Synthesis and calcium modulatory activity of 3-alkyloxy-carbonyl-4- (disubstituted)aryl-5-oxo-1,4,5,6,7,8-hexahydroquinoline derivatives

Article abstract of DOI:10.1002/ardp.200700087

In this study, twelve hexahydroquinoline derivatives which are condensed analogs of the 1,4-DHP molecule were synthesized and evaluated for their calcium-antagonistic activity. The results indicated that all compounds and nifedipine produced concentration-dependent relaxation in rabbit gastric fundus smooth muscle strips. The relaxant effects of the compounds on the tissues were expressed as percentage of the precontraction using Ca²⁺. The maximum response (E_max) and pD₂ values (the negative logarithm of the concentration for the half-maximal response (EC₅₀)) were calculated. It is generally believed that introduction of a second electron-withdrawing substituent into the phenyl ring increases the mentioned activity. Methyl-2,7,7-trimethyl-4-(2-nitro-5-chlorophenyl)-5-oxo-1,4,5,6,7,8- hexahydroquinoline-3-carboxylate 2a has been found to be the most active compound in this serie.

Full text of DOI:10.1002/ardp.200700087

Arch. Pharm. Chem. Life Sci. 2008, 341, 55–60
R. S˛ ims˛ek et al.
55
Full Paper
Synthesis and Calcium Modulatory Activity of 3-Alkyloxy-
carbonyl-4-(disubstituted)aryl-5-oxo-1,4,5,6,7,8-hexa-
hydroquinoline Derivatives
1
2
2
1
2
˙
ˇ
Rahime S˛ ims˛ek , Gꢀkꢁe S. ꢂztꢃrk , I. Mert Vural , Miyase G. Gꢃndꢃz , Yusuf Sarıoglu ,
and Cihat S˛ afak1
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
2 Department of Pharmacology, Faculty of Medicine, Gazi University, Ankara, Turkey
In this study, twelve hexahydroquinoline derivatives which are condensed analogs of the 1,4-
DHP molecule were synthesized and evaluated for their calcium-antagonistic activity. The
results indicated that all compounds and nifedipine produced concentration-dependent relaxa-
tion in rabbit gastric fundus smooth muscle strips. The relaxant effects of the compounds on
the tissues were expressed as percentage of the precontraction using Ca2+. The maximum
response (Emax) and pD2 values (the negative logarithm of the concentration for the half-maximal
response (EC50)) were calculated. It is generally believed that introduction of a second electron-
withdrawing substituent into the phenyl ring increases the mentioned activity. Methyl-2,7,7-tri-
methyl-4-(2-nitro-5-chlorophenyl)-5-oxo-l,4,5,6,7,8-hexahydroquinoline-3-carboxylate 2a has been
found to be the most active compound in this serie.
Keywords: Calcium antagonist activity / 1,4-DHP / Gastric fundus / Hexahydroquinoline / Nifedipine /
Received: April 25, 2007; accepted: September 14, 2007
DOI 10.1002/ardp.200700087
The cytosolic calcium concentration regulates many
cellular functions. In striated and smooth muscle,
increasing of the intracellular calcium triggers the onset
of contraction. Calcium influx through voltage-gated
channels contributes to this rise in cytosolic calcium.
Voltage-sensitive calcium channels have become a target
for the drug therapy of cardiovascular and gastrointesti-
nal diseases [2].
Calcium channel antagonists inhibit muscle contrac-
tion by blocking the influx of Ca2+ through calcium chan-
nels and are used as anti-anginal and antihypertensive
drugs [3–11]. 1,4-Dihydropyridine (DHP) derivatives, of
which nifedipine (Fig. 1) is the prototype, are the most
studied calcium channel antagonists.
Introduction
Voltage-gated calcium channels are integral membrane
proteins that open via electrical depolarization of the
cell membrane and mediate the entry of calcium ions
into the cell. Calcium channels are expressed in every
excitable cell, including neurons, myocytes, and pancre-
atic ß-cells and form the most efficient molecular link
between membrane depolarization and intracellular bio-
chemical signaling. Ca2+ entering the cell through volt-
age-gated Ca2+ channels serves as the second messenger
of electrical signaling, initiating cellular events such as
contraction, secretion, synaptic transmission, and gene
expression [1].
