Synthesis, antiproliferative activity and tubulin targeting effect of acridinone and dioxophenothiazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.10.051

The synthesis of new acridinone and dioxophenothiazine derivatives along with their tubulin polymerization inhibitory and antiproliferative activities is reported. The analysis of correlation for cytotoxic and antitubulin potential of tested compounds sho

Full text of DOI:10.1016/j.ejmech.2012.10.051

European Journal of Medicinal Chemistry 59 (2013) 39e47
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative activity and tubulin targeting effect of acridinone
and dioxophenothiazine derivatives
Valérie Verones a, Nathalie Flouquet a, Marie Lecoeur b, Amelie Lemoine c, Amaury Farce a,
Brigitte Baldeyrou d, Christine Mahieu d, Nicole Wattez d, Amélie Lansiaux d, Jean-François Goossens b,
,
Pascal Berthelot a, Nicolas Lebegue a
*
a Université Lille Nord de France, Faculté des Sciences Pharmaceutiques et Biologiques, Laboratoire de Chimie Thérapeutique, EA 4481, IFR 114, 3 rue du Professeur Laguesse, BP-83,
F-59006 Lille, France
b Université Lille Nord de France, Faculté des Sciences Pharmaceutiques et Biologiques, Laboratoire de Chimie Analytique, EA 4481, IFR 114, 3 rue du Professeur Laguesse, BP-83,
F-59006 Lille, France
c Université Lille-Nord de France, Institut de Chimie Pharmaceutique Albert Lespagnol, EA4481, IFR114, 3 Rue du Pr Laguesse, BP-83, F-59006 Lille, France
d Inserm U837, Centre Oscar Lambret, Université Nord de France, IMPRT et IRCL, 1 Place de Verdun, 59045 Lille cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of new acridinone and dioxophenothiazine derivatives along with their tubulin poly-
merization inhibitory and antiproliferative activities is reported. The analysis of correlation for cytotoxic
and antitubulin potential of tested compounds showed that 4-methoxyphenylethyl derivatives 18a and
19a were highly cytotoxic but were regarded to have no significant antitubulin activity. However, the
introduction of a 3-hydroxy substituent leading to compounds 18e and 19e, strongly increased the
antitubulin potential but was associated with a loss of the antiproliferative activity. Modeling studies,
topoisomerase inhibition assays and cell cycle analysis have been performed to better investigate the
mechanism of action of such compounds.
Received 25 July 2012
Received in revised form
19 October 2012
Accepted 29 October 2012
Available online 8 November 2012
Keywords:
Acridinone
Phenothiazine
Tubulin polymerization
Cytotoxicity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
much effort has been devoted to the identification of ligands binding
at the colchicine site of tubulin derived not only from natural sources
Antimitotic agents are one of the major classes of cytotoxic drugs
for cancer treatment, and tubulin is the target for numerous small
natural and synthetic molecules that inhibit the formation of the
mitotic spindle [1]. Microtubules play an important role in the
formation and function of the spindle apparatus, which realizes the
controlled segregation of the chromosomes in dividing cells.
Microtubules are also directly involved in a variety of other cellular
functions, such as cell movement, maintenance of the cell shape, as
well as the transport of organelles inside the cell. Interfering with the
dynamic instability of microtubules, spindle poisons arrest dividing
cells in G2/M phases of the cell cycle, causing mitotic catastrophy and
finally apoptotic cell death [2]. There are two major groups of these
antitumor agents, microtubule stabilizers such as paclitaxel [3] and
microtubule destabilizers such as vinca alkaloids [4], colchicine [5],
combretastatin A-4 [6], and sulfonamide E-7010 [7]. In recent years,
but also by screening compound libraries in combination with
traditional medicinal chemistry [8e10]. However, no representatives
of this class have yet been approved for use in cancer chemotherapy.
We have earlier described a series of benzopyridooxathiazepine-
derived tubulin polymerization inhibitors, that could be considered
as constraint analogs of E-7010, with potent in vitro and moderately
in vivo antitumor activity (Fig. 1) [11,12]. The relatively low in vivo
antitumor activity of compound 1, has been explained by chemical
stability study [13]. Results from this study showed that compound 1
suffered from a lack of stability of the oxathiazepine ring which could
have some consequences on the storage conditions. Continuing our
search strategy for novel potent tubulin-targeting compounds, we
extended our studies to tricyclic heterocycles, with improved
chemical stability, which were important scaffolds in medicinal
chemistry and were in agreement with the structureeactivity rela-
tionships previously established [12]. 10H-Acridin-9-one and its
sulfonyl analog, 10H-phenothiazine 5,5-dioxide, have shown diverse
biological activities including antimalarial [14], antineoplastic [15],
antiasthmatic [16,17] and antidiabetic properties [18]. In addition,
* Corresponding author. Tel.: þ33 320964977; fax: þ33 320964913.
E-mail address: nicolas.lebegue@univ-lille2.fr (N. Lebegue).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.051

40
V. Verones et al. / European Journal of Medicinal Chemistry 59 (2013) 39e47
following an ‘one-pot’ two steps procedure previously described
[22]. Basic hydrolysis of amide bond of 7 with a 30% aqueous
solution of sodium hydroxide followed by oxidative cleavage of the
a-ketoacid intermediate mediated by addition of 35% aqueous
hydrogen peroxide solution did not provide the desired 10H-
phenothiazine-1-carboxylic acid as described in the literature [23]
but led to the formation of its 5-oxide analog 8. Additional oxida-
tion into sulfone 9 was realized by refluxing sulfoxide 8 in acetic
acid with an excess of 35% aqueous hydrogen peroxide solution in
67% yield. The final target compounds 18aee and 19aee were
synthesized following the same procedure described in Scheme 2.
Carboxylic acids 5 and 9 were first reacted with thionyl chloride in
refluxing toluene followed by a palladium-catalyzed reduction of
the formed acyls chlorides 10 and 11 with tributyltin hydride to
provide exclusively the corresponding aldehydes 12 and 13 under
very mild conditions and quantitative yields [24]. The systematic
investigation of substituted acridinone or dioxo-phenothiazine
derivatives bearing various arylethyl substituents was realized by
condensation of aldehydes 12 and 13 under Wittig conditions with
several phosphonium salt intermediates 15aee giving a Z/E-
isomeric mixture of the corresponding olefins which were not
separated. The phosphonium salts were prepared by refluxing in
toluene the corresponding commercially available 14aed or
described 14e [25] benzyl chlorides with triphenylphosphine.
Finally, catalytic hydrogenation allowed reduction of the double
bond and hydrogenolysis of benzyloxy group for compounds 16e
and 17e.
Fig. 1. Structures of reference and synthesized compounds.
N-benzoylated phenothiazines have recently been described as
potent inhibitors of tubulin polymerization. The antitubulin activity
of the most active compounds was comparable or superior to those
of the reference compounds, such as nocodazole, podophyllotoxin,
and colchicine [19].
This article reported the synthesis and biological evaluation of
10H-acridin-9-one 18aee and 10H-phenothiazine 5,5-dioxide
19aee derivatives bearing a phenyl-substituted-ethyl side chain
on the benzenic part of the tricycle as potential antitumor agents
(Fig. 1). The newly synthesized compounds have been tested
together with their tubulin polymerization inhibitory activities and
their cytotoxicities toward the human colon adenocarcinoma HT29
cell line.
