β-Lactams with antiproliferative and antiapoptotic activity in breast and chemoresistant colon cancer cells

Journal Pre-proof β-Lactams with antiproliferative and antiapoptotic activity in breast and chemoresistant colon cancer cells Azizah M. Malebari, Darren Fayne, Seema M. Nathwani, Fiona O'Connell, Sara Noorani, Brendan Twamley, Niamh M. O'Boyle, Jacintha O'Sullivan, Daniela M. Zisterer, Mary J. Meegan PII:

S0223-5234(20)30017-9

DOI:

https://doi.org/10.1016/j.ejmech.2020.112050

Reference:

EJMECH 112050

To appear in:

European Journal of Medicinal Chemistry

Received Date: 1 October 2019 Revised Date:

20 December 2019

Accepted Date: 8 January 2020

Please cite this article as: A.M. Malebari, D. Fayne, S.M. Nathwani, F. O'Connell, S. Noorani, B. Twamley, N.M. O'Boyle, J. O'Sullivan, D.M. Zisterer, M.J. Meegan, β-Lactams with antiproliferative and antiapoptotic activity in breast and chemoresistant colon cancer cells, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112050. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

β-Lactams with Antiproliferative and Antiapoptotic Activity in Breast and Chemoresistant Colon Cancer Cells

Azizah M. Malebaria,b*, Darren Faynec, Seema M. Nathwanic, Fiona O'Connelle, Sara Nooranib, Brendan Twamleyd, Niamh M. O’Boyleb, Jacintha O'Sullivane, Daniela M. Zistererc and Mary J. Meeganb

a

Department of Pharmaceutical Chemistry, College of Pharmacy, King Abdulaziz

University, Jeddah, Saudi Arabia b

School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Trinity

Biomedical Sciences Institute, 152-160 Pearse Street, Dublin 2, Ireland c

School of Biochemistry and Immunology, Trinity College Dublin, Trinity Biomedical

Sciences Institute, 152-160 Pearse Street, Dublin 2, Ireland d

School of Chemistry, Trinity College Dublin, Dublin 2, Ireland

e

Trinity Translational Medicine Institute, Department of Surgery, Trinity College Dublin,

Ireland, Dublin 2, Ireland.

*Corresponding author: Azizah M. Malebaria E-mail: [email protected]

1

Abstract A series of novel 1,4-diaryl-2-azetidinone analogues of combretastatin A-4 (CA-4) have been designed, synthesized and evaluated in vitro for antiproliferative activity, antiapoptotic activity and inhibition of tubulin polymerization. Glucuronidation of CA-4 by uridine 5-diphosphoglucuronosyl transferase enzymes (UGTs) has been identified as a mechanism of resistance in cancer cells. Potential sites of ring B glucuronate conjugation are removed by replacing the B ring meta-hydroxy substituent of selected series of βlactams with alternative substituents e.g. F, Cl, Br, I, CH3. The 3-phenyl-β-lactam 11 and 3-hydroxy-β-lactam 46 demonstrate improved activity over CA-4 in CA-4 resistant HT29 colon cancer cells (IC50 = 9 nM and 3 nM respectively compared with IC50 = 4.16 µM for CA-4), while retaining potency in MCF-7 breast cancer cells (IC50 = 17 nM and 22 nM respectively compared with IC50 = for 4 nM for CA-4). Compound 46 binds at the colchicine site of tubulin, and strongly inhibits tubulin assembly at micromolar concentrations comparable to CA-4. In addition, compound 46 induced mitotic arrest at low concentration in both cell lines MCF-7 and HT-29 together with downregulation of expression of antiapoptotic proteins Mcl-1, Bcl-2 and survivin in MCF-7 cells. These novel antiproliferative and antiapoptotic β-lactams are potentially useful scaffolds in the development of tubulin-targeting agents for the treatment of breast cancers and chemoresistant colon cancers. Keywords Combretastatin A-4 Antiproliferative activity 1,4-Diaryl-2-azetidinone Tubulin polymerization Cell cycle arrest Microtubule targeting agent

2

1. Introduction

Microtubules are highly dynamic structures consisting of α and β-tubulin heterodimers that are involved in cellular processes such as cell division and mitosis. Thus, the microtubule is an essential target for design and development of natural and synthetic agents that inhibit the formation of microtubules of the mitotic spindle [1-3]. Microtubule targeting agents (MTAs) such as paclitaxel and vinca alkaloids are among the most successful classes of chemotherapeutic agents. The tubulin stabilizing compound colchicine (1, Figure 1) binds at the interface of the α and β-subunits of tubulin, forms a colchicine-tubulin complex, and depolymerises the α,β-heterodimer. The cis stilbene combretastatin A-4 (CA-4), (2a, Figure 1), is one of the most effective agents among the naturally occurring antimitotic drugs. CA-4 inhibits tubulin assembly causing mitotic arrest and cell death, by strongly binding to the colchicine site on β-tubulin [4-6], while non-microtubule targets have also been identified for CA-4 [4, 7]. The use of CA-4 as a clinical antitumour agent is limited by its low aqueous solubility, low bioavailability, instability as well as susceptibility to rapid clearance [8-15]. A water soluble phosphate prodrug of CA-4 (CA-4P, Fosbretabulin) (2b, Figure 1) having potent activity as a vascular disrupting agent [8] is currently in clinical trials [16-19]. A phosphate prodrug of combretastatin A-1 (2c), namely OXi4503 (2d) [20] and a serine prodrug Ombrabulin (AVE8062) (2f) [21] of the amino CA-4 analogue 2e have also been clinically evaluated (Figure 1). Many CA-4 analogues have been prepared with modifications to the chemical features, double bond geometry and substitution pattern of rings A and B [22-28]. The 3,4,5-trimethoxyphenyl is a common structural feature of many colchicine binding site inhibitors such as CA-4, CA-1, podophyllotoxin (3a), phenstatin (3b) [29] and the 4benzoylimidazole VERU-III (3c)[30] (Figure 1). However, the trimethoxy phenyl moiety is not always necessary[25] and many structurally diverse compounds such as chalcones 4a (TUB091) and 4b (TUB092)[31], triazoles 4c and 4d [27], BAL27862 (4e) [32], pyridine-chalcone derivatives [33], quinoline-chalcone derivatives [34] and 1-phenyl-1(quinazolin-4-yl)ethanols [35] have been reported as potent colchicine site-binding MTAs (Figure 1).

3

The instability of the phosphate prodrug CA-4P, 2b (figure 1) is attributed to the isomerization of the cis-double bond in vivo to the thermodynamically more stable and biologically less active trans isomer [36, 37]. Therefore, many conformationally restricted analogues of CA-4 have been reported e.g. those based on diverse heterocycles such as indoles, imidazoles, pyrroles and triazoles [2, 38-40]. The β-lactam ring scaffold has attracted much attention in the design of CA-4 analogues [14, 40-49] and has resulted in the discovery of chiral β-lactam bridged CA-4 analogues having potent antitumour properties [50, 51]. We have reported the potent antiproliferative activity of β-lactam compounds 5a and 5b (Figure 1) in human breast cancer MCF-7 and MDA-MB-231 and leukaemia cell lines [43, 52]. However, CA-4 and β-lactam compounds containing a Bring meta-hydroxy substituent group were not active in the combretastatin refractory uridine 5-diphosphoglucuronosyl transferase (UGT) expressing HT-29 colon cancer cells [14]. It is suggested that CA-4 and its analogues undergo direct glucuronidation by UGT at the meta-hydroxy position of ring B leading to inactivation in HT-29 cells [12]. Removal of the ring B meta-hydroxy group significantly increased the antiproliferative activity in the combretastatin refractory HT-29 cells e.g. IC50 for 5b of 22 nM compared to 4.16 µM for CA-4 [14]. A similar result was also reported for 1,5-diaryltetrazole and 4,5-disubstituted oxazole rigid analogues of CA-4 lacking the ring B meta-hydroxy group [26, 53]. We now report a series of novel β-lactam CA-4 analogues in which the meta-hydroxy group of ring B is replaced with a methyl or halogen substituent. This modification is designed to block metabolism at this site, thus preventing the glucuronidation metabolism pathway in HT-29 cells and subsequently improving the cytotoxic effect against both MCF-7 human breast cancer cell lines and the UGT expressing HT-29 colon cancer cells. The substituents at the C-3 position of these novel β-lactam compounds (aryl, amino, phenoxy, halogen, hydroxyl, vinyl) are chosen to optimise the efficacy of the compounds in the cancer cell lines. The characteristic 3,4,5-trimethoxyaryl ring A of CA-4

is

optimally located at N-1 of the β-lactams [2, 22, 25, 54]. Having previously investigated structure-activity relationships of β-lactam CA-4 analogues [14, 43], this study is designed to provide additional information on the role of both the meta substituent in

4

Ring B and the β-lactam C-3 substituent to form strategic interactions with the colchicine binding site. All β-lactam compounds were initially screened for their antiproliferative activity against two cell lines: CA-4 resistant human colon cancer HT-29 cells and CA-4sensitive breast cancer MCF-7, and to act as potential antimitotic agents.

