Iodination of verapamil for a stronger induction of death, through GSH efflux, of cancer cells overexpressing MRP1

Article abstract of DOI:10.1016/j.bmc.2010.07.031

The multidrug resistance protein 1 (MRP1), involved in multidrug resistance (MDR) of cancer cells, was found to be modulated by verapamil, through stimulation of GSH transport, leading to apoptosis of MRP1-overexpressing cells. In this study, various iodi

Full text of DOI:10.1016/j.bmc.2010.07.031

Bioorganic & Medicinal Chemistry 18 (2010) 6265–6274
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Iodination of verapamil for a stronger induction of death, through GSH efflux,
of cancer cells overexpressing MRP1
Régis Barattin b, Thomas Perrotton a, Doriane Trompier a, Doriane Lorendeau a, Attilio Di Pietro a,
Amaury du Moulinet d’Hardemare b, Hélène Baubichon-Cortay a,
*
a Equipe Labellisée Ligue 2009, Institut de Biologie et Chimie des Protéines, UMR5086 CNRS-Université Lyon 1, IFR128, 7 passage du Vercors, Lyon F-69367, France
b Département de Chimie Moléculaire, UMR 5250, CNRS/Université Joseph Fourier-Grenoble I, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The multidrug resistance protein 1 (MRP1), involved in multidrug resistance (MDR) of cancer cells, was
found to be modulated by verapamil, through stimulation of GSH transport, leading to apoptosis of
MRP1-overexpressing cells. In this study, various iodinated derivatives of verapamil were synthesized,
including iodination on the B ring, known to be involved in verapamil cardiotoxicity, and assayed for
the stimulation of GSH efflux by MRP1. The iodination, for nearly all compounds, led to a higher stimu-
lation of GSH efflux. However, determination of concomitant cytotoxicity is also important for selecting
the best compound, which was found to be 10-fold more potent than verapamil. This will then allow us to
design original anti-cancer compounds which could specifically kill the resistant cancer cells.
Ó 2010 Published by Elsevier Ltd.
Received 12 April 2010
Revised 13 July 2010
Accepted 14 July 2010
Available online 17 July 2010
Keywords:
Verapamil iodination
MRP1
Apoptosis
GSH efflux
1. Introduction
inhibiting MRP1. MK571, a leukotriene antagonist, is known as a ref-
erence MRP1 inhibitor.3 Argosterol and analogs, which are sterols
The development of multidrug resistance (MDR) is a major cause
of chemotherapy failure. This MDR phenotype is mostly related to an
increased expression of ATP-binding cassette (ABC) transporters in
cancer cell membranes, like P-glycoprotein (P-gp), multidrug resis-
tance protein 1 (MRP1) and/or breast cancer resistance protein
(BCRP).1 These transporters actively expel anti-cancer drugs out of
the cell by using the ATP hydrolysis energy, but the efflux mecha-
nism remains unclear. MRP1 transports many anti-cancer drugs
and organic anions: a broad range of substrates are usually conju-
gated either to glutathione (GSH), such as the proinflammatory cys-
teinyl leukotriene C4 (LTC4), or to glucuronate or sulfate.2 Some
cytotoxic drugs like vincristine and vinblastine are co-transported
with GSH, and MRP1 can transport the GSH alone. A lot of efforts
has been made to identify compounds able to reverse this MDR phe-
notype. Most of the modulators used for P-gp inhibition failed on
MRP1, since P-gp transports cationic/neutral hydrophobic mole-
cules, whereas MRP1 substrates are anionic and rather hydrophilic.
Fortunately, a great number of modulators have been discovered for
from marine sponge, also inhibit MRP1.4 Another class of modula-
tors, the flavonoids, including genistein, was shown to inhibit
daunorubicin efflux from MRP1-overexpressing cells, while biofla-
vonoids were reported to stimulate GSH efflux. Another interesting
MRP1 modulator is verapamil, which is known as a calcium channel
blocker, used in therapy for cardiovascular diseases. This molecule
was first described as a reference P-gp inhibitor.5–8 No reversal in
drug sensitivity of MRP1-overexpressing cells was observed with
verapamil,9,10 but it was shown to stimulate GSH efflux by
MRP1.11 Verapamil alone poorly inhibited Leukotriene C4 transport
by MRP1, but this inhibition was markedly increased in the presence
of GSH.11–13 We have shown that verapamil acts as a modulator of
MRP1 since it specifically kills MRP1-overexpressing cells, by trig-
gering apoptosis through MRP1-mediated glutathione extrusion.14
A similar phenomenon was described later on.15,16 Thus, verapamil
activity might be used to elaborate a novel anti-cancer chemothera-
peutic strategy in order to target and eliminate MRP1-overexpress-
ing resistant cells within a tumor, taking advantage that it is not
transported.11 This would constitute an original strategy to selec-
tively eradicate resistant cancer cells by comparison to the classical
approach using efflux inhibitors. However, one major difficulty in
the clinical use of verapamil is its cardiovascular activity. Thus, the
design of new verapamil analogs was carried out, we modified the
verapamil structure in order to increase its effectiveness on MRP1.
The useful amounts of active compound could thus be decreased.
Some works17,18 have shown that modifications on the B moiety of
* Corresponding author. Tel.: +33 4 72 72 26 91; fax: +33 4 72 72 26 04.
E-mail addresses: regis_barattin@yahoo.fr (R. Barattin), thomas.perrotton@
yahoo.fr (T. Perrotton), doriane.trompier@u-bourgogne.fr (D. Trompier), doriane.
lorendeau@ibcp.fr (D. Lorendeau), a.dipietro@ibcp.fr (A. Di Pietro), amaury.
d-harde mare@ujf-grenoble.fr (Amaury du Moulinet d’Hardemare), h.cortay@
ibcp.fr (H. Baubichon-Cortay).
URL: http://www.ibcp.fr (H. Baubichon-Cortay).
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.07.031

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R. Barattin et al. / Bioorg. Med. Chem. 18 (2010) 6265–6274
verapamil (Fig. 1), particularly the phenyl ring, lead to a lower car-
diovascular activity. Iodine atoms were introduced with the aim to
potentially increase the affinity for MRP1, as this was observed for
iodinated derivatives of chalcones in the case of P-gp.19
We then evaluated the ability of new iodinated verapamil deriv-
atives to stimulate MRP1-mediated GSH efflux and to decrease the
proliferation of MRP1-overexpressing cells. Our results show that
iodination greatly enhances verapamil effectiveness, while length
modification of the verapamil linker seems also to be critical. More
potent and safer verapamil analogs could then be designed, and
further develop in the frame of a new therapeutic strategy aimed
at eliminating resistant cancer cells.
cially available tyramine and the mesylate 1 and subsequent iodin-
ation (method D1).
The synthetic way to the methoxy compounds follows the same
general route, which is depicted in Scheme 3. It was shorter since
the direct mesylation of the starting compound 4-methoxypheny-
lethanol 16 could be performed. Alkylation of the amine 2 with the
mesylate 21 afforded the desired compound 24a in a satisfactory
yield (51%). The iodinated analogs 24b–c could not be directly ob-
tained from 24a, because both rings in A and B showed very similar
reactivity towards iodinating agents. Therefore, the iodine atom
was introduced earlier in the synthesis before the condensation
of the B moiety, on the 4-methoxyphenylethanol 16. We found that
chloramine-T or iodine in ammonia was inefficient to perform
iodination; it was necessary to generate the electrophilic I+ with io-
dine and sodium periodate in acetic acid to achieve the iodination
step (method D2). However, in such conditions, acetylation of the
alcohol function was noted (compounds 17 and 18) and it was nec-
essary to cleave the ester bond with potassium carbonate in meth-
anol to afford the desired iodinated alcohols 19 and 20, which were
easily mesylated. The target compounds 24b–c were finally pre-
pared in moderate yield.
2. Results and discussion
Verapamil was shown to specifically kill mrp1-transfected
BHK-21 cells by triggering apoptosis though MRP1-mediated glu-
tathione extrusion.14 This constitutes a promising approach since
verapamil could act as a therapeutic drug able to kill MRP1-overex-
presing resistant cancer cells. One of the major difficulties in using
verapamil is related to its cardiovascular activity by blocking cal-
cium channels. Thus, as soon as verapamil was discovered to mod-
ulate P-glycoprotein activity, a lot of efforts were spent to
synthesize new verapamil analogs with low cardiovascular activity
and high affinity for the ABC transporter.17,18,23–25 We modified
verapamil structure (Fig. 1) according to a previous study on the ef-
fects of iodinated chalcones derivatives on P-gp,19 which showed
that substitution by various halogen atoms gradually enhanced
the binding affinity: I > Br > Cl > F. Furthermore, modifications of
the B moiety of verapamil might decrease its cardiovascular activ-
ity.17 In this study, synthesis of modified verapamil derivatives,
particularly by iodination of the B ring, has been performed. We
have evaluated their activity on both glutathione efflux and cell
proliferation, using a cell line transfected by mrp1.
