Serine- and threonine-derived diamine equivalents for site-specific incorporation of platinum centers in peptides, and the anticancer potential of these conjugates

Article abstract of DOI:10.1039/c7nj03999a

A modular strategy that allows introduction of one or more reactive platinum units at chosen locations along a peptide sequence is presented. This makes use of diazides generated from serine and threonine as diamine equivalents which can be conjugated to the peptide under standard coupling conditions. Reduction of these diazides using Pd/C and H₂ followed by platination affords the final products in good yields. Following this, we prepared a new class of peptide-platinum conjugates and carried out preliminary cytotoxicity evaluation and DNA interaction studies. Inclusion of lysine residues in the sequence was found to improve DNA interaction and anticancer activities compared to analogous conjugates with hydrophobic side chains.

Full text of DOI:10.1039/c7nj03999a

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Serine- and threonine-derived diamine equivalents
for site-specific incorporation of platinum centers
in peptides, and the anticancer potential of these
conjugates†
Cite this:DOI: 10.1039/c7nj03999a
Sateeshkumar Kumbhakonam,‡a Kasipandi Vellaisamy,‡a Soumya Saroj,a
b
a
Nalini Venkatesan,b Karunagaran D.
and Muraleedharan Kannoth Manheri
*
A modular strategy that allows introduction of one or more reactive platinum units at chosen locations
along a peptide sequence is presented. This makes use of diazides generated from serine and threonine
as diamine equivalents which can be conjugated to the peptide under standard coupling conditions.
Reduction of these diazides using Pd/C and H2 followed by platination affords the final products in good
yields. Following this, we prepared a new class of peptide–platinum conjugates and carried out preliminary
cytotoxicity evaluation and DNA interaction studies. Inclusion of lysine residues in the sequence was found
to improve DNA interaction and anticancer activities compared to analogous conjugates with hydrophobic
side chains.
Received 17th October 2017,
Accepted 5th January 2018
DOI: 10.1039/c7nj03999a
rsc.li/njc
Analogs of cisplatin, in general, are designed making use of
simple diamines, peptide- or non-peptidic oligomers with built-in
Introduction
Cisplatin exemplifies the relevance of metal complexes in complexation sites, heterocyclic scaffolds and polymeric
therapy and is widely used for the treatment of testicular-, templates for Pt-coordination.14–20 While chemotherapy with
ovarian-, head and neck-, bladder-, and cervical cancers. Its cisplatin analogs has significantly improved patient survival,
dose-limiting toxicity is a concern which reduces the success new approaches which enable site-directed drug delivery,
rates in many cases, especially in the event of resistance.1–4 Its modulation of DNA interaction or type of cross-linking, and
analogs namely, carboplatin and oxaliplatin, offer some advantage use of pro-drugs that release reactive Pt after cellular activation are
in terms of toxicity, which have also been introduced for treatment currently being explored to improve and retain the effectiveness of
worldwide; newer members of this series like nedaplatin, lobaplatin, this class of drugs.21 Multinuclear Pt-complexes, characterized by
and heptaplatin at the same time are approved only in specific the presence of more than one Pt center in one molecular frame-
countries.5,6 After cellular activation, they are known to cross-link work, have gained attention as they could bring distinct types of
DNA, predominantly through N-7 of guanine. Efforts towards DNA crosslinks compared to cisplatin.22,23 A review by Wheate
exploring the therapeutic utility of other metal complexes have et al. has compared the activities of a large number of cisplatin
led to the identification of a number of leads from Ru(II), Ru(III), analogs, in both mono- and multi-nuclear forms. In the case of the
Os(II), Ir(I), Co(III), Co(II), Au(III), Au(I), etc.7 Depending on the latter group, factors like spacing between reactive metal centers,
nature of metal, its oxidation state and the ligand, these their relative orientations, flexibility of the spacer, secondary
complexes could elicit different cellular responses including interaction possibilities and charge are seen as the major
redox imbalance, and target specific effects like DNA cross- contributory factors. These studies tend to show that a higher
linking/binding/intercalation, affecting the expression levels of degree of flexibility in the spacer could increase the reactivity/
functional proteins like p53, p21 and cyclin D1, inhibition of extent of DNA cross-linking but at the expense of higher
HIF-1a-p300 interaction, etc.8–13
toxicity. Increased rigidity in the spacer, at the same time, leads
to lower potency. In this context, a balanced flexibility could
bring optimal reactivity with a better safety profile.24 Since
the conformation and flexibility of peptide segments can be
controlled by varying the nature and sequence of building
blocks, they offer an opportunity to study DNA cross-linking
propensities and reactivities in a systematic manner.25,26 They
are also ideal for modulating tissue localization, cellular uptake
a Department of Chemistry, Indian Institute of Technology Madras, Chennai,
600 036, India. E-mail: mkm@iitm.ac.in; Fax: +91-44 2257 4202;
Tel: +91-44 2257 4233
b Department of Biotechnology, Indian Institute of Technology Madras, Chennai,
600 036, India. Fax: +91-44 2257 4102; Tel: +91-44 2257 4126
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nj03999a
‡ These authors contributed equally to this work.
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and DNA interaction.27 In this regard, synthetic protocols that
allow introduction of platinum units at desired locations along
the peptide sequences are important and are the theme of this
report. In terms of mode of action, the peptide–Pt conjugates
discussed in this manuscript are expected to be more close to
cisplatin but the presence of peptide segments would allow
fine-tuning of target interaction or cellular uptake.
Scheme 2 Reagents and conditions: (a) free-amine from 3a and b, EDC,
HOBt, Et3N, CH2Cl2, 0 1C to rt, 70–80%; and (b) (i) 10% Pd/C, H2, MeOH;
and (ii) K2PtCl4 (1 eq.), H2O, 76–94%.
HRMS (ESI), which gave an isotope pattern for the mono-
platinum complex with the molecular formula C8H19Cl2N3O2PtK
corresponding to [M + K]+ with m/z at 493.0149 (calcd 493.0139).
It also showed a signal at À2970 ppm in 195Pt NMR.
