Design, modeling, synthesis and biological activity evaluation of camptothecin-linked platinum anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2013.02.022

The design, modeling, synthesis and biological activity evaluation of two hybrid agents formed by 7-oxyiminomethylcamptothecin derivatives and diaminedichloro-platinum (II) complex are reported. The compounds showed growth inhibitory activity against a panel of human tumor cell lines, including sublines resistant to topotecan and platinum compounds. The derivatives were active in all the tested cell lines, and compound 1b, the most active one, was able to overcome cisplatin resistance in the osteosarcoma U2OS/Pt cell line. Platinum-containing camptothecins produced platinum-DNA adducts and topoisomerase I-mediated DNA damage with cleavage pattern and persistence similar to SN38, the active principle of irinotecan. Compound 1b exhibited an appreciable antitumor activity in vivo against human H460 tumor xenograft, comparable to that of irinotecan at lower well-tolerated dose levels and superior to cisplatin. The results support the interpretation that the diaminedichloro-platinum (II) complex conjugated via an oxyiminomethyl linker at the 7-position of the camptothecin resulted in a new class of effective antitumor compounds.

Full text of DOI:10.1016/j.ejmech.2013.02.022

European Journal of Medicinal Chemistry 63 (2013) 387e400
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, modeling, synthesis and biological activity evaluation of
camptothecin-linked platinum anticancer agents
,
b
c
Raffaella Cincinelli a, Loana Musso a, Sabrina Dallavalle a , Roberto Artali , Stella Tinelli ,
Donato Colangelo d, Franco Zunino c, Michelandrea De Cesare c, Giovanni Luca Beretta c,
Nadia Zaffaroni c
*
a Department of Food, Environmental and Nutritional Sciences, Division of Chemistry and Molecular Biology, Università di Milano, Via Celoria 2,
20133 Milano, Italy
b Scientia Advice, Via Galileo Ferraris 28, 20851 Lissone (MB), Italy
c Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCSS Istituto Nazionale Tumori,
Via Amadeo 42, 20133 Milan, Italy
d Università del Piemonte Orientale, Via Solaroli 17, 28100 Novara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, modeling, synthesis and biological activity evaluation of two hybrid agents formed by
7-oxyiminomethylcamptothecin derivatives and diaminedichloro-platinum (II) complex are reported.
The compounds showed growth inhibitory activity against a panel of human tumor cell lines, including
sublines resistant to topotecan and platinum compounds. The derivatives were active in all the tested cell
lines, and compound 1b, the most active one, was able to overcome cisplatin resistance in the osteo-
sarcoma U2OS/Pt cell line. Platinum-containing camptothecins produced platinum-DNA adducts and
topoisomerase I-mediated DNA damage with cleavage pattern and persistence similar to SN38, the active
principle of irinotecan. Compound 1b exhibited an appreciable antitumor activity in vivo against human
H460 tumor xenograft, comparable to that of irinotecan at lower well-tolerated dose levels and superior
to cisplatin. The results support the interpretation that the diaminedichloro-platinum (II) complex
conjugated via an oxyiminomethyl linker at the 7-position of the camptothecin resulted in a new class of
effective antitumor compounds.
Received 4 December 2012
Received in revised form
14 February 2013
Accepted 19 February 2013
Available online 1 March 2013
Keywords:
Antitumor compounds
Docking
Camptothecin
Diaminedichloro-platinum (II)
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
interactions; d) relatively poor pharmacokinetic profiles and e)
increased DNA repair capacity [3e5].
The antitumor activity of cis-diaminedichloro-platinum (II)
(cisplatin or cDDP, Chart 1) was first reported by Rosenberg et al. in
1969 [1]. The success of cisplatin paved the way for the second and
third-generation platinum(II) drugs, carboplatin and oxaliplatin
(Chart 1). Presently, platinum-based coordination complexes are
among the most widely used antitumor agents in the clinic [2]. The
effectiveness of cisplatin lies in its ability to covalently bind DNA,
leading to important changes in the helical structure. In spite of the
efficacy of platinum-based treatment regimens, long-term cure is
difficult to obtain. The major drawbacks include a) severe and
sometimes life-threatening toxic side effects; b) activation of drug
resistance mechanisms by tumor cells; c) inadequate intratumor
concentration of the drug and tumor microenvironmental
Recently, major effort has been made to develop new platinum
complexes. Often, the strategies were aimed to increase cellular
uptake and/or facilitate targeting of the compounds to DNA [6]. The
latter approach involves the incorporation of a functional group into
the platinum complex that interacts or intercalates with DNA.
Numerous conjugates have been prepared in which the Pt moiety
has been tethered to high-affinity nucleic acid ligands, such as
doxorubicin [7], acridine [8], amines or peptides [9], anthraquinones
[10] and short oligonucleotides [11]. In particular, a mixed plati-
num(II) complex with doxorubicin demonstrated antitumor activity
against a variety of tumor cell lines, including doxorubicin- and
cisplatin-resistant cell lines. Moreover, in contrast tothe highly toxic
doxorubicin/cisplatin combination, the doxorubicineplatinum
complex was not associated to an increase in toxicity [7].
As part of our program aimed to advance DNA as a drug target,
we were interested in devising novel platinum containing dual
* Corresponding author. Tel.: þ39 (2)50316818; fax: þ39 (2)50316801.
E-mail address: sabrina.dallavalle@unimi.it (S. Dallavalle).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.022

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R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
compounds that interacted in an effective way with DNA and in
addition accomplished the requirements of solubility in the body
fluids and improved cellular uptake. In this context, a promising
approach seemed to be the conjugation of platinum to analogues of
the natural antitumor compound camptothecin (CPT).
CPTs [12] are widely used for the treatment of human cancers
[13]. They exhibit a unique mechanism of action because they
target the topoisomerase I (Topo I), forming a ternary complex with
the enzyme and DNA [14]. Stabilization of the cleavable complex
and collision with the replication fork results in lethal double-
strand breaks during DNA replication [15]. On the basis of this
mechanism of action, the reversibility of drug-target interaction
represents a limitation that results in resistance of slowly growing
tumors. Intensive efforts in medicinal chemistry over the past de-
cades have provided a large number of CPT analogues, of which
topotecan (TPT) and irinotecan (prodrug of SN-38, Chart 2) are
those so far approved for the clinical therapy [16].
Given the chemical interest of these agents [17], we planned to
incorporate the two active compounds in a single “hybrid” molecule
with the intention of exerting dual action and/or increase the drug-
target interaction. The potential advantages of using CPTePt con-
jugates are multiple. On one hand, they could promote adequate
cellular accumulation and nuclear localization of the Pt(II)-complex
by virtue of the hydrophobicity of CPTs. On the other hand, the
incorporation of a chemical function able to covalently bind to DNA,
like a Pt moiety, could stabilize the CPTeDNAeenzyme ternary
complex, thereby improving the drug-target interaction.
derivatives, even when bulky [19] and long-chain [20] substituents
were introduced. Some compounds gave promising results in pre-
clinical [21] and clinical [22] studies. In contrast, incorporation of the
same groups in position 9 caused a drop in cytotoxic activity [23].
For this reason, (E)-7-oxyminomethyl CPTs were selected for
conjugation to Pt(II) [19]. Initially, we focused on the most prom-
ising 7-substituted compound in our hands, 3a (Fig. 1). As the
molecule is characterized by a terminal amino group, we planned to
link the CPT moiety to Pt(II) through an amide bond. Thus, the
synthesis of compound 1a (Fig. 1), designed as our first conjugate,
was devised to proceed by coupling of 3a with 2,3-
diaminopropionic acid followed by complexation of the derived
diamine (2a) with a suitable platinum salt.
Prior to start the synthesis, we studied the binding mode of
compound 1a to the Topo I covalent complex with DNA [24]. A two-
step protocol was used to obtain a better estimate of the orientation
of the ligands within the binding site. First, the non-platinated li-
gands were correctly placed within the binding site using the
molecular docking technique. On the basis of the geometry of
interaction thus obtained, the platinated ligands were built and
analyzed using the QM/MM approach. To gain insight into the role
of the linker on the interaction with the target, we also modeled the
binding of a series of derivatives 1 with different carbon chain
length (n ¼ 1e4). Molecular modeling showed that the compound
with six carbons (n ¼ 3, 1b) demonstrated the best interactions.
Thus, for the sake of clarity, here we describe only the target-drug
interactions of compound 1b compared to 1a.
