Antitumor agents 6. Synthesis, structure-activity relationships, and biological evaluation of spiro[imidazolidine-4,3′-thieno[2,3-g]quinoline]- tetraones and spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]triazinane]- tetraones with potent antiproliferative activity

Article abstract of DOI:10.1021/jm8007689

Two series of quinolinquinone derivatives, 2′H-spiro[imidazolidine-4, 3′-thieno[2,3-g]quinoline]-2,4′,5,9′-tetraones (2a-n) and 2H-spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]triazinane]-3′,4, 6′,9-tetraones (3a-e), were designed and synthesized using the previously described ethyl 3-amino-4,9-dioxo-2,3,4,9-tetrahydrothieno[2,3-g]quinoline-3- carboxylate (1) as a starting material. All compounds were evaluated for their antiproliferative activity against a panel of representative liquid and solid human tumor cell lines and exhibit IC₅₀ values in the micromolar/submicromolar range. Series 2 displayed higher cytotoxicity than did series 3. The nature of the substituents on both imidazoline and triazinane N1 nitrogen markedly affected the activity profile of these series. Spectrophotometric and fluorescence measurements as well as unwinding assays performed on the most cytotoxic compounds, 2c, 2g, and 2k, showed that they are nonintercalative DNA agents and inhibit the catalytic activity of Topo II in a concentration-dependent mode. 2g was the most active Topo II inhibitor with activity levels comparable to those of VP-16.

Full text of DOI:10.1021/jm8007689

8148
J. Med. Chem. 2008, 51, 8148–8157
Antitumor Agents 6. Synthesis, Structure-Activity Relationships, and Biological Evaluation of
Spiro[imidazolidine-4,3′-thieno[2,3-g]quinoline]-tetraones and Spiro[thieno[2,3-g]quinoline-3,5′-
[1,2,4]triazinane]-tetraones with Potent Antiproliferative Activity†
Adele Bolognese,*,‡,| Gaetano Correale,‡ Michele Manfra,‡ Anna Esposito,‡ Ettore Novellino,§ and Antonio Lavecchia*,§,|
Dipartimento di Chimica Organica e Biochimica, UniVersita` di Napoli “Federico II”, Via Cynthia 6, Monte Sant’Angelo,
I-80126 Napoli, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersita` di Napoli “Federico II”, Via D. Montesano 49,
I-80131 Napoli, Italy
ReceiVed June 23, 2008
Two series of quinolinquinone derivatives, 2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quinoline]-2,4′,5,9′-
tetraones (2a-n) and 2H-spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]triazinane]-3′,4,6′,9-tetraones (3a-e), were
designed and synthesized using the previously described ethyl 3-amino-4,9-dioxo-2,3,4,9-tetrahydrothieno[2,3-
g]quinoline-3-carboxylate (1) as a starting material. All compounds were evaluated for their antiproliferative
activity against a panel of representative liquid and solid human tumor cell lines and exhibit IC50 values in
the micromolar/submicromolar range. Series 2 displayed higher cytotoxicity than did series 3. The nature
of the substituents on both imidazoline and triazinane N1 nitrogen markedly affected the activity profile of
these series. Spectrophotometric and fluorescence measurements as well as unwinding assays performed on
the most cytotoxic compounds, 2c, 2g, and 2k, showed that they are nonintercalative DNA agents and
inhibit the catalytic activity of Topo II in a concentration-dependent mode. 2g was the most active Topo II
inhibitor with activity levels comparable to those of VP-16.
Chart 1. Chemical Structures of Quinolindiones 1 and 1′
Introduction
Current chemotherapy commonly uses quinone-containing drugs
to interfere with DNA replication and induce apoptosis in replicat-
ing cells.1-4 Numerous quinone derivatives are powerful anticancer
agents characterized by a DNA intercalating mechanism of action
because of the planar structure of the R,ꢀ-unsaturated system.
Furthermore, they can induce the formation of semiquinone
radicals, which can transfer an electron to oxygen and produce a
superoxide anion originating the ROS cascade.5-8 This process is
catalyzed by flavoenzymes such as NADPH-cytochrome P-450
reductase. Both superoxide and semiquinone radical anions can
generate the hydroxyl radical, which is known to cause DNA strand
breaks and enzyme modification, devastating effects leading to
determine cell death. These multimodal activities of quinone drugs,
using different weapons to attack the neoplastic cells contempo-
raneously, give high therapeutic relevance to this class of antitumor
drugs. Very significant examples of these compounds are the well-
known actinomicyn D and doxoa.3
In a previous paper, we reported the synthesis of ethyl
3-amino-4,9-dioxo-2,3,4,9-tetrahydrothieno[2,3-g]quinoline-3-
carboxylate (1, Chart 1 and Scheme 1) and of ethyl 3-amino-
4,9-dioxo-2,3,4,9-tetrahydrothieno[3,2-g]quinoline-3-carboxy-
late (1′), two quinolindione derivatives arising from a 1,4-
Michael reaction of quinolin-5,8-dione with a thiolate species
formed by the opening of 2-aryl-1,3-thiazolidine ethyl esters in
basic medium.9 Both 1 and 1′, which can be regarded as cyclic
cysteine derivatives, were suspected of interacting with
topoisomerases.9-12
Moreover, the antiproliferative activity of 1 and 1′, evaluated
against representative leukemia and solid human neoplastic cell
lines, exhibited a significantly high in vitro cytotoxicity, with 1
(IC50 ≈ 0.01 µM) being 10 times more active than 1′.9 Because
of our ongoing interest in the search for new antitumor
compounds containing the quinolin-5,8-dione motif, we explored
structural modifications of the dihydrothiophene ring of the most
active quinone 1, keeping the quinolin-5,8-dione moiety un-
changed. Because some imidazoline-diones,13 pyrimidine-diones
(ER-37328),14 and piperazine-diones (ICRF-159)15 (Chart 2) are
potent antitumor drugs whose mechanisms of action include
metal chelation16 and inhibition of the enzymatic activity of
Topo II,17 four new compounds, two 2′H-spiro[imidazolidine-
4,3′-thieno[2,3-g]quinoline]-2,4′,5,9′-tetraones, (+)-2 and (-)-
2, and two 2H-spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]triazinane]-
3′,4,6′,9-tetraones, (+)-3 and (-)-3 (Chart 3), were designed
and synthesized to be scaffolds for the development of new
potential antitumor drugs useful in chemotherapy.
†
Dedicated to Professor Rodolfo A. Nicolaus, a pioneer of the melanin
chemistry.
* Corresponding authors. (A.B.) Tel: +39-081-674121. Fax: +39-081-
674393. E-mail: bologne@unina.it. (A.L.) Tel: +39-081-678613. Fax: +39-
081-678613. E-mail: lavecchi@unina.it.
‡
Dipartimento di Chimica Organica e Biochimica.
Dipartimento di Chimica Farmaceutica e Tossicologica.
These authors equally contributed to this work.
