Synthesis and antiplatelet activity of gemfibrozil chiral analogues

Article abstract of DOI:10.1016/S0960-894X(02)00021-5

The chiral analogues of gemfibrozil 5-(2,5-dimethylphenoxy)-2-methylpentanoic acid and 5-(2,5-dimethylphenoxy)-2-ethylpentanoic acid were synthesized in optically active form using (S)-4-(1-methylethyl)-2-oxazolidinone as chiral auxiliary. All compounds inhibit human platelet aggregation. From these data, one can surmise that all tested compounds and gemfibrozil act at the platelet level with different mechanism than that of ASA, even if with a different potency.

Full text of DOI:10.1016/S0960-894X(02)00021-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 817–821
Synthesis and Antiplatelet Activity of Gemfibrozil
Chiral Analogues
Alessandra Ammazzalorso,a Rosa Amoroso,a Mario Baraldi,b Giancarlo Bettoni,a,*
Daniela Braghiroli,a Barbara De Filippis,a Andrea Duranti,c Marco Moretti,d
Paolo Tortorella,a Maria Luisa Triccaa and Francesca Vezzalinib
a
`
Dipartimento di Scienze del Farmaco, Universita degli Studi ‘‘G. D’Annunzio’’, Via dei Vestini, 66100 Chieti, Italy
b
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy
c
`
Istituto di Chimica Farmaceutica, Universita di Urbino, P.zza Rinascimento 6, 61029 Urbino, Italy
dLaboratorio di Analisi Chimico Cliniche, Ospedale Civile S. Agostino, P.le S. Agostino 228, 41100 Modena, Italy
Received 19 September 2001; accepted 3 January 2002
Abstract—The chiral analogues of gemfibrozil 5-(2,5-dimethylphenoxy)-2-methylpentanoic acid and 5-(2,5-dimethylphenoxy)-2-
ethylpentanoic acid were synthesized in optically active form using (S)-4-(1-methylethyl)-2-oxazolidinone as chiral auxiliary. All
compounds inhibit human platelet aggregation. From these data, one can surmise that all tested compounds and gemfibrozil act at
the platelet level with different mechanism than that of ASA, even if with a different potency. # 2002 Elsevier Science Ltd. All
rights reserved.
Introduction
phenolate (2) in EtOH at reflux. Treatment of 1 with
LDA and alkylation of the anion formed with methyl
iodide or ethyl iodide gave in good chemical yield the
monoalkylated compounds rac-4a–b, that were easily
hydrolyzed in the presence of KOH to give the racemic
acids rac-5a–b.
Gemfibrozil, 5-(2,5-dimethylphenoxy)-2,2-dimethylpenta-
noic acid, is a widely used hypolipidemic drug related to
fibrates.1,2 In previous works, the synthesis of chiral ana-
logues of clofibrate was described3 and it exhibited differ-
ent biological activity related to the stereochemistry.4
Likewise, as part of an ongoing research on fibrates, chiral
analogues of gemfibrozil were prepared with an asym-
metric carbon atom next to the carboxyl group (Fig. 1).
Recent studies have shown that in addition to its effects
on lipids, gemfibrozil may modulate the fibrinolytic
system.5 Therefore, the optical pure analogues were tes-
ted in biological experiments in order to evaluate their
capability to inhibit the human platelet aggregation.
The (S)-4-isopropyl-2-oxazolidinone seems to be very
efficient as chiral auxiliary in the optical resolution of
the a-aryloxyacetic acids6 because of its well established
capacity of resolution via silica gel column and its abil-
ity to hydrolyze the N-acylated heterocycles under mild
conditions.
Chemistry
The 5a–b acids were synthesized as outlined in Scheme 1.
The ethyl 5-(2,5-dimethylphenoxy)pentanoate (3) was
obtained by SN2 reaction from the commercially available
ethyl 5-bromopentanoate (1) and sodium 2,5-dimethyl-
*Corresponding author. Tel.: +39-0871-355-5213; fax: +39-0871-
355-5267; e-mail: bettoni@unich.it
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00021-5

818
A. Ammazzalorso et al. / Bioorg. Med. Chem. Lett. 12 (2002) 817–821
After their transformation into the corresponding im-
ides with (S)-4-isopropyl-2-oxazolidinone, the chroma-
tographic resolution of the gemfibrozil chiral analogues
5a–b was performed. The chiral auxiliary was N-acyl-
ated7 with acyl chlorides rac-6a–b. The equimolar dia-
stereomeric mixtures of chiral imides 7a–b were
obtained in good chemical yield, detected by TLC and
GC and separated on silica gel;8 unfortunately, only the
less polar isomer of 7b mixture was obtained in diastereo-
merically pure form. The cleavage with lithium hydro-
peroxyde9 led to both enantiomers of optically active
acid 5a and only dextrorotatory isomer of 5b (the more
polar imide of 7b was not hydrolyzed).10 Unfortunately,
the levorotatory enantiomer of acid 5b was not obtained
in enantiomerically pure form (Scheme 2).
