Design, synthesis and antiproliferative activity of novel aminosubstituted benzothiopyranoisoindoles

Article abstract of DOI:10.1016/j.bmcl.2011.03.021

The synthesis of a number of new benzothiopyrano[4,3,2-cd]isoindole aminoderivatives designed as structural analogues of the key metabolite of the anticancer agent Ledacrine (nitracrine) and their in vitro cytotoxic activity evaluation against HCT-116, ME

Full text of DOI:10.1016/j.bmcl.2011.03.021

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3110–3112
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and antiproliferative activity of novel aminosubstituted
benzothiopyranoisoindoles
Antonios Christodoulou a, Ioannis K. Kostakis a, Vassilios Kourafalos a, Nicole Pouli a, Panagiotis Marakos a,
,
⇑
Ioannis P. Trougakos b, Ourania E. Tsitsilonis c
a Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
b Department of Cell Biology & Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens 15784, Greece
c Department of Animal & Human Physiology, Faculty of Biology, University of Athens, Panepistimiopolis, Athens 15784, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a number of new benzothiopyrano[4,3,2-cd]isoindole aminoderivatives designed as
structural analogues of the key metabolite of the anticancer agent Ledacrine (nitracrine) and their
in vitro cytotoxic activity evaluation against HCT-116, MES-SA, and MES-SA/Dx cancer cell lines is
Received 19 January 2011
Revised 4 March 2011
Accepted 5 March 2011
Available online 11 March 2011
reported. The majority of the derivatives possessed noticeable cytotoxicity in a low
an interesting structure–activity relationship.
lM range indicating
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Thioxanthone analogues
Fuzed isoindoles
Nitracrine
Cytotoxic activity
Multi-drug resistance
The supply of blood to large solid tumors is disorganizedand hyp-
oxic cells exist in the non-vasculated internal regions of such tu-
mors. These cells are resistant to ionizing radiation, relatively
inaccessible to chemotherapeutic drugs and furthermore, are refrac-
tory to treatment by many of these agents. Therefore, the need for
effective therapeutic strategies designed to eliminate hypoxic can-
cer cells is widely recognized.1 Among the studied compounds, are
certain nitroaromatics or heterocyclic nitroderivatives which can
be reduced in hypoxic tissues to produce active intermediate spe-
cies. These are relatively stable in the absence of oxygen and selec-
tively cytotoxic to the hypoxic cancer cells. The 1-nitroacridine
derivatives are potent DNA binding agents presenting significant
antiproliferative activity against hypoxic solid tumors2,3 and were
developed as anticancer agents that may not affect the bone mar-
row.1 1-Nitro-9-[30-(dimethylamino)propylamino]acridine (nitra-
crine, I, Fig. 1) has been used clinically since the early 1980s in
Poland for the treatment of ovarian, lung, colon and breast cancers.4
Although further development of this drug was discontinued due to
severe side effects, a second generation of 1-nitroacridine deriva-
tives has been synthesized. These modified compounds retain the
anticancer activity of the parent drug and enhance its therapeutic
efficacy.5–7 Since the metabolism of nitracrine is too rapid to allow
efficient distribution of the active metabolites through hypoxic
tumor areas and in order to decrease the ability of the 1-nitro group
to undergo metabolic reduction, the introduction of an electron-
donating methyl group at C4 of the acridine core was effected.8 In-
deed, C1748 (Fig. 1), is a new clinical candidate, as it induces apop-
tosis, necrosis or senescence in human cancer cell lines, and
possesses high antitumor activity in nude mice while demonstrating
low mutagenicity9 and low systemic toxicity.10 Like nitracrine,
C1748 binds covalently to DNA upon activation by cellular enzymes
and induces DNA crosslinking leading to cell cycle perturbation.11
While the bioreduction pathway of both drugs is generally un-
known, the study of nitracrine metabolites revealed a number of
HO
(CH3)2N
NH NO2
NH NO2
N
N
CH3
Nitracrine (I)
(CH3)2N
C1748
N
N
N
H
Nitracrine metabolite (II)
⇑
Corresponding author. Tel.: +30 210 7274830; fax: +30 210 7274747.
E-mail address: marakos@pharm.uoa.gr (P. Marakos).
Figure 1. Structure of nitracrine and bioactive analogues.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.021

