Synthesis and Pro-Apoptotic Effects of Nitrovinylanthracenes and Related Compounds in Chronic Lymphocytic Leukaemia (CLL) and Burkitt’s Lymphoma (BL)

Chronic lymphocytic leukaemia (CLL) is a malignancy of the immune B lymphocyte cells and is the most common leukaemia diagnosed in developed countries. In this paper, we report the synthesis and antiproliferative effects of a series of (E)-9-(2-nitrovinyl)anthracenes and related nitrostyrene compounds in CLL cell lines and also in Burkitt’s lymphoma (BL) cell lines, a rare form of non-Hodgkin’s immune B-cell lymphoma. The nitrostyrene scaffold was identified as a lead structure for the development of effective compounds targeting BL and CLL. The series of structurally diverse nitrostyrenes was synthesised via Henry–Knoevenagel condensation reactions. Single-crystal X-ray analysis confirmed the structure of (E)-9-chloro-10-(2-nitrobut-1-en-1-yl)anthracene (19f) and the related 4-(anthracen-9-yl)-1H-1,2,3-triazole (30a). The (E)-9-(2-nitrovinyl)anthracenes 19a, 19g and 19i–19m were found to elicit potent antiproliferative effects in both BL cell lines EBV−MUTU-1 (chemosensitive) and EBV+ DG-75 (chemoresistant) with >90% inhibition at 10 μM. Selected (E)-9-(2-nitrovinyl)anthracenes demonstrated potent antiproliferative activity in CLL cell lines, with IC50 values of 0.17 μM (HG-3) and 1.3 μM (PGA-1) for compound 19g. The pro-apoptotic effects of the most potent compounds 19a, 19g, 19i, 19l and 19m were demonstrated in both CLL cell lines HG-3 and PGA-1. The (E)-nitrostyrene and (E)-9-(2-nitrovinyl)anthracene series of compounds offer potential for further development as novel chemotherapeutics for CLL.


