Design, Synthesis and Biochemical Evaluation of Novel Ethanoanthracenes and Related Compounds to Target Burkitt’s Lymphoma

Lymphomas (cancers of the lymphatic system) account for 12% of malignant diseases worldwide. Burkitt’s lymphoma (BL) is a rare form of non-Hodgkin’s lymphoma in which the cancer starts in the immune B-cells. We report the synthesis and preliminary studies on the antiproliferative activity of a library of 9,10-dihydro-9,10-ethanoanthracene based compounds structurally related to the antidepressant drug maprotiline against BL cell lines MUTU-1 and DG-75. Structural modifications were achieved by Diels-Alder reaction of the core 9-(2-nitrovinyl)anthracene with number of dienophiles including maleic anhydride, maleimides, acrylonitrile and benzyne. The antiproliferative activity of these compounds was evaluated in BL cell lines EBV− MUTU-1 and EBV+ DG-75 (chemoresistant). The most potent compounds 13j, 15, 16a, 16b, 16c, 16d and 19a displayed IC50 values in the range 0.17–0.38 μM against the BL cell line EBV− MUTU-1 and IC50 values in the range 0.45–0.78 μM against the chemoresistant BL cell line EBV+ DG-75. Compounds 15, 16b and 16c demonstrated potent ROS dependent apoptotic effects on the BL cell lines which were superior to the control drug taxol and showed minimal cytotoxicity to peripheral blood mononuclear cells (PBMCs). The results suggest that this class of compounds merits further investigation as antiproliferative agents for BL.


