Synthesis and biological evaluation of new clofibrate analogues as potential PPARα agonists

Article abstract of DOI:10.1016/j.ejmech.2004.09.018

Clofibrate is a lipid-profile modifying agent belonging to the fibrate class of drugs. Fibrates are known to exhibit their beneficial effects by activating peroxisome proliferator-activated receptor-α (PPARα) and used in the treatment of dyslipidemia and atherosclerosis and for the prevention of heart failure. Hereby, the preparation of two new sets of clofibrate analogues, ethyl 2-(4-chlorophenoxy)-3-oxoalkanoates and ethyl 2-(4-chlorophenoxy)-3-hydroxyalkanoates is described starting from commercially available 3-oxoalkanoates in fair to good yields. Treatment of 3-oxoalkanoates with SO₂Cl₂ yielded the corresponding 2-chloro-3- oxoalkanoates, that were then converted into 2-(4-chlorophenoxy)-3-oxoalkanoates by reacting with sodium or caesium 4-chlorophenate. Reduction of the keto group with NaBH₄ afforded the corresponding 2-(4-chlorophenoxy)-3- hydroxyalkanoates in very high yields and with variable diastereoselectivity. Biological evaluation of the compounds was performed by a transactivation assay in a transiently transfected monkey kidney fibroblast cell line. The newly synthesised clofibrate analogues failed to show noticeable levels of PPAR activation at concentrations where clofibrate showed an evident activity, suggesting that the structural modifications caused the loss of PPAR activity.

Full text of DOI:10.1016/j.ejmech.2004.09.018

European Journal of Medicinal Chemistry 40 (2005) 143–154
www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of new clofibrate analogues
as potential PPARa agonists
Maria Grazia Perrone a, Ernesto Santandrea a, Natalina Dell’Uomo b, Fabio Giannessi b,
Ferdinando Maria Milazzo b, Anna Floriana Sciarroni b, Antonio Scilimati a,*,
Vincenzo Tortorella a
a Dipartimento Farmaco-Chimico, Università degli Studi di Bari, Via E.Orabona 4, 70125 Bari, Italy
b Ricerca e Sviluppo, Sigma tau Industrie Farmaceutiche S.p.A., Via Pontina km 30.400, 00040 Pomezia, Italy
Received 23 July 2004; received in revised form 20 September 2004; accepted 23 September 2004
Available online 26 November 2004
Abstract
Clofibrate is a lipid-profile modifying agent belonging to the fibrate class of drugs. Fibrates are known to exhibit their beneficial effects by
activating peroxisome proliferator-activated receptor-a (PPARa) and used in the treatment of dyslipidemia and atherosclerosis and for the
prevention of heart failure. Hereby, the preparation of two new sets of clofibrate analogues, ethyl 2-(4-chlorophenoxy)-3-oxoalkanoates and
ethyl 2-(4-chlorophenoxy)-3-hydroxyalkanoates is described starting from commercially available 3-oxoalkanoates in fair to good yields.
Treatment of 3-oxoalkanoates with SO2Cl2 yielded the corresponding 2-chloro-3-oxoalkanoates, that were then converted into 2-(4-
chlorophenoxy)-3-oxoalkanoates by reacting with sodium or caesium 4-chlorophenate. Reduction of the keto group with NaBH4 afforded the
corresponding 2-(4-chlorophenoxy)-3-hydroxyalkanoates in very high yields and with variable diastereoselectivity. Biological evaluation of
the compounds was performed by a transactivation assay in a transiently transfected monkey kidney fibroblast cell line. The newly synthesised
clofibrate analogues failed to show noticeable levels of PPAR activation at concentrations where clofibrate showed an evident activity, sug-
gesting that the structural modifications caused the loss of PPAR activity.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Clofibrate analogues; Dyslipidemia; Ethyl 2-(4-chlorophenoxy)-3-oxoalkanoates; Ethyl 2-(4-chlorophenoxy)-3-hydroxyalkanoates
1. Introduction
have been identified as natural ligands for PPARs. More potent
synthetic PPAR ligands, including fibrates and thiazolidinedi-
ones, have been proven to be effective in the treatment of
dyslipidemia and type 2 diabetes through activation of the a-
and c-PPAR isoform, respectively. Use of such ligands has
helped unveil many potential roles for PPARs in pathological
states including atherosclerosis, inflammation, cancer, infer-
tility and demyelination [1].
Fibrates, such as clofibrate (1, R = C2H5, Fig. 1), are gen-
erally effective in lowering elevated plasma triglycerides [2].
They are well tolerated in the clinic, and enjoy favourable
safety profiles. A rare incidence of fibrate-associated toxicity
has been reported in almost every organ system [3]. The most
pronounced contraindication is the liver and renal insuffi-
ciency [4,5]. Fibrate toxicities include myalgia, myotonia,
sporadic rhabdomyolysis (may be aggravated by combina-
tion with statins), elevated transaminases and gallstone for-
mation [6,7]. The magnitude of lipid changes depends on the
The peroxisome proliferator-activated receptors (PPARs)
are a group of nuclear receptors. To date, three isoforms
(namely PPARa, PPARc and PPARd) have been identified.
PPARs are ligand-activated transcription factors and control
the gene expression by binding to specific response elements
(PPREs) within the promoter regions of several target genes.
Binding of a ligand to PPARs causes the heterodimerisation
of PPARs with the 9-cis retinoic acid-activated receptor
(RXR). The heterodimer then recruits a number of specific
cofactors. This series of events ultimately results in the regu-
lation of the transcription rate of the target genes. PPARs play
a critical physiological role as lipid sensors in the regulation
of lipid and energy metabolism. Fatty acids and eicosanoids
* Corresponding author. Tel.: +39 080 544 2762; fax: +39 080 544 2231.
E-mail address: ascilimati@farmchim.uniba.it (A. Scilimati).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.09.018

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M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
Fig. 1. Clofibrate (1, R= Et) and some of its acyclic (2) and cyclic (3–5) analogues.
patient’s lipoprotein status [8] as well as the relative potency
of the fibrate used in the treatment [9].
2. Chemistry
Clofibrate analogues 8a–f and 9a–f were synthesised as
reported in the scheme of Table 1.
2-Chloro-3-oxoalkanoates 7a–f were prepared in fair to
very good yields (51–98%) by treating the corresponding
commercially available 3-oxoalkanoates 6a–f with SO2Cl2
(scheme of Table 1) [21].
It has been reported that stereochemistry of clofibrate
analogues bearing a stereogenic centre (2, Fig. 1) affects their
pharmacological profile (therapeutic and adverse side ef-
fects) [10–14]. In fact, many clofibrate derivatives have been
synthesised and tested both as racemates and in enantiomeri-
cally pure form [15].
Then, 2-(4-chlorophenoxy)-3-oxoalkanoates 8a–f were
obtained by reacting 7a–f with sodium or caesium
4-chlorophenate. Initially, the nucleophilic displacement of
chloride was performed by using sodium 4-chlorophenate in
toluene, DMF or without any solvent. In contrast, when
caesium 4-chlorophenate was used instead of corresponding
sodium salt, reaction at 50 °C in the absence of solvent gave
the desired product in a similar yield but the work-up became
easier. The keto group of 8a–f was reduced to the corre-
sponding alcohols 9a–f by treating with NaBH4 in methanol
at 0 °C (Scheme in Table 1).
In our previous investigation, a number of racemic and
optically active acyclic (2, Fig. 1) [16] and cyclic clofibrate
(3–5, Fig. 1) [15] analogues were synthesised and pharmaco-
logically evaluated on PPAR [17] and muscle tissue chloride
channels [18–20] for their therapeutic activity and toxic side
effects, respectively, with the aim of discriminating the struc-
tural determinants responsible for the different activities.
Herein, synthesis of new and more highly functionalised
clofibrate derivatives, together with the investigation of their
potential activity as PPARa ligands, is described.
Table 1
Yields of each step of the synthetic route to 2-chloro-3-oxoalkanoates 7a–f, 2-(4-chlorophenoxy)-3-oxoalkanoates 8a–f, 2-(4-chlorophenoxy)-3-
hydroxyalkanoates 9a–f and 2-(4-chlorophenoxy)-3-hydroxyalkanoic acids 10a–f
R
Yield (%)
9a–f b (d.e.) c
90 (11)
7a–f a
65
8a–f b
69
10a–f d (d.e.) c
79 (15)
a:CH3
b:C2H5
c:n-C3H7
d:i-C3H7
e:t-C4H9
f:C6H5
60
76
88 (7)
78 (11)
64
70
73 (22)
71 (4)
51
68
93 (25)
44 (27)
98
65
94 (8)
14 (41)
92
78
96 (34)
84 (32)
a Yields refer to the product isolated by chromatography.
b GC yield based on the starting 7a–f (see Section 6).
c The d.e. was determined by 1H NMR by comparing the integration value of the signals due to the CHOC6H4Cl of the two diastereomeric couples.
d Yields refer to the product crystallised.

