Anticancer Activity and Cisplatin Binding Ability of Bis-Quinoline and Bis-Isoquinoline Derived [Pd2L4]4+ Metallosupramolecular Cages

New bis-quinoline (L_q) and bis-isoquinoline-based (L_iq) ligands have been synthesized, along with their respective homoleptic [Pd₂(L_q or L_iq)₄]⁴⁺ cages (C_q and C_iq). The ligands and cages were characterized by ¹H, ¹³C and diffusion ordered (DOSY) NMR spectroscopies, high resolution electrospray ionization mass spectrometry (HR-ESIMS) and in the case of the bis-quinoline cage, X-ray crystallography. The crystal structure of the C_q architecture showed that the [Pd₂(L_q)₄]⁴⁺ cage formed a twisted meso isomer where the [Pd(quinoline)₄]²⁺ units at either end of the cage architecture adopt the opposite twists (left and right handed). Conversely, Density Functional Theory (DFT) calculations on the C_iq cage architecture indicated that a lantern shaped conformation, similar to what has been observed before for related [Pd₂(L_tripy)₄]⁴⁺ systems (where L_tripy = 2,6-bis(pyridin-3-ylethynyl)pyridine), was generated. The different cage conformations manifest different properties for the isomeric cages. The C_iq cage is able to bind, weakly in acetonitrile, the anticancer drug cisplatin whereas the C_q architecture shows no interaction with the guest under the same conditions. The kinetic robustness of the two cages in the presence of Cl⁻ nucleophiles was also different. The C_iq cage was completely decomposed into free L_iq and [Pd(Cl)₄]²⁻ within 1 h. However, the C_q cage was more long lived and was only fully decomposed after 7 h. The new ligands (L_iq and L_q) and the Pd(II) cage architectures (C_iq and C_q) were assessed for their cytotoxic properties against two cancerous cell lines (A549 lung cancer and MDA-MB-231 breast cancer) and one non-cancerous cell line (HDFa skin cells). It was found that L_q and C_q were both reasonably cytotoxic (IC_50S ≈ 0.5 μM) against A549, while C_iq was slightly less active (IC₅₀ = 7.4 μM). L_iq was not soluble enough to allow the IC₅₀ to be determined against either of the two cancerous cell lines. However, none of the molecules showed any selectivity for the cancer cells, as they were all found to have similar cytotoxicities against HDFa skin cells (IC₅₀ values ranged from 2.6 to 3.0 μM).

Introduction

Metallosupramolecular architectures (MSAs) (Cook and Stang, 2015) have been attracting increasing attention over the past two decades due to their many potential applications including catalysis (Yoshizawa et al., 2009; Yoshizawa and Fujita, 2010; Martí-Centelles et al., 2018), storage (Mal et al., 2009), and sensing (Wang et al., 2011). Inspired by the success of cisplatin and other metallodrugs (Mjos and Orvig, 2014) there is emerging interest in exploiting MSAs for biomedical purposes (Cook et al., 2013; Therrien, 2015; Casini et al., 2017; Zhou et al., 2017). Several groups have examined MSAs as drug delivery vectors (Therrien et al., 2008; Schmitt et al., 2012; Yi et al., 2012; Zheng et al., 2015; Samanta et al., 2016, 2017; Bhat et al., 2017; Xu et al., 2017; Wang J.-F. et al., 2018; Yue et al., 2018). Additionally, MSAs have been shown to bind DNA (Oleksy et al., 2006; Garci et al., 2017; Zhao et al., 2017) and RNA (Phongtongpasuk et al., 2013; Malina et al., 2015), interact with proteins (Li et al., 2014; Mitchell et al., 2017) and have anticancer (Hotze et al., 2008; Faulkner et al., 2014; Grishagin et al., 2014; Dubey et al., 2015; Zheng et al., 2016; Allison et al., 2018) and antibacterial (Richards et al., 2009; Howson et al., 2012; Wang H. et al., 2018) properties.

