Synthesis of N-substituted-N-acylthioureas of 4-substituted piperazines endowed with local anaesthetic, antihyperlipidemic, antiproliferative activities and antiarrythmic, analgesic, antiaggregating actions

Article abstract of DOI:10.1016/S0014-827X(03)00132-0

Three series of N-acyl and N-cyclohexyl- or N-methyl or N-phenyl-thioureas of 4-substituted (methyl, phenyl, 2-pyridyl)piperazines (4-12) were synthesised according to a highly convergent one-pot procedure and tested in vivo (local anaesthetic, anti-hyper

Full text of DOI:10.1016/S0014-827X(03)00132-0

Il Farmaco 58 (2003) 765Á780
/
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Synthesis of N-substituted-N-acylthioureas of 4-substituted
piperazines endowed with local anaesthetic, antihyperlipidemic,
antiproliferative activities and antiarrythmic, analgesic,
antiaggregating actions
Angelo Ranise a,*, Andrea Spallarossa a, Olga Bruno a, Silvia Schenone a, Paola Fossa a,
Giulia Menozzi a, Francesco Bondavalli a, Luisa Mosti a, Annalisa Capuano b,
Filomena Mazzeo b, Giuseppe Falcone b, Walter Filippelli b
a Dipartimento di Scienze Farmaceutiche, Facolta` di Farmacia, Universita´ degli Studi di Genova, Viale Benedetto XV 3, I-16132 Genova, Italy
b Dipartimento di Medicina Sperimentale-Sezione di Farmacologia ‘L. Donatelli’, II Universita` degli Studi, Facolta` di Medicina e Chirurgia,Via S.
Andrea delle Dame 8, I-80138 Napoli, Italy
Received 16 November 2002; accepted 18 January 2003
Abstract
Three series of N-acyl and N-cyclohexyl- or N-methyl or N-phenyl-thioureas of 4-substituted (methyl, phenyl, 2-
pyridyl)piperazines (4Á12) were synthesised according to a highly convergent one-pot procedure and tested in vivo (local
/
anaesthetic, anti-hyperlipoproteinemic, analgesic, anti-inflammatory, antiarrythmic activities) and in vitro (antiaggregating and, for
some selected derivatives, antiproliferative activities) experiments. All the test compounds showed local anaesthesia in particular
4Ar4, 5Ar4, 12Ar3 (after 5 min) and 5Ar2, 5Ar3, 9Ar4 (after 30 min) were equipotent to lidocaine. In lowering triglyceride levels,
compounds 6Ar4 and 7Ar3 were more active than nicotinic acid, whereas 7Ar4 and 11Ar4 were approximately equipotent. As
concerns analgesic activity, 5Ar2 and 5Ar4 were as active as indomethacin. Appreciable anti-inflammatory activity was found in
8Ar1, 5Ar2 and 11Ar2, but inferior to that of indomethacin. High levels of antiarrythmic activity, comparable with that of quinidine,
were found in derivatives 4Ar2 and 10Ar1. Compounds 4Ar2 and 8Ar2, assayed in antitumor in vitro screening system at National
Cancer Institute (NCI), showed significant antiproliferative activity against ACHN cell line (GI50: 0.13 mM) and NCI-H226 cell line
(GI50: 1.03 mM), respectively.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 3,3-Disustituted-1-acylthioureas;
N-substituted-N-acyl
4-substituted
piperazine-1-thiocarboxamides;
Antihyperlipidemic; Analgesic; Anti-inflammatory; Antiarrhythmic; Platelet antiaggregating and antiproliferative activities
Local
anaesthetic,
1. Introduction
N-substituted-N-acylthioureas of 4-methyl-, 4-phenyl-
and 4-(2-pyridyl) piperazine 4Á12, as new analogues of
/
In previous papers [1,2], 3,3-disubstituted 1-phe-
nyl(thio)ureas (A, B, Chart 1, embodying moieties of
pharmacologically useful secondary amines, were re-
ported to be endowed with various and interesting
pharmacological properties. In order to expand the
SAR studies on acylthioureas (ATUs) and confirm their
pharmacological potential, we designed and synthesised
A and B, incorporating three variable structural regions
(R, R1Á3, Ar1Á4, Chart 2). These portions were designed
with differing electronic, lipophilic and steric properties,
so that the products are featured by a common scaffold
with different shapes and different substitution patterns.
The title compounds were assayed in some in vivo and in
vitro tests. Additional goal of the present investigation is
to validate ATUs as sources of new hit/lead compounds,
exploiting an analogue design approach, even if there is
no mechanistic understanding of the target(s) at all.
* Corresponding author.
E-mail address: ranise@unige.it (A. Ranise).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00132-0

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A. Ranise et al. / Il Farmaco 58 (2003) 765Á780
/
sodium hydride), and, owing to the presence of the 4-
basic nitrogen, the primary reaction products were
hydrochlorides 3R1Á3Ar2Á4. Indeed, the use of bases is
not only unnecessary, but ineffective too, causing a
decrease of yields and tedious and time-consuming
work-up procedures. On the other hand, the isolation
of the hydrochlorides indicates that the 4-nitrogen atom
of the piperazine nucleus acts as an intramolecular
scavenger of hydrogen chloride, thus playing the same
role of tertiary base (pyridine). It is noteworthy that the
three synthetic variants are featured by high atom
economy, because either only one molecule of HCl is
formally lost in the whole process (Scheme 1b and c) or
all chemical features of the building blocks are incorpo-
rated into the products (Scheme 1a). Furthermore, no
intermediate needs to be isolated and in general the
overall yields are good to high. Owing the variety of the
chemically accessible building blocks (the employed
ones are commercially available), maximum need mole-
cular diversity can be obtained in the smallest number of
steps and under the mildest conditions possible. For
these reasons, the above procedures could be easily
adaptable to combinatorial synthesis of ATU libraries.
The reaction conditions of route (a) were somewhat
similar to acylation (acetylation/benzoylation) of
thiourea in aprotic medium, to afford N-acyl thiourea
via isolable S-acylisothiouronium salt intermediate [3],
which underwent sulphur-to-nitrogen acyl migration in
consequence of basic treatment (NaHCO3) or heating.
Chart 1. The shared structures of prieviously described acyl(thio)ureas
[1,2].
Chart 2. The variable structural regions of the title compounds for
SAR studies.
2. Chemistry
The methodology employed for the preparation of the
new ATUs is a highly convergent one-pot procedure
achieved through three steps by sequentially combining
three types of building blocks (amines, isothiocyanates,
acyl chlorides, respectively).
The synthesis of 7Á
according to the previously described synthetic variants
(Scheme 1, paths b and c) [1], whereas 4Á6 were
obtained (path a) in the absence of base (pyridine,
/
9 and 10Á
/
12 was accomplished
/
Scheme 1. One-pot synthesis of the title compounds.

A. Ranise et al. / Il Farmaco 58 (2003) 765Á
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767
Moreover, a similar intermediate was also proposed for
acylation of carbothioamides, since benzoylation of
thioformamide gives the corresponding isolable S-ben-
zoyl derivative [4]. Starting from these observations, we
hypothesised the following mechanism for formation of
mediate IV. This implies a syn configuration of IV, with
the nitrogen lone pair in a proper steric alignment for
attack upon the acyl carbonyl with a request of a major
activation entropy. A further example of this sort of acyl
cationotropy in thiourea derivatives, is given by nitro-
gen-to-nitrogen acyl migration, as proved by some of us
[8]. Therefore, with the aim to verify existence of III, we
isolated the primary reaction products as salts, before
basic treatment to avoid any possible acyl transposition.
Actually, all these products were soluble in water and
two of them, being deliquescent, could not be charac-
terised (i.e. 3R2Ar3 and 3R2Ar4, see Section 4). Their
saline nature were also confirmed by the IR spectra,
which exhibit typical absorption bands corresponding to
ATUs 4Á6 (Scheme 2). Preliminarily, the formation of
/
complexes II might occur between intermediate Ia and
acyl chlorides, since it is well known that bases (as
pyridine and triethylamine) having tertiary nitrogen
form reversible complexes with acyl chlorides [5], at
the same time enhancing reactivity of the latter. It is
noteworthy that 3,3-disubstituted thioureas not having
basic nitrogens cannot be acylated under base-free
conditions and the slow release of hydrogen chloride
from unreacted acyl reagents decomposes the key
thiourea intermediate, yielding starting materials
(amines and isothiocyanates) [6] due to reversibility of
the reaction [7]. After this initial step, the sulphur atom
would undergo a kinetically-controlled acyl attack,
yielding resonance-stabilised isothiouronium salts III.
Then, as reported above for simple thiourea [3], a
subsequent in situ basic treatment should cause a
the quaternary ammonium groups in the range of 2720Á
/
2100 cmꢀ1. Nevertheless, in order to make possible
clear assignment of the acyl group, and in this way to
confirm either the S-acyl or N-acyl structure (III or 3,
respectively), we achieved the 13C NMR spectrum of
one (R1Á3: C6H5, Ar1Á4: 2-thienyl) of these salts,
assumed as a prototype. The presence of the thione
group at d 185.30 excludes the structure of type III, and
therefore salts 3 possess the N-acyl structure. As a
consequence, the subsequent treatment with sodium
thermodynamically-assisted S0
/
N acyl transposition
through a transient four-centre Chapman-type inter-
Scheme 2. The proposed mechanism for formation of hydrochlorides 3.

