Design, synthesis, and biological activity of a novel non-cisplatin-type platinum-acridine pharmacophore

Article abstract of DOI:10.1021/jm010293m

Platinum-acridine conjugates were prepared from [PtCl₂(ethane-1,2-diamine)] and the novel acridinylthioureas MeHNC(S)NMeAcr (6) and MeHNC(S)NMe(CH₂CH₂)NHAcr (15) by replacing one chloro leaving group in the cisplatin analogue with thiourea sulfur. In HL-60 leukemia cells, IC₅₀ values for 7 (Pt-tethered 6) and 16 (Pt-tethered 15) were 75 and 0.13 μM, respectively. In the ovarian cell lines 2008 and C13*, 16 was active at micromolar concentrations and showed only partial cross-resistance with clinical cisplatin. Possible structure-activity relationships are discussed.

Full text of DOI:10.1021/jm010293m

4492
J . Med. Chem. 2001, 44, 4492-4496
Brief Articles
Design , Syn th esis, a n d Biologica l Activity of a Novel Non -Cisp la tin -typ e
P la tin u m -Acr id in e P h a r m a cop h or e
Elizabeth T. Martins,† Hemanta Baruah,† J aroslaw Kramarczyk,† Gilda Saluta,‡ Cynthia S. Day,†
Gregory L. Kucera,‡ and Ulrich Bierbach*,†
Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109, and Comprehensive Cancer Center
of Wake Forest University, Medical Center Boulevard, Winston-Salem, North Carolina 27157
Received J une 27, 2001
Platinum-acridine conjugates were prepared from [PtCl2(ethane-1,2-diamine)] and the novel
acridinylthioureas MeHNC(S)NMeAcr (6) and MeHNC(S)NMe(CH2CH2)NHAcr (15) by replac-
ing one chloro leaving group in the cisplatin analogue with thiourea sulfur. In HL-60 leukemia
cells, IC50 values for 7 (Pt-tethered 6) and 16 (Pt-tethered 15) were 75 and 0.13 µM, respectively.
In the ovarian cell lines 2008 and C13*, 16 was active at micromolar concentrations and showed
only partial cross-resistance with clinical cisplatin. Possible structure-activity relationships
are discussed.
In tr od u ction
developed to enhance the DNA affinity of well-estab-
lished therapeutics, such as nitrogen mustards8 and
platinum drugs.9 In the case of targeted platinum, the
classical cis-[PtX2A2] (X ) leaving group, A2 ) diamine
nonleaving group) unit has been linked to various
intercalating groups via flexible alkyl chains of varying
length.9 Temple et al.10 recently demonstrated that the
cis-diaminedichloroplatinum(II) moiety in 9-aminoacri-
dine-platinum complexes has a tendency to dictate the
sequence specificity of covalent attachment of platinum
on DNA. This results in cisplatin-like binding of the
conjugates to adjacent purines, unless prohibited by
steric strain in the polymethylene linkage. Our working
hypothesis for the discovery of structurally novel Pt-
containing pharmacophores demands that, to produce
non-cisplatin behavior, the metal needs to be completely
prevented from forming cross-links within extended
runs of consecutive purine bases on DNA. Toward this
objective, we have developed platinum complexes de-
rived from [PtCl2(en)] (en ) ethane-1,2-diamine), a
cisplatin analogue, by replacing one of the chloro leaving
groups with novel acridinylthioureas. The use of biden-
tate en instead of simple NH3 prevents trans-labilization
and undesired displacement of the nonleaving group by
sulfur and nitrogen donors.11 Thiourea sulfur was used
to covalently link acridine to platinum. The thermo-
dynamic stability of the Pt-S bond renders thiourea a
typical nonleaving group that should not be displaced
by DNA nucleophiles.11 As a consequence, platinum is
turned into a monofunctional DNA metalating group.
