodilation produced by directing the blockade of the calcium
channels in vascular smooth muscle cells. Endothelial nitric oxide
2. Results and discussion
2.1. Chemistry
The hexahydroquinoline (HHQ) derivatives (4a–n) were
prepared by a modification of the Hantzsch synthesis [31]. Thus,
4,4-dimethyl (or 5,5-dimethyl)-1,3-cyclohexanedione was treated
with the 2-fluoro-3-chloro-5-trifluoromethyl benzaldehyde to
yield the benzylidine derivatives which, when condensed with
alkyl acetoacetate, yielded the corresponding HHQ derivatives
(Scheme 1).
The furoquinoline derivatives (5a,b) were obtained by using the
procedure described by S¸ims¸ek and coworkers [10,23] In this
procedure, the appropriate HHQ derivative reacted in the presence
of pyridinium bromide perbromide (Scheme 2).
synthase (eNOS) converts substrate L-arginine to nitric oxide, which
The reaction of the HHQ derivative and pyridinium bromide
perbromide results in allylic bromination and forms 2-bromome-
thylhexahydroquinoline derivatives. This intermediate could not be
isolable. Then intermolecular cyclisation of 2-bromomethyl deriv-
atives gives lactone analogs.
diffuses to the vascular smooth muscle cells and promotes vascular
relaxation [37–39]. The nitric oxide releasing actions of the 1,4-
DHPs appear to be a class action, to be exerted at therapeutic
The structures of the compounds were elucidated by IR, 1H NMR,
13C NMR, COSY, DEPT, mass spectra and elemental analyses. In the
IR spectra, characteristic N–H and C]O stretching bonds were
1
CCDC-677108 contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge Crystallographic
Data Centre via, www.ccdc.cam.ac.uk/data_request/cif.

2054
B. Bu¨lbu¨l et al. / European Journal of Medicinal Chemistry 44 (2009) 2052–2058
S
O
C
O
CH3
H3CO
H3CO
CH3
CH3
N
CN
C
O
CH2
CH2
N
(CH2)2
N
(CH2)3
CH3
CH
CH3
CH3
H3C
CH3
Verapamil
Diltiazem
Fig. 2. Verapamil and Diltiazem.
concentrations and to involve the stimulation of calcium influx into
endothelial cells through a non-L-type process [40–42].
To investigate whether relaxation induced by the test
compounds was due to interaction with the cyclooxygenase (COX),
adrenergic system or nitric oxide pathways tissues, were pretreated
with indomethacin (COX inhibitor), guanethidine (an adrenergic
equation for drug–receptor interaction. Agonist pD2 values
(apparent agonist affinity constants) were calculated from each
agonist concentration–response curve by linear regression of the
linear part of the curve and taken as a measure of the sensitivity of
the tissues to each agonist. All data are expressed as mean -
ꢁ standard error. Statistical comparison between groups was per-
formed using general linear models by Scheffe’s F-test and P values
of less than 0.05 were considered to be statistically significant.
nerve blocker) or N
hydrochloride (the nitric oxide synthase inhibitor), respectively.
Pretreatment of the strips with indomethacin, guanethidine and
u-nitro-L-arginine methyl ester (L-NAME)
L-
NAME did not significantly alter the relaxant responses to the
compounds indicating that cyclooxygenase, adrenergic and nitric
oxide (NO) pathways do not play a role in relaxations evoked by
these substances.
3. Conclusions
In this study, new condensed 1,4-DHP derivatives have been
synthesised by the reaction of substituted cyclohexanedione
derivatives, various alkyl acetoacetate esters and trisubstituted
aromatic aldehydes according to modified Hantzsch synthesis. In
pharmacological screening tests, the obtained results showed that
condensed 1,4-DHP derivatives exerted calcium channel blocking
effects. The inclusion of two or three electron-withdrawing
substituents on aromatic ring does not change the mentioned
activity. The investigation examined the influence of ester groups in
the 3-position of the HHQ ring and the 2-methoxyethyl analogs
were found to be the most active derivatives. The metabolite of the
HHQ derivative (compound 4a), which has a furoquinoline struc-
ture, (compound 5a) was found less active.