2.2. Structure–activity relationships
The results of glutathione efflux shown in Figure 2 are ex-
pressed as changes in the time for which 50% of the intracellular
glutathione is lost. Indeed, verapamil was previously described to
activate MRP1,11 leading to a strong and rapid GSH efflux, with
50% of intracellular GSH lost in less than 30 min.14 Thus, the effec-
tiveness of the verapamil analogs was studied by measuring GSH
efflux kinetics, allowing the determination of the time for which
50% of intracellular glutathione is lost. As expected, verapamil,
here used at 5 lM, induces a lost of 50% cellular GSH in about
20 min (Fig. 2), whereas no GSH efflux is detected in control
BHK-21 cells under the same conditions (not shown here). To
investigate the role of B ring substituents, the methoxy group at
position R3 of verapamil has been suppressed, leading to the
24a–c family of compounds (Table 1). This modification does not
change effectiveness, when compared to verapamil, since glutathi-
one efflux is similar in the presence of derivative 24a. The addi-
tional substitution of the methoxy group at position R2 by a
hydroxyl group (Table 1: compounds 13a–c) was also without ef-
fect, since the efflux in presence of derivative 13a was the same
as that obtained with verapamil (Fig. 2). In contrast, the methyl
group on the central nitrogen atom at position R1 is quite impor-
tant, since we observed a GSH efflux twofold lower in the presence
of derivative 15a (where this methyl group is absent) by compari-
son with verapamil (Table 1). The length of verapamil linker also
appears critical, since compounds 14a (containing a longer linker
than verapamil: n = 3) and 12a (containing a shorter linker: n = 1)
than verapamil (n = 2) (Table 1) failed to strongly activate MRP1,
as 50% extrusion of intracellular GSH required up to 50–60 min.
The cytotoxicity of these compounds was checked, as shown in
Table 2. We must distinguish non-specific cytotoxicity, as moni-
tored on control BHK-21 cells, from that produced on BHK-MRP1
which results from both non-specific and specific cytotoxicity
through MRP1. As a reference, verapamil exhibits an IC50 of
2.1. Chemistry
Synthesis of the phenol derivatives was achieved according to
Scheme 1. Starting from commercial 4-hydroxyalkylphenols 3–5,
the key step of this synthesis is the selective protection of the phenol
group with ethylchloroformate at ꢀ78 °C to prepare compounds
6–8. This allowed a smooth and quantitative O-mesylation of the
alcohol function to afford 9–11. Those protected phenolic B moieties
were finally condensed with the amine 220 in methanol with potas-
sium iodide and bicarbonate as a proton scavenger. Surprisingly, in
such conditions the protecting group of the phenol was cleanly re-
moved affording the desired compounds 12a–14a. Iodination was
then attempted with chloramine-T and NaI.21 This method proved
to be unsatisfactory because great difficulties were encountered to
obtain a pure material. Therefore, we decided to perform this
reaction simply with iodine and ammonia in acetonitrile as de-
scribed by Flanagan et al. (method D1).22 The target monoiodinated
12b–14b and diiodinated 12c–14c compounds were then isolated as
pure forms. The demethylated analogs 15a–c were obtained easily
in a similar manneras shownin Scheme 2 starting fromthe commer-
10.6 0.5 lM for BHK-MRP1 cells, and up to 100 lM for control
cells (Table 2 and Fig. 3), as previously shown.14 The specific cyto-
toxicity for BHK-MRP1 of 24a and 13a derivatives is not higher
than that observed with verapamil (Table 2). Those of 15a and
14a derivatives are four times and two times lower, respectively,
than that of verapamil. While compound 12a was slightly more
cytotoxic for BHK-MRP1, it was much more cytotoxic for control
BHK-21 cells. Specific cytotoxicity of these various compounds
showed a close relationship with GSH efflux results. The more
CN
MeO
MeO
N
OMe
OMe
B
A
Figure 1. Chemical structure of verapamil.

R. Barattin et al. / Bioorg. Med. Chem. 18 (2010) 6265–6274
6267
a
HO
O
O
O
O
b
OH
n
O
OMs
n
OH
n
O
3: n=1
4: n=2
5: n=3
6: n=1
9
: n=1
7: n=2
8: n=3
10
: n=2
11: n=3
R3
OH
CN
OH
R4
MeO
MeO
NH
CN
CN
MeO
MeO
N
MeO
MeO
N
d
2
c
12b
12c
: n=1, R3=H, R4=I
: n=1, R3=R4=I
12a: n=1
13a: n=2
14a: n=3
13b: n=2, R3=H, R4=I
13c: n=2, R3=R4=I
14b
14c
: n=3, R3=H, R4=I
: n=3, R3=R4=I
Scheme 1. Preparation of 12a–c, 13a–c, and 14a–c (Table 1). Reagents and conditions: (a) EtOCOCl, NEt3/CH3CN, ꢀ80 °C; (b) MeSO2Cl, NEt3/CH2Cl2, 0 °C, 2h; (c) NaHCO3/KI,
MeOH, 65 °C, 3 days; (d) I2, CH3CN/NH4OH.
H
N
CN
CN
MeO
MeO
O
MeO
MeO
a
S
O O
OH
1
15a
H
N
CN
MeO
MeO
R3
b
OH
R4
15b: R3=H, R4=I
15c: R3=R4=I
Scheme 2. Preparation of 15a–c (Table 1). Reagents and condition: (a) tyramine, CH3CN, 82 °C, 20 h; (b) I2, CH3CN/NH4OH.
R3
CN
MeO
MeO
R3
MeO
MeO
N
c
d
OH
R4
OMs
OMe
R4
24a: R3=R4=H
21: R3=R4=H
24b:
24c:
R3=I, R4=H
R3=R4=I
22:
23:
R3=I, R4=H
R3=R4=I
a
c
R3
R3
MeO
R4
MeO
R4
b
OAc
OH
17: R3=I, R4=H
18: R3=R4=I
19: R3=I, R4=H
20: R3=R4=I
Scheme 3. Preparation of 24a–c (Table 1). Reagents and conditions: (a) I2/NaIO4, AcOH/H2SO4/H2O, 60 °C, 1–2 days; (b) K2CO3, MeOH, rt, 3 h; (c) MeSO2Cl, pyridine, 0 °C, 3 h;
(d) amine 2, CH3CN/NEt3, 82 °C, 6 days.

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R. Barattin et al. / Bioorg. Med. Chem. 18 (2010) 6265–6274
Figure 2. Effects of verapamil derivatives on the GSH content of BHK-21-MRP1 cells. MRP1-BHK-21 cells were cultured with or without 5 lM racemic verapamil or
derivatives during various incubation times, from 10 to 120 min. Then total intracellular glutathione levels were determined as described in Section 4. Differences in GSH
levels between cells cultured in the absence of compound (taken as 100% intracellular GSH) and cells cultured in the presence of compound (% residual intracellular GSH),
corresponding to GSH efflux, were determined at each time. Data represent the values after 30 min incubation, and are the mean of triplicate determinations in a typical
experiment; bars, SD.
Table 1
Table 2
Verapamil derivatives
Cytotoxic effects of verapamil derivatives on BHK-21 and BHK-21-MRP1 cells
R4
Compound
IC50 (lM)
R2
R3
BHK-21-MRP1
BHK-21
R1
N
CN
Verapamil
24a
24b
24c
13a
13b
13c
14a
14b
14c
12a
12b
12c
10.6 0.5
17.2 1.0
4.7 0.6
1.7 0.9
11.7 0.3
8.6 1.2
1.1 0.9
20.9 1.4
9.3 1.4
6.0 0.8
6.6 0.9
13.0 1.2
6.6 1.2
38.1 0.3
0.78 0.7
1.96 0.4
P100
MeO
33.6 7.7
13.0 0.5
8.9 0.3
55.9 0.8
32.8 0.9
54.7 7.1
P100
9.4 0.2
33.1 5.1
28.5 1.6
14.1 0.3
27.4 2.6
P100
n
MeO
Compound
R1
R2
R3
R4
n
12a
12b
12c
13a
13b
13c
14a
14b
14c
15a
15b
15c
24a
24b
24c
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
Me
Me
Me
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
I
I
H
I
I
H
I
I
H
I
I
H
H
I
H
H
I
H
H
I
H
H
I
1
1
1
2
2
2
3
3
3
2
2
2
2
2
2
15a
15b
15c
14.2 0.3
13.8 0.5
Data are means SD of experiments performed in triplicate.
OMe
OMe
OMe
H
I
I
H
H
I
with respective IC50 values of 1.1 0.9 and 54.7 7.1
MRP1 and BHK-21 control cell lines, and a glutathione efflux
l
M for BHK-
strongly enhanced in BHK-MRP1 cells.
R1 is the substituent linked to the central nitrogen atom. R2, R3, and R4 are the
substituents of the B moiety. n is the length of the linker hydrocarbon chain
between the central nitrogen atom and the benzene ring B.
3. Conclusions
rapidly intracellular GSH was extruded, the more cytotoxic for
BHK-MRP1 was the compound. These results confirm the assump-
tion that the rapidity of GSH efflux indeed induces the death of
cells.
Only few works have been reported on novel MRP1 verapamil
analogs. Structure–activity relationship of dithiane compounds
showed that the most lipophilic were the best inhibitors of LTC4
transport.26 We have designed here new verapamil analogs in
order to enhance glutathione transport activity and to decrease pro-
liferation of mrp1-transfected cells. When studies of MRP1 modula-
tion are undertaken, it is crucial to measure in parallel GSH efflux
and cytotoxicity in order to only select the MRP-effective com-
pounds. We have modified the B ring of verapamil in order to get
compounds with lower cardiovascular activity. The informations
collected with these data suggest that iodination of the B ring of
verapamil could be crucial for an increased activity. We previously
demonstrated that verapamil directly binds to MRP127 and thus in-
duces GSH transport stimulation, results which are in agreement
with the fact that verapamil is not transported by MRP1.11 We could
These effects were highly modulated by iodination. With the
24a–c family compounds, iodination was without effect on GSH ef-
flux, but it strongly increased cytotoxicity for control cells. Di-iodin-
ation of compound 13a, leading to compound 13c, led to a higher
GSH efflux and an increased cytotoxic effect on BHK-MRP1, despite
a slightly increased cytotoxic effect on control BHK-21 cells. The spe-
cific cytotoxic effects produced on BHK-MRP1 by iodinated 12b–c,
14b–c, 15b–c derivatives increased as GSH was extruded more rap-
idly. However, cytotoxic effect on control BHK-21 was also stronger.