A. Christodoulou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3110–3112
3111
active species, including dihydropyrazoloacridine II (Fig. 1), which
Et2N
Et2N
results from the intramolecular cyclization of the initially gener-
ated 1-aminoderivative of nitracrine. This key metabolite under-
goes further transformations in the presence of electrophilic
carbon atoms, resulting in the formation of a six-membered ring
attached to positions 1- and 9- of the acridine core.12
N
N
S
H
N
NO2
NEt2
a
S
O
O
O
O
Pursuant to our study of new aminosubstituted xanthenon-
es13,14 and related fused xanthones15,16 as cytotoxic agents, we
have reported on the synthesis of some benzopyrano[4,3,2-cd]iso-
indoles with a remarkable profile of cytotoxicity against cancer
cells.17 With this in mind we decided to investigate activity within
the closely related benzothiopyranoisoindoles, in order to better
understand structure–activity relationships around this scaffold.
For the synthesis of the target derivatives we have used com-
mercial thiosalicylic acid (1, Scheme 1) which was converted to
the corresponding benzoic acid 2 upon treatment of potassium
thiosalicylate with 5-chloro-2-nitrotoluene. Compound 2 was then
ring-closed with the use of Eaton’s reagent, providing the isomeric
thioxanthenones 3 and 4 (in a 3:1 ratio), which were separated by
column chromatography and spectroscopically identified. Bromide
5 was prepared by the reaction of 3 with NBS in the presence of
benzoyl peroxide and was then used for the reaction with suitable
ethanediamines to provide in one step and in high yield the isoin-
dole derivatives 6a–d. As reported,18 the presence of the 2-nitro
group in compound 5 is necessary for the effective ring closure
of the intermediate diamines, which result from the nucleophilic
substitution of the bromine atom by the dialkylaminoethylamines,
giving rise, in turn, to spontaneous cyclization, also evident from
the observed characteristic violet color of the reaction solution.
In order to synthesize of the target S,S-dioxides, we initially oxi-
dized the nitro derivative 3, but our attempts to achieve the subse-
quent bromination of the resulting dioxide were not successful.
Consequently, we oxidized the bromide 5 using chromium trioxide
to obtain the rather unstable compound 7, which was then con-
verted into compounds 8a–d upon reaction with the suitable
diamines.
8b
9
Scheme 2. Reagents and conditions: (a) N,N-diethylethylenediamine, EtOH, reflux,
24 h, 67%.
group. This reaction was effective only in the case of dioxides 8,
probably due to the electron withdrawing character of the sulfonyl
group of these derivatives. On the basis of this fact, we treated 8b
with N,N-diethylethylenediamine and prepared amine 9 in high
yield (Scheme 2).
As previously reported,18 the corresponding benzopyrano[4,3,2-
cd]isoindoles possess a highly acidic 2-aromatic proton and in the
corresponding 1H NMR spectra (recorded in 1/9 solution of CD3OD/
CDCl3), a H–D exchange occurred readily under mild conditions.
This phenomenon was also observed in the series of compounds
6 and 8, although the exchange was very slow in the case of 6;
on the contrary, it was extremely rapid for compounds 8, presum-
ably facilitated by the strong inductive effect of the sulfonyl group.
The in vitro cytotoxic activity of the new compounds was eval-
uated using the MTT dye reduction assay20 in three human solid
tumor cell lines, namely the colorectal adenocarcinoma HCT-116,
the uterine sarcoma MES-SA as well as its variant MES-SA/Dx5, re-
ported to be 100 fold resistant to doxorubicin.21 The 50% inhibitory
concentrations (IC50) of the compounds on each cell line, including
as reference compounds doxorubicin (Dx), and mitoxantrone (Mx),
are presented in Table 1.
As observed, the target aminoderivatives 6a–d and 8a–d exhib-
ited remarkable cytotoxicity against the tested cell lines HCT-116
and MES-SA and for the majority of them (6a–c, 8a–d), the IC50 val-
In order to verify our previous results, concerning the signifi-
cance of the nitro group for biologic activity, we also considered
preparing an analogue where this group would have been replaced
by a second aminosubstituted side chain. The corresponding ana-
logues of the benzopyranoisoindole series showed considerably re-
ues varied typically within the range of 1.15–7.04 lM. It is of inter-
est that the benzothiopyranoisoindoles 6 are 2–3 times more
active than the corresponding dioxides 8, with the exception of
the piperidine analogue 6d, which is the less active compound of
the series. The pyrrolidine analogue 6c is the most potent cytotoxic
compound in both cell lines; this observation is in accordance with
our previously reported data, where synthetic analogues of the
benzopyranoisoindole series were tested against the same (MES-
SA) or of similar origin (HT-29) cancer cells.17
duced cytotoxicity when compared to the nitroderivatives.17
A
possible synthetic approach for the preparation of this kind of
analogues could proceed through reduction of the nitro group, fol-
lowed by suitable treatment of the resulting aminoderivative.
However, such a reduction is troublesome, due to the ease of
reduction of the isoindole nucleus.19 Thus we decided to insert
the side-chain through the nucleophilic displacement of the nitro
By evaluating the cytotoxic activity of all compounds in the Dx-
sensitive (MES-SA) and Dx-resistant (MES-SA/Dx5) cell lines, we
found that they could exert a cytotoxic activity in both cell lines
O
O
CH3
COOH
NO2
CH3
COOH
a
NO2
+
CH3
NO2
b
S
S
SH
S
a R= N(CH3 2
b R= N(CH2CH3)2
c R= N(CH2)4
)
2
3
4
1
c
d R= N(CH2)5
R
R
Br
Br
N
S
O
O
N
NO2
NO2
NO2
NO2
e
d
d
S
S
S
O
O
O
O
8a-d
6a-d
5
7
Scheme 1. Reagents and conditions: (a) 5-chloro-2-nitrotoluene, KOH, DMSO, 3 h, 110 °C, 94%; (b) CH3SO2OH/P2O5, 4 h, 60 °C, 31% for 3, 10% for 4; (c) NBS, benzoyl peroxide,
CCl4, 40 min, 70 °C, 51%; (d) EtOH, RCH2CH2NH2, rt, 30–60 min for 6a–d, 12 h for 8a–d, 69–78%; (e) H2SO4/CH3COOH (1:4 v/v), CrO3, 110 °C, 90 min, 56%.