Monofunctional platinum chloroam(m)ine complexes,
such as [PtCl(NH3)3]+, however, are known to be
therapeutically inactive,6 and the cytotoxic potential of
sulfur-modified platinum would greatly rely on alterna-
tive lesions, such as a dual-binding mode involving
covalent and intercalative association.12
Platinum coordination compounds, such as cis-diam-
minedichloroplatinum(II) (cisplatin) and cis-diammine-
1,1-cyclobutanedicarboxylatoplatinum(II) (carboplatin),
are in clinical use for the treatment of malignancies of
the urogenital tract and other cancers.1 The overall
clinical success of cisplatin, as evidenced by the number
of long-term survivors among individuals afflicted with
advanced testicular cancer,2 is somewhat diminished by
intrinsic and acquired tumor resistance.3 The develop-
ment of thousands of direct cisplatin derivativessmost
recently accomplished by high-throughput synthesis and
screening methods4sreflects the common notion, that
the formation of bifunctional covalent adducts on DNA
is a prerequisite for cytotoxicity in platinum-based
agents.5 The 1,2 intrastrand cross-link between adjacent
purine bases formed by cisplatin is considered the
principal cytotoxic lesion of the drug.5 Structural ana-
logues of cisplatin, however, damage DNA in a way
similar to the parent drug and therefore cause similar
biological effects.6 In fact, to be considered for clinical
evaluation, a new platinum drug would need to dem-
onstrate unique biological properties that eliminate
multifactorial drug resistance and lead to a spectrum
of activity different from that of the clinical agents.
Therefore, future platinum drug candidates are unlikely
to be discovered by following the classical structure-
activity relationships for cisplatin analogues.
Duplex DNA is the biological target of the acridinium
group with intercalation being the predominant mode
of association.7 Intercalator-based drug conjugates were
* Corresponding author phone: (336)758-3507; fax: (336)758-4656;
e-mail: bierbau@wfu.edu.
† Department of Chemistry.
‡ Comprehensive Cancer Center.
10.1021/jm010293m CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/09/2001

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4493
Sch em e 1a
linker. The synthesis involved selective protection13 of
the amino groups in 8 and subsequent transformation
of the dangling, deprotected secondary amine in inter-
mediate 14 into thiourea. The rationale behind the use
of 8 instead of en was that the resulting trialkylated
thiourea derivative would show greater chemical ro-
bustness than an analogous dialkylated species. In the
presence of transition metal ions, mono- and disubsti-
tuted thioureas have been shown to undergo deproto-
nation14 and desulfurization15 of the SCN2 framework.
To produce the platinum-acridines, 2, 6, and 15a (the
HNO3 salt of 15) were reacted with the monoactivated
form of [PtCl2(en)], [PtCl(DMF-O)(en)]+. Bidentate en
instead of simple NH3 was introduced to avoid trans-
labilization of the nonleaving group by sulfur. The
reaction of 6 and 15a with platinum gave the desired
conjugates 7 and 16 in 62% and 58% yield, respectively.
S,N chelate formation precluded the isolation of an
analogous complex of 2 (Scheme 1). The decomposition
of target compound 3 to 3a in reaction mixtures of 6
and platinum was detected by 195Pt NMR spectroscopy.
a
Counter ions not shown. Reagents and conditions: (i) MeNCS/
EtOH/∆. (ii) [PtCl2(en)], AgNO3/DMF, dark, rt. (iii) aqueous
solution.
1H NMR spectroscopy, elemental analyses, and 195Pt
NMR spectroscopy, where applicable, were used to
identify the structures of the target compounds (see
Experimental Section). In addition, the single-crystal
X-ray structure of conjugate 7 was determined (Scheme
2). The structure confirms the formation of a 1:1
conjugate with 6 being linked to square-planar platinum
through thiourea sulfur. 195Pt NMR spectra taken over
a period of 2 days indicate that the [PtN2SCl] coordina-
tion (for both 7 and 16) persists in solution.11 In the solid
state, the sterically demanding acridin-9-yl group on N1
is oriented cis to sulfur and perpendicular to the planar
SCN2 thiourea framework (angle between planes is
Resu lts a n d Discu ssion
Ch em istr y. The synthetic chemistry used in this
study is summarized in Schemes 1 and 2. Two types of
acridine-thioureas were prepared. The derivatives 1-acri-
din-9-yl-3-methylthiourea (2) and 1-acridin-9-yl-1,3-
dimethylthiourea (6) were obtained by reaction of the
9-amino nitrogen in the appropriate 9-aminoacridines
with isothiocyanate. 1-[2-(Acridin-9-ylamino)ethyl]-1,3-
dimethylthiourea (15) was prepared by attaching the
primary amino nitrogen in N-methylethylenediamine
(8) to the planar chromophore via nucleophilic aromatic
substitution. In 15, the thiourea group and the 9-amino
nitrogen on acridine are separated by an ethylene
Sch em e 2a
a
Counter ions and crystal solvent not shown for clarity. Reagents and conditions: (i) MeNH2/MeOH/2 M NH3. (ii) MeNCS/EtOH/∆.
