Products of metabolic activation of the antitumor drug ledakrin (Nitracrine) in vitro

Article abstract of DOI:10.1021/tx000081c

The aim of this work was to characterize the products of metabolic activation of the antitumor drug ledakrin (Nitracrine) in model metabolic systems, where formation of drug - DNA adducts was previously discovered. The metabolic products obtained in different biological systems were compared with those obtained in experiments where chemical reducing agents were applied. Therefore, activation products were obtained in the presence of the microsomal fraction of rat liver and in the experiments with the reducing agents dithiothreitol, hydrazine hydrate, and SnCl₂. Furthermore, transformations of the drug with oxidoreductase enzymes DTdiaphorase and xanthine oxidase were observed. The ledakrin transformation products were separated and analyzed by HPLC with diode array detection. Structural studies of the products were performed by means of ESI-MS and NMR. Proton, carbon, and nitrogen assignments were made based upon DQF-COSY, ROESY, TOCSY, HSQC, and HMBC experiments. It was demonstrated during the reduction of ledakrin that a key metabolite, a compound with an additional five-membered ring attached to positions 1 and 9 of the acridine core and with the retained 9-aminoalkyl side chain, was formed in all the systems that were studied. It was determined that the reactive nitrogen atoms of this additional ring underwent further transformations resulting in the formation of a six-membered ring produced by the addition of a carbon atom to the dihydropyrazoloacridine ring. Furthermore, it was observed that positions 2 and 4 of ledakrin's acridine ring are susceptible to nucleophilic substitution as revealed by the studies with dithiothreitol. Additionally, although most products from the reduction of ledakrin were-extremely unstable, 1-aminoacridinone, produced enzymatically and with dithiothreitol, exhibited persistent stability under the studied conditions.

Full text of DOI:10.1021/tx000081c

J ANUARY 2001
VOLUME 14, NUMBER 1
©
Copyright 2001 by the American Chemical Society
Articles
P r od u cts of Meta bolic Activa tion of th e An titu m or Dr u g
Led a k r in (Nitr a cr in e) in Vitr o
Katarzyna Gorlewska, Zofia Mazerska,* Paweł Sowi n´ ski, and J erzy Konopa
Department of Pharmaceutical Technology and Biochemistry, Technical University of Gda n´ sk,
8
0-952 Gda n´ sk, Poland
Received April 11, 2000
The aim of this work was to characterize the products of metabolic activation of the antitumor
drug ledakrin (Nitracrine) in model metabolic systems, where formation of drug-DNA adducts
was previously discovered. The metabolic products obtained in different biological systems
were compared with those obtained in experiments where chemical reducing agents were
applied. Therefore, activation products were obtained in the presence of the microsomal fraction
of rat liver and in the experiments with the reducing agents dithiothreitol, hydrazine hydrate,
2
and SnCl . Furthermore, transformations of the drug with oxidoreductase enzymes DT-
diaphorase and xanthine oxidase were observed. The ledakrin transformation products were
separated and analyzed by HPLC with diode array detection. Structural studies of the products
were performed by means of ESI-MS and NMR. Proton, carbon, and nitrogen assignments
were made based upon DQF-COSY, ROESY, TOCSY, HSQC, and HMBC experiments. It was
demonstrated during the reduction of ledakrin that a key metabolite, a compound with an
additional five-membered ring attached to positions 1 and 9 of the acridine core and with the
retained 9-aminoalkyl side chain, was formed in all the systems that were studied. It was
determined that the reactive nitrogen atoms of this additional ring underwent further
transformations resulting in the formation of a six-membered ring produced by the addition
of a carbon atom to the dihydropyrazoloacridine ring. Furthermore, it was observed that
positions 2 and 4 of ledakrin’s acridine ring are susceptible to nucleophilic substitution as
revealed by the studies with dithiothreitol. Additionally, although most products from the
reduction of ledakrin were extremely unstable, 1-aminoacridinone, produced enzymatically
and with dithiothreitol, exhibited persistent stability under the studied conditions.
In tr od u ction
an antitumor drug that has been used clinically for
several years (1-4). It belongs to a group of 1-nitro-9-
1
Ledakrin, 1 (WHO, recommended Nitracrine), 1-nitro-
9
-[3′-(dimethylamino)propylamino]acridine (Figure 1), is
1
Abbreviations: DQF-COSY, double-quantum-filtered correlation
spectroscopy; DTT, dithiothreitol; ESI-MS, electrospray ionization mass
spectrometry; HMBC, heteronuclear multibond correlation; HSQC,
heteronuclear single-quantum correlation; ROESY, rotating frame
enhanced spectroscopy; TOCSY, total correlation spectroscopy; RP-
HPLC, reversed-phase HPLC; WHO, World Health Organization.
*
To whom correspondence should be addressed. E-mail: mazerska@
altis.chem.pg.gda.pl. Phone: (48 58) 347 24 07. Fax: (48 58) 347 15
6.
1
1
0.1021/tx000081c CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/14/2000

2
Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Gorlewska et al.
ing the aminoalkyl side chain in position 9 of the acridine
core. Reduction of the drug with sodium borohydride (26)
and hydrazine hydrate (27) was also investigated; how-
ever, no clear evidence of the existence of the N-hydroxy-
1
-amino analogue of ledakrin was provided.
The results described above, although insightful, have
yet to substantiate the mode of action of ledakrin due to
insufficient structural evidence of the drug’s metabolites.
This prompted our group to attempt to isolate and
structurally identify the products from reductive meta-
bolic activation of ledakrin obtained in such systems
where the formation of drug-DNA adducts has been
observed (13). The following questions were posed: (i)
what are the chemical structures of the metabolites; (ii)
which elements of those structures could be responsible
for the high reactivity of ledakrin under reducing condi-
tions; and (iii) which elements of the metabolites’ struc-
ture might create the covalent bonds between ledakrin
and DNA?
F igu r e 1. Tautomeric structures of ledakrin (Nitracrine),
-nitro-9-[3′-(dimethylamino)propylamino]acridine.
1
aminoacridine derivatives developed in our laboratory,
which exhibit exceptionally high cytotoxic and antitumor
properties (5-8). This unusual biological activity relates
only to 1-nitroacridine derivatives, whereas the remain-
ing nitro isomers exhibit significantly weaker action (5).