3112
A. Christodoulou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3110–3112
Table 1
the molecular level and the induced effects in DNA damage and
repair signaling pathways as well as on the cell cycle and apoptotic
cellular machineries is currently under active investigation in our
laboratories.
Inhibition of proliferation induced after a 72 h incubation with the thioxanthenone
derivatives (IC50 values in
l
Ma)
Compound
HCT-116
MES-SA
MES-SA/Dx5
RFb
6a
6b
6c
6d
8a
8b
8c
8d
9
2.10 0.36
2.14 0.28
1.15 0.07
8.09 0.43
5.42 0.55
6.02 0.53
7.04 0.47
5.47 2.12
60.37 3.57
0.16 0.07
0.022 0.003
4.44 1.85
1.93 0.13
1.72 0.10
16.74 2.75
3.52 0.45
3.98 0.94
2.30 0.13
3.98 0.10
68.17 2.99
0.030 0.008
0.012 0.003
1.70 0.05
5.08 0.26
2.15 0.26
9.16 0.90
5.18 0.26
2.61 0.37
1.45 0.31
2.55 0.51
>100
0.38
2.63
1.25
0.55
1.47
0.66
0.63
0.64
––
Acknowledgments
The authors would like to thank Dr. H. Pratsinis and Dr. D. Klet-
sas, who kindly provided MES-SA and MES-SA/Dx5 cell lines as
well as Dr. N. Cacoullos for helpful discussions.
Supplementary data
Dx
Mx
2.398 0.162
0.080 0.016
79.93
6.67
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.021.
a
The results represent the mean ( standard deviation) of 3–5 independent
experiments and are expressed as IC50, the concentration that reduced by 50% the
optical density of treated cells with respect to untreated controls.
b
IC50 resistant cells / IC50 sensitive cells.
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resistant factor (RF) of each compound revealed that, with the
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chemo-sensitive and chemo-resistant cells (RF less or close to 1),
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more sensitive, as compared to Dx-sensitive MES-SA cells, to the
cytotoxic effects of the compounds under study (Table 1).
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replaced by a second basic side-chain, lacks cytotoxicity. As previ-
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