The derivatives having a 1,4-DHP structure have
attracted attention and many active compounds have
been obtained by making modifications on the nifedi-
pine molecule. One of these modifications is to replace
the ester function by various acyl groups such as amide,
nitro, nitril, or ketone [8, 12–18]. In ester derivatives,
when one of the R groups is methyl or ethyl ester, the
Correspondence: Assoc. Prof. Rahime S˛ ims˛ek, Hacettepe University,
Faculty of Pharmacy, Department of Medicinal Chemistry, 06100 Ankara,
Turkey.
E-mail: rsimsek@hacettepe.edu.tr
Fax: +90 312 305-1872
Abbreviations: Dihydropyridine (DHP); nitric oxide (NO); cyclooxyge-
nase (COX); Nw-nitro-L-arginine methyl ester (L-NAME)
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R. S˛ ims˛ek et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 55–60
Figure 1. Formula of nifedipine.
activity increases due to the lipophilic properties [19, 20].
In addition, to fix one carbonyl group in an antiperipla-
nar position, 1,4-DHP structure could be annelated. Thus,
condensed 1,4-DHP derivatives have been obtained and
these analogs showed calcium antagonistic activity [21–
30].
The derivatives carrying a phenyl group with two elec-
tron-withdrawing substituents also showed calcium-
modulatory activity [14, 31–33]. Many studies have
shown that the compounds having a dichlorophenyl
moiety in their structure have been more active than
nicardipine on isolated rat ileum and lamb carotid artery
[18].
Scheme 1. Synthesis of the presented compounds.
The object of this study is to show the contribution of
two different electron-withdrawing substituents in the
phenyl ring with respect to the calcium-modulatory
activity. Therefore, the compounds having nitro and
chlorine substitution in four different positions of the
hexahydroquinoline ring were synthesized and eval-
uated for their calcium antagonistic activity in the rabbit
gastric fundus smooth muscle strips.
ysis. In the IR spectra, N-H, C=O (ester), and C=O (amide)
stretching bands (only for compounds 1c–4c) were seen
at the expected values. In the 1H-NMR spectra six or seven
methyl protons of the hexahydroquinoline ring were
seen at about 0.90–1.00 ppm as separate singlets. The
peaks belonging to the aromatic protons and the protons
of the alkyl groups were seen at the expected chemical
shift values. The N-H signal of the compounds was found
in their spectra except for compounds 1c and 2c. The
mass spectra of the compounds was recorded using the
electron impact technique. Molecular ion peaks were
seen for all compounds. In further fragmentation, the
peaks were forming by cleavage of the aryl ring from the
parent molecule. Aromatization of the DHP ring to the
pyridine analog was also realized (Scheme 2). These find-
ings are in accordance with the literature [25]. In addi-
tion, the structure of the compounds was confirmed by
elemental analysis.
Results and discussion
Chemistry
In this study, the hexahydroquinoline derivatives were
obtained by modified Hantzsch synthesis [34]. The ester
derivatives 1a–4a, 1b–4b were synthesized by the reac-
tion of 4,4- or (5,5)-dimethyl-1,3-cyclohexanedione,
appropriate aromatic aldehyde and methyl (or ethyl)
aminocrotonoate. In order to synthesize amide deriv-
atives 1c–4c, 4,4- or (5,5)-dimethyl-1,3-cyclohexanedione
and appropriate aromatic aldehyde derivatives were
reacted with N,N-diethylacetoacetamide (Scheme 1).
Compound 2a and 4a are mentioned in the literature,
but there without chemical and pharmacological infor-
mation. Therefore, compounds 2a and 4a were also syn-
thesized. The structures of the compounds were eluci-
dated by IR, 1H-NMR, mass spectra, and elemental anal-
Pharmacology
The maximum relaxant effects (Emax) and pD2 values of
the compounds and nifedipine on isolated strips of rab-
bit gastric fundus smooth muscle are given in Table 1.
The results indicate that all compounds produced con-
centration-dependent relaxation in rabbit gastric fundus
smooth muscle strips. Compounds and nifedipine
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Arch. Pharm. Chem. Life Sci. 2008, 341, 55–60
Calcium Modulatory Activity of Quinoline Derivatives
57
Scheme 2. Mass fragmentation of the presented compounds.
