Synthesis and antitumor activity of novel amsacrine analogs: The critical role of the acridine moiety in determining their biological activity

Article abstract of DOI:10.1016/j.bmc.2008.11.072

A new series of N-[4-(2′-oxo-2H-pyrano[2,3-b]quinolin-5′-ylamino)-phenyl]-methanesulfonamides was prepared and analyzed as novel amsacrine-like derivatives. Our preliminary biological evaluation has shown that the replacement of the acridine moiety with t

Full text of DOI:10.1016/j.bmc.2008.11.072

Bioorganic & Medicinal Chemistry 17 (2009) 523–529
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antitumor activity of novel amsacrine analogs: The critical role
of the acridine moiety in determining their biological activity
a
a
a
a
Adriana Chilin a, , Giovanni Marzaro , Christine Marzano , Lisa Dalla Via , Maria Grazia Ferlin ,
*
Giovanni Pastorini b, Adriano Guiotto a
a Department of Pharmaceutical Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy
b Istituto di Chimica Biomolecolare del CNR, Sezione di Padova, Via Marzolo 3, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
A new series of N-[4-(20-oxo-2H-pyrano[2,3-b]quinolin-50-ylamino)-phenyl]-methanesulfonamides was
prepared and analyzed as novel amsacrine-like derivatives. Our preliminary biological evaluation has
shown that the replacement of the acridine moiety with the analogous 2-oxo-2H-pyrano[2,3-b]quinoline
system drastically reduced both their anticancer activity and their propency to intercalate into double
stranded DNA.
Article history:
Received 27 August 2008
Revised 26 November 2008
Accepted 29 November 2008
Available online 6 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Amsacrine analogs
Pyranoquinoline
Anticancer drugs
DNA
1. Introduction
minants for the sequence-specific recognition of the covalent to-
poII–DNA complexes.5 The bisantrene/amsacrine hybrid was able
Chemotherapy is often the treatment of choice for many types
of cancer and the search for new chemotherapeutic agents still
plays a major role in the fight against cancer.
to poison topoII with an intermediate activity between those of
bisantrene and m-AMSA, but with a different base preferences of
DNA cleavage.
A worthwhile approach in this area deals with the use of com-
pounds interacting with DNA and/or inhibiting enzymes critical for
cell life and replication. A good example of such a compound is m-
amsacrine (m-AMSA, Fig. 1), a well known antiproliferative agent
used to treat some types of cancers including acute adult leuke-
mia.1,2 m-AMSA and, in general, 9-aminoacridine derivatives work
inhibiting DNA topoisomerase II (topoII).3 Their strong activity was
due to the ability of acridine nucleus to intercalate into DNA base
pair, stabilizing the DNA–topoII cleavable complex, and forming
the so-called ‘ternary complex’ which involve DNA, intercalated
compound and topoII. The poisoning of topoII activity inhibits
the religation process and causes lethal double-strand breaks in
DNA, leading to cell cycle arrest and apoptosis.4 The intercalative
property was referred to the planar aromatic system of the acridine
moiety.
In this way we planned to synthesize new mixed molecules to
complete the structural studies in order to determine the role of
the planar part in the amsacrine series of topoII inhibitors, in terms
of base pair selectivity.
As new amsacrine-like compounds we projected the synthesis
of a series of N-[4-(20-oxo-2H-pyrano[2,3-b]quinolin-50-ylamino)-
phenyl]-methanesulfonamides (Fig. 1).
In particular, the planar and aromatic acridine moiety of AMSA
has been replaced by the analogous 2-oxo-2H-pyrano[2,3-b]quino-
line system. In fact, the 2-pyrone (2H-pyran-2-one) moiety, a six-
membered cyclic unsaturated ester which shares chemical and
MeO
HN
NHSO2Me
R1
NHSO2Me
In the past some authors have used partial structures of two dif-
ferent drugs to obtain hybrid molecules sharing the activity of the
two parent compounds. For example, m-AMSA acridine nucleus
was substituted with bisantrene (an anthracenyl bishydrazone
with antitopoII activity) side chain, to identify the structural deter-
HN
R2
N
N
O
O
R = H, OMe
m
-AMSA
* Corresponding author. Tel.: +39 049 827 5349; fax: +39 049 827 5366.
E-mail address: adriana.chilin@unipd.it (A. Chilin).
Figure 1. Chemical structures of m-AMSA (on the left) and of newly synthesized
analogs (on the right).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.072

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A. Chilin et al. / Bioorg. Med. Chem. 17 (2009) 523–529
physical properties reminiscent of alkene and aromatic com-
pounds, is present in several natural compounds, such as couma-
rins and furocoumarins, with a very well documented biological
activities.6,7 In particular furocoumarins exhibited interesting
DNA intercalative properties with good association constants8,9
and thus we supposed that the replacement of one benzene of
the acridine nucleus with 2-pyrone ring allowed to maintain favor-
able intercalative features due to the planarity of the pyranoquin-
olinone system.
OH
O
R1
R2
R1
R2
Ph
a
N
H
N
H
O
N
H
O
R3
R3
1a-d
2a-d
b
Cl
Cl
R1
COOEt
c
R1
R2
CHO
O
R2
N
H
O
N
H
R3
R3
2. Results and discussion
3a-d
4a-d
d
2.1. Molecular modelling studies
Cl
N
SO2Me
HN
R1
R2
R4
To assess the structure of the ligand–DNA complex, molecular
modelling investigations were performed with m-AMSA and m-
AMSA analog 6e. These calculations show that the intercalation
of these ligands into DNA is an exergonic process. In Figure 2 the
best docking pose of m-AMSA (on the top) and 6e (on the bottom)
intercalated into [poly(dGdC)]2 duplex is shown.
Docking studies have shown that the intercalative requirements
are favourable for both m-AMSA and 6e analog. Indeed, the theo-
retical energies of intercalation support that both compounds
could be efficiently accommodated into the GC intercalation site
e
O
O
NH
R3
R1
R2
5a-d
N
O
O
R3
6a-g
R1
R2
H
H
OMe
H
H
R3
H
H
H
OMe
H
H
R
4 (only for 6)
H
H
H
H
H
a
OMe
H
b
c
d
e
f
(D
Eintꢀm-AMSA = ꢀ11.2 kcal/mol vs
D
Hintꢀ6e = ꢀ10.5 kcal/mol).
