Further structure-activity relationships in the series of tropanyl esters endowed with potent antinociceptive activity

Article abstract of DOI:10.1016/S0014-827X(98)00106-2

Several analogs of the α-tropanyl esters of 2-(4-chlorophenoxy)butyric acid (SM21) and 2-phenylthiobutyric acid (SM32), endowed with potent antinociceptive and cognition enhancing activity, were synthesized, aimed at obtaining more potent and safe drug candidates. Variation of the acyl moiety, as well as the conformational restriction of atropine to give the α-tropanyl ester of 2,3-dihydrobenzofurane-3-carboxylic acid (18), practically abolished activity. In the case of 18, the antimuscarinic activity was also severely affected by the conformation restrain. On the contrary, conformational restriction of phenoxybutyric and phenylthiobutyric acid derivatives to give the α-tropanyl ester of 2,3-dihydro-benzofurane-2-carboxylic acid and 2,3-dihydro-benzothiophene-2-carboxylic acid, afforded potent analgesic drugs that unfortunately were too toxic to be reliable drug candidates. A series of related esters of benzofurane-3-carboxylic acid and benzothiophene-3-carboxylic acid were also studied and found to be potent but toxic analgesics.

Full text of DOI:10.1016/S0014-827X(98)00106-2

Il Farmaco 53 (1998) 764–772
Further structure–activity relationships in the series of tropanyl
esters endowed with potent antinociceptive activity
Serena Scapecchi a,*, Antonella Giorgi a, Cristina Bellucci a, Silvia Dei a,
Carla Ghelardini b, Dina Manetti a, M. Novella Romanelli a, Elisabetta Teodori a
a Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Firenze, Via G. Capponi 9, 50121 Florence, Italy
b Dipartimento di Farmacologia Preclinica e Clinica, Uni6ersita` di Firenze, Viale Morgagni 65, 50134 Florence, Italy
Received 7 January 1998; accepted 30 November 1998
Abstract
Several analogs of the a-tropanyl esters of 2-(4-chlorophenoxy)butyric acid (SM21) and 2-phenylthiobutyric acid (SM32),
endowed with potent antinociceptive and cognition enhancing activity, were synthesized, aimed at obtaining more potent and safe
drug candidates. Variation of the acyl moiety (4–11), as well as the conformational restriction of atropine to give the a-tropanyl
ester of 2,3-dihydrobenzofurane-3-carboxylic acid (18), practically abolished activity. In the case of 18, the antimuscarinic activity
was also severely affected by the conformation restrain. On the contrary, conformational restriction of phenoxybutyric and
phenylthiobutyric acid derivatives to give the a-tropanyl ester of 2,3-dihydro-benzofurane-2-carboxylic acid and 2,3-dihydro-ben-
zothiophene-2-carboxylic acid (12–17), afforded potent analgesic drugs that unfortunately were too toxic to be reliable drug
candidates. A series of related esters of benzofurane-3-carboxylic acid (20–27) and benzothiophene-3-carboxylic acid (28) were
also studied and found to be potent but toxic analgesics. © 1998 Elsevier Science S.A. All rights reserved.
Keywords: Tropanyl esters; Antinociceptive activity; Structure–activity relationships
tivity relationships [6], particularly in the series of 2-
phenoxy- (2) and 2-phenylthio-butyric acid esters (3); in
1. Introduction
this paper we also comment on the results obtained.
In the past few years we have developed a new series
of compounds derived from R-(+)-hyoscyamine that
are endowed with strong antinociceptive activity and
which also show cognition enhancing properties, possi-
bly due to their cholinergic mechanism of action [1–5].
As a matter of fact, these compounds, as it has been
shown by microdialysis studies [3,4], enhance the cen-
tral release of acetylcholine which in turn is responsible
of antinociceptive (hot plate) and cognition enhancing
(passive avoidance) activities. The mechanism by which
acetylcholine is released is still unclear even if presynap-
tic muscarinic and 5-HT4 receptors seem to be involved
[3,4].
