Cytotoxic activities and metabolic studies of new combretastatin analogues

Article abstract of DOI:10.1007/s00044-015-1363-3

A new series of combretastatin analogues with B-ring modifications were synthesized and evaluated for their cytotoxicity against one endothelial (HUVEC) and three tumor cell lines, e.g., the LoVo colon, the PC-3 prostate, and the U373 glioma cancer models. These new combretastatin analogues showed differential cytotoxic activities, cis derivatives 13 5-(2-Z-trimethoxyphenylethenyl)benzo[1,2-c]1,2,5-oxadiazole N ¹-oxide and 14 5-(2-Z-trimethoxyphenylethenyl)benzo[1,2,5]thiadiazole exhibiting interesting cytotoxicity both on endothelial and on tumor cells. Unlike the cis benzofurazan 12 5-(2-Z-trimethoxyphenylethenyl)benzo[1,2-c]1,2,5-oxadiazole, induction of apoptosis by 13 appeared to be through caspase-3 activation. Metabolic investigations showed a positive correlation between highly metabolized compounds and cytotoxic activity, suggesting that highly cytotoxic derivatives may act as pro-drug via a reductive metabolization to more active metabolites.

Full text of DOI:10.1007/s00044-015-1363-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1363-3
ORIGINAL RESEARCH
Cytotoxic activities and metabolic studies of new combretastatin
analogues
1
2
3
3
4
•
Yohan Mace´
•
Emilie Bony
Olivier Feron
•
David Delvaux
Jo e¨ lle Quetin-Leclercq
•
Adan Pinto
2
•
V e´ ronique Mathieu
4
3
1
Robert Kiss
•
•
•
Olivier Riant
Received: 3 November 2014 / Accepted: 10 March 2015
Ó Springer Science+Business Media New York 2015
Abstract A new series of combretastatin analogues with
B-ring modifications were synthesized and evaluated for
their cytotoxicity against one endothelial (HUVEC) and
three tumor cell lines, e.g., the LoVo colon, the PC-3
prostate, and the U373 glioma cancer models. These new
combretastatin analogues showed differential cytotoxic
activities, cis derivatives 13 5-(2-Z-trimethoxyphenylethenyl)-
activation. Metabolic investigations showed a positive cor-
relation between highly metabolized compounds and cyto-
toxic activity, suggesting that highly cytotoxic derivatives
may act as pro-drug via a reductive metabolization to more
active metabolites.
Keywords Benzofuroxan derivates ꢀ Benzothiadiazole ꢀ
Cytotoxicity ꢀ Metabolism ꢀ Stilbenes
1
benzo[1,2-c]1,2,5-oxadiazole N -oxide and 14 5-(2-Z-
trimethoxyphenylethenyl)benzo[1,2,5]thiadiazole exhibiting
interesting cytotoxicity both on endothelial and on
tumor cells. Unlike the cis benzofurazan 12 5-(2-Z-
trimethoxyphenylethenyl)benzo[1,2-c]1,2,5-oxadiazole, in-
duction of apoptosis by 13 appeared to be through caspase-3
Introduction
Combretastatin initially isolated and characterized by Pettit
et al. from the bark of South African tree Combretum
caffrum is known for its anti-tumor activity due to its anti-
tubulin properties (Simoni et al., 2006). It also showed
cytotoxic (Cushman et al., 1992; Pettit et al., 1995a) and
anti-angiogenic (Dark et al., 1997; Liekens et al., 2001)
activities, but its major drawback is its low solubility. The
ability to create strong damage in tumor cells and structural
simplicity of CA-4 1, a combretastatin analogue (Fig. 1),
stimulated the search of new and more efficient compounds
with better pharmacological properties. Many efforts have
led to the preparation of more soluble analogues (Bedford
et al., 1996), for example, combretastatin CA-4 phosphate
Yohan Mac e´ and Emilie Bony have contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1363-3) contains supplementary
material, which is available to authorized users.
&
&
Olivier Feron
olivier.feron@uclouvain.be
Olivier Riant
olivier.riant@uclouvain.be
1
2
3
4
Institute of Condensed Matter and Nanosciences Molecules,
Solids and Reactivity (IMCN/MOST), Universit e´ catholique
de Louvain, Louvain-la-Neuve, Belgium
(CA-4P) 2 or amine 3 pro-drugs (Pettit et al., 1995b). They
exert antineoplastic activity through disruption of the blood
flow within the tumor leading to tumor cell death (Chaplin
and Dougherty, 1999). In particular, the water soluble CA-
Pharmacognosy Research Group (GNOS), Louvain Drug
Research Institute (LDRI), Universit e´ catholique de Louvain,
Brussels, Belgium
4P has shown great clinical promise. In a phase I clinical
Pole of Pharmacology and Therapeutics (FATH), Institut de
Recherche Exp e´ rimentale et Clinique (IREC), Universit e´
catholique de Louvain, Brussels, Belgium
trial, 15 patients with advanced cancer received CA-4P
with bevacizumab, and this combination appeared safe and
well tolerated. It was shown in this study that CA-4P in-
duced profound vascular changes, which were maintained
by the presence of bevacizumab (Nathan et al., 2012). In a
Laboratoire de Canc e´ rologie et de Toxicologie
Exp e´ rimentale, Facult e´ de Pharmacie, Universit e´ Libre de
Bruxelles, Brussels, Belgium
123