Results and discussion
At first, we focused on identifying a bidentate ligand with a
suitable anchoring point for peptide conjugation. Of different
possibilities, diazides 3a–b (Scheme 1), accessible from ortho-
gonally protected serine and threonine through a reduction–
mesylation–azidation sequence, seemed attractive as they can
be attached to the peptide templates of interest through their
carboxyl units. There have been reports on the use of 1,2,3-
triaminopropane for preparing fluorescently labelled cisplatin
analogs or similar conjugates with peptides, but these approaches
involved the use of triaminopropane-1,3-dicarbamate, requiring
acid-treatment followed by neutralization before platinum
complexation.28–32 In comparison, the diazides used in this
report can be accessed and introduced into peptides rather
easily and converted to the required diamines just before the
complexation step. To obtain the simplest cisplatin analogs
based on these ligands, azides 3a–b were reduced under Pd/C
and H2 conditions and the diamines generated were reacted with
K2PtCl4 in water to obtain complexes 4a–b in 90–95% yields. The
formation of 4a was monitored by 1H NMR experiments by
noting shifts in CH and CH2 signals after addition of K2PtCl4
(1 equiv.) in D2O. There were considerable shifts in these peak
positions, indicative of complexation, as shown in Fig. 1. The
presence of platinum in the product was further confirmed by
The feasibility of attaching the diamine equivalents 3a and b
to various amino acids and peptides is demonstrated in
Scheme 2. Deprotection of the Boc group from 3a and b and
coupling of the free amine with appropriate Boc-protected
amino acids or peptides (5) under EDC/HOBt conditions led
to conjugates with the general structure 6a–k in 70–80% yields.
They were individually reduced and treated with K2PtCl4 in
water to obtain the corresponding platinum complexes 7a–k
shown in Fig. 2.
Among them, the ornithine- and lysine based Pt conjugates
7d–7k (Fig. 2) were prepared to make use of the electrostatic
interaction of the side chain with the phosphate backbone
of DNA to achieve additional stabilization to the Pt-complex.
In fact, Reedijk et al. previously reported similar peptide–Pt
conjugates from lysine and arginine, where the complexation
was done through an ethylenediamine unit attached at the
N-terminus.33 The synthesis of 7h starting from bis-Boc lysine
(8) and e-Boc LysOMe is shown in Scheme 3 as a representative
example. The C-terminal deprotection and chain elongation of
the initial adduct 9 can be done to introduce an appropriate
number of lysine residues in the backbone and then capped with the
diamine equivalent 3a before taken for reduction and platination.
Scheme 1 Reagents and conditions: (a) NaBH4/MeOH, 0 1C–rt, 24 h,
80–85%; (b) MsCl/Et3N, CH2Cl2, 0 1C to rt; 2 h; (c) NaN3, DMF, 60 1C, 12 h,
75–80%; (d) 10% Pd/C, H2, MeOH, 2 h, 95–96%; and (e) K2PtCl4 (1 eq.),
H2O, 24 h, 90–95%.
Fig. 1 1H NMR spectra of the 1,3-diamine derived from L-serine (a) and its
platinum complex (b).
Fig. 2 Peptide platinum conjugates 7a–k.
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Scheme 3 Reagents and conditions: (a) H2N-Lys(Boc)-OMe, EDC, HOBt,
NEt3, CH2Cl2, 68%; (b) LiOH, H2O/THF (1 : 2), rt, 2 h, 94%; (c) free amine
from 3a, EDC, HOBt, NEt3, CH2Cl2, 81%; (d) Pd/C, H2, MeOH; (e) K2PtCl4,
H2O, 75%; and (f) CF3CO2H (neat), quantitative.
Scheme 5 Reagents and conditions: (a) free-amine from 3a, EDC, HOBt,
NEt3, CH2Cl2, 0 1C to rt, 76%; (b) NH3, methanol, rt, 6 h, 63%; (c) (i) TFA,
CH2Cl2, rt; and (ii) Boc-Ileu-OH, EDC, HOBt, NEt3, CH2Cl2, 0 1C to rt, 57%;
and (d) (i) 10% Pd/C, H2, MeOH; and (ii) K2PtCl4 (1 eq.), H2O, 63%.
The next higher homologue 7j was prepared following a similar
sequence and the details are presented in the ESI.† The Boc-
protected complexes 7d, 7e, 7h and 7j were individually treated
with TFA to release the side chains as TFA salts which were also
characterized by HRMS.
To introduce the ligand at any chosen location in the
peptide sequence, we considered the aspartic acid side chain
as a suitable anchoring point. Towards this, the key intermediate
12 (Scheme 4) was prepared by reacting N- and C-protected
aspartic acid 11 with the free-amine from 3a, and was subjected
to azide reduction. Contrary to our expectation, diamine 13
underwent spontaneous cyclization to give imide 14, which
thwarted our progress through this route. For complete char-
acterization, 14 was converted to its dinitrobenzamide derivative
15 and the spectral details are included in the ESI.†
Cyclization in peptides containing aspartic acid residues has
previously been studied by Miklos Bodanszky and co-workers
and is often a side reaction during the synthesis of aspartyl
peptides.34 We envisaged that amidation of the carboxyl terminus
would decrease the cyclization propensity during the azide
reduction step due to decreased electrophilicity. To know whether
such an approach would work in the present context, BocAsp-
(b-COOH)COOBn 16 (Scheme 5) was first prepared by RuO4
oxidation of Boc-Phe-OBn35 and coupled with the free-amine
from 3a under EDC/HOBt conditions to obtain 17 in 76% yield,
and then treated with methanolic ammonia to obtain the C-terminal
amide 18. Subsequently, it was subjected to Boc-deprotection and
coupled with Boc-Ileu-OH to obtain intermediate 20, which upon
reduction and platination gave the target compound 21 in 63%
yield. It is also possible to reduce 18 and then prepare its platinum
conjugate 19 in 67% without the problem of cyclization.
To prepare conjugate 26 (Scheme 6) having a platinum unit
on the N-terminus residue, BocAsp(b-OMe)OH was first coupled
with the free amine 23 (prepared from BocNHValPheOMe via
amidation and Boc-deprotection). Here, the C-terminus is used
in the form of carboxamide to discriminate it during the
hydrolysis of N-terminal Asp-b-OMe. After treatment of 24 with
LiOH, the resulting acid was coupled with the free-amine from
3a and taken through reduction–platinum complexation steps
to obtain product 26 in 80% yield.
Extension of this methodology to place multiple platinum
centers on a peptide sequence is demonstrated in Scheme 7.