768
A. Ranise et al. / Il Farmaco 58 (2003) 765Á780
/
hydroxide simply converts salts 3 into free bases 4, 5 and
6 (Scheme 2, paths a and b, and also see Scheme 1, route
a).
values are reported (in parentheses: fiducial limits): 91.6
(30.7Á277.2), 98.5 (44.6Á208.9) and 63.6 mg/kg (33.4Á
121.3) for total, free and esterified hypercholesterolemia,
respectively; 50.3 mg/kg (30.9Á81.8) for hypertriglycer-
/
/
/
Both mechanistic paths a and b of Scheme 2 can
explain the outcome of the reaction. The intermediate
III is likely unstable and, even though no base has been
added, spontaneously undergoes sulphur-to-nitrogen
rearrangement via IV, with concomitant internal proton
transfer from the less basic nitrogen of the NHR1Á3
group to the more basic 4-nitrogen. Alternatively (route
b), the acyl attack would be directed towards the
nitrogen atom and hydrogen chloride released was
trapped by the 4-tertiary nitrogen. Unlike simple
/
idemia.
3.3. Analgesic activity
In the hot plate test, 5Ar2, 5Ar4, and 9Ar2 at the dose
of 50 mg/kg were about equipotent to indometacin (5
mg/kg) (Table 3). Noteworthy activity was showed also
by 5Ar3, 10Ar3, and 12Ar2. Conversely, 4Ar2, 4Ar4,
6Ar3, 7Ar2, 8Ar3 and 8Ar4 were moderately active,
whereas 9Ar3, 10Ar4, 12Ar3, 12Ar4 were poorly active
or practically inactive.
thiourea, intermediate Ia bears the groupings R1Á3
,
which might enhance the reactivity of the NH group,
due to either an increase of nucleophilicity (Me, C6H11)
or acid ionizability (C6H5). Moreover, this reaction
pattern might be further justified by the HSAB principle
[9]. From this point of view, Ia shows two basic reactive
sites: one soft (S), the other hard (N). Since acyl
chlorides are well-known ‘hard acids’, they would
preferentially react with the hard site. It is noteworthy
that of these two mechanisms the former, (exemplified
by path a, Scheme 2), enjoyed the widest support [10].
3.4. Anti-inflammatory activity
In the carrageenan-induced paw oedema in rats, only
8Ar1, 5Ar3, 11Ar2 turned out to be active, but to a lesser
extent than indomethacin (Table 4). Some compounds
exerted a better anti-inflammatory effect at the 2nd h
(5Ar2, 10Ar3) or the 3rd h (8Ar2, 8Ar3, 9Ar3, 9Ar4,
10Ar2) after administration. Compounds 4Ar3, 6Ar2,
6Ar3, 7Ar2, 7Ar4, 9Ar2, 12Ar3 were poorly inactive or
practically inactive.
3. Pharmacology
3.5. Antiarrhythmic activity
The title compounds were evaluated for infiltration
anaesthesia, antihyperlipidemic, analgesic, anti-inflam-
matory, antiarrhythmic, platelet antiaggregating and
antiproliferative activities.
4Ar3 and 10Ar1 were as approximately active as
quinidine (Table 5). Moreover, 6Ar4 and 7Ar4 exhibited
good activity levels. Some compounds (5Ar4, 11Ar2,
11Ar3) significantly protracted the appearance time of
extrasystoles only. Compounds 4Ar4, 5Ar2, 6Ar2, 6Ar3,
7Ar2, 8Ar2, 11Ar1, 12Ar3, 12Ar4 were poorly inactive or
practically inactive.
3.1. Infiltration anaesthesia
All test compounds were active (Table 1). In parti-
cular, 4Ar4, 5Ar4, 12Ar3 and 5Ar2, 5Ar3, 9Ar4, were
about equipotent to lidocaine (after 5 and 30 min,
respectively). Also 8Ar1, 10Ar2, 10Ar3, and 12Ar2
showed good activity levels, whereas 4Ar3, 6Ar2, 7Ar4,
were moderately active.
3.6. Platelet antiaggregation activity
In the inhibition test of aggregation induced by
collagen, only 5Ar2 was approximately equipotent to
acetylsalicylic acid. Other prominent derivatives were
4Ar3, 5Ar3, 6Ar4, 7Ar3, 9Ar4, 10Ar3. The other reported
compounds were poorly active (Table 6).
3.2. Antihyperlipidemic activity
Compounds 4Ar4, 6Ar2, 6Ar4, 7Ar3, 8Ar3, 9Ar3, 11Ar1
and 11Ar2 were capable of lowering the triglyceride
levels rather than the cholesterol ones, and were more
active than nicotinic acid (Table 2). Moreover, 7Ar4 and
11Ar4 were about equipotent to nicotinic acid and 5Ar4,
8Ar4, 10Ar, and 11Ar3 were still appreciably active,
displaying the same activity trend. Conversely, 7Ar1,
7Ar2, 8Ar1, 9Ar2 10Ar3, and 12Ar4 displayed an
appreciable antihypercholesterolemic activity, causing
smaller or no (only for 12Ar4) reduction of the
triglyceride levels. In order to assess the antihyperlipi-
demic potency of 7Ar3, the following calculated ED50
3.7. Antiproliferative activity
Some of compounds selected by NCI showed an
interesting antiproliferative activity against subpanel cell
lines. In particular, 4Ar2 and 8Ar2 were significantly
active at very low concentrations against ACHN cell line
(GI50: 0.13 mM) and NCI-H226 cell line (GI50: 1.03 mM),
derived from renal cancer and non-small cell lung
cancer, respectively. 7Ar2, 11Ar2, 11Ar4, 12Ar2 and, to
a lesser extent, 5Ar2 exhibited an appreciable, broad

A. Ranise et al. / Il Farmaco 58 (2003) 765Á
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769
Table 1
Infiltration anaesthesia by pinch-tail test a, induced by the compounds of series 4
/
12
Á
b
Comp.
R
R1 Á 3
Ar1 Á 4
Dose
Activity c
5 min
30 min
4Ar2
4Ar3
CH3
CH3
CH3
CH3
p-ClC6H5
2-furyl
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
60
20
40
60
40
30
60
40
20
10
20
20
10
40
40
20
40
20
10
20
40
20
40
40
30
20
20
10
20
40
60
20
80
40
40
40
80
80
60
40
20
20
40
60
30
40
60
40
20
20
20
40
80
40
60
60
50
40
20
40
60
60
40
20
4Ar4
5Ar2
CH3
CH3
CH3
2-thienyl
p-ClC6H5
2-furyl
C6H11
C6H11
C6H11
C6H5
C6H5
C6H5
CH3
5Ar3
5Ar4
CH3
CH3
2-thienyl
p-ClC6H5
2-furyl
6Ar2
6Ar3
CH3
CH3
6Ar4
7Ar1
CH3
2-thienyl
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
7Ar2
7Ar3
CH3
CH3
p-ClC6H5
2-furyl
7Ar4
8Ar1
CH3
2-thienyl
C6H5
C6H11
C6H11
C6H11
C6H11
C6H5
C6H5
C6H5
CH3
8Ar2
8Ar3
p-ClC6H5
2-furyl
8Ar4
9Ar2
2-thienyl
p-ClC6H5
2-furyl
9Ar3
9Ar4
2-thienyl
C6H5
10Ar1
10Ar2
10Ar3
10Ar4
11Ar1
11Ar2
11Ar3
11Ar4
12Ar2
12Ar3
12Ar4
Lidocaine
CH3
CH3
p-ClC6H5
2-furyl
CH3
2-thienyl
C6H5
C6H11
C6H11
C6H11
C6H11
C6H5
C6H5
C6H5
p-ClC6H5
2-furyl
2-thienyl
p-ClC6H5
2-furyl
2-thienyl
0.2
a
Ten mice (20Á
/
25 g)/group.
Percent of glycofurol solution (0.2 ml).
Percent of animals showing anaesthesia, 5 and 30 min after infiltration of test compounds into the tail root. Dieffenbach tweezers were applied
b
c
for 10 s.
activity. The most sensitive cell lines are here reported,
as follows (Table 7).
4. Experimental
For 5Ar2: SNB-75 (GI50: 20.1 mM) and HS 578T
(GI50: 27.4 mM); for 7Ar2: UO-31 (GI50: 20.1 mM) and
HS 578T (GI50: 19.0 mM); for 11Ar2: HL-60(TB) (GI50:
17.3 mM), MOLT-4 (GI50: 24.3 mM), RPMI-8226 (GI50:
25.5 mM), NCI-H226 (GI50: 18.1 mM), and XF 498
(GI50: 8.2 mM); for 11Ar4: NCI-H226 (GI50: 19.5 mM),
COLO 205 (GI50: 22.4 mM) and RXF-393 (GI50: 22.5
mM); finally, for 12Ar2: RXF-393 (GI50: 15.6 mM).
4.1. Chemistry
All the building blocks used are commercially avail-
able. Piperazines, isothiocyanates and acyl chlorides,
60% sodium hydride dispersion were purchased by
Aldrich Chemical, Milan (Italy). Solvents (chloroform,
pyridine) were reagent grade. Organic solutions were
dried over anhydrous sodium sulphate and evaporated

770
A. Ranise et al. / Il Farmaco 58 (2003) 765Á780
/
Table 2
Effects of the compounds of series 4Á12 on hypercholesterolemia and hypertriglyceridemia induced by Triton in rats
/
Comp.
R
R1 Á 3
Ar1 Á 4
Dose (mg/kg p.o.)
Serum cholesterol, mg%9/SE a
Serum triglycerides, mg%9/SE a
Total
Free
Esterified
4Ar2
4Ar3
4Ar4
5Ar2
5Ar3
5Ar4
6Ar2
6Ar3
6Ar4
7Ar1
7Ar2
7Ar3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5
CH3
CH3
CH3
p -ClC6H5
2-furyl
50
50
50
50
50
50
50
50
50
50
50
25
50
100
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
3029/79* (ꢀ/33.2)
4189/28 (ꢀ/7.5)
1229/7* (ꢀ/18.7)
1789/12 (ꢁ/18.7)
1189/15 (ꢀ/21.3)
1689/18 (ꢁ/12.0)
1299/5* (ꢀ/14.0)
1639/12 (ꢁ/8.7)
1129/6** (ꢀ/25.3)
1669/2 (ꢁ/10.7)
1069/8** (ꢀ/29.3)
1149/6** (ꢀ/24.0)
1229/12 (ꢀ/18.7)
1539/12 (ꢁ/2.0)
1049/8** (ꢀ/30.7)
979/9** (ꢀ/35.3)
1569/8 (ꢁ/4.0)
1809/63* (ꢀ/40.4)
2409/47 (ꢀ/20.5)
1659/21** (ꢀ/45.4)
2529/46 (ꢀ/16.6)
2589/48 (14.6)
2909/31* (ꢀ/19.4)
3109/20 (ꢀ/13.9)
2129/44** (ꢀ/41.1)
3579/21 (ꢀ/0.8)
2-thienyl
2839/31** (ꢀ/37.4)
4209/27 (ꢀ/7.1)
C6H11 p -ClC6H5
C6H11 2-furyl
3879/55 (ꢀ/14.4)
4039/56 (ꢀ/10.8)
2779/19** (ꢀ/38.7)
3839/27* (ꢀ/15.3)
2419/19** (ꢀ/46.7)
3019/42** (ꢀ/33.4)
3159/36** (ꢀ/30.3)
3919/21 (ꢀ/13.5)
2559/19** (ꢀ/43.6)
2009/13** (ꢀ/55.7)
3849/50 (ꢀ/15.0)
3039/39** (ꢀ/33.0)
4709/40 (ꢁ/4.0)
2789/25* (ꢀ/22.8)
2689/24** (ꢀ/25.6)
2019/19** (ꢀ/44.2)
2699/16** (ꢀ/25.3)
1409/27** (ꢀ/61.1)
2759/32* (ꢀ/23.6)
2809/28* (ꢀ/22.2)
2799/22** (ꢀ/22.5)
1439/20** (ꢀ/60.3)
1129/14** (ꢀ/68.9)
2519/19** (ꢀ/30.3)
2719/32** (ꢀ/24.7)
3589/25 (ꢀ/0.6)
C6H11 2-thienyl
2409/64 (ꢀ/20.5)
1659/41** (ꢀ/45.4)
2179/35* (ꢀ/28.1)
1359/17** (ꢀ/55.3)
1879/61* (ꢀ/38.1)
1939/43* (ꢀ/36.1)
2389/10* (ꢀ/21.2)
1519/19** (ꢀ/50.0)
1039/12** (ꢀ/65.9)
2389/58 (ꢀ/21.2)
1819/63* (ꢀ/40.1)
3289/46 (ꢁ/8.6)
2869/18 (ꢀ/5.3)
2529/55 (ꢀ/16.6)
1369/39** (ꢀ/55.0)
2139/28 (ꢀ/29.5)
1819/49* (ꢀ/40.1)
2409/51 (ꢀ/20.5)
1609/22** (ꢀ/47.0)
1979/28** (ꢀ/34.8)
2659/32 (ꢀ/12.2)
1909/25** (ꢀ/37.1)
1759/13** (ꢀ/42.0)
2429/60 (ꢀ/19.9)
2359/60 (ꢀ/22.2)
3229/15 (ꢁ/6.6)
1679/46 (ꢀ/44.7)
1759/33* (ꢀ/42.0)
619/15
C6H5
C6H5
C6H5
CH3
CH3
CH3
p -ClC6H5
2-furyl
2-thienyl
C6H5
p -ClC6H5
2-furyl
7Ar4
C6H5
CH3
2-thienyl
8Ar1
C6H5
C6H11 C6H5
1229/1** (ꢀ/18.7)
1429/7 (ꢀ/5.3)
8Ar2
C6H5
C6H11 p -ClC6H5
C6H11 2-furyl
C6H11 2-thienyl
8Ar3
C6H5
4139/28 (ꢀ/8.6)
1279/12 (ꢀ/15.3)
1169/15* (ꢀ/22.7)
1209/7* (ꢀ/20.0)
1939/12 (ꢁ/28.7)
1379/16 (ꢀ/8.7)
1689/11 (ꢁ/12.0)
1339/6* (ꢀ/11.3)
1249/12 (ꢀ/17.3)
1469/13 (ꢀ/2.7)
1739/16 (ꢁ/15.3)
1489/12 (ꢀ/1.3)
1659/11 (ꢁ/10.0)
1589/10 (ꢁ/5.3)
1369/17 (ꢀ/9.3)
1619/8 (ꢁ/7.3)
2169/14** (ꢀ/40.0)
2779/23* (ꢀ/23.1)
3189/23 (ꢀ/11.7)
2229/18** (ꢀ/38.3)
2879/19* (ꢀ/20.3)
2669/24** (ꢀ/26.1)
3239/18 (ꢀ/10.3)
2749/30* (ꢀ/23.9)
3069/26 (ꢀ/15.0)
2139/18** (ꢀ/40.8)
2169/18* (ꢀ/40.0)
2669/21 (ꢀ/26.1)
2489/19 (ꢀ/31.1)
3369/21 (ꢀ/6.7)
8Ar4
C6H5
3689/30* (ꢀ/18.6)
2569/38** (ꢀ/43.4)
4069/43 (ꢀ/10.2)
3189/41** (ꢀ/29.6)
4089/46 (ꢀ/9.7)
9Ar2
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
CH3
p -ClC6H5
2-furyl
9Ar3
C6H5
9Ar4
C6H5
2-thienyl
C6H5
10Ar1
10Ar2
10Ar3
10Ar4
11Ar1
11Ar2
11Ar3
11Ar4
12Ar2
12Ar3
12Ar4
Controls
Triton
Tritonꢁ/nicotinic acid
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
C5H4N
p -ClC6H5
2-furyl
2939/21** (ꢀ/35.2)
3219/42** (ꢀ/29.0)
4119/28 (ꢀ/9.1)
2-thienyl
C6H11 C6H5
3639/31** (ꢀ/19.7)
3239/14** (ꢀ/28.5)
4079/55 (ꢀ/10.0)
3939/51 (ꢀ/13.0)
4589/32 (ꢁ/1.3)
C6H11 p -ClC6H5
C6H11 2-furyl
C6H11 2-thienyl
C6H5
C6H5
C6H5
p -ClC6H5
2-furyl
3289/41 (ꢀ/27.4)
2889/26** (ꢀ/36.3)
1029/12
3019/18 (ꢀ/16.4)
3869/15 (ꢁ/7.2)
2-thienyl
1139/16 (ꢀ/24.7)
419/9
1309/9
400
50
4529/13
1509/10
3029/15
3609/21
1789/18** (ꢀ/60.6)
799/10** (ꢀ/47.3)
999/12** (ꢀ/67.2)
2439/18** (ꢀ/32.5)
*, **Statistically significant value calculated in comparison with the test performed with Triton only (P B
In parentheses, % variation compared with the group treated with Triton only.
/
0.05 and P B0.01, respectively) [23].
/
a
using a rotatory evaporator operating at reduced
Melting points were determined on a FisherÁ
apparatus and are uncorrected. IR spectra were re-
corded on a PerkinÁElmer 398 spectrometer as KBr
discs.
1H and 13C NMR spectra were recorded on a Varian
Gemini 200 instrument, chemical shifts were reported in
d (ppm) units relative to the internal reference tetra-
methylsilane. Coupling constant values were given in
Hertz. Elemental analyses for C, H, N were performed
/
Johns
pressure of about 10Á20 Torr.
/
Thin layer chromatography system for routine mon-
itoring the course of reactions and confirming the purity
of analytical samples employed aluminium-backed silica
gel plates (Merck DC-Alufolien Kieselgel 60 F254):
chloroform was used as a developing solvent and
detection of spots was made by UV light and/or by
iodine vapours.
/