Iodination of the compounds tends to increase their toxicity. How-
ever, one of them, the diiodinated compound 13c is very efficient,

R. Barattin et al. / Bioorg. Med. Chem. 18 (2010) 6265–6274
6269
4. Experimental
4.1. Materials and methods
1H and 13C NMR spectra were recorded on a Bruker Avance 300
(300 MHz for 1H, 75 MHz for 13C) instrument. Chemical shifts were
reported as d values (ppm) relative to Me4Si as an internal standard.
DCI mass spectra were recorded on a ThermoFinnigan Polaris Q
instrument. Melting points were determined on a Büchi 300 appara-
tus and are uncorrected. IR spectra were recorded on a Nicolet Im-
pact 530 spectrometer. Elemental analyses were performed by the
Analytical Department of CNRS, Vernaison, France. Thin-layer chro-
matography (TLC) was carried out using E. Merck silica gel F-254
plates (thickness 0.20 mm). Flash chromatography was carried out
using Merck or Macherey-Nagel silica gel 60 (0.04–0.063 mm). All
solvents were distilled prior to use. Chemical and reagents were
obtained either from Aldrich or ACROS companies and used
without modification. Racemic verapamil, reduced glutathione
(GSH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT), 5.50-dithiobis(2-nitrobenzoic acid) (DTNB), b-nicotin-
amide adenine dinucleotide phosphate reduced (b-NADPH),
penicillin–streptomycin, and fetal bovine serum were obtained
from Sigma (Saint Quentin Fallavier, France), Triton X-100 was from
Merck (Darmstadt, Germany), methotrexate from Rhône-Poulenc
(Montrouge, France). Glutathione Reductase (EC 1.6.4.2) was from
Roche Applied Sciences (Meylan, France).
4.2. Chemistry
4.2.1. General procedure for the protection of phenol with
ethylchloroformate (method A)
A solution of the appropriate phenol in CH3CN (40 mL) and NEt3
(1 equiv) was cooled at ꢀ80 °C, under argon. A solution of ethyl-
chloroformate (1 equiv) in CH3CN (10 mL) was added and the stir-
red reaction mixture was allowed to warm to room temperature.
After adding water (80 mL), the aqueous solution was extracted
with ether (3ꢁ 70 mL). The combined organic layers were washed
with brine (70 mL) and dried over anhydrous Na2SO4. Removal of
the solvent under reduced pressure gave an oily residue which
was purified by silica gel column chromatography (CH2Cl2/
AcOEt = 1:1) to afford protected phenol.
4.2.2. General procedure for mesylation of alcohols in
dichloromethane and triethylamine (method B1)
Figure 3. Cytotoxicity of verapamil derivatives on BHK-21 and BHK-21-MRP1 cells.
BHK-21 control cells (circles) and MRP1-BHK-21 cells (squares) were cultured in
the presence of increasing concentrations of racemic verapamil or verapamil
derivative for 96 h. Cytotoxicity was measured by the MTT assay, and data were
analyzed by using a sigmoidal fitting. Results are the mean of triplicate experi-
mental points; bars, SD.
Mesyl chloride (1.3 equiv) was added to an ice-bath cooled
solution of the appropriate alcohol in CH2Cl2 (30 mL) and NEt3
(1 equiv). After stirring at 0 °C for 2 h, the reaction mixture was al-
lowed to warm at room temperature, then washed successively
with 0.2 M NaHCO3 solution (2ꢁ 30 mL), water (30 mL), brine
(30 mL) and dried over anhydrous Na2SO4. Removal of the solvent
in vacuo gave the mesylated alcohol, which was used as such in the
next step.
make the assumption that iodination of verapamil enhances its
affinity for MRP1. Upon iodine addition, cell proliferation might be
highly decreased and glutathione efflux strongly enhanced in
BHK-MRP1. Cytotoxicity remains low in control BHK-21 cells when
methoxy group in position R2 of the B ring is suppressed, as for com-
pound 13c. Such an information is important for designing new
verapamil analogs. Furthermore, the length modification of verapa-
mil linker seems to be critical for MRP1, whereas it was not for P-gly-
coprotein since a large range in length was tolerated.23 An important
feature was brought by Perrotton et al.27 who showed that only the
S-isomer of verapamil is responsible of the GSH transport stimula-
tion. From the new findings described in this paper, synthesis of
the 13c derivative of S-verapamil is to be considered; a very high
efficiency towards MRP1 might be expected for further experiments
with such a new derivative.
4.2.3. General procedure for mesylation of alcohols in pyridine
(method B2)
Mesyl chloride was carefully added to an ice-bath cooled solu-
tion of the appropriate alcohol in pyridine (10–15 mL), (1.4 equiv).
The mixture was stirred at 0 °C for 3 h and at room temperature
overnight. After adding 0.1 M sulfuric acid solution (50 mL), the
aqueous phase was extracted with ether (3ꢁ 50 mL). Combined or-
ganic phases were washed successively with saturated CuSO4 solu-
tion (3ꢁ 50 mL), water (2ꢁ 50 mL), brine (50 mL), then dried with
anhydrous Na2SO4 and concentrated in vacuo, yielding mesylated
alcohol.

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R. Barattin et al. / Bioorg. Med. Chem. 18 (2010) 6265–6274
4.2.4. General procedure for alkylation of amine using
NaHCO3/KI in MeOH (method C1)
4.2.9. Carbonic acid ethyl ester 4-(2-hydroxy-ethyl)-phenyl ester
(7)
The appropriate mesylate (1.2 equiv), sodium hydrogenocar-
bonate NaHCO3 (1.8 equiv), and potassium iodide KI (0.5 equiv)
were added to a solution of amine 7 in methanol (20 mL). The reac-
tion mixture was then refluxed under argon for 3 days. After add-
ing water (60 mL), the resulting aqueous solution was successively
acidified to pH 1 by adding concentrated HCl, washed with ether
(3ꢁ 60 mL), basified to pH 10 by adding 22% ammonia and finally
extracted with ether (3ꢁ 60 mL). These last combined organic lay-
ers were dried over anhydrous Na2SO4 and concentrated under re-
duced pressure. The oily residue was then purified by silica gel
column chromatography to afford alkylated amine.
4-(2-hydroxy-ethyl)-phenol 4 (2.77 g, 20 mmol) was treated
with ethylchloroformate according to method A to afford com-
pound 7 (4.16 g) as yellow oil, in quantitative yield. 1H NMR
(CDCl3) d 1.38 (t, 3H, J = 7.17 Hz), 2.85 (t, 2H, J = 6.53 Hz), 3.82 (q,
2H, J = 6.53 and 6.27 Hz), 4.30 (q, 2H, J = 7.17 Hz), 7.11 (d, 2H,
J = 8.58 Hz), 7.23 (d, 2H, J = 8.58 Hz); 13C NMR (CDCl3) d 14.1,
38.5, 63.4, 64.8, 121.1, 129.9, 136.4, 149.7, 153.7; IR (KBr, cmꢀ1
)
3100–3600, 3050, 2984, 2930, 2875, 1761, 1508, 1370, 1256,
1217, 1200, 1057, 1018; MS (m/z) 193, 228 (M þ NHþ4 , 100%); Anal.
Calcd for C11H14O4: C, 62.85; H, 6.71; O, 30.44. Found: C, 62.71; H,
6.78; O, 30.05.
4.2.5. General procedure for alkylation of amine using
CH3CN/NEt3 (method C2)
4.2.10. Carbonic acid ethyl ester 4-(3-hydroxy-propyl)-phenyl
ester (8)
A mixture of the amine 7 and of the appropriate mesylated alco-
hol (1 equiv) in acetonitrile (10 mL), and triethylamine (3 equiv)
was refluxed, under argon, for 6 days. (Reaction followed by TLC,
elution with CH2Cl2/iPrNH2 = 9.6:0.4.) The reaction was then
poured into water (30 mL) and extracted with ether (3ꢁ 30 mL).
Combined organic phases were washed with brine (40 mL), dried
over anhydrous Na2SO4 and concentrated in vacuo. The oily resi-
due was purified by silica gel column chromatography (CH2Cl2/
iPrNH2 = 9.6:0.4) to provide alkylated amine.
Compound 8 was prepared following method A from 4-(3-hy-
droxy-propyl)-phenol 5 (4.34 g, 28 mmol) and was obtained as a
pale yellow oil (6.30 g, 98% yield). 1H NMR (CDCl3) d 1.38 (t, 3H,
J = 7.17 Hz), 1.5–1.7 (sl, OH), 1.86 (m, 2H), 2.70 (t, 2H,
J = 7.43 Hz), 3.65 (t, 2H, J = 6.4 Hz), 4.30 (q, 2H, J = 7.17 Hz), 7.08
(d, 2H, J = 8.7 Hz), 7.19 (d, 2H, J = 8.7 Hz); 13C NMR(CDCl3) d 14.1,
31.3, 34.1, 62.0, 64.7, 120.8, 129.3, 139.5, 149.2, 153.8; IR (KBr,
cmꢀ1) 3100–3600, 3053, 2987, 2938, 2865, 1763, 1510, 1371,
1257, 1206, 1069, 1020; MS (m/z) 107, 135, 242 (M þ NHþ4 ,
100%); Anal. Calcd for C12H16O4: C, 64.27; H, 7.19; O, 28.54. Found:
C, 64.26; H, 7.44; O, 28.25.
4.2.6. General procedure for the iodination of phenol with I2 in
CH3CN/NH4OH (method D1)
A 0.05 M iodine solution in acetonitrile (1.1 equiv for monoio-
dination and 2.3 equiv for diiodination), and 22% ammonia (1 mL
for 5 mL of iodine solution) were added to the appropriate phenol.