In the present study, we describe the synthesis, molecular
modeling and biological evaluation of CPTePt complexes with
different Pt-containing linkers. To the best of our knowledge, there
are no examples in the literature of diaminedichloro-platinum (II)
complexes tethered to CPT analogues.
The intercalation binding site for all of the studied compounds
was located between the þ1 (upstream) and ꢀ1 (downstream) base
pairs of the uncleaved strand, which effectively “open” the DNA
duplex. Comparative analysis of the best poses obtained for the four
non-platinum-containing compounds (2aeb, 3aeb) revealed their
common behavior of intercalating at the site of DNA cleavage,
forming base-stacking interactions with both the ꢀ1 and þ1 base
pairs. There is evidence of two direct hydrogen bonds between the
Asp533 and Arg364 residues of the enzyme and the planar skeleton
of the investigated derivatives, in perfect agreement with the CPTe
DNA complex structure [24]. The greatest differences were found
by analyzing the behavior of the side chains. For 2a, the side chain
was too short and allows only a partial interaction with the DNA
duplex, leading one of the two amino groups to interact with O6
and N7 of G11, whereas the immine nitrogen interacts weakly with
N6A113. For both 2b and 3b, the greater length of their side chains
allows them to interact more efficiently with the DNA duplex. The
amino group of 3b forms three strong hydrogen with N7G11,
whereas 2b forms a hydrogen bond to N6A113, two between the
amino group and O6G12 and the last with N7G11. The interactions
between derivatives 2a and 2b and the DNA-Topo I complex are
2. Results and discussion
2.1. Chemistry and molecular modeling
The following criteria have guided the design of the CPTePt(II)
conjugates: a) a CPT moiety substituted in a suitable position to
maintain cytotoxic activity; b) a linker between the Pt center and
the CPT skeleton, chosen to ensure enough conformational flexi-
bility while maintaining proximity of the reactive metal center to
the DNA-selective scaffold; c) a chelating diamine selected to
secure Pt(II) coordination to the CPT moiety.
Recently, a series of CPTs substituted in position 7 have been
synthesized in our laboratory. The compounds exhibited potent
cytotoxic activity in vitro and in vivo comparable or superior to TPT
[18e20]. The highest activity was shown by oxyiminomethyl
Cl
Pt
Cl
H2N
NH2
NH2
NH
n
NH2
NH
n
NH2
O
O
N
O
n
N
O
O
N
O
O
O
N
N
N
N
N
N
O
O
O
1a n = 1
1b n= 3
3a n = 1
3b n= 3
2a n = 1
2b n= 3
OH
O
OH
OH
O
O
Fig. 1. Retrosynthetic approach to compounds 1.

R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
389
base for 1a is even more solvent exposed than for 1b, giving rise to
an additional source of instability of the 1a complex.
It should be noted that in Figs. 2 and 3, the best poses for de-
rivatives 1 and 2 with only S absolute configuration at the stereo-
genic center on the diamine moiety are reported. The best poses for
the corresponding R diastereomers did not show striking differ-
ences with regard to chirality, indicating that the stereochemistry
of the diamine moiety does not seem to play a role in the interac-
tion with DNA.
On the basis of molecular modeling results, the synthesis of
compounds 1a and 1b was carried out.
Compound 1a was prepared from the corresponding
7-oxyminomethylCPT 3a [19], which in turn was obtained from
7-formylCPT (4). The oxime was then condensed with N,N-bis-t-
butoxycarbonyl-2,3-diaminopropanoic acid in the presence of WSC
and HOBt as condensing agents. The Boc-diamine 5a was quanti-
tatively deprotected with TFA in CH2Cl2 to obtain the bistri-
fluoroacetate 6a. Reaction with K2PtCl4 [25] afforded 1a in 69%
yield (Scheme 1).
Fig. 2. Interactions between the derivatives (2a and 2b, on the right) and the exper-
imental DNAeTopoisomerase I complex (left). The binding site is located inside the
region delimited by the white rectangle (left) and the dotted green lines (right side)
represent the intermolecular hydrogen bonds. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
The synthesis of compound 1b required the preparation of the
longer-chain hydroxylamine
8 (Scheme 2). Protection of 6-
given in Fig. 2 (together with the experimental DNA-Topo I struc-
ture (PBD code 1T8I)), where the interactions listed as dotted green
lines represent the intermolecular hydrogen bonds.
aminohexanol with (Boc)2O, followed by Mitsunobu reaction with
N-hydroxyphthalimide, afforded compound 7. Hydrazinolysis and
subsequent deprotection from the Boc group with 5.9 M HCl at 0 ꢁC
Orientations of the systems described above (2a and 2b) were
used to assemble the two Pt complexes (1a and 1b), which were
further analyzed using the QM/MM mixed approach on the plati-
nated complexes. This QM/MM mixed approach allowed us to
obtain a better and more complete description of the interactions of
the two Pt complexes with the DNA-Topo I system, as well as an
evaluation of the structural changes induced in the complex by the
binding of ligands 1a and 1b. Major differences were observed at
the level of the side chain, whereas the CPT ring maintained its
position inside the DNA-Topo I system (Fig. 3).
afforded hydroxylamine
7-formylCPT (4) by refluxing in ethanol in the presence of pyridine
afforded oxime 3b.
8 hydrochloride. Condensation with
Following the same synthetic approach described for compound
1a, the bistrifluoroacetate 6b was obtained in 65% overall yield
from 3b. Disappointingly, in this case the reaction with K2PtCl4
gave compound 1b in low yield and unsatisfactory purity, due to
a troublesome separation of the desired compound from the
salt 6b. Thus, a different strategy was followed. The cis-dichlor-
obis(diaminopropionic acid hydrochloride) platinum (II) complex
was first prepared in high yield by reacting 2,3-diaminopropionic
acid hydrochloride with K2PtCl4 in H2O [26] and then condensed
with 3b to give 1b in 89% yield (Scheme 2).
The compound with the longer spacer (1b) is more effective in
its interaction with the DNA duplex than the one with the shorter
side chain (1a). Both complexes are stabilized by the formation of
two hydrogen bonds between O6G12 and an amino group bound to
the platinum atom that lies in the square planar coordination plane
with a small distortion of the PteN7 bond due to the destacking of
the bases. Compound 1b is further stabilized by two interactions
with N7G11 and N6A113, the same as those previously discussed for
2b. Indeed, the complex formation induces a distortion of the DNA
duplex, with a partial destacking of the nucleotide bases, which is
less marked in the case of compound 1b. The shift of G12 in the
2.2. Antiproliferative activity studies
The antiproliferative effect of the derivatives 1aeb and 6aeb
was determined at 1 h exposure on different human tumor cell
lines, including cell lines resistant to TPT and platinum compounds
(Table 1). TPT and cDDP were used as reference drugs. All the tested
compounds were generally more potent than cDDP on all the
selected cell lines, including those resistant to cDDP, oxaliplatin and
TPT. In contrast, a comparison between the new CPTePt derivatives
and TPT revealed cell line-dependent drug potency. A superior
cytotoxicity with respect to TPT was evidenced for 1b and 6b on the
H460 cell line, since 6a and 1a are less potent than TPT. All the
compounds were less potent that TPT in the squamous cell carci-
noma A431 cell line, including the corresponding sublines resistant
to TPT (A431/TPT) and cDDP (A431/Pt). The compounds were less
potent than TPT in the osteosarcoma U2OS cell line and, with the
exception of 1b, such behavior was observed also in the corre-
sponding cDDP-resistant variant (U2OS/Pt). A general reduced po-
tency with respect to TPT was observed in ovarian carcinoma
IGROV-1 and A2780 cell lines. This was also evident in the corre-
sponding cDDP and oxaliplatin-resistant sublines with the excep-
tion of 1b in IGROV-1/Pt and 1b and 6b in IGROV-1/OHP cells.