Introduction
Burkitt's lymphoma (BL) is an aggressive non-Hodgkin lymphoma occurring with high incidence in developing areas such as equatorial Africa and Papua New Guinea [1]. Within these tropical regions of high incidence (40-50 per 10 6 ), BL accounts for approximately 50% of childhood cancers and up to 90% of diagnosed childhood lymphomas [2]. The treatment of BL in these regions is difficult and the development of new, safe and cost-effective therapeutics are of current interest. For developed countries, the sporadic form represents 1-2% of adult lymphomas [3]. In contrast, the endemic BL is commonly associated with infection by the oncogenic Epstein-Barr virus (EBV), which interrupts cellular pathways regulating cell proliferation and thus prevents apoptosis [4]. Treatment of BL Nitrostyrenes and related nitrovinyl compounds induce anti-cancer effects and stimulate an apoptotic response in cancer cell lines e.g., oral and colon cancers, osteosarcoma and Erlich ascetic tumour cell lines [37][38][39][40][41][42], modulating tumorigenesis in colon and breast cancer via reactive oxygen species (ROS) mediated pathways [43][44][45]. Inhibition of telomerase, protein tyrosine phosphatase (PTP), phospholipase A2 and tubulin have been demonstrated for simple nitrostyrenes, while In the present work, a structurally diverse library of 9,10-dihydro-9,10-ethanoanthracene compounds related in structure to the previous lead nitrostyrene compounds 10a-c and the tetracyclic antidepressant maprotiline 8 were synthesised. This approach will facilitate the identification of potent and selective compounds which may be useful in the design of proapoptotic agents. These compounds are synthesised by Diels-Alder cycloaddition reaction of the required anthracene-diene system and hence modification of the ethano-bridgehead could be achieved by variation of the dienophile employed. The dienophiles chosen for the study included maleic anhydride, maleimide and N-substituted maleimides together with benzyne, acrylate esters and acrylonitrile. Variation of the anthracene substitution at C-9 from nitrovinyl to alternative double bonded systems such as cyanovinyl, imine and oxime are also of interest for biological activity, as is the introduction of maleimide linkers and aryl-substituted maleimides. Structural modifications of the nitrovinyl unit by reduction and extension of the alkyl chain length at C-2 were also explored, (see target structure, Figure 1). The compounds were evaluated in the EBV − MUTU-1 cell line and chemoresistant EBV + DG-75 cell line to establish the structure-activity relationships for these ethanoanthraenes and to optimize the antiproliferative and proapoptotic effects in BL cell lines.
Single crystal X-ray structure determination was completed on (E)-9-(2-nitrovinyl)-9,10-dihydro-9,10-[3,4]epipyrroloanthracene-12,14-diones 16c, 16j, 17l and 17n ( Figure 2) and selected data is summarised in Tables 4 and 5. The core dihydroethanoanthracene moiety is rigid, possessing analogous conformations in the different structures; the packing structure displayed by the products is centrosymmetric and monoclinic. From the asymmetric unit, it was possible to confirm the (E)-configuration of the nitrovinyl unit. The angle A o was calculated for each compound as the angle between the centroid of phenyl ring 1 and the centroid of phenyl ring 2-ranging from 122.05 • to 129.35 • . This angle is 119.46 • in the crystal structure of maprotiline [52]. The flat succinimide ring is fused to the ethano bridge, so that it is tilted through almost exactly 60 • with respect to the plane of the C-(4)-C(18)-C(22)-C(11) bridging atoms. The angle B o (maleimide substituent centroid to centroid of main axis) was also calculated and ranged from 118.05-121.71 • . The distance between the carbons in the ethano bridge (d 1 ) ranged from 1.542-1.563 Å, comparing well to that of maprotiline 1.54 Å [52] and related inclusion complexes [53]. The distance between the carbon at C-10 and the nearest carbon of the ethano bridge (d 2 ) for the series was 1.555-1.573 Å, while in maprotiline, this distance is 1.546 Å [52].
X-ray crystallographic analysis of the novel Diels-Alder adduct (E)-10-(2-nitrovinyl)-9,10-dihydro-9,10-ethanoanthracene-11-carbonitrile (19a) confirmed the regioisomer obtained, (Figure 2, Tables 4 and 5), and the packing structure of the product was centrosymmetric and monoclinic. The angle A o between the centroid of phenyl ring 1 and the centroid of phenyl ring 2 was determined as 126.94 o . This angle is reported as 119.46 o in the crystal structure of maprotiline [52]. The distance between the carbons in the ethano bridge (d 1 ) was 1.565 Å, comparing well to that of maprotiline 1.540 Å. The distance between the carbon at C-10 and the nearest carbon of the ethano bridge (d 2 ) was 1.556 Å, in maprotiline this distance is 1.546 Å [52].  Compound To investigate the effect of increased rigidity on the ethanoanthracene bridge, the (E)-9-(2-nitrovinyl)anthracenes (12a-12d) were converted to the corresponding (E)-9-(2-nitrovinyl)-9,10dihydro-9,10-[1,2]benzenoanthracenes (20a-d) in modest yields (Scheme 5, Series V, Table 7). The Diels-Alder reaction was achieved with benzyne as the dienophile generated in situ by thermal decomposition of benzenediazonium-2-carboxylate, (prepared from anthranilic acid and isoamyl nitrite) [56][57][58][59][60]. Diphenyl and triphenyl side products have been reported using in situ benzyne generation [57], but these were not isolated during the current work. The benzyne adduct 20f was similarly prepared from anthracene carboxaldehyde. Sodium borohydride reduction of 20a and 20f afforded the alcohol and nitroalkane analogues 20e and 20g, respectively. The library of novel triptycene analogues synthesised are summarised in Table 7. The 1 H NMR spectrum of compound 20d shows two multiplets occurring at 7.58-7.69 ppm and 7.72-7.82 ppm both integrating for three protons each. These signals were assigned to the two groups of three equivalent aromatic protons H-1, H-8, H-3" and H-4, H-5, H-6" respectively. The assignments were confirmed from the C-H COSY, HMBC and DEPT 90 NMR spectra, (see Supplementary Information).
The structure of 20a was confirmed by X-ray crystallographic analysis ( Figure 2, Tables 4 and 5), showing that the packing structure assumed by the product was monoclinic and confirming the (E)-configuration of the nitrovinyl unit. The angles between the centroids of each of the three phenyl rings and the centroid of the central axis were calculated from the asymmetric unit, these were A o (118. The distance between the carbons in the ethano bridge (d 1 ) was 1.400 Å, in maprotiline this distance is 1.540 Å [52]. The distance between the carbon at C-10 and the nearest carbon of the ethano bridge (d 2 ) was 1.518 Å, in maprotiline this distance is 1.546 Å [52], hence the X-ray structures indicate a comparable configuration for 20a and maprotiline.
A preliminary stability study of the representative ethanoanthracene compound 16a was carried out at acidic, neutral and basic conditions (pH 4, 7.4 and 9) using HPLC. The half-life (t 1 2 ) was determined to be 11 h at pH 4, 10.5 h at pH 7.4 and greater than 24 h at pH 9. Based on this stability study the compound would be suitable for further preclinical investigation.

Preliminary Evaluation of In Vitro Anti-Proliferative Activity of the Ethanoanthracenes in Burkitt Lymphoma EBV − MUTU-1 and EBV + DG-75 (Chemoresistant) Cell Lines
The panel of compounds synthesised (Series I-VII) based on the 9,10-dihydro-9,10ethanoanthracene scaffold was then initially screened at two concentrations (1 µM and 10 µM) for antiproliferative activity in the BL EBV − MUTU-1 and EBV + DG-75 (chemoresistant) cell lines to determine the structure-activity relationship for these maprotiline analogues. The results obtained from this preliminary screen in the MUTU-I and DG-75 cell lines (at 10 µM and 1 µM) are displayed in Tables 1-3 and Tables 6-9, with maprotiline and taxol used as the control drugs. Maprotiline induced a modest anti-proliferative effect at 10 µM in the MUTU-I and DG-75 BL cell lines, while taxol was more effective with 10% cell viability at 10 µM in both cell lines. The results obtained for these novel ethanoanthracene compounds (Series I-VII) are discussed by structural type.