The first variation in the structure concerned the
nature of the acyl group. It is known that the hypolipi-
daemic compounds related to clofibrate have a definite
hepatic toxicity in the rat [7]. Since SM21, on pro-
longed treatment, also presents this problem, it is possi-
ble that part of the toxicity of the compound could be
connected with the 4-chloro-phenoxyalkanoic moiety.
We have therefore designed and synthesized a series of
Three compounds, labeled PG9 (1), SM21 (2), and
SM32 (3), respectively, have emerged from these re-
searches and are now undergoing preclinical studies. In
the meantime we have continued to study structure–ac-
* Corresponding author.
0014-827X/98/$ - see front matter © 1998 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(98)00106-2

S. Scapecchi et al. / Il Farmaco 53 (1998) 764–772
765
lower analgesic activity [2]. It is known that restriction
of conformational freedom can induce receptor selectiv-
ity in flexible drugs acting with multiple mechanisms of
action [8]; since the release of acetylcholine of 1–3
apparently involves both muscarinic and serotonergic
receptors [3,4], we reasoned that this modification could
induce selectivity for only one of the receptors involved,
thus reducing unwanted side-effects. This was also the
rationale for the synthesis of compounds 18 and 19,
which are positional isomers of compounds 12 and 16
and can be regarded as a frozen analog (18) and an
isoster thereof (19) of atropine (the racemic form of
R-(+)-hyoscyamine).
Finally, compounds 20–28 were synthesized as aro-
matic analogs of 18 and 19 on the basis of the potent
antinociceptive activity of the previously studied com-
pound 20, which seems also to act by the same acetyl-
choline-releasing mechanism [9].
2. Chemistry
The synthesis of compounds 4–11 was performed by
standard methods according to Scheme 1; most of the
compounds were obtained through the key intermediate
29, which had been already used by us [2] but not
characterized.
The synthesis of compounds 12–17 is reported in
Scheme 2. Benzofurane-2-carboxylic acid is commer-
cially available; the other aromatic acids used as start-
ing materials were obtained as described in the
literature: 5-chlorobenzofurane-2-carboxylic acid [10];
3-methylbenzofurane-2-carboxylic acid [11]; 3-methyl-5-
chlorobenzofurane-2-carboxylic acid [12]; benzothio-
phene-2-carboxylic acid [13]; 3-methylbenzothiophene-
2-carboxylic acid [14]. The aromatic acids were reduced
to the corresponding 2,3-dihydro derivatives with Na/
Hg. 2,3-Dihydrobenzofurane-2-carboxylic acid [15],
2,3-dihydro-5-chlorobenzofurane-2-carboxylic acid [16]
analogs lacking this group and presenting isosteric or
bioisosteric alkyl, cycloalkyl, alkenyl, alkynyl and hete-
rocyclic moieties (general structure A; compounds 4–
11).
The second variation regarded the reduction of flexi-
bility of the parent compounds to give the unsubsti-
tuted dihydrobenzofurane and dihydrobenzothiophene
derivatives 15 and 17. The other compounds with gen-
eral structure B (12,13,14,16) correspond to frozen
analogs of compounds related to 2 and 3, but with
Scheme 1. (a) Anhydrous C6H6, reflux; (b) NaOH, C2H5OH, R–XH; (c) NaOH, C2H5OH; (d) CDI, a-tropanol.

766
S. Scapecchi et al. / Il Farmaco 53 (1998) 764–772
Scheme 2. (a) Na/Hg; (b) SOCl2; (c) a-tropanol.
and 2,3-dihydrobenzothiophene-2-carboxylic acid [17]
have already been described. Their 3-methyl homologs
(30–32) are described here for the first time.
The final esters 12–17 were obtained by standard
esterification with a-tropanol; compounds 15 and 17
(Scheme 2, R1=CH3) are mixtures of cis/trans isomers
that have not been separated in consideration of the
unsatisfactory biological results obtained.
