5-[ω-aminoalkyl)amino]-4-[N-(ω-aminoalkyl)carbamoyl]-9-ox-9,10- dihydroacridines as intercalating cytotoxic agents: Synthesis, DNA binding, and biological evaluation

Article abstract of DOI:10.1021/jm970114u

A series of DNA-intercalating potential antitumor agents, 1-[(ω- aminoalkyl)amino]-4-[N-(ω aminoalkyl) carbamoyl]-9-oxo-9,10- dihydroacridines, has been prepared by a aminolysis of the corresponding 4- [N-(ω-aminoalkyl)carbamoyl]-1-chloro derivative with

Full text of DOI:10.1021/jm970114u

J . Med. Chem. 1997, 40, 3749-3755
3749
1-[(ω-Am in oa lk yl)a m in o]-4-[N-(ω-a m in oa lk yl)ca r ba m oyl]-9-oxo-9,10-d ih yd r o-
a cr id in es a s In ter ca la tin g Cytotoxic Agen ts: Syn th esis, DNA Bin d in g, a n d
Biologica l Eva lu a tion
Ippolito Antonini,*, Paolo Polucci, Terence C. J enkins,† Lloyd R. Kelland,‡ Ernesto Menta,§ Nicoletta Pescalli,§
Barbara Stefanska,| J an Mazerski,| and Sante Martelli
Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino, Italy, Cancer Research
Campaign Biomolecular Structure Unit and Cancer Research Campaign Cancer Therapeutics Centre, The Institute of Cancer
Research, Cotswold Road, Sutton, Surrey SM2 5NG, U.K., Boehringer Mannheim Italia, Research Center, 20052 Monza, Italy,
and Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, 80952 Gdansk, Poland
Received February 25, 1997X
A series of DNA-intercalating potential antitumor agents, 1-[(ω-aminoalkyl)amino]-4-[N-(ω-
aminoalkyl)carbamoyl]-9-oxo-9,10-dihydroacridines, has been prepared by aminolysis of the
corresponding 4-[N-(ω-aminoalkyl)carbamoyl]-1-chloro derivative with a suitable ω-aminoalkyl-
amine. The noncovalent DNA-binding properties of these bis-functionalized compounds have
been examined using a combination of fluorometric and thermal denaturation techniques and
are compared with the behaviors for established DNA intercalants and cationic minor groove
ligands. The results indicate that (i) the agents are considerably more DNA-affinic than less
functionalized acridinones, with ‘apparent’ binding constants of (0.1-2.1) × 107 and (0.3-7.5)
× 107 M-1 at pH 5 and 7, respectively, (ii) overall affinity is sensitive to both the length of the
flexible side chain and the complexity of the attached amine substituents, and (iii) the pendant
side chains effect a switch to moderate AT-preferential binding. In vitro cytotoxic potencies
toward six tumor cell lines broadly parallel the observed DNA affinities, although poor
correlation is evident for certain compounds. The octanol/water partition coefficients have been
also calculated, but there is no correlation with cytotoxicity values. Two highly DNA-affinic
analogs, 10 and 13, have been identified with a useful broad spectrum of cytotoxic activity.
In tr od u ction
Antitumor cytotoxic agents with DNA-intercalative
properties are characterized by the presence of a planar
chromophore, generally a tri- or tetracyclic ring system,
and one or two flexible basic side chains. Some mean-
ingful examples include anthrapyrazoles,1 pyrazolo-
acridines,2 acridine-4-carboxamides,3 mitoxantrone,4 and
several of its aza analogs.5
F igu r e 1. Structures and ring numbering for the tetracyclic
pyrimido[5,6,1-de]acridines 1 and the tricyclic 4-carbamoyl-
9(10H)-acridinones 2.
We recently described the synthesis and the antitu-
mor properties of some tetracyclic pyrimido[5,6,1-de]-
acridines (1; Figure 1) with one or two basic side
chains.6,7 This family of drugs belongs to the group in
9(10H)-acridones 2 appear to intercalate DNA but are
which two side chains are essential for anticancer
activity. In fact, while the derivatives with only one side
chain [1: R ) (alkylamino)alkyl; X ) H, Cl, or NO2]
possess marginal or no antineoplastic activity, the
derivatives with a second side chain in position 6 [1: R
) (alkylamino)alkyl; X ) [(alkylamino)alkyl]amino]
comprise a novel group of potential intercalating agents
devoid of significant anticancer activity.8
In order to establish if the introduction of a second
basic side chain in tricyclic compounds 2 could effect a
marked increase of antitumor activity, as seen for the
tetracyclic derivatives 1, we have synthesized and
evaluated a series of bis-functionalized 1-[(ω-amino-
alkyl)amino]-4-[N-(ω-aminoalkyl)carbamoyl]-9(10H)-acri-
endowed with remarkable anticancer properties both in
dinones 10-20. The second substituent at ring position
1 of the 9-oxo-9,10-dihydroacridine chromophore here
corresponds with that in position 6 of the pyrimido[5,6,1-
de]acridine system (cf. 2 and 1, respectively, in Figure
1).
vitro and in vivo.6
Certain 9-oxo-9,10-dihydroacridine-4-carboxamides (‘ac-
ridone 4-carboxamides’, 2: X ) H or NO2; Figure 1),
which can be considered as ring-opened models or
precursors of tetracyclic structure 1, have been inves-
tigated previously.8 While the corresponding acridine-
4-carboxamides are promising antitumor agents,3 the
DNA-binding results from thermal denaturation and
fluorescence-based studies are reported for these com-
pounds with calf thymus DNA and two polyoligonucle-
otide duplexes. In vitro cytotoxicity data against six
tumor cell lines are described.
