Bis(acridinylthiourea)platinum(II) complexes: Synthesis, DNA affinity, and biological activity in glioblastoma cells

Article abstract of DOI:10.1016/S0960-894X(02)01078-8

The preparation of two novel bis(acridine)platinum(II) complexes is reported. The 4+ charged conjugates associate strongly with double-stranded native DNA (K_i>10⁶), possibly through bisintercalation. A cell viability assay was used to demonstrate that both compounds are capable of mediating cytotoxicity at micromolar concentrations in SNB19 brain tumor cells.

Full text of DOI:10.1016/S0960-894X(02)01078-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 855–858
Bis(acridinylthiourea)platinum(II) Complexes: Synthesis, DNA
Affinity, and Biological Activity in Glioblastoma Cells
Todd M. Augustus,a Joel Anderson,b Suzanne M. Hessb and Ulrich Bierbacha,*
aDepartment of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA
bDepartment of Radiation Oncology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
Received 9 November 2002; revised 16 December 2002; accepted 16 December 2002
Abstract—The preparation of two novel bis(acridine)platinum(II) complexes is reported. The 4+ charged conjugates associate
strongly with double-stranded native DNA (Ki>106), possibly through bisintercalation. A cell viability assay was used to demon-
strate that both compounds are capable of mediating cytotoxicity at micromolar concentrations in SNB19 brain tumor cells.
# 2003 Elsevier Science Ltd. All rights reserved.
Recently, we have reported a new class of DNA-tar-
geted hybrid platinum–acridine agents that show cyto-
toxic activity at nano-to-micromolar concentrations in
solid tumor and leukemia cancer cell lines.1 The proto-
type, conjugate 1, was synthesized by replacing one
chloro leaving group in [PtCl2(en)] (en=ethane-1,2-dia-
mine), a cisplatin analogue, with the novel 9-amino-
acridine derivative, 1-[2-(acridin-9-ylamino)ethyl]-1,3-
dimethylthiourea (2a).1 Unlike intercalator-tethered cis-
second chloride by 2a (Scheme 1). The formation of 3a
in the above reaction mixtures is most likely due to the
high nucleophilicity of thiourea sulfur.8 Sulfur donors
exhibit a high affinity to divalent platinumand are
known to replace chloro ligands without prior solvolysis
of the Pt-Cl bond.9
To investigate the DNA interactions and possible bio-
logical effects of bis(acridinylthiourea)platinum(II)
complexes we synthesized 3a and the new derivative, 3b,
containing an acridinylthiourea with an extended pro-
pylene flexible linker chain (2b). The tethering of two
acridine moieties to platinum was achieved after
removal of chloride in the precursor with two equiva-
lents of silver nitrate, affording both complexes as their
nitrate salts (Scheme 2). The new acridine derivative,
1-[3-(acridin-9-ylamino)propyl]-1,3-dimethylthiourea (2b,
HNO3 salt), was generated using the synthetic scheme
developed for the prototype, 2a.1 Briefly, the synthesis
involved selective protection of the primary and
diaminedichloro complexes reported previously,2ꢀ6
1
does not induce bifunctional covalent adducts (cross-
links) in DNA but acts through a mechanism that
involves monofunctional platination and intercalation
of the planar chromophore into the DNA base stack.
This type of adduct, which causes local unwinding of
double-stranded DNA by 21ꢁ, is considered a potential
cytotoxic lesion of the drug.7 The sequence and groove
specificity of platinumbinding are currently under
investigation.
In unpurified preparations of conjugate 1, obtained
from reactions of equimolar amounts of platinum pre-
cursor and acridine, a minor impurity was observed. We
were now able to identify the side product as the corres-
ponding bis(acridinylthiourea)platinum(II) complex, 3a.
Monoactivation of [PtCl2(en)], achieved by abstraction
of one chloro ligand with silver ion, obviously does not
completely prevent the unwanted substitution of the
*Corresponding author. Tel.: + 1-336-758-3507; fax: + 1-336-758-
4656; e-mail: bierbau@wfu.edu
Scheme 1.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01078-8

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T. M. Augustus et al. / Bioorg. Med. Chem. Lett. 13 (2003) 855–858
secondary amino groups in N1-methylpropane-1,3-dia-
mine and attachment of the deprotected primary amino
nitrogen to C(9) in the acridine precursor, 9-phenoxy-
acridine. After removal of the tert-butyl carbamate
(Boc) group, the secondary amino group was reacted
with methylisothiocyanate followed by treatment of the
reaction mixture with HNO3 to give the desired hydro-
nitrate salt of the acridinylthiourea, 2b (Scheme 3).