As regards the resistance index (ratio between IC50 values of
resistant and sensitive cells), the contribution of the Pt(II) complex
was demonstrated by the reduced resistance indexes observed for
CPTePt derivatives with respect to cDDP and TPT in several human
tumor cell lines. The resistance index of CPTePt (1a and 1b) was
ꢀ
complex with 1a is significantly larger (7.1 A) than in the complex
ꢀ
with 1b (5.4 A), in poor agreement with the guanine position in the
crystal structure. Moreover, the hydrophobic face of the guanine
Fig. 3. Interactions between the Pt-derivatives (1a and 1b) and the experimental
DNAetopoisomerase I complex. The main structural changes induced in the complex
by the presence of platinum were observed at the level of the side chain, whereas the
CPT ring maintained its position inside the DNAetopoisomerase I system. Again, the
dotted green lines represent the intermolecular hydrogen bonds. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)

390
R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
NHBoc
NHBoc
NH2
NH
O
O
N
O
N
O
CHO
O
a
O
b
N
N
N
N
N
N
O
O
O
3a
5a
4
OH
O
OH
OH
O
O
Cl
Pt
-OOCCF3
Cl
+
-OOCCF3
H2N
NH3
+
NH2
NH3
NH
O
NH
O
O
N
O
N
O
O
d
c
N
N
N
N
O
O
1a
6a
OH
O
OH
O
Scheme 1. Reagents and conditions: (a) H2NeOeCH2CH2NH2$2HCl, ethanol, Py, reflux, 1 h, 63%; (b) N,N-bis-t-butoxycarbonyl-2,3-diaminopropanoic acid, WSC, HOBt, DIPEA,
DMF, rt, 2.5 h, 77%; (c) TFA/CH2Cl2 1:1, from 0 ꢁC to rt, 3 h, quantitative; (d) K2PtCl4, H2O, N2, rt, 3h, 69%.
O
a,b
c,d
HO
NH2
N
O
NHBoc
O
NH2
. 2 HCl
4
H2N
4
4
O
8
7
O
NH3+CF3COO-
NH3+CF3COO-
O
N
NH2
4
O
N
NH
4
4
e
f, g
4
O
O
N
N
N
N
O
O
3b
6b
OH
O
OH
O
h
i
O
NH2
NH2
Cl
Cl
O
N
NH
4
Pt
4
O
N
N
O
OH
O
1b
Scheme 2. Reagents and conditions: (a) (Boc)2O, CH2Cl2, rt, overnight, 99%; (b) N-hydroxyphthalimide, triphenylphosphine, DIAD, THF, from 0 ꢁC to rt, 1 h, 50%; (c) hydrazine
hydrate, methanol, reflux, 1 h, quantitative; (d) HCl 5.9 M in ethyl acetate, ethyl acetate, 0 ꢁC, 1 h, 97%; (e) 4, Py, ethanol, reflux, 1 h, 77%; (f) N,N-bis-t-butoxycarbonyl-2,3-
diaminopropanoic acid, WSC, HOBt, DIPEA, DMF, rt, 2.5 h, 65%; (g) TFA/CH2Cl2 1:1, from 0 ꢁC to rt, 3 h, quantitative; (h) K2PtCl4, H2O, N2, rt, 3h, 20%. (i) cis-dichlorobis[(d,l)-
diaminopropionic acid hydrochloride]platinum(II), EDC, TEA, DMF, rt, overnight, 89%.

R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
391
Table 1
Cellular sensitivity of human tumor cell lines to CPT derivatives.a
Cell line
TPT
cDDP
6a
1a
6b
1b
H460
U2OS
U2OS/Pt
A431
A431/TPT
A431/Pt
IGROV-1
IGROV-1/Pt
IGROV-1/OHP
A2780
1.37 ꢂ 0.34
22 ꢂ 6.3
6.47 ꢂ 2.82
4.1 ꢂ 0.59
0.44 ꢂ 0.017
0.4 ꢂ 0.016
1.09 ꢂ 0.54
0.48 ꢂ 0.4
20.5 ꢂ 9.2
4.2 ꢂ 2.26
5.39 ꢂ 2.4
1.36 ꢂ 0.34
2.34 ꢂ 0.86 (4.87)
0.075 ꢂ 0.067
0.29 ꢂ 0.01 (3.86)
0.27 ꢂ 0.29 (3.6)
1.03 ꢂ 0.8
107.8 ꢂ 24 (5)
19.57 ꢂ 1.37
16.84 ꢂ 2.19 (0.86)
64.6 ꢂ 7.6 (3.3)
14.8 ꢂ 2.54
10.9 ꢂ 4.44 (2.6)
0.99 ꢂ 0.35
14.2 ꢂ 5.6 (2.6)
1.68 ꢂ 0.29
2.59 ꢂ 1.58 (1.9)
0.28 ꢂ 0.099
0.82 ꢂ 0.25 (0.75)
0.185 ꢂ 0.035
8 ꢂ 1.4 (8)
14.2 ꢂ 0.28 (8.4)
3.79 ꢂ 1.7 (2.25)
5.79 ꢂ 0.29
2.87 ꢂ 1.94 (10.3)
0.72 ꢂ 0.176 (2.57)
2.24 ꢂ 0.3
1.23 ꢂ 0.89 (6.6)
0.67 ꢂ 0.19 (3.62)
1.24 ꢂ 0.077
4.27 ꢂ 1.07 (4.3)
17.8 ꢂ 4.5
3.9 ꢂ 0.96 (3.9)
10.9 ꢂ 1.34 (10.5)
0.036 ꢂ 0.0035
0.275 ꢂ 0.01 (7.6)
211 ꢂ 19 (14)
120.8 ꢂ 20 (8)
4.35 ꢂ 0.49
11.8 ꢂ 1.42 (0.66)
28 ꢂ 7.5 (1.57)
0.69 ꢂ 0.44
14.2 ꢂ 10.2 (2.5)
15.8 ꢂ 2.6 (2.7)
2.16 ꢂ 0.23
4.1 ꢂ 2.14 (1.8)
2.38 ꢂ 0.18 (1.06)
0.08 ꢂ 0.028
3.03 ꢂ 1.32 (2.4)
3.17 ꢂ 0.75 (2.6)
0.22 ꢂ 0.064
A2780/CP
41.5 ꢂ 9.2 (9.5)
2.55 ꢂ 0.35 (3.7)
4.8 ꢂ 1.4 (2.2)
0.79 ꢂ 0.45 (9.8)
0.73 ꢂ 0.31 (3.3)
a
Cell sensitivity to drug was assessed by growth-inhibition assay. Twenty-four hours after seeding, cells were exposed to drug for 1 h and then, after an additional 72 h,
M (drug concentration inhibiting growth by 50%). In parenthesis is reported the resistance index
(ratio between IC50 values of resistant and sensitive cells). Data represent mean values ꢂ SD from at least 3 independent experiments.
counted using a cell counter. The reported value is the IC50 expressed in
m
higher than that of cDDP and TPT in A431/TPT cells. Conversely, a
reduced resistant index with respect to TPT and cDDP was observed
for the two hybrid compounds in A431/Pt, U2OS/Pt, IGROV-1/Pt,
IGROV-1/OHP and A2780/CP cells.
effect of the combination of cDDP and our derivatives (6aeb) on
antiproliferative activity was evaluated on the H460 cell line (Fig. 5).
To this purpose, cells were simultaneously exposed for 1 h to
increasing concentrations of CPTs and to subtoxic concentration
It is noteworthy that the most active derivative 1b was effective
in overcoming cDDP resistance in osteosarcoma cDDP-resistant
U2OS cells (Table 1 and Fig. 4).
(10 mM) of cDDP (Fig. 5). The data demonstrated that, as for the
combination of cDDP with TPT, the combination of 6aeb with cDDP
resulted in a synergistic interaction, thus supporting the rational of
joining the two moieties into a single molecule.
To clarify the role of the Pt(II) complex in drug activity, the ratio
between the IC50 of compounds not containing Pt and the IC50 of
compounds containing Pt (6a/1a or 6b/1b) was considered. A ratio
higher than 1 indicates the positive effect produced by the Pt atom.
As reported in Fig. 4, the role of the Pt atom appears more impor-
tant for the potency of 1b than for 1a. Indeed, considering the 11
cell lines evaluated, the ratios were <1 in six cell lines for 6a/1a and
only in two cell lines for 6b/1b.
2.4. Topoisomerase I-dependent DNA cleavage assay
Topo I-mediated DNA cleavage experiments were performed to
investigate the ability of the new compounds to stimulate DNA
damage. SN38 was used as reference compound. As reported in
Fig. 6, a dose-dependent precipitation of labeled DNA in the wells
was observed for CPTePt derivatives (1a and 1b). This behavior was
drug- and enzyme-dependent since it was not observed in the
presence of cDDP, 6b, or in the absence of Topo I.