Med Chem Res
analyzed. Then, we performed metabolism investigations
to determine whether these compounds act directly or via
metabolites. Metabolites formed by in vitro microsomal
degradation were analyzed by high-performance liquid
chromatography (HPLC). Structural determination of
metabolites was realized by high-resolution mass spec-
trometry (HRMS) and by LC–MS comparison with au-
thentic standards of expected metabolites whose synthesis
is described here.
Fig. 1 Chemical structures of combretastatin A-4 and studied
analogues
phase II trial on 44 patients with platinum-resistant ovarian
cancer, the addition of CA-4P to paclitaxel and carboplatin
was well tolerated and appeared to produce a higher re-
sponse rate in this patient population than if the che-
motherapy was given without CA-4P (Zweifel et al., 2011).
In addition to their effect on the vasculature network within
the tumors, it was shown that CA-4P as well as other cis-
combretastatin A-4 derivatives had a strong inhibition ac-
tivity on several cancer cell lines including multi-drug-
resistant cells (Dark et al., 1997). However, these new
analogues were found to have significant side effects such
as cardiovascular toxicity and neurotoxicity (Rustin et al.,
Results and discussion
Chemistry
The synthetic route to obtain the combretastatin analogues is
described in Scheme 1. Trimethoxybenzyltriphenylphos-
phonium bromide was synthesized from corresponding
aldehyde by reduction with NaBH4 followed by an
exchanging of alcohol by bromide with PBr as reported in
3
the literature. The last step consists in the formation of
phosphonium salt in the presence of triphenylphosphine in
tetrahydrofuran (THF) (Maya et al., 2005).
2
003). For these reasons, it is necessary to elaborate new
The 5-formylbenzofuroxan was easily synthesized in
one-pot process from 4-chloro-3-nitrobenzaldehyde via
SNAr with azide and subsequent cyclization in dimethyl
sulfoxide (DMSO) (Scheme 2) (Porcal et al., 2008). The
5-formylbenzothiadiazole 7 was prepared from the com-
mercially available methyl-3,4-diaminobenzoate 4, which
was treated with thionyl chloride in the presence of tri-
ethylamine in dichloromethane (DCM) as solvent, afford-
ing the corresponding benzothiadiazole 5 (DaSilveira Neto
et al., 2005). Reaction with LiI in pyridine and subsequent
esterification/reduction in the presence of N,N-diisopropy-
lethylamine (DIPEA) and ClCOOEt following a treatment
and more specific combretastatin analogues for tumor and
endothelial cells. Previous structure–activity relationship
(
SAR) studies established that the cis-configuration of the
double bond and the methoxy groups in position 3, 4, and 5
0
in the A-ring and position 4 in the B-ring is essential for an
actual biological activity (Nam, 2003). In order to optimize
both their activity on tumor and endothelial cells, we
synthesized new combretastatin derivatives that cover a
wide range of chemical structures.
We maintained the 3,4,5-trimethoxy-substituted pattern
on A-ring and examined the effects due to replacement of
the B-ring with different benzoheterocycles. Recently, Si-
moni et al. examined the effects regarding replacement of
the B-ring of CA-4 with benzofuran or benzothiophene
with NaBH led to benzothiadiazol-5-ylmethanol 6 (Pou-
4
lain et al., 2001), which can be oxidized to the corre-
sponding aldehyde 7 with MnO (Carroll et al., 2004).
2
(
Simoni et al., 2006; Simoni et al., 2008). These com-
Wittig reaction between phosphonium bromides and the
different aryl aldehydes was carried out in THF with
potassium bis(trimethylsilyl)amide (KHMDS) in the pres-
ence of hexamethylphosphoramide (HMPA) (Scheme 1).
Use of KHMDS as the base and HMPA as chelating agent
gave the best results of desired Z-isomers with a Z/E ratio
of 60:40. The mixture of the Z and E stilbene isomers was
separated using flash column chromatography, leading to
the corresponding cis-stilbenes and trans-stilbenes identi-
fied by the difference of the coupling constants of vinylic
proton in NMR (around 12 Hz for Z-isomers and 16 Hz for
E-isomers).
pounds possess potent cytotoxic activity while also show-
ing potent binding to tubulin and inhibition of tubulin
polymerization. In this study, we investigated stilbenes by
extending the replacement of the B-ring by a new class of
benzoheterocycle pro-drugs such as benzofuroxan, benzo-
furazan, or benzothiadiazole (Fig. 2).
The present report summarizes the synthesis of cis- and
trans-stilbenes of these new types of combretastatin ana-
logues. We analyzed the in vitro activity of the synthesized
compounds on endothelial and cancer cell lines and
showed that some molecules exert both a very strong anti-
angiogenic (inhibition of endothelial cell growth) and anti-
tumoral (inhibition of cancer cell growth) effects. The pro-
apoptotic effect of the most active derivatives was also
Finally, the other analogues were prepared directly from
the benzofuroxan stilbenes (Scheme 2). The use of triph-
enyl phosphine in the presence of ethanol led to the
1
23

Med Chem Res
Fig. 2 Heterocyclic derivates
of combretastatin
Scheme 1 General pathway for
the preparation of stilbenes.
i NaBH
iii PPh
HMPA/THF
4
, MeOH; ii PBr
, THF; iv KHMDS,
3
, DCM;
3
Scheme 2 Synthesis of
heterocyclic compounds.
i SOCl
pyridine; iii DIPEA, ClCOOEt,
THF; iv NaBH , H O; v MnO
CHCl ; vi NaN , DMSO; vii
2 3
, Et N, DCM; ii LiI,
4
2
2
,
3
3
Arylphophosnium bromide salt,
KHMDS, HMPA/THF; viii
2
-nitropropane, piperidine,
THF; ix PPh , EtOH;
x FeSO .7H O, DMSO; xi
SnCl O, AcOEt
ꢀH
3
4
2
2
2
deoxygenation of benzofuroxan in benzofurazan (Aguirre
et al., 2005). The formation of benzimidazole dioxide was
obtained following the Abu El-Haj (Abu El-Haj, 1972)
method using 2-nitropropane in the presence of piperidine
as base in THF (Boiani et al., 2006).
afford references for the metabolic investigations. Only the
metabolites from the reduction process were prepared fol-
lowing two different methods (other metabolites were al-
ready prepared by the reaction described above). On the
one hand, the reduction of benzofuroxan was performed
Some of the expected metabolites of heterocyclic deri-
vates of combretastatin (Scheme 3) were synthesized to
with FeSO in ethyl acetate (AcOEt) to obtain the corre-
4
sponding nitro amine stilbenes (Porcal et al., 2008), on the
123

Med Chem Res
Scheme 3 Some of the
expected metabolites
other hand with SnCl in DMSO to obtain the diamine
2
that occurs after the final lytic event (Collins et al., 1997).
To quantitate the relative proportion of apoptotic popula-
tion of cells following a 72-h treatment with 12, 13, and 3,
U373 glioma cells were analyzed by flow cytometry after
propidium iodide and TUNEL staining. As shown in
Fig. 3, the apoptotic population, as indicated by pre-G1
phase, was much higher in treated cells than in control.
TUNEL staining confirmed that 12, 13, and 3 does in-
deed induce apoptosis in U373 glioma cells (Fig. 4a). In
contrast to propidium iodide staining, TUNEL assay nev-
ertheless highlighted a dose-dependent apoptotic response
in U373 cells treated with 13, which is not the case with 12
and 3. To determine the mechanism of apoptosis-induced
effects, we also analyzed caspase-3 activation (J a¨ nicke
et al., 1998). The differential effect between 13 and the
other tested compounds is probably due to the fact that 13
is the only compound that induces apoptosis through cas-
pase-3 activation (Fig. 4b).
stilbene (Scheme 3).
Biological studies
For most products of the two groups 8–11 and 12–15, we
did not observe striking differences regarding the cyto-
toxicity (IC ) between HUVEC cells and cancer cells
5
0
(
Table 1). We nevertheless observed a higher activity on
HUVEC cells for 14. The group 12–15 contains the more
active products; their IC50 are distributed on a 3 log order
range. Previously reported importance of cis-configuration
was only observed here with benzofuroxan and 2,1,3-
benzothiadiazole substitutions. 3 was used as reference
compound since this combretastatin analogue already
showed potent anti-tumor activity in vitro and in vivo
(
Ohsumi et al., 1998).
Pro-apoptotic effects
Metabolic stability studies
Within the most active group (cis-stilbenes), we decided to
investigate the pro-apoptotic effect of 13, a highly cyto-
toxic derivative, compared to 12, that is less cytotoxic, and
To determine whether the synthesized compounds were
effective as such or via metabolites on cell proliferation,
and to try to explain the observed cytotoxic effects, we
evaluated the phase I metabolism of stilbenes both on
3 which is used as a reference compound. Internucleosomal
DNA fragmentation is one of the hallmarks of apoptosis
Table 1 Cytotoxic activity (IC50 lM) of the compounds
Compound
A-ring
B-ring
HUVECs
LoVo
PC-3
U373
8
9
Benzofurazan
[100
60
37
3
83
6
43
10
Benzofuroxan
1
1
1
1
1
1
3
0
1
2
3
4
2,1,3-benzothiadiazole
2,2-dimethyl-benzimidazole dioxide
Benzofurazan
87
38
4
88
7
[100
4
4.3
30
24
0.5
1
61
2
30
Benzofuroxan
1.5
0.1
6.2
0.02
0.9
3
2,1,3-benzothiadiazole
2,2-dimethyl-benzimidazole dioxide
4-methoxy-3-aminobenzene
7
5
a
4
19
\0.01
5
\0.01
\0.01
a
Reference compound
1
23

