Isoindolo[2,1-a]quinoxaline derivatives, novel potent antitumor agents with dual inhibition of tubulin polymerization and topoisomerase I

Article abstract of DOI:10.1021/jm070834t

Isoindoloquinoxalines 4 and 5 were obtained by refluxing 2-(2′-aminoaryl)-1-cyanoisoindoles 3a-e in acetic or formic acid. All derivatives were screened by the National Cancer Institute (Bethesda, MD) for the in vitro one dose primary anticancer assay aga

Full text of DOI:10.1021/jm070834t

J. Med. Chem. 2008, 51, 2387–2399
2387
Isoindolo[2,1-a]quinoxaline Derivatives, Novel Potent Antitumor Agents with Dual Inhibition of
Tubulin Polymerization and Topoisomerase I
,
†
†
†
†
†
†
Patrizia Diana,* Annamaria Martorana, Paola Barraja, Alessandra Montalbano, Gaetano Dattolo, Girolamo Cirrincione,
‡
‡
‡
§
‡
Francesco Dall’Acqua, Alessia Salvador, Daniela Vedaldi, Giuseppe Basso, and Giampietro Viola
Dipartimento Farmacochimico Tossicologico e Biologico, UniVersità degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy,
Dipartimento di Scienze Farmaceutiche, UniVersità degli Studi di PadoVa, Via Marzolo 5, 35131 PadoVa, Italy, and Dipartimento di Pediatria,
UniVersità degli Studi di PadoVa, Via Giustiniani 3, 35131 PadoVa, Italy
ReceiVed July 10, 2007
Isoindoloquinoxalines 4 and 5 were obtained by refluxing 2-(2′-aminoaryl)-1-cyanoisoindoles 3a-e in acetic
or formic acid. All derivatives were screened by the National Cancer Institute (Bethesda, MD) for the in
vitro one dose primary anticancer assay against a 3-cell line panel. Compounds 4a-e, screened against a
panel of about 60 human tumor cell lines, showed remarkable antineoplastic activity; they had GI50 values
in the low micromolar or submicromolar range and reached, in the case of 4c, nanomolar concentrations on
8
8% of the 59 tested cell lines. Flow cytometric analysis of cell cycle after treatment with 4c demonstrated
an arrest of the cell cycle in G2/M phase. This effect was accompanied with apoptosis of the cells,
mitochondrial depolarization, generation of reactive oxygen species, and activation of caspase-3 and caspase-
9
. Moreover, 4c induced a clear increase in the mitotic index, inhibited microtubule assembly in vitro, and
interestingly also acted as a topoisomerase I inhibitor.
Introduction
biochemical mechanism-based screens (cdc2 kinase and cdc25
phosphatase) with IC50 of 70 and 25 µM, respectively.
5
DNA represents one of the most important targets for several
chemotherapeutic drugs. Polycyclic nitrogen heterocycles with
planar structure can be good pharmacophores for classes of
antitumor drugs because they can intercalate between the base
pairs of double-stranded DNA. Well-known cancer chemothera-
peutic agents, such as anthracyclines, camptothecin, and amsa-
crine (Chart 1), characterized by planar polycyclic systems, are
able to interfere with DNA-processing enzymes (topoisomerases
I and II) by forming a ternary complex involving the drug, the
Continuing our research on polycyclic heterocycles containing
6
the pyrrole or indole moieties with antineoplastic activity, and
in our attempts to search for novel antitumor compounds, we
extended our interest to the isoindole system and planned to
synthesize isoindolo[2,1-a]quinoxaline derivatives in order to
evaluate their antiproliferative activity and their targets at cellular
and molecular levels.
Chemistry
1
DNA, and the enzyme.
Our approach to the isoindoloquinoxaline nucleus involved
the annelation of a quinoxaline ring on the isoindole moiety by
using suitably substituted 2-(2′-aminoaryl)-1-cyano-isoindoles
of type 3 as key intermediates. This synthetic route allowed
easy functionalization of the quinoxaline moiety, low cost and
easily available starting material, and high yields. Only two
isoindoloquinoxaline derivatives of type 5 were incidentally
obtained during attempts to synthesize the benzoimidazo[2,1-
a]isoquinoline system. As a matter of fact, reaction of 2-alkynyl-
arylaldehydes with 1,2-phenylendiamine led to an inseparable
mixture of the desired system and the unexpected isoindolo[2,1-
a]quinoxaline in low yields. The components of the mixture,
Quinoxalines and the structurally related quinoxalinones
represent an important class of compounds that are found in a
variety of biologically and medicinally useful agents. A large
number of synthetic quinoxalines showed antineoplastic activ-
2
ity. For instance, substituted (phenoxymethyl)quinoxalinones
demonstrated excellent antagonism of P-glycoprotein and mul-
a
3
tidrug resistant protein (MRP1) in drug-resistant cell lines. A
quinoxaline ring condensed with a pyridine moiety led to
benzo[f]pyrido[4,3-b] and pyrido[3,4-b]quinoxalines derivatives
4
which showed topoisomerases inhibition. Incorporation of an
indole ring gave 5-substituted 2-bromoindolo[3,2-b]quinoxalines
that showed a broad spectrum of antitumor activity with two
7
rather sensitive, could be characterized only as HBF4 salts.
The isolation of derivative 3 was possible by a Strecker-type
synthesis between substituted 1,2-phenylendiamine 1 and ph-
thalaldehyde 2 in water and in the presence of potassium cyanide
and sodium hydrogensulfite. In the case of 3-methyl-1,2-
phenylendiamine 1b, along with 3b was also isolated the isomer
*
Corresponding author. Phone: +39-091-6161606. Fax: +39-091-
6
169999. E-mail: diana@unipa.it.
†
8
Dipartimento Farmacochimico Tossicologico e Biologico, Università
degli Studi di Palermo.
‡
Dipartimento di Scienze Farmaceutiche, Università degli Studi di
1
-cyano-2-(2′-amino-6′-methylphenyl)isoindole. On the other
Padova.
§
Dipartimento di Pediatria, Università degli Studi di Padova.
MRP1, multidrug resistant protein; st-DNA, salmon testes DNA; ICD,
hand, in the case of 4-methoxy-1,2-phenylendiamine 1c, only
derivative 3c was isolated because of the enhanced reactivity
of the amino group placed in the para position with respect to
the methoxy substituent (Scheme 1).
Isoindole quinoxalines 4a-e were prepared by reaction of
the isoindole intermediates 3a-e. Refluxing these latter in acetic
acid brought about the reaction of the amino group with the
carbonitrile group, leading to the quinoxaline ring system. In
a
induced circular dichroism; PI, propidium iodide; LD, linear dichroism;
LDr, reduced LD; PS, phosphatidylserine; ROS, reactive oxygen species;
HE, hydrohethidine; NAO, 10-nonyl acridine orange; FITC, fluorescein
isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate; JC-1, 5,5′,6,6′-
tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine; NCI, National Can-
cer Institute; TGI, total growth inhibition; MG_MID, mean graph midpoint;
P-gp, P-glycoprotein; CD, circular dichroism; HBSS, Hank’s balanced salt
solution; BSA, bovine serum albumin.
1
0.1021/jm070834t CCC: $40.75

2008 American Chemical Society
Published on Web 03/27/2008

2
388 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Diana et al.
Scheme 1.
Chart 1
Table 1. One Dose Primary Anticancer Assay for Isoindoloquinoxalines
situ hydrolysis of the formed imine moiety gave isoindolo-
quinoxalinones 4a-e in excellent yields. Derivatives of the same
ring system of type 5 were obtained, in excellent yields, by
reaction of intermediates 3 with refluxing formic acid (Scheme
4
a–e and 5a,c–e
nonsmall cell-lung
breast
MCF7
CNS
SF-268
compd selected
for 60 cell
compd
NCI-H460
4
4
4
b
b
c
0
2
0
0
0
91
97
78
104
6
27
0
0
10
107
115
97
9
55
1
Y
Y
Y
Y
Y
N
N
N
N
1
). However, the reaction of 3b with formic acid originated a
very complex mixture from which it was impossible to isolate
b. The structure of all synthesized compounds was confirmed
5
4d
0
1
13
4
5
5
5
5
e
a
c
d
e
13
94
96
83
104
by their analytical and spectral data (IR, H NMR, and
C
NMR).
108
Biological Results and Discussion
Antitumor Activity (in Vitro Growth Inhibition and
Cytotoxicity). The isoindoloquinoxaline derivatives 4a-e and
inhibition), and LC50 (concentration leading to 50% net cell
death). The average values of mean graph midpoint (MG_MID)
5
a,c-e were evaluated by the National Cancer Institute (NCI,
Bethesda, MD) in the 3-cell line (MCF7-breast, NCI-H460-
10
were calculated for each of these parameters. Isoindoloqui-
nonsmall cell lung, and SF-268-CNS), one dose primary
-4
noxaline derivatives 5, at the dose of 10 M, showed to be
-
4
anticancer assay (10
M) (Table 1). A compound was
inactive against the three cell lines mentioned above, likely
because of the lack of lactam moiety in the quinazoline nucleus.
An evaluation of the data reported in the Table 2 points out
that compounds 4a-e exhibited antineoplastic activity against
all the human cell lines. The most active compound was the
considered as active when it reduced the growth of any of the
cell lines to 32% or less (negative numbers indicate cell kill).
The compounds which satisfied the criteria set by the NCI for
activity in this assay were scheduled automatically for evaluation
9
against the full panel of tumor cell lines. All isoindoloqui-
3
4
-methoxy derivative 4c (MG_MID ) 7.32), followed by
-methyl 4b, unsubstituted 4a, and 2,3-dimethyl 4d derivatives,
noxaline derivatives of type 4 were selected for evaluation
against the full NCI panel of approximately 60 human cancer
cell lines grouped in disease subpanels including leukemia,
nonsmall-cell lung, colon, central nervous system, melanoma,
ovarian, renal, prostate, and breast tumors cell lines. The
following parameters were determined for every cell line: GI50
which showed similar MG_MID values (5.28, 5.17, and 4.99,
respectively). The 2,3-dichloro derivative 4e (MG_MID ) 4.74)
was the least active. From the available data, it seems that
substituents that enriched the π-electron density of the benzene
moiety of the quinoxaline system improved the antineoplastic
(concentration inhibiting 50% net cell growth), TGI (total growth

