2,7-Dihydro-3H-pyridazino[5,4,3-kl]acridin-3-one derivatives, novel type of cytotoxic agents active on multidrug-resistant cell lines. Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2005.01.023

We have earlier postulated that the presence of a pyridazone ring fused with an anthracenedione moiety resulted in the analog's ability to overcome multidrug resistance of tumor cells [J. Med. Chem. 1999, 42, 3494]. High cytotoxic activity of obtained ant

Full text of DOI:10.1016/j.bmc.2005.01.023

Bioorganic & Medicinal Chemistry 13 (2005) 1969–1975
2,7-Dihydro-3H-pyridazino[5,4,3-kl]acridin-3-one derivatives,
novel type of cytotoxic agents active on multidrug-resistant
cell lines. Synthesis and biological evaluation
a
Barbara Stefanska, Maria M. Bontemps-Gracz, Ippolito Antonini, Sante Martelli,
a
b
b
´
Małgorzata Arciemiuk,a Agnieszka Piwkowska,a Dorota Rogackaa and
Edward Borowskia,*
a
´ ´
Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 80-952 Gdansk, Poland
bDepartment of Chemistry, University of Camerino, 62033 Camerino, Italy
Received 21 September 2004; accepted 12 January 2005
Abstract—We have earlier postulated that the presence of a pyridazone ring fused with an anthracenedione moiety resulted in the
analogÕs ability to overcome multidrug resistance of tumor cells [J. Med. Chem. 1999, 42, 3494]. High cytotoxic activity of obtained
anthrapyridazones [Bioorg. Med. Chem. 2003, 11, 561] toward the resistant cell lines, prompted us to synthesize the similarly modi-
fied acridine compounds. A series of pyridazinoacridin-3-one derivatives (2b–h) were prepared from the reaction of 9-oxo-9,10-
dihydroacridine-1-carboxylate with POCl3, followed by addition of the appropriate (alkylamino)alkylhydrazines. In vitro cytotoxic
activity toward sensitive and resistant leukemia cell lines: L1210, K562, K562/DX, HL-60, HL-60/VINC, and HL-60/DX, with var-
ious type of multidrug resistance (MDR and MRP) was determined. The compounds studied exhibited in comparison to the refer-
ence cytostatics (DX, MIT) desirable very low resistance indexes (RI). Variations have been observed depending upon the
substituent and the type of drug exporting pump. The cytotoxic activities of examined compounds, as well as of model anthrapyri-
dazone derivative PDZ, were lower than those of reference drugs (DX, MIT) due to their diminished affinity to DNA.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ular antitumor agents of anthraquinone and acridine
groups. We have earlier postulated that the essential
structural factor in anthracenedione and acridine cyto-
statics, enabling the compounds to overcome multidrug
resistance of tumor cells, is the presence of a fused five
or six-membered heterocyclic ring(s) as part of the mole-
cule.4–8 We have evidenced that the mechanism of
this effect is based on the increased rate of diffusive drug
influx surpassing its export mediated by efflux pumps in
the multidrug-resistant tumor cells.9–11 Some of these
analogs of the anthraquinone family are anthrapyridaz-
ones (1), characterized by the presence of an additional
pyridazone ring fused to the anthracenedione moiety
(Fig. 1).5 These compounds exhibit good activity on
multidrug-resistance tumor cell lines as of yet, the analo-
gous tetracyclic acridine compounds have not been
prepared. Known acridine polycyclic analogs active on
multidrug-resistant tumor cell lines comprise com-
pounds with fused pyrazole and dihydropyrimidine
rings in tetracyclic and pentacyclic systems.7,8 Now, we
report the synthesis and the biological evaluation of
The prolonged use of antitumor agents often leads to the
appearance of cell populations, which are crossresistant
to many cytostatic agents.1–3 This effect known as mul-
tidrug resistance has recently become a major cause of
failures in antitumor chemotherapy. This phenomenon
depends on the overexpression of plasma membrane
drug efflux pumps. Many efforts have been directed to-
ward the design of new cytotoxic agents with the ability
to overcome multidrug resistance, including rather pop-
Abbreviations: MIT, Mitoxantrone; DX, Doxorubicin; VINC, Vin-
cristine; PDZ, 6-chloro-2-[2-(dimethylamino)ethyl]-2,7-dihydro-3H-
dibenzo[de,h]-cinnoline-3,7-dione; MDR, P-gp dependent multidrug
resistance; MRP, multidrug resistance associated protein dependent
resistance.
Keywords: Antitumor compounds; Acridine analogs; Cytotoxic activ-
ity; Multidrug resistance.