Since the pioneering work of McMorran and Steel (McMorran and Steel, 1998) interest in [M₂(L)₄]ⁿ⁺ cage-type structures has burgeoned (Schmidt et al., 2014). Some time ago now we reported that a [Pd₂(L_tripy)₄]⁴⁺ cage (where L_tripy = 2,6-bis(pyridin-3-ylethynyl)pyridine) could host two molecules of the anticancer drug cisplatin within the cavity of the cage (Lewis et al., 2012), and thus had potential as a drug delivery vector. Disappointingly, the binding event, which was governed mainly by hydrogen bonding interactions, was weak (Preston et al., 2015). The host-guest complex formed in acetonitrile (CH₃CN) and dimethylformamide (DMF) but unfortunately, in more hydrogen bond competitive solvents such as water and dimethyl sulfoxide (DMSO) no host-guest interaction was observed. Additionally, the parent Pd(II) based cage decomposed rapidly in the presence of nucleophiles (Lewis et al., 2012; McNeill et al., 2015). Thus, in order to exploit these [Pd₂(L)₄]⁴⁺ cages as cisplatin delivery vehicles these issues need to be addressed. We and others have examined a range of modifications to the parent [Pd₂(L)₄]⁴⁺ cage system in order to improve the solubility (Lewis and Crowley, 2014; Preston et al., 2015; Han et al., 2017) and other properties (Lewis et al., 2013, 2014; Kaiser et al., 2016; Schmidt et al., 2016a) of the cage. Efforts have also been made to enhance the strength of the host-guest interaction (Kim et al., 2015) and the stability of the cages in the presence of biologically revelant nucleophiles (Preston et al., 2016). However, while some improvements have been made these [Pd₂(L)₄]⁴⁺ cages still require further modifcations in order to be useful drug delivery vectors.

The [Pd₂(L)₄]⁴⁺ cages have also been examined for their cytotoxic properties. We showed that the parent [Pd₂(L_tripy)₄]⁴⁺ was modestly cytotoxic (IC₅₀ values range from 40 to 70 μM) against a range of cancer cell lines but was less active than related bis-1,2,3-triazole [Pd₂(L)₄]⁴⁺ helicates (McNeill et al., 2015). We also examined the cytotoxicity of related amino substituted [Pd₂(L)₄]⁴⁺ cages against the same panel of cancer cells and found that they exhibited similar cytotoxic properties as the parent systems (Preston et al., 2016). Casini, Kühn and co-workers (Kaiser et al., 2016; Schmidt et al., 2016b) have measured the cytotoxicity of a series of related [Pd₂(L)₄]⁴⁺ cages and observed similar IC₅₀ values (10–70 μM). Additionally, they have measured the cytotoxicity of mixtures of the cages and cisplatin and unsurprisingly have found that those mixtures are more cytotoxic than cage alone (IC₅₀ = 2–13 μM). Yoshizawa, Ahmedova and co-workers have also found that [M₂(L)₄]⁴⁺ (M = Pd²⁺ or Pt²⁺) cages with similar, but more hydrophobic, dipyridyl anthracenyl ligands (L_anthracene) display high anticancer activity (IC₅₀ values range from 0.9 to 37.4 μM against HL-60, HL-60/Dox, HT-29, T-24, SKW-3 cancer cell lines) (Ahmedova et al., 2016; Anife et al., 2016).

The majority of the [Pd₂(L)₄]⁴⁺ cages examined to date feature pyridyl donors, as part of our efforts to improve the biological properties of these systems herein we describe the use of isoquinoline and quinoline-derived ligands for the assembly of two new [Pd₂(L)₄]⁴⁺ cages. While is well-known that isoquinoline and quinoline ligands can bind with palladium(II) and platinum(II) (Bondy et al., 2004) their use as donor systems in ligands for the generation MSAs has not been extensively explored (Bloch et al., 2016, 2017). These quinoline derived systems feature different electronic and steric properties compared to the parent pyridyl systems thus we also examine the effect these changes have on the host-guest chemistry with cisplatin, the stability of the cages in the presence of nucleophiles and the antiproliferative properties of the cages.

