Inhibitors of tubulin polymerization: Synthesis and biological evaluation of hybrids of vindoline, anhydrovinblastine and vinorelbine with thiocolchicine, podophyllotoxin and baccatin III

Article abstract of DOI:10.1016/j.bmc.2008.04.025

A series of novel hybrid compounds obtained by the attachment of anhydrovinblastine, vinorelbine, and vindoline to thiocolchicine, podophyllotoxin, and baccatin III are described. Two types of diacyl spacers are introduced. The influence of the hybrid compounds on tubulin polymerization is reported. The results highlight the importance of the length of the spacer. Immunofluorescence microscopy and flow cytometry measurements that compound with the best in vitro activity could disrupt microtubule networks in cell and prevent the formation of the proper spindle apparatus, thereby causing cell cycle arrest in the G2/M phase. The newly synthesized compounds were tested in the human lung cancer cell line A549.

Full text of DOI:10.1016/j.bmc.2008.04.025

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6269–6285
Inhibitors of tubulin polymerization: Synthesis and biological
evaluation of hybrids of vindoline, anhydrovinblastine and
vinorelbine with thiocolchicine, podophyllotoxin and baccatin III
Daniele Passarella,a,* Alessandra Giardini,a Bruno Peretto,a Gabriele Fontana,b
Alessandro Sacchetti,a Alessandra Silvani,a Cristina Ronchi,d Graziella Cappelletti,c
Daniele Cartelli,c Jurgen Borlake,ꢀ and Bruno Danielia,ꢀ
a
`
Dipartimento di Chimica Organica e Industriale, Via Venezian 21, Universita degli Studi di Milano, 20133 Milano, Italy
bIndena S.p.a., Via Ortles 19, Milano, Italy
c
`
Dipartimento di Biologia, Universita degli Studi di Milano, Via Celoria 26, Milano, Italy
`
d
D.I.P.A.V., Sezione di Biochimica, Universita degli Studi di Milano, Via Celoria 10, Milano, Italy
eFraunhofer Institute of Toxicology and Experimental Medicine, Medical School of Hannover, Germany
Received 15 November 2007; revised 4 April 2008; accepted 11 April 2008
Dedicated to Prof. Joan Bosch (Univ. Barcelona) in the occasion of his 60th birthday
Abstract—A series of novel hybrid compounds obtained by the attachment of anhydrovinblastine, vinorelbine, and vindoline to thi-
ocolchicine, podophyllotoxin, and baccatin III are described. Two types of diacyl spacers are introduced. The influence of the hybrid
compounds on tubulin polymerization is reported. The results highlight the importance of the length of the spacer. Immunofluo-
rescence microscopy and flow cytometry measurements that compound with the best in vitro activity could disrupt microtubule net-
works in cell and prevent the formation of the proper spindle apparatus, thereby causing cell cycle arrest in the G2/M phase. The
newly synthesized compounds were tested in the human lung cancer cell line A549.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ing the aggregation (colchicines, vincristine, and vinblas-
tine), while others inhibit the depolymerization of
tubulin (paclitaxel and epothilones). Furthermore, it
has been recently established that some agents interact-
ing with tubulin are also able to target the vascular sys-
tem of tumors1 causing vasculature recession (induction
of apoptosis by a classical anti-angiogenic mechanism)
or vasculature ‘normalization’. 2 The vasculature nor-
malization favors the cell penetration of anticancer
drugs, and apoptosis can be induced by administration
of a combination of drugs. The structural diversity of
the antitubulin drugs reflects the diversity of the mecha-
nisms of interaction with tubulin.3 For example, the bin-
ary Vinca alkaloids vincristine, vinblastine, and the
semisynthetic vinorelbine (Fig. 1) induce the destabiliza-
tion of polymerized tubulin, by binding to a site local-
ized on b-tubulin.4 Taxanes, and in particular
paclitaxel5 (Fig. 1), lower the dissociation constant at
both the plus and minus end of the microtubules, thereby
promoting microtubule assembly and reducing the
amount of free tubulin in the cell. Colchicine6 (Fig. 1)
Microtubules are complex polymeric structures gener-
ated by the circular arrangement of linear polymers
(protofilaments) of tubulin, a dimeric protein formed
by the combination of two non-identical chains
(a- and b-tubulin). As components of the mitotic spin-
dle, microtubules have emerged as a strategic target in
anticancer drug discovery. The discovery of substances
able to inhibit the formation or the disaggregation of
microtubules has always been regarded as a great
achievement in the fight against cancer. Among natural
products, a few substances have been discovered that are
able to interfere with microtubules, and therefore block
the cell cycle. Some of these compounds act by inhibit-
Keywords: Bivalent inhibitors; Tubulin polymerization; Vinca
alkaloids.
*
Corresponding author. Tel.: +39 02 50314080; fax: +39 02 50314078;
e-mail: Daniele.Passarella@unimi.it
Contributed equally as senior authors.
ꢀ
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.025

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D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
OH
N
N
N
N
H
H
N
H
N
H
H
H
MeOOC
MeOOC
N
Me
OAc
COOMe
MeO
O Ac
COOMe
N
R
MeO
H
HO
H
HO
Vinorelbine
(Navelbine)
Vinblastine R = CH3
Vincristine R = CHO
O
OH
MeO
MeO
Ph
O
O
H
N
NH
HO
AcO
OH
O
H
O
O
OH
COCH3
H
Ph
O
O
OMe
O
O
OAc
MeO
OMe
OBz
XMe
X = O Colchicine
OMe
Paclitaxel
Podophyllotoxin
X = S Thiocolchicine
Figure 1. Well-known drugs that disrupt the dynamic tubulin-microtubule equilibrium.
induces various effects on tubulin, the most important of
which is the modification of the secondary structure of
the protein, followed by inhibition of tubulin
polymerization.
antimitotic compounds. Here we report the synthesis
of hybrids with anhydrovinblastine and vinorelbine19
as scaffolds and tiocolchicine, podophyllotoxin and
baccatin III as reagents separated by succinyl and deca-
noyl spacers. The chemistry involved was rather com-
plex. In the case of vinorelbine we could obtain two
hybrids by direct attachment of the spacer at position
17 of deacetylvinorelbine, while in the case of the hy-
brids based on anhydrovinblastine we had first to func-
tionalize vindoline portion at position 17 and then to
introduce the ‘upper’ carboxyvelbanamine part. There-
after we tested the efficacy of the different hybrids
against the polymerization of tubulin. This enabled us
to compare not only anhydrovinblastine and vinorelbine
but also vindoline. In addition, we studied the antican-
cer activity of the hybrids in the human lung cancer cell
line, A549. For one of new compounds we investigated
the effects of inhibition of tubulin polymerization on cell
cycle regulation and the structure and distribution of
microtubules in cultured cells via immunofluorescence
microscopy.
Such an effect has been explained through the strong
binding to the ‘colchicine site’ of tubulin that induces
an alteration of the secondary structure of b-tubulin at
the carboxy terminal.7 The same mechanism has been
reported for thiocolchicine8 and podophyllotoxin9
(Fig. 1). Despite the great potential of antitubulin drugs,
therapy based on antimitotic agents is still associated
with undesired side effects10 which have a negative im-
pact on patient compliance. The side effects are usually
associated with the lack of the selectivity.11 The discov-
ery of selective, non-toxic, and drug-like active princi-
ples is therefore still an urgent demand.
Among the different approaches to the design of new
antitubulin agents12 we explored the possibilities offered
by the concept of multivalency,13 an area which has been
reviewed by Whitesides.14 The theory of multivalency is
based on the observation that in Nature the activity and
selectivity of some lead compounds are enhanced
through the formation of their homo- or heterodimers.15
As a consequence, the concept of multivalency can be
successfully applied as an effective approach for design-
ing ligands, inhibitors, and drugs.
2. Results and discussion
The subsequent Scheme highlights the general strategy
that has been used for the synthesis of the hybrid com-
pounds. The synthesis are discussed below.
During our research on the synthesis of antitumor com-
pounds,16 a few years ago we reported the synthesis of
taxoid–thiocolchicine hybrids17 and more recently a dy-
namic combinatorial library of thiocolchicine–podo-
phyllotoxin derivatives.18 From a mechanistic point of
view, we have shown that these hybrids of antitubulin
agents behave as multifunctional tubulin inhibitors.
The compounds served as probes to investigate the biol-
ogy of tubulin and the complex relationships between
tubulin inhibition and cytotoxicity. The results obtained
encouraged us to develop a broad program directed to-
ward the synthesis of hybrids of naturally occurring
2.1. Preparation of vindoline-based hybrids (vindoline-C4-X)
(8, 9, and 10, Fig. 3)
17-Deacetylvindoline 1a (Fig. 2), obtained by careful
hydrolysis of vindoline with K2CO3 in MeOH, was re-
acted with succinic anhydride in the presence of pyri-
dine to give the 17-hemisuccinate 1b that was used as
starting material for the preparation of several adducts
(path A). The presence of the succinic spacer was
confirmed by HR-ESI-MS data and by the chemical
shift of H-17 that appeared at d 5.25 with respect to
d 4.17 in 17-deacetylvindoline. The reaction of 1b with

