Discovery of azaisoerianin derivatives as potential antitumors agents

Article abstract of DOI:10.1016/j.ejmech.2014.03.032

A series of N-methyl-diarylamines 2 was designed and synthesized as a novel class of CA-4 and isoCA-4 analogues. Compounds 2b and 2m showed excellent antiproliferative activity with mean GI₅₀ values at a nanomolar level in a diverse set of huma

Full text of DOI:10.1016/j.ejmech.2014.03.032

European Journal of Medicinal Chemistry 78 (2014) 178e189
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of azaisoerianin derivatives as potential antitumors agents
Mohamed Ali Soussi a, Olivier Provot a, Guillaume Bernadat a, Jérome Bignon b,
Joanna Wdzieczak-Bakala b, Déborah Desravines b, Joëlle Dubois b, Jean-Daniel Brion a,
,
a,
*
Samir Messaoudi a , Mouad Alami
*
a Univ Paris-Sud, CNRS, BioCIS-UMR 8076, Laboratoire de Chimie Thérapeutique, LabEx LERMIT, Equipe Labellisée Ligue Contre le Cancer, Faculté de
Pharmacie, 5 rue J.-B. Clément, Châtenay-Malabry F-92296, France
b Institut de Chimie des Substances Naturelles, UPR 2301, CNRS avenue de la terrasse, Gif sur Yvette F-91198, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-methyl-diarylamines 2 was designed and synthesized as a novel class of CA-4 and isoCA-4
analogues. Compounds 2b and 2m showed excellent antiproliferative activity with mean GI50 values at a
nanomolar level in a diverse set of human cancer cells. These compounds also inhibited tubulin assembly
at a micromolar range, arrested the cellular cycle in the G2/M phase and induced apoptosis at very low
concentrations. Preliminary in vitro results revealed that 2b and 2m displayed substantial efficacy as
potent antivascular agents. Docking studies indicates that these lead compounds showed a binding mode
similar to those observed with isoCA-4 at the colchicine binding site of tubulin.
Received 21 January 2014
Received in revised form
11 March 2014
Accepted 12 March 2014
Available online 13 March 2014
Keywords:
Combretastatin A-4
IsoCA-4
Ó 2014 Elsevier Masson SAS. All rights reserved.
Isoerianin
AzaisoCA-4
Cytotoxicity
Antimitotic
Tubulin
1. Introduction
Despite their remarkable anticancer properties, the main
problem associated with CA-4 and its prodrugs is the ready
Combretastatin A-4 (CA-4) a natural stilbene isolated from the
African willow tree, Combretum caffrum in 1989 by Pettit [1] has
been found to be a potent anticancer agent which strongly inhibits
tubulin assembly by binding to the colchicine site. [2] CA-4 also
shows potent cytotoxicity against a wide range of human cancer
cells including multi drugs resistant (MDR) cancer cells [3]. More-
over, CA-4 has been demonstrated to selectively target the vascular
network of tumors, inducing irreversible shutdown of blood flow to
neoplastic cells [4]. Currently, two water-soluble prodrug de-
rivatives are in clinical trials: disodium phosphate CA-4P (fos-
bretabulin, ZybrestatÔ) [5] and AVE8062 (ombrabulin) [6]. To date,
CA-4P [7,8] either as a single agent or in combination therapy is
undergoing several advanced clinical trials worldwide for the
treatment of age-related macular degeneration or anaplastic thy-
roid cancer.
isomerization of the Z-double bond into the inactive E-form dur-
ing storage, administration and metabolism [9,10]. In an ongoing
project aimed at developing stable CA-4 analogues [11e19], we
recently solved the stability problem of CA-4 by the discovery of
the non-isomerizable isocombretastatin A-4 (isoCA-4) which
holds biological activities comparable to that of CA-4 [20,21]. This
structural isomer of the natural product, having
a 1,1-
diarylethylene scaffold, is chemically and metabolically stable
[22] and easy to synthesize [23e25] at a multigrams scale without
the need to control the olefin geometry. By chemical modifications
on the B-ring of isoCA-4, we have also identified other stable and
promising antiproliferative agents [21,26] such as isoNH2CA-4 and
isoFCA-4. We next have demonstrated that the reduction of the
1,1-ethylene bond of isoCA-4 led to (ꢀ)-isoerianin, in which
racemic and both enantiomers displayed excellent anti-cancer
activities comparable to that of the natural isomer erianin [27].
It seems that the carbon (Csp3) linker in isoerianin is not involved
in the interaction in the colchicine binding site. It serves only to
position correctly the two aromatic rings of the molecule [27].
Herein, we planned to prepare a new class of isoerianin de-
rivatives, named azaisoerianins (Fig. 1), in which the two aromatic
* Corresponding authors.
E-mail addresses: samir.messaoudi@u-psud.fr (S. Messaoudi), mouad.alami@u-
psud.fr (M. Alami).
http://dx.doi.org/10.1016/j.ejmech.2014.03.032
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
179
Fig. 1. Structures of CA-4 and synthetic prodrugs, isoCA-4 and derivatives, erianin, isoerianin and targeted azaisoerianin.
rings are connected through a nitrogen atom in place of the car-
bon linker in isoerianin compounds. Indeed, the diphenylamine
skeleton is a common privileged structure found in biologically
active compounds, and compounds derived from this scaffold
manifest diverse activities, including anticancer activity [28,29].
This design has several advantages as follow: (i) the ease in syn-
thesis where various side-arms can be introduced on nitrogen
atom in a convergent access, (ii) the absence of a chiral center, (iii)
the rapid access to weakly basic and chargeable derivatives in a
solubilization strategy, (iv) the discovery of a third generation of
candidates having promising antiproliferative and antitubulin
activities and a better understanding of structureeactivity re-
lationships (SAR). In this article are described the synthesis and
the biological evaluation of novel azaisoerianin derivatives 2 and
3. Preliminary in vitro efficacy of these compounds in terms of
cytotoxicity, inhibition of tubulin assembly, cell cycle, apoptosis
and in vitro antivascular activity is described. The possible binding
mode of the most bioactive substrates 2b and 2m on tubulin is
also reported.
2j was synthesized from 4-chloro derivative 2i using dimethyl-
amine in the presence of Pd2(dba)3.CHCl3 (5 mol%), and Johnphos
(10 mol%) as the ligand and tBuONa as the base in hot toluene [35].
Finally, the removal of the N-protected tosyl group of 2p was
accomplished by the use of TBAF in DMF at 100 ꢁC to afford 2q
(49%).
2.2. Biological results
New synthesized azaisoerianin compounds were evaluated for
their cytotoxic effects against HCT116 human carcinoma cell line
and for tubulin polymerization inhibitory activity. Isoerianin [27],
isoCA-4 [21] and CA-4 [36] were included as reference compounds.
The cytotoxicity and inhibition of tubulin polymerization (ITP) re-
sults are presented in Table 1. Without any exception, all secondary
diarylamines 1 were not cytotoxic (data not shown).
In the series of compounds 2 having a benzene substituted B-
ring, our findings showed that compound 2b, having a fluorine
atom at the C30-position, displayed the best cytotoxicity with a GI50
values of 20 nM comparable to that of the reference isoerianin.
Additionally, the water-soluble hydrochloride salt 2b-HCl displayed
a high level of cytotoxicity against HCT116 cells (GI50 ¼ 28 nM)
comparable to that of the parent molecule 2b (GI50 ¼ 22 nM).
Replacement of the F atom with H or a NH2 on the C30-position
generated compounds 2a and 2d (GI50 ¼ 64 nM and 66 nM,
respectively) which were three fold less active than 2b. Contrary to
our expectations, a dramatic loss of antiproliferative activity was
observed with azaisoerianin 2g, having a OH group on C30-posi-
tion.This compound and its corresponding acetate 2h displayed
only modest cytotoxicity against HCT116 cells (GI50 ¼ 650e
700 nM). We next showed that the introduction of a pyridine or a
quinoline nucleus as B-ring resulted in products 2k (GI50 ¼ 650 nM)
and 2l (GI50 ¼ 2100 nM), respectively, with a significant loss of
potency. We next investigated the role on cytotoxicity of derivatives
having an indole nucleus which was found useful as B-ring in the
CA-4 series [37]. It is significant to note that in the azaisoerianin
series, N-methyl-indol-5-yl compound 2m was consistently the
more potent compound. This lead compound (2m) was about four
times more cytotoxic than the reference isoerianin with a low GI50
value of 7 nM. The absence of the trimethoxyphenyl-ring in the 5-
indoyl series reduced the cytotoxicity as it was observed with
2. Results and discussion
2.1. Chemistry
The synthesis of azaisoerianin derivatives 2aeq and 3aec, is
summarized in Scheme 1. Diarylanilines 1ael were prepared from
commercially available methoxylated anilines which were coupled
in a sealed tube with a variety of (hetero)aryl halides under our
previously reported conditions (Pd(OAc)2 as the catalyst, Xantphos
as the ligand, Cs2CO3 as the base in hot dioxane) [30e33]. Treat-
ment of the resulting diarylamines 1 with sodium hydride followed
by reaction with MeI gave the best results for the N-alkylation
process furnishing the desired trisubstituted amines 2. By analogy
with highly cytotoxic N-propylbenzylanilines, [34] N-propyl de-
rivatives 3 were prepared using a large excess of nPrBr as the
alkylating agent and NaH as the base. Reduction of the nitro group
of 2c using HCl and iron powder yielded azaisoerianin derivative 2d
with 3-amino-4-methoxy groups on the B-ring identical to the B-
ring of AVE8062 drug. Debenzylation of the phenol function of 2f
was carried out by hydrogenation to afford azaisoerianin 2g having
the greatest resemblance with isoerianin and isoCA-4. Compound

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M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
aReagents and conditions: (a) Pd(OAc)2, Xantphos, ArX, Cs2CO3, dioxane, 100-130 °C, sealed tube. (b) H2, Pd/C, AcOEt. (c) NaH, MeI,
DMF, RT. (d) HCl, MeOH, Et2O, RT. (e) Fe, HCl, EtOH/H2O. (f) Pyridine, DMAP, (Ac)2O, CH2Cl2. (g) Pd2(dba)3.CHCl3, t-Bu2P(o-
biphenyl), NHMe2, tBuONa, toluene, 100 °C. (h) TBAF, DMF, 100 °C. (i) NaH, nPrBr, DMF, RT.
