Influence of Terminal Functionality on the Crystal Packing Behaviour and Cytotoxicity of Aromatic Oligoamides

The synthesis and characterization of three aromatic oligoamides, constructed from the same pyridyl carboxamide core but incorporating distinct end groups of acetyl (Ac) 1, tert-butyloxycarbonyl (Boc) 2 and amine 3 is reported. Single crystal X-ray diffraction analysis of 1–3 and a dimethylsulfoxide (DMSO) solvate of 2 (2-DMSO), has identified the presence of a range of intra- and intermolecular interactions including N-H⋯N, N-H⋯O=C and N-H⋯O=S(CH3)2 hydrogen-bonding interactions, C-H⋯π interactions and off-set, face-to-face stacking π-π interactions that support the variety of slipped stack, herringbone and cofacial crystal packing arrangements observed in 1–3. Additionally, the cytotoxicity of this series of aromatic oligoamides was assessed against two human ovarian (A2780 and A2780cisR), two human breast (MCF-7 and MDA-MB-231) cancerous cell lines and one non-malignant human epithelial cell line (PNT-2), to investigate the influence of the terminal functionality of these aromatic oligoamides on their biological activity. The chemosensitivity results highlight that modification of the terminal group from Ac to Boc in 1 and 2 leads to a 3-fold increase in antiproliferative activity against the cisplatin-sensitive ovarian carcinoma cell line, A2780. The presence of the amine termini in 3 gave the only member of the series to display activity against the cisplatin-resistance ovarian carcinoma cell line, A2780cisR. Compound 2 is the lead candidate of this series, displaying high selectivity towards A2780 cancer cells when compared to non-malignant PNT-2 cells, with a selectivity index value >4.2. Importantly, this compound is more selective towards A2780 (cf. PNT-2) than the clinical platinum drugs oxaliplatin by > 2.6-fold and carboplatin by > 1.6-fold.

Systematic solid-state studies of aromatic oligoamides have identified that small structural variations in these molecules can have a profound influence on their conformational behavior and such studies can to help deepen our understanding of their structure-activity relationships (SARs). A crystallographic study of aromatic oligoamides by Nissinen and co-workers (Suhonen et al., 2012) showed that modification of the aromatic ring from benzene to pyridine results in marked changes in the folding behavior of these compounds resulting in the adoption of curved molecular structures. Gunnlaugsson and co-workers described a crystallographic analysis of a series of cytotoxic pyridine-based aromatic oligoamides, showing that they adopted curved molecular structures with a supramolecular arrangement that could potentially promote interaction with DNA. (Frimannsson et al., 2010). The pyridine-based aromatic oligoamides were identified as DNAtargeting supramolecular binders and displayed cytotoxicity against the drug-resistant chronic myeloid leukaemia, K562 cell line. Fletcher and co-workers determined SARs on a series of short chain aromatic oligoamides, highlighting that relaxation of the rigidity of the backbone of the scaffold lead to increased cytotoxicity. (Yap et al., 2012). The lead candidate of the series displays low IC 50 values (1.1-4.3 μM) against the human colon carcinoma (DLD-1), mesothelioma (I45), lung carcinoma (A549), and human non-small cell lung carcinoma (H1299).
Gaining an understanding of the influence of the structure of an aromatic oligoamide on its biological activity is central to the development of new molecules within this class that have the potential to demonstrate improved cytotoxicity towards cancerous cells. To probe the influence of the terminal group on the solid-state structure and antiproliferative activity of these aromatic oligoamides, we undertook the synthesis, crystallographic analysis and cytotoxicity studies of three aromatic oligoamides based on the same pyridyl carboxamide core but including different end groups; acetyl (Ac) 1, tertbutyloxycarbonyl (Boc) 2 and amine 3 (Figure 1). We report on the solid-state properties of 1-3 and solvatomorph 2-DMSO, and employ single-crystal X-ray diffraction analysis to identify the presence of a range of non-covalent interactions which support the diverse crystal packing behavior of these aromatic oligoamides. We describe the influence of varying the terminal functionality in compounds 1-3 on their cytotoxicity against breast and ovarian cancer cell lines, and report the chemosensitivity studies against a non-malignant cell type. The results show that the most promising compound, a Boc-terminated aromatic oligoamide, is non-toxic towards non-malignant cells, unlike all cisplatin (CDDP), carboplatin (CARB) and oxaplatin (OXA), which all demonstrate high cytotoxicity.

