Synthesis and biological evaluation of new camptothecin derivatives obtained by modification of position 20

Article abstract of DOI:10.1016/j.bmc.2010.09.069

The preparation and biological evaluation of a novel series of dimeric camptothecin derivatives are described. All the new compounds showed a significant ability to inhibit human tumor cell growth with IC₅₀ values ranging from 0.03 to 12.2 μM. The interference with the activity of the nuclear enzymes topoisomerases has been demonstrated, highlighting the poison effect of one of the obtained byproducts toward topoisomerase I. A moderate antiangiogenic activity has been demonstrated for one of the obtained compounds. Moreover, the effects of four new compounds on caspases activity and ROS generation have been studied on transgenic mouse cell.

Full text of DOI:10.1016/j.bmc.2010.09.069

Bioorganic & Medicinal Chemistry 18 (2010) 8660–8668
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of new camptothecin derivatives
obtained by modification of position 20
Elena Riva a, Daniela Comi a, Stella Borrelli a, Francesco Colombo a, Bruno Danieli a, Jurgen Borlak b,
Lasse Evensen c, James B. Lorens c, Gabriele Fontana d, Ornella Maria Gia e, Lisa Dalla Via e,
Daniele Passarella a,
⇑
a Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy
b Fraunhofer Institute of Toxicology and Experimental Medicine Medical School of Hannover, Nikolai-Fuchs-Str 1, 30625 Hannover, Germany
c Department of Biomedicine, University of Bergen, Bergen, Norway
d Indena S.p.a., Via Ortles 19, Milano, Italy
e Dipartimento di Scienze Farmaceutiche, Università degli Studi di Padova, Via F. Marzolo, 5-35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2010
Revised 27 September 2010
Accepted 29 September 2010
Available online 7 October 2010
The preparation and biological evaluation of a novel series of dimeric camptothecin derivatives are
described. All the new compounds showed a significant ability to inhibit human tumor cell growth with
IC50 values ranging from 0.03 to 12.2 lM. The interference with the activity of the nuclear enzymes topoi-
somerases has been demonstrated, highlighting the poison effect of one of the obtained byproducts
toward topoisomerase I. A moderate antiangiogenic activity has been demonstrated for one of the
obtained compounds. Moreover, the effects of four new compounds on caspases activity and ROS gener-
ation have been studied on transgenic mouse cell.
Keywords:
Camptothecin derivative
Topoisomerase inhibition
Inhibition of angiogenesis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
spacers taking into consideration the possibility to improve the
interaction with the biological targets.10
Camptothecin (1, CPT), a pentacyclic alkaloid1 isolated in 1958
from Camptotheca acuminata,2 acts as a selective poison of the nu-
clear enzyme topoisomerase I by forming a ternary complex with
topoisomerase I and DNA.3 Therapeutic applications of CPT have
been hindered by its poor aqueous solubility, and by severe toxic-
ities identified in early clinical studies performed in the 1970’s.4
The toxicity is partially due to the instability of lactone ring (E),
that results in the formation of the inactive but toxic carboxylate
species.5 Since then, academic and industrial research groups have
focussed their attention on the synthesis of the alkaloids of the
camptothecin family and their analogues as promising agents for
the treatment of human cancers.6 The compounds which have a
20-OH substituted groups can increase the stability of the drugs–
DNA–Topo I ternary complex,7 decrease the toxicity and inhibit
the opening of the lactone ring.8
2. Results and discussion
2.1. Chemistry
We decided to synthesize the dimeric compounds according to
the approach described in Scheme 1.
The presence of hydroxyl group at C-20 campthotecin position
offered the opportunity for the direct introduction of a spacer moi-
ety. We selected the use of a urethane function for the junction of
the spacer to the CPT scaffold. Reaction of CPT (1) with triphosgene
(bis(trichloromethyl) carbonate) and subsequently with t-butyl-N-
(2-aminoethyl)carbamate11 afforded unsuccessful results. Derivate
212 was prepared by an alternative method using PNP-chlorofor-
mate. Subsequent reaction with N-Boc monoprotected ethylendi-
amine afforded compound 3. The protective group was removed
by treatment with HCl (dioxane saturated solution) to give 4 that
we used for the preparation of several dimers by condensation
with appropriate dicarboxylic acids. The reaction in the presence
of DCC and DMAP gave the products 5–8 accompanied by the
1,3-dioxoazacyclo derivatives 9–12. All the obtained compounds,
but 5 and 9, appeared sufficiently stable is solution13 and showed
a better solubility (0.5–0.8 mg/mL, MeOH/H2O v/v 8:2) if compared
with campthotecin (CH3Cl/MeOH v/v 1:1).14
In the frame of our ongoing project devoted to the synthesis of
bivalent compounds using natural products as building blocks,9 we
planned the formation of dimeric compounds of CPT with the aim
to modulate the activity, to improve the solubility and to increase
the stability of the lactone ring. We selected the use of polyamide
⇑
Corresponding author. Tel.: +39 02 50314081; fax: +39 02 50314078.
