Synthesis, physicochemical studies, molecular dynamics simulations, and metal-ion-dependent antiproliferative and antiangiogenic properties of cone ICL670-substituted calix[4]arenes

DOI: 10.1002/cplu.201200141

Synthesis, Physicochemical Studies, Molecular Dynamics

Simulations, and Metal-Ion-Dependent Antiproliferative

and Antiangiogenic Properties of Cone ICL670-Substituted

Calix[4]arenes

Pascal Rouge,^[a]Alexandra Dassonville-Klimpt,^[a]Christine Cꢀzard,^[a]

Stꢀphanie Boudesocque,^[b]Roger Ourouda,^[c]Carole Amant,^{[c, g]}FranÅois Gaboriau,^[d]

Isabelle Forfar,^[e]Jean Guillon,^[e]Emmanuel Guillon,^[b]Enguerran Vanquelef,^[a]Piotr Cieplak,^[f]

FranÅois-Yves Dupradeau,^[a]Laurent Dupont,^[b]and Pascal Sonnet*^[a]

In memory of Enguerran Vanquelef

Iron chelators, through their capacity to modulate the iron

concentration in cells, are promising molecules for cancer che-

motherapy. Chelators with high lipophilicity easily enter into

cells and deplete the iron intracellular pool. Consequently,

iron-dependent enzymes, such as ribonucleotide reductase,

which is over-expressed in cancer cells, become nonfunctional.

A series of calix[4]arene derivatives substituted at the lower

rim by ICL670, a strong Fe^IIIchelator, have been synthesized.

Physicochemical properties and antiproliferative, angiogenesis,

and tumorigenesis effects of two calix[4]arenes mono- (5a) or

disubstituted (5b) with ICL670 have been studied. These com-

pounds form metal complexes in a ratio of one to two ligands

per Fe^IIIatom as shown by combined analyses of the proto-

metric titration curves and ESIMS spectra. The grafting of an

ICL670 group on a calix[4]arene core does not significantly

alter the acid–base properties, but improves the iron-chelating

and lipophilicity properties. The best antiproliferative and anti-

angiogenic results were obtained with calix[4]arene ligand 5a,

which possesses the highest corresponding properties. Analy-

ses of molecular dynamics simulations performed on the two

calix[4]arenes provide three-dimensional structures of the com-

plexes and proved 5a to be the most stable upon complexa-

tion.

[a] Dr. P. Rouge, Dr. A. Dassonville-Klimpt, Dr. C. Cꢀzard, Dr. E. Vanquelef,

Prof. Dr. F.-Y. Dupradeau, Prof. Dr. P. Sonnet

Laboratoire des Glucides, CNRS FRE 3517, UFR de Pharmacie

Universitꢀ de Picardie Jules Verne

Introduction

Iron chelators such as deferoxamine, deferiprone, or deferasir-

ox/ICL670, initially developed for iron-overload therapy, have

shown antiproliferative effects.^[1]The chemical structure of

these iron chelators influences their capacity to modulate iron

uptake in cells. Indeed, deficiency of labile iron in the cyto-

plasm results in inhibition of deoxyribonucleotide biosynthesis,

which is the limiting step in DNA synthesis, and subsequently

leads to the inhibition of cell proliferation.^[2]Ribonucleotide re-

ductase (RR) is over-expressed in rapidly dividing cancer cells,

and is critical for deoxyribonucleotide biosynthesis and cell di-

vision. RR is the main target of iron chelators and during the

last two decades, a new generation of iron chelators has been

synthesized and studied in cancer chemotherapy. Triapine,^[3]o-

trensox,^[4]tachpyridine,^[5]di-2-pyridyl thiosemicarbazones like

Dp44mT,^[6]and the pyridoxal isonicotinoyl hydrazone (PIH)

family^[7]currently appear to be the most promising molecules.

In the case of PIH analogues, Richardson and Lovejoy identi-

fied a significant chelator structure–activity relationship depen-

dent on lipophilicity.^[8]Within a series or family of ligands, the

most lipophilic compounds have generally the best iron-clear-

ing efficiency. Moreover, lipophilicity has a profound effect on

organ distribution of the chelators.^[9]Chelators with a high lip-

ophilicity can easily enter cells and subsequently deplete the

1, rue des Louvels, 80037 Amiens cedex 1 (France)

Fax: (+33)322827469

E-mail: pascal.sonnet@u-picardie.fr

[b] Dr. S. Boudesocque, Prof. Dr. E. Guillon, Prof. Dr. L. Dupont

Institut de Chimie Molꢀculaire de Reims (ICMR)

UMR CNRS 7312, UFR des Sciences Exactes et Naturelles

Universitꢀ de Reims Champagne-Ardenne, Reims (France)

[c] Dr. R. Ourouda, Dr. C. Amant

Hꢀmostase et remodelage vasculaire post-ischꢀmique, EA3801

Universitꢀ de Picardie Jules Verne

1, rue des Louvels, 80037 Amiens cedex 1 (France)

[d] Dr. F. Gaboriau

Inserm U991 (EA/MDC), Universitꢀ de Rennes I

Hꢁpital Pontchaillou, Rennes (France)

[e] Dr. I. Forfar, Prof. Dr. J. Guillon

CNRS FRE 3396 (Pharmacochimie), UFR de Pharmacie

Universitꢀ de Bordeaux Segalen, Bordeaux (France)

[f] Dr. P. Cieplak

Sandford-Burnham Medical Research Institute

10901 North Torrey Pines Road

La Jolla, CA 92037 (USA)

[g] Dr. C. Amant

Laboratoire d’Oncobiologie Molꢀculaire, CHU d’Amiens

Amiens (France)

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cplu.201200141.

ChemPlusChem 2012, 77, 1001 – 1016

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1001

P. Sonnet et al.

iron intracellular pool, which is necessary for the RR catalytic

activity.

Owing to the development of supramolecular chemistry,

a number of calixarene-based ligands have been extensively

developed for their coordination properties,^[10]in particular to-

wards iron.^[11]We have previously prepared new calix[4]arene

compounds as selective cesium chelators.^[12]In 2001, Fe^IIIcom-

plexes of some amide-substituted calix[4]arenes were report-

ed.^[13]Later Rao et al. reported for the first time the synthesis

and characterization of Mn^II, Fe^III, Co^II, Ni^II, Cu^II, and Zn^IIcom-

plexes of p-tert-butylcalix[4]arene-1,3-diacid derivatives.^[14]

More recently, Wang et al. described selective Fe^IIIand Cr^IIIrec-

ognition by fluorescent calix[4]arene sensors.^[15]Many pharma-

cological properties have been described for calix[4]arenes in-

cluding anticancer, antiviral, antibacterial, antifungal, and an-

tithrombotic activities.^[16]Sebti et al. have shown that a calix[4]-

arene called GFA-116 is capable of selectively disrupting the

binding of vascular endothelial growth factor (VEGF) to its re-

ceptor.^[17]Consequently, the VEGF-dependent signaling is in-

hibited, which suppresses angiogenesis and tumorigenesis. We

have shown recently that calix[4]arenes 1–4 exhibit interesting

antiproliferative activities close to the ICL670 reference, and

the correlation between the cytostatic effect and iron deple-

tion was established clearly for tetra-acid 4.^[18]

Results and Discussion

Ligand design and synthesis

The synthesis strategy for the new calix[4]arene derivatives

5a,b starts from unsubstituted calix[4]arene 6 and iodo com-

pound 7 to obtain the mono- and diaminocalix[4]arenes 9a,b

(Scheme 1). The peptidic coupling of the 9a,b key intermedi-

ates with ICL670 leads to the corresponding calix[4]arenes

5a,b in a cone conformation.

Calix[4]arenes 5a,b were synthesized in three steps starting

from 6 and 7 (Scheme 1). Unsubstituted calix[4]arene 6 was

first prepared according to a two-step synthesis developed by

Gutsche et al.^[19]from p-tert-butylphenol, whereas iodo com-

pound 7 was synthesized as described previously.^[20]Two main

strategies are described to prepare monoalkylated calix[4]arene

derivatives: either by a method of protection/deprotection^[21]

or by a selective approach.^[22]In our case, the best result was

obtained by using the CsF base (1.3 equiv.) in dimethylforma-

mide (DMF) and monoalkylated calix[4]arene 8a in a cone con-

1

formation was isolated in a 62% yield. The H NMR spectrum

for 8a shows two singlet peaks at d=9.05 (2H) and 9.68 ppm

(1H), which correspond to the phenolic hydroxyl protons and

thereby confirm the monoalkylation. Calix[4]arene 8b was pre-

pared in a 74% yield by dialkylation of calix[4]arene 6 with 7

(4 equiv.) in the presence of K₂CO₃in acetonitrile and heated at

reflux for seven days (Scheme 1).

Herein, we report on the synthesis, physicochemical and

molecular dynamics (MD) studies, as well as on the antiprolifer-

ative and antiangiogenic effects of new calix[4]arenes 5a,b

substituted at the lower rim with ICL670, a strong Fe^IIIcom-

plexant.

The alkylation selectively led to diametrically substituted cal-

ix[4]arene 8b in the cone conformation, as indicated by the

¹H NMR spectrum. A typical “AB” pattern was observed for the

methylene bridge Ar-CH₂-Ar (Ar=aryl) protons (²J=12.9 Hz) at

d=3.06 ppm for the equatorial protons and d=3.97 ppm for

the axial protons. The treatment of phthalimidocalix[4]arenes

8a,b with an excess amount of aqueous hydrazine in ethanol

at reflux gave the corresponding aminocalix[4]arenes 9a,b in

the cone conformation in good yields (88–99%; Scheme 1).

Aminocalix[4]arenes 9a,b were then coupled to the ICL670

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ChemPlusChem 2012, 77, 1001 – 1016

Cone ICL670-Substituted Calix[4]arenes

backbone resulted in a signifi-

cant increase of the lipophilicity

for 5a and 5b relative to

ICL670. The introduction of

a second ICL670 substituent on

5b, in comparison with 5a,

yielded a substantial reduction

of the lipophilicity by about 12-

fold. There is a clear relationship

between the fact that chelators

presenting a high lipophilicity

can easily enter cells and de-

plete the intracellular iron that

is necessary for the catalytic ac-

tivity of RR. Compound 5a

should present a better antipro-

liferative active profile than cal-

ix[4]arene 5b.

