Preparation, DNA binding, and in vitro cytotoxicity of a pair of enantiomeric platinum(II) complexes, [(R)- and (S)-3- aminohexahydroazepine]dichloroplatinum(II). Crystal structure of the S enantiomer

Article abstract of DOI:10.1021/jm9607966

A pair of enantiomeric Pt(II) complexes, [Pt(R-ahaz)Cl₂] and [Pt(S- ahaz)Cl₂] (ahaz = 3-aminohexahydroazepine), has been investigated for their ability to bind enantioselectively to DNA. Improved synthetic procedures were developed for preparing both the ligands and the Pt complexes. The structure of the complex of the S enantiomer was determined by X-ray crystallographic methods. Crystals of [Pt(S-ahaz)C1₂] are orthorhombic, space group P2₁2₁2₁, with a = 6.917(1) ?, b = 11.167(1) ?, c = 12.373(2) ?, Z = 4, and the structure was refined to R = 0.023 (1505F). Molecular modeling techniques were used to investigate the role of steric interactions between the ligand and DNA in influencing the bifunctional binding of the two enantiomers, and it was found that the S enantiomer should bind more readily. The binding of the S enantiomer, to calf thymus DNA, was indeed found to be slightly greater than that for the R enantiomer though slightly less than that for cis-DDP. Assays of the proportion of monofunctional adducts showed that a substantially greater proportion of monofunctional adducts remained for the R enantiomer and cisplatin than for the S enantiomer. Each of the enantiomers was subjected to in vitro cytotoxicity assays using cultures of human bladder (BL13/0), lung and resistant lung (PC9 and PC9cisR), and prostate (DU145) cancer cells. The R enantiomer was found to be slightly more cytotoxic in the bladder cell line and may be less cytotoxic in the lung cell line but there were no significant differences in the resistant cell line nor in the prostate cell line. The two enantiomers were taken up equally by the bladder cancer cells.

Full text of DOI:10.1021/jm9607966

1090
J . Med. Chem. 1997, 40, 1090-1098
P r ep a r a tion , DNA Bin d in g, a n d in Vitr o Cytotoxicity of a P a ir of En a n tiom er ic
P la tin u m (II) Com p lexes, [(R)- a n d (S)-3-Am in oh exa h yd r oa zep in e]d ich lor o-
p la tin u m (II). Cr ysta l Str u ctu r e of th e S En a n tiom er
Ronald R. Fenton,1a Warren J . Easdale,1b H. Meng Er,1a Shaun M. O’Mara,1b Mark J . McKeage,1b
Pamela J . Russell,1b and Trevor W. Hambley*,1a
School of Chemistry, University of Sydney, NSW 2006, Oncology Research Centre, Prince of Wales Hospital,
NSW 2031, Australia
Received November 18, 1996X
A pair of enantiomeric Pt(II) complexes, [Pt(R-ahaz)Cl2] and [Pt(S-ahaz)Cl2] (ahaz ) 3-ami-
nohexahydroazepine), has been investigated for their ability to bind enantioselectively to DNA.
Improved synthetic procedures were developed for preparing both the ligands and the Pt
complexes. The structure of the complex of the S enantiomer was determined by X-ray
crystallographic methods. Crystals of [Pt(S-ahaz)Cl2] are orthorhombic, space group P212121,
with a ) 6.917(1) Å, b ) 11.167(1) Å, c ) 12.373(2) Å, Z ) 4, and the structure was refined to
R ) 0.023 (1505F). Molecular modeling techniques were used to investigate the role of steric
interactions between the ligand and DNA in influencing the bifunctional binding of the two
enantiomers, and it was found that the S enantiomer should bind more readily. The binding
of the S enantiomer, to calf thymus DNA, was indeed found to be slightly greater than that for
the R enantiomer though slightly less than that for cis-DDP. Assays of the proportion of
monofunctional adducts showed that a substantially greater proportion of monofunctional
adducts remained for the R enantiomer and cisplatin than for the S enantiomer. Each of the
enantiomers was subjected to in vitro cytotoxicity assays using cultures of human bladder (BL13/
0), lung and resistant lung (PC9 and PC9cisR), and prostate (DU145) cancer cells. The R
enantiomer was found to be slightly more cytotoxic in the bladder cell line and may be less
cytotoxic in the lung cell line but there were no significant differences in the resistant cell line
nor in the prostate cell line. The two enantiomers were taken up equally by the bladder cancer
cells.
In tr od u ction
cis-DDP (cis-[Pt(NH3)2Cl2]) and related compounds
are believed to effect their anticancer activity by forming
bifunctional adducts with DNA.2-4 However, which of
the bifunctional adducts is primarily responsible for the
anticancer activity has not been unequivocally estab-
lished. Our approach has been to design complexes to
F igu r e 1. Schematic representation of the enantiomers of [Pt-
(ahaz)Cl2].
interact stereoselectivity with DNA, forming only a
subset of the adducts and thereby acting as probes of
the Pt/DNA interactions.5-9 For example, we recently
was too flexible to impose the chirality at the nitrogen
reported on the DNA binding of a compound designed
atoms that we sought and have now turned our atten-
to bind interstrand but not intrastrand.9 The low
tion to chiral complexes with more rigid ligand frame-
works.
activity of this compound in vitro provides evidence that
the interstrand adduct is not the adduct primarily
In this context the ahaz ligand (shown in Figure 1)
was considered to be ideal because it contains an
endocyclic nitrogen atom and consequently has limited
possibilities for conformational rearrangement once
bound to a metal ion. The stereochemical properties of
Cu complexes of ahaz have been studied extensively.14-16
The complex [Pt(L-ahaz)Cl2] has been reported to have
antitumour activity,17 but no details have been provided.
responsible for effecting cell death and indirect support
for the notion that the intrastrand adduct is responsible.
