A safe and efficacious Pt(ii) anticancer prodrug: Design, synthesis,: In vitro efficacy, the role of carrier ligands and in vivo tumour growth inhibition

Article abstract of DOI:10.1039/c8cc06586a

Diaminocyclohexane-Pt(ii)-phenalenyl complexes (1 and 2) showed an appropriate balance between efficacy and toxicity. Compound 2 showed nearly two-fold higher tumour growth inhibition than oxaliplatin in a murine NSCLC tumour model, when a combined drug development approach was used. The fluorescent properties of phenalenone were utilized to understand the mechanistic details of the drug.

Full text of DOI:10.1039/c8cc06586a

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A safe and efficacious Pt(II) anticancer prodrug:
design, synthesis, in vitro efficacy, the role of
carrier ligands and in vivo tumour growth
inhibition†
Cite this:DOI: 10.1039/c8cc06586a
Pradip Kumar Dutta,‡a Rupali Sharma,‡a Smita Kumari,‡a Ravindra Dhar Dubey,a
Received 13th August 2018,
Accepted 20th December 2018
b
Sujit Sarkar,a Justin Paulraj,a Gonela Vijaykumar,
Manoj Pandey,a L. Sravanti,§c
Mallik Samarla,a Hari Sankar Das,b Yashpal,a Heeralal B.,a Ravinder Goyal,a
DOI: 10.1039/c8cc06586a
ac
b
Nimish Gupta,
Arindam Sarkar
Swadhin K. Mandal,
Aniruddha Sengupta*ac and
ac
rsc.li/chemcomm
*
Diaminocyclohexane-Pt(II)-phenalenyl complexes (1 and 2) showed oxalate group, in oxaliplatin, leading to 75–85% non-specific
an appropriate balance between efficacy and toxicity. Compound 2 binding to plasma and cytosolic proteins; deactivating the drug
showed nearly two-fold higher tumour growth inhibition than before it reaches its target.1 A subtle and appropriate balance
oxaliplatin in a murine NSCLC tumour model, when a combined between aquation (efficacy) and deactivation (toxicity) should
drug development approach was used. The fluorescent properties solve this problem. Although numerous complexes have been
of phenalenone were utilized to understand the mechanistic details prepared and tested thus far,8,9 only a few additional com-
of the drug.
pounds are approved for regional use in Asia.7 Thus, a properly
tuned Pt coordination displaying increased antitumour activity
Platinum-based drugs play an important role in cancer chemo- and reduced cross-resistance and toxicity is still being sought.
therapy.1 Despite their prevalence in cancer treatment regimens, Phenalenone is a natural product10 and can be easily sub-
inherent adverse effects and limitations of platinum compounds stituted by heteroatoms,11 which can be coordinated to platinum.
continue to plague the field.2 Numerous novel platinum and Phenalenone based metallodrugs with an oxaliplatin backbone
other metal complexes have been designed and synthesized to have not been studied till date although the application of
develop new antitumour drugs in the past decades, with cisplatin, phenalenyl (PLY) in materials science is now well-documented.12
carboplatin and oxaliplatin representing the 1st, 2nd and 3rd Significance of heteroatom substituted platinum phenalenone
generation of platinum based anticancer drugs respectively.3 compounds could be as follows: (1) easily tunable heteroatoms,
Despite its better tolerability compared to cisplatin, oxaliplatin containing a lone pair of electrons, could facilitate and regulate
is also associated with side effects such as neurotoxicity, hemato- the aquation procedure in the acidic environment of tumour
logic toxicities, neutropenia, nausea, and vomiting that limit its tissues where the heteroatoms can be protonated and the carrier
doses.4,5 Oxaliplatin and cisplatin have a similar rate of aquation ligand(phenalenone) be released from the prodrug; (2) a six
which is faster than that of carboplatin,6 with both drugs under- membered chelate of PLY to Pt could stabilize the coordination;
going extensive nonenzymatic biotransformation in vivo, leading (3) PLY can impart additional hydrophobicity which could be
to non-specific toxicity.7 Nucleophiles, such as endogenous pro- helpful for liposomal formulation and subsequent cellular
teins and small molecules containing thiol groups, displace the uptake; and (4) inherent emissive characteristics of phenale-
none, due to intramolecular charge transfer (ICT),12c can give
a Invictus Oncology Pvt. Ltd., 465 Patparganj Industrial Area, Delhi 110092, India.
