Evaluation of myorelaxant activity of 7-substituted hexahydroquinoline derivatives in isolated rabbit gastric fundus

Article abstract of DOI:10.1016/j.ejmech.2007.04.012

In this article, 16 new methyl(ethyl) 4-(dichlorophenyl)-2,7-dimethyl-5-oxo-l,4,5,6,7,8-hexahydroquinoline-3-carboxylates and methyl(ethyl) 2-methyl-4-(dichlorophenyl)-5-oxo-7-phenyl-l,4,5,6,7,8-hexahydroquinoline-3-carboxylate derivatives have been synthesized by the Hantzsch reaction and screened for their myorelaxant and potassium channel opening activities. The maximum relaxant effects (E_max) and pD₂ values on exogenous noradrenaline precontracted tissues and inhibitory effects on cholinergic neurotransmission of the compounds and pinacidil were determined on isolated strips of rabbit gastric fundus smooth muscle. Obtained results indicated that some compounds and pinacidil produced concentration-dependent relaxation on rabbit gastric fundus smooth muscle strips in the two test conditions.

Full text of DOI:10.1016/j.ejmech.2007.04.012

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 562e568
http://www.elsevier.com/locate/ejmech
Original article
Evaluation of myorelaxant activity of 7-substituted hexahydroquinoline
derivatives in isolated rabbit gastric fundus
a
Miyase Gozde Gunduz , Gokc¸e Sevim Ozturk , Ismail Mert Vural ,
b
b
_
¨
¨
¨
¨
¨
¨
a
b
a,
*
˘
Rahime Sximxsek , Yusuf Sarıoglu , Cihat Sxafak
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey
b Department of Pharmacology, Faculty of Medicine, Gazi University, Ankara, Turkey
Received 3 July 2006; received in revised form 6 March 2007; accepted 19 April 2007
Available online 13 May 2007
Abstract
In this article, 16 new methyl(ethyl) 4-(dichlorophenyl)-2,7-dimethyl-5-oxo-l,4,5,6,7,8-hexahydroquinoline-3-carboxylates and methyl(ethyl)
2-methyl-4-(dichlorophenyl)-5-oxo-7-phenyl-l,4,5,6,7,8-hexahydroquinoline-3-carboxylate derivatives have been synthesized by the Hantzsch
reaction and screened for their myorelaxant and potassium channel opening activities. The maximum relaxant effects (Emax) and pD2 values
on exogenous noradrenaline precontracted tissues and inhibitory effects on cholinergic neurotransmission of the compounds and pinacidil
were determined on isolated strips of rabbit gastric fundus smooth muscle. Obtained results indicated that some compounds and pinacidil
produced concentration-dependent relaxation on rabbit gastric fundus smooth muscle strips in the two test conditions.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Hexahydroquinoline; Myorelaxant activity; Potassium channel opener; Cholinergic neurotransmission; Rabbit gastric fundus
1. Introduction
to modulate physiological processes such as insulin secretion,
leptin release, synaptic transmission and excitability of car-
diac, vascular and nonvascular smooth muscles [7].
It is well known that ion channels are very important for
cell function and responsible for physiological effects. Potas-
sium channels regulate some functions in both excitable and
non-excitable cells. Potassium channel opening is a physiolog-
ical mechanism by which excitable cells exploit to maintain or
restore their resting state [1e5].
KATP channel (ATP-sensitive Kþ channel) openers are
a structurally diverse group of drugs with a broad spectrum
of potential therapeutic usages. These drugs interact with
KATP channels in numerous tissues and increase their activity,
thereby hyperpolarizing the plasma membrane and reducing
electrical excitability [6]. These channels play critical roles
K
ATP channel openers have a relaxation effect due to lower
cellular membrane potential and inhibit calcium influx. There
has been considerable interest in exploring KATP channel
openers in the treatment of various diseases such as cardiovas-
cular, cerebrovascular, and urinary system disease and prema-
ture labor [8,9].
The properties of KATP channels in guinea pig gastric myo-
cytes were similar to those of KATP channels in other smooth
muscles [10].
Many 1,4-dihydropyridine (1,4-DHP) derivatives are known
to be calcium channel blockers. In addition it has been shown
that some of these derivatives have potassium channel opening
activity [11]. For example, niguldipine, which is a 1,4-DHP
derivative has increased Kþ flux in isolated vascular smooth
muscle by opening Ca2þ-activated potassium channels [3].
* Corresponding author. Tel.: þ90 312 3052341; fax: þ90 312 3051872.
E-mail address: csafak@hacettepe.edu.tr (C. Sxafak).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.04.012

M.G. Gu¨ndu¨z et al. / European Journal of Medicinal Chemistry 43 (2008) 562e568
563
NO2
cleavage of the aryl ring from the parent molecule. In further
fragmentation, the ions are formed by the rupture of the cyclo-
hexene ring and acylium ions are formed by the cleavage of
the ester group. Aromatisation of the DHP ring to the pyridine
analog was also observed (Scheme 2). These findings are in
accordance with the literature [15e17].
CH3OOC
H3C
COOCH2CH2CH2
CH3
N
N
H
In addition, X-ray analysis of compounds 3c and 3k was re-
alised and published elsewhere [18]. X-ray analysis results
showed that the 2,3-dichlorophenyl ring is oriented such that
the chloro substituents are in a synperiplanar orientation
with respect to the 1,4-DHP ring plane and the oxocyclohex-
ene ring has a slightly distorted envelope conformation.
