Substituted 9-aminoacridine-4-carboxamides tethered to platinum(II)diamine complexes: Chemistry, cytotoxicity and DNA sequence selectivity

Article abstract of DOI:10.1016/j.jinorgbio.2010.03.011

Three platinum complexes in which substituted (7-OMe, 9-NH₂; 7-F, 9-NH₂; and 7-H, 9-NH(CH₂)₂OH) 9-aminoacridine-4-carboxamides were tethered to a platinum(II)diamine moiety were synthesised and characterised at the chemical and biological level. These variants showed a decrease in cytotoxicity, as measured by IC₅₀ values in HeLa cells, when compared with the parent 7-H, 9-NH₂ compound. The 7-F and 9-NH(CH₂)₂OH substituents gave rise to a small decrease in cytotoxicity, and the 7-OMe substituent resulted in a larger decrease in cytotoxicity. Their binding to purified pUC19 plasmid DNA was investigated and it was found that the addition of 7-F, 9-NH(CH₂)₂OH and especially the 7-OMe substituents, resulted in reduced DNA binding. This correlated well with the IC₅₀ cytotoxicity values. However, the DNA sequence selectivity was unaffected by the addition of these moieties.

Full text of DOI:10.1016/j.jinorgbio.2010.03.011

Journal of Inorganic Biochemistry 104 (2010) 815–819
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Substituted 9-aminoacridine-4-carboxamides tethered to platinum(II)diamine
complexes: Chemistry, cytotoxicity and DNA sequence selectivity
Michael Carland a, Martin J. Grannas a, Murray J. Cairns b,1, Vanessa J. Roknic b, William A. Denny c,
W. David McFadyen a, Vincent Murray b,
⁎
a
School of Chemistry, The University of Melbourne, Grattan Street, Parkville, Victoria 3052, Australia
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia
Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, The University of Auckland, Private Bag 92019, Auckland, New Zealand
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Three platinum complexes in which substituted (7-OMe, 9-NH2; 7-F, 9-NH2; and 7-H, 9-NH(CH2)2OH)
9-aminoacridine-4-carboxamides were tethered to a platinum(II)diamine moiety were synthesised and
characterised at the chemical and biological level. These variants showed a decrease in cytotoxicity, as measured
by IC50 values in HeLa cells, when compared with the parent 7-H, 9-NH2 compound. The 7-F and 9-NH(CH2)2OH
substituents gave rise to a small decrease in cytotoxicity, and the 7-OMe substituent resulted in a larger decrease
in cytotoxicity. Their binding to purified pUC19 plasmid DNA was investigated and it was found that the addition
of 7-F, 9-NH(CH2)2OH and especially the 7-OMe substituents, resulted in reduced DNA binding. This correlated
well with the IC50 cytotoxicity values. However, the DNA sequence selectivity was unaffected by the addition of
these moieties.
Received 17 December 2009
Received in revised form 16 February 2010
Accepted 19 March 2010
Available online 27 March 2010
Keywords:
Acridine-4-carboxamides
Substituents
Platinum(II)
Synthesis
Cytotoxicity
© 2010 Elsevier Inc. All rights reserved.
