Synthesis, cytotoxicity and structure-activity relationships between ester and amide functionalities in novel acridine-based platinum(II) complexes

Article abstract of DOI:10.1016/j.jinorgbio.2012.02.006

In order to improve the pharmacological profile of the anticancer drug cisplatin, several new acridine-based tethered (ethane-1,2-diamine)platinum(II) complexes connected by a polymethylene chain were synthetized. Activity-structure relationship between amide or ester functionalities was explored by changing acridine-9-carboxamide into acridine-9-carboxylate chromophore. The in vitro cytotoxicity of these new complexes was assessed in human colic HCT 116, SW480 and HT-29 cancer cell lines. Series of complexes bearing the acridine-9-carboxylate chromophore displayed higher cytotoxic effect than acridine-9-carboxamide complexes, with gradual effect according to the size of the polymethylene linker.

Full text of DOI:10.1016/j.jinorgbio.2012.02.006

Journal of Inorganic Biochemistry 110 (2012) 51–57
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis, cytotoxicity and structure-activity relationships between ester and amide
functionalities in novel acridine-based platinum(II) complexes
Florence Bouyer a, Johnny Moretto a, David Pertuit b, Anna Szollosi b, Marie-Aleth Lacaille-Dubois b,
Yves Blache c, Bruno Chauffert a, Nicolas Desbois a,
⁎
a
INSERM UMR U866, Facultés de Médecine & Pharmacie, 7 boulevard Jeanne d'Arc, BP 89700, 21079 Dijon cedex, France
Unité de Molécules d'Intérêt Biologique, EA 3660, Faculté de Pharmacie, 7 boulevard Jeanne d'Arc, BP 89700, 21079 Dijon cedex, France
Laboratoire Matériaux, Polymères, Interfaces, Environnement Marin, EA4323, ISITV, Avenue G. Pompidou, BP 56, 83162 La Valette Cedex, France
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 July 2011
Received in revised form 9 February 2012
Accepted 9 February 2012
Available online 20 February 2012
In order to improve the pharmacological profile of the anticancer drug cisplatin, several new acridine-based
tethered (ethane-1,2-diamine)platinum(II) complexes connected by a polymethylene chain were synthe-
tized. Activity-structure relationship between amide or ester functionalities was explored by changing
acridine-9-carboxamide into acridine-9-carboxylate chromophore. The in vitro cytotoxicity of these new
complexes was assessed in human colic HCT 116, SW480 and HT-29 cancer cell lines. Series of complexes
bearing the acridine-9-carboxylate chromophore displayed higher cytotoxic effect than acridine-9-
carboxamide complexes, with gradual effect according to the size of the polymethylene linker.
© 2012 Elsevier Inc. All rights reserved.
Keywords:
Acridine
Platinum(II) complexes
Cytotoxicity
Structure–activity relationship
1. Introduction
very few of them were successfully transferred to clinic. Such disap-
pointments focused basic scientists towards new concepts of drug de-
Platinum (Pt) compounds, such as cisplatin and oxaliplatin (Fig. 1)
exhibit antitumor activity by formation of DNA-Pt adducts, which led
to DNA structural alterations and activation of various cell death path-
ways such as apoptosis [1,2]. However, the development of cellular re-
sistance to platinum complexes is a common feature in mammalian
cells [3,4]. Mechanisms of resistance are multifactorial and may involve
low cellular uptake of the drug, or inactivation by high levels of cellular
low molecular weight thiol containing molecules, such as glutathione or
metallothioneine, and/or enhancement of DNA repair systems [4]. In
clinic, Pt complexes are involved in chemotherapeutic protocols against
various types of solid tumors including colorectal cancers (CRC) [1,5].
CRC is one of the most common cancer in western industrialized coun-
tries and a major cause of cancer death [6]. Most CRC develop through
ordered multi-step carcinogenic pathways which combine specific his-
tological lesions with genetic alterations [5–8]. Oxaliplatin, in associa-
tion with fluoropyrimidines, represent a mainstay of colorectal cancer
chemotherapy in both adjuvant and metastatic settings, whereas cis-
platin is poorly active in clinic [1,4,5,9].
sign, for example, on increasing DNA targeting by incorporation of
platinum-binding functionalities into suitable “carrier” ligands such as
9-anilinoacridine [12], 9-aminoacridine [13] or 1,10-phenanthroline
[14]. Several families of bifunctional molecules containing both
entities were developed such as bis(carboxylate)platinum(IV) [15]
complexes or [PtCl(ethylenediamine)-(1-[2-(acridin-9-ylamino)ethyl]-
1,3-dimethylthiourea)] (PT-ACRAMTU) [16] (Fig. 1), in which acridine
core drives platination into DNA minor groove [17]. Acridine orange-
ring has been rapidly recognized as a potential “carrier” [18–20], as it
could easily go across cell membrane, and intercalate into DNA struc-
ture [18]. Bifunctional molecules containing acridine orange moiety
tethered to cisplatin-like group by a polymethylene linker were
shown to have a dual mode of action in which the platinum moiety
binds covalently to DNA while the acridine orange moiety is intercalat-
ed one or two base pairs away [20]. Structure-activity relationship stud-
ies highlighted the influence of the chemical structure and substituted
state of the intercalator pharmacophore, as well as the size of the linker
chain connecting the two functionalities on the cytotoxicity of Pt
complexes.
Resistance to and high toxicity of oxaliplatin and cisplatin could be
circumvent by better understanding of the pathogenesis of colorectal
cancer as well as the development of novel platinum-based agents
[10,11]. Among the thousands of compounds already synthesized,
In our study, the synthesis, characterization and cytotoxic activity
of novel acridine-based tethered (ethane-1,2-diamine)platinum(II)
complexes connected by a polymethylene chain were reported.
