Novel 9′-substituted-noscapines: Synthesis with Suzuki cross-coupling, structure elucidation and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2014.07.050

Tubulin is a major molecular target for anticancer drugs. The dynamic process of microtubule assembly and disassembly can be blocked by various agents that bind to distinct sites on tubulin, usually its β-subunit. Among the antimitotic agents that perturb microtubule dynamics, noscapinoids represent an emerging class of agents. In particular, 9′-bromonoscapine (EM011) has been identified as a potent noscapine analog. Here we present high yielding, efficient synthetic methods based on Suzuki coupling of 9′-alkyl and 9′-arylnoscapines and an evaluation of their antiproliferative properties. Our results showed that 9′-alkyl and 9′-aryl derivatives inhibit proliferation of human cancer cells. The most active compounds were the 9′-methyl and the 9′-phenyl derivatives, which showed similar cytotoxic potency in comparison to the 9′-brominated derivative. Interestingly these newly synthesized derivatives did not induce cell death in normal human lymphocytes, suggesting that the compounds may be selective against cancer cells. All of these derivatives, except 9′-(2-methoxyphenyl)- noscapine, efficiently induced a cell cycle arrest in the G2/M phase of the cell cycle in HeLa and Jurkat cells. Furthermore, we showed that the most active compounds in HeLa cells induced apoptosis following the mitochondrial pathway with the activation of both caspase-9 and caspase-3. In addition, these compounds significantly reduced the expression of the anti-apoptotic proteins Mcl-1 and Bcl-2.

Full text of DOI:10.1016/j.ejmech.2014.07.050

European Journal of Medicinal Chemistry 84 (2014) 476e490
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 90-substituted-noscapines: Synthesis with Suzuki cross-
coupling, structure elucidation and biological evaluation
,
,
Elena Porcù a 1, Attila Sipos e 1, Giuseppe Basso a, Ernest Hamel b, Ruoli Bai b,
Verena Stempfer , Antal Udvardy , Attila Cs. Benyei e, Helmut Schmidhammer c,
Sandor Antus , Giampietro Viola
c
d
d,
ꢀ
f
a, *
ꢀ
a Department of Woman's and Child's Health, Laboratory of Oncohematology, University of Padova, Via Giustiniani 2, Padova 35128, Italy
b Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for
Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
c Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Austria
d Department of Physical Chemistry, University of Debrecen, Hungary
e Department of Pharmaceutical Chemistry, Medical and Health Science Center, University of Debrecen, Hungary
f Department of Organic Chemistry, University of Debrecen, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Tubulin is a major molecular target for anticancer drugs. The dynamic process of microtubule assembly
and disassembly can be blocked by various agents that bind to distinct sites on tubulin, usually its b-
Received 4 January 2014
Received in revised form
14 July 2014
Accepted 15 July 2014
Available online 16 July 2014
subunit. Among the antimitotic agents that perturb microtubule dynamics, noscapinoids represent an
emerging class of agents. In particular, 90-bromonoscapine (EM011) has been identified as a potent
noscapine analog. Here we present high yielding, efficient synthetic methods based on Suzuki coupling
of 90-alkyl and 90-arylnoscapines and an evaluation of their antiproliferative properties. Our results
showed that 90-alkyl and 90-aryl derivatives inhibit proliferation of human cancer cells. The most active
compounds were the 90-methyl and the 90-phenyl derivatives, which showed similar cytotoxic potency in
comparison to the 90-brominated derivative. Interestingly these newly synthesized derivatives did not
induce cell death in normal human lymphocytes, suggesting that the compounds may be selective
against cancer cells. All of these derivatives, except 90-(2-methoxyphenyl)-noscapine, efficiently induced
a cell cycle arrest in the G2/M phase of the cell cycle in HeLa and Jurkat cells. Furthermore, we showed
that the most active compounds in HeLa cells induced apoptosis following the mitochondrial pathway
with the activation of both caspase-9 and caspase-3. In addition, these compounds significantly reduced
the expression of the anti-apoptotic proteins Mcl-1 and Bcl-2.
Keywords:
Noscapine
Antimicrotubule agents
Apoptosis
Cell cycle arrest
Suzuki reaction
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
dynamics, whether their detailed mechanism of action involves
inhibition of tubulin assembly (vinca alkaloids, eribulin, estra-
Antimitotic agents, primarily of natural origin, are a class of
compounds that have been used for the treatment of a variety of
malignancies for many years. Although they are sometimes
considered “old chemotherapeutics” with respect to current anti-
cancer approaches [1,2], at the present time they still represent
valuable drugs that retain high scientific interest. Their impressive
success in patients is due to their potent anti-proliferative effects
and to their particular mechanism of action of altering microtubule
mustine, drugeantibody complexes with dolastatin 10 and may-
tansine analogues) or inhibition of microtubule disassembly
(taxoids, epothilones). The importance of microtubules in mitosis
and cell division, as well as the clinical success of microtubule
targeting drugs, has made these dynamic organelles one of the
most attractive targets for anticancer therapy [3]. In 1997, Ye et al.
[4] discovered the anti-cancer effect of noscapine [1, (ꢀ)-
a-nosca-
pine, (S)-6,7-dimethoxy-3-((R)-40-methoxy-60-methyl-50,60,70,80-
tetrahydro[1,3]dioxolo-[4,5]isoquinolin-50-yl)isobenzofuran-
1(3H)-one], formerly known as narcotine (Fig. 1), a phthalide iso-
quinoline alkaloid constituting 1e10% of the alkaloid content of
opium. The antimitotic activity of noscapine was characterized, and
it was demonstrated that the compound could inhibit tubulin
* Corresponding author.
E-mail address: Giampietro.viola.1@unipd.it (G. Viola).
Equal contributing authors.
1
http://dx.doi.org/10.1016/j.ejmech.2014.07.050
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
477
2. Chemistry
The literature procedure for the preparation of 90-bromono-
scapine (2) was published in 2003 by Zhou et al. [7]. This procedure
began with noscapine free base and used the 48% HBr-bromine
water reactant mixture for the bromination of noscapine at posi-
tion 90. Afterward, the alkalinization of the reaction mixture was
performed with cc. NH3 solution. The filtration of the crude product
was followed by recrystallization from 96% ethanol. In our hands,
this procedure gave rise to the desired 90-bromonoscapine in a
considerably lower yield (<10%), so we optimized the procedure in
several steps. The application of extraction after filtration of the
aqueous phase of the crude product resulted in a significant
amount of off-white product that was a mixture of 90-bromono-
scapine (2) and a large amount of unreacted noscapine (1).
Recrystallization from 96% ethanol yielded only a very small
amount of 2. The separation of noscapine 1 and its bromo derivative
2 was also attempted several times by column chromatography, but
the highly similar retention times of the two compounds prevented
their separation, even after trying many different mobile phases. 90-
Bromonoscapine (2) was prepared by a modified procedure [7] and
used as a starting material for the synthesis of new analogues
through the application of modern Pd-catalyzed cross-coupling
reactions.
This approach was found to be superior as solvation required
less HBr, and this led to an increase in product purity and yield. The
yield was further improved by processing the mother liquor and
using less base for the neutralization step. The concentration of the
mother liquor resulted in a significant amount of a light yellow
residue that was found to be 90-bromonoscapine (2) containing a
small amount of noscapine impurity. These modifications com-
bined afforded a total yield of 50% on average. The pure 90-bro-
monoscapine (2) we obtained, confirmed by 1H NMR, showed a
higher melting point than the reported value (174e175 ꢁC instead
of 169e170 ꢁC). The 90-bromonoscapine (2) obtained was used as a
starting agent for its transformation by the application of modern
Pd-catalyzed cross-coupling reactions (Suzuki reaction) into new
90-noscapine derivatives. The application of Suzuki cross-coupling
reaction in the synthesis and derivatization of alkaloids is an
excellent choice for the formation of new CeC bonds. For example,
substitutions in morphinans and aporphinoids were successfully
realized at different positions of the two alkaloid backbones
[14e16]. The first tested protocol with 2 was adapted from these
earlier procedures using K2CO3 as a base and Pd[P(Ph)3]4 as the Pd-
source combined with ligands. As summarized in Table 1, this
combination of reagents and reaction conditions (heating at 90 ꢁC
for 2 h) led to 4e12% percent yields after isolation by means of
column chromatography. After testing other generally used
Fig. 1. The structure of natural noscapine (1) and its 90-bromo derivative 2.
assembly. These workers therefore embarked on an intensive
search for more potent analogues of noscapine [4,5].
Most importantly, 90-bromonoscapine (2, EM011) was patented
[6], characterized, and introduced into clinical trials against non-
Hodgkin's lymphoma and chronic lymphocytic leukemia. A few
other 90-substituted noscapine derivatives were reported recently
[7e10]. For instance 90-nitronoscapine was found to be active
against drug-resistant ovarian cancer and T-cell lymphoma cells [8],
and its reduced congener, 90-aminonoscapine, has good activity as a
tubulin inhibitor [10].
Considering the increased cytotoxic activity of the 90-bromo
derivative of 1 as compared with the parent molecule, it is
important to know whether the substitution of the phtalideiso-
quinoline backbone in position 90 contributes to a conformational
change. Besides that, it is known from previous reports that
phtalideisoquinoline derivatives are sensitive to acidic or basic
media, and in numerous cases epimerization occurs [11,12].
Therefore, it was considered important to unambiguously
determine whether or not there is an epimerization reaction
during the relatively harsh bromination procedure. Thus, we
obtained crystals of 90-bromonoscapine (2) for X-ray crystallog-
raphy and investigated the circular dichroism of compound 2 in
solution. In addition, 90-bromonoscapine (2) prepared by a
modified procedure, was identified as a starting agent for the
synthesis of new analogues through the application of modern
Pd-catalyzed cross-coupling reactions (Scheme 1) [13]. These
new 90-alkyl and 90-arylnoscapines were evaluated for their
antiproliferative and anti-tubulin properties, as well as their
apoptotic mechanism of action.
Scheme 1. Synthesis of targeted 90-alkyl- and arylnoscapines 3e9.

