Synthesis of new troglitazone derivatives: Anti-proliferative activity in breast cancer cell lines and preliminary toxicological study

Article abstract of DOI:10.1016/j.ejmech.2012.02.044

Breast cancer is the most prevalent cancer in women. The development of resistances to therapeutic agents and the absence of targeted therapy for triple negative breast cancer motivate the search for alternative treatments. With this aim in mind, we synth

Full text of DOI:10.1016/j.ejmech.2012.02.044

European Journal of Medicinal Chemistry 51 (2012) 206e215
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Original article
Synthesis of new troglitazone derivatives: Anti-proliferative activity in breast
cancer cell lines and preliminary toxicological study
Stéphane Salamone a, Christelle Colin b, Isabelle Grillier-Vuissoz b, Sandra Kuntz b, Sabine Mazerbourg b,
,
Stéphane Flament b, Hélène Martin c, Lysiane Richert c, Yves Chapleur a, Michel Boisbrun a
*
a Groupe SUCRES, UMR 7565, Nancy-Université-CNRS, BP 70239, F-54506 Vandoeuvre-lès-Nancy, France
b EA 4421 Signalisation, Génomique et Recherche Translationnelle en Oncologie (SIGRETO), Faculté des Sciences, Université Henri Poincaré, Nancy-Université, BP 70239,
54506 Vandoeuvre-lès-Nancy Cedex, France
c Laboratoire de Toxicologie Cellulaire, EA 4267, IFR133, UFR des Sciences Médicales et Pharmaceutiques, 25030 Besançon Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Breast cancer is the most prevalent cancer in women. The development of resistances to therapeutic
agents and the absence of targeted therapy for triple negative breast cancer motivate the search for
alternative treatments. With this aim in mind, we synthesised new derivatives of troglitazone,
a compound which was formerly used as an anti-diabetic agent and which exhibits anti-proliferative
activity on various cancer cell lines. Among the compounds prepared, some displayed micromolar
activity against hormone-dependent and hormone-independent breast cancer cells. Furthermore, the
influence of the compounds on the viability of primary cultures of human hepatocytes was evaluated.
This enabled us to obtain for the first time interesting structureetoxicity relationships in this family of
compounds, resulting in 6b and 8b, which show good anti-proliferative activities and poor toxicity
towards hepatocytes, compared to troglitazone.
Received 9 November 2011
Received in revised form
20 February 2012
Accepted 21 February 2012
Available online 28 February 2012
Keywords:
Troglitazone
Chromane
Breast cancer
Hepatotoxicity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
including troglitazone, rosiglitazone, and pioglitazone [5]. Among
these drugs, which were used in the treatment of type II diabetes,
Breast cancer is the most prevalent cancer in women and
represents the second highest leading cause of cancer death in this
population after lung cancer. Approximately 75% of tumours
only pioglitazone is still therapeutically used in some countries; the
others were withdrawn from the market because of side-effects
such as Drug-Induced Liver Injury (DILI) [6]. Regarding breast
express the oestrogen receptor ER
a
that allows for treatment with
cancer, several PPARg ligands of the TZD family have been shown to
the anti-oestrogen tamoxifen. Additionally, human epidermal
growth factor receptor 2 (HER2) expressing tumours may be
treated with the monoclonal antibody trastuzumab [1]. Neverthe-
less, de novo or acquired resistance to these therapeutic agents
often lead to treatment failure [2,3]. Therefore, there is an urgent
need to develop alternative therapeutics. Moreover, there is no
targeted therapy for the treatment of breast tumours known as
possess anticancer properties in vitro and in vivo [7,8]. Among these,
troglitazone (TGZ) seems to be particularly interesting and has even
been tested in clinical trials for breast cancer before its withdrawal
[9]. The anti-proliferative modes of action have been extensively
studied, but are still not fully elucidated. Actually, increasing data
suggest that its activity is mainly related to PPAR
mechanisms [10e12]. Cyclin D1 ablation [13,14], inhibition of Bcl-
xL/Bcl-2 functions [15], TGF signalling [16], ER disruption [10],
telomerase activity suppression [17], interaction with oestrogen-
related receptor and [18], energy restriction [19], and calcium
and ERK-dependent expression of the early growth response gene 1
[20] are examples of TZD-induced PPAR -independent events. A
major aspect of this new therapeutic strategy lies in the fact that
non-malignant cells seem to be resistant to these PPAR -inde-
g-independent
triple negative: ER
regard, ligands of peroxisome proliferator-activated receptor
gamma (PPAR ) and especially thiazolidinediones (TZD) were
envisioned as a therapeutic alternative towards advanced (or
relapsing) breast cancer [4]. PPAR is a nuclear receptor associated
a
-, PR- (progesterone receptor), HER2-. In this
b
a
g
a
g
g
g
with glucose homeostasis. It can be activated by endogenous
ligands such as 15d-PGJ(2) or synthetic compounds like TZD,
g
pendent antitumour effects, which holds the translational potential
of these agents [12].
New TGZ derivatives have already been prepared. In particular,
the introduction of a double bond adjacent to the thiazolidinedione
* Corresponding author. Tel.: þ33 383 68 43 63; fax: þ33 383 68 47 80.
E-mail address: michel.boisbrun@univ-lorraine.fr (M. Boisbrun).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.044

S. Salamone et al. / European Journal of Medicinal Chemistry 51 (2012) 206e215
207
ring yielded
D
2-troglitazone (
D
2TGZ) which was devoid of PPAR
g
3. Biological studies and discussion
activity but exhibited an anti-proliferative ability similar to the one
of TGZ [13,21]. Additional structural modulations were made on the
All of the TGZ derivatives were tested for their anti-proliferative
activity against hormone-dependent (MCF-7) and hormone-
independent (MDA-MB-231) breast cancer cell lines. Their hepa-
totoxicity was also evaluated by incubation with primary cultures
of human hepatocytes. The results are summarised in Table 1.
Regarding cell proliferation, it can first be noticed that the
hormone-independent cell line (MDA-MB-231) is more sensitive to
the synthesised molecules than the hormone-dependent cell line
(MCF-7), with the exception of compound 8c. These are interesting
data that could be helpful for understanding the mechanism of
action of TGZ derivatives in future studies. Besides, MDA-MB-231
cells display less variability than MCF-7 in their sensitivity to the
D2TGZ template, especially by chromane phenol functionalisation,
arising in low micromolar active molecules [22]. Nevertheless,
a major drawback of TGZ derivatives lies in the fact that TGZ itself
exhibits hepatotoxicity. The mechanisms underlying TGZ DILI are
not fully understood. Yokoi [23] has recently reviewed the
proposed mechanisms and suggested that the presence of the
chromane moiety could be of importance in the induction of DILI
since it can lead to toxic derivatives. Besides, Bharatam [24]
recently reported a theoretical study to better understand the
formation of these derivatives.
The goal of the present work was to synthesise new
D2TGZ
derivatives, aiming at increasing anti-proliferative activity towards
breast cancer cell lines, and decreasing hepatotoxicity, as compared
to TGZ.
various compounds: the IC50 values range from 3.2 to 5.0
most of the active compounds (see 6a, 8aed) in MDA-MB-231
whereas in the MCF-7 cell line they are ranging from 3.2 M for
the most active 8c to 26.0 M for the least active 8d.
mM for
m
m
2. Chemistry
Dealing with structureeactivity relationships, one can first
notice that the double bond has an influence on the activity of the
compounds. If we compare 6a to 9a, 6b to 9b, and 8a to 11, a slight
positive effect is observed on both cell lines: unsaturated deriva-
tives are more active than their saturated counterparts. Comparing
Since phenolic functionalisation of D2TGZ seems to have a great
influence on its biological activity, we envisioned the synthesis of
many ester derivatives of this molecule. Thus, we first needed
a straightforward access to
[25], its synthesis is not trivial. Taking into account reported
methods dealing with the synthesis of 2TGZ and derivatives
D
2TGZ. As mentioned by Halperin et al.
D2TGZ to TGZ, a positive effect is also observed in MCF-7, but not in
MDA-MB-231 cell line. Besides, phenol functionalisation with
succinoyl residues (see 8e, 8f, 12) appears deleterious to the anti-
proliferative activity. This might be due to the increase of polarity
that such substituents induce in the proximity of the chromane
core. In the case of compound 8c, the polar ammonium moiety is
rather distant from the chromane, since it is linked to this hetero-
cycle by seven apolar methylene groups, resulting in one of the
most active molecules. Such methylene groups are also present in
the case of biotinoyl derivatives 8a and 8d and in the case of Boc-
linker intermediate 8b, which are also active. Furthermore,
strongly apolar Boc residue directly linked to the chromane affor-
ded very active 6a. A confirmation of the role of polarity came from
compounds 6b and 9b in which the polar hydroxyl moiety was
replaced by a hydrogen atom. Even if they are not the most active
compounds, it resulted in a clear increase in potency. Furthermore,
one can notice that the presence of the biotin residue (with or
without a caprylic linker: see 8a and 8d) does not provide additive
value compared to other substituents, unlike what we expected.
D
[22,25e29], we started from commercial racemic TroloxÒ 1, but
intended to proceed without protection of the phenol of the chro-
mane moiety with usual groups (i.e. Bn, TBDMS, MOM, MEM) whose
introduction and removal proved to be troublesome. Therefore, after
a simple esterification of the starting material [30,31] to give 2,
we smoothly introduced a tert-butyloxycarbonyl group to get
compound 3a (Scheme 1). After reduction to 4a, triflate formation
followed by substitution with p-hydroxybenzaldehyde gave 5a.
Then, condensation with 2,4-thiazolidinedione gave O-protected
D2TGZ 6a in 65% overall yield. In order to study the influence of the
phenolic hydroxyl group on both activity and toxicity, we intended
to remove this moiety. Thus, based on a method reported by Sal-
vatore et al. [32] to get new tocopheryl derivatives, we prepared
triflate 3b in almost quantitative yield, followed by catalytic
hydrogenation under basic conditions to get the deoxygenated
compound 3c in very good yield. The previously mentioned
sequence afforded deoxygenated
Smooth removal of the phenolic protecting group of 6a in acidic
medium afforded 2TGZ 7 in 86% yield afterchromatography, which
D
2TGZ 6b in 39% overall yield.
