Enhanced kinetic stability of [Pd₂L₄]⁴⁺ cages through ligand substitution

Abstract

There is considerable interest in exploiting metallosupramolecular cages as drug delivery vectors. Recently, we developed a [Pd₂L₄]⁴⁺ cage capable of binding two molecules of cisplatin. Unfortunately, this first generation cage was rapidly decomposed by common biologically relevant nucleophiles. In an effort to improve the kinetic stability of these cage architectures here we report the synthesis of two amino substituted tripyridyl 2,6-bis(pyridin-3-ylethynyl)pyridine (tripy) ligands (with amino groups either in the 2-(2A-tripy) or 3-(3A-tripy) positions of the terminal pyridines) and their respective [Pd₂(L_tripy)₄]⁴⁺ cages. These systems have been characterised by ¹H, ¹³C and DOSY NMR spectroscopies, high resolution electrospray mass spectrometry, elemental analysis and, in one case, by X-ray crystallography. It was established, using model palladium(II) N-heterocyclic carbene (NHC) probe complexes, that the amino substituted compounds were stronger donor ligands than the parent system (2A-tripy > 3A-tripy > tripy). Competition experiments with a range of nucleophiles showed that these substitutions lead to more kinetically robust cage architectures, with [Pd₂(2A-tripy)₄]⁴⁺ proving the most stable. Biological testing on the three ligands and cages against A549 and MDA-MB-231 cell lines showed that only [Pd₂(2A-tripy)₄]⁴⁺ exhibited any appreciable cytotoxicity, with a modest IC₅₀ of 36.4 ± 1.9 μM against the MDA-MB-231 cell line. Unfortunately, the increase in kinetic stability of the [Pd₂(L_tripy)₄]⁴⁺ cages was accompanied by loss of cisplatin-binding ability.

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Introduction

Interest in self-assembled coordination complexes of well-defined two- and three-dimensional geometries, or metallosupramolecular architectures,¹ continues to grow due to their potential in a range of applications. The molecular recognition properties of these systems have been used to develop molecular reactions flasks,² catalysts³ and drug delivery agents.⁴ Systems have also been used to sequester reactive species,⁵ and environmental pollutants.⁶ Additionally, the biological,⁷ photophysical,⁸ electronic⁹ and redox¹⁰ properties of these metallosupramolecular architectures have been studied.

As part of our interest in the biological properties¹¹ of metallosupramolecular architectures we have previously reported the synthesis of a tripyridyl (tripy) [Pd₂L₄]⁴⁺ cage capable of binding cisplatin (cis-[Pt(NH₃)₂Cl₂]).^11d We hoped to exploit these [Pd₂L₄]⁴⁺ cages as metallosupramolecular drug delivery vectors, unfortunately the cage architecture was rapidly decomposed when exposed to chloride (Cl⁻),^11a,d,12 histidine (his)^11a and cysteine (cys)^11a (common nucleophiles in biological systems). In order to use these cisplatin binding [Pd₂L₄]⁴⁺ cages as drug delivery agents a method for increasing the kinetic stability of these metallosupramolecular architectures against nucleophiles was required.¹³ There are two obvious approaches to enhance the kinetic stability of the cage architectures: (1) assemble the cage using more kinetically inert metal ions or (2) sterically and electronically tune the tripy ligand framework. Exploiting kinetically inert metals for the generation of metallosupramolecular architectures can be difficult due to the formation of kinetically “trapped” intermediate structures that cannot “error-correct” into the desired system. Therefore we chose to generate more electron-donating tripyridyl ligands in an effort to obtain more kinetically robust [Pd₂L₄]⁴⁺ cages. Herein we report the synthesis of two new amino substituted tripyridyl ligands (2A-tripy and 3A-tripy) and their respective cages (Scheme 1). The addition of the amino groups to the 2- and 3-positions of the terminal ligating pyridyl units of the ligand framework was expected to increase the donor properties of the ligands and lead to more kinetically robust cage architectures. Additionally, the presence of the 2-amino units within the [Pd₂(2A-tripy)₄]⁴⁺ cage architecture was expected to sterically reduce access to the palladium(II) ions potentially further enhancing the cage lifetime in the presence of biological nucleophiles. Competition experiments with Cl⁻, his and cys nucleophiles showed that the [Pd₂(2A-tripy)₄]⁴⁺ cage was more stable than the [Pd₂(3A-tripy)₄]⁴⁺ cage which in turn was more stable than the [Pd₂(tripy)₄]⁴⁺ cage. Unfortunately, the structural and electronic changes imposed through amino substitution in the 2-position circumscribe the ability of the cage to encapsulate cisplatin. Preliminary biological testing on these systems indicated that only [Pd₂(2A-tripy)₄]⁴⁺ exhibited an IC₅₀ < 50 μM against either of the two investigated cell lines.

Scheme 1 Synthesis of the [Pd₂(L_tripy)₄]⁴⁺ cages from the tripyridyl ligands (tripy, 2A-tripy, 3A-tripy). Conditions: (i) [Pd(CH₃CN)₄](BF₄)₂, solvent (CH₃CN, DMF or DMSO), RT or 50 °C.

Results and discussion

Synthesis and characterisation

The electronically and sterically tuned tripyridyl ligands (tripy, 2A-tripy, and 3A-tripy, Scheme 1) were synthesised using standard methods^11d,12,14 (ESI†) and characterised using NMR spectroscopy, high resolution electrospray mass spectrometry (HR-ESMS), and elemental analysis (Experimental section and ESI†).

Addition of one of the ligands (tripy, 2A-tripy, and 3A-tripy) to a solution of [Pd(CH₃CN)₄](BF₄)₂ in a 2 : 1 ratio resulted in the formation of the desired cages (Scheme 1). While the tripy and 3A-tripy containing cages formed instantaneously at room temperature, the formation of [Pd₂(2A-tripy)₄](BF₄)₄ required heating in DMSO solution at 50 °C for six hours to complete the assembly. The cages were characterised using ¹H and DOSY NMR spectroscopy, HR-ESMS, and elemental analysis, (Experimental and ESI†). The ¹H NMR spectra (Fig. 1 and ESI†) of the [Pd₂(L_tripy)₄]⁴⁺ cages show a single set of peaks. The proton resonances due to the terminal pyridyl units of the cages (H_c–f) are shifted downfield (Δδ = 0.19–0.59 ppm) relative to the free tripy ligands, consistent with complexation to palladium(II) ions. Interestingly, the proton resonance of the 2-amino group in the [Pd₂(2A-tripy)₄](BF₄)₄ cage displays a very large (Δδ(H_NH) = 1.56 ppm) shift relative to the free ligand (Fig. 1). Presumably, this large shift is caused by a combination of coordination to the palladium(II) ions and intra-ligand hydrogen bonding interactions between the amino groups on the adjacent ligands. Consistent with this postulate, the shift of the corresponding proton resonance in the [Pd₂(3A-tripy)₄](BF₄)₄ cage is much smaller (Δδ(H_NH) = 0.81 ppm, ESI†). The sharp, uncomplicated signals observed in the ¹H NMR spectra of the [Pd₂(L_tripy)₄]⁴⁺ systems are similar to what was previously observed for the formation of other [Pd₂(L)₄]⁴⁺ cages and are consistent with the formation of complexes of high symmetry in solution.^11a,d,12

Fig. 1 Partial ¹H NMR spectra (298 K, 500 MHz, d₆-DMSO) of (a) 2A-tripy and (b) the cage [Pd₂(2A-tripy)₄](BF₄)₄.

