Synthesis and antiplatelet activity of thioaryloxyacids analogues of clofibric acid

Article abstract of DOI:10.1016/j.ejmech.2005.03.022

The thiophene-, benzothiazole- and pyridine-thioaryloxyacids analogues of clofibric acid were synthesized and their antiplatelet activity was screened. Some compounds exhibited antiaggregating properties. The platelet-related haemostasis was measured on a PFA-100 analyzer using bull blood.

Full text of DOI:10.1016/j.ejmech.2005.03.022

European Journal of Medicinal Chemistry 40 (2005) 918–921
www.elsevier.com/locate/ejmech
Short communication
Synthesis and antiplatelet activity of thioaryloxyacids
analogues of clofibric acid
a
a,
b
a
Alessandra Ammazzalorso , Rosa Amoroso *, Mario Baraldi , Giancarlo Bettoni ,
a
a
a
Daniela Braghiroli , Barbara De Filippis , Letizia Giampietro ,
a
b
Maria L. Tricca , Francesca Vezzalini
a
Dipartimento di Scienze del Farmaco, Università degli Studi “G. D’Annunzio”, via dei Vestini, 66100 Chieti, Italy
b
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Modena e Reggio Emilia, via Campi 183, 41100 Modena, Italy
Received 4 November 2004; received in revised form 17 March 2005; accepted 22 March 2005
Available online 13 June 2005
Abstract
The thiophene-, benzothiazole- and pyridine-thioaryloxyacids analogues of clofibric acid were synthesized and their antiplatelet activity
was screened. Some compounds exhibited antiaggregating properties. The platelet-related haemostasis was measured on a PFA-100® ana-
lyzer using bull blood.
©
2005 Elsevier SAS. All rights reserved.
Keywords: Clofibrate bioisosters; Antiplatelet activity; Fibrates; Thioaryloxyacids; PFA-100®
1
. Introduction
a dependence of biological activity on chirality, with the (R)-
compounds having greater antiplatelet effect and less toxic-
ity than (S)-isomers [5]. Subsequently, chiral analogues of
gemfibrozil were synthesized and all the tested compounds
inhibit the human platelet aggregation [6].
The fibrates are a widely used class of lipid-modifying
agents that decrease plasma triglycerides [1]; they activate
the peroxisome proliferator-activated receptors (PPARs),
belonging to the nuclear hormone receptor superfamily, induc-
ing alterations in transcription of genes encoding for proteins
that control lipoprotein metabolism [2]. In addition to the anti-
lipidemic effect, the fibrates and the related drug gemfibrozil
act on plasma concentration of proteins involved in coagula-
tion, fibrinolysis and platelet action; several studies indicate
that fibrates decrease the levels of factors promoting coagu-
lation and increase fibrinolysis [3]. This dual anti-
lipidemic/antiplatelet effect of fibrates makes them interest-
ing candidates for the prevention and treatment of thrombotic
disorders.
Herein we report the synthesis and the pharmacological
evaluation of analogues of clofibric acid with a heterocycle
instead of benzene ring and with a thioisobutyrate side-chain
(Fig. 1). A simple and efficient method for platelet-related
haemostasis analysis, involving the use of animal blood
samples, was adopted.
2. Chemistry
Reported in previous publications, the synthesis of chiral
analogues of clofibric acid, the active metabolite of clofi-
brate, with the asymmetric carbon next to the carbonyl group
was described [4]; the two enantiomers in which the substitu-
ents on stereogenic center are methyl and ethyl groups showed
The 4a–e acids were synthesized as outlined in Schemes
and 2. The compounds 3a–d were readily obtained by reac-
tion of ethyl 2-bromoisobutyrate (1) with sodium salts of com-
mercially available thiols 2a–d, in refluxing EtOH. The com-
pound 3e was synthesized by chlorination of 3a with
1
N-chlorosuccinimide in glacial CH COOH at room tempera-
3
ture.All the esters 3a–e were hydrolyzed in presence of KOH
to give the acids 4a–e.
*
Corresponding author. Tel.: +39 0871 355 5386; fax: +39 0871 355 5267.