2. Results and discussion 2.1 Chemistry The preparation of the β-lactam compounds is shown in Schemes 1 and 2. Imines 6-10 were obtained in high yields by condensation of the appropriate amines and aldehydes under reflux conditions in ethanol (Scheme 1). The preparation of the β−lactam compounds 11-40 utilised a modified Staudinger reaction which afforded almost exclusively trans isomer products (step a, Scheme 2). The trans stereochemistry was confirmed from the 1H NMR spectrum e.g. for compound 12trans, H3 was identified as a doublet at δ 5.07 (J = 1.66 Hz) coupled to H4 at δ 4.90. The minor cis isomer was isolated for one 3-phenoxy compound 12cis, and confirmed by 1H NMR spectrum where H3 was identified as a doublet at δ 5.51 (J = 4.98 Hz) coupled to H4 at δ 5.27. Superior antiproliferative potency is reported for trans substituted β-lactams when compared with the cis substituted compounds [41]. 3-Unsubstituted β-lactams 41-45 were successfully synthesised by the microwave assisted Reformatsky reaction in good yields (step b, Scheme 2). The 3-hydroxy-β-lactam compounds 46-50 were obtained by treatment of the initially isolated 3-acetoxy-β-lactams with hydrazine or with sodium bicarbonate (step c, Scheme 2). [55]. Staudinger reaction using Mukaiyama’s reagent (2-chloro-1methylpyridinium iodide) was applied successfully for the preparation of 3-amino βlactams 51-52 and also resulted in the exclusive formation of trans isomer product (steps d and e, Scheme 2). The stability of the representative β-lactams 18, 29 and 52 was investigated in acidic, neutral and basic conditions (pH 4, 7.4 and 9). Each β-lactam was incubated in the appropriate phosphate buffer at 37 oC and examined by analytical HPLC at selected time intervals for 24 h. The half-life (t1⁄2) at pH 4, 7.4 and 9 was found to be greater than 15 h for compound 18 and greater than 24 h for compound 52 while t1⁄2 for compound 29 at pH

5

values of 4, 7.4 and 9 was 15 h, 14 h and 5 h respectively. Based on this stability study, these classes of β-lactams would be the suitable for further development.

2.1.1. X-ray structural study for ring B meta-fluorophenyl β-lactam compounds An X-ray crystallography study was carried out on the 3-fluoro-4-methoxyphenyl ring B containing compounds 11, 12trans, 12cis, 13 and 46 with substituents C6H5, OC6H5, Cl and OH at C-3 of β-lactam ring to confirm the stereochemical assignments (Table 1, Table S1 Supplementary information). Generally, the β-lactam ring forms a rigid scaffold for the planar hydrophobic aryl rings A and B required for interaction with the colchicine binding site of tubulin. As shown in Table 2, the X-ray structures demonstrated a configuration for all the β-lactams where the rings A and B are not coplanar. For comparison, the torsional angles (Ring A/B) for colchicine [56] and CA-4 [57, 58] were 53° and 55° respectively while the torsional angles (Ring A/B) observed for compounds 11, 12trans, 12cis, 13 and 46 were determined to be 69°, -57°, 65°, 62° and -63° respectively (Table 2). The main difference in these compounds is the substituent at C-3 of β-lactam ring. For the 3-phenoxy-β-lactam 12cis, the Ring B/C and H-3/H-4 torsional angle values were 9.7° and 4.7° respectively compared to the trans isomer (Ring B/C and H-3/H-4 torsional angles 123.6° and 140.9° respectively. Similarly, the torsional angle value between ring B and phenyl ring C of compound 11 is -121.9° which is similar to the 3-phenoxy β-lactam 12trans -123.6°. The torsional angle value between ring B and 3hydroxyl group of compound 46 is 117.0° while that observed for the 3-chloro compound 13 is 114.4° (Table 2).

2.2 Biological results and discussion 2.2.1 Antiproliferative activity The antiproliferative potential of the β-lactams 11-52 was evaluated in two cell lines: CA-4 sensitive MCF-7 human breast cancer and CA-4 resistant HT-29 colon cancer cells using the AlamarBlue assay, with CA-4 and our previously reported lead β-lactams compounds (5a) and (5b) as reference compounds, (Table 3). Replacing the metahydroxy group of ring B with a methyl or halogen substituent was examined in an effort to block metabolism at this site, preventing the glucuronidation metabolism pathway in

6

HT-29 cells and subsequently improving the cytotoxic effect against both MCF-7 human breast cancer cell lines and the UGT expressing HT-29 colon cancer cells [14]. From the initial screen of compounds 11-52 for antiproliferative activity in MCF-7 and HT-29 cells (grouped according to the Ring B substituent X), the effects of the substituents on aryl ring B at C-4 of the β-lactam ring is evident. The 3-hydroxy-4methoxyphenyl (5a) was one of the most active compounds in MCF-7 cells (IC50 = 4 nM) while its activity decreased significantly in HT-29 cells (IC50= 430 nM). Compound 5b, with deletion of hydroxyl group at the meta-position maintained similar activity to 5a in MCF-7 (IC50=0.015 µM), with significantly improved cytotoxicity in HT-29 cells (IC50=22 nM) [14]. The introduction of fluorine may improve the pharmacological and physicochemical properties of a compound, such as metabolic stability, lipophilicity and ligand binding [59-61]. The fluoro analogues 11-16, 41, 46, 51 (Table 3) exhibited significant anticancer activity in both cell lines MCF-7 and HT-29 in the nanomolar range (17-483 nM in MCF7 cells and 3-678 nM in HT-29 cells), demonstrating tolerance of a variety of substituents at C-3 position of the β-lactam ring. Compounds 11 and 46 were the most active analogues in this series with IC50 values of 17 and 22 nM in MCF-7 cells and with potent activity in CA-4 resistant HT-29 cells (IC50=9 nM and 3 nM respectively). Compounds 13 (3-chloro), 15 (3-vinyl) and 41 (3-unsubstituted) were also significantly more potent than CA-4 in HT-29 cells with IC50 values of 15 nM, 37 nM and 0.015 nM respectively. The 4-(3-chloro-4-methoxyphenyl) analogues 17-22, 42, 47, 52 all exhibited improved antiproliferative activity in the chemoresistant HT-29 cells when compared with CA-4 (Table 3). 3-Hydroxy β-lactam compound 47 was the most potent analogue in this series in both cell lines (IC50=12 nM in MCF-7, IC50=18 nM in HT-29) with the 3-chloro compound 17 and the 3,3-dichloro analogue 19 also displaying good activity. The most potent of the 4-(3-bromo-4-methoxyphenyl) Ring B analogues were identified as 27 (3vinyl), 43 (3-unsubstituted) and 48 (3-hydroxy) with impressive antiproliferative activity (IC50 values of 88, 60, 44 nM in MCF-7 cells and 91, 64, 9 nM in HT-29 cells respectively) (Table 3). The 4-(3-iodo-4-methoxyphenyl)-β-lactams 29-33, 44, 49 also displayed submicromolar antiproliferative effects. 3-Hydroxy-β-lactam 49 was the most 7

potent compound in this series with IC50 value of 21 nM in HT-29 cells. The 4-(3-methyl4-methoxyphenyl) analogues 35-40, 45, 50 displayed similar activity in the chemoresistant HT-29 cells with IC50 values in the range (7-358 nM). The C-3 hydroxy compound 50 demonstrated significant activity in this series, with nearly 600-fold more potency than CA-4 in chemoresistant HT-29 cells. In summary, the 3-hydroxy β-lactams 46-50 were identified as the most potent group in the series synthesized, with the ring B meta-methyl compound 50 (IC50=5 nM for MCF-7 cells and IC50=7 nM for HT-29 cells). The ring B meta-fluoro compound 46 (IC50 = 22 nM for MCF-7 cells and IC50 = 3 nM for HT-29 cells) also demonstrated notable activity, The order of activity for 3-hydroxy β-lactams 46-50 related to the Ring B 3’-substituent is F > CH3 > Br > Cl > I for HT-29 cells with some correlation with the clog P values, (see Table 3). Comparing the 3-phenyl substituted compounds 11, 17, 23, 29, 35 shown in Figure 2, the order of activity related to the Ring B 3’-substituent is F>Cl>CH3>Br >I for HT-29 cells. Replacing the ring B meta-hydroxy substituent of lead compound 5a with fluorine maintains the nanomolar antiproliferative activity in MCF-7 cell lines and greatly improves the activity compared to 5a in HT-29 cells. Introduction of halogen (Cl, Br and I) or methyl at Ring B C-3 also results in significant activity in HT-29 cells, potentially suggesting effective blocking of a glucuronidation site in the β-lactams in HT29 cells. Five novel β-lactam compounds from the present work (11, 12, 19, 29 and 37) were selected for evaluation in the NCI 60 cell line screen [62], following initial analysis of the drug-like (Lipinski) properties from the Tier-1 profiling screen and also predictions of the ADMET properties of metabolic stability, permeability, blood-brain barrier partition, plasma protein binding and human intestinal absorption properties, (Table S2 and Table S3 Supplementary information). The compounds are predicted to be moderately lipophilic-hydrophilic and are potentially suitable candidates for further investigation. Compound 11 demonstrated impressive antiproliferative effects in the nanomolar range for most of the cancer cell lines in the NCI 60 cell line screen, (Table 4). The mean GI50 value across all cell lines tested was 59 nM and compared very favourably with CA-4 (GI50=99 nM). The GI50 values for 11 were in the nanomolar range for all except 5 of the