The influence of ester or amide function at the 9-position of the acri-
dine moiety and the influence of the length of polymethylene chain
having n=2 to n=6 in relation to the cytotoxic activity were also
investigated.
⁎
Corresponding author. Tel.: +33 3 80 39 32 52; fax: +33 3 80 39 33 00.
E-mail address: nicolas.desbois@u-bourgogne.fr (N. Desbois).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2012.02.006

52
F. Bouyer et al. / Journal of Inorganic Biochemistry 110 (2012) 51–57
O
Cl
Cl
NH3
NH3
O
NH2
NH2
Pt
Pt
O
O
Oxaliplatin
Cisplatin
O
NH2
H2N
Cl
2+
H2
Pt
O
COO(CH2)3CH3
COO(CH2)3CH3
N
S
Cl
Cl
H
Pt
O
N
H3CHN
N
N
CH3
H2
NH
O
PT-ACRAMTU
Bis(carboxylate)platinum(IV) complex
Fig. 1. Chemical structures of anticancer platinum complexes either used in clinic: cisplatin, oxaliplatin ; or under preclinical assessment: PT-ACRAMTU, bis(carboxylate)platinum(IV)
complex.
2. Experimental section
75 MHz) δ 169.3, 149.6, 143.8, 132.3, 129.6, 128.2, 126.6,
123.7, 60.6, 38.2, 33.2.
2.1. Material and methods
2c: Yield : 46%. mp 139–141 °C. 1H NMR (CDCl3, 300 MHz) δ 7.98
(d, 2H, J=9 Hz), 7.92 (d, 2H, J=8 Hz), 7.65 (m, 2H), 7.46
(m, 2H), 3.59 (m, 4H), 1.76 (m, 2H), 1.63 (m, 2H). 13C NMR
(CDCl3, 75 MHz) δ 167.3, 148.0 141.7, 130.6, 128.7, 126.7,
125.2, 122.2, 61.6, 39.9, 29.8, 26.0.
All column chromatography was performed with Merck neutral
aluminum oxide 90 standardized (63–200 μm) or silica gel (Acros or-
ganic, 60 Å, 35–70 μm). All thin layer chromatography was performed
on Fluka aluminum oxide plates (with fluorescent indicator 254 nm)
or Merck silica gel 60 F254 plates. Melting points were determined on
a Reichert-Jung-Koffler apparatus and were not corrected. NMR spec-
tra (300 MHz for 1H, 75 MHz for 13 C, and 128 MHz for 195Pt) were
recorded on Bruker Avance 300 and 600 instruments using the indi-
cated solvents. Chemical shifts were reported in ppm (δ). High
resolution mass spectrometry (HRMS) was obtained with a Brucker
MicroTOF-Q spectrometer. Elemental analyses were performed with
an Elemental Analyser Thermo electron Flash EA 1112 at the
“Plateforme d'Analyse Chimique et de Synthèse Moléculaire de l'Université
de Bourgogne (PACSMUB).”
2d: [21] Yield : 47%. mp 169–171 °C.
2e: [22]Yield : 80%. mp 108–110 °C.
2f: [23]Yield : 54%. mp 190–192 °C (lit.[23] 184–186 °C).
2g: [24]Yield : 69%. mp 133–135 °C (lit.[24] 133–135 °C).
2h: Yield : 72%. mp 96–98 °C. 1H NMR (CDCl3, 300 MHz) δ 8.27
(d, 2H, J=9 Hz), 8.02 (d, 2H, J=9 Hz), 7.81 (m, 2H), 7.61
(m, 2H), 4.69 (t, 2H, J=7 Hz), 3.74 (t, 2H, J=7 Hz), 2.01
(m, 2H), 1.75 (m, 2H). 13C NMR (CDCl3, 75 MHz) δ 167.6, 148.6,
137.2, 130.4, 129.8, 127.2, 125.1, 122.3, 66.3, 62.0, 29.1, 25.3.
2i: Yield 45%. mp 219–221 °C. 1H NMR (CDCl3, 300 MHz) δ 8.25 (d,
2H, J=9 Hz), 8.00 (d, 2H, J=9 Hz), 7.78 (m, 2H), 7.57 (m, 2H),
4.50 (2H, J=7 Hz), 3.67 (t, 2H, J=7 Hz), 1.94 (m, 2H), 1.67 (m,
4H). 13C NMR (CDCl3, 75 MHz) δ 167.6, 148.7, 137.2; 130.3,
129.9, 127.1, 125.1, 122.3, 66.3, 62.5, 32.2, 28.5, 22.3.
2j: Yield 56%. mp 55–57 °C. 1H NMR (CDCl3, 300 MHz) δ 8.27 (d,
2H, J=9 Hz), 8.02 (d, 2H, J=9 Hz), 7.80 (m, 2H), 7.60 (m,
2H), 4.65 (t, 2H, J=7 Hz), 3.65 (t, 2H, J=7 Hz), 1.90 (p, 2H,
J=7 Hz), 1.48 (m, 6H). 13C NMR (CDCl3, 75 MHz) δ 167.6,
148.6, 137.3; 130.4, 129.9, 127.1, 125.1, 122.3, 66.4, 62.6,
32.6, 28.7, 25.8, 25.4.