Med Chem Res
Fig. 3 Pre-G1 accumulation of
U373 glioma cells after 72-h
treatment with 12, 13, and 3
Fig. 4 a TUNEL; b Immunoblot of caspase-3 cleavage in U373 cells following 24 h of treatment with different concentrations of the indicated
compounds
mouse liver and on human liver S9 microsomes. On mouse
liver microsomes, most tested compounds showed a rapid
degradation with t1/2 from 0.1 to 6.0 min, 8 and 10 with t1/2
of 13.4 and 10.8 min, respectively, being the more stable
compounds. On human liver S9 microsomes, all com-
pounds appeared more stable and 8 and 10 exhibited again
a better metabolic stability profile with t1/2 of 114 and
as 13 and 14, which would not be the case with slowly
metabolized compounds.
In order to see whether a correlation exists between cy-
totoxic activity and metabolization for 8–15 derivatives,
metabolite identification was performed by HPLC–Orbi-
2
trap–MS (Table 3) by comparing retention times and MS
mass spectra with expected reduced metabolites (16, 17, 18,
19, and 20). Cytotoxic activity of synthesized metabolites
was also investigated, and the data are given in Table 4.
HPLC–MS profiles of furazan metabolites (8 and 12)
only revealed the presence of oxidized metabolites. Me-
tabolization of 8 only produced an unstable metabolite at
7
3.6 min, respectively (Table 2). Interestingly, 8 and 10
which are the more stable compounds in this study also
showed the lowest cytotoxicity. As metabolization in cul-
tured cells is much slower than on purified microsomes, it
is possible that produced metabolites are responsible for
cytotoxic activities of highly metabolized compounds such
?
?
m/z 347.1226 corresponding to [C H N O ] ([M?H]
17 19 2 6
123

Med Chem Res
Table 2 Stability (t1/2 and CLint) of combretastatin analogues both in mouse liver microsomes and in human liver S9
Compound
Mouse liver microsomes
1/2 (min)
Human liver S9
1/2 (min)
t
CLint (mL/min/g)
t
CLint (mL/min/g)
8
9
13.4
6
51.7
115.5
64.2
114
13.8
73.6
5.2
6.1
50.2
9.4
1
1
1
1
1
1
0
1
2
3
4
5
10.8
1.3
1.7
0.1
0.1
1.9
533.1
407.6
6930.0
6930.0
364.0
133.3
15.6
216.6
42.5
77.9
44.5
3.2
16.3
8.9
n = 2 for each compound
1/2: half-life of the drug; CLint intrinsic clearance
t
calcd: 347.1238), which probably results from the oxida-
tion of the furazan group, whereas produced metabolites
from 12 corresponded to several demethylated metabolites
at m/z 299.1018 and hydroxylated metabolites at m/z
compared to 9 may be explained by its fast reduction to
more cytotoxic cis metabolites, i.e., 18, 19, and 20 as
illustrated in Fig. 5 and Table 4. Indeed, 18 and 19 are
rapidly observed since they already appeared on the
chromatogram at 0 min of incubation (directly when the
compound is solubilized).
?
?
3
29.1121 with raw formula [C H N O ] ([M?H]
16 15 2 4
?
?
calcd: 299.1026) and [C H N O ] ([M?H] calcd:
1
7 17 2 5
3
29.1132), respectively.
The metabolization of benzofuroxans produced both
Compounds 11 and 15 showed similar cytotoxicity and
metabolic stability. However, HPLC–MS metabolite pro-
file of 11 showed only the reduced metabolite 17, whereas
15 metabolization produced two reduced metabolites 17
and 20, and various primary or secondary oxidized
metabolites at m/z 285.1223, 287.1377, 341.1482, and
oxidized and reduced metabolites. By comparison with
expected metabolites, HPLC–MS profile of 9 allowed us
to identify two reduced metabolites 16 and 17, 16 being
the main metabolite, and an oxidized metabolite at m/z
?
?
?
?
3
47.1217 corresponding to [C H N O ] ([M?H]
1
355.1637 correspond to [C H N O ] ([M?H] calcd:
16 17 2 3
7
19
2
6
?
285.1234), [C H N O ] ([M?H] calcd: 287.1390),
?
calcd: 347.1238), the same metabolite as 8. The me-
tabolization of 13 produced oxidized metabolites at m/z
1
6 19 2 3
?
?
[C H N O ]
1
([M?H]
calcd: 341.1496), and
9 21 2 4
?
[C H N O ] ([M?H] calcd: 355.1652), respectively.
?
3
33.1067 and m/z 317.1120 corresponding to demethy-
2
0 23 2 4
?
lated ([C H N O ] , [M?H] calcd: 333.1081) and
?
Even if 20 is the major metabolite produced, other identi-
fied compounds still represent a significant proportion of
produced metabolites.
1
6 17 2 6
?
hydroxylated metabolites ([C H N O ] , [M?H] cal-
?
1
6 17 2 5
cd: 317.1132). Moreover, four reduced metabolites have
been identified: 16, 18, 19, and 20, with 18 being the main
metabolite. It appeared that the higher cytotoxicity of 13
The metabolization of thiadiazole isomers 10 and 14 is
similar to 11 and 15. Indeed, 10 metabolization produced
Table 3 Major identified metabolites of combretastatin analogues by HPLC–Orbitrap–HRMS (m/z at 10 mmu) and relative intensities after
6
0-min incubation with mouse liver microsomes
a
Tr (min)
?
Compound
[M?H]
Identified compounds m/z (relative intensity %)
8
9
16.4
15.8
17.1
9.9
313.1170
329.1115
329.0941
371.1585
313.1171
329.1121
313.1226 8 (10.6), 347.1226 (100)
301.1531 17 (78.4), 331.1271 16 (100), 347.1217 (2.7)
301.1532 17 (100), 329.0941 10 (23.8)
301.1530 17 (100)
1
1
1
1
0
1
2
3
16.1
15.5
299.1018, 329.1121 (100)
301.1535 20 (5.3), 317.1120 (26.5), 331.1278 16 (2.2), 331.
277 18 (100), 331.1277 19 (0.3), 333.1067 (4.1)
1
1
4
5
16.8
9.8
329.0941
371.1585
315.0787 (7.3), 301.1535 17 (5.5), 301.1534 20 (100), 363.0996 (16.5)
1
285.1223 (5.7), 287.1377 (44.0), 301.1534 17 (33.1), 301.
1
535 20 (100), 341.1482 (36.1), 355.1637 (42.9)
a
Retention time of initial compounds obtained by HPLC–UV
1
23

Med Chem Res
Table 4 Cytotoxic activity (IC50 lM) of reduced metabolites
Compound
A-ring
B-ring
HUVECs
LoVo
PC-3
U373
a
1
6
3-Nitro-4-aminobenzene
3,4-Diaminobenzene
46
24
4
22
5
38
4
1
7
3.7
1
1
2
8
9
0
3-Nitro-4-aminobenzene
4-Nitro-3-aminobenzene
3,4-Diaminobenzene
0.08
0.6
0.05
0.6
0.23
9
0.15
0.7
1.1
0.3
1.2
0.5
a
Ratio 72/28 3-nitro-4-aminobenzene/4-nitro-3-aminobenzene
Fig. 5 Comparison of obtained chromatograms from 9 to 13 in the presence of mouse liver microsome at 0 and after 10-min incubation
detection at 313 nm)
(
the reduced metabolite 17, whereas the metabolization of
4 produced diverse oxidized metabolites and the reduced
which is very unstable, leading rapidly to the production of
the more cytotoxic major metabolite 20 as shown in
Table 4.
1
metabolites 17 and 20; unlike 15, the main metabolite of 14
is 20, which is much cytotoxic than 17. The oxidized
metabolites identified at m/z 315.0787 and 363.0996 cor-
?
?
respond to [C H N O S ] ([M?H] calcd: 315.0798)
1
Conclusion
6 15 2 3 1
?
and [C H N O S ] ([M?H] calcd: 363.1009), re-
?
1
7 19 2 5 1
spectively. However, the cis-isomer 14 is much more cy-
totoxic than the trans-isomer 10. This difference may be
due to the good metabolic stability of 10 compared to 14,
In this study, we reported the synthesis of new combre-
tastatin analogues by replacing the B-ring with different
benzofuroxan and benzothiadiazole derivatives as well as
123

