Synthesis and in vitro characterization of platinum(II) anticancer coordinates using FTIR spectroscopy and NCI COMPARE: A fast method for new compound discovery

Article abstract of DOI:10.1016/j.bmc.2014.04.017

Platinum-based drugs have been used for several decades to treat various cancers successfully. Cisplatin is the original compound in this class; it cross-links DNA, resulting in cell cycle arrest and cell death via apoptosis. Cisplatin is effective against several tumor types but exhibits toxic side effects; in addition, tumors often develop resistance. An original in vitro approach is proposed to determine whether platinum-based research compounds are good candidates for further study by comparing them to marketed drugs using FTIR spectroscopy and the COMPARE analysis from the NCI. Both methods can produce fingerprints and highlight differences between the compounds, classifying the candidates and revealing promising derivatives.

Full text of DOI:10.1016/j.bmc.2014.04.017

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro characterization of platinum(II) anticancer
coordinates using FTIR spectroscopy and NCI COMPARE: A fast
method for new compound discovery
b
c
a
c
a
Gilles Berger a, , Hélène Leclercqz , Allison Derenne , Michel Gelbcke , Erik Goormaghtigh , Jean Nève ,
⇑
Véronique Mathieu b, François Dufrasne a
a Laboratoire de Chimie Pharmaceutique Organique, Campus Plaine CP205/5, Université Libre de Bruxelles, Bd du Triomphe, B1050 Brussels, Belgium
b Laboratoire de Toxicologie et Cancérologie Expérimentale, Faculté de Pharmacie, Campus Plaine CP205/1, Université Libre de Bruxelles, Bd du Triomphe, B1050 Brussels, Belgium
c Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine CP206/2, Université Libre de Bruxelles,
Bd du Triomphe, B1050 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Platinum-based drugs have been used for several decades to treat various cancers successfully. Cisplatin
is the original compound in this class; it cross-links DNA, resulting in cell cycle arrest and cell death via
apoptosis. Cisplatin is effective against several tumor types but exhibits toxic side effects; in addition,
tumors often develop resistance. An original in vitro approach is proposed to determine whether plati-
num-based research compounds are good candidates for further study by comparing them to marketed
drugs using FTIR spectroscopy and the COMPARE analysis from the NCI. Both methods can produce fin-
gerprints and highlight differences between the compounds, classifying the candidates and revealing
promising derivatives.
Received 13 November 2013
Revised 26 February 2014
Accepted 10 April 2014
Available online xxxx
Keywords:
Platinum
Chemotherapy
Cytotoxicity
Infrared spectroscopy
NCI
Ó 2014 Elsevier Ltd. All rights reserved.
COMPARE
1. Introduction
approval in 1989) and oxaliplatin (FDA approval in 2002). While
carboplatin exhibited an improved toxicity profile, oxaliplatin
The cytotoxic activity of platinum salts was discovered in the
laboratory of Barnett Rosenberg more than 40 years ago.1 While
studying the effect of electromagnetic radiation on bacterial
growth and the possible involvement of electromagnetic fields in
mitosis, an unexpected inhibition of cell division was observed.
Later, this effect was revealed to be unrelated to electromagnetic
radiation and was attributed to the platinum(II) salts produced
via electrolysis. The most active compound was cis-diamminedi-
chloroplatinum(II); this compound is better known as cisplatin
and has significantly impacted cancer treatment, particularly tes-
ticular and ovarian malignancies.2,3
Forty years after its discovery, cisplatin is still incorporated in
most cancer chemotherapies. However, the severe drawbacks of
cisplatin limit its use and efficacy. The poor pharmacokinetics, high
toxicity and the development of resistance have challenged medic-
inal chemists for decades. Some improvements were introduced
with the two next drugs marketed worldwide: carboplatin (FDA
extended the spectrum of activity for platinum derivatives.4,5 If
the first goal has been roughly achieved by carboplatin which
retains the antitumor effects of cisplatin but with a modified tox-
icity profile (particularly highly reduced nephrotoxicity while
exhibiting higher myelosuppression), the latter has become a
major focus over the past ten years: current research regarding
platinum compounds is driven by the need to expand the group
of tumors that respond to treatment while overcoming resistance
toward platinum chemotherapy. Much effort has been spent to
elucidate the molecular basis for both the anticancer properties
and the natural or acquired resistance to platinum drugs,
paving the way for the rational design of new compounds.
Currently, the chemical diversity of platinum derivatives has
grown (trans isomers, orally active platinum(IV), bi- and tri-
nuclear coordinates6–8), important clinical trials were conducted
and are still.9–12 Structurally-related derivatives to the compounds
here tested have been reported very recently.13
The search for new drugs that overcome resistance while exhib-
iting broadened or shifted activities requires activity profiling with
better sensitivity. Highlighting the subtle differences in the
⇑
Corresponding author. Tel.: +32 2 650 5248; fax: +32 2 650 5249.
E-mail address: giberger@ulb.ac.be (G. Berger).
http://dx.doi.org/10.1016/j.bmc.2014.04.017
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Berger, G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.017

2
G. Berger et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
underlying molecular mechanisms of action for platinum com-
plexes could be very useful when identifying hits that differ from
classical platinum drugs. Consequently, fingerprinting the bio-
chemical effects on cells may help identify good candidates for fur-
ther work and in vivo experiments. Fourier transform infrared
(FTIR) spectroscopy with complex systems including living cells
and tissues has undergone rapid development over the last two
decades, growing alongside the increasing accuracy of spectrome-
ters and modern computational tools able to treat large datasets.
