Structure - Activity relationships in 2,2-diphenyl-2-ethylthioacetic acid esters: Unexpected agonistic activity in a series of muscarinic antagonists

Article abstract of DOI:10.1016/S0968-0896(00)00332-1

As a continuation of previous research on anticholinergic drugs derived from 2,2-diphenyl-2-ethylthioacetic acid, several 5,5-diphenyl-5-ethylthio-2-pentynamines (2-11) were synthetised and their antimuscarinic activity on M_1-4 receptor subtypes was evaluated by functional tests and binding experiments. One of the compounds obtained showed unexpected agonistic activity in functional experiments on M₂ receptors. Since the compound carried a phenylpiperazine moiety, other similar compounds (12-17) were prepared and found to be endowed with similar behaviour. These ligands, although possessing the bulky structure characterising muscarinic antagonists, display agonistic activity at M₂ subtypes while, as expected, behaving as antagonists on M₃ and M₄ subtypes. On M₁ subtypes, they show agonistic activity which, however, is not blocked by atropine. The peculiar pharmacological profile of these compounds is of interest for studying muscarinic receptor subtypes.

Full text of DOI:10.1016/S0968-0896(00)00332-1

Bioorganic & Medicinal Chemistry 9 (2001) 1165±1174
Structure±Activity Relationships in 2,2-Diphenyl-2-ethylthioacetic
Acid Esters: Unexpected Agonistic Activity in a Series of
Muscarinic Antagonists
S. Scapecchi,a G. Marucci,b R. Matucci,c P. Angeli,b C. Bellucci,a M. Buccioni,b
S. Dei,a F. Gualtieri,a,* D. Manetti,a M. N. Romanellia and E. Teodoria
aDipartimento di Scienze Farmaceutiche, Universita' di Firenze, Via Gino Capponi 9, 50121 Firenze, Italy
bDipartimento di Scienze Chimiche, Universita' di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
Á
Dipartimento di Farmacologia Preclinica e Clinica, Universita di Firenze, viale Pieraccini 6, 50139 Firenze, Italy
c
Received 6 October 2000; accepted 4 December 2000
AbstractÐAs a continuation of previous research on anticholinergic drugs derived from 2,2-diphenyl-2-ethylthioacetic acid, several
5,5-diphenyl-5-ethylthio-2-pentynamines (2±11) were synthetised and their antimuscarinic activity on M1À4 receptor subtypes was
evaluated by functional tests and binding experiments. One of the compounds obtained showed unexpected agonistic activity in
functional experiments on M2 receptors. Since the compound carried a phenylpiperazine moiety, other similar compounds (12±17)
were prepared and found to be endowed with similar behaviour. These ligands, although possessing the bulky structure character-
ising muscarinic antagonists, display agonistic activity at M2 subtypes while, as expected, behaving as antagonists on M3 and M4
subtypes. On M1 subtypes, they show agonistic activity which, however, is not blocked by atropine. The peculiar pharmacological
pro®le of these compounds is of interest for studying muscarinic receptor subtypes. # 2001 Elsevier Science Ltd. All rights
reserved.
Introduction
we report the synthesis, binding and functional activity
of the compounds shown in Figure 1 as well as a pre-
liminary characterisation of the in vitro pharmacologi-
cal pro®le of the members with agonistic activity.
For some time, we have been engaged in a research on
muscarinic antagonists derived from 2,2-diphenyl-2-
ethylthioacetic acid exempli®ed by compound 1, one of
the most potent and selective among those studied.1À3
In an e€ort to obtain more potent and selective com-
pounds and to improve SARs in this class of muscarinic
antagonists, we designed and synthetised the alkyne
analogues (2±11) shown in Figure 1. Obtained com-
pounds were tested with binding experiments and func-
tional assays. Much to our surprise, one of the prepared
compounds (11), a methyl iodide, behaved as an agonist
in functional tests on guinea pig atria, while being an
antagonist on the other muscarinic receptor models
studied. Unlike the other members of the family, 11
carries a phenylpiperazine ring. We therefore designed
the analogues 12±17 to verify whether this feature was
responsible for this unexpected behaviour and found
that, indeed, these compounds show agonistic activity
on the same M2 receptor model. In the present paper,
Chemistry
The compounds of the alkyne series (2±11) were
obtained as shown in Scheme 1. Diphenylmethane was
submitted to a double metallation with butyllithium to
introduce in the benzylic position a thioethyl (18) and
then a propargyl group (19). The acetylenic compound
obtained was transformed into the desired amines (2, 4,
6, 8, 10) by Mannich reaction. Compounds 3, 5, 7, 9 and
11 were obtained from the corresponding amines with
methyl iodide. Their chemical and physical character-
istics are reported in Table 1.
The esters of 2,2-diphenyl-2-ethylthioacetic acid (12±17)
were obtained by standard esteri®cation of compounds
20±224,5 with 2,2-diphenyl-2-ethylthioacetic acid chloride,2
according to Scheme 2. Their chemical and physical
characteristics are reported in Table 2. The chemical
*Corresponding author. Tel.: +39-5-5275-7295; fax: +39-5-524-0776;
e-mail: gualtieri@farm®.scifarm.uni®.it
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00332-1

1166
S. Scapecchi et al. / Bioorg. Med. Chem. 9 (2001) 1165±1174
Figure 1.