Compounds 6a, 8a, and reference compounds 7 (D2TGZ) and TGZ
were selected to exhibit proliferation curves for both cell lines, in
Figs. 1 and 2.
D
enabled us to functionalise the molecule via an ester linkage.
It has been reported [33,34] that conjugation of molecules with
biotin might increase their uptake in tumour cells since the latter
often over-express biotin-specific receptors on their surface. Thus,
we prepared the biotinoyl derivative 8a, albeit in moderate yield. In
order to facilitate recognition by the biotin receptor, we envisioned
the introduction of a linker. Hence, Boc-protected aminocaprylic
compound 8b was obtained in good yield. Acidic deprotection
afforded 8c which was in turn linked to biotin to give 8d.
Furthermore, it has been reported that the succinoyl moiety linked
In terms of hepatotoxicity assessment, it is important to take
into account the pharmacokinetic parameters [23] and even more
importantly, the potential tissue concentration in the liver, an organ
potentially able to accumulate xenobiotics. As reported by Loi et al.
[36], the maximum plasma concentration of TGZ in patients taking
a dose of 600 mg/day reached 6.3
mM, while Sahi et al. [37]
demonstrated that in rats the concentration in liver was 10e12
fold higher than that in the plasma. For this reason, in the
present study, the cytotoxicity of TGZ and its derivatives were
assessed using human hepatocytes at concentrations up to 100 mM,
to
a
-tocopherol induces antiadhesion properties on breast cancer
as reported by Yamamoto [38] for the evaluation of TGZ metabolites
toxicity. Our study was conducted by measuring cell viability using
the MTT assays on primary cultures of human hepatocytes and for
clarity, we present the results as percentage of surviving cells after
cells [35]. We then synthesised the succinoyl derivative 8e, along
with non-acidic derivative 8f.
In order to study the influence of the double bond of these
molecules on the anti-proliferative activity and toxicity, we
prepared saturated analogues. Thus, as reported by Ramachandran
[28], 70 psi hydrogen afforded 9a and 9b in good yield from 6a and
6b respectively. Acidic treatment of 9a gave TGZ 10 which was in
turn converted into biotinoyl and succinoyl derivatives (11 and 12
respectively).
incubation with 100
mM of compound (Table 1). The study shows
that, with the exception of non-active succinoyl derivatives, all
D2TGZ derivatives are much less toxic than TGZ ones: compare
D2TGZ (7) to TGZ, 6a to 9a, 6b to 9b, and 8a to 11. It has been
reported that TGZ toxicity is at least partly due to glutathione
depletion because of conjugation through linkage with the

208
S. Salamone et al. / European Journal of Medicinal Chemistry 51 (2012) 206e215
Scheme 1. Reagents and conditions: i EtOH, cat. p-TSA, reflux, 95%; ii Boc2O, cat. DMAP, 99%; iii Tf2O, Pyr., 99%; iv TEA, H2 70 psi, 10% Pd/C, 96%; v LiAlH4, 91% (4a), 94% (4b); vi 1)
Tf2O, Pyr. 2) 4-hydroxybenzaldehyde, K2CO3 86% (5a), 84% (5b); vii 2,4-thiazolidinedione, piperidine, , 88% (6a), 54% (6b); viii TFA, 86% (7), 94% (8c); ix D-biotin, TEA, IBCF, cat.
D
DMAP, 35% (8a and 11), 64% (8d); x Boc-aminocaprylic acid, TEA, IBCF, cat. DMAP, 75%; xi Succinic anhydride, cat. DMAP, 43% (8e), 63% (12); xii TEA, IBCF, N-acetylethylenediamine,
29%; xiii H2 70 psi, 10% Pd/C, 99% (9a), 70% (9b); xiv HCl, quant.
thiazolidinedione ring [23,29,39]. The presence of a conjugated
double bond might decrease the electrophilicity of the heterocycle,
then disfavour conjugation with glutathione, and thus decrease the
toxicity.
for these compounds up to 50 mM but evidences that 6b and 8b are
less cytotoxic, as compared to TGZ and D2TGZ at 100 mM.
4. Conclusion
In addition, one can notice that
D2TGZ derivatives bearing
a lipophilic substituent on the chromane (see 6a and 8b) exhibit
much less toxicity towards hepatocytes than others. Such residues
might disfavour formation of reported toxic quinone-type metab-
olites [23,39]. From that point of view, compounds 6b and 9b
devoid of hydroxyl group and thus unable to be transformed into
that type of metabolite, show less toxicity towards hepatic cells
A series of new TGZ and D2TGZ derivatives were prepared and
tested for their anti-proliferative activity against hormone-
dependent and hormone-independent breast cancer cell lines.
These molecules were obtained via functionalisation or removal of
the hydroxyl group of the chromane moiety. The most active
molecules were
range activity. Some of them also showed much less toxicity on
primary cultured hepatocytes than parents TGZ and 2TGZ. This is
a new step in the way to get more active and less toxic molecules to
D2TGZ derivatives, exhibiting low-micromolar
than
value of the whole series is observed for 6b. Fig. 3 shows the
cytotoxicity profiles for TGZ, 2TGZ, 6b, and 8b which are similar
D2TGZ and TGZ respectively. Furthermore, the best viability
D
D

S. Salamone et al. / European Journal of Medicinal Chemistry 51 (2012) 206e215
209
Table 1
Anti-proliferative activity and hepatocyte viability related to prepared compounds
and TGZ.
IC50 ꢀ SEMb
Hepatocyte
viabilityf (%)
a
Compound
MCF-7c
MDA-MB-231d
6ae
6b
(
7.7 ꢀ 0.5
12.5 ꢀ 0.9
29.7 ꢀ 2.0
11.2 ꢀ 1.8
13.0 ꢀ 1.5
3.2 ꢀ 0.1
26.0 ꢀ 5.7
>50
3.3 ꢀ 0.1
10.8 ꢀ 0.7
16.6 ꢀ 1.0
3.5 ꢀ 0.1
3.2 ꢀ 0.3
5.0 ꢀ 0.7
3.7 ꢀ 0.7
>25
75
84
63
66
79
57
61
54
75
47
66
50
76
52
7e
D2TGZ)
8ae
8be
8ce
8d
8e
8f
9a
9b
11
12
>50
>25
11.2 ꢀ 0.3
20.0 ꢀ 1.0
15.9 ꢀ 0.8
>50
3.8 ꢀ 0.4
12.3 ꢀ 0.5
6.5 ꢀ 0.4
>25
Fig. 2. Cytotoxicity of troglitazone (TGZ) and derived compounds 7 (D2TGZ), 6b and 8b
TGZ
35.4 ꢀ 1.3
15.7 ꢀ 0.1
in MDA-MB-231 cells. Data are means þ/ꢁ SEM of triplicate determinations. IC50 of 6b
a
Concentration (mM) required to inhibit tumour cell proliferation by 50%.
Standard Error of the Mean.
Hormone-dependent breast cancer cell line.
Hormone-independent breast cancer cell line.
Anti-proliferative activity of these molecules on MCF-7 and MDA-MB-231 cell
and 8b were significantly different from those measured for TGZ (p < 0.05) and
(p < 0.05).
D2TGZ
b
c
d
e
13C NMR spectra were mainly recorded on a Bruker spectrometer
DPX250 (250 MHz and 62.9 MHz, respectively). A few spectra were
recorded on a Bruker spectrometer DRX400 (400 MHz for 1H and
lines have already been reported [21].
Percentage of surviving primary hepatocytes as compared to non-treated cells,
after 90 min of incubation with compounds, at 100 mM.
f
100.6 MHz for 13C). Chemical shifts (
d) are given in ppm relative to
the solvent residual peak. The following abbreviations are used for
multiplicity of NMR signals: s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quadruplet, m ¼ multiplet, br ¼ broad signal. The J values given
refer to apparent multiplicities and do not represent the true
coupling constants. Mass spectra were obtained on a VG-Platform
Micromass-Waters (ESIþ/quad). Elemental analyses were per-
formed on a Thermofinnigan FlashEA 1112 apparatus.
treat breast cancer. Synthesis of new molecules aiming at reaching
this goal is currently ongoing by our team.
5. Experimental protocols
Solvents and liquid reagents were purified and dried according to
recommended procedures. Troglitazone used for anti-proliferative
and hepatotoxicity assays was purchased from SigmaeAldrich. TLC
analyses were performed using standard procedures on Kieselgel 60
F254 plates (Merck). Compounds were visualised using UV light
(254 nm) and a solution of cerium sulfate tetrahydrate and phos-
phomolybdic acid in 10% aqueous sulfuric acid as developing agent.
Column chromatography was performed on Silica Gel SI 60
5.1. Synthesis
5.1.1. (ꢀ) 6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic
acid ethyl ester (2)
To a solution of (ꢀ) 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-
carboxylic acid 1 (4.46 g, 18.82 mmol) in EtOH (175 mL) was
added p-TSA (361 mg, 1.90 mmol), and the mixture was heated at
reflux for 12 h. The solution was concentrated under vacuum, and
the residue was dissolved in EtOAc (150 mL). The resulting solution
(63e200
m
m) (Merck). Some chromatographic purifications were
performed on Silica Gel 60H (5e40
m
m) (Merck) using an AxxialÒ
Modul Prep apparatus, working at 8 bars, with a 20 mm diameter
column. FTIR spectra were recorded on a PerkineElmer spectrum
1000 apparatus on NaCl windows or KBr pellets. Melting points
were determined with a Kofler bench and are uncorrected. 1H and
Fig. 3. Cytotoxicity of troglitazone (TGZ) and derived compounds 7 (D2TGZ), 6b and 8b
Fig. 1. Cytotoxicity of troglitazone (TGZ) and derived compounds 7 (D2TGZ), 6b and 8b
in human hepatocytes. Data are means þ/ꢁ SEM of triplicate determinations.
in MCF-7 cells. Data are means þ/ꢁ SEM of triplicate determinations. IC50 of 6b and 8b
*Statistical significantly different from TGZ (*p < 0.05, **p < 0.01). #Statistical signif-
were significantly different from those measured for TGZ (p < 0.05) and
(p < 0.05).