E-mail address: ramoroso@unich.it (R. Amoroso).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.03.022

A. Ammazzalorso et al. / European Journal of Medicinal Chemistry 40 (2005) 918–921
919
Fig. 1. Analogues of clofibric acid.
3
. Pharmacological evaluation and discussion
cates the platelet function in the sample. If an occluding plate-
let plug does not form after 300 s, the analysis is stopped.
Table 1 shows the results of the experiments on platelet
aggregation induced by ADP, detected at concentrations of
4a–e ranging from 1.0 to 5.0 mM. An inhibitory effect was
found for the basic compound gemfibrozil (entry 6). TheASA
was screened as negative control, because COL/ADP test car-
tridges detect platelet dysfunctions not mediated by ASA
(entry 7) [8]. Compound 4d showed the best activity among
all the tested compounds (entry 4), revealing a valuable dose-
dependent pattern. Compound 4e is only effective at high con-
centrations (entry 5). The others have low effect and no dose-
effect relationships (entries 1–3). The presence of a chlorine
atom on the heterocyclic system seems to increase the activ-
ity: compounds without this substituent showed lower anti-
aggregating properties (entries 1–3) than corresponding sub-
Effects of compounds 4a–e on platelet aggregation were
monitored with a PFA-100® instrument [7] using anticoagu-
lated bull blood. Gemfibrozil and acetylsalicilic acid (ASA)
were used as reference drugs.
The PFA-100® is a platelet function analyzer which allows
to study reactivity of blood platelets under conditions similar
to those in circulation. In this apparatus, blood samples are
introduced into a disposable cartridge and aspirated through
a small aperture into a membrane coated with collagen–ADP
(COL/ADP) or collagen–epinephrine (COL/EPI). In response
to stimulation by the components of membrane, platelets form
a stable plug that occludes the aperture and blood flow stops.
The time from the beginning of the test until formation of an
occluding platelet plug is called “closure time” (CT) and indi-
Scheme 1
.
Scheme 2
.

920
A. Ammazzalorso et al. / European Journal of Medicinal Chemistry 40 (2005) 918–921
Table 1
Antiplatelet effect of compounds 4a–e
reduced pressure. The residue was purified by column chro-
matography on silica gel (eluent cyclohexane/ethyl acetate).
Percentage inhibition
Entry Compound
1 mM 2 mM
3 mM
2.27
0
4 mM
0.45
1.82
6.82
100
5 mM
4.1
0
5
.2.1. Ethyl 2-methyl-2-(thien-2-ylthio)propanoate (3a)
Yellow oil, 3.849 g, 16.7 mmol, 97% yield; H NMR
1
2
3
4
5
6
7
4a
4b
4c
4d
4e
0.91
0
0.45
0.91
2.27
6.82
0
1
(CDCl ): d 1.25 (t, 3H, CH CH ), 1.50 (s, 6H, (CH ) C), 4.14
3 2 3 3 2
2.27
0.91
0
4.51
11.36
0
15
(q, 2H, CH CH ), 7.01–7.04 (dd, 1H, Ar), 7.16 (d, 1H, Ar),
100
100
100
5.4
2 3
–
1
7
.45 (d, 1H, Ar); IR (neat) 1724 cm .
36.82
100
Gemfibrozil 0.91
ASA
0.91
0
26.36
0.91
0
0.45
5.2.2. Ethyl 2-methyl-2-(pyridin-2-ylthio)propanoate (3b)
1
Yellow oil, 3.555 g, 14.8 mmol, 92% yield; H NMR
stituted ones (entries 4–5). The size of heterocycle may also
influence the results, with benzothiazolthioaryloxyacids
(CDCl ): d 1.18 (t, 3H, CH CH ), 1.67 (s, 6H, (CH ) C), 4.16
3
2
3
3 2
(q, 2H, CH CH ), 6.97–7.01 (dd, 1H, Ar), 7.20 (d, 1H, Ar),
2
3
–
1
(
entries 3–4) more active than thiophene- and pyridine-
7.48 (dt, 1H, Ar), 8.38 (dd, 1H, Ar); IR (neat) 1730 cm .
derivatives (entries 1–2). The more active compound 4d fits
in each of two conditions.