Series I and II, Compounds 13a-n, 14a-c and 15
The initial lead 9-(2-nitrovinyl)anthracene 12a demonstrated activity (< 14% cell viability) in both BL cell lines at 10 µM, ( Table 1). The effects of the maleic anhydride adducts 13a-f and maleimide adducts (13g-n) of the lead nitrovinyl anthracene compound 12a on cell viability were first investigated ( Table 1). The 9-(2-nitrovinyl)-9,10-dihydro-9,10-ethanoanthracenes 13a, 13b produced a significant anti-proliferative effect at both 1 and 10 µM concentrations in the MUTU-I cell line (7-24% cell viability), (Table 1). In the DG-75 cell line the maleic anhydride derivative 13a gave slightly improved results compared to maprotiline at both concentrations. C-10 chloro substitution e.g., 13d resulted in decreased activity in both cell lines. The anhydride 13b and the imide 13g demonstrated potent antiproliferative effects in DG-75 cell line (1% cell viability at 1 µM), with the imide 13g more potent than the anhydride 13a.  ., 13f, 13i, 13l). Reduction of the C-9 nitrostyrenes 13a, 13g to afford the C-9 nitroalkane substituted 14a and 14b (Series II) resulted in significant reduction in activity, indicating the essential requirement of the nitrostyrene functional group for activity. The dimer compound 15 was identified as a potential lead compound with significant activity in both cell lines (< 5% viable cells at 1 µM).

Series IIIA, Compounds 16a-n
Compounds 16a-n (Series IIIA, Table 2) were designed to investigate the effect of N-arylsubstitution (halogen, ether, phenol, ketone, ester, amine) on antiproliferative activity of the lead ethanoanthracene scaffold structure 13a. All analogues in the series elicited a very potent anti-proliferative action in the MUTU-I cell line at 1 µM (<5% cell viability, except 16a, 10%). In the DG-75 cell line, the most potent activity was produced by compounds 16a and 16b, with <6% viable cells remaining at 1 µM. The 9-chloro compound 16d also produced a promising result at 1 µM with <14% cell viability. Significantly reduced activity was observed when comparing compounds 16b (p-Cl) and 16k (m-Cl) in the DG-75 cell line. In a study of para phenyl substitution of this series of compounds 16a-16n in the DG-75 cell line, the unsubstituted compound 16a and p-chloro compound 16b were found to elicit more favourable antiproliferative effects than the bromo (16g), fluoro (16e), amino (16n), ketone (16m) and methoxy (16c) analogues (Table 2). Maleimide (13g) and phenyl maleimide (16a) adducts produce superior activity in the two BL cell lines than the maleic anhydride adduct 13a. C-9 chloro substituted compounds 13j and 16d possessed significant anticancer effects in both cell lines (0.4-13.5% cell viability).

Series IIIB, Compounds 17a-n
Compounds 17a-n were designed to investigate the effect of the alkyl substitution on the nitrostyrene group, and also to investigate the introduction of a benzyl or phenylethylamine substitution on the heterocyclic nitrogen ( Table 3). The deactivating effect of extended alkyl chain length (methyl and ethyl) at C-2 on anticancer activity can once again be observed (70-90% cell viability). With the exception of compounds 17i (N-benzyl) and 17n (N-phenylethylamine), these analogues elicited poor anti-proliferative action in both MUTU-I and DG-75 cell lines confirming that alkyl substitution (methyl or ethyl) on the nitrostyrene group dramatically reduces activity e.g., comparing adducts 16a with 17a and 17b in both DG-75 and MUTU-I cell lines at 10 µM, (Table 3). Compound 18 was less potent at the lower concentration in both BL lines than 16a, indicating that the ethanoanthracene structure was more favourable than the triazole-anthracene bridged system for the desired anticancer effect.

Series IV, Compounds 19a-f
The effect of ethano-bridge substitution on antiproliferative effects was assessed in compounds 19a-f using a range of different dienophiles for the Diels-Alder reaction, together with the ring opening reaction of the anhydride 13a to introduce nitrile and ester groups on the ethano-bridge, Table 6. All Pharmaceuticals 2020, 13, 16 20 of 58 but one of these simpler Diel-Alder adducts possessed enhanced anti-proliferative activity compared to the parent nitrovinylanthracene compound 12a. The most promising compounds identified from this cohort were 19a, 19c and 19f showing good anti-proliferative effects in both cell lines with cell viability of 0-14% at 10 µM. The inclusion of the ester group on the ethano-bridged system in 19d reduced the potency observed for compound 19a, (Table 6).