NMR data confirm the formation of symmetrical bisin-
tercalator complexes with the two intercalators linked to
the metal center via thiourea sulfur.
Under physiological conditions, compounds 3a and 3b
exist as 4+ cations with protonated acridine moieties
(pKaꢂ9.81) and a 2+ charge localized on the divalent
platinumcenter. Strong DNA binding is predicted for
these compounds despite their inability to undergo
covalent DNA interactions due to the lack of substitu-
tion-labile chloride. Both agents have the potential to
associate with double-stranded DNA via bisintercala-
tion. Additional electrostatic forces and hydrogen
bonding involving thiourea- and en-NH groups may
also contribute to the DNA binding. Spectro-
photometric equilibrium binding titrations12 showed
that 3a and 3b bind to calf thymus DNA with high affi-
nity. Upon addition of nucleic acid to buffered solutions
of 3a and 3b, a red shift and decrease in absorbance of
1H NMR spectroscopy, combustion analyses, and,
where appropriate, 195Pt NMR spectroscopy were used
to characterize the new compounds, 2b, 3a, and 3b.10
195Pt chemical shifts of ꢀ3362 and ꢀ3345 ppmwere
found for 3a and 3b, respectively, characteristic of a
[N2S2] environment of platinum(II).11 The combined
acridine-based bands in the 350–450 nmregion was
13
observed. FromScatchard-type plots
we determined
the association constants (Ki) and the size of the binding
sites (n, number of nucleotides; n/2, number of base
pairs) for both complexes. The binding isotherms in the
intercalation region (at low drug-to-DNA base pair
ratios, r) were fitted to the neighbor exclusion model of
McGhee and von Hippel.14 The data are summarized in
Table 1. The bisacridines prove to be stronger binders
than the simple monointercalators 9-(methylamino)
acridine (9-MeAA) and 2a.7 Complex 3a showed an
approx. 5-fold higher affinity than 3b, possibly indicat-
ing more favorable intercalative DNA interactions of
the n=2 derivative. Two interesting trends emerged for
the set of compounds listed in Table 1 that deserve fur-
ther discussion. First, an increase in n is noted for 3a
and 3b compared to the simple acridines, 9-MeAA and
2a.7 While the values observed for the latter species are
consistent with each drug molecule occupying 4 nucleo-
tides (2 base pairs), indicative of classical intercala-
tion,13 the increased (approximately doubled) binding
site size for 3a and 3b suggests a bisintercalative binding
mode:
Scheme 2.
On the other hand, the notably reduced red shift and
hypochromicities found for 2a and the platinum-
bridged species 3a and 3b compared to simple 9-MeAA
possibly indicate that groove interactions of the
thiourea and platinum–thiourea linker groups allow
only partial intercalation of the planar chromophores
into the DNA base stack. Minor groove interactions
have recently been established for the thiourea residue
in 2a (distamycin A competition displacement assays,7
2-D NMR spectroscopy15). In summary, it appears that
alternative (non-specific) DNA–drug interactions com-
pensate for the reduced tendency of the acridine moiety
Scheme 3. Reagents and conditions: (i) 1. CF3COOEt, 2. (BOC)2O,
THF/0 ꢁC; (ii) OHꢀ/MeOH; (iii) 1. 9-PhOAcr/THF; 2. HCl/AcOH, 3.
2 M NH3; (iv) 1. MeNCS, 2. HNO3.

T. M. Augustus et al. / Bioorg. Med. Chem. Lett. 13 (2003) 855–858
857
Table 1. DNA binding data for acridines and bisacridines in calf
thymus DNA deduced from UV–visible equilibrium titrationsa
showed IC50 values in the low micromolar concen-
tration range.
