Design, synthesis, and biological evaluation of the first podophyllotoxin analogues as potential vascular-disrupting agents

Article abstract of DOI:10.1002/cmdc.201000305

We designed and synthesized two novel series of azapodo-phyllotoxin analogues as potential antivascular agents. A linker was inserted between the trimethoxyphenyl ring E and the tetracyclic ABCD moiety of the 4-aza-1,2-didehydropodophyllotoxins. In the first series, the linker enables free rotation between the two moieties; in the second series, conformational restriction of the E nucleus was considered. We have identified several new compounds with inhibitory activity toward tubulin polymerization similar to that of CA-4 and colchicine, while displaying low cytotoxic activity against normal and/or cancer cells. An aminologue and a methylenic analogue were shown to disrupt endothelial cell cords on Matrigel at subtoxic concentrations, and an original assay of drug washout allowed us to demonstrate the rapid reversibility of this effect. These two new analogues are promising leads for the development of vascular-disrupting agents in the podophyllotoxin series.

Full text of DOI:10.1002/cmdc.201000305

MED
DOI: 10.1002/cmdc.201000305
Design, Synthesis, and Biological Evaluation of the First
Podophyllotoxin Analogues as Potential Vascular-
Disrupting Agents
Raphaꢀl Labruꢁre,[a, b] Benoꢂt Gautier,[b] Marlꢁne Testud,[a] Johanne Seguin,[c]
Christine Lenoir,[b] Stꢃphanie Desbꢁne-Finck,[a] Philippe Helissey,[a] Christiane Garbay,[b]
Guy G. Chabot,[c] Michel Vidal,[b] and Sylviane Giorgi-Renault*[a]
We designed and synthesized two novel series of azapodo-
phyllotoxin analogues as potential antivascular agents. A linker
was inserted between the trimethoxyphenyl ring E and the tet-
racyclic ABCD moiety of the 4-aza-1,2-didehydropodophyllo-
toxins. In the first series, the linker enables free rotation be-
tween the two moieties; in the second series, conformational
restriction of the E nucleus was considered. We have identified
several new compounds with inhibitory activity toward tubulin
polymerization similar to that of CA-4 and colchicine, while dis-
playing low cytotoxic activity against normal and/or cancer
cells. An aminologue and a methylenic analogue were shown
to disrupt endothelial cell cords on Matrigel at subtoxic con-
centrations, and an original assay of drug washout allowed us
to demonstrate the rapid reversibility of this effect. These two
new analogues are promising leads for the development of
vascular-disrupting agents in the podophyllotoxin series.
Introduction
The recognition that tumor vasculature can be used as a spe-
cific target for cancer therapy has been a breakthrough for
cancer treatment.[1] Indeed, tumor vasculature is needed to
provide oxygen and nutrients to tumor cells, and is also the
main route for metastatic spread. In addition, because a single
vessel can support the survival of millions of tumor cells, the
targeting of tumor vasculature is crucial for killing the maxi-
mum number of tumor cells.
tors of tubulin depolymerization that bind to the taxane site.[5]
Early discovered VDAs are active only at doses near their maxi-
mum tolerated dose (MTD). Agents that disrupt the tumor vas-
culature at doses well below their MTD are currently undergo-
ing preclinical and clinical investigations, including the phos-
phate prodrugs of combretastatin A-4 (fosbretabulin) and A-1
(Oxi4503), AVE8062, and ABT-751, among others.[6] These com-
pounds are used in combination with cytotoxic chemotherapy
or radiotherapy.
The antivascular approach aims to selectively cause the col-
lapse of newly formed capillaries by using small-molecule vas-
cular-disrupting agents (VDAs), as opposed to antiangiogenic
therapy, which aims to prevent the formation of new tumor
blood vessels from the pre-existing vasculature. The specificity
of VDAs is mainly due to major differences between normal
and tumor vessels, as the latter are highly disorganized. Tumor
vessels are fragile, have abnormal permeability and diameter,
and are characterized by unstable endothelial intercellular
junctions.[2]
Although the mechanism of action of VDAs has not been
fully elucidated, it involves a rapid cytoskeletal remodeling of
endothelial cells through interphase microtubule disruption,
leading to shutdown of tumor blood flow within 2–6 h post-
treatment. Simultaneous activation of the RhoA/Rho signaling
[a] Dr. R. Labruꢀre, M. Testud, Dr. S. Desbꢀne-Finck, Dr. P. Helissey,
Prof. Dr. S. Giorgi-Renault
Laboratoire de Chimie Thꢁrapeutique, UMR CNRS No. 8638
Universitꢁ Paris Descartes
Existing small-molecular-weight VDAs are predominantly tu-
bulin-binding agents.[3] However, some derivatives, including
flavone acetic acid and ASA404, belong to a second class that
acts through a pro-inflammatory pathway. Microtubules are es-
sential structural components of the cytoskeleton and are in-
volved in several important cellular processes such as mitosis,
vesicular transport, cell signaling, and shape maintenance. Mi-
crotubules are dynamic structures, formed of a- and b-tubulin
heterodimers.[4] Historically, the search for tubulin-binding
agents has attracted much attention for the development of
anticancer drugs. Anti-microtubule agents can be divided into
two classes: inhibitors of tubulin polymerization that bind to
either the colchicine site or the vinca alkaloid site, and inhibi-
Facultꢁ des Sciences Pharmaceutiques et Biologiques
4 avenue de l’Observatoire, 75270 Paris Cedex 06 (France)
Fax: (+33)1-43-29-14-03
E-mail: sylviane.giorgi-renault@parisdescartes.fr
[b] Dr. R. Labruꢀre, Dr. B. Gautier, C. Lenoir, Prof. Dr. C. Garbay,
Prof. Dr. M. Vidal
Laboratoire de Pharmacochimie Molꢁculaire et Cellulaire, INSERM U648
Universitꢁ Paris Descartes, UFR Biomꢁdicale
45 rue des Saints Pꢀres, 75006 Paris (France)
[c] J. Seguin, Dr. G. G. Chabot
Laboratoire de Pharmacologie Chimique, Gꢁnꢁtique et Imagerie
INSERM U 1022-CNRS UMR 8151
4 avenue de l’Observatoire, 75270 Paris Cedex 06 (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000305.
2016
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Podophyllin Analogues for Vascular Disruption
pathway allows endothelial cells to adopt a rounded morphol-
ogy and form actin stress fibers, promoting disruption of VE
cadherin junctions and membrane blebbing. These changes
enhance endothelial monolayer permeability and cause blood
leakage and vessel collapse, leading to blood flow shutdown
and central necrosis of the tumor.[7]
results suggest that podophyllotoxin analogues may be of
value as potential new VDAs.