*
Corresponding author. Tel.: +48 58 347 2523; fax: +48 58 347
1144; e-mail: borowski@altis.chem.pg.gda.pl
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.023

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B. Stefanska et al. / Bioorg. Med. Chem. 13 (2005) 1969–1975
1970
R1
N
R
for two major types of drug exporting pumps, MDR
O
N
2
O
(P-gp dependent multidrug resistance) and MRP (mul-
tidrug resistance associated protein dependent resis-
tance). The applied panel was comprised of leukemia
cell lines: sensitive murine L1210 and human K562
and HL-60, as well as resistant sublines K562/DX
(MDR-type resistance), HL-60/VINC (MDR-type resis-
tance), and HL-60/DX (MRP-type resistance). For the
examined compounds the DNA binding test was
performed.
N
N
N
6
O
1
H
2
NHR2
X
Figure 1. The structures of anthrapyridazones (1) and pyridazoacr-
idines (2).
pyridazinoacridines (2), in which an acridine moiety is
fused with the pyridazone ring to form a tetracyclic
system (Fig. 1). The pyridazone ring is substituted, as
in active anthrapyridazones, at position 2 with different
(alkylamino)alkyl side chains. Also an analog with
a chlorine atom at the C6 has been synthesized. The
2. Chemistry
Schemes 1 and 2 show the applied synthetic pathways.
The 2-[3-(carboxy)anilino]benzoic acid 3 is most conve-
niently prepared by the Jourdan–Ullmann condensation
of 2-chlorobenzoic acid and 3-aminobenzoic acid.12
9-Oxo-9,10-dihydro-1-acridinecarboxylic 4a and 9-oxo-
9,10-dihydro-3-acridinecarboxylic acids 4b were pre-
pared together as an isomeric mixture from 3 by acid
catalyzed cyclization with concentrated H2SO4. An ini-
tial attempt to separate the two isomers by recrystalliza-
tion was rather tedious because of their insolubility
properties. Therefore, to facilitate the separation of iso-
model,
analogous
anthrapyridazone
compound
(PDZ), was also used in these studies for comparative
purposes.
Pyridazinoacridin-3-one derivatives (2b–g) were evalu-
ated for their in vitro cytotoxic potency toward sensitive
and multidrug resistant tumor cell lines representative
COOH
O
COOH
COOH
COOH
(a)
(b)
+
N
H
N
Cl
H2N
COOH
H
3
4a,4b
R
N
O
COOC2H5
O
O
N
(c)
(d)
(e)
N
H
N
H
N
H
COOC2H5
5a,5b
5a
2a-2f
Scheme 1. Synthetic route for compounds 2a–f. Reagents and conditions: (a) Cu powder, K2CO3, i-C5H11OH, reflux/3 h; (b) concd H2SO4, 100 ꢁC/
1 h; (c) EtOH/dry HCl, reflux/4 h; (d) chromatography on silica gel; (e) for 2a: NH2NH2xH2O, 125 ꢁC/6 h; for 2b–f: 1. POCl3, 125 ꢁC/1 h; 2.
NH2NHR, 125 ꢁC/2.5–3 h.
COOCH3
COOCH3
COOCH3
(a)
O
COOH
COOH
Cl
(b)
+
H2N
N
H
6
N
H
Cl
Cl
Cl
7
R
N
O
N
(c)
N
H
Cl
2g-h
Scheme 2. Synthetic route for compounds 2g–h. Reagents and conditions: (a) N-methylpyrrolidinone and N,N-diisopropylethylamine,
Cu(OAc)2xH2O, 130 ꢁC/3 h; (b) PPE, 100 ꢁC/1 h; (c) for 2g: 1. POCl3, reflux/0.5 h, 2. NH2NH(CH2)3N(CH3)2, DMA, 130 ꢁC/3 h; for 2h:
NH2NH2xH2O, DMA, 125 ꢁC/2 h.

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B. Stefanska et al. / Bioorg. Med. Chem. 13 (2005) 1969–1975
1971
Table 1. Structure and characteristics of compounds 2b–g
R
N
O
N
N
H
X
Compound
R
X
Yield (%)
Mp (ꢁC)
Molecular formula
2b
2c
2d
2e
2f
CH2CH2N(CH3)2
H
H
H
H
H
Cl
45
40
51
43
35
25
258–259
251–253
248–250
236–238
231–233
185–186
C18H18N4O
C19H20N4O
C20H22N4O
C20H20N4O2
C21H22N4O
C19H19N4OCl
CH2CH2CH2N(CH3)2
CH2CH2N(CH2CH3)2
CH2CH2-c-N(CH2)4O
CH2CH2-c-N(CH2)5
CH2CH2CH2N(CH3)2
2g
mers, isomeric mixture of 4a and 4b was esterified with
acidic ethanol under anhydrous conditions. The isomer
esters were separated by chromatography with silica
gel as adsorbent using CHCl3/MeOH as eluent to give
5a and 5b.
(DX), Mitoxantrone (MIT), and representative anthra-
pyridazone closely related to 2g, the 6-chloro-2-[2-(di-
methylamino)ethyl]-2,7-dihydro-3H-dibenzo[de,h]-cinno-
line-3,7-dione (PDZ),5 were used as reference
compounds.
