Synthesis and antiproliferative activity of substituted benzopyranoisoindoles: A new class of cytotoxic compounds

Article abstract of DOI:10.1016/j.bmcl.2006.06.074

A series of novel aminosubstituted benzopyranoisoindoles possessing structural analogy to an active nitracrine metabolite are reported. The compounds exhibited interesting cytotoxic activity against a panel of cell lines, which was maximized by the presen

Full text of DOI:10.1016/j.bmcl.2006.06.074

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4822–4825
Synthesis and antiproliferative activity of substituted
benzopyranoisoindoles: A new class of cytotoxic compounds
a
a
a
a,
*
Christiana Hadjipavlou, Ioannis K. Kostakis, Nicole Pouli, Panagiotis Marakos,
b
b
Harris Pratsinis and Dimitris Kletsas
a
Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens, Panepistimiopolis-Zografou,
Athens 15771, Greece
b
Laboratory of Cell Proliferation and Ageing, Institute of Biology, NCSR ‘‘Demokritos’’, 15310 Athens, Greece
Received 25 May 2006; revised 21 June 2006; accepted 21 June 2006
Available online 7 July 2006
Abstract—A series of novel aminosubstituted benzopyranoisoindoles possessing structural analogy to an active nitracrine metabo-
lite are reported. The compounds exhibited interesting cytotoxic activity against a panel of cell lines, which was maximized by the
presence of both 1-dialkylaminoethyl and 3-nitro substituents.
ꢀ
2006 Elsevier Ltd. All rights reserved.
0
1
-Nitro-9-[3 -(dimethylamino)propylamino]acridine
I, nitracrine, Fig. 1) has been used clinically for several
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.
(
years in Poland for the treatment of mammary, ovarian,
lung, and colon tumors. A number of in vitro studies
5
1
have indicated that this drug undergoes metabolic
reduction of the 1-nitro group to form a reactive species,
while inhibition of this reaction by oxygen transforms
the drug into an extremely potent, hypoxia selective
During our work directed toward the synthesis of vari-
ous xanthenone derivatives and the evaluation of their
antiproliferative activity, we have prepared aminosubsti-
tuted pyranoxanthenones, their pyrazole-fused counter-
parts, and some related benzopyranoindazoles, which
have shown interesting cytotoxicity against a panel of
2
cytotoxic agent. Intercalative DNA binding does not
appear to be directly responsible for cytotoxicity, but
it may contribute to the high potency of the drug, by
serving to target reactive, reduced cellular metabolites
6
–8
tumor cell lines. As part of our ongoing efforts in this
field, we have recently reported on the synthesis of a
3
9
to the DNA. Unfortunately, the in vivo effectiveness
of nitracrine on solid tumors is limited, probably be-
cause reductive metabolism is too rapid to allow efficient
novel benzopyranoisoindole ring system. This scaffold
could potentially provide bioisosters of the active nitra-
crine metabolite (II) and with this prospect in mind, we
report the preparation of a number of related aminosub-
stituted derivatives and the evaluation of their in vitro
cytotoxic activity.
4
distribution through hypoxic tumor areas. In an effort
to elucidate the bioreduction pathway, extensive struc-
tural studies of the cellular metabolites performed in
various biological systems revealed the presence of
highly reactive intermediates. The initially generated
For the synthesis of the target compounds commer-
cial 2-iodobenzoic acid (1, Scheme 1) was first con-
1
-aminoderivative of nitracrine is susceptible to
intramolecular cyclization, giving rise to the dihydropy-
razoloacridine II (Fig. 1). This key metabolite was
found to be a reactive species, as it easily undergoes
(CH ) N
3
2
NH NO2
(CH ) N
3 2
N
N
N
N
H
Keywords: Aminosubstituted benzopyrano[4,3,2-c,d]isoindoles; Anti-
proliferative activity; DNA-content analysis.
Nitracrine (I)
II
*
Corresponding author.
marakos@pharm.uoa.gr
Fax:
+30
210
7274747; e-mail:
Figure 1. Structures of Nitracrine and its active metabolite.
