Synthesis and biological properties of Quilamines II, new iron chelators with antiproliferative activities

Article abstract of DOI:10.1021/bc4004734

To selectively target tumor cells expressing an overactive Polyamine Transport System (PTS), we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, derived from the lead compound HQ1-44, which we named Quilamines II. The structures of four new antiproliferative agents were developed. They differ in the size of the linker (HQ0-44 and HQ2-44) or in the nature of the linker (HQCO-44 and HQCS-44) between a hydroxyquinoline moiety (HQ) and a homospermidine (44) chain, the best polyamine vector. The Quilamines II were obtained after 6 to 9 steps by Michael addition, peptide linkage, and reductive amination or by using the Willgerodt-Kindler reaction. The biological evaluation of these second-generation Quilamines showed that modifying the size of the linker increased the selectivity of these compounds for the PTS. In addition, measurement of the toxicity of Quilamines HQ0-44 and HQ2-44 highlighted their marked antiproliferative nature on several cancerous cell lines as well as a differential activity on nontransformed cells (fibroblasts). In contrast, Quilamines HQCO-44 and HQCS-44 presented low selectivity for the PTS, probably due to a loss of electrostatic interaction. We also demonstrated that the HCT116 cell line, originating from a human colon adenocarcinoma, was the most responsive to the various Quilamines. As deduced from the calcein and HVA assays, the higher iron chelating capacity of HQ1-44 could explain its higher antiproliferative efficiency.

Full text of DOI:10.1021/bc4004734

Article
pubs.acs.org/bc
Synthesis and Biological Properties of Quilamines II, New Iron
Chelators with Antiproliferative Activities
†
,‡,§
́ ́
hanie Renaud, Isabelle Cannie, Karine Julienne, Seb
astien G. Gouin,†
‡,§
,‡,§
‡,§
†
Vincent Corce,
́
Step
‡
,§
,†
Olivier Lorea
́
l, Franc o̧ is Gaboriau,* and David Deniaud*
†
LUNAM Universite,
́
CEISAM, Chimie Et Interdisciplinarite,
e, BP 92208, 44322 Nantes Cedex 3, France
INSERM, UMR 991, CHRU Pontchaillou, 35033 Rennes, France
§Universite
de Rennes 1, 35043 Rennes, France
́ ̀ ́
Synthese, Analyse, Modelisation, UMR CNRS 6230, UFR des Sciences
et des Techniques, 2, rue de la Houssinier
̀
‡
́
ABSTRACT: To selectively target tumor cells expressing an overactive Polyamine
Transport System (PTS), we designed, synthesized, and evaluated the biological activity
of a new generation of iron chelators, derived from the lead compound HQ1−44, which we
named Quilamines II. The structures of four new antiproliferative agents were developed.
They differ in the size of the linker (HQ0−44 and HQ2−44) or in the nature of the linker
(
HQCO-44 and HQCS-44) between a hydroxyquinoline moiety (HQ) and a
homospermidine (44) chain, the best polyamine vector. The Quilamines II were obtained
after 6 to 9 steps by Michael addition, peptide linkage, and reductive amination or by using
the Willgerodt-Kindler reaction. The biological evaluation of these second-generation
Quilamines showed that modifying the size of the linker increased the selectivity of these
compounds for the PTS. In addition, measurement of the toxicity of Quilamines HQ0−44 and HQ2−44 highlighted their
marked antiproliferative nature on several cancerous cell lines as well as a differential activity on nontransformed cells
(
fibroblasts). In contrast, Quilamines HQCO-44 and HQCS-44 presented low selectivity for the PTS, probably due to a loss of
electrostatic interaction. We also demonstrated that the HCT116 cell line, originating from a human colon adenocarcinoma, was
the most responsive to the various Quilamines. As deduced from the calcein and HVA assays, the higher iron chelating capacity
of HQ1−44 could explain its higher antiproliferative efficiency.
INTRODUCTION
Polyamine metabolism (biosynthesis and uptake), detrimen-
■
tal to cell proliferation, is also particularly amplified in tumor
Iron is a cofactor of various enzymes and thus plays a key role
in biological processes including energy generation by oxidative
10
cells. The Polyamine Transport System (PTS), which can
deal with natural and synthetic polyamines, is increased in
1
phosphorylation and cell growth and proliferation by
11
tumor cells. This property is of potential interest for targeting
regulating the activity of ribonucleotide reductase (RR)
12−21
antitumor agents.
This concept of tumor vectorization by
2
,3
involved in DNA synthesis. In tumoral cells, the DNA
synthesis rate is higher than in healthy cells and it has been
shown that both the activity and the expression of RR increase
polyamines has also been developed to inhibit their own
22,23
transporter,
to vectorize cytotoxic compounds such as
24
25,26
dimethylsilane polyamine derivatives or molecules carrying
boron atoms for boronotherapy,
4
during tumor growth.
and to monitor the
There are a number of alterations in iron metabolism in
tumor cells leading to increased iron ingress via the transferrin5
receptor (TfR1) and to decreased iron egress by ferroportin.
In most cancerous cells, the expression of the transferrin 1
receptor (TfR1) is higher than in healthy cells and is
intracellular traffic of polyamines toward cells by using
1
8,27−29
fluorescent polyamine analogues.
More recently,
F14512, a novel spermine conjugate associated with etoposide
30
(
epipodophyllotoxin), an inhibitor of DNA-topoisomerase II,
was selected for clinical development in AML phase I.
6
,7
proportional to the stage of progress of the tumor. Recent
studies have shown that levels of ferroportin and hepcidin are8
disrupted in cancerous cells leading to an accumulation of iron.
In breast cancer cells, the expression of ferroportin is reduced,
or even absent, at the gene and protein level and is
accompanied by a simultaneous increase in the expression of
Even though neoplastic cells require more iron than normal
cells, it seems possible to use a method targeting the chelator
selectively toward the tumor cells. To target tumor cells
expressing an overactive PTS compared to normal cells, we
designed and synthesized a new generation of iron chelators,
which we named Quilamines, based on an 8-hydroxyquinoline
8
,9
31
hepcidin. Moreover, this drop in ferroportin is associated
with a concomitant rise in the labile iron pool (LIP) and a
reduction in the level of ferritin. This enhanced requirement for
iron by growing tumor cells provides a new research route for
(8-HQ) scaffold linked to linear polyamine vectors. A set of
Received: October 14, 2013
Revised: January 14, 2014
Published: January 14, 2014
5
iron ligands with antiproliferative effects.
©
2014 American Chemical Society
320
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Bioconjugate Chemistry
Article
Quilamines bearing variable polyamine chains was designed and
assessed for their ability to interact with iron. Quilamines were
also screened for their cytostatic/cytotoxic effects and their
selective uptake by the PTS in the CHO cell line. HQ1−44, the
most promising Quilamine identified, contains a homospermi-
dine moiety and was shown to display an efficient
antiproliferative activity in the micromolar range. The
homospermidine cargo (44) was shown to be the most
selectively transported by the PTS and to be involved in iron
coordination. We also demonstrated the high complexation
capacity of HQ1−44 with iron, while much weaker complexes
were formed with other cations, indicative of a high selectivity.
The HQ1−44 ligand utilizes an O,N,N-donor set and, in view
of the stoichiometry, it could be assumed that the iron complex
rings (Figure 2, top). Ligands HQCO-44 and HQCS-44 differ
only in the nature of their heteroatom, which is either oxygen
or sulfur. As these ligands contain amide or thioamide
functions, the iron would be chelated by nitrogen, oxygen, or
sulfur atoms by simple rotation (Figure 2, bottom). Our
hypothesis was that, depending on the “hard/soft” nature of the
heteroatom, the ligands should lead to notable differences in
complexation, and thus change the antiproliferative activity.
EXPERIMENTAL SECTION
■
Chemistry. NMR spectra were recorded at room temper-
ature with a Bruker Avance 300 Ultra Shield or a Bruker Avance
III 400 spectrometer. Chemical shifts are reported in parts per
million (ppm); coupling constants are reported in units of
Hertz [Hz]. Infrared (IR) spectra were recorded with a Bruker
Vector 22 FTIR using KBr films or KBr pellets. Low-resolution
mass spectra (MS) were recorded with a Thermo Electron
DSQ spectrometer. High-resolution mass spectra (HRMS)
were recorded with a Thermo Fisher Hybrid LTQ-orbitrap
[
Fe/HQ1−44] forms three five-membered chelate rings. In
fact, the ferric complex adopted a 1:2 stoichiometry at pH = 7.4
in which the iron was probably coordinated by two vicinal N-
atoms and a hydroxyl group (Figure 1).
+
spectrometer (ESI ) and a Bruker Autoflex III SmartBeam
spectrometer (MALDI). Microanalyses were performed on a
Thermo Scientific FLASH 2000 Series CHNS/O Analyzer.
Melting points were determined in open capillary tubes and are
uncorrected. All reagents were purchased from Acros Organics
or Aldrich and were used without further purification. Column
chromatographs were conducted on silica gel Kieselgel SI60
Figure 1. Hypothetical structure of the HQ1−44/Fe complex.
(
40−63 μm) from Merck. Reactions requiring anhydrous
In terms of chemistry, we would like to improve the iron-
chelating efficiency in order to increase the antiproliferative
activity of HQ1−44 by modifying the length and the nature of
the spacer between the chelating moiety and the homospermi-
dine chain.
Our objective was to develop new polyaminoquinoline iron
chelators, derived from the lead HQ1−44, which we named
Quilamines II. The structures of the four new antiproliferative
agents developed differ in the size of the linker (HQ0−44 and
HQ2−44) or in the nature of the linker (HQCO-44 and
HQCS-44) between a hydroxyquinoline moiety (HQ) and a
homospermidine (44) chain, the best vector. For HQ0−44 and
HQ2−44, the introduction of zero or two carbon atoms will
probably have a significant effect on the stability of the iron
complexes formed by modification of the size of the chelate
conditions were performed under argon. Dichloromethane,
acetonitrile, and MeOH were distilled over CaH , THF over
Na/benzophenone. Dichloroethane, DMSO, and DMF were
used dry from secure sealed bottles.