2. Results and discussion
2.2. In vitro antiproliferative and tubulin polymerization assays
2.1. Chemistry
Antiproliferative effects of 10H-acridin-9-one and 10H-pheno-
thiazine 5,5-dioxide derivatives 18 and 19 and the reference
compounds (E-7010 and 1) were evaluated on human colon
adenocarcinoma HT29 cell line. The results presented in Table 1
showed that the nature and the location of the substituents at the
phenylethyl side chain played a critical role for antiproliferative
potency. Only 4-methoxy-substituted derivatives 18a and 19a
showed IC50 values in the submicromolar range with respectively
10H-Acridin-9-one-4-carboxylic acid 5 and 10H-phenothiazine-
1-carboxylic acid 5,5-dioxide 9 were synthesized according to the
general methods described in Scheme 1. The N-phenylanthranilic
acid 4 was prepared by the JourdaneUllmann copper-catalyzed
condensation of 2-chlorobenzoic acid 3 and 2-aminobenzoic acid
2 in 82% yield [20]. Cyclization into the corresponding acridinone 5
was performed by acid catalyzed reaction with concentrated H2SO4
[21]. The 10H-phenothiazine-1-carboxylic acid 5,5-dioxide 9 was
0.051 and 0.87
mM. Interestingly, introduction of a 3,4,5-
trimethoxyphenyl group in 18c and 19c, a typical pharmacophore
found in many inhibitors of tubulin polymerization [26] led to a total
loss of antiproliferative activity as documented by our earlier work
[12,27] and as confirmed herein. Introduction of a 3-hydroxy
synthesized using phenothiazine
construction of the tetracyclic isatin intermediate 7 consisted in
treatment of 6 with oxalyl chloride and aluminum chloride,
6 as starting material. The
Scheme 1. Synthesis of 10H-Acridin-9-one-4-carboxylic acid 5 and 10H-phenothiazine-1-carboxylic acid 5,5-dioxide 9.

V. Verones et al. / European Journal of Medicinal Chemistry 59 (2013) 39e47
41
Scheme 2. Synthesis of target compounds 18aee and 19aee.
substituent at the aryethyl side chain of 18a or 19a led to weaker
the same cytotoxic profile but was found less potent than its
cytotoxicities in the micromolar range (IC50 of respectively 12.0 and
carbonyl counterpart.
7.2 mM for compounds 18e and 19e). Furthermore, quite other vari-
ations of substituents within the aryl moiety of 18a or 19a resulted
either to reduced activities (18b, 18d, 19b) or made new compounds
2.3. Topoisomerase I and II inhibition
almost inactives (18c, 19c, 19d) at concentrations as high as 100 mM.
Numerous acridinone and phenothiazine derivatives were
described in the literature to act as DNA binders and topoisomerase
(Topo) inhibitors [30]. For these reasons, compounds 18a, 18c and
19a were tested for their topoisomerase I and II inhibition. As
shown in Fig. 2A, supercoiled plasmid DNA was treated with human
Topo I in the presence of the reference camptothecin (CPT) or
To characterize the possible interaction with the microtubule
system of this novel series of 10H-acridin-9-one and 10H-pheno-
thiazine 5,5-dioxide derivatives, compounds 18aee and 19aee
were evaluated for their in vitro inhibition of tubulin polymeriza-
tion. For comparison, E-7010 and compound 1 were examined in
contemporaneous experiments (Table 1). Namely, some recent
reports suggest that nonlinear relationship between anti-
proliferative activity and the effect on tubulin polymerization of
described inhibitors, as combretastatin A-4, may occur, where
highly cytotoxic compounds are not necessarily potent inhibitors of
tubulin polymerization and vice versa [6,28,29]. In this assembly
assay, only compounds 18e and 19e displayed potent inhibitory
activities and were more actives than the reference compound E-
synthesized compounds at 10 and 20
mM. The DNA relaxation/
cleavage products were resolved by electrophoresis. Inhibition of
Topo I was clearly specifically detected with the reference CPT,
which produced a marked level of DNA double stranded breaks
while no increase in the nicked band or modification of the relax-
ation of the DNA was observed with the tested compounds.
The same tests, with Topo I being replaced with Topo II, were
performed to evaluate effects on the catalytic activity of Topo II. In
these experiments supercoiled plasmid DNA was treated with Topo
7010 and compound 1, with IC50 values of 0.51, 1.25, 2.2 and 2.4 mM,
respectively. Compounds 18aed and 19aed seemed to be inactive
as inhibitors of tubulin polymerization and did not inhibit tubulin
assembly at concentrations as high as 10 mM. These findings indi-
II in the presence of 50 mM of the tested drugs or 20 and 50 mM of
the reference etoposide (ETO) which produced a marked level of
cated that the 3-hydroxy-4-methoxyphenyl was crucial moiety for
inhibition of tubulin polymerization and was preferential partial
structure seen with many colchicine site inhibitors. The analysis of
correlation for cytotoxic and antitubulin potential of tested
compounds showed that 4-methoxyphenylethyl derivatives 18a
and 19a were highly cytotoxic but were regarded to have no
significant antitubulin activity. However, the introduction of a 3-
hydroxy substituent strongly increased the antitubulin potential
but was associated with a loss of the antiproliferative activity. A
possible explanation for the significantly lower cytotoxicities of 18e
and 19e in respect to E7010 and compound 1 might be the bad
physicochemical properties (such as solubility, membrane perme-
ability, log P) that influence pharmacokinetic parameters in part did
not allow compounds to reach tubulin itself within cells.
Next, to further investigate the antiproliferative potential of
tumor cells, the effects of the highly active 4-methoxyphenylethyl
analogs 18a and 19a as well as the negative control 18c were
investigated against four other cell lines derived from head and
neck carcinoma (HNSCC), leukemia (HL60) and prostate tumors
(PC3, DU145) (Table 2). Acridinone 18a showed potent nanomolar
cytotoxic effects on HNSCC and DU145 cell lines with IC50 of
respectively 37 and 50 nM and was found less active on HL60
and PC3 cell lines with IC50 values in the submicromolar range
Table 1
Structures, inhibition of tubulin polymerization and in vitro antiproliferative activ-
ities against human colon adenocarcinoma HT29 cell line of compounds 18aee and
19aee.
A
R1
R2
N
H
R3
Compd
A
R1
R2
R3
IC50
(m
M)a
IC50 TPI (m
M)b
E-7010
Cpd 1
18a
18b
18c
18d
18e
19a
19b
19c
19d
19e
e
e
e
e
0.21
2.2
2.4
e
e
e
e
0.024
0.051
>50
>100
>50
12.0
0.87
>50
>100
>100
7.2
CO
CO
CO
CO
CO
SO2
SO2
SO2
SO2
SO2
H
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OH
H
H
OCH3
H
H
H
H
OCH3
H
H
>10
>10
>10
>10
0.51
>10
>10
>10
>10
1.25
OCH3
OCH3
OH
OH
H
OCH3
OCH3
OH
OH
OCH3
a
% of inhibition at 10 mM or concentration inhibiting HT29 cell proliferation by
50% relative to untreated controls after 72 h of drug exposure.
b
(0.1 and 0.75
mM respectively). Dioxophenothiazine 19a displayed
Inhibition of tubulin polymerization.

42
V. Verones et al. / European Journal of Medicinal Chemistry 59 (2013) 39e47
Table 2
2.4. Cell cycle analysis and apoptosis (flow cytometry)
Antiproliferative activities (IC50
tumor cell lines.
mM) of compounds 18a, 18c and 19a on different
The effects of the compounds 18a, 18c and 19a and reference
antimitotic compounds (vinorelbine, vincristine, docetaxel) on cell
cycle progression of HNSSC CAL33, colon HT29 and leukemia HL60
cells lines were investigated by flow cytometric analysis and the
data are disclosed in Table 3 which shows the percentage of cells in
each phase of the cell cycle. Compound 18a allowed a large increase
a
Cell type
IC50
(
m
M)
HNSCCb
Leukemia
Prostate
PC3
Prostate
DU145
Cell line
CAL33
N.t.c
HL60
Cpd 1
18a
18c
N.t.