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.074

C. Hadjipavlou et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4822–4825
4823
O
CH3
O
COOR
J
b
d
+
O
CH3
O
O
CH3
COOR
5
6
1
2
R= H
R= Et
3 R= Et
4 R= H
a
c
e
Br
O
O
CH3
NO2
NO2
f
O
O
8
7
Scheme 1. Synthesis of compound 8. Reagents and conditions: (a) HCl/EtOH, reflux, 8 h, 93%; (b) m-cresol, Cs
9%; (c) 1—NaOH (40%), CH OH, rt, 2 h; 2—HCl (36%), 99% (d) (CF CO) O, CH Cl , rt, 3 h, 57%; (e) HNO 65%, H
NBS, benzoyl peroxide, CCl , reflux, 18 h, 82%.
2
CO
3
, CuCl, pyridine, reflux, 3 h,
8
3
3
2
2
2
3
2
SO
4
98%, 0 ꢁC, 2 h, 56%; (f)
4
verted to the corresponding ethyl benzoate 2 and
then treated with m-cresol in the presence of cesium
carbonate and cuprous chloride to provide the diary-
lether 3 in 89% yield. This diarylether has been pre-
compound 8 is necessary for effective ring closure.
1
1
Indeed, when 1-bromomethylxanthen-9(9H)-one was
heated at reflux with an ethanolic solution of 2-diethyla-
0
minoethylamine,
xanthen-1-yl)methyl]ethane-1,2-diamine was obtained.
only
N,N-diethyl-N -[(9-oxo-9H-
1
0
viously prepared, but the yield of the reaction was
clearly improved, through the alterations reported
herein. Compound 3 was first saponified and then
ring-closed, upon treatment with trifluoroacetic anhy-
dride, to result in a mixture of the isomeric xanthe-
nones 5 and 6, which were separated by column
chromatography (silica gel, cyclohexane/EtOAc 15:1)
and identified. The use of trifluoroacetic anhydride,
as an alternative to the reported acetic anhydride/
The nitro group of 9 could be displaced easily and upon
treatment with 2-dialkylaminoethylamines resulted in
1
2
the target derivatives 10a–c. Similarly, when the bro-
mide 8 was treated with suitable ethanediamines, it pro-
1
3
vided in one step the amines 11a–c, which upon
nucleophilic substitution of the nitro group yielded the
1
4
target derivatives 12a–c.
1
0a
10b
sulfuric acid,
or PPA,
improved the yield of
both isomers 5 and 6, and provided a larger amount
of the required compound 5 (57%, instead of the
reported 42%). 1-Methylxanthenone (5) was then
The in vitro cytotoxic activity of the new compounds
6
–8
was evaluated by using the MTT assay in the colorec-
tal adenocarcinoma cell line HT-29, the uterine sarcoma
MES-SA as well as its variant MES-SA/Dx5, reported
9
nitrated to provide only the 2-nitroderivative 7,
1
5
which was treated with NBS in the presence of a cat-
alytic amount of benzoyl peroxide to give the 1-bro-
momethyl analogue 8.
to be 100-fold resistant to doxorubicin. The results,
including reference compounds mitoxantrone and doxo-
rubicin, are presented in Table 1.
Reaction of the bromide 8 with ethylamine provided
quantitatively, in one step, the isoindole derivative 9
The nitroderivative 9 is inactive against the HT-29 cell
line and exhibits moderate activity against MES-SA
uterine sarcoma cells, as well as the corresponding
9
(
Scheme 2). The presence of the nitro group in
Br
O
N
O
N
H
N
NO2
NO2
a
NRR
b
O
O
8
9
10a-c
c
NRR
RRN
RRN
N
N
O
H
a: N(CH3)2
b: N(C2H5)2
c: N(CH2)4
NO2
N
NRR
b
O
11a-c
12a-c
Scheme 2. Synthesis of compounds 10a–c, 11a–c, and 12a–c. Reagents and conditions: (a) ethylamine/CH
-dialkylaminoethylamine, DMSO, 90 ꢁC, 2 h, 78–85%; (c) 2-dialkylaminoethylamine, EtOH, rt, 1 h, 92–95%.
