Synthesis, characterization and cytotoxicity studies of platinum(II) complexes with amino acid ligands in various coordination modes

Article abstract of DOI:10.1016/j.ica.2012.08.034

Reactions of [Pt(CO₃)(PPh₃)₂] ·CH₂Cl₂ (1) with non-substituted and alkyl substituted amino acids, NH(R)CH(R′)CO₂H (R/R′ = H/Me, L1; H/ⁱPr, L2; H/CH₂CHMe₂, L3; Me/H, L4; Et/H, L5), in the presence of Tl[PF₆] in methanol afforded with liberation of CO₂ the formation of platinum(II) complexes of the type [Pt(PPh₃)₂{NHR-CHR^′-C(O)O-κN, κO}][PF₆] (R/R′ = H/Me, 2; H/ⁱPr, 3; H/CH ₂CHMe₂, 4; Me/H, 5; Et/H, 6). Single-crystal X-ray diffraction analysis of complex 4 exhibited a square-planar coordination of the platinum atom having coordinated two triphenylphosphane ligands and a deprotonated κN,κO-coordinated leucine ligand (L3_-H). On varying the pK_a value of the amino group, platinum(II) complexes with different coordination modes of amino acid ligands were obtained. Thus, treatment of complex 1 with N-acetyl l-alanine (L6), possessing a comparatively highly acidic NH proton, in 1:1 ratio in methanol resulted in the formation of [Pt(PPh₃)₂{N(COMe)-CHMe-C(O)O-κN,κO}] (7), while reacting N-phenyl glycine (L7) having a moderately acidic NH proton with complex 1 afforded a mixture of complexes [Pt(PPh₃) ₂{NPh-CH₂-C(O)O-κN,κO}] (8) and [Pt(PPh ₃)₂{NHPh-CH₂-C(O)O-κO}₂] (10). Treatment of complex 1 with two equivalents of L6/L7 in dichloromethane resulted in the formation of [Pt(PPh₃)₂{NHR-CHR′- C(O)O-κO}₂] (R/R′ = COMe/Me, 9; Ph/H, 10). An analogous reactivity was observed for l-lactic acid on treating with complex 1 in 1:1 and 2:1 ratio resulting in [Pt(PPh₃)₂{O-CHMe-C(O)O-κO, κO′}] (11) and [Pt(PPh₃)₂{HO-CHMe-C(O)O- κO}₂] (12). The identities of all complexes have been proven by NMR (¹H, ¹³C, ³¹P) spectroscopic and high-resolution ESI mass-spectrometric investigations. In vitro cytotoxicity studies against human tumor cell lines (8505C, A2780, HeLa, SW480, and MCF-7) showed the highest activities for the neutral complex 7. Furthermore, complexes 7 and 9 against the A2780 cell line induced an apoptotic mode of cell death, which was further supported by morphological investigation and DNA laddering. Cell cycle perturbation studies showed that both complexes induced faster cell death than cisplatin.

Full text of DOI:10.1016/j.ica.2012.08.034

Inorganica Chimica Acta 394 (2013) 472–480
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, characterization and cytotoxicity studies of platinum(II) complexes
with amino acid ligands in various coordination modes
Sangeeta Roy Chaudhuri a,b, Goran N. Kaluderovic a,b, Martin Bette a, Jürgen Schmidt c, Harry Schmidt a,
-
´
Reinhard Paschke b, Dirk Steinborn a,
⇑
a Institut für Chemie – Anorganische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 2, D-06120 Halle, Germany
b Biozentrum, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, D-06120 Halle, Germany
c Abteilung für Natur- und Wirkstoffchemie, Leibniz-Institut für Pflanzenbiochemie, Weinberg 3, D-06120 Halle, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2012
Received in revised form 28 August 2012
Accepted 31 August 2012
Available online 13 September 2012
Reactions of [Pt(CO3)(PPh3)2]ꢀCH2Cl2 (1) with non-substituted and alkyl substituted amino acids,
NH(R)CH(R0)CO2H (R/R0 = H/Me, L1; H/iPr, L2; H/CH2CHMe2, L3; Me/H, L4; Et/H, L5), in the presence of
Tl[PF6] in methanol afforded with liberation of CO2 the formation of platinum(II) complexes of the type
[Pt(PPh3)2{NHR–CHR0–C(O)O-
j j
O}][PF6] (R/R0 = H/Me, 2; H/iPr, 3; H/CH2CHMe2, 4; Me/H, 5; Et/H, 6).
N,
Single-crystal X-ray diffraction analysis of complex 4 exhibited a square-planar coordination of the plat-
inum atom having coordinated two triphenylphosphane ligands and a deprotonated N, O-coordinated
leucine ligand (L3ꢁH). On varying the pKa value of the amino group, platinum(II) complexes with different
coordination modes of amino acid ligands were obtained. Thus, treatment of complex 1 with N-acetyl
alanine (L6), possessing a comparatively highly acidic NH proton, in 1:1 ratio in methanol resulted in the
formation of [Pt(PPh3)2{N(COMe)–CHMe–C(O)O- N, O}] (7), while reacting N-phenyl glycine (L7) having
a moderately acidic NH proton with complex 1 afforded a mixture of complexes [Pt(PPh3)2{NPh–CH2–
C(O)O- N, O}] (8) and [Pt(PPh3)2{NHPh–CH2–C(O)O- O}2] (10). Treatment of complex 1 with two equiv-
O}2] (R/
-lactic acid on treating with com-
O,
O0}] (11) and [Pt(PPh3)2{HO–
j
j
Keywords:
Platinum carbonato complexes
Platinum amino acid complexes
Cytotoxicity
DNA laddering
Cell cycle
L-
j
j
j
j
j
alents of L6/L7 in dichloromethane resulted in the formation of [Pt(PPh3)2{NHR–CHR0–C(O)O-
j
R0 = COMe/Me, 9; Ph/H, 10). An analogous reactivity was observed for
plex 1 in 1:1 and 2:1 ratio resulting in [Pt(PPh3)2{O–CHMe–C(O)O-
L
j
j
CHMe–C(O)O-j
O}2] (12). The identities of all complexes have been proven by NMR (1H, 13C, 31P) spectro-
scopic and high-resolution ESI mass-spectrometric investigations. In vitro cytotoxicity studies against
human tumor cell lines (8505C, A2780, HeLa, SW480, and MCF-7) showed the highest activities for the
neutral complex 7. Furthermore, complexes 7 and 9 against the A2780 cell line induced an apoptotic
mode of cell death, which was further supported by morphological investigation and DNA laddering. Cell
cycle perturbation studies showed that both complexes induced faster cell death than cisplatin.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
Amino acids, like other biomolecules, can have a multitude of
coordination modes. They can be monocoordinated (
dicoordinated in a chelating ( O,
N or j2O,O0) or bridging (
or O:
tion of the amino acid as anionic, dianionic or neutral (zwitter-
ionic) ligand [3–7].