Isoindoloquinoxalines, NoVel Potent Antitumor Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2389
a
Table 2. Overview of the Results of the in Vitro Antitumor Screening for Compounds 4a–e : Number of Cell Lines Giving Positive pGI50, pTGI, and
pLC50
b
c
d
pGI50
pTGI
range
pLC50
e
f
g
f
g
f
g
cpd
no.
N
range
MG_MID
N
MG_MID
N
range
MG_MID
4
4
4
4
4
a
a
b
c
d
e
57
57
59
57
56
57
57
59
57
56
6.28–4.60
>8.00–4.51
8.39–5.29
5.80–4.60
6.09–4.48
5.17
5.28
7.32
4.99
4.74
49
48
59
55
53
5.20–4.05
5.38–4.03
7.68–4.51
5.51–4.22
4.73–4.12
b
4.5
30
22
44
50
43
4.50–4.07
4.90–4.02
6.30–4.06
5.21–4.01
4.27–4.03
4.11
4.12
5.20
4.21
4.16
4.53
5.87
4.57
4.42
Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen. pGI50 is the -log of the molar concentration that inhibits 50% net
c
d
cell growth. pTGI is the -log of the molar concentration giving total growth inhibition. pLC50 is the -log of the molar concentration leading to 50% net
e
f
g
cell death. Number of the cell lines investigated. Number of cell lines giving positive GI50, TGI, and LC50. MG_MID (mean graph midpoint) is the
arithmetical mean value for all tested cancer cell lines. If the indicated effect was not attainable within the used concentration interval, the highest tested
concentration was used for the calculation.
activity. The fact that the 2,3-disubstituted derivatives were the
least active indicates that substituents in position 2 were not
tolerated.
The isoindoloquinoxalines showed inhibitory effects in the
growth of all cancer cell lines from micromolar to nanomolar
concentration (Table 3). Derivatives 4, with the exception of
Thus, the antiproliferative effects of isoindolo[2,1-a]quinoxa-
lines 4a-e were evaluated in three human cancer cell lines
expressing high level of 170-kDa P-glycoprotein (P-gp) drug
efflux and MRP. As shown in Table 5, the examined compounds
are equally potent toward (i) parental cells, (ii) cells resistant
to vinblastine and doxorubicine, and (iii) cell sublines resistant
to different drugs.
4
e, were particularly effective against the leukemia subpanel.
In fact, the calculated MG_MID values for the leukemia
subpanel were always higher than the overall cell lines MG_
MID values (∆MG_MID ) 0.24-0.51).
Cell Cycle Analysis. In order to clarify the mechanism(s) of
action of the isoindoloquinoxalines, the effects of different
concentrations of the most active compound 4c on cell cycle
progression of Jurkat cells (pGI50 ) 8.39) after 24 h of drug
exposure were studied. The cell cycle histograms, based on flow
cytometric analyses of 4c at different concentrations, are
illustrated in Figure 1. It can be noted that treatment of Jurkat
cells with 4c at low concentrations led to profound changes of
the cell cycle profile. Untreated cells showed a classical pattern
of proliferating cells proportionally distributed in G1 (48%), S
Table 3 shows that compound 4c was selective toward 88%
-
8
of the tested cell lines and had GI50 values at the 10 M level.
The most sensitive cell lines were HL-60 (TB) (pGI50 7.94),
SR (pGI50 8.00), and K562 (pGI50 8.05), belonging to the
leukemia subpanel; MALME-3 M (pGI50 8.00) and M14 (pGI50
8
8
.00) of the melanoma subpanel; and MDA-MB-435 (pGI50
.39), belonging to the breast cancer subpanel.
Compound 4b, bearing a methyl group in position 4, showed
(
37%), and G2/M (15%) phases. On the contrary, a clear and
excellent response in the breast cancer subpanel, besides
exhibiting selectivity with respect to the leukemia subpanel. The
most sensitive cell lines were MCF7 (pGI50 > 8.00), MDA-
MB-231/ATCC (pGI50 5.62), and MDA-MB-435 (pGI50 5.50).
Derivative 4a was selectively active with respect to breast cancer
and especially active against HS 578T (pGI50 6.28) and MDA-
MB-231/ATCC (pGI50 6.02).
The dichloro derivative 4e had a low pGI50 mean value (4.74)
but showed a good selectivity with respect to the HCC-2998
cell line (pGI50 6.09), belonging to the colon cancer subpanel.
In Table 4, the pTGI and pLC50 values of compounds 4a-e
are reported.
rapid G2/M arrest pattern (lower panel) was observed with a
concomitant decrease of the G1 phase. It was also interesting
to note the appearance of a hypodiploid peak (sub-G1),
indicative of apoptosis, which reached the value of 35% at the
concentration of 62.5 nM.
Mitotic Index. Because 4c caused a massive cell accumula-
tion in the G2/M phase, the appearance of mitotic cells was
quantitatively analyzed. The cells were recognized by optical
microscopy by the presence of dispersed chromosomes in the
cytoplasm and by the disappearance of the nuclear membrane.
It was found that the percentage of mitotic cells (the mitotic
index) increased in a concentration-dependent manner upon
treatment (Figure 2), well in accordance with the cell cycle
analysis data. The concentration needed to arrest 50% of Jurkat
cells at mitosis was about 0.5 µM for compound 4c and was
similar to that of colchicine used as reference drug.
At TGI level, the most interesting compound was 4c, both
in terms of pTGI values (MG_MID ) 5.87) and in terms of
activity against the total number of cell lines (59), followed by
4
d, 4b, 4a, and 4e (MG_MID in the range 4.42-4.57 and
number of sensitive cell lines in the range 48-55). Compound
c showed good selectivity with respect to the MDA-MB-435
pTGI 7.68) and NCI/ADR-RES (pTGI 7.24) cell lines of the
Immunofluorescence Microscopy. As further proof of these
new derivatives interfering with the microtubule network,
compound 4c was checked by immunofluorescence microscopy.
For these experiments, we used a nonsmall cell lung carcinoma
4
(
breast cancer subpanel, the HCC-2998 (pTGI 7.31) cell lines
of the colon subpanel, the HL-60(TB) (pTGI 7.22) of the
leukemia subpanel, OVCAR-3 (pTGI 7.19) of the ovarian
subpanel, and SF-539 (pTGI 7.15) and SNB-75 (pTGI 7.08) of
the CNS subpanel.
At LC50 level, the best response was observed in the case of
the HCC-2998 (pLC50 6.30) colon cancer cell lines obtained
by 4c.
Effect of Isoindolo[2,1-a]quinoxalines on Multidrug Re-
sistant Cell Lines. Although many anticancer drugs in clinical
use are effective in the treatment of different kinds of tumors,
their potential is limited by the development of drug resistance.
Resistance can be intrinsic or acquired, but in either case, tumors
become refractory to a variety of structurally different drugs.
(
A-549) cell line. As shown in Figure 3 (panel A), the
microtubule network in control cells exhibited normal organiza-
tion and arrangement. On the contrary, compound 4c, at different
concentrations and after 24 h of incubation, (panels C 5 µM
and D 2.5 µM) completely disrupted the tubulin network. Cells
showed an evident characteristic rounded-up morphology caused
by disaggregation of microtubules in both interphase and mitotic
phases after 24 h of treatment. These effects resembled those
of vinblastine chosen as reference compound (panel B).
Effect of Isoindolo[2,1-a]quinoxaline 4a–e on Microtu-
bule Polymerization in Vitro. As the immunofluorescence
studies suggested that 4c impaired the formation or the stability