2.3. Drug combination studies
Drug combination experiments using platinum compounds with
CPTs are widely documented [27]. Synergism was evidenced in
various human tumor cell lines [27]. Indeed, the presence of Pte
DNA adducts in the substrate DNA inhibited Topo I activity,
whereas it enhanced CPT action on the cleavable complex [28]. The
Stabilization of the ternary cleavable complex was evaluated by
adding a high salt concentration (0.6 M NaCl) after 30 min of in-
cubation in the presence of the tested compound. The high salt
concentration favors the dissociation of the drugeenzymeeDNA
complex, thus producing evidence of the stability of the ternary
A
B
6b/1b
4,0
6a/1a
TPT
cDDP
1a
15
14
13
12
11
10
9
3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
1b
8
7
6
5
4
3
2
1
0
Fig. 4. (A) Graphic representation of the resistance indexes observed in the tested cell lines. The ratio between IC50 values of resistant and sensitive cells is reported for 1a and 1b.
The resistant indexes of TPT and cDDP are reported for comparison. (B) Graphic representation of the ratio between the IC50 of the compound not containing Pt (6b or 6a) and the
IC50 of the compound containing Pt (1b or 1a). A ratio higher than 1 (dashed line) indicates the positive effects produced by the Pt atom for drug potency.

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R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
2,2
2,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
stability with respect to SN38. Such behavior was less evident when
CPTePt compounds were compared with the corresponding de-
rivatives lacking Pt (6a and 6b) (Fig. 7).
TPT
6b
6a
2.5. Drug uptake and DNA platination
Drug uptake and DNA platination were evaluated in H460 cells
exposed for 1 h to equitoxic (IC50 cDDP ¼ 22
m
M; 1a ¼ 4.1
mM;
S
1b ¼ 0.4
m
M) or equimolar (0.4 M) concentrations of compounds.
m
cDDP was used as reference drug. As reported in Fig. 8A, at equi-
toxic concentrations a marked cellular uptake of 1a was observed. It
is noteworthy that the uptake of 1b was comparable to that of cDDP,
in spite of its 55-fold lower concentration. As regards the exposure
A
to equimolar drug concentration (0.4 mM), a significantly higher
cellular Pt accumulation was evidenced for 1a (P < 0.02) and 1b
(P < 0.01) with respect to cDDP (Fig. 8B).
0
5
10
15
20
25
30
DNA platination was studied in H460 cells exposed for 1 h to
equitoxic (IC50) concentrations of compounds. PteDNA adducts
were revealed in the DNA extracted from cells treated with both 1a
and 1b (Fig. 8C). DNA-bound Pt was significantly higher in cells
exposed to cDDP than 1a and 1b (P < 0.02). The finding is expected,
because the low potency of cDDP required the use of substantially
Drug concentration (µM)
Fig. 5. Interaction between camptothecins and cDDP. Antiproliferative activity of the
combination was determined after a 1 h exposure to the camptothecin and cDDP using
a cell counter. Dose-response curves for each CPT were determined in the presence of
subtoxic concentrations of cDDP (10 mM; i.e., under conditions that did not produce
antiproliferative effects of the cDDP). The combination index was calculated according
to the method of Kern. S, synergism; A, antagonism.
higherconcentrations (IC50 cDDP ¼ 22
m
M). In contrast, the exposure
of H460 cells to equimolar concentration of drugs (0.4
mM) resulted
in DNA platination only in cells treated with 1b (data not shown).
complex. The stability of the cleavage complex was evaluated at
2.6. Viability and DNA platination of yeast cells expressing human
DNA topoisomerase I
1
mM drug concentration. It was not possible to evaluate the sta-
bility of the cleavable complex at 10 M drug concentration since
m
the accumulation of the labeled DNA into the wells was not
reversed (data not shown). The cleavage persistence evaluated at
1 mM indicated that CPTePt 1b and 1a induced a persistent cleavage
The S. cerevisiae JN2-134 strain, which lacks endogenous DNA
Topo I, was used. Yeast cells transformed with the empty vector
(pEMBLyex4) or with the same vector containing the entire ORF of
the human DNA Topo I (pEZ-2hTop1) were treated with CPTs (1ae
b, 6aeb) for 24 h and then plated according to the yeast spot test
(Fig. 9). CPT and cDDP were used as references. cDDP exhibited
similar growth inhibitory potency on both pEMBLyex4 and pEZ-
2hTop1 transformed yeasts, indicating that cDDP potency was
unaffected by the presence/absence of Topo I. Conversely, cell
growth of yeast cells expressing human Topo I was affected by the
exposure to CPTs (1aeb, 6aeb). No important change in cell growth
was observed for yeast cells transformed with the empty vector and
treated with CPTs. Interestingly, a significant increase (P < 0.01) in
DNA platination was documented in yeasts expressing human Topo
I and treated with CPTePt with respect to yeasts transformed with
empty vector (Fig. 9B). Although less relevant, a significant change
(P < 0.02) in the amount of PteDNA adducts was also observed for
transformed yeasts treated with cDDP.
2.7. Effect of DNA Topoisomerase I on the formation of PteDNA
adducts
CPT derivatives (1aeb, 6aeb) were tested for their capability to
bind the DNA by evaluating the migration of pCMV6neo plasmid in
agarose gel electrophoresis after drug exposure (Fig. 10). In com-
parison to cDDP and SN38, which did not produce any effect on DNA
migration, the exposure of plasmid DNA to 1a revealed a signifi-
cantly reduced migration of the vector. Such behavior was likely
dependent on the major capability of 1a to produce PteDNA ad-
ducts. The effect was less marked for 1b. Moreover, the simultaneous
exposure of pCMV6neo plasmid toTopo I and 1a or 1b produced DNA
accumulation in the wells, thus resembling that observed in cleavage
assays. Such behavior was dependenton the presence of Topo I, since
it was not observed in the absence of enzyme or in the presence of
human serum albumin (BSA). Interestingly, degradation of the
protein by SDS and proteinase K before loading on agarose gel
Fig. 6. Topoisomerase I-mediated DNA cleavage by SN38 and CPT derivatives. Samples
were reacted with 1, 10 and 50 m
M drug at 37 ꢁC for 30 min. Reaction was then stopped
by adding 0.5% SDS and 0.3 mg/ml of proteinase K and incubating for 45 min at 42 ꢁC
before loading on a denaturing 8% polyacrylamide gel. C, control DNA; T, reaction
without drug; M, purine markers.

R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
393
Fig. 7. Persistence of topoisomerase I-mediated DNA cleavage in the presence of SN38 and camptothecin derivatives. Time-course analysis of DNA cleavage induced by SN38 and
camptothecin analogs. The samples were reacted for 30 min with 1 M drug. DNA cleavage was then reversed by adding 0.6 M NaCl. The 100% value refers to the extent of DNA
m
cleavage at 30 min of incubation. C, control DNA; T, reaction without drug. Cleavage persistence was measured by densitometric analysis. Data represent mean values from 3
independent experiments.
Fig. 8. Cellular Pt accumulation and DNA platination in the H460 cell line. Platinum accumulation was determined after a 1 h exposure to equitoxic (IC50) (A) and equimolar (0.4 mM)
(B) concentrations of cDDP and CPT derivatives. (C) DNA-bound platinum after a 1 h exposure to equitoxic concentration (IC50) of cDDP and camptothecin derivatives. Accumulation
was assessed by atomic absorption spectroscopy, and DNA platination was measured by inductively coupled plasma mass spectroscopy. Mean values (ꢂSD) of triplicate de-
terminations are shown.

394
R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
Fig. 9. (A) sensitivity of S. cerevisiae JN2-134 strain in spot test. Yeast cells transformed with the empty vector (pEMBLyex4) or with the same vector containing the entire ORF of the
human DNA topoisomerase I (pEZ-2hTop1) were used. Yeasts were grown to an A595 of 0.3 and then treated for 24 h with CPT derivatives at 1, 10 and 50 M. Aliquots of 10 l were
spotted onto plates and incubated for 3 days at 30 ꢁC. Two references (CPT and cDDP) were included in the test. (B) DNA-bound platinum after overnight exposure of the
transformed JN2-134 strain to 50 M cDDP and CPT derivatives. After treatment, genomic DNA was extracted and PteDNA adducts were measured by inductively coupled plasma
m
m
m
mass spectroscopy. Mean values (ꢂSD) of triplicate determinations are shown.
Fig. 10. (A) DNA binding of CPT containing Pt. EcoRV linearized pCMV6neo plasmid (200 ng) and CPT analogues (50 m
M) were incubated for 1 h at 37 ꢁC before loading on 1% agarose
gel. Where indicated, before loading on agarose gel, samples were treated with 0.5% SDS and 0.3 mg/ml of proteinase K. (B) Platination of pCMV6neo vector after exposure to CPT
derivatives. pCMV6neo plasmid (10 M of cDDP or 1b in the presence or absence of topoisomerase I. DNA concentration was
m
g sample) was treated for 1 h at 37 ꢁC with 50
m
determined spectrophotometrically and platinum content was measured by flameless atomic absorption spectroscopy.