Compound 5b did not induce relaxation responses in the pres-
ence of tetraethylammonium (TEA) but induced relaxation
responses (%49.19 ꢁ 6.83) in the absence of TEA. Similarly,
compound 4a-induced relaxation responses in the absence of TEA
(%36.87 ꢁ4.52) were higher than in the presence of TEA
(%17.19 ꢁ 3.15). These results suggest that compounds 5b and 4a
had a potency for relaxing isolated rabbit sigmoid colon circular
smooth muscle, due to the activation of Kþ channels. The absence of
TEA did not alter the relaxation responses induced by Nifedipine
and the other compounds. These results suggest that the tested
compounds, other than 5b and 4a, have a potency for relaxing
isolated rabbit sigmoid colon circular smooth muscle, a result of the
possible blockade of Ca 2þ channels. To ascertain the ability of these
compounds to block Ca2þ channels further investigation is needed.
The in vitro hepatic microsomal biotransformation of compound
4a gives its lactone derivatives. This phenomenon was verified by
HPLC. The retention times of the HHQ (4a) and the furoquinoline
derivatives (5a) were 10.68 and 16.69 min, respectively. This study
gives evidence that HHQ derivatives converted to their less active
lactone derivatives during in vitro hepatic microsomal biotrans-
formation. These findings are in accordance with the literature.
The relaxant effects of the compounds were expressed as
percentage of the precontraction using Caþ2. To evaluate the effects
of the compounds, the maximum response (Em) and pD2 values [the
negative logarithm of the concentration for the half-maximal
response (EC50)] were calculated, as predicted from the Scatchard
4. Experimental protocol
4.1. Chemistry
Melting points were determined on a Thomas Hoover Capillary
Melting Point Apparatus and uncorrected values. Infrared spectra
were recorded on a Perkin Elmer FT-IR Systems Spectrum BX. 1H
NMR and 13C NMR spectra were obtained for DMSO solutions on
a Varian Mercury 400, 400 MHz High Performance Dijital FT-NMR
Spectrometer. Chemical shifts are reported in parts per million
relative to tetramethylsilane, spin multiplicities are given as s
(singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Mass
spectra were obtained on a Waters 2996 Photoiodide array
dedector. Microanalysis was obtained on
a Leco CHNS-932
CF3
Cl
F
CHO
O
O
F
COOR
O
C
O
C
CH3COONH4
H3C
H3C
H3C
H3C
+
+
CH3
CH2
OR
CF3
Cl
N
H
CH3
O
R: -CH3, -C2H5, -CH2CH=CH2, -CH2CH2OCH3,
-CH2CH(CH3)2, -C(CH3)3, -CH2C6H5
4a-4n
Scheme 1. Synthesis of hexahydroquinoline derivatives.

B. Bu¨lbu¨l et al. / European Journal of Medicinal Chemistry 44 (2009) 2052–2058
2055
CF3
Cl
CF3
Cl
F
-
Br3
O
F
+
O
O
N
COOR
H3C
H3C
H
H3C
H3C
O
N
H
CH3
N
H
5a-5b
Scheme 2. Synthesis of furoquinoline derivatives.
Elemental Analyzer and the results were within ꢁ0.4% of the
4.1.1.3. Allyl 2,6,6-trimethyl-4-(2-fluoro-3-chloro-5-trifluoromethyl-
phenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate
theoretical values.
(4c). Yield 29%. M.p. 194 ꢂC. IR 3213, 1709, 1652, 860. 1H NMR
d 0.80
4.1.1. Alkyl 2,6,6-(2,7,7)-trimethyl-4-(2-fluoro-3-chloro-5-
trifluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylates (4a–n)
The ester derivatives were prepared by the reaction of an
equimolar amount of alkyl acetoacetate, 4,4-(5,5)-dimethyl-1,3-
cyclohexanedione, and 2-fluoro-3-chloro-5-trifluoromethyl benz-
aldehyde with ammonium acetate in methanol and crystallised
from methanol–water.