Diffusion-ordered ¹H NMR spectroscopy (DOSY) provided additional strong support for the selective formation of the cages in solution. ¹H DOSY spectra (d₆-DMSO, 298 K) were obtained for ligands (tripy, 2A-tripy, and 3A-tripy) and cages ([Pd₂(tripy)₄]⁴⁺, [Pd₂(2A-tripy)₄]⁴⁺ and [Pd₂(3A-tripy)₄]⁴⁺ (ESI†)). Each of the proton signals in the individual spectra show the same diffusion coefficient (D), indicating that there is only one species present in solution (ESI†). The D_complex/D_ligand ratios of ∼0.50 : 1 are similar to those observed for related literature compounds^11a,d,12 and suggest that the palladium(II) cage species are stable in solution (ESI†).

Mass spectra (HR-ESMS) of the [Pd₂(L_tripy)₄]⁴⁺ cage systems in DMF–CH₃CN solution obtained under pseudo cold-spray conditions displayed overlapping peaks due to [Pd₂L₄]⁴⁺ and [PdL₂]²⁺ ions. Additionally, the spectrum of the 3A-tripy based cage displayed a peak due to the [Pd₂(3A-tripy)₄(BF₄)₂]²⁺ ion.

After considerable effort (>50 crystallisations and data collections) the solid state structure of the [Pd₂(2A-tripy)₄](BF₄)₄ complex was obtained using X-ray crystallography (Fig. 2 and ESI†). Small weakly diffracting X-ray quality crystals were generated by vapour diffusion of diethyl ether into a CH₃CN solution of the [Pd₂(2A-tripy)₄](BF₄)₄ cage. Although the weak diffraction was, at least in part, due to the presence of multiple disordered solvent molecules and counter anions within the crystal lattice (vide infra), the cationic framework of the cage was readily identified (Fig. 2).

Fig. 2 Molecular structure of [Pd₂(2A-tripy)₄]⁴⁺ cations determined using X-ray crystallography: (a) a ball-and-stick representation of [Pd₂(2A-tripy)₄]⁴⁺ cation; (b) a partial view of the [Pd₂(2A-tripy)₄]⁴⁺ cation from the top showing the hydrogen bonding interactions between the 2-amino substituents of the ligands. Solvent molecules and counterions omitted for clarity. Colours: magenta = palladium, grey = carbon, white = hydrogen, blue = nitrogen, red = oxygen.

The solid state structure of the [Pd₂(2A-tripy)₄]⁴⁺ cage confirms that the coordination of the 2-amino pyridyl units to the palladium(II) ions was monodentate through the pyridyl nitrogen as expected.¹⁵ Additionally, the 2-amino units of the ligands are engaged in intra-ligand hydrogen bonding interactions (N⋯N 3.78(3) Å, N–H⋯N 2.94 Å, Fig. 2b and ESI†) consistent with ¹H NMR data described above. In contrast to the [Pd₂(tripy)₄]⁴⁺ cage,¹⁶ the [Pd₂(2A-tripy)₄]⁴⁺ cation adopts a more twisted structure in which the ligands of the 2A-tripy cages are significantly bent out of planarity and this appears to be caused by hydrogen bonding interactions between the amino groups on the exo faces of the architecture. This is quite different to what has been previously observed in the solid state structures of unsubstituted [Pd₂(tripy)₄]⁴⁺ cations, these cages without the 2-amino groups all were found to adopt a lantern shape, with essentially planar tripy ligands.^11a,d,12 The coordinated 2A-tripy ligand distorts in two ways: a swivelling of the coordinating pyridine rings relative to the principal rotation axis of the molecule (θ = 34.44°–34.64°, compared with θ = 3.47°–9.42° for [Pd₂(tripy)₄]⁴⁺) and a twisting of the central pyridine out of the plane through which the ligand coordinates to the two Pd(II) centres (φ = 35.10° compared with φ = 5.61° for [Pd₂(tripy)₄]⁴⁺). The cavity dimensions also differ (a Pd⋯Pd distance of 11.530(9)–11.610(9) Å compared with 11.201(1) Å for [Pd₂(tripy)₄]⁴⁺, and a core-to-core pyridyl N⋯N distance of 10.711(9)–10.732(9) Å compared with 11.07(1)–11.26(1) Å for [Pd₂(tripy)₄]⁴⁺, Table 1).

Table 1 Selected crystallographic distances and angles from [Pd₂(2A-tripy)₄]⁴⁺ and [Pd₂(tripy)₄]^4+ 16

	[Pd2(2A-tripy)4]^4+	[Pd2(tripy)4]^4+ ^16
a θ: the swivelling of the coordinating pyridine rings relative to the principal rotation axis of the molecule. b φ: twisting of the central pyridine out of the plane through which the ligand coordinates to the two Pd(II) centres.
Θ (°)^a	34.44°–34.64°	3.47°–9.42°
Φ (°)^b	35.10°	5.61°
d(Pd(II)⋯Pd(II)) (Å)	11.53(9)–11.610(9) Å	11.201(1) Å
d(Ncore⋯Ncore) (Å)	10.71(9)–10.73(9) Å	11.07(1)–11.26(1) Å

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Interestingly, the central cavity of the [Pd₂(2A-tripy)₄]⁴⁺ cage is filled in the solid state. The exo-methylene alcohol (CH₂OH) substituents from the four neighbouring cages in the crystal lattice penetrate into the cavity of each cage and form a hydrogen bonding interaction (O⋯N 2.74(3) Å, O–H⋯N 1.91 Å, Fig. 3 and ESI†) with the endo-pyridyl unit. These interactions generate 2D supramolecular sheets of cages through the solid state structure (Fig. 3 and ESI†).

Fig. 3 The crystal packing of [Pd₂(2A-tripy)₄]⁴⁺ cations showing (a) the interdigitation of the methylene alcohol substituents on adjacent [Pd₂(2A-tripy)₄]⁴⁺ cations into the central cavity of the [Pd₂(2A-tripy)₄]⁴⁺ cation (cage shown in ball-and-stick, methylene alcohol substituents in spacefilling view), and (b) the extended lattice.