478
E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
Table 1
Optimization of the Suzuki coupling of compound 2.
Compounds
Pd-source
Ligand
Base
Solvents
Yields (%)
3
3
4
4
5
5
6
6
7
7
8
8
9
9
Pd[P(Ph)3]4
Pd(OAc)2
Pd[P(Ph)3]4
Pd(OAc)2
Pd[P(Ph)3]4
Pd(OAc)2
Pd[P(Ph)3]4
Pd(OAc)2
Pd[P(Ph)3]4
Pd(OAc)2
Pd[P(Ph)3]4
Pd(OAc)2
Pd[P(Ph)3]4
Pd(OAc)2
e
K2CO3
K3PO4
K2CO3
K3PO4
K2CO3
K3PO4
K2CO3
K3PO4
K2CO3
K3PO4
K2CO3
K3PO4
K2CO3
K3PO4
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
THF/MeOH
4
44
12
53
8
61
4
41
9
55
4
42
7
XPhosa
e
Xphosa
e
Xphosa
e
XPhosa
e
Xphosa
e
XPhosa
e
Xphosa
57
a
XPhos: (2-biphenylyl)-dicyclohexyl-phosphine.
reagents and conditions, we found no significant improvement
with these common coupling methodologies. After examining the
single crystal structure of compound 2 (vide infra), especially the
steric hindrance in the proximity of the bromine substituent, we
turned to methods developed for the cross-coupling of hindered
aromatic bromides [17]. The application of Pd(OAc)2 as the Pd-
source, K3PO4 as the base and XPhos, a specific biphenyl phos-
phine, made possible the isolation of the desired products 3e8 in
significantly higher yields (Table 1).
2.1. Structural analysis of 90-bromonoscapine (2) and Suzuki
products 3e8
The structure of compound 2 in solution was investigated
with the use of circular dichroism (CD). In Fig. 2 (Panel A) the CD
spectra of parent compound 1 and its brominated congener 2 are
presented. The spectrum of (ꢀ)-
a-isomer 1 shows Cotton effects
Fig. 2. Panel A. CD spectra for natural noscapine 1 and its 90-bromo derivative 2 in
around 320 and 225 nm. These data are in accordance with the
most relevant and detailed evaluation of the circular dichroism
characteristics of phthalide isoquinoline alkaloids by Snatzke
et al. [18]. As concluded in this fundamental work, the configu-
ration of the C50 asymmetric centre could be associated with
Cotton effects at around 290 (1LB transition) and 205 nm (1B
aromatic transition), while the data related to the configuration
of the C3 carbon appear in the regions of 320 and 225 nm (ar-
omatic transitions). Comparing the CD spectra obtained for
compounds 1 and 2, it can be unambiguously stated that the
characteristics, the types and the positions of Cotton-effects
confirm the high conformational similarity between the two
molecules. On the basis of the X-ray crystal structure, the torsion
angle between the H3eC3eC50eH50 atoms can be measured
(Fig. 3, panel A). These data are considered an efficient indicator
for showing the relative positions of the tetrahydroisoquinoline
and isobenzofuranone ring systems of the phthalide isoquinoline
derivatives. The angle for natural noscapine base (1) was re-
ported to be ꢀ66ꢁ [19]. Interestingly, the protonation of the
tertiary amino function (formation of noscapine hydrochloride) is
followed by a remarkable conformational change characterized
by the important torsional angle of þ78ꢁ for the H3eC3eC50eH50
atomic connections [20]. As presented in Fig. 3 (panel B), the
acetonitrile. Panel B. CD spectra for natural (ꢀ)-
a-noscapine 1 and for representative
novel 90-alkyl and 90-arylnoscapines in acetonitrile.
was ꢀ80.1ꢁ. A recent study on the three dimensional chemical
space of a pharmacophore model for noscapinoids [21] allows us
to determine that the relative positions of hydrogen bond
acceptor O10 and C7eOCH3 oxygen atoms and the hydrophobic
C40eOCH3 and C6eOCH3 methyl centres are within 1.2 Å of
maximal distance. This was done after performing the overlay of
the X-ray structures of the free bases of noscapine (1) and 90-
bromonoscapine (2) by overlaying the structures of both the
tetrahydroisoquinoline and isobenzofuranone ring systems. In
order to prove that the Suzuki cross-coupling conditions did not
lead to epimerization of the original phthalide isoquinoline
structure (i.e., that the conformations of the synthesized com-
pounds were similar to the pharmacologically promising pre-
cursor 2), the circular dichroism spectra of compounds 3e8 were
recorded. The a-isomers of phthalide isoquinoline alkaloids show
Cotton effects typically around 320 and 225 nm. It can be
concluded from the set of CD spectra presented in Fig. 2 (panel
B), that in these regions the spectra show similar characteristics,
which is a proof of the retention of configuration at the two
chiral centers.
protonation evokes
a
significant twisting of the iso-
benzofuranone moiety relative to the tetrahydroisoquinoline
group. The modified synthesis of 90-bromonoscapine (2) allowed
us to obtain crystals of the compounds appropriate for single
crystal X-ray structural analysis (CCDC deposition # 955643)
(Fig. 3, panel C). It was determined that the indicative torsion
angle between H3eC3eC50eH50 for the 90-bromonoscapine
3. Results and discussion
3.1. Evaluation of antiproliferative activity
The newly synthesized noscapine derivatives, in comparison
with reference compound 2, were tested for their antiproliferative

