Design, synthesis, and antiproliferative activity of some novel aminosubstituted xanthenones, able to overcome multidrug resistance toward MES-SA/Dx5 cells

Article abstract of DOI:10.1016/j.bmcl.2005.07.079

A series of novel xanthenone aminoderivatives and their pyrazole-fused counterparts possessing structural analogy to the potent anticancer agent 9-methoxypyrazoloacridine (PZA) reported. These compounds exhibited an interesting cytotoxic activity against

Full text of DOI:10.1016/j.bmcl.2005.07.079

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5057–5060
Design, synthesis, and antiproliferative activity of some novel
aminosubstituted xanthenones, able to overcome
multidrug resistance toward MES-SA/Dx5 cells
Ioannis K. Kostakis,a Roxane Tenta,a, Nicole Pouli,a Panagiotis Marakos,a,*
Alexios-Leandros Skaltsounis,b Harris Pratsinisc and Dimitris Kletsasc
aDivision of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens, Panepistimiopolis-Zografou,
Athens 15771, Greece
bDivision of Pharmacognosy, Department of Pharmacy, University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
cLaboratory of Cell Proliferation and Ageing, Institute of Biology, NCSR ‘‘Demokritos,’’ 15310 Athens, Greece
Received 17 June 2005; revised 22 July 2005; accepted 29 July 2005
Available online 19 September 2005
Abstract—A series of novel xanthenone aminoderivatives and their pyrazole-fused counterparts possessing structural analogy to the
potent anticancer agent 9-methoxypyrazoloacridine (PZA) reported. These compounds exhibited an interesting cytotoxic activity
against a panel of cell lines. Most noticeably, they retain activity against the multidrug resistant MES-SA/Dx5 subline, showing
resistant factors close to 1.
Ó 2005 Elsevier Ltd. All rights reserved.
DNA-interacting agents are widely used against a num-
H
N
CH3
ber of malignant diseases. Among them, the anthracy-
cline glycosides doxorubicin and daunomycin are
extensively used in clinical practice.1
OH
OH
O
O
HN
HN
OH
OH
N
N
N
H3CO
CH3
.2 HCl
N
H
Structure–activity studies of the anthracyclines rational-
ized the use of aminoanthraquinone congeners as antitu-
mor agents and resulted in the development of the
clinically useful anthracenedione mitoxantrone (1,
Fig. 1).2,3 Numerous analogues of this agent are studied
to eliminate its side effects, mainly cardiotoxicity4 and
the development of multidrug resistance (MDR).5
Cumulative cardiotoxicity has limited the prolonged
use of quinone derivatives and led to the development
of anthrapyrazoles,6 which due to the presence of a qua-
si-iminoquinone group, possess reduced cardiotoxicity.7
Many compounds belonging to this class are currently
under preclinical and clinical evaluation,8 and a number
NO2
N
H
1 Mitoxantrone dihydrochloride
2 PZA
Figure 1. Structures of mitoxantrone and PZA.
of structurally related acridine derivatives have also
been developed.9
Unfortunately, as most anticancer drugs behave,
anthrapyrazoles are subjected to the development of ac-
quired or intrinsic resistance (MDR) of certain tumor
cell populations, which results in a limited potential
for cancer cure.10 One of the most investigated strategies
to reverse MDR is the development of pharmacological
agents that are able to modulate the function of P-glyco-
protein (Pgp), an ATP-binding membrane protein,
responsible for the active efflux of cytotoxic drugs out
of resistant cells.11 For anthracenediones and the related
acridine cytostatics, it has been postulated that the
incorporation into the chromophore of a fused five- or
Keywords: Aminoxanthenones; Aminobenzopyrano[4,3,2-cd]indazoles;
Cytotoxic activity; Cell-cycle selectivity; Multidrug resistance.