DNA sequence selectivity
1. Introduction
9-aminoacridine-4-carboxamide platform (compound 1) are of
particular note, showing promising activity against cisplatin resistant
Expanding the spectrum of activity of platinum-based antitumour
agents is an important goal which has motivated the continued
development of drugs of this type [1]. One approach which has been
explored is to develop platinum compounds with an altered pattern of
DNA damage to that caused by cisplatin and carboplatin. This strategy
has been a significant driving force in the development of bi- and
trinuclear complexes exemplified by BBR3464 [2,3]. It has also been a
key feature in the development of: (i) platinum–acridinylthiourea
conjugates [4,5]; (ii) minor groove targeted multinuclear complexes
derived from the 4,4′-dipyrazoylmethane ligand [6]; (iii) complexes
in which platinum is tethered to the 5′-terminus of an oligothymidine
sequence [7]; (iv) binuclear complexes based on a 1,4-diaminoan-
thraquinone intercalating chromophore [8,9]; and (v) platinum(II)
derivatives of distamycin and netropsin analogues [10,11]. We have
also explored this concept using intercalating chromophores such as
acridine and 9-anilino-acridine [12,13], phenazine [14] and the
phenanthridinium cation [15] and extended this work to include a
more comprehensive investigation of 9-aminoacridine carboxamides
[16–18]. Of the complexes we have studied to date, those based on the
cells [16] and exhibiting a different DNA sequence specificity to that of
cisplatin [17], shifted away from runs of consecutive guanines (the
main binding site for cisplatin). This sequence specificity was shown
to be dependent on linker chain length, with the shorter chain length
homologues (n=2, 3) showing the greatest alteration in DNA
sequence specificity, whereas the longer chain length homologues
(n=4, 5) more closely resembled cisplatin and PtenCl2 in their
behaviour [17]. The latter study was the first occasion that an altered
sequence specificity was convincingly demonstrated for a cisplatin
analogue of the diamine type. We have also shown that the members of
the class displayed an altered sequence specificity in intact human cells
[18], preferentially targeting GA sequences rather than consecutive
G bases. The DNA sequencingresults suggest that it is thepresenceof the
9-amino group in the 9-aminoacridine-4-carboxamide chromophore
which results in this positional targeting of platinum. The presence of
the intercalator is also expected to rapidly localise compounds similar to
1 on DNA and we have previously shown that these compounds show a
marked increase in the rate of DNA platination when compared with
cisplatin or PtenCl2 [17].
Since the 9-aminoacridine-4-carboxamide Pt complex (1, n=2)
showed the best activity in earlier studies, we initiated structure/
activity studies on substituted 9-aminoacridine complexes, with the
ultimate aim of optimizing antitumour activity. We chose to
investigate the set of analogues shown in Fig. 1, with an n=2 linker
⁎
Corresponding author. Tel.: +61 2 9385 2028; fax: +61 2 9385 1483.
E-mail address: v.murray@unsw.edu.au (V. Murray).
Current address: Schizophrenia Research Institute and School of Biomedical
1
Sciences, The University of Newcastle, NSW 2308, Australia.
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.03.011

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M. Carland et al. / Journal of Inorganic Biochemistry 104 (2010) 815–819
Fig. 1. The structure of the 9-aminoacridine-4-carboxamide Pt complex (1) and related compounds used in this study.
chain, since this linker length previously showed the most significant
alterations to DNA specificity in the set of parent compounds. We
examined the effects of substituents at the 7-position of the acridine
ring and alterations to the amino group at the 9-position on the
behaviour of these compounds, and report the synthesis, cytotoxicity
and sequence specificity of these compounds.
2.2. In vitro cytotoxicity
The cytotoxicity of the acridine Pt complexes was compared to that
of cisplatin and PtenCl2. The IC50 values were determined in the
human cell line HeLa using an amylar blue assay [21,22] and are
presented in Table 1. Compound 1 (n=2) was the most potent in
killing HeLa cells with an IC50 value of 0.4 µM (Table 1), with 6 and 7
having IC50 values between 1.1 and 1.3 µM. Cisplatin, PtenCl2, and
compound 5 were the least potent, with IC50 values of approximately
10 µM. Thus the addition of substituents to the 9-aminoacridine ring
at different positions and of varied nature (fluoro in 6, 9-NH(CH2)2OH
in 7, and especially methoxy in 5) reduced the ability of the compound
to kill HeLa cells.
2. Results and discussion
2.1. Chemistry
The acridine ligands 2–4 were prepared as shown in Scheme 1. The
differentially protected triamine 13 was synthesised by first selec-
tively protecting one of the primary amine groups of diethylene-
triamine by reaction with ethyltrifluoroacetate at −78 °C [19]
followed by Boc protection of the remaining two amine functional
groups. The triamine 13 was reacted selectively with the appropri-
ately substituted 9-chloroacridine-4-carbonyl chloride to give the 9-
chloro compounds 8a, 9a, and 10a. These compounds were then
activated by in situ conversion to the 9-phenoxy compounds which on
treatment with ammonia (8a and 9a) or ethanolamine (10b) gave the
Boc-protected ligand precursors 8b, 9b, and 10b respectively.
