Synthesis, β-adrenergic blocking activity and β-receptor binding affinities of 1-substituted-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalates

Article abstract of DOI:10.1016/S0014-827X(03)00083-1

The compounds 1-isopropylamino-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalate (5) and 1-tert-butylamino-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalate (6) were synthesized from thymol (1), a naturally occurring agent in Thymus vulgaris L. Phar

Full text of DOI:10.1016/S0014-827X(03)00083-1

Il Farmaco 58 (2003) 557Á562
/
www.elsevier.com/locate/farmac
Synthesis, b-adrenergic blocking activity and b-receptor binding
affinities of 1-substituted-3-(2-isopropyl-5-methyl-phenoxy)-propan-
2-ol oxalates
Dharam Paul Jindal a,*, Mohane S. Coumar a, K. Nandakumar b,
Subhash Laxmanrao Bodhankar b, Prasad Gopal Purohit b,
Kakasaheb Ramoo Mahadik b, Giancarlo Bruni c, Elga Collavoli c, Paola Massarelli c
a University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India
b Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Pune 411038, India
c Dipartimento di Farmacologia ‘Giorgio Segre’ 6, via delle Scotte-53100 Siena, Italy
Received 15 January 2002; accepted 15 May 2002
Abstract
The compounds 1-isopropylamino-3-(2-isopropyl-5-methyl-phenoxy)-propan-2-ol oxalate (5) and 1-tert-butylamino-3-(2-isopro-
pyl-5-methyl-phenoxy)-propan-2-ol oxalate (6) were synthesized from thymol (1), a naturally occurring agent in Thymus vulgaris L.
Pharmacological evaluation of 5 and 6 were carried out using mouse ECG and isolated rat uterus models. Pretreatment of 5 (100 mg/
kg, i.v.) and 6 (50 mg/kg, i.v.) antagonized isoprenaline (2 mg/kg, i.v.) induced tachycardia, similar to that of atenolol (CAS 29122-68-
7, 20 mg/kg, i.v.) pretreatment in mouse ECG experiments as measured by R-R interval. Pretreatment of 5 and 6 blocked
isoprenaline and adrenaline induced relaxation of isolated rat uterus (unprimed). Also the compounds 5 and 6 were subjected to in
vitro b1- and b2-adrenergic receptor binding assay using turkey erythrocyte membrane (b1) and lung homogenate of rats (b2). Both 5
and 6 showed b-adrenergic receptor affinity comparable with that of propranolol (propranolol hydrochloride, CAS 318-98-9) with
out selectivity to any one b-adrenergic receptor. These results suggest that both the compounds possess non-selective b-adrenergic
blocking activity, with the tert-butyl derivative 6 being more active than the isopropyl derivative 5.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: b-Adrenergic blocking activity; b-Adrenergic receptor binding affinity; Aryloxypropanolamines; Atenolol; Propranolol; Thymol
1. Introduction
to study and treat hypertension. Worldwide many drug
classes including diuretics, b-adrenergic blocking agents,
calcium antagonist, angiotensin converting enzyme in-
hibitors, angiotensin II antagonists and a-adrenergic
blockers are being used for the control of blood pressure
[3,4].
b-Adrenergic blocking agents are safe, cheap and
effective for use as monotherapy or in combination with
diuretics, calcium antagonists and a-adrenergic blockers
[4]. Also b-adrenergic blocking agents are proven
effective in reducing the symptoms of angina pectoris
and in reducing morbidity and mortality after myocar-
dial infarction [5]. Moreover, recent clinical trial results
support the beneficiary action of b-adrenergic blocking
agents in patients with mild to moderate congestive
heart failure [6,7]. In addition to their usefulness in
Cardiovascular diseases such as angina pectoris,
myocardial infarction, cardiac arrhythmias and conges-
tive heart failure are the major cause of deaths world-
wide, even surpassing deaths due to cancer. Of the
various cardiovascular diseases, hypertension is of
particular concern, since elevated blood pressure for
prolonged periods of time increase the risks of develo-
ping angina pectoris, myocardial infarction, cerebral
hemorrhage, stroke, kidney failure [1,2]. Implication of
hypertension in the pathogenesis of coronary heart
disease and stroke had made the health professionals
* Corresponding author.
