Cis-Dichloroplatinum(II) complexes tethered to dibenzo[c,h][1,6] naphthyridin-6-ones: Synthesis and cytotoxicity in human cancer cell lines in vitro

Article abstract of DOI:10.1016/j.ejmech.2013.09.037

A novel family of cisplatin-type complexes tethered to dibenzo[c,h][1,6] naphthyridin-6-one topoisomerase inhibitor via a polymethylene chain and their nonplatinated counterparts were prepared. Their potential cytotoxicity was assessed in three human colo

Full text of DOI:10.1016/j.ejmech.2013.09.037

European Journal of Medicinal Chemistry 69 (2013) 719e727
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
cis-Dichloroplatinum(II) complexes tethered to dibenzo[c,h][1,6]
naphthyridin-6-ones: Synthesis and cytotoxicity in human cancer cell
lines in vitro
,
b
a
c
d
Nicolas Desbois a , David Pertuit , Johnny Moretto , Claire Cachia , Bruno Chauffert ,
**
Florence Bouyer a
,
*
a INSERM UMR U866 “Lipides/Nutrition/Cancer”, Université de Bourgogne, UFR Médecine, 7 bd Jeanne d’Arc, BP 89700, 21079 Dijon Cedex, France
b EA 3660 Unité de Molécules d’Intérêt Biologique, UFR Pharmacie, 7 bd Jeanne d’Arc, BP 89700, 21079 Dijon Cedex, France
c Université de Bourgogne, UFR Pharmacie, 7 bd Jeanne d’Arc, BP 89700, 21079 Dijon Cedex, France
d Service d’Oncologie Médicale, CHU Amiens Sud, Av Laennec, 80054 Amiens Cedex 1, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel family of cisplatin-type complexes tethered to dibenzo[c,h][1,6]naphthyridin-6-one topoisom-
erase inhibitor via a polymethylene chain and their nonplatinated counterparts were prepared. Their
potential cytotoxicity was assessed in three human colorectal cancer cell lines HCT 116, SW480 and HT-
29 and compared to the reference molecules cisplatin and oxaliplatin. Platinated compounds were poorly
active whilst nonplatinated dibenzo[c,h][1,6]naphthyridin-6-one moieties exhibited higher cytotoxic
properties than cisplatin and oxaliplatin whatever the length of the polymethylene chain; molecules
containing the tri- and hexamethylene chain length were the most cytotoxic.
Received 16 May 2013
Received in revised form
10 September 2013
Accepted 16 September 2013
Available online 26 September 2013
Keywords:
Topoisomerase I targeting agents
Cisplatin
Ó 2013 Elsevier Masson SAS. All rights reserved.
5H-Dibenzo[c,h][1,6]naphthyridin-6-one
Cytotoxicity
Colorectal cancer cell
1. Introduction
restricted by intrinsic toxicities or resistance occurrence [11e13];
and despite the thousands of Pt derivates already synthetized, only
Platinum (Pt) derivatives represent an old family of anticancer
drugs, that still play a pivotal role in the treatment of many solid
tumors, including lung, ovarian, colon and bladder cancers [1e3].
The transition metal complex cis-diaminedichloroplatinum(II), also
known as cisplatin (Fig. 1), consists of the leader molecule. Oxali-
platin (Fig. 1), a third-generation derivative with antitumor activity
against cisplatin resistant tumor cells, represents the reference Pt
compound used in the management of colorectal cancer [4e6]. In
cancer cells, Pt drugs target DNA to establish coordination bonds
especially at the N-7 of guanine base of DNA [7e9]. These so-called
Pt-DNA adducts generate both intra- and interstrand crosslinks
which, in final, lead to inhibition of DNA replication and RNA
transcription [8e10]. In clinical practice, Pt drug efficacy could be
a few Pt drugs are available in clinic.
Several strategies have been intensively explored to develop
new compounds, especially by combining in a same molecule a Pt
moiety to another entity exhibiting cytotoxic properties [14]. Such
approach could have several advantages: to combine 2 modes of
action which could help to circumvent drug resistance; to decrease
toxicity of each drug used separately and to increase DNA targeting
by incorporation of Pt into suitable “carrier” molecules, such as
acridine derived-pharmacophore [15,16], anthraquinone [17], bis-
naphthalimide [18], camptothecin [19]..
Our approach was focused on developing Pt compounds coupled
to a cytotoxic moiety exhibiting topoisomerase I inhibiting proper-
ties. Human DNA topoisomerases are nuclear enzymes that control
DNA topology, allowing, for example, DNA supercoiling relaxation
during replication and transcription [20,21]. A few inhibitors of
topoisomerases I are commercially available in clinic, especially
those derived from camptothecin, a cytotoxic quinoline alkaloid
extracted from a Chinese tree Camptotheca acuminata. Camptothe-
cin exhibited too cytotoxic properties to be used in clinic, but
* Corresponding author. Tel.: þ33 03 80 39 34 50.
** Corresponding author. Tel.: þ33 03 80 39 32 52.
E-mail addresses: nicolas.desbois@u-bourgogne.fr (N. Desbois), florence.bouyer@
u-bourgogne.fr (F. Bouyer).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.037

720
N. Desbois et al. / European Journal of Medicinal Chemistry 69 (2013) 719e727
Fig. 1. The structure of the cisplatin, oxaliplatin and dibenzo[c,h]naphthyridin-6-one derivative.
2 analogs, irinotecan and topotecan, have been commercially
approved [22e24]. However, these two camptothecin-derived
topoisomerase 1 inhibitors displayed two major limits [23]: they
appearance in both spectra of ionic pattern corresponding to
[M þ Na]þ.
suffer from partial hydrolytic inactivation due to the presence of a d-
2.2. Cytotoxicity in cancer cell lines
lactone in the E-ring of their molecular structure. And, they are
substrates for efflux pumps such as MDR1 (Multi Drug Resistance 1)
and BCRP (Breast Cancer Resistance Protein) which play a major role
in anticancer drug resistance. Since, many non-camptothecin and
novel camptothecin derived-topoisomerase I inhibitors have been
developed [25e28].
Thus, we described novel compounds associating a Pt moiety
with a promising non-camptothecin topoisomerase I inhibitor
fragment. Many families of such inhibitors have already been
identified, such as indolocarbazoles [29], benzophenazines [30],
Cytotoxicity of 5H-dibenzo[c,h][1,6]naphthyridin-6-one-based
Pt(II) complexes (8aee) and their nonplatinated counterparts (7ae
e) was performed in three human colic cancer cell lines HCT 116,
HT-29 and SW480, as colorectal cancer is a clinical indication of Pt
compounds [5,6]. Based on clinical trials, oxaliplatin in combination
with 5-FU and folinic acid, has been approved for many years for
the treatment of advanced metastatic colorectal cancer. Consid-
ering that topoisomerase I inhibitors represent also firmly estab-
lished first-line treatment drugs of some chemotherapeutic
protocols of colorectal cancer, we were interesting in synthesizing
new compounds combining the Pt moiety to a topoisomerase I
inhibitor carrier, anticipating that such compounds would localize
in the vicinity of DNA and would carry out powerful cytotoxic ef-
fects. In the literature, the potential benefit of the concomitant use
of a topoisomerase inhibitor and a Pt drug has already been
underlined in different types of cancer [37,38]. However, the effi-
cacy of such association, tested in human colorectal cell lines, was
dependant of the schedule of administration applied [39]. At the
molecular level, Pt-DNA adducts could act as endogenous DNA
topoisomerase I poisons to enhance the stability of covalent
Topoisomerase I-DNA complexes [40].
benz[a]acridines [31], or benzo[c]phenanthridines [32]. Previous
studies outlined the good correlation between topoisomerase I
targeting activity and cytotoxicity of 5H-dibenzo[c,h]naphthyridin-
6-one derivatives [33,34] (Fig. 1).
We designed cisplatin type complexes tethered to promising
topoisomerase inhibitor dibenzo[c,h]naphthyridin-6-one deriva-
tive by a variable polymethylene linker (Fig. 2) and their potential
cytotoxic properties were assessed in a panel of human colorectal
cancer cell lines, as clinical protocols of colorectal cancer commonly
include Pt or anti-topoisomerase drugs [5,6]. Cytotoxicity was dis-
cussed according to the size of the alkyl chain bond and to the
presence or absence of the platinum fragment.
In our biological evaluation, cytotoxicity of newly synthesized
compounds was thus compared to the approved Pt drug in clinic,
oxaliplatin, but also to the reference Pt drug, cisplatin. In our
experimental conditions, oxaliplatin was more cytotoxic than
cisplatin in the three cell lines tested, as highlighted by IC50 values
(Table 1). High IC50 values could be attributed to stringent condi-
tions of cytotoxic assay, as drug treatment was applied on sub-
confluent cells. The platinated 8aee and nonplatinated 7aee
derivates of 2,3-methylenedioxy-8-nitro-5H-dibenzo[c,h][1,6]
naphthyridin-6-one displayed very different cytotoxic properties
(Table 1; Figs. 3 and 4) in the three colic cancer cell lines. Unfor-
tunately, the platinated complexes 8aee did not exhibit significant
cytotoxicity, which remained much lower than 7aee and oxali-
platin and cisplatin in the three cell lines studied, especially in HCT
116 (Table 1 and Fig. 3). 8aee displayed cytotoxic properties 20
2. Results and discussion
2.1. Preparation and spectroscopic properties
The compounds described in this study and the methods of
synthesis are outlined in Schemes 1 and 2.