We have synthesized aza-analogues of podophyllotoxin (1)
as a new class of antimitotic agents, with 4-aza-2,3-didehydro-
podophyllotoxin S 26711 (3) and N-methyl analogue S 26390
(4) as the hit compounds for this cytotoxic series (Figure 2).[14]
Identification of microtubule-binding drugs with greater
therapeutic antivascular selectivity, relative to their cancer cell
cytotoxicity, is an important objective for the next generation
of VDAs.[3a] In this paper, we present our investigation of a
series of nitrogen analogues of podophyllotoxin (Figure 1).
Figure 1. Structures of podophyllotoxin and deoxypodophyllotoxin.
Figure 2. Design of carbon homologated analogues 5–10 and aminologue
11 as potential VDAs.
Podophyllotoxin (1), a naturally occurring cyclolignan isolat-
ed from Podophyllum species, is a well-known cytotoxic deriva-
tive that acts as a potent anti-microtubule agent.[8] In spite of
potential uses as a medicinal drug, human trials with podo-
phyllotoxin were discontinued due to its systemic toxicity.[9]
Over the last 20 years, extensive structural modifications of po-
dophyllotoxin have led to the synthesis of etoposide, a glyco-
sylated epimer of 4’-demethylpodophyllotoxin, which is pres-
ently in clinical use for the treatment of small cell lung cancer,
testicular carcinoma, acute leukemia, and lymphoma, among
others. However, etoposide is a topoisomerase II inhibitor and,
therefore, has an entirely different mechanism of action than
that of the parent compound. Many structural podophyllotoxin
analogues have been described, although research in this area
has mainly focused on the development of new topoisomera-
se II inhibitors.[10]
From a chemical point of view, the 4-aza-2,3-didehydro ana-
logues present the advantages of possessing only one chiral
center and a stable, unsaturated, lactone ring. Indeed, under
physiological conditions, epimerization at C2 of the trans-fused
g-lactone of podophyllotoxin leads to the thermodynamically
stable but inactive cis-epimer. We therefore aimed to design
and synthesize novel 4-azapodophyllotoxin analogues as po-
tential selective antivascular agents that maintain tubulin affin-
ity but exhibit lower cytotoxicity against normal and/or cancer
cells, in other words, compounds that are active as VDAs at
subtoxic concentrations.
Within the 4-aza-2,3-didehydropodophyllotoxin series,[14] sev-
eral derivatives were found to exhibit low cytotoxicity and
high tubulin affinity, making them good candidates for antivas-
cular purposes. Taking into account these results and the SAR
study in the podophyllotoxin series,[10c] coupled with the X-ray
structure of the a,b-tubulin–podophyllotoxin complex,[12] we
designed new azapodophyllotoxins with the E ring farther
from the ABCD tetracycle, with complete or partial conforma-
tional restriction of the E nucleus.
Podophyllotoxin acts at the colchicine binding site of tubu-
lin.[11] The binding mode of colchicine was recently confirmed
by an X-ray structure of a,b-tubulin complexed with DAMA-
colchicine.[12] Colchicine and podophyllotoxin bind to b-tubulin
at its interface with a-tubulin, with the trimethoxyphenyl nu-
cleus hidden within the b-subunit.
Vascular disruption properties reported for antitubulin
agents have surprisingly not been exploited to date in podo-
phyllotoxin analogue studies. Recently, the antiangiogenic ac-
tivity of deoxypodophyllotoxin (2) (Figure 1), a naturally occur-
ring analogue of 1 without a hydroxy group at the C4 position,
was reported.[13] Compound 2 inhibits the tube-like formation
of human umbilical vein endothelial cells (HUVEC) at non-cyto-
toxic concentrations. Moreover, deoxypodophyllotoxin is sever-
al-fold more potent against HUVEC than against several cancer
cell lines (A549, SK-OV, SK-Mel-2, HCT15, and B16F10).[13] These
The aim of the present work was to study two new series of
azapodophyllotoxins. In the first series, the linker inserted be-
tween the E and ABCD moieties enabled free rotation around
single bonds C9-X and X-C1’ (Figure 2). Modifications to these
carbon homologues involved bond length and degree of bond
saturation, resulting in homologues 5 and 6, ethynologue 7,
benzologue 8, and vinylogues 9 and 10. In the second series,
conformational restriction of the E nucleus was considered, re-
sulting in aminologue 11,[15] in which the C9 hybridization is
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S. Giorgi-Renault et al.
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sp2 and rotation is blocked by creation of an additional pseu-
docycle (hydrogen bond between the amino moiety and the
lactone carbonyl, Figure 2). A majority of analogues 5–11 are
N-methylated for synthetic and stability purposes, as N-substi-
tution of the 1,4-dihydroquinoline nucleus prevents aromatiza-
tion. In the first study, the methylenedioxy moiety, the lactone,
and the three methoxy groups of the E ring were maintained
in order to compare biological results with parent compounds
3 and 4. The potential antivascular activity of the synthesized
compounds was assessed by several biological tests, including
in vitro cytotoxicity, a tubulin polymerization inhibition assay,
analysis of endothelial cell morphology, and a cord disruption/
reorganization assay.
subsequent oxidation by Dess–Martin periodinane. Benzologue
8 was obtained in four steps from commercially available tri-
methoxyaniline 16. Diazotation of 16, followed by substitution
with potassium iodide, yielded trimethoxyiodobenzene 17.[18]
Carbaldehyde 19 was then synthesized by a palladium-cata-
lyzed biaryl coupling between iodobenzene 17 and boronic
acid 18.[19]
Vinylogue derivative 10 was used as a common precursor
for both analogues 6 and 7. Application of our one-pot, three
component procedure to the preparation of vinylogues 9 and
10 resulted in poor yields, and subsequent isolation of pure
compounds was very difficult. To circumvent this hurdle, we
split this procedure into two distinct steps: condensation of tri-
methoxycinnamaldehyde 21 with tetronic acid 15, followed by
reaction of diene 22 with the aniline.
The preparation of 21 involved two-carbon homologation of
the 3,4,5-trimethoxybenzaldehyde using either a Wittig reac-
tion[20] or an aldolization-crotonization with the acetic acid
vinyl ester.[21] We were unable to reproduce the latter synthesis,
even under a variety of experimental conditions. We optimized
the preparation of aldehyde 21 adopting the procedure de-
scribed for the 3,4-dimethoxy analogues,[22] that is, reaction of
the commercially available acid 20 with thionyl chloride, fol-
lowed by reduction of the resulting acyl chloride by LiAlH-
(OtBu)3 in dry THF. In a second step, three equivalents of 21
were reacted with one equivalent of tetronic acid 15 in hydro-
chloric acid, according to a procedure described for the unsub-
stituted cinnamaldehyde.[23] Benzylidene 22 was obtained as a
mixture of stereoisomers and in only 10% yield, likely due to
the instability of the compound in acidic medium. Indeed, stoi-
chiometric condensation between 15 and 21 under neutral
conditions afforded 22 in 69% yield, but as a single stereoiso-
mer which was not further identified. Further reaction of 22
(as a mixture or as a single stereoisomer) and aniline
Results and Discussion
Synthesis
We previously reported an original three component one-pot
reaction for the synthesis of 4-aza-2,3-didehydropodophyllo-
toxins.[16] The notable advantages of this method are mild con-
ditions without activation, fast reaction times, and tolerance
for structural diversity. Therefore, we decided to use this reac-
tion for the preparation of the new carbon homologated ana-
logues using the corresponding aldehydes. Using this one-pot
procedure, involving tetronic acid 15, N-methyl-3,4-methylene-
dioxyaniline (14), and either aldehyde 13 or 19, the monome-
thylenic analogue 5 and benzologue 8 were synthesized in
50% and 97% yields, respectively (Scheme 1).