8

panel cell lines investigated, with low toxicity, (LC50>95 µM). The 3-chloro β-lactams (19 and 37) and the 3-phenoxy compound 12 also demonstrated nanomolar antiproliferative activities with mean GI50 values of 112 nM, 190 nM and 190 nM across the 60 cancer cell lines respectively, Table 5. The detailed results are contained in the supplementary information, Table S4. 2.2.3. Antiproliferative activity in non-carcinogenic human cells Non-tumourigenic cell line HEK-293T (normal human embryonic kidney) was chosen to investigate the toxicity and selectivity of 46 towards normal cells. As shown in Figure 3, the IC50 value of 46 was greater than 50 µM in HEK-293 cells which was significantly higher than that observed against the MCF-7 and HT-29 cancer cell lines (IC50=22 nM and 3 nM, respectively), demonstrating that β-lactam 46 was less toxic to human normal cells than cancer cells, providing a window of selectivity. 2.2.4. Inhibition of tubulin polymerisation As the tubulin-microtubule system has a major role in maintaining cellular function and morphology [63], and considering the structural similarity of the novel compounds to known colchicine-site binders (Figure 1), it was important to determine the effect of these compounds on microtubule dynamics. Firstly, the in vitro inhibition of tubulin polymerisation by the novel β-lactam analogues 46, 47, 48 and 50 (all at 10 µM concentration) was determined using isolated purified bovine tubulin (Figure 4). CA-4 and paclitaxel were used as positive controls. Paclitaxel, a known microtubule-stabilising agent, increased the Vmax and the final polymer mass as shown in Figure 4. Incubation with either CA-4 or β-lactam analogues resulted in various degrees of inhibition of tubulin polymerisation. All tested β-lactam compounds strongly inhibited tubulin assembly while 46 was the most potent inhibitor with a 5.4 fold reduction in Vmax compared to the control, mimicking the effects of CA-4. This result is correlated with the excellent antiproliferative activity observed for the meta-fluoro substituted β-lactam 46. The other three compounds (meta-chloro analogue 47, meta-bromo analogue 48 and meta-methyl analogue 50), showed inhibition of tubulin polymerization (~2.4 fold reduction in Vmax) when compared to the vehicle. This indicates that the β-lactams are microtubule-destabilising agents. 9

2.2.5. Immunofluorescence studies for visualisation of microtubule network in MCF-7 cells treated with compound 46 The effect of compound 46 on the microtubule network of MCF-7 cells was evaluated by tubulin immunostaining to further identify the cellular effects that are possibly relevant to its antiproliferative activity and inhibition of tubulin polymerisation. A microscopic analysis of MCF-7 cells stained with α-tubulin antibodies indicated a well-organised microtubular network in the control cells (Figure 5). The figure clearly demonstrated depolymerisation and solubilisation of the cell membrane of microtubules in CA-4 (10 nM)-treated cells (positive control). In contrast, substantial stabilisation of microtubules in paclitaxel (1.0 µM) treated cells is observed. Compound 46 significantly impacts the microtubule network in MCF-7 cells and induces cell rounding, detachment and a loss of the radial distribution of structured microtubules at high concentration of 100 nM. A similar effect was observed with CA-4. At the low concentration of compound 46 (10 nM), the minor effect of depolymerisation and less distinct abnormalities of the spindle formation was observed and this effect was partially increased at 50 nM concentration. These results indicate that compound 46 destabilises microtubules supporting the findings from the tubulin polymerisation assay. 2.2.6. Colchicine-site binding assay Due to the structural similarity between our compounds and known drugs which target the colchicine-binding site of tubulin (Figure 1), further studies were performed to investigate the interaction of compound 46 at the colchicine binding site of tubulin using N,N-ethylenbis(iodoactamide) (EBI) in a whole-cell based assay. EBI forms adducts with tubulin which can be detected by Western blotting. Microtubule destabilizing agents that bind at the colchicine site, such as colchicine and CA-4, prevent the formation of the βtubulin-EBI adduct by occupying the binding site [64, 65]. In our assay, MCF-7 cells were treated with vehicle control [ethanol 0.1% (v/v)], CA-4 (10 µM) or 46 (10 µM) for 2 h, followed by EBI (100 µM) for an additional 1.5 h. Control samples show the presence of the β-tubulin-EBI adduct at a lower position indicating that EBI has crosslinked Cys239 and Cys354 amino acids on the β-tubulin (Figure 6). In contrast, tubulin EBI-adduct formation was inhibited in MCF-7 cells treated with CA-4 and 46, confirming that both CA-4 and 46 bind to the colchicine binding site of tubulin. 10

2.2.7. Effect on cell cycle arrest In general, G2/M cell cycle arrest is strongly associated with inhibition of tubulin polymerisation [66]. It is well established that CA-4 causes G2/M arrest in cell cycle [17, 67]. The effect of compound 46 was investigated in MCF-7 and HT-29 cells by flow cytometry followed by propidium iodide (PI) staining of the cells at different time points. For MCF-7 cells the percentage of cells in G2/M phase was 66%, 75% and 60% when the cells were treated with compound 46 at a concentration of 1 µM for 24 h, 48h and 72 h, respectively (Figure 7A). In the control sample, 24% of accumulation in G2M phase was observed. There was also an increase in the number of cells in sub-G1 following treatment with compound 46, with 15% at 72 h indicative of apoptosis. Compound 46 caused a significant G2/M arrest in a time dependent manner and induction of apoptosis, consistent with the behaviour of tubulin binding agents. These findings are in agreement with CA-4 and related analogues which significantly induce apoptosis and cell cycle arrest at the G2/M phase in MCF-7 cells [14, 41, 44]. The cell cycle effect of compound 46 was also examined in the HT-29 cells. Compound 46 at 1 µM induced gradual accumulation of cells in the G2/M phase of the cell cycle in a time dependent manner with 52%, 57% and 64% at 24 h, 48 h and 72 h respectively compared to the vehicle control (11%). In addition, there was an increase in the population in the sub-G1 phase (apoptotic cells) at 48 h with 11% compared with the control (2.5%), (Figure 7B) suggesting other mechanisms could be involved in the cell death process for HT-29 cells such as necrosis or autophagy [68-72]. CA-4 and its βlactam analogue 5a induce autophagy in colon cancer cells as a novel mode of cell death which enhances the therapeutic efficacy [73, 74]. 2.2.8. Apoptosis quantification by Annexin V-FITC/PI assay Programmed cell death, (such as apoptosis, autophagy and necroptosis) plays a crucial role in cancer metastasis [68, 75, 76]. To determine the role of apoptosis in the inhibition of MCF-7 and HT-29 cell growth, cells were treated with compound 46 and stained with Annexin V-FITC/PI and then were analyzed by flow cytometry. Dual staining with Annexin-V and PI permits discrimination between live cells (annexin-V−/PI−), early apoptotic cells (annexin-V+/PI−), late apoptotic cells (annexin-V+/PI+) and necrotic cells