Deprotection of these was carried out using methanolic HCl to form
ligands 2, 3, and 4 as their hydrochloride salts. Each of these was
characterised by 1H NMR, ESI-MS and elemental analysis.
Preparation of the complexes was carried out by addition of an
aqueous solution of K2PtCl4 to a solution of the ligand trihydrochlor-
ides in water which had been adjusted to pH 8–9 by the addition of
carbonate. The complexes 5 and 6 were obtained in pure form directly
from the reaction mixture although in the case of 7 purification was
necessary. The 195Pt chemical shift values for the complexes fall at
about δ −2350 ppm and are indicative of a platinum coordination to a
chelating diamine and two chloride donors in accord with the
structures of the complexes. These shift values are similar to those
obtained for the previously reported complexes in this series 1 [16]
and for Pt(en)Cl2 (−2345 ppm) [20]. ESI-MS was also used to
characterise the platinum complexes 5–7. The complexes were
sprayed from H2O/DMF mixtures and the spectra showed the
presence of the [M+H]+, [M−Cl]+ and [M−HCl2]+ ions, each of
which displayed the expected isotopic distribution.
2.3. The interaction of the complexes with purified plasmid DNA
The interaction of the analogues with purified DNA was investi-
gated using plasmid DNA. Compounds 6 and 7 required a slightly
higher concentration of drug to that of 1 (n=2) in order to achieve a
comparable level of DNA damage, whereas 5 required about a 2-fold
higher concentration. The presence of the electron donating methoxy
substituent in 5 and electron withdrawing fluoro substituent in 6
affects the charge distribution and the steric outline of the acridine
intercalating moiety, and both of these factors could influence the
DNA binding of these compounds compared with the unsubstituted
parent. Because of the known [23] parallel intercalation mode of
compounds of this class, it seems likely that the steric changes are a
more important influence on binding, with the larger OMe group
having the greater deleterious effect. In contrast, the 9-NH(CH2)2OH
substituent in 7 did not produce a major decrease in the binding of the
compound to DNA, possibly because this substituent will lie in the
minor groove where its OH group can be accommodated, and may
form stabilising H-bond interactions. The decreased DNA binding of 5
(with a methoxy substituent) correlated with the decreased cyto-
toxicity of the compound.
2.4. The DNA sequence selectivity in purified plasmid DNA
Preliminary experiments to determine the DNA sequence selec-
tivity of these cisplatin analogues in purified plasmid DNA indicated
that compounds 5, 6 and 7 had a very similar profile to that of 1

M. Carland et al. / Journal of Inorganic Biochemistry 104 (2010) 815–819
817
Scheme 1. aReagents: (i) CF3CO2Et, −78 °C, MeOH; (ii) (t-BuO)2CO; (iii) NH3(aq), 25%, RT; (iv) 8a: 9-chloro-7-methoxyacridine-4-carbonyl chloride; 9a: 9-chloro-7-
fluoroacridine-4-carbonyl chloride, 10a: 9-chloroacridine-4-carbonyl chloride, phenol/NH2CH2CH2OH/120 °C; (v) 8b: phenol/NH3/120 °C, 9b: phenol/NH3/120 °C, 10b: phenol/
NH2CH2CH2OH/120 °C, (vi) conc. HCl/MeOH/CH2Cl2; (vii) Na2CO3, K2PtCl4.
(n=2). In these experiments damage at sites other than consecutive
guanines were prominent as previously found for 1 (n=2) [17,24–
26]. The sequence selectivity of 1 (n=2), 5, 6 and 7 was different to
that of cisplatin as the damage sites were shifted away from
consecutive guanines. This also indicated that despite a change in
the efficiency of damaging DNA, the DNA sequence selectivity of
compound 5 was unaffected and similar to that of 1 (n=2), 6 and 7.
The similar DNA sequence specificity of compound 7 (with the
9-NH(CH2)2OH substitution) compared to compounds 1 (n=2), 5,
and 6 was interesting as previously we had shown that the addition of
the 9-amino substituent to the acridine was crucial for the alteration in
the DNA sequence selectivity compared to cisplatin [17,24]. This
indicated that the 9-amino group can be modified to 9-NH(CH2)2OH
without affecting the DNA sequence selectivity of the compound,
despite the presence of the extra steric bulk of the latter substituent.