Introduction
Chronic lymphocytic leukaemia (CLL) is a B-cell malignancy and is the most common form of leukaemia of the adult population in developed countries, accounting for 25% of all cases of leukaemia and 1.3% of all cancers [1].CLL is largely a disease of the elderly [2,3], with greater than 70% of patients aged over 65 years at time of clinical diagnosis.However, the disease is now increasingly common in younger patients [4].Ireland (along with Australia, Italy and the US) shows the highest incidence rates for CLL worldwide [5] of approximately 4.5 per 100,000 in males and 2 per 100,000 in females [6][7][8].CLL is a clinically heterogeneous lymphoproliferative disorder characterised by the clonal expansion of CD5 + mature B-lymphocytes, usually involving the bone marrow, spleen, lymph nodes and peripheral blood [9][10][11].The disease is clinically classified according to the mutational status of the immunoglobulin heavy chain gene (IGVH); CLL patients with mutated IGVH (M-IGVH) usually have an indolent form of the disease, while unmutated IGVH (UM-IGVH) is associated with a more aggressive disease course [12].The confirmation of a clonal population of B cells greater than 5000/mL of blood is diagnostic in most cases of CLL together with the expression of surface CD5 and CD23.Genomic and molecular markers are useful in assessing CLL prognosis; TP53 mutations, UM-IGVH, del(17p) and del(11q), together with complex karyotype, are associated with a poor prognosis.A favorable CLL prognosis is associated with del(13q), while normal karyotype and trisomy 12 are regarded as intermediate prognostic factors for CLL.A number of emerging prognostic markers for CLL are now identified, including mutations in Notch receptor 1 (NOTCH1), splicing factor 3B subunit 1 (SF3B1), baculoviral IAP repeat-containing 3 (BIRC3) and ATM serine/threonine kinase (ATM) [13][14][15].
Chemotherapeutic drugs used for CLL include the cytostatic nucleoside prodrug fludarabine phosphate 1 (Figure 1) and the alkylating agents cyclophosphamide, chlorambucil and bendamustine [16]; however, a number of very effective oral targeted therapies (such as ibrutinib 2, idelalisib 3 and venetoclax 4) are now available.Ibrutinib 2, a Bruton's tyrosine kinase (BTK) inhibitor approved for CLL [17], forms a covalent bond with cysteine residue Cys481 at the ATP binding site of BTK.Idelalisib 3 is a phosphoinositide 3-kinase δ (PI3Kδ) inhibitor, which inhibits B-cell receptor signalling and is approved for the treatment of relapsed CLL [18] and mantle cell lymphoma [19].Venetoclax 4 is a highly potent, orally bioavailable selective inhibitor of the anti-apoptotic B-cell lymphoma-2 protein (Bcl-2) [20].Many non-covalent BTK inhibitors have been reported to be effective in CLL and multiple other B-cell malignancies, e.g., GDC-0853 [21] and pirtobrutinib 5 [22].The second-generation BTK inhibitor acalabrutinib 6 forms a covalent bond with the key cysteine residue (Cys481) of BTK, resulting in inhibition of BTK.[23] Zanubrutinib 7, a nextgeneration BTK inhibitor, was approved by the FDA in 2023 for treatment of patients with CLL or small lymphocytic lymphoma (SLL) [24].Immunotherapies such as the anti-CD20 monoclonal antibodies rituximab, obinutuzumab, ofatumumab and the anti-CD52 antibody alemtuzumab have proven successful in treating CLL [16,25,26].Richter's transformation (RT) of CLL to an aggressive B-cell lymphoma is a complication of CLL; however, the introduction of the PD-1-blocking antibodies pembrolizumab and nivolumab [27] show selective efficacy in CLL patients with RT [28].
Despite the recent advancements in the targeted clinical treatment of CLL, there still remains an urgent requirement for the discovery and development of novel therapeutic agents to combat acquired disease resistance as opposed to maintenance alone [29].Examples of preclinical development of CLL-directed small-molecule therapies include the novel tubulin-targeting agent pyrrolo-1,5-benzoxazepine-15 (PBOX-15), which induces apoptosis in poor prognostic subgroups of CLL patients [30], while 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) inhibitors have demonstrated efficacy in primary chronic CLL cells [31].
Burkitt's lymphoma (BL) is a rare, aggressive non-Hodgkin's lymphoma (NHL) that affects B-lymphocytes.The endemic form has a high incidence in equatorial Africa [32], whereas the sporadic form is identified in 1-2% of adult lymphomas globally and up to 40% of pediatric lymphomas in US and western Europe [33].Treatments for BL include a combination of rituximab with chemotherapeutics such as vincristine, methotrexate, doxorubicin and cyclophosphamide [34,35].With increased reports of immunodeficient HIV-linked BL [36] and the association of endemic BL with Epstein-Barr virus (EBV) [37], selective and potent treatments for BL are required as prognosis for relapsed BL is extremely poor [38].Burkitt's lymphoma (BL) is a rare, aggressive non-Hodgkin's lymphoma (NHL) that affects B-lymphocytes.The endemic form has a high incidence in equatorial Africa [32], whereas the sporadic form is identified in 1-2% of adult lymphomas globally and up to 40% of pediatric lymphomas in US and western Europe [33].Treatments for BL include a combination of rituximab with chemotherapeutics such as vincristine, methotrexate, doxorubicin and cyclophosphamide [34,35].With increased reports of immunodeficient HIVlinked BL [36] and the association of endemic BL with Epstein-Barr virus (EBV) [37], selective and potent treatments for BL are required as prognosis for relapsed BL is extremely poor [38].
Molecules 2023, 28, x FOR PEER REVIEW 4 of 43 potent antiproliferative agent in the BL cell lines MUTU-I (IC50 = 3 µM) and DG-75 (IC50 = 1.5 µM), which induced apoptosis in both BL cell lines [41,42].The (E)-(2-nitrovinyl)benzene pharmacophore was thus identified as a scaffold that has demonstrated relevant anticancer activity [45].The biological macromolecular target(s) of these compounds, driving the antiproliferative response, is as yet unknown, so we focussed our work on phenotypic cellular responses.We wished to investigate the preclinical potential of a panel of related (E)-9-(2-nitrovinyl)anthracenes as antiproliferative compounds in CLL, which is a more common but related B-cell malignancy.Nitro-group-containing compounds may induce selective cancer cell toxicity by diverse mechanisms [46] such as topoisomerase inhibition [47], histone deacetylase inhibition [48], DNA alkylation [49] or tubulin polymerisation inhibition [50][51][52], while anthracene-containing compounds are reported to interact selectively with G-quadruplex structures and inhibit telomerase [53].The objective of this research was the investigation of a series of (E)-9-(2-nitrovinyl)anthracenes and related nitrostyrene compounds for antiprolifertive evaluation in BL and CLL cell lines and is arranged as follows: i.
The synthesis of a panel of nitrovinylanthracenes for initial BL evaluation to optimise the core structure for further CLL investigation; ii.
The identification of a focussed panel of nitrostyrenes to confirm the efficacy of nitrostyrene pharmacophore in CLL; iii.
The evaluation and optimisation of the antiproliferative activity of the selected nitrovinylanthracenes in CLL and related cell lines; iv.
The determination of the pro-apoptotic effects of nitrovinylanthracenes in CLL cells.In this work, we initially prepared a series of novel halogenated nitrostyrenes for evaluation in BL to confirm the requirement of the nitrostyrene pharmacophore for antiproliferative activity in CLL, based on our previous investigations [39].Subsequently, a series of substituted 9-anthraldehydes were synthesised from the appropriate anthracenes, anthrones and anthraquinones including chloro, bromo, methyl, phenyl, methoxy and isopropyl substitutions at C-10, from which the required (E)-9-(2-nitrovinyl)anthracenes were synthesised.The reduction of (E)-9-(2-nitrovinyl)anthracene was also investi- The nitrostyrene-containing compounds such as 10a-c reduced cell viability effectively in both BL and CLL cell lines and were superior to the clinical drugs fludarabine phosphate and taxol [39,45].The IC 50 values in the CLL cell lines were in the low-micromolar range (2-5 µM) irrespective of IGVH mutational status (I83 and PGA-1: mutated-IGVH; HG-3 and CII: unmutated-IGVH).This result compared favourably with IC 50 values of 20-50 µM obtained for fludarabine phosphate (a current CLL frontline treatment) in these cell lines.We also identified the related (E)-9-(2-nitrovinyl)anthracene 19a as a potent antiproliferative agent in the BL cell lines MUTU-I (IC 50 = 3 µM) and DG-75 (IC 50 = 1.5 µM), which induced apoptosis in both BL cell lines [41,42].The (E)-(2-nitrovinyl)benzene pharmacophore was thus identified as a scaffold that has demonstrated relevant anticancer activity [45].The biological macromolecular target(s) of these compounds, driving the antiproliferative response, is as yet unknown, so we focussed our work on phenotypic cellular responses.We wished to investigate the preclinical potential of a panel of related (E)-9-(2-nitrovinyl)anthracenes as antiproliferative compounds in CLL, which is a more common but related B-cell malignancy.Nitro-group-containing compounds may induce selective cancer cell toxicity by diverse mechanisms [46] such as topoisomerase inhibition [47], histone deacetylase inhibition [48], DNA alkylation [49] or tubulin polymerisation inhibition [50][51][52], while anthracene-containing compounds are reported to interact selectively with G-quadruplex structures and inhibit telomerase [53].
The objective of this research was the investigation of a series of (E)-9-(2-nitrovinyl)anthracenes and related nitrostyrene compounds for antiprolifertive evaluation in BL and CLL cell lines and is arranged as follows: i.
The synthesis of a panel of nitrovinylanthracenes for initial BL evaluation to optimise the core structure for further CLL investigation; ii.The identification of a focussed panel of nitrostyrenes to confirm the efficacy of nitrostyrene pharmacophore in CLL; iii.The evaluation and optimisation of the antiproliferative activity of the selected nitrovinylanthracenes in CLL and related cell lines; iv.The determination of the pro-apoptotic effects of nitrovinylanthracenes in CLL cells.
In this work, we initially prepared a series of novel halogenated nitrostyrenes for evaluation in BL to confirm the requirement of the nitrostyrene pharmacophore for antiproliferative activity in CLL, based on our previous investigations [39].Subsequently, a series of substituted 9-anthraldehydes were synthesised from the appropriate anthracenes, anthrones and anthraquinones including chloro, bromo, methyl, phenyl, methoxy and isopropyl substitutions at C-10, from which the required (E)-9-(2-nitrovinyl)anthracenes were synthesised.The reduction of (E)-9-(2-nitrovinyl)anthracene was also investigated together with inclusion of the phenanthrene system and the extension of carbon chain between the nitrovinyl unit and the anthracene ring.Other unsaturated systems introduced at C-9 include oximes, cyanovinyl and nitrone systems to assess their effect on the activity of the series.The target substituted anthracene structures identified for investigation are summarised in Figure 3.
Molecules 2023, 28, x FOR PEER REVIEW 5 of 43 the activity of the series.The target substituted anthracene structures identified for investigation are summarised in Figure 3.
A series of diversely substituted (E)-9-(2-nitrovinyl)anthracenes were prepared from the substituted-9-anthraldehyde library described above, together with some related commercially available anthracene aldehydes (Scheme 3).A piperidine-catalysed Henry-Knoevenagel condensation reaction was utilised to obtain the panel of (E)-9-(2-nitrovinyl)anthracenes 19a-m (piperidine acetate, excess nitroalkane, 1.5 h 90 • C) with a significant increase in the product yield (up to 99%) when compared with the alternative method (cyclohexylamine, acetic acid, excess nitroalkane, 20 min, MW) [62] and was particularly useful for preparation of 19c.The yields for compounds 19a, 19b and 19c for the piperidine-catalysed method were 99%, 73% and 60% compared with 25%, 10% and trace, respectively, for the microwave method.The (E)-9-(2-nitrovinyl)anthracenes 19a-m included a range of C-9 and C-10 halogen, alkyl and aryl substitutions and the previously unreported compounds 19h-m.In the 1 H NMR spectrum of 19f, the downfield singlet occurring at δ 8.49 ppm was characteristic of H-1 ′ , the proton on C-1 ′ of the nitrovinyl group.In the 13 C NMR spectrum, the signal at 130.1 ppm was allocated to the nitrovinyl C-1 ′ , while the quaternary signal at 157.0 ppm was assigned to C-2 ′ due to the adjacent electron-withdrawing nitro group (Supplementary Information, Figures S6-S8).