(iii) [PtCl2(en)], AgNO3/DMF/dark, rt. (iv) 1, CF3COOEt/THF/<10 °C; 2, (Boc)2O/THF/<10 °C. (v) dilute NaOH/48 h, 35 °C. (vi) NaOC6H5,
∆. (vii) THF/∆. (viii) HCl, CH3COOH/rt. (ix) 2 M NH3. (x) 15, MeNCS/EtOH/∆; 15a ,b, from 15 and 1 M HNO3 and 1 M HCl, respectively/
MeOH. (xi) 15a , [PtCl2(en)], AgNO3/DMF/dark, rt.

4494 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Brief Articles
Ta ble 1. Cytotoxicity Data for 7, 15b, and 16
IC50, µM
carries a rigidly linked, low-pKa acridine. Although the
mechanistic basis of this observation is still to be
explored, we suggest that the reason for the inactivity
of 7 may lie at the DNA level. The inability of acridine
in 7 to efficiently interact with the duplex, for electro-
static and/or steric reasons discussed above, may lead
to only minor structural alterations in DNA that
ultimately render the monofunctional adducts repair-
able and noncytotoxic. Thus, the ethylene linker in 16
may be a structural prerequisite for a sterically feasible
and electronically favored dual-binding mode. The
magnitude of changes in DNA conformation resulting
from such “quasibifunctional” adducts may ultimately
trigger downstream effects that lead to cell death.
Tumor resistance to cisplatin is multifactorial in
nature and is usually mediated by elevated levels of
glutathione, enhanced DNA repair, and impaired cel-
lular accumulation of the drug.3 The cisplatin-resistant
cell line chosen, C13*, exhibits a ca. 12-fold resistance
as compared to the parent cell line, 2008.18 Unmodified
acridine, 15b, and platinum-acridine, 16, partially
circumvent acquired resistance to cisplatin. Decreased
drug accumulation and reduced ability to form intra-
strand cross-links on DNA are the major determinants
of cisplatin resistance in C13*.19 The level of cross-
resistance established for 15b and 16 (1.7- and 2.5-fold,
respectively) appears to be in agreement with the
observed ca. 2-fold impairment of drug uptake in the
C13* subline.20 In the case of impaired uptake being
the only contributor to the residual cross-resistance, our
data would indicate that 16 acts through an alternative
mechanism (possibly at the DNA level) not susceptible
to cisplatin-specific detoxification and repair.
In summary, we have developed a prototypical plati-
num-acridine conjugate, 16, that proved to be cytotoxic
at micromolar concentrations. We have demonstrated
that through simple modification of chemical structure,
i.e., introduction of an ethylene spacer, an inactive
derivative can be turned into a deadly cell poison.
Although the cytotoxicity of 16 in 2008/C13* ovarian cell
lines does not indicate any advantage over that observed
for unmodified acridine, 15b, the reduced level of cross-
resistance indicates that 16 partially circumvents ac-
quired resistance to cisplatin in vitro. This finding and
the high potency of 16 in HL-60 leukemia cells suggest
potential clinical utility of this type of conjugate and
warrant further DNA binding and structure-activity
relationship studies within an extended series of struc-
tural derivatives and a broader range of cell lines.
compd
HL-60
2008
C13* a
7
15b
16
75
11
0.13
>100
>100
2.2
3.8
3.7 (1.7)b
9.6 (2.5)
a
C13* is the cisplatin-resistant variant of the 2008 cell line.
