Investigation of myorelaxant activity of 9-aryl-3,4,6,7-tetrahydroacridine- 1,8-(2H,5H,9H,10H)-diones in isolated rabbit gastric fundus

Article abstract of DOI:10.1007/s00044-011-9698-x

In this study, twelve compounds having 9-aryl- 3,4,6,7-tetrahydroacridine- 1,8-(2H,5H,9H,10H)-dione structure were synthesized by reaction of 5-methyl-1,3-cyclohexanedione, the appropriate aromatic aldehydes, and ammonium acetate in methanol. The structur

Full text of DOI:10.1007/s00044-011-9698-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1817–1824
DOI 10.1007/s00044-011-9698-x
ORIGINAL RESEARCH
Investigation of myorelaxant activity of 9-aryl-3,4,6,7-tetrahydroacridine-
1,8-(2H,5H,9H,10H)-diones in isolated rabbit gastric fundus
¨
Gokc¸e Sevim Ozturk Fincan Miyase Gozde Gunduz
•
•
¨
Ismail Mert Vural Rahime S¸ims¸ek
¨
¨
¨
¨
˙
•
•
•
˘
Yusuf Sarıoglu Cihat S¸afak
Received: 23 February 2011 / Accepted: 8 June 2011 / Published online: 25 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this study, twelve compounds having 9-aryl-
3,4,6,7-tetrahydroacridine-1,8-(2H,5H,9H,10H)-dione
structure were synthesized by reaction of 5-methyl-1,3-
cyclohexanedione, the appropriate aromatic aldehydes, and
ammonium acetate in methanol. The structures of the
compounds were elucidated by infrared, 1H- and 13C-
nuclear magnetic resonance spectroscopy (-NMR), mass
spectroscopy, and elemental analysis. The maximum
relaxant effects (Emax) and pD2 values of the compounds
3a–l and pinacidil were tested on isolated strips of rabbit
gastric fundus smooth muscle.
gastric myocytes were similar to those of KATP channels
in other smooth muscles (Sim et al., 2002).
Potassium channel opening is a physiological mecha-
nism by which excitable cells exploit to maintain or restore
their resting state (Grissmer, 1997; Jaggar et al., 1998;
¨
Klockner and Trieschmann, 1989; Lawson, 2000; Firth
et al., 2000; Loussouarn et al., 2001; Robertson and
Steinberg, 1990). Potassium ion channels are now classi-
fied into ten types according to their electrophysiological
and pharmacological properties (Sanguinetti and Spector,
1997). Several types of potassium ion channels including
voltage-activated potassium ion channels (Kv channel),
calcium-activated potassium ion channels (KCa channel)
and adenosine 50-triphosphate (ATP)-sensitive potassium
ion channels (KATP channel) are found in central and
peripheral nervous system (Roeper and Pongs, 1996;
Aronson, 1992). Potassium ion channels play an important
role in a number of different aspects of the electrical
responses of the nervous system (Nicoll et al., 1990; Cook,
1988; Mathie et al., 1998). For example, potassium ion
channel opener activity is involved in setting the membrane
resting potential, determining the frequency of action-
potentials and the shape of the action-potential wave forms.
Most of these channels permit the potassium ion efflux
from the neurons, thereby tending to oppose depolarization
or to use repolarization or hyperpolarization and resulting
in a decrease of neurotransmitter release (Nakamura et al.,
2004).
Keywords Myorelaxant activity ꢀ Acridinedione ꢀ
Pinacidil
Introduction
Ion channels are very important for cell function and
responsible for physiological effects. Potassium ion chan-
nels regulate some functions in both excitable and non-
excitable cells. With respect to the effects of potassium ion
channels on autonomic nerve functions, different research
groups have recently shown that big conductance Ca2?
-
activated potassium ion channels operate in the release of
acetylcholine from vagal nerve terminals (Tagaya et al.,
1998). The properties of KATP channels in guinea pig
It was shown that these KATP channels play critical
roles in modulating physiological processes such as insulin
secretion, leptin release, synaptic transmission, and excit-
ability of cardiac, vascular, and nonvascular smooth mus-
cle (Davis-Taber et al., 2003).