Both structures exhibit the same intermolecular N/H/O
hydrogen bonding motif in which the molecules are linked
into chains by interactions involving the carbonyl oxygen
atom of the oxocyclohexene ring. Finally, the elemental anal-
ysis results are also consistent with the postulated structures.
Although all compounds have two asymmetric centers in
their 4 and 7 positions of HHQ ring, their isomers could not
be separated due to technical shortage. Therefore the effect
of isomerism on the mentioned activity could not be
determined.
Niguldipine
It is well known that bicyclo (quinoline) and tricyclo (acri-
dine) analogs of 1,4-DHP also have potassium channel open-
ing activities [12,13]. The object of this study is to
investigate the effects of quinoline derivatives in rabbit gastric
fundus smooth muscle strips.
In this article, 16 annelated 1,4-DHP derivatives have been
synthesized to take into one of the carbonyl groups in con-
densed ring system. The contribution of dichlorophenyl in 4
position and methyl or phenyl substituent in 7 position of
quinoline ring to the activity was also studied.
2. Results and discussion
2.1. Chemistry
2.2. Pharmacology
The hexahydroquinoline (HHQ) derivatives were prepared
by the Hantzsch reaction [14]. The reaction of a dichloroben-
zaldehyde derivative, alkyl aminocrotonate and 5-methyl (or
5-phenyl)-l,3-cyclohexanedione in methanol yielded the re-
spective HHQ derivative (Scheme 1).
2.2.1. Relaxant effects of compounds and pinacidil on the
tissues precontracted by noradrenaline
The maximum relaxant effects (Emax) and pD2 values of
compounds 3aep and pinacidil on isolated strips of rabbit gas-
tric fundus smooth muscle are given in Table 1.
The structures of the compounds were elucidated by IR, 1H
NMR, 13C NMR, mass spectra and elemental analysis. In the
IR spectra, characteristic NeH and C]O stretching bands
The results indicated that compounds 3aej, 3l, 3m pro-
duced concentration-dependent relaxation in rabbit gastric
fundus smooth muscle strips. Compounds 3k, 3nep dis-
played slightly relaxant responses which were not statistically
significant from control relaxations produced by dimethyl-
sulphoxide (DMSO). Compounds and pinacidil exerted con-
centration-dependent relaxation responses precontracted
with submaximal concentration of noradrenaline in the gas-
tric fundus smooth muscle strips with the efficacy order:
3b > 3l ꢀ 3c ꢀ pinacidil ꢀ 3m ꢀ 3j > 3d > 3h > 3i ꢀ 3g > 3a
> 3e ꢀ 3f. It is interesting that Emax value of compound 3b
was higher than that of pinacidil. Also Emax values of com-
pounds 3c and 3l have been found similar to that of pinacidil.
Glibenclamide did not reverse the myorelaxant effects of the
1
were seen. In H NMR spectra, 7-CH3 protons of the HHQ
ring were seen at 1.05 ppm for compounds 3aei. H-7 protons
of all compounds, were seen at 2.00e3.00 ppm. The chemical
shifts of the aromatic, 2-methyl, methylene and methine pro-
tons of the compounds have expected values. The NeH sig-
nals were seen at 8.80 ppm as a broad singlet. The 13C
NMR spectra of the compounds displayed the appropriate
number of resonances that exactly fitted the number of carbon
atoms. The mass spectra of the compounds were recorded us-
ing the electron impact technique. Molecular ion peaks were
seen for all molecules. Also the base peak is formed by
Ar
Ar
O
O
O
COOR1
CH
CH3C(=NH)CH2COOR1
ArCHO
+
R
O
R
N
R
O
CH3
H
3a-p
1
2
R: CH3, C6H5
R1: CH3, C2H5
Ar: Dichlorophenyl
Scheme 1. Synthesis of HHQ derivatives.

564
M.G. Gu¨ndu¨z et al. / European Journal of Medicinal Chemistry 43 (2008) 562e568
+
O
O
Ar
N
Ar
O
+
COOR
CH3
COOR
CH3
COOR
CH3
- Ar
N
H
M-Ar +
N
H
R1
R1
R1
Scheme 2. Main fragmentation of the compounds.
compounds. These results suggested that myorelaxant effects
of the compounds were not mediated by KATP channels. Cal-
cium channels or other types of potassium channels may me-
diate these effects of the compounds. While compounds 3a,
3i and 3l had a direct relaxant effect on exogenous noradren-
aline precontracted tissues, they did not alter EFS induced
contractile responses (Table 1).
To investigate whether relaxation induced by the test com-
pounds was due to interaction with the cyclooxygenase, adren-
ergic system or nitric oxide pathways, tissues were pretreated
with indomethacin (cyclooxygenase inhibitor), propranolol
(b-adrenergic receptor blocker) or N-u-nitro-L-arginine
methyl ester (L-NAME) hydrochloride (the nitric oxide syn-
thase inhibitor), respectively. Pretreatment of the strips with
indomethacin, propranolol and L-NAME did not significantly
alter the relaxant responses to the compounds. This situation
explained that cyclooxygenase, adrenergic and nitric oxide
(NO) pathways do not play a role in relaxations evoked by
these substances.