5.2.3. Ethyl 2-(1,3-benzothiazol-2-ylthio)-2-methyl-
propanoate (3c)
1
Yellow oil, 3.630 g, 12.9 mmol, 75% yield: H NMR
4. Conclusions
(CDCl ): d 1.20 (t, 3H, CH CH ), 1.75 (s, 6H, (CH ) C), 4.20
3
2
3
3 2
(
q, 2H, CH CH ), 7.33 (t, 1H, Ar), 7.43 (t, 1H, Ar), 7.78 (d,
2 3
–
1
In conclusion, we synthesized analogues of clofibric acid
1H, Ar), 7.92 (d, 1H Ar); IR (neat) 1731 cm .
with a thioisobutyrate side-chain bound to a heterocyclic
group. Some compounds revealed a dose-dependent antiplate-
let activity, measured on a PFA-100® instrument, using bull
blood. The simplicity of method and the easy availability of
animal blood allow a quick and cheap measurement of a stan-
dardized in vitro bleeding time, reflecting platelet function.
5.2.4. Ethyl 2-[(5-Chloro-1,3-benzothiazol-2-yl)thio]-2-
methylpropanoate (3d)
1
Oil, 2.879 g, 9.12 mmol, 53% yield; H NMR (CDCl ): d
3
1.21 (t, 3H, CH CH ), 1.75 (s, 6H, (CH ) C), 4.20 (q, 2H,
2
3
3 2
CH CH ), 7.28–7.32 (dd, 1H, Ar), 7.67 (d, 1H, Ar), 7.88 (d,
2
3
–
1
1
H, Ar); IR (neat) 1733 cm .
5. Experimental protocols
5.3. Experimental procedure for the synthesis of ethyl
2
-[(5-chlorothien-2-yl)thio]-2-methylpropanoate (3e)
5.1. Chemistry
To a solution of ethyl 2-methyl-2-(thien-2-ylthio)propa-
Melting points were determined on an Electrothermal IA
100 apparatus and are uncorrected. The infrared spectra were
noate (3a) (1.843 g, 18.0 mmol) in glacial CH COOH (20 ml),
N-chlorosuccinimide (1.068 g, 8.0 mmol) was added under
3
9
recorded on a FT-IR 1600 Perkin–Elmer spectrometer. The
NMR spectra were run at 300 MHz on aVarian spectrometer;
chemical shifts (d) are reported in ppm. Microanalyses were
carried out with a Eurovector Euro EA 3000 model analyzer
and the analytical results are within 0.4% of the theoretical
values. GC analyses were run on an autosystem GC Perkin
Elmer apparatus using a fused silica capillary column (30 m,
N atmosphere. After stirring for 3 h at room temperature,
the solvent was removed under reduced pressure. The resi-
due was dissolved in ether (15 ml) and washed with 2 M
2
NaOH solution (15 ml) and with H O (15 ml). The organic
2
layer was dried over Na SO and the solvent removed under
2
4
reduced pressure. The residue was purified by column chro-
matography on silica gel (eluent cyclohexane/chloroform 7:3)
to give the ester 3e (yellow oil, 635 mg, 2.39 mmol, 30%
0.53 mm ID), SPB-5 Supelco. Commercial reagents were used
–
1 1
as received from Aldrich.
yield). IR (neat) 1725 cm ; H NMR (CDCl ): d 1.27 (t, 3H,
3
CH CH ), 1.50 (s, 6H, (CH ) C), 4.15 (q, 2H, CH CH ), 6.86
2
3
3 2
2
3
5.2. General experimental procedure for the synthesis
(d, 1H, Ar), 6.96 (d, 1H, Ar).
of esters 3a–d
5
.4. General experimental procedure for the synthesis
To a solution of sodium (395 mg, 17.2 mmol) in absolute
of acids 4a–e
EtOH (15 ml), ethyl bromoisobutyrate (1) (3.355 g,
1
7.2 mmol) and the heterocyclic thiol 2a–d (17.2 mmol), both
To a solution of 1% alcoholic KOH (7 ml), the ester 3a–e
was added (0.87 mmol) and the mixture was refluxed for 2–4 h
under stirring. The reaction was quenched with 2 M KOH
(25 ml) and the solvent was removed under reduced pressure.