Table 1. Maximum relaxant responses (Emax) and pD2 values
and molecular structures of the compounds and nifedipine on
isolated strips of rabbit gastric fundus smooth muscle.
Compound
pD2
Emax
1a
2a
3a
4a
1b
2b
3b
4b
1c
2c
3c
4c
4.69 l 0.48
5.10 l 0.08
4.35 l 0.19
6.87 l 0.83
4.88 l 0.50
5.66 l 0.31
5.94 l 0.41
5.92 l 0.42
3.96 l 0.14
4.30 l 0.34
6.25 l 0.33
5.10 l 0.29
7.94 l 0.25
83.21 l 8.63
99.94 l 0.06
80,82 l 1.16
96.76 l 3.24
88,21 l 2.09
95.35 l 4.65
84.19 l 5.03
82.12 l 6.45
87.33 l 7.00
67.28 l 6.20
98.40 l 1.60
67.55 l 7.88
99.30 l 0.70
Figure 2. Typical traces showing the relaxant effects of nifedi-
pine (10– 9 M to 10– 4 M) (Fig. 2a) and compound 2a (10– 8 M to
10– 4 M) (Fig. 2b) on the precontracted with Ca2+ (2.5 mM) in the
gastric fundus smooth muscle strips.
Nifedipine
Relaxation is expressed as a percentage of the precontraction
induced by Ca2+ (2.5 mM). The negative logarithm of the concen-
tration for the half-maximal response (pD2) and Emax values rep-
resent mean value l S. E.
phosphorylation, the latter often having a negative effect
on smooth muscle [35, 38].
Considering the overall powerful selectivity of action
* p a 0.05, compared with vehicle responses (n = 6).
exerted concentration-dependent relaxation responses of the clinically available 1,4-DHPs for the L-type channel,
precontracted with Ca2+ (2.5 mM) in the gastric fundus some evidence suggests that a component of their vascu-
smooth muscle strips with the efficacy order: 2a = nifedi- lar selectivity and vasodilating properties may arise from
pine A 3c F 4a F 2b A 1b F 1c F 3b F 1a F 4b F 3a A 4c = an ability to stimulate nitric oxide (NO) release from vas-
2c. While the efficacy of the compounds 2a, 4a, and 3c cular endothelial cells [39]. Such an action would add to
have been found the same as nifedipine, potency of com- the vasodilation produced by direct blockade of the cal-
pounds 2a and 3c have been found less than nifedipine cium channels in vascular smooth muscle cells. Endothe-
(Fig. 2). There is no difference between ester and amide lial NO synthase (eNOS) converts substrate L-arginine to
derivatives with respect to the mentioned activity.
NO, which diffuses to the vascular smooth muscle cells
The main site of action of the Ca2+-channel blockers is and promotes vascular relaxation [40–42]. NO releasing
believed to be the voltage-dependent channels, where actions of the 1,4-DHPs appear to be a class action, to be
inhibition of the influx of extracellular Ca2+ results in exerted at therapeutic concentrations and to involve the
uncoupling of excitation and contraction. Calcium-chan- stimulation of calcium influx into endothelial cells
nel blockers may not only directly decrease the concen- through a non-L-type process [43–45].
tration of cytoplasmic Ca2+, but also cause a decrease in
Ca2+ release from intracellular stores [35].
To investigate whether relaxation induced by the test
compounds was due to interaction with the cyclooxyge-
The Ca2+-channel types described for smooth muscle nase (COX), adrenergic system, or NO pathways, tissues
consist of the L- and T-types, with slow and fast inactiva- were pretreated with indomethacin (COX inhibitor), pro-
tion characteristics, respectively [36]. L-Type Ca2+ chan- pranolol (beta-adrenergic receptor blocker), or Nw-nitro-L-
nels are inhibited by Mg2+ and by dihydropyridines such arginine methyl ester (L-NAME) hydrochloride (the NO
as nifedipine, nimodipine, and isradipine [37]. The func- synthase inhibitor), respectively. Pretreatment of the
tion of L-type Ca2+ channels can be regulated both by gua- strips with indomethacin, propranolol, and L-NAME did
nosine 59-triphosphate (GTP)-binding proteins and by not significantly alter the relaxant responses to the com-
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R. S˛ ims˛ek et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 55–60
Ar H-6), 7.20 (dd, 1H, J = 8.9, 2.4 Hz, Ar H-4), 7.80 (d, 1H, J = 8.9 Hz,
Ar H-3), 9.00 (s, 1H, NH). MS: (m/z) 406, 405, 404, 403, 248 (100),
57. Analysis for C20H21ClN2O5: calculated C: 59.34, H: 5.23, N:
6.92; found C: 59.77 , H: 5.38, N: 6.85.