The reaction mixture was stirred at room temperature under argon
for 2 days. The solution was then poured into water (30 mL) and
the aqueous solution was extracted with ether (3ꢁ 40 mL). Com-
bined organic layers were washed successively with 0.2 M Na2S2O3
solution (3ꢁ 40 mL), brine (50 mL) and were dried over anhydrous
Na2SO4. Removal of the solvent under reduced pressure provided
an oily residue which was purified by silica gel column chromatog-
raphy to afford mono- or diiodinated phenol.
4.2.11. Methanesulfonic acid 4-ethoxycarbonyloxy-benzyl ester
(9)
Alcohol 6 (5.05 g; 26 mmol) was treated with mesyl chloride
according to method B1 to afford compound 9 (7.03 g, 100% yield)
as a white solid. Mp 60–61 °C; 1H NMR (CDCl3) d 1.39 (t, 3H,
J = 7.17 Hz), 2.94 (s, 3H), 4.33 (q, 2H, J = 7.17 Hz), 5.23 (s, 2H),
7.23 (d, 2H, J = 8.7 Hz), 7.45 (d, 2H, J = 8.7 Hz); 13C NMR (CDCl3) d
14.1, 38.4, 65.0, 70.5, 121.6, 130.1, 131.1, 151.8, 153.3; IR (KBr,
cmꢀ1) 3100–3600, 3040, 2993, 2935, 1755, 1511, 1364, 1270,
1238, 1175, 1060, 985; MS (m/z) 107, 179, 196, 292 (M þ NHþ4 ,
100%); Anal. Calcd for C11H14O6Sꢂ1/3H2O: C, 47.14; H, 5.27. Found:
C, 47.26; H, 5.68.
4.2.7. General procedure for the iodination with I2/NaIO4 in
AcOH/H2SO4/H2O (method D2)
4.2.12. Methanesulfonic acid 2-(4-ethoxycarbonyloxy-phenyl)-
ethyl ester (10)
Iodine I2 and sodium periodate were added to a solution of the
appropriate compound in acetic acid (12 mL), water (2 mL), and
sulfuric acid (0.2 mL). The reaction mixture was heated at 60 °C,
under argon, for 1–2 days, then allowed to warm at room tempera-
ture and poured into water (50 mL). The aqueous solution was then
extracted with ether (4ꢁ 50 mL). Combined organic layers were
washed successively with 0.2 M NaHCO3 solution (3ꢁ 50 mL),
0.2 M Na2S2O3 solution (3ꢁ 50 mL), brine (60 mL), and were dried
over anhydrous Na2SO4. Removal of the solvent under reduced pres-
sure provided an oily residue which was purified by silica gel column
chromatography (pentane/AcOEt = 9.6:0.4) to afford the iodinated
compound.
Alcohol 7 (3.50 g, 16 mmol) was converted into mesylate 10
(4.78 g) as described in method B1 and was obtained as a brown
solid, in quantitative yield. Mp 33–34 °C; 1H NMR (CDCl3) d 1.39
(t, 3H, J = 7.17 Hz), 2.86 (s, 3H), 3.06 (t, 2H, J = 6.79 Hz), 4.31 (q,
2H, J = 7.17 Hz), 4.41 (t, 2H, J = 6.79 Hz), 7.14 (d, 2H, J = 8.58 Hz),
7.25 (d, 2H, J = 8.58 Hz); 13C NMR (CDCl3) d 14.2, 35.0, 37.4, 64.8,
69.9, 121.3, 130.0, 134.1, 150.2, 153.6; IR (KBr, cmꢀ1) 3100–3600,
3044, 2995, 2930, 1755, 1510, 1363, 1273, 1240, 1175, 1061,
987; MS (m/z) 306 (M þ NHþ, 100%); Anal. Calcd for C12H16O6S:
4
C, 49.99; H, 5.59; O, 33.30. Found: C, 50.00; H, 5.70; O, 32.99.
4.2.13. Methanesulfonic acid 3-(4-ethoxycarbonyloxy-phenyl)-
propyl ester (11)
4.2.8. Carbonic acid ethyl ester 4-hydroxymethyl-phenyl ester
(6)
A solution of alcohol 8 (6.29 g; 28 mmol) was treated according
to method B1 to afford compound 11 (8.06 g, 95% yield) as a white
solid. Mp 40–41 °C; 1H NMR (CDCl3) d 1.38 (t, 3H, J = 7.17 Hz), 2.06
(m, 2H), 2.75 (t, 2H, J = 7.16 Hz), 2.98 (s, 3H), 4.23 (t, 2H,
J = 6.14 Hz), 4.31 (q, 2H, J = 7.17 Hz), 7.11 (d, 2H, J = 8.44 Hz), 7.19
(d, 2H, J = 8.44 Hz); 13C NMR (CDCl3) d 14.2, 30.6, 30.9, 37.4, 64.8,
68.9, 121.1, 129.4, 138.1, 149.6, 153.7; IR (KBr, cmꢀ1) 3045, 3004,
2947, 2938, 2865, 1755, 1518, 1346, 1257, 1216, 1167, 1004,
930; MS (m/z) 107, 135, 207, 303 (M+H+), 320 (M þ NH4þ, 100%);
A solution of 4-hydroxymethylphenol 3 (4.03 g, 32 mmol) was
treated according to method A to afford compound 6 (5.06 g, 79%
yield) as a yellow oil. 1H NMR (CDCl3) d 1.39 (t, 3H, J = 7.17 Hz),
1.6–1.7 (OH), 4.32 (q, 2H, J = 7.17 Hz), 4.68 (s, 2H), 7.17 (d, 2H,
J = 8.19 Hz), 7.38 (d, 2H, J = 8.19 Hz); 13C NMR (CDCl3) d 14.2,
64.7, 64.8, 121.2, 128.0, 138.6, 150.5, 153.6; IR (KBr, cmꢀ1) 3100–
3600, 3030, 3012, 2987, 2938, 2914, 2873, 1763, 1600, 1510,
1371, 1257, 1210 1061, 1020; MS (m/z) 107, 179; 196 (M+); 214
(M þ NHþ4 , 100%).

R. Barattin et al. / Bioorg. Med. Chem. 18 (2010) 6265–6274
6271
Anal. Calcd for C13H18O6S: C, 51.64; H, 6.00; O, 31.75. Found: C,
51.54; H, 6.15; O, 31.68.
2.00–2.16 (m, 2H), 2.19 (s, 3H), 2.30–2.42 (m, 2H), 2.43–2.53 (m,
2H), 2.60–2.68 (m, 2H), 3.87 (s, 6H), 6.71 (d, 2H, J = 8.32 Hz), 6.82
(d 1H, J = 8.32 Hz), 6.85 (d, 1H, J = 2.31 Hz), 6.90 (dd, 1H, J = 8.32
and 2.31 Hz), 6.98 (d, 2H, J = 8.32 Hz); 13C NMR (CDCl3) d 18.6,
18.9, 23.1, 32.4, 35.6, 37.9, 41.8, 53.3, 55.9, 56.0, 56.8, 59.4,
109.6, 111.1, 115.3, 118.7, 121.4, 129.7, 130.6, 132.1, 148.3,
149.0, 154.2; IR (KBr, cmꢀ1) 3100–3500, 2963, 2930, 2873, 2840,
2236, 1591, 1518, 1469, 1412, 1257, 1151, 1028; MS (m/z) 107,
303 (100%), 411 (M+H+); Anal. Calcd for C25H34N2O3: C, 73.14; H,
8.35; N, 6.82. Found: C, 72.97; H, 8.29; N, 7.22.
4.2.14. 2-(3,4-Dimethoxy-phenyl)-5-[(4-hydroxy-benzyl)-
methyl-amino]-2-isopropyl-pentanenitrile (12a)