The studies on the “mode of action” showed that
ledakrin is the latent form of the drug and requires
metabolic activation before it exhibits any cytotoxic and
antitumor activity. Activation results in covalent binding
of the drug to DNA and other cellular macromolecules
In this work, we present the results from activation of
ledakrin in the presence of the microsomal fraction of rat
liver and with a chemical reducing agent dithiothreitol.
These systems were considered ideal since they have been
shown to activate ledakrin, resulting in the formation of
DNA-drug adducts (13). We also investigated the activa-
tion of ledakrin with two oxidoreductase enzymes: DT-
diaphorase [NAD(P)H:quinone oxidoreductase] and xan-
thine oxidase. Furthermore, comparative studies were
performed on products obtained in the reaction of ledakrin
with chemical reducing agents. The reactions were fol-
lowed and their products isolated with liquid chroma-
tography. Structural studies of the reaction products were
accomplished by employing ESI-MS and the multidimen-
sional NMR techniques of DQF-COSY, ROESY, TOCSY,
HSQC, and HMBC.
(
10-12). The formation of the adduct between DNA and
32
the activated form of ledakrin was shown by P-postla-
beling analyses in cells as well as in other activation
systems (13). Specifically, ledakrin was able to induce
covalent interstrand cross-links in the DNA of mam-
malian and bacterial cells (14) as well as DNA-protein
cross-links in leukemia cells (15). It was suggested that
these cross-links play a crucial role in the mode of
antitumor action of this drug (15, 16).
Previously, it was shown that the inhibitory effect of
ledakrin on DNA transcription with RNA polymerase
significantly increased in the presence of reducing agents
such as thiols (17). Likewise, the covalent binding of
ledakrin to DNA was observed to take place under the
reducing environment of microsomes (10, 11). Further-
more, DNA adduct formation was demonstrated not only
in experiments performed in the cell but also after
reducing activation occurring with microsomal enzymes
and after reduction with dithiothreitol (DTT) (13). Wilson
et al. suggested that nitro reduction is the major route
of ledakrin metabolism in hypoxic cells, as the drug is
selectively toxic to the AA8 cell line growing under
hypoxic conditions (18, 19).
Exp er im en ta l P r oced u r es
Ch em ica ls a n d En zym es. Ledakrin (Nitracrine), 1-nitro-
9
-[3′-(dimethylamino)propylamino]acridine (C-283), was syn-
thesized as a dihydrochloride in the Department of Pharma-
ceutical Technology and Biochemistry, Technical University of
Gda n´ sk. The following enzymes and chemicals were obtained
from Sigma Chemical Co. (St. Louis, MO): DT-diaphorase (EC
1.6.99.2), xanthine oxidase (EC 1.1.3.22), glucose-6-phosphate
dehydrogenase (EC 1.1.1.49), dithiothreitol (DTT), tris(hy-
droxymethyl)aminomethane (Trizma base), 3-methylcholan-
threne, nicotinamide, glucose 6-phosphate, NADPH, and xan-
thine. Polyethylene glycol 6000 was obtained from Serva
All of the results listed above clearly indicated that the
reduction of ledakrin is the necessary activation step that
leads to the covalent binding of the drug to DNA and/or
proteins in cellular systems.
Several attempts at synthesis, isolation, and identifica-
tion all the reduced metabolites of ledakrin have been
undertaken. It was expected that the N-hydroxy-1-amino
analogue of ledakrin would be formed, based on previous
observations from metabolic reduction of the majority of
nitroaromatic compounds (20, 21). Accordingly, Wilson
et al. presented the HPLC peak of one main metabolite
obtained after anaerobic incubation of AA8 cells with
ledakrin, and its structure was suspected to also be the
N-hydroxyamino analogue of the drug (19). Reduction of
ledakrin with glutathione resulted in several acridine-
containing products; however, none were characterized
due to their high level of instability (22).
Feinbiochemica , and DMSO, hydrazine hydrate, SnCl
2
, THF,
FeSO , Pd/C, and HPLC grade methanol were obtained from
4
Fluka (Buchs, Switzerland). BHT (2,6-di-tert-buthyl-p-krezol)
and ammonium formate (AnalaR) were obtained from BDH Ltd.
(Poole, England). All chemicals were used without further
purifications.
In str u m en ta tion . Reversed-phase HPLC with UV/vis detec-
tion was performed with a Waters Associates HPLC system
equipped with a model 600E solvent delivery system and a
model 991 UV/vis photodiode array detector. HPLC analyses
were carried out with the following systems: (A) an isocratic
elution at 40% methanol in ammonium formate (50 mM, pH
3
.5) for 15 min followed by a linear gradient from 40 to 100%
Common reducing agents such ethanethiol in pyridine
methanol in ammonium formate for 10 min, followed by a linear
gradient from 100 to 40% methanol in ammonium formate, and
(23), mercaptoethanol (24), and hydrogen sulfide (25)
were used to simulate metabolic reduction of ledakrin.
It turned out to be strongly reactive under the applied
reducing conditions. The majority of the products ob-
tained in these reactions were 1-amino derivatives miss-
(
(
B) an isocratic elution at 40% methanol in ammonium formate
50 mM, pH 3.5) for 15 min followed by a linear gradient from
40 to 100% methanol in ammonium formate for 15 min, followed
by a linear gradient from 100 to 40% methanol in ammonium

Products of Ledakrin Metabolic Activation
Chem. Res. Toxicol., Vol. 14, No. 1, 2001
3
formate at a flow rate of 1 mL/min using a 5 µm Suplex pKb
column with water and methanol as the mobile phase. The
samples were lyophilized and redissolved in DMSO for NMR
analysis.
Red u ction of Led a k r in by Hyd r a zin e Hyd r a te. Ledakrin
(1 mM) was dissolved in 25 mL of THF, and 50 mg of Pd/C (10%
v/w) was added. The reaction mixture was placed in the ice bath
and bubbled with argon for 5 min. Two hundred microliters of
the hydrazine hydrate solution was added in small portions of
1
00 analytical column (0.46 cm × 25 cm) (Supelco, Bellefonte,
PA).
The products for NMR analysis were isolated by means of
low-pressure liquid chromatography using a reversed stationary-
phase C18 from Merck (Darmstadt, Switzerland).