E-mail address: dharampjindal@yahoo.co.in (D.P. Jindal).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00083-1

558
D.P. Jindal et al. / Il Farmaco 58 (2003) 557Á562
/
various cardiovascular diseases they are also used in the
treatment of various non-cardiovascular disorders such
as migraine, hyperthyroidism, anxiety, tremor and
glaucoma [5]. The usefulness of b-adrenergic blocking
agents in various cardiovascular and non-cardiovascular
disorders prompted the present investigators to under-
taken a research programme to search compounds with
b-adrenergic blocking activity.
Recently many b-adrenergic blocking agents were
reported which were synthesized from natural products
[8Á15], such as capsaicin [10], vanillin [12], gingerone
/
[14] and eugenol [15]. Keeping this in view, we had
planned to use other naturally occurring compounds as
starting materials to synthesize b-adrenergic blocking
agents. The aim of this study was to develop b-
adrenergic blocking agents starting from thymol (1),
which is a natural product obtained from Thymus
vulgaris L., Monarda punctata L. or Carum copticum
Benth. & Hook. f., and is mainly used for its antiseptic
action [16]. Herein we report the synthesis, preliminary
pharmacological screening and in vitro b1-, b2-adrener-
gic receptor binding assay of the aryloxypropanolamine
derivatives of thymol.
Fig. 1. Synthetic procedure to compounds 3 and 5. Reagents and
conditions: (i) epichlorohydrin/K2CO3, reflux; (ii) isopropylamine or
tert-butylamine, reflux; and (iii) oxalic acid/methanol, reflux.
using tetramethylsilane (TMS) as the internal standard
(chemical shifts in d, ppm). IR spectra were recorded on
PerkinÁ
/
Elmer 882 spectrophotometer model (PerkinÁ
/
Elmer Ltd., Beaconsfield, Buckinghamshire, England).
IR spectra were obtained with potassium bromide
pellets (g max in cmꢀ1). The purity of the compounds
were established by thin layer chromatography (TLC)
and by elemental analysis (C, H, N). Elemental analyses
2. Chemistry
were carried out on a PerkinÁ
/
Elmer-2400 (PerkinÁ
/
Thymol (1) was condensed with epichlorohydrin in
the presence of potassium carbonate to give the oxirane
Elmer corporation, Norwalk, CT). Plates for TLC
were prepared with silica gel G using ethyl acetate.
Iodine vapors were used to develop the plates. Silica gel
1
(2). H NMR spectrum showed a one proton double
doublet (dd) at d 2.75 and a one proton triplet at d 2.87
(100Á200 mesh) for column chromatography was pur-
/
for Á
lamine gave 3, which in 1H NMR spectrum showed a six
proton doublet at d 1.09 for ÁNHCH(CH3)2 and
/CH2 of oxirane ring. Reaction of 2 with isopropy-
chased from Acme synthetic chemicals, Mumbai, India.
/
3.1.1. 2-(2-Isopropyl-5-methyl-phenoxymethyl)-oxirane
(2)
another six proton doublet at d 1.20 for ArCH(CH3)2.
While reaction of 2 with tert-butylamine gave 4, which
in 1H NMR spectrum showed a nine proton singlet at d
Thymol (1) (5.0 g, 33.3 mmol), epichlorohydrin (50
ml) and potassium carbonate (5.0 g) were refluxed for 12
h while stirring. TLC confirmed completion of reaction.
Filtered the residue and distilled off the excess epichlor-
ohydrin under reduced pressure to give an oily residue,
which was chromatographed over silica gel, using
chloroform as the eluent to give an oily residue 2 (4.0
1.13 for ÁNHC(CH3)3 and a six proton doublet at d
/
1.20 for ArCH(CH3)2.
The compounds 3 and 4 were converted to their
oxalates 5 and 6, respectively, by treatment with oxalic
acid in refluxing methanol (Fig. 1). Their structures were
confirmed by 1H NMR, IR spectroscopy and by
elemental analyses. Nomenclature of the compounds
were done through internet at the website www.chem-
web.com/autonom/.
g, 58%). 1H NMR (CDCl3): d 1.20 (d, 6H, Á
2.30 (s, 3H, ÁCH3), 2.75 (dd, 1H, ÁCH2 of oxirane),
2.87 (t,1H, ÁCH2 of oxirane), 3.31 (m, 2H, ÁCH of
oxirane and ÁCH(CH3)2), 3.95 (dd, 1H, ÁOCH2Á), 4.20
(dd, 1H, ÁOCH2Á), 6.64 (s, 1H, Ar), 6.75 (d,1H, Jꢁ7.7
Hz, Ar) and 7.09 (d, 1H, Jꢁ7.7 Hz, Ar). M/S: m/z: 206
[Mꢂ].