M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
145
Scheme 1
.
Reagents and conditions: (a) Cs2CO3, CH3I, r.t., 90% yield; (b) NaBH4, MeOH, 0 °C, 87% yield; (c) KOH, THF–H2O, 64% yield.
The reduction proceeded in very high chemical yields.
The loss of activity of 8a and 9a could be attributed to the
The diastereomeric excess (d.e.) depended on the group (R)
bonded to carbonyl (Table 1). It was around 10% for R = CH3
(9a) and C2H5 (9b). D.e was increased almost twofold for R
= C3H7 (9c and 9d) and was again around 10% for R = t-C4H9
(9e). It was increased threefold (d.e. = 34%) when the group
bonded to the carbonyl is a phenyl (9f).
presence of the ester group, that prevents interaction with the
four amino acids (Ser280, Tyr314, His440, Tyr464) [22]
present in the ligand-binding pocket of PPARa.
As far as the low activity of the other compounds is
concerned, at least three aspects should be taken into consid-
eration. First, it is well known that the differences in the
pharmacological properties of the stereoisomers depend on
their ability to interact with the receptor, which possesses its
stereospecificity. All of the compounds were biologically
evaluated as mixtures of four stereoisomers (9a and 10a–g)
with the exception of 8a (existing only as a pair of enanti-
omers). Each stereoisomer could have a different behaviour
with respect to PPARa and this could explain the observed
activity of the racemates 8a, 9a, and 10a–g.
Ethyl
2-(4-chlorophenoxy)-2-methyl-3-hydroxybuta-
noate (9g) was prepared by treating 8a with CH3I in the
presence of Cs2CO3 affording 8g in 90% yield, that was
reduced to 9g with NaBH4 in methanol at 0 °C (87% yield,
and d.e. = 60%; Scheme 1).
Ethyl esters 9a–g were hydrolysed to the corresponding
acids 10a–g by KOH in THF–H2O (14–84% yield, Table 1
and Scheme 1).
Secondly, it could be that the hydroxy group, present in
almost all the molecules, is responsible for the not suitable
interaction between the compounds and the LBD of the
receptor.
3. Biology
Evaluation of the PPAR functional activities of newly
synthesised compounds was performed by a transactivation
assay in eukaryotic cells.
Thirdly, another reason for the loss of activity could reside
in the higher size of the moiety linked to the C-2 of 8a, 9a and
10a–g than the two hydrogens of WY-14,643 and the two
methyls of the clofibrate [23].
COS-7 cells were transiently transfected with an expres-
sion vector encoding a fusion protein between the DNA
binding domain (DBD) of the yeast GAL4 transcription
factor and the ligand-binding domain (LBD) of the mouse
PPARa (GAL4DBD/PPARaLBD). The reporter vector, con-
taining five copies of the high affinity binding site for GAL4
(named UAS, upstream activating sequence) upstream of a
strong viral promoter linked to the reporter gene
chloramphenicol-acetyl transferase (CAT), was co-
transfected. Beside expression and reporter vectors, cells
were transfected with a control vector pCH110 that encodes
the b-galactosidase enzyme to correct for differences in
transfection efficiency. Following transfection, cells were
treated for 48 h with increasing concentrations (50, 150 and
300 µM) of the test compounds 8a, 9a, 10a–g, and of clofi-
brate. CAT activity, normalised to b-galactosidase activity,
was determined as “fold activation” relative to cells treated
with vehicle (DMSO 0.1%) alone. Finally, results were ex-
pressed as percentage activation of the CAT reporter gene
compared to that measured in the presence of positive control
WY-14,643 (2 µM), conventionally taken as equal to 100%.
5. Conclusion
In summary, new clofibrate analogues have been prepared
in good yields. Their behaviour as PPARa ligands was evalu-
ated by a transactivation assay in comparison with clofibrate.
Unfortunately, they failed to show PPAR activity. The loss of
activity might be attributed to the steric congestion near the
carboxylic acid moiety, which presumably binds to the helix
12 of the LBD of the receptor.
6. Experimental protocols
6.1. General
Melting points were taken on electrothermal apparatus
and are uncorrected. Reaction progress was monitored by
TLC or GC analysis. Thin-layer chromatography (TLC) was
performed on silica gel sheets with a fluorescent indicator
(Statocrom SIF, 60 F254 MERK); TLC spots were observed
under ultraviolet light or visualised with I2 vapour. Column
chromatography was conducted using silica gel MERK 60
(0.063–0.200 µm).
4. Results and discussion
All the new synthesised compounds were biologically
evaluated to determine their ability to activate PPARa. Re-
sults of transactivation assays are shown in Table 2.
GC analyses were performed by using a HP1 column
(methyl silicone gum; 30 m × 0.25 mm × 250 µm film

146
M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
Table 2
Compound
50 µM
(%)
150 µM
(%)
300 µM
(%)
Percentage of activation of the CAT reporter gene by 8a, 9a and 10a–g at the
concentration of 50, 150 and 300 µM, compared to that measured in the
presence of reference compound WY-14,643 (2 µM), conventionally taken
as equal to 100%
12.6
11
89.9
Compound
50 µM
(%)
150 µM
(%)
300 µM
(%)
Clofibrate
5.2
2.7
4.0
2.7
4.2
1.5
5.6
3.4
4.2
1.5
7.3
9.0
100 (2 µM)
–
–
WY-14,643
thickness) on a Hewlett Packard 5890 model, SERIES II.
GC–MS analyses were performed on a Hewlett Packard
6890-5793MSD, and microanalysis on an Elemental Analy-
ser 1106-Carlo Erba-instrument.
1H NMR spectra were recorded in CDCl3 on VARIAN
EM-390 or Mercury 300 MHz spectrometers and the chemi-
cal shifts were reported in parts per million (d). Absolute
values of the coupling constant (J) are reported. The extent of
enolisation of 7a–f and 8a–f was measured by 1H NMR. IR
spectra were recorded on a Perkin–Elmer 681 spectrometer.
3-Oxoalkanoates, ethyl 2-chloro-3-oxobutanoate (also
prepared by us, as depicted in the scheme of Table 1) and all
other chemicals and solvents were purchased from Aldrich
Chemical Co.
3.4
4.6
5.9
One hundred to 500 mg of biologically tested compounds
(Table 1 and Scheme 1) were prepared.
6.2. General procedure for the synthesis of ethyl
2-chloro-3-oxoalkanoates 7a–f [21]
3.2
3.7
3.2
3.7
10.8
4.3
5.9
13.0
5.1
Sulphuryl chloride (SO2Cl2) (139 mg, 5 mmol) was added
dropwise to the oxoalkanoate (5 mmol) kept under stirring at
0 °C for 10 min. Then, the mixture was allowed to warm at
room temperature. The reaction progress was monitored by
TLC, and reactant/products visualised by exposure to iodine
vapour. After the time indicated below (see each compound),
the reaction mixture was washed with a saturated solution of
Na2CO3. The aqueous layer was extracted three times with
ethyl acetate. The combined organic layers were dried over
anhydrous Na2SO4 and the solvent evaporated under reduced
pressure. The product was isolated by chromatography.
In CDCl3 solution, the ethyl 2-chloro-3-oxoalkanoates
7a–f are in equilibrium with the corresponding enol forms
(Scheme 2). The extent of enolisation is between 12% and
22% for 7a–d (see each compound), and less than 3% for 7e
5.1
8.8
17.2
86.5
Not tested
Not tested
Clofibric acid
Scheme 2
.