2
390 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Diana et al.
of microtubules in the cells and microtubules are the major
apparatus responsible for mitotic division, the effects of isoin-
dolo[2,1-a]quinoxaline 4a-e on the polymerization of micro-
tubule protein isolated from porcine brain were examined. The
assay utilized a fluorescent compound which binds to tubulin
and microtubules. Polymerization was followed by fluorescent
enhancement because of incorporation of a fluorescent reporter
Table 3. Inhibition of in Vitro Cancer Cell Lines by
Isoindolo[2,1-a]quinoxalines 4a–e
a
b
pGI50
cell line
CCRF-CEM
4a
4b
4c
4d
4e
Leukemia
5.23
ND
5.43
5.44
5.54
ND
5.92
ND
5.70
5.74
5.81
ND
7.52
7.94
7.44
7.11
8.00
8.05
5.00
ND
5.17
5.25
5.59
ND
4.66
ND
4.65
4.73
4.76
ND
c
HL-60 (TB)
RPMI-8226
MOLT-4
SR
11
into the microtubules as polymerization occurred. Figure 4
shows that in the control sample (tubulin alone without addition
of any test agent), the fluorescence increased over time. On the
contrary, the tested compounds 4a-e clearly induced a con-
centration-dependent inhibition of tubulin polymerization. In
particular, a complete inhibition of microtubule assembly was
observed for 4c at a concentration of 5 µM. Colchicine, the
reference drug, completely inhibited the tubulin polymerization
at a concentration of 3 µM, whereas at the same concentration,
Taxol significantly promoted tubulin polymerization (data not
shown). The other derivatives presented a similar behavior,
although a complete inhibition of the tubulin polymerization
was observed at concentrations higher than 10 µM. In further
experiments, the effects of 4c on preassembled microtubules
were investigated. None of the tested compounds were able to
induce microtubule depolymerization (data not shown), indicat-
ing that the action of these compounds was strictly related to
the interference with microtubule assembly.
K-562
Nonsmall Cell Lung Cancer
A549/ATCC
EKVX
5.23
5.32
5.36
5.40
5.54
4.95
4.62
5.11
4.86
5.30
5.25
5.45
5.40
5.49
4.76
4.87
5.72
5.19
7.17
7.04
7.51
6.00
7.34
7.37
7.08
7.46
7.22
4.83
4.87
5.57
4.81
4.82
4.83
4.78
5.35
4.84
4.72
4.66
ND
4.74
4.73
4.76
4.87
4.73
4.99
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO-205
HCC-2998
HCT-116
HCT-15
HT29
5.34
5.13
5.34
5.57
5.28
5.66
5.47
5.24
7.39
7.74
7.40
7.58
7.47
7.48
7.58
4.82
5.39
5.80
5.21
5.22
5.15
5.42
4.53
6.09
4.68
4.78
4.54
4.76
4.80
5.23
5.67
5.48
5.36
4.72
5.43
KM12
SW-620
CNS Cancer
DNA Binding Studies. As isoindolo[2,1-a]quinoxaline 4a-e
exhibit a planar structure, the interaction of compound 4c, as
representative, with DNA was evaluated with a series of
spectrophotometric studies. The binding of 4c to DNA in
buffered aqueous solution was monitored by absorption and
fluorescence spectroscopy (Figure 5). The absorption of the
compound was recorded both in the absence and in the presence
of salmon testes DNA (st-DNA). The maximum absorption of
compound 4c exhibited a weak bathochromic shift of about 2
nm with respect to the free drug in the presence of nucleic acid
(Figure 5A); 4c also showed a hypochromism of the signal
intensity, suggesting the association of the drug with DNA. Most
notably, isosbestic points appeared in the titration, indicating
that one type of drug-DNA complex was almost exclusively
formed. The spectrophotometric titration data for 4c bound to
the DNA were used to estimate the binding constant as well as
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
4.67
5.52
5.58
5.41
4.81
4.51
5.18
7.13
7.57
7.64
7.41
7.56
7.36
4.81
4.91
4.95
4.79
4.81
5.39
4.62
4.79
4.72
4.66
4.67
4.79
4.75
5.29
5.05
4.87
5.49
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
5.52
5.21
5.08
4.90
4.84
5.16
4.93
4.78
5.16
7.15
8.00
8.00
6.80
7.28
7.55
5.29
7.13
5.69
4.65
4.87
4.74
4.83
4.87
4.69
4.78
4.76
4.77
4.64
4.60
4.48
4.70
4.69
4.78
4.60
5.19
4.69
4.65
5.72
4.82
5.49
Ovarian Cancer
IGROV1
4.99
4.73
5.14
4.98
5.28
4.91
5.43
5.32
5.17
4.75
5.45
4.56
7.13
7.65
7.04
7.00
7.24
7.41
4.91
4.86
4.87
4.86
5.15
4.74
4.76
4.73
4.69
4.77
4.73
4.74
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
12
binding-site size according to McGhee and von Hippel. The
corresponding binding isotherm, represented as a Scatchard
Renal Cancer
1
3
plot, is presented in the inset of Figure 5A). The binding
7
86–0
5.37
5.23
4.79
5.39
5.66
5.04
5.74
4.57
5.25
7.38
7.65
6.90
7.45
7.69
7.24
6.29
7.10
4.97
4.67
4.99
5.27
4.92
5.22
4.70
4.84
4.60
4.80
4.81
4.69
4.76
4.80
4.78
4.75
A498
4.95
5.74
4.75
5.20
5.78
4.69
4.90
constants K revealed a high binding affinity of 4c (K ) 2.7 ×
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
5
-1
1
0 M ), and the binding-site size n was 3.6 (in bases), in
accordance with the intercalative binding mode used in the
12
neighbor-exclusion model. Spectrofluorometric titrations of st-
DNA to compound 4c were also performed (Figure 5B). The
emission intensity of the free drug was quenched upon addition
of st-DNA without any shift of the emission maximum.
Prostate Cancer
PC-3
DU-145
5.24
5.12
5.41
5.28
7.43
7.58
4.92
4.86
4.76
4.75
The Stern-Volmer plot derived from the fluorimetric titration
of st-DNA exhibited linear behavior of the titration curve up to
a concentration of 40 µM (Figure 5B). From these data,
quenching efficiency was estimated by the Stern-Volmer
Breast Cancer
MCF7
4.98
>8.00
4.92
5.62
4.66
5.50
4.92
4.94
5.28
7.55
7.67
7.25
7.29
8.39
7.26
7.32
7.32
4.93
4.87
5.08
4.60
5.22
4.71
4.81
4.99
4.67
4.75
4.76
4.70
4.77
4.58
4.51
4.74
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
5.19
6.02
6.28
4.79
4.72
5.13
5.17
-1
constant KSV (4c: KSV ) 31765 M ).
MDA-MB-435
BT-549
T-47D
MG_MID
To further study the interaction of 4c with DNA, circular
dichroism (CD) spectra of DNA in the presence of increasing
concentration of 4c were recorded. The CD spectra of DNA
revealed a significant perturbation of the DNA signal because
the positive CD band of the DNA at 275 nm increased upon
successive addition of 4c (Figure 6, upper panel). Also,
significant positive induced CD (ICD) signals were observed
in the chromophore absorption region of 4c (300-400 nm),
d
a
Data obtained from NCI’s in vitro disease-oriented tumor cells screen.
b
pGI50 is the -log of the molar concentration causing 50% growth inhibition
c
d
of tumor cells. ND: not determined. MG_MID (mean graph midpoint)
is the arithmetical mean value for all tested cancer cell lines. If the indicated
effect was not attainable within the used concentration interval, the highest
tested concentration was used for the calculation.

Isoindoloquinoxalines, NoVel Potent Antitumor Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2391
a
Table 4. Inhibition of in Vitro Cancer Cell Lines by Isoindolo[2,1-a]quinoxalines 4a–e
b
c
pTGI
pLC50
cell line
CCRF-CEM
RPMI-8226
MOLT-4
SR
HL-60 (TB)
K-562
4a
4b
4c
4d
4e
4a
4b
4c
4d
4e
Leukemia
4.22
4.65
5.06
5.11
4.90
5.15
5.25
5.38
ND
5.84
5.85
5.67
5.69
7.22
5.99
4.51
4.53
4.71
5.24
ND
4.31
4.31
4.43
4.41
ND
<4.00
<4.00
<4.00
<4.00
ND
4.13
4.22
4.68
4.90
ND
<4.00
5.16
4.12
4.12
5.45
5.29
4.01
<4.00
4.15
4.31
ND
<4.00
<4.00
4.14
4.06
ND
d
ND
ND
ND
ND
ND
ND
ND
ND
ND
Nonsmall Cell Lung Cancer
A549/ATCC
EKVX
4.49
4.16
4.75
4.73
4.86
4.44
4.33
<4.00
4.49
<4.00
4.35
4.82
4.49
4.56
4.41
4.34
5.00
4.19
5.78
4.53
5.91
4.80
6.28
5.47
5.59
5.95
5.64
4.42
4.50
5.09
4.52
4.44
4.49
4.52
4.69
4.51
4.46
4.36
ND
4.46
4.45
4.51
4.57
4.48
4.51
<4.00
<4.00
4.28
<4.00
<4.00
4.29
5.22
<4.00
5.41
4.02
4.12
4.54
4.23
4.05
4.16
4.26
4.20
4.18
4.20
4.06
ND
4.18
4.16
4.25
4.26
4.24
4.03
HOP-62
HOP-92
4.29
<4.00
<4.00
4.06
4.40
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
4.26
<4.00
<4.00
5.11
5.29
5.00
<4.00
4.05
<4.00
<4.00
4.12
4.50
<4.00
Colon Cancer
COLO-205
HCC-2998
HCT-116
HCT-15
HT29
<4.00
4.68
4.46
4.79
4.93
4.50
4.88
4.64
4.75
5.94
7.31
5.88
5.94
5.83
5.82
5.85
<4.00
4.40
4.73
4.45
4.52
<4.00
4.51
4.53
<4.00
4.24
<4.00
4.39
5.24
6.30
<4.00
4.30
<4.00
4.36
4.78
5.51
4.66
<4.00
4.67
4.48
4.91
4.31
4.47
5.35
5.21
4.23
4.73
4.03
<4.00
5.34
4.21
4.26
<4.00
<4.00
4.44
<4.00
<4.00
<4.00
KM12
4.40
4.08
<4.00
4.37
5.22
4.28
4.25
SW-620
<4.00
<4.00
5.38
<4.00
4.27
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
4.06
4.45
4.65
<4.00
4.13
4.87
5.07
4.60
4.80
4.32
<4.00
4.71
5.53
5.98
7.15
5.39
7.08
5.82
4.49
4.55
4.57
4.44
4.52
4.85
4.37
4.52
4.40
4.25
4.27
4.52
<4.00
4.15
4.12
4.06
5.38
4.16
4.18
4.19
4.08
4.23
4.32
4.12
<4.00
4.26
4.12
4.39
5.56
4.09
<4.00
<4.00
4.15
<4.00
<4.00
4.34
<4.00
<4.00
5.30
<4.00
<4.00
4.26
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
4.92
4.33
4.64
4.41
<4.00
4.83
4.48
4.80
4.74
5.75
5.47
4.89
5.60
5.60
5.93
4.51
5.72
5.40
4.43
4.57
4.46
4.45
4.53
4.45
4.50
4.51
4.49
4.40
4.27
<4.00
4.38
4.31
4.52
4.07
4.07
4.19
4.14
<4.00
4.22
4.14
4.34
4.37
5.35
5.07
5.44
5.11
5.18
5.46
4.13
5.36
5.10
4.20
4.28
4.18
4.08
4.19
4.20
4.22
4.25
4.61
4.17
4.20
4.51
4.11
4.16
<4.00
<4.00
4.62
<4.00
<4.00
4.31
<4.00
<4.00
4.06
4.03
4.55
<4.00
<4.00
4.02
4.26
Ovarian Cancer
IGROV1
4.59
4.44
4.05
4.45
4.48
4.45
<4.00
4.55
5.64
7.19
5.29
5.10
5.63
5.79
4.57
4.55
4.56
4.56
4.59
4.46
4.51
4.49
4.30
4.51
4.49
4.48
4.20
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
5.07
5.54
4.24
4.24
4.25
4.26
4.09
4.19
4.25
4.24
<4.00
4.26
4.24
4.21
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
4.14
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.18
<4.00
<4.00
4.11
<4.00
5.07
Renal Cancer
7
86–0
4.74
4.57
5.14
4.45
4.52
5.20
4.43
4.57
4.49
4.38
4.65
5.06
4.42
4.72
4.06
<4.00
5.73
5.87
5.74
5.70
5.50
5.68
5.61
5.65
4.64
4.40
4.52
4.54
4.46
4.51
4.53
4.52
4.48
4.26
4.19
4.46
4.15
<4.00
4.50
4.18
4.25
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
5.34
5.40
4.32
4.14
4.27
4.31
4.20
4.25
4.23
4.27
4.20
4.25
4.27
4.23
4.25
4.26
4.26
4.22
A498
4.40
4.63
4.75
4.56
4.69
4.47
4.55
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
5.36
5.29
<4.00
<4.00
5.18
5.21
Prostate Cancer
PC-3
DU-145
4.54
4.42
4.35
4.76
5.59
4.94
4.59
4.52
4.51
4.50
<4.00
<4.00
<4.00
<4.00
4.27
4.19
4.25
4.25
4.38
5.37
Breast Cancer
MCF7
<4.00
4.49
4.99
4.46
4.51
4.19
<4.00
4.5
4.90
4.46
4.76
4.19
4.94
4.23
4.31
4.53
5.84
7.24
6.13
5.57
7.68
5.69
5.34
5.87
<4.00
4.40
4.50
4.49
4.28
4.52
4.24
4.12
4.42
<4.00
<4.00
4.23
4.40
5.26
5.00
<4.00
4.13
4.14
NCI/ADR-RES
MDA-MB-31/ATCC
HS 578T
4.50
4.63
4.22
4.70
4.39
4.30
4.57
<4.00
<4.00
<4.00
4.02
4.25
5.51
4.22
4.21
<4.00
<4.00
<4.00
5.20
<4.00
<4.00
MDA-MB-435
BT-549
T-47D
MG_MID
4.23
4.30
4.26
<4.00
<4.00
4.11
<4.00
<4.00
4.12
4.07
<4.00
<4.00
4.16
<4.00
<4.00
e
5.2
4.21
a
b
Data obtained from NCI’s in vitro disease-oriented tumor cells screen. pTGI is the -log of the molar concentration giving total growth inhibition.
c
d
e
pLC50 is the -log of the molar concentration leading to 50% net cell death. ND: not determined. MG_MID (mean graph midpoint) is the arithmetical
mean value for all tested cancer cell lines. If the indicated effect was not attainable within the used concentration interval, the highest tested concentration
was used for the calculation.
1
7,18
indicating an orientation of the transition dipole of the molecule
perpendicular to the long axis of the binding pocket.
Linear Dichroism. By measuring the flow linear dichroism
of the different drug-nucleic acid complexes, it was possible
to elucidate the binding geometry of 4c bound to DNA.
1
4–16
Figure 6 (lower panel) shows absorbance, LD, and reduced LD
(LDr) spectra for 4c at different [drug]/[DNA] ratios. Examina-
tion of the figures revealed that the LD spectra of 4c complexed