§
|
a Abbreviations: VP-16, etoposide; doxo, doxorubicin; CCRF-CEM,
human acute T-lymphoblastic leukemia; CCRF-SB, human acute B-
lymphoblastic leukemia; MT-4, human CD4+ T cells expressing the TAT
gene of HTLV-1; HT-29, human colon adenocarcinoma; MCF-7, human
breast adenocarcinoma; HeLa, cervix carcinoma; ACHN, human renal
adenocarcinoma;MRC-5,normalhumanlungfibroblasts;SAR,structure-activity
relationships; LNR, linear DNA; CC, circularized DNA; SC, supercoiled
DNA; RLX, relaxed DNA; ATCC, American Type Culture Collection; FCS,
fetal calf serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide; BSA, bovine serum albumin; Topo, topoisomerase; HBTU,
O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate; HOBt,
N-hydroxybenzotriazole; HBT, 1-hydroxybenotriazole; DIEA, diisopropy-
lethylamine.
10.1021/jm8007689 CCC: $40.75
2008 American Chemical Society
Published on Web 11/20/2008

Antitumor Agents 6
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8149
Scheme 1. Synthesis of Compounds (()-1 and (()-1′a
a Reagent and conditions: (i) Ag2CO3, DBU, acetonitrile, rt, 12 h; (ii) 1N HCl, 1 h, H2O/CHCl3.
Table 1. Cytotoxic Activity of 2 and 3 Enantiomers
Chart 2. Chemical Structures of Some Potent Antitumor Dione
IC50 (µM)a
Compounds
b
compd
MT-4
HT-29
(+)-2
(-)-2
(+)-3
(-)-3
doxob
0.13 ( 0.02
0.12 ( 0.004
0.55 ( 0.03
0.50 ( 0.09
0.003 ( 0.001
0.19 ( 0.02
0.18 ( 0.04
1.58 ( 0.05
1.60 ( 0.03
0.002 ( 0.001
a Data represent mean values ((SD) for three independent determinations.
b doxo was used as control.
1), showed a promising cytotoxicity against both leukemia/
lymphoma and carcinoma cells, independently of their stereo-
chemical difference. With the aim of increasing the compound
lypophilicity, a substituted pendant phenyl group was introduced
to scaffolds 2 and 3, and the effect of a set of electron-donating
and -withdrawing substituents at the four-position of the phenyl
ring was investigated to better delineate SAR (compounds 2a-e
and 3a-e). Positively chargeable polar substituents (compounds
1f-i) and highly lypophilic neutral groups (compounds 1j-n)
were also placed on the pendant phenyl of the more active series
1. The synthesis and assessment of the in vitro antiproliferative
activity of quinolinediones 1 and 2 (Chart 3) are described in
the present article.
Chart 3. Chemical Structures of the Newly Designed and
Synthesized Quinolindione Derivatives
Chemistry
The racemic mixture of ethyl 3-amino-4,9-dioxo-2,3,4,9-
tetrahydrothieno[2,3-g]quinoline-3-carboxylate ((()-1) was ob-
tained from the in situ 1,4-Michael addition of a thiolate species
arising from the ethyl 2-phenylthiazolidine-4-carboxylate demo-
lition in basic medium, followed by hydrolysis, as reported in
a previous article.9 A small amount of isomer (()-1′ was also
formed in the reaction mixture (Scheme 1).
(()-1 was resolved according to the Evans method (Scheme
2),18 a useful alternative procedure to the salt crystallization
technique. (()-1 was coupled to boc-L-Phe in a THF/DMF (3:1
v/v) anhydrous solution (using the coupling reagents HBTU,
HOBT, and DIEA) to give the mixture of protected diastereomer
phenylalanylamides 1a and 1a′ (1:1). Removal of the boc group,
followed by neutralization, gave the diastereomer amides 1b
and 1b′, which were separated by flash chromatography. The
diasteroisomers 1b and 1b′ were warmed, respectively, with
phenylisothiocyanate to form the corresponding thiocarbamoyl
derivatives 1c and 1c′, which in turn yielded (+)-1 and (-)-1
by TFA treatment.
The pure enantiomers (+)-1 and (-)-1 were used to synthe-
size the couples (+)-2 and (-)-2 and (+)-3 and (-)-3,
respectively, as reported in Scheme 3.
The enantiomeric couples of 2 and 3 (Chart 3), which were
preliminarily tested against MT-4 and HT-29 cell lines (Table

8150 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Scheme 2. Resolution of (+)-1 and (-)-1 Enantiomersa
Bolognese et al.
Scheme 3. Synthesis of Enantiomers (+)-2, (-)-2, (+)-3, and
(-)-3a
a Reagents and conditions: (i) (CHCl3)2CO, TEA, THF, rt, 30 min, then
NH4Cl, TEA; (i′) (CHCl3)2CO, TEA, THF, rt, 30 min, then NH2-NH2;
(ii) EtOH, TEA, reflux (1 or 2 h).
a Reagents and conditions: (i) Boc-L-Phe, HBTU, HOBt, DIEA, THF/
DMF, rt, 48 h; (ii) TFA, CH2CH2, rt, 2 h, then TEA; (iii) flash
chromatography separation; (iv) CSNHPh, CH2Cl2, reflux 1 h; (v) TFA,
CH2Cl2, rt, 1 h.
active compounds were also tested on MRC-5, a normal human
lung fibroblast cell line. Data for doxo and VP-16 were included
as control.
(+)-1 and (-)-1 were treated, respectively, with bis(trichlo-
romethyl)carbonate, a very handy phosgene analog, in presence
of TEA and ammonium chloride or hydrazine in THF. The
parenthesized urea intermediates were not isolated and afforded
(+)-2 and (-)-2 and (+)-3 and (-)-3, respectively, by the
subsequent addition of EtOH and TEA at reflux temperature.
This synthetic pathway consists of a sequence of three imidation
reactions. The first two steps involve the highly favored
nucleophilic attack of an amine group on the electron-poor
carbonyl carbon of the bis(trichloromethyl)carbonate. The third
step, an ester-urea cyclization, needed further heating. Series
2a-n and 3a-e were prepared according to this general
procedure by the use of appropriate anilines and aminopyridine
or hydrazines (Scheme 4), and their structures were determined
on the basis of the physical-chemical and spectroscopic
properties and elemental analysis.
The cytotoxicities of compounds reported in Tables 1 and 2
revealed that lead 3 was 10 times less active than 2 and that
the substituted phenyl derivatives 2a-e and 3a-e were more
cytotoxic than 2 and 3, respectively. In general, series 2a-e
was more active than series 3a-e. On the basis of the median
of the IC50 values, the cytotoxic activity seems to decrease
according to the nature of the substituent in the order 4-NO2Ph
< 4-OCH3Ph < Ph E 4-CH3Ph < 4-ClPh in both series 2a-e
and 3a-e. We note that the human CNS SF-268 and XF 498
cells were 10-1000 times less sensitive than the other tumor
lines to the antiproliferative effect of compounds 2a-e and 3a-
e. In series 2a-e, the phenyl derivative 2a (median IC50 ) 0.02
µM), the 4-methylphenyl derivative 2b (median IC50
)
0.02 µM), and the 4-chlorophenyl derivative 2c (median IC50
) 0.01 µM) were the most cytotoxic compounds, exhibiting an
activity comparable or superior to that of doxo (median IC50
)
0.03 µM) and superior to that of VP-16 (median IC50 ) 2.15
µM). Compounds 2a-d displayed better specificity as antipro-
liferative agents against carcinoma than against leukemia cell
lines. A selective inhibitory effect was observed for the
proliferation of HT-29 cells with IC50 values ranging from 0.001
to 0.002 µM. Surprisingly, the 4-nitrophenyl derivative 2e,
which is weakly cytotoxic against carcinoma cells, showed a
selective activity against lymphoma/leukemia cells (range of
0.001 to 0.008 µM).