M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
147
and 7f. Hence, IR and NMR spectral descriptions, whenever
possible, are given for both keto and enol forms.
J = 7.28 Hz, 2H, CH2COH, enol form); 1.64–1.51 (sextet,
J = 7.28 Hz, 2H, CH2CH2, enol form); 1.60–1.47 (sextet,
J = 7.28 Hz, 2H, CH2CH2, keto form); 1.26–1.23 (t,
J = 7.14 Hz, 3H, CH3CH2O, enol form); 1.23–1.17 (t,
J = 7.14 Hz, 3H, CH3CH2O, keto form); 0.89–0.84 (t,
J = 7.28 Hz, 3H, CH3CH2CH2, enol form); 0.89–0.84 (t,
J = 7.28 Hz, 3H, CH3CH2CH2, keto form). 13C NMR
(CDCl3, d): 199.02, 165.23, 63.22, 61.13, 40.94, 17.13,
14.09, 13.53. GC–MS (70 eV) (m/z) (rel. int.) 194 [M(37Cl)+,
1], 192 [M(35Cl)+, 3], 177 (0.2), 164 (2), 146 (4), 118 (5), 94
(12), 71 (100), 43 (58), 41 (16). Anal. Calc. for C8H13ClO3:
C, 50.00; H, 6.77. Found: C, 50.01; H, 6.78.
6.2.1. Ethyl 2-chloro-3-oxobutanoate (7a) [21]
The product was isolated from the crude reaction mixture
as an oil (b.p. = 145 °C/22 mmHg) [21] by chromatography:
silica gel, mobile phase: petroleum ether/ethyl acetate
= 9.5:0.5; reaction time = 1 h; 65% yield; keto form/enol
form = 80:20. IR (neat): 3449 (weak band due to the enol
form), 2986, 2942, 2912, 2874, 1761, 1733, 1639, 1619,
1469, 1447, 1364, 1303, 1256, 1096, 1069, 1032, 882, 851,
1
771 cm–1. H NMR (CDCl3, d): 12.29 (s, 1H, enol OH:
exchanges with D2O); 4.74 (s, 1H, CHCl); 4.30–4.22 (q,
J = 7.14 Hz, 4H, CH2OCO, 2H of enol form and 2H of keto
form); 2.35 (s, 3H, CH3CO, keto form); 2.14 (s, 3H,
CH3COH, enol form); 1.34–1.29 (t, J = 7.14 Hz, 3H,
CH3CH2O, enol form); 1.30–1.26 (t, J = 7.14 Hz, 3H,
CH3CH2O, keto form). 13C NMR (CDCl3, d): 196.78,
165.14, 63.33, 61.53, 26.41 14.11. GC–MS (70 eV) (m/z)
(rel. int.) 166 [M(37Cl)+, 4], 164 [M(35Cl)+, 12], 136 (11),
124 (17), 122 (49), 119 (14), 118 (25), 96 (16), 94 (48), 76
(22), 69 (7), 43 (100).Anal. Calc. for C6H9ClO3: C, 43.90; H,
5.49. Found: C, 43.89; H, 5.48.
6.2.4. Ethyl 2-chloro-3-oxo-4-methylpentanoate (7d)
The product was isolated from the crude reaction mixture
as an oil by chromatography: silica gel, mobile phase: petro-
leum ether/ethyl acetate = 9:1; reaction time = 3 h; 51%
yield; keto form/enol form = 81:19. IR (neat): 3435 (enol
form), 2979, 2937, 2873, 1763, 1735, 1632, 1595, 1467,
1448, 1386, 1367, 1295, 1255, 1177, 1096, 1022, 871,
1
809 cm–1. H NMR (CDCl3, d): 12.49 (s, 1H, enol OH:
exchanges with D2O); 4.90 (s, 1H, CHCl); 4.27–4.19 (q,
J = 7.14 Hz, 4H, CH2O, 2H of enol form and 2H keto form,
two completely overlapped quartets); 3.12–2.98 (heptet,
J = 6.87 Hz, 2H, CH(CH3)2, 1H of enol form and 1H of keto
form, two completely overlapped heptets); 1.28–1.23 (t,
J = 7.14 Hz, 6H, CH3CH2, 3H of enol form and 3H of keto
form, two completely overlapped triplets); 1.14–1.10 (2d
partially overlapped, J = 6.87 Hz, 6H, (CH3)2CH, keto form);
1.13–1.08 (2d partially overlapped, J = 6.87 Hz, 6H,
(CH3)2CH, enol form). 13C NMR (CDCl3, d): 202.80,
165.26, 63.22, 59.79, 38.17, 18.85, 18.66, 14.12. GC–MS
(70 eV) (m/z) (rel. int.) 194 [M(37Cl)+, 2], 192 [M(35Cl)+, 6],
146 (8), 122 (6), 121 (8), 96 (11), 94 (35), 76 (9), 71 (100), 69
(10), 43 (96), 41 (24). Anal. Calc. for C8H13ClO3: C, 50.00;
H, 6.77. Found: C, 50.00; H, 6.75.
6.2.2. Ethyl 2-chloro-3-oxopentanoate (7b) [21]
The product was isolated from the crude reaction mixture
as an oil (b.p. = 125 °C/17 mmHg) [21] by chromatography:
silica gel, mobile phase: petroleum ether/ethyl acetate = 9:1;
reaction time = 1 h; 60% yield; keto form/enol form = 88:12.
IR (neat): 3660–3400 (enol form), 2978, 2943, 2908, 1768,
1733, 1644, 1606, 1462, 1369, 1250, 1180, 1104, 1068,
1019, 947, 879, 822, 737 cm–1. 1H NMR (CDCl3, d): 12.38
(s, 1H, enol OH: exchanges with D2O); 4.77 (s, 1H, CHCl);
4.29–4.22 (q, J = 7.14 Hz, 4H, CH2OCO, 2H of keto form
and 2H of enol form); 2.75–2.68 (q, J = 7.28 Hz, 2H,
CH2CO, keto form); 2.55–2.46 (q, J = 7.55 Hz, 2H,
CH2COH, enol form); 1.31–1.26 (t, J = 7.14 Hz, 6H,
CH3CH2O, 3H of keto form and 3H of enol form); 1.18–1.13
(t, J = 7.55 Hz, 3H, CH3CH2, enol form); 1.11–1.07 (t,
J = 7.28 Hz, 3H, CH3CH2, keto form). 13C NMR (CDCl3, d):
199.89, 165.33, 63.28, 60.94, 32.62, 14.12, 7.84. GC–MS
(70 eV) (m/z) (rel. int.) 180 [M(37Cl)+, 2], 178 [M(35Cl)+, 5],
132 (10), 94 (12), 69 (6), 57 (100), 41 (4). Anal. Calc. for
C7H11ClO3: C, 47.19; H, 6.18. Found: C, 47.20; H, 6.19.
6.2.5. Ethyl 2-chloro-3-oxo-4,4-dimethylpentanoate (7e)
The product was isolated from the crude reaction mixture
as an oil by chromatography: silica gel, mobile phase: petro-
leum ether/ethyl acetate = 9:1; reaction time = 1 h; 98%
yield. IR (neat): 3427 (very weak band due to almost 3% of
enol form as by NMR, see below), 2977, 2839, 1769, 1721,
1478, 1467, 1398, 1369, 1299, 1278, 1225, 1182, 1057,
1
1031, 1004, 877, 812 cm–1. H NMR (CDCl3, d): 12.39 (s,
1H, enol OH: exchanges with D2O); 5.17 (s, 1H, CHCl);
4.16–4.09 (q, J = 7.14 Hz, 2H, CH2CH3); 1.24–1.21 (t,
J = 7.14 Hz, 3H, CH3CH2); 1.18 (s, 9H, (CH3)3C). 13C NMR
(CDCl3, d): 203.90, 165.26, 63.01, 54.66, 45.24, 26.39,
13.99. GC–MS (70 eV) (m/z) (rel. int.) 208 [M(37Cl)+, 0.1],
206 [M(35Cl)+, 0.2], 122 (21), 96 (6), 94 (19), 85 (28), 69 (7),
57 (100), 41 (25). Anal. Calc. for C9H15ClO3: C, 52.43; H,
7.28. Found: C, 52.40; H, 7.29.
6.2.3. Ethyl 2-chloro-3-oxohexanoate (7c)
The product was isolated from the crude reaction mixture
as an oil by chromatography: silica gel, mobile phase: petro-
leum ether/ethyl acetate = 9:1; reaction time = 3 h; 64%
yield; keto form/enol form = 78:22. IR (neat): 3442 (enol
form), 2969, 2933, 2877, 1733, 1720, 1466, 1400, 1372,
1299, 1251, 1176, 1023, 851, 733 cm–1. 1H NMR (CDCl3, d):
12.30 (s, 1H, enol OH: exchanges with D2O); 4.72 (s, 1H,
CHCl); 4.21–4.14 (q, J = 7.14 Hz, 2H, CH2OCO, enol form);
4.20–4.13 (q, J = 7.14 Hz, 2H, CH2OCO, keto form); 2.60–
2.55 (t, J = 7.28 Hz, 2H, CH2CO, keto form); 2.40–2.35 (t,
6.2.6. Ethyl 2-chloro-3-oxo-4-phenylpropanoate (7f) [21]
The product was isolated from the crude reaction mixture
as an oil (b.p. = 150 °C/22 mmHg) [21] by chromatography:

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M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
Table 3
silica gel, mobile phase: petroleum ether/ethyl acetate = 9:1;
reaction time = 2 h; 92% yield. IR (neat): 3467 (very weak
band due to almost 3% of enol form as by NMR, see below),
3066, 2984, 1764, 1692, 1597, 1581, 1468, 1450, 1369,
1303, 1269, 1184, 1025, 1002, 950, 875, 824, 763, 735,
4-Chlorophenate/ethyl 2-chloro-3-oxoalkanoates ratios and reaction times
for the preparation of 8a–f
Compound 4-Chlorophenate/substrate
Reaction time a (h)
8a
8b
8c
8d
8e
1.1
1.1
1.5
1.4
1.1
1.5
5 a
3 b
24 c
1
689 cm–1. H NMR (CDCl3, d): 12.40 (s, 1H, enol OH:
18 c (30 h, 62% yield) d
exchanges with D2O); 7.95–7.92 (m, 2H, aromatic protons);
7.59–7.53 (m, 1H, aromatic proton); 7.45–7.40 (t, 2H, aro-
matic protons); 5.64 (s, 1H, CHCl); 4.24–4.16 (q,
J = 7.14 Hz, 2H, CH2CH3); 1.17–1.13 (t, J = 7.14 Hz, 3H,
CH3CH2). 13C NMR (CDCl3, d): 188.57, 165.47, 134.64,
133.51, 129.43, 129.14, 63.36, 58.11, 14.05. GC–MS
(70 eV) (m/z) (rel. int.) 228 [M(37Cl)+, 0.5], 226 [M(35Cl)+,
1.5], 125 (3), 106 (13), 105 (100), 77 (44), 51 (11). Anal.
Calc. for C11H11ClO3: C, 58.41; H, 4.87. Found: C, 58.42; H,
4.87.
3 c
24 c (6) d
8f
a Unless otherwise indicated, reaction time was almost the same in the
several conditions: reactants solubilised in toluene or DMF, the use of either
sodium or caesium chlorophenate (see Sections 6.3.3. and 6.3.4.).
b It refers to the reaction accomplished with the ethyl alkanoate and
sodium chlorophenate solubilised in toluene or DMF.
c It refers to the reaction accomplished with the ethyl alkanoate and
sodium chlorophenate solubilised in toluene.
d The reaction was performed by using caesium chlorophenate in the
absence of any solvent (Section 6.3.4.).
6.3. Synthesis of ethyl
2-(4-chlorophenoxy)-3-oxoalkanoates 8a–g
(Table 3). The mixture was treated with ethyl acetate and then
washed with a saturated solution of NaCl. The organic ex-
tract was dried over anhydrous Na2SO4 and the solvent
evaporated under reduced pressure. The product was isolated
by chromatography.
6.3.1. Sodium 4-chlorophenate
To a solution of 4-chlorophenol (10.51 g, 8.17 mmol) in
absolute ethanol (10 ml) was slowly added a solution of
NaOEt (0.56 g, 8.17 mmol) in absolute EtOH (110 ml). The
mixture protected from light and moisture was stirred at
room temperature for 1.5 h. Then, the EtOH was removed
under reduced pressure. A white, light and moisture sensitive
solid was obtained.
6.3.4.1. Ethyl 2-(4-chlorophenoxy)-3-oxobutanoate (8a)
[24]. The product was isolated from the crude reaction mix-
ture as an oil by chromatography: silica gel, mobile phase:
petroleum ether/ethyl acetate = 9:1; 69% yield; keto
form/enol form = 68:32. IR (neat): 3600–3150, 3100, 3073,
2985, 2940, 2875, 1758, 1735, 1661, 1629, 1587, 1489,
1446, 1414, 1371, 1268, 1216, 1167, 1089, 1013, 827,
6.3.2. Caesium 4-chlorophenate
1
665 cm–1. H NMR (CDCl3, d): 11.32 (s, 1H, OH enol:
A solution of 4-chlorophenol (10.51 g, 8.17 mmol) in
absolute ethanol (15 ml) was slowly added to a suspension of
Cs2CO3 (2.7 g, 8.17 mmol) in absolute EtOH (35 ml). The
mixture was stirred for 3 h at room temperature. Then, the
EtOH was removed under reduced pressure. The obtained
white powder was more stable and less hygroscopic than
sodium 4-chlorophenate.
exchanges with D2O); 7.26–7.20 (m, 4H, aromatic protons,
2H of keto form and 2H of enol form); 6.87–6.81 (m, 4H,
aromatic protons, 2H of keto form and 2H of enol form); 5.03
(s, 1H, CH); 4.30–4.23 (q, J = 7.14 Hz, 2H, CH2CH3, keto
form); 4.22–4.15 (q, J = 7.14 Hz, 2H, CH2CH3, enol form);
2.35 (s, 3H, COCH3, keto form); 1.95 (s, 3H, COCH3, enol
form); 1.29-1.24 (t, J = 7.14 Hz, 3H, CH3CH2, keto form);
1.17–1.13 (t, J = 7.14 Hz, 3H, CH3CH2, enol form). 13C
NMR (CDCl3, d): 200.66, 165.88, 155.44, 129.95, 129.63,
127.87, 116.71, 116.05, 83.16, 62.79, 26.56, 14.22. GC–MS
(70 eV) (m/z) (rel. int.) 258 [M(37Cl)+, 10], 256 [M(35Cl)+,
29], 216 (10), 214 (30), 147 (15), 143 (32), 141 (100), 139
(29), 129 (8), 128 (9), 113 (11), 111 (22), 75 (15), 43 (37).
Anal. Calc. for C12H13ClO4: C, 56.25; H, 5.08. Found: C,
56.21; H, 5.06.
6.3.3. Procedure A
A mixture of sodium 4-chlorophenate (Table 3) in anhy-
drous toluene or anhydrous DMF (32 ml) was stirred at 80 °C
for 30 min. Then, the reaction was cooled to 60 °C and a
solution of ethyl 2-chloro-3-oxoalkanoate (2.968 g,
18.1 mmol) in anhydrous toluene or anhydrous DMF (5.5 ml)
was added dropwise. The reaction mixture was stirred at
80 °C. The reaction was monitored by GC and stopped at the
time indicated in Table 3. The mixture was treated with ethyl
acetate and then washed with a saturated solution of NaCl.
The organic extract was dried over anhydrous Na2SO4 and
the solvent evaporated under reduced pressure. The product
was isolated by chromatography.
6.3.4.2. Ethyl 2-(4-chlorophenoxy)-3-oxopentanoate (8b).
The product was isolated from the crude reaction mixture as
an oil by chromatography: silica gel, mobile phase: petro-
leum ether/ethyl acetate = 9:1; 76% yield; keto form/enol
form = 69:31. IR (neat): 3418 (enol form), 3065, 2983, 2935,
1732, 1590, 1493, 1433, 1217, 1162, 1091, 1017, 827, 757,
6.3.4. Procedure B
1
Ethyl 2-chloro-3-oxoalkanoate and caesium 4-chloro-
phenate, in the ratio 1:1.4, were stirred at 50 °C. The reaction
was monitored by GC and stopped at the indicated time
643 cm–1. H NMR (CDCl3, d): 11.39 (s, 1H, OH enol:
exchanges with D2O); 7.30–7.18 (m, 4H, aromatic protons,
2H of keto form and 2H of enol form); 6.88–6.79 (m, 4H,