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
6271
20'
15'
N
N
H
N
N
N
H
H
N
H
N
H
17
COOMe
H
H
MeOOC
O
R
N
MeO
MeOOC
17
COOMe
17
COOMe
H
HO
Me
N
Me
O
R
O
R
MeO
N
MeO
H
HO
H
HO
Me
3
R = COCH3
3a R = H
1
R = COCH3
1a R = H
1b R = CO(CH2)2COOH
2
R = COCH3
2a R = H
3b R = CO(CH2)2COOH
R
AcO
O
O
OR'
MeO
MeO
4
H
N
O
N
O
R
7
O
13
O
H
R
N
H
O
H
O
OMe
OH
OAc
OBz
COOMe
O
7
MeO
OMe
6
R = H
R' = H
SCH3
6a R = H
R' = TES
OMe
4
R = COCH3
4a R = H
4b R = CO(CH2)8COOH
5
R = H
6b R = CO(CH)2COOCH2CCl3 R' = TES
5a R = CO(CH2)2COOH
5b R = CO(CH2)8COOCH2CCl3
5c R = CO(CH2)8COOH
6c R = CO(CH2)2COOH
6d R = CO(CH2)8CH=CH2 R' = TES
6e R = CO(CH2)8COOH R' = TES
R' = TES
Figure 2. Chemical structure of compounds 1–7.
deacetylthiocolchicine in the presence of DCC and
DMAP permitted the formation of the first vindoline–
thiocolchicine adduct 8 (Fig. 3). The introduction of
the thiocolchicine nucleus was confirmed by the appear-
the thiocolchicine moiety and in particular by the mul-
tiplet signal due to H-7 of the thiocolchicine nucleus in
the range 4.68–4.59 ppm (thiocolchicine d 4.35). The
same type of condensation reaction was used to gener-
ate the vindoline–podophyllotoxin adduct 9. In this
case diagnostic change in the NMR spectrum was the
chemical shift of the H-4 of the acylated podophyllo-
toxin that occurred in the region between d 6.05 and
5.98, overlapping the dioxymethylene protons, in com-
parison to d 4.85–4.77 in podophyllotoxin. Compound
9 was obtained in better yield by reaction of podophyl-
lotoxin with succinic anhydride to give derivative 5a
and subsequent introduction of the vindoline nucleus
by acylation (Scheme 1, path B). A different approach
was necessary for the preparation of vindoline–baccatin
adduct 10 as a consequence of the particular reactivity
of OH-13 of baccatin III. 7-TES-baccatin 6a20 was first
reacted with trichloroethylsuccinic acid to give the cor-
responding derivative 6b that was deprotected with
activated Zn under acidic conditions to give the carbox-
ylic derivative 6c.21 The usual condensation reaction
with deacetylvindoline 1a gave the vindoline–baccatin
adduct 10 in 61% yield. The chemical shifts of the
signals due to H-17 (d 5.43) and H-130 (d 6.20–6.15)
confirmed the presence of the diacyl spacer at the
expected positions.
1
ance of the H NMR spectrum of the typical signals of
N
H
OMe
O
O
H
17
7'
O
OMe
N
H
N
( CH2)n
MeO
H
COOMe
HO
Me
OMe
8
n = 2
11 n = 8
O
SCH3
N
H
O
O
O
O
N
( CH2)n
MeO
H
COOMe
O
O
HO
Me
4'
O
O
9
n = 2
12 n = 8
OMe
O
MeO
OMe
AcO
2.2. Preparation of vindoline-based hybrids (vindoline-
C10-X) (11, 12, and 13, Fig. 3)
N
H
OTES
We were unable to prepare in acceptable yield 17-hemi-
sebacoylvindoline from 17-deacetylvindoline and seba-
cic acid and therefore for the synthesis of the
heterodimers with a C10 spacer we used the alternative
strategy developed before, based on the introduction
of the vindoline portion on the preformed hemisebacoyl
derivatives of podophillotoxin, N-deacetylthiocolchi-
cine, and 7-TES-baccatin (4b, 5c, and 6e, respectively,
O
O
O
13'
OH
O
N
( CH2)n
MeO
H
COOMe
O
HO
Me
H
OAc
OBz
10 n = 2
13 n = 8
Figure 3. Vindoline-based derivatives 8–13.

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D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
N
H
O
O
O
N
OH
MeO
(CH2)n
H
COOMe
HX
HO
Me
1b
path A
O
O
N
H
X
HO
(CH2)n
17
OR
N
MeO
R = COCH3
path B
path C
7
H
COOMe
HO
Me
1
1a R = H
N
N
N
H
H
N
H
H
O
O
MeOOC
X
O
N
MeO
(CH2)n
OAc
COOMe
N
MeO
H
2
H
COOMe
HO
Me
HO
Me
8 - 13
N
N
H
7
COOMe
N
N
N
N
H
H
N
H
H
N
H
H
O
O
MeOOC
MeOOC
MeO
O
X
N
Me
O
R
N
MeO
(CH2)n
H
H
COOMe
COOMe
HO
Me
HO
3
R = COCH3
14 - 16
3a R = H
N
H
N
N
N
H
HX
H
N
N
H
H
H
MeOOC
MeOOC
O
O
O
O
N
Me
X
MeO
N
Me
O
COOMe
MeO
O
OH
(CH2)2
(CH2)2
H
HO
H
HO
COOMe
3b
18, 19
Scheme 1. General strategy for the preparation of hybrid compounds 8–19. For the particular structures see Figures 3–5 and for HX
Figure 2.
see
Fig. 2). The hemisebacoyl derivative of podophillotoxin
(5c) was obtained by condensation of 5 with the mono-
trichloroethylester of sebacic acid and subsequent
hydrolysis of the trichloroethyl ester (5b) in the presence
of activated Zn. In the case of thiocolchicine this proce-
dure was not possible due to the sensitivity of thiocolch-
icine to acidic treatment. For this reason we had to
accomplish the preparation of the desired adduct by
monoamination of the sebacoyl acid with N-deac-
etylthiocolchicine to give 4b, which was then submitted
to a condensation reaction with deacetylvindoline to
give 11. 7-TES-13-sebacoylbaccatin III 6e was obtained
by NaIO4–RuCl 3 oxidation22 of the corresponding 7-
TES-13-undecenylbaccatin III 6d.
2.3. Preparation of anhydrovinblastine-based hybrids
(anhydrovinblastine-C4-X) (14, 15, and 16, Fig. 4)
Conceivably, the access to adducts that incorporate the
anhydrovinblastine (AVBL) entity could be obtained

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
6273
20'
N
15'
N
H
N
H
OMe
OMe
H
O
O
H
MeOOC
O
N
H
N
( CH2)n
MeO
H
COOMe
HO
Me
OMe
14 n = 2
17 n = 8
O
SCH3
N
N
H
N
H
H
O
O
MeOOC
O
O
N
( CH2)2
MeO
H
COOMe
O
O
HO
Me
O
O
15
OMe
AcO
MeO
OMe
N
N
H
O
OTES
N
H
H
O
O
MeOOC
O
O
N
( CH2)2
MeO
H
HO
COOMe
O
Me
H
OH
OAc
OBz
16
Figure 4. Anhydrovinblastine-based derivatives 14–17.
by acylation of the 17-deacetylanhidrovinblastine. This
type of approach is problematic due to the instability
of the C200–C150 double bond to many experimental
conditions. For this reason we chose to introduce the
upper portion (the carbomethoxyvelbanamine nucleus)
deriving from 7, on the vindoline-adducts (8, 9) using
the biomimetic approach studied by Szantay and
Kutney that we successfully applied to the synthesis
of vinblastine analogs.16 The use of FeCl3 in glycine-
buffered solution induces an oxidative coupling that
proceeds in a stereocontrolled way. The proper stereo-
chemistry at position C160 of the heterodimers 14 and
15 thus obtained was confirmed by considering the
signs for the Cotton effect curves that were as expected
for the natural configuration (negative in the range
205–210 and positive in the range 220–235).23 Applica-
tion of the same methodology for the preparation of
compound 16 was unsuccessful, probably as a result
of the low solubility of the substrate 10. For this rea-
son we turned instead to a Polonowsky–Potier reac-
tion.24 The use of m-CPBA to generate the N-oxide
of catharanthine and the subsequent reaction with
compound 10 in the presence of TFAA generated the
expected compound 16 after the addition of NaBH4.
Compounds 14, 15, and 16 were fully characterized
spectroscopically. Despite the interesting chemical re-
sult obtained for the introduction of the velbanamine
nucleus on complex substrates, we were disappointed
to note that compounds 14, 15, and 16 were insuffi-
ciently stable to be stored for longer than 2 or 3 days,
probably as a consequence of the sensitivity of C150–
C200 double bond.
2.4. Preparation of anhydrovinblastine-based hybrids
(anhydrovinblastine-C10-X) (17, Fig. 4)
The reaction of 11 with catharanthine in the presence
of FeCl3 in glycine-buffered solution induced the for-
mation of compound 17. Unfortunately compound
17 suffered from the same instability as compounds
14–16.
2.5. Preparation of vinorelbine-based hybrids (vinorelbine-
C4-X) (18 and 19, Fig. 5)
The semisynthetic alkaloid vinorelbine (Navelbine)25
appeared an interesting entity to be involved in the for-
mation of bivalent structures with higher stability. We
submitted anhydrovinblastine 2 (path C)26 to reaction
with NBS and TFA at ꢀ60 ꢁC to generate the bromoin-
dolenine on the indole nucleus of the velbanamine upper
part. The subsequent rearrangement that resulted in the
loss of a carbon atom between N40 and C70 was induced
by reaction with AgBF4.27 Vinorelbine was then sub-
mitted to reaction with Na2CO3 in MeOH to remove
the acetyl group from OH-17 to give 3a. The usual reac-
tion with succinic anhydride gave 3b. The subsequent re-
action with podophyllotoxin and thiocolchicine gave,
respectively, compounds 18 and 19, which were stable
and easy to manipulate.

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D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
N
N
H
N
H
OMe
OMe
H
O
O
MeOOC
H
O
N
MeO
N
H
( CH2)2
H
HO
COOMe
Me
OMe
18
O
SCH3
N
N
H
N
H
H
O
O
MeOOC
O
N
MeO
O
( CH2)2
H
HO
COOMe
Me
O
O
O
O
19
OMe
MeO
OMe
Figure 5. Vinorelbine-based derivatives 18–19.
3. Biological evaluation
inhibited tubulin polymerization whereas 11 was almost
ineffective. Analogously, the dimers 13 and 10 contain
vindoline and 7-TES-baccatin moieties but the dimer
13 inhibited tubulin polymerization whereas 10 was al-
most ineffective. We report in Table 1 the ratios of unpo-
lymerized/polymerized tubulin obtained in the presence
of the different compounds. By comparing the results,
we confirm the significant effect of the dimers 8 and 13
on tubulin polymerization.
To get an insight into the potential biological activity of
the novel heterodimers we investigated their ability to
affect tubulin polymerization in vitro. In order to assess
the effect of the compounds on tubulin assembly, por-
cine tubulin (purified from brain) was mixed with a stan-
dard solution of each sample in the absence of GTP. The
solutions were then incubated at 37 ꢁC, and after 15 min
GTP was added to allow slow binding drugs to bind to
the tubulin. After 30 min the polymerized and the unpo-
lymerized fractions were separated by centrifugation
and analyzed by densitometry.
Furthermore, the anticancer activities of the new com-
pounds (Fig. 9) were examined in human lung carci-
noma A549.30 All the new compounds exerted
The behavior of the monomers was assumed as baseline
(Fig. 6). High red bars mean high content of non-poly-
meric tubulin, while high blue bars stand for high con-
tent of polymeric tubulin. By analyzing the different
moieties (Fig. 6) we confirmed that thiocolchicine, pod-
ophyllotoxin, and vinorelbine induced more or less a sig-
nificant shift of tubulin from the polymerized to the
unpolymerized pool, whereas vindoline appeared to be
unable to affect tubulin polymerization. An unexpected
behavior resulted in the case of 7-TES-baccatin when
compared with the taxane stabilization of the polymer-
ized form.28 The test compounds 8–13, 18 and 19 were
then analyzed. In the conditions of the assay compounds
14–17 were not stable enough to be tested.29 The results
are summarized in Figure 7. These experiments show
that a significant inhibition of tubulin polymerization
occurred in the presence of compounds 8 and 13. Inter-
estingly, heterodimers built up with the same moieties
connected by different spacers showed different effects
on tubulin polymerization. The dimers 8 and 11 contain
vindoline and thiocolchicine nucleus but compound 8
Figure 6. Tubulin polymerization in the presence of 10 lM thiocolch-
icine (4), podophyllotoxin (5), vinorelbine (3) or vindoline (1); (c):
control. In red unpolymerized fraction, in blue the polymerized
fraction. Bars indicate SEM (standard error of mean). *P < 0.05,
**
P < 0.001 versus control, according to Student’s unpaired t-test.