* Address correspondence to this author.
University of Camerino.
† Cancer Research Campaign Biomolecular Structure Unit.
‡ Cancer Research Campaign Cancer Therapeutic Centre.
§ Boehringer Mannheim Italia.
Ch em istr y
Schemes 1 and 2 show the synthetic pathway used,
and the physical data for compounds 9-20 are given in
| Technical University of Gdansk.
X Abstract published in Advance ACS Abstracts, October 1, 1997.
S0022-2623(97)00114-3 CCC: $14.00 © 1997 American Chemical Society

3750 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Antonini et al.
Sch em e 1a
Reaction of the appropriate amine with carboxamide 9
afforded the 1-[(ω-aminoalkyl)amino]-4-[N-(ω-amino-
alkyl)carbamoyl]-9(10H)-acridinones 10-19; cleavage of
19 with HBr gave the hydroxy derivative 20.
In order to examine the DNA-binding properties and
the in vitro antineoplastic activity of these agents, the
free base forms of 10-15 and 17-20 were converted into
their water-soluble dihydrochloride or maleate addition
salts by the usual methods. This was not necessary for
16 as the free base is appreciably water-soluble.
Resu lts a n d Discu ssion
DNA-Bin d in g P r op er ties. Thermal denaturation
data for interaction of the acridinones 10-20, simpler
acridinone derivatives 21 and 22,11 acridines, and
selected DNA-binding ligands with calf thymus DNA
(CT-DNA) at three [ligand]:[DNAp] molar ratios are
shown in Table 3. The results show that these agents
strongly stabilize the thermal helix f coil transition
(positive Tm increase) for the DNA duplex, although
none is as effective as mitoxantrone. However, all
derivatives are considerably more DNA-affinic than
acridine, proflavine (an established DNA intercalant),
or acridinones 21 and 22, suggesting that the function-
alized side chains are necessary for effective interaction.
Melting profiles were monophasic at low [ligand]:[D-
NAp] molar ratios although biphasic behavior was found
for 10, 16, and 18-20 at higher ratios, indicating
possible secondary modes of interaction for these drugs.
Examination of the ligand-induced effects upon the low-
and high-temperature portions (AT and GC segments,
respectively) of the melting curves11 revealed no clear
evidence for either base site- or sequence-preferential
binding.
a
Reagents: (i) for 3a (MeCOO)2Cu‚H2O/[(Me)2CH]2NEt/1-meth-
yl-2-pyrrolidinone, for 3b Cu/K2CO3/isopentyl alcohol; (ii) for 4a
PPE, for 4b POCl3/xylene; (iii) NaOH/EtOH.
Sch em e 2a
The ∆Tm data in Table 3 reveal a clear 18 > 10 ∼ 16
> 12 ∼ 13 ∼ 20 > 15 ∼ 19 > 17 > 11 ∼ 14 rank order
for differential thermal stabilization of the DNA duplex
that can be interpreted in terms of the extent and
strength of ligand binding. The poor effect of 14
suggests that the protonation status of the pendant side
chain(s) influences overall DNA binding, since the
morpholine residue provides the least basic amine
residue within this series and would be only ∼10-20%
protonated at pH 7. Comparison of the relative effects
induced by homologs 10 (n ) 2) and 12 (n ) 3) indicates
that homologous extension of the side chains results in
poorer stabilization. In general, side chain elaboration
in terms of steric load appears to effect a decrease in
relative binding strength, such that less crowded mol-
ecules provide superior DNA affinity. Similarly, the
results determined for 10 and 17 show that N-methyl-
ation of the 1-amino group markedly weakens the
binding efficiency, presumably as a result of reduced
overall chromophore planarity due to elimination of the
favorable intramolecular hydrogen-bonded interaction
with the 9-carbonyl residue in 10. The introduction of
electron-releasing ring substituents (e.g., X ) OMe or
OH, 19 and 20) leads to weaker overall binding, whereas
the electron-withdrawing nitro group in 18 provides a
small enhancement of stabilization.
a
Reagents: (i) for 6 ClCOOEt/H2NCH2CH2R, for 7 and 8 1,1′-
carbonyldiimidazole/H2NCH2CH2R; (ii) HCl/dioxane; (iii)
HN(R1)CH2CH2R2; (iv) HBr. Substituents for compounds 9 and
10-20 are shown in Tables 1 and 2, respectively.