We have also designed probes of the major intrastrand
adducts which are to GpG and ApG sequences.10 These
adducts are chiral, and chiral Pt complexes therefore
have the potential to react enantioselectively when
forming such adducts. Some years ago Kidani and
colleagues reported modest enantioselectivity for the
pair [Pt(R,R-1,2-chxn)Cl2] and [Pt(S,S-1,2-chxn)Cl2]
(1,2-chxn ) 1,2-cyclohexanediamine).11-13 Our first
approach to the design of chiral probes, [Pt(R,R-eap)-
Cl2] and [Pt(S,S-eap)Cl2] (eap ) N,N-diethyl-2,4-pen-
tanediamine) met with only partial success with enan-
tioselectivity based on in vitro cytotoxicity of less than
2:1.10 We found that the pentanediamine framework
Exp er im en ta l Section
Sp ectr oscop y. 1H and 13C NMR spectra were recorded at
200.13 and 50.239 MHz, respectively, on a Bruker AC 200F
spectrometer with solvents D2O and CDCl3 of 99.6% isotopic
purity. Chemical shifts are reported in ppm (δ) relative to
TMS or known solvent resonances used as internal calibrants.
Infrared spectra were obtained on a Biorad FTS-40 spectro-
photometer. Mass spectral data were recorded on Kratos
MS902 mass spectrometer. Optical rotations were measured
X Abstract published in Advance ACS Abstracts, March 1, 1997.
S0022-2623(96)00796-0 CCC: $14.00 © 1997 American Chemical Society

Enantiomeric Platinum(II) Complexes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1091
at the sodium D line (589 nm) on an Optical Activity POLAAR
2001 polarimeter at ambient temperatures.
P r ep a r a tion s: (R)- a n d (S)-3-Am in oh exa h yd r oa zep in e.
(R)-3-Aminohexahydroazepine was used as supplied by J PS
Chimie, Bevaix, Switzerland. (S)-3-Aminohexahydroazepine
was prepared by a modification of the method described by
Saburi et al.14 (S)-3-Aminohexahydroazepine (12.5 g, 98 mmol)
was dissolved in sodium-dried THF (250 mL) and cooled to 0
°C in an ice bath. Freshly crushed LiAlH4 (28.8 g) was added
in small portions, and after all the LiAlH4 had been added,
the mixture was left to stand until it reached room tempera-
ture, whereupon the reaction was refluxed for 48 h. After
being cooled to room temperature, the reaction mixture was
transferred to a large conical flask and then further cooled in
an ice bath. A solution of 57 mL of water in 100 mL of THF
was then added very slowly to the reaction mixture, and the
resultant mixture refluxed gently for 0.5 h. The mixed
hydroxide solids were removed by suction filtration, and the
filter cake was washed with boiling THF. The filter cake was
then broken up, refluxed in 1,4-dioxane for 1 h, and then
refiltered. All filtrates and washings were then combined, and
the solvents were removed at reduced pressure to leave the
crude ligand as a pale yellow oil. The crude product was then
distilled at reduced pressure (ca 0.05 mmHg) to give a colorless
oil in 60-65% yield.
F igu r e 2. ORTEP plot of [Pt(S-ahaz)Cl2] giving the crystal-
lographic atom numbering; 30% probability ellipsoids are
shown.
[(R)- a n d (S)-3-Am in oh exa h yd r oa zepin e]dich lor opla ti-
n u m (II). The complexes were first prepared by the Dhara
method.18 Briefly, K2[PtCl4] (Aldrich, 0.50 g, 1.2 mmol) was
dissolved in water (20 mL), and the solution was heated to 60
°C on a steam bath. Potassium iodide (0.83 g, 5 mmol) was
added, and the solution was stirred for a few minutes. The
ligand (1.2 mmol) was dissolved in the minimum volume of
methanol and added to the [PtI4]2- solution. The mixture was
stirred for 1 h and allowed to stand overnight. The product
was collected, washed with cold water, and transferred damp
to a flask containing silver nitrate (0.41 g, 2.41 mmol). After
12 h of stirring in the absence of light, the silver iodide was
removed. Aqueous sodium chloride (0.5 M) was added to the
filtrate until no further silver chloride precipitated. Solid
lithium chloride (1 g) was added, and the solutions were left
to slowly evaporate over silica gel. The dark yellow-orange
crystals were collected and washed with ice-cold water and
ethanol. Yields: [Pt(S-ahaz)Cl2] 0.32 g, 69%, [Pt(R-ahaz)Cl2]
0.26 g, 57%. Anal. (C6H14N2Cl2Pt) C, 19.0; H, 3.7, N, 7.4.
Found: [Pt(R-ahaz)Cl2] C, 19.0; H, 3.7; N 7.4; [Pt(S-ahaz)Cl2]
C, 19.4; H, 3.7; N, 7.4. Infrared spectra (recorded on a BIO-
RAD FTS spectrometer as a KBr pellet): νmax (cm-1) 3225 s,
3163 s, 3108 s, 2925 s, 2850 m, 1575 s, 1448 m, 1426 m, 1390
m, 1221 m, 1170 s, 1147 m, 1092 m, 1074 m, 1000 m, 960 m,
918 m, 836 m, 328 vs, 315 vs, 271 s.