Series V, Compounds 20a-g
A series of triptycene based Diel-Alder adducts were prepared to assess the effect of the rigid triptycene moiety on the anti-proliferative activity ( Table 7). The C-9 functionalities such as nitrovinyl 20a, 20b, 20c, 20d, nitroalkane 20e, aldehyde 20f and alcohol 20g were included to assess their impact on the anticancer properties of the series. The most promising results were obtained by nitrovinyl compounds 20a and 20d (10-chloro) having potent effects (<6% cell viability) in the MUTU-I cell line. A potent effect was also observed for 20d in DG-75 at 10 µM with no viable cells detected. Reduction of the nitrovinyl bond was once again detrimental to anti-proliferative effects in both cell lines (e.g., 20e).
Alkyl substituent at C-2 of the nitrovinyl unit resulted in diminished activity demonstrated for methyl (20b) and ethyl (20c) compounds. The aldehyde based triptycene (20f) exhibited good activity at the higher concentration in the MUTU-I cell line (<5%), while alcohol (20g) was inactive (Table 7).
In this initial evaluation of the ethanoanthracenes (Series I-VII) for antiproliferative activity, a number of key structural requirements were identified. The nitrovinyl pharmacophore was demonstrated to be critical for antiproliferative effect, epipyrrolo bridgeheads were also found to exert a more potent effect than simple ethano and furan-based bridgeheads. Introduction of a chloro substituent at C-9 of the anthracene core also contributed to potency for many compounds.

In Vitro Anti-Proliferative Activity of Selected Potent Ethanoanthracenes
Based on the results obtained from the cell viability study above, the following potent compounds were identified for further investigation in the MUTU-I cell line: maleimide 13j, N-hydroxymethylmaleimide 13n, and maleimide dimer 15 (Series I), N-arylmaleimides 16a-16j, 16m, 16n (Series IIIA), N-benzylmaleimide 17n (series IIIB), acrylonitrile adduct 19a, acrylate ester adduct 19c and diester 19f (Series IV) and triptycene 20d (Series V). The IC 50 values were determined in the sub-micromolar range (0.09-0.55 µM), with compound 15 identified as the most potent (IC 50 = 0.09 µM), Table 10. The MUTU-1 IC 50 values combined with the data provided by the preliminary screen in the DG-75 cell line were used to select the following compounds for subsequent IC 50 determination in the chemoresistant DG-75 cell line: 13j, 15 (Series I), 16a, 16b, 16c, 16d (Series II) and 19a (Series IV), Table 11.  compounds with maprotiline highlights the presence of three main shared molecular features: the (E)-configuration nitrovinyl pharmacophore located at C-9, the 9,10-dihydroanthracene core structure and the presence of the 9,10-ethanoanthracene bridge, unsubstituted as in maprotiline, having a nitrile substituent as in the acrylonitrile adduct 19a or forming part of the heterocyclic structure as in the maleimide adducts 13j, 16a and 16b.    (Table 11). These novel compounds were selected for further investigation also based on analysis of their drug-like properties (Lipinski) from a Tier-1 profiling screen, together with predictions of blood brain barrier partition, permeability, plasma protein binding, metabolic stability and human intestinal absorption properties which confirmed that these compounds are moderately lipophilic-hydrophilic drugs and are suitable candidates for further investigation (Tables S1 and S2, Supporting information) .  Compounds 12a, 13j, 16a-d and 19a were found to satisfy all the Lipinski rule of five criteria with logP values in the range 3.31-5.26, indicating their potential as lead compounds for further development. Examples of the potent compounds 13j, 16a, 16b and 19a (displayed as yellow in their respective overlays) were flexibly aligned with the lead compound maprotiline 8 (cyan) using MOE (Molecular Operating Environment) 2016.V8, (Figure 3). The close correspondence between overlays of these compounds with maprotiline highlights the presence of three main shared molecular features: the (E)-configuration nitrovinyl pharmacophore located at C-9, the 9,10-dihydroanthracene core structure and the presence of the 9,10-ethanoanthracene bridge, unsubstituted as in maprotiline, having a nitrile substituent as in the acrylonitrile adduct 19a or forming part of the heterocyclic structure as in the maleimide adducts 13j, 16a and 16b.