In Vitro Antiproliferative Activity of Nitrosytrenes, Nitrovinylanthracenes and Related Compounds in Burkitt Lymphoma
A panel of 58 anthracene-based compounds were initially evaluated for anti-proliferative activity in the Burkitt lymphoma EBV -MUTU-I (chemosensitive) and EBV + DG-75 (chemoresistant) cell lines at 10 µM and 1 µM using an alamarBlue viability assay to determine the structure-activity relationships for these anthracene compounds and to identify the most potent compounds for further investigation.We previously reported the antiproliferative effects of a panel of nitrovinylstyrenes in the BL MUTU-I and DG-75 cell lines X-ray crystallography confirmed the structure of 4-(anthracen-9-yl)-1H-1,2,3-triazole 30a, (Figure 5 and Supplementary Information, Figures S22 and S23).The triazole bond lengths and angles were within the reported ranges [73].To assess the antiproliferative effect of alternative isosteric imine systems to replace the nitrostyrene, the anthracen-9-ylmethanimines 32a-d were prepared by reaction of 9anthraldehyde, 9-chloroanthraldehyde or 2-(anthracen-9-yl)acetaldehyde 18 with the appropriate amine in yields of 85-90%, (Scheme 5).A series of anthracene amides 33a-f were obtained by coupling anthracene-9-carboxylic acid and selected amines using the Mukaiyama reaction (75-90%), as shown in Scheme 5.In the 1 H NMR spectrum of the novel anthracen-9-yl(azepan-1-yl)methanone 33c, a triplet at δ 3.09 (J = 6.1 Hz) and a multiplet (δ 4.02) were assigned to the C1′ and C6′ methylene protons due to the non-equivalent nature of these homopiperidine protons, with signals at 45.1 ppm and 49.0 ppm in the 13 C NMR spectrum assigned to C1′ and C6′ (see Supplementary Information Figures S19 and  S20).
In total, a panel of 58 anthracene-based compounds and 12 halo-substituted nitrostyrenes were designed and synthesised for this study.Reaction of a number of substituted anthraldehydes with the required nitroalkanes in a Henry-Knoevenagel condensation reaction afforded a series of 16 substituted (E)-9-(2-nitrovinyl)anthracenes and phenanthrenes.Anthracene ring substitutions included methoxy, isopropy, alkyl (methyl, ethyl), phenyl and halogen (chlorine and bromine).The alkyl substituent at C-2 was varied by using different nitroalkanes (nitromethane, nitroethane and nitropropane).Extension of the carbon linker between the vinyl unit and the anthracene moiety was also achieved using a Wittig reaction.Related vinyl functionalities were introduced on the anthracene including cyanovinyl, oxime, hydrazone and alkyl analogues, together with a selection of amines and amides.The compounds synthesised were initially screened for biological activity in the BL cell lines DG-75 and MUTU-I with subsequent screening of the more potent compounds in the CLL cell lines PGA-1 and HG-3.The results of this preliminary screen are discussed in the following section.To assess the antiproliferative effect of alternative isosteric imine systems to replace the nitrostyrene, the anthracen-9-ylmethanimines 32a-d were prepared by reaction of 9-anthraldehyde, 9-chloroanthraldehyde or 2-(anthracen-9-yl)acetaldehyde 18 with the appropriate amine in yields of 85-90%, (Scheme 5).A series of anthracene amides 33a-f were obtained by coupling anthracene-9-carboxylic acid and selected amines using the Mukaiyama reaction (75-90%), as shown in Scheme 5.In the 1 H NMR spectrum of the novel anthracen-9-yl(azepan-1-yl)methanone 33c, a triplet at δ 3.09 (J = 6.1 Hz) and a multiplet (δ 4.02) were assigned to the C 1 ′ and C 6 ′ methylene protons due to the non-equivalent nature of these homopiperidine protons, with signals at 45.1 ppm and 49.0 ppm in the 13 C NMR spectrum assigned to C 1 ′ and C 6 ′ (see Supplementary Information Figures S19 and S20).In total, a panel of 58 anthracene-based compounds and 12 halo-substituted nitrostyrenes were designed and synthesised for this study.Reaction of a number of substituted anthraldehydes with the required nitroalkanes in a Henry-Knoevenagel condensation reaction afforded a series of 16 substituted (E)-9-(2-nitrovinyl)anthracenes and phenanthrenes.Anthracene ring substitutions included methoxy, isopropy, alkyl (methyl, ethyl), phenyl and halogen (chlorine and bromine).The alkyl substituent at C-2 was varied by using different nitroalkanes (nitromethane, nitroethane and nitropropane).Extension of the carbon linker between the vinyl unit and the anthracene moiety was also achieved using a Wittig reaction.Related vinyl functionalities were introduced on the anthracene including cyanovinyl, oxime, hydrazone and alkyl analogues, together with a selection of amines and amides.The compounds synthesised were initially screened for biological activity in the BL cell lines DG-75 and MUTU-I with subsequent screening of the more potent compounds in the CLL cell lines PGA-1 and HG-3.The results of this preliminary screen are discussed in the following section.

In Vitro Antiproliferative Activity of Nitrosytrenes, Nitrovinylanthracenes and Related Compounds in Burkitt Lymphoma
A panel of 58 anthracene-based compounds were initially evaluated for anti-proliferative activity in the Burkitt lymphoma EBV − MUTU-I (chemosensitive) and EBV + DG-75 (chemoresistant) cell lines at 10 µM and 1 µM using an alamarBlue viability assay to determine the structure-activity relationships for these anthracene compounds and to identify the most potent compounds for further investigation.We previously reported the anti-proliferative effects of a panel of nitrovinylstyrenes in the BL MUTU-I and DG-75 cell lines and identified halogenated compounds 11c, 11f-l with cell viability at 10 µM in the range 2-26% and 0-16%, respectively, and with IC 50 values in the range 0.82-2.18µM (MUTU-I) and 2.05-3.11µM (DG-75) [39] (Supplementary Information Table S7), suggesting that the nitrovinylstyrene pharmacophore may be suitable for further study.In this work, the antiproliferative activity of the more potent selected nitrostyrene compounds 11i, 11h, 11g and 11j was further investigated in the following BL cell lines: Ramos (BL, EBV-negative) and Bjab (BL, EBV-negative) together with HeLa (cervical), MCF-7 (ER-positive breast cancer) and HL-60 (promyelocytic leukaemia) cell lines (Table 2).The compounds elicited good anti-proliferative effects at a 10 µM concentration in all cell lines, e.g., 4.4-13.9%viability in the BL Ramos and 6.5-16.55% in the leukaemia HL60 cell line, exerting a more potent effect than taxol in all of these cell lines, apart from MUTU-I.(E)-1-Chloro-2-(2-nitrobut-1-en-1-yl)benzene 11j was particularly potent at 10 µM in the Ramos BL cell line (4.4% cell viability) and 9.1% viability in the HL60 cell line.

Effect of Nitrostyrene 11h on the Viability of PBMCs
The nitrostyrene 11h was evaluated for its cytotoxic effect on healthy donor peripheral blood mononuclear cells (PBMCs) to determine the selective toxicity of compounds containing the nitrostyrene pharmacophore on malignant BL cell lines over normal blood cells.Compound 11h was evaluated at 1 µM and 10 µM concentrations over a 24 h treatment time (Supplementary Information, Table S8).Compound 11h demonstrated a low toxicity in PBMCs at 1 µM (74% viable cells remaining).In comparison, compound 11h induced a significant anti-proliferative effect in MUTU-I cells, with 39.8% viable cells remaining at 1 µM.A similar response was observed in DG-75 cells at the higher concentration (10 µM); a potent anti-proliferative effect (0.038% viable cells remaining) was observed, in comparison to 34.1% of viable PBMCs, indicating that compound 11h is selectively toxic to these BL cell lines.