Again compound 5 was the least efficient at damaging DNA in intact
cells, with 6 and 7 the next most efficient, and 1 (n=2) the most
efficient. This pattern of damage correlates with the IC50 values.
Preliminary DNA sequence selectivity experiments indicated that the
DNA sequence selectivity profiles of compounds 5, 6 and 7 were very
similar to that of 1 (n=2) and different to cisplatin. Again sites that
did not consist of consecutive guanines, were found to be prominently
damaged as found previously [18,24,27].
2.6. Conclusions
The variation of substituents on the 9-aminoacridine carboxamide
platform, (fluoro in compound 5, 9-NH(CH2)2OH in 7, and methoxy in
5) reduced the cytotoxicity of these compounds as measured by IC50
values when compared to the parent compound. The presence of the
fluoro and 9-NH(CH2)2OH substituents gave rise to a small decrease in
cytotoxicity, while the methoxy substituent resulted in a large
decrease in cytotoxicity. The DNA binding studies produced evidence
that the reduction in cytotoxicity (on addition of a substituent), was
due to a reduction in interaction with DNA. However, the DNA
sequence selectivity was unaffected by the addition of these moieties.
2.5. The DNA sequence selectivity in intact human cells
The interaction of the compounds in intact human cells has also
been investigated using the linear amplification assay [24,27,28].
Table 1
3. Experimental
The IC50 of cisplatin and analogues in HeLa cells.
3.1. Chemistry
Compound
Substituent on acridine ring
IC50 (µM)
Cisplatin
PtenCl2
1 (n=2)
5
6
7
–
10
11
0.4
10
1.3
1.1
1H and 195Pt NMR spectra were measured on Varian UnityPlus
400 MHz or Unity 300 spectrometers. 1H chemical shifts were
measured relative to the residual hydrogens of bulk solvent (δ 4.85
for D2O, 7.26 for CDCl3 and 2.49 for DMSO) and quoted relative to
TMS. The 195Pt reference was a 0.1 M solution of K2PtCl4 in D2O
–
None
Methoxy
Fluoro
9-NH(CH2)2OH

818
M. Carland et al. / Journal of Inorganic Biochemistry 104 (2010) 815–819
(δ −1630). ESI-MS spectra were recorded on a quadrupole ion trap
Finnigan-MAT LCQ mass spectrometer equipped with electrospray
ionisation. Triethylamine was redistilled from toluene-4-sulfonyl
chloride. N,N-Dimethyl formamide and N-methyl pyrrolidinone
were dried over 4 Å molecular sieves.
The compounds 9-acridone-4-carboxylic acid, 7-methoxy-9-acridone-
4-caboxylic acid and 7-fluoro-9-acridone-4-caboxylic acid were pre-
pared by reported methods [29].
J=8.5 Hz), 8.37 (d, 1H, J=8.7 Hz), 8.31 (d, 1H, J=7.5 Hz), 7.97 (t, 1H,
J=7.8 Hz), 7.79 (d, 1H, J=8.6 Hz), 7.58 (t, 2H, J=8.0 Hz), 4.28
(t, 2H, J=4.9 Hz, CH2–O), 4.08 (t, 2H, J=4.9 Hz, Acr–N–CH2–), 3.91
(t, 2H, J=5.5 Hz, CONH–CH2), 3.58−3.46 (m, 6H, CH2–N). ESI-MS
(H2O) m/z (relative intensity), 368.2 (70, [M+H]+); 308.2 (100,
[M+H–NHCH2CH2OH]+).
Anal. Calc. for C20H25N5O2.3HCl.0.75H2O requires: C, 48.63;
H, 6.27; N, 13.83; Cl, 21.00. Found: C, 48.74; H, 6.24; N, 13.49;
Cl, 20.80.