LC/MS analysis of the products was accomplished by elec-
trospray ionization with positive ion detection performed on a
Finnigan MAT TSQ 700 tandem mass spectrometer equipped
with a Finnigan electrospray ionization source interfaced with
5
0 µL every 2 min. The reaction was monitored spectrophoto-
metrically, and the products were separated and analyzed by
HPLC and off-line mass spectrometry. One of the products (6)
was isolated by means of low-pressure liquid chromatography
and analyzed by NMR.
2
the HPLC system and a Dionex GP40 gradient pump. N was
used as the sheath gas (386 kPa/56 psi). The HPLC apparatus
was a Waters Associates System model 600-MS with a 484-MS
tuneable absorbance detector operated at 254 nm.
Red u ction of Led a k r in by Sn Cl
2 2 4
. SnCl (1.75 g) and FeSO
(0.35 g) were dissolved in 20 mL of concentrated HCl. The
1H NMR measurements with COSY and ROESY techniques,
temperature was raised to 70 °C, and 0.5 g of ledakrin (dissolved
in concentrated HCl) was added after 15 min. The high reaction
temperature was maintained for 30 min and then slowly reduced
to -10 °C. The pellet was dissolved in water and alkalized with
NaOH. The pellet was extracted with ether, and the extract was
1
3
C NMR measurements with HSQC and HMBC techniques,
1
5
and N NMR measurements were carried out at 500.13 MHz
in deuterated dimethyl sulfoxide (DMSO-d ) using a Varian 500
spectrometer. Chemical shifts are reported in parts per million
6
1
3 4
for H relative to the internal standard (CH ) Si.
2 5
dried over P O . Recrystallization was performed on the solution
Activa tion of Led a k r in w ith th e Micr osom a l F r a ction
of Ra t Liver . Six-week-old male rats (Wistar) were injected
with 3-methylcholanthrene (4 mg/animal) for 3 days before
decapitation. Livers were removed, homogenized in a Potter-
Elvehjem homogenizer, and centrifuged for 30 min at 10000g.
The supernatant was mixed 1:1 with 20 mM sodium pyrophos-
phate. Polyethylene glycol (50%) was added to obtain a concen-
tration of 5% (w/v) and mixed for 10 min. The mixture was then
centrifuged for 10 min at 10000g. The resulting pellet was then
placed in a homogenizer with a solution of 0.1 M Tris acetate,
of methanol and ether, and the product was subjected to NMR
analysis. Product 7 was identified with a combination of UV/
vis spectra, NMR spectra (including COSY, ROESY, HSQC,
HMBC, and N), and electrospray ionization MS/MS.
Sp ectr a l Ch a r a cter iza tion of th e Rea ction P r od u cts. (1)
15
1
1
2
7
1
-Am in o-9-a cr id in on e (2): H NMR (in DMSO) δ 6.25 (d, 1H,
-H), 6.45 (d, 1H, 4-H), 7.18 (m, 1H, 7-H), 7.28 (m, 1H, 3-H),
.4 (d, 1H, 5-H), 7.6 (m, 1H, 6-H), 8.17 (d, 1H, 8-H), 11.27 (s,
H, 10-NH); UV (in MeOH) λmax 245, 260, 320, 420 nm; MS [M
O m/z 211 (found).
2) N -[3′-(Dim et h yla m in o)p r op yl]-4-(1′′,4′′-d isu lfa n yl-
+
+
H] for C13
H
10
N
2
1
mM EDTA, and 20% glycerine (pH 7.4) and homogenized. All
2
(
of the operations were carried out between 0 and 4 °C. Protein
concentrations were determined by the Lowry method.
-
2′′,3′′-d i h y d r o x y b u t a n e )-1,9-d i a z a n e -1,10-d i h y d r o -
1
a cr id in e (3): H NMR (in DMSO) δ 2.04 (m, 2H, 2′-H), 2.24 (d,
H, 4′-CH ), 2.42 (m, 2H, 3′-H), 2.54 (m, 2H, 4′′-H), 2.64 (dd, J
5.3 Hz, 1H, 1′′-H), 2.83 (dd, J ) 5.3 Hz, 1H, 1′′-H), 3.61 (m,
H, 2′′-H), 3.63 (m, 1H, 3′′-H), 4.6 (m, J ) 6.8 Hz, 2H, 1′-H), 6.5
d, J ) 8.7 Hz, 1H, 2-H), 7.04 (m, 1H, 3-H), 7.06 (m, J ) 7.3 Hz,
The activation of ledakrin with the microsomal fraction of
rat liver was carried out in 0.16 M Trizma base buffer (pH 7.4).
The incubation mixture consisted of 125 mM nicotinamide, 100
mM glucose 6-phosphate, 10 mM NADPH, glucose-6-phosphate
dehydrogenase (40 units), rat liver microsomes (5 mg/mL), and
ledakrin (1 mmol). The incubation was carried out at 37 °C in
air. RgThe reaction was initiated by addition of the compound
from the initial incubation after 5 min. After appropriate periods
of time, the incubation mixture was centrifuged for 5 min at
6
)
1
(
1
1
3
H, 7-H), 7.32 (m, J ) 5.8 Hz, 1H, 6-H), 7.54 (d, J ) 8.3 Hz,
13
H, 5-H), 7.8 (d, J ) 7.3 Hz, 1H, 8-H), 9.85 (s, 1H, 10-NH);
C
NMR δ 26.0 (4′′-C), 26.8 (2′-C), 39.0 (1′′-C), 44.8 (4′-C), 48.7 (1′-
C), 54.0 (3′-C), 70.0 (3′′-C), 72.5 (2′′-C), 93.0 (4-C), 103.0 (2-C),
1
1
14.0 (12-C), 117.0 (5-C), 118.5 (13-C), 121.0 (7-C), 122.5 (8-C),
1
2000g. The supernatant was analyzed directly by means of
15
29.0 (6-C), 131 (9-C), 137.5 (3-C), 140.0 (1-C), 141.0 (11-C);
N
HPLC/UV/Vis, HPLC/MS, and MS/MS. The pellet was extracted
with chloroform, dried, then dissolved in water and methanol
NMR δ -354.7 (4′-N), -272.5 (10-N), -175 (9-N), -95.5 (1-N);
UV [in a 60:40 ammonium formate (pH 3.5)/MeOH mixture] λmax
(1:1), and analyzed by HPLC and MS.