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
6275
accord with the trend of their influence on tubulin poly-
merization. By contrast, compounds 10 and 13 showed a
similar behavior and this was not the case in the tubulin
polymerization test.
On the grounds that compound 8 displayed structural
simplicity, maintained the ability to inhibit tubulin poly-
merization, and, interestingly, was more effective as an
antiproliferative agent than its moiety thiocolchicine,
we moved to obtain a deeper understanding of its behav-
ior in vitro and in cells. We first studied the effect of
compound 8 on tubulin polymerization at different con-
centrations in comparison with thiocolchicine (Fig. 8).
The ratio of unpolymerized/polymerized tubulin in-
creases in a dose-dependent manner in the presence of
compound 8 and thiocolchicine (see Table 1). In addi-
tion, we followed the kinetics of tubulin polymerization
in the presence of compound 8 and thiocolchicine (Fig.
10a). We found that both the compounds markedly and
similarly interfere with it by lowering the initial rate of
assembly rather than the final extent of assembly. Look-
ing at microtubule morphology, we collected the assem-
bled microtubules at the end of polymerization and
analyzed them by DIC microscopy (Fig. 10b). Our re-
sults show that microtubules assembled in the presence
of compound 8 as well as thiocolchicine are shorter than
controls.
Figure 7. Tubulin polymerization in the presence of 10 lM solution of
hybrid compounds; (c): control. In red unpolymerized fraction, in blue
the polymerized fraction. Bars indicate SEM (standard error of mean).
*P < 0.05, ** P < 0.001 versus control, according to Student’s unpaired
t-test.
antiproliferative activity, albeit at different concentra-
tions. Compound 19 showed the highest cytotoxicity at
10 lM concentration as determined by the MTT assay
and when compared with those of vinorelbine and pod-
ophyllotoxin. Compound 8 was clearly distinguished
from compound 11 despite their structural similarity in
Figure 8. Anticancer activities for vindoline, thiocolchicine, 7-TES-baccatin III, podophyllotoxin, and vinorelbine in human lung carcinoma A549.

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D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
Table 1. Ratio of unpolymerized/polymerized tubulin as obtained
from data presented in Figures 6 and 7
pounds on tubulin polymerization in vitro has been
described. The antiproliferative study on A549 showed
low activities but almost confirmed the trend noticed
in the tubulin polymerization test. Relevant differences
in the behavior of compounds that differ in the length
of the spacer emerged, in particular compound 8 inhibits
the polymerization and 11 is ineffective. Anhydrovin-
blastine derivatives (14–17) were not stable and there-
fore not interesting. On the other hand, vinorelbine
derivatives (18 and 19) were stable enough to be studied
but had low efficacy for the inhibition of tubulin assem-
bly, with an interesting cytotoxicity that suggests a dif-
ferent biological target. Compound 8 was submitted to
further studies in order to confirm its interaction with
tubulins. Dose-dependence and the kinetics of the inhi-
bition of tubulin polymerization together with the influ-
ence on the cell cycle furnished consistent results. The
ability of compound 8 to disrupt microtubules in cells
has been demonstrated by fluorescence microscopy.
Further analyses would be necessary to discover in de-
tail the mechanisms by which this compound affects
tubulin polymerization in vitro. In fact, microtubule-tar-
geted drugs affect microtubule system in several different
ways, including changing microtubule mass by binding
to multiple distinct sites in tubulin dimers or microtu-
bule or by suppressing microtubule dynamics.31 Finally,
the nature and the biological responses of compounds 8
and 19 are the evidence that the creation of conjugates
remains a valuable and not trivial strategy to discover
new interesting activities.
Unpolymerized/polymerized
tubulin (mean SEM)
Control
Thiocolchicine
0.93 0.08
10 lM: 2.55 0.23**
5 lM: 1.34 0.09**
1.68 0.12**
Podophyllotoxin
Vinorelbine
Vindoline
8
1.19 0.02*
0.76 0.01 n.s.
10 lM: 2.21 0.02**
5 lM: 1.20 0.03*
0.79 0.02 n.s.
0.60 0.18 n.s.
0.77 0.11 n.s.
0.78 0.07 n.s.
2.33 0.63**
11
9
12
10
13
18
19
0.74 0.02*
0.79 0.01 n.s.
*P < 0.05, **P < 0.001 versus control according to Student’s unpaired
t-test. Drug concentration 10 lM if not specified.
In order to establish whether the antiproliferative activ-
ity of compound 8 is based on cell cycle arrest due to
damage to the microtubular cytoskeleton we applied a
flow cytometry analysis (Fig. 11). By 24 h, a substantial
number of cells (74%) treated with 10 lM compound 8
were in cell cycle arrest at the G2/M phases. This finding
was highly significant, when compared to the DMSO
vehicle control.
Finally, we investigated microtubule structure and dis-
tribution in human lung carcinoma cell line A549 ex-
posed to vindoline, thiocolchicine and compound 8 by
indirect immunofluorescence using anti-a-tubulin anti-
bodies (Fig. 12). In control cells (Fig. 12a), we observed
a widespread network of long microtubules other than
the typical accumulation of microtubules at one side
of the nucleus in the region called the microtubule orga-
nizing center (MTOC). This conventional microtubule
distribution was not significantly changed in the pres-
ence of vindoline (Fig. 12b) but underwent dramatic
rearrangements in the presence of thiocolchicine (Fig.
12c) and the dimer 8 (Fig. 12d) showing the evident dis-
organization of the network, fragmented microtubules,
and abnormal accumulation in the perinuclear region.
The results of the indirect immunofluorescent detection
of the microtubular cytoskeleton are consistent with the
data coming from the in vitro assay: vindoline is unable
to affect the microtubule system in cells as previously ob-
served in tubulin assembly tests, whereas thiocolchicine
and the hybrid 8 significantly interfere with tubulin
organization in cells as well as with tubulin polymeriza-
tion in vitro.
5. Experimental
All reagents and solvents were of reagent grade or were
purified by standard methods before use. Melting points
were determined in open capillaries on a Buchi melting
point apparatus and are uncorrected. Column chroma-
tography was carried out on flash silica gel (Merck
230–400 mesh). NMR spectra were recorded at 300/
400 MHz (1H) and at 75/100 MHz (13C). FAB+ mass
spectra were recorded at an ionizing voltage of 6 keV.
ESI mass spectra were recorded on FT-ICR instrument.
5.1. Chemistry
5.1.1. Preparation of 17-deacetylvindoline (1a). A mix-
ture of vindoline (1, 505 mg, 1.11 mmol), Na2CO3
(5.53 mmol) in MeOH (16 ml) was stirred for 4 days at
65 ꢁC. After the evaporation of the solvent the residue
was chromatographed on alumina (AcOEt/MeOH/
MeOH 85:15:2) to give 1a (348 mg, 76%). Rf: 0.49
1
(AcOEt/MeOH 8.5:1.5); [a]D ꢀ22 (MeOH, c = 0.6). H
NMR (CDCl3, 300 MHz) selected signals: 6.85 (1H, d,
J = 8), 6.28 (1H, dd, J = 8.2), 6.05 (1H, d, J = 2), 5.85
(1H, dd, J = 11, 4.5), 5.70 (1H, d, J = 11), 4.08 (1H, s),
3.85 (3H, s), 3.78 (3H, s), 3.70 (1H, s), 3.35–3.49 (2H,
m), 2.85 (1H, d, J = 16), 2.70 (3H, s), 2.62 (1H, s),
2.48–2.60 (1H, m), 2.20 (2H, dd, J = 10.5, 7), 1.38–
1.52 (1H, m), 0.91–1.06 (1H, m), 0.67 (3H, t, J = 7).
13C NMR (CDCl3, 75 MHz) selected signals: 174.0,
161.8, 154.8, 131.5, 125.8, 124.4, 123.5, 105,0, 96.5,
83.8, 81.5, 74.6, 68.9, 56.0, 53.6, 52.1, 45.3, 43.5, 39.3,
4. Conclusions
The preparation of 12 hybrid compounds that contain
Vinca alkaloids and other antitubulin entities connected
by a diacyl spacer has been described. A concise biolog-
ical test to evaluate the inhibitory effect of these com-

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
6277
Figure 9. Anticancer activities for compounds 8–13, 18, and 19 in human lung carcinoma, A549.
33.2, 8.5. ESI positive MS: Anal. Calcd for
C23H30O5N2+Na+ 437.2047, found 437.2048.
3.26 (1H, m), 2.80 (1H, d, J = 16), 2.68 (1H, s), 2.62
(3H, s), 2.56–2.40 (5H, m), 2.38–2.28 (2H, m), 1.58–
1.43 (1H, m), 1.07–0.92 (1H, m), 0.45 (3H, t, J = 7.5).
ESI positive MS: Anal. Calcd for C27H34O8N2+Na+
537.2207, found 537.2209.
5.1.2. 17-Deacetyl-17-O-hemisuccinylvindoline (1b). A
mixture of 1a (190 mg, 0.458 mmol), succinic anhydride
(92 mg, 0.917 mmol), and pyridine (8 ml) was stirred for
12 h at room temperature. The solution was concen-
trated. The addition of CH2Cl2, the evaporation, and
the treatment of the residue with MeOH gave after sub-
sequent evaporation a residue that was submitted to
chromatography (AcOEt/MeOH 8:2) to give compound
1b (225 mg, 95%). 1H NMR (300 MHz, CD3COCD3) se-
lected signals: d 7.05 (1H, d, J = 12), 6.30 (1H, dd,
J = 12, 2), 6.20 (1H, d, J = 2), 5.85 (1H, dd, J = 15, 5),
5.25 (1H, s), 5.20 (1H, d, J = 15), 3.74 (3H, s), 3.69
(3H, s), 3.60 (1H, s), 3.45 (1H, dd, J = 16, 5.5), 3.37–
5.1.3. 17-Deacetyl-17-O-(N-deacetyl-N-succinylthiocol-
chicinyl)vindoline (8).
A
mixture of
8
(225 mg,
0.437 mmol), DMAP (53 mg, 0.437 mmol), DCC
(180 mg, 0.875 mmol), and deacetylthiocolchicine 4a
(163 mg, 0.437 mmol) was stirred at room temperature
for 10 h. The mixture was filtered on Celite and submit-
ted to chromatography (AcOEt/MeOH/NEt3 94:6:2) to
give 8 (75 mg, 20%) as a yellow solid. Mp: 165 ꢁC; Rf:
1
0.13 (AcOEt/acetone 6:4). H NMR (400 MHz, CDCl3)
selected signals: d 7.27 (1H, d, J = 9), 7.24 (1H, s), 7.03

