Cavity-Containing [Fe2L3]4+ Helicates: An Examination of Host-Guest Chemistry and Cytotoxicity

Two new di(2,2′-bipyridine) ligands, 2,6-bis([2,2′-bipyridin]-5-ylethynyl)pyridine (L1) and bis(4-([2,2′-bipyridin]-5-ylethynyl)phenyl)methane (L2) were synthesized and used to generate two metallosupramolecular [Fe2(L)3](BF4)4 cylinders. The ligands and cylinders were characterized using elemental analysis, electrospray ionization mass spectrometry, UV-vis, 1H-, 13C and DOSY nuclear magnetic resonance (NMR) spectroscopies. The molecular structures of the [Fe2(L)3](BF4)4 cylinders were confirmed using X-ray crystallography. Both the [Fe2(L1)3](BF4)4 and [Fe2(L2)3](BF4)4 complexes crystallized as racemic (rac) mixtures of the ΔΔ (P) and ΛΛ (M) helicates. However, 1H NMR spectra showed that in solution the larger [Fe2(L2)3](BF4)4 was a mixture of the rac-ΔΔ/ΛΛ and meso-ΔΛ isomers. The host-guest chemistry of the helicates, which both feature a central cavity, was examined with several small drug molecules. However, none of the potential guests were found to bind within the helicates. In vitro cytotoxicity assays demonstrated that both helicates were active against four cancer cell lines. The smaller [Fe2(L1)3](BF4)4 system displayed low μM activity against the HCT116 (IC50 = 7.1 ± 0.5 μM) and NCI-H460 (IC50 = 4.9 ± 0.4 μM) cancer cells. While the antiproliferative effects against all the cell lines examined were less than the well-known anticancer drug cisplatin, their modes of action would be expected to be very different.

While there are only a few reports on the biological properties of double-and quadruple-stranded helicates, the related triplestranded analogues have been extensively examined. These triply-stranded supramolecular structures are assembled from an octahedral metal ion and di(bidentate) linker ligands [M 2 L 3 ]; this combination of building blocks can generate three isomeric complexes the chiral M (ΛΛ) and P (ΔΔ), and the meso (ΛΔ). Pioneering work by Hannon and co-workers described the synthesis of the first [Fe 2 (L Pyim ) 3 ] 4+ helicates (where L Pyim (1E,1′E)-N,N′-[methylenebis(4,1-phenylene)]bis[1-(pyridin-2-yl) methanimine, Figure 1] obtained from pyridylimine binding motifs, octahedral Fe(II) ions and a diphenylmethylene spacer unit (Hannon et al., 1997). The mechanical coupling exerted by the spacer unit meant that a racemic (rac) mixture of the M (ΛΛ) and P (ΔΔ) [M 2 (L Pyim ) 3 ] 4+ helicates (where M Fe(II) or Ni(II)) was formed and the authors went on to show that the M and P helicates could be resolved by chiral chromatography (Hannon et al., 2001a). The interaction of the M− and P−[Fe 2 (L Pyim ) 3 ] 4+ helicates with DNA has been extensively examined. The complexes have been shown to bind in the major grove of duplex DNA (Moldrheim et al., 2002), and at the center of three-way DNA (Oleksi et al., 2006;Cerasino et al., 2007;Malina et al., 2007;Cardo et al., 2011) and RNA (Phongtongpasuk et al., 2013) junctions (3WJ). More recently, the [Fe 2 (L Pyim ) 3 ] 4+ helicates were also shown to bind to DNA and RNA bulges (Malina et al., 2014;Malina et al., 2016 (Cardo et al., 2018) and the Ni(II) and Fe(II) helicates have been demonstrated to interact with G-quadruplexes (Zhao et al., 2013;Zhao et al., 2016) and the β-amyloid polypeptide (Aβ) (Yu et al., 2012;Li et al., 2015). Furthermore, the interaction of the iron(II) helicate with duplex DNA induces intramolecular DNA coiling (Hannon et al., 2001b;Malina et al., 2008) and it has been shown to display anti-cancer (Hotze et al., 2008), antibacterial (Richards et al., 2009) and anti-fungal (Vellas et al., 2013) properties, but is not mutagenic or genotoxic. This remarkable range of biological properties has been obtained without investigating changes to either the metal binding (pyridylimine) or spacer units of the helicates, suggesting that the system could potentially be improved by further tuning of the molecular scaffold.