Here, dipeptide 29 was first prepared from orthogonally protected
coupling partners 27 and 28 in the solution phase. Methyl esters
on N- and C-terminal residues were then hydrolysed using LiOH
and the resulting diacid 30 was coupled with 2 equivalents of the
free-amine from 3a to obtain 31 in 69% yield. As done in the
previous cases, reduction of 31 and platinum complexation
afforded product 32a as a brown coloured solid. The formation
Scheme 4 Reagents and conditions: (a) free-amine from 3a (1.2 eq.),
EDC, HOBt, Et3N, CH2Cl2, 0 1C to rt, 24 h, 70%; (b) 10% Pd/C, H2, MeOH,
5 h, 95%; and (c) 3,5-dinitrobenzoyl chloride (2.2 eq.), Et3N, CH2Cl2, 0 1C to
rt, 5 h, 70%.
Scheme 6 Reagents and conditions: (a) EDC, HOBt, Et3N, CH2Cl2, 0 1C to
rt, 73%; (b) LiOH, THF/H2O (2 : 1), rt, 85%; (c) free-amine from 3a (1.2 eq.),
EDC, HOBt, Et3N, CH2Cl2, 0 1C to rt, 66%; (d) 10% Pd/C, H2, MeOH, 94%;
and (e) K2PtCl4 (1 eq.), H2O, 78%.
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Scheme 7 Reagents and conditions: (a) EDC, HOBt, NEt3, CH2Cl2, 12 h,
0 1C; (b) LiOH, H2O/THF (1 : 2), 24 h; (c) free-amine from 3a, EDC, HOBt,
NEt3, CH2Cl2, 0 1C to rt, 62%; (d) (i) Pd/C, H2, MeOH, (ii) K2PtCl4, H2O, 71%;
and (e) TFA (neat).
of this complex was confirmed by HRMS (ESI), which gave a peak
at m/z = 1098.6023 corresponding to the molecular formula
C26H53Cl3N9O7Pt2 [M À Cl]+ with two platinum atoms. The isotope
pattern in agreement with the theoretical values is included in the
ESI.†
DNA interaction studies: ethidium bromide displacement assay
Ethidium bromide (EtBr) displacement assay is commonly
followed to assess the DNA binding affinity of various inter-
calators and binders from natural and synthetic sources.36–38
This cationic dye exhibits strong emission at around 600 nm
(when excited at 526 nm) on intercalation between the base
pairs of DNA, but displacement with other molecules leads to
decrease in emission intensity.39–41 At first, a stock solution of
calf thymus DNA was prepared in Tris–HCl buffer (50 mM Tris,
10 mM NaCl, pH 7.4). The ratio of absorbance of this solution at 260
and 280 nm (A260/A280) was found to be in the 1.8–1.9 range,
indicating that the influence from proteins is minimal.42 The
concentration of this stock solution was determined on the basis
of UV absorbance at 260 nm and the reported molar extinction
coefficient of 6600 MÀ1 cmÀ1 for CT DNA, and was estimated to be
3.32 Â 10À4 M.43 For fluorescence studies, the peptide–Pt conju-
gates were dissolved in 10% DMSO in Tris–HCl buffer (50 mM Tris,
10 mM NaCl, pH 7.4), whereas an ethidium bromide solution was
prepared in Millipore water. Fluorescence measurements were
performed using a Jasco spectrofluorometer at an excitation wave-
length of 526 nm and emission was observed at 605 nm. Emission
spectra were recorded with increasing amounts of the Pt complex by
adding 10 mL of 10 mM solution each time. In these experiments,
the intensity of the emission at 605 nm was found to decrease with
increase in the concentration of peptide–Pt conjugates. The
emission profiles of 4a, 7e, 32a and 7i are presented in Fig. 3
and those of others are included in the ESI† (Fig. S88). The
peptide–Pt conjugates 4a–b, 7a–e, 7h, 7j and 7f, 7g, 7i, 7k allow us
Fig. 3 Variation in fluorescence intensity from ethidium bromide upon
increasing the concentration of 4a (a), 7e (b), 32a, (c) and 7i (d); [DNA] =
1.5 Â 10À5 M, [EtBr] = 1.5 Â 10À5 M. Comparison of Stern–Volmer plots
obtained from various experiments showing relatively better binding
profiles of positively charged Pt conjugates as indicated by their slopes (e).
(7f, 7g, 7i and 32b) are greater than those of the analogous
uncharged ones (7d, 7e, 7h and 32a), which supports the fact
that an increase in the number of charges has a positive
influence on DNA interaction which is expected based on the
electrostatic interaction with the phosphate backbone. Apparent
binding constants are useful for comparing compounds which
interact with DNA. A Kapp value above 106 MÀ1 indicates the classical
intercalation like EtBr, whereas the value in the 104–105 MÀ1 range
represents the groove binding. Although cisplatin analogs interact
with DNA through a different mechanism, their relative affinities
44
with DNA have been correlated using Kapp
.
To make some
comparative assessment, the apparent binding constants (Kapp
)
of the complexes were calculated using the equation
KEB[EB] = Kapp[complex]
to understand the effect of positive charge on the side chain to where KEB is the DNA binding constant of EtBr (1.0 Â 107 MÀ1),
their DNA interaction. The former group, having the side chain in [EB] is the concentration of ethidium bromide and [complex] is
the Boc-protected form, was relatively less efficient compared to the concentration of the Pt complex at 50% reduction of the
the latter, which have free amino groups as triflate salts.
initial fluorescence emission intensity of EB bound DNA. The
As evident from Fig. 4(e) the slopes of the Stern–Volmer Kapp values for various complexes are given in Table 1. It is very
plots in the case of systems with positively charged side chains interesting to note that at least two compounds (7i and 7k) from
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compounds to DNA solution (r = 0, 0.5, where r = [complex]/
[DNA]) confirm that these complexes interact well with DNA and
induce changes in its conformation (Fig. 4).
Anticancer assay
Based on the promising results from the ethidium bromide
displacement assay, the cytotoxicities of these platinum con-
jugates were evaluated on human cervical cancer cells (SiHa)
following Alamar blue assay.45,46 The dose–response plots of
selected compounds are shown in Fig. 5 and those of others are
included in the ESI† (Table S1). As can be seen, there was a
dose-dependent decrease in cell-viability with increase in the
concentration of these conjugates. In general, the presence of
positively charged side chains led to a better cytotoxic response
compared to those containing hydrophobic groups (7i, 7k vs.