Tables 1 and 2. Condensation of 2-chloro-5-nitrobenzoic
acid (3b) with 2-amino-4-chlorobenzoic acid afforded
diphenylamine 4b; cyclization by reflux treatment with
POCl3 gave an isomeric mixture of the 1-chloro deriva-
tive 6 and 6-chloro-2-nitro-9-oxo-9,10-dihydroacridine-
1
4-carboxylic acid in a ∼2:1 ratio, as judged from H NMR
analysis. This mixture proved difficult to separate by
usual methods but could be used for subsequent steps
without purification. Analogous reaction of 2-bromo-5-
methoxybenzoic acid (3a ) with methyl 2-amino-4-chlo-
robenzoate9 afforded 4a , which was cyclized with PPE
(polyphosphoric acid ethyl ester) to the ester 5; subse-
quent hydrolysis with aqueous NaOH provided the
carboxylic acid 7.
Treatment of either 1-chloro-7-methoxy-9-oxo-9,10-
dihydroacridine-4-carboxylic acid (7) or 1-chloro-9-oxo-
9,10-dihydroacridine-4-carboxylic acid (8)9 with the
required amine, after prior reaction of the acid with 1,1′-
carbonyldiimidazole at room temperature in DMF, gave
the 1-chloro carboxamides 9a -g. The amide 9h was
prepared from carboxylic acid 6 by the “mixed anhy-
dride” method,10 while 9i was obtained by deprotection
of 9g with HCl in dioxane solution at room temperature.
Competitive displacement (C50) and quenching (Q)
fluorometric assays with DNA-bound ethidium can be
used11 to (i) determine ‘apparent’ equilibrium constants
(Kapp) for drug binding, as the C50 value is approximately
inversely proportional to the binding constant,12 (ii)

Novel Bis(amine-functionalized) Acridinones
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3751
Ta ble 1. Substituents, Melting Point, Yield, and Formula of Compounds 9 (Scheme 2)
no.
9a a
9b
9c
9d
9e
9f
9g
X
R
mp, °C
yield, %
formula
H
H
H
H
H
N(Me)2
N(Et)2
185-187
157-158
150-151
188-190
158-161
170-172
oil
87
74
66
83
54
66
85
65
73
C18H18ClN3O2
C20H22ClN3O2
C19H20ClN3O2
C20H20ClN3O3
C21H22ClN3O2
C19H20ClN3O3
C28H34ClN3O6
C18H18Cl2N4O4
C18H18ClN3O3
CH2N(Me)2
4-morpholinyl
1-piperidyl
N(Me)2
OMe
H
NO2
H
N(Boc)(CH2)2OZb
N(Me)2
9h ‚HCl
9i
295-297
145-146
NH(CH2)2OH
a
b
Data taken from ref 6. Boc ) tert-butyloxycarbonyl; Z ) 2-tetrahydropyranyl.
Ta ble 2. Substituents, Melting Point, log P, Yield, and Formula of Compounds 10-20 (Scheme 2)
no.
X
R1
R2
R
mp, °C
log Pa
yield, %
formulab
10
11
12
13
14
15
16
17e
18
19
20
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
N(Me)2
N(Et)2
N(Me)2
N(Et)2
151-152 (259-260)c
96-97 (>350)d
5.12 ( 0.65
7.25 ( 0.65
5.76 ( 0.63
4.09 ( 0.66
4.42 ( 0.73
7.73 ( 0.68
3.15 ( 0.68
4.21 ( 0.62
5.68 ( 0.65
5.32 ( 0.66
4.77 ( 0.66
72
63
67
57
51
33
39
66
87
82
80
C22H29N5O2
C26H37N5O2
C24H33N5O2
C22H29N5O3
C26H33N5O4
C28H37N5O2
C22H29N5O4
C27H35N5O6
C22H28N6O4
C23H31N5O3
C22H29N5O3
CH2N(Me)2
NH(CH2)2OH
4-morpholinyl
1-piperidyl
NH(CH2)2OH
N(Me)2
N(Me)2
N(Me)2
N(Me)2
CH2N(Me)2
N(Me)2
4-morpholinyl
1-piperidyl
NH(CH2)2OH
N(Me)2
N(Me)2
N(Me)2
N(Me)2
108-110 (272-273)c
120-121 (218-221)c
193-195 (124-126)d
128-130 (210-212 d)d
160-161
oil (159-160)d
NO2
OMe
OH
207-208 (187-188 d)d
167-168 (151-152 d)d
236-237 (204-206 d)d
a
b
log P is the calculated value (log10) of the octanol/water partition coefficient. Analyses for C, H, and N. c In parentheses is the melting
point of the dihydrochloride. In parentheses is the melting point of the maleate; d ) decomposition. e Isolated and characterized as the
maleate.
d
distinguish intercalating agents from nonintercalative
ligands,13 and (iii) establish possible base- or sequence-
preferential binding.14
> 10 ∼ 20 > 19 > 17 reinforces conclusions for this
subset of related compounds with regard to the effects
of 7-substitution and N-methylation.