Trimethoxyphenylethanal 13 was prepared according to a
two-step sequence developed by Nicolaou et al.[17] from com-
mercially available trimethoxyphenylacetic acid 12: reduction
in the presence of LiAlH4 to the corresponding alcohol, with
23 in ethanol at reflux led to vinylogue 9 in 24%
yield. The Z conformation was established by NMR
3
spectroscopy, in particular, by the 16 Hz J coupling
constant between the two ethylenic hydrogens (d=
6.20 and 6.30 ppm). To avoid formation of the corre-
sponding quinoline, the reaction was stopped imme-
diately after disappearance of the starting materials
as determined by TLC. Dihydroquinoline 9 was puri-
fied by recrystallization from a dichloromethane/
methanol mixture, due to aromatization of the com-
pound during flash column chromatography on silica
gel or alumina.
For N-methylated analogue 10, the yield was en-
hanced to 50% by treating aldehyde 22 with aniline
14 at reflux in dichloromethane instead of ethanol
(Scheme 2). The two-carbon homologated analogue
6 was obtained in a very good yield by palladium-
catalyzed hydrogenation of the extracyclic double
bond of vinylogue 10 without any reduction of the
lactone double bond (Scheme 2). This regioselectivity
Scheme 1. Synthesis of azapodophyllotoxin analogues 5 and 8: a) LiAlH4, anhydrous THF,
0–58C, 3 h, 90%; b) Dess–Martin periodinane, CH2Cl2, 258C, 1 h, 65%; c) NaNO2, KI,
H2SO4/H2O, 508C, 1 h, 70%; d) 18, Pd(PPh3)4, K2CO3, DMF, 658C, 15 h, 80%; e) EtOH,
reflux, 1 h, 50% for 5 and 4 h, 97% for 8.
can be explained by the strong electron delocaliza-
tion of the vinylogous carbamate function, which
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Podophyllin Analogues for Vascular Disruption
Biological evaluation
In order to determine the vascular-disrupting poten-
tial of these novel azapodophyllotoxin analogues, we
first evaluated their inhibitory activity toward tubulin
polymerization. These inhibition of tubulin polymeri-
zation (ITP) values are presented in Table 1 and are
expressed as the ratio of the IC50 value of a given de-
rivative over the IC50 value of colchicine. The inhibito-
ry activities of combretastatin A-4 (CA-4) and S26711
(3) are also presented for comparison purposes. As
expected, reference compounds CA-4 (ITP=0.72) and
3 (ITP=0.47) were both strong anti-microtubule
agents, with IC50 values lower than that of colchicine.
Compounds 5–8, 10, and 27 were considered inac-
tive for tubulin polymerization inhibition because
they presented ITP values greater than 40. In the
carbon homologue series, only N-unsubstituted vi-
nylogue 9 showed significant anti-microtubule activi-
ty (ITP=0.75). Insertion of a double bond between
the tetraline moiety and the trimethoxyphenyl ring
of 3 is therefore possible, whereas introduction of
both vinylogation and N-methylation (10, ITP>40)
resulted in complete loss of anti-microtubule activity.
Furthermore, the combination of both a spacer
group and N-methylation led to a complete loss in
ITP activity, as shown for derivatives 5–8 and 10. In
contrast, with an amino spacer, N-methylation was
well tolerated as aminologue 11 (ITP=2.89) was
found to exhibit significant ITP activity. Collectively,
these results suggest that the tetracyclic N-substitu-
tion is not, in itself, detrimental to tubulin binding. In
Scheme 2. Synthesis of azapodophyllotoxin analogues 6, 7, 9, and 10: a) SOCl2, 1,2-di-
chloroethane, reflux, 2 h; b) LiAlH(OtBu)3, anhydrous THF, 0–58C, 2 h, 59% over two
steps; c) 15, HCl trituration, 258C, 15 min, 10%, 22 (obtained as a mixture of stereoiso-
mers); d) 15, EtOH, reflux, 10 h, 69%, 22 (obtained as a single stereoisomer); e) 23, EtOH,
reflux, 5 min, 24%; f) 14, CH2Cl2, reflux, 1 h, 50%; g) 10% Pd/C, 10 bar H2, MeOH, 258C,
2 h, 98%; h) OsO4, NMO, CH3CN/H2O: 4/1, 258C, 24 h, 80%; i) MsCl, Et3N, anhydrous
CH2Cl2, 08C, 15 min, then LiBr, 258C, 48 h, 60%; j) tBuOK, anhydrous THF,
258C, 3 h, 71%.
causes partial loss of the ethylenic character of the intracyclic
double bond.
In regards to ethynologue 7, the general 4-azapodophyllo-
toxin synthetic route, either in one step or in two sequential
steps, was unsuccessful for its preparation from vinylogue 10.
Attempts at direct deshydrogenation of 10 into 7 using man-
ganese dioxide, selenium dioxide, or dichlorodicyanoquinone
Figure 3. Structures of quinoline 26 and imine 27.
as oxidizing agents failed. Consequently, we elaborated an
original pathway involving an addition-elimination procedure
for the synthesis of ethynologue 7 (Scheme 2). In the first step,
10 was combined with osmium tetroxide to give diol 24 with-
out hydroxylation of the intracyclic double bond for the same
reason as mentioned above. An in situ, two-step procedure,
i.e., mesylation of diol 24 and subsequent addition of LiBr, re-
sulted in a 60% yield of compound 25 upon optimization of
the reaction conditions. Finally, treatment of 25 with potassium
tert-butoxide in THF at room temperature afforded ethyno-
logue 7 in a 71% yield. Amino analogue 11 was synthesized
according to our previously reported procedure.[15] Two struc-
turally relevant synthetic intermediates of this process, quino-
line 26[15] and imine 27[15] (Figure 3), were also biologically eval-
uated.
this series, it is noteworthy that the presence of a hydrogen
bond between the amino spacer and the lactone carbonyl
seems to be critical for tubulin inhibition activity (e.g. amino-
logue 11 and quinoline 26, as compared with inactive imine
27).