H
H
OMe
OMe
2.2. Chemistry
OMe
H
H
H
OMe OMe
g
The title compounds were prepared firstly synthesizing the aro-
matic tricyclic nucleus and then functionalizing it with the AMSA
side chain, that is (4-aminophenyl)methanesulfonamide or (4-ami-
no-3-methoxyphenyl)methanesulfonamide. The tricyclic pyrano-
quinolinone system was built condensing the pyrone ring on the
appropriate quinolinone nucleus (Scheme 1), identifying 4-
chloro-3-formylquinolones as key intermediates. These crucial
synthons were obtained from 3-anilinomethylenequinolin-2,4-
dione intermediates, using a known synthetic approach,10 but with
improved yields.
Scheme 1. Reagents and conditions: (a) aniline, trimethyl orthoformate, ethylene-
glycol, 175 °C; (b) POCl3, DMF, K2CO3, rt; (c) (ethoxycarbonylmethylene)triphenyl-
phosphorane, N,N-diethylaniline, 190 °C; (d) PPA, 150 °C; (e) 4’-amin-
omethanesulfonanilide, DMF, reflux.
Starting 4-hydroxyquinolin-2-ones11 (1a–d) were reacted with
trimethyl orthoformate and aniline to give 3-phenylaminomethy-
lenquinolin-2,4-diones (2a–d), which were directly transformed
into 4-chloro-3-formylquinolin-2-ones (3a–d) by phosphorus oxy-
Figure 2. Molecular models of m-AMSA (on the top) and m-AMSA analog 6e (on the bottom) intercalated into [poly(dGdC)]2 duplex (see Section 4 for details); ds DNA is
represented by its Connolly’s surface. Intercalation binding energies ( Eint) are reported in kcal/mol.
D

A. Chilin et al. / Bioorg. Med. Chem. 17 (2009) 523–529
525
chloride in DMF at room temperature. Subsequent Wittig olefin-
ation12 of derivatives
with (ethoxycarbonylmethylene)-
a
b
0.000
-0.005
-0.010
-0.015
-0.020
-0.025
3
x 3
c
triphenyl-phosphorane in N,N-diethylaniline afforded the ethyl
3-(40-chloro-20-oxoquinolin-30-yl)-acrylates (4a–d), which were
cyclized with PPA at 150 °C to 5-chloro-2H-pyrano[2,3-b]quino-
lin-2-ones (5a–d). The 5-chloropyranoquinolinones 5 were finally
coupled with the 40-aminomethanesulfonanilides to provide the
desired compounds 6a–g.
a
b
2.3. Biological activity
c
The AMSA analogs 6a–g and the tricyclic base compounds
5a–d were examined for their cytotoxic properties in a panel
of four human tumor cell lines containing examples of ovarian
(2008), cervix (A431), lung (A549), breast (MCF-7) cancer and
leukaemia (HL60). For comparison purpose, the cytotoxicity of
m-AMSA was evaluated under the same experimental conditions.
IC50 values, calculated from the dose–survival curves obtained
after 72 h drug treatment from MTT test, are shown in the Table
1. All the derivatives showed a very low or no activity in all tu-
mor cell lines.
To evaluate the ability of the new derivatives to form a
molecular complex with DNA, flow linear dichroism (LD) exper-
iments were performed. In detail, salmon testes DNA was incu-
bated in the presence of increasing amounts of test compounds
and the LD spectra were recorded in the 230–400 nm region.
In Figure 3 the spectra of DNA (trace a) and compound 6e (trace
b) at [drug]/[DNA] = 0.08 is reported as an example, with
m-AMSA (trace c) as reference compound. Compound 6e was
choosen as it is the m-AMSA analog in the newly synthesized
series, but all the compounds show the same behaviour. The
DNA spectrum shows a strong negative signal at 260 nm, typical
of the macromolecule. It is known that for compounds able to
intercalate between base pairs the appearance of a further neg-
ative LD signal at higher wavelengths (300–500 nm), where only
the chromophore belonging to the added drug can absorb,
occurs. The spectra obtained in the presence of the new com-
pounds show none significant variation with respect to the
DNA spectrum in this spectral range, while m-AMSA induces a
significant negative dichroic signal in the same experimental
conditions. Thus, these results suggest for the considered chro-
mophore the inability to give rise to an efficient intercalative
mode of binding with the macromolecule.
250
300
350
400
450
500
Wavelength (nm)
Figure 3. Linear flow dichroism spectra for DNA alone (a) and in the presence of
compound 6e (b) and m-AMSA (c). [DNA] = 1.5 ꢁ 10-3 M, [drug]/[DNA] = 0.08.
planar nucleus on the biological activity. As clearly described,
our preliminary biological evaluation has shown that the
replacement of the acridine moiety with the analogous 2-oxo-
2H-pyrano[2,3-b]quinoline system drastically reduced both their
anticancer activity and their propensity to intercalate with dou-
ble stranded DNA, despite the small structural difference be-
tween the parent m-AMSA and the pyranoquinolinone
derivatives.
Even though computational study revealed that the structures
of m-AMSA and synthesized analogs were nearly superimposable
and both could be easily inserted in DNA groove, our present ef-
forts did not yield compounds suitable for therapeutic purposes,
but gave evidence that the integrity of the acridine nucleus in
the amsacrine class of anticancer agents is crucial for the biological
activity.
4. Experimental
4.1. Computational methodologies
TM
All modeling studies were carried out on a 16 CPU (Intel Core 2
Quad CPU 2.40 GHz) linux cluster.
Geometry optimization studies were performed using the
Molecular Operating Environment (MOE, ver. 2007.09) suite.13 All
structures were fully optimized without geometry constraints
using RHF/AM1 semiempirical calculations. The software package
MOPAC (ver.7),14 implemented in MOE suite, was utilized for all
quantum mechanical calculations.
