Antitumor Agents. 3. Design, Synthesis, and Biological Evaluation of New Pyridoisoquinolindione and Dihydrothienoquinolindione Derivatives with Potent Cytotoxic Activity

Article abstract of DOI:10.1021/jm030918b

New antiproliferative compounds, the 1-aryl-3-ethoxycarbonyl-pyrido[2,3-g]isoquinolin-5,10-diones (PIQDs, 1-7), were designed on the basis of a molecular model obtained by aligning the common quinolinquinone substructure of 5H-pyrido[3,2-a]phenoxazin-5-one (PPH) and some known anticancer agents. A Diels-Alder reaction between quinolin-5,8-dione (QD) and a 2-azadiene, formed by demolition of 2-aryl-1,3-thiazolidine ethyl esters (T compounds), was used to produce 1-7 and the isomeric 1-aryl-3-ethoxycarbonylpyrido[3,2-g]isoquinolin-5,10-diones (8-14). Two other compounds, the 3-amino-3-ethoxycarbonyldihydrothieno[2,3-g]-quinolin-4,9-dione (15) and the 3-amino-3-ethoxycarbonyldihydrothieno[3,2-g]quinolin-4,9-dione (16), arising from a 1,4 Michael reaction of QD with a thiolate species formed by opening of T compounds, were recovered from the reaction mixture. The antiproliferative activity of 1-16 was evaluated against representative human liquid and solid neoplastic cell lines. The IC₅₀ of these compounds had median values in the range 2.00-0.01 μM, with 2-4 and 15 exhibiting significantly higher in vitro cytotoxic activity. Compound 2, also evaluated against KB subclones (KB^MDR, KB^7D, and KB^V20C), was shown to be scarcely subject to the MDR1/P-glycoprotein drug efflux pump responsible for drug resistance. The noncovalent DNA-binding properties of PIQDs were examined using UV-vis and ¹H NMR spectroscopy experiments. Accordingly, these compounds were confirmed to have an ability to intercalate into double-stranded DNA by topoisomerase I superhelix unwinding assay. Interesting structure-activity relationships were found. Three important features seem to contribute to the cytotoxic activity of these anticancer ligands: (i) the DNA intercalating capability of the three-cyclic quinonic system, typical of this class of compounds, (ii) the position of the pendant phenyl ring that, according to the superimposition model, must occupy the same area of the corresponding benzo-fused ring A of PPH, and (iii) the effect of electron-withdrawing substituents on the phenyl ring, which can contribute improving the π-π stacking interactions between ligand and DNA base pairs. Besides, a mechanism of action suspected to involve topoisomerases could be hypothesized to interpret the antiproliferative activity of the thienoquinolindione 15, which can be regarded as a cyclic cysteine derivative.

Full text of DOI:10.1021/jm030918b

J . Med. Chem. 2004, 47, 849-858
849
An titu m or Agen ts. 3. Design , Syn th esis, a n d Biologica l Eva lu a tion of New
P yr id oisoqu in olin d ion e a n d Dih yd r oth ien oqu in olin d ion e Der iva tives w ith
P oten t Cytotoxic Activity
,
†
†
†
§
§
§
Adele Bolognese,* Gaetano Correale, Michele Manfra, Antonio Lavecchia, Orazio Mazzoni, Ettore Novellino,
‡
‡
‡
Paolo La Colla, Giuseppina Sanna, and Roberta Loddo
Dipartimento di Chimica Organica e Biochimica, Universit a` di Napoli “Federico II”, Via Cynthia 6, Monte Sant’Angelo,
I-80126 Napoli, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, Universit a` di Napoli “Federico II”,
Via D. Montesano 49, I-80131 Napoli, Italy, and Dipartimento di Biologia Sperimentale, Sezione di Microbiologia,
Universit a` di Cagliari, Cittadella Universitaria SS 554, I-09042 Monserrato, Cagliari, Italy
Received J une 6, 2003
New antiproliferative compounds, the 1-aryl-3-ethoxycarbonyl-pyrido[2,3-g]isoquinolin-5,10-
diones (PIQDs, 1-7), were designed on the basis of a molecular model obtained by aligning
the common quinolinquinone substructure of 5H-pyrido[3,2-a]phenoxazin-5-one (PPH) and some
known anticancer agents. A Diels-Alder reaction between quinolin-5,8-dione (QD) and a
2
-azadiene, formed by demolition of 2-aryl-1,3-thiazolidine ethyl esters (T compounds), was
used to produce 1-7 and the isomeric 1-aryl-3-ethoxycarbonylpyrido[3,2-g]isoquinolin-5,10-
diones (8-14). Two other compounds, the 3-amino-3-ethoxycarbonyldihydrothieno[2,3-g]-
quinolin-4,9-dione (15) and the 3-amino-3-ethoxycarbonyldihydrothieno[3,2-g]quinolin-4,9-dione
(16), arising from a 1,4 Michael reaction of QD with a thiolate species formed by opening of T
compounds, were recovered from the reaction mixture. The antiproliferative activity of 1-16
was evaluated against representative human liquid and solid neoplastic cell lines. The IC50 of
these compounds had median values in the range 2.00-0.01 µM, with 2-4 and 15 exhibiting
significantly higher in vitro cytotoxic activity. Compound 2, also evaluated against KB subclones
MDR
7D
V20C
(KB
, KB , and KB
), was shown to be scarcely subject to the MDR1/P-glycoprotein drug
efflux pump responsible for drug resistance. The noncovalent DNA-binding properties of PIQDs
1
were examined using UV-vis and H NMR spectroscopy experiments. Accordingly, these
compounds were confirmed to have an ability to intercalate into double-stranded DNA by
topoisomerase I superhelix unwinding assay. Interesting structure-activity relationships were
found. Three important features seem to contribute to the cytotoxic activity of these anticancer
ligands: (i) the DNA intercalating capability of the three-cyclic quinonic system, typical of
this class of compounds, (ii) the position of the pendant phenyl ring that, according to the
superimposition model, must occupy the same area of the corresponding benzo-fused ring A of
PPH, and (iii) the effect of electron-withdrawing substituents on the phenyl ring, which can
contribute improving the π-π stacking interactions between ligand and DNA base pairs.
Besides, a mechanism of action suspected to involve topoisomerases could be hypothesized to
interpret the antiproliferative activity of the thienoquinolindione 15, which can be regarded
as a cyclic cysteine derivative.
5
In tr od u ction
binding to other substrates such as topoisomerases and
6
transition metals. The redox activity of quinones has
also been seen to have a role in the cleavage of DNA
Quinone derivatives, containing planar polycyclic
systems, represent a large number of antitumor drugs
that target DNA. Most of these antiproliferative agents
7
-9
1
mediated by oxygen or sulfur radicals.
Actinomycin
2
D (AMD), doxorubicin (doxo), mitoxantrone, and strep-
tonigrin are significant examples of these DNA damag-
ing agents, and their side effects, such as myelosup-
exhibit cytostatic activity through DNA intercalation,
which causes enzymatic blockade and reading errors
during the replication process.2,3 Intercalators contain
1
pression and cardiotoxicity, are related to quinone
a planar chromophore with two to four fused aromatic
rings. A positive charge, generally needed for activity,
4
redox activity.
Recently we described10,11 the synthesis and the
structure-activity relationships (SARs) of a series of
pyridophenoxazinones, which exhibit an antiprolifera-
tive activity on a large panel of lymphoblastoid and
solid-tumor derived cells at submicromolar concentra-
tions. Molecular modeling studies and NMR investiga-
tions suggested that the 5H-pyrido[3,2-a]phenoxazin-
is usually provided by a quaternized or protonable
aromatic nitrogen of the chromophore. The primary
nucleic acid intercalation may be further modulated by
*
To whom correspondence should be addressed. Phone: +39 081
6
74121. Fax: +39 081 674393. E-mail: bologne@unina.it.
†
Dipartimento di Chimica Organica e Biochimica, Universit a` di
Napoli “Federico II”.