Studies in the late 1980s used FTIR spectra to classify and identify
bacterial pathogens, enabling strain determination and resistance
detection.14 Shortly afterward, disease detection was first
attempted: malignancies in cervical cells were observed through
their FTIR signature. Consequently, FTIR spectroscopy provides a
sensitive tool for identifying the fingerprint of the metabolic
changes caused by drug treatment. This approach provides a global
signature of all molecule types present in the cells: proteins,
nucleic acids, lipids, carbohydrates, small metabolites and others.
FTIR-based classifications of cancer cells in cultures exposed to
various cytotoxic agents have been reported for cardenolides, poly-
phenols and platinum compounds.15–17 Importantly, when using
well-characterized anticancer drugs, FTIR-based classifications
were separated according to the documented modes of action for
these compounds.18,19 That observation was later validated using
seven different cell lines.20 This approach is well adapted for plat-
inum complexes because their mechanism of action remains
poorly understood; the data rely on a panel of partially identified
or unidentified targets.
The present paper describes an efficient method for
characterizing a series of platinum compounds. Based on previous
studies,21–23 five chiral platinum(II) coordinates were prepared
through the convenient opening of aziridinium intermediates.
The synthetic products were initially assessed for their anticancer
properties through growth inhibition assays. After determining the
in vitro anticancer potency, the products were fingerprinted and
compared using infrared spectroscopy, acquiring a global signature
for all of the effects induced by the cytotoxic agent. In addition, the
two most interesting compounds were evaluated using a 60 cancer
cell-line panel from the NCI, comparing their mechanism(s) of
action to the compounds already available in the NCI database.
Indeed, the COMPARE algorithm was used to reveal any similarities
in the activity pattern relative to cisplatin and oxaliplatin.
Carboplatin was not used because it is essentially a toxicity-
modified cisplatin analog with slower activation kinetics.24
The best candidates for this study were selected based on cyto-
toxicity assays performed previously by our group.18–20 This
screening phase involved performing crystal violet assays.20 These
first studies revealed the strong dependence of the anti-prolifera-
tive potency on the stereochemistry. Even when little enantiomeric
effect was observed, the diastereomeric selectivity was important,
trans compounds were considerably more active than cis isomers,
and adding a second aromatic substituent on the Pt-containing five
membered ring decreased the activity. Table 1 summarizes the T/C
values obtained from these studies.18–20 The growth inhibition is
expressed as the ratio between the growth of the treated and con-
trol samples (T/C values). Negative values are obtained when the
compounds are cytocidal.
2. Synthesis
Compounds were prepared using an asymmetric process begin-
ning with enantiopure
a-amino acids to form b-amino alcohols
intermediates.25 These intermediates were transformed to b-dia-
mines via chiral aziridines, and the diamines were complexed with
Pt(II) to afford the final cytotoxic compounds.
Two distinct pathways were used to produce syn- and anti-con-
figured diamines from their common amino alcohol precursors (1);
these compounds were prepared as described by Reetz et al.26 anti-
Configured diamine 10a was obtained through a dibenzylaziridini-
um intermediate that was regiospecifically opened at the benzylic
position by NH3. To produce the the syn isomers, aziridines 4a–d
were prepared as previously published22 and activated with HCl
to produce the protonated aziridinium species in preparation for
a nucleophilic ring-opening. This efficient activation was enabled
using azide (pKa = 4.7) as the nucleophile. Indeed, the relatively
low basicity of Nꢀ3 facilitates the selective protonation of the nitro-
gen on the aziridine. The protonated aziridines (4a) preferentially
opened at the benzylic position because the phenyl group activates
the electrophilic carbons of the three-membered ring. These effects
have been extensively discussed and rationalized using reactivity
descriptors and conceptual density functional theory.27 The syn-
thetic pathways are summarized in Scheme 1.
3. Results
3.1. In vitro growth inhibitory concentration—IC50
The IC50 value represents the concentration that reduces the cell
growth by 50% after 72 h of drug exposure. The values for each syn-
thesized compound and the marketed drugs (cisplatin and oxalipl-
atin) are given in Table 2.
Table 2 shows that all trans-configured compounds (7a–d) dis-
play similar to higher antiproliferative effects than cisplatin and
oxaliplatin, while the cis-configured compound 10a shows a much
lower antigrowth activity, as expected due to the T/C values. Enan-
tiomers 7a and 7b are the most potent compounds in the series
and are significantly more active than cisplatin and oxaliplatin.
As outlined in previous papers with another assay,18–20 compounds
7a and 7b bear a fluorine substituent on the aromatic ring and an
isopropyl group on the diaminoalkane side chain and show higher
activities in this cancer cell panel than compounds 7c and 7d. Com-
pounds 7a and 7b were evaluated by the Developmental Therapeu-
tic Program (DTP) from the National Cancer Institute (see
Section 3.3).
Table 1
Antiproliferative effects determined using the T/C (%) values obtained for the whole compounds series on the MCF-7 cell line
Stereochemistry
Pt1
33
Pt2
6
Pt3
7
Pt4
Pt5
0
(1R,2S)
26
5
ꢀ27
ꢀ51
(1S,2R)
(1S,2S)
29
10
6
ꢀ52
ꢀ41
28
0
ꢀ5
0
Pt1
Pt2
Pt3
: R2= 4-H, R1= Me
: R2= 4-F, R1= Me
ꢀ32
ꢀ23
R1
R2
∗
∗
(1R,2R)
ꢀ2
: R2= 4-F, R1= Et
i
NH2
Cl
H2N
Cl
Pt4: R2= 4-F, R1= Pr
Pt5: R2= 4-F, R1= 4-F-Ph
Pt
Cytostatic effect (positive values): T/Ccorr (%) = [(T ꢀ C0)/(C ꢀ C0)] ꢁ 100; Cytocidal effect (negative values):
s (%) = [(T ꢀ C0)/C0] ꢁ 100. For more details on the growth
inhibition screening, see Ref. 20. Underlined values indicate that the compound was chosen for the present study.