Although there is a multitude of ways to synthesize these com-
plexes, but to acquire control over the coordination mode may be a
challenge. Carbonato platinum(II) complexes which can react with
acidic ligands under release of carbon dioxide may open a way to a
targeted deprotonation of the amino acid. In this respect, they have
been used so far to react with various acidic substrates. Thus,
j
O or
j
j
N) or
O:
Platinum complexes with bioligands attract attention both from
the viewpoint of bioactivity, mainly due to their carcinostatic prop-
erties, and from the coordination chemistry perspective, because in
many cases bioligands have various donor sites of similar proper-
ties at their disposal [1,2]. This may result into many different
coordination modes and a detailed knowledge on them is a
requirement for a deeper understanding of the role of metals in
biochemistry. Furthermore, to get control over the coordination
mode of bioligands to metals may be a prerequisite for a targeted
development of metal-containing drugs.
j
j
jN
j
j
O0) fashion. A further versatility results from the coordina-
bis(phosphane)carbonatoplatinum(II)
(2 L = 2 PR3, dppe) were found to react with polyols containing at
least one vicinal diol moiety (such as ethane-1,2-diol, -ascorbic
acid, -mannitol) with formation of diolato complexes of the type
complexes
[Pt(CO3)L2]
L
⇑
Corresponding author. Tel.: +49 345 5525620; fax: +49 345 5527028.
E-mail address: steinborn@chemie.uni-halle.de (D. Steinborn).
D
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.08.034

S.R. Chaudhuri et al. / Inorganica Chimica Acta 394 (2013) 472–480
473
[Pt(O
BINOL,
O)L2] [8–10]. Analogously, aromatic 1,2-diols (such as
1,8,9,16-tetrahydroxytetraphenylene) [11–16] and
those of the unreacted amino acids and the unreacted complex 1,
respectively.
silasesquioxane [17] reacted with deprotonation of phenolic OH
groups and silanol groups, respectively. Reactions of [Pt(CO3)L2]
complexes with dicarboxylic acids having a rigid backbone (e.g., C6-
H4(CO2H)2-1,4; trans,trans-HO2CCH@CHCH@CHCO2H) gave rise to
the formation of dinuclear complexes with bridging dicarboxylato
ligands, whereas in the presence of NH4+, additionally, under its
deprotonation ammine complexes were formed [18]. Reactions of
triketones having an enolizable b-diketone unit (heptane-2,4,6-tri-
one, 1,5-diphenylpentane-1,3,5-trione) led to the deprotonation of
either the enolic OH proton or the methylene group, thus forming
platinum(II) complexes with b-diketonato type ligands or platina-
cyclobutan-3-one complexes, respectively [19–21]. Furthermore,
reactions with trifluoromethyl sulfonamides (CF3SO2NH–
CHPhCHPh–NHSO2CF3, CF3SO2NH–CHPhCHPhOH) under deproto-
nation of the relatively highly acidic NH groups [15,22,23] and,
very recently, with 1,3-dithiols (HSCH2–CH(OH)–CH2SH, HSCH2–
CH(OSiMe2CMe3)–CH2SH) under deprotonation of SH groups have
been reported [24].
Herein, we report reactions of the bis(triphenylphosphane)car-
bonatoplatinum(II) complex [Pt(CO3)(PPh3)2]ꢀCH2Cl2 (1) with
unsubstituted and substituted amino acids which proceed with
deprotonation of the carboxylic group and, in dependence on the
NH acidity and the solvent, of the amino group, too. In analogous
reactions with lactic acid, a pronounced solvent dependence on
the deprotonation of the alcoholic OH group was found. Finally,
the in vitro cytotoxicity of the amino acid complexes and the cor-
responding ligand precursors against five human tumor cell lines
has been investigated.
In contrast, on treating complex 1 with N-acetyl alanine (L6)
which possesses a comparatively highly acidic NH proton in 1:1 ra-
tio in methanol afforded the neutral platinum(II) complex 7 con-
taining
jN,jO-chelating dianionic amino acid ligand (Scheme 2,
route a). On the other hand, on treating complex 1 with N-phenyl
glycine (L7), an amino acid having a moderately acidic NH proton,
in 1:1 ratio in methanol resulted in the formation of a mixture of
complexes 8 and 10 (Scheme 2, routes a/b), bearing one dianionic
jN,jO-chelating amino acid ligand and two anionic monodentate-
ly
jO-coordinated ligands, respectively. However, due to similar
solubilities, complex 8 could not be prepared on a pure state.
Unlike the case described for L1–L5, changing the molar ratios
of the platinum complex 1 and the amino acids with more acidic
NH protons (L6, L7) resulted in a different course of reaction. Thus,
treatment of complex 1 with L6/L7 in 1:2 ratio in a protic solvent
like methanol gave the complexes 9 and 10 with two anionic
monodentately
jO-coordinated amino acid ligands along with
unreacted complex 1 in case of L7. However, these latter com-
plexes could be obtained as solely reaction product when working
in an aprotic solvent like dichloromethane (Scheme 2, route b). The
complexes 7, 9, and 10 were isolated in yields between 60% and
70% as air-stable white solids. They were characterized by 1H,
13C, and 31P NMR spectroscopy and by HRMS-ESI measurements.
Quite analogously, L-lactic acid (L8), an a-hydroxy acid with a
moderately acidic OH group, was found to react with complex 1.
Using a 1:1 and 1:2 ratio in methanol, also afforded a mixture of
two neutral platinum(II) complexes, one bears a chelating j2O,O0-
coordinated dianionic lactato ligand (11) and the other one is a
bis(lactato) complex with monodentately
jO-coordinated ligands
(12). Due to its sparing solubility in methanol, complex 11 could
be isolated in a pure state as white solid in 52% yield (Scheme 3,
route a). On the other hand, reactions of complex 1 with L8 in
1:1 and 1:2 ratio in an aprotic solvent (dichloromethane) proceed
differently. Using a 1:1 ratio resulted in formation of complex 12
along with an unidentified platinum complex having non-equiva-
lent phosphane ligands, whereas using a 1:2 ratio resulted in the
formation of complex 12 exclusively (isolated yield 65%) (Scheme 3,
route b). The identities of the two complexes 11 and 12 have been
proven by NMR spectroscopy (1H, 13C, 31P) and HRMS-ESI
measurements.
2. Results and discussion
2.1. Synthesis
Treatment of the complex [Pt(CO3)(PPh3)2]ꢀCH2Cl2 (1) with
unsubstituted a-amino acids (L-alanine, L1; DL-valine, L2; l-leucine,
L3) and alkyl substituted amino acids (N-methyl glycine, L4; N-
ethyl glycine, L5) in 1:1 ratio in methanol, afforded the mononu-
clear cationic platinum(II) complexes 20–60 (Scheme 1, route a).
Addition of 1.2 equivalents of Tl[PF6] to the reaction solutions re-
sulted in the formation of Tl2CO3 and cationic platinum(II) com-
2.2. Spectroscopic investigations
plexes 2–6 with anionic
jN,jO-coordinated amino acid ligands
(Scheme 1, route b). The complexes obtained are found to be air-
stable white powders, which were isolated in yields between 73%
and 86%, and they were characterized by 1H, 13C, and 31P NMR
spectroscopy, HRMS-ESI measurements, and a single crystal X-
ray diffraction analysis (4).
However, no other mode of coordination of the amino acid li-
gands was observed on treating complex 1 with excess or in defi-
ciency of L1–L5 (0.5–4 equivalents) in methanol. In all cases,
formation of complexes 20–60 was observed. 1H and 31P NMR spec-
troscopy studies showed the signals of these complexes along with
Selected NMR spectroscopic parameters of complexes 2–12 are
given in Table 1. All spectra show the expected signals in the ex-
pected shift range. The 1H NMR spectra give unequivocally proof
of the 1:1 and 1:2 stoichiometry, respectively, as given in Table 1.