§
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita`
5
-one (PPH, I; Figure 1), the prototype of the series,
di Napoli “Federico II”.
‡
Universit a` di Cagliari.
intercalates between the 5′-GC-3′ base pairs of double-
1
0.1021/jm030918b CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/20/2004

8
50 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Bolognese et al.
F igu r e 1. Structures of known anticancer DNA binding agents, with the common quinonic motif highlighted in bold: PPH (I),
1
2
13
14
meridine (II), LY83583 (III), and wakayin (IV) together with 1 representing the PIQD series.
Ch a r t 1
stranded DNA, involving two strong hydrogen bonds
between the hydrogen of the charged pyridine nitrogen
and (i) the O4′ in the deoxyribose ring of the cytosine
C5 residue and (ii) the O5′ of the phosphate backbone
located between the G4 and C5 residues. Additional
stability of the complex arises from π-π stacking
interactions between the tetracyclic pyridophenoxazi-
none system and the aromatic base rings.
In pursuing our research in the field of anticancers,
we have investigated a series of 1-aryl-3-ethoxycarbo-
nylpyrido[2,3-g]isoquinolin-5,10-diones (PIQDs, 1-7 in
Chart 1) that fulfill the requirements for DNA interca-
lating binding of pyridophenoxazinones.
Molecular modeling studies were performed to verify
whether compound 1, selected as a representative of the
PIQD series, possessed the pharmacophoric elements
of PPH and of some other antitumor agents that share
their common quinonic structural motif (compounds 1
F igu r e 2. Energy-minimized structures of compounds I-IV
(by atom) and 1 (red) overlayed on the quinonic substructure
shown in orange.
1
2-14
and I-IV
in Figure 1). This common motif could
immediately suggests that the phenyl ring at the 6
position of 1 occupies the same position as ring A of PPH
and of the other antitumor agents.
Prompted by the above rationale, we synthesized
compounds 1-7 and evaluated their antineoplastic
represent a possible pharmacophore for DNA recogni-
tion, a hypothesis that will clearly require more testing
for verification. An inspection of the molecular align-
ment of compounds I-IV and 1 shown in Figure 2

Quinolindione Derivatives with Cytotoxic Activity
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 851
Sch em e 1
Sch em e 2
(1 mmol), in anhydrous acetonitrile, at -20 °C was
added to a solution of QD (1 mmol) in anhydrous aceto-
nitrile. After 15 min, 1,8-diazobicyclo[5.4.0]undec-7-ene
(DBU, 1 mmol) was added to generate, in situ, the
corresponding 2-azadiene. The reaction mixtures, stirred
for 2 h at -20 °C and then kept at room temperature
overnight, yielded the corresponding 1-aryl-3-ethoxy-
carbonylpyrido[2,3-g]isoquinolin-5,10-dione (1-7) to-
gether with the isomeric 1-aryl-3-ethoxycarbonylpyrido-
[3,2-g]isoquinolin-5,10-dione (8-14). The dihydrothieno-
quinolindiones 15 and 16 were obtained as a racemic
mixture in all reactions.
activity against some human cell lines representative
of liquid and solid human tumors. The isomeric 1-aryl-
3
(
-ethoxycarbonylpyrido[3,2-g]isoquinolin-5,10-diones
8-14) and the products, the 3-amino-3-ethoxycar-
bonyldihydrothieno[2,3-g]quinolin-4,9-dione (15) and
Schemes 1 and 2 report a reasonable hypothesis for
the formation mechanism of compounds 1-16. In the
basic medium, the breakdown of T takes place, yielding
3
4
-amino-3-ethoxycarbonyldihydrothieno[3,2-g]quinolin-
,9-dione (16), arising from the same synthetic pathway
1
7
a thiolic intermediate and a 2-azadiene. The azadiene
used to obtain 1-7, were also evaluated for their
antiproliferative activity. The noncovalent DNA-binding
properties of these compounds were examined using
attacks the QD double bond via a hetero Diels-Alder
1
8
reaction, affording two dihydro intermediates that, in
turn, yield the corresponding quinone derivatives 1-7
and 8-14 by oxidation (Scheme 1).
1
UV-vis, H NMR, and gel-electrophoresis methods.
As illustrated in Scheme 2, in the 1,4 Michael addi-
tion, the thiolate, formed from breakdown of T, attacks
the 6 and 7 positions of the R,â-unsaturated system of
QD, yielding the corresponding diphenol intermediates.
After oxidation, an intramolecular cyclization of a
carbanion species generates the three-cyclic reduced
systems, which yield 15 and 16 according to the posi-
tion of the heterocyclic nitrogen, by hydrolysis and
oxidation. The carbanion species may be formed in the
basic medium because of the electron-withdrawing effect
of the benzalimine and the carbethoxy group. The
benzalimine derivatives a and b, isolated and identi-
fied in the reaction mixture (not described in this
paper), support such a hypothesis for the formation
mechanism.
Resu lts a n d Discu ssion
Ch em istr y. According to the widely reported reactiv-
ity of the quinone system toward enophiles in the Diels-
Alder reaction and toward nucleophiles in 1,4 Michael
addition, the compounds 1-16 seem to be formed by the
competitive attack of different species, arising from the
demolition of 2-aryl-1,3-thiazolidine ethyl esters (T com-
pounds), on quinolin-5,8-dione (QD), through the above-
mentioned pathways (Schemes 1 and 2).
T compounds were prepared according to the reported
1
5
procedure from the L-cysteine ethyl ester and the
corresponding benzaldehydes. QD was prepared by
hydroxylation followed by oxidation of 5-hydroxyquino-
1
6
line. A mixture of each T (1 mmol) and silver carbonate

8
52 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Bolognese et al.
Ta ble 1. Yields of the Reaction between QD and T
Compounds
stituents in the para position of the pendant phenyl ring
A (Figure 1) increase cytotoxic activity. As evident from
Table 2, the five solid tumor cell lines were substantially
less sensitive to compounds 1-7.
Diels-Alder products
1,4 Michael products
R
yield, %
yield, %
According to the molecular alignment depicted in
Figure 2, ring A of PIQDs 1-7 occupies the same area
as the corresponding benzo-fused ring A of PPH, which
was found to play a crucial role in the π-π stacking
H
NO2
Cl
Br
Me
1/8
2/9
15/10
5/4
8/8
15/16
15/16
15/16
15/16
15/16
15/16
15/16
18/4
7/2
8/2
8/4
6/2
3/10
4/11
5/12
6/13
7/14
9/8
20/15
12/10
22/20
1
0
interactions with the base pairs of DNA. Following the
OMe
N(Me)2
10/8
7/3
previously described intercalation model of PPH,1
0,11
the
difference in potency between compounds 2-4 and
compounds 1 and 5-7 could be ascribed to electronic
effects. In fact, substitution by the electron-withdrawing
groups, which pull electrons out of ring A and lower the
LUMO energy, could improve stacking interactions with
purine and pyrimidine bases in the DNA active site. In
contrast, the electron-donating methyl, methoxy, and
dimethylamino groups, enriching the electron density
of ring A, could weaken the stacking interactions. This
would be in agreement with the activity levels of 1-7,
which decrease according to the nature of the substitu-
ent in the order NO2 < Cl < Br < H < Me < OMe <
N(Me)2.
The other PIQD derivatives 8-14 and the thieno-
quinolindiones 15 and 16 were also evaluated for their
antiproliferative activity against the same panel of
human tumor cells (Table 2). Compounds 8-14 were
less active than 1-7 with IC50 median values ranging
between 0.10 and 2.0 µM, and they exhibited a cytotox-
icity about 10- to 100-fold lower than that of 2-4.
However, nitroderivative 9 showed interesting activity
and among the tested cell lines; the CCRF-SB cells were
particularly sensitive (IC50 ) 0.015 µM). By combination
of the present findings with the above-mentioned su-
perimposition model, the low cytotoxic activity of 8-14
could be related to the position of the pendant phenyl
ring, which, taking the 9 position rather than the 6
position, no longer occupies the same area as the benzo-
fused ring A of PPH.
