Full text of DOI:10.1038/sj.bjp.0704611

British Journal of Pharmacology (2002) 135, 1513 ± 1523
ã
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Anthrapyridones, a novel group of antitumour non-cross resistant
anthraquinone analogues. Synthesis and molecular basis of the
cytotoxic activity towards K562/DOX cells
1
1
1
3
2
*,1,4J. Tarasiuk, B. Stefanska, I. Plodzich, K. Tkaczyk-Gobis, O. Seksek, S. Martelli,
3A. Garnier-Suillerot & E. Borowski
1
2
1Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, Poland; Department of
Chemical Sciences, University of Camerino, I-62032 Camerino, Italy and Laboratoire de Physicochimie Biomoleculaire et
Cellulaire (ESA Centre National de la Recherche Scienti®que 7033), Universite Paris Nord, France
3
1
Multidrug resistance (MDR) to antitumour agents, structurally dissimilar and having di€erent
intracellular targets, is the major problem in cancer therapy. MDR phenomenon is associated with
the presence of membrane proteins which belong to the ATP-binding cassette family transporters
responsible for the active drug e‚ux leading to the decreased intracellular accumulation.
2
The search of new compounds able to overcome MDR is of prime importance.
3 Recently we have synthesized a new family of anthrapyridone compounds. The series contained
derivatives modi®ed with appropriate hydrophobic or hydrophylic substituents at the side chain.
4
The interaction of these derivatives with erythroleukemia K562 sensitive and K562/DOX
resistant (overexpressing P-glycoprotein) cell lines has been examined. The study was performed
using a spectro¯uorometric method which allows to continuously follow the uptake and e‚ux of
¯uorescent molecules by living cells.
5
It was demonstrated that the increase in the lipophilicity of anthrapyridones favoured the very
fast cellular uptake exceeding the rate of P-gp dependent e‚ux out of the cell. For these derivatives,
very high accumulation (the same for sensitive and resistant cells) was observed and the in vitro
biological data con®rmed that these compounds exhibited comparable cytotoxic activity towards
sensitive and P-gp resistant cell line. In contrast, anthrapyridones modi®ed with hydrophylic
substituents exhibited relatively low kinetics of cellular uptake.
6
For these derivatives decreased accumulation in resistant cells was observed and the in vitro
biological data demonstrated that they were much less active against P-gp resistant cells in
comparison to sensitive cells.
7
We also studied, using confocal microscopy, the intracellular distribution of anthrapyridones in
NIH-3T3 cells. Our data showed that these compounds were strongly accumulated in the nucleus
and lysosomes.
British Journal of Pharmacology (2002) 135, 1513 ± 1523
Keywords: Multidrug resistance; active e‚ux; P-glycoprotein; non-cross resistant agents; anthrapyridones; lipophilicity
Abbreviations: CO, anthrapyridone; DOX, doxorubicin; MDR, multidrug resistance; MRP1, multidrug resistance protein;
PIRA, pirarubicin; P-gp, P-glycoprotein
Introduction
The ability of neoplastic cells to develop multi-drug
resistance (MDR) to chemotherapeutic agents (e.g. anthra-
cyclines, vinca alkaloids, podophylotoxins, colchicine),
structurally dissimilar and having di€erent intracellular
targets, constitutes the major problem in cancer therapy
(Chaudhary & Roninson, 1993). MDR is a well-character-
ized phenomenon (Beck, 1987; Gottesman & Pastan, 1993),
associated with the presence of membrane proteins
belonging to the ATP-binding cassette protein family (P-
glycoprotein, MRP1, c-MOAT proteins) (Roninson, 1992;
Germann, 1996; Loe et al., 1996; Kool et al., 1997). These
transporters are responsible for the active ATP-dependent
e‚ux of drugs out of resistant cells resulting in the
decreased intracellular accumulation insucient to inhibit
resistant cell proliferation (Zaman et al., 1994; Paul et al.,
1996).
Current strategies to reverse MDR are based: (i) on the
synthesis of new non-cross resistant cytostatics (Antonini et
al., 1995; Bassan et al., 1997; Kubota et al., 1998; Stefanska
et al., 1999); (ii) on the identi®cation of e€ective and more
selective MDR reversing agents (chemosensitizers) (Raderer
& Scheithaner, 1993; Pereira et al., 1994; Mankhetkorn &
Garnier-Suillerot, 1996; Pascaud et al., 1998; Berger et al.,
1999; Teodori et al., 1999); (iii) on the selective inhibition of
gene expression encoding ATP-dependent export pump using
antisense oligonucleotides (Alahari et al., 1996; Stewart et al.,
*Author for correspondence at: University of Szczecin, Department
of Biochemistry, 3a Felczaka st, 71-412 Szczecin, Poland;
E-mail: tarasiuk@univ.szczecin.pl
4Current address: Department of Biochemistry, University of
Szczecin, Poland

1514
J. Tarasiuk et al
Non-cross resistant anthrapyridones
1996; Garcia-Chaumont et al., 2000) or ribozymes (Palfner et
al., 1995).