7a, 7b). The bis-platinum complex in the Boc-protected form
(32a) and as salt (32b) showed comparable activities (IC50
values 75.9 mM and 83.4 mM), which could be due to the
formation of distinct types of DNA cross-links with less influence
from the side chains. The advantage of having multiple positive
charges becomes evident if we compare the activity of 7i with
three positive charges (IC50 44.5 mM, Table 1) with that of 7g (IC50
98.1 mM), which is the lower homologue; increase in cellular
uptake and better phosphate binding could be responsible. In
fact, this is in agreement with the observations from the
ethidium bromide displacement assay where an increase in
the number of lysine residues was found to positively influence
DNA interaction. In comparison with cisplatin (IC50 25.3 mM),
7i and 7k showed IC50 values of 44.5 mM and 51.1 mM upon
incubation for 48 h (Table 2, entries 6 and 7). To rule out the
possibility of ligands alone contributing to the cytotoxic effect,
the free amines of 4a, 7d, 7e, 32a, 7f, 7g, and 32b were also
tested for their anti-cancer activities against SiHa cell lines at
24 h, 48 h and 72 h using the Alamar blue assay. Their IC50
values were 4200 mM, and distinctly different from those of the
corresponding Pt conjugates (Fig. S86, ESI†). We also tested
these compounds against normal mouse fibroblastic cell lines
(NiH3t3) for assessing safety. Their therapeutic index (TI)
values were either comparable to or better than that of cisplatin
(Fig. S87 and Table S2, ESI†). Although the relative activity is
Fig. 4 Results obtained from CD analysis indicative of changes in the DNA
conformation in the presence of Pt-complexes 7f, 7g, 7i, 7k and 32b;
r = [complex]/[DNA]; r = 0 represents only DNA.
Table 1 Apparent binding constants (Kapp) of peptide–platinum conjugates
Pt conjugate
Kapp (Â105 MÀ1
)
Pt conjugate
Kapp (Â105 MÀ1
)
4a
4b
7a
7b
7c
7d
7e
7f
0.82
1.12
2.09
1.52
2.03
2.19
2.51
32.03
7g
7h
7i
30.45
2.99
106.70
5.90
115.20
8.01
3.99
7j
7k
26
32a
32b
62.80
this series have Kapp values in the order of 107 that are
comparable with EtBr.
CD spectroscopic studies
Circular dichroism is a sensitive technique to study the con-
formational changes of DNA during interaction with a drug or
binding agent. Normally, the CD spectrum of calf thymus DNA
shows a positive CD signal at 275 nm, and a negative CD signal
at 245 nm. This is attributed to base-stacking and right handed
helicity, respectively. Disturbance in the helicity of base stacking in
the presence of a chemical compound can be monitored from the
changes in its CD spectrum. CD measurements for Pt-complexes
7f, 7g, 7i, 7k and 32b, which are soluble in water, were performed
using a Chirascan CD spectrometer. A 10 mm quartz cuvette was
used for the study. Platinum complexes and DNA were dissolved
in Tris–HCl buffer (50 mM Tris, 10 mM NaCl, pH 7.4). Sig-
nificant changes in the CD spectrum upon addition of these
Fig. 5 Dose–response plots of the peptide–Pt conjugates in the assay
against SiHa cells after incubation for 48 h.
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Table 2 IC50 values of the peptide–platinum conjugates (after incubation
for 48 h)
on a Waters Q-Tof microt spectrometer with a LockSpray source.
Infrared spectra were recorded using a Nicolet 6700 FT-IR spectro-
photometer. Fluorescence measurements were performed using a
Jasco spectrofluorometer and circular dichroism measurements
were performed using a Chirascan CD spectrometer.
Synthesis of diazides 3a and 3b. Methyl esters of L-serine and
L-threonine were first prepared in quantitative yields by stirring
their MeOH solutions with SOCl2 (2 eq.) under ice-cold conditions.
N-Boc protection of these esters was done by treatment with
Boc-anhydride (1.1 eq.) in the presence of triethylamine (2 eq.)
in dichloromethane to obtain 1a and 1b in 95% yields. These
Boc-protected esters were reduced to the corresponding alco-
hols 2a and 2b (85–95%) using NaBH4 (2 eq.) in methanol
(Chem. Pharm. Bull., 1985, 33, 1342). Mesylation of 2a and 2b
using mesyl chloride, followed by azide substitution with
sodium azide (2.5 eq.), gave diazides 3a and 3b in 80–95%
yields.
Pt
IC50 (mM) Æ std
Pt
IC50 (mM) Æ std
Sl no. conjugate dev.
Sl no. conjugate dev.
1
2
3
4
5
4a
4b
7a
7b
7g
77.7 Æ 0.0041
80.5 Æ 0.0038
99.7 Æ 0.0085
116.5 Æ 0.0017
6
7
8
9
7i
7k
32a
32b
44.5 Æ 0.0101
51.1 Æ 0.0028
75.9 Æ 0.0046
83.4 Æ 0.0062
98.1 Æ 0.0144 10
Cisplatin 25.3 Æ 0.0041
lower, the methodology developed here offers an opportunity to
fine-tune the backbone composition and position of the Pt
centers, which should allow us to identify better leads. The
cytotoxicities of these compounds and related derivatives
against different types of cancer cell lines will be investigated
in detail and a complete structure–activity relationship study
will be communicated in due course.
General procedure for the preparation of peptide–platinum
complexes. To a stirred solution of the diamine (1 eq.) in HPLC
grade water (1 mL/10 mg), K2PtCl4 (1 eq.) was added, and the
mixture was allowed to stir for 24–48 h under dark conditions.
Once the starting materials were consumed, a yellow or light
brown precipitate formed was filtered under vacuum under
protection from light, and washed with water and diethyl ether
to obtain the platinum complex in 55–95% yields.
Cell culture. Cells (SiHa and NiH3t3) were maintained as
adherent monolayers in a tissue culture flask fed with complete
DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum
(FBS) containing 100 mg LÀ1 penicillin and 66.6 mg LÀ1
streptomycin. The cells were incubated in a humidified incubator
at 37 1C with 5% CO2. During the experiment the cells were washed
using 1Â PBS–EDTA, trypsinized using 0.25% trypsin, neutralized
with complete DMEM, and centrifuged at 2000 rpm for 5 min, and
the supernatant was aspirated, whereas the pellet was suspended in
complete DMEM for seeding or culturing.