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G. Berger et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Example
from 1a
OH
NH2
NH2
S
S
S
f
R1
g
R1
R1
R
R
R
R2
R2
R2
NBn2
NBn2
NH2
S
S
PhR2
N+
Bn Bn
R1
MsO-
1a-anti
8a-anti
9a-anti
R2= p-F, R1 = iPr, (1R,2S)
a,b,c
N3
NH2
R2
d
S
S
e
R1
R1
S
R
R1
S
S
R2
R2
NH2
NH2
N
S R
R1
PhR2
Cl-
H
N
5a-
syn
6a-syn
4a-cis
+
H2
trans compounds:
i
7a
7b
: R2= p-F, R1 = Pr, (1S,2S)
: R2= p-F, R1 = Pr, (1R,2R)
i
R1
NH2
R2
S
7c: R2= p-F, R1 = Me, (1R,2R)
7d: R2= p-H, R1 = Me, (1R,2R)
R2
h
S
S
S
R1
NH2
H2N
Cl
Pt
cis compound:
NH2
6a-syn
Cl
i
10a:
R2= p-F, R1 = Pr, (1R,2S)
7a-trans
Scheme 1. Reagents and conditions: (a) H2 (Patm.), Pd(OH)2/C, HCl, MeOH, rt; (b) SOCl2, toluene, 40 °C; (c) NaOH, MeOH, 50 °C; (d) NaN3, HCl, MeCN, 80 °C; (e) LAH, Et2O, rt; (f)
MsCl, NH4OH, toluene, 0 °C/rt; (g) H2 (Patm.), Pd(OH)2/C, HCl, MeOH, rt; (h) K2PtCl4, NaOH, H2O (pH 7).
Table 2
IC50 determined through MTT assay for 6 cell lines
A549
U373
SKMEL28
11
3.7 0.9
OE21
Hs683
B16F10
Mean
Cisplatin
Oxaliplatin
7a
7b
7c
7.0 0.6
2.3 0.9
0.36 0.03
1.3 0.4
2.7 0.7
4.0 0.1
1.7 0.3
0.9 0.5
0.17 0.03
0.37 0.03
0.7 0.2
4
3.3 0.3
0.83 0.09
0.47 0.07
1.6 0.4
2.7 0.3
7
3.3 0.3
3
3.0 0.6
0.17 0.07
0.10 0.01
0.10 0.01
0.27 0.03
0.70 0.06
6.7 0.9
5
1
1
1.8 0.5
0.8 0.4
1.3 0.4
2.7 0.9
3.7 0.8
42 11
3
2
0.47 0.09
1.5 0.8
3.7 0.9
4.3 0.9
57 22
3.0 0.6
6
4
3
1
7d
10a
2.0 0.6
19
2
49
9
7
58 17
60 10
Data are expressed as mean SEM in lM.
3.2. FTIR spectroscopy
of lipids. The shoulder present at 1745 cmꢀ1 is assigned to the ester
(C@O) stretching vibration. The region between 1700 and
1300 cmꢀ1 is dominated by protein absorption. The carbonyl
stretching vibration from the peptide bond is observed at
1650 cmꢀ1 (i.e., amide I), and the bending mode of the amide
NAH bond (amide II) is found at 1540 cmꢀ1. Between 1480 and
1300 cmꢀ1, the resonances arise from various amino acid side
chains and fatty acids. Between 1300 and 900 cmꢀ1, the absorption
bands are largely attributed to carbohydrates and phosphates, par-
ticularly phosphate groups related to nucleic acids (DNA and RNA).
The peaks at 1241 cmꢀ1 and 1085 cmꢀ1 are typical of the asymmet-
ric and symmetric phosphodiester vibration of nucleic acids.31,32
FTIR spectroscopy has been used to compare the above selected
products to cisplatin and oxaliplatin in parallel with the NCI data
discussed in the later sections. Indeed, comparing the impact of
various drugs on cancer cells through perturbation of their infrared
fingerprints would help obtain a global view on the subtle differ-
ences within the modes of action. Platinum drugs might be
multi-targeted drugs because they react with numerous molecular
species inside cells, even though DNA is likely the major target pre-
dominantly responsible for the cytotoxic effect.17,28 Nevertheless,
only 5–10% of covalently bound cell-associated cisplatin effectively
forms DNA adducts, while approximately 80% of the drug is bound
to proteins or peptides.29,30
3.2.1. Difference spectra and Student t-test
IR spectra of A549 cells were measured after 6 h of treatment.
Four independent cell cultures were produced and four spectra
were taken per culture flask. During the first step, the preprocessed
spectra (see Section 5) were averaged for each drug, and the stan-
dard deviation was calculated. Figure 1 displays the mean spectra
(solid lines) and standard deviations calculated at every cmꢀ1 (dot-
ted lines). These spectra seem very similar at first glance; they can
be divided approximately into four main regions. The bands found
between 2800 and 3000 cmꢀ1 arise from the CAH bonds stretches
primarily present in lipids (membrane phospholipids and fatty
acids). The region between 1800 and 1700 cmꢀ1 is also characteristic
To reveal the spectral variations between treated samples and
controls, the mean control spectrum (i.e., untreated A549 cells)
was subtracted from the mean of each condition, generating differ-
ence spectra. The statistical significance was determined using a
Student t-test (a = 0.01) at every wavenumber and the thicker lines
indicate the locations of a significant difference (Fig. 2).