In the present study, fluorescence displacement as-
says were performed at pH 5, to ensure that the added
compounds were present as the fully amine-protonated
cationic species, as in previous studies,11,12b,14 and at pH
7 to enable comparison in more biologically relevant
systems. The C50 values (Table 3) determined for
ethidium displacement from CT-DNA by acridinones
10-20 indicate that these molecules, with the exception
of 11 and 14, are strong DNA-binding ligands at both
pH 5 and 7, with greater affinity than simpler acridines
or acridinones (e.g., 22), m-AMSA12b,14 or established
minor groove-binding cationic ligands, including berenil
and distamycin.11,13,15,16 However, no compound is as
efficient as mitoxantrone. It is noteworthy that 3-9-
fold poorer binding of 10-20 is evident at the lower pH
as compared to pH 7, presumably due to protonation of
the host DNA bases and/or pH-dependent conforma-
tional effects. Opposite behavior is seen for mitox-
antrone and acridine (Table 3), although in the latter
case this reflects the poor basicity of the chromophore.
On this basis, reported competitive C50 data determined
for ligands at pH 5 (e.g., refs 11, 12b) should be treated
with caution as this may not correctly predict the
binding affinity at physiological pH.
The fluorescence quenching (Q) values for 10-20 with
[poly(dA-dT)]2 at pH 5 are much smaller than expected
for ‘classical’ DNA intercalants, including acridine and
proflavine, and more closely resemble values for minor
groove-binding ligands (e.g., berenil and distamycin in
Table 3).11,12b,14 This distinction arises from the larger
DNA binding site sizes associated with nonintercalative
drugs compared to planar intercalators,12b,14 suggesting
that the flexible protonated side chains in acridinones
10-20 are accommodated within the groove conduits
of the host DNA duplex upon binding. Similar behavior
is evident for mitoxantrone, where the small Q values
indicate more efficient quenching of the bound ethidium
fluorophore (under conditions of minimum displace-
ment) than could be achieved by a strict intercalation
process. However, such data do not preclude involve-
ment of an intercalative binding mode. Compounds 10-
20 show significantly different binding behavior to
polyamines, which bind to DNA in a nonspecific manner
by bridging the minor groove and by inter- and intras-
trand electrostatic contacts to the phosphate residues.17
Thus, for example, all derivatives are both considerably
more affinic than spermidine (C50 ) 37.5 µM at pH 7)
and more efficient quenching ligands, suggesting deep
penetration of the groove tracts. On this basis, we infer
that DNA binding with these molecules involves both
intercalative and groove-binding modes. Equivalent
dual binding behavior has been established for hybrid
ligands and combilexins.11,14,18,19
The relative Kapp values of (0.1-2.1) × 107 M-1 at pH
5 and (0.3-7.5) × 107 M-1 at pH 7 determined for
binding of 10-20 with CT-DNA compare with 3.4 × 108
M-1 (pH 7) for mitoxantrone and (2-4) × 104 M-1 for
the unsubstituted acridine and 3,6-diaminoacridinone
22, again indicating that the functionalized side chains
are required for effective duplex binding. The 18 > 15
> 10 ∼ 12 ∼ 13 ∼ 16 ∼ 20 > 19 > 17 > 11 ∼ 14 rank
order determined for binding is closely similar to that
predicted from the DNA melting experiments, although
the piperidine derivative 15 has greater affinity than
expected from its induced ∆Tm (Table 3). The order 18
The Q values determined for 10-20 using CT-DNA,
[poly(dA-dT)]2, and [poly(dG-dC)]2 indicate a moderate
preference for binding to AT-rich duplexes. This AT >
GC behavior contrasts with the GC > AT preference
shown by acridine, proflavine, and m-AMSA (Table 3),
where DNA binding is likely to involve strict ‘classical’
intercalation. Acridinones 21 and 22 also appear to

3752 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Antonini et al.

Novel Bis(amine-functionalized) Acridinones
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3753
an asterisk. Elemental analyses were performed on a Model
1106 elemental analyzer (Carlo Erba Strumentazione); all
analytical values for C, H, and N were within (0.4% of the
theoretical values.
favor GC sequences although binding is considerably
weaker. These data indicate that the binding behavior
of 10-20 is largely dictated by the functionalized side
chains, since AT-preferential binding is characteristic
for most cationic minor groove-binding ligands,15,16
including berenil and distamycin (Table 3). In contrast,
the binding behavior of mitoxantrone appears to be
base- or sequence-neutral.
2-[[5-Ch lor o-2-(m e t h oxyca r b on yl)p h e n yl]a m in o]-5-
m eth oxyben zoic Acid (4a). A suspension of methyl 2-amino-
4-chlorobenzoate9 (0.8 g, 4.3 mmol), commercially available
2-bromo-5-methoxybenzoic acid (3a ; 1 g, 4.3 mmol), and Cu-
(OAc)2‚H2O (0.86 g, 4.3 mmol) in 1-methyl-2-pyrrolidinone (5
mL) and N,N-diisopropylethylamine (10 mL) was stirred for
6 h at 160 °C. After cooling, dilution with water, and
acidification to pH 2, the dried collected precipitate was stirred
with Et2O and then filtered to give 4a (0.75 g, 52%): mp 176-
177 °C (MeOH).