0.475 ꢀ 0.05
0.75 ꢀ 0.1
>100
0.017 ꢀ 0.02
0.05 ꢀ 0
0.037 ꢀ 0
0.1 ꢀ 0.01
of cells in the sub-G1 and G2 phases at concentrations of 0.5
mM
>100
>50
>100
19a
0.627 ꢀ 0.02
1.65 ꢀ 0.07
>50
0.81 ꢀ 0.05
and 5 M after 24 h of exposure and it was accompanied by a cor-
m
a
responding reduction of cells in the G1 phase. The marked accu-
mulation of cells in sub-G1 phase indicated the induction of cellular
apoptosis as for the reference compounds. Compound 19a dis-
played the same cell cycle profile but only at a concentration of
Concentration inhibiting cell proliferation by 50% relative to untreated controls
after 48 h of drug exposure.
b
Head and neck squamous cell carcinoma.
Not tested.
c
5 mM, while compound 18c had no effects compared to the controls.
DNA double stranded breaks, corresponding to linear DNA (Fig. 2B).
No inhibition of Topo II was also detected with our new derivatives.
The results from this Topo I or II assay formally established that
compounds 18a, 18c and 19a were not topoisomerase poisons and
did not interact with DNA.
These results indicated that the induction of cellular apoptosis by
18a and 19a could be by virtue of their antimitotic activities as there
was an increase in cell cycle arrest at G2 phase with parallel
increase in apoptotic cells at sub-G1 phase.
2.5. Modeling studies
To explain marked difference between cytotoxicity and anti-
tubulin potential of compounds 18a, 18e, 19a and 19e, docking
studies have been performed on the colchicine binding site of
tubulin to explore the interaction mechanism and understand the
structural features responsible for their activity (Fig. 3). Compounds
18e and 19e only showed a single conformation occupying the
binding site and engaging in the same hydrogen bonds with Ser
178, Thr 179 and Ala 250. The tricyclic core of 18e and 19e was
oriented near the residues Ala 250 and Lys 254 and interacted via
hydrogen bonding with the carbonyl or sulfonyl moieties while the
2-(3-hydroxy-4-methoxyphenyl)ethyl adopted a folded up confor-
mation stabilized by hydrogen bonds formed between the hydroxy
and methoxy groups and Ser 178 and Thr 179. Docking studies
performed with others compounds, as 18a and 19a, revealed that
the scoring functions employed were not able to clearly indicate
which would be the preferred conformations. These compounds
could not be docked successfully in the colchicine binding site and
the molecular modeling studies were in good agreement with the
biological data. These results suggested that 3-hydroxy-4-
methoxyphenyl substitution might be crucial for binding interac-
tions and provided a possible explanation why compounds 18e and
19e displayed potent tubulin inhibitory activities with regard to
their counterparts 18a and 19a.
3. Conclusion
We have designed and synthesized acridinone and dioxopheno-
thiazine analogs of the oxathiazepine 1, bearing various substituted
arylethyl chains. The 3-hydroxy-4-methoxyphenylethyl derivatives
18e and 19e were potent in vitro tubulin polymerization inhibitors
but with weak cytotoxicities toward the human colon adenocarci-
noma HT29 cell line, in the micromolar range. This antitubulin
activity could be explained by modeling studies which have shown
that the aryethyl chain adopted a folded up conformation stabilized
by hydrogen bonds which were not recovered with the others
analogs. Determination of the physicochemical properties as solu-
bility, membrane permeability or log P is necessary to understand
the bad cellular activity compared to the potent in vitro tubulin
polymerization inhibition. The 4-methoxyphenylethyl counterparts
18a and 19a seemed to be inactive as inhibitors of tubulin poly-
merization and did not inhibit tubulin assembly at concentrations as
Fig. 2. (A) Effects of compounds 18a and 19a on the relaxation of plasmid DNA by
human topoisomerase I. Native supercoiled PUC18 (130 ng, lane DNA) was incubated
with 4 units topo I in the absence (lane Topo I) or presence of tested compounds at the
indicated concentration (10 or 20
compound. DNA samples were separated by electrophoresis on a 1% agarose gel
containing 1 g/mL ethidium bromide. (B) Effects of compounds 18a and 19a on the
mM). Camptothecin (CPT) was used as reference
m
relaxation of plasmid DNA by human topoisomerase II. Native supercoiled pUC19
(350 ng, lane DNA) was incubated with 8 units topo II in the absence (lane Topo II) or
presence of tested compounds at the indicated concentration 50
was used at 20 and 50 M. DNA samples were separated by electrophoresis on a 1%
agarose gel containing 1 g/mL ethidium bromide. Gels were photographed under UV
light. Nck, nicked; Sc, supercoiled; Lin: linear; Rel, relaxed.
mM. Etoposide (ETO)
m
m
high as 10
mM. Nevertheless, compound 18a displayed very inter-
esting antiproliferative activities over a panel of five tumor cell lines

V. Verones et al. / European Journal of Medicinal Chemistry 59 (2013) 39e47
43
Fig. 3. Docking studies into the colchicine binding site of tubulin of (a) compound 18e, (b) compound 19e, (c) compound 18a and (d) compound 19a.
in the submicromolar range for all of the histological types tested
and flow cytometric studies performed on selected tumor cells
showed an accumulation of the cells in the sub-G1 and G2 phases
indicating induction of cellular apoptosis. Further experiments are
therefore ongoing to better investigate the mechanism of action of
such compounds and confirm the improvement of pharmacokinetic
properties.
4. Experimental protocols
4.1. Chemistry and chemical methods
General methods: All reagents and solvents were purchased
and used without further purification Melting points were deter-
mined on a BÜCHI B-540 apparatus and are uncorrected. Mass
Table 3
Cell cycle analysis of selected tumor cell lines exposed to compounds 18a, 18c and 19a.
Phase of cell
cycle
Cell type
Cell line
Control cell
Vinoa 0.5
m
M
Vincb 0.5
m
M
Docec 0.5
mM
18a
0.5
18c
19a
0.5
m
M
5
m
M
5
m
M
m
M
5 mM
Sub G1 (%)
HNSCCd
Colon
Leukemia
HNSCC
Colon
Leukemia
HNSCC
Colon
Leukemia
HNSCC
Colon
CAL33
HT29
HL60
CAL33
HT29
HL60
CAL33
HT29
HL60
CAL33
HT29
HL60
0.92
0.81
6.84
68.89
78.2
56.81
16.01
8.67
21.94
14.21
12.2
24.44
1.39
N.t.
9.96
21.42
N.t.
17.62
3.41
N.t.
49.16
72.85
N.t.
20.5
5.97
N.t.
14.29
16.43
N.t.
17.13
4.65
N.t.
49.2
72.53
N.t.
17.11
0.86
N.t.
4.99
7.85
N.t.
15.41
2.76
N.t.
64.1
88.24
N.t.
9.84
0.65
37.16
4
14.55
25.15
28.46
2.75
22.73
0.83
34.34
8.35
21.11
24.4
33.12
4.6
1.43
1.58
8.46
N.t.e
1.63
8.22
N.t.
73.56
57.66
n.t.
8.63
21.83
n.t.
15.95
12.4
N.t.
0.47
34.12
N.t.
8.63
26.83
n.t.
2.46
24.77
n.t.
87.92
14.77
G1 (%)
64.33
74.22
58.11
16.17
10.72
21.55
21.55
13.43
11.87
S (%)
18.5
26.55
35.16
73.1
G2 (%)
57.42
81.81
19.58
Leukemia
14.54
15.2
a
Vino: vinorelbine.
Vinc: vincristine.
Doce: docetaxel.
Head and neck squamous cell carcinoma.