Values in parentheses are resistance factors, IC50,resistant
IC50,sensitive
b
/
.
88.0°). The bulkiness of the planar residue has a
pronounced effect on the molecular dynamics of 7, which
is slow on the NMR time scale, as evidenced by the
multiplicity and temperature dependence of the signals
in the 1H and 195Pt NMR spectra (see Supporting
Information). This effect is not observed for conjugate
16.
A critical difference between the acridinylthioureas
6 and 15 emerged with respect to their acid-base
chemistry. The basicity of endocyclic acridine nitrogen
is an important parameter that determines the proto-
nation state of the chromophore at physiological pH.
Cationic planar heterocycles usually exhibit high DNA
affinity.16 We determined a pKa of 9.8 (( 0.1) for 15. In
contrast, the apparent pKa of 6 in 25% MeOH was found
to be 3.6 (( 0.2), which extrapolates to a pKa around
4.0 in water.17 The lowered basicity of 6 as compared
to 15 is ascribed to the less efficient conjugation between
the “anilino” and endocyclic nitrogens as a result of the
orthogonal orientation of the thiourea and acridine
π-systems (see Scheme 2). Under physiological condi-
tions, 6 will be deprotonated, while 15 will exist as the
cationic acridinium form. This crucial difference should
be reflected in the DNA binding and biological activity
of the acridines and the corresponding platinum conju-
gates.
Cytotoxicity. 7, 15b, and 16 were studied for poten-
tial antitumor activity against HL-60 leukemia cells and
human ovarian 2008 (wild type) and C13* (cisplatin-
resistant) cell lines. 2 and 6 could not be solubilized with
N-dimethylformamide and dimethyl sulfoxide and were
not included in this study. The results are summarized
in Table 1. Conjugate 7 was inefficient at inhibiting cell
growth in the cell lines tested. In contrast, 15b (the
acridinium chloride salt of 15) and its platinum conju-
gate, 16, proved to be cytotoxic at micromolar concen-
trations. While 15b appears to be only moderately active
in HL-60 cells, tethering of the [PtCl(en)]+ fragment to
thiourea sulfur (giving 16) causes a dramatic increase
in cytotoxicity (approximately 85-fold) in this cell line.
15b was 5-fold more potent in the 2008 ovarian cell line
than in HL-60 cells. The opposite effect was observed
for 16, which was slightly less cytotoxic in the 2008 cell
line than the unmodified acridine, 15b. In C13*, a cell
line possessing acquired cisplatin resistance, 15b and
16, were markedly active and showed only low levels of
cross resistance with the clinical agent.
Exp er im en ta l Section
Ch em ist r y. (a ) Ma t er ia ls. 9-Chloroacridine (4) and 9-
phenoxyacridine (11) were synthesized according to known
procedures.21 All other reagents were obtained from common
vendors and used as supplied. [PtCl2(en)] was prepared
following the method described by Dhara22 for cisplatin by
simply replacing aqueous ammonia with ethane-1,2-diamine
(en).
1
(b) Gen er a l P r oced u r es. H NMR data were acquired on
From the structures of 7 and 16, it can be inferred
that the DNA binding of these agents must be distinctly
different from that of cisplatin, potentially involving
platination of nucleophilic sites and interactions of the
planar acridines with the base stack. Interestingly, 16,
which contains a high-pKa, flexibly linked acridine, is
ca. 580 times more potent in HL-60 cells than 7, which
a Bruker Avance 300 spectrometer. Chemical shifts (δ, ppm)
were referenced to residual solvent peaks. 195Pt NMR spectra
of 7 and 16 were recorded on a Bruker Avance 500 spectrom-
eter at 107.5 MHz. Aqueous K2[PtCl4] was used as external
standard, and 195Pt chemical shifts are reported vs [PtCl6]2-
.
The decomposition of 3 in D2O was followed by 195Pt NMR
spectroscopy using a similar setup and a sweep width of

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4495
500 000 Hz. The pKa values of 6 and 15 were determined
spectrophotometrically. Melting points were determined on a
Mel-Temp II apparatus and are uncorrected. Elemental analy-
ses were performed by Quantitative Technologies, Inc., Madi-
son, NJ .