The 31P NMR spectra of the complexes (2–7, 11) exhibit the pres-
ence of two chemically non-equivalent P nuclei. They show AB spin
patterns with the corresponding ABX satellite spectra (A, B = 31P;
X = 195Pt). The signal assignment is based on the large differences
1
in the two JPt,P coupling constants. The much higher trans influ-
ence of the amide and alcoholate groups over that of the carboxyl-
O
R'
O
N
Ph3P
Ph3P
O
N
O
Ph3P
Ph3P
O
O
1
Tl[PF6]
OH
MeOH
Pt
[PF6]
Ph3P
Ph3P
Pt
Pt
O.
CH2Cl2
HN
R
R'
R'
b
a
O
H
R
H
R
2'−6'
L1−L5
2−6
L1, 2', 2 L2, 3', 3
R/R' H/Me
H/iPr
L3, 4', 4
H/CH2CHMe2
L4, 5', 5
L5, 6', 6
Me/H
Et/H
Scheme 1. Reactivity of [Pt(CO3)(PPh3)2]ꢀCH2Cl2 toward amino acids.

474
S.R. Chaudhuri et al. / Inorganica Chimica Acta 394 (2013) 472–480
(1:1)
O
O
N
Ph3P
Pt
Pt
Ph3P
R'
MeOH
R'
a
b
O
O
Ph3P
Ph3P
R
OH
Pt
O.
CH2Cl2
7, 8
HN
R
1
O
NHR
R'
L6, L7
O
(1:2)
O
O
Ph3P
Ph3P
R'
CH2Cl2
L6, 7, 9
L7, 8, 10
NHR
O
R/R'
Ph/H
COMe/Me
9, 10
Scheme 2. Reactivity of [Pt(CO3)(PPh3)2]ꢀCH2Cl2 toward N-substituted amino acids.
O
Ph3P
Ph3P
O
O
(1:1)
Pt
Me
a
b
MeOH
Me
11
OH
O
O
Ph3P
Ph3P
O.
CH2Cl2
HO
Pt
OH
Me
O
(1:2)
O
O
O
Ph3P
Ph3P
Me
Pt
CH2Cl2
OH
O
12
Scheme 3. Reactions of [Pt(CO3)(PPh3)2]ꢀCH2Cl2 with
L-lactic acid.
Table 1
Selected NMR spectroscopic data (d in ppm, J in Hz) of complexes [Pt(PPh3)2{NHR–CHR0 –C(O)O-
j
N,
j
O}][PF6] (2–6), [Pt(PPh3)2{NHCOMe–CHMe–C(O)O-
j
N,
jO}] (7), [Pt(PPh3)2
{NHR–CHR0–C(O)O-
j
O}2] (9, 10), [Pt(PPh3)2{O0–CHMe–C(O)O-j2O,O0}] (11), and [Pt(PPh3)2{HO–CHMe–C(O)O-
j
O}2] (12).
a
a
00
dP (1JPt,P
)
dP (1JPt,P
)
d
(2JH,H
)
-H
dC (COO)
0
0
00
Complexes
a
2 (R/R0 = H/Me)
3 (R/R0 = H/iPr)
5.6 (3663)
6.4 (3643)
7.2 (3570)
7.4 (3703)
6.5 (3703)
6.4 (4091)
6.8 (3865)
6.8 (3826)
12.3 (3911)
5.7 (3856)
7.7 (3508)
7.3 (3471)
7.6 (3486)
8.2 (3381)
6.4 (3353)
9.3 (3068)
–
3.86
3.65
3.69
186.0
184.6
187.0
181.0
183.0
190.0
175.6b
174.1
194.1
178.8
4 (R/R0 = H/CH2CHMe2)
5 (R/R0 = Me/H)
6 (R/R0 = Et/H)
3.31 (16.7), 4.38 (16.7)
3.46 (16.8), 4.29 (16.8)
7
4.41
3.64
3.03
4.66
3.4
9 (R/R0 = COMe/Me)
10 (R/R0 = Ph/H)
–
11
12
10.6 (3241)
–
a
P0 and P00 are trans to a carboxylate oxygen atom and nitrogen atom, respectively. In case of 11 P00 is trans to the alcoholate oxygen atom.
Assignment of the signal is uncertain.
b
ate groups gives rise to smaller (3068–3508 Hz) and higher (3570–
4091 Hz) 1JPt,P coupling constants, respectively. The 31P NMR spec-
tra of the bis(amino acid) complexes 9, 10, and 12 show, as
expected, a singlet phosphorus resonance with 195Pt satellites.
of carboxylate C atoms to phosphorus (3/4JP,C) through the hetero-
atoms have been observed, although any coupling to 195Pt is not
discernible. Analogous couplings were observed in some other
amino acid complexes of platinum [30].
Anionic chelating amino acid ligands produce invariably a
downfield shift of the proton resonances, relative to their corre-
sponding amino acid anions [31,32]. Furthermore, the resonances
1
The magnitude of the JPt,P coupling constants (3826–3865 Hz)
and the comparison with requisite data of carboxylato complexes
(both in jO and jO,j
O0 coordination) having a PPh3 ligand in trans
position [25] (cf. also with the carbonato complex 1: 1JPt,P = 3702 -
Hz [26]) show that the anionic amino acid ligands are coordinated
through the carboxylate groups.
of the
monodentately coordinated (
bidentately coordinated ( O,
complexes 7/9 and 11/12). Thus, the downfield shift of
a
protons were found to be more shielded in complexes with
O) amino acid ligands than with
N; j2O,O0) amino acid ligands (cf.
protons
O,
j
j
j
The resonances of the carboxylate C atoms of the complexes 2–
a
7 and 11 having anionic/dianionic
j
O,j
N coordinated amino acid
in complexes 2–7 and 11 gives an additional proof for a j jN
ligands fall in the range of d 181.0–194.1 ppm, which is typical
for such complexes [27,28]. The carboxylate C atoms of the com-
plexes 9, 10, 12 gave signals in the range d 174.1–178.8 ppm. Thus,
they are significantly more shielded than in the chelate complexes,
coordination.
Furthermore, as expected, the
a-methylene protons of the N-
substituted glycine complexes 5 and 6 are not equivalent and
two separate doublets of doublet resonances are observed due to
coupling to each other (2JH,H = 16.7/16,8 Hz) and to NH protons
(3JH,H = 3.6–5.2 Hz) [27].
which is typical for complexes with monocoordinated (
jO) amino
acid ligands [27,29]. Noteworthy, in complexes 3 and 11 couplings

S.R. Chaudhuri et al. / Inorganica Chimica Acta 394 (2013) 472–480
475
2.3. Molecular structure of the leucinato complex 4
Crystals of [Pt(PPh3)2{NH2–CH(CH2CHMe2)–C(O)O-
jN,jO}][BF4]
(4; obtained as described before but using Ag[BF4] instead of
Tl[PF6]) suitable for X-ray diffraction analysis were obtained from
dichloromethane solutions with a layer of diethyl ether at room
temperature. In the asymmetric unit, two symmetry-independent
cations and anions of very similar structure are found. The
structure of one of the two cations is shown in Fig. 1 and selected
structural parameters are given in the figure caption. The platinum
atom is square-planar coordinated by two PPh3 ligands and the
jN,jO-chelating leucinato ligand (sum of angles around Pt1:
360.0/360.2°1). Deviations originate from the restricted bite of the
chelating ligand (O1–Pt1–N1 80.3(3)/80.0(2)°). One of the trans
0
angles (P2–Pt1–O1) is nearly linear (178.4(2)/176.3(2) AÅ), whereas
0
the other one (P1–Pt1–N1) is severely bent (162.2(2)/163.0(2) AÅ).