6278
D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
Figure 10. (a) Kinetics of microtubule polymerization in the absence and presence of 10 lM each of thiocolchicine and compound 8. Tubulin
(2.7 mg/ml) was polymerized in the absence and presence of 10 lM of thiocolchicine and 8 and the change in absorbance at 340 nm was monitored.
(b) At the end of polymerization, microtubules were pelletted and observed by DIC microscopy (B). Bar = 1 lm.
3.47–3.45 (1H, m), 3.45–3.42 (1H, m), 2.79 (1H, d,
J = 15), 2.65 (3H, s), 2.63 (1H, s), 2.57–2.48 (1H, m),
2.49–2.44 (2H, m), 2.42 (3H, s), 1.92–1.82 (2H, m),
1.64–1.57 (1H, m), 1.13–1.10 (1H, m), 0.35 (3H, t,
J = 8.5). 13C NMR (100 MHz, CDCl3) selected signals:
d 182.8, 173.1, 172.4, 171.2, 161.6, 158.6, 154.1, 153.9,
151.7, 150.9, 142.1, 138.3, 134.8, 134.7, 130.8, 128.9,
126.5, 126.3, 125.5, 124.3, 123.1, 107.7, 104.9, 96.2,
83.8, 79.9, 77.1, 67.5, 61.9, 61.8, 56.5, 55.8, 53.2, 52.7,
52.5, 52.4, 51.5, 44.4, 43.3, 38.6, 37.0, 31.3, 31.2, 30.3,
29.8, 15.5, 8.1. ESI positive MS: Anal. Calcd for
C47H55O11N3S+Na+ 892.3450, found 892.3448.
5.1.4. 4-O-Hemisuccinylpodophyllotoxin (5a). A mixture
of 5 (780 mg, 1.88 mmol), DMAP (92 mg, 0.75 mmol),
imidazole (153 mg, 2.25 mmol), and succinic anhydride
(395 mg, 3.95 mmol) was stirred at room temperature
for 13 h. The mixture was submitted to chromatography
(CH2Cl2/MeOH 40: 1) to give 5a (880 mg, 91%) as a yel-
low solid. Mp: 165 ꢁC; Rf: 0.23 (CH2Cl2/MeOH 40:1).
1H NMR (400 MHz, CD3COCD3) selected signals: d
6.96 (s, 1H), 6.59 (s, 1H), 6.41 (s, 2H), 6.02 (d, 1H,
J = 9.2 Hz), 4.63 (d, 1H, J = 4.6), 4.32 (t, 1H,
Figure 11. A549 cell cycle analysis for compound 8 (10 lM).
(1H, d, J = 9), 6.89 (1H, d, J = 8.5), 6.64 (1H, d,
J = 6.5), 6.55 (1H, s), 6.30 (1H, dd, J = 8.5, 2), 6.07
(1H, d, J = 2), 5.72 (1H, dd, J = 8, 4.5), 5.43 (1H, s),
5.15 (1H, d, J = 8), 4.68–4.59 (1H, m), 3.95 (3H, s),
3.91 (3H, s), 3.79 (3H, s), 3.73 (3H, s), 3.65 (3H, s),
J = 9.1 Hz), 4.15 (t, 1H, J = 9.1 Hz), 3.71 (s, 1H), 3.67
(s, 1H), 3.61 (s, 1H), 3.27 (dd, 1H, J = 10.1, 4.6 Hz),
2.91–2.80 (m, 1H), 2.71–2.63 (m, 2H), 2.61–2.51 (m,
2H). ESI positive MS: Anal. Calcd for C26H26O11+Na+
537.1367, found 537.1365.
Figure 12. Microtubule organization in human lung carcinoma cell line A549 exposed for 1 h to solvent vehicle alone (a) or 10 lM vindoline 1 (b),
thiocolchicine 4 (c), compound 8 (d), as revealed by immunofluorescence localization of a-tubulin. Bar: 5 lm.

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
6279
5.1.5. 17-Deacetyl-17-O-(4-O-succinylpodophyllotoxinyl)
vindoline (9). A mixture of 5a (150 mg, 0.362 mmol),
DCC (89 mg, 0.43 mmol), DMAP (18 mg, 0.14 mmol),
and 1b (150 mg, 0.362 mmol) and CH2Cl2 (10 ml) was
stirred for 12 h. The mixture was submitted to chroma-
tography (AcOEt/hexane 20:1) to give 9 (160 mg, 49%)
as amorphous yellow solid. Rf: 0.35 (AcOEt). 1H
NMR (400 MHz, CD3COCD3) selected signals: d 8.58
(1H, s), 7.08 (1H, d, J = 8.5), 7.05 (1H, s), 6.58 (1H,
s), 6.46 (2H, s), 6.31 (1H, dd, J = 8.5, 2.0), 6.18 (1H,
d, J = 2.0), 6.05 - 5.98 (3H, m), 5.81 (1H, dd, J = 8.5,
4.0), 5.41 (1H, s), 5.20 (1H, d, J = 8.5), 4.63 (1H,d,
J = 4.5), 4.38 (1H, t, J = 9.0), 4.18 (1H, t, J = 9.0),
3.75 (3H, s), 3.71 (6H, s), 3.68 (6H, s), 3.63 (1H, s),
3.53–3.43 (2H, m), 3.25 (1H, dd, J = 13.0, 4.5, H-20),
2.92–2.83 (1H, m, H-3b), 2.86–2.55 (1H, m, H-5b),
2.81–2.72 (1H, m, H-30), 2.68 (3H, m, NMe), 2.65 (1H,
m, H-21), 2.38–2.26 (m, 2H, CH2(6)), 1.72 (1H, m, H-
19a), 1.21–1.05 (1H, m, H-19b), 0.45 (3H, t, J = 5.5,
CH3(18)). 13C NMR (100 MHz, CD3COCD3) selected
signals: d 173.8, 173.8, 171.79(2C), 161.1, 153.6,
152.63(2C), 148.1, 147.6, 137.1, 134.9, 130.4, 124.2,
124.2, 123.1, 109.3, 108.6(2C), 107.3, 104.7, 101.5,
95.6, 83.5, 79.5, 76.8, 73.3, 70.9, 67.3, 59.5, 55.5(2C),
54.6, 52.5, 51.9, 51.2, 51.0, 44.7, 43.9, 43.7, 43.1, 38.8,
38.6, 31.9, 7.2. ESI positive MS: Anal. Calcd for
C49H54O15N2+Na+ 933.3416, found 933.3417.
Anal. Calcd for C41H56O14Si+Na+ 823.3332, found
823.3333.
5.1.7. 13-O-(17-Deacetyl-17-O-succinylvindolinyl)-7-O-
(triethylsilyl)baccatin III (10). A solution of 6c (50 mg,
0.062 mmoli), DMAP (3 mg, 0.025 mmol), DCC
(15.5 mg, 0.075 mmol), and 1a (26 mg, 0.063 mmol) in
CH2Cl2 (3 ml) was stirred for 16 h at room temperature
and then filtered on Celite. The evaporation of the sol-
vent gave a mixture that was submitted to chromatogra-
phy (AcOEt/hexane 6:1 Et3N 1%) to give 10 (46 mg,
61%) as amorphous solid. [a]D ꢀ51.2 (CHCl3, c = 0.3).
1H NMR (400 MHz, CD3OD) selected signals: d 8.11
(2H, d, J = 7.5), 7.65 (1H, t, J = 7.5), 7.53 (2H, t,
J = 7.5), 7.03 (1H, d, J = 8.23), 6.51 (1H, s), 6.37 (1H,
dd, J1=8.2, J2=2.2), 6.17 (2H, m), 6.10–5.75 (1H, m),
5.69 (1H, d, J = 7.1), 5.43 (1H, s), 5.22 (1H, d,
J = 10.16), 5.02 (1H, d, J = 7.8), 4.55 (1H, dd,
J = 10.4, 6.7), 4.21 (2H, AB-syst), 3.89 (1H, d,
J = 7.15), 3.80 e 3.77 (6H, s), 3.62 (1H, s), 3.51.3.44
(1H, m), 3.38 (1H, td, J = 9, 3.8), 2.87 (1H, m), 2.73
(1H, s), 2.7–2.58 (4H, m) 2.67 (3H, s), 2.60–2.5 (2H,
m), 2.4–2.2 (4H, m), 2.37 (3H, s), 2.16 (3H, s), 2.04
(3H, s), 1.86 (1H, m), 1.7 (3H, s), 1.60 (1H, m), 1.20
(3H, s), 1.18 (3H, s), 1.15–1.08 (1H, m), 0.96 (9H, t),
0.62 (6H, q, J = 7.5), 0.5 (3H, s). 13C NMR (100 MHz,
CD3OD) selected signals: d 204.8, 174.13(2C), 173.9,
172.1, 171.1, 167.9, 163.1, 155.4, 142.5, 135.3, 134.8,
131.8(2C),131.4, 130.0(2C), 126.8, 126.0, 124.6, 106.4,
97.3, 85.8, 85.1, 82.3, 81.2, 79.3, 78.4, 77.8, 77.0, 76.5,
74.2, 71.6, 68.5, 60.2, 56.1, 54.4, 53.3, 53.1, 52.4, 45.3,
44.7, 39.4, 38.7, 37.3, 35.1, 32.4, 30.4, 27.1, 23.3, 22.0,
21.1, 15.4, 10.9, 8.4, 7.5, 6.6. ESI positive MS: Anal.
Calcd for C64H84O18N2Si+Na+ 1219.5381, found
1219.5383.
5.1.6. 13-O-Hemisuccinoyl-7-O-(triethylsilyl)baccatin III
(6c). A mixture of 6a (1.97 g, 2.81 mmol), DMAP
(514 mg, 4.21 mmol,), DCC (1.74 g, 8.43 mmol), and
hemytrichloroethylester of succynic acid (2.1 g,
8.43 mmol) in toluene (20 ml) was stirred for 60 h at
90 ꢁC. The mixture was filtered, concentrated, and sub-
mitted to chromatography (AcOEt/hexane 1:4) to give
1
6b (2.24 g, 85%). H NMR (300 MHz, CDCl3) selected
5.1.8. 7-N-Deacetyl-N-hemisebacoylthiocolchicine (4b).
A solution of 4a (500 mg, 1.34 mmol), sebacoyl acid
(271 mg, 1.34 mmol), BOPCl (409 mg, 1.608 mmol),
and Et3N (221 ll, 1.608 mmol) in CH2Cl2 (10 ml) was
stirred at room temperature for 2 days. After the addic-
tion of HCl (1 N, 10 ml) the organic phase was concen-
trated and purified by chromatography (AcOEt/MeOH
30: 1) to give 4b (212 mg, 28%) as amorphous solid.
signals: d 8.07 (2H, d, J = 7.7), 7.62 (1H, t, J = 7.7),
7.50 (2H, t, J = 7.7), 6.44 (1H, s), 6.18 (1H, t, J = 8.7),
5.66 (1H, d, J = 7), 4.94 (1H, d, J = 8.4), 4.76 (2H, sist.
AB), 4.47 (1H, dd, J = 10.5, 6.7), 4.30 (1H, d, J = 8.3),
4.14 (1H, d, J = 8.3), 3.83 (1H, d, J = 7), 2.9–2.75 (4H,
m), 2.15 (1H, ddd, J = 6.7, 9.6, 15), 2.34 (3H, s), 2.23
(2H, sist. AB), 2.16 (3H, s), 2.03 (3H, s), 1.92–1.83
(1H, m), 1.67 (3H, s), 1.21 (3H, s), 1.16 (3H, s), 0.91
(9H, m), 0.56 (6H, m). A mixture of Zn (1 g) and HCl
(2%, 10 ml) was stirred for 5 min. After septum-filtra-
tion, the solid was washed with water (pH 7), acetone
and ethyl ether. A mixture of activated Zn (2.2 g,
33.82 mmol), 6b (2.1 g, 2.25 mmol) in AcOH/MeOH
(1:1, 20 ml) was stirred for 10 h to convert all the start-
ing material. After Celite filtration the solution was
washed with water and extracted with AcOEt. The or-
ganic phase was washed with water and evaporated to
give 6c (1.31 g, 73%) as white amorphous solid. 1H
NMR (200 MHz, CDCl3) selected signals: d 8.10 (2H,
d, J = 7.7), 7.62 (1H, t, J = 7.7), 7.50 (2H, t, J = 7.7),
6.48 (1H, s), 6.21 (1H, t, J = 8.7), 5.70 (1H, d, J = 7),
4.96 (1H, d, J = 8.4), 4.49 (1H, dd, J = 10.5, 6.7), 4.32
(1H, d, J = 8.3), 4.18 (1H, d, J = 8.3), 3.87 (1H, d,
J = 7), 2.90–2.70 (4H, m), 2.60–2.50 (1H, m), 2.38 (3H,
s), 2.32–2.22 (2H, m), 2.20 (3H, s), 2.07 (3H, s), 1.95–
1.85 (1H, m), 1.71 (3H, s), 1.21 (3H, s), 1.19 (3H, s),
0.91 (9H, t), 0.67–0.56 (6H, m). ESI positive MS:
1
1H NMR H NMR (300 MHz, CDCl3) selected signals:
d 7.58 (1H, s), 7.34 (1H, d, J = 9), 7.10 (1H, d, J = 9),
6.95 (1H, d, J = 5), 6.52 (1H, s), 4.75–4.63 (1H, m),
3.91 (3H, s), 3.88 (3H, s), 3.62 (3H, s), 2.56–2.40 (1H,
m), 2.40 (3H, s), 2.40–2.23 (4H, m), 2.23–2.15 (2H, m),
1.92–1.78 (1H, m), 1.68–1.49 (4H, m), 1.38–1.18 (8H,
m); ESI positive MS: Anal. Calcd for C30H39O7NS+Na+
580.2339, found 580.2346.
5.1.9. 17-Deacetyl-17-O-(N-deacetyl-N-sebacoylthiocol-
chicinyl)vindoline (11). A solution of 4b (230 mg,
0.412 mmol), DMAP (230 mg, 0.412 mmol), DCC
(170 mg, 0.825 mmol) and 1a (170 mg, 0.412 mmol) in
CH2Cl2 (10 ml) was stirred for 16 h at room tempera-
ture. After Celite filtration, the mixture was submitted
to chromatography (AcOEt/Et3N 0.1%) to give 11
(212 mg, 54%) as amorphous solid. 1H NMR
(400 MHz, CD3OD) selected signals: d 7.38–7.31 (1H,
m), 7.19 (1H, s), 7.02 (1H, d, J = 8), 6.71 (1H, s), 6.35