/CH(CH3)2),
/
/
/
/
/
/
/
/
/
/
/
3. Experimental
3.1. Chemistry
3.1.2. 1-Isopropylamino-3-(2-isopropyl-5-methyl-
phenoxy)-propan-2-ol (3)
2-(2-Isopropyl-5-methyl-phenoxymethyl)-oxirane (2)
(2.0 g, 9.7 mmol) and isopropylamine (10 ml), were
refluxed for 12 h. Completion of reaction was confirmed
Melting points (melting point apparatus MP I, Veego,
Mumbai, India) reported are uncorrected. 1H NMR
spectra were recorded on Bruker AC-300F, 300 MHz
NMR instrument (Brucker AG, Fallanden, Switzerland)
¨

D.P. Jindal et al. / Il Farmaco 58 (2003) 557Á
/562
559
by TLC. Removal of excess isopropylamine under
reduced pressure gave an only residue 3 (2.5 g, 97%).
d 1.19 (d, 6H, ArCH(CH3)2), 1.40, (s. 9H, Á
2.31 (s, 3H, ArCH3), 3.01 (t, 1H, ÁCH2NHÁ
2H, ÁCH2NHCHꢀ), 3.91 (m, 1H, ÁOCH2Á
1H, ÁOCH2Á), 4.41 (m, 1H, ÁCH(OH)Á
Ar), 6.75 (d, 1H, Jꢁ7.7 Hz, Ar), 7.07 (d, 1H, Jꢁ
/
C(CH3)3),
/
/
), 3.24 (m,
), 4.10 (m,
), 6.66 (s, 1H,
7.7
1H NMR (CDCl3): d 1.09 (d, 6H, Á
/
NHCH(CH3)2), 1.20
(d, 6H, ArCH(CH3)2), 2.30 (s, 3H, ArCH3), 2.7Á3.02
(m, 5H, ÁCH(OH)CH2NHCHꢀ, two protons ex-
changed in D2O), 3.27 (m, 1H, ArCH(CH3)2), 3.92 (m,
1H, ÁOCH2Á), 4.00 (m, 1H, ÁOCH2Á), 4.08 (bm, 1H,
CH(OH)Á), 6.67 (s, 1H, Ar), 6.75 (d, 1H, Jꢁ7.7 Hz,
Ar) and 7.09 (d, 1H, Jꢁ7.7 Hz, Ar). M/S: m/z: 265
[Mꢂ].
/
/
/
/
/
/
/
/
/
/
/
Hz, Ar). Anal Calc. for C19H31NO6: C, 61.76; H, 8.46;
N, 3.79. Found: C, 61.95; H, 8.60; N, 3.89%.
/
/
/
/
Á
/
/
/
/
3.2. Pharmacological methods
Swiss albino mice of either sex (20Á/30 g) and female
3.1.3. 1-Isopropylamino-3-(2-isopropyl-5-methyl-
phenoxy)-propan-2-ol oxalate (5) [DPJ-576]
rats of Wistar strain (200Á250 g) were purchased from
/
National Toxicology Centre, Pune, India. All animals
were housed in clean environment under 12 hours light
and 12 hours dark cycle. The animals had free access to
food pellets (Chakan oil mills, Pune, India) and water
was made available ad libitum.
Isoprenaline (ISOLIN†, Samarth Pharma Pvt. Ltd.,
Mumbai, India), Adrenaline (Adrenaline inj. I.P., Walk-
er’s Pharmaceuticals Pvt. Ltd., Pune, India), Atenolol
hydrochloride (Khandewall labs ltd, Mumbai, India)
and Urethane (Wilson laboratories, Mumbai, India)
were diluted/dissolved to appropriate concentrations
with physiological saline. Fresh drug solutions were
prepared for each day’s work.