Scheme 1. Synthesis of compounds 1e3.
compounds 2n (GI50 ¼ 1100 nM) and 2o (GI50 ¼ 70 nM). Contrary to
other observations, [37] the N-methyl substitution of the indole
moiety in 2m plays an important role for the antiproliferative ac-
tivity. Indeed, its des-methyl analogue 2q showed a 25-fold
reduction in cytotoxicity. Finally, a marked loss in cytotoxicity
was observed with compounds 3b (GI50 ¼ 1400 nM) and 3c
(GI50 ¼ 200 nM) having a N-propyl group in comparison with their
N-methyl analogues (e.g. 3c vs 2m; 3b vs 2g). To further charac-
terize the cytotoxicity profiles of these compounds, we next
investigated the effect of the most active substances 2a, 2b, 2d and
2m on the proliferation of three tumor cell lines (hormone-inde-
pendent breast cancer (MDA-MB231)), human glioblastoma (U87-
MG), myelogenous leukemia (K562) and on the normal primary
human umbilical vein endothelial (HUVEC). As shown in Table 2, all
examined compounds displayed very potent activity against all cell
lines tested, in the nanomolar range. Again, compound 2b and
indole 2m displayed the highest level of cytotoxicity with GI50
values ranging from 2 to 39 nM.
To investigate whether compounds 2 and 3 exert their activities
by interacting with microtubules, their effect on the in vitro poly-
merization of purified tubulin was next examined (see Table 1). It is
of interest to note that highly cytotoxic derivatives 2b and N-
methylindole 2m were also very potent as tubulin inhibitors and
were comparable to isoerianin with IC50
mM values (2.7 mM and
1.3 M for 2b and 2m, respectively). Interestingly, derivative 2g,
m
having the greatest resemblance to isoCA-4 and isoerianin which
was poorly cytotoxicity against HCT116 cells was one of the more
potent agent as tubulin inhibitor with an IC50 value of 1.5
mM.
Similarly, N-propyl derivatives 3b and 3c which displayed a modest
level of cytotoxicity were very potent in the tubulin assay with IC50
values ranging from 2.8 to 3.6
mM. These results showed an
important discrepancy between the potency of antitubulin activity
and cytotoxicity of N-propyl derivatives 3, indicating that with
hindered substituents on the nitrogen atom of derivatives 3, it
might be possible to design news series of antivascular agents with
low cytotoxicity.

M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
181
Table 1
Cytotoxicity against HCT 116 cellsa and ITP of selected compounds 2 and 3.
Compound
2a
2b
2b.HCl
28 ꢀ 2
3 ꢀ 0.2
2c
Cytotoxicity GI50b [nM]
64 ꢀ 4
20 ꢀ 1.5
190 ꢀ 15
c
ITP IC50
[mM)
5.5 ꢀ 0.5
2.7 ꢀ 0.2
28 ꢀ 2
Compound
2d
2e
2g
2h
Cytotoxicity GI50b [nM]
66 ꢀ 5
170 ꢀ 12
650 ꢀ 55
700 ꢀ 55
c
ITP IC50
[mM)
3.0 ꢀ 0.2
14 ꢀ 1.8
1.5 ꢀ 0.2
8.0 ꢀ 0.5
Compound
2j
2k
2l
2m
Cytotoxicity GI50b [nM]
250 ꢀ 25
650 ꢀ 62
2100 ꢀ 180
7 ꢀ 0.6
ITP IC50
[m
M]
18 ꢀ 1.2
NAd
NAd
1.3 ꢀ 0.1
c
Compound
2n
2o
2q
3b
Cytotoxicity GI50b [nM]
1100 ꢀ 120
70 ꢀ 5
180 ꢀ 14
1400 ꢀ 145
ITP IC50
[m
M]
NAd
3.4 ꢀ 0.5
NAd
2.8 ꢀ 0.3
c
Compound
3c
isoCA-4
2 ꢀ 0.2e
2.0 ꢀ 0.3
isoerianin
28 ꢀ 2e
CA-4
Cytotoxicity IC50b [nM]
200 ꢀ 16
2 ꢀ 0.2e
ITP IC50
[m
M]
3.6 ꢀ 0.5
3.0 ꢀ 0.2e
1.0 ꢀ 0.2e
c
a
HCT116 Human colon carcinoma.
b
c
GI50 is the concentration of compound needed to reduce cell growth by 50% following 72 h cell treatment with the tested drug (average of three experiments).
IC50 is the concentration of compound required to inhibit 50% of the rate of microtubule assembly (average of three experiments).
NA not active.
d
e
The GI50 values for isoCA-4, isoerianin and CA-4 were determined in this study.
To gain further insight the mechanism of action of azaisoerianin
24 h. The results presented in Fig. 2, demonstrate that there was an
accumulation of K562 and HCT116 cells in the G2/M phase of the
cellular cycle after treatment with 2b and 2m at very low con-
centrations. One note that there is a better perturbation of cycle cell
progression in K562 cells after treatment with these drugs, and
indolic derivative 2m seems to be slightly more effective than 2b in
K562 and HCT116 cells.
derivatives 2, the most cytotoxic compounds 2b and 2m were
assayed for their effects on cell cycle distribution. K562 and HCT116
cells were treated with 2b and 2m at different concentrations for
Table 2
The ability of 2b and 2m to induce apoptosis in HCT116 cells
was further characterized using caspase-3 and caspase-7 assays.
The enzymatic activity of these caspases was measured by
monitoring the cleavage of the fluorogenic substrate Z-DEVD-
R110 in cancer cells. The results presented in Table 3 show a
significant dose-dependent increase in proteolytic activity of
caspases in the cells treated for 24 h with diarylmethylamines
2b and 2m. A spectacular 4-fold increase in apoptosis was evi-
denced by 2m in HCT116 cells at a low concentration of 7.5 nM.
These results indicate that, in addition to their antiproliferative
and antitubulin effects, the treatment of cancer cells with 2b
and 2m activate caspases system leading to programmed cell
death.
Cytotoxicity against human cancer cell lines, HCT116, MDA-MB231, U87-MG, K562
and HUVEC.
Compounds
Cytotoxicity (GI50 nM)a
HCT116b
MDA-MB231c
U87-MGd
K562e
HUVECf
2a
2b
2d
64 ꢀ 4
20 ꢀ 1.5
66 ꢀ 5
7 ꢀ 0.6
28 ꢀ 2
55 ꢀ 4
24 ꢀ 2
80 ꢀ 7
16 ꢀ 1.8
35 ꢀ 3
30 ꢀ 2
7 ꢀ 0.6
20 ꢀ 1.7
2.2 ꢀ 0.3
30 ꢀ 3
39 ꢀ 4
39 ꢀ 5
62 ꢀ 5
8 ꢀ 0.7
25 ꢀ 2
35 ꢀ 3
22 ꢀ 2
25 ꢀ 3
7 ꢀ 0.5
45 ꢀ 5
2m
Isoerianing
a
GI50 is the concentration of compound needed to reduce cell growth by 50%
following 72 h cell treatment with the tested drug (average of three experiments).
b
HCT116, colon carcinoma.
MDA-MB-231 hormone-independent breast cancer.
U87-MG Glioblastoma.
K562, myelogenous leukaemia.
HUVEC, Human umbilical vein endothelial cells.
The GI50 values of isoerianin was determined in this study.
c
d
To identify compounds that disrupt the neovascularization for-
mation, an assay using endothelial colony-forming cells (ECFCs)
was performed with a panel of diarylamines 2 and 3. In the
e
f
g

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M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
Fig. 2. Effects of 2b and 2m on cell cycle distribution in HCT116 and K562 cells determined by flow cytometry analysis. DNA content was assessed via propidium iodide staining.
presence of the vascular endothelial growth factor (VEGF), ECFCs
form tube-like structures that express the endothelial marker
(CD31) when co-cultured on an ADSC feeder layer [38]. The inhi-
bition of endothelial tube formation (IC50) was determined after
96 h of treatment using ECFCs, VEGF and compounds 2a-b, 2d and
2m as angiogenesis inhibitors.