Not tested.
b
c
d
e

44
V. Verones et al. / European Journal of Medicinal Chemistry 59 (2013) 39e47
spectra were performed on a Finnigan MAT SSQ 710 Advantage
spectrometer and were recorded in the ESþ mode. 1H NMR
(300 MHz) and 13C NMR (75 MHz) spectra were recorded on
4.1.5. 10H-Phenothiazine-1-carboxylic acid 5,5-dioxide (9)
To a suspension of sulfoxide 8 (2.0 g, 7.7 mmol) in anhydrous
acetic acid (40 mL) was added a 35% H2O2 solution (2 mL,
13.6 mmol) and the mixture was stirred at 70 ꢁC for 3 h. After
cooling, the precipitate was filtered, washed with water, EtOH and
a Bruker AC 300 P (chemical shifts
d in parts per million, the
following abbreviations are used: singlet (s), broad singlet (br s),
doublet (d), doubled doublet (dd), triplet (t)). Elemental analyses
were performed by C.N.R.S-Vernaison, and were in agreement with
the calculated values within ꢀ0.2%.
dried to afford 1.6 g (67%) of pale pink solid; 1H NMR (DMSO)
d: 7.35
(m, 2H), 7.56 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 1.4 Hz), 7.70 (td, 1H,
3J ¼ 8.4 Hz, 4J ¼ 1.4 Hz), 7.98 (dd, 1H, 3J ¼ 7.95 Hz, 4J ¼ 1.4 Hz), 8.25
(dd, 1H, 3J ¼ 7.85 Hz, 4J ¼ 1.4 Hz), 8.35 (dd, 1H, 3J ¼ 7.8 Hz,
4J ¼ 1.4 Hz), 11.55 (br s, 1H); Anal. Calcd for C13H9NO4S: C, 56.72; H,
3.30; N, 5.09. Found: C, 56.83; H, 3.19; N, 5.14.
4.1.1. 2-[(2-Carboxyphenyl)amino]benzoic acid (4)
A mixture of 2-chlorobenzoic acid (10.0 g, 64 mmol), 2-
aminobenzoic acid (17.5 g, 130 mmol), potassium carbonate
(8.8 g, 64 mmol) and copper powder (30 mg) were refluxed for 2 h
in DMF (100 mL). After cooling, 6 N HCl aqueous solution (200 mL)
was added and the mixture was stirred for 12 h and filtered. The
resulting solid was washed with water and methanol, then dried to
afford 13.5 g (82%) of a gray solid: mp 317e319 ꢁC; 1H NMR (DMSO)
4.1.6. General procedure for synthesis of acyl chlorides 10 and 11
SOCl2 (2.35 mL, 30 mmol) was added dropwise to a suspension
of carboxylic acid 5 or 9 (6 mmol) in toluene (60 mL) and the
mixture was stirred at 80 ꢁC for 12 h. After cooling, the solution was
filtered and the filtrate evaporated under reduced pressure. The
residue was taken up into Et2O and the resulting precipitate
collected by filtration to give the corresponding acyl chlorides 10
and 11.
d: 6.94 (m, 2H), 7.40e7.55 (m, 4H), 7.90 (m, 2H), 10.80 (br s, 1H),
13.00 (br s, 2H).
4.1.2. 10H-Acridin-9-one-4-carboxylic acid (5)
A solution of diacid 4 (6.0 g, 23 mmol) in concentrated H2SO4
(50 mL) was stirred at 100 ꢁC for 4 h. The reaction mixture was
cooled, poured into ice-cold water and the resulting precipitate was
filtered and washed with water. The solid was dissolved in 2 N
aqueous NaOH solution (100 mL) and the solution was filtered. The
filtrate was then acidified with glacial acetic acid until precipita-
tion. The precipitated product was collected and washed with EtOH
to give 3.95 g (72%) of yellow solid; mp 338e340 ꢁC; 1H NMR
4.1.6.1. 10H-Acridin-9-one-4-carbonyl chloride (10). Yellow solid;
80% yield; mp 224e226 ꢁC; 1H NMR (DMSO)
d: 7.30e7.37 (m, 2H),
7.75e7.79 (m, 2H), 8.23 (dd, 1H, 3J ¼ 8.2 Hz, 4J ¼ 1.75 Hz), 8.43 (dd,
1H, 3J ¼ 7.6 Hz, 4J ¼ 1.75 Hz), 8.52 (dd, 1H, 3J ¼ 7.95 Hz, 4J ¼ 1.75 Hz),
11.95 (br s, 1H); Anal. Calcd for C14H8ClNO2: C, 65.26; H, 3.13; N,
5.44. Found: C, 65.38; H, 3.04; N, 5.50.
4.1.6.2. 10H-Phenothiazine-1-carbonyl chloride 5,5-dioxide (11).
(DMSO)
d
: 7.27e7.37 (m, 2H), 7.76 (m, 2H), 8.23 (d, 1H, 3J ¼ 8.0 Hz),
Red solid; 91% yield; mp 233e236 ꢁC; 1H NMR (DMSO)
d: 7.25 (dd,
8.44 (dd, 1H, 3J ¼ 7.6 Hz, 4J ¼ 1.75 Hz), 8.50 (dd, 1H, 3J ¼ 8.0 Hz,
1H, 3J ¼ 8.2 Hz, 4J ¼ 1.4 Hz), 7.37 (m, 2H), 7.65 (td, 1H, 3J ¼ 8.2 Hz,
4J ¼ 1.65 Hz), 8.10 (dd, 1H, 3J ¼ 8.15 Hz, 4J ¼ 1.4 Hz), 8.44 (dd, 1H,
3J ¼ 7.9 Hz, 4J ¼ 1.4 Hz), 8.62 (dd, 1H, 3J ¼ 8.0 Hz, 4J ¼ 1.65 Hz), 10.60
(br s, 1H); Anal. Calcd for C13H8ClNO3S: C, 53.16; H, 2.75; N, 4.77.
Found: C, 53.25; H, 2.68; N, 4.84.
4J ¼ 1.75 Hz), 11.1 (br s, 1H), 12.3 (br s, 1H).
4.1.3. 1,2-Dihydropyrrolo[3,2,1-kl]-phenothiazin-1,2-dione (7)
To a boiling solution of oxalyl chloride (5.1 g, 40 mmol) in THF
(20 mL) was added dropwise over a period of 1 h a solution of
phenothiazine (4.0 g, 20 mmol) in THF (30 mL) and the reaction
mixture was boiled under reflux an additional 4 h and then
concentrated under reduced pressure. The resulting green solid
was dissolved in CS2 (100 mL) and AlCl3 (5.3 g, 40 mmol) was
added portion wise over a period of 30 min. When addition was
complete, the reaction mixture was boiled under reflux an
additional 12 h. After cooling and decantation of the CS2 solution,
0.6 N HCl aqueous solution was added at 0 ꢁC to the residue and
the mixture was stirred until precipitation. The solid was filtered
and washed respectively with water, EtOH and Et2O to afford
4.8 g (95%) of purple powder; mp 199e200 ꢁC; 1H NMR (DMSO)
4.1.7. General procedure for synthesis of carboxaldehydes 12 and 13
A solution of acyl chloride 10 or 11 (5 mmol) and Pd(PPh3)4
(57.7 mg, 0.05 mmol) in deoxygenated benzene (30 mL) was stirred
under argon in a Schlenck tube. Bu3SnH (1.06 mL, 5.4 mmol) was
then added in three portions at intervals of 10 min. After comple-
tion of the reaction (monitored by TLC), the solvent was evaporated,
and petroleum ether was added to the residue until precipitation.
The precipitate was collected by filtration and washed with Et2O to
afford the corresponding carboxaldehydes 12 and 13.
4.1.7.1. 10H-Acridin-9-one-4-carboxaldehyde (12). Yellow solid; 98%
d
: 6.95 (dd, 1H, 3J ¼ 8.1 Hz, 4J ¼ 1.5 Hz), 7.0e7.20 (m, 4H), 7.33
yield; mp 215e217 ꢁC; 1H NMR (DMSO)
d: 7.35 (m, 1H), 7.50 (t, 1H,
(dd, 1H, 3J ¼ 7.3 Hz, 4J ¼ 1.3 Hz), 8.65 (dd, 1H, 3J ¼ 8.4 Hz,
4J ¼ 1.5 Hz).