A. Ranise et al. / Il Farmaco 58 (2003) 765Á
/780
771
by an EA1110 Analyser, Fison Instruments (Milan) and
were within 90.4% of theoretical values.
C19H21Cl2N3OS, MM 410.36, Yield 96%, m.p. 191Á
193 8C (MeOH/Et2O). IR (KBr): 2600Á2180, 1655
cmꢀ1; H NMR (CDCl3): d 2.90 (s, 3H, CH3), 2.95Á
3.30 (m, 4H, 2CH2/N-4), 3.95Á5.00 (m, 4H, 2CH2/N-1),
7.05Á7.80 (m, 9H, arom. H), 12.45 (very bs, 1H,
exchangeable, NH). After change with D2O: d 2.88 (s,
3H, CH3), 3.06Á3.67 (m, 4H, 2CH2/N-4), 4.30Á4.81 (m,
4H, 2CH2/N-1).
/
/
/
1
/
4.1.1. General procedure for preparation of
/
hydrochlorides 3
/
To a chloroform solution (15 ml) of 1-methylpiper-
azine (1.50 g, 15 mmol) and proper isothiocyanate
(methyl-, cyclohexyl-, phenyl-isothiocyanate, 15
mmol), stirred for 10 min at room temperature (r.t.),
neat acyl chloride (15 mmol) was added in a single
portion. The resulting reaction mixture was kept under
vigorously stirring at r.t. for 4 h. At different times,
some of the hydrochlorides precipitated. Afterwards,
/
/
4-{[2-Furoyl(phenyl)amino]carbonothioyl}-1-methyl-
piperazin-1-ium chloride (3R3Ar3). C17H20ClN3O2S,
MM 365.88, Yield 96%, m.p. 182Á
Et2O). IR (KBr): 2600Á
2180, 1640 cmꢀ1
(DMSO): d 2.82 (s, 3H, CH3), 3.00Á3.78 (m, 4H, 2CH2/
N-4), 4.10Á5.18 (m, 4H, 2CH2/N-1), 6.56Á6.76 (m, 1H,
H-4 fur.), 6.79Á6.95 (d, Jꢂ4 Hz, H-3 fur), 7.20Á7.70
(m, 5H, arom H.), 7.98Á8.14 (m, 1H, H-5 fur.), 12.50
/
183 8C (MeOH/
/
;
1H NMR
/
small volumes of diethyl ether (0.5Á/1.0 ml) were added
/
/
to the solution/suspension till no more turbidity formed.
The solid was allowed to stand overnight, collected by
filtration and recrystallized from proper solvent(s). The
isolated hydrochlorides 3R2Ar3 and 3R2Ar4, owing to
their deliquescence, could not be characterised.
/
/
/
/
(very bs, 1H, exchangeable, NH).
4-{[Phenyl(thien-2-ylcarbonyl)amino]carbonothioyl}-
1-methylpiperazin-1-ium
C17H20ClN3OS2, MM 381.94, Yield 85%, m.p. 196Á
199 8C (CHCl3/Et2O). IR (KBr): 2680Á2200, 1655
cmꢀ1; H NMR (DMSO): d 2.90 (s, 3H, CH3), 3.10Á
3.70 (m, 4H, 2CH2/N-4), 4.02Á5.30 (m, 4H, 2CH2/N-1),
7.75 (m, 8H, arom H and H-3, H-4, H-5 thioph.),
chloride
(3R3Ar4).
4-{[4-Chlorobenzoyl(methyl)amino]carbonothioyl}-1-
/
methylpiperazin-1-ium
C14H19Cl2N3OS, MM 348.29, Yield 98%, m.p. 203Á
204 8C (MeOH/Et2O). IR (KBr): 2700Á2100, 1655
cmꢀ1; H NMR (CDCl3): d 2.82 (s, 3H, CH3), 3.10Á
4.10 (m, 4H, 2CH2/N-4), 3.44 (s, 3H, CH3), 4.30Á4.95
(m, 4H, 2CH2/N-1), 7.35Á7.82 (m, 4H, arom. H), 12.55
chloride
(3R1Ar2).
/
1
/
/
/
/
1
/
6.80Á
/
/
12.88 (very bs, 1H, exchangeable, NH). 13C NMR
(DMSO): d 85.30, 141.38, 137.33, 134,50, 134,29,
130,13, 129.27, 128.46, 52.01, 47.24, 42.28, 41.54.
/
(very bs, 1H, exchangeable, NH).
4-{[2-Furoyl(methyl)amino]carbonothioyl}-1-methyl-
piperazin-1-ium chloride (3R1Ar3). C12H18ClN3O2S,
4.1.2. General procedure for the synthesis of ATU
MM 303.81, Yield 97%, m.p. 205Á
Et2O). IR (KBr): 3100, 2700Á
2200, 1655 cmꢀ1
NMR (CDCl3): d 2.92 (s, 3H, CH3), 3.11Á3.73 (m, 4H,
2CH2/N-4), 3.34 (s, 3H, CH3), 3.98Á5.00 (m, 4H, 2CH2/
N-1), 6.48Á6.73 (m, 1H, H-4 fur.), 7.23 (d, Jꢂ4 Hz, H-3
fur), 7.56Á7.82 (m, 1H, H-5 fur.), 12.93 (very bs, 1H,
exchangeable, NH).
4-{[Methyl(thien-2-ylcarbonyl)amino]carbo-
nothioyl}-1-methylpiperazin-1-ium chloride (3R1Ar4).
/
207 8C (MeOH/
derivatives 4Á6
/
/
;
1H
To a stirred chloroform solution (40 ml) of 1-
methylpiperazine (1.00 g, 10 mmol) and proper isothio-
cyanate (methyl-, cyclohexyl-, phenyl-isothiocyanate, 10
mmol) neat acyl chloride (11 mmol) was added in a
single portion after 10 min at r.t. The resulting mixture
was allowed to react at r.t. for 4 h under stirring; then
water (50 ml) was added and the chloroform layer was
separated and washed two times with water (25 ml). The
combined aqueous layers were made alkaline by treat-
ment of 1 M NaOH (45 ml) and extracted with chloro-
/
/
/
/
/
C12H18ClN3OS2×
188Á189 8C (hot CHCl3). IR (KBr): 3600Á
2180, 1655 cmꢀ1; H NMR (DMSO): d 2.80 (s, 3H,
CH3), 3.01Á3.80 (m, 4H, 2CH2/N-4), 3.25 (s, 3H, CH3),
3.90Á5.00 (m, 4H, 2CH2/N-1), 7.08Á7.32 (m, 1H, H-3
thioph.), 7.48Á7.58 (m, 1H, H-4 thioph.), 7.80Á8.05 (m,
/H2O, MM 337.88, Yield 97%, m.p.
/
/
3300, 2720Á
/
1
form (3ꢃ15 ml). The organic phase was dried and
/
/
evaporated under reduced pressure to dryness to afford
an oily or solid residue, which was crystallized by proper
solvents.
/
/
/
/
1H, H-5 thioph.), 12.15 (very bs, 1H, exchangeable,
NH).
4-Chloro-N-methyl-[(4-methylpiperazin-1-yl)carbo-
nothioyl]benzamide (4Ar2). C14H18ClN3OS, MM
4-{[4-Chlorobenzoyl(cyclohexyl)amino]carbo-
nothioyl}-1-methylpiperazin-1-ium chloride (3R2Ar2).
311.84, Yield 71%, m.p. 111Á112 8C (CH2Cl2/Et2O).
/
IR (KBr): 1660 cmꢀ1; H NMR (CDCl3): d 2.00Á
(m, 4H, 2CH2/pip), 2.25 (s, 3H, CH3N/pip), 3.30Á
(m, 4H, 2CH2N/pip), 3.50 (s, 3H, CH3N), 7.44Á
4H, arom. H).
/2.55
1
C19H27Cl2N3OS, MM 416.41, Yield 85%, m.p. 186Á
187 8C (MeOH/Et2O). IR (KBr): 2700Á2200, 1690
2.32 (m, 10H, 5CH2
cyclohexyl), 2.74 (s, 3H, CH3), 3.20Á5.02 (m, 9H, 4CH2/
pip and CH), 7.23Á7.94 (m, 4H, arom. H), 13.38 (very
/
/3.95
/
/
7.76 (m,
1
cmꢀ1; H NMR (CDCl3): d 0.92Á
/
/
N-Methyl-[(4-methylpiperazin-1-yl)carbonothioyl]-2-
/
furamide (4Ar3). C12H17N3O2S, MM 267.35, Yield
bs, 1H, exchangeable, NH).
4-{[4-Chlorobenzoyl(phenyl)amino]carbonothioyl}-1-
60%, m.p. 81Á
/
83 8C (CH2Cl2/Et2O). IR (KBr): 1660
cmꢀ1; H NMR (CDCl3): d 2.22Á
2.65 (m, 4H, 2CH2/
pip), 2.30 (s, 3H, CH3N/pip), 3.32 (s, 3H, CH3N), 3.80Á
1
/
methylpiperazin-1-ium
chloride
(3R3Ar2).
/