M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
149
aromatic protons, 2H of keto form and 2H of enol form); 5.04
(s, 1H, CHOC6H4Cl); 4.30–4.23 (q, J = 7.14 Hz, 2H,
CH2OCO of keto form); 4.22–4.15 (q, J = 7.14 Hz, 2H,
CH2OCO of enol form); 2.79–2.71 (dq, J = 18.95 and
7.28 Hz, 2H, CH2CO of keto form); 2.34–2.26 (q,
J = 7.28 Hz, 2H, CH2COH of enol form); 1.29–1.24 (t,
J = 7.14 Hz, 6H, CH3CH2OCO, 3H of keto form and 3H of
enol form); 1.17–1.25 (t, J = 7.28 Hz, 3H, CH3CH2COH,
enol form); 1.10–1.06 (t, J = 7.28 Hz, 3H, CH3CH2CO, keto
form). 13C NMR (CDCl3, d): 203.32, 166.10, 155.56, 129.94,
129.60, 127.80, 116.69, 116.05, 82.85, 62.68, 32.41, 14.25,
7.30. GC–MS (70 eV) (m/z) (rel. int.) 272 [M(37Cl)+, 8], 270
[M(35Cl)+, 25], 216 (15), 214 (48), 197 (6), 168 (4), 161 (9),
143 (29), 141 (100), 139 (22), 128 (10), 111 (19), 75 (15), 57
(90). Anal. Calc. for C13H15ClO4: C, 57.78; H, 5.56. Found:
C, 57.75; H, 5.58.
form); 5.13 (s, 1H, CHOC6H4Cl); 4.31–4.23 (q, J = 7.14 Hz,
2H, CH2CH3 of keto form); 4.22–4.15 (q, J = 7.14 Hz, 2H,
CH2CH3 of enol form); 3.24–3.13 (heptet, J = 6.87 Hz, 1H,
CH(CH3)2 of enol form); 2.91–2.81 (heptet, J = 6.87 Hz, 1H,
CH(CH3)2 of keto form); 1.29–1.24 (t, J = 7.14 Hz, 3H,
CH3CH2 of keto form); 1.17–1.15 (d, J = 6.87 Hz, 6H,
(CH3)2CH of enol form); 1.11–1.07 (2d, J = 6.87 Hz, 6H,
(CH3)2CH of keto form); 1.12–1.07 (t, J = 7.14 Hz, 3H,
CH3CH2 of enol form). 13C NMR (CDCl3, d): 206.26,
166.31, 155.68, 129.94, 129.58, 127.83, 116.73, 116.01,
81.99, 62.63, 37.32, 19.20, 18.38, 14.25. GC–MS (70 eV)
(m/z) (rel. int.) 286 [M(37Cl)+, 7], 284 [M(35Cl)+, 20], 216
(12), 214 (37), 143 (12), 141 (43), 139 (17), 128 (13), 111
(17), 75 (13), 71 (84), 43 (100), 41 (11). Anal. CH for
C14H17ClO4.
6.3.4.5. Ethyl 2-(4-chlorophenoxy)-3-oxo-4,4-dimethylpen-
tanoate (8e). The product was isolated from the crude reac-
tion mixture as an oil by chromatography: silica gel, mobile
phase: petroleum ether/ethyl acetate = 10:1; 65% yield; enol
form content in CDCl3 solution measured by NMR was less
than 3%. IR (neat): 3429 (very weak band, enol form), 3099,
2975, 2873, 1756, 1719, 1646, 1586, 1491, 1396, 1369,
1339, 1215, 1086, 1009, 825, 646 cm–1. 1H NMR (CDCl3, d):
12.01 (bs, 1H, OH enol: exchanges with D2O; the extent of
enolisation found was approximately 3%); 7.22–7.18 (m,
2H, aromatic protons); 6.85–6.80 (m, 2H, aromatic protons);
5.35 (s, 1H, CHOC6H4Cl); 4.26–4.19 (q, J = 7.14 Hz, 2H,
CH2CH3); 1.25–1.20 (t, J = 7.14 Hz, 3H, CH2CH3); 1.21 (s,
9H, (CH3)3CH). 13C NMR (CDCl3, d): 205.77, 166.83,
155.92, 129.83, 127.72, 116.75, 78.99, 62.35, 44.94, 26.52,
14.21. GC–MS (70 eV) (m/z) (rel. int.) 300 [M(37Cl)+, 4],
298 [M(35Cl)+, 13], 214 (14), 143 (18), 141 (53), 139 (8), 128
(10), 111 (11), 85 (23), 57 (100), 41 (13). Anal. CH for
C15H19ClO4.
6.3.4.3. Ethyl 2-(4-chlorophenoxy)-3-oxohexanoate (8c).
The product was isolated from the crude reaction mixture as
an oil by chromatography: mobile phase: petroleum
ether/ethyl ether = 7:3; 70% yield; keto form/enol form
= 60:40. IR (neat): 3441 (enol form), 2967, 2933, 2876,
1752, 1728, 1658, 1622, 1490, 1266, 1216, 1090, 1025, 826,
1
677 cm–1. H NMR (CDCl3, d): 11.39 (s, 1H, OH enol:
exchanges with D2O); 7.28–7.21 (m, 4H, aromatic protons,
2H of enol form and 2H of keto form); 6.88–6.82 (m, 4H,
aromatic protons, 2H of enol form and 2H of keto form); 5.03
(s, 1H, CHOC6H4Cl); 4.31–4.24 (q, J = 7.14 Hz, 2H, CH2O
of keto form); 4.23–4.16 (q, J = 7.14 Hz, 2H, CH2O of enol
form); 2.79–2.68 (dt, 1H, J = 7.14 and 18.13 Hz, CHHCO of
keto form); 2.70–2.59 (dt, 1H, J = 7.14 and 18.13 Hz,
CHHCO of keto form); 2.29–2.24 (t, J = 7.49 Hz, 2H,
CH2COH of enol form); 1.70–1.53 (m, 4H, CH2CH2, 2H of
enol form and 2H of keto form); 1.30–1.25 (t, J = 7.14 Hz,
3H, CH3CH2O of keto form); 1.18–1.13 (t, J = 7.14 Hz, 3H,
CH3CH2O of enol form); 0.94–0.89 (t, J = 7.28 Hz, 3H,
CH3CH2CH2 of keto form); 0.90–0.85 (t, J = 7.28 Hz, 3H,
CH3CH2CH2 of enol form). 13C NMR (CDCl3, d): 202.64,
166.03, 155.53, 129.91, 129.55, 127.75, 116.68, 116.09,
83.00, 62.66, 40.76, 31.78, 16.64, 14.02. GC–MS (70 eV)
(m/z) (rel. int.) 286 [M(37Cl)+, 9], 284 [M(35Cl)+, 26], 216
(18), 214 (53), 175 (11), 143 (28), 141 (97), 139 (22), 128
(15), 113 (10), 111 (23), 75 (16), 71 (100), 43 (77), 41 (13).
Anal. CH for C14H17ClO4.
6.3.4.6. Ethyl 2-(4-chlorophenoxy)-3-oxo-3-phenylpropa-
noate (8f). The product was isolated from the crude reaction
mixture as an oil by chromatography: silica gel, mobile
phase: petroleum ether/ethyl ether = 9.5:0.5; 78% yield; enol
form content in CDCl3 solution measured by NMR was less
than 3%. M.p. = 60.2–61.3 °C (hexane), white crystals. IR
(CHCl3): 3449 (very weak band, enol form), 3072, 2978,
2942, 2913, 1735, 1697, 1596, 1581, 1493, 1447, 1372,
1
1223, 1209, 1075, 1010, 820, 706, 685 cm–1. H NMR
(CDCl3, d): 11.99 (bs, 1H, OH enol: exchanges with D2O; the
extent of enolisation found was approximately 3%); 8.09–
8.05 (m, 2H, aromatic protons); 7.64–7.58 (m, 1H, aromatic
proton); 7.51–7.45 (m, 2H, aromatic protons); 7.26–7.21 (m,
2H, aromatic protons); 6.92–6.87 (m, 2H, aromatic protons);
5.70 (s, 1H, CHOC6H4Cl); 4.32–4.25 (q, J = 7.14 Hz, 2H,
CH2CH3); 1.25–1.20 (t, J = 7.14 Hz, 3H, CH3CH2). 13C
NMR (CDCl3, d): 191.16, 166.49, 155.68, 134.53, 134.00,
129.88, 129.72, 129.00, 127.84, 117.03, 81.38, 62.74, 14.19.
GC–MS (70 eV) (m/z) (rel. int.) 320 [M(37Cl)+, 2], 318
[M(35Cl)+, 6], 245 (1), 209 (3), 128 (3), 105 (100), 77 (22),
51 (4). Anal. CH for C17H15ClO4.
6.3.4.4. Ethyl 2-(4-chlorophenoxy)-3-oxo-4-methylpenta-
noate (8d). The product was isolated from the crude reaction
mixture as an oil by chromatography: silica gel, mobile
phase: petroleum ether/ethyl ether = 7:3; 68% yield; keto
form/enol form = 46:54. IR (neat): 3440 (enol form), 3060,
2979, 2927, 2869, 1755, 1725, 1657, 1621, 1592, 1489,
1469, 1411, 1376, 1342, 1263, 1230, 1169, 1091, 1028,
1014, 826, 692 cm–1. 1H NMR (CDCl3, d): 11.45 (s, 1H, OH
enol: exchanges with D2O); 7.28–7.20 (m, 4H, aromatic
protons, 2H of enol form and 2H of keto form); 6.88–6.81
(m, 4H, aromatic protons, 2H of enol form and 2H of keto