3
3
stability of compounds 11 and 12±17 under experi-
mental conditions was evaluated on samples kept at
30 ꢀC in DMSO, or in a DMSO/H2O solution adjusted
to pH=7.5, running 1H NMR spectra from time to time
(up to 24 h). Under these conditions, no changes in the
spectra were observed.
ventricle homogenates using [H]-NMS and [H]-OXO
as labelled ligand and expressed as Ki.
Results
The results obtained in binding and functional experi-
ments are reported in Tables 3±6.
Pharmacology
Muscarinic antagonism
All the compounds were tested on M1 (rabbit vas defer-
ens), M2 (guinea-pig atria), M3 (guinea-pig ileum) and
M4 (guinea-pig lung) muscarinic receptor subtypes for
their muscarinic and antimuscarinic activity, using p-Cl-
McN-A-343 and arecaidine propargyl ester (APE) as
agonists at M1 and M2À4 subtypes, respectively. Agonist
and antagonist potencies were expressed as ÀlogED50
and as Àlog of dissociation constant (pKb), respectively.
The muscarinic binding properties of the compounds
were evaluated on rat cerebral cortex and domestic pig
Alkyne derivatives 2±10 possess rather modest antag-
onistic activity on M1±M3 receptors (Table 3) which is
even lower as far as M4 receptors are concerned. The
most interesting compounds are 5, which shows selec-
tivity for M1 receptors (M1/M2=76; M1/M3=20 and
M1/M4=95), and 7 which shows selectivity for M1 and
M3 receptors (M1/M2=16; M1/M4=37; M3/M2=20
and M3/M4=44). With regard to antagonism, esters 12±
14 (Table 4) show the same behaviour on M2, M3 and
Scheme 1. (a) BuLi, Et2S2; (b) BuLi, propargylbromide; (c) CH2O, NHR1R2; (d) CH3I.

S. Scapecchi et al. / Bioorg. Med. Chem. 9 (2001) 1165±1174
Table 2. Physical characteristics of compounds 12±17
1167
Table 1. Chemical and physical characteristics of compounds 2±11
N
n
R
X
Mp (ꢀC) (recr. solv.)a
Analysisb
N
Y
Salt
Mp (ꢀC) (recr. solv.)a
90±92 (A)
Analysisb
12
13
14
15
16
17
2
3
4
2
3
4
H
H
H
CH3
CH3
CH3
Cl
Cl
Cl
I
I
I
149±151 (A)
178 dec. (A)
169±172 (A)
191±193 (B)
99±102 (B)
78±80 (B)
C28H33ClN2O2S
C29H35ClN2O2S
C30H37ClN2O2S
C29H35IN2O2S
C30H37IN2O2S
C31H39IN2O2S
2
Oxalate
C25H31NO4S
3
Iodide
Hydrochloride
Iodide
80±82 (B)
C24H32INS
C23H28ClNS
C24H30INS
4
134±137 (A)
157±160 (B)
143±145 (A)
171±173 (B)
141±142 (A)
175±178 (B)
145±148 (A)
171±173 (B)
a(A) Abs ethanol + anhydrous ether. (B) Abs ethanol.
bAll compounds were analysed for C, H and N and the results are
within 0.4% of the theoretical value. Their IR and 1H NMR spectra
are consistent with the proposed structures.
5
6
Hydrochloride
Iodide
C24H30ClNS
C25H32INS
ments on rat cerebral cortex con®rmed the modest a-
nity of such compounds (Table 5).
7
Muscarinic agonism
8
Hydrochloride
Iodide
C23H28ClNOS
C24H30INOS
C29H33ClN2S
C30H35IN2S
Table 3 shows that compound 11 is a weak antagonist
on M1, M3, and M4 muscarinic receptor models and
behaves like an agonist on the M2 receptor model.
Much like compound 11, compounds 15±17 (Table 4)
behave as muscarinic agonists on M2 and as antagonists
on M3 and M4 receptor subtypes. Agonism on M2 sub-
types is antagonised by atropine. On stimulated rabbit
vas deferens (M1), while compounds 12±14 behave as
weak antagonists, compounds 15±17 behave as agonists,
but their action is not antagonised by atropine or pir-
enzepine. The potency of all compounds examined on
M2 (force) is de®nitely higher when an incubation time
of 3 h is adopted (Table 6). In contrast, on M2 (rate) the
9
10
11
Hydrochloride
Iodide
a(A) Abs ethanol + anhydrous ether. (B) Abs ethanol.
bAll compounds were analysed for C, H and N and the results are
within 0.4% of the theoretical value. Their IR and 1H NMR spectra
are consistent with the proposed structures.
same compounds are inactive up to the dose of 10À7
10À6 M.
±
M4 receptors where they are modest antagonists with
poor selectivity. In general, the pharmacological pro®le
of all compounds studied in the present work as
antagonists is much less interesting than that of 1 and of
the esters reported previously.1À3 The binding experi-
Binding experiments on rat cerebral cortex against
[3H]NMS, show that compounds 11 and 15±17 possess
anity for muscarinic receptors comparable with that
Scheme 2. (a) Br±(CH2)n±OH; (b) 2,20-diphenyl-2-ethylthioacetic acid chloride; (c) CH3I.