2
392 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Diana et al.
Table 5. Effect of Isoindolo[2,1-a]quinoxalines 4a–e against Different
Drug-Resistant Cell Lines
a
growth inhibition (GI50) [µM]
wt
MDR
b
cpd
MCF-7
MCF-7
RI
4
4
4
4
4
a
b
c
d
e
6.5 ( 0.7
3.7 ( 0.4
0.17 ( 0.02
5.1 ( 0.5
3.3 ( 0.2
10.6 ( 1.4
8.1 ( 1.2
0.15 ( 0.02
9.2 ( 0.8
7.6 ( 0.6
1.6
2.2
0.9
1.8
2.3
wt
Vbl-100
b
cpd
CEM
CEM
RI
4
4
4
4
4
a
b
c
d
e
4.7 ( 0.5
2.9 ( 0.3
0.02 ( 0.002
15.5 ( 1.6
2.2 ( 0.31
3.8 ( 0.4
3.0 ( 0,3
0.02 ( 0.002
15.1 ( 1.6
1.9 ( 0.2
0.80
1.04
1.0
0.97
0.86
Figure 2. Mitotic index determinations. Jurkat cells were treated with
4
c at the indicated concentrations. A total of 400 cells/treatment was
wt
Doxo
b
scored for the presence of mitotic figures by contrast phase microscopy,
and the mitotic index was calculated as the proportion of cells with
mitotic figures. Colchicine was used as reference compound. Data are
expressed as mean ( SEM of three independent experiments.
cpd
LoVo
LoVo
RI
c
4
4
4
4
4
a
b
c
d
e
ND
ND
ND
0.13 ( 0.02
ND
ND
ND
1.0
ND
ND
ND
0.13 ( 0.013
ND
ND
ND
a
Concentration causing 50% growth inhibition of tumor cells determined
by MTT test after 72 h of incubation. Data represented as mean ( SEM of
b
c
three independent experiments. RI: resistance index. ND: not determined.
Figure 3. Immunofluorescences images of A-549 cells treated with
anti-ꢀ-tubulin antibody and with a secondary antibody tetramethyl
rhodamine isothiocyanate (TRITC)-conjugated and then observed by
confocal microscopy (panel A). Cells were exposed to concentrations
of 5 µM (panel C) and 2.5 µM (panel D) of compound 4c for 24 h. As
reference, vinblastine (panel B) at the concentration of 1 µM was used.
suggesting that the DNA became better oriented in the hydro-
dynamic field because of a stiffening of the helix upon binding
1
7
of the drug. The negative LD signals in the long-wavelength
absorption (S0-S1 transition, 300-450 nm) of 4c revealed that
it was oriented perpendicularly to the flow field, usually caused
by an intercalation into the DNA helix. This assumption was
further corroborated by the observation that the intensity of the
DNA signal increased on addition of the drug.
Figure 1. Histograms of flow cytometry data of Jurkat cells treated at
the indicated concentrations of 4c. After 24 h of treatment, cells were
labeled with propidium iodide and analyzed by flow cytometry as
described in the Experimental Section.
The LDr spectrum provided further information on the average
orientation of the transition moment of the dye relative to those
of the DNA bases and allowed us to distinguish between
homogeneous and heterogeneous binding. A nearly constant
value of LDr over the range 310-400 nm was observed (Figure
6, lower panel), which unambiguously proved an almost
exclusive intercalation of 4c into the DNA.
with DNA were negative in the 230-300 nm region where DNA
absorbed. A significant increase in the values of LD of the DNA
band at 260 nm for these drug-DNA complexes was observed,

Isoindoloquinoxalines, NoVel Potent Antitumor Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2393
Figure 4. Effects of compound 4c on microtubule assembly. Purified tubulin was incubated in the absence or in the presence of the compound at
the indicated concentrations. Fluorescence measurement (λex ) 355 nm and λem) 450 nm) was determined every minute for 60 min at 37 °C.
Polymerization was initiated by the addition of tubulin.
Topoisomerases Assay. DNA relaxation experiments were
performed with both topoisomerase I and II. Supercoiled plasmid
pBR322 was incubated with human topoisomerase I and 4a-e
in concentrations ranging from 0.5 to 200 µM. The unwinding
of the plasmid was monitored by agarose gel electrophoresis.
For compound 4c, the picture of the gel (Figure 7, panel A3)
indicated that at low concentrations, the intensity of the slowest
migrating band (corresponding to nicked + fully relaxed DNA)
increased significantly, whereas at higher concentrations, the
relaxation of DNA was inhibited, as observed with the reference
topoisomerase I poison camptothecin used at a concentration
of 20 µM. To exclude a nonspecific effect due to a direct
interaction of 4c with DNA, the relaxation assay was repeated
on ethidium bromide containing agarose gel, as shown in Figure
Analogous experiments were performed with human topoi-
somerase II, and the results indicated no effect of isoindolo[2,1-
a]quinoxaline 4a-e on this enzyme (data not shown).
Loss of Plasma Membrane Asymmetry during Apopto-
sis. To better characterize drug-induced apoptosis, a biparametric
cytofluorimetric analysis, using propidium iodide (PI) and
AnnexinV-fluorescein isothiocyanate (FITC) which stain DNA
19
and phosphatidylserine (PS) residues, was performed. Because
externalization of PS occurs in the first stages of apoptosis,
Annexin-V staining identifies apoptosis at an earlier stage than
sub-G1 appearance, which represents a later event of apoptosis
as a consequence of nuclear changes such as DNA fragmenta-
tion. After drug treatment for 24 h, Jurkat cells were labeled
with the two dyes and washed, and the resulting red (PI) and
green (FITC) fluorescence was monitored by flow cytometry.
It can be observed from Figure 8 that 4c provoked a significant
induction of apoptotic cells in a concentration-dependent manner
after 24 h of treatment. These findings prompted us to further
investigate the apoptotic machinery after treatment with the test
compound.
7
, panel B3. Under these conditions, the presence of nicked
DNA molecules was observed, indicating that compound 4c may
stimulate DNA cleavage by topoisomerase I.
As compared to compound 4c, the other derivatives 4a, b,
d, and e exhibited a less pronounced influence on the topoi-
somerase activity (Figure 7, panels A1 and A2). The topoi-
somerase-induced relaxation was inhibited only at relatively high
concentrations. Moreover, the analysis of the relaxation assay
with an ethidium bromide-containing agarose gel revealed that
over the whole concentration range of compounds 4a, b, d, and
e (1-200 µM), a small and insignificant amount of single strand
breaks were formed, suggesting that these derivatives did not
efficiently stabilize the topoisomerase cleavage complex but
acted only as catalytic topoisomerase I inhibitors.
Variations of the Mitochondrial Potential. Mitochondria
2
0,21
play an essential role in the propagation of apoptosis.
It is
well established that, at an early stage, apoptotic stimuli alter
the mitochondrial transmembrane potential (∆ψmt). To address
whether 4c affected the ∆ψmt, it was monitored by fluorescence
of the dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-
carbocyanine (JC-1), which is considered as a reliable probe to
2
2
assess ∆ψmt. JC-1 has the unique property of forming red