E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
479
Fig. 3. The X-ray crystal structures of natural noscapine free base (1, Panel A), its HCl salt (Panel B) and 90-bromonoscapine (2, Panel C).
activity in a panel of human cancer cell lines. As shown in Table 2,
90-aryl derivatives 4 and 5 and the 90-alkyl derivative 3 present
higher values of IC50 respect to with 90-bromonoscapine (2).
Generally less active than 2, the three methoxyphenyl derivatives
6e8, as well as the 90-alkyl derivative 9, showed similar potencies
to each other. To obtain more insight into the potential cytotoxic
activity of these new compounds for normal human cells, they
were assayed in vitro against peripheral blood lymphocytes (PBL)
from healthy donors (Table 3). All the compounds were ineffective
3.2. Inhibition of tubulin polymerization and colchicine binding
The series of new noscapine analogues was first evaluated for
potential inhibition of tubulin assembly in comparison with the
potent colchicine site agent combretastatin A-4 (CA-4), using a
GTP- and glutamate-dependent polymerization assay that
measured extent of assembly after 20 min at 30 ꢁC (Table 4) [22]. It
was quickly apparent that, while some activity was observed with
several of the noscapine analogues, they were far less potent than
in resting lymphocytes, having IC50 values over 200
m
M, in well
CA-4, which yielded an IC50 value of 1.2
centrations up to 400 M were evaluated, an IC50 value was ob-
tained only with compound 5. The inhibition observed with 5 (IC50
120 M) was 100-fold weaker than the inhibition observed with
CA-4. However, besides IC50 values based on inhibition of the extent
of assembly, it is also possible to change the parameter measured
from the extent to the rate of assembly [22e24]. In our experience,
mM. In fact, when con-
agreement with previous reports [5,7]. Only some compounds (2, 4
and 6) proved cytotoxic in PHA-stimulated PBL, but only at higher
concentrations (generally, 5e30 fold higher) than against the
lymphoblastic cell lines Jurkat and CCRF-CEM. Together, these data
suggest these compounds may have cancer cell selective killing
properties.
m
,
m
Table 2
In vitro cell growth inhibitory effects of compounds 2e9.
a
IC50
(mM)
Compounds
HeLa
2.6 0.4
5.2 0.8
7.4 1.6
12.8 3.8
30.8 1.9
8.9 0.3
15.6 5.3
27.4 3.5
Jurkat
SEM
1.8 0.3
RS4; 11
CEM
2.8 0.19
8.8 3.1
5.2 0.8
11.8 2.2
6.9 1.5
22.3 4.7
25.8 1.7
23.3 3.4
LoVo
HT29
A549
IGROV-1
2
3
4
5
6
7
8
9
3.0 0.6
5.9 0.8
5.4 2.1
5.1 0.9
33.6 5.1
13.2 4.2
11.3 4.7
20.0 4.7
1.4 0.6
15.8 2.0
2.4 0.7
16.4 3.5
13.8 5.4
7.1 2.9
42.8 16.7
66.3 10.1
42.8 2.8
41.8 3.6
57.1 4.4
29.4 2.7
28.8 1.5
60.3 4.5
18.7 7.0
34.0 10.4
36.5 7.1
24.9 7.3
72.7 12.0
36.1 5.8
47.8 4.2
58.5 10.3
73.9 7.6
>100
55.3 3.2
22.2 7.9
59.7 5.3
>100
78.2 6.9
62.4 3.5
68.1 6.1
66.3 7.3
90.9 11.7
57.2 9.3
55.6 2.8
67.0 8.4
4.3 0.7
3.2 0.4
7.4 1.1
22.2 3.2
13.0 4.0
15.1 3.9
8.4 2.1
12.6 1.9
30.8 0.9
>100
48.1 6.2
a
IC50 ¼ compound concentration required to inhibit tumor cell proliferation by 50%. Data are presented as the mean SEM from the doseeresponse curves of at least three
independent experiments.

480
E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
Table 3
no significant activity when they were added to the reaction
mixture at 5 M (data not shown). However, we decided to evaluate
the compounds for potential inhibition at 500 M (Table 4). We did
observe inhibition reasonably concordant with the effects on
tubulin assembly. Thus, the best inhibition was observed with 5,
and the worst with the three compounds that had assembly rate
Cytotoxicity of noscapine derivatives for human peripheral blood lymphocytes
(PBL).
m
m
Compound
IC50
(
m
M)a
b
c
PBLresting
PBLPHA
2
3
4
5
6
7
8
9
>200
>200
>200
>200
>200
>200
>200
>200
92.2 13.8
>200
54.9 7.6
>200
42.4 5.0
>200
IC50 values over 400 mM (2, 4, and 6). We should note here that this
assembly assay uses reaction conditions that strongly stabilize the
colchicine binding activity of tubulin [16]. Nevertheless, we
generally measure inhibition of colchicine binding at short reaction
times, such as 10 min, when the reaction is 40e60% complete,
because we have found that inhibition by weakly active compounds
is maximal at shorter incubation times [27]. This is probably
because most colchicine site agents dissociate from tubulin much
more rapidly than colchicine itself [27,28]. Consequently, once
bound, [3H]colchicine in effect locks other agents out of the binding
site. Under the reaction conditions used here, the half-life of the
colchicineetubulin complex is about 24 h [28]. The difference in
reaction conditions and incubation time probably explains the
limited effects reported for noscapine on the binding of [3H]
colchicine to tubulin [4]. We should note, however, that a recent
report [29] described inhibition of colchicine binding, measured by
inhibition of the fluorescence that occurs when colchicine binds to
tubulin [30], by 90-bromonoscapine (2), although noscapine (1) it-
self had no activity at the highest concentration examined
>200
>200
a
Compound concentration required to reduce cell growth by 50%.
PBL not stimulated with PHA.
PBL stimulated with PHA. Values are the mean
b
c
SEM for three separate
experiments.
this has the effect of reducing the IC50 value for compounds 2e4-
fold, thus permitting measurements of IC50's for weakly active
compounds and, potentially, for compounds that induce aberrant
polymer formation at higher concentrations, which is generally
associated with turbidity development that can only be distin-
guished from “normal” assembly by electron microscopy [25,26].
Our concentration studies with the noscapine analogues did not
provide any evidence for an aberrant assembly reaction, since we
only observed progressive inhibition, albeit very weak. In addition,
it should be noted that the typical microtubule assembly curve, as
measured by turbidimetry, has a sigmoidal shape. We therefore
measured the maximum rate of assembly at the inflection points of
the turbidity curves. This resulted in our obtaining 3-fold re-
(100 mM).
3.3. Effects of 90-noscapine derivatives on the cellular microtubule
network
We investigated the effects of the new derivatives on the cyto-
skeletal microtubule and microfilament networks by immunoflu-
orescence in HeLa cells. As shown in Fig. 4, the microtubule
network exhibited normal arrangement and organization in HeLa
cells in the absence of drug treatment. In contrast, 24 h of exposure
ductions in the IC50 values for CA-4 (0.44 mM) and 5 (45 mM), but, in
addition, we obtained maximum rate IC50 values for two com-
pounds (3 and 7) (Table 4). Only, 2, 4, and 6 failed to yield rate IC50
values.
Because noscapine has structural similarity to colchicine, it was
evaluated without success as a potential inhibitor of [3H]colchicine
binding to tubulin [4]. With most active colchicine site inhibitors,
we have been able to demonstrate significant inhibition of the
to compounds 2, 3 or 7 at 25 mM caused extensive microtubule
rearrangement, with induction of spherical morphology in 70e80%
of the cells. In addition, the treatment also induced mitotic arrest,
characterized at the concentrations and time studied, by an in-
crease in the number of cells with typical bipolar spindles with
chromosomes arranged along the metaphase plate. We also
observed abnormal microtubule spindles with astral or multipolar
configurations as well as a disorganized or spherical arrangement
of chromosomes. In good agreement with microtubule rearrange-
ment, we also observed PCM1 alteration, after treatment of cells
with the new compounds (effects of compounds 2, 3 and 5 shown
in Fig. 5). PCM1 is a pericentriolar protein involved in recruiting
proteins necessary for centrosome replication, and it dynamically
fluctuates during the cell cycle. Late in G2, the protein dissociates
from the centrosome remaining dispersed throughout the cell
during mitosis. The mechanism is cell cycle dependent, with PCM1
aggregates disassembling during mitosis and reassembling in
interphase [31,32].
As shown in Fig. 5, PCM1 staining revealed an accumulation of
the pericentriolar material in some treated cells, similar to the ef-
fects observed after treatment with nocodazole and other antimi-
totics [33]. Moreover, cells arrested in mitosis showed the
characteristic rounded shape, and PCM1 was scattered and
dispersed throughout the cell. The derivatives induced a depletion
of PCM1 function, affecting its localization and the organization of
cell cycle machinery, resulting in microtubules anchoring to the
binding of [3H]colchicine to tubulin, with tubulin at 1
m
M and
colchicine and the inhibitor at 5
mM. This is shown in Table 4 for CA-
4, although few compounds are as potent as CA-4 in inhibiting this
reaction. With the noscapine analogues, in agreement with the
findings with noscapine, [4] in a preliminary experiment we found
Table 4
Inhibition of tubulin polymerization and colchicine binding by compounds 1e7 and
CA-4.
Compound
Tubulin assemblya
Colchicine bindingb
IC50 S.D (
m
M)
%
S.D.
Extent
Rate
5
m
M drug
500
44
mM drug
1
2
3
4
5
6
7
CA-4
>400
>400
>400
>400
170
>400
220 50
>400
4
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
4
34 4c
39 2c
14 1c
120
3
45
8
64
4
>400
>400
1.2 0.09
>400
170 20
0.44 0.03
13 5c
38
4
99 0.4
n.d.
a
Inhibition of tubulin polymerization. Tubulin was at 10
m
M.
b
Inhibition of [3H]colchicine binding. Tubulin and colchicine were at 1 and 5
m
M,
respectively, and the tested compound was at the indicated concentration. n.s., not
shown, and n.d., not determined.
centrosome. The derivatives also induced
a change in cell
morphology detectable by staining with phalloidin (Fig. 5). Actin
filaments were intact but disorganized as compared with the
control cells, contributing to impairment of cell organization.
c
In the indicated samples, turbidity was observed in the reaction mixtures,
implying compound precipitation and a saturating, but unknown, concentration of
the added compound.