2
Compounds 1, 3−5, 13, and 14 have already been described
in the literature.
2-(2-oxoethyl)quinolin-8-yl Acetate (2). Oxalyl chloride
(1.2 equiv, 0.36 mmol) in dichloromethane (3 mL) was added
to a solution of dimethylsulfoxide (2.4 equiv, 0.72 mmol) in
dichloromethane (150 μL), and the mixture was stirred at −78
°C for 30 min under Ar. Alcohol 7 (1 equiv, 0.3 mmol) in
dichloromethane (1 mL) was added dropwise, and the resulting
mixture was then stirred for 30 min at −78 °C. Triethylamine
(4.8 equiv, 1.44 mmol) was added dropwise, and the resulting
mixture was then stirred for 15 min at −78 °C; then, the
Figure 2. Hypothetical structures of iron complexes with different sizes of chelate rings or different heteroatoms.
3
21
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Bioconjugate Chemistry
Article
temperature was increased to room temperature over a period
of 1 h. The mixture was quenched by addition of water (4 mL)
and extracted with CH Cl . The organic extracts were dried
dichloromethane, dried over Na SO , filtered, and concentrated
2
4
under reduced pressure and the residue was purified by
chromatography on silica gel (CH Cl ) to give compound 8 as
2
2
2
2
over Na SO , filtered, and concentrated under reduced pressure
and the residue was purified by chromatography on silica gel
a white solid (96%). IR (KBr): γ = 3064, 1614, 1561, 1514,
1436, 1377, 1339, 1293, 1243, 1091, 984 cm . H NMR
2
4
−1
1
3
2
(
(
5
=
CH Cl /EtOAc (9:1)) to give compound 2 as a red solid
(CDCl , 400 MHz): δ = 5.64 (dd, 1H, J = 10.85 Hz, J = 0.9
2
2
3
1
3
2
75%). H NMR (CDCl , 300 MHz): δ = 2.59 (s, 3H, CH ),
Hz, CH ), 6.31 (dd, 1H, J = 17.7 Hz, J = 0.9 Hz, CH ), 6.99
2 2
3
3
3
3
3
3
3
.41 (d, 1H, J = 3.6 Hz, H), 6.81 and 7.69 (system AB, 2H, J
9 Hz, H and H ), 7.27 (m, 1H), 7.23−7.47 (m, 3H), 8.75 (s-
(dd, 1H, J = 17.7 Hz, J = 10.85 Hz, CH), 7.16 (dd, 1H, J =
7.64 Hz, J = 1.2 Hz, H ), 7.28 (dd, 1H, J = 8.25 Hz, J = 1.2
3
4
4
7
3
4
ar
1
3
5
3
3
6
e, 1H, H), 15.21 (s-e, 1H, OH) ppm. C NMR (CDCl , 75
Hz, H ), 7.40 (dd, 1H, J = 7.64 Hz, J = 8.25 Hz, H ), 7.57
ar ar
3
3
3
4
13
MHz): δ = 21.1 (CH ), 95.1, 121.8, 123.4, 124.6, 124.8, 131.2,
and 8.09 (system AB, 2H, J = 8.4 Hz, H and H ) ppm. C
3
ar ar
7
5
1
36.3, 139.2, 153.6, 169.0, 179.9 (CO) ppm. HRMS
NMR (CDCl , 100 MHz): δ = 110.2 (C ), 117.7 (C ), 119.5
3
(C ), 119.8 (CH ), 127.6 (C ), 127.8 (C ), 136.5 (C ), 137.3
+
3
4a
6
4
(
MALDI): calcd. for C H NO [M+H] 229.2313; found
1
3
12
3
2
8
a
8
2
2
29.2351.
(CH), 137.9 (C ), 152.3 (C ), 153.8 (C ) ppm. HRMS (ESI
+
2-(2-Hydroxyethyl)quinolin-8-ol (6). n-Butyllithium (2.2
+): calcd. for C H NO [M+H] ; 172.07569; found
1
1
10
equiv, 13.86 mmol) was added dropwise to a stirring solution
of 8-hydroxyquinaldine (1 equiv, 6.3 mmol) in tetrahydrofuran
172.07562. Anal. Calcd. for C H NO: C, 77.17; H, 5.30; N,
8.18. Found: C, 77.10; H, 5.41; N, 8.09.
11 9
(
20 mL) at −78 °C for 1 h under Ar. Paraformaldehyde (4
2-[4-tert-Butyloxycarbonyl-(4-N-tert-butyloxycarbonyl-
aminobutyl)aminobutyl]amino ethylquinolin-8-ol (9). 2-
Vinylquinolin-8-ol (8) (1 equiv, 0.52 mmol) was added to a
stirring solution of amine 1 (1 equiv, 0.52 mmol) in methanol
(5 mL), and then 3 drops of acetic acid were added and the
mixture was stirred at 70 °C for 24 h. It was then concentrated
under reduced pressure, dichloromethane was added, and the
equiv, 50.4 mmol) was added at −78 °C, and the temperature
was increased to room temperature over a period of 1 h. The
mixture was quenched by addition of sodium hydrogen
carbonate (20 mL) at −78 °C and extracted with diethylether.
The organic extracts were dried over Na SO , filtered, and
2
4
concentrated under reduced pressure and the residue was
purified by chromatography on silica gel (CH Cl /MeOH
mixture was washed with saturated aqueous NaHCO and
2
2
3
(
(
1
3
7
9.6:0.4)) to give compound 6 as a white solid (88%). IR
KBr): γ = 3156, 3086, 2950, 1565, 1436, 1383, 1322, 1269,
water. The organic extracts were dried over Na SO , filtered,
and concentrated under reduced pressure; and the residue was
2
4
−1 1
088, 1055, 1036, 820 cm . H NMR (CDCl , 400 MHz): δ =
purified by chromatography on silica gel (CH Cl/MeOH/
3
3
3
3
.20 (t, 2H, J = 6.2 Hz, CH ), 4.14 (t, 2H, J = 6.2 Hz, CH ),
.16 (dd, 1H, J = 8 Hz, J = 3.2 Hz, H ), 7.28 (dd, 1H, J = 8
NH OH: (8.5:0.4:0.1)) to give compound 9 as a yellow oil
2
2
4
3
4
7
3
(64%). IR (KBr): γ = 3383, 1668, 1455, 1390, 1251, 1089, 869
ar
4
5
ar
3
−1 1
Hz, J = 3.2 Hz, H ), 7.34 and 8.08 (system AB, 2H, J = 8.4
cm . H NMR (CD OD, 400 MHz): δ = 1.37−1.65 (m, 26H,
3
Hz, H3 and H ), 7.39 (t, 2H, J = 8 Hz, H ) ppm. C NMR
4
3
6
13
3
2 CH CH and 3 C(CH ) ), 2.82 (br s, 2H), 3.03 (t, 2H, J =
ar
ar
ar
2
2
3 3
7
3
(
1
1
CDCl , 100 MHz): δ = 40.7 (CH ), 61.5 CH ), 110.6 (C ),
17.9 (C ), 122.8 (C ), 127.2 (C ), 127.3 (C ), 136.8 (C ),
37.7 (C ), 151.7 (C ) 158.1 (C ) ppm. HRMS (ESI+): calcd.
6.8 Hz, BocNHCH ), 3.12−3.29 (m, 8H), 7.09 (dd, 1H, J =
3
2
2
2
5
3
8a
6
4
4 7 3 4
7.4 Hz, J = 1.5 Hz, H ), 7.32 (dd, 1H, J = 8.25 Hz, J = 1.5
ar
4a
2
8
5
3
ar
6
3
Hz, H ), 7.35−7.44 (m, 2H, H and H ), 8.18 (d, 1H, J =
ar
ar
+
4 13
for C H NO [M+H] 190.08680; found 190.08614.
8.25 Hz, H ) ppm. C NMR (CD OD, 100 MHz): δ = 26.9
1
1
12
2
ar 3
2-(2-Hydroxyethyl)quinolin-8-yl acetate (7). A solution of
(3 CH ), 28.3 (CH ), 28.7 (C(CH ) ), 28.8 (C(CH ) ), 36.9
2 2 3 3 3 3
sodium hydroxide 1 M (30 mL) and acetic anhydride (2 equiv,
5
2
stirred at r.t. for 3 h. The mixture was extracted with
dichloromethane, dried over Na SO , filtered, and concentrated
under reduced pressure and the residue was purified by
chromatography on silica gel (CH Cl /EtOAc: (9:1)) to give
(CH ), 40.9 (CH ), 48.1 (3 CH ), 49.0 (CH ), 79.8
2
2
2
2
7
5
.28 mmol) was added to a solution of compound 6 (1 equiv,
.64 mmol) in dichloromethane (30 mL), and the mixture was
(C(CH ) ), 80.9 (C(CH ) ), 112.3 (C ), 118.9 (C ), 123.2
(C ), 128.1 (C ), 129.1 (C ), 138.1 (C ), 139.4 (C ), 153.9
3 3 3 3
3
4a
6
8a
4
2
8
(C ), 157.4 (C ), 158,5 (CO) ; 159,2 (CO) ppm. HRMS
+
2
4
(MALDI): calcd. for C H N O [M+H] ; 531.3541; found
29 47
4
5
531.3544.