3.2. N-(2-aminoethyl)-N,N′-bis(tert-butoxycarbonyl)ethylenediamine (13)
3.4. (N-[2-[(2-aminoethyl)amino]ethyl]-9-[(2-hydroxyethyl)amino]
A solution of diethylenetriamine (11.4 g, 111 mmol) in methanol
(50 mL) was cooled to −78 °C under nitrogen. Ethyl trifluoroacetate
(15.7 g, 111 mmol) in methanol (50 mL) was added dropwise over
10 min and then the mixture was allowed to warm to room
temperature over a period of 3 h followed by the addition of
NaHCO3 (40 g). A solution of di-tert-butyldicarbonate (53.1 g,
243 mmol) in methanol (20 mL) was then added over a 5 min period,
the addition being accompanied by some effervescence. The mixture
was then sonicated for 20 min and set aside to stir overnight. The
solvent was then removed and the residue partitioned between water
and ethyl acetate. The organic phase was dried (Na2SO4) and the
solvent removed to yield a viscous pale yellow oil. (1H NMR confirmed
this product as the Boc-protected trifluoroacetamide 12). This
material was taken up in ethanol (100 mL) and water (20 mL) and
then potassium carbonate (40 g) was added. The mixture was heated
on the steam bath for 6 h then stirred for a further 12 h at room
temperature. The solids were then removed by filtration and the
solvents removed from the filtrate under reduced pressure. The
residue was partitioned between water and ethyl acetate and the
organic phase was dried (Na2SO4). The solvent was removed under
reduced pressure to afford a viscous oil that solidified over several
days to form a white waxy solid (26.2 g, 78%). 1H NMR (CDCl3)
δ=1.41 (m, 18H), 2.62 (s, 2H), 2.8–3.4 (m, 8H), 5.1 (bs, 1H); ESI-MS
m/z (relative intensity), 304.3 (100, [M+H]+), 204.1 (40), 104.1 (85).
Anal. Calc. for C14H29N3O4: C, 55.42; H, 9.63; N, 13.85. Found: C 55.38;
H, 9.74; N, 13.80.
acridine-4-carboxamide)dichloroplatinum(II) (7)
A solution of K2PtCl4 (87 mg, 0.21 mmol) in H2O (1 mL) was
filtered into a stirred solution of compound 4 (100 mg, 0.21 mmol) in
H2O (1.5 mL) that had been brought to pH 8 with aqueous Na2CO3
solution (1 M). An orange amorphous solid separated almost
immediately. The suspension was stirred rapidly at ambient temper-
ature overnight. LiCl (89 mg, 2.1 mmol) was then added and the
mixture again brought to pH 8 with aqueous Na2CO3 (1 M). After
another day stirring at ambient temperature the solid was collected,
washed with water and dried at 50 °C. This solid was then dissolved in
hot (90 °C) DMF (3 mL) and the solution was filtered into aqueous LiCl
(33 mL, 1 M). The precipitated solid was collected by centrifugation,
the supernatant liquid decanted off and the solid washed with water
(2×10 mL) and then dried for 4 h under reduced pressure (44 mg,
29%). 195Pt NMR (NMP) δ −2351.4. ESI-MS m/z (relative intensity),
634.3 (25, [M+H]+), 594.4 (10, [M−Cl]+), 560.3 (100, [M−HCl2]+).
Anal. Calc for C20H25N5O2PtCl2.HCl.3H2O requires: C, 33.3; H, 4.5; N,
9.7. Found: C, 33.3; H, 4.4; N, 9.8.
3.5. 9-amino-N-[2-[(N,N′-bis(tert-butoxycarbonyl)ethylenediamino)]-
ethyl]-7-methoxyacridine-4-carboxamide (8b)
7-Methoxy-9-acridone-4-caboxylic acid (1.30 g, 4.83 mmol) was
heated in thionyl chloride (20 mL) containing one drop of DMF for 1 h.
Excess thionyl chloride was removed under reduced pressure and the
residue mixed with toluene, followed by removal of volatiles under
reduced pressure. The residue was suspended with stirring in CH2Cl2
(20 mL) and to this was added compound 13 (1.21 g, 4.0 mmol)
dissolved in triethylamine (10 mL) and DCM (10 mL) with a
darkening in color. The mixture was stirred overnight at room
temperature and then DCM (50 mL) was added. The mixture was
washed with 10% NaHCO3 solution, then dried (MgSO4) and the
solvent removed under reduced pressure. To the residue was added
benzene (10 mL) and phenol (20 g) and the mixture was heated with
stirring to 90 °C, with most of the benzene being allowed to boil off.
