Structure-activity relationship at α-adrenergic receptors within a series of imidazoline analogues of cirazoline

Article abstract of DOI:10.1016/S0968-0896(00)00030-4

Several analogues of cirazoline (2), a selective α₁-adrenoreceptor agonist, were prepared and their pharmacological profiles studied. Although at the α₁-adrenoreceptor all the compounds displayed a significant agonist activity, at the α₂-adrenoreceptor they showed either agonist or antagonist activity depending on the nature of the phenyl substituent. The qualitative structure-activity relationship led us to the conclusion that the oxygen atom in the side-chain is essential for α₁-agonist activity, while the cyclopropyl ring is not, and may be replaced by several groups. Of the groups studied, isopropoxy appears to be the best. Instead, the same substitution (i.e., isopropoxy for the cyclopropyl ring) at α₂-adrenoreceptors causes a reversal of activity. On the other hand, the cyclopropyl ring seems to be important for α₁-selectivity. Compound 20 is the most potent α₁-agonist of the series, being equiactive with cirazoline on rat vas deferens and in pithed rat. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00030-4

Bioorganic & Medicinal Chemistry 8 (2000) 883±888
Structure±Activity Relationship at ꢀ-Adrenergic Receptors Within
a Series of Imidazoline Analogues of Cirazoline
a
a
a
a
Maria Pigini, Wilma Quaglia, Francesco Gentili, Gabriella Marucci,
c
b
c
c,
Franco Cantalamessa, Silvia Franchini, Claudia Sorbi and Livio Brasili *
aDipartimento di Scienze Chimiche, Via S. Agostino 1, UniversitaÁ degli Studi di Camerino, 62032 Camerino (MC), Italy
bDipartimento di Scienze Farmacologiche e Medicina Sperimentale, Via Scalzino, UniversitaÁ degli Studi di Camerino,
62032 Camerino (MC), Italy
Dipartimento di Scienze Farmaceutiche, Via Campi 183, Universit aÁ degli Studi di Modena, 41100 Modena, Italy
c
Received 3 August 1999; accepted 1 November 1999
AbstractÐSeveral analogues of cirazoline (2), a selective a
1
-adrenoreceptor agonist, were prepared and their pharmacological pro-
®
les studied. Although at the a -adrenoreceptor all the compounds displayed a signi®cant agonist activity, at the a -adrenoreceptor
1
2
they showed either agonist or antagonist activity depending on the nature of the phenyl substituent. The qualitative structure±
activity relationship led us to the conclusion that the oxygen atom in the side-chain is essential for a -agonist activity, while the
cyclopropyl ring is not, and may be replaced by several groups. Of the groups studied, isopropoxy appears to be the best. Instead,
1
the same substitution (i.e., isopropoxy for the cyclopropyl ring) at a -adrenoreceptors causes a reversal of activity. On the other
2
1 1
hand, the cyclopropyl ring seems to be important for a -selectivity. Compound 20 is the most potent a -agonist of the series, being
equiactive with cirazoline on rat vas deferens and in pithed rat. # 2000 Elsevier Science Ltd. All rights reserved.
6
Introduction
agonist activity. These results, together with the struc-
tural similarity between 7 and cirazoline (2), prompt us
Since the discovery of a-adrenergic receptor subtypes,
an intense e€ort has been mounted to develop speci®c
agonists and antagonists as potential therapeutic agents
and a signi®cant amount of structure±activity data has
to investigate further the e€ects of other substituents in
the ortho-position of the phenyl ring. In preceeding
papers1
compounds 4±7, 10±18 and 20.
1,12
we reported on the a-adrenergic activity of
1�3
emerged in pursuit of these goals.
Instead, here we report on the synthesis of compounds
8, 9, 19 and 21±24 and on the a-adrenergic activity
obtained, on the rat, of the imidazolines 4±24 (Chart 2).
One of the most studied classes of compounds is the
imidazolines, which have been shown to be very pecu-
liar in that small structural modi®cations can alter the
balance between agonist and antagonist, as well as the
2
,4
a /a selectivity.