As illustrated in Table 1, the normal human MRC-5 lung
fibroblasts were about 100 times less sensitive to the inhibitory
activity of the most cytotoxic compounds, 2c, 2g, and 2k. This
finding points to the highly differential (selective) effect between
tumor and normal cells of the aforementioned compounds. It is
Results and Discussion
In Vitro Cytotoxicity. Table 1 shows the cytotoxic activity
of the separated enantiomers of lead compounds 2 and 3 tested
against the lymphoid MT-4 and the carcinoma HT-29 cell lines
compared with doxo. The reported data suggest that both
enantiomers of 2 and 3 are active at micromolar concentration
and that the activity is independent of the compound chirality.
Because no substantial difference was observed between the
activities of the enantiomeric couples of 2 and 3, the further
synthesized derivatives were tested as racemic mixtures. Table
2 shows the in vitro cytotoxic activity of compounds 2, 2a-e,
3, and 3a-e tested on a panel of nine human tumor cell lines
representative of leukemia and solid human tumors. The most

Antitumor Agents 6
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8151
Scheme 4. Synthesis of Compounds (()-2a-n and (()-3a-ea
a Reagents and conditions: (i) (CHCl3)2CO, TEA, THF, rt, 30 min, then substituted anilines; (i′) (CHCl3)2CO, TEA, THF, rt, 30 min, then aminopyridine;
(i′′) (CHCl3)2CO, TEA, THF, rt, 30 min, then substituted hydrazines; (ii) EtOH, TEA, reflux (1 or 2 h).
noteworthy that 2c, 2g, and 2k showed significantly stronger
activity than VP-16 against normal cells but showed lower
activity in comparison with doxo.
erative activity. Table 2 reports the cytotoxic activity of the
new derivatives 2f-n.
Compounds 2f, 2g, and 2h, which hold, respectively, a
pyridine-, 4-N-dimethylphenyl-, and 4-N-diethylphenyl sub-
stituent, showed a nanomolar cytotoxic activity against lym-
phoma/leukemia cells and a lower antiproliferative effect on
carcinoma cells compared with compounds 2a-e. Only com-
pound 2f displayed high cytotoxicity against HT-29 carcinoma
cells (IC50 ) 0.002 µM). The 4-morfolinophenyl derivative 2i
was the least active compound of the series, suggesting that an
electronegative oxygen far from the phenyl ring and the further
bulky six-membered ring decreases the activity of the molecule.
For the highly lipophilic derivatives 2j-n, the ethyl and
propyl derivatives 2j and 2k showed the highest potency against
almost all tested cell lines, including the CNS cells, SF-268
and XF-498. Extending the chain length from methyl to ethyl
and propyl chain (compounds 2b, 2j, and 2k), an increase in
potency was recorded, whereas longer side chains resulted in a
dramatic loss of potency (compounds 2l, 2m, and 2n). This
result could mean that too many lipophilic compounds, trapped
in the outer lipid bilayer, could not reach their target to exert
biological activity.
In regards to the less-active series 3a-e, Table 2 shows that
the compounds were more active against lymphoma/leukemia
than carcinoma cells. Among the lymphoma cell lines, the
CCRF-CEM and the CCRF-SB cells were most prone to the
antiproliferative effect of 3a-e. The 4-nitrophenyl derivative
3e revealed a distinct specificity against CCRF-CEM, CCRF-
SB, and MT-4 cells. The activity on lymphoma/leukemia cells
of both 2e and 3e was 10 times higher than that of doxo (range
of 0.002 to 0.005 µM). These compounds, both containing a
reductable nitro group, could give rise to cell-damaging reactive
amine species generated by some bioreductive processes.19
However, to sustain this hypothesis, a more detailed investiga-
tion should be carried out.
Because series 2a-e was found to be more active than series
3a-e, the scaffold of 2 was selected to develop the derivatives
2f-n further (Chart 3). Compounds 2f-i, which have cationic
groups, were designed on the basis of the fact that many
quinones or polycarbonylic compounds bearing a cationic side
chain such as intoplicine,20 TAS-103,21 and BBR-277822 (Chart
4) are potent inhibitors of Topo I and II, important targets of
numerous antiproliferative drugs.
DNA Binding Properties. To test the propensity of the most
active series 2 to interact with DNA, we carried out spectro-
photometric and fluorescence measurements. Moreover, un-
winding assays were undertaken to observe whether these
compounds could intercalate DNA. The most representa-
tive compounds of series 2, 2c, 2g, and 2k, were examined.
Moreover, given that the lipophilicity is a physical parameter
that can contribute to in vitro cytotoxicity,23 the 4-substituted
phenyl derivatives 2j-n, which contain highly apolar substit-
uents, were also synthesized and evaluated for their antiprolif-

8152 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Bolognese et al.
Table 2. Antiproliferative Activity of Compounds (()-2, (()-2a-e, (()-3, and (()-3a-e
IC50 (µM)a
compd
R
CCRF-CEM CCRF-SB MT-4 HT-29 MCF-7 HeLa ACHN SF-268 XF-498 MRC-5 normal cells
2
H
Ph
0.12
0.01
0.02
0.01
0.05
0.001
0.002
0.001
0.020
0.60
0.01
0.001
0.15
0.20
0.25
0.45
0.09
0.07
0.05
0.035
0.002
0.02
1.20
0.15
0.03
0.02
0.01
0.13
0.19
0.002
0.001
0.002
0.002
0.25
0.002
0.02
0.05
0.75
0.001
0.001
0.05
0.15
0.25
1.60
0.55
0.20
0.22
0.65
1.01
0.01
2.50
0.12
0.001
0.002
0.004
0.05
0.35
0.01
0.01
0.03
0.75
0.05
0.002
0.07
0.13
0.25
1.25
0.15
0.41
0.11
0.23
0.55
0.05
1.00
0.15
0.003
0.002
0.002
0.001
0.10
0.01
0.01
0.05
0.20
0.005
0.005
0.09
0.10
0.20
0.98
0.21
0.21
0.15
0.15
0.61
0.07
3.00
0.08
0.021
0.012
0.004
0.05
0.10
0.08
0.09
0.02
0.14
0.005
0.001
0.05
0.15
0.25
1.05
0.94
0.50
0.36
0.43
0.65
0.04
2.15
3.10
1.70
0.66
0.58
0.65
1.52
1.10
0.95
0.96
1.98
0.45
0.30
0.40
0.50
0.52
4.82
2.20
2.10
2.00
2.85
3.55
0.20
3.25
3.12
1.51
1.35
0.59
0.65
1.03
1.12
0.80
0.73
1.70
1.20
0.45
0.95
0.85
0.83
5.00
4.55
3.80
2.50
3.80
4.80
0.16
3.45
2a
2b
2c
2d
2e
0.025
0.027
0.01
4-CH3Ph
4-ClPh
4-OCH3Ph
4-NO2Ph
4-Py
4-N(CH3)2Ph
4-N(C2H5)2Ph
4-N-morpholinylPh
4-ethylPh
4-propylPh
4-butylPh
4-t-butylPh
4-pentylPh
H
0.80
1.00
0.85
0.01
0.07
0.002
0.001
0.001
0.009
0.65
0.01
0.002
0.19
0.008
0.003
0.015
0.008
0.80
0.001
0.001
0.20
0.20
0.19
0.55
0.15
0.10
0.15
0.08
0.005
0.01
2f
2g
2h
2i
2j
2k
2l
2m
2n
3
3a
3b
3c
0.20
0.15
0.048
0.039
0.037
0.051
0.035
0.002
0.03
Ph
4-CH3Ph
4-ClPh
4-OCH3Ph
4-NO2Ph
3d
3e
doxob
VP-16
0.30
3.90
0.10
1.25
a Compound concentration required to reduce cell proliferation by 50%, as determined the MTT method, under conditions allowing untreated controls to
undergo at least three consecutive rounds of multiplication. Data represent mean values for three independent determinations. b Control.