Investigations into the Pro-Apoptotic Effect of the Most Potent Ethanoanthracenes-FITC Annexin V/PI FACS Analysis
The potential pro-apoptotic effects of the most potent maprotiline analogues were determined by Annexin V/PI FACS analysis of a subset of the most potent compounds identified e.g., compounds 12a, 13j, 15, 16a-d and 19a. The study was carried out over a concentration range (0.  Figure 4A). The p-chlorophenyl compound 16b induced a more potent effect than the unsubstituted compound 16a with a response of >80% apoptosis at all concentrations in the MUTU-I cell line. The p-methoxyphenyl compound 16c induced >90% apoptosis at 0.5 µ M. Similar activity was observed for the 10-chloro compound 16d with 63% apoptosis at 0.2 µM, ( Figure 4B). Compounds 13j and 19a were also found to possess potent apoptotic activity at 0.5 µ M > 90%. The most potent compound investigated was the dimer 15, demonstrating > 90% apoptosis at 0.2 μM, ( Figure 4C). In summary, compounds 15 and 16b not only possessed the most potent antiproliferative activity but were found to induce the most favourable pro-apoptotic response in the MUTU-I cell line.
In the chemoresistant DG-75 cell line, taxol was found to elicit a pro-apoptotic effect at both 10 µ M (73%) and 1 µ M (21%), ( Figure 5A). Compound 12a elicits similar effects to taxol at both 10 µ M (82%) and 1 µ M (10%). The unsubstituted maleimide adduct 16a shows a potent apoptotic effect at 10 µ M (92%) but is inactive at lower concentrations (<5% apoptosis). The p-chlorophenylmaleimide 16b and p-methoxyphenylmaleimide compound 16c exhibits improved pro-apoptotic activity when compared to the unsubstituted compound 16a in the DG-75 cell line. Compound 16b and 16c induce 61% and 29% apoptosis at 1 µ M respectively. The 10-chloro compound 16d induced 44% apoptosis at 10 µ M and a modest 15% at 1 µ M ( Figure 5B). The maleimide Diels-Alder adduct 13j and acrylonitrile adduct 19a induced 90% and 87% apoptosis at 10 µ M. The leading compound 15 induced dose dependent apoptosis at all concentrations assayed with 95% (10 µ M) in the DG-75 cell line, Figure 5C. Overall compounds 15, 16b and 16c were shown to induce a superior potent pro-apoptotic response in both the MUTU-I and DG-75 BL cell lines than other selected compounds in this grouping and merit further study.

Evaluation of In Vitro Cytotoxicity of Ethanoanthracenes
Compounds 15, 16b and 16c were found to elicit the most potent anti-proliferative and proapoptotic activity of the series. In vitro cytotoxicity of these compounds was evaluated using a lactate

Investigations into the Pro-Apoptotic Effect of the Most Potent Ethanoanthracenes-FITC Annexin V/PI FACS Analysis
The potential pro-apoptotic effects of the most potent maprotiline analogues were determined by Annexin V/PI FACS analysis of a subset of the most potent compounds identified e.g., compounds  12a, 13j, 15, 16a-d and 19a. The study was carried out over a concentration range (0.2-10 µM) in both the BL cell lines (DG-75 and MUTU-I), Figure 4. Taxol was used as a positive control. Taxol was found to elicit a pro-apoptotic effect in the MUTU-I at both 10 µM (87%) and 1 µM (63%). The initial anthracene-nitrostyrene compound 12a demonstrated potent apoptotic activity at 10 µM (90%), with little effect at 1 µM [27] ( Figure 4A). Compound 16a induced over 80% apoptosis at 10 µM, 1 µM and 0.5 µM with 46% apoptosis at 0.2 µM ( Figure 4A). The p-chlorophenyl compound 16b induced a more potent effect than the unsubstituted compound 16a with a response of >80% apoptosis at all concentrations in the MUTU-I cell line. The p-methoxyphenyl compound 16c induced >90% apoptosis at 0.5 µM. Similar activity was observed for the 10-chloro compound 16d with 63% apoptosis at 0.2 µM, ( Figure 4B). Compounds 13j and 19a were also found to possess potent apoptotic activity at 0.5 µM > 90%. The most potent compound investigated was the dimer 15, demonstrating > 90% apoptosis at 0.2 µM, ( Figure 4C). In summary, compounds 15 and 16b not only possessed the most potent antiproliferative activity but were found to induce the most favourable pro-apoptotic response in the MUTU-I cell line.

Effect of Compounds 15, 16b and 16c on the Viability of PBMCs
The 2-nitrovinyl-9,10-dihydro-9,10-ethanoanthracenes compounds 15, 16b and 16c were evaluated for their toxicity on peripheral blood mononuclear cells (PBMCs) to determine the selective toxicity of these compounds on malignant BL cell lines over normal lymphatic cells.

Evaluation of In Vitro Cytotoxicity of Ethanoanthracenes
Compounds 15, 16b and 16c were found to elicit the most potent anti-proliferative and pro-apoptotic activity of the series. In vitro cytotoxicity of these compounds was evaluated using a lactate dehydrogenase (LDH) assay. LDH is released during mechanisms of cell death associated with loss of cell membrane integrity (necrosis). The MUTU-I and DG-75 BL cell lines were treated at 10 µM and 1 µM for the desired treatment period and the results are presented as percentage of total LDH release, (Figure 6). In the MUTU-I cell line low levels of LDH release were obtained (2-17%) at 10 µM and 1 µM concentration, indicating low cytotoxicity. The lowest LDH release was observed by compound 16b with 2% and 5% cytotoxicity (at 10 µM and 1 µM concentrations respectively). The