Methods
Because of the continuous identi®cation of new proteins
involved in multidrug resistance (Doyle et al., 1995; Sche€er
et al., 1995; Gaj & Yung-Chi, 1998) the approaches (ii) and
(iii) seem very far from being resolved. For this reason the
search for new compounds able to overcome MDR is of
prime importance. The cytotoxic activity depends on drug
concentration in the compartment where its cellular target is
located. The intracellular drug concentration is determined by
the kinetics of cellular uptake and the kinetics of ATP-
dependent drug export of the drug (Frezard & Garnier-
Suillerot, 1991; Garnier-Suillerot, 1995). For anthracyclines,
it was found that the rate of passive di€usion was
comparable to the rate of P-gp and MRP1 mediated e‚ux
leading to the signi®cant decrease in drug accumulation in
resistant cells (Marbeuf-Gueye et al., 1998). The increase in
drug accumulation in resistant cells could be achieved by an
augmentation of kinetics of its passive uptake into the cells or
by the reduction of their anity to exporting pumps resulting
in the decrease of kinetics of active e‚ux. It seems that the
rational design of compounds having molecular properties
which allow the fast kinetics of passive di€usion across the
membrane is easier to realise (Mankhetkorn et al., 1996;
Marbeuf-Gueye et al., 1999; Tkaczyk-Gobis et al., 2001).
Recently we have synthesized a new family of anthra-
pyridone compounds. The series of obtained derivatives
contained six anthrapyridones modi®ed at the side chain
with appropriate hydrophobic or hydrophylic substituents.
The interaction of these derivatives with erythroleukemia
K562 sensitive and K562/DOX resistant cell lines (over-
expressing P-gp) has been examined in this study. Our data
showed that the increase in the lipophilicity of anthrapyri-
done derivatives favoured their fast passive cellular uptake
exceeding the rate of P-gp dependent e‚ux out of the cell.
For these derivatives, very high accumulation (the same for
sensitive and resistant cells) were observed and the in vitro
biological data con®rmed that these compounds exhibited
comparable cytotoxicity towards sensitive and P-gp resistant
cell lines. In contrast, anthrapyridones modi®ed with
hydrophylic substituents exhibited relatively low kinetics of
cellular uptake. For these derivatives decreased accumulation
in resistant cells was observed and the in vitro biological data
showed that they were much less active against P-gp resistant
cells in comparison to sensitive cells. In addition, we studied
the intracellular distribution of anthrapyridones in NIH-3T3
cells using confocal microscopy. It was found that these
compounds were strongly accumulated in the nucleus and
lysosomes.
General procedure for the synthesis of anthrapyridone
compounds CO-1, CO-3 ± CO-7
4-[[2-(dimethylamino)ethyl]amino]-N-methyl-1,9-anthrapyri-
done (CO-1) A sample of 0.38 g (1 mmol) of 4-bromo-N-
methyl-1,9-anthrapyridone was dissolved in a mixture of 2 ml
of 2-(dimethylamino)ethyl amine and was stirred in 808C
under nitrogen atmosphere for 0.5 h. The progress of the
reaction was followed by TLC (silica gel) in the solvent
system CHCl3/MeOH (5 : 1). The reaction mixture was
diluted with CHCl3 and washed with diluted HCl followed
by water to remove the excess of amine. The organic layer
was dried over Na2SO4 and evaporated under reduced
pressure. The residue was chromatographed on a silica gel
column using CHCl3/MeOH (5 : 1) as eluent followed by
CHCl3/MeOH/25% aqueous NH4OH (5 : 1 : 1).
The CO-1 pure product was converted into its hydro-
chloride salt using an etheral solution of hydrogen chloride,
which was then crystallized from MeOH to give CO-1 as a
dark red powder (240 mg, 60%): mp 285-78C dec. IR (KBr,
1
major peaks cm71): 1470, 1510, 1620. H NMR (as free base,
CDCl3): d 2.4 (s, 6H); 2.75 (t, 2H, J=6.5 Hz); 3.55 (q, 2H,
I=6.5 Hz); 3.85 (s,3H); 7.22 (d, 1H, I=9); 7.4 (s, 1H); 7.68
(d, 1H, I=9); 7.72 ± 7.78 (m, 2H); 8.22 (dd, 1H); 8.5 (dd, 1H);
10.5 (t, 1H). MS-FD m/z (relative intensity, per cent) 348
([M+1]+, 100) ([M]+, 30). Anal. (C21H21N3O26HCl)
C,H,N. Compounds CO-3 ± CO-7 were obtained in the same
way using respective aminoalkylamine RNH2 (for R, see
Table 1).
Melting points were determinated with
a
Boeticus
PHMK05 apparatus and were uncorrected. Analyses were
within 0.4% of the theoretical values and were carried out on
a Carlo Erba CHNS-O-EA1108 instrument. IR spectra were
1
recorded on a Brucker IFS66 spectrometer in KBr pellets: H
NMR spectra were taken on a Varian 300 MHz spectrometer
using tetramethylsilane as an internal standard. Molecular
weights were determined by mass spectrometry (®eld
desorption technique, MS-FD) on
a Varian MAT 711
instrument. Column chromatography was performed on silica
gel Merck (70 ± 230 mesh).
Stock solutions of anthrapyridones in deionized double-
distilled water were prepared just before use. Concentrations
were determined by diluting stock solutions to approximately
10 mM
495=6013 M cm71. Absorption spectra were recorded on
a Beckman spectrophotometer.
and
using
an
extinction
coecient
of
71
e
Table 1 Synthesis and physicochemical properties of 4-[(aminoalkyl)amino]-N-methyl-1,9 anthrapyridones(a)
Reaction conditions
Time (hrs) temp (8C)
Yield
(%)
Mp
(8C dec.)
