Design, synthesis, and evaluation of the antiproliferative activity of a series of novel fused xanthenone aminoderivatives in human breast cancer cells

Article abstract of DOI:10.1021/jm061410m

Derivatives of two novel, structurally related heterocyclic ring systems, xantheno[3,4-d]imidazole and chromeno[4,3,2-c,d]imidazo[4,5-f]indazole, bearing aminoalkyl side chains, have been synthesized, and their antiproliferative activity has been studied

Full text of DOI:10.1021/jm061410m

1716
J. Med. Chem. 2007, 50, 1716-1719
Design, Synthesis, and Evaluation of the Antiproliferative Activity of a Series of Novel Fused
Xanthenone Aminoderivatives in Human Breast Cancer Cells
Vasiliki Giannouli,† Ioannis K. Kostakis,† Nicole Pouli,*,† Panagiotis Marakos,† Olga Ch. Kousidou,‡
George N. Tzanakakis,§ and Nikos K. Karamanos‡
Department of Pharmacy, DiVision of Pharmaceutical Chemistry, UniVersity of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece,
Laboratory of Biochemistry, Department of Chemistry, UniVersity of Patras, 26110, Patras, Greece, and Department of Histology, Medical
School, UniVersity of Crete, 71110 Herakleion, Crete, Greece
ReceiVed December 8, 2006
Derivatives of two novel, structurally related heterocyclic ring systems, xantheno[3,4-d]imidazole and
chromeno[4,3,2-c,d]imidazo[4,5-f]indazole, bearing aminoalkyl side chains, have been synthesized, and their
antiproliferative activity has been studied against the aggressive human breast MDA-MB-231 cell line. The
pyrazole-fused analogue 27a possesses a pronounced antiproliferative effect on the tested cell line, evident
at 1 µM, and achieves an IC50 of 6.5 µM.
Introduction
Many compounds based on tricyclic planar chromophore
framework, fully or partially consisting of anthraquinone,
anthrapyrazole, or acridine, show interesting cytostatic and
antitumor properties.1 The presence of a five- or six-membered
heterocyclic ring fused to the anthracenedione or acridine moiety
usually increases the activity and enables the compounds to
overcome multidrug resistance of tumor cells.2 Among different
acridone derivatives, rationally designed imidazoacridones
exhibit high cytotoxic and antitumor properties and the most
active compound in the series, C-1311 (Figure 1), has recently
entered phase I clinical trials for the treatment of patients with
advanced solid tumors.3
Figure 1. Structures of C-1311, acronycine, and previously prepared
xanthenone derivatives.
We have been involved in the design, synthesis, and cytotoxic
activity evaluation of a number of pyrano(thio)xanthenone
derivatives4 possessing structural similarity with the pyrano-
(methyl)imidazole ring and to study the effect of this structural
acridone alkaloid acronycine (Figure 1). This compound has
modification on the tumor cell growth inhibitory activity of the
shown promising antitumor properties on several murine solid
new compounds.
tumor models5 and has been used as a lead for the synthesis of
analogues with markedly improved pharmacological properties.6
Results and Discussion
During the exploration of the structure-activity relationship in
the pyranoxanthenone series, we have found that the replacement
of the 6-methoxy group by a flexible dialkylaminoethylamino
side chain substitution (I, Figure 1) results in a clear improve-
ment of the antiproliferative activity toward the murine leukemia
L1210 cell line.7 The pyrazole-fused counterparts of these
molecules (II, Figure 1) are effective against leukemia and solid
tumor cell lines.8
Prompted by the above results, we performed the synthesis
of two novel fused heterocycles, namely, xantheno[3,4-d]-
imidazole and chromeno[4,3,2-cd]imidazo[4.5-f]indazole, that
possess a suitable aminosubstituted side chain at the position
para to the imidazole nitrogen atom. In addition, we have
prepared the corresponding analogues incorporating the electron-
releasing methyl group in the vacant 2-position of the imidazole
ring. The objective of this investigation was to replace the pyran
moiety of the previously prepared xanthenone derivatives by a
Chemistry. For the synthesis of the target derivatives we have
used the nitroderivative 1 (Scheme 1) that resulted from the
nitration of 1,3,5-trichlorobenzene with fuming nitric acid.9
Treatment of 1 with sodium azide provided a mixture of the
azides 2 and 3. These azides were not separated because of their
close polarity in a variety of solvent systems; consequently, their
mixture was subjected to reduction with sodium borohydride
in the presence of CuSO410 to provide the anilines 4 and 5. The
mixture of 4 and 5 was separated by column chromatography,
and the aniline 5 reacted with acetic anhydride to provide 3,5-
dichloro-2-nitroacetanilide (6). The preparation of the acetanilide
6 has been reported previously through a different procedure
in low yield (approximately 20%);11 however, the method
reported herein is simple and high-yielding (83% overall yield
starting from commercially available 1,3,5-trichlorobenzene).