11

(annexin-V−/PI+). Compound 46 induced both early and late apoptosis in MCF-7 cells in a concentration dependent manner when compared to the untreated control cells (Figure 8A). When MCF-7 cells were treated with 46 (0.01 µM, 0.1 µM and 1 µM) for 48 h, the average proportion of Annexin V-staining positive cells (total apoptotic cells) increased from 10% in control cells to 13%, 20% and 35% respectively. As supported from flow cytometry findings above (Figure 7A), these results suggested that compound 46 induces apoptosis of MCF-7 cells in a dose dependent manner. Treatment of the HT-29 cells with 46 (1µM) at time points of 24, 48 and 72 h demonstrated moderate cell apoptosis at 24 h (21%) and 48 h (25%), which decreased to 2% at 72 h (Figure 8B). A small increase of Annexin-V- / PI + cells (necrotic cells) from 8% at 24 h to 11% at 48 h was also observed which increased further at 72 h (22%) in comparison with untreated HT-29 cells (3%), (Figure S5, supplementary information). This result indicates that in HT-29 cells, 46 induced cellular death by necrosis at 72 h in comparison to its effect in MCF-7 cells which mainly involved apoptosis as mechanism of cell death. 2.2.9. Effect of compound 46 on expression levels of anti-apoptotic proteins in MCF7 cells Due to the increase in apoptotic cell population induced by 46 in MCF-7 cells as confirmed by cell cycle assay, PI staining and Annexin V-FITC, further effects of 46 on anti-apoptotic proteins of the Bcl-2 protein family were investigated, specifically Bcl-2 and Mcl-1. MCF-7 cells were treated with compound 46 (0.05, 0.1 and 0.5 µM) for 48 and 72 h (Figure 9). Expression levels of both Mcl-1 and Bcl-2 undergo a moderate to dramatic decrease respectively after 48 and 72 h treatment at all compound concentrations examined in a dose dependent manner, indicating that 46 induced downregulation of these proteins to disable their anti-apoptotic function. Survivin is an apoptotic protein that inhibits caspase activation and can be used as an indicator of apoptosis process [77, 78]. Survivin expression is regulated by cell cycle stages and is absent in most normal cells, until accumulation in G2/M phase whereas it displays high levels of expression in cancer cells such as breast and leukemia [70, 79, 80]. Treatment of MCF-7 cells with compound 46 (0.05, 0.1 and 0.5 µM) caused some down-regulation of the expression of survivin as demonstrated by Western blotting (figure 9), also indicating the pro-apoptotic effect of 46 in MCF-7 cells.

12

2.2.10. Effect of compounds 5a, 41, 46, 50 on angiogenic, vascular injury, inflammatory cytokine and chemokine secretions from HT-29 cells To further investigate the molecular mechanism of action of the β-lactam CA-4 related compounds, the angiogenic, vascular injury, inflammatory cytokine and chemokine secretions from HT-29 cells treated with compounds 5a, 41, 46, 50 and the antimitotic compound phenstatin [29] were determined. Levels of VEGF when measured through ELISA showed some increased secretion, which may indicate that these compounds may not affect the classical VEGF pathway of angiogenesis. IL-1

and IL-1 , also showed

some increased levels of secretion with both cytokines being associated with proinflammatory response and IL-1 associated with regulating VEGF secretion, (Figure S7, Supplementary information). ELISA data also showed significantly increased secretion of IL-1RA, the inhibitor of IL-1 and IL-1 , when compared to the vehicle control Ethanol. IL-1RA which has been shown to inhibit tumour-mediated angiogenesis [81] and has also been shown to inhibit IL-1

induced VEGF secretion [82] may be linked to anti-

angiogenic effects in these compounds. However, further mechanistic studies are currently being pursued on these compounds to indicate the metabolic pathway upon which they act 2.2.11. In vitro metabolism of compounds 11, 46 and CA-4 in human liver microsomes The metabolic stability of compound 11, 46 and CA-4 in human liver microsomes was investigated in the presence of alamethicine (a pore forming peptide used to activate UGTs in human liver microsomes) and uridine-diphosphate-glucuronic acid trisodium salt (UDPGA). We observed that CA-4 is rapidly metabolised by glucuronidation with t1/2 of 2.71 min and intrinsic clearance of 645 µL/min/mg protein. In contrast, the 3hydroxy-β-lactam compound 46 demonstrated considerable metabolic stability to glucuronidation with t1/2 of 98 min and intrinsic clearance of 14.2 µL/min/mg protein which is significantly longer than CA-4 (2.71 min). The control compound 11 with replacement of the m-hydroxy group of ring B with fluorine resulted in enhanced metabolic stability with t1/2 of 1410 min and intrinsic clearance of 0.98 µL/min/mg protein. These results indicate that compounds 11 and 46 displayed considerable stability

13

toward hepatic enzymes and could be considered as promising compounds for further development. 2.3. Molecular modelling The X-ray structure of Combretastatin A-4 co-crystallised with tubulin has been determined suggesting that cis-CA-4 inhibits tubulin polymerization by preventing the transition from curved to straight tubulin.[83] The X-ray structure of cis and trans stereoisomers of a 3-methyl-1,4-diarylazetidinone [42] co-crystallised with tubulin was reported by Zhou et al (PDB ID 5GON and 5XAF) [50, 51]. In the present study, the 5GON X-ray structure was initially examined to determine suitability for docking calculations and compound binding mode analysis for the 3-phenyl-β-lactams 11, 17, 23 29 and 35. The similarities in the overlay of compounds in the two X-ray structures 5GON [50, 51] and 1SA0 [56] are evident but significant changes in the position of Lys β352 and Thr α179 are apparent, (see Supplementary Information, Figure S6). An amino acid change was noted below the 3,4,5-trimethoxyphenyl binding region where Val β318 was mutated to Ile β318 in the 5GON structure. An important hydrogen bonding (HB) binding interaction between Lys β352 and the carbonyl oxygen atom of colchicine, as observed in the 1SA0 X-ray [56], was not possible in the 5GON structure, (Supplementary information, figure S6). The orientation of the side chain of Lys β352 was significantly changed, with the nitrogen atom moving by 5 Å. It is now suggested that the meta-substituent on the 4-aryl B ring of compounds 11, 17, 23, 29 and 35 would co-locate with the colchicine carbonyl group to form the required HB acceptor (HBA) interaction with Lys β352, so all subsequent calculations for these compounds were performed on the 1SA0 X-ray structure which is comparable to previous work from our group and others [84]. Docking calculations using MOE 2016.0802 [85] were undertaken on both the 3S/4R and 3R/4S enantiomers of the β–lactams 11, 17, 23, 29 and 35 using the tubulin cocrystallised with DAMA-colchicine X-ray crystal structure (Figure 10, PDB entry 1SA0) [56] but only results for the 3S/4R studies will be discussed as these stereoisomers were more highly ranked than the 3R/4S enantiomer and this is also in line with the crystallographic evidence [50, 51]. Both the 3S/4R enantiomers of the fluorine 11 and chlorine 17 14

substituted analogues overlay their B-rings on the C-ring of DAMA-colchicine, co-locate the trimethoxyphenyl substituents, overlap the halogens onto the DAMA-colchicine carbonyl oxygen atom and form HBA interactions with Lys β352 as shown in figure 10A. Analogue 35 also mapped well to the B- and C-rings but the methyl substituted 3-phenyl ring was rotated 180° as compared to 11 and 17. We postulate that this is because the methyl group prefers a more hydrophobic environment so is orientated away from Lys β352. Due to their larger size and clashing with Lys β352, the bromine 23 and iodine 29 substituted analogues were unable to position their B-rings deeper within the binding pocket leading to a flipped orientation which directs the B-ring outside the pocket (figure 10B). This alternative binding mode is reflected in lower potency of the in vitro data presented. The 3,4,5-trimethoxyphenyl groups of all analogues are able to make favourable van der Waals contacts within the lower subpocket delineated by Val β318 and Cys β241. The β-lactam carbonyl oxygen atom can make a HBA interaction with the backbone amine of Asp β251 for the fluoro 11, chloro 17 and methyl 35 substituted analogues which is not possible for compounds 23 and 29. Interestingly, the C-ring of the fluoro, chloro and methyl analogues (11, 17 and 35) and the B-ring of the bromo and iodo analogues (23 and 29) can make favourable van der Waals interactions with the carbon side-chain of Lys β254. For all compounds, the trans geometry at C3/C4 facilitates a more favourable interaction of rings A and B with the residues of the β-tubulin colchicine binding site. The overlay of the X-ray structure of tubulin co-crystallised with DAMA-colchicine (PDB entry 1SA0) on the best ranked docked pose of compound 46 demonstrates the colocation of the trimethoxyphenyl substituent of 46 with that of DAMA-colchicine, overlaps the fluorine atom onto the DAMA-colchicine carbonyl oxygen atom and forms a HBA interaction with Lys β352, (Figure 11, showing 3S/4S enantiomer). While not found in the docking procedure as the protein structure was kept rigid, it is likely that the hydroxyl group on the β-lactam ring can form a HDB interaction with Lys254.