Alternatively, the less polar diastereomers of the mix-
tures 7a–b were synthesized in diastereomerically en-
riched form by diastereoselective alkylation (Scheme 3).
After basic hydrolysis of ester 3, the acid 8 was treated
with thionyl chloride, to give the 9 acyl chloride. The
coupling with the Evans (S)-oxazolidinone afforded the
imide 10. The asymmetric alkylation11 of 10 was per-
formed using methyl iodide and ethyl iodide as electro-
philes, giving only the less polar diastereomers of the
mixtures 7a–b, which, as illustrated above, were
obtained by chromatographic separation. The diaster-
eomeric excesses of 98 and 99%, respectively, were
evaluated by means of GC analysis, by assuming that
the ratio of areas were equivalent to the molar ratios.
After purification on silica gel of the less polar imides
Scheme 1. Reagents and conditions: (a) EtOH, reflux; (b) Mel or Etl, LDA, HMPA, THF, À78 ꢀC/À20 ꢀC; (c) KOH, EtOH, reflux.
Scheme 2. Reagents and conditions: (a) SOCl2, CH2Cl2, 40 ꢀC, 98%; (b) BuLi, oxazolidinone, THF, À78 ꢀC, 81–85%; (c) LiOOH, THF/H2O, rt,
88–97%.
Scheme 3. Reagents and conditions: (a) KOH, EtOH, 70 ꢀC, 91%; (b) SOCl2, CH2Cl2, 40 ꢀC, 97%; (c) BuLi, oxazolidinone, THF, À78 ꢀC, 94%; (d)
LDA, RI, THF, À78 ꢀC, 52–69%; (e) LiOOH, THF/H2O, rt, 88–97%.

A. Ammazzalorso et al. / Bioorg. Med. Chem. Lett. 12 (2002) 817–821
819
of platelet function13,14 on small samples of antico-
agulated whole blood based on work described by
Kratzer and Born.15,16 This method has been designed
to provide an in vitro measure of primary platelet-rela-
ted hemostasis simply, quickly, quantitatively and
accurately to aid in the routine screening of patients
with potential hemorrhagic risk due to abnormal plate-
let plug formation.17 Herein we report the results
obtained by comparing acids rac-5a, rac-5b, (+)-5a,
(À)-5a, (+)-5b with gemfibrozil and acetylsalicylic acid
(ASA).18 Unfortunately, (À)-5b was not supplied and
thus not tested. All tested compounds revealed a dose-
dependent inhibitory activity toward human platelet
aggregation. Moreover, a similar inhibitory effect is
detectable in their precursor, the well-known gemfi-
brozil (Fig. 2) used as basic reference compound. The
inhibitory activity of these compounds is generally
detectable at concentrations ranging from 1 to 5 mM.
Their activity is probably affected by the size of the
substituents: in this case compound rac-5b seems to be
the most active, probably because of the substitution of
the methyl group with an ethyl group. As shown in
Table 1, the two racemates rac-5a and rac-5b and the
enantiomer (À)-5a are more active (their IC50 being
2.47, 2.42 and 2.61 mM for, respectively, entries 1, 2 and
4), when compared with the two (+)-enantiomers which
have an IC50 of 3.09 mM for (+)-5a (entry 3) and
4.12 mM for (+)-5b (entry 5). Considering the well-
known activity of ASA against platelet aggregation, we
used ASA as a well established reference compound.
Utilizing the PFA-1001 assay, we confirmed that ASA
exerts an anti-aggregating activity at low doses, being its
IC50 0.05 mM (entry 7) as described with other meth-
odologies.19 These data clearly indicate that ASA is still
the most potent compound in comparison with the new
synthesized analogues of gemfibrozil. All these results
were obtained using COL/EPI Cartridges, able to detect
platelet dysfunction induced by exposure to platelet
inhibiting agents. The other test was performed using
COL/ADP Cartridges which are able to indicate if an
abnormal result obtained with the COL/EPI Test Car-
tridge may have been caused by the presence of ASA in
the blood of the patient (Table 2). In accordance with
our expectation, when the test was performed on ASA
(entry 7) and on compounds rac-5a, rac-5b and (+)-5a
(entries 1, 2 and 3), Test Cartridges detected the effect of
ASA or similar medications. On the contrary when the
test was performed on compounds (À)-5a and (+)-5b
and on gemfibrozil, (entries 4, 5 and 6) we detected an
inhibitory effect on platelet aggregation also by ADP
Cartridges. From these data we can surmise a different
Figure 2. Effect of gemfibrozil analogues on human platelet aggregation.