Recently, a common pharmacophore for colchicine site in-
hibitors has been proposed to explain the structure-inhibition
of tubulin assembly relationships.[24] Six pharmacophoric fea-
tures have been identified for podophyllotoxin (1), such as two
hydrophobic centers (trimethoxyphenyl and methylenedioxy
groups), one planar group (phenyl nucleus from the tetra-
cycle), two hydrogen bond acceptors (the lactonic C=O and
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EA.hy926 cells). We did not ob-
serve preferential selectivity
toward a particular cell line, with
the possible exception that fi-
broblast NIH 3T3 cells were more
vulnerable to compounds 9, 10,
and 11, as compared to B16 and
EA.hy926 cells. It is also note-
worthy that ITP and cell growth
inhibition values correlated rela-
tively well for most of the new
compounds, with the exception
of vinylogue 10, which was sig-
nificantly cytotoxic against the
three cell lines but devoid of ITP
effect. Therefore, the cytotoxicity
of 10 is likely due to binding to
a different cellular target.
Table 1. Inhibition of tubulin polymerization, cytotoxicity, and morphological effects on EA.hy926 endothelial
cells caused by azapodophyllotoxin analogues.
Compd
ITP[a]
Cytotoxicity: IC50 [mm][b]
Rounding [mm][c]
B16
NIH 3T3
EA.hy926
CA-4
3 (S 26711)
0.72
0.47
>40
>40
>40
>40
0.75
>40
2.89
1.17
>40
0.010Æ0.003
0.016Æ0.001
0.020Æ0.005
0.15Æ0.07
>30
>30
>30
0.03Æ0.01
0.15Æ0.05
0.01
0.01
5
6
7
8
9
10
11
26
27
>30
>30
2.35
28.46
>30
>10
0.12
>30
>30
>10
>30
>30
>10
>10
0.41Æ0.21
0.08Æ0.02
7.5Æ2.1
2.1Æ0.8
0.5Æ0.1
>30
0.77Æ0.05
14.1Æ2.9
38.4Æ10.1
0.40Æ0.01
19.1Æ1.8
8.6Æ2.4
2.8Æ1.5
2.29
0.12
1.95
>30
>30
>30
[a] Inhibition of tubulin polymerization (ITP) is expressed as the ratio of IC50 compd/IC50 colchicine. IC50 compd is the con-
centration of compound required to inhibit 50% of the rate of microtubule assembly (average of three experi-
ments), and the average IC50 value for colchicine was 0.36 mm under our conditions. [b] Concentration of com-
pound corresponding to 50% growth inhibition after 48 h incubation (average of three experiments ÆSEM).
[c] Morphological effects (rounding up) on modified HUVEC (EA.hy926) are expressed as the lowest concentra-
tion at which cell rounding was observed following a 2 h incubation period with the test compound; experi-
ments were done in triplicate.
The active compounds were
less cytotoxic than reference
compounds CA-4 and 3. Indeed,
the antiproliferative effects of vi-
the meta-OCH3 from the trimethoxyphenyl ring), and one hy-
drogen bond donor (hydroxy group).
nylogue 9 and quinoline 26 against B16 cells were 40-fold
lower than those of CA-4, whereas their anti-microtubule activ-
ities were similar. Notably, aminologue 11 displayed high ITP
activity associated with poor B16 cytotoxicity. This profile
(good ITP activity and low cytotoxicity) is considered a good
indicator for antivascular activity.[3] Indeed, this could indicate
that the cytotoxicity of our new derivatives could be attributed
to specific binding to tubulin, rather than binding to several
other targets which activate apoptotic pathways, or could be
due to rapidly reversible kinetic binding to tubulin.
Our new derivatives display most of the pharmacophoric
features reported for podophyllotoxin. In a recent publication,
using isothermal titration calorimetry, this pharmacophoric
proposition was only partially confirmed, with the hydrogen
bond between the hydroxy group and Thr179 of a-tubulin
found to be unnecessary for the podophyllotoxin–tubulin in-
teraction.[25] This is in agreement with our observations that
both N-methylated analogues 4 (data not shown) and 11 re-
tained significant tubulin affinity. This series of novel 4-azapo-
dophyllotoxin analogues is the first demonstration that a
spacer group can be introduced between the trimethoxyphen-
yl and tetracycle ABCD (vinylogue 9 and aminologue 11). As
the trimethoxyphenyl group of podophyllotoxin is buried
within a hydrophobic pocket of b-tubulin, the additional
double bond may reinforce the van der Waals interactions be-
tween vinylazapodophyllotoxin 9 and tubulin.
Our overall goal was to obtain microtubule-binding drugs
with high therapeutic antivascular selectivity relative to their
cancer cell and/or normal cell cytotoxicity. In order to achieve
the necessary results, the cytotoxicity of the new derivatives
was evaluated using a solid tumor-derived murine B16 melano-
ma cell line. These results for each compound were compared
with the cytotoxicity against a murine fibroblast NIH 3T3 cell
line as an example of normal cells and against the EA.hy926
cell line, which is considered one of the best immortalized
HUVE cell (HUVEC) lines, because these cells express most of
the biochemical markers of parental HUVEC.[26] The results pre-
sented in Table 1 reveal that the cytotoxic activities of refer-
ence compounds CA-4 and compound 3 are in the nanomolar
range, while the new podophyllotoxin analogues 9–11 and 26
are less toxic (IC50 values in the micromolar range), as expect-
ed. The compounds showed similar activity toward B16 mela-
noma cells and in the other cell lines tested (i.e., NIH 3T3 and
The new derivatives were also tested for their effects on the
morphology of endothelial cells after a brief exposure time.
Successful antivascular agents were shown to induce rapid en-
dothelial cell retraction, due to both tubulin and actin cytoske-
leton remodeling, leading to neovessel destructuring in vivo at
concentrations below those required to block mitosis.[27] The
morphological effects of the new compounds on EA.hy926 en-
dothelial cells is presented in Table 1 and was expressed as the
lowest concentration at which cell rounding up was observed
following a 2 h-incubation period with test compounds. As ex-
pected, both cytotoxic reference compounds CA-4 and 3
caused the rounding up of endothelial cells at nanomolar con-
centrations. Among the newly synthesized derivatives, com-
pounds 5, 9–11, and 26 were found to change the morpholo-
gy of EA.hy926 endothelial cells after a 2 h-exposure period at
either non toxic or sub-cytotoxic concentrations in the low mi-
cromolar range for the same cell line (Table 1). Figure 4 shows
representative photographs of EA.hy926 cells exposed to the
morphologically active compounds. Control cells exposed to
the solvent (1% DMSO) were not affected and presented an
elongated morphology, whereas active compounds induced
the typical rounding up effect observed with antivascular
agents. Vinylogue 9 and aminologue 11 caused rounding up
at concentrations 6- and 72-fold lower than their IC50 values
against this cell line, respectively. Interestingly, homologues 5
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Podophyllin Analogues for Vascular Disruption
were also quite effective, leading to tube disruption
at 10 mm. Treatment with 10 at concentrations up to
20 mm did not modify endothelial cell tubular struc-
ture (Figure 5 and Supporting Information). It is note-
worthy that compounds 5 and 11 significantly disor-
ganized HUVEC assembly at sub-cytotoxic doses in
B16 cells.