Compounds 18 and 19 and 20–28 were obtained
according to Scheme 3, starting from benzofurane-3-
carboxylic acid [18], benzothiophene-3-carboxylic acid
[19], 2,3-dihydrobenzofurane-3-carboxylic acid [20] and
2,3-dihydrobenzothiophene-3-carboxylic acid [17], re-
spectively. Even in this case, due to the poor pharmaco-
logical profile of the compounds, no attempts were
made to separate the enantiomers of 18 and 19.
toxicity of the compounds, ED50 values were not deter-
mined, except for the most efficacious ones and for
comparative purposes. Analgesic efficacy was evaluated
with respect to the effect of 8 mg/kg of morphine.
Whenever available the minimal dose giving evident
toxic effects (convulsions and death) was reported.
Muscarinic antagonism of 18 was evaluated on rabbit
vas deferens (M1), guinea pig atrium (M2), guinea pig
ileum (M3) and guinea pig immature uterus (putative
M4) tissues, using McN-A-343, charbacol and ACh as
agonists, as reported in Section 5. The results are
reported in Table 2 and are expressed as Schild’s pA2
[22] for the reference compound atropine and as pKb
(−log Kb), calculated according to Van Rossum [23],
for 18. Each concentration was tested between four and
seven times. No reduction in the maximum effect was
observed at the doses used.
Although the active compounds would very likely
show the non-atropic action of their parent com-
pounds, considering their toxicity, no experiments to
evaluate cognitive effects were made.
3. Pharmacology
The analgesic activity of the compounds was tested
on mice, subcutaneously (s.c.) or intraperitoneally (i.p.),
with the hot-plate test [21], using the previously re-
ported methods [1]. The results are reported in Table 1
and are expressed as the dose (mg/kg) giving the maxi-
mal analgesic effect; as a rule, because of the high
4. Results and discussion
The data regarding analgesic activity, reported in
Table 1, show that the modifications introduced into
the parent molecules have different consequences on
their activity.
The first modification, concerning the acyl moiety,
was unsuccessful as, among the compounds synthesized
and studied (4–11), only 10 and 11 possess a weak
analgesic activity which, however, is not reversed by
atropine and therefore is of non-cholinergic nature.
Conformationally restricted analogs gave mixed re-
sults. The frozen analog of atropine (18) is somehow
more efficacious, but definitely less potent than atropine
(Tables 1 and 2). It is interesting to observe that
conformational restriction has major consequences also
on the antimuscarinic activity of atropine, as 18 is some
100 times less potent on the tissues studied (Table 2).
Its isoster 19 is fairly efficacious, much less potent, and
highly toxic, showing a trend that unfortunately is
characteristic of most of the compounds studied.
Scheme 3. (a) Na/Hg; (b) SOCl2; (c) aminoalcohol of Table 7; (d)
a-tropanol.

S. Scapecchi et al. / Il Farmaco 53 (1998) 764–772
767
Table 1
Analgesic activity of compounds 2,3 and 10–28 on the hot-plate test (mouse)
Compound
Analgesic activity
Maximal effect dose (MED)a
(mg/kg, i.p.)
ED50 (9SE)
(mg/kg, i.p.)
Efficacyb (%)
Maximal non-toxic dosec
(mg/kg, i.p.)
2 (SM21)
3 (SM32)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
40
40
50d
50d
50
50
50
50e
25
30f
25
50
50
40
50
50
50
50
50
50
50
21.2 (1.8)
16.6 (1.6)
–
–
–
–
–
17.6 (1.3)
11.1 (2.5)
14.3 (1.9)
–
34.2 (3.8)
26.3 (2.7)
–
–
114.4
107.5
–
–
–
400
380
–
–
–
–
–
80
48.4
78.7
79.7
100.9
73
111.9
129.5
75.3
48.4
83.6
59.4
48.4
47.9
21.0
132.6
100
40
40f
40
80
100
50
n.d.
70
B75
n.d.
n.d.
\200
100
–
–
–
–
25
26
27
28
–
32.7 (2.6)
a The maximal dose reported was limited to 50 mg/kg, even if some compounds (12,13) show some activity at higher doses.
b Efficacy is expressed as % of the effect of MED respect to that of a dose of 8 mg/kg of morphine (i.p.).
c The toxic dose is that at which the animals present convulsions and death. Even in the cases where the maximal non-toxic dose has not been
evaluated (n.d.) the compounds show toxicity at doses very close to those tested.
d The analgesic effect is not atropine sensitive and for this reason the compound has not been further studied.
e The analgesic effect produced by this dose is almost completely reversed by SDZ-205557.
f The compound was tested subcutaneously (s.c.).