RESULTS AND DISCUSSION
Aromatic oligoamides, 1-3, which all have the same pyridyl carboxamide core, but incorporate different terminal groups of Ac 1, Boc 2 and NH 2 3 ( Figure 1) have been prepared according to known or modified literature procedures, (Annala et al., 2017;Suhonen et al., 2016;Suhonen et al., 2012) (Suhonen et al., 2012;Suhonen et al., 2016;Annala et al., 2017), and were all characterized by 1 H and 13 C{ 1 H} NMR spectroscopy, melting point analysis, FTIR spectroscopy, high-resolution mass spectrometry and single crystal X-ray diffraction. The 1 H and 13 C NMR spectra of 1-3 indicate that these compounds are symmetrical, with the 1 H NMR spectra showing only one resonance for the NHs in the amide bonds of the terminal Ac and Boc group of 1 and 2 at δ 10.93 and δ 10.73 ppm respectively. Whilst the 13 C NMR spectrum of 3 displays only one resonance for the 2 C atoms in the carbonyl groups adjacent to the pyridine ring at δ 161.2 ppm (see Supporting Information

Crystallographic Studies
Single crystals suitable for X-ray diffraction were obtained for 1-3 and for a DMSO solvatomorph of 2 (2-DMSO). Table 1 summarizes selected crystallographic data for 1-3 and 2-DMSO (for full crystallographic tables, see Supporting Information). X-ray diffraction analysis identified the nature of the non-covalent interactions present in the solid state for each of the studied aromatic oligoamides.

Crystallographic Analysis of 1
Single crystals of 1 were grown by vapor diffusion of diethyl ether into a dimethylformamide solution at ambient temperature. 1 crystallizes in a monoclinic crystal system and solution refinement was performed in the P2 1 /c space group ( Table 1). The molecular structure of 1 is shown in Figure 2A, with displacement ellipsoids placed at 50% probability level. 1 displays three sets of bifurcated intramolecular hydrogen-bonding interactions, firstly, involving the pyridyl N atom and the two NH's of the adjacent amide group (i.e., N (2/4)-H (2/4)/N (1) (2.6583(17)-3.2310(16) Å, Table 2) and, additionally, two bifurcated interactions exist between each of the NH's of a central amide group and the adjacent pyridyl N atom and the O atom of the terminal amide group (N (2/4)-H (2/4A)/N (1) and N (2/4)-H (2/4A)/O (4) (1.97(2)-2.39(2) Å, Figure 2A). (Rozas et al., 1998) 1 displays a slipped stack crystal packing arrangement, (Yao et al., 2018), aligned along the b axis ( Figure 2B), which is supported by two sets of intermolecular hydrogen-bonding interactions and one set of edge-to-face π-π stacking interactions. One of the intermolecular hydrogen-bonding interactions is present between one of the NH's of an terminal Ac group and an O atom on the carbonyl of the central amide group (N (3)-H    Compounds   Figure S8). 1 also displays an edge-to-face π-π stacking interaction between the terminal 2acylaminophenyl rings on neighboring molecules, further supporting the slipped stack crystal packing arrangement (Supplementary Figure S9). (Nishio, 2011).

Crystallographic Analysis of 2 and 2-DMSO
Single crystals of compound 2 and the dimethylsulfoxide (DMSO) solvate, 2-DMSO, were grown from two different crystallization conditions at ambient temperature, firstly, through the slow evaporation of chloroform to give 2 and secondly, through the slow evaporation of a 9:1 chloroform: DMSO solvent mixture to generate 2-DMSO. In the former conditions, 2 crystallizes in an orthorhombic crystal system and solution refinement was performed in the P2 1 2 1 2 1 space group (Table 1) and in the latter conditions, 2 crystallizes, as the DMSO solvate, in a monoclinic crystal system and solution refinement was performed in the P2 1 /c space group ( Table 1). The molecular structures of 2 and the 2-DMSO solvate are shown in Figure 3, with displacement ellipsoids placed at 50% probability level. Both 2 and 2-DMSO display two sets of bifurcated intramolecular hydrogen bonding interactions, firstly, between the pyridyl N atom and the two NH's of the adjacent amide groups (N (2/4)-H (2/4A)/N (1) 2.34(4)-2.36(4) Å, Figures 3A,B) and, secondly, between one of the NH's in a central amide group and the adjacent pyridyl N atom and the O atom of the terminal amide group (N (2)-H (2A)/N (1) and N (2)-H (2)/O (2) (1.88(4)-2.36(4) Å, Figures 3A,B). (Arifuzzaman et al., 2013).