772
A. Ranise et al. / Il Farmaco 58 (2003) 765Á
/780
Table 3
Analgesic activity of the compounds of series 4Á
12, evaluated by hot plate test a
/
Comp.
R
R1 Á 3 Ar1 Á 4
Dose (mg/kg p.o.) Mean reaction time (s9
/
SE) at the following times (hours) after treatment (in
parentheses per cent change relative to 0 value) b
0
1
2
3
4
4Ar2
4Ar3
CH3
CH3
CH3 p-ClC6H5 50
99
89
99
79
/
1.2
2.3
1.2
1.8
109
/
1.0 (11)
119
992.6 (12)
109
129
149
149
109
99
109
109
109
99
109
129
109
119
99
109
991.9 (0)
109
119
99
129
119
129
99
109
991.8 (12)
1591.0* (50)
/
0.8* (22)
119
991.4 (12)
129
149
159
169
119
109
129
119
129
109
129
149
109
119
109
129
99
119
119
99
139
129
129
109
129
99
179
992.3 (29)
1092.5 (25)
790.9 (0)
1590.9** (87)
/
0.7* (22)
139
119
139
169
189
179
129
109
139
129
139
109
139
159
119
129
119
149
109
129
129
99
159
129
139
119
139
109
179
99
129
791.1 (0)
1691.7** (100)
/
1.6* (44)
CH3 2-furyl 50
CH3 2-thienyl 50
C6H11 p-ClC6H5 50
/
89
99
99
139
129
109
89
99
99
99
89
99
109
992.1 (0)
1090.9* (25)
/
1.9 (0)
2.1 (0)
1.6 (29)
/
/
/3.8 (37)
4Ar4
5Ar2
CH3
CH3
/
/
/1.8 (11)
/
1.1* (33)
1.2** (100)
2.1* (50)
1.7** (100)
1.8 (10)
/
1.6* (44)
2.1** (129)
1.7** (80)
2.3** (112)
2.6 (20)
/
/
/2* (71)
/
/
5Ar3
5Ar4
CH3
CH3
C6H11 2-furyl 50
C6H11 2-thienyl 50
C6H5 p-ClC6H5 50
109
891.5
1091.2
/1.7
/
0.9 (30)
1.6* (50)
2.0 (0)
/
1.0* (40)
2.0* (75)
1.5 (0)
/
/
/
/
/
/
/
6Ar2
CH3
/
/
/
/
/
6Ar3
6Ar4
CH3
CH3
C6H5 2-furyl
C6H5 2-thienyl 50
CH3 C6H5 50
CH3 p-ClC6H5 50
CH3 2-furyl 50
CH3 2-thienyl 50
C6H11 C6H5 50
C6H11 p-ClC6H5 50
C6H11 2-furyl 50
50
79
99
99
89
89
99
/
1.9
2.1
1.4
1.3
1.1
1.9
/
1.4 (14)
1.7 (0)
1.5 (0)
1.2 (12)
1.0 (0)
1.1 (0)
/
1.6 (29)
/3.1 (43)
/2.6 (43)
/
/
/
1.8 (11)
1.2 (11)
1.9 (25)
/3.2 (33)
/1.7 (44)
7Ar1
7Ar2
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
/
/
/
/1.9 (22)
/2.3 (33)
/
/
/
/1.2* (50)
/2.0* (62)
7Ar3
/
/
/
0.9 (12)
/1.2 (25)
/1.6 (25)
7Ar4
8Ar1
/
/
/
1.9 (11)
2.1* (20)
2.7 (11)
1.1* (37)
/2.3 (33)
/1.4 (44)
109
99
89
79
79
99
89
99
79
99
109
109
/0.9
/1.7 (0)
/
/1.4* (40)
/1.3** (50)
8Ar2
8Ar3
/
1.8
0.7
1.9
2.1
1.5
1.1
2.1
0.9
1.5
/
/
/2.3 (11)
/1.5 (22)
/
/
/
/1.2* (37)
/1.6* (50)
8Ar4
9Ar2
C6H11 2-thienyl 50
C6H5 p-ClC6H5 50
/
99
99
99
99
/
2.1 (29)
1.7 (29)
1.6 (0)
/
1.8 (29)
/1.9 (43)
/2.4 (57)
/
/
/2.0 (43)
/1.8* (71)
/1.9** (100)
9Ar3
9Ar4
C6H5 2-furyl
C6H5 2-thienyl 50
50
C5H4N CH3 p-ClC6H5 50
C5H4N CH3 2-furyl 50
C5H4N CH3 2-thienyl 50
C5H4N C6H11 C6H5 50
C5H4N C6H11 p-ClC6H5 50
C5H4N C6H11 2-furyl 50
50
/
/
/
/
1.6 (0)
/2.0 (11)
/
/
1.2 (12)
/1.4 (25)
/0.8* (37)
/1.1* (50)
10Ar1
10Ar2
10Ar3
10Ar4
11Ar1
11Ar2
11Ar3
11Ar4
12Ar2
12Ar3
12Ar4
Control
Indomethacin
C5H4N CH3 C6H5
/
109
892.1 (14)
109
109
119
/1.9 (11)
/2.1 (22)
/1.8 (22)
/1.7 (33)
/
/
/
1.4 (29)
/
0.9 (29)
/
1.3 (29)
/
/1.4 (11)
/
1.8 (33)
1.7 (10)
2.4 (20)
/
1.0* (44)
2.5 (20)
2.1 (20)
2.5 (25)
3.1 (33)
/
1.2** (67)
1.9 (20)
1.9 (30)
3.1 (37)
2.7 (44)
1.5 (25)
1.8* (70)
/1.4
/1.5 (0)
/
/
/
/2.1
/1.6 (10)
/
/
/
89
99
89
/
1.9
2.7
1.4
89
99
89
/
2.0 (0)
2.3 (0)
1.7 (0)
/
2.3 (12)
/
/
/
/
/1.8 (11)
/
/
C5H4N C6H11 2-thienyl 50
/
/
/
/
2.1 (12)
/
C5H4N C6H5 p-ClC6H5 50
C5H4N C6H5 2-furyl 50
C5H4N C6H5 2-thienyl 50
109
/1.7
129
/
1.4 (20)
/
/2.1* (70)
/
79
89
79
89
/
1.2
2.4
0.2
1.2
79
89
79
/
2.5 (0)
1.8 (0)
1.0 (0)
89
99
79
139
/
1.8 (14)
1.9 (12)
0.8 (0)
/
/
1.9 (29)
3.1 (50)
/
/
/
/
/
/
/
/
/
/
5
/
109
/
0.8* (25)
/
1.0** (62)
/
/
*, ** Statistically significant values calculated in comparison with the test performed with basis value (P B
/
0.05, P B0.01, respectively) [23].
/
a
Ten mice (20Á
/
25 g)/group.
Mean value of five determinations.
b
1
4.20 (m, 4H, 2CH2N/pip), 6.47Á
/
6.59 (m, 1H, H-4 fur.),
IR (KBr): 1630 cmꢀ1; H NMR (CDCl3): d 1.00Á
(m, 14H, 5CH2 and 2CH2/pip), 2.20 (s, 3H, CH3N/pip),
3.05Á4.00 (m, 4H, 2CH2N/pip), 4.05Á4.80 (m, 1H,
CHN), 7.37Á7.82 (m, 4H, arom. H).
/2.50
7.20 (d, 1H, Jꢂ4 Hz, H-3 fur.), 7.59 (bs, 1H, H-5 fur.).
/
N-Methyl-[(4-methylpiperazin-1-yl)carbo-
nothioyl]thiophene-2-carboxamide
/
/
(4Ar4).
/
C12H17N3OS2, MM 283.42, Yield 75%, m.p. 105Á
106 8C (CH2Cl2/Et2O). IR (KBr): 1630 cmꢀ1 1H
NMR (CDCl3): d 2.10Á2.55 (m, 4H, 2CH2/pip), 2.24
(s, 3H, CH3N/pip), 3.42 (s, 3H, CH3N), 3.65Á4.10 (m,
4H, 2CH2N/pip), 7.00Á7.25 (m, 1H, H-4 thioph.), 7.50Á
/
N-Cyclohexyl-[(4-methylpiperazin-1-yl)carbo-
nothioyl]-2-furamide (5Ar3). C17H25N3O2S, MM
;
/
335.47, Yield 82%, m.p. 104Á
/
105 8C (CH2Cl2/Et2O).
IR (KBr): 1615 cmꢀ1; H NMR (CDCl3): d 1.00Á
2.60
(m, 14H, 5CH2 and 2CH2/pip), 2.25 (s, 3H, CH3N/pip),
3.65Á4.15 (m, 4H, 2CH2N/pip), 4.10Á4.60 (m, 1H,
CHN), 6.44Á6.54 (m, 1H, H-4 fur.), 7.25 (d, 1H, Jꢂ
Hz, H-3 fur.), 7.57 (bs, 1H, H-5 fur.).
1
/
/
/
/
7.90 (m, 2H, H-3 and H-5 thioph.)
4-Chloro-N-cyclohexyl-[(4-methylpiperazin-1-yl)car-
/
/
/
/
4
bonothioyl]benzamide (5Ar2). C19H26ClN3OS, MM
379.25, Yield 65%, m.p. 133Á134 8C (CH2Cl2/Et2O).
/