3. Conclusion
A series of new amsacrine-like derivatives was prepared and
evaluated with the aim to study the influence of the tricyclic
Table 1
Cytotoxicity on human tumor cell linesa
Compound
IC50
(l
M) S.D
A431
A549
2008
MCF-7
HL60
5a
5b
5c
5d
6a
6b
6c
6d
54.33 2.58
96.99 7.15
>100
55.31 2.81
94.14 5.07
55.63 3.25
92.73 2.03
>100
62.91 2.60
>100
53.40 3.55
75.42 2.59
>100
>100
>100
>100
52.6 3.69
>100
>100
60.62 2.11
>100
68.24 4.56
>100
>100
>100
39.53 3.87
>100
50.12 2.13
54.14 1.25
>100
60.61 3.15
>100
60.32 2.11
37.12 3.61
92.10 2.90
47.28 2.06
>100
>100
>100
>100
6e
>100
>100
>100
>100
>100
6f
6g
85.69 4.17
>100
5.89 1.20
>100
81.52 2.66
3.52 1.31
>100
>100
4.31 0.53
>100
>100
5.13 0.50
95.23 3.21
>100
1.15 0.49
Amsacrine
a
IC50 values were calculated by probit analysis (P < 0.05, v2 test). Cells (3–8 ꢁ 104 mLꢀ1) were treated for 72 h with increasing concentrations of tested compounds.
Cytotoxicity was assessed by MTT test. S.D. = standard deviation.

526
A. Chilin et al. / Bioorg. Med. Chem. 17 (2009) 523–529
4.1.1. Duplex intercalation site preparation
4.2.1.2. 3-Phenylaminomethylen-6-methoxyquinolin-2,4-dione
(2b). Yield 65%; mp 297 °C; 1H NMR (DMSO-d6) d 12.93 (d, 1H,
J = 12.5 Hz, –NH), 10.80 (s, 1H, –NH), 8.89 (d, 1H, J = 12.5 Hz, –
CH), 7.60–7.30 (m, 5H, phenyl protons), 7.42 (d, 1H, J = 2.8 Hz, 5-
H), 7.20 (dd, 1H, J = 8.8, 2.8 Hz, 7-H), 7.13 (d, 1H, J = 8.8 Hz, 8-H),
3.79 (s, 3H, –OCH3). Anal. Calcd for C17H14N2O3: C, 69.38; H,
4.79; N, 9.52. Found: C, 69.41; H, 4.82; N, 9.50.
The present study involved the use of consensus dinucleotide
intercalation geometry d(GpC) initially obtained using Nucleic
Acid MOdeling Tool (NAMOT2, ver. 2) software.15 d(GpC) Inter-
calation sites was contained in the centre of a decanucleotide
duplex of d(50-GCGCG-30)2 sequence. Decamer in B-form was
built using the ‘DNA Builder’ module of MOE.13 The decanucle-
otide was minimized using Amber94 all-atom force field, imple-
mented by MOE modeling package, until the rms value of
4.2.1.3. 3-Phenylaminomethylen-7-methoxyquinolin-2,4-dione
(2c). Yield 87%; mp 297 °C; 1H NMR (DMSO-d6) d 12.66 (d, 1H,
J = 13.3 Hz, –NH), 10.81 (s, 1H, –NH), 8.80 (d, 1H, J = 13.3 Hz, –
CH), 7.88 (d, 1H, J = 8.7 Hz, 5-H), 7.51–7.28 (m, 5H, phenyl pro-
tons), 6.73 (dd, 1H, J = 8.7, 2.4 Hz, 6-H), 6.67 (d, 1H, J = 8.7 Hz, 8-
H), 3.82 (s, 3H, –OCH3). Anal. Calcd for C17H14N2O3: C, 69.38; H,
4.79; N, 9.52. Found: C, 69.42; H, 4.78; N, 9.52.
Truncated Newton method (TN) was <0.1 kcal molꢀ1 Åꢀ1
model the effects of solvent more directly, a set of electrostatic
interaction corrections are used. MOE suite implemented
modified version of GB/SA contact function described by Still
and co-authors.16 These terms model the electrostatic contribu-
tion to the free energy of solvation in a continuum solvent
model.
. To
a
4.2.1.4. 3-Phenylaminomethylen-8-methoxyquinolin-2,4-dione
(2d). Yield 78%; mp 202 °C; 1H NMR (DMSO-d6) d 12.50 (d, 1H,
J = 13.3 Hz, –NH), 9.84 (s, 1H, –NH), 9.00 (d, 1H, J = 13.3 Hz, –CH),
7.70–7.12 (m, 8H, aromatic protons), 3.96 (s, 3H, –OCH3). Anal.
Calcd for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52. Found: C, 69.37;
H, 4.80; N, 9.49.
4.1.2. Molecular docking protocol
m-AMSA and its analogs were docked into both intercalation
sites using flexible MOE-Dock methodology. The purpose of
MOE-Dock is to search for favorable binding configurations be-
tween a small, flexible ligand and a rigid macromolecular target.
Searching is conducted within a user-specified 3D docking box,
using ‘tabù search’ protocol and MMFF94 force field. Charges for
m-AMSA and its analogs were imported from the MOPAC output
files. MOE-Dock performs a user-specified number of independent
docking runs (55 in the presented case) and writes the resulting
conformations and their energies to a molecular database file.
The resulting intercalative complexes were subjected to MMFF94
all-atom energy minimization until the rms of conjugate gradient
was <0.1 kcal molꢀ1 Åꢀ1. GB/SA approximation has been used to
model the electrostatic contribution to the free energy of solvation
in a continuum solvent model.
4.2.2. General procedure for 4-chloro-3-formylquinolin-2-ones
(3)
To a solution of 2 (10.0 mmol) in anhydrous DMF (25 mL) POCl3
(1.4 mL, 15.0 mmol) was slowly added and the mixture was stirred
at room temperature for 20 h. The mixture was poured into ice and
water (250 mL), the precipitate was collected and crystallized from
toluene to give 3.
4.2.2.1. 4-Chloro-3-formylquinolin-2-one (3a). Yield 98%; mp
273 °C; 1H NMR (DMSO-d6) d 12.45 (s, 1H, –NH), 10.31 (s, 1H,
CHO), 8.11 (dd, 1H, J = 7.8, 1.4 Hz, 5-H), 7.73 (td, 1H, J = 7.8,
1.1 Hz, 6-H), 7.40 (dd, 1H, J = 7.8, 1.1 Hz, 8-H), 7.37 (td, 1H,
J = 7.8, 1.4 Hz, 7-H). Anal. Calcd for C10H6ClNO2: C, 57.85; H, 2.91;
Cl, 17.08; N, 6.75. Found: C, 57.82; H, 2.93; Cl, 17.10; N, 6.73.