The frozen analogs of SM21 (15) and SM32 (17),
which have been studied in more detail, are practically
equiactive with their parent compounds, but their acute
toxicity is much higher (Table 1). The same happens for
the analog 16, while 12, 13, 14 are practically inactive at
the dose tested.
Among the analogs of 20, which have already been
studied by us [9], only the sulfur isoster 28 is interest-
ing, since it shows a remarkable efficacy as analgesic.
We have previously shown that the antinociceptive
activity of 20, despite its structural similarity with 5-
HT3 antagonists, is not due to interaction with this kind
of receptors but, more likely, to activation of central
5-HT4 receptors. It is possible that its isoster 28 behaves
in the same way. However, due to its high toxicity, that
would prevent any useful use as analgesic, we did not
investigate any further its mechanism of action.
In conclusion, it appears that reduction of molecular
flexibility of the lead compounds maintains or slightly
reduces analgesic activity but, at the same time, in-
creases their toxicity. During our researches in this field
we have noticed [9] that toxicity somehow parallels
5-HT4 affinity. As a matter of fact, the analgesic activ-
ity of 15 at the maximal effect dose (MED=50 mg/kg)
is almost completely reversed (66%) by SDZ-205557, a
5-HT4 antagonist. The reversal of the analgesic activity
of SM21 (2) was, under the same conditions, quite
lower (44%) [3]. We may conclude that conformational
restriction of atropine, SM21 and SM32, apparently
and contrary to our expectations, favors interaction
with the 5-HT4, respect to presynaptic muscarinic
receptors.
5. Experimental
5.1. Chemistry
All melting points were taken on a Bu¨chi 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
column by gravity chromatography (Kieselgel 40,
0.063–0.200 mm, Merck) or flash chromatography
(Kieselgel 40, 0.040–0.063 mm, Merck). Yields are
given after purification, unless otherwise stated. Where

768
S. Scapecchi et al. / Il Farmaco 53 (1998) 764–772
Table 2
Antimuscarinic and analgesic activity of atropine and 18
Comp.
pKb (9SE)
Analgesic activity
a
b
c
M1
M2
M3
Mud
MEDe (mg/kg)
Efficacyf (%)
Atropine
18
9.46g
8.92g
9.00g
9.50g
0.005
25
42
73
7.43h (0.03)
6.55h (0.01)
6.57h (0.09)
7.25i (0.02)
a Rabbit vas deferens.
b Guinea pig atrium (force).
c Guinea pig ileum.
d Guinea pig uterus.
e Maximal effect dose (i.p.).
f Effect of the maximal effect dose compared to that of 8 mg/kg of morphine.
g pA2 calculated according to Arunlakshana and Schild [22].
h 10−6 M.
i 3×10−7 M.
analyses are indicated by symbols, the analytical re-
sults are within 90.4% of the theoretical values.
Table 3 shows the elemental analyses for compounds
4–32.
the eluent, yielding the desired product, that was trans-
formed into the salt reported in Table 4.
5.4. Synthesis of the esters 5 and 4
5.2. 3-h-Tropanyl 2-bromopropionate (29)
A total of 2 g (20 mmol) of cyclohexanol was added
to a solution of 20 mmol of NaOH in 15 ml of abs.
ethanol; 3.2 g (30 mmol) of 2-chloropropionic acid were
then added to this solution and the mixture refluxed for
4 h and evaporated to dryness. The residue was dis-
A 1.5 ml sample of 2-bromopropionyl bromide was
added to a solution of a-tropanol (2 g) in 20 ml of
anhydrous benzene, and the solution refluxed for 13 h.
The resulting mixture was cooled and evaporated to
dryness. The residue was dissolved in CH2Cl2, and the
organic layer shaken with a saturated solution of
Na2CO3, washed with water and dried. Evaporation of
the solvent gave a white solid (3.6 g), m.p. 178–180°C.