6280
D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
(1H, dd, J = 8, 2), 6.18 (1H, d, J = 2), 5.85 (1H, dd,
J = 10, 4), 5.39 (1H, s), 5.15 (1H, d, J = 10), 4.55–
4.48 (1H, m), 3.91 (3H, s), 3.89 (3H, s), 3.77 (3H, s),
3.76 (3H, s), 3.64 (3H, s), 3.62 (1H, s), 3.48 (1H, dd,
J = 16, 5), 3.42–3.35 (1H, m), 2.85 (1H, d, J = 16),
2.73 (1H, s), 2.65 (3H, s), 2.62–2.56 (1H, m), 2.47
(3H, s), 2.45–2.13 (8H, m), 2.32–2.25 (2H, m), 2.00–
1.90 (1H, m), 1.65–1.54 (4H, m), 1.27–1.37 (8H, m),
1.18–1.09 (1H, m), 0.50 (3H, t, J = 5.5). 13C NMR
(100 MHz, CD3OD) selected signals: d 182.4, 174.3,
173.7, 172.1, 161.5, 158.5, 154.0, 153.8, 152.1, 150.9,
141.5, 138.7, 135.0, 134.7, 130.0, 127.6, 127.2, 125.6,
125.2, 124.3, 122.8, 107.6, 104.7, 95.8, 83.4, 79.7,
76.1, 66.9, 60.6, 60.2, 55.4, 54.5, 52.7, 52.0(2C), 51.4,
50.6, 43.5, 43.0, 37.6, 35.9, 35.4, 33.7, 30.6, 29.2,
28.8(4C), 28.7, 25.3, 13.6, 6.6. ESI positive MS: Anal.
Calcd for C53H67O11N3S+Na+ 976.4389, found
976.4387.
5.1.12. 13-O-Hemisebacoyl-7-O-(triethylsilyl)baccatin III
(6e). A solution of 6a (900 mg, 1.29 mmol), undecenoyl
acid (1.56 ml, 7.74 mmol), DCC (1.59 g, 7.74 mmol),
and DMAP (0.645 mmol) in toluene (10 ml) was stirred
at room temperature for 14 h. The mixture was filtered
and the solvent evaporated. The crude mixture was sub-
mitted to chromatography (AcOEt/hexane 1:9) to give
6d (970 mg, 87%) as a white amorphous solid. 1H
NMR (300 MHz, CDCl3) selected signals: d 8.10 (2H,
d, J = 7.5), 7.61 (1H, t, J = 7.5), 7.46 (2H, t, J = 7.5),
6.47 (1H, s), 6.20–6.10 (1H, m), 5.89–5.72 (1H, m),
5.66 (1H, d, J = 7), 5.01–4.93 (3H, m), 4.46 (1H, dd,
J = 10, 7), 4.30 (1H, d, J = 8.3), 4.13 (1H, d, J = 8.3),
3.83 (1H, d, J = 7), 2.56–2.33 (3H, m), 2.31 (3H, s),
2.20 (2H, d, J = 8.9), 2.16 (3H, s), 2.02 (3H, s), 1.92–
1.84 (1H, m), 1.8–1.55 (4H, m) 1.67 (3H, s), 1.43–1.20
(8H, m), 1.21 (3H, s), 1.17 (3H, s), 0.91 (9H, s), 0.59
(6H, s). 6d (918 mg, 1.06 mmol) was dissolved in
CH3CN/CCl4/H2O (1:1:2) (20 ml) and treated with
NaHCO3 (890 mg, 10.6 mmol), and NaIO4 (1.93 g,
9.01 mmol). After 5 min RuCl3 (33 mg, 0.159 mmol)
was added and the mixture was stirred for 10 days at
room temperature in the dark. Every 2 days a quote
of RuCl3 (22 mg, 0.106 mmol) was added. The mixture
was diluted with AcOEt and treated with H2SO42N.
The organic phase was washed with a saturated solution
of Na2SO3 (100 ml) and ice. The mixture was submitted
to chromatography (AcOEt/hexane 1:4) to give 6e
(766 mg, 82%) as white amorphous solid. 1H NMR
(CDCl3, 300 MHz) selected signals: d 8.1 (2H, d,
J = 7.5), 7.6 (1H, t, J = 7.5), 7.46 (2H, t, J = 7.5), 6.45
(1H, s), 6.16 (1H, br t, J = 8.8), 5.67 (1H, d, J = 7),
4.93 (1H, d, J = 10), 4.47 (1H, dd, J = 10.2, 7), 4.29
(1H, d, J = 8.3), 4.14 (1H, d, J = 8.3), 3.82 (1H, d,
J = 7), 2.59–2.33 (5H, m), 2.31 (3H, s), 2.20 (2H, d,
J = 8.9), 2.16 (3H, s), 2.02 (3H, s), 1.95–1.80 (1H,
m), 1.78–1.58 (4H, m) 1.67 (3H, s), 1.44–1.30 (8H,
m), 1.21 (3H, s), 1.17 (3H, s), 0.91 (9H, s), 0.59 (6H,
s). ESI positive MS: Anal. Calcd for C47H68O14Si+-
Na+ 907.4271, found 907.4272.
5.1.10. 4-O-Hemisebacoylpodophyllotoxin (5c). A mix-
ture of 5 (159 mg, 0.384 mmol), DMAP (19 mg,
0.154 mmol), DCC (95 mg, 0.461 mmol), and hemytri-
chloroethylester of sebacic acid (155 mg, 0.461 mmol)
in CH2Cl2 (16 ml) was stirred for 13 h at room temper-
ature. The mixture was submitted to chromatography
(AcOEt/hexane 1:3) to give 5b (244 mg, 87%) as a white
amorphous solid. A mixture of Zn (1 g) and HCl (2%,
10 ml) was stirred for 5 min. After septum-filtration
the solid was washed with water, acetone, and ethyl
ether. A mixture of activated Zn (306 mg, 4.69 mmol),
and 5b (229 mg, 0.312) in AcOH/MeOH (1:1, 5 ml)
was stirred for 10 h. After Celite filtration the solution
was washed with water and extracted with AcOEt.
The evaporation of the solvent gave 5c (176 mg, 94%)
as white amorphous solid. 1H NMR (300 MHz, CDCl3)
selected signals: d 7.07 (1H, s), 6.57 (1H, s), 6.45 (2H, s),
6.06–5.97 (3H, m), 4.66 (1H, d, J = 4.5), 4.38 (1H, t,
J = 9.0), 4.19 (1H, t, J = 9.0), 3.27 (1H, dd, J = 13.0,
4.5), 2.81–2.72 (1H, m), 2.40–2.23 (2H, m), 2.23–2.15
(2H, m), 1.66–1.47 (4H, m), 1.35–1.15 (8H, m). ESI po-
sitive MS: Anal. Calcd for C32H38O11+Na+ 621.2306,
found 621.2307.
5.1.13. 13-O-(17-Deacetyl-17-O-sebacoylvindolinyl)-7-O-
(triethylsilyl)baccatin III (13). A solution of 6e (40 mg,
0.045 mmoli), DMAP (2.2 mg, 0.018 mmol), DCC
(11.2 mg, 0.054 mmoli), and 1a in CH2Cl2 (3 ml) was
stirred for 16 h at room temperature and then filtered
on Celite. The evaporation of the solvent gave a mixture
5.1.11. 17-Deacetyl-17-O-(4-O-sebacoylpodophyllotoxi-
nyl)vindoline (12).
A
mixture of 5c (168 mg,
0.281 mmol), DCC (69 mg, 0.337 mmol), DMAP
(14 mg, 0.112 mmol), and1b (116 mg, 0.281 mmol)
and CH2Cl2 (10 ml) was stirred for 12 h. The mixture
was submitted to chromatography (AcOEt/hexane
20:1) to give 12 (130 mg, 46%) as amorphous yellow
solid. Rf: 0.35 (AcOEt). 1H NMR (400 MHz,
CD3COCD3) selected signals: d 8.58 (1H, s), 7.10
(1H, d, J = 8.5), 6.92 (1H, s), 6.58 (1H, s), 6.42 (2H,
s), 6.32 (1H, dd, J = 8.5, 2.0), 6.18 (1H, d, J = 2.0),
6.04 (2H, s), 5.98 (1H, d, J = 9), 5.81 (1H, dd,
J = 8.5, 4.0), 5.39 (1H, s), 5.16 (1H, br d, J = 8.5),
4.63 (1H,d, J = 4.5), 4.38 (1H, dd, J = 9.0, 9.1), 4.18
(1H, dd, J = 9.0, 9.1), 3.75 (3H, s), 3.71 (6H, s),
3.68 (6H, s), 3.64 (1H, s), 3.48 (1H, dd, J = 14, 4),
3.39 (1H, td, J = 4, 8), 3.25 (1H, dd, J = 14, 4.6),
2.92–0.45 (23H, m), 2.68 (3H, m, NMe); ESI positive
MS: Anal. Calcd for C55H66O15N2+Na+ 1017.4355,
found 1017.4361.
that was submitted to chromatography (AcOEt/hexane
25
2:1, 0.5% Et3N) to give 13 (39 mg, 69%). ½aꢁ +133
D
(CHCl3, c = 0.3). 1H NMR (400 MHz, CD3OD) selected
signals: d 8.11 (2H, d, J = 7), 7.65 (1H, t, J = 7.5), 7.53
(2H, t, J = 7.5), 7.03 (1H, d, J = 8.2), 6.51 (1H, s), 6.35
(1H, dd, J = 8.2, 2.2), 6.17 (2H, m), 5.87 (1H, ddd,
J = 6.6, 4.7, 1.7), 5.69 (1H, d, J = 7.1), 5.41 (1H, s),
5.16 (1H, d, J = 10.2), 5.02 (1H, d, J = 8), 4.55 (1H,
dd, J = 10.5, 6.7), 4.21 (2H, AB-syst, J = 8.4), 3.89
(1H, d, J = 7.2), 3.78 e 3.77 (6H, s), 3.62 (1H, s), 3.49
(1H, m), 3.4 (1H, m), 2.89 (1H, m), 2.75 (1H, s), 2.66
(3H, s), 2.61–2.57 (2H, m), 2.35–2.27 (8H, m), 2.36
(3H, s), 2.17 (3H, s), 2.03 (3H, s), 1.95–1.80 (1H, m),
1.7 (3H, s), 1.62 (5H, m, 1), 1.45 - 1.30 (8H, m), 1.22
(3H, s), 1.18 (3H, s), 0.96 (9H, t), 0.62 (6H, q,
J = 7.5), 0.5 (3H, s). 13C NMR (100 MHz, CD3OD) se-