D2TGZ
icantly different from
D2TGZ (#p < 0.05).

210
S. Salamone et al. / European Journal of Medicinal Chemistry 51 (2012) 206e215
was washed with 5% aqueous NaHCO3 solution (2 ꢂ 50 mL) and
brine (2 ꢂ 50 mL). The organic layer was dried (MgSO4) and the
solvent evaporated. The crude product was dried to give 4.98 gꢁo1f
purchased from Fisher/ACROS (catalogue #19503-0500). A solution
of 4.96 g (12.09 mmol) of 3b in THF (40 mL) was added to the flask,
followed by MeOH (80 mL), and TEA (7.4 mL, 53.22 mmol). The
suspension was shaken under 70 psi hydrogen pressure for 20 h at
room temperature, then filtered on celiteÒ and the resulting solu-
tion was concentrated. Column chromatography (Hexane/EtOAc,
90:10) afforded 3.03 g (11.55 mmol, 96% yield) of colourless liquid
which upon storage at 4 ꢃC gave low melting point (# 30 ꢃC) white
crystals. IR (film) cmꢁ1: 2981, 2935, 1753, 1732, 1462, 1312, 1202,
white crystals (17.90 mmol, 95% yield). M.p. 124 ꢃC. IR (film) cm
:
3532, 2985, 2927, 1731, 1185, 1107. 1H NMR (CDCl3)
d: 1.18
(t, J ¼ 7.1 Hz, 3H, COOCH2CH3), 1.60 (s, 3H, CH3), 1.81e1.94 (m, 1H,
chromane 3-HaHb), 2.06 (s, 3H, ArCH3), 2.16 (s, 3H, ArCH3), 2.19 (s,
3H, ArCH3), 2.38e2.69 (m, 3H, chromane 3-HaHb and 4-H2), 4.12 (q,
J ¼ 7.1 Hz, 2H, COOCH2CH3), 4.24 (s, 1H, OH). 13C NMR (CDCl3)
d: 11.4
(ArCH3), 11.9 (ArCH3), 12.3 (ArCH3), 14.2 (CH3), 21.1 (CH2), 25.5
1178, 1137, 1108, 1023. 1H NMR (CDCl3)
d: 1.19 (t, J ¼ 7.1 Hz, 3H,
(CH3), 30.7 (CH2), 61.1 (OCH2), 77.0, 117.0, 118.5, 121.3, 122.7, 145.4,
COOCH2CH3), 1.62 (s, 3H, CH3), 1.82e1.95 (m, 1H, chromane
3-HaHb), 2.13 (s, 3H, ArCH3), 2.16 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3),
2.38e2.69 (m, 3H, chromane 3-HaHb and 4-H2), 4.14 (q, J ¼ 7.1 Hz,
145.8, 174.0 (C]O). ESI-MS (pos. mode): m/z
¼
205.42
[M ꢁ CO2Et]þ, 301.30 [M þ Na]þ, 317.27 [M þ K]þ. Anal. Calcd for
C16H22O4 (278.3): C, 69.04; H, 7.97. Found: C, 68.70; H, 8.08.
2H, COOCH2CH3), 6.58 (s, 1H, Harom). 13C NMR (CDCl3)
d: 11.5, 14.2,
18.8, 19.8, 20.5, 25.4, 30.4, 61.2, 77.4, 116.6, 122.0, 123.4, 133.2, 134.9,
151.6, 173.8 (ester C]O). ESI-MS (pos. mode): m/z ¼ 285.21
[M þ Na]þ. Anal. Calcd for C16H22O3 (262.4): C, 73.25; H, 8.45.
Found: C, 73.63; H, 8.20.
5.1.2. (ꢀ) 6-tert-Butoxycarbonyloxy-2,5,7,8-tetramethyl-chroman-
2-carboxylic acid ethyl ester (3a)
To a solution of 2 (4.72 g, 16.96 mmol) in CH2Cl2 (40 mL) were
added di-tert-butyldicarbonate (4.08 g, 18.96 mmol) and 4,4-
dimethylaminopyridine (207 mg, 1.69 mmol) under argon atmo-
sphere. The solution was stirred at room temperature for 2 h and
the solvent was evaporated. The residue was dissolved in EtOAc
(100 mL), washed with a 1:1 solution of brine and 1 N aqueous HCl
(60 mL), and a saturated aqueous solution of NaHCO3 (2 ꢂ 40 mL).
The organic layer was dried (MgSO4) and the solvent evaporated.
The crude product was purified by column chromatography
(Hexane/EtOAc, 90:10) to give the titled compound (6.39 g,
16.88 mmol, 99% yield) as colourless liquid. IR (film) cmꢁ1: 2981,
5.1.5. (ꢀ) Carbonic acid tert-butyl ester 2-hydroxymethyl-2,5,7,8-
tetramethyl-chroman-6-yl ester (4a)
To a suspension of LiAlH4 (558 mg, 14.70 mmol) in anhydrous
THF (15 mL) at 0 ꢃC, was slowly added under argon, a solution of 3a
(6.34 g,16.75 mmol) in anhydrous THF (40 mL). The suspension was
stirred for 1 h (not more, to avoid reduction of the carbonate pro-
tecting group) at 0 ꢃC, then, allowed to reach room temperature.
The mixture was poured into a saturated aqueous solution of NH4Cl
(60 mL) and extracted with EtOAc (3 ꢂ 30 mL). The combined
organic layers were washed with water (1 ꢂ 60 mL), brine
(1 ꢂ 60 mL), dried (MgSO4) and the solvent was evaporated. The
crude product was purified by column chromatography (Hexane/
EtOAc, 80:20) to give a colourless gum which was dissolved in
hexane. Evaporation of the solvent afforded the titled compound
(5.14 g, 15.28 mmol, 91% yield) as a white solid. M.p. 78e80 ꢃC. IR
(film) cmꢁ1: 3486, 2980, 2932, 1754, 1281, 1236, 1156, 1097. 1H NMR
2934, 1754, 1731, 1279, 1238, 1157, 1105. 1H NMR (CDCl3)
d: 1.17 (t,
J ¼ 7.1 Hz, 3H, COOCH2CH3), 1.54 (s, 9H, t-Bu), 1.59 (s, 3H, CH3),
1.79e1.92 (m,1H, chromane 3-HaHb),1.99 (s, 3H, ArCH3), 2.08 (s, 3H,
ArCH3), 2.16 (s, 3H, ArCH3), 2.36e2.69 (m, 3H, chromane 3-HaHb
and 4-H2), 4.12 (q, J ¼ 7.1 Hz, 2H, COOCH2CH3). 13C NMR (CDCl3)
d:
11.8 (ArCH3 ꢂ2),12.7 (ArCH3),14.1 (CH3), 20.8 (CH2), 25.3 (CH3), 27.7
(t-Bu), 30.2 (CH2), 61.1 (OCH2), 77.1, 82.7 (t-Bu), 117.1, 123.0, 125.1,
127.3, 141.6, 149.3, 152.2 (carbonate C]O), 173.6 (ester C]O). ESI-
MS (pos. mode): m/z ¼ 401.25 [M þ Na]þ. Anal. Calcd for
C21H30O6 (378.5): C, 66.65; H, 7.99. Found: C, 66.24; H, 7.76.
(CDCl3) d: 1.22 (s, 3H, CH3), 1.55 (s, 9H, t-Bu), 1.64e1.77 (m, 1H,
chromane 3-HaHb), 1.87e2.02 (m, 1H, chromane 3-HaHb), 2.05 (s,
3H, ArCH3), 2.09 (br s, 6H, 2ꢂ ArCH3), 2.65 (m, 2H, chromane 4-H2),
3.62 (m, 2H, CH2OH). 13C NMR (CDCl3)
d
: 12.0 (2ꢂ ArCH3), 12.8
5.1.3. (ꢀ) 2,5,7,8-Tetramethyl-6-trifluoromethanesulfonyloxy-
chroman-2-carboxylic acid ethyl ester (3b)
(ArCH3), 20.2 (CH2), 20.6 (CH3), 27.5 (CH2), 27.8 (t-Bu), 69.4 (OCH2),
75.6, 82.9 (t-Bu), 117.4, 123.0, 125.5, 127.4, 141.3, 148.8, 152.3 (C]O).
ESI-MS (pos. mode): m/z ¼ 359.28 [M þ Na]þ. Anal. Calcd for
C19H28O5 (336.4): C, 67.83; H, 8.39. Found: C, 67.97; H, 8.19.
To a stirred solution of 2 (500 mg, 1.80 mmol) in dry CH2Cl2
(10 mL) under argon was added pyridine (872
m
L, 10.78 mmol) and
the solution was cooled to 0 ꢃC. Then, triflic anhydride (453
m
L,
2.69 mmol) was added dropwise and the solution was stirred for
1 h at room temperature. The solution was diluted with CH2Cl2
(20 mL) and washed with water (2 ꢂ 30 mL), dried (MgSO4) and
concentrated to dryness. The liquid residue was purified by column
chromatography (Hexane/EtOAc, 85:15) to give 735 mg (1.79 mmol,
99% yield) of colourless liquid. IR (film) cmꢁ1: 2922, 1457, 1417,
5.1.6. (ꢀ) (2,5,7,8-Tetramethyl-chroman-2-yl)methanol (4b)
To a suspension of LiAlH4 (85 mg, 2.24 mmol) in anhydrous THF
(5 mL) at 0 ꢃC, was added dropwise under argon, a solution of 3c
(623 mg, 2.37 mmol) in anhydrous THF (10 mL). The suspension
was stirred for 1 h at 0 ꢃC, then LiAlH4 (81 mg, 2.13 mmol) was anew
added to the mixture. After 30 min the mixture was poured into
a saturated aqueous solution of NH4Cl (20 mL) and extracted with
EtOAc (3 ꢂ 30 mL). The combined organic layers were washed with
brine (2 ꢂ 10 mL), dried (MgSO4) and the solvent was evaporated.