4.2. Chemistry
Melting points were determined on a Gallenkamp MFB-595-
010M melting point apparatus and are uncorrected. Analytical
TLC was performed on pre-coated 60 F254 silica gel plates
4.2.2.2. 4-Chloro-3-formyl-6-methoxyquinolin-2-one (3b). Yield
99%; mp 262 °C; 1H NMR (DMSO-d6) d 12.39 (s, 1H, –NH), 10.32 (s,
1H, CHO), 7.45 (dd, 1H, J = 8.2, 2.6 Hz, 7-H), 7.41 (d, 1H, J = 2.6 Hz,
5-H), 7.37 (d, 1H, J = 8.2 Hz, 8-H), 3.86 (s, 3H, –OCH3). Anal. Calcd
for C11H8ClNO3: C, 55.60; H, 3.39; Cl, 14.92; N, 5.89. Found: C,
55.60; H, 3.42; Cl, 14.89; N, 5.93.
(0.25 mm; Merck) developing with
a CHCl3/MeOH mixture
(9:1) unless otherwise indicated. Preparative column chromatog-
raphy was performed using silica gel 60 (0.063–0.100 mm;
Merck), eluting with CHCl3. 1H NMR spectra were recorded on
a Bruker AMX300 spectrometer with TMS as internal standard.
HRMS spectra were obtained using an ESI-TOF Mariner 5220
(Applied Biosystem) mass spectrometer (in the positive ion
mode). Elemental analyses were obtained on all intermediates
and are within 0.4% of theoretical values. Starting 4-hydroxy-
quinolinones 1a–d were prepared according to literature
methods.11
4.2.2.3. 4-Chloro-3-formyl-7-methoxyquinolin-2-one (3c). Yield
78%; mp >300 °C; 1H NMR (DMSO-d6) d 12.29 (s, 1H, –NH), 10.28
(s, 1H, CHO), 8.02 (d, 1H, J = 9.2 Hz, 5-H), 6.99 (dd, 1H, J = 9.2,
2.4 Hz, 6-H), 6.86 (d, 1H, J = 2.4 Hz, 8-H), 3.88 (s, 3H, –OCH3). Anal.
Calcd for C11H8ClNO3: C, 55.60; H, 3.39; Cl, 14.92; N, 5.89. Found: C,
55.61; H, 3.43; Cl, 14.92; N, 5.90.
4.2.1. General procedure for 3-phenylaminomethylenquinolin-
2,4-diones (2)
4.2.2.4. 4-Chloro-3-formyl-8-methoxyquinolin-2-one (3d). Yield
92%; mp 210 °C; 1H NMR (DMSO-d6) d 11.49 (s, 1H, –NH), 10.08
(s, 1H, CHO), 7.46 (dd, 1H, J = 7.8, 1.5 Hz, 5-H), 7.14 (dd, 1H,
J = 7.8, 1.5 Hz, 7-H), 7.12 (t, 1H, J = 7.8 Hz, 6-H), 3.71 (s, 3H,
–OCH3). Anal. Calcd for C11H8ClNO3: C, 55.60; H, 3.39; Cl, 14.92;
N, 5.89. Found: C, 55.60; H, 3.40; Cl, 14.91; N, 5.87.
A mixture of 110 (20.0 mmol), aniline (1.82 mL, 20.0 mmol), and
trimethyl orthoformate (2.19 mL, 20.0 mmol) in ethyleneglycol
(20 mL) was heated to 70 °C under stirring. The temperature was
increased to 175 °C (10 °C/5 min), then kept at 175 °C for 2 h. After
cooling the solid was collected and washed with EtOH to give 2.
4.2.1.1. 3-Phenylaminomethylenquinolin-2,4-dione (2a). Yield
60%; mp 267 °C; 1H NMR (DMSO-d6) d 10.87 (br s, 1H, –NH), 8.89
(br s, 1H, –CH), 7.96 (dd, 1H, J = 7.6, 1.2 Hz, 5-H), 7.59–7.45 (m,
5H, phenyl protons), 7.29 (t, 1H, J = 7.6 Hz, 6-H), 7.18 (d, 1H,
J = 7.6 Hz, 8-H), 7.13 (td, 1H, J = 7.6, 1.2 Hz, 7-H). Anal. Calcd for
C16H12N2O2: C, 72.72; H, 4.58; N, 10.60. Found: C, 72.75; H, 4.55;
N, 10.63.
4.2.3. General procedure for ethyl 3-(40-chloro-20-oxoquinolin-
30-yl)-acrylates 4
A mixture of 3 (12.0 mmol), (ethoxycarbonylmethylene)tri-
phenylphosphorane (5.0 g, 14.4 mmol) and N,N-diethylaniline
(60 mL) was heated at 190 °C under nitrogen for 4 h. After cooling
the precipitate was collected, washed with diethyl ether and
crystallized from MeOH to give 4.

A. Chilin et al. / Bioorg. Med. Chem. 17 (2009) 523–529
527
4.2.3.1. Ethyl 3-(40-chloro-20-oxoquinolin-30-yl)-acrylates (4a). Yield
70%; mp 210 °C; 1H NMR (CDCl3-d) d 11.02 (s, 1H, –NH), 8.11 (d,
1H, J = 15.8 Hz, 3-H), 8.09 (dd, J = 7.5, 1.1 Hz, 50-H), 7.63 (td, 1H,
J = 7.5, 1.1 Hz, 70-H), 7.61 (d, 1H, J = 15.8 Hz, 2-H), 7.34 (td, 1H,
J = 7.5, 1.1 Hz, 60-H), 7.32 (dd, 1H, J = 7.5, 1.1 Hz, 80-H), 4.31 (q,
2H, J = 7.1 Hz, CH2CH3), 1.37 (t, 3H, J = 7.1 Hz, CH2CH3). Anal. Calcd
for C14H12ClNO3: C, 60.55; H, 4.36; Cl, 12.77; N, 5.04. Found: C,
60.53; H, 4.38; Cl, 12.80; N, 5.01.