150
M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
6.3.5. Synthesis of ethyl 2-(4-chlorophenoxy)-2-methyl-3-
oxobutanoate (8g)
1.34–1.32 (d, J = 6.45 Hz, 3H, CHCH3 of one stereoisomer
couple); 1.33–1.31 (d, J = 6.45 Hz, 3H, CHCH3 of the other
stereoisomer couple); 1.25–1.21 (t, J = 7.14 Hz, 3H, CH3CH2
of one stereoisomer couple); 1.24–1.20 (t, J = 7.14 Hz, 3H,
CH3CH2 of the other stereoisomer couple). 13C NMR
(CDCl3, d): 169.76, 156.45, 129.76, 127.29, 116.88, 81.52,
81.06, 68.65, 68.60, 61.95, 19.10, 14.37. GC–MS (70 eV)
(m/z) (rel. int.) 260 [M(37Cl)+, 5], 258 [M(35Cl)+, 17], 214
(21), 143 (30), 141 (100), 139 (16), 130 (17), 128 (52), 111
(13), 75 (12), 43 (13). Anal. CH for C12H15ClO4.
To a solution of 8a (700 mg) in anhydrous DMF (2.8 ml)
kept in an ice bath, Cs2CO3 (891 mg) was added. The mixture
was stirred at 0 °C for 15 min, then CH3I (1.7 ml) in DMF
(5.7 ml) was added dropwise. The reaction mixture was
brought to room temperature and stirred for further 15 h.
Then, brine was added and the product was extracted three
times with ethyl ether. The organic phase was washed three
times with brine, then with aqueous saturated Na2S2O3 and
aqueous saturated NaHCO3. The organic layer was dried
with anhydrous Na2SO4 and then the solvent was removed
under reduced pressure. The product was isolated as colour-
less oil (90% yield) by chromatography (silica gel, petroleum
ether/ethyl acetate = 10:1). IR (neat): 3064, 2984, 2933,
1752, 1728, 1593, 1490, 1456, 1374, 1356, 1263, 1232,
6.3.6.2. Ethyl 2-(4-chlorophenoxy)-3-hydroxypentanoate
(9b). Substrate/NaBH4 used in the reaction was 1:1.3; reac-
tion time = 30 min; 88% yield; d.e. = 7%. Oil. IR (neat):
3432–3237, 3050, 2964, 2920, 2872, 1730, 1642, 1490,
1
1458, 1262, 1216, 1092, 1017, 800, 760 cm–1. H NMR
1
1130, 1098, 1012, 828, 669 cm–1. H NMR (CDCl3, d):
(CDCl3, d): 7.18–7.14 (m, 4H, aromatic protons, 2H for each
stereoisomer couple); 6.80–6.71 (m, 4H, aromatic protons,
2H for each stereoisomer couple); 4.51–4.49 (d, J = 4.54 Hz,
1H, CHOC6H4Cl of one stereoisomer couple); 4.43–4.41 (d,
J = 3.98 Hz, 1H, CHOC6H4Cl of the other stereoisomer
couple); 4.20–4.13 (q, J = 7.14 Hz, 4H, CH2OCO, 2H for
each stereoisomer couple); 3.98–3.89 (m, 2H, CHOH, 1H for
each stereoisomer couple); 2.40–2.00 (bs, 1H for each couple
of stereoisomers, OH: exchange with D2O); 1.62–1.57 (m,
4H, CH2CHOH, 2H for each stereoisomer couple); 1.20–
1.15 (t, J = 7.14 Hz, 6H, CH3CH2O, 3H for each stereoiso-
mer couple); 0.99–0.92 (m, 6H, CH3CH2CHOH, 3H for each
stereoisomer couple). 13C NMR (CDCl3, d): 170.00, 156.60,
129.72, 127.30, 116.84, 80.29, 79.88, 73.92, 73.92, 61.86,
61.78, 26.46, 25.78, 14.32. GC–MS (70 eV) (m/z) (rel. int.)
274 [M(37Cl)+, 4], 272 [M(35Cl)+, 13], 216 (8), 214 (24), 168
(7), 143 (33), 142 (9), 141 (100), 139 (11), 130 (13), 129 (9),
128 (38), 111 (10), 99 (9), 75 (8), 57 (8), 43 (6). Anal. CH for
C13H17ClO4.
7.25–7.19 (m, 2H, aromatic protons); 6.85–6.80 (m, 2H,
aromatic protons); 4.27–4.20 (q, J = 7.14 Hz, 2H, CH2CH3);
2.40 (s, 3H, COCH3); 1.61 (s, 3H, CH3Cq); 1.25–1.21 (t,
J = 7.14 Hz, 3H, CH3CH2). 13C NMR (CDCl3, d): 204.25,
169.13, 153.32, 129.64, 128.46, 120.61, 87.84, 62.54, 25.45,
18.68, 14.12. GC–MS (70 eV) (m/z) (rel. int.) 272 [M(37Cl)+,
11], 270 [M(35Cl)+, 31], 230 (32), 229 (27), 228 (91), 227
(42), 199 (33), 197 (29), 183 (14), 181 (42), 157 (33), 155
(100), 153 (40), 147 (16), 139 (15), 130 (13), 129 (30), 128
(33), 113 (11), 112 (10), 111 (30), 99 (11), 75 (21), 43 (83).
Anal. CH for C13H15ClO4.
6.3.6. General procedure for the reduction of ethyl
2-(4-chlorophenoxy)-3-oxoalkanoates 8a–g by
NaBH4/MeOH
To a solution of 2-(4-chlorophenoxy)-3-oxoalkanoate
(0.406 mmol) in methyl alcohol (5 ml) kept in an ice bath was
added NaBH4 (see each compound). The reaction mixture
was stirred at 0 °C for 10 min, then at room temperature and
stopped at the indicated time (see each compound). Water
(3 ml) was added and the methyl alcohol was evaporated
under vacuum. The residue was extracted three times with
EtOAc (30 ml). The extracts were dried over anhydrous
Na2SO4 and the solvent evaporated under reduced pressure.
The product (9a–g) was obtained in very high yield (Table 1).
6.3.6.3. Ethyl 2-(4-chlorophenoxy)-3-hydroxyhexanoate
(9c). Substrate/NaBH4 used in the reaction was 1:1.5; reac-
tion time = 3 h; 73% yield; d.e. = 22%. Oil. IR (neat):
3500–3150, 3065, 2961, 2934, 2872, 1737, 1595, 1492,
1465, 1379, 1282, 1238, 1199, 1094, 1075, 1030, 825,
668 cm–1. 1H NMR (CDCl3, d): 7.18–7.13 (m, 4H, aromatic
protons, 2H for each stereoisomer couple); 6.77–6.73 (m,
4H, aromatic protons, 2H for each stereoisomer couple);
4.50–4.49 (d, J = 4.40 Hz, 1H, CHOC6H4Cl of one stereoi-
somer couple); 4.42–4.40 (d, J = 3.98 Hz, 1H, CHOC6H4Cl
of the other stereoisomer couple); 4.19–4.10 (2q, J = 7.14 Hz,
4H, CH2O, 2H for each stereoisomer couple); 4.09–3.95 (m,
2H, CHOH, 1H for each stereoisomer couple); 2.28–2.10
(bs, 1H for each couple of stereoisomers, OH: exchange with
D2O); 1.65–1.42 (m, 8H, CH2CH2CHOH, 4H for each stere-
oisomer couple); 1.27–1.22 (2t, J = 7.14 Hz, 6H, CH3CH2O,
3H for each stereoisomer couple); 0.94–0.82 (m, 6H,
CH3CH2CH2, 3H for each stereoisomer couple). 13C NMR
(CDCl3, d): 169.98, 156.53, 129.73, 116.89, 116.84, 80.61,
80.22, 72.35, 72.23, 61.93, 35.44, 19.00, 14.33, 14.08.
6.3.6.1. Ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate
(9a). Substrate/NaBH4 used in the reaction was 1:1.3; reac-
tion time = 20 min; 90% yield; d.e. = 11%. Oil. IR (neat):
3500–3100, 3061, 2982, 2933, 1738, 1596, 1584, 1492,
1
1375, 1283, 1238, 1200, 1094, 1023, 825 cm–1. H NMR
(CDCl3, d): 7.26–7.18 (m, 4H, aromatic protons, 2H for each
stereoisomer couple); 6.88–6.78 (m, 4H, aromatic protons,
2H for each stereoisomer couple); 4.56–4.53 (d, J = 4.29 Hz,
1H, CHOC6H4Cl of a stereoisomer couple); 4.43–4.41 (d,
J = 4.94 Hz, 1H, CHOC6H4Cl of the other stereoisomer
couple); 4.30–4.17 (m, 6H, 3H for each couple of stereoiso-
mers: 1H of CHCH3 and 2H of CH2CH3); 2.90–2.50 (bs, 1H
for each couple of stereoisomers, OH: exchange with D2O);