Med Chem Res
1
13
reduced metabolites and analyzed their cytotoxic activity
on one endothelial and three tumor cell lines. We also
determined their metabolic stability and identified
metabolites by LC-HRMS. Two newly synthesized
derivatives 13 and 14 exhibited interesting levels of cyto-
toxicity both on endothelial and on tumor cells, with 14
being more toxic to HUVEC cells showing a higher anti-
angiogenic potential. We also showed that these com-
pounds possess pro-apoptotic properties and that the mode
of induction of apoptosis could differ according to the
chemical structure. In fact, we showed that 13 induced
apoptosis through caspase-3 activation, whereas the less
cytotoxic derivative of this series 12 and the reference
compound 3 did not induce apoptosis through this pathway.
Investigation of metabolic stability indicated that the
most cytotoxic cis compounds 13 and 14 are rapidly me-
tabolized to more cytotoxic reduced cis derivatives,
whereas the less cytotoxic trans compounds 9 and 10 ap-
peared more stable both on human and on mouse micro-
somes and also produced less cytotoxic trans metabolites.
These results indicated that some of these compounds may
act as pro-drug via reductive activation and that highly
cytotoxic cis-configuration can be maintained during re-
ductive metabolization.
1998) copy of H and C NMR spectra are given in the
electronic supplementary material.
5
-Formylbenzo[1,2-c]1,2,5-oxadiazole N-Oxide A mix-
ture of 4-chloro-3-nitrobenzaldehyde (1.9 g, 10.3 mmol)
and NaN3 (0.63 g, 9.7 mmol) in DMSO (12 mL) was
heated at 75 °C during 30 min and subsequently at 100 °C
during 1.5 h. The residue was diluted with H O (20 mL)
2
and extracted with EtOAc (3 9 10 mL). After the workup,
the organic layer was dried with MgSO and evaporated in
4
vacuum. The residue was purified by chromatographic
1
column (DCM) to give a yellow solid (1.65 g, 98 %). H
NMR (300 MHz, CDCl ) d 10.00 (s, 1H), 7.95 (br s, 1H),
.83 (br s, 1H), 7.71 (br s, 1H).
3
7
3,4-Diaminobenzoic acid methyl ester (4) H SO (6 mL,
2
4
113 mmol) was slowly added dropwise at 0 °C to a solu-
tion of 3,4-diaminobenzoic acid (3 g, 19.7 mmol) in
methanol (60 mL) with stirring; then, the mixture heated at
reflux for 3 h. Saturated solution of NaHCO was added
3
until the mixture was basic; then, the mixture was extracted
with DCM. The extract was dried and evaporated in
vacuum to give the product 4 (2.98 g, 91 %) as a brown
1
crystalline solid. H NMR (300 MHz, CDCl ) d 7.46 (dd,
3
J = 8.1, 1.8 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H), 6.67 (d,
J = 8.1 Hz, 1H), 3.85 (s, 3H), 3.53 (s, 4H).
Benzo[1,2,5]thiadiazole-5-carboxylic acid methyl ester
(
5) 3,4-Diaminobenzoic acid methyl ester 4 (1.5 g, 9 m-
Experimental
Chemistry
mol) was added to a solution of DCM (50 mL) and tri-
ethylamine (5.1 mL, 36.6 mmol). The solution was stirred
until total dissolution of the diamine. Then, thionyl chlo-
ride (1.32 mL, 18 mmol) was added dropwise very slowly,
and the mixture refluxed for 4 h. The solvent was removed
under reduced pressure and 20 mL of water added. Con-
centrated HCl was added at 0 °C until pH = 2. Then, the
mixture was extracted with DCM (3 9 20 mL), dried over
All starting materials were purchased from commercial
suppliers (Acros, Sigma-Aldrich, ABCR or TCI) and used
without further purification. Solvent used was anhydrous,
and the reactions were carried out under an argon atmosphere
1
13
using standard glassware. H and C spectra were recorded
1
on a Brucker-300 MHz spectrometer. Chemical shifts for H
MgSO
pure compound 5 in quantitative yield (1.75 g). H NMR
300 MHz, CDCl ) d 8.76 (d, J = 0.6 Hz, 1H), 8.22 (dd,
, and filtered. The solvent was removed, affording
4
1
1
and C NMR acquired in CDCl are reported in ppm relative
3
3
(
3
to residual solvent proton (d = 7.26 ppm) and carbon
J = 9.3, 1.3 Hz, 1H), 8.05 (d, J = 9.2 Hz, 1H), 4.01 (s,
(
d = 77 ppm) signals, respectively. Multiplicity is indicated
follows: s (singlet); d (doublet); t (triplet); m (multiplet); dd
doublet of doublet); ddd (doublet of doublet of doublet); and
3
H).
(
Benzo[1,2,5]thiadiazol-5-ylmethanol (6) A mixture of
benzo[1,2,5]thiadiazole-5-carboxylic acid methyl ester 5
(1.75 g, 9 mmol) in pyridine (25 mL) and LiI (7.25 g,
54.2 mmol) was refluxed overnight. The solvent was re-
moved under reduced pressure, and the resulting oil was
br (broad). Coupling constants are reported in hertz (Hz).
High- and low-resolution mass spectra were acquired using
electrospray as the ionization technique in positive ion mode
as stated all MS analysis samples were prepared as solutions
in methanol. Column chromatography was carried out on
silica gel (ROCC 60, 40–63 lm). TLC analyses were per-
formed on commercial aluminium plates bearing a 0.25-mm
layer of Merck Silica gel 60F254. The purity of all
biologically evaluated compounds was confirmed to be
dissolved in H O (25 mL). After the addition of HCl (3 N)
2
at 0 °C, the mixture was extracted with DCM
(3 9 25 mL), dried over MgSO , and filtered. The solvent
4
was removed. The resulting solid was dissolved in THF
(30 mL) and DIPEA (1.8 mL, 10.33 mmol). The mixture
was cooled to 0 °C, and ClCOOEt (1 mL, 10.46 mmol)
was added. After the resulting mixture was stirred for 1.5 h
[95 % by HPLC. Combretastatin analogue 3 was prepared
by the method as reported in the literature (Ohsumi et al.,
1
23