Metal-binding properties

The chelating abilities of ICL670,

5a, and 5b towards Fe^IIIwere

estimated on the basis of the

global formation constants of

their complexes, which were de-

termined by UV-visible spectro-

photometric and potentiometric

(Hyperquad program^[27]

) tech-

niques. Because of the poor sol-

ubility of the chelators in an

aqueous medium, all experi-

ments were monitored in

a

water/DMSO media (v/v=

30:70; x_DMSO=0.5). The global

stability constant b_mlhof each

ferric complex is defined by

Equations (1) and (2):

*

mM þ lL þ hH)ꢀM_mL_lH_h

ð1Þ

ð2Þ

Scheme 1. Synthesis of the cone calix[4]arene derivatives 5a,b.

½M_mL_lH_hꢂ

b_mlh

¼

m

l

h

½Mꢂ ½Lꢂ ½Hꢂ

moieties, subsequently synthesized in two steps according to

Steinhauser et al.,^[23]using 1-(3-dimethylaminopropyl)-3-ethyl-

carboiimide/N-hydroxybenzotriazole (EDCI/HOBt) to afford ami-

docalix[4]arenes 5a,b in moderate yields (37–51%).

in which m, l, and h are values in the general complex formula

[M_mL_lH_h]. M, L, and H correspond to the Fe^IIImetal ion, the

ligand (L¹=ICL670, L²=5a, L³=5b), and the protons, respec-

tively. For the sake of clarity, charges are omitted. The HYSS

computer program^[27]was used to obtain the species distribu-

tion curves.

Analysis of the hydrophobicity of the ligands

The determination of the logarithm of the distribution coeffi-

cient (logD) was achieved by using HPLC for calix[4]arenes

5a,b at pH 7.^[24]In the case of ICL670, reported values for the

logarithm of the partition coefficient P (logP) and logD are

3.8^[25]and ꢀ0.1,^[26]respectively. Compounds 5a,b were found

to be highly lipophilic with logD values of 4.59 (ꢁ0.06) and

3.49 (ꢁ0.05), respectively. The introduction of the calixarene

Analysis of neutralization curves of the ligand solution

shows that ICL670 and 5a have three sites able to be proton-

ated in the 2.5–13 pH range, whereas 5b has five such sites. In

the case of ICL670, pK_a1is ascribed to the deprotonation of the

carboxylic functions, whereas pK_a2and pK_a3correspond to the

deprotonation of the two phenolic groups. Although the two

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1003

P. Sonnet et al.

OH groups are structurally equivalent, a difference of two units

is observed between pK_a2and pK_a3. Such a difference has been

observed already by Steinhauser et al.,^[23]and can be explained

by a hydrogen bond between the proton of one phenolic

group and a nitrogen atom of the triazole moiety making the

phenolic group more basic. Nevertheless, it appears highly

probable that the two hydroxyl groups are deprotonated si-

multaneously. The observed pK_avalues thus represent averages

of the individual (microscopic) acidity constants. On the other

hand, pK_a2and pK_a3of ICL670 and 5a show that the grafting

of the ICL670 group on a calix[4]arene core does not signifi-

cantly alter the acid–base properties. In the case of 5b, depro-

tonation of the ICL670 phenolic groups corresponds to values

in the range of pK_a2to pK_a5. Finally, the pK_a1value for 5a and

5b is ascribed to the deprotonation of the phenolic groups of

the calix[4]arene core. This assignation is corroborated by the

study of the acid–base properties of calix[4]arene 6 in solution,

which shows that the latter possesses a site able to be easily

deprotonated with a pK_avalue of 4.6. The relatively low value

of pK_a1relative to that of an isolated phenolic function is indi-

cative of strong cooperation between all the phenolic groups

of the calix[4]arene crown by means of hydrogen bonding,

which stabilizes the negative charge of the phenolate moiety.

The higher pK_a1values for 5a and 5b than calix[4]arene 6 are

related to the decreasing number of free hydroxyl groups on

the crown.^[28]

The protonation constants were determined from the refine-

ment of neutralization curves. b_11ꢀ2for FeL¹and b_11ꢀ3for FeL²

were determined from spectrophotometric titrations and were

used without further adjustment in the refinement of potentio-

metric titration. All the results are reported in Table 1. The cor-

responding species distribution curves are reported in

Figure 1.

Table 1. Logarithmic values of the overall protonation constants of the li-

gands (b_01h) and of the stability constant of the Fe^IIIcomplexes (b_m1h).^[a]

Logb_mlh

pK_a

ICL670 [L¹]^[b]

5a [L²]^[b]

5b [L³]^[b]

0 1ꢀ1

0 1ꢀ2

0 1ꢀ3

0 1ꢀ4

0 1ꢀ5

1 1ꢀ2

1 1ꢀ3

1 2ꢀ5

1 2ꢀ6

pK_a1

pK_a2

pK_a3

pK_a4

pK_a5

–

ꢀ5.56(5) 5.56

ꢀ16.55(7) 10.99

ꢀ29.53(7) 12.98

–

ꢀ6.67(2) 6.67

ꢀ9.88(3) 9.88

ꢀ21.11(3) 11.23

ꢀ33.21(3) 12.10

ꢀ46.38(6) 13.17

ꢀ59.85(4) 13.47

ꢀ2.35(8)^[c]

ꢀ5.08(6)

ꢀ17.12(2) 10.45

ꢀ29.63(2) 12.51

–

ꢀ1.06(9)^[c]

ꢀ6.48(9)

ꢀ1.74(1)^[c]

ꢀ13.72(9)

ꢀ20.35(8)

–

ꢀ19.23(8)

–

[a] The calculated uncertainties for logb_m1hwere determined on the basis

of the standard deviation. Values in parentheses refer to estimated stan-

dard deviations for the last significant digit. [b] Italic numbers correspond

to the pK_avalues. [c] Determined by potentiometric and spectrophoto-

metric titrations; I=0.1m (KCl); T=298 K, H₂O/DMSO media (v/v 30:70).

The complexation study of L¹(ICL670), L²(5a), and L³(5b)

with iron was then studied. In the case of Fe–L¹and Fe–L²sol-

utions, at the beginning of the titration (pHꢃ2.5) Fe^IIIis almost

The stoichiometry of the complexes was confirmed by ESI-

MS analysis of Fe–L solutions (spectra are reported in the Sup-

porting Information). For the Fe^III–L¹solution the spectrum re-

corded at pH 3 in the positive mode shows only 1:1 complex

species detected as proton adducts at m/z 505

[Fe+L¹+DMSOꢀ2H]⁺, 523 [Fe+L¹+DMSO+H₂Oꢀ2H]⁺, and

583 [Fe+L¹+2DMSOꢀ2H]⁺, which is compatible with the ex-

completely complexed as FeL¹H and FeL²H_ꢀ3. For these spe-

ꢀ2

cies the formation constant was calculated on the basis of

spectrophotometric titration monitored in the pH range 1 to

6.5 (see Figure SI1 in the Supporting Information). These data

were then incorporated in the refinements of potentiometric

curves to determine the formation constant of the whole spe-

cies. In the pH range 1–3, complexation reactions induce a pro-

gressive change in the solution coloration from yellow to

violet. In this pH range, a change in the solution absorbance

induced by a pH increase reflects a single equilibrium between

clusive formation of FeL¹H . The spectrum recorded at pH 9 in

ꢀ2

the negative mode corroborates the addition of a second

ligand to the metal center and the formation of FeL¹H de-

2

ꢀ6

tected at m/z 798 [Fe+2L¹ꢀ4H]^ꢀ. For Fe–L²solutions, the ESI-

MS analysis shows a similar complexation scheme to the Fe–L¹

system. The complex species detected at pH 4 at m/z 998.2

the free Fe^IIIion and the 1:1 species (corresponding to FeL¹H ,

ꢀ2

FeL²H ). The electronic spectra of 1:1 species show similar fea-

[Fe+L²+DMSOꢀ2H]⁺correspond to the formation of FeL²H

,

ꢀ3

tures and are characterized by a single band centered at

whereas those detected at pH 9 at m/z 1787.5 [Fe+2L²ꢀ2H]⁺

510 nm and

a

molar extinction coefficient around

are ascribed to FeL²H (Figure 1b). In the case of Fe–L³solu-

2

ꢀ6

3200 mol^ꢀ1Lcm^ꢀ1. This transition, corresponding mainly to

a charge-transfer transition between the metal center and the

deprotonated phenolic groups, is indicative of the binding of

Fe^IIIto ICL670 moieties. Above pH 4, the solutions turn orange,

which results in the appearance of a new band at 400 nm. In

this pH range, the spectral characteristics of the solution do

not vary with the Fe^IIIconcentration, even when the amount of

iron increases, thereby leading to a decrease of the [L]/[M]

ratio below a value of 2. This indicates that the spectrum

modifications of Fe^III–L solutions cannot be ascribed to the for-

mation of 1:2 species as postulated by Steinhauser et al.^[23]This

makes the spectrum more difficult to interpret and does not

allow the calculation of stability constants for 1:2 species.

tions, only 1:1 species have been detected at m/z 1384

[Fe+L³+Naꢀ3H]⁺and 1406 [Fe+L³+2Naꢀ4H]⁺, which corre-

spond to the formation of FeL³H

.

ꢀ3

In designing iron chelators for clinical applications, metal se-

lectivity and ligand–metal complex stability are of paramount

importance. The most suitable way to compare the Fe^IIIchelat-

ing ability between ligands 5a, 5b, and ICL670 is to determine

pFe, since, unlike stability constants, pFe takes into account

the effects of ligand basicity, ligand protonation, and metal hy-

drolysis as well as differences in metal–ligand stoichiometries.

pFe is defined as the negative logarithm of the free Fe^IIIcon-

centration in solution under defined conditions. The pFe was

derived from ligand protonation and Fe-complex formation

constants. For clinically relevant conditions, pFe values are typ-

1004

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Cone ICL670-Substituted Calix[4]arenes

which validates our calculation procedure for water-insoluble

systems. The pFe values determined in this study for ICL670-

based calix[4]arene ligands (pFe>20) met the criteria for appli-

cation as iron chelation therapy agents.^[30]The stability of [FeL]

at physiological pH decreases in the order ICL670<5b<5a.

The higher Fe-chelating ability at physiological pH of ligand 5a

relative to 5b is probably due to the coordination of two

ICL670 moieties, which leads to a more stable bis-tridentate

structure similar to the one published in the case of ICL670.^[23]

Force-field development for molecular dynamics simulations

of the iron complexes

Efforts to obtain crystalline samples of the ferric complexes for

X-ray diffraction experiments have been unsuccessful to date.