Chemistry
1
2
Compounds 8, 9,13 1913 and 21±24 were synthesized as
shown in Scheme 1, through the alkylation of appro-
priate phenol with 25 or 26.14 The free bases were
transformed into the corresponding hydrochloride (8, 9,
19) or hydrogen oxalate salts (21±24). In the case of
compound 24, the free base was obtained by oxidation
Idazoxan (1) has been described as a selective a -adre-
noreceptor blocking agent. Extensive structure±activity
2
5
relationship studies have been carried out on 1 to
determine the e€ect of substituents on both aromatic
and imidazoline rings, as well as of structural modi®ca-
6�10
tion of these moieties.
Opening the benzodioxane
of the 2-thiomethyl precursor with H O /CH COOH.
2
2
3
ring of 1 to give 7 (Chart 1) resulted in a drop in a2-
adrenoreceptor antagonist potency and, interestingly,
disclosure of a selective postsynaptic a -adrenoreceptor
1
Pharmacology
The compounds in Chart 2, as hydrogen oxalate salts (4,
5, 11, 16, 20±24) or hydrochloride salts (6±10, 12±15,
*Corresponding author. Tel.: +39-59-378-570; fax: +39-59-378-560;
e-mail: brasili@unimo.it
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00030-4

8
84
M. Pigini et al. / Bioorg. Med. Chem. 8 (2000) 883±888
Chart 1.
Scheme 1. (a) Na/C OH; (b) (CO
2
H
5
2 2 2 5 2
H) /(C H ) O; (c) HCl concd/
2
C H
5
OH; (d) H /CH COOH.
2
O
2
3
Table 1. Antagonist potency of prazosin against a few selected agonists
a
Compd
pA
2
Noradrenaline
2
7
8.85‹0.04
8.93‹0.03
9.01‹0.07
8.94‹0.09
8.85‹0.08
8.98‹0.07
9.15‹0.11
1
1
1
2
5
6
9
0
apA
values were calculated according to Arunlakshana and Schild17
2
and are the means plus or minus SEM of, in each case, a minimum of
six experiments.
Results and Discussion
In the present investigation, the a - and a -adreno-
1
2
receptor activities of the compounds 4±24 were deter-
mined and compared with those of idazoxan (1),
cirazoline (2) and tolazoline (3). The results are shown
in Tables 2 and 3. It can be seen that opening the ben-
zodioxane ring of idazoxan (1) to give 7 results in a
Chart 2.
3
site a reversal of activity is seen, since compound 7 is a
0-fold reduction of a -antagonist potency while at a
2
1
1
7±19), were tested for a - and a -adrenergic activity
nearly full agonist. These ®ndings con®rm the results
6
1
2
using epididymal and prostatic portions, respectively, of
isolated rat vas deferens, following standard testing
procedures.
expressed as pD . In the case of antagonists, pA values,
previously reported by Chapleo et al. Compound 7
may be regarded as a very close derivative of cirazoline
(2) where the cyclopropyl substituent has been replaced
by a methoxy function. The meta (8) and para (9) iso-
mers are less active than the ortho one, which in turn is
slightly more active than unsubstituted compound 6,
indicating that a substituent in the ortho position may
positively contribute to drug±receptor interaction.
1
5,16
Agonist potency of the drugs was
2
2
17
calculated according to Arunlakshana and Schild or
8
1
van Rossum, were used. In order to determine the
interaction at a -adrenoreceptor, antagonism with pra-
1
zosin was studied for a few selected compounds. Prazo-
sin displaced the dose±response curves to the right in
parallel manner and to a very similar extent to the
displacement of noradrenaline and cirazoline dose±
Tolazoline (3) is a 10-fold selective a -adrenoreceptor
2
antagonist. The introduction of a second methylene
group between the phenyl and imidazoline rings (4)
causes a 10-fold decrease in anity at both subtypes. If
bridge lengthening involves a heteroatom, such as sul-
phur or oxygen, as in 5 or in 6 the antagonist activity at
a site is maintained, while at a site there is a reversal
response curves (Table 1). Interaction at a -adreno-
2
receptor was determined by the ability of idazoxan or
yohimbine to reverse the twitch inhibition of electrically
stimulated rat vas deferens caused by the agonist under
study. Compounds 10, 14±16, 19 and 20 were also studied
in pithed rat, and the contribution of a and a vascular
2
1
of activity, since compound 5 is a partial agonist and
1
2
1
9
adrenoreceptors to the increase of mean arterial pres-
sure was determined by using selective antagonists.
compound 6 a nearly full agonist. Ru€olo et al.,
studying the e€ect of the bridge length in another series

M. Pigini et al. / Bioorg. Med. Chem. 8 (2000) 883±888
885
Table 2.
activity (ia) values in the isolated rat vas deferens
a
1
- And a
2
-adrenoreceptor pD
2
(or pA
2
) and intrinsic
of the receptor-coupling mechanism. In fact, compound
6 is an a -agonist while its congener tolazoline (3) and 4
1
are a -antagonists.