Molecular
formula
Compound
R
CO-1
CO-3
CO-4
CO-5
CO-6
CO-7
(CH2)2N(CH3)2
(CH2)3N(CH3)2
(CH2)2NHCH2CH3
(CH2)2N(CH2CH3)2
(CH2)2NH(CH2CH2OH)
(CH2)2NH2
0,5
80
60
55
45
50
35
45
285-7
276-8
270-3
283-5
278-80
280-2
C21H21N3O2xHC1
C22H23N3O2xHCIx0,5H2O
C21H21N3O2xHCI
C23H25N3O2xHC1
C21H21N3O3xHC1x0,5H2O
C19H17N3O2xHC1
1
1
1
1
1
80
60
80
60
60
(a)
The structures of examined compounds were con®ned by their spectral data (1H NMR, MS-FD) and by elemental analysis (C,H,N).
British Journal of Pharmacology vol 135 (6)

J. Tarasiuk et al
Non-cross resistant anthrapyridones
1515
DNA preparation
Determination of the rates of energy-dependent processes
(non-nuclear accumulation and P-glycoprotein-mediated
efflux)
High molecular weight calf thymus DNA (from Sigma) was
dissolved in PBS bu€er for 3 h under vigorous stirring. DNA
concentration was determined from absorbance measure-
Cells (16106 ml71) are incubated for 30 min in HEPES
bu€er in the presence of 10 mM NaN3 and in the absence of
glucose and then incubated with anthrapyridone (CT=1 mM).
At the steady state (Fn) 5 mM glucose was added yielding a
new steady state (F'n). The concentration of anthrapyridone
were respectively Cn=CT(F0 ± Fn)/F0 and Cn=CT(F0 ± F'n)/F0.
The global rate of energy-dependent processes was deter-
mined using the equation VE=(dF/dt)glu´CT/F0. The rate of
energy-dependent non-nuclear accumulation was determined
as VE for sensitive cells [VE(S)] and P-glycoprotein-mediated
e‚ux was calculated as VP-gp=VE(S)-VE(R) for anthrapyridones
CO-1, CO-3, CO-5 (Figure 4) and as VP-gp=VE(S)+VE(R) for
anthrapyridones CO-4, CO-6, CO-7 (Figure 5).
ments at 260 nm using an extinction coecient of
71
e
260=13200 M cm71 (b.p.).
Cell lines and cultures
Anthracycline-sensitive K562 and anthracycline-resistant ery-
throleukemia K562/DOX cells (Tsuruo et al., 1986; Mankhet-
korn et al., 1996) were grown in RPMI medium supplemented
with L-glutamine and 10% foetal calf serum in a humidi®ed
atmosphere of 95% air and 5% CO2. The resistant K562/DOX
cells were cultured with 400 nM doxorubicin until 1 ± 4 weeks
before experiments. Cultures initiated at a density of 105
cells ml71 grew exponentially to about 106 cells ml71 in 3 days.
For the experiments, culture was initiated at 56105 cells ml71
and cells were used 24 h later in order to assure an exponential
growth phase. They were counted with a Coulter counter,
immediately before use in the assay. Cell viability was assessed
by trypan blue exclusion. NIH/3T3 cells were obtained from
the American Type Culture Collection (Manassas, VA,
U.S.A.). They were grown in Dulbecco's modi®ed Eagle's
medium (DMEM) supplemented with 10% foetal calf serum in
a humidi®ed incubator with 5% CO2. Before each experiment,
cells were plated on 18 mm diameter glass coverslips and
allowed to grow overnight to reach 75% con¯uence. The
cytotoxicity of the compounds was determined by incubating
cells (36105) with six di€erent concentrations of the
compound for 72 hours in standard 48-well plates. The IC50
value de®ned as the concentration which reduced by 50% the
growth rate attained by control culture. The resistance factor
(RF) was de®ned as the IC50 for the resistant cells divided by
the IC50 for the sensitive cells.
All ¯uorescence measurements were made on a Perkin-
Elmer LS 5B spectro¯uorometer.
Lysosomes and trans-Golgi labelling
Lysosomes were stained with LysoTrackerTM Green DND-26
(from Molecular Probes) after incubation with 1 mM CO-1 for
5 min at 378C. Cells were incubated with 50 nM of the probe
(from 1 mM stock solution in dimethyl sulphoxide (DMSO)
in complemented DMEM medium for 30 min at 378C and
washed with PBS prior to observation. The trans-Golgi were
stained according to Panago et al. (1989). 50 nmol of C6-
NBD-ceramide (6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ami-
no) caproyl) sphingosine from Molecular Probes) was
dissolved in 200 ml of ethanol and injected into 10 ml of
10 mM HEPES-bu€ered minimal essential medium containing
0.34 mg of defatted bovine serum albumin. The solution was
dialysed overnight at 48C against HEPES-bu€ered minimal
essential medium and aliquoted. After incubation with 1 mM
CO-1, cells were incubated with C6-NBD-ceramide-bovine
serum albumin complex for 5 min at 378C and washed with
PBS prior to observation.
Cellular drug accumulation
The cellular uptake of anthrapyridones was followed by
monitoring the decrease of the ¯uorescence signal at 562 nm
(566 nm) (lex=495 nm). This spectro¯uorometric method
has been largely described previously (Tarasiuk et al., 1989;
Frezard & Garnier-Suillerot, 1991; Mankhetkorn et al., 1996;
Marbeuf-Gueye et al., 1998). The incubation of the cells with
the compound proceeds without compromising cell viability.
All experiments were conducted in 1 cm quartz cuvettes
containing 2 ml of 20 mM HEPES bu€er plus (mM): NaCl
132, KCl 3.5, CaCl2 1, MgCl2 0.5, glucose 5 (pH 7.3) at 378C.