E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
481
Fig. 4. Effects of compounds 2, 3 and 7 on microtubule networks in HeLa cells. Cells were incubated with 25
antibody and secondary Alexa-conjugated antibody and then observed by confocal microscopy (magnification 20ꢂ, bar ¼ 10
visualize the nuclei. Arrows indicate cells with disorganized spherical arrangements of chromosomes or multipolar microtubules.
m
M compound for 24 h and then stained with anti-
b
-tubulin primary
m
m). Cells were also counterstained with DAPI to
3.4. 90-Noscapine derivatives induce G2/M arrest of the cell cycle
CA-4, 38% with compound 3, and 28% with compound 7 indi-
cating that they acted like antitubulin agents. We next studied
the effects of compounds 2e5, on alterations in the expression of
proteins that regulate cell division. The cdc2/cyclin B complex
controls both entry into and exit from mitosis. Phosphorylation
of cdc2 on Tyr15 and phosphorylation of cdc25c phosphatase on
Ser216 negatively regulate the activation of the cdc2/cyclin B
complex [34e36]. Thus, dephosphorylation of these proteins is
needed to activate the cdc2/cyclin B complex. Cdc25c is a major
phosphatase that dephosphorylates the site on cdc2 and auto-
dephosphorylates itself. Phosphorylation of cdc25C directly
stimulates both its phosphatase and autophosphatase activities, a
condition necessary to activate cdc2/cyclin B on entry of cells
into mitosis [34e36]. As shown in Fig. 8 in HeLa cells, treatment
The effects of a 24 h treatment with different concentrations
of compounds 2e7 on cell cycle progression in HeLa (Fig. 6) and
Jurkat cells (Fig. 7), were determined by flow cytometry. All
compounds, except 6, caused a significant G2/M arrest in a
concentration-dependent manner in both cell lines, with the new
compounds having effects essentially identical to those observed
with the reference compound 2. In HeLa cells (Fig. 6), the rise in
G2/M cells occurred maximally at a concentration between 10
and 25 mM, while at higher concentrations more than 80% of the
cells were arrested in G2/M. A similar behavior was observed
with the Jurkat cells (Fig. 7), but, except with compound 2,
maximal effects required higher compound concentrations. As
would be expected, the cell cycle arrest in G2/M phase was
accompanied by a commensurate reduction in cells in the other
phases of the cell cycle. We also examined several of the com-
pounds, in comparison with CA-4, in human Burkitt lymphoma
CA46 cells since this cell line generally yield a very high mitotic
index when treated with antitubulin agents. Compounds 3 and 7
with 3, 4 or 5 at 25
mM for 24 or 48 h caused an increased
expression of cyclin B at 24 h, in particular for compounds 5,
followed by its disappearance at 48 h. Similarly, slower migrating
forms of phosphatase cdc25C appeared at 24 h following treat-
ment with 4 or 5, indicating changes in the phosphorylation state
of this protein, while at 48 h with these compounds, as well as
compound 2, the expression of cdc25c strongly decreased. We
also observed a dramatic reduction in the expression of the
phosphorylated form of cdc2 (Tyr15) with all tested compounds.
We also examined the effect of the new derivatives on the
expression of aurora kinase A and its phosphorylated form at
yielded IC50 values of 18
8
m
M and 20
4 mM, respectively (the
contemporaneously obtained value for CA-4 was 20 7 nM). At
five times the IC50 concentrations, there was over 80% G2/M cells
with all three compounds. Morphological examination of parallel
cultures stained with Giemsa yielded mitotic indices of 46% with