Chart 4. Chemical Structures of Potent Inhibitors of Topo I and
II
Figure1. Quenchingoffluorescenceintensityofethidiumbromide-DNA
complex by compound 2c. Experimental conditions: 20 µM calf thymus
DNA, 2 µM ethidium bromide (λexc ) 546 nm, λem ) 595 nm), in
0.01 M ionic-strength buffer (9.3 mM NaCl, 2 mM Na-acetate, and
0.1 mM EDTA).
The spectroscopic properties of the selected compounds, in the
presence and absence of calf thymus DNA, were studied by
conventional optical spectroscopy. Characterization of this
mixture was made by comparing the UV-vis absorption
spectrum of the complex with the spectra of 2c, 2g, and 2k,
respectively.
The absorption spectrum of compound 2c (data not shown)
was totally unaffected by the addition of DNA. The absorption
maximum of the free drug at 425 nm remains unchanged, and
the hypochromism is extremely weak (3%), suggesting that 2c
has no significant interaction with the DNA double helix.
Moreover, we used fluorescence spectroscopy to examine the
abilities of the test drugs to compete with a DNA-binding ligand
for available binding sites. In these experiments, the fluorescent
ligand used was the intercalating drug ethidium bromide, which
is highly fluorescent in the presence of DNA. Almost no
quenching was observed with compounds 2c, 2g, and 2k. Figure
1 shows the fluorescence quenching of 2c. The results of the
fluorescence quenching experiments are totally consistent with
the absorption measurements and demonstrate that compound
2c has minimal, if any, interaction with DNA. Similar spectro-
photometric and fluorescence results were recorded for com-
pounds 2g and 2k.
To further investigate whether compounds belonging to series
2 intercalate in DNA, we performed an unwinding assay using
linearized pBR322 and T4 DNA ligase. In this assay, DNA
intercalators first unwind the linearized DNA, which results in
a change in the twist of the duplex helix. Circulation of drug-
bound DNA by T4 DNA ligase freezes the linking number of
the unwound DNA. Upon drug removal, the twist changes back
to normal, whereas the linking number remains constant, which
causes the introduction of negative superhelicity to the DNA.
Figure 2 shows the separation of the products on an agarose
gel. In lanes 3-5, the inhibition of the activity of the T4 ligase
by doxo was observed, which was due to the strong intercalation

Antitumor Agents 6
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8153
Figure 2. Effect of compounds 2c, 2g, and 2k on the DNA unwinding assay with T4 DNA ligase. In this assay, linearized plasmid DNA was
incubated with T4 DNA ligase in the presence of testing compounds. Linear DNA is shown for reference. The positions of LNR and CC DNA are
indicated.
Figure 3. Effect of 2c, 2g, and 2k on the DNA unwinding assay with DNA Topo I. In this assay, supercoiled plasmid DNA was incubated with
Topo I. Negatively supercoiled pBR322 (DNA Std) is shown for reference. The positions of SC and RLX DNA are indicated.
ability of doxo.24 However, the negative control VP16 and the
compounds 2c, 2g, and 2k, which were chosen to be representa-
tive of series 2, had no significant influence on the DNA
topoisomers, even at concentrations of 100 µM.
4, 2c, 2g, and 2k displayed significant inhibition of this reaction
in a concentration-dependent mode. It appears that compounds
2c, 2g, and 2k are potent Topo II inhibitors, with 2g being the
most potent. This result suggests that the cationic group
promotes more favorable interactions with the enzyme.
To verify the results, another unwinding assay was performed.
In this assay, supercoiled DNA is relaxed by Topo I in the
presence of the testing compounds. The intercalation of the
compounds into a closed circular DNA reduces the twisting
number of the DNA. Upon Topo I addition, the linking number
is adjusted to the twist of the intercalated DNA. Following
relaxation, samples are phenol-extracted to remove the inter-
calator, and the twisting number is restored, thus leaving the
DNA in a new supercoiled state. As shown in Figure 3, doxo
converts the topoisomers to a less-relaxed form at a low
concentration (5 µM) and to a complete supercoiled form at a
higher concentration (25 µM). On the contrary, VP16, 2c, 2g,
and 2k have no effect. From these two experiments, we
concluded that compound 2c, 2g, and 2k and the whole series
2 are nonintercalative agents.
Inhibition of the Activity of Topo II by 2c, 2g, and 2k.
Topo II is an essential enzyme that plays an important role in
DNA replication, repair, transcription, and chromosome segre-
gation. Topo II alters DNA topology by catalyzing the passing
of an intact DNA double helix through a transient double-
stranded break made in a second helix.25,26 A number of
antitumor drugs, including quinonic structures, are thought to
be cytotoxic by virtue of their ability to stabilize a covalent
Topo II-DNA intermediate, the cleavable complex.27
Conclusions
This study reports the synthesis and the SARs of two series
of quinolindione derivatives, the spiro[(dihydroimidazo-2,4-
dione)-5,3′-(2′,3′-dihydrothieno[2,3-b]quinoline-4′,9′-diones)]
(2a-n) and the spiro[(1,2,4-triazinane-3,6-dione)-6,3′-(2′,3′-
dihydrothieno[2,3-g]quinoline-4′,9′-dione)] (3a-e), analogues
of the 3-amino-3-(ethoxycarbonyl)-2,3-dihydrothieno[2,3-b]quin-
oline-2,5-dione (1), which exhibits a broad spectrum of cyto-
toxicity against leukemia and solid tumor cell lines at micro-
molar and submicromolar concentrations. Series 2 displays
higher cytotoxicity compared with the corresponding series 3.
Among the compounds 2a-e, the 4-nitrophenyl derivative 2e
showed potent activity against lymphoma/leukemia cells (range
of 0.001 to 0.008 µM). The positively charged compounds 2f,
2g, and 2h displayed nanomolar cytotoxic activity against
lymphoma/leukemia cells and, when compared with compounds
2a-e, a lower antiproliferative effect on carcinoma cells. Among
the lipophilic compounds 2j-n, the propyl derivative 2k showed
the highest potency against almost all tested cell lines, including
the CNS cells, SF-268 and XF-498.