Med Chem Res
at 0 °C, a solution of NaBH (0.8 g, 21.14 mmol) in H O
7.65–7.54 (m, 6H), 6.47 (d, J = 2.5 Hz, 2H), 5.39 (d,
J = 14.1 Hz, 2H), 3.75 (s, 3H), 3.49 (s, 6H).
4
2
(
4 mL) was added dropwise. The mixture was stirred for
h at room temperature and evaporated under reduced
pressure. The orange residue was dissolved in AcOEt
2
General procedure for Wittig reactions (9, 10, 13 and 14)
(
(
20 mL) and washed with a saturated solution of Na CO3
2
20 mL) and brine (20 mL). The organic layer was dried
The required phosphonium bromide (ca. 0.1 M) was sus-
pended in dry THF and cooled to 0 °C under Ar. KHMDS
(0.7 M in toluene 1.1 equiv) was added dropwise, and the
resulting solution was stirred at room temperature for 1 h.
Then, dry HMPA (2 equiv) was added, and the mixture was
stirred at the same temperature for an additional 30 min.
Then, a solution of aldehyde (1 equiv) in THF (ca. 0.2 M)
was added dropwise at -78 °C. The mixture was stirred
overnight at room temperature, quenched with 10 mL of
over MgSO , filtered, evaporated under reduced pressure,
4
and purified by flash chromatography (petroleum ether/
AcOEt 5:5 to AcOEt) to yield 1.06 g (71 %) of 6 a yellow
1
powder. H NMR (300 MHz, CDCl ) d 7.84 (s, 1H), 7.83
3
(
d, J = 9.1 Hz, 1H), 7.46 (dd, J = 8.9, 1.7 Hz, 1H), 4.80
(
br s, 2H), 3.42 (br s, 1H).
Benzo[1,2,5]thiadiazol-5-carboxaldehyde (7) Benzo[1,2,
]thiadiazol-5-ylmethanol 6 (115 mg, 0.69 mmol) and
MnO (241 mg, 2.8 mmol) in CHCl (10 mL) were stirred
5
aqueous solution of NaHCO (5 %), and extracted with
3
2
3
AcOEt (3 9 20 mL). The organic layer was dried over
at room temperature overnight. The reaction mixture was
filtered on a plug of Celite and the filtrate evaporated to
MgSO and evaporated under reduce pressure. The residue
4
was purified with flash chromatography on silica gel using
petroleum ether and AcOEt as eluant to afford the E- and
Z-isomers in variable yields.
provide 111 mg of pure 2,1,3-benzothiadiazole-5-carbox-
1
aldehyde 7 (98 %): H NMR (300 MHz, CDCl ) d 10.19
3
(
s, 1H), 8.49 (s, 1H), 8.09 (s, 2H).
5
-(2-Z-Trimethoxyphenylethenyl)benzo[1,2-c]1,2,5-oxadiazole
1
N -oxide (13) and 5-(2-E-Trimethoxyphenylethenyl)benzo
3
,4,5-Trimethoxybenzyl
zaldehyde (5 g, 25.5 mmol) was dissolved in methanol
50 mL) and cooled to 0 °C. NaBH (1.45 g, 38 mmol)
alcohol 3,4,5-Trimethoxyben-
1
1,2-c]1,2,5-oxadiazole N -oxide (9) Purification by flash
chromatography (PE/AcOEt, 80:20) afforded 87 % yield of
[
(
4
1
separable isomers Z/E (56/44 ratio). 13 (Z-isomer): H
was added in small portion. Then, the reaction mixture was
stirred 1 h at room temperature. The solvent was removed
under reduce pressure, and the crude was diluted in water
NMR (300 MHz, CDCl ) d 7.49–7.01 (m, 3H), 6.77 (d,
J = 12.2 Hz, 1H), 6.53 (d, J = 12.1 Hz, 1H), 6.45 (s, 2H),
3
1
3
3
1
5
.86 (s, 3H), 3.71 (s, 6H); C NMR (75 MHz, CDCl ) d
3
(
20 mL) and extracted with AcOEt (3 9 20 mL). The or-
53.18, 138.18, 134.34, 131.29, 126.91, 106.17, 60.93,
?
6.01; MS (APCI) m/z: 301.1 ([M4H-2O] ), 314.1 ([M2H-
ganic phases were combined, dried over MgSO , filtered,
4
and concentrated to give colorless oil in quantitative yield
1
(
?
?
O) ], 329.1 ([MH] ); HRMS calcd for C17H O
17 5
1
N 329.11320, found 329.11298. 9 (E-isomer): H NMR
5.05 g, 25.5 mmol). H NMR (300 MHz, CDCl ) d
ppm): 6.60 (s, 2H), 4.63 (d, J = 5.4 Hz, 2H), 3.86 (s, 6H),
3
2
(
(300 MHz, CDCl ) d 7.70–7.20 (m, 3H), 7.14 (d,
3
3
.83 (s, 3H).
J = 16.3 Hz, 1H), 6.99 (d, J = 16.3 Hz, 1H), 6.76 (s, 2H),
1
3
3
1
6
.93 (s, 6H), 3.89 (s, 3H); C NMR (75 MHz, CDCl ) d
3
3
,4,5-Trimethoxybenzyl bromide 3,4,5-Trimethoxybenzyl
53.49, 139.04, 133.03, 131.49, 125.43, 105.93, 104.13,
?
0.96, 56.14. MS (APCI) m/z: 301.1 ([M4H-2O] ), 314.1
alcohol (5.05 g, 25.5 mmol) was diluted in dry DCM
20 mL) and cooled to 0 °C. PBr (2.5 mL, 26.3 mmol)
(
3
?
[M2H-O) ], 329.1 ([MH] ); HRMS calcd for
?
(
was added dropwise. The mixture was stirred 2 h at room
temperature, poured onto water (20 mL), and extracted
with DCM (3 9 20 mL). The organic layer was dried over
C H O N 329.11320, found 329.11308.
17 17 5 2
5-(2-Z-Trimethoxyphenylethenyl)benzo[1,2,5]thiadiazole (14)
and 5-(2-E-Trimethoxyphenylethenyl) benzo[1,2,5]thiadiazole
MgSO and evaporated under reduce pressure to give white
4
1
solid in quantitative yield (6.66 g, 25.5 mmol). H NMR
(10) Purification by flash chromatography (PE/Et O,
2
(
300 MHz, CDCl ) d (ppm): 6.58 (s, 2H), 4.42 (s, 2H),
3
80:20) afforded 94 % yield of separable isomers Z/E (39/61
3
.82 (s, 6H), 3.80 (s, 3H).
ratio) (Isomerization of Z-isomer during the treatment and
1
purification). 14 (Z-isomer): H NMR (300 MHz, CDCl ) d
3
3,4,5-Trimethoxybenzyltriphenylphosphonium bromide Triph-
7.91 (s, 1H), 7.80 (d, J = 9.1 Hz, 1H), 7.53 (d, J = 9.2 Hz,
enylphosphine (10.5 g, 30.6 mmol) and 3,4,5-trimethoxy-
benzyl bromide (6.66 g, 25.5 mmol) were dissolved in
1H), 6.72 (s, 2H), 6.49 (s, 2H), 3.85 (s, 3H), 3.63 (s, 6H);
3
1
C NMR (75 MHz, CDCl ) d 155.13, 154.05, 153.06,
3
6
0 mL of THF. The mixture was refluxed with stirring
138.99, 137.80, 132.68, 131.82, 131.45, 128.26, 120.96,
overnight. The resulting white solid was filtered and washed
120.36, 106.19, 60.94, 55.92; MS (APCI) m/z: 329.1
?
([MH] ); HRMS calcd for C H O N S 329.09544,
with hexane to afford the phosphonium salt in 91 % yield
1
1
7 17 3 2
1
found 329.09519. 10 (E-isomer): H NMR (300 MHz,
(
12.5 g). H NMR (300 MHz, CDCl ) d 7.80–7.70 (m, 9H),
3
123