E-mail address: Daniele.Passarella@unimi.it (D. Passarella).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.069

E. Riva et al. / Bioorg. Med. Chem. 18 (2010) 8660–8668
8661
O
O
N
O
A
B
C
N
N
D
N
N
N
a
b
E
O
O
O
O
2
3 R = Boc
4, R = H
20
c
HO
1 camptothecin
O
O
O
O
O
NH
O
O
NHR
NO2
O
O
n
X
n
X
O
OH
O
HO
O
N
d
N
N
N
O
O
+
O
O
O
O
O
O
O
O
X
n
H
N
H
N
O
X
O
N
X
O
N
N
N
H
X
N
H
H
n
n
n
O
O
O
9 - 12
5 - 8
N
Compound
5 and 9
6 and 10
7 and 11
8 and 12
n
1
2
3
3
X
S
S
S
CH2
Scheme 1. Reagents and conditions: (a) PNP-chloroformate, DMAP, CH2Cl2, 0 °C, rt; (b) 1-(tert-butyloxycarbonyl)ethyldiamine (13), DMF; (c) HCl/dioxane; (d) dicarboxylic
acid, DCC, DMAP, CH2Cl2.
2.2. Antiproliferative activity
propyl seems to be the most convenient spacer. Indeed, it is possi-
ble to evidence in both classes a similar behavior where 7 > 8 > 6
and 11 > 12 > 10. In particular, the dimeric derivative 7, is the most
active in all the tested cell lines.
The ability of new derivatives and their byproducts to inhibit
cell growth was evaluated by means of an in vitro assay performed
on three human tumor cell lines, HeLa (cervix adenocarcinoma),
HL-60 (promyelocytic leukemia), and A-431 (epithelial carci-
2.3. Interaction with DNA
noma).15 The IC50 values were in the range 0.03–12
72 h incubation as reported in Table 1.
lM after
The noticeable antiproliferative activity shown by the new cam-
ptothecin derivatives, prompted us to study their mechanism of
action. In the first place, we investigated the ability of compounds
to form a molecular complex with DNA by linear dichroism (LD)
experiments.17 The obtained results showed that the DNA spec-
trum is not significantly modified by the addition of the examined
compounds at different [drug]/[DNA] ratios (data not shown),
according to the behavior of camptothecin.
The antiproliferative activity of the new compounds was evalu-
ated also toward 11 transformed mouse cell lines from lung
tumors of double c-myc and c-raf transgenic mice (A2C12, yB8,
yD12, bD10, yA7, yA3, B3, bD5, A2B1, yD1, and Craf/Cmyc). Nota-
bly, the transgenic cell lines display epigenetic plasticity and ex-
press several tumor stem cell markers providing a mechanistic
insight into lung cancer as recently reported by us.16 The murine
cell lines were more resistant, with IC50 values typically in the
region of 1
lM. A comparison between the compounds that
2.4. Effect on DNA topoisomerases
include one unit of camptothecin (10–12) and the ones with two
units (6–8), evidenced for these latter a higher antiproliferative
activity. Moreover, inside each series of derivatives the sulfur
DNA topoisomerases have been shown to be the molecular
target of many anticancer drugs: camptothecin, in particular, acts
Table 1
Compound concentration (
triplicate samples each SD
l
M) that causes 50% reduction in cell number with respect to the control culture; data are means of at least three independent experiments with
Compound IC50
Cell lines
CPT
0.011 0.002
0.0089 0.0016
0.017 0.0028
6
7
8
10
2.97 0.78
11.96 0.37
8.1
11
12
1.88 0.39
8.3 0.29
12.25 0.17
A-431
HeLa
HL-60
0.15 0.06
0.7 0.38
0.92 0.06
0.03 0.01
0.17 0.07
0.29 0.09
0.08 0.01
0.30 0.05
0.42 0.07
1.21 0.04
0.57 0.17
1.55 0.26
1

8662
E. Riva et al. / Bioorg. Med. Chem. 18 (2010) 8660–8668
as topoisomerase I poison, that is, stabilizing the covalent ternary
complex with enzyme in the cutting phase, and preventing the
rejoining of the filament.18,19
different from the starting CPT. In Figure 4 we report the result
we obtained in the case of compound 7. In the case of cmyc/craf
cell line the mean rhodamine 110 fluorescence was 13-fold signif-
In order to investigate the ability of new compounds (6–8 and
10–12) to interact with the nuclear enzyme, we performed topoi-
somerase I relaxation assay on supercoiled plasmid pBR322 DNA.
The results did not show any inhibitory effects on topoisomerase
I activity (data not shown). To study the influence of new com-
pounds on the formation of the cleavable complex between DNA
and topoisomerase I a further test was performed.15 The obtained
results indicated only for the monomer 12 the ability to stabilize
the formation of the cleavable complex, as demonstrated by the
appearance of the ‘nicked’ band, according to the reference drug
CPT (Fig. 1).
icantly increased (7, 10 lmol/L). On the same cell line compound 8
induced an eightfold increasing of the rhodamine 110 fluorescence.
In the case compounds 10 and 12 we did not observe any relevant
activity.
Compounds 7, 8, 10, and 12 were additionally investigated on
ROS (reactive oxygen species) generation in the transgenic mouse
cells on the base that ROS are connected with induction of apopto-
sis. The influence of the new compounds on ROS generation devi-
ates from the behavior of CPT. Compound 7 showed a significant
increase of ROS production in the majority of the cell lines with
exception of A2B1 (10-fold increasing in cmyc/craf, Fig. 5).20 Com-
pound 8 resembled the behavior of compound 7 in all the cell lines
To further investigate the ability of all compounds to interfere
with the enzymatic activity of DNA topoisomerase II, a relaxation
assay was performed.
apart from bD10,
cA7, cB8 and cD12. Compounds 10 and 12
showed a reduced efficacy.