¨
˙
˘
G. S. O. Fincan ꢀ I. M. Vural ꢀ Y. Sarıoglu
Department of Pharmacology, Faculty of Medicine,
Gazi University, Ankara, Turkey
M. G. Gu¨ndu¨z ꢀ R. S¸ims¸ek (&) ꢀ C. S¸afak
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Hacettepe University, 06100 Ankara, Turkey
e-mail: rsimsek@hacettepe.edu.tr
KATP channel openers are a structurally diverse group
of drugs with a broad spectrum of potential therapeutic
123

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Med Chem Res (2012) 21:1817–1824
applications. These drugs interact with KATP channels in
numerous tissues and increase their activity, thereby
hyperpolarizing the plasma membrane and reducing elec-
trical excitability (Ashcroft and Gribble, 2000). Potassium
channel openers might be useful as therapeutic agents in
the treatment of various diseases such as hypertension,
asthma, peripheral vascular disease, right heart failure,
congestive heart failure, angina, ischemic heart disease,
cerebrovascular disease, glaucoma, renal cholic, disorders
associated with kidney stones, overactive bladder, irritable
bowel syndrome, male pattern baldness, premature labor,
and peptic ulcers (Ohnmacht et al., 1995; Carroll et al.,
2004a, b). Calcium channel blockers having 1,4-dihydro-
pyridine (DHP) structure which nifedipine is the prototype
and their condensed analogs such as bicyclo (quinoline)
and tricyclo (acridine) have vasodilator and antihyperten-
(Philadelphia, PA, USA); the values are uncorrected.
Infrared (IR) spectra were recorded by using a Perkin
Elmer Fourier-transform FT–IR Systems Spectrum BX.
1H-nuclear magnetic resonance (1H-NMR) and 13C-nuclear
magnetic resonance (13C-NMR) spectra were recorded by
using a Varian Mercury 400, 400 MHz high-performance
digital FT–NMR spectrometer (methanol-d4; tetramethyl-
silane as internal standard). Chemical shift values are given
as ppm. Mass spectra (MS) were recorded by using a
Waters 2996 photodiode array detector. Thin layer chro-
matography was run on aluminum sheets Merck silica gel
60 F254, and short wavelength ultraviolet (UV) light
(254 nm) was used to detect the UV absorption spots.
Elemental analysis was carried out by using a Leco CHNS-
932 elemental analyzer. The elemental analysis results
were within 0.4% of the theoretical values.
¨
sive effects (Ozturk et al., 2008; Simsek et al., 2008, 2004;
Sarac¸ et al., 2002; Berkan et al., 2002; Carroll et al., 2004a,
b; Ashworth et al., 2002). Some DHP derivatives have also
¨
potassium channel opener activity (Klockner and Triesch-
General procedure for 9-aryl-3,4,6,7-tetrahydroacridine-
1,8-(2H,5H,9H,10H)-diones (3a–3l)
mann, 1989; Davis-Taber, et al., 2003; Grissmer and
Cahalan, 1989; Mannhold, 2004; Gopalakrishnan et al.,
2003). Frank and coworkers synthesized 9-(3-nitrophenyl)-
3,4,6,7-tetrahydroacridine-1,8-(2H,5H,9H,10H)-dione and
showed its potassium channel opener activity (Frank et al.
1993). It has been shown that imidazolylacridinedione
derivatives possess potassium channel modulatory activity
(Hadizadeh and Mehri, 2006). In addition, we synthesized
some compounds having similar structure in our previous
work (Gu¨ndu¨z et al., 2009). Although those compounds
have disubstituted phenyl ring in 3,4,6,7-tetrahydroacri-
dine-1,8-dione structure, monosubstituted analogs of them
were synthesized in this study. In this study, we aimed to
synthesize twelve derivatives and investigate their effects
on isolated strips of rabbit gastric fundus smooth muscle.
Compounds 3a and 3c were synthesized earlier by Bossert
and Vater (1971). Since there was no data about potassium
channel modulatory activity of these two compounds, we
also synthesized them in order to investigate their activities
in this study. Therefore, we planned to synthesize new
tricyclic analog of 1,4-DHP to explain the contribution of
the position of electron withdrawing substituents on phenyl
ring of acridine and to the methyl groups substituted to
acridine ring to the mentioned activity.
The mixture of 5-methyl-1,3-cyclohexanedione (0.252 g,
0.002 mol) and appropriate aromatic aldehyde (0.001 mol)
were refluxed in methanol (20 ml) in 65°C in the presence
of ammonium acetate (0.385 g, 0.005 mol) for 4 h. Then
the forming crystals were filtered off and crystallized from
alcohol.