Med Chem Res
CDCl ) d 7.95 (d, J = 9.4 Hz, 1H), 7.94 (s, 1H) 7.86 (d,
J = 9.6 Hz, 1H), 7.21 (d, J = 16.2 Hz, 1H), 7.13 (d,
chromatography on silica gel (petroleum ether/AcOEt
50/50 to AcOEt) to afford the corresponding benzimida-
zole in variable yields.
3
J = 16.3 Hz, 1H), 6.79 (s, 2H), 3.93 (s, 6H), 3.89 (s, 3H);
1
3
C NMR (75 MHz, CDCl ) d 155.48, 154.48, 153.43,
3
(
E)-2,2-Dimethyl-5-(3,4,5-trimethoxyphenylethenyl)-2H-
1
1
38.55, 132.20, 131.61, 127.78, 126.74, 121.32, 118.65,
?
03.88, 60.94, 56.12; MS (APCI) m/z: 329.1 ([MH] );
benzimidazole 1,3-dioxide (11) Compound was isolated
1
in 82 % yield. H NMR (300 MHz, CDCl ) d 7.24 (s, 2H),
3
HRMS calcd for C H O N S 329.09544, found
17 17 3 2
7.16 (s, 1H), 7.07 (d, J = 16.2 Hz, 1H), 6.85 (d,
3
29.09513.
J = 16.2 Hz, 1H), 6.73 (s, 2H), 3.92 (s, 6H), 3.88 (s, 3H),
1
3
1
1
1
.73 (s, 6H); C NMR (75 MHz, CDCl ) d 153.44, 139.53,
3
General procedure for the preparation of benzofurazan (8
and 12)
39.05, 136.46, 135.55, 132.75, 131.56, 129.53, 125.64,
15.65, 111.86, 104.13, 97.42, 60.91, 56.10, 24.11; MS
?
APCI) m/z: 355.2 ([MH-O] ), 371.2 ([MH] ); HRMS
?
(
The corresponding N-oxidebenzoxadiazole 9 or 13
0.4 mmol) and triphenylphosphine (93 mg, 0.4 mmol)
calcd for C H O N 371.16015, found 371.16000.
20 23 5 2
(
were dissolved in 30 mL of EtOH and refluxed for 2 h. The
solvent was evaporated under reduced pressure and then
purified with flash chromatography on silica gel using
petroleum ether and AcOEt as eluant to afford benzofu-
razan in variable yields.
(Z)-2,2-Dimethyl-5-(3,4,5-trimethoxyphenylethenyl)-2H-
benzimidazole 1,3-dioxide (15) Compound was isolated
1
in 90 % yield. H NMR (300 MHz, CDCl ) d 7.11 (s, 1H),
3
7.00 (d, J = 9.7 Hz, 1H), 6.77 (d, J = 9.8 Hz, 1H), 6.71
(d, J = 12.2 Hz, 1H), 6.47 (s, 2H), 6.36 (d, J = 12.2 Hz,
1
H), 3.86 (s, 3H), 3.75 (s, 6H), 1.69 (s, 6H); C NMR
3
1
5
-(2-E-Trimethoxyphenylethenyl)benzo[1,2-c]1,2,5-oxa-
(
75 MHz, CDCl ) d 153.15, 140.32, 138.25, 136.27,
3
diazole (8) Purification by flash chromatography (PE/
AcOEt, 90:10) afforded pure benzofurazan 8 (85 % yield).
1
1
35.21, 134.40, 132.94, 131.63, 127.24, 114.71, 114.22,
06.37, 104.16, 97.38, 60.96, 56.11, 24.16; MS (APCI) m/
?
1
H NMR (300 MHz, CDCl ) d 7.81 (d, J = 9.7 Hz, 1H),
3
?
z: 355.2 ([MH-O] ), 371.2 ([MH] ); HRMS calcd for
7
.72 (d, J = 9.5 Hz, 1H), 7.71 (s, 1H), 7.19 (d,
C H O N 371.16015, found 371.15995.
20 23 5 2
J = 16.2 Hz, 1H), 7.06 (d, J = 16.2 Hz, 1H), 6.78 (s, 2H),
1
3
3
1
1
.93 (s, 6H), 3.89 (s, 3H); C NMR (75 MHz, CDCl ) d
3
General procedure for the preparation of diamine
53.48, 149.76, 148.78, 140.21, 138.94, 133.02, 131.64,
derivatives (17 and 20)
30.10, 126.04, 116.42, 112.54, 104.10, 60.95, 56.14; MS
?
APCI) m/z: 298.1 ([M2H-O) ], 313.1 ([MH] ); HRMS
?
(
The corresponding benzoxadiazole N-oxide 9 or 13
calcd for C H O N 313.11828, found 313.11806.
7 17 4 2
1
(
100 mg, 0.305 mmol) and SnCl ꢀH O (350 mg, 1.52 m-
2
2
mol) in AcOEt (10 mL) were refluxed overnight. Then, the
mixture was treated with 10 mL of aqueous solution of
5-(2-Z-Trimethoxyphenylethenyl)benzo[1,2-c]1,2,5-oxadiazole
(
12) Purification by flash chromatography (PE/AcOEt,
1
0:10) afforded pure benzofurazan 12 (96 % yield). H
NaHCO (5 %) and extracted with AcOEt (3 9 10 mL).
3
9
The organic layer was dried over MgSO and evaporated
4
NMR (300 MHz, CDCl ) d 7.72 (s, 1H), 7.63 (d,
3
under reduce pressure. The residue was purified with flash
chromatography on silica gel (petroleum ether/AcOEt
J = 9.4 Hz, 1H), 7.28 (d, J = 9.5 Hz, 1H), 6.78 (d,
J = 12.1 Hz, 1H), 6.63 (d, J = 12.2 Hz, 1H), 6.45 (s, 2H),
70/30 to AcOEt) to afford the corresponding benzimida-
1
3
3
1
1
.86 (s, 3H), 3.67 (s, 6H); C NMR (75 MHz, CDCl ) d
3
zole in variable yields.
53.13, 149.40, 148.27, 140.94, 138.08, 134.09, 133.95,
31.39, 127.45, 115.15, 114.96, 106.21, 60.92, 55.94; MS
?
APCI) m/z: 313.1 ([MH] ); HRMS calcd for
5-(2-E-Trimethoxyphenylethenyl)benzene-1,2-diamine
1
(17) Compound was isolated in 79 % yield. H NMR
(
C H O N 313.11828, found 313.11803.
7 17 4 2
(300 MHz, CDCl
3
) d 6.98–6.80 (m, 4H), 6.73–6.68 (m,
H), 6.69 (s, 2H), 3.90 (s, 6H), 3.86 (s, 3H), 3.31 (br s, 4H);
1
1
1
3
C NMR (75 MHz, CDCl ) d 153.25, 137.26, 134.94,
3
General procedure for the preparation of benzimidazole
,3-dioxide derivatives (11 and 15)
1
1
34.59, 133.68, 129.42, 128.41, 125.27, 119.34, 116.52,
1
14.26, 103.07, 60.88, 55.99.; MS (ESI) m/z: 301.2
?
[MH] ); HRMS calcd for C H O N 301.15467, found
(
1
7 21 3 2
The corresponding benzoxadiazole N-oxide 9 or 13
301.15494.
(
(
0.22 mmol), 2-nitropropane (23 lL, 1.2 equiv), piperidine
26 lL, 1.2 equiv) in THF (5 mL) were stirred at room
5-(2-Z-Trimethoxyphenylethenyl) benzene-1,2-diamine
1
(20) Compound was isolated in 87 % yield. H NMR
temperature until disappearance of starting material was
observed by TLC (24–72 h). The solvent was evaporated
under reduce pressure. The residue was purified with flash
(300 MHz, CDCl ) d 6.71–6.66 (m, 2H), 6.59–6.54 (m,
1H), 6.57 (s, 2H), 6.43 (d, J = 12.2 Hz, 1H), 6.32 (d,
3
1
23