The Pt1–N1 distances (2.102(6)/2.080(7) Å) and the Pt1–O1 dis-
tances (2.067(5)/2.024(6) Å) are approximately as long as those in
structurally similar amino acid platinum(II) complexes (Pt–N:
median: 2.044 Å, lower/higher quartile: 2.060/2.094 Å; Pt–O: med-
ian: 2.074 Å, lower/higher quartile: 2.033/2.073 Å; n = 11, n = num-
ber of observations) [33]. In accordance with the higher trans
influence of the amino group over the carboxylate group [34] the
Pt1–P1 bond (2.264(2)/2.272(2) Å) is much longer than the Pt1–
P2 bond (2.243(2)/2.249(2) Å). The five-membered PtNOC2 ring is
twisted on C1–C2 and Pt1–N1, respectively.
Fig. 1. Structure of one of the two symmetry-independent cations in crystals of
[Pt(PPh3)2{NH2–CH(CH2CHMe2)–C(O)O0- O0}][BF4] (4). The ellipsoids are drawn
jN,j
with a probability of 30%. Hydrogen atoms were omitted for clarity. Selected
structural parameters (distances in Å, angles in °); the values for the two symmetry-
independent molecules are separated by a slash: Pt1–P1 2.264(2)/2.272(2), Pt1–P2
2.243(2)/2.249(2), Pt1–N 2.102(6)/2.080(7), Pt1–O1 2.067(5)/2.024(6), C1–O1
1.30(1)/1.317(9), C1–O2 1.21(1)/1.217(9), Pl–Pt1–P2 98.02(8)/98.74(7), P1–Pt1–
O1 83.6(2)/84.1(2), O1–Pt1–N1 80.3(3)/80.0(2), N1–Pt1–P2 98.1(2)/97.4(2), P1–
Pt1–N1 162.2(2)/163.0(2), O1–Pt1–P2 178.4(2)/176.3(2).
Crystals of 4 are threaded by N1–Hꢀ ꢀ ꢀF hydrogen bonds which
connect the cationic platinum complexes with the [BF4]ꢁ anions.
0
The N1ꢀ ꢀ ꢀF distances are between 2.94(1) and 3.15(1) ÅA.
more active than the cationic complex 2 having coordianted L-ala-
nine. In comparison to cisplatin, complexes 1–12 are less active
against investigated cell lines, except complex 7 which showed a
comparable cytotoxicity against 8505C cell line.
2.4. In vitro studies
2.4.1. Cytotoxicity studies
In order to find possible structure–activity relationships, in vitro
cytotoxicities of complexes 2–12 against the human tumor cell
lines 8505C anaplastic thyroid cancer, A2780 ovarian cancer, HeLa
epithelial cancer, SW480 colon cancer, and MCF-7 breast cancer
were determined by using the sulforhodamine-B microculture col-
orimetric assay. The cytotoxicities of the amino acids/lactic acid,
starting platinum(II) carbonato complex (1) and cisplatin [35,36]
are included for comparison.
2.4.2. Mode of cell death
The mode of cell death was investigated for the two most active
platinum complexes (7, 9) against ovarian A2780 cancer cell line.
Discrimination of the living cells from dead ones can be done on
the basis of membrane integrity using acridine orange/ethidium
bromide (AO/EB) staining. Microphotographs of AO/EB double
stained A2780 cells, which were pretreated for 24 h with com-
plexes 7 and 9 and cisplatin (for comparison) are presented in
Fig. 3. Control cells (non treated) have green nuclei and uniform
chromatin with an intact cell membrane. In contrast, complexes
7 and 9, as well as cisplatin induced apoptosis as seen by conden-
sation and/or fragmentation of the nuclei. In addition, rounding of
the cells was also observed when treated with complexes 7, 9, and
cisplatin.
Furthermore, to confirm findings from AO/EB double staining
assay, floating A2780 cells after treatment with IC90 concentrations
were collected and analyzed by laddering technique (Fig. 3). For
A2780 cells treated with platinum(II) compounds 7 and 9, DNA ap-
peared as characteristic ladder-like fragments which is the bio-
chemical hallmark of apoptosis. The results are compared to the
negative control (untreated cells) where no laddering pattern or
smear (indicating necrosis) was seen and to the positive control
cisplatin (induces apoptosis).
Treatment with L1–L6 and L8 did not show any toxicity in the
dose range used (0.1–150 lM) except L7 (IC50 P 78 lM). On the
other hand, their corresponding complexes showed much higher
activity (Table 2). The platinum(II) complexes 1–12 showed
dose–dependent antiproliferative effect toward investigated can-
cer cell lines, see Fig. 2 as example. The highest and lowest activity
of these complexes was observed in A2780 and HeLa cell lines,
respectively. A medium activity was observed against 8505C,
SW480, and MCF-7 cell lines except complexes with N-alkylated
amino acid ligands (5 and 6) which showed maximum activity
against MCF-7 cell line. Thus, alkylation of the amino group leads
to a change in the selectivity of the complexes. Cytotoxicities of
the cationic complexes 2–6 were found to be comparable with
those of the starting platinum(II) precursor complex 1. Only com-
plexes 5 and 6 against MCF-7 cell line and complex 3 against
A2780 cell line show cytotoxicities which are increased and re-
duced, respectively, roughly by factor two compared to complex
1 (see Table 2). Of all the studied complexes, complex 7 seems to
2.4.3. Cell cycle investigation
be the most active against selected cell lines (IC50: 3.4–9.9
Explicitly, the neutral complex 7 having coordinated a deproto-
nated N-acetyl derivative of L-alanine was found to be 3–7 times
l
M).
To investigate the effects of platinum(II) complexes 7 and 9 (cis-
platin used as a positive control) on cell cycle progression, A2780
cells were treated for 24 h and 48 h with IC50 and IC90 concentra-
tions and analyzed by flow cytometry. The obtained results (see
Fig. 4 for example and Supplemental material) indicated that when
A2780 cells were exposed for 24 h to IC50 and IC90 concentrations
of 7 and 9 drastically decreased the cell population in all phases
1
Here and in the following the values for the two symmetry independent
molecules are given separated by a slash.