1-Isopropylamino-3-(2-isopropyl-5-methyl-phenoxy)-
propan-2-ol (3) (2.0 g, 7.5 mmol) was dissolved in
acetone (50 ml) by warming, added a hot solution of
oxalic acid (1.25 g, 9.9 mmol) in acetone (10 ml) and
refluxed for 0.5 h. The reaction mixture was concen-
trated and left for crystallization to afford oxalate 5
(1.72 g, 64%), m.p. 143Á
3246 (NÁH), 2963 (aliphatic CÁ
CÁ
H) cmꢀ1 1H NMR (CDCl3): d 1.17 (q, 6H,
NHCH(CH3)2) 1.39 (d, 6H, ArCH(CH3)2), 2.28 (s,
3H, ÁCH3), 3.21(m, 2H, 2ꢃ CH(CH3)2), 3.39 (m, 1H,
CH2NHÁ), 3.51 (m, 1H, ÁCH2NHÁ), 3.90 (m, 1H,
OCH2Á), 4.03 (m, 1H, ÁOCH2Á), 6.58 (s, 1H, Ar),
6.75 (d, 1H, Jꢁ7.7 Hz, Ar), 7.08 (d, 1H, Jꢁ7.7 Hz,
/
146 8C. IR (KBr): 3396 (OÁ
/H),
/
/H) and 811 (aromatic
/
.
Á
/
/
/
Á
/
Á
/
/
/
/
Á
/
/
/
/
/
/
3.2.1. Mouse ECG experiments
Ar). Anal Calc. for C18H29NO6: C, 60.82; H, 8.22; N,
3.94. Found: C, 60.29; H, 8.36; N, 4.06%.
Mice of either sex weighing 25Á
with freshly prepared urethane (1.75 g/kg i.p.) and
stainless steel needles of gauge 21ꢃ1/2 were inserted
/
35 g were anesthetized
/
3.1.4. 1-tert-Butylamino-3-(2-isopropyl-5-methyl-
phenoxy)-propan-2-ol (4)
2-(2-Isopropyl-5-methyl-phenoxymethyl)-oxirane (2)
(2.0 g, 9.7 mmol) and tert-butylamine (10 ml), were
refluxed for 12 h. Completion of reaction was confirmed
by TLC. Removal of excess tert-butylamine under
subcutaneously in the flexor aspect of limbs. The needles
were clipped to SS2L electrodes of BIOPAC Student lab
pro (Model MP 30, BIOPAC Systems, Inc., Santa
Barba, CA).
The drugs in the appropriate doses were administered
intravenously though the tail vein. ECG recordings
(Lead I) were taken at 5, 15, 30, 45 min, 1 hour and
then hourly response till the action of the drug ends. At
the end of the experiment the needles were unclipped
and detached from the limbs of the animals and the
animals were observed intermittently till full recovery.
1
reduced pressure gave an oily residue 4 (2.5 g, 92%). H
NMR (CDCl3): d 1.13 (s, 9H, Á
ArCH(CH3)2), 2.71Á2.92 (m, 4H, Á
two protons exchanged in D2O), 3.26 (m, 1H,
CH(CH3)2), 3.97 (m, 3H, ÁOCH2CH(OH)Á), 6.68 (s,
/
C(CH3)3), 1.20 (d, 6H,
/
/
CH(OH)CH2NH Á,
/
Á
/
/
/
1H, Ar), 6.75 (d, 1H, Jꢁ
/
7.7 Hz, Ar), 7.09 (d, 1H, Jꢁ
/
7.7 Hz, Ar). M/S: m/z: 279 [Mꢂ].
3.2.2. Isolated rat uterus preparation
Isolated rat uterus preparation was carried as de-
scribed by Levy and Tozzi [17]. Spontaneous motility of
the unprimed rat uterus was recorded by means of force
transducer of Student’s Physiograph (Bio-Devices, Am-
bala, India). The drugs were injected in to the isolated
organ bath (INCO, Ambala, India) and the effects on
the contraction of the uterus were recorded.
3.1.5. 1-tert-Butylamino-3-(2-isopropyl-5-methyl-
phenoxy)-propan-2-ol oxalate (6) [DPJ-577]
1-tert-Butylamino-3-(2-isopropyl-5-methyl-phenoxy)-
propan-2-ol (4) (1.5 g, 5.4 mmol) was dissolved in
acetone (50 ml) by warming, added a hot solution of
oxalic acid (1.0 g, 7.3 mmol) in acetone (10 ml) and
refluxed the mixture for 0.5 h. Removal of excess solvent
under reduced pressure gave oily residue, which was
given washing with dry ether. Crystallized the residue
from acetone to afford oxalate 6 (0.91 g, 46%). m.p.