Table 2
Antitumor activity of 1b against human H460 tumors xenografted s.c. in athymic
nude mice.
Compound Days of treatment Dose (mg/kg) TVI% (20)a BWL%b Toxc
1b
cDDP
Irinotecan
3, 7, 11, 15
3, 10, 17
3, 7, 11, 15
25
5
60
82**
58**
88**
0
11
8
0/6
1/5
0/8
O
O
H2
N
H3N
H3N
O
O
Cl
Cl
O
H3N
Pt
H3N
O
Pt
Pt
**P < 0.01 by two-tailed Student’ t test vs control mice.
a
N
O
Tumor volume inhibition % in treated over control mice; in parentheses the day
it was assessed. Tumor fragments were implanted in both flanks at day 0. Treatment
started when tumors were just palpable. CPTs were administered i.v. according to
the q4dx4 schedule. cDDP was administered i.v. at the maximum tolerated dose and
according to the schedule q7dx3.
O
H2
Cisplatin
Carboplatin
Oxaliplatin
Chart 1. Chemical structures of the clinically approved platinum dugs: cisplatin, car-
b
Body weight loss % induced by drug treatment; the highest change is reported.
Dead/treated mice.
boplatin and oxaliplatin.
c

R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
395
drugs overlapped TPT in the cleavable complex, independently of
the absolute configuration of the stereogenic center on the diamine
moiety. The most efficient interaction with duplex DNA was
observed for compound 1b, thus supporting a critical role of the
linker to provide drugeDNA interaction. CPTePt derivatives were
effective antiproliferative agents, with potency similar/superior to
TPT and in general more potent than cDDP. The potential advantage
of the Pt(II) complex was also supported by the reduced resistance
indexes observed for CPTePt derivatives with respect to cDDP and
TPT in several human tumor cell lines. It is noteworthy that the
most active derivative 1b was effective in overcoming cDDP resis-
tance in osteosarcoma cDDP-resistant U2OS cells. More impor-
tantly, CPTePt exhibited activity both as a Topo I poison and as a
DNA-platinating molecule, thus indicating that the conjunction of
the two components in a one hybrid molecule did not negatively
impact on their properties as single drugs. Interestingly, results
obtained in a cell-free system and in experiments involving yeast
cells documented two relevant findings produced by the presence
of Topo I: i) DNA platination produced by the CPTePt molecules was
increased in the presence of Topo I, and ii) the presence of Topo I,
but not BSA, produced a dose-dependent accumulation of DNA in
the wells of agarose and acrylamide gels, thus evoking a network
organization involving DNA, CPTePt and Topo I. It could be spec-
ulated that the network effect observed at a high concentration
might be the result of CPTePt acting outside, rather than inside, the
active site of Topo I. Thus, a scenario in which the Pt atoms form
covalent bounds linking the DNA and the Topo I outside the active
site should be considered as a possible alternative/additional
mechanism of drug action. This aspect remains to be properly
investigated.
N(CH3)2
HO
O
O
N
N
N
N
O
O
OH
O
OH
O
Camptothecin
Topotecan
C2H5
N
RO
O
N
O
OH
O
Irinotecan R =
SN 38 R = H
N
N CO
Chart 2. Chemical structures of camptothecin and of the two clinically approved de-
rivatives topotecan and irinotecan. The structure of SN38, the active metabolite of
irinotecan, is reported.
produced a different behavior depending on the compound. No
change in DNA migration was evidenced with respect to un-
proteolized sample for plasmid exposed to 1b, whereas a migra-
tion similar to the control was obtained when DNA treated with 1a
and Topo I was exposed to SDS/proteinase K before loading.
To evaluate the effect of Topo I on platination of the plasmid DNA,
pCMV6neowas exposed to 1b orcDDP in the presenceand absence of
Topo I in a cell-free system. As reported in Fig. 10B, the PteDNA ad-
ducts were significantly increased by the addition of purified human
Topo I in the presence of both cDDP (P < 0.05) and 1b (P < 0.02).
Concerning antitumor activity, preliminary in vivo data indicated
that compound 1b was well tolerated and exhibited promising
antitumor potency, comparable to that of irinotecan and superior to
that of cDDP. The widely used combinations of CPTs with platinum
compounds exhibit synergism in terms of both efficacy and toxicity.
Thus, the good profile of tolerability of the CPTePt complex supports
additional therapeutic advantages over the drug combinations.
In conclusion, results of the study provide evidence that CPT and
a Pt(II) complex joined in a single molecule via an oxyiminomethyl
linker could be considered a new class of effective antitumor
compounds.
2.8. In vivo antitumor activity
On the basis of the promising results observed in vitro, com-
pound 1b was selected for further preclinical development. Mice
bearing non-small cell lung cancer H460 carcinoma tumor xeno-
graft were treated with 1b administered intravenously (i.v.) ac-
cording to the intermittent q4dx4 schedule (Table 2). Compound 1b
exhibited antitumor activity comparable to that of irinotecan and
higher than that of cDDP, both delivered at their maximum toler-
ated doses. Considering the toxicity profile, 1b was well tolerated
(no lethal toxicity and no body weight loss) with respect to cDDP
(1/5 toxic deaths).
4. Experimental section
4.1. Chemistry. General methods
All reagents and solvents were reagent grade or were purified by
standard methods before use. Melting points were determined in
open capillaries. NMR spectra were recorded with a Bruker AMX
300 spectrometer using TMS as an internal standard. Chemical
shifts are given in ppm (d). Mass spectra were recorded with a
Fourier transform ion cyclotron resonance (FT-ICR) mass spec-
trometer APEX II & Xmass software (Bruker Daltonics)-4.7T Magnet
(Magnex). The elemental analyses were recorded with a CARLO
ERBA EA 1108 instrument. Solvents were routinely distilled prior to
use; anhydrous tetrahydrofuran (THF) and ether (Et2O) were ob-
tained by distillation from sodiumebenzophenone ketyl; dry
dichloromethane was obtained by distillation from phosphorus
pentoxide. All reactions requiring anhydrous conditions were per-
formed under a positive nitrogen flow, and all glassware were oven
dried and/or flame dried. Isolation and purification of the com-
pounds were performed by flash column chromatography on silica
gel 60 (230e400 mesh). Analytical thin-layer chromatography (TLC)
was conducted on TLC plates (silica gel 60 F254, aluminum foil).
3. Conclusions
Platinum drug administration is associated with dose-limiting
side effects, off-target effects and relatively poor pharmacokinetic
profiles. In particular, the low cellular uptake and the rapid meta-
bolic inactivation of platinum compounds imply that the amount of
active drug reaching the target is low. As regards CPTs, the
reversibility of the DNA cleavage may represent a limitation of drug
efficacy, resulting in resistance of slowly growing tumors. In an
attempt to overcome such drawbacks, hybrid 7-oxyiminomethyl
CPTs containing a Pt(II) complex were synthesized. Molecular
modeling studies evidenced that the CPT moiety of the hybrid

396
R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
4.1.1. {2-tert-Butoxycarbonylamino-1-[20-(camptothecin-7-
ylmethyleneaminooxy)ethylcarbamoyl]ethyl}carbamic acid tert-
butyl ester (5a)
4.1.4. [6-(1,3-Dioxo-1,3-dihydro-isoindol-2-yloxy)hexyl]carbamic
acid tert-butyl ester (7)
To a solution of (6-hydroxyhexyl)carbamic acid tert-butyl ester
[29] (730 mg, 3.36 mmol), triphenylphosphine (1.173 g, 4.48 mmol)
and N-hydroxyphthalimide (731 mg, 4.48 mmol) in THF (45 mL) at
To a solution of N,N-bis-t-butoxycarbonyl-2,3-d,l-diaminopropa-
noic acid [25] (134 mg, 0.441 mmol) in anhydrous DMF (6.6 mL) at
0 ꢁC under nitrogen, HOBt (103 mg, 0.750 mmol) and WSC (103 mg,
0.523 mmol) were added. After stirring for 30 min at 0 ꢁC, the so-
lution was added with 3a (157 mg, 0.362 mmol) and DIPEA (165 mg,
1.28 mmol) and stirred for 2.5 h at room temperature. The solvent
was evaporated and water was added (4 mL). The precipitate was
filtered and dried. Purification by flash chromatography (CH2Cl2/
MeOH 95:5) afforded the title compound as a yellow solid (yield
0
ꢁC, diisopropyl azodicarboxylate (906 mg, 4.48 mmol) was drop-
ped. After stirring overnight at room temperature, the solvent was
evaporated and the residue was purified by flash chromatography
(hexane/ethylacetate 3:7 then hexane/ethylacetate 2:8) and succes-
sively crystallized from MeOH to give the title compound as a white
solid (yield 82%); mp 166 ꢁC. Rf 0.34 (CH2Cl2/MeOH 95:5). 1H NMR
(CDCl3)
d
: 7.89e7.67 (m, 4H); 4.61 (brs, 1H); 4.15 (t, J ¼ 6.7 Hz 2H);
77%); mp 148 ꢁC; Rf 0.36 (CH2Cl2/MeOH 95:5). 1H NMR (DMSO-d6)
d:
3.18e3.00 (m, 2H); 1.41 (s, 9H); 1.85e1.20 (m, 8H). 13C NMR (CDCl3)
9.30 (s, 1H); 8.60 (d, J ¼ 8.6 Hz,1H); 8.25 (d, J ¼ 8.6 Hz, 1H); 8.11 (brs,
1H); 7.98e7.85 (m, 1H); 7.85e7.70 (m, 1H); 7.39 (s, 1H); 6.80e6.60
(m, 2H); 6.50 (s, 1H); 5.48 (s, 2H); 5.39 (s, 2H); 4.38 (t, J ¼ 6.9 Hz
2H); 4.03e3.97 (m,1H); 3.70e3.50 (m, 2H); 3.25e3.10 (m, 2H); 198e
d
163.6,156.0,134.4 (ꢃ2),128.9 (ꢃ2),123.4 (ꢃ2), 78.3, 70.0, 40.4, 29.8,
28.4, 28.2, 28.0, 26.3, 25.2, 21.9. HRMS (ESIþ): [M þ Na]þ calcd for
C
19H26N2NaO5 385.1734; found 385.1779. Anal. calcd for C19H26N2O5:
C, 62.97; H, 7.23; N, 7.73. Found: C, 62.81; H, 7.02; N, 7.98.
