Synthesis and Biological Evaluation of 1-(2, 4-dimethoxyphenacyl)-4-acetyl-4-phenylpiperidinium bromide in Intestinal and Cardiovascular Tissues
Syed I.H. Taqvi,
Mohammad T. Aftab,
Muhammad N. Ghayur,
Anwar H. Gilani
Zafar S. Saify
1-(2, 4-dimethoxyphenacyl)- 4-acetyl-4-phenylpiperidinium
bromide is a synthetic piperidine compound which was screened in isolated intestinal
and aortic smooth muscle and Langendorffs heart preparations. The test
compound exhibited dose-dependent spasmolytic effect on the spontaneous and
high K+ (75 mM) contracted isolated rabbit jejunum with respective
EC50 values of 0.08 mM (0.05-0.13, 95% CI) and 0.12 mM (0.06-0.21).
The Ca++ channel blocking (CCB) activity was confirmed when the test
compound (0.05-0.1 mM) shifted the Ca++ dose-response curves to the
right, similar to that produced by verapamil (0.3-1.0 μM), a standard CCB.
In the isolated rabbit aorta, the test compound showed a dose-dependent vasodilator
effect on high K+ (75 mM)-induced contractions with an EC50
value of 0.04 mM (0.02-0.08) while also suppressed the norepinephrine (1 μM)
peak responses with an EC50 value of 0.04 mM (0.03-0.06). When tested
in Langendorff perfused rabbit heart preparation, it exhibited a negative inotropic
effect in the ventricles with EC50 value of 0.59 mM (0.04-8.86) with
negligible effect on the rate of ventricular contractions. The results show
inhibitory effects of the test compound on intestinal and vascular smooth muscle
preparations and a cardio-suppressant effect, possibly mediated via blockade
of voltage and receptor-operated Ca++ channels.
to cite this article:
Syed I.H. Taqvi, Mohammad T. Aftab, Muhammad N. Ghayur, Anwar H. Gilani and Zafar S. Saify, 2006. Synthesis and Biological Evaluation of 1-(2, 4-dimethoxyphenacyl)-4-acetyl-4-phenylpiperidinium bromide in Intestinal and Cardiovascular Tissues. Journal of Pharmacology and Toxicology, 1: 126-133.
Piperidine derivatives are known to exhibit antihypertensive activities (Maillard
et al., 1972; Clark et al., 1983). According to recent reports
of pharmacological piperidine nucleus in their skeletons (Saeed et al.,
1997, 1998; Saify et al., 2005). This led effects of substituted piperidines,
there is still an increasing interest in synthesizing and evaluating new compounds
having us to synthesize the test compound: 1-(2, 4-dimethoxyphenacyl)-4-acetyl-4-phenylpiperidinium
bromide, belonging to phenylpiperidine class (North, 1986) and this was evaluated
on isolated intestinal and vascular smooth muscles as well as isolated Langendorff
perfused heart preparation. The results showed spasmolytic, vasodilator and
specific negative inotropic activities of the test compound possibly mediated
via calcium channel blockade (CCB).
Materials and Methods
Synthesis of the Test Compound
Equimolar quantity of 4-acetyl-4-phenylpiperidine and 2, 4-dimethoxyphenacyl
bromide were dissolved in acetone (Fig. 1) and refluxed on
a water bath and the reaction was continuously monitored by TLC using the solvent
system CHCl3-MeOH in the ratio of 9:1.When all the starting material
changed into product, the resulting solid material or the precipitate was collected
by filtration and thoroughly washed to remove traces of reactant. It was then
dissolved and recrystallized from ethyl alcohol: yield 67%, m.p. 224-226°C.
1H-NMR (CDCl3, 400 MHZ) δ: 7.88 (1H. d. J = 8.78 Hzm H-6),
7.31 (5H, m, H, 2-6), 6.53 (1 H, dd, J = 8.78. 2.29 Hz, H-5).
