Novel tumor-targeted RGD peptide-camptothecin conjugates: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2009.11.019

Five RGD peptide-camptothecin (CPT) conjugates were designed and synthesized with the purpose to improve the therapeutic index of this antitumoral drug family. New RGD cyclopeptides were selected on the basis of their high affinity to α_v integr

Full text of DOI:10.1016/j.bmc.2009.11.019

Bioorganic & Medicinal Chemistry 18 (2010) 64–72
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel tumor-targeted RGD peptide–camptothecin conjugates:
Synthesis and biological evaluation
a
a
b
b
Alma Dal Pozzo a, , Ming-Hong Ni , Emiliano Esposito , Sabrina Dallavalle , Loana Musso ,
Alberto Bargiotti b, Claudio Pisano c, Loredana Vesci c, Federica Bucci c, Massimo Castorina c,
Rosanna Foderà c, Giuseppe Giannini c, Concetta Aulicino c, Sergio Penco c
*
a Istituto di Ricerche Chimiche e Biochimiche G.Ronzoni, via G.Colombo, 81, 20133 Milano, Italy
b Dipartimento di Scienze Molecolari Agroalimentari, Università di Milano, via Celoria 2, 20133 Milano, Italy
c Area of Oncology-Research & Development Sigma-Tau, Pomezia, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 May 2009
Revised 29 October 2009
Accepted 7 November 2009
Available online 12 November 2009
Five RGD peptide–camptothecin (CPT) conjugates were designed and synthesized with the purpose to
improve the therapeutic index of this antitumoral drug family. New RGD cyclopeptides were selected
on the basis of their high affinity to av integrin receptors overexpressed by tumor cells and their meta-
bolic stability. The conjugates can be divided in two groups: in the first the peptide was attached to the
drug through an amide bond, in the second through a hydrazone bond. The main difference between the
two spacers lies in their acid stability. Affinity to the receptors was maintained for all conjugates and
their internalization into tumor cells was demonstrated. The first group conjugates showed lower
in vitro and in vivo activity than the parent drug, probably due to the excessive stability of the amide
bond, even inside the tumor cells. Conversely, the hydrazone conjugates exhibited in vitro tumor cell
inhibition similar to the parent drug, indicating high conversion in the culture medium and/or inside
the cells, but their poor solubility hampered in vivo experiments. On the basis of these results, informa-
tion was acquired for additional development of derivatives with different linkers and better solubility
for in vivo evaluation.
Keywords:
Tumor-targeting
Integrins
Camptothecin conjugates
RGD cyclopeptides
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
limited amount of antigens are overexpressed on the tumor cell
surface and a limited number of molecules can be loaded on each
Traditional cancer chemotherapy is based on the assumption
that rapidly proliferating cancer cells are more likely to be killed
than quiescent cells of physiological tissues. However, cytotoxic
agents have very poor specificity, which leads to systemic toxicity,
causing severe side effects. Recent improvements in the knowledge
of typical receptors overexpressed by cancer cells during their pro-
liferation allow the exploitation of selective ligands, which, conju-
gated with cytotoxic agents, are able to preferentially address
them to the tumors.
Advances in tumor-targeting drug conjugates include monoclo-
nal antibodies (mAb), polyunsaturated fatty acids, hyaluronic acid,
folic acid and small peptides as ligands of tumor associated recep-
tors.1 At present, several immunoconjugates are undergoing clini-
cal evaluation,2 and among them, the immunoconjugate of
calicheamicin3 (MylotargÒ) has been approved by FDA against
acute myeloid leukemia. Nevertheless, the practical use of immu-
noconjugates is only suitable for highly potent drugs, because a
mAb without decreasing the binding affinity and increasing the
immunogenicity.
In the last few years, conjugates of cytotoxic agents with small
peptides, addressed to different receptors overexpressed by tumor-
al cells, have been studied as potential selective antitumoral che-
motherapeutics. Somatostatin and bombesin have been
intensively studied as tumor-targeting molecules in conjugation
with different cytotoxic agents.4–8
Among selective receptor-targeting small peptides, integrin-
mediated RGD peptides appear attractive candidates. The argi-
nine–glycine–aspartic acid (RGD) is a cell adhesion motif present
in many proteins of the extracellular matrix (ECM).9 Through their
RGD sequence ECM proteins recognize
receptors, which play an important role in angiogenesis and tumor
growing. v Integrins are expressed on the luminal surface of neo-
avb3 and avb5 integrin
a
vasculature, but are not found on the mature capillaries. In addi-
tion, they have been shown to be upregulated in tumor blood
vessels that undergo continuous angiogenesis and have been
implicated in metastasis.10 Inhibition of angiogenesis has been
shown to prevent tumor growth and even cause tumor regression
* Corresponding author. Tel.: +39 02 70600223; fax: +39 02 70641634.
E-mail address: dalpozzo@ronzoni.it (A. Dal Pozzo).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.019

A. Dal Pozzo et al. / Bioorg. Med. Chem. 18 (2010) 64–72
65
in various experimental models.11 However, antiangiogenic ther-
2. Results and discussion
apy alone usually is not sufficient to eradicate tumors.12 On the
other hand, RGD peptides conjugated with cytotoxic agents are
likely to exhibit a tumor-targeting and antiangiogenic synergetic
effect. During the last few years, a number of RGD-cytotoxic drugs
were developed and showed promising activities in vitro and in
vivo.13–18
2.1. Chemistry
2.1.1. Synthesis of camptothecin derivatives
Camptothecin derivatives 1a (ST1968, Namitecan) and 1b were
synthesized according to methods already published.19–21 Com-
pound 1c was obtained as illustrated in Scheme 1: the aldehyde
1b was transformed into the unsaturated aldehyde 3 by a Wittig
reaction and, after protection as the acetal 4, the double bond
was reduced giving 5. Finally, acidic deprotection afforded the
camptothecin derivative 1c.