1168
S. Scapecchi et al. / Bioorg. Med. Chem. 9 (2001) 1165±1174
Table 3. Functional and binding activity of compounds 1 and 2±11
N
Bindinga KiÆSEM (mM)
Functional activity pKbÆSEM
b
c
d
e
M1
M2
M3
M4
Ð
1f
2
3
4
5
6
7
8
9.97Æ0.06
6.35Æ0.06
6.63Æ0.05
7.10Æ0.17
7.54Æ0.09j
5.87Æ0.2
7.01Æ0.16
<5
9.12Æ0.03
6.01Æ0.06
6.96Æ0.13
6.79Æ0.10g
5.71Æ0.09
5.30Æ0.14
5.82Æ0.20
<5
6.50Æ0.07
<5
2.70Æ0.35
0.78Æ0.13
0.40Æ0.038
0.36Æ0.039
4.67Æ0.89
0.85Æ0.17
>5
<5
6.30Æ0.25
6.11Æ0.11h
6.31Æ0.19
6.62Æ0.20
7.08Æ0.19g
5.63Æ0.13g
5.93Æ0.14g
<5k
5.66Æ0.03
5.27Æ0.20i
5.56Æ0.11
<5
5.44Æ0.11
5.11Æ0.15
5.58Æ0.11
5.48Æ0.20
5.06Æ0.08m
9
10
11
2.41Æ0.29
6.43Æ0.07
5.84Æ0.15
>5
2.38Æ0.31
<5
<5
<5
a=0.92 4.80Æ0.15 (Àlog ED50
)
<5l
aRat cerebral cortex homogenate, [3H]NMS. Each experimental value is calculated from at least three determinations performed in duplicate.
bRabbit stimulated vas deferens.
cGuinea-pig atria.
dGuinea-pig ileum.
eGuinea-pig lung strips.
fSee ref 2.
g10% reduction of maximal e€ect.
h25% reduction of maximal e€ect.
i19% reduction of maximal e€ect.
jNon competitive (n signi®cantly di€erent from 1).
k69% reduction of maximal e€ect.
lAt 10À5 there is an 85% reduction of maximal e€ect.
m57% reduction of maximal e€ect.
of carbachol, and pilocarpine,6 16 and 17 being the most
anitive. The NMS/OXO ratio, which according to
Freedman7 is an evaluation of ecacy, predicts that
compounds 15±17, on rat cerebral cortex as well as on
domestic pig ventricle receptors, would behave as
antagonists. However the agonistic activity shown by
these compounds on M2 tissue preparations seems to
indicate that the method does not have general validity
or does not apply to these compounds.
However, in the last few years, some potent agonists
with somewhat larger size have appeared, and this has
required a rede®nition of the binding site of muscarinic
ligands.11 Xanomeline,12 PD15183213 and SK-94614 are
representative of such compounds (Chart 1). Moreover,
a few molecules showing strong muscarinic agonism and
characterised by an even larger size have been reported
in the literature. Their structures challenge the currently
accepted picture of the muscarinic receptor active site
and are therefore quite interesting for muscarinic recep-
tor studies. This group of compounds is exempli®ed by
SR-95639A and SR 96095A, which are representative of
a series of M1 selective muscarinic agonists developed
by the group of Wermuth15À17 by the acylhydrazone 23,
claimed to be a M1/M3 selective muscarinic agonist,18
and by clozapine which has been reported to be a potent
and selective M4 receptor agonist19À21 (Chart 1).
Discussion
Muscarinic antagonism
With regard to antagonism, all the compounds studied
show modest potency and selectivity when compared
with the compounds reported previously. Therefore, the
molecular modi®cations introduced, in the present work,
into the parent compounds2 are detrimental for their
pharmacological pro®le.
The pharmacological pro®le of compounds 11±17
appears fairly complex. So far, our work has been
aimed at a better de®nition of their properties, leaving
interpretation of their complex behaviour to further
studies.
Muscarinic agonism
The muscarinic agonism of 11 and subsequently of 12±
17 is quite unexpected. As a matter of fact, muscarinic
agonists are a fairly well known class of ligands whose
molecular characteristics have been studied extensively,8,9
and it is well known that increasing their size usually
results in the appearance of muscarinic antagonism.10
Since the agonistic action of compounds 11±17 is hardly
explainable, given their molecular structure, we wanted
to be sure that activity was not due to molecular frag-
mentation as a consequence of poor chemical or meta-
bolic stability. Therefore, the stability of 11±17, under
pharmacological experiment conditions, was checked:

S. Scapecchi et al. / Bioorg. Med. Chem. 9 (2001) 1165±1174
Table 4. Functional muscarinic activity (1 h incubation time) of compounds 12±17
1169
a
b
c
d
N
M1
M2
M3
M4
Forcee
Ratee
af
Àlog ED50 ÆSEM
af
Àlog ED50 ÆSEM
af
Àlog ED50 ÆSEM
pKb ÆSEM
pKb ÆSEM
g
g
g
h
h
i
i
l
j
12
13
14
15
16
17
0
0
0
1
1
1
0.9
1
0.7
1
1
0.7
5.54Æ0.24
6.17Æ0.23
5.59Æ0.15
6.07Æ0.14
6.54Æ0.06
4.08Æ0.24
1
1
1
1
1
1
5.74Æ0.21
6.24Æ0.20
5.51Æ0.11
6.46Æ0.25
6.64Æ0.20
4.51Æ0.13
5.37Æ0.14
5.15Æ0.01
5.36Æ0.02
5.51Æ0.06
5.32Æ0.05
<5
k
m
5.67Æ0.19n
6.00Æ0.20n
5.69Æ0.23n
6.78Æ0.14
6.66Æ0.08
5.53Æ0.12
aRabbit stimulated vas deferens.