6.41 (1H, d, J = 2.29 Hz, H-3), 6.40 (2H, s, H-α). 3.87 (3H, s, Ar-OCH3),
3.84 (3H, s, Ar-OCH3), 3.83-3.80 (4H, m, H-2.6), 2.57-2.40 (4H, m,
H-3.5) and 1.93 (3H s, COCH3, C-4).
EIMS m/z (relative int.,%): 381 (M + -HBr, C 23 H27 NO4, 1), 336 (4), 259 (3), 217 (15), 216 (100), 202 (18), 173 (6), 165 (30), 159 (7), 129 (6) and 82 (37).
IR Vmax (KBr) cm-1: 2800, 2500, 1570, 1430, 1240 and 940.
UV λmax MeOH) nm: 351, 256 and 203.
C23H28BrNO4, Formula Weight: 467.13
Drugs and Chemicals
Acetylcholine (ACh), norepinephrine (NE) and verapamil were obtained from
Sigma Chemical Company, St. Louis, MO, USA while heparin injections BP were
purchased from Rotex Medica, Trittau, Germany. The following chemicals were
used to make the physiological salt solutions: potassium chloride (Sigma Chemical
Company, St. Louis, MO, USA), calcium chloride, glucose, magnesium chloride,
magnesium sulphate, potassium dihydrogen phosphate, sodium bicarbonate, sodium
chloride, sodium dihydrogen phosphate (E. Merck, Darmstadt, Germany) and Ethylenediaminetetra-acetic
Acid (EDTA) from BDH Laboratory Supplies, Poole, England. Stock solutions of
all the chemicals were made in saline fresh on the day of the experiment.
Experiments performed complied with the rulings of the Institute of Laboratory
Animal Resources, Commission on Life Sciences, National Research Council (NRC,
1996). Local male rabbits (around 1 kg) used in the study were housed in the
animal house of the Aga Khan University under a controlled environment (23-25°C).
Animals were given tap water ad libitum and a standard diet consisting
of (g/kg): flour 380, fibre 380, molasses 12, NaCl 5.8, nutrivet L 2.5, potassium
metabisulphate 1.2, vegetable oil 38, fish meal 170 and powdered milk 150.
||The chemical structure and formula of the test compound1-(2,
Isolated Rabbit Jejunum
Experiments were performed as described earlier (Gilani and Cobbin, 1986).
Segments of rabbit jejunum tissue 2 cm long were suspended in 10 mL tissue baths
containing Tyrodes solution, aerated with a mixture of 95% oxygen and
5% carbon dioxide (carbogen) and maintained at 37°C. The composition of
Tyrodes solution in mM was: KCl 2.68, NaCl 136.9, MgCl2 1.05,
NaHCO3 11.90, NaH2PO4 0.42, CaCl2
1.8 and glucose 5.55. Intestinal responses were recorded isotonically using
Harvard student oscillographs and isotonic transducers. Each tissue was allowed
to equilibrate for at least 30 min before the addition of any drug. Under these
conditions, rabbit jejunum exhibits spontaneous rhythmic contractions, allowing
testing of relaxant (spasmolytic) activity directly with out the use of an agonist.
Determination of Ca++ Antagonist Activity in Rabbit Jejunum
To assess whether the spasmolytic activity of the test compound was mediated
through CCB, K+ (75 mM) was used to depolarize the preparations (Farre
et al., 1991). High K+ (75 mM) was added to the tissue bath,
which produced a sustained contraction. The test compound was then added in
a cumulative fashion to obtain concentration-dependent inhibitory responses.
The relaxation of intestinal preparations, precontracted with K+
(75 mM) was expressed as percent of the control response mediated by K+.
Contraction of smooth muscle induced by K+ is known to be mediated,
via influx of Ca++ from extracellular fluid and a substance, which
inhibits this contraction, is considered to act through blockade of Ca++
channels (Bolton, 1979).