Compound 2a (R = H) was obtained by prolonged heat-
ing of 5a with hydrazine by a procedure similar to that
described for the synthesis of anthrapyridazones.5 How-
ever, this procedure failed to give the 2-N-alkyl deriva-
tives of the pyridazinoacridin-3-one chromophore.
Therefore, a different procedure was required for pro-
ducing compounds 2b–f from 5a. Compound 5a was
dissolved in POCl3 and heated to reflux to afford the
9-chloroderivative, which was immediately treated with
the respective (alkylamino)alkylhydrazine.
All compounds tested, depending on their structure,
exhibited varied cytotoxic activities on three sensitive
cell lines (L1210, K562, HL-60). These activities, includ-
ing those of PDZ, is generally lower than those of refer-
ence compounds DX and MIT. Cytotoxic activity of
compounds are influenced by the type of side chain.
The highest activity was recorded for compounds with
terminal dimethylamino moiety (2c, 2b). The length of
alkyl moiety (two or three methylene groups; com-
pounds 2b and 2c, respectively) has no significant influ-
ence. The lowest activity is exhibited by compounds 2d
and 2f with terminal N-diethyl and piperydyl moieties,
respectively. Somewhat higher activity is exhibited by
2e, bearing terminal morpholine moiety in the side
chain. The replacement of hydrogen atom by chlorine
(2g vs 2e) does not seem to have an effect on cytotoxic-
ity. We did not find any essential correlation between
the cytotoxic activity of examined tetracyclic com-
pounds and DNA binding measured by ethidium bro-
mide displacement method. The essential difference
was observed only between these compounds and highly
active reference DX and MIT.
Compound 2g, the 6-chloro analog of 2c, was prepared
from 7 under similar reaction conditions described for
the preparation of 2b–f. The last one was prepared in
the following way: 2-chlorobenzoic acid and methyl-3-
amino-4-chlorocarboxylate were condensed in a modi-
fied Jourdan–Ullmann reaction13 to give the half ester
6, followed by ring closure with polyphosphate ester to
afford compound 7. Finally, cyclization was achieved
with hydrazine to give 2h.
Attempts to substitute the chlorine atom at position 6
with an (aminoalkyl)amino nucleophile in compound
2g or 2h under various conditions were unsuccessful.
The novel group of pyridazinoacridines, however of
rather low cytotoxic activity against sensitive cell lines,
was designed to investigate the effect of structural fac-
tors of tetracyclic acridine analogs on their ability to
overcome multidrug resistance of tumor cells. In this re-
spect the dramatic improvement of cytotoxicity against
resistant K562/DX, HL-60/VINC, and HL-60/DX cell
lines was noticed in comparison with the activity of ref-
erence DX and MIT. It is of importance that these novel
compounds are able to overcome MDR, as well as,
MRP types of resistance. However, the activity against
the MRP cell line is somewhat lower. The cytotoxic
activity of tested pyridazinoacridines toward cells with
MDR-type resistance is not significantly influenced by
modification of substituents. The resistance indexes are
within range ca. 1–4. Much more interesting is the
activity of these compounds toward tumor cells with
The structure, molecular formulas, and melting points
of compounds 2b–g are presented in Table 1. For bio-
logical evaluations, the free bases were converted into
the hydrochloride salts.
3. Results and discussion
Tetracyclic anthraquinone analogs with fused pyridaz-
one ring (anthrapyridazones) are able to overcome mul-
tidrug resistance of tumor cells.5 Presently we have
synthesized and examined a group of analogous tetracy-
clic acridine analogs (pyridazinoacridines). The results
of cytotoxic activities and DNA binding data for com-
pounds 2b–g are presented in Table 2. Doxorubicin

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B. Stefanska et al. / Bioorg. Med. Chem. 13 (2005) 1969–1975
1972
Table 2. Cytotoxic activity in vitro in a panel of sensitive and resistant leukemia cell lines and DNA binding of compounds 2b–g, in comparison with
reference compounds: PDZ, Doxorubicin (DX), and Mitoxantrone (MIT)
Compound
Tested
2e
Reference
DX
2b
2c
2d
2f
2g
PDZa
MIT
Cytotoxic activity
Cell lineb
L1210
IC50 (nM) SEMd/RIe
c
48
4
39
5
398 54
1189 69
93 13
380 64
574 79
300 23
960 44
2294 211
2.39
15
1
839 40
2018 373
4140 388
2.05
19
42
1
3
1.4 0.1
21
554 50
26.4
K562
K562/DX
RI
308 68
256 16
0.83
234 20
178 24
320 36
451 62
1.41
4
2188 313
1.84
7031 542
167
0.76
73
255 15
3.49
1.51
91
407 20
4.47
HL-60
HL-60/VINC
RI
79
245
3.10
2
7
4
1484 36
3097 185
2.09
4
755 59
1419 89
1.88
49
110
2.24
3
9
666 69
472 39
0.71
18
523 33
29.0
1
2.2 0.3
56.5 4.2
25.7
HL-60/DX
RI
931 54
11.78
1035 53
14.2
3801 364
2.56
1061 85
11.7
2159 129
2.86
744 76
15.2
710 98
1.07
3869 59
215
1179 203
536
DNA binding
f
C50 (lM)
Kapp
2.95
0.43
3.75
0.34
6.24
0.20
3.12
0.40
2.92
0.43
2.83
0.44
1.76
0.72
0.08
15.00
0.9933
0.05
25.20
0.9677
g
R2h
0.9939
0.9935
0.9936
0.9895
0.9812
0.9797
0.9850
a 6-Chloro-2-[2-(dimethylamino)ethyl]-2,7-dihydro-3H-dibenzo[de,h]-cinnoline-3,7-dione.5
b L1210—murine lymphocytic leukemia; K562—human myelogenous leukemia and Doxorubicin resistant (MDR type) subline K562/DX; HL60—
human promyelocytic leukemia, and Vincristine resistant (MDR type) subline HL-60/VINC, and Doxorubicin resistant (MRP type) subline HL-60/
DX.