3,6-Dimethyl-9-(2-nitrophenyl)-3,4,6,7-tetrahydroacridine-
1,8-(2H,5H,9H,10H)-dione (3a) M.p. [ 300°C. Yield:
1
0.278 g (76%). IR (cm-1): 3302, 1616. H-NMR d 0.93,
0.97 (3H; d; J: 6.0 Hz, 3-CH3, 6-CH3), 1.85–2.50 (10H; m;
H-2,3,4,5,6,7), 5.55 (1H; s; 9-H), 7.24–7.76 (4H; m; Ar–
H), 9.40 (1H; s; NH). 13C-NMR d 20.4, 20.7, 27.9, 28.3,
33.1, 33.9, 38.2, 49.6, 50.0, 105.8, 106.2, 124.6, 126.3,
127.8, 131.5, 140.5, 146.8, 153.3, 153.6, 196.5, 196.7. MS
(m/z) 366, 365, 320, 244. Analysis for C21H22N2O4: cal-
culated C: 68.84, H: 6.05, N: 7.65; found C: 68.52, H: 5.99,
N: 7.48.
3,6-Dimethyl-9-(3-nitrophenyl)-3,4,6,7-tetrahydroacridine-
1,8-(2H,5H,9H,10H)-dione (3b) M.p. 274°C Yield:
1
0.292 g (80%). IR (cm-1): 3319, 1613. H-NMR d 0.93,
0.97 (3H; d; J: 6.2 Hz, 3-CH3, 6-CH3), 1.99–2.56 (10H; m;
H-2,3,4,5,6,7), 5.00 (1H; s; 9-H), 7.46-8.03 (4H; m; Ar–H),
9.52 (1H; s; NH). 13C-NMR d 19.7, 20.1, 27.4, 27.8, 32.3,
32.8, 39.2, 52.2, 52.9, 104.8, 105.4, 126.3, 127.2, 128.4,
132.6, 142.6, 147.2, 151.0, 151.8, 195.1, 195.9. MS (m/z)
366, 365, 320, 244. Analysis for C21H22N2O4: calculated
C: 68.84, H: 6.05, N: 7.65; found C: 69.10, H: 6.01, N:
7.56.
Materials and methods
Chemistry
All chemicals used in this study were purchased from
Sigma-Aldrich. Melting points were determined by using a
Thomas Hoover capillary melting point apparatus
3,6-Dimethyl-9-(4-nitrophenyl)-3,4,6,7-tetrahydroacridine-1,8-
(2H,5H,9H,10H)-dione (3c) M.p. 186°C. Yield: 0.314 g
123

Med Chem Res (2012) 21:1817–1824
1819
1
(86%). IR (cm-1): 3298, 1611. H-NMR d 0.93, 0.95 (3H;
d; J: 6.1 Hz, 3-CH3, 6-CH3), 1.94–2.55 (10H; m; H-
2,3,4,5,6,7), 4.95 (1H; s; 9-H), 7.38 (2H; d; J: 8.60 Hz, Ar–
H2, Ar–H6), 8.17 (2H; d; J: 8.60 Hz, Ar–H3, Ar–H5), 9.52
(1H; s; N–H). 13C-NMR d 21.2, 21.8, 28.7, 29.0, 32.5,
32.7, 39.9, 50.6, 50.8, 106.2, 106.7, 125.8, 127.4, 129.4,
132.8, 142.4, 147.1, 150.2, 150.8, 194.3, 194.6. MS (m/z)
366, 365, 320, 244. Analysis for C21H22N2O4: calculated
C: 68.84, H:6.05, N: 7.65; found C: 68.55, H: 5.97, N: 7.52.
(4H; m; Ar–H), 9.35 (1H; s; NH). 13C-NMR d 19.4, 19.8,
28.9, 29.2, 33.9, 34.2, 40.1, 51.2, 51.9, 105.8, 106.3, 121.3,
124.2, 125.7, 128.2, 132.7, 134.5, 146.8, 153.2, 153.7,
196.4, 196.8 MS (m/z) 389, 388, 320, 244. Analysis for
C22H22F3NO2: calculated C: 67.86, H: 5.69, N: 3.60; found
C: 67.55, H: 5.54, N: 3.48.
3,6-Dimethyl-9-(3-trifluoromethylphenyl)-3,4,6,7-tetrahy-
droacridine-1,8-(2H,5H,9H,10H)-dione (3h) M.p. 287°C.