Oxaliplatin and cisplatin display weaker and less significant dif-
ferences when compared to the control. However, cisplatin treat-
ment induces specific bands in the region related to asymmetric
and symmetric stretching of lipids (between 3000 and
2800 cmꢀ1, arrows 1); conversely, oxaliplatin shows no effect,
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4
G. Berger et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 1. Mean spectra (solid lines) standard deviations (dotted lines) of the A549 cells exposed to the drug indicated in the right margin during 6 h at their IC50
concentration. Raw spectra were first normalized for equal area between 1582 and 1492 cmꢀ1 (amide II) with a baseline correction. The mean spectra were calculated using
spectra that passed for signal-to-noise ratio and absorbance thresholds (see Methods). At least 9 spectra were used for calculating the mean. For better readability, the mean
spectra were offset along the absorbance axis (axis is omitted).
and compounds 7a–d and 10a tend to diminish the lipid content of
the A549 cells. The differential effect between our compounds and
cisplatin is also supported by the increase in amide I and II bands in
cisplatin-treated cells, while the other conditions resulted in a
decreased absorbance in these regions (arrows 2). Additionally,
no significant changes are observable with cisplatin in the carbo-
hydrate and DNA/RNA region, but a strong disruption is observed
in these spectral regions for compounds 7, as well as compound
10a and oxaliplatin to a lesser extent. In summary, we mainly
observe spectral differences between cisplatin and our compounds,
while oxaliplatin seems to have a biochemical signature more
similar to 7 and 10. These observations were corroborated by
hierarchical clustering and the COMPARE algorithm (see
Section 3.3.1). Interestingly, the COMPARE process confirmed a
stronger correlation to oxaliplatin (PCC = 0.662), although cisplatin
shows a very different activity pattern in the NCI60 panel and thus
a low PCC (0.165).
3.2.2. Hierarchical cluster analysis
To compare and classify the compounds, a hierarchical classifi-
cation of the difference spectra was built using the Ward’s algo-
rithm. This linkage method enables the hierarchical clustering of
groups with a minimal loss of information; it is based on the sim-
ilarity of group members relative to many variables.33 Clustering
Figure 2. Differences between the mean spectra obtained for the A549 cells treated over 6 hours with the drug indicated in the right margin and control cells. The spectra
have been normalized to the same area under 1582 and 1492 cmꢀ1 before subtraction. The spectra have been offset along the absorbance axis for better readability (axis is
omitted). A Student t-test was computed at every wavenumber with a significance level of
a = 0.01. Each marked wavenumber (thicker lines) indicates a statistically
significant difference between the means. The numbered arrows highlight major differences between spectra (see text).
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G. Berger et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
supports the DTP antitumor drug discovery program at the NCI.
This screening operation uses 60 different human tumor cell lines,
representing leukemia and melanoma, as well as cancers of the
lung, colon, brain, ovary, breast, prostate, and kidney.35,36 In the
first stage, the compound is evaluated against the 60 cell line NCI
panel at a single high dose of 10 lM. Afterward, the output from
the single dose screen is reported as a mean graph of the percent
growth of the treated cells; the results for 7a are represented in
Figure 4. Briefly, these data indicate the following: (1) compound
7a is particularly active against leukemia cells, (2) interesting
results are obtained on several cells lines of lung (A549 and NCI-
H460 cell lines), colon (HCT-116, HT29 and SW-621) and breast
(MCF-7 and MDA-MB-468) cancers, (3) complex 7a exhibits high
variability with moderate activity on CNS and prostate cancer cell
lines, and (4) ovarian cancers are the most resistant. The activities
against the other cell lines are not as interesting because they are
either weak or very low. NCI data for 7b is not presented because it
does not differ significantly from 7a (see Section 3.3.1).
Figure 3. Hierarchical clustering using the Ward’s algorithm.
A second stage occurs if the inhibitory potency is sufficient; a
compound is tested on the same cell line panel using five doses
to build dose-response curves and estimate the IC50 values (or
GI50). The data for these 5-doses were then used for the COMPARE
analysis.
was achieved for the biologically relevant infrared ranges: 3000–
2800 cmꢀ1 and 1800–950 cmꢀ1
.
For the clustering in the overall spectral range (Fig. 3), marketed
drugs form a group apart from the other complexes, as empirically
expected from difference spectra. The only unfluorinated com-
pound (7d) is also separated, while all fluorinated compounds pro-
duce a third cluster. Within this group, enantiomers 7a and 7b
form a close group. The same pattern is found when examining
spectral regions one by one, except for the proteins resonances;
7d becomes separated.