3J ¼ 7.7 Hz), 7.76e7.82 (m, 2H), 7.90 (dd, 1H, 3J ¼ 7.8 Hz, 4J ¼ 1.4 Hz),
8.39 (dd, 1H, 3J ¼ 7.7 Hz, 4J ¼ 1.75 Hz), 8.58 (dd, 1H, 3J ¼ 8.5 Hz,
4J ¼ 1.75 Hz), 10.2 (s, 1H), 11.8 (br s, 1H); Anal. Calcd for C14H9NO2: C,
75.33; H, 4.06; N, 6.27. Found: C, 75.41; H, 3.98; N, 6.32.
4.1.4. 10H-Phenothiazine-1-carboxylic acid 5-oxide (8)
A 30% NaOH aqueous solution (30 mL) was added at 30 ꢁC to
a suspension of isatin 7 (2.2 g, 8.7 mmol) and the mixture was
stirred at room temperature for 1 h. A 35% H2O2 solution (5 mL,
34 mmol) in water (50 mL) was added and the reaction mixture was
stirred for 2 h at room temperature before acidification with
concentrated HCl until precipitation. The solid was collected by
filtration, washed with water, EtOH and dried to provide 2.1 g (93%)
4.1.7.2. 10H-phenothiazine-1-carboxaldehyde
5,5-dioxide
(13).
: 7.25e
Orange solid; 97% yield; mp 198e200 ꢁC; 1H NMR (DMSO)
d
7.40 (m, 3H), 7.63 (td, 1H, 3J ¼ 7.9 Hz, 4J ¼ 1.6 Hz), 7.99 (dd, 1H,
3J ¼ 7.8 Hz, 4J ¼ 1.4 Hz), 8.11 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 1.45 Hz), 8.37
(dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 1.45 Hz), 10.10 (s, 1H), 11.45 (br s, 1H); Anal.
Calcd for C13H9NO3S: C, 60.22; H, 3.50; N, 5.40. Found: C, 60.38; H,
3.39; N, 5.33.
of pale brown solid; mp 159e162 ꢁC; 1H NMR (DMSO)
d: 7.30 (m,
2H), 7.60 (dd, 1H, 3J ¼ 8.2 Hz, 4J ¼ 1.4 Hz), 7.67 (td, 1H, 3J ¼ 8.2 Hz,
4J ¼ 1.4 Hz), 8.00 (dd, 1H, 3J ¼ 7.85 Hz, 4J ¼ 1.4 Hz), 8.25 (dd, 1H,
3J ¼ 7.7 Hz, 4J ¼ 1.7 Hz), 8.32 (dd, 1H, 3J ¼ 7.8 Hz, 4J ¼ 1.7 Hz), 11.7 (br
s, 1H); Anal. Calcd for C13H9NO3S: C, 60.22; H, 3.50; N, 5.40. Found:
C, 60.35; H, 3.41; N, 5.47.
4.1.8. General procedure for synthesis of phosphonium salts 15aee
A solution of commercially available or described benzyl chlo-
rides 14aee (85 mmol) and PPh3 (26.2 g, 100 mmol) in toluene
(110 mL) was stirred at reflux for 24 h. After cooling, the precipitate

V. Verones et al. / European Journal of Medicinal Chemistry 59 (2013) 39e47
45
was collected by filtration, washed with Et2O and dried to give the
corresponding phosphonium salts 15aee.
4.1.9.3. 10H-4-[2-(3,4,5-Trimethoxyphenyl)ethyl]acridin-9-one (18c).
White solid; 53% yield; mp 198e201 ꢁC; 1H NMR (DMSO)
d: 2.98 (t,
2H, 3J ¼ 6.9 Hz), 3.31 (t, 2H, 3J ¼ 7.3 Hz), 3.52 (s, 3H), 3.72 (s, 6H),
6.53 (s, 2H), 7.23 (m, 2H), 7.57 (d, 1H, 3J ¼ 8.1 Hz), 7.69 (t, 1H,
3J ¼ 7.9 Hz), 7.88 (d, 1H, 3J ¼ 8.1 Hz), 8.15 (dd, 1H, 3J ¼ 8.1 Hz,
4.1.8.1. 4-Methoxybenzyl-triphenylphosphonium chloride (15a).
White solid; 79% yield; mp 243e245 ꢁC; 1H NMR (DMSO)
d: 3.69 (s,
3H), 5.11 (m, 2H), 6.80 (m, 2H), 6.90 (m, 2H), 7.60 (m, 6H), 7.75 (m,
6H), 7.95 (m, 3H).
4J ¼ 1.3 Hz), 8.20 (dd,1H, 3J ¼ 8.0 Hz, 4J ¼ 1.4 Hz),10.50 (br s,1H); 13
C
NMR (DMSO) d: 31.8, 35.8, 56.2, 60.3,106.4,118.6, 120.6, 121.2, 121.6,
124.6,126.1,129.3,133.4,133.6,136.1,137.2,139.4,141.5,153.0,177.5;
Anal. Calcd for C24H23NO4: C, 74.02; H, 5.95; N, 3.60. Found: C,
74.18; H, 5.85; N, 3.49.
4.1.8.2. (3,4-Dimethoxybenzyl)triphenylphosphonium chloride (15b).
White solid; 70% yield; mp 224e226 ꢁC; 1H NMR (DMSO)
d: 3.55 (s,
3H), 3.81 (s, 3H), 5.08 (s, 2H), 6.62e6.70 (m, 3H), 7.66e7.73 (m,
15H).
4.1.9.4. 10H-4-[2-(3,4-Dihydroxyphenyl)ethyl]acridin-9-one (18d).
Off-white solid; 60% yield; mp 206e208 ꢁC; 1H NMR (DMSO)
d: 2.92
4.1.8.3. (3,4,5-Trimethoxybenzyl)triphenylphosphonium
chloride
(t, 2H, 3J ¼ 7.3 Hz), 3.27 (t, 2H, 3J ¼ 7.3 Hz), 6.62 (d, 1H, 3J ¼ 8.2 Hz),
6.80 (m, 2H), 7.19 (t,1H, 3J ¼ 7.5 Hz), 7.25 (t,1H, 3J ¼ 7.5 Hz), 7.57 (dd,
1H, 3J ¼ 8.1 Hz, 4J ¼ 2.1 Hz), 7.73 (td, 1H, 3J ¼ 7.5 Hz, 4J ¼ 1.7 Hz), 7.90
(d,1H, 3J ¼ 8.1 Hz), 8.14 (dd,1H, 3J ¼ 8.1 Hz, 4J ¼ 1.7 Hz), 8.20 (dd,1H,
3J ¼ 8.1 Hz, 4J ¼ 1.7 Hz), 8.85 (s, 2H), 10.55 (br s, 1H); Anal. Calcd for
C21H17NO3: C, 76.12; H, 5.17; N, 4.23. Found: C, 76.05; H, 5.10;
N, 4.32.
(15c). White solid; 70% yield; mp 222e223 ꢁC; 1H NMR (DMSO)
3.37 (s, 9H), 4.97 (s, 2H), 6.18 (m, 2H), 7.68e7.73 (m, 15H).
d:
4.1.8.4. (3,4-Dibenzyloxybenzyl)triphenylphosphonium
chloride
(15d). White solid; 80% yield; mp 238e240 ꢁC; 1H NMR (DMSO)
d:
4.54 (s, 2H), 4.56 (s, 2H), 5.01 (s, 2H), 6.50 (d, 1H, 4J ¼ 2.1 Hz), 6.83
(d, 1H, 3J ¼ 8.2 Hz), 7.25 (dd, 1H, 3J ¼ 8.2 Hz, 4J ¼ 2.1 Hz), 7.32e7.43
(m, 10H), 7.75e7.80 (m, 15H).