c IC50—the concentration of compound causing 50% inhibition of cell growth after 48 h (L1210) or 72 h (other lines) continuous exposure.
d SEM—standard error of the mean.
e RI—resistance index; the ratio of IC50 value for resistant cell line to IC50 value for sensitive cell line.
f C50—the micromolar drug concentration of compound to give a 50% drop in fluorescence of ethidium bound to DNA.
g Kapp (·10À7 MÀ1)—apparent equilibrium binding constant.
h R2—curvefit coefficient.
MRP-type resistance. In this case the effect of structure
on RI value is much more expressed. The compounds 2d
and 2f, as more lypophilic, exhibited the highest activity
(lowest RI). These compounds exhibit most dramatic
differences in the ability to overcome MRP-type resis-
tance in regard to reference DX and MIT.
spectra were recorded on a UR 10 Zeiss spectrometer
from KBr pellets; H NMR spectra were taken on a
1
Varian 300-MHz or 500-MHz spectrometer using tetra-
methylsilane as an internal standard. The following
NMR abbreviations are used: ex (exchangeable with
D2O), d ex (exchangeable with D2O, but with difficulty).
MS spectra were recorded on a Quadrupolic Mass Spec-
trophotometer Trio-3 (FAB technique). Thin layer chro-
matography (TLC) was carried out on silica gel
(Kieselgel 60 plates, Merck), column chromatography
on silica gel (Kieselgel 70–230 mesh Merck, and Sepha-
dex LH-20, Pharmacia).
Comparison of pyridazinoacridines with analogous
anthrapyridazones concerning their ability to overcome
multidrug resistance of tumor cells (PDZ vs 2g) point to
the latter group as more effective in this respect.
The results obtained broaden our knowledge on
cytotoxic activity of acridine compounds with fused
heterocyclic rings and bring new data supporting our
earlier hypothesis4 that anthraquinone and acridine ana-
logs with fused heterocyclic rings, when properly substi-
tuted, are able to overcome multidrug resistance of
tumor cells.
4.2. Typical procedure for the preparation of hydrochlo-
ride salt of 2b–g
To a stirred solution of the compound (as free base) in
CHCl3 or CHCl3/MeOH, a slightly molar excess of
hydrogen chloride in absolute ethanol was added drop-
wise at 5 ꢁC. Anhydrous diethyl ether was added and a
precipitate formed, which was collected by filtration.
The solid was purified by column chromatography
(Sephadex LH-20) eluting with MeOH and crystallized
from MeOH/Et2O.
4. Experimental
4.1. Chemistry
Melting points were determined with a Boeticus
PHMK05 apparatus and are uncorrected. Combustion
analyses are within 0.4% of the theoretical values and
were carried out on a Carlo Erba CHNS-O-EA1108
instrument for C, H, N. A Beckman 3600 spectropho-
tometer was used for UV spectral determination. IR
4.3. Ethyl 9-oxo-9,10-dihydro-acridine-1-carboxylate (5a)
A suspension of 2-chlorobenzoic acid (7.8 g, 50 mmol),
3-aminobenzoic acid (6.8 g, 50 mmol), and K2CO3
(7 g, 60.6 mmol) in isopentyl alcohol (150 mL) was stir-
red at reflux point for 0.5 h during which time one-tenth

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B. Stefanska et al. / Bioorg. Med. Chem. 13 (2005) 1969–1975
1973
of the solvent was allowed to boil off. Copper powder
4.5. Methyl 4-chloro-9-oxo-9,10-dihydro-acridine-1-car-
boxylate (7)
(0.25 g, 3.9 mmol) was added and then the reaction mix-
ture was heated at reflux for 3 h. After that time 1 M
aqueous K2CO3 (100 mL) was added and then the
reaction mixture was filtered to remove insoluble
material. The aqueous layer was separated and acidified
to pH 5 with 2 N HCl to form a precipitate, which
was filtered, suspended in boiling water (200 mL),
filtered again, and washed with EtOH to give crude
product 3 (60% yield), which was used for the next
step.