2
394 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Diana et al.
Figure 5. Spectrophotometric titration (A) of st-DNA to 4c in ETN
buffer (0.01 M, pH ) 7.0, T ) 25 °C). The Scatchard plot of the
spectrophotometric titration is presented in the inset. The solid lines
represent the best fit of the McGhee and von Hippel equation to the
experimental data. Fluorometric titration (B) of st-DNA to 4c in ETN
buffer (0.01 M, pH ) 7.0, T ) 25 °C). The Stern-Volmer plot is
presented in the inset.
Figure 6. Upper panel: circular dichroism spectra of 4c complexed to
st-DNA at different [dye]/[DNA] ratios from a to f (a ) 0.00, b )
0
.02, c ) 0.04, d ) 0.08, e ) 0.2, and f ) 0.4) in ETN buffer (0.01 M,
pH ) 7.0, T ) 25 °C). Lower panel: absorbance A, linear dichroism
LD, and reduced linear dichroism LD spectra of mixtures of st-DNA
r
fluorescent aggregates locally and spontaneously under high
mitochondrial ∆ψmt, whereas the monomeric form fluoresces
in green. Treated Jurkat cells in the presence of 4c exhibited a
dramatic shift in fluorescence in a dose-dependent manner
compared to the control cells, indicating depolarization of
mitochondrial membrane potential. The percentage of cells with
low mitochondrial potential for 4c is depicted in Figure 9 (upper
panel). It is interesting to note that the disruption of ∆ψmt is
associated with the appearance of Annexin-V positivity in the
treated cells. As a matter of fact, the dissipation of ∆ψmt is
characteristic of apoptosis and has commonly been observed
with a variety of anticancer drugs, irrespective of the cell type.
Mitochondrial Generation of Reactive Oxygen Species
and 4c at different [drug]/[DNA] ratios (a ) 0.00, b ) 0.04, c ) 0.08,
and d ) 0.2) in ETN buffer (0.01 M, pH ) 7.0).
specifically binds to cardiolipin, and its binding affinity and
fluorescence properties depend on the oxidation state of car-
diolipin. NAO binds with high affinity to a non-oxidized
cardiolipin in a 2:1 ratio, whereas in the case of oxidized
cardiolipin, NAO has been reported to bind this phospholipid
with a decreased affinity reflected by a lower fluorescence
26
intensity. Therefore, oxidative stress localized in mitochondria
can be assessed by measuring the fluorescence of NAO. Jurkat
treated cells showed (Figure 9, lower panel) a progressive and
remarkable decrease in mean NAO fluorescence with increasing
concentration of 4c,consistent with a loss in cardiolipin content.
Caspase Activation. The role of the caspases in apoptosis
induction was also studied. In fact, programmed cell death is
(
ROS). Mitochondrial membrane depolarization is associated
23
with mitochondrial production of ROS. It was therefore
investigated whether ROS production had increased after
treatment with the test compound. The probe hydrohethidine
2
7,28
associated with activation of this large family of proteases.
24
(HE), the fluorescence of which appears if ROS are generated,
Caspases are synthesized as proenzymes activated by cleavage.
Caspase-8 and caspase-9, called apical caspases, are usually the
first to be stimulated in the apoptotic process, and then, they
activate effector caspases such as caspase-3.
was utilized in order to investigate the effects of the test
compound on the production of oxygen species during apoptosis.
HE is oxidized by superoxide anion into an ethidium ion, which
emits red fluorescence. Superoxide is produced by mitochondria
because of a shift from the normal 4-electron reduction of O2
to a 1-electron reduction when cytochrome C is released from
mitochondria. The results presented in Figure 9 (middle panel)
show that 4c significantly induced the production of ROS in
comparison to the control cells.
Among the apical caspases, caspase-9 is activated in response
to DNA damage, whereas caspase-8 is one effector of the death
2
9
receptor Fas (CD95, APO-1). Caspase-3, in particular, is
essential to the propagation of the apoptotic signal after the
exposure to many DNA-damaging agents and anticancer drugs,
and in many cases, its activation is associated with a dissipation
3
0,31
Moreover, the damage produced by ROS in mitochondria by
assessing the oxidation state of cardiolipin, a phospholipid
restricted to the inner mitochondrial membrane, was evaluated
of the mitochondrial potential.
The results showed that compound 4c was able to induce
the activation of caspase-3 and in particular caspase-9. A modest
activity of caspase-8 was also recorded, suggesting that the
2
5
by using the specific dye NAO. This fluorescent probe

Isoindoloquinoxalines, NoVel Potent Antitumor Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2395
Figure 7. Effect of increasing concentrations of compounds 4a-e on the relaxation of pBR322 plasmid DNA by human topoisomerase I. Supercoiled
pBR322 DNA was incubated with five units of topoisomerase I in the absence or in the presence of the indicated concentrations of compounds
4
a-e expressed in µM. Camptothecin was used as reference compound at a concentration of 20 µM. DNA samples were separated by electrophoresis
on agarose gel without (panels A1, A2, and A3) or with (panels B1, B2, and B3) ethidium bromide. Sc, supercoiled; Rel, relaxed; Nick, nicked.
In our cell cycle study, cells treated with 4c accumulated in
G2-M phase with concomitant loss of the G1 phase. By using in
vitro tubulin polymerization assay, it was found that 4c inhibited
tubulin polymerization in a concentration-dependent manner, in a
way similar to colchicine and vinblastine, indicating that these
compounds can be classified as microtubule depolymerizing agents.
Moreover, the mitotic index determinations and the immunofluo-
rescence studies clearly confirmed the interaction with tubulin. It
is interesting to note that, to the best of our knowledge, the structure
of isoindolo[2,1-a]quinoxaline derivatives does not resemble any
of the known antimitotic drugs.
The spectrophotometric results indicate that 4c bound to DNA
by intercalation, and consequently, the macromolecule may be
considered as a potential target for this class of compounds.
Figure 8. Percentage of Annexin-V positive cells after 24 h of
incubation at the indicated concentrations of compound 4c. Data are
expressed as mean ( SEM of three independent experiments.
Many reports showed that DNA intercalators could be
32,33
topoisomerase I and/or topoisomerase II inhibitors.
By using
action of the test compound is integrated at the mitochondrial
level (Figure 10).
a DNA relaxation assay, it was determined that compound 4c
inhibited topoisomerase I catalytic activity but not topoisomerase
II activity.
Conclusion
Further studies show that 4c is a potent inducer of apoptosis in
Jurkat cell line. The induction of apoptosis is associated with (i)
dissipation of the mitochondrial transmembrane potential, (ii)
production of reactive oxygen species and cardiolipin oxidation,
and (iii) activation of caspase-3 and caspase-9. All of these effects
are associated with both antimitotic drugs and topoisomerase I
The present results show that isoindolo[2,1-a]quinoxaline deriva-
tives, obtained in very good yields with a straightforward synthesis,
exhibit high cytotoxic activity against a very wide range of human
tumor cell lines with GI50 values reaching nanomolar concentra-
tions. More importantly, these compounds are also found to be
active in cells overexpressing P-gp, suggesting that these derivatives
might be useful in treating drug-refractory patients. At the molecular
level, such compounds act through inhibition of tubulin polymer-
ization and topoisomerase I activity.
34–37
inhibitors.
In conclusion, these results, in their whole, suggest that isoin-
dolo[2,1-a]quinoxaline derivative 4c acts on both tubulin and