J = 8.2 Hz, 7-H), 7.23 (dd, 1H, J = 8.2, 1.0 Hz, 8-H), 6.64 (d, 1H,
J = 9.8 Hz, 3-H), 4.10 (s, 3H, –OCH3). Anal. Calcd for C13H8ClNO3:
C, 59.67; H, 3.08; Cl, 13.55; N, 5.35. Found: C, 59.65; H, 3.10; Cl,
13.59; N, 5.30.
4.2.5. General procedure for N-[4-(20-oxo-2H-pyrano[2,3-
b]quinolin-50-ylamino)-phenyl]-methane-sulfonamides 6
A solution of 5 (1.0 mmol), N-(4-aminophenyl)-methanesulfon-
amide or N-(4-amino-3-methoxyphenyl)methanesulfonamide17
(1.0 mmol) and DMF (10 mL) was refluxed until starting product
disappeared (4–6 h, TLC). After cooling the mixture was diluted
with water (50 mL), the precipitate was collected, washed with
water, and purified by column chromatography to give 6.
4.2.3.2. Ethyl 3-(40-chloro-60-methoxy-20-oxoquinolin-30-yl)-acry-
lates (4b). Yield 75%; mp 279 °C; 1H NMR (CDCl3-d) d 12.33 (s, 1H,
–NH), 7.94 (d, 1H, J = 15.7 Hz, 3-H), 7.78–7.74 (m, 2H, 70-H and 80-
H), 7.55 (d, 1H, J = 15.7 Hz, 2-H), 7.38 (d, 1H, J = 2.2 Hz, 50-H), 4.21
(q, 2H, J = 7.1 Hz, CH2CH3), 3.86 (s, 3H, –OCH3), 1.27 (t, 3H,
J = 7.1 Hz, CH2CH3). Anal. Calcd for C15H14ClNO4: C, 58.55; H,
4.59; Cl, 11.52; N, 4.55. Found: C, 58.54; H, 4.56; Cl, 11.56; N, 4.57.
4.2.5.1. N-[4-(20-oxo-2H-pyrano[2,3-b]quinolin-50-ylamino)-
phenyl]-methanesulfonamide (6a). Pale yellow needles (MeOH);
yield 42%; mp >300 °C; 1H NMR (DMSO-d6) d 11.94 (s, 1H, –
NHSO2CH3), 10.20 (br s, 1H, –NH), 8.28 (d, 1H, J = 9.6 Hz, 40-H),
7.39 (t, 1H, J = 8.3 Hz, 80-H), 7.36 (d, 1H, J = 8.3 Hz, 90-H), 7.33 (s,
4H, 2-H, 3-H, 5-H and 6-H), 6.72 (t, 1H, J = 8.3 Hz, 70-H), 6.70 (d,
1H, J = 9.6 Hz, 30-H), 6.53 (d, 1H, J = 8.3 Hz, 60-H), 3.07 (s, 3H, –
SCH3); 13C NMR (DMSO-d6) d 162.71 (C-20), 159.11 (C-100a),
145.27 (C-90a), 139.45 (C-1 or C-4), 139.10 (C-1 or C-4), 137.48
(C-40), 135.28 (C-50), 130.85 (C-80), 129.80 (C-3 and C-5), 125.97
(C-90), 120.77 (C-60 or C-70), 120.01 (C-2 and C-6), 119.65 (C-30),
116.70 (C-60 or C-70), 112.07 (C-50a), 109.87 (C-40a), 38.80 (–
SCH3); HRMS (ESI) calcd for C19H16N3O4S (M+1)+ 382.0862, found
382.0926. Anal. Calcd for C19H15N3O4S: C, 59.83; H, 3.96; N,
11.02; S, 8.41. Found: C, 59.78; H, 3.90; N, 11.05; S, 8.37.
4.2.3.3. Ethyl
3-(40-chloro-70-methoxy-20-oxoquinolin-30-yl)-
acrylates (4c). Yield 59%; mp 294 °C; 1H NMR (CDCl3-d) d 12.24
(s, 1H, –NH), 7.92 (d, 1H, J = 9.2 Hz, 50-H), 7.91 (d, 1H, J = 15.4 Hz,
3-H), 7.47 (d, 1H, J = 15.4 Hz, 2-H), 6.97 (dd, 1H, J = 9.2, 2.4 Hz, 60-
H), 6.87 (d, 1H, J = 2.4 Hz, 80-H), 4.20 (q, 2H, J = 7.1 Hz, CH2CH3),
3.88 (s, 3H, –OCH3), 1.26 (t, 3H, J = 7.1 Hz, CH2CH3). Anal. Calcd
for C15H14ClNO4: C, 58.55; H, 4.59; Cl, 11.52; N, 4.55. Found: C,
58.57; H, 4.58; Cl, 11.55; N, 4.52.
4.2.3.4. Ethyl 3-(40-chloro-80-methoxy-20-oxoquinolin-30-yl)-
acrylates (4d). Yield 48%; mp 262 °C; 1H NMR (CDCl3-d) d 9.36 (s,
1H, –NH), 8.08 (d, 1H, J = 15.8 Hz, 3-H), 7.65 (d, 1H, J = 8.1 Hz, 50-
H), 7.61 (d, 1H, J = 15.8 Hz, 2-H), 7.25 (t, 1H, J = 8.1 Hz, 60-H), 7.06
(d, 1H, J = 8.1 Hz, 70-H), 4.29 (q, 2H, J = 7.1 Hz, CH2CH3), 4.01 (s,
3H, –OCH3), 1.34 (t, 3H, J = 7.1 Hz, CH2CH3). Anal. Calcd for
C15H14ClNO4: C, 58.55; H, 4.59; Cl, 11.52; N, 4.55. Found: C,
58.55; H, 4.57; Cl, 11.56; N, 4.55.
4.2.5.2. N-[4-(70-Methoxy-20-oxo-2H-pyrano[2,3-b]quinolin-50-
ylamino)-phenyl]methanesulfonamides (6b). Pale yellow nee-
dles (MeOH); yield 10%; mp >300 °C; 1H NMR (DMSO-d6) d 11.86
(s, 1H, –NHSO2CH3), 10.11 (s, 1H, –NH–), 8.29 (d, 1H, J = 9.6 Hz,
40-H), 7.38 (s, 4H, 2-H, 3-H, 5-H and 6-H), 7.28 (d, 1H, J = 9.0 Hz,
90-H), 7.10 (dd, 1H, J = 9.0, 2.6 Hz, 80-H), 6.71 (d, 1H, J = 9.6 Hz, 30-
H), 6.14 (d, 1H, J = 2.6 Hz, 60-H), 3.27 (s, 3H, –OCH3), 3.12 (s, 3H,
4.2.4. General procedure for 5-chloro-2H-pyrano[2,3-b]quinolin-2-
ones 5
A mixture of 4 (5.0 mmol) and PPA (10.0 g) was heated at 150 °C
under nitrogen for 2 h. After cooling the solution was poured into
ice and water (500 mL). The solid was collected, washed with
water and crystallized from MeOH to give 5.