4.1.9.5. 10H-4-[2-(3-Hydroxy-4-methoxyphenyl)ethyl]acridin-9-one
(18e). Off-white solid; 48% yield; mp 212e214 ꢁC; 1H NMR (DMSO)
4.1.8.5. (3-Benzyloxy-4-methoxybenzyl)triphenylphosphonium chlo-
d
: 2.90 (t, 2H, 3J ¼ 7.1 Hz), 3.25 (t, 2H, 3J ¼ 7.1 Hz), 3.72 (s, 3H), 6.66
ride (15e). White solid; 74% yield; mp 237e239 ꢁC; 1H NMR (DMSO)
(dd, 1H, 3J ¼ 8.2 Hz, 4J ¼ 1.9 Hz), 6.75 (d, 1H, 3J ¼ 1.9 Hz), 6.82 (d, 1H,,
3J ¼ 8.2 Hz), 7.18 (t, 1H, 3J ¼ 7.9 Hz), 7.26 (t, 1H, 3J ¼ 7.9 Hz), 7.54 (dd,
1H, 3J ¼ 7.3 Hz, 4J ¼ 1.5 Hz), 7.71 (td, 1H, 3J ¼ 7.25 Hz, 4J ¼ 1.5 Hz),
7.92 (dd, 1H, 3J ¼ 8.3 Hz, 4J ¼ 1.55 Hz), 8.13 (dd, 1H, 3J ¼ 7.9 Hz,
4J ¼ 1.55 Hz), 8.21 (dd, 1H, 3J ¼ 8.15 Hz, 4J ¼ 1.55 Hz), 8.80 (s, 1H),
d
: 3.71 (s, 3H), 4.56 (s, 2H), 5.04 (s, 2H), 6.50 (d,1H, 4J ¼ 2.05 Hz), 6.60
(m, 2H), 6.85 (d, 1H, 3J ¼ 8.2 Hz), 7.25 (dd, 1H, 3J ¼ 8.2 Hz,
4J ¼ 2.05 Hz), 7.30e7.40 (m, 3H), 7.75e7.80 (m, 12 H), 7.90 (m, 3H).
4.1.9. General procedure for synthesis of target compounds 18aee
and 19aee
10.60 (br s, 1H); 13C NMR (DMSO)
d: 32.0, 34.4, 56.2, 112.6, 116.5,
118.6, 119.5, 120.6, 121.2, 121.3, 121.7, 124.5, 126.1, 129.3, 133.3, 133.6,
134.3, 139.4, 141.5, 146.4, 146.7, 177.5; Anal. Calcd for C22H19NO3: C,
76.50; H, 5.54; N, 4.06. Found: C, 76.42; H, 5.63; N, 4.17.
A mixture of carboxaldehyde 12 or 13 (2 mmol), phosphonium
chloride 15aee (2.5 mmol) and DBU (0.65 mL, 4.35 mmol) in
acetonitrile was stirred at reflux for 3 h. The reaction mixture was
hydrolyzed and extracted twice with ethyl acetate. The combined
organic extracts were dried over MgSO4, evaporated under reduced
pressure and the resulting residue was purified via flash column
chromatography with CH2Cl2 as eluent to afford a Z/E-isomeric
mixture of the corresponding olefins 16aee and 17aee. A solution
of the previously prepared olefins (0.55 mmol) in EtOH (100 mL)
was stirred in the presence of 10% palladium on charcoal under an
atmosphere of hydrogen at room temperature until reaction was
completed as judged by TLC (12e24 h). The solution was filtered
through Celite, the filtrate was evaporated under reduced pressure
and the resulting residue was recrystallized from EtOH to provide
the final compounds 18aee and 19aee.
4.1.9.6. 10H-1-[2-(4-Methoxyphenyl)ethyl]phenothiazine 5,5-dioxide
(19a). Gray solid; 51% yield; mp 188e190 ꢁC; 1H NMR (CDCl3)
d:
2.91 (m, 4H), 3.70 (s, 3H), 6.72 (dd, 1H, 3J ¼ 8.4 Hz, 4J ¼ 1.6 Hz), 6.78
(d, 2H, 3J ¼ 8.1 Hz), 6.96 (s, 1H), 7.03 (d, 2H, 3J ¼ 8.1 Hz), 7.17 (m, 2H),
7.37 (m, 2H), 7.98 (m, 2H); 13C NMR (CDCl3)
d: 32.7, 34.9, 55.2, 114.1,
117.0, 120.7, 121.4, 121.6, 121.8, 122.8, 127.7, 129.3, 132.4, 132.5, 133.3,
135.7, 137.5, 158.2; Anal. Calcd for C21H19NO3S: C, 69.02; H, 5.24; N,
3.83. Found: C, 69.13; H, 5.41; N, 3.91.
4.1.9.7. 10H-1-[2-(3,4-Dimethoxyphenyl)ethyl]phenothiazine 5,5-
dioxide (19b). White solid; 59% yield; mp 186e188 ꢁC; 1H NMR
(CDCl3)
6.95 (s, 1H), 7.17 (m, 2H), 7.37 (m, 2H), 7.99 (m, 2H); 13C NMR
(CDCl3) : 32.4, 35.5, 55.7, 55.9, 111.1, 111.5, 116.9, 120.2, 120.4, 121.2,
d: 2.92 (m, 4H), 3.77 (s, 3H), 3.80 (s, 3H), 6.59e6.73 (m, 4H),
4.1.9.1. 10H-4-[2-(4-Methoxyphenyl)ethyl]acridin-9-one
(18a).
: 3.03 (t,
d
Yellow solid; 56% yield; mp 251e253 ꢁC; 1H NMR (CDCl3)
d
121.3, 121.5, 121.7, 122.5, 127.9, 132.4, 132.9, 133.4, 135.8, 137.5, 147.5,
149.0; Anal. Calcd for C22H21NO4S: C, 66.82; H, 5.35; N, 3.54. Found:
C, 69.93; H, 5.23; N, 3.62.
2H, 3J ¼ 7.3 Hz), 3.17 (t, 2H, 3J ¼ 7.3 Hz), 3.70 (s, 3H), 6.81 (d, 2H,
3J ¼ 8.2 Hz), 7.08 (m, 3H), 7.23 (td, 1H, 3J ¼ 7.9 Hz, 4J ¼ 2.1 Hz), 7.50e
7.63 (m, 3H), 8.42 (m, 2H); 13C NMR (CDCl3)
d: 33.3, 35.1, 55.2, 114.2,
116.7,120.9,121.3,121.6,125.6,127.0,127.2,129.4,133.0,133.2,133.5,
138.7, 140.1, 158.3, 178.6; Anal. Calcd for C22H19NO2: C, 80.22; H,
5.81; N, 4.25. Found: C, 80.36; H, 5.74; N, 4.17.
4.1.9.8. 10H-1-[2-(3,4,5-Trimethoxyphenyl)ethyl]phenothiazine 5,5-
dioxide (19c). White solid; 59% yield; mp 218e220 ꢁC; 1H NMR
(CDCl3) d: 2.94 (m, 4H), 3.70 (s, 3H), 3.77 (6H), 6.32 (s, 2H), 6.71 (d,
1H, 3J ¼ 8.2 Hz), 6.99 (s, 1H), 7.18 (m, 2H), 7.39 (m, 2H), 7.99 (m, 2H);
4.1.9.2. 10H-4-[2-(3,4-Dimethoxyphenyl)ethyl]acridin-9-one (18b).
13C NMR (CDCl3)
d: 32.4, 36.3, 56.1, 60.7, 77.2, 105.2, 116.7, 120.7,
Off-white solid; 60% yield; mp 206e208 ꢁC; 1H NMR (CDCl3)
d
: 3.05
121.4, 121.5, 121.9, 122.7, 127.7, 132.7, 133.3, 135.8, 136.1, 136.5, 137.4,
153.3; Anal. Calcd for C23H23NO5S: C, 64.92; H, 5.45; N, 3.29. Found:
C, 70.07; H, 5.35; N, 3.34.