The appearance of supercoiled DNA with a concurrent decrease
of the relaxed one demonstrated a detectable inhibition on the
relaxation activity only for 6 and 10. In details, compounds 6 and
10 showed a dose dependent effect at concentration ranging from
2.6. Anti-angiogenic activity
We considered that the new compounds are characterized by
modulated biological activity in comparison with CPT. As recent
publications describe the antiangiogenic activity of topotecan22
and irinotecan,23 we evaluated our new compounds for anti-angio-
genic activity. Compounds 7, 10, and 11 were assessed for effects
on organotypic capillary-like vessel formation using co-culture of
primary human endothelial cells (EC) and vascular smooth muscle
cells (vSMC) according to a procedure developed by our labora-
tory.24 CPT (1, Fig. 6) showed a potent inhibitory effect on capil-
lary-like network formation at the second lowest concentration
tested (25 nM). Compound 7 had an intermediate inhibitory effect
and reduced the total tube length by 50% at concentrations exceed-
ing 125 nM. However, image analysis revealed that the morphol-
ogy of the endothelial cells is not affected; instead, there were
overall fewer tubules formed. Compounds 10 and 11 had no effect
in the concentration range tested (2–200 nM, in Fig. 6 is repre-
sented the behavior of compound 11).
12.5 to 100
lM (Figs. 2 and 3). In particular, the inhibition at 50 lM
and 100 M for compound 6 and 10, respectively, are similar to
l
that obtained by incubating the enzyme in the presence of 8 lM
amsacrine (m-AMSA), taken as referenced drug.20
On the other hand, the new compounds appeared unable to in-
duce the formation of the cleavable complex in the presence of
topoisomerase II, indicating that they are not a poison for the en-
zyme (data not shown). Overall these results suggest that the com-
pounds 6 and 10 act as catalytic inhibitors of topoisomerase II.
2.5. Effects on the induction of apoptosis
In light of the induction of cell death, we investigate the activa-
tion of caspases (a family of cysteine proteases) to assess the ability
of test compounds to trigger the apoptotic pathway. Upon activa-
tion, these enzymes cleave specific substrates and thereby mediate
many of the typical biochemical and morphological changes in
apoptotic cells, such as cell shrinkage, chromatin condensation,
DNA fragmentation and plasma membrane blebbing. Hence, the
detection of activated caspases can be used as a biochemical mar-
ker for apoptosis. We selected compounds 7, 8, 10, and 12 to inves-
tigate their effects on caspase activity. We used a cell-permeable
caspase substrate, (Asp)2-rhodamine 110. Upon caspase cleavage,
the nonfluorescent substrate is converted to rhodamine 110 lead-
ing to an increase in fluorescence.21 The new compounds appear
3. Conclusions
The convenient synthesis of four new dimeric compounds of
CPT is reported with three of them stable enough to be considered
for the biological evaluation on cancer cells. The condensation
reaction induces the formation of the corresponding cyclic imides
that have been considered as well for the biological evaluation.
In particular we achieved an improvement in the solubility due
to the polyamide character of the linker. Three different human
cancer cells have been used for the evaluation and the results
moved us toward the evaluation on eleven different murine stem
cell lines. The obtained results showed a general dissimilar activity
with respect to CPT. The dimeric derivative 7, is the most active in
all the tested cell lines. The interference with the activity of the nu-
clear enzymes topoisomerases has been demonstrated, highlight-
ing the poison effect of compound 12 toward topoisomerase I.
Compounds 6 and 10 act as catalytic inhibitors of topoisomerase
II. The effects of compounds 7, 8, 10, and 12 on caspases activity
and ROS generation evidenced an interesting activity of compound
7. The observed general diversity of the biological activity drove us
to study the influence of the obtained compounds on angiogenesis.
Compound 7 showed a moderate antiangiogenic activity. In con-
clusion, the interesting behavior of dimeric derivative 7 allows us
to consider this scaffold as a promising lead worth to be further
developed in order to obtain more precise structure–activity rela-
tionships. We are convinced that the use of natural products as
building blocks for the creation of new entities is a noteworthy
Figure 1. Effect of compound 12 on the stabilization of covalent DNA–topoiso-
merase I complex: (a) supercoiled pBR322 DNA; (b) supercoiled DNA incubated
with topoisomerase I in the presence of compound (100
incubated with topoisomerase alone; (d) supercoiled DNA incubated with
topoisomerase I in the presence of CPT (100 M); (e) supercoiled DNA incubated
with topoisomerase I in the presence of solvent.
lM); (c) supercoiled DNA
I
l

E. Riva et al. / Bioorg. Med. Chem. 18 (2010) 8660–8668
8663
Figure 2. Effect of compound 6 on relaxation of supercoiled pBR322 DNA of topoisomerase II: (a) supercoiled pBR322; (b) same as lane (a) in the presence of 1 U
topoisomerase II; (c–f) same as lane (b) in the presence of 12.5, 25, 50, and 100
same as lane (b) in the presence of solvent alone.
lM test compound, respectively; (g) same as lane (b) in the presence of 8 lM m-AMSA; (h)
Figure 3. Effect of compound 10 on relaxation of supercoiled pBR322 DNA of topoisomerase II: (a) supercoiled pBR322; (b) same as lane (a) in the presence of 1 U
topoisomerase II; (c–f) same as lane (b) in the presence of 12.5, 25, 50, and 100
same as lane (b) in the presence of solvent alone.
lM test compound, respectively; (g) same as lane (b) in the presence of 8 lM m-AMSA; (h)
Figure 4. Effects of 7 on caspase activity. Cells were pretreated with 7 (10 lmol/L) for 2 h. The caspase substrate (Asp)2-rhodamine 110 was added at 10 lmol/L for additional
2 h.
strategy to improve the exploration of the chemical space for the
sake of finding new biological activities.25
(CDCl3) and methanol-d4 (CD3OD). Chemical shifts are reported
in parts per million (d) downfield from tetramethylsilane (TMS)
as internal standard. EI mass spectra were recorded at an ionizing
voltage of 6 Kev on a VG 70-70 EQ. ESI mass spectra were recorded
on FT-ICR APEXII (Bruker Daltonics). All reactions were carried out
in dry solvents.