13
–SCH3); C NMR (DMSO-d6) d 162.81 (C-20), 158.75 (C-100a),
152.65 (C-70), 144.76 (C-90a), 139.00 (C-1), 137.63 (C-40), 135.26
(C-50), 133.95 (C-40), 130.06 (C-3 and C-5), 120.15 (C-2 and C-6),
119.72 (C-30), 117.85 (C-80 and C-90), 112.35 (C-50a), 110.18 (C-
40a), 108.60 (C-60), 54.66 (–OCH3), 38.88 (–SCH3); HRMS (ESI) calcd
for C20H18N3O5S (M+1)+ 412.0962, found 412.1049. Anal. Calcd for
C20H17N3O5S: C, 58.38; H, 4.16; N, 10.21; S, 7.79. Found: C, 58.42;
H, 4.14; N, 10.26; S, 7.85.
4.2.4.1. 5-Chloro-2H-pyrano[2,3-b]quinolin-2-one
(5a). Yield
84%; mp 210 °C; 1H NMR (CDCl3-d) d 8.31 (dd, 1H, J = 8.1, 1.1 Hz,
6-H), 8.25 (d, 1H, J = 9.8 Hz, 4-H), 8.09 (d, 1H, J = 8.1 Hz, 9-H),
7.97 (td, 1H, J = 8.1, 1.1 Hz, 8-H), 7.69 (td, 1H, J = 8.1, 1.1 Hz, 7-H),
6.63 (d, 1H, J = 9.8, 3-H). Anal. Calcd for C12H6ClNO2: C, 62.22; H,
2.61; Cl, 15.31; N, 6.05. Found: C, 62.25; H, 2.64; Cl, 15.29; N, 6.02.
4.2.5.3. N-[4-(80-Methoxy-20-oxo-2H-pyrano[2,3-b]quinolin-50-
ylamino)-phenyl]-methanesulfonamides (6c). Pale yellow nee-
dles (MeOH); yield 15%; mp >300 °C; 1H NMR (DMSO-d6) d 11.80
(s, 1H, –NHSO2CH3), 10.08 (s, 1H, –NH–), 8.23 (d, 1H, J = 9.5 Hz,
40-H), 7.36 (d, 2H, J = 9.1 Hz, 2-H and 6-H or 3-H and 5-H), 7.33
(d, 2H, J = 9.1 Hz, 2-H and 6-H or 3-H and 5-H), 6.83 (d, 1H,
J = 2.3 Hz, 90-H), 6.61 (d, 1H, J = 9.5 Hz, 30-H), 6.43 (d, 1H,
J = 9.6 Hz, 60-H), 6.33 (d, 1H, J = 9.6 Hz, 70-H), 3.74 (s, 3H, –OCH3),
4.2.4.2. 5-Chloro-7-methoxy-2H-pyrano[2,3-b]quinolin-2-one
(5b). Yield 21%; mp 251 °C; 1H NMR (DMSO-d6) d 8.35 (d, 1H,
J = 9.8 Hz, 4-H), 7.94 (d, 1H, J = 9.2 Hz, 9-H), 7.63 (dd, 1H, J = 9.2,
2.7 Hz, 8-H), 7.51 (d, 1H, J = 2.7 Hz, 6-H), 6.75 (d, 1H, J = 9.8 Hz, 3-
H), 3.98 (s, 3H, –OCH3). Anal. Calcd for C13H8ClNO3: C, 59.67; H,
3.08; Cl, 13.55; N, 5.35. Found: C, 59.70; H, 3.11; Cl, 13.54; N, 5.32.
13
3.10 (s, 3H, –SCH3); C NMR (DMSO-d6) d 162.75 (C-20), 160.69
(C-80), 159.37 (C-100a), 145.60 (C-90a), 141.62 (C-50), 138.90 (C-1),
137.66 (C-40), 135.40 (C-4), 129.81 (C-3 and C-5), 127.67 (C-70),
119.95 (C-2 and C-6), 118.14 (C-30), 109.48 (C-90), 107.67 (C-40a),
105.76 (C-50a), 99.30 (C-60), 55.30 (–OCH3), 40.10 (–SCH3); HRMS
(ESI) calcd for C20H18N3O5S (M+1)+ 412.0962, found 412.0941.
Anal. Calcd for C20H17N3O5S: C, 58.38; H, 4.16; N, 10.21; S, 7.79.
Found: C, 58.40; H, 4.12; N, 10.25; S, 7.80.
4.2.4.3. 5-Chloro-8-methoxy-2H-pyrano[2,3-b]quinolin-2-one
(5c). Yield 77%; mp 237 °C; 1H NMR (DMSO-d6) d 8.32 (d, 1H,
J = 9.7 Hz, 4-H), 8.20 (d, 1H, J = 9.2 Hz, 6-H), 7.42 (dd, 1H, J = 9.2,
2.4 Hz, 7-H), 7.40 (d, 1H, J = 2.4 Hz, 9-H), 6.67 (d, 1H, J = 9.7 Hz, 3-
H), 3.99 (s, 3H, –OCH3). Anal. Calcd for C13H8ClNO3: C, 59.67; H,
3.08; Cl, 13.55; N, 5.35. Found: C, 59.69; H, 3.07; Cl, 13.57; N 5.36.