476
S.R. Chaudhuri et al. / Inorganica Chimica Acta 394 (2013) 472–480
Table 2
Cytotoxicity of investigated compounds against 8505C (anaplastic thyroid carcinoma), A2780 (ovarian carcinoma), HeLa (human epithelial carcinoma), SW480 (human colon
carcinoma), and MCF7 (breast carcinoma) cell lines represented by the IC50 values [
l
M].a
Compound
IC50 SD
8505C
A2780
HeLa
SW480
MCF-7
L1–L6, L8
L7
1
2
3
4
5
6
>150.0
>150.0
66.9 3.5
50.4 2.4
57.8 2.3
39.9 2.7
42.7 4.7
41.0 3.1
9.9 1.5
135.2 1.7
36.6 0.5
26.0 1.9
39.9 0.5
27.7 1.1
32.6 1.4
31.3 1.6
7.2 0.5
104.7 3.7
17.3 1.3
17.3 0.3
31.1 1.4
12.9 0.5
23.4 2.1
23.8 2.2
3.4 0.6
121.2 1.7
29.4 2.7
27.3 1.3
46.3 2.8
25.8 63.8
29.4 2.4
27.9 1.6
7.4 0.6
77.7 4.6
23.0 1.9
22.0 2.1
29.1 1.4
21.9 1.7
10.7 1.4
13.4 1.2
8.7 4.3
7
9
10
11
12
18.0 0.2
30.9 1.3
15.0 0.4
24.2 0.8
5.02 0.23
10.0 0.1
28.2 1.5
15.4 0.3
15.7 0.4
0.55 0.03
54.5 1.9
28.4 3.1
50.8 6.5
64.1 1.8
4.6 0.34
27.6 2.1
28.2 3.5
17.1 0.1
25.4 0.3
3.2
28.9 1.2
16.0 1.4
18.5 1.5
27.6 1.3
2.03 0.11
Cisplatin
a
Mean values SD (standard deviation) from three experiments.
Fig. 2. Representative graph showing survival after 96 h of the A2780 cell line treated with L6 and platinum(II) complexes 1, 7, and 9.
Fig. 3. Left: AO/EB double stained A2780 cells: untreated (C – control) and treated with cisplatin (Pt) and complexes 7 and 9. Right: DNA laddering of A2780 cells untreated
(C – control) and treated with IC90 concentrations of cisplatin (Pt), complexes 7 and 9. Arrows indicate shrinkage of nuclei and chromatin condensation.

S.R. Chaudhuri et al. / Inorganica Chimica Acta 394 (2013) 472–480
477
under aerobic conditions. The solvents used in these reactions
were dried by standard methods and freshly distilled under argon
prior to use. NMR spectra (1H, 13C, 31P; 13C and 31P NMR spectra are
generally broadband proton decoupled) were recorded at 27 °C on
Varian Gemini 200, VXR 400, and Unity 500 spectrometers. Chem-
ical shifts are relative to solvent signals (CDCl3; dH 7.24, dC 77.0) as
internal references. H3PO4 (85%) was used as external reference for
31P NMR spectra. High-resolution ESI mass spectra were obtained
from a Bruker Apex III Fourier transform ion cyclotron resonance
(FT-ICR) mass spectrometer (Bruker Daltonics) equipped with an
Infinity cell, a 7.0 T superconducting magnet (Bruker), an rf-only
hexapole ion guide, and an external APOLLO electrospray ion
source (Agilent, off-axis spray). The sample solutions were intro-
duced continuously via a syringe pump with a flow rate of
Fig. 4. Cell cycle analysis of A2780 cell line treated for 24 h to IC50 concentrations of
cisplatin and complexes 7 and 9; control – untreated cells.
120
l
L ꢀ hꢁ1. cis-[PtCl2(PPh3)2] was prepared as reported in litera-
ture [37]. [Pt(CO3)(PPh3)2]ꢀCH2Cl2 (1) was prepared according to
the procedure given in literature [26], but using a 7-fold excess
of Ag2CO3 and a much longer (5 days) reaction time (yield 75%).
All other chemicals were commercially available and used as sup-
plied without any further purification.
of the cell cycle. Consequently, an increasement in the number of
cells in sub-G1 phase was observed. Cisplatin slightly increased
the relative number of A2780 cells in the G2/M phases when trea-
ted for 24 h at IC50 and IC90 concentrations. On treatment of A2780
cells with IC50 and IC90 concentration of complexes 7 and 9 for 48 h
also show a decrease in the cell population in G1, S, and G2/M
phases and relative increasement in the cells in sub-G1 phase. Un-
like cisplatin, platinum complexes 7 and 9 did not induce a signif-
icant cell cycle arrest in any cell cycle phase but rather directly
provoked an increase in the number of apoptotic cells with con-
comitant decline of all other cell cycle phases in A2780 cells. Thus,
the cell cycle perturbation studies indicate that complex 7 induced
apoptosis (see Section 2.4.2) of the A2780 cells faster than complex
9 and both complexes faster than cisplatin.
4.2. Synthesis of the platinum(II) complexes
4.2.1. Synthesis of [Pt(PPh3)2{NHR–CHR0–C(O)O-
jN,jO}][PF6] (2–6)
Amino acids (L1–L5, 0.22 mmol) were added to a suspension of
[Pt(CO3)(PPh3)2]ꢀCH2Cl2 (1; 160.0 mg, 0.19 mmol) in methanol
(5 mL). The reaction mixture was stirred overnight at room tem-
perature resulting in a colorless solution. Then, Tl[PF6] (76.9 mg,
0.22 mmol) was added and the reaction mixture was further stir-
red for 2 h. A white precipitate started appearing within 10 min.
Dichloromethane (5 mL) was added and the reaction mixture
was stirred for about 30 min resulting in a dissolution of the major-
ity of the precipitate. The reaction mixture was then passed
through Celite and the Celite pad was washed with CH2Cl2
(6 ꢂ 5 mL). The combined organic phases were concentrated under
reduced pressure almost to dryness. Addition of diethyl ether
(10 mL) resulted in a white precipitate, which was filtered off,
washed with diethyl ether (3 ꢂ 2 mL) and dried under vacuum.
R/R0 = H/Me (2). Yield: 146.0 mg (83%). HRMS (ESI): m/z Calc. for
[C39H36O2NP2Pt]+: 807.1868; found for [M]+: 807.1864. 1H NMR
3. Summary and conclusions
The carbonato platinum complex [Pt(CO3)(PPh3)2] ]ꢀCH2Cl2 (1)
has been found to react with amino acids and lactic acid, without
adding an external base, with liberation of CO2 yielding platinum
complexes with monodeprotonated amino acid/lactic acid ligands
of type I ([Pt(PPh3)2{HX
type II ([Pt(PPh3)2{HRN
C(O)O-
j
O}2]; HX = NHR, OH) or
C(O)O- N,
j
jO}][PF6]) and with dou-
bly deprotonated amino acid/lactic acid ligands of type III
3
(400 MHz, CDCl3): d 1.12 (d, JH,H = 6.8 Hz, 3H, CH3), 3.22 (s (br),
([Pt(PPh3)2(X C(O)O- X, O}]), respectively. The type of
j
j
2H, NH2), 3.86 (m, 1H, CH), 7.16–7.55 (m, 30H, Ph). 13C NMR
complex formed does not depend primarily on the ratio of starting
carbonato platinum complex (1) and amino acid/lactic acid used in
the synthesis, but on the solvent and the acidity of the NHR group
tuned via the substituent R. A deprotonation of the carboxylic
group takes place both in aprotic (dichloromethane) and protic sol-
vents (methanol), whereupon the formation of type I complexes is
favored in dichloromethane and that of type II complexes in meth-
anol. An additional deprotonation of the OH group in lactic acid
and of the NHR group in amino acids leading to type III complexes
was only found in methanol and for amino acids with more acidic
amino groups (R = COMe, Ph). Furthermore, the in vitro cytotoxici-
ties of these complexes against five human cancer cell lines indi-
cated that the neutral type III complex 7 is the most active one.
It proved to be only slightly less active than cisplatin and, more-
over, to induce apoptosis (shown for the cell line A2780), but faster
than cisplatin.