1
Compd
a
1
a
2
a
iac
pD b
iac
pD
2
2
Cirazoline (2) has been known for several years to be a
very potent and selective a -adrenoreceptor agonist,
5.99‹0.10b
(Cirazoline) 7.33‹0.27
(Tolazoline) 5.42‹0.06
8.02‹0.08b
5.50‹0.11b
6.55‹0.07
5.60‹0.06b
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
(Idazoxan)
Ð
1.00
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
possessing also an, albeit modest, a -antagonist activity
2
b
b
(ref 20 and present study). Opening the cyclopropyl
moiety (14) causes a slight decrease of a -agonist activ-
4.51‹0.07b
4.69‹0.16
5.26‹0.17
5.74‹0.33
4.37‹0.15
2.78‹0.16
3.76‹0.08
6.01‹0.13
5.54‹0.08
6.09‹0.09
6.62‹0.09
6.88‹0.12
6.55‹0.02
5.63‹0.08
5.84‹0.12
6.50‹0.05
7.23‹0.07
6.97‹0.18
6.41‹0.18
6.75‹0.09
5.31‹0.11
Ð
0.60‹0.09 6.03‹0.05
0.95‹0.06 6.71‹0.10b
0.80‹0.08 6.51‹0.04b
1
b
ity and a 10-fold increase as a -antagonist. These ®nd-
2
ings show that the cyclopropyl ring is not an essential
structural feature for a -agonist activity, while it
b
0.57‹0.07 5.72‹0.07
1
0.33‹0.05 5.84‹0.14b
0.56‹0.04 6.67‹0.15
0.68‹0.04 6.61‹0.09
0.81‹0.04 6.67‹0.24
0.77‹0.06 7.06‹0.36
1.00‹0.04 6.43‹0.06b
0.77‹0.05 7.31‹0.13
0.93‹0.07 6.84‹0.07
0.85‹0.05 6.40‹0.08b
Ð
appears to be important for a selectivity.
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
0.75‹0.08
0.96‹0.20
0.88‹0.03
0.50‹0.13
Ð
0.94‹0.03
0.92‹0.02
Ð
Isosteric replacement of oxygen atom with sulphur was
also studied as a substituent in the ortho position and
compound 22 prepared. Its activity as an a -agonist is
1
about ®ve times higher than the activity of compound 7.
At a -site, the O/S replacement causes a reversal of
activity: compound 7 behaves as an antagonist while
2
b
0.80‹0.07 6.65‹0.07
Ð
Ð
1.00‹0.03 6.34‹0.17b
1.00‹0.05 7.25‹0.08
0.77‹0.08 7.44‹0.07
0.97‹0.06 5.94‹0.03
0.79‹0.04 7.78‹0.07
0.84‹0.07 4.58‹0.10
compound 22 is an a -agonist. Furthermore, oxidation
2
0.83‹0.03
1.00‹0.04
0.88‹0.03
1.00‹0.06
0.40‹0.11
of the sulphur atom to give the sulphoxide 24 decreases
both a - and a -activities. This ®nding is in agreement
1
2
with our earlier studies with 1,4-benzodioxane-based
antagonists where the same isosteric substitutions were
investigated.1
0,15
a
pD
pA
2
is � log ED50
.
values, calculated according to van Rossum, using cirazoline
b
18
2
Compound 20 is the most potent a -agonist (pD =7.23)
of the series, equiactive with cirazoline. Although it
lacks selectivity, being equiactive as a2-agonist
1
2
(
a
1
) or clonidine (a
2
) as agonist.
c
Intrinsic activity (ia) is the maximum e€ect obtained with the agonist
under study, expressed as percentage of those of cirazoline and cloni-
dine, both taken equal to 1. Values are means plus or minus SEM of,
in each case, a minimum of six experiments.