Compound 6 was then coupled with ethyl salicylate in the
presence of potassium carbonate and copper(II) oxide to result
in a mixture of the isomeric diaryl ethers 7 and 8 that were
isolated in pure form by column chromatography. The structure
* To whom correspondence should be addressed. Phone: 30-1-7274185.
Fax: 30-1-7274747. E-mail: pouli@pharm.uoa.gr.
† University of Athens.
1
of each isomer was unambiguously established by H and 13C
‡ University of Patras.
§ University of Crete.
NMR spectroscopy, using both direct and long-range hetero-
10.1021/jm061410m CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/03/2007

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1717
Scheme 3a
Scheme 1a
a Reagents: (a) NaN3, dry DMSO, room temp, 20 h; (b) NaBH4,
CuSO4‚2H2O, 1 h, room temp; (c) Ac2O, AcOH, reflux, 3 h; (d) ethyl
salicylate, K2CO3, CuO, dry pyridine, reflux, 36 h; (e) 40% NaOH, dioxane,
2 h, room temp; (f) Ac2O, 98% H2SO4, 90 °C, 50 min.
Scheme 2a
a Reagents: (a) 2-hydroxyethylhydrazine (excess), dry DMSO, room
temp, 1 h; (b) 2-hydroxyethylhydrazine, DMAP, dry THF, room temp, 20
h; (c) MsCl, dry pyridine, 0 °C, room temp, 10 min; (d) 9% HCl solution,
dioxane, reflux, 3 h; (e) H2, Pd/C, 50 psi, absolute EtOH, 45 °C, 10 h; (f)
AcOH, dry toluene, reflux, 1 h; (g) triethyl orthoformate, 36% HCl, room
temp, 12 h; (h) dialkylamine, EtOH, reflux, 4 h.
ethylhydrazine to provide only a small amount of the desired
carbinol 18, together with the substituted hydrazine 17 that was
the main product of the reaction (Scheme 3). Compound 17
should have resulted from the nucleophilic substitution of the
nitro group by 2-hydroxyethylhydrazine, presumably upon initial
formation of 18. In this regard, we have avoided the formation
of the side product 17 and have improved the yield of 18 through
treatment of xanthenone 10 with an equimolar amount of
2-hydroxyethylhydrazine in the presence of 4-dimethylamino-
pyridine (DMAP). The structural assignment for the carbinol
18 was confirmed using NOESY experiments. The side chain
methylene, which is adjacent to the pyrazole ring, exhibited
NOEs only with the 3-aromatic proton. Compound 18 was
subsequently converted into the corresponding mesylate 19,
which was subjected to catalytic hydrogenation over palladium
on activated carbon, followed by ring closure of the resulting
acetanilide 20 to furnish 21. The target derivatives 22a,b were
prepared by the nucleophilic substitution of the readily displaced
mesyl group of 21 with the appropriately substituted secondary
amines.