(125 MHz, CDCl3): d 19.0 (s, CH3), 53.5 (s, CH), 125.0 (d, 1JP,C = 66.5 -
1
3
Hz, i-C, Ph), 126.1 (d, JP,C = 63.2 Hz, i-C, Ph), 129.2 (d, JP,C = 11.5 -
3
Hz, m-C, Ph), 129.4 (d, JP,C = 11.7 Hz, m-C, Ph), 133.8 (d,
4JP,C = 2.7 Hz, p-C, Ph), 134.2 (d, JP,C = 2.7 Hz, p-C, Ph), 134.0 (d,
4
2JP,C = 10.9 Hz, o-C, Ph), 134.6 (d, JP,C = 10.9 Hz, o-C, Ph), 186.0 (s,
2
COO). 31P NMR (162 MHz, CDCl3): d ꢁ142.7 (sept, JP,F = 713.1 Hz,
1
2
1
PF6ꢁ), 5.6 (d + d, JP ,P = 24.2 Hz, JPt,P’ = 3663 Hz, P0), 7.7 (d + d,
0
00
2
1
JP ,P = 24.2 Hz, JPt,P = 3508 Hz, P00).
0
00
00
R/R0 = H/iPr (3). Yield: 156.0 mg (86%). HRMS (ESI): m/z Calc. for
[C41H40O2NP2Pt]+: 835.2180; found for [M]+: 835.2177. 1H NMR
3
(400 MHz, CDCl3): d 0.71 (d, JH,H = 7.0 Hz, 3H, CH(CH3)2), 0.84 (d,
3JH,H = 6.9 Hz, 3H, CH(CH3)2), 2.13 (m, 1H, CH(CH3)2), 2.37 (m, 1H,
NH2), 3.65 (m, 1H, CHNH2), 3.94 (m, 1H, NH2), 7.23–7.62 (m,
30H, Ph). 13C NMR (125 MHz, CDCl3): d 16.9 (s, CH(CH3)CH3),
18.8 (s, CH(CH3)CH3), 31.0 (s, CH(CH3)2), 61.9 (s, CHCOO), 124.8
1
1
(d, JP,C = 64.8 Hz, i-C, Ph), 125.8 (d, JP,C = 63.7 Hz, i-C, Ph), 129.1
(d, JP,C = 11.5 Hz, m-C, Ph), 129.5 (d, JP,C = 11.6 Hz, m-C, Ph),
3
3
2
4. Experimental section
132.3 {br (s), p-C, Ph}, 132.6 {br (s), p-C, Ph}, 133.9 (d, JP,C = 10.1 -
Hz, o-C, Ph), 134.5 (d, 2JP,C = 10.7 Hz, o-C, Ph), 184.6 (d, 3/4JP,C = 5.3 -
4.1. General remarks
Hz, COO). 31P NMR (162 MHz, CDCl3):
d
ꢁ142.7 (sept,
1
0
1
2
JP,F = 712.1 Hz, PF6ꢁ), 6.4 (d + d, JP ,P = 24.1 Hz, JPt,P = 3643 Hz,
0
00
2
1
0
00
00
All reactions were carried out in an atmosphere of dry argon
using standard Schlenk and vacuum line techniques, but the work-
up procedures and the spectral measurements could be conducted
P0), 7.3 (d + d, JP ,P = 24.1 Hz, JPt,P = 3471 Hz, P00).
R/R0 = H/CH2CH(CH3)2 (4). Yield: 140 mg (76%). HRMS (ESI): m/z
Calc. for [C42H42O2NP2Pt]+: 849.2340; found for [M]+: 849.2333. 1H

478
S.R. Chaudhuri et al. / Inorganica Chimica Acta 394 (2013) 472–480
3
NMR (400 MHz, CDCl3): d 0.60 (d, JH,H = 6.4 Hz, 3H, CH(CH3)CH3),
lid which was filtered off, washed with diethyl ether (3 ꢂ 2 mL)
and dried under vacuum.
3
0.71 (d, JH,H = 6.8 Hz, 3H, CH(CH3)CH3), 0.98 (m, 1H, CH(CH3)2),
1.63 (m, 2H, CH2), 2.9 (br, 1H, NH2), 3.69 (br, 2H, NH + CHCH2),
7.16–7.63 (m, 30H, Ph). 13C NMR (125 MHz, CDCl3): d 21.5 (s,
CH(CH3)CH3), 23.3 (s, CH(CH3)CH3), 25.7 (s, CH(CH3)2), 43.7 (s,
R/R0 = C(O)Me/Me, (9). Yield: 109 mg (60%). HRMS (ESI): m/z
Calc. for [C41H38O3P2NPt]+: 849.1971; found for [MꢁL6ꢁH]+:
3
849.1969. 1H NMR (400 MHz, CDCl3): d 0.79 (d, JH,H = 6.8 Hz, 6H,
1
CH2), 56.2 (s, CHCH2), 125.3 (d, JP,C = 66.7 Hz, i-C, Ph), 127.4 (d,
CHCH3), 1.75 (s, 6H, C(O)CH3), 3.64 (dq, 3JH,H = 5.8 Hz, 3JH,H = 6.8 Hz,
1JP,C = 64.3 Hz, i-C, Ph), 129.9 (d, JP,C = 11.6 Hz, m-C, Ph), 130.5 (d,
2H, CH), 6.12 (d, 3JH,H = 5.8 Hz, 2H, NH), 7.15–7.54 (m, 30H, Ph). 13
C
3
4
3JP,C = 11.6 Hz, m-C, Ph), 133.3 (d, JP,C = 2.4 Hz, p-C, Ph), 133.8 (d,
NMR (100 MHz, CDCl3): d 18.8 (s, CHCH3), 23.4 (s, COCH3), 50.5 (s,
CH), 127.5 (m⁄,2 i-CPh), 128.2 (‘t’⁄,3 m-CPh), 131.1 {br (s), p-CPh}, 134.5
(‘t’⁄, o-CPh), 168.6/175.6 (s/s, COCH3, COO). 31P NMR (162 MHz, CD2-
4JP,C = 2.9 Hz, p-C, Ph), 135.4 (d, JP,C = 11.1 Hz, o-C, Ph), 135.8 (d,
2
2JP,C = 10.6 Hz, o-C, Ph), 187.0 (s, COO). 31P NMR (162 MHz, CDCl3):
1
2
1
d ꢁ142.7 (sept, JP,F = 712.1 Hz, PF6ꢁ), 7.2 (d + d, JP ,P = 24.7 Hz,
Cl2): d 6.8 (s + d, JPt, P = 3865 Hz).
0
00
1
2
1
JPt,P = 3570 Hz, P0), 7.6 (d + d, JP ,P = 24.6 Hz, JPt,P = 3486 Hz, P00).
R/R0 = Ph/H (10). Yield: 132 mg (70%). HRMS (ESI): m/z Calc. for
[C44H38O2P2NPt]+: 869.2016; found for [MꢁL7ꢁH]+: 869.2020. 1H
NMR (200 MHz, CD2Cl2): d 3.03 (s, 4H, CH2), 4.1 (br, 2H, NH),
6.25–7.60 (m, 40H, Ph). 13C NMR (125 MHz, CDCl3): 47.6 (s, CH2),
112.6–147.9 (aromatic C), 174.1 (s, COO). 31P NMR (81 MHz, CD2-
0
0
00
00
R/R0 = Me/H (5). Yield: 140 mg (79%). HRMS (ESI): m/z Calc. for
[C39H36O2NP2Pt]+: 807.1865; found for [M]+: 807.1864. 1H NMR
(200 MHz, CDCl3): d 2.33 (br, 3H, CH3), 3.1 (br, 1H, NH), 3.31 (dd,
2JH,H = 16.7 Hz, JH,H = 4.0 Hz, 1H, CH2), 4.38 (dd, JH,H = 16.7 Hz,
3JH,H = 5.2 Hz, 1H, CH2), 7.24–7.65 (m, 30H, Ph). 13C NMR
(125 MHz, CDCl3): 41.4 (s, CH3), 57.3 (s, CH2), 125.4 (d, 1JP,C = 65.0 -
3
2
1
Cl2): d 6.8 (s + d, JPt,P = 3826 Hz).