4. Experimental section
4.1. General
Thin-layer chromatography (TLC) was performed on Merck pre-
coated 60F254 plates. Reactions were monitored by TLC on silica
gel, with detection by UV light (254 nm) or by charring with sulfu-
ric acid. Flash chromatography was performed using silica gel
(240–400 mesh, Merck). 1H NMR spectra were recorded with Bruc-
ker 200, 300, and 400 MHz spectrometers using chloroform-d
4.2. Preparation of (2)
(p-Nitrophenyl camptothecin carbonate): camptothecin (1)
(228 mg, 0.65 mmol) and PNP-chloroformate (460 mg, 2.29 mmol)
were dissolved in CH2Cl2 (30 mL) at 0 °C, followed by the addition
of DMAP (444 mg, 3.60 mmol). The resulting clear solution was

8664
E. Riva et al. / Bioorg. Med. Chem. 18 (2010) 8660–8668
Figure 5. Effects of 7 (10
l
mol/L) on ROS production. ROS production was measured by flow cytometry with DCFH-DA.
Figure 6. Anti-angiogenesis assay—(a) SMC and fluorescent HUVEC were seeded in a 96-well plate and treated with dilution series for the compounds 11, 7, 10 and
camptothecin (CPT) in the concentration range 2–200 nM.
stirred at room temperature for 1 h. The reaction was monitored by
TLC (CH2Cl2/MeOH 60:1). After completion, the mixture was di-
luted with 50 mL of CH2Cl2 and washed with 10 mL HCl 0.1 N.
The organic layer was dried over Na2SO4, concentrated under re-
duced pressure. The remaining residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH 80:1), affording com-
pound 2 in the form of yellow powder (292 mg; yield 87%). 1H
NMR (300 MHz, CDCl3): d (ppm) = 8.44 (1H, s), 8.24 (1H, d,
J = 7.5 Hz), 8.20 (2H, d, J = 10.0 Hz); 7.99 (1H, d, J = 7.5 Hz), 7.85
(1H, t, J = 7.5 Hz); 7.69 (1H, t, J = 7.5 Hz), 7.40 (2H, d, J = 10 Hz),
7.36 (1H, s), 5.70 (1H, d, J = 16.0 Hz); 5.40 (1H, d, J = 16.0 Hz),
5.31 (2H, s), 1.99–2.44 (2H, m), 1.06 (3H, t, J = 6.5 Hz). Anal. Calcd
for C27H19N3O8: C, 63.18; H, 3.73; N, 8.18. Found: C, 63.21; H, 3.79;
N, 8.11; ESIMS: 536.20 (MNa+); ½a D25
ꢀ
= +20.57 (c 1, CHCl3).

E. Riva et al. / Bioorg. Med. Chem. 18 (2010) 8660–8668
8665
4.3. Preparation of (3)
(2C), 42.9 (2C), 39.1 (2C), 36.4 (2C), 36.5 (2C), 34.8 (2C), 8.6 (2C).
Anal. Calcd for C52H50N8O12S2: C, 59.87; H, 4.83; N, 10.74; S,
6.15. Found: C, 59.83; H, 4.85; N, 10.76; S, 6.11; HR-ESIMS. Anal.
p-Nitrophenyl camptothecin carbonate (2) (70 mg, 0.14 mmol)
was dissolved in DMF (7 mL), and 1-(tert-butyloxycarbonyl)ethyl-
diamine (13) (43 mg, 0.27 mmol) was added at room temperature.
The mixture was stirred for 10 min, and the DMF was removed un-
der reduced pressure. AcOEt was added to the crude residue and
then extracted with water. The organic layer was dried over so-
dium sulfate and concentrated under reduced pressure to give 3
(68 mg, yield 90%). 1H NMR (300 MHz, CDCl3): d (ppm) = 8.18
(1H, d, J = 7.8 Hz), 8.08 (1H, s), 8.83 (2H, s), 7.80 (1H, t,
J = 7.8 Hz), 7.65 (1H, d, J = 7.8 Hz), 7.53 (1H, t, J = 7.8 Hz), 5.5 (1H,
br s), 5.19 (2H, s), 5.06 (2H, s), 3.86 (2H, m), 3.47–3.55 (2H, m),
2.36–2.51 (2H, m), 1.47 (9H, s), 1.05 (3H, t, J = 7.3 Hz). Anal. Calcd
for C28H30N4O7: C, 62.91; H, 5.66; N, 10.48. Found: C, 62.87; H,
Calcd for
= +20.9 (c 1, CHCl3).