The crude product was purified by column chromatography
(Hexane/EtOAc, 90:10) to give the titled compound (490 mg,
2.22 mmol, 94% yield) as a white solid. M.p. 81e82 ꢃC. IR (film)
1403, 1228, 1208, 1140, 1111, 1034, 878. 1H NMR (CDCl3)
d: 1.20 (t,
J ¼ 7.1 Hz, 3H, COOCH2CH3), 1.66 (s, 3H, CH3), 1.84e1.97 (m, 1H,
chromane 3-HaHb), 2.19 (s, 3H, ArCH3), 2.22 (s, 3H, ArCH3), 2.27 (s,
3H, ArCH3), 2.44e2.74 (m, 3H, chromane 3-HaHb and 4-H2), 4.17
(dq, Ja ¼ 7.1 Hz, Jb ¼ 1.0 Hz, 2H, COOCH2CH3). 13C NMR (CDCl3)
d:
12.1, 13.3, 14.2 (2 peaks), 21.1, 25.4, 30.2, 61.4, 77.7, 118.4, 118.8 (q,
J ¼ 318 Hz, CF3), 124.6, 126.8, 128.6, 140.5, 151.1, 173.2 (ester C]O).
ESI-MS (pos. mode): m/z ¼ 433.14 [M þ Na]þ. Anal. Calcd for
C17H21O6F3S (410.4): C, 49.75; H, 5.16. Found: C, 49.87; H, 5.39.
cmꢁ1: 3367, 2931, 1458, 1310, 1106, 1049. 1H NMR (CDCl3)
d: 1.24 (s,
3H, CH3), 1.68e1.78 (m, 1H, chromane 3-HaHb), 1.89 (t, J ¼ 7.5 Hz,
1H, OH), 1.95e2.05 (m, 1H, chromane 3-HaHb), 2.08 (s, 3H, ArCH3),
2.17 (s, 3H, ArCH3), 2.21 (s, 3H, ArCH3), 2.62e2.68 (m, 2H, chromane
5.1.4. (ꢀ) 2,5,7,8-Tetramethyl-chroman-2-carboxylic acid ethyl
ester (3c)
In a Parr hydrogenator flask was first introduced 10% palladium
on carbon (3.00 g), in order to avoid any ignition of the solvent.
Please note that following reported procedure [32], Pd/C was
4-H2), 3.63 (m, 2H, CH2OH), 6.89 (s, 1H, Harom); 13C NMR (CDCl3)
d:
11.5, 18.9, 19.7, 19.9, 20.7, 27.7, 69.6, 75.8, 116.9, 122.1, 123.0, 133.7,
135.0, 151.1. ESI-MS (pos. mode): m/z ¼ 243.06 [M þ Na]þ. Anal.
Calcd for C14H20O2 (220.3): C, 76.33; H, 9.15. Found: C, 76.52;
H, 9.03.

S. Salamone et al. / European Journal of Medicinal Chemistry 51 (2012) 206e215
211
5.1.7. (ꢀ) Carbonic acid tert-butyl ester 2-(4-
J ¼ 9.3 Hz, 2H, CH2O), 6.59 (s, 1H, Harom), 7.03 (d, J ¼ 8.8 Hz, 2H,
Harom), 7.83 (d, J ¼ 8.8 Hz, 2H, Harom), 9.89 (s, 1H, CHO). 13C NMR
formylphenoxymethyl)-2,5,7,8-tetramethyl-chroman-6-yl ester (5a)
Preparation of the triflate intermediate: to an ice-cooled solu-
tion of anhydrous pyridine (2.8 mL, 34.50 mmol) in dry CH2Cl2
(20 mL), was added dropwise trifluoromethanesulfonic anhydride
(1.39 mL, 8.26 mmol) under argon. A solution of 4a (1.95 g,
5.80 mmol) in dry CH2Cl2 (20 mL) was added to the reaction
mixture. The solution was stirred at 0 ꢃC for 30 min and the solvent
was evaporated. The excess of pyridine was co-evaporated with
toluene and the residue so obtained was diluted in EtOAc (50 mL).
The organic layer was washed with 5% aqueous NaHCO3 solution
(2 ꢂ 30 mL), brine (2 ꢂ 30 mL), dried (MgSO4) and the solvent was
removed under vacuum to give a brown liquid residue.
(CDCl3)
d 11.5, 18.9, 19.8, 19.9, 22.9, 28.5, 73.1, 74.6, 115.2, 116.7,
122.2, 123.0, 130.3, 132.1, 133.6, 135.2, 151.1, 164.2, 190.9 (CHO). ESI-
MS: m/z ¼ 347.14 [M þ Na]þ. Anal. Calcd for C21H24O3 (324.4): C,
77.75; H, 7.46. Found: C, 77.05; H, 6.98.
5.1.9. (ꢀ) Carbonic acid tert-butyl ester 2-{4-[2,4-dioxo-
thiazolidin-(5Z)-ylidenemethyl]phenoxymethyl}-2,5,7,8-
tetramethyl-chroman-6-yl ester (6a)
To a solution of 5a (2.20 g, 4.99 mmol) in absolute EtOH (50 mL,
which may be replaced by dry toluene), was added thiazolidine-
2,4-dione (1.17 g, 9.99 mmol) and piperidine (247 mL, 2.50 mmol).
Substitution with 4-hydroxybenzaldehyde: to a solution of
the above triflate in anhydrous DMF (12 mL) were added
4-hydroxybenzaldehyde (709 mg, 5.81 mmol) and K2CO3 (1.61 g,
11.65 mmol). The reaction mixture was stirred under argon at room
temperature for two days, and then water (30 mL) was added. The
resulting solution was extracted with EtOAc (30 mL). The organic
layer was washed with water (30 mL) dried (MgSO4) and concen-
trated under vacuum. The crude product was purified by column
chromatography (Hexane/EtOAc, 90:10) to give a colourless syrup
which was dissolved in diethyl ether and concentrated under
vacuum to give the titled compound as a white solid (2.20 g,
4.99 mmol, 86% yield over two steps). M.p. 124e125 ꢃC. IR (film)
cmꢁ1: 2980, 2934, 1752, 1693, 1601, 1278, 1237, 1157, 1096. 1H NMR
The mixture was heated at reflux under argon for 12 h. The solution
was concentrated under vacuum, and the residue was dissolved in
EtOAc (100 mL). The resulting solution was washed with 5%
aqueous NaHCO3 solution (2 ꢂ 50 mL) and water (2 ꢂ 50 mL). The
organic layer was dried (MgSO4) and the solvent evaporated to give
a yellow solid. The crude product was purified by column chro-
matography (Hexane/EtOAc, 80:20) to give the titled compound
(2.37 g, 4.39 mmol, 88% yield) as a white solid. M.p. 195e196 ꢃC. IR
(film) cmꢁ1: 3189, 2981, 2933, 1747, 1705, 1595, 1510, 1245, 1154. 1H
NMR (CDCl3) d: 1.41 (s, 3H, CH3), 1.56 (s, 9H, t-Bu), 1.81e1.93 (m, 1H,
chromane 3-HaHb), 2.04 (s, 3H, ArCH3), 2.05 (s, 3H, ArCH3), 2.07 (s,
3H, ArCH3), 2.08e2.16 (m, 1H, chromane 3-HaHb), 2.64 (br t,
J ¼ 6.7 Hz, 2H, chromane 4-H2), 3.94, 4.01 (AB system, J ¼ 9.3 Hz, 2H,
CH2O), 6.99 (d, J ¼ 8.8 Hz, 2H, Harom), 7.43 (d, J ¼ 8.8 Hz, 2H, Harom),
(CDCl3) d: 1.43 (s, 3H, CH3), 1.55 (s, 9H, t-Bu), 1.80e1.95 (m, 1H,
chromane 3-HaHb), 2.04 (s, 3H, ArCH3), 2.06 (s, 3H, ArCH3), 2.08 (s,
3H, ArCH3), 2.11e2.19 (m, 1H, chromane 3-HaHb), 2.64 (m, 2H,
chromane 4-H2), 3.97, 4.08 (AB system, J ¼ 9.3 Hz, 2H, CH2O), 7.03
(d, J ¼ 8.7 Hz, 2H, Harom), 7.83 (d, J ¼ 8.7 Hz, 2H, Harom), 9.89 (s, 1H,
7.80 (s, 1H, ArCH]). 13C NMR (CDCl3)
d: 11.9 (ArCH3), 12.0 (ArCH3),
12.8 (ArCH3), 20.2 (CH2), 22.7 (CH3), 27.8 (t-Bu), 28.4 (CH2), 73.0,
74.5, 83.1 (t-Bu), 115.6 (CHarom), 117.3, 119.6, 123.3, 125.6, 125.9,
127.7, 132.4 (CHarom), 134.2 (]CH), 141.4, 148.8, 152.6 (carbonate
C]O), 161.1, 167.0 (C]O), 167.5 (C]O). ESI-MS (pos. mode):
m/z ¼ 562.08 [M þ Na]þ. Anal. Calcd for C29H33NO7S (539.6): C,
64.54; H, 6.16; N, 2.60. Found: C, 64.38; H, 6.20; N, 2.65.
CHO). 13C NMR (CDCl3)
d: 11.9 (ArCH3), 12.0 (ArCH3), 12.8 (ArCH3),
20.2 (CH2), 22.8 (CH3), 27.8 (t-Bu), 28.3 (CH2), 72.8, 74.4, 82.9 (t-Bu),
115.1 (CHarom), 117.2, 123.2, 125.5, 127.5, 130.2, 132.0 (CHarom), 141.4,
148.8, 152.3 (carbonate C]O), 164.1, 190.9 (CHO). ESI-MS (pos.
mode): m/z ¼ 463.22 [M þ Na]þ, 441.22 [M þ H]þ Anal. Calcd for
C26H32O6 (440.5): C, 70.89; H, 7.32. Found: C, 70.77; H, 7.29.