12 Yield: 108 mg (65%). HRMS (ESI): m/z Calc. for [C39H35P2O3-
Pt]+: 808.1701; found for [MꢁL8ꢁH]+: 808.1704. 1H NMR
(400 MHz, CDCl3): d 0.8 (br, 6H, CH3), 3.4 (br, 2H, CH), 4.2 (br,
OH), 7.18–7.60 (m, 30H, Ph). 13C NMR (125 MHz, CDCl3): d 20.5
(s, CH3), 68.3 (m, CH), 128.1 (‘t’⁄, m-CPh), 131.1 (br, p-CPh), 134.5
(‘d’⁄, o-CPh), 178.8 (s, COO); i-C resonance not found. 31P NMR
1
3
Hz, i-C, Ph), 126.0 (d, JP,C = 64.2 Hz, i-C, Ph), 128.9 (d, JP,C = 11.6 -
Hz, m-C, Ph), 129.7 (d, JP,C = 11.5 Hz, m-C, Ph), 132.1 {br (s), p-C,
3
2
Ph}, 132.7 {br (s), p-C, Ph}, 133.9 (d, JP,C = 10.8 Hz, o-C, Ph), 134.5
(d, JP,C = 10.5 Hz, o-C, Ph), 181.0 (s, COO). 31P NMR (81 MHz,
2
1
CDCl3):
d
ꢁ142.7 (sept, JP,F = 712.1 Hz, PF6ꢁ), 7.4 (d + d,
2
1
2
1
JP ,P = 23.7 Hz, JPt,P = 3703 Hz, P0), 8.2 (d + d, JP ,P = 23.7 Hz,
(162 MHz, CDCl3): d 5.7 (s + d, JPt,P = 3856 Hz).
0
00
0
0
00
1
JPt,P = 3381 Hz, P00).
00
R/R0 = Et/H (6). Yield: 131 mg (73%) HRMS (ESI): m/z Calc. for
[C40H38O2NP2Pt]+: 821.2011; found for [M]+: 821.2020. 1H NMR
4.2.4. Synthesis of [Pt(PPh3)2{O–CHMe–C(O)O-
Anhydrous -lactic acid (L8) (17.5 mg, 0.19 mmol) was added to
jO,j
O0}] (11)
3
(400 MHz, CDCl3): d 0.60 (t, JH,H = 6.2 Hz, 3H, CH3), 2.76 (br, 1H,
NH), 2.86 (br, 2H, CH2CH3), 3.46 (dd, JH,H = 16.8 Hz, JH,H = 3.6 Hz,
L
2
3
a suspension of [Pt(CO3)(PPh3)2]ꢀCH2Cl2 (1; 140.0 mg, 0.16 mmol)
in MeOH (5 mL). The reaction mixture was stirred overnight at
room temperature. The white solid precipitated during the reac-
tion was filtered off, washed with diethyl ether (3 ꢂ 2 mL) and
dried under vacuum.
2
3
1H, CH2), 4.29 (dd, JH,H = 16.8 Hz, JH,H = 4.5 Hz, 1H, CH2), 7.24–
7.68 (m, 30H, Ph). 13C NMR (125 MHz, CDCl3): 12.0 (s, CH3), 48.8
(s, CH2CH3), 52.5 (s, CH2), 124.9, 125.4, 125.6, 126.1, 129.2 (d,
3
3JP,C = 10.7 Hz, m-C, Ph), 129.8 (d, JP,C = 10.9 Hz, m-C, Ph), 132.3
4
4
(d, JP,C = 1.6 Hz, p-C, Ph), 132.7 (d, JP,C = 1.8 Hz, p-C, Ph), 133.9
Yield: 78 mg (52%). HRMS (ESI): m/z Calc. for [C39H34NaP2O3-
2
2
Pt]+: 830.1524; found for [M]+: 830.1523. 1H NMR (200 MHz,
(d, JP,C = 9.9 Hz, o-C, Ph), 134.5 (d, JP,C = 9.6 Hz, o-C, Ph), 183.0 (s,
COO). 31P NMR (81 MHz, CDCl3): d ꢁ142.7 (sept, JP,F = 712.1 Hz,
1
3
CDCl3): d 1.35 (d, JH,H = 6.7 Hz, 3H, CH3), 4.66 (m, 1H, CH), 7.11–
2
1
PF6ꢁ), 6.4 (d + d, JP ,P = 24.0 Hz, JPt,P = 3353 Hz), 6.5 (d + d,
7.52 (m, 30H, Ph). 13C NMR (125 MHz, CDCl3): d 24.9 (s, CH3),
75.7 (s, CH), 127.9 (d, 3JP,C = 11.3 Hz, m-C, Ph), 128.0 (d, 1JP,C = 63.7 -
Hz, i-C, Ph), 128.1 (d, 3JP,C = 11.0 Hz, m-C, Ph), 129.1 (d, 1JP,C = 59.7 -
0
00
00
2
1
0
00
0
JP ,P = 23.1 Hz, JPt,P = 3703 Hz).
4
4
Hz, i-C, Ph), 130.7 (d, JP,C = 2.2 Hz, p-C, Ph), 130.8 (d, JP,C = 2.4 Hz,
4.2.2. Synthesis of [Pt(PPh3)2{N(COMe)–CHMe–C(O)O-
jN,jO}] (7)
2
2
p-C, Ph), 134.4 (d, JP,C = 10.9 Hz, o-C, Ph), 134.6 (d, JP,C = 10.8 Hz,
N-Acetyl -alanine (L6) (27.2 mg, 0.21 mmol) was added to a
L
o-C, Ph), 194.1 (m, COO). 31P NMR (81 MHz, CDCl3): d 10.6 (d + d,
suspension of [Pt(CO3)(PPh3)2]ꢀCH2Cl2 (1; 150.0 mg, 0.17 mmol)
in MeOH (5 mL). The reaction mixture was stirred overnight at
room temperature. Then the solvent was completely removed un-
der reduced pressure. The residue was dissolved in CH2Cl2 (0.5 mL)
and addition of diethyl ether (10 mL) resulted in precipitation of a
white solid which was filtered off, washed with diethyl ether
(3 ꢂ 2 mL) and dried under vacuum.
2
1
1
2
JP ,P = 24.8 Hz, JPt,P = 3241 Hz, P00), 12.3 (d + d, JP ,P = 24.8 Hz,
0
00
00
00
0
JPt,P = 3911 Hz, P0).
0
4.3. X-ray crystallography
Single crystals suitable for X-ray diffraction measurements of 4
were obtained from CH2Cl2 solutions with a layer of diethyl ether.