3
OH, rt, 2 h, 99%; (b)
2

4824
C. Hadjipavlou et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4822–4825
a
Table 1. Inhibition of proliferation induced after incubation for 72 h with the xanthenone derivatives (IC50 values in lM)
b
Compound
NRR
HT-29
MES-SA
MES-SA/Dx5
RF
9
—
>100
19.8 (±9.12)
11.5 (±5.74)
31.9 (±8.83)
15.7 (±7.12)
15.5 (±6.94)
0.8 (±0.12)
0.6
0.7
0.5
0.6
0.9
1.3
1.0
0.8
10a
10b
10c
11a
11b
11c
12a
12b
12c
N(CH
N(CH
N(CH
N(CH
N(CH
N(CH
N(CH
N(CH
N(CH
3
2
2
3
2
2
3
2
2
)
2
44.6 (±6.12)
19.9 (±5.31)
46.6 (±4.02)
4.2 (±0.78)
6.8 (±4.39)
3.3 (±0.38)
65.1 (±9.71)
29.8 (±8.57)
50.5 (±3.67)
0.025 (±0.008)
0.153 (±0.076)
45.8 (±4.18)
31.0 (±1.20)
24.7 (±6.05)
0.9 (±0.03)
1.1 (±0.35)
0.6 (±0.09)
50.4 (±7.30)
CH
3
3
3
)
2
)
2
)
2
)
)
4
2
CH
1.4 (±0.26)
)
)
4
0.6 (±0.11)
2
40.3 (±8.56)
20.8 (±7.80)
17.7 (±4.59)
0.028 (±0.002)
0.704 (±0.337)
CH
>100
—
)
4
60.5 (±8.57)
0.003 (±0.001)
0.0097 (±0.0012)
0.3
9.3
Mx
Dx
72.6
a
The results represent mean (±standard deviation) of three independent experiments and are expressed as IC50, the concentration that reduced by
0% the optical density of treated cells with respect to untreated controls.
IC50 resistant cells/IC50 sensitive cells.
5
b
a
Table 2. DNA-content analysis
Cell-cycle perturbations induced after incubation of
exponentially growing MES-SA uterine sarcoma cells
with the new compounds for 24 h were studied by
b
Compound
G0/G1
S
G2/M
Apoptosis
9
62.14
74.70
77.69
74.33
57.44
55.57
64.04
68.91
68.09
73.58
63.68
37.86
21.55
21.34
23.53
20.65
15.33
33.73
25.87
28.13
26.42
33.19
0.00
3.75
0.97
2.14
21.91
29.10
2.23
5.22
3.78
0.00
3.14
2.51
1.17
0.79
0.52
4.15
7.36
14.10
0.55
0.54
1.44
0.29
8
flow-cytometric analysis of DNA content. The sub-
1
1
1
1
1
1
1
1
1
0a
0b
0c
1a
1b
1c
2a
2b
2c
diploid peak observed during this analysis was used
for the assessment of cell death due to apoptosis. As
shown in Table 2, compounds 11a and 11b provoke
a significant G2/M arrest, as well as cell death by
apoptosis. Furthermore, compound 11c does not seem
to block the cell-cycle in the G2 phase at the specific
time-point selected for the FACS analysis, however it
possesses a strong apoptotic effect, which is in accor-
dance with its high cytotoxic activity. Finally, com-
pounds 10a–c and 12a–c induce to a greater or a
lesser extent, a G1 block.
Control
a
Values represent cell-cycle phase distribution (%), while apoptosis has
been calculated as percentage of the total number of events. One out
of two similar experiments is depicted.
b
All compounds were administered at concentration equal to their
IC50, except for 12b and 12c which were used at 50 lM.
In conclusion, we have prepared a series of novel
bioactive xanthenone aminoderivatives. This class of
compounds constitute new potential anticancer leads
and the observed variation of in vitro activity against co-
lon HT-29 and MES-SA uterine sarcoma cell lines of-
fered the opportunity to determine the substitution
pattern favorable for cytotoxicity.