Consequently, it was decided that MD studies would be per-

formed to provide further insight into both of the ferric com-

plex structures. In this study, the four phenolic ICL670 groups

(corresponding to pK_a2to pK_a5) of the Fe^III–calix[4]arene 5a and

5b complexes were deprotonated, whereas the phenolic

groups of the calix[4]arene crown (pK_a1) were conserved since

they are not involved in the iron complexation. The resulting

total charge of each complex is negative (Fe^III–4H=ꢀ1). Conse-

quently, three molecular complexes have been studied by

means of quantum chemistry (QC) and/or MD methods: the

complex composed of two ICL670 molecules and Fe^III, abbrevi-

ated as [Fe–ICL670]^ꢀ; the complex composed of two 5a mole-

cules and Fe^III, abbreviated as [Fe–5a]^ꢀ; and the complex com-

posed of one 5b molecule and Fe^III, abbreviated as [Fe–5b]^ꢀ.

The following approach was used:

1) The complexation sites in 5a and 5b were assumed to

adopt a similar conformation to that observed in the octa-

hedral [Fe–ICL670]^ꢀcomplex, therefore the [Fe–5a]^ꢀand

[Fe–5b]^ꢀmolecular systems were built accordingly.

2) [Fe–ICL670]^ꢀwith a covalently bound Fe^IIIwas used for pa-

rameterization of iron-related force-field parameters.

3) MD simulations of complexes 5a and 5b with bound and

nonbound iron were performed in explicit water.

The geometry of [Fe–ICL670]^ꢀwas taken from the Cam-

bridge Structural Database (CSD)^[31]under the SAJFAL refer-

ence.^[23]In this study, two empirical force fields for the Fe^III-

based complexes, namely, “q4md-FeF” and “q4md-FeB”, were

developed in association with the Amber99SB force field,^[32]

which correspond to approaches in which the iron is nonbond-

ed and bonded, respectively. Atomic charges were derived at

the B3LYP/6-31G* level of theory using both the RESP and ESP

methods using the latest version of the R.E.D. IV program avail-

able through “R.E.D. Server Development”^[33](see the Support-

ing Information for additional details about the charge deriva-

tion procedures). ESP and RESP charge values embedded in

force-field libraries, and DFT-optimized geometries as well as

the molecular fragments required to build the Fe^IIIcomplexes

in this study were submitted to the R.E.DD.B. database. The

corresponding projects are available under the “F-88” (ESP

charges) and “F-89” (RESP charges) R.E.DD.B. codes.^[34]The

Figure 1. Distribution curves for the FeL systems (C_L=2.10^ꢀ3m; C_M=10^ꢀ3m):

a) FeL¹, b) FeL², c) FeL³.

ically calculated in pure water at pH 7.45 for total ligand and

Fe concentrations equal to 10^ꢀ5and 10^ꢀ6m, respectively. Under

our conditions, taking into account the extended pH range

due to DMSO, the free Fe^IIIconcentration was calculated at

pH 9.16 (see the Experimental Section). The pFe values are

23.12, 26.44, and 23.23 for Fe–ICL670, Fe–5a, and Fe–5b, re-

spectively. The ICL670 pFe value determined in this study is

close to the one reported in the literature (pFe=23.5),^[29]

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1005

P. Sonnet et al.

method developed by Seminario^[35]was used to extract miss-

ing bond and angle force constants.

Complex [Fe–ICL670]^ꢀ

We performed 50 ns MD simulations using the q4md-FeB and

q4md-FeF force fields. In both cases, the molecular system re-

mained quite rigid with an RMSD mean value of (0.84ꢁ0.17)

and (0.86ꢁ0.22) ꢂ for q4md-FeB and q4md-FeF, respectively,

using the heavy atoms only and with the crystallographic

structure taken as the reference. These values indicate that

both force fields are suitable to study the Fe^III–5a and –5b

complexes.

The [Fe–5a]^ꢀand [Fe–5b]^ꢀsystems

Owing to the lack of any experimental three-dimensional struc-

ture of 5a and/or 5b complexed with Fe^III, preliminary MD sim-

ulations were performed with the q4md-FeB force field to get

more insight into a consistent structure. Hence, for each com-

plex, we performed a series of MD simulations using different

conditions accounting for 1 ms of simulation consisting of 30

independent trajectories. Analyses of the different trajectories

for [Fe–5b]^ꢀindicated that, after a few picoseconds, the struc-

ture converges to a stable conformation. Superimposition of

the different structures obtained after each independent MD

run shows that they belong to the same family of conforma-

tion. A representative structure of this family in represented in

Figure 2a. RMSD analyses performed for the [Fe–5a]^ꢀsystem

with the q4md-FeB and q4md-FeF force fields indicate that,

contrary to the [Fe–5b]^ꢀsystem, [Fe–5a]^ꢀdoes not converge

towards a unique family of conformations (see Figure SI7 in

the Supporting Information). Indeed, the [Fe–5a]^ꢀcomplex is

composed of two calix[4]arene cavities, which, opposed to

those in [Fe–5b]^ꢀ, are not constrained and hence fluctuate

around the Fe(ICL670)₂center. The superimposition of 30 struc-

tures randomly extracted from six 50 ns MD simulations is rep-

resented in Figure 2b. The Fe(ICL670)₂center fluctuates a bit

more than that in the [Fe–5b]^ꢀsystem but remains neverthe-

less rigid relative to the calix[4]arene part of the complex.

Many stable structures of the [Fe–5a]^ꢀsystem are observed

because the two calix[4]arene cavities can adopt different

stable positions and yet share a similar Fe(ICL670)₂geometry.

The nonbonded model demonstrated a robust performance,

as no disruption of the coordination center and no significant

conformational changes were observed throughout the simula-

tion, neither for [Fe–5b]^ꢀnor for [Fe–5a]^ꢀ. The obtained struc-

tures globally remained in the same conformation, which

makes us confident in the reliability of the proposed [Fe–5b]^ꢀ

structure (its Cartesian coordinates are provided in the Sup-

porting Information). The [Fe–5b]^ꢀand [Fe–5a]^ꢀcomplexes

optimize their structure by forming stabilizing intramolecular

interactions between aromatic cycles of the ICL670 moiety and

those of the calix[4]arene(s).^[36]The calix[4]arene part of the

complex remains in a cone conformation throughout the simu-

lation, but is significantly distorted from its C₄symmetry. The

cone conformation, however, adopts an approximate C₂sym-

Figure 2. Representative structure of the a) [Fe–5b]^ꢀand b) [Fe–5a]^ꢀcom-

plex obtained after MD simulations using the q4md-FeB force field. Similar

geometries were obtained with the q4md-FeF force field after a B3LYP/6-

31G* geometry optimization.

metry. The rings bound to the ligands are more vertical with

respect to the cone axis. Relative to its crystallographic state,

the [Fe(ICL670)₂]^ꢀpart of the two calix[4]arene complexes is

more distorted as demonstrated in Figure 3. Because of smaller

constraints, the Fe(ICL670)₂center is able to adopt a conforma-

tion closer to its “native” state in the [Fe–5a]^ꢀcomplex.

In the [Fe–ICL670]^ꢀcomplex, aromatic intramolecular inter-

actions are weak and two sandwich interactions between the

two ICL670 fragments are observed. When grafted on the cal-

ix[4]arene scaffold, aromatic rings of the ICL670 moieties are

able to interact with the newly added phenolic rings to form

T-shaped aromatic interactions, thus stabilizing the entire

system. Even though one of the ICL670 fragments moves

around the FeꢀN pseudoaxis, the octahedral conformation is

kept as shown in Table SI2 (see the Supporting Information),

which compares the distances and angles associated with the

position of the Fe^IIIion. It has to be stressed that, independent-

ly of the system, the octahedral conformation is always kept

during any MD simulation performed with the q4md-FeF force

field.

1006

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ChemPlusChem 2012, 77, 1001 – 1016

Cone ICL670-Substituted Calix[4]arenes

Table 2. Biological effect of a 72 h treatment of HepaRG cells with ICL670

and calix[4]arenes 5a and 5b. SDH activity (MTT assay) expressed by IC₅₀

[mm] is given as a measurement of cell viability and LDH leakage in the

cell supernatant for compound concentrations of 10 and 100 mm was

used as an index of membrane damage (cytotoxicity). Data expressed as

a percentage of the control (absence of compound) are averaged over

three independent experiments.

Compound SDH, IC₅₀

SDH, IC₅₀

with Fe^III

20 mm

%LDH/control %LDH/control

without Fe^III[a]with Fe^III20 mm^[a]

without

Fe^III

5a

6ꢁ1

90ꢁ7

11ꢁ4

37ꢁ3

125ꢁ5

61ꢁ7

105ꢁ11

200ꢁ15

67 ꢁ6

187ꢁ5

5b

76ꢁ4

91ꢁ11

69ꢁ6

103ꢁ2

ICL670

87ꢁ7

80ꢁ5

88ꢁ1

Figure 3. Superimposition of the Fe(ICL670)₂moiety of the [Fe–5a]^ꢀstruc-

ture (blue) and of the [Fe–5b]^ꢀstructure (green) with the crystallographic

structure of [Fe–ICL670]^ꢀ(pink).

101ꢁ13

[a] Compound concentrations of 10 and 100 mm

Binding energies between Fe^IIIand the ligands were evaluat-

ed using the MM-GBSA method^[37](see Table SI3 in the Sup-

Ligand 5a and 5b effects on angiogenesis and on HUVEC

and MDA-MB-231 cell proliferation

porting Information). The binding energy DH(GB_tot

: total

energy from generalized Born implicit solvation) favors [Fe–

5a]^ꢀ, which is in agreement both with experimental results

and with the better flexibility of this molecular system to rear-

range around the ion binding site.