(3H; s; 6-CH3), 0.95 (3H; s; 6-CH3), 1.65–1.72 (4H; m; H 7–8), 2.27
(3H; s; 2-CH3), 4.40–4.44 (2H; d; OCH2), 5.03 (1H; s; H-4), 5.10 (2H;
d; CH]CH2), 5.80 (1H; m; CH]CH2), 7.40–7.42 (1H; d; Ar-H), 7.76–
7.78 (1H; d; Ar H), 9.28 (1H; s; NH). 13C NMR
d 18.2, 22.7, 24.0, 24.4,
33.1, 33.8, 63.8, 100.3, 107.4, 117.3, 120.7, 121.6, 124.3, 125.3, 126.4,
132.6, 137.4, 146.9, 150.4, 155.7, 158.2, 165.7 204.0. Mass (m/z) 472,
235, 229, 158, 141. Anal. calcd. C23H22ClF4NO3 (C, H, N).
4.1.1.4. 2-Methoxyethyl
2,6,6-trimethyl-4-(2-fluoro-3-chloro-5-tri-
4.1.1.1. Methyl 2,6,6-trimethyl-4-(2-fluoro-3-chloro-5-trifluoromethyl-
phenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate
fluoromethyl phenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carbox-
ylate (4d). Yield 23%. M.p. 183 ꢂC. IR 3186, 1711, 1652, 862. 1H NMR
(4a). Yield 61%. M.p. 197 ꢂC. IR 3288, 1709, 1652, 860. 1H NMR
d
0.81
d 0.82 (3H; s; 6-CH3), 0.97 (3H; s; 6-CH3), 1.65–1.76 (4H; m; H 7–8),
(3H; s; 6-CH3), 0.96 (3H; s; 6-CH3), 1.67–1.74 (4H; m; H 7-8), 2.27
(3H; s; 2-CH3), 3.49 (3H; s; OCH3), 5.08 (1H; s; H-4), 7.40–7.42 (1H;
2.28 (3H; s; 2-CH3), 3.19 (2H; t; CH2-OCH3), 3.42 (3H; s; OCH3), 4.40–
4.44 (2H; t; OCH2), 5.08 (1H; s; H-4), 7.45–7.59 (1H; d; Ar-H), 7.73–
d; Ar-H), 7.80–7.82 (1H; d; Ar H), 9.30 (1H; s; NH). 13C NMR
d
18.6,
7.80 (1H; d; Ar H), 9.41 (1H; s; NH). 13C NMR
d 18.7, 23.3, 23.5, 24.8,
24.6, 24.9, 33.5, 39.4, 39.6, 51.0, 101.3, 108.0, 121.0, 125.3, 126.0, 127.7,
147.0, 151.0, 158.7, 167.3, 199.7. Mass (m/z) 446, 413, 248, 163, 136, 101,
88, 73, 59. Anal. calcd. C21H20ClF4NO3 (C, H, N).
33.6, 39.3, 40.1, 58.3, 62.7, 70.1, 100.9, 109.0, 121.1, 121.3, 122.2, 125.0,
125.2,126.9,127.7,150.7,158.9,166.6,199.7. Mass (m/z) 490, 414, 271,
136, 101, 59. Anal. calcd. C23H24ClF4NO4 (C, H, N).
4.1.1.2. Ethyl 2,6,6-trimethyl-4-(2-fluoro-3-chloro-5-trifluoromethyl-
phenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate
4.1.1.5. Isobutyl 2,6,6-trimethyl-4-(2-fluoro-3-chloro-5-trifluoromethyl-
phenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate
(4b). Yield 46%. M.p. 210 ꢂC0. IR 3285,1702,1651, 861. 1H NMR
d
0.82
(4e). Yield 39%. M.p. 179 ꢂC. IR 3086, 1703, 1652, 860. 1H NMR
d 0.69–
(3H; s; 6-CH3), 0.97 (3H; s; 6-CH3), 1.02 (3H; t; CH2CH3), 1.64-1. (4H;
m; H 7–8), 2.27 (3H; s; 2-CH3), 3.90 (2H; q; CH2CH3), 5.06 (1H; s; H-
4), 7.30–7.44 (1H; d; Ar-H), 7.79–7.90 (1H; d; Ar H), 9.16 (1H; s; NH).