The aqueous residue was extracted with ether (2 × 25 ml),
acidified with 2 N HCl and re-extracted with CH Cl (3 ×
dissolved in absolute EtOH (10 ml), were added under N2
atmosphere. After stirring for 2–5 h at reflux, the solvent was
removed under reduced pressure. The residue was poured into
H O (45 ml) and extracted with ethyl ether (3 × 40 ml). The
organic layer was dried over Na SO and concentrated under
2
2
4
2
2

A. Ammazzalorso et al. / European Journal of Medicinal Chemistry 40 (2005) 918–921
921
2
5 ml). The organic layer was washed with H O (65 ml), dried
reservoirs containing 3.8% (0.129 M) buffered sodium cit-
rate (one part anticoagulant to nine parts blood). The haema-
tocrit value of anticoagulated bull blood ranged from 36% to
38%.Aliquots of 900 µl were incubated with 100 µl of known
concentrations of 4a–e acids at room temperature for 30 min.
Test compounds and gemfibrozil were dissolved in NaOH
2
over Na SO and concentrated under reduced pressure. The
residue was purificated by crystallization using hexane as sol-
vent.
2
4
5.4.1. 2-Methyl-2-(thien-2-ylthio)propanoic acid (4a)
White solid, 141 mg, 0.70 mmol, 80% yield; m.p. 50–
1
N and saline solution (1:10 v/v) and monitored at the final
–1 1
52 °C; IR (neat) 1692 cm ; H NMR (CDCl ): d 1.52 (s, 6H,
3
concentrations of 1, 2, 3, 4 and 5 mM. ASA was dissolved in
DMSO and saline solution (1:20 v/v) and tested at the same
concentrations. Experiments showed that there was no differ-
ence using NaOH or DMSO as vehicles in the aggregation
assays.
(CH ) C), 7.04–7.07 (dd, 1H, Ar), 7.23 (d, 1H, Ar), 7.48 (d,
3 2
1
3
1
1
H, Ar); C NMR (CDCl ): d 25.28 (CH ) , 52.37 (C),
3 3 2
27.95, 132.34 and 138.06 (Ar), 179.58 (C=O); MS m/z 201.3
–
(M ).
5
.4.2. 2-Methyl-2-(pyridin-2-ylthio)propanoic acid (4b)
5
.5.2. Antiplatelet assays
The tests were performed on PFA-100® instrument (Dade-
White solid, 103 mg, 0.52 mmol, 60% yield; m.p. 81–
–1 1
83 °C; IR (neat) 1701 cm ; H NMR (CDCl ): d 1.65 (s, 6H,
3
Behring, Milano, Italy) using only COL/ADP test cartridges,
that revealed a better sensitivity and data reproducibility with
bull blood respect to COL/EPI test cartridges. Blood samples
were gently mixed and aliquots of 800 µl were pipetted into
the smaller opening (sample reservoir opening) of the test car-
tridge, avoiding air entrapment in the sample reservoir. The
CT was determined and expressed in seconds. The percentage
of platelet aggregation inhibition was calculated in compari-
son with a control sample additioned with the proper vehicle.
(
CH ) C), 7.27 (t, 1H, Ar), 7.43 (d, 1H, Ar), 7.73 (dt, 1H,
3 2
13
Ar), 8.95 (dd, 1H, Ar); C NMR (CDCl ) d 26.27 (CH ) ,
5
3
3 2
2.58 (C), 121.81, 125.98, 138.85 and 147.05 (Ar), 157.11
+
+
(Ar), 174.84 (C=O); MS m/z 198.0 (MH ), 220.0 (M + Na).