ICL670, 5a, and 5b (10^ꢀ8to 10^ꢀ5m) angiogenesis activity was

evaluated by testing human umbilical vein endothelial cell

(HUVEC) migration and tube capillary formation. ICL670, 5a,

and 5b (10^ꢀ8to 10^ꢀ5m) effects on HUVEC and MDA-MB-231

cell survival and proliferation were determined by using the

BrdU assay as described in the Experimental Section. The rela-

tive cell proliferation and survival in the presence of ICL670,

5a, or 5b was normalized to the untreated controls after back-

ground subtraction. No statistical differences were found be-

tween the control and ICL670 for HUVEC or MDA-MB231 cell

proliferation and survival. It was found that, after 48 h of treat-

ment, 5a at 10^ꢀ7to 10^ꢀ5m decreased HUVEC survival signifi-

cantly by 28% for 10^ꢀ7m (p: statistical significance from Stu-

dent’s test<0.05), 56% for 10^ꢀ6m (p<0.05), and 65% for

10^ꢀ5m (p<0.001) versus the control (data shown in the Sup-

porting Information). For MDA-MB-231 survival, 5a at 10^ꢀ6to

Antiproliferative effects in the human hepatocarcinoma

HepaRG cell cultures

In the proliferating HepaRG cells, a dose-dependent decrease

of the cell viability, measured by the succinate dehydrogenase

(SDH) activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-

um bromide (MTT) assay), was observed after a 72 h cell treat-

ment in the presence of increasing concentrations of the L¹, L²,

and L³ligands (see Figure SI8 in the Supporting Information, 0

to 400 mm). As deduced from the dose–effects curves, com-

pound 5a is cytostatic for concentrations lower than 10 mm,

whereas it induces a cytotoxic effect, associated with mem-

brane damages and lactate dehydrogenase (LDH) leakage for

concentrations higher than 10 mm, which suggests a narrow

therapeutic index. These two effects were partly reversed in

the presence of exogenous iron. At 10 mm of compound 5a,

SDH and LDH values were 6 and 105% relative to the control

(Table 2), respectively. The decrease in cell viability for chelator

concentrations higher than 100 mm was associated with LDH

leakage in the cell supernatants and was probably due to the

cytotoxic effects of both the chelators and DMSO. In this range

of concentrations, the addition of 20 mm of exogenous Fe^IIIwas

ineffective in reversing the cytotoxic effect of the various com-

pounds. Experiments of membrane diffusion were carried out

on HepaRG cells that had been incubated with 5a and 5b.

After cellular lysis, the two ligands were found in the intracellu-

lar medium by LC–MS analyses. These antiproliferative-effect

results are consistent with the fact that 5a possesses a better

antiproliferative active profile due to its higher iron-chelating

abilities and lipophilicity than those of ICL670 and 5b.

10^ꢀ5m decreased cell survival significantly by 51% for 10^ꢀ6

m

(p<0.001) and 58% for 10^ꢀ5m (p<0.001) versus the control.

Ligand 5a at 10^ꢀ7to 10^ꢀ5m reduced the HUVEC proliferation

significantly by 46% for 10^ꢀ7m (p<0.05), 51% for 10^ꢀ6m (p<

0.001), and 74% for 10^ꢀ5m (p<0.001) versus the control. For

the MDA-MB-231 cell proliferation, 5a showed close results:

from 10^ꢀ7to 10^ꢀ5m cell proliferation decreased by 33 (p<

0.01), 62 (p<0.001), and 73% (p<0.001), respectively, versus

the control. ICL670 and 5b did not show any effect on HUVEC

and MDA-MB-231 cell survival and proliferation. ICL670, 5a,

and 5b effects on HUVEC migration have been evaluated

using the wound-healing assay. As shown in Figure 4, the mi-

gration capacity of HUVECs was examined by counting cells

that migrated through the wound after 16 h of treatment.

Ligand 5a at 10^ꢀ6to 10^ꢀ5m reduced HUVEC migration signifi-

cantly by 51 (p<0.01) and 59% (p<0.01) versus the control,

whereas ICL670 and 5b did not show any effect on HUVEC mi-

gration versus the control.

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1007

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Figure 4. Effects of ICL670, 5a and 5b on HUVEC migration. 16 h after the wound was performed, cell migration was reduced by treatment with 5a at 10^ꢀ5

and 10^ꢀ6m but not by treatment with ICL670 or 5b versus the control. Two controls were used: genistein (Gen) at 30 mgmL^ꢀ1as an inhibitor of HUVEC migra-

tion and EGF (50 ngmL^ꢀ1) as an activator of HUVEC migration (**p<0.01, ***p<0.001 versus the control).

1008

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Cone ICL670-Substituted Calix[4]arenes

with a UV lamp (254 nm) and phosphomolybdic acid in EtOH. Melt-

ing points were determined on a Stuart SMP3 apparatus and were

reported uncorrected. Infrared measurements were performed on

an FT/IR-4200 Jasco fitted with an ATR-golden gate allowing analy-

To examine whether ICL670, 5a, or 5b can induce morpho-

genetic changes resembling capillary-like structure formation,

HUVECs were plated on Matrigel (Figure 5). After 16 h of treat-

ment, 5a at 10^ꢀ6to 10^ꢀ5m decreased the ability of HUVEC to

form capillary tubes significantly by 42 (p<0.01) and 48% (p<

0.01), respectively, versus the control. At the same concentra-

tions, ICL670 and 5b did not show any effect on the HUVEC

capillary tube formation versus the control. The most antian-

giogenic efficient molecule tested in this study is 5a, which

shows an inhibition of HUVEC and MDA-MB-231 survival and

proliferation, as well as an inhibition of HUVEC migration and

capillary-tube formation abilities.

1

sis of solids and liquids. H and ¹³C NMR spectra were recorded on

a Bruker AC600, 500, or 300 spectrometer. Chemical shifts (d) are

reported in parts per million and the signals are quoted as s (sin-

glet), brs (broad singlet), d (doublet), t (triplet), brt (broad triplet),

q (quartet), or m (multiplet). Coupling constant (J) values are given

in hertz. Signal assignment was made using HMBC, HSQC, COSY,

and NOESY experiments when necessary. Mass spectra and high-

resolution mass spectra (electrospray in positive mode, ESI+) were

recorded on a Waters Q-TOF Ultima apparatus. Elemental analyses

were performed on an elementary analyzer Flash EA 1112 series

Thermo Finnigan. All commercially available products were used

without further purification unless otherwise specified.

Conclusion

Iron(III)-chelating ligands 5a and 5b have been synthesized by

grafting one or two ICL670 moieties, respectively, onto a cal-

ix[4]arene core locked into a cone conformation. Ligand 5a is

more lipophilic than ICL670 or 5b with a logD value of 4.59.

The higher iron-chelating ability of the 5a ligand (pFe=26.4)

relative to that of 5b (pFe=23.2) is likely due to the coordina-

tion of two ICL670 moieties, which lead to a more stable bis-

tridentate structure similar to that published in the case of

ICL670. These results are in agreement with the binding ener-

gies extracted from MD trajectories, the trend of which is as

follows: [Fe–5a]^ꢀ<[Fe–5b]^ꢀ<[Fe–ICL670]^ꢀ. The higher chelat-

ing ability of 5a along with its stability result from a better

flexibility of the ICL670 moieties and from stabilizing intramo-

lecular interactions with aromatic rings of the calix[4]arene.

Furthermore, two force-field models for the [Fe–ICL670]^ꢀ, [Fe–

5a]^ꢀ, and [Fe–5b]^ꢀmolecular systems, namely, “q4md-FeB”

and “q4md-FeF”, were developed for this study. Binding-

energy calculations performed on the calix[4]arene complexes

proved 5a to be the most stable by 7 kcalmol^ꢀ1, likely because

of the better flexibility and structural adaptation, hence dem-

onstrating that the grafting of two ICL670 moieties onto a cal-

ix[4]arene does not afford any cooperativity effect upon iron

binding.

Synthesis

25-{1-[2-(2-Phthalimidoethoxy)ethoxy]}-26,27,28-trihydroxyca-

lix[4]arene (8a): A solution of iodide 7 (8.14 g, 23.6 mmol) in anhy-

drous DMF (25 mL) was added at RT under an argon atmosphere

to a slurry of calix[4]arene 6 (5 g, 11.8 mmol) and CsF (2.32 g,

15.3 mmol) in anhydrous DMF (15 mL). The reaction was stirred at

508C for 10 days. After cooling to RT, CH₂Cl₂(300 mL) and water

(300 mL) were added. The organic phase was collected and

washed with water and brine. The organic phase was dried over

Na₂SO₄, filtered, and then evaporated over silica (50 mL). The crude

product was purified by chromatography (gradient elution CH₂Cl₂

to CH₂Cl₂/AcOEt 8:2). Some starting material was recovered

(950 mg) and the desired product 8a was obtained as a white

1

solid (4.7 g, 62%). M.p. 230–2328C; H NMR (500 MHz, CDCl₃): d=

3.27 (d, ²J(H,H)=13.7 Hz, 2H; C_qArꢀCH₂ꢀC_qAr), 3.36 (d, ²J(H,H)=

13.1 Hz, 2H; C_qArꢀCH₂ꢀC_qAr), 3.98–4.12 (m, 8H; CH₂ꢀOꢀCH₂ꢀCH₂ꢀN,

C_qArꢀCH₂ꢀC_qAr), 4.26–4.30 (m, 2H; CH₂ꢀOꢀC_qAr), 4.36 (d, ²J(H,H)=

13.0 Hz, 2H; C_qArꢀCH₂ꢀC_qAr), 6.59 (t, ³J(H,H)=7.5 Hz, 2H; CH_{Ar calix}),

6.65 (t, ³J(H,H)=7.5 Hz, 1H; CH_{Ar calix}), 6.83–6.88 (m, 3H; CH_{Ar calix}),

3

6.93–6.98 (m, 4H; CH_{Ar calix}), 7.05 (d, J(H,H)=7.5 Hz, 2H; CH_{Ar calix}),

7.50–7.55 (m, 2H; CHꢀCHꢀC_qArꢀC=O), 7.58–7.61 (m, 2H; CHꢀC_qAr

ꢀ

C=O), 9.05 (s, 2H; OH), 9.68 ppm (s, 1H; OH); ¹³C NMR (125 MHz,

CDCl₃): d=31.53 (2C), 32.23 (2C), 38.32 (1C), 69.12 (1C), 70.31

(1C), 75.57 (1C), 121.39 (2C), 122.01 (1C), 123.27 (2C), 126.48 (1C),

128.71 (2C), 128.86 (2C), 129.03 (6C), 129.13 (2C), 129.64 (2C),

132.23 (2C), 133.99 (2C), 135.10 (2C), 150.01 (1C), 150.60 (2C),

151.66 (1C), 168.77 ppm (2C); IR (ATR): n˜ =3317, 3171 (OH), 2920,

2873 (CH), 1764, 1701 cm^ꢀ1(C=O); MS: m/z: 664 [M+Na]⁺; HRMS:

m/z calcd for [C₄₀H₃₅NO₇Na]⁺: 664.2311; found: 664.2282.