1
Yield: 0.311 g (80%). IR (cm-1): 3298, 1612. H-NMR d
3,6-Dimethyl-9-(2-fluorophenyl)-3,4,6,7-tetrahydroacridine-
1,8-(2H,5H,9H,10H)-dione (3d) M.p. [ 300°C. Yield:
0.93, 0.96 (3H; d; J: 6.5 Hz, 3-CH3, 6-CH3), 1.95–2.56
(10H; m; H-2,3,4,5,6,7), 4.90 (1H; s; 9-H), 7.36–7.96 (4H;
m; Ar–H), 9.47 (1H; s; NH). 13C-NMR d 20.3, 20.7, 27.8,
28.0, 35.2, 35.7, 42.3, 52.2, 52.7, 109.3, 109.5, 122.4,
125.8, 127.2, 129.0, 132.5, 134.7, 139.8, 150.1, 150.7,
194.2, 194.9. MS (m/z) 389, 388, 320, 244. Analysis for
C22H22F3NO2: calculated C: 67.86, H: 5.69, N: 3.60; found
C: 67.45, H: 5.43, N: 3.52.
1
0.261 g (77%). IR (cm-1): 3275, 1608. H-NMR d 0.93,
0.97 (3H; d; J: 6.1 Hz, 3-CH3, 6-CH3), 1.91–2.56 (10H; m;
H-2,3,4,5,6,7), 4.97 (1H; s; 9H), 6.88–7.62 (4H; m; Ar–H),
9.42 (1H; s; N–H) 13C-NMR d 20.3, 20.8, 28.2, 28.9, 33.2,
33.8, 40.2, 49.5, 50.2, 99.8, 100.2, 115.6, 124.3, 126.7,
131.5, 143.8, 153.1, 153.6, 160.8, 195.9, 196.1. MS (m/z)
339, 338, 320, 244. Analysis for C21H22FNO2: calculated
C: 74.31, H: 6.53, N: 4.13; found C: 74.04, H: 6.46, N:
3.87.
3,6-Dimethyl-9-(4-trifluoromethylphenyl)-3,4,6,7-tetrahy-
droacridine-1,8(2H,5H,9H,10H)-dione (3i) M.p. 128°C.
1
Yield: 0.346 g (89%). IR (cm-1): 3295, 1608. H-NMR d
3,6-Dimethyl-9-(3-fluorophenyl)-3,4,6,7-tetrahydroacridine-
1,8-(2H,5H,9H,10H)-dione (3e) M.p. [ 300°C. Yield:
0.93, 0.96 (3H; d; J: 6.5 Hz, 3-CH3, 6-CH3), 1.96–2.53
(10H; m; H-2,3,4,5,6,7), 4.91 (1H; s; 9-H), 7.35 (2H; d; J:
8.32 Hz, Ar–H2, Ar–H6), 7.71 (2H; d; J: 8.32 Hz, Ar–H3,
Ar–H5), 9.46 (1H; s; N–H). 13C-NMR d 21.1, 21.8, 28.3,
28.9, 31.9, 32.3, 43.6, 50.4, 50.8, 104.3, 104.7, 122.6,
125.8, 128.4, 129.2, 135.7, 136.8, 140.4, 150.2,150.9,
193.5, 193.9. MS (m/z) 389, 388, 320, 244. Analysis for
C22H22F3NO2: calculated C: 67.86, H: 5.69, N: 3.60; found
C: 67.55, H: 5.50, N: 3.49.
1
0.288 g (85%). IR (cm-1): 3284, 1611. H-NMR d 0.93,
0.97 (3H; d; J: 6.0 Hz, 3-CH3, 6-CH3), 1.97–2.56 (10H; m;
H-2,3,4,5,6,7), 4.91 (1H; s; 9-H), 6.89–7.71 (4H; m; Ar–
H), 9.49 (1H; s; NH). 13C-NMR d 21.2, 21.7, 27.9, 28.1,
32.9, 33.5, 41.3, 50.1, 50.6, 100.8, 101.2, 118.9,
125.4,126.8, 133.4, 141.7, 150.2, 150.8, 159.3, 196.4,
196.6. MS (m/z) 339, 338, 320, 244. Analysis for-
C21H22FNO2: calculated C: 74.31, H: 6.53, N: 4.13; found
C: 73.93, H: 6.27, N: 4.05.
9-(2-Cyanophenyl)-3,6-dimethyl-3,4,6,7-tetrahydroac-
ridine-1,8-(2H,5H,9H,10H)-dione
(3j) M.p. [ 300°C.