15

3. Conclusion Glucuronidation of CA-4 by UGTs has been identified as a mechanism of resistance in cancer cells. Increased expression of UGTs in HT-29 chemoresistant colon cancer cells results in glucuronidation of CA-4 at the meta-hydroxy position of Ring B leading to CA4 resistance for this cell line. A novel panel of β-lactam analogues of CA-4 with modification at meta-position of ring B is described. These compounds contain a nonisomerisable β-lactam heterocycle, replacing the alkene bridge of CA-4, and linking the A and B aromatic rings of CA-4. The efficacy of these compounds toward CA-4 chemoresistant HT-29 cells is significantly improved by preventing the susceptibility of the meta-position in ring B to phase I hydroxylation and subsequently to the glucuronidation. We have now demonstrated that replacing the B ring meta-hydroxy substituent of the selected series of β-lactams with alternative substituents e.g. F, Cl, Br, I, CH3 is effective in removal of this potential site of Ring B glucuronate conjugation. These compounds 11-52 contain the 3,4,5-trimethoxyphenyl moiety (ring A of CA-4), together with the cis-olefin configuration at the CA-4 olefin bridge considered essential for maximum tubulin binding activity while some B-ring structural modification is tolerated by the target. Potent activity was obtained for the meta-fluoro, meta-chloro and meta-methyl ring B derivatives of β-lactam compounds 11, 17, 19, 41 and 46 in HT-29 cells while also retaining useful activity in the MCF-7 cell line. Importantly, compound 46 showed low cytotoxicity in HEK-293T cell lines suggesting its preferential toxicity to proliferating cancer cells such as HT-29 and MCF-7 cells. Compound 46 induced mitotic arrest at low concentration in both cell lines MCF-7 and HT-29 and strongly inhibited tubulin assembly exhibiting similar potency to CA-4. In addition, compound 46 downregulated the expression of antiapoptotic proteins Mcl-1 and Bcl-2 in MCF-7 cells and binds in the colchicine site of tubulin. These results were supported by the molecular modelling study showing that the compounds interact with tubulin at the same site as colchicine with similar binding mode. Compounds 11 and 46 displayed considerable stability toward hepatic enzymes. Collectively, the data presented in this study support further preclinical development of these novel 1,4-diaryl-2-azetidinone CA-4 analogues in the treatment of chemoresistant colon cancers and other malignancies. 16

4. Experimental Section 4.1 Chemistry All reagents were commercially available and were used without further purification unless otherwise indicated. Anhydrous solvents were obtained by distillation from the indicated systems immediately prior to use: Dichloromethane from calcium hydride and toluene from sodium. Uncorrected melting points were measured on a Gallenkamp apparatus. Infra-red (IR) spectra were recorded on a Perkin Elmer FT-IR Paragon 1000 spectrometer. 1H and 13C nuclear magnetic resonance spectra (NMR) were recorded on a Brucker DPX 400 spectrometer (400.13 MHz, 1H; 100.61 MHz, 13C) in CDCl3 (internal standard tetramethylsilane (TMS)) at 27 oC. 1H-NMR spectra were assigned relative to the TMS peak at 0.00 ppm and

13

C-NMR spectra were assigned relative to the middle

CDCl3 peak at 77.0 ppm. 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, USA) in acetonitrile: water (60:40 %v/v) at 200 µL/min. The capillary voltage of the mass spectrometer was at 3kV. The sample cone (de-clustering) voltage was set at 40V. For exact mass determination, the instrument was externally calibrated for the mass range m/z 100 to m/z 1000. A lock (reference) mass (m/z 556.2771) was used. TLC was performed using Merck Silica gel 60 TLC aluminium sheets with fluorescent indicator visualizing with UV light at 254 nm. Flash chromatography was carried out using standard silica gel 60 (230-400 mesh) obtained from Merck. All products isolated were homogenous on TLC. The purity of the tested compounds was determined by HPLC with purity level was >95%. Analytical high-performance liquid chromatography (HPLC) was performed using a Waters 2487 Dual Wavelength Absorbance detector, a Waters 1525 binary HPLC pump and a Waters 717 plus Autosampler. The column used was a Varian Pursuit XRs C18 reverse phase 150 x 4.6 mm chromatography column. Samples were detected using a wavelength of 254 nm. All samples were analyzed using acetonitrile (70%): water (30%) over 10 min and a flow rate of 1 mL/min. Microwave experiments were carried using a Biotage Discover SP4 and

17

CEM microwave synthesisers on standard power setting (maximum power supplied is 300 watts) unless otherwise stated. 4.1.1. General method I: preparation of imines The appropriately substituted benzaldehyde (10 mmol) and substituted aniline (10 mmol) were heated together at reflux in ethanol (40 mL) for 4 h. The reaction mixture was reduced by evaporation in vacuo and the resulting solid product was recrystallised from ethanol. 4.1.1.1. (E)-1-(3-Fluoro-4-methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)methanimine (6):

was

obtained

using

the

general

method

I

above

from

3-fluoro-4-

methoxybenzaldehyde and 3,4,5-trimethoxyaniline as white crystals; yield: 85%, Mp: 121 °C, purity (HPLC): 98%, IRνmax (ATR): 1575.9 (C=N) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 3H, OCH3), 3.88 (s, 9H, OCH3), 6.45 (s, 2H, ArH), 7.01 (t, J = 8.29 Hz, 1H, ArH), 7.54 (d, J = 8.29 Hz, 1H, ArH), 7.70 (d, J = 13.68 Hz, 1H, ArH), 8.35 (s, 1H, CH=N). 13C NMR (100 MHz, CDCl3): δ 56.1, 56.3, 61.0, 98.2, 112.8, 115.2, 126.1, 129.6, 136.4, 147.7, 153.6, 157.8, 158.0, 161.5 (HC=NC). HRMS: calculated for C17H19FNO4 [M + H]+ 320.1298; found 320.1296. 4.1.1.2. (E)-1-(3-Chloro-4-methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)methanimine (7):

was

obtained

using

the

general

method

I

above

from

3-chloro-4-

methoxybenzaldehyde and 3,4,5-trimethoxyaniline as yellow crystals; yield: 91%, Mp: 123 °C, purity (HPLC): 100%, IRνmax (ATR): 1583.0 (C=N) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 3H, OCH3), 3.88 (s, 6H, OCH3), 3.94 (s, 3H OCH3) 6.45 (s, 2H, ArH), 6.99 (d, J = 8.71 Hz, 1H, ArH), 7.71 (d, J = 8.29 Hz, 1H, ArH), 7.96 (d, J = 1.66 Hz, 1H, ArH), 8.34 (s, 1H, CH=N).

C NMR (100 MHz, CDCl3): δ 56.3, 58.8, 61.0, 101.0,

13

111.7, 123.4, 128.9, 129.8, 130.1, 138.7, 147.7, 153.6, 157.5 (HC=NC). HRMS: calculated for C17H1935ClNO4 [M + H]+ 336.1003; found 336.1017. 4.1.1.3. (E)-1-(3-Bromo-4-methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)methanimine (8):

was

obtained

using

the

general

method

I

above

from

3-bromo-4-

methoxybenzaldehyde and 3,4,5-trimethoxyaniline as yellow crystals; yield: 83%, Mp: 146 °C, purity (HPLC): 100%, IRνmax (ATR): 1583.0 (C=N) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.77 (s, 3H, OCH3), 3.92 (s, 6H, OCH3), 3.95 (s, 3H, OCH3), 6.45 (s, 2H,

18

ArH), 6.96 (d, J = 8.29 Hz, 1H, ArH), 7.76 (dd, J = 8.29, 2.07 Hz, 1H, ArH), 7.91 (d, J = 2.07 Hz, 1H, ArH), 8.34 (s, 1H, CH=N). 13C NMR (100 MHz, CDCl3): δ 56.1, 56.4, 61.0, 98.2, 111.6, 112.4, 128.6, 129.6, 130.3, 137.3, 147.7, 153.6, 157.4, 158.2 (HC=NC). HRMS: calculated for C17H1979BrNO4 [M + H]+ 380.0497; found 380.0480. 4.1.1.4.

(E)-1-(3-Iodo-4-methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)methanimine

(9): was obtained using the general method I above from 3-iodo-4-methoxybenzaldehyde and 3,4,5-trimethoxyaniline as yellow crystals; yield: 90%, Mp: 152 °C, purity (HPLC): 100%, IRνmax (ATR): 1577.9 (C=N) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 3H, OCH3), 3.88 (s, 6H, OCH3), 3.93 (s, 3H, OCH3), 6.45 (s, 2H, ArH), 7.17 (d, J = 8.29 Hz, 1H, ArH), 7.80 (dd, J = 8.50, 1.87 Hz, 1H, ArH), 8.02 (s, 1H, ArH), 8.35 (s, 1H, CH=N). C NMR (100 MHz, CDCl3): δ 56.1, 56.6, 61.0, 86.4, 92.7, 110.5, 129.6, 130.6, 137.0,

13

139.6, 147.6, 153.2, 160.2 (HC=NC). HRMS: calculated for C17H19INO4 [M + H]+ 428.0359; found 428.0365. 4.1.1.5. (E)-1-(4-Methoxy-3-methylphenyl)-N-(3,4,5-trimethoxyphenyl)methanimine (10): was obtained using the general method I above from 4-methoxy-3methylbenzaldehyde and 3,4,5-trimethoxyaniline as yellow crystals; yield: 75%, Mp: 112 °C, purity (HPLC): 98%, IRνmax (ATR): 1587.6 (C=N) cm-1. 1H NMR (400 MHz, CDCl3): δ 2.26 (s, 3H, OCH3), 3.82 (s, 6H, OCH3), 3.94 (s, 6H, OCH3), 6.48 (s, 2H, ArH), 6.88 (d, J = 8.71 Hz, 1H, ArH), 7.63 (dd, J = 8.29, 2.07 Hz, 2H, ArH), 8.36 (s, 1H, CH=N).