E-mail: asarkar@indiainnovationcenter.org, asengupta@indiainnovationcenter.org
b Department of Chemical Sciences, Indian Institute of Science Education and
Research-Kolkata, Mohanpur – 741252, India
insight into the mechanistic details of cytotoxicity. Additionally,
1,2-diaminocyclohexane (DACH) substitution to the other two
coordination sites could potentially prevent the cross resistance
of new drug molecules (Scheme S1, ESI†).13
c India Innovation Research Center, 465 Patparganj Industrial Area, Delhi 110092,
In recent years, different strategies have been embraced to
India
† Electronic supplementary information (ESI) available. Experimental procedures, incorporate platinum compounds into nanodelivery or targeted
designs.14 Some of these designs utilize the enhanced perme-
ability and retention (EPR) effect, which results in preferential
characterization data, TEM images, TUNEL assay, DLS data, emission plots of ligands
and complexes, and crystal data table. CCDC 1849788 and 1849789. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/c8cc06586a
loading of the anticancer drugs into cancer tissues, owing to
‡ These authors contributed equally.
their leaky vasculature. Thus, the liposomal formulation of a
carefully designed prodrug could be an effective approach to
§ All authors (except L. Sravanti and Hari Sankar Das) are either employees/
collaborators with Invictus Oncology Pvt. Ltd and/or hold equity of IOPL.
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overcome some of the above-mentioned toxicity issues asso-
ciated with platinum anti-cancer therapeutics.
In the current work, three different approaches of anticancer
drug development have been combined: unique molecular
designing, novel drug delivery systems and understanding
drug-function using inherent fluorescent properties of PLY. Here
we illustrate the kinetic inertness of platinum to PLY coordina-
tion in the presence of deactivating thiols, as well as demon-
strated the release of a carrier ligand in cancer cells. Fluorescent
‘‘on–off’’ properties of the carrier ligand and its complexed forms,
respectively, have been utilized to trace the released carrier ligand
inside the cancer cells. Entrapment of small molecules into
Fig. 1 Molecular structures of (a) (OO)PtPLY (1) and (b) (NO)PtPLY (2)
(hydrogen atoms are not shown for the sake of clarity).
liposomal nanoparticles resulted in higher cellular uptake and considered, a significant reduction in the activation energy
improved in vitro efficacy in comparison to oxaliplatin. In vivo barrier has been observed for 1 and 2, indicating that the acidic
results indicate that tumour growth inhibition of the novel tumour microenvironment would facilitate the aquation process
compound in a murine lung cancer model was significantly of these two compounds.
better than oxaliplatin.
In order to understand whether the carrier ligand was
Compounds 1 and 2 were synthesized by refluxing the released in the cancer cell, we treated A549 cells with 5 mM of
corresponding phenalenone ligands 1a and 2a,11 respectively, compound 2 and visualized the cells using an epifluorescence
with aquated DACH platinum compounds (Scheme S1, ESI†) in microscope to determine the release of PLY at designated time
an ethanol–water mixture and purified by recrystallization. The points. Representative images clearly indicated the release of
molecular structures of these compounds were confirmed by carrier ligand in a time dependent manner (Fig. 2a). Different
elemental analysis, single crystal X-ray crystallography and staining patterns and intensities, obtained for uncomplexed
NMR spectroscopy. In the 1H NMR spectrum, the absence of PLY treated A549 cells, indicate that 2 is dissociated inside the
any OH or NH signal around the 11 to 13 ppm region supported cells (Fig. S9, ESI†). Using ‘‘on–off’’ switching to trace the
the fact that anionic form of the ligand was bound to Pt. ligand release was not shown in previously reported fluorescent
1H NMR signals for compound 1 established the fact that the Pt-drugs.17 It has already been established that the interaction
PLY moiety was symmetrically coordinated to platinum. 195Pt of intra and extracellular proteins or small molecules contain-
NMR signals at Bꢀ1500 and ꢀ1800 ppm for compounds 1 and 2 ing sulfhydryl groups inactivates platinum drugs. Thiols bind
indicated O,O and N,O coordination, respectively, to platinum.15 with platinum, resulting in a reduction of active platinum
In order to understand the change in emission properties due to available for DNA binding.18 The reactivity of thiols towards 1
complexation, comparative analyses of the emission spectra were and 2 was examined using glutathione and results in Fig. 2b
performed for the ligand and corresponding metal complexes and c indicate the kinetic inertness of Pt to phenalenyl coordina-
(1 and 2). We establish that 1 and 2 were not emissive in tion in the presence of glutathione (t1/2 4 72 h). Fast degradation
solution at room temperature whereas the uncomplexed forms (t1/2 o 2 h) of oxaliplatin or carboplatin in the presence of
(1a, 2a) were highly emissive [l(em) 440–475 (1a) and 480–520 nm glutathione has already been reported19 and our results indicate
(2a)] (Fig. S8, ESI†).