Scott R. Wilson (School of Chemical Sciences, University
of Illinois, Urbana-Champaign) for the collection of
CCD X-ray data.
Su p p or tin g In for m a tion Ava ila ble: Detailed X-ray crys-
tallographic data for 7; synthetic procedures, including inter-
mediates and compounds not included in the cytotoxicity
studies; details of the pKa measurements and clonogenicity
assays; listing of analytical data for target compounds 7, 15b,
and 16. This material is available free of charge via the
Internet at http://pubs.acs.org.
(c) Syn th esis of Dr u g P r ototyp es. 1-[2-(Acr id in -9-
yla m in o)eth yl]-1,3-d im eth ylth iou r ea (15), 1-[2-(Acr id in -
9-ylam in o)eth yl]-1,3-dim eth ylth iou r ea Hydr on itr ate (15a),
a n d 1-[2-(Acr id in -9-yla m in o)eth yl]-1,3-d im eth ylth iou r ea
Hyd r och lor id e (15b). A solution of methylisothiocyanate
(2.60 g, 35.6 mmol) in 50 mL of absolute ethanol was added
dropwise within 15 min to a solution of 7.04 g (28.0 mmol) of
14 in 350 mL of absolute ethanol. The mixture was heated at
reflux for 6 h and passed, while hot, through a Celite pad.
Ethanol was removed using a rotary evaporator, yielding 10
g of a brownish yellow crystal mass. 5.00 g (15.4 mmol) of the
crude product was recrystallized from 150 mL of methanol. A
fraction of a golden yellow, microcrystalline 15 was obtained
after the solution was kept at 4 °C for 12 h. Yield: 3.30 g (66%);
mp 196 °C (dec). UV-Vis (MeOH): 395 (9143), 413 (12357),
Refer en ces
(1) Wong E.; Giandomenico, C. M. Current Status of Platinum-based
Antitumor Drugs. Chem. Rev. 1999, 99, 2451-2466.
(2) Oliver, R. T. D. Testicular Cancer. Curr. Opin. Oncol. 2001, 13,
191-198.
(3) Singh, G.; Dorward, A.; Moorehead, R.; Sweet, S. Novel Mech-
anisms of Resistance to Cancer Chemotherapy. Cancer J . 1995,
8, 304-307.
(4) Ziegler, C. J .; Silverman, A. P.; Lippard, S. J . High-throughput
Synthesis and Screening of Platinum Drug Candidates. J . Biol.
Inorg. Chem. 2000, 5, 774-783.
(5) J amieson, E. R.; Lippard, S. J . Structure, Recognition, and
Processing of Cisplatin-DNA Adducts. Chem. Rev. 1999, 99,
2467-2498.
(6) Farrell, N. Nonclassical Platinum Antitumor Agents: Perspec-
tives for Design and Development of New Drugs Complementary
to Cisplatin. Cancer Invest. 1993, 11, 578-589.
(7) Adams, A.; Guss, J . M.; Collyer, C. A.; Denny, W. A.; Wakelin,
L. P. G. Crystal Structure of the Topoisomerase II Poison
9-Amino-[N-(2-dimethylamino)ethyl]acridine-4-carboxamideBound
to the DNA Hexanucleotide d(CGTACG)2. Biochemistry 1999,
38, 9221-9233.
(8) Kohn, K. W.; Orr, A.; O’Connor, P. M.; Guziec, L. J .; Guziec, F.
S., J r. Synthesis and DNA Sequence Selectivity of a Series of
Mono- and Difunctional 9-Aminoacridine Nitrogen Mustards. J .
Med. Chem. 1994, 37, 67-72.
(9) See, for example: Pe´rez, J . M.; Lo´pez-Solera, I.; Montero, E. I.;
Bran˜a, M. F.; Alonso, C.; Robinson, S. P.; Navarro-Ranninger,
C. Combined Effect of Platination and Intercalation upon DNA
Binding of Novel Cytotoxic Pt-Bis(naphthalimide) Complexes.