1.80 (m, 2H); 1.38 (s, 18H); 0.87 (t, J ¼ 7.0 Hz, 3H). 13C NMR (CDCl3)
d:
173.6, 171.0, 157.3, 156.9, 156.1, 152.0, 150.1, 149.3, 148.6, 145.7, 144.1,
130.8,130.5,128.4,125.8,125.1,122.7,118.8, 97.9, 73.9, 73.5, 72.7, 66.2,
55.8, 42.5, 38.9, 31.5, 28.2, 24.8, 7.7. HRMS(ESIþ): [M þ Na]þ calcd for
4.1.5. O-(6-Aminohexyl)hydroxylamine dihydrochloride (8)
To a solution of compound 7 (1.260 g, 3.48 mmol) in MeOH
(10.2 mL), hydrazine hydrate (0.522 g, 10.44 mmol) was added. The
resulting mixture was refluxed for 1 h. The white solid was filtered,
the solvent evaporated and the residue purified by flash chroma-
tography (CH2Cl2: MeOH 99:1) to give (6-aminooxyhexyl)carbamic
acid tert-butyl ester as a colorless oil in quantitative yield. Rf
C36H45N6O10 721.3192; found 721.3163. Anal. calcd for C36H44N6O10:
C, 59.99; H, 6.15; N, 11.66. Found: C, 59.76; H, 5.93; N, 11.81.
4.1.2. 2,3-d,l-Diamoniumtrifluoroacetate-N-[20-(camptothecin-7-
ylmethyleneaminooxy)-ethyl]-propionamide (6a)
0.25 (CH2Cl2/MeOH 95:5). 1H NMR (CDCl3)
(t, J ¼ 6.6 Hz 2H); 3.15e3.02 (m, 2H); 1.44 (s, 9H), 1.89e1.20 (m, 8H).
d: 6.32 (brs, 1H); 4.01
TFA (2.75 mL) was dropped into a solution of 5a (138 mg,
0.27 mmol) in CH2Cl2 (2.7 mL) at 0 ꢁC. The resulting orange solution
was stirred for 3 h at room temperature. The solvent was evapo-
rated and Et2O was added to obtain a precipitate. The solid was
filtered and washed three times with Et2O then dried to give the
title compound as a yellow solid in quantitative yield; mp 120 ꢁC. 1H
13C NMR (CDCl3)
d: 156.1, 77.8, 75.2, 44.6, 29.8, 28.7 (ꢃ2), 28.6, 26.5,
25.7, 22.1. HRMS (ESIþ): [M þ Na]þ calcd for C11H25N2O3 233.1860;
found 233.1845.
To a solution of the above tert-butyl ester (389 mg, 1.67 mmol)
in AcOEt (2.8 mL) at 0 ꢁC, a 5.9 M solution of HCl in AcOEt was
dropped. After 1 h at 0 ꢁC the solvent was evaporated to obtain the
title compound as a white solid (yield 97%); Rf 0.15 (CH2Cl2/MeOH
NMR (DMSO-d6)
d
: 9.35 (s, 1H); 8.90 (brs, 1H); 8.60 (d, J ¼ 8.6 Hz,
1H); 8.50e8.00 (brm, 7H); 7.99e7.92 (m, 1H); 7.90e7.82 (m, 1H);
7.39 (s,1H); 6.60 (brs, 1H); 5.50 (s, 2H); 5.35 (s, 2H); 4.44 (t, J ¼ 7 Hz,
2H); 4.22e4.12 (m, 1H); 3.70e3.65 (m, 1H); 3.65e3.50 (m, 1H);
9:1). 1H NMR (DMSO-d6)
d
: 10.90 (brs, 3H); 7.91 (brs, 3H); 3.97
(t, J ¼ 6.7 Hz, 2H); 2.80e2.65 (m, 2H); 1.61e1.43 (m, 4H); 1.37e1.21
(m, 4H). 13C NMR (DMSO-d6)
: 74.2, 27.3, 27.1, 25.7, 24.9 (one peak
3.35e3.20 (m, 2H); 1.95e1.80 (m, 2H); 0.87 (t, J ¼ 7.0 Hz, 3H). 13
C
d
NMR (DMSO-d6)
d
: 172.8, 166.9, 166.0, 159.3, 158.8, 157.1, 152.8,
missing due to overlapping with solvent signal). The compound
150.5, 149.0, 146.1, 145.5, 131.2, 130.9, 130.3, 128.8, 127.2, 125.3,
124.4, 119.7, 97.4, 97.0, 73.4, 72.8, 65.6, 65.2, 52.5, 50.6, 40.1, 30.7,
8.2. HRMS(ESIþ): [M ꢀ 2CF3COOꢀ]þ calcd for C26H30N6O6 522.2221;
found 522.2287. Anal. calcd for C30H30F6N6O10: C, 48.13; H, 4.04; N,
11.23. Found: C, 47.90; H, 3.91; N, 10.85.
was used without further purification for the next step.
4.1.6. 7-(6-Aminohexyloxyiminomethyl)camptothecin (3b)
To a suspension of camptothecin-7-carbaldehyde (4) (150 mg,
0.39 mmol) in EtOH (3.8 mL) compound 8 (163 mg, 0.79 mmol) and
pyridine (0.65 mL) were added. The resulting mixture was heated
at reflux for 1 h, then the solvent was evaporated. The pure title
compound was obtained as a yellow solid after crystallization from
Et2O/EtOH (yield 77%); mp 80 ꢁC; Rf 0.20 (CH2Cl2/MeOH 90:10). 1H
4.1.3. Dichloro{2,3-d,l-diamino-N-[20-(camptothecin-7-
ylmethyleneaminooxy)-ethyl]-propionamide}platinum (II) (1a)
A solution of K2PtCl4 (44 mg, 0.11 mmol) in water (0.40 mL) was
prepared under a stream of nitrogen. The aqueous solution was
added to a solution of compound 2a (80 mg, 0.11 mmol) in water
(2 mL) and the resulting mixture was stirred in the dark at room
temperature under nitrogen for 3 h. The orange precipitate formed
was filtered and washed twice with water, then with methanol and
with Et2O. After drying under vacuum the title compound was ob-
tained as a yellow solid (yield 69%); mp 255 ꢁC; Rf 0.30 (CH2Cl2/
NMR (DMSO-d6)
d
: 9.31 (s, 1H); 8.60 (d, J ¼ 8.5 Hz, 1H); 8.20 (d,
J ¼ 8.5 Hz, 1H); 7.98e7.61 (m, 4H); 7.34 (s, 1H); 6.53 (brs, 1H); 5.42
(s, 2H); 5.29 (s, 2H); 4.34 (t, J ¼ 6.7 Hz, 2H); 2.86e2.63 (m, 2H);
1.96e1.75 (m, 4H); 1.69e1.40 (m, 6H); 0.87 (t, J ¼ 6.7 Hz, 3H). 13
C
NMR (75 MHz, DMSO-d6)
d 168.6, 166.8, 157.1, 152.9, 150.5, 149.1,
146.5, 145.6, 131.0, 130.3, 128.7, 127.4, 125.4, 124.7, 119.7, 97.2, 74.4,
72.8, 65.6, 52.6, 44.0, 31.3, 30.7, 28.2, 27.5, 26.8, 8.2. HRMS (ESIþ):
[M þ Hþ] calcd for C27H31N4O5 491.2289; found 491.2266. Anal.
calcd for C27H30N4O5: C, 66.11; H, 6.16; N, 11.42. Found: C. 66.32; H.