Spectrophotometric and fluorescence measurements as well
as unwinding assays performed on the most active compounds
2c, 2g, and 2k, showed that they are nonintercalative DNA
agents. The inhibition of these compounds on the catalytic
activity of Topo II was also examined, and 2g was the most
The effect of compounds 2c, 2g, and 2k on the strand passage
activity of Topo II was determined by the enzyme-mediated
negatively supercoiled pBR322 relaxation. As shown in Figure

8154 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Bolognese et al.
Figure 4. Effect of compounds 2c, 2g, and 2k on Topo-II-mediated supercoiled pBR322 relaxation. Negatively supercoiled pBR322 (DNA Std)
and relaxed pBR322 (no drug) are shown for reference. The positions of SC and RLX are reported.
CDCl3): 8.90 (1H, dd, J ) 4.3, 2.2 Hz), 8.36 (1H, dd, 7.3, 2.2 Hz),
7.70 (1H, bs, NH), 7.57 (1H, dd, J ) 7.3, 4.3 Hz), 7.35 (5H, m),
7.10 (6H, m, 5H + NH), 6.32 (1H, bs, NH), 5.25 (1H, m, RCH),
4.25 (2H, q, CH2), 3.81 (1H, d, J ) 12.5 Hz), 3.70 (1H, d, J )
12.5 Hz), 3.10 (2H, m, ꢀCH2), 1.22 (3H, t, CH3). Anal.
(C30H26N4O5S2) C, H, N, S.
active compound, showing an inhibitory effect comparable to
that of VP-16. An attempt to understand the mechanism of action
of this series of new nonintercalative Topo II inhibitors further
is being undertaken in our laboratory.
Experimental Section
Ethyl 4,9-Dioxo-3-(3-phenyl-2-(3-phenylthioureido)propanamido)-
2,3,4,9-tetrahydrothieno[2,3-g]quinoline-3-carboxylates (1c′). Yel-
low solid (70%). mp: dec > 220. MS m/z: 586 (M+).1H NMR (δ,
CDCl3): 8.90 (1H, dd, J ) 4.3, 2.2 Hz), 8.36 (1H, dd, 7.3, 2.2 Hz),
7.71 (1H, bs, NH), 7.57 (1H, dd, J ) 7.3, 4.3 Hz), 7.35 (5H, m),
7.10 (6H, m, 5H + NH), 6.32 (1H, bs, NH), 5.27 (1H, m, RCH),
4.25 (2H, q, CH2), 3.81 (1H, d, J ) 12.5 Hz), 3.70 (1H, d, J )
12.5 Hz), 3.12 (2H, m, ꢀCH2), 1.22 (3H, t, CH3). Anal.
(C30H26N4O5S2) C, H, N, S.
General. Reagents, starting material, and solvents were pur-
chased from commercial suppliers and were used as received.
Analytical TLC was performed on a 0.25 mm layer of silica gel
60 F254 from Merck, and preparative TLC was performed on 20
× 20 cm2 glass plates coated with a 2 mm layer of silica gel PF254
from Merck. Silica gel 60 (300-400 mesh), Merck, was used for
flash chromatography. Melting points were taken on a Kofler
apparatus and are uncorrected. Optical rotations were determined
1
with a Perkin-Elmer-241 MC polarimeter. H NMR spectra were
2′H-Spiro[imidazolidine-4,3′-thieno[2,3-g]quinoline]-2,4′,5,9′-tet-
raones (2, 2a-n) and 2H-Spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]tri-
azinane]-3′,4,6′,9-tetraones (3, 3a-e). General Procedure. Bis-
(trichloromethyl)carbonate (118 mg, 0.4 mmol) was added to a
solution of the quinoline-dione 1, (300 mg, 1 mmol) in dry THF
(25 mL) at room temperature. TEA (0.3 mL, 2 mmol) was added
dropwise under stirring. After 10 min, a solution in dry THF (5
mL) of TEA (0.3 mL, 2 mmol) and ammonium chloride, aniline,
substituted anilines (4-methyl-, 4-chloro-, 4-methoxy-, and 4-nitro),
or 4-aminopyridine (2 equiv) was added under stirring. After 1 h,
the reaction mixtures were diluted with chloroform, washed with
water, dried over Na2SO4, and evaporated to dryness and afforded
the crude compounds 2 and 2a-n, respectively. All compounds
were purified by flash chromatography (80% CHCl3, 20 MeOH).
To prepare compounds (3, 3a-e), according to the above-reported
procedure, hydrazine, phenylhydrazine, and the corresponding
substituted phenylhydrazines (4-methyl, 4-chloro, 4-methoxy, and
4-nitro) were used instead of the titled anilines.
recorded with a Bruker-500 spectrometer. Chemical shifts are
reported in δ relative to internal Me4Si, and J values are reported
in Hz. Mass spectra were obtained using an EI at 70 eV on a ZAB
2F spectrometer and a FABMS spectrometer.
Resolution of the (+)-1 and (-)-1 Enantiomers. (()-1 (1 mmol,
304 mg) was dissolved in a solution of THF/DMF (20 mL; 2:1
v/v), and L-Boc-Phe (1 equiv), HBTU (1.2 equiv), HOBt (1.2
equiv), and DIEA (2 equiv) were added to the stirring solution at
room temperature for 48 h. The reaction was evaporated in vacuo,
and the solid residue, dissolved in CHCl3, was washed with a 10%
citric acid (2 × 25 mL), 10% NaHCO3 (2 × 25 mL), and H2O (25
mL) solutions. The organic layer was dried over Na2SO4 and
evaporated. Flash chromatography (20-50% gradient of EtOAc
in n-hexane) of the solid residue gave the diastereoisomeric mixture
of the ethyl 3-(2-(tert-butoxycarbonylamino)-3-phenylpropana-
mido)-4,9-dioxo-2,3,4,9-tetrahydrothieno[2,3-g]quinoline-3-carbox-
ylates (1a, 1a′). The mixture, which was treated with TFA in CH2Cl2
at room temperature for 2 h, was neutralized by TEA, and the
solvent was evaporated to dryness. Flash chromatography of the
residue by the use of AcOEt as eluent yielded the ethyl 3-(2-amino-
3-phenylpropanamido)-4,9-dioxo-2,3,4,9-tetrahydrothieno[2,3-g]quin-
oline-3-carboxylates (1b, Rf less) and (1b′, Rf greater). Phenyl-
isothiocyanate (60 mg, 0.44 mmol) and TEA (88 mg, 0.8 mmol)
were added to the respective stirring solutions of 1b and 1b′ (170
mg, 0.35 mmol) in CH2Cl2. After 1 h at reflux temperature, the
solvent was evaporated to dryness, and the residues were analyzed
by flash chromatography using CHCl3 as eluent. The yellow solids
ethyl 4,9-dioxo-3-(3-phenyl-2-(3-phenylthioureido)propanamido)-
2,3,4,9-tetrahydrothieno[2,3-g]quinoline-3-carboxylates 1c and 1c′
(∼106 mg) were treated with TFA (2 mL). After 1 h at room
temperature, the reaction mixtures were neutralized with TEA and
evaporated to dryness. Flash chromatography, using ethyl ether as
eluent, yielded the crude residues of the enantiomers (+)-1 (orange
2′H-Spiro[imidazolidine-4,3′-thieno[2,3-g]quinoline]-2,4′,5,9′-tet-
1
raone (2). Pale yellow solid (43%). mp: 188-189 °C. H NMR
(500 MHz, CDCl3, δ): 10.5 (1H, s, NH-1), 9.02 (1H, d, J ) 4.2
Hz, 6′-H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7,
4.2 Hz, 7′-H), 5.90 (1H, s, NH-3), 3.40 (1H, d, J ) 12.9 Hz, 2′a-
H), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H). MS [M+] Calcd for
C13H7N3O4S: 301; Found. Anal. (C13H7N3O4S) C, H, N, S. (+)-2
([R]D20 ) +29.9° (c 1.2, MeOH)), (-)-2 ([R]2D0 ) -30.1° (c 1.1,
MeOH)).