Effect of Pre-Treatment with N-Acetylcysteine and Caspase Inhibitor Z-VAD-FMK on Induction of Apoptosis by Compound 11h
Additional annexin V/PI FACS analysis was carried out in the presence of a reactive oxygen species (ROS) inhibitor (N-acetylcysteine) and a pan-caspase inhibitor (Z-VAD-FMK) in order to study their effects (if any) on the pro-apoptotic effects of 11h (Figure 6).In the presence of NAC (5 µM), the apoptosis induced by compound 11h decreased from 68% to 21% at 2.5 µM and from 88% to 33% at 5 µM in the PGA1 cell line.Similar results were obtained in the HG-3 cell line at 2.5 µM (90% to 31%) and 5 µM of 11h (84% to 51%).In the presence of the caspase inhibitor Z-VAD-FMK (5 µM), the apoptosis induced by compound 11h decreased from 63% to 25% at 2.5 µM and from 79% to 43% at 5 µM in the PGA1 cell line.Similar results were obtained in the HG-3 cell line at 2.5 µM (84% to 29%) and 5 µM (74% to 50%).These findings indicate that both caspases and ROS may be involved in the mechanism of apoptosis for compound 11h.
Molecules 2023, 28, x FOR PEER REVIEW 15 of 43 PGA1 cell line.Similar results were obtained in the HG-3 cell line at 2.5 µM (84% to 29%) and 5 µM (74% to 50%).These findings indicate that both caspases and ROS may be involved in the mechanism of apoptosis for compound 11h.

In Vitro Antiproliferative Activity of the Nitrovinylanthracenes and Related Compounds in Burkitt's Lymphoma
As a further development, the anticancer effects of the panel of nitrovinylanthracene and related compounds synthesised together with the C-9 substituted anthracenes such as amines, carboxamides, cyanovinyl and hydrazone derivatives were investigated.The effects of additional C-10 substitution (e.g., alkyl, alkoxy, halogen, aryl) on the anti-proliferative effects of the anthracene compounds were also evaluated and are discussed by structural type.The results obtained from this preliminary screen in the MUTU-I and DG-

In Vitro Antiproliferative Activity of the Nitrovinylanthracenes and Related Compounds in Burkitt's Lymphoma
As a further development, the anticancer effects of the panel of nitrovinylanthracene and related compounds synthesised together with the C-9 substituted anthracenes such as amines, carboxamides, cyanovinyl and hydrazone derivatives were investigated.The effects of additional C-10 substitution (e.g., alkyl, alkoxy, halogen, aryl) on the anti-proliferative effects of the anthracene compounds were also evaluated and are discussed by structural type.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 Figures 6 and 7, with maprotiline and taxol used as the positive controls.Maprotiline induced a modest anti-proliferative effect at 10 µM in the MUTU-I and DG-75 BL cell lines (72% and 65% viable cells, respectively).Treatment with taxol resulted in a 7% cell viability at 10 µM and 32% at 1 µM in MUTU-I cell line, while a 40% and >90% cell viability was observed at 10 and 1 µM treatment concentrations, respectively, in the more chemoresistent DG-75 cells.The lead nitrovinylanthracene compound 19a (IC 50 8A,B) and anthracene amides 33a-f (Figure 8A,B) exhibited poor antiproliferative effects in the MUTU-I and DG-75 cell lines at 10 µM (>80% and >60% viability, respectively), indicating the requirement of the nitrovinyl functionality for activity.The 4-(anthracen-9-yl)-1H-1,2,3-triazoles 30a, 30b and 31 with a constrained (E)-configuration for the vinyl system and compounds 24 and 25 with alternative vinyl functionalities were inactive in both the BL cell lines (>90% viability) (Figure 7A,B).However, the C-9 dicyanovinyl compound 23b (synthesised to evaluate the effects of alternative substituents at the C-2 carbon of the vinyl unit) was effective at 10 µM in the MUTU-I cell line (~20% viability) with moderate activity at 10 µM in the DG-75 cell line (~55%) Figure 7A,B).(See Supplementary Information, Tables S3-S6 for complete cell viability data for all compounds).

Physicochemical, ADME, Pharmacokinetic and Stability Properties of (E)-9-(2-Nitrovinyl)Anthracenes and Related Compounds
The physicochemical, ADME and pharmacokinetic properties of the most potent synthesised compounds (19a-19m, 20b, 21, 22, 23a, 23b) were initially investigated using Pipeline Pilot Professional [74] (see Supplementary Information for details of the Tier 1 profiling screen, Tables S1 and S2).These anthracene compounds complied with Lipinski and Veber rules with a molecular weight less of than 500 Da (within the range of 249-328 Da) and with fewer than 10 rotatable bonds, fewer than 10 hydrogen bond acceptors, fewer than 5 hydrogen bond donors and a logP of less than 5 (in the range 2.68-4.00)(Supplementary Information, Table S2).The topological polar surface area (TPSA) of the compounds was found in the range 45.82-47.58Å 2 , within the desirable limit of 140 Å 2 for good intestinal absorption.The compounds were predicted not to inhibit CYP2D6, while high blood-brain barrier (BBB) absorption levels and good plasma protein binding properties (greater than 90%) were predicted for all compounds (Supplementary Information, Tables S1 and S2).
The synthesised compounds 19a-19m, 20b, 21, 23a and 23b are predicted to be un-ionised at physiological pH, with the theoretical pKaH value for compound 22 calculated with a Marvin of 8.22.However, low aqueous solubility is predicted for the panel of compounds in the range logSw = −7.0840 to −5.3960, e.g., the 10-methoxy compound 19g is predicted with greatest solubility in the series (logSw = −5.3960mol/L) (see Supplementary Information, Table S1).The nitrovinylanthracene compounds 19a-19m, 20b, 21, 22, 23a and 23b were not signalled in a filter for pan-assay interference compounds (PAINS) [75] and are predicted to have good drug-like physicochemical properties within the appropriate range for oral bioavailability [76,77].Additional biochemical studies are described in the following sections to determine their mechanism of action.

Physicochemical, ADME, Pharmacokinetic and Stability Properties of (E)-9-(2-Nitrovinyl)Anthracenes and Related Compounds
The physicochemical, ADME and pharmacokinetic properties of the most potent synthesised compounds (19a-19m, 20b, 21, 22, 23a, 23b) were initially investigated using Pipeline Pilot Professional [74] (see supplementary information for details of the Tier 1 profiling screen, Tables S1 and S2).These anthracene compounds complied with Lipinski and Veber rules with a molecular weight less of than 500 Da (within the range of 249-328 A preliminary HPLC stability study was performed on a representative nitrovinylanthracene compound 19m (isopropyl) in various biologically relevant pH systems (acidic pH 4 found in the stomach, basic pH 9 found in the intestine and pH 7.4 in the plasma).The half-life (t 1⁄2 ) was determined to be 19 h at pH 9 (42% remaining at 24 h) and greater than 24 h at both pH 4 and pH 7.4, with 55% and 56% remaining, respectively.Based on the results of this stability study, the compound 19m was determined to be suitable for further preclinical investigation.The panel of nitrostyrenes and anthracene-based maprotiline analogues was next evaluated for in vitro anti-proliferative activity in CLL.The HG-3 cell line was established from an in vitro EBV (Epstein-Barr virus) infection from an IGVH1-2 unmutated B1 lymphocyte origin CLL patient clone and is representative of poor patient prognosis [78].The PGA-1 cell line is a cell line that was established from leukemic B cells of a Caucasian male with CLL with a mutated IGVH1-2 and is representative of good patient prognosis [79].Fludarabine phosphate was used as a comparative control for CLL cell lines [45] (IC 50 values of 28.1 µM and 32.0 µM in HG-3 and PGA-1 cell lines, respectively, with cell viability of HG-3 60%, PGA-1 65% at 10 µM concentration).From our previous work, we have identified a number of nitrostyrene-containing compounds demonstrating antiproliferative activity in BL cells, e.g., compounds (10a-c), with IC 50 values of 0.45, 0.47 and 2.97 µM in MUTU-I and IC 50 values of 1.41, 1.92 and 6.39 µM in DG-75, respectively, while anthracene (19a) also demonstrated activity in BL cell lines with IC 50 values of 3.0 µM (MUTU) and 1.5 µM (DG75) [41], suggesting that the nitrostyrene pharmacophore is relevant in the antiproliferative activity of the series.