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E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
Fig. 5. Effects of indicated compounds on PCM1 in HeLa cells. Cells were incubated for 24 h with the indicated compounds (25
and secondary Alexa-conjugated antibody, phalloidin-tetramethylrhodamine B to visualize actin microfilaments and DAPI. Images were captured by confocal microscopy
(magnification 60ꢂ, bar ¼ 10 m). Arrows indicate points of PCM1 accumulation or diffused in mitotic cells.
mM) and stained with anti-PCM1-primary antibody
m
T288 since the phosphorylation of aurora kinase A is a hallmark
of the G2/M phase [37]. As shown in Fig. 8, aurora kinase levels
increased after a 24 h treatment with compounds 3, 4 and 5, but
not 2. Altogether these data suggest, as did the immunofluores-
cence studies, that the observed G2/M arrest was not due to a
defect in G2 to M phase progression but instead was caused by
aberrant execution of mitosis.
3.6. 90-Noscapine derivatives induce apoptosis through the
mitochondrial pathway
Mitochondria play an essential role in the propagation of
apoptosis [41]. It is well established that, at an early stage, apoptotic
stimuli alter the mitochondrial transmembrane potential (Djmt
)
[42]. Djmt was monitored by the fluorescence of the dye JC-1. As
shown in Fig. 10 (Panels AeD), compounds 2e5 induced a time and
concentration-dependent increase in the proportion of cells with
depolarized mitochondria. Mitochondrial membrane depolariza-
tion is associated with mitochondrial production of ROS [43].
Therefore, we investigated whether ROS production increased after
treatment with 2e5. We analyzed the production of ROS by flow
cytometry utilizing the fluorescence indicator H2-DCFDA. The re-
sults presented in Fig. 11 (Panels AeD) show that all the tested
compounds induced the production of significant amounts of ROS
in comparison with control cells, which agrees with the previously
described dissipation of Djmt. Altogether, these results indicate
that these compounds induced apoptosis through the mitochon-
drial pathway in good agreement with previous reports [44e46].
Prolonged mitotic arrest can lead to DNA damage [38,39]. We
identified DNA damage through the detection of the phosphory-
lated histone
increase in the expression of
g
-H2A.X. Compounds 3, 4 and 5 induced a marked
-H2A.X after a 24 h treatment, while
g
a similar increase was observed after 48 h with compound 2, sug-
gesting that these compounds induce major DNA damage during
mitotic arrest that could contribute to their antiproliferative
activity.
3.5. 90-Noscapine derivatives induce apoptosis
To characterize the mode of cell death induced by these com-
pounds, a biparametric cytofluorimetric analysis was performed
using PI, which stains DNA and enters only dead cells, and fluo-
rescent immunolabeling of the protein annexin-V, which binds to
PS in a highly selective manner [40]. Compounds 2e5 were incu-
bated with HeLa cells for 24 or 48 h and then stained with the two
dyes. Dual staining with annexin-V and with PI permits discrimi-
nation between live cells (annexin-Vꢀ/PIꢀ), early apoptotic cells
(annexin-Vþ/PIꢀ), late apoptotic cells (annexin-Vþ/PIþ) and
necrotic cells (annexin-Vꢀ/PIþ). As depicted in Fig. 9 (Panels AeE),
the treated HeLa cells showed an accumulation of annexin-V pos-
itive cells in comparison with the control, in a concentration and
time-dependent manner. In good agreement with MTT data, the
reference compound 2 was slightly more active than the other
three compounds.
3.7. Effect of 90-alkyl and 90-arylnoscapines on caspase activation
The activation of caspases plays a central role in the process of
apoptotic cell death [47]. Synthesized as proenzymes, caspases are
themselves activated by specific proteolytic cleavage reactions.
Caspases-2, -8, -9, and -10 are termed initiator caspases and are
usually the first to be activated in the apoptotic process. Following
their activation, they in turn activate effector caspases, in particular
caspase-3 [48]. As shown in Fig. 12 (Panel A), all tested compounds
induced proteolytic cleavage of caspase-9 and caspase-3, in good
agreement with the mitochondrial depolarization described above.
The DNA repair enzyme PARP is cleaved by caspase-3 from its full

E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
483
Fig. 6. Percentage of cells in each phase of the cell cycle in HeLa cells, treated with the indicated compound at different concentrations for 24 h. Cells were fixed and labeled with PI
and analyzed by flow cytometry as described in the experimental section. Data are presented as mean SEM of three independent experiments.
length 116 kDa form to an inactive 85 kDa form. We also observed
that PARP cleavage was detectable after 24 h and 48 h treatments.
Altogether, these results showed that apoptosis induced by the 90-
noscapine derivatives was caspase-dependent and followed the
intrinsic (mitochondrial) pathway. These findings are in good
agreement with those of Aneja et al. who found that 90-bromono-
scapine induces mitochondrial depolarization followed by caspase-
dependent apoptosis in both human prostate cancer cells and
leukemia cells [49,50].
treatment, however. Bcl-2 expression was decreased with all four
compounds (2e5). The pro-apoptotic protein Bax was essentially
unchanged after either 24 or 48 h treatments. Mcl-1 is an anti-
apoptotic member of the Bcl-2 family, and recently it was re-
ported that sensitivity to antimitotic drugs is regulated by Mcl-1
levels [51,52]. As shown in Fig. 12 (Panel B), the Mcl-1 band was
strongly reduced after the 24 h treatment with compounds 2, 3 and
4, but not 5. After the 48 h treatment, the Mcl-1 band disappeared
with all four compounds. It has recently emerged that Mcl-1 acts as
a controller of the apoptotic timing response during mitotic arrest
[51,52]. When Mcl-1 levels fall, Bak and Bax form pores in the
mitochondrial membrane, resulting in the release of cytochrome c,
mitochondrial depolarization and caspase activation that ulti-
mately lead to apoptosis [52].
3.8. Effect on proapoptotic proteins and IAP expression
There is increasing evidence that regulation of the Bcl-2 family
proteins shares the signaling pathways induced by antimicrotubule
compounds [44]. Several pro-apoptotic family proteins (e.g., Bax,
Bid, Bim and Bak) promote the release of cytochrome c, whereas
anti-apoptotic members (Bcl-2, Bcl-XL and Mcl-1) are capable of
antagonizing the pro-apoptotic proteins and preventing the loss of
mitochondrial membrane potential [44]. As shown in Fig. 12 (Panel
B), after a 24 h treatment, Bcl-2 expression was reduced with
compounds 2 and 3, but not with compounds 4 and 5. After a 48 h
4. Conclusion
We devised an efficient new synthesis for noscapine derivatives
modified at position 90, using Suzuki cross-coupling, a specific
palladium-catalyzed carbonecarbon bond formation reaction, on
the phthalide isoquinoline backbone. The ligands examined here

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E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
Fig. 7. Percentage of cells in each phase of the cell cycle in Jurkat cells, treated with the indicated compound at different concentrations for 24 h. Cells were fixed and labeled with PI
and analyzed by flow cytometry as described in the experimental section. Data are presented as mean SEM of three independent experiments.
were chosen to study the general utility of the reaction and, equally
importantly, to obtain new, potentially cytotoxic noscapine de-
rivatives. As shown above, 90-bromonoscapine (2) has a very similar
conformation in solid phase as the noscapine free base (1). This
suggests that cytotoxic activity is a consequence of electronic rather
than structural effects.
We also showed that the new 90-substituted noscapines shared
common properties with the lead compound 2 and are efficacious
as antiproliferative agents in different cancer cell lines. It is
worthwhile to note that our compounds present higher cytotox-
icity in comparison to other noscapine derivatives recently syn-
thesized such as 60-substituted [53] but similar to that of a series of
analogues modified in position 7 of the benzofuranone ring [54].
Moreover, they are also strong apoptosis inducers that follow the
mitochondrial intrinsic pathway. They did not show any appre-
ciable activity on normal human lymphocytes, suggesting a low
toxicity profile. It is worth noting that the replacement of bromine
Fig. 8. Effects of 2e5 on G2/M regulatory proteins. HeLa cells were treated for 24 or
with a methyl group or a more bulky substituent such as 4-
48 h with the indicated compound at 25
detection of cyclin B, p-cdc2Y15
cdc25C and
analysis. To confirm equal protein loading, each membrane was stripped and reprobed
with anti- -actin antibody.
mM. The cells were harvested and lysed for the
methylphenyl (compound 5) did not substantially modify the
cytotoxic potency in comparison with 2, suggesting the existence of
a large binding pocket in tubulin. From the point of view of the
,
g
H2A.X expression by western blot
b

E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
485
Fig. 9. Flow cytometric demonstration of apoptosis by compounds 2, 3, 4 and 5. A. Representative biparametric histograms obtained after a 24 h incubation of HeLa cells with the
indicated compound at 25 mM. The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow cytometry. In these histograms, the lower left-hand segment
represents the annexin-Vꢀ/PIꢀ cells, the lower right-hand segment the annexin-Vþ/PIꢀ cells, the upper right-hand segment the annexin-Vþ/PIþ cells, and the upper left-hand
segment the annexin-Vꢀ/PIþ cells. Percentage of cells found in the different regions of the biparametric histograms shown in panel A and analogous histograms obtained after
48 h incubations are shown for compounds 2 (Panel B), 4 (panel C), 5 (panel D) or 3 (panel E) at the indicated concentrations. Data shown in panels BeE are presented as
mean SEM of three independent experiments.
inhibition of tubulin polymerization in vitro, 90-bromonoscapine
and related derivatives did not show potent activity. The most
active compound had an IC50 of 120 mM, 100 fold weaker than that
observed for CA-4. However, our results indicate, as have those
presented by other workers, that noscapine and, in particular, 90-
bromonoscapine may produce subtle effects on microtubule dy-
namics that could interfere, for example, with the proper attach-
ment of chromosomes to the kinetochore microtubules [55] instead
of strongly binding to tubulin as do most well-studied antimitotic
drugs. In accord with this idea, it was recently shown [55] that 90-
bromonoscapine did not perturb the morphology of microtubules
but rather induced alterations in the centrosome duplication cycle
and caused inappropriate centrosome amplification. Our data
showed that 90-substituted noscapine induce accumulation of
round cells with condensed DNA indicative of mitotic arrest and in
addition, PCM1 alteration disrupted the radial organization of mi-
crotubules. We also found that compounds 3e5 induced a sub-
stantial increase in the expression of the phosphorylated form of
H2AX that is indicative of DNA damage. This could be due to a
protracted mitotic arrest that ultimately led to cell death. In