2
2
4-tert-Butyloxycarbonyl-(4-tert-butyloxycarbonyl-
aminobutyl)aminobutanal (10). Bis acetoxyiodobenzene (1.1
equiv, 1.1 mmol) and 2,2,6,6-tetramethylpiperidine-1-oxyl (0.1
equiv, 0.1 mmol) were added to a stirring solution of
aminoalcohol 14 (1 equiv, 1 mmol) in dichloromethane (1
mL) at r.t. for 5 h under Ar. Dichloromethane was added and
the organic solution was washed with saturated aqueous
Na S O , NaHCO , brine, and water. The organic extracts
1
compound 7 as a yellow solid (90%). H NMR (CDCl , 300
MHz): δ = 2.50 (s, 3H, CH ), 3.20 (t, 2H, J = 6.2 Hz, CH ),
3
3
3
2
3
4
.14 (t, 2H, J = 6.2 Hz, CH ), 7.31 and 8.13 (system AB, 2H,
2
3
3
4
3
4
J = 8.4 Hz, H and H ), 7.44 (dd, 1H, J = 9 Hz, J = 3 Hz,
ar
ar
5
3
6
3
4
H ), 7.51 (t, 2H, J = 8 Hz, H ), 7.70 (dd, 1H, J = 9 Hz, J =
3
ar
ar
7
13
Hz, H ) ppm. C NMR (CDCl , 75 MHz,): δ = 20.9
a
r
3
5
3
(
(
(
CH ), 38.9 (CH ), 61.0 (CH ), 121.9 (C ), 122.7 (C ), 125.8
C ), 126.0 (C ), 128.1 (C ), 136.9 (C ), 139.8 (C ), 146.7
C ), 161.6 (C ), 169.9 (CO) ppm. HRMS (ESI+): calcd. for
3 2 2
2
2
3
3
7
6
8a
4
4a
were dried over Na SO , filtered, and concentrated under
2 4
2
8
reduced pressure; and the residue was purified by chromatog-
raphy on silica gel (CH Cl /EtOAc: (8:2)) to give compound 9
+
C H NO [M+H] 232.09737; found 232.09769.
13
14
3
2
2
2
-Vinylquinolin-8-ol (8). Triethylamine (5 equiv, 15.85
as a colorless oil (75%). IR (film): γ = 2974, 2930, 2867, 1694,
−1 1
mmol) and methanesulfonyl chloride (3 equiv, 9.51 mmol)
were added to a solution of compound 6 (1 equiv, 3.17 mmol)
in dichloromethane (30 mL) at 0 °C and the mixture was
stirred at r.t. for 3 h. Solvent was evaporated, the resulting solid
was solubilized in the minimum of EtOH, and an aqueous
solution of sodium hydroxide (1 M, 30 mL) was added. The
mixture was stirred for 5 h at reflux and then neutralized with
HCl 1 M to pH = 7. The solution was extracted with
1479, 1390, 1168 cm . H NMR (CDCl , 400 MHz): δ =
3
3
1.35−1.62 (m, 22H, CH CH and 2 C(CH ) ), 1.83 (qt, 2H, J
2
2
3 3
3
3
2
= 7.1 Hz, CH ), 2.44 (t, 2H, J = 7.1 Hz, CH ), 3.04−3.27
2
2
1
3
(m, 6H), 4.56 (br s, 1H, NH), 9.77 (s, 1H, CHO) ppm.
C
NMR (CDCl , 100 MHz): δ = 21.2 (CH ), 25.8 (CH ), 27.6
3
2
2
(CH ), 28.5 (C(CH ) ), 28.6 (C(CH ) ), 40.3 (CH ), 41.2
2
3 3
3 3
2
(CH ),46.3 (CH ), 46.8 (CH ), 79.3 (C(CH ) ), 79.7
2
2
2
3 3
(C(CH ) ), 155.7 (CO), 156.1 (CO), 201.7 (CHO)
3
3
3
22
dx.doi.org/10.1021/bc4004734 | Bioconjugate Chem. 2014, 25, 320−334

Bioconjugate Chemistry
ppm. HRMS (MALDI): calcd. for C H NaN O [M+Na] ;
Article
+
7
5
3
80.8 (C(CH ) ), 111.2 (C ), 114.3 (C ), 118.8 (C ), 122.7
(C ), 124.3 (C ), 137.7 (C ), 138.6 (C ), 151.3 (C ), 157.5
1
8
34
2
5
3 3
4
a
6
8a
4
2
381.2360; found 381.2366.
8
4
-(4-Hydroxybutyl)aminobutanenitrile (11). Potassium
(C ), 157.7 (CO), 158.5 (CO) ppm. HRMS (MALDI):
+
carbonate (1.5 equiv, 15 mmol) and 4-bromobutanenitrile (1
equiv, 10 mmol) were added to a stirring solution of 4-
aminobutanol (1 equiv, 10 mmol) in acetonitrile (100 mL) at
calcd. for C H N O [M+H] ; 503.3228; found 503.3228.
2
7
43
4
5
N-[4-tert-Butyloxycarbonyl-(4-tert-butyloxycarbonyl-
aminobutyl)aminobutyl]-8-hydroxyquinolin-2-carboxamide
(16). To a stirred solution of 8-hydroxyquinaldinic acid (1
equiv, 0.94 mmol) in dichloromethane (5 mL) at room
temperature under argon was added N,N′-dicyclohexylcarbo-
diimide (1.1 equiv, 1.034 mmol). After 20 min, hydroxybenzo-
triazole (1.1 equiv, 1.034 mmol) was added and the mixture
was stirred at r.t. for 2 h under argon. Then, a solution of amine
1 (1 equiv, 0.94 mmol) was added in dichloromethane (3 mL)
at room temperature under argon and the mixture was stirred
for 18 h. The mixture was filtered, concentrated under reduced
pressure, and the residue purified by chromatography on silica
5
0 °C for 16 h under Ar. After filtration, the solvent was
removed under reduced pressure and the residue was purified
by chromatography on silica gel (CH Cl/MeOH/NH OH
3
4
(
8.9:1.0:0.1)) to give compound 11 as a colorless oil (56%). IR
(film): γ = 3357, 2936, 2865, 1694, 1652, 1542, 1375, 1249,
−
1 1
1
4
=
059 cm . H NMR (CDCl , 300 MHz): δ = 1.57−1.69 (m,
3
3
3
3
H, CH CH ), 1.84 (qt, 2H, J = 7.1 Hz, CH ), 2.44 (t, 2H, J
2
2
2
2
3
7.2 Hz, CH ), 2.66 (t, 2H, J = 5.4 Hz, CH NH), 2.76 (t,
H, J = 6.9 Hz, CH ), 3.57 (t, 2H, J = 5.1 Hz, CH OH) ppm.
C NMR (CDCl , 75 MHz): δ = 15.2 (CH ), 25.6 (CH ),
2
2
3
4
3
2
2
2
1
3
2
2
3
3
2
4
2
8.4 and 32.3 (CH CH ), 47.9 (CH ), 49.8 (CH NH), 62.7
gel (CH Cl /MeOH: (9.7:0.3)) to give compound 16 as a
yellowish solid (80%). IR (film): γ = 3324, 2974, 2932, 1674,
1568, 1503, 1391, 1246, 1165 cm . H NMR (CD OD, 400
2 2
2
2
2
2
(
CH OH), 119.5 (CN) ppm. HRMS (ESI+): calcd. for
2
+
−1 1
C H N O [M+H] ; 157.13354; found 157.13348.
8
17
2
3
4
-tert-Butyloxycarbonyl-(4-hydroxybutyl)amino-
MHz): δ = 1.31−1.74 (m, 26H, 2 CH CH and 2 C(CH ) ),
2
2
3 3
3
3
butanenitrile (12). A solution of di-tert-butyldicarbonate (1.5
equiv, 1.34 mmol) was added to a stirring solution of 11 (1
equiv, 0.896 mmol) in a solution of methanol and 10%
triethylamine (10 mL). After stirring for 24 h at room
temperature, solvents were removed under reduced pressure to
give a colorless oil which was dissolved in dichloromethane,
washed with saturated NaHCO and water, dried over Na SO ,
3.01 (t, 2H, J = 6.7 Hz, BocNHCH ), 3.19 (t, 2H, J = 7.2 Hz),
2
3
3.24 (t-e, 2H), 3.51 (t, 2H, J = 6.7 Hz, CONHCH ), 7.16 (dd,
2
3
4
7
ar
3
4
1H, J = 7.6 Hz, J = 1 Hz, H ), 7.42 (dd, 1H, J = 8.2 Hz, J =
1 Hz, H ), 7.53 (dd, 1H, J = 8.2 Hz, J = 7.6 Hz, H ), 8.18
5
ar
3
3
6
ar
3
3
ar
4
13
and 8.39 (system AB, 2H, J = 8.8 Hz, H and H ) ppm. C
ar
NMR (CD OD, 100 MHz): δ = 27.0 (2 CH ), 27.9 (CH ),
3
2
2
28.3 (CH ), 28.7 (C(CH ) ), 28.8 (C(CH ) ), 40.3 (CH ),
3
2
4
2
3 3
3 3
2
filtered, and concentrated under reduced pressure. The residue
40.9 (BocNHCH ), 48.1 (2 CH ), 79.8 (C(CH ) ), 80.8
2 2 3 3
7
5
4
6
was purified by chromatography on silica gel (CH Cl /EtOAc
C(CH ) ), 112.7 (C ), 118.9 (C ), 119.9 (C ), 130.5 (C ),
2
2
3 3
4a
8a
3
2
8
(
3
6:4)) to give compound 12 as a colorless oil. IR (film): γ =
442, 2974, 2931, 1685, 1479, 1420, 1296, 1167, 1057 cm . H
131.4 (C ), 138.4 (C ), 138.8 (C ), 148.8 (C ), 155.0 (C ),
−1 1
157.4 (CO), 158.5 (CO), 166.6 (CONH) ppm. HRMS
+
NMR (CDCl , 300 MHz): δ = 1.45 (s, 9H, C(CH ) ), 1.49−
(MALDI): calcd. for C H N NaO [M+Na] ; 553.2997;
3
3 3
28 42
4
6
3
3
2
1
(
.66 (m, 4H, CH CH ), 1.89 (qt, 2H, J = 7.1 Hz, CH ), 2.35
t, 2H, J = 7.1 Hz, CH ), 3.22 (br t, 2H, CH NBoc), 3.31 (t,
H, J = 6.9 Hz, CH ), 3.66 (t, 2H, J = 6 Hz, CH OH) ppm.
C NMR (CDCl , 75 MHz): δ = 14.9 (CH ), 24.7 (2 CH ),
found 553.2971.