Amine 2 (233 mg, 0.77 mmol) was treated with compound 9
(320 mg, 1.17 mmol) according to method C1 (heating for only
20 h). Purification by column chromatography (elution CH2Cl2/
iPrNH2 = 4.8:0.2) provided compound 12a (0.230 g, 76% yield) as
a pale yellow oil. 1H NMR (CDCl3) d 0.79 (d, 3H, J = 6.66 Hz), 1.17
(d, 3H, J = 6.66 Hz), 1.15–1.30 (m, 1H), 1.47–1.62 (m, 1H, 1.78–
1.90 (m, 1H), 2.00–2.20 (m, 2H), 2.07 (s, 3H), 2.23–2.36 (m, 2H),
3.32 (s, 2H), 3.85 (s, 3H), 3.87 (s, 3H), 6.71 (m, 2H), 6.82 (d, 1H,
J = 8.45 Hz), 6.83 (d, 1H, J = 2.18 Hz), 6.91 (dd, 1H, J = 8.45 and
2.18 Hz), 7.07 (m, 2H); 13C NMR (CDCl3) d 18.6, 18.9, 23.2, 35.5,
37.9, 41.9, 53.3, 55.9, 56.0, 56.4, 61.4, 109.5, 111.1, 115.1, 118.8,
121.5, 130.3, 130.7, 148.3, 149.0, 154.9; IR (KBr, cmꢀ1) 3300–
3600, 3059, 2964, 2938, 2875, 2837, 2236, 1613, 1517, 1465,
1415, 1260, 1148, 1025, 914, 732; MS (m/z) 291, 397 (M+), 398
(M+H+); Anal. Calcd for C24H32N2O3ꢂ1/2H2O: C, 71.08; H, 8.20; N,
6.91. Found: C, 71.30; H, 8.27; N, 7.22.
4.2.18. 2-(3,4-Dimethoxy-phenyl)-5-{[2-(4-hydroxy-3-iodo-
phenyl)-ethyl]-methyl-amino}-2-isopropyl-pentanenitrile
(13b)
A mixture of compound 13a (130 mg, 0.32 mmol), iodine solu-
tion in CH3CN (10 mL, 1.4 equiv) and ammonia (3 mL) was treated
following method D1. Purification by column chromatography
(CH2Cl2/iPrNH2 = 9.6:0.4) provided monoiodinated phenol 13b
(37 mg, 22% yield) as a yellow oil. 1H NMR (CDCl3) d 0.78 (d, 3H,
J = 6.66 Hz), 1.17 (d, 3H, J = 6.66 Hz), 1.10–1.30 (m, 1H), 1.50–1.60
(m, 1H), 1.87 (m, 1H, J = 4.61 Hz), 2.00–2.20 (m, 2H), 2.21 (s, 3H),
2.35–2.50 (m, 2H), 2.50–2.60 (m, 2H), 2.60–2.70 (m, 2H), 3.87 (s,
3H), 3.88 (s, 3H), 4.0–5.0 (OH), 6.75–6.95 (m, 4H), 6.99 (dd, 1H,
J = 8.19 and 1.92 Hz), 7.46 (d, 1H, J = 1.92 Hz); 13C NMR (CDCl3) d
18.6, 18.9, 22.9, 31.7, 35.4, 37.9, 41.5, 53.3, 55.9, 56.0, 56.7, 58.9,
85.4, 109.6, 111.1, 115.0, 118.7, 121.4, 130.3, 130.5, 133.9, 138.2,
148.3, 149.0, 153.5; IR (KBr, cmꢀ1) 3030, 2971, 2930, 2881, 2865,
2236, 1600, 1518, 1469, 1436, 1265, 1142, 1028; MS (m/z) 303,
411, 537 (M+H+, 100%); Anal. Calcd for C25H33IN2O3ꢂH2O:
C, 54.15; H, 6.36; N, 5.05. Found: C, 54.00; H, 6.46; N, 5.13.
4.2.15. 2-(3,4-Dimethoxy-phenyl)-5-[(4-hydroxy-3-iodo-
benzyl)-methyl-amino]-2-isopropyl-pentanenitrile (12b)
Phenol 12a (260 mg, 0.66 mmol) was iodinated by an I2 solution
in CH3CN (12 mL, 1 equiv) and ammonia (2 mL), following method
D1. Purification by column chromatography (AcOEt/CH2Cl2/
MeOH = 5:4:1) afforded compound 12b (105 mg, 31% yield) as a
pale yellow oil. 1H NMR (CDCl3) d 0.79 (d, 3H, J = 6.65 Hz), 1.19
(d, 3H, J = 6.65 Hz), 1.11–1.28 (m, 1H), 1.45–1.62 (m, 1H), 1.78–
1.92 (m, 1H), 2.00–2.20 (m, 2H), 2.07 (s, 3H), 2.21–2.33 (m, 2H),
3.28 (s, 2H), 3.87 (s, 3H), 3.88 (s, 3H), 6.81–6.95 (m, 4H), 7.10
(dd, 1H, J = 8.45 and 1.92 Hz), 7.58 (s, 1H); 13C NMR (CDCl3) d
18.6, 18.9, 23.3, 35.5, 38.0, 41.9, 53.3, 55.9, 56.0, 56.4, 60.7, 85.5,
109.6, 111.1, 114.6, 118.7, 121.5, 130.7, 133.9, 138.4, 148.3,
149.0, 153.5; IR (KBr, cmꢀ1) 3300–3600, 3053, 2971, 2938, 2881,
2832, 2791, 2236, 1600, 1510, 1477, 1420, 1265, 1159, 1036,
751; MS (m/z) 291, 523 (M+H+, 100%); Anal. Calcd for
4.2.19. 2-(3,4-Dimethoxy-phenyl)-5-{[2-(4-hydroxy-3,5-diiodo-
phenyl)-ethyl]-methyl-amino}-2-isopropyl-pentanenitrile
(13c)
Compound 13c was prepared using method D1 from phenol 13a
(90 mg, 0.22 mmol), iodine (12 mL of 0.05 M solution in CH3CN,
2.7 equiv) and ammonia (2 mL). Purification by column chroma-
tography (CH2Cl2/iPrNH2 = 9.6:0.4) afforded diiodinated phenol
13c (60 mg, 41% yield) as a pale orange oil. 1H NMR (CDCl3) d
0.78 (d, 3H, J = 6.66 Hz), 1.17 (d, 3H, J = 6.66 Hz), 1.10–1.27 (m,
1H), 1.45–1.62 (m, 1H), 1.78–1.91 (m, 1H, J = 4.61 Hz), 2.00–2.15
(m, 2H), 2.17 (s, 3H), 2.30–2.43 (m, 2H), 2.45–2.53 (m, 2H), 2.55–
2.64 (m, 2H), 3.88 (s, 3H), 3.89 (s, 3H), 6.8–6.92 (m, 3H), 7.49 (s,
2H); 13C NMR (CDCl3) d 18.7, 18.9, 23.1, 31.4, 35.4, 37.9, 41.4,
53.3, 55.9, 56.0, 58.8, 60.4, 82.3, 109.7, 111.2, 118.6, 121.4, 130.6,
136.3, 139.3, 148.3, 149.0, 152.1; IR (KBr, cmꢀ1) 3053, 2963,
2930, 2881, 2873, 2236, 1600, 1510, 1461, 1273, 1142, 1028,
742; MS (m/z) 303 (100%), 411, 537, 663 (M+H+); Anal. Calcd for
C
24H31IN2O3ꢂ1/2H2O: C, 54.24; H, 6.07; N, 5.27. Found: C, 54.11;
H, 6.06; N, 5.15.
4.2.16. 2-(3,4-Dimethoxy-phenyl)-5-[(4-hydroxy-3,5-diiodo-
benzyl)-methyl-amino]-2-isopropyl-pentanenitrile (12c)
Phenol 12a (206 mg, 0.52 mmol) was treated by I2 (30 mL of
0.05 M I2 solution in CH3CN, 3 equiv) and ammonia (7 mL) according
to method D1. Purification by column chromatography (ether) affor-
ded compound12c (102 mg, 30% yield) as a whitesolid. Mp 88 °C; 1H
NMR (CDCl3) d 0.81 (d, 3H, J = 6.65 Hz), 1.21 (d, 3H, J = 6.65 Hz),
1.08–1.25 (m, 1H), 1.45–1.62 (m, 1H), 1.78–1.92 (m, 1H), 2.00–
2.20 (m, 2H), 2.07 (s, 3H), 2.20–2.35 (m, 2H), 3.25 (s, 2H), 3.88 (s,
6H), 6.83–6.86 (m, 2H), 6.92 (dd, 1H, J = 8.19 and 1.92 Hz), 7.59 (s,
2H); 13C NMR (CDCl3) d 18.7, 19.0, 23.3, 35.5, 38.0, 41.9, 53.3, 55.9,
56.0, 56.4, 60.1, 82.0, 109.6, 111.2, 118.7, 121.4, 130.7, 133.9,
139.4, 148.3, 149.0, 153.5; IR (KBr, cmꢀ1) 3200–3600, 3030, 2959,
2931, 2878, 2835, 2236, 1517, 1464, 1259, 1146, 1025, 803; MS
(m/z) 291 (100%), 649 (M+H+); Anal. Calcd for C24H30I2N2O3: C,
44.46; H, 4.66; N, 4.32. Found: C, 44.66; H, 4.79; N, 4.31.
C25H32I2N2O3: C, 45.33; H, 4.87; N, 4.23. Found: C, 45.53; H, 5.06;
N, 4.03.
4.2.20. 2-(3,4-Dimethoxy-phenyl)-5-{[3-(4-hydroxy-phenyl)-
propyl]-methyl-amino}-2-isopropyl-pentanenitrile (14a)
Amine 2 (2.02 g, 7 mmol) was alkylated with compound 11
(2.77 g, 9.2 mmol) according to method C1. Purification by column
chromatography (AcOEt/CH2Cl2/MeOH = 5:3.5:1.5) provided com-
pound 14a (0.970 g, 33% yield) as a yellow oil. 1H NMR (CDCl3) d
0.77 (d, 3H, J = 6.66 Hz), 1.14 (d, 3H, J = 6.66 Hz), 1.10–1.20 (m,
1H), 1.45–1.62 (m, 1H), 1.73 (m, 2H), 1.78–1.90 (m, 1H), 1.98–
2.10 (m, 2H), 2.11 (s, 3H), 2.25–2.40 (m, 4H), 2.49 (m, 2H), 3.86
(s, 3H), 3.87 (s, 3H), 6.61 (d, 2H, J = 8.45 Hz), 6.70–6.86 (m, 2H,
J = 8.32 and 2.11 Hz), 6.88–6.92 (dd, 1H, J = 8.32 and 2.11 Hz),
6.94 (d, 2H, J = 8.45 Hz); 13C NMR (CDCl3) d 18.5, 18.9, 22.7, 28.6,
32.5, 35.6, 37.8, 41.4, 53.3, 55.8, 55.9, 56.8, 56.9, 109.6, 111.1,
115.3, 118.7, 121.4, 129.3, 130.5, 133.1, 148.3, 149.0, 154.4; IR
(KBr, cmꢀ1) 3100–3600, 3010, 2963, 2938, 2873, 2840, 2800,
4.2.17. 2-(3,4-Dimethoxy-phenyl)-5-{[2-(4-hydroxy-phenyl)-
ethyl]-methyl-amino}-2-isopropyl-pentanenitrile (13a)
Mixture of amine 2 (590 mg, 2.03 mmol) and compound 10
(706 mg, 2.45 mmol) was treated following method C1. Purifica-
tion of crude product (elution AcOEt/CH2Cl2/MeOH = 5:3.5:1.5)
afforded compound 13a (0.250 g, 30% yield) as a yellow oil. 1H
NMR (CDCl3) d 0.78 (d, 3H, J = 6.78 Hz), 1.17 (d, 3H, J = 6.78 Hz),
1.10–1.30 (m, 1H), 1.46–1.62 (m, 1H), 1.83 (m, 1H, J = 4.47 Hz),

6272
R. Barattin et al. / Bioorg. Med. Chem. 18 (2010) 6265–6274
2236, 1591, 1518, 1469, 1420, 1265, 1159, 1036, 806, 734; MS (m/
z) 425 (M+H+, 100%); Anal. Calcd for C26H36N2O3ꢂ1/2H2O: C, 70.56;
H, 8.65; N, 6.33. Found: C, 70.52; H, 8.55; N, 6.15.