In the study described herein, we investigated the usefulness of
RGD peptides as carriers for antitumoral drugs belonging to the
family of camptothecins (CPT). Potent CPT analogues 1a–c were
chosen among a large series prepared in our laboratory,19–22 bear-
ing at 7-position functional groups suitable for conjugation
(Fig. 1a). As targeting device we chose cyclic peptide analogues
of c(RGDfV) developed by Kessler and co-worker.23 We synthe-
sized a series of novel pseudopeptides,24 all containing at the 5-po-
sition a trifunctional pseudoamino acid, consisting of a non-
proteinogenic amino acid with a side chain bearing a suitable func-
tional group for the attachment of cytotoxic drugs. Among them,
four cyclopeptides, 2a–d, were selected, which proved to have
the optimal characteristics of affinity to av integrins in vitro and
metabolic stability, increased by the presence of the non-natural
amino acid in their sequence (Fig. 1b). The linker between drug
and targeting device has critical significance for the efficacy of
the conjugates, its prerequisites being stability into the body circu-
lation and lability into the tumor cell. Unlike the linkers used for
the immunoconjugates, that are shielded from plasma peptidases
by the bulky antibody moiety, the choice of the linker for conju-
gates with small peptides is very challenging. Accordingly, we syn-
thesized two classes of conjugates, that differ in the chemical
nature of the bond between drug and peptide. The first is charac-
terized by a stable amide bond, that should guarantee stability in
the body circulation; for this purpose we avoided the use of ester
bonds prone to the attack of esterases. The second class of conju-
gates is characterized by a hydrazone bond which is supposed to
be stable at neutral pH of the body fluids, but promptly cleaved
at the low pH inside the tumor cells. The use of hydrazones for
the release of drugs at low pH has been widely documented in
the literature.25,26
2.1.2. Synthesis of cyclopeptides
The linear peptides were synthesized on solid-phase following
the standard Fmoc-protocol. The four peptides differ from each
other for the pseudoamino acid at the 5-position. The commer-
cially available pseudoaminoacids
x-benzyl Fmoc-adipic acid es-
ter[Fmoc-Aad(Bn)-OH] and Fmoc-aminomethylphenylalanine
[Fmoc-Amp(Cbz)-OH] were introduced in the cyclopeptides 2a0
and 2b0–c0, respectively, while the pseudoaminoacid 6, introduced
in the cyclopeptide 2d, was synthesized in our laboratory accord-
ing to the method described in Scheme 2: the trioxaundecanedioic
acid monobenzyl ester was submitted to hydrazinolysis and the
monoacid hydrazide was protected with Boc, activated with N-
hydroxysuccinimide and coupled with Fmoc-Amp-OH to afford 6.
After cleavage from the resin, the peptides were cyclized in solu-
tion and orthogonally deprotected at the 5-position. Then, the
Amp-containing peptides were further treated with succinic anhy-
dride or 3,6,9-trioxaundecanedioic acid to obtain 2b0 and 2c0,
respectively. Only 2d was obtained after total deprotection, be-
cause the successive formation of the hydrazone conjugates is
chemoselective.
2.1.3. Synthesis of conjugates
Five conjugates were synthesized. For the three conjugates 7a–
c, cyclopeptides in their partially protected form were attached to
CPT via an amide bond, using DCC/HOAT as condensing reagents,
followed by total acidic deprotection (Scheme 3). For the two con-
jugates 8a–b, cyclopeptide 2d was attached to CPT via hydrazone
bond, after removal of the protecting groups from arginine and
aspartic acid. Conjugates 8a and 8b were easily obtained by adding
the corresponding CPT aldehydes to a solution containing the pep-
The binding assays of the cyclopeptides and of the correspond-
ing conjugates to the isolated am integrins, the study of their effect
on cell adhesion and in vitro cytotoxicity are described. Further-
more, the in vivo activity of one of the conjugates was evaluated
against A2780 human ovarian carcinoma in nude mice.
R
a
7
O
1a R = -CH=NO(CH2)2NH2 (ST1968, Namitecan)
1b R = -CHO
N
N
O
1c R = -(CH2)2-CHO
OH
O
OH
O
R1, R2 = H
R1, R2 = H
O
2a R =
R
5
O
N
H
OH
O
b
HN
O
N
HN
2b R =
H
H
O
R1OOC
N
NHR2
NH
O
O
O
O
NH
O
O
R1, R2 = H
R1, R2 = H
N
H
O
OH
NH
2c R =
2d R =
O
O
O
O
N
O
NH-NH2
H
2a', 2b', 2c' R1 = tBu R2 = Pmc
Figure 1. (a) Structure of Camptothecin analogues; (b) structure of cyclopeptides.

66
A. Dal Pozzo et al. / Bioorg. Med. Chem. 18 (2010) 64–72
O
O
CHO
CHO
N
O
O
O
b
a
N
N
N
N
N
3
O
O
4
O
1b
c
OH
O
OH
O
OH
O
O
O
N
O
CHO
O
d
N
N
N
1c
O
O
5
OH
O
OH
O
Scheme 1. Reagents and conditions: (a) Ph3P@CH–CHO, CHCl3, reflux, 68%; (b) CH2OHCH2OH, PTSA, MgSO4, toluene, reflux Dean–Stark, 56%; (c) H2, Pd/C 10%, MeOH, 99%; (d)
80% AcOH, 80 °C, 39%.
O
O
COOH
a
O
COOBn
O
O
C
b
HOOC
c
O
HOOC
O
HOOC
O
NH-NH
2
2
2
2
O
O
O
N
O
C
O
2
C
C
O
O
NH-NH Boc
HOOC
O
NH-NH Boc
Fmoc
2
O
H N
2
HN
COOH
e
Amp=
O
C
H
N
O
C
O
O
NH-NH Boc
2
H N
2
6
COOH
Scheme 2. Reagents and conditions: (a) BnOH, DCC, TEA, DMAP, DCM; (b) NH2-NH2.H2O, EtOH; (c) (Boc)2O, 1 N NaOH, MeOH, 76%; (d) N-hydroxysuccinimide, DCC, DCM,
86%; (e) Fmoc-Amp-OH, TEA, DCM/DMF, 52%.
tide hydrazide in MeOH and stirring under nitrogen at room tem-
perature for 36 h (Scheme 4).
was observed on PC3 prostate carcinoma cells endowed with low
levels of integrins.
We also demonstrated the internalization of the conjugates
into the tumor cells: the compounds were isolated from the whole
cell extracts and analyzed by HPLC with fluorometric detection.
From the results illustrated in Figure 2, it is evident that the con-
jugates 7a and 7c accumulate inside the cell at a much lower rate
than the free parent drug. In fact, CPT shows a rapid intracellular
uptake with the highest value at 5 h and a slow decrease until
72 h, while the conjugates reach a concentration peak at 48 h,
suggesting a mechanism dependent on integrins interaction and/
or turnover. This experiment also shows that only small detect-
able amounts of CPT are released at the end of the treatment,
proving the high stability of the conjugates both in culture med-
ium and inside the tumor cells. This is consistent with their lower
cytotoxicity compared to the free drug, as reported in Table 2.
Conjugates 7a–c cytotoxicity resulted more than one order of
magnitude lower than CPT alone, and we attributed these results
mainly to the amide bond, which is too stable to promptly release
the drug.
2.2. Biological evaluation
2.2.1. In vitro evaluation of 7a–c
Firstly, the binding of the three conjugates to the isolated
receptors and adhesion to three tumoral cell lines (PC3 prostatic
carcinoma, A498 renal carcinoma, A2780 ovarian carcinoma)
were measured, showing that peptides effectively mediate the
binding to the cell surface in a dose-dependent manner, whereas
CPTs alone do not bind to integrins or the cell surface (Table 1).