2
2
3
2
2
N-[4-tert-Butyloxycarbonyl-(4-tert-butyloxycarbonyl-
aminobutyl)aminobutyl]-8-hydroxyquinolin-2-carbo-
thioamide (17). To a stirred suspension of sulfur (1.6 equiv,
0.45 mmol as elemental sulfur) in dimethylformamide (1.5 mL)
was added sodium sulfide (0.15 equiv, 0.042 mmol). The
mixture was warmed until the solution appeared blue and was
added via cannula to a stirring mixture of amine 1 (1 equiv, 0.28
mmol) and 2-formylquinolin-8-ol (1 equiv, 0.28 mmol) in
dimethylformamide (1.5 mL) at 115 °C. After 2 h the mixture
was quenched by addition of saturated ammonium chloride (20
mL) and extracted with diethyl ether. The organic extracts were
2
3
4
3
2
2
2
1
3
2
2
3
2
4
2
6
8.5 (C(CH ) ), 29.8 (CH ), 45.9 (CH ), 47.5 (CH NBoc),
3 3 2 2 2
2.6 (CH OH), 80.2 (C(CH ) ), 119.5 (CN), 155.7 (CO)
2
3 3
+
ppm. HRMS (MALDI): calcd. for H NaN O [M+Na] ;
1
3
24
2
3
279.1679; found 279.1690.
2
-[4-tert-Butyloxycarbonyl-(4-tert-butyloxycarbonyl-
aminobutyl)aminobutyl]aminoquinolin-8-ol (15). To a
stirred solution of polyamine 10 (1 equiv, 0.555 mmol) in
1
,2-dichloroethane (5 mL) at room temperature under argon
was added 2-aminoquinolin-8-ol (1.2 equiv, 0.66 mmol). After
5 min, sodium triacetoxyborohydride (3 equiv, 1.65 mmol)
dried over Na
pressure and the residue was purified by chromatography on
silica gel (CH Cl /EtOAc (9:1)) to give compound 17 as a
yellow solid (52%). IR (film): γ = 3419, 3267, 2930, 1676,
SO , filtered, and concentrated under reduced
2
4
1
was added and the mixture was stirred for 12 h. The reaction
mixture was quenched by adding 1 M NaOH (5 mL), stirred
for 30 min; the product was extracted with dichloromethane,
dried over Na SO , filtered, and concentrated under reduced
2
2
−1 1
1522, 1503, 1464, 1365, 1165 cm . H NMR (CD OD, 400
3
MHz): δ = 1.33−1.59 (m, 22H, CH CH and 2 C(CH ) ),
2
4
2
2
3 3
3
pressure; and the residue was purified by chromatography on
1.62−1.73 (m, 2H), 1.78−1.88 (m, 2H), 3.00 (t, 2H, J = 6.8
3
3
silica gel (CH Cl /EtOAc (8:2)) to give compound 12 as a
Hz, BocNHCH ), 3.19 (t, 2H, J = 6.8 Hz), 3.26 (t, 2H, J = 6.8
2
2
2
3
3
yellow oil (73%). IR (film): γ = 3366, 2974, 2930, 1683, 1615,
Hz), 3.6 (t, 2H, J = 6.8 Hz, CSNHCH ), 7.16 (dd, 1H, J = 7.6
2
−1
1
4
7
3
4
5
1
478, 1391, 1249, 1169 cm . H NMR (CD OD, 400 MHz):
Hz, J = 1 Hz, H ), 7.40 (dd, 1H, J = 8.2 Hz, J = 1 Hz, H ),
ar ar
7.51 (dd, 1H, J = 8.2 Hz, J = 7.6 Hz, H ), 8.32 and 8.80
(system AB, 2H, J = 8.8 Hz, H and H ) ppm. C NMR
ar ar
3
3
3
6
ar
δ = 1.32−1.55 (m, 22H, CH CH and 2 C(CH ) ), 1.59−1.72
2
2
3 3
3
3
3
4
13
(
(
6
=
=
m, 4H, CH CH ), 2.99 (t, 2H, J = 6.7 Hz, BocNHCH ), 3.15
t, 2H, J = 7.3 Hz), 3.24 (br t, 2H), 3.53 (br t, 2H, NHCH ),
2
2
2
3
(CD OD, 100 MHz): δ = 26.2 (CH ), 27.1 (CH ), 28.3 (2
2
3
2
2
3
3
3
4
.72 (d, 1H, J = 8.9 Hz, H ), 6.91 (dd, 1H, J = 7.5 Hz and J
CH ), 28.7 (C(CH ) ), 28.8 (C(CH ) ), 41.0 (BocNHCH ),
2 3 3 3 3 2
ar
7
3
6
3
1.3 Hz, H ), 7.00 (t, 1H, J = 7.7 Hz, H ), 7.06 (dd, 1H, J
46.3 (CH ), 48.1 (2 CH ), 79.8 (C(CH ) ), 80.9 (C(CH ) ),
ar
ar
2
2
3 3
3 3
4a
4
5
3
4
ar
7
5
4
6
8 Hz and J = 1.3 Hz, H ), 7.75 (d, 1H, J = 8.9 Hz, H )
112.9 (C ), 118.9 (C ), 122.9 (C ), 130.4 (C ), 131.1 (C ),
ar
13
8a
3
2
8
ppm. C NMR (CD OD, 100 MHz): δ = 26.9 (2 CH ), 27.7
137.3 (C ), 137.9 (C ), 150.4 (C ), 155.2 (C ), 157.5 (CO),
3
2
(
(
CH ), 28.3 (CH ), 28.7 (C(CH ) ), 28.8 (C(CH ) ), 40.9
158.5 (CO), 192.4 (CS) ppm. HRMS (MALDI): calcd. for
2 2 3 3 3 3
+
BocNHCH ), 41.6 (CH ), 47.9 (2 CH ), 79.8 (C(CH ) ),
C H N NaO S [M+Na] ; 569.2768; found 569.2775.
2
2
2
3 3
28 42
4
5
3
23
dx.doi.org/10.1021/bc4004734 | Bioconjugate Chem. 2014, 25, 320−334

Bioconjugate Chemistry
Article
6
3
3
General Procedure for Boc Removal (HQX-44). To a stirred
solution of polyamines 9, 15−17 (1 equiv, 0.2 mmol) dissolved
in ethanol (2 mL) at 0 °C was added 6 M HCl (2 mL). After
= 7.9 Hz, H ), 8.06 and 8.25 (system AB, 2H, J = 8.4 Hz, H
ar ar
4
13
and H ) ppm. C NMR (D O, 100 MHz,): δ = 22.7 (CH ),
23.5 (CH ), 23.9 (CH ), 24.2 (CH ), 38.8 (CH ), 45.1 (CH ),
ar
2
2
2
2
2
2
2
7
5
4
12 h at room temperature, the solution was concentrated in
46.8 (CH ), 47.3 (CH ), 112.3 (C ), 118.8 (C ), 120.9 (C ),
2 2
6
4a
8a
3
2
vacuo and the resulting solid was washed with cold ethanol
129.4 (C and C ), 134.9 (C ), 137.4 (C ), 148.1 (C ), 151.4
8
(
−20 °C), filtered, and dried in vacuo to afford Quilamine
(C ), 190.5 (CS) ppm. HRMS (MALDI): calcd. for
+
HQX-44 as a yellow solid (HQ2−44, HQCO-44), a white
solid (HQ0−44), and an orange solid (HQCS-44), its
hydrochloride salt.
C H N OS [M+H-3HCl] ; 347.1900; found 347.1897.
1
8
27
4
Anal. Calcd. for C H Cl N OS.0.3C H O.1H O: C, 45.81;
H, 6.78; N, 11.49; S, 6.57. Found: C, 45.56; H, 7.15; N, 11.09;
18
29
3
4
2
6
2
(
2-[4-(4-Aminobutyl)aminobutyl]aminoethylquinolin-8-
S, 6.98.
ol)ammonium Tetrachloride (HQ2−44). (96%). Mp sup 250
Biological Methods. Chelator Solutions. All reagents,
which were of the highest available grade, were obtained from
Sigma-Aldrich (Saint Quentin Fallavier, France). All synthe-
sized Quilamines were compared to the tridentate iron-chelator
hydroxyphenyltriazole ICL670 as a reference (Deferasirox,
Exjade from Novartis Pharma) as well as to 8-hydroxyquino-
line. Stock solutions of each chelator (10 mM) were prepared
in water. The high solubility of each derivative in culture
medium in the concentration range 0−400 μM was first verified
by turbidimetry measurement. Three controls were prepared
for each experiment: one with the standard culture medium,
and the other with culture medium.
Cell Selectivity for the PTS. The involvement of the PTS in
the selective uptake of Quilamines was investigated by using
Chinese Hamster Ovary cells (CHO) exhibiting high PTS
activity and the mutated derived-cell line CHO-MG cells,
devoid of PTS (provided by Dr. W. Flintoff, University of
°
C. IR (KBr): γ = 2955, 2933, 2809, 2778, 2778, 1636, 1606,
−1 1
1
1
3
591, 1457, 1298, 838 cm . H NMR (D O, 400 MHz): δ =
2
.79−1.95 (m, 8H, 2 CH CH ), 3.07−3.23 (m, 6H, 3 CH ),
2
2
2
.25−3.33 (m, 2H, CH ), 3.68−3.76 (m, 2H, CH ), 3.77−3.86
2
2
3
4
7
(
m, 2H, CH ), 7.58 (dd, 1H, J = 6.5 Hz, J = 2.34 Hz, H ),
2
a
r
5
6
7
2
.78−7.86 (m, 2H, H and H ), 8.02 and 9.05 (system AB,
ar
ar
3 3 4 13
H, J = 8.4 Hz, H and H ) ppm. C NMR (D O, 100
ar
ar
2
MHz,): δ = 22.8 (3 CH ), 23.9 (CH ), 30.4 (CH ), 38.9 (CH ),
2
2
2
2
7
4
1
1
5.8 (CH ), 46.8 (CH ), 46.9 (CH ), 47.3 (CH ), 117.1 (C ),
2 2 2 2
5
3
4a
6
8a
19.6 (C ), 122.6 (C ), 129.1 (C ), 129.9 (C ), 130.4 (C ),
47.0 (C ), 147.1 (C ), 153.6 (C ) ppm. HRMS (MALDI):
calcd. for C H N O [M+H-4HCl] ; 331.2492; found
4
2
8
+
1
9
31
4
3
0
6
31.2497. Anal. Calcd. for C H Cl N O·0.25C H O·
.95HCl: C, 44.83; H, 7.03; N, 10.72. Found: C, 44.91; H,
.97; N, 10.64.