C
52H50N8O12S2Na+, 1065.2882. Found 1065.2908;
½ ꢀ
a 2D5
4.5.3. Compound (7)
1H NMR (400 MHz, CD3OD-d4, 50 °C): d 8.48 (2H, s), 8.07 (2H, m),
7.94 (2H, m), 7.70 (4H, m), 7.43 (2H, s), 5.16 (2H, m), 5.05 (6H, m),
3.78 (4H, m), 3.50 (4H, m), 2.50–2.05 (16H, m), 1.01 (6H, m); 13C
NMR (100.6 MHz, CD3OD-d4, 50 °C): d 173.6 (2C); 169.2 (2C);
167.9 (2C); 157.4 (2C); 153.5 (2C); 148.1 (2C); 145.9 (2C); 144.7
(2C); 131.7 (2C); 131.4 (2C); 131.1 (2C); 130.7 (2C); 130.1 (2C);
129.4 (2C); 128.9 (2C); 128.5 (2C); 99.7 (2C); 89.6 (2C); 55.47
(2C); 51.52 (2C); 42.56 (2C); 37.70 (2C); 34.84 (2C); 33.84 (2C);
32.76 (2C); 30.95 (2C); 7.40 (2C). Anal. Calcd for C54H54N8O12S2: C,
60.55; H, 5.08; N, 10.46; S, 5.99. Found: C, 60.51; H, 5.06; N, 10.44;
5.62; N, 10.45; ESIMS: 536.20 (MNa+); ½a D25
ꢀ
= +24.5 (c 1, CHCl3).
4.4. Preparation of (4)
S, 6.01; ESIMS: 1093.70 (MNa+); ½a D25
ꢀ
= +20.7 (c 1, CHCl3).
Compound (3) (452 mg, 0.85 mmol) was dissolved in dioxane
(10 mL). A freshly prepared saturated solution of HCl in dioxane
(8 mL) was added portion wise at 0 °C for 1 h. The reaction was
monitored by TLC (CH2Cl2/MeOH v/v 10:1). After completion, the
reaction mixture was worked-up with saturated solution of NaH-
CO3 obtaining a basic pH and then was extracted with CH2Cl2.
The organic layer was dried over Na2SO4, concentrated under re-
duced pressure to afford compound 3 (365 mg; quantitative yield).
1H NMR (300 MHz, CDCl3): d (ppm) = 8.42–7.19 (6H, m), 5.23 (2H,
br s), 5.10 (2H, br s), 5.02 (2H, br s), 3.84 (2H, m), 3.11 (2H, m), 2.35
(2H, m), 1.1 (3H, t, J = 7.1 Hz). Anal. Calcd for C23H22N4O5: C, 63.59;
H, 5.10; N, 12.90. Found: C, 63.57; H, 5.07; N, 12.92; ESIMS: 457.20
4.5.4. Compound (8)
1H NMR (400 MHz, DMSO-d6, 80 °C): d 8.65 (2H, s), 8.19 (2H, m,
J = 7.2 Hz), 8.09 (2H, m, J = 7.3 Hz), 7.85 (2H, m, J = 7.3 Hz), 7.77
(2H, m, J = 7.2 Hz), 7.46 (2H, s), 6.82 (br s, 4H), 5.28 (4H, m), 4.89
(4H, m), 3.62 (4H, m), 3.34 (4H, m), 2.20 (4H, m), 1.92–1.25
(16H, m), 0.98 (6H, m); 13C NMR (100 MHz, DMSO-d6, 80 °C): d
174.0 (2C), 172.8 (2C), 165.5 (2C), 155.3 (2C), 151.4 (2C), 148.1
(2C), 146.1 (2C), 144.3 (2C), 130.7 (2C), 130.4 (2C), 130.2 (2C),
130.1 (2C), 129.7 (2C), 129.4 (2C), 127.8 (2C), 127.4 (2C), 99.3
(2C), 88.9 (2C), 57.2 (2C), 50.3 (2C), 40.8 (2C), 37.5 (2C), 36.6
(1C), 33.6 (2C), 32.0 (1C), 29.9 (1C), 29.6 (1C), 29.2 (1C), 28.7
(1C), 28.5 (1C), 25.1 (1C); 7.5 (2C). Anal. Calcd for C56H58N8O12
:
(MNa+); ½a 2D5
ꢀ
= +21.57 (c 1, CHCl3).
C, 64.98; H, 5.65; N, 10.83. Found: C, 64.93; H, 5.01; N, 10.40;
ESIMS 1057.70 (MNa+); ½a D25
ꢀ
= +20.4 (c 1, CHCl3).
4.5. General procedure for preparation of compounds (5–12)
4.5.5. Compound (9)
DMAP (0.57 mmol), (4) (1.14 mmol) and DCC (1.70 mmol) were
added to a solution of diacid (0.57 mmol) in CH2Cl2 dry at room
temperature. The mixture was stirred for 13 h then concentrated
under reduced pressure. The crude residue was filtered on Celite,
the organic layer was concentrated and the crude residue was puri-
fied by flash chromatography (silica gel, CH2Cl2/MeOH 40:1) to
give the dimeric compounds (yields: 42–43%).