M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
151
GC–MS (70 eV) (m/z) (rel. int.) 288 [M(37Cl)+, 4], 286
[M(35Cl)+, 12], 216 (11), 214 (32), 168 (8), 143 (37), 142
(13), 141 (100), 139 (11), 130 (14), 128 (38), 113 (10), 111
(11), 75 (8), 71 (1), 43 (11). Anal. CH for C14H19ClO4.
3], 300 [M(35Cl)+, 9], 227 (2), 216 (13), 214 (39), 168 (6),
143 (35), 141 (100), 139 (8), 130 (15), 128 (33), 111 (11), 57
(27), 41 (10). Anal. CH for C15H21ClO4.
6.3.6.6. Ethyl 2-(4-chlorophenoxy)-3-hydroxy-3-phenyl-
propanoate (9f). Substrate/NaBH4 used in the reaction was
1:1.3; reaction time = 3 h; 96% yield; d.e. = 34%. M.p.
= 82.3–83.3 °C (hexane). IR (KBr): 3550–3250, 3225, 2955,
2926, 2845, 1733, 1596, 1582, 1490, 1450, 1375, 1261,
6.3.6.4. Ethyl 2-(4-chlorophenoxy)-3-hydroxy-4-methyl-
pentanoate (9d). Substrate/NaBH4 used in the reaction was
1:1.5; reaction time = 3 h; 93% yield; d.e. = 25%. Oil. IR
(neat): 3500–3100, 2926, 2848, 1739, 1491, 1462, 1375,
1237, 1162, 1130, 1097, 953, 874, 800, 860 cm–1. 1H NMR
(CDCl3, d): 7.27–7.20 (m, 4H, aromatic protons, 2H for each
stereoisomer couple); 6.86–6.80 (m, 4H, aromatic protons,
2H for each stereoisomer couple); 4.67–4.66 (d, J = 3.29 Hz,
1H, CHOC6H4Cl of one stereoisomer couple); 4.63–4.61 (d,
J = 5.36 Hz, 1H, CHOC6H4Cl of the other stereoisomer
couple); 4.28–4.21 (2q, J = 7.14 Hz, 4H, CH2CH3, 2H for
each stereoisomer couple); 3.86–3.82 (t, J = 5.36 Hz, 1H,
CHOH of one stereoisomer couple); 3.75–3.72 (dd,
J = 3.29 and 7.62 Hz, 1H, CHOH of the other stereoisomer
couple); 2.36–2.28 (m, 1H, CH(CH3)2 of one stereoisomer
couple); 2.05–1.97 (m, 1H, CH(CH3)2 of the other stereoiso-
mer couple); 1.70–1.50 (bs, 1H for each couple of stereoiso-
mers, OH: exchange with D2O); 1.29–1.22 (t, J = 7.14 Hz,
6H, CH3CH2, 3H for each stereoisomer couple); 1.09–1.03
(2d, J = 6.73 Hz, 6H, (CH3)2CH of one stereoisomer couple);
1.00–0.92 (2d, J = 6.73 Hz, 6H, (CH3)2CH of the other
stereoisomer couple). 13C NMR (CDCl3, d): 170.25, 156.00,
129.79, 127.15, 116.67, 78.54, 78.14, 70.78, 61.94, 31.21,
29.38, 27.44, 14.34. GC–MS (70 eV) (m/z) (rel. int.) 288
[M(37Cl)+, 3], 286 [M(35Cl)+, 9], 216 (10), 214 (31), 168(6),
143 (32), 141 (100), 130 (12), 128 (34), 113 (10), 111 (12),
75 (8), 71 (9), 43 (17), 41 (9). Anal. CH for C14H19ClO4.
1
1235, 1216, 1192, 1093, 1050, 1026, 824, 760 cm–1. H
NMR (CDCl3, d): 7.40–7.16 (m, 10H, aromatic protons, 5H
for each stereoisomer couple); 7.13–7.02 (m, 4H, aromatic
protons, 2H for each stereoisomer couple); 6.74–6.67 (m,
4H, aromatic protons, 2H for each stereoisomer couple);
5.13–5.11 (d, J = 5.84 Hz, 1H, CHOH of one stereoisomer
couple); 5.09–5.08 (d, J = 5.42 Hz, 1H, CHOH of the other
stereoisomer couple); 4.65–4.63 (d, J = 5.84 Hz, 1H,
CHOC6H4Cl of one stereoisomer couple); 4.61–4.59 (d,
J = 5.42 Hz, 1H, CHOC6H4Cl of the other stereoisomer
couple); 4.11–4.04 (q, J = 7.14 Hz, 2H, CH2CH3 of one
stereoisomer couple); 4.01–3.96 (q, J = 7.14 Hz, 2H,
CH2CH3 of the other stereoisomer couple); 3.10–2.70 (bs,
1H for each couple of stereoisomers, OH: exchange with
D2O); 1.08–1.03 (t, J = 7.14 Hz, 3H, CH3CH2 of one stere-
oisomer couple); 1.02–0.97 (t, J = 7.14 Hz, 3H, CH3CH2 of
the other stereoisomer couple). 13C NMR (CDCl3, d):
169.47, 139.12, 129.73, 129.66, 128.73, 128.61, 126.93,
126.89, 117.19, 82.34, 81.45, 75.03, 74.38, 61.82, 14.17.
GC–MS (70 eV) (m/z) (rel. int.) 322 [M(37Cl)+, 0.1], 320
[M(35Cl)+, 0.2], 302 [M(35Cl)+ – 18, 0.3], 247 (2), 216 (22),
214 (68), 143 (34), 141 (100), 128 (20), 113 (16), 111 (25),
107 (19), 106 (27), 105 (36), 91 (14), 79 (16), 77 (49), 75
(15), 51 (16), 50 (10). Anal. CH for C17H17ClO4.
6.3.6.5. Ethyl 2-(4-chlorophenoxy)-3-hydroxy-4,4-dimethyl-
pentanoate (9e). Substrate/NaBH4 used in the reaction was
1:1.2; ethanol replaced methanol as reaction solvent; reac-
tion time = 1.5 h; 94% yield; d.e. = 8%. Oil. IR (neat):
3550–3100, 3062, 2960, 2872, 1739, 1595, 1584, 1491,
1397, 1370, 1337, 1283, 1237, 1094, 1064, 1021, 825,
667 cm–1. 1H NMR (CDCl3, d): 7.25–7.21 (m, 4H, aromatic
protons, 2H for each stereoisomer couple); 6.86–6.77 (m,
4H, aromatic protons, 2H for each stereoisomer couple);
4.76–4.75 (d, J = 1.65 Hz, 1H, CHOC6H4Cl of one stereoi-
somer couple); 4.62–4.60 (d, J = 5.91 Hz, 1H, CHOC6H4Cl
of the other stereoisomer couple); 4.27–4.20 (q, J = 7.14 Hz,
4H, CH2CH3, 2H for each stereoisomer couple); 3.81–3.79
(d, J = 5.91 Hz, 1H, CHOH of one stereoisomer couple);
3.75–3.74 (d, J = 1.65 Hz, 1H, CHOH of the other stereoiso-
mer couple); 2.60–2.20 (bs, 1H for each couple of stereoiso-
mers, OH: exchange with D2O); 1.27–1.22 (t, J = 7.14 Hz,
6H, CH3CH2, 3H for each stereoisomer couple); 1.01 (s, 9H,
(CH3)3CH of one stereoisomer couple); 1.00 (s, 9H,
(CH3)3CH of the other stereoisomer couple).
6.3.6.7. Ethyl 2-(4-chlorophenoxy)-2-methyl-3-hydroxy-
butanoate (9g). Substrate/NaBH4 used in the reaction was
1:1.3; reaction time = 30 min; 87% yield; d.e. = 60%. Oil. IR
(neat): 3500–3100, 3062, 2983, 2936, 1736, 1593, 1490,
1
1443, 1376, 1239, 1094, 1047, 1012, 850, 823 cm–1. H
NMR (CDCl3, d): 7.21–7.18 (m, 4H, aromatic protons, 2H
for each stereoisomer couple); 6.85–6.82 (m, 4H, aromatic
protons, 2H for each stereoisomer couple); 4.28–4.21 (q,
J = 7.14 Hz, 4H, CH2OCO, 2H for each stereoisomer
couple); 4.18–4.12 (q, J = 6.41 Hz, 2H, CHOH, 1H for each
stereoisomer couple); 2.80–2.40 (bs, 1H for each stereoiso-
mer couple, OH: exchange with D2O); 1.46 (s, 3H, CH3Cq of
one stereoisomer couple); 1.40 (s, 3H, CH3Cq of the other
stereoisomer couple); 1.28–1.17 (m, 12 H, 3H of CH3CH2
and 3H of CH3CH for each stereoisomer couple, respec-
tively). 13C NMR (CDCl3, d): 172.55, 153.82, 129.48,
128.13, 121.27, 121.18, 85.80, 72.74, 72.47, 61.91, 16.85,
15.47, 14.27. GC–MS (70 eV) (m/z) (rel. int.) 274 [M(37Cl)+,
3], 272 [M(35Cl)+, 8], 228 (23), 199 (13), 157 (11), 155 (34),
130 (34), 129 (13), 128 (100), 111 (9), 99 (19), 43 (36). Anal.
CH for C13H17ClO4.
13C NMR (CDCl3, d): 170.66, 155.89, 129.91, 127.06,
116.66, 79.37, 76.75, 62.02, 61.78, 35.71, 35.24, 26.74,
26.18, 14.33. GC–MS (70 eV) (m/z) (rel. int.) 302 [M(37Cl)+,