6.00; N. 11.63.
MeOH 7:1); 1H NMR (DMSO-d6)
d
: 9.33 (s, 1H); 8.62 (d, J ¼ 8.6 Hz,
1H); 8.50 (brs,1H); 8.25 (d, J ¼ 8.6 Hz,1H); 8.00e7.79 (m,1H); 7.85e
7.75 (m, 1H); 7.40 (s, 1H); 6.58 (brs, 1H); 5.82 (brs, 1H); 5.49 (s, 2H);
5.40 (s, 2H); 5.11 (brs,1H); 4.40 (t, J ¼ 7.0 Hz, 2H); 3.70e3.50 (m, 3H);
3.40e3.30 (m, 2H), 1.95e1.80 (m, 2H); 0.87 (t, J ¼ 7.0 Hz, 3H). 13
C
4.1.7. Dichloro{2,3-d,l-diamino-N-[20-(camptothecin-7-
NMR (DMSO-d6)
d
172.9, 167.9, 167.0, 157.1, 152.9, 150.5, 149.1, 146.3,
ylmethyleneaminooxy)-hexyl]-propionamide}platinum (II) (1b)
To a solution of 7-(6-aminohexyloxyiminomethyl)CPT (70 mg,
0.14 mmol) and dichloro[(d,l)-diaminopropionic acid]platinum(II)
[26] (106 mg, 0.29 mmol) in dry DMF (7 mL), EDC (137 mg,
0.71 mmol) and TEA (5 drops) were added under nitrogen. The
resulting mixture was stirred at room temperature for 24 h in the
145.6, 131.0, 130.3, 128.8, 127.4, 125.4, 124.7, 119.7, 97.2, 73.5, 72.8,
65.6, 61.7, 52.6, 40.7, 39.0, 30.6, 8.2. HRMS(ESIþ): [M þ Na]þ calcd for
C
C
26H28Cl2N6NaO6Pt 808.0987; found: 808.0958. Anal. calcd for
26H28 Cl2N6O6Pt: C, 39.70; H, 3.59; Cl, 9.02; N, 10.69, Pt 24.80.
Found: C, 39.82; H, 3.44; Cl, 9.35; N, 10.33; Pt 24.40.

R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
397
ꢀ
dark. The solvent was evaporated and water was added. The sus-
pension was heated to 60 ꢁC, and the yellow precipitate was filtered
(yield 89%); mp 197 ꢁC. Rf 0.37 (CH2Cl2/MeOH/TEA 90:10:0.1). 1H
QM subsystem consisting of the ligand and bases within 3.5 A,
whereas the rest of the system (the MM subsystem) was treated
using the AMBER force field, together with a low memory conver-
gence algorithm. The boundary problem between the QM and MM
subsystems was treated using the pseudo-bond approach. With
this QM/MM system, an iterative optimization procedure was
applied to the QM/MM system, using BLYP/PW QM/MM calcula-
tions, leading to an optimized structure for the reactants. BLYP-STB
and BP-STB calculations were carried out in order to check the
dependence of the relative energies on the basis set and exchange
correlation functional. The core electrons were frozen up to 4f for
Pt, 2p for Cl and 1s for N. For Pt, scalar relativistic effects were taken
into account. The convergence criterion used was set to obtain an
energy gradient of <10ꢀ4, using the twin-range cutoff method for
NMR 600 MHz (DMSO-d6)
d
: 9.32 (s, 1H); 8.59 (d, J ¼ 8.6 Hz, 1H);
8.20 (d, J ¼ 8.6 Hz, 1H); 8.11 (brs, 1H), 7.95e7.80 (m, 1H); 7.80e7.72
(m, 1H); 7.34 (s, 1H); 6.52 (s, 1H); 5.79 (brs, 1H); 5.45e5.35 (m, 3H);
5.37 (s, 2H); 5.00 (brs, 1H); 4.33 (t, J ¼ 7.0 Hz, 2H); 3.40e3.25
(m, 2H); 3.18e3.00 (m, 2H); 1.90e1.75 (m, 5H); 1.60e1.10 (m, 5H);
0.87 (s, 3H). 13C NMR 150 MHz (DMSO-d6)
d 187.2,172.8,166.5,157.1,
152.9,150.4,149.1,145.6,145.2,131.5,130.9,130.2,128.7,127.2,125.4,
124.6, 119.6, 97.1, 75.3, 72.8, 65.6, 61.4, 52.6, 50.0, 30.7, 29.1, 29.0,
26.5, 25.4, 8.1. HRMS(ESIþ): [M þ Na]þ calcd for C30H36Cl2N6NaO6Pt
864.1613; found: 864.1601. Anal. calcd for C30H36Cl2N6O6Pt: C,
42.76; H, 4.31; Cl, 8.41; N, 9.97; Pt, 23.15; found C, 42.91; H, 406; Cl,
8.25; N, 9.69; Pt, 23.30.
ꢀ
non-bonded interactions, with a long-range cutoff of 14 A and a
ꢀ
short-range cutoff of 8 A.
4.2. Molecular modeling
4.3. Assessment of cytotoxic activity on human cancer cell lines
The five studied camptothecin derivatives were assembled and
refined using a systematic conformer search followed by geometry
optimization of the lowest energy structure with MOPAC (PM3
Method, RMS gradient 0.0100) [30]. The macromolecule (PDB
deposition code: 1T8I) was prepared by removing the camptothe-
cin molecule and adding polar hydrogen atoms, while the side
chains of the disordered amino acid residues of the macromolecule
complex were checked and rebuilt in DeepView [31]. The individual
compounds were pre-positioned based on the coordinates of
camptothecin from the X-ray structure.
The non-small cell lung cancer H460 cell line, the squamous
carcinoma A431 cell line and the corresponding TPT-resistant
A431/TPT and cisplatin-resistant A431/Pt sublines, the osteosar-
coma U2OS cell line and the corresponding cDDP-resistant U2OS/Pt
subline, the ovarian carcinoma IGROV-1 and A2780 cell lines and
the corresponding Pt-resistant IGROV-1/Pt, IGROV-1/OHP and
A2780/CP sublines were used. Cells were cultured in RPMI 1640
containing 10% FCS. Cytotoxicity was assessed by growth inhibition
assay after a 1 h drug exposure. Compounds 1a, 1b, 6a and 6b were
initially dissolved in dimetylsulphoxide and then diluted in sterile
water. TPT and cDDP were dissolved and diluted in saline solution
(0.9% sodium chloride). Twenty-four hours after seeding in well
plates (from 30 ꢃ 103 to 120 ꢃ 103 cells/plate, depending on cell
line), cells were exposed to the drugs and counted 72 h later by a
Coulter Counter. Each experimental sample was run in triplicate.
The IC50 was defined as the drug concentration causing 50% cell
growth inhibition determined by dose response curves generated
by testing at least 5 different drug concentrations. Combination
studies were performed using cDDP concentrations producing 80e
90% of cell growth.
The small molecule compounds and the macromolecule were
further processed using the Autodock Tool Kit (ADT) [32].