Results and Discussion

The synthesis of the new quinoline (L_q) and isoquinoline (L_iq) based ligands was facile (Supplementary Material). Using sequential Sonogashira carbon-carbon cross coupling reactions from commercially available building blocks the ligands were generated in good yields (L_q = 86% and L_iq = 78%). ¹H and ¹³C NMR spectroscopic data were consistent with the formation of the ligands which was supported by high-resolution electrospray mass spectrometry (HR-ESIMS) (Figure 1 and Supplementary Material). Peaks corresponding to protonated and sodiated ligand were observed at m/z = 382.1320 and 404.1132, respectively, for L_iq and similar peaks were observed for L_q (Supplementary Material).

FIGURE 1

Figure 1. (A) General scheme for formation of [Pd₂(L)₄]⁴⁺ cages (C_q and C_iq) and partial ¹H NMR spectra (400 MHz, CD₃CN, 298 K) of (B) L_q and C_q, and (C) L_iq and C_iq.

With the ligands in hand, the complexation with [Pd(CH₃CN)₄](BF₄)₂ in acetonitrile was examined (Figure 1). The cage formation was monitored using ¹H NMR spectroscopy (CD₃CN, 298 K) and showed that mixing [Pd(CH₃CN)₄](BF₄)₂ and the L_iq ligand at room temperature (RT) in a 1:2 ratio led to the rapid (< 2 min) and quantitative formation of the expected C_iq cage (Figure 1), similar to what was observed with the parent L_tripy system (Lewis et al., 2012). Interestingly, the behavior of L_qwith [Pd(CH₃CN)₄](BF₄)₂ at room temperature was very different. After 5 min at RT the reaction mixture displayed multiple proton resonances, none of which were due to free ligand, consistent with the formation of a mixture of different cage isomers. The reaction was monitored using ¹H NMR spectroscopy for 24 h at RT however little to no changes were observed after the first hour and the spectrum still displayed multiple proton resonances. A ¹H DOSY experiment (CD₃CN, 298 K) on the mixture showed that all the different proton resonances had the same diffusion co-efficient consistent with the postulate that the reaction mixture contains a series of cage isomers (Supplementary Material).

The assembly reaction between L_qwith [Pd(CH₃CN)₄](BF₄)₂ was then carried out at 65°C, in CD₃CN and again monitored using ¹H NMR spectroscopy (Figure 2). After 5 min the same complicated series of proton resonance were observed. However, with continued heating this slowly resolved into a single series of resonances (after 7 h), consistent with the formation of a single cage isomer (Figure 2). Pleasingly, both cages (C_iq and C_q) could be isolated by adding diethyl ether into the acetonitrile reaction mixtures providing the cages as colorless/tan precipitates in 88% (C_iq) or 92% (C_q) yield, respectively. ¹H NMR spectroscopy (CD₃CN, 298 K) exhibited the expected downfield shifts of the signals pertaining to protons H_a, H_b and H_f as well as the anticipated downfield shifts of the rest of the isoquinoline and quinoline protons resonances (Figure 1). HR-ESIMS data also supported the formation of the cages, showing ions corresponding to the loss of 2, 3 and 4 tetrafluoroborate ($B{F}_4^{-}$) counterions (m/z = 956.1610 (2⁺), 608.4424 (3⁺), and 434.5832 (4⁺), Supplementary Material). ¹H DOSY experiments (CD₃CN, 298 K) on the ligands (Diffusion coefficients (D) = 13.1 (L_q) and 15.0 (L_iq) x 10⁻¹⁰ m² s⁻¹) and cages (D = 4.1 (C_q) and 4.3 (C_iq) × 10⁻¹⁰ m² s⁻¹ were also consistent with the formation of the larger [Pd₂(L)₄]⁴⁺ cages (Supplementary Material).