Meth yl 1-Ch lor o-7-m eth oxy-9-oxo-9,10-dih ydr oacr idin e-
4-ca r boxyla te (5). Compound 4a (1 g, 3 mmol) was refluxed
with PPE (40 g) in CHCl3 (50 mL) until all solid was dissolved.
The reflux condenser was removed, and the CHCl3 was allowed
to evaporate, to give an oil which was heated for 1 h at 100
°C. The mixture was diluted cautiously with MeOH (5 mL)
and water (10 mL) and then extracted with CHCl3 (3 × 20
mL). The concentrated chloroform extracts were flash-chro-
matographed on silica gel using CHCl3/C6H6 (3:2, v/v) to yield
the ester 5 (0.58 g, 61%): mp 219-220 °C.
Cytotoxic Activity. In vitro cytotoxic potency was
determined for acridinones 10-20 and related DNA-
binding compounds using six cell lines, including murine
leukemia (L1210), human ovarian carcinoma (A2780,
CH1, and SKOV-3), human melanoma (G-361), and
human colon adenocarcinoma (HT29) cells. Table 3
reveals that all compounds show excellent or good
cytotoxic activity, but no compound approaches the
potency of mitoxantrone. In qualitative agreement with
both our spectroscopic and binding data, the cytotoxic
potencies generally parallel the experimental DNA
affinity, although there are clear discrepancies and
differences among the cell lines examined. Thus, for
example, derivatives 11, 14, and 17 are uniformly poorly
cytotoxic, as would be expected from their high C50 (i.e.,
low Kapp) and low ∆Tm values, whereas the highly affinic
compound 18 is of only average potency within this
series. This behavior suggests that other factors (e.g.,
cellular uptake) must also influence the cytotoxic ef-
ficiency. The octanol/water partition coefficients for
compounds 10-20 have also been calculated (Table 2)
using ACD/Labs software (Advanced Chemistry Devel-
opment, Inc., Toronto, Ontario M5H 2L3, Canada).
However, attempts to correlate log10 of partition coef-
ficients with in vitro cytotoxic activity failed. Two DNA-
affinic compounds from this series, 10 and 13, provide
a useful degree of biological potency toward a range of
tumor cell lines.
1-Ch lor o-7-m eth oxy-9-oxo-9,10-d ih yd r oa cr id in e-4-ca r -
boxylic Acid (7). The ester 5 (1 g, 3.1 mmol) was suspended
in EtOH (100 mL) and 2 M NaOH (100 mL) and heated under
reflux for 30 min. The resulting mixture was acidified with 4
M HCl and stirred at room temperature for 20 min; the
precipitate was collected and washed with water, MeOH, and
1
Et2O to give pure 7 (0.72 g, 75%): mp 340-341 °C; H NMR
(DMSO-d6) δ 3.89 (s, 3H, CH3), 7.28 (d, 1H, 2-H, ar), 7.43 (m,
1H, 6-H, ar), 7.59 (d, 1H, 8-H, ar), 7.75 (d, 1H, 5-H, ar), 8.30
(d, 1H, 3-H, ar), 12.35 (s, 1H, NH, ex).
1-Ch lor o-7-n itr o-9-oxo-9,10-d ih yd r oa cr id in e-4-ca r box-
ylic Acid (6). 2-Amino-4-chlorobenzoic acid (7.0 g, 40.8
mmol), 2-chloro-5-nitrobenzoic acid (3b; 7.0 g, 34.7 mmol), K2-
CO3 (7 g, 50.6 mmol), and Cu powder (0.25 g, 3.9 mmol) were
suspended in isopentyl alcohol (150 mL). After refluxing and
stirring for approximately 20 min, a red-orange mass precipi-
tated; reaction was continued for 4-5 h, after which time 1 M
aqueous K2CO3 (150 mL) was added and insolubles were
removed from the hot mixture by filtration. The aqueous layer
was separated and acidified to pH 5 with 2 M HCl to
precipitate the product, which was filtered, suspended in
boiling MeOH (100 mL), suspended in boiling water (200 mL),
filtered again, and washed with EtOH to give adduct 4b. The
crude diacid was treated with POCl3 (55 mL) in xylene (55
mL) under reflux for 2 h. The precipitate that appeared upon
cooling was collected, boiled with MeOH, filtered, washed with
Et2O, and dried to give a mixture (5.8 g, 52%) of 6 and 6-chloro-
2-nitro-9-oxo-9,10-dihydroacridine-4-carboxylic acid, in an ap-
proximate 2:1 ratio by NMR. The isomeric mixture was used
for the next step without further purification.