To confirm the Ca++ antagonist activity of the test compound, the
tissue was allowed to stabilize in normal Tyrodes solution, which was
then replaced with Ca++-free Tyrodes solution containing EDTA
(0.1 mM) for 30 min in order to remove Ca++ from the tissues. This
solution was further replaced with K+-rich and Ca++-free
Tyrodes solution, having the following composition (mM): KCl 50, NaCl
91.04, MgCl2 1.05, NaHCO3 11.90, NaH2PO4
0.42, glucose 5.55 and EDTA 0.1. Following an incubation period of 30 min, control
Dose-Response Curves (DRCs) of Ca++ were obtained. When the control
DRCs of Ca++ were found super-imposable (usually after two cycles),
the tissue was pretreated with the test compound for 60 min to test the possible
CCB effect. The DRCs of Ca++ were reconstructed in the presence of
different concentrations of the test compound while verapamil was used as a
Isolated Rabbit Aorta
Rabbits were sacrificed and the descending thoracic aorta was removed and
cut into 2-3 mm wide rings which were individually mounted in 20 mL tissue baths
containing Krebs-Henseliet solution (composition in mM: NaCl 11.50, KCl
4.70, CaCl2 2.50, NaHCO3 25.0, MgSO4.7H2O
1.50, K2H2PO4. 2H2O 1.20 and glucose
11.0) at 37°C and aerated with carbogen gas. A resting tension of 2 g was
applied to each tissue and an equilibrium period of 1 h was allowed before any
experimentation. The changes in isometric tensions of the rings were measured
via a force-displacement transducer (FT-03) using a Grass Model 7 Polygraph.
Following an equilibrium period of 1 h, the tissues were stabilized with a fixed
dose of NE (1 μM). The tissues were considered stable only when similar
responses were obtained from the repeated doses of NE (1 μM). Effect of
the test compound was first determined on the resting baseline of the tissue
to see if it has any vasoconstrictor effect. Later it was tested for any ability
to relax the high K+ (75 mM)-induced contractions or control NE (1
μM) peak responses. The ability of the extract to relax K+ (80
mM)-induced contractions would indicate L-type voltage-dependent CCB mode of
vasodilation while inhibition of the NE-peak responses would signify the blockade
of the Ca++ influx through the receptor-operated Ca++
channels (Karaki, 2004). Procedure for the latter possibility involved incubating
the control NE responses with increasing doses (0.01-0.5 mM) of the test compound
for 1 h.
Langendorff Perfused Rabbit Heart
Whole hearts were obtained from healthy rabbits (male, 1 kg). Heparin (5000
IU) was injected (ip) 1 h prior to isolation of the whole hearts. After cervical
dislocation, hearts were excised rapidly and mounted on Langendorff apparatus
as quickly as possible. Krebs-Henseliet solution perfused the heart retrogradely,
aerated by carbogen at thermostatically controlled temperature (37°C) with
pH of 7.4. Ventricular activity was recorded by Harvard isotonic transducers.
Approximately 60 min were allowed to each heart to adapt to the new environment
and to exhibit sino atrial nodal pattern of the cardiac activity. Heart showing
abnormal patterns were discarded. After taking 10 min of equilibrium period,
the test compound was added in ascending order. For each dose, 10 min were allowed
to achieve the peak effect. Changes in ventricular activity were calculated
when maximal effect persisted for 5 min or more (Staff Department of Pharmacology,
University of Edinburgh, 1970).
All the data expressed are mean±standard error of mean (SEM, n =
number of experiments) and the median effective concentrations (EC50
values) with 95% confidence intervals (CI). Concentration-response curves were
analyzed by non-linear regression (GraphPAD program, Graph PAD, San Diego, CA,
Results and Discussion
When tested on the spontaneously contracting isolated rabbit jejunum, the test
compound caused a dose-dependent (0.005-0.2 mM) relaxant effect (Fig.