FIGURE 2

Figure 2. Partial ¹H NMR (500 MHz, CD₃CN, 338 K) spectra showing the formation of C_q over time at 65°C. (A) L_q, (B) initial complexation (t = 0), (C) t = 30 min, (D) t = 1 h, (E) t = 2 h, (F) t = 3 h, (G) t = 4 h, (H) t = 5 h, (I) t = 6 h, (J) t = 7 h, (K) isolated C_q.

Crystals of C_q suitable for X-ray diffraction formed during the cooling of an acetonitrile solution of the cage from 65°C to room temperature. The structure was solved in the tetragonal space group P4/mnc with the asymmetric unit containing one eighth of the cage and one quarter of a $B{F}_4^{-}$ counterion (Figure 3 and Supplementary Material). The other $B{F}_4^{-}$ anions and some acetonitrile molecules could not be modeled sensibly thus the SQUEEZE routine was employed to account for the diffuse electron density (Supplementary Material). The data revealed the expected [Pd₂(L_q)₄]⁴⁺ cage structure. The Pd-N bond lengths (Pd1-N2 2.045 Å) were similar to what have been previously observed for the related [Pd₂(L_tripy)₄]⁴⁺ cages where the Pd-N bond lengths range from 2.016 to 2.027 Å (Lewis et al., 2012; Lewis and Crowley, 2014). The L_q ligands of the cage are twisted giving a V-shaped conformation where the terminal quinoline and central pyridyl heterocyclic units are not co-planar which is quite different to what was observed with the [Pd₂(L_tripy)₄]⁴⁺ cages. In X-ray structures of the parent [Pd₂(L_tripy)₄]⁴⁺ cages the L_tripy ligands were found in a linear conformation with the heterocyclic units coplanar. The twisting also alters the Pd1-Pd1′ distance within C_q related to the [Pd₂(L_tripy)₄]⁴⁺ cages. The Pd1-Pd1′ distances for the parent [Pd₂(L_tripy)₄]⁴⁺ cages range from 11.49 to 12.24 Å, whereas the Pd1-Pd1′ distance was found to be longer (12.506 Å) suggesting that the C_qcage has a larger cavity despite featuring the same 2,6-diethynylpyridine linker units. The [Pd(quinoline)₄]²⁺ units at the top and bottom of C_q are twisted in opposite directions, the top cationic unit has a right handed twist while the bottom cationic unit has a left handed twist giving an overall meso structure (Figures 3B,C and Supplementary Material). Despite extensive efforts we were unable to obtain X-ray diffraction quality single crystals of C_iq. Thus, to gain further insight into the structure of C_iq we modeled the cage using Density Functional Theory (DFT) calculations (Figures 3D,E). Energy minimization of C_iq (DFT, BP86 def2-SVP, acetonitrile solvation, Supplementary Material) showed that the cage adopted a lantern shape similar to what was previously observed for [Pd₂(L_tripy)₄]⁴⁺ cages (Lewis et al., 2012; Lewis and Crowley, 2014). The calculated Pd – N bond distances (2.049 Å) and the Pd-Pd′ distance (11.758 Å) match well with those observed crystallographically for the related [Pd₂(L_tripy)₄]⁴⁺ cages. The L_iq ligand adopts a linear conformation with all the heterocyclic units coplanar. The DFT calculations indicated that the C_iq is structurally very similar to the parent [Pd₂(L_tripy)₄]⁴⁺ cages whereas the C_q is more twisted and provided a cavity of different size and shape to the parent cages and the C_iq system.

FIGURE 3

Figure 3. Molecular structures of C_q and C_iq. X-ray structure of C_q: (A) ellipsoid side view, (B) tube side view, and (C) tube top view showing paddle-like array of quinoline panels over palladium(II) center. Solvent molecules and counterions have been omitted for clarity. Ellipsoids are shown at 50% probability. DFT optimized (BP86 def2-SVP) model of C_iq; (D) side view and (E) top view showing lantern-shaped structure. Colors: carbon gray, nitrogen blue, palladium magenta, hydrogen white.