The antiproliferative effects of these ligands on HepaRG cells

show that 5a is more active than ICL670 and 5b is less active.

The decrease in cell viability is reversed for 5a and ICL670

upon addition of 20 mm of exogenous Fe^III. Only 5a shows in-

teresting angiogenesis activity: at 10^ꢀ6to 10^ꢀ5m it reduces

HUVEC proliferation, migration, and capillary-tube formation

abilities. In addition, MDA-MB-231 viability and proliferation

was reduced by 5a at 10^ꢀ6to 10^ꢀ5m. These results indicate

that 5a, relative to ICL670 and 5b, presents the best antitu-

moral profile due to its higher iron-chelating ability and lipo-

philicity.

25,27-Bis{1-[2-(2-phthalimidoethoxy)ethoxy]}-26,28-dihydroxyca-

lix[4]arene (8b): A slurry of calix[4]arene 6 (10 g, 23.6 mmol), anhy-

drous K₂CO₃(6.5 g, 47.2 mmol), and iodide 7 (32.5 g, 94.4 mmol) in

dry CH₃CN (150 mL) was heated to reflux for 1 week. After removal

of CH₃CN, the residue was dissolved in CH₂Cl₂(250 mL). The organ-

ic phase was washed successively with water (30 mL), of 1m aque-

ous HCl (2ꢃ20 mL), and brine (20 mL). After drying over Na₂SO₄,

CH₂Cl₂was removed by rotary evaporation and the crude product

was purified by crystallization in CH₂Cl₂/MeOH (2:3). White crystals

1

were obtained (14.9 g, 74%). M.p. 199.5–2008C; H NMR (500 MHz,

CDCl₃): d=3.06 (d, ²J(H,H)=12.9 Hz, 4H; C_qArꢀCH₂ꢀC_qAr), 3.97 (d,

²J(H,H)=12.8 Hz, 4H; C_qArꢀCH₂ꢀC_qAr), 4.02–4.07 (m, 16H; CH₂ꢀN,

CH₂ꢀO), 6.47 (t, ³J(H,H)=7.4 Hz, 2H; CH_{Ar calix}), 6.65 (t, J=7.6 Hz,

2H; CH_{Ar calix}), 6.79 (d, ³J(H,H)=7.5 Hz, 4H; CH_{Ar calix}), 6.82 (d,

³J(H,H)=7.6 Hz, 4H; CH_{Ar calix}), 7.38–7.42 (m, 4H; CHꢀCHꢀC_qArꢀC=O),

7.47–7.51 (m, 4H; CHꢀC_qArꢀC=O), 7.88 ppm (s, 2H; OH); ¹³C NMR

Experimental Section

Chemicals and analysis

Column chromatography was performed on Kielselgel 60 (40–

63 mm) ASTM (Merck). Reactions were analyzed on precoated silica

gel 60 F254 plates (Merck) and the compounds were visualized

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P. Sonnet et al.

Figure 5. ICL670, 5a, and 5b effects on capillary-tube formation. The network length of the capillary-tube formation was quantified by using a CFM-S130T

Digital Imaging System. 16 h after HUVECs were seeded directly on the Matrigel, they were treated with 10^ꢀ6or 10^ꢀ5m ICL670, 5a, or 5b. Ligand 5a but nei-

ther 5b nor ICL670 impaired endothelial cell differentiation. Untreated cells were used as a control (Ct). Two controls were used: genistein (30 mgmL^ꢀ1) as an

inhibitor of capillary-tube formation, and EGF (50 ngmL^ꢀ1) as an activator (**p<0.01, ***p<0.001 versus the control).

1010

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ChemPlusChem 2012, 77, 1001 – 1016

Cone ICL670-Substituted Calix[4]arenes

(125 MHz, CDCl₃): d=31.43 (4C), 38.53 (2C), 68.98 (2C), 70.44 (2C),

75.66 (2C), 119.3 (2C), 123.12 (4C), 125.71 (2C), 128.54 (4C), 128.62

(4C), 129.14 (4C), 132.24 (4C), 133.89 (4C), 134.24 (4C), 151.88

(2C), 153.06 (2C), 168.89 ppm (4C); IR (ATR): n˜ =3320 (OH), 1773,

1709 cm^ꢀ1(C=O); MS: m/z: 881 [M+Na]⁺; HRMS: m/z calcd for

[C₅₂H₄₆N₂O₁₀Na]⁺: 881.3050; found: 881.3008.

rated by rotary evaporation. The crude product was purified by

chromatography.

25-{1-[2-(2-{4-[3,5-Bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]phe-

nyl}carbamoylethoxy)ethoxy]}-26,27,28-trihydroxycalix[4]arene

(5a): According to the general procedure this compound was pre-

pared from aminocalix[4]arene 9a (0.20 g, 0.391 mmol). The resi-

due was purified by flash chromatography (CH₂Cl₂/AcOEt 9:1)),

then by recrystallization in EtOH, to yield 5a as a beige solid

General procedure for the preparation of compounds 9a,b: A

35% aqueous solution of hydrazine (10 mmol per mmol of phthali-

midocalix[4]arene 8a,b) was added to a slurry of phthalimidoca-

lix[4]arene 8a,b in absolute EtOH (50 mL per mmol^ꢀ1of phthalimi-

docalix[4]arene 8a,b). The reaction was heated to reflux for 7 h.

Solvents were removed under reduced pressure. The residue was

stirred in ethyl acetate (50 mL per mmol). The resulting precipitate

was filtered and washed with small volumes of AcOEt. The com-

bined organic phases were washed successively with a 2m NaOH

aqueous solution, water, and brine. The organic phase was dried

over Na₂SO₄and concentrated by rotary evaporation. After stirring

in Et₂O, a white gum 8a,d solidified after several days in air.

1

(172 mg, 51%). M.p. 195–2008C; H NMR (600 MHz, CDCl₃): d=3.35

(d, ²J(H,H)=13.7 Hz, 2H; C_qArꢀCH₂ꢀC_qAr), 3.46 (d, ²J(H,H)=13.0 Hz,

2H; C_qArꢀCH₂ꢀC_qAr), 3.95–3.98 (m, 4H; OꢀCH₂ꢀCH₂ꢀN), 4.12 (d,

²J(H,H)=13.7 Hz, 2H; C_qArꢀCH₂ꢀC_qAr), 4.18–4.20 (m, 2H; CH₂ꢀO),

2

4.38–4.40 (m, 2H; CH₂ꢀOꢀC_qAr), 4.43 (d, J(H,H)=13.0 Hz, 2H; C_qAr

ꢀ

CH₂ꢀC_qAr), 6.62–6.70 (m, 4H; CH_{Ar ICL}+CH_{Ar calix}), 6.86 (d, ³J(H,H)=

3

6.9 Hz, 1H; CH_{Ar ICL}), 6.93 (t, J(H,H)=7.6 Hz, 1H; CH_{Ar calix}), 6.95 (d,

³J(H,H)=7.5 Hz, 2H; CH_{Ar calix}), 6.99–7.02 (m, 4H; CH_{Ar calix}), 7.08 (t,

³J(H,H)=7.7 Hz, 1H; CH_{Ar ICL}), 7.11–7.16 (m, 3H; CH_{Ar calyx}+CH_{Ar ICL}),

3

7.18 (d, J(H,H)=8.2 Hz, 1H; CH_{Ar ICL}), 7.21 (d, J(H,H)=8.4 Hz, 2H;

CH_{Ar ICL}), 7.37 (dt, J(H,H)=7.8 Hz, J(H,H)=1.5 Hz, 1H; CH_{Ar ICL}), 7.43

(dt, ³J(H,H)=7.8 Hz, ⁴J(H,H)=1.6 Hz, 1H; CH_{Ar ICL}), 7.47 (brt, 1H;

3

4

25-{1-[2-(2-Aminoethoxy)ethoxy]}-26,27,28-trihydroxycalix[4]ar-

ene (9a): According to the general procedure, this compound was

prepared from phthalimidocalix[4]arene 8a (2.7 g, 4.21 mmol). For

solubility reasons, AcOEt was replaced by hot CHCl₃. Compound

9a was obtained as a white solid (1.9 g, 88%). M.p. 195–1978C;

¹H NMR (500 MHz, CDCl₃): d=2.83 (brt, 2H; CH₂ꢀN), 3.37–3.41 (m,

3

NH); 7.83 (d, J(H,H)=8.3 Hz, 2H; CH_{Ar ICL}), 8.17 (dd, J(H,H)=7.8 Hz,

⁴J(H,H)=1.5 Hz, 1H; CH_{Ar ICL}), 9.41 (s, 2H; OH_calix), 9.72 (s, 1H; OH_ICL),

9.68 (s, 1H; OH_calix), 11.41 ppm (s, 1H; OH_ICL); ¹³C NMR (125 MHz,

CDCl₃): d=31.32 (2C), 31.77 (2C), 40.52 (1C), 69.81 (1C), 70.25

(1C), 75.48 (1C), 109.91 (1C), 113.23 (1C), 117.21 (1C), 118.40 (1C),

119.06 (1C), 119.91 (1C), 121.70 (2C), 122.00 (1C), 126.01 (2C),

126.47 (1C), 127.56 (1C), 127.66 (1C), 128.48, 128.51, 128.64,

128.67, 128.80, 128.84, 128.98, 129.01, 129.45, 131.85 (1C), 132.97

(1C), 134.43 (2C), 135.85 (1C), 139.97 (1C), 149.34 (1C), 149.91

(2C), 151.19 (1C), 152.07 (1C), 156.57 (1C), 158.08 (1C), 159.44

(1C), 166.32 ppm (1C); IR (ATR): n˜ =3277 (OH), 1461 cm^ꢀ1(C=O);

MS: m/z: 889 [M+Na]⁺; HRMS: m/z calcd for [C₅₃H₄₆N₄O₈Na]⁺:

889.3213; found: 889.3246; elemental analysis calcd (%) for

C₅₃H₄₆N₄O₈: C 73.43, H 5.35, N 6.46; found: C 73.32, H 5.37, N 6.49.