Antiproliferative Activity of Nitrostyrenes in HG-3 and PGA-1 CLL Cell Lines
The halogenated nitrostyrenes 11a-l were initially screened in CLL cells PGA-1 and HG-3 at 1 and 10 µM together with lead nitrostyrene compound 10a from our previous study in BL.All compounds displayed low PGA1 and HG3 viability at 10 µM, while the control drug fludarabine 1 demonstrated 89% (HG-3) and 94% (PGA-1) viability at 10 µM.
The most potent compounds from the series 19a, 19g, 19i, 19l and 19m were chosen
The most potent compounds from the series 19a, 19g, 19i, 19l and 19m were chosen for IC 50 determination and evaluation in the CLL cell lines PGA-1 and HG-3.

In Vitro IC 50 Determination of the Most Potent (E)-9-(2-nitrovinyl)anthracene Derivatives in HG-3 Cells and PGA-1 Cells
The IC 50 values at 24 h for the selected (E)-9-(2-nitrovinyl)anthracene compounds 19a, 19g, 19i, 19l and 19m in HG-3 cells and PGA-1 cells were determined using a concentration range of 10 µM-0.01 µM (Table 3).The compounds demonstrated a more potent effect than the fludarabine control (5-40 fold greater in the HG-3 cells; 4-25 fold greater in the PGA-1 cells) across both the HG-3 and PGA-1 cell lines with IC 50 ranges of 0.70-3.85µM and 1.29-9.10µM, respectively.In the HG-3 cells, the most potent compounds identified were the 10-methoxy derivative 19g (IC 50 0.17 µM) and the 10-isopropyl derivative 19m (IC 50 0.70 µM), while in the PGA-1 cells, the most potent compounds were 19g (IC 50 1.29 µM) and the 10-ethyl derivative 19l (IC 50 1.30µM).These results suggest that alkyl and alkoxy substituents present at the C-10 position on the anthracene core can lead to a greater anti-proliferative activity in CLL compared to the unsubstituted 19a.The 10-isopropyl substituent of 19m was selective, causing a 13-fold greater response in the HG-3 as opposed to PGA-1 cells (0.7 µM vs. 9.1 µM, possibly due to increased lipophilic/steric effects) and approximately a 3.5-fold increased activity compared to 19a in both cell lines.Furthermore, 19g (10-methoxy) resulted in a 7.8-fold improvement in the IC 50 value in the HG-3 compared to PGA-1 cells and a similar activity to 19l (10-ethyl) in the PGA-1 cells (IC 50 = 1.29 µM).These results suggest the potential for similar, yet distinct, compound attributes for potent antiproliferative activity in the main two CLL disease cell subtypes.

In Vitro Antiproliferative Activity of Nitrovinylanthracenes in Estrogen-Receptor-Positive Breast Cancer Cell Lines MCF-7 and MDA-MB-231
Selected compounds were also evaluated in the estrogen-receptor-positive (ER+) breast cancer cell line MCF-7 and the triple-negative breast cancer (TNBC) cell line MDA-MB-231.TNBC accounts for 10-15% of breast cancers that do not express estrogen and progesterone receptors (ER/PR) and are HER2-negative.TNBCs are not responsive to hormone therapies, e.g., the selective estrogen receptor modulator tamoxifen, the aromatase inhibitor anastrozole or the monoclonal antibody herceptin, which targets the HER2 receptor (human epidermal growth factor receptor 2).Fewer treatment options are available for TNBC compared with ER+, PR+ and HER2+ breast cancers, and the outcome is uncertain [80].Five compounds were screened in MCF-7 and MDA-MB-231 breast cancer cells at 1 and 10 µM concentrations (Figure 11), and based on the results, the IC 50 values for three of the five compounds were determined (Table 4).The nitrovinylanthracenes 19a, 19g and 19i were found to display moderate antiproliferative activity in MCF-7 breast cancer cells with an IC 50 value of 1.85 µM for the most potent example 19a, which compared favourably with tamoxifen (IC 50 = 4.12 µM).The compounds also displayed a low micromolar activity when evaluated in the TNBC cell line MDA-MB-231 with IC 50 values in the range 3.26-3.82µM, suggesting that they were not selective for ER+ breast cancer cells.To examine the potential anti-proliferative effects of these nitrostyrene-type compounds, the ability of the most potent compounds identified from the cell viability study to induce apoptosis in the MUTU-I and DG-75 cell lines was investigated using Annexin V and propidium iodide staining at a 10 µM concentration.The chemotherapeutic drug taxol was used as a positive control.The pro-apoptotic effects of the selected nitrostyrene compounds 11g, 11h, 11i, 11j, 11k and 11l (10 µM) were determined using FITC (fluorescein isothiocyanate), Annexin V/PI (propidium iodide) staining and FACS (fluorescence-activated cell sorting) analysis to characterise the mode of cellular death induced by the synthesised compounds.Four populations were produced in this assay: Annexin-V-and PI-negative (Q4, healthy cells), Annexin-V-positive and PI-negative (Q3, early apoptosis), Annexin-Vand PI-positive (Q2, late apoptosis) and Annexin-V-negative and PI-positive (Q1, necrosis), which were easily identified and quantified.Apoptosis was assessed as % total apoptosis by a combination of early and late apoptosis (Q3 and Q2, respectively).In the MUTU-I cell line, the selected nitrostyrene compounds 11g, 11h, 11i, 11j, 11k and 11l demonstrated a significant increase in apoptosis (80-91%), with the most potent effect being observed for the 3-chloro compound 11k (91%) and was comparable to the effect induced by taxol (87%) at the same concentration (Table 5).In the DG-75 cell line, the 3-chloro compound 11h (10 µM) produced a significant increase in apoptosis (92%), while a significant increase in apoptosis (70-92%) in the DG-75 cells was observed upon treatment with the compounds 11g, 11h, 11i, 11j, 11k and 11l and compared favourablely with taxol (72.7%) at 10 µM.The identification of compounds that can induce apoptosis in cancer cells is required in the development of potential lead structures for anticancer drugs.The pro-apoptotic effects of the selected nitrovinylanthracene compounds 19a, 19g, 19i, 19l and 19m after 48 h were next determined using FITC (fluorescein isothiocyanate), Annexin V/PI (propidium iodide) staining and FACS (fluorescence-activated cell sorting) analysis to characterize the mode of cellular death induced.Apoptosis was assessed as above using the cell lines HG-3 and PGA-1 and using nitrovinylanthracene compound treatment concentrations of 1 µM and 10 µM.We previously reported that fludarabine phosphate (50 µM) induces an increase of 24.6% in apoptosis of cancer cells isolated from CLL patients [45].