Cytotoxicity assay. The cell viability was assessed using the
Alamar blue reagent, resazurin (7-hydroxy-10-oxidophenoxazin-
10-ium-3-one). Briefly, 4000 cells per well were seeded into
96 well plates and allowed to adhere overnight. The cells were
treated with different concentrations of cisplatin and analogs
for 24 h, 48 h and 72 h. At the end of each time point, 10 mL of
Alamar blue (1 mg mLÀ1) was added and mixed well. The plate
was incubated at 37 1C with 5% CO2 for 3 h. The colour
Conclusions
Knowing the importance of cisplatin in cancer chemotherapy
and the need to develop new analogs to overcome resistance, the
objective of the present study was to use diamines generated
from serine and threonine as ligands for platinum complexation
in a peptide environment. Towards this, the side-chain and
a-carboxyl groups of Ser and Thr were modified to azidomethylene
units and then connected to various peptides through the central
amino group. The resulting diazide derivatives of peptides were
subjected to reduction and platinum complexation to obtain
the target compounds in good yields. This included peptide–Pt
conjugates with the metal at the C-terminus and N-terminus
and on both ends. These compounds induced dose-dependent
changes in DNA, which was studied by ethidium bromide
displacement assay. The presence of multiple positive charges
on the peptide side chains was found to favor DNA interaction,
which was also reflected in their cytotoxic response. The
methodology presented here allows the synthesis of new analogs
with better cytotoxic and selectivity profiles for development.
Experimental
General methods and materials
All amino acids and reagents were purchased from SRL Pvt. Ltd developed was read as optical density (OD) using a Bio-Rad
or Spectrochem Pvt. Ltd and used as received. Potassium 680 model colorimeter at 570 nm with 595 nm as a background
tetrachloroplatinate, calf thymus DNA and ethidium bromide reference.
were purchased from Sigma Aldrich. Solvents were dried following
Diazide 3a. Colorless liquid; yield = 80%; Rf (EtOAc/hexane,
standard procedures. 1H NMR and 13C NMR spectra were recorded 1 : 3), 0.3; H NMR (CDCl3, 500 MHz): d 4.83 (bs, 1H), 3.87 (bs,
on Bruker Avance 400 MHz and 500 MHz instruments. The 1H), 3.53–3.50 (dd, 2H, J = 12.38, 4.57 Hz), 3.43–3.39 (dd, 2H,
chemical shifts are reported in parts per million (ppm) relative J = 12.38, 5.91 Hz), 1.43 (s, 9H) ppm; 13C NMR (CDCl3, 125
to tetramethylsilane, with J values in Hertz. 13C NMR spectral data MHz): d 155.0, 80.4, 51.8 (2C), 49.4, 28.4 (3C) ppm; IR (KBr):
are reported with the solvent peak (CDCl3 at 77.16 ppm and 2923, 2896, 2102, 1703, 1683, 1354, 1195 cmÀ1; HRMS (ESI)
DMSO-d6 at 39.52 ppm) as the internal standard. 195Pt NMR was exact mass calcd for C8H15N7O2Na [M + Na]+ 264.1185, found
recorded on a 500 MHz instrument and chemical shifts were 264.1199.
1
reported relative to K2PtCl4 (d = À1617 ppm) as an external
Pt-Conjugate 4a. Orange solid; yield = 95%; 1H NMR (DMSO-d6,
standard. High-resolution mass spectra (HRMS) were recorded 400 MHz): d 7.19–6.97 (m, 1H), 5.85–5.68 (bs, 2H), 5.60–5.43 (bs, 1H),
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5.40–4.94 (m, 1H), 3.83–3.60 (bs, 1H), 2.76–2.66 (m, 1H), 2.65–2.53
Diazide 6d (precursor of 7d). White solid; yield = 63%; Rf
1
(m, 2H), 2.48–2.34 (m, 1H), 1.38 (s, 9H) ppm; 195Pt NMR (DMSO-d6, (EtOAc/hexane, 1 : 3), 0.2; H NMR (400 MHz, CDCl3): d 6.94–
107 MHz): d = À2970 ppm; HRMS (ESI) exact mass calcd for 6.75 (bs, 1H), 5.25–5.11 (bs, 1H), 4.78–4.65 (m, 1H), 4.27–4.11
C8H19Cl2N3O2PtK [M + K]+ 493.0139, found 493.0149, isotopic (m, 2H), 3.58–3.39 (m, 4H), 3.35–3.31 (m, 1H), 3.13–3.01 (m,
peaks m/z 492, 493, 494, 495, 496.
1H), 1.87–1.84 (bs, 1H), 1.63–1.50 (m, 3H), 1.43 (s, 18H) ppm;
Diazide 3b. Colourless liquid; yield = 80%; Rf (EtOAc/hexane, 13C NMR (100 MHz, CDCl3): d 172.5, 156.7, 156.0, 80.4, 79.6,
1 : 3), 0.3; 1H NMR (CDCl3, 500 MHz): d 4.80 (d, 1H, J = 7.4 Hz), 53.6, 51.5, 51.4, 48.3, 39.6, 29.8, 28.5 (3C), 28.4 (3C), 26.5 ppm;
3.71–3.56 (m, 3H), 3.46–3.43 (dd, 1H, J = 12.5, 3.7 Hz), 1.45 (s, IR (neat): 2926, 2102, 1654, 1169 cmÀ1; HRMS (ESI) exact
9H), 1.34 (d, 3H, J = 6.5 Hz) ppm; 13C NMR (CDCl3, 125 MHz): d mass calculated for C18H33N9O5Na [M + Na]+ 478.2502, found
155.3, 80.4, 58.3, 53.8, 51.5, 28.4 (3C), 16.2 ppm; IR (KBr): 2936, 478.2522.
2889, 2102, 1700, 1526, 1519 cmÀ1; HRMS (ESI) exact mass
calcd for C9H17N7O2Na [M + Na]+ 256.1522, found 256.1493.