4.2.4.4. 5-Chloro-9-methoxy-2H-pyrano[2,3-b]quinolin-2-one
(5d). Yield 55%; mp 225 °C; 1H NMR (CDCl3-d) d 8.23 (d, 1H,
J = 9.8 Hz, 4-H), 7.86 (dd, 1H, J = 8.2, 1.0 Hz, 6-H), 7.60 (t, 1H,
4.2.5.4. N-[4-(90-Methoxy-20-oxo-2H-pyrano[2,3-b]quinolin-50-
ylamino)-phenyl]-methanesulfonamides (6d). Pale yellow nee-
dles (MeOH); yield 33%; mp >300 °C; 1H NMR (DMSO-d6) d 10.87

528
A. Chilin et al. / Bioorg. Med. Chem. 17 (2009) 523–529
(s, 1H, –NHSO2CH3), 10.08 (s, 1H, –NH–), 8.29 (d, 1H, J = 9.6 Hz, 40-
H), 7.34 (s, 4H, 2-H, 3-H, 5-H and 6-H), 7.06 (d, 1H, J = 8.4 Hz, 60-H),
6.71 (d, 1H, J = 9.6 Hz, 30-H), 6.69 (t, 1H, J = 8.4 Hz, 70-H), 6.14 (d,
1H, J = 8.4 Hz, 80-H), 3.88 (s, 3H, –OCH3), 3.09 (s, 3H, –SCH3); 13C
NMR (DMSO-d6) d 162.73 (C-20), 158.72 (C-100a), 146.14 (C-90),
145.46 (C-90a), 138.86 (C-1), 137.58 (C-40), 135.46 (C-4), 129.88
(C-50), 129.63 (C-3 and C-5), 120.44 (C-70), 119.94 (C-2 and C-6),
119.82 (C-30), 117.63 (C-50a), 112.55 (C-60 or C-80), 111.22 (C-60
or C-80), 110.23 (C-40a), 56.17 (–OCH3), 38.85 (–SCH3); HRMS
(ESI) calcd for C20H18N3O5S (M+1)+ 412.0962, found 412.0892.
Anal. Calcd for C20H17N3O5S: C, 58.38; H, 4.16; N, 10.21; S, 7.79.
Found: C, 58.40; H, 4.12; N, 10.25; S, 7.80.
4.3. Biology
4.3.1. Experiments with human cells
Compounds were dissolved in dimethyl sulfoxide just before
the experiments; calculated amounts of drug solution were added
to the growth medium containing cells to a final solvent concentra-
tion of 0.5% which had no discernible effect on cell killing.
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide, and m-amsacrine were obtained from Sigma Chemical Co.,
St. Louis, USA.
4.3.2. Cell cultures
Human lung (A549) and breast (MCF-7) carcinoma cell lines
along with promyelocytic leukemia (HL60) cells were obtained
by ATCC (RocKville, MD). 2008 Human ovarian cancer cell line
was kindly provided by Prof. G. Marverti (Dept. of Biomedical Sci-
ence of Modena University, Italy) and A431 human cervix carci-
noma were kindly provided by Prof. Zunino (Division of
Experimental Oncology B, Istituto Nazionale dei Tumori, Milan,
Italy). Cell lines were maintained in the logarithmic phase at
37 °C in a 5% carbon dioxide atmosphere using the following cul-
ture media: (i) RPMI-1640 medium (Euroclone, Celbio, Milan, Italy)
containing 10% foetal calf serum (Biochrom-Seromed GmbH&Co,
4.2.5.5. N-[4-(20-Oxo-2H-pyrano[2,3-b]quinolin-50-ylamino)-3-
methoxyphenyl]methanesulfonamide (6e). pale yellow needles
(MeOH); yield 18%; mp >300 °C; 1H NMR (CD3OD-d4) d 8.45 (d,
1H, J = 9.5 Hz, 40-H), 7.44 (td, 1H, J = 8.1, 1.8 Hz, 80-H), 7.34 (dd,
1H, J = 8.1, 1.6 Hz, 90-H), 7.25 (d, 1H, J = 8.6 Hz, 5-H), 7.13 (d, 1H,
J = 1.9 Hz, 2-H), 7.05 (dd, 1H, J = 8.1, 1.8 Hz, 60-H), 6.94 (dd, 1H,
J = 8.6, 1.9 Hz, 6-H), 6.82 (td, 1H, J = 8.1, 1.6 Hz, 70-H), 6.79 (d, 1H,
J = 9.5 Hz, 30-H), 3.66 (s, 3H, –OCH3), 3.10 (s, 3H, –SCH3); 13C
NMR (DMSO-d6) d 162.12 (C-20), 159.10 (C-100a), 155.19 (C-3),
145.64 (C-90a), 140.73 (C-1), 139.27 (C-4), 137.53 (C-40), 131.05
(C-50), 130.29 (C-80), 124.62 (C-90), 124.02 (C-5), 121.13 (C-60 or
C-70), 119.54 (C-30), 116.78 (C-60 o C-70), 112.14 (C-6), 111.39 (C-
50a), 109.46 (C-40a), 103.59 (C-2), 55.70 (–OCH3), 39.00 (–SCH3);
HRMS (ESI) calcd for C20H18N3O5S (M+1)+ 412.0967, found
412.1066. Anal. Calcd for C20H17N3O5S: C, 58.38; H, 4.16; N,
10.21; S, 7.79. Found: C, 58.44; H, 4.10; N, 10.21; S, 7.81.
Berlin, Germany) and supplemented with 25 mM HEPES buffer,
glutamine and with antibiotics penicillin (50 units mLꢀ1) and
streptomycin (50
g mLꢀ1) for 2008, A431, MCF7 and HL60 cells;
L-
l
(ii) F-12 Ham’s (Sigma Chemical Co.) containing 15% foetal calf ser-
um, penicillin (50 units mLꢀ1) and streptomycin (50
A549 cells.