Reaction of 4-chloro-6,7-methylenedioxyquinoline [35] (1) with
the corresponding amines 3aee yielded the 4-substituted quino-
lines 2aee respectively (Scheme 1). Phenol was utilized to form a
phenolic intermediate of heightened reactivity to 4-chloroquinoline
1. Treatment of commercially 2-bromo-5-nitrobenzoic acid with
thionyl chloride provided the acid chloride 4 which was treated
with 2aee to yield the benzamide derivatives 5aee. Initially,
Sharma [36] protocol using 2-iodo-5-nitrobenzoic acid was applied,
but unfortunately, this method was not reproducible in our condi-
tion: the iodine atom was more bulky than the bromine atom. The
Heck coupling reaction of amide 5aee with Pd(OAc)2, a phosphine
ligand (PBu3), and a base (Ag2CO3) in DMF reflux afforded 6aee
formation in good yields (36e68%). Deprotection of diboc de-
rivatives 6aee with trifluoroacetic acid provided 7aee. Intermedi-
ate TFA salts which were converted with HCl 1 N diethylether to 7ae
e 3HCl for biological study.
The Pt complexes 8aee (Scheme 2) were prepared by addition of
an aqueous solution of K2PtCl4 to a basic solution of 7aee ligands
and the products were isolated directly from the reaction mixture.
In each complex, coordination of Pt to diamine was confirmed by
ElectroSpray Ionization (ESI) mass spectrometry (HR-MS), with the
Fig. 2. Compounds of interest.

N. Desbois et al. / European Journal of Medicinal Chemistry 69 (2013) 719e727
721
Scheme 1. Reagents and conditions: (i) a) phenol,
D
; b) NH2(CH2)nNBoc(CH2)2NHBoc (3aee), 110 ꢀC; (ii) CH2Cl2, NEt3; (iii) Pd(OAc)2, PBu3, Ag2CO3, DMF,
D
; (iv) a) TFA, 0 ꢀC then RT;
b) HCl 1 N/diethylether.
times lower than 7aee, suggesting that, in cell culture medium, the
Pt drugs were not cleaved to generate the 5H-dibenzo[c,h][1,6]
naphthyridin-6-one derivative and the Pt moiety. In addition, the
presence of a polymethylene spacer in platinated complexes did
not play a role in the cytotoxic potential of the complexes. The
absence of significant activity may imply that the (N-(aminoalkyl)-
(ethane-1,2-diamine))Pt(II) linker in 5-position interfered with
dibenzo[c,h][1,6]naphthyridin-6-one targeting topoisomerase I ac-
tivity, and in final failed to exhibit high cytotoxicity. Other hy-
potheses could involve lower cellular uptake of platinated
complexes, or disturbance of cell death mechanism by steric hin-
drance. Thus, instead of combining 2 modes of action, the presence
of the two different entities e the topoisomerase I inhibiting and
the Pt moiety e in a same complex was detrimental to its cytotoxic
activity. Nevertheless, drugs combining a Pt moiety to a bioactive
carrier ligand did not always support enhanced cytotoxic proper-
ties. Previous testing of cisplatin-type complexes tethered to a
both properties of single molecules: the topoisomerase I poisoning
activity and the DNA-platination [19], corroborating the idea of
combining several mechanisms of action in a unique derivative.
In spite of that, nonplatinated derivates of 2,3-methylenedioxy-
8-nitro-5H-dibenzo[c,h][1,6]naphthyridin-6-one (7aee) displayed
potent cytotoxic properties, as highlighted by IC50 values, which
could be 5e10 times lower than reference compounds as observed
in HCT 116 cells (Table 1 and Fig. 4). The powerful cytotoxic prop-
erties of the substituted 5H-dibenzo[c,h][1,6]naphthyridin-6-one
were previously described by LaVoie and co-workers who exten-
sively investigated such compounds in term of cytotoxicity, topo-
isomerase I targeting activity [33,43e45] and antitumor activity
in vivo, mainly in athymic nude mice bearing human xenografts
[33,46e48]. For many 5H-dibenzo[c,h][1,6]naphthyridin-6-one
derivatives, cytotoxicity correlated with their topoisomerase I
inhibitory activity [33,35,36,46,49,50], the ceto-group in position 6
playing a key role in enhanced topoisomerase I targeting and
cytotoxic activities [33].
substituted acridine chromophore containing
a carboxamide
functionality [15,16] also displayed low cytotoxicity in human
cancer cells, including human colorectal cancer cell lines.
However, several Pt complexes of differently substituted acridine-
4-carboxamide [41,42] or acridine-9-carboxylate chromophore
were reported as promising anticancer drugs active in different cell
lines sensitive or resistant to cisplatin [41,42] including human
colon cell lines HCT 116, SW480 and HT-29 [15]. Recently, the
conjugation of Pt to a 7-substituted camptothecin moiety generated
compounds with growth inhibitory activity against a panel of hu-
man tumor cancer cell lines, and with appreciable antitumor ac-
tivity in mice bearing nonsmall cell lung cancer H460 tumor
xenograft [19]. Interestingly, such hybrid compounds cumulated
5H-Dibenzo[c,h][1,6]naphthyridin-6-one derivatives were poten-
tially efficacious in the treatment of colorectal cancer, as ARC-111, a
well-known and extensively studied derivative, was highly potent in
scid (severe combined immunodeficient) mice carrying human HCT-
8 colon adenocarcinoma tumor xenografts [48]; and Genz-644282,
selected for phase I clinical trial, exhibited potent cytotoxicity to-
wards human colorectal cancer cells HCT 116 and HT-29, and signif-
icative antitumor activity in many human colon carcinoma tumor
xenografts (HCT 116, HT-29, HCT-15, DLD-1) [47].
The originality of 7aee leads in the chemical substituent in
position 5, as a polymethylene spacer (n ¼ 2e6) connected the 2,3-
methylenedioxy-8-nitro-5H-dibenzo[c,h][1,6]naphthyridin-6-one
Scheme 2. Reagents and conditions: a) Na2CO3$2N, b) K2PtCl4, H2O, c) KCl 5%.

722
N. Desbois et al. / European Journal of Medicinal Chemistry 69 (2013) 719e727
Table 1
potentially with the topoisomerase 1-targeting activity of the
dibenzo[c,h][1,6]naphthyridin-6-one chromophore, in which the
ceto-group in position 6 is highly involved.
Determination of IC50 values assessed by cytotoxic assay of naphthyridin-6-one-
based derivatives platinated (8aee) or not (7aee), the vehicle DMF, and the refer-
ence drugs, cisplatin and oxaliplatin, in human cancer colorectal HCT 116, HT-29 and
Compared to the literature, especially to ARC-111, 7aee dis-
played another distinctive feature. The presence of a nitro substit-
uent in the 8-position supplied derivatives with potent cytotoxic
properties. Such findings were in agreement with previous work
performed on ARC-111, in which the replacement of the 8,9-
dimethoxy groups with a 8,9-dinitro substituent allowed for
retention of potent topoisomerase I activity and cytotoxicity [34].
The nature of the substituent in position 8- and 9- of the dibenzo
[c,h][1,6]naphthyridin-6-one significantly influenced the cytotox-
icity of compounds. Thus, in ARC-111 analogs, the replacement of 8-
nitro by 8-amino, 8-arylamine [36] or by a 8,9-diethoxy [46] sub-
stituent decreased cytotoxic and topoisomerase I activities.
Further works will assess the topoisomerase I targeting activity
of 7aee, as such inhibitory activity could be expected from data of
the literature. Optimization of the nitro group in position 8 and/or 9
will also be performed in a way to design compounds with
enhanced cytotoxicity.
SW480 cell lines after
a 72 h-treatment. Each IC50value represented the
mean ꢁ standard deviation of at least 3 different experiments (nd ¼ not deter-
minable). Statistical analyses compared IC50 values of newly synthetized drugs
versus cisplatin (*) or oxaliplatin (y) (*/y: p < 0.05; **/yy: p < 0.01; ***/yyy: p < 0.001).