1H NMR (CDCl3) l 1.82 (d, J=6.1 Hz, 3H, CHCH3);
1.85–1.95 (m, 2H, tropanol); 2.08–2.22 (m, 4H,
tropanol); 2.49 (s, 3H, N–CH3); 3.34–3.41 (m, 2H, CH
bridge); 4.36 (q, J=6.1 Hz, 1H, CHCH3); 5.10 (t, 1H,
CH–O) ppm.
Table 3
Elemental analyses of compounds 4–19 and 21–32
Comp.
Calculated (%)
Found (%)
C
H
N
C
H
N
4
5
6
7
8
59.44
59.19
59.19
59.50
59.50
56.82
54.52
51.36
55.40
61.36
56.40
57.99
58.94
63.04
60.07
64.37
69.42
62.43
60.90
62.02
60.90
60.49
60.42
67.40
56.48
61.82
8.53
8.12
8.12
7.64
7.64
6.37
6.11
5.84
5.39
6.45
5.69
5.90
6.20
6.86
6.54
6.62
6.09
5.91
6.15
6.52
6.15
6.78
5.98
5.67
4.27
5.20
4.08
59.25
58.80
59.41
59.26
59.87
57.10
54.36
51.05
55.18
61.22
56.74
58.23
59.18
62.85
59.81
64.51
69.11
62.71
61.21
61.78
61.18
60.80
60.71
67.21
56.91
62.18
8.86
8.27
7.80
7.91
7.31
6.05
6.25
6.03
5.31
5.91
5.37
5.61
5.92
7.05
6.81
6.85
5.81
6.07
5.89
6.79
5.88
6.55
6.21
5.81
4.01
4.85
3.89
3.63
3.63
3.65
3.65
7.37
7.07
10.57
3.40
3.58
3.29
3.56
3.44
4.33
4.12
4.17
3.52
4.55
4.74
4.52
4.74
4.70
4.15
–
3.85
3.91
3.87
3.95
7.59
7.33
10.23
3.72
3.28
3.51
3.72
3.71
4.02
3.95
3.88
3.77
4.27
4.98
4.71
4.51
4.91
3.83
–
9
5.3. General method for the synthesis of the esters
6–11
10
11
13
14
15
16
17
18
19
21
22
23
24
25
26
27
28
30
31
32
A solution of 2.5 mmol of the appropriate commer-
cial alcohol or thioalcohol in 2 ml of absolute (abs.)
ethanol were added to a solution of 2.5 mmol of NaOH
in 2 ml of abs. ethanol. The mixture was refluxed for
0.5 h and then evaporated to dryness. To the white
residue dissolved in anhydrous DMF a solution of 29
(0.5 g, 1.8 mmol) dissolved in anhydrous DMF was
added. The mixture was refluxed for 18–30 h, depend-
ing from the reagent used. In every case, the solvent
was removed at the end of the reaction and the residue
dissolved in CHCl3 and washed with a saturated solu-
tion of Na2CO3. Evaporation of the solvent gave an oil
that was purified by flash chromatography with
CHCl3:petrol ether:abs. EtOH:conc. NH4OH=
340:60:65:8 (6–9) or CHCl3:MeOH=75:25 (10–11) as
–
–
–
–

S. Scapecchi et al. / Il Farmaco 53 (1998) 764–772
769
Table 4
and physical characteristics of these compounds are
reported in Table 4.
Chemical and physical characteristics of compounds 4–11
5.5. General method for the synthesis of esters 13–28
A 0.5 mmol portion of the appropriate carboxylic
acid, synthesized as reported below or in the literature,
was transformed into the chloride by reaction with
SOCl2 (1 ml) at 60°C for 5 h, and purified by fractional
distillation. Acyl chlorides were then added to a solution
of a-tropanol (0.16 g, 1.1 mmol) in 5 ml anhydrous
benzene and the solution refluxed for 12 h. The solvent
was removed at the end of the reaction and the residue
dissolved in CHCl3, washed with NaHCO3 sat. solution
and dried over Na2SO4. Evaporation of the solvent gave
a residue that was transformed into the salts reported in
Tables 5–7.
No.