2.2.2. Effects of compounds and pinacidil on EFS evoked
neurogenic contractions of the rabbit gastric fundus smooth
muscle strips
Pharmacological analysis of field stimulation-induced me-
chanical responses in isolated smooth muscle strips is a useful
in vitro technique for clarifying neurotransmitter release. Pre-
synaptic potassium channels play an important role on the EFS
evoked neurotransmitter release. EFS evoked contractile re-
sponses in rabbit gastric fundus. Tetrodotoxin (TTX) abolished
these responses. Cadmium (Cd2þ) ions reduced the contrac-
tion responses evoked by EFS. In this study, atropine (a mus-
carinic receptor blocker), abolished the contraction responses
evoked by EFS and neostigmine (acetylcholinesterase inhibi-
tor), potentiated responses indicating that EFS evoked contrac-
tile responses due to cholinergic neurotransmission.
Compounds 3be3h, 3j, 3k, 3m, 3o, 3p and pinacidil
caused a concentration-dependent inhibition of EFS evoked
contractions of rabbit gastric fundus smooth muscle strips as
given in Table 1.
Table 1
Maximum relaxant responses (Emax*) and pD2* values on precontracted tissues by noradrenaline and maximum inhibitory responses (Emax**) and pD2** values of
compounds 3aep and pinacidil on EFS evoked contractile responses on isolated strips of rabbit gastric fundus smooth muscle
Ar
O
COOR1
N
H
R
CH3
R
R1
Ar
Emax*
pD2*
E
max**
pD2**
3a
CH3
CH3
CH3
2,3-Dichlorophenyl
2,3-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,5-Dichlorophenyl
2,5-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,3-Dichlorophenyl
2,3-Dichlorophenyl
2,4-Dichlorophenyl
2,4-Dichlorophenyl
2,5-Dichlorophenyl
2,5-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
53.99 ꢁ 13.01a
89.47 ꢁ 8.77a
71.2 ꢁ 9.41a
63.65 ꢁ 5.41a
47.99 ꢁ 3.81a
45.78 ꢁ 5.99a
56.01 ꢁ 9.55a
59.75 ꢁ 6.12a
56.29 ꢁ 5.89a
66.35 ꢁ 13.95a
No effect
5.26 ꢁ 0.58a
6.02 ꢁ 0.68a
5.62 ꢁ 0.76a
5.96 ꢁ 0.27a
5.54 ꢁ 0.31a
5.75 ꢁ 0.36a
5.91 ꢁ 0.61a
5.93 ꢁ 0.23a
5.62 ꢁ 0.46a
6.06 ꢁ 0.73a
No effect
No effect
No effect
3b
3c
C2H5
CH3
52.87 ꢁ 12.08a
57.71 ꢁ 3.30a
52.04 ꢁ 8.62a
45.95 ꢁ 21.69a
47.74 ꢁ 4.40a
69.62 ꢁ 1.29a
52.07 ꢁ 21.31a
No effect
6.02 ꢁ 0.62a
5.23 ꢁ 0.79a
5.27 ꢁ 0.32a
5.03 ꢁ 0.36a
6.16 ꢁ 0.38a
6.26 ꢁ 0.55a
6.05 ꢁ 0.27a
No effect
CH3
CH3
3d
3e
C2H5
CH3
CH3
CH3
3f
3g
C2H5
CH3
CH3
CH3
3h
3i
C2H5
CH3
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
3j
C2H5
CH3
41.25 ꢁ 12.17a
41.31 ꢁ 4.84a
No effect
5.15 ꢁ 0.69a
6.35 ꢁ 0.47a
No effect
3k
3l
C2H5
CH3
72.46 ꢁ 12.61a
67.72 ꢁ 13.76a
No effect
7.06 ꢁ 0.93a
6.54 ꢁ 1.06a
No effect
3m
3n
3o
53.56 ꢁ 11.29a
7.05 ꢁ 0.97a
C2H5
CH3
No effect
No effect
No effect
No effect
71.15 ꢁ 5.39a
No effect
No effect
5.03 ꢁ 0.12a
88.61 ꢁ 5.87a
75.08 ꢁ 8.45a
76.33 ꢁ 3.44a
5.35 ꢁ 0.38a
5.05 ꢁ 0.57a
7.49 ꢁ 0.15a
3p
Pinacidil
C2H5
Relaxation is expressed as a percentage of the precontraction induced by noradrenaline. The inhibitory effects of the compounds and pinacidil on EFS evoked
contractions were expressed as percentage of the control contraction. The negative logarithm of the concentration for the half-maximal response (pD2) and
Emax values represent mean ꢁ S.E.
a
p < 0.05, compared with control responses (n ¼ 6).

M.G. Gu¨ndu¨z et al. / European Journal of Medicinal Chemistry 43 (2008) 562e568
565
1
Compounds 3a, 3i, 3l and 3n displayed inhibitory responses,
which were not statistically significant from control inhibition
produced by DMSO. Compounds and pinacidil exerted con-
centration-dependent inhibition of EFS induced contractile
responses in the gastric fundus smooth muscle strips with the ef-
ficacy order: 3o > pinacidil ¼ 3p > 3g > 3c ꢀ 3m ¼ 3b ¼
3d ¼ 3h ꢀ 3f ꢀ 3e ꢀ 3k ¼ 3j.