Compounds 2k, 2l having a pyridine and quinoline B-rings
respectively, as well as 2n were found to be inactive as angiogenesis
inhibitors (Table 4). All other selected compounds inhibit the
endothelial tube formation in a concentration-dependent manner
(data not shown) with remarkable low IC50 values as for examples
with 2b or 2m (CI50 ¼ 25 and 8 nM, respectively). One can note that
N-propyl derivatives 3b and 3c which displayed a low level of
cytotoxicity (Table 1) inhibit angiogenesis with nanomolar IC50
values of 73 and 50 nM, respectively.
These results were next confirmed by a second in vitro model
of angiogenesis [39] in which human umbilical vein endothelial
cells (HUVECs), when seeded on MatrigelÔ, aggregate to form a
reticular vascular network of capillary-like vessels. The results
presented in Table 2 (see above) revealed that after 72 h of

M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
183
Table 3
4. Experimental
Apoptotic effects of 2b and 2m in HCT116 cells.
Rhodamine 110 relative fluorescence intensitya (% of control)
4.1. General considerations
2b 25 nM
(300)
2b 50 nM
(452)
2m 7.5 nM
(393)
2m 15 nM
(471)
The compounds were all identified by usual physical methods,
i.e. 1H NMR, 13C NMR, IR, MS. 1H and 13C NMR spectra were
measured in CDCl3 with a Bruker Avance 300. 1H chemical shifts are
reported in ppm from an internal standard TMS or of residual
chloroform (7.27 ppm). The following abbreviations are used: m
(multiplet), s (singlet), d (doublet), br s (broad singlet), t (triplet), dd
(doublet of doublet), td (triplet of doublet). 13C chemical shifts are
reported in ppm from the central peak of CDCl3 (77.14). IR spectra
were measured on a Bruker Vector 22 spectrophotometer (neat,
cmꢂ1). Mass spectra were obtained with a LCT Micromass spec-
trometer. Analytical TLC was performed on Merck precoated silica
gel 60F plates. Merck silica gel 60 (230e400 mesh) was used for
column chromatography.
a
The results are expressed in the percentage of apoptotic cells detected following
24 h of treatment with 2b and 2m at different concentrations.
incubation, 2b and 2m exhibited good growth inhibition activity
against HUVEC cells with GI50 values of 22 and 7 nM, respec-
tively. To evaluate whether 2b and 2m could affect models of
newly formed blood vessels, the HUVEC tube disruption assay
was performed. Addition of 2b at a concentration of 10 nM, and
more significantly at 100 nM, rapidly disrupted the integrity of
the network (Fig. 3). This effect was visible after only 3 h of
treatment at doses of 10 and 100 nM which were not cytotoxic
for such incubation time (data not shown). This antivascular
effect is even more pronounced with indole derivative 2m
which destroyed neovascularization at a very low dose of 5 nM.
All together these results suggested that azaisoerianin de-
rivatives 2, and particularly 2m and 2b, might be considered and
evaluated as vascular disrupting agents for further in vivo
studies.
4.1.1. 3,4,5-Trimethoxy-N-(4-methoxyphenyl)-N-methylaniline 2a
In a sealed tube and under an argon atmosphere were added
successively, Pd(OAc)2 (11 mg; 5 mol%), Xantphos (29 mg; 5 mol%),
1-bromo-4-methoxybenzene (187 mg;
1
mmol), 3,4,5-
trimethoxyaniline (275 mg; 1.5 mmol), Cs2CO3 (652 mg; 2 mmol)
in dioxane (2 mL). After heating at 100 ꢁC for 12 h, the resulting
suspension was cooled to room temperature and filtered through a
pad of Celite eluting with ethyl acetate, and the inorganic salts were
removed. The filtrate was concentrated and a rapid purification by
silica gel column chromatography of the residue to eliminate the
excess of 3,4,5-trimethoxyaniline gave the desired product 1a
which was immediately engaged in the alkylation step. 1a: (75 mg,
2.3. Docking study
The biological IPT and antivascular results demonstrated that
tubulin is the target of these diarylamines 2 and 3. Molecular
docking calculations were performed with 2b and 2m in order to
investigate their possible binding mode in the colchicine binding
site of tubulin. For this purpose, the X-ray structure of tubulin
DAMA-colchicine complex (accession code 1SA0) [40] was used.
Fig. 4 illustrates the docking-derived superimposition of 2b and
2m, with isoCA-4 (green). As expected, these diarylamines showed
a binding pose matching the one calculated under the same con-
ditions for isoCA-4, used as a reference compound with its A-tri-
methoxyphenyl ring placed in the proximity of Cys241.
Additionally, the 40-OMe substituent of 2b accepts a stabilizing H-
bond donated by a polar hydrogen atom belonging to the side-
chain amide moiety of Asn101.
26%); 1H NMR (300 MHz, CDCl3)
d
7.04 (d, 2H, J ¼ 9.0 Hz), 6.85 (d,
2H, J ¼ 9.0 Hz), 6.15 (s, 2H), 5.52 (s, 1H), 3.79 (s, 6H), 3.76 (s, 3H). 13
C
NMR (75 MHz, CDCl3) d 155.1, 153.9 (2), 141.6, 136.1, 131.5, 121.8 (2),
114.7 (2), 93.7 (2), 61.1, 55.9 (2), 55.6. IR (neat) nmax/cmꢂ1: 3278,
2993, 1600, 1501, 1224, 1124, 1007. MS (APCI) m/z: 290 [M þ H]þ. To
a diarylamine 1a (50 mg; 0.17 mmol) solution in DMF (2 mL) was
added at 0 ꢁC, NaH (8 mg; 0.34 mmol), and iodomethane (48 mg;
28 mL, 0.34 mmol). The mixture was stirred at r.t. for a night and
diluted in EtOAc (2 mL) and washed with aqueous NaHCO3. The
organic layer was dried, evaporated, and the residue was subjected
to flash chromatography to give 2a as a yellow oil (35 mg, 67%). 1H
NMR (300 MHz, CDCl3)
J ¼ 8.9 Hz), 6.05 (s, 2H), 3.81 (s, 3H), 3.79 (s, 3H), 3.76 (s, 6H), 3.25 (s,
3H). 13C NMR (75 MHz, CDCl3)
155.9, 153.6 (2), 146.5, 142.5, 131.6,
d
7.05 (d, 2H, J ¼ 8.9 Hz), 6.88 (d, 2H,
3. Conclusions
d
125.1 (2), 114.8 (2), 95.1 (2), 61.2, 56.1 (2), 55.6, 41.0. IR (neat) nmax
/
We designed and synthesized a series of original N-methyl- and
N-propyldiarylamines 2 and 3, respectively as aza-analogues of
isoerianin and isoCA-4. High inhibition of cell growth was observed
with compounds 2b and 2m in a range of human cancer cells. These
potent drugs also inhibited tubulin assembly with micromolar IC50
values. Cell cycle analysis of K562 and HCT116 cells treated with
nanomolar concentrations of 2b and 2m indicated that cells were
arrested at the G2/M phase. Moreover, 2b and 2m induced
apoptosis in HCT116 cells and destroyed in vitro models of neo-
vasculature at very low doses. Docking studies revealed that these
compounds adopted an orientation similar to that of isoCA-4 in the
colchicine binding-site of tubulin.
cmꢂ1: 1575, 1503, 1465, 1419, 1242, 1127. MS (APCI) m/z: 304
[M þ H]þ. HRMS calcd for C17H22NO4 [M þ H]þ 304.1549, obsd.
304.1548.
4.1.2. N-(3-Fluoro-4-methoxyphenyl)-3,4,5-trimethoxy-N-
methylaniline 2b
By following the general procedure described above for 1a.
Heating at 100 ꢁC 3,4,5-trimethoxyaniline (275 mg; 1.5 mmol) and
4-bromo-2-fluoro-1-methoxybenzene (205 mg; 1 mmol) for 5 h
gave 1b (181 mg; 59%). 1H NMR (300 MHz, CDCl3)
d
6.92e6.84 (m,
2H), 6.76 (d, 1H, J ¼ 8.7 Hz), 6.22 (s, 2H), 5.45 (s, 1H), 3.87 (s, 3H),
Table 4
Inhibition of endothelial tube formation using ECFCs.
Inhibition of endothelial colony tube formation
Compounds
(IC50) nM
2a
56
2b
25
2c
300
2k
2000
2l
2m
8
2n
2o
67
2q
250
3b
73
3c
50
isoerianin
70
isoCA-4
7
NAa
NAa
a
Not active.

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M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
Fig. 3. Inhibition of HUVEC tube formation on a Matrigel by 2b (at 10 and 100 nM) and 2m (at 5 and 10 nM). Images were taken 3 h after addition of compound 2b and 2m.