1
3,6-Dimethyl-9-(4-fluorophenyl)-3,4,6,7-tetrahydroacridine-
1,8-(2H,5H,9H,10H)-dione (3f) M.p. 279°C. Yield:
Yield: 0.262 g (76%). IR (cm-1): 3288, 1615. H-NMR d
0.94, 0,97 (3H; d; J: 6.4 Hz, 3 CH3, 6-CH3), 1.87–2.32
(10H; m; H-2,3,4,5,6,7), 5.04 (1H; s; 9-H), 7.18–7.96 (4H;
m; Ar–H), 9.59 (1H; s; NH). 13C-NMR d 20.2, 20.7, 28.1,
28.6, 33.9, 34.4, 37.9, 49.6, 50.1, 98.7, 99.2, 107.8, 112.9,
126.6, 128.2, 132.5, 134.8, 143.1, 153.1, 153.6, 196.0,
196.4. MS (m/z) 346, 345, 244. Analysis for C22H22N2O2:
calculated C: 76.28, H: 6.40, N: 8.09; found C: 76.51, H:
6.00, N: 8.03.
1
0.298 g (88%). IR (cm-1): 3306, 1618. H-NMR d 0.94,
0.99 (3H; d; J: 6.1 Hz, 3-CH3, 6-CH3), 1.92–2.55 (10H; m;
H-2,3,4,5, 6,7), 5.01 (1H; s; 9-H), 7.02 (2H; d; J: 8.72 Hz,
Ar–H2, Ar–H6), 7.35 (2H; d; J: 8.72 Hz, Ar–H3, Ar–H5),
9.60 (1H; s; N–H). 13C-NMR d 19.5, 20.0, 26.3, 26.8, 31.8,
32.1, 42.5, 51.2, 51.8, 102.3, 102.9, 121.6, 126.8, 128.2,
132.6, 139.8, 150.4, 150.6, 156.7, 196.0, 196.4. MS (m/z)
339, 338, 320, 244. Analysis for C21H22FNO2: calcu
lated C:74.31, H: 6.53, N: 4.13; found C: 74.23, H: 6.46, N:
4.08.
9-(3-Cyanophenyl)-3,6-dimethyl-3,4,6,7-tetrahydroacridine-
1,8-(2H,5H,9H,10H)-dione (3k) M.p. 277°C. Yield:
0.276 g (80%). IR (cm-1): 3290, 1614. 1H-NMR d 0.92, 0.97
(3H; d; J: 6.2 Hz, 3-CH3, 6-CH3), 1.94–2.55 (10H; m; H-
2,3,4,5,6,7), 4.84 (1H; s; 9-H), 7.35–7.52 (4H; m; Ar–H),
9.50 (1H; s; NH). 13C-NMR d 21.3, 21.8, 27.4, 27.9, 32.8,
33.5, 38.1, 50.6, 51.2, 99.7, 100.3, 108.3, 113.6,125.8, 128.9,
133.5, 135.7, 143.8, 152.2, 152.8, 195.2, 195.9. MS (m/z)
3,6-Dimethyl-9-(2-trifluoromethylphenyl)-3,4,6,7-tetrahy-
droacridine-1,8-(2H,5H,9H,10H)-dione
(3g) M.p. [
1
300°C. Yield: 0.280 g (72%). IR (cm-1): 3303, 1610. H-
NMR d 0.92, 0.97 (3H; d; J: 6.4 Hz, 3-CH3, 6-CH3), 1.81–
2.50 (10H; m; H-2,3,4,5,6,7), 5.35 (1H; s; 9-H), 7.04–7.58
123

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Med Chem Res (2012) 21:1817–1824
346, 345, 244. Analysis for C22H22N2O2: calculated C:
76.28, H: 6.40, N: 8.09; found C: 75.90, H: 6.27, N: 7.78.
Rabbit fundus smooth muscle strips were precontracted
with submaximal concentration of noradrenaline (10-5 M).
Concentration–relaxation for compounds and pinacidil
were obtained by adding into the bath in cumulative
manner. Dimethylsulfoxide (DMSO) was also tested in
noradrenaline precontracted rings. Cumulative concentra-
tion–response curve was constructed in a stepwise manner
after the response to previous concentration had reached a
plateau. The same experimental procedure was performed
in presence of glibenclamide (KATP channel blocker,
10-6 M), tetraethylammonium (TEA) (Ca2?-activated K?
channel blocker, 10-4 M), 4-aminopyridine (4AP) (KV
blocker, 10-4 M), apamine (a selective blocker of small
conductance Ca2?-activated potassium ion channels,
10-6 M), iberiotoxin (large-conductance Ca2?-activated
potassium ion channels-blocker, 10-8 M).