19 34 4 4 2 6
32
(
2-[4-(4-Aminobutyl)aminobutyl]aminoquinolin-8-ol)-
ammonium Trichloride (HQ0−44). (95%). Mp sup 250 °C. IR
33
Western Ontario). The CHO-MG cells were selected for
(
KBr): γ = 3461, 3104, 2935, 1669, 1608, 1570, 1467, 1281,
−1
1
growth resistance to methylglyoxalbis-(guanylhydrazone), using
a single-step selection after mutagenesis with ethyl methanesul-
fonate. Therefore, highly selective PTS ligands should give high
(CHO-MG/CHO) IC50 ratios.
The two cell lines were grown in RPMI 1640 medium,
supplemented with 10% fetal calf serum (Eurobio, Les Ulis,
France), 2 mM glutamine (Bio Media, Boussens, France), 100
units/mL penicillin, and 50 μg/mL streptomycin, at 37 °C in
1
219, 1050 cm . H NMR (D O, 400 MHz): δ = 1.64−2.11
2
(
m, 8H, 2 CH CH ), 3.05−3.33 (m, 6H, 3 CH ), 3.65 (br t,
2
2
2
2
H, CH ), 7.00−7.16 (m, 1H), 7.24−7.32 (m, 1H), 7.38−7.51
2
13
(
m, 2H), 8.22−8.31 (m, 1H) ppm. C NMR (D O, 100
2
MHz,): δ = 22.8 (CH ), 23.1 (CH ), 23.9 (CH ), 38.8 (CH ),
2
2
2
2
ar
4
1.5 (CH ), 46.9 (CH ), 47.2 (CH ), 116.6 (2 C ), 119.7 (2
2 2 2
ar
ar
C ), 122.1, 125.6 (2 C ), 144.1, 152.4 ppm. HRMS (MALDI):
calcd. for C H N O [M+H-3HCl] ; 303.2179; found
+
1
7
27
4
5
% humidified CO . L-Proline (2 μg/mL) was added to the
culture medium for CHO-MG cells.
2
3
03.2183. Anal. Calcd. for C H Cl N O.0.6C H O·1.15HCl:
17 29 3 4 2 6
C, 45.41; H, 7.07; N, 11.64. Found: C, 45.42; H, 7.07; N, 11.64.
Cell Treatment. Cells were harvested with trypsin and
seeded for 24 h before polyamine treatment in 96-well
microplates (Becton Dickinson, Oxnard, CA) at a density of
2000 cells per well. In these conditions, cells reached
confluency in 5−6 days. Chelator exposure was performed
one day after cell seeding in proliferating CHO and CHO-MG
cells. To prevent oxidation of exogenous polyamine by the
serum amine oxidase present in the fetal calf serum,
aminoguanidine (1 mM), an inhibitor of this enzyme, was
added to the medium. The various derivatives were compared
to the tridentate iron chelator hydroxyphenyltriazole ICL670
(
N-[4-(4-Aminobutyl)aminobutyl]-8-hydroxyquinolin-2-
carboxamide)ammonium Trichloride (HQCO-44). (98%).
Mp sup 250 °C. IR (KBr): γ = 3444, 2351, 2976, 2859,
678, 1596, 1537, 1440, 1296, 1143 cm . H NMR (D O, 400
MHz): δ = 1.73−1.91 (m, 8H, 2 CH CH ), 3.03−3.23 (m,
H), 3.48 (t, 2H, J = 6.6 Hz, CH ), 7.07 (dd, 1H, J = 7.7 Hz,
J = 1.1 Hz, H ), 7.29 (dd, 1H, J = 8.4 Hz, J = 1.1 Hz, H ),
.45 (dd, 1H, J = 8.4 Hz, J = 7.7 Hz, H ), 7.74 and 8.16
system AB, 2H, J = 8.8 Hz, H and H ) ppm. C NMR
D O, 100 MHz): δ = 22.7 (CH ), 23.2 (CH ), 23.9 (CH ),
2 2 2 2
−1 1
1
2
2
2
3
3
6
2
4
7
3
4
5
ar
ar
3
3
6
7
(
(
2
1
1
ar
3
3
4
13
ar ar
5.7 (CH ), 38.8 (CH ), 38.9 (CH ), 46.8 (CH ), 47.3 (CH ),
12.2 (C ), 118.3 (C ), 118.9 (C ), 129.4 (C ), 129.5 (C ),
2 2 2 2 2
7
5
4
6
4a
(
Deferasirox, Exjade) and to the bidentate 8-hydroxyquinoline.
8a
3
2
8
Measurement of Cytostatic and Cytotoxic Effects.
35.4 (C ), 138.4 (C ), 145.8 (C ), 150.9 (C ), 165.5 (CO)
Chelator exposure was performed one day after cell seeding
in proliferating CHO and CHO-MG cells. After 72 h of
incubation at 37 °C, cell supernatants were collected for
cytotoxicity analysis by measuring extracellular LDH activity
(cytotoxicity detection kit − LDH, Roche, Penzberg,
Germany). Extracellular LDH activity was measured as
described by the manufacturer on a 20 μL aliquot of cell-free
medium obtained by centrifugation (2500 rpm/min for 5 min).
LDH activities were detected by reading absorbance at 485 nm.
They were reported as a percentage of extracellular LDH
activity with respect to the control value.
ppm. HRMS (MALDI): calcd. for C H N NaO [M+Na-
1
8
26
4
2
+
3
HCl] ; 353.1948; found 353.1942. Anal. Calcd. for
C H Cl N O .2.35H O: C, 44.84; H, 7.05; N, 11.62.
1
8
29
3
4
2
2
Found: C, 45.06; H, 7.05; N, 11.34.
N-[4-(4-Aminobutyl)aminobutyl]-8-hydroxyquinolin-2-
(
carbothioamide)ammonium Trichloride (HQCS-44). (94%).
Mp sup 250 °C. IR (KBr): γ = 3431, 2958, 1569, 1522, 1502,
386, 1275, 1156, 1087, 892 cm . H NMR (D O, 400 MHz):
−1 1
1
2
3
δ = 1.76−2.01 (m, 8H, 2 CH CH ), 3.09 (t, 2H, J = 6.9 Hz),
2
2
3
3
.13−3.25 (m, 4H), 3.90 (t, 2H, J = 6.9 Hz, CH ), 7.05 (d, 1H,
2
3
7 3 5 3
J = 7.5 Hz, H ), 7.25 (d, 1H, J = 8.3 Hz, H ), 7.42 (t, 1H, J
ar
ar
3
24
dx.doi.org/10.1021/bc4004734 | Bioconjugate Chem. 2014, 25, 320−334

Bioconjugate Chemistry
Article
Cell viability was determined by counting the cell nuclei after
staining with the fluorescent DNA intercalating dye Hoechst
and IC50 values of each sigmoid, which were obtained from
these fits.
3
3342. The treated CHO and CHO-MG cells were washed
Three independent replicates were performed for each
experiment, which was repeated three times.
twice with PBS (50 mM pH 7) and fixed with ethanol/acetic
acid for 20 min at 4 °C. Cells were counterstained for 10 min
with Hoechst 33342 dye diluted by 1/1000 (5 μg/mL).
Hoescht fluorescence (exc = 340 nm, em = 450 nm) was
measured in a multiwell microplate fluorometer. The
fluorescence intensity, which is proportional to the number of
cell nuclei, was reported as a percentage of the control value.
Parameters of the dose−response curves were deduced from a
Physicochemical Methods. Comparison of Chelator
Efficiency in the Aqueous Phase and Competition with
Calcein. In solution, calcein, a fluoresceinated analogue of
35
EDTA, binds iron(II) and, more slowly, iron(III). The
fluorescence of this metallosensor dye is quenched during its
interaction with iron and, conversely, is restored if iron is
removed from the [calcein-iron] complex by a more efficient
competitive iron chelator. The rate and extent of fluorescence
recovery depend on the chelator concentration, the kinetics and
stoichiometry of iron binding, and the relative binding affinity.
The fluorescence emission (λExc = 485 nm, λEm = 520 nm) of
calcein (100 nM) in a HEPES buffer (20 mM HEPES, 150 mM
NaCl, pH 7.3) was measured at room temperature in a
microplate fluorescence reader (Polarstar Omega, BMG
Labtech), equipped with orbital stirring. Iron(III) (1 μM)
slowly reacted with calcein and maximal quenching of its
fluorescence was observed at a time longer than 6 h. The
fluorescence recovery was monitored after 4 h incubation in the
presence of various chelator concentrations. In the absence of
iron, some chelators slightly modulated the calcein fluorescence
intensity. The results were expressed as a percentage of
fluorescence recovery with respect to the free calcein
fluorescence intensity measured in the absence of iron. The
calcein test is qualitative, but the concentration of the chelator
for which 50% of the fluorescence is restored is indicative of
chelator affinity (RC ). These RC values were deduced from
34
4-parameter curve fit according to Rodbard
y = (
Acmin − Acmax
)
+ Acmax
⎛
⎞
C
1
+
⎜
⎟
P
ip
⎝
Cip
⎠
in which y is the percentage of the fluorescence intensity with
respect to the control, ACmin and ACmax are the y values observed
for minimal and maximal chelator concentrations, respectively,
C is the chelator concentration (C at the inflection point) and
ip
Pip is the slope at the inflection point of the sigmoid curve. Due
to their biphasic feature, the dose−response curves were fitted
as the sum of two sigmoids (double 4-parameter fit).
Percentages of cells involved in each viability response were
deduced from the ACmin, ACmax, and IC50 values of each sigmoid,
which were obtained from these fits.
Three independent replicates were performed for each
50
50
experiment, which was repeated three times.
the four-parameter curve fitting of the dose−effect curves.
HVA Assay. We have previously developed a simple,
sensitive, and reliable test to measure the total antioxidative
Screening of the Cytostatic/Cytotoxic Effects in Tumor
Cell Lines. The various derivatives were compared to the
tridentate iron chelator hydroxyphenyltriazole ICL670 (Defer-
asirox, Exjade) and to the bidentate 8-hydroxyquinoline.