pounds indicating that COX, adrenergic, and NO path-
ways do not play a role in relaxations evoked by these
substances.
Our results showed that these compounds had potency
for relaxing isolated rabbit gastric fundus smooth
muscle, possibly due to the blockade of Ca2+ channels,
similar to the action of nifedipine.
Methyl 2,7,7-trimethyl-4-(2-nitro-5-chlorophenyl)-5-oxo-
l,4,5,6,7,8-hexahydroquinoline-3-carboxylate 2a
Mp. 2088C. Yield 74%. IR (cm– 1) 3282, 1701, 1611, 741. 1H-NMR d
0.80 (s, 3H, 7-CH3), 0.95 (s, 3H, 7-CH3), 1.90–2.60 (m, 7H, 2-CH3, H-
6,8), 3.40 (s, 3H, COOCH3), 5.55 (s, 1H, H-4), 6.90 (d, 1H, J = 2.3 Hz,
Ar H-6), 7.15 (dd, 1H, J = 8.8, 2.3 Hz, Ar H-4), 7.75 (d, 1H, J = 8.8 Hz,
Ar H-3), 9.25 (s, 1H, NH). MS: (m/z) 406, 405, 404, 403, 387, 356,
301, 248 (100), 232, 192, 55. Analysis for C20H21ClN2O5: calculated
C: 59.34, H: 5.23, N: 6.92; found C: 59.24, H: 5.66, N: 6.99.
The authors have declared no conflict of interest.
Experimental
Methyl 2,6,6-trimethyl-4-(3-nitro-4-chlorophenyl)-5-oxo-
l,4,5,6,7,8-hexahydroquinoline-3-carboxylate 3a
Chemistry
All chemicals used in this study were purchased from Aldrich
(Steinheim, Germany) and Fluka (Buchs, Switzerland). Melting
points are determined by a Thomas Hoover capillary melting
point apparatus (Philadelphia, PA, USA); the values are uncor-
rected. UV spectra: Shimadzu UV-160A UV-visible spectropho-
tometer (Shimadzu Co., Kyoto, Japan). IR spectra: Perkin Elmer
FT-IR spectrophotometer 1720 X (Perkin Elmer, Beaconsfield,
Mp. 2238C. Yield 69%. IR (cm– 1) 3250, 1700. 1H-NMR d 0.85 (s, 3H,
6-CH3), 0.95 (s, 3H, 6-CH3), 1.75 (t, 2H, H-7), 2.30 (s, 3H, 2-CH3), 2.50
(t, 2H, H-8) 3.50 (s, 3H, COOCH3), 4.95 (s, 1H, H-4), 7.45 (dd, 1H, J =
2.0, 7.3 Hz, Ar H-6), 7.60 (d, 1H, J = 7.3 Hz, Ar H-5), 7.80 (d, 1H, J =
2.0 Hz, Ar H-2), 9.30 (s, 1H, NH). MS: (m/z) 407, 406, 405, 404 403,
248 (100), 76, 53. Analysis for C20H21ClN2O5: calculated C: 59.34,
H: 5.23, N: 6.92; found C: 59.17, H: 4.99, N: 6.96.
UK) (KBr disc) (c, cm– 1). H-NMR spectra: Bruker DPX-400 MHz
1
digital FT NMR spectrophotometer (Bruker, Karlsruhe, Germany)
(DMSO-d6; tetramethylsilane as internal standard). Chemical
shift values are given as ppm. Mass spectra: Hewlett Packard Ser-
ies II Plus 5890; Gas cromatograph Hewlett Packard 5972 series
mass selective detector (Hewlett Packard, Philadelphia, PA, USA).