A. Ranise et al. / Il Farmaco 58 (2003) 765Á
/780
773
Table 4
Anti-inflammatory activity of the compounds of series 4Á
/
12, evaluated by carrageenan-induced rat paw oedema test a
Comp.
R
R1 Á 3 Ar1 Á 4
Dose (mg/kg p.o.) % Oedema inhibition relative to control at the following times (h) after
administration
1st h
2nd h
18
3rd h
4th h
4Ar2
4Ar3
CH3
CH3
CH3 p-ClC6H5 50
CH3 2-furyl 50
CH3 2-thienyl 50
ꢀ
/
18
ꢀ/
ꢀ
/
33
15
25
44
34
34
25
28
25
25
18
19
36
67
61
40
25
15
40
45
25
35
46
37
25
45
25
36
33
10
34
67
63
ꢀ
/
39
20
32
44
50
38
16
12
30
30
25
39
36
63
55
55
30
ꢀ
/
36
ꢀ
/2
ꢀ
/
ꢀ
/
4Ar4
5Ar2
CH3
CH3
ꢀ
/9
ꢀ
/
32
43
50
25
16
61
16
16
0
ꢀ
/
ꢀ
/
C6H11 p-ClC6H5 50
C6H11 2-furyl 50
C6H11 2-thienyl 50
ꢀ
ꢀ/
/
31
50
0
ꢀ
/
ꢀ
/
ꢀ
/
5Ar3
5Ar4
CH3
CH3
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
6Ar2
6Ar3
CH3
CH3
C6H5 p-ClC6H5 50
ꢁ
/
14
50
14
45
0
ꢀ
/
ꢀ
/
ꢀ
/
C6H5 2-furyl
C6H5 2-thienyl 50
CH3 C6H5 50
CH3 p-ClC6H5 50
CH3 2-furyl 50
CH3 2-thienyl 50
C6H11 C6H5 50
C6H11 p-ClC6H5 50
C6H11 2-furyl 50
50
ꢁ
/
ꢁ
/
ꢁ
/
ꢁ
/
6Ar4
7Ar1
CH3
ꢁ
/
ꢀ
/
ꢀ
/
ꢀ
/
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
ꢀ/
ꢀ/
ꢀ
/
ꢀ
/
7Ar2
7Ar3
ꢀ
/
ꢀ
/
ꢀ
/
18
36
50
18
ꢀ
/
18
36
50
31
31
16
ꢀ
/
ꢀ
/
7Ar4
8Ar1
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
8Ar2
8Ar3
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/9
ꢀ
/
ꢀ
/
ꢀ
/
8Ar4
9Ar2
C6H11 2-thienyl 50
C6H5 p-ClC6H5 50
ꢀ
/
45
ꢀ/
ꢀ
/
ꢀ/
ꢁ
/
27
ꢀ
/2
ꢀ
/
ꢀ5
/
9Ar3
9Ar4
C6H5 2-furyl
C6H5 2-thienyl 50
50
C5H4N CH3 p-ClC6H5 50
C5H4N CH3 2-furyl 50
C5H4N CH3 2-thienyl 50
C5H4N C6H11 C6H5 50
C5H4N C6H11 p-ClC6H5 50
C5H4N C6H11 2-furyl 50
50
ꢀ
/9
ꢀ
/
32
32
16
25
39
25
14
16
ꢀ
/
ꢀ
/
44
44
15
50
49
25
25
44
32
36
29
20
38
89
70
ꢀ
ꢀ/
/
45
45
0
ꢀ
/
ꢀ
/
ꢀ
/
10Ar1
10Ar2
10Ar3
10Ar4
11Ar1
11Ar2
11Ar3
11Ar4
12Ar2
12Ar3
12Ar4
Control
Indomethacin
C5H4N CH3 C6H5
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
18
23
50
45
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢁ
/
ꢁ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/9
ꢀ
/9
ꢀ
/
ꢀ
/
C5H4N C6H11 2-thienyl 50
C5H4N C6H5 p-ClC6H5 50
ꢀ
/
41
18
27
0
ꢀ
/
36
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
39
ꢀ
/
ꢀ
/
C5H4N C6H5 2-furyl
C5H4N C6H5 2-thienyl 50
Carr. 1% sol
50
ꢁ/
ꢀ
/2
ꢀ
/
ꢀ
/
ꢀ
/
25
44
43
ꢀ
/
ꢀ
/
ꢁ
/
22
ꢁ
/
ꢁ
/
ꢁ
/
5
ꢀ
/
45
ꢀ/
ꢀ/
ꢀ/
a
Each compound was tested on a group of five albino rats (180Á250 g) and given by gastric probe 30 min before carrageenan.
/
N-Cyclohexyl-[(4-methylpiperazin-1-yl)carbo-
nothioyl]thiophene-2-carboxamide
CH3N/pip), 3.80Á
/
4.40 (m, 4H, 2CH2N/pip), 7.05Á
/7.80
(5Ar4).
and 8.00Á8.30 (m, 9H, arom. H).
/
N-Phenyl-[(4-methylpiperazin-1-yl)carbonothioyl]-2-
furamide (6Ar3). C17H19N3O2S, MM 329.42, Yield
C17H25N3OS2, MM 351.54, Yield 70%, m.p. 127Á
128 8C (CH2Cl2/Et2O). IR (KBr): 1615 cmꢀ1 1H
NMR (CDCl3): d 1.00Á2.65 (m, 14H, 5CH2 and
2CH2/pip), 2.25 (s, 3H, CH3N/pip), 3.55Á4.10 (m, 4H,
2CH2N/pip), 4.15Á4.75 (m, 1H, CHN), 6.90Á7.25 (m,
1H, H-4 thioph.), 7.40Á7.60 (m, 1H, H-3 thioph.), 7.75Á
/
;
78%, m.p. 126Á
/
127 8C (CH2Cl2/Et2O). IR (KBr): 1665
cmꢀ1; H NMR (CDCl3): d 2.25 (s, 3H, CH3N/pip),
2.25Á2.70 (m, 4H, 2CH2/pip), 3.85Á4.40 (m, 4H,
2CH2N/pip), 6.40Á6.65 (m, 1H, H-4 fur.), 6.85 (d, 1H,
Jꢂ4 Hz, H-3 fur.), 7.25Á7.78 (m, 9H, arom. H and H-5
fur.).
/
1
/
/
/
/
/
/
/
/
/
/
8.00 (m, 1H, H-5 thioph.).
4-Chloro-N-phenyl-[(4-methylpiperazin-1-yl)carbo-
N-Phenyl-[(4-methylpiperazin-1-yl)carbo-
nothioyl]thiophene-2-carboxamide
nothioyl]benzamide (6Ar2). C19H20ClN3OS, MM
373.91, Yield 65% (Py/TEA), 73% (CHCl3), m.p. 191Á
193 8C (CH2Cl2/Et2O). IR (KBr): 1675 cmꢀ1; 1H NMR
(CDCl3): d 2.25Á2.75 (m, 4H, 2CH2/pip), 2.30 (s, 3H,
(6Ar4).
/
C17H19N3OS2, MM 345.49, Yield 95%, m.p. 148Á
/
149 8C (CH2Cl2/Et2O). IR (KBr): 1615 cmꢀ1; IR
/

774
A. Ranise et al. / Il Farmaco 58 (2003) 765Á780
/
Table 5
Antiarrhythmic activity of the compounds of series 4Á
12, evaluated as protection index against ecgraphic effects from aconitine in albino rats a
/
b
Comp.
R
R1 Á 3 Ar1 Á 4
Dose (mg/kg p.o.) Appearance time (s9
/
SE) of extrasystoles b Death time (sec9
SE)
/
4Ar2
4Ar3
CH3
CH3
CH3
CH3
CH3
p-ClC6H5 50
2-furyl 50
2-thienyl 50
2609
3459
2109
2209
1929
2749
1939
1859
2629
2079
1779
162916.4 (ꢀ
2709
1949
2209
1949
2369
2109
1899
2109
3259
2079
2279
2419
1929
2709
2929
2409
2429
1889
1849
1809
3609
/
24.1** (44)
42.0** (92)
28.9 (17)
16.8 (22)
14.5 (7)
7609
9809
6329
7209
7409
6829
6419
4939
9459
6929
5809
4479
8929
5869
9649
6469
8929
6449
7049
9049
10589
/
10.1** (31)
17.2** (69)
30.3 (9)
/
/
4Ar4
CH3
/
/
5Ar2
5Ar3
CH3
CH3
C6H11 p-ClC6H5 50
C6H11 2-furyl 50
C6H11 2-thienyl 50
/
/
44.3* (24)
47.9** (28)
31.8 (18)
/
/
5Ar4
6Ar2
CH3
CH3
/
40.4* (52)
9.8** (7)
10.9 (3)
/
C6H5 p-ClC6H5 50
/
/54.3 (10)
6Ar3
6Ar4
CH3
CH3
C6H5 2-furyl 50
C6H5 2-thienyl 50
/
/9.4 (ꢀ15)
/
/30.3* (46)
/
30** (63)
51* (19)
18.3 (0)
7Ar1
7Ar2
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
CH3
C6H5
p-ClC6H5 50
2-furyl 50
2-thienyl 50
50
C6H11 p-ClC6H5 50
C6H11 2-furyl 50
50
/45.2 (15)
/
/8.6 (ꢀ
/2)
/
7Ar3
7Ar4
/
/10)
/10.4 (ꢀ23)
/
/24.3** (50)
/20.4** (54)
8Ar1
C6H11 C6H5
/8.3 (8)
/10.2 (1)
8Ar2
8Ar3
/19.2 (22)
/
17.4** (66)
20.7 (11)
44* (54)
/16.8 (8)
/
8Ar4
9Ar2
C6H11 2-thienyl 50
C6H5 p-ClC6H5 50
/
17.4* (31)
24.6 (17)
12.3 (5)
/
/
/
22.3 (11)
36.9* (21)
11.2 (56)
9Ar3
C6H5 2-furyl
50
/
/
9Ar4
C6H5 2-thienyl 50
/7.9 (17)
/
10Ar1
10Ar2
10Ar3
10Ar4
11Ar1
11Ar2
11Ar3
11Ar4
12Ar2
12Ar3
12Ar4
Controls (aconitine HCl)
Quinidine
C5H4N CH3
C5H4N CH3
C5H4N CH3
C5H4N CH3
C6H5
p-ClC6H5 50
2-furyl 50
2-thienyl 50
50
C5H4N C6H11 p-ClC6H5 50
C5H4N C6H11 2-furyl 50
50
/
42.1** (81)
11.4 (15)
15.6* (26)
33.4 (34)
10.8 (7)
/28.1** (82)
/
6109
6529
6929
5969
6069
6749
6569
7469
5329
6069
5809
/
17.4 (5)
/
/
18.2* (12)
20.2** (19)
12.8 (3)
/
/
C5H4N C6H11 C6H5
/
/
/
36.4* (50)
45.6 (62)
20.1* (33)
49.6 (34)
20.5 (4)
/29.1 (4)
/
/
25.1 (16)
15.4* (13)
46.4 (29)
C5H4N C6H11 2-thienyl 50
/
/
C5H4N C6H5 p-ClC6H5 50
/
/
C5H4N C6H5 2-furyl 50
C5H4N C6H5 2-thienyl 50
/
/26.4 (ꢀ8)
/
/12.1 (2)
/27.4 (4)
c
/15.9
/27.2
25
/20.1** (100)
10209
/
14.3* (76)
*, ** Statistically significant values calculated in comparison with the test performed with aconitine alone (P B
/
0.05, P B0.01, respectively) [23].
/
a
b
c
Five animals (200Á250 g)/group.
/
In parentheses % time increase as regard to the control.
15 mg/kg i.v. until death time.
1
(KBr): 1680 cmꢀ1; H NMR (CDCl3): d 2.30 (s, 3H,
residue, which was treated with water. The resulting
suspension was extracted with dichloromethane (3ꢃ20
ml), washed three times with water (40 ml), dried,
filtered through a plug of Florisil and finally evaporated
under reduced pressure to yield a residue, which was
crystallised from proper solvents.
CH3N/pip), 2.20Á
4H, 2CH2N/pip), 6.70Á
H-4, H-5 thioph.).
/
2.70 (m, 4H, 2CH2/pip), 3.85Á
/4.40 (m,
/
/
7.70 (m, 8H, arom. H and H-3,
4.1.3. General procedure for preparation of ATU
derivatives 7Á
N-Methyl-[(4-phenylpiperazin-1-yl)carbo-
/
9
nothioyl]benzamide (7Ar1). C19H21N3OS, MM 339.46,
In a non-anhydrous pyridine solution (20 ml) 1-
phenylpiperazine (1.62 g, 10 mmol) and proper isothio-
cyanate (methyl-, cyclohexyl-, phenyl-isothiocyanate, 10
mmol) were allowed to react for 10 min at r.t. under
stirring. After adding neat acyl chloride (11 mmol) in a
single portion at r.t., the reaction mixture was stirred for
4 h at r.t. Evaporating pyridine in vacuo gave a crude
Yield 77%, m.p. 115Á
(KBr): 1665 cmꢀ1 1H NMR (CDCl3): d 2.90Á
(m, 4H, 2CH2/pip), 3.52 (s, 3H, CH3N), 3.60Á4.10 (m,
4H, 2CH2N/pip), 6.70Á8.10 (m, 10H, arom. H).
/
116 8C (CH2Cl2/MeOH). IR
;
/3.35
/
/
4-Chloro-N-methyl-[(4-phenylpiperazin-1-yl)carbo-
nothioyl]benzamide (7Ar2). C19H20ClN3OS, MM