A sample of 6 (1 g, 3.3 mmol) and PPE (polyphosphate
ester) (40 g) in CHCl3 (50 mL) were stirred at reflux until
all solid was dissolved. The CHCl3 was allowed to evap-
orate to give oil, which was heated for 1 h at 100 ꢁC. The
cooled mixture was diluted with water and basified to
pH 9. The insoluble product was collected, washed with
water, dried and crystallized from EtOH/ether. Yield
55%. Mp 228–229 ꢁC.
A sample of 3 (2.6 g, 10 mmol) was heated with concen-
trated H2SO4 (10 mL) for 1 h at 90 ꢁC. The mixture was
poured on ice. A precipitate formed, which was filtered
and washed with hot water, followed with hot MeOH
to give the crude isomeric mixture of 4a and 4b (yield
85%). The isomeric mixture was suspended in EtOH sat-
urated with dry HCl (150 mL) and refluxed for 4 h. The
reaction mixture was concentrated in vacuo and then the
residue was poured into a cold excess aqueous ammonia
solution to provide a precipitate, which was filtered,
washed with water, and dried (yield 95%). The isomeric
mixture (ca. 1.8:1 ratio of 5a:5b) was chromatographed
three times on silica gel using CHCl3/MeOH (10:1, v/v;
10:1, v/v; 5:1,v/v) as eluent.
1H NMR (DMSO-d6): d 3.84 (s, 3H); 7.19 (d, 1H,
J = 7.3 Hz); 7.35 (t, 1H, J = 7.5 Hz); 7.8 (t, 1H,
J = 7.2 Hz); 7.97 (d, 1H, J = 7.3 Hz); 8.06 (d, 1H, J =
8.4 Hz); 8.16 (dd, 1H, J = 8.1 Hz, J = 1.1 Hz); 11.2 (s,
1H, d ex).
4.6. 2,7-Dihydro-3H-pyridazino[5,4,3-kl]acridin-3-one
(2a)
To a solution of 5a (0.26 g, 1 mmol) in DMA (3 mL)
hydrazine hydrate (NH2NH2xH2O) (3 mL) was added
and the reaction mixture was heated for 6 h at 125 ꢁC.
The mixture was neutralized with dilute HCl to form a
precipitate, which was filtered, washed with acetone,
and crystallized from DMF/acetone to give 2a as a light
yellow powder (yield 30%). Mp >300 ꢁC.
Compound 5a was isolated as a pale brown solid with
strong fluorescence at 366 nm. Mp 218–219 ꢁC. 1H
NMR (DMSO-d6): d 1.25 (t, 3H, J = 3.5 Hz); 4.28 (q,
2H, J = 3.5 Hz); 7.15 (dd, 1H, J = 6.8 Hz, J = 1.1 Hz);
7.28 (t, 1H, J = 7.0 Hz); 7.56 (d, 1H, J = 8.0 Hz); 7.68
(dd, 1H, J = 9.0 Hz, J = 1.1 Hz); 7.68–7.8 (m,
2H); 8.18 (dd, 1H, J = 8.0 Hz, J = 1.1 Hz); 11.95 (s,
1H, ex).
1H NMR (DMSO-d6): d 7.08 (t, 1H, J = 7.3 Hz); 7.16 (d,
1H, J = 7.3 Hz); 7.3 (d, 1H, J = 7.8 Hz); 7.42 (t, 1H,
J = 7.5 Hz); 7.50 (d, 1H, J = 7.3 Hz); 7.70 (t, 1H,
J = 8.2 Hz); 8.04 (d, 1H, J = 7.8 Hz); 10.75 (s, 1H, d
ex); 12.23 (s, 1H, d ex). MS m/z (relative intensity, %):
235 [(M)+, 100].
The faster eluting compound 5b is a yellow solid. Mp
301–302 ꢁC. 1H NMR (DMSO-d6): d 1.65 (t, 3H,
J = 3.5 Hz); 4.39 (q, 2H, J = 3.5 Hz); 7.31 (t, 1H,
J = 3.6 Hz); 7.55 (d, 1H, J = 4.0 Hz); 7.71–7.86 (m,
2H); 8.21 (d, 1H, J = 2.5 Hz); 8.26 (s, 1H); 8.33 (1H,
d, J = 4 Hz); 12.0 (s, 1H, ex).