Compounds IC50 (mM)
HCT 116
HT-29
SW480
7a
24.97 ꢁ 2. 73
(**) (yy)
43.02 ꢁ 3.83 (***) (yy) 40.70 ꢁ 4.42 (y)
7b
7c
12.16 ꢁ 0.58 (**) (yy) 22.20 ꢁ 2.19 (**) (yy) 30.13 ꢁ 5.34 (y)
30.71 ꢁ 11.51
(**) (yy)
41.20 ꢁ 4.34 (***) (yy) 43.74 ꢁ 2.22 (*) (y)
7d
7e
25.15 ꢁ 2.64
(**) (yy)
47.60 ꢁ 1.92 (***) (yy) 50.73 ꢁ 4.52
15.44 ꢁ 4.71
(**) (yy)
30.84 ꢁ 5.51 (**) (yy) 24.00 ꢁ 0.28 (**) (y)
8a
8b
387.68 ꢁ 11.74
nd
319.57 ꢁ 22.28
428.26 ꢁ 9.87
446.38 ꢁ 13.29
384.06 ꢁ 34.46
nd
188.86 ꢁ 13.07
143.16 ꢁ 33.96
418.48 ꢁ 7.99
440.22 ꢁ 50.33
460.14 ꢁ 8.87
nd
8c
nd
8d
nd
nd
8e
Cisplatin
491.07 ꢁ 3.16
364.13 ꢁ 34.34
185.76 ꢁ 65.82
58.64 ꢁ 11.02
409.42 ꢁ 7.32
208.50 ꢁ 9.97
3. Conclusion
Oxaliplatin 126.19 ꢁ 25.33
DMF
nd
Two series of substituted dibenzo[c,h][1,6]naphthyridin-6-one
derivatives were synthetized either platinated (8aee) or not (7ae
e). Platinated complexes 8aee did not exhibit significant cytotoxic
properties in human colic cancer cell lines HCT 116, SW480 and HT-
29. But, nonplatinated compounds 7aee displayed higher cytotoxic
effect than 8aee and reference molecules, cisplatin and oxaliplatin.
Their cytotoxicity was influenced by the length of the poly-
methylene linker (n ¼ 2e6), compounds bearing n ¼ 3 (7b) and
n ¼ 6 (7e), disclosing the most powerful capacities. In final, 7aee
could potentially generate promising anticancer drugs.
Future investigations will examine whether topoisomerase I
inhibitory activity could contribute to the observed cytotoxic
activity of these compounds, and whether changes in 8- or/and 9-
substituents could optimize cytotoxicity. Alternatively, conforma-
tional structures of 8aee will be examined to determine the causes
of low cytotoxicity of Pt complexes.
moiety to the 5-[N-[aminoalkyl]-ethane-1,2-diamine] linear chain.
The ability of an alkylamino group on the side chain attached to the
N-5 position to generate powerful cytotoxic compounds was ex-
pected, even though molecules combining such 5-substituent to an
8-nitro group have never been explored before. Actually, the
presence of a N-5-alkylamino group was already associated with
potent topoisomerase 1 targeting and cytotoxic activities [33,35].
On the contrary, additional substituents on the carbon directly
attached to the N-5 position [33] has been shown to decrease
topoisomerase I targeting activity and cytotoxicity, as well as
increased steric bulk in close proximity to the N-5 heteroatom
[33,35,46,49]. Recently, a series of dibenzo[c,h][1,6]naphthyridin-6-
one derivatives with varied ethylenediamine substituent in posi-
tion 5- were proved to be more active when both amino group were
tertiary [50]. In our study, we demonstrated that nonsubstituted
amino group in substituent in position-5 could also retain high
cytotoxic properties.
4. Experimental
4.1. Chemistry
Moreover, we clearly showed that the length of the poly-
methylene linker at the 5-position influence the cytotoxicity of the
2,3-methylenedioxy-8-nitro-5H-dibenzo[c,h][1,6]naphthyridin-6-
one derivates (7aee). The propylene and hexamethylene spacers
generated the most powerful compounds whatever the cell line
tested (Fig. 5). High cytotoxicity of compounds 7b and e came out in
nonplatinated derivatives compared to their platinated counter-
parts. These data are quite new, as until now, the high cytotoxic
dibenzo[c,h][1,6]naphthyridin-6-one derivatives were character-
ized by compounds connected at the 5-position, to a two-carbon
linker to an alkylamine [33]. Even though a complete series of
compounds bearing a polymethylene linker (n ¼ 2e6) was not
investigated in the literature, partial data derived from ARC-111
analogs corroborated our findings: insertion of a methylene
group into the side chain in position-5 of ARC-111 e resulting in a
trimethylene substituent e did allow for retention of high topo-
isomerase I and cytotoxic activity [33], as well as compounds
containing 6 atoms linear alkyl side from the N-5 position [33]. The
high cytotoxicity of the derivative connected to the hexamethylene
linker could be due to the substantial conformational flexibility of
the linker that would not interfere with the cytotoxic activity, and
4.1.1. Materials and methods
All column chromatography was performed with Merck neutral
aluminum oxide 90 standardized (63e200
mm) or silica gel (Acros
ꢀ
organic, 60 A, 35e70
mm). All thin layer chromatography was per-
formed on Fluka aluminum oxide plates (with fluorescent indicator
254 nm) or Merck silica gel 60F254 plates. Melting points were
determined on a ReicherteJungeKoffler apparatus and were not
corrected. NMR spectra (300 MHz for 1H, and 75 MHz for 13C) were
recorded on a Bruker Avance 300 and 600 instruments using the
indicated solvents. Chemical shifts were reported in ppm (d). All the
coupling constants (J) are in Hertz. Accurate mass measurements
(HR-MS) were obtained with a LTQ Orbitrap XL (THERMO) instru-
ment in ESI mode. Elemental analyses were performed with an
Elemental Analyser Thermo electron Flash EA 1112. Infrared spectra
were recorded on IR FT BRUKER Vertex 70v.
The compound 4-chloro-6,7-methylenedioxyquinoline (1) was
prepared according to reported methods [35]. The compounds N,N0-
(tert-butoxycarbonyl)-N-[aminoalkyl]-ethane-1,2-diamines (3aee)
were prepared by a method described by Holmes et al. [41] that
consisted in modification of the diboc procedure to avoid monoboc
product: for that,
a mixture of di-tert-butyldicarbonate and

N. Desbois et al. / European Journal of Medicinal Chemistry 69 (2013) 719e727
723
Fig. 3. Concentrationeresponse curves of cytotoxicity of platinated complexes (8aee) connected by a di- to hexamethylene chain 8a ( ) 8b ( ) 8c ( ) 8d ( ) 8e ( ), and of their
vehicle DMF ( ), as well as of the reference molecules cisplatin ( ) and oxaliplatin ( ) in human cancer colorectal HCT 116 (A), HT-29 (B) and SW480 (C) cell lines after a treatment
of 72 h. Cytotoxicity values are represented by the mean ꢁ standard deviation of 3 independent experiments. IC50 values of each compound evaluated in HCT 116 (G), HT-29 ( ) and
SW480 (
) are reported in (D).
appropriate N-[[(benzyloxycarbonyl)amino]-alkyl]-ethane-1,2-dia
Yield: 33%. Brown oil. 1H NMR (CDCl3, 300 MHz)
d 8.37 (d, 1H,
mine in dry dichloromethane was stirred for 5 days.
J ¼ 5.5 Hz), 7.28 (s, 1H), 7.26 (s, 1H), 6.34 (d, 1H, J ¼ 5.5 Hz), 6.05 (s,
2H), 3.30 (m, 8H), 1.72 (m, 4H), 1.46 (s, 9H), 1.43 (s, 9H); 13C NMR
4.1.2. Synthesis of 4-[N,N0-(tert-butoxycarbonyl)-N-[aminoalkyl]-
ethane-1,2-diamine]-6,7-methylenedioxyquinolines (2aee)
A mixture of 4-chloro-6,7-methylenedioxyquinoline (1) (1.35 g,
6.50 mmol) inphenol (3.23 g, 34.4 mmol) was heated to reflux for 3 h.
The bath temperature was lowered to 110 ꢀC, and the appropriate
N,N0-(tert-butoxycarbonyl)-N-[aminoalkyl]-ethane-1,2-diamine (3)
(7.82 mmol) was added. The mixture was stirred at this temperature
for 18 h.
(CDCl3, 75 MHz) d 156.0 (2C), 150.0, 149.9, 148.3, 147.2, 146.8, 114.2,
106.1, 101.5, 98.5, 96.7, 80.2, 79.2, 52.6, 46.8, 43.2, 39.7, 28.4 (6CH3),
27.3, 26.5. 2d: Yield: 80%. mp 64e66 ꢀC. 1H NMR (CDCl3, 300 MHz)
d
8.38 (d, 1H, J ¼ 5.5 Hz), 7.35 (s, 1H), 7.28 (s, 1H), 7.04 (br, 1H), 6.36
(d, 1H, J ¼ 5.5 Hz), 6.06 (s, 2H), 3.28 (m, 8H), 1.72e1.79 (m, 6H), 1.46
(s, 9H), 1.43 (s, 9H); 13C NMR (CDCl3, 75 MHz)
155.9 (2C), 149.9,
d
149.4, 148.7, 146.7, 146.2, 114.1, 106.4, 101.5, 98.6, 95.9, 79.9 (2C),
53.0, 46.6, 43.1, 39.6, 27.8e28.6 (6CH3, 3CH2). 2e: Yield: 85%. mp
The residue was partitioned between dichloromethane
(100 mL) and 10% aqueous NaOH (100 mL). The aqueous layer was
extracted with dichloromethane. The organic phase was dried
(Na2SO4) and evaporated to dryness. The crude product was puri-
fied on aluminum oxide eluting dichloromethane. 2a: Yield: 78%.