In a typical experiment, 26106 cells were suspended in 2 ml
of HEPES bu€er under vigorous stirring. 20 ml of the stock
anthrapyridone solution was quickly added to this suspension
yielding an anthrapyridone concentration equal to CT
(CT=1 mM). The decrease of the ¯uorescence intensity F at
562 nm (or 566 nm) was followed as a function of time until
the curve F=f(t) reached a plateau and the ¯uorescence
intensity was equal to Fn. The initial rate of uptake V+ was
determined using the equation V+=(dF/dt)t=0´CT/F0 where
(dF/dt)t=0 is the slope of the tangent to the curve F=f(t) and
F0 is the ¯uorescence intensity at t=0. The overall cellular
concentration of anthrapyridone at the steady state was
Cn=CT(F0 ± Fn)/F0 (see Figure 2).
Fluorescence microscopy was carried out on a Nikon
Optiphot-2 epi¯uorescence microscope equipped with
a
Nipkow wheel coaxial-confocal attachment (Technical In-
struments, San Francisco, CA, U.S.A.). Cells were mounted
in a perfusion chamber and viewed with a Nikon Plan-Apo
X60 oil immersion chamber objective (numerical aperture,
1.4). Confocal ¯uorescence images were detected with a
cooled CCD camera (Micromax; Princeton Instruments,
Evry, France) with a 12-bit detector (RTEA-1317 K; Kodak).
C6-NBD-ceramide or LysoTrackerTM Green DND-26 and
CO-1 were visualized with standard ¯uorescein and rhoda-
mine ®lter sets, respectively. Image pairs were acquired
(exposure time, 500 ms) for the same ®eld containing one
or more cells. Analysis and display were performed using
IPLab software (Scanalytics, Fairfax, VA, U.S.A.).
Lipophilicity measurements
HPLC column containing as stationary phase 1-myristoyl, 2-
[13-carbonylimidazolide-tridecanoyl]-sn-3-glycerophospholine
(lecithin-imidazolide) bonded to silica-propylamine with the
unreacted propylamine moieties end-capped with C10 and C3
alkyl chains (IAM.PC.DD2) was purchased from Regis
British Journal of Pharmacology vol 135 (6)

1516
J. Tarasiuk et al
Non-cross resistant anthrapyridones
Technologies Inc. (Morton Grove, Ill, U.S.A.). The
IAM.PC.DD2 column was 3 cm64.6 mm; particle diameter
12 mm; pore diameter 300 A. For all studies the injection
volume was ca. 10 ml of a solute aqueous solution of
anthrapyridone at concentration *100 mM. Acetonitrile/
0.1 M Sorensen bu€er (K2HPO4/KH2PO4) pH 7.2 eluent
was used in a proportion 50 : 50%, 35 : 65%, 25 : 75%,
20 : 80% and 15 : 85% (v v71). The ¯ow-rate was 1 ml min71
and solute detection was at 495 nm. The chromatographic
system consisted of a Model L-6200A pump, a Model L-4250
UV-VIS detector and a Model D-2500 chromatointegrator
(all from Merck-Hitachi, Vienna, Austria).
Capacity factors, k'IAM, were calculated as k'IAM=tr-t0/t0.
tr=retention time; t0=dead time of the column. The dead
time of the column was the signal given by 50 mg/ml citric
acid solution. A standard, commercially available statistical
package for regression analysis was employed on a personal
computer.
yielded 50% of the anthrapyridone binding Cb (Table 2). As
can be seen, anthrapyridones have a high anity for DNA.
This anity was comparable for all examined derivatives as
well as for reference compounds, doxorubicin and pirarubi-
cin.
Accumulation of anthrapyridones in erythroleukemia
K562 sensitive and K562/DOX resistant cells
The accumulation of anthrapyridones has been studied
comparatively in control cells and in energy-depleted cells.
Figures 2 and 3 present the typical ¯uorescence signals
observed when drug-sensitive and drug-resistant K562 cells
were incubated in glucose-containing HEPES bu€er respec-
tively with 1 mM anthrapyridone of type I (CO-1, CO-3, CO-5)
or anthrapyridone of type II (CO-4, CO-6, CO-7). For
anthrapyridones of type I, very fast and important decrease
of the ¯uorescence signal was observed with K562 sensitive as
well as resistant cells due to the very rapid cellular uptake of
anthrapyridone of type I (Figure 2). A steady state was
obtained for these compounds within about 5 min. The
concentration bound in the cells at the steady state was almost
the same for sensitive and resistant lines and equal to
0.80+0.05 mM. In contrast, for anthrapyridones of type II,
very low decrease of ¯uorescence signal was observed with
K562 sensitive and resistant cells due to the slow cellular uptake
of these compounds. A steady state for derivatives of type II
was obtained within about 1 h (Figure 3). Their accumulation
in resistant cells was signi®cantly reduced in comparison to
sensitive cells mostly for anthrapyridones CO-6 and CO-7.