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E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
Fig. 10. Assessment of mitochondrial membrane potential (Djmt) after treatment of HeLa cells with compounds 2e5. Cells were treated with different concentration of the
indicated compounds for 24 or 48 h and then stained with the fluorescent probe JC-1. Data are expressed as mean SEM for three independent experiments.
conclusion, all these findings indicate that the noscapine de-
rivatives have good therapeutic potential and merit further
investigation.
with a Kofler hot-stage apparatus and are uncorrected. Thin-layer
chromatography was performed on pre-coated Merck 5554 Kie-
selgel 60 F254 foils using CH2Cl2:methanol ¼ 8:2 mobile phase.
Column chromatography was performed on silica gel 60 (Mesh
230e400 ASTM, grading 0.04e0.063) of the Merck VWR company
5. Experimental protocol
as stationary phase. As
a mobile phase the mixture of
CH2Cl2:methanol ¼ 9:1 was used for each product.
5.1. Chemistry
The spots were visualized with Dragendorff's reagent. 1H NMR
spectra were recorded on a Bruker Avance DRX 400 spectrometer,
5.1.1. Materials and methods
chemical shifts are reported in parts per million (
TMS, and coupling constants (J) are measured in Hertz. Elemental
d) from internal
Starting materials were purchased from SigmaeAldrich and
used without further purification. Melting points were determined
Fig. 11. Mitochondrial production of ROS in HeLa cells following treatment with compounds 2e5. After 24 or 48 h incubations with the indicated compounds at the indicated
concentration, HeLa cells were stained with H2-DCFDA and analyzed by flow cytometry. Data are expressed as mean SEM of three independent experiments.

E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
487
(C7a); 118.0 (C5); 116.4 (C4); 116.3 (C90); 100.7 (C20); 82.1 (C3); 69.7
(C50); 61.2 (OCH3); 59.7 (OCH3); 57.1 (OCH3); 49.4 (C70); 46.0
(NCH3); 24.1 (C80); 11.2 (C90-CH3); IR (KBr pellet, cmꢀ1): 3448;
2963; 1762; 1262; 1092; 1023; calculated for C23H25NO7: C, 64.63;
H, 5.90; N, 3.28; found: C, 64.69; H, 5.99; N, 3.34.
5.2.2. 90-Phenylnoscapine (4)
Light yellow crystals; m.p.: 78e80 ꢁC; 1H NMR (CDCl3)
d
¼ 7.45e7.21 (5H, m, ar); 7.10 (1H, d, H5, J4e5 ¼ 6.7); 6.15 (1H, d, H4,
J4e5 ¼ 6.7); 5.98 (2H, d, 2H20, J ¼ 18.8); 5.58 (1H, d, H3, J5 e3 ¼ 4.9);
0
4.52 (1H, d, H50, J5 e3 ¼ 4.9); 4.13 (3H, s, OCH3); 4.12 (3H, s, OCH3);
0
3.92 (3H, s, OCH3); 2.58 (3H, s, NCH3); 2.38e2.18 (4H, m, H70 , H70
a b,
H80a, H80
b
); 13C NMR (CDCl3)
d
¼ 173.3 (C]O); 157.6 (C6); 154.0
(C10a); 148.2 (C40); 148.0 (C7); 142.3 (ar); 137.9 (C3a); 135.8 (C30a);
132.8 (C80a); 130.2 (2 ar); 129.5 (2 ar); 127.8 (ar); 118.2 (C40a); 117.6
(C7a); 115.9 (C5); 112.1 (C4); 108.4 (C90); 101.1 (C20); 82.2 (C3); 68.2
(C50); 61.4 (OCH3); 59.8 (OCH3); 57.2 (OCH3); 51.1 (C70); 47.0
(NCH3); 24.2 (C80); IR (KBr pellet, cmꢀ1): 3443; 2963; 1762; 1261;
1091; 1035; calculated for C28H27NO7: C, 68.70; H, 5.56; N, 2.86;
found: C, 68.69; H, 5.61; N, 2.91.
5.2.3. 90-(4-Methylphenyl)-noscapine (5)
Yellow amorphous solid; 1H NMR (CDCl3)
d
¼ 8.14 (2H, d, ar);
Fig. 12. Effect of 2e5 on caspase activation (panel A) and on Bcl-2, Mcl-1 and Bax
proteins (panel B) in HeLa cells. Cells were treated for 24 or 48 h with the indicated
compounds at 25 mM. The cells were harvested and lysed for the detection of pro-
caspase-9, pro-caspase-3, PARP, Mcl-1, Bcl-2 and Bax expression by western blot
analysis. To confirm equal protein loading, each membrane was stripped and reprobed
with anti-b-actin antibody.
7.35e7.16 (2H, m, ar); 7.04 (1H, d, H5, J4e5 ¼ 7.1); 6.17 (1H, d, H4,
J4e5 ¼ 7.1); 5.95 (2H, d, 2H20, J ¼ 15.2); 5.61 (1H, d, H3, J5 e3 ¼ 4.5);
0
4.47 (1H, d, H50, J5 e3 ¼ 4.5); 4.14 (3H, s, OCH3); 4.11 (3H, s, OCH3);
0
3.93 (3H, s, OCH3); 2.59 (3H, s, NCH3); 2.41 (3H, s, C400-CH3);
2.34e2.07 (4H, m, H70 , H70 , H80a, H80 ); 13C NMR (CDCl3)
a b b
d
¼ 167.4 (C]O); 152.3 (C6); 151.7 (C10a); 151.1 (C40); 148.3 (C7);