5.1.10. (ꢀ) 5-[1-[4-(2,5,7,8-Tetramethyl-chroman-2-ylmethoxy)-
phenyl]-meth-(Z)-ylidene]-thiazolidine-2,4-dione (6b)
To a solution of 5b (437 mg, 1.35 mmol) in absolute EtOH
(13 mL), were added thiazolidine-2,4-dione (317 mg, 2.71 mmol)
5.1.8. (ꢀ) 4-(2,5,7,8-Tetramethyl-chroman-2-ylmethoxy)-
benzaldehyde (5b)
and piperidine (67
mL, 0.68 mmol). The mixture was heated at
Preparation of the triflate intermediate: to an ice-cooled solu-
reflux under argon for 24 h. The solution was concentrated under
vacuum, and the residue was dissolved in EtOAc (40 mL). The
resulting solution was washed with 5% aqueous NaHCO3 solution
(2 ꢂ 10 mL) and brine (2 ꢂ 10 mL). The organic layer was dried
(MgSO4) and the solvent evaporated. The crude product was puri-
fied by column chromatography (Hexane/EtOAc, 80:20) to give the
titled compound (308 mg, 0.73 mmol, 54% yield) as a yellow
amorphous solid. M.p. 129e134 ꢃC. IR (film) cmꢁ1: 3190, 3050,
2934, 1740, 1690, 1595, 1511, 1249, 1178, 1111, 1042, 827, 738. 1H
tion of anhydrous pyridine (930
(10 mL), was added dropwise trifluoromethanesulfonic anhydride
(480 L, 2.85 mmol) under argon. A solution of 4b (422 mg,
mL, 11.50 mmol) in dry CH2Cl2
m
1.92 mmol) in dry CH2Cl2 (10 mL) was added to the reaction
mixture. The solution was stirred at 0 ꢃC for 20 min and the solvent
was evaporated. The excess of pyridine was co-evaporated with
toluene and the residue so obtained was dissolved in EtOAc
(50 mL). The organic layer was washed with 5% aqueous NaHCO3
solution (2 ꢂ 10 mL), brine (1 ꢂ 10 mL), dried (MgSO4) and the
solvent was removed under vacuum to give a brown oily residue.
Substitution with 4-hydroxybenzaldehyde: to a solution of the
above triflate in anhydrous DMF (5 mL) were added 4-
hydroxybenzaldehyde (236 mg, 1.93 mmol) and K2CO3 (528 mg,
3.82 mmol). The reaction mixture was stirred under argon at room
temperature for two days, then water (10 mL) was added. The
resulting solution was extracted with EtOAc (2 ꢂ 30 mL). The
organic layer was dried (MgSO4) and concentrated under vacuum.
The crude product was purified by column chromatography
(Hexane/EtOAc, 90:10) to give the titled compound as a colourless
NMR (CDCl3) d: 1.44 (s, 3H, CH3), 1.85e1.96 (m, 1H, chromane 3-
HaHb), 2.05 (s, 3H, ArCH3), 2.08e2.16 (m, 1H, chromane 3-HaHb),
2.16 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 2.63 (br t, J ¼ 6.2 Hz, 2H,
chromane 4-H2), 3.98, 4.07 (AB system, J ¼ 9.3 Hz, 2H, 2-CH2O), 6.59
(s, 1H, Harom), 7.02 (d, J ¼ 9.0 Hz, 2H, Harom), 7.44 (d, J ¼ 9.0 Hz, 2H,
Harom), 7.81 (s, 1H, ArCH]), 8.28 (br s, 1H, NH). 13C NMR (CDCl3)
d
11.5, 18.9, 19.8, 19.9, 22.9, 28.5, 73.0, 74.6, 115.7, 116.8, 119.4, 122.2,
123.0, 125.9, 132.4, 133.6, 134.5, 135.2, 151.1, 161.3, 166.5 (C]O),
167.2 (C]O). ESI-MS (pos. mode): m/z ¼ 446.17 [M þ Na]þ. Anal.
Calcd for C24H25NO4S (423.5): C, 68.06; H, 5.95; N, 3.31. Found: C,
67.82; H, 5.95; N, 3.34.
syrup (521 mg,1.61 mmol, 84% yield over two steps). IR (film) cmꢁ1
:
2933, 1693, 1600, 1577, 1310, 1246, 1157. 1H NMR (CDCl3)
d
: 1.45 (s,
5.1.11. (ꢀ) 5-[1-[4-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-
ylmethoxy)phenyl]meth-(Z)-ylidene]thiazolidine-2,4-dione (7)
To a solution of 6a (601 mg, 1.11 mmol) in CH2Cl2 (15 mL), was
added trifluoroacetic acid (5 mL). The mixture was stirred for 1 h
3H, CH3), 1.86e1.97 (m, 1H, chromane 3-HaHb), 2.05 (s, 3H, ArCH3),
2.08e2.17 (m, 1H, chromane 3-HaHb), 2.17 (s, 3H, ArCH3), 2.20 (s,
3H, ArCH3), 2.63 (m, 2H, chromane 4-H2), 4.00, 4.09 (AB system,

212
S. Salamone et al. / European Journal of Medicinal Chemistry 51 (2012) 206e215
and the solvent was evaporated. The crude product was dissolved
in EtOAc (50 mL) and washed with 5% aqueous NaHCO3 solution
(2 ꢂ 50 mL) and water (2 ꢂ 50 mL). The organic layer was dried
(MgSO4) and the solvent evaporated. The crude product was puri-
fied by column chromatography (Hexane/EtOAc, 80:20) and then
recrystallised in MeOH to give the titled compound (422 mg,
0.95 mmol, 86% yield) as a yellow solid. M.p. 130e132 ꢃC. IR (film)
cmꢁ1: 3321, 3181, 2927, 1738, 1690, 1594, 1510, 1253, 1178. 1H NMR
(266 mL, 2.05 mmol) was added, the solution was stirred for 1 h at
the same temperature, and a solution of 7 (751 mg, 1.71 mmol) and
DMAP (25 mg, 0.21 mmol) in CH2Cl2 (40 mL) was added. The
reaction mixture was stirred for 12 h at room temperature, then
washed with 5% aqueous NaHCO3 solution (2 ꢂ 40 mL), 5% aqueous
citric acid solution (2 ꢂ 40 mL), brine (2 ꢂ 40 mL), dried (MgSO4)
and concentrated to dryness. Column chromatography (CH2Cl2/
EtOAc, 95:5) using AxxialÒ Modul Prep apparatus (see above)
afforded 873 mg (1.28 mmol, 75% yield) of light yellow amorphous
low melting point solid. IR (film) cmꢁ1: 3375, 3181, 2932,1747,1705,
(CDCl3) d: 1.42 (s, 3H, CH3), 1.85e1.96 (m, 1H, chromane 3-HaHb),
2.05e2.20 (m, 1H, chromane 3-HaHb), 2.08 (s, 3H, ArCH3), 2.11 (s,
3H, ArCH3), 2.16 (s, 3H, ArCH3), 2.65 (br t, J ¼ 6.5 Hz, 2H, chromane
4-H2), 3.96, 4.05 (AB system, J ¼ 9.3 Hz, 2H, CH2O), 4.31 (br s, 1H,
OH), 7.02 (d, J ¼ 8.7 Hz, 2H, Harom), 7.44 (d, J ¼ 8.7 Hz, 2H, Harom),
1597, 1511, 1250, 1178. 1H NMR (CDCl3)
d: 1.42 (s, 3H, CH3), 1.45 (s,
9H, t-Bu), 1.32e1.55 (m, 8H, caprylic CH2ꢂ 4), 1.80 (m, 2H, caprylic
CH2), 1.92 (m, 1H, chromane 3-HaHb), 1.97 (s, 3H, ArCH3), 2.01 (s, 3H,
ArCH3), 2.05 (s, 3H, ArCH3), 2.10 (m, 1H, chromane 3-HaHb), 2.62 (m,
4H, chromane 4-CH2 þ caprylic CH2), 3.12 (br q, J ¼ 6.4 Hz, 2H,
caprylic CH2), 3.99 (m, 2H, CH2O), 4.53 (br s, 1H, carbamate NH),
6.99 (d, J ¼ 8.7 Hz, 2H, Harom), 7.43 (d, J ¼ 8.7 Hz, 2H, Harom), 7.79 (s,
7.81 (s, 1H, ArCH]), 8.75 (br s, 1H, NH). 13C NMR (CDCl3)
d: 11.5
(ArCH3), 12.0 (ArCH3), 12.4 (ArCH3), 20.4 (CH2), 22.7 (CH3), 28.8
(CH2), 72.9, 74.1,115.7 (CHarom),117.3,118.7,119.3,121.5,122.9,125.8,
132.4 (CHarom), 134.5 (]CH), 145.0, 145.2, 161.3, 166.8 (C]O), 167.4
(C]O). ESI-MS (pos. mode): m/z ¼ 440.15 [M þ H]þ. Anal. Calcd for
C24H25NO5S,1/4H2O (444.0): C, 64.92; H, 5.79; N, 3.16. Found: C,
64.84; H, 5.62; N, 3.35.
1H, ArCH]), 8.87 (br s, 1H, NH). 13C NMR (CDCl3)
d: 12.0, 12.3, 13.1,
20.3, 22.8, 25.2, 26.8, 28.5, 28.6, 29.1, 29.4, 30.2, 34.2, 40.8, 73.1, 74.5,
77.4, 79.3, 115.7, 117.4, 119.7, 123.3, 125.3, 126.0, 127.4, 132.3, 134.1,
141.2, 148.8, 156.2, 161.1, 166.8, 167.3, 172.6. ESI-MS (pos. mode):
m/z ¼ 703.27 [M þ Na]þ. Anal. Calcd for C37H48N2O8S (680.8): C,
65.27; H, 7.11; N, 4.12. Found: C, 64.99; H, 6.89; N, 4.14.