Compd
Ki (Mꢀ1
)
n
Ál (nm)
% H
Literature
In conclusion, we have synthesized a novel type of plat-
inum-based bisacridine agent that strongly associates
with double-stranded DNA, possibly through bisinter-
calation. The preliminary biological data suggest that 3a
and 3b may have potential utility as DNA-targeted agents
in cancer chemotherapy. Platinum drugs have been
among the primary chemotherapeutic agents used against
malignant gliomas but have shown poor response rates.21
The platinum-acridines 1, 3a, and 3b are likely to act
through a mechanism different from that of current
platinum-based cross-linkers and may be more effective
treatments against this aggressive type of brain cancer.
Although high-molecular-weight polycationic com-
pounds such as 3a and 3b are unlikely to pass the
blood–brain barrier, novel strategies of local drug
delivery (interstitial chemotherapy) hold considerable
promise of improving the clinical potential of such
agents in neuro-oncology.22 Multimodality therapy,
even though not curative, is still the best strategy for the
long-termmanagement of this disease. 21 Another future
goal will therefore be to explore the use of the above
conjugates as radiation sensitizers in combination
chemotherapy.
9-MeAA
(1.1ꢃ0.2)ꢄ105
(1.5ꢃ0.2)ꢄ106
(1.0ꢃ0.1)ꢄ107
(2.2ꢃ0.3)ꢄ106
4.4
3.8
7.3
7.4
6
5
3
3
46
36
32
32
Ref 7
Ref 7
This work
This work
2a
3a
3b
aData were fitted to r/Cf=Ki(1ꢀnr)[(1ꢀnr)/(1ꢀ(nꢀ1)r]nꢀ1 with n,
number of nucleotides occupied by a single drug molecule; r, occupied
binding sites/total number of sites; Cf, concentration of free drug; Ki,
intrinsic binding constant, giving standard deviations (values are
means of two experiments). Spectral changes were followed at
lmax=413 nm; Ál=bathochromic shift; H=hypochromicity.
in 2a, 3a, and 3b to insert into the DNA base stack.
Cooperative binding of the acridine chromophores in 3a
and 3b, as previously reported for simple poly-
methylene-bridged bisacridines,16 was not observed.
Reversible DNA intercalators and bisintercalators are
of potential interest for their use as anticancer thera-
pies.17 The cytotoxic effect of most intercalators
belonging to the acridine family is closely related to the
ability of these compounds to inhibit (poison) DNA
topoisomerases.17 Previous studies have shown that 2a
and the corresponding conjugate 1 produce topo-medi-
ated DNA damage.18 To assess the biological activity of
the complexes 3a and 3b in cancer cells, we studied the
effect of both compounds on the viability of SNB19
glioblastoma cells using the trypan blue exclusion
assay.19 In a parallel set of experiments, rat astrocytes20
were exposed to drug to assess the effect of the new
agents on normal brain cells. Cisplatin, the clinical
agent, was included in this study for comparison of
effectiveness. Both cell lines were incubated at a drug
concentration of 2 mM. At this concentration, 3a and 3b
decreased the cell viability by 67 and 60%, respectively,
while leaving the astrocytes unaffected. Cisplatin was
markedly less active in SNB19, producing only 20%
non-viable cells. The data are summarized in Figure 1.
The cytotoxic effect observed in this assay has now been
confirmed in clonogenic survival assays performed in
our laboratory. In these experiments, which will be
reported in detail elsewhere, compounds 3a and 3b
Acknowledgements
This research was in part supported by the Cross-Cam-
pus Collaborative Research Fund of Wake Forest Uni-
versity and the American Cancer Society (Grant IRG
93-035-6).
References and Notes
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10. Synthesis and characterization of compounds. 1-[3-(Acri-
.
din-9-ylamino)propyl]-1,3-dimethylthiourea HNO3 (2b). This
derivative was synthesized fromthe precursor N-acridin-9-yl-
N0-methylpropane-1,3-diamine analogous to the procedure
Figure 1. Assessment of cell viability using the trypan blue exclusion
assay. Columns represent the average of two experiments. NT denotes
untreated cells. The incubation concentration was 2 mM in all cases.