Analogously, the acetanilide 18 was hydrolyzed and the
resulting 23 was converted to the mesylate 24 (Scheme 3). This
derivative was subjected to catalytic hydrogenation over pal-
ladium on activated carbon, followed by ring closure of the
intermediate unstable dianiline 25 upon treatment with triethyl
orthoformate in the presence of hydrochloric acid to provide
the mesylate 26. This compound was used for the preparation
of the target amines 27a,b,
For biological evaluation purposes, the free base forms of
the amines were converted into their water-soluble hydrochloride
or fumarate addition salts by treatment with hydrochloric or
fumaric acid, respectively, in methanol.
a Reagents: (a) N,N-dialkylethylenediamine, dry DMSO, reflux, 1 h; (b)
H2, Pd/C, 50 psi, absolute EtOH, 4 h, room temp; (c) AcOH, dry toluene,
reflux, 1 h; (d) 40% NaOH, EtOH, 60 °C, 1 h; (e) triethyl orthoformate,
36% HCl, room temp, 12 h.
nuclear correlation experiments (HMBCa and HMQC se-
quences). Structural discrimination resulted from the observation
2
that C-1′ of 7 exhibits J coupling with two aromatic protons,
namely, H-2′ and H-6′, while in the case of 8 the corresponding
2
C-1′ possesses J coupling only with H-6′. Compound 8 was
subsequently saponified, and the resulting carboxylic acid 9 was
ring-closed upon treatment with concentrated sulfuric acid in
the presence of acetic acid anhydride to afford the substituted
xanthenone 10.
Compound 10 was converted in the amino derivatives 11a,b
through nucleophilic substitution of the chlorine atom by
appropriately substituted diamines (Scheme 2). The 4-nitro
group of 11a,b was then easily reduced by hydrogenation over
palladium on activated carbon, and the resulting unstable
aminoderivatives 12a,b were converted into the target xan-
thenoimidazoles 13a,b upon treatment with acetic acid in boiling
toluene. Following an analogous procedure, the acetanilides
11a,b were first converted through alkaline hydrolysis to the
corresponding anilines 14a,b, which were then reduced, and
the intermediate amines 15a,b were treated with triethyl
orthoformate in the presence of hydrochloric acid to provide
the target amino derivatives 16a,b (Scheme 2).
Biological Assessment. The tested compounds were studied
on the breast cancer cells MDA-MB-231 cultured in serum-
containing medium. We chose to grow the cells in the presence
of serum in order for our experimental model to be more similar
to physiological conditions, since serum in the culture media
stimulates the cells via numerous active components present
therein.12 Mitoxantrone was used as reference compound and
showed an IC50 of 0.96 µM.
For the preparation of the corresponding pyrazole-fuzed
derivatives, xanthenone 10 reacted with excess 2-hydroxy-
a Abbreviations: HMBC, heteronuclear multiple bond correlation;
HMQC, heteronuclear multiple quantum correlation; WST-1, water-soluble
tetrazolium salt-1.

1718 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Brief Articles
through a Celite pad, and the filtrate was evaporated to dryness to
afford the amine 12. This amine, without further purification, was
dissolved in anhydrous toluene (30 mL). Glacial acetic acid (9
mmol) was added, and the resulting solution was refluxed for 1 h.
The solvent was vacuum-evaporated, and the residue was dissolved
in CH2Cl2, washed with 10% Na2CO3 solution, and dried (Na2SO4),
and the solvent was evaporated to dryness. Flash chromatography
on silica gel using a mixture of 10:1 CH2Cl2/MeOH provided 13
in 81-82% yield.
Synthesis of Target Compounds 16. The amines 15 were first
prepared by a procedure analogous to that of 12. Without further
purification each amine was suspended in triethyl orthoformate (4
mL). Hydrochloric acid 36% (3 drops) was added, and the resulting
mixture was stirred at room temperature for 12 h. The mixture was
then made alkaline with a 10% NaHCO3 solution and extracted
with CH2Cl2 (3 × 40 mL). The combined organic extracts were
washed with brine, dried (Na2SO4), and concentrated to dryness,
and the residue was recrystallized from diethyl ether to afford pure
16 in 80-81% yield.
Synthesis of Target Compounds 22. A solution of methane-
sulfonyl chloride (121 µL, 1.55 mmol) in CH2Cl2 (5 mL) was added
dropwise at 0 °C to a suspension of 18 (500 mg, 1.412 mmol) in
dry pyridine (6 mL), and the mixture was stirred at room
temperature for 10 min. The mixture was poured into ice/water
and acidified with hydrochloric acid 9%, and the resulting solid
was filtered, washed with water, and air-dried to give the mesylate
19 (550 mg, 90%). Compound 19 was then converted into the
analogue 21 in 84% yield by a procedure analogous to the one
described for 13. Compound 21 (0.26 mmol) was then added to a
33% solution of the suitable dialkylamine in ethanol (4 mL), and
the resulting solution was heated at reflux for 4 h. The solvent was
vacuum-evaporated, and the residue was purified by column
chromatography (silica gel, 8:1 CH2Cl2/MeOH) to furnish 22 in
90-92% yield.