With regard to the reorganization of tube-like
structures 3 h after drug washout of unbound com-
pound, a rapid network reconstruction was noted for
compounds 5 and 11 (Figure 5). This particular reor-
ganization effect was observed with the same inten-
sity at higher concentrations (20 mm) for analogue 5,
whereas the amino analogue 11 was a very potent
disruptor at 10 mm and, therefore, only partial recon-
struction was observed after 3 h (see supporting in-
formation). Compound 9 also disrupted the cords at
a highly cytotoxic concentration (10 mm), and its
effect was not reversible after drug washout. This
suggests that 9 and 11 may exhibit different tubulin
binding kinetics which cause distinct vascular disrup-
tion activity. It has been shown that, unlike CA-4, col-
chicine and podophyllotoxin can elicit their effects
on neovasculature at doses close to the maximum
tolerated dose.[29] The wider therapeutic window of
CA-4 has, therefore, been attributed to differences in
the association/dissociation rates of tubulin bind-
ing[30] and to the pseudo-irreversibility of tubulin
binding for colchicine and podophyllotoxin.[25,31] It is
noteworthy that methylenic analogue 5 displayed a
Figure 4. Morphological effects of selected azapodophyllotoxin analogues on EA.hy926
endothelial cells. Exponentially growing cells were exposed to the indicated compound
and incubated at 378C for 2 h at the indicated concentrations. Representative photo-
graphs were taken at 360ꢅ magnification. Scale bar, 20 mm.
and 10 produce a potent morphological effect which is not re-
lated to their antitubulin activity. In addition, the methane
bridge homologue 5 was less morphologically active (2.35 mm)
but was devoid of cytotoxic activity for the three cell lines. In
contrast, quinoline 26, which was effective toward EA.hy926
rounding up, could be considered as a cytotoxic agent. In fact,
cytotoxic concentration in EA.hy926 cells (IC50 =2.8 mm) was in
the same range as the rounding up effect (IC50 =1.95 mm).
To further evaluate the antivascular potency of the four new
podophyllotoxin analogues 5, 9, 10, and 11, their ability to dis-
rupt newly formed endothelial cell cords on Matrigel was eval-
uated, as well as the reorganization of the tube-like network
following drug washout. The latter assay is an important pa-
rameter, because it is recognized that rapidly reversible bind-
ing would lead to prompt drug clearance in vivo.[28] The dose-
effect response of the selected 4-azapodophyllotoxins toward
in vitro capillary tube disruption was evaluated by exposing
pre-plated HUVEC on Matrigel to increasing concentrations of
the compounds for 3 h. After 3 h exposure to various drug
concentrations (shown in Supporting Information) covering
the cytotoxic IC50 range of the B16 cell line (up to 20 mm and
below the limit of solubility), the lowest dose causing tube-like
disruption was determined for each derivative (Figure 5). Refer-
ence compound CA-4 was used as a cord disruptive control.
Among the new azapodophyllotoxins, the amino analogue 11
was the most potent, altering the HUVEC cord network at
1 mm, with complete inhibition at 10 mm. Homologues 5 and 9
reversible antivascular effect in vitro, even though this com-
pound is not a tubulin binding agent and is devoid of cytotox-
icity at concentrations as high as 30 mm.
Conclusions
We have designed and synthesized two novel series of azapo-
dophyllotoxin analogues as potential antivascular agents. In
the first series, the linker inserted between the E and ABCD
moieties enabled free rotation around single bonds C9-X and
X-C1’. In the second series, conformational restriction of the E
nucleus was pursued. The potential antivascular activity of the
synthesized compounds has been assessed by several biologi-
cal tests, including a tubulin polymerization inhibition assay, in
vitro cytotoxicity, analysis of endothelial cell morphology, and
a cords disruption/reorganization assay. Our results allowed
identification of the first two podophyllotoxin analogues, 5
and 11, as potential vascular disrupting agents with low toxici-
ty. Compound 5 was of particular interest due to its activity
toward endothelial cord disruption, while it was surprisingly
found inactive toward tubulin polymerization. Aminologue 11
was shown to be particularly active as a potential new antivas-
cular compound, as it displayed excellent ITP activity, low cyto-
toxicity, and a rapidly reversible endothelial cell cord disruption
effect at subtoxic doses, as hypothesized. Combined, these re-
sults demonstrate that 4-azapodophyllotoxin analogues are
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S. Giorgi-Renault et al.
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Elemental analyses were performed at the CNRS Analysis Laborato-
ry (Gif-sur-Yvette, France) and were found to be within Æ0.4% of
theoretical values. Compounds 11, 26, and 27 were prepared as
previously described.[15]
(9RS)-4-Methyl-6,7-(methylenedioxy)-9-(3,4,5-trimethoxybenzyl)-
4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (5). An equimolar mix-
ture (1 mmol) of tetronic acid 15, aldehyde 13,[17] and aniline 14 in
EtOH (10 mL) was held at reflux for 1 h. The resulting solid was re-
moved by filtration and recrystallized from MeOH to afford 5 as a
white powder (217 mg, 50%): Rf =0.13 (CH2Cl2/EtOAc 9:1); mp:
1
2008C; H NMR (300 MHz, CDCl3): d=2.80 (s, 3H), 2.85 (dd, J=13
and 5 Hz, 1H), 3.00 (dd, J=13 and 5 Hz, 1H), 3.65 (s, 6H), 3.80 (s,
3H), 4.25 (t, J=5 Hz, 1H), 4.55 (d, J=15 Hz, 1H), 4.65 (d, J=15 Hz,
1H), 5.90 (s, 2H), 5.95 (s, 2H), 6.35 (s, 1H), 6.65 ppm (s, 1H);
13C NMR (75 MHz, CDCl3): d=33.2, 36.2, 43.4, 55.9, 61.3, 65.0, 94.7,
99.7, 101.5, 106.7, 109.8, 118.6, 133.6, 134.3, 136.4, 143.9, 147.2,
152.2, 159.9, 173.1 ppm; IR (KBr): n˜ =1742, 1662, 1590, 1500, 1483,
1421, 1245, 1230, 1209, 1128, 1035, 1007 cmÀ1; Anal. calcd for
C23H23NO7·0.5 H2O: C 63.59, H 5.57, N 3.22; found: C 63.83, H 5.37,
N 3.28.