(pD =7.25), it shows the role of isopropoxy function,
2
which may replace the cyclopropyl ring. On the other
hand, the most potent a -agonist is compound 23
2
(
pD =7.78), while compound 10 shows the highest a -
2
2
Table 3. Characterization of a-adrenergic receptor-mediated pressor
response in pithed rats
selectivity (about three orders of magnitude). The e€ect
of the insertion of a methyl group in the bridge separ-
ating the phenyl and imidazoline rings was not constant:
in some cases no variation of the e€ect was seen, while
in some others an increase was observed, together with
an interesting change of activity, from antagonist to
a
Dose ratiob
Compd
ED50 (mg/kg, iv)
Prazosin
0.1 mg/kg, iv)
Yohimbine
(1 mg/kg, iv)
(
agonist at a site. It appears that the e€ect of the methyl
2
BHT-920
3.79‹1.26
75.2‹0.04
657‹204
7.90‹0.08
8.06‹1.08
20.2‹1.3
4.62‹0.51
2.03‹2.2
29.5‹5.5
1.9‹0.2
1.7‹0.6
12.3‹1.2
3.6‹0.5
2.1‹0.5
1.4‹0.5
2.2‹0.6
2.3‹04
2
group in the bridge depends on the agonist potency of
the desmethyl parent compound: the lower the potency
of the latter the larger the increase (10 versus 11 and 22
10
14
15
16
19
20
23.2‹7.1
18.0‹2.0
10.3‹1.2
21.3‹5.9
41.0‹5.8
21.3‹5.9
versus 23). In an earlier work,11 made on a -adreno-
1
receptors of the rabbit aorta, where the two parameters
that govern agonist potency, anity and ecacy were
separately studied using fewer compounds (2, 6±18 and
1.2‹0.1
a
Dose required to increase diastolic blood pressure by 50 mmHg.
ED50 of agonist following pretreatment with antagonist divided by
control ED50
2
0), we showed that the methyl group in the oxymethy-
b
lene side chain enhances agonist ecacy. Therefore, the
increase of agonist potency by a methyl group, found in
the present investigation, is most likely related also to
an increase of ecacy. The fact that compound 14 is an
.
of tolazoline-like imidazolines, showed a second meth-
ylene group to be detrimental for both potency and
anity. The present results, while con®rming these
antagonist (zero ecacy) at a -adrenoreceptor subtype,
while its methyl derivative 15 is an agonist, seems to
support this explanation.
2
®
ndings, indicate the peculiar role of the oxygen in the
bridge. Its presence, probably via interaction with an
additional binding site, may overcome the decrease of
anity due to the variation of the bridge length, and at
a1 site such an interaction may trigger the biochemical
events giving rise to contraction, that is to say the oxy-
gen atom plays a crucial role in determining the ecacy
In addition, although the a -adrenoreceptor activities
1
found in that study, with few exceptions, were higher
than those found in the present investigation, the trend
is much the same. In both studies, however, with the
exception of compound 20, all the 2-phenoxymethyl-
imidazolines were less active a -agonists than cirazoline.
1

8
86
M. Pigini et al. / Bioorg. Med. Chem. 8 (2000) 883±888
Some selected compounds were also studied in pithed
rat. The results are reported in Table 3, together with
those obtained with the reference compounds, cirazoline
Varian EM-390 instruments, respectively. Although the
IR and NMR spectra data are not included (because of
the lack of unusual features), they were obtained for all
compounds reported and were consistent with the
assigned structures. The microanalyses were performed
by the Microanalytical Laboratory of our department
and the elemental compositions of the compounds agreed
to within ‹0.4% of the calculated value. The term
`dried' refers to the use of anhydrous sodium sulphate.
(
a -selective agonist) and BHT 920 (a -selective ago-
1
2
nist). The most active derivative is again compound 20
with an ED50 of 2.03 mg/kg which shows the same potency
as cirazoline (ED =3.79), con®rming the peculiar role
5
0
of isopropoxy group. Since the pressor responses eli-
cited by drugs in pithed rats may be due to a mixed
population of post-synaptic vascular a - and a -adren-
1
2
2
1�23
ergic receptors,
the e€ect of compounds was exam-
Synthesis of 23. 2-Thiomethylphenol (1.0 g, 7.1 mmol)
was added to a solution of Na (0.3 g, 13.04 mmol) in
ined after pretreatment with the a -adrenoreceptor-
1
1
4
selective antagonist prazosin (0.1 mg/kg, iv) and the a2-
adrenoreceptor selective antagonist yohimbine (1 mg/
kg, iv) in order to determine the contribution of each
subtype to the pressor activity. The dose±ratio values
obtained show that the compounds studied produce
abs EtOH (15 mL). After 1 h, imidazoline 26 as
hydrochloride (1.55 g, 10.0 mmol) was added to the
reaction mixture, which was heated to re¯ux for 6 h
under vigorous stirring. The solution was evaporated to
dryness to give a residue which was taken up in chloro-
form (100 mL) and washed with water (25 mL).