Pt-Conjugate 7d. Brown solid, yield = 70%, ESI-MS m/z, 711
[M À Cl + DMSO]+; isotopic pattern m/z: 710, 711, 712, 713, 714;
Pt-Conjugate 4b. Brown solid; yield = 90%; HRMS (ESI) exact 195Pt NMR (107 MHz) d À2953.9 ppm.
mass calcd for C9H21Cl2N3O2PtNa [M + Na]+ 491.0556, found
Pt-Conjugate 7f. Brown-coloured hygroscopic solid; yield =
491.0570; isotopic peaks: 489, 490, 491, 492, 493; 195Pt NMR quantitative; ESI-MS: m/z 470 corresponding to C8H22Cl2N5OPt
(107 MHz, DMSO-d6): d = À2992 ppm.
[M + H]+, isotopic mass 468, 469, 470, 471, 472; m/z 434
Diazide 6a (precursor of 7a). White solid; yield = 94%; (C8H21ClN5OPt [M À Cl]+), isotopic mass 432, 433, 434,
1H NMR (CDCl3, 400 MHz): d 6.96 (bs, 1H), 5.30 (bs, 1H), 4.18 435, 436.
(bs, 2H), 3.57–3.40 (m, 4H), 1.46 (s, 9H), 1.36 (d, 3H, J = 4.6 Hz)
Diazide 6e (precursor of 7e). Colorless liquid; yield = 69%;
ppm; 13C NMR (CDCl3, 100 MHz): d 173, 156, 80.6, 51.5, 51.4, Rf (EtOAc/hexane, 1 : 3), 0.2; 1H NMR (500 MHz, CDCl3):
50.2, 48.1, 28.4 (3C), 17.7 ppm; IR (KBr): 2929, 2879, 2099, 1715, d 6.85–6.56 (bs, 1H), 5.40–5.23 (bs, 1H), 4.66 (bs, 1H),
1679, 1348 cmÀ1; HRMS (ESI) exact mass calcd for C11H21N8O3 4.18–4.16 (m, 1H), 4.02 (bs, 1H), 3.58–3.40 (m, 4H), 3.17–3.03
[M + H]+ 313.1731, found 313.1719.
(bd, 2H), 1.89–1.81 (bs, 2H), 1.68–1.55 (bs, 2H), 1.46–1.40 (m,
Pt-Conjugate 7a. Yellow solid; yield = 94%; 195Pt NMR 20H) ppm; 13C NMR (125 MHz, CDCl3): d 172.5, 156.5, 156.1,
(107 MHz, DMSO-d6): d = À2992 ppm; HRMS (ESI) exact mass 80.5, 79.4, 54.7, 51.5, 51.4, 48.1, 39.7, 31.4, 29.8, 28.6 (3C), 28.4
calcd for C11H24Cl2N4O3PtK [M + K]+ 564.0510, found 564.0524, (3C), 22.6 ppm; IR (neat): 3316, 2925, 2856, 2102, 1690,
m/z 563, 564, 565, 566, 567.
1523, 1169 cmÀ1; HRMS (ESI) exact mass calculated for
Diazide 6b (precursor of 7b). White solid; yield = 85%; Rf
C
19H35N9O5Na [M + Na]+ 492.2659, found 492.2672.
1
(EtOAc/hexane, 1 : 3), 0.2; H NMR (CDCl3, 400 MHz): d 7.15–
Pt-Conjugate 7e. Brown solid; yield = 76%, 195Pt NMR
7.00 (bs, 1H), 6.77–6.11 (bs, 1H), 5.14–4.97 (bs, 1H), 4.50–4.38 (107 MHz) d À2953.9 ppm; HRMS (ESI): exact mass calculated
(m, 1H), 4.18–4.01 (m, 2H), 3.57–3.34 (m, 4H), 1.75–1.48 (m, for C19H39Cl2N5O5PtNa [M + Na]+ 705.1874, found 705.1854;
6H), 1.44 (s, 9H), 0.96–0.85 (m, 12H); 13C NMR (CDCl3, 100 ESI-MS: m/z 679 [M À Cl + CH3OH]+, isotopic peaks 677, 678,
MHz): d 172.8, 171.9, 155.7, 80.6, 51.9, 51.2, 48.4, 40.6, 40.4, 679, 680, 681.
28.3 (3C), 24.8 (2C), 24.7 (2C), 23.0, 22.9, 21.8 (2C) ppm;
Pt-Conjugate 7g. Brown color hygroscopic solid; yield =
IR (KBr): 3096, 2927, 2102, 1711, 1671, 1630, 1542, 1345, quantitative; HRMS (ESI) exact mass calculated for C9H24N5OCl2Pt
1297, 1254, 1202, 1163, 1078, 918, 728, 664 cmÀ1; HRMS (ESI) [M + H]+ 483.1006, found 483.0995, isotopic peaks m/z 482, 483, 484,
exact mass calcd for C20H37N9O4Na [M + Na]+ 490.2866, found 485, 486, 487, 488.
490.2872.
Diazide 6h (precursor of 7h). White solid; yield = 61%; Rf
Pt-Conjugate 7b. Yellow solid; yield = 84%; 195Pt NMR (107 (EtOAc/hexane, 1 : 1), 0.3; 1H NMR (400 MHz, CDCl3): d 7.06 (bs,
MHz, DMSO-d6): d = À2962 ppm; HRMS (ESI) exact mass calcd 1H), 6.67 (bs, 1H), 5.56 (bs, 1H), 4.88–4.63 (m, 2H), 4.38–4.30
for C20H41Cl2N5O4PtNa [M + Na]+ 703.2081, found 703.2065, (m, 1H), 4.19–4.10 (m, 1H), 4.05–3.95 (bs, 1H), 3.57–3.39 (m,
m/z 702, 703, 704, 705, 706.