We and others have previously shown that other similar [Pd₂(L_tripy)₄]⁴⁺ cages can encapsulate cisplatin through hydrogen bonding interactions in CH₃CN and DMF solvents (Lewis et al., 2012, 2013, 2014; Kaiser et al., 2016; Preston et al., 2016, 2017; Schmidt et al., 2016b). Therefore, we examined the ability of C_iq and C_q to interact with cisplatin in CH₃CN using ¹H NMR spectroscopy. Addition of an excess of cisplatin to a CD₃CN solution of the C_iq cage resulted in a downfield shift and broadening (Δδ = 0.03 ppm) of the internally directed cage proton H_a (Figures 4A,B) indicative of cisplatin binding within the cage cavity, albeit weakly. A similar ¹H NMR experiment was carried out with the C_q cage (Figures 4C,D). However, with the C_q cage no shifts were observed for any of the cage proton resonances in the presence of an excess of cisplatin suggesting that the more twisted C_q cage does not interact with the anticancer agent. The behavior was similar to what has been observed with a related twisted [Pd₂(L_2Atripy)₄]⁴⁺ cage (where L_2Atripy = 2,6-bis[2-(6-amino-3-pyridinyl)ethynyl]-4-pyridinemethanol) (Preston et al., 2016). The [Pd₂(L_2Atripy)₄]⁴⁺ cage did not bind cisplatin in DMF solvent and the lack of binding was ascribed to the twisted cage cavity which was not as preorganised as those of the related lantern shaped [Pd₂(L_tripy)₄]⁴⁺ cages. Presumably the different sized cavity and different spatial arrangement of the hydrogen bond donors and acceptors caused by the twisting observed in the crystal structure of C_q impedes the cisplatin-C_q interaction in this case.

FIGURE 4

Figure 4. Partial ¹H NMR (500 MHz, CD₃CN, 298 K) stacked plot of (A) C_iq (B) C_iq + cisplatin (10 eq.) (C) C_q, and (D) C_q + cisplatin (10 eq.).

The kinetic robustness of the related [Pd₂(L_tripy)₄]⁴⁺ architectures in the presence of common biological nucleophiles (chloride (Cl⁻), histidine and cysteine) has been determined using ¹H NMR competition experiments. When the parent [Pd₂(L_tripy)₄]⁴⁺ architectures were treated with 8 equivalents of tetrabutylammonium chloride the pyridyl substituted cages were rapidly and quantitatively decomposed (in < 5 min). To examine the effect of substituting the pyridyl donor units for quinoline heterocycles time-course ¹H NMR competition experiments were carried out in d₆-DMSO where 2 mM solutions of each cage (C_qor C_iq) were treated with 8 equivalents of tetrabutylammonium chloride at 298 K (Figure 5 and Supplementary Material). Within 30 s of adding Cl⁻ to the C_iq cage, there were multiple species observed in the ¹H NMR spectrum. These were attributed to the C_iq cage, [Cl⊂C_iq]³⁺, the [Pd₂(L_iq)₂Cl₄] macrocycle and free ligand based on our own previous results (Preston et al., 2015) and related literature. After 50 min, only uncoordinated ligand was visible in the ¹H NMR spectrum (Supplementary Material).

FIGURE 5

Figure 5. Partial ¹H NMR (500 MHz, d₆-DMSO, 298 K) spectra showing the stability of C_q in the presence of 8 eq. Cl⁻ anions. (A) L_q, (B) C_q, (C) C_q with 8 eq. Cl⁻ (t = 2 min), (D) t = 10 min, (E) t = 20 min, (F) t = 30 min, (G) t = 1 h, (H) t = 3 h, (I) t = 5 h, (J) t = 7 h.