J . Med. Chem. 1999, 42, 5482-5486.
(10) Temple, M. D.; McFadyen, W. D.; Holmes, R. J .; Denny, W. A.;
Murray, V. Interaction of Cisplatin and DNA-Targeted 9-Ami-
noacridine Platinum Complexes with DNA. Biochemistry 2000,
39, 5593-5599.
(11) Bierbach, U.; Roberts, J . D.; Farrell, N. Modification of Platinum-
(II) Antitumor Complexes with Sulfur Ligands. 2. Reactivity and
Nucleotide Binding Properties of Cationic Complexes of the
Types [PtCl(diamine)(L)]NO3 and [{PtCl(diamine)}2(L-L)](NO3)2
(L ) Monofunctional Thiourea Derivative; L-L ) Bifunctional
Thiourea Derivative) in Relation to Their Cytotoxicity. Inorg.
Chem. 1998, 37, 717-723.
(12) Sundquist, W. I.; Bancroft, D. P.; Lippard, S. J . Synthesis,
Characterization, and Biological Activity of cis-Diammineplati-
num(II) Complexes of the DNA Intercalators 9-Aminoacridine
and Chloroquine. J . Am. Chem. Soc. 1990, 112, 1590-1596.
(13) Xu, D.; Prasad, K.; Repic, O.; Blacklock, T. J . Ethyl Trifluoro-
acetate: A Powerful Reagent for Differentiating Amino Groups.
Tetrahedron Lett. 1995, 36, 7357-7360.
(14) Okeya, S.; Fujiwara, Y.; Kawashima, S.; Hayashi, Y.; Isobe, K.;
Nakamura, Y.; Shimomura, H.; Kushi, Y. Novel Bis(triph-
enylphosphine)platinum(II) Complexes Containing a Thiourea
or a 1,3-Diethylthiourea Dianion as an S,N-Chelating Ligand.
Chem. Lett. 1992, 1823-1826.
1
436 (9964). H NMR (MeOH-d4): δ 2.98 (3H, s), 3.03 (3H, s),
4.18 (2H, t), 4.38 (2H, br m), 7.32 (2H, t), 7.64 (2H, t), 7.82
(2H, d), 8.40 (2H, d). Anal. (C18H20N4S) C, H, S. N: calcd, 17.27;
found, 16.71.
The corresponding acridinium salts were generated by
adding 14 mL of a 1 M solution of the appropriate acid to 5.00
g (15.4 mmol) of crude 15 in 150 mL of methanol. 15a and
15b precipitated spontaneously as bright yellow needles, which
were filtered off, washed with small amounts of diethyl ether,
and dried in a vacuum.
15a . Yield: 3.90 g (66%); mp 230 °C (dec). 1H NMR (MeOH-
d4): δ 3.06 (3H, s), 3.09 (3H, s), 4.51 (4H, m), 7.55 (2H, t),
7.78 (2H, d), 7.96 (2H, t), 8.69 (2H, d). Anal. (C18H21N5O3S) C,
H, N, S.
15b. Yield: 3.51 g (63%); mp 240 °C (dec). 1H NMR (MeOH-
d4): δ 3.03 (3H, s), 3.10 (3H, s), 4.51 (4H, m), 7.55 (2H, t),
7.79 (2H, d), 7.96 (2H, t), 8.70 (2H, d). Anal. (C18H21N4ClS) C,
H, N, S.
[P tCl(en )(C18H21N4S)](NO3)2‚MeOH (16). A mixture of
0.652 g (2.00 mmol) of [PtCl2(en)] and 0.338 g (2.00 mmol) of
AgNO3 in 10 mL of anhydrous DMF was stirred at room
temperature in the dark for 14 h. Precipitated AgCl was
filtered off through a Celite pad, 0.740 g (1.91 mmol) of 15a
was added to the filtrate, and the suspension was stirred for
3 h in the dark. The solution was evaporated to dryness in a
vacuum at 30 °C yielding a yellow residue, which was dissolved
in 1.8 L of dry methanol. Activated carbon was added, and
the solution was stirred for 15 min. Carbon was filtered off,
and the solution was concentrated to a final volume of 150
mL. Crude 16 was obtained as a bright yellow solid after the
solution was stored for 24 h at 4 °C. The crude batch was
recrystallized from hot methanol. The solution was stored in
the refrigerator for 48 h to afford 16 as a microcrystalline
yellow solid, which was dried at 60 °C in a vacuum for 4 h.