+
2
30, 260, 295, 305, 405 nm; MS [M + H] for C22
H
28
N
4
O
2
S
2
m/z
Activa tion of Led a k r in w ith DT-Dia p h or a se. Activation
444 (found), fragment ions at m/z 400 and 372.
of ledakrin (1 mM) with DT-diaphorase (500 µg/mL) in the
presence of 5 mM NADPH was carried out in 0.16 M Trizma
base buffer (pH 7.4) at 37 °C in air. The reaction was initiated
by the addition of ledakrin. The products were separated by
means of HPLC (elution system A) and analyzed by mass
spectrometry.
2
(
3) N -[3′-(Dim eth yla m in o)p r op yl]-2-(1′′,4′′-d isu lfa n yl-
2
′′,3′′-d ih yd r oxyb u t a n e )-1,9-d ia za n e -1,10-d ih yd r oa cr i-
1
d in e (4): H NMR (in DMSO) δ 2.04 (m, 2H, 2a′-H), 2.24 (d,
6
1
1
3
H, 4a′-CH ), 2.42 (m, 2H, 3a′-H), 2.5 (m, 2H, 4a′′-H), 2.89 (dd,
H, 1a′′-H), 3.0 (dd, aH, 1a′′-H), 3.60 (m, 1H, 2a′′-H), 3.60 (m,
H, 3a′′-H), 4.60 (m, 2H, 1a′-H), 5.79 (d, J ) 7.3 Hz, 1H, 4a-H),
Activa tion of Led a k r in w ith Xa n th in e Oxid a se. Activa-
tion of ledakrin (1 mM) by xanthine oxidase (5.3 mg/mL) in the
presence of xanthine (0.3 mg/mL) was carried out in 0.16 M
Trizma base buffer (pH 7.4) at 37 °C in air. The products were
separated by HPLC and analyzed by mass spectrometry.
7.01 (m, 1H, 7a-H), 7.07 (m, 1H, 3a-H), 7.11 (d, J ) 7.8 Hz, 1H,
5a-H), 7.30 (m, J ) 5.8 Hz, 1H, 6a-H), 7.78 (d, J ) 7.3 Hz, 1H,
8a-H), 10.3 (s, 1H, 10a-NH); 13C NMR δ 36.0 (1a′′-C), 69.0 (4a′′-
C), 70.0 (3a′′-C), 72.5 (2a′′-C), 93.0 (4a-C), 104.5 (2a-C), 113.5
(12a-C), 116.0 (5a-C), 118.5 (13a-C), 120.5 (7a-C), 122.5 (8a-C),
1
5
1
29.0 (6a-C), 131.0 (9a-C), 135.0 (3a-C), 141.0 (11a-C); N NMR
Red u ction of Led a k r in by Dith ioth r eitol. The reaction
of ledakrin (1 mM) with DTT (20 mM) was carried out in 0.16
M Trizma base buffer (pH 7.4) at room temperature for 45 min.
The products of the reaction were separated by HPLC (elution
system B). Mass spectra were recorded directly from the
incubation mixture during the reaction progress, after 5, 15,
δ -354.7 (4a′-N), -272.5 (10a-N), -97.9 (1a-N); UV [in a 60:40
ammonium formate (pH 3.5)/MeOH mixture] λmax 230, 260, 295,
+
3
05, 405 nm; MS [M + H] for C22
H
28
N
4
O
2
S
2
m/z 444 (found),
fragment ions at m/z 400 and 372.
(4) N -[3′-(Dim eth yla m in o)p r op yl]-1,9-d ia za n e-1,10-d i-
h yd r oa cr id in e (5): UV [in a 60:40 ammonium formate (pH
3.5)/MeOH mixture] λmax 225, 365, 430 nm; MS [M + H] for
18 20 4
C H N m/z 293 (found), fragment ions at m/z 248 and 220.
(5) N -[3′-(Dim et h yla m in o)p r op yl]-N ,N -d ia m in oet h -
3
yla cr id in e (6): H NMR δ 1.36 (m, 3H, b-CH ), 2.38 (m, 2H,
2
3
0, and 60 min. Products 2-4 were isolated from the reaction
+
mixture after separation by means of low-pressure liquid
chromatography. Products 3 and 4 were eluted from the column
with an isocratic elution at 40% methanol in ammonium formate
2
1
2
1
(50 mM, pH 3.5), and product 2 was eluted at 50% methanol in
ammonium formate (50 mM, pH 3.5). Ammonium formate was
removed from the isolated fractions by separation on the same
2′-H), 3.1 (m, 2H, 3′-H), 4.15 (m, 2H, 1′-H), 5.4 (m, 1H, a-H),
6.6 (d, 1H, 2-H), 7.12 (d, 1H, 4-H), 7.5 (t, 1H, 7-H), 7.68 (t, 1H,

4
Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Gorlewska et al.
3
(
-H), 7.95 (t, 1H, 6-H), 8.02 (d, 1H, 5-H), 8.2 (d, 1H, 8-H), 8.3
s, 1H, 1-NH), 10.95 (m, 1H, 4′-NH), 14.38 (s, 1H, 10-NH); 13
C
NMR δ 19.8 (b-C), 53 (1′-C), 66 (a-C), 103.9 (4-C), 104 (2-C),
06 (13-C), 112.5 (12-C), 119 (5-C), 124 (7-C), 127 (8-C), 136 (6-
1
C), 137.5 (3-C), 139 (14-C), 141 (11-C), 143 (1-C), 155 (9-C); MS
+
[
M + H] for C20
(
H
24
N
4
m/z 320 (found).
2
1
2
6) N -[3′-(Dim et h yla m in o)p r op yl]-N ,N -d ia m in o-1′′-
1
m eth yleth yla cr id in e (7): H NMR δ 1.68 (s, 6H, b-H), 2.32
m, 2H, 2′-H), 2.72 (m, 6H, 5′-H), 3.1 (m, 2H, 3′-H), 4.04 (m,
(
2
7
1
H, 1′-H), 6.62 (d, 1H, 2-H), 7.1 (d, 1H, 4-H), 7.56 (t, 1H, 7-H),
.72 (t, 1H, 3-H), 7.95 (d, 1H, 5-H), 7.95 (t, 1H, 6-H), 8.0 (s, 1H,
-NH), 8.2 (d, 1H, 8-H), 10.9 (m, 1H, 4′-NH), 14.04 (s, 1H, 10-
1
3
NH); C NMR δ 24 (2′-C), 24 (b-C), 41 (5′-C), 48 (1′-C), 52 (3′-
C), 104 (2-C), 104 (4-C), 106 (13-C), 111 (12-C), 119 (5-C), 123
(
7-C), 127 (8-C), 135 (6-C), 137 (3-C), 138 (1-C), 140 (11-C), 143
+
(14-C), 155 (9-C); MS [M + H] for C21
26 4
H N m/z 334 (found).