Table 1 reports the yields and the product distribution
of the reaction between QD and the substituted T com-
pounds. Electron-donating substituents on the 2-aryl
system of T increase the yields of the Diels-Alder
compounds to the detriment of 1,4 Michael addition
products 15 and 16. This result suggests that electron-
donating groups in the para position of the aryl group
contribute to the stability of the 2-azadiene formed in
situ. The greater amount of 15 compared to 16 is in
agreement with both the charge density and orbital
coefficient of QD at the 6 and 7 positions (for C6, charge
-
density is 0.0139|e | and LUMO energy is -0.458 91 eV;
-
for C7, charge density is 0.0076|e | and LUMO energy
1
0
is 0.324 92 eV), as predicted from the frontier orbital
theory.19
A quantitative interpretation of the reactivity of QD
vs the 2-azadiene and thiol derivatives using theoretical
quantum mechanics computations is currently in
progress. The Experimental Section reports general
synthetic procedures and characterization of 1-16 on
the basis of spectral data and elemental analysis.
Structural assignments were accomplished through
extensive 2D NMR spectroscopy, HMQC, and HMBC.
An tip r olifer a tive Activity. The antiproliferative
activity of the PIQD derivatives 1-7 was evaluated
using a panel of nine human cell lines representative
of liquid and solid human tumors. The results are shown
in Table 2. Data for the anticancer agents PPH, doxo,
and AMD are included for comparison.
The thienoquinolindione 15 (IC50 ) 0.08 µM) was 14-
fold more active than the isomeric 16 (IC50 ) 0.9 µM).
It is interesting also to note that 15 was more potent
against lymphoblastoid cell lines, having cytotoxic
activities comparable to that of PPH and doxo. Among
the solid human tumors, prostate carcinoma (DU-145)
cells were the most sensitive with an IC50 value of 0.08
µM.
The median of the IC50 values (the drug concentration
inhibiting the mean growth value of cell lines by 50%)
shows that the nitro, chloro, and bromo derivatives 2
(
(
median ) 0.01 µM), 3 (median ) 0.05 µM), and 4
median ) 0.09 µM) were the most cytotoxic compounds,
exhibiting an activity comparable with that of PPH
median ) 0.04 µM) and doxo (median ) 0.03 µM). A
(
In light of these results, the position of the fused
dihydrothieno ring with respect to the quinolinquinonic
moiety seems to affect the activity dramatically. This
ring, which contains a sulfur atom and both the amino
and carbethoxy susbstituents, may be regarded as a
cyclized cysteine fragment. If we assume that 1-16
intercalate to DNA in the same mode as PPH, one may
imagine that the amino and carbethoxy groups of 15,
emerging from the drug/DNA complex, interact with the
active site of the topoisomerase enzymes. This putative
but attractive hypothesis needs further investigation.
To prove whether the ester function might be hydro-
lyzed during the biological activity tests, compounds
1-16 were transformed in the corresponding acids and
tested for their cytotoxicity. No remarkable activity was
observed (data not shown), suggesting that the acid
function is not involved in the antiproliferative process.
particular inhibitory effect was observed on the prolif-
eration of human liquid tumor cells, particularly on
acute B-lymphoblastic leukemia (CCRF-SB) and on
+
CD4 T-cells containing an integrated HTLV-1 genome
(MT-4), with IC50 values ranging between 0.01 and 0.001
µM. Compound 2 also demonstrated high potency
against the splenic B-lymphoblastoid (Wil2-NS, IC50 )
0
.001µM) and against a solid human tumor, skin
melanoma (SKMEL-28, IC50 ) 0.001µM). It is interest-
ing to note that the cell lines of solid tumors are more
susceptible to the cytotoxic action of 2 than of 3 and 4.
Compounds 2 (median ) 0.01 µM), 3 (median ) 0.05
µM), and 4 (median ) 0.09 µM) showed IC50 values
lower than those of 1 (median ) 0.50 µM), methyl
derivative 5 (median ) 0.3 µM), 6 (median ) 0.4 µM),
and 7 (median ) 1.0 µM), suggesting that the presence
of electron-withdrawing nitro, chloro, and bromo sub-

Quinolindione Derivatives with Cytotoxic Activity
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 853
Ta ble 2. Antitumor Activity of 1-16 and PPH Compared to Doxo and AMD
IC50 (µM)a
leukemia/lymphoma
carcinoma
SKMES-1
normal cells
CRL-7065
Wil2-NS
CCRF-CEM
CCRF-SB
MT-4
SKMEL-28
MCF-7
HepG-2
DU-145
c
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
0.02
0.001
0.01
0.02
0.03
0.09
1.4
1.0
0.03
2.0
2.1
2.5
2.1
2.5
0.06
3.1
0.20
0.05
0.15
0.20
0.02
0.1
1.2
0.7
0.23
0.8
1.3
1.0
1.5
2.3
0.07
2.0
0.01
0.002
0.007
0.01
0.09
0.1
0.9
0.6
0.015
0.7
0.8
1.0
0.9
2.2
0.01
0.6
0.015
0.001
0.006
0.01
0.3
0.4
0.9
0.7
0.01
0.7
0.7
1.0
1.0
2.0
0.02
0.5
0.8
0.001
0.2
0.2
0.2
0.3
0.9
0.8
0.2
0.8
0.9
1.3
1.0
1.9
0.2
1.1
0.5
0.08
0.3
0.2
1.0
1.5
1.0
0.3
0.2
0.5
0.6
1.6
0.9
1.6
0.2
0.5
0.5
2.0
0.07
0.4
0.3
0.9
1.0
0.9
1.0
0.5
2.0
2.3
1.9
2.0
2.2
0.6
0.5
1.5
nd
nd
c
0.01
0.05
0.09
1.0
1.6
1.0
0.3
0.08
0.5
0.5
1.2
0.01
0.03
0.05
1.0
1.5
1.0
1.0
0.1
1.5
1.3
1.9
1.5
1.9
0.08
1.2
0.04
0.05
0.07
c
nd
nd
nd
nd
c
c
c
0
1
2
3
4
5
6
0.9
1.3
c
nd
1.2
1.8
0.1
0.9
1.5
c
nd
0.09
1.7
PPH
doxo
AMD
0.05
0.02
0.0030
0.01
0.02
0.001
0.009
0.03
0.002
0.01
0.01
0.0009
0.21
0.06
0.002
0.23
0.05
0.006
0.04
0.03
0.004
0.05
0.12
0.010
0.04
0.03
0.008
0.50
0.50
0.010
b
b
a
Compound concentration required to reduce cell proliferation by 50%, as determined by the MTT method, under conditions allowing
untreated controls to undergo at least three consecutive rounds of multiplication. Data represent the mean values of three independent
+
determinations. Abbreviations are the following: Wil2-NS, human splenic B-lymphoblastoid cells; CCRF-CEM, CD4 human acute
+
T-lymphoblastic leukaemia; CCRF-SB, human acute B-lymphoblastic cells; MT-4, CD4 human acute T-lymphoblastic leukemia; SKMEL-
2
8, MCF-7, human breast adenocarcinoma; SKMES-1, human lung squamous carcinoma; HepG-2, human hepatocellular carcinoma;
b
c
DU-145, human prostate carcinoma; CRL-7065, human foreskin fibroblasts. The doxo and AMD compounds were used as controls. nd
not determined.
)
doxorubicin, vincristine, and etoposide. (iii) KB7D was
selected under uninterrupted treatment with etopo-
side. The resistance is due to overexpression of the
Ta ble 3. Effect of 2 on the Proliferation of Wild-Type and
Drug-Resistant KB Cells
IC50 (µM)a
24
KBWTb
KBMDR c
KB7D d
KBV20C e
mrp gene, which codes for a membrane glycoprotein
compd
(
MRP) capable of mediating the efflux of etoposide,
2
0.08 ((0.01)
0.3 ((0.01) 0.4 ((0.01) 0.2 ((0.02)
V20C
doxorubicin 0.06 ((0.02)
AMD
vincristine 0.004 ((0.001) 1 ((0.2)
etoposide
1.8 ((0.1) 3.0 ((0.2)
0.4 ((0.1)
doxorubicin, and vincristine. Interestingly, KB
KB
respectively, more resistant to 2 relative to the parental
KB cell line, thus suggesting that this compound is
scarcely subject to the pump mediating the efflux of
many antitumor drugs.