2.2. Synthesis
2.2.1. Synthesis of alcohols 2a–j
A mixture of acridine-9-carboxylic acid hydrate (1) (97%, 0.50 g,
2.17 mmol), thionyl chloride (10 mL) and dry N,N-dimethylforma-
mide (one drop) was heated to reflux for 1 h. The thionyl chloride
was then removed in vacuo. For 2a–e, the residue was dissolved in
dry dichloromethane (10 mL), the solution cooled to 0 °C, a solution
of aminoalcohol (2.82 mmol) and triethylamine (0.90 mL,
6.51 mmol) in dry dichloromethane (10 mL) was added under N2
and stirred to room temperature for 2 h. For 2f–j, the residue was dis-
solved in diol (52.0 mmol) and stirred to 100 °C for 2 h. The reaction
was diluted with dichloromethane (30 mL), washed with 10% sodium
bicarbonate solution (2×20 mL) and dried (Na2SO4). The solvent was
removed in vacuo to provide crude product which was purified on
aluminum oxide eluting with 96% dichloromethane and 4% methanol
(for 2a–e) or on aluminum oxide eluting with 98% dichloromethane
and 2% methanol (for 2f–j).
2.2.2. Synthesis of ligands 3a–j1
The above alcohols 2a–j (0.55 mmol) were dissolved in dry pyridine
(5 mL) and methanesulfonylchloride (0.1 mL, 1.29 mmol) was added.
The solution was stirred at 20 °C under N2 for 1 h then chilled on ice
and treated with water (0.16 mL), followed by 1,2 diaminoethane
(1.5 mL). The mixture was stirred 20 h at 20 °C and then the solvents
were removed. The residue was dissolved in dichloromethane
(30 mL), and the organic phase was washed with 1 M Na2CO3 (50 mL)
and water (50 mL). After drying over Na2SO4, the solvent was
removed and the residue was chromatographed on silica eluting with
MeOH/NH4OH (97/3, v/v). The pure product was converted to the
trihydrochloride.
2a: Yield : 65%. mp 244–246 °C. 1H NMR (CDCl3, 300 MHz) δ 8.25
(d, 2H, J=8.5 Hz), 8.11 (d, 2H, J=8.5 Hz), 7.81 (m, 2H), 7.61
(m, 2H), 4.02 (m, 2H), 3.91 (m, 2H). 13C NMR (DMSO,
75 MHz) δ 166.2, 148.2, 142.6, 130.7, 129.2, 126.7, 125.9,
121.9, 59.8, 42.1.
3a: Yield 20%. mp 220–222 °C.1H NMR (D2O, 300 MHz) δ 8.30 (m,
6H), 7.96 (m, 2H), 4.08 (t, 2H, J=7 Hz), 3.58 (m, 4H), 3.47 (m,
2b: Yield : 88%. mp 212–216 °C. 1H NMR (CD3OD, 300 MHz) δ 8.17
(d, 2H, J=9 Hz), 8.07 (d, 2H, J=9 Hz), 7.89 (m, 2H), 7.66 (m,
2H), 3.74 (m, 4H), 1.99 (p, 2H, J=7 Hz). 13C NMR (CD3OD,
1
Although the elemental analysis values for “C” are somewhat unsatisfactory for
most of the compounds, the spectroscopic analysis data (1H, 13C, 195Pt NMR, HRMS
presented in the supporting information) reasonably support the formula.)

F. Bouyer et al. / Journal of Inorganic Biochemistry 110 (2012) 51–57
53
Scheme 1. Reagents and conditions : (i) 1) SOCl2, DMF, Δ, 2) NH2(CH2)nOH, CH2Cl2, NEt3, room temperature or OH(CH2)nOH, 100 °C; (ii) 1) MsCl, Pyridine, Room temperature,
2) NH2(CH2)2NH2, room temperature, 3) HCl 1 N in diethyl ether; (iii) H2O, Na2CO3, K2PtCl4, room temperature.
2H). 13C NMR (D2O, 75 MHz) δ 167.8, 155.5, 142.1, 136.1,
128.9, 125.8, 122.2, 119.4, 46.7, 44.5, 36.4, 35.4. Anal. Calc. for
13 C NMR (D2O, 75 MHz) δ 165.7, 146.8, 139.8, 137.9, 129.5,
126.1, 122.1, 119.8, 67.7, 47.6, 44.1, 35.4, 24.9, 22.3. Anal.
Calc. for C20H23N3O2.3HCl.3H2O requires: C, 47.96; H, 6.44; N,
8.39. Found: C, 48.14; H, 6.32; N, 8.51.
C18H20N4O.3HCl.3H2O requires: C, 45.82; H, 6.20; N, 11.87.
Found: C, 46.02; H, 5.99; N, 11.74.
3b: Yield 55%. mp 199–201 °C. 1H NMR (D2O, 300 MHz) δ 8.23 (m,
6H), 7.93 (m, 2H), 3.82 (t, 2H, J=7 Hz), 3.51 (m, 6H), 2.27
(p, 2H, J=7 Hz). 13C NMR (D2O, 75 MHz) δ 165.7, 149.9,
139.5, 137.8, 129.4, 126.1, 121.8, 119.9, 46.1, 44.4, 37.5, 35.7,
25.6. Anal. Calc. for C19H22N4O.3HCl.3H2O requires: C, 46.97;
H, 6.43; N, 11.53. Found: C, 47.10; H, 6.59; N, 11.97.
3i: Yield 31%. mp 208–210 °C. 1H NMR (D2O, 300 MHz) δ 8.20
(m, 4H), 8.14 (d, 2H, J=9 Hz), 7.86 (m, 2H), 4.79 (m, 2H),
3.37 (m, 4H), 3.11 (t, 2H, J=7 Hz), 1.95 (p, 2H, J=7 Hz), 1.78
(p, 2H, J=7 Hz), 1.53 (p, 2H, J=7 Hz). 13 C NMR (D2O,
75 MHz) δ 165.9, 146.7, 139.8, 137.7, 129.4, 126.0, 122.0,
119.9, 68.3, 47.9, 44.0, 35.4, 27.2, 25.2, 22.2. Anal. Calc.
for C21H25N3O2.3HCl.H2O requires: C, 52.67; H, 6.31; N, 8.78.