0.70 (3H; d; CH-CH3), 0.77–0.79 (3H; d; CH-CH3), 0.81 (3H; s; 6-CH3),
0.96 (3H; s; 6-CH3), 1.66–1.78 (4H; m; H 7-8), 2.28 (3H; s; 2-CH3), 2.29
(3H; s; 2-CH3), 3.35 (2H; d; OCH2), 3.63–3.77 (1H; m; CH2-CH), 5.08
(1H; s; H-4), 7.43–7.45 (1H; d; Ar-H), 7.81–7.83 (1H; d; Ar H), 9.32 (1H;
13C NMR
d 14.1, 23.3, 24.9, 33.6, 39.5, 39.6, 101.2, 107.9, 121.0, 121.2,
122.3, 124.9, 125.9, 126.2, 126.8, 127.6, 147.0, 151.0, 158.7, 166.6,
199.7. Mass (m/z) 460, 381, 363, 364, 152, 150, 136, 73, 70, 59. Anal.
calcd. C22H22ClF4NO3 (C, H, N).
s; NH). 13C NMR
d 18.8, 19.2, 23.3, 24.5, 25.0, 27.6, 33.6, 39.3, 39.6, 69.9,
Table 1
Maximum relaxant responses (Emax) and pD2 values of the compounds 4a–n, 5a,b
and Nifedipine on isolated strips of rabbit sigmoid colon circular smooth muscle.
Relaxation responses are expressed as a percentage of the relaxations evoked by
papaverine (10ꢃ4 M). The negative logarithm of the concentration for the half-
maximal response (pD2) and Emax values represents mean value ꢁ S.E.M. (*p < 0.05,
compared with control responses) (n ¼ 6).
Compound
Emax
pD2
4a*
4b*
4c
4d*
4e
4f
4g
4h*
4i*
4j
4k*
4l
4m
4n
5a
5b
17.19 ꢁ 3.15
22.97 ꢁ 7.59
No activity
43.67 ꢁ 5.01
No activity
No activity
No activity
32.22 ꢁ 4.90
22.83 ꢁ 4.88
No activity
44.99 ꢁ 4.07
No activity
No activity
No activity
No activity
No activity
94.02 ꢁ 3.58
4.94 ꢁ 0.21
5.06 ꢁ 0.22
No activity
4.89 ꢁ 0.14
No activity
No activity
No activity
4.84 ꢁ 0.07
4.74 ꢁ 0.12
No activity
5.11 ꢁ 0.06
No activity
No activity
No activity
No activity
No activity
6.92 ꢁ 0.05
Nifedipine
Fig. 3. X-ray diagram of compound 4f.

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B. Bu¨lbu¨l et al. / European Journal of Medicinal Chemistry 44 (2009) 2052–2058
101.0, 107.9, 121.1, 121.3, 122.2, 125.8, 125.9, 126.8, 147.3, 150.9, 158,7
166.7, 199.7. Mass (m/z) 488, 441, 413, 148, 136, 104, 101, 88, 73, 59.
Anal. calcd. C24H26ClF4NO3 (C, H, N).
3.32–3.37 (2H; s; H-8), 3.43 (3H; s; OCH3), 3.93–4.08 (2H, t, CH2-
CH2), 5.05 (1H; s; H-4), 7.20–7.75 (1H; d; Ar-H), 7.80–8.20 (1H; d;
ArH), 9.40 (1H; S; NH). 13C NMR
d 18.8, 26.2, 29.5, 32.0, 32.6, 49.0,
50.3, 58.3, 62.8, 70.1, 101.4, 108.7, 119.5, 121.3, 122.2, 126.2, 126.9,
127.6, 147.5, 150.8, 158.6, 166.6, 194.5. Mass (m/z) 490, 441, 414, 413,
136, 101, 73, 59. Anal. calcd. C23H24ClF4NO4 (C, H, N).