[(S)-Am in oh exa h yd r oa zep in e]d ich lor op la t in u m (II).
The S enantiomer was subsequently prepared by an alterna-
tive method based on that described by Fanizzi et al.19
[Pt(DMSO)2Cl2] was prepared by literature methods.20
[Pt(DMSO)2Cl2] (0.42 g, 1.0 mmol) was suspended in methanol
(40 mL), and the ligand (0.20 g, 1 mmol) in 20 mL of methanol
was added. The resulting mixture was stirred until a clear
pale-yellow solution resulted (∼1.5 h) and then stirred for a
further 1.5 h. The solvent was removed on a rotary evaporator
at 40 °C. The product was dissolved in water (20 mL), excess
lithium chloride (0.5 g) was added, and the mixture was gently
warmed on a steam bath until the volume was reduced to 10
mL. The fine pale yellow crystals that resulted were filtered
off and washed with ice-cold water and ethanol. Further
evaporation of the solution yielded additional crops of product.
Total yield: 0.22 g, 57%. The IR spectrum obtained for each
of the crops was identical to that obtained from the product
synthesized via the [PtI4]2- intermediate.
Ta ble 1. Crystallographic Dataa
chemical formula C6H14Cl2N2Pt fw
380.18
P212121
21
a, Å
b, Å
c, Å
V, Å3
Z
6.917(1)
11.167(1)
12.373(2)
955.7(2)
4
space group
T, °C
λ, Å
0.71069
absorp coeff, cm-1 152.38
R
Rw
0.023
0.024
D
calcd, g cm-3
2.642
a
2 1/2
R ) ∑(||Fo| - |Fc||)/∑|Fo|, Rw ) (∑w(|Fo| - |Fc|)2/∑wFo
) .
Ta ble 2. Bond Lengths (Å) for [Pt(S-ahaz)Cl2]
Pt(1)-Cl(1)
Pt(1)-N(1)
N(1)-C(1)
N(2)-C(2)
C(2)-C(3)
C(4)-C(5)
2.320(2)
2.044(6)
1.48(1)
1.51(1)
1.54(1)
1.52(1)
Pt(1)-Cl(2)
Pt(1)-N(2)
N(1)-C(6)
C(1)-C(2)
C(3)-C(4)
C(5)-C(6)
2.312(2)
1.995(7)
1.517(9)
1.52(1)
1.50(1)
1.49(1)
Ta ble 3. Bond Angles (deg) for [Pt(S-ahaz)Cl2]
Cl(1)-Pt(1)-Cl(2)
Cl(1)-Pt(1)-N(2)
Cl(2)-Pt(1)-N(2)
Pt(1)-N(1)-C(1)
C(1)-N(1)-C(6)
N(1)-C(1)-C(2)
N(2)-C(2)-C(3)
C(2)-C(3)-C(4)
C(4)-C(5)-C(6)
94.52(9)
174.9(2)
89.6(2)
108.6(5)
112.4(6)
110.6(7)
109.9(7)
116.9(7)
116.4(6)
Cl(1)-Pt(1)-N(1)
Cl(2)-Pt(1)-N(1)
N(1)-Pt(1)-N(2)
Pt(1)-N(1)-C(6)
Pt(1)-N(2)-C(2)
N(2)-C(2)-C(1)
C(1)-C(2)-C(3)
C(3)-C(4)-C(5)
N(1)-C(6)-C(5)
91.6(2)
172.9(2)
84.1(3)
116.7(5)
112.1(5)
105.4(7)
114.9(7)
113.7(7)
116.5(7)
of Lorentz, polarization, and analytical absorption corrections
were carried out using the teXsan system.21
Str u ctu r e Solu tion . The structure was solved by direct
methods using SHELXS-86.22 Hydrogen atoms were included
at calculated sites with fixed isotropic thermal parameters.
All other atoms were refined anisotropically. Full-matrix
least-squares methods were used to refine an overall scale
factor, positional, and thermal parameters. Neutral atom
scattering factors were taken from Cromer and Waber.23
Anomalous dispersion effects were included in Fc;24 the values
for ∆f′ and ∆f′′ were those of Creagh and McAuley.25 The
values for the mass attenuation coefficients are those of Creagh
and Hubbell.26 All calculations were performed using the
teXsan21 crystallographic software package of Molecular Struc-
ture Corp., and plots were drawn using ORTEP.27
Cr ysta llogr a p h y: Str u ctu r e Deter m in a tion . For dif-
fractometry a crystal was mounted on a glass fiber with
cyanoacrylate resin. Lattice parameters at 21 °C were deter-
mined by least-squares fits to the setting parameters of 25
independent reflections, measured, and refined on an Enraf-
Nonius CAD4F four-circle diffractometer employing graphite
monochromated Mo KR radiation. Intensity data were col-
lected in the range 1 θ < 27.5°. Data reduction and application
The atom numbering scheme is given in Figure 2, and bond
lengths and angles are given in Tables 2 and 3. Observed and
calculated structure factors, non-hydrogen atom positional and
thermal parameters, hydrogen atom coordinates and thermal
parameters, torsion angles, close nonbonded contacts, and

1092 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Fenton et al.