5.1.12. (ꢀ) 5-((3aS,4S,6aR)-2-Oxo-hexahydro-thieno[3,4-d]
imidazol-4-yl)-pentanoic acid 2-{4-[2,4-dioxo-thiazolidin-(5Z)-
ylidenemethyl]-phenoxymethyl}-2,5,7,8-tetramethyl-chroman-6-yl
ester (8a)
5.1.14. (ꢀ) 8-Amino-octanoic acid 2-{4-[2,4-dioxo-thiazolidin-(5Z)-
ylidenemethyl]-phenoxymethyl}-2,5,7,8-tetramethyl-chroman-6-yl
ester (8c)
To a solution of D(þ)-biotin (46 mg, 0.19 mmol) in DMF (4 mL)
under an argon atmosphere were added CH2Cl2 (2 mL) and trie-
thylamine (26
m
L, 0.19 mmol). The solution was cooled to 0 ꢃC and
To a solution of 8b (306 mg, 0.45 mmol) in CH2Cl2 (15 mL) was
added trifluoroacetic acid (5 mL) and the mixture was stirred at
room temperature for 45 min. The solution was concentrated to
dryness. The residue was dissolved in CH2Cl2 (10 mL) and Et2O
(10 mL) was added to the solution. The suspension was concen-
trated to dryness and carefully dried to give 290 mg (0.42 mmol,
94% yield) of light yellow solid. M.p. 114 ꢃC. IR (KBr) cmꢁ1: 3423,
isobutyl chloroformate (24
mL, 0.19 mmol) was added. The mixture
was stirred 45 min at this temperature, then 7 (76 mg, 0.17 mmol)
and DMAP (2 mg, 0.017 mmol) dissolved in CH2Cl2 (6 mL) were
added. The solution was stirred at room temperature for 12 h. The
CH2Cl2 was evaporated and the liquid residue was diluted with
EtOAc (30 mL). The solution was washed with 5% aqueous NaHCO3
solution (2 ꢂ 20 mL), 5% aqueous citric acid solution (2 ꢂ 20 mL),
water (2 ꢂ 20 mL), dried (MgSO4) and concentrated to dryness.
Column chromatography (CH2Cl2/MeOH, 97:3) using AxxialÒ
Modul Prep apparatus (see above) afforded a colourless residue
which was suspended in a 1:1 mixture of water and CH3CN (5 mL)
and freeze dried to give 40 mg (0.06 mmol, 35% yield) of white
powder. M.p. 196 ꢃC. IR (KBr) cmꢁ1: 3422, 2928, 1736, 1702, 1598,
2930,1745, 1686, 1598, 1252, 1204,1179, 1147. 1H NMR (DMSO-d6)
d:
1.33 (br s, 9H, CH3 þ caprylic CH2ꢂ 3), 1.52 (m, 2H, caprylic CH2),
1.66 (m, 2H, caprylic CH2), 1.85 (m, 1H, chromane 3-HaHb), 1.91 (s,
6H, 2ꢂ ArCH3), 1.96 (s, 3H, ArCH3), 2.03 (m, 1H, chromane 3-HaHb),
2.63 (m, 4H, chromane 4-CH2 þ caprylic CH2), 2.78 (m, 2H, caprylic
CH2), 4.12 (s, 2H, OCH2), 7.15 (d, J ¼ 8.9 Hz, 2H, Harom), 7.54 (d,
J ¼ 8.9 Hz, 2H, Harom), 7.68 (br s, 3H, NHþ3 ), 7.75 (s, 1H, ArCH]),
1509, 1250, 1160. 1H NMR (DMSO-d6, 400 MHz)
d: 1.28 (s, 3H, CH3),
12.54 (br s, 1H, NH). 13C NMR (DMSO-d6)
d: 11.6, 11.9, 12.7, 19.4, 21.5,
1.35e1.70 (m, 6H, biotin CH2), 1.75e2.00 (m, 2H, chromane 3-CH2),
1.86 (s, 6H, ArCH3), 1.91 (s, 3H, ArCH3), 2.50e2.65 (m, 5H,
CH2CO þ chromane 4-CH2 þ CHaHbS), 2.78 (A part of an ABX
system, J ¼ 5.1, 12.4 Hz, 1H, CHaHbS), 3.07 (m, 1H, CHS), 4.08 (m, 3H,
OCH2 þ CHNH), 4.26 (m, 1H, CHNH), 6.32 (s, 1H, NH), 6.41 (s, 1H,
NH), 7.10 (d, J ¼ 8.8 Hz, 2H, Harom), 7.48 (d, J ¼ 8.8 Hz, 2H, Harom),
7.69 (s, 1H, ArCH]), 12.45 (br s, 1H, thiazolidinedione NH). 13C NMR
24.4, 25.7, 27.0, 27.6, 28.2, 28.3, 30.7, 33.1, 72.6, 74.5, 115.7, 117.4,
120.4, 122.0, 125.0, 125.7, 126.5, 131.8, 132.0, 140.6, 148.2, 160.5,
167.4, 167.9, 171.6. ESI-MS (pos. mode): m/z ¼ 581.28 [M]þ. Anal.
Calcd for C34H41N2O8SF3, 1/2H2O (703.8): C, 58.02; H, 6.02; N, 3.98.
Found: C, 57.79; H, 5.91; N, 3.91.
5.1.15. (ꢀ) 8-[5-((3aR,6R,6aS)-2-Oxo-hexahydro-thieno[2,3-d]
imidazol-6-yl)-pentanoylamino]-octanoic acid 2-{4-[2,4-dioxo-
thiazolidin-(5Z)-ylidenemethyl]-phenoxymethyl}-2,5,7,8-
tetramethyl-chroman-6-yl ester(8d)
(DMSO-d6,100 MHz) d: 11.7 (ArCH3),12.0 (ArCH3),12.8 (ArCH3),19.4
(CH2), 21.5 (CH3), 24.6 (biotin CH2), 27.6 (CH2), 28.1 (biot CH2), 28.2
(biot CH2), 33.0 (CH2), 40.2 (CH2S), 55.4 (CHS), 59.2 (CHNH), 61.1
(CHNH), 72.6 (OCH2), 74.5 (OCCH3), 115.7 (CHarom), 117.4, 120.4,
122.0, 125.0, 125.7, 126.5, 131.8 (]CH), 132.0 (CHarom), 140.6, 148.2,
160.5, 162.7, 167.5, 168.0, 171.6. ESI-MS (pos. mode): m/z ¼ 666.21
[M þ H]þ, 688.19 [M þ Na]þ. Anal. Calcd for C34H39N3O7S2, 1/2H2O
(674.8): C, 60.51; H, 5.97; N, 6.23. Found: C, 60.75; H, 6.21; N, 6.21.
To an argon-flushed solution of biotin (35 mg, 0.14 mmol) in dry
DMF (4 mL) were added dry CH2Cl2 (1 mL) and TEA (50
0.36 mmol). The solution was cooled in an ice bath, isobutyl chlor-
oformate (19 L, 0.14 mmol) was added, and the solution was stirred
mL,
m
for 45 min. Then a solution of 8c (100 mg, 0.14 mmol) in DMF (2 mL)
was added and the mixture was stirred for 12 h. The solution was
diluted with EtOAc (50 mL) and washed with 5% aqueous NaHCO3
solution (2 ꢂ 30 mL), 5% aqueous citric acid solution (2 ꢂ 30 mL),
brine (2 ꢂ 30 mL), dried (MgSO4) and concentrated to dryness.
Column chromatography (CH2Cl2/MeOH, 97:3 to 90:10) using
AxxialÒ Modul Prep apparatus (see above) afforded 72 mg
5.1.13. (ꢀ) 8-tert-Butoxycarbonylamino-octanoic acid 2-{4-[2,4-
dioxo-thiazolidin-(5Z)-ylidenemethyl]-phenoxymethyl}-2,5,7,8-
tetramethyl-chroman-6-yl ester (8b)
To an argon-flushed solution of 8-(tert-butoxycarbonylamino)
caprylic acid (532 mg, 2.05 mmol, prepared as previously reported
[40]) in CH2Cl2 (40 mL) was added TEA (286
mL, 2.05 mmol) and the
(0.089 mmol, 64% yield) of white powder. M.p.147 ꢃC. IR (KBr) cmꢁ1
3422, 2927, 1741, 1699, 1597, 1510, 1250, 1151. 1H NMR (DMSO-d6,
:
mixture was cooled with an ice bath. Then, isobutyl chloroformate

S. Salamone et al. / European Journal of Medicinal Chemistry 51 (2012) 206e215
213
400 MHz)
d
: 1.12e1.42 (m, 15H, CH3 þ 6ꢂ CH2), 1.50 (m, 2H, CH2),
4.12 (s, 2H, OCH2), 7.15 (d, J ¼ 8.6 Hz, 2H, Harom), 7.54 (d, J ¼ 8.6 Hz,
1.65 (m, 2H, CH2), 1.80e2.02 (m, 2H, chromane 3-CH2), 1.91 (s, 6H,
2ꢂ ArCH3), 1.96 (s, 3H, ArCH3), 2.04 (m, 2H, caprylic CH2), 2.40e2.70
(m, 5H, chromane 4-CH2 þ CH2CONH þ CHaHbS), 2.80 (dd, J ¼ 4.7,
12.8 Hz, 1H, CHaHbS), 3.01 (m, 2H, caprylic CH2), 3.08 (m, 1H, CHS),
4.11 (m, 3H, OCH2 þ CHNH), 4.29 (m, 1H, CHNH), 6.36 (s, 1H, NH),
6.43 (s, 1H, NH), 7.15 (d, J ¼ 8.4 Hz, 2H, Harom), 7.54 (d, J ¼ 8.4 Hz, 2H,
Harom), 7.74 (m, 2H, ArCH] þ CONH), 12.50 (br s, 1H, NH). 13C NMR
2H, Harom), 7.82 (br t, 1H, NH), 7.94 (br t, 1H, NH), 12.49 (br s, 1H,
NH). 13C NMR (DMSO-d6)
d: 11.6,11.9, 12.7, 19.4, 21.5, 22.6, 27.6, 28.7,
29.8, 38.3, 38.4, 72.6, 74.5, 115.7, 117.3, 120.7, 121.8, 125.1, 125.7,
126.7, 131.6, 132.0, 140.6, 148.1, 160.4, 167.7, 168.1, 169.3, 170.7, 171.1.
ESI-MS (pos. mode): m/z ¼ 646.39 [M þ Na]þ. Anal. Calcd for
C32H37N3O8S, 1/2H2O (632.7): C, 60.74; H, 6.05; N, 6.64. Found: C,
60.83; H, 5.92; N, 6.50.
(DMSO-d6,100 MHz) d: 12.5 (CH3),12.8 (CH3),13.6 (CH3), 20.3 (CH2),
22.4 (CH3), 25.3 (CH2), 26.2 (CH2), 27.1 (CH2), 28.5 (CH2), 28.9 (CH2),
29.1 (CH2), 29.3 (CH2), 29.4 (CH2), 30.0 (CH2), 34.0 (CH2), 36.1 (CH2),
39.2 (CH2), 55.8 (CH2), 56.3 (CH), 60.0 (CH), 61.9 (CH), 73.5, 75.4,
116.5 (CHarom),118.2,121.6,122.8,125.8,126.7,127.4,132.2 (ArCH]),
132.8 (CHarom), 141.4, 149.0, 161.2, 163.6, 168.7, 169.0, 172.5, 172.6.