4.2.24. 2-(3,4-Dimethoxy-phenyl)-5-[2-(4-hydroxy-3-iodo-
phenyl)-ethylamino]-2-isopropyl-pentanenitrile (15b)
Compound 15a (238 mg, 0.6 mmol) was treated by an iodine solu-
tion in CH3CN (13 mL, 1.06 equiv) and NH4OH (3 mL) according to
method D1. Purification of crude product by column chromatography
(CH2Cl2/iPrNH2 = 9.5:0.5) provided monoiodinated phenol 15b
(100 mg, 32% yield) as a yellow oil. 1H NMR (CDCl3) d 0.78 (d, 3H,
J = 6.66 Hz), 1.18 (d, 3H, J = 6.66 Hz), 1.2–1.3 (m, 1H, J = 12.03 Hz),
1.5–1.6 (m 1H, J = 12.03 Hz), 1.90 (m, 1H, J = 12.03 and 4.48 Hz),
2.07 (m, 1H, J = 6.66 Hz), 2.19 (m, 1H, J = 12.03 and 4.48 Hz), 2.67
(m, 2H), 2.7–2.9 (m, 4H), 3.87 (s, 3H), 3.89 (s, 3H), 6.84 (m, 3H),
6.90 (dd, 1H, J = 2.31 and 8.32 Hz), 7.01 (dd, 1H, J = 2.05 and
8.32 Hz), 7.47 (d, 1H, J = 2.05 Hz); 13C NMR (CDCl3) d 18.6, 18.9,
25.0, 33.8, 35.4, 37.9, 48.7, 50.2, 53.3, 55.9, 56.1, 85.7, 109.6, 111.2,
115.0, 118.7, 121.3, 130.3, 130.4, 132.8, 138.2, 148.4, 149.0, 153.8;
IR (KBr, cmꢀ1) 2500–3500, 3030, 3000, 2963, 2930, 2881, 2832,
2236, 1591, 1518, 1469, 1412, 1257, 1151, 1028; MS (m/z). 276,
289, 397 (100%), 523 (M+H+); Anal. Calcd for C24H31IN2O3: C, 53.34;
H, 6.15; N, 5.18. Found: C, 53.41; H, 6.34; N, 5.27.
4.2.21. 2-(3,4-Dimethoxy-phenyl)-5-{[3-(4-hydroxy-3-iodo-
phenyl)-propyl]-methyl-amino}-2-isopropyl-pentanenitrile
(14b)
Phenol 14a (181 mg, 0.43 mmol) was monoiodinated by an
0.05 M I2 solution (8 mL, 1 equiv) and ammonia (2 mL) as described
in method D1. Purification of crude product (elution AcOEt/CH2Cl2/
MeOH = 5:4:1) provided compound 14b (60 mg, 27% yield) as a pale
yellow oil. 1H NMR (CDCl3) d 0.79 (d, 3H, J = 6.66 Hz), 1.18 (d, 3H,
J = 6.66 Hz), 1.17–1.2 (m, 1H), 1.48–1.62 (m, 1H), 1.67–1.80 (m,
2H), 1.85–1.98 (m, 1H), 2.00–2.20 (m, 2H), 2.15 (s, 3H), 2.28–2.40
(m, 3H), 2.48 (t, 2H, J = 7.55 Hz), 3.88 (s, 3H), 3.89 (s, 3H), 6.82–
6.89 (m, 3H), 6.90–6.95 (dd, 1H, J = 8.32, 2.17 Hz), 6.98–7.03 (dd,
1H, J = 8.19, 2.05 Hz), 7.45 (d, 1H, J = 2.05 Hz); 13C NMR (CDCl3) d
18.6, 18.9, 22.9, 28.3, 32.0, 35.5, 37.9, 41.4, 53.3, 55.9, 56.0, 56.6,
56.8, 85.5, 109.6, 111.2, 118.8, 121.4, 130.0, 130.5, 135.8, 137.8,
148.3, 149.1, 153.2; IR (KBr, cmꢀ1) 3200–3500, 3053, 2971, 2938,
2881, 2236, 1591, 1518, 1412, 1265, 1151, 1028, 742; MS (m/z)
551 (M+H+, 100%); Anal. Calcd for C26H35IN2O3: C, 56.73; H, 6.41;
N, 5.09. Found: C, 56.80; H, 6.63; N, 4.96.
4.2.25. 2-(3,4-Dimethoxy-phenyl)-5-[2-(4-hydroxy-3,5-diiodo-
phenyl)-ethylamino]-2-isopropyl-pentanenitrile (15c)
A 0.05 M I2 solution in CH3CN (22 mL, 2 equiv) and 22% ammonia
(4 mL) were added to compound 15a (207 mg, 0.52 mmol). The mix-
ture was stirred at room temperature, under argon, for 16 h. The pre-
cipitate formed was filtered and washed with CH3CN (20 mL). The
solid obtained was stirred for 12 h in cyclohexane (20 mL), filtered
and dried on KOH in vacuo to afford compound 15c (147 mg, 44%
yield) as a marronsolid, which was used withno further purification.
1H NMR (DMSO) d 0.72 (d, 3H, J = 6.60 Hz), 1.14 (d, 3H, J = 6.60 Hz),
1.0–1.2 (m, 1H), 1.4–1.6 (m, 1H), 2.0–2.2 (m, 2H), 2.2–2.3 (m, 1H,
J = 6.60 Hz), 2.6–2.7 (m, 2H), 2.7–2.9 (m, 2H), 2.9–3.0 (m, 2H), 3.79
(s, 3H), 3.80 (s, 3H), 6.9–7.1 (m, 3H), 7.60 (s, 2H); 13C NMR (DMSO)
d 19.3, 19.7, 25.2, 32.5, 35.0, 37.5, 48.5, 50.4, 53.8, 56.4, 56.7, 88.6,
110.3, 112.4, 119.8, 122.2, 131.1, 133.2, 139.8, 149.1, 149.9, 154.8;
IR (KBr, cmꢀ1) 2500–3500, 3030, 2961, 2934, 2875, 2837, 2236,
1590, 1517, 1457, 1415, 1260, 1143, 1024, 868, 811, 737, 698; MS
(m/z). 289, 397, 523, 649 (M+H+, 100%); Anal. Calcd for
4.2.22. 2-(3,4-Dimethoxy-phenyl)-5-{[3-(4-hydroxy-3,5-diiodo-
phenyl)-propyl]-methyl-amino}-2-isopropyl-pentanenitrile
(14c)
Ammonia (4 mL) and iodine solution (16 mL, 2.2 equiv) were
added to phenol 14a (165 mg, 0.39 mmol) following method D1.
Purification by column chromatography (AcOEt/CH2Cl2/MeOH =
5:3.5:1.5) afforded compound 14c (133 mg, 51% yield) as a yellow
oil. 1H NMR (CDCl3) d 0.80 (d, 3H, J = 6.66 Hz), 1.19 (d, 3H,
J = 6.66 Hz), 1.20–1.40 (m, 1H), 1.50–1.70 (m, 3H), 1.87–2.10 (m,
1H), 2.50–2.19 (m, 2H), 2.20 (s, 3H), 2.30–2.50 (m, 6H), 6.83–6.97
(m, 3H, J = 8.32 and 2.18 Hz), 7.46 (s, 2H); 13C NMR (CDCl3) d 18.6,
18.9, 22.6, 28.2, 31.4, 35.5, 37.9, 41.2, 53.3, 55.9, 56.1, 56.4, 56.7,
83.7, 109.7, 111.2, 118.8, 121.4, 130.4, 137.3, 138.9, 148.4, 149.1,
152.7; IR (KBr, cmꢀ1) 3010, 2955, 2930, 2865, 2236, 1591, 1518,
1469, 1412, 1265, 1142, 1028, 808; MS (m/z) 291, 418, 551, 677
(M+H+, 100%); Anal. Calcd for C26H34I2N2O3: C, 46.17; H, 5.07; N,
4.14. Found: C, 46.20; H, 5.23; N, 4.10.
C
24H30I2N2O3ꢂ3/2H2O: C, 42.68; H, 4.93; N, 4.15. Found: C, 42.50;
H, 4.43; N, 4.38.
4.2.26. Acetic acid 2-(3-iodo-4-methoxy-phenyl)-ethyl ester (17)
2-(4-Methoxyphenyl)ethan-1-ol 16 (260 mg, 1.7 mmol) was
treated by I2 (0.216 g, 2 equiv) and NaIO4 (0.055 g, 0.15 equiv) fol-
lowing method D2, to afford compound 16 (192 mg, 35% yield) as a
yellow oil. 1H NMR (CDCl3) d 2.03 (s, 3H), 2.83 (t, 2H, J = 7.05 Hz),
3.85 (s, 3H), 4.22 (t, 2H, J = 7.05 Hz), 6.75 (d, 1H, J = 8.31 Hz), 7.50
(dd, 1H, J = 2.19 and 8.31 Hz), 7.63 (d, 1H, J = 2.19 Hz); 13C NMR
(CDCl3) d 20.8, 33.6, 56.3, 64.7, 85.8, 110.8, 129.8, 131.9, 139.7,
156.8, 170.8; IR (KBr, cmꢀ1) 3030, 3000, 2963, 2930, 2900, 2840,
1745, 1600, 1493, 1363, 1240, 1053, 808; MS (m/z). 338
4.2.23. 2-(3,4-Dimethoxy-phenyl)-5-[2-(4-hydroxy-phenyl)-
ethylamino]-2-isopropyl-pentane nitrile (15a)
Tyramine (265 mg, 1.9 mmol) was added to a stirred solution of
mesylated alcohol 1 (330 mg, 0.93 mmol) in dry CH3CN (10 mL).