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
6281
lected signals: d 204.8, 173.7, 173.4, 172.5, 170.2, 169.4,
166.2, 161.4, 153.8, 140.6, 133.8, 133.2, 130.0, 129.7,
128.3, 125.1, 124.3, 122.9, 104.7, 95.7, 84.1, 83.4, 80.7,
79.6, 77.5, 76.1, 76.1, 75.3, 74.7, 72.5, 69.5, 66.8, 58.5,
54.4, 52.7, 51.5, 51.5, 50.6, 46,8, 43.5, 43.2, 43.0, 37.7,
37.0, 35.6, 33.9, 33.7, 33.4, 30.7, 28.7, 25.5, 24.6, 21.6,
20.4, 19.4, 13.7, 9.2, 6.7, 5.8, 4.9. ESI positive MS: Anal.
Calcd for C70H96O18N2Si+Na+ 1303.6320, found
1303.6319.
J = 7.4), 0.77 (3H, t, J = 7.3). 13C NMR (125 MHz,
CD3COCD3) selected signals: d 173.5, 173.1,173.0,
171.9, 161.2, 156.1, 152.7(2C), 147.9, 147.5, 137.5,
136.5, 135.6, 135.0, 132.8, 130.9, 130.6, 129.4, 129.1,
125.9, 125.0, 124.3, 124.2, 121.7, 121.2, 118.5, 118.4,
117.3, 111.2, 109.3, 108.7, 107.3, 101.6, 94.1, 83.3,
79.3, 76.9, 73.4, 70.9, 65.2, 59.5, 55.6(3C), 54.2, 53.1,
53.1, 51.1(2C), 50.6, 50.2(2C), 50.2, 46.0, 44.7, 43.7,
42.6, 38.9, 37.7, 34.1, 33.5, 30.7, 28.9–28.7, 27.1, 25.4,
8.2, 7.8. MSFAB (M++1) 1247. ESI positive MS: Anal.
CalcdforC70H78O17N4+Na+ 1269.5254, found 1269.5257.
5.1.14. 17-Deacetyl-17-O-(N-deacetyl-N-succinylthiocol-
chicinyl)anhydrovinblastine (14). A solution of catharan-
thineÆHCl (55.5 mg, 0.149 mmol), FeCl36H2O (270 mg,
0.747 mmol), glycine buffer (4 ml), and HCl (4 ml, 0.1
N) was stirred at room temperature for 2.5 h. 8 (130 mg,
0.149 mmol). After 2.5 h NaBH4 (6.1 mg, 0.160 mmol)
was dissolved in NH4OH (0.74 ml). The solution was
extracted with CH2Cl2. After chromatographic purifica-
tion (AcOEt/MeOH/Et3N 20:2:0.4) 14 (40 mg, 22%)
5.1.16. 13-O-(17-Deacetyl-17-O-succinylanhydrovinblas-
tinyl)-7-O-(triethylsilyl)baccatin III (16). A solution of
catharanthine (143 mg, 0.425 mmol) in CH2Cl2 was
treated with m-CPBA (110 mg, 0.64 mmol in CH2Cl2)
at 0 ꢁC. The solution was maintained at the same tem-
perature for 10 min and poured in NaHCO3 (20 ml,
10%). The extraction with CH2Cl2 gave the N-oxide-
catharanthine (141 mg) which was immediately dis-
solved in CH2Cl2 (2 ml) and compound 10 (186 mg,
0.155 mmol) was added. TFAA (165 ll, 1.17 mmol)
was added at ꢀ78 ꢁC. After 1 h the solvent was removed
and the mixture dissolved in MeOH (8 ml). NaBH4
(64 mg, 1.7 mmol) was added at 0 ꢁC. After 20 min the
solution was poured in water and extracted with
CH2Cl2. The evaporation of the solvent gave a mixture
that was submitted to chromatography (AcOEt/MeOH
7:1) to give 16 (53 mg, 22 %). 1H NMR (400 MHz,
CD3COCD3) selected signals: d 8.14 (2H, d, J = 7.5),
7.68 (1H, t, J = 7.5), 7.57 (2H, t, J = 7.5), 7.46 (1H, d,
J = 7.8), 7.38 (1H, d, J = 7.8), 7.08 (1H, t, J = 7.8),
7.01 (1H, t, J = 7.8), 6.82 (1H, s), 6.52 (1H, s), 6.37
(1H, s), 6.25 (1H, t, J = 8.6), 5.81 (1H, dd, J = 10.2,
3.9), 5.72 (1H, d, J = 7), 5.41 (1H, br d, J = 5.3), 5.36
(1H, d, J = 10.2), 5.00 (1H, d, J = 8.5), 4.59 (1H, dd,
J = 10, 6.7), 4.18 (2H, br s), 3.93 (1H, d, J = 7), 3.68
(1H, s), 2.78 (1H, s), 2.74 (3H, s), 2.47–2.32 (2H, m),
0.97 (9H, t, J = 9), 0.64 (6H, q, J = 9). 13C NMR
(100 MHz, CD3COCD3) selected signals: d 133.2,
130.6, 130.0, 128.6, 124.3, 124.1(2C), 121.6, 118.5,
117.9, 111.1, 94.2, 77.7, 76.8, 75.9, 75.1, 74.8, 72.5,
69.6, 58.3, 54.3, 52.8, 51.3, 50.2, 46.9, 37.9, 37.3, 36.1,
26.0, 22.1, 20.6, 19.9, 14.1, 9.6, 6.2, 5.0. ESI positive
MS: Anal. Calcd for C85H108O20N4Si+Na+ 1555.7218,
found 1555.7221.
1
was obtained as a yellow amorphous solid. H NMR
(300 MHz, CD3COCD3) selected signals: d 9.18 (1H, br
s), 8.48 (1H, s), 7.80 (1H, d, J = 6.5), 7.45 (1H, d, J = 7),
7.40 (1H, d, J = 7), 7.20 (1H, s), 7.20 (1H, s), 7.12
(1H, s), 7.11 (1H, t, J = 7), 7.01 (1H, t, J = 7), 6.78 (1H,
s), 6.32 (1H, s), 5.51 (1H, dd, J = 8, 4.5), 5.35–5.45
(1H, m), 5.30 (1H, s), 5.25 (1H, d, J = 8), 4.61–4.50 (1H,
m), 3.88 (3H, s), 3.83 (3H, s), 3.80 (3H, s), 3.65 (3H, s),
3.62 (3H, s), 3.50 (3H, s), 2.68 (3H, s), 2.41 (3H, s), 0.99
(1H, t, J = 7.5), 0.62 (3H, t, J = 5.5). 13C NMR (100
MHz, CD3COCD3) selected signals: d 184.1, 177.2,
173.9(2C), 173.5, 160.2, 159.9, 155.5, 154.8, 153.7,
152.6, 143.0, 140.3, 137.0, 136.8, 136.5, 132.0, 131.9,
130.6, 129.3, 129.0, 127.3, 125.6, 125.4, 125.1, 123.8,
123.7(2C), 120.3, 119.3, 117.7, 112.2, 109.1(2C), 95.6,
84.6, 81.6, 78.0, 66.7, 62.3, 62.0, 56.9, 56.9, 56.2, 55.9,
54.9, 54.1, 53.3, 53.1, 52.3, 51.7, 51.1, 47.0, 46.1, 44.4,
39.1, 37.5, 36.1, 34.1, 32.1, 31.3, 30.1, 30.0, 29.3, 25.1, 15.4,
12.9,9.3.FABMS:1205(M+). ESI positive MS: Anal. Calcd
for C68H79O13N5S+Na+ 1228.5287, found 1228.5288.
5.1.15. 17-Deacetyl-17-O-(4-O-succinylpodophyllotoxi-
nyl)anhydrovinblastine (15). The use of the same proce-
dure described for the preparation of 14 and the
addition of compound 9 as electrophile gave 15 (28%)
(chromatographic purification CH2Cl2/MeOH 30:1,
1
24%). H NMR (500 MHz, CD3COCD3) selected sig-
nals: d 7.47 (1H, d, J = 7.5), 7.38 (1H, d, J = 7.5), 7.08
(1H, t, J = 7.5), 7.04 (1H, s), 7.01 (1H, t, J = 7.5), 6.84
(1H, s), 6.62 (1H, s), 6.48 (2H, s), 6.38 (1H, s), 6.05
(1H, d, J = 9.1), 6.04 (2H, AB-syst), 5.77 (1H, dd,
J = 8.5, 4.0), 5.45–5.39 (1H, m), 5.38 (1H, s), 5.37 (1H,
d, J = 8.5), 4.65 (1H, d, J = 4.5), 4.42 (1H, dd, J = 8.7,
7.2), 4.22 (1H, dd, J = 10.5, 8.7), 3.73 (3H, s), 3.72
(3H, s), 3.72 (3H, s), 3.68 (6H, s), 3.68 (3H, s), 3.40–
3.30 (1H, m), 3.57–3.49 (1H, m), 3.38–3.20 (2H, m),
3.37–3.21 (1H, m), 3.35–3.29 (1H, m), 2.75–2.68 (1H,
m), 3.20–3.12 (1H, m), 2.48–2.32 (1H, m), 3.20 (1H, s),
3.10–3.00 (1H, m), 3.37–3.21 (1H, m), 2.90–2.76 (1H,
m), 2.90–2.75 (4H, m), 2.82 (1H, s), 2.71 (3H, s), 2.55
(1H, br d, J = 12.5), 3.45–3.38 (1H, m), 2.45–2.35
(1H, m), 3.37–3.21 (1H, m), 2.00–1.90 (2H, m), 2.00–
1.89 (1H, m), 2.19–2.10 (1H, m), 1.48–1.35 (1H, m),
1.72–1.59 (1H, m), 1.26–1.21 (1H, m), 1.00 (3H, t,
5.1.17. 17-Deacetyl-17-O-(N-deacetyl-N-sebacoylthiocol-
chicinyl)anhydrovinblastine (17). The use of the same
procedure described for the preparation of 14 and the
addition of compound 11 as electrophile gave 17
(Chrom. AcOEt/MeOH/Et3N 20:2:0.4) (24 %). 1H
1
NMR H NMR (300 MHz, CD3COCD3) selected sig-
nals: d 9.15 (1H, br s), 8.70–8.40 (1H, large), 7.75 (1H,
d, J = 6.5), 7.47 (1H, d, J = 7), 7.38 (1H, d, J = 7),
7.18 (2H, s), 7.15 (1H, s), 7.11 (1H, t, J = 7), 7.00 (1H,
t, J = 7), 6.31 (1H, s), 6.78 (1H, s), 6.32 (1H, s), 5.85
(1H, dd, J = 8, 4.5), 5.41–5.32 (1H, m), 5.31 (1H, s),
5.28 (1H, d, J = 8), 4.61–4.48 (1H, m), 3.88 (3H, s),
3.83 (3H, s), 3.80 (3H, s), 3.65 (3H, s), 3.62 (3H, s),
3.50 (3H, s), 2.68 (3H, s), 2.41 (3H, s), 3.90–0.70 (32H,
m). 13C NMR (100 MHz, CD3COCD3) selected signals:
d 181.5, 177.2, 172.0(2C), 171.5, 160.2, 157.9, 153.5,