92e94 ꢀC. 1H NMR (CDCl3, 300 MHz)
d
8.34 (d, 1H, J ¼ 5.5 Hz), 7.31
(s, 1H), 7.26 (s, 1H), 6.34 (d, 1H, J ¼ 5.5 Hz), 6.05 (s, 2H), 3.28 (m, 8H),
1.41e1.72 (m, 24H); 13C NMR (CDCl3, 75 MHz)
156.3, 155.9, 149.9,
d
149.3, 148.9, 146.7, 146.3, 114.1, 106.5, 101.5, 98.5, 95.8, 79.8, 79.2,
53.7, 46.4, 42.9, 39.6, 26.3e28.4 (6CH3, 4CH2).
mp 60e62 ꢀC. 1H NMR (CDCl3, 300 MHz)
d
8.55 (d, 1H, J ¼ 5.5 Hz),
7.26 (s, 1H), 7.13 (s, 1H), 6.39 (br, 1H), 6.24 (d, 1H, J ¼ 5.5 Hz), 6.03 (s,
2H), 3.28 (m, 8H), 1.43 (m, 18H), 13C NMR (CDCl3, 75 MHz)
157.8,
4.1.3. Synthesis of N-(6,7-methylenedioxyquinolin-4-yl)-N-[N,N0-
(tert-butoxycarbonyl)-N-[aminoalkyl]-ethane-1,2-diamine]-2-
bromo-4-nitrobenzamides (5aee)
d
156.0, 149.9, 149.7, 148.8, 146.7, 146.3, 114.2, 106.3, 101.4, 97.6, 96.7,
81.2, 79.6, 53.4, 46.5, 43.8, 39.3, 28.4 (6CH3). 2b: Yield: 54%. mp
A solution of commercially 2-bromo-5-nitrobenzoic acid (0.56 g,
2.27 mmol) in thionyl chloride (10 mL) was refluxed with stirring
under nitrogen for 3 h. The mixture was evaporated. The solution of
crude chloride acid 4 in dry dichloromethane (29 mL) was added to a
solution of appropriate 4-[N,N0-bis (tert-butoxycarbonyl)-N-[amino-
alkyl]-ethane-1,2-diamine]-6,7-methylenedioxyquinoline (2) (0.60 g,
110e112 ꢀC. 1H NMR (CDCl3, 300 MHz)
d
8.33 (d, 1H, J ¼ 5.5 Hz), 7.26
(m, 2H), 6.32 (d, 1H, J ¼ 5.5 Hz), 6.12 (br, 1H), 6.04 (s, 2H), 3.31 (m,
8H), 1.88 (m, 2H), 1.50 (s, 9H), 1.43 (s, 9H); 13C NMR (CDCl3, 75 MHz)
d
156.3, 156.0, 149.9, 149.5, 148.8, 146.7, 146.5, 114.5, 106.3, 101.4,
98.1, 96.8, 80.6, 79.5, 53.4, 46.8, 44.4, 39.4, 28.4 (6CH3, 1CH2). 2c:

724
N. Desbois et al. / European Journal of Medicinal Chemistry 69 (2013) 719e727
Fig. 4. Concentrationeresponse curves of cytotoxicity of substituted 5H-dibenzo[c,h][1,6]naphthyridin-6-ones trihydrochlorides (7aee) connected by a di- to hexamethylene chain
7a ( ) 7b ( ) 7c ( ) 7d ( ) 7e ( ), and of reference molecules cisplatin ( ) and oxaliplatin ( ) in human cancer colorectal HCT 116 (A), HT-29 (B) and SW480 (C) cell lines after a
treatment of 72 h. Cytotoxicity values are represented by the mean ꢁ standard deviation of 3 independent experiments. IC50 values of each compound evaluated in HCT 116 (G), HT-
29 ( ) and SW480 (
) are reported in (D).
1.19 mmol) and triethylamine (1.32 mL, 9.52 mmol) in dry dichloro-
methane (10 mL). The mixture was refluxed with stirring under ni-
trogen for 16 h. At room temperature, a saturated aqueous Na2CO3
solution (50 mL) was added and the aqueous layer was extracted with
dichloromethane (3 ꢂ 40 mL). The combined organic phases were
dried (Na2SO4) and evaporated to dryness. The crude mixture was
chromatographed on silica in 2:1 ethyl acetate-cyclohexane. 5a:
2H), 7.56 (d, 1H, J ¼ 9 Hz), 7.31 (s, 1H), 7.28 (s, 1H), 7.19 (d, 1H,
J ¼ 5.5 Hz), 6.19 (m, 2H), 4.47 (m,1H), 3.41 (m,1H), 3.25 (m, 6H),1.52e
1.78 (m, 6H), 1.43 (m, 18H), 13C NMR (CDCl3, 75 MHz)
d 166.2, 156.0
(2C), 151.5, 149.7, 148.5, 148.1, 145.9, 144.2, 138.9, 134.1, 127.3, 124.7,
122.3, 121.9, 119.0, 106.6, 102.4, 97.5, 79.7 (2C), 60.4, 48.9, 47.5, 39.5,
29.7, 28.4 (7C), 27.6, 24.0. 5e: Yield: 72%. mp 42e44 ꢀC. 1H NMR
(CDCl3, 300 MHz)
d
8.52 (d, 1H, J ¼ 5.5 Hz), 7.79 (m, 2H), 7.57 (d, 1H,
Yield: 26%. mp 90e92 ꢀC. 1H NMR (CDCl3, 300 MHz)
d
8.43 (d, 1H,
J ¼ 9 Hz), 7.32 (s,1H), 7.28 (s,1H), 7.20 (d,1H, J ¼ 5.5 Hz), 6.19 (m, 2H),
J ¼ 5.5 Hz), 7.76 (m, 2H), 7.52 (m,1H), 7.21e7.26 (m, 3H), 6.13 (m, 2H),
4.47 (m, 1H), 3.40 (m, 1H), 3.23 (m, 6H), 1.52e1.75 (m, 8H), 1.43 (m,
4.61 (m, 1H), 3.52 (m, 1H), 3.17e3.35 (m, 6H), 1.36 (m, 18H), 13C NMR
18H), 13C NMR (CDCl3, 75 MHz)
d 166.2,155.9 (2C), 151.5,149.7, 148.5,
(CDCl3, 75 MHz)
d
166.8, 156.0 (2C), 151.5, 148.6, 148.2, 148.0, 146.0,
148.1, 145.9, 144.3, 138.9, 134.0, 127.3, 124.7, 122.4, 121.9, 119.1, 106.7,
102.4, 97.5, 79.7 (2C), 60.4, 49.0, 46.4, 39.6, 28.4 (7C), 27.8, 26.6, 26.4.
144.4, 138.4, 134.1, 127.1, 124.8, 123.1, 122.1, 119.5, 106.7, 102.4, 97.8,
80.5, 79.4, 60.5, 47.3, 45.4, 39.4, 28.5 (6CH3). 5b: Yield: 65%. mp 70e
72 ꢀC.1H NMR (CDCl3, 300 MHz)
d
8.53 (d,1H, J ¼ 5.5 Hz), 7.78 (m, 2H),
4.1.4. Synthesis of 2,3-methylenedioxy-8-nitro-5-[N,N0-(tert-
butoxycarbonyl)-N-[aminoalkyl]-ethane-1,2-diamine]-5H-dibenzo
[c,h][1,6]naphthyridin-6-ones (6aee)
7.56 (d, 1H, J ¼ 9 Hz), 7.32 (s, 1H), 7.26 (m, 2H), 6.19 (m, 2H), 4.49 (m,
1H), 3.31 (m, 1H), 3.25 (m, 6H), 1.72 (m, 2H), 1.43 (m, 18H), 13C NMR
(CDCl3, 75 MHz)
d
166.4, 155.9 (2C), 151.5, 149.1, 148.5, 148.2, 146.0,
To a solution of appropriate N-(6,7-methylenedioxyquinolin-4-
yl)-N-[N,N0-(tert-butoxycarbonyl)-N-[aminoalkyl]-ethane-1,2-
diamine]-2-bromo-4-nitrobenzamide (5) (0.60 mmol) in dry DMF
(11 mL) were successively added Pd(OAc)2 (26.9 mg, 0.12 mmol),
Bu3P (0.06 mL, 0.24 mmol) and Ag2CO3 (0.33 g, 1.20 mmol). The
mixture was refluxed for 45 min and the reaction mixture was
diluted with ether and the precipitate was removed by filtration.
The filtrate was diluted with dichloromethane (30 mL) and washed
with water (3 ꢂ 30 mL) and brine (30 mL). The organic layer was
then dried with anhydrous sodium sulfate, filtered and evaporated.