Figures 4 and 5 show the records of experiments performed
respectively for anthrapyridones of type I and anthrapyridones
of type II incubated with sensitive and resistant K562 cells in
HEPES bu€er in the presence of 10 mM NaN3 and in the
absence of glucose. The incorporation of anthrapyridones at
the steady state in these energy-depleted cells was signi®cantly
modi®ed in comparison to control cells. After the addition of
5 mM glucose giving rise to ATP synthesis via the glycolysis
pathway, a further decrease of ¯uorescence signal was observed
for anthrapyridones of type I, more distinct in the case of
sensitive cells. For anthrapyridones of type II whereas, the
addition of glucose to sensitive cells yielded to a slight decrease
of ¯uorescence signal while for resistant cells an increase of this
signal was observed. This is related to the occurrence of two
Results
The structures of new synthesized anthrapyridones are
presented in Figure 1. As can be seen the series of obtained
compounds contained two types of anthrapyridones: (a)
ANTHRAPYRIDONES OF TYPE I having hydrophobic
substituents in the side chain: CO-1, CO-3, CO-5; (b)
ANTHRAPYRIDONES OF TYPE II having hydrophylic
substituents in the side chain: CO-4, CO-6, CO-7
Interaction of anthrapyridones with naked DNA
In order to determine an anity of examined compounds to
isolated DNA, the ¯uorometric titration of anthrapyridones
(1 mM) with naked DNA was performed at 378C in HEPES
bu€er (pH 7.3). The addition of DNA to a compound
solution yielded a quenching of the ¯uorescence intensity.
For each compound, two parameters have been determined:
Cb ± the concentration of anthrapyridone bound to DNA at
the equilibrium state, CDNA50
± the DNA concentration
Table 2 Anity of anthrapyridones, doxorubicin and
pirarubicin to naked DNA
Compound
Cb [mM]
CDNA 50 [mM]
CO-1
CO-3
CO-4
CO-5
CO-6
CO-7
DOX
PIRA
0.80
0.86
0.73
0.75
0.72
0.66
0.92
0.83
4.6
5.6
5.7
4.8
4.8
5.4
2.6
2.9
The reaction mixture (2 ml) contained: 1 mM examined
compound, 0 ± 72 mM DNA, 9.5 mM HEPES, 132 mM NaCl,
3.5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, pH 7.25. Cb ± the
concentration of compound bound to DNA at the equili-
brium state (+5%); CDNA 50 ± the DNA concentration
yielded 50% of the compound binding Cb (+5%).
Figure 1 Structures of anthrapyridone derivatives.
British Journal of Pharmacology vol 135 (6)

J. Tarasiuk et al
Non-cross resistant anthrapyridones
1517
Figure 3 Uptake of anthrapyridone of type II (CO-7) by sensitive
(S) and resistant (R) K562 cells. Fluorescence intensity at 566 nm
(lex=495 nm) was recorded as a function of incubation time until the
steady state. Cells, 26106, either sensitive (S) or resistant (R), were
suspended in a cuvette ®lled with 2 ml bu€er at pH=7.25 under
vigorous stirring. At t=0, 20 ml of a 100 mM stock anthrapyridone
solution was added to the cells yielding a CT=1 mM anthrapyridone
solution.
Figure 2 Uptake of anthrapyridone of type I (CO-1) by sensitive (S)
and resistant (R) K562 cells. Fluorescence intensity at 562 nm
(lex=495 nm) was recorded as a function of incubation time until the
steady state. Cells, 26106, either sensitive (S) or resistant (R), were
suspended in a cuvette ®lled with 2 ml bu€er at pH=7.25 under
vigorous stirring. At t=0, 20 ml of a 100 mM stock anthrapyridone
solution was added to the cells yielding a CT=1 mM anthrapyridone
solution.
simultaneous energy-dependent processes: non-nuclear accu-
mulation (Vnn) which causes an autoquenching of ¯uorescence
signal due to the small volume of non-nuclear organelles and P-
glycoprotein-mediated e‚ux (VP-gp) which gives an increase of
¯uorescence signal due to the release of anthrapyridones out of
the cells. It is possible to estimate the contribution of each of
these processes because, for energy-depleted sensitive cells after
the addition of glucose, only non-nuclear accumulation was
observed (VE(S)=Vnn(S)). It seems also very probable that the
rate of non-nuclear accumulation limited by the step of the
passive passage through intracellular membranes do not
depend on cell line (Vnn(S)%Vnn(R)), since it was found that
the kinetics of cellular uptake determined by the step of a
passive passage of anthrapyridone through the plasma
membrane was the same for sensitive and resistant cells. Taking
into account these assumptions, the P-glycoprotein-mediated
e‚ux contribution in the summary rate of energy dependent
processes VE(R) can be calculated using following expressions:
of P-gp-mediated e‚ux of anthrapyridones. We have found
that 10 mM verapamil inhibited the e‚ux of these compounds
to about 70% (to the same extent that for reference
compound pirarubicin, 75%) (data not presented).
Figure
6 and Figure 8b show diagrams representing
anthrapyridone accumulation in control (cells+E) and in
energy deprived (cells-E) K562 cells. Figure 7 and Figure 8a
show comparative diagrams representing the values of
kinetics of passive cellular uptake, kinetics of active P-gp-
mediated e‚ux and the rate of non-nuclear accumulation for
all examined anthrapyridones.
Intracellular distribution of anthrapyridones in NIH-3T3
cells
The above data showed that, beside an energy-independent
accumulation of anthrapyridones in nucleus, an energy-
dependent accumulation of these compounds occurred in
non-nuclear compartments. In order to de®ne the intracel-
lular distribution of anthrapyridones, NIH-3T3 cells were
used, because they have relatively small nucleus and thus
their intracellular organelles are easy visualized. Cells were
incubated with 1 mM CO-1 and then with LysoTrackerTM
Green DND-26 to stain lysosomes or with C6-NBD-
ceramide-bovine serum albumin complex to stain trans-Golgi.