Effect of Pre-Treatment with Antioxidants on Cellular Viability
Reactive oxygen species (ROS) are short lived diffusible entities containing oxygen such as hydroxy, nitroxyl, alkoxy, superoxide or peroxyl radicals. ROS are generated as metabolites of oxygen and are utilised for signalling events for essential cell functions. ROS are often associated with the induction of cell death and apoptosis. A potential role for ROS in the mechanism of cell death induced by selected potent compounds 15, 16b and 16c was investigated. A viability assay was used to investigate the effects of pre-incubation with an antioxidant on ROS levels in DG-75 BL cells with the compound of interest. DG-75 cells were pre-treated with N-acetylcysteine (NAC), a known ROS scavenger and subsequently treated with the selected compounds 15, 16b and 16. Viability was monitored using the alamarBlue assay, (Figure 8). From the results, obtained it is evident that in the presence of the reactive oxygen species inhibitor NAC-the anti-proliferative effects of compounds

Effect of Pre-Treatment with Antioxidants on Cellular Viability
Reactive oxygen species (ROS) are short lived diffusible entities containing oxygen such as hydroxy, nitroxyl, alkoxy, superoxide or peroxyl radicals. ROS are generated as metabolites of oxygen and are utilised for signalling events for essential cell functions. ROS are often associated with the induction of cell death and apoptosis. A potential role for ROS in the mechanism of cell death induced by selected potent compounds 15, 16b and 16c was investigated. A viability assay was used to investigate the effects of pre-incubation with an antioxidant on ROS levels in DG-75 BL cells with the compound of interest. DG-75 cells were pre-treated with N-acetylcysteine (NAC), a known ROS scavenger and subsequently treated with the selected compounds 15, 16b and 16. Viability was monitored using the alamarBlue assay, (Figure 8). From the results, obtained it is evident that in the presence of the reactive oxygen species inhibitor NAC-the anti-proliferative effects of compounds 15, 16b and 16 at 1 µM was reduced. Overall the anti-proliferative effects previously observed by compounds 16b, 15 and 16c increased from 6-23% viable cells to 73-83% viable cells in the presence of 5 mM NAC, indicating that ROS may be involved in the mechanism of cell death induced by these compounds, (Figure 8). Cell proliferation of DG-75 cells was determined with an alamarBlue assay (seeding density 2 × 10 4 cells/mL per well for 96-well plates), with vehicle control ethanol 1% (v/v). Cells were retained for 24 h and then pre-treated with NAC (5 mM) for 1 h, followed by compounds 15, 16b and 16c at 1 μM for 48 h. Cell viability was measured by alamarBlue assay (mean of three independent experiments).

Conclusions
A series of 9,10-dihydro-9,10-ethanoanthracene based maprotiline analogues were synthesised and evaluated for potential antiproliferative activity in the MUTU-I and chemoresistant DG-75 BL cell lines. Substitution at C-9 and C-10 of the 9,10-dihydro-9,10-ethanoanthracene compounds was achieved by modification of the diene system to include functionalities such as nitrovinyl, nitroalkyl, aldehyde, imine, carboxylic acid, alcohol, oxime, cyanovinyl and hydrazone on the anthracene scaffold. The effect of a number of 9,10-dihydro-9,10-ethanoanthracene structural modifications on activity was also investigated; these modifications included ethano bridge modifications, phenyl substitutions, maleimide substitutions and extension of alkyl chain length at C-2 of the nitrovinyl unit. The most promising 9,10-dihydro-9,10-ethanoanthracene based maprotiline analogues were identified and all included a nitrovinyl substituent at C-9. The structure-activity relationships for the series of ethanoanthracenes synthesised in this study are summarised in Figure 9. The preliminary screen of the 9,10-dihydro-9,10-ethanoanthracenes identified the maleimide compounds 15, 16b and 16c as the lead compounds from this study. Cell proliferation of MUTU-1 and DG-75 cells was determined with an alamarBlue assay (seeding density 1-5 × 10 4 cells/mL per well for 96-well plates). Compound concentrations of either 1 µM or 0.5 µM for 24 h (MUTU-1) or 48 h (DG-75) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol (1% v/v). The mean value for three independent experiments is shown.
Cell proliferation of DG-75 cells was determined with an alamarBlue assay (seeding density 2 × 10 4 cells/mL per well for 96-well plates), with vehicle control ethanol 1% (v/v). Cells were retained for 24 h and then pre-treated with NAC (5 mM) for 1 h, followed by compounds 15, 16b and 16c at 1 µM for 48 h. Cell viability was measured by alamarBlue assay (mean of three independent experiments).