144.1,138.8,134.0,127.3,124.7,122.3 (2C),119.1,106.7,102.5, 97.5, 80.2
(2C), 60.4, 46.8 (2C), 39.5, 28.4 (6C). 5c: Yield: 59%. mp 66e68 ꢀC. 1H
NMR (CDCl3, 300 MHz)
d
8.52 (d,1H, J ¼ 5.5 Hz), 7.80 (m, 2H), 7.56 (d,
1H, J ¼ 9 Hz), 7.33 (s,1H), 7.26 (m, 2H), 6.19 (m, 2H), 4.52 (m,1H), 3.43
(m, 1H), 3.25 (m, 6H), 1.45e1.75 (m, 4H), 1.43 (m, 18H), 13C NMR
(CDCl3, 75 MHz)
d 166.3, 155.4 (2C), 151.6, 149.8 (2C), 148.1, 146.0,
144.2, 134.0, 130.6, 127.3, 124.7, 122.3, 120.9, 119.0, 106.6, 102.5, 97.4,
79.9 (2C), 60.4, 48.6, 46.0, 39.5, 28.4 (6C), 25.2, 21.1. 5d: Yield: 50%. mp
47e49 ꢀC.1H NMR (CDCl3, 300 MHz)
d
8.52 (d,1H, J ¼ 5.5 Hz), 7.79 (m,

N. Desbois et al. / European Journal of Medicinal Chemistry 69 (2013) 719e727
725
Fig. 5. Influence of the polymethylene linker chain on IC50 values of substituted 5H-dibenzo[c,h][1,6]naphthyridin-6-ones trihydrochlorides (7aee) in human cancer colorectal HCT
116, HT-29 and SW480 cell lines after a 72 h-treatment. Statistics: *: p < 0.05; **: p < 0.01 (A). Ranking of IC50 values from the most to the less powerful derivatives according to the
size of the polymethylene linker; < expressed significant differences between compounds (B).
The mixture was purified using column chromatography (SiO2,
removed by rotary evaporation. The crude product was purified
using column chromatography (SiO2, CH2Cl2/MeOH/NH4OH, 90/15/
3). The instable product was diluted with dry dichloromethane
(2 mL) and treated with an anhydrous 1 N hydrochloric acideether
solution (2 mL). The solution was concentrated under vacuo started
again with dry ether (3 mL). The suspension was stirred overnight
and filtered. 7a: Yield: 49%. mp 223e225 ꢀC. 1H NMR (DMSO,
AcOEt).
6a: Yield: 63%. mp 230e232 ꢀC. 1H NMR (CDCl3, 300 MHz)
d 9.37
(s, 1H), 9.26 (s, 1H), 8.54 (d, 1H, J ¼ 9 Hz), 8.46 (d, 1H, J ¼ 9 Hz), 7.40
(m, 2H), 6.16 (s, 2H), 4.74 (t, 2H, J ¼ 7 Hz), 3.04e3.40 (m, 6H), 1.34
(m, 18H), 13C NMR (CDCl3, 75 MHz)
d 163.5, 156.1 (2C), 151.5, 148.5,
148.4, 147.2, 143.6, 143.3, 137.8, 127.4, 125.5, 124.8, 122.9, 114.4,
110.6, 106.7, 102.7, 101.3, 80.7 (2C), 50.3, 47.4, 45.6, 39.3, 28.4 (6C).
300 MHz)
d 9.94 (s, 1H), 9.62 (se, 2H), 9.00 (m, 2H), 8.67 (d, 1H,
6b: Yield: 56%. mp 109e111 ꢀC. 1H NMR (CDCl3, 300 MHz)
d
9.45 (s,
J ¼ 8 Hz), 9.37 (se, 3H), 7.78 (s, 1H), 7.75 (s, 1H), 6.41 (s, 2H), 5.00 (t,
1H), 9.31 (s, 1H), 8.58 (d, 1H, J ¼ 9 Hz), 8.49 (d, 1H, J ¼ 9 Hz), 7.53 (m,
2H, J ¼ 7 Hz), 3.44 (m, 2H), 3.13 (m, 4H), 13C NMR (DMSO, 75 MHz)
2H), 6.20 (s, 2H), 4.48 (t, 2H, J ¼ 7 Hz), 3.27e3.40 (m, 6H), 2.32 (t,
d
162.4, 152.3, 148.6, 147.1, 145.1, 141.1, 140.4, 137.1, 127.6, 125.1, 125.0,
2H, J ¼ 7 Hz), 1.42 (m, 18H), 13C NMR (CDCl3, 75 MHz)
d
163.0, 155.9
123.2, 114.5, 111.6, 103.6, 102.0, 101.8, 49.9, 46.9, 44.2, 35.1. HR-MS
(ESI) m/z ¼ 422.1450 [M þ H]þ 422.1459 calcd for C21H20N5O5.
Anal. Calc. for C21H19N5O5$3HCl$2H2O requires: C, 44.50; H, 4.62; N,
12.36. Found: C, 46.30; H, 4.72; N, 12.17. 7b: Yield: 80%. mp 240e
(2C), 151.1, 148.8, 148.1, 147.0, 143.9, 143.1, 137.5, 127.2, 125.5, 124.8,
122.8, 114.3, 110.3, 107.3, 102.6, 100.6, 80.2 (2C), 49.1, 46.4, 44.6,
39.3, 29.7, 28.3 (6C). 6c: Yield: 36%. mp 163e165 ꢀC. 1H NMR (CDCl3,
300 MHz)
d
9.46 (s, 1H), 9.33 (s, 1H), 8.59 (d, 1H, J ¼ 9 Hz), 8.50 (d,
242 ꢀC. 1H NMR (D2O, 300 MHz)
d 9.30 (s, 1H), 8.87 (s, 1H), 8.54 (d,
1H, J ¼ 9 Hz), 7.51 (s, 1H),7.48 (s, 1H), 6.21 (s, 2H), 4.50 (t, 2H,
1H, J ¼ 8 Hz), 8.38 (d, 1H, J ¼ 8 Hz), 7.38 (s, 1H), 7.24 (s, 1H), 6.33 (s,
J ¼ 7 Hz), 3.23 (m, 6H), 1.66e2.08 (m, 4H), 1.41 (m, 18H), 13C NMR
2H), 4.43 (m, 2H), 3.24e3.46 (m, 6H), 2.44 (m, 2H), 13C NMR (DMSO,
(CDCl3, 75 MHz)
d
163.0, 156.1 (2C), 151.3, 148.3, 148.1, 146.9, 143.7,
75 MHz) d 162.2, 152.8, 149.0, 147.2, 145.9, 140.3, 139.3, 136.4, 127.6,
143.1, 137.3, 127.3, 125.3, 124.7, 122.8,114.3, 110.5,106.7, 102.6,100.4,
125.2, 124.8, 123.1, 114.5, 110.9, 104.0, 101.6, 99.6, 48.3, 44.4, 44.0,
35.2, 24.8. HR-MS (ESI) m/z ¼ 436.1608 [M þ H]þ 436.1616 calcd for
C22H22N5O5. Anal. Calc. for C22H21N5O5$3HCl$OEt2$2H2O requires:
C, 47.68; H, 5.85; N, 10.69. Found: C, 48.96; H, 6.09; N, 10.21. 7c:
80.2 (2C), 50.7, 46.9, 46.4, 39.1, 28.1 (6C), 26.0 (2C). 6d: Yield: 62%.
mp 189e191 ꢀC. 1H NMR (CDCl3, 300 MHz)
d 9.42 (s, 1H), 9.29 (s,
1H), 8.56 (d, 1H, J ¼ 9 Hz), 8.47 (d, 1H, J ¼ 9 Hz), 7.50 (s, 1H), 7.45 (s,
1H), 6.20 (s, 2H), 4.45 (t, 2H, J ¼ 7 Hz), 3.24 (m, 6H), 1.66e2.08 (m,
Yield: 85%. mp 259e261 ꢀC. 1H NMR (D2O, 300 MHz)
d 9.44 (s, 1H),
6H), 1.40 (m, 18H), 13C NMR (CDCl3, 75 MHz)
d
163.0, 156.1 (2C),
8.92 (s, 1H), 8.60 (d, 1H, J ¼ 8 Hz), 8.43 (d, 1H, J ¼ 8 Hz), 7.53 (s, 1H),
7.37 (s, 1H), 6.38 (s, 2H), 4.42 (t, 2H, J ¼ 7 Hz), 3.42 (m, 4H), 3.20 (t,
2H, J ¼ 7 Hz), 2.08 (m, 2H), 1.78 (m, 2H), 13C NMR (D2O, 75 MHz)
151.1, 148.5, 148.1, 147.1, 143.8, 143.2, 137.4, 127.2, 125.5, 124.8, 122.8,
114.4, 110.4, 106.9, 102.6, 100.6, 80.0 (2C), 51.0, 47.4, 46.2, 39.2, 29.7,
28.5, 28.3 (6C), 24.0. 6e: Yield: 68%. mp 72e74 ꢀC. 1H NMR (CDCl3,
d
163.6, 153.9, 149.7, 147.4, 146.7, 139.7, 138.6, 135.8, 128.2, 124.5,
300 MHz)
d
9.43 (s, 1H), 9.30 (s, 1H), 8.57 (d, 1H, J ¼ 9 Hz), 8.48 (d,
123.9, 123.7, 114.8, 110.8, 104.5, 101.3, 99.8, 51.0, 47.4, 44.0, 35.3, 25.0,
1H, J ¼ 9 Hz), 7.52 (s, 1H), 7.45 (s, 1H), 6.21 (s, 2H), 4.45 (t, 2H,
22.9. HR-MS (ESI) m/z ¼ 450.1752 [M þ H]þ 450.1772 calcd for
J ¼ 7 Hz), 3.24 (m, 6H), 1.66e2.07 (m, 6H), 1.41 (m, 18H), 13C NMR
C23H24N5O5. Anal. Calc. for C23H23N5O5$3HCl$H2O requires: C, 47.89;
(CDCl3, 75 MHz)
d
163.0, 156.1 (2C), 151.2, 148.8, 148.1, 147.1, 144.0,
H, 4.89; N,12.14. Found: C, 47.74; H, 4.73; N,11.98. 7d: Yield: 50%. mp
143.3, 137.6, 127.2, 125.6, 124.9, 122.8, 114.4, 110.3, 107.3, 102.6,
100.7, 79.9 (2C), 51.1, 47.4, 46.9, 39.7, 28.8, 28.6, 28.5 (7C), 26.6, 26.4.