Med Chem Res
J = 12.2 Hz, 1H), 3.84 (s, 3H), 3.70 (s, 6H), 3.53 (br s,
1
(d, J = 12.2 Hz, 1H), 6.21 (br s, 2H), 3.85 (s, 3H), 3.70 (s,
13
3
4
H); C NMR (75 MHz, CDCl ) d 152.73, 136.78,
6H); C NMR (75 MHz, CDCl ) d 153.02, 145.22,
3
3
1
34.20, 134.02, 133.18, 130.13, 128.97, 127.59, 121.39,
133.40, 131.46, 127.63, 126.02, 118.39, 118.09, 106.12,
?
60.96, 55.98; MS (APCI) m/z: 331.1 ([MH] ); HRMS
1
17.11, 116.05, 105.87, 60.86, 55.86; MS (APCI) m/z:
?
01.2 ([MH] ); HRMS calcd for C H O N 301.15467,
3
calcd for C H O N 331.12885, found 331.12860.
17 19 5 2
1
7
21
3
2
found 301.15498.
Cell models and in vitro treatments
General procedure for the preparation of nitroaniline
derivatives (16, 18, and 19)
The histological types and origins of the three cancer cell
lines that were used for the MTT colorimetric assay are as
follows. The cell lines were obtained from the American
Type Culture Collection (ATCC, Manassas, VA), the
European Collection of Cell Culture (ECACC, Salisbury,
UK), and the Deutsche Sammlung von Mikroorganismen
und Zellkulturen (DSMZ, Braunschweig, Germany). The
analyzed cell lines include the U373 (ECACC code
89081403) glioma, the LoVo (DSMZ code ACC350) colon
carcinoma, and the PC-3 (ATCC code CRL1435) prostate
carcinoma cell lines. All cell lines are from human origin.
The cancer cell lines were cultured in DMEM. Human
Umbilical Vein Endothelial cells (HUVECs, Sigma-
Aldrich) were cultured in endothelial cell growth medium
The corresponding benzoxadiazole N-oxide 9 or 13
(
100 mg, 0.305 mmol) and FeSO .7H O (6 equiv) in
4 2
DMSO (10 mL) were stirred for 1 h at room temperature.
The mixture was treated with water (10 mL) and extracted
with AcOET (3 9 10 mL). The organic layer was dried
over MgSO and evaporated under reduce pressure. The
4
residue was purified with flash chromatography on silica
gel (petroleum ether/AcOEt 80/20) to afford the corre-
sponding benzimidazole in variable yields.
4
-(2-E-Trimethoxyphenylethenyl)-2-nitroaniline (16p) and
-(2-E-Trimethoxyphenylethenyl)-2-nitroaniline (16o) An
5
inseparable mixture of two isomers was isolated in 41 %
1
yield (16p/16o 72/28 ratio). C NMR (75 MHz, CDCl ) d
(Sigma), in 0.2 % gelatinized plates. After 24-h period of
3
3
incubation at 37 °C, the tested compounds (dissolved in
sterile DMSO) are diluted in culture medium in order to
have a 109 concentration. The dilutions are made in a
1
1
1
1
2
53.42, 153.37, 145.07, 144.49, 143.94, 138.79, 137.85,
33.14, 132.74, 132.01, 131.90, 131.01, 127.49, 126.85,
26.66, 125.88, 123.83, 119.21, 116.23, 114.62, 104.07,
03.31, 60.94, 56.12, 56.07; MS (ESI) m/z: 301.2 ([M3H-
96-well plate with a round bottom. Twenty microlitre of
each dilution is mixed with the 180 lL of culture medium
?
?
?
O] ), 331.1 ([MH] ), 353.1([MNa] ); HRMS calcd for
-
8
in the appropriate well. The final concentrations are 10
,
C H O N 331.12885, found 331.12888. Data for 16p
1
7
19
5
2
-8
-7
-7
-6
-6
-5
.10 , 10 , 5.10 , 10 , 5.10 , 10 , 5.10 , and
-5
5
1
1
(
NH para (4) H NMR (300 MHz, CDCl ) d 8.20 (d,
2
3
-4
0
g/mL. Each experiment is made with an n = 6. Cells
J = 2.0 Hz, 1H), 7.58 (dd, J = 8.7, 2.1 Hz, 1H), 6.90 (s,
are incubated in a humidified atmosphere and a CO con-
2
2
H), 6.82 (d, J = 8.7 Hz, 1H), 6.70 (s, 2H), 6.18 (br s, 2H),
1
.91 (s, 6H), 3.87 (s, 3H). Data for 16o (NH ortho (5) H
centration fixed at 5 % in a Binder incubator. After 72-h
incubation at 37 °C, with or without the compound to be
tested, the medium was replaced by 100 lL of HBSS
3
2
NMR (300 MHz, CDCl ) d 8.10 (d, J = 9.0 Hz, 1H), 7.12
3
(
d, J = 16.2 Hz, 1H), 6.90 (s, 1H), 6.89 (d, J = 9.1 Hz,
(without phenol red) containing MTT at the concentration
1
2
H), 6.88 (d, J = 16.2 Hz, 1H), 6.74 (s, 2H), 6.18 (br s,
of 1 mg/mL. The micro-wells are subsequently incubated
during 3 h at 37 °C and centrifuged at 1600 rpm during
4 min. Medium is removed, and formazan crystal formed is
dissolved in 100 lL non-sterile DMSO. The plate is in-
cubated in the dark during 10 min. The micro-wells are
shaken for 1 min and read on a spectrophotometer at
wavelengths of 570 nm (maximum of formazan ab-
sorbance) and 650 nm (background). For each ex-
perimental condition, the mean optical density is
calculated, allowing the determination of the percentage of
living cells in comparison with the control.
H), 3.91 (s, 6H), 3.88 (s, 3H).
4
5
-(2-Z-Trimethoxyphenylethenyl)-2-nitroaniline (18) and
A
-(2-Z-Trimethoxyphenylethenyl)-2-nitroaniline (19)
mixture of two isomers was isolated in 37 % yield of
1
separable isomers 26/27 (80/20 ratio). Isomer 26: H NMR
(
300 MHz, CDCl ) d 8.07 (d, J = 1.8 Hz, 1H), 7.28 (dd,
3
J = 9.1, 2.4 Hz, 1H), 6.63 (d, J = 8.7 Hz, 1H), 6.51 (d,
J = 12.2 Hz, 1H), 6.49 (s, 2H), 6.40 (d, J = 12.2 Hz, 1H),
1
.13 (br s, 2H), 3.85 (s, 3H), 3.72 (s, 6H); C NMR
3
6
(
75 MHz, CDCl ) d 153.10, 143.66, 137.35, 136.42,
3
1
1
32.42, 131.70, 129.82, 127.58, 126.45, 126.27, 118.17,
?
05.78, 60.95, 55.98; MS (APCI) m/z: 331.1 ([MH] );
Metabolic stability studies
HRMS calcd for C H O N 331.12885, found
1
7 19 5 2
1
3
31.12871. Isomer 27: H NMR (300 MHz, CDCl ) d 8.00
Compounds at 10 lM (stock solution in DMSO at 5 mM)
were preincubated at 37 °C for 5 min in tubes with
NADPH regenerating system (1.3 mM NADP, 3.3 mM
3
(
d, J = 8.9 Hz, 1H), 6.73 (s, 1H), 6.63 (dd, J = 8.9,
1
.7 Hz, 1H),, 6.65 (d, J = 12.2 Hz, 1H), 6.48 (s, 2H), 6.43
123