NMR and H AMX 600 MHz FT NMR Spectrophotometer
(Karlsruhe, Germany) (DMSO-d6; tetramethylsilane as internal
standard). 13C NMR Spectra: 13C AMX 150 MHz FT NMR
Spectrophotometer. Chemical shift values are given as ppm.
Mass spectra: Hewlett Packard Series II Plus 5890 GAS Chro-
matographeHewlett Packard 5972 Series Mass Selective De-
tector (Philadelphia, USA). Thin Layer Chromatography
(TLC) was run on precoated silica gel (E. Merck, Darmstadt,
Germany) and short wave UV light (254 nm) was used to detect
the UV absorbing spots. Elemental analysis was carried out on
a Leco 932 CHNS-O Elemental Analyzer (Philadelphia, USA)
(TUBITAK, Ankara, Turkey). The elemental analysis results
were within 0.4% of theoretical values.
It is interesting to note that efficacy of compound 3o is
greater than that of pinacidil but potency of this compound
is lower than that of pinacidil. Efficacy of compound 3p is
similar to that of pinacidil but pinacidil is 100 fold more potent
than this compound (Table 1). In our study, Glibenclamide,
a KATP channel blocker, at 10ꢂ6 M reversed the inhibition ef-
fects of compounds 3c (16.14%), 3e (11.24%), 3g (27.35%),
3h (17.16%) and pinacidil (32.18%) on EFS evoked contrac-
tions. This result suggests that inhibitory effects of mentioned
compounds were possibly mediated, at least in part, by presyn-
aptic KATP channels. Surprisingly, compounds 3o and 3p
which had no direct relaxant effect on exogenous noradrena-
line precontractile tissues, have inhibited EFS induced con-
tractile responses. On the other hand, it is also surprising to
note that the most potent compound on isolated strips of rabbit
gastric fundus smooth muscle (compound 3l with a pD2 value
of 7.06) was completely inactive on EFS evoked neurogenic
contraction of the same smooth muscle.
4.1.1. Synthesis of methyl(ethyl) 4-(dichlorophenyl)-2,7-
dimethyl-5-oxo-l,4,5,6,7,8-hexahydroquinoline-3-
carboxylates (3aeh)
A mixture of methyl or ethyl aminocrotonate (0.001 mol)
5-methyl-1,3-cyclohexanedione (0.001 mol), and appropriate
aromatic aldehyde (0.001 mol) in 20 mL methanol was re-
fluxed for 4 h. The solvent was evaporated and the residue
was crystallized from alcohol.
4.1.1.1. Methyl 4-(2,3-dichlorophenyl)-2,7-dimethyl-5-oxo-1,
4,5,6,7,8-hexahydroquinoline-3-carboxylate (3a). Mp. 245 ꢃC.
IR (cmꢂ1) 3288, 1703, 1616, 776. H NMR d 1.05 (d, 3H, 7-
1
3. Conclusion
CH3), 2.05e2.70 (m, 5H, H-6, 7, 8 HHQ), 2.35 (s, 3H,
2-CH3), 3.60 (s, 3H, COOCH3), 5.41 (s, 1H, H-4 HHQ),
7.00e7.50 (m, 3H, Ar), 8.90 (broad, 1H, NH ). 13C NMR
d 19.1, 20.7, 28.8, 35.2, 36.9, 51.0, 53.3, 102.3, 106.1, 126.7,
127.3, 128.4, 131.7, 133.1, 133.7, 143.5, 146.0, 167.3, 197.4.
MS (m/z) 234 (%100). Anal. calcd. (C19H19Cl2NO3) C, H, N.
Our results showed that several hexahydroquinoline deriva-
tives have a myorelaxant activity in isolated rabbit gastric fun-
dus smooth muscle precontracted by noradrenaline similar to
pinacidil. Glibenclamide did not reverse the myorelaxant ef-
fects of the compounds. This result suggests that myorelaxant
effects of compounds were not mediated by KATP channels.
Several other compounds have an inhibitory activity on the
EFS evoked contractions (cholinergic neurotransmission) in
isolated rabbit gastric fundus smooth muscle similar to pinaci-
dil. Glibenclamide partially reversed the inhibitory effects of
compounds 3c, 3e, 3g, 3h and pinacidil on EFS evoked con-
tractions. This result suggests that inhibitory effects of com-
pounds 3c, 3e, 3g and 3h were possibly mediated at least in
part by KATP channels. But the potassium channel opening
ability of these compounds needs further investigations by
using radioisotopic or electrophysiological experiments.
4.1.1.2. Ethyl 4-(2,3-dichlorophenyl)-2,7-dimethyl-5-oxo-1,4,5,
6,7,8-hexahydroquinoline-3-carboxylate (3b). Mp. 246 ꢃC. IR
1
(cmꢂ1) 3290, 1702, 1616, 769. H NMR d 1.05 (d, 3H, 7-
CH3), 1.20 (t, 3H, CH2CH3), 2.35 (s, 3H, 2-CH3), 2.00e2.55
(m, 5H, H-6,7,8 HHQ), 4.05 (q, 2H, CH2CH3), 5.45 (s, 1H,
H-4 HHQ), 7.00e7.40 (m, 3H, Ar), 8.90 (broad, 1H, NH ).
13C NMR d 14.2, 19.2, 20.7, 28.8, 35.3, 35.5, 37.2, 59.9,
103.1, 106.0, 126.5, 126.6, 128.3, 130.5, 131.8, 133.0,
143.7, 145.2, 167.0, 195.7. MS (m/z) 248 (%100). Anal. calcd.