R
X
O
M.p. (°C)a Formulab (salt)
c
5
C17H29NO3 · C2H2O4
4
6
CH2 158–160
C15H27NO2 · C2H2O4
C17H29NO3 · C2H2O4
O
68–71
7
8
O
O
90–91
78–81
C17H27NO3 · C2H2O4
C17H27NO3 · C2H2O4
5.6. 2,3-Dihydro-3-methylbenzofurane-2-carboxylic
acid (30)
9
O
S
140–143
142–144
138–140
C16H22N2O3 · C2H2O4
C16H22N2O2S · C2H2O4
C15H21N3O2S · C2H2O4
A 0.2 g (0.9 mmol) sample of 3-methylbenzofurane-2-
carboxylic acid [12] was suspended in a solution of 15 ml
of water containing 0.3 g of KOH. 1 g of Na/Hg 5%
amalgam was then added, the mixture kept at r.t. for 4
h and then filtered. The yellow solution obtained was
acidified giving 30 as a white solid (150 mg of a 1:2
diastereomeric mixture; m.p. 145–147°C from water).
1H NMR (DMSO) l 1.32 (d, J=6.5 Hz, 3H, CH3) and
1.51 (d, J=6.5 Hz, 3H, CH3) in a 1:2 ratio; 3.60–3.88
(m, 1H, CH–CH3); 4.55 (d, J=6.3 Hz, 1H, CH–O)
10
11
S
a All compounds are oxalic acid salts and were crystallized from
ethylacetate.
b The compounds were analyzed after vacuum drying for 5 h at a
temperature below the melting point. The analytical results were
within 90.4 of the theoretical values. Their IR and proton NMR are
consistent with the proposed structure.
Table 5
Chemical and physical characteristics of compounds 12–17
c The compound melts at r.t.
solved in CHCl3 and washed with 2 N HCl. Evaporation
of the solvent gave 1.8 g of 2-cyclohexyloxypropionic
acid that was used as such in the following reaction.
A 0.48 g (3 mmol) sample of 1,1%-carbonyldiimida-
zole (CDI) was dissolved in anhydrous CH2Cl2, 0.5 g (3
mmol) of 2-cyclohexyloxypropionic acid was added,
and the mixture was kept at room temperature (r.t.) for
2 h. Evaporation of the solvent gave a residue that was
dissolved in 5 ml of anhydrous acetone and added to a
solution of 0.45 g (3.2 mmol) of a-tropanol in anhy-
drous acetone. The mixture was refluxed for 22 h and
evaporated to dryness. The residue was dissolved in
CH2Cl2 and the organic layer washed with water and
Na2CO3 sat. solution. Evaporation of the solvent gave
an oil that was purified by flash chromatography using
CHCl3:petrol ether:abs. EtOH:conc. NH4OH=
340:60:65:8 as the eluent, yielding 0.45 g of 5.
No.
R1
R2
X
M.p. (°C)a
Formulab (salt)
12c
13
14
15
16
17
H
H
H
Cl
H
O
O
O
O
S
–
–
156–159
170–171
154–157
195–198
190–191
C17H20ClNO3 · C2H2O4
C18H23NO3 · C2H2O4
C18H22ClNO3 · C2H2O4
C17H21NO2S · C2H2O4
C18H23NO2S · C2H2O4
CH3
CH3 Cl
H
CH3
H
H
S
a All compounds are oxalic acid salts and were crystallized from
ethylacetate except 17 that was crystallized from abs. ethanol.
b The compounds were analyzed after vacuum drying for 5 h at a
temperature below the melting point. The analytical results were
within 90.4 of the theoretical values. Their IR and proton NMR
spectra are consistent with the proposed structures.
In the same way, starting from commercial 2-methyl-
hexanoic acid, compound 4 was prepared. The chemical
c See Ref. [9].

770
S. Scapecchi et al. / Il Farmaco 53 (1998) 764–772
Table 6
Table 7
Chemical and physical characteristics of compounds 18 and 19
Chemical and physical characteristics of compounds 20–28
No.
X
M.p. (°C)a
Formulab (salt)
No.