6282
D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
152.9, 151.1, 150.8, 141.7, 137.8, 134.6, 134.5, 133.8,
132.0, 131.9, 130.6, 129.3, 129.0, 127.3, 125.6, 125.4,
125.1, 123.8, 123.7(2C), 120.3, 119.3, 117.7, 111.1,
109.1, 107.7, 95.6, 84.6, 81.6, 78.0, 66.7, 62.3, 62.0,
56.9, 56.9, 56.2, 55.9, 54.9, 54.1, 53.3, 53.1, 52.3, 51.7,
51.1, 47.0, 46.1, 44.4, 39.1, 37.5, 36.1, 35.4, 34.1, 33.7,
32.1, 31.3, 30.1, 30.0, 29.3, 28.8(4C), 28.7, 25.3, 25.1,
15.4, 12.9, 9.3; ESI positive MS: Anal. Calcd for
C74H91O13N5S+H+ 1290.6407, found 1290.6419.
nals: d 175.2, 172.9,158.6, 153.0, 134.9, 134.7, 134.1,
130.5, 128.5, 123.8, 123.4, 122.6, 122.3, 119.5, 119.3,
117.6, 110.8, 93.2, 82.6, 81.1, 74.0, 65.5, 54.9, 54.9,
53.1, 51.8, 51.4, 50.3, 49.5, 46.5, 44.4, 44.3, 42.6, 37.4,
35.8, 31.8, 28.7, 27.4, 11.2, 7.3. ESI positive MS: Anal.
Calcd for C43H52O7N4+Na+ 759.3728, found 759.3728.
5.1.20. 17-Deacetyl-17-O-(N-deacetyl-N-succinylthiocol-
chicinyl)vinorelbine (18). A mixture of 3 (100 mg,
0.136 mmol), DMAP (7 mg, 0.054 mmol), imidazole
(11 mg, 0.163 mmol), and succinic anhydride (20 mg,
0.204 mmol) in CH2Cl2 (8 ml) was stirred for 12 h at
room temperature. The solution was washed with water.
The evaporation of the solvent gave a crude compound
(3b, 110 mg, 97%) that was directly used for the succeed-
ing preparation. A solution of 3b (55 mg, 0.066 mmol),
DCC (33 mg, 0.161 mmol), DMAP (7 mg, 0.054 mmol),
and 4a (55 mg, 0.147 mmol) in CH2Cl2 (15 ml) was stir-
red for 24 h. Evaporation of the solvent and chromatog-
5.1.18. Vinorelbine (3). AVBL (196 mg, 0.248 mmol) was
dissolved in CH2Cl2 (1 ml). NBS (49.5 mg, 0.278 mmol)
and CF3COOH (27.5 ll, 0.357 mmol) in CH2Cl2 (1 ml)
were added at ꢀ60 ꢁC and the solution was stirred for
2 h at the same temperature. CH3COONH4 (51.68 mg,
0.67 mmol in 400 ll of H2O) and AgBF4 (53.86 mg,
0.277 mmol in THF/H2O 1:1) were added at room tem-
perature. After 20 h NaHCO3 (1.6 ml, 10%) was added
and the solution was extracted with CH2Cl2. The crude
mixture was submitted to chromatography (AcOEt/
MeOH/Et3N 98.25:0.75:1) to give 3 (101 mg, 53%) as a
amorphous solid. 1H NMR (500 MHz, CD3COCD3) se-
lected signals: d 9.72 (1H, s), 8.45 (1H, s), 7.76 (1H, d,
J = 10), 7.45 (1H, d, J = 10), 7.13 (1H, t, J = 10), 7.09
(1H, t, J = 10), 6.58 (1H, s), 6.36 (1H, s), 5.81–5.78 (2H,
m), 5.30 (1H, br d, J = 10), 5.28 (1H, s), 4.53–4.44 (1H,
m), 4.40 (1H, d, J = 12.86), 3.87 (3H, s), 3.78–3.74 (1H,
m), 3.70 (3H, s), 3.63 (3H, s), 3.60 (1H, s), 3.54 (1H, br
raphy (AcOEt/MeOH/Et3N 10:1:0.2) gave 18 (24 mg,
25
30%) as an amorphous solid. ½aꢁ ꢀ66.3 (CHCl3,
D
c = 0.8). 1H NMR (400 MHz, CD3COCD3) selected sig-
nals: d 9.64 (1H, s), 8.42 (1H, s), 7.83 (1H, d, J = 6.9),
7.69 (1H, d, J = 7.8), 7.43 (1H, d, J = 7.8), 7.22 (2H,
AB-syst), 7.16 (1H, s), 7.15 (1H, td, J = 7.8. 1.2), 7.09
(1H, t, J = 7.8), 6.79 (1H, s), 6.52 (1H, s), 6.33 (1H, s),
5.75 (1H, br s), 5.49 (1H, dd, J = 10, 4), 5.25 (1H, s),
5.30–5.19 (1H, m), 4.60–4.50 (1H, m), 4.41–4.28 (2H,
m), 3.89 (3H, s), 3.88 (3H, s), 3.85 (3H, s), 3.74–3.60
(1H, m), 3.65 (6H, s), 3.62 (3H, s), 3.56 (1H, s), 3.46
(1H, d, J = 15), 3.25–3.13 (3H, m), 2.90 (1H, dd,
J = 15.4, 7.4), 2.75–2.66 (1H, m), 2.71 (3H, s), 2.67
(1H, s), 2.66–2.49 (5H, m), 2.49–2.40 (2H, m), 2.44
(3H, s), 2.40–2.26 (2H, m), 2.27–2.18 (1H, m), 2.10–
1.98 (3H, m), 1.98–1.89 (1H, m), 1.89–1.79 (1H, m),
1.54–1.43 (2H, m), 1.29–1.19 (1H, m), 1.08 (3H, t,
J = 7.5), 0.56 (3H, t, J = 7.2). 13C NMR (100 MHz,
CD3COCD3) selected signals: d 182.6, 175.3, 172.8,
172.5, 171.4, 159.6, 159.0, 154.9; 154.0, 152.2, 151.7,
142.9, 137.3, 136.3, 135.6, 135.3, 134.8, 131.9, 130.0,
129.3, 127.2, 126.8, 125.1, 124.8, 124.6, 124.1, 122.9,
121.5, 120.2, 119.2, 112.3, 108.9; 94.8, 84.1, 80.8, 77.4,
66.0, 61.8, 61.4, 56.6, 56.4, 56.1, 55.9, 54.2, 53.1, 52.7,
52.0, 51.4, 50.9, 48.5, 46.0, 45.6, 43.9, 38.7, 38.0, 37.3,
31.5, 31.3, 30.8, 30.6–29.3(2C), 28.8, 15.1, 12.9, 8.6.
ESI positive MS: Anal. Calcd for C67H77O13N5S+Na+
1214.5131, found 1214.5132.
d,
J1 = 13.92,
J = 14.01), 3.33–3.27 (1H, m), 3.28 (1H, dd,
J2 = 4.23), 3.20 (1H, dt, J1 = 9.7,
J2 = 5.6), 2.95 (1H, dd, J1 = 15.76, J2 = 7.32), 2.74 (3H,
s), 2.73 (1H, s), 2.35 (1H, dd, J1 = 13.92, J2 = 4.23), 2.66
(1H, d, J = 15.76), 2.41–2.38 (1H, m), 2.35 (1H, dt,
J1 = 9.7, J2 = 5.6), 2.11–2.05 (3H, m), 1.99 (3H, s), 1.95–
1.89 (1H, m), 1.63–1.56 (2H, m), 1.39–1.29 (1H, m), 1.10
(3H, t, J = 7.46), 0.68 (3H, t, J = 7.34). 13C NMR
(100 MHz, CD3COCD3) selected signals: d 174.1, 170.0,
171.5, 158.4, 153.1, 135.3, 130.6, 128.8, 124.1, 123.8,
123.6, 123.1, 122,0, 120.1, 120.1, 118.4, 111.3, 93.7, 83.0,
79.7, 76.4, 64.6, 55.4, 54.9, 53.2, 51.7,51.0, 50.2, 49.7,
47.2, 44.3, 44.6, 42.7, 37.7, 36.6, 30.6, 27.5, 20.2, 11.7,
7.6. ESI positive MS: Anal. Calcd for C45H54O8N4+Na+
801.3834, found 801.3835.
5.1.19. 17-O-Deacetylvinorelbine (3a). A solution of 3
(265 mg, 0.341 mmol) and Na2CO3 in MeOH (10 ml)
was stirred at 65 ꢁC for 14 days. Evaporation of the sol-
vent and chromatography (alumina, AcOEt/MeOH/
25
Et3N 10:1:0.1) 3a (149 mg, 59%). ½aꢁ +98.2 (CHCl3,
5.1.21. 17-Deacetyl-17-O-(4-O-succinylpodophyllotoxinyl)
vinorelbine (19). A solution of crude 3b (55 mg,
D
c = 0.61). 1H NMR (400 MHz, CD3OD) selected sig-
nals: d 7.73 (1H, d, J = 7.12), 7.34 (1H, dd, J1 = 7.12,
J = 1.2), 7.18 (1H, td, J = 7.1, 1.2), 7.14 (1H, td,
J = 7.1, 1.2), 6.29 (2H, s), 5.83 (1H, dd, J = 10, 3.7),
5.78 (1H, d, J = 4.9), 5.64 (1H, d, J = 10), 4.42–4.36
(2H, m), 4.03 (1H, br d, J = 15.3), 3.96 (1H, s), 3.88–
3.79 (1H, m), 3.86 (3H, s), 3.82 (3H, s), 3.75 (3H, s),
3.53 (1H, s), 3.43 (1H, d, J = 14), 3.31–3.26 (1H, m),
3.20 (1H, dt, J = 10.3, 3. 9), 3.06 (1H, dd, J = 15.5,
7.64), 2.76 (3H, s), 2.67–2.59 (2H, m), 2.52 (1H, s),
2.37 (1H, dd, J = 15.5, 4.), 2.28 (1H, dt, J = 10.3, 4.5),
2.14–1.99 (3H, m), 1.79–1.69 (1H, m), 1.66–1.56 (2H,
m), 1.35–1.22 (1H, m), 1.11 (3H, t, J = 7.5), 0.81 (3H,
t, J = 7.3). 13C NMR (100 MHz, CD3OD) selected sig-
0.066 mmol), DCC (33 mg, 0.161 mmol), DMAP (7 mg,
0.054 mmol), and 5 (56 mg, 0.132 mmol) in CH2Cl2
(15 ml) was stirred for 24 h. Evaporation of the solvent
and chromatography (CH2Cl2/MeOH 15:1) gave 19
(25 mg, 31%) as an amorphous solid. 1H NMR
(400 MHz, CD3COCD3) selected signals: d 9.75 (1H, s),
7.81 (1H, d, J = 7.8), 7.45 (1H, d, J = 7.8), 7.14 (1H, t,
J = 7.8), 7.09 (1H, t, J = 7.8), 7.01 (1H, s), 6.58 (1H, s),
6.56 (1H, s), 6.45 (2H, s), 6.33 (1H, s), 6.06–5.99 (3H,
m), 5.83(1H, d, J = 5.3), 5.74 (1H, dd, J = 10.2, 3.9),
5.34 (1H, d, J = 10.2), 5.29 (1H, s), 4.68 (1H, A portion
of AB-syst), 4.65 (1H, d, J = 4.6), 4.49 (1H, B portion
of AB-syst), 4.40 (1H, dd, J = 8.7, 7.2), 4.21 (1H, t,