bGuinea-pig atria.
cGuinea-pig ileum.
dGuinea-pig lung strips.
eAntagonized by atropine (pA2 ꢁ9.0).
fa=intrinsic activity, measured by the ratio between the maximum response of the compound and the maximum response of the reference agonist.
gED50=dose producing 50% of maximal e€ect.
hCalculated from the equation pKb=log(DRÀ1)Àlog[B].
iOn this tissue the compound is actually an antagonist with pKb<5.
jOn this tissue, at 10À5 M, there is a 75% reduction of the maximum e€ect of APE.
kOn this tissue, at 10À5 M, there is a 53% reduction of the maximum e€ect of APE.
lOn this tissue the compound is actually an antagonist with pKb=5.59Æ0.02.
mOn this tissue, at 10À5 M, there is a 76% reduction of the maximum e€ect of APE.
nNot antagonized by atropine nor pirenzepine.
Table 5. Muscarinic binding of compounds 11±17
N
Rat cerebral cortexa
Domestic pig ventriclea
3[H]-NMS
3[H]-OXO
NMS/OXO
3[H]-NMS
3[H]-OXO
NMS/OXO
KiÆSEM (mM)
KiÆSEM (mM)
KiÆSEM (mM)
KiÆSEM (mM)
11
12
2.38Æ0.31
ndb
Ð
Ð
Ð
Ð
Ð
Ð
Ð
nd
Ð
Ð
Ð
nd
Ð
Ð
Ð
Ð
Ð
>100
>100
>10
13
14
Ð
Ð
15
16
17
6.16Æ0.91
1.79Æ0.27
2.18Æ0.33
41.32Æ7.0c
0.00016cÆ0.00002
8.05Æ1.29c
8.47Æ1.45
0.96Æ0.26
1.37Æ0.35
0.0045Æ0.0008
0.00011Æ0.00002
0.00031Æ0.00006
0.7
1.9
1.6
8.78Æ1.34
1.99Æ0.49
2.98Æ0.57
4.38Æ0.83
0.0002Æ0.000044
0.22Æ0.06
19.0Æ4.6
0.5
2.6
3.4
1622
2
0.78Æ0.20
0.88Æ0.23
0.0027Æ0.0005
0.000095Æ0.000023
0.00058Æ0.00013
Carbachol
NMS
OXO
>4000
1.5
>4000
379
aKi values are reported in mMÆSEM (n53).
bnd=not determined.
cSee ref 6.
apparently the compounds do not undergo any mod-
i®cation after solubilisation (see Chemistry). To exclude
metabolic cleavage, agonism experiments were run in
the presence of physostigmine, an acetylcholinesterase
inhibitor, and we found no di€erences with previous
experiments.
Table 6. M2 muscarinic activity of compounds 11±17 after 3 h incu-
bation time
a
N
M2
Forceb
Rate
d
d
ac
Àlog ED50 ÆSEM
ac
Àlog ED50 ÆSEM
Binding experiments show that only the ammonium
salts 11 and 15±17 have anity for muscarinic brain
receptors similar to that of carbachol. This is an initial
unexpected result, as compounds 12±14 do show ago-
nistic activity on M2 receptors as reported in Table 4.
The NMS/OXO ratio,7 on the other hand, indicates an
antagonistic behaviour on the rat cerebral cortex. This
result, however, is of little help as their agonistic activity
appears only on M2 receptors while they have antag-
onistic action on other muscarinic subtypes. For this
reason, we tested compounds 11 and 15±17 also on
domestic pig ventricles where only M2 receptors are
present. In this preparation, the binding anity appears
e
e
e
e
e
e
e
11
12
13
14
15
16
17
0.94
1
0.9
0.9
1
8.10Æ0.17
7.65Æ0.08
7.96Æ0.17
8.59Æ0.03
7.62Æ0.04
8.07Æ0.19
7.62Æ0.15
Ð
Ð
Ð
Ð
Ð
Ð
Ð
0.9
0.9
aGuinea-pig atria.
bAntagonized by atropine (pA2ꢁ9.0).
ca=intrinsic activity, measured by the ratio between the maximum
response of the compound and the maximum response of APE.
dED50=dose producing 50% of maximal e€ect.
eInactive up to the dose range of 10À7±10À6M.

1170
S. Scapecchi et al. / Bioorg. Med. Chem. 9 (2001) 1165±1174
to be fairly similar to that on rat brain, but again the
NMS/OXO ratio suggests an antagonistic kind of
activity. At present, there is not an explanation to
account for the peculiar behaviour of this class of drugs.
Despite the complexity of the pharmacological pro®le
of this series of molecules (a fact that obviously
requires further studies), there is little doubt that they
can mimic agonistic activity on M2 receptors. Therefore,
these compounds should be added to the ligands repor-
ted in Chart 1 showing muscarinic agonism, although
their molecular structure is odd with respect to the
currently-accepted picture of the muscarinic receptor
active site.