Found: C, 50.91; H, 6.38; N, 9.02.
3c: Yield 39%. mp 130–132 °C. 1H NMR (CD3OD, 300 MHz) δ 8.21
(d, 2H, J=9 Hz), 8.10 (d, 2H, J=9 Hz), 8.02 (m, 2H), 7.82 (m,
2H), 3.82 (m, 2H), 3.48 (m, 2H), 3.27 (m, 4H), 2.00 (m, 4H).
13C NMR (CD3OD, 75 MHz) δ 169.7, 148.5, 142.2, 132.6, 128.7,
128.5, 125.7, 122.8, 47.6, 41.5, 39.7, 38.2, 27.0, 25.5. Anal.
Calc. for C20H24N4O.3HCl.2H2O requires: C, 49.85; H, 6.48; N,
11.63. Found: C, 49.01; H, 6.43; N, 11.41.
3j: Yield 31%. mp 139–141 °C. 1H NMR (D2O, 300 MHz) δ 8.15
(m, 4H), 8.03 (d, 2H, J=9 Hz), 7.80 (m, 2H), 4.72 (t, 2H,
J=7 Hz), 3.40 (m, 4H), 3.11 (t, 2H, J=7 Hz), 1.90 (p, 2H,
J=7 Hz), 1.71 (p, 2H, J=7 Hz), 1.48 (m, 4H). 13C NMR (D2O,
75 MHz) δ 165.6, 146.7, 139.4, 137.8, 129.4, 125.9, 121.7,
119.7, 68.7, 48.1 44.1, 35.4, 27.5, 25.4, 25.3, 24.7. Anal. Calc.
for C22H27N3O2.3HCl.4H2O requires: C, 48.31; H, 7.00; N, 7.68.
Found: C, 46.44; H, 7.06; N, 7.88.
3d: Yield 25%. mp 186–188 °C. 1H NMR (D2O, 300 MHz) δ 8.31
(m, 6H), 7.97 (m, 2H), 3.72 (t, 2H, J=7 Hz), 3.41 (m, 4H),
3.18 (m, 2H), 1.84 (m, 4H), 1.59 (p, 2H, J=7 Hz). 13C NMR
(D2O, 75 MHz) δ 166.1, 150.7, 140.0, 137.9, 129.3, 126.1,
122.4, 119.9, 48.0, 44.0, 40.1, 35.4, 27.8, 25.2, 23.3. Anal. Calc.
for C21H26N4O.3HCl.2H2O requires: C, 50.87; H, 6.71; N, 11.30.
Found: C, 48.40; H, 6.79; N, 11.83.
2.2.3. Synthesis of complexes 4a–j1
The trihydrochloride 3a–j (0.20 mmol) was added to a solution of
K2PtCl4 (84 mg, 0.20 mmol) in water (4 mL). The pH was adjusted to
8 with 2 M sodium bicarbonate solution and the mixture was stirred
in the dark for 24 h. A solution of 5% aqueous KCl (20 mL) was then
added, and the mixture was stirred for 90 min. The resulting precipi-
tate was collected, washed several times with water, acetone and
dried to give pure product 4a–j.
3e: Yield 40%. mp 137–139 °C. 1H NMR (D2O, 300 MHz) δ 8.16
(m, 6H), 7.87 (m, 2H), 3.68 (t, 2H, J=7 Hz), 3.43 (m, 4H),
3.17 (t, 2H, J=7 Hz), 1.78 (m, 4H), 1.51 (m, 4H). 13C NMR
(D2O, 75 MHz) δ 165.7, 150.7, 139.7, 137.9, 129.3, 126.1,
122.1, 119.8, 48.2, 44.1, 40.3, 35.4, 28.0, 25.8, 25.5, 25.3. Anal.
Calc. for C22H28N4O.3HCl.1.5H2O requires: C, 52.75; H, 6.84;
N, 11.19. Found: C, 51.43; H, 6.95; N, 11.11.
4a: Yield 69%. mp 221–223 °C. HRMS calcd. for C18H20Cl2N4OPt
[M+Na]+ 596.0555 found 596.05613.195Pt NMR (DMF,
600 MHz) not obtained. Anal. Calc. for C18H20Cl2N4OPt.H2O re-
quires: C, 36.50; H, 3.74; N, 9.46. Found: C, 35.03; H, 3.64; N,
9.02.
4b: Yield 70%. mp >260 °C. HRMS calcd. for C19H22Cl2N4OPt
[M+Na]+ 610.06963 found 610.07147. 195Pt NMR (DMF,
128 MHz) δ−2342. Anal. Calc. for C19H22Cl2N4OPt.2H2O re-
quires: C, 36.55; H, 4.20; N, 8.97. Found: C, 35.50; H, 4.02;
N, 8.69.
4c: Yield 70%. mp 250–252 °C. HRMS calcd. for C20H24Cl2N4OPt
[M+Na]+ 624.08687 found 624.08651. 195Pt NMR (DMF,
128 MHz) δ−2341. Anal. Calc. for C20H24Cl2N4OPt.3H2O re-
quires: C, 36.59; H, 4.61; N, 8.53. Found: C, 34.95; H, 4.48; N,
8.83.
3f: Yield 20%. mp 214–216 °C. 1H NMR (D2O, 300 MHz) δ 8.26 (m,
6H), 7.89 (m, 2H), 5.10 (t, 2H, J=7 Hz), 3.71 (t, 2H, J=7 Hz),
3.46 (m, 2H), 3.34 (m, 2H). 13C NMR (D2O, 75 MHz) δ 164.7,
145.7, 139.6, 138.0, 129.6, 126.1, 122.2, 119.8, 63.0, 46.3, 44.6,
35.3. Anal. Calc. for C18H19N3O2.3HCl.3H2O requires: C, 45.73;
H, 5.97; N, 8.89. Found: C, 47.01; H, 6.04; N, 8.99.