4-[N-[2-(Diet h yla m in o)et h yl]ca r b a m oyl]-1-ch lor o-9-
oxo-9,10-d ih yd r oa cr id in e (9b). Exa m p le of th e Gen er a l
Meth od . The carboxylic acid 8 (1 g, 3.65 mmol) and 1,1′-
carbonyldiimidazole (1.1 g, 6.8 mmol) in DMF (10 mL) were
stirred, warming if necessary, until homogeneous. The mix-
ture was cooled to 10 °C, and N,N-diethylethylenediamine
(1.16 g, 10 mmol) was added. After 15 min at room temper-
ature, the mixture was partitioned between CHCl3 and aque-
ous 1 M Na2CO3. The organic layer was worked up to give
the crude carboxamide 9b, which solidified on washing with
Et2O and was used for the next step: 1H NMR (CDCl3) δ 1.08
(t, 6H, 2 × CH3), 2.60 (q, 4H, 2 × CO-N-C-C-N-CH2), 2.72 (t,
2H, CO-N-C-CH2), 3.55 (q*, 2H, CO-N-CH2), 7.20 (d, 1H, 2-H,
ar), 7.23-7.40 (m, 3H, 2 ar + CO-NH ex), 7.60-7.72 (m, 2H,
ar), 8.42 (dd, 1H, 8-H or 5-H, ar), 12.77 (br s, 1H, 10-H, ex).
The 4-[N-(ω-aminoalkyl)carbamoyl]-9(10H)-acridinones 9c-g
were prepared in a similar manner by analogous treatment
of an appropriate acid with the corresponding amine. All
amine reagents were commercially available, with the excep-
tion of O-tetrahydropyranyl-2-[[(2-aminoethyl)-N-tert-butoxy-
carbonyl]amino]ethanol.20 Amides 9b-f were solidified by
washing with Et2O, whereas 9g was chromatographed on a
Con clu sion s
The introduction of a second basic side chain in
position 1 of tricyclic compounds 2 results in an excellent
increase of cytotoxicity, as predicted. In fact, derivative
10 is about 40 times more cytotoxic against the L1210
cell line (Table 3) than the corresponding unsubstituted
acridonecarboxamide 2a . Acridinones 10-20 show a
usefully wide spectrum of cytotoxic activity toward a
variety of tumor cell lines, although no compound
approaches the potency of mitoxantrone. Generally,
there is qualitative agreement between cytotoxic po-
tency and the DNA affinity although there are some
discrepancies among the various cell lines examined.
Two compounds, 10 and 13, have been identified as
worthwhile leads for the development of more potent
cytotoxins in this family of bis(amine-functionalized)
acridinone carboxamides.
Exp er im en ta l Section
Syn th etic Ch em istr y. Melting points were determined on
a Bu¨chi 510 apparatus and are uncorrected. Thin-layer
chromatography (TLC) was accomplished using plates pre-
1
coated with silica gel 60 F-254 (Merck). All H NMR spectra
were recorded on a Varian VXR 300 instrument. Chemical
shifts are reported as δ values (ppm) downfield from internal
Me4Si in the solvent shown. The following NMR abbreviations
are used: br (broad), s (singlet), d (doublet), dd (double
doublet), t (triplet), q (quartet), m (multiplet), ar (aromatic
proton), ex (exchangeable with D2O). Quartets that are
transformed into triplets by addition of D2O are labeled with

3754 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23
Antonini et al.
silica gel column eluted with CHCl3/C6H6/MeOH (10:10:1, v/v)
to obtain a dense oil. All the carboxamide products were used
for subsequent steps without further purification.
an Omega DP4-TC high-performance temperature probe.
Heating was applied at a rate of 1 °C min-1 in the 45-98 °C
range, with absorbance (260 nm) and temperature data
sampling at 8-s intervals. DNA helix f coil transition
temperatures (Tm) were determined at the midpoint of the
normalized melting profiles. Drug-induced alterations in DNA
melting behavior are given by: ∆Tm ) Tm(DNA + drug) - Tm-
(DNA), where the Tm for the drug-free DNA control is 67.8 (
0.1 °C; results are given as the mean from three determina-
tions.
4-[N-[2-(Dim eth yla m in o)eth yl]ca r ba m oyl]-1-ch lor o-7-
n itr o-9-oxo-9,10-d ih yd r oa cr id in e Hyd r och lor id e (9h ). To
a stirred and cooled (ice-water, 0 °C) isomeric mixture of 6
and 6-chloro-2-nitro-9-oxo-9,10-dihydroacridine-4-carboxylic
acid (1 g, 3.14 mmol of isomeric mixture, ∼2.1 mmol of 6) and
anhydrous Et3N (0.314 g, 3.14 mmol) in CHCl3 (30 mL) was
added a solution of EtOCOCl (0.34 g, 3.14 mmol) in CHCl3
(20 mL) dropwise. After stirring at room temperature for 1 h
and cooling to 0 °C, the mixture was treated with N,N-
dimethylethylenediamine (0.32 g, 3.14 mmol) and then stirred
overnight at room temperature. The solid precipitate was
filtered and washed sequentially with MeOH, CHCl3, and then
Et2O to give 9h (0.58 g, ∼65% based upon 6): 1H NMR (DMSO-
d6) δ 2.88 (s, 6H, 2 × CH3), 3.34 (t, 2H, CO-N-C-CH2), 3.75
(q*, 2H, CO-N-CH2), 7.33 (d, 1H, 2-H, ar), 7.90 (d, 1H, 5-H,
ar), 8.30 (d, 1H, 3-H, ar), 8.49 (dd, 1H, 6-H, ar), 8.93 (d, 1H,
8-H, ar), 9.47 (t, 1H, CO-NH, ex), 10.03 (br s, 1H, 10-H, ex),
11.03 (br m, 1H, N+-H, ex).