X
O
Y
M.p. (°C)a
–
Formulab (salt)
18
19
O
S
220–222
235 (dec.)
C17H21NO3 · HCl
C17H21NO2S · HCl
20c
a The compounds are hydrochlorides and were crystallized from
anhydrous ether/abs. ethanol.
21
22
O
O
201–204
226–228
C18H21NO3 · HCl
C23H23NO3 · HCl
b The compounds were analyzed after vacuum drying for 5 h at a
temperature below the melting point. The analytical results were
within 90.4 of the theoretical values. Their IR and proton NMR
spectra are consistent with the proposed structures.
23
O
209–211
C16H17NO3 · HCl
and 5.26 (d, J=6.3 Hz, 1H, CH–O); 6.82–7.20 (m, 4
H, aromatics); 10.20–10.65 (bs, 1H, OH) ppm. IR
(Nujol) w 3600–2600 (OH); 1740 (CO) cm−1. Anal.
C10H10O3 (C, H).
With the same procedure were also synthesized the
other dihydro derivatives; among them the following
have not been reported previously.
24
25
O
O
170–172
180–183
C15H17NO3 · HCl
C16H19NO3 · HCl
5.7. 2,3-Dihydro-3-methyl-5-chlorobenzofurane-2-
carboxylic acid (31)
26
27
O
O
194–196
C15H17NO3 · HCl
C15H19NO3 · HCl
Starting from 3-methyl-5-chlorobenzofurane-2-car-
boxylic acid [12] (1:3 diastereomeric mixture; m.p. 145–
147°C from water).1H NMR (CDCl3) l 1.35 (d, J=6.3
Hz, 3H, CH3) and 1.51 (d, J=6.3 Hz, 3H, CH3) in a
1:3 ratio; 3.60–3.85 (m, 1H, CH–CH3); 4.82 (d, J=6.6
Hz, 1H, CH–O) and 5.28 (d, J=11.9 Hz, 1H, CH–O);
7.41–7.65 (m, 3H, aromatics) ppm. IR (Nujol) w 3600–
2500 (OH); 1740 (CO) cm−1. Anal. C10H9ClO3 (C, H).
134 (dec.)
28
S
260 (dec.)
C17H19NO2S · HCl
a The compounds have been recrystallized from abs. ethanol/anhy-
drous ether.
5.8. 2,3-Dihydro-3-methylbenzothiophene-2-carboxylic
acid (32)
b The compounds were analyzed after vacuum drying for 5 h at a
temperature below the melting point. The analytical results were
within 90.4 of the theoretical values. Their IR and proton NMR are
consistent with the proposed structure.
Starting from 3-methylbenzothiophene-2-carboxylic
acid [14] (1:1 diastereomeric mixture; m.p. 120–122°C
c See Ref. [9].
1
from water). H NMR (CDCl3) l 1.35–1.52 (m, 3H,
CH3); 3.80–3.93 (m, 1H, CH–CH3); 4.05 (d, J=5.7
Hz, 1H, CH–O) and 4.47 (d, J=10.8 Hz, 1H, CH–O)
in a 1:1 ratio; 7.01–7.31 (m, 4H, aromatics); 10.10 (bs,
1H, COOH) ppm. IR (Nujol) w 3300–2500 (OH); 1720
(CO) cm−1. Anal. C10H10O2S (C, H).
of mice treated in each test varied from 8 to 20. The
level of analgesia reached was evaluated comparing the
analgesic effect of the maximal effect dose of each
compound to that of morphine, taken as the reference
compound, and injected at 8 mg/kg s.c., a dose that
does not alter animal behavior.
5.9. Pharmacology
Calculations were performed using the following for-
mula: analgesic efficacy of X expressed as percentage of
that of morphine · HCl (8 mg/kg s.c.)=(maximum re-
action time of X−pretest reaction of X)/(maximum
5.9.1. Analgesic acti6ity
The plate temperature was fixed at 52.590.1°C. An
arbitrary cutoff time of 45 s was adopted. The number

S. Scapecchi et al. / Il Farmaco 53 (1998) 764–772
771
reaction time of morphine−pretest reaction of
morphine)×100.