3g: Yield 29%. mp 167–169 °C. 1H NMR (D2O, 300 MHz) δ 8.11
(m, 4H), 8.01 (d, 2H, J=9 Hz), 7.77 (m, 2H), 4.83 (t, 2H,
J=7 Hz), 3.39 (m, 4H), 3.27 (t, 2H, J=7 Hz), 2.34 (p, 2H,
J=7 Hz). 13C NMR (D2O, 75 MHz) δ 165.0, 146.1, 139.2,
137.9, 129.5, 125.9, 121.7, 119.7, 65.0, 45.0, 44.3, 35.4, 24.8.
Anal. Calc. for C19H21N3O2.3HCl.3H2O requires: C, 46.88; H,
6.21; N, 8.63. Found: C, 45.13; H, 6.02; N, 8.74.
3h: Yield 25%. mp 205–207 °C. 1H NMR (D2O, 300 MHz) δ 8.23
(m, 6H), 7.90 (m, 2H), 4.81 (m, 2H), 3.37 (m, 4H), 3.19 (t, 2H,
J=7 Hz), 2.01 (p, 2H, J=7 Hz), 1.90 (p, 2H, J=7 Hz).
4d: Yield 71%. mp 254–256 °C. HRMS calcd. for C21H26Cl2N4OPt
[M+Na]+ 638.10253 found 638.10332. 195Pt NMR (DMF,

54
F. Bouyer et al. / Journal of Inorganic Biochemistry 110 (2012) 51–57
128 MHz) δ−2339. Anal. Calc. for C21H26Cl2N4OPt.0.5H2O re-
acetic acid. The intensity of coloration was determined by the mea-
surement of absorbance by spectrophotometry (UVM 340, Bioserv)
at λ=570 nm. Each concentration measurement was conducted in
triplicate from three independent experiments. Results were
expressed as concentration-response curves, representing the per-
centage of cytotoxicity according to the concentration of the drug.
From these curves, the 50% Inhibitory Concentration (IC50), repre-
senting the concentration which inhibits 50% of cell growth, was cal-
culated after linearization.
quires: C, 40.33; H, 4.35; N, 8.96. Found: C, 38.51; H, 4.56; N,
9.36.
4e: Yield 72%. mp 248–250 °C. HRMS calcd. for C22H28Cl2N4OPt
[M+Na]+ 652.11670 found 652.11796. 195Pt NMR (DMF,
128 MHz) δ−2341. Anal. Calc. for C22H28Cl2N4OPt.3H2O re-
quires: C, 38.60; H, 5.01; N, 8.18. Found: C, 37.45; H, 4.91; N,
8.12.
4f: Yield 43%. mp 239–241 °C. HRMS calcd. for C18H19Cl2N3O2Pt
[M+Na]+ 597.03958 found 597.03897. 195Pt NMR (DMF,
128 MHz) δ−2343. Anal. Calc. for C18H19Cl2N3O2Pt.2H2O re-
quires: C, 35.36; H, 3.79; N, 6.87. Found: C, 33.68; H, 3.63; N, 6.57.
4g: Yield 46%. mp 207–209 °C. HRMS calcd. for C19H21Cl2N3O2Pt
[M+Na]+ 611.05524 found 611.05716. 195Pt NMR (DMF,
128 MHz) δ−2343. Anal. Calc. for C19H21Cl2N3O2Pt.H2O re-
quires: C, 37.57; H, 3.82; N, 6.92. Found: C, 36.06; H, 3.72; N, 6.94.
4h: Yield 68%. mp 163–165 °C. HRMS calcd. for C20H23Cl2N3O2Pt
[M+Na]+ 625.07090 found 625.07319. 195Pt NMR (DMF,
128 MHz) δ−2342. Anal. Calc. for C20H23Cl2N3O2Pt requires:
C, 39.81; H, 3.84; N, 6.96. Found: C, 40.35; H, 3.68; N, 7.17.
4i: Yield 60%. mp 156–158 °C. HRMS calcd. for C21H25Cl2N3O2Pt
[M+Na]+ 639.08656 found 639.08972. 195Pt NMR (DMF,
128 MHz) δ−2338. Anal. Calc. for C21H25Cl2N3O2Pt requires:
C, 40.85; H, 4.08; N, 6.81. Found: C, 42.64; H, 4.04; N, 6.96.
4j: Yield 52%. mp 129–131 °C. HRMS calcd. for C22H27Cl2N3O2Pt
[M+Na]+ 653.10221 found 653.10475. 195Pt NMR (DMF,
128 MHz) δ−2338. Anal. Calc. for C22H27Cl2N3O2Pt.H2O re-
quires: C, 40.67; H, 4.50; N, 6.47. Found: C, 41.34; H, 4.35; N, 6.66.
3. Results and discussion
3.1. Preparation and spectroscopic properties
Synthesis of alcohols was carried out as shown in Scheme 1.
Acridine-9-carboxylic acid 1 was coupled selectively with appropriate
aminoalcohol or diol via the thionyl chloride. To avoid dimerization
with diols, it was necessary to heat at 100 °C during 2 h. The resulting
alcohols 2a–j were activated with methanesulfonyl chloride and trea-
ted with an excess of 1,2-diaminoethane to give the free instable
bases of the ligands 3a–j. After quick chromatography purification
on silica gel, these diamines were isolated as trihydrochloride salts
3a–j by treatment of HCl 1 M in diethyl ether. Each ligand was charac-
terized by 1H and 13C NMR spectroscopic methods. The platinum
complexes 4a–j were prepared by addition of an aqueous solution
of K2PtCl4 to a solution of the ligand trihydrochlorides 3a–j in water
adjusted to basic pH by addition of sodium bicarbonate. Pure prod-
ucts were isolated directly by precipitation from the reaction mixture.