3.3.1. COMPARE algorithm analysis
The NCI COMPARE program37–40 consists of an online database
and a comparison tool that analyzes cytotoxicity data from the 60
cell-line panel, searching for activity profiles similar to compounds
previously screened by the DTP. A probe compound is entered into
the program and the COMPARE algorithm delivers a list of agents
with similar cytotoxicity patterns based on a calculated similarity
coefficient. The similarity between the probe and any database
compound is expressed through a Pearson Correlation Coefficient
(PCC):
3.2.3. Manova
To investigate the significance of the differences observed
between the compounds, a multivariate analysis of variance
(MANOVA) analysis was conducted using reduced data sets. The
data were reduced via principal components analysis (PCA), and
the scores for the first six principal components (PC) were used
to compute the MANOVA p values. These first six PCs represent
more than 99% of the variance in the unreduced data. This proce-
dure allows the significance between each pairs of tested com-
pounds to be highlighted. Statistically significant differences (p
<5 ꢁ 10ꢀ2) are observed for almost every pair of cytotoxic com-
pounds (see Table 3). A high significance is found for all com-
pounds against cisplatin (p <5 ꢁ 10ꢀ3); oxaliplatin shows a global
FTIR signature closer to our compounds, parallel to the COMPARE
results (see Section 3.3.1).
ꢀ
ꢀ
COVðX; YÞ
ðxi ꢀ xÞ ꢂ ðyi ꢀ yÞ
rxy
¼
¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ; ꢀ1 ꢃ rxy ꢃ þ1
P
P
rx ry
ꢂ
2
2
ꢀ
ꢀ
ðxi ꢀ xÞ ꢂ ðyi ꢀ yÞ
ð1Þ
The higher the correlation coefficient, the higher the similarity
between X and Y. Close correlations (i.e., high similarity) between
compounds may imply a common intracellular target or a similar
mechanism for cytotoxicity despite dissimilarity in the structure.
Therefore, a high correlation between compounds with known bio-
logical mechanism for cytotoxicity is often predictive and helps
identify the target(s) or mechanisms of action. For platinum-based
cytotoxic agents, a first step toward discovering new compounds
that may partially circumvent the limitations of current drugs
would be to obtain low PCC’s between these new compounds
and the common Pt drugs. These values would suggest a different
activity pattern regarding to the intracellular targets of the drug
3.3. NCI-60 human tumor cell line screen
The two most potent compounds in terms of their IC50 values
(i.e., 7a and 7b) were analyzed by the NCI to confirm their
in vitro activity with the help of DTP.34 The In Vitro Cell Line
Screening Project (IVCLSP) is a dedicated service that directly
Table 3
p-Values calculated by Multivariate Analysis of Variance (MANOVA) on the first 6 principal components between each pair of treatment after 6 h of incubation
Control
Cisplatin
Oxaliplatin
7a
7b
7c
7d
Control
Cisplatin
Oxaliplatin
7a
1
2.55 ꢁ 10ꢀ2
9.28 ꢁ 10ꢀ5
2.17 ꢁ 10ꢀ5
1
6.53 ꢁ 10ꢀ4
1
8.25 ꢁ 10ꢀ4
1
3.09 ꢁ 10ꢀ1
6.04 ꢁ 10ꢀ3
1.78 ꢁ 10ꢀ5
1.07 ꢁ 10ꢀ5
1
3.40 ꢁ 10ꢀ1
7b
7c
7d
5.57 ꢁ 10ꢀ5
4.10 ꢁ 10ꢀ6
7.74 ꢁ 10ꢀ4
3.97 ꢁ 10ꢀ5
1.67 ꢁ 10ꢀ2
2.30 ꢁ 10ꢀ2
2.76 ꢁ 10ꢀ3
4.82 ꢁ 10ꢀ2
1
6.07 ꢁ 10ꢀ3
1.05 ꢁ 10ꢀ2
1
9.23 ꢁ 10ꢀ2
1.63 ꢁ 10ꢀ2
3.26 ꢁ 10ꢀ3
2.34 ꢁ 10ꢀ3
4.91 ꢁ 10ꢀ3
1.64 ꢁ 10ꢀ2
3.82 ꢁ 10ꢀ2
1.05 ꢁ 10ꢀ1
10a
The principal components were computed between 3000–2800 cmꢀ1 and 1800–950 cmꢀ1 the scores on the first 6 principal components of all the spectra for each compound
were submitted to MANOVA. Underlined values indicate non-significant differences between spectra (p >0.05).
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6
G. Berger et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 4. The NCI one-dose assay data for compound 7a. The reported values for the one-dose assay represent the observed growth relative to the no-drug control, allowing
the detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). The colored bars indicate the difference relative to the mean growth
inhibition over the entire cell line panel.
with the possibility of a modified activity spectrum and/or toxicity
profile.
neither returned a correlation coefficient higher than 0.70 with cis-
platin, oxaliplatin or carboplatin. However, a 0.722 PCC was found
with a cyclohexanyl Pt(IV) salt, and a correlation coefficient of
0.713 was obtained with a dimethylsilylated, cisplatin-like com-
pound (Table 4). The highest observed correlation (0.726) with
any of the NCI database was obtained with a copper-based metallo-
drug. The COMPARE correlation data are summarized in Table 4.
The 5-dose NCI-60 results for compounds 7a and 7b were used
as probes and were run through the COMPARE program with a r
limit of 0.7; a lower value might be non-specific, generating numer-
ous answers. Values over 0.8 strongly suggest a similar mode of
action. Although 7a and 7b showed high similarity (r = 0.91),
Please cite this article in press as: Berger, G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.017

G. Berger et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Table 4
Full database COMPARE analysis using compound 7a (NSC 765876) as the probe
Chemical structure and NSC reference
Pearson correlation coefficient
iPr
0.91
(n = 56)
R
NH
2Cl
Pt
Cl
R
N
H2
F
7b (NSC 765877)
Br
S
0.726
(n = 43)
Cu+
N
N
N
NH
NSC 635450
O
O
S
0.722
(n = 47)
N
S
O
O
N
Pt2+
O
O
H2N
NH2
O
O
NSC 614887
H2
0.713
(n = 42)
N
Cl
Cl
Si
Pt
N
H2
NSC 625299
O
0.71
(n = 55)
N
OH
O
NSC 692758
O
N
0.703
(n = 53)
NH
N
N
NSC 691081
The number of common cell lines used for the calculation of the PCC is also pro-
vided (n).