208e210 ꢀC. 1H NMR (D2O, 300 MHz)
d
9.46 (s,1H), 8.92 (s,1H), 8.58
(d,1H, J ¼ 8 Hz), 8.43 (d,1H, J ¼ 8 Hz), 7.55 (s,1H), 7.39 (s,1H), 6.37 (s,
2H), 4.38 (t, 2H, J ¼ 7 Hz), 3.38 (m, 4H), 3.11 (t, 2H, J ¼ 7 Hz), 2.00 (m,
4.1.5. Synthesis of 2,3-methylenedioxy-8-nitro-5-[N-[aminoalkyl]-
ethane-1,2-diamine]-5H-dibenzo[c,h][1,6]naphthyridin-6-ones
trihydrochlorides (7aee)
Boc deprotection of 6 was attained by dropwise addition of tri-
fluoroacetic acid (TFA) (1.2 mL, 16.2 mmol) at 0 ꢀC to a solution of 6
(0.15 mmol) in distilled CH2Cl2 (5 mL). The mixture was stirred at
room temperature under nitrogen for 1 h. The solvents were then
2H),1.73 (m, 2H),1.42 (m, 2H). 13C NMR (D2O, 75 MHz)
d 163.6,154.1,
149.8,147.5, 147.2, 139.4, 139.1, 135.6, 128.2, 124.5, 124.0, 123.9, 114.9,
110.7, 104.6, 101.5, 99.4, 51.6, 47.8, 44.0, 35.3, 27.3, 25.0, 22.8. HR-MS
(ESI) m/z ¼ 469.1919 [M þ H]þ 469.1929 calcd for C24H26N5O5. Anal.
Calc. for C24H25N5O5$3HCl$2H2O requires: C, 47.37; H, 5.30; N, 11.50.
Found: C, 47.80; H, 5.15; N, 11.21. 7e: Yield: 64%. mp 241e243 ꢀC. 1H
NMR (D2O, 300 MHz)
d
9.35 (s,1H), 8.83 (s,1H), 8.57 (d,1H, J ¼ 9 Hz),

726
N. Desbois et al. / European Journal of Medicinal Chemistry 69 (2013) 719e727
8.38 (d,1H, J ¼ 9 Hz), 7.43 (s,1H), 7.34 (s,1H), 6.42 (s, 2H), 4.28 (t, 2H,
J ¼ 7 Hz), 3.47 (m, 4H), 3.18 (t, 2H, J ¼ 7 Hz), 1.96 (m, 2H), 1.77 (m,
containing a polymethylene spacer in position ꢃ5 (7aee), cisplatin
(SigmaeAldrich, France), and oxaliplatin (Sanofi-Aventis, France)
were diluted in sterile physiological serum (Aguettant Co., France).
The 5H-dibenzo[c,h][1,6]naphthyridin-6-one-based tethered [N-
(aminoalkyl)-(ethane-1,2-diamine)]Pt(II) complexes connected by
a polymethylene chain with n ¼ 2 to 6 (8aee) were diluted into
dimethylformamide (DMF) (Sigma, France), that’s why the vehicle
DMF was also tested alone. However, in any case, the maximal
concentration of DMF in cell culture medium did not exceed 3%.
Cytotoxic assays were conducted on HCT 116, SW480 and HT-29
in 96-well plates. Subconfluent cells were treated for 72 h by
2H), 1.43 (m, 4H), 13C NMR (DMSO, 75 MHz)
d 162.3, 152.2, 148.5,
147.0, 145.0, 143.9, 141.9, 136.7, 127.6, 124.9, 124.8, 123.1, 114.2, 110.5,
103.9, 102.8, 101.2, 50.9, 46.8, 44.1, 35.5, 25.7, 25.6, 25.5, 25.3 HR-MS
(ESI) m/z ¼ 478.2063 [M þ H]þ 478.2085 calcd for C25H28N5O5. Anal.
Calc. for C25H27N5O5$3HCl$H2O requires: C, 49.64; H, 5.33; N, 11.58.
Found: C, 50.50; H, 5.14; N, 11.21.
4.1.6. Synthesis of (2,3-methylenedioxy-8-nitro-5-[N-[aminoalkyl]-
ethane-1,2-diamine]-5H-dibenzo[c,h][1,6]naphthyridin-6-ones)
dichloroplatinums (II) (8aee)
increasing concentrations from 0 to 500 mM of the newly synthe-
To a solution of K2PtCl4 (84 mg, 0.20 mmol) in water (4 mL), the
trihydrochloride 7 (0.20 mmol) was added. The pH was adjusted to
8 with 2 N 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 pre-
cipitate was collected, washed several times with water, acetone
and dried to give pure product 8. 8a: Yield 76%. mp >260 ꢀC. IR:
1653 (CO), 1457 (NO2), 1336 (NO2) cmꢃ1. 1H NMR (DMSO, 300 MHz)
tized or reference molecules. After treatment, cytotoxicity was
assessed by crystal violet staining. Cells were washed in Phosphate
Buffered Saline (PBS) 1ꢂ, fixed in pure ethanol, stained with 1%
crystal violet, and eluted in 33% acetic acid. The intensity of color-
ation was determined by the measurement of absorbance by
spectrophotometry (UVM 340, Bioserv) at
l
¼ 570 nm. Each con-
centration measurement was conducted in triplicate from three
independent experiments.
d
9.69 (s, 1H), 9.00 (s, 1H), 8.96 (m, 1H), 8.62 (m, 1H), 7.70 (s, 1H),
7.50 (s, 1H), 6.31 (s, 2H), 4.55 (m, 2H), 2.50e3.33 (m, 6H). HR-MS
(ESI) m/z 709.0274 [M
Na]þ 709.0305 calcd for
21H19Cl2N5NaO5Pt. Anal. Calc. for C21H19Cl2N5O5Pt$CH3COCH3 re-
Results were expressed as concentrationeresponse curves,
representing the percentage of cytotoxicity according to the con-
centration of the drug. The 50% Inhibitory Concentration (IC50),
representing the concentration which inhibits 50% of cell growth,
was calculated for each compound.
Statistical analyses were performed to examine the significance
of differences in IC50 values between newly synthetized derivatives
and reference Pt drugs (cisplatin and oxaliplatin) using the Manne
Whitney test. Moreover, in each cell line, comparison of IC50 ac-
cording to the length of the polymethylene linker was assessed
using KruskaleWallis rank sum test. Differences were considered
statistically significant when p-value was <0.05.
¼
þ
C
quires: C, 38.67; H, 3.38; N, 9.39. Found: C, 38.16; H, 3.48; N, 9.20.
8b: Yield 67%. mp >260 ꢀC. IR: 1655 (CO), 1458 (NO2), 1336 (NO2)
cmꢃ1. 1H NMR (DMSO, 300 MHz)
d 9.67 (s, 1H), 9.00 (s, 1H), 8.94 (m,
1H), 8.60 (m, 1H), 7.67 (s, 1H), 7.48 (s, 1H), 6.31 (s, 2H), 4.37 (m, 2H),
2.50e3.33 (m, 6H), 2.21 (m, 2H). HR-MS (ESI) m/z ¼ 723.0470
[M þ Na]þ 723.0461 calcd for C22H21Cl2N5NaO5Pt. Anal. Calc. for
C
22H21Cl2N5O5Pt$2ꢂ CH3COCH3 requires: C, 41.13; H, 4.07; N, 8.57.
Found: C, 41.71; H, 3.93; N, 8.73. 8c: Yield 66%. mp >260 ꢀC. IR:
1656 (CO), 1458 (NO2), 1337 (NO2) cmꢃ1. 1H NMR (DMSO, 300 MHz)
d
9.63 (s, 1H), 8.97 (s, 1H), 8.94 (m, 1H), 8.58 (m, 1H), 7.63 (s, 1H),
Acknowledgments
7.45 (s, 1H), 6.31 (s, 2H), 4.48 (m, 2H), 2.50e3.33 (m, 6H), 1.84 (m,
4H). HR-MS (ESI) m/z ¼ 737.0637 [M þ Na]þ 737.0618 calcd for
The Nievre committee of the French National League against
cancer supported this work. We are really thankful to Marie-José
Penouilh for the HR-MS mass spectra and Marcel Soustelle for
elemental analyses at the “Welience, Pôle Chimie Moléculaire de
l’Université de Bourgogne (WPCM)”.