Conclusions
A series of 9,10-dihydro-9,10-ethanoanthracene based maprotiline analogues were synthesised and evaluated for potential antiproliferative activity in the MUTU-I and chemoresistant DG-75 BL cell lines. Substitution at C-9 and C-10 of the 9,10-dihydro-9,10-ethanoanthracene compounds was achieved by modification of the diene system to include functionalities such as nitrovinyl, nitroalkyl, aldehyde, imine, carboxylic acid, alcohol, oxime, cyanovinyl and hydrazone on the anthracene scaffold. The effect of a number of 9,10-dihydro-9,10-ethanoanthracene structural modifications on activity was also investigated; these modifications included ethano bridge modifications, phenyl substitutions, maleimide substitutions and extension of alkyl chain length at C-2 of the nitrovinyl unit. The most promising 9,10-dihydro-9,10-ethanoanthracene based maprotiline analogues were identified and all included a nitrovinyl substituent at C-9. The structure-activity relationships for the series of ethanoanthracenes synthesised in this study are summarised in Figure 9. The preliminary screen of the 9,10-dihydro-9,10-ethanoanthracenes identified the maleimide compounds 15, 16b and 16c as the lead compounds from this study. The dimer compound 15 displayed potent anticancer activity, in both BL lines with IC 50

Chemistry
All commercially available reagents were used without further purification. Solvents were dried prior to use; tetrahydrofuran (THF) by distillation from sodium/benzophenone under nitrogen, toluene was distilled from sodium, dichloromethane was distilled from calcium hydride. Melting points were recorded on a Gallenkamp SMP 11 melting point apparatus and are uncorrected. Infrared (IR) spectra were obtained on a Perkin Elmer FT-IR Spectrum 100 spectrometer. 1 H and 13 C NMR spectra were obtained on a Bruker Avance DPX 400 spectrometer operating at 400.13 MHz, ( 1 H) and 100.61 MHz ( 13 C) at 20 °C in either CDCl3 or DMSO-d6 with appropriate solvent peaks as reference standards. Mass spectrometry (ESI-MS) was performed on a Micromass LCT instrument with mass measurement accuracies of <±5 ppm. Low resolution mass spectra (LRMS) were obtained on a Hewlett-Packard 5973 MSD GC-MS system. Preparative separations were performed using flash column chromatography on silica gel (Merck Kieselgel 60, particle size 0.040-0.063 mm). Chromatographic separations were also performed on Biotage SP4 instrument. All reactions and products were monitored on thin layer chromatography (TLC) using Merck silica gel 60 F254. HPLC was used to determine the purity of the compounds (2487 Dual Wavelength Absorbance detector (Waters), 1525 binary HPLC pump, In-Line Degasser AF and Waters 717plus Autosampler), together with a Varian Pursuit XRs C18 reverse phase 150 × 4.6 mm chromatography column. Samples were detected using a wavelength of 254 nm. Details for the preparation of compounds 11a-11n, 11p-11r,  20a, 20f, 20g, 21a-j, 22a-c, 22e is contained in the Supplementary Information. Maleimides (11a-11s) To a solution of maleic anhydride (20 mmol) in diethyl ether (25 mL) was added the appropriate benzyl or aryl amine (20 mmol) in diethyl ether (10 mL). The reaction mixture was stirred under Figure 9. Summary of SAR for 9,10-dihydro-9,10-ethanoanthracenes.

Chemistry
All commercially available reagents were used without further purification. Solvents were dried prior to use; tetrahydrofuran (THF) by distillation from sodium/benzophenone under nitrogen, toluene was distilled from sodium, dichloromethane was distilled from calcium hydride. Melting points were recorded on a Gallenkamp SMP 11 melting point apparatus and are uncorrected. Infra-red (IR) spectra were obtained on a Perkin Elmer FT-IR Spectrum 100 spectrometer. 1 H and 13 C NMR spectra were obtained on a Bruker Avance DPX 400 spectrometer operating at 400.13 MHz, ( 1 H) and 100.61 MHz ( 13 C) at 20 • C in either CDCl 3 or DMSO-d 6 with appropriate solvent peaks as reference standards. Mass spectrometry (ESI-MS) was performed on a Micromass LCT instrument with mass measurement accuracies of <±5 ppm. Low resolution mass spectra (LRMS) were obtained on a Hewlett-Packard 5973 MSD GC-MS system. Preparative separations were performed using flash column chromatography on silica gel (Merck Kieselgel 60, particle size 0.040-0.063 mm). Chromatographic separations were also performed on Biotage SP4 instrument. All reactions and products were monitored on thin layer chromatography (TLC) using Merck silica gel 60 F254. HPLC was used to determine the purity of the compounds (2487 Dual Wavelength Absorbance detector (Waters), 1525 binary HPLC pump, In-Line Degasser AF and Waters 717plus Autosampler), together with a Varian Pursuit XRs C18 reverse phase  compounds 11a-11n, 11p-11r, 20a, 20f, 20g, 21a-j, 22a-c, 22e is contained in the Supplementary Information. (11a-11s) To a solution of maleic anhydride (20 mmol) in diethyl ether (25 mL) was added the appropriate benzyl or aryl amine (20 mmol) in diethyl ether (10 mL). The reaction mixture was stirred under reflux at 20 • C for 1 h. The precipitated solid was filtered and washed with diethyl ether. This solid was immediately used in the next step and placed in a conical flask containing sodium acetate (8.5 mmol, 0.7 g) and acetic anhydride (10 mL). The mixture was heated at 90 • C for 0.5 h, then poured over ice water (100 mL). The solid was filtered and recrystallised from ethanol.