MES-SA/Dx5 variant. Derivatives 10a–c and 12a–c,
that possess two side chains in positions 1- and 3-,
show a low profile of cytotoxicity, in all cell lines. In
contrast, the 3-nitro aminoderivatives, 11a–c, appear
to be highly active against both HT-29 and MES-SA
cell lines and their IC50 values vary typically within
the range of 0.6–6.8 lM. This could be considered an
important finding, since it is evident that the insertion
of an aminosubstituted side chain in position 1- of the
molecule in conjunction with the existence of a 3-nitro
group substantially increases cytotoxic activity. This
improvement is not so pronounced when the nitro
group is replaced by an additional aminosubstituted
side chain. Among the compounds 11, the pyrrolidine
analogue 11c is the most cytotoxic derivative, followed
by the dimethylamino analogue 11a. From a direct
comparison of activity toward sensitive and resistant
cell lines, it is evident that the compounds appear to
be active against MES-SA and also possess generally
comparable cytotoxicity against the doxorubicin
resistant MES-SA/Dx5 cell line. The ability of com-
pounds 11a–c to overcome multidrug resistance to
the MES-SA/Dx5 cell line is clearly indicated by the
resistant factor (RF) values, which are practically all
equal to 1. These results suggest that the novel com-
pounds are hardly recognized by the protein machinery
governing multidrug resistance.
Acknowledgment
The present study was supported in part from the
Special Account for Research Grants Committee of
the National and Kapodistrian University of Athens
(Program Kapodistrias).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.06.074.
References and notes
1
. (a)
Kwasniewska-Rokicinska,
Wieczorkiewicz, A. Arch. Immunol. Ther. Exp. 1973, 21,
C.;
Swiecki,
J.;
863; (b) Kwasniewska-Rokicinska, C.; Sawicki, J.; Drosik,

C. Hadjipavlou et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4822–4825
4825
K. Mater. Med. Pol. (Engl. Ed.) 1976, 8, 289; (c)
Kwasniewska-Rokicinska, C.; Drosik, K. Mater. Med.
Pol. (Engl. Ed.) 1983, 15, 43.
. Wilson, W. R.; Denny, W. A.; Twigden, S. J.; Baguley, B.
C.; Probert, J. C. Br. J. Cancer 1984, 49, 215.
56.18 (CH
2
CH NMe
2
2
), 96.86 (C-5), 113.79 (C-10c),
114.02 (C-2), 117.11 (C-2a), 117.37 (C-7), 117.62 (C-
10b), 120.55 (C-9), 120.58 (C-10a), 128.19 (C-3), 128.80
(C-4), 131.42 (C-8), 132.36 (C-10), 150.56 (C-5a), 155.92
2
3
4
5
6
23 3
(C-6a). Anal. Calcd for C20H N O; calcd: C, 74.74; H,
. Pawlak, K.; Matuszkiewicz, A.; Pawlak, J. W.; Konopa, J.
Chem. Biol. Interact. 1983, 43, 131.
. Wilson, W. R.; Denny, W. A.; Stewart, G. M.; Fenn, A.;
Probert, J. C. Int. J. Radiat. Oncol. Biol. Phys. 1986, 12, 1235.
. Gorlewska, K.; Mazerska, Z.; Sowinski, P.; Konopa, J.
Chem. Res. Toxicol. 2001, 14, 1.
7.21; N, 13.07. Found: C, 74.97; H, 7.12; N, 12.84.
13. N,N-Dimethyl-2-(3-nitro-1H-benzopyrano[4,3,2-c,d]isoin-
dol-1-yl)ethylamine (11a). A solution of the bromide 8
(400 mg, 1.2 mmol) and 2-dimethylaminoethylamine
(850 lL, 6 mmol) in absolute ethanol (7 mL) was stirred
at room temperature for 1 h. The reaction mixture was
. (a) Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos,
P.; Kletsas, D.; Pratsinis, H. Bioorg. Med. Chem. 2003, 11,
vacuum-evaporated, extracted with CH
organic layer was dried (Na SO ) and evaporated to
dryness. The residue was purified by column chromatog-
raphy (silica gel, CH Cl /MeOH 98:2) to furnish the target
compound (380 mg, 92%); mp 189–192 ꢁC (CH Cl /diethyl
, 400 MHz) d (ppm) 2.32 (s, 6H,
2 2
Cl and water, the
2
4
4
591; (b) Kolokythas, G.; Kostakis, I. K.; Pouli, N.;
Marakos, P.; Skaltsounis, A.-L.; Pratsinis, H. Bioorg.