141.9 (ar); 139.9 (C3a); 138.8 (ar); 135.6 (C30a); 131.8 (C80a); 129.8
(2 ar); 129.0 (2 ar); 117.9 (C40a); 117.7 (C7a); 115.0 (C5); 111.8 (C4);
109.8 (C90); 100.8 (C20); 82.0 (C3); 69.9 (C50); 61.1 (OCH3); 59.6
(OCH3); 56.9 (OCH3); 51.0 (C70); 46.8 (NCH3); 25.1 (C80); 21.7 (C400-
CH3); IR (KBr pellet, cmꢀ1): 3448; 2962; 1760; 1262; 1084; 1034;
calculated for C29H29NO7: C, 69.17; H, 5.80; N, 2.78; found: C, 69.30;
H, 5.91; N, 2.71.
analyses (C, H, N) were obtained on a Carlo Erba EA1108 analyzer.
Optical rotation data were obtained from a PerkineElmer 341
polarimeter. CD spectra were recorded on
a J-810 spec-
tropolarimeter. The CD spectra were measured in millidegrees and
normalized into
grade CH3CN.
D l [nm] units in spectroscopic
εmax [l molꢀ1 cmꢀ1]/
Crystallography data for compound 2 were collected on a Bru-
kereNonius MACH3 diffractometer at 293 K, Mo K radiation
¼ 0.71073 Å, motion. All non-hydrogen atoms were refined
anisotropically.
a
5.2.4. 90-(2-Methoxyphenyl)-noscapine (6)
l
u
Pale brown amorphous product; m.p.: 192e195 ꢁC; 1H NMR
(CDCl3)
d
¼ 7.34e6.89 (5H, m, ar, H5); 6.26 (1H, d, H4, J4e5 ¼ 7.1);
5.85 (2H, s, 2H20); 5.58 (1H, d, H3, J5 e3 ¼ 5.0); 4.41 (1H, d, H50,
0
5.2. General procedure for the Suzuki coupling of 90-
bromonoscapine (2)
0
J5 e3 ¼ 5.0); 4.04 (6H, s, 2 OCH3); 3.81 (3H, s, OCH3); 3.75 (3H, s,
OCH3); 2.49 (3H, s, NCH3); 2.21e1.98 (4H, m, H70 , H70 , H80a,
a b
H80
b
); 13C NMR (CDCl3)
d
¼ 168.7 (C]O); 157.9 (ar); 153.1 (C6);
A mixture of 150 mg 90-bromonoscapine (2, Mr 430.1 g/mol;
0.35 mmol), 0.452 mmol of the respective boronic acid, 3 mg pal-
ladium(II)acetate (Mr 177.4 g/mol; 0.016 mmol) and 6 mg (2-
biphenylyl)-dicyclohexylphosphine (Mr 350.2 g/mol; 0.016 mmol)
was dissolved in 6.35 mL THF/methanol (4:1). To the stirred solu-
151.8 (C10a); 148.3 (C40); 147.9 (C7); 145.0 (ar); 139.0 (C3a); 134.3
(C30a); 132.4 (C80a); 130.8 (ar); 128.4 (ar); 119.6 (2 ar); 117.5 (C40a);
117.3 (C7a); 116.8 (C5); 111.6 (C4); 110.0 (C90); 99.8 (C20); 81.4 (C3);
66.4 (C50); 60.2 (OCH3); 58.5 (OCH3); 55.9 (OCH3); 54.5 (OCH3);
50.0 (C70); 45.9 (NCH3); 25.0 (C80); IR (KBr pellet, cmꢀ1): 3447;
2962; 1753; 1261; 1114; 1083; 1036; calculated for C29H29NO8: C,
67.04; H, 5.63; N, 2.70; found: C, 67.12; H, 5.58; N, 2.54.
tion, 300 ml K3PO4 solution (2 M) was added at room temperature.
The temperature was increased to 80 ꢁC, and the reaction mixture
was refluxed for 2e5 h. Every hour the progress of the reaction was
followed by TLC. The reaction was stopped when there was no
further progress, and the mixture was evaporated under reduced
pressure. Afterward, the solid mixture was separated using column
chromatography. The product was then analyzed.
5.2.5. 90-(3-Methoxyphenyl)-noscapine (7)
Off-white amorphous product; m.p.: 155e157 ꢁC; 1H NMR
(CDCl3)
d
¼ 7.47e6.95 (5H, m, ar, H5); 6.24 (1H, d, H4, J4e5 ¼ 8.0);
5.83 (2H, s, 2H20); 5.58 (1H, d, H3, J5 e3 ¼ 4.8); 4.44 (1H, d, H50,
0
5.2.1. 90-Methylnoscapine (3)
J5 e3 ¼ 4.8); 4.07 (6H, s, 2 OCH3); 3.81 (3H, s, OCH3); 3.71 (3H, s,
0
Amorphous yellow solid; m.p.: 147e149 ꢁC; 1H NMR (CDCl3)
OCH3); 2.44 (3H, s, NCH3); 2.29e2.00 (4H, m, H70 , H70 , H80a,
a b
d
¼ 6.98 (1H, d, H5, J4e5 ¼ 7.0); 6.13 (1H, d, H4, J4e5 ¼ 7.0); 5.93 (2H,
H80
b
); 13C NMR (CDCl3)
d
¼ 166.4 (C]O); 161.8 (ar); 152.8 (C6);
s, 2H20); 5.56 (1H, d, H3, J5 e3 ¼ 4.5); 4.40 (1H, d, H50, J5 e3 ¼ 4.5);
151.8 (C10a); 148.4 (C40); 147.3 (C7); 144.4 (ar); 138.6 (C3a); 134.4
(C30a); 134.1 (C80a); 131.0 (ar); 128.4 (ar); 120.6 (2 ar); 118.0 (C40a);
117.6 (C7a); 117.0 (C5); 112.4 (C4); 110.9 (C90); 99.5 (C20); 81.8 (C3);
66.6 (C50); 61.0 (OCH3); 59.4 (OCH3); 56.7 (OCH3); 55.5 (OCH3); 50.9
(C70); 44.4 (NCH3); 23.0 (C80); IR (KBr pellet, cmꢀ1): 3441; 2955;
0
0
4.09 (3H, s, OCH3); 3.96 (3H, s, OCH3); 3.87 (3H, s, OCH3); 2.76e2.33
(7H, m, NCH3, H70 , H70 , H80a, H80 ); 2.06 (3H, s, C90-CH3); 13C
a b b
NMR (CDCl3)
d
¼ 168.4 (C]O); 153.1 (C6); 151.7 (C10a); 150.1 (C40a);
147.4 (C7); 140.1 (C3a); 135.9 (C30a); 131.0 (C80a); 121.7 (C40a); 118.3