2
396 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Diana et al.
Figure 10. Caspase activity induced by compound 4c. Jurkat cells
were incubated in the presence of 4c at a concentration of 500 nM.
After 24 h of treatment, cells were harvested and assayed for caspase-
3
, caspase-8, and caspase-9 activity by using the cell-permeable
substrates for caspase-3 (FITC-DEVD-fmk), caspase-8 (FITC-IETD-
fmk), and caspase-9 (FITC-LEDH-fmk). After 1 h of incubation at 37
°
C, the cells were washed and analyzed by flow cytometry. Data are
represented as fold increase of enzymes activity, in comparison to the
control.
as internal reference). Column chromatography was performed with
Merck silica gel 230-400 mesh ASTM or with Sepacore Buchi
apparatus. Elemental analyses were within (0.4% of the theoretical
values and were performed with a VARIO EL III elemental
analyzer.
Synthesis of Substituted 1-Cyano-2-(2′-aminophenyl)isoin-
doles 3a–e. To a solution of sodium hydrogen sulfite (1.56 g, 0.015
mol) in water (38 mL), phthalaldehyde 2 (2 g, 0.015 mol) was
added. The mixture was stirred until the solid was dissolved, and
the appropriate 1,2-phenylenediamine 1a-d (0.015 mol) was added.
The reaction was heated on a steam bath for 30 min, then KCN
(
3.39 g, 0.052 mol) in water (8.0 mL) was added, and the mixture
was heated for an additional 90 min. The solid formed upon cooling
was filtered and purified by chromatography. In the case of
derivative 3e, the 1,2-phenylenediamine 1e was first dissolved in
DMF (15 mL) and then added to the reaction mixture.
1-Cyano-2-(2′-aminophenyl)isoindole 3a. This compound was
purified by flash chromatography by using dichloromethane as
eluent to give a solid: yield 65%; mp 150-151 °C; IR 3473, 3363
1 1
2 6 2
NH ) δ 5.19 (s, 2H, NH ),
), 2202 (CN) cm- ; H NMR (DMSO-d
(
6
7
7
.70 (t, 1H, J ) 7.2 Hz, H-5′), 6.94 (d, 1H, J ) 7.2 Hz, H-3′),
.15-7.19 (m, 3H, H-4′, H-5, H-6′), 7.31 (t, 1H, J ) 8.2 Hz, H-6),
.66 (d, 1H, J ) 8.2 Hz, H-4), 7.78 (d, 1H, J ) 8.2 Hz, H-7), 7.86
Figure 9. Assessment of mitochondrial dysfunction after treatment
with compound 4c. Upper panel: induction of loss of mitochondrial
membrane potential after 24 h of incubation of Jurkat cells by 4c at
the indicated concentrations. Cells were stained with the fluorescent
probe JC-1 and analyzed by flow cytometry. Middle panel: mitochon-
drial production of ROS in Jurkat cells. After 24 h of incubation with
13
(
(
6
s, 1H, H-3); C NMR (DMSO-d ) δ 93.9 (s), 114.0 (s), 115.8
d), 116.1 (d), 117.5 (d), 121.5 (d), 122.2 (d), 122.4 (d), 122.5 (s),
24.1 (s), 125.5 (d), 127.8 (d), 130.4 (d), 131.2 (s), 144.1 (s). Anal.
) C, H, N.
-Cyano-2-(2′-amino-3′-methylphenyl)isoindole 3b. This com-
1
15 11 3
(C H N
1
pound was purified by a Sepacore Buchi apparatus by using
4c, cells were stained with HE and analyzed by flow cytometry. Lower
dichloromethane:cyclohexane (7:3) as eluent to give compound 3b:
panel: decrease of mean fluorescence intensity of the cardiolipin-binding
dye 10-N-nonyl-acridine orange (NAO) in Jurkat cells after treatment
for 24 h with 4c at the indicated concentrations and analyzed by flow
cytometry. Data are expressed as mean ( SEM of three independent
experiments.
-
1
yield 40%; mp 62-63 °C; IR 3481, 3388 (NH
2
), 2200 (CN) cm
), 3.55 (s, 2H, NH ), 6.80 (t,
H, J ) 7.6 Hz, H-5′), 7.10 (d, 1H, J ) 7.6 Hz, H-6′), 7.14 (d, 1H,
J ) 7.6 Hz, H-4′), 7.23 (t, 1H, J ) 8.1 Hz, H-5), 7.31 (t, 1H, J )
.1 Hz, H-6), 7.44 (s, 1H, H-3), 7.69 (d, 1H, J ) 8.1 Hz, H-4),
;
1
H NMR (CDCl
3
) δ 2.25 (s, 3H, CH
3
2
1
8
1
3
topoisomerase I. Considering their interesting biochemical mech-
anism of action and the potent activity against a very wide range
of human cell lines, these new derivatives deserve further inves-
tigations as promising antitumor agents. In particular, it will be
interesting to determine whether tubulin or topoisomerase I is the
principal target of isoindolo[2,1-a]quinoxaline in tumor cells.
7.73 (d, 1H, J ) 8.1 Hz, H-7); C NMR (CDCl
(
3
) δ 17.6 (q), 92.2
s), 113.0 (s), 117.9 (d), 118.3 (d), 120.5 (d), 120.9 (d), 123.2 (d),
1
23.8 (s), 124.1 (s), 124.6 (s), 125.5 (d), 125.9 (d), 131.8 (s), 131.9
) C, H, N.
Further elution gave 1-cyano-2-(2′-amino-6′-methylphenyl)isoin-
dole: yield 45%; mp 87-88 °C; IR 3483, 3390 (NH ), 2202 (CN)
), 3.45 (s, 2H, NH ),
.69 (d, 1H, J ) 8.1 Hz, H-3′), 6.75 (d, 1H, J ) 8.1 Hz, H-5′),
.16 (t, 1H, J ) 8.1 Hz, H-4′), 7.20 (t, 1H, J ) 7.6 Hz, H-5), 7.26
(
13 3
d), 140.5 (s). Anal. (C16H N
2
-1
1
cm ; H NMR (CDCl
3
) δ 1.98 (s, 3H, CH
3
2
6
7
Experimental Section
Chemistry. All melting points were taken on a Buchi-Tottoli
(t, 1H, J ) 7.6 Hz, H-6), 7.35 (s, 1H, H-3), 7.71 (d, 1H, J ) 7.6
13
capillary apparatus and are uncorrected; IR spectra were determined
3
Hz, H-4), 7.74 (d, 1H, J ) 7.6 Hz, H-7); C NMR (CDCl ) δ
1
in bromoform with a Jasco FT/IR 5300 spectrophotometer; H and
17.0 (q), 94.7 (s), 113.6 (s), 114.1 (d), 118.5 (d), 120.0 (d), 120.3
(d), 121.0 (d), 123.2 (d), 123.6 (s), 124.8 (s), 125.9 (d), 130.3 (s),
1
3
C NMR spectra were measured at 200 and 50.3 MHz, respec-
tively, by using a Bruker AC series 200 MHz spectrometer (TMS
13 3
130.6 (d), 136.3 (s), 143.0 (s). Anal. (C16H N ) C, H, N.