(C20H21Cl2NO3) C, H, N.
4.1.1.3. Methyl 4-(2,4-dichlorophenyl)-2,7-dimethyl-5-oxo-1,4,
4. Experimental
5,6,7,8-hexahydroquinoline-3-carboxylate (3c). Mp. 250 ꢃC.
1
IR (cmꢂ1) 3284, 1708, 1615, 761. H NMR d 1.05 (d, 3H,
4.1. Chemistry
7-CH3), 2.00e2.55 (m, 5H, H-6,7,8 HHQ), 2.35 (s, 3H, 2-
CH3), 3.60 (s, 3H, COOCH3), 5.35 (s, 1H, H-4 HHQ),
7.05e7.35 (m, 3H, Ar), 8.90 (broad, 1H, NH ). 13C NMR
d 19.0, 20.8, 28.7, 35.2, 44.7, 50.9, 53.4, 104.9, 105.1,
126.8, 127.1, 129.1, 132.1, 132.6, 133.7, 143.2, 144.3,
167.7, 196.1. MS: (m/z) 234 (%100). Anal. calcd.
(C19H19Cl2NO3) C, H, N.
All chemicals used in this study were purchased from
Aldrich (Steinheim, Germany), Fluka (Buchs, Switzerland)
and Sigma (Germany).
Melting points were determined using a Thomas Hoover
Capillary Melting Point Apparatus (Philadelphia, PA, USA)
and are uncorrected. IR spectra: Perkin Elmer FT-IR Spectro-
photometer 1720 X (Beaconsfield, UK) (KBr disc) (n, cmꢂ1).
1H NMR Spectra: Bruker GMB HD PX-400 MHz Digital FT
4.1.1.4. Ethyl 4-(2,4-dichlorophenyl)-2,7-dimethyl-5-oxo-1,4,5,
6,7,8-hexahydroquinoline-3-carboxylate (3d). Mp. 218 ꢃC. IR

566
M.G. Gu¨ndu¨z et al. / European Journal of Medicinal Chemistry 43 (2008) 562e568
1
(cmꢂ1) 3275, 1703, 1608, 761. H NMR d 1.05 (d, 3H, 7-
CH3), 1.20 (t, 3H, t; CH2CH3), 2.35 (s, 3H, 2-CH3), 2.00e
2.55 (m, 5H, H-6,7,8 HHQ), 4.10 (q, 2H, CH2CH3), 5.35 (s,
1H, H-4 HHQ), 7.00e7.40 (m, 3H, Ar), 8.85 (broad, 1H,
NH ). 13C NMR d 14.2, 19.3, 20.6, 28.8, 35.3, 35.9, 44.7,
60.0, 111.2, 111.4, 126.7, 129.3, 132.2, 133.1, 133.3, 133.9,
142.6, 143.7, 167.1, 195.7. MS: (m/z) 248 (%100). Anal.
calcd. (C20H21Cl2NO3) C, H, N.
aromatic aldehyde (0.001 mol) in 20 mL methanol was re-
fluxed for 4 h. The solvent was evaporated and the residue
was crystallized from alcohol.
4.1.2.1. Methyl 4-(2,3-dichlorophenyl)-2-methyl-5-oxo-7-phe-
nyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (3i). Mp.
228 ꢃC. IR (cmꢂ1) 3292, 1707, 1612, 740. 1H NMR d 2.35 (s,
3H, 2-CH3), 2.45e2.85 (m, 5H, H-6,7,8 HHQ), 3.60 (s, 3H,
COOCH3), 5.45 (s, 1H, H-4 HHQ), 7.00e7.45 (m, 8H, Ar),
8.65 (broad, 1H, NH ). 13C NMR d 19.3, 34.9, 37.1, 39.3,
43.6, 50.9, 104.9, 112.2, 126.7, 127.1, 127.3, 128.9, 130.0,
130.2, 131.8, 133.1, 142.4, 146.2, 149.4, 151.2, 167.6,194.7.
MS (m/z) 296 (%100). Anal. calcd. (C24H21Cl2NO3) C, H, N.
4.1.1.5. Methyl 4-(2,5-dichlorophenyl)-2,7-dimethyl-5-oxo-1,4,
5,6,7,8-hexahydroquinoline-3-carboxylate (3e). Mp. 229 ꢃC.
IR (cmꢂ1) 3282, 1701, 1611, 741. H NMR d 1.05 (d, 3H,
1
7-CH3), 2.00e2.60 (m, 5H, H-6,7,8 HHQ), 2.40 (s, 3H, 2-
CH3), 3.60 (s, 3H, COOCH3), 5.35 (s, 1H, H-4 HHQ),
7.00e7.35 (m, 3H, Ar), 8.95 (broad, 1H, NH ). 13C NMR
d 19.2, 20.7, 28.9, 35.3, 35.8, 44.5, 50.9, 105.8, 111.7,
127.5, 130.4, 130.6, 131.2, 131.7, 132.3, 143.7, 145.9,
167.4, 195.6. MS: (m/z) 234 (%100). Anal. calcd.
(C19H19Cl2NO3) C, H, N.
4.1.2.2. Ethyl 4-(2,3-dichlorophenyl)-2-methyl-5-oxo-7-phenyl-
1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (3j). Mp.131 ꢃC.