Figure 5. Apoptosis level (a) and cell cycle analysis (b) for A549 and B16F10 after
72 h of treatment with 7a and cisplatin at their IC50 concentration. Kitꢀ and Kit+ are
kit-included controls; however, proper functioning was confirmed by treating PC3
cells with Narciclasin. Cell cycle was divided in three phases, G0/G1 phase (black),
S-phase (light grey) and G2-M phase (dark grey).
The COMPARE program was also run using a standard database
of chemotherapeutic agents, allowing a direct comparison with
marketed anticancer drugs. A correlation coefficient of 0.662 was
found with oxaliplatin, supporting the findings of the IR experi-
ments. Indeed, oxaliplatin showed rather high p values against
our compounds after the MANOVA. Conversely, a PCC value of
0.165 was found for cisplatin, for which the spectral signatures
on A549 cells are clearly dissimilar.
These results validated the FTIR fingerprinting technique for
classifying and highlighting the differences in the global cellular
effects of a drug, suggesting that FTIR is appropriate for rapidly
screening platinum derivatives.
cells (60%) are found for 7a, while cisplatin is not more apoptotic
when compared to the data for the A549 cells. In addition, almost
all of the cells are blocked in the G2/M phase, indicating a high level
of DNA alteration; the cellular checkpoints block the division. These
results support FTIR fingerprints: at the IC50, 7a produces stronger
cellular impairments than cisplatin, particularly to the DNA/RNA
content of the cell. Although the A549 cells seem to be able to man-
age this damage, the apoptosis sensitive cell line B16F10 does not.
4. Discussion
3.4. Flow cytometry
Platinum anticancer agents are valuable compounds because
they remain one of the most used and useful drugs for fighting can-
cers, even 40 years after the fortuitous discovery of cisplatin.
Unfortunately, toxicity and resistance limit their clinical use and
compromise the treatment outcomes. Carboplatin and oxaliplatin
provided some clinical improvements; the former is as effective
as cisplatin while displaying a more acceptable toxicity profile,
while the latter exhibits a broadened activity profile and effective-
ness against colorectal cancer. Similar goals led to the regionally
marketed lobaplatin, heptaplatin and nedaplatin, as well as picopl-
atin and satraplatin, which are still under trial (satraplatin is the
first orally available Pt drug with a promising response on prostate
cancer, which is intrinsically resistant to cisplatin). Due to the rel-
atively disappointing outcomes of promising chemotherapeutic
To prove the differences between compound 7a and cisplatin
observed from their FTIR fingerprints and the COMPARE analysis,
apoptosis levels and cell cycle were analyzed using flow cytometry.
The analysis was conducted on A549 and B16F10 cell lines against
cisplatin; the former was used for the spectroscopic measure-
ments, and the latter is apoptosis sensitive, highlighting the differ-
ential impact on the cells. The PC3 cell line and Narciclasin were
used as the positive controls (see Fig. 5).
Although
a stronger disruption of cellular metabolism is
observed for A549 during the FTIR examination between 7a and cis-
platin, this result is not reflected by an increase in the apoptosis level
when using a TUNEL analysis after 72 h. However, after examining
the result obtained on B16F10, a very high percentage of apoptotic
Please cite this article in press as: Berger, G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.017

8
G. Berger et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
classes, such as anti-angiogenic agents and growth factor inhibi-
tors, the research over the last decade has re-focused on plati-
num-based compounds.41
be needed to expand the applicability of these early findings. How-
ever, previous reports on seven cell lines (A549, A427, PC-3,
DU145, T98G, U373 and Hs683) revealed that the clustering of
antitumor drugs displaying well characterized modes of action
was strictly independent of the cell line.17
Moreover, many specific features of this class hinder the
development of new drugs with a better profile. The complex
chemistry associated with their anticancer properties (i.e., activa-
tion kinetics and thermodynamics, dissociation equilibria and sta-
bility of activated species), their multiple targets, the relative
composition and structure of their DNA adducts must be under-
stood and controlled. The multitude of factors influencing their
anticancer properties makes classical structure activity-relation-
ships difficult to elucidate. A recent attempt to develop a quanti-
tative structure-activity relationship (QSAR) model for novel
cytotoxic platinum(IV) and platinum(II) complexes has yet to be
mentioned.42,43
Therefore, alternative methods for platinum-drug discovery
are necessary. Moreover, the interest in fingerprinting techniques
is justified by the rapid profiling of these compounds for the rea-
sons cited above. In this paper, innovative methods were used to
profile new drug candidates. After selecting the most promising
compounds with prescreening tests,18–20 a profile of the com-
pounds was acquired using two techniques that reflected the glo-
bal impact and mode of action on cancer cells, avoiding the
characterization of the cytotoxic agent at the molecular level.
Promising Pt compounds for future investigations could be
selected based on two major criteria: in vitro growth inhibitory
potency with an acceptable value and an atypical profile after fin-
gerprinting using FTIR spectroscopy and the COMPARE algorithm.