(9RS)-4-Methyl-6,7-(methylenedioxy)-9-(3’,4’,5’-trimethoxybi-
phenyl-4-yl)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (8). An
equimolar mixture (0.73 mmol) of tetronic acid 15, aldehyde 19,[19]
and aniline 14 in EtOH (7 mL) was held at reflux for 4 h. The result-
ing solid was removed by filtration, purified by flash chromatogra-
phy (EtOAc/CH2Cl2, 1:9), and then recrystallized from EtOH to
afford 8 as a white solid (345 mg, 97%): Rf =0.54 (EtOAc); mp:
1
>2608C; H NMR (300 MHz, [D6] DMSO): d=3.25 (s, 3H), 3.87 (s,
3H), 3.90 (s, 6H), 4.80 (d, J=15 Hz, 1H), 4.90 (d, J=15 Hz, 1H), 5.10
(s, 1H), 5.90 (s, 1H), 5.95 (s, 1H), 6.55 (s, 1H), 6.60 (s, 1H), 6.70 (s,
2H), 7.25 (d, J=8 Hz, 2H), 7.45 ppm (d, J=8 Hz, 2H); 13C NMR
(75 MHz, [D6] DMSO): d=33.6, 40.2, 56.1, 60.9, 65.0, 95.0, 97.5,
101.6, 104.4, 110.8, 118.6, 127.3, 128.2, 133.0, 137.0, 137.4, 139.8,
144.2, 144.9, 147.5, 153.3, 158.0, 173.2 ppm; IR (KBr): n˜ =1748,
1663, 1589, 1482, 1345, 1249, 1204, 1126, 1036, 1002 cmÀ1; Anal.
calcd for C28H25NO7: C 68.98, H 5.17, N 2.87; found: C 68.90, H 5.17,
N 2.89.
(2E)-3-(3,4,5-Trimethoxyphenyl)prop-2-enal (21). A solution of (E)-
3,4,5-trimethoxycinnamic acid (20) (2.38 g, 10 mmol) and SOCl2
(2.2 mL, 30 mmol) in 1,2-dichloroethane (100 mL) was held at
reflux for 2 h. The solvent was eliminated under reduced pressure,
and the crude resulting yellow solid was dissolved in anhydrous
THF (100 mL). A suspension of LiAlH(OtBu)3 (2.8 g, 11 mmol) in an-
hydrous THF (50 mL) was added dropwise to the stirred reaction
mixture at 0–58C. The reaction mixture was then warmed to room
temperature and stirring was continued for another 2 h. After addi-
tion of a solution of 1% aqueous HCl (300 mL), the mixture was ex-
tracted by CH2Cl2 (3ꢅ50 mL). The combined organic layers were
dried over Na2SO4 and evaporated to give a crude residue, which
was purified by flash chromatography (CH2Cl2/EtOAc, 9:1), followed
by recrystallization from heptane to afford 21 as a white powder
(1.3 g, 59%): mp: 110–1118C; spectra and melting point are identi-
cal to published values.[32]
Figure 5. After allowing HUVE cells to form cords on Matrigel for 16 h, vari-
ous concentrations of tested compounds were added to the media. The
same microscopic field was recorded 3 h after drug addition and again 3 h
after drug washout. A reversible effect in vitro was observed for 5, 11, and
CA-4. The effect induced by 9 was not reversible, and 10 was not effective
against tubular structures. Arrowheads indicate disrupted or reformed cords.
promising new leads for the development of selective and
nontoxic vascular-disrupting agents.
Experimental Section
Chemistry
Commercial reagents (Fluka, Aldrich) were used without further pu-
rification, except for 3,4-methylenedioxyaniline which was recrystal-
lized from cyclohexane. Solvents were distillated prior to use. Thin
layer chromatography analyses were carried out on Merck GF 254
silica gel plates. Flash chromatography was carried out on silica
gel 70 (30–70 mm). Melting points were determined with a Kçfler
apparatus and are uncorrected. IR spectra were recorded on a Per-
kinElmer 1600 spectrometer. NMR spectra were recorded on a
(3E) or (3Z)-3-[(2E)-3-(3,4,5-Trimethoxyphenyl)prop-2-enylidene]-
furane-2,4(3H,5H)-dione (22).
A suspension of aldehyde 21
(2.22 g, 10 mmol) and tetronic acid 15 (1 g, 10 mmol) in EtOH
(10 mL) was held at reflux for 10 h. The resulting solid was re-
moved by filtration, washed with EtOH, and recrystallized from
EtOH to afford 22 as a red powder (2.16 g, 69%): Rf =0.25 (CH2Cl2/
EtOAc 95:5); mp: 1728C; 1H NMR (300 MHz, CDCl3): d=3.95 (s,
9H), 4.60 (s, 2H), 6.90 (s, 2H), 7.40 (d, J=15 Hz, 1H), 7.70 (d, J=
1
Bruker AC spectrometer at 300 MHz for H and at 75 MHz for 13C.
2022
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Podophyllin Analogues for Vascular Disruption
12 Hz, 1H), 8.15 ppm (dd, J=15 and 12 Hz, 1H); 13C NMR (75 MHz,
CDCl3): d=56.4, 61.1, 72.0, 107.0, 114.7, 122.0, 130.0, 142.5, 151.6,
153.6, 155.9, 168.2, 195.5 ppm; IR (KBr): n˜ =1752, 1706, 1601, 1578,
1560, 1509, 1500, 1458, 1420, 1374, 1327, 1249, 1166, 1131, 1044,
1002 cmÀ1; Anal. calcd for C6H16O6·0.5 H2O: C 61.34, H 5.47; found:
C 61.39, H 5.43.
1.32 mmol) in a 4:1 mixture of CH3CN/H2O (150 mL). The reaction
mixture was stirred at room temperature for 24 h, and then ex-
tracted with EtOAc (3ꢅ40 mL). The combined organic layers were
dried over Na2SO4 and concentrated. The crude solid was purified
by flash chromatography (CH2Cl2/EtOAc, 6:4 to 0:10), followed by
recrystallization from MeOH to afford 24 as a white powder
(248 mg, 80%): Rf =0.32 (EtOAc); mp: 1928C; 1H NMR (300 MHz,
CDCl3): d =3.05 (s, 3H), 3.75 (s, 3H), 3.80 (s, 6H), 3.85 (m, 2H), 4.00
(d, J=9 Hz, 1H), 4.30 (d, J=5 Hz, 1H), 4.50 (m, 1H), 4.90 (d, J=
15 Hz, 1H), 5.00 (d, J=15 Hz, 1H), 5.90 (s, 1H), 5.95 (s, 1H), 6.25 (s,
1H), 6.35 (s, 2H), 6.80 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=
33.6, 39.4, 56.1, 60.7, 66.5, 74.7, 81.5, 93.5, 95.2, 101.7, 104.7, 110.3,
116.5, 132.7, 135.4, 137.5, 144.2, 147.3, 152.7, 160.4, 173.3 ppm; IR
(KBr): n˜ =3400, 1717, 1657, 1508, 1487, 1419, 1324, 1244, 1199,
1126, 1044 cmÀ1; Anal. calcd for C24H25NO9: C 61.14, H 5.34, N 2.97;
found: C 61.37, H 5.74, N 2.66.