F igu r e 3. (a) Concentration dependent platinum-DNA binding curves for cisplatin (+), [Pt(R-ahaz)Cl2] (O), and [Pt(S-ahaz)Cl2]
(b). (b) Time-dependent platinum DNA binding curves for cisplatin (+), [Pr(R-ahaz)Cl2] (O), and [Pt(S-ahaz)Cl2] (b) at an Rt
value of 3.3 × 10-2
.
details of least-squares planes calculations (Tables S1-S7) are
available as Supporting Information.
solution at 4 °C. Dialysis was carried out using a BRL
microdialysis system.
Graphite furnace atomic absorption spectroscopy (GFAAS)
was performed on a Varian SpectrAA-20 absorption spectrom-
eter equipped with a GTA-96 graphite tube atomizer and a
PSC-56 autosampling system. UV absorbance was measured
using a Hewlett-Packard diode array spectrometer.
The solutions of the platinum complexes were prepared
immediately before being used by dissolving in TE buffer (10
mM Tris, 0.1 mM EDTA, pH 7.4 adjusted with 10 M HCl) with
heating and sonication. These were filtered through 0.22 µm
cellulose acetate filters (MFS).
Calf thymus DNA (25 µg) was reacted with the platinum
complexes in TE buffer in a total volume of 500 µL. The final
platinum concentrations ranged from 0 to 150 µM, which
corresponded to R (molar ratio of platinum added to nucleotide)
of 0 and 0.98, respectively. The reaction mixtures were
incubated at 37 °C for 24 h and were then stopped by the
addition of 56 µL of 2 M NaCl. Any unbound platinum species
were removed by dialysis against 3 L of TE buffer at 4 °C.
The concentration of DNA in each reaction mixture was
determined by measuring the absrobance at 260 nm (ú260
{DNA} ) 6600 M-1 cm-1). The bound platinum concentration
was analyzed using GFAAS. The results are presented as
plots of the Rb (molar ratio of platinum bound to nucleotide)
Molecu la r Mod elin g. DNA starting models (B-DNA,
d(GpGpGpG*pG*pGpGpG):d(CpCpCpCpCpCpCpC)) were con-
structed using the HyperChem program.28 A Pt atom was first
bonded to the N7 atom of the 5′-guanine using HyperChem.
A bifunctional adduct was then produced by reducing the
distance between the Pt atom of the N7 of the 3′-guanine using
constrained minimization in MOMEC-91.29 Models of [Pt(R-
ahaz)Cl2] and [Pt(S-ahaz)Cl2] were generated using Hyper-
Chem and were energy minimized using MOMEC-91 prior to
docking with the bifunctionally platinated DNA in the ap-
propriate orientations to produce the different isomers. These
adducts were then energy minimized until the RMS shift was
less than 0.05 Å. The force fields used for modeling the Pt
complexes and the DNA were those described previously.7
DNA Bin d in g. Con cen tr a tion Dep en d en ce. Calf thy-
mus DNA and Tris [tris(hydroxymethyl)aminomethane] were
obtained from Sigma. The disodium salt of EDTA dihydrate
was obtained from Merck. The 12-14 kDa dialysis membrane
was purchased from Selby and was prepared by boiling in a
solution containing 2% w/v sodium bicarbonate and 1 mM
EDTA (pH 8.0) for 10 min, followed by rinsing thoroughly in
distilled water and further boiling in 1 mM EDTA (pH 8.0)
for 10 min. The membrane was cooled and stored in this

Enantiomeric Platinum(II) Complexes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1093
against the treated platinum concentrations in Figure 3a.
Cisplatin was included in the assay for comparison.
Tim e Dep en d en t Stu d ies. The rates of platination of
DNA by cisplatin and the enantiomers of [Pt(ahaz)Cl2] were
measured using a similar method to that described above.
Briefly, calf thymus DNA (25 µg) was treated with 5 µM of
the platinum complexes (which corresponded to an Rt value
of 3.3 × 10-2) in TE buffer. The mixtures were incubated at
37 °C for various time periods between 0 and 24 h, after which
the reactions were terminated by the addition of NaCl (2 M,
56 µL). The samples were then dialyzed, and the Rb values
shown in Figure 3b were determined as described above.
Ra te of Mon oa d d u ct to Dia d d u ct Con ver sion . The rate
of monoadduct to diadduct conversion was measured using a
method reported by Chaney et al.30 Salmon sperm DNA
(Sigma) (1 mg mL-1, 2 mL) was incubated with freshly
prepared platinum complexes (600 µM, 2 mL) in 25 mM NaCl,
0.2 mM EDTA, and 2 mM Tris-HCl, pH 7.4, at 37 °C for 30
min. The reactions were stopped by adding NaCl (2.5 M, 1
mL) and cooling to 4 °C. The samples were dialyzed against
1.5 L of high-salt TEN 7.4 buffer (10 mM Tris-HCl, 10 mM
NaCl, 1 mM EDTA, pH 7.4, plus 0.5 M NaCl) at 4 °C (BRL
Microdialysis System, Selby 12-14 Kdal dialysis membrane)
for 2 h. The DNA samples were then precipitated with 4 mL
of 2-propanol (Sigma, molecular biology grade) and centrifuged
at 10 000 rpm at 4 °C, and the supernatants were decanted.
These were resuspended in 2 mL of high-salt TEN 7.4 buffer
and dialyzed against 1.5 L of 10 mM NaClO4 at 4 °C for 2 h.