1
IR (cmꢂ1) 3292, 1699, 1614, 765. H NMR d 1.15 (t, 3H,
CH2CH3), 2.30 (s, 3H, 2-CH3), 2.45e2.85 (m, 5H, H-6,7,8
HHQ), 4.05 (q, 2H, CH2CH3), 5.45 (s, 1H, H-4 HHQ), 7.00e
7.45 (m, 8H, Ar), 8.65 (broad, 1H, NH ). 13C NMR d 14.2,
19.2, 34.6, 37.2, 39.3, 43.6, 59.9, 105.6, 111.4, 126.5, 126.7,
127.1, 128.7, 130.4, 131.1 131.8, 133.0, 142.3, 143.6, 145.9,
146.1, 167.1, 194.9. MS (m/z) 310 (%100). Anal. Calcd.
(C25H23Cl2NO3) C, H, N.
4.1.1.6. Ethyl 4-(2,5-dichlorophenyl)-2,7-dimethyl-5-oxo-1,4,5,
6,7,8-hexahydroquinoline-3-carboxylate (3f). Mp. 236 ꢃC. IR
1
(cmꢂ1) 3285, 1699, 1612, 746. H NMR d 1.05 (d, 3H, 7-
CH3), 1.15 (t, 3H, CH2CH3), 2.35 (s, 3H, 2-CH3), 2.00e2.60
(m, 5H, H-6,7,8 HHQ), 4.05 (q, 2H, CH2CH3), 5.35 (s, 1H,
H-4 HHQ), 7.00e7.35 (m, 3H, Ar), 8.95 (broad, 1H, NH ).
13C NMR d 14.1, 19.3, 20.8, 28.9, 35.3, 36.3, 44.6, 60.0,
111.1, 111.3, 127.4, 129.2, 130.6, 130.9, 131.8, 132.1,
143.8, 145.7, 167.0, 195.7. MS (m/z) 248 (%100). Anal. calcd.
(C20H21Cl2NO3) C, H, N.
4.1.2.3. Methyl 4-(2,4-dichlorophenyl)-2-methyl-5-oxo-7-phe-
nyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (3k). Mp.
1
148 ꢃC. IR (cmꢂ1) 3303, 1704, 1618, 766. H NMR d 2.30
(s, 3H, 2-CH3), 2.45e2.85 (m, 5H, H-6,7,8 HHQ), 3.60 (s,
3H, COOCH3), 5.40 (s, 1H, H-4 HHQ), 7.00e7.45 (m, 8H,
Ar), 8.65 (broad, 1H, NH ). 13C NMR d 19.0, 34.5, 38.8,
39.3, 43.5, 50.9, 105.3, 111.6, 126.7, 127.1, 127.4, 128.9,
129.1, 132.1, 132.6, 133.1, 133.8, 142.3, 143.1, 144.3,
167.6, 195.1. MS (m/z) 296 (%100). Anal. calcd.
(C24H21Cl2NO3) C, H, N.
4.1.1.7. Methyl 4-(2,6-dichlorophenyl)-2,7-dimethyl-5-oxo-1,4,
5,6,7,8-hexahydroquinoline-3-carboxylate (3g). Mp. 243 ꢃC.
IR (cmꢂ1) 3301, 1704, 1616, 780. H NMR d 1.05 (d, 3H,
1
7-CH3), 1.95e2.50 (m, 5H, H-6,7,8 HHQ), 2.25 (s, 3H, 2-
CH3), 3.55 (s, 3H, COOCH3), 5.85 (s, 1H, H-4 HHQ),
6.90e7.35 (m, 3H, Ar), 9.05 (broad, 1H, NH ). 13C NMR
d 18.9, 20.7, 28.5, 35.1, 35.6, 43.9, 50.6, 103.1, 106.8,
127.3, 132.1, 133.3, 138.8, 144.8, 145.3, 167.6, 195.3. MS
(m/z) 234 (%100). Anal. calcd. (C19H19Cl2NO3) C, H, N.
4.1.2.4. Ethyl 4-(2,4-dichlorophenyl)-2-methyl-5-oxo-7-phenyl-
1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (3l). Mp. 130 ꢃC.
1
IR (cmꢂ1) 3293, 1702, 1618, 766. H NMR d 1.25 (t, 3H,
CH2CH3), 2.35 (s, 3H, 2-CH3), 2.45e2.85 (m, 5H, H-6,7,8
HHQ), 4.10 (q, 2H, CH2CH3), 5.40 (s, 1H, H-4 HHQ), 7.00e
7.55 (m, 8H, Ar), 8.65 (broad, 1H, NH ). 13C NMR d 14.2,
19.3, 34.9, 38.7, 39.4, 43.6, 59.9, 104.9, 111.7, 126.7, 126.9,
127.1, 128.7, 129.2, 132.9, 133.1, 134.0, 142.3, 143.6, 149.7,
158.9, 167.1, 194.7. MS (m/z) 310 (%100). Anal. calcd.
(C25H23Cl2NO3) C, H, N.
4.1.1.8. Ethyl 4-(2,6-dichlorophenyl)-2,7-dimethyl-5-oxo-1,4,
5,6,7,8-hexahydroquinoline-3-carboxylate (3h). Mp. 244 ꢃC.