Resu lts
Act iva t ion of Led a k r in w it h t h e Micr osom a l
F r a ction of Ra t Liver . Studies on formation of DNA-
ledakrin adducts with 32P-postlabeling methods showed
that identical adducts were formed in tumor cells and
after activation of ledakrin with the microsomal fraction
of rat liver (13). Therefore, we began the studies on the
metabolism of ledakrin from the experiments with the
microsomal fraction of rat liver.
The products from various periods of incubation of
ledakrin with this enzymatic system were separated by
HPLC and identified by ESI-MS operating in the positive
ion mode. The chromatograms displaying the contents
of the incubation mixture after activation for 5 and 30
min are shown in panels A and B of Figure 2, respec-
tively. The peaks with retention times of 8.6 (M1) and
F igu r e 2. RP-HPLC (see Experimental Procedures for condi-
tions) profile of products obtained by incubation of ledakrin with
the microsomal fraction of rat liver after incubation for (A) 5
and (B) 30 min.
2
8.6 (M2) min primarily exhibited ions in the mass
spectrum at m/z 293 and 211, respectively (data not
shown). However, the M1 HPLC fraction exposed to air
revealed many other molecular species with MS ions at
m/z 307 (M3), 321 (M4), and 335 (M5) (Figure 3).
Further investigations of the structure of M1 (m/z 293)
were performed by means of ESI-MS/MS. The mass
spectrum in Figure 4 shows a fragment ion at m/z 248
formed by the loss of 45 mass units. These data will be
applied later to determine the structure of M1.
The spectral data (MS and UV/vis) of product M2
turned out to be identical to those of the standard
1
1
-aminoacridinone. Its structure (2) was confirmed by H
NMR as presented in Figure 5.
Product M3 was formed when M1 was exposed to air
+
for 10 min in solution. M3 displays an [M + H] value at
m/z 307, 14 units greater than the M1 ion at m/z 293.
Products M4 and M5 gave ions at m/z 321 and 335, 28
and 42 units greater than M1, respectively. Therefore,
M4 and M5 are possibly the result of addition of two and
three methylene groups to M1, respectively. The analysis
of the structures of M4 and M5 will be discussed later.
Activa tion of Led a k r in w ith DT-Dia p h or a se a n d
Xa n th in e Oxid a se. The second enzymatic system ap-
plied for studies on the activation of ledakrin was DT-
diaphorase [NAD(P)H:quinone oxidoreductase], an en-
zyme commonly used in activation of nitroaromatic
compounds (28, 29). The incubation of the drug with DT-
diaphorase resulted in two main products. Analysis of
the HPLC retention times and UV/vis spectra of chro-
matographic peaks D1 and D2 (Figure 6) revealed that
they were identical to products M1 and M2, respectively,
from microsomal activation (see Figure 2B). Likewise,
F igu r e 3. Positive ion ESI-MS spectrum of the products formed
during incubation of ledakrin with the microsomal fraction of
+
rat liver. Ions [M + H] at m/z 293, 307, 321, and 335 correspond
to products M1, M3, M4, and M5, respectively.
activation of ledakrin with the third enzymatic system,
metalloflavoprotein enzyme, xanthine oxidase (30, 31),
gave a product (out of several others), named O1, which
exhibits chromatographic and spectral properties identi-
cal to those of the microsomally activated product, M1
(data not shown).
The quantities of the products formed during enzy-
matic activation of ledakrin were not sufficient for
structural elucidation by NMR. What is more, standards
for the reduced products of ledakrin were not available

Products of Ledakrin Metabolic Activation
Chem. Res. Toxicol., Vol. 14, No. 1, 2001
5
F igu r e 7. RP-HPLC (see Experimental Procedures for condi-
tions) profile of products obtained by reaction of ledakrin with
DTT for 30 min. Products P1 and P2 (ions at m/z 293 and 211,
respectively) correspond to products M1 (D1) and M2 (D2),
respectively. Product P3 is an ion at m/z 445.
F igu r e 4. Positive ion ESI-MS/MS spectrum of product M1
(
ion at m/z 293). The mass spectrum shows fragment ions at
m/z 248 and 220 formed by the loss of 45 and 73 mass units,
respectively.
Preliminary experiments have successfully established
2
the activation conditions for ledakrin with DTT (32). The
HPLC analysis (Figure 7) shows that, of the activation
products, two compounds, P1 and P2, were identical to
products M1 and M2 formed in the microsomal fraction
of rat liver and in the presence of reducing-oxidizing
enzymes. The MS spectra were obtained from the samples
taken after reaction for 5 (Figure 8A), 15 (Figure 8B), 30
F igu r e 5. Structure of 1-amino-9-acridinone, the final product
of reduction under the studied conditions.
(Figure 8C), and 60 min (Figure 8D). The MS spectrum
taken after 5 min contains the intensive ion of ledakrin
at m/z 325 and two other ions at m/z 293 and 445 of low
abundance. In the MS spectrum taken after 15 min,
besides the two ions at m/z 293 and 445, an additional
ion at m/z 295 was observed. The third spectrum recorded
after reaction for 30 min consists of previously mentioned
ions at m/z 293, 295, and 445 with one added ion at m/z
4
2
47. Last, after reaction for 60 min, an extra ion at m/z
11 appeared. This product, P2, was isolated, and by
NMR, its structure was determined to be identical to M2,
which was determined above to be 1-amino-9-acridinone
(2).
The product termed P1, identical to products M1, D1,
and O1 formed in enzymatic systems, could not be
isolated due to its instability.
F igu r e 6. RP-HPLC (see Experimental Procedures for condi-
tions) profile of products obtained by incubation of ledakrin with
DT-diaphorase for 30 min. Product D1 (ion at m/z 293) corre-
sponds to product M1 shown in Figure 2, and product D2 (ion
at m/z 211) corresponds to product M2 shown in Figure 2.
However, product P3 (m/z 445) could be isolated from
the reaction mixture and showed a mass difference of 152
from P1 (m/z 293). This suggested that P3 was formed
as a result of nucleophilic substitution of DTT on P1.