1H NMR (400 MHz, DMSO-d6, 80 °C): d 8.71 (1H, s), 8.33 (1H, m),
8.13 (1H, m), 8.03 (br s, 1H), 8.00 (1H, m), 7.75 (1H, m), 7.53 (1H, s),
5.47 (1H, m), 5.34 (3H, m), 3.39 (4H; m), 2.38 (2H, m), 2.01–1.10
(4H, m), 0.98 (3H, t, J = 7.1 Hz); 13C NMR (100.6 MHz, DMSO-d6,
80 °C): d 176.2 (1C); 174.8 (1C); 172.2 (2C); 163.6 (1C); 156.7
(1C), 153.2 (1C); 152.3 (1C); 148.1 (1C); 131.6 (1C); 130.2 (1C);
129.6 (1C); 128.7 (1C); 127.9 (1C); 127.7 (1C); 127.5 (1C); 125.9
(1C); 115.7 (1C); 88.7 (1C); 50.4 (2C); 42.3 (1C); 39.1 (1C); 33.2
(1C); 32.2 (1C); 25.3 (1C); 7.3 (1C). Anal. Calcd for C27H24N4O7S2:
C, 55.85; H, 4.17; N, 9.65; S, 11.04. Found: C, 55.83; H, 4.14; N,
4.5.1. Compound (5)
1H NMR (400 MHz, DMSO-d6, 80 °C): d 8.56 (2H, s), 8.18 (2H, m),
8.11 (2H, m), 7.84 (2H, m), 7.74 (2H, m), 7.45 (2H, s), 6.72 (4H, br s),
5.25 (4H, m), 4.86 (4H, s), 3.40 (8H, m), 2.43 (4H, m), 2.14 (4H, m),
0.95 (6H, m); 13C NMR (100.6 MHz, DMSO-d6, 80 °C): d 172.4 (2C);
168.2 (2C); 167.6 (2C); 156.6 (C); 153.5 (2C) 148.1 (2C); 145.7
(2C); 144.0 (2C); 131.6 (2C); 131.4 (2C); 130.2 (2C); 129.7 (2C);
129.6 (2C); 129.0 (2C); 128.0 (2C); 127.5 (2C); 97.2 (2C); 88.6 (2C)
55.5 (2C); 50.5 (2C); 42.4 (2C); 36.4 (2C); 31.2 (2C); 30.9 (2C); 7.40
(2C). Anal. Calcd for C50H46N8O12S2: C, 59.16; H, 4.57; N, 11.04.
Found: C, 59.14; H, 4.55; N, 11.06; HR-ESIMS. Anal. Calcd for
9.67; S, 11.06; HR-ESIMS. Anal. Calcd for
C
27H24N4O7S2Na+,
= +27.7 (c 1, CHCl3).
603.0984. Found 603.0978; ½a D25
ꢀ
4.5.6. Compound (10)
1H NMR (400 MHz, CD3OD-d4, 50 °C): d 8.64 (1H, s), 8.22 (1H, d,
J = 8.3 Hz), 8.07 (1H, d, J = 8.4 Hz), 8.03 (1H, s), 7.87 (1H, t,
J = 7.1 Hz), 7.72 (1H, t, J = 7.2 Hz), 5.16 (1H, m), 5.05 (3H, m), 3.83
(2H, m), 3.47 (2H, m), 3.04–2.77 (6H, m), 2.43 (4H, m), 1.02 (3H,
t, J = 7.3 Hz); 13C NMR (100.6 MHz, CD3OD-d4, 50 °C): d 171.4
(1C), 167.2 (1C), 167.9 (2C), 156.5 (1C), 153.5 (1C), 148.5 (1C),
145.5 (1C), 143.9 (1C), 132.0 (1C), 131.3 (1C), 130.2 (1C), 130.1
(1C), 129.3 (1C), 128.9 (1C), 128.1 (1C), 127.4 (1C), 96.1 (1C),
81.6 (1C), 55.5 (1C), 51.5 (1C), 42.9 (1C), 39.1 (1C), 36.3 (2C),
36.5 (2C), 34.8 (1C), 8.6 (1C). Anal. Calcd for C29H28N4O7S2: C,
57.22; H, 4.69; N, 9.20; S, 10.54. Found: C, 57.23; H, 4.67; N,
C
50H46N8O12S2Na+, 1037.2569. Found 1037.2581; ½a 2D5
ꢀ
= +24.5 (c 1,
CHCl3).
4.5.2. Compound (6)
1H NMR (400 MHz, CD3OD-d4, 50 °C): d 8.27 (2H, s), 7.81 (2H,
m), 7.79 (2H, m), 7.76 (2H, m), 7.74 (2H, m), 7.28 (2H, s), 5.32
(2H, m), 5.19 (2H, m), 5.06 (4H, m), 3.73 (4H, m), 3.54 (4H, m),
3.02–2.66 (8H, m), 2.44 (4H, m), 1.0 (6H, m); 13C NMR
(100.6 MHz, CD3OD-d4, 50 °C): d 171.4 (2C), 167.2 (2C), 167.9
(2C), 156.57 (2C), 153.5 (2C), 148.5 (2C), 145.5 (2C), 143.9 (2C),
132.0 (2C), 131.3 (2C), 130.2 (2C), 130.1 (2C), 129.4 (2C), 128.9
(2C), 128.1 (2C), 127.5 (2C), 96.1 (2C), 81.6 (2C), 55.6 (2C), 51.6
9.23; S, 10.57; ESIMS: 631.13 (MNa+); ½a D25
ꢀ
= +26.6 (c 1, CHCl3).