,
MDR
7D
0.004 ((0.001) 0.2 ((0.1) 0.2 ((0.1)
0.05 ((0.01)
, and KB cells were 2.5-fold, 3.7-fold, and 5-fold,
0.05 ((0.01) 0.2 ((0.1)
0.8 ((0.1)
>20
>20
2.4 ((1.2)
wt
a
Compound concentration required to reduce cell proliferation
by 50% as determined by the MTT method under conditions
allowing untreated controls to undergo at least three consecutive
rounds of multiplication. Data represent mean values ((SD) of
three independent determinations. KB human nasopharyngeal
carcinoma. KB subclones passaged in the presence of doxorubicin
DNA-Bin d in g P r op er ties. To test the propensity of
b
PIQD compounds to interact with DNA, spectrophoto-
metric measurements and a preliminary H NMR
c
1
d
0
.09 µM. KB subclones passaged in the presence of etoposide 7
e
investigation were carried out. The spectroscopic prop-
erties of 1 and 15, selected as representative of series,
in the presence and absence of calf thymus DNA were
studied by conventional optical spectroscopy. Charac-
terization of this mixture was made by comparing the
ultraviolet absorption spectrum of the complex with the
spectra of 1 and 15. The absorption spectra of the free
and DNA-bound drugs showed well-defined bathochro-
mic and hypochromic effects. The two absorption bands
of 1, centered at 290 and 360 nm, are red-shifted by
about 4 nm, and the hypochromic effect exceeds 30%.
The visible absorption maximum of 15, centered at 418
nm, is red-shifted by about 5 nm, while the hypochromic
effect exceeds 28%. These spectral changes suggest the
binding of the compounds with the base pairs, while the
presence of isosbestic points, at 296 and 370 nm for 1
and at 330 nm for 15, indicate the existence of a single
binding mode with DNA. Similar results were recorded
for 2-7, while no red shift was observed for compounds
8-14 and 16.
µM. KB subclones passaged in the presence of vincristine
0
.02 µM.
An tip r olifer a tive Activity a ga in st Dr u g-Resis-
ta n t Tu m or Cell Lin es. Drug resistance is a relevant
therapeutic problem caused by the emergence of tumor
cells that confer resistance to a variety of anticancer
drugs. The most common mechanisms of drug resistance
are related to the overexpression of glycoproteins ca-
pable of mediating the efflux of different drugs such as
doxorubicin, vincristine, and etoposide and to the al-
tered contents of target enzymes (topoisomerases I and
II). Therefore, the inhibitory activity of 2 was evaluated
MDR
against the following KB subclones (Table 3): (i) KB
was obtained by infection of KBwt with a retroviral
vector carrying the human mdr-1 gene and maintained
under uninterrupted treatment with doxorubicin. These
cells express a membrane glycoprotein (Pgp) responsible
for the efflux of many unrelated drugs (multidrug
resistance or MDR).2
0,21
(ii) KB
V20C
was selected under
1
uninterrupted treatment with vincristine. These cells
Compound 3 was chosen for the H NMR investiga-
possess an MDR phenotype2
2,23
related to the overex-
tion because it gives easily distinguishable resonance
signals and may serve as a reference structure for the
pression of the mdr-1 gene and mediate the efflux of

8
54 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Bolognese et al.
Ta ble 4. Proton Chemical Shift Assignments of Unbound 3
and of Guanine G4 and Cytosine C5 in the Free Octamer
d(GAAGCTTC)]2 and Their Comparison with the
[
3
/[d(GAAGCTTC)]2 Complex
unbounded 3, δ
3/[d(GAAGCTTC)]2, δ
∆δ
H-1
H-2
H-3
H-6
H-8
H-9
H-10
H-11
8.96
7.88
9.02
6.54
7.51
7.63
7.41
7.88
8.56
7.58
8.71
6.25
7.10
7.42
7.22
7.43
-0.60
-0.30
-0.31
-0.29
-0.41
-0.21
-0.19
-0.45
guanine G4 in
[d(GAAGCTTC)]2, δ
3/[d(GAAGCTTC)]2, δ
∆δ
H-8
7.65
7.55
-0.10
cytosine C5 in
d(GAAGCTTC)]2, δ
[
3/[d(GAAGCTTC)]2, δ
∆δ
H-5
H-6
5.52
7.36
5.53
7.31
+0.01
-0.05
F igu r e 3. 1H NMR chemical shift variations of the aromatic
region of (A) free octamer [d(GAAGCTTC)] and (B) 1:1
/[d(GAAGCTTC)] complex were recorded at 10 °C on a 500
MHz spectrometer.
2
3
2
other active compounds. The [d(GAAGCTTC)]2 octamer,
used as the canonical sequence for NMR structural
2
5,10
studies of AMD/DNA and PPH/DNA complexes,
was
expressly synthesized to analyze the 3/DNA binding.
A titration of the [d(GAAGCTTC)]2 octamer was
carried out in the probe of a Brucker AMX 500 MHz
spectrometer at 10 °C by addition of small aliquots of 3
F igu r e 4. Influence of compound 3 on circular DNA topology.
Native supercoiled SV40 DNA (lane A) was reacted with an
excess of topoisomerase I in the absence of the drug (lane B)
or in the presence of increasing concentrations of 3 (lanes
C-H) for 60 min at 37 °C. Reactions were stopped, samples
were run on 1% agarose gel in TAE buffer, and DNA topoiso-
mers were visualized after staining with ethidium bromide.
1
until a 1:1 ratio of 3/octamer was reached. The H NMR
spectra were recorded with 5 min time intervals be-
tween the additions of 3 to allow binding equilibration.
A comparison of the chemical shifts of the aromatic
protons of 3 and [d(GAAGCTTC)]2 with those of the
3
/[d(GAAGCTTC)]2 complex showed that all resonances
shifted toward higher fields upon drug addition (Figure
). All the chemical shifts of 3 in the complex are
During the reaction, the enzyme relaxes positive
supercoils generated by drug-induced duplex unwinding.
In this way, topoisomerase I changes DNA linking
number in a manner proportional to the magnitude of
drug-induced unwinding. Following drug removal by
phenol extraction, these altered linking number values
result in rewinding of topoisomers from drug-treated
samples, whereas control DNA remains relaxed. Under
the condition of electrophoresis, the fully relaxed DNA
incubated in the absence of drug migrates as slightly
3
shielded compared to those of the free drug, with the
protons H1, H8, and H11 exhibiting shields of 0.60, 0.41,
and 0.45 ppm, respectively. The most significant shifts
of the octamer are due to the proton H8 of guanine G4
(
from 7.62 to 7.59 ppm) and to the protons H6 and H5
of cytosine C5 (from 7.36 to 7.31 ppm and from 5.77 to
.74 ppm, respectively). Table 4 reports the proton
chemical shift assignments of 3, G4, and C5 in the
/[d(GAAGCTTC)]2 complex and their comparison with
5
2
6
3
positively supercoiled. Topoisomer distributions in
samples treated with 0.3 and 0.6 µM of 3 (lanes C and
D, respectively) are consistent with a transition to
negative supercoiling that progresses further with in-
creasing concentrations of 3, as reflected by the acceler-
ated DNA migration. These characteristic patterns of
topoisomer distribution provide the unequivocal evi-
dence for the intercalative mode of 3 binding to DNA,
as well as of the other derivatives belonging to this
series (not shown).
both the unbounded ligand and the free octamer. These
results provide further evidence that 3 binds to DNA
and that intercalation occurs, altering the base sequence
GC.