The Rb values for the total platinum bound were measured as
described above.
The above samples were divided into 200 µL aliquots and
incubated at 37 °C for various time periods between 0 and 24
h. The level of monoadduct at each time point was determined
by treatment with 20 µL of [14C]thiourea (Sigma) (110 mM,
5.23 µCi µmol-1, deionized over Bio-Rad AG501X8 resin) at
37 °C for 10 min. The reactions were stopped by adding 2 mL
of cold 5% TCA (Sigma) and cooling on ice. The samples were
then centrifuged at 10 000 rpm, and the supernatants were
removed by aspiration.
The DNA pellets were redissolved in 200 µL of 0.1 M NaOH
and then reprecipitated with 2 mL of cold 5% TCA. After the
supernatants were removed by aspiration, the samples were
dissolved in 200 µL of 0.1 M NaOH. Aqueous scintillant
(Ultima Gold XR Scintillant) (1 mL) was added to each sample
and mixed thoroughly, and the radioactivity of [14C]thiourea
bound to DNA was counted on a Wallac 1410 liquid scintilla-
tion counter. The background count was determined by an
unplatinated DNA sample treated according to the procedure
above.
F igu r e 4. Schematic representation of the possible isomers
formed when Pt(R-ahaz)2+ and Pt(S-ahaz)2+ bind to intra-
strand GpG sequences of DNA.
phosphate-buffered saline (PBS) followed by 200 µL of warm
tissue culture medium. The viable cell concentrations were
determined using either the MTT assay34 or the SRB (Sulfo-
rhodamine B) assay.35
MTT Assa y. Cell viability was measured as the activity of
the mitochondrial succinate dehydrogenase.34 MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in FCS-
free RPMI tissue culture medium (1 mg mL-1, 200 µL) was
added into each well, and the plates were incubated at 37 °C
for 4 h. After centrifugation and removal of the MTT solution,
the formazan formed at the bottom of the wells was solubilized
with 2-propanol (200 µL). The optical density at 570 nm for
the solution in each well was determined using a Dynatech
MR 5000 plate reader.
SRB Assa y. In this assay, sulforhodamine B was used to
stain the basic amino acids.35 After the cells in each well were
fixed with 200 µL of ice-cold 10% trichloroacetic acid for 30
min at 4 °C, the plates were washed five times with tap water.
The fixed cells were stained with 100 µL of 0.4% sulfo-
rhodamine B dissolved in 1% acetic acid for 15 min at room
temperature. The stain was then removed, and the plates
were washed five times with 1% acetic acid and left to air dry
overnight. Tris (10 mM, 100 µL) was added to solubilize the
dye in each well, and the absorbance at 570 nm was read using
a Dynatech MR 5000 plate reader.
In both assays, the percentage of viable cells was calculated
by comparing the absorbance of the platinum-treated cells to
that of the untreated controls. Data were represented as the
percentage of control versus the concentration of complex. The
cell viability for each concentration was an average value for
four or six replicates. The IR50 values for the drugs (the
concentration required to kill 50% of the cell population) was
determined from the dose-response curves.
Cellu la r Up ta k e of P la tin u m Com p lexes. The uptake
of platinum complexes in human bladder tumour cells (BL13/
0) was measured using a modification of a previously described
method.36 The cells were grown to 80% confluence in 60 × 15
mm tissue culture dishes. The freshly prepared solutions of
platinum complexes in 0.9% (w/v) NaCl (filtered through sterile
0.22 µm cellulose acetate filters) were diluted to concentrations
ranging from 0 to 100 µM with RPMI 1640 tissue culture
medium supplemented with 10% FCS, penicillin, and strep-
tomycin. Three tissue culture dishes were set up for each
platinum concentration.
Cytotoxicity Assa ys. The cell lines tested were human
bladder tumor cells (UCRU BL 13/0),31 lung cancer cells (PC9
and PC9cisR), and prostate cancer cells (DU145). (Note: PC9
is a cisplatin-sensitive cell line and its resistant variant,
PC9cisR, has decreased intracellular drug accumulation and
elevated cellular glutathione levels.32,33
)
The tumor cells were grown as a monolayer culture in RPMI
1640 culture medium, which was supplemented with 10%
foetal calf serum (FCS), penicillin, and streptomycin. Expo-
nentially growing cells were maintained in a humidified
incubator at 37 °C in an atmosphere of 7.5% CO2, 5% O2, and
85% N2 until 60-80% confluence. The pH of the culture
medium was monitored with phenol red and maintained at
7.2. For subsequent drug assays, the cell monolayers were
washed with Hanks buffered saline and treated with trypsin-
EDTA to produce single cell suspensions. The cells were
seeded at a concentration of 5000 cells per well in 100 µL of
tissue culture medium in 96-well microtitre plates and incu-
bated at 37 °C for 24 h.
The platinum complexes were dissolved in 0.9% (w/v) NaCl
saline with gentle heating and sonication immediately prior
to use and filtered through 0.22 µm cellulose acetate filters.
The solutions were diluted with the tissue culture medium,
and 100 µL portions were added into the wells so that the final
concentrations in the wells ranged from 0 to 200 µM. After
96 h of drug exposure at 37 °C, the drug-containing medium
was removed and the cells were washed with 200 µL of warm
After the tissue culture medium was removed by aspiration,
the monolayered cells in each tissue culture dish were treated
with 5 mL of the drug solutions at 37 °C for 2 h. The drug-
containing tissue culture medium was then removed, and the

1094 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Fenton et al.