A. Ranise et al. / Il Farmaco 58 (2003) 765Á
/780
775
Table 6
Inhibition test of platelet aggregation induced by collagen in human plasma versus the compounds of series 4Á
/
12
Comp.
R
R1 Á 3
CH3
Ar1 Á 4
Final concentration
% Aggregation9
/
SE
% D vs. collagen
4Ar2
CH3
p-ClC6H5
10ꢀ6 g/ml
10ꢀ5
77.09
74.59
75.09
73.29
74.59
71.09
76.59
70.59
78.49
76.59
79.29
74.59
77.09
69.59
76.09
72.49
79.29
78.69
79.69
79.09
79.59
70.19
76.99
72.49
76.49
70.59
79.49
73.99
77.99
71.39
79.99
74.69
81.59
74.59
68.99
/
2.5
3.8
5.6
4.1
5.5
7.5
2.6
4.3
5.9
2.4
3.5
4.3
2.7
5.7
5.0
4.9
2.4
5.1
3.7
4.1
3.7
4.8
5.3
3.3
7.6
8.9
4.8
3.7
5.1
3.9
3.7
4.9
2.3
1.0
1.1
ꢀ
/
5.5
8.6
8.0
/
ꢀ
/
4Ar3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5
C6H5
C6H5
CH3
2-furyl
10ꢀ6 g/ml
10ꢀ5
/
ꢀ/
/
ꢀ
ꢀ
/
10.2
8.6
12.9
6.1
13.5
5Ar2
C6H11 p-ClC6H5
C6H11 2-furyl
10ꢀ6 g/ml
10ꢀ5
/
/
/
ꢀ
/
5Ar3
10ꢀ6 g/ml
10ꢀ5
/
ꢀ
ꢀ/
/
/
5Ar4
C6H11 2-thienyl
10ꢀ6 g/ml
10ꢀ5
/
ꢀ
/
3.8
6.1
2.8
8.6
5.5
/
ꢀ
/
6Ar3
C6H5
C6H5
CH3
2-furyl
2-thienyl
2-furyl
10ꢀ6 g/ml
10ꢀ5
/
ꢀ
/
/
ꢀ
/
6Ar4
10ꢀ6 g/ml
10ꢀ5
/
ꢀ/
/
ꢀ
ꢀ
ꢀ/
/
14.7
6.7
11.2
7Ar3
10ꢀ6 g/ml
10ꢀ5
/
/
/
8Ar2
C6H11 p-ClC6H5
10ꢀ6 g/ml
10ꢀ5
/
ꢀ
/
2.8
3.6
2.3
3.1
2.4
/
ꢀ
/
8Ar4
C6H11 2-thienyl
10ꢀ6 g/ml
10ꢀ5
/
ꢀ/
/
9Ar2
C6H5
C6H5
p-ClC6H5
2-thienyl
2-furyl
10ꢀ6 g/ml
10ꢀ5
/
ꢀ/
/
ꢀ
ꢀ
/
14.0
5.6
11.2
6.3
13.5
9Ar4
10ꢀ6 g/ml
10ꢀ5
/
/
/
ꢀ
/
10Ar3
11Ar2
11Ar3
12Ar4
Collagen
C5H4N CH3
10ꢀ6 g/ml
10ꢀ5
/
ꢀ
ꢀ/
/
/
C5H4N C6H11 p-ClC6H5
10ꢀ6 g/ml
10ꢀ5
/
ꢀ
/
2.6
9.3
4.4
/
ꢀ
/
C5H4N C6H11 2-furyl
10ꢀ6 g/ml
10ꢀ5
/
ꢀ/
/
ꢀ12.5
/
C5H4N C6H5
2-thienyl
10ꢀ6 g/ml
10ꢀ5
/
ꢀ
/
2.0
/
ꢀ
/
8.5
10ꢀ5 g/ml
10ꢀ6 g/ml
10ꢀ5
/
Acetylsalicylic Acidꢁ
/Collagen
/
ꢀ
/
8.6
/
ꢀ/15.5
373.91, Yield 78%, m.p. 161Á
/
163 8C (CH2Cl2/MeOH).
IR (KBr): 1640 cmꢀ1; H NMR (CDCl3): d 2.90Á
3.30
(m, 4H, 2CH2/pip), 3.50 (s, 3H, CH3N), 3.65Á4.10 (m,
4H, 2CH2N/pip), 6.70Á7.95 (m, 9H, arom. H).
(s, 3H, CH3N), 3.75Á
7.95 (m, 8H, arom. H and H-3, H-4, H-5 thioph.).
N-Cyclohexyl-[(4-phenylpiperazin-1-yl)carbo-
/
4.20 (m, 4H, 2CH2N/pip), 6.70Á
/
1
/
/
/
nothioyl]benzamide (8Ar1). C24H29N3OS, MM 407.58,
Yield 53%, m.p. 164Á166 8C (CH2Cl2/MeOH). IR
(KBr): 1640 cmꢀ1 1H NMR (CDCl3): d 1.00Á
2.70
(m, 10H, 5CH2), 2.80Á3.25 (m, 4H, 2CH2/pip), 3.40Á
4.20 (m, 4H, 2CH2N/pip), 4.25Á4.90 (m, 1H, CHN),
6.60Á8.15 (m, 10H, arom. H).
N-Methyl-[(4-phenylpiperazin-1-yl)carbonothioyl]-2-
furamide (7Ar3). C17H19N3O2S, MM 329.42, Yield
/
;
/
64%, m.p. 159Á
/
160 8C (CH2Cl2/MeOH). IR (KBr):
1665 cmꢀ1; H NMR (CDCl3): d 3.10Á
3.30 (m, 4H,
2CH2/pip), 3.40 (s, 3H, CH3N), 3.82Á4.62 (m, 4H,
2CH2N/pip), 6.40Á6.65 (m, 1H, H-4 fur.), 6.80Á7.70
/
/
1
/
/
/
/
/
/
4-Chloro-N-cyclohexyl-[(4-phenylpiperazin-1-yl)car-
(m, 7H, arom. H and H-3, H-5 fur.).
N-Methyl-[(4-phenylpiperazin-1-yl)carbo-
nothioyl]thiophene-2-carboxamide
bonothioyl]benzamide (8Ar2). C24H28N3ClOS, MM
442.02, Yield 66%, m.p. 119Á
/
120 8C (CH2Cl2/MeOH).
IR (KBr): 1660 cmꢀ1; H NMR (CDCl3): d 1.00Á
2.65
(m, 10H, 5CH2), 2.75Á3.30 (m, 4H, 2CH2/pip), 3.35Á
4.85 (m, 5H, 2CH2N/pip and CHN), 6.65Á7.65 and
7.75Á8.15 (m, 9H, arom. H).
1
(7Ar4).
/
C17H19N3OS2, MM 345.49, Yield 58%, m.p. 123Á
124 8C (CH2Cl2/MeOH). IR (KBr): 1650 cmꢀ1 1H
NMR (CDCl3): d 2.90Á3.35 (m, 4H, 2CH2/pip), 3.50
/
/
/
;
/
/
/

776
A. Ranise et al. / Il Farmaco 58 (2003) 765Á780
/
Table 7
Antiproliferative activity of selected compounds
GI50 (mM) a
Panel/cell line
4Ar2
5Ar2
7Ar2
8Ar2
11Ar2
11Ar4
12Ar2
Leukemia
CCRF-CEM
HL-60(TB)
K-562
72.5
61.6
51.3
63.1
56.6
35.5
42.1
58.4
64.3
35.2
48.5
17.3
37.6
24.3
25.5
41.1
81.1
94.6
58.2
54.2
63.5
67.6
32.6
41.7
43.6
82.5
39.3
47.0
33.1
43.1
36.6
MOLT-4
RPMI-8226
SR
58.8
Non-small cell lung cancer
A549/ATCC
HOP-62
89.1
45.1
71.8
43.7
94.2
24.9
HOP-92
89.6
47.6
66.2
19.5
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
LXFL 529
Small cell lung cancer
DMS 114
DMS 273
Colon cancer
COLO 205
DLD-1
1.03
18.1
96.8
45.3
30.0
38.7
75.6
37.8
51.5
54.6
66.2
58.6
55.2
70.0
46.9
34.9
34.3
36.9
43.1
42.4
78.4
51.0
44.4
81.6
36.6
43.8
55.3
68.0
74.2
83.6
22.4
63.8
49.3
78.0
52.0
60.6
70.7
HCT-116
HCT-15
45.7
97.5
87.5
77.6
53.2
HT29
KM12
KM20L2
SW-620
CNS cancer
SF-268
27.3
74.1
61.6
96.5
SF-295
99.3
SF-539
SNB-19
SNB-75
20.1
46.7
85.2
79.4
U251
37.1
8.2
79.1
XF 498
Melanoma
LOX IMVI
MALME-3M
M14
56.1
77.4
36.3
76.4
87.2
58.6
M19-MEL
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
OVCAR-3
Renal cancer
786-0
35.5
71.5
92.4
38.7
30.8
31.3
51.0
39.7
66.3
91.1
45.9
57.2
36.4
43.6
ACHN
0.13
69.2
76.3
22.5
44.4
CAKI-1
RXF-393
UO-31
35.7
48.3
15.6
68.8
20.1
Breast cancer
MCF7
79.0
19.0
HS 578T
27.4
a
The notation GI50 refers to the tested compound concentration that produced 50% growth inhibition. Only the values B
/
100 mM are reported.