4.1.1.6. Terbutyl 2,6,6-trimethyl-4-(2-fluoro-3-chloro-5-tri-
fluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (4f). Yield 23%. M.p. 227 ꢂC. IR 3286, 1702,1652, 862. 1H
NMR
d
0.80 (3 H; s; 6-CH3), 0.94 (3H; s; 6-CH3), 1.25 (9H; s;
4.1.1.12. Isobutyl 2,7,7-trimethyl-4-(2-fluoro-3-chloro-5-tri-
fluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (4l). Yield 43%. M.p. 180 ꢂC. IR 3314, 1708, 1650, 862. 1H
C(CH3)3), 1.65–1.69 (4H; m; H7-8), 2.21 (3H; s; 2-CH3), 4.99 (1H; s;
H-4), 7.41–7.43 (1H; d; Ar-H), 7.79–7.81 (1H; d; Ar H), 9.15 (1H; s;
NH). 13C NMR
d
18.8, 23.5, 24.8, 25.3, 28.3, 28.3, 28.3, 34.5, 34.6,
NMR d 0.69 (3H; d; CH-CH3), 0.79 (3H; d; CH-CH3), 0.82 (3H; s; 7-
39.5, 79.8, 102.8, 107.6, 121.3, 121.5, 125.9, 127.3, 138.2, 146.1, 151.3,
156.6, 159.15, 166.4, 199.9. Mass (m/z) 488, 413, 148, 136, 101, 88, 73,
59. Anal. calcd. C24H26ClF4NO3 (C, H, N).
CH3),1.01 (3H; s; 7-CH3),1.76–2.26 (2H; m; H-8), 2.31 (3H; s; 2-CH3),
2.44–2.52 (2H; m; H-6), 3.36 (2H; s; OCH2), 3.64–3.79 (1H; m; CH2-
CH), 5.11 (1H; s; H-4), 7.44–7.46 (1H; d; Ar-H), 7.81–7.83 (1H; d; Ar-
H), 9.32 (1H; s; NH). 13C NMR
d 18.9, 19.1, 19.2, 26.2, 27.5, 29.5, 32.6,
4.1.1.7. Benzyl 2,6,6-trimethyl-4-(2-fluoro-3-chloro-5-tri-
fluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (4g). Yield 21%. M.p. 198 ꢂC. IR 3291, 1708, 1653, 860. 1H
33.0, 40.6, 50.3, 69.9, 101.6, 108.6, 121.2, 121.4, 122.2, 124.9, 125.8,
125.9, 126.8, 147.4, 150.8, 166.7, 194.5. Mass (m/z) 488, 441, 414, 290,
163, 136, 101, 88, 73, 59. Anal. calcd. C24H26ClF4NO3 (C, H, N).
NMR
7-8), 2.30 (3H; s; 2-CH3), 4.92 (2H; s; OCH2), 5.11 (1H; s; H-4), 7.14–
7.81 (7H; m; Ar-H), 9.31 (1H; s; NH). 13C NMR
18.9, 23.3, 24.5, 24.9,
34.3, 39.3, 40.0, 40.2, 40.4, 40.6, 65.4, 100.8, 108.0, 121.1, 121.3, 122.1,
125.7, 126.8, 128.1, 128.6, 136.8, 137.9, 138.1, 147.6, 150.87, 166.5,
199.7. Mass (m/z) 522, 441, 413, 150, 136, 101, 90. Anal. calcd.
C27H24ClF4NO3 (C, H, N).
d 0.80 (3H; s; 6-CH3), 0.95 (3H; s; 6-CH3), 1.63–1.73 (4H; m; H
4.1.1.13. Terbutyl 2,6,6-trimethyl-4-(2-fluoro-3-chloro-5-tri-
d
fluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (4m). Yield 51%. M.p.183 ꢂC. IR 3286,1702,1652, 860. 1H
NMR 0.77 (3H; s; 7-CH3), 0.96 (3H; s; 7-CH3), 1.24 (9H; s; C(CH3)3),
1.88–2.41 (2H; dd; H-8), 2.22 (3H; s; 2-CH3), 2.26–2.47 (2H; dd; H-
6), 5.02 (1H; s; H-4), 7.41–7.42 (1H; d; Ar-H), 7.76–7.77 (1H; d; Ar-H),
9.16 (1H; s; NH). 13C NMR
d 18.8, 26.5, 28.3, 28.3, 28.3, 29.6, 32.7,
4.1.1.8. Methyl 2,7,7-trimethyl-4-(2-fluoro-3-chloro-5-tri-
fluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (4h). Yield 39%. M.p. 199 ꢂC. IR 3285, 1709, 1651, 861. 1H
33.8, 40.6, 50.6, 79.8, 103.3, 108.3, 121.5, 122.4, 125.1, 125.6, 126.0,
127.2, 146.2, 151.2, 158.9, 166.3, 194.7. DEPT-135 18.8 (CH3),
26.5(CH3), 28.3(CH3), 28.3(CH3), 28.3(CH3), 29.6 (CH3), 32.7, 33.8
(CH), 40.6 (CH2), 50.6 (CH2), 126.0 (CH), 127.2 (CH). Mass (m/z) 488,
413, 148, 136, 101, 88, 73, 59. Anal. calcd. C24H26ClF4NO3 (C, H, N).