Thin layer chromatography (TLC) was run on precoated (0.2 mm)
silica gel Merck 254 + 366 (E. Merck, Darmstadt, Germany) and
short wave UV light (254 nm) was used to detect the UV absorb-
ing spots. Elemental analysis was carried out an a Leco 932
CHNS. Elemental analyzer (Leco, Philadelphia, PA, USA) (TUBI-
TAK, Ankara, Turkey). The elemental analysis results were
within 0.4% of theoretical values.
Methyl 2,7,7-trimethyl-4-(3-nitro-4-chlorophenyl)-5-oxo-
l,4,5,6,7,8-hexahydroquinoline-3-carboxylate 4a
Mp. 2118C. Yield 77%. IR (cm– 1) 3240, 1700. 1H-NMR d 0.90 (s, 3H,
7-CH3), 1.00 (s, 3H, 7-CH3), 1.90-2.60 (m, 7H, 2-CH3, H-6,8), 3.50 (s,
3H, COOCH3), 5.00 (s, 1H, H-4), 7.50 (dd, 1H, J = 2.1, 7.4 Hz, Ar H-
6), 7.65 (d, 1H, J = 7.4 Hz, Ar H-5), 7.80 (d, 1H, J = 2.1 Hz, Ar H-2),
9.30 (s, 1H, NH). MS: (m/z) 406, 405, 404, 403, 248 (100), 55. Anal-
ysis for C20H21ClN2O5: calculated C: 59.34, H: 5.23, N: 6.92; found
C: 59.72, H: 5.49, N: 6.43.
Ethyl 2,6,6-trimethyl-4-(2-nitro-5-chlorophenyl)-5-oxo-
l,4,5,6,7,8-hexahydroquinoline-3-carboxylate 1b
Drugs
Mp. 1078C. Yield 66%. IR (cm– 1) 3250, 1705. 1H-NMR d 0.90 (s, 3H,
6-CH3), 1.10 (s, 3H, 6-CH3), 1.20 (t, 3H, CH2CH3), 2.00–2.60 (m, 7H,
2-CH3, H-7,8), 4.00 (q, 2H, CH2CH3), 4.90 (s, 1H, H-4), 6.95 (d, 1H, J =
2.4 Hz, Ar H-6), 7.20 (dd, 1H, J = 8.9, 2.4 Hz, Ar H-4), 7.80 (d, 1H, J =
8.9 Hz, Ar H-3), 9.20 (s, 1H, NH). MS (m/z) 420, 419, 418, 417, 262
(100), 152. Analysis for C21H23ClN2O5: calculated C: 60.22, H: 5.53,
N: 6.69; found C: 60.62, H: 5.38, N: 6.38.
Nx-nitro-L-arginine-methylester (L-NAME) hydrochloride, indo-
methacin, propranolol hydrochloride, and nifedipine were sup-
plied by Sigma (Sigma, Taufkirchen, Germany). L-NAME, propra-
nolol hydrochloride, and nifedipine were dissolved in distilled
water. Compounds and indomethacin were dissolved in DMSO.
DMSO was supplied by Sigma.
General procedure for methyl (and ethyl) 2,6,6-
(2,7,7)-trimethyl-4-disubstituted-aryl-5-oxo-
l,4,5,6,7,8-hexahydroquinoline-3-carboxylates 1a–4a,
Ethyl 2,7,7-trimethyl-4-(2-nitro-5-chlorophenyl)-5-oxo-
l,4,5,6,7,8-hexahydroquinoline-3-carboxylate 2b
Mp. 1548C. Yield 81%. IR (cm– 1) 3240, 1700. 1H-NMR d 0.80 (s, 3H,
7-CH3), 0.95 (s, 3H, 7-CH3), 1.00 (t, 3H, CH2CH3), 1.85-2.50 (m, 7H,
2-CH3, H-6,8), 3.90 (q, 2H, CH2CH3), 5.60 (s, 1H, H-4), 6.95 (d, 1H, J =
2.4 Hz, Ar H-6), 7.20 (dd, 1H, J = 8.9, 2.4 Hz, Ar H-4), 7.80 (d, 1H, J =
8.9 Hz, Ar H-3), 9.20 (s, 1H, NH). MS (m/z) 421, 420, 419, 418, 350,
262 (100), 152, 58. Analysis for C21H23ClN2O5: calculated C: 60.22,
H: 5.53, N: 6.69; found C: 59.87, H: 5.20, N: 6.70.