Molecular Modelling
The designed 9-nitrovinylanthracenes 19a-m and related compounds are structurally related to maprotiline and may drive their cellular antiproliferative effect through a similar mechanism of action.In order to examine the structural similarity in more detail, all the compounds described in this work were overlaid on maprotiline using two separate but complementary methodologies.MOE flexible alignment [84] was used in our previous work [85] and is based on several similarity terms, such as hydrogen bond donor/acceptor, aromaticity and partial charge.A stochastic search procedure was used to flexibly align and superimpose similar functional groups in each molecule while sampling the full conformational flexibility of each structure.OpenEye fastROCS [86] is a GPU-based 3D shape similarity method that takes a low-energy conformation of maprotiline as the query molecule and aligns to it each conformation of the compounds in this paper by a solid body optimisation process to maximise the volume overlap.Both colour (feature) and shape similarity were measured with a Tanimoto score with a maximum (best) score of 1, and the overall overlay quality was giving by the Tanimoto combo (Tc) score, which is the sum of these two scores with a maximum (best) value of 2. All databases and reference structures are provided in the Supplementary Information as sdf or mdb files.

Molecular Modelling
The designed 9-nitrovinylanthracenes 19a-m and related compounds are structurally related to maprotiline and may drive their cellular antiproliferative effect through a similar mechanism of action.In order to examine the structural similarity in more detail, all the compounds described in this work were overlaid on maprotiline using two separate but complementary methodologies.MOE flexible alignment [84] was used in our previous work [85] and is based on several similarity terms, such as hydrogen bond donor/acceptor, aromaticity and partial charge.A stochastic search procedure was used to flexibly align and superimpose similar functional groups in each molecule while sampling the full conformational flexibility of each structure.OpenEye fastROCS [86] is a GPU-based 3D shape similarity method that takes a low-energy conformation of maprotiline as the query molecule and aligns to it each conformation of the compounds in this paper by a solid body optimisation process to maximise the volume overlap.Both colour (feature) and shape similarity were measured with a Tanimoto score with a maximum (best) score of 1, and the overall overlay quality was giving by the Tanimoto combo (Tc) score, which is the sum of these two scores with a maximum (best) value of 2. All databases and reference structures are provided in the Supplementary Information as sdf or mdb files.The overlay results obtained for all the compounds in this study are inconclusive but generally indicate that the compounds in this paper may have a similar mechanism of action as maprotiline (Supplementary Information, Tables S9 and S10).In the fastROCS study, considering the cell viability of the BL MUTU-1 cell lines treated at 10 µM, only 5 of the top 12 overlaid compounds decreased the cell viability by over 50% (Supplementary Information, Table S9).A similar result was obtained in the analysis of the CLL HG-3 cell viability.The MOE flexible alignment also demonstrated a lack of correlation between the lowest (best) scored compounds and those with the most promising cellular data, both for the CLL and BL cell lines (Supplementary Information, Tables S9 and S10).
A selection of the best (lowest)-scored overlaid structures for the most potent anthracene compounds 19a, 19g, 19i, 19l and 19m (displayed as green in their respective overlays) with the lead compound maprotiline 9 (pink) is provided in Table 6, together with the antiproliferative activity in CLL cells.Shared molecular features were clearly identified, e.g., the (E)-configuration nitrovinyl pharmacophore located at C-9 that overlays with the methylpropylamine-containing substituent of maprotiline, and the aromatic anthracene structure that overlays with the 9,10-dihydroanthracene core structure of maprotiline.A se-lection of the best-scored overlaid structures for the most potent nitrostyrene compounds 11c, 11d, 11j and 11k is provided in Table 7.The nitrostyrenes mapped closely to the cyclic core of maprotiline rather than to the central ring and along the sidechain as for the anthracenes; again, the correlation with cellular activity in CLL cell lines was not conclusive (See Supplementary Information, Tables S9 and S10).

Experimental Section
Uncorrected melting points were measured on a Gallenkamp apparatus.Infra-red (IR) spectra were recorded on a Perkin Elmer Spectrum FT-IR 100 spectrometer (Waltham, MA, USA). 1 H, 13 C and 19 F nuclear magnetic resonance spectra (NMR) were recorded at 27 • C on a Bruker DPX 400 spectrometer (Bruker UK Limited, Coventry, UK) (400.13MHz, 1 H; 100.61MHz, 13 C; 376.47 MHz, 19 F) in either CDCl 3 (internal standard tetramethylsilane (TMS)) or CD 3 OD or DMSO-d 6 .For CDCl 3 , 1 H-NMR spectra were assigned relative to the TMS peak at 0.00 ppm, and 13 C-NMR spectra were assigned relative to the middle CDCl 3 peak at 77.0 ppm.For CD 3 OD, 1 H and 13 C-NMR spectra were assigned relative to the center peaks of the CD 3 OD multiplets at 3.30 ppm and 49.00 ppm, respectively.Coupling constants are reported in Hertz.For 1 H-NMR assignments, chemical shifts are reported as the shift value (number of protons, description of absorption and coupling constant(s), where applicable).Electrospray ionisation mass spectrometry (ESI-MS) was performed in the positive ion mode on a liquid chromatography time-of-flight mass spectrometer (Micromass LCT, Waters Ltd., Manchester, UK).The samples were introduced to the ion source by an LC system (Waters Alliance 2795, Waters Corporation, Milford, MA, USA) in acetonitrile: water (60:40% v/v) at 200 µL/min.The capillary voltage of the mass spectrometer was at 3 kV.The sample cone (de-clustering) voltage was set at 40 V.For exact mass determination, the instrument was externally calibrated for the mass range from m/z 100 to m/z 1000.A lock (reference) mass (m/z 556.2771) was used.Mass measurement accuracies of <±5 ppm were obtained.R f values are quoted for thin-layer chromatography on silica gel Merck F-254 plates unless otherwise stated.Flash column chromatography was carried out on Merck Kieselgel 60 (particle size 0.040-0.063mm).Microwave experiments were carried out using a Discover CEM microwave synthesiser on the standard power setting (300 watts) unless otherwise stated.See Supplementary Information for preparation and characterisation of compounds previously reported: 11a-l, 13a-e, 14a, 14c-e, 14f-g, 15, 16, 18, 19a-g, 21, 23c, 24-28, 29a-d, 30a, 32a-e, 33a, 33d-f [39, 40,55,57,58,66,67,71,.

AlamarBlue Cell Viability Assay
Cells were seeded at a density of 2.5 × 10 4 cells/well (MCF-7, MDA-MB-231 cells), 1 × 10 4 cells/well (HL-60 cells), 1-5 × 10 4 cells per well (BL MUTU-I, DG-75 cells) and 2 × 10 5 cells/well [HG-3, PGA-1 (CLL cells)], Ramos, BJAB (BL cells) (200 µL per well), in 96-well plates.Cells were treated with the desired drug concentration for the appropriate time for each cell type, and the samples were incubated as required.alamarBlue (20 µL) was then added to each well and the samples further incubated in the dark at 37 • C for 4 h.Wells containing only reagent and cell culture medium in the absence of cells were used as blank controls.Ethanol was used as a vehicle, and cells were treated with 1% ethanol (v/v) in all experiments.The 96-well plates were analysed on a fluorescence plate reader (SpectraMax Gemini, Molecular Devices) (emission and excitement wavelengths of 590 nm and 544 nm, respectively), and the fluorescence was recorded.The decrease in cell viability was then calculated with reference to the vehicle samples (100% viability).EMEM medium with the addition of alamarBlue was used as a blank.Vehicle-treated cells were considered to be 100% viable, and the viabilities of each compound was calculated accordingly.The transformed data (Final Concentration = Log (Final Concentration)) was used to plot a non-linear, sigmoidal dose-response curve, and the concentration of drug resulting in a 50% reduction in cell survival (IC 50 values) was obtained using the software package Prism (GraphPad Software, Inc., La Jolla, CA, USA).Taxol (10 µM) was used as an internal standard and resulted in a 90% cytotoxicity in each of the cell lines.All biochemical assays were performed in triplicate on at least three independent occasions, and the mean values were determined.