1-Phenyl-2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quinoline]-
2,4′,5,9′-tetraone (2a). Pale yellow solid (45%). mp: 193-194 °C.
1H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2 Hz, 6′-H),
8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz,
7′-H), 7.64 (2H, d, J ) 7.2 Hz), 7.57 (2H, t, J ) 6.9 Hz), 7.30
(1H, t, J ) 6.5 Hz), 5.90 (1H, s, NH-3), 3.40 (1H, d, J ) 12.9 Hz,
2′a-H), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H). MS [M+] Calcd for
C19H11N3O4S: 377; Found. Anal. (C19H11N3O4S) C, H, N, S.
oil, [R]D20 ) +31.0° (c 1.2, MeOH)) and (-)-1 (orange oil, [R]D20
-30.1° (c 1.0, MeOH).
)
Ethyl 4,9-Dioxo-3-(3-phenyl-2-(3-phenylthioureido)propanamido)-
2,3,4,9-tetrahydrothieno[2,3-g]quinoline-3-carboxylates (1c). Yel-
low solid (70%). mp: dec > 220. MS m/z: 586 (M+).1H NMR (δ,
1-p-Tolyl-2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quinoline]-
2,4′,5,9′-tetraone (2b). Pale yellow solid (44%). mp: 196-197 °C.
1H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2 Hz, 6′-H),

Antitumor Agents 6
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8155
8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz,
7′-H), 7.58 (2H, d, J ) 7.2 Hz), 7.35 (2H, d, J ) 7.2 Hz), 5.90
(1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.40 (1H, d, J )
12.9 Hz, 2′a-H), 2.32 (3H, s). MS [M+] Calcd for C20H13N3O4S:
391; Found. Anal. (C20H13N3O4S) C, H, N, S.
1-(4-Propylphenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quin-
oline]-2,4′,5,9′-tetraone (2k). Pale yellow solid (44%). mp: 196-197
1
°C. H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2 Hz, 6′-
H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz,
7′-H), 7.58 (2H, d, J ) 7.2 Hz), 7.35 (2H, d, J ) 7.2 Hz), 5.90
(1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.40 (1H, d, J )
12.9 Hz, 2′a-H), 2.54 (2H, t, J ) 6.7 Hz), 1.60 (2H, m), 0.98 (3H,
t, J ) 6.7 Hz). MS [M+] Calcd for C22H17N3O4S: 419; Found.
Anal. (C22H17N3O4S) C, H, N, S.
1-(4-Chlorophenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quin-
oline]-2,4′,5,9′-tetraone (2c). Pale yellow solid (43%). mp: 197-198
1
°C. H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2 Hz, 6′-
H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz,
7′-H), 7.50 (2H, d, J ) 7.2 Hz), 7.30 (2H, d, J ) 7.2 Hz), 5.90
(1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.40 (1H, d, J )
12.9 Hz, 2′a-H). MS [M+] Calcd for C19H10ClN3O4S: 411, 413
(M + 2); Found. Anal. (C19H10ClN3O4) C, H, N, S.
1-(4-Butylphenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quin-
oline]-2,4′,5,9′-tetraone (2l). Pale yellow solid (44%). mp: 198-199
1
°C. H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2 Hz, 6′-
H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz,
7′-H), 7.58 (2H, d, J ) 7.2 Hz), 7.35 (2H, d, J ) 7.2 Hz), 5.90
(1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.40 (1H, d, J )
12.9 Hz, 2′a-H), 2.60 (2H, t, J ) 6.7 Hz), 1.58 (2H, m), 1.36 (2H,
m), 0.90 (3H, t, J ) 6.7 Hz). MS [M+] Calcd for C23H19N3O4S:
433; Found. Anal. (C23H19N3O4S) C, H, N, S.
1-(4-Methoxyphenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-
g]quinoline]-2,4′,5,9′-tetraone (2d). Pale yellow solid (46%). mp:
1
>200 °C. H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2
Hz, 6′-H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7,
4.2 Hz, 7′-H), 7.40 (2H, d, J ) 7.2 Hz), 7.15 (2H, d, J ) 7.2 Hz),
5.90 (1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.75 (3H, s),
3.40 (1H, d, J ) 12.9 Hz, 2′a-H). MS [M+] Calcd for C20H13N3O5S:
407; Found. Anal. (C20H13N3O5S) C, H, N, S.
1-(4-tert-Butylphenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-
g]quinoline]-2,4′,5,9′-tetraone (2m). Pale yellow solid (44%). mp:
1
198-199 °C. H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J )
4.2 Hz, 6′-H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J )
7.7, 4.2 Hz, 7′-H), 7.48 (2H, d, J ) 7.2 Hz), 7.30 (2H, d, J ) 7.2
Hz), 5.90 (1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.40
(1H, d, J ) 12.9 Hz, 2′a-H), 1.35 (9H, s). MS [M+] Calcd for
C23H19N3O4S: 433; Found. Anal. (C23H19N3O4S) C, H, N, S.
1-(4-Pentylphenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quin-
oline]-2,4′,5,9′-tetraone (2n). Pale yellow solid (44%). mp: 198-199
1-(4-Nitrophenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quin-
oline]-2,4′,5,9′-tetraone (2e). Orange solid (23%). mp: >200 °C.
1H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2 Hz, 6′-H),
8.42 (1H, d, J ) 7.7 Hz, 8′-H), 8.05 (2H, d, J ) 7.2 Hz), 7.70 (2H,
d, J ) 7.2 Hz), 7.65 (1H, dd, J ) 7.7, 4.2 Hz, 7′-H), 5,90 (1H, s,
NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H). 3.40 (1H, d, J ) 12.9
Hz, 2′a-H), MS [M+] Calcd for C19H10N4O6S: 422; Found. Anal.
(C19H10N4O6S) C, H, N, S.
1
°C. H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2 Hz, 6′-
H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz,
7′-H), 7.58 (2H, d, J ) 7.2 Hz), 7.35 (2H, d, J ) 7.2 Hz), 5.90
(1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.40 (1H, d, J )
12.9 Hz, 2′a-H), 2.62 (2H, t, J ) 6.7 Hz), 1.60 (2H, m), 1.34 (4H,
m), 0.95 (3H, t, J ) 6.7 Hz). MS [M+] Calcd for C24H21N3O4S:
447; Found. Anal. (C24H21N3O4S) C, H, N, S.
1-(Pyridin-4-yl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quino-
line]-2,4′,5,9′-tetraone (2f). Pale yellow solid (45%). mp: >200 °C.