All the conjugates maintained a good receptor affinity. More-
over, the cell adhesion resulted increased in some case, compared
to the peptides alone; this behavior should be attributed to co-
operative conformational effects of the whole molecule. It is inter-
esting to notice that either peptides or the corresponding conju-
gates efficiently inhibit the adhesion of A2780 ovarian carcinoma
cells overexpressing
avb5 integrin as well as of A498 renal carci-
noma overexpressing
avb3 and vb5 integrins, while a minor effect
a

A. Dal Pozzo et al. / Bioorg. Med. Chem. 18 (2010) 64–72
67
1a + 2a'
a,b
H
N
N
O
O
N
H
O
O
O
HN
HN
NH
H
O
N
HOOC
N
NH2
O
N
NH
NH
O
O
7a
OH
O
1a + 2b'
a,b
O
HN
O
H
N
N
H
O
HN
O
N
HN
NH
O
H
N
O
HOOC
NH2
O
NH
O
NH
7b
N
O
N
O
1a + 2c'
OH
O
a,b
O
H
N
O
2
O
O
O
O
N
N
H
N
O
O
HN
H
O
HN
H
N
NHR2
NH
HOOC
N
O
NH
N
NH
O
O
7c
OH
O
Scheme 3. Synthesis of camptothecin–cyclopeptide conjugates with amide bond. Reagents: (a) DCC, HOAT, DIEA, DMF; (b) TFA/DCM, 1:1.
To overcome this problem, novel conjugates were designed con-
taining the acid-labile hydrazone linker. Because hydrazones are
known to be stable at neutral pH, these conjugates were expected
to have a sufficiently long lifetime under normal physiological con-
ditions to undergo internalization and localization in the acidic
lysosomal environment, releasing the active drug. Accordingly,
two CPT analogues 1b and 1c were synthesized and linked to the
RGD cyclopeptide 2d bearing a hydrazide group, to obtain the con-
jugates 8a and 8b.
of 25 h. (Table 3) The resistance to acid hydrolysis exhibited by the
latter hydrazone, derived from the aromatic aldehyde, can be
attributed to the conjugation of the
p-bond of the C@N– group
with the
p-bond of the CPT aromatic ring, as observed by Kale
et al.26 for their hydrazone derivatives.
2.2.3. In vitro evaluation of 8a–b
Biological results of the conjugates and the corresponding CPT
analogues are reported in Table 4. Both conjugates showed high
cytotoxic activity. Considering the poor stability found at pH 7.4,
we can hypothesize that this activity is partially due to the CPT re-
leased in the culture medium during the experiments. On the other
hand, the difference in cytotoxicity found between the two conju-
gates can be attributed to the difference in their acidic stability in
lysosomal medium.
2.2.2. Stability of the conjugates
In order to assess the stability of these conjugates at different
pH, they were dissolved with DMSO and diluted with phosphate
or acetate buffers at 37 °C, in order to reach pH 7.4 or 5, respec-
tively, and a concentration of 100 lM (1% DMSO). Disappearance
of the starting material was monitored by HPLC (see Supplemen-
tary data). Conjugates 7a–c proved to be stable at both pH (half-life
>72 h), whereas conjugates 8a and 8b showed half-lives of about
6 h at pH 7.4. Compound 8b underwent rapid hydrolysis at pH 5
(half-life less than 2 min) as expected, while 8a showed a half-life
2.2.4. In vivo antitumor activity and tumor distribution
The antitumor effect of the conjugate 7c was investigated on
A2780 human ovarian carcinoma xenograft in nude mice. The mol-
ecule, delivered bis in die intraperitoneally or subcutaneously, pro-

68
A. Dal Pozzo et al. / Bioorg. Med. Chem. 18 (2010) 64–72
1b + 2d
a
O
H
N
N
O
2
O
O
NH
O
O
N
H
O
HN
O
N
HN
NH
O
H
N
N
HOOC
8a
NH
2
O
NH
O
NH
1c + 2d
OH
O
a
O
H
N
O
2
N
O
O
N
H
O
N
O
O
HN
H
N
HN
O
H
N
HOOC
8b
NH
2
N
O
NH
NH
NH
O
O
OH
O
Scheme 4. Synthesis of camptothecin–cyclopeptide conjugates with hydrazone bond. Reagents and conditions: (a) MeOH, N2, room temperature.
Table 1
Table 2
Binding of cyclopeptides 2a–c and the corresponding conjugates 7a–c to the isolated
av integrins and their effect on cell adhesion in the presence of vitronectin after 3 h
treatment
Cytotoxicity of the conjugates 7a–c and the corresponding cyclopeptides 2a–c on
different tumor cell lines after 72 h treatment, IC50
, lM
Entry
PC3
A498
A2780
Entry
Binding assay, IC50 nM
vb3 vb5
Adhesion assay, IC50
A498
lM
1a
2a
7a
2b
7b
2c
7c
0.36 0.06
>1000
15 1.5
>1000
4.7 0.4
>1000
27 3.9
0.064 0.01
>1000
3.2 0.4
>1000
4.3 0.1
>1000
0.74 0.1
0.0049 0.001
>1000
0.4 0.03
>1000
0.16 0.001
>1000
0.18 0.01
a
a
PC3
A2780
1a
2a
7a
2b
7b
2c
7c
No binding
28.6 0.76
0.59 0.01
37.6 0.99
7.27 0.06
4.0 0.1
No adhesion
38.8 8.5
5.8 0.6
33.7 7.8
>100
0.17 0.01
0.37 0.01
5.1 0.07
8.39 0.07
0.35 0.09
6.5 0.03
6.5 0.6
3.7 0.3
34 2.2
5.4 1.3
10.7 0.4
3.1 0.3
5.2 0.2
3.1 0.5
9.5 1.9
18.8 4.4
8.3 1.0
12.3 0.9
1.8 0.2
12.5 2.1
Table 3
Half-lives of hydrazone conjugates 8a and 8b
7a
0.16
7c
1a in 7c
1a
Entry
pH 7.4, 37 °C
pH 5, 37 °C
t1/2, h
0.14
0.12
0.1
t1/2,
h
8a
8b
6
5.8
25
<2 min
0.08
0.06
0.04
0.02
0
extrapolated to infinity) for 7c and 1a: 82 and 2
l
g/h/mL were
found, respectively. This means that 2.5% of the free drug is re-
leased from the conjugate in the blood circulation. In fact, after
administration of 0.42 mg/Kg of 1a, equivalent to the amount of
free drug, the tumor volume inhibition of 43% and a body loss of
0% were found. These data strongly suggest that the antitumor
activity found for 7c could be due to the small circulating plasma
levels of free CPT released from the conjugate and to CPT tumor
distribution.