The 1 H NMR spectra (500 MHz, CD 3 CN, 298 K) for the complexes of L1 and L2 were significantly distinct ( Frontiers in Chemistry | www.frontiersin.org July 2021 | Volume 9 | Article 697684 aromatic region (δ 8.5−7.0 ppm) consistent with the formation of a racemic (rac) mixture of the helical [Fe 2 (L1) 3 ] 4+ isomers, P ΔΔ and M ΛΛ. Conversely, the 1 H NMR spectrum of [Fe 2 (L2) 3 ](BF 4 ) 4 was more complex with several broad overlapping resonances in the aromatic region. However, the methylene protons of the spacer backbone (H j, δ 4.0−3.8 ppm) were clearly split into two distinct resonances; a singlet and an AB quartet. This suggests that in solution [Fe 2 (L2) 3 ](BF 4 ) 4 forms a mixture of helicate rac-ΔΔ/ΛΛ and mesocate meso-ΔΛ isomers. Others (Goetz and Kruger, 2006;Vellas et al., 2013) have observed this behavior in solution with related [Fe 2 L 3 ] 4+ systems that feature the diphenylmethylene spacer unit. This is in contrast to observations with the pyridyl imine helicate, [Fe 2 (L Pyim ) 3 ] 4+ of Hannon and co-workers (Hannon et al., 1997). Those helicates have the same spacer unit and are found to exclusively form rac-helicates in solution and the solid state. The difference appears to be related to the larger size of [Fe 2 (L2) 3 ] 4+ compared to [Fe 2 (L Pyim ) 3 ] 4+ . In [Fe 2 (L Pyim ) 3 ] 4+ , the aryl rings of the spacer unit are in close contact and interdigitate, mechanically locking the complex into the helical arrangement. The larger size of L2 lessens this steric interdigitation of the spacer aryl groups, therefore making the mesocate arrangement more energetically accessible.
The molecular structures of [Fe 2 (L1) 3 ](BF 4 ) 4 and [Fe 2 (L2) 3 ](BF 4 ) 4 were confirmed by X-ray crystallography with crystals grown by slow vapour diffusion of diethyl ether into nitromethane solutions (Figure 2, Supplementary Figures S27,  S28). The [Fe 2 (L1) 3 ](BF 4 ) 4 structure was solved in the P1 space group and the asymmetric unit contains two iron ions, three L1 ligands, four tetrafluoroborate anions and two nitromethane solvent molecules. Each iron(II) ion is coordinated to three bipy units generating a triple-stranded helicate architecture. The Fe-Fe distance of 14.2 Å confirmed that the system is elongated in comparison to the parent Hannon helicate. The compound crystallized as a racemic mixture in the solid state with both the ΔΔ (P) and ΛΛ (M) isomers (see the structural representations in Figure 2) present in the crystal.
The [Fe 2 (L2) 3 ](BF 4 ) 4 structure was solved in the P2 1 /n space group and the asymmetric unit was occupied by one [Fe 2 (L2) 3 ] 4+ unit, two tetrafluoroborate anions and seven co-crystallized nitromethane molecules. [Fe 2 (L2) 3 ](BF 4 ) 4 was shown to crystallize as a rac mixture of the ΔΔ (P) and ΛΛ (M) helicates. The meso-form detected in solution by 1 H NMR spectroscopy was not observed in the solid state, presumably due to crystal packing effects (Vellas et al., 2013). The combination of the two 5-ethynyl-2,2′-bipyridine units and the diphenylmethylene spacer in L2 led to a large Fe-Fe distance MSA cage systems have been used extensively as hosts for small molecule guests (Yoshizawa et al., 2009;Cook and Stang, 2015;Hong et al., 2018;Saha et al., 2018;Bardhan and Chand, 2019;Gao et al., 2019;Rizzuto et al., 2019;Percastegui et al., 2020). In contrast, the use of [Fe 2 L 3 ] 4+ helicate architectures as hosts is far less common, presumably because the vast majority of reported MSAs do not contain a central cavity. Recently, there have been a few reports of guest binding [anions (Goetz and Kruger, 2006;Cui et al., 2012), sugars (Yang et al., 2021) and small aromatic molecules (Fazio et al., 2018;Jiang et al., 2019)] within [Fe 2 L 3 ] 4+ helicates that feature small cavities. As the cavities of Frontiers in Chemistry | www.frontiersin.org July 2021 | Volume 9 | Article 697684 the [Fe 2 (L1) 3 ] 4+ and [Fe 2 (L2) 3 ] 4+ helicates are both lined by functional groups that could interact with guests through either hydrogen bonding or π-interactions, we sought out some small drug molecules that could potentially interact with the helicates using those non-covalent interactions. Therefore, the guest molecules 1,4-benzoquinone, nalidixic acid, acridine (as an analogue of proflavine), cisplatin and 5-fluorouracil were selected as they are either known or analogues of known anticancer and antibacterial drugs (Figure 3; Supplementary Material). The host-guest (HG) interactions were examined using 1 H NMR spectroscopy and ESIMS. One of the potential guest molecules (2 equiv.) was combined with one of the helicates (1 equiv.) in CD 3 CN at 298 K and the 1 H NMR spectrum acquired (Supplementary Material). 