3.80 (s, 9H). 13C NMR (75 MHz, CDCl3)
d
154.0 (2), 153.0 (d,
isolated as green amorphous solid after filtration. 1H NMR
(300 MHz, DMSO)
J ¼ 244.5 Hz), 142.4 (d, J ¼ 11.2 Hz), 140.1, 137.6 (d, J ¼ 9.0 Hz), 132.7,
d
7.06 (t, J ¼ 9.4 Hz, 1H), 6.83 (dd, J ¼ 13.8, 2.5 Hz,
115.1 (d, J ¼ 2.2 Hz), 114.5 (d, J ¼ 2.2 Hz), 107.8 (d, J ¼ 20.2 Hz), 95.4
1H), 6.73 (d, J ¼ 8.9 Hz, 1H), 6.60 (s, 1H), 6.25 (s, 2H), 3.78 (s, 3H),
(2), 61.1, 57.1, 56.1 (2). 19F NMR (188 MHz, CDCl3)
d
ꢂ131.0. IR (neat)
3.69 (s, 6H), 3.61 (s, 3H), 3.19 (s, 3H). 13C NMR (75 MHz, DMSO)
nmax/cmꢂ1: 1604, 1502, 1464, 1262, 1129, 1030. MS (APCI) m/z: 308
[M þ H]þ. By following the general procedure described above for
2a. Diarylamine 1b (102 mg; 0.33 mmol), NaH (12 mg; 0.5 mmol)
and CH3I (71 mg; 0.5 mmol) gave 2b as a yellow oil (80 mg; 75%). 1H
d
153.86 (2C), 152.19 (d, J ¼ 233.9 Hz), 145.37, 143.40 (d, J ¼ 8.5 Hz),
141.91 (d, J ¼ 11.1 Hz), 133.19, 116.20, 115.32, 108.60 (d, J ¼ 20.2 Hz),
99.00 (2C), 60.56, 56.88, 56.28 (2C), 41.02. IR (neat) nmax/cmꢂ1
:
2999, 1612, 1521, 1488, 1275, 1122, 1005. MS (APCI) m/z: 322 [M]þ.
NMR (300 MHz, CDCl3)
d
6.87 (dd, 1H, J ¼ 9.3 Hz, J ¼ 9.0 Hz), 6.75
HRMS calcd for C17H21FNO4 [M]þ 322.1449, obsd. 322.1442.
(dd, 1H, J ¼ 13.3 Hz, J ¼ 2.7 Hz), 6.71e6.66 (m, 1H), 6.18 (s, 2H), 3.84
(s, 3H), 3.80 (s, 3H), 3.76 (s, 6H), 3.22 (s, 3H). 13C NMR (75 MHz,
4.1.4. 3,4,5-Trimethoxy-N-(4-methoxy-3-nitrophenyl)-N-
methylaniline 2c
CDCl3)
d
153.6 (2), 152.7 (d, J ¼ 243.7 Hz), 145.4,143.4 (d, J ¼ 8.4 Hz),
142.3 (d, J ¼ 11.1 Hz), 133.2, 115.9 (d, J ¼ 2.7 Hz), 114.6 (d, J ¼ 2.3 Hz),
By following the general procedure described above for 1a.
Heating at 100 ꢁC 3,4,5-trimethoxyaniline (275 mg; 1.5 mmol) and
4-iodo-1-methoxy-2-nitrobenzene (279 mg; 1 mmol) in the pres-
ence of Pd(OAc)2 (22 mg; 10 mol%), Xantphos (58 mg; 10 mol%) for
109.1 (d, J ¼ 20.5 Hz), 98.2 (2), 61.0, 56.9, 56.1 (2), 40.8. 19F NMR
(188 MHz, CDCl3)
d
ꢂ133.4. IR (neat) nmax/cmꢂ1: 2999, 1604, 1581,
1503, 1242, 1125. MS (APCI) m/z: 322 [M þ H]þ. HRMS calcd for
C
17H21FNO4 [M þ H]þ 322.1455, obsd. 322.1450.
1 h 30 gave 1c (307 mg; 92%). 1H NMR (300 MHz, CDCl3)
d 7.55 (d,
1H, J ¼ 2.8 Hz), 7.21 (dd, 1H, J ¼ 9.0 Hz, J ¼ 2.8 Hz), 7.01 (d, 1H,
4.1.3. N-(3-fluoro-4-methoxyphenyl)-3,4,5-trimethoxy-N-
methylbenzenaminium chloride 2b.HCl
J ¼ 9.0 Hz), 6.25 (s, 2H), 5.59 (s, 1H), 3.92 (s, 3H), 3.81 (s, 3H), 3.80 (s,
6H). 13C NMR (75 MHz, CDCl3)
d 154.2 (2), 147.6, 140.2, 139.0, 137.3,
To a solution of 2b (97 mg; 1 mmol) in MeOHeEt2O (1:1)
(10 mL) was added HClg within 10 min and then the mixture was
stirred for 1 h at RT. The precipitated salt 2b.HCl (92 mg; 86%) was
133.4, 124.2, 115.3, 114.9, 96.3 (2), 61.2, 57.2, 56.2 (2). IR (neat) nmax/
cmꢂ1: 1600, 1535, 1347, 1302, 1230, 1122, 1021. MS (APCI) m/z: 335
[M þ H]þ.
Fig. 4. Calculated binding mode for 2b and 2m in the colchicine binding site of tubulin and superimposition of the latter with that of isoCA-4. (Central nitrogen atom and (if
applicable) fluorine atom are represented as spheres in order to increase their visibility.)

M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
185
By following the general procedure described above for 2a.
Diarylamine 1c (167 mg; 0.5 mmol), NaH (18 mg; 0.75 mmol) and
CH3I (106 mg; 0.75 mmol) gave 2c as an orange oil (172 mg; 99%).
NMR (300 MHz, CDCl3)
d
6.79 (d, 1H, J ¼ 8.7 Hz), 6.67 (d, 1H,
J ¼ 2.7 Hz), 6.53 (dd, 1H, J ¼ 8.7 Hz, J ¼ 2.7 Hz), 6.14 (s, 2H), 5.64 (s,
1H), 3.87 (s, 3H), 3.80 (s, 3H), 3.77 (s, 6H), 3.22 (s, 3H). 13C NMR
1H NMR (300 MHz, CDCl3)
d
7.41 (d, 1H, J ¼ 2.9 Hz), 7.09 (dd, 1H,
(75 MHz, CDCl3) d 153.6 (2), 146.3, 146.1, 143.7, 142.3, 132.4, 113.4,
J ¼ 9.1 Hz, J ¼ 2.9 Hz), 6.97 (d, 1H, J ¼ 9.1 Hz), 6.26 (s, 2H), 3.91 (s,
111.5, 109.2, 97.0 (2), 61.1, 56.4, 56.2 (2), 41.0. IR (neat) nmax/cmꢂ1
:
3H), 3.83 (s, 3H), 3.79 (s, 6H), 3.27 (s, 3H). 13C NMR (75 MHz, CDCl3)
2959, 1581, 1501, 1447, 1249. MS (APCI) m/z: 342 [M þ Na]þ. HRMS
d
154.0 (2), 146.9, 144.6, 143.0, 140.1, 134.6, 124.6, 115.0, 114.8, 100.3
calcd for C17H22NO5 [M þ H]þ 320.1498, obsd. 320.1494.
(2), 61.1, 57.2, 56.2 (2), 40.9. IR (neat) nmax/cmꢂ1: 1589, 1525, 1502,
1443, 1364, 1254, 1234, 1122. MS (APCI) m/z: 349 [M þ H]þ. HRMS
calcd for C17H21N2O6 [M þ H]þ 349.1400, obsd. 349.1392.
4.1.9. 2-Methoxy-5-(methyl(3,4,5-trimethoxyphenyl)amino)phenyl
acetate 2h
4.1.5. 4-Methoxy-N-methyl-N-(3,4,5-trimethoxyphenyl)benzene-
1,3-diamine 2d
At 0 ꢁC, to a solution of 2g (101 mg; 0.316 mmol) in CH2Cl2
(1 mL) were added pyridine (54
mL), and DMAP (0.016 mmol) and
To a (EtOH/H2O: 4/1) solution (1.5 mL) of 2c (140 mg; 0.4 mmol)
were added iron (225 mg; 4 mmol) and a drop of HCl 12N. After
refluxing for 2 h, the mixture was filtered through a pad of Celite,
concentrated to give a residue which was subjected to flash chro-
matography to give 2d as a brown oil (96 mg, 76%). 1H NMR
Ac2O (42 L). After one hour of stirring, H2O (3 mL) was added to
m
the mixture. After extraction with EtOAc (3 ꢃ 3 mL), the organic
layer was dried, evaporated, and the residue was subjected to flash
chromatography to give 2h as a yellow oil (76 mg, 67%). 1H NMR
(300 MHz, CDCl3)
(s, 2H), 3.80 (s, 6H), 3.77 (s, 6H), 3.24 (s, 3H), 2.28 (s, 3H). 13C NMR
(75 MHz, CDCl3) 169.0, 153.7 (2), 146.4, 145.7, 142.9, 140.2, 132.8,
d
6.92e6.84 (m, 2H), 6.75 (d, J ¼ 2.4 Hz, 1H), 6.17
(300 MHz, CDCl3)
(s, 2H), 3.84 (s, 3H), 3.79 (s, 3H), 3.76 (s, 6H), 3.21 (s, 3H), NH2 not
seen. 13C NMR (75 MHz, CDCl3)
153.6 (2), 146.6, 143.8, 143.2, 136.9,
d
6.74 (d, 1H, J ¼ 8.3 Hz), 6.49e6.44 (m, 2H), 6.09
d
d
119.3, 116.8, 113.3, 97.4 (2), 61.1, 56.4, 56.2 (2), 40.9, 20.7. IR (neat)
nmax/cmꢂ1: 1721,1574,1452,1375,1204. MS (ESI) m/z: 362 [M þ H]þ.