1b–4b
Methyl (or ethyl) aminocrotonate (0.001 mol, 0.001 mol), 4,4- (or
5,5)-dimethyl-1,3-cyclohexanedione and 0.001 mol aromatic
aldehyde was refluxed in 20 mL methanol for 4 h. The resulting
precipitate was filtered and crystallized from methanol.
Methyl 2,6,6-trimethyl-4-(2-nitro-5-chlorophenyl)-5-oxo-
Ethyl 2,6,6-trimethyl-4-(3-nitro-4-chlorophenyl)-5-oxo-
l,4,5,6,7,8-hexahydroquinoline-3-carboxylate 1a
Mp.1698C. Yield 68%. IR (cm– 1) 3280, 1700. 1H-NMR d 0.90 (s, 3H,
6-CH3), 1.10 (s, 3H, 6-CH3), 1.60–1.70 (m, 4H, H-7,8), 2.45 (s, 3H, 2-
CH3), 3.35 (s, 3H, COOCH3), 5.25 (s, 1H, H-4), 6.95 (d, 1H, J = 2.4 Hz,
l,4,5,6,7,8-hexahydroquinoline-3-carboxylate 3b
Mp. 2208C. Yield 71%. IR (cm– 1) 3290, 1695. 1H-NMR d 0.90 (s, 3H,
6-CH3), 1.00 (s, 3H, 6-CH3), 1.10 (t, 3H, CH2CH3), 1.70 (t, 2H, H-7),
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Arch. Pharm. Chem. Life Sci. 2008, 341, 55–60
Calcium Modulatory Activity of Quinoline Derivatives
59
2.30 (m, 5H, 2-CH3, H-8), 4.00 (q, 2H, CH2CH3), 4.90 (s, 1H, H-4),
7.50 (dd, 1H, J = 2.1, 7.4 Hz, Ar H-6), 7.65 (d, 1H, J = 7.4 Hz, Ar H-5),
7.80 (d, 1H, J = 2.1 Hz, Ar H-2), 9.25 (s, 1H, NH). MS (m/z) 421, 420,
418,417, 404, 350, 262 (100), 58. Analysis for C21H23ClN2O5: calcu-
lated C: 60.22, H: 5.53, N: 6.69; found C: 59.89, H: 5.49, N: 6.64.
N,N-Diethyl-2,7,7-trimethyl-4-(3-nitro-4-chlorophenyl)-5-
oxo-l,4,5,6,7,8-hexahydroquinoline-3-carboxamide 4c
Mp. 1378C. Yield 70%. IR (cm– 1) 3210, 1690, 1600. 1H-NMR d 0.90
(t, 6H, NCH2CH3), 0.95 (s, 3H, 6-CH3), 1.00 (s, 3H, 6-CH3), 1.70 (s,
3H, 2-CH3), 1,90 (s, 2H, H-8), 2.50 (s, 2H, H-6), 3.10 (q, 4H,
NCH2CH3), 4.60 (s, 1H, H-4), 7.50 (dd, 1H, J = 2.0, 7.3 Hz, Ar H-6),
7.65 (d, 1H, J = 7.3 Hz, Ar H-5), 7.75 (d, 1H, J = 2.0 Hz, Ar H-2), 8.80
(s, 1H, NH). MS (m/z) 446, 445, 423, 372, 352, 289 (100), 265, 193,
51. Analysis for C23H28ClN3O4: calculated C: 61.95, H: 6.33, N:
9.42; found C: 61.69, H: 6.12, N: 9.02.