the release of a ligand upon reaction of 2 with 50-GMP (Fig. S10,
Crystals of compounds 1 and 2, suitable for X-ray investigation, ESI†). Combining this result with epifluorescence images, we
were obtained from methanol/toluene and methanol/diethylether establish that the compound successfully internalizes in cancer
solvent mixtures, respectively. Single crystal X-ray structures are cells, following which the carrier ligand releases active platinum
shown in Fig. 1. Both complexes adopted roughly a square planar for interaction with DNA, thus generating the necessary balance
coordination geometry around the platinum center (as suggested between stability and efficacy of compound 2. We postulate that
from the bond angle data shown in Table S2, ESI†). The Pt–O this could be a putative mechanism of action, however, other
bond lengths were around 197 pm, which is 3 pm less than the mechanisms cannot be ruled out.
Pt–O bond length (200 pm) found in structurally related clinically
approved drugs, such as oxaliplatin.16 Shorter Pt–O bonds justi- medium, a liposomal formulation was developed for in vivo
fied the stability of the prodrug. administration of the compounds. Significant enhancement of
Owing to low solubility of the compound in aqueous
Activation energy barriers for the 1st and 2nd aquation steps drug solubility was observed, enabling administration of the
of the three proposed compounds (1, 2 and 3, Scheme S1, ESI†) drug in required dosage (see the ESI,†). The formulation of 2
have been computationally evaluated for better understanding was characterized by HRTEM, (Fig. S12a, ESI†) which revealed
the aquation procedure and evaluation of in vitro efficacy.6 The the formation of predominant liposomal structures, less than
results depicted in Table S3 (ESI†) show that compounds 1 and 150 nm in diameter. Dynamic light scattering (DLS) further
2 did not significantly differ in terms of their activation energy confirmed the homogeneous size distribution of the liposomal
barrier of aquation, but compound 3 had higher energies with particles with a mean hydrodynamic diameter of 117.6 ꢁ 2 nm
respect to 1 and 2 for 1st and 2nd aquation under acidic for 1 and 121 ꢁ 9 nm for 2 (Fig. 2d and Tables S4 and S5, ESI†),
conditions. In the 1st aquation, where protonation has been which would ensure that the drug is compartmentalized away
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Fig. 3 (a) Cellular uptake of compounds 1 and 2, oxaliplatin and carbo-
platin in A549 cells, measured by AAS and represented as ng Pt/105 cells.
(b) The DNA–Pt adduct formation following 24 h incubation with Pt-equivalent
doses of oxaliplatin or compound 2 in A549 cells, measured by AAS and
represented as ng Pt/mg of DNA. Values are mean ꢁ SD (*, p r 0.05;
**, p r 0.005; ***, p r 0.0005).
Hence, for subsequent studies, a liposomal formulation of 2 was
selected. Increased intracellular uptake of 2 may result in an
elevated amount reaching the DNA. We evaluated the interaction
of compound 2 and DNA through DNA platination following
exposure of A549 cells to liposomal 2 or oxaliplatin for 24 h.
Fig. 3(b) indicates higher interaction of 2 with DNA, in compar-
ison to oxaliplatin at Pt-equivalent concentrations of compounds.