ESI-MS (pos. mode): m/z ¼ 829.33 [M þ Na]þ. Anal. Calcd for
C42H54N4O8S2, 1/2H2O (816.0): C, 61.81; H, 6.79; N, 6.87. Found: C,
61.72; H, 6.80; N, 6.68.
5.1.18. (ꢀ) Carbonic acid tert-butyl ester 2-[4-(2,4-dioxo-
thiazolidin-5-ylmethyl)-phenoxymethyl]-2,5,7,8-tetramethyl-
chroman-6-yl ester (9a)
In a Parr hydrogenator flask was first introduced 10% palladium
on carbon (400 mg), in order to avoid any ignition of the solvent. A
solution of 6a (500 mg, 0.93 mmol) in dioxane (40 mL) was then
added and the suspension was shaken under 70 psi hydrogen
pressure for 20 h at room temperature, then filtered on celiteÒ and
the resulting solution was concentrated. Thorough drying of the
residue afforded 499 mg (0.92 mmol, 99% yield) of amorphous
solid. M.p. 143 ꢃC. IR (film) cmꢁ1: 3218, 2928, 1755, 1704, 1514, 1239,
5.1.16. (ꢀ) Succinic acid mono-(2-{4-[2,4-dioxo-thiazolidin-(5Z)-
ylidenemethyl]-phenoxymethyl}-2,5,7,8-tetramethyl-chroman-6-yl)
ester (8e)
1156. 1H NMR (CDCl3)
d: 1.43 (s, 3H, CH3), 1.55 (s, 9H, t-Bu), 1.89 (m,
To a solution of 7 (82 mg, 0.18 mmol) in CH2Cl2 (10 mL) were
added succinic anhydride (37 mg, 0.37 mmol) and DMAP (2 mg,
0.018 mmol), and the mixture was refluxed for 12 h. Succinic
anhydride (18 mg, 0.18 mmol) was anew added and the mixture
was refluxed for another 12 h. The solvent was evaporated, the
residue was dissolved in EtOAc (20 mL) and the solution was
washed with 5% aqueous citric acid solution (3 ꢂ 10 mL), water
(3 ꢂ 10 mL), dried (MgSO4) and concentrated to dryness. Column
chromatography (CH2Cl2/MeOH, 98:2 then 96:4) using AxxialÒ
Modul Prep apparatus (see above) afforded a residue which was
suspended in a 1:1 mixture of water and CH3CN (5 mL) to give after
freeze drying 43 mg (0.08 mmol, 43% yield) of light yellow powder.
M.p. 131e136 ꢃC. IR (KBr) cmꢁ1: 3433, 2926, 1736, 1686, 1598, 1510,
1H, chromane 3-HaHb), 2.04 (s, 3H, ArCH3), 2.07 (s, 6H, 2ꢂ ArCH3),
2.14 (m, 1H, chromane 3-HaHb), 2.63 (t, J ¼ 6.3 Hz, 2H, chromane 4-
CH2), 3.09 (dd, A part of an ABX system, J ¼ 9.5, 14.1 Hz, 1H, PhCH2),
3.45 (dd, B part of an ABX system, J ¼ 3.8, 14.1 Hz, 1H, PhCH2),
3.86,3.97 (AB system, J ¼ 9.2 Hz, 2H, CH2O), 4.48 (dd, X part of an
ABX system, J ¼ 3.8 Hz, 9.5 Hz, 1H, CH), 6.87 (d, J ¼ 8.6 Hz, 2H,
Harom), 7.13 (d, J ¼ 8.6 Hz, 2H, Harom), 7.26 (s, 1H, NH). 13C NMR
(CDCl3) d: 11.9, 12.0, 12.8, 20.3, 23.0, 27.9, 28.5, 37.9, 53.8, 72.8, 74.7,
82.9, 115.2, 117.5, 123.2, 125.5, 127.5, 128.0, 130.4, 141.4, 149.0, 152.5,
158.7, 170.1, 173.9. ESI-MS (pos. mode): m/z ¼ 564.29 [M þ Na]þ.
Anal. Calcd for C29H35NO7S (541.6): C, 64.30; H, 6.51; N, 2.59.
Found: C, 64.49; H, 6.96; N, 2.44.
1253, 1151. 1H NMR (DMSO-d6)
d
: 1.34 (s, 3H, CH3), 1.92 (s, 6 H,
5.1.19. (ꢀ) 5-[4-(2,5,7,8-Tetramethyl-chroman-2-ylmethoxy)-
benzyl]-thiazolidine-2,4-dione (9b)
ArCH3ꢂ 2), 1.96 (s, 3H, ArCH3), 1.78e2.11 (m, 2H, chromane 3-CH2),
2.61 (m, 4H, CH2), 2.83 (m, 2H, CH2), 4.12 (s, 2H, OCH2), 7.15 (d,
J ¼ 8.9 Hz, 2H, Harom), 7.54 (d, J ¼ 8.9 Hz, 2H, Harom), 7.73 (s, 1H,
ArCH]), 12.29 (s, 1H, NH), 12.47 (br s, 1H, COOH). 13C NMR (DMSO-
In a Parr hydrogenator flask was first introduced 10% palladium
on carbon (102 mg), in order to avoid any ignition of the solvent. A
solution of 6b (98 mg, 0.23 mmol) in dioxane (20 mL) was then
added and the suspension was shaken under 70 psi hydrogen
pressure for 20 h at room temperature, then filtered on celiteÒ and
the resulting solution was concentrated. The crude product was
purified by column chromatography (Hexane/EtOAc, 80:20) to give
a colourless syrup which was dissolved in diethyl ether and
concentrated under vacuum to give 67 mg of white solid (0.16 mmol,
70% yield). Low melting point solid. IR (film) cmꢁ1: 3212, 2924,1755,
d6) d: 11.6,11.8,12.6,19.4, 21.5, 27.6, 28.4, 28.7, 72.6, 74.5,115.7,117.3,
120.6, 121.9, 125.1, 125.7, 126.7, 131.6, 132.0, 140.6, 148.1, 160.4, 167.6,
168.0, 170.9, 173.3. ESI-MS (pos. mode): m/z ¼ 562.20 [M þ Na]þ.
Anal. Calcd for C28H29NO8S, 1/2H2O (548.6): C, 61.30; H, 5.51; N,
2.55. Found: C, 60.98; H, 5.40; N, 2.58.
5.1.17. (ꢀ) N-(2-Acetylamino-ethyl)-succinamic acid 2-{4-[2,4-
dioxo-thiazolidin-(5Z)-ylidenemethyl]-phenoxymethyl}-2,5,7,8-
tetramethyl-chroman-6-yl ester (8f)
1699,1512,1310,1243,1159. 1H NMR (CDCl3)
d: 1.43 (s, 3H, CH3),1.88
(m, 1H, chromane 3-HaHb), 2.06 (s, 3H, ArCH3), 2.13 (m, 1H, chro-
mane 3-HaHb), 2.16 (s, 3H, ArCH3), 2.20 (s, 3H, ArCH3), 2.62 (br t,
J ¼ 7.0 Hz, 2H, chromane 4-H2), 3.11 (dd, A part of an ABX system,
J ¼ 9.3,14.1 Hz,1H, PhCH2), 3.45 (dd, B part of an ABX system, J ¼ 3.8,
14.1 Hz, 2H, PhCH2), 3.88, 3.98 (AB system, J ¼ 9.1 Hz, 2H, CH2O), 4.50
(dd, X part of an ABX system, J ¼ 3.8, 9.3 Hz, 1H, CH), 6.57 (s, 1H,
Harom), 6.87 (d, J ¼ 8.7 Hz, 2H, Harom), 7.13 (d, J ¼ 8.7 Hz, 2H, Harom),
To an argon-flushed solution of 8e (101 mg, 0.18 mmol) in
CH2Cl2 (6 mL) was added TEA (28
mL, 0.20 mmol). The solution was
cooled with an ice bath, then isobutyl chloroformate (26
mL,
0.20 mmol) was added. The mixture was stirred at this temperature
for 45 min, then it was added to a solution of N-acetylethylenedi-
amine (23 mg, 0.20 mmol) in MeOH (10 mL), and the mixture was
stirred at room temperature for 12 h. The solvents were evaporated.
Column chromatography (CH2Cl2/MeOH, 98:2 then 96:4) using
AxxialÒ Modul Prep apparatus (see above) afforded a residue which
was suspended in a 1:1 mixture of water and CH3CN (5 mL) to give
after freeze drying 34 mg (0.054 mmol, 29% yield) of white powder.
M.p. 150e154 ꢃC. IR (KBr) cmꢁ1: 3432, 2930, 1743, 1698, 1648, 1598,
7.74 (br s,1H, NH). 13C NMR (CDCl3)
d: 11.5,18.9,19.8,19.9, 23.1, 28.5,
37.9, 53.8, 72.8, 74.7, 115.2, 116.9, 122.2, 122.9, 127.9, 130.4, 133.6,
135.0, 151.2, 158.8, 170.0, 173.7. ESI-MS (pos. mode): m/z ¼ 448.22
[M þ Na]þ. Anal. Calcd for C24H27NO4S (425.5): C, 67.74; H, 6.40; N,
3.29. Found: C, 67.53; H, 6.28; N, 3.37.
1511, 1251, 1153. 1H NMR (DMSO-d6)
d
: 1.33 (s, 3H, CH3), 1.78 (s, 3H,
5.1.20. (ꢀ) 5-((3aS,4S,6aR)-2-Oxo-hexahydro-thieno[3,4-d]imidazol-
4-yl)-pentanoic acid 2-[4-(2,4-dioxo-thiazolidin-5-ylmethyl)-
phenoxymethyl]-2,5,7,8-tetramethyl-chroman-6-yl ester (11)
Synthesis of troglitazone 10: A suspension of 9a (100 mg,
0.18 mmol) in CH3CN (10 mL) and 3 M HCl (5 mL) was refluxed for
CH3CO), 1.87 (m, 1H, chromane 3-HaHb), 1.91 (s, 6H, 2ꢂ ArCH3), 1.96
(s, 3H, ArCH3), 2.01 (m, 1H, chromane 3-HaHb), 2.46 (t, J ¼ 6.7 Hz,
2H, succinic CH2), 2.63 (br t, 2H, chromane 4-CH2), 2.82 (t,
J ¼ 6.7 Hz, 2H, succinic CH2), 3.07 (m, 4H, ethylenediamine CH2),

214
S. Salamone et al. / European Journal of Medicinal Chemistry 51 (2012) 206e215
45 min. The mixture of solvents was evaporated, the residue was
dissolved in EtOAc (20 mL) and the solution was washed with water
(3 ꢂ 20 mL), dried (MgSO4) and concentrated to dryness to give
a colourless residue.