1H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2 Hz, 6′-H),
8.70 (2H, d, J ) 5.8 Hz), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H,
dd, J ) 7.7, 4.2 Hz, 7′-H), 7.61 (2H, d, J ) 5.8 Hz), 5.90 (1H, s,
NH-3), 3.40 (1H, d, J ) 12.9 Hz, 2′a-H), 4.12 (1H, d, J ) 12.9
Hz, 2′b-H). MS [M+] Calcd for C18H10N4O4S: 378; Found. Anal.
(C18H10N4O4S) C, H, N, S.
2H-Spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]triazinane]-3′,4,6′,9-
tetraone (3). Pale yellow solid (40%). mp: 198-199 °C. 1H NMR
(500 MHz, CD3OD, δ): 9.01 (1H, d, J ) 4.2 Hz, 6′-H), 8.40 (1H,
d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz, 7′-H), 3.52
(1H, d, J ) 12.9 Hz, 2′a-H), 4.20 (1H, d, J ) 12.9 Hz, 2′b-H). MS
[M+] Calcd for C13H8N4O4S: 316; Found. Anal. (C13H8N4O4S) C,
1-(4-(Dimethylamino)phenyl)-2′H-spiro[imidazolidine-4,3′-
thieno[2,3-g]quinoline]-2,4′,5,9′-tetraone (2g). Pale yellow solid
(40%). mp: >200 °C. 1H NMR (500 MHz, CD3OD, δ): 9.03 (1H,
d, J ) 4.2 Hz, 6′-H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.67 (1H, dd,
J ) 7.7, 4.2 Hz, 7′-H), 7.50 (2H, d, J ) 7.2 Hz), 7.15 (2H, d, J )
7.2 Hz), 5.90 (1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H),
3.40 (1H, d, J ) 12.9 Hz, 2′a-H), 2.32 (6H, s). MS [M+] Calcd for
C21H16N4O4S: 420; Found. Anal. (C21H16N4O4S) C, H, N, S.
1-(4-(Diethylamino)phenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-
g]quinoline]-2,4′,5,9′-tetraone (2h). Pale yellow solid (39%). mp:
H, N, S. (+)-3 ([R]2D0 ) +27.5° (c 1.2, MeOH)), (-)-3 ([R]D20
-28.1° (c 1.2, MeOH)).
)
1′-Phenyl-2H-spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]triazinane]-
3′,4,6′,9-tetraone (3a). Yellow solid (32%). mp: >200 °C. 1H NMR
(500 MHz, CD3OD, δ): 9.01 (1H, d, J ) 4.2 Hz, 6′-H), 8.40 (1H,
d, J ) 7.7 Hz, 8′-H), 7.65 (3H, m), 7.57 (2H, t, J ) 6.9 Hz), 7.30
(1H, t, J ) 6.5 Hz), 3.52 (1H, d, J ) 12.9 Hz, 2′a-H), 4.20 (1H, d,
J ) 12.9 Hz, 2′b-H). MS [M+] Calcd for C19H12N4O4S: 392; Found.
Anal. (C19H12N4O4S) C, H, N, S.
1′-p-tolyl-2H-spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]triazinane]-
3′,4,6′,9-tetraone (3b). Yellow solid (32%). mp: >200 °C. 1H NMR
(500 MHz, CD3OD, δ): 9.01 (1H, d, J ) 4.2 Hz, 6′-H), 8.40 (1H,
d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz, 7′-H), 7.38
(2H, d, J ) 7.2 Hz), 7.20 (2H, d, J ) 7.2 Hz), 4.20 (1H, d, J )
12.9 Hz, 2′b-H), 3.52 (1H, d, J ) 12.9 Hz, 2′a-H), 2.30 (3H, s).
MS [M+] Calcd for C20H14N4O4S: 406; Found. Anal.
(C20H14N4O4S) C, H, N, S.
1′-(4-Chlorophenyl)-2H-spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]tri-
azinane]-3′,4,6′,9-tetraone (3c). Yellow solid (32%). mp: >200 °C.
1H NMR (500 MHz, CD3OD, δ): 9.01 (1H, d, J ) 4.2 Hz, 6′-H),
8.40 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz,
7′-H), 7.43 (2H, d, J ) 7.2 Hz), 7.20 (2H, d, J ) 7.2 Hz), 4.20
(1H, d, J ) 12.9 Hz, 2′b-H), 3.52 (1H, d, J ) 12.9 Hz, 2′a-H). MS
[M+] Calcd for C19H11ClN4O4S: 426, 428 (M + 2); Found. Anal.
(C19H11ClN4O4S) C, H, N, S.
1
>200 °C. H NMR (500 MHz, CD3OD, δ): 9.03 (1H, d, J ) 4.2
Hz, 6′-H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.67 (1H, dd, J ) 7.7,
4.2 Hz, 7′-H), 7.50 (2H, d, J ) 7.2 Hz), 7.15 (2H, d, J ) 7.2 Hz),
5.90 (1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.40 (5H,
m), 1.15 (6H, t, J ) 7.4 Hz). MS [M+] Calcd for C23H20N4O4S:
448; Found. Anal. (C23H20N4O4S) C, H, N, S.
1-(4-Morpholinophenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-
g]quinoline]-2,4′,5,9′-tetraone (2i). Pale yellow solid (39%). mp:
1
>200 °C. H NMR (500 MHz, CD3OD, δ): 9.03 (1H, d, J ) 4.2
Hz, 6′-H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.67 (1H, dd, J ) 7.7,
4.2 Hz, 7′-H), 7.50 (2H, d, J ) 7.2 Hz), 7.15 (2H, d, J ) 7.2 Hz),
5.90 (1H, s, NH-3),4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.65 (4H,
m), 3.40 (1H, d, J ) 12.9 Hz, 2′a-H), 3.32 (4H, m). MS [M+]
Calcd for C23H18N4O5S: 462; Found. Anal. (C23H18N4O5S) C, H,
N, S.
1-(4-Ethylphenyl)-2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quin-
oline]-2,4′,5,9′-tetraone (2j). Pale yellow solid (44%). mp: 198-199
1
°C. H NMR (500 MHz, CDCl3, δ): 9.02 (1H, d, J ) 4.2 Hz, 6′-
H), 8.42 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7, 4.2 Hz,
7′-H), 7.58 (2H, d, J ) 7.2 Hz), 7.35 (2H, d, J ) 7.2 Hz), 5.90
(1H, s, NH-3), 4.12 (1H, d, J ) 12.9 Hz, 2′b-H), 3.40 (1H, d, J )
12.9 Hz, 2′a-H), 2.58 (2H, q, J ) 6.7 Hz), 1.20 (3H, t, J ) 6.7
Hz). MS [M+] Calcd for C21H15N3O4S: 405; Found. Anal.
(C21H15N3O4S) C, H, N, S.
1′-(4-Methoxyphenyl)-2H-spiro[thieno[2,3-g]quinoline-3,5′-
[1,2,4]triazinane]-3′,4,6′,9-tetraone (3d). Yellow solid (32%). mp:
1
>200 °C. H NMR (500 MHz, CD3OD, δ): 9.01 (1H, d, J ) 4.2
Hz, 6′-H), 8.40 (1H, d, J ) 7.7 Hz, 8′-H), 7.65 (1H, dd, J ) 7.7,
4.2 Hz, 7′-H), 7.43 (2H, d, J ) 7.2 Hz), 7.20 (2H, d, J ) 7.2 Hz),

8156 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Bolognese et al.