Using NMR, the full proton assignment of P3 was made
on the basis of DQF-COSY, ROESY, and TOCSY experi-
ments (see Experimental Procedures for detailed results).
The 13C assignments were based upon HSQC and HMBC
experiments. The results showed that product P3 existed
as two isomers, 3 and 4. Their structures are shown in
Figure 9. The DQF-COSY experiment indicated that the
nucleophilic substitution at position 4 of the acridine core
occurred in compound 3, whereas substitution in com-
pound 4 was at position 2a. The aliphatic region of the
COSY spectrum revealed the presence of both an ami-
noalkyl side chain and a DTT-derived substituent. Ap-
plication of ROESY NMR spectra showed that products
3 and 4 possess a hydrogen atom attached to nitrogen at
position 10. Furthermore, HSQC and HMBC NMR
techniques confirmed the presence of a 9-aminoalkyl side
due to their lack of stability under these conditions, and
the expected products of ledakrin reduction, N-hydroxy-
1
-amino and 1-amino analogues, have yet to be isolated.
Therefore, studies were undertaken on the reductive
transformation of ledakrin with the other reducing
systems in attempts to produce yields large enough for
NMR analysis.
Rea ction of Led a k r in w ith DTT. Like the microso-
mal fraction of rat liver, it was found by 32P-postlabeling
methods that incubation of ledakrin with DTT in the
presence of DNA led to the same adduct patterns as that
obtained in tumor cells (13). Therefore, DTT was chosen
as the first nonenzymatic reducing system for the studies
on ledakrin metabolic activation.
2
L. Szmigiero, personal communication.

6
Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Gorlewska et al.
F igu r e 9. Structures of the two main products formed in the
2
reaction of ledakrin with DTT, N -[3′-(dimethylamino)propyl]-
4
-(1′′,4′′-disulfanyl-2′′,3′′-dihydroxybutane)-1,9-diazane-1,10-di-
2
hydroacridine (P3) (3) and N -[3′-(dimethylamino)propyl]-2-
1′′,4′′-disulfanyl-2′′,3′′-dihydroxybutane)-1,9-diazane-1,10-
(
dihydroacridine (P3a) (4).
F igu r e 10. (A) Positive-ion ESI-MS/MS spectrum of product
P3 (ion at m/z 445). The mass spectrum shows fragment ions
at m/z 400 and 372 formed by the loss of 45 and 73 mass units,
respectively. (B) Ion formation process observed in the ESI-MS/
MS spectrum.
15N spectrum clearly indicate the presence of a double
bond between N1 and C1.
This evidence for a five-membered ring in compounds
3
and 4 clarified the spectral data obtained from the MS
analysis of P3. Namely, the MS/MS spectrum obtained
for P3 (Figure 10A) shows that the fragment ion at m/z
F igu r e 8. Positive ion ESI-MS spectra of the products of
4
00 was formed by loss of dimethylamine from the species
ledakrin reaction with DTT after reaction for (A) 5, (B) 10, (C)
at m/z 445. The driving force for such fragmentation is
the formation of a highly stable fragment ion at m/z 400,
the structure of which is shown in Figure 10B. The
formation of this fragment ion was only possible when
the additional five-membered ring was present in posi-
tions 1 and 9 of the acridine ring.
3
0, and (D) 60 min.
chain and a DTT-derived substituent in positions 4 and
a for compounds 3 and 4, respectively. The spectral data
2
1
5
extracted from N NMR (HSQC and HMBC experi-
ments) were diagnostic for the assignment of the struc-
ture of the additional ring. The chemical shifts of
appropriate nitrogen atoms [N1, -95.5; N1a, -97.9; and
N9, -175] associated with the long-range heteronuclear
connectivity of N1 (N1a) to H1′ (H1a′) and N9 (N9a) to
H2′ (H2a′) revealed the presence of the five-membered
ring. Furthermore, the chemical shift values of N1 in the
In light of the results described above, it is apparent
that the ledakrin activation product M1, formed with the
microsomal fraction of rat liver (Figures 2 and 3),
undergoes loss of the dimethylamine moiety (Figure 4).
The proposed structure of M1 (5) is shown in Figure 11.
On the basis of the HPLC retention time and UV/vis and

Products of Ledakrin Metabolic Activation
Chem. Res. Toxicol., Vol. 14, No. 1, 2001
7
F igu r e 13. Proposed structure of M3 microsome metabolite
2
1
2
8
, N -[3′-(dimethylamino)propyl]-N ,N -diaminomethylacridine.
F igu r e 11. Structure of the key product P1 (M1 and D1), N2-
[
3′-(dimethylamino)propyl]-1,9-diazane-1,10-dihydroacridine (5),
Ta ble 1. Ch em ica l Str u ctu r e Id en tifica tion of th e
P r od u cts F or m ed d u r in g Activa tion of Led a k r in by th e
Micr osom a l F r a ction of Ra t Liver (M), DT-Dia p h or a se
obtained with microsomes, DT-diaphorase, and DTT.
(
D), Xa n th in e Oxid a se (O), a n d DTT (P )
number
symbol name
number
symbol name
1
2
3
4
ledakrin
M2, D2, P2
P3
5
6
7
8
M1, D1, O1, P1
M4
M5
M3
P3a
as a reducing agent of ledakrin. The reduction product
1
of the drug was isolated and analyzed by H NMR and
1
3
C NMR. The assignment of the structure of M5 was
based on the same NMR experiments as presented above
for compound M4. The structure of M5 was proven to be
7
and is presented in Figure 12. The ROESY experiment
showed the presence of a proton on N1 and the methyl
protons on C . The C proton, in turn, also coupled with
H1′, confirming that the aminoalkyl side chain re-
mainded on this structure. The coupling of C to the
b
b
F igu r e 12. Structures of the products formed by reduction of
2
ledakrin with hydrazine hydrate, N -[3′-(dimethylamino)propyl]-
1
2
2
a
2
N ,N -diaminoethylacridine (6), and SnCl , N -[3′-(dimethylami-
1
2
no)propyl]-N ,N -diamino-1′′-methylethylacridine (7), related to
protons on the side chain (C1′) was observed by HMBC.
M4 and M5, respectively, obtained with microsomes (see Figure
Considering that the formation of six-membered ring
structures above was evidence for two products of ledakrin
reduction, 6 and 7, we postulate that product M3 found
in the incubation mixture with microsomes (m/z 307, 14
units lower than that of 6) might also contain the six-
membered ring in its structure. The proposed structure
of M3 (8) is presented in Figure 13.