In each complex, 195Pt NMR confirmed Pt coordination to the di-
amine function. The 195Pt chemical shift values (Table 1) are charac-
teristic of platinum(II) complexes with a diamine associated with
two chloride atoms. The HRMS were consistent with the proposed
empirical formulas. For compound 4a, 195Pt NMR signal was not
obtained, probably because of poor solubility but the presence of plat-
inum in the chemical structure of compound 4a was clearly con-
firmed by accurate mass measurement.
2.3. Cell lines and culture conditions
Human colon cancer cell lines HCT 116, SW480 and HT-29 were
obtained from the American Type Culture Collections (Manassas, VA,
United States), and were cultured in RPMI 1640 medium (Biowhittaker,
France) supplemented with 10% fetal bovine serum (Biowhittaker,
France) in a 5% CO2 atmosphere. All cell lines were maintained as expo-
nentially growing monolayers in mycoplasma free culture condition
checked by polymerase chain reaction (PCR) analysis (PCR Mycoplasma
Test Kit I/C, PromoKine, PromoCell France).
Table 2
Cytotoxicity of acridine-based derivates platinated (4a–j) or not (3a–j), cisplatin and
oxaliplatin, and DMF in human colon HCT116, SW480 and HT29 cells after a 72 h-treat-
ment. Results are expressed as IC50, each value representing the mean SD of at least 3
different experiments (statistics: acridine compounds versus cisplatin or oxaliplatin:
** : pb0.01; *** pb0.001) (nd=not determinable).
2.4. Assay of cytotoxicity in cancer cell lines
The newly synthetized acridine-based tethered (ethane-1,2-
diamine) derivatives connected by a polymethylene spacer (3a–j), cis-
platin (Sigma-Aldrich, France), and oxaliplatin (Sanofi-Aventis, France)
were diluted in sterile physiological serum (Aguettant Co., France),
whilst new acridine-based tethered (ethane-1,2-diamine)Pt(II) com-
plexes connected by a polymethylene chain (4a–j) were diluted into
dimethylformamide (DMF) (Sigma, France). In cell culture, maximum
concentration of DMF did not exceed 3% in the medium.
HCT 116, SW480 and HT-29 were seeded in 96-well plates at a
density of 20,000 cells per well. At sub-confluence, cells were treated
for 72 h by increasing concentrations (from 0 to 500 μM) of the differ-
ent compounds. After incubation, cells were washed in PBS 1×, fixed
in pure ethanol, stained with crystal violet (1%), and eluted in 33%
Compounds
IC50 (μM)
HCT116
SW480
HT29
3a
nd
nd
nd
3b
nd
nd
nd
3c
nd
nd
nd
3d
nd
nd
nd
3e
nd
nd
nd
4a
nd
nd
nd
4b
nd
nd
nd
4c
4d
4e
nd
nd
nd
232.3 15.8
229.6 24.6
nd
nd
nd
nd
3f
3g
3h
3i
3j
4f
4g
4h
352.2 13.2 *
126.3 9.2 **
153.1 6.1 **
51.8 4.6 ***
37.4 7.1 ***
nd
242.0 10.9 **
198.7 9.8 **
249.8 12.2 **
128.6 7.9 **
453.2 6.9
478.3 9.3
372.3 8.9
465.6 14.5
108.2 5.6
113.4 7.4
34.5 3.2
17.3 6.2
nd
nd
114.3 6.1 **
201.4 4.5 **
49.8 3.1 ***
46.8 7.1 ***
76.3 6.2 ***
193.2 4.2 **
187.3 7.4 **
243.6 6.0
190.3 7.8 **
373.2 6.4
308.1 2.4
313.9 7.1
Table 1
195
NMR
Pt of platinum complexes 4a–j.
Compounds
X=NH
195Pt NMR
δ (ppm)
Compounds
X=O
195Pt NMR
δ (ppm)
nd
231.1 10.6
nd
93.4 6.6
nd
4a, n=2
4b, n=3
4c, n=4
4d, n=5
4e, n=6
Not obtained
−2342
4f, n=2
4g, n=3
4h, n=4
4i, n=5
4j, n=6
−2343
−2343
−2342
−2338
−2338
4i
4j
−2341
Cisplatin
oxaliplatin
DMF
−2339
−2341
nd
352.8 6.9

F. Bouyer et al. / Journal of Inorganic Biochemistry 110 (2012) 51–57
55
3.2. Cytotoxicity in cancer cell lines
transplanted into mice. From our results, we could suggest that
short linkage connecting the acridine chromophore to Pt-moiety
was probably not the major parameter determining poor biological
effect as compounds with hexamethylene linker did not always ex-
hibit higher cytotoxicity. Thus, in such series of compounds, the size
of the spacer link might not modulate the noncovalent intercalation,
as well as selectivity of platination. However, different series
of acridine-2- and -4-carboxamide linked analogues of (ethane-1,2-
diamine)Pt(II) complexes have also been synthetized, and the platinum
complexes were generally more cytotoxic in vitro than nonplatinated
analogues [13,26]. Acridine-4-carboxamide compounds were more cy-
totoxic than acridine-2-carboxamide tethered Pt(II) complexes in
P388 leukemia cells in vitro [26] ; and acridine-4-carboxamide com-
plexes with the shorter linker chain lengths were generally the most ac-
tive [13] in different cell lines sensitive or resistant to cisplatin [13,26].
In spite of that, addition of 9-amino substituent on acridine-4-
carboxamide led to decreased cytotoxic activities in HeLa cells [27].