IR (cmꢂ1) 3292, 1703, 1652, 776. H NMR d 1.05 (d, 3H,
1
7-CH3), 1.15 (t, 3H, CH2CH3), 2.25 (s, 3H, 2-CH3), 1.95e
2.65 (m, 5H, H-6,7,8 HHQ), 4.05 (q, 2H, CH2CH3), 5.85 (s,
1H, H-4 HHQ), 6.95e7.45 (m, 3H, Ar), 9.05 (broad, 1H,
NH ). 13C NMR d 14.1, 19.1, 20.7, 28.5, 35.2, 35.5, 49.6,
59.8, 103.6, 106.8, 127.4, 132.1, 132.3, 138.6, 143.9, 145.7,
166.9, 194.8. MS (m/z) 248 (%100). Anal. Calcd.
(C20H21Cl2NO3) C, H, N.
4.1.2.5. Methyl 4-(2,5-dichlorophenyl)-2-methyl-5-oxo-7-phe-
nyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (3m). Mp.
1
184 ꢃC. IR (cmꢂ1) 3293, 1701, 1611, 755. H NMR d 2.35
(s, 3H, 2-CH3), 2.45e2.85 (m, 5H, H-6,7,8 HHQ), 3.65 (s,
3H, COOCH3), 5.40 (s, 1H, H-4 HHQ), 7.00e7.45 (m, 8H,
Ar), 8.65 (broad, 1H, NH ). 13C NMR d 19.0, 34.6, 38.8,
39.2, 43.3, 51.0, 105.6, 111.6, 126.7, 127.2, 127.4, 127.6,
128.9, 130.2, 130.6, 131.2, 132.5, 142.1, 144.0, 146.0,
167.4, 194.9. MS (m/z) 296 (%100). Anal. calcd.
(C24H21Cl2NO3) C, H, N.
4.1.2. Synthesis of methyl(ethyl) 4-(dichlorophenyl)-2-
methyl-5-oxo-7-phenyl-l,4,5,6,7,8-hexahydroquinoline-3-
carboxylates (3iep)
A mixture of methyl(ethyl) aminocrotonate (0.001 mol) 5-
phenyl-1,3-cyclohexanedione (0.001 mol) and appropriate

M.G. Gu¨ndu¨z et al. / European Journal of Medicinal Chemistry 43 (2008) 562e568
567
4.1.2.6. Ethyl 4-(2,5-dichlorophenyl)-2-methyl-5-oxo-7-phenyl-
NaH2PO4 0.9; glucose 10.04. The solution was gassed with
95% O2 and 5% CO2 during the study and temperature
was maintained at 37 ꢃC by a thermoregulated 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 measure-
ment of isometric force, which was continuously displaced
and recorded on an online computer via four-channel trans-
ducer 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 ana-
lyze the data. After mounting, each strip was allowed to
equilibrate with a basal tension of 1 g. for 60 min. KHS
was replaced with fresh solution every 15 min. during this
period. N-u-nitro-L-arginine methyl ester (L-NAME) hydro-
chloride (the nitric oxide synthase inhibitor, 10ꢂ4 M), indo-
methacin (COX inhibitor, 10ꢂ5 M) and propranolol (b-
adrenergic receptor blocker, 10ꢂ6) were added into the organ
bath 20 min before the precontraction in order to eliminate
the effects of nitric oxide, prostaglandins and adrenergic ag-
onists. Rabbit gastric fundus smooth muscle strips were pre-
contracted with submaximal concentration of noradrenaline
(10ꢂ5 M). Concentrationerelaxation responses for com-
pounds 3aep, pinacidil and dimethylsulphoxide (DMSO)
were obtained by adding these into the bath in a cumulative
manner. A cumulative concentrationeresponse curve was
constructed in a stepwise manner after the response to the
previous concentration had reached a plateau. This procedure
was repeated in the presence of glibenclamide (10ꢂ6 M).
The relaxant effects of the compounds and pinacidil were
expressed as percentage of the precontraction using submaxi-
mal concentration of noradrenaline.
1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (3n). Mp. 129 ꢃC.
1
IR (cmꢂ1) 3302, 1696, 1616, 763. H NMR d 1.15 (t, 3H,
CH2CH3), 2.35 (s, 3H, 2-CH3), 2.45e2.95 (m, 5H, H-6,7,8
HHQ), 4.10 (q, 2H, CH2CH3), 5.45 (s, 1H, H-4 HHQ), 6.90e
7.70 (m, 8H, Ar), 8.70 (broad, 1H, NH). 13C NMR d 14.2,
19.2, 34.7, 38.7, 39.2, 43.9, 60.0, 105.6, 111.4, 126.7, 127.2,
128.7, 129.4, 130.4, 131.7, 132.2, 133.0, 142.2, 143.8, 144.0,
145.7, 167.0, 194.9. MS (m/z) 310 (%100). Anal. calcd.
(C25H23Cl2NO3) C, H, N.
4.1.2.7. Methyl 4-(2,6-dichlorophenyl)-2-methyl-5-oxo-7-phe-
nyl-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (3o). Mp.