13

C NMR (100 MHz, CDCl3): δ16.2, 55.5, 60.1, 98.1, 109.7, 126.5, 127.4,

128.9, 130.3, 136.0, 148.4, 153.5, 159.5 (HC=NC), 160.6. HRMS: calculated for C18H22NO4 [M + H]+ 316.1549; found 316.1548. 4.1.2. General method II: synthesis of 2-azetidinones (Staudinger reaction) The appropriate imine (5 mmol) and the appropriate acid chloride (7 mmol) were dissolved in dry toluene (50 mL), under nitrogen with stirring at 0 °C. The solution was allowed to reach room temperature and then warmed to 100 °C. Dry triethylamine TEA (9 mmol) was added dropwise. The mixture was warmed at 100 °C for 5 h and stirred at room temperature overnight until the starting material had disappeared as monitored by TLC. The reaction mixture was washed with water (2

100 mL), dried over anhydrous

Na2SO4 and solvent was removed by evaporation in vacuo. The crude product was purified by flash chromatography over silica gel (eluent: 5:1; n-hexane: ethyl acetate).

19

4.1.2.1. 4-(3-Fluoro-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin2-one (11): was synthesised using the general procedure II above from imine 6, and phenylacetyl chloride to afford the product as white powder; yield: 25%, Mp: 125 °C, purity (HPLC): 96%, IRνmax (ATR): 1746.1 (C=O) cm-1.1H NMR (400MHz, CDCl3): δ 3.72 (s, 6H, OCH3), 3.77 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.24 (d, J = 2.49 Hz, 1H, H3), 4.82 (d, J = 2.49 Hz, 1H, H4), 6.58 (s, 2H, ArH), 6.94 - 7.01 (m, 1H, ArH), 7.12 (s, 1H, ArH), 7.13 - 7.16 (m, 1H, ArH), 7.28 - 7.34 (m, 3H, ArH), 7.34 - 7.41 (m, 2H, ArH). C NMR (100 MHz, CDCl3): δ 56.1, 56.3, 60.9, 63.3, 65.1, 94.9, 114.0, 121.9, 127.4,

13

128.0, 129.1, 130.3, 133.5, 134.4, 148.1, 151.5, 153.6, 165.3 (C=O).

19

F NMR (376

MHz, CDCl3): δ ppm: - 132.06 (ArF). HRMS: calculated for C25H24FNNaO5 [M + Na]+ 460.1536; found 460.1538. 4.1.2.2. Trans-4-(3-Fluoro-4-methoxyphenyl)

-3-phenoxy-1-(3, 4,

5-trimethoxy

phenyl)azetidin-2-one (12): was synthesised using the general procedure II above from imine 6, and phenoxyacetyl chloride to afford the product as yellow powder; yield: 33%, Mp: 126 °C, purity (HPLC): 100%, IRνmax (ATR): 1745.6 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.69 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.90 (d, J = 1.66 Hz, 1H, H4), 5.07 (d, J = 1.66 Hz, 1H, H3), 6.53 (s, 2H, ArH), 6.84 (dd, J = 8.71, 1.24 Hz, 2H, ArH), 6.96 - 7.03 (m, 2H, ArH), 7.10 - 7.16 (m, 2H, ArH), 7.24 (dd, J = 8.71, 7.46 Hz, 2H, ArH).

13

C NMR (100 MHz, CDCl3): δ 56.0, 60.9, 63.5, 87.2, 95.3,

114.0, 115.4, 122.4, 128.3, 129.7, 132.8, 135.1, 148.4, 151.5, 153.5, 156.9, 162.3 (C=O). HRMS: calculated for C25H25FNO6 [M + H]+ 454.1666; found 454.1669. 4.1.2.3. Cis-4-(3-Fluoro-4-methoxyphenyl) -3-phenoxy-1-(3, 4, 5-trimethoxyphenyl) azetidin-2-one (12cis): was synthesised using the general procedure II above from imine 6, and phenoxyacetyl chloride to afford the product as yellow powder; yield: 18%, Mp: 110 °C, purity (HPLC): 100%, IRνmax (ATR): 1745.87 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.71 (s, 6H, OCH3), 3.76 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 5.27 (d, J = 4.98 Hz, 1H, H4), 5.51 (d, J = 4.98 Hz, 1H, H3), 6.58 (s, 2H, ArH), 6.74-6.82 (m, 2H, ArH), 6.83 - 6.96 (m, 2H, ArH), 7.07 - 7.19 (m, 4H, ArH).

13

C NMR (100 MHz, CDCl3): δ

56.1, 60.9, 81.0, 95.3, 113.1, 115.6, 122.3, 124.2, 125.4, 129.3, 132.9, 148.1, 150.9, 153.6, 156.8, 162.7 (C=O). HRMS: calculated for C25H25FNO6 [M + H]+ 454.1666; found 454.1663.

20

4.1.2.4. 3-Chloro-4-(3-fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin2-one (13): was synthesised using the general procedure IV above from imine 6, and chloroacetyl chloride to afford the product as pale yellow powder; yield: 31%, Mp: 122 °C, purity (HPLC): 98%, IRνmax (ATR): 1733.1 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.71 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.57 (d, J = 2.07 Hz, 1H, H4), 4.88 (d, J = 2.07 Hz, 1H, H3), 6.50 (s, 2H, ArH), 6.98 (t, J = 8.50 Hz, 1H, ArH), 7.06 - 7.14 (m, 2H, ArH). 19F NMR (376 MHz, CDCl3): δ ppm: - 132.4 (ArF). 13C NMR (100 MHz, CDCl3): δ 56.1, 60.9, 63.1, 65.5, 95.3, 114.0, 122.3, 127.7, 132.7, 135.3, 148.7, 151.5, 153.6, 160.4 (C=O). HRMS: calculated for C19H2035ClFNO5 [M + H]+ 396.1014; found 396.1008. 4.1.2.5.

3,3-Dichloro-4-(3-fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (14): was synthesised using the general procedure II above from imine 6, and dichlorocetyl chloride to afford the product as a grey powder; yield: 63%, Mp: 127° C, purity (HPLC): 100%, IRνmax (ATR): 1747.2 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.72 (s, 6H, OCH3), 3.78 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 5.38 (s, 1H, H4), 6.52 (s, 2H, ArH), 6.96 - 7.03 (m, 1H, ArH), 7.03 - 7.09 (m, 2H, ArH).

13

C NMR (100

MHz, CDCl3): δ 56.2, 61.0, 73.4, 84.0, 95.9, 113.4, 115.6, 124.0, 131.6, 135.9, 148.9, 151.1, 153.7, 158.1 (C=O). 19F NMR (376 MHz, CDCl3): δ ppm: - 132.6 (ArF). HRMS: calculated for C19H1935Cl2FNO5 [M + H]+ 430.0624; found 430.0633. 4.1.2.6. 4-(3-Fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin-2one (15): was synthesised using the general procedure II above from imine 6, and crotonyl chloride to afford the product as brown solid; yield: 70%, Mp: 105 °C, purity (HPLC): 95%, IRνmax (ATR): 1746.7 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.71 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.68 (d, J = 2.49 Hz, 1H, H3), 5.28 - 5.41 (m, 3H, H6, H4), 5.92 - 6.07 (m, 1H, H5), 6.52 (s, 2H, ArH), 6.91 - 7.00 (m, 1H, ArH), 7.04 - 7.12 (m, 2H, ArH).13C NMR (100 MHz, CDCl3): δ 56.1, 60.9, 63.91, 94.7, 113.6, 120.1, 121.8, 130.2, 133.6, 147.9, 151.5, 153.5, 164.9 (C=O). HRMS: calculated for C21H23FNO5 [M + H]+ 388.1560; found 388.1571. 4.1.2.7. 4-(3-Fluoro-4-methoxyphenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-trimethoxyphenyl) azetidin-2-one (16): was synthesised using the general procedure II above from imine 6 and dimethylacryloyl chloride to afford the product as a brown oil; yield: 32%, Mp: 119

21

°C, purity (HPLC): 99%, IR νmax (ATR): 1746.3 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 1.85 (s, 3H, H7), 3.66 (d, J = 2.07 Hz, 1H, C3), 3.71 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.71 (d, J = 2.49 Hz, 1H, H4), 5.02 (d, J = 18.66 Hz, 2H, H6), 6.52 (s, 2H, ArH), 6.96 (t, J = 8.50 Hz, 1H, ArH), 7.05 - 7.11 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ 20.5, 56.0, 59.7, 60.9, 67.0, 94.7, 113.6, 114.6, 121.8, 130.5, 133.5, 134.6, 137.8, 147.8, 151.5, 153.5, 164.9 (C=O). HRMS: calculated for C22H25FNO5 [M + H]+ 402.1717; found 402.1710. 4.1.2.8. 4-(3-Chloro-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin -2-one (17): was synthesised using the general procedure II above from imine 7 and phenylacetyl chloride to afford the product as yellow powder; yield: 42%, Mp: 118 °C, purity (HPLC): 100%, IRνmax (ATR): 1746.1 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.77 (s, 6H, OCH3), 3.81 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 4.29 (d, J = 2.01 Hz, 1H, H3), 4.86 (d, J = 2.01 Hz, 1H, H4), 6.62 (s, 2H, ArH), 6.99 (d, J = 8.53 Hz, 1H, ArH), 7.30 - 7.37 (m, 4H, ArH), 7.41 (d, J = 6.53 Hz, 2H, ArH), 7.47 (d, J = 2.01 Hz, 1H, ArH). C NMR (100 MHz, CDCl3): δ 56.1, 56.2, 60.9, 63.1, 65.1, 94.9, 112.6, 123.4, 125.3,

13

127.4, 127.9, 128.0, 129.1, 130.5, 153.6, 165.3 (C=O). HRMS: calculated for C25H2535ClNO5, 454.1421 [M + H]+ found 454.1431. 4.1.2.9.