Med. Chem. Lett. 2002, 12, 1443.
2
2
2
2
1
7
. Kostakis, I. K.; Magiatis, P.; Pouli, N.; Marakos, P.;
Skaltsounis, A.-L.; Pratsinis, H.; Leonce, S.; Pierre, A.
J. Med. Chem. 2002, 45, 2599.
ether); H NMR (CDCl
3
2· CH ), 2.79 (t, J = 7.32 Hz, 2H, Me NCH CH ), 4.49 (t,
3
2
2
2
2 2 2
J = 7.32 Hz, 2H, Me NCH CH ), 6.11 (d, J = 8.3 Hz, 1H,
8
. (a) Kostakis, I. K.; Tenta, R.; Pouli, N.; Marakos, P.;
Skaltsounis, A.-L.; Pratsinis, H.; Kletsas, D. Bioorg. Med.
Chem. Lett. 2005, 15, 5057; (b) Kostakis, I. K.; Pouli, N.;
Marakos, P.; Skaltsounis, A.-L.; Pratsinis, H.; Kletsas, D.
Bioorg. Med. Chem. 2006, 14, 2910.
H-5), 7.16–7.32 (m, 3H, H-7, H-8, H-9), 7.42 (s, 1H, H-2),
7.53 (dd, J = 7.1, 1.7 Hz, 1H, H-10), 8.08 (d, J = 8.3 Hz.
1
3
1H, H-4); C NMR (CDCl , 50 MHz) d (ppm) 46.01 (2·
3
3 2 2 2 2 2 2
CH ), 48.80 (Me NCH CH ), 58.61 (Me NCH CH ),
98.27 (C-5), 115.25 (C-2a), 116.61 (C-2), 118.19 (C-10a),
118.78 (C-7), 119.63 (C-10b), 120.20 (C-10c), 120.51 (C-
10), 125.51 (C-9), 128.19 (C-8), 130.32 (C-4),131.90 (C-3),
153.07 (C-6a), 157.67 (C-5a). Anal. Calcd for
9
. Hadjipavlou, C.; Kostakis, I. K.; Pouli, N.; Marakos, P.;
Mikros, E. Tetrahedron Lett. 2006, 47, 3681.
0. (a) Goldberg, A. A.; Wragg, A. H. J. Chem. Soc. 1958,
1
4
3
227; (b) Pickert, M.; Frahm, A. W. Arch. Pharm. 1998,
11, 177; (c) Pellon, R. F.; Carrasco, R.; Milian, V.;
Rodes, L. Synth. Commun. 1995, 25, 1077.
18 17 3 3
C H N O ; calcd: C, 66.86; H, 5.30; N, 13.00. Found:
C, 67.03; H, 5.22; N, 12.79.
0
14. N,N-Dimethyl-N -[1-(2-dimethylaminoethyl)-1H-benzo-
pyrano[4,3,2-c,d]isoindol-3-yl]-1,2-ethanediamine (12a).