4H), 3.15–3.04 (m, 4H), 1.83–1.79 (bs, 5H), 1.69–1.66 (m, 2H),
Diazide 6c (precursor of 7c). White solid; yield = 82%; Rf 1.50–1.39 (m, 32H) ppm; 13C NMR (100 MHz, CDCl3): d 172.7,
(EtOAc/hexane, 1 : 3), 0.2; 1H NMR (CDCl3, 500 MHz): d 6.61 (bs, 171.8, 156.8, 156.3, 80.8, 79.5, 55.5, 53.5, 51.5, 51.4, 48.8, 40.13,
1H), 4.94 (bs, 1H), 4.06–3.99 (m, 2H), 3.66–3.61 (m, 2H), 39.3, 31.2, 30.0, 29.8, 29.6, 28.6 (3C), 28.5 (3C), 28.4 (3C), 22.9,
3.46–3.43 (m, 1H), 1.91–1.86 (m, 1H), 1.67–1.66 (m, 2H), 1.44 22.3 ppm; IR (neat): 3304, 2976, 2931, 2102, 1693, 1170 cmÀ1
;
(s, 9H), 1.31 (d, 3H, J = 6.4 Hz), 0.94 (t, 6H, J = 6.2 Hz) ppm; 13
C
HRMS (ESI) exact mass calculated for C30H55N11O8Na [M + Na]+
NMR (CDCl3, 125 MHz): d 172.7, 156.0, 80.5, 57.8, 53.4, 52.3, 720.4133, found 720.4122.
51.4, 40.5, 28.4 (3C), 24.9, 22.9, 22.2, 16.1 ppm; IR (KBr): 2961,
Pt-Conjugate 7h. Dark brown solid; yield = 55%; 195Pt NMR
2929, 2102, 1682, 1366, 1286, 1055, cmÀ1; HRMS (ESI) exact (107 MHz): d À2953.435 ppm; HRMS (ESI): exact mass calculated
mass calcd for C15H28N8O3Na [M + Na]+ 391.4346, found for C30H59N7O8Cl2PtNa [M + Na]+ 933.3348, found 933.3351,
391.4326.
Pt-Conjugate 7c. Orange-yellow solid; yield = 95%; 195Pt
isotopic peaks 932, 933, 934, 935, 936, 937.
Pt-Conjugate 7i. Brown color hygroscopic solid; yield =
NMR (107 MHz, DMSO-d6): d = À2967 ppm; HRMS (ESI) exact quantitative; HRMS (ESI) exact mass calculated for C15H36N7O2Cl2Pt
mass calcd for C15H32Cl2N4O3PtK [M + K]+ 620.1136, found [M + H]+ 611.1955, found 611.1977, isotopic peaks 610, 611, 612,
620.1150, m/z 619, 620, 621, 622, 623.
613, 614, 615, 616.
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Diazide 6j (precursor of 7j). Gummy liquid; yield = 55%; Rf 1.91–1.77 (bs, 1H), 1.45 (s, 9H), 1.31–1.16 (m, 2H), 0.97–0.86 (m,
1
(EtOAc/hexane, 7 : 3), 0.4; H NMR (400 MHz, CDCl3): d 7.17– 6H) ppm; 13C NMR (CDCl3, 125 MHz): d 172.5, 171.5, 168.6,
7.06 (bs, 1H), 6.86–6.65 (bs, 1H), 5.67–5.31 (bs, 1H), 5.04–4.65 153.8, 80.1, 60.8, 52.0 (2C), 50.5, 49.1, 37.7, 29.1 (3C), 25.6, 16.4,
(m, 3H), 4.48–3.84 (m, 4H), 3.59–3.34 (m, 4H), 3.23–2.96 (m, 12.3, 11.9 ppm; IR (KBr): 2984, 2986, 2105, 1749, 1717, 1672,
6H), 1.94–1.36 (m, 54H) ppm; IR (neat): 3219, 2988, 2936, 2102, 1445, 1422, 1048, 895, 667, 580 cmÀ1; HRMS (ESI) exact mass
1677, 1207 cmÀ1; HRMS (ESI) exact mass calculated for calcd for C18H33N10O5 [M + H]+ 469.2635, found 469.2646.
C
41H75N13O11Na [M + Na]+ 948.5607, found 948.5637.
Pt-Conjugate 21. Orange colour solid; yield = 62%; ESI-MS,
Pt-Conjugate 7j. Brown color solid; ESI-MS: m/z 1177 m/z 646 [M À Cl]+ with mol. formula [C18H36ClN6O5Pt]+, isotopic
(C41H79Cl2N9O11PtK [M + K]+), isotopic peaks 1176, 1177, peaks 645, 646, 647, 648, 649.
1178, 1179, 1180, 1181.
Diazide 25 (precursor of 26). White solid; yield = 62%; Rf
Pt-Conjugate 7k. Brown color hygroscopic solid; yield = (EtOAc/hexane, 9 : 1), 0.2; 1H NMR (DMSO-d6, 500 MHz): d
quantitative; HRMS (ESI) exact mass calculated for C21H48N9O3Cl2Pt 8.26–8.06 (m, 3H), 7.79–7.78 (m, 1H), 7.26–7.22 (m, 5H),
[M + H]+, 739.2905, found 739.2933, isotopic peaks 738, 739, 740, 7.17–7.16 (m, 1H), 7.05–7.04 (m, 1H), 4.57–4.50 (m, 1H),
741, 742, 743, 744.
4.42–4.41 (m, 1H), 4.27–4.25 (m, 3H), 3.42–3.38 (m, 5H), 3.05–
Diazide 12. White solid; yield = 70%; Rf (EtOAc/hexane, 1 : 3), 3.04 (m, 1H), 2.83–2.82 (m, 1H), 1.94–1.90 (m, 1H), 1.37 (s, 9H),
0.5; 1H NMR (CDCl3, 500 MHz): d 6.89 (bs, 1H), 5.70 (d, 1H, 0.85–0.52 (m, 6H) ppm; 13C NMR (125 MHz, DMSO-d6): d 173.0,
J = 6.8 Hz), 4.45 (bs, 1H), 4.13–4.12 (m, 1H), 3.68 (s, 3H), 172.2, 165.3, 155.2, 129.5 (2C), 128.5 (2C), 126.5, 125.1, 80.5,
3.53–3.49 (m, 2H), 3.43–3.40 (m, 2H), 2.97–2.91 (m, 1H), 2.67 54.6 (2C), 40.8, 39.2, 38.6 (3C), 38.2, 37.7 (2C), 28.6 (3C), 19.6,
(dd, 1H, J = 17.0, 5.9 Hz), 1.43 (s, 9H) ppm; 13C NMR (CDCl3, 17.7 ppm; IR (KBr): 3116, 2911, 2102, 1675, 1384, 1276, 915;
125 MHz): d 172.4, 171.0, 155.8, 80.9, 52.2 (2C), 51.3, 50.7, 48.2, 195Pt NMR (107 MHz, DMSO-d6): d À2960 ppm; HRMS (ESI)
35.6, 28.3 (3C) ppm; IR (KBr, neat): 2970, 2933, 2102, 1641, exact mass calcd for C26H40N11O6 [M + H]+ 602.3163, found
1528, 1447, 1367, 1269, 968, 743, 699, 672 cmÀ1; HRMS (ESI) 602.3184.
exact mass calcd for C13H22N8O5Na [M + Na]+ 393. 1611, found
393.1624.