3.3. b-Adrenergic receptor binding assay
All the used solvents and powders were for analysis
(J.T. Baker, Deventer, Holland). Propranolol hydro-
chloride and ICI 118551 were purchased from Sigma
193Á
/
1948C. IR (KBr): 3289 (NÁ
/
H), 2981 (aliphatic CÁ
/
1
H) and 845 (aromatic CÁ
/
H) cmꢀ1. H NMR (CDCl3):

560
D.P. Jindal et al. / Il Farmaco 58 (2003) 557Á562
/
Chemical Co., St. Louis, MO, USA. [3H]Dihydroalpre-
nolol ([3H]DHA) (New England Nuclear, Boston, MA,
USA), having a specific activity of 99.9 Ci/Mol and
Three hundred microliters of membrane of lung
(ꢁ13.05 mg of fresh tissue, dilution 1:10) were incu-
/
bated for 30 min at 37 8C with 100 ml of 6 nM [3H]DHA,
100 ml of ketanserine 10ꢀ7 M 5HT antagonist and 100 ml
of various concentrations of the test compounds (dis-
radiochemical purity ꢀ98.5%, was used as ligand.
/
3.3.1. b1-Adrenergic receptor binding assay
Pellets containing b1 type adrenergic receptors were
obtained from turkey erythrocyte membranes as de-
scribed in the literature [18].
solved in water and diluted with buffer) and 75 M TrisÁ
/
HCl (pH 7.65), 25 M MgCl2 (total vol. 1 ml). The
samples were subsequently washed with 4.5 ml of the
same buffer and placed into scintillation vials. 10 ml of
Filter-Count liquid scintillation cocktail (Packard
BioScience s.r.l., Pero, Milan, Italy) was then added to
each vial and counting was carried out by scintillation
spectrometer (Packard TRI-CARB† 2000CA-Packard
BioScience s.r.l., Pero, Milan, Italy).
b1-Adrenergic receptor binding assay was determined
as follows: 300 ml of membrane (ꢁ/1.2 mg/ml of protein,
dilution 1: 8 v/v) were incubated for 15 min at 37 8C with
100 ml of 4 nM [3H]DHA and 100 ml of various
concentrations of the test compounds (dissolved in
water and diluted with saline buffer) and 12 M TrisÁ
HCl, pH 7.5 (total vol. 1 ml). The incubations were
stopped adding 4 ml of cold buffer (12 M TrisÁHCl)
/
Non-specific binding was measured in the presence of
100 ml of 10 mM unlabelled ICI 118551, and specific
binding as the difference between total and non-specific
binding.
The concentration of the test compounds that in-
hibited [3H]DHA binding by 50% (IC50) was determined
as above reported.
/
followed by rapid filtration through glass fiber filter
Whatman GF/B disks (Brandel Biomedical Research
and Laboratories Inc., Gaithersburg, MD, USA). The
samples were subsequently washed three times with 4.5
ml of the same buffer and placed into scintillation vials
10 ml of Filter-Count liquid scintillation cocktail
(Packard BioScience s.r.l., Pero, Milan, Italy) was then
added to each vial and counting was carried out by
scintillation spectrometer (Packard TRI-CARB†
2000CA-Packard BioScience s.r.l., Pero, Milan, Italy).
Non-specific binding was defined as non-displaceable
binding in the presence of 100 ml of 10 mM propranolol.
Competition experiments were analyzed by the ‘Easy
4. Results
Pharmacological evaluation of newly synthesized
compounds 5 (DPJ-576) and 6 (DPJ-577) have been
carried out using mouse E.C.G and isolated rat uterus.
Table 1 shows the effect of isoprenaline, atenolol, 5 and
6 alone, and the effect of isoprenaline after pretreatment
with atenolol, 5 and 6 on heart rate, on mouse ECG
recordings. Both 5 and 6 antagonized the relaxant effect
of isoprenaline and adrenaline on isolated unprimed rat
uterus, the results are shown in Table 2. Also both the
compounds were subjected to in vitro b1- and b2-
adrenergic receptor binding assay using turkey erythro-
cyte membrane (b1) and lung homogenate of rats (b2)
and the results are shown in Table 3. The binding
affinities were compared with that of propranolol
(propranolol hydrochloride, CAS 318-98-9), a non-
selective b-adrenergic blocking agent and atenolol, a
cardioselective b-adrenergic blocking agent.