A. Ranise et al. / Il Farmaco 58 (2003) 765Á
/780
777
N-Cyclohexyl-[(4-phenylpiperazin-1-yl)carbo-
nothioyl]-2-furamide (8Ar3). C22H27N3O2S, MM
(s, 3H, CH3N), 6.40Á
(m, 9H, arom. H and H-3, H-4, H-5 pyr.).
4-Chloro-N-methyl-[(4-pyridin-2-ylpiperazin-1-yl)car-
/
6.85 and 7.25Á
/
8.00 and 8.10Á
/
8.40
397.54, Yield 43%, m.p. 132Á
/
133 8C (CH2Cl2/MeOH).
IR (KBr): 1635 cmꢀ1; H NMR (CDCl3): d 1.00Á
2.55
(m, 10H, 5CH2), 2.95Á3.55 (m, 4H, 2CH2/pip), 3.80Á
4.70 (m, 5H, 2CH2N/pip and CHN), 6.40Á6.65 (m, 1H,
H-4 fur.), 6.70Á7.70 (m, 7H, arom. H and H-3, H-5
fur.).
1
/
bonothioyl]benzamide (10Ar2). C18H19ClN4OS, MM
/
/
374.89, Yield 74%, m.p. 153Á
/
154 8C (CH2Cl2/MeOH).
IR (KBr): 1640 cmꢀ1; H NMR (CDCl3): d 3.35Á
4.00
(m, 8H, 4CH2/pip), 3.50 (s, 3H, CH3N), 6.48Á6.90 and
7.25Á7.95 and 8.10Á8.40 (m, 8H, arom. H and H-3, H-4,
H-5 pyr.).
1
/
/
/
/
/
/
N-Cyclohexyl-[(4-phenylpiperazin-1-yl)carbo-
nothioyl]thiophene-2-carboxamide
(8Ar4).
N-Methyl-[(4-pyridin-2-ylpiperazin-1-yl)carbo-
nothioyl]-2-furamide (10Ar3). C16H18N4O2S, MM
C22H27N3OS2, MM 413.61, Yield 68%, m.p. 135Á
136 8C (CH2Cl2/MeOH). IR (KBr): 1615 cmꢀ1 1H
NMR (CDCl3): d 0.90Á2.70 (m, 10H, 5CH2), 2.80Á
3.50 (m, 4H, 2CH2/pip), 3.65Á4.80 (m, 5H, 2CH2N/pip
and CHN), 6.65Á7.650 and 7.80Á8.05 (m, 8H, arom H.
and H-3, H-4, H-5 thioph.).
/
;
330.41, Yield 67%, m.p. 104Á
IR (KBr): 1660 cmꢀ1; 1H NMR (CDCl3): d 3.38 (s, 3H,
CH3N), 3.50Á3.85 (m, 4H, 2CH2/pip), 3.90Á4.30 (m,
4H, 2CH2N/pip), 6.40Á6.95 (m, 3H, H-4 fur. and 2H
pyr.), 7.25 (d, Jꢂ4 Hz, 1H, H-3 fur.), 7.45Á7.85 (m, 2H,
H-5 fur; and 1H pyr.), 8.15Á8.45 (m, 1H, H-6 pyr.).
N-Methyl-[(4-pyridin-2-ylpiperazin-1-yl)carbo-
nothioyl]thiophene-2-carboxamide (10Ar4).
C16H18N4OS2, MM 346.48, Yield 82%, m.p. 149Á
150 8C (CH2Cl2/MeOH). IR (KBr): 1640 cmꢀ1 1H
NMR (CDCl3): d 3.38Á3.78 (m, 4H, 2CH2/pip), 3.48
(s, 3H, CH3N), 3.80Á4.25 (m, 4H, 2CH2N/pip), 6.50Á
6.90 and 7.00Á7.25 and 7.35Á7.90 and 8.15Á8.45 (m,
7H, pyr. and thioph. arom. H.).
N-Cyclohexyl-[(4-pyridin-2-ylpiperazin-1-yl)carbo-
nothioyl]benzamide (11Ar1). C23H28N4OS, MM
408.57, Yield 59%, m.p. 136Á137 8C (CH2Cl2/MeOH).
IR (KBr): 1660 cmꢀ1; H NMR (CDCl3): d 1.00Á
2.80
(m, 10H, 5CH2), 3.15Á4.05 (m, 8H, 4CH2/pip), 4.20Á
4.90 (m, 1H, CHN), 6.35Á6.95 and 7.10Á8.05 (m, 8H,
arom. H and H-3, H-4, H-5 pyr.), 8.10Á8.35 (m, 1H, H-
6 pyr.).
/
105 8C (CH2Cl2/MeOH).
/
/
/
/
/
/
/
/
/
/
4-Chloro-N-phenyl-[(4-phenylpiperazin-1-yl)carbo-
nothioyl]benzamide (9Ar2). C24H22ClN3OS, MM
/
435.98, Yield 71%, m.p. 145Á
/
147 8C (CH2Cl2/MeOH).
IR (KBr): 1670 cmꢀ1; H NMR (CDCl3): d 3.00Á
3.50
(m, 4H, 2CH2/pip), 3.90Á4.50 (m, 4H, 2CH2N/pip),
6.65Á7.80 (m, 14H, arom. H).
1
/
/
/
;
/
/
N-Phenyl-[(4-phenylpiperazin-1-yl)carbonothioyl]-2-
furamide (9Ar3). C22H21N3O2S, MM 391.49, Yield
/
/
/
/
/
67%, m.p. 141Á
/
142 8C (CH2Cl2/MeOH). IR (KBr):
1675 cmꢀ1; H NMR (CDCl3): d 3.05Á
3.50 (m, 4H,
2CH2/pip), 4.00Á4.45 (m, 4H, 2CH2N/pip), 6.40Á6.60
(m, 1H, H-4 fur.), 6.70Á7.75 (m, 12H, arom. H and H-3,
H-5 fur.).
1
/
/
/
/
/
1
/
N-Phenyl-[(4-phenylpiperazin-1-yl)carbo-
nothioyl]thiophene-2-carboxamide
/
/
(9Ar4).
/
/
C22H21N3OS2, MM 407.56, Yield 92%, m.p. 165Á
167 8C (CH2Cl2/MeOH). IR (KBr): 1650 cmꢀ1 1H
NMR (CDCl3): d 3.15Á3.55 (m, 4H, 2CH2/pip), 4.10Á
4.50 (m, 4H, 2CH2N/pip), 6.80Á7.75 (m, 13H, arom. H
and H-3, H-4, H-5 thioph.).
/
/
;
/
/
4-Chloro-N-cyclohexyl-[(4-pyridin-2-ylpiperazin-1-
yl)carbonothioyl]benzamide (11Ar2). C23H27ClN4OS,
/
MM 443.01, Yield 59%, m.p. 117Á
/
119 8C (CH2Cl2/
MeOH). IR (KBr): 1660 cmꢀ1; H NMR (CDCl3): d
1.00Á2.80 (m, 10H, 5CH2), 3.10Á4.90 (m, 8H, 4CH2/pip
and CHN), 6.35Á7.00 and 7.20Á8.40 (m, 8H, arom. H
and H-3, H-4, H-5, H-6 pyr.).
1
/
/
4.1.4. General procedure for preparation of ATU
derivatives 10Á12
/
/
/
In an anhydrous pyridine solution (20 ml) 1-(2-
pyridyl)piperazine (1.63 g, 10 mmol) and proper iso-
thiocyanate (methyl-, cyclohexyl-, phenyl-isothiocya-
nate, 10 mmol) were allowed to react for 10 min at r.t.
under stirring. After adding a 60% sodium hydride
N-Cyclohexyl-[(4-pyridin-2-ylpiperazin-1-yl)carbo-
nothioyl]-2-furamide (11Ar3). C21H26N4O2S, MM
398.53, Yield 65%, m.p. 139Á
/
140 8C (CH2Cl2/MeOH).
IR (KBr): 1640 cmꢀ1; H NMR (CDCl3): d 1.00Á
2.60
(m, 10H, 5CH2), 3.35Á3.80 (m, 4H, 2CH2/pip), 3.85Á
4.80 (m, 5H, 2CH2N/pip and CHN), 6.35Á6.95 and
7.10Á7.80 (m, 6H, H-3, H-4, H-5 fur. and H-3, H-4, H-5
pyr.), 8.05Á8.35 (m, 1H, H-6 pyr.).
N-Cyclohexyl-[(4-pyridin-2-ylpiperazin-1-yl)carbo-
nothioyl]thiophene-2-carboxamide (11Ar4).
C21H26N4OS2, MM 414.50, Yield 60%, m.p. 118Á
119 8C (CH2Cl2/MeOH). IR (KBr): 1620 cmꢀ1 1H
NMR (CDCl3): d 1.00Á2.65 (m, 10H, 5CH2, 3.20Á
3.68 (m, 4H, 2CH2/pip), 3.72Á4.80 (m, 5H, 2CH2N/pip
and CHN), 6.45Á7.20 and 7.30Á8.00 (m, 6H, arom H.
1
/
/
/
dispersion in mineral oil (0.4 g, Â10 mmol) portion wise
/
/
at r.t., the reaction mixture was kept under stirring, until
hydrogen evolution subsided. Then, neat acyl chloride
(10 mmol) was added in a single portion at r.t. The
/
/
reaction mixture was heated at 55Á60 8C for 4 h. For
/
work-up procedure see Section 4.1.3.N-Methyl-[(4-pyr-
idin-2-ylpiperazin-1-yl)carbonothioyl]benzamide
(10Ar1)
/
;
/
/
C18H20N4OS, MM 340.45, Yield 63%, m.p. 101Á
103 8C (CH2Cl2/MeOH). IR (KBr): 1655 cmꢀ1 1H
NMR (CDCl3): d 3.80Á4.10 (m, 8H, 4CH2/pip), 3.52
/
/
;
/
/
/