HRMS calcd for C19H23NO6Na [M þ Na]þ 384.1423, obsd. 384.1426.
131.7, 113.3, 111.3, 110.8, 95.7 (2), 61.1, 56.2 (2), 55.9, 41.0. IR (neat)
nmax/cmꢂ1: 1612, 1581, 1502, 1148, 1248, 1126. MS (APCI) m/z: 319
[M þ H]þ. HRMS calcd for C17H23N2O4 [M þ H]þ 319.1658, obsd.
319.1654.
4.1.10. N-(4-Chlorophenyl)-3,4,5-trimethoxy-N-methylaniline 2i
By following the general procedure described above for 1a.
Heating at 100 ꢁC 3,4,5-trimethoxyaniline (275 mg; 1.5 mmol) and
1-chloro-4-iodobenzene (59 mg; 0.25 mmol) in the presence of
Pd(OAc)2 (22 mg; 10 mol%), Xantphos (58 mg; 10 mol%) for 12 h
gave 1f as an orange solid (40 mg; 55%). m.p. ¼ 109 ꢁC. 1H NMR
4.1.6. N-Methyl-N-(3,4,5-trimethoxyphenyl)naphthalen-2-amine
2e
By following the general procedure described above for 1a.
Heating at 100 ꢁC 3,4,5-trimethoxyaniline (275 mg; 1.5 mmol) and
2-iodonaphtalene (254 mg; 1 mmol) in the presence of Pd(OAc)2
(22 mg; 10 mol%), Xantphos (58 mg; 10 mol%) for 3 h gave 1d
(300 MHz, CDCl3)
d
7.20 (d, 2H, J ¼ 8.9 Hz), 6.95 (d, 2H, J ¼ 8.9 Hz),
(213 mg; 69%). 1H NMR (300 MHz, CDCl3)
d
7.77 (d, 2H, J ¼ 8.5 Hz),
6.30 (s, 2H), 3.82 (s, 3H), 3.80 (s, 6H), NH not seen. 13C NMR
7.67 (d,1H, J ¼ 8.2 Hz), 7.46e7.42 (m, 2H), 7.36e7.22 (m, 2H), 6.45 (s,
(75 MHz, CDCl3) d 154.0 (2), 142.5, 138.9, 133.3, 129.4 (2), 125.3,
2H), 5.86 (s, 1H), 3.89 (s, 3H), 3.84 (s, 6H). 13C NMR (75 MHz, CDCl3)
118.6 (2), 96.8 (2), 61.1, 56.1 (2). IR (neat) nmax/cmꢂ1: 1611, 1590,
1508, 1228, 1119. MS (APCI) m/z: 294, 296 [M þ H]þ. By following
the general procedure described above for 2a. Diarylamine 1f
(29 mg; 0.1 mmol), NaH (5 mg; 0.2 mmol) and CH3I (28 mg;
0.2 mmol) gave 2i as a yellow-orange solid (27 mg; 88%).
d
153.9 (2), 141.4, 139.2, 134.7, 133.2, 129.2, 129.1, 127.7, 126.5, 126.5,
123.5, 119.9, 111.5, 96.4 (2), 61.1, 56.1 (2). IR (neat) nmax/cmꢂ1: 1631,
1596, 1503, 1465, 1223, 1127, 1006. MS (APCI) m/z: 310 [M þ H]þ. By
following the general procedure described above for 2a. Diaryl-
amine 1d (155 mg; 0.5 mmol), NaH (18 mg; 0.75 mmol) and CH3I
(106 mg; 0.75 mmol) gave 2e as a yellow oil (140 mg; 87%). 1H NMR
m.p. ¼ 91 ꢁC. 1H NMR (300 MHz, CDCl3)
d
7.17 (d, 2H, J ¼ 8.5 Hz),
6.82 (d, 2H, J ¼ 8.5 Hz), 6.31 (s, 2H), 3.84 (s, 3H), 3.79 (s, 6H), 3.26 (s,
(300 MHz, CDCl3)
d
7.72 (t, 2H, J ¼ 7.8 Hz), 7.66 (d, 1H, J ¼ 9.0 Hz),
3H). 13C NMR (75 MHz, CDCl3)
d 153.9 (2), 147.9, 144.8, 134.6, 129.0
7.45e7.39 (td, 1H, J ¼ 8.6 Hz, J ¼ 1.3 Hz), 7.33e7.28 (m, 1H), 7.25 (d,
(2), 124.6, 118.9 (2), 101.0 (2), 61.0, 56.1 (2), 40.6. IR (neat) nmax/
1H, J ¼ 2.5 Hz), 7.17 (dd, 1H, J ¼ 8.9 Hz, J ¼ 2.4 Hz), 6.39 (s, 2H), 3.87
cmꢂ1: 2999, 1609, 1579, 1510, 1451, 1237.
(s, 3H), 3.79 (s, 6H), 3.41 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 153.9
(2), 146.9, 145.4, 134.8, 134.4, 128.7, 128.5, 127.6, 126.7, 126.4, 123.5,
121.0, 112.5, 100.9 (2), 61.1, 56.2 (2), 41.0. IR (neat) nmax/cmꢂ1: 1629,
1589, 1502, 1240, 1128, 1005. MS (APCI) m/z: 324 [M þ H]þ. HRMS
calcd for C20H22NO3 [M þ H]þ 324.1600, obsd. 324.1602.
4.1.11. N,N,N0-Trimethyl-N0-(3,4,5-trimethoxyphenyl)benzene-1,4-
diamine 2j
In a sealed tube and under an argon atmosphere were added
successively, Pd2(dba)3.CHCl3 (52 mg; 5 mol%), Johnphos (30 mg;
10 mol%), dimethylamine (large excess), 2i (307 mg; 1 mmol),
NaOtBu (134 mg; 1.4 mmol) in toluene (10 mL). After heating at
100 ꢁC for 24 h, the resulting suspension was cooled to room
temperature and filtered through a pad of Celite eluting with ethyl
acetate, and the inorganic salts were removed. The filtrate was
concentrated and a rapid purification by silica gel column chro-
matography of the residue to eliminate the excess of 3,4,5-
trimethoxyaniline gave the desired product 2j as a white solid
4.1.7. N-(3-(benzyloxy)-4-methoxyphenyl)-3,4,5-trimethoxy-N-
methylaniline 2f
Compound 2f was prepared from diarylaniline 1e (yield 61%)
according to literature [41]. 1H NMR (300 MHz, CDCl3)
d 7.40e7.27
(m, 5H), 6.86 (d,1H, J ¼ 8.4 Hz), 6.67e6.62 (m, 2H), 6.06 (s, 2H), 5.08
(s, 2H), 3.88 (s, 3H), 3.81 (s, 3H), 3.72 (s, 6H), 3.20 (s, 3H). 13C NMR
(75 MHz, CDCl3) d 153.6 (2),148.7,146.1,145.8,142.9,137.1,128.6 (2),
127.9, 127.4 (2), 115.1, 112.9, 110.1, 96.2 (2), 71.1, 61.1, 56.5, 56.1 (2),
41.0. IR (neat) nmax/cmꢂ1: 1579, 1502, 1452, 1411, 1230, 1125. MS
(ESI) m/z: 432.3 [M þ Na]þ. HRMS calcd for C24H28NO5 [M þ H]þ
410.1967, obsd. 410.1964.
(98 mg, 31%). m.p. ¼ 75 ꢁC. 1H NMR (300 MHz, CDCl3)
d 7.05 (d, 2H,
J ¼ 7.3 Hz), 6.75 (d, 2H, J ¼ 7.3 Hz), 6.01 (s, 2H), 3.78 (s, 3H), 3.76 (s,
6H), 3.24 (s, 3H), 2.95 (s, 6H). 13C NMR (75 MHz, CDCl3)
d 153.6 (2),
147.9, 147.8, 146.9, 139.1, 126.0 (2), 113.9 (2), 93.8 (2), 61.2, 56.1 (2),
41.1 (2), 41.0. IR (neat) nmax/cmꢂ1: 1611, 1583, 1504, 1128. MS (ESI)
m/z: 339 [M þ Na]þ. HRMS calcd for C18H25N2O3 [M þ H]þ 317.1865,
obsd. 317.1860.