152
M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
6.3.7. General procedure for the preparation of 2-(4-chlo-
rophenoxy)-3-hydroxyalkanoic acids 10a–g
3200, 3035, 2964, 2876, 1723, 1595, 1584, 1491, 1415,
1
1344, 1227, 1175, 1133, 1066, 1008, 826 cm–1. H NMR
(CD3OD, d): 7.28–7.20 (m, 4H, aromatic protons, 2H for
each stereoisomer couple); 6.94–6.87 (m, 4H, aromatic pro-
tons, 2H for each stereoisomer couple); 5.00–4.80 (bs, 4H,
OH and COOH, 2H for each stereoisomer couple: exchange
with D2O); 4.61–4.59 (2d partially overlapped, J = 4.67 and
3.57 Hz, 2H, CHOC6H4Cl, 1H of each stereoisomer couple);
4.12–3.99 (m, 2H, CHOH, 1H of each stereoisomer couple);
1.73–1.33 (m, 8H, CH2CHOH and CH3CH2, 4H for each
stereoisomer couple); 0.97–0.94 (m, 6H, CH3CH2, 3H for
each stereoisomer couple). 13C NMR (CD3OD, d): 172.08,
171.86, 157.36, 157.14, 129.14, 125.25, 126.12, 116.68,
116.51, 80.97, 79.95, 71.59, 71.50, 35.09, 34.33, 18.82,
13.08. Anal. CH for C12H15ClO4.
0.45 M aqueous KOH (substrate/KOH = 1/2) was added to
0.08 M solution of ethyl 2-(4-chlorophenoxy)-3-
a
hydroxyalkanoate in THF. The reaction mixture was stirred
at room temperature and when no more substrate was
observed by TLC (mobile phase: petroleum ether/ethyl ac-
etate = 8:2), the solvent was removed under reduced pres-
sure. The alkaline aqueous layer was washed three times with
ethyl ether, then acidified with 2 N HCl and extracted three
times with ethyl ether. The combined organic extracts were
dried over anhydrous Na2SO4, and the solvent was evapo-
rated under reduced pressure to give a colourless oil which
was crystallised from CHCl3/hexane.
6.3.7.1. 2-(4-Chlorophenoxy)-3-hydroxybutanoic acid
(10a). Reaction time = 1 h; 79% yield; d.e. = 15%. White
solid. M.p. = 105.9–107 °C (CHCl3/hexane). IR (KBr):
3650–3200, 3030, 2985, 2940, 1718, 1594, 1586, 1495,
1455, 1409, 1384, 1230, 1171, 1157, 1080, 1009, 878, 826,
799 cm–1. 1H NMR (CD3OD, d): 7.27–7.22 (m, 4H, aromatic
protons, 2H for each stereoisomer couple); 6.94–6.89 (m,
4H, aromatic protons, 2H for each stereoisomer couple);
5.10–4.90 (bs, 4H, OH and COOH, 2H for each stereoisomer
couple: exchange with D2O); 4.61–4.59 (d, J = 4.26 Hz, 1H,
CHOC6H4Cl of one stereoisomer couple); 4.54–4.52 (d,
J = 4.12 Hz, 1H, CHOC6H4Cl of the other stereoisomer
couple); 4.28–4.19 (m, 2H, CHOH, 1H for each stereoisomer
couple); 1.34–1.32 (d, J = 6.46 Hz, 3H, CH3CHOH of one
stereoisomer couple); 1.33–1.30 (d, J = 6.59 Hz, 3H,
CH3CHOH of the other stereoisomer couple). 13C NMR
(CD3OD, d): 171.88, 171.74, 157.31, 157.14, 129.15,
126.26, 126.19, 116.71, 116.57, 81.21, 81.09, 67.90, 18.19,
17.28. Anal. CH for C10H11ClO4.
6.3.7.4. 2-(4-Chlorophenoxy)-3-hydroxy-4-methylpentanoic
(10d). Reaction time = 1.5 h; 44% yield; d.e. = 27%. White
solid. M.p. = 144.4–145.7 °C (CHCl3/hexane). IR (KBr):
3600–3200, 3035, 2961, 2872, 1715, 1598, 1583, 1490,
1410, 1388, 1226, 1175, 1136, 1064, 1007, 900, 965, 823,
1
799, 708 cm–1. H NMR (CD3OD, d): 7.27–7.23 (m, 4H,
aromatic protons, 2H for each stereoisomer couple); 6.93–
6.89 (m, 4H, aromatic protons, 2H for each stereoisomer
couple); 5.10–4.83 (bs, 4H, OH and COOH, 2H for each
stereoisomer couple: exchange with D2O); 4.77–4.76 (d,
J = 2.74 Hz, 1H, CHOC6H4Cl of one stereoisomer couple);
4.62–4.60 (d, J = 6.04 Hz, 1H, CHOC6H4Cl of the other
stereoisomer couple); 3.79–3.75 (t, J = 5.77 Hz, 1H, CHOH
of one stereoisomer couple); 3.72–3.68 (dd, J = 8.10 Hz,
J = 2.74 Hz, 1H, CHOH of the other stereoisomer couple);
2.08–1.92 (m, 2H, CH(CH3)2, 1H for each stereoisomer
couple); 1.07–1.05 (d, J = 6.59 Hz, 3H, CH3 of one stereoi-
somer couple); 1.04–1.01 (d, J = 7.14 Hz, 3H, CH3 of the
other stereoisomer couple); 0.97–0.94 (d, J = 6.59 Hz, 3H,
CH3 of one stereoisomer couple); 0.89–0.87 (d, J = 7.14 Hz,
3H, CH3 of the other stereoisomer couple). 13C NMR
(CD3OD, d): 172.44, 172.34, 157.31, 156.81, 129.22,
129.15, 126.32, 126.04, 116.54, 116.30, 78.91, 77.93, 77.51,
76.23, 30.65, 30.00, 18.71; 18.44; 18.12; 16.16.Anal. CH for
C12H15ClO4.
6.3.7.2. 2-(4-Chlorophenoxy)-3-hydroxypentanoic acid
(10b). Reaction time = 1.5 h; 78% yield; d.e. = 11%. White
solid. M.p. = 103–104.2 °C (CHCl3/hexane). IR (KBr):
3650–3200, 3060, 2971, 2884, 1723, 1595, 1584, 1491,
1411, 1341, 1233, 1174, 1146, 1070, 1006, 875, 827, 800,
741 cm–1. 1H NMR (CD3OD, d): 7.27–7.20 (m, 4H, aromatic
protons, 2H for each stereoisomer couple); 6.94–6.85 (m,
4H, aromatic protons, 2H for each stereoisomer couple);
5.10–4.75 (bs, 4H, OH and COOH, 2H for each stereoisomer
couple: exchange with D2O); 4.63–4.55 (m, 2H,
CHOC6H4Cl, 1H of each stereoisomer couple); 4.02–3.88
(m, 2H, CHOH, 1H of each stereoisomer couple); 1.74–1.60
(m, 4H, CH3CH2, 2H for each stereoisomer couple); 1.05–
6.3.7.5. 2-(4-Chlorophenoxy)-4,4-dimethyl-3-hydroxypen-
tanoic acid (10e). Reaction time = 48 h; 14% yield;
d.e. = 41%. White solid. M.p. = 151.8–152.3 °C
1
(CHCl3/hexane). H NMR (CD3OD, d): 7.28–7.23 (m, 4H,
aromatic protons, 2H for each stereoisomer couple); 6.93–
6.87 (m, 4H, aromatic protons, 2H for each stereoisomer
couple); 4.95–4.80 (m, 5H, OH and COOH for each stereoi-
somer couple (4H, exchange with D2O), completely over-
lapped to CHOC6H4Cl of one stereoisomer couple); 4.62–
4.59 (d, J = 6.59 Hz, 1H, CHOC6H4Cl of the other
stereoisomer couple); 3.81–3.80 (d, J = 1.38 Hz, 1H, CHOH
of one stereoisomer couple); 3.70–3.68 (d, J = 6.59 Hz, 1H,
CHOH of the other stereoisomer couple); 1.00 (s, 9H, 3 CH3
of one stereoisomer couple); 0.99 (s, 9H, 3 CH3 of the other
stereoisomer couple). Anal. CH for C13H17ClO4.
0.95 (m, 6H, CH3CH2, 3H for each stereoisomer couple). 13
C
NMR (CD3OD, d): 176.02, 175.83, 161.29, 161.03, 133.07,
130.18, 130.04, 120.59, 120.42, 84.58, 83.44, 77.33, 77.24,
29.90, 29.16, 13.38. Anal. CH for C11H13ClO4.
6.3.7.3. 2-(4-Chlorophenoxy)-3-hydroxyhexanoic acid
(10c). Reaction time = 3 h; 71% yield; d.e. = 4%. White solid.
M.p. = 101.5–103 °C (CHCl3/hexane). IR (KBr): 3650–