1 H NMR spectra of the host-guest mixtures were then compared to the 1 H NMR spectra of the corresponding "free" host and guest compounds (Supplementary Figures S23, S24). Disappointingly, no complexation induced shifts were observed for either the host or the guest resonances suggesting that none of the guests bound within the cavities of the helicates. Molecular models (SPARTAN16, MMFF, Supplementary Figures S25, S26) showed that there are no obvious steric interactions that would prevent host-guest formation for the majority of the examined HG pairs. Thus, the lack of guest binding in the cases examined appears to be due to the absence of the correct combination of complementary noncovalent and solvaphobic interactions. Additionally, the BF 4 − counter-anions may be competing for the cavity as has been observed in other cationic MSA systems (August et al., 2016). Related [Fe 2 L 3 ] 4+ helicates have shown excellent anticancer activity (Song et al., 2021). Therefore, we examined the cytotoxicity of [Fe 2 (L1) 3 ] 4+ and [Fe 2 (L2) 3 ] 4+ against a panel of cancer cell lines. As the [Fe 2 (L1) 3 ](BF 4 ) 4 and [Fe 2 (L2) 3 ](BF 4 ) 4 complexes were only soluble in polar organic solvents (DMSO, CH 3 CN, CH 3 NO 2, and acetone) we attempted to render the helicates water soluble by exchanging the BF 4 − counter anions with Cl − or OTf − (Supplementary Material). While we were able to generate the new [Fe 2 (L1) 3 ](X) 4 and [Fe 2 (L2) 3 ](X) 4 (where X Cl − , OTf − ) salts, they proved even less soluble than the original BF 4 − salts. The OTf − salts were soluble in CH 3 CN and DMSO, however, the Cl − salts were only soluble in DMSO with none of the systems showing any appreciable water solubility ( Supplementary Figures S18-S21). Due to these complications, we carried out the cytotoxicity experiments with [Fe 2 (L1) 3 ](BF 4 ) 4 and [Fe 2 (L2) 3 ](BF 4 ) 4 dissolved in DMSO and these solutions were then diluted with biological media to the required concentrations.
The helicates were more long lived in 1:19 v/v DMSO:water mixtures. A 72 h UV-visible stability study of [Fe 2 (L1) 3 ](BF 4 ) 4 and [Fe 2 (L2) 3 ](BF 4 ) 4 in 1:19 v/v DMSO:water was conducted to replicate the timeframe of the biological testing ( Supplementary  Figures S16, S17). Interestingly, the smaller [Fe 2 (L1) 3 ](BF 4 ) 4 showed no signs of decomposition (within the uncertainty of the measurement) whereas the larger [Fe 2 (L2) 3 ](BF 4 ) 4 did slowly degrade, approximately 43% of the [Fe 2 (L2) 3 ](BF 4 ) 4 was still present in solution after 72 h. Given the moderate to good stability of the helicates under conditions similar to those required for the cytotoxicity assay we proceeded to measure the in vitro antiproliferative activity of the compounds.
Presumably the higher activity of the helicates is associated with the different molecular shape and higher charge. While the in vitro activity of the [Fe 2 (L1) 3 ](BF 4 ) 4 helicate is promising we note that the widely used anti-cancer drug cisplatin is more active in all the cell lines examined (Table 1). However, the mode of action of this covalent DNA binder, in comparison to supramolecular structures that are more likely to form noncovalent interactions with biological targets, will be very different, making any direct comparison difficult. Cytotoxicity assays demonstrated that both helicates were active against the four cell lines examined. The smaller rac-[Fe 2 (L1) 3 ](BF 4 ) 4 helicate was more cytotoxic than the larger rac/ meso-[Fe 2 (L2) 3 ](BF 4 ) 4 analogue and displayed promising low μM antiproliferative activity against HCT116 (IC 50 7.1 ± 0.5 μM) and NCI-H460 (IC 50 4.9 ± 0.4 μM) human cancer cells. Although both helicates were less active than the widely used anti-cancer drug cisplatin, these results suggest that helicates constructed from di(2,2′bipyridine) ligands have potential as anti-cancer agents in their own right. The combination of a cytotoxic supramolecular structure with encapsulated drugs may result in synergistic activity. However, the poor aqueous solubility and modest stability in biological media of the current [Fe 2 (L1) 3 ](BF 4 ) 4 helicates means that the properties of these compounds will need to be fine-tuned to overcome these shortfalls. This could potentially be achieved by using more kinetically inert metal ions such as Ru(II) (Glasson et al., 2008;Kumar et al., 2015) or Co(III) (Symmers et al., 2015;Burke et al., 2018;Crlikova et al., 2020) to assemble the helicates.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
Frontiers in Chemistry | www.frontiersin.org July 2021 | Volume 9 | Article 697684 analyzed the data. CH and SJ oversaw the cytotoxicity studies and analyzed the data. All authors provided feedback on the manuscript drafts and approved the submission.

ACKNOWLEDGMENTS
All the authors thank Charlotte Dobson, Pauline Lane and Oceanbridge for their kind donations which supported this work. We are grateful to Tasha Steel for the support with the cytotoxicity assays.