3
).
MS data, product M1 was identical to product P1 formed
during reduction of ledakrin with DTT. Similarly, the D1
(Figure 6) and O1 (data not shown) products described
above were also identical to the M1 metabolite. Thus,
activation product 5 appeared to be a key product, which
was observed in all the activation systems that were
studied.
Discu ssion
In conclusion, studies on the reaction of ledakrin with
DTT allowed us to obtain the structures of the rat liver
microsomal metabolites, M1 and M2. The structures of
compounds 5 and 2 were determined for M1 and M2,
respectively. It was also found that two compounds
The aim of this work was to characterize the products
from metabolic activation of ledakrin in systems, where
the formation of drug-DNA adducts was previously
demonstrated (13).
We showed that ledakrin easily underwent metabolic
transformations in all studied activation systems. It was
reactive in the presence of the microsomal fraction of rat
liver and with DTT, a chemical reducing agent. Both
activation systems were previously shown to result in
similar patterns of drug-DNA adducts. Ledakrin was
also easily metabolized with oxidoreductase enzymes:
DT-diaphorase and xanthine oxidase. The numbers and
the related symbols of the obtained products are pre-
sented in Table 1. The structures of the identified
metabolites are shown in Scheme 1. The probable path-
way of enzymatic and chemical reduction of ledakrin was
also proposed in Scheme 1.
(3 and 4) are isomeric products from the nucleophilic
substitution occurring with the reductive transformation
of ledakrin by DTT.
R ed u ct ion of Led a k r in b y H yd r a zin e H yd r a t e.
The conditions for the formation of activation product M4
were achieved by reducing ledakrin with hydrazine
hydrate in the presence of Pd/C. Although preliminary
identification of M4 was made with MS (m/z 321),
reduction of ledakrin with hydrazine hydrate provided a
large enough yield of M4 to be analyzed by NMR. DQF-
COSY, ROESY, HSQC, and HMBC experiments were
used to determine the structure of M4 (6) which is shown
in Figure 12. The presence of the additional carbon atom
between N1 and N9 was demonstrated by DQF-COSY
and HMBC techniques. The DQF-COSY spectrum re-
One product of metabolic transformations (5) was
observed in all studied systems. It is an unstable com-
pound obtained as a result of incomplete reduction of
ledakrin. A new heterocyclic ring, formed between nitro-
gen atoms in positions 1 and 9 of the acridine core,
distinguishes the structure of this metabolite from the
structures of metabolites reported for other biologically
active aromatic nitro compounds (20, 21). Furthermore,
in contrast to previously reported products of ledakrin
reduction (23-25), this new product (5) retained the
vealed the correlation of the C
the HMBC technique was able to observe the coupling
signals between C and H1′. The aminoalkyl side chain
a b
H with N1H and C H, and
a
in position 9 was proven by heterocorrelation HMBC
experiments to be a signal between C9 and the ami-
noalkyl aliphatic protons.
Red u ction of Led a k r in by Sn Cl
35) was formed in experiments where SnCl
2
. Product M5 (m/z
was used
3
2

8
Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Sch em e 1. P r op osed P a th w a y for Red u cin g Tr a n sfor m a tion s of Led a k r in a
Gorlewska et al.
a
It was based on the structures determined for the products observed with rat liver microsomes and with chemical reducing agents.
The structures, which are drawn in bold lines, were proven in this paper, whereas the structures of the remaining ones related to the
hypothesized intermediates were not determined. The most possible directions for the attack of cellular nucleo- and electrophiles to ledakrin
metabolites are marked with circle arrows.
aminoalkyl side chain of the parent drug. Product 5
seems to be a key compound of the ledakrin reduction
pathway.
The N-hydroxyamino derivative, which is usually the
common metabolic product of the majority of nitroaro-
matic agents (20, 21), was not isolated in our studies of
ledakrin activation. Nevertheless, the mass ion, m/z 311
oxidation of Tris to one-carbon compounds was also
shown in microsomes (34), and with dithiothreitol and
xanthine oxidase (35). When it is considered that Tris
buffer was applied in our experiments, the source of the
one-carbon element in 8 might have the same origin.
The reactive dihydropyrazoloacridine (5) could also be
the precursor for metabolites 6 and 7. These metabolites
were found during microsomal activation as well as from
chemical reduction of ledakrin. The source of the two-
carbon unit in 6 in the mixture of microsomal enzymes
might be an acetyl group transported by acetyl-CoA. The
three-carbon unit of 7 might originate from the natural
transformation of glucose 6-phosphate to pyruvate, be-
cause the former compound was added to the incubation
mixture as a component of the reducing-oxidizing sys-
tem. In conclusion, the transformation of 5 to 6-8 likely
results from electrophilic attack of the formaldehyde,
acetyl, and puryvate groups, respectively, on the nitrogen
atom of the dihydropyrazoloacridine ring.
It was established that 1-aminoacridinone 2 was the
second-most common product of all the studied reactions.
This product seemed to be the result of exhaustive
reduction of the nitro group and detachment of the
aminoalkyl side chain. However, the appearance of the
acridinone derivative under the studied conditions (pH
7.4 and 37 °C) did not result from the hydrolysis of the
aminoalkyl side chain of ledakrin, which usually occurs
slowly even in boiling acidic medium (36). It was shown
earlier that the origin of the acridinone oxygen atom in
such a reaction was the nitro group of ledakrin (37).
Therefore, the obtained product had to be a result of
intramolecular substitution by the oxygen atom at posi-
tion 9 of the acridine core. Thus, we observed another
(data not shown), which may relate to 1-hydroxyamino
analogue of this drug, b, was observed to persist for a
short time during chemical and microsomal reductions.
On the other hand, incubation of ledakrin with DTT gave
rise to the 1-amino analogue of the drug, c (m/z 295,
Figure 8), which disappeared after incubation for 60 min.
The structure of 5 allowed us to explain why neither the
N-hydroxy-1-amino, b, nor the 1-amino, c, derivative of
ledakrin was isolated from the mixture of the reduction
products. The proximity of nitrogen atoms at positions 1
and 9 of the acridine core seems to make the N-hydroxy
intermediate, b, susceptible to the following cyclization
to the dihydropyrazoloacridine ring, 5, which, in turn,
inhibits the formation of the 1-amino derivative.