The same xenograft model was used to evaluate pharmacoki-
netics and tumor distribution. As shown in Figure 3, high levels
of compound 7c were found both in plasma and in tumor, demon-
strating that the conjugate attains in vivo tumor homing and re-
leases free CPT inside tumors. Moreover, 7c concentration
profiles versus time were similar in plasma and tissue and sug-
gested a good and comparable apparent half-life, as evident by
the curves slope. As formerly observed during the in vitro uptake
5
24
48
72
treatment time (h)
Figure 2. Uptake of conjugates 7a, 7c and 1a in A498 tumor cells.
duced a tumor volume inhibition of about 40%. Moreover, the
product showed to be well tolerated at a dose of 48 mg/Kg, since
no body weight loss or lethal toxicity occured (Table 5). Neverthe-
less, CPT 1a given alone at 16.3 mg/Kg, corresponding to the equi-
molar dose present in the conjugate, resulted to be toxic, because
all mice died. In a subsequent experiment, we used a non toxic
dose of 1a comparable to the free drug found in plasma after
administration of the conjugate. This dose was estimated from
measurements of AUCINF (area under plasma concentration curve

A. Dal Pozzo et al. / Bioorg. Med. Chem. 18 (2010) 64–72
69
Table 4
Binding to av integrins, adhesion to tumor cells and cytotoxicity of the cyclopeptide 2d, CPT analogues 1b and 1c, and the corresponding conjugates 8a and 8b
Entry
Binding assay IC50 nM
vb5
Adhesion assay IC50
l
M
Cytotoxicity IC50 lM
a
vb3
3.8 0.1
a
PC3
A2780
PC3
A2780
2d
1b
8a
1c
8b
0.65 0.1
59.9 1.07
22.95 1.18
15.6 2.4
11.8 0.9
>1000
>1000
0.13 0.04
0.22 0.02
0.12 0.04
0.42 0.03
0.003 0.00006
0.09 0.01
0.015 0.002
0.017 0.002
11.66 2.5
6.01 0.22
4.8 0.5
11 0.8
6.7 0.7
12.9 0.5
Table 5
Antitumor activity of the novel conjugate 7c and CPT 1a delivered subcutaneously or intraperitoneally bis in die against A2780 human ovarian carcinoma xenografted in nude
mice
Compound
Single dose mg/Kg
Route
Treatment schedule
TVI%a SE
BWL%b
Lethal toxicityc
7c
7c
1a
1a
48
48
16.3
0.42
ip
sc
sc
sc
q4 d ꢀ 4
qd ꢀ 5
qd ꢀ 5
qd ꢀ 5
43 6* (+16)
37 5* (+17)
ne
2
0
30
0
0/8
0/8
8/8
0/8
43 12* (+17)
ne = not evaluable for lethal toxicity.
a
Tumor volume inhibition percentage versus control mice (in parenthesis the day upon the tumor injection).
Maximum body weight loss percentage due to the drug treatment.
Dead/treated animals.
b
c
*
P 60.05 versus vehicle-treated group (Mann–Whitney).
7c in plasma
100000
taining conjugates demonstrated poor stability in buffer solution at
pH 7.4 (half-life 6 h). Hydrazone linkers have been often used for
conjugating drugs with macromolecules such as antibodies or bio-
polymers, which, thanks to their steric hindrance, can shield the
bond from hydrolysis. This is not the case of conjugates containing
a small molecule, as our compounds, being the linker between
drug and targeting device much more accessible. As expected, at
pH 5 the conjugate 8b was totally hydrolyzed within 2 min, while
8a, synthesized from the aromatic aldehyde 1b, was hydrolyzed
more slowly in the same conditions. Considering in vitro cytotox-
icity of these two conjugates, we can hypothesize that the high
activity found is partially due to the CPT released in the culture
medium during the experiment. Further studies or in vivo experi-
ments were hampered by the high insolubility of these molecules.
Based on the information provided by the experiments described
in this article, the future research will aim to systematically design
and evaluate new RGD-CPT conjugates in order to find the optimal
linker that warrants acceptable stability in the body circulation
and prompt release inside the tumor cell, together with improved
solubility.
1a found in plasma of 7c treated mice
7c in tumor
10000
1000
100
10
1a found in tumor of 7c treated mice
1
0
5
10
15
20
25
time (h)
Figure 3. Plasma and tumor concentration profiles versus time of conjugate 7c in
A2780 human ovarian carcinoma xenografted nude mice treated at a dose of 48 mg/
Kg.
study, free CPT is very slowly released by the conjugate, confirming
also in vivo its stability.
4. Experimental
4.1. Materials and methods
3. Conclusions
All reagents and solvents were reagent grade and were distilled
prior to use. All amino acids and resin were purchased from Ba-
chem (Switzerland). Flash chromatography was carried out on sil-
ica gel (Merck 230–400 mesh). TLC analysis was conducted on
silica gel plates (Merck 60F254). Products were characterized by
HPLC, Waters 600 instrument, with an analytical column Puro-
Our studies demonstrate the potential applicability of suitable
RGD peptides for CPTs tumor-targeting. We obtained conjugates
with high affinity to av integrin receptors overexpressed by tumor
cells and demonstrated their internalization through the cell mem-
brane. Amide bond containing conjugates are very stable in buffer
solution at pH 7.4 and in culture medium, but are less active than
the parent drug either in vitro or in vivo, most probably because
the linker between the targeting peptide and CPT is too stable
and does not release sufficient amounts of the drug inside the tu-
mor cells. The in vivo experiments were carried out using only
one dose of 7c, which corresponds to the maximum solubility of
the compound. Since none toxicity was observed, we may argue
that its antitumor profile could be enhanced using a dose corre-
sponding to the MTD (Maximum Tolerated Dose). Hydrazone-con-
sphere, STARÒ, Merck, C18, 5
l, 4.6 ꢀ 250 mm, using acetonitrile/
water buffered with 0.1% trifluoroacetic acid as mobile phase. UV
detection was performed at k = 220 nm for peptides and 360 nm
for conjugates. When necessary, compounds were purified by
semi-preparative HPLC system equipped with column AlltimaÒ,
Alltech, RP18, 10
l
, 22 ꢀ 250 mm. 1H NMR spectra were recorded
on AC300 Bruker instrument. Chemical shifts (d values) are given
in ppm. Mass spectra were recorded on Bruker Autoflex Maldi-
Tof or Micro-Tof Q. av integrins were purchased from Chemicon.

70
A. Dal Pozzo et al. / Bioorg. Med. Chem. 18 (2010) 64–72
125I-Echistatin was purchased from Amersham. Vitronectin was
purchased from Cal-biochem.
(m, 13H), 6.55 (s, 1H), 5.85–5.60 (m, 4H), 4.56–3.81 (m, 20H),
3.70–2.79 (m, 10H), 2.05–1.10 (m, 8H), 0.91(t, 3H).
4.4. Synthesis of 1c
4.2. Synthesis of the conjugates 7a–c
To a suspension of 3 (obtained from 1b by Wittig reaction,27
40 mg, 0.1 mmol) in 5 mL of dry toluene in a flask equipped with
a Dean–Stark apparatus, MgSO4 (24 mg, 0.2 mmol), PTSA (5 mg,
0.026 mmol) and ethylene glycol (40 mg, 0.72 mmol) were added.