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T. M. Augustus et al. / Bioorg. Med. Chem. Lett. 13 (2003) 855–858
reported for 2a (see ref 1). The purity of intermediates was
assessed by 1H NMR spectroscopy. 2b: Yield (final step, Scheme
3, iv): 66%. 1H NMR (300 MHz, MeOH-d4) d 2.27 (2H, q), 2.95
(3H, s), 3.07 (3H, s), 4.11 (2H, t), 4.19 (2H, t), 7.54 (2H, t), 7.76
(2H, d), 7.94 (2H, t), 8.50 (2H, d). Elemental analysis for
11. Bierbach, U.; Roberts, J. D.; Farrell, N. Inorg. Chem.
1998, 37, 717.
12. Spectrophotometric titrations–conditions: 10 mM Tris–
HCl buffer (pH 7.3), 150 mM NaCl, T=20 ꢁC, [3a, 3b]=40
mM. For a detailed description of the procedure see ref 7.
13. Jenkins, T. C. In Drug-DNA Interaction Protocols; Fox,
K. R., Ed.; Humana: Totowa, NJ, 1997; p 195.
14. McGhee, J. D.; von Hippel, P. H. J. Biol. Chem. 1974, 86,
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19. Trypan blue exclusion assay. Rat astrocytes and SNB19
cells were plated at densities of 5ꢄ104 cells in 60-mm dishes.
After reaching 70–80% confluency, cells were treated in
duplicate with 3a, 3b, and cisplatin at a concentration of 2
mM. Cells were incubated with drug for 48 h and then har-
vested. Both media and cells were collected. Cells were pelleted
and resuspended in 200 mL of trypan blue. The per cent cell
viability (viable cells/total cells) was determined for each
treatment group.
20. The preliminary data were generated with rat astrocytes
due to difficulties with identifying optimized growth media for
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.
C19H23N5O3S 0.5H2O (410.49), Found: C, 55.90; H, 5.84; N,
16.96; S, 7.75. Calcd: C, 55.59; H, 5.89; N, 17.06; S, 7.81.
Complexes 3a and 3b (tetranitrate salts). A mixture of 326 mg
(1.00 mmol) of [PtCl2(en)] and 338 mg (2.00 mmol) of AgNO3
in 10 mL DMF was stirred for 20 h in the dark. AgCl was fil-
tered off, and 2.00 mmol of the appropriate acridinylthiourea
was added to the filtrate. The mixture was allowed to stir for 6
h in the dark. DMF solvent was distilled off in vacuumat
roomtemperature, and the oily residue was redissolved in
approximately 250–300 mL of hot dry MeOH. The solution was
treated with activated carbon (100 mg) for 10 min and filtered
through Celite while hot. After the solutions were concentrated
to a final volume of 50 mL and stored at 4 ꢁC for 12 h, a brown-
ish-yellow crystal mass was obtained. The crude products were
recrystallized twice fromhot methanol to afford bright-yellow
needles, which were collected and dried at 60 ꢁC in vacuum. 3a:
Yield: 550 mg (45%). 1H NMR (300 MHz, D2O) d 2.66 (4H, t),
3.04 (6H, s), 3.31 (6H, s), 3.80 (4H, t), 4.17 (4H, t), 7.24 (4H, d),
7.40 (4H, t), 7.72 (4H, t), 7.90 (4H, d). 195Pt NMR (107.5MHz,
D2O): d ꢀ3362 (vs. Na2[PtCl6] standard). Elemental analysis for
C38H50N14O12PtS2 (1154.10), Found: C, 38.80; H, 4.48; N,
16.50; S, 5.40. Calcd: C, 39.55; H, 4.37; N, 16.99; S, 5.56. 3b:
Yield: 520 mg (47%). 1H NMR (300 MHz, D2O) d 2.01 (4H, q),
3.05 (6H, s), 3.27 (6H, s), 3.61 (4H, t), 3.84 (4H, t), 7.20 (4H, d),
7.27 (4H, t), 7.66 (4H, t), 7.77 (4H, d). 195Pt NMR (107.5MHz,
D2O) d ꢀ3345 (vs Na2[PtCl6] standard). Elemental analysis for
C40H54N14O12PtS2 (1182.15), Found: C, 40.39; H, 4.64; N,
16.26; S, 5.05. Calcd: C, 40.64; H, 4.60; N, 16.59; S, 5.43.