F igu r e 5. Energy-minimized models of the isomers formed by the binding of Pt(R-ahaz)2+ and Pt(S-ahaz)2+ to GpG sequences of
DNA.
cells were washed three times with 25 mL of ice-cold PBS. The
cells were scraped with 2 × 250 µL of ice-cold PBS, and the
cell suspensions were lysed using a probe sonicator. The
protein concentrations in the cell lysates were determined in
duplicates using the Lowry assay,37 and the platinum contents
were analyzed using GFAAS.
To reduce the background absorption in the GFAAS mea-
surements, the cell extracts were centrifuged for 5 min at
10 000 rpm. The supernatants were premixed with one
volume of modifier (0.01% Triton X-100, 0.01% HNO3 solution)
in order to reduce the sample viscosity. The [K2PtCl4] stan-
dards were introduced with one volume of modifier into the
graphite partition tube such that the final concentrations of
the Pt standards ranged from 0.0 to 141.0 ppb. Three
replicates were performed for the standards, and the samples
were measured in duplicates. 2-Propanol (10%) was included
into the sample dispenser cleaning fluid to prevent sample
carry over.
Resu lts a n d Discu ssion
Syn th eses. Reduction of the caprolactam, (S)-3-
aminohexahydroazepinone, with LiAlH4 proved to be a

Enantiomeric Platinum(II) Complexes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1095
Ta ble 4. Minimized Strain Energies for the Isomers Formed
When Pt(R-ahaz)2+ and Pt(S-ahaz)2+ Bind to Intrastrand GpG
Sequences of DNA
highly effective means of generating the ahaz ligand in
high yield and purity. The complexes were initially
synthesized via the [PtI4]2- intermediate method first
described by Dhara.18 However, this is a relatively
involved and time-consuming method and is less ef-
ficient when the ligand has secondary amine donor
groups. In addition solubility problems were encoun-
tered when using the Dhara method. Therefore, we also
refined the preparation of the S enantiomer using cis-
[Pt(DMSO)2Cl2] as a starting material. The method
used is based on that described by Fanizzi et al.19 and
produces a [PtL(DMSO)Cl] intermediate. Care must be
taken to avoid contamination by this intermediate in
the final complex; however, we found that addition of
excess lithium chloride yielded the pure [PtLCl2] com-
plex in high yield. IR spectroscopy is a sensitive method
for establishing that no DMSO remains in the final
product. Reliable, high-yield synthesis of the complex
sought were obtained in 3-4 h.
Descr ip tion of th e Str u ctu r e. The structure con-
sists of neutral [Pt(S-ahaz)Cl2] molecules with only one
weak H-bond between a H(amine) atom of one molecule
and a chloro ligand of a symmetry related molecule
[H(2)‚‚‚Cl(2)i 2.47 Å, i ) 0.5 + x, 0.5 - y, -z]. There
are more distant intramolecular contacts between the
H(amine) atoms and the chloro ligands (H‚‚‚Cl 2.93-
3.14 Å). The situation is reversed in [Pt(R,R-eap)Cl2]
where the intramolecular H‚‚‚Cl contacts are shorter
(H‚‚‚Cl 2.37-2.57 Å) than the closest of the intermo-
lecular contacts (H‚‚‚Cl 2.83-3.06 Å) because the
H(amine) atoms lie close to the coordination plane.10 We
suggested that the close intramolecular contacts in [Pt-
(R,R-eap)Cl2] stabilized the particular diastereomer
observed.10 Evidently, such interactions do not influ-
ence the conformational geometry in the present struc-
ture. The least-squares plane through the Pt atom and
the four donor atoms shows deviations up to 0.11 Å. The
deviations from planarity are unusual in that the four
donor atoms lie on the same side of the Pt atom, with
the deviations being greatest for the N atoms. The five-
membered chelate ring adopts a slightly flattened,
asymmetric conformation as indicated by the N-C-
C-N torsion angle of -48.1(9)° and the unequal deiva-
tions of C(1) (0.50 Å) and C(2) (-0.16 Å) from the
aforementioned least-squares plane. The seven-mem-
bered hexahydroazepine ring adopts a “skew chair”
conformation in which four atoms, N(1), C(2), C(3), and
C(6), define the approximately planar seat (deviations
less than 0.12 Å), C(1) defines the back, and C(4) and
C(5) lie skew to the plane. Similar conformations have
been observed in other complexes of the ahaz ligand.14-16
Somewhat surprisingly this conformation results in a
close contact (2.72 Å) between the Pt atom and one of
the H atoms bonded to C(4). Given the flexibility of the
seven-membered ring (gross disorder has been observed
in some structures), this contact might have been
avoided if an alternative conformation had been adopted.
This leads the question as to whether the contact
represents a favorable (agostic) interaction or a weakly
unfavorable nonbonded interaction. We have used
molecular modeling techniques to investigate such
interactions in this and related complexes and have
concluded that the observations are consistent with the
interaction being a weakly unfavorable nonbonded
interaction if the van der Waals radius of Pt is in the
isomer
strain energy (kJ mol-1
)
R(a)
R(b)
S(a)
S(b)
-2468
-2476
-2478
-2483
range 1.6-1.8 Å.38 The Pt-Cl bond lengths are not
unusual, but the two Pt-N amine bond lengths differ
significantly from one another. The Pt-N bond to the
primary amine is at the short end of the range and that
to the secondary amine is at the long end of the range.