The reaction was kept under reflux for 5 h and diluted with DCM,
washed with water and dehydrated with Na2SO4. After removal
of the solvent, the residue was purified by flash chromatography
(DCM/MeOH 95:5) to give the acetal 4 with 56% yield. To a solution
of 4 (25 mg, 0.055 mmol) in 10 mL of MeOH, 8 mg of Pd/C 10%
were added and the suspension was hydrogenated for 3 h. Evapo-
ration of the solvent followed by flash chromatography (DCM/
MeOH 97:3) gave compound 5 with 99% yield. Deprotection of
the acetal was carried out in 80% acetic acid under reflux for 6 h.
After evaporation and flash chromatography (DCM/MeOH 95:5)
1c was obtained with 39% yield. ESI mass [MH]+: 403.12; 1H
NMR (CDCl3) d: 9.83 (s, 1H), 8.22 (d, 1H), 8.00 (d, 1H), 7.79 (m,
1H), 7.66 (m, 2H), 5.53 (m, 2H), 5.73–5.29 (m, 4H), 3.46 (m, 2H),
3.02 (m, 2H), 1.88 (m, 2H), 1.01 (t, 3H).
General procedure. To a solution of the cyclopeptides 2a0–c0 in
DMF at 0 °C, 1a (1.5 equiv) was added followed by 2.2 equiv of
DIEA, HOAT and DCC and the mixture was stirred at room temper-
ature for 24 h. After evaporation of the solvent under reduced pres-
sure, the residue was dissolved with DCM and the solution washed
with water, dehydrated (Na2SO4) and evaporated to dryness. The
residue was purified by standard flash chromatography (DCM/
MeOH, 95:5?80:20) and total deprotection was carried out with
TFA/DCM, 1:1 for 2 h.
After evaporation of the solvent, the residue was redissolved in
a minimum quantity of TFA and precipitated with anhydrous ether,
repeating the operation several times, to obtain the final conju-
gates as trifluoroacetate salts with high purity and good yield.
4.2.1. Conjugate 7a
Total yield 75% with >98% HPLC purity (34% CH3CN in
H2O + 0.1% TFA): Rt = 6.92 and 9.65 min, corresponding to E/Z iso-
mers of CPT derivatives; Maldi mass = 1035.40 [MH]+; 1H NMR
(DMSO-d6) d: 9.29 (s, 1 H), 8.60 (d, 1H), 8.22 (d, 1H), 7.91 (t, 1H),
7.76 (t, 1H), 7.37 (s, 1H), 7.20–7.10 (m, 5H), 5.39 (d, 4H), 4.60 (m,
1H), 4.44 (m, 1H), 4.36 (t, 2H), 4.10–3.95 (m, 4H), 3.53 (d, 2H),
3.08–2.30 (m, 8H), 2.05 (t, 2H), 2.03–1.88 (m, 2H), 1.87–1.65 (m,
1H), 1.60–1.37 (m, 5H), 0.88 (t, 3H).
4.5. Synthesis of cyclopeptides
General procedure. The linear peptides were synthesized on so-
lid-phase starting from Fmoc-Gly-SasrinÒ. Protected amino acids
were added to the resin in the following order: Fmoc-Arg(Pmc)-
OH, psuedoamino acid, Fmoc-D-Phe-OH, Fmoc-Asp(OtBu)-OH. Each
4.2.2. Conjugate 7b
coupling step was monitored for completion by the Kaiser test.
Fmoc deprotection was carried out with 20% piperidine. Cleavage
from the resin was performed with 1% TFA in DCM; the suspension
of the resin was stirred for 15 min at room temperature, filtered,
the filtrate immediately neutralized with pyridine and this opera-
tion repeated for 5 times. The collected filtrates were concentrated
under vacuum until 5% of the initial volume and the final linear
peptide isolated by precipitation with cold water. After drying,
the residue was dissolved with acetonitrile containing 1% DIEA to
obtain a 1.5 ꢀ 10ꢁ3 M solution. Three equivalents of TBTU/HOBT
were added and the solution stirred for 1 h. After evaporation of
the acetonitrile, the residue was dissolved with DCM and washed
(water, 0.1 M HCl, water). Solvent was evaporated and the totally
protected cyclopeptides were purified by flash chromatography.
Total yield 61% with 95% HPLC purity (38% CH3CN in H20 + 0.1%
TFA): Rt = 3.20 and 3.98 min; Maldi mass = 1168.24 [MH]+; 1H NMR
(DMSO-d6) d: 9.30 (s, 1H), 8.62 (m, 1H), 7.61–8.40 (m, 11H), 7.35 (s,
1H), 6.82–7.25 (m, 12H), 6.55 (s, 1H), 5.30–5.50 (m, 4H), 4.02–4.60
(m, 10H), 3.50 (m, 2H), 2.20–3.25 (m, 12H), 1.20–1.95 (m, 6H), 0.89
(t, 3H).
4.2.3. Conjugate 7c
Total yield 72% with 97% HPLC purity (34% CH3CN in H2O + 0.1%
TFA): Rt = 6.6 and 9.33 min; ESI mass = 1272.27; 1H NMR (DMSO-
d6) d 9.39 (s, 1H), 8.68 (d, 1H), 8.35 (d, 1H), 8.01 (t, 1H), 7.87 (t,
1H), 7.48 (s, 1H), 7.23–7.12 (m, 9H), 5.50 (d, 4H), 4.65 (m, 1H),
4.51 (t, 2H), 4.36–4.10 (m, 7H), 4.01 (d, 4H), 3.75–3.59 (m, 10H),
3.19 (t, 2H), 3.10–2.68 (m, 6H), 2.02–1.90 (m, 2H), 1.89–1.40 (m,
4H), 0.99 (t, 3H).
4.5.1. c[Arg(Pmc)-Gly-Asp(OtBu)-D
-Phe-Aad-OH] (2a0)
The protected cyclopeptide was purified by flash chromatogra-
phy (DCM/MeOH 95:5?92:8), total yield 47.5%. 713 mg
(0.69 mmol) were dissolved with 30 mL DMF/MeOH 1:1, under
Ar, and selectively deprotected by Catalytic Transfer Hydrogena-
tion (CTH), with ammonium formate (3.45 mmol) and 0.8 g of
Pd/C 10%, during 2 h. The suspension was filtered through Celite,
the filtrate evaporated to dryness and the residue triturated several
times with water. The title compound (650 mg) was obtained as a
white solid, 99% purity, with 91% yield. HPLC (50% CH3CN in
H2O + 0.1% TFA,): Rt = 16.49 min; ESI mass: [MꢁH+] 939.4. 1H
NMR (DMSO-d6) d: 8.16 (br, 1H), 8.05–7.80 (m, 3H), 7.65 (d, 1H),
7.25–7.15 (m, 5H), 6.90 (br, 1H), 6.51 (br, 2H), 4.58 (q, 1H), 4.50
(q, 1H), 4.10–3.85 (m, 4H), 3.10–2.25 (m, 16H), 2.08 (t, 2H), 2.02
(s, 3H), 1.80–1.35 (m, 8H), 1.34 (s, 9H), 1.21 (s, 6H).