4H; C_qArꢀCH₂ꢀC_qAr), 3.64 (brt, 2H; OꢀCH₂ꢀCH₂ꢀN), 3.95 (brt, 2H;

2

CH₂ꢀO), 4.25 (brt, 2H; CH₂ꢀO), 4.28 (d, J(H,H)=13.4 Hz, 2H; C_qAr

ꢀ

CH₂ꢀC_qAr), 4.42 (d, ²J(H,H)=12.8 Hz, 2H; C_qArꢀCH₂ꢀC_qAr), 6.53 (brt,

1H; CH_Ar), 6.65 (t, ³J(H,H)=7.4 Hz, 2H; CH_Ar), 6.78 (t, ³J(H,H)=

7.2 Hz, 1H; CH_Ar), 6.95 (d, ³J(H,H)=7.3 Hz, 4H; CH_Ar), 7.01 (d,

3

³J(H,H)=7.2 Hz, 2H; CH_Ar), 7.06 ppm (d, J(H,H)=7.2 Hz, 2H; CH_Ar);

¹³C NMR (125 MHz, CDCl₃): d=31.61 (2C), 33.55 (2C), 41.64 (1C),

69.64 (1C), 72.76 (1C), 75.09 (1C), 120.16 (1C), 125.78 (1C), 128.56,

128.96, 129.20, 129.54, 129.86, 129.95, 134.08 (18C), 152.83 ppm

(4C); IR (ATR): n˜ =2917 cm^ꢀ1(CH); MS: m/z: 512 [M+H]⁺. HRMS:

m/z calcd for [C₃₂H₃₃NO₅Na]⁺: 534.2256; found: 534.2272.

25,27-Bis-{1-[2-(2-{4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-

phenyl}carbamoylethoxy)ethoxy]}-26,28-dihydroxycalix[4]arene

(5b): According to the general procedure, this compound was pre-

pared from aminocalix[4]arene 9b (0.10 g, 0.167 mmol). The resi-

due was purified by flash chromatography (CH₂Cl₂/MeOH 98:2),

then by recrystallization in EtOH, to yield 5b (80 mg; 37%) as

a beige solid. M.p. 1738C; ¹H NMR (600 MHz, CDCl₃): d=3.26 (d,

²J(H,H)=12.9 Hz, 4H; C_qArꢀCH₂ꢀC_qAr), 3.85–3.93 (m, 8H; OꢀCH₂ꢀ

CH₂ꢀN), 4.02–4.04 (m, 4H; CH₂ꢀO), 4.20–4.22 (m, 4H; CH₂ꢀOꢀC_qAr),

4.27 (d, ²J(H,H)=12.9 Hz, 4H; C_qArꢀCH₂ꢀC_qAr), 6.57 (t, ³J(H,H)=

25,27-Bis-{1-[2-(2-aminoethoxy)ethoxy]}-26,28-dihydroxycalix[4]-

arene (9b): According to the general procedure, this compound

was prepared from phthalimidocalix[4]arene 8b (1.5 g, 1.75 mmol)

to yield 9b as a white gum (1.03 g, 99%). M.p. 1048C; ¹H NMR

(500 MHz, CDCl₃): d=2.07 (brs, 4H; NH₂), 2.98 (brt, 4H; CH₂ꢀN),

3.38 (d, ²J(H,H)=13.0 Hz, 4H; C_qArꢀCH₂ꢀC_qAr), 3.73 (brt, 4H; Oꢀ

CH₂ꢀCH₂ꢀN), 4.01 (brt, 4H; CH₂ꢀO), 4.22 (brt, 4H; CH₂ꢀOꢀC_qAr),

4.43 (d, ²J(H,H)=13.0 Hz, 4H; C_qArꢀCH₂ꢀC_qAr), 6.68 (t, ³J(H,H)=

7.4 Hz, 2H; CH_Ar), 6.76 (t, ³J(H,H)=7.5 Hz, 2H; CH_Ar), 6.92 (d,

3

4

3

³J(H,H)=7.5 Hz, 4H; CH_Ar), 7.08 ppm (d, J(H,H)=7.4 Hz, 4H; CH_Ar);

7.5 Hz, 2H; CH_{Ar calix}), 6.64 (td, J(H,H)=7.6 Hz, J(H,H)=0.9 Hz, 2H;

CH_{Ar ICL}), 6.78 (t, ³J(H,H)=7.6 Hz, 2H; CH_{Ar calix}), 6.88 (dd, ³J(H,H)=

¹³C NMR (125 MHz, CDCl₃): d=31.58 (4C), 42.39 (2C), 70.11 (2C),

73.92 (2C), 75.86 (2C), 119.51 (2C), 125.81 (2C), 128.54 (4C), 128.88

(4C), 129.38 (4C), 133.84 (4C), 152.20 (2C), 153.45 ppm (2C); IR

(ATR): n˜ =3359 (OH, NH), 2922, 2868 cm^ꢀ1(CH); MS: m/z: 599

[M+H]⁺; HRMS: m/z calcd for [C₃₆H₄₃N₂O₆]⁺: 599.3121; found:

599.3147.

4

8.0 Hz, J(H,H)=1.4 Hz, 2H; CH_{Ar ICL}), 6.93 (m, 8H; CH_{Ar ICL}+CH_{Ar calix}),

7.05 (t, ³J(H,H)=7.1 Hz, 2H; CH_{Ar ICL}), 7.09 (d, ³J(H,H)=8.3 Hz, 2H;

CH_{Ar ICL}), 7.14 (d, ³J(H,H)=8.5 Hz, 2H; CH_{Ar ICL}), 7.21 (d, ³J(H,H)=

8.5 Hz, 4H; CH_{Ar ICL}), 7.35 (t, ³J(H,H)=7.1 Hz, 2H; CH_{Ar ICL}), 7.40 (t,

3

³J(H,H)=7.8 Hz, 2H; CH_{Ar ICL}), 7.74 (brt, 2H; NH), 7.81 (d, J(H,H)=

3

4

8.4 Hz, 4H; CH_{Ar ICL}), 8.15 (dd, J(H,H)=7.8 Hz, J(H,H)=1.5 Hz, 2H;

CH_{Ar ICL}), 8.24 (s, 2H; OH_calix), 9.69 (s, 2H; OH_ICL), 11.26 ppm (s, 2H;

OH_ICL); ¹³C NMR (CDCl₃, 125 MHz): d=31.10 (4C), 40.53 (2C), 69.95

(2C), 70.20 (2C), 75.47 (2C), 110.10 (2C), 113.26 (2C), 117.22 (2C),

118.40 (2C), 119.07 (2C), 119.95 (2C), 120.66 (2C), 125.86 (2C),

125.99 (4C), 127.67 (2C), 127.70 (2C), 128.66, 128.76, 128.98,

129.15, 131.87 (2C), 132.98 (2C), 133.64 (4C), 135.56 (2C), 140.13

(2C), 151.69 (2C), 151.73 (2C), 152.10 (2C), 156.59 (2C), 158.04

(2C), 159.54 (2C), 165.58 ppm (2C); IR (ATR): n˜: 3296 (OH),

1459 cm^ꢀ1(C=O); MS: m/z: 1331 [M+Na]⁺; HRMS: m/z calcd for

[C₇₈H₆₈N₈O₁₂Na]⁺: 1331.4854; found: 1331.4827; elemental analysis

General coupling procedure for preparation of compounds

5a,b: A solution of carboxylic acid (1.1 equiv. per equiv. of NH₂

group), HOBt (1.1 equiv. per equiv. of NH₂group), and EDCI ( equiv.

per equiv. of NH₂group) in CH₂Cl₂(10 mL per mmol of aminoca-

lix[4]arene) was stirred at RT for 15 min. The aminocalix[4]arene

9a,b was added and the reaction was stirred at RT overnight. The

reaction was diluted with CH₂Cl₂(100 mL per mmol of aminoca-

lix[4]arene 9a,b) and this organic phase was washed successively

with 1m aqueous HCl, saturated aqueous NaHCO₃, water, and

brine. The organic phase was dried over Na₂SO₄and then evapo-

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1011

P. Sonnet et al.

calcd (%) for C₇₈H₆₈N₈O₁₂: C 71.55, H 5.23, N 8.56; found: C 71.68, H

5.20, N 8.59.

was kept constant (I=0.1) by addition of potassium chloride (Pro-

labo) of the highest purity (>99%). The titrating solutions of car-

bonate-free base KOH and hydrochloric acid 0.1m were prepared

from standardized 1m solutions (Prolabo). All solutions were pre-

pared with glass-distilled, deionized water and were degassed by

argon saturation to remove all dissolved CO₂. Protometric titrations

were carried out with an automatic titrator composed of a micro-

processor burette (Metrohm Dosimat 665), a glass electrode (met-

rohm 6 204100) with an incorporated Ag/AgCl reference (filled

with KCl 3 moldm^ꢀ3in H₂O/DMSO 30:70 (v/v)) and a pH meter

(Metrohm 713) connected to a computer. The combined electrode

has a very low alkaline error. The titration was fully automated. All

measurements were performed within a thermoregulated cell at

258C under an argon stream to avoid the dissolution of carbon di-

oxide. For a classical titration, a total of 120 to 150 points (volume

of titrant, pH) was recorded. All equilibrium measurements were

carried out in 5.00 mL sample volumes with magnetic stirring. The

ionic strength was adjusted to 0.1 with potassium chloride solu-

tion. An HCl solution at exactly 10^ꢀ2m was used to calibrate the

electrode. The electrode slope was checked by titration with an

HCl solution, and no correction was necessary in the pH range of

2–15.3. The titrant, a carbonate-free KOH solution (Normadose),

was standardized against a 0.05m potassium hydrogen phthalate

solution by pH potentiometry. The ionic product of water was de-

termined under these conditions (pK_w=17.16) and was used in the

calculations. For the determination of Fe^IIIformation constants,

metal/ligand mixtures of 1:3, 1:2, and 1:1 molar ratios were stud-

ied. For spectrophotometric titrations, Fe^III–ligand solutions were

prepared with an excess amount of hydrochloric acid and were ti-

trated with potassium hydroxide until pH 6.5. UV-visible spectra

were recorded after each base addition at (25.0ꢁ0.1)8C on a Cary

500 spectrophotometer equipped with a thermostated cell carrier.

Protonation and formation constants were calculated from poten-

tiometric and spectrophotometric data by the HYPERQUAD^[27]soft-

ware. The computer program HYSS^[27]was used to obtain the spe-

cies distribution curves.