Interestingly, the replacement of amide by ester functionalities
in the series of novel acridine-9-carboxylate tethered (ethane-1,2-
diamine) complexes platinated (4f–j) or not (3f–j) and connected by
a polymethylene chain increased their cytotoxic effect which was
higher than cisplatin or oxaliplatin in the three colic HCT 116, SW480
and HT-29 cell lines (Table 2). The influence of the polymethylene link-
er in platinated complexes was dependent on the cell line: in HCT 116
and SW480, 4j, having the hexamethylene chain, remained the most cy-
totoxic, whilst in HT-29, the most sensitive cell line to reference Pt com-
pounds, 4f, with the dimethylene linker was the most active (Table 2). A
relationship between cytotoxicity and polymethylene chain length in
platinum compounds was also reported by Silva et al. [28]. Furthermore,
the non platinated ligands (3f–j) displayed powerful cytotoxic effect com-
pared to cisplatin and oxaliplatin, with a gradual effect according to the
Acridine-based derivatives platinated or not were tested in three
human colic cancer cell lines HCT 116, SW480 and HT-29, as colorec-
tal cancer is a clinical indication of Pt compounds. Oxaliplatin was
used as the reference molecule as referred to clinical protocols of
CRC, and cisplatin as the reference of Pt derivate family. Despite dif-
ferences in mechanisms of action and DNA-Pt adducts formation, cis-
platin and oxaliplatin displayed similar cytotoxic properties in each
cell line tested in our experimental conditions (Table 2). Indeed, our
cytotoxic assay was performed under stringent conditions, as treat-
ments were administrated in subconfluent cells. According to this
protocol, the two series of acridine-based complexes (3a–j and 4a–
j) displayed various cytotoxic properties (Table 2 and Fig. 2) in the
three colic cancer cell lines, which are differently sensitive to the ref-
erence molecules. SW480 was quite resistant to cisplatin and oxali-
platin compared to HT-29 and HCT 116 (Table 2). HT-29 was the
most sensitive cell line, with oxaliplatin disclosing more cytotoxic ef-
fect than cisplatin. HCT 116 exhibited intermediate sensibility, with
similar IC50 for both molecules (Table 2).
This study reported, for the first time, the synthesis and in vitro
evaluation of series of compounds containing the acridine-9-
carboxamide chromophore (3a–e and 4a–e). Unfortunately,
acridine-9-carboxamide tethered (ethane-1,2-diamine)Pt(II) com-
plexes (4a–e) did not display significant cytotoxicity, which remained
lower than cisplatin and oxaliplatin, especially in HCT 116 and HT-29
cells (Table 2). The non platinated counterparts (3a-e) were totally
inactive. Such findings corroborate previous testing of a cisplatin-
type complex tethered to acridine intercalator via one methylene
chain (at the 9-position of acridine skeleton) [25]. Such compound
demonstrated only low antitumor activity against P388 cells
Fig. 2. Concentration-response curves of cytotoxicity of acridine-9-carboxylate tethered (ethane-1,2-diamine) derivates connected by di- to hexamethylene chain (A) 3f (●) 3 g (○)
3 h (▼) 3i (△) 3j (■), and of reference molecules cisplatin (□) and oxaliplatin (♦) in human colic HCT116 (B), SW480 (C) and HT29 (D) cell lines after 72 h of drug incubation.
Cytotoxicity values are represented by the mean SD of 3 independent experiments.

56
F. Bouyer et al. / Journal of Inorganic Biochemistry 110 (2012) 51–57
Fig. 3. Relationship between polymethyleme chain length (n) of acridine-9-carboxylate tethered (ethane-1,2-diamine) derivates connected by di- to hexamethylene chain (A) and
IC50 values calculated from cytotoxic assay after 72 hours of drug incubation in human colic HCT116 (B), SW480 (C) and HT29 (D). Pearson's correlation coefficients were indicated
on each graph. IC50 values are represented by the mean SD of 3 independent experiments.
size of the polymethylene spacer (Table 2 and Fig. 2). Cytotoxicity in-
creased with the length of the polymethylene spacer, as highlighted by
the negative correlation between the number of carbon in the alkyl
chain and IC50 of compounds in all cell lines (r²=−0.8828; r²=
−0.8394; r²=−0.6299 in HCT 116, SW480, and HT-29 respectively)
(Fig. 3). Likewise, cytotoxic properties were high whatever the sensitivity
of the cell line studied (Table 2). In final, theses new derivatives with
acridine-9-carboxylate chromophore could potentially generate promis-
ing anticancer drugs. Nevertheless, no synergy between platination
and intercalation were described in acridine-9-carboxylate tethered
(ethane-1,2-diamine)platinum (II) complexes compared to unplati-
nated ligands. Physical and molecular analyses will be conducted to un-
derstand such phenomenon at the DNA level.
Future investigations will examine interactions at the DNA levels and
antitumor activity in vivo.
5. Abbreviations
CRC
Colorectal cancer
DMF
HRMS
IC50
Dimethylformamide
High resolution mass spectrometry
50% inhibitory concentration
Polymerase chain reaction
PCR
Acknowledgments
4. Conclusions
This work was supported by the Nievre committee of the French
National League against cancer. We are really thankful to Marie-José
Penouilh for the HRMS mass spectra and 195RMN Pt at the “Plateforme
d'Analyse Chimique et de Synthèse Moléculaire de l'Université de Bourgogne
(PACSMUB)”.