Competition experiments, cisplatin binding and cytotoxicity studies

The relative pK_a values of 2-aminopyridine (6.82), 3-aminopyridine (6.04), and pyridine (5.23) indicate that 2-aminopyridine is the most basic ligand.¹⁷ Evidence that 2-aminopyridine was also the strongest nucleophile was obtained using the palladium(II)-N-heterocyclic carbene (NHC) probe system developed by Huynh and coworkers (Table 2 and ESI†).¹⁸ Consistent with the pK_a values, the probe complexes indicated that 2-aminopyridine (161.2 ppm) is a stronger donor than 3-aminopyridine (159.8 ppm) which is a stronger donor than pyridine (159.3 ppm). The chemical shift observed for the 2-aminopyridine ligand is very similar to that previously reported for N-methylimidazole (161.1 ppm)^18b suggesting that the donor strength of these ligands are similar. To allow direct comparison to the literature pK_a values (Table 2) and for synthetic convenience^11a we have examined the probe complexes of the simpler pyridine rather than the tripyridyl ligands. However, these pyridine model systems can serve as proxies for their respective tripyridyl ligands (2A-tripy, 3A-tripy, tripy) and provide indirect experimental evidence for the donor properties of the tripy ligands because the steric and electronic changes on going from the pyridine to tripy ligands are the same across the series. Thus the model complexes provide a qualitative ranking of the substituents' effects present in the tripy ligands and strongly suggest that the donor properties of the tripyridyl ligands follow the order 2A-tripy > 3A-tripy > tripy.

Table 2 pK_a values, ¹³C NMR chemical shifts of NHC carbene of the model probe complexes, and half-lives (t_1/2) for the decomposition of the [Pd₂(L_tripy)₄]⁴⁺ architectures against selected biologically relevant nucleophiles (3 : 2 d₆-DMSO/D₂O, 298 K, 500 MHz) as measured through time-course ¹H NMR spectroscopy

Compound	pKa	^13C δ^a (ppm)	Cl^− (8 eq.)	his (4 eq.)	cys (4 eq.)
a Chemical shift (ppm) of the NHC carbene carbon in the model Pd(II) probe complexes (ESI).
[Pd2(tripy)4](BF4)4	5.23^17	159.3^a^,^18	>1 min	18 min	6 min
[Pd2(2A-tripy)4](BF4)4	6.82^17	161.2^a	2 h	46 h	3 h
[Pd2(3A-tripy)4](BF4)4	6.04^17	159.8^a	10 min	25 min	10 min

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The kinetic stability of the [Pd₂(L_tripy)₄]⁴⁺ architectures in the presence of common biological nucleophiles (Cl⁻, his and cys) was determined using ¹H NMR competition experiments (Table 2 and ESI†). Time-course ¹H NMR competition experiments were carried out in 3 : 2 d₆-DMSO/D₂O where 3 mM solutions of each cage were treated with 8 equivalents of tetramethylammonium chloride or 4 equivalents of his or cys. Under these conditions the unsubstituted [Pd₂(tripy)₄]⁴⁺ cage was rapidly decomposed by all the nucleophiles. The half-life for the decomposition of the [Pd₂(tripy)₄]⁴⁺ complex with his was 18 minutes. Despite the [Pd₂(3A-tripy)₄]⁴⁺ architecture containing the slightly more electron rich 3A-tripy ligand the cage was still quickly decomposed by each of the nucleophiles. However, the t_1/2 were subtly increased against all the nucleophiles (for his t_1/2 = 25 min) suggesting that the enhanced ligand donor properties of the 3A-tripy ligand does lead to increased cage stability relative to the unsubstituted system.

The [Pd₂(2A-tripy)₄]⁴⁺ cage displayed markedly higher stability against all the nucleophiles studied. The half-lives for the 2A-tripy cage decomposition against each nucleophile were all over 2 h, whereas the corresponding t_1/2 for the other cages were all less than 30 min. 2A-tripy is only a modestly stronger donor ligand than the 3A-tripy, and thus the observed large difference in stability is presumably not predominantly due to the increase donor ability of the ligand. A more important element is likely to be the presence of the 2-amino groups on the exo-faces of the [Pd₂(2A-tripy)₄]⁴⁺ cage which sterically protect the palladium(II) ions from the incoming nucleophiles. Additionally, as observed in the X-ray structure (Fig. 3), the hydrogen bonding interactions between the eight amino groups of the four 2A-tripy ligands may further enhance the stability of the [Pd₂(2A-tripy)₄]⁴⁺ cage relative to the other tripy architectures. However, against the stronger nucleophiles (Cl⁻ and cys) the half-lives for the decomposition of the [Pd₂(2A-tripy)₄]⁴⁺ are less than 3 h suggesting that these systems would need further tuning in order to be useful in a biological setting.

Cisplatin binding

We^11d,12,19 and others¹⁶ have previously shown that other similar [Pd₂(tripy)₄]⁴⁺ cages can encapsulate cisplatin through hydrogen bonding interactions in CH₃CN and DMF solvents.²⁰¹H NMR spectroscopy and recently reported crystallographic evidence^16b confirms that despite the presence of the potential hydrogen bond donor CH₂OH units of the exo surface of the [Pd₂(tripy)₄]⁴⁺ cage the system retains the ability to bind cisplatin. Addition of an excess of cisplatin to a d₇-DMF solution of [Pd₂(tripy)₄]⁴⁺ resulted in a large downfield shift (Δδ = 0.24 ppm) of the internally directed cage proton H_c (Fig. 4a and b) indicative of cisplatin binding within the cage cavity. The solid state structure^16b of the [(cisplatin)₂⊂Pd₂(tripy)₄]⁴⁺ host–guest adduct has recently been reported by Casini, Kuhn and co-workers and is very similar to what we have previously observed in related systems.^11d,19b Consistent with the ¹H NMR evidence, the [Pd₂(tripy)₄]⁴⁺ cation binds two molecules of cisplatin within the cavity of the cage.¹⁶ The guest molecules are rotated 180° with respect to each other; hydrogen bonds between the guests and cage (N–H⋯N_Py and Cl⋯H–C_Py) as well as a metal–metal interaction between the platinum atoms of the guests were observed (Fig. 3a).^11d,16b,19b

Fig. 4 Partial ¹H NMR spectra (298 K, 500 MHz, d₇-DMF) of (a) [Pd₂(tripy)₄](BF₄)₄, (b) [(cisplatin)₂⊂Pd₂(tripy)₄](BF₄)₄ and (c) [Pd₂(3A-tripy)₄](BF₄)₄ and (d) [(cisplatin)₂⊂Pd₂(3A-tripy)₄](BF₄)₄.