1
128 ꢃC. IR (cmꢂ1) 3315, 1703, 1615, 764. H NMR 2.25 (s,
3H, 2-CH3), 2.40e2.95 (m, 5H, H-6,7,8 HHQ), 3.55 (s, 3H,
COOCH3), 5.85 (s, 1H, H-4 HHQ), 6.85e7.60 (m, 8H, Ar),
8.65 (broad, 1H, NH ). 13C NMR d 18.9, 34.7, 38.6, 39.3,
44.0, 50.7, 105.6, 111.6, 126.7, 127.2, 127.4, 127.6, 128.9,
130.6, 131.2, 132.5, 142.1, 144.3, 146.0, 167.3, 194.2. MS (m/z)
296 (%100). Anal. calcd. (C24H21Cl2NO3) C, H, N.
4.1.2.8. Ethyl 4-(2,6-dichlorophenyl)-2-methyl-5-oxo-7-phenyl-
1,4,5,6,7,8-hexahydroquinoline-3-carboxylate
(3p). Mp.
113 ꢃC. IR (cmꢂ1) 3319, 1699, 1615, 769. H NMR d 1.15
(t, 3H, CH2CH3), 2.30 (s, 3H, 2-CH3), 2.40e3.00 (m, 5H,
H-6,7,8 HHQ), 4.05 (q, 2H, CH2CH3), 5.85 (s, 1H, H-4
HHQ), 6.90e7.75 (m, 8H, Ar), 8.70 (broad, 1H, NH ). 13C
NMR d 14.1, 19.1, 34.6, 38.9, 39.3, 43.9, 59.7, 102.9, 109.5,
126.7, 127.2, 128.7, 129.4, 130.4, 132.2, 133.0, 142.2,
143.8, 144.9, 152.2, 167.3, 194.9. MS (m/z) 310 (%100).
Anal. calcd. (C25H23Cl2NO3) C, H, N.
1
4.2. Pharmacology
In another set of experiments, the inhibitory effects of the
compounds and pinacidil were tested on electrical field stim-
ulation (EFS) induced neurotransmitter release. Electrical
stimulation of gastric fundus smooth muscle strips was pro-
vided by two parallel platinum electrodes (diameter 3 mm,
separation 10 mm.). Pulses of 1 ms duration with a voltage
of 60 V were delivered by a stimulator (may STPT 03 Re-
search stimulator, COMMAT Iletisim Co., Ankara, Turkey).
Frequencyeresponse contractions of gastric fundus smooth
muscle strips at 8 Hz. were obtained. To determinate the
EFS induced contractile responses TTX (a blocker of Naþ
channels, 10ꢂ6 M), Cadmium (Cd2þ, a voltage-gated cal-
cium channel blocker, 10ꢂ4 M), atropine (a muscarinic
receptor blocker, 10ꢂ6 M) and neostigmine (acetylcholines-
terase inhibitor, 10ꢂ6 M) were applied (Fig. 1). The EFS in-
duced contractile responses were repeated in the presence of
compounds 3aep, pinacidil and DMSO at cumulative con-
centrations (10ꢂ9e3 ꢄ 10ꢂ4 M). The initial response to
EFS was used as the control. This experimental protocol
was repeated in the presence of glibenclamide (10ꢂ6 M)
(Fig. 2).
Cadmium chloride, TTX, atropine sulphate, glibenclamide,
L-NAME, indomethacin, propranolol hydrochloride, neostig-
mine hydrochloride, noradrenaline hydrate, pinacidil and
DMSO were supplied by Sigma. Stock solution of TTX, cad-
mium, atropine, L-NAME, propranolol, neostigmine and nor-
adrenaline were dissolved in distilled water. Compounds,
pinacidil and indomethacin were dissolved in DMSO.
DMSO has no effect on experiments.
New Zealand white rabbits, weighing 2.5e3 kg were used
in this study. The study was approved by the Ethics Commit-
tee at Gazi University Medical School. Procedures involving
animals and their care were conducted in conformity with in-
ternational laws and policies. At time of study, rabbits were
sacrificed with i.v. injection of sodium pentobarbital (30e
40 mg/kg, i.v.), followed by removal of the stomach through
abdominal incision. The fundal part of the stomach was then
dissected parallel to the longitudinal muscle wall. One mus-
cle strip with approximately 15e20 mm length and 2 mm
width was obtained and allowed to equilibrate for a period
of 60 min in 20 mL organ baths filled with normal
Krebs’eHenseleit solution (KHS). The composition of the
KHS used in the study was as follows (in mmol/l): NaCl
118; KCl 4.7; CaCl2 1.26; NaHCO3 25; Mg Cl2 0.54;
4.2.1. Data analysis
The relaxant effects of the compounds on the tissues, pre-
contracted with noradrenaline, were expressed as percentage

568
M.G. Gu¨ndu¨z et al. / European Journal of Medicinal Chemistry 43 (2008) 562e568
compounds or pinacidil. To evaluate the effects of the com-
pounds, the maximum response (Em) [each drug’s Em value
has been established at 3 ꢄ 10ꢂ4 M concentration] and pD2
values [the negative logarithm of the concentration for the
half-maximal response (EC50)] were calculated, as predicted
from the Scatchard equation for drugereceptor interaction.
Agonist pD2 values (apparent agonist affinity constants)
were calculated from each agonist concentrationeresponse
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. While Emax is the parameter for efficacy, pD2 is the
parameter for potency. All data are expressed as
mean ꢁ standard error.
4.2.2. Statistical analysis
Statistical comparison between groups were performed
using general linear models by Scheffe’s F-test and p
values less than 0.05 were considered to be statistically
significant.
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