P la sm id DNA Un w in d in g Assa y. Since compound
3
seems to intercalate at the middle 5′-GC-3′ base pairs
of the octamer [d(GAAGCTTC)]2 based on the above-
1
described H NMR experiments, a classic topoisomerase
I DNA unwinding assay was employed to determine
whether 3, binding to DNA, could cause unwinding of
a supercoiled plasmid. Figure 4 shows a representative
gel electrophoresis of SV40 DNA prerelaxed with topo-
isomerase I and then further incubated with 3 (0-10
µM) in the presence of topoisomerase I.
Con clu sion s
In conclusion, we have described a new series of
1-arylpyridoisoquinolindiones (PIQDs), some of which
have levels of cytotoxic activity comparable to that of

Quinolindione Derivatives with Cytotoxic Activity
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 855
doxo against a panel of some liquid and solid tumor
cells. The structure-based design was guided by an
overlay of compound 1 on some well-known antitumor
agents. The IC50 values of these compounds had median
values in the range 1.0-0.01 µM, with 2-4 and 15
exhibiting significantly higher in vitro cytotoxic activity.
Compound 2 was also evaluated against KB subclones
the UXNMR software package. NMR spectra were measured
1
13
at 500 MHz ( H) and 125 MHz ( C). The chemical shifts are
referenced to 13CDCl
and CDCl solvent signals at 77.0 and
.26 ppm, respectively. Standard pulses sequences were
3
3
7
employed for magnitude COSY. HMQC and HMBC experi-
ments were optimized for 1
2,3
J
C-H ) 135 Hz and
J C-H ) 10
Hz, respectively. Me Si was used as an internal reference.
4
Su bstitu ted P h en yl-3-eth oxyca r bon ylp yr id oisoqu in o-
lin -5,10-d ion e (1-14), 3-Am in o-3-eth oxyca r bon yld ih y-
d r oth ien o[2,3-g]qu in olin -4,9-d ion e (15), a n d 3-Am in o-3-
et h oxyca r bon yld ih yd r ot h ien o[3,2-g]q u in olin -4,9-d ion e
MDR
7D
V20C
(KB
, KB , and KB
), which overexpress the
MDR1/P-glycoprotein drug efflux pump responsible for
drug resistance. All the above KB subclones showed
scarcely altered sensitivity to the antiproliferative activ-
ity of 2. The noncovalent DNA-binding properties of
(
16). Gen er a l P r oced u r e. A mixture of 2-aryl-1,3-thiazolidine
ethyl esters (1 mmol) and silver carbonate (1 mmol), in
anhydrous acetonitrile, at -20 °C was added to a solution of
quinolin-5,8-dione (1 mmol) in anhydrous acetonitrile. After
15 min, 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU, 1 mmol) was
added. The reaction mixture, stirred for 2 h at -20 °C and
kept at room temperature overnight, yielded the corresponding
1
PIQDs were examined using UV-vis and H NMR
spectroscopy experiments. Accordingly, these com-
pounds followed the “classical” pattern observed for
intercalation, as provided by the DNA topoisomerase I
unwinding assays. Three important features seem to
contribute to the cytotoxic activity of these anticancer
ligands: (i) the DNA intercalating capability of the
three-cyclic quinonic system, typical of this class of
compounds, (ii) the position of the pendant phenyl ring,
which must occupy the same area of the corresponding
benzo-fused ring A of PPH, according to the superim-
position model, and (iii) the effect of electron-withdraw-
ing substituents on the phenyl ring, which can contrib-
ute to the improvement of the π-π stacking interactions
between the ligand and DNA base pairs. The highly
active thienoquinolindione 15 (IC50 ) 0.08 µM), which
can be regarded as a cyclic cysteine derivative, is
suspected of interacting with topoisomerases, since both
the amino and carbethoxy susbstituents may be in a
favorable position for forming hydrogen bonds with the
enzyme/DNA complex.
1
-aryl-3-ethoxycarbonylpyrido[2,3-g]isoquinolin-5,10-dione
(1-7) together with the isomeric 1-aryl-3-ethoxycarbonylpy-
rido[3,2-g]isoquinolin-5,10-dione (8-14) separated chromato-
graphically on silica gel plates using a mixture of carbon
tetrachloride and ethyl acetate (1:1 v/v) as eluent. The dihy-
drothienoquinolindiones 15 and 16 were recovered from the
reaction as racemic mixture.
1
-P h en yl-3-eth oxyca r bon ylp yr id o[2,3-g]isoqu in olin -5,-
0-d ion e (1): mp, 205-6 °C; UV-vis (CHCl ) λmax (log ꢀ) nm,
3
58 (2.6); H NMR (CDCl ) δ 9.13 (1H, dd, J ) 4.8, 1.2 Hz),
1
3
8
7
3
1
.82 (1H, s), 8.64 (1H, dd, J ) 8.6, 1.2 Hz), 7.79 (1H, dd, J )
.8, 4.8 Hz), 7.59 (2H, d, J ) 7.0 Hz), 7.47 (3H, d + t, J ) 7.0
13
Hz), 4.53 (2H, q, J ) 7.2 Hz), 1.50 (3H, t, J ) 7.2 Hz);
C
NMR (CDCl ) δ 181.7 (s, C5), 180.9 (s, C10), 163.6 (s, COO),
3
162.5 (s, C1), 156.1 (d, C7), 151.8 (s, C3), 149.7 (s, C5a), 141.3
s, C10a), 138.9 (d, C1′), 135.2 (d, C9), 128.8 (d, C8), 128.7 (s,
C9a), 128.5 (2C d, C2′6′), 128.0 (2C d, C3′5′), 126.9 (d, C4a),
(
+
1
26.8 (d, C4′), 119.3 (d, C4), 62.6 (t), 14.2 (q); MS m/z 358 (M ).
Anal. (C21 ) C, H, N.
-(4-Nit r op h en yl)-3-et h oxyca r b on ylp yr id o[2,3-g]iso-
qu in olin -5,10-d ion es (2): mp, 308-9 °C °C; UV-vis (CHCl
14 2 4
H N O
1
Whether the cytotoxic activity of the class of com-
pounds that we synthesized is exerted through DNA
intercalating binding and/or through other mechanisms
3
)
1
λ
max
(log ꢀ) nm, 369 (2.8); H NMR (CDCl ) δ 9.20 (1H, dd, J )
3
4.8, 1.2 Hz), 9.02 (1H, s), 8.52 (1H, dd, J ) 8.6, 1.2 Hz), 8.37
(2H, d, J ) 8.6 Hz), 7.81 (1H, dd, J ) 7.8, 4.8 Hz), 7.69 (2H,
d, J ) 8.6 Hz), 4.56 (2H, q, J ) 7.2 Hz), 1.48 (3H, t, J ) 7.2
1
is not yet known. Nevertheless, it has been suggested
that DNA intercalating antraquinones can generate
disrupting free radical intermediates and alkylating
agents. This evidence could account for the cytotoxic
activity of 1-16, which possess a quinone function
capable of a one-electron redox reaction. Moreover, these
compounds, having an electron-withdrawing carbethoxy
substituent on the quinone system, could be more prone
structurally to single-electron reduction toward the free
radical state. In fact, the capacity of quinones to accept
electrons is due to the electron-attracting or -donating
substituents that modulate the redox properties respon-
sible for oxidative stress.
Hz); 13C NMR (CDCl
) δ 181.5 (s, C5), 180.5 (s, C10), 163.2 (s,
COO), 161.8 (s, C1), 156.4 (d, C7), 151.7 (s, C3), 148.2 (s, C5a),
42.4 (s, C10a), 138.9 (s, C1′), 136.7 (d, C4′), 135.4 (d, C9),
3
1
132.2 (s, C9a), 130.1 (2C d, C3′5′), 129.0 (d, C8), 123.2 (2C d,
C2′6′), 126.1 (s, C4a), 120.7 (d, C4), 62.7 (t), 14.2 (q); MS m/z
+
4
03 (M ). Anal. (C21
H
13
N
3
O
6
) C, H, N.