The series of images obtained by confocal microscopy (Figure
9) showed the accumulation of anthrapyridones in nucleus as
well as punctuated zones of strong ¯uorescence signal have
been detected. Confocal microscopy analysis using Lyso-
TrackerTM Green DND-26, the speci®c probe for lysosomes,
showed that these punctuated zones corresponded to the
lysosomal accumulation. Additional confocal microscopy
For anthrapyridones of type I:
. in the case of sensitive cells:
VE/(S)=Vnn(S)
. in the case of resistant cells: VE(R)=Vnn(R)-VP-gp
Vnn(S)&Vnn(R), Vnn4VP-gp
VP-gp=VE(S)-VE(R)
For anthrapyridones of type II:
. in the case of sensitive cells: VE(S)=Vnn(S)
. in the case of resistant cells: VE(R)=VP-gp-Vnn(R)
Vnn(S)&Vnn(R), VP-gp4Vnn
,
,
VP-gp=VE(S)+VE(R)
Additional studies have been done with verapamil, a well-
known P-glycoprotein blocking agent to support the existence
British Journal of Pharmacology vol 135 (6)

1518
J. Tarasiuk et al
Non-cross resistant anthrapyridones
Figure 5 Uptake of anthrapyridone of type II (CO-7) by energy-
deprived sensitive (S) and resistant (R) K562 cells. Fluorescence
intensity at 566 nm (lex=495 nm) was recorded as a function of
incubation time. Cells, 26106, either sensitive (S) or resistant (R),
were suspended in a cuvette ®lled with 2 ml bu€er at pHe=7.25
under vigorous stirring and preincubated for 30 min with 10 mM
NaN3 in the absence of glucose. At t=0, 20 ml of a 100 mM stock
anthrapyridone solution was added to the cells yielding a CT=1 mM
anthrapyridone solution. When the steady state was reached 5 mM
glucose was added yielding a new steady state. The global rate of
energy-dependent processes (VE) was determined from the slope of
the tangent to the curve after the addition of glucose.
Figure 4 Uptake of anthrapyridone of type I (CO-1) by energy-
deprived sensitive (S) and resistant (R) K562 cells. Fluorescence
intensity at 526 nm (lex=495 nm) was recorded as a function of
incubation time. Cells, 26106, either sensitive (S) or resistant (R),
were suspended in a cuvette ®lled with 2 ml bu€er at pHe=7.25
under vigorous stirring and preincubated for 30 min with 10 mM
NaN3 in the absence of glucose. At t=0, 20 ml of a 100 mM stock
anthrapyridone solution was added to the cells yielding a CT=1 mM
anthrapyridone solution. When the steady state was reached, 5 mM
glucose was added yielding a new steady state. The global rate of
energy-dependent processes (VE) was determined from the slope of
the tangent to the curve after the addition of glucose.
analysis done with trans-Golgi probe (C6-NBD-ceramide-
bovine serum albumin complex) showed that there was no
accumulation of anthrapyridones in trans-Golgi organelles.
The lysosomal accumulation of anthrapyridones being weak
bases is related to an energy-dependent pH gradient and was
observed in our transport studies as an energy-dependent
accumulation. From all these data, we can infer that
lysosomes are a second site of anthrapyridones accumulation.
doxorubicin and pirarubicin have been added. As can be
seen, for anthrapyridones of type I the RF values are low
(1.172.8) while for anthrapyridones of type II the signi®cant
decrease of cytotoxic activity against K562 resistant cells was
observed. The RF values for CO-4 and for CO-6 and CO-7,
the less active derivatives against K562 resistant cells, were
respectively 6, 67 and 34. These in vitro results corroborate
with the accumulation data presented above. The decrease in
accumulation of CO-4 in resistant cells was much less
important (47%) than for CO-6 (88%) and CO-7 (73%)
(Figure 8b). We have found that nearly linear relation exists
between the resistance factor RF and the parameter Cn(S)/
Cn(R) values re¯ecting the decrease in anthrapyridones
accumulation in resistant cells in comparison to sensitive
parent line (r=0.976) (Figure 11). Although the resistance
factors for anthrapyridone of type I were near 1, they
exhibited relatively low cytostatic activity on sensitive cells in
comparison to doxorubicin and pirarubicin.
Determination of lipophilic properties of anthrapyridones
Measurements of lipophilicity parameters of anthrapyridones
have been done by RF-HPLC method using an immobilized
arti®cial membrane column (IAM). This method seems to be
more accurate in comparison to standard measure of
lipophilicity parameters as a partition coecient in n-
octanol/bu€er system (Kaliszan et al., 1994).
For all examined anthrapyridones, the capacity factors
k'IAM (k'IAM=tr-to/to; tr- retention time, to-dead time of the
column) have been determined. It was found that there was
nearly a linear correlation between the lgk'IAM value of this
lipophilicity parameters and lgV+ as well as between lgk'IAM
and lgknn (were: knn-coecient of non-nuclear accumulation
rate knn=Vnn/Ci) (Figure 10).
Discussion
The rational design of antitumour agents able to circumvent
multidrug resistance is one of the most important questions
for an e€ective cancer therapy. The reduced drug accumula-
tion, due to the active export of cytostatics out of resistant
cells is the main cause of multidrug resistance (Garnier-
Suillerot, 1995).