2) with an EC50 value of 0.08 mM (0.05-0.13, 95% CI, n = 4).
To elucidate the possible mechanism of this spasmolytic effect, the test compound
was tested on high K+ (75 mM)-induced contractions. It exhibited
a dose-dependent (0.01-0.5 mM) inhibition (Fig. 2) of these
induced contractions with an EC50 value of 0.12 mM (0.06-0.21, n
= 4). The contractions of smooth muscles, including that of rabbit jejunum,
are dependent upon an increase in the cytoplasmic free Ca++, which
activates the contractile elements (Karaki, 2004).
||Dose-response curves showing the dose-dependent spasmolytic
effect of the test compound in spontaneous and high K+ (75 mM)-contracted
isolated rabbit jejunum (values shown are mean±SEM, n = 4)
While contraction induced by high K+ is dependent upon the entry
of Ca++ into the cells through the voltage-operated Ca++
channels and thus inhibition of high K+-induced contraction is due
to the result of blocked Ca++ entry through these channels (Bolton,
1979), a characteristic of Ca++ antagonists. The interaction with
Ca++ channels was further studied in jejunum, which is known to be
quick in responding to spasmolytic activity. The test compound dose-dependently
(0.05-0.1 mM, n = 4) shifted the Ca++ dose-response curves to the
right (Fig. 3A), similar to that produced by verapamil (0.3-1.0
μM, n = 4, Fig. 3B), a standard CCB (Bolton, 1979; Godfraind,
1987), thus confirming the CCB activity.
Keeping in mind the widespread utility of CCBs in cardiovascular disorders such as hypertension (Godfraind et al., 1986; Triggle, 1992), the test compound was tested in isolated aorta and whole heart preparations. The test compound was found devoid of any contractile effect on the resting baseline of rabbit aorta but showed a dose-dependent vasodilator effect (Fig. 4) on high K+ (75 mM)-induced contractions and control peak responses of NE (1 μM), after pretreating the peaks with each dose of test compound for 1 h, with EC50 values of 0.04 mM (0.02-0.08, n = 5) and 0.04 mM (0.03-0.06, n = 5), respectively, thus reiterating the already observed CCB activity. This suggested non-specific Ca++-antagonist activity of the test compound on voltage-and receptor-operated channels (Karaki, 2004). The vasodilator activity of the test compound is in accordance with the earlier findings of other piperidine derivatives shown to have vasodilator and hypotensive activities (Clark et al., 1983; Takai et al., 1985).
In Langendorff perfused rabbit heart, the test compound exhibited a dose-dependent negative inotropic effect (Fig. 5) on the ventricular contractility with an EC50 value of 0.59 mM (0.04-8.86, n = 5). The reduction in rate of contraction is known to be due to decrease in trans-sarcolemmal Ca++ influx (Malecot and Trautwein, 1987) while reduction in force of contraction is the result of inhibition of transmembrane Ca++ influx through L-type Ca++ channels (Fleckenstein, 1977; Conti et al., 1985).
||Dose-response curves showing the inhibitory effect of increasing
doses of [A] test compound and (B) verapamil on Ca++ concentration-response
curves, constructed in a Ca++-free medium, in rabbit jejunum
preparations (values shown are mean±SEM, n = 4)
||The inhibitory effect of increasing doses of the test compound
on high K+ (75 mM)-induced contractions and norepinephrine(NE,
1 μM) control peak responses in isolated rabbit aorta (values shown
are mean±SEM, n = 5)
||Dose-response curves showing the inhibitory effect of increasing
doses of the test compound on rate and force of ventricular contractions
of rabbit whole heart perfused preparation (values shown are mean±SEM,
n = 5)
The results showed intestinal spasmolytic, vasodilator and cardio-suppressant activities of the test compound mediated possibly through blockade of voltage- and receptor-operated Ca++ channels.
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