Isoindoloquinoxalines, NoVel Potent Antitumor Agents
-Cyano-2-(2′-amino-4′-methoxyphenyl)isoindole 3c. This com-
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2397
2,3-Dimethyl-5H-isoindolo[2,1-a]quinoxalin-6-one 4d. Yield
1
-
1 1
pound was purified by flash chromatography by using dichlo-
96%; mp 370-371 °C; IR 3365, 3286 (NH), 1631 (CO) cm ; H
romethane as eluent to give a solid: yield 95%; mp 116-117 °C;
NMR (DMSO-d ) δ 2.30 (3H, s, CH ), 2.35 (3H, s, CH ), 7.31 (s,
6
3
3
-
1 1
IR 3465, 3375 (NH
bs, 2H, NH ), 3.80 (s, 3H, CH
J ) 7.9 Hz, H-5′), 7.12 (d, 1H, J ) 7.9 Hz, H-6′), 7.14 (t, 1H, J
2
), 2198 (CN) cm ; H NMR (CDCl
3
) δ 3.64
1H, H-4), 7.46 (t, 1H, J ) 7.8 Hz, H-9), 7.57 (t, 1H, J ) 7.8 Hz,
H-8), 8.00 (d, 1H, J ) 7.8 Hz, H-10), 8.09 (s, 1H, H-1), 8.41 (d,
(
2
3
), 6.38 (bs, 1H, H-3′), 6.42 (d, 1H,
1
3
1H, J ) 7.8 Hz, H-7), 9.02 (s, 1H, H-11), 13.14 (bs, 1H, NH); C
)
8.6 Hz, H-5), 7.29 (t, 1H, J ) 8.6 Hz, H-6), 7.41 (s, 1H, H-3),
6
NMR (DMSO-d ) δ 19.5 (q), 19.6 (q), 116.7 (d), 117.4 (d), 118.7
1
3
7
.66 (d, 1H, J ) 8.6 Hz, H-4), 7.70 (d, 1H, J ) 8.6 Hz, H-7);
C
(s), 119.1 (d), 119.6 (d), 121.0 (s), 122.0 (d), 125.3 (s), 125.4 (d),
125.8 (s), 127.6 (d), 127.9 (s), 135.9 (s), 139.0 (s), 147.2 (s). Anal.
3
NMR (CDCl ) δ 55.4 (q), 95.4 (s), 101.4 (d), 104.5 (d), 113.9 (s),
1
1
17.5 (s), 118.2 (d), 120.8 (d x 2), 123.1 (d), 124.5 (s), 125.8 (d),
28.7 (d), 131.7 (s), 143.3 (s), 161.5 (s). Anal. (C16 O) C,
(C17
H
14
N
2
O) C, H, N.
13 3
H N
2,3-Dichloro-5H-isoindolo[2,1-a]quinoxalin-6-one 4e. Yield
-
1 1
8
7%; mp 340-343 °C; IR 3350, 3302 (NH), 1645 (CO) cm ; H
NMR (DMSO-d ) δ 7.43 (t, 1H, J ) 7.8 Hz, H-9), 7.54 (t, 1H, J
7.8 Hz, H-8), 7.86 (s, 1H, H-4), 7.95 (d, 1H, J ) 7.8 Hz, H-10),
.52 (d, 1H, J ) 7.8 Hz, H-7), 8.85 (s, 1H, H-1), 9.32 (s, 1H,
H-11), 12.46 (bs, 1H, NH); C NMR (DMSO-d
d), 118.7 (d), 119.2 (d), 119.4 (d), 121.1 (d), 122.0 (s), 124.7 (s),
H, N.
-Cyano-2-(2′-amino-4′,5′-dimethylphenyl)isoindole 3d. This
1
6
)
8
compound was purified by flash chromatography by using dichlo-
romethane as eluent to give a solid: yield 85%; mp 119-120 °C;
13
-
1 1
6
) δ 105.1 (s), 117.1
IR 3465, 3375 (NH
s, 3H, CH ), 2.22 (s, 3H, CH
H-3′), 6.95 (s, 1H, H-6′), 7.12 (t, 1H, J ) 7.6 Hz, H-5), 7.27 (t,
H, J ) 7.6 Hz, H-6), 7.38 (s, 1H, H-3), 7.64 (d, 1H, J ) 7.6 Hz,
2
), 2202 (CN) cm ; H NMR (CDCl
3
) δ 2.17
(
1
(
(
3
), 3.44 (s, 2H, NH ), 6.64 (s, 1H,
3 2
24.8 (d), 126.5 (s), 126.9 (s), 127.0 (d), 127.2 (s), 130.3 (s), 147.0
Cl O) C, H, N.
General Procedure for the Synthesis of 11-Cyanoisoin-
s). Anal. (C15
H
8
2 2
N
1
1
3
H-4), 7.68 (d, 1H, J ) 7.6 Hz, H-7); C NMR (CDCl
q), 19.7 (q), 95.1 (s), 114.0 (s), 118.1 (d), 118.2 (d), 120.6 (d),
3
) δ 18.6
(
dolo[2,1-a]quinoxalines 5a,c–e. 1-Cyano-2-(2′-aminophenyl)isoin-
doles 3a,c–e (3 mmol) were dissolved in formic acid 99% (10 mL)
and refluxed until disappearance of the starting material (10-30
h). The reaction mixture was poured into ice water (150 mL), and
the resulting precipitate was collected by filtration. The solid was
purified by flash chromatography to give the products 5a,c-e.
1
20.9 (d), 121.8 (s), 123.1 (d), 124.5 (s), 125.8 (d), 127.0 (s), 128.2
) C, H, N.
-Cyano-2-(2′-amino-4′,5′-dichlorophenyl)isoindole 3e. This
(
15 3
d), 131.7 (s), 139.6 (s), 139.7 (s). Anal. (C17H N
1
compound was purified by flash chromatography by using dichlo-
romethane as eluent to give a solid: yield 77%; mp 71-72 °C; IR
-
1 1
1
1-Cyanoisoindolo[2,1-a]quinoxaline 5a. This compound was
3
(
7
394, 3340 (NH
2 6
), 2200 (CN) cm ; H NMR (DMSO-d ) δ 5.75
eluted by using dichloromethane: yield 70%; mp 237-238 °C; IR
2H, s, NH ), 7.12 (1H, s, H-3′), 7.16 (1H, t, J ) 8.4 Hz, H-5),
2
1 1
192 (CN) cm- ; H NMR (DMSO-d
2
6
) δ 7.55 (t, 1H, J ) 7.2 Hz,
.32 (1H, t, J ) 8.4 Hz, H-6), 7.58 (1H, s, H-6′), 7.66 (1H, d, J )
.4 Hz, H-4), 7.78 (1H, d, J ) 8.4 Hz, H-7), 7.91 (1H, s, H-3);
H-8), 7.68 (t, 1H, J ) 7.2 Hz, H-9), 7.81 (d, 1H, J ) 7.2 Hz, H-7),
8
1
3
7
(
1
8
(
1
.87 (t, 1H, J ) 8.0 Hz, H-2), 7.90 (t, 1H, J ) 8.0 Hz, H-3), 8.20
6
C NMR (DMSO-d ) δ 94.0 (s), 113.8 (s), 115.8 (s), 116.2 (d),
d, 1H, J ) 7.2 Hz, H-10), 8.57 (d, 1H, J ) 8.0 Hz, H-1), 9.06 (d,
1
17.5 (d), 121.6 (d), 121.7 (s), 122.4 (d), 122.6 (d), 124.2 (s), 125.8
1
3
H, J ) 8.0 Hz, H-4), 9.79 (s, 1H, H-6); C NMR (DMSO-d
6
) δ
(
d), 129.6 (d), 131.2 (s), 132.9 (s), 144.7 (s). Anal. (C15
H
9
Cl N )
2 3
9.4 (s), 115.4 (s), 115.7 (d), 116.9 (d), 119.8 (s), 120.3 (d), 121.7
C, H, N.
s), 124.7 (d), 127.2 (s), 128.4 (d), 128.5 (d), 129.0 (d), 139.7 (d),
32.2 (s), 138.5 (s), 143.3 (d). Anal. (C16 ) C, H, N.
1-Cyano-3-methoxyisoindolo[2,1-a]quinoxaline 5c. This com-
pound was eluted by using dichloromethane: yield 68%; mp
General Procedure for the Synthesis of 5H-Isoindolo[2,1-
a]quinoxalin-6-ones 4a–e. 1-Cyano-2-(2′-aminophenyl)isoindoles
9 3
H N
1
3
a–e (3 mmol) were dissolved in acetic acid (10 mL) and refluxed
for 30 min. The reaction mixture was poured into ice water (150
mL). The resulting precipitate was collected by filtration and
recrystallized from ethanol.
-1 1
2
(
8
7
9
d
1
20-221 °C; IR 2193 (CN) cm ; H NMR (DMSO-d
6
) δ 3.97
s, 3H, CH
3
), 7.31 (dd, 1H, J ) 9.6, 3.0 Hz, H-2), 7.46 (t, 1H, J )
.1 Hz, H-8), 7.53 (s, 1H, H-4), 7.58 (t, 1H, J ) 8.1 Hz, H-9),
5
H-Isoindolo[2,1-a]quinoxalin-6-one 4a. Yield 99%; mp
.90 (d, 1H, J ) 8.1 Hz, H-10), 8.19 (d, 1H, J ) 8.1 Hz, H-7),
-1 1
2
(
2
55-256 °C; IR 3390, 3203 (NH), 1685 (CO) cm ; H NMR
13
.06 (d, 1H, J ) 9.6 Hz, H-1), 8.81 (s, 1H, H-6); C NMR (DMSO-
DMSO-d ) δ 7.75 (dt, 1H, J ) 7.0, 1.6 Hz, H-2), 7.78-7.91 (m,
6
6
) δ 55.8 (q), 90.2 (s), 111.1 (d), 115.6 (s), 117.2 (d), 117.6 (d),
H, H-3 and H-4), 7.93 (s, 1H, H-11), 7.96 (t, 1H, J ) 7.8 Hz,
18.6 (d), 118.8 (d), 120.2 (s), 121.2 (s), 122.0 (s), 124.8 (d), 127.9
O)
H-9), 8.00 (t, 1H, J ) 7.8 Hz, H-8), 8.27 (dd, 1H, J ) 7.0, 1.6 Hz,
(
d), 132.6 (s), 140.5 (s), 142.3 (d), 159.2 (s). Anal. (C17
H
11
N
3
H-1), 8.34 (d, 1H, J ) 7.8 Hz, H-10), 8.84 (d, 1H, J ) 7.8 Hz,
C, H, N.
13
H-7), 12.35 (bs, 1H, NH); C NMR (DMSO-d
6
) δ 124.1 (d), 127.0
1
1-Cyano-2,3-dimethylisoindolo[2,1-a]quinoxaline 5d. This
(
d), 127.5 (d), 127.8 (d), 127.9 (d), 128.9 (d), 130.9 (d), 131.4 (d),
compound was eluted by using dichloromethane:ethyl acetate (98:
1
33.1 (s), 133.4 (d), 135.3 (s), 138.9 (s), 141.0 (s), 143.9 (s), 161.7
O) C, H, N.
-Methyl-5H-isoindolo[2,1-a]quinoxalin-6-one 4b. Yield 98%;
-1 1
2
(
)
): yield 71%; mp 220-221 °C; IR 2193 (CN) cm ; H NMR
DMSO-d ) δ 2.45 (s, 3H, CH ), 2.54 (s, 3H, CH ), 7.49 (t, 1H, J
7.5 Hz, H-8), 7.60 (t, 1H, J ) 7.5 Hz, H-9), 7.85-7.88 (m, 2H,
(
10 2
s). Anal. (C15H N
6
3
3
4
-1 1
mp 338-339 °C; IR 3360, 3284 (NH), 1632 (CO) cm ; H NMR
DMSO-d ) δ 2.62 (s, 3H, CH ), 7.32-7.40 (m, 2H, H-9 and H-3),
.45 (t, 1H, J ) 6.8 Hz, H-2), 7.51 (t, 1H, J ) 8.8 Hz, H-8), 7.96
d, 1H, J ) 8.8 Hz, H-10), 8.27 (d, 1H, J ) 6.8 Hz, H-1), 8.47 (d,
H-10 and H-4), 8.33 (d, 1H, J ) 7.5 Hz, H-7), 8.85 (s, 1H, H-1),
1
3
(
7
(
6
3
9
(
1
(
.44 (s, 1H, H-6); C NMR (DMSO-d
s), 113.1 (s), 115.2 (d), 116.6 (d), 119.0 (d), 119.4 (s), 121.0 (s),
24.0 (d), 124.9 (s), 127.4 (d), 130.0 (d), 132.0 (s), 136.7 (s), 137.3
s), 138.2 (s), 141.0 (d). Anal. (C18 ) C, H, N.
1-Cyano-2,3-dichloroisoindolo[2,1-a]quinoxaline 5e. This
compound was eluted by using dichloromethane: yield 75%; mp
6
) δ 19.2 (q), 20.1 (q), 111.2
1
3
1
H, J ) 8.8 Hz, H-7), 9.24 (s, 1H, H-11), 12.53 (bs, 1H, NH); C
) δ 17.8 (q), 104.8 (s), 114.8 (d), 117.3 (d), 119.2
d), 121.5 (d), 122.3 (s), 125.0 (d), 125.1 (s), 125.2 (d), 125.3 (s),
13 3
H N
NMR (DMSO-d
(
6
1
-1 1
1
27.1 (d), 127.3 (s x 2), 130.0 (d), 147.0 (s). Anal. (C16
H
12
N
2
O)
279-281 °C; IR 2196 (CN) cm ; H NMR (CDCl ) δ 7.57 (t,
3
C, H, N.
-Methoxy-5H-isoindolo[2,1-a]quinoxalin-6-one 4c. Yield 75%;
1H, J ) 8.3 Hz, H-8), 7.68 (t, 1H, J ) 8.3 Hz, H-9), 8.00 (d, 1H,
3
J ) 8.3 Hz, H-10), 8.29 (d, 1H, J ) 8.3 Hz, H-7), 8.31 (s, 1H,
-1
1
13
mp 293-294 °C; IR 3363, 3280 (NH), 1635 (CO) cm ; H NMR
H-1), 9.35 (s, 1H, H-4), 9.46 (s, 1H, H-6); C NMR (CDCl ) δ
3
(
)
DMSO-d ) δ 3.84 (3H, s, CH ), 7.04 (s, 1H, H-4), 7.05 (d, 1H, J
6
3
86.9 (s), 96.5 (s), 112.5 (s), 117.6 (d x 2), 119.1 (s), 121.7 (s),
125.8 (s), 128.8 (d x 2), 129.7 (s), 131.8 (d), 143.3 (d x 2), 145.1
(s), 152.3 (s). Anal. (C H Cl N ) C, H, N.
9.8 Hz, H-2), 7.40 (t, 1H, J ) 7.8 Hz, H-9), 7.51 (t, 1H, J ) 7.8
Hz, H-8), 7.95 (d, 1H, J ) 7.8 Hz, H-10), 8.36 (d, 1H, J ) 9.8 Hz,
H-1), 8.51 (d, 1H, J ) 7.8 Hz, H-7), 9.18 (s, 1H, H-11), 13.50 (bs,
1
6
7
2
3
Biology. Colchicine and vinblastine were purchased from Sigma-
Aldrich (Milan, Italy), Taxol from Cytoskeleton (USA), and
Camptothecin from Alexis Biochemicals (Switzerland).
1
3
1
1
6
H, NH); C NMR (DMSO-d ) δ 55.7 (q), 101.5 (d), 104.6 (s),
12.7 (d), 115.7 (d), 116.2 (s), 118.2 (d), 119.1 (d), 120.9 (d), 124.3
(
d), 124.4 (s), 126.2 (d), 126.6 (s), 128.5 (s), 147.1 (s), 158.9 (s).
Cell Cultures. Human lymphoblastic leukemia cells (Jurkat) and
12 2 2
Anal. (C16H N O ) C, H, N.
human lymphoblastoid cells (CEM) were grown in RPMI-1640