4.5.7. Compound (11)
1H NMR (400 MHz, CD3OD-d4, 50 °C): d 8.67 (1H, s); 8.25 (1H, d,
J = 8.3 Hz); 8.09 (1H, d, J = 8.2 Hz), 7.96 (1H, s); 7.90 (1H, t,
J = 7.4 Hz); 7.74 (1H, t, J = 7.2 Hz); 5.48 (1H, m); 5.37 (3H, m);

8666
E. Riva et al. / Bioorg. Med. Chem. 18 (2010) 8660–8668
3.79 (2H, m); 3.48 (2H, m); 2.95–2.44 (12H, m); 2.19 (2H, m); 1.08
(3H, t, J = 7.2 Hz); 13C NMR (100.6 MHz, CD3OD-d4, 50 °C): d 175.8
(1C), 174.5 (1C), 173.3 (2C), 164.0 (1C), 156.4 (1C), 154.1 (1C),
151.3 (1C), 147.8 (1C), 133.6 (1C), 132.5 (1C), 131.6 (1C), 131.0
(1C), 130.8 (1C), 130.3 (1C), 130.1 (1C), 125.8 (1C), 100.1 (1C),
89.5 (1C), 61.4 (1C), 52.9 (1C), 42.5 (1C), 39.1 (1C), 36.9 (2C),
36.2 (2C), 36.1 (2C), 35.0 (1C), 8.65 (1C). Anal. Calcd for
reduction in cell number compared with control cultures. A2B1,
A2C12, B3, bD5, bD10, c-myc-c-raf, A3, A7, B8, D1, and
D12 cells were harvested and plated in 96-well flat-bottomed
microplates at a density of 103 cells/well. Assays were performed
in quintuplicates. Cells were allowed to attach for 24 h. The drugs
were prepared in medium at different concentrations and were
c
c
c
c
c
added to the plates at a volume of 100
ll/well. After 24 (or 96) h
C
31H32N4O7S2: C, 58.47; H, 5.07; N, 8.80; S, 10.07. Found: C,
58.42; H, 5.05; N, 8.82; S, 10.05; ESIMS: 659.16 (MNa+);
= +28.7 (c 1, CHCl3).
incubation 20
l
l of the CellTiter 96Ò AQueous One Solution Re-
agent (Promega Corporation, Madison, WI, USA) were added to
each well and the plates were incubated for 1 h at 37 °C. The CellT-
iter 96Ò AQueous One Solution Reagent contains a tetrazolium
compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phe-nyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and
an electron coupling reagent (phenazine ethosulfate; PES). PES
has a high chemical stability, which allows it to form a stable solu-
tion with MTS. The absorbance was read at 490 nm on a plate spec-
trophotometer (VictorTM 1420 Multilabel Counter, Perkin–Elmer
Instruments, Shelton, USA). Cell cytotoxicity was expressed as
the percentage of the controls.
½ ꢀ
a 2D5
4.5.8. Compound (12)
1H NMR (400 MHz, CD3OD-d4, 50 °C): d 8.65 (1H, s); 8.25 (1H, d,
J = 8.3 Hz); 8.24 (1H, d, J = 8.2 Hz); 7.93 (1H, s); 7.89 (1H, t,
J = 7.5 Hz); 7.73 (1H, t, J = 7.4); 5.52 (1H, m); 5.39 (3H, m); 3.73
(2H, m); 3.50 (2H, m); 2.38–2.16 (12H, m); 1.85–1.10 (6H, m);
0.94 (3H, t, J = 7.2 Hz); 13C NMR (100.6 MHz, CD3OD-d4, 50 °C): d
174.6 (1C), 173.1 (1C), 172.6 (2C), 162.5 (1C), 156.1 (1C), 153.4
(1C), 149.5 (1C), 133.9 (1C), 133.4 (1C), 132.6 (1C), 132.0 (1C),
131.0 (1C), 130.4 (1C), 130.1 (1C), 122.7 (1C), 100.6 (1C), 88.8
(1C), 60.7 (1C), 52.8 (1C), 39.2 (1C), 37.5 (1C), 34.6 (1C), 35.6
(1C), 31.5 (1C), 30.9 (2C), 30.3 (1C), 30.1 (1C), 27.6 (1C), 26.8
(1C), 25.5 (1C), 9.1 (1C). Anal. Calcd for C33H36N4O7: C, 65.99; H,
6.04; N, 9.33. Found: C, 65.95; H, 6.02; N, 9.34; ESIMS: 623.25
4.8. Linear flow dichroism
LD measurements were performed on a Jasco J500A circular
dichroism spectropolarimeter converted for LD and equipped with
an IBM PC and a Jasco J interface. Linear dichroism is defined as:
(MNa+); ½a 2D5
ꢀ
= +25.8 (c 1, CHCl3).
LDðkÞ ¼ AkðkÞ ꢂ A?ðkÞ
4.6. Preparation of (13)
where Ak and A? correspond to the absorbances of the sample when
polarized light is oriented parallel or perpendicular to the flow
direction, respectively. The orientation is produced by a device
A solution of di-tert-butyl dicarbonate (1.21 g, 5.55 mmol) in
dichloromethane (80 mL) was added dropwise at 0 °C to a solution
of ethylenediamine (2 g, 33.3 mmol) in dichloromethane (10 mL)
over 1 h with vigorous stirring. The reaction mixture was stirred
at room temperature for 19 h. After concentration to an oily resi-
due, the reaction mixture was dissolved in aqueous sodium car-
bonate (60 mL) and extracted with dichloromethane (2 ꢁ 60 mL).