*
Corresponding author. Tel.: +30 210 7274830; fax: +30 210
7274747; e-mail: marakos@pharm.uoa.gr
Present address: Department of Experimental Physiology, Medical
School, University of Athens, 75 Mikras Asias 11527 Goudi, Athens,
Greece.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.079

5058
I. K. Kostakis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5057–5060
six-membered heterocyclic ring has led to compounds
that are able to overcome MDR.12 9-Methoxypyrazolo-
acridine (PZA, 2, Fig. 1) was selected for clinical evalu-
ation from a series of compounds that combine the
DNA-complexing activity of the acridine chromophore
with the potential hypoxic cell-selectivity derived from
a reducible nitro group substitution on the ring.13,14
PZA retains full activity against resistant cell lines that
exhibit the MDR phenotype.15 Recent findings suggest
that this compound targets both topoisomerases simul-
taneously, but without stabilization of the topoisomer-
ase-DNA cleavable complexes.16 The results obtained
from different studies suggest a novel mechanism of ac-
tion, which needs further exploitation and maybe addi-
tional clinical combination studies of PZA with other
chemotherapeutic agents, with the aim of delaying or
circumventing drug resistance.8,13,17
and long-range heteronuclear correlation experiments
(HMBC and HMQC sequences). Structural discrimina-
tion resulted from the observation that C-10 of com-
pound 6 exhibits 2J coupling with two aromatic
3
protons, namely H-20 and H-60, and J coupling with
H-50, while in the case of compound 5 the correspond-
2
3
ing C-10 possesses J coupling only with H-60 and J
coupling with H-30. Compound 5 was then saponified
under mild conditions since the presence of a nitro
group facilitates the easy cleavage of the ether linkage.
The resulting carboxylic acid
7 was isolated in
good purity and was ring-closed upon treatment with
PPA, to provide 1-chloro-4-nitro-9H-xanthen-9-one
(8) in good yield. The target compounds 9a–c were
prepared by the nucleophilic substitution of the chloro
group of
8
(dialkylamino)ethylamines.
by appropriately substituted 2-
We have previously studied the synthesis and antipro-
liferative activity study of a number of cytotoxic xan-
thenone aminoderivatives.18–20 On the other hand,
some pyrazole-fused pyranoxanthenones, bearing a
dialkylaminoethyl substitution, showed strong cyto-
toxic activity against the murine leukemia L1210 cell
line, as well as against some human solid tumor cell
lines.21 As a continuation of this study, we present
here the synthesis and biological evaluation of some
novel xanthenone aminoderivatives and their pyra-
zole-fused counterparts, with direct structural similar-
ity to PZA.
The next step concerns the preparation of the
corresponding pyrazole-fuzed aminoderivatives. For
this purpose, 8 was reacted with commercially available
2-hydroxyethylhydrazine to provide the carbinol 10
(Scheme 1). The structural assignment for the carbinol
was confirmed using NOESY experiments. The side-
chain methylene, which is adjacent to the pyrazole ring,
exhibited NOEs only with the 3-aromatic proton. Addi-
tional evidence for the structure of compound 10 was
obtained by a gradient inverse-detected long-range
1H–15N correlation experiment at natural abundance,
where clear cross-peaks were observed, correlating N-2
with the adjacent side-chain methylene and the
3-aromatic proton, confirming the N-2 NMR signal
assignment (N-2, H-3 cross-peak) and the position of
the side-chain. Conversion of the carbinol 10 to the mes-
ylate 11, followed by nucleophilic substitution of the
readily displaced mesyloxy group by appropriately
substituted secondary amines, resulted in the benzopyr-
ano[4,3,2-cd]indazoles 12a–c.