7a–b, their hydrolysis was carried out with lithium
hydroperoxyde and led to the (+)-enantiomers of 5a–b
acids in enantiomerically pure form. Studies are in pro-
gress to use the (R)-oxazolidinone as chiral auxiliary in
asymmetric alkylation to obtain the levorotatory acids
5a–b.
Pharmacology
Traditionally optical aggregometry12 was used to assess
platelet function, however this assay is highly dependent
on sample preparation and technical procedure; so, as a
result, data from various laboratories can be quite vari-
able. Hence we decided to assess platelet aggregation
using a recently developed Platelet Function Analyzer
PFA-1001, a system which allows for rapid evaluation
Table 1. Inhibitory effect of gemfibrozil analogues against human
platelet aggregation by COL/EPI cartridges
a
Compd
IC50
Entry
1
2
3
4
5
6
7
rac-5a
rac-5b
(+)-5a
2.47
2.42
3.09
2.61
4.12
2.67
0.05
(À)-5a
(+)-5b
Gemfibrozil
Acetylsalicilic acid
aAverage 50% inhibitory concentration (mM).
Table 2. Inhibitory effect of gemfibrozil analogues against human platelet aggregation by COL/ADP cartridges
% Inhibition
Entry
Compd
1Â10À3 M
2Â10À3 M
3Â10À3 M
4Â10À3 M
5Â10À3 M
1
2
3
4
5
6
7
rac-5a
rac-5b
(+)-5a
0
0
0
0
0
0
0
0
0
0
0
0
2.5
0
0.49
3.30
0.97
0
0
17
4.39
5.51
0
0
0
24.39
2.57
5.36
100
100
100
(À)-5a
(+)-5b
Gemfibrozil
Acetylsalicilic acid
100
6.76
0
17.12

820
A. Ammazzalorso et al. / Bioorg. Med. Chem. Lett. 12 (2002) 817–821
mechanism of action of these last three compounds in
comparison with rac-5a, rac-5b and (+)-5a which seem
to work as ASA.
3H, CH3 aromatic), 2.28 and 2.30–2.38 (s and m, 4H, CH3
aromatic and (CH3)2CH), 3.77–3.85 (m, 1H, CH2CHCH3),
3.87–3.98 (m, 2H, ArOCH2), 4.18 (dd, 1H, NCHCHH), 4.25
(t, 1H, NCHCHH), 4.42–4.48 (m, 1H, NCH), 6.59 (s, 1H,
aromatic), 6.63 (d, 1H, aromatic), 6.97 (d, 1H, aromatic). Less
polar diastereomer of 7b: oil, GC 270 ꢀC rt 6.8min; [a]D +6.80 (c
1.9, CHCl3); IR (neat) 1610, 1694, 1774 cmÀ1; MS 240.20, 361.30
m/z; 1H NMR (CDCl3) d 0.85–0.95 (m, 9H, (CH3)2CH) and
CH3CH2CH), 1.56–1.89 (m, 4H, ArOCH2(CH2)2), 2.15 (s, 3H,
CH3 aromatic), 2.28 and 2.30–2.40 (s and m, 4H, CH3 aromatic
and (CH3)2CH), 3.79–3.84 (m, 1H, CH3CH2CH), 3.85–3.94
(m, 2H, ArOCH2), 4.14–4.18 (m, 2H, NCHCH2), 4.40–4.45
(m, 1H, NCH), 6.59 (s, 1H, aromatic), 6.63 (d, 1H, aromatic),
6.97 (d, 1H, aromatic).
In conclusion, this work describes a simple method for
the synthesis and covalent resolution of chiral analogues
of gemfibrozil. This procedure offers various advantages
over the conventional resolution methods such as a
cheaper access to the enantiomers, and their preparation
on a multigram scale. However, the simplicity of the
PFA-1001 system would facilitate its use in preliminary
screening tests to find new anti-aggregating compounds.
The use of this method allows us to demonstrate that
the synthesized gemfibrozil analogues exert an anti-
aggregating effect.
9. Evans, D. A.; Britton, T. C. Tetrahedron Lett. 1987, 28,
6141.