Data were collected with a STOE IPDS diffractometer at 200(2) K
Yield: 99 mg (63%). HRMS (ESI): m/z Calc. for [C41H37NaO3P2-
NPt]: 871.1798; found for [M+Na]+: 871.1789. 1H NMR (400 MHz,
3
CD3OD): d 1.42 (s, 3H, C(O)CH3), 1.71 (d, JH,H = 7.0 Hz, 3H, CH3),
using Mo K
a radiation (k = 0.71073 A, graphite monochromator).
4.41 (m, 1H, CH), 7.11–7.84 (m, 30H, Ph). 13C NMR (125 MHz, CD3-
OD) d 19.4/21.8 (s/s, C(O)CH3, CHCH3), 62.9 (s, CH), 126.3–135.8
(aromatic C), 173.3 (s, CO), 190.0 (COO) 31P NMR (81 MHz, CD3OD):
A summary of the crystallographic data, the data collection param-
eters, and the refinement parameters is given in Table 3. Absorp-
tion correction was applied empirically with the PLATON
program package (Tmin/Tmax 0.13/0.16) [38]. The structure was
solved with direct methods using SHELXS-97 [39] and refined using
full-matrix least-square routines against F2 with SHELXL-97 [40].
All non-hydrogen atoms were refined with anisotropic displace-
ment parameters and hydrogen atoms with isotropic ones. Hydro-
gen atoms were placed in calculated positions according to the
riding model. ORTEP III was used for graphical presentation [41].
2
1
0
00
0
d
6.4 (d+d, JP ,P = 24.7 Hz, JPt,P = 4091 Hz, P0), 9.3 (d+d,
2
1
JP ,P = 24.7 Hz, JPt,P = 3068 Hz, P00).
00
0
00
4.2.3. Synthesis of [Pt(PPh3)2{NHR–CHR0–C(O)O-
jO}2] (9, 10) and
[Pt(PPh3)2{HO–CHMe–C(O)O-jO}2] (12)
The requisite amino acid L6/L7 and lactic acid L8, respectively,
(0.41 mmol) were added to a solution of [Pt(CO3)(PPh3)2]ꢀCH2Cl2
(1; 160.0 mg, 0.19 mmol) in CH2Cl2 (5 mL) and the reaction mix-
ture was stirred overnight at room temperature. The solution
was concentrated under reduced pressure almost to dryness. Addi-
tion of diethyl ether (10 mL) resulted in precipitation of a white so-
2
Here and in the following a star (⁄) represents the X part of a ABX spin system.
Here and in the following ‘t’ and ‘d’ represent the pseudo triplet and pseudo
3
doublet, respectively.

S.R. Chaudhuri et al. / Inorganica Chimica Acta 394 (2013) 472–480
479
Table 3
4.4.3. DNA fragmentation assay
Crystallographic data, data collection parameters, and refinement parameters for
Determination of apoptotic cell death was performed by DNA
gel electrophoresis. Briefly, 1 ꢂ 106 A2780 cells were treated with
the respective IC90 doses of complexes 7 and 9, respectively, for
48 h. Floating cells induced by drug exposure were collected,
washed with PBS, and lysed with lysis buffer (100 mM Tris–HCl
pH 8.0; 20 mM EDTA; 0.8% SDS; all from Sigma Aldrich). Then they
were treated with RNAase A at 37 °C for 2 h and proteinase K at
50 °C (both from Roche Diagnostics, Mannheim, Germany). Control
sample was obtained by harvesting untreated A2780 cells and iso-
lating DNA according to the same procedure described above. DNA
laddering was observed by running the samples on 2% agarose gel
followed by ethidium bromide (Sigma Aldrich) staining.
complex 4.
Empirical formula
C42H42BF4NO2P2Pt
Mr
936.61
monoclinic
P21
12.7036(9)
23.773(2)
13.5624(9)
106.322(7)
3930.8(5)
4
Crystal system
Space group
a (Å)
b (Å)
c (Å)
b (°)
V (Å3)
Z
Dcalc (g cmꢁ1
)
1.583
3.707
l
(Mo K ) (mmꢁ1
)
a
F(000)
1864
h range (°)
Reflection collected
1.88–26.08
35449
4.4.4. Cell cycle analysis
For this assay, 1 ꢂ 105 A2780 cells were seeded in a six-well
plate with 5 mL of medium. After 24 h-incubation, cisplatin, 7
and 9 were added at their respective IC50 and IC90 concentrations.
Following a 24- and 48 h-incubation, cells were harvested by mild
trypsinization, collected by centrifugation, and washed with PBS.
Reflection observed [I > 2
r
(I)]
)
13222
Reflection independent (Rint
Data/restraints/parameter
Goodness-of-fit (GOF) on F2
15350 (0.0451)
15350/1/953
0.954
0.0324, 0.0649
0.0425, 0.0676
1.097 and ꢁ0.849
R1, wR2 [I > 2
r(I)]
R1, wR2 (all data)
Largest difference in peak and hole (e Åꢁ3
)
Adherent and floating cells were both resuspended in 100 lL of
PBS and fixed with 2 mL of 70% ethanol at 4 °C for at least 1 h.
The fixed samples were then centrifuged, and the cell pellet was
washed with 2 mL of staining buffer (PBS + 2% FCS + 0.01% NaN3)
and again centrifuged. The cell pellet was then resuspended in
4.4. In vitro studies
100
At the end of incubation, the samples are treated with propidium
iodide (20 g/mL of staining buffer) and allowed to stand for at
lL of RNase A (1 mg/mL) and incubated for 30 min at 37 °C.
4.4.1. Cell cultures and cytotoxicity assay
l
The human tumor cell lines: human anaplastic thyroid cancer
(8505C), ovarian cancer (A2780), human colon cancer (SW480),
human epithelial cancer (HeLa), and human breast cancer (MCF-
7) were maintained as monolayers in nutrient medium at 37 °C
in a humidified atmosphere with 5% CO2. Nutrient medium was
RPMI-1640 (PAA Laboratories) supplemented with 10% fetal bo-
vine serum (Biochrom AG) and penicillin/streptomycin (PAA
Laboratories).
least 30 min before analysis. The fluorescence intensity was deter-
mined by an AttuneÒ Acoustic Focusing Cytometer equipped with
AttuneÒ Cytometric Software. Each analysis was carried out using
about 1 ꢂ 104 events.
Acknowledgments
The authors would like to acknowledge Mrs. Renate Herzog for
technical support and Mrs. Bianka Siewert for the help in cell cul-
ture laboratory as well as BioSolutions Halle GmbH for providing
cell culture facilities.
The cytotoxic activity of the investigated compounds was mea-
sured by the sulforhodamine-B (SRB, Sigma Aldrich) microculture
colorimetric assay after 96 h [42]. Stock solutions of investigated
amino acids, platinum complexes, and of the reference compound
(cisplatin) were made in dimethyl sulfoxide (DMSO) at concentra-
tion of 20 mM and diluted by nutrient medium to the various
working concentrations. Final concentrations achieved in treated
Appendix A. Supplementary material
wells were in the range from 0.1 to 150 lM and tested in quadru-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2012.08.034.
plicate. All experiments were done in triplicate. The final concen-
trations (<0.1%) of DMSO were non-toxic to the cells. Absorbance
was measured at 570 nm using a 96-well plate reader (SpectraFlu-
or Plus Tecan, Germany). The IC50 and IC90 values, defined as the
concentrations of the compound at which 50% or 90% cell inhibi-
tion was observed, were estimated from the dose response curves.
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