NMR d 0.80 (3H; s; 7-CH3), 1.01 (3H; s; 7-CH3), 2.26–2.29 (2H; s; H-
6), 2.27 (3H; s; 2-CH3), 2.45–2.51 (4H; m; H 7-8), 3.49 (3H; s; OCH3),
3.87–4.00 (2H; s; H-8), 5.10 (1H; s; H-4), 7.36–7.43 (1H; d; Ar-H),
7.80–7.81 (1H; d; Ar H), 9.27 (1H; s; NH). 13C NMR
d
18.7, 26.1, 29.5,
4.1.1.14. Benzyl 2,6,6-trimethyl-4-(2-fluoro-3-chloro-5-tri-
fluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (4n). Yield 36%. M.p.161 ꢂC. IR 3291, 1708, 1653, 860. 1H
32.5, 32.8, 40.4, 50.3, 51.1 101.8, 108.7, 121.1, 121.3, 122.2, 124.9,
125.4, 126.0, 138.2, 147.0 158.4, 167.1, 194.5. Mass (m/z) 446, 413,
248, 148, 136, 104, 101, 70, 59. Anal. calcd. C21H20ClF4NO3 (C, H, N).
NMR
6-8), 2.31 (3H; s; 2-CH3), 4.92–4.97 (2H, s, OCH2), 5.13 (1H; s; H-4),
7.09–7.74 (7H; m; Ar-H), 9.31 (1H; s; NH). 13C NMR
18.9, 26.2, 29.5,
d 0.74 (3H; s; 7-CH3), 0.99 (3H; s; 7-CH3), 1.79–2.26 (4H; m; H
4.1.1.9. Ethyl 2,7,7-trimethyl-4-(2-fluoro-3-chloro-5-tri-
d
fluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
32.5, 33.0, 40.4, 40.6, 50.3, 65.4, 101.3, 108.7, 121.2, 121.4, 124.8,
125.8, 126.0, 126.8, 128.2, 128.6, 136.7, 137.8, 137.9, 147.8, 150.8,
158.6, 166.5, 194.5. Mass (m/z) 522, 441, 413, 148, 136, 101, 90, 59.
Anal. calcd. C27H24ClF4NO3 (C, H, N).
carboxylate (4i). Yield 51%. M.p. 209 ꢂC. IR 3297, 1709, 1651, 862. 1H
NMR
d 0.81 (3H; s; 7-CH3), 1.01 (3H; s; 7-CH3), 1.05–1.08 (3H; t;
CH2CH3), 2.26–2.29 (2H; s; H-6), 2.30 (3H; s; 2-CH3), 3.87–4.00 (2H;
s; H-8), 3.87–4.00 (2H; q; CH2-CH3), 5.10 (1H; s; H-4), 7.43–7.44
(1H; d; Ar-H), 7.79–7.81 (1H; d; Ar H), 9.26 (1H; s; NH). 13C NMR
4.1.2. 9-(3-Chloro-2-fluoro-5-trifluoromethylphenyl)-6,6(7,7)-
dimethyl-6,7-dihydrofuro [3,4-b]quinoline-1,8-diones (5a,b)
The mixture of 0.532 mmol methyl 2,6,6-(2,7,7)-trimethyl-4-(2-
fluoro-3-chloro-5-trifluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hex-
ahydroquinoline-3-carboxylate and 0.6 mmol pyridinium bromide
perbromide in chloroform was stirred in ice-water for 1 h. Then, the
mixture was heated under reflux for 4 h. The precipitate was
crystallised from methanol.
d
14.1, 18.5, 18.7, 26.2, 29.5, 32.6, 33.0, 49.0, 50.3, 59.6, 101.7, 108.6,
122.2, 124.9, 125.7, 126.8, 127.6, 147.1, 150.9, 158.5, 166.5, 194.5. Mass
(m/z) 460, 441, 413, 262, 136, 104, 101, 73, 59. Anal. calcd.