Although ledakrin was strongly susceptible to cycliza-
tion under reducing conditions, the resulting product 5,
bearing a five-membered ring added to the acridine core,
also turned out to be the reactive compound, and it was
easily transformed to product 8. The structure of 8 has
not been proved by NMR. However, the fact that product
8
5
exhibited a molecular ion 14 mass units greater than
suggests that metabolite 8 possesses an additional one-
carbon group. It indicates, in turn, that a six-membered
ring was formed between positions 1 and 9. Reactions of
this type were reported earlier as a result of radical
oxidation of Tris to formaldehyde (33). Furthermore,

Products of Ledakrin Metabolic Activation
Chem. Res. Toxicol., Vol. 14, No. 1, 2001
9
case where the “proximity effect” of positions 1 and 9
dictated the exceptional properties of ledakrin.
the case of the N-hydroxy-1-amino derivative of ledakrin
the reaction pathway might be similar. On the other
hand, carbon atoms 2 and 4, activated during metabolic
reduction, might create the C-C bond with the C carbon
atom of guanine as previously reported (44, 45).
The reaction of ledakrin with DTT as a reducing agent
yielded not only the two metabolites, 5 and 2, presented
above but also compounds 3 and 4, which were the
products of substitution by the DTT residue at positions
ortho and para to the nitro group. Therefore, the nucleo-
philic attack of DTT on ledakrin most likely occurred
before the final reduction of the nitro group, or the
reduction and nucleophilic substitution happened simul-
taneously. Had nucleophilic substitution resulted after
the reduction of ledakrin to 5, it would have substituted
into the position meta to the 1-imine group and the
nucleophilic attack would have been much more difficult,
if at all possible.
In summary, the structural studies presented in this
paper have revealed three characteristic chemical prop-
erties of ledakrin, which seem to be crucial for its
exceptional biological activity. First, ledakrin turned out
to be extremely reactive under enzymatic and chemical
reducing conditions. We suggest that this trait results
more from the specific reactivity of the reduced interme-
diate species than from the susceptibility of the 1-nitro
group to reduction. This was based on the fact that the
reduction potential of ledakrin is not significantly lower
than that of other nitroacridines (38, 39). The high
reactivity of the reduction intermediates seems to result
from the proximity of nitrogen atoms in positions 1 and
8
The above discussion indicates that two reactive re-
gions of the ledakrin molecule are able to bind with
guanine. Two bonds might be formed between one
activated molecule of ledakrin and two guanine moieties,
possibly resulting in cross-links between the drug and
DNA. DNA cross-links with ledakrin were discovered
earlier in our laboratory (14) and were postulated to have
a significant impact on the cytotoxic and antitumor action
of this drug (15, 16). Because similar patterns of DNA
adducts were previously observed under reducing condi-
tions in vitro and in the cell (13), we postulate that two
reactive centers of activated ledakrin presented above
might also react with cellular DNA.
Ack n ow led gm en t. This work was supported by the
Committee for Scientific Research (KBN), Poland, via
Grant P05 F01215. We are grateful to Dr. J an Pawlak
for his fruitful discussion and to Andrzej Gorlewicz for
his skillful technical assistance. We also thank Kenneth
P. Roberts for his assistance with the manuscript.
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(
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9
of the acridine core. This proximity, in turn, makes the
9
inhibiting compounds. XXIX. Some N -derivatives of 1-, 2-, 3-, and
intermediate molecule strongly susceptible to intra-
molecular cyclization, giving rise to metabolite 5. Second,
the crucial product (5) was also found to be a reactive
compound as it easily undergoes transformation in the
presence of electrophilic carbon atoms to six-membered
ring products (6-8). The third characteristic chemical
feature of ledakrin relates to its high susceptibility to
nucleophilic substitution para and ortho to the nitro
group.
The specific reduction pathway of ledakrin (Scheme 1)
described above should have a significant impact on the
reaction of this drug with DNA. The formation of inter-
strand cross-links between DNA and the activated form
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(
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(
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(
(
(
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possible reactive sites of ledakrin that might participate
in bond formation with DNA. The first one appeared on
the level of the two-electron reduction intermediate,
nitroso derivative, a. Carbon atoms at position 2 or 4 of
this intermediate are capable of reaction with DNA. The
second reactive site was formed on the reduction level of
four electrons. It is the nitrogen atom of the N-hydroxy-
4
0, 472-477.
(
9) Mazerska, Z., Mazerski, J ., and Led o´ chowski, A. (1990) QSAR of
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(
10) Pawlak, J . W., and Konopa, J . (1979) In vitro binding of metaboli-
1
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1
4
14
cally activated ( C)-Ledakrin, or 1-nitro-9- C-(3′-dimethylamino-
N-propylamino)acridine, a new antitumor and DNA cross-linking
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vated ledakrin molecule are the electrophilic centers.
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obtained, whereas in the presence of DNA, the nucleo-
philic attack of the 2-amino group or the N7 atom of
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action of cytotoxic and antitumor 1-nitroacridines. II. In vitro
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guanine seems to be preferable. The reactions such as
these were demonstrated earlier for carcinogenic com-
2
pounds, where the formation of N -guanyl adducts with
(
12) Szmigiero, L., and Studzian, K. (1989) DNA damage and cyto-
toxicity of nitracrine in cultured HeLa cells. Biochim. Biophys.
Acta 1008, 339-345.
an aromatic carbon atom (40) as well as with a N-
7
hydroxyamino group (41, 42) was shown. The N -guanyl
8
(13) Bartoszek, A., and Konopa, J . (1989) 32P-post-labeling analysis
of DNA adduct formation by antitumor drug Nitracrine (Ledakrin)
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adduct was postulated to be the intermediate of the C -
guanyl adduct, which was observed for activated aryl-
amines (43, 44). In this case, the nitrenium ion formed
from N-hydroxyamine was the reactive species able to
(
14) Konopa, J ., Pawlak, J . W., and Pawlak, K. (1983) The mode of
8
bind with the C atom of guanine. We postulate that in
action of cytotoxic and antitumor 1-nitroacridines. III. In vivo

1
0
Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Gorlewska et al.
interstrand cross-linking of DNA of mammalian or bacterial cells
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(
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(
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interstrand DNA cross-linking. Cancer Res. 44, 4289-4296.
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