1-(4-Ch lor op h en yl)-3-eth oxyca r bon ylp yr id o[2,3-g]iso-
qu in olin -5,10-d ion es (3): mp, 208-9 °C; UV-vis (CHCl
3
) λmax
1
(
log ꢀ) nm, 369 (2.8); H NMR (CDCl
3
) δ 9.17 (1H, dd, J ) 4.8,
1
.2 Hz), 8.94 (1H, s), 8.54 (1H, dd, J ) 8.6, 1.2 Hz), 7.79 (1H,
dd, J ) 7.8, 4.8 Hz), 7.55 (2H, d, J ) 8.6 Hz), 7.45 (2H, d, J )
8
NMR (CDCl ) δ 181.5 (s, C10), 180.8 (s, C5), 163.5 (s, COO),
161.1 (s, C1), 156.2 (d, C7), 151.8 (s, C3), 149.5 (s, C5a), 141.8
13
.6 Hz), 4.52 (2H, q, J ) 7.2 Hz), 1.43 (3H, t, J ) 7.2 Hz);
C
3
From a synthetic point of view, a Diels-Alder reaction
and a 1,4 Michael addition originally use the quinoline-
(
(
(
(
s, C10a), 137.5 (2C d, C2′6′), 135.6 (s, C4′), 135.2 (d, C9), 132.1
d, C9a), 131.6 (s, C1′), 128.7 (d, C8), 128.4 (2C d, C3′5′), 126.8
5
,8-dione and various substituted thiazolidines, enabling
+
s, C4a), 119.6 (d, C4), 62.5 (t), 14.1 (q); MS m/z 392 (M ), 394
1
-16 to be synthesized.
+
M + 2, 32% M ). Anal. (C21
13 2 4
H N O Cl) C, H, N.
1
-(4-Br om op h en yl)-3-eth oxyca r bon ylp yr id o[2,3-g]iso-
q u in olin -5,10-d ion es (4): mp, 205-6 °C; UV-vis (CHCl
max (log ꢀ) nm, 368 (2.8); 1H NMR (CDCl
) δ 9.17 (1H, dd,
Exp er im en ta l Section
3
)
Ch em istr y. Melting points were determined by a Kofler
apparatus and are uncorrected. The elemental analysis (C, H,
and N) of reported compounds agrees with the calculated
values and was within (0.4% of theoretical values. Electron
impact (EI) mass spectra were obtained at 70 eV on a ZAB 2F
spectrometer. UV spectra were recorded on Perkin-Elmer 550S
UV-visible spectrophotometer. The purity of compounds was
checked by ascending TLC on Merck’s silica gel plates (0.25
mm) with fluorescent baking. NMR measurements (data
reported in ppm) were performed on a Bruker AMX-500
spectrometer equipped with a Bruker X-32 computer, using
λ
3
J ) 4.8, 1.2 Hz), 8.94 (1H, s), 8.54 (1H, dd, J ) 8.6, 1.2 Hz),
7.79 (1H, dd, J ) 7.8, 4.8 Hz), 7.58 (2H, d, J ) 8.6 Hz), 7.34
(2H, d, J ) 8.6 Hz), 4.52 (2H, q, J ) 7.2 Hz), 1.43 (3H, t,
J ) 7.2 Hz); 13C NMR (CDCl
) δ 181.5 (s, C10), 180.8 (s,
3
C5), 163.5 (s, COO), 161.1 (s, C1), 156.2 (d, C7), 151.8 (s, C3),
149.5 (s, C5a), 141.8 (s, C10a), 135.2 (d, C9), 132.4 (2C d, C3′5′),
132.1 (d, C9a), 131.6 (s, C1′), 130.5 (2C d, C2′6′), 128.7 (d, C8),
126.8 (s, C4a), 124.6 (s, C4′), 119.6 (d, C4), 62.5 (t), 14.1 (q);
+
+
MS m/z 437 (M ), 439 (M + 2, 97% M ). Anal. (C21
13 2 4
H N O Br)
C, H, N.

8
56 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Bolognese et al.
1
-(4-Meth ylp h en yl)-3-eth oxyca r bon ylp yr id o[2,3-g]iso-
λ
max (log ꢀ) nm, 368 (2.8); 1H NMR (CDCl
3
) δ 9.15 (1H, dd,
qu in olin -5,10-d ion es (5): mp, 203-4 °C; UV (CHCl
log ꢀ) nm, 389 (3.3); H NMR (CDCl
3
) λmax
J ) 4.8, 1.2 Hz), 8.86 (1H, s), 8.59 (1H, dd, J ) 8.6, 1.2 Hz),
7.80 (1H, dd, J ) 7.8, 4.8 Hz), 7.55 (2H, d, J ) 8.6 Hz), 7.35
(2H, d, J ) 8.6 Hz), 4.52 (2H, q, J ) 7.2 Hz), 1.43 (3H, t,
1
(
1
3
) δ 9.15 (1H, dd, J ) 4.8,
.2 Hz), 8.89 (1H, s), 8.53 (1H, dd, J ) 8.6, 1.2 Hz), 7.76 (1H,
dd, J ) 7.8, 4.8 Hz), 7.46 (2H, d, J ) 8.6 Hz), 7.30 (2H, d, J )
J ) 7.2 Hz); 13C NMR (CDCl
) δ 181.5 (s, C10), 180.8 (s, C5),
3
8
7
(
.6 Hz), 4.53 (2H, q, J ) 7.2 Hz), 2.42 (3H, s), 1.47 (3H, t, J )
163.5 (s, COO), 161.9 (s, C1), 157.2 (d, C7), 151.8 (s, C3),
149.5 (s, C5a), 141.8 (s, C10a), 135.2 (d, C9), 132.2 (2C d, C3′5′),
132.1 (d, C9a), 132.0 (s, C1′), 130.5 (2C d, C2′6′), 128.7 (d, C8),
126.8 (s, C4a), 124.6 (s, C4′), 119.6 (d, C4), 62.5 (t), 14.1 (q);
1
3
.2 Hz); C NMR (CDCl ) δ 181.7 (s, C5), 180.5 (s, C10), 163.7
3
s, COO), 162.4 (s, C1), 156.0 (d, C7), 151.5 (s, C3), 149.5 (s,
C5a), 141.3 (s, C10a), 139.4 (s, C1′), 136.0 (s, C4′), 135.1 (d,
C9), 131.6 (s, C9a), 129.1 (2C d, C2′6′), 128.8 (2C d, C3′5′),
27.9 (d, C8), 125.6 (s, C4a), 118.9 (d, C4), 62.5 (t), 21.4 (q),
4.1 (q); MS m/z 372 (M ). Anal. (C22
1
+
+
MS m/z 437 (M ), 439 (M + 2, 97% M ). Anal. (C21
C, H, N.
13 2 4
H N O Br)
1
1
+
H
16
N
2
O
4
) C, H, N.