The latter criterion is likely more important because we would
expect atypical compounds to overcome the current limitations
of platinum drugs. Good in vitro growth inhibitory activity might
be necessary for a good drug candidate, but it is not sufficient
alone.
While ‘omic’ techniques report the cell composition for a spe-
cific type of molecule (proteins, lipids, small metabolites and oth-
ers) FTIR spectroscopy offers the opportunity to observe all
metabolic modifications induced by a drug in all types of mole-
cules. Drug-induced metabolic disorder should be amenable to
classification just as the bacterial gender, species, and strains can
be classified based on their FTIR spectra. While FTIR spectroscopy
presents several advantages, one limitation of the approach is that
assigning spectral changes to a specific molecule is difficult. For
instance, the amide I and II region (roughly 1700–1500 cmꢀ1) is
usually assigned to proteins, but weak absorption from some lipids
(phosphatidylserine, sphingomyelin), nucleic acids and metabo-
lites containing aromatic or amine groups are also present in this
part of the spectrum. In turn, the molecular origin of the spectral
signatures remains difficult to assess unless the molecules can be
extracted and investigated further.
The accuracy of this technique when classifying drug modes of
action cannot yet be assessed. Distinct spectral signatures for the
metabolic changes induced by a series of anticancer molecules
have already been obtained: various antimetabolites, anti-topoi-
somerases and agents blocking polymerization or depolymeriza-
tion of microtubules, cardenolide derivatives. The results
obtained here indicate that platinum derivatives can also be classi-
fied using this method.
In aqueous solutions or media, such as blood and cytoplasm,
chlorido-platinum complexes reacts with water to produces mono-
and diaqua species that may lose protons to form hydroxo com-
pounds. After hydrolysis, platinum compounds become much
more reactive toward nucleophiles, such as DNA nucleobases.46
Hydrolysis of platinum derivatives is therefore considered as a nec-
essary footstep prior to their reaction with DNA; this step has been
presumed to occur inside cells where the chloride concentration
drops (from 100 mM in plasma to 3–20 mM in cytoplasm).47,48
Consequently, these crucial properties represent key parameters
for the activity and toxicity-related behaviors of platinum com-
pounds, possibly contributing to the differences observed in the
antiproliferative potencies and the FTIR spectra.
The results obtained from the IVCLSP program (Fig. 4) agree
with those previously obtained on MCF-7 (Table 1) and A549
(Table 2).18–20 In contrast, we showed that complex 7a retains
excellent activity against U373 (glioblastoma astrocytoma) and
B16F10 (melanoma) cells, even if the results for the other cell lines
from the IVCLSP are disappointing. Finally, the excellent activity
against leukemia cells corroborated the results obtained with very
close analogs of our complexes by Zhang et al.49
For the compounds spotted using NCI COMPARE, the following
conclusions can be drawn, apart from the strong resemblance
between enantiomers 7a and 7b: (1) two Pt(II) complexes (NSC
614887 and NSC 625299) are closely related to compound 7a.
This similarity is not surprising because they certainly share
DNA as main target. However, despite their common metal cen-
ters, cisplatin and oxaliplatin also display discrepancies in their
mechanism of action. The structural and pharmacological rela-
tionships between compound 7a and complexes NSC 614887
and NSC 625299 indicate a parallel mechanism of action between
our compound and oxaliplatin derivatives (no biological informa-
tion was found for NSC 625299). In previous work by Rixe and
colleagues using COMPARE,50 NSC 614887 has been assigned a
different mechanism of action than cisplatin. (2) Copper complex
NSC 635450 is notable for its anti-leukemic properties.51
Although it is still under investigation, the mechanism of action
for Cu(II)–thiosemicarbazone complexes seems to be based on
the inhibition of the NF-jB pathway and proteasome rather than
DNA binding.52 (3) No information has been found regarding NSC
692758. However, because it bears a hydroxamic acid moiety, this
compound might target at least one of the identified targets for
its anticancer parents: histone deacetylase and matrix metallo-
proteinase.53 The first enzyme is also linked to nucleic acid
metabolism in cancer cells because it triggers DNA unwrapping
before replication. No information has been found regarding
NSC 691081.
Complex 7a differs significantly from cisplatin in term of biolog-
ical response and cell cycle disruption, particularly in B16F10 cell
lines.
The present study shows that the proposed workflow could
reveal significant differences between our synthetic compounds
and the marketed drugs. The data obtained from the NCI60 panel
and the COMPARE PCCs are consistent with the FTIR experiments.
Based on the preliminary studies (T/C values from crystal violet
assays), physicochemical properties (especially solubility) and FTIR
spectroscopic data, we could select a lead compound: 7a. This lead
shows high cytotoxicity; its biochemical impact on A549 cells is
different from that of cisplatin and oxaliplatin. Furthermore, no
strong correlation was found with the marketed drugs based on
the NCI COMPARE data.
To summarize the information obtained from the FTIR analysis:
(1) oxaliplatin and cisplatin may have significantly different mech-
anisms of action than our platinum derivatives;44,45 (2) the spectral
signatures of coordinates 7a–d and 10a are closer to oxaliplatin
than cisplatin; (3) non-F-substituted complexes give different IR
fingerprints;13 (4) the F-substituted complexes present similar IR
signatures.