4-(3-Chloro-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (18): was synthesised using the general procedure II above from imine 7 and phenoxyacetyl chloride to afford the product as yellow solid; yield: 28%, Mp: 129 °C, purity (HPLC): 100%, IRνmax (ATR): 1748.1 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.75 (s, 6H, OCH3), 3.80 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 4.94 (s, 1H, H4), 5.13 (s, 1H, H3), 6.58 (s, 2H, ArH), 6.90 (d, J = 8.53 Hz, 2H, ArH), 6.92 - 7.09 (m, 3H, ArH), 7.30 - 7.35 (m, 2H, ArH), 7.48 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ 56.1, 56.3, 60.9, 63.3, 64.9, 95.4, 112.6, 114.6, 115.4, 122.4, 125.9, 128.3, 129.5, 153.6, 162.4 (C=O). HRMS: calculated for C25H2535ClNO6 [M + H]+ 470.1370; found 470.1369. 4.1.2.10.

3-Chloro-4-(3-chloro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (19): was synthesised using the general procedure II above from imine 7 and chloroacetyl chloride to afford the product as an oil; yield: 42%, purity (HPLC): 99%, IRνmax (ATR): 1758.6 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.76 (s, 6H, OCH3), 3.81 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 4.63 (d, J = 2.20 Hz, 1H, H4), 4.93 (d, J 22

= 1.47 Hz, 1H, H3), 6.55 (s, 2H, ArH), 6.99 (d, J = 8.07 Hz, 1H, ArH), 7.27 (d, J = 2.20 Hz, 1H, ArH), 7.45 (d, J = 2.20 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ 56.1, 60.9, 63.1, 65.4, 95.3, 112.6, 123.7, 125.6, 128.0, 132.7, 153.6, 155.9, 160.4 (C=O). HRMS: calculated for C19H1935Cl2NaNO5 [M+Na]+ 434.0538; found 434.0521. 4.1.2.11

3,3-Dichloro-4-(3-chloro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (20): was synthesised using the general procedure IV above from imine 7, and dichloroacetyl chloride to afford the product as white powder; yield: 63%, Mp: 132 °C, purity (HPLC): 98%, IRνmax (ATR): 1759.1 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.77 (s, 6H, OCH3), 3.83 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 5.42 (s, 1H, H4), 6.56 (s, 2H, ArH), 7.01 (d, J = 8.53 Hz, 1H, ArH), 7.20 - 7.24 (m, 1H, ArH), 7.41 (d, J = 2.51 Hz, 1H, ArH).

13

C NMR (100 MHz, CDCl3): δ 56.2, 60.9, 68.1, 84.1, 95.9, 124.5,

127.3, 128.8, 129.6, 130.8, 131.6, 153.7, 156.2, 158.1, 167.7 (C=O). HRMS: calculated for C19H1835Cl4NO5 [M + Cl]+ 479.9939; found 479.9915. 4.1.2.12 4-(3-Chloro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin2-one (21): was synthesised using the general procedure II above from imine 7 and crotonyl chloride to afford the product as yellow solid; yield: 61%, Mp: 104 °C, purity (HPLC): 95%, IRνmax (ATR): 1746.5 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 3.74 (s, 6H, OCH3), 3.77 (s, 3H, OCH3), 3.83 (s, 1H, H3), 3.91 (s, 3H, OCH3), 4.68 - 4.76 (m, 1H, H4), 5.31 - 5.44 (m, 2H, H6), 5.94 - 6.09 (m, 1H, H5), 6.54 (s, 2H, ArH), 6.95 (d, J = 8.03 Hz, 1H, ArH), 7.20 - 7.31 (m, 1H, ArH), 7.39 - 7.43 (m, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ 55.6, 55.8, 60.2, 63.5, 94.2, 112.1, 119.7, 122.8, 124.9, 127.4, 129.8, 133.2, 153.1, 164.6 (C=O). HRMS: calculated for C21H2235ClNNaO5 [M + Na]+ 426.1084; found 426.1067. 4.1.2.13.

4-(3-Chloro-4-methoxyphenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-

trimethoxyphenyl)azetidin-2-one (22): was synthesised using the general procedure II above from imine 7, and dimethylacryloyl chloride to afford the product as an oil; yield: 68%, purity (HPLC): 94%, IRνmax (ATR): 1745.4 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 1.88 (s, 3H, H7), 3.71 (s, 1H, H3), 3.75 (s, 6H, OCH3), 3.78 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 4.74 (d, J = 2.01 Hz, 1H, H4), 5.05 (d, J = 19.58 Hz, 2H, H6), 6.56 (s, 2H, ArH), 6.96 (d, J = 8.53 Hz, 1H, ArH), 7.22 - 7.31 (m, 1H, ArH), 7.42 (d, J = 2.51 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ 20.5, 56.0, 56.2, 59.6, 60.9, 66.9, 94.7, 23

112.5, 114.7, 123.3, 125.3, 127.9, 130.7, 133.5, 137.8, 153.5, 155.2, 164.9 (C=O). HRMS: calculated for C22H2435ClNNaO5 [M + Na]+ 440.1241; found 440.1219. 4.1.2.14.

4-(3-Bromo-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (23): was synthesised using the general procedure II above from imine 8 and phenylacetyl chloride to afford the product as a brown oil; yield: 43%, purity (HPLC): 94%, IRνmax (ATR): 1745.1 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.72 (s, 6H, OCH3), 3.76 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.25 (d, J = 2.49 Hz, 1H, H3), 4.81 (d, J = 2.49 Hz, 1H, H4), 6.58 (s, 2H, ArH), 6.91 (d, J = 8.29 Hz, 1H, ArH), 7.28 - 7.33 (m, 4H, ArH), 7.33 - 7.43 (m, 2H, ArH), 7.60 (d, J = 2.49 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 56.1, 56.3, 60.9, 63.0, 65.1, 94.9, 112.4, 126.12, 127.4, 128.0, 129.1, 130.9, 131.0, 133.5, 134.4, 153.6, 165.3 (C=O). HRMS: calculated for C25H2579BrNO5 [M + H]+ 498.0916; found 498.0922. 4.1.2.15.

4-(3-Bromo-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (24): was synthesised using the general procedure II above from imine 8, and phenoxyacetyl chloride to afford the product as yellow oil; yield: 61%, purity (HPLC): 100%, IRνmax (ATR): 1748.1 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.71 (s, 6H, OCH3), 3.76 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.89 (d, J = 1.66 Hz, 1H, H4), 5.09 (d, J = 1.66 Hz, 1H, H3), 6.54 (s, 2H, ArH), 6.81 - 6.90 (m, 1H, ArH), 7.19 7.29 (m, 4H, ArH), 7.29 - 7.38 (m, 2H, ArH), 7.61 (d, J = 2.49 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 56.2, 56.4, 60.9, 63.2, 64.2, 87.2, 95.4, 112.4, 114.88, 115.4, 122.4, 126.7, 129.4, 131.5, 132.8, 153.6, 156.6, 162.3 (C=O). HRMS: calculated for C25H2579BrNO6 [M + H]+ 514.0865; found 514.0881. 4.1.2.16.