The cytostatic and cytotoxic effects of Quilamines were
tested on various cell lines from human tumors and obtained
from the ECACC collection. They included HuH7 (hepato-
carcinoma), Caco-2 and HCT-116 (colon cancers), MDA-
MB231 (breast cancer), PC3 (prostate cancer), NCI-H727
36
efficiency of biological fluids. The autoxidation of homo-
vanillic acid (HVA) gives rise to a fluorescent dimer (λexc.= 315
nm; λem = 425 nm). Its appearance, monitored by its
fluorescence increase, follows a linear kinetic pattern for a
time longer than 5 h. This Fenton-like reaction, which requires
2+
divalent metal ions such as Fe , can be transiently stopped by
various ROS scavengers inducing a delay in the HVA
autoxidation. In contrast, metal chelating agents such as 8-
hydroxyquinoline (HQ) and Quilamines only reduce the HVA
autoxidation rate without introducing a detectable delay. These
characteristics constitute the basis of the HVA test which
enables, in a single run, the investigation of both components of
the antioxidant property: ability to prevent the formation of
ROS by chelation and/or to scavenge the free radicals giving
rise to HVA autoxidation.
(
lung cancer), and Hacat, a human immortalized keratinocyte
cell line. These cells were grown according to ECACC
recommendations. This study was extended to skin diploid
fibroblastic cells which were provided by BIOPREDIC
International Company (Rennes, France).
The cytostatic/cytotoxicity tests of the compounds on these
cells were as follows: 2.103 cells/well for the HCT-116 cell line
or 4.103 cells/well for the other cell lines were seeded in 96-
well microplates (Becton Dickinson, Oxnard, CA). In these
conditions, cells reached confluency in 5−6 days. Twenty-four
hours after seeding, cells were exposed to increasing
concentrations of the compounds (0.1−200 μM). To prevent
oxidation of exogenous polyamine by the serum amine oxidase
present in the fetal calf serum, aminoguanidine (1 mM), an
inhibitor of this enzyme, was added to the medium. After 48 h
of treatment, 20 μL of cell supernatants were collected for
cytotoxicity analysis by measuring extracellular LDH activity
The final reaction mixture for the HVA assay contained 3 ×
−4
10 M HVA in 0.1 M borate buffer (pH 9.0). Quilamines, at
various final concentrations ranging from 0 to 200 μM, were
introduced in 200 μL of this reaction mixture in a 96-well
microplate. They were incubated at 37 °C under gentle stirring
and fluorescence was measured every minute (λexc. = 340 nm;
λem = 440 nm) using a microplate fluorescence reader
(Polarstar Omega, BMG Labtech). The autoxidation level in
the absence (R ) or presence of an effector (R ) was calculated
0
a
(
see above).
from the slope of the fluorescence kinetics. Each kinetic
determination was performed in triplicate.
Cell viability was determined by counting the cell nuclei after
staining with the fluorescent DNA intercalating dye Hoechst
3342 as described above. Fits of the dose−effect curves were
performed as described above and percentages of cells involved
in each viability responses were deduced from the ACmin, ACmax
RESULTS AND DISCUSSION
Chemistry. As for the first-generation Quilamines, the
synthesis of ligand HQ2−44 was envisaged by a reductive
3
■
,
3
25
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Bioconjugate Chemistry
Article
Figure 3. Retrosynthesis of Quilamine HQ2−44.
a
Scheme 1. Synthesis of Protected Homospermidine 1
a
Reagents: (a) (Boc) O (0.33 equiv), Et N, MeOH, 0 °C to r.t., 20 h; (b) 4-bromobutyronitrile (1 equiv), K CO , CH CN, 50 °C, 12 h; (c)
2
3
2
3
3
(
Boc) O (2.5 equiv), Et N, MeOH, r.t., 20 h; (d) H , Raney Ni, EtOH, NH OH, 20 atm, r.t., 12 h.
2 3 2 4
a
Scheme 2. Synthesis of 2-(8-Hydroxyquinolin-2-yl)acetaldehyde 2
a
Reagents: (a) nBuLi (2.2 equiv), paraformaldehyde (4 equiv), THF, −78 °C to r.t., 3 h; (b) (CH CO) O (1.2 equiv), NaOH 1 M, CH Cl , r.t., 3 h;
3
2
2
2
(
c) (COCl) (1.2 equiv), DMSO (2.4 equiv), Et N (4.8 equiv), CH Cl , −78 °C to r.t., 2 h.
2
3
2
2
Figure 4. Retrosynthesis of Quilamine HQ2−44.
amination between the protected homospermidine 1 and the
protected 2-(8-hydroxyquinolin-2-yl)acetaldehyde 2 (Figure 3).
The homospermidine analogue 1 was prepared according to
a modified four-step procedure providing good yields (Scheme
The reductive amination of this aldehyde 2 with homo-
spermidine 1 was achieved in one pot by using sodium
triacetoxyborohydride to reduce the imine generated in situ.
39
Unfortunately, it proved impossible to form the expected
compound, and only the product of the deprotection of the
phenol function could be recovered at the end of the reaction.
This lack of reactivity may be due to the strong stabilization of
the enol form by hydrogen bonding between the enol, the
nitrogen of pyridine and acetate. In fact, NMR analysis revealed
that the enol is by far the major form in solution (90/10). The
hydrogen of the hydroxyl group has a chemical shift of 15 ppm,
which appears as a broad singlet even in polar solvents, while
3
1
1). The first step consisted of reacting putrescine with di-tert-
butyl dicarbonate (Boc O) leading to monoprotected diamine
2
3
, which was reacted with 4-bromobutyronitrile to give
secondary amine 4, followed by quantitative Boc-protection
giving rise to compound 5. The nitrile was reduced by
hydrogenation with Raney Ni to give masked homospermidine
2
1,37
1
with an overall yield of 63%.
The synthesis of aldehyde 2 was carried out in three steps
from commercial 8-hydroxyquinaldine with an overall yield of
0% (Scheme 2). 2-(2-Hydroxyethyl)quinolin-8-ol 6 was
3
the ethylenic hydrogens have a low coupling constant J of 3.2
6
Hz, characteristic of the Z geometry of the double bond. Lastly,
obtained by hydroxymethylation of 8-hydroxyquinaldine,
a DEPT analysis showed that no CH was present in the
2
using n-butyl lithium in THF in the presence of paraformalde-
structure.
38
hyde as an electrophile to trap the anion formed. The
hydroxyl of phenol 6 was then protected in acetate form, in the
presence of acetic anhydride in a two-phase volume to volume
solution of dichloromethane and an aqueous solution of 1 M
sodium hydroxide, to lead to product 7. Finally, the primary
alcohol function of compound 7 was oxidized in Swern
conditions to give acetaldehyde 2 which tautomerizes with the
enol form.
The synthesis of Quilamine HQ2−44 was thus envisaged by
Michael addition between homospermidine 1 and 2-vinyl-
quinolin-8-ol 8 (Figure 4).
2-Vinylquinolin-8-ol 8 was synthesized by intramolecular
dehydration of alcohol 6. Following this reaction, the use of 2
equiv of methanesulfonyl chloride, in the presence of
triethylamine in dichloromethane at room temperature,
protected the phenolic hydroxyl and activated the primary
3
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Bioconjugate Chemistry
Article
alcohol. Basic treatment of this intermediate led simultaneously
to the deprotection of the phenol group and the elimination of
methanesulfonic acid thus giving the desired vinylic compound
as oxidant and of bis(acetoxy)iodobenzene (BAIB) as co-
oxidant, aldehyde 10 was isolated in good yield (75%).
45
The reductive amination of this aldehyde with 2-amino-
quinolin-8-ol was achieved in one pot by using sodium
triacetoxyborohydride to reduce the imine generated in situ.
Protected intermediate 15 was isolated in good yield (73%) and
removal of the Boc-protecting groups under acidic conditions at
room temperature gave Quilamine HQ0−44 as the hydro-
chloride salt (Scheme 6).
8
in 96% yield (Scheme 3). This strategy proved to be much
a
Scheme 3. Synthesis of 2-Vinylquinolin-8-ol 8
The second modification envisaged concerned the nature of
the spacer, i.e., the introduction of a carbonyl (HQCO-44) and
a thiocarbonyl (HQCS-44) between homospermidine and
hydroxyquinoline.
Protected Quilamine HQCO-44 (16) was obtained in 80%
yield by peptide linkage between homospermidine 1 and
commercial 8-hydroxyquinaldinic acid in the presence of N,N′-
dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole
(HOBt) in dichloromethane (Scheme 7). Deprotection of the
amine functions of compound 16, by aqueous hydrochloric acid
in ethanol, led to Quilamine HQCO-44 as the hydrochloride
salt.
For Quilamine HQCS-44, the most obvious strategy is to
carry out a sulfuration of Quilamine HQCO-44. With this aim,
we began with amide 16 to obtain the corresponding thioamide
17. Different attempts were made by varying the sulfuration
reagent (Lawesson’s reagent (LR), phosphorus pentasulfide,
Davy’s reagent), the nature of the solvent (toluene,
tetrahydrofuran), and the temperature (from room temperature
to reflux). Unfortunately, regardless of the experimental
conditions used, thioamide 17 could not be obtained and
only an extremely insoluble salt was recovered. Several attempts
at sulfuration were also carried out by first protecting phenol
with different groups (acetate, ether, sulfonate), but only the
deprotection products were recovered at the end of the reaction
and the use of an excess of sulfuration reagent proved
ineffective.
a
Reagents: (a) MsCl (3 equiv), Et N (5 equiv), CH Cl , r.t., 2 h; (b)
3
2
2
NaOH (1 M), EtOH, reflux, 4 h.
more efficient than the method described by Yoneda who
obtained 2-vinylquinolin-8-ol 8 by a Wittig reaction between 2-
formylquinolin-8-ol and methyltriphenylphosphonium bromide
40
with a yield of only 30%.
Homospermidine 1 was then reacted with vinyl 8 in a
Michael addition carried out in methanol, in the presence of a
catalytic quantity of acetic acid, which led to the formation of
compound 9 with a yield of 64%. Deprotection of amines by
aqueous hydrochloric acid in ethanol at room temperature
resulted in the formation of Quilamine HQ2−44, in hydro-
chloride form (Scheme 4).