Under the same conditions, C_q was stable for 1 h before showing signs of decomposition (Figure 5). After 3 h, there was no evidence of the C_q cage, and the ¹H NMR spectrum displayed peaks corresponding to free ligand and a second metal-containing species, which based on the observed chemical shifts was most likely the neutral [Pd₂(L_q)₂Cl₄] macrocycle (Figure 5H). This degradation behavior has been seen before with the [Pd₂(L_tripy)₄]⁴⁺ system in DMF (Preston et al., 2015). After 7 h, only free ligand could observed in the ¹H NMR spectrum indicating that all the ligand containing metal complexes had been completely decomposed into [Pd(Cl)₄]²⁻(Figure 5J).

In comparison to the previously reported [Pd₂(L_tripy)₄]⁴⁺ cage (τ_1/2 = 2 min), the isoquinoline cage displayed an identical half-life (τ_1/2 = 2 min), whereas the quinoline system was considerably more robust (τ_1/2 = 2 h). Presumably the observed results reflect the different steric profiles of the two quinoline substituted cages (C_qor C_iq). The C_q cage has the quinoline moieties protecting the external face of the palladium, providing more impediment to nucleophilic attack from that face (Figure 6). The C_iq does not feature the same steric impediment as the benzene units of the isoquinoline heterocycles do not block the top face of the C_iq cage as much as they do in the quinoline C_q(Figure 6).

FIGURE 6

Figure 6. Top down views of (A) and (B) the DFT optimized model of C_iq, and (C) and (D) the X-ray structure of C_q.

To assess biological activity, the cytotoxic effect (as half-maximal inhibitory concentrations (IC₅₀)) of the ligands and cages were determined against three different cell lines: cisplatin resistant MDA-MB-231 (breast cancer) (Lehmann et al., 2011), A549 (lung cancer) and non-cancerous primary cells: adult human dermal fibroblasts (HDFa) (Table 1 and Supplementary Material). The ligands L_q and L_iq exhibited limited solubility, and so data above the concentration of 1 μM was unattainable. Below this threshold, L_iq displayed minimal cytotoxic activity against both cell lines, while L_q was shown to be cytotoxic against A549 (IC₅₀ = 0.5 μM). Both cages were observed to be cytotoxic against the malignant cell lines, with C_q showing the same level of toxicity as its ligand against lung cancer cells (IC₅₀ = 0.5 μM). C_q was slightly less cytotoxic against MDA-MB-231 (IC₅₀ = 1.7 μM), whereas C_iq was less cytotoxic than the quinoline analog, with the IC₅₀ values ranging from 4.0 to 7.4 μM against the cancer cells. Both quinoline cages were found to be considerably more active than the related parent [Pd₂(L_tripy)₄]⁴⁺ cage system (IC₅₀ = 41.4 and 56.7 μM against A549 and MDA-MB-231, respectively) (McNeill et al., 2015). The quinoline cages were also more active than cisplatin against the two cancer lines examined (cisplatin IC₅₀ values = 41.2 and 9.4 μM, against MDA-MB-231 and A549, respectively) (Lo et al., 2015; McNeill et al., 2015). The quinoline cages C_q and C_iq were more cytotoxic than all the [Pd₂(L_tripy)₄]⁴⁺ cage systems reported in the literature (IC₅₀ values for the L_tripy based systems ranged from 10 to 100 μM) (McNeill et al., 2015; Kaiser et al., 2016; Schmidt et al., 2016b). Additionally, C_q was also more active, albeit against different cancer cell lines (HL-60, HL-60/Dox, HT-29, T-24, SKW-3), than the hydrophobic [Pd₂(L_anthracene)₄]⁴⁺ cages of Yoshizawa and Ahmedova (IC₅₀ values ranged from 0.9 to 37.4 μM) (Ahmedova et al., 2016; Anife et al., 2016). C_q was also more cytotoxic than a hydrophobic bis-hexyl-1,2,3-triazole substituted [Pd₂(L_hextrz)₄]⁴⁺ helicate, C_hextrz, we developed previously (IC₅₀ values = 6.9 and 6.0 μM against A549 and MDA-MB-231, respectively) (McNeill et al., 2015). We presume that the favorable combination of high hydrophobicity and the kinetic robustness against biological nucleophiles leads to the higher observed activity of C_q relative to the other [Pd₂(L)₄]⁴⁺ architectures. Disappointingly, neither of the cages (C_q and C_iq) showed any selectivity for the cancer cells, they were all found to have similar cytotoxicity against HDFa skin cells (IC₅₀ values ranged from 2.6 to 3.0 μM).