Significantly high DNA-platination of compound 2 is not observed
probably due to a slow reaction rate with DNA as suggested by the
50-GMP reaction. The interaction of compound 2 with genomic-
DNA was evaluated in vitro, and the results indicated that inter-
Fig. 2 (a) Cellular internalization of a liposomal formulation of compound 2
in A549 cells and time dependent release of PLY. Scale bar represents
10 mm; (b) reactivity of 1 with glutathione in PBS shows that 88% com-
pound remains unreacted after 72 h; (c) reactivity of 2 with glutathione
in PBS shows that 89% compound remains unreacted after 72 h; (d) DLS
histogram of formulated 2 shows uniform size distribution.
from healthy tissues and delivered preferentially to the tumours action with 2 retarded the migration of genomic-DNA on an
by EPR effect.14
agarose gel, through the formation of a high molecular weight
Low PDI (o0.2) indicated narrow particle size distribution complex (Fig. S13, ESI†).
and stability of formulations. In vitro release-kinetics data
Hence, these data show that in contrast to the clinically
exhibited a pH-dependent sustained release of 2 (Fig. S12b, approved 2nd and 3rd generation platinum drugs, designing
ESI†) with minimal release in PBS at physiological pH, ensuring compounds such as 1 and 2 may be a promising approach, as
higher availability of the drug in an acidic environment of their higher cellular uptake and enhanced DNA interaction would
cancer cells. Liposomal formulations of compounds 1 and 2 improve the therapeutic index of platinum anticancer agents.
when evaluated for cellular cytotoxicity across human cancer
Assessment of apoptosis is an important parameter for
lines, responsive to standard platinum therapy, showed signifi- evaluating the response to therapy, as platinum drugs induce
cantly improved efficacy for 2 (Table S6, ESI†) compared to DNA adduct formation, leading to apoptosis, which is asso-
clinically approved platinum drugs oxaliplatin and carboplatin. ciated with the cytotoxicity of these drugs.1 We evaluated com-
Liposomal formulations enhance efficacy of the synthesized pound mediated apoptosis for oxaliplatin and 2 at equivalent
compounds, possibly through preferential delivery and higher Pt-concentrations in A549 cells. TUNEL results indicated that the
accumulation in cells, hence we examined the cellular accumu- treatment with compound 2 leads to higher apoptosis (B60%) in
lation of liposomal formulations of 1 and 2 in comparison to A549 cells in comparison to oxaliplatin (B20%) under identical
oxaliplatin and carboplatin in A549 cells. Data reveal signifi- experimental conditions (Fig. S14, ESI†).
cantly higher uptake for the formulated compounds in compar-
The antitumour activity of compound 2 was compared to
ison to the clinically approved drugs (Fig. 3a). The increased oxaliplatin in a murine NSCLC tumour model (LLC tumour).
cellular accumulation of 1 and 2, in comparison to oxaliplatin Chemotherapy remains the standard first-line therapy for the
and carboplatin is consistent with the premise that hydrophobic majority of NSCLC patients with an advanced stage of the
rather than hydrophilic platinum compounds penetrate the disease, of which platinates are considered the most efficacious
cytoplasmic membrane more easily (log P values are shown in option.20 The goal for new platinum agents to be tested in NSCLC
Table S7, ESI†). Higher intracellular uptake and cytotoxicity of 2 is to maintain or improve the efficacy and/or reduce toxicity.
can be explained by more drug loading (3 mmol of 2 vs. 2 mmol Oxaliplatin based first line therapy for NSCLC tumour models
of 1) in the liposomes. The hydrophobic nature of the compound has been undergoing clinical trials and showing similar results
assists in liposome formation, which in turn enhances the uptake. to that of other clinically approved platinum therapeutics.21
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or splenomegaly (toxicities commonly associated with oxaliplatin),
which could potentially enable higher dosage administration,
thereby improving the clinical outcome.
This work was financially supported by the Science and
Engineering Research Board (SERB) (Project EMR/2016/000357).
The authors gratefully acknowledge the use of the services and
analytical facility of IISER Kolkata funded by IISER-IOPL
technical grant.
Conflicts of interest
There are no conflicts to declare.
Fig. 4 (a) Antitumour activity of compound 2 and oxaliplatin was evaluated
in syngeneic LLC tumour model. The mice were implanted with 1 ꢂ 106 LLC
cells and grouped as untreated (control), oxaliplatin and compound 2
treated. The tumour volume and body weight of the treated animals were
recorded. (b) Tumour growth inhibition measurement for mice treated with
compound 2 and oxaliplatin. The biodistribution of the tested compounds
was recorded in (c) tumour, (d) plasma and blood, and (e) other tissues
(*, p r 0.05; **, p r 0.005; ***, p r 0.0005).
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