(t, 2H, 3J ¼ 7.7 Hz), 3.21 (t, 2H, 3J ¼ 7.7 Hz), 3.72 (s, 3H), 3.77 (s, 3H),
6.70e6.90 (m, 3H), 7.10 (d, 1H, 3J ¼ 7.9 Hz), 7.25 (td, 1H, 3J ¼ 7.8 Hz,
4J ¼ 1.9 Hz), 7.52e7.68 (m, 3H), 8.44 (m, 2H); 13C NMR (CDCl3)
d:
32.8, 36.5, 55.8, 55.9, 105.3, 116.7, 120.8, 121.3, 121.4, 121.7, 125.6,
127.0, 127.3, 133.4, 136.5, 136.7, 138.9, 140.2, 153.3, 178.4; Anal. Calcd
for C23H21NO3: C, 76.86; H, 5.89; N, 3.90. Found: C, 76.98; H, 5.80;
N, 4.01.
4.1.9.9. 10H-1-[2-(3,4-Dihydroxyphenyl)ethyl]phenothiazine 5,5-
dioxide (19d). Off-white solid; 57% yield; mp 246e248 ꢁC; 1H
NMR (DMSO)
d
: 2.78 (t, 2H, 3J ¼ 7.3 Hz), 3.06 (t, 2H, 3J ¼ 7.3 Hz), 6.47
(d, 1H, 3J ¼ 7.8 Hz), 6.65 (m, 2H), 7.06 (t, 1H, 3J ¼ 7.7 Hz), 7.12 (t, 1H,

46
V. Verones et al. / European Journal of Medicinal Chemistry 59 (2013) 39e47
3J ¼ 7.7 Hz), 7.31 (d, 1H, 3J ¼ 7.2 Hz), 7.44 (t, 1H, 3J ¼ 7.8 Hz), 7.54 (d,
1H, 3J ¼ 8.3 Hz), 7.76 (d,1H, 3J ¼ 7.8 Hz), 7.84 (d,1H, 3J ¼ 7.8 Hz), 8.06
varying concentrations of the drug under study. Reactions were
terminated by adding SDS to 0.25% and proteinase K to 250 g/mL.
DNA samples were then added to the electrophoresis dye mixture
(3 mL) and electrophoresed in a 1% agarose gel containing ethidium
bromide (1 mg/mL), at room temperature for 2 h at 120 V. Gels
were washed and photographed under UV light.
m
(br s, 2H), 9.18 (s, 1H); 13C NMR (DMSO)
d
: 32.1, 34.4, 115.8, 116.5,
118.7, 119.6, 120.7, 121.1, 121.6, 122.2, 122.5, 129.4, 132.2, 133.4, 133.5,
136.5, 138.9, 143.8, 145.4; Anal. Calcd for C20H17NO4S: C, 65.38; H,
4.66; N, 3.81. Found: C, 65.47; H, 4.57; N, 3.91.
4.1.9.10. 10H-1-[2-(3-Hydroxy-4-methoxyphenyl)ethyl]phenothia-
4.2.4. Cell cycle analysis
zine 5,5-dioxide (19e). White solid; 51% yield; mp 199e201 ꢁC; 1H
Cells (2.5e4 ꢂ 105) were seeded into a 6-well plate for 24 h
incubation and then treated with compounds at different concen-
trations for 24 h. Supernatants and cells were collected and fixed
with cold 70% ethanol for 24 h. The cells were then stained with
NMR (CDCl3) d: 2.91 (m, 4H), 3.74 (s, 3H), 5.64 (br s, 1H), 6.43 (dd,
1H, 3J ¼ 8.2 Hz, 4J ¼ 2.1 Hz), 6.62 (d, 1H, 3J ¼ 8.2 Hz), 6.77 (d, 1H,
3J ¼ 8.3 Hz), 6.84 (d, 1H, 4J ¼ 2.1 Hz), 6.94 (s, 1H), 7.17 (m, 2H), 7.38
(m, 2H), 7.98 (m, 2H); 13C NMR (CDCl3)
d: 33.0, 35.5, 55.9, 110.8,
propidium iodide (50
mg/mL, Interchim, France) and RNase solution
114.0, 116.8, 120.1, 121.2, 121.6, 121.7, 121.8, 122.0, 123.1, 127.4, 132.5,
133.4,133.6,136.0,137.6,145.3,146.0; Anal. Calcd for C21H19NO4S: C,
66.12; H, 5.02; N, 3.67. Found: C, 66.23; H, 4.94; N, 3.76.
(100 g/mL, Sigma Aldrich) in PBS for 30 min in the dark at room
m
temperature. Cell cycle distribution was determined on a Bectone
Dickinson FACScan flow cytometer using the Cell QuestPro soft-
ware and analyzed by WinMDI software.
4.2. Biological assay methods
4.3. Molecular modeling
4.2.1. Cell growth inhibitory activity
Cytotoxicity of selected drugs was tested using the MTT (thia-
zolyl blue tetrazolium bromide) assay based on the cellular uptake
of MTT (Sigma Aldrich) and its subsequent reduction in the mito-
chondria of living cells to dark blue MTT formazan crystals (Mos-
mann, 1983). Cells were seeded on 96-well plates ((1.6e
1.8) ꢂ 104 cells/well) in the corresponding medium supple-
mented with 10% FBS. Seventy-two hours later, cells were deprived
of FBS. The next day, they were treated with various concentrations
of each tested compound. Following the 72 h exposure to the
cytotoxic drugs, cell survival was determined by incubation with
MTT (4 mg/mL) for 4 h (37 ꢁC, 5% CO2). The cells were then lyzed in
SDS/HCl and plates left in the incubator to dissolve the purple
formazan crystals. The color intensity reflecting cell viability was
read with a spectrophotometer Elx 800 (Biotek Instruments Inc.) at
570 nm.
All the calculations were realized under the Sybyl 6.9.2.
molecular modeling package [33]. The tubulin structure was
downloaded from the PDB data bank (http://www.rcsb.org/-PDB
code: 1SA0) [34]. The studied compounds were built from the
internal fragments library of Sybyl then their geometry was opti-
mized by the Powell method available in the Maximin2 procedure
ꢀ
to a gradient of 0.001 kcal/mol A using the Tripos force field [35]. In
order to implicitly represent a biological middle, the dielectric
constant was set to 4. Atomic charges were attributed following the
GasteigereHückel method. They were subsequently docked into
the enzyme with GOLD 5.0.1 [36]. The best of the 30 conformations
generated was determined by an in house consensus scoring
function base on Goldscore [36] and X-Score [37] and visual
inspection of the consistency of the chosen solution.
Appendix A. Supplementary data
4.2.2. In vitro microtubule polymerization assay
In-vitro tubulin polymerization assays were performed on
a fluorescence spectrometer (Varioskan flash, Thermo Scientific,
Courtaboeuf, France), equipped with a half area 96 wells microplate
(Corning Costar). Excitation and emission wavelengths were 360
and 420 nm, respectively. All reagents were purchased from Cyto-
skeleton (Denver, CO, USA). For each inhibitor investigated, a series
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.10.051.
References
[1] A.L. Risinger, F.J. Giles, S.L. Mooberry, Cancer Treat. Rev. 35 (2009) 255e261.
[2] S. Honore, E. Pasquier, D. Braguer, Cell. Mol. Life Sci. 62 (2005) 3039e3056.
[3] P.B. Schiff, J. Fant, S.B. Horwitz, Nature 277 (1979) 665e667.
[4] M.A. Jordan, J.A. Hadfield, N.J. Lawrence, A.T. McGown, Med. Res. Rev. 18
(1998) 259e296.
[5] Q. Li, H. Sham, S. Rosenberg, Annu. Rep. Med. Chem. 34 (1999) 139e148.