A similar ¹H NMR experiment with cisplatin and [Pd₂(3A-tripy)₄]⁴⁺ indicated that the 3-amino substituted cage is also able to bind cisplatin in solution, albeit more weakly (Δδ = 0.07 ppm for H_c, Fig. 4c and d) than the parent [Pd₂(tripy)₄]⁴⁺ cage. Conversely, ¹H NMR spectra of the [Pd₂(2A-tripy)₄]⁴⁺ cage in the presence of cisplatin acquired in either deuterated CH₃CN or DMF solvents displayed no shifts relative to the free cage indicating that the 2-amino substituted cage is not able to bind cisplatin. We have previously shown that cisplatin binding is very weak¹² and that subtle changes to the size, steric profile and electronic properties of the cage cavity²¹ are enough to completely turn off cisplatin binding. Presumably, the lack of cisplatin binding in this system can be ascribed to two factors. Firstly, the presence of the eight amino groups on the exo faces of the cage has caused a twisting of the architecture (as indicated in the crystal structure, Fig. 3, and discussed and listed in Table 1). This twisting subtly alters both the size of the cisplatin binding cavity and the orientations of the hydrogen-bond acceptors and donors groups within the cage cavity, weakening the interaction between the host and the cisplatin guest. Secondly, the electron donating 2-amino units push electron density back onto the terminal pyridyl rings of the tripy ligand. This would reduce the polarisation of the acidic H_c protons of the pyridyl unit, weakening the hydrogen bonding interaction with chloride ligands of the cisplatin guest. These effects, in concert, appear to be enough to fully circumscribe the already weak cisplatin–cage interaction.²²

Cytotoxicity

While the improvement of kinetic stability of the [Pd₂(2A-tripy)₄]⁴⁺ cage has been achieved at the expense of host–guest capacity, recent work has shown that [Pd₂(L)₄]⁴⁺ architectures can act as biological agents in their own right.^11a,23 Accordingly, a preliminary investigation of the cytotoxic properties of the three cages (and the corresponding ligands) was undertaken against two cell lines: A549 (lung cancer) and cisplatin-resistant MDA-MB-231 (breast cancer) (Table 3). We have previously determined the IC₅₀ values for cisplatin against these two cell lines, which were 9.4 ± 0.3 μM (A549)²⁴ and 41.2 ± 3.9 μM) (MDA-MB-231)^11b respectively. None of the ligands exhibited significant cytotoxicity (IC₅₀ > 200 μM) against these cell-lines. Similarly, neither of the unstabilised cages, [Pd₂(tripy)₄](BF₄)₄ or [Pd₂(3A-tripy)₄](BF₄)₄ (IC₅₀ > 50 μM) nor the palladium(II) tetrafluoroborate salt (IC₅₀ > 100 μM))^11a [Pd(CH₃CN)₄](BF₄)₄ displayed any appreciable cytotoxic effect. The most kinetically stable cage [Pd₂(2A-tripy)₄](BF₄)₄ displayed low cytotoxicity against the A549 cell line (IC₅₀ > 50 μM), and slightly higher cytotoxicity against the MDA-MB-231 cell line (IC₅₀ = 36.4 ± 1.9 μM). We have previously shown that a more kinetically robust and hydrophobic [Pd₂(L_a)₄]⁴⁺ helicate (L_a = 1,3-bis(1-hexyl-1H-1,2,3-triazol-4-yl)benzene) was considerably more cytotoxic (IC₅₀ = 6.9 ± 0.9 and 6.0 ± 0.6 μM) against both the A549 and MDA-MB-231 cell lines.^11a Additionally, Yoshizawa and co-workers have recently reported that a hydrophobic [Pd₂(L_b)₄]⁴⁺ cage (L_b = 3,3′-((4,5,6-tris(2-methoxyethoxy)-1,3-phenylene)bis(anthracene-10,9-diyl))dipyridine) is highly cytotoxic (IC₅₀ = 1.8 and 1.9 μM) against HL-60 and SKW-3 cancer cell lines.²³ Thus it is presumed that low cytotoxicity of the systems described here is connected to the combination of both high kinetic lability and the more hydrophilic nature of the [Pd₂(L_tripy)₄]⁴⁺architectures and ligands.

Table 3 IC₅₀ values for [Pd₂(L_tripy)₄]⁴⁺ and related literature [Pd₂(L)₄]⁴⁺ compounds against selected cell lines

Compound	IC50 (μM)
	A549 (lung)		MDA-MB231 (breast)		HL-60 (leukemia)		SKW-3 (leukemia)
	Ligand	Cage	Ligand	Cage	Ligand	Cage	Ligand	Cage
[Pd2(tripy)4](BF4)4	>200	>50	>200	>50	—	—	—	—
[Pd2(2A-tripy)4](BF4)4	>200	>50	>200	36.4 ± 1.9	—	—	—	—
[Pd2(3A-tripy)4](BF4)4	>200	>50	>200	>50	—	—	—	—
[Pd2(La)4](BF4)4 (La = hextrz)^11a	28.5 ± 2.6	6.9 ± 0.9	89.8 ± 10.7	6.0 ± 0.6	—	—	—	—
[Pd2(Lb)4](BF4)4 (Lb = dipy-dianthracenyl)^23	—	—	—	—	>100	1.9 ± 0.2	>80	1.8 ± 0.2
Cisplatin	—	9.4 ± 0.3	—	41.2 ± 3.9	—	8.1 ± 1.4	—	9.3 ± 2.1

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Conclusions

Two amino substituted tripyridyl 2,6-bis(pyridin-3-ylethynyl)pyridine (tripy) ligands (with amino groups either in the 2-(2A-tripy) or 3-(3A-tripy) positions of the terminal pyridines) and their respective [Pd₂(L_tripy)₄]⁴⁺ cages were synthesised. These systems have been characterised by ¹H, ¹³C and DOSY NMR spectroscopies, high resolution electrospray mass spectrometry, elemental analysis and, in one case, by X-ray crystallography. It was established, using palladium(II) NHC carbene probe model complexes, that the amino substituted compounds were moderately stronger donor ligands than the parent pyridyl system (2A-tripy > 3A-tripy > tripy). Competition experiments with common biological nucleophiles (Cl⁻, his and cys) showed that the [Pd₂(2A-tripy)₄]⁴⁺ cage proved the most kinetically stable, presumably due to a favourable combination of enhanced ligand donor strength, and probably more importantly, intramolecular hydrogen bonding and steric shielding. Preliminary biological investigations against two cell lines (A549 and MDA-MB-231) found that all ligands and cages had IC₅₀ values >50 μM, with the exception of [Pd₂(2A-tripy)₄]⁴⁺ which against MDA-MB-231 has an IC₅₀ of 36.4 ± 1.9 μM.

However, while the ligand tuning resulted in more robust [Pd₂(L_tripy)₄]⁴⁺ architectures the half-lives of the systems against the stronger nucleophiles were still modest (t_1/2 = 2–3 h). Furthermore, the subtle structural changes in the most stabilised cage, [Pd₂(2A-tripy)₄]⁴⁺ were found to completely destroy the ability of the system to bind cisplatin. Thus, it appears that in order to exploit these types of metallosupramolecular cage architectures as drug delivery vectors, systems assembled from more kinetically inert metals ions such as Pt(II)^4h and Ru(II)^4e–g and Co(III)²⁵ will be required. Efforts to generate more robust systems, composed of kinetically inert metal ions, capable of binding drug molecules are underway.