5.2. Anti-proliferative activity measurement
5.2.1. Cell culture and reagents
MCF-7 and MDA-MB231 human breast cancer cell lines were
obtained from American Type Culture Collection (Rockville, MD).
Both cell lines were grown at 37 ꢃC, according to American Type
Culture Collection recommendations, in phenol red Dulbecco’s
modified Eagle medium (DMEM, Invitrogen, Cergy Pontoise,
France) for MCF-7 and in L-15 medium (Invitrogen) for MDA-MB-
231. These media were supplemented with 10% fetal calf serum
Coupling with biotin: To a solution of D(þ)-biotin (50 mg,
0.20 mmol) in DMF (4 mL) were added CH2Cl2 (2 mL) and TEA
(28
mL, 0.20 mmol). The solution was cooled with an ice bath,
isobutyl chloroformate (26
mL, 0.20 mmol) was added under an
argon atmosphere, then the mixture was stirred for 45 min, and
a solution of 10 and DMAP (2 mg, 0.02 mmol) in CH2Cl2 (6 mL)
was added. Stirring was continued at room temperature for 12 h,
and CH2Cl2 was evaporated. The residue was diluted with EtOAc
(30 mL) and the solution was washed with 5% aqueous NaHCO3
solution (2 ꢂ 20 mL), 5% aqueous citric acid solution (2 ꢂ 20 mL),
water (2 ꢂ 20 mL), dried (MgSO4) and concentrated to dryness.
Column chromatography (CH2Cl2/MeOH, 97:3) using AxxialÒ
Modul Prep apparatus (see above) afforded a residue which was
suspended in a 1:1 mixture of water and CH3CN (5 mL) to give
after freeze drying 43 mg (0.064 mmol, 35% yield) of white
powder. M.p. 133 ꢃC. IR (KBr) cmꢁ1: 3423, 2926, 1752, 1702, 1511,
(SigmaeAldrich, Lyon, France) and 2 mM L-glutamine (Invitrogen).
5.2.2. Cell proliferation assay
Cells were seeded in 6-well plates at the density of 8.104
cells/well in 2 mL of medium supplemented with 10% FCS and
2 mM
replaced by fresh medium supplemented with 1% FCS and 2 mM
-glutamine. Cell proliferation was studied after 24 h of treatment.
L-glutamine. After 24 h of cell attachment, the medium was
L
Control wells received 0.1% DMSO. At the end of the treatment, cells
were washed with PBS, trypsinized and counted with the CellTiter-
GloTM Luminescent Cell Viability Assay (Promega, Charbonnieres,
France). Each treatment was performed in triplicate. For the
different compounds, the concentration leading to a decrease of
50% in the number of viable cells (IC50) was measured.
1459, 1244, 1160. 1H NMR (DMSO-d6)
d: 1.32 (s, 3H, CH3), 1.46 (m,
2H, biotin CH2), 1.70 (m, 2H, biotin CH2), 1.86 (m, 1H, chromane
3-HaHb), 1.91 (s, 6H, 2ꢂ ArCH3), 1.98 (s, 6H, ArCH3), 2.03 (m, 1H,
3-HaHb), 2.62 (m, 5H, CH2CO þ chromane 4-CH2 þ CHaHbS), 2.84
(A part of an ABX system, J ¼ 5.0, 12.4 Hz, 1H, CHaHbS), 3.06 (dd, A
part of an ABX system, J ¼ 8.9, 14.3 Hz, 1H, PhCH2), 3.13 (m, 1H,
biotin CHS), 3.29 (m, 1H, PhCH2, overlapped with H2O from
solvent), 3.99 (m, 2H, OCH2), 4.16 (m, 1H, CHNH), 4.31 (m, 1H,
CHNH), 4.86 (dd, X part of an ABX system, J ¼ 4.4, 8.9 Hz, 1H,
PhCH2CH), 6.35 (br s, 1H, biotin NH), 6.43 (br s, 1H, biotin NH),
6.92 (d, J ¼ 8.5 Hz, 2H, Harom), 7.14 (d, J ¼ 8.5 Hz, 2H, Harom), 11.98
5.3. Hepatotoxicity evaluation
5.3.1. Cell culture and reagents
Cryopreserved human hepatocytes were provided by Kaly-cell
(Illkirch, France). They were distributed in 96-well plates at the
density of 0.1 ꢂ 106 cells/well in 50
ml of DMEM (Gibco, Invitrogen,
UK) supplemented with gentamycin (50 mg/L, Sigma Aldrich,
France), insulin (4 mg/L, Sigma Aldrich, France) and dexamethasone
(s, 1H, NH). 13C NMR (DMSO-d6)
d: 11.6, 11.9, 12.7, 19.4, 21.7, 24.6,
27.7, 28.0, 28.1, 32.9, 36.2, 39.8, 53.0, 55.3, 59.2, 61.1, 72.4, 74.5,
114.7, 117.4, 122.0, 124.9, 126.5, 128.8, 130.3, 140.5, 148.2, 157.8,
162.7, 171.5, 171.6, 175.7. ESI-MS (pos. mode): m/z ¼ 690.23
[M þ Na]þ. Anal. Calcd for C34H41N3O7S2 (667.8): C, 61.14; H, 6.19;
N, 6.29. Found: C, 60.87; H, 6.52; N, 6.31.
(10
atmosphere at 37 ꢃC. After 30 min of equilibration, under stirring at
900 rpm, 50 L of test compounds or DMSO (0.4%) were added.
Compounds were tested at 7 concentrations (0.5, 2.5, 5, 12.5, 25, 50
and 100 M) in triplicate.
mM, Sigma Aldrich, France) under a CO2/air (5%/95%) humidified
m
m
5.1.21. (ꢀ) Succinic acid mono-{2-[4-(2,4-dioxo-thiazolidin-5-
ylmethyl)-phenoxymethyl]-2,5,7,8-tetramethyl-chroman-6-yl}
ester (12)
5.3.2. Cytotoxicity assay (MTT assay)
After 90 min of treatment, MTT (10 mL/well at 10 mg/mL, Sigma
Aldrich, France) was added and incubated during 30 min at 37 ꢃC.
To a solution of 10 (82 mg, 0.19 mmol, obtained from 9a as
reported above) in CH2Cl2 (14 mL) were added succinic anhydride
(37 mg, 0.37 mmol) and DMAP (2 mg, 0.019 mmol). The solution
was refluxed for 2 days. The solvent was removed under reduced
pressure and the residue was dissolved in EtOAc (30 mL). The
solution was washed with 5% aqueous citric acid solution
(2 ꢂ 20 mL), water (2 ꢂ 20 mL), dried (MgSO4) and concentrated to
dryness. The solid residue was suspended in CH2Cl2 (5 mL), soni-
cated, filtered and washed with CH2Cl2. Careful drying afforded
63 mg (0.12 mmol, 63% yield) of white powder. M.p. 204 ꢃC. IR (KBr)
cmꢁ1: 3418, 3189, 2926, 1743, 1680, 1515, 1246, 1142. 1H NMR
Plates were centrifuged, MTT was removed and 100 mL DMSO was
distributed per well. The absorbance was read at 595 nm by
microplate spectrophotometry. Cell viability was expressed as
percentage over controls (DMSO).
Acknowledgements
This article is dedicated to the memory of Professor Alain Olivier
(17th September 1942e19thFebruary2010). Wethank Sanofi-Aventis
for a studentship to Stéphane Salamone, and “Ministère de l’en-
seignement supérieur et de la recherche” for a studentship to Chris-
telle Colin. Many thanks to Kaly-Cell (Illkirch) for providing human
hepatocytes and for viability measurements. We acknowledge Bri-
gitte Fernette, Sandrine Adach and François Dupire for their technical
assistance. Thiswork was performed with grantsof theLiguecontrele
cancer, Conseil Régional de Lorraine, and Université Henri Poincaré.
Special thanks to Jason M. Hargreaves for help with the English.
(DMSO-d6) d: 1.32 (s, 3H, CH3), 1.85 (m, 1H, chromane 3-HaHb), 1.91
(s, 6H, 2ꢂ ArCH3), 1.97 (s, 6H, ArCH3), 2.00 (m, 1H, 3-HaHb), 2.61 (m,
4H, succinic CH2CH2), 2.83 (m, 2H, chromane 4-CH2), 3.05 (dd, A
part of an ABX system, J ¼ 9.0, 14.1 Hz, 1H, CH2CH), 3.29 (dd, B part
of an ABX system, J ¼ 4.3, 9.0 Hz, 1H, CH2CH), 3.98 (m, 2H, OCH2),
4.86 (dd, X part of an ABX system, J ¼ 4.3, 9.0 Hz, 1H, CH2CH), 6.92
(d, J ¼ 8.4 Hz, 2H, Harom), 7.14 (d, J ¼ 8.4 Hz, 2H, Harom), 12.04 (br s,
1H), 12.29 (br s, 1H). 13C NMR (DMSO-d6)
d: 11.7, 11.9, 12.7, 19.5, 21.7,
27.7, 28.5, 28.7, 36.3, 53.1, 72.4, 74.6, 114.7, 117.4, 122.0, 125.1, 126.7,
128.9, 130.3, 140.5, 148.2, 157.8, 171.0, 171.8, 173.4, 175.9. ESI-MS
References
(pos. mode): m/z
¼
564.05 [M
þ
Na]þ. Anal. Calcd for
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