5.9.2.2. Guinea pig stimulated left atria. The heart was
rapidly removed and the left atria were excised and
mounted under 1 g of tension in PSS with the following
composition (mM): NaCl (137), KCl (2.7), CaCl2 (1.8),
MgCl2 (1.05), NaH2PO4 (0.42), NaHCO3 (11.9), glucose
(5.6). The solution was maintained at 30°C and stimu-
lated through platinum electrodes by square-wave
pulses (1 ms, 1 Hz, 4–10 V). Inotropic activity was
recorded isometrically. Tissues were equilibrated for 1 h
and a cumulative dose–response curve to cabachol was
plotted [25].
Standard errors (SEs) on the values expressed as
percentage were not evaluated. Original data however,
have been statistically processed by employing Dun-
nett’s two-tailed test in order to verify the significance
of the differences between the means shown by treated
mice at the maximum reaction time and the pretest
reaction time. Differences were considered statistically
significant when P50.05. Percent values were calcu-
lated only for those differences that were statistically
significant; in all other cases, the drugs were considered
inactive. Since the reaction time was measured with an
accuracy of 915%, the errors on the percent values
calculated through the formula reported above are in
the same range.
5.9.2.3. Guinea pig ileum. Portions of terminal ileum (2
cm) were removed at about 5 cm from the ileum–ce-
cum junction and mounted under 1 g of tension in PSS
(the same used for atria) at 37°C. Tension changes were
recorded isotonically. Tissues were equilibrated for 1 h
and a dose–response curve to acetylcholine was ob-
tained [26].
5.9.2. Antimuscarinic acti6ity
Male guinea pigs (200–300 g), female guinea pigs
(150–200 g) and male New Zealand white rabbits (2.5–
3 kg) were killed by cervical dislocation and the organs
were set up under the appropriate tension (see below) in
13 ml organ baths containing physiological salt solution
(PSS) kept at an appropriate temperature (see below)
and treated with 5% CO2–95% O2. Dose–response
curves were constructed by addition of the agonist
(cumulative curves in the case of rabbit vas deferens
and guinea pig atria).
The concentration of agonist in the organ bath was
increased approximately 3-fold each step, each addition
being made only after the response to the previous
addition had attained a maximal level and remained
steady. Tissues were incubated with the antagonist for 1
h and a new dose–response curve to the agonist was
obtained. Contractions were recorded by means of a
force transducer connected to a single channel recorder
(U. Basile).
5.9.2.4. Guinea pig uterus. Uterine horns were divided
into four portions and mounted under 1 g of tension in
PSS with the following composition (mM): NaCl (154),
KCl (5.63), CaCl2 (0.54), MgCl2 (0.95), NaHCO3 (5.95),
glucose (2.78). The preparations were maintained at
30°C and after a 1 h equilibration period, isotonic
contractions to carbachol were recorded. Initially the
tissues were exposed to a single concentration of carba-
chol (3 mM) to check the responsiveness to the agonist,
then a dose–response curve for carbachol was obtained
[27].
Acknowledgements
The authors would like to thank A. Bartolini and F.
Gualtieri who suggested and supervised this research
that was partially supported by Fidia s.p.a. (Abano
Terme) and Manetti & Roberts (Firenze).
5.9.2.1. Rabbit stimulated 6as deferens. Surrounding tis-
sues were carefully removed from vasa deferentia which
were then divided into four segments, two prostatic
portions of 1 cm and two epididymal portions approxi-
mately 1.5 cm long. The four segments were mounted
under 0.75 g tension in PSS with the following compo-
sition (mM): NaCl (118.4), KCl (4.7), CaCl2 (1.8),
MgCl2 (0.6), KH2PO4 (1.18), NaHCO3 (25), glucose
(11.1); 10−6 M yohimbine was included to block a2-
adrenoceptors. The solution was maintained at 32°C
and tissues were stimulated through platinum electrodes
by square-wave pulses (2 ms, 0.1 Hz, 10–30 V). Con-
tractions were measured isometrically after tissues had
been equilibrated for 1 h, then a cumulative dose–re-
sponse curve for the inhibitory effect of McN-A-343
was plotted [24].
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