Fit’ program (EasyFit 1.4, 1989Á1991, Matteo Vaccari
/
and Mario Negri Institute Milan, Italy) to obtain the
concentration of unlabeled drug that caused 50%
inhibition of ligand binding (IC50), with six concentra-
tions or displacers, each performed in triplicate.
The IC50 values obtained were used to calculate
apparent inhibition constants (Ki) by the method of
Cheng and Prussoff [19], from the following equation:
Kiꢁ
/
IC50/(1ꢂS/KD) where S represents the concentra-
/
tion of the ligand used and KD its receptor dissociation
constant (KD values, obtained by Scatchard analysis
[20], for [3H]DHA is 3.6ꢃ10ꢀ9 M).
/
3.3.2. b2-Adrenergic receptor binding assay
Preparation of lung homogenate: male SpragueÁ
Dawley rats (Harlan Italy s.r.l., Correzzana, Milan)
were sacrificed by decapitation. The right lung was
removed free of the major bronchi. Lungs were homo-
genized with Polytron† Apparatus-Kinematica GmbH,
Littau, Switzerland (setting 5 for 15 s) in 50 volumes
/
5. Discussion
In mouse ECG experiment administration of isopre-
naline (2 mg/kg i.v.) decreased the R-R interval repre-
senting increase in the heart rate. The onset of action
was 5 min and the peak was observed between 15 and 30
min. Complete restoration of normal heart rate was
observed at 60 minutes. Administration of atenolol, 5
and 6 produced a dose dependent decrease in mouse
heart rate. Administration of isoprenaline (2 mg/kg i.v.)
failed to produce tachycardia in atenolol (20 mg/kg i.v.)
pretreated mice after a pretreatment time of 3 hours,
buffer, 75 M TrisÁ
centrifuged at 30,000ꢃ
pellets were resuspended in 100 volumes of buffer 75 M
TrisÁHCl (pH 7.65), 25 M MgCl2, then were frozen at
80 8C before being assayed [21,22]. [3H]dihydroalpre-
nolol was used as ligand.
/HCl (pH 7.65), 25 M MgCl2 and then
/g for 10 min twice. The resulting
/
ꢀ
/

D.P. Jindal et al. / Il Farmaco 58 (2003) 557Á
/562
561
Table 1
Effect of isoprenaline, atenolol, 5 (DPJ-576) and 6 (DPJ-577) on mouse ECG parameters
Sr. no.
Drug treatment and dose
1
Time (min)
Heart Rate
2
1
2
3
4
5
6
7
isoprenaline (2 mg/kg)
atenolol (20 mg/kg)
30
180
180
60
617.469
321.56*9
327.00*9
337.40*9
414.70*9
371.60*9
375.25*9
/
39.68
/
24.99
20.40
65.00
20.76
36.73
10.49
atenolol (20 mg/kg)
isoprenaline (2 mg/kg)
isoprenaline (2 mg/kg)
isoprenaline (2 mg/kg)
/
5 (DPJ-576) (100 mg/kg)
5 (DPJ-576) (100 mg/kg)
6 (DPJ-577) (50 mg/kg)
6 (DPJ-577) (50 mg/kg)
/
60
/
60
/
60
/
* P B0.05 level significance applying paired Student’s t-test.
/
indicating blockade of b1-adrenergic receptors. Similarly
isoprenaline (2 mg/kg i.v.) injected after pretreatment of
5 (100 mg/kg i.v.) in mice failed to produce tachycardia.
Also isoprenaline (2 mg/kg i.v.) injected after 6 (50 mg/kg
i.v.) pretreatment in mice failed to produce tachycardia,
indicating blockade of b1-adrenergic receptors. In the
present experiment atenolol (20 mg/kg i.v.) was more
effective than 5 (100 mg/kg i.v.) or 6 (50 mg/kg i.v.) as a
b1-adrenergic blocking agent as it showed better inhibi-
tion of isoprenaline induced increase in heart rate. Out
of the two compounds the tert-butyl derivative 6 was
more active as a b1-adrenergic blocker than the isopro-
pyl derivative 5.