The mixture was then cooled to 45 °C and ammonia gas was bubbled
through the stirred mixture which was then heated to 110 °C for
10 min followed by further addition of ammonia gas. The mixture was
then cooled to room temperature and CH2Cl2 added. After washing
twice with 10% NaOH solution the organic phase was dried (MgSO4)
then volatiles removed under reduced pressure. The residue was
subjected to silica chromatography (EtOAc) to afford compound 8b as
a yellow powder (1.26 g, 57%). 1H NMR (d6-DMSO) δ=1.28 (m, 18H),
3.02 (m, 2H), 3.21 (m, 2H), 3.37 (m, 2H), 3.58 (m, 2H), 3.84 (s, 3H),
6.25 (bs, exch), 6.60 (bs, exch), 7.35 (m, 2H), 7.55 (s, 1H), 7.70 (m,
1H), 8. 41 (d, 1H, J=5.2 Hz), 8.48 (d, 1H, J=2.0 Hz).
3.3. N-[2-[(2-aminoethyl)amino]ethyl]-9-[(2-hydroxyethyl)amino]
acridine-4-carboxamide (4)
Acridone-4-carboxylic acid (2.39 g, 10 mmol) was converted to
9-chloroacridine-4-carbonyl chloride by a reported procedure [30].
The acid chloride was dissolved into a mixture of CH2Cl2 (50 mL) and
Et3N (5 mL), then a solution of compound 13 (2.79 g, 10 mmol) in
CH2Cl2 (20 mL) was added and the mixture was stirred at ambient
temperature overnight. The volatiles were removed under reduced
pressure and the residue of crude 9-chloroacridine-4-carboxamide was
suspended in N-methyl pyrrolidinone (5 mL) containing phenol
(1.412 g, 15 mmol). Heating the rapidly stirred mixture was com-
menced and ethanolamine (0.92 g, 15 mmol) was added once 80 °C was
reached. The mixture was heated between 80 and 100 °C for 3 h, and
then let cool. It was then partitioned between EtOAc and 1 M aq. NaOH.
The organic phase was dried (Na2SO4) and evaporated to yield an
impure compound 10b as a brown oil. Chromatography on SiO2 eluting
with EtOAc removed the mobile impurities and Me2CO eluted the
desired component as a bright yellow band. The solvent was removed to
yield compound 10b (2.85 g). The Boc groups were removed by stirring
this material in methanolic HCl (44 mL) for 3 h. (The HCl solution was
generated by adding acetyl chloride (4 mL) to ice cold MeOH (40 mL)
and then allowed the solution to stand for 10 min). Following solvent
removal at reduced pressure, the residue was recrystallised from MeOH/
EtOH. The hydrochloride salt obtained in this way is a hygroscopic
amorphous yellow solid that becomes gummy upon exposure to
atmospheric moisture but solidifies again upon standing in a sealed
vial over a few days (1.53 g, 31%). 1H NMR (D2O) δ=8.57 (d, 1H,
3.6. 9-amino-N-[2-[(2-aminoethyl)amino]ethyl]-7-methoxyacridine-4-
carboxamide (2)
Acetyl chloride (5 mL) was added dropwise to stirred methanol
(30 mL) at room temperature with the evolution of heat. Compound
8b (0.45 g) was dissolved in methanolic HCl (35 mL) (prepared by
adding acetyl chloride (5 mL) to methanol 30 mL) and stirred for 3 h

M. Carland et al. / Journal of Inorganic Biochemistry 104 (2010) 815–819
819
at room temperature at which time TLC indicated the absence of
3.11. Cytotoxicity assays
starting material. Volatiles were removed under reduced pressure and
diethyl ether (20 mL) was added. The resulting suspension was
sonicated, and the solid collected by centrifugation. The product was
dried under reduced pressure to afford 2 as the trihydrochloride salt
which was a hygroscopic orange powder (0.35 g, 86%). 1H NMR (D2O)
δ=3.38–3.55 (m, 6H), 3.77 (s, 3H), 3.83 (t, 2H, J=6.4 Hz), 7.01 (s,
1H), 7.41 (d, 1H, J=9.2 Hz), 7.44 (d, 1H, J=9.2 Hz), 7.50 (t, 1H,
J=7.6 Hz), 8.21 (d, 1H, J=8.4 Hz), 8.25 (d, 1H, J=7.2 Hz). ESI-MS m/z
(relative intensity), 354.4 (95, [M+H]+), 303.3 (44). Anal. Calc. for
The IC50 values were determined in the human cell line HeLa using
an amylar blue assay as previously described [21,22]. Briefly this
involved seeding of 4×106 HeLa cells per well, growth for 24 h,
addition of amylar blue, further growth for 48 h, and measurement of
fluorescence at 590 nm after excitation at 544 nm. At least three
independent experiments were performed for each IC50 value. Solvent
only controls were also employed in each experiment.