Experimental

General

Unless otherwise stated, all reagents were purchased from commercial sources and used without further purification. 2,5-Dibromo-4-(hydroxymethyl)pyridine,²⁶ 5-iodo-2-aminopyridine,^6a the dimeric dibromobis(benzimidazolin-2-ylidene)dipalladium(II) complex,²⁷ and trans-dibromo(1,3-diisopropylbenzimidazolin-2-ylidene)(pyridine)palladium(II)^18b were synthesised according to literature procedures. Solvents were laboratory reagent grade. Petroleum ether refers to the fraction of petrol boiling in the range 40–60 °C, isopropyl alcohol (IPA), methanol (CH₃OH), dichloromethane (CH₂Cl₂), ethylenediaminetetraacetate (EDTA), ethynyltrimethylsilane (TMS-acetylene), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF). ¹H and ¹³C NMR spectra were recorded on either a 400 MHz Varian 400-MR or Varian 500 MHz AR spectrometer. Chemical shifts are reported in parts per million and referenced to residual solvent peaks (CDCl₃: ¹H δ 7.26 ppm, ¹³C δ 77.16 ppm; CD₃CN: ¹H δ 1.94, ¹³C δ 1.32, 118.26 ppm, d₆-DMSO: ¹H δ 2.50 ppm; ¹³C δ 39.52 ppm). Coupling constants (J) are reported in Hertz (Hz). Standard abbreviations indicating multiplicity were used as follows: m = multiplet, q = quartet, t = triplet, dt = double triplet, d = doublet, dd = double doublet, s = singlet, br = broad. Full ¹H and ¹³C NMR spectra, together with structural labelling are included in the ESI.† IR spectra were recorded on a Bruker ALPHA FT-IR spectrometer with an attached ALPHA-P measurement module. Microanalyses were performed at the Campbell Microanalytical Laboratory at the University of Otago. Electrospray mass spectra (ESMS) were collected on a Bruker microTOF-Q spectrometer.

Synthesis of 5-iodopyridin-2-acetamide (1). A solution of 5-iodopyridin-2-amine (2.5 g, 11.4 mmol) in triethylamine (10 mL) and CH₂Cl₂ (50 mL) was degassed with N₂ for 15 minutes. Acetic anhydride (10.7 mL, 110 mmol) was added and the mixture was stirred at room temperature for 18 hours. After removal of solvents under vacuum, the crude mixture was dissolved in 3 : 1 CHCl₃/IPA (150 mL), and washed with saturated aqueous NaHCO₃ solution (2 × 75 mL), and brine (75 mL). The organic layer was dried over MgSO₄, filtered and the solvent removed under vacuum. Column chromatography on silica (1 : 19 acetone/CH₂Cl₂) gave the product as a brown solid (2.3 g, 8.8 mmol, 77%). ¹H NMR (400 MHz, d₆-DMSO, 298 K) δ: 10.58 (1H, s, H_NH), 8.50 (1H, d, J = 2.3 Hz, H_a), 8.07 (1H, d, J = 8.8 Hz, H_b), 7.94 (1H, d, J = 8.8 Hz, H_c), 2.08 (3H, s, H_d). ¹³C NMR (100 MHz, d₆-DMSO, 298 K) δ: 169.4, 153.3 (C_a), 151.2, 145.8 (C_b), 115.3 (C_c), 85.7, 23.9 (C_d). HR ESI-MS (CHCl₃) m/z = 284.95 [M + Na]⁺ (calc. For C₇H₇IN₂NaO, 284.95). IR: ν (cm^-1) 3215, 3139, 3079, 3018, 1676, 1660, 1520, 1362, 1298, 829, 799. Anal. calcd for: C₇H₇IN₂O: C, 32.08; H, 2.69; N, 10.69%. Found: C, 32.30; H, 2.55; N, 10.71%.

Synthesis of N-(5-((trimethylsilyl)ethynyl)pyridin-2-yl)acetamide (2). A round bottom flask containing 1 (1.00 g, 3.82 mmol), CuI (0.07 g, 0.38 mmol) and Pd(PPh₃)₂Cl₂ (0.19 g, 0.27 mmol) was purged with N₂. Triethylamine (20 mL) was added via syringe and the solution was degassed with N₂ for 15 minutes. After adding TMS-acetylene (0.68 g, 0.93 mL, 6.87 mmol) via syringe, the reaction was heated at reflux under N₂ for 48 hours. The solvent was removed under vacuum, and the resulting solid was taken up in 3 : 1 CHCl₃/IPA (40 mL). The solution was stirred with aqueous 0.1 M EDTA/NH₄OH solution (40 mL) for 1.5 hours. After washing with aqueous 0.1 M EDTA/NH₄OH (50 mL) and brine (100 mL), the organic layer was dried with Na₂SO₄, filtered, and the solvent removed under vacuum. The solid was purified through column chromatography on silica (1 : 19 acetone/CH₂Cl₂), giving the product as a brown solid (0.86 g, 3.72 mmol, 97%). ¹H NMR (500 MHz, CDCl₃, 298 K) δ: 8.34 (1H, d, J = 1.6 Hz, H_d), 8.17 (1H, d, J = 8.7 Hz, H_b), 8.07 (1H, br, H_NH), 7.77 (1H, dd, J = 8.7 Hz, 2.1 Hz, H_c), 2.21 (3H, s, H_a), 0.25 (15H, s, H_e). ¹³C NMR (125 MHz, CDCl₃, 298 K) δ: 168.5, 150.9 (C_d), 150.3, 141.4 (C_c), 116.0, 112.9 (C_b), 101.4, 97.1, 24.8 (C_a), −0.1 (C_e). HR ESI-MS (CHCl₃) m/z = 255.09 [M + Na]⁺ (calc. for C₁₂H₁₆N₂NaOSi, 255.09). IR: ν (cm⁻¹) 3243, 2955, 2161, 1697, 1579, 1526, 1380, 1303, 1246, 1030. Anal. calcd for C₁₂H₁₆N₂OSi·0.5 acetone: C, 62.03; H, 7.14; N, 11.35%. Found: C, 61.94; H, 7.29; N, 11.35%.