4.3. Synthesis of the conjugates 8a and 8b
To a solution of the cyclopeptide 2d in MeOH, CPT aldehydes
were added under stirring and the solution kept at room tempera-
ture under N2 for 36 h. Dilution with diethylether gave a precipi-
tate, which was filtered and repeatedly triturated with
diethylether/DCM to give the final conjugates.
4.3.1. Conjugate 8a
Yield 52% with 92% HPLC purity (28% CH3CN in H2O):
Rt = 13.33 min; Maldi mass = 1228.13 [MH]+; 1H NMR (DMSO-d6)
d: 8.09–8.40 (m, 7H), 7.78–8.00 (m, 5H), 7.84 (s, 1H), 6.81–7.75
(m, 13H), 6.55 (s, 1H), 5.39–5.55 (m, 4H), 3.85–4.70 (m, 16H),
3.75–2.78 (m, 10H), 2.03–1.02 (m, 8H), 0.90 (t, 3H).
4.5.2. c[Arg(Pmc)-Gly-Asp(OtBu)-
D
-Phe-Amp(CO–CH2–CH2–
-Phe-
COOH)] (2b0)
4.3.2. Conjugate 8b
Yield 88% with 86% HPLC purity (28% CH3CN in H2O):
Rt = 8.87 min; Maldi mass = 1255.77 [MH]+; 1H NMR (DMSO-d6)
d: 8.45–8.11 (m, 7H), 7.96–7.63 (m, 5H), 7.35 (s, 1H), 7.25–6.80
The protected cyclopeptide c[Arg(Pmc)-Gly-Asp(OtBu)-
D
Amp(Cbz)] was selectively deprotected with CTH as described for
2a0, and the product purified on preparative HPLC (50% CH3CN in

A. Dal Pozzo et al. / Bioorg. Med. Chem. 18 (2010) 64–72
71
H2O + 0.1% TFA) with 44% yield. 120 mg (0.11 mmol) of the latter
were dissolved with 4 mL of DCM/DMF 2:1, succinic anhydride
(11 mg, 0.11 mmol) and TEA (0.11 mmol) were added and the
reaction mixture stirred at room temperature for 1 h. After dilution
with 30 mL DCM, the solution was washed with water (4 ꢀ 20 mL)
and taken to dryness. 100 mg of a white solid were obtained with
84.7% yield, HPLC (50% CH3CN in H2O + 0.1% TFA): Rt = 11.75 min;
Maldi mass [MH]+: 1074.47, 1H-NMR (DMSO-d6) d: 8.30–8.25 (m,
2H), 8.01 (m, 2H), 7.87–7.79 (m, 2H), 7.17–7.03 (m, 9H), 6.79 (b,
1H), 6.51 (br, 1H), 4.60 (q, 1H), 4.46 (q, 1H), 4.32 (q, 1H), 4.22 (d,
2H), 4.07–3.99 (m, 2H), 3.22 (s, 1H), 3.05–2.31 (m, 14H), 2.43 (s,
6H), 2.01 (s, 3H), 1.75 (t, 2H), 1.73–1.30 (m, 4H), 1.34 (s, 9H),
1.24 (s, 6H).
with EtOAc (5 ꢀ 30 mL) to remove organic impurities, then acidi-
fied with cold 0.5 N HCl to pH 2 and extracted with EtOAC
(5 ꢀ 250 mL). 10.2 g of trioxaundecanedioic acid mono-Boc-hydra-
zide were obtained with 76% yield.
To the latter monoacid (8.47 g, 25 mmol), dissolved with
260 mL DCM at 0 °C, hydroxysuccinimide (3.47 g, 30.2 mmol) and
DCC (6.24 g, 30.2 mmol) were added. After 3 h at room tempera-
ture, the suspension was filtered, washed with water (3 ꢀ 40 mL)
and solvent evaporated to give the activated ester with 86% yield.
This ester was dissolved with 135 mL DCM together with TEA
(26 mmol) and added to a solution containing Fmoc-Amp-OH
(12.06 g, 22 mmol) in 42 mL in DMF. After 2.5 h at room tempera-
ture, the solvent was evaporated, the residue dissolved with
400 mL DCM and washed with water (1 ꢀ 50 mL), 0.1 N HCl
(1 ꢀ 30 mL) and water (3 ꢀ 50 mL). The crude product was purified
by flash chromatography (DCM/MeOH 95:5?90:10) to give 8.34 g
of pure 6 with 52% yield. HPLC (52% CH3CN in H2O + 0.1% TFA):
Rt = 9.06 min; Maldi mass [M+Na] 756.9; 1H NMR (DMSO-d6) d:
8.65 (m, 1H), 8.09 (t, 1H), 7.94 (s, 1H), 7.86 (d, 2H), 7.64–7.57 (m,
2H), 7.43 (t, 2H), 7.30 (t, 2H), 7.17 (s, 4H), 4.28–4.20 (m, 6H),
3.92 (d, 4H), 3.57–3.55 (m, 8H), 3.10–2.80 (m, 2H), 1.38 (s, 9H).
4.5.3. c[Arg(Pmc)-Gly-Asp(OtBu)-
CH2)2–O–CH2–COOH)] (2c0)
D-Phe-Amp(CO–CH2–(OCH2–
The same cyclopeptide described for 2b0, after selective depro-
tection, (800 mg, 0.737 mmol) and DIEA (2.2 mmol) were dissolved
with DCM/DMF 3:1 and excess of 3,6,9-trioxaundecanedioic acid
(5 g, 24.3 mmol) was added followed by DCC (304 mg,
1.472 mmol). The reaction mixture was stirred at room tempera-
ture overnight, then diluted with 250 mL of CHCl3, washed with
water (6 ꢀ 30 mL), dehydrated and concentrated to dryness. The
crude residue was purified by flash chromatography (DCM/MeOH
7:3?DCM/MeOH 7:3 + 1% AcOH). 628 mg of pure 2c0 were ob-
tained with 72.4% yield. HPLC (50% CH3CN in H2O+0.1% TFA):
Rt = 13.96 min; Maldi mass: [MH]+ 1178, 1H NMR (DMSO-d6) d:
8.04–7.89 (m, 5H), 7.15–6.99 (m, 9H), 4.58 (q, 1H), 4.44–4.30 (m,
4H), 4.11–3.99 (m, 3H), 3.91 (d, 4H), 3.60–3.51 (m, 8H), 3.00 (t,
2H), 2.98–2.28 (m, 14H), 2.01 (s, 3H), 1.76–1.72 (m, 3H), 1.54–
1.45 (m, 1H), 1.40–1.31 (m, 4H), 1.33 (s, 9H), 1.24 (s, 6H).
4.7. Affinity to av integrin receptors
96-Well plates were subjected overnight to coating with 0.5
lg/
mL of integrin vb3 or 1 g/mL of vb5, respectively. On the next
a
l
a
day, the wells were washed and incubation was performed with
0.05 nM 125I-Echistatin in the presence or absence of the sample
compounds. After 3 h incubation and washing, the integrins from
each well were solubilized with 2 N NaOH and the radioactivity
measured on a gamma counter. Non-specific binding was deter-
mined in the presence of 1 lM Echistatin. The affinity of the sam-
4.5.4. c[Arg-Gly-Asp-
CO–NH–NH2)] (2d)
D-Phe-Amp(CO–CH2–(OCH2–CH2)2–O–CH2–
ples was expressed as IC50 SD value (nM), elaborated using
‘ALLFIT’ software.