For the synthesis of the target compounds, we have
used ethyl salicylate (3) as starting material (Scheme
1), which was treated with 2,4-dichloronitrobenzene
(4) to result in a mixture of the isomeric esters 5 and
6. These esters were separated by column chromatogra-
phy and their structure was unambiguously established
1
by H and 13C NMR spectroscopy, using both direct
Cl
CO2Et
CO2R
O
CO2Et
a
+
+
OH
Cl
O
1'
2'
NO2
NO2
3
6'
5'
4
Cl
Cl
NO2
5 R= Et
7 R= H
b
6
c
(from 7)
R1
R
O
O
HN
O
Cl
N
O
N
e
d
O
NO2
NO2
NO2
8
9a-c
a: R1= N(CH3)2
b: R1= N(CH2)4
c: R1= N(CH2)5
10 R= OH
11 R= OMs
12a-c R= R1
f
g
Scheme 1. Synthesis of compounds 9a–c and 12a–c. Reagents and conditions: (a) K2CO3, Cu2O, DMF, 110 °C, 8 h; (b) EtOH, NaOH 40%, rt,
30 min; (c) PPA 110 °C, 1 h; (d) H2NCH2CH2R1, pyridine, reflux, 1–3 h; (e) H2NNHCH2CH2OH, pyridine, rt 12h; (f) CH3SO2Cl, CH2Cl2, Et3N, rt,
4 h; (g) secondary amine, EtOH abs., reflux, 10–12 h.

I. K. Kostakis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5057–5060
5059
For comparative reasons, as regards the structure–activ-
ity relationship studies, we have also prepared the corre-
sponding aminoderivatives that lack the nitro group. We
have thus used salicylic acid (13, Scheme 2), which was
treated with 1,3-cyclohexanedione to result in the
diketone 14. The preparation of 14 has already been
reported and was effective through the ring closure
of 2-fluorobenzoic acid 3-oxo-cyclohex-1-enyl ester.22
compounds mitoxantrone and doxorubicin, are pre-
sented in Tables 1 and 2.
The derivatives possess strong cytotoxic activity against
all the tested cell lines, and their IC50 values vary typical-
ly within the range of 0.8–20 lM.
The aminoderivatives bearing the nitro group (9a–c) ap-
pear to be considerably more active when compared
with the corresponding non-substituted analogues
(17a–c). On the other hand, the difference in activity be-
tween the substituted and non-substituted derivatives in
the pyrazole-fused analogues is less pronounced.
However, the procedure presented herein is very simple
and high-yielding. The diketone 14 was subsequently
oxidized by the use of DDQ in boiling toluene to pro-
vide the phenol 15, which was then converted to the cor-
responding tosylate 16. Compound 16 was used for the
synthesis of the amines 17a–c upon reaction with the
suitably substituted diamines. On the other hand 16
was treated with 2-hydroxyethylhydrazine, the resulting
carbinol 18 was converted to the mesylate 19, which
reacted with the suitable secondary amines to provide
the pyrazole-fused derivatives 20a–c.
With regard to the activity against the L1210 cell line,
compounds 9b, 12a, 20a, and 20b possess interesting
cytotoxicity, with IC50 values within the range of 1.17–
1.81 lM, while the corresponding value for PZA has
been reported to be 0.424 lM.26
The new derivatives, with the exception of compound
9b, possess a rather diminished antiproliferative activity
against the colorectal adenocarcinoma HT-29 cell line.
For biological evaluation purposes, the free base forms
of the target amines were converted into their water-sol-
uble hydrochloride addition salts by treatment with
hydrochloric acid in methanol.
On the contrary, the nitro substituted derivatives exhibit
cytotoxicity against the uterine sarcoma MES-SA cell
line. The majority of them possess an interesting profile
of IC50 values, below 2 lM, the most cytotoxic compound
being 9b, which shows an antiproliferative activity in
the submicromolar range (IC50 value: 0.78 lM). Further-
more, from a direct comparison of the activity toward
the sensitive and resistant cell lines, it is evident that
the compounds appear to be active against MES-SA,
The in vitro cytotoxic activity of the new compounds
was evaluated in the established model of the murine
leukemia cell line L1210, and in three human solid
tumor cell lines: colorectal adenocarcinoma HT-29,
uterine sarcoma MES-SA, as well as its variant
MES-SA/Dx5, reported to be 100-fold resistant to
doxorubicin.23 The results, including reference
R
O
O
O
R
N
N
e
CO2H
OH
(from 16)
a
b
O
O
O
18 R= OH
19 R= OMs
20a-d R= R1
15 R= OH
16 R= OTs
17a-d R= NHCH2CH2R1
14
13
c
f
g
d
a: R1= N(CH3)2
b: R1= N(CH2)4
c: R1= N(CH2)5
Scheme 2. Synthesis of compounds 17a–c and 20a–c. Reagents and conditions: (a) PPA, 1,3-cyclohexanedione, 150 °C, 2h; (b) DDQ, toluene, 90 °C,
30 min; (c) 4-toluenesulfonylchloride, acetone, Na2CO3, reflux, 6 h; (d) H2NCH2CH2R1, pyridine, reflux, 16 h; (e) H2NNHCH2CH2OH, pyridine,
reflux 10 h; (f) CH3SO2Cl, CH2Cl2, Et3N, rt, 4 h; (g) secondary amine, EtOH abs., reflux, 10 h.