4.1.8. AzaisoCA-4 2g
Compound 2g was prepared from diarylaniline 2f using H2 and
Pd/C (10 mol%) in EtOAc (yield 98%) according to literature [1]. 1H

186
M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
4.1.12. 6-Methoxy-N-methyl-N-(3,4,5-trimethoxyphenyl)pyridin-3-
amine 2k
93.3 (2), 61.2, 56.1 (2), 33.1. IR (neat) nmax/cmꢂ1: 3370, 1605, 1505,
1247, 1125, 1009. MS (APCI) m/z: 313 [M þ H]þ. By following the
general procedure described above for 2a. Diarylamine 1i (31 mg;
0.1 mmol), NaH (12 mg; 0.5 mmol) and CH3I (71 mg; 0. 5 mmol)
gave 2m as a yellow oil (21 mg; 65%). 1H NMR (300 MHz, CDCl3)
By following the general procedure described above for 1a.
Heating at 100 ꢁC 3,4,5-trimethoxyaniline (275 mg; 1.5 mmol) and
5-bromo-2-methoxypyridine (187 mg; 1 mmol) in the presence of
Pd(OAc)2 (11 mg; 5 mol%), Xantphos (29 mg; 5 mol%) for 5 h gave
d
7.43 (d, 1H, J ¼ 2.0 Hz), 7.30 (d, 1H, J ¼ 8.6 Hz), 7.08e7.05 (m, 2H),
6.45 (d, 1H, J ¼ 3.0 Hz), 6.03 (s, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.74 (s,
6H), 3.33 (s, 3H). 13C NMR (75 MHz, CDCl3)
153.5 (2), 147.4, 141.7,
1g as a yellow oil (160 mg; 54%). 1H NMR (300 MHz, CDCl3)
d 7.98 (d,
1H, J ¼ 2.3 Hz), 7.42 (dd, 1H, J ¼ 8.8 Hz, J ¼ 2.8 Hz), 6.72 (d, 1H,
d
J ¼ 8.8 Hz), 6.10 (s, 2H), 5.36 (s, 1H), 3.92 (s, 3H), 3.79 (s, 3H), 3.78 (s,
134.4, 130.7, 129.6, 129.3, 120.7, 117.4, 110.1, 101.0, 93.6 (2), 61.1, 56.1
(2), 41.5, 33.1. IR (neat) nmax/cmꢂ1: 1602, 1581, 1505, 1490, 1418,
1234, 1123. MS (APCI) m/z: 327 [M þ H]þ. HRMS calcd for
6H). 13C NMR (75 MHz, CDCl3)
d 160.1, 154.1 (2), 141.2, 139.0, 133.5,
133.1, 132.2, 111.2, 93.9 (2), 61.2, 56.2 (2), 53.8. IR (neat) nmax/cmꢂ1
:
1599, 1486, 1367, 1230, 1125, 1028. MS (APCI) m/z: 291 [M þ H]þ. By
following the general procedure described above for 2a. Diaryl-
amine 1g (290 mg; 1 mmol), NaH (48 mg; 2 mmol) and CH3I
(284 mg; 2 mmol) gave 2k as a yellow oil (267 mg; 88%). 1H NMR
C
19H23N2O3 [M þ H]þ 327.1709, obsd.327.1698.
4.1.15. N-(4-Methoxyphenyl)-N,1-dimethyl-1H-indol-5-amine 2n
By following the general procedure described above for 1i.
Heating at 130 ꢁC under microwave irradiation 4-methoxyaniline
(123 mg; 1 mmol) and 6-bromo-1-methyl-1H-indole (105 mg;
0.5 mmol) for 3 h gave 1j as a yellow oil (50 mg; 40%). 1H NMR
(300 MHz, CDCl3)
J ¼ 2.8 Hz), 6.68 (d, 1H, J ¼ 8.8 Hz), 6.03 (s, 2H), 3.90 (s, 3H), 3.76 (s,
3H), 3.74 (s, 6H), 3.22 (s, 3H). 13C NMR (75 MHz, CDCl3)
160.2,
d
7.93 (d, 1H, J ¼ 2.8 Hz), 7.34 (dd, 1H, J ¼ 8.8 Hz,
d
153.7 (2), 145.8, 141.2, 139.7, 134.9, 132.2, 111.2, 95.6 (2), 61.0, 56.1
(2), 53.6, 41.0. IR (neat) nmax/cmꢂ1: 1587, 1491, 1275, 1235, 1127,
1028. MS (APCI) m/z: 305 [M þ H]þ. HRMS calcd for C16H21N2O4
[M þ H]þ 305.1501, obsd. 305.1501.
(300 MHz, CDCl3)
7.08e7.02 (m, 4H), 6.94e6.89 (m, 2H), 6.47 (d, 1H, J ¼ 3.0 Hz), 5.31
(br s, 1H), 3.86 (s, 3H), 3.79 (s, 3H). 13C NMR (75 MHz, CDCl3)
153.7,
139.5, 136.9, 133.2, 129.4, 129.2, 118.6 (2), 116.2, 114.7 (2), 110.6,
109.8, 100.3, 55.7, 32.9. IR (neat) nmax/cmꢂ1: 1618, 1571, 1509, 1422,
1240. MS (APCI) m/z: 253 [M þ H]þ. By following the general pro-
cedure described above for 2a. Diarylamine 1j (63 mg; 0.25 mmol),
NaH (12 mg; 0.5 mmol) and CH3I (71 mg; 0.5 mmol) gave 2n as a
white solid (34 mg; 51%). m.p. ¼ 113 ꢁC; 1H NMR (300 MHz, CDCl3)
d
7.37 (d, 1H, J ¼ 1.7 Hz), 7.30 (d, 1H, J ¼ 8.7 Hz),
d
4.1.13. N-Methyl-N-(3,4,5-trimethoxyphenyl)quinolin-3-amine 2l
By following the general procedure described above for 1a.
Heating at 100 ꢁC 3,4,5-trimethoxyaniline (275 mg; 1.5 mmol) and
3-bromoquinoline (207 mg; 1 mmol) in the presence of Pd(OAc)2
(44 mg; 20 mol%), Xantphos (29 mg; 5 mol%) and Cs2CO3 (534 mg;
1.64 mmol) for 3 h gave 1h as a yellow oil (167 mg; 54%). 1H NMR
d
7.25 (d, 1H, J ¼ 1.9 Hz), 7.17e7.14 (m, 1H), 6.94 (d, 1H, J ¼ 2.9 Hz),
6.90 (dd, 1H, J ¼ 8.7 Hz, J ¼ 2.0 Hz), 6.78e6.69 (m, 4H), 6.32 (d, 1H,
(300 MHz, CDCl3)
(d, 1H, J ¼ 2.7 Hz), 7.61e7.58 (m, 1H), 7.50e7.40 (m, 2H), 6.41 (s, 3H),
3.84 (s, 3H), 3.77 (s, 6H). 13C NMR (75 MHz, CDCl3)
154.0 (2), 144.9,
d
8.69 (d, 1H, J ¼ 2.7 Hz), 8.00e7.97 (m, 1H), 7.64
J ¼ 3.0 Hz), 3.68 (s, 6H), 3.20 (s, 3H). 13C NMR (75 MHz, CDCl3)
d
153.4, 144.9, 143.0, 133.7, 129.5, 129.3, 119.2 (2), 119.1, 114.8, 114.6
d
(2), 110.0, 100.8, 55.8, 41.8, 33.0. IR (neat) nmax/cmꢂ1: 1620, 1508,
1487, 1422, 1234, 1031. MS (APCI) m/z: 267 [M þ H]þ. HRMS calcd
for C17H19N2O [M þ H]þ 267.1497, obsd. 267.1494.
143.4, 138.2, 137.8, 133.6, 129.0, 129.0, 127.2, 126.4 (2), 116.7, 97.1 (2),
61.1, 56.1 (2). IR (neat) nmax/cmꢂ1: 3338, 1602, 1506, 1237, 1124. MS
(APCI) m/z: 311 [M þ H]þ. By following the general procedure
described above for 2a. Diarylamine 1h (155 mg; 0.5 mmol), NaH
(18 mg; 0.75 mmol) and CH3I (107 mg; 0.75 mmol) gave 2l as a
4.1.16. N-(3,5-Dimethoxyphenyl)-N,1-dimethyl-1H-indol-5-amine
2o
yellow oil (110 mg; 68%). 1H NMR (300 MHz, CDCl3)
d
8.62 (d, 1H,
By following the general procedure described above for 1i.
Heating at 130 ꢁC under microwave irradiation 3,5-
dimethoxyaniline (153 mg; 1 mmol) and 6-bromo-1-methyl-1H-
indole (105 mg; 0.5 mmol) for 3 h gave 1k as a yellow oil (35 mg;
J ¼ 2.8 Hz), 8.00e7.94 (m, 1H), 7.68e7.63 (m, 1H), 7.51e7.42 (m, 2H),
7.39 (d, 1H, J ¼ 2.8 Hz), 6.38 (s, 2H), 3.85 (s, 3H), 3.78 (s, 6H), 3.38 (s,
3H). 13C NMR (75 MHz, CDCl3)
d 154.1 (2), 145.4, 144.2, 142.7, 142.6,
135.2, 129.0, 127.1, 126.5, 126.3, 117.3, 101.5 (2), 61.1, 56.2 (2), 40.82
(one C missing). IR (neat) nmax/cmꢂ1: 1588, 1504, 1245, 1229, 1128.
MS (APCI) m/z: 325 [M þ H]þ.