C22H22ClF4NO3 (C, H, N).
4.1.1.10. Allyl 2,7,7-trimethyl-4-(2-fluoro-3-chloro-5-tri-
fluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (4j). Yield 28%. M.p. 172 ꢂC. IR 3282, 1688, 1616, 862. 1H
NMR
d
0.80 (3H; s; 7777-CH3), 1.00 (3H; s; 7-CH3), 2.19 (2H; s; H-6),
4.1.2.1. 9-(3-Chloro-2-fluoro-5-trifluoromethylphenyl)-7,7-dimethyl-
2.48–2.50 (2H; s; H-8), 2.30 (3H; s; 2-CH3), 4.38–4.49 (2H; d;
OCH2), 5.04 (1H; s; H-4), 5.12 (2H; d; CH]CH2), 5.74 (1H; m;
CH]CH2), 7.43–7.44 (1H; d; Ar-H), 7.75–7.76 (1H; d; Ar-H), 9.29
6,7-dihydrofuro [3,4-b]quinoline-1,8-dione (5a). Yield 11%. M.p.
>300 ꢂC. IR 3194, 3084, 1722, 1671, 861. 1H NMR
d 0.84 (3H; s; 7-
CH3), 0.94 (3H; s; 7-CH3), 1.72–1.80 (2H; t; H-6), 2.50–2.55 (2H; t;
H-5), 4.86 (2H; s; H-3), 4.97 (1H; s; H-9), 7.42–7.44 (1H; d; Ar-H),
(1H; s; NH). 13C NMR
d 18.8, 26.2, 29.5, 32.5, 33.1, 39.8, 40.1, 50.3,
64.4, 101.4, 108.6, 117.6, 121.2, 124.9, 125.8, 126.0, 126.9, 137.8, 147.5,
150.8, 158.6, 166.2, 194. Mass (m/z) 472, 413, 163, 136, 101, 73, 59.
Anal. calcd. C23H22ClF4NO3 (C, H, N).
7.82–7.84 (1H; d; Ar-H),10.17 (1H; s; NH). 13C NMR
d 24.4, 24.7, 24.9,
29.9, 34.5, 39.5, 66.1, 100.5, 108.9, 119.6, 119.7, 121.1, 121.3, 122.4,
125.1, 153.7, 157.5, 158.9, 171.7, 200.3. Mass (m/z) 522, 452, 441, 413,
153, 136, 101, 88, 70, 59. Anal. calcd. C20H16ClF4NO3 (C, H, N).
4.1.1.11. 2-Methoxyethyl 2,7,7-trimethyl-4-(2-fluoro-3-chloro-5-tri-
fluoromethylphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (4k). Yield 38%. M.p. 92 ꢂC. IR 3287, 1686, 1653, 861. 1H
4.1.2.2. 9-(3-Chloro-2-fluoro-5-trifluoromethylphenyl)-6,6-dimethyl-
6,7-dihydrofuro [3,4-b]quinoline-1,8-dione (5b). Yield 13%. M.p.
NMR
d
0.81 (3H; s; 7-CH3), 1.01 (3H; s; 7-CH3), 0.97 (3H; s; 6-CH3),
>300 ꢂC. IR 3197, 17251666 1491, 862. 1H NMR
d
0.93 (3H; s; 6-CH3),
1.92–2.20 (2H; dd; H-6), 2.30 (3H; s; 2-CH3), 3.18 (2H; t; CH2-OCH3),
1.03 (3H; s; 6-CH3), 2.01–2.24 (2H; t; H-5), 2.38–2.56 (2H; t; H-7),

B. Bu¨lbu¨l et al. / European Journal of Medicinal Chemistry 44 (2009) 2052–2058
2057
4.87 (2H; s; H-3), 4.99 (1H; s; H-9), 7.45–7.47 (1H; d; Ar-H), 7.88–
7.90 (1H; d; Ar-H), 10.25 (1H; s; NH). 13C NMR
26.7, 29.4, 29.6, 32.8,
39.58, 50.6, 66.2, 100.9, 109.7, 121.3, 121.5, 122.3, 125.0, 125.9, 127.8,
136.2, 152.3, 157.8, 171.7, 195.2. Mass (m/z) 522, 452, 441, 413, 153,
136, 101, 88, 70, 59. Anal. calcd. C20H16ClF4NO3 (C, H, N).