Generation of Human Peripheral Blood Mononuclear Cells (PBMCs)
Peripheral blood was obtained from healthy donors (n = 2) after informed consent was received.The blood was then placed into a 50 mL falcon tube and diluted with an equal volume of phosphate-buffered saline (PBS).PBMCs were isolated using density gradient centrifugation using LymphoPrep as described previously [117] Approval for this study was obtained from the School of Pharmacy and Pharmaceutical Sciences Trinity College Dublin Research Ethics Committee (2020-06-01-MS).

Inhibitor Studies
The HG-3 and PGA-1 CLL cells (5 × 10 4 cells/mL) were pre-treated at 37 • C with either 5 mM N-acetylcysteine (NAC) for 1 h or 40 µM caspase inhibitor (Z-VAD-FMK; MBL International, Co., Woburn, MA, USA) for 4 h prior to treatment with (11 h at 5 µM) for 48 h.Annexin V/PI FACS analysis was then carried out as described above in Section 4.5.

X-ray Experimental Procedure
Data for 19f were measured on a Bruker D8 Quest ECO device using Mo Ka radiation (λ = 0.71073 Å) with an Oxford Cryostream low-temperature device, and data for 30a were collected on a Bruker APEX DUO device using Cu Kα radiation (λ = 1.54178Å).Each sample was mounted on a MiTeGen cryoloop, and data were collected at 100(2) K.The Bruker APEX [118] software was used to collect and reduce data.Absorption corrections were applied using SADABS [119].Structures were solved with the SHELXT structure solution program [120] using Intrinsic Phasing.All were refined using the least squares method on F 2 with SHELXL [121].All non-hydrogen atoms were refined anisotropically.Hydrogen atoms were assigned to calculated positions using a riding model with appropriately fixed isotropic thermal parameters.Molecular graphics were generated using OLEX2 [122].Crystal data, details of data collection and refinement are given in Table S1.
Compound 30a was a weakly diffracting sample, especially at high angle, and it had two independent molecules in the asymmetric unit.One complete anthracene-triazole molecule was disordered over two locations (50% occupancy) and modelled with displacement restraints (SIMU).
Crystallographic data for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos.2171050-2171051.Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).

Computational Overlay Study
For the MOE 2022.02work, all the compounds were opened in a database viewer.The compounds were washed with default values and explicit hydrogens were added.For each compound, MMFF94x partial charges were calculated, and each was minimised to a gradient of 0.001 kcal/mol/Å.The compounds were then overlaid individually on 3D structure of maprotiline using flexible alignment on MOE with default values.In the fastROCS [86] 3D similarity study, 200 conformations were generated for each compound using OpenEye Omega [84,123].This conformation file was then used to search for compound conformations that map to a low-energy conformation of maprotiline.The output hit list was sorted according to the combined Tanimoto scores.

Conclusions
The treatment of hematological malignancies such as CLL is evolving as the underlying mechanisms of disease are understood and new immune pathways are discovered.Patients with CLL do not need treatment with chemotherapy until they become symptomatic or display evidence of rapid progression of the disease.The drug treatments available for patients with CLL has improved considerably and now include very effective oral targeted therapies (such as ibrutinib 2, idelalisib 3 and venetoclax 4).While immunotherapies such as obinutuzumab have proven successful in treating CLL, more research is required to optimise the current chemotherapies and immunotherapies for blood cancers, the combination and sequencing of treatments and the development of personalised and targeted agents.Many recent developments have been reported in the targeted clinical treatment of CLL; however, the discovery of novel therapeutic agents that are designed to be effective in acquired disease resistance and provide a curative treatment rather than maintenance alone is required.
We investigated the synthesis, structure−activity relationship (SAR) studies and biological activity of a series of nitrostyrenes and nitrovinylanthracenes in a phenotypic drug discovery approach.Potential preclincial applications of the novel anthracene nitrostyrene compounds in BL and CLL (a more translational but related B-cell malignancy) were identified.A library of 58 anthracene-based compounds structurally related to maprotiline were initially evaluated for antiproliferative activity in the BL EBV − MUTU-I (chemosensitive) and EBV + DG-75 (chemoresistant) cell lines.The selected (E)-9-(2-nitrovinyl)anthracenes demonstrated potent antiproliferative activity in the CLL cell lines, with IC 50 values of 0.17 µM (HG-3) and 1.3 µM (PGA-1) for compound 19g, superior to the chemotherapeutic drug fludarabine with IC 50 values of 28.1 µM (HG-3) and 32.0 µM (PGA-1).The pro-apoptotic effects of the most potent compounds 19a, 19g, 19i, 19l and 19m were demonstrated in both the CLL cell lines.The mechanism of cell death in these cell lines was identified as apoptotic, and the lead compounds elicited significant apoptotic effects which were comparable to taxol in the BL cell lines MUTU-I and DG-75 and fludarabine in the CLL cell lines HG-3 and PGA-1.The nitroalkenes reported in this study are attractive substrates for Michael addition reactions with nucleophiles present in biological systems.A molecular modelling study demonstrated close correspondence between overlays of these compounds with maprotiline with shared molecular features; however, correlation with cellular activity was inconclusive.The (E) nitrostyrene and (E)-9-(2-nitrovinyl)anthracene series of compounds offer potential for further development as novel chemotherapeutics for chronic lymphocytic leukaemia (CLL) and suggest the suitability of this group of selected (E)-9-(2-nitrovinyl)anthracenes compounds for further preclinical development.

Figure 4 .
Figure 4. (A) Molecular structure of 19f.(B) Molecular structure of 19f with atomic displacement shown at 50% probability with only heteroatoms labelled.(C) π-π interaction orientation with a perpendicular distance of 3.4278 Å, with a slippage of 1.630 Å from the centroid of the central rings.

Figure 4 .
Figure 4. (A) Molecular structure of 19f.(B) Molecular structure of 19f with atomic displacement shown at 50% probability with only heteroatoms labelled.(C) π-π interaction orientation with a perpendicular distance of 3.4278 Å, with a slippage of 1.630 Å from the centroid of the central rings.

Figure 5 .
Figure 5. (A) Molecular structure of compound 30a.(B) Asymmetric unit of compound 30a showing two independent molecules, with one completely disordered.Atomic displacement shown at 50% probability and heteroatoms labelled only.(C) π-π interactions indicated by dashed lines to plane centroids in 30a with A = 4.1284 Å with slippage 2.230 Å and B = 3.4176 Å, slippage 1.944 Å.Similar interactions were seen for the disordered molecule.

Figure 5 .
Figure 5. (A) Molecular structure of compound 30a.(B) Asymmetric unit of compound 30a showing two independent molecules, with one completely disordered.Atomic displacement shown at 50% probability and heteroatoms labelled only.(C) π-π interactions indicated by dashed lines to plane centroids in 30a with A = 4.1284 Å with slippage 2.230 Å and B = 3.4176 Å, slippage 1.944 Å.Similar interactions were seen for the disordered molecule.