2
398 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Diana et al.
medium (Sigma-Aldrich), human intestinal adenocarcinoma (LoVo)
were grown in HAM’S F12 medium (Sigma-Aldrich), whereas
human nonsmall cell lung carcinoma cells (A-549) and breast
adenocarcinoma cells (MCF-7) were grown in DMEM medium
bated with a TRITC-conjugated rat antimouse IgG antibody (diluted
1:200 in 2% BSA) at 37 °C for a further 1 h. Slides were then
washed repeatedly with PBS, mounted with mounting medium, and
analyzed by confocal microscopy (SP-2, Leica) under green light.
Tubulin Polymerization. The effects of test compounds on the
polymerization of microtubule protein isolated from porcine brain were
analyzed by using a microtubule polymerization assay kit (Cytoskel-
eton) following the recommended protocol. Polymerization was
followed by fluorescent enhancement because of the incorporation of
a fluorescent reporter into microtubules as polymerization occurred.
The assay was done in 96-well microtiter plates, and the reaction was
initiated by the addition of tubulin. The plate was incubated at 37 °C
in a fluorescence microplate reader (Fluoroskan Ascent FL Lab-
systems), and the fluorescence measurement (λex ) 355 nm and λem
(
Sigma, USA). All were supplemented with 115 units/mL of
penicillin G (Invitrogen, Milan, Italy), 115 µg/mL streptomycin
Doxo
(
Invitrogen), and 10% fetal bovine serum (Invitrogen). LoVo
38
are doxorubicin-resistant subclones of LoVo cells and were grown
in complete HAM’S F12 medium supplemented with doxorubicin
Vbl-100
(
0.1 µg/mL). CEM
are a multidrug-resistant line selected
39
MDR
against vinblastine. MCF-7
are human mammary carcinoma
40
exhibiting multidrug resistance and high level of P-gp expression.
They were grown in complete DMEM medium supplemented with
doxorubicin (0.1 µg/mL).
Cell Cycle Analysis. For flow cytometric analysis of DNA
)
450 nm) was determined every minute for 60 min. As reference
5
content, 5 × 10 Jurkat cells in exponential growth were treated at
compounds, Taxol and colchicine were used.
different concentrations of the test compound for 24 h. After this
incubation period, cells were centrifuged and fixed with ice-cold
ethanol (70%), treated with lysis buffer containing RNaseA, and
then stained with propidium iodide. Samples were analyzed on a
Becton Coulter Epics XL-MCL flow cytometer. For cell cycle
analysis, DNA histograms were analyzed by using MultiCycle for
Windows (Phoenix Flow Systems, San Diego, CA).
DNA-Binding Studies. Light absorption spectra were recorded
with a Perkin-Elmer Lambda 15 spectrophotometer. Fluorescence
spectra were recorded with a Perkin-Elmer LS50B spectrofluorim-
eter. Measurements were carried out in ETN buffer (10 mM Tris,
1
mM EDTA, and 10 mM NaCl) at 25 °C. To obtain the intrinsic
binding constant and the binding-site size as well, absorption spectra
of solutions with different [drug]/[DNA] ratios and a constant drug
concentration were recorded. The binding isotherms obtained were
Externalization of Phosphatidylserine. Surface exposure of
phosphatidylserine (PS) by apoptotic cells was measured by flow
cytometry with a Coulter Cytomics FC500 (Becton Dickinson, USA)
by adding Annexin V-FITC to cells according to the manufacturer’s
instructions (Annexin-V Fluos, Roche Diagnostic, Indianapolis, IN).
Simultaneously, the cells were stained with PI. Excitation was set at
13
represented as Scatchard plots 12and evaluated according to the
McGhee and von Hippel model.
Linear Dichroism. LD measurements were performed with a
Jasco J500A spectropolarimeter equipped with an IBM PC and a
41
Jasco J interface as reported previously. The sample orientation
4
88 nm, and the emission filters were at 525 and 585 nm.
Assessment of Mitochondrial Changes. The mitochondrial
42
was produced by a device designed by Wada and Kozawa at a
shear gradient of 700 rpm in ETN buffer at pH ) 7.0.
membrane potential was measured with the lipophilic cation
Topoisomerase I Assay. Supercoiled pBR322 DNA (Fermentas,
Glen Burnie, MD) was incubated with 5 units of human topoisomerase
I (Calbiochem, Gibbstown, NJ) at 37 °C for 30 min in relaxation buffer
5
1
2
,5′,6,6′-tetrachlo-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine (JC-
, Molecular Probes, USA), as described elsewhere. Briefly, after
4 h of treatment, the cells were collected by centrifugation and
21
2
(50 mM Tris-HCl pH ) 7.8, 50 mM KCl, 10 mM MgCl , 1 mM
resuspended in Hank’s balanced salt solution (HBSS) containing
the JC-1 at a concentration of 1 µM. The cells were then incubated
for 10 min at 37 °C, centrifuged, and resuspended in HBSS.
The production of ROS and the oxidation of cardiolipin were
measured by flow cytometry by using HE (Molecular Probes, USA)
dithiothreitol, and 1 mM EDTA) in the presence of various concentra-
tions of the drug. Reactions were terminated by adding 1% SDS and
an additional digestion with 50 µg/mL proteinase K at 37 °C for 30
min. DNA samples were then added to loading buffer and electro-
phoresed in a 1% agarose gel at room temperature overnight at 20 V.
After staining in ethidium bromide solution, the gel was washed with
water, and the DNA bands were detected under UV radiation with a
UV transilluminator. Photographs were taken with a digital photo-
camera Kodak DC256, and the quantization of the bands was achieved
by image analyzer software Quantity One (BIO RAD, Milan, Italy).
Similar experiments were performed by using ethidium-containing
agarose gels.
2
3,24
and NAO (Molecular Probes, USA), respectively.
After 24 h of treatment, the cells were collected by centrifugation
and resuspended in HBSS containing the fluorescence probes HE
or NAO at concentrations of 2.5 µM and 100 nM, respectively.
The cells were then incubated for 30 min at 37 °C, centrifuged,
and resuspended in HBSS. The fluorescence was directly recorded
with the flow cytometer by using as excitation wavelength 488 nm
and emission at 585 and 530 nm for HE and NAO, respectively.
Mitotic Index Determinations. Exponentially growing Jurkat
cells were incubated with test compound for 24 h prior to
centrifugation at 400g for 5 min and resuspension of the resultant
cell pellet in 1 mL of KCl 75 mM at 4 °C. After 20 min, 1 mL of
methano--acetic acid (3:l) as fixative was slowly added under
constant mild agitation. Slides were prepared after the cells were
repelled, washed twice with 1 mL of fixative, and resuspended in
the fixative. After drying, samples were stained with Giemsa
solution. A total of 400 cells/treatment was scored for the presence
of mitotic figures by optical microscopy, and the mitotic index was
calculated as the proportion of cells with mitotic figures.
Topoisomerase II Assay. Assay mixtures were prepared by
using 125 ng of supercoiled pBR322 plasmid, 2 units of human
topoisomerase IIR (Sigma), and 0-50 µM of selected compounds
in aqueous solution (50 mM Tris-HCl pH ) 8.0, 10 mM MgCl
2
,
1
20 mM KCl, 0.5 mM DTT, and 30 µg of BSA). Reactions were
carried out and then stopped after an incubation period of 30 min.
Gel electrophoresis was performed as described above for topoi-
somerase I.
Caspase Assay. Jurkat cells were treated in the presence of the
test compounds, and after 24 h, the cells were harvested, washed,
and resuspended in HBSS buffer containing the cell-permeable
substrates for caspase-3 (FITC-DEVD-fmk, Bender Medsystem,
Burlingame, CA), caspase-8 (FITC-IETD-fmk, Bender Medsystem),
and caspase-9 (FITC-LEDH-fmk, Bender Medsystem). After 1 h
of incubation at 37 °C, the cells were washed and analyzed by flow
cytometry by using the FL-1 channel.
Immunofluorescence Detection of Microtubule Perturba-
tion. A549 cells were seeded on sterile microscope coverslips. After
2
4 h, the test compound (5 and 2.5 µM) and vinblastine (1 µM), as
reference compound, were added to the culture medium, and cells
were then incubated for 18 h. Afterward, cells were fixed with 4%
formaldehyde in PBS for 10 min at room temperature, washed three
times with PBS, permeabilized in 0.2% Triton X-100 in PBS for
Acknowledgment. This work was financially supported by
Ministero dell’Istruzione dell’Università e della Ricerca. We
thank the National Cancer Institute (Bethesda, MD) and
especially Dr. V. L. Narayanan and his team for the antitumor
tests reported in this paper. G.V. thanks Fondazione Città della
Speranza for its financial support.
1
0 min at room temperature, and placed in methanol at -20 °C
for 30 min. They were then washed with PBS and incubated for
h at 37 °C with 2% bovine serum albumin (BSA) and sub-
sequently, with a mouse monoclonal anti-ꢀ-tubulin antibody for
h at 37 °C. Slides were washed three times with PBS and incu-
1
1

Isoindoloquinoxalines, NoVel Potent Antitumor Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2399
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Mitochondria and cell death. Mechanistic aspects and methodological
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