9-(4-Cyanophenyl)-3,6-dimethyl-3,4,6,7-tetrahydroacridine-
1,8-(2H,5H,9H,10H)-dione (3l) M.p. 261°C. Yield:
1
0.290 g (84%). IR (cm-1): 3284, 1618. H-NMR d 0.93,
0.97 (3H; d; J: 6.1 Hz, 3-CH3, 6-CH3), 1.91–2.50 (10H; m;
H-2,3,4,5,6,7), 5.03 (1H; s; 9-H), 7.43 (2H; d; J: 8.80 Hz,
Ar–H2, Ar–H6), 7.64 (2H; d; J: 8.80 Hz, Ar–H3, Ar–H5),
9.54 (1H; s; N–H). 13C-NMR d 210.4, 20.9, 26.6, 26.9,
33.2, 34.1, 38.4, 51.3, 51.6, 100.6, 100.9, 109.0, 112.7,
126.1, 129.4, 138.1, 139.3, 142.5, 151.1, 151.4, 195.3,
195.7. MS (m/z) 346, 345, 244. Analysis for C22H22N2O2:
calculated C: 76.28, H: 6.40, N: 8.09; found C: 76.08, H:
6.11, N: 7.96.
Pharmacology
Drugs
New Zealand white rabbits (weighing 2.5–3 kg.) were used
in this study. The study was approved by the Ethics
Committee at Gazi University, Faculty of Medicine. Rab-
bits were killed with i.v. injection of sodium pentobarbital
(30–40 mg/kg, i.v.) then stomachs were removed by
abdominal incision. The fundal part of the stomach was
dissected parallel to the longitudinal muscle wall. One
muscle strip which is 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 baths containing normal
Krebs’–Henseleit solution (KHS). The composition of the
Krebs’ solution was as follows (in mmol/l): NaCl (118);
KCl (4.7); CaCl2 (1.26); NaHCO3 (25); MgCl2 (0.54);
NaHPO4 (0.9); glucose (10.04). The solution was gassed
with 95% O2 and 5% CO2 and temperature was maintained
at 37°C by a thermoregulated water circuit during the
study. 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. It was displaced contin-
uously and recorded on online computer via 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.). 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. N-(c)-nitro-L-arginine methyl ester (L-NAME)
hydrochloride (the nitric oxide synthase inhibitor, 10-4 M),
indomethacin (COX inhibitor, 10-5 M), and guanethidine
(adrenergic blocker, 10-6 M) were added into the organ
bath 20 min before the precontraction in order to eliminate
the effects of nitric oxide, prostaglandins, and adrenergic
agonists. These contribute to the gastric fundus smooth
muscle relaxation induced by compounds and pinacidil.
L-NAME, indomethacin, guanethidine, noradrenaline, gli-
benclamide, TEA, 4AP, apamine, iberiotoxin, pinacidil,
and DMSO were supplied by Sigma. Stock solutions of
L-NAME, guanethidine, TEA, 4-AP, iberiotoxin, and nor-
adrenaline were prepared in distilled water. Compounds,
pinacidil, glibenclamide, apamine, and indomethacin were
dissolved in DMSO.
Data analysis
The relaxant effects of the compounds and pinacidil on the
tissues precontracted with noradrenaline were expressed as
percentage of the obtained precontraction by using nor-
adrenaline. To evaluate the effects of the compounds, the
maximum response (Emax) and pD2 values [the negative
logarithm of the concentration for the half-maximal
response (EC50)] were calculated according to Scatchard
equation for drug–receptor interaction. While Emax is the
parameter for efficacy, pD2 is the parameter for potency.
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.
This value is taken as a measure of the sensitivity of
the tissues to each agonist. All data are expressed as
mean standard error.
Statistical analysis
Statistical comparison between groups was performed
using general linear models by Scheffe’s F test and P
values of less than 0.05 were considered to be statistically
significant.
123

Med Chem Res (2012) 21:1817–1824
1821
Results and discussion
distilled water or DMSO, their relaxant responses could not
be tested. To investigate whether relaxation induced by test
compounds was due to interaction with the cyclooxygenase,
In this study, acridinedione derivatives were obtained by
the reaction of 5-methyl-1,3-cyclohexanedione (1) with the
appropriate aromatic aldehydes (2) (Scheme 1). The
1
structures of the compounds were elucidated by IR, H-
NMR, 13C-NMR, mass spectra, and elemental analysis. In
Table 1 Maximum relaxant responses(Emax) and pD2 values of the
compounds and pinacidil on isolated strips of rabbit gastric fundus
smooth muscle
the IR spectra, N–H and C=O stretching bands were seen at
1
the expected values. In the H-NMR spectra, protons in
three and six positions of the acridinedione ring were seen
about 0.92–0.97 ppm as doublet. The peaks belonging to
the protons of aromatic ring and alkyl groups were seen at
the expected chemical shift values. The N–H signal of the
compounds was observed at about 9.35–9.59 ppm. The
13C-NMR spectra of the compounds displayed the appro-
priate number of resonances that exactly fitted the number
of carbon atoms. The mass spectra of the compounds were
recorded using the electrospray ionization technique.