[6] N.H. Nam, Curr. Med. Chem. 10 (2003) 1697e1722.
[7] K. Yoshimatsu, A. Yamaguchi, H. Yoshino, N. Koyanagi, K. Kitoh, Cancer Res. 57
(1997) 3208e3213.
[8] B.R. Hearn, S.J. Shaw, D.C. Myles, D.C. Compr, Med. Chem. 7 (2007) 81e110.
[9] Q. Li, H.L. Sham, Expert Opin. Ther. Pat. 12 (2002) 1663e1702.
[10] J.A. Hadfield, S. Ducki, N. Hirst, A.T. McGown, Prog. Cell. Cycle Res. 5 (2003)
309e325.
[11] S. Gallet, N. Lebegue, N. Flouquet, P. Berthelot, A. Pierre, P. Renard, B. Pfeiffer,
Int Patent PCT FR 04/00235, 2004.
[12] S. Gallet, N. Flouquet, P. Carato, B. Pfeiffer, P. Renard, S. Léonce, A. Pierré,
P. Berthelot, N. Lebegue, Bioorg. Med. Chem. 17 (2009) 1132e1138.
[13] M. Lecoeur, V. Vérones, C. Vaccher, J.P. Bonte, N. Lebegue, J.F. Goossens, Eur. J.
Pharm. Sci. 45 (2012) 559e569.
[14] A. Fernández-Calienes, R. Pellón, M. Docampo, M. Fascio, N. D’Accorso, L. Maes,
J. Mendiola, L. Monzote, L. Gille, L. Rojas, Biomed. Pharmacother. 65 (2011)
210e214.
of ten concentrations (from 0.1 to 10 mM) was prepared in 100 mg
purified bovine tubulin and buffer containing 20% glycerol, 1 mM
GTP, 80 mM PIPES (pH 6.9), 2.0 mM MgCl2, 0.5 mM EGTA and 5 nM
fluorescent reporter and kept at 0 ꢁC. Then, the medium was
incubated in the plate reader, at 37 ꢁC, for 1 h. A mixture without
inhibitor exhibited an increase in fluorescent intensity as a function
of incubation time corresponding to the inclusion of the probe
inside the tubulin polymer. On the contrary, compounds which
strongly inhibited microtubule formation were identified by
a fluorescent signal remaining negligible along the experiment. The
effect of inhibitor concentration on tubulin polymerization rate was
studied and the IC50 value was defined as the concentration of
product which inhibits the rate of polymerization by a factor 50. It
was determined from three independent assays using GraphPad
Prism software (version 4).
4.2.3. Topoisomerase inhibition assay
[15] P. Belmont, I. Dorange, Exp. Opin. Ther. Pat. 18 (2008) 1211e1224.
[16] K. Moriya, J. Rivera, S. Odom, Y. Sakuma, K. Muramato, T. Yoshiuchi,
M. Miyamoto, K. Yamada, Proc. Natl. Acad. Sci.U. S. A. 94 (1997) 12539e12544.
[17] H.M. Eggenweiler, R. Jonas, M. Wolf, M. Gassen, T. Welge, Deutch Patent DE
54707, 1999.
The assays were performed as previously described [31,32].
Supercoiled puC19 DNA (0.5
mg) was incubated with 4 units of
human topoisomerase I or II (TopoGen Inc.) at 37 ꢁC for 30 min in
relaxation buffer (50 mM Tris pH 7.8, 50 mM KCl, 10 mM MgCl2,
1 mM dithiothreitol, 1 mM EDTA and ATP) in the presence of
[18] S. Keil, E. Defossa, K. Schoenafinger, D. Schmoll, A. Deitrich, J. Kuhlmann-
Gottke, K.C. Engel, Deutch Patent DE 5045, 2007.

V. Verones et al. / European Journal of Medicinal Chemistry 59 (2013) 39e47
47
[19] H. Prinz, C. Behfar, K. Vogel, K.J. Böhm, B. Aicher, M. Gerlach, E.G. Günther,
P. Amon, I. Ivanov, K. Müller, J. Med. Chem. 54 (2011) 4247e4263.
[20] J.A.Lowe,R.L.Archer,D.S.Chapin,J.B.Cheng,D.Helweg,J.L.Johnson,B.K.Koe,L.A.Lebel,
P.F. Moore, J.A. Nielsen, L.L. RUSSO, J.T. Shirley, J. Med. Chem. 34 (1991) 624e628.
[21] A. Kamal, O. Srinivas, P. Ramulu, G. Ramesh, P. Praveen Kumar, Bioorg. Med.
Chem. Lett. 14 (2004) 4107e4111.
[22] V. Boekelheide, R.A. Hollins, J. Org. Chem. 36 (1971) 2437e2440.
[23] G.A. Silva, L.M.M. Costa, F.C.F. Brito, A.L.P. Miranda, E.J. Barreiroa, C.A.M. Fraga,
Bioorg. Med. Chem. 12 (2004) 3149e3158.
[29] K. Gaukroger, J.A. Hadfield, N.J. Lawrence, S. Nolan, A.T. McGown, Org. Biomol.
Chem. 1 (2003) 3033e3037.
[30] (a) M. Koba, T. Baczek, Med. Chem. Res. 20 (2011) 1385e1393;
(b) C. Gao, F. Liu, X. Luan, C. Tan, H. Liu, Y. Xie, Y. Jin, Y. Jiang, Bioorg. Med.
Chem. 18 (2010) 7507e7514;
(c) K. Pluta, B. Morak-M1odawska, M. Jelen, Eur. J. Med. Chem. 46 (2011)
3179e3189.
[31] L. Dassonneville, K. Bonjean, M.C. De Pauw-Gillet, P. Colson, C. Houssier,
J. Quetin-Leclercq, L. Angenot, C. Bailly, Biochemistry 38 (1999) 7719e7726.
[32] L. Dassonneville, N. Wattez, B. Baldeyrou, C. Mahieu, A. Lansiaux, B. Banaigs,
I. Bonnard, C. Bailly, Biochem. Pharmacol. 60 (2000) 527e537.
[33] Sybyl 6.9.2, Tripos Inc., 1699South Hanley Road, St. Louis, MO 63144e2913, U.S.A.
[34] R.B.G. Ravelli1, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel,
M. Knossow, Nature 428 (2004) 198e202.
[35] M. Clark, R.D. Cramer III, N. van Opdenbosch, J. Comput. Chem. 10 (1989)
982e1012.
[36] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, J. Mol. Biol. 267 (1997)
727e748.
[24] P. Four, F. Guibe, J. Org. Chem. 46 (1981) 4439e4445.
[25] V. Lisowski, C. Enguehard, J.C. Lancelot, D.H. Caignard, S.Lambel, S. Leonce,A.Pierre,
G. Atassi, P. Renard, S. Rault, Bioorg. Med. Chem. Lett. 11 (2001) 2205e2208.
[26] T.L. Nguyen, C. McGrath, A.R. Hermone, J.C. Burnett, D.W. Zaharevitz,
B.W. Day, P. Wipf, E. Hamel, R. Gussio, J. Med. Chem. 48 (2005) 6107e6116.
[27] N. Lebegue, S. Gallet, N. Flouquet, P. Carato, B. Pfeiffer, P. Renard, S. Léonce,
A. Pierré, P. Chavatte, P. Berthelot, J. Med. Chem. 48 (2005) 7363e7373.
[28] D. Alloatti, G. Giannini, W. Cabri, I. Lustrati, M. Marzi, A. Ciacci, G. Gallo,
M.O. Tinti, M. Marcellini, T. Riccioni, M.B. Guglielmi, P. Carminati, C. Pisano,
J. Med. Chem. 51 (2008) 2708e2721.
[37] R. Wang, L. Lai, S.J. Wang, J. Comput. Aid. Mol. Des. 16 (2002) 11e26.