Table 3
Inhibition of [3H]DHA binding on b1- and b2-adrenergic receptor and
selectivity ratio (b1/b2) of compounds 5 (DPJ-576)and 6 (DPJ-577)
Compound Kib19
/
SD (M)
Kib29
/
SD (M)
Selectivity ratio
(b1/b2)
5 (DPJ-576) 1.39ꢃ
/
10ꢀ109
10ꢀ109
/
1.21ꢃ
0.454
/
10ꢀ99
10ꢀ109
/
8.70
0.56
1.56
0.052
6 (DPJ-577) 4.77ꢃ
/
/
2.66ꢃ
/
/
1.312
1.141
propranolol 1.60ꢃ
/
10ꢀ99
/
2.50ꢃ
/
10ꢀ99
/
0.134
0.177
atenolol
2.70ꢃ
/
10ꢀ89
/
0.401
Isolated rat uterus (unprimed) is an experimental
model specific for adrenergic b2-receptor. Both 5 and 6
blocked isoprenaline (0.02 mg/ml) and adrenaline (0.02
mg/ml) induced relaxation of isolated rat uterus The
rank order of potency of inhibition of relaxation was, 6
show a lesser b1-adrenergic blocking activity as mea-
sured by the ability to inhibit the tachycardia induced by
isoprenaline in mouse ECG experiments when com-
pared to atenolol, but the binding affinities of 5 and 6
are better than that of atenolol. This difference in
functional and receptor binding studies may be due to
pharmacokinetic factors of the newly synthesized com-
pounds that may also be responsible for the activity of
the drug in vivo.
In conclusion the results of functional and receptor
binding studies indicate that both the newly synthesized
compounds 5 and 6 starting from thymol possessed non-
selective b-adrenergic receptor blocking activity, with
the tert-butyl derivative (6) being more active than the
isopropyl derivative (5). Further modification of these
compounds are underway to obtain more potent and
cardioselective b-adrenergic blocking agents.
(2.5 mg/ml)ꢀ5 (5 mg/ml). This experiment shows that
/
both 5 and 6 possess b2-adrenergic blockade, with 6
being a better b2-adrenergic blocker.
The b-adrenergic receptor binding assay of 5 and 6
(Table 3), show that both the compounds possess b-
adrenergic receptor affinity comparable to that of
propranolol without selectivity to any one receptor,
while atenolol has a selective affinity to b1-adrenergic
receptor. Both the compounds 5 and 6 have similar
binding profile with the isopropyl derivative (5) being
slightly selective for b1-adrenergic receptor, compared to
the tert-butyl derivative (6). Thus the results of the
functional studies are in accordance with the b-adrener-
gic receptor binding affinity for both 5 and 6. They both
Table 2
Effect of adrenaline, isoprenaline alone and in presence of 5 (DPJ 576) and 6 (DPJ 577) on isolated rat uterus (unprimed)
Drug
Height (mm)
Normal
Adrenaline (0.02 mg/ml) Drugꢂ
/
adrenaline Normal
Isoprenaline (0.02 mg/ml) Drugꢂisoprenaline
/
DPJ-576 (5 mg/ml)
22.009
/
1.73 0.009
/
0.00*
21.339
/
1.15
12.679
/
6.51 0.009
/
0.00*
12.009
/
5.29
DPJ-577 (2.5 mg/ml) 23.00911.53 4.009
/
/
6.93*
24.33911.24
/
22.3396.08 3.679
/
/
3.52*
23.3395.36
/
* P B0.05 level significance applying paired Student’s t-test.
/

562
D.P. Jindal et al. / Il Farmaco 58 (2003) 557Á562
/
[11] Y.T. Lin, B.N. Wu, J.R. Wu, Y.C. Lo, L.C. Chen, I.J. Chen,
Vasomolol: an ultra short-acting and vasorelaxant vanilloid type
b1-adrenoceptor antagonist, J. Cardiovasc. Pharmacol. 28 (1996)
Acknowledgements
We wish to thank Council of Scientific and Industrial
Research, New Delhi, India and PARR by University of
Siena, Italy for financial supports.
149Á157.
/
[12] B.N. Wu, T.L. Hwang, C.F. Liao, I.J. Chen, Vaninolol: a new
selective b1-adrenoceptor antagonist derived from vanillin, Bio-
chem. Pharmacol. 48 (1994) 101Á109.
/
[13] B.N. Wu, C.R. Yang, J.M. Yang, I.J. Chen, A new b-adreno-
ceptor blocking agent derived from dehydrozingerone, Gen.
References
Pharmacol. 4 (1994) 651Á659.