Molecu la r Mod elin g. The GpG adducts formed
when Pt(R-ahaz)2+ and Pt(S-ahaz)2+ bind to the starred
site in the B-DNA sequences d(GpGpGpG*pG*pGp-
GpG): d(CpCpCpCpCpCpCpC) were modeled. Pt(R-
ahaz)2+ and Pt(S-ahaz)2+ can bind to intrastrand GpG
sequences of DNA in two ways: with the primary amine
cis to the 5′ N7(guanine) atom (hereafter referred to as
isomer a) or with the primary amine trans to this atom
(isomer b). The four possibilities are shown schemati-
cally in Figure 4, and the energy minimized adducts are
shown in Figure 5. The minimized strain energies are
given in Table 4. The two isomers formed by the R
enantiomer are less stable than those formed by the S
enantiomer and the isomers with the primary amine cis
to the 5′-guanine are less stable than the isomers where
it is cis to the 3′-guanine. In the case of isomer R(a),
no hydrogen bonding is possible between an amine
proton and the 3′ O6 (guanine); instead there are close
contacts (2.62, 2.65 Å) between H(carbon) atoms of the
ahaz ring and this O atom. Isomer R(b) has the ahaz
ring disposed away from the floor of the major groove,
but the ring makes a close H‚‚‚H contact (2.52 Å) with
H8 of the 5′-guanine and a second longer contact (2.76
Å) to the H8 of the guanine on the 5′ side of the GpG
pair. There is a hydrogen bond between the primary
amine and O6 of the 3′-guanine (H(N)‚‚‚O, 2.04 Å).
Isomer S(a) also has the ahaz ring disposed away from
the floor of the major groove, but because the bulk of
the ring is oriented in the 3′ direction, no contact with
H8 of the 5′-guanine or any other part of the DNA is
observed. Indeed it is clear from Figure 5 that this
orientation gives the least encumbered “fit” of the ahaz
ring into the major groove. There is a hydrogen bond
between the secondary amine and the O6 atom of the
3′-guanine. Isomer S(b) has a hydrogen bond between
the primary amine and the 3′ O6 atom, but the ahaz
ring is disposed toward the floor of the major groove.
There are three close contacts between the ring and the
DNA; 2.72 and 2.88 Å to the O6 and N7 atoms
respectively of the 5′-guanine and 2.76 Å to the N7 of
the guanine base at the 5′ side of the GpG pair. These
observations are in general accord with the minimized
strain energies, except that we might have expected
isomer S(a) to be more stable than isomer S(b). The
precision and accuracy of strain energies from calcula-
tions of molecules of this size is unknown; however,
taken with the detailed observations, there is a clear
indication that the S enantiomer should bind more
readily than the R.
DNA Bin d in g. The concentration and time depend-
ent DNA binding assays reveal that, at all concentra-
tions, cisplatin binds most readily, S-ahaz next, and

1096 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Fenton et al.
F igu r e 6. Monoadduct to diadduct conversion for cisplatin (+), [Pt(R-ahaz)Cl2] (O), and [Pt(S-ahaz)Cl2] (b).
Ta ble 5. Cytotoxicity (IC50) Values (µm) Determined in
R-ahaz least readily. Thus, these results accord with
the predictions based on molecular mechanics modeling.
That cisplatin binds more readily than either [Pt(S-
ahaz)Cl2] or [Pt(R-ahaz)Cl2] is not unexpected, given its
lesser steric bulk. What is surprising is that the
difference is not greater; however, differences in the
rates of hydrolysis that are dependent on the degree of
substitution at the N donor atoms may play a role. Also,
these assays are measuring total Pt binding and not
discriminate between monofunctional and bifunctional
binding.
Human Tumor Cell Linesa
IC50 (µM)
platinum
complexes
UCRU BL13/0
PC9
PC9cisR DU145
MTT Assay
cisplatin
[Pt(R-ahaz)Cl2]
[Pt(S-ahaz)Cl2]
1.9(5)
1.3(2)
2.0(2)
1.4(2)
5.9(1)
3.0(4)
11.7(31) 1.8(8)
16.0(10) 2.9(1)
15.5(13) 3.4(3)
SRB Assay
cisplatin
[Pt(R-ahaz)Cl2]
[Pt(S-ahaz)Cl2]
9.5(18)
7.2(4)
12.3(7)
5.0(5)
11.0(15)
11.7(9)
>17.5
27.3(35) 3.3(2)
25.0(15) 3.7(3)
1.0(2)
Mon oa d d u ct to Dia d d u ct Con ver sion . Studies
were carried out to determine whether the enantiomers
[Pt(ahaz)Cl2] form different proportions of monofunc-
tional adducts and to determine how rapidly these
monofunctional adducts are converted to the bifunc-
tional adducts. In these experiments, salmon-sperm
DNA was incubated at 37 °C with the platinum com-
plexes for 30 min. The DNA was precipitated with
2-propanol, and the ratio of the total bound platinum
per nucleotide (Rb) was determined. The platinum-
treated DNA was further incubated at 37 °C for 0-24
h to permit rearrangement of the monoadducts to
diadducts. The monoadduct level at each time point
was assessed by the incorporation of [14C]thiourea under
conditions that saturated the monofunctional adducts
without causing reversal of DNA platination. The
percentage of monoadduct was calculated as the ratio
of the concentrations of monofunctionally bound plati-
num to total bound platinum.