10. Compound (+)-5a: white solid, mp 78 ꢀC (recrystallized
from petroleum ether); [a]D +10.4 (c 1.3, MeOH); IR (nujol)
1
1463, 1702 cmÀ1; MS 122.05, 236.15 m/z; H NMR (CDCl3) d
Acknowledgements
1.22 (d, 3H, CH3), 1.62–1.70 (m, 1H, CHHCHCH3), 1.79–1.92
(m, 3H, CHHCHCH3 and OCH2CH2), 2.16 (s, 3H, CH3 aro-
matic), 2.29 (s, 3H, CH3 aromatic), 2.49–2.59 (m, 1H,
CHCH3), 3.94 (t, 2H, ArOCH2), 6.60 (s, 1H, aromatic), 6.64
(d, 1H, aromatic.), 6.98 (d, 1H, aromatic). Compound (À)-5a:
white solid, mp 78 ꢀC (recrystallized from petroleum ether);
[a]D À9.7 (c 0.7, MeOH). Compound (+)-5b: white solid; mp
85 ꢀC (recrystallized from petroleum ether); [a]D +7.5 (c 2.0,
MeOH); IR (nujol) 1453, 1697 cmÀ1; MS 122.05, 250.15 m/z;
1H NMR (CDCl3) d 0.95 (t, 3H, CHCH2CH3), 1.48–1.94 (m,
6H, CHCH2CH3 and ArOCH2(CH2)2), 2.15 (s, 3H, CH3 aro-
matic), 2.28 (s, 3H, CH3 aromatic), 2.36–2.44 (m, 1H,
CH2CHCH3), 3.91–3.93 (m, 2H, ArOCH2), 6.60 (s, 1H, aro-
matic), 6.64 (d, 1H, aromatic), 6.98 (d, 1H, aromatic).
This research was supported by Italian MURST. The
authors gratefully acknowledge the collaboration of Dr.
Barbara Casolari and Dr. Anna Maria Cenci (Labora-
torio Analisi Chimico Cliniche, Ospedale Civile S.
Agostino, Modena).
References and Notes
1. Spencer, C. M.; Barradel, L. B. Drugs 1996, 51, 982.
2. Corton, J. C.; Bocos, C.; Moreno, E.; Merritt, A.; Cattley,
R. C.; Gustagsson, J. A. Biochimie 1997, 79, 151.
3. Amoroso, R.; Bettoni, G.; Tricca, M. L. Synlett 1998, 363.
4. Bettoni, G.; Loiodice, F.; Tortorella, V.; Conte Camerino,
D.; Mambrini, M.; Ferranini, E.; Bryant, S. H. J. Med. Chem.
1987, 30, 1267.
11. Evans, D. A. In Asymmetric Synthesis; Morrison, J. D.,
Ed.; Academic: New York, 1984; Vol. 3, p 1.
12. Nicholson, N. S.; Panzer-Knodle, S. G.; Haas, N. F.;
Taite, B. B.; Szalony, J. A.; Page, J. D.; Feigen, L. P.; Lansky,
D. M.; Salyers, A. K. Am. Heart J. 1998, 135, 170.
5. Fujii, S.; Sobel, B. E. Circulation 1992, 85, 1888.
6. Amoroso, R.; Bettoni, G.; Tricca, M. L.; Loiodice, F.;
Ferorelli, S. Il Farmaco 1998, 53, 73.