C19H26N5O2.3HCl.MeOH requires: C, 48.54; H, 6.11; Cl, 21.49; N, 14.15.
3.12. Sequence selectivity studies
Found: C, 48.74; H, 6.24; Cl, 20.89; N, 13.79.
The DNA sequence specificity of the Pt complexes was determined as
previously described for purified pUC19 plasmid DNA [17,24–26] and
intact human cells [24,27,28]. The DNA sequence specificity studies
were performed on at least three occasions.
3.7. (9-amino-N-[2-[(2-aminoethyl)amino]ethyl]-7-methoxyacridine-4-
carboxamide)dichloroplatinum(II) (5)
Compound 2 (200 mg, 0.432 mmol) was dissolved in water
(10 mL) and the pH was adjusted to 8–9 with 1 M aqueous Na2CO3.
A solution of K2PtCl4 (179 mg, 0.432 mmol) in water (4 mL) was
added and the mixture was stirred in the dark for 24 h at room
temperature. A solution of 5% aqueous KCl (30 mL) was then added,
and the mixture was stirred for a further 2 h. The resulting solid was
collected and washed several times with distilled water. After
drying at 80 °C this material was ground to a fine powder, washed
several times with distilled water and dried to give 2 as an orange
powder (214 mg, 80%). 195Pt NMR (DMF) δ −2353.2. ESI-MS m/z
(relative intensity), 619.3 ([M+H]+, 22), 682.4 ([M−Cl]+, 45), 645.1
([M−HCl2]+, 12). Anal. Calc. for C19H23Cl2N5O2Pt requires C, 36.84;
H, 3.74; Cl, 11.45; N, 11.31. Found: C, 36.67; H, 3.69, Cl, 11.51; N, 11.27.
Acknowledgments
Support of this work by The National Health and Medical Research
Council (WDM, VM, WAD) is gratefully acknowledged.
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3.8. 9-amino-N-[2-[(N,N′-bis(tert-butoxycarbonyl)ethylenediamino)-
ethyl]-7-fluoroacridine-4-carboxamide (9b)
[9] G.V. Kalayda, B.A.J. Jansen, P. Wielaard, H.J. Tanke, J. Reedijk, J. Biol. Inorg. Chem.
10 (2005) 305–315.
This was prepared in a similar way to compound 8b but in this case
by reaction between 7-fluoro-9-acridone-4-caboxylic and compound
13. 1H NMR (DMSO) δ=1.2–1.4 (m, 18H), 3.10 (m, 2H), 3.35 (m, 2H),
3.54 (m, 2H), 3.71 (m, 2H), 6.82 (bs, exch), 7.49 (t, 1H, J=7.1 Hz),
7.72 (m, 1H), 8. 01 (m, exch), 8.28 (dd, 1H, J=1.8,7.1 Hz), 8.60 (d, 1H,
J=7.1 Hz), 8.66 (d, 1H, J=4.4 Hz). ESI-MS m/z (relative intensity),
542.2 ([M+H]+, 100). Anal. Calc. for C28H36FN5O5 requires: C, 62.09;
H, 6.70; N, 12.93. Found: C, 62.07; H, 6.84; N, 12.86.
[10] M. Lee, J.E. Simpson, A.J. Burns, S. Kupchinsky, N. Brooks, J.A. Hartley, L.R. Kelland,
Med. Chem. Res. 6 (1996) 365–371.
[11] H. Loskotova, V. Brabec, Eur. J. Biochem. 266 (1999) 392–402.
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2983–2987.