Synthesis of Target Compounds 27. HCl (9%, 1 mL) was added
to a stirred solution of the acetamide 18 (1.13 mmol) in dioxane
(15 mL), and the resulting mixture was heated at reflux for 3 h.
The solvent was vacuum-evaporated, the aqueous layer was
extracted with CH2Cl2 (3 × 70 mL), and the organic extracts were
dried (Na2SO4) and concentrated to dryness. The resulting solid
was recrystallized from ethanol to give 23 (94%), which was
converted to the corresponding mesylate 24 in 96% yield according
to the procedure described for the preparation of the analogue 19.
The mesylate 24 was then converted to 26 in 83% yield through
initial hydrogenation followed by treatement with triethyl ortho-
formate, as described for the preparation of 16. The mesylate 26
(0.32 mmol) was refluxed for 5 h with an ethanolic solution of the
appropriate dialkylamine (4 mL), the solvent was vacuum-
evaporated, and the residue was purified by column chromatography
(silica gel, 9:1 CH2Cl2/MeOH) to furnish 27 in 83-91% yield.
Figure 2. Effects of new amino derivatives on human breast cancer
cells. The MDA-MB-231 cell line was incubated in serum-containing
medium for 72 h in the presence of increasing concentrations of
aminoderivatives. Cell proliferation was determined by measuring the
absorbance at 450 nm (WST-1 method). Data are representative of three
individual experiments, performed in three replicates. Control values
did not exhibit significant changes compared to the dimethyl sulfoxide
(DMSO) vehicle. Asterisks indicate the statistically significant changes
of treated cells compared to control at the level of 0.01.
Compounds 16a,b showed significant inhibitory effect on cell
growth only at the highest concentration tested (100 µM). At
this concentration a large number of cells lost contact with the
culture flask and the remaining adherent cells underwent
morphological changes suggestive of apoptosis. The insertion
of a 2-methyl group in compounds 16a,b, providing the
analogues 13a,b, did not alter the observed effect.
The effects of the pyrazole-fused derivatives 22a,b and 27a,b
on cell growth are shown in Figure 2. A dose-dependent
inhibitory effect on cell growth was observed. It is worth
noticing that for concentrations up to 30 µM the breast cancer
cells do not present any morphological changes, suggesting a
cytostatic rather than a cytotoxic effect. The obtained results
indicate that the incorporation of a pyrazole ring fusion into
16a resulting in compound 27a significantly increased the
antiproliferative activity. The later effect was profound even
from 1 µM (Figure 2). The IC50 for 27a was 6.5 µM. The
insertion of a 9-methyl group in 27a resulting in 22a did not
improve the growth inhibitory effect of 27a and gave an IC50
of 17 µM. It is plausible to suggest that the higher inhibitory
effect of 27a compared to 22a may due to the enhanced
imidazole tautomerism of 27a.
The pyrazole-fused analogue 27b showed a significant
antiproliferative activity. However, a higher IC50 (8.5 µM)
compared to that for 27a was obtained. Similar with 22a, the
9-methyl analogue 22b did not improve the antiproliferative
activity of 27b (IC50 ) 18 µM).
Previous studies concerning the antiproliferative activity of
the structurally related pyranobenzopyranoindazoles8 (II, Figure
1) on MDA-MB-231 breast cancer cells had shown that they
possess IC50 in the range 9-50 µM. The replacement of the
pyran moiety of those derivatives by an imidazole ring resulted
only in a slight improvement of their activity. However, the
involvement of the imidazole tautomerism in the biological
activity of the compounds could suggest that this novel scaffold
may constitute a new lead for the development of antiprolif-
erative agents.
Supporting Information Available: Experimental procedures
and characterization data for the new compounds, cell culture
conditions, details for cell proliferation assays, and elemental
analysis results. This material is available free of charge via the
Internet at http://pubs.acs.org.
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