5
.4.3. 2-(1,3-Benzothiazol-2-ylthio)-2-methylpropanoic
acid (4c)
White solid, 38 mg, 0.35 mmol, 40% yield; m.p. 154–
–1 1
1
57 °C; IR (neat) 1714 cm ; H NMR (CDCl ): d 1.76 (s,
3
6
H, (CH ) C), 7.43 (t, 1H, Ar), 7.52 (t, 1H, Ar), 7.82 (d, 1H,
3
2
13
Ar), 7.94 (d, 1H, Ar); C NMR (CDCl ): d 25.98 (CH ) ,
3
3 2
5
1
2
4.54 (C), 127.57, 125.96 and 127.22 (Ar), 134.90 (=C–S),
Acknowledgements
51.15 (=C–N), 158.69 (N=C–S), 173.69 (C=O); MS m/z
+
+
54.0 (MH ), 276.0 (M + Na).
This investigation was supported by Italian MIUR. The
authors gratefully acknowledge Dr. Leonardo Bigi and Dr.
Chiara Vendola of the Dideco S.r.L. (Mirandola, Modena,
Italy) for the technical collaboration.
5.4.4. 2-[(5-Chloro-1,3-benzothiazol-2-yl)thio]-2-methyl-
propanoic acid (4d)
White solid, 133 mg, 0.46 mmol, 53% yield; m.p. 125–
–1 1
1
6
1
1
1
2
30 °C; IR (neat) 1712 cm ; H NMR (CDCl ): d 1.78 (s,
H, (CH ) C), 7.37 (dd, 1H, Ar), 7.71 (d, 1H, Ar), 7.92 (d,
3
3
2
References
1
3
H, Ar); C NMR (CDCl ): d 26.02 (CH ) , 54.68 (C),
3
3 2
21.60, 122.15 and 126.27 (Ar), 123.70 (=C–S), 133.40 (CCl),
52.30 (=C–N), 168.20 (N=C–S), 174.11 (C=O); MS m/z
[
[
1] B. Staels, J. Dallongeville, J. Auwerx, K. Schoonjans, E. Leitersdorf,
J.C. Fruchart, Circulation 98 (1998) 2088–2093.
+
+
88.0 (MH ), 309.0 (M + Na).
2] T.M. Willson, P.J. Brown, D.D. Sternbach, B.R. Henke, J. Med.
Chem. 43 (2000) 527–550.
5
.4.5. 2-[(5-Chlorothien-2-yl)thio]-2-methylpropanoic acid
[
[
3] G. Shonfeld, Atherosclerosis 111 (1994) 161–174.
4] G. Bettoni, F. Loiodice, V. Tortorella, D. Conte-Camerino, M. Mam-
brini, E. Ferrannini, S.H. Bryant, J. Med. Chem. 30 (1987) 1267–
(4e)
Yellow solid, 123 mg, 0.52 mmol, 60% yield; IR (neat)
–
1 1
1
269.
1699 cm ; H NMR (CDCl ) d 1.52 (s, 6H, (CH ) C), 6.87
3 3 2
13
[5] K.J. Romsted, L. Lei, D.R. Feller, D.T. Witiak, F. Loiodice, V. Tor-
torella, Farmaco 51 (1996) 107–114.
[6] A.Ammazzalorso, R.Amoroso, M. Baraldi, G. Bettoni, D. Braghiroli,
B. De Filippis, A. Duranti, M. Moretti, P. Tortorella, M.L. Tricca,
F. Vezzalini, Bioorg. Med. Chem. Lett. 12 (2002) 817–821.
(d, 1H, Ar), 7.03 (d, 1H, Ar); C NMR (CDCl ): d 25.24
3
(CH ) , 52.58 (C), 127.33 (Ar), 128.52 (Ar), 135.02 (Ar),
3
2
–
1
37.95 (Ar), 179.50 (C=O); MS m/z 235 (M ).
5
5
.5. Pharmacology
[
7] S.H. Kundu, E.J. Eilmann, R. Sio, C. Garcia, R.M. Davidson,
R.A. Ostgaard, Semin. Thromb. Haemost 21 (1995) (Suppl. 2) 106–
.5.1. Preparation of blood samples
1
12.
Bull blood samples were supplied by a slaughter-house in
Pegognaga (Mantova, Italy) and drawn directly into plastic
[
8] V. Pengo, M. Boschello, A. Marzari, M. Baca, L. Schivazappa,
S. Dalla Volta, Thromb. Haemost. 56 (1986) 147–150.