A particular aspect of M2 muscarinic agonist activity of
compounds 11±17 is that, when the incubation time is
prolonged up to 3 h, the potency of the compounds is
de®nitely higher. In this case, however, the recovery of
tissue functionality is reduced to some 40% while
recovery ranged around 80% using the more usual 1 h
incubation time. The reasons for this behaviour are not
understood at present; they could be related to the high
lipophilicity of compounds 11±17 (which show poor
water solubility while being soluble in chloroform).
Alternative explanations for the peculiar activity of
compounds 11±17, such as an indirect agonistic e€ect,
seem less probable. In fact, since their behaviour is
similar on stimulated M1 and M2 subtypes and their
binding anity is poor to modest, they could con-
Chart 1.

S. Scapecchi et al. / Bioorg. Med. Chem. 9 (2001) 1165±1174
1171
ceivably be involved in the mobilisation of Ca++ or
ATP, producing a reduction of tissue contraction which
can mimic a muscarinic agonistic activity. However,
while this explanation ®ts well for the rabbit vas defe-
rens model, where activity of 15±17 is not antagonised
by muscarinic antagonists, it is in contrast with the fact
that the action of compounds 11±17 on atria is blocked
by the classical muscarinic antagonist atropine.
SCH2CH3); 2.07 (t, 1H, CH); 2.35 (q, 2H, SCH2CH3);
3.41 (d, 2H, CH2); 7.32±7.48 (m, 6H, aromatics); 7.52±
7.62 (m, 4H, aromatics) ppm.
N,N-Diethyl (5-ethylthio-5,5-diphenylpent-2-ynyl)amine
(2). A solution of formaldehyde (0.25 mL of a 40%
solution in water), diethylamine (3.0 mmol) and CuSO4
(0.05 g) were added to a solution of 19 (2.3 mmol) in 4
mL of EtOH/H2O (1/1). The pH of the solution was
adjusted to 8 with 50% sulphuric acid. The mixture was
heated to re¯ux for 24 h, then 15 mL of NH4OH were
added and the solution was extracted with Et2O. The
organic phase was dried and evaporated under reduced
pressure. The oily mixture was puri®ed by ¯ash chro-
matography using CH2Cl2/MeOH 95/5 as eluting sys-
tem. Yield 75% of a thick oil. 1H NMR (CDCl3): d 0.92
(t, 6H, NCH2CH3); 1.18 (t, 3H, (SCH2CH3); 2.35 (q,
2H, SCH2CH3); 2.29 (q, 4H, NCH2CH3); 3.22 (m, 2H,
CH2); 3.31 (m, 2H, CH2); 7.22±7.33 (m, 6H, aromatics);
7.42±7.50 (m, 4H, aromatics) ppm. The oily amine was
transformed into the oxalate and recrystallized from
abs. EtOH and dry ether. Mp 90±92 ꢀC. Anal.
(C25H31NO4S) C, H, N.
From a medicinal chemistry point of view, compounds
like 15±17 can be useful leads for designing classical and
non-classical muscarinic agonists. It is tempting to con-
sider that the phenylpiperazine nucleus, which is present in
all active compounds, is responsible for their activity and
therefore use it as a pharmacophore for new muscarinic
ligands. At present, we are exploring this possibility.
Experimental
Chemistry
All melting points were taken on a Buchi apparatus and
are uncorrected. Infrared spectra were recorded with a
Perkin-Elmer 681 spectrophotometer in a Nujol mull
for solids and neat for liquids. NMR spectra were
recorded on a Gemini 200 spectrometer. Chromato-
graphic separations were performed on a silica gel col-
umn by gravity chromatography (Kieselgel 40, 0.063±
0.200 mm, Merck) or ¯ash chromatography (Kieselgel
40, 0.040±0.063 mm, Merck). Yields are given after
puri®cation, unless otherwise stated. Where analyses are
indicated by symbols, the analytical results are within
Æ0.4% of the theoretical values.
Compounds 4, 6, 8, and 10 were made in the same way,
starting from the appropriate amine and transformed
into the salt indicated. Their chemical and physical
1
characteristics are reported in Table 1. Their IR and H
NMR spectra are consistent with the proposed struc-
tures.
N,N-Diethyl-N-methyl (5-ethylthio-5,5-diphenylpent-2-yn-
yl)amonium iodide (3). An excess of methyl iodide (2
mL) was added to 0.2 g of 2 dissolved in anhydrous
ether (10 mL) and the solution left overnight at room
temperature in the dark. The pale yellow solid obtained
was recrystallised from abs. EtOH; yield 90%. Mp 80±
82 ꢀC. 1H NMR(CDCl3): d 1.05 (t, 3H, SCH2CH3); 1.15
(t, 6H, +NCH2CH3); 2.12 (q, 2H, (SCH2CH3); 3.06 (s,
3H, +NCH3); 3.18 (q, 4H, +NCH2CH3); 3.22 (m, 2H,
CH2); 4.12 (m, 2H, CH2); 7.09±7.22 (m, 10H, aromatics)
ppm. Anal. (C24H32INS) C, H, N.