1-(4-Meth ylp h en yl)-3-eth oxyca r bon ylp yr id o[3,2-g]iso-
qu in olin -5,10-d ion es (12): mp, 208-9 °C; UV-vis (CHCl
max (log ꢀ) nm, 391 (3.5). 1H NMR (CDCl
) δ 9.16 (1H, dd,
-(4-Meth oxylp h en yl)-3-eth oxyca r bon ylp yr id o[2,3-g]-
3
)
isoqu in olin -5,10-d ion es (6): mp, 199-200 °C; UV (CHCl
max (log ꢀ) nm, 391 (3.2); H NMR (CDCl
3
)
λ
3
1
λ
4
(
3
) δ 9.15 (1H, dd, J )
J ) 4.8, 1.2 Hz), 8.78 (1H, s), 8.64 (1H, dd, J ) 8.6, 1.2 Hz),
7.80 (1H, dd, J ) 7.8, 4.8 Hz), 7.51 (2H, d, J ) 8.6 Hz), 7.27
(2H, d, J ) 8.6 Hz), 4.53 (2H, q, J ) 7.2 Hz), 2.45 (3H, s), 1.46
.8, 1.2 Hz), 8.89 (1H, s), 8.53 (1H, dd, J ) 8.6, 1.2 Hz), 7.76
1H, dd, J ) 7.8, 4.8 Hz), 7.42 (2H, d, J ) 8.6 Hz), 7.05 (2H,
d, J ) 8.6 Hz), 4.53 (2H, q, J ) 7.2 Hz), 3.85 (3H, s), 1.47 (3H,
(3H, t, J ) 7.2 Hz); 13C NMR (CDCl
) δ 181.8 (s, C10), 181.1
3
1
3
t, J ) 7.2 Hz); C NMR (CDCl
63.7 (s, COO), 162.4 (s, C1), 156.0 (d, C7), 151.5 (s, C3), 149.5
s, C5a), 141.3 (s, C10a), 148.0 (s, C4′), 135.1 (d, C9), 131.6 (s,
3
) δ 181.7 (s, C5), 180.5 (s, C10),
(s, C5), 163.6 (s, COO), 162.3 (s, C1), 155.3 (d, C8), 151.8 (s,
C3), 147.7 (s, C9a), 141.7 (s, C10a), 139.5 (s, C1′), 136.5 (s,
C4′), 136.5 (d, C6), 129.9 (s, C5a), 129.3 (2C d, C2′6′), 128.9
1
(
C9a), 131.4 (s, C1′), 129.1 (2C d, C2′6′), 127.9 (d, C8), 125.6 (s,
(2C d, C3′,5′), 128.7 (d, C7), 126.9 (s, C4a), 119.7 (d, C4), 62.5
+
C4a), 120.8 (2C d, C3′5′), 118.9 (d, C4), 62.5 (t), 54.1 (q), 21.4
(t), 21.3 (q), 14.2 (q); MS m/z 372 (M ). Anal. (C22
H
16
N
2
O
4
) C,
+
(
q); MS m/z 388 (M ). Anal. (C22
H
16
N
2
O
5
) C, H, N.
H, N.
1
-(4-Dim eth yla m in op h en yl)-3-eth oxyca r bon ylp yr id o-
1-(4-Meth oxylp h en yl)-3-eth oxyca r bon ylp yr id o[3,2-g]-
isoqu in olin -5,10-d ion es (13): mp, 205-6 °C; UV (CHCl
[
λ
4
2,3-g]isoqu in olin -5,10-dion es (7): mp, 191-2 °C; UV (CHCl
max (log ꢀ) nm, 390 (3.2); H NMR (CDCl
3
)
3
) λmax
1
1
3
) δ 9.15 (1H, dd, J )
3
(log ꢀ) nm, 395 (3.1); H NMR (CDCl ) δ 9.14 (1H, dd, J ) 4.8,
.8, 1.2 Hz), 8.89 (1H, s), 8.53 (1H, dd, J ) 8.6, 1.2 Hz), 7.76
1.2 Hz), 8.87 (1H, s), 8.55 (1H, dd, J ) 8.6, 1.2 Hz), 7.75 (1H,
dd, J ) 7.8, 4.8 Hz), 7.41 (2H, d, J ) 8.6 Hz), 7.07 (2H, d, J )
(
1H, dd, J ) 7.8, 4.8 Hz), 7.15 (2H, d, J ) 8.6 Hz), 6.80 (2H,
d, J ) 8.6 Hz), 4.53 (2H, q, J ) 7.2 Hz), 2.85 (6H, s), 1.47 (3H,
8.6 Hz), 4.53 (2H, q, J ) 7.2 Hz), 3.85 (3H, s), 1.47 (3H, t, J )
1
3
13
t, J ) 7.2 Hz); C NMR (CDCl
63.7 (s, COO), 162.4 (s, C1), 156.0 (d, C7), 151.5 (s, C3), 149.5
s, C5a), 149.0 (s, C4′), 141.3 (s, C10a), 135.1 (d, C9), 131.6 (s,
3
) δ 181.7 (s, C5), 180.5 (s, C10),
7.2 Hz); C NMR (CDCl
3
) δ 181.6 (s, C5), 180.5 (s, C10), 163.9
1
(
(s, COO), 162.3 (s, C1), 156.2 (d, C7), 151.5 (s, C3), 149.0 (s,
C5a), 140.8 (s, C10a), 148.0 (s, C4′), 135.1 (d, C9), 131.6 (s,
C9a), 132.4 (s, C1′), 129.8 (2C d, C2′6′), 127.9 (d, C8), 125.6 (s,
C9a), 129.0 (2C d, C2′6′), 127.9 (d, C8), 127.4 (s, C1′), 125.6 (s,
C4a), 118.9 (d, C4), 115.8 (2C d, C3′5′), 62.5 (t), 40.3 (2C, q),
C4a), 121.5 (2C d, C3′5′), 118.9 (d, C4), 62.5 (t), 54.3 (q), 21.4
+
+
2
1.4 (q); MS m/z 401 (M ). Anal. (C23
H
19
N
3
O
4
) C, H, N.
(q); MS m/z 388 (M ). Anal. (C22
H
16
N
2
O
5
) C, H, N.
1
-P h en yl-3-eth oxyca r bon ylp yr id o[3,2-g]isoqu in olin -5,-
0-d ion e (8): mp, 209-10 °C; UV (CHCl ) λmax (log ꢀ) nm, 360
) δ 9.15 (1H, dd, J ) 4.8, 1.2 Hz), 8.92
1H, s), 8.52 (1H, dd, J ) 8.6, 1.2 Hz), 7.77 (1H, dd, J ) 7.8,
.8 Hz), 7.54 (2H, d, J ) 7.0 Hz), 7.51 (2H, t, J ) 7.2 Hz), 7.47
1H, t, J ) 7.0 Hz), 4.53 (2H, q, J ) 7.2 Hz), 1.47 (3H, t, J )
1-(4-Dim eth yla m in op h en yl)-3-eth oxyca r bon ylp yr id o-
1
(
(
4
(
7
3
[3,2-g]isoqu in olin -5,10-d ion es (14): mp, 200-1 °C; UV
1
1
2.9); H NMR (CDCl
3
(CHCl
3
) λmax (log ꢀ) nm, 388 (3.2); H NMR (CDCl
3
) δ 9.11 (1H,
dd, J ) 4.8, 1.2 Hz), 8.87 (1H, s), 8.58 (1H, dd, J ) 8.6, 1.2
Hz), 7.72 (1H, dd, J ) 7.8, 4.8 Hz), 7.14 (2H, d, J ) 8.6 Hz),
6.82 (2H, d, J ) 8.6 Hz), 4.53 (2H, q, J ) 7.2 Hz), 2.84 (6H, s),
1
3
13
.2 Hz); C NMR (CDCl
s, COO), 162.2 (s, C1), 155.4 (d, C8), 151.6 (s, C3), 147.7 (s,
C9a), 141.7 (s, C10a), 139.4 (s, C1′), 135.8 (d, C6), 131.5 (s,
C5a), 129.2 (2C d, C2′6′), 129.0 (2C d, C3′5′), 128.7 (d, C4′),
3
) δ 181.6 (s, C10), 180.5 (s, C5), 163.5
1.47 (3H, t, J ) 7.2 Hz); C NMR (CDCl
3
) δ 181.6 (s, C5), 180.0
(
(s, C10), 163.7 (s, COO), 162.4 (s, C1), 156.0 (d, C7), 151.5 (s,
C3), 149.5 (s, C5a), 149.0 (s, C4′), 141.3 (s, C10a), 135.1 (d,
C9), 131.6 (s, C9a), 129.0 (2C d, C2′6′), 127.9 (d, C8), 127.4 (s,
1
28.5 (d, C7), 125.5 (s, C4a), 120.0 (d, C4), 62.6 (t), 14.2 (q);
C1′), 125.6 (s, C4a), 118.9 (d, C4), 115.8 (2C d, C3′5′), 62.5 (t),
+
+
MS m/z 358 (M ). Anal. (C21
H
14
N
2
O
4
) C, H, N.