Med Chem Res
D-glucose 6-phosphate, 3.3 mM MgCl , and 0.4 U/mL
2
by the formula of in vitro t1/2 = ln(2)/k, regarded as first-
order kinetics (GraphPad Prism, version 5). In vitro in-
glucose-6-phosphate dehydrogenase) in 0.1 M potassium
phosphate buffer pH 7.4 (BD Biosciences, Woburn, MA).
The reaction was initiated by the addition of pooled human
liver S9 or pooled mouse liver microsomes (1 mg protein/
mL, BD Biosciences, Woburn, MA). Aliquots (100 lL)
were removed at 0, 10, 30, 60, and 120 min and mixed with
trinsic clearance (CL ) of compounds was calculated us-
int
ing the formula CLint = (ln(2)/t1/2) 9 (V/P), where V is the
incubation volume, and P is the mass of microsomal pro-
teins in the incubation mixture.
1
1
00 lL acetonitrile, vortexed, and centrifuged for 5 min at
0,000 rpm. The supernatants were then subjected to UV–
Metabolite identification
HPLC analysis. Controls consisted of: (a) incubation of
tested compound (positive control) in 0.1 M potassium
phosphate buffer and (b) incubation of all components
except NADPH regenerating system. All incubations were
assayed in duplicate.
For metabolites identification, chromatographic ex-
periments were carried out in gradient mode in order to
separate each newly formed metabolite. Mobile phases
were A: 0.1 % trifluoroacetic acid in water and B: 0.1 %
trifluoroacetic acid in acetonitrile. Gradient elution was
from 20 to 100 % phase B in 20 min and 5 min at 100 %
UV–HPLC analysis
B
at 1 mL/min on
a
RP-18e 250 mm 9 4 mm
Ò
LiChroCART column (5 lM) equipped with a guard
column. Metabolites identification was performed by LC–
MS analysis on an Accela HPLC system hyphenated with
a LTQ-Orbitrap XL mass spectrometry system (Thermo
Fisher Scientific, Bremen, Germany) from UCL MASS-
MET platform. The system was equipped with an APCI
interface that was used in positive ionization. Identifica-
tion of major metabolites was achieved by comparing
Methanol HPLC grade was purchased from Prolabo, VWR
(
Leuven, Belgium), and acetonitrile HPLC grade from
Fisher Scientific (Tournai, Belgium). Analysis was per-
formed on a LaChrom Elite HPLC integrated system
(
Merck Hitachi, VWR, Leuven, Belgium) equipped with a
L-2450 UV detector, a L-2300 oven, L-2130 autosampler,
and L-2130 pump all piloted by EZChrom software.
Chromatographic experiments were carried out in isocratic
2
their retention time and mass spectra (MS and MS ) with
Ò
mode on a RP-18e 250 mm 9 4 mm LiChroCART col-
synthetic metabolic standards.
umn (5 lM) equipped with a guard column. The mobile
phase consisted of a mixture of acetonitrile and water in
isocratic mode (see Table 5). The flow rate was 1 mL/min,
and the injected volume was 20 lL. Analysis was carried
out at room temperature at different wavelengths (see
Table 5). To determine metabolic stability, results were
expressed as the remaining percentage of compound
against time by dividing the peak area of test compound at
each time point by the peak area at 0 min multiplied by
Cell treatment for caspase-3 activation
U373 (human brain tumor cells), plated in a P60 at full
confluence, were treated with the compounds at the given
concentrations. After a 24-h treatment, the cells were lysed
with ripa buffer containing proteases inhibitors.
Immunoblot anti-caspase 3
1
00. The slope of the linear regression from log percentage
remaining versus incubation time relationships (-k) was
Cell extracts were separated by SDS–PAGE and trans-
ferred onto PVDF membranes before incubation with
used to calculate the in vitro half-life (t1/2) of compounds
Table 5 HPLC conditions (mobile phase and wavelength of detection) for each compound
Compound
Mobile phase (v/v)
Wavelength (nm)
8
9
ACN/H
ACN/H
ACN/H
ACN/H
ACN/H
ACN/H
ACN/H
ACN/H
2
O 60/40
2
O 60/40
2
O 70/30
2
O 40/60
2
O 60/40
2
O 60/40
2
O 70/30
2
O 40/60
298
313
298
365
277
258
298
292
1
1
1
1
1
1
0
1
2
3
4
5
1
23

Med Chem Res
primary antibody as previously described (Seront et al.,
Holladay MW, King LL, Kort ME, Kym PR, Milicic I, Tang R,
Turner SC, Whiteaker KL, Yi L, Zhang H, Sullivan JP (2004)
Synthesis and structure–activity relationships of a novel series of
2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-8(4H)-one 1,1-diox-
ide K(ATP) channel openers: discovery of (-)-(9S)-9-(3-bromo-
2
013). Antibody against total and active caspase-3 was
purchased from Cell Signaling (Danvers, MA, USA).
Flow cytometry
4
8
-fluorophenyl)-2,3,5,6,7,9-hexahydrothieno[3,2-b]quinolin-
(4H)-one 1,1-dioxide (A-278637), a potent K(ATP) opener that
U373 cells (ECACC catalogue number 08061901) were
seeded in T25 flasks and treated with compounds at the
given concentrations during 72 h. A the end of the treat-
ment periods, cells were detached with trypsin–EDTA,
pooled with their supernatant, centrifuged, washed with
PBS, and fixed for 1 h in paraformaldehyde 1 % in PBS at
selectively inhibits spontaneous bladder contractions. J Med
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°C. After permeabilization overnight with 70 % ice-cold
(
1992) Synthesis and evaluation of analogues of (Z)-1-(4-
ethanol, cells were stained by TUNEL technic as well as
propidium iodide with the APO AF apoptosis detection kit
according to the manufacturer’s instructions (BD, Erem-
bodegem, Belgium). Cells were then analyzed for their
fluorescence levels with a Cell Lab Quanta flow cytometer
methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene as potential
cytotoxic and antimitotic agents. J Med Chem 35:2293–2306
Dark GG, Hill SA, Prise VE, Tozer GM, Pettit GR, Chaplin DJ (1997)
Combretastatin A-4, an agent that displays potent and selective
toxicity toward tumor vasculature. Cancer Res 57:1829–1834
DaSilveira Neto BA, Lopes AS, Ebeling G, Gon c¸ alves RS, Costa
VEU, Quina FH, Dupont J (2005) Photophysical and electro-
chemical properties of p-extended molecular 2,1,3-benzothia-
diazoles. Tetrahedron 61:10975–10982
Dumont P, Ingrassia L, Rouzeau S, Ribaucour F, Thomas S, Roland I,
Darro F, Lefranc F, Kiss R (2007) The Amaryllidaceae
isocarbostyril narciclasine induces apoptosis by activation of
the death receptor and/or mitochondrial pathways in cancer cells
but not in normal fibroblasts. Neoplasia 9:766–776
(
Beckman Coulter, ANalis, Suarlee, Belgium). MCF7
breast cancer cells (DSMZ code ACC115) left untreated or
treated with 1 lM narciclasine for 72 h were used as
negative and positive controls (Dumont et al., 2007). Ex-
periment was conducted once in tetraplicate, and results are
presented as mean ± SEM.
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required for alpha-fodrin cleavage but dispensable for cleavage of
other death substrates in apoptosis. J Biol Chem 273:15540–15545
Liekens S, De Clercq E, Neyts J (2001) Angiogenesis: regulators and
clinical applications. Biochem Pharmacol 61:253–270
Acknowledgments The authors thank Dr. Marie-France H e´ rent for
her skillful assistance on the MASSMET platform. The authors
gratefully thank the R e´ gion Bruxelles-Capitale (INNOVIRIS) for fi-
nancial support on this research. This work was supported by the
Fonds National de la Recherche Scientifique (F.R.S.-FNRS, Belgium)
FRFC Grant No. 2.4555.08 and the Fonds Sp e´ cial de Recherche
Maya ABS, P e´ rez-Melero C, Mateo C, Alonso D, Fernandez JL,
Gajate C, Mollinedo F, Pelaez R, Caballero E, Medarde M
(FSR) of the Faculty of Medecine (UCL) as part of the MASSMET
platform (LC-HRMS Orbirtap).
(
2005) Further naphthylcombretastatins. An investigation on the
role of the naphthalene moiety. J Med Chem 48:556–568
Nam N-H (2003) Combretastatin A-4 analogues as antimitotic
antitumor agents. Curr Med Chem 10:1697–1722
Nathan P, Zweifel M, Padhani AR, Koh DM, Ng M, Collins DJ,
Harris A, Carden C, Smythe J, Fisher N, Taylor NJ, Stirling JJ,
Lu SP, Leach MO, Rustin GJS, Judson I (2012) Phase I trial of
combretastatin A4 phosphate (CA4P) in combination with
bevacizumab in patients with advanced cancer. Clin Cancer
Res Off J Am Assoc Cancer Res 18:3428–3439
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