Synthesis of 5-ethynylpyridin-2-amine (3). A solution of 2 (0.63 g, 2.70 mmol) and NaOH (1.08 g, 27.0 mmol) in methanol (30 mL) was heated at reflux for 1.5 hours before removal of solvent under vacuum, with the resultant residue taken up in 3 : 1 CHCl₃/IPA (100 mL) and washed with water (100 mL) and brine (100 mL). After drying with Na₂SO₄ and filtration, the solvent was removed under vacuum, and purification through column chromatography on silica (1 : 8 acetone/CH₂Cl₂) gave the product as a brown solid (0.22 g, 1.90 mmol, 70%). ¹H NMR (500 MHz, CDCl₃, 298 K) δ: 8.22 (1H, dd, J = 2.2 Hz, 0.8 Hz, H_c), 7.53 (1H, dd, J = 8.6 Hz, 2.3 Hz, H_b), 6.46 (1H, dd, J = 8.6 Hz, 0.8 Hz, H_a), 4.69 (2H, br, H_NH), 3.06 (s, H_d). ¹³C NMR (125 MHz, CDCl₃, 298 K) δ: 157.6, 151.6 (C_c), 141.0 (C_b), 108.7, 108.0 (C_a), 81.2, 77.8 (C_d). IR: ν (cm⁻¹) 3326, 3295, 3163, 2148, 1645, 1585, 1424, 1329, 1246, 1156. Anal. calcd for C₇H₆N₂·0.1 acetone: C, 70.74; H, 5.37; N, 22.60%. Found: C, 70.44; H, 5.17; N, 22.56%.

Synthesis of N-(5-((trimethylsilyl)ethynyl)pyridin-3-yl)acetamide (4). A glass tube containing diisopropylamine (7 mL) degassed with N₂ was charged with 5-bromopyridin-3-amine (500 mg, 2.89 mmol), CuI (55 mg, 0.29 mmol), Pd(dppf)₂Cl₂ (85 mg, 0.17 mmol) and TMS-acetylene (1.173 mL, 852 mg, 8.67 mmol) against a positive N₂ flow. The tube was sealed and stirred at 60 °C for 72 hours. 3 : 1 CHCl₃/IPA (100 mL) and aqueous 0.1 M EDTA/NH₄OH solution (100 mL) were added and the mixture stirred for one hour. The organic layer was washed with water (50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and the solvent was removed under vacuum. The resultant solid was purified by column chromatography (1 : 8 acetone/CH₂Cl₂) to give the product as a brown solid (512 mg, 2.71 mmol, 94%). ¹H NMR (500 MHz, CDCl₃, 298 K) δ: 8.10 (1H, d, J = 1.5 Hz, H_c), 8.01 (1H, d, J = 2.7 Hz, H_a), 7.04 (1H, dd, J = 2.7 Hz, 1.7 Hz, H_b), 0.25 (9H, s, H_d). ¹³C NMR (125 MHz, CDCl₃, 298 K) δ: 142.9 (C_c), 141.9, 136.8 (C_a), 124.1 (C_b), 120.3, 101.9, 97.6, 0.0 (C_d). HR ESI-MS (MeOH) m/z = 191.10 [M + H]⁺ (calc. for C₁₀H₁₅N₂Si, 191.10). Anal. calcd for C₁₀H₁₄N₂OSi: C, 63.11; H, 7.41; N, 14.62%; found: C, 63.40; H, 7.48; N, 14.62%. IR: ν (cm⁻¹) 3327, 3295, 3162, 2147, 1585, 1424, 1329, 1245.

Synthesis of 5-ethynylpyridin-3-amine (5). A solution of 4 (0.43 g, 2.3 mmol) in methanol (30 mL) with Na₂CO₃ (0.48 g, 4.5 mmol) was stirred at room temperature for 1 h. After filtration, the solvent was removed under vacuum, and the resultant solid was purified by column chromatography on silica (1 : 8 acetone/CH₂Cl₂) to give the product as a brown solid (0.25 g, 2.1 mmol, 96%). ¹H NMR (500 MHz, CDCl₃, 298 K) δ: 8.13 (1H, d, J = 2.7 Hz, H_c), 8.05 (1H, d, J = 2.7 Hz, H_a), 7.06 (1H, dd, J = 2.7 Hz, 2.7 Hz, H_b), 3.71 (2H, br, H_NH), 3.14 (1H, s, H_d). ¹³C NMR (125 MHz, CDCl₃, 298 K) δ: 143.0 (C_c), 141.8, 137.1 (C_a), 124.0 (C_b), 119.1, 80.7, 79.8 (C_d). IR: ν (cm⁻¹) 3328, 3295, 3163, 3020, 2148, 1644, 1585, 1424, 1329, 1246. Anal. calcd for C₇H₆N₂·0.1 (acetone): C, 70.74; H, 5.37; N, 22.60%. Found: C, 70.96; H, 5.14; N, 22.80%. Mass spectral analysis was not successful for this compound.

Synthesis of tripy. A round bottom flask containing (2,6-dibromopyridin-4-yl)methanol (336 mg, 1.26 mmol), 3-ethynylpyridine (325 mg, 3.15 mmol), CuI (24 mg, 0.12 mmol) and Pd(PPh₃)₂Cl₂ (35 mg, 0.05 mmol) was purged with N₂, before addition of triethylamine (25 mL) and dry THF (25 mL). The reaction was stirred in the absence of light for 24 hours. CH₂Cl₂ (50 mL) and aqueous 0.1 M EDTA/NH₄OH solution (100 mL) were added and the mixture stirred at room temperature for 2 hours. After extraction into CH₂Cl₂ (2 × 100 mL), the combined organic layers were washed with brine (100 mL), dried over MgSO₄, and then filtered. The solvent was removed under vacuum. Column chromatography (2 : 3 CH₂Cl₂/acetone) gave the product as a colourless solid (280 mg, 0.90 mmol, 71%). ¹H NMR (500 MHz, CD₃CN, 298 K) δ: 8.81 (2H, dd, J = 2.1 Hz, 0.8 Hz, H_c), 8.61 (2H, dd, J = 4.9 Hz, 1.7 Hz, H_f), 7.98 (2H, ddd, J = 7.9 Hz, 2.2 Hz, 1.7 Hz, H_d), 7.58 (2H, t, J = 0.9 Hz, H_b), 7.41 (2H, ddd, J = 7.9 Hz, 4.9 Hz, 0.9 Hz, H_e), 4.67 (2H, dt, J = 5.9 Hz, 0.8 Hz, H_a), 3.55 (1H, t, J = 5.9 Hz, H_OH). ¹³C NMR (500 MHz, CD₃CN, 298 K) δ: 154.1, 153.3 (C_c), 150.6 (C_f), 143.8, 139.9 (C_d), 125.3 (C_b), 124.4 (C_e), 119.9, 91.8, 86.2, 62.4 (C_a). HR ESI-MS (acetone) m/z = 645.21 [2M + Na]⁺ (calc. for C₄₀H₂₆N₆NaO₂, 645.20), 334.10 [M + Na]⁺ (calc. for C₂₀H₁₃N₃NaO, 334.10). IR: ν (cm⁻¹) 3204, 3032, 2923, 2213, 1685, 1596, 1547, 1475, 1415, 1189, 1076, 1023. Anal. calcd for C₂₀H₁₃N₃O·0.25 acetone: C, 76.48; H, 4.49; N, 12.90%. Found: C, 76.70; H, 4.49; N, 13.06%.