Ethyl 2,7,7-trimethyl-4-(3-nitro-4-chlorophenyl)-5-oxo-
l,4,5,6,7,8-hexahydroquinoline-3-carboxylate 4b
Mp. 1418C. Yield 63%. IR (cm– 1) 3300, 1690. 1H-NMR d 1.00 (s, 3H,
7-CH3), 1.05 (s, 3H, 7-CH3), 1.15 (t, 3H, CH2CH3), 2.25 (s, 3H, 2-CH3),
1.95–2.65 (m, 4H, H-6,8), 4.05 (q, 2H, CH2CH3), 5.85 (s, 1H, H-4),
7.45 (dd, 1H, J = 2.0, 7.3 Hz, Ar H-6), 7.60 (d, 1H, J = 7.3 Hz, Ar H-5),
9.05 (s, 1H, NH). MS (m/z) 419, 418, 417, 262 (100), 57. Analysis for
C21H23ClN2O5: calculated C: 60.22, H: 5.53, N: 6.69; found C:
59.98, H: 5.41, N: 6.89.
Pharmacology
New Zealand white rabbits, weighing 2.5–3 kg were used in this
study. Experiments were performed in agreement with the ethi-
cal standards in the Helsinki declaration and this study was
approved by Gazi University Ethics Committee for animals. Pro-
cedures involving animals and their care were conducted in con-
formity with international laws and policies. At time of study,
rabbits were sacrificed with injection of sodium pentobarbital
(30–40 mg/kg, i.v.), followed by removal of the stomach through
abdominal incision.
General procedure for N,N-diethyl-2,6,6 -(and 2,7,7)-
trimethyl-4-aryl-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxamides 1c–4c
The mixture of 0.001 mol of 4,4-dimethyl-1,3-cyclohexanedione
(I), 0.001 mol of aromatic aldehyde derivative (II), and 0.001 mol
of N,N-diethylacetoacetamide (III) with 1 mL ammonia in 25 mL
methanol was refluxed for 4 h. The solution was poured into ice
water. The precipitate was filtered, dried, and crystallized from
ethanol.
Organ chamber experiments
The fundal part of the stomach was then dissected parallel to
the longitudinal muscle wall. One muscle strip approximately
15–20 mm long and 2 mm wide was obtained, and allowed to
equilibrate for a period of 60 min in 20 mL organ bath filled
with Ca2+-free Krebs'–Henseleit solution (Ca2+-free KHS). The com-
position of the Ca2+-free KHS used was as follows (in mmol/L):
NaCl 118; KCl 4.7; NaHCO3 25; MgCl2 0.54; NaHPO4 0.9; glucose
10.04. The solution was gassed with 95% O2 and 5% CO2 during
the study and temperature was maintained at 378C by a thermo-
regulated water circuit. 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 dis-
placed and recorded on an online computer via four-channel
transducer data acquisition system (MP30B-CE, BIOPAC Systems
Inc., Santa Barbara, CA, USA) using software (BSL PRO v 3.6.7, BIO-
PAC Systems Inc.) which also had the capacity to analyze the
data. After mounting, each strip was allowed to equilibrate with
a basal tension of 1 g for 60 min. Ca2+-free KHS was replaced with
fresh solution every 15 min during this time period.
N,N-Diethyl-2,6,6-trimethyl-4-(2-nitro-5-chlorophenyl)-5-
oxo-l,4,5,6,7,8-hexahydroquinoline-3-carboxamide 1c
Mp. 1398C. Yield 56%. IR (cm– 1) 3200, 1690, 1590. 1H-NMR d 0.95
(s, 3H, 6-CH3), 1.00 (s, 3H, 6-CH3), 1.05 (t, 6H, NCH2CH3), 1,50–2.20
(m, 4H, H-7,8), 2.50 (s, 3H, 2-CH3), 3.10 (s, 4H, NCH2CH3), 4.60 (s,
1H, H-4), 7.00 (d, 1H, J = 2.3 Hz, Ar H-6), 7.20 (dd, 1H, J = 8.7,
2.3 Hz, Ar H-4), 7.80 (d, 1H, J = 8.7 Hz, Ar H-3). MS (m/z) 446, 445,
444, 312, 289 (100), 261, 83, 55. Analysis for C23H28ClN3O4: calcu-
lated C: 61.95, H: 6.33, N: 9.42; found C: 61.56, H: 6.75, N: 9.46.