Table 1. Inhibition of proliferation of the aminosubstituted xanthenone derivatives (IC50 values in lMa)
Compound
R1
L1210
HT-29
MES-SA
MES-SA/Dx5
RFb
9a
9b
N(CH3)2
N(CH2)4
N(CH2)5
N(CH3)2
N(CH2)4
N(CH2)5
3.13 ( 0.31)
1.48 ( 0.67)
5.88 ( 1.47)
6.58 ( 1.65)
9.00 ( 1.88)
4.59 ( 0.97)
0.077 ( 0.010)
0.080 ( 0.005)
4.66 ( 0.84)
2.53 ( 0.39)
6.49 ( 2.29)
21.6 ( 7.51)
26.6 ( 2.55)
26.5 ( 5.11)
0.020 ( 0.004)
0.320 ( 0.180)
1.56 ( 0.62)
0.78 ( 0.11)
1.59 ( 0.18)
9.03 ( 2.61)
14.61 ( 4.16)
8.31 ( 2.19)
0.003 ( 0.000)
0.016 ( 0.008)
0.95 ( 0.20)
0.55 ( 0.07)
1.56 ( 0.12)
6.62 ( 1.96)
7.76 ( 1.80)
6.04 ( 1.75)
0.030 ( 0.020)
1.56 ( 0.10)
0.61
0.70
9c
0.98
0.73
17a
17b
17c
Mx
Dx
0.53
0.73
11.55
98.25
a The results represent means ( standard deviation) of three 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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I. K. Kostakis et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5057–5060
Table 2. Inhibition of proliferation of the pyrazole-fuzed xanthenone derivatives (IC50 values in lMa)
Compound
R1
L1210
HT-29
MES-SA
MES-SA/Dx5
RFb
12a
12b
12c
20a
20b
20c
Mx
Dx
N(CH3)2
N(CH2)4
N(CH2)5
N(CH3)2
N(CH2)4
N(CH2)5
1.81 ( 0.41)
3.52 ( 0.35)
10.7 ( 1.85)
1.17 ( 0.20)
1.79 ( 0.21)
2.74 ( 0.70)
0.077 ( 0.010)
0.080 ( 0.005)
7.49 ( 0.31)
15.8 ( 2.38)
21.6 ( 7.99)
20.9 ( 9.45)
11.5 ( 1.89)
9.92 ( 3.41)
0.020 ( 0.004)
0.320 ( 0.180)
1.03 ( 0.58)
1.13 ( 0.07)
4.05 ( 0.75)
2.79 ( 0.69)
1.75 ( 0.16)
4.40 ( 0.90)
0.003 ( 0.000)
0.016 ( 0.008)
0.63 ( 0.25)
1.47 ( 0.21)
5.38 ( 1.26)
2.76 ( 1.08)
1.58 ( 0.12)
3.88 ( 0.35)
0.030 ( 0.020)
1.56 ( 0.10)
0.61
1.29
1.33
0.99
0.90
0.88
11.55
98.25
a The results represent means ( standard deviation) of three 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.
Table 3. Cell cycle-phase distribution (%)a
3. Wiseman, L. R.; Spencer, C. M. Drug Aging 1997, 10, 473.
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Pharmacol. 2004, 68, 1815.
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1992, 35, 4770.
15. Keshelava, N.; Tsao-Wei, D.; Reynolds, C. P. AntiCancer
Drug Des. 1999, 14, 451.