These results might be cell line dependent. Therefore, a compre-
hensive study including a comparison over different cell lines may
Please cite this article in press as: Berger, G.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.017

G. Berger et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
The spectral data were analyzed as follows. The vapor water
contribution was removed as previously described,64,65 and the
spectra were baseline corrected. Normalization was achieved for
equal area between 1492 and 1582 cmꢀ1, corresponding to the
NAH bending peak of amides. Finally, the spectra were smoothed
to a 4 cmꢀ1 resolution through the apodization of their Fourier
transform with a Gaussian curve. The minimum and maximum
absorbance (0.02 and 1.1), as well as the signal to noise ratio
(>400), were checked to satisfy the fixed values; otherwise, the
spectra were discarded. The normality of the absorbance distribu-
tion was checked for each group at every wavenumber with a
Kolmogorov-Smirnov test through a comparison with a standard
5. Experimental details
5.1. Chemistry methods
General procedures and spectral data can be found in the pro-
vided Supporting information.
5.2. In vitro growth inhibitory concentration
The growth inhibitory potency of the five compounds was
determined using a colorimetric MTT (3-[4,5-dimethylthiazol-
2yl]-2,5-diphenyltetrazolium bromide, Sigma, Bornem, Belgium)
assay.54,55 Gliomas U373 (ECACC, Salisbury, UK, code 89081403)
and HS683 (ATCC, Manassas, VA, US, code HTB-138),56,57 non-small
cell lung cancer A549 (DSMZ, Braunschweig, Germany, code
ACC107),58 Caucasian esophageal squamous cell carcinoma OE21
(ECACC, code 96062201),59 human melanoma SKMEL28 (ATCC,
code HTB-72)60 and murine metastatic melanoma B16F10 (ATCC,
code CRL-6475)61 cell lines were used. All of the cell lines were
maintained under a special atmosphere (37 °C, 5% CO2) in culture
medium containing RPMI1640 supplemented with heat-inacti-
vated (56 °C, 1 h) fetal bovine serum (10%), glutamine (0.6 mg/
ml), penicillin (200 IU/ml), streptomycin (200 IU/ml) and gentami-
cin (0.1 mg/ml); all of these reagents were purchased from Lonza
(Verviers, Belgium). The cells were seeded in 96-microwell plates
(the seeding density varied between 800 and 1200 cells in
normal distribution for a confidence level where
shown).
a = 0.5% (not
The spectral corrections, the Kolmogorov–Smirnov test, the
Student t-test, the PCA, the confidence ellipses, the MANOVA and
the hierarchical classifications were carried out by Kinetics; this
custom made program ran under Matlab 7.1. (Matlab, MathWorks
Inc.).
5.4. Flow cytometry
The B16F10 and A549 cancerous cells were seeded in 25 cm2
flasks for 24 h before the treatments with or without 7a, 7b or
cisplatin; these treatments were applied to the cells over 72 h
at their IC50 concentrations. The IC50 concentrations were deter-
mined using an MTT colorimetric assay (see Table 2). The apop-
totic cells and cell cycle were analyzed simultaneously with a
TUNEL coupled with propidium iodide staining in presence of
RNAse. Stainings were performed by the use of the APO-Direct
TUNEL detection kit (BD Biosciences, Erembodegem, Belgium)
according to the manufacturer’s instructions. Fluorescence mea-
surements were undertaken with a Cell Lab Quanta cytometer
(Beckman Coulter, Analis, Suarlee, Belgium). Briefly, cells of each
flask were detached by trypsin as detailed above and pooled
with their own supernatant that could contain late apoptotic
detached cells. Cells are then fixed with paraformaldehyde 1%
in phosphate buffer (pH 7.4), permeabilized in ice-cold ethanol
70% diluted in water overnight and conserved at ꢀ20 °C till
staining. 30-OH extremities of single or double strand DNA
appearing during apoptotic process were labelled through enzy-
matic addition of FITC-dUTP while cell cycle profile was analyzed
simultaneously by propidium iodide staining in presence of
RNAse. All the staining solutions are provided with the kit with-
out concentration informations. Detailed protocol is available on
the data sheet.
100
treatments to ensure adequate cell adhesion. The anticancer com-
pounds were assayed from 10 nM up to 100 M for 72 h (from
ll/well, depending on the cell line) 24 h before applying the
l
10 mM stock solutions in dimethylformamide); the cell population
growth in the control and treated samples were determined
according to the capability of living cells to reduce the MTT yellow
product (0.5 mg/ml in white RPMI1640 medium; 100 ll/well) dur-
ing a minimum of 3 h into formazan blue crystals in their mito-
chondria.62 After removing of the MTT solution and
centrifugation at 200g for 8 min, the crystals are solubilized in
DMSO (100 ll/well). The cells surviving after 72 h of culture in
the presence or absence of the various compounds is directly pro-
portional to the intensity of the blue color. The optical density is
quantitatively measured via spectrophotometry with a Biorad
Model 680XR (Biorad, Nazareth, Belgium) at 570 nm (with a refer-
ence of 630 nm). Each experiment was conducted three times
independently.
5.3. FTIR spectroscopy
The A549 cells were cultured to sub-confluence (80% conflu-
ence) in 25 cm2 flasks63 and exposed to the compounds for 6 h at
their IC50, as determined by the MTT assay. Afterward, the culture
medium was removed and the cultured cells were quickly washed
with a trypsin/EDTA 0.5/0.2 g lꢀ1 buffer (1 ml per flask; Lonza,
Verviers, Belgium) before being harvested through a 5-minute
treatment with the same mix (1 ml per flask). The cells were cen-
trifuged at 200g and washed twice with 1 ml isotonic NaCl. After
the last centrifugation, the cell pellet is resuspended in isotonic
NaCl at a cell concentration adjusted to be between 5000 and
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.017.
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