For Quilamine HQ0−44, we envisaged a reductive
amination between commercial 2-aminoquinolin-8-ol and
homospermidine 10 carrying an aldehyde function at the end
of the chain obtained by reduction of compound 5 (Figure 5).
Different attempts were made to reduce polyaminonitrile 5
by diisobutylaluminum hydride by varying the temperature of
the reaction (from −78 °C to room temperature) or the
number of reductor equivalents (from 1 to 3) but aldehyde 10
was never obtained. Treatment with sodium hypophosphite
41,42
and Raney nickel was also unsuccessful.
method we tried which was based on the reduction of nitrilium
So was the last
Kanbara has recently described the formation of several
thioamides from aldehydes and aromatic amines using the
43
46
ion by triethylsilane. We thus changed strategy and opted for
the synthesis of aldehyde 10 by oxidation of the corresponding
alcohol (Scheme 5).
The nitrogen atom of 4-aminobutan-1-ol was monoalkylated
by 4-bromobutanenitrile in acetonitrile in the presence of
potassium carbonate, at a temperature which must not exceed
Willgerodt-Kindler reaction. Sulfur was used as the
sulfuration agent and a catalytic quantity of sodium sulfide
47
was introduced as a base in order to start the reaction. By
applying these experimental conditions, we obtained the
desired thiocarbonylated derivative 17 starting from commer-
cial 2-formylquinolin-8-ol and homospermidine 1 (Scheme 8).
As for the other Quilamines, thioamide 17 was deprotected to
afford Quilamine HQCS-44 as the hydrochloride salt.
5
0 °C to avoid disubstitution (monitored by TLC). Amine 11
was protected by a Boc group in order to isolate compound 12.
The latter was hydrogenated under a pressure of 20 bar in the
presence of Raney nickel and ammonium hydroxide in ethanol
leading to aminoalcohol 13. A new protection of the amine
function gave alcohol 14. Oxidation of the hydroxyl in aldehyde
These four new second-generation Quilamines were fully
characterized by a combination of the usual techniques (IR,
44
13
1
MS, elemental analysis, C and H NMR) and fulfilled the
required purity for the biological evaluations.
1
0 was first attempted in Swern conditions or with pyridinium
Biological Evaluations. Selectivity for the Polyamine
Transport System (PTS). This second generation of Quil-
amines, HQX-44 (HQ2−44, HQ0−44, HQCO−44, and
HQCS−44) were screened for their antiproliferative activity
chlorochromate, but only degradation products were recovered
at the end of the reaction. In contrast, when a catalytic quantity
of (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) was used
a
Scheme 4. Synthesis of Quilamine HQ2-44
a
Reagents: (a) AcOH (cat), MeOH, 70 °C, 24 h; (b) HCl (6 M), EtOH, r.t., 12 h.
3
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Figure 5. Retrosynthesis of Quilamine HQ0−44.
a
Scheme 5. Synthesis of Aldehyde 10
a
Reagents: (a) 4-bromobutyronitrile (1 equiv), K CO (1.5 equiv), CH CN, 50 °C, 12 h; (b) (Boc) O (1.5 equiv), Et N, MeOH, r.t., 24 h; (c) H ,
2
3
3
2
3
2
Raney Ni, EtOH, NH OH, 20 atm, r.t., 12 h; (d) (Boc) O (2 equiv), Et N, MeOH, r.t., 24 h; (e) TEMPO (0.1 equiv), BAIB (1.1 equiv), CH Cl ,
4
2
3
2
2
r.t., 5 h.
a
Scheme 6. Synthesis of Quilamine HQ0-44
a
Reagents: (a) NaBH(OAc) (3 equiv), C H Cl , r.t., 12 h; (b) HCl (6 M), EtOH, r.t., 12 h.
3
2
4
2
a
Scheme 7. Synthesis of Quilamine HQCO-44
a
Reagents: (a) DCC (1.1 equiv), HOBt (1.1 equiv), CH Cl , r.t., 20 h; (b) HCl (6 M), EtOH, r.t., 12 h.
2
2
a
Scheme 8. Synthesis of Quilamine HQCS-44
a
Reagents: (a) S (1.6 equiv), Na S (0.15 equiv), DMF, 115 °C, 2 h; (b) HCl (6 M), EtOH, r.t., 12 h.
8
2
3
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Article
and cytotoxicity in CHO (Chinese Hamster Ovary) cells, which
Studies carried out by Phanstiel and Delcros using
anthracene−polyamine and naphthalene−polyamine deriva-
tives showed that the charge and the size of polyamines were
key parameters for the synthesis of compounds targeting the
48
exhibit high PTS activity, and the mutated derived-cell line
CHO-MG cells, devoid of the PTS (provided by Dr. W.
33
Flintoff, University of Western Ontario). CHO cells and the
CHO-MG cell line were chosen to evaluate the potential role of
the PTS as a selective transport system. We assessed the
antiproliferative activity of the various compounds by
measuring DNA content after Hoescht staining. The dose−
effect curves reflected both the PTS efficiency and the
cytostatic/cytotoxic effects of each compound. The release of
the cytoplasmic protein lactate dehydrogenase (LDH) into cell
supernatants was used as a marker of cell membrane disruption
correlated to cytotoxicity. The IC50 values obtained by fitting
the dose−effect curves are recorded in Table 1. In this study, 8-
27,28
PTS.
They put forward a model enabling the design of
1
cytotoxic N -substituted triamines specifically targeting the PTS
49
(Figure 6). Their work showed that the best selectivity was
a
Table 1. Selectivity of Quilamines for the PTS
Figure 6. Model of the structural constraints of the PTS proposed by
Phanstiel and Delcros.
4
9
a
IC50 (μM)
CHO-MG
IC50 ratio
Compound
CHO
CHO-MG/CHO
obtained with triamines having butyl or pentyl chains between
1
2
HQ1−44
HQ2−44
HQ0−44
HQCO−44
HQCS−44
1.4
1.8
0.4
35
344.5
>800
130
249
>500
325
>30
5.6
1
the N and N nitrogen atoms, and propyl or butyl chains
2
3
between the N and N nitrogen atoms. In fact, this model
shows three negative charges (probably amino acids deproto-
nated at physiological pH) at the PTS level with a spacing
corresponding to that of the alkyl chains between the nitrogen
atoms of the triamine (Figure 6). Moreover, the size of the
>800
140
25
8
-HQ
5.0
9.2
6.5
ICL670
8.0
1
1
spacer between the N nitrogen and the substituent R also plays
a
Effect of Quilamines HQX-44, 8-HQ, and ICL670 on cell viability in
CHO and CHO-MG cells. This denotes the (CHO-MG/CHO) IC50
a role in the selectivity. These authors suggested the presence
of a hydrophobic pocket within the recognition site of the PTS
which would favor affinity (Figure 6). Bergeron finally
confirmed these results in a study of the vectorization of iron
chelators by polyamines, in which he also showed that the
a
ratio, a measurement of PTS selectivity.
5
0,51
hydroxyquinoline and ICL670 (tridentate iron chelator,
Deferasirox, Exjade from Novartis Pharma) were used as
reference chelators, while Quilamine HQ1−44 was introduced
to compare the activities of second-generation Quilamines.
Quilamines differing in the length of the spacer (HQ0−44,
HQ1−44, and HQ2−44) showed increased antiproliferative
activities, with IC values between 0.4 and 1.8 μM, while those
differing in the chemical nature of the spacer (HQCO-44 and
HQCS-44) were less effective, with higher IC50 values. A
weaker effect of all the Quilamines on the proliferation of
CHO-MG cells was observed with IC50 values ranging from
charge of the chelating group was an essential factor.
This model could explain the drop in selectivity observed for
Quilamines HQCO-44 and HQCS-44 in relation to other
Quilamines. Indeed, the introduction of carbonyl and
thiocarbonyl groups prevents the nitrogen of the (thio)amide
function from being protonated at physiological pH. Thus, the
loss of an electrostatic interaction, required for recognition,
could be the reason for the decreased affinity of these
Quilamines for the PTS (Figure 7).
5
0
1
30 to more than 800 μM (Table 1). These results are
consistent with a low uptake of Quilamines into CHO-MG cells
and thus a weak activity in the absence of the PTS. The
reference chelators, 8-hydroxyquinoline and ICL670, with no
polyamine vector, enter cells by a route other than the PTS and
thus presented identical IC50 values for both CHO and CHO-
MG cells.
The ratio between the IC50 obtained for the CHO-MG and
CHO cell lines gives an indication of the degree of selectivity of
uptake by the PTS (Table 1). Two groups of Quilamines can
be clearly distinguished: Quilamines HQCO-44 and HQCS-44
with an IC50 ratio between 1 and 30, implying an average
selectivity for the PTS; and Quilamines HQ0−44, HQ1−44,
and HQ2−44 with an IC ratio higher than 200, indicating
Figure 7. Hypothesis to explain the loss of selectivity of Quilamines
HQCO-44 and HQCS-44 for the PTS according to the model of
Phanstiel and Delcros.
49
Quilamine HQ0−44 has an amidine function within its
structure. At physiological pH, this function is monocationic
with a distribution of the positive charge between the two
nitrogen atoms. This delocalization could be responsible for a
higher electrostatic interaction and thus a gain in selectivity
(Figure 8, left). Assuming that there is a hydrophobic pocket
within the PTS structure, increasing the size of the spacer in
Quilamine HQ2−44 could result in the heterocycle being
5
0
strong selectivity for the PTS.
Thus, although they contain the same polyamine vector
homospermidine chain), the second-generation Quilamines
(
display differences in selectivity for the PTS. Changing the size
of the spacer increased the selectivity while a decrease in
selectivity was observed when its chemical nature was changed.
Several factors can explain these results.
3
29
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Figure 8. Hypothetical modeling of the structure−selectivity relationship between the PTS and Quilamines HQ0−44 and HQ2−44 according to
49
the model of Phanstiel and Delcros.
closer to this cavity, thus increasing its interaction (Figure 8,
right).
biphasic for the three Quilamines with a cytostatic effect in the
concentration range 1−5 μM and a cytotoxic effect at higher
concentration marked by arrows in the figure. In contrast, only
one cytotoxic effect was observed with the reference chelator
HQ.