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E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
1750; 1270; 1111; 1080; 1036; calculated for C29H29NO8: C, 67.04; H,
5.63; N, 2.70; found: C, 67.00; H, 5.69; N, 2.81.
and varying compound concentrations were preincubated 15 min
at 30 ꢁC in 0.24 mL. The mixtures were then chilled on ice, and 10
m
L
of 10 mM GTP was added (final concentration, 0.4 mM). All con-
centrations refer to the final volume of 0.25 mL. The samples were
transferred to 0 ꢁC cuvettes in the spectrophotometers, and the
temperature was jumped to 30 ꢁC over about 1 min. Either the
maximum reaction rate (occurring at varying times) or the extent of
assembly after 20 min was measured. The colchicine binding assay
was measured as described in detail previously [57]. Reaction
5.2.6. 90-(4-Methoxyphenyl)-noscapine (8)
Off-white amorphous product; m.p.: 167e169 ꢁC; 1H NMR
(CDCl3)
d
¼ 7.62e7.55 (2H, m, ar); 7.12e7.05 (3H, m, ar, H5), 6.28
(1H, d, H4, J4e5 ¼ 7.0); 5.81 (2H, s, 2H20); 5.54 (1H, d, H3, J5 e3 ¼ 5.0);
0
4.47 (1H, d, H50, J5 e3 ¼ 5.0); 4.00 (6H, s, 2 OCH3); 3.81 (3H, s, OCH3);
0
3.78 (3H, s, OCH3); 2.44 (3H, s, NCH3); 2.30e1.94 (4H, m, H70 , H70
a b,
H80a, H80
b
); 13C NMR (CDCl3)
d
¼ 168.4 (C]O); 159.1 (ar); 153.1
mixtures (0.1 mL) contained 1.0
mM tubulin, 5.0
m
M [3H]colchicine
M, 5%
(C6); 152.0 (C10a); 148.7 (C40); 148.0 (C7); 145.4 (ar); 139.0 (C3a);
134.3 (C30a); 132.4 (C80a); 130.8 (2 ar); 128.4 (ar); 119.6 (ar); 117.5
(C40a); 117.3 (C7a); 116.8 (C5); 111.6 (C4); 110.0 (C90); 99.8 (C20);
81.4 (C3); 66.4 (C50); 60.2 (OCH3); 58.5 (OCH3); 55.9 (OCH3); 54.5
(OCH3); 50.0 (C70); 45.4 (NCH3); 23.8 (C80); IR (KBr pellet, cmꢀ1):
3441; 2960; 1761; 1255; 1114; 1087; 1039; calculated for
(from PerkineElmer), the potential inhibitor at 5 or 500
m
DMSO, and the tubulin stabilizing components described previ-
ously [58,59]. Incubation was for 10 min at 37 ꢁC, and the bound
colchicine was trapped through the tubulin on a stack of two
Whatman DEAE-cellulose filters (from GE Healthcare Life Sciences)
and quantitated by liquid scintillation counting.
C
29H29NO8: C, 67.04; H, 5.63; N, 2.70; found: C, 66.95; H, 5.54; N,
2.84.
5.3.3. Immunofluorescence analysis
Cells were fixed in cold 4% formaldehyde for 15 min, rinsed and
stored prior to analysis. Primary antibody staining was performed
for b-tubulin (mouse, monoclonal 1:1000, SigmaeAldrich, Milano,
5.2.7. 90-(2-Naphthyl)-noscapine (9)
Pale brown amorphous product; m.p.: 101e103 ꢁC; 1H NMR
(CDCl3)
d
¼ 8.03e7.53 (7H, m, ar); 7.01 (1H, d, H5, J4e5 ¼ 7.0); 6.21
Italy). After incubation, cells were washed and incubated with an
Alexa conjugated secondary antibody (1:2000, Life Technologies,
Monza, Italy). Cells were counterstained with 40,6-diamidin-2-
fenilindole (DAPI) (1:10,000, SigmaeAldrich, Milano, Italy). Im-
ages were obtained on a video-confocal microscope (Vico, Eclipse
Ti80, Nikon), equipped with a digital camera.
(1H, d, H4, J4e5 ¼ 7.0); 5.87 (2H, s, 2H20); 5.53 (1H, d, H3, J5 e3 ¼ 4.7);
0
4.33 (1H, d, H50, J5 e3 ¼ 4.7); 4.01 (6H, s, 2 OCH3); 3.84 (3H, s, OCH3);
0
2.47 (3H, s, NCH3); 2.24e1.99 (4H, m, H70 , H70 , H80a, H80 ); 13C
a b b
NMR (CDCl3)
d
¼ 168.7 (C]O); 152.8 (C6); 151.7 (C10a); 148.1 (C40);
148.0 (C7); 138.8 (C3a); 137.4 (ar), 136.9 (ar), 136.5 (ar) 135.7 (2 ar),
134.2 (C30a); 131.9 (C80a); 130.2 (2 ar); 128.7 (2 ar); 119.9 (ar); 118.0
(C40a); 117.2 (C7a); 116.5 (C5); 111.4 (C4); 109.8 (C90); 99.5 (C20);
81.5 (C3); 66.9 (C50); 60.1 (OCH3); 59.1 (OCH3); 56.2 (OCH3); 55.0
(OCH3); 50.4 (C70); 44.4 (NCH3); 23.2 (C80); IR (KBr pellet, cmꢀ1):
3448; 2959; 1758; 1252; 1110; 1087; 1035; calculated for
5.3.4. Evaluation of mitotic index
The Burkitt lymphoma CA46 cells were grown in RPMI 1640
medium supplemented with 17% fetal bovine serum and 2 mM L-
glutamine at 37 ꢁC 5% CO2 atmosphere. The mitotic index in the
Burkitt cell cultures was determined at 16 h, the time that produces
a near-maximal value after treatment with antitubulin drugs.
About 4.5 ml of cell culture medium was centrifuged at 1000 rpm
for 1 min. The pelleted cells were resuspended in 5 ml of
phosphate-buffered saline at room temperature, and the cells were
harvested by centrifuging the suspension as before. The cell pellet
was suspended in 0.5 ml of half-strength phosphate-buffered sa-
line, and the cells were allowed to swell for 10 min. The cells were
then fixed by adding 6 ml of 0.5% acetic acid-1.5% ethanol. After
30 min, the cells were harvested by centrifuging as before. The cells
were resuspended in 25% acetic acid/75% ethanol, and a droplet of
the cell suspension was spread on the slide. The slide was air-dried
and stained with Giemsa. The slide was examined under a light
microscope, with mitotic cells defined as those with condensed
chromosomes and no nuclear membrane. At least 200 cells were
counted for each condition examined.
C
32H29NO7: C, 71.23; H, 5.42; N, 2.60; found: C, 71.12; H, 5.53; N,
2.54.
5.3. Biology
5.3.1. Antiproliferative assays
Human T-cell (Jurkat) and B-cell leukemia cell lines (SEM and
RS4; 11) were grown in RPMI-1640 medium (Gibco, Milano, Italy).
Human breast adenocarcinoma (MCF7), non-small cell lung carci-
noma (A549), cervix carcinoma (HeLa), ovarian carcinoma (IGROV-
1) and colon adenocarcinoma (HT-29) cells were grown in DMEM
medium (Gibco, Milano, Italy), all supplemented with 115 units/mL
of penicillin G (Gibco, Milano, Italy), 115
mg/mL of streptomycin
(Invitrogen, Milano, Italy) and 10% fetal bovine serum (Invitrogen,
Milano, Italy). Stock solutions (10 mM) of the different compounds
were prepared by dissolving them in dimethyl sulfoxide (DMSO).
Individual wells of a 96-well tissue culture microtiter plate were
inoculated with 100
m
L
of complete medium containing
5.3.5. Flow cytometric analysis of cell cycle distribution
8 ꢂ 103 cells. The plates were incubated at 37 ꢁC in a humidified 5%
For flow cytometric analysis of DNA content, 5 ꢂ 105 HeLa or
Jurkat cells were treated with different concentrations of the test
compounds for 24 or 48 h. After the incubation period, the cells
were collected, centrifuged and fixed with ice-cold ethanol (70%).
The cells were then treated with lysis buffer containing RNAse A
and 0.1% Triton X-100 and stained with propidium iodide (PI).
Samples were analyzed on a Cytomic FC500 flow cytometer
(Beckman Coulter). DNA histograms were analyzed using Multi-
Cycle® for Windows (Phoenix Flow Systems).
CO2 incubator for 18 h prior to compound addition. After medium
removal, 100 mL of fresh medium containing the test compound at
different concentrations was added to each well, and the plates
were incubated at 37 ꢁC for 72 h. Cell viability was assayed by the
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
test as previously described [56].
5.3.2. Effects on tubulin polymerization and on colchicine binding
to tubulin
Tubulin assembly was measured by turbidimetry at 350 nm in
Gilford model 250 spectrophotometers equipped with electronic
temperature controllers. Reaction mixtures containing purified
5.3.6. Annexin-V assay
Surface exposure of phosphatidyl serine (PS) on apoptotic cells
was measured by flow cytometry with a Coulter Cytomics FC500
(Beckman Coulter) by adding annexin-V-FITC to cells according to
the manufacturer's instructions (Annexin-V Fluos, Roche
bovine brain tubulin [15] at 10
mM (1.0 mg/mL), 0.8 M monosodium
glutamate (pH 6.6 with HCl in 2 M stock solution), 4% (v/v) DMSO,

E. Porcù et al. / European Journal of Medicinal Chemistry 84 (2014) 476e490
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Diagnostic). Simultaneously, the cells were stained with PI. Exci-
tation was set at 488 nm, and the emission filters were at 525 and
585 nm, respectively, for FTIC and PI.
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HeLa cells were incubated in the presence of test compounds
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two times with ice cold phosphate-buffered saline (PBS). The pellet
was then resuspended in lysis buffer. After the cells were lysed on
ice for 30 min, lysates were centrifuged at 15,000 ꢂ g at 4 ꢁC for
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A as an
This research was supported by the European Union and the
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stration of microtubule-like structures formed with (e)-rhazinilam from pu-
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of analog activity, Arch. Biochem. Biophys. 407 (2009) 98e104.
State of Hungary (OTKA Grant No. 101372), co-financed by the Eu-
ropean Social Fund in the framework of TAMOP 4.2.4. A/2-11-1-
2012-0001 ‘National Excellence Program’.
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