4.7. 2-[3-(Dimethylamino)ethyl]-2,7-dihydro-3H-pyridaz-
ino[5,4,3-kl]acridin-3-one (2b)
A sample of 5a (0.26 g, 1 mmol) was refluxed in POCl3
(2 mL) for 0.5 h. The reaction mixture was evaporated
in vacuo to give an oily residue, which was dissolved
in DMA (2 mL). An excess of 2-(dimethylamino)ethyl-
hydrazine (0.5 mL) was added dropwise and the solution
of reagents were heated for 3 h at 130 ꢁC. The reaction
mixture was diluted with CHCl3 and washed carefully
with water. The organic layer was dried over Na2SO4,
evaporated in vacuo and the crude product purified by
column chromatography (eluent CHCl3/MeOH 5:1) to
afford 2b as pale yellow powder (yield 45%). Mp 258–
259 ꢁC (xHCl 293–295 ꢁC).
4.4. 3-(2-Carboxy-phenylamino)-4-chlorobenzoic acid
methyl ester (6)
A
suspension of methyl 3-amino-4-chlorobenzoate
(1.6 g, 8.6 mmol), 2-chlorobenzoic acid (1.3 g, 8.6 mmol)
and Cu(OAc)xH2O (1.7 g, 8.6 mmol) in a mixture of
methyl-2-pyrrolidinone (10 mL) and N,N-diisopropyl-
ethylamine (20 mL) was stirred for 3 h at 130 ꢁC. After
cooling, dilution with water and acidification to pH 2,
the precipitate was washed with hot water and chro-
matographed (eluent CHCl3/MeOH 15:1) to give 6
(yield 52%). Mp 216 ꢁC.
1H NMR (DMSO-d6): d 2.20 (s, 6H, 2 · CH3); 2.69 (t,
2H, J = 6.5 Hz); 4.29 (t, 2H, J = 6.5 Hz); 7.09 (t, 1H, J =
7.3 Hz); 7.17 (d, 1H, J = 8.3 Hz); 7.29 (d, 1H, J = 8.3
Hz); 7.43 (t, 1H, J = 7.5 Hz); 7.51 (d, 1H, J = 7.3 Hz);
7.70 (t, 1H, J = 8.0 Hz); 8.07 (d, 1H, J = 7.8); 10.83
(s, 1H, d ex). MS m/z (relative intensity, %): 306
[(M)+, 100]. Found: C, 70.26; H, 5.83; N, 18.01. Calcd
for C18H18N4O: C, 70.57; H, 5.92; N, 18.29.
1H NMR (DMSO-d6): d 3.82 (s, 3H); 6.95 (t, 1H,
J = 7.0 Hz); 7.28 (d, 1H, J = 8.0 Hz); 7.48 (d, 1H,
J = 8.5 Hz); 7.56 (dd, 1H, J = 8.3 Hz, J = 1.8 Hz); 7.66
(d, 1H, J = 6.5 Hz); 7.97 (dd, 1H, J = 8.0 Hz,
J = 1.5 Hz); 8.02 (d, 1H, J = 8.0 Hz); 9.95 (s, 1H, ex);
13.2 (s, 1H, ex).
Compounds 2c–g were prepared by the procedure de-
scribed for the preparation of 2b using the appropriate

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B. Stefanska et al. / Bioorg. Med. Chem. 13 (2005) 1969–1975
1974
(alkylamino)alkylhydrazines. Compounds 2c–f and 2g
were prepared from 5a and 7, respectively.
J = 8.2 Hz); 7.85 (dd, 1H, J = 8.2 Hz, J = 1.1 Hz); 8.09
(d, 1H, J = 8.2 Hz); 10.17 (s, 1H, ex). Found: C, 64.25;
H, 5.42; N, 15.71. Calcd C19H19N4OCl for: C, 64.31;
H, 5.40; N, 15.79.
4.8. 2-[3-(Dimethylamino)propyl]-2,7-dihydro-3H-pyr-
idazino[5,4,3-kl]acridin-3-one (2c)
4.13. 6-Chloro-2,7-dihydro-3H-pyridazino[5,4,3-kl]acri-
din-3-one (2h)
1
Yield 40%. Mp 251–253 ꢁC (xHCl 271–272 ꢁC dec). H
NMR (DMSO-d6): d 1.89 (m, 2H); 2.15 (s, 6H,
2 · CH3); 2.28 (t, 2H, J = 7.0 Hz); 4.12 (t, 2H, J =
7.0 Hz); 7.07 (t, 1H, J = 7.3 Hz); 7.15 (d, 1H, J = 8.3
Hz); 7.27 (d, 1H, J = 8.3 Hz); 7.40 (t, 1H, J = 7.5 Hz);
7.49 (d, 1H, J = 7.3 Hz); 7.68 (t, 1H, J = 8.1 Hz); 8.05
(d, 1H, J = 7.7); 10.80 (s, 1H, ex). Found: C, 70.92; H,
6.14; N, 17.26. Calcd for C19H20N4O: C, 71.23; H,
6.29; N, 17.49.