Synthesis of 2A-tripy. In a round bottom flask, diisopropylamine (20 mL) and THF (20 mL) were degassed with N₂, before addition of 3 (400 mg, 3.39 mmol), (2,6-dibromopyridin-4-yl)methanol (362 mg, 1.35 mmol), CuI (25 mg, 0.14 mmol), and Pd(PPh₃)₂Cl₂ (38 mg, 0.050 mmol) against a positive N₂ flow. The solution was heated at 50 °C for 48 hours. After removal of the solvent under vacuum, the resultant solid was taken up in 3 : 1 CHCl₃/IPA (150 mL) and aqueous 0.1 M EDTA/NH₄OH solution (50 mL) and stirred for 40 minutes. The organic layer was washed with brine, dried with Na₂SO₄, filtered and then the solvent was removed under vacuum. Purification of the resultant solid on a silica column deactivated with 3 : 97 triethylamine/CH₂Cl₂ (0.5/4.5/95 then 1/9/90 saturated aqueous NH₄OH solution/MeOH/CH₂Cl₂) gave the product as a brown solid (364 mg, 1.07 mmol, 79%). ¹H NMR (500 MHz, d₆-DMSO, 298 K) δ: 8.18 (2H, dd J = 2.3 Hz, 0.5 Hz, H_c), 7.57 (2H, dd, J = 8.6 Hz, 2.3 Hz, H_d), 7.42 (2H, s, H_b), 6.55 (4H, s, H_NH), 6.49 (2H, dd, J = 8.6 Hz, 0.6 Hz, H_e), 5.51 (1H, t, J = 5.8 Hz, H_OH), 4.54 (2H, d, J = 5.7 Hz, H_a). ¹³C NMR (500 MHz, d₆-DMSO, 298 K) δ: 159.7, 153.0, 152.0 (C_c), 142.9, 139.7 (C_d), 122.6 (C_b), 107.7 (C_e), 104.9, 88.8, 88.0, 60.9 (C_a). HR ESI-MS (CH₃CN) m/z = 683.26 [2M + H]⁺ (calc. for C₄₀H₃₁N₁₀O₂, 683.26), 342.13 [M + H]⁺ (calc. for C₂₀H₁₆N₅O, 342.14). IR: ν (cm⁻¹) 3307, 3145, 2920, 2201, 1670, 1634, 1593, 1543, 1505, 1393, 1171, 1140, 1055, 1016. Anal. calcd for C₂₀H₁₅N₅O·CH₂Cl₂: C, 69.00; H, 4.38; N, 20.02%. Found: C, 69.12; H, 4.51; N, 20.33%.

Synthesis of 3A-tripy. In a round bottom flask, diisopropylamine (20 mL) and THF (20 mL) were degassed with N₂, before 5 (365 mg, 3.09 mmol), (2,6-dibromopyridin-4-yl)methanol (275 mg, 1.03 mmol), CuI (20 mg, 0.10 mmol), and Pd(PPh₃)₂Cl₂ (36 mg, 0.05 mmol) were added against a positive N₂ flow. The solution was heated at 40 °C for 48 hours. After removal of the solvent under vacuum, the resultant solid was taken in 3 : 1 CHCl₃/IPA (150 mL) and aqueous 01. M EDTA/NH₄OH solution (50 mL) and stirred for 40 minutes. The organic layer was washed with brine (100 mL), dried with Na₂SO₄, filtered, and then the solvent was removed under vacuum. Purification of the resultant solid in a silica column deactivated with 3 : 97 triethylamine/CH₂Cl₂ (0.5/4.5/95 then 1/9/90 saturated aqueous NH₄OH solution/MeOH/CH₂Cl₂) gave the product as a brown solid (197 mg, 0.58 mmol, 56%). ¹H NMR (500 MHz, d₆-DMSO, 298 K) δ: 7.99 (2H, d, J = 2.9 Hz, H_e), 7.96 (2H, d, J = 1.6 Hz, H_c), 7.59 (2H, s, H_b), 7.09 (2H, t, J = 2.2 Hz, H_d), 5.60 (5H, m, H_NH & H_OH), 4.59 (2H, d, J = 5.8 Hz, H_a). ¹³C NMR (125 MHz, d₆-DMSO, 298 K) δ: 153.6, 144.5, 142.2, 139.2 (C_c), 136.9 (C_e), 124.2 (C_b), 121.3 (C_d), 117.8, 89.8, 86.4, 60.8 (C_a). HR ESI-MS (CH₃CN) m/z = 340.1190 [M + H]⁺ (calc. for C₂₀H₁₄N₅O, 340.1204). IR: ν (cm⁻¹) 3326, 3294, 3163, 3020, 2148, 1644, 1585, 1424, 1329, 1246, 1156. Anal. calcd for C₂₀H₁₅N₅O: C, 70.37; H, 4.43; N, 20.52%. Found: C, 70.21; H, 4.54; N, 20.52%.

Synthesis of [Pd₂(tripy)₄](BF₄)₄. A solution of tripy (62 mg, 0.20 mmol) and [Pd(CH₃CN)₄](BF₄)₂ (44 mg, 0.10 mmol) in acetonitrile (6 mL) was stirred for 1 hour. Vapor diffusion with diethyl ether gave a white solid (76 mg, 0.040 mmol, 85%). ¹H NMR (500 MHz, CD₃CN, 298 K) δ: 9.32 (8H, d, J = 1.8 Hz, H_c), 9.05 (8H, dd, J = 6.1 Hz, 1.1 Hz, H_f), 8.17 (8H, dt, J = 8.0 Hz, 1.4 Hz, H_d), 7.65 (8H, ddd, J = 8.0 Hz, 5.9 Hz, 0.6 Hz, H_e), 7.64 (8H, t, J = 0.8 Hz, H_b), 4.63 (8H, dd, J = 5.6 Hz, 0.8 Hz, H_a), 3.57 (4H, t, J = 5.6 Hz, H_OH). ¹³C NMR (125 MHz, CD₃CN, 298 K) δ: 154.5, 154.4 (C_c), 151.4 (C_f), 144.5 (C_d), 143.2, 128.5 (C_e), 126.5 (C_b), 124.1, 94.8, 83.2, 62.2 (C_a). HR ESI-MS (CH₃CN) m/z = 816.1313 [M − (BF₄)₂]²⁺ (calc. for C₈₀H₅₂N₁₂O₄Pd₂, 816.1313). IR: ν (cm⁻¹) 3211, 3097, 2976, 2921, 2852, 2220, 1685, 1595, 1545, 1483, 1420, 1043, 1027. Anal. calcd for C₈₀H₅₂B₄F₁₆N₁₂O₄Pd₂·3H₂O: C, 51.67; H, 3.14; N, 9.04%. Found: C, 51.87; H, 3.09; N, 8.71%.