Ethylthiodiphenylmethane (18). Diphenylmethane (3 g;
0.018 mol) was dissolved in THF (15 mL) and cooled at
0 ꢀC. Butyllithium in hexane (12.25 mL of a 1.6 M
solution) was then added and the mixture stirred for 2 h
at 0 ꢀC. Diethyldisulphide (0.02 mol) was then added
and the mixture warmed up to room temperature, trea-
ted with water, extracted with ether and the organic
phase dried. The oily mixture (3.5 g) obtained after
evaporation was puri®ed by ¯ash chromatography
using cyclohexane/ethyl acetate 98/2 as eluting system.
Yield 67% of a thick oil that was used as such in the
Compounds 5, 7, 9, and 11 were made in the same way,
starting from the appropriate amine. Their chemical and
physical characteristics are reported in Table 1. Their
1
1
following reaction. H NMR (CDCl3): d 1.25 (t, 3H,
IR and H NMR spectra are consistent with the pro-
posed structures.
SCH2CH3); 2.42 (q, 2H, SCH2CH3); 5.20 (s, 1H, CH);
7.08±7.32 (m, 6H, aromatics); 7.40±7.51 (m, 4H, aro-
matics) ppm.
2-(4-Phenylpyperazinyl)ethyl 2,2-diphenyl-2-ethylthio ace-
tate (12). 2,2-Diphenyl-2-ethylthio acetyl chloride2 (2
mmol) was re¯uxed for 8 h with 4 mmol of 2-(4-phe-
nylpyperazinyl)ethanol (20)5 in 20 mL of EtOH-free
CHCl3. The solution was washed with 10% Na2CO3
solution and the organic layer anhydri®ed and evapo-
rated to give an oil that was puri®ed by ¯ash chroma-
tography on silica gel (eluent CHCl3/MeOH 99/1).
1-Ethylthio-1,1-diphenylbut-3-yne (19). Butyllithium in
hexane (3 mL of a 1.6 M solution) was added to 1 g (4.4
mmol) of 18 dissolved in THF (15 mL) and cooled at
0 ꢀC and the mixture stirred for 2 h at 0 ꢀC. Propargyl-
bromide (0.57 g, 4.8 mmol) was then added and the
mixture warmed up to room temperature, treated with
water, extracted with ether and the organic phase dried.
The oily mixture was puri®ed by ¯ash chromatography
using CH2Cl2/hexane 15/85 as eluting system.
Yield 59%. IR (neat) n 1740 (CO) cmÀ1 1H NMR
.
(CDCl3): d 1.15 (t, 3H, SCH2±CH3); 2.42 (q, 2H, SCH2±
CH3); 2.51±2.58 (m, 4H, piperazine protons); 2.62 (t,
2H, OCH2±CH2±N); 3.09±3.19 (m, 4H, piperazine pro-
tons); 4.41 (t, 2H, OCH2±CH2±N); 6.85±6.98 (m, 2H,
aromatics); 7.21±7.52 (m, 8H, aromatics) ppm.
Yield 47% of a thick oil that was used as such in the
1
following reaction. H NMR (CDCl3): d 1.18 (t, 3H,

1172
S. Scapecchi et al. / Bioorg. Med. Chem. 9 (2001) 1165±1174
were set up rapidly under 1 g of tension in 20 mL organ
baths containing physiological salt solution (PSS)
maintained at an appropriate temperature (see below)
and aerated with 5% CO2±95% O2. Two dose±response
curves were constructed by cumulative addition of the
reference agonist. The concentration of agonist in the
organ bath was increased approximately 3-fold at each
step, with each addition being made only after the
response to the previous addition had attained a max-
imum level and remained steady. When agonism was
studied, following 30 min of washing, a new dose±
response curve for the agonist under study was
obtained. Responses were expressed as a percentage of
the maximal response obtained in the control curve. The
results are expressed in terms of Àlog ED50, the con-
centration of agonist required to produce 50% of the
maximum contraction. The dose±response curve for the
agonist under study was obtained at two di€erent incu-
bation times, 1 and 3 h, with the cardiac tissue, in order
to examine the kinetics of the transduction mechanism
which is responsible for the slow tissue response. When
a 1 h incubation time with cardiac tissue was used, a 75±
80% of functional recovery was observed after 1 h of
washing; this percentage drops to 40±45% when a 3 h
incubation time and persistent washings were used.
When antagonism was studied, following 30 min of
washing, tissues were incubated with the antagonist for
1 h, and a new dose±response curve for the agonist was
obtained. To quantify antagonist potency, pKb values
were calculated from the equation pKb=log(DRÀ1)
Àlog[B], where DR is the ratio of ED50 values of ago-
nist after and before treatment with one or two antago-
nist concentrations [B].22
Table of elemental analyses of compounds 2±17 and 19
Calculated Found
Comp. C % H % N % C % H % N %
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
19
68.00 7.08
58.41 6.54
71.57 7.31
58.65 6.15
72.06 7.56
59.40 6.38
68.72 7.02
56.80 5.96
73.01 6.97
61.85 6.06
67.65 6.69
68.15 6.90
68.61 7.10
57.80 5.85
58.44 6.05
59.04 6.23
81.15 6.81
3.17
2.84
3.63
2.85
3.50
2.77
3.48
2.76
5.87
4.81
5.64
5.48
5.33
4.65
4.54
4.44
67.83 7.31
58.20 6.32
71.81 7.52
58.93 5.90
72.33 7.89
59.71 6.05
68.45 7.38
57.03 5.81
73.31 6.72
62.13 5.84
67.31 6.40
68.41 7.15
68.87 6.85
58.08 5.66
58.71 5.88
58.77 6.51
81.40 6.58
3.44
3.07
3.38
3.05
3.30
2.61
3.59
2.97
5.63
4.67
5.31
5.67
5.07
4.37
4.85
4.16
The oily product was transformed into the hydrochlo-
ride and crystallised from abs. EtOH and an. ether. Mp
149±151 ꢀC. Anal. (C28H33ClN2O2S) C, H, N.