(9RS)-6,7-(Methylenedioxy)-9-[(1E)-2-(3,4,5-trimethoxyphenyl)-
ethenyl]-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (9). A suspen-
sion of 22 (939 mg, 3 mmol) and 3,4-methylenedioxyaniline 23
(411 mg, 3 mmol) in EtOH (30 mL) was stirred under reflux for
5 min. The resulting solid was removed by filtration and recrystal-
lized from a 7:3 mixture of CH2Cl2/MeOH to afford 9 as a white
powder (300 mg, 24%): Rf =0.36 (CH2Cl2/EtOAc, 8:2); mp: >2608C;
1H NMR (300 MHz, [D6] DMSO): d=3.60 (s, 3H), 3.75 (s, 6H), 4.50
(d, J=7 Hz, 1H), 4.80 (d, J=15 Hz, 1H), 4.90 (d, J=15 Hz), 5.90 (s,
1H), 5.95 (s, 1H), 6.20 (dd, J=16 and 7 Hz, 1H), 6.30 (d, J=16 Hz,
1H), 6.50 (s, 1H), 6.65 (s, 2H), 6.70 (s, 1H), 9.80 ppm (s, 1H);
13C NMR (75 MHz, [D6] DMSO): d=38.5, 56.9, 61.1, 65.9, 93.5, 102.3,
104.6, 110.7, 116.3, 129.4, 131.7, 133.1, 133.5, 138.0, 144.4, 144.7,
154.0, 159.6, 173.3 ppm; IR (KBr): n˜ =3280, 1718, 1647, 1624, 1560,
1508, 1483, 1347, 1248, 1192, 1129, 1035, 1007 cmÀ1; Anal. calcd
for C23H21NO7: C 65.24, H 5.00, N 3.31; found: C 65.21, H 4.92, N
3.30.
(9RS)-9-[1,2-Dibromo-2-(3,4,5-trimethoxyphenyl)ethyl]-4-methyl-
6,7-(methylenedioxy)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one
(25). Et3N (500 mL, 3.6 mmol) was added to a stirred solution of
compound 24 (170 mg, 0.36 mmol) maintained under nitrogen in
the minimal quantity of anhydrous CH2Cl2. Methanesulfonyl chlo-
ride (280 mL, 3.6 mmol) was added dropwise at 08C while stirring.
A solution of LiBr (313 mg, 3.6 mmol) in anhydrous acetone (2 mL)
was added after 15 min, and the reaction was stirred at room tem-
perature for another 48 h. The reaction mixture was poured into
water and then extracted with CH2Cl2. The combined layers were
dried over Na2SO4 and concentrated. The crude solid was purified
by flash chromatography (CH2Cl2/EtOAc, 9:1) to afford 25 as a
white powder (129 mg, 60%): Rf =0.60 (EtOAc); mp: 2108C;
1H NMR (300 MHz, CDCl3): d=3.10 (s, 3H), 3.80 (s, 3H), 3.85 (s,
6H), 3.95 (m, 1H), 4.10 (d, J=7 Hz, 1H), 4.65 (s, 2H), 5.10 (d, J=
7 Hz, 1H), 5.95 (s, 1H), 6.00 (s, 1H), 6.50 (s, 1H), 6.60 (s, 2H),
6.80 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=33.6, 38.7, 56.1,
60.8, 65.0, 80.2, 87.5, 93.6, 95.1, 101.7, 104.4, 111.0, 116.0, 134.0,
134.6, 137.6, 144.0, 147.7, 153.1, 161.2, 173.2 ppm; IR (KBr): n˜ =
(9RS)-4-Methyl-6,7-(methylenedioxy)-9-[(1E)-2-(3,4,5-trimethoxy-
phenyl)ethenyl]-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (10). A
solution of 22 (1.25 g, 4 mmol) and N-methyl-3,4-methylenedioxy-
aniline 14 (604 mg, 4 mmol) in CH2Cl2 (40 mL) was held at reflux
for 1 h. The solvent was removed, and the residue was purified by
flash chromatography (CH2Cl2/EtOAc, 9:1) to afford 10 as a white
powder (910 mg, 50%): Rf =0.43 (CH2Cl2/EtOAc, 8:2); mp: >2608C;
1H NMR (300 MHz, [D6] DMSO): d=3.10 (s,3H), 3.60 (s, 3H), 3.75 (s,
6H), 4.50 (d, J=7 Hz, 1H), 4.95 (d, J=15 Hz, 1H), 5.05 (d, J=15 Hz,
1H), 5.95 (s, 1H), 6.00 (s, 1H), 6.20 (dd, J=16 and 7 Hz, 1H), 6.30
(d, J=16 Hz, 1H), 6.65 (s, 2H), 6.80 (s, 1H), 6.90 ppm (s, 1H);
13C NMR (75 MHz, [D6] DMSO): d=34.7, 38.5, 56.9, 61.1, 66.2, 94.0,
97.1, 102.5, 104.6, 110.9, 118.4, 129.4, 133.0, 133.4, 134.0, 138.1,
144.4, 148.1, 154.0, 161.4, 173.3 ppm; IR (KBr): n˜ =1733, 1654, 1612,
1735, 1654, 1508, 1486, 1238, 1193, 1155, 1126, 1037, 1007 cmÀ1
;
Anal. calcd for C24H23Br2NO7: C 48.26, H 3.88, N 2.35; found: C
48.45, H 3.76, N 2.36.
1582, 1507, 1484, 1418, 1332, 1242, 1193, 1123, 1038, 1009 cmÀ1
;
(9RS)-4-Methyl-6,7-(methylenedioxy)-9-[2-(3,4,5-trimethoxyphe-
nyl)ethynyl]-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (7). A sus-
pension of tBuOK (24 mg, 0.2 mmol) and compound 25 (50 mg,
0.084 mmol) in anhydrous THF (10 mL) was stirred under nitrogen
atmosphere for 3 h. After dilution with H2O, the reaction mixture
was extracted with CH2Cl2. The combined layers were dried over
Na2SO4 and concentrated. The crude product was purified by prep-
arative thin-layer chromatography on silica gel (EtOAc) to afford 7
as a white powder (26 mg, 71%): Rf =0.46 (EtOAc); mp: >2608C;
1H NMR (300 MHz, CDCl3): d=3.20 (s, 3H), 3.80 (s, 9H), 4.80 (d, J=
15 Hz, 1H), 4.85 (d, J=15 Hz, 1H), 5.00 (s, 1H), 5.95 (s, 2H), 6.40 (s,
2H), 6.56 (s, 1H), 6.57 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=
26.8, 33.4, 56.0, 60.6, 64.6, 84.5, 86.9, 93.6, 94.1, 101.9, 104.2, 110.8,
115.7, 134.5, 134.6, 137.4, 143.8, 147.5, 152.9, 161.0, 173.0 ppm; IR
(KBr): n˜ =2211, 1737, 1686, 1638, 1561, 1544, 1509, 1477, 1460,
1128 cmÀ1; Anal. calcd for: C24H21NO7: C 66.20, H 4.86, N 3.22;
found: C 65.91, H 4.84, N 3.11.
Anal. calcd for C24H23NO7·H2O: C 63.29, H 5.53, N 3.07; found: C
62.95, H 5.37, N 3.07.