M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
153
6.3.7.6. 2-(4-Chorophenoxy)-3-hydroxy-3-phenylpropanoic
6.4.2. Transfection assay
acid (10f). Reaction time = 1 h; 84% yield; d.e. = 32%. White
solid. M.p. = 110.3–111.4 °C (CHCl3/hexane). IR (KBr):
3600–3200, 3032, 2980, 1729, 1595, 1584, 1489, 1454,
1399, 1278, 1235, 1172, 1121, 1079, 1058, 1006, 821, 769,
Monkey kidney fibroblasts (COS-7) were seeded at 1.2 ×
105 cells per well, in 12-well plates, and cultured in Dulbec-
co’s modified Eagle’s medium (DMEM) containing 10%
fetal bovine serum, overnight at 37 °C. Two hours before
transfection, the culture medium was replaced by fresh
serum-free medium and then transfection was performed
with the multicomponent lipid-based FuGENE6 Transfec-
tion Reagent (F. Hoffmann-La Roche; Basel, Switzerland)
according to the instructions of the manufacturer.
The transfection mixture containing (for each well) 0.8 µg
of the expression vector, 1.6 µg of the reporter vector, 0.8 µg
of the control vector and 9 µl of FuGENE6 Transfection
Reagent was added directly to the cells in the presence of
serum-free medium. After 5 h the transfection medium was
removed and transfected cells were treated with three differ-
ent concentrations (50, 150 and 300 µM) of the test mol-
ecules 8a, 9a and 10a–g, at 37 °C for 48 h in the complete
culture medium. 2 µM WY-14,643, a known PPARa ligand,
was used as positive control.
Cells were then washed twice with phosphate-buffered
saline (PBS) and then dissolved in lysis buffer (0.25 M
Tris–HCl, pH 8) and lysed by three rapid freeze/thaw cycles
(three 5-min cycles). Cell debris was then removed by cen-
trifugating at 4 °C, for 15 min at 15,000 rpm. Glycerol (final
10% v/v) and b-mercaptoethanol (final 5 mM) were then
added (final volume 75 µl) and the cell extracts were stored at
–80 °C until assayed.
1
743, 705, 672 cm–1. H NMR (CD3OD, d): 7.49–7.46 (m,
4H, aromatic protons, 2H for each stereoisomer couple);
7.35–7.22 (m, 6H, aromatic protons, 3H for each stereoiso-
mer couple); 7.21–7.14 (m, 4H, aromatic protons, 2H for
each stereoisomer couple); 6.86–6.78 (m, 4H, aromatic pro-
tons, 2H for each stereoisomer couple); 5.22–5.21 (d,
J = 4.12 Hz, 1H, CHOH of one stereoisomer couple); 5.14–
5.09 (d, J = 6.32 Hz, 1H, CHOH of the other stereoisomer
couple); 5.00–4.80 (bs, 4H, OH and COOH, 2H for each
stereoisomer couple: exchange with D2O); 4.80–4.79 (d,
J = 4.12 Hz, 1H, CHOC6H4Cl of one stereoisomer couple);
4.78–4.76 (d, J = 6.32 Hz, 1H, CHOC6H4Cl of the other
stereoisomer couple). 13C NMR (CD3OD, d): 171.81,
171.44, 157.25; 156.91; 140.58; 140.36; 129.11; 129.03,
128.02, 127.91, 127.82, 127.71, 127.19, 126.70, 126.41,
126.28, 116.83, 81.80, 81.46, 74.24, 73.93. Anal. CH for
C15H13ClO4.
6.3.7.7. 2-(4-Chlorophenoxy)-3-hydroxy-2-methylbutanoic
acid (10g). Reaction time = 21 h; 64% yield; d.e. = 46%.
White solid. M.p. = 125.6–126.8 °C (CHCl3/hexane). IR
(KBr): 3600–3200, 3030, 2999, 2943, 2818, 1745, 1593,
1581, 1491, 1459, 1408, 1285, 1240, 1182, 1149, 1129,
1
1091, 1027, 927, 825, 761 cm–1. H NMR (CD3OD, d):
7.25–7.19 (m, 4H, aromatic protons, 2H for each stereoiso-
mer couple); 6.97–6.92 (m, 4H, aromatic protons, 2H for
each stereoisomer couple); 5.10–4.75 (bs, 4H, OH and
COOH, 2H for each stereoisomer couple: exchange with
D2O); 4.13–4.05 (m, 2H, CHOH, 1H of each stereoisomer
couple); 1.40 (s, 3H, CH3COC6H4Cl of one stereoisomer
couple); 1.38 (s, 3H, CH3COC6H4Cl of the other stereoiso-
mer couple); 1.31–1.29 (d, J = 6.32 Hz, 3H, CH3CHOH of
one stereoisomer couple); 1.24–1.22 (d, J = 6.32 Hz, 3H,
CH3CHOH of the other stereoisomer couple). 13C NMR
(CD3OD, d): 175.07, 174.69, 154.56, 128.84; 127.36,
127.17; 121.45, 121.14, 85.40, 84.53, 72.24, 71.66, 16.17;
15.36; 14.83; 14.27. Anal. CH for C11H13ClO4.
6.4.3. Assays to determine CAT and b-galactosidase
activity
The CAT activity assay was performed as follows: 20 µl of
cell lysate (prewarmed at 65 °C for 10 min, to deactivate
internal deacetylase enzymatic activity) were added to 10 µl
of 3.5 mg ml–1 n-butirryl-CoA, 5 µl (0.25 µCi) of [14C]-
chloramphenicol and 65 µl of distilled water and incubated
2 h at 37 °C. Reaction was blocked by adding 200 µl of the
solution xylene/2,6,10,14-tetramethylpentadecane (1:2 v/v).
After a vigorous vortexing and centrifugation for 5 min at top
speed, 150 µl of supernatant were transferred to scintillation
vial in the presence of 5 ml of scintillation liquid, and the
relative radioactivity was measured by a b-counter.
The b-galactosidase activity was measured as follows:
20 µl of cellular extracts were added to 750 µl of reaction
buffer consisting of 1 vol. of 2 mg ml–1 ONPG (o-
nitrophenyl-b-galactopyranoside) and 3 vol. of “Z buffer”
(10 mM potassium chloride, 1 mM magnesium chloride, and
50 mM b-mercaptoethanol in phosphate buffer). Reaction
was performed at 37 °C and blocked by adding 200 µl of 1 M
Na2CO3 when a typical yellow colour became appreciable.
Samples were incubated for 10 min at room temperature and
then the absorbance at 420 nm (A420) was spectrophoto-
metrically measured.
6.4. PPAR␣ transactivation assay
6.4.1. Plasmids
The reporter construct pG5-CAT, containing five copies of
the high affinity binding site for GAL4 (UAS) and used for
CAT assay, was purchased from BD Biosciences Clontech
(Palo Alto, CA). GAL4/mousePPARaLBD receptor plasmid
was prepared by fusing the mouse PPARa LBD to the DBD
of yeast GAL4, and then subcloning the fusion into the
pSG5 expression vector. pCH110, that encodes the
b-galactosidase enzyme to correct for differences in transfec-
tion efficiency, was purchased from Pharmacia Biotech (Pis-
cataway, NJ).
Finally, the CAT activity was normalised to the
b-galactosidase activity.

154
M.G. Perrone et al. / European Journal of Medicinal Chemistry 40 (2005) 143–154
[13] N. Inomata, H. Yoshida, Y. Aoki, M. Tsunoda, M. Yamamoto,
Acknowledgements
J. Tohoku, Exp. Med. 165 (1991) 171–182.
[14] E. Ciolek, M. Dauca, Biol. Cell. 71 (1991) 313–320.
[15] S. Ferorelli, C. Franchini, F. Loiodice, M.G. Perrone, A. Scilimati,
M.G. Sinicropi, P. Tortorella, Tetrahedron: Asymmetry 12 (2001)
853–862 (and references cited therein).
Work carried out under the framework of the National
Project “Progettazione, Sintesi e Valutazione Biologica di
Nuovi Farmaci Cardiovascolari” supported by the Ministero
dell’Istruzione, dell’Università e della Ricerca (MIUR,
Rome). Thanks are also due to the University of Bari and
CNR-Istituto di Chimica dei Composti OrganoMetallici-
ICCOM/Sezione di Bari (Italy).
[16] S. Ferorelli, F. Loiodice, V. Tortorella, R. Amoroso, G. Bettoni,
D. Conte-Camerino, A. De Luca, Farmaco 52 (1997) 367–374 (and
references cited therein).
[17] S.M. Rangwala, M.L. O’Brien, V. Tortorella, A. Longo, F. Loiodice,
D.J. Noonan, D.R. Feller, Chirality 9 (1997) 37–47.
[18] S. Ferorelli, F. Loiodice, V. Tortorella, D. Conte-Camerino, A. De
Luca, Farmaco 56 (2001) 239–246.
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[21] 7a–f were prepared modifying (Scheme in Table 1) the procedure
reported by C. Trebaul, J. Teste, Bull. Soc. Chim. 7 (1969) 2456–2462
(the modified procedure requires shorter reaction time, 1–3 h instead
of one night. If the reaction mixture was left (under stirring or not) for
more than 3 h others mono- and di-halogenated compounds were the
main reaction products).
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