4-(3-Bromo-4-methoxyphenyl)-3-chloro-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (25): was synthesised using the general procedure II above from imine 8, and chloroacetyl chloride to afford the product as an oil; yield: 50%, purity (HPLC): 98%. IRνmax (ATR): 1743.3 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.72 (s, 6H, OCH3), 3.76 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.58 (d, J = 1.66 Hz, 1H, H4), 4.87 (d, J = 2.07 Hz, 1H, H3), 6.50 (s, 2H, ArH), 6.91 (d, J = 8.71 Hz, 2H, ArH), 7.28 (dd, J = 8.50, 2.28 Hz, 1H, ArH), 7.58 (d, J = 2.49 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 56.1, 56.4, 60.9, 63.1, 65.3, 95.3, 97.9, 110.0, 112.4, 126.3, 128.3, 131.2, 153.6, 156.8,

24

160.4 (C=O). HRMS: calculated for C19H2079Br35ClNO5 [M + H]+ 456.0213; found 456.0208. 4.1.2.17.

4-(3-Bromo-4-methoxyphenyl)-3,3-dichloro-1-(3,4,5-trimethoxyphenyl)

azetidin -2-one (26): was synthesised using the general procedure II above from imine 8 and dichloroacetyl chloride to afford the product as yellow oil; yield: 45%, purity (HPLC): 96%, IRνmax (ATR): 1736.1 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.70 (s, 6H, OCH3), 3.77 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 5.37 (s, 1H, H4), 6.54 (s, 2H, ArH), 6.83 (d, J = 8.29 Hz, 1H, ArH), 7.04 - 7.12 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 55.3, 56.1, 60.9, 74.2, 96.0, 109.9, 112.4, 122.8, 126.6, 127.4, 129.9, 132.0, 153.6, 158.0 (C=O). HRMS: calculated for C19H1979Br35Cl2NO5 [M + H]+ 489.9824; found 489.9818. 4.1.2.18. 4-(3-Bromo-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin2-one (27): was synthesised using the general procedure II above from imine 8 and crotonyl chloride to afford the product as a brown oil; yield: 63%, purity (HPLC): 95%, IRνmax (ATR): 1736.7 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.70 (s, 6H, OCH3), 3.74 (s, 3H, OCH3), 3.75 - 3.83 (m, 1H, H3), 3.91 (s, 3H, OCH3), 4.66 (d, J = 2.49 Hz, 1H, H4), 5.25 - 5.42 (m, 2H, H6), 5.98 (d, J = 10.26 Hz, 1H, H5), 6.51 (s, 2H, ArH), 6.88 (d, J = 8.71 Hz, 1H, ArH), 7.26 (dd, J = 8.29, 2.07 Hz, 1H, ArH), 7.54 (d, J = 2.49 Hz, 1H, ArH).

13

C NMR (100 MHz, CDCl3): δ ppm 56.31, 60.6, 60.9, 63.9, 94.7,

112.4, 120.1, 126.0, 130.8, 131.0, 133.6, 153.5, 156.1, 164.9 (C=O). HRMS: calculated for C21H2279BrNNaO5 [M + Na]+ 470.0579; found 470.0574. 4.1.2.19.

4-(3-Bromo-4-methoxyphenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-

trimethoxyphenyl)azetidin-2-one (28): was synthesised using the general procedure II above from imine 8 and dimethylacryloyl chloride to afford the product as yellow oil; yield: 47%, purity (HPLC): 96%, IRνmax (ATR): 1678.2 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 1.85 (s, 3H, H7), 3.68 (d, J = 2.07 Hz, 1H, H3), 3.72 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.70 (d, J = 2.49 Hz, 1H, H4), 5.02 (d, J = 18.25 Hz, 2H, H6), 6.53 (s, 2H, ArH), 6.89 (d, J = 8.29 Hz, 1H, ArH), 7.27 (dd, J = 8.50, 2.28 Hz, 2H, ArH), 7.56 (d, J = 2.49 Hz, 1H, ArH).

13

C NMR (100 MHz, CDCl3): δ ppm 20.5,

56.1, 56.3, 59.5, 60.9, 67.0, 94.7, 112.4, 114.7, 126.0, 131.0, 131.2, 133.5, 137.8, 153.6,

25

156.1, 164.9 (C=O). HRMS: calculated for C22H2479BrNNaO5 [M + Na]+ 484.0736; found 484.0730. 4.1.2.20. 4-(3-Iodo-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin2-one (29): was synthesised using the general procedure II above from imine 9 and phenylacetyl chloride to afford the product as as white powder; yield: 62%, Mp: 145 °C, purity (HPLC): 97%, IRνmax (ATR): 1742.9 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.69 (s, 6H, OCH3), 3.74 (s, 3H, OCH3), 3.83 (s, 3H, OCH3) 4.24 (d, J = 2.49 Hz, 1H, H3), 4.80 (d, J = 2.49 Hz, 1H, H4), 6.57 (s, 2 H, ArH), 6.80 (d, J = 8.29 Hz, 1H, ArH), 7.26 (s, 1H, ArH), 7.28 (s, 1H, ArH), 7.29 - 7.37 (m, 4H, ArH), 7.81 (d, J = 2.07 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 49.5, 50.6, 54.5, 60.9, 65.0, 66.2, 78.6, 94.8, 111.4, 127.4, 129.1, 131.4, 133.5, 134.4, 136.3, 137.2, 138.7, 153.5, 158.5, 171.1 (C=O). HRMS: calculated for C25H25INO5 [M + H]+ 546.0777; found 546.0789. 4.1.2.21.

4-(3-Iodo-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (30): was synthesised using the general procedure II above from imine 9 and phenoxyacetyl chloride to afford the product as an oil; yield: 67%, purity (HPLC): 98%. IRνmax (ATR): 1744.3 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.70 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.85 - 4.89 (m, 1H, H4), 5.09 (d, J = 1.66 Hz, 1H, H3), 6.53 (s, 2H, ArH), 6.84 (d, J = 8.29 Hz, 3H, ArH), 7.00 (t, J = 7.26 Hz, 1H, ArH), 7.20 - 7.31 (m, 3H, ArH), 7.35 (dd, J = 8.50, 2.28 Hz, 1H, ArH), 7.83 (d, J = 2.07 Hz, 1H, ArH).

13

C NMR (100 MHz, CDCl3): δ ppm 56.5, 60.9, 63.1, 86.8, 87.1,

95.3, 111.3, 114.6, 115.4, 122.4, 127.7, 129.7, 132.8, 137.7, 153.5, 156.9, 158.8, 162.4 (C=O). HRMS: calculated for C25H25INO6 [M + H]+ 562.0727; found 562.0721. 4.1.2.22. 3-Chloro-4-(3-iodo-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin2-one (31): was synthesised using the general procedure II above from imine 9 and chloroacetyl chloride to afford the product as yellow oil; yield: 54%, purity (HPLC): 100%. IRνmax (ATR): 1730.1 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.71 (s, 6H, OCH3) 3.75 (s, 3H, OCH3) 3.87 (s, 3H) 4.59 (d, J = 2.07 Hz, 1H, H4) 4.85 (d, J = 2.07 Hz, 1H, H3) 6.50 (s, 2H, ArH) 6.78 - 6.86 (m, 2H, ArH) 7.30 (dd, J = 8.50, 2.28 Hz, 1H, ArH) 7.80 (d, J = 2.07 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 56.1, 56.5, 60.9, 63.0, 65.1, 95.3, 105.9, 111.3, 127.3, 128.9, 131.4, 137.4, 153.6, 154.3, 168.8 (C=O). HRMS: calculated for C19H2035ClINO5 [M + H]+ 504.0075; found 504.0070.

26

4.1.2.23.

3,3-Dichloro-4-(3-iodo-4-methoxyphenyl)-1-(3,4,5-

trimethoxyphenyl)azetidin-2-one (32): was synthesised using the general procedure II above from imine 9 and dichloroacetyl chloride to afford the product as brown oil; yield: 34%, purity (HPLC): 100%, IRνmax (ATR): 1728.0 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.70 (s, 6H, OCH3), 3.76 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 5.36 (s, 1H, H4), 6.50 (s, 2H, ArH), 6.82 (d, J = 8.29 Hz, 1H, ArH), 7.23 (d, J = 7.05 Hz, 1H, ArH), 7.75 (d, J = 2.07 Hz, 1H, ArH).

13

C NMR (100 MHz, CDCl3): δ ppm 56.2, 56.5, 61.0,

86.2, 95.9, 110.8, 125.4, 129.1, 131.6, 138.8, 153.7, 158.2, 159.3 (C=O). HRMS: calculated for C19H1935Cl2INO5 [M + H]+ 537.9685; found 537.9680. 4.1.2.24. 4-(3-Iodo-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin-2one (33): was synthesised using the general procedure II above from imine 9 and crotonyl chloride to afford the product as yellow oil; yield: 51%, purity (HPLC): 100%, IRνmax (ATR): 1728.8 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.72 (s, 7H, OCH3, H3), 3.77 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.65 (d, J = 2.49 Hz, 1H, H4), 5.26 - 5.42 (m, 2H, H6), 5.91 - 6.05 (m, 1H, H5), 6.52 (s, 2H, ArH), 6.80 (d, J = 8.71 Hz, 1H, ArH), 7.06 (dd, J = 14.93, 7.05 Hz, 