l
g mLꢀ1) for
4.2.5.6. N-[4-(70-Methoxy-20-oxo-2H-pyrano[2,3-b]quinolin-50-
4.3.3. Cytotoxicity assay
ylamino)-3-methoxyphenyl]-methane-sulfonamide
(6f). Pale
The growth inhibitory effect towards tumor cell lines was eval-
uated by means of MTT (tetrazolium salt reduction) assay.18
Briefly, between 3 and 8 ꢁ 10ꢀ3 cells, dependent upon the growth
characteristics of the cell line, were seeded in 96-well microplates
yellow needles (MeOH); yield 12%; mp >300 °C; 1H NMR (DMSO-
d6) d 11.86 (s, 1H, –NHSO2CH3), 9.57 (s, 1H, –NH–), 7.52 (d, 1H,
J = 9.8 Hz, 40-H), 7.27 (d, 1H, J = 9.0 Hz, 5-H), 7.24 (dd, 1H, J = 9.0,
2.5 Hz, 6-H), 7.12 (d, 1H, J = 8.3 Hz, 90-H), 6.96 (d, 1H, J = 2.2 Hz,
60-H), 6.85 (dd, 1H, J = 8.3, 2.2 Hz, 80-H), 6.74 (d, 1H, J = 2.5 Hz, 2-
H), 6.61 (d, 1H, J = 9.8 Hz, 30-H), 3.70 (s, 3H, –OCH3), 3.63 (s, 3H,
in growth medium (100 lL) and then incubated at 37 °C in a 5%
carbon dioxide atmosphere. After 24 h, the medium was removed
and replaced with a fresh one containing the compound to be stud-
ied at the appropriate concentrations. Quadruplicate cultures were
established for each treatment. Seventy-two hours later, each well
13
–OCH3), 2.98 (s, 3H, –SCH3); C NMR (DMSO-d6) d 158.74 (C-20),
156.49 (C-100a), 154.31 (C-70), 151.16 (C-3), 148.24 (C-90a),
135.08 (C-1), 133.07 (C-50), 131.45 (C-40), 131.32 (C-4), 122.80
(C-90), 122.18 (C-80), 118.95 (C-30), 117.42 (C-5), 112.32 (C-6),
112.06 (C-50a), 107.34 (C-40a), 104.64 (C-2), 101.44 (C-60), 55.35
(–OCH3), 55.14 (–OCH3), 38.74 (–SCH3); HRMS (ESI) calcd for
C21H20N3O6S (M+1)+ 442.1067, found 442.1017. Anal. Calcd for
C21H19N3O6S: C, 57.13; H, 4.34; N, 9.52; S, 7.26. Found: C, 57.15;
H, 4.30; N, 9.55; S, 7.22.
was treated with 10
after 5 h of incubation, 100
l
L of a 5 mg mLꢀ1 MTT saline solution, and
L of a sodium dodecylsulfate (SDS)
l
solution in HCl 0.01 M was added. After overnight incubation, the
inhibition of cell growth induced by the tested complexes was de-
tected by measuring the absorbance of each well at 540 nm using a
Camberra–Packard microplate reader. Mean absorbance for each
drug dose was expressed as a percentage of the control untreated
well absorbance and plotted versus drug concentration. IC50 values
represent the drug concentrations that reduced the mean absor-
bance at 540 nm to 50% of those in the untreated control wells.
4.2.5.7. N-[4-(90-Methoxy-20-oxo-2H-pyrano[2,3-b]quinolin-50-
ylamino)-3-methoxyphenyl]-methane-sulfonamide
(6g). Pale
yellow needles (EtOAc); yield 10%; mp >300 °C; 1H NMR (DMSO-
d6) d 10.84 (s, 1H, –NHSO2CH3), 9.95 (s, 1H, –NH–), 8.30 (d, 1H,
J = 9.5 Hz, 40-H), 7.23 (d, 1H, J = 8.5 Hz, 5-H), 7.09 (d, 1H,
J = 8.6 Hz, 60-H), 7.03 (d, 1H, J = 1.9 Hz, 2-H), 6.94 (dd, 1H, J = 8.5,
1.9 Hz, 6-H), 6.73 (t, 1H, J = 8.6 Hz, 70-H), 6.69 (d, 1H, J = 9.5 Hz,
30-H), 6.35 (d, 1H, J = 8.6 Hz, 80-H), 3.88 (s, 3H, –OCH3), 3.60 (s,
3H, –OCH3), 3.12 (s, 3H, –SCH3); 13C NMR (DMSO-d6) d 162.16
(C-20), 158.70 (C-100a), 155.19 (C-3), 146.11 (C-90), 145.88 (C-90a),
140.83 (C-1), 137.60 (C-40), 130.36 (C-5), 129.42 (C-50), 124.03
(C-4), 120.79 (C-70), 119.70 (C-30), 116.24 (C-6), 112.61 (C-50a),
111.42 (C-60 or C-80), 111.34 (C-60 or C-80), 109.77 (C-40a), 103.57
(C-2), 56.14 (–OCH3), 55.72 (–OCH3), 39.27 (–SCH3); HRMS (ESI)
calcd for C21H20N3O6S (M+1)+ 442.1067, found 442.1008. Anal.
Calcd for C21H19N3O6S: C, 57.13; H, 4.34; N, 9.52; S, 7.26. Found:
C, 57.14; H, 4.30; N, 9.51; S, 7.21.
4.3.4. Statistical analysis
All the values are the means SD of not less than five measure-
ments. IC50 values were calculated by the Probit model. Statistical
analysis was performed using Chi-square test. P < 0.05 was consid-
ered statistically significant.
4.3.5. Experiments with nucleic acid
Salmon testes DNA was purchased from Sigma Chemical Com-
pany. The concentration was determined using extinction coeffi-
cient 6600 Mꢀ1 cmꢀ1 at 260 nm.
4.3.6. Linear flow dichroism
Linear dichroism (LD) measurements were performed on a Jasco
J500A circular dichroism spectropolarimeter converted for LD and

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equipped with an IBM PC and a Jasco J interface. Linear dichroism is
defined as:
LD ¼ AkðkÞ ꢀ A
ðkÞ
?ðkÞ
where A
and A
correspond to the absorbances of the sample
kðkÞ
?ðkÞ
when polarized light is oriented parallel or perpendicular to the
flow direction, respectively. The orientation is produced by a device
designed by Wada and Kozawa19 at a shear gradient of 500–
700 rpm and each spectrum was accumulated four times. A solution
of salmon testes DNA (1.5 ꢁ 10ꢀ3 M) in ETN buffer (containing
10 mM Tris, 10 mM NaCl, and 1 mM EDTA, pH 7) was used. Spectra
were recorded at 25 °C at [drug]/[DNA] ratios: 0, 0.02, 0.04, 0.08.
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13. MOE (The Molecular Operating Environment) Version 2007.09, Software
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Acknowledgements
The present work has been carried out with financial supports
of the University of Padova (Italy) and the Italian Ministry for Uni-
versity and Research (MIUR), Rome (Italy). We thanks the Molecu-
lar Modeling Section (MMS) of our Department for the modeling
studies.
References and notes
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L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48,
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