Removal of the dried solvents gave 23 as the free base
which was puri®ed through column chromatography.
Eluting with CHCl :EtOH:33% NH OH (13.8:0.2:0.05)
their e€ect through a -adrenoreceptors. This is appar-
1
ently in contrast with the results found in vitro where a
high a -adrenoreceptor activity was detected. However,
2
vascular postsynaptic a -adrenoreceptors might be dif-
2
3
4
ferent from the presynaptic ones present in the vas
4
gave the free base which was transformed into the
.
2
deferens.
hydrogen oxalate salt. Anal. (C H N OS H C O ) C,
2
12
16
2
2
4
H, N.
In conclusion we have shown that the oxygen atom in
the side-chain of cirazoline is essential for a -agonist
Synthesis of 24. A solution of 23 (0.1 g, 0.423 mmol)
and 30% H O (0.2 mL) in AcOH (1.25 mL) was left at
1
activity, while the cyclopropyl ring is not, and may be
replaced by several groups. Isopropoxy, among the
groups studied, appears to be the best. The same sub-
2
2
room temperature for 0.5 h. The mixture was made
basic with 2 N NaOH and extracted with CHCl3.
Removal of the dried solvent gave an oil which was
transformed into the hydrogen oxalate salt and crystal-
stitution at a -adrenoreceptors causes a reversal of
2
activity. On the other hand, the cyclopropyl ring seems
.
to be important for a -selectivity. These results con®rm
lized from EtOH. Anal. (C H N O S H C O ) C, H, N.
4
1
12 16
2
2
2
2
the peculiarity of this class of compounds since small
structural variations change antagonist activity to ago-
nist activity and a /a selectivity.
Compounds 8, 913 and 1913 as hydrochloride salts and
21 and 22 as hydrogen oxalate salts were obtained
following the same procedure used for 23, from the
corresponding phenols (Scheme 1). The physical char-
acteristics of all the compounds are reported in Table 4.
1
2
Experimental
Chemistry
Pharmacology
Melting points were taken in glass capillary tubes on a
Buchi SMP-20 apparatus and are uncorrected. IR and
NMR spectra were recorded on Perkin±Elmer 297 and
In all cases, contractions were recorded isometrically by
means of a force transducer connected to a two-channel
Gemini polygraph (Basile, Comerio, Italy).
Table 4. Physical±chemical characteristics of compounds 8, 9, 19, 21±24
0
a
ꢀ
Analysesb
Compd
R
R
Formula
Recrytn solvent
Mp ( C)
Yield (%)
3
3
H
H
H
CH
H
CH
CH
C
C
C
11
11
12
H
H
H
14
14
16
N
N
N
2
2
2
O
O
O
2.
2.
2.
HCl
HCl
HCl
EtOH
i-PrOH
i-PrOH
i-PrOH
i-PrOH
i-PrOH
EtOH
158±161
80±83
49±50
50
45
19
48
43
42
52
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
8
9
3-OCH
4-OCH
1
2
2
2
2
9
1
2
3
4
OC
Oi-C
2
H
5
3
H
7
3
14 20 2 2 4 2
2.
C H N O C O H
79±81
.
.
.
SCH
SCH
3
C
C
11
H
H
14
N
N
2
OS C
2
O
O
4
H
2
120±122
98±100
145±147
3
3
3
12
16
2
OS C
2
4
H
2
SOCH
3
12 16 2 2 2 4 2
C H N O S C O H
a
ꢀ
The heating rate was 1 C/min.
Analyses for C, H, N were within ‹0.4% of the theoretical value required.
b

M. Pigini et al. / Bioorg. Med. Chem. 8 (2000) 883±888
887
Rat vas deferens. Male albino rats (175±200 g) were
killed by a sharp blow on the head and both vasa
deferentia were carefully removed, freed from adhering
connective tissue and transversally bisected.
cycle. Food and water were available ad libitum.