This compound was prepared by a procedure analogous
1
1. Rewcastle, G. W.; Atwell, G. J.; Baguley, B. C.; Calveley,
S. B.; Denny, W. A. J. Med. Chem. 1989, 32, 793.
2. N,N-Dimethyl-N -(1-ethyl-1H-benzopyrano[4,3,2-c,d]iso-
indol-3-yl)-1,2-ethanediamine (10a). A solution of 9
0
1
to 10a. Yield: 78%; mp 71–74 ꢁC (diethyl ether/n-pentane);
1
H NMR (CDCl
CH N(CH
2.25 (m, 2H, HNCH CH N(CH ) ), 2.65 (m, 1H,
3
, 400 MHz) d (ppm) 1.96 (s, 6H,
(
(
40 mg, 0.14 mmol) and 2-dimethylaminoethylamine
200 lL, 1.4 mmol) in dry dimethylsulfoxide (6 mL) was
HNCH
2
2
3
)
2
), 2.11 (s, 6H, (CH
3
)
2
NCH
2
CH
2
),
2
2
3 2
heated at 90 ꢁC for 2 h. Upon cooling, the reaction
mixture was poured into ice/water and extracted with
(CH
3.11 (m, 2H, HNCH
(CH NCH CHH), 4.07 (m, 1H, (CH
3
)
2
NCHHCH
2
), 3.04 (m, 1H, (CH
CH N(CH ), 3.91 (m, 1H,
NCH CHH),
3 2 2
) NCHHCH
),
2
2
3 2
)
CH
washed with brine, dried over Na SO , and concentrated
2
Cl
2
(3· 40 mL). The combined organic extracts were
3
)
2
2
3
)
2
2
5.53 (br s, 1H, D O exchangeable, NH), 5.82 (d,
2
2
4
to dryness. The residue was purified by column chroma-
tography (silica gel, CH Cl /MeOH 95:5) to furnish the
J = 8.7 Hz, 1H, H-5), 6.99 (t, J = 7.2 Hz, 1H, H-9),
7.08–7.13 (m, 2H, H-7, H-10), 7.42 (ddd, J = 1.7, 7.2,
8.1 Hz, 1H, H-8), 7.86 (s, 1H, H-2), 8.23 (d, J = 8.7 Hz,
2
2
target compound (40 mg, 85%); mp 167–170 ꢁC (diethyl
1
13
ether/n-pentane); H NMR (CDCl , 400 MHz) d (ppm)
1H, H-4); C NMR (CDCl , 50 MHz) d (ppm) 39.80
3
3
1
.31 (t, J = 7.32 Hz, 3H, CH
2
CH ), 2.00 (s, 6H, N(CH
3
3
)
), 2.36 (m, 1H,
2
),
(HNCH
46.18 ((CH
2
CH
2
N(CH
)
3
)
2
), 44.46 (HNCH
2
CH ), 46.35 ((CH
2
CH N(CH
NCH
2
3
)
2
),
2.30 (m, 1H, HCHCH
HCHCH NMe ), 3.18 (m, 2H, HNCH CH NMe ), 3.94
2
NMe
2
3
2
NCH
2
3
)
2
2
CH
2
),
56.21 (HNCH CH N(CH ) ), 60.72 ((CH ) NCH CH ),
2 2 3 2 3 2 2 2
2
2
2
2
2
(
NH), 5.81 (d, J = 8.7 Hz, 1H, H-5), 6.96 (t, J = 7.5 Hz,
m, 2H, CH CH ), 5.72 (br s, 1H, D O exchangeable,
97.09 (C-5), 113.02 (C-2), 113.04 (C-10c), 118.23 (C-2a),
119.89 (C-10a), 120.36 (C-7), 121.03 (C-9), 123.95 (C-10b),
127.54 (C-3), 129.64 (C-4), 131.95 (C-8), 132.62 (C-10),
151.94 (C-5a), 157.27 (C-6a). Anal. Calcd for C H N O;
2
3
2
1
H, H-9), 7.04 (d, J = 7.9 Hz, 1H, H-7), 7.16 (dd, J = 1.7,
.5 Hz, 1H, H-10), 7.36 (ddd, J = 1.7, 7.2, 8.1 Hz, 1H,
7
H-8), 7.76 (s, 1H, H-2), 8.05 (d, J = 8.7 Hz, 1H, H-4);
2
2
28
4
1
3
C
calcd: C, 72.50; H, 7.74; N, 15.37. Found: C, 72.32; H,
7.69; N, 15.12.
15. Harker, W. G.; Sikic, B. I. Cancer Res. 1985, 45, 4091.
NMR (CDCl
(
3
, 50 MHz) d (ppm) 16.66 (CH
2
CH
3
), 39.56
),
CH CH NMe
2
2
2
), 42.91 (CH CH
2
3
), 44.47 (2· NCH
3