Synthesis of [Pd₂(2A-tripy)₄](BF₄)₄. A solution of 2A-tripy (40 mg, 0.12 mmol) and [Pd(CH₃CN)₄](BF₄)₄ (26 mg, 0.060 mmol) in 1.5 mL d₆-DMSO in a tube was purged with N₂ and was heated at 50 °C for 6 hours. Diethyl ether (30 mL) was added and the solution shaken vigorously. After decanting the liquid portion, the precipitate was suspended in CH₂Cl₂ (5 mL) and isolated by filtration. After washing with CH₂Cl₂ (5 mL), the solid was dried under vacuum at 60 °C for 4 days to give the product as a red solid (32 mg, 0.017 mmol, 57%). ¹H NMR (500 MHz, d₆-DMSO, 298 K) δ: 8.77 (8H, d, J = 1.6 Hz, H_c), 8.11 (8H, s, H_NH), 7.84 (8H, dd, J = 8.9 Hz, 1.5 Hz, H_d), 7.56 (8H, s, H_b), 6.73 (8H, d, J = 8.9 Hz, H_e), 4.53 (8H, s, H_a). ¹³C NMR (500 MHz, d₆-DMSO, 298 K) δ: 159.8, 153.5, 149.4 (C_c), 142.7 (C_d), 142.1, 125.3 (C_b), 112.6 (C_e), 107.7, 90.5, 84.6, 60.6 (C_a). ESI-MS (DMSO/CH₃CN) m/z = 394.60 [M − 4(BF₄)]⁴⁺ (calc. for C₈₀H₆₀N₂₀O₄Pd₂, 394.58), 394.10 [Pd(2A-tripy)₂]²⁺ (calc. for C₄₀H₃₀N₁₀O₂Pd, 394.08). IR: ν (cm⁻¹) 337, 3194, 2210, 1637, 1546, 1513, 1411, 1025. Anal. calcd for C₈₀H₆₀B₄F₁₆N₂₀O₄Pd₂·5H₂O·4DMSO: C, 45.50; H, 4.07; N, 12.03%. Found: C, 45.24; H, 3.78; N, 12.29%.

Synthesis of [Pd₂(3A-tripy)₄](BF₄)₄. A solution of 3A-tripy (60 mg, 0.18 mmol) and [Pd(CH₃CN)₄](BF₄)₄ (39 mg, 0.090 mmol) in d₆-DMSO (0.75 mL) was sonicated for five minutes. Addition of ethyl acetate (20 mL)) resulted in precipitation of the product. The precipitate was collected by filtration, and washed with ethyl acetate (15 mL) and diethyl ether (15 mL) to give a brown solid (64 mg, 0.03 mmol, 75%). OR A solution of 3A-tripy (60 mg, 0.18 mmol) and [Pd(CH₃CN)₄](BF₄)₄ (39 mg, 0.090 mmol) in DMF (2 mL) was sonicated for five minutes. Addition of ethyl acetate (20 mL) resulted in precipitation of the product. The precipitate was collected by filtration, and washed with ethyl acetate (15 mL) and diethyl ether (15 mL) to give a brown solid (60 mg, 0.03 mmol, 70%). ¹H NMR (500 MHz, d₆-DMSO, 298 K) δ: 8.36 (8H, s, H_e), 8.26 (8H, s, H_c), 7.63 (8H, s, H_b), 7.30 (2H, s, H_d), 6.41 (8H, s, H_NH), 5.59 (4H, br, H_OH), 4.55 (8H, s, H_a). ¹³C NMR (125 MHz, d₆-DMSO, 298 K) δ: 154.0, 146.9, 141.4, 138.3 (C_c), 135.9 (C_e), 125.3 (C_d), 125.1 (C_b), 120.7, 119.1, 91.9, 83.3, 60.7 (C_a). HR ESI-MS (DMSO/CH₃CN) m/z = 394.5820 [M − 4(BF₄)₄]⁴⁺ (calc. for C₈₀H₆₀N₂₀O₄, 394.5797), 342.1401 [3A-tripy + H]⁺ (calc. for C₂₀H₁₆N₅O, 342.1401). IR: ν (cm⁻¹) 3468, 3384, 3230, 2220, 1637, 1592, 1547, 1443, 1020. Anal. calcd for C₈₀H₆₀B₄F₁₆N₂₀O₄Pd₂·5H₂O·4DMSO: C, 45.50; H, 4.07; N, 12.03%. Found: C, 45.20; H, 3.77; N, 12.28%.

X-ray crystallography [Pd₂(2A-tripy)₄](BF₄)₄

Yellow block crystals of [Pd₂(2A-tripy)₄](BF₄)₄ were grown by vapour diffusion of diethyl ether in a solution of MeCN. X-ray data were collected at 100(1) K on an Agilent Technologies Supernova system using Cu Kα radiation with exposures over 1.0°, and data were treated using CrysAlisPro²⁸ software. The structure was solved using Sir-97 ²⁹ and weighted full-matrix refinement on F² was carried out using SHELXL-97 ³⁰ running within the WinGX package.³¹ All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were placed in calculated positions and refined using a riding model. The structure was solved in the primitive tetragonal space group P4/m and refined to an R₁ value of 14.4%. The asymmetric unit contains two half ligands and two quarter occupancy palladium ions. The counter-anions and solvent molecules present in the solvent lattice were severely disordered and could not be appropriately modelled. The SQUEEZE routine within PLATON was employed to resolve this problem, resulting in ten void spaces (total of 460 electrons), variously assigned to tetrafluoroborate anions (8 in total), H₂O (5 in total) and MeCN (3 in total) solvent molecules (total of 444 electrons), as described below. Despite repeated efforts (>50 crystallisations and data collections over a two year period) to crystallise the compound, the most suitable candidate was small and a poor diffractor. The data quality is poor, with two A alerts (a large Hirshfield difference and high MainMol U_eq. compared to neighbours) and many B alerts, and we emphasise that metric data cannot be reliably extracted from the structure and should be treated with caution. However, the connectivity of the cationic framework is readily apparent. The methylene alcohol substituents from the four neighbouring cages in the lattice interpenetrate the cavity of each cage (Fig. 3 and ESI†), interpenetrating groups shown in spacefilling mode (yellow, green, blue and pink) and preclude cisplatin encapsulation in the solid state. Around the coordinating pyridine rings, the amino groups form a hydrogen bonding network. SQUEEZE details and crystallographic parameters can be found in the ESI.†

Acknowledgements

The authors wish to thank Dr Dave McMorran and Dr Warrick Lo for fruitful discussions, and Christopher Anderson for early synthetic efforts. DP and JEML thank the University of Otago for PhD scholarships. JDC (Laurenson Award, LA307) and DP (MacQueen Summer Scholarship) thank the Otago Medical Research Fund for financial support. The authors thank the Department of Pharmacology and Toxicology and the Department of Chemistry, University of Otago for additional funding.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: The experimental procedures, ¹H, ¹³C and DOSY NMR, HR-ESMS, crystallographic and computational data. CCDC 1439952. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6dt00133e

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