16. Adjei, A. A.; Charron, M.; Rowinski, E. K.; Svingen,
P. A.; Miller, J.; Reid, J. M.; Sebold-Leopold, J.;
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683.
17. Hu, Y.; Krishan, A.; Nie, W. J.; Sridhar, K. S.; Mayer,
L. D.; Bally, M. Clin. Cancer Res 2004, 10, 1160.
18. Kostakis, I. K.; Ghirtis, K.; Pouli, N.; Marakos, P.;
Skaltsounis, A. L.; Leonce, S.; Gaignard, D. H.; Atassi, G.
Il Farmaco 2000, 55, 455.
19. Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos, P.;
Skaltsounis, A. L.; Pratsinis, H. Bioorg. Med. Chem. Lett.
2002, 12, 1443.
20. Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos, P.;
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4591.
21. Kostakis, I.; Magiatis, P.; Pouli, N.; Marakos, P.;
Skaltsounis, A. L.; Pratsinis, H.; Leonce, S.; Pierre, A.
J. Med. Chem. 2002, 45, 2599.
Compound
G0/G1
S
G2/M
9b
41.70 ( 2.12)
59.41 ( 3.71)
30.00 ( 4.90)
33.06 ( 3.08)
22.63 ( 1.19)
20.79 ( 3.67)
32.66 ( 4.12)
43.40 ( 8.12)
20.87 ( 4.21)
14.81 ( 0.91)
37.41 ( 5.51)
7.93 ( 1.49)
26.60 ( 5.75)
46.08 ( 3.60)
62.57 ( 3.88)
17b
12a
20a
20b
Control
54.70 ( 4.80)
35.57 ( 2.84)
9.74 ( 1.96)
a Mean ( standard deviation) of three independent experiments.
simultaneously possess generally comparable cytotoxicity
against the doxorubicin resistant MES-SA/Dx5 cell line.
The ability of all the tested compounds to overcome mul-
tidrug resistance of the MES-SA/Dx5 cell line is clearly
indicated by the resistant factor (RF) values, which are
all practically equal to 1. These results suggest that the
novel compounds seem to be hardly recognized by the
protein mechanisms controlling multidrug resistance.
Worth mentioning, the RF to doxorubicin was found to
be 98.25, as expected,23 while the RF to mitoxantrone
was 11.55.
Cell-cycle perturbations induced after incubation of
exponentially growing MES-SA uterine sarcoma cells
with a number of new compounds for 24 h are given
in Table 3. The studied compounds provoke, in general,
a G2/M arrest, expected on account of their structural
similarity to pyrazoloacridines and mitoxantrone, which
have also been reported to block the cell cycle in the G2
phase.24,25 Compound 17b, with an IC50 higher than the
concentration used for FACS analysis, had, indeed, no
significant effect on the cell cycle-phase distribution.
In conclusion, we have prepared a novel series of xan-
thenone aminoderivatives, bearing structural analogy
to PZA, which inhibit the proliferation of several cancer
cell lines. Most noticeably, this class of compounds
exhibits a very promising ability to overcome the multi-
drug resistance of the MES-SA/Dx5 cell line.
22. Gabbutt, C. D.; Hepworth, J. D.; Urquhart, M. W. J.;
Vazquez de Miguel, L. M. J. Chem. Soc., Perkin Trans. 1
1997, 1819.
23. Harker, W. G.; Sikic, B. I. Cancer Res. 1985, 45, 4091.
24. Grem, J. L.; Politi, P. M.; Berg, S. L.; Benchekroun, N.
M.; Patel, M.; Balis, F.; Sinha, B. K.; Dahut, W.; Allegra,
C. J. Biochem. Pharmacol. 1996, 51, 1649.
References and notes
1. Malonne, H.; Atassi, G. AntiCancer Drugs 1997, 8, 811.
2. Dunn, C. J.; Goa, K. L. Drug Aging 1996, 9, 122.
25. Traganos, F.; Evenson, D. P.; Staiano-Coico, L.; Dar-
zynkiewicz, Z.; Melamed, M. R. Cancer Res. 1980, 40, 671.