(9RS)-4-Methyl-6,7-(methylenedioxy)-9-[2-(3,4,5-trimethoxyphe-
nyl)ethyl]-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (6).
A sus-
pension of 10 (210 mg, 0.46 mmol) in MeOH (200 mL) was hydro-
genated for 2 h under 10 bar pressure and in the presence of 10%
Pd/C (100 mg). The reaction mixture was filtered through a pad of
Celite, and the solvent was removed. The crude solid was recrystal-
lized from Et2O to afford 6 as a white powder (205 mg, 98%): Rf =
1
0.81 (CH2Cl2/EtOAc 8:2); mp: 1988C; H NMR (300 MHz, CDCl3): d=
1.90 (m, 1H), 2.25 (m, 1H), 2.45 (m, 2H), 3.10 (s, 3H), 3.80 (s, 3H),
3.85 (s, 6H), 4.30 (t, J=5 Hz, 1H), 4.70 (s, 2H), 5.95 (s, 1H), 6.00 (s,
1H), 6.30 (s, 2H), 6.50 (s, 1H), 6.70 ppm (s, 1H); 13C NMR (75 MHz,
CDCl3): d=31.7, 33.4, 33.9, 37.9, 56.1, 60.7, 65.0, 94.9, 96.4, 101.6,
105.3, 109.3, 118.9, 133.8, 135.9, 137.7, 144.1, 147.2, 152.9, 159.6,
173.2 ppm; IR (KBr): n˜ =3310, 1735, 1685, 1654, 1560, 1508, 1478,
1458, 1420, 1239, 1128, 1034 cmÀ1; Anal. calcd for C24H25NO7·H2O: C
63.01, H 5.95, N 3.06; found: C 62.90, H 5.89, N 2.67.
Biology
(9RS)-9-[(1,2-Dihydroxy-2-(3,4,5-trimethoxyphenyl)ethyl]-4-
methyl-6,7-(methylenedioxy)-4,9-dihydrofuro[3,4-b]quinolin-
1(3H)-one (24). A 2.5% solution of OsO4 in tBuOH (500 mL,
0.05 mmol) was slowly added to a suspension of alkene 10
(300 mg, 0.66 mmol) and N-methylmorpholine-N-oxide (154 mg,
Inhibition of tubulin polymerization assay. Tubulin assembly in mi-
crotubules was evaluated using the fluorescent dye DAPI (4’,6-di-
amidino-2-phenylindole)[33] in 96-well black plates and observed
using a Victor plate reader as previously described by Barron
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S. Giorgi-Renault et al.
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et al.[34] and Bane et al.[35] The standard assay was performed as fol-
lows: wells were charged with tubulin (Cytoskeleton, 97% pure,
final concentration: 1 mgmLÀ1) in PME buffer (100 mm PIPES,
1 mm MgSO4, 2 mm EGTA) with 10 mm DAPI and varying concentra-
tions of compounds or colchicine as an internal control. After pre-
incubation for 45 min at room temperature, 5 mL of 1 mm GTP was
added to each well to initiate tubulin polymerization, and the
plate was then transferred to the temperature-controlled Victor
plate reader at 378C for an additional 2 h. Fluorescence was read
at excitation and emission wavelengths of 360 nm and 450 nm, re-
spectively. Percent inhibition was determined as follows:
HUVECs in 100 mL EGM-2 (2ꢅ104 cellswellÀ1) were plated in 96-well
plates on a thick layer of Matrigel (Becton Dickinson; 75 mLwellÀ1
)
and incubated for 16 h at 378C in a 5% CO2 atmosphere to allow
the cells to form tube-like structures. Test compounds were dis-
solved in DMSO (less than 0.1% in each preparation; 100 mLwellÀ1
)
at various concentrations and were added to the formed cords
and incubated for a 3 h period. Each well was then carefully
washed with medium and incubated for an additional 3 h. The ef-
fects of compounds on capillary tube disruption and eventual reor-
ganization was evaluated by light microscopy (40ꢅ magnification)
at three indicated times (0 h, 3 h after addition of compound, and
3 h after washout). Experiments were done in triplicate.
[1ÀDFsample/DFcontrol]ꢅ100, where DFcontrol =Fno inhibitionÀFcomplete inhibition
,
and DFsample =FsampleÀFcomplete colchicine. The IC50 for drug-in-
inhibition with
Dose–effect responses of CA-4 and four new azapodophyllotoxins
(5, 9, 10, and 11) on endothelial tube-like disruption/reorganization
are provided in the Supporting Information.
duced inhibition of tubulin polymerization is the concentration of
drug at which the extent of polymerization inhibition is 50% of
the maximum value as determined from the semi-logarithmic plot
of percent inhibition as a function of the drug concentration using
the nonlinear regression software SigmaPlot (Jandel Scientific). The
results are presented as the inhibition of tubulin polymerization
(ITP), which is the ratio of the IC50 of the compound of interest
over the IC50 of colchicine. In our experimental conditions, the
average IC50 for the ITP of colchicine was 0.36 mm. Because the in-
trinsic fluorescence of compounds could interfere with this assay,
fluorescence readings were routinely taken at the same concentra-
tions as used in the tubulin polymerization assay.
Acknowledgements
R.L. was supported by the Ministꢀre de l’Education Supꢁrieure et
de la Recherche. The authors thank Dr. Claude Monneret for pro-
viding us with combretastatin A-4. This study was supported by a
grant from the Institut National du Cancer (INCa, Boulogne-Bill-
ancourt, France). C.G. thanks the Ligue Nationale contre le
Cancer for financial support.
Cytotoxicity assay. Murine B16 melanoma cells, murine fibroblasts
NIH 3T3, and EA.hy926 endothelial cells [originally obtained from
Dr. Cora-Jean S. Edgell and used with her permission][36] were
grown in Dulbecco’s modified essential medium (DMEM) contain-
ing 2 mmL-glutamine, 10% fetal bovine serum, 100 U/mL penicillin
and 100 mgmLÀ1 streptomycin. The cells were maintained at 378C
in a humidified atmosphere containing 5% CO2. Exponentially
growing cells were plated onto 96-well plates at 5000 cells per
well in 200 mL DMEM. After 24 h, the cells were exposed to the sol-
vent alone (DMSO) or to the compounds of interest at the indicat-
ed concentrations for an additional 48 h. Viability was assessed
using the MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium)
test, and absorbance was read at 562 nm in a microplate reader
(BioKinetics Reader, EL340).[37] Stock solutions were prepared in di-
methyl sulfoxide (DMSO) and kept at 48C in the dark. Experiments
were run in triplicate and repeated three times. Results are pre-
sented as the inhibitory concentrations for 50% of cells (IC50) for a
48 h exposure time. The IC50 values were determined using Graph-
Pad Prism software (GraphPad Software, Inc., San Diego, CA, USA).
Keywords: antitumor agents
·
antivascular agents
·
azapodophyllotoxins · nitrogen heterocycles · podophyllotoxin
analogues
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Received: July 22, 2010
Revised: September 17, 2010
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