Compound 2h was prepared from 7 and NH2NH2xH2O
(98%) by the procedure described for the preparation of
2b and was worked up by the procedure described for
the preparation of 2a. Yield 55%. Mp >300 ꢁC. 1H
NMR (DMSO-d6): d 7.15 (t, 1H, J = 7.5 Hz); 7.45 (t,
1H, J = 7.5 Hz); 7.53 (d, 1H, J = 8.0 Hz); 7.66 (d, 1H,
J = 8.0 Hz); 7.84 (d, 1H, J = 8.0 Hz); 8.06 (d, 1H,
J = 8.0 Hz); 10.0 (s, 1H, ex); 12.45 (s, 1H, d ex).
4.9. 2-[2-(Diethylamino)ethyl]-2,7-dihydro-3H-pyridaz-
ino[5,4,3-kl]acridin-3-one (2d)
5. Biophysical and biological evaluation
5.1. Fluorescence binding studies
1
Yield 51%. Mp 248–250 ꢁC (xHCl 273–275 ꢁC dec). H
NMR (DMSO-d6): d 1.12 (t, 6H, J = 3.5 Hz); 2.60–
2.75 (m, 6H); 4.25 (t, 2H, J = 6.5 Hz); 7.09 (t, 1H, J =
7.3 Hz); 7.15 (d, 1H, J = 8.3 Hz); 7.28 (d, 1H, J = 8.3
Hz); 7.39 (d, 1H, J = 7.5 Hz); 7.50 (t, 1H, J = 7.3 Hz);
7.70 (t, 1H, J = 8.0 Hz); 8.06 (d, 1H, J = 7.9 Hz); 10.81
(s, 1H, ex). Found: C, 71.47; H, 6.53; N, 16.48. Calcd
for C20H22N4O: C, 71.83; H, 6.63; N, 16.75.
The fluorometric assays were performed as described
previously.14 The C50 values for ethidium displacement
from CT-DNA were determined using aqueous buffer
(10 mM Na2HPO4, 10 mM Na2HPO4, 1 mM EDTA,
pH 7.0, room temperature) containing 1.26 lM ethi-
dium bromide and 1 lM CT-DNA (type II, Sigma).14–16
Examined compounds were added to this solution
to yield several concentrations. Measurements were
made using a PerkinElmer LS55 instrument (excitation
at 546 nm; emission at 595 nm). The C50 values are de-
fined as the drug concentrations, which reduce the fluo-
rescence of the DNA-bound ethidium by 50%. Data
from three independent determinations were fitted to
second polynomial order curves or linear curve in the
case of MIT by Microsoft Excel Program, and C50 val-
ues were calculated. Apparent equilibrium binding con-
stants were calculated from the C50 (in lM) values using
the following equation: Kapp = (1.26/C50) · Kethidium
where Kethidium = 107 MÀ1 for ethidium bromide15 and
1.26 is concentration (in lM) of ethidium in ethidium–
DNA complex.
4.10. 2-(2-Morpholinoethyl)-2,7-dihydro-3H-pyridaz-
ino[5,4,3-kl]acridin-3-one (2e)
1
Yield 43%. Mp 236–238 ꢁC (xHCl 260–262 ꢁC dec). H
NMR (DMSO-d6): d 2.6 (4H, t, J = 4.6 Hz); 2.75 (4H,
t, J = 6.3 Hz); 3.24 (2H, t, J = 5.8 Hz); 4.5 (2H, t,
J = 5.8 Hz); 7.10 (t, 1H, J = 7.2 Hz); 7.15 (d, 1H,
J = 8.2 Hz); 7.27 (d, 1H, J = 8.3 Hz); 7.40 (t, 1H, J =
7.5 Hz); 7.50 (d, 1H, J = 7.2 Hz); 7.68 (t, 1H,
J = 8.0 Hz); 8.08 (d, 1H, J = 7.9 Hz); 10.86 (s, 1H, ex).
Found: C, 68.75; H, 5.81; N, 16.07. Calcd for
C20H20N4O2: C, 68.95; H, 5.79; N 16.08.
4.11. 2-(2-Piperidinoethyl)-2,7-dihydro-3H-pyridaz-
ino[5,4,3-kl]acridin-3-one (2f)
1
Yield 35%. Mp 231–233 ꢁC (xHCl 225–227 ꢁC dec). H
5.2. Biological evaluation
NMR (DMSO-d6): d 1.38–1.62 (m, 6H); 2.47 (m, 4H);
2.70 (t, 2H, J = 6.5 Hz); 4.30 (t, 2H, J = 6.5 Hz); 7.12
(t, 1H, J = 7.3 Hz); 7.17 (d, 1H, J = 8.3 Hz); 7.27 (d,
1H, J = 8.5 Hz); 7.45 (t, 1H, J = 7.5 Hz); 7.55 (d, 1H,
J = 7.4 Hz); 7.72 (t, 1H, J = 8.0 Hz); 8.10 (d, 1H,
J = 7.9 Hz); 10.78 (s, 1H, ex). Found: C, 72.50; H,
6.45; N, 16.01. Calcd for C21H22N4O: C, 72.81; H,
6.40; N, 16.17.