The organic layer was dried (anhydrous Na2SO4) and the solvent
evaporated under reduced pressure to yield 13 (1.44 g, 100%) as
a colorless viscous liquid. 1H NMR (300 MHz, CDCl3): d 4.89 (br s,
1H), 3.18 (q, J = 5.9 Hz, 2H), 2.80 (t, J = 6.1 Hz, 2H), 1.60 (br s, 2H),
1.44 (s, 9H). 13C NMR (75 MHz, CDCl3): d 156.2 (1C), 79.2 (1C),
43.3 (1C), 41.8 (1C), 28.4 (3C).
designed by Wada and Kozawa51 at
a shear gradient of
500–700 rpm and each spectrum was accumulated four times.
A solution of salmon testes DNA (1.9 ꢁ 10ꢂ3 M) in ETN buffer
(containing 10 mM TRIS, 10 mM NaCl, and 1 mM EDTA, pH 7) was
used. Spectra were recorded at 25 °C at different[drug]/[DNA] ratios.
4.9. Topoisomerases-mediated DNA relaxation
Supercoiled pBR322 plasmid DNA (0.25
Sciences) was incubated with 1 U topoisomerase II (human recom-
binant topoisomerase II , USB) or 2 U topoisomerase I (calf thymus
topoisomerase I, USB) and the test compounds as indicated for
60 min at 37 °C in 20 l of reaction buffer. Reactions were stopped
by adding 4 l of stop buffer (5% sodium dodecyl sulfate (SDS),
0.125% bromophenol blue, and 25% glycerol) and 50 g/mL protein-
lg, Fermentas Life
a
4.7. Inhibition growth assay
l
l
HL-60 (human myeloid leukemic cells) and HeLa (human cervix
adenocarcinoma cells) were grown in RPMI 1640 (Sigma Chemical
Co.) supplemented with 15% heat-inactivated fetal calf serum (Bio-
logical Industries) and in Nutrient Mixture F-12 [HAM] (Sigma
Chemical Co.) supplemented with 10% heat-inactivated fetal calf
serum (Biological Industries), respectively. A-431 (skin carcinoma
squamous cell) were grown in Dulbecco’s Modified Eagle’s Medium
(Sigma) supplemented with 10% fetal bovine serum (Seromed).
l
ase K (Sigma) and incubating for a further 30 min at 37 °C. The sam-
ples were separated by electrophoresis on a 1% agarose gel at room
temperature. The gels were stained with ethidium bromide 1 lg/mL
in TAE buffer (0.04 M Tris–acetate and 0.001 M EDTA), transillumi-
nated by UV light, and fluorescence emission was visualized by a
CCD camera coupled to a Bio-Rad Gel Doc XR apparatus.
100 U/mL penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL
4.10. Topoisomerase II-mediated DNA cleavage
amphotericin B (Sigma Chemical Co.) were added to the media.
The cells were cultured at 37 °C in a moist atmosphere of 5% car-
bon dioxide in air. HL-60 cells (4 ꢁ 104) were seeded into each well
of a 24-well cell culture plate. After incubation for 24 h, various
concentrations of the test agents were added to the complete med-
ium and incubated for a further 72 h. HeLa and A-431 (4 ꢁ 104)
cells were seeded into each well of a 24-well cell culture plate.
After incubation for 24 h, the medium was replaced with an equal
volume of fresh medium, and various concentrations of the test
agents were added. The cells were then incubated in standard con-
ditions for a further 72 h. A Trypan blue assay was performed to
determine cell viability. Cytotoxicity data were expressed as IC50
values, that is, the concentration of the test agent inducing 50%
Reaction mixtures (20
50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15
bovine serum albumin (BSA), 1 mM ATP, 0.25 g pBR322 plasmid
DNA, 10 U topoisomerase II (human recombinant topoisomerase
II , USB), and test compounds were incubated for 60 min at
37 °C. Reactions were stopped by adding 4 l of stop buffer (5%
SDS, 0.125% bromophenol blue, and 25% glycerol) and 50 g/mL
l
l) containing 10 mM Tris–HCl (pH 7.9),
l
g/mL
l
a
l
l
proteinase K (Sigma) and incubating for a further 30 min at
37 °C. The samples were separated by electrophoresis on a 1% aga-
rose gel containing ethidium bromide 0.5
lg/mL at room tempera-
ture in TBE buffer (0.09 M Tris–borate and 0.002 M EDTA).

E. Riva et al. / Bioorg. Med. Chem. 18 (2010) 8660–8668
8667
4.11. Topoisomerase I-mediated DNA cleavage
Reaction mixtures (20 l) containing 35 mM Tris–HCl (pH 8.0),
72 mM KCl, 5 mM MgCl2, 5 mM DTT, 5 mM spermidine, 0.01%
bovine serum albumin (BSA), 10 ng pBR322 plasmid DNA, 5 U
topoisomerase I (calf thymus topoisomerase I, USB), and test com-
pounds were incubated for 60 min at 37 °C. Reactions were
Supplementary data
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.09.069. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
stopped by adding 4
blue, and 25% glycerol) and 0.6
l
l of stop buffer (5% SDS, 0.125% bromophenol
References and notes
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This research has been developed under the umbrella of CM
0602 COST Action ‘Inhibitors of Angiogenesis: Design, Synthesis
and Biological Exploitation’. The authors are grateful to INDENA
for providing camptothecin. D.P. and D.C. are grateful to the finan-
cial support provided by Ministero dell’Istruzione, dell’Università e
della Ricerca (MIUR) PRIN 2007—Program ‘Sviluppo e caratterizz-
azione di nuovi inibitori di tirosine chinasi cellulari con attività
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