a
Numbers in parentheses are standard errors in units of the
last significant figure quoted.
both orientations of the R enantiomer lead to clashes
with the DNA but one of the orientations of the S
enantiomer does not lead to any clashes. The mono-
functional adducts formed by [Pt(S-ahaz)Cl2] and [Pt-
(R-ahaz)Cl2] then disappear at similar rates and even-
tually level off with a larger proportion of monofunctional
adducts remaining for the R enantiomer. A similar
difference in the proportions of monofunctional adducts
for [Pt(S-ahaz)Cl2] and [Pt(R-ahaz)Cl2] has emerged
from preliminary studies of the adduct profiles using
enzymatic digestion and HPLC analysis.39
A more rapid initial drop off is observed for cisplatin,
but after 24 h there remains a greater proportion of
monofunctional adducts than is observed for [Pt(S-
ahaz)Cl2]. Interpretation of these results is complicated
by the likelihood that two processes are taking place:
closure to bifunctional adducts where initial attack was
at a GpG or ApG site and migration from isolated G
sites to GpG or ApG sites followed by closure. If this is
the case, then differences between the three compounds
will result from both differences in reaction rates trans
to NH3 and primary or secondary amines and differ-
ences in steric bulk.
At the platinum treatment concentration used in
these experiments, the Rb values for cisplatin, [Pt(R-
ahaz)Cl2], and [Pt(S-ahaz)Cl2] are 4.0 × 10-2, 3.0 × 10-2
,
and 3.3 × 10-2, respectively. The monoadduct levels
over a 24 h time period for these complexes are
displayed in Figure 6.
At the first timepoint (30 min after the initial reac-
tion), similar numbers of monofunctional adducts re-
main for cisplatin and [Pt(R-ahaz)Cl2] but only half this
number remain for [Pt(S-ahaz)Cl2]. This suggests that
a large proportion of the bound [Pt(S-ahaz)Cl2] has
rapidly formed bifunctional adducts. The more rapid
closure to the bifunctional adduct by the S enantiomer
is in accord with the molecular models that show that
Cytotoxicity Stu d ies. Both enantiomers of [Pt-
(ahaz)Cl2] show cytotoxic activity comparable with that
of cisplatin toward cultures of human bladder tumor
cells (Table 5). The R enantiomer is more active than
the S enantiomer, by factors of 1.5 and 1.7 according to
the MTT and SRB assays, respectively. In the PC9,
PC9cisR, and DU145 cell lines, both enantiomers are

Enantiomeric Platinum(II) Complexes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1097
F igu r e 7. Uptake in BL13/0 cells for cisplatin (+), [Pt(R-ahaz)Cl2] (O), and [Pt(S-ahaz)Cl2] (b).
less active than cisplatin although they are not fully
cross resistant in the PC9cisR cell line and there is no
clear enantioselectivity.
simple measurements of DNA binding or the rate of
closure of monofunctional adducts, we need to consider
other possibilities. Cellular uptake does differ slightly
for the two enantiomers, but not at a level that is likely
to contribute to the difference in activities. Enantiose-
lective differences in cytotoxicity might arise from
differences in deactivation by intracellular components,
interactions with DNA, or repair of DNA adducts.
Alternatively, the profile of adducts produced on DNA
binding or the bending induced in the DNA by each
enantiomer could be important. Experiments are un-
derway to investigate the role of each of these possible
factors.
Cellu la r Up ta k e of P la tin u m Com p lexes. In
order to establish whether the difference between the
in vitro cytotoxic activities of the enantiomers of [Pt-
(ahaz)Cl2] in the BL 13/0 cell line is caused by the
differences in the efficiency of transportation across the
cell membrane, cellular uptake studies of these com-
plexes in the human bladder cells were carried out and
the results are shown in Figure 7.
Higher amounts of the enantiomeric [Pt(ahaz)Cl2]
complexes entered the cells than cisplatin over the
entire platinum treatment concentration range. This
can be explained by the increased lipophilicity of these
complexes due to the presence of the ahaz ligand. The
cellular uptake curves reveal that the R enantiomer was
transported across the cell membrane at a slightly
higher level than the S enantiomer at all platinum
treatment concentrations. It is unlikely that this
marginal difference was a significant factor in the
difference observed in the in vitro cytotoxic activities of
these enantiomers in the human bladder cancer cell line.
Ack n ow led gm en t. The support of the Sydney Uni-
versity Cancer Research Fund, the University of Sydney
Research Grants Scheme, and the Australian Research
Council is gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Tables of crystal
data, positional and thermal parameters, bond lengths and
angles, torsion angles, least-squares planes, and nonbonded
distances (8 pages); observed and calculated structure factors
(6 pages). Ordering information is given on any current
masthead.
Refer en ces
Con clu sion s
(1) (a) School of Chemistry, University of Sydney. (b) Oncology
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readily to DNA than the R enantiomer, and this was
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DNA. Also, we found that closure of monofunctional
adducts occurred more rapidly for the S enantiomer,
again in accord with the molecular models. Modest but
reproducible enantioselective differences in cytotoxicity
were observed in cultures of human bladder cancer cells,
but surprisingly, it was the R enantiomer that was
found to be the more active. In lung and prostate cell
lines, no reproducible differences were observed. Since
the differences in cytotoxicity do not correlate with
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