[13] B.D. Palmer, H.H. Lee, P. Johnson, B.C. Baguley, G. Wickham, L.P.G. Wakelin, W.D.
McFadyen, W.A. Denny, J. Med. Chem. 33 (1990) 3008–3014.
[14] L.C. Perrin, C. Cullinane, W.D. McFadyen, D.R. Phillips, Anticancer Drug Des. 14 (1999)
243–252.
[15] J. Whittaker, W.D. McFadyen, G. Wickham, L.P.G. Wakelin, V. Murray, Nucl. Acid.
Res. 26 (1998) 3933–3939.
3.9. 9-amino-N-[2-[(2-aminoethyl)amino]ethyl]-7-fluoroacridine-4-
carboxamide (3)
[16] R.J. Holmes, M.J. McKeage, V. Murray, W.A. Denny, W.D. McFadyen, J. Inorg.
Biochem. 85 (2001) 209–217.
[17] M.D. Temple, W.D. McFadyen, R.J. Holmes, W.A. Denny, V. Murray, Biochemistry
39 (2000) 5593–5599.
This was prepared in a similar way to 2 but in this case by reaction
between compound 9b (0.45 g) and HCl in methanol (0.33 g, 83%).
1H NMR (D2O) δ 3.25–3.35 (m, 6H), 3.70 (t, 2H, J=5.6 Hz), 7.36 (t, 1H,
J=7.2 Hz), 7.56 (m, 3H), 8.10 (d, 1H, J=8.4 Hz), 8.13 (d, 1H, J=7.2 Hz).
Anal. Calc. for C18H20FN5O.3HCl.MeOH requires: C, 47.27; H, 5.64;
N, 14.51; Cl, 22.03. Found: C, 46.95; H, 5.31; N, 14.53; Cl, 21.85.
[18] M.D. Temple, P. Recabarren, W.D. McFadyen, R.J. Holmes, W.A. Denny, V. Murray,
Biochim. Biophys. Acta 1574 (2002) 223–230.
[19] A.J. Geall, I.S. Blagbrough, Tetrahedron Lett. 39 (1998) 443–446.
[20] S.J. Kerrison, P. Sadler, J. Inorg. Chim. Acta 104 (3) (1985) 197–201.
[21] M. Carland, B.F. Abrahams, T. Rede, J. Stephenson, V. Murray, W.A. Denny, W.D.
McFadyen, Inorg. Chim. Acta 359 (2006) 3252–3256.
[22] M. Carland, K.J. Tan, J.M. White, J. Stephenson, V. Murray, W.A. Denny, W.D.
McFadyen, J. Inorg. Biochem. 99 (2005) 1738–1743.
[23] A. Adams, M. Guss, C. Collyer, W.A. Denny, L.P.G. Wakelin, Biochemistry 38 (1999)
9221–9233.
[24] V. Murray, Prog. Nucl. Acid Res. Mol. Biol. 63 (2000) 367–415.
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McFadyen, J. Biol. Chem. 267 (1992) 18805–18809.
3.10. (9-amino-N-[2-[(2-aminoethyl)amino]ethyl]-7-fluoroacridine-4-
carboxamide)dichloroplatinum(II) (6)
[26] V. Murray, J. Whittaker, M.D. Temple, W.D. McFadyen, Biochim. Biophys. Acta
1354 (1997) 261–271.
This was prepared in a similar way to 5 but in this case by reaction
between compound 3 (200 mg) and K2PtCl4 in water (209 mg, 78%).
195Pt NMR (NMP) δ −2351.4. ESI-MS m/z (relative intensity), 608.3
(100, [M+H]+), 571.3 (15, [M−Cl]+), 536 (12, [M−HCl2]+). Anal.
Calc. for C18H20Cl2FN5OPt.HCl requires: C, 33.58; H, 3.29; Cl, 16.52;
N, 10.88. Found: C, 33.51; H, 3.34; N, 10.92.
[27] V. Murray, H. Motyka, P.R. England, G. Wickham, H.H. Lee, W.A. Denny, W.D.
McFadyen, Biochemistry 31 (1992) (1817) 11812–11817.
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[29] G.W. Rewcastle, W.A. Denny, Synthesis (1985) 217–220.
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1481.