Pt-Conjugate 26. Yellow solid; yield = 80%; 195Pt NMR
(107 MHz, DMSO-d6): d À2960 ppm; HRMS (ESI) exact mass
Compound 15. White solid; yield = 70%; Rf (EtOAc/hexane, calcd for C26H43Cl2N7O6PtK [M + K]+ 853.1937, found 853.1947,
1 : 3), 0.2; [a]2D5 À14.05 (c 1, CHCl3); 1H NMR (DMSO-d6, m/z 852, 853, 854, 855, 856, 857.
500 MHz): d 9.35 (t, J = 5.5 Hz, 1H), 9.30–9.26 (m, 1H), 9.01
Tetraazide 31 (precursor of 32a). White solid; Rf (EtOAc/
1
(bs, 2H), 8.98–8.97 (m, 2H), 8.96–8.94 (m, 2H), 7.42 (d, hexane, 7 : 3), 0.3; H NMR (CDCl3, 400 MHz): d 7.58–7.37 (bs,
J = 7.9 Hz, 1H), 4.57–4.50 (m, 1H), 4.24–4.20 (q, J = 7.6 Hz, 1H), 7.36–7.28 (bs, 1H), 7.24–7.00 (bs, 1H), 6.40–5.95 (bs, 1H),
1H), 3.93–3.88 (m, 1H), 3.81–3.79 (t, J = 12.25 Hz, 2H), 3.68–3.59 4.97–4.74 (bs, 1H), 4.53–4.34 (bs, 2H), 4.24–4.05 (bs, 2H), 3.56–
(m, 1H), 2.93 (dd, J = 17.45, 9.25 Hz, 1H), 2.56 (dd, J = 17.50, 3.37 (m, 8H), 3.16–3.00 (m, 2H), 3.15–3.00 (bs, 1H), 2.55–2.46
5.90 Hz, 1H), 1.20 (s, 9H) ppm; 13C NMR (125 MHz, DMSO-d6): d (bs, 1H), 1.98–1.78 (m, 1H), 1.73–1.57 (m, 1H), 1.49–1.33 (m,
176.7, 175.1, 162.5 (2C), 162.3 (2C), 154.9, 148.19 (2C), 148.15 22H) ppm; 13C NMR (CDCl3, 100 MHz): d 172.1, 171.8, 171.0,
(2C), 136.8(2), 136.7(2), 127.4, 127.3, 120.9, 120.8, 78.7, 50.9, 156.6, 156.0, 80.8, 79.3, 53.3, 51.6, 51.5 (2C), 51.4, 49.0, 48.8,
48.9, 34.9, 27.7 (3C) ppm; IR (KBr): 3096, 2927, 1711, 1671, 48.4, 39.9, 37.6, 31.4, 29.6, 28.5 (3C), 28.3 (3C), 22.6 ppm; 3216,
1630, 1542, 1345, 1297, 1254, 1202, 1163, 1078, 918, 728, 664 2925, 2102, 1675, 1544; HRMS calculated for C26H45N17O7Na [M
cmÀ1; HRMS (ESI) exact mass calcd for C26H26N8O14Na + Na]+ 730.3586, found 730.3596.
[M + Na]+ 697.1466, found 697.1467.
Diplatinum conjugate (32a). Colour: brown; yield = 71%;
Diazide 18 (precursor of 19). White solid; yield = 55%; Rf ESI-MS m/z 1100 [M À Cl]+, isotopic peaks 1096, 1097, 1098,
(EtOAc/hexane, 9 : 1), 0.2; 1H NMR (CDCl3, 400 MHz): d 7.46 and 1099, 1100, 1101, 1102, 1103, 1104, 1105.
7.00 (bs, 1H), 6.91 and 6.34 (bs, 1H), 6.28–5.93 (m, 2H), 4.63–
4.36 (bs, 1H), 4.23–4.02 (bs, 1H), 3.60–3.35 (m, 4H), 2.93–2.78 brown; hygroscopic solid; HRMS (ESI-MS) calculated for
(m, 1H), 2.65–2.55 (dd, 1H), 1.44 (s, 9H) ppm; 13C NMR (CDCl3, 16H38N9O3Cl4Pt2 [M + H]+ 934.1147, found 934.1149, isotopic
Diplatinum conjugate (32b). Yield = quantitative; colour =
C
100 MHz): d 174.2, 171.1, 155.8, 80.6, 51.3 (2C), 48.2, 37.6, 36.9, peaks m/z 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942.
28.3 (3C) ppm; IR (neat): 3304, 2976, 2931, 2102, 1693,
1170 cmÀ1; HRMS (ESI) exact mass calcd for C12H22N5O4 [M +
H]+ 356.1795, found 356.1811.
Conflicts of interest
Pt-Conjugate 19. Orange coloured solid; yield = 49%; HRMS
(ESI) calculated for C12H25N5O4Cl2PtK [M + K]+, 607.0569,
found 607.0582, isotopic peaks 605, 606, 607, 608, 609; m/z
533, [M À Cl]+, isotopic peaks 532, 533, 534, 535, 536.
There are no conflicts to declare.
Acknowledgements
Diazide 20 (precursor of 21). White solid; yield = 56%; Rf
1
(EtOAc/hexane, 3 : 1), 0.2; H NMR (CDCl3, 500 MHz): d 8.10– Financial support of this work from the Indian Institute of
7.93 (bs, 2H), 7.33–7.06 (bs, 1H), 6.41–6.16 (bs, 1H), 6.16–5.87 Technology Madras, India (CHY/14-15/831/RFER/KMMU), and
(m, 1H), 4.83–4.67 (m, 1H), 4.16–4.06 (bs, 1H), 4.01–3.83 (m, SERB, DST (EMR/2016/003973), is gratefully acknowledged. We,
1H), 3.56–3.35 (m, 4H), 2.89–2.74 (m, 1H), 2.68–2.57 (m, 1H), in addition, thank the Department of Chemistry and DST (FIST)
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