4.20 (1H, d, J ) 12.9 Hz, 2′b-H), 3.98 (3H,s), 3.52 (1H, d, J )
12.9 Hz, 2′a-H). MS [M+] Calcd for C20H14N4O5S: 422; Found.
Anal. (C20H14N4O5S) C, H, N, S.
HCl (pH 7.8), 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, 0.1 µg
of linearized DNA, and testing compounds were equilibrated at 20
°C for 30 min and were then incubated with excess amounts of T4
DNA ligase at 20 °C for 30 min. The reactions were stopped by
shifting the temperature to 55 °C. Afterward, the compounds were
removed from the reaction mixture by extraction with phenol. DNA
bands were analyzed by 1% agarose gel electrophoresis.
Another unwinding assay was employed according to the Topo-
I-mediated supercoiled pBR322 relaxation except that the DNA-
compound complexes were dissociated by extraction of the reaction
mixture with an equal volume of phenol/chloroform/isoamyl alcohol
(25:24:1 v/v/v). Then, the samples were subjected to electrophoresis
and were photographed under UV light.
Topo-II-Mediated Supercoiled pBR322 Relaxation. DNA re-
laxation assays were based on the procedure of Osheroff et al.30
Reaction buffer contained 10 mM Tris-HCl (pH 7.9), 50 mM KCl,
50 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 15 µg/mL BSA, 1
mM ATP, 0.15 µg supercoiled pBR322, and 4 units of Topo II in
a total of 20 µL. Relaxation was employed at 37 °C for 6 min and
stopped by the addition of 3 µL of stop solution (100 mM EDTA,
0.5% SDS, 50% glycerol, 0.05% bromophenol blue). Electrophore-
sis was carried out in a 1% agarose gel in 0.5× TBE (89 mM Tris
base, 89 mM boric acid, and 2 mM EDTA) at 4 V/cm for 1 h.
DNA bands were stained with 0.5 µg/mL ethidium bromide solution
and were photographed under UV light.
1′-(4-Nitrophenyl)-2H-spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]tri-
azinane]-3′,4,6′,9-tetraone (3e). Orange solid (32%). mp: >200 °C.
1H NMR (500 MHz, CD3OD, δ): 9.01 (1H, d, J ) 4.2 Hz, 6′-H),
8.40 (1H, d, J ) 7.7 Hz, 8′-H), 8.20 (2H, t, J ) 7.2 Hz),7.65 (1H,
dd, J ) 7.7, 4.2 Hz, 7′-H), 7.53 (2H, d, J ) 7.2 Hz), 4.20 (1H, d,
J ) 12.9 Hz, 2′b-H), 3.52 (1H, d, J ) 12.9 Hz, 2′a-H). MS [M+]
Calcd for C19H11N5O6S: 437; Found. Anal. (C19H11N5O6S) C, H,
N, S.
Biology. Compounds. Test compounds were dissolved in DMSO
at an initial concentration of 200 µM and were then serially diluted
in culture medium.
Cells. Cell lines were from American type Culture Collection
(ATCC). MRC-5 normal human lung fibroblasts and leukemia- and
lymphoma-derived cells were grown in RPMI 1640 containing 10%
fetal calf serum (FCS), 100 U/mL penicillin G, and 100 µg/mL
streptomycin. Solid-tumor-derived cells were grown in their specific
media supplemented with 10% FCS and antibiotics. Cell cultures
were incubated at 37 °C in a humidified 5% CO2 atmosphere. Cell
cultures were periodically checked for the absence of mycoplasma
contamination by the Hoechst staining method.
Antiproliferative Assays. Exponentially growing normal, leuke-
mia, and lymphoma cells were resuspended at a density of 1 ×
105 cells/mL in RPMI containing serial dilutions of the test drugs.
Cell viability was determined after 96 h at 37 °C by the MTT
method. The activity against solid-tumor-derived cells was evaluated
in exponentially growing cultures seeded at 5 × 104 cells/mL and
allowed to adhere for 16 h to culture plates before the addition of
drugs. Cell viability was determined by the MTT method 4 days
later.
Acknowledgment. We thank the Centro Interdipartimentale
di Metodologie Chimico-Fisiche CIMCF and the Centro Inter-
dipartimentale di Analisi Strumentale dell’Universita` di Napoli
“Federico II”. We thank Dr. Maria Teresa Buonanno for her
contribution in performing biological assays. This work was
supported by a grant from Italian MIUR (PRIN 2006).
Linear Regression Analysis. Tumor cell growth at each drug
concentration was expressed as the percentage of untreated controls,
and the concentration resulting in 50% (IC50) growth inhibition was
determined by linear regression analysis.
Supporting Information Available: Elemental analysis data for
compounds 1c, 1c′,2, 2a-n, 3, and 3a-e. This material is available
free of charge via the Internet at http://pubs.acs.org.
Spectrophotometry. Molar extinction coefficients at the wave-
length of maximum absorption in the visible spectrum were
determined for 2c free in solution, bound to calf thymus DNA
(Sigma-Aldrich, Milano, Italy) using a Cary 5000 UV-vis-NIR
spectrophotometer. Measurements were made in 0.1 SHE buffer
(2 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mM
EDTA, and 99.4 mM NaCl, pH 7.0) at 25 °C. Titrations of 2c, 2g,
and 2k with DNA, covering a large range of phosphate-DNA/drug
ratios, were performed by the addition of aliquots of a concentrated
DNA solution to a drug solution at constant ligand concentration
(20 µM). DNA blanks at the same nucleotide concentrations were
concomitantly prepared and used as a reference in the recording of
absorption spectra.
Fluorescence Measurements. Fluorescence measurements were
carried out on a Perkin-Elmer LS50B spectrofluorimeter. All
measurements were made using a 10 mm lightpath cuvette in a
0.01 M ionic-strength buffer (9.3 mM NaCl, 2 mM Na acetate,
and 0.1 mM EDTA) using 20 µM DNA and 2 µM ethidium
bromide. The DNA-ethidium complex was excited at 546 nm, and
the fluorescence was measured at 595 nm.28
Materials. Supercoiled pBR322 DNA, EcoRI restriction endo-
nuclease, T4 DNA ligase, and SDS were purchased from the New
England Biolab (U.K.). DNA Topo I and II were purchased from
TopoGen (Columbus, OH). VP16 and doxo were obtained from
Sigma-Aldrich (Milano, Italy). 2c, 2g, 2k, and control drugs were
solubilized at 104 µM in DMSO as stock solution. All stock
solutions were stored at -20 °C and diluted with double-distilled
water just before the test. Testing compound (or DMSO for control
reactions to control for the solvent’s effect) (2 µL) was added to
the reaction mixture.
Unwinding Assay. DNA unwinding effects of the compounds
2c, 2g, and 2k were assayed according to the method described by
Yamashita et al.29 Plasmid DNA was linearized by EcoRI restriction
endonuclease and recovered by phenol extraction and ethanol
precipitation. Reaction mixtures (20 µL) containing 30 mM Tris-
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