C
23H23Cl2N5NaO5Pt. Anal. Calc. for C23H23Cl2N5O5Pt$H2O requires:
C, 37.66; H, 3.44; N, 9.55. Found: C, 37.66; H, 3.27; N, 9.23. 8d: Yield
43%. mp >260 ꢀC. IR: 1654 (CO), 1458 (NO2), 1337 (NO2) cmꢃ1. 1H
NMR (DMSO, 300 MHz) d 9.69 (s, 1H), 9.01 (s, 1H), 8.97 (m, 1H), 8.63
(m, 1H), 7.67 (s, 1H), 7.49 (s, 1H), 6.31 (s, 2H), 4.48 (m, 2H), 2.50e
3.33 (m, 6H), 1.90 (m, 4H), 1.55 (m, 2H). HR-MS (ESI) m/z ¼ 751.0810
[M þ Na]þ 751.0774 calcd for C24H25Cl2N5NaO5Pt. Anal. Calc. for
Appendix A. Supplementary data
C
24H25Cl2N5O5Pt requires: C, 39.52; H, 3.45; N, 9.60. Found: C,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.037.
41.81; H, 3.41; N, 9.21. 8e: Yield 69%. mp >260 ꢀC. IR: 1660 (CO),
1457 (NO2), 1335 (NO2) cmꢃ1. 1H NMR (DMSO, 300 MHz)
d
9.69 (s,
1H), 9.01 (s, 1H), 8.96 (m, 1H), 8.62 (m, 1H), 7.68 (s, 1H), 7.50 (s, 1H),
6.32 (s, 2H), 4.47 (m, 2H), 2.50e3.33 (m, 6H), 1.91 (m, 2H), 1.76 (m,
2H), 1.48 (m, 4H). HR-MS (ESI) m/z ¼ 765.0947 [M þ Na]þ 765.0931
calcd for C25H27Cl2N5NaO5Pt. Anal. Calc. for C25H27Cl2N5O5Pt$H2O
requires: C, 39.43; H, 3.84; N, 9.20. Found: C, 39.44; H, 3.73; N, 8.88.
References
[1] A.M. Florea, D. Busselberg, Metals and breast cancer: risk factors or healing
agents? J. Toxicol. 2011 (2011) 159619.
[2] I. Kostova, Platinum complexes as anticancer agents, Recent Pat. Anticancer
Drug Discov. 1 (2006) 1e22.
[3] D. Lebwohl, R. Canetta, Clinical development of platinum complexes in cancer
therapy: an historical perspective and an update, Eur. J. Cancer 34 (1998)
1522e1534.
4.2. Cell lines, growth and cytotoxic assays
[4] B. Desoize, C. Madoulet, Particular aspects of platinum compounds used at
present in cancer treatment, Crit. Rev. Oncol. Hematol. 42 (2002) 317e325.
[5] R.J. Cersosimo, Management of advanced colorectal cancer, part 1, Am. J.
Health Syst. Pharm. 70 (2013) 395e406.
[6] J.J. Marin, F. Sanchez de Medina, B. Castano, L. Bujanda, M.R. Romero,
O. Martinez-Augustin, R.D. Moral-Avila, O. Briz, Chemoprevention, chemo-
therapy, and chemoresistance in colorectal cancer, Drug Metab. Rev. 44 (2012)
148e172.
[7] M.D. Hall, M. Okabe, D.W. Shen, X.J. Liang, M.M. Gottesman, The role of cellular
accumulation in determining sensitivity to platinum-based chemotherapy,
Annu. Rev. Pharmacol. Toxicol. 48 (2008) 495e535.
[8] L. Kelland, The resurgence of platinum-based cancer chemotherapy, Nat. Rev.
Cancer 7 (2007) 573e584.
The three human colorectal cancer cell lines HCT 116, SW480
and HT-29 were obtained from the American Type Culture Collec-
tions (Manassas, VA, United States). They were cultured in RPMI
1640 medium (Biowhittaker, France) supplemented with 10% fetal
bovine serum (Biowhittaker, France) in a 5% CO2 atmosphere. Ex-
periments were carried out on exponentially growing monolayer
cells which were mycoplasma free. The absence of mycoplasma was
checked by polymerase chain reaction (PCR) analysis (PCR Myco-
plasma Test Kit I/C, PromoKine, PromoCell France).
For cytotoxic assays, molecules were prepared as followed: the
substituted 5H-dibenzo[c,h][1,6]naphthyridin-6-one derivatives
[9] D. Wang, S.J. Lippard, Cellular processing of platinum anticancer drugs, Nat.
Rev. Drug Discov. 4 (2005) 307e320.

N. Desbois et al. / European Journal of Medicinal Chemistry 69 (2013) 719e727
727
[10] Y.P. Ho, S.C. Au-Yeung, K.K. To, Platinum-based anticancer agents: innovative
design strategies and biological perspectives, Med. Res. Rev. 23 (2003) 633e655.
[11] B. Chauffert, M. Correia, C. Sergent, Update on mechanisms of drug resistance,
Bull. Cancer 86 (1999) 97e103.
[36] L. Sharma, Y.C. Tsai, A.A. Liu, L.F. Liu, E.J. LaVoie, Cytotoxicity and TOP1-
targeting activity of 8- and 9-amino derivatives of 5-butyl- and 5-(2-N,N-
dimethylamino)ethyl-5H-dibenzo[c,h][1,6]naphthyridin-6-ones, Eur. J. Med.
Chem. 44 (2009) 1471e1476.
[12] P. Heffeter, U. Jungwirth, M. Jakupec, C. Hartinger, M. Galanski, L. Elbling,
M. Micksche, B. Keppler, W. Berger, Resistance against novel anticancer metal
compounds: differences and similarities, Drug Resist. Updates 11 (2008) 1e16.
[13] D.J. Stewart, Mechanisms of resistance to cisplatin and carboplatin, Crit. Rev.
Oncol. Hematol. 63 (2007) 12e31.
[14] N. Margiotta, N. Denora, R. Ostuni, V. Laquintana, A. Anderson, S.W. Johnson,
G. Trapani, G. Natile, Platinum(II) complexes with bioactive carrier ligands
having high affinity for the translocator protein, J. Med. Chem. 53 (2010)
5144e5154.
[15] F. Bouyer, J. Moretto, D. Pertuit, A. Szollosi, M.A. Lacaille-Dubois, Y. Blache,
B. Chauffert, N. Desbois, Synthesis, cytotoxicity and structure-activity re-
lationships between ester and amide functionalities in novel acridine-based
platinum(II) complexes, J. Inorg. Biochem. 110 (2012) 51e57.
[16] M. Carland, M.J. Grannas, M.J. Cairns, V.J. Roknic, W.A. Denny, W.D. McFadyen,
V. Murray, Substituted 9-aminoacridine-4-carboxamides tethered to plati-
num(II)diamine complexes: chemistry, cytotoxicity and DNA sequence
selectivity, J. Inorg. Biochem. 104 (2010) 815e819.
[17] D. Gibson, K.F. Gean, R. Ben-Shoshan, A. Ramu, I. Ringel, J. Katzhendler,
Preparation, characterization, and anticancer activity of a series of cis-PtCl2
complexes linked to anthraquinone intercalators, J. Med. Chem. 34 (1991)
414e420.
[18] J.M. Perez, I. Lopez-Solera, E.I. Montero, M.F. Brana, C. Alonso, S.P. Robinson,
C. Navarro-Ranninger, Combined effect of platination and intercalation upon
DNA binding of novel cytotoxic Pt-bis(naphthalimide) complexes, J. Med.
Chem. 42 (1999) 5482e5486.
[19] R. Cincinelli, L. Musso, S. Dallavalle, R. Artali, S. Tinelli, D. Colangelo, F. Zunino,
M. De Cesare, G.L. Beretta, N. Zaffaroni, Design, modeling, synthesis and bio-
logical activity evaluation of camptothecin-linked platinum anticancer agents,
Eur. J. Med. Chem. 63C (2013) 387e400.
[20] S.J. Brill, S. DiNardo, K. Voelkel-Meiman, R. Sternglanz, Need for DNA topo-
isomerase activity as a swivel for DNA replication for transcription of ribo-
somal RNA, Nature 326 (1987) 414e416.
[37] T. Sunami, K. Nishio, F. Kanzawa, K. Fukuoka, S. Kudoh, J. Yoshikawa, N. Saijo,
Combination effects of TAS-103, a novel dual topoisomerase I and II inhibitor,
with other anticancer agents on human small cell lung cancer cells, Cancer
Chemother. Pharmacol. 43 (1999) 394e401.
[38] E. Wasserman, W. Sutherland, E. Cvitkovic, Irinotecan plus oxaliplatin: a
promising combination for advanced colorectal cancer, Clin. Colorectal Cancer
1 (2001) 149e153.
[39] C. Plasencia, A. Abad, E. Martinez-Balibrea, M. Taron, Antiproliferative effects
of ZD0473 (AMD473) in combination with 5-fluorouracil or SN38 in human
colorectal cancer cell lines, Invest. New Drugs 22 (2004) 399e409.