25%). 1H NMR (300 MHz, CDCl3)
d
7.45 (d, 1H, J ¼ 1.9 Hz), 7.30 (d, 1H,
J ¼ 9.4 Hz), 7.13e7.07 (m, 2H), 6.45 (d, 1H, J ¼ 3.0 Hz), 6.14 (d, 2H,
J ¼ 2.1 Hz), 6.01 (t,1H, J ¼ 2.1 Hz), 5.68 (br s,1H), 3.81 (s, 3H), 3.76 (s,
6H). 13C NMR (75 MHz, CDCl3)
d 161.2 (2), 148.7, 134.2, 134.1, 129.6,
4.1.14. N,1-Dimethyl-N-(3,4,5-trimethoxyphenyl)-1H-indol-5-
amine 2m
129.1, 118.6, 114.5, 109.9, 100.7, 93.6 (2), 91.3, 55.3 (2), 33.0. IR (neat)
nmax/cmꢂ1: 1592, 1518, 1448, 1154. MS (APCI) m/z: 305 [M þ Na]þ.
By following the general procedure described above for 2a. Dia-
rylamine 1k (28 mg; 0.1 mmol), NaH (12 mg; 0.5 mmol) and CH3I
(71 mg; 0.5 mmol) gave 2o as a yellow oil (27 mg; 93%). 1H NMR
To an Emrys Optimizer were added 3,4,5-trimethoxyaniline
(260 mg; 1.42 mmol), 6-bromo-1-methyl-1H-indole (150 mg;
0.71 mmol), Pd(OAc)2 (16 mg; 10 mol%), Xantphos (41 mg; 10 mol
%), Cs2CO3 (463 mg; 1.42 mmol) in dioxane (5 mL). The reaction
vessel was then placed in the Emrys Optimizer and exposed to
microwave irradiation according to the following specifications:
temperature: 130 ꢁC, time: 3 h, fixed hold time: on, sample ab-
sorption: high, pre-stirring: 60 s. After cooling to room tempera-
ture, the mixture was filtered through a pad of Celite eluting with
ethyl acetate, and the inorganic salts were removed. The filtrate was
concentrated and a rapid purification by silica gel column chro-
matography of the residue to eliminate the excess of 3,4,5-
trimethoxyaniline gave the desired product 1i as a yellow oil
which was immediately engaged in the alkylation step. 1i: (108 mg,
(300 MHz, CDCl3)
d
7.47 (d, 1H, J ¼ 2.0 Hz), 7.32 (d, 1H, J ¼ 8.6 Hz),
7.09 (d, 2H, J ¼ 6.9 Hz, J ¼ 2.5 Hz), 6.46 (d, 1H, J ¼ 3.1 Hz), 5.94 (s,
3H), 3.81 (s, 3H), 3.70 (s, 6H), 3.32 (s, 3H). 13C NMR (75 MHz, CDCl3)
d
161.4 (2), 152.5, 141.2, 134.8, 129.6, 129.3, 121.5, 118.9, 110.2, 101.1,
93.6 (2), 89.6, 55.3 (2), 41.2, 33.1. IR (neat) nmax/cmꢂ1: 3433, 1608,
1578,1419,1236,1138. MS (APCI) m/z: 297 [M þ H]þ. HRMS calcd for
C
18H21N2O2 [M þ H]þ 297.1603, obsd. 297.1601.
4.1.17. N-Methyl-1-(phenylsulfonyl)-N-(3,4,5-trimethoxyphenyl)-
1H-indol-5-amine 2p
By following the general procedure described above for 1i.
Heating at 130 ꢁC under microwave irradiation 3,4,5-
trimethoxyaniline (183 mg; 1 mmol) and 5-bromo-1-(phenyl-
sulfonyl)-1H-indole (167 mg; 0.5 mmol) for 4 h gave 1l as a yellow
49%); 1H NMR (300 MHz, CDCl3)
7.02 (m, 2H), 6.38 (d, 1H, J ¼ 3.0 Hz), 6.15 (s, 2H), 5.50 (brs, 1H), 3.77
(s, 3H), 3.76 (s, 3H), 3.74 (s, 6H). 13C NMR (75 MHz, CDCl3)
154.0
(2), 143.0, 135.1, 133.8, 131.3, 129.6, 129.2, 117.9, 113.4, 110.0, 100.6,
d 7.35 (s, 1H), 7.23 (s, 1H), 7.05e
d
oil (162 mg; 74%). 1H NMR (300 MHz, CDCl3)
d 7.89e7.84 (m, 3H),

M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
187
7.53e7.47 (m, 2H), 7.43e7.37 (m, 2H), 7.17 (d, 1H, J ¼ 2.0 Hz), 7.02
4.1.21. 1-Methyl-N-propyl-N-(3,4,5-trimethoxyphenyl)-1H-indol-5-
amine 3c
(dd, 1H, J ¼ 2.1 Hz, J ¼ 8.8 Hz), 6.53 (dd, 1H, J ¼ 3.6 Hz, J ¼ 0.5 Hz),
6.26 (s, 2H), 5.74 (s, 1H), 3.80 (s, 3H), 3.74 (s, 6H). 13C NMR (75 MHz,
CDCl3) d 153.9 (2), 140.3,139.8,138.2,133.8,132.5,131.9, 130.2,129.2
3c yellow oil (120 mg; 34%) was prepared from 1i by following
the general procedure described above for 3a. 1H NMR (300 MHz,
(2), 127.0, 126.7 (2), 117.4, 114.3, 109.9, 109.3, 95.6 (2), 61.0, 56.0 (2).
IR (neat) nmax/cmꢂ1: 2927, 1600, 1504, 1450, 1336, 1227. MS (ESI) m/
z: 461 [M þ Na]þ. By following the general procedure described
above for 2a. Diarylamine 1l (110 mg; 0.25 mmol), NaH (12 mg;
0.5 mmol) and CH3I (71 mg; 0. 5 mmol) gave 2p as a yellow oil
CDCl3)
d
7.45 (d,1H, J ¼ 1.7 Hz), 7.32 (d,1H, J ¼ 8.6 Hz), 7.08e7.05 (m,
2H), 6.47 (d, 1H, J ¼ 3.0 Hz), 5.99 (s, 2H), 3.81 (s, 6H), 3.73 (s, 6H),
3.66 (t, 2H, J ¼ 6.5 Hz), 1.76e1.60 (m, 2H), 0.97 (t, 3H, J ¼ 7.4 Hz). 13
C
NMR (75 MHz, CDCl3)
d 153.5 (2), 146.7, 139.9, 134.5, 130.2, 129.4,
129.3,121.8,118.7,110.1,100.9, 93.3 (2), 61.1, 56.0 (2), 55.0, 33.0, 21.0,
11.6. IR (neat) nmax/cmꢂ1: 1606, 1580, 1507, 1487, 1237, 1125. MS
(ESI) m/z: 377 [M þ Na]þ. HRMS calcd for C21H27N2O3 [M þ H]þ
355.2022, obsd. 355.2018.
(81 mg; 72%). 1H NMR (300 MHz, CDCl3)
d 7.89e7.86 (m, 3H), 7.55e
7.50 (m, 2H), 7.42 (dd, 2H, J ¼ 10.4 Hz, J ¼ 4.7 Hz), 7.13 (d, 1H,
J ¼ 2.2 Hz), 7.02 (dd, 1H, J ¼ 8.9 Hz, J ¼ 2.2 Hz), 6.57 (d, 1H,
J ¼ 3.6 Hz), 6.16 (s, 2H), 3.82 (s, 3H), 3.73 (s, 6H), 3.28 (s, 3H). 13
C
NMR (75 MHz, CDCl3)
d 153.7 (2), 146.1, 145.7, 138.3, 133.8, 133.0,
4.2. Biology
131.9, 130.4, 129.3 (2), 127.0, 126.8 (2), 119.6, 114.2, 112.7, 109.4, 98.0
(2), 61.1, 56.1 (2), 41.2. IR (neat) nmax/cmꢂ1: 1586, 1504, 1446, 1368,
1123. MS (ESI) m/z: 453 [M þ H]þ, 475 [M þ Na]þ. HRMS calcd for
4.2.1. Cell culture and proliferation assay
Cancer cell lines were obtained from the American type Culture
Collection (Rockville, MD) and were cultured according to the
supplier’s instructions. Briefly, A549 lung carcinoma, U87-MG hu-
man glioblastoma, MDA-MB231 cells were grown in Dulbecco
minimal essential medium (DMEM) containing 4.5 g/L glucose
supplemented with 10% FCS and 1% glutamine. Human K562 leu-
kemia and HCT116 colorectal carcinoma cells were grown in RPMI
1640 containing 10% FCS and 1% glutamine. Human umbilical vein
endothelial cells (HUVECs) were obtained from Clonetics (Lonza,
Walkersville, MD, USA) and cultured according to the supplier’s
instructions. Briefly, HUVECs from three to six passages were sub-
cultured to confluence onto 0.2% gelatin coated tissue culture flasks
in endothelial cell growth medium (EGM2) containing growth
factors and 2% FCS. All cell lines were maintained at 37 ꢁC in a
humidified atmosphere containing 5% CO2. Cell viability was
assessed using Promega CellTiter-Blue TM reagent according to the
manufacturer’s instructions. Cells were seeded in 96-well plates
C
25H27N2O5S [M þ H]þ 453.1484, obsd.453.1487.