HPLC pump (Gilson 302, Villiers le Bel, France), injection valve
(Rheodyne 7125, Rohnert Park, CA, USA), UV detector (Hewlett
Packard HP 1050 Variable Wavelength Dedector, USA), HPLC inte-
grator (C-R8A Chromatopac, Shimadzu, Kyoto, Japan), reverse
phase column (Waters, m-Bondapak C18 column, USA). Mobile
phase: 50:50 (acetonitrile:water v/v), flow velocity: 1 ml/min,
maximum absorption: 254 nm.
d
4.2. Pharmacology
The calcium channel blocker effect of compounds 4a–n and 5a,b
was tested on isolated rabbit sigmoid colon. New Zealand white
rabbits, weighing 2.5–3 kg were used in this study. At the time of
study, rabbits were sacrificed with i.v. injection of sodium pento-
barbital (30–40 mg/kg, i.v.), followed by removal of the sigmoid
colon through abdominal incision. The fundal part of the sigmoid
colon was then dissected parallel to the circular muscle. The strips
(n: 4–8 from each rabbits) were mounted in tissue baths (20 ml)
containing Ca2þ-free Krebs–Henseleit solution at 37 ꢂC and gassed
with % 95O2 and % 5CO2. The pH of the saturated solution was 7.4.
Each strip was connected to a force transducer (FDT 10-A, May IOBS
99, COMMAT Iletisim Co., Ankara, Turkey) for the measurement of
isometric force, which was continuously displaced and recorded on
an online computer via a four-channel transducer data acquisition
system (MP30B-CE, BIOPAC Systems Inc., Santa Barbara, CA) using
software (BSL PRO v 3.6.7, BIOPAC Systems Inc.) which also had the
capacity to analyze the data.
4.3.1. Method of microsomal preparations [44,45]
The animals were sacrificed by cervical dislocation and the liver
was removed, cut into small pieces and homogenized with sucrose
solution (0.25 mol/l) to make a 25% v/v homogenate. The homog-
enate was first centrifuged at 10,500 rpm at 4 ꢂC for 30 min to
remove cell debris, intact cells, nuclei and mitochondria. The
supernatant containing the microsomal and soluble cell fractions
was carefully decanted and its volume measured. Calcium chloride
solution (80 mmol/l) was added to the supernatant (1 ml for 9 ml
supernatant) and the mixture was then centrifuged at 15,500 rpm
for 15 min. For the washing step, the pellet containing microsomes
was resuspended in KCl solution (0.15 mol/l), equal volume with
the first supernatant. Finally, the washed microsomes were
prepared by centrifuging this homogenate at 15,500 rpm for
15 min. The pellet (washed microsomes) was resuspended in
phosphate buffer (0.2 mol/l, pH 7.4) and the final suspension con-
tained 1 g original liver per 1 ml. This was used freshly or stored at
ꢃ80 ꢂC until required.
After mounting, each strip was allowed to equilibrate with
a basal tension of 1 g for 60 min. Ca2þ-free KHS was replaced with
fresh solution every 15 min during this time period.
The production of NADPH was effected by incubation of cofac-
tors. Substrates and microsomal preparations were incubated in
a water bath for 30 min. Metabolic extracts were injected into
a reversed HPLC system.
To eliminate whether relaxation induced by the test compounds
was due to an interaction with cyclooxygenase, adrenergic, potas-
sium channel or nitric oxide pathways, all experiments were done
in the presence of indomethacin (COX inhibitor, 10ꢃ5 M),
guanethidine (an adrenergic nerve blocker, 10ꢃ6 M), tetraethy-
lammonium (TEA) (Ca2þ-activated potassium channels (KCa)
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