TABLE 1

Table 1. Half-maximal inhibitory concentrations (IC₅₀) of ligands L_q and L_iq, and cages C_q and C_iq architectures at 24 h.

Conclusion

We have herein reported the synthesis, characterization, cisplatin binding, kinetic robustness and cytotoxicity of two new bis-isoquinoline and bis-quinoline derived [Pd₂(L)₄]⁴⁺ cage complexes. The crystal structure of C_q architecture showed that the [Pd₂(L)₄]⁴⁺ cage formed a twisted meso isomer where the [Pd(quinoline)₄]²⁺ units at either end of the cage architecture adopt the opposite twists (left and right handed). Conversely, Density Functional Theory (DFT) calculations on the C_iq cage architecture indicated that a lantern shaped conformation similar to what has been observed before for related [Pd₂(L_tripy)₄]⁴⁺ systems was generated. The different cage conformations resulted in different properties for the isomeric cages. The C_iq cage is able to bind, weakly in acetonitrile, the anticancer drug cisplatin whereas the C_q architecture shows no interaction with the guest under the same conditions. The kinetic robustness of the two cages in the presence of Cl⁻ nucleophiles was also different. The C_iq cage was completely decomposed into free L_iq and [Pd(Cl)₄]²⁻within 1 h. However, the C_q cage was more long lived and was only fully decomposed after 7 h. The ligands (L_iq and L_q) and cages (C_iq and C_q) were assessed for their cytotoxic properties against two cancerous cell lines (A549 lung cancer cells and MDA-MB-231 breast cancer cells) and one non-cancerous cell line (HDFa skin cells). It was found that L_q and C_q were both reasonably cytotoxic against A549, while C_iq was slightly less active. The higher observed cytotoxicity of C_q relative to the other [Pd₂(L)₄]⁴⁺ architectures was presumed to be due the favorable combination of high hydrophobicity and the kinetic robustness against biological nucleophiles. However, none of the new molecules showed any selectivity for cancer cells, they were all found to have similar cytotoxicity against HDFa skin cells. A range of [Pd₂(L)₄]⁴⁺ cage systems have now been shown to be cytotoxic. However, in order to advance this class of MSA as anticancer agents more in depth mode of action/mechanistic studies on the origins of the cytotoxic activity are required. Studies to this effect are now underway.

Author Contributions

RV and JC conceived the idea, analyzed the data and wrote the manuscript. RV and LG conducted the synthesis. RV and DP conducted stability studies. RV, DP, and JJ conducted cytotoxicity studies. GG oversaw the cytotoxicity studies and analyzed the data. All authors provided feedback on the manuscript drafts and approved the submission.

Funding

JC thanks the University of Otago, Department of Chemistry and the MacDiarmid Institute for funding. RV was supported by a Claude McCarthy Fellowship. All the authors thank Charlotte Dobson, Pauline Lane and Oceanbridge for their kind donations which supported this work.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

RV and JJ acknowledge the University of Otago for doctoral stipends. Dr. Anna Garden and Dr. David McMorran are thanked for useful discussions. Professor Sally McCormick, Department of Biochemistry, University of Otago is thanked for kindly providing a sample of the HDFa skin cells. The authors acknowledge the contribution of the NeSI high performance computing facilities to the results of this research. New Zealand's national facilities are provided by the New Zealand eScience Infrastructure and funded jointly by NeSI's collaborator institutions and through the Ministry of Business, Innovation and Employment's Research Infrastructure program. URL: https://www.nesi.org.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2018.00563/full#supplementary-material

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