778
A. Ranise et al. / Il Farmaco 58 (2003) 765Á780
/
and H-3, H-4, H-5 thioph. and H-3, H-4, H-5 pyr.),
8.10Á8.40 (m, 1H, H-6 pyr.).
4-Chloro-N-phenyl-[(4-pyridin-2-ylpiperazin-1-yl)car-
a time showing an outstanding reduction of hypercho-
lesterolemia. Interestingly, also some aryloxyalkylthioi-
midazoles, inhibitors of acyl-CoA: cholesterol-O-
acyltransferase, gave similar effects [20]. The activity
trend in the 4-methylpiperazine derivatives is in the
/
bonothioyl]benzamide (12Ar2). C23H21ClN4OS, MM
436.96, Yield 57%, m.p. 125Á
/
126 8C (CH2Cl2/MeOH).
IR (KBr): 1670 cmꢀ1; H NMR (CDCl3): d 3.50Á
3.85
(m, 4H, 2CH2/pip), 3.90Á4.50 (m, 4H, 2CH2N/pip),
6.50Á6.95 and 7.05Á7.80 (m, 12H, arom. H and H-3, H-
4, H-5 pyr.), 8.10Á8.40 (m, 1H, H-6 pyr.).
1
/
order: 6Ar2ꢀ
/
4Ar4ꢀ
/
5Ar4:
/
6Ar3ꢀ5Ar3, indicating
/
/
that the best results are obtained, when R1Á3 and
Ar1Á4 are aliphatic and prevalently heteroaromatic,
respectively. Also in the 4-(hetero)arylpiperazines,
when R1Á3, is an aliphatic function (compounds
11Ar1, 8Ar3, 11Ar2, 9Ar3), an enhanced activity was
observed, whereas a major tolerability to the nature of
the acyl substituent is allowed. In some cases, it is
worthwhile noting how activity of the close analogues
could be deeply affected by alteration of only one of the
/
/
/
N-Phenyl-[(4-pyridin-2-ylpiperazin-1-yl)carbo-
nothioyl]-2-furamide (12Ar3). C21H20N4O2S, MM
392.48, Yield 76%, m.p. 127Á
/
128 8C (CH2Cl2/MeOH).
IR (KBr): 1670 cmꢀ1; H NMR (CDCl3): d 3.50Á
3.90
(m, 4H, 2CH2/pip), 3.95Á4.40 (m, 4H, 2CH2N/pip),
6.40Á7.00 and 7.30Á7.80 (m, 11H, arom H. and H-3, H-
4, H-5 fur. and H-3, H-4, H-5 pyr.), 8.10Á8.40 (m, 1H,
H-6 pyr.).
N-Phenyl-[(4-pyridin-2-ylpiperazin-1-yl)carbo-
nothioyl]thiophene-2-carboxamide (12Ar4).
C21H20N4OS2, MM 408.55, Yield 94%, m.p. 159Á
160 8C (CH2Cl2/MeOH). IR (KBr): 1650 cmꢀ1 1H
NMR (CDCl3): d 3.50Á3.90 (m, 4H, 2CH2/pip), 4.00Á
4.40 (m, 4H, 2CH2N/pip), 6.50Á7.10 and 7.20Á7.80 and
8.10Á8.40 (m, 11H, arom H. and H-3, H-4, H-5 thoph.
and H-3, H-4, H-5 pyr.), 8.10Á8.40 (m, 1H, H-6 pyr.).
1
/
/
/
/
/
variable groups, in particular R1Á3 or Ar1Á4, (6Ar4ꢁ
/
4Ar4; 7Ar3ꢀ8Ar3ꢀ9Ar3; 6Ar4ꢁ6Ar2). Interestingly,
/
/
/
some compounds showed a certain degree of selectivity.
Thus, 8Ar3 caused a 40% reduction of triglycerides,
scarcely influencing the total, free and esterified choles-
terol levels. On the contrary, 9Ar2 and 12Ar4 signifi-
cantly decreased hypercholesterolemia, causing about
12% (9Ar2) or no (12Ar4) reduction of hypertriglycer-
idemia. Interestingly, R and R1Á3 of these ATUs are
(hetero)aromatic (phenyl for 8Ar3 and 9Ar2; 2-pyridyl
for 12Ar4) and cyclic (ciclohexyl for 8Ar3, phenyl for
9Ar2 and 12Ar4), respectively. Therefore, selectivity
might depend on fine-tuning these substituents. Because
in some forms of dysliproteinemia, only one of the
above serum lipid parameters is altered, it is important
to find new agents, which reduce either hypercholester-
olemia or hypertriglyceridemia. Moreover, the pharma-
cological protocol available for the treatment of
hypertriglyceridemia is quite small [21].
/
;
/
/
/
/
/
/
4.2. Pharmacology
All the compounds were assayed for local anaesthetic
[11], anti-iperlipidemic [12], antiarrythmic [13], analgesic
[14], anti-inflammatory [15], platelet antiaggregating [16]
and antiproliferative [17Á19] activities, evaluated ac-
/
cording to previously described standard procedures.
As concerns analgesic activity (Table 3), the best
results were obtained (5Ar2, 5Ar4, 9Ar2 and 5Ar3, 10Ar3
5. Discussion and conclusion
12Ar2), when R1Á3 is cyclic (C6H11ꢀC6H5), and Ar1Á4
/
The SARs will be discussed taking into account the
principal structural parameters, relevant to each of the
tested activities.
is heteroaryl or 4-chlorophenyl. Taken together, the
data indicate that electronic factors are not critical for
activity.
Widespread local anaesthetic activity, reported in
Table 1, seems to be due to the features of the whole
molecule rather than of one variable region. Never-
theless, it emerges that the benzoyl group does not
favour activity. When compared to derivatives B (Chart
1), the tested compounds are in general more active,
indicating that their substitution patterns elicit better
anaesthetic effects.
In evaluating their antihyperlipidemic properties (Ta-
ble 2), several compounds (4Ar4, 6Ar2, 6Ar4, 7Ar3, 8Ar3,
9Ar3, 11Ar1 and 11Ar2) belonging to the three series
were more active than nicotinic acid in lowering
trygliceride levels, but they showed lower efficacy in
decreasing serum hypercholesterolemia. Notably, 6Ar4
and 7Ar3, the most active compounds, reduced the
almost tripled triglyceride levels to near normality, at
Only few compounds, 5Ar3, 8Ar1 and 11Ar2, were
endowed with significant anti-inflammatory activity
(Table 4), that seems to depend on the presence of the
cyclohexyl substituent. When compared to piperidinoa-
cylureas B, the title compounds turn out to be less active
as anti-inflammatory agents. This indicates that isosteric
replacements of the urea carbonyl group and the
piperidine nucleus of B with the thione group and the
4-substituted piperazine ring, respectively, are not
beneficial to anti-inflammatory activity.
The SARs concerning antirrhythmic activity (Table 5)
display that the most active compounds 4Ar2, 10Ar1 are
featured by the same group R1Á3(CH3), and by the
benzoyl and 4-chlorobenzoyl moieties as substituents of
the thiourea nitrogen, respectively. In these two series,
replacement of the methyl group with the cyclohexyl

A. Ranise et al. / Il Farmaco 58 (2003) 765Á
/780
779
ring gives either derivatives still capable of protracting
the appearance time of extrasystoles (11Ar2) or poorly
active (5Ar2).
In the inhibition test of aggregation (Table 6), a
number of the most active compounds belongs to the 4-
methylpiperazine series (4Ar3, 5Ar2, 6Ar4). More in
general, the best combination of the variable substitu-
ents is R1Á3: CH3, C6H11 and Ar1Á4: 4-chlorophenyl or
heteroaryl, respectively.
References
[1] A. Ranise, F. Bondavalli, O. Bruno, S. Schenone, C. Losasso, M.
Costantino, M.L. Cenicola, D. Donnoli, E. Marmo, 3-Disubsti-
tuted 1-acyl-1-phenylthioureas with platelet antiaggregating and
other activities, Farmaco 46 (1991) 317Á338.
/
[2] A. Ranise, S. Schenone, O. Bruno, F. Bondavalli, W. Filippelli,
G. Falcone, B. Rivaldi, N-Acyl-N-phenyl ureas of piperidine and
substituted piperidines endowed with anti-inflammatory and anti-
proliferative activities, Farmaco 56 (2001) 647Á
[3] A.E. Dixon, J. Hawthorne, The action of acyl chlorides on
thiourea, J. Chem. Soc. 91 (1907) 122Á146.
[4] Series Editor S. Patai, The chemistry of amide, In: J. Zabicky, W.
Walter, J. Voss (Eds.), The Chemistry of Thioamide, Interscience
Publishers, NY, 1970, chapter 8, p. 445.
/
657.
Finally, it is apparent that the 4-chlorophenyl and, in
a lesser degree, 2-thienyl substituents are molecular
determinants for antiproliferative activity (4Ar2, 8Ar2,
5Ar2, 7Ar2, 11Ar2, 11Ar2) (Table 7), the other variable
groups playing a secondary role. The cytotoxic effects of
these ATUs were approximately similar to that of some
isosteres B. This can be reasonably related to the
presence of the acyl(thio)urea moiety in both the series.
The NCI anticancer drug discovery screen has been
designed to distinguish between broad spectrum anti-
tumor compounds and subpanel-selective antiprolifera-
tive agents. In the present study, 4Ar2 and, to a lesser
extent, 8Ar2 showed a distinctive pattern of selectivity
(ACHN and NCI-H226 cell lines, respectively), whereas
5Ar2, 7Ar2, 11Ar2, 11Ar2 and 12Ar2 exhibited a broad
spectrum antitumor activity, even if at higher concen-
trations. Provided that only mechanistic investigations
can explain this differential response, selective cytotoxi-
city of 4Ar2 and 8Ar2 might be mediated, at least in part,
by their preferential transport and accumulation in the
above sensitive cell lines. Effectively, these phenomena
can influence cytotoxicity, as proved for some selective
cytotoxic agents [22].
/
[5] Editor S. Patai, The chemistry of acyl halides. D.P.N. Satchell,
R.S. Satchell, AcidÁbase Behaviour and Complex Formation,
Interscience Publisher, NY, 1972, chapter 4, p. 122.
[6] A. Ranise, Unpublished results.
/
[7] P.A.S. Smith, The Chemistry of Open-chain Organic Nitrogen
Compounds, vol. I, W.A. Benjamin, NY, 1965, p. 275 (chapter
VI).
[8] A. Ranise, F. Bondavalli, O. Bruno, S. Schenone, D. Donnoli, C.
Parrillo, M.L. Cenicola, F. Rossi, 1-Acyl-, 3-acyl- and 1,3-diacyl-
3-furfuryl-1-phenylthioureas with platelet antiaggregating and
other activities, Farmaco 46 (1991) 1203Á
[9] (a) R.G. Pearson, Hard and soft acids and bases, J. Am. Chem.
Soc. 85 (1963) 3533Á3539;
(b) R.G. Pearson, Chemical hardness and bond dissociation
energies, J. Am. Chem. Soc. 110 (1988) 7684Á7690.
/
1216.
/
/
[10] H. Kohn, M.J. Cravey, J.H. Arceneaux, R.L. Cravey, M.R.
Willcott, Synthesis and spectral properties of substituted imida-
zolidones and imidazolines, J. Org. Chem. 42 (1977) 941Á
[11] C. Bianchi, A simple new quantitative methods for testing local
anaesthetics, Br. J. Pharmacol. 11 (1956) 104Á106.
/948.
/
[12] E. Marmo, C. Vacca, L. Giordano, R. Petrarca, L. Barone, C.
Visone, F. Del Vecchio, Chenic acid, ursic acid and experimental
hyperlipidemia, Boll. Chim. Farm. 117 (1978) 416Á423.
/
In conclusion, we have synthesised and evaluated
three series of ATUs sharing the 4-substituted piper-
azine scaffold. The results emerged from in vivo and in
vitro tests show that several compounds are endowed
with various interesting pharmacological properties. In
particular, local anaesthetic, antihyperlipemic and anti-
proliferarive activities turn out to be prominent. There-
fore, further SAR studies of ATU derivatives are in
progress.
[13] E. Marmo, G. Lampa, F. Rossi, A.P. Caputo, S. Chieppa, C.
Vacca, C. Giordano, P. Pedone, Ricerche sperimentali sulla
specificita` ed aspecificita` di un b-adrenolitico, il bunitrololo,
Arch. Sci. Med. 135 (1978) 15Á16.
/
[14] N.B. Eddy, C. Fuhrmeister Touchberry, J.E. Lieberman, Syn-
thetic analgesics. I. Methadon isomers and derivatives, J. Phar-
macol. Expt. Therap. 98 (1950) 121Á137.
/
[15] C.A. Winter, E.A. Risley, G.W. Nuss, Carragenin-induced edema
in hind paw of the rat as an assay for anti-inflammatory drugs,
Proc. Soc. Exp. Biol. Med. 111 (1956) 104Á106.
/
[16] A. Bargagna, M. Longobardi, E. Mariani, P. Schenone, S. Russo,
S. Vilagliano, V. De Novellis, E. Marmo, N,N-Disubstituted 4-
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