Crystallographic Analysis of 3
Single crystals of compound 3 were grown through the slow evaporation of chloroform at ambient temperature. 3 crystallizes in a monoclinic space group and solution refinement was performed in the P2 1 /c space group (Table 1). In the unit cell of 3, there are two distinct molecules present and the molecular structure is shown in Figure 4 with displacement ellipsoids placed at 50% probability level. Both molecules show the presence of a bifurcated intramolecular hydrogen-bonding interactions involving the pyridyl N atom and the adjacent amide NHs (N (2/4)-H (2/4)/N (1) and N (7/9)-H (7/9)/N (6), 2.08(5)-2.36 (4 Å, Figure 4). (Arifuzzaman et al., 2013).
3 adopts a combination of cofacial and slipped stack layered crystal packing arrangement (Chang et al., 2008;Kobayashi et al., 2006) orientated along the c axis ( Figure 5A) and this is supported by a series of intermolecular hydrogen-bonding interactions and parallel displaced π-π stacking interactions. In  Figure S23). The second of which adopts reciprocal intermolecular hydrogenbonding interactions between two adjacent molecules, giving rise to the formation of a hydrogen-bonded dimer ( Figure 5B). Additionally, there are two sets of intermolecular parallel displaced π-π stacking interactions present which support the cofacial and slipped stacking crystal packing arrangement of 3 (Supplementary Figure S24, 25). (Egli et al., 2003).

Chemosensitivity Studies
Cisplatin (CDDP), carboplatin (CARB) and oxaliplatin (OXA) and compounds 1-3 were screened for their cytotoxicity against human cell lines: cisplatin-sensitive ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780cisR) and breast adenocarcinomas (MCF-7 and MDA-MB-231). The IC 50 values were obtained via the MTT assay after a 96 h incubation period of each compound with the cells at 37°C and 5% CO 2 (Table 2; Figure 6). The Ac-terminated compound 1 was found be moderate to non-cytotoxic against all cell lines, with IC 50 values ranging from 63 ± 4 μM to >100 μM. Similarly, the Boc-terminated analogue 2 was found to be moderate to non-cytotoxic against A2780cisR, MCF-7 and MDA-MB-231. However, a significant increase in cytotoxicity is observed when comparing compounds 1 with 2 against A2780, with the potency of 2 increasing by up to 3-fold (77 ± 5 μM for 1 cf. 24 ± 0.9 μM for 2). The amine-terminated compound 3 in non-toxic towards the breast adenocarcinomas cell lines (MCF-7 and MDA-MB-231), with IC 50 values greater than the tested threshold (>100 μM). Notably, 3 is non-toxic against the cisplatin-sensitive ovarian carcinoma A2780 but is the only one in the library which displays any level of antiproliferative activity against the cisplatin-resistant ovarian carcinoma cell line, A2780cisR, with a moderate IC 50 value of 61 ± 1 μM. On analysis of these results no definite structure-activity relationship can be established but a general observation that the nature of the terminal group on these short aromatic oligoamides has a marked effect on determining their cytotoxicity against ovarian carcinomas (A2780 and A2780cisR) and breast adenocarcinomas (MCF-7 and MDA-MB-231). Results highlight the Boc-terminated compound 2 displays the highest activity, with moderate sensitivity against A2780 and the amine-terminated compound 3, is the only compound to display any level of cytotoxicity against A2780cisR.