Cytotoxic activity of anthrapyridones
The anthrapyridone concentrations required for
a 50%
inhibition (IC50) of respectively sensitive and resistant K562
cell growth are shown in Table 3 with the resistance factor
(RF) values. For comparison, the values obtained for
Our approach to search for non-cross resistant antitumour
agents is based on the fact that the intracellular drug
concentration attainable in resistant cells depends on the
British Journal of Pharmacology vol 135 (6)

J. Tarasiuk et al
Non-cross resistant anthrapyridones
1519
Figure 6 Anthrapyridone accumulation in control (cells+E) and energy-deprived (cells-E) K562 cells. Cn=anthrapyridone
concentration bound in the cells at the steady state; C'n=anthrapyridone concentration bound in the cells at the new steady state
after the addition of 5 mM glucose to energy-deprived cells. The values are given in mM (+10%). CT=total anthrapyridone
concentration; CT=1 mM; S-sensitive cells; R-resistant cells.
di€erence between the kinetics of passive cellular uptake and
kinetics of active ABC transporter-mediated e‚ux (Mankhet-
korn et al., 1996; Marbeuf-Gueye et al., 1998; 1999). It might be
possible to design derivatives which, for the reason of their
physicochemical properties, di€use very rapidly into the cell and
in such cases the protein transporter responsible for the e€ective
drug export out of the cell essentially operates in a futile cycle.
We have recently demonstrated that benzoperimidines (Stefaska
et al., 1999; Tkaczyk-Gobis et al., 2001) and properly substituted
pyrimidoacridones (Antonini et al., 1995), exhibiting very high
kinetics of cellular uptake, were able to overcome multidrug
resistance mediated either by P-gp or MRP1. We postulate
further that the presence of additional heterocyclic ring (®ve or
six membered), fused to the anthracenedione or acridine ring
system, is the structural factor which extremely favours the
passive di€usion of obtained derivatives across the plasma
membrane. In order to further support this hypothesis, a novel
family of anthrapyridones was obtained and examined in regard
to erythroleukemia K562 sensitive and K562/DOX resistant cells
with the overexpression of P-glycoprotein. Anthrapyridones,
carboquinoid derivatives of anthracenediones, contain an
additional six-membered ring condensed to the anthracenedione
chromophore. A series of six compounds was properly designed.
Anthrapyridones of type I have hydrophobic substituents on the
side chain favouring a priori the kinetics of cellular uptake and
anthrapyridones of type II, designed as a negative control, have
hydrophylic substituents on the side chain disturbing a priori the
kinetics of cellular uptake. The data presented in this paper
indicate that: (i) all examined anthrapyridones have a high
anity to naked DNA; (ii) the modulation of the kinetics of
passive cellular uptake of anthrapyridones V+ is much easier to
perform by suitable structure modi®cations of the side chain.
The kinetics of P-gp-dependent active e‚ux of anthrapyridones
VP-gp is the parameter much less susceptible to modulate by
British Journal of Pharmacology vol 135 (6)

1520
J. Tarasiuk et al
Non-cross resistant anthrapyridones
Figure 7 Diagrams representing the values of V+, VP-gp and Vnn for anthrapyridone derivatives: V+=kinetics of passive cellular
uptake; VP-gp=kinetics of active P-gp-mediated e‚ux; Vnn=rate of non-nuclear accumulation. The values are given in 10718
mole cell71 s71 (+10%).
chemical modi®cations, pointing to the similarity of the
substrate spectrum of all tested compounds for P-gp; (iii)
introduction of hydrophobic substituents to the side chain of
anthrapyridones [CO-1, CO-3, CO-5] favours the fast passive
cellular uptake (V+44VP-gp) (Figure 8a) and causes that their
cellular accumulation determining the cytotoxic activity are very
high and comparable for sensitive and resistant cells
(Cn(R)&Cn(S)) (Figure 8b); (iv) introduction of hydrophylic
substituents to the side chain of anthrapyridones [CO-4, CO-6,
lipophilicity parameter lgk'IAM and lgV+ as well as between
lgk'IAM and lgknm corroborating the very important role of the
hydrophobic-hydrophylic character of examined anthrapyri-
dones in their ability to cross biological membranes (cytoplasmic
and membranes surrounding cellular organelles). In contrast, it
seems that the hydrophobic-hydrophylic character of anthrapyr-
idones does not determine their anity for P-gp exporting pump.
It is well demonstrated for anthracyclines that there is a
good correlation between the short-term measurements of
drug accumulation in living cells and the long-term growth
CO-7] disturbs the fast passive cellular uptake (V+&VP-gp
)
(Figure 8a) and causes a drastic decrease of cellular accumula-
tion in resistant cells (Cn(R)5Cn(S)) (Figure 8b); (v) cellular
accumulation of anthrapyridones occurs in the nucleus,
probably through intercalation of the ¯at conjugated rings
between the bases pairs. Our data show that these compounds
being weak bases accumulate also in lysosomes in an energy-
dependent manner; (vi) a nearly linear relation exists between
inhibition (Pereira & Garnier-Suillerot, 1994; Garnier-
Suillerot, 1995). Our in vitro biological data con®rm that
anthrapyridones of type I conserving very high accumulation
in resistant cells exhibit comparable cytotoxic activity against
K562 sensitive and K562/DOX resistant cells, while
anthrapyridones of type II are much less active against
K562/DOX resistant cells in comparison to sensitive cells.