/
[14] B.N. Wu, W.C. Ho, L.C. Chiang, I.J. Chen, Zingeronolol: a
newly developed b-adrenergic blocking agent derived from
zingerone, a pungent principle of ginger, Asia Pac. J. Pharmacol.
[1] J.H. Pinkey, J.S. Yudkin, Antihypertensive drugs: Issues beyond
blood pressure control, Prog. Cardiovasc. Dis. 36 (1994) 397Á
[2] J.G.F. Cleland, Progression from hypertension to heart failure,
Cardiology 92 (Suppl. 1) (1999) 10Á19.
/415.
11 (1996) 5Á12.
/
/
[15] S.J. Chen, Y.C. Huang, B.N. Wu, I.J. Chen, Eugenolol: an
eugenol-derived b-adrenoceptor blocker with partial b2-agonist
and calcium mobilization inhibition associated vasorelaxant
[3] E.D. Freis, V. Papademetriou, Current drug treatment and
treatment patterns with antihypertensive drugs, Drugs 52 (1996)
1Á16.
/
activities, Drug Dev. Res. 40 (1997) 239Á
[16] W.C. Evans (Ed.), Trease and Evans’ Pharmacognosy, 14th ed.,
W.B. Saunders Co., Ltd., London, UK, 1989, pp. 255Á292.
[17] B. Levy, S. Tozzi, The adrenergic receptive mechanism of the rat
uterus, J. Pharmacol. Exp. Ther. 142 (1963) 178Á184.
/250.
[4] Joint National Committee on Prevention, Detection, evaluation
and treatment of high blood pressure. The sixth report, Arch.
/
Intern. Med. 157 (1997) 2413Á46.
/
[5] B.G. Main, H. Tucker, Recent advances in b-adrenergic blocking
agents, in: G.P. Ellis (Ed.), Progress in Medicinal Chemistry, vol.
/
[18] K.P. Minneman, G.A. Weilland, P.B. Molinoff, A comparison of
the beta-adrenergic receptor of the turkey erythrocyte with
mammalian beta1 and beta2 receptors, Mol. Pharmacol. 17
22, Elsevier, Amsterdam, 1985, pp. 121Á
[6] CIBIS II Investigators and Committees, The Cardiac Insufficiency
Bisoprolol Study II (CIBIS-II), Lancet 353 (1999) 9Á13.
[7] MERIT-HF Study Group, Effect of metoprolol CR/XL in
chronic heart failure: metoprolol CR/XL Randomized Interven-
tion Trail in Congestive Heart Failure (MERIT-HF), Lancet 353
/
164.
/
(1980) 1Á7.
/
[19] Y.C. Cheng, W.H. Prussoff, Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes 50%
inhibition (IC50) of an enzymatic reaction, Biochem. Pharmacol.
(1999) 2001Á7.
/
22 (1973) 3099Á
[20] G. Scatchard, The attractions of proteins for small molecules and
ions, Ann. N. Y. Acad. Sci. 51 (1949) 660Á672.
/
3108.
[8] C.R. Kinsolving, B.E. Watkins, A.R. Borrelli, F.C. Kaiser, E.S.
Wu, Flavodilol: a new antihypertensive agent, J. Cardiovasc.
/
Pharmacol. 14 (1989) 127Á141.
/
[21] K.P. Minneman, L.R. Hegstrand, P.B. Molinoff, The pharmaco-
[9] I.J. Chen, S.J. Liou, S.S. Liou, C.N. Lin, Xanthonolol: a calcium
channel and beta-adrenoceptor blocker with vasodilating proper-
logical specificity of beta-1 and beta-2 adrenergic receptors in rat
heart and lung in vitro, Mol. Pharmacol. 16 (1979) 21Á33.
/
ties, Gen. Pharmacol. 24 (1993) 1425Á1433.
/
[22] R.D. Aarons, P.B. Molinoff, Changes in the density of beta
adrenergic receptors in rat lymphocytes, heart and lung after
chronic treatment with propranolol, J. Pharmacol. Exp. Ther. 221
[10] I.J. Chen, J.L. Yeh, S.J. Liou, A.Y. Shen, Guaiacoxypropanola-
mine derivatives of capsaicin: a new family of b-adrenoceptor
blockers with intrinsic cardiotonic properties, J. Med. Chem. 37
(1994) 938Á/943.
(1982) 439Á443.
/