4-[N-[2-[(2-H yd r oxyet h yl)a m in o]et h yl]ca r b a m oyl]-1-
ch lor o-9-oxo-9,10-d ih yd r oa cr id in e (9i). A mixture of 9g
(0.45 g, 0.83 mmol) in dioxane (30 mL) and aqueous HCl (3
mL of 37%, w/w) was stirred for 30 min at room temperature.
The reaction mixture was partitioned between CHCl3 and an
excess of 1 M aqueous Na2CO3. The organic layer was worked
up to give the crude carboxamide 9i, which solidified upon
washing with Et2O: 1H NMR (DMSO-d6) δ 2.70 (t, 2H, CH2),
2.85 (t, 2H, CH2), 3.42-3.57 (m, 4H, CO-N-CH2 + O-CH2), 4.65
(br m, 1 H, ex), 7.27-7.38 (m, 2H, ar), 7.62-7.80 (m, 2H, ar),
8.12-8.23 (m, 2H, ar), 9.31 (br s, 1H, 10-H, ex). This material
was used for the next step without further purification.
1-[[2-(Dim et h yla m in o)et h yl]a m in o]-4-[N-[2-(d im et h -
yla m in o)et h yl]ca r b a m oyl]-9-oxo-9,10-d ih yd r oa cr id in e
(10). Exa m p le of Gen er a l P r oced u r e for th e P r ep a r a tion
of 10-19. A suspension of the carboxamide 9a (0.48 g, 1.4
mmol) in DMF (10 mL) and the 2,2′-dimethylethylenediamine
(0.53 g, 6 mmol) were stirred for 2 h at 120 °C. The reaction
mixture was partitioned between CHCl3 (70 mL) and an excess
of 1 M aqueous Na2CO3 (50 mL). The organic layer was
worked up to give a residue which was chromatographed on a
silica gel column eluted with CHCl3/MeOH (7:3, v/v) and 32%
aqueous NH3 (10 mL for 1 L of eluent) to afford pure 10: 1H
NMR (CDCl3) δ 2.34 (s, 6H, 2 × CH3), 2.36 (s, 6H, 2 × CH3),
2.60 (t, 2H, C-CH2), 2.70 (t, 2H, C-CH2), 3.44 (q*, 2H, 1-N-
CH2), 3.55 (q*, 2H, CO-N-CH2), 6.22 (d, 1H, 2-H, ar), 6.92 (t,
1H, CO-NH, ex), 7.23 (t, 1H, 7-H or 6-H, ar), 7.36 (d, 1H, 5-H
or 8-H, ar), 7.62 (t, 1H, 6-H or 7-H, ar), 7.70 (d, 1H, 3-H, ar),
8.38 (d, 1H, 8-H or 5-H, ar), 11.02 (t, 1H, 1-NH, slow ex), 13.42
(s, 1H, 10-H, ex).
The 1-[(ω-aminoalkyl)amino]-4-[N-(ω-aminoalkyl)carbam-
oyl]-9(10H)-acridinones 10-19 were prepared in a similar
manner by analogous treatment of the appropriate compound
9 with the suitable amine. Compound 17, obtained as a dense
oil, was converted into a maleate, crystallized from EtOH-
Et2O, and then characterized.
1-[[2-(Dim et h yla m in o)et h yl]a m in o]-4-[N-[2-(d im et h -
yla m in o)et h yl]ca r b a m oyl]-7-h yd r oxy-9-oxo-9,10-d ih y-
d r oa cr id in e (20). Compound 19 (0.26 g, 0.61 mmol) in 48%
HBr (10 mL) was refluxed for 1 h. The reaction mixture was
partitioned between CHCl3 and 0.5 M aqueous Na2CO3.
Workup of the organic phase and column chromatography on
silica gel eluted with CHCl3/MeOH (4:1, v/v) and 32% aqueous
NH3 (10 mL for 1 L of eluent) afforded 20: 1H NMR (CDCl3)
δ 2.35 (s, 6H, 2 × CH3), 2.52 (s, 6H, 2 × CH3), 2.68 (t, 2H,
C-CH2), 2.90 (t, 2H, C-CH2), 3.40 (q*, 2H, 1-N-CH2), 3.66 (q*,
2H, CO-N-CH2), 6.03 (d, 1H, 2-H, ar), 6.72 (dd, 1H, 6-H, ar),
6.82 (d, 1H, 5-H, ar), 6.86 (m, 1H, CO-NH, ex), 7.35 (d, 1H,
8-H, ar), 7.54 (d, 1H, 3-H, ar), 10.90 (t, 1H, 1-NH, slow ex),
12.58 (s, 1H, 10-H, ex).