The protected cyclopeptide was obtained with 45% yield after
flash chromatography (DCM/MeOH 94:6?92:8). Total deprotec-
tion was performed with 95% TFA in water for 1 h. After concentra-
tion to small volume, the product was precipitated with dry and
cool diethylether, affording pure 2d with 99% purity and 97.8%
yield. HPLC (20% CH3CN in H2O + 0.1% TFA): Rt = 9.14 min; Maldi
mass [MH]+ = 870.13. 1H NMR (DMSO-d6 + D2O) d: 7.14–7.09 (m,
5H), 7.02–6.97 (m, 4H), 4.57–4.52 (m, 2H), 4.46–4.44 (m, 2H),
4.26 (s, 2H), 4.00 (d, 1H), 3.95–3.90 (m, 2H), 3.57–3.51 (m, 8H),
3.26 (d, 1H), 3.04 (t, 2H), 2.87–2.64 (m, 5H), 2.38–2.33 (m, 1H),
1.75–1.72 (m, 1H), 1.51–1.47 (m, IH), 1.34–1.32 (m, 2H).
4.8. Cell adhesion assay
96-well plates were pretreated with 5 lg/ml of human vitro-
nectin for 2 h at room temperature. Vitronectin was removed and
1% bovine serum albumin (BSA) was added and the plates left for
1 h at room temperature. Plates were washed with medium culture
without fetal calf serum (FCS). The molecules were assayed at sca-
lar concentrations ranging from 0.1 to 100 lM. Tumor cells were
re-suspended in medium culture containing 1% BSA without FCS
and plated on vitronectin substrate. After 1 h of incubation, the
cells were washed once with PBS. The adhering cells were fixed
with 4% paraformaldehyde solution for 10 min at room tempera-
ture and then stained with a 1% toluidine blue solution for
10 min at room temperature. After staining, the cells were washed
with double-distilled water, dried and solubilized with a 1% SDS
solution, and quantified by means of absorbance on a Victor mul-
tilabel plate counter (Wal-lac) at 600 nM. The effect of tumor cell
4.6. Fmoc-Amp[CO–CH2–(O–CH2–CH2)2–O–CH2–CO–NH–NH2]–
OH (6) (pseudoamino acid incorporated in 2d)
To
a solution of 3,6,9-trioxaundecanedioic acid (22 mg,
100 mmol) and DCC (12.4 g, 60 mmol) in 600 mL of DCM, at 0 °C,
benzyl alcohol (4.32 g, 40 mmol), DMAP (977 mg, 8 mmol) and
TEA (14 mL, 100 mmol) were added and the reaction mixture stir-
red at room temperature for 1 h, then filtered. The filtrate was
washed with water (1 ꢀ 50 mL) 0.1 N HCl (1 ꢀ 50 mL) and water
(3 ꢀ 50 mL). After evaporation of the solvent, the crude residue
was dissolved with 500 mL of dry EtOH and NH2–NH2ꢂH2O
(13.6 mL, 280 mmol) was added. After refluxing for 1 h, the solu-
tion was evaporated under vacuo until excess hydrazine was re-
moved. The residue was dissolved with 300 mL MeOH containing
50 mL of 1 N NaOH; then a solution of (Boc)2O (26 mg, 120 mmol)
in 30 mL MeOH was added dropwise and the reaction mixture stir-
red at room temperature for 4 h, maintaining the pH around 8 with
additions of NaOH. After evaporation of methanol, the residue was
dissolved with 300 mL of EtOAc and extracted with water
(2 ꢀ 100 mL). The collected aqueous phases were washed back
adhesion was expressed as IC50 SD value (
‘ALLFIT’ software.
lM), elaborated using
4.9. Drug uptake
6.2 ꢀ 105 A-498 cells were seeded in 100 ꢀ 20 mm cell culture
dishes. After 24 h, cells were treated for several times (5 h, 24 h,
48 h and 72 h) using as dose their IC50 evaluated after 72 h of treat-
ment. At each time point, each drug-containing medium was col-
lected and stored at ꢁ20 °C, and cells were washed with
phosphate buffered saline (PBS), collected by scraping, and centri-
fuged at 1000g at 4 °C for 100. Pellets were re-suspended in 100
lL
of PBS and lysed with five cycles of freeze-and-thaw (3 min each).
After centrifugation at 14,000g for 10 min, supernatant was placed

72
A. Dal Pozzo et al. / Bioorg. Med. Chem. 18 (2010) 64–72
in new eppendorf tubes and processed adding 200
l
L of methanol/
were kept on ice for 10 min and then centrifuged at 4000g for
10 min at 4 °C. Supernatants were analyzed by HPLC-FL as de-
scribed above. All data were expressed as mean (ng of drug/mL
plasma or ng/g tissue) SE.
acetonitrile mixture (1:1 v/v). After vortexing, samples were placed
on ice for 10 min, then centrifuged at 14,000g for 200 at 4 °C and
supernatants were transferred into auto-sampler vials. Protein
concentration of cell lysates was determined using Comassie
(Bradford) Protein Assay kit (Pierce). Drug containing cell culture
Acknowledgements
medium was processed as described adding 700 lL of extraction
mixture. Supernatants were analyzed by HPLC-FL (Beckman Instru-
ments) and analytical data were acquired and processed by a com-
puter system (32Karat Software, Beckman-Coulter). The separation
was performed by isocratic elution (30% CH3CN, 70% 0.1 M AcOH
containing 0.1%TEA, pH ꢃ3.5) with a flow rate of 1 mL/min on a
This work was supported by Sigma-Tau, Roma, Italy. We thank
Patrizia Tobia and Matilde Cati for technical assistance.
Supplementary data
discovery HS-F5 column, 100 ꢀ 4.6 mm,
5 lm (Supelco). The
Figures S1 and S2 describing decreasing rates of 8a at different
pHs. Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2009.11.019.
detection was performed with a spectrofluorometric detector
(RF-10AXL, Shimadzu) at kex 370 nm and kem 510 nm. All data were
expressed as mean (nmol of drug/mg of total proteins) SE.
References and notes
4.10. Cytotoxicity assay in vitro
1. Jaracz, S.; Chen, J.; Kuznetsova, L. V.; Ojima, I. Bioorg. Med. Chem. 2005, 13, 5043.
2. Chari, R. V. J. Acc. Chem. Res. 2008, 41, 98.
3. Hamann, P. R.; Hinman, L. M.; Hollander, I.; Beyer, C. F.; Lindh, D.; Holcomb, R.;
Hallett, W.; Tsou, H.; Upeslacis, J.; Shochat, D.; Mountain, A.; Flowers, D. A.;
Bernstein, I. Bioconjugate Chem. 2002, 13, 47–58.