Selectivity Index
CDDP, OXA, and CARB and compounds 1-3 were also screened against the non-malignant prostate cell line (immortalized with SV40), PNT-2, to determine any cancer cell selectivity. The results for CDDP, OXA, and CARB show that these clinical platinum drugs have high to moderately cytotoxicity towards PNT-2, with IC 50 values of 1.3 ± 0.2 μM (OXA), 8.5 ± 0.4 μM (CDDP) and 27 ± 2 μM (CARB). Unlike the clinical platinum drugs, compounds 1-3 are non-toxic towards PNT-2 (IC 50 values >100 μM) The selectivity index (SI) values were calculated for all the compounds, using the IC 50 values obtained against PNT-2 and dividing by the IC 50 value against the cancer cell line in parenthesis in Table 2). A SI value >1 indicates increased selectivity for the cancerous cell line over the nonmalignant one, whilst a SI value <1 indicates the inverse (i.e., increased selectivity for the non-malignant cell line over the cancerous one). Compound 1 shows only slight increases in selectivity, with an SI > 1.3* (p < 0.05, where * indicates the minimum SI value due to the PNT-2 IC 50 value >100 μM, Table 2 footnote) for A2780. 1 However, an SI > 1.6* (p < 0.05) for this compound against the triple negative breast cancer (TNBC) cell line, MDA-MB-231, is higher than those observed for the clinical platinum anticancer drugs OXA and CARB (of 0.5 and 0.8  Frontiers in Chemistry | www.frontiersin.org June 2021 | Volume 9 | Article 709161 6 respectively). 1 Compound 2 displays a notable SI > 4.2* (p < 0.05) against A2780 and a very moderate increase in selectivity towards MCF-7 (SI > 1.2*, p < 0.05) and MDA-MB-231 (SI > 1.4*). 1 The amine-terminated compound 3, is the only compound to display increased selectivity for A2780cisR when compared to PNT-2, with a SI > 1.6* (p < 0.05), which, albeit is very modest, is higher than the SI values observed for CDDP (0.6), OXA (0.6) and CARB (0.3*). Overall, these results highlight that small structural changes to the terminal groups of these aromatic oligoamides can have a marked effect on their biological activity against the tested ovarian and breast cancer cell lines. Herein, it is shown that modification of the terminal groups from Ac to Boc leads to a notable increase in the SI against A2780 but similar SI values are observed against MDA-MD-231, whilst variation of the terminal group to NH 2 leads to a change in the SI of the aromatic oligoamide against A2780cisR with a slight increase in SI (>1.6*, p < 0.05) being observed by this amine-terminated compound.

CONCLUSION
In conclusion, we have synthesized and characterized a series of aromatic oligoamides based on a common pyridyl carboxamide core but incorporating distinct end groups: acetyl (Ac) 1, tertbutyloxycarbonyl (Boc) 2 and amine 3. Single crystal X-ray diffraction analysis of 1-3 and 2-DMSO has identified the presence of an array of non-covalent interactions including N-H/N and N-H/O C hydrogen-bonding interactions, a series of C-H/π and π-π stacking interactions that support the diverse crystal packing arrangements present in these aromatic oligoamides including slipped stack (1), herringbone (2) and cofacial/slipped stacked (3). The crystal packing of 3 also reveals the presence of hydrogen-bonded dimer formed by the presence of reciprocal intermolecular N-H/O C hydrogen bonding interactions formed between the NH of the terminal amine groups and the O atom on the carbonyl group in the amide group of an adjacent molecule.
To understand SARs, the cytotoxicity of the compound 1-3 (and CDDP, OXA and CARB) were obtained via a 96 h MTT assay, and screening against human ovarian carcinomas (A2780 and A2780cisR), human breast adenocarcinomas (MCF-7 and MDA-MB-231) and non-malignant prostrate cell line (PNT-2). Generally, compounds 1-3 display either moderate cytotoxicity or are non-toxic against A2780cisR, MCF-7 and MDA-MB-231 cancer cell lines. The Bocterminated compound, 2, is the lead candidate of the tested aromatic oligoamides displaying an IC 50 value of 24 ± 0.9 μM against A2780. Unlike the tested clinical platinum anticancer drugs, compound 2 is non-toxic towards PNT-2 (IC 50 > 100 µM), meaning it displays an SI value >4.2*-fold towards A2780 (cf. PNT-2), making it more selective towards ovarian cancer than the platinum drugs CDDP and OXA (SI values against A2780: 1.6 (CDDP), 6.5 (CARB); 2.6 (OXA)). The insights gained from this study, regarding the importance of small structural modifications on influencing the biological activity of aromatic oligoamides, will facilitate the future design of related compounds with improved cytotoxicity against ovarian and breast cancer cell lines.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
PD was responsible for the synthesis and characterization of compounds 1-3. CS was responsible for the collection of crystallographic data and solving crystal structures of 2, 2-DMSO and 3 and for the analysis of crystallographic data. LM was responsible for the collection of crystallographic data and solving crystal structure of 1. RL was responsible for conducting the biological assays on the tested compounds, analysis of biological data and contributed to the manuscript. SP conceived the project and was responsible for analysis of crystallographic and biological data, manuscript preparation and project supervision.

FUNDING
RL and SP thank the University of Bradford Research Development Fund for financial support. SP acknowledges the University of Birmingham for a Birmingham Fellowship. RL is a UKRI Future Leaders Fellow, and this work is supported by a UKRI Future Leaders Fellowship (MR/T041315/1). SP is a UKRI Future Leaders Fellow, and this work is supported by a UKRI Future Leaders Fellowship (MR/S035486/2).