Yield: 0.850 g (58%). 1H NMR (D2O): δ 2.62 (4H, s, broad base
due to unresolved Pt satellites), 2.87 (3H, s), 3.08 (3H, s), 3.37
(3H, s), 4.38 (2H, m) 4.42, (2H, m), 7.57 (2H, d), 7.62 (2H, t),
7.94 (2H, t), 8.21 (2H, d). 195Pt NMR (DMF-d7): δ -2873. Anal.
(C21H33N8ClO7PtS) C, H, N, Cl, S.
Cytotoxicity. Cytotoxicity assays were carried out as
described previously.23 Solutions of the drugs in saline were
prepared immediately before the incubations from 0.100 mM
stocks, which were protected from light and stored at -20 °C.
IC50 data (drug concentration at which colony growth was
inhibited by 50%) were calculated as a percentage of control
cells from logarithmic plots of drug concentration versus colony
counts. IC50 values are averages of two individual experiments,
with each incubation performed in quadruplicate.
(15) Brader, M. L.; Ainscough, E. W.; Baker, E. N.; Brodie, A. M.
Copper(II) Promoted Desulphurization of N-Phenylthiourea. The
Synthesis and X-ray Structure of [{Cu(bipy)(pc)2}2] (bipy ) 2,2′-
Bipyridine, pc ) Phenylcyanamide). Polyhedron 1989, 8, 2219-
2221.
(16) Neidle, S. DNA Structure and Recognition; IRL Press: Oxford,
UK, 1994; pp 74-83.
(17) Avdeef, A.; Comer, J . E. A.; Thomson, S. J . pH-Metric log P. 3.
Glass Electrode Calibration in Methanol-Water, Applied to pKa
Determination of Water-Insoluble Substances. Anal. Chem.
1993, 65, 42-49.
(18) IC50 values for cisplatin in the 2008 and C13* ovarian cell lines
used in this study were 3.1 and 37 µM, respectively (Chaney, S.
G., personal communication).
Ack n ow led gm en t. This work was supported by the
American Cancer Society (Grant IRG-93-035-6). We are
grateful to Dr. Stephen G. Chaney (University of North
Carolina, Chapel Hill) for providing the ovarian cancer
cell lines and for stimulating discussions. We thank Dr.
(19) J ekunen, A. P.; Hom, D. K.; Alcaraz, J . E.; Eastman, A.; Howell,
S. B. Cellular Pharmacology of Dichloro(ethylenediamine)-
platinum(II) in Cisplatin-Sensitive and Resistant Human Ova-
rian Carcinoma Cells. Cancer Res. 1994, 54, 2680-2687.

4496 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Brief Articles
(20) Mann, S. C.; Andrews, P. A.; Howell, S. B. Modulation of cis-
Diamminedichloro-platinum(II) Accumulation and Sensitivity by
Forskolin and 3-Isobutyl-1-methylxanthine in Sensitive and
Resistant Human Ovarian Carcinoma Cells. Int. J . Cancer 1991,
48, 866-872.
(21) Raulins, N. R. Acridines. In The Chemistry of Heterocyclic
Compounds, 2nd ed.; Vol. 9, Acridines; Acheson, R. M., Ed.;
Wiley: New York, 1973; pp 9-108 and literature cited therein.
(22) Dhara, S. C. A Rapid Method for the Synthesis of cis-[Pt(NH3)2-
Cl2]. Indian J . Chem. 1970, 8, 193-194.
(23) Cartee, L.; Kucera, G. L. Gemcitabine Induces Programmed Cell
Death and Activates Protein Kinase C in BG-1 Human Ovarian
Cancer Cells. Cancer Chemother. Pharmacol. 1998, 41, 403-412.
J M010293M