N,N-Diethyl-2,7,7-trimethyl-4-(2-nitro-5-chlorophenyl)-5-
oxo-l,4,5,6,7,8-hexahydroquinoline-3-carboxamide 2c
Mp. 1988C. Yield 58%. IR (cm– 1) 3220, 1680, 1600. 1H-NMR d 0.90
(s, 3H, 7-CH3), 0.95 (s, 3H, 7-CH3), 1.05 (t, 6H, NCH2CH3), 1.90 (s,
2H, H-8), 2.45 (s, 3H, 2-CH3), 2.80 (s, 2H, H-6), 3.20 (q, 4H,
NCH2CH3), 4.60 (s, 1H, H-4), 7.00 (d, 1H, J = 2.3 Hz, Ar H-6), 7.20
(dd, 1H, J = 8.7, 2.3 Hz, Ar H-4), 7.80 (d, 1H, J = 8.7 Hz, Ar H-3). MS
(m/z) 446, 445, 397, 289 (100), 264, 246, 97, 69, 41. Analysis for
C23H28ClN3O4: calculated C: 61.95, H: 6.33, N: 9.42; found C:
61.73, H: 6.62, N: 9.75.
To determine whether a calcium-antagonistic activity plays a
role in the relaxation induced by compounds, nifedipine and
dimethylsulfoxide (DMSO), the following procedure was
applied. Preparations were placed in a Ca2+-free, high K+-contain-
ing (80 mM) solution. When Ca2+ (2.5 mM) was added to the
organ bath, a contraction developed. At the plateau level of con-
traction, compounds (10– 8 to 3610– 4 M), nifedipine (10– 9 to
3610– 6 M), and DMSO (10– 9 to 3610– 4 M) were applied. Concen-
tration-relaxation for compounds, nifedipine and DMSO were
obtained by adding them into the bath in a cumulative manner
[46].
The following antagonists and inhibitors were used: Nx-nitro-
L-arginine methyl ester (L-NAME) hydrochloride (the nitric oxide
synthase inhibitor, 10– 4 M), indomethacin (COX inhibitor,
10–5 M), propranolol (beta-adrenergic receptor blocker, 10– 6 M).
The relaxant effects of the compounds, nifedipine and DMSO
were expressed as percentage of the precontraction using Ca2+
(2.5 mM).
N,N-Diethyl-2,6,6-trimethyl-4-(3-nitro-4-chlorophenyl)-5-
oxo-l,4,5,6,7,8-hexahydroquinoline-3-carboxamide 3c
Mp. 1398C. Yield 66%. IR (cm– 1) 3220, 1680, 1600. 1H-NMR d 0.90
(t, 6H, NCH2CH3), 0.95 (s, 3H, 6-CH3), 1.00 (s, 3H, 6-CH3), 1.70 (s,
3H, 2-CH3), 1,80–2,50 (m, 4H, H-7, 8), 3.10 (q, 4H, NCH2CH3), 4.70
(s, 1H, H-4), 7.60 (dd, 1H, J = 2.0, 7.3 Hz, Ar H-6), 7.70 (d, 1H, J =
7.3 Hz, Ar H-5), 7.80 (d, 1H, J = 2.0 Hz, Ar H-2), 8.75 (s, 1H, NH). MS
(m/z) 446, 445, 350, 289 (100), 51. Analysis for C23H28ClN3O4: cal-
culated C: 61.95, H: 6.33, N: 9.42; found C: 61.88, H: 6.55, N: 9.03.
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

60
R. S˛ ims˛ek et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 55–60
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neim. Forsch. Drug Res. 2004, 54, 371–375.
Data analysis
The relaxant effects of the compounds on the tissues, precon-
tracted with Ca2+ (2.5 mM), were expressed as percentage of the
precontraction using Ca2+ (2.5 mM). To evaluate the effects of an
agonist, the maximum response (Emax), the concentration for a
half-maximal response (EC50) and pD2 values were calculated
from the concentration-response curve (CRC) obtained in each
experiment, as predicted from the Scatchard equation for drug-
receptor interaction, where:
[19] A. Davood, N. Mansouri, D. A. Rerza, H. Shafaroudi, et al.,
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Response/concentration = –1/EC506response
ˇ
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aco 2000, 55, 665–668.
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parameter for efficacy, pD2 is the parameter for potency. All data
are expressed as mean l standard error. Statistical comparison
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