Calf thymus (CT-DNA) [Sigma; 42% G + C content, ꢀ260
)
6600 (M phosphate)-1 cm-1] was dialyzed extensively against
water before use. Solutions were prepared in aqueous phos-
phate buffer (10 mM Na2HPO4/NaH2PO4, 1 mM EDTA, pH
7.00 ( 0.01); working solutions (100 µM) were prepared by
dilution of a stock solution. DNA-drug solutions were pre-
pared by addition of the compound in DMSO to give a final
drug concentration of 5, 10, or 20 µM. It was established that
the highest [drug]:[DNAp] molar ratio used did not effect
saturation of the DNA duplex by any candidate ligand. Linear
correction factors were applied to correct for the effects of
DMSO cosolvent (max 2.5%, v/v) used in the DNA-drug
studies.11
2. F lu or escen ce Bin d in g Stu d ies. The fluorometric
assays have been described previously.11 Quenching Q values
were determined for CT-DNA, [poly(dA-dT)]2, and [poly(dG-
dC)]2 for solutions (20 µM DNAp) in 0.01 M ionic strength
aqueous buffer (9.3 mM NaCl, 2 mM NaOAc, 0.1 mM EDTA,
pH 5.0) containing 2 µM ethidium bromide.14 These concen-
trations effect minimal displacement of the ethidium fluor-
ophore and maximum drug-induced quenching of the fluores-
cence due to the DNA-bound ethidium intercalant.12 Molar
extinction values of ꢀ260 ) 6550 and 8400 (M phosphate)-1
cm-1, respectively, were used for the AT and GC oligonucle-
otides (Sigma). The C50 values for ethidium displacement were
determined using solutions in either the same buffer (for pH
5.0 studies) or aqueous TES buffer (10 mM TES, 0.1 mM
EDTA, pH 7.0) containing 1.26 µM ethidium bromide and 1
µM CT-DNA.11,12,14,21
All measurements were made in 10-mm quartz cuvettes at
20 °C using a Perkin-Elmer LS5 instrument (excitation at 546
nm; emission at 595 nm) following serial addition of aliquots
of a stock drug solution (∼5 mM in DMSO). The Q and C50
values are defined as the drug concentrations which reduce
the fluorescence of the DNA-bound ethidium by 50% and are
reported as the mean from three determinations. Apparent
equilibrium binding constants were calculated from the C50
values (in µM) using: Kapp ) (1.26/C50) × Kethidium, and with a
value of Kethidium ) 107 M-1 for ethidium bromide.12a
3. In Vitr o Cyt ot oxicit y. A. L1210 E xp er im en t a l
P r otocol. Drug solutions of appropriate concentration were
added to a culture containing mouse L1210 leukemic cells at
5 × 104 cells/mL of medium.11 Stock aqueous drug solutions
(e0.5%, v/v, DMSO) were used, and it was separately estab-
lished that this level of DMSO was tolerated. Cells were
exposed to the compounds for 48 h at 37 °C, and the IC50 values
were calculated by counting (Coulter counter) the number of
remaining living cells and comparison with drug-free controls.
All assays were performed in duplicate.
B. Hu m a n Ova r ia n Ca r cin om a a n d Mela n om a Cell
Lin es. Human melanoma cell line G-361 was obtained from
the European Collection of Animal Cell Cultures, Porton Down,
Salisbury, U.K., via the American Type Culture Collection.
Establishment details and biological properties of human
ovarian carcinoma cell lines (A2780, CH1, and SKOV-3) have
been described previously.22 The sulforhodamine B (SRB)
experimental protocol used has been described previously.11,22
Cells were plated (100-5000 cells) in 96-well microtiter plates
and left overnight to adhere prior to drug treatment. Aqueous
drug solutions at pH 7.0 were then added to the cells at various
concentrations following dilution of a stock DMSO solution.
After 96 h continuous drug exposure at 37 °C, growth inhibi-
tion was assessed using SRB protein staining. IC50 values
(drug dose required for 50% growth inhibition compared to
drug-free controls) were determined by comparing treated and
untreated cells.
Biop h ysica l Eva lu a tion . 1. Th er m a l DNA Den a tu r -
a tion Stu d ies. The experimental protocol used has been
outlined previously.11 Briefly, melting studies were performed
in stoppered quartz cuvettes using a Shimadzu UV-2101PC
spectrophotometer fitted with a SPR-8 heating controller and

Novel Bis(amine-functionalized) Acridinones
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 23 3755
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C. HT29 Hu m a n Colon Ad en oca r cin om a Exp er im en -
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