[40] R.C. van Waardenburg, L.A. de Jong, M.A. van Eijndhoven, C. Verseyden,
D. Pluim, L.E. Jansen, M.A. Bjornsti, J.H. Schellens, Platinated DNA adducts
enhance poisoning of DNA topoisomerase I by camptothecin, J. Biol. Chem.
279 (2004) 54502e54509.
[41] R.J. Holmes, M.J. McKeage, V. Murray, W.A. Denny, W.D. McFadyen, cis-
Dichloroplatinum(II)
complexes
tethered
to
9-aminoacridine-4-
carboxamides: synthesis and action in resistant cell lines in vitro, J. Inorg.
Biochem. 85 (2001) 209e217.
[42] H.H. Lee, B.D. Palmer, B.C. Baguley, M. Chin, W.D. McFadyen, G. Wickham,
D. Thorsbourne-Palmer, L.P. Wakelin, W.A. Denny, DNA-directed alkylating
agents. 5. Acridinecarboxamide derivatives of (1,2-diaminoethane)dichlor-
oplatinum(II), J. Med. Chem. 35 (1992) 2983e2987.
[43] A.L. Ruchelman, S.K. Singh, X. Wu, A. Ray, J.M. Yang, T.K. Li, A. Liu, L.F. Liu,
E.J. LaVoie, Diaza- and triazachrysenes: potent topoisomerase-targeting
agents with exceptional antitumor activity against the human tumor xeno-
graft, MDA-MB-435, Bioorg. Med. Chem. Lett. 12 (2002) 3333e3336.
[44] A.L. Ruchelman, J.E. Kerrigan, T.K. Li, N. Zhou, A. Liu, L.F. Liu, E.J. LaVoie, Nitro
and amino substitution within the A-ring of 5H-8,9-dimethoxy-5-(2-N,N-
dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: influence on
topoisomerase I-targeting activity and cytotoxicity, Bioorg. Med. Chem. 12
(2004) 3731e3742.
[45] A.L. Ruchelman, S. Zhu, N. Zhou, A. Liu, L.F. Liu, E.J. LaVoie, Dimethoxy benzo[i]
phenanthridine-12-carboxylic acid derivatives and 6H-dibenzo[c,h][2,6]
naphthyridin-5-ones with potent topoisomerase I-targeting activity and
cytotoxicity, Bioorg. Med. Chem. Lett. 14 (2004) 5585e5589.
[46] W. Feng, M. Satyanarayana, L. Cheng, A. Liu, Y.C. Tsai, L.F. Liu, E.J. LaVoie,
Synthesis of N-substituted 5-[2-(N-alkylamino)ethyl]dibenzo[c,h][1,6]naph-
thyridines as novel topoisomerase I-targeting antitumor agents, Bioorg. Med.
Chem. 16 (2008) 9295e9301.
[21] K.C. Neuman, Single-molecule measurements of DNA topology and top-
oisomerases, J. Biol. Chem. 285 (2010) 18967e18971.
[22] K. Legarza, L.X. Yang, New molecular mechanisms of action of camptothecin-
type drugs, Anticancer Res. 26 (2006) 3301e3305.
[23] Y. Xu, M.A. Villalona-Calero, Irinotecan: mechanisms of tumor resistance and
novel strategies for modulating its activity, Ann. Oncol. 13 (2002) 1841e1851.
[24] Y. Pommier, Topoisomerase I inhibitors: camptothecins and beyond, Nat. Rev.
Cancer 6 (2006) 789e802.
[25] S. Basili, S. Moro, Novel camptothecin derivatives as topoisomerase I in-
hibitors, Expert Opin. Ther. Pat. 19 (2009) 555e574.
[47] L.S. Kurtzberg, S. Roth, R. Krumbholz, J. Crawford, C. Bormann, S. Dunham,
M. Yao, C. Rouleau, R.G. Bagley, X.J. Yu, F. Wang, S.M. Schmid, E.J. Lavoie,
B.A. Teicher, Genz-644282, a novel non-camptothecin topoisomerase I in-
hibitor for cancer treatment, Clin. Cancer Res. 17 (2011) 2777e2787.
[48] T.K. Li, P.J. Houghton, S.D. Desai, P. Daroui, A.A. Liu, E.S. Hars, A.L. Ruchelman,
E.J. LaVoie, L.F. Liu, Characterization of ARC-111 as a novel topoisomerase I-
targeting anticancer drug, Cancer Res. 63 (2003) 8400e8407.
[26] G.L. Beretta, V. Zuco, P. Perego, N. Zaffaroni, Targeting DNA topoisomerase I
with non-camptothecin poisons, Curr. Med. Chem. 19 (2012) 1238e1257.
[27] C. Sheng, Z. Miao, W. Zhang, New strategies in the discovery of novel non-
camptothecin topoisomerase
I inhibitors, Curr. Med. Chem. 18 (2011)
4389e4409.
[49] W. Feng, M. Satyanarayana, Y.C. Tsai, A.A. Liu, L.F. Liu, E.J. Lavoie, Facile for-
mation of hydrophilic derivatives of 5H-8,9-dimethoxy-5-[2-(N,N-dimethy-
lamino)ethyl]-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one
(ARC-111) and its 12-aza analog via quaternary ammonium intermediates,
Bioorg. Med. Chem. Lett. 18 (2008) 3570e3572.
[50] W. Feng, M. Satyanarayana, Y.C. Tsai, A.A. Liu, L.F. Liu, E.J. LaVoie, Novel topo-
isomerase I-targeting antitumor agents synthesized from the N,N,N-trimethy-
lammonium derivative of ARC-111, 5H-2,3-dimethoxy-8,9-methylenedioxy-
5-[(2-N,N,N-trimethylammonium)ethyl]dibenzo[c,h][1,6]naphthyridin-6-one
iodide, Eur. J. Med. Chem. 44 (2009) 3433e3438.
[28] Y. Pommier, DNA topoisomerase I inhibitors: chemistry, biology, and inter-
facial inhibition, Chem. Rev. 109 (2009) 2894e2902.
[29] Y. Yamashita, N. Fujii, C. Murakata, T. Ashizawa, M. Okabe, H. Nakano, In-
duction of mammalian DNA topoisomerase I mediated DNA cleavage by
antitumor indolocarbazole derivatives, Biochemistry 31 (1992) 12069e12075.
[30] N. Vicker, L. Burgess, I.S. Chuckowree, R. Dodd, A.J. Folkes, D.J. Hardick,
T.C. Hancox, W. Miller, J. Milton, S. Sohal, S. Wang, S.P. Wren, P.A. Charlton,
W. Dangerfield, C. Liddle, P. Mistry, A.J. Stewart, W.A. Denny, Novel angular
benzophenazines: dual topoisomerase I and topoisomerase II inhibitors as
potential anticancer agents, J. Med. Chem. 45 (2002) 721e739.
[31] D. Makhey, C. Yu, A. Liu, L.F. Liu, E.J. LaVoie, Substituted benz[a]acridines and
benz[c]acridines as mammalian topoisomerase poisons, Bioorg. Med. Chem. 8
(2000) 1171e1182.
List of captions
[32] Y.L. Janin, A. Croisy, J.F. Riou, E. Bisagni, Synthesis and evaluation of new 6-
amino-substituted benzo[c]phenanthridine derivatives, J. Med. Chem. 36
(1993) 3686e3692.
BCRP: Breast Cancer Resistance Protein
DMF: dimethylformamide
DMSO: dimethylsulfoxide
[33] A.L. Ruchelman, S.K. Singh, A. Ray, X.H. Wu, J.M. Yang, T.K. Li, A. Liu, L.F. Liu,
E.J. LaVoie, 5H-Dibenzo[c,h]1,6-naphthyridin-6-ones: novel topoisomerase I-
targeting anticancer agents with potent cytotoxic activity, Bioorg. Med. Chem.
11 (2003) 2061e2073.
[34] S.K. Singh, A.L. Ruchelman, T.K. Li, A. Liu, L.F. Liu, E.J. LaVoie, Nitro and amino
substitution in the D-ring of 5-(2-dimethylaminoethyl)-2,3-methylenedioxy-
5H-dibenzo[c,h][1,6]naphthyridin-6-ones: effect on topoisomerase-I targeting
activity and cytotoxicity, J. Med. Chem. 46 (2003) 2254e2257.
[35] A.L. Ruchelman, P.J. Houghton, N. Zhou, A. Liu, L.F. Liu, E.J. LaVoie, 5-
(2-Aminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: variation of n-alkyl
substituents modulates sensitivity to efflux transporters associated with
multidrug resistance, J. Med. Chem. 48 (2005) 792e804.
ESI: electrospray ionization
HR-MS: high resolution-mass spectrometry
IC50: 50% inhibitory concentration
MDR1: multi drug resistance 1
mp: melting point
NMR: nuclear magnetic resonance
PBS: phosphate buffered saline
PCR: polymerase chain reaction
Pt: platinum
Scid: severe combined immuno deficiency
TFA: trifluoroacetic acid