Two series of new acridine-based tethered (ethane-1,2-diamine)
platinum(II) complexes connected by an appropriate polymethylene
chain were prepared in a three step synthesis, starting from 1. In
human colic cancer cell lines HCT 116, SW480 and HT-29, series of
complexes bearing the acridine-9-carboxamide chromophore dis-
played either no cytotoxic effect for non-platinated derivatives
(3a–e), either cytotoxic properties similar or less potent than cisplat-
in and oxaliplatin for platinated complexes (4a–e). Interestingly, de-
rivatives bearing the acridine-9-carboxylate chromophore (3f–j and
4f–j) were more active: platinated complexes (4f–j) exhibited higher
cytotoxicity than reference molecule cisplatin and oxaliplatin, whilst
unplatinated acridine-9-carboxylate intercalators (3f–j) exhibited
tremendous cytotoxicity, with increasing intensity from compounds
connected to a polymethylene linker having n=2 to n=6. These ex-
periments suggest that acridine-9-substituted chromophore could be
of great interest, especially with acridine-9-carboxylate functionality.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.jinorgbio.2012.02.006.
References
[1] L. Kelland, Nat. Rev. Cancer 7 (2007) 573–584.
[2] D. Wang, S.J. Lippard, Nat. Rev. Drug Discov. 4 (2005) 307–320.
[3] P. Heffeter, U. Jungwirth, M. Jakupec, C. Hartinger, M. Galanski, L. Elbling, M.
Micksche, B. Keppler, W. Berger, Drug Resist. Updat. 11 (2008) 1–16.
[4] D.J. Stewart, Crit. Rev. Oncol. Hematol. 63 (2007) 12–31.

F. Bouyer et al. / Journal of Inorganic Biochemistry 110 (2012) 51–57
57
[5] D. Cunningham, W. Atkin, H.J. Lenz, T. Lynch Henry, B. Minsky, B. Nordlinger, N.
Starling, Lancet 375 (2010) 1030–1047.
[19] A. Valentini, D. Pucci, A. Crispini, G. Federici, S. Bernardini, Chem. Biol. Interact.
161 (2006) 241–250.
[6] A. Walther, E. Johnstone, C. Swanton, R. Midgley, I. Tomlinson, D. Kerr, Nat. Rev.
Cancer 9 (2009) 489–499.
[20] B.E. Bowler, K.J. Ahmed, W.I. Sundquist, L.S. Hollis, E.E. Whang, S.J. Lippard, J. Am.
Chem. Soc. 111 (1989) 1299–1306.
[7] T. Tanaka, J. Carcinog. 8 (2009) 5.
[21] A. Kuzuya, K. Machida, R. Mizoguchi, M. Komiyama, Bioconjug. Chem. 13 (2002)
365–369.
[22] M. Matsukura, T. Okamoto, T. Miike, H. Sawai, K. Shinozuka, Biochem. Biophys.
Res. Commun. 293 (2002) 1341–1347.
[23] K. Agiamarnioti, T. Triantis, D. Dimotikali, K. Papadopoulos, J. Photochem. Photo-
biol. 172 (2005) 215–221.
[24] K. Agiamarnioti, T. Triantis, K. Papadopoulos, D. Dimotikali, Acta Chim. Slov. 51
(2004) 67–76.
[25] Y. Mikata, K. Mogami, M. Kato, I. Okura, S. Yano, Bioorg. Med. Chem. Lett. 7 (1997)
1083–1086.
[26] H.H. Lee, B.D. Palmer, B.C. Baguley, M. Chin, W.D. McFadyen, G. Wickham, D.
Thorsbourne-Palmer, L.P.G. Wakelin, W.A. Denny, J. Med. Chem. 35 (1992)
2983–2987.
[27] M. Carland, M.J. Grannas, M.J. Cairns, V.J. Roknic, W.A. Denny, W.D. McFadyen, V.
Murray, J. Inorg. Biochem. 104 (2010) 815–819.
[28] H. Silva, C. Valerio Barra, C. Franca da Costa, M. Vieira de Almeida, E.T. Cesar, J.N.
Silveira, A. Garnier-Suillerot, F.C. Silva de Paula, E.C. Pereira-Maia, A.P.S. Fontes,
J. Inorg. Biochem. 102 (2008) 767–772.
[8] S. Bhushan, H. McLeod, C.M. Walko, Clin. Colorectal Cancer 8 (2009) 15–21.
[9] B. Desoize, C. Madoulet, Crit. Rev. Oncol. Hematol. 42 (2002) 317–325.
[10] M. Skander, P. Retailleau, B. Bourrie, L. Schio, P. Mailliet, A. Marinetti, J. Med.
Chem. 53 (2010) 2146–2154.
[11] A.R. Kheradi, G. Saluta, G.L. Kucera, C.S. Day, U. Bierbach, Bioorg. Med. Chem. Lett.
19 (2009) 3423–3425.
[12] B.D. Palmer, H.H. Lee, P. Johnson, B.C. Baguley, G. Wickham, L.P.G. Wakelin, W.D.
McFadyen, W.A. Denny, J. Med. Chem. 33 (1990) 3008–3014.
[13] R.J. Holmes, M.J. McKeage, V. Murray, W.A. Denny, W.D. McFadyen, J. Inorg. Biochem.
85 (2001) 209–217.
[14] C.R. Brodie, J.G. Collins, J.R. Aldrich-Wright, Dalton Trans. (2004) 1145–1152.
[15] M.R. Reithofer, A. Schwarzinger, S.M. Valiahdi, M. Galanski, M.A. Jakupec, B.K.
Keppler, J. Inorg. Biochem. 102 (2008) 2072–2077.
[16] E.T. Martins, H. Baruah, J. Kramarczyk, G. Saluta, C.S. Day, G.L. Kucera, U. Bierbach,
J. Med. Chem. 44 (2001) 4492–4496.
[17] Z. Ma, J.R. Choudhury, M.W. Wright, C.S. Day, G. Saluta, G.L. Kucera, U. Bierbach,
J. Med. Chem. 51 (2008) 7574–7580.
[18] B.E. Bowler, L.S. Hollis, S.J. Lippard, J. Am. Chem. Soc. 106 (1984) 6102–6104.