2. 6 .
Evaluation of In Vitro Antiproliferative Activity of Nitrostyrenes and Anthracene-Based Maprotiline Analogues in Chronic Lymphocytic Leukaemia (CLL)

Figure 9 .
Figure 9. Preliminary cell viability data for nitrostyrenes in CLL (A) HG-3 and (B) PGA-1 cells.Cell proliferation of HG-3 and PGA-1 cells was determined with an alamarBlue assay.Compound concentrations of either 1 µM or 10 µM for 24 h 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.(Map = maprotiline).Statistical significance was evaluated using the Kruskal-Wallis test.* Indicates a p value between 0.01 and 0.05; ** indicates a p value between 0.001 and 0.01; *** indicates a p value between 0.0001 and 0.001.

Figure 9 .
Figure 9. Preliminary cell viability data for nitrostyrenes in CLL (A) HG-3 and (B) PGA-1 cells.Cell proliferation of HG-3 and PGA-1 cells was determined with an alamarBlue assay.Compound concentrations of either 1 µM or 10 µM for 24 h 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.(Map = maprotiline).Statistical significance was evaluated using the Kruskal-Wallis test.* Indicates a p value between 0.01 and 0.05; ** indicates a p value between 0.001 and 0.01; *** indicates a p value between 0.0001 and 0.001.

Figure 10 .
Figure 10.Preliminary cell viability data for nitrovinylanthracenes and related compounds in CLL: (A) HG-3 cells (1 and 10 µM) and (B) PGA-1 cells (1 and 10 µM).Cell proliferation of HG-3 and PGA-1 cells was determined with an alamarBlue assay.Compound concentrations of either 1 µM or 10 µM for 24 h were used to treat the cells (in triplicate) with control wells containing vehicle ethanol (1% v/v).Map = maprotiline, Flu = fludarabine The mean value for three experiments is shown.Statistical significance was evaluated using the Kruskal-Wallis test.* Indicates a p value between 0.01 and 0.05; ** indicates a p value between 0.001 and 0.01.
are half-maximal inhibitory concentrations required to block the growth stimulation of MCF-7 and MDA-MB-231 human breast cancer cells.Treatment at eight different concentrations (0.001-50 µM) was used for the determination of the IC50 values for each compound.Values represent the mean ± SEM (error values × 10 −6 ) for three independent experiments performed in triplicate.bThe IC50 values for tamoxifen were in good agreement with the reported IC50 values in MCF-7 cells[81] and MDA-MB-231cells[82,83].

Figure 11 .
Figure 11.Antiproliferative effects of selected nitrovinylanthracenes 19a, 19g, 19i, 19l and 19m in MCF-7 and MDA-MB-231 human breast cancer cell lines.Cell proliferation of MCF-7 and MDA-MB-231 cells was determined with an alamarBlue assay.Compound concentrations of either 1 µM or 10 µM for 72 h (MCF-7 and MDA-MB-231) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol (1% v/v).Values represent the mean ± SEM for three independent

Figure 11 .Table 4 .
Figure 11.Antiproliferative effects of selected nitrovinylanthracenes 19a, 19g, 19i, 19l and 19m in MCF-7 and MDA-MB-231 human breast cancer cell lines.Cell proliferation of MCF-7 and MDA-MB-231 cells was determined with an alamarBlue assay.Compound concentrations of either 1 µM or 10 µM for 72 h (MCF-7 and MDA-MB-231) were used to treat the cells (in triplicate) with control wells containing vehicle ethanol (1% v/v).Values represent the mean ± SEM for three independent experiments performed in triplicate.Statistical significance was evaluated using the Kruskal-Wallis test.* Indicates a p value between 0.01 and 0.05; ** indicates a p value between 0.001 and 0.01.Table 4. Antiproliferative effects of selected nitrovinylanthracenes 19a, 19g and 19i in MCF-7 and MDA-MB-231 human breast cancer cell lines.

Figure 13 .
Figure 13.Compounds 19a and 19m induced cell apoptosis in HG-3 and PGA-1 leukaemia cells.HG-3 and PGA-1 leukaemia cells were treated with 19a and 19m (10 and 1.0 µM) and control vehicle (1% DMSO (v/v)) at 48 h.(A) Compound 19a (10 µM concentration) treatment in HG-3 cells; (B) compound 19a (1 µM concentration) treatment in HG-3 cells; (C) HG-3 cells treatment with vehicle 1% DMSO; (D) compound 19m (10 µM concentration) in PGA-1 cells; (E) compound 19m (1 µM concentration) in PGA-1 cells; (F) PGA-1 cells treatment with vehicle 1% DMSO.The % of apoptotic cells was determined by staining with Annexin V-FITC and PI.The lower left quadrant shows cells that are negative for both Annexin V-FITC and PI, and upper left shows only PI cells that are necrotic.The lower right quadrant shows Annexin-positive cells that are in the early apoptotic stage, and the upper right shows cell lines that are both Annexin-and PI-positive, which are in the late apoptosis stage.

Figure 13 .
Figure 13.Compounds 19a and 19m induced cell apoptosis in HG-3 and PGA-1 leukaemia cells.HG-3 and PGA-1 leukaemia cells were treated with 19a and 19m (10 and 1.0 µM) and control vehicle (1% DMSO (v/v)) at 48 h.(A) Compound 19a (10 µM concentration) treatment in HG-3 cells; (B) compound 19a (1 µM concentration) treatment in HG-3 cells; (C) HG-3 cells treatment with vehicle 1% DMSO; (D) compound 19m (10 µM concentration) in PGA-1 cells; (E) compound 19m (1 µM concentration) in PGA-1 cells; (F) PGA-1 cells treatment with vehicle 1% DMSO.The % of apoptotic cells was determined by staining with Annexin V-FITC and PI.The lower left quadrant shows cells that are negative for both Annexin V-FITC and PI, and upper left shows only PI cells that are necrotic.The lower right quadrant shows Annexin-positive cells that are in the early apoptotic stage, and the upper right shows cell lines that are both Annexin-and PI-positive, which are in the late apoptosis stage.

Table 6 .a
Overlay of potent anthracene compounds on maprotiline with their overlay scores.Overlay of most potent nitrovinylanthracene compound series 19a, 19g, 19i, 19l and 19m on maprotiline 9 with their overlay scores (F and S) in MOE 2022 and 2D images.The F column contains the similarity score (the lower the better) of the alignment.The S column has the sum of the U (not shown but is the average internal energy of the ligand) and F values.b The compounds shown represent those with the best IC 50 or percentage viability values on the HG-3 and PGA-1 cell lines.

a
Overlay of most potent nitrostyrene compound series on maprotiline with their overlay scores (F and S) in MOE 2022 and 2D images.The F column contains the similarity score (the lower the better) of the alignment.The S column has the sum of the U (not shown but is the average internal energy of the ligand) and F values.b The compounds shown represent those with the best IC 50 or percentage viability values on the HG-3 and PGA-1 cell lines.

Table 1 .
Crystal data and structure refinement for compounds 19f and 30a.

Table 1 .
Crystal data and structure refinement for compounds

IC 50 (µM) a PGA-1 IC 50 (µM) a logP b
IC 50 values are half-maximal inhibitory concentrations required to block the growth stimulation of HG3 and PGA-1 cells.Values represent the mean ± SEM (error values × 10 −6 ) for at least three experiments performed in triplicate at 24 h.Treatment at eight different concentrations (0.001-50 µM) was used for the determination of the IC 50 values for each compound.
a b Predicted property using Chemaxon.

Table 7 .
Overlay of potent nitrostyrene compound series on maprotiline with overlay scores.