40.3 (2C, q), 21.4 (q); MS m/z 401 (M ). Anal. (C23
H
19
N
3
O
4
) C,
1
-(4-Nit r op h en yl)-3-et h oxyca r b on ylp yr id o[3,2-g]iso-
H, N.
qu in olin -5,10-d ion es (9): mp, 311-2 °C; UV-vis (CHCl
3
) λmax
3-Am in o-3-eth oxyca r bon yld ih yd r oth ien o[2,3-g]qu in o-
lin -4,9-d ion e (15): mp > 200 °C dec; UV (CHCl ) λmax (log ꢀ)
) δ 9.01 (1H, dd, J ) 6.3, 1.6
1
(
log ꢀ) nm, 371 (3.0); H NMR (CDCl
3
) 9.17 (1H, dd, J ) 4.8,
3
1
1
.2 Hz), 8.93 (1H, s), 8.68 (1H, dd, J ) 8.6, 1.2 Hz), 8.34 (2H,
nm, 418 (3.6); H NMR (CDCl
3
d, J ) 8.6 Hz), 7.82 (1H, dd, J ) 7.8, 4.8 Hz), 7.73 (2H, d, J )
.6 Hz), 4.56 (2H, q, J ) 7.2 Hz), 1.48 (3H, t, J ) 7.2 Hz); 13
NMR (CDCl ) δ 182.1 (s, C10), 181.9 (s, C5), 163.3 (s, COO),
62.5 (s, C1), 155.9 (d, C8), 152.1 (s, C3), 148.9 (s, C9a), 142.9
Hz), 8.40 (1H, dd, J ) 8.2, 1.6 Hz), 7.64 (2H, d, J ) 8.2 Hz),
8
C
4.27 (2H, q, J ) 7.2 Hz), 3.87 (1H, d, J ) 12.4 Hz), 3.34 (1H,
d, J ) 12.4 Hz), 1.27 (3H, t, J ) 7.2 Hz); 13C NMR (CDCl
) δ
3
3
1
180.7 (s, C5), 179.9 (s, C9), 172.5 (s, COO), 154.9 (s, C5a), 154.3
(d, C2), 146.7 (s, C9a), 142.0 (s, C8a), 139.1 (s, C4a), 136.7 (d,
C4), 128.2 (d, C3), 73.3 (s, C8), 62.6 (t), 42.4 (t, C7), 14.0 (q);
(
(
(
s, C10a), 139.3 (s, C1′), 136.5 (d, C4′), 135.9 (d, C6), 130.0
2C d, C3′5′), 129.5 (d, C5a), 128.4 (d, C7), 125.5 (s, C4a), 123.2
2C d, C2′6′), 121.3 (d, C4), 63.0 (t), 14.1 (q); MS m/z 403 (M ).
+
+
+
+
MS m/z 304 (M ), 306 (M + 2, 11% M ), 308 (M + 4, 4% M ).
Anal. (C14 S) C, H, N.
3-Am in o-3-eth oxyca r bon yld ih yd r oth ien o[3,2-g]qu in o-
lin -4,9-d ion e (16): mp > 200 °C dec; UV (CHCl ) λmax (log ꢀ)
) δ 9.0 (1H, dd, J ) 6.3, 1.6
Anal. (C21
H
13
N
3
O
6
) C, H, N.
12 2 4
H N O
1
-(4-Ch lor op h en yl)-3-eth oxyca r bon ylp yr id o[3,2-g]iso-
qu in olin -5,10-d ion es (10): mp, 217-8 °C; UV-vis (CHCl
3
)
3
1
1
λ
max (log ꢀ) nm, 370 (2.9); H NMR (CDCl
4
3
) δ 9.17 (1H, dd, J )
nm, 413 (3.2); H NMR (CDCl
3
.8, 1.2 Hz), 8.84 (1H, s), 8.63 (1H, dd, J ) 8.6, 1.2 Hz), 7.82
Hz), 8.38 (1H, dd, J ) 8.2, 1.6 Hz), 7.62 (2H, d, J ) 8.2 Hz),
(
1H, dd, J ) 7.8, 4.8 Hz), 7.50 (2H, d, J ) 8.6 Hz), 7.47 (2H,
4.27 (2H, q, J ) 7.2 Hz), 3.85 (1H, d, J ) 12.4 Hz), 3.36 (1H,
d, J ) 8.6 Hz), 4.55 (2H, q, J ) 7.2 Hz), 1.46 (3H, t, J ) 7.2
d, J ) 12.4 Hz), 1.27 (3H, t, J ) 7.2 Hz); 13C NMR (CDCl
) δ
3
1
3
Hz); C NMR (CDCl
3
) δ 181.6 (s, C10), 180.2 (s, C5), 163.4 (s,
180.1 (s, C9), 180.0 (s, C5), 171.9 (s, COO), 155.5 (d, C2), 154.7
(s, C8a), 146.8 (s, C9a), 142.2 (s, C5a), 138.9 (s, C4a), 135.8
(d, C4), 128.5 (d, C3), 72.0 (s, C6), 62.5 (t), 43.1 (t, C7), 14.1
COO), 160.9 (s, C1), 155.5 (d, C7), 152.3 (s, C3), 147.7(s, C9a),
1
1
1
3
41.3 (s, C10a), 137.1 (2C d, C2′6′), 135.8 (d, C6), 135.6 (s, C4′),
30.4. (s, C1′), 129.4 (s, C5a), 128.6 (d, C7), 128.5 (2C d, C3′5′),
+
+
(q); MS m/z 304 (M ), 306 (M + 2, 11% M ), 308 (M + 4, 4%
+
+
25.7 (s, C4a), 120.3 (d, C4), 62.7 (t), 14.1 (q); MS m/z 392 (M ),
12 2 4
M ). Anal. (C14H N O S) C, H, N.
+
94 (M + 2, 32% M ). Anal. (C21
H
13
N
2
O
4
Cl) C, H, N.
Biology. Test compounds were dissolved in DMSO at an
initial concentration of 200 µM and then were serially diluted
in culture medium.
1
-(4-Br om op h en yl)-3-eth oxyca r bon ylp yr id o[3,2-g]iso-
qu in olin -5,10-d ion es (11): mp, 209-10 °C; UV-vis (CHCl
3
)

Quinolindione Derivatives with Cytotoxic Activity
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 857
Cells. Cell lines were from American Type Culture Collec-
tion (ATCC). Leukemia- and lymphoma-derived cells were
grown in RPMI 1640 containing 10% fetal calf serum (FCS),
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00 U/mL penicillin G, and 100 µg/mL streptomycin. Solid-
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V20C
7D
subclones KB
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(
KB cell lines were maintained in growth medium supple-
MDR
mented with 0.09 µM doxorubicin for KB
, 0.02 µM vinc-
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7D
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2
atmosphere.
2
79.
Cell cultures were checked periodically for the absence of
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(
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(
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×
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1
995, 48, 499-504.
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(
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2
6
I assay. Supercoiled SV40 DNA (0.4 µg) was incubated with
5
U of topoisomerase I (Life Technologies Inc.) in 30 µL of Topo
I reaction buffer. After 5 min at 37 °C, a compound to be tested
was added and the incubation continued for 60 min. Reactions
were stopped by addition of dodecyl sulfate (SDS) and pro-
(
-
1
teinase K (final concentrations of 1% and 0.5 mg mL
,
respectively). After an additional 15 min at 37 °C, the samples
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-
1
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mM EDTA, pH 8.0). DNA bands were visualized with ethidium
bromide (0.5 µg mL-1) and photographed under UV
3
282.
Sp ectr op h otom etr y. Molar extinction coefficients at the
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hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mM EDTA,
and 99.4 mM NaCl (pH 7.0)]/DMSO (1:1 v/v) at 25 °C. Spectra
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(
(
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2
7
ducted using the software package SYBYL running on a
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8
29
mechanics methods AM1, available in the MOPAC program
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(
1
15.
superimposed by minimization of the root-mean-square dis-
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(
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(
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the Centro Interdipartimentale di Analisi Strumentale
dell’Universit a` di Napoli “Federico II”. This work was
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J M030918B