Taken together, our data suggest that changes made to the
size of the spacer have improved the selectivity of Quilamines
for the PTS unlike those made to the nature of the spacer.
Antiproliferative Action of Quilamines HQ0−44, HQ1−44,
and HQ2−44 on Human Tumor Cell Lines. In this work, as
the antiproliferative activity of Quilamines HQCO-44 and
HQCS-44 was shown to be low (Table 1), only HQ0−44 and
HQ2−44 were assessed on different human tumor cell lines
and compared to HQ1−44 as a biological activity reference.
The dose−effect curves, like that obtained after treatment of
HCT-116 cells by HQ1−44 presented in Figure 9, can be
The percentages of each of these two components and their
apparent IC values (IC50app-1 and IC50app-2), calculated from a
50
sigmoid fit of the curves, are given in Table 2. The minimum
Quilamine concentration triggering a rise in LDH release
compared to the control is also given.
As for Quilamine HQ1−44, CHO cells, which have the most
effective PTS, show the highest percentage of antiproliferative
component (C1) after treatment with Quilamines HQ0−44
and HQ2−44. For MDA, NCI-H727, and PC3 cells, the three
Quilamines exhibit no antiproliferative effect and only their
cytotoxicity is observed at higher concentrations.
The antiproliferative effect of Quilamines is most marked on
HCT-116, HuH7, and Caco-2 cell lines, which show
component C1 percentages between 14% (HQ0−44, Caco-
2
) and 62% (HQ1−44, HCT-116) and IC
-1 values
50app
between 1.3 μM (HQ1−44, HCT-116) and 5 μM (HQ0−
4
4, HCT-116). HCT-116 cells respond most effectively to
Quilamine HQ1−44 (component 1 of 62% and IC50app-1 of 1.3
μM) while for Caco-2, HuH7, and Hacat cells, the first
component only represents 35%, 30%, and 25%, respectively, of
the total effect. In contrast, the low antiproliferative component
observed for fibroblasts (25%), along with a higher IC50app-1 of
7
.5 μM, demonstrates the weak activity of Quilamine HQ1−44
on nontransformed cells.
The toxicity of Quilamines HQ0−44 and HQ2−44 seems
more marked than that of HQ1−44. In fact, for CHO cells, the
antiproliferative components of the three Quilamines are of the
same order (about 80%), but the minimum concentration
triggering a rise in LDH activity compared to the control
Figure 9. Cytostatic and cytotoxic effects of Quilamines HQ0−44
_
_
__
_ _ _ _
(
▲
), HQ1−44 (
⧫
), and HQ2−44 (- -■- -) compared to the
(
membrane damage) is observed at lower concentrations of 1.5
__ __
reference chelator 8-hydroxyquinoline, HQ (
◊
). The effect on cell
and 10 μM for Quilamines HQ0−44 and HQ2−44,
respectively, while Quilamine HQ1−44 is not toxic below 30
μM. Thus, these three Quilamines clearly present a marked
antiproliferative effect, but the difference in their toxicity
suggests that other phenomena are involved for each molecule.
In addition, the antiproliferative effect seems different
between the three Quilamines as they present different
percentages of component 1 for the lines studied. For example,
for Caco-2 cells, Quilamines HQ0−44, HQ1−44, and HQ2−
44 have a component 1 representing 14%, 35%, and 50%,
respectively, of the total effect. This is consistent with these
three Quilamines having different modes of action within the
cell.
viability was determined by measuring DNA content after Hoescht
staining and fluorescence analysis. The cytotoxic effect leading to cell
membrane damage was evaluated by measuring LDH leakage into the
cell supernatant. The arrows indicate the minimum Quilamine
concentration triggering a rise in LDH activity compared to the
untreated control.
parametrized into two components. The first indicates the
antiproliferative effect of the Quilamine (component 1, C1,
cytostatic) and the second its cytotoxicity (component 2, C2,
cytotoxic), linked to membrane damage leading to a release of
lactate dehydrogenase (LDH) into the supernatant. The
viability curves of Quilamines HQ0−44, HQ1−44, and
HQ2−44 were compared in the HCT116 cell line to that of
the reference iron chelator 8-hydroxyquinoline (HQ). The
dose−effect curves reported in Figure 9 were shown to be
Physicochemical Evaluations. Calcein Assay. The
cellular labile iron pool (LIP) consists of chelatable and
redox-active iron, which plays a key role as a crossroads of cell
3
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Article
Table 2. Percentages of the Two Components and Apparent IC Values Calculated for Quilamines HQ0-44, HQ1-44, and
5
0
a
HQ2-44 on Different Human Cell Lines
HQ0−44
HQ1−44
HQ2−44
b
b
b
cell lines
%C1
IC50app-1 (μM)
LDH (μM)
%C1
IC50app-1 (μM)
LDH (μM)
%C1
IC50app-1 (μM)
LDH (μM)
CHO
85
0.4
5
1.5
30
22
66
66
22
22
66
22
85
1.2
1.3
30
30
66
66
66
22
66
66
66
83
1.4
5
10
100
200
66
HCT-116
HuH7
55
62
36
18
5
30
2.7
25
15
Caco-2
Fibroblast
Hacat
14
1
35
4
50
2
25
7.5
25
7.5
25
15
66
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
25
1.5
25
1.5
N.D.
N.D.
N.D.
200
200
22
MDA
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
NCI-H727
PC3
200
a
b
N.D.: not detectable. Minimum Quilamine concentration triggering a rise in LDH activity compared to the untreated control.
iron metabolism. The ability of iron chelators to mobilize this
temporary iron pool bound to low molecular weight and low
iron affinity chelators (citrate, ascorbate, phosphate, and
adenosine triphosphate) is an essential factor influencing their
biological efficiency. According to previous work, the calcein
5
2,53
test can predict the iron-chelating efficiency of chelators.
Thus, an acellular calcein test was performed to compare the
potential ability of Quilamines bearing various chain length
linkers, HQ0−44, HQ1−44, HQ2−44, and the reference iron
chelator 8-hydroxyquinoline (HQ), to chelate this labile iron
pool at physiological pH. Recovered calcein fluorescence was
measured 4 h after the addition of the competitive chelators
and is reported as a function of their concentrations in Figure
31
1
0A. As previously reported, we confirmed the chelating
efficiency of Quilamine HQ1−44 (RC = 1 μM) is higher than
5
0
that of the reference chelator, HQ (RC = 3.7 μM). The ability
5
0
of Quilamine HQ2−44 (RC = 3 μM) to transchelate iron
5
0
from calcein was similar to that of 8-hydroxyquinoline while
that of HQ0−44 was lower (RC = 7.5 μM). Due to their
5
0
amino groups, polyamines are also able to chelate metallic
5
4,55
cations.
The stability constants of these chelates increase
with the number of amino groups and with the chain length of
5
6
the polyamine. Our previous results suggested the involve-
ment of the amine group of the polyamine chain in the
formation of iron complexes, as expected during the design and
31
synthesis of this Quilamine concept. Our present study
showed that the involvement of the first secondary amine in
Quilamines, in iron coordination, depended on the chain length
of the linker. When this amine was directly connected to the
hydroxyquinoline moiety (HQ0−44), the compound was
fluorescent and its chelating capacity was lower than that of
the reference chelator HQ. When this amine was linked by a
longer spacer (2 carbon atoms) to the chelating moiety, it did
not appear to be involved in iron coordination, since the
chelating ability of HQ2−44 was shown to be similar to that of
HQ. With an intermediary chain length spacer of one carbon
atom, our results suggested the involvement of the first amine
in iron coordination. So, HQ1−44 could exhibit the best iron
transchelating ability from calcein which could explain its
higher cytostatic effect.
Figure 10. Comparison of the iron-chelating efficiency of Quilamines
HQ0−44, HQ1−44, and HQ2−44 with the reference chelator HQ in
a cell-free system (A, calcein fluorescence measurements; B, HVA
autoxidation test).
HVA Assay. The effects of Quilamines and HQ on HVA
autoxidation are shown in Figure 10B. None of these
Quilamines induced a delay in the autoxidation reaction, but
all of them induced a significant decrease in the autoxidation
level in relation to their concentration. This behavior is
characteristic of metal chelating agents with very low (or no)
scavenging capability against the ROS (mainly the hydroxyl
36
radicals) involved in the HVA oxidation process.
3
31
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Article
HQ1−44 totally inhibited HVA autoxidation at concen-
trations higher than 100 μM, while only a partial decrease in
this process was observed with all other compounds in this
range of concentrations. The efficiency of the Quilamines to
inhibit HVA autoxidation was in the following order: HQ1−44
Nationale Contre le Cancer (LNCC, Ille et Vilaine/Loire
Atlantique), to the Association pour la Recherche sur le Cancer
(ARC), and to the Centre National de la Recherche
Scientifique (CNRS) for financial support. The cell viability
measurements on the different tumor cell lines were carried out
by the analytical platform ImPACcell (Imagerie pour une
>
HQ2−44 = HQ > HQ0−44. These results indirectly
confirmed the chelating efficiency of the various Quilamines
deduced from the calcein assay. The best chelating ability of
HQ1−44 to chelate iron could explain its higher antiprolifer-
ative efficiency.
analyse du contenu cellulaire) of the Structure Fed
Recherche BIOSIT in Rennes (BiogenOuest and Cancer
́
er
́
ative de
opole
́
̂
Grand Ouest network).
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■
CONCLUSION
■
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AUTHOR INFORMATION
Corresponding Authors
Phone: 00-33-2-23233861. Fax: 00-33-2-99540137. E-mail:
to cancer therapy. Retrospect and prospect. Part 1. Selective enzyme
■
*
inhibitors. Curr. Drug Targets 4, 537−564.
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francois.gaboriau@univ-rennes1.fr.
Phone: 00-33-2-51125406. Fax: 00-33-2-51125402. E-mail:
*
david.deniaud@univ-nantes.fr.
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Notes
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The authors declare no competing financial interest.
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■
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on the cytotoxicity and cellular transport of acridine. J. Med. Chem. 45,
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