GasteigereMarsili charges [33] were loaded on the small molecules
in ADT and Cornell parameters used for the phosphorus atoms in
the DNA. Solvation parameters were added to the final macro-
molecule structure using Addsol utility of Autodock. All docking
runs were performed with Autodock 4 [34], using an empirical free
energy function and Lamarckian Genetic Algorithm, with an initial
population of 50 randomly placed individuals, a maximum number
of 200 energy evaluations, a mutation rate of 0.02, a crossover rate
of 0.80, and an elitism value of 1. For the local search, the so-called
pseudo-Solis and Wets algorithm was applied using a maximum of
250 iterations per local search. The probability of performing local
search on an individual in the population was 0.06, and the
maximum number of consecutive failures before doubling or
halving the local step size was 4. Fifty independent docking runs
4.4. Saccharomyces cerevisiae yeast strain and yeast spot test
JN2-134top1-1 S. cerevisiae strain (MATa rad52::LEU2, trp1,
ade2-1, his7, ura3-52, ise1, top1, leu2), which lacks the endogenous
TOP1 gene, was transformed with the empty vector (pEMBLyex4)
or with the same vector containing the wild-type human DNA
topoisomerase IB (pEZ-2hTop1) [36]. Yeast cells were maintained at
30 ꢁC in synthetic complete medium lacking uracil (uracil-) and
supplemented with 2% glucose. For the yeast spot test, cells were
growth at 30 ꢁC in uracil medium to an OD595 of 0.3. The yeast
culture was then treated with camptothecins or cisplatin for 24 h.
ꢀ
were carried out for each ligand. Results differing by less than 1.0 A
in positional root-mean-square deviation (rmsd) were clustered
together and represented by the result with the most favorable free
energy of binding. The grid maps representing the protein in the
actual docking process were calculated with Autogrid. The grids
(one for each atom type in the ligand, plus one for electrostatic
interactions) were chosen to be sufficiently large to include the
entire width of the DNA fragment in which the original inhibitor
was posed, a portion of minor and major grooves, and the active site
residues of Topoisomerase I. The dimensions of the grids were thus
After treatment, aliquots of 10 ml were spotted onto plate uracil-.
Following 3 days of incubation at 30 ꢁC, images were acquired using
the IMAGE MASTER VDS (Amersham Pharmacia Biotech).
ꢀ
80 ꢃ 80 ꢃ 50, with a spacing of 0.200 A between the grid points and
the center close to the cavity left by the ligand after its removal. The
simpler intermolecular energy function based on the Weiner force
field16 in Autodock was used to score the docking results.
4.5. Topoisomerase I-dependent DNA cleavage assay
A 30-end labeled gel purified 751-bp BamHIeEcoRI fragment of
SV40 DNA was used for the cleavage assay. SV40 plasmid was first
linearized with BamHI enzyme and then 30-labeled by using DNA
polymerase I large (klenow) fragment (Invitrogen, Paisley, UK) in
4.2.1. Hybrid QM/MM calculations
In the current study, we used the pseudo-bond ab-initio QM/
MM approach as implemented in Gaussian-03 [35]. For the QM/
MM calculations, the DNAeTopoisomerase I-ligand system result-
ing from the docking study was first partitioned into a QM sub-
system and an MM subsystem. The reaction system used a smaller
the presence of a
32P dGTP. The labeled DNA was then restricted
with EcoRI enzyme, and the corresponding 751-bp was purified on
agarose gel. Topoisomerase I DNA cleavage reactions (20,000 cpm/
sample) were performed in 20
ml of 10 mM TriseHCl (pH 7.6),

398
R. Cincinelli et al. / European Journal of Medicinal Chemistry 63 (2013) 387e400
150 mM KCl, 5 mM MgCl2, 0.1 mM dithiothreitol, and human re-
combinant enzyme (full length topoisomerase I) for 30 min at
37 ꢁC [36]. Reactions were stopped by 0.5% SDS and 0.3 mg/ml of
proteinase K for 45 min at 42 ꢁC. Persistence of DNA cleavage at
different time points was examined by adding 0.6 M NaCl after
30 min of incubation. After precipitation DNA was resuspended in
denaturing buffer (80% formamide, 10 mM NaOH, 0.01 M EDTA
and 1 mg/ml dyes) before loading on a denaturing 8% poly-
acrylamide gel in TBE buffer. Overall DNA cleavage levels were
4.9. Antitumor activity
All experiments were carried out using female athymic Swiss
nude mice, 7e10 weeks old (Charles River, Calco, Italy). Mice were
maintained in laminar flow rooms keeping temperature and hu-
midity constant. Mice had free access to food and water. Experi-
ments were approved by the Ethics Committee for Animal
Experimentation of the Istituto Nazionale Tumori of Milan according
to institutional guidelines.
measured with
Dynamics).
a
PhosphoImager 425 model (Molecular
The compound was dissolved in DMSO/cremophor ELP. The
solution was maintained at 4 ꢁC. Before the treatment, the drug
solution was suspended in cold saline (5 þ 2.5 þ 92.5 of final vol-
ume) under magnetic stirring. Drug was delivered in a volume of
10 ml/kg of body weight.
Cisplatinum Teva (clinical) was ready to use. Irinotecan was
dissolved in sterile, distilled water keeping it under magnetic stir-
ring for about 2 h.
4.6. Drug accumulation studies
Exponentially growing H460 cells (1 ꢃ106) were seeded in 5 cm
diameter dishes in triplicate and, 24 h later, they were exposed to
equitoxic (IC50) or equimolar (0.4 mM) concentrations of the drugs
for 1 h. After treatment, cell monolayers were washed with ice-cold
PBS, scraped, harvested and dissolved in 1 N NaOH. Total cellular Pt
content was determined by flameless atomic absorption spectros-
copy [37] (Model 3300, Perkin Elmer) or by inductively coupled
plasma-mass spectrometry [38]. Cellular Pt levels were expressed
as ng/106 cells, with cell number determined by counting parallel
cultures. For each type of treatment at least three independent
experiments were performed.
Exponentially growing tumor cells (107 cells/mouse) were s.c.
injected into the right flank of athymic nude mice. The tumor line
was achieved by serial s.c. passages of fragments (about
2 ꢃ 2 ꢃ 6 mm) from growing tumors into healthy mice as previously
described [39]. Tumor fragments were implanted on day 0, and tu-
mor growth was followed by biweekly measurements of tumor di-
ameters with a Vernier caliper. Tumor volume (TV) was calculated
according to the formula: TV (mm3) ¼ d2 ꢃ D/2 where d and D are
the shortest and the longest diameter, respectively. Drugs were
delivered i.v. every fourth day for four times (q4dx4) starting when
tumors were just palpable. The efficacy of the drug treatment was
assessed as tumor volume inhibition percentage (TVI%) in treated
versus control mice, calculated as: TVI% ¼ 100 ꢀ (mean TV treated/
mean TV control ꢃ 100). The toxicity of the drug treatment was
determined as body weight loss and lethal toxicity. Deaths occurring
in treated mice before the death of the first control mouse were
ascribed to toxic effects.
4.7. DNA platination studies
Exponentially growing H460 cells (3 ꢃ 106) were seeded in 5 cm
diameter dishes in triplicate and, 24 h later, they were exposed to
the drugs for 1 h at equitoxic (IC50) or equimolar (0.4 mM) con-
centrations. DNA was then extracted according to standard pro-
cedures involving lysis in the presence of 1 mg/ml proteinase K
overnight at 37 ꢁC. DNA was then isolated following phenol
extraction, ethanol precipitation, RNase treatment, and reprecipi-
tation and finally was dissolved in 10 mM TriseHCl (pH 7.4) and
1 mM EDTA. DNA content was determined spectrophotometrically
and platinum content was measured by flameless atomic absorp-
tion spectroscopy (Model 3300, Perkin Elmer) or by inductively
coupled plasma-mass spectrometry.
4.10. Statistical analysis
Two-tailed Student’s t test was used for statistical comparison of
drug uptake and DNA platination data as well as of tumor volumes
in mice.
For the experiments with yeast cells, JN2-134top1-1 S. cerevisiae
strain transformed with pEMBLyex4 or with pEZ-2hTop1 vectors
was treated over night with 50 mM drug. Genomic DNA was ob-
Acknowledgments
tained using MasterPure Yeast DNA purification kit (Epicentre
biotechnologies, Wisconsin, USA) and the platinum content
measured by inductively coupled plasma-mass spectrometry.
We are indebted to MIUR (PRIN 2009 project) and to the Asso-
ciazione Italiana per la Ricerca sul Cancro, Milan, for financial sup-
port. We thank Ms. Betty Johnston for editorial assistance.
For cell free experiments, pCMV6neo vector (10
m
g sample) was
M) in the
treated for 1 h at 37 ꢁC with different compounds (50
m
Appendix A. Supplementary data
presence and absence of Topo I. After treatment, DNA was isolated
following phenol extraction and ethanol precipitation. DNA con-
centration was determined spectrophotometrically, and platinum
content was measured by flameless atomic absorption spectros-
copy (Model 3300, Perkin Elmer).
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.02.022.
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