General Procedure 2: Preparation of Nitrovinyl Anthracenes 12a-12f
To a solution of 9-anthraldehyde (2 g, 9.7 mmol) in the appropriate nitroalkane (nitromethane, nitroethane, nitropropane) (15 mL) was added piperidinium acetate (1.5 g, 10.3 mmol). (Piperidinium acetate was prepared from piperidine 6.6 mL and acetic acid 3 mL). The solution was heated at 90 • C for 1.5 h under nitrogen for 1 h, then cooled to room temperature and poured onto 100 mL of ice cold H 2 O. Following DCM extraction, the organic layers were combined, dried (Na 2 SO 4 ) and solvent removed. The product was recrystallised from an appropriate solvent.

(E)-9-(2-nitrovinyl)anthracene (12a)
Compound 12a was prepared from 9-anthraldehyde (9.7 mmol, 2 g) and nitromethane (15 mL) as outlined in the general procedure 2. The product was recrystallized from methanol and diethyl ether as red crystals 2. Compound 12b was prepared from 9-anthraldehyde (9.7 mmol, 2 g) and nitroethane (15 mL) following the method in the general procedure 2. The product was recrystallized from ethanol and diethyl ether as orange crystals, 1 Compound 12c was prepared from 9-anthraldehyde (9.7 mmol, 2 g) and nitropropane (15 mL) as described in the general procedure 2. The product was recrystallized from ethanol and diethyl ether as gold crystals, 1.

Molecular Modelling
Using MOE (Molecular Operating Environment) 2016.V8 [71], the structures of interest compounds 13j, 16a, 16b and 19a were flexibly aligned with maprotiline 1 for structural comparison. 13j, 16a, 16b and 19a (displayed as yellow in their respective overlays) were flexibly aligned with the lead compound Maprotiline 1 (cyan), Figure 8. The molecular structures were processed using the MMFF94s force field, commonly used for small molecule modelling. Flexible alignment was conducted on each compound at 1000 iterations per run. The chirality of the stereogenic centres of the compounds was not defined. Default parameters were utilised for other settings. The proposed alignments featured as the top ranked alignment of generated poses, ranked in order of ascending S score (flexible alignment score comprising of both molecular strain energy and configuration similarity inputs).

X-ray Crystallography
Data for samples 16c, 16j, 17l, 17n, 19a and 20a were collected on a Bruker APEX DUO Kappa system with Mo Kα (λ = 0.71073 Å). Samples were mounted on a MiTeGen microloop and data collected at 100(2) K using an Oxford Instruments Cobra low temperature device. Bruker APEX [72] software was used for collection and reducing data and determination of the space group. Structures were solved employing direct methods (XT [73]) and subsequently refined using least squares minimization procedures (XL [74]) in Olex2 [75]. SADABS [76] was used to apply absorption corrections. Details of the crystal data, data collection and refinement are presented in Table S1. The trimethoxyphenyl group are disordered in 17l. The phenyl ring was modelled in two positions with the ipso carbon constrained by EXYZ/EADP. Occupancies are 65:35%. Two of the methoxy groups were further disordered and modelled in three positions in total. O30/O30c were constrained to the same position using EXYZ/EADP with O30/C31 34%; O30c/C31c 31% and O30b/C31b 34% occupied and restrained with DFIX, SIMU. O32/C32 34%; O32c/C32c 32%; O32b/C32b 34% occupied and restrained with DFIX and SIMU. The disordered trimethoxyphenyl atoms were restrained by SIMU. In 17n, the diffuse contribution of approximately 4 MeOH molecules per unit cell have been removed from the overall scattering by using SQUEEZE/PLATON. [77] CCDC 1938150-1938155 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Materials
The DG-75 BL cell line was kindly provided by Dr. Dermot Walls (School of Biotechnology, Dublin City University, Ireland). The MUTU-I (c179) cell line was provided by Professor Martin Rowe, (Division of Cancer Studies, The University of Birmingham, Birmingham, UK). alamarBlue was obtained from BioSource, Belgium and Fetal Bovine Serum (FBS) was sourced from Invitrogen, U.K. RPMI 1640 medium, HEPES and sodium pyruvate were sourced from Biosciences, Ireland. Cell culture consumables were purchased from Greiner Bio-One Ltd., U.K., while all reagents used were obtained from Sigma-Aldrich, Arklow, Ireland.

Cell Culture
The DG-75 Burkitt's lymphoma cell line used in these experiments is a B-lymphocyte cell line which is derived from a metastatic pleural effusion (lung) isolated from a sporadic case of Burkitt's lymphoma. The MUTU-I (c179) cell line is an isogenic stable group I BL cell line derived from a BL biopsy. MUTU-I and chemoresistant DG-75 cell lines were cultured in RPMI 1640 (Glutamax)