3-(4-Phenylpyperazinyl)propyl 2,2-diphenyl-2-ethylthio
acetate (13) and 4-(4-phenylpyperazinyl)butyl 2,2-diphe-
nyl-2-Ethylthio acetate (14). These were obtained in the
same way from 2,2-diphenyl-2-ethylthio acetyl chloride
and the proper alcohols 21 and 224 and transformed
into the corresponding hydrochloride. Their chemical
and physical characteristics are reported in Table 2.
1
Their IR and H NMR spectra are consistent with the
proposed structures.
Contractions were recorded by means of a force dis-
placement transducer connected to the MacLab system
PowerLab/800 and to a two-channel Gemini polygraph
(U. Basile).
2-(4-Phenylpyperazinyl)ethyl 2,2-diphenyl-2-ethylthio ace-
tate methyliodide (15). An excess (2 mL) of CH3I was
added to 2 mmol of the free base of 12 in 20 mL of
anhydrous ether and the solution was left at room tem-
perature overnight in the dark. The white solid was ®l-
tered and crystallised from abs. ethanol. Yield 90%. Mp
In all cases, parallel experiments in which tissues
received only the reference agonist were run in order to
check any variation in sensitivity.
191±193 ꢀC. IR (nujol) n 1740 (CO) cmÀ1. H NMR
Guinea-pig ileum. Two-cm-long portions of terminal
ileum were taken at about 5 cm from the ileum±cecum
junction and mounted in PSS, at 37 ꢀC, with the fol-
lowing composition (mM): NaCl 118, NaHCO3 23.8,
KCl 4.7, MgSO4.7H2O 1.18, KH2PO4 1.18, CaCl2 2.52,
and glucose 11.7. Tension changes were recorded iso-
tonically. Tissues were equilibrated for 30 min and dose±
response curves for arecaidine propargyl ester (APE)
were obtained at 30 min intervals, the ®rst one being
discarded and the second one being taken as the control.
1
(CDCl3): d 1.10 (t, 3H, SCH2CH3); 2.32 (q, 2H, SCH
CH3); 3.15±3.26 (m, 4H, piperazine protons); 3.32 (s,
3H, +NCH3); 3.42±3.52 (m, 4H, piperazine protons);
4.22±5.00 (m, 2H, CH2CH2O); 4.70±4.78 (m, 2H,
CH2CH2O); 6.82 (d, 2H, aromatics); 6.98±7.18 (m, 1H,
aromatic); 7.29±7.49 (m, 12H, aromatics) ppm. Anal.
(C29H35IN2O2S) C, H, N.
Compounds 16 and 17 were obtained in the same way
starting from 13 and 14, respectively. Their chemical
and physical characteristics are reported in Table 2.
Guinea-pig stimulated left atria. The heart was rapidly
removed, and the right and left atria were separately
excised. Left atria were mounted in PSS (the same used
for ileum ) at 30 ꢀC and stimulated through platinum
electrodes by square-wave pulses (1 ms, 1 Hz, 5±10 V)
(Tetra Stimulus, N. Zagnoni). Inotropic activity was
recorded isometrically. Tissues were equilibrated for 2 h
and a cumulative dose±response curve using APE was
constructed.
1
Their IR and H NMR spectra are consistent with the
proposed structures.
Pharmacology
Functional in vitro tests
General considerations. Male guinea pigs (200±300 g)
were killed by cervical dislocation. The organs required

S. Scapecchi et al. / Bioorg. Med. Chem. 9 (2001) 1165±1174
1173
Right atria. Right atria were equilibrated for 2 h at the
above conditions (see preceding paragraph for PSS and
temperature). Contractions were recorded isometrically.
Binding experiments
Rat cerebral cortex. The muscarinic receptors were
labelled with 0.1 nM [3H]N-methyl scopolamine
([3H]NMS, 79.5 Ci/mmol from NEN-Dupont). The
frozen pellet was suspended in Krebs-Hepes bu€er pH
7.4 (composition, mM: NaCl 118, KCl 4.7, NaHCO3 5,
KH2PO4 1.2, glucose 11, MgSO4 1.2, CaCl2 2.5 and
Hepes 20), at 4 ꢀC, as previously described in Freedman
et al.7 Aliquots of cerebral membranes at a protein
concentration of 100±150 mg/mL were incubated at
30 ꢀC for 60 min in a ®nal volume of 1 mL with the
marker ligand and with di€erent concentrations of
unlabeled ligands (0.1 nM±0.1 mM); the incubation was
terminated by ®ltration through Whatman GF/B glass
®ber ®lters presoaked in 0.1% polyethyleneimine (PEI),
using a Brandel M-48R 48 well cell harvester. Filters
were washed twice with 10 mL of ice-cold 0.9% saline
solution.