The reaction mixture was refluxed under argon for 20 h and then al-
lowed to warm at room temperature. After adding water (20 mL),
the aqueous solution was acidified to pH 1 by adding concentrated
HCl and washed with ether (3ꢁ 30 mL). The resulting aqueous phase
was then basified to pH 10 by adding 22% ammonia and extracted
with ether (3ꢁ 30 mL). These combined organic layers were dried
with anhydrous Na2SO4 and concentrated in vacuo to provide com-
pound 15a (200 mg, 55% yield) as a yellow oil. 1H NMR (CDCl3) d 0.77
(d, 3H, J = 6.79 Hz), 1.13 (d, 3H, J = 6.79 Hz), 1.2–1.3 (m, 1H,
J = 12.03 Hz), 1.53 (m, 1H, J = 12.03 Hz, 1.82 (m, 1H, J = 12.03 and
4.48 Hz), 2.03 (m, 1H, J = 6.79 Hz), 2.10 (m, 1H, J = 12.03 and
4.48 Hz), 2.60 (m, 2H), 2.69 (m, 2H), 2.76 (m, 2H), 3.85 (s, 6H), 3.9–
4.3 (OH), 6.68 (d, 2H, J = 8.34 Hz), 6.80 (d, 1H, J = 8.34 Hz), 6.82 (d,
1H, J = 2.16 Hz), 6.87 (dd, 1H, J = 8.34 and 2.16 Hz), 6.97 (d, 2H,
J = 8.34 Hz); 13C NMR (CDCl3) d 18.5, 18.9, 25.7, 34.8, 35.5, 37.9,
49.1, 50.7, 53.3, 55.9, 56.0, 109.6, 111.2, 115.7, 118.7, 121.3, 129.7,
129.9, 130.5, 148.3, 149.0, 155.1; IR (KBr, cmꢀ1) 3500, 3030, 2963,
2930, 2881, 2832, 2236, 1591, 1518, 1469, 1412, 1257, 1151,
1028; MS (m/z) 397 (M+H+, 100%); Anal. Calcd for C24H32N2O3ꢂ2H2O:
C, 66.64; H, 8.39; N, 6.48. Found: C, 66.94; H, 8.02; N, 6.44.
(M þ NHþ, 100%); Anal. Calcd for C11H13IO3: C, 41.27; H, 4.09; I,
4
39.64. Found: C, 41.15; H, 4.17; I, 39.34.
4.2.27. Acetic acid 2-(3,5-diiodo-4-methoxy-phenyl)-ethyl ester
(18)
2-(4-Methoxyphenyl)ethan-1-ol 16 (0.538 g, 3.53 mmol) was
diiodinated by I2 (0.648 g, 3 equiv) and NaIO4 (0.390 g, 0.5 equiv)
as described in method D2, to provide compound 18 (0.387 g,
25% yield) as a white solid. Mp 62–63 °C; 1H NMR (CDCl3) d 2.05
(s, 3H), 2.81 (t, 2H, J = 6.93 Hz), 3.84 (s, 3H), 4.22 (t, 2H,
J = 6.93 Hz), 7.62 (s, 2H); 13C NMR (CDCl3) d 20.9, 33.2, 60.7, 64.2,
90.4, 137.8, 140.1, 157.6, 170.9; IR (KBr, cmꢀ1) 3030, 2986, 2950,
2934, 2878, 2845, 1744, 1456, 1390, 1226, 1027, 707; MS (m/z)
464 (M þ NHþ, 100%); Anal. Calcd for C11H12I2O3: C, 29.62; H,
4
2.71. Found: C, 29.64; H, 2.59.

R. Barattin et al. / Bioorg. Med. Chem. 18 (2010) 6265–6274
6273
4.2.28. 2-(3-Iodo-4-methoxy-phenyl)-ethanol (19)
(CDCl3) d 33.8, 37.6, 60.7, 69.1, 90.6, 136.2, 140.2, 158.1; IR (KBr,
cmꢀ1) 2970, 2938, 2840, 1575, 1534, 1461, 1420, 1355, 1248,
1167, 1061, 955; MS (m/z) 387, 482 (M+), 500 (M þ NH4þ, 100%);
Anal. Calcd for C10H12I2O4S: C, 24.91; H, 2.51. Found: C, 25.43; H,
2.60.
K2CO3 (10 equiv) was added to a solution of compound 17
(325 mg, 1 mmol) in methanol (15 mL). The mixture was stirred
at room temperature for 3 h and then poured into water (30 mL).
The aqueous solution was extracted with ether (3ꢁ 40 mL) and
combined organic layers were dried over anhydrous Na2SO4. Re-
moval of the solvent under reduced pressure provided alcohol 19
(282 mg, 100% yield) as a white oil. This product was used as such
for further transformations. 1H NMR (CDCl3) d 1.4–1.7 (OH), 2.77 (t,
2H, J = 6.54 Hz), 3.81 (t, 2H, J = 6.54 Hz), 3.86 (s, 3H), 6.77 (d, 1H,
J = 8.31 Hz), 7.17 (dd, 1H, J = 2.07 and 8.31 Hz), 7.65 (d, 1H,
J = 2.07 Hz); 13C NMR (CDCl3) d 37.6, 56.4, 63.5, 86.1, 110.9,
130.0, 132.7, 139.7, 156.8; IR (KBr, cmꢀ1) 3100–3600, 2938, 2850,
2810, 1600, 1567, 1493, 1461, 1270, 1257, 1183, 1053, 1020; MS
(m/z) 247, 261, 278 (M)+, 296 (M þ NHþ4 , 100%); Anal. Calcd for
C9H11IO2: C, 38.87; H, 3.99; I, 45.63. Found: C, 39.40; H, 4.03; I,
44.26.
4.2.33. 2-(3,4-Dimethoxy-phenyl)-2-isopropyl-5-{[2-(4-
methoxy-phenyl)-ethyl]-methyl-amino}-pentanenitrile (24a)
Compound 24a was prepared from mesylate 21 (175 mg,
0.76 mmol) and amine 2 (214 mg, 0.74 mmol) according to method
C2, and was obtained as a yellow oil (160 mg, 51% yield). 1H NMR
(CDCl3) d 0.78 (d, 3H, J = 6.65 Hz), 1.05–1.21 (m, 1H), 1.17 (d, 3H,
J = 6.65 Hz), 1.40–1.60 (m, 1H), 1.81 (m, 1H, J = 4.48 Hz), 2.00–
2.15 (m, 2H), 2.17 (s, 3H), 2.30–2.40 (m, 2H), 2.40–2.50 (m, 2H),
2.60–2.70 (m, 2H), 3.78 (s, 3H), 3.88 (s, 6H), 6.80–6.93 (m, 5H),
7.08 (d, 2H, J = 8.7 Hz); 13C NMR (CDCl3) d 18.6, 18.9, 23.4, 32.7,
35.6, 37.9, 42.0, 53.3, 55.2, 55.9, 56.0, 56.9, 59.5, 109.6, 111.1,
113.7, 118.6, 121.4, 129.5, 130.7, 132.5, 148.2, 149.0, 157.9; IR
(KBr, cmꢀ1) 2963, 2840, 2791, 2236, 1616, 1518, 1461, 1412,
1257, 1151, 1036; MS (m/z) 303, 425 (M+, 100%); Anal. Calcd for
4.2.29. 2-(3,5-Diiodo-4-methoxy-phenyl)-ethanol (20)
K2CO3 (10 equiv) was added to a solution of compound 18
(300 mg, 0.6 mmol) in methanol (15 mL). The mixture was stirred
at room temperature for 3 h and then poured into water (30 mL).
The aqueous solution was extracted with ether (3ꢁ 40 mL) and
combined organic layers were dried over anhydrous Na2SO4. Re-
moval of the solvent under reduced pressure provided alcohol 20
(257 mg, 100% yield) as a white solid. This product was used as
such for further transformations. Mp 62–63 °C; 1H NMR (CDCl3) d
2.05 (s, 3H), 2.81 (t, 2H, J = 6.93 Hz), 3.84 (s, 3H), 4.22 (t, 2H,
J = 6.93 Hz), 7.62 (s, 2H); 13C NMR (CDCl3) d 20.9, 33.2, 60.7, 64.2,
90.4, 137.8, 140.1, 157.6, 170.9; IR (KBr, cmꢀ1) 3030, 2986, 2950,
2934, 2878, 2845, 1744, 1456, 1390, 1226, 1027, 707; MS (m/z)
C
26H36N2O3ꢂH2O: C, 70.56; H, 8.65; N, 6.33. Found: C, 70.52; H,
8.50; N, 6.54.