4. Nagy, A.; Schally, A. V.; Armatis, P.; Szepeshazi, K.; Halmos, G.; Kovacs, M.;
Zarandi, M.; Groot, K.; Miyazaki, M.; Jungwirth, A.; Horvath, J. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 7269.
Tumor cells were seeded in 96-well tissue culture plates. The
day after, different concentrations of drugs were added to each
well. The plates were incubated for 72 h. The number of surviving
cells was determined by staining with sulforodamine B. The cyto-
toxic activity of the compounds was expressed as IC50 SD value
(
l
M), elaborated using ‘ALLFIT’ software.
5. Nagy, A.; Schally, A. V.; Halmos, G.; Armatis, P.; Cai, R. Z.; Csernus, V.; Kovacs,
M.; Koppan, M.; Szepeshazi, K.; Kahan, Z. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
1794.
6. Safavy, A.; Raisch, K. P.; Khazaeli, M. B.; Buchsbaum, D. J.; Bonner, J. A. J. Med.
Chem. 1999, 42, 4919.
4.11. Nude mouse xenograft assay
A2780 ovarian carcinoma cells (3 ꢀ 106/mouse) were implanted
subcutaneously into right flank of nude mice on day 0. Compound
7c was dissolved in 12% 1-methyl-2-pyrrolidinone and was intra-
peritoneally administered at a concentration of 48 mg/kg bis in
die every fourth day for four times (q4 d ꢀ 4) or subcutaneously
bis in die every day for 5 days (qd ꢀ 5/w) in a volume of 10 mL/
kg, starting two days after tumor injection. Tumor volumes TV
(mm3) were estimated as d ꢀ D/2, where d and D are the shortest
diameter and the longest diameter measured in mm upon 16 or
17 days after tumor injection. To determine toxicity of the com-
pound, the body weight of tumor-bearing mice was also measured.
All data are presented as means SE. Mice were sacrificed when
tumor volumes reached around 2 cm3. The statistical differences
compared with vehicle-treated group were calculated by Mann–
Whitney’s test. Animal experiments were performed under the
permission in accordance with European Directives no. 86/609,
and with Italian DL 116, January 27th, 1992.
7. Fuselier, J. A.; Sun, L.; Woltering, S. N.; Murphy, W. A.; Vasilevich, N.; Coy, D. H.
Bioorg. Med. Chem. Lett. 2003, 13, 799.
8. Moody, T. W.; Mantey, S. A.; Pradhan, T. K.; Schumann, M.; Nakagawa, T.;
Martinez, A.; Fuselier, J. A.; Coy, D. H.; Jensen, R. T. J. Biol. Chem. 2004, 279,
23580.
9. Pierschbacher, M. D.; Ruoslathi, E. Nature 1984, 309, 30.
10. Brooks, P. C.; Clarks, R. A.; Cheresh, D. A. Science 1994, 264, 569.
11. Molema, G. Pharm. Res. 2002, 19, 1251.
12. Cristofanilli, M.; Charnsangavey, C.; Hortobagyi, G. N. Nat. Rev. Drug Disc. 2002,
1, 415.
13. Arap, W.; Pasqualini, R.; Ruoslathi, E. Science 1998, 279, 377.
14. de Groot, F. M. H.; Broxterman, H. J.; Hans, P. H. M.; Adams, H. P.; van Vliet, A.;
Tesser, G. I.; Elderkamp, Y. W.; Schraa, A. J.; Kok, R. J.; Grietje Molema, G.;
Pinedo, H. M.; Scheeren, H. W. Mol. Cancer Ther. 2002, 1, 901.
15. Curnis, F.; Gasparri, A.; Sacchi, A.; Longhi, R.; Corti, A. Cancer Res. 2004, 64,
565.
16. Chen, X.; Plasencia, C.; Hou, Y.; Neamati, N. J. Med. Chem. 2005, 48, 1098.
17. Temming, K.; Meyer, D. L.; Zabinski, R.; Dijkers, E. C.; Poelstra, K.; Molema, G.;
Kok, R. J. Bioconjugate Chem. 2006, 17, 385.
18. Ryppa, C.; Mann-Steinberg, H.; Fichtner, I.; Weber, H.; Satchi-Fainaro, R.;
Biniossek, M. L.; Kratz, F. Bioconjugate Chem. 2008, 19, 1414.
19. Dallavalle, S.; Merlini, L. In Modern Alkaloids; Fattorusso, E., Taglialatela, O.,
Eds.; Wiley-VCH: Weinheim, 2008; pp 503–520.
20. Dallavalle, S.; Delsoldato, T.; Ferrari, A.; Merlini, L.; Penco, S.; Carenini, N.;
Perego, P.; De Cesare, M.; Pratesi, G.; Zunino, F. J. Med. Chem. 2000, 43, 3963.
21. Dallavalle, S.; Ferrari, A.; Merlini, L.; Penco, S.; Carenini, N.; Perego, P.; De
Cesare, M.; Pratesi, G.; Zunino, F. Bioorg. Med. Chem. Lett. 2001, 11, 291.
22. Dallavalle, S.; Ferrari, A.; Biasotti, B.; Merlini, L.; Penco, S.; Gallo, G.; Marzi, M.;
Tinti, M. O.; Martinelli, R.; Pisano, C.; Carminati, P.; Carenini, N.; Beretta, G.;
Perego, P.; De Cesare, M.; Pratesi, G.; Zunino, F. J. Med. Chem. 2001, 44,
3264.
23. Haubner, R.; Finsinger, D.; Kessler, H. Angew. Chem., Int. Ed. 1997, 36, 1374.
24. Dal Pozzo, A.; Penco, S.; Giannini, G.; Tinti, O.; Pisano, C.; Vesci, L.; Ni, M.H. PCT
WO 2005/111064 A1, 2005.
25. Langer, M.; Kratz, F.; Rothen-Rutishauser, B.; Wunderli-Allenspach, H.; Beck-
Sinckinger, A. G. J. Med. Chem. 2001, 44, 1341.
4.12. In vivo plasma concentrations and tumor distribution
Tumors were established in nude mice as described above. Ani-
mals (3 mice/group) were treated intravenously with the conjugate
7c at a dose of 45 mg/kg and sacrificed at different times (0.5, 1, 2,
4, 7, 24 h) after intravenous injection. Plasma (100
lL) was pro-
cessed by adding 400 L of a cold mixture of 0.1% acetic acid and
l
methanol (1:5, vol/vol). Samples were kept on ice for 10 min, then
centrifuged at 1000g for 10 min at 4 °C. Tumors were homogenized
with a Potter using the same extraction mixture and applying a tis-
sue weight/mixture volume ratio of 1:5. Homogenized samples
26. Kale, A. A.; Torchilin, V. P. Bioconjugate Chem. 2007, 18, 363.
27. Eilers, J. D.; Horton, A. L.; Grissom, R. A. J. Org. Chem. 2004, 69, 8987.