D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
6283
J = 8.7), 3.92–3.82 (1H, m), 3.85 (3H, s), 3.70 (6H, s),
3.69–3.61 (1H, m) 3.68 (3H, s), 3.65 (3H, s), 3.48–3.38
(1H, m), 3.28 (1H, dd, J = 10.2, 4.7), 3.24–3.14 (2H, m),
3.04–2.82 (2H, m), 2.82–2.60 (6H, m), 2.74 (4H, s),
2.60–2.52 (1H, m), 2.41–2.32 (1H, m), 2.15–2.07 (2H,
m), 2.07–2.02 (1H, m), 1.98–1.89 (1H, m), 1.82–1.70
(1H, m), 1.65–1.53 (1H, m), 1.40–1.29 (1H, m), 1.11
(3H, t, J = 7.4), 0.67 (3H, t, J = 7.3). 13C NMR
(100 MHz, CD3COCD3) selected signals: d 174.5, 174.1,
173.9, 172.8, 172.7, 159.5, 154.2, 153.8(2C), 148.1,
147.6, 136.5, 136.2, 131.5, 130.2, 125.2, 124.6(2C),
123.4, 120.6, 119.6, 112.2, 110.3, 110.1(2C), 108.2,
102.5, 94.9, 84.1, 80.6, 78.0, 74.5, 72.0, 66.1, 60.6,
56.7(2C), 56.4, 54.2(2C), 52.8, 52.0, 51.3, 51.0, 47.9,
45.8, 45.4, 45.0, 44.8, 43.9, 40.0, 38.6, 36.9, 31.7, 30.4,
29.2, 28.3, 12.5, 8.5. ESI positive MS: Anal. Calcd for
C69H76O17N4+Na+ 1255.5098, found 1255.5099.
pounds, the kinetics of tubulin polymerization was fol-
lowed turbidimetrically at 340 nm in Ultraspec 300
spectrophotometer (Pharmacia) equipped with tempera-
ture controller. Reaction mixture contained 2.7 mg/ml
tubulin, different compounds (final concentration 10 lM)
or equal volume of the solvent (final 0.2% DMSO), in
assembly buffer (80 mM K-Pipes pH 6.9, 2 mM EGTA,
1 mM MgCl2, and 10% glycerol). After addition of GTP
(final concentration 1 mM), reaction mixture was trans-
ferred to cuvettes in spectrophotometer and the poly-
merization reaction was followed at 37 ꢁC for 40 min.
5.2.2. DIC microscopy. Microtubules were collected by
centrifugation at 30,000 g for 30 min at 30 ꢁC, fixed
with 10% glycerol, 0.5% glutaraldehyde in BRB 80,
and put onto coverslips. Image acquisition was per-
formed using a Zeiss Axiovert 200 equipped with differ-
ential interference contrast (DIC) optics, an 63· oil
objective, and a digital image recording system (Axio-
cam HRM Rev. 2 camera driven by Axiovision soft-
ware rel. 4.4, Zeiss).
5.2. Biology
5.2.1. Tubulin assembly assay. Tubulin was purified from
porcine brain purchased from a local slaughterhouse,
conserved before use in ice-cold Pipes buffer (100 mM
K-Pipes, pH 6.9, 2 mM EGTA, and 1 mM MgCl2)
and used as soon as possible. Pure tubulin was obtained
by three cycles of warm-cold polymerization–depoly-
merization followed by anion-exchange chromatogra-
phy (MonoQ column, Pharmacia) to separate tubulin
from MAPs.32 Protein concentration was determined
by MicroBCA assay kit (Pierce). Stock solutions of thi-
ocolchicine, podophyllotoxin, TES-baccatin, vinorel-
bine, vindoline, and all the obtained hydrids were
prepared by dissolving the powders at a concentration
of 5 mM in DMSO. To assess their effects on tubulin
assembly, porcine tubulin (2.7 mg/ml) was mixed with
different compounds (final concentration 10 lM) or
equal volume of the solvent (final 0.2% DMSO) at room
temperature in an assembly buffer minus GTP (100 mM
K-Pipes, pH 6.9, 2 mM EGTA, 1 mM MgCl2, and 10%
glycerol). The reaction mixtures were incubated at 37 ꢁC
for 15 min to allow slow binding drugs to bind to the
tubulin. The reaction mixtures were then chilled on
ice, GTP (final concentration, 1 mM) was added, and
they were incubated for 30 min at 37 ꢁC. At the end of
polymerization, unpolymerized and polymerized frac-
tions of tubulin were separated by centrifugation at
30,000g for 30 min at 30 ꢁC. The collected microtubules
were resuspended in SDS–PAGE sample buffer (2% w/v
SDS, 10% v/v glycerol, 5% v/v b-mercaptoethanol,
0.001% w/v bromophenol blue, and 62.5 mM Tris, pH
6.8) and the unpolymerized tubulin was diluted 3:1 with
4· SDS–PAGE sample buffer. Equal proportions of
each fraction were resolved by a 7.5% SDS-gel and
stained with Coomassie blue. Densitometric analyses
of stained gels were performed by using the ImageMas-
ter VDS Software (Pharmacia Biotech), and the ob-
tained data were elaborated using SigmaPlot 8.0
program (Systat Software Inc., Point Richmond, CA,
USA). At least three independent experiments were per-
formed with each compound. Differences between the
effects of the different compounds were evaluated by a
Student’s t-test for unpaired data with a confidence level
of 95%. To further analyze the effects of selected com-
5.2.3. Cytotoxicity by MTS assay. A549 cells were har-
vested 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.
Vindoline, vinorelbine, podophylotoxin, thiocholchi-
cine, 7-TES-baccatin III, 8–13, 18, and 19 were prepared
in medium at three different concentrations (1 lM,
10 lM, and 100 lM) and were added to the plates at a
volume of 100 ll/well. After 24 h of incubation 20 ll
of the CellTiter 96ꢂ and AQueous One Solution Reagent
(Promega Corporation, Madison, WI, USA) were added
to each well and the plates were incubated for 1 h at
37 ꢁC. The CellTiter 96ꢂ AQueous One Solution Reagent
contains a tetrazolium compound [3-(4,5-dimethylthi-
azol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-
nyl)-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 solution with MTS. The absorbance was read
at 490nm on a plate spectrophotometer (Victor3
TM
1420 Multilabel Counter, Perkin Elmer Instruments,
Shelton, USA). Cell cytotoxicity was expressed as
the percentage of the controls.
5.2.4. Cell cycle analysis. The effects of DMSO and 8 on
the cell cycle were studied using flow cytometry analysis.
Cells were plated in six-well sterile plastic plates at a
density of 105 ꢀ 2·105cells/well and were allowed to at-
tach for 24 h. After drug treatment cells were incubated
for 24 h at 37 ꢁC and in 5% CO2 atmosphere. Cells were
collected by trypsinization and DNA staining was per-
formed with the CycleTEST PLUS DNA Reagent Kit
(Becton Dickinson Immunocytometry Systems, San
Jose, CA, USA). According to the manufacturers’
instructions cells were washed with a buffer solution
containing sodium citrate, sucrose, and dimethyl sulfox-
ide (DMSO). Then cells were incubated with a sequence
of three steps: (a) 10 min at room temperature with
Solution A containing trypsin in a spermine tetrahydro-
chloride detergent buffer (to digest cell membranes and
cytoskeleton); (b) 10 min at room temperature with

6284
D. Passarella et al. / Bioorg. Med. Chem. 16 (2008) 6269–6285
Solution B containing a trypsin inhibitor and ribonucle-
ase A in citrate-stabilizing buffer with spermine tetrahy-
drochloride (to inhibit the trypsin activity and to digest
RNA); (c) 15 min in the refrigerator with Solution C
containing propidium iodide and spermine tetrahydro-
chloride in citrate-stabilizing buffer. Analysis was per-
formed using a FACScan (Becton Dickinson GmbH
Immunzytometrische Systeme, Heidelberg, Germany)
and data analysis was carried out with CELLQuest soft-
ware, while cell cycle distribution was determined using
Modifit software (Verity Software House, Inc.).
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The authors are grateful for the financial support pro-
`
vided by Ministero dell’Istruzione, dell’Universita e del-
la Ricerca (MIUR) COFIN 2001—2003 Program
`
‘Sostanze Naturali ed Analoghi Sintetici con Attivita
Antitumorale’. A.G. expresses her gratitude to INDE-
NA S.p.A. (Milano) for the PhD fellowship. This re-
search has been developed under the umbrella of CM
0602 COST Action ‘Inhibitors of Angiogenesis: design,
synthesis and biological exploitation’.
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Supplementary data
Supplementary data associated with this article can be
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