4.2.34. 2-(3,4-Dimethoxy-phenyl)-5-{[2-(3-iodo-4-methoxy-
phenyl)-ethyl]-methyl-amino}-2-isopropyl-pentanenitrile
(24b)
Mixture of amine 2 (135 mg, 0.46 mmol) and compound 22
(165 mg, 0.046 mmol) was treated following method C2 to afford
compound 24b (97 mg, 38% yield) as a yellow oil. 1H NMR (CDCl3)
d
0.78 (d, 3H, J = 6.66 Hz), 1.05–1.21 (m, 1H), 1.17 (d, 3H,
J = 6.66 Hz), 1.45–1.61 (m, 1H), 1.81 (m, 1H, J = 4.35 Hz), 2.00–2.12
(m, 2H), 2.15 (s, 3H), 2.28–2.36 (m, 2H), 2.42–2.50 (m, 2H),
2.56–2.65 (m, 2H), 3.85 (s, 3H), 3.88 (s, 3H), 3.89 (s, 3H), 6.73 (d,
1H, J = 8.32 Hz), 6.81–6.96 (m, 3H), 7.08 (dd, 1H, J = 8.32 and
2.04 Hz), 7.59 (d, 1H, J = 2.04 Hz); 13C NMR (CDCl3) d 18.6, 18.9,
23.4, 32.1, 35.5, 37.9, 41.8, 53.3, 56.0, 56.4, 56.9, 59.2, 85.8, 109.6,
110.8, 110.9, 118.7, 121.5, 129.7, 130.7, 134.8, 139.5, 148.2, 149.0,
156.4; IR (KBr, cmꢀ1) 3030, 3000, 2963, 2930, 2840, 2800, 2236,
1600, 1518, 1461, 1412, 1248, 1053, 1028, 914; MS (m/z) 291, 303,
425, 551 (M+H+, 100%); Anal. Calcd for C26H35IN2O3: C, 56.73; H,
6.41; N, 5.05. Found: C, 56.99; H, 6.47; N, 5.12.
464 (M þ NHþ, 100%); Anal. Calcd for C11H12I2O3: C, 29.62; H,
4
2.71. Found: C, 29.64; H, 2.59.
4.2.30. Methanesulfonic acid 2-(4-methoxy-phenyl)-ethyl ester
(21)
A
solution of 2-(4-methoxyphenyl)ethan-1-ol 16 (498 mg;
3.3 mmol) was treated according to method B2 to afford compound
21 (500 mg, 66% yield) as a yellow oil. 1H NMR (CDCl3) d 2.85 (s,
3H), 2.99 (t, 2H, J = 6.91 Hz), 3.79 (s, 3H), 4.38 (t, 2H, J = 6.91 Hz),
6.85 (d, 2H, J = 8.71 Hz), 7.15 (d, 2H, J = 8.71 Hz); 13C NMR (CDCl3)
d 34.7, 37.3, 55.2, 70.6, 114.1, 128.2, 130.0, 158.6; IR (KBr, cmꢀ1
)
3030, 3020, 3000, 2970, 2938, 2900, 2840, 1600, 1518, 1469,
1355, 1241, 1175, 1028, 955; MS (m/z) 121, 135, 248 (M þ NHþ4 ,
100%); Anal. Calcd for C10H14O4S: C, 52.16; H, 6.13. Found: C,
52.44; H, 6.14.
4.2.35. 5-{[2-(3,5-Diiodo-4-methoxy-phenyl)-ethyl]-methyl-
amino}-2-(3,4-dimethoxy-phenyl)-2-isopropyl-pentanenitrile
(24c)
Amine 2 (133 mg, 0.46 mmol) was alkylated by compound 23
(244 mg, 0.51 mmol), as described in method C2, to provide com-
pound 24c as a yellow oil (93 mg, 30% yield). 1H NMR (CDCl3) d
0.78 (d, 3H, J = 6.70 Hz), 1.05–1.12 (m, 1H), 1.18 (d, 3H,
J = 6.70 Hz), 1.42–1.60 (m, 1H), 1.82 (m, 1H, J = 4.48 Hz), 2.00–
2.10 (m, 2H), 2.12 (s, 3H), 2.20–2.40 (m, 2H, 2.40–2.51 (m, 2H),
2.52–2.63 (m, 2H), 3.83 (s, 3H), 3.88 (s, 6H), 6.86 (m, 3H), 7.59 (s,
2H); 13C NMR (CDCl3) d 18.7, 18.9, 23.4, 31.9, 35.5, 37.9, 41.6,
53.4, 55.9, 56.0, 57.0, 58.7, 60.7, 90.1, 109.7, 111.1, 118.6, 121.4,
130.7, 140.0, 140.7, 148.2, 149.0, 157.0; IR (KBr, cmꢀ1) 3060,
2955, 2930, 2840, 2791, 2236, 1591, 1518, 1469, 1412, 1257,
1151, 1025, 1070; MS (m/z) 303, 551, 677 (M+H+, 100%); Anal.
Calcd for C26H34I2N2O3: C, 46.17; H, 5.07; N, 4.14. Found: C,
45.95; H, 5.07; N, 4.06.
4.2.31. Methanesulfonic acid 2-(3-iodo-4-methoxy-phenyl)-
ethyl ester (22)
Alcohol 19 (174 mg; 0.6 mmol) was treated with mesyl chloride
according to method B2 to afford compound 22 (213 mg, 96%
yield) as a white oil. 1H NMR (CDCl3) d 2.90 (s, 3H), 2.95 (t, 2H,
J = 6.93 Hz), 3.87 (s, 3H), 4.36 (t, 2H, J = 6.93 Hz), 6.77 (d, 1H,
J = 8.34 Hz), 7.18 (dd, 1H, J = 2.16 and 8.34 Hz), 7.65 (d, 1H,
J = 2.16 Hz); 13C NMR (CDCl3) d 34.2, 37.5, 56.4, 69.9, 86.1, 110.9,
130.1, 130.4, 139.8, 157.3; IR (KBr, cmꢀ1) 2938, 2840, 1600, 1567,
1493, 1355, 1257, 1175,þ1051, 955, 816; MS (m/z) 135, 248, 261,
356 (M+), 374 (M þ NH4 , 100%); Anal. Calcd for C10H13IO4S: C,
33.72; H, 3.68. Found: C, 34.29; H, 3.73.
4.2.32. Methanesulfonic acid 2-(3,5-diiodo-4-methoxy-phenyl)-
ethyl ester (23)
4.3. Biology
Alcohol 20 (164 mg, 0.4 mmol) was transformed into mesylate
23 (157 mg) as a yellow oil, with a yield of 80%, as described in
method B2. 1H NMR (CDCl3) d 2.94 (t, 2H, J = 6.78 Hz), 2.96 (s,
3H), 3.85 (s, 3H), 4.36 (t, 2H, J = 6.78 Hz), 7.64 (s, 2H); 13C NMR
4.3.1. Cell lines
BHK-21 cells stably transfected with wild type MRP1 has been
described previously. Cells were grown at 37 °C in 5% CO2 in DMEM
culture medium containing 1% penicillin–streptomycin and 5%

6274
R. Barattin et al. / Bioorg. Med. Chem. 18 (2010) 6265–6274
fetal bovine serum, in the presence of 200
transfected cells.
l
M methotrexate for
sur le Cancer (ARC 4853) and the Ligue Nationale contre le Cancer
(Equipe Labellisée 2009). The technical assistance of Mélanie Her-
bert is acknowledged.
4.3.2. Cell proliferation determined by MTT
The MTT colorimetric assay, as described previously, was used
to assess the sensitivity of control cells and MRP1-transfected
BHK-21 cells to verapamil derivatives. Briefly, growth inhibition
References and notes
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9675.
(IC50
) assays were performed by plating cells at density
1.0 ꢁ 104 cells/well in 96-well plates. Cells were cultured for 17 h
before addition of verapamil derivatives diluted in complete cul-
ture medium, DMSO concentration was fixed at 0.5%. The cells
were then incubated for 96 h in a wet tissue-culture chamber
(37 °C, 5% CO2). Surviving cells were detected by the bromo-
3(4,5-dimethyl-2-thiazoyl)2,5-diphenyltetrazolium (MTT) assay.
The MTT solution was prepared to 0.5 mg/mL, and 100
added to each well and incubated for 4 h at 37 °C. Thereafter, the
MTT dye was aspirated and 200 l of isopropanol was added to
ll was
l
each well. The formazan, resulting from the reaction, was photo-
metrically measured at 570 nm. IC50 values were calculated from
dose–response curves obtained from triplicate experiments.
13. Cullen, K. V.; Davey, R. A.; Davey, M. W. Biochem. Pharmacol. 2001, 62, 417.
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20. The synthesis of mesylate 1 and amine 2 was performed according to the
previously reported procedure: Teodori, E.; Dei, S.; Quidu, P.; Budriesi, R.;
Chiarini, A.; Garnier-Suillerot, A.; Gualtieri, F.; Manetti, D.; Novella Romanelli,
M.; Scapecchi, S. J. Med. Chem. 1999, 42, 1687. For convenient reasons, phase
transfer catalysis was chosen to achieve the successive alkylations of
4.3.3. Total cellular glutathione determination
The total cellular glutathione content (GSH + oxidized glutathi-
one) was measured using the enzymatic method described by Tie-
zte, as modified by Anderson to the microtiter plate. Cells were
seeded into 96-well plates at a density of 1.0 ꢁ 105 cells/well and
cultured for 17 h before addition of 5 lM verapamil derivatives;
DMSO concentration did not exceed 0.5%. After various incubation
times, from 10 to 120 min, cells were twice washed with 200
cold PBS, and then lysed by 50 l PBS supplemented with 0.1% Tri-
ton X-100. The lysate was then homogenized and 10 l was trans-
ferred to a new plate for GSH titration, and 5 l to another plate for
protein quantification. GSH determination was performed by
adding 120 l of reaction buffer containing 221.3 M NADPH,
462.6 M DTNB, 11 U/mL GSH reductase, and PBS. The plate was
ll
l
l
l
l
l
l
homoveratronitrile
with
2-bromopropane,
and
bromopropyl
then placed in a microplate reader, and the absorbance was read
at 412 nm every 30 s for 2 min. The content of total cellular gluta-
thione was quantified by comparison with known glutathione
standards. The slope was determined for each sample, and allowed
us to determine the GSH concentration. Protein titration was per-
formed on the remaining 5 ll by the Bradford method assay. The
measured total glutathione was expressed in nanomole per milli-
gram protein, and the results obtained were issued from triplicates.
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Acknowledgments
This work was funded by the CNRS and the Université Lyon 1
(UMR 5086), and grants from the Association pour la Recherche