British Journal of Pharmacology vol 135 (6)

J. Tarasiuk et al
Non-cross resistant anthrapyridones
1521
a
Our preliminary results done on small-cell lung cancer cell
line GLC4/DOX overexpressing MRP1 show that anthra-
pyridones of type I exhibit also comparable cytotoxic activity
towards sensitive and MRP1 resistant cells, while for
reference compound ± doxorubicin RF(GLC4) is equal to
74. The presented results show that it is possible to design
non-cross resistant antitumour agents, simply by the fact that
they penetrate very rapidly into the cell. The con®rmation of
this postulate was the main aim of this study. Although the
resistance factors for anthrapyridones of type I are near 1, it
seems hardly probable that they could ®nd a clinical
application as potential cytostatics in the treatment of
resistant types of cancers because of their relatively low
cytostatic activity on sensitive cells. In fact, the cytotoxic
activity of anticancer agents depends on intracellular
concentration and for resistant cells is determined by the
kinetics of cellular uptake and kinetics of an energy-
b
Figure 8 Comparative diagrams representing: (a) the values of V+
VP-gp and Vnn for anthrapyridone derivatives; V+=kinetics of
passive cellular uptake; P-gp=kinetics of active P-gp-mediated
e‚ux; Vnn=rate of non-nuclear accumulation; The values are given
in 10718 mole cell71 s71 (+10%):
compound CO-1,
compound CO-3, 5 ± compound CO-5, 4 ± compound CO-4, 6 ±
compound CO-6, compound CO-7. (b) the intracellular
,
V
1
±
3 ±
7
±
anthrapyridone accumulation (Cn) in sensitive (S) and resistant (R)
K562 cells; Cn=anthrapyridone concentration bound in the cells at
the steady state. The values are given in mM (+10%). CT=total
anthrapyridone concentration; CT=1 mM.
a
b
c
d
Figure 10 Dependence of IgV+ and lgknn of anthrapyridone
compounds on the lipophilicity parameter lgk'IAM: V+=kinetics of
passive cellular uptake; knn=coecient of non-nuclear accumulation
rate knn=Vnn/Ci: Vnn ± rate of non-nuclear accumulation, Ci=free
anthrapyridone concentration in the cytoplasm of energy-deprived
(cells-E), K562 cells at the steady state, Ci=CT-Cn. k'IAM=the
capacity factor measured on an immobilized arti®cial membrane
column IAM (k'IAM=tr-t0/t0; tr-retention time, t0 ± dead time of the
column). The lines drawn have been least-squares ®tted to the data.
The correlation coecients are higher than 0.92.
Figure 9 Colocalization of CO-1 with lysosomes and trans-Golgi
probes in NIH/3T3 cells. Cells were labelling according to the
procedure described in Methods with 1 mM CO-1 (A and C), speci®c
lysosome probe: LysoTrackerTM Green DND-26 (B), speci®c trans-
Golgi probe: C6-NBD-ceramide-bovine serum albumin complex (D).
British Journal of Pharmacology vol 135 (6)

1522
J. Tarasiuk et al
Non-cross resistant anthrapyridones
Table 3 Cross-resistance pattern of doxorubicin-resistant
K562 cells
K562
IC50(S)[mM]
K562/DOX
IC50(R)[mM]
Compound
RF
CO-1
CO-3
CO-5
CO-4
CO-6
CO-7
DOX
PIRA
0.7+0.2
11.0+0.1
0.8+0.3
0.5+0.1
0.95+0.04
1.8+0.1
2.0+0.5
12.0+0.1
2.1+0.2
3.0+0.2
64+9
62+12
0.34+0.03
0.050+0.003
2.8
1.1
2.6
6.0
67
34
34
8
0.010+0.002
0.006+0.001
Figure 11 Dependence of resistance factor RF on the Cn(S)/Cn(R)
parameter for anthrapyridones compounds. The values Cn(S)/Cn(R)
re¯ect the decrease in anthrapyridones accumulation at the steady
state in resistant cells Cn(R) in comparison to sensitive parent line
Cn(S).
IC50(S) and IC50(R) are the compound concentrations
required to inhibit 50% of respectively sensitive and resistant
cell growth. Resistance factor (RF) was calculated as
RF=IC50(R)/IC50(S). The values represent mean+s.d. of
triplicate determinations.
of these derivatives, would operate in a futile cycle mode. It
creates the possibility to reverse the multidrug-resistance to
anthracycline antitumour agents by the combinated admin-
istration of these drugs with anthrapyridone compounds.
Studies on the ability of anthrapyridones to inhibit the P-gp
and MRP1-dependent export of anthracyclines and in
consequence to restore their cytotoxic activity against
K562/DOX and GLC4/DOX resistant cell lines is under
progress in our laboratory.
dependent export. Nevertheless, the cytotoxic activity
depends highly on mechanism(s) of interaction of anticancer
agents with DNA machinery (DNA intercalation, alkylation
or cross-linking mechanisms, inhibition of topoisomerase I
and/or II etc). The examination of these mechanisms could
be the subject of another study in a further step. In this stage
of our work we can only suggest that the relatively low
cytostatic activity of anthrapyridones against sensitive cells
would be related in part to their accumulation not only in
nucleus (as in the case of doxorubicin) but also in lysosomes.
Nevertheless, presented data suggest that: ®rstly ± non-
nuclear accumulation of cytostatics is not necessarily very
toxic for the cell and in consequence do not exclude their
clinical use; secondly ± we consider the potential application
of anthrapyridones of type I as e€ective modulators of
multidrug resistance exporting pumps which, in the presence
This work was supported by the State Committee for Scienti®c
Research Warsaw (grants No 4 P05A 062 16 and 4P05F 035 19), the
Chemical Faculty, Technical University of Gdansk, Universite Paris
Nord and Centre National de la Recherche Scienti®que (CNRS).
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(Received November 19, 2001
Accepted January 15, 2002)
British Journal of Pharmacology vol 135 (6)