Reagents

Collagenase was purchased from Roche Diagnostic (Meylan,

France); and M199 medium and phosphate-buffered saline (PBS)

from Eurobio (Les Ulis, France). Endothelial cell grow supplement

(ECGS) and endothelial growth factor (EGF) were from Promo Cell

GmbH (Heidelberg, Germany); penicillin–streptomycin, gelatin 2%,

and genistein were from Sigma (Saint-Quentin Fallavier, France);

fetal bovine serum (FBS) was purchased from AbCys (Paris, France),

and Dulbecco’s modified Eagle’s medium (DMEM) low glucose was

from Invitrogen (Cergy Pontoise, France). The BrdU kit was from

Chemikon (Paris, France). Matrigel (Basement Membrane Matrix)

was purchased from Becton Dickinson (Le pont de Claix, France).

Partition coefficients

In this study, the relative logD values were assessed at pH 7 by the

micro-HPLC method. Determinations were performed with a chro-

matographic apparatus (Spectra Series, San Jose, USA) equipped

with a model P1000XR pump and a model SCM 1000 vacuum

membrane degasser, a model UV 150 ultraviolet detector (l=

280 nm), and a ChromJet data module integrator (ThermoFinnigan,

San Jose, USA). The reversed-phase column used was an Equisorb

C8 (4.6ꢃ150 mm; 5 mm particle size, C.I.L. Cluzeau-France) with

a mobile phase consisting of acetonitrile with trifluoroacetic acid

(1%, v/v)–water with trifluoroacetic acid (1%, v/v): 75:25, v/v (5b)

and 65:35, v/v (5a). The compounds were partitioned between n-

octanol (HPLC grade) and phosphate buffer (pH 7). Octanol was

presaturated with the adequate phosphate buffer (1%), and con-

versely. An amount of each compound (1 mg) was dissolved in an

adequate volume of methanol to produce 1 mgmL^ꢀ1stock solu-

tions. Then, an appropriate aliquot of these methanolic solutions

was dissolved in buffer to obtain final concentrations of

50 mgmL^ꢀ1. Under the above-described chromatographic condi-

tions, the aqueous phase (50 mL) was injected into the chromato-

graph, leading to the determination of a peak area before parti-

tioning (W₀). In screw-capped tubes, the aqueous phase (V_aq;

1000 mL) was then added to n-octanol (V_oct; 10 mL). The mixture

was shaken by mechanical rotation for 20 min, followed by centri-

fugation at 3000 rpm for 10 min. An amount of the lower phase

(20 mL) was injected into the chromatograph column. This led to

the determination of a peak area after partitioning (W₁). For each

compound, the logD value was calculated by using the formula

[Eq. (3)]:

ESI-MS

All experiments (MS and MS/MS) were performed on a hybrid

tandem quadrupole/time-of-flight (Q-TOF) instrument, equipped

with a pneumatically assisted electrospray (Z-spray) ion source (Mi-

cromass, Manchester, UK) operated in positive mode. The electro-

spray potential was set to 3 kV in positive- and negative-ion mode

and the extraction cone voltage was usually varied between 30

and 90 V to obtain optimized mass spectra. The FeL solutions were

prepared in similar conditions to those set out for the potentio-

metric study and were introduced using a syringe pump with

a flow rate of 5 mLmin^ꢀ1. The stoichiometries of the molecular as-

sociations were determined in accordance with the greater isotopic

peak (corresponding to species with ⁵⁶Fe) and further checked by

simulating the different parts of the spectrum with the Isopro 3.0

software.^[38]The differences between the experimental and calcu-

lated m/z values were less than or equal to 0.1 unit.

log D ¼ log ½ðW₀ꢀW₁ÞV_aq=W₁V_oct

ꢂ

ð3Þ

Protonation and iron-binding constants in solution

All commercial reagents used were of the highest purity (>99%)

and were used without further purification. All stock solutions

were used fresh. Because of the poor solubility of the chelators in

aqueous media, all experiments were monitored in H₂O/DMSO

media (v/v=30:70; x_DMSO=0.5). Ligand solutions were prepared in

pu_ꢀre₅DMSO. These solutions were then diluted to the 10^ꢀ4to

10 m range. FeCl₃solutions were prepared at a concentration of

10^ꢀ2m in H₂O media in the presence of an excess amount of hy-

drochloric acid (0.5m). Their concentrations were determined by

ethylenediaminetetraacetic acid (EDTA) titration. The ionic strength

pFe determination

For ligands that have a low water solubility, the classical methodol-

ogy to determine pFe consists of determining the overall forma-

tion constant (logb) values in a water/solvent mixture by varying

the molar fraction of the used solvent (x_solvent). Then, the values are

extrapolated in a pure H₂O medium by taking into account a linear

dependence approximation between logb and x_solvent

[23]

.

The com-

1012

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ChemPlusChem 2012, 77, 1001 – 1016

Cone ICL670-Substituted Calix[4]arenes

plex and ligand insolubility in a water/DMSO medium for x_DMSO

<

Molecular dynamics simulations: The SANDER module of the

AMBER10 program suite was used to perform MD simulations on

the aforementioned complexes.^[42]The systems were solvated in

a truncated octahedral box with a buffer distance of 10.0 ꢂ along

with one sodium counterion to neutralize the complex net charge.

The parameters used for water were taken from the TIP3P

model.^[43]After minimization, the systems were brought to the

target temperature by increasing the temperature over periods of

25 ps followed by a run of 200 ps to relax and equilibrate the

system. Classical MD simulations of 50 ns were then performed

using the NPT ensemble at a pressure of 1 atm and a temperature

of 300 K. The weak coupling algorithm^[44]was used to regulate the

temperature and pressure. The temperature was maintained close

to the intended value by weak coupling to an external tempera-

ture bath with a relaxation time of 2 ps and the pressure to an ex-

ternal pressure bath of 1 atm with a coupling constant of 2 ps. The

SHAKE algorithm^[45]was used to constrain CꢀH bonds, and a time

step of 2 fs was used to integrate the equations of motion. Period-

ic boundary conditions were imposed during the simulation. A dis-

tance cutoff of 9.0 ꢂ was applied to nonbonded interactions and

the PME method^[46]was used to compute long-range interactions.

Configurations of the systems were stored at intervals of 1 ps.

Analyses of the trajectories were performed using the PTRAJ

module available in AmberTools 1.4. Visualization of the trajectories

was achieved by using the VMD package.^[47]The Hornak et al.^[32]

all-atom model force field parameters (i.e., Amber99SB) were used

to model the [Fe–ICL670]^ꢀ, [Fe–5a]^ꢀ, and [Fe–5b]^ꢀsystems when

available, and those of GAFF^[48]otherwise. Additional parameters

relevant to iron, not available within Amber99SB or GAFF, are re-

quired. The van der Waals parameters for iron were taken from

Ref. [49].

0.2 renders such a methodology irrelevant to the pFe determina-

tion in the case of Fe–5a and Fe–5b systems. This problem can be

overcome by performing pFe calculations with data collected in

a H₂O/DMSO medium (x_DMSO=0.5) at a pH corresponding to the

same [OH^ꢀ]/[H₃O⁺] ratio as that in pure water (pH 7.45). For x_DMSO

equal to 0.5, the pH scale is extended from 0 to 17.2, and a neutral

solution has a pH value of 8.6. Consequently, a pH of 7.45 in water

corresponds to 9.16 in a H₂O/DMSO medium (x_DMSO=0.5). This

value was experimentally confirmed by pH measurements of

a phosphate buffer solution prepared with the same H₂PO₄

ꢀ

/

HPO₄^2ꢀratio in water and H₂O/DMSO medium (x_DMSO=0.5). pFe cal-

culations were monitored by using the HYSS software.^[27]The pro-

tonation constants were determined from nonlinear least-squares

refinements of potentiometric titration in H₂O/DMSO (x_DMSO=0.5)

media using the Hyperquad software.^[27]Iron-complex formation

was studied by spectrophotometric and potentiometric titrations

in the 1–13 pH range. The stability constants were calculated using

Hyperquad refinements. ESIMS analyses were also used to confirm

the stoichiometry of the major complexes.

Molecular modeling

Models: The initial geometry for [Fe–ICL670]^ꢀwas taken from the

Cambridge structural database (CSD)^[31]under the SAJFAL refer-

ence.^[25]The LEaP program from the AmberTools 1.4 distribution

was used to build the topologies of the Fe^III–calix[4]arene systems.

QC calculations: Calculations were performed using the Gaussi-

an 03 (revision E.01) program.^[39]The [Fe–ICL670]^ꢀ, [Fe–5a]^ꢀ, and

[Fe–5b]^ꢀstructures were optimized with the hybrid B3LYP^[40]DFT

method using the 6-31G* basis set. According to Steinhauser

et al.,^[23]we assumed the iron complex to be in a high-spin state.

This was confirmed by DFT calculations as well, as the sextet state

was found to be the most stable relative to the doublet and quar-

tet. Frequencies were calculated after geometry optimization at

the same level of theory to confirm that the optimized structures

were true energy minima and to provide the Hessian matrix neces-

sary for developing force-field parameters for iron by means of the

FUERZA procedure.^[35]

Chelator solutions

Biological properties of the various calix[4]arenes were compared

to tridentate hydroxyphenyltriazole ICL670 (Exjade from Novartis

Pharma).^[50]Stock solutions of each molecule (10 mm) were pre-

pared in DMSO. The 100 mm chelator concentration corresponds to

an addition in the culture medium of 1% DMSO, which was pre-

liminary verified to be inefficient on HepaRG cell viability. However,

a low but significant cytotoxicity (20%) was observed with 2%

DMSO. This percentage was reached for chelator concentrations of

200 mm. The solubility of each derivative in the culture medium in

the concentration range of 0–200 mm was preliminary verified by

turbidity measurements. Two controls have been performed for

each experiment: one with the standard culture medium, and the

other with a culture medium supplemented with DMSO at the

concentration used for ICL670 and calix[4]arenes tests.