4.1.18. NN-Methyl-N-(3,4,5-trimethoxyphenyl)-1H-indol-5-amine
2q
A THF (1 mL) solution of 2p (47 mg; 0.1 mmol) was added 0.5 mL
of a TBAF solution in THF (0.5 mmol) and stirred at 100 ꢁC for 6 h.
After cooling, the solution was filtered through a pad of Celite,
eluting with ethyl acetate. The filtrate was concentrated and a
purification by silica gel column chromatography gave the desired
product 2q as a white solid (15 mg, 49%). m.p. 166 ꢁC. 1H NMR
(300 MHz, CDCl3)
d
8.34 (s, 1H), 7.44 (s, 1H), 7.36 (d, 1H, J ¼ 8.6 Hz),
7.22 (t, 1H, J ¼ 2.7 Hz), 7.02 (dd, 1H, J ¼ 8.6 Hz, J ¼ 1.9 Hz), 6.52e6.51
(m, 1H), 6.04 (s, 2H), 3.80 (s, 3H), 3.74 (s, 6H), 3.33 (s, 3H). 13C NMR
(75 MHz, CDCl3)
d 153.6 (2), 147.4, 142.1, 133.4, 130.8, 128.8, 125.1,
121.0, 117.1, 112.0, 102.7, 93.8 (2), 61.2, 56.1 (2), 41.5. IR (neat) nmax
/
cmꢂ1: 3433, 1608, 1578, 1505, 1450, 1419, 1236. MS (ESI) m/z: 335
[M þ Na]þ. HRMS calcd for C18H21N2O3 [M þ H]þ 313.1552, obsd.
331.1548.
(5 ꢃ 103 cells/well) containing 50
culture, the cells were supplemented with 50
compound dissolved in DMSO (less than 0.1% in each preparation).
After 72 h of incubation, 20 L of resazurin was added for 2 h before
recording fluorescence ( em ¼ 590 nm) using a
ex ¼ 560 nm,
mL growth medium. After 24 h of
mL of the tested
4.1.19. N-(3-(benzyloxy)-4-methoxyphenyl)-3,4,5-trimethoxy-N-
propylaniline 3a
m
l
l
To a diarylamine 1e (197 mg; 0.5 mmol) solution in DMF (5 mL)
was added at 0 ꢁC, NaH (24 mg; 1 mmol), and 1-bromopropane
(122 mg; 1 mmol). The mixture was stirred at r.t. for 2 h, diluted
in EtOAc (5 mL) and washed with aqueous NaHCO3. The organic
layer was dried, evaporated, and the residue was subjected to flash
chromatography to give 3a as a brown oil (208 mg, 95%). 1H NMR
Victor microtiter plate fluorimeter (PerkineElmer, USA). The IC50
corresponds to the concentration of the tested compound that
caused a decrease of 50% in fluorescence of drug treated cells
compared with untreated cells. Experiments were performed in
triplicate.
(300 MHz, CDCl3)
6.59 (m, 2H), 6.00 (s, 2H), 5.09 (s, 2H), 3.89 (s, 3H), 3.80 (s, 3H), 3.71
d 7.40e7.27 (m, 5H), 6.86e6.83 (m, 1H), 6.63e
4.2.2. Tubulin binding assay
Sheep brain tubulin was purified according to the method of
Shelanski [42] by two cycles of assembly-disassembly and then
diluted in the assembly buffer containing 0.1 M MES, 0.5 mM
MgCl2, 1 mM EGTA, and 1 mM GTP, pH 6.6 to a final concentration
around 2e3 mg/mL. Tubulin assembly was monitored by fluo-
rescence according to reported procedure [43] using DAPI as
fluorescent molecule. Assays were realized on 96-well plates
prepared with Biomek NKMC and Biomek 3000 from Beckman
Coulter and read at 37 ꢁC on Wallac Victor fluorimeter from Perkin
Elmer. The IC50 value of each compound was determined as the
concentration required to decrease the maximum assembly rate
of tubulin by 50% compared to the rate in the absence of com-
pound. The IC50 values for all compounds were compared to the
IC50 of isoCA-4 and isoerianin measured the same day under the
same conditions.
(s, 6H), 3.50e3.44 (m, 2H), 1.61e1.59 (m, 2H), 0.89 (t, 3H, J ¼ 7.4 Hz).
13C NMR (75 MHz, CDCl3)
d 153.7 (2), 148.7, 145.8, 145.4, 141.6, 137.1,
131.8, 128.6 (2), 127.9, 127.4 (2), 116.0, 112.8, 111.0, 96.3 (2), 71.1, 61.2,
56.5, 56.1 (2), 54.7, 21.0, 11.6. IR (neat) nmax/cmꢂ1: 2925, 1610, 1502,
1464, 1225, 1127. MS (APCI) m/z: 438 [M þ H]þ. HRMS calcd for
C
26H32NO5 [M þ H]þ 438.2280, obsd. 438.2285.
4.1.20. 2-methoxy-5-(propyl(3,4,5-trimethoxyphenyl)amino)
phenol 3b
3b brown oil (140 mg; 99%) was prepared by following the
general procedure described above for 2g. 1H NMR (300 MHz,
CDCl3)
d
6.78 (d, 1H, J ¼ 8.7 Hz), 6.65 (d, 1H, J ¼ 2.6 Hz), 6.51 (dd, 1H,
J ¼ 8.7 Hz, J ¼ 2.6 Hz), 6.11 (s, 2H), 5.60 (s, 1H), 3.88 (s, 3H), 3.81 (s,
3H), 3.77 (s, 6H), 3.57e3.52 (m, 2H), 1.70e1.60 (m, 2H), 0.94 (t, 3H,
J ¼ 7.4 Hz). 13C NMR (75 MHz, CDCl3)
d 153.7 (2), 146.3, 145.2, 142.6,
142.2,132.3,114.1,111.6, 109.7, 97.5 (2), 61.2, 56.4, 56.2 (2), 54.7, 21.0,
11.6. IR (neat) nmax/cmꢂ1: 2959, 1581, 1501, 1466, 1261, 1126, 1027.
MS (ESI) m/z: 370 [M þ Na]þ. HRMS calcd for C19H26NO5 [M þ H]þ
348.1811, obsd. 348.1812.
4.2.3. Cell cycle analysis
Exponentially growing cancer cells (K562, HCT116) were incu-
bated with tested compound or DMSO for 24 h. Cell-cycle profiles

188
M.A. Soussi et al. / European Journal of Medicinal Chemistry 78 (2014) 178e189
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were determined by flow cytometry on a FC500 flow cytometer
(BeckmaneCoulter, France) as described previously [44].
4.2.4. Apoptosis assay
Apoptosis was measured by the Apo-one homogeneous
caspase-3/7 assay (Promega Co, WI) according to the manufac-
turer’s recommendations. Briefly, cells were subcultured on a 96-
well plate with 5 ꢃ 104 cells/well in 100
mL medium. After 24 h of
incubation, the medium in the 96-well plate was discarded and
replaced with medium containing different concentrations of 2b
and 2m (7.5, 15, 25 and 50 nM). The treated cells were incubated for
24 h, each well then received 100 mL of a mixture of caspase sub-
strate and Apo-one caspase 3/7 buffer. After 1 h of incubation, the
fluorescence of sample was measured using a Victor microtiter
plate fluorimeter (PerkineElmer, USA) at 527 nm.
4.2.5. Inhibition of endothelial tube formation using ECFCs
The protocol was achieved according to ref. [38].
4.2.6. Cord disruption assay
HUVECs (2 ꢃ 104 cells per well) were plated in 96-well plates on
a thick layer of Matrigel (Becton Dickinson; 10 mg mLꢂ1, 60
mL per
well) and allowed to align for 24 h. 2b, 2m or vehicle (DMSO) were
added to the formed cords and left for 3 h. Images were taken 3 h
after the addition of compounds.
4.2.7. Molecular modelling
Coordinates for compounds 2b, 2m and isoCA-4 were generated
using CORINA v3.44 software [45]. Molecules were then freely
docked in the colchicine binding site between chains C and D from
PDB structure 1SA0 using GOLD v5.1 software [46]. CHEMPLP with
default parameters was used as an objective function [47]. Struc-
tures of the complexes were exported for further examination and
depiction with Chimera v1.8.1 software [48], including hydrogen
bonds detection, close contact analysis and representation of the
solvent-accessible surface colored according to polarity.
Acknowledgments
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The CNRS (Centre National de la Recherche Scientifique) is
gratefully acknowledged for financial support of this research. We
also thank ARC (association pour la recherche sur le cancer), for
their financial support of this research (doctoral fellowship to M.A.
Soussi). OIDD screening data supplied courtesy of Eli Lilly and
Company e used with Lilly’s permission. Our laboratory BioCIS-
UMR 8076 is a member of the Laboratory of Excellence LERMIT
supported by a grant from ANR (Agence Nationale de la Recherche,
ANR-10-LABX-33). This work on tubulin was supported by a grant
from ANR (ANR-09-BLAN-0071).
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
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