13. Bick, R. Semin. Thromb. Haemost. 1992, 18, 167.
14. As a measure of platelet function in the PFA-1001
system, the process of platelet aggregation builds a platelet
thrombus thereby gradually diminishing and finally arresting
the blood flow. The PFA-1001 instrument (supplied by Dade
Behring-Milano), determines the time from the start of the
test until the platelet plug occludes the aperture, and
reports that time interval as the Closure Time (CT): results
of the PFA-1001 test are reported by the instrument as CT in
seconds. The CT is an indicator of platelet function in the
analyzed whole blood sample. It uses two disposable car-
tridges: a Collagen/Epinephrine (COL/EPI) Test Cartridge
7. Gage, J. R.; Evans, D. A. Org. Synth. 1989, 68, 77.
8. General procedure: BuLi (10 mL, 1.6 M in hexane,
16.0 mmol) was added to a solution of (S)-4-(1-methylethyl)-2-
oxazolidinone (2.06 g, 16.0 mmol) in dry THF (40 mL), under
N2 atmosphere, at À78 ꢀC. After 15 min, rac-6a–b (16.0 mmol)
in dry THF (20 mL) was added dropwise and the mixture was
stirred for 1 h at À78 ꢀC. After quenching with saturated aqu-
eous NH4Cl (15 mL), the THF was evaporated and the mix-
ture was extracted twice with CH2Cl2 (30 mL). The combined
organic layers were washed with saturated solutions of
NaHCO3 (30 mL) and NaCl (30 mL), dried and concentrated
under reduced pressure. The residue was purified on silica gel
(cyclohexane/ethyl acetate 9:1) to give the separated diaster-
eomers of 7a–b mixtures. Less polar diastereomer of 7a: oil,
GC 270 ꢀC rt 6.0 min; [a]D +69.02 (c 0.9, CHCl3); IR (neat)
1700, 1792 cmÀ1; MS 226.15, 347.20 m/z; 1H NMR (CDCl3) d
0.87 (dd, 6H, (CH3)2CH), 1.23 (d, 3H, CH2CHCH3), 1.54–
1.69 (m, 1H, CHHCHCH3), 1.71–1.97 (m, 3H, CHHCHCH3
and ArOCH2CH2), 2.15 (s, 3H, CH3 aromatic), 2.28 and 2.30–
2.49 (s and m, 4H, CH3 aromatic and (CH3)2CH), 3.73–3.84
(m, 1H, CH2CHCH3), 3.85–3.97 (m, 2H, ArOCH2), 4.13–4.22
(m, 2H, NCHCH2), 4.37–4.42 (m, 1H, NCH), 6.60 (s, 1H,
aromatic), 6.63 (d, 1H, aromatic), 6.98 (d, 1H, aromatic).
More polar diastereomer of 7a: oil, GC 270 ꢀC rt 6.3 min; [a]D
+25.07 (c 0.7, CHCl3); IR (neat) 1700, 1776 cmÀ1; MS 226.15,
347.20 m/z; 1H NMR (CDCl3) d 0.87 (dd, 6H, (CH3)2CH),
1.17 (d, 3H, CH2CHCH3), 1.57–1.68 (m, 1H, CHHCHCH3),
1.77–1.97 (m, 3H, CHHCHCH3 and ArOCH2CH2), 2.14 (s,
and
a Collagen/ADP (COL/ADP) Test Cartridge (pur-
chased from Dade Behring-Milano). The COL/EPI Test
Cartridge is the primary cartridge used to detect platelet dys-
function induced by intrinsic platelet defects, von Willebrand
disease or exposure to platelet inhibiting agents. The COL/
ADP Test Cartridge is used to indicate if an abnormal
result obtained with the COL/EPI Test Cartridge may have
been caused by the effect of ASA or medications containing
ASA.
15. Kratzer, M. A. A.; Born, G. V. R. Haemostasis 1985, 5,
357.
16. Kratzer, M. A. A.; Bellucci, S.; Caen, J. P. Haemostasis
1985, 15, 363.
17. Kundu, S. K.; Heilmann, E. J.; Sio, R.; Garcia, C.; Ost-
gaard, R. A. Clin. Appl. Thromb. Haemost. 1996, 2, 241.
18. Healthy volunteers (males and females) free from any
pharmacological treatment for at least 10 days before the
experiment, participated in the study. Blood samples were
taken by venipuncture in the forearm and were drawn directly

A. Ammazzalorso et al. / Bioorg. Med. Chem. Lett. 12 (2002) 817–821
821
into an evacuated plastic tube containing 3.8% (0.129 M)
buffered sodium citrate (1 part anticoagulant to 9 parts
blood). 900 mL aliquots were incubated with 100 mL of known
concentrations of test compounds at 37 ꢀC for 30 min. Test
compounds, dissolved in NaOH 0.1 N and water (1:10; v/v),
were tested at the following final concentrations: 5, 4, 3, 2 and
1 mM. The test was performed using acetylselicylic acid (ASA)
too, as known platelet aggregation inhibitor. ASA was dis-
solved in DMSO and water (3:100; v/v) and was tested from
5 mM to 1Â10À5 mM. Experiments conducted to test possible
interference of the two used different vehicles on the assay,
showed that there was no difference between NAOH and
DMSO at the used concentrations. Blood sample was gently
mixed and the test was performed pipetting 800 mL of blood
into the smaller opening (sample reservoir opening) of the test
cartridge avoiding air entrapment in the sample reservoir. The
closure time was determined and expressed in seconds. The
percentage of inhibition of platelet aggregation in treated
blood (that is additioned with compounds to be tested) was
calculated in comparison with control blood additioned with
the proper vehicle.
19. Dohi, M.; Sakata, Y.; Seki, J.; Namikawa, Y.; Fujisaki, J.;
Tanaka, A.; Takasugi, H.; Motoyama, Y.; Yoshida, K. Eur. J.
Pharmacol. 1993, 243, 179.