Charge derivation and force-field topology database building:

The geometries of the different building blocks constituting the

ICL670- and calix[4]arenes–ICL670-based Fe^IIIchelators were opti-

mized by using the B3LYP/6-31G* theory level with the 2003 (ver-

sion E.01) program.^[39]For each building block the lowest minimum

found after a conformational search was selected except for 2-me-

thoxyethanol, for which two conformations were chosen. The MEP

computation involved the Connolly surface algorithm, a radius of

1.8 ꢂ for iron, and the B3LYP/6-31G* theory level implemented in

the Gaussian 2003 (version E.01) program. As defined by Steinhaus-

er et al. the high-spin state (S=5/2) of Fe^IIIwas considered in DFT

calculations.^[23]For each building block two molecular orientations

based on the rigid-body reorientation algorithm implemented in

the R.E.D. program were involved in the MEP computation, which

ensured the reproducibility of the charge values.^[33]The molecular

fragments required for MD simulations were constructed by setting

specific intra- and intermolecular charge constraints between fully

characterized connecting groups during the charge fitting step

(Figure SI6 in the Supporting Information). An additional intramo-

lecular charge constraint of +3.0 e was used to define iron as an

individual bound ion. ESP charge fitting was carried out by using

a stand-alone version of the RESP program and by following

a single-stage fitting procedure.^[41]

Solubility of the Fe^IIIchelators

Solubility of the new calix[4]arenes were estimated in PBS solutions

and in cell culture media containing 10% FCS. Solutions for the

various compounds (200, 100, 50, 25 mm) were prepared in 96-well

microplates by diluting the 2.5 mm stock solutions in DMSO in PBS

(200 mL) and a culture medium. The absorbance (turbidity) of the

solutions was measured at 590 nm, out of the absorption range of

the chromophores.

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1013

P. Sonnet et al.

curves were fitted as the sum of two sigmoids (double four-param-

eter fit). Percentages of HepaRG cells involved in each viability re-

sponse were deduced from the A_Cmin, A_Cmax, and IC₅₀values of each

sigmoid, which were obtained from these fits. Statistical analysis

was performed using the nonparametric Mann–Whitney test. The

significant level was set to 0.01.

HepaRG cell cultures

HepaRG cells were isolated from a liver tumor of a female patient

suffering from hepatocarcinoma and a hepatitis C infection.^[51]

HepaRG cells plated at 2ꢃ10⁴cellscm^ꢀ2in the presence of insulin

and corticosteroids proliferated for 3 to 4 days with a doubling

time of 24 h. During the proliferation phase, undifferentiated

HepaRG cells appeared as a homogeneous epithelial phenotype

cell population without regular structural organization. Progressive

morphological changes, including the appearance of granular

polygonal hepatocyte-like cells and flat, clear epithelial cells, oc-

curred after reaching confluency two weeks after plating. In this

study, chelator exposures were performed one day after cell seed-

ing in proliferating HepaRG cells. HepaRG cells were cultured as

previously described.^[1h]They were maintained in William’s E

medium supplemented with 10% fetal bovine serum, penicillin

(100 UmL^ꢀ1), streptomycin (100 mgmL^ꢀ1), hydrocortisone hemisuc-

cinate (5ꢃ10^ꢀ5m), and insulin (5 mgmL^ꢀ1). Cells were seeded at

2ꢃ10⁴cellscm^ꢀ2in 96-well microplates for viability measurements

including LDH and cell-nuclei counting after Hoescht staining.

Chelator-uptake measurement

Chelator uptake was investigated in HepaRG cell cultures seeded

in 25 cm²culture flasks (20000 cellscm^ꢀ2) according to a previously

reported procedure.^[1h]Cell treatments were performed in triplicate,

in proliferating cells (D4) or after differentiation (D21), in the ab-

sence (control) or in the presence of the various compounds

(50 mm). After 72 h of treatment, supernatants were removed and

cells were then washed three times with ice-cold PBS solutions

(3 mL). They were collected by scraping after adding water (1 mL)

and sonication for 30 s at 08C. The protein content in these cell ex-

tracts was measured by using the Bradford method.^[52]Samples for

calibration were prepared in triplicate by adding various com-

pound concentrations (0.5, 1, and 5 mm) to cell extracts obtained

from untreated control cells. Ultrafiltration of the cell lysates

(200 mL) was performed by centrifugation for 20 min at 15000g in

a NANOSEP 3K centrifugal device (Pall Filtron Co). A volume of

1 mL of each intracellular medium was injected into a liquid chro-

matograph mass spectrometer (LCMS 2020 Shimadzu) with a flow

rate of 0.5 mLmin^ꢀ1for 10 min. This chromatograph was equipped

with a diode-array detector (SPD-M20A) and the reversed column

used was a Shim pack XR-ODSII (2.0ꢃ75 mm) with a mobile phase

consisting of acetonitrile/water 70:30 (v/v) until 100:0 (v/v) (5a,

t_R=3.4 min) or acetonitrile/water 80:20 (v/v) (5b, t_R=4.6 min).

Cytostatic and cytotoxic effects measurements

The viability assay of the compounds on cells was as follows: 2ꢃ

10⁴cellscm^ꢀ2were seeded in 96-multiwell plates and were left for

24 h for attachment, spreading, and growth. Then, they were ex-

posed for 72 h to increasing concentrations of the compounds,

ranging from 0.1 to 400 mm in a final volume of 100 mL culture

medium. The cytotoxic and cytostatic effects of the various com-

pounds were evaluated by measuring extracellular LDH activity (cy-

totoxicity detection kit–LDH, Roche, Penzberg, Germany) and mito-

chondrial SDH activity by the tetrazolium colorimetric assay (MTT,

Sigma, St Louis, MO). Extracellular LDH activity was measured as

described by the manufacturer on a 20 mL aliquot of cell-free

medium obtained by centrifugation (2500 rpmmin^ꢀ1for 5 min).

LDH activities were detected by reading the absorbance at

485 nm. An LDH standard curve (0 to 4000 mUmL^ꢀ1) was used to

quantify the enzyme activity (l-lactate dehydrogenase, Sigma, St

Louis, MO) and the values were corrected from the intracellular

protein content measured according to the method of Bradford.^[52]

Extracellular LDH activity was reported as a percentage of the con-

trol value. After a 72 h treatment, cells supernatants were removed

and the HepaRG cells were washed twice with PBS (50 mm, pH 7).

SDH activity was detected after 3 h of incubation in a 100 mL

serum-free medium containing MTT (0.5 mgL^ꢀ1). Formazan salts

were dissolved in DMSO and the absorbance was read at 535 nm

in a microplate fluorescence reader (Packard, Fusion). Data are re-

ported as a percentage of SDH activity with respect to the control

value. Three independent replicates were performed for each ex-

periment and each experiment was repeated three times. Data re-

ported are the average results of three independent experiments.

Parameters of the dose–response curves were deduced from

a four-parameters curve fit according to Rodbar [Eq. (4)]:^[53]

HUVEC cultures

HUVECs were freshly isolated from umbilical veins of newborn

babies by collagenase digestion.^[54]The umbilical cords were ob-

tained from the Centre de Gynꢀcologie Obstꢀtrique d’Amiens.

HUVECs were seeded in a 25 cm²plastic flask coated with 0.2%

gelatin in M199 medium supplemented with penicillin–streptomy-

cin (100 unitsmL^ꢀ1, 20 mgmL^ꢀ1), 20% FBS, and ECGS (10 ngmL^ꢀ1).

Plastic culture flasks were maintained at 378C in a humidified at-

mosphere of 5% CO₂. For cell-culture experiments, HUVECs were

used between the second and the fifth passage.

BrdU assay

For cell-culture experiments, ICL, 5a and 5b were dissolved in

methanol at a 10^ꢀ3m concentration, removed as aliquots, and then

frozen at ꢀ208C until further utilization. The 5a and 5b effects on

HUVEC and MDA-MB-231 cell survival and proliferation were tested

using “BrdU Cell Proliferation Assay Kit” as per the manufacturer

description.^[55]HUVECs were seeded, in triplicate, at 2ꢃ10⁴cells per

well and MB-231 cells at 1.5ꢃ10⁴cells per well in a 96-well micro-

plate, in the appropriate cell-culture medium. The day after, cells

were synchronized in the G1 phase for 24 h with serum-free

medium (100 mL) without red phenol. Cells were then incubated in

the presence or absence of 5a or 5b (10^ꢀ8-–10^ꢀ5m) freshly pre-

pared in growth medium (100 mL). We tested two experimental

conditions: low serum conditions, 0% FBS (for MDA-MB-231) or

1% FBS (for HUVEC) for cell survival determination, and 10% FBS

conditions for cell-proliferation determination. After 48 h of incuba-

tion, BrdU (20 mL) was added to each well at a final concentration

ðA_{C min}ꢀ A_{C max}

Þ

y ¼

ꢀ ꢁ

þ A_{C max}

P_ip

C

ð4Þ

1 þ

C_ip

in which y represents the SDH activity with respect to the control;

A_Cminand A_Cmaxare the minimal and maximal chelator concentra-

tions, respectively; C is the chelator concentration (C_ipat the inflec-

tion point); and P_ipis the slope at the inflection point of the sig-

moid curve. Due to their biphasic feature, the dose–response

1014

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ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemPlusChem 2012, 77, 1001 – 1016

Cone ICL670-Substituted Calix[4]arenes

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Matrigel assay and wound-healing assay

A Matrigel assay allows in vitro endothelial cell differentiation in

capillary tubes. This technique gives a simple initial assessment of

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Forty thousand HUVECs were seeded directly on top of the Matri-

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(500 mL) with different concentrations of ICL670, 5a and 5b (10^ꢀ5

to 10^ꢀ6m) and incubated for 16 h.^[57]To determine whether ICL670

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5a and 5b (10^ꢀ5to 10^ꢀ6m) freshly prepared in M199 medium

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both cases, pictures were taken after 16 h of incubation by using

a JVC video camera connected to the VisioL software and mounted

on a Leica microscope. The results are given as an average of at

least three separate (replicate) experiments for each treatment. Re-

sults were presented as means plus or minus the SEM. The statisti-

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ed and control cells was determined by ANOVA and the test of stu-

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were considered significant when p is <0.05.

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This study was supported by Rꢀgion Picardie (TsFer project). P.R.

was the recipient of a grant from the Rꢀgion Picardie. We thank

Dr. D. Harakat for mass spectra recording. We also thank P.

Gans, A. Sabatini, and A. Vacca for the free distribution of the

HYSS program. We thank “Fonds Europꢀen de Dꢀveloppement

Rꢀgional et la Rꢀgion Picardie” and “La ligue nationale contre le

cancer” for financial support. We also thank the “Service de gynꢀ-

cologie obstꢀtrique et mꢀdecine de la reproduction du Centre de

Gynꢀcologie Obstꢀtrique d’Amiens (CGO)” for providing the um-

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Keywords: calixarenes

· chelates · iron · lipophilicity ·

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Received: June 1, 2012

Revised: August 20, 2012

Published online on September 23, 2012

1016

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Synthesis, physicochemical studies, molecular dynamics simulations, and metal-ion-dependent antiproliferative and antiangiogenic properties of cone ICL670-substituted calix[4]arenes

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