5.2.1. Cell lines. Murine L1210 lymphocytic leukemia
cells were grown in RPMI 1640 medium supplemented
with 5% FBS (foetal bovine serum), penicillin G
(100,000 units/L), and streptomycin (100 mg/L). Human
myelogenous leukemia sensitive cell line K562 and
Doxorubicin resistant subline K562/DX (ICIG, Ville-
juif, France) were grown in RPMI 1640 medium supple-
mented with 10% FBS, penicillin G (100,000 units/L),
streptomycin (100 mg/L), and 2 mM L-glutamine. Rese-
lection of the resistant cell lines was performed once a
month by exposure to 500 nM Doxorubicin. Human
promyelocytic leukemia sensitive cell line HL-60 and
resistant sublines: Vincristine resistant HL-60/VINC
and Doxorubicin resistant HL-60/DX (Kansas State
University, Manhattan, KS, USA), were grown in
RPMI 1640 medium supplemented with 10% FBS peni-
4.12. 6-Chloro-2-[2-(dimethylamino)propyl]-2,7-dihydro-
3H-pyridazino[5,4,3-kl]acridin-3-one (2g)
1
Yield 25%. Mp 185–186 ꢁC (xHCl 257–258 ꢁC dec). H
NMR (DMSO-d6): d 1.92 (m, 2H); 2.18 (s, 6H,
2 · CH3); 2.68 (t, 2H, J = 6.5 Hz); 4.27 (t, 2H,
J = 6.5 Hz); 7.16 (t, 1H, J = 7.5 Hz); 7.45 (t, 1H,
J = 7.5 Hz); 7.54 (d, 1H, J = 8.3 Hz); 7.68 (d, 1H,

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B. Stefanska et al. / Bioorg. Med. Chem. 13 (2005) 1969–1975
1975
cillin G (100,000 units/L), streptomycin (100 mg/L).
References and notes
Reselection of the resistant cell lines was performed once
a month by exposure to 200 nM Doxorubicin and
1 lM vincristine for HL-60/DX and HL-6-/VINC,
respectively. Cell lines were grown in a controlled (air–
5% CO2) humidified atmosphere at 37 ꢁC and were
transplanted three times a week. For the experiments
the cells in logarithmic growth were suspended in the
growth medium to give a final required density.
The resistant cell lines were maintained without the
reselection drugs at least one week before the
experiments.
1. Dicato, M.; Duhem, C.; Pauly, M.; Ries, F. Cytokines
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3. van den Heuvel-Eibrink, M. M.; Sonneveld, P.; Pieters, R.
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5.2.2. In vitro cytotoxic evaluation. Cells of required den-
sity were seeded and different concentrations of the
drugs were added. The experiments were carried out in
a controlled (air–5% CO2) humidified atmosphere at
37 ꢁC. The exposure times were: 48 h for L1210 cells
and 72 h for other cell lines. The cytotoxic activity
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vitro concentrations causing 50% inhibition of cell
growth after continuous exposure to the drug, as mea-
sured by cell counting with Z2 Cell Analyzer (Beckman
Coulter) or by the protein content of the cells as de-
scribed previously.17 Results are given as the mean of
at least three independent experiments standard error
of the mean (SEM). The resistance index was defined as
the ratio of IC50 value for resistant cell line to IC50 value
for sensitive cell line.
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12. Albert, A. The Acridines, 2nd ed.; Edward Arnold:
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13. Rewcastle, G. W.; Denny, W. A. Synthesis 1985, 220.
14. McConnaughie, A. W.; Jenkins, T. C. J. Med. Chem.
1995, 38, 3488.
15. Morgan, A. R.; Lee, J. S.; Pulleyblank, D. E.; Murray, N.
L.; Evans, D. H. Nucleic Acids Res. 1979, 7, 547.
16. Baguley, B. C.; Denny, W. A.; Atwell, G. J.; Cain, B. F. J.
Med. Chem. 1981, 24, 170.
Acknowledgements
The authors acknowledge the financial support of these
studies by the State Committee for Scientific Research
(KBN), Warsaw, Poland (grant nos. 4 P05F 035 19
and 3 P05F 003 24), and in part by Italian Ministry of
´
Foreign Affairs and the Chemical Faculty Gdansk Uni-
versity of Technology, Poland.
17. Bontemps-Gracz, M. M.; Milewski, S.; Borowski, E. Acta
Biochim. Pol. 1991, 38, 229.