Molecular ion peaks were seen for all compounds. In fur-
ther fragmentation, the peaks formed by cleavage of the
aryl ring from the parent molecule. These findings are in
accordance with the literature (Mathie et al., 1998). In
addition, the structure of the compounds was confirmed by
elemental analysis.
R
O
O
H3C
N
H
CH3
Compounds
R
Emax
pD2
3a
2-Nitro
3-Nitro
4-Nitro
2-Fluoro
3-Fluoro
4-Fluoro
No effect
62.08 5.81
53.93 7.49
No effect
–
No effect
4.57 0.35
4.66 0.42
No effect
–
3b*
3c*
3d
3ea
3f
37.23 9.23
No effect
No effect
33.76 8.54
–
4.60 0.24
No effect
No effect
4.68 0.51
–
The maximum relaxant effects (Emax) and pD2 values of
the compounds 3a–l and pinacidil on isolated strips of rabbit
gastric fundus smooth muscle are given in Table 1. The
values indicate that compounds 3b, 3c, 3f, 3i, 3k, 3l, and
pinacidil produced concentration-dependent relaxation in
rabbit gastric fundus smooth muscle strips. Compounds 3a,
3d, 3g, and 3h did not show any relaxation effect. DMSO
had no significant relaxant effect. Some compounds and
pinacidil exerted concentration-dependent relaxation
responses precontracted with submaximal concentration of
noradrenaline in the gastric fundus smooth muscle strips
with efficacy order: pinacidil C 3b > 3k C 3c > 3l > 3f C
3i. Compound 3b has similar relaxation activity with
pinacidil. Since compounds 3e and 3j did not dissolve in
3g
2-Trifluoromethyl
3-Trifluoromethyl
4-Trifluoromethyl
2-Cyano
3h
3i*
3ja
3k*
3l*
Pinacidil
3-Cyano
56.02 7.5
45.99 4.29
73.55 6.11
4.70 0.29
4.57 0.33
4.90 0.15
4-Cyano
–
Relaxant responses are expressed as a percentage of the precontrac-
tion induced by noradrenaline. The negative logarithm of the con-
centration for the half-maximal response (pD2) value represent mean
value SEM
* P \ 0.05, compared with control responses (n = 6)
a
Since it did not dissolve in distilled water or DMSO, their relaxant
responses could not be tested
Scheme 1 Synthesis of compounds 3a–l
123

1822
Med Chem Res (2012) 21:1817–1824
adrenergic or nitric oxide pathways, tissues were pretreated
with indomethacin (COX inhibitor), guanethidine (adren-
ergic blocker) or L-NAME hydrochloride (the nitric oxide
synthase inhibitor), respectively. Pretreatment of the strips
with indomethacin, guanethidine and L-NAME did not sig-
nificantly alter the relaxant responses of the compounds.
These findings indicate that cyclooxygenase, adrenergic and
nitric oxide (NO) pathways do not play a role in relaxations
evoked by these substances. When the Emax values were
investigated, it was seen that the compounds having sub-
stituent in 2 position of the phenyl ring do not possess any
response. The compounds having nitrophenyl (3a–c) and
cyanophenyl groups) (3j–l) showed similar activity pattern.
Among these two groups, 3-substituted derivatives show
higher activity than their 2-substituted analogs. The pD2 and
dimethyl analog of KATP channel activators. The intro-
duction of dimethyl groups to the acridine ring might have
changed the action characteristics of this agent on potassium
channels. All potassium ion channel blockers used in this
study did not alter 3l induced relaxation response. The
results of the study showed that the introduction of the
electron withdrawing substituent to 3 position of the phenyl
ring increased mentioned activity. The opening of the
potassium (K?) channels, causing hyperpolarisation of the
cell membrane, is a physiological means of decreasing cell
excitability. Thereby, drugs having this property will dem-
onstrate a broad clinical potential. New molecules evoke
physiological responses such as smooth muscle relaxation
by the opening of potassium ion channels led to a new
direction in the pharmacology of ion channels. The term
‘‘potassium channel openers’’ was initially associated with a
group of chemically diverse agents (for example, cro-
makalim, pinacidil, and nicorandil) that evoke potassium ion
efflux through KATP channels. KATP channels are inhib-
ited by intracellular ATP. These channels play a role in
linking cell metabolic state to membrane potential. KATP
channels are associated with diverse cellular functions such
as shortening of action-potential duration in cardiac myo-
cytes, insulin release in pancreatic b-cells, regulation of
excitability in skeletal muscle, and regulation of vascular
smooth muscle contractility (Aronson, 1992; Ashcroft and
Gribble, 2000; Noma, 1983; Ashcroft and Rorsman, 1989).