Guinea-pig lung strips. The lungs were rapidly
removed and strips of peripheral lung tissue were cut
either from the body of a lower lobe with the long-
itudinal axis of the strip parallel to the bronchus or
from the peripheral margin of the lobe.23 The prepara-
tions were mounted, with a preload of 0.3 g, in PSS with
the following composition (mM): NaCl (118.78), KCl
(4.32), CaCl2.2H2O (2.52), MgSO4.7H2O (1.18),
KH2PO4 (1.28), NaHCO3 (25), glucose (5.55). Contrac-
tions were recorded isotonically at 37 ꢀC after tissues
were equilibrated for 1 h, then two cumulative dose±
response curves using APE (0.01, 0.1, 1, 10, 100 mM)
were obtained at 45 min intervals, the ®rst one being
discarded and the second one being taken as the control.
3
Rabbit stimulated vas deferens. This preparation was
set up according to Eltze.24 Vasa deferentia were care-
fully dissected free of surrounding tissue and were divi-
ded into four segments, two prostatic portions of 1 cm
and two epididymal portions approximately 1.5 cm
long. The four segments were mounted in PSS with the
following composition (mM): NaCl (118.4), KCl (4.7),
CaCl2 (2.52), MgCl2 (0.6), KH2PO4 (1.18), NaHCO3
(25), glucose (11.1); 10À6 M yohimbine and 10À8 M tri-
pitramine were included to block alpha2-adrenoceptors
and M2 muscarinic receptors, respectively. The solution
was maintained at 30 ꢀC and tissues were stimulated
through platinum electrodes by square-wave pulses (0.1
ms, 2 Hz, 10±15 V). Contractions were measured iso-
metrically after tissues were equilibrated for 1 h, then a
cumulative dose±response curve using pCl-McN-A-343
was constructed.
For [H]oxotremorine (3[H]-OXO, 87.5 Ci/mmol from
NEN-DuPont) binding studies, frozen pellet was
washed by suspending in Hepes bu€er, pH=7.4, and
centrifuged for 15 min at 17,000g as described by
Freedman et al.7 Incubations were initiated by adding
100 mL of membrane suspension (1±1.5 mg/mL) with
3
0.3 nM [H]-OXO and di€erent concentrations of cold
ligands (0.1 nM±0.1 mM) at 30 ꢀC for 40 min in a ®nal
volume of 1 mL. After incubation, membranes were ®l-
tered through Whatman GF/C ®lters strips presoaked
in 0.1% PEI solution; the ®lters were rinsed with 10 mL
of ice-cold saline solution. Protein determinations were
performed by the method of Bradford26 using bovine
serum albumine as the standard.
Domestic pig ventricles. [3H]NMS (0.2 nM) and [H]-
3
OXO (0.3 nM) were carried out using thawed pellet
suspended in Krebs±Hepes and in Hepes bu€er respec-
tively. Incubation mixtures (1 mL) contained 150±300
mg of membranes in [3H]NMS competition experiments,
Statistical analysis. The results are expressed as the
mean+SEM. Student's t-test was used to assess the
statistical signi®cance of the di€erence between two means.
3
while with [H]-OXO the amount of membranes was
600±800 mg; mixtures were incubated as previously
described. Protein concentration was estimated using
the Pierce protein assay reagent (Pierce Chemical Co.,
Rockford, IL) based on the method of Bradford26 with
bovine serum albumine as standard.
Binding tests
Membrane preparation
Rat cerebral cortex. Male rats (Wistar, 200 g) were
killed by decapitation and the brains were removed and
immediately placed on ice. The brain was dissected,
homogenised in 10 vol 0.32 M sucrose with a glass-
te¯on homogeniser. The homogenate was then centri-
fuged at 1000g for 15 min; the resulting supernatant was
further centrifuged at 17,000g for 20 min and the pellet
was stored at À80 ꢀC until assayed.7
In all experiments, the radioactivity retained by ®lters
was measured in a liquid scintillation counter (TRI-
CARB 1900TR, Packard) after the addition of 4 mL of
scintillation ¯uid (Filter Count, Packard) and all mea-
surements were obtained in duplicate.
The binding data were evaluated quantitatively with
non-linear least-square curve ®ttings using the computer
programs ALLFIT27 and LIGAND.28
Domestic pig ventricles. Preparation of porcine heart
homogenates was carried out as described by Burgmer et
al.25 Brie¯y, porcine hearts were obtained from the local
slaughterhouse and stored at À80 ꢀC. Thawed ventri-
cular tissue (40 g) was minced and homogenised in 20 vol
of 0.32 M sucrose solution with the aid of Ultra-Turrax
T25 IKA and then with a motor driven glass-te¯on
homogeniser. The homogenate was then centrifuged for
11 min at 300g and the resulting supernatant centrifuged
for 41 min at 80,000g; the pellet was stored at À80 ꢀC.
Acknowledgements
This research was ®nanced with funds from the Italian
Ministry of University and Scienti®c and Technological
Research (MURST).

1174
S. Scapecchi et al. / Bioorg. Med. Chem. 9 (2001) 1165±1174
13. Tecle, H.; Barret, S. D.; Lau€er, D. J.; Augelli-Szafran,
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