The animals were anesthetized with equithesin (9.6 g
nembutal sodium, 42.6 g choral hydrate, 21.2 g MgSO4,
400 mL propylene glycol, 50 mL ethyl alcohol and
water to 1000 mL) 3 mL/kg bw ip. The right jugular
vein was cannulated (PESO polyethylene tubing) for
drug administration. Blood pressure was measured from
the left common carotid artery through a PESO catheter
connected to a pressure transducer (P23 ID, Staham,
Hato Rey, Puerto Rico). The rats were then pithed by
insertion of a steel rod (1.5 mm in diameter) through the
skull and foramen magnum down into the spinal
The epididymal portion was mounted in 20 mL organ
baths containing a physiological salt solution (PSS) of
the following composition (mM): NaCl 118, KCl 4.7,
CaCl 2.5, MgCl 1.2, KH PO 1.2, NaHCO 25, glu-
2
2
2
4
3
cose 11.1.
The initial loading tension was set at 1 g. The medium
was maintained at 37 C and was aerated with 95% O ±
ꢀ
% CO . The tissues were allowed to equilibrate for at
25
canal. The tracheal cannula was inserted immediately
2
5
after pithing and the animals were respired arti®cially
by means of a Harvard Apparatus model 681 Roden
Respirator at a frequency of 35 cycles/min with a
volume of 2 mL/100 g. Temperature was maintained at
2
least 1 h before the addition of any drug. Cirazoline
concentration±response (contracture) curves were
obtained cumulatively and taken as a control.
ꢀ
approximately 37 C. The pithed rats received heparin iv
A 1 h washing period was followed and the tissues were
challenged with the agonist under study. Responses
were expressed as a percentage of the maximal response
obtained in the control curve. It was veri®ed in parallel
experiments that the second cirazoline concentration±
response curve was identical to the ®rst because the
change in ratio of equi-e€ective concentrations was less
than 2, which usually entails a minimal correction. The
results are expressed in terms of pD , � log ED , where
(150 iu/kg). The preparation was allowed to equilibrate
for at least 30 min before drug administration. The
increase in diastolic blood pressure was measured for
single doses of the agonists given iv (0.5 mL/kg). Full
recovery from the pressor e€ects with return to pre-
injection values was ascertained between subsequent
doses. After drug injection, the venous cannula was ¯u-
shed with 50 mL of isotonic saline solution. The results
are expressed in terms of ED , the concentration of the
50
2
50
ED is the concentration of agonist required to pro-
agonist required to increase the diastolic blood pressure
by 50 mm Hg. The pressor response of the test com-
pounds were evaluated after pretreatment with the a1-
adrenoreceptor selective antagonist prazosin (0.1 mg/
5
0
duce 50% of the maximum contraction.
When studying prazosin, dose-ratios at the ED50 values
of the agonists were calculated at three antagonist con-
centrations (30 min incubation) and each concentration
kg, iv) or a -adrenoreceptor-selective antagonist yohim-
2
bine (1 mg/kg, iv), in order to establish the contribution
made by each a-adrenoreceptor subtype to the pressor
activity observed. In this case antagonists were given 15
min earlier than the agonist dose.
was tested six times. Dissociation constants (pA values,
2
Table 1) were estimated by Schild plot17 constrained to
slope � 1.0. When applying this method, it was always
veri®ed that the experimental data generated a line
whose derived slope did not signi®cantly di€er from
unity (P>0.05).
Statistical evaluation. The results are expressed as the
means‹SEM. Student's-test was used to assess the sta-
tistical signi®cance of the di€erence between two means.
The prostatic portion was used to assess the agonistic
activity on a -adrenoreceptors. PSS composition was as
2
given above for the epididymal portions, except that the
MgCl concentration was reduced to 0.54 mM and that
it contained prazosin (0.1 mM) in order to block post-
Acknowledgements
2
This work was supported by a grant from the University
of Camerino and the National Research Council
synaptic a -adrenoreceptors. The tissues were stimu-
1
lated electrically at 0.1 Hz with square pulses of 3 ms
duration at a voltage of 10±15 V. When the twitch
response became constant, cumulative concentration±
response curves were run in each tissue, which was
treated only once with the agonist under study. The
results are expressed in terms of pD , � log ED , where
(
Rome, Italy).
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50
ED is the concentration required to produce 50%
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