But KATP blocker glibenclamide did not alter compounds-
induced relaxation responses in this study. The physiological
role of potassium ion channels is not clear in gastrointestinal
smooth muscle. KATP channels may play a role in regula-
tion in membrane potential and/or in change of contractility
E
max values of the compounds 3b, 3c, 3f, 3i, 3k, and 3l are
closer to pinacidil. The results obtained in the presence
of potassium channel blockers (TEA, glibenclamid, 4AP,
apamine, and iberitoxin) are in accordance with the Emax
value of the compounds compared with pinacidil (Table 2).
The relaxant response of 3b was significantly inhibited by
apamine. Compound 3c induced relaxation response was
altered by either TEA or iberiotoxin. The enhancement of 3c
induced relaxation responses driven by these mentioned
antagonists points out the possibility that 3c exerts its effect
via mechanisms other than potassium channels. This
requires further investigation. Either 4AP or TEA inhibited
3f induced relaxation responses. Compound 3i induced
relaxation response was inhibited by 4AP. Compound 3k
induced relaxation response was inhibited by 4AP or ibe-
riotoxin. Relaxation responses induced by 3k were not
inhibited by glibenclamide despite the fact that 3k is a
Table 2 In the presence of potassium channel blockers, maximum relaxant responses (Emax) values of the compounds on isolated strips of rabbit
gastric fundus smooth muscle
Compounds
Control
In the presence of potassium channel blockers, Emax values of compounds
Emax
TEA (10-4 M)
Glibenclamide
(10-6 M)
4-Aminopyridine
(10-4 M)
Apamine
(10-6 M)
Iberiotoxin
(10-8 M)
3a
3b
3c
3d
3f
No effect
No effect
No effect
No effect
No effect
No effect
62.08 5.81
53.93 7.49
No effect
54.63 8.43
74.43 4.63*
No effect
51.82 7.64
51.97 9.72
No effect
54.08 6.88
51.37 4.53
No effect
35.30 5.55*
54.45 5.65
No effect
54.44 5.23
82.53 2.40*
No effect
37.23 9.23
No effect
18.83 6.82*
No effect
46.68 5.31
No effect
21.84 7.72
No effect
39.3 5.99
No effect
39.08 5.11
No effect
3g
3h
3i
No effect
No effect
No effect
No effect
No effect
No effect
33.76 8.54
56.02 7.5
45.99 4.29
35.11 7.39
44.61 7.00
45.19 6.27
43.75 4.22
66.05 5.72
45.24 8.24
14.04 6.21*
32.26 4.08*
51.08 7.88
26.74 6.34
60.6 8.01
45.74 5.01
43.3 5.44
30.88 4.57*
52.02 6.09
3k
3l
Relaxant responses are expressed as a percentage of the precontraction induced by noradrenaline. The maximum relaxant responses (Emax) value
represents mean value SEM
* P \ 0.05, compared with control responses (n = 6)
123

Med Chem Res (2012) 21:1817–1824
1823
Cook NS (1988) The pharmacology of potassium channels and their
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mediated by neurotransmitters in guinea pig stomach (Sim
et al., 2002). In the present study, Ca2?-activated potassium
ion channel blocker TEA, KV blocker 4AP, small conduc-
tance Ca2?-activated potassium ion channel blocker ap-
amine, large-conductance Ca2?-activated potassium ion
channel blocker iberiotoxin significantly inhibited com-
pounds-induced relaxation responses. Every blocker
showed different effect on different compounds. It has been
shown that several types of potassium ion channels,
including voltage sensitive potassium ion channels, big
conductance Ca2?-activated potassium ion channels and
small conductance Ca2?-activated potassium ion channels
are present on nerve cells (MacKinnon and Yellen, 1990;
Reinhart et al., 1989; Blatz and Magleby, 1986). Most of
these channels permit the potassium ion efflux from within
the neurons, thereby tending to oppose depolarization or to
cause repolarization or hyperpolarization, and resulting in a
decrease of neurotransmitter release (Nakamura et al., 2004).
In conclusion, there is need of more direct evidence to
enlighten the potassium channel opening mechanisms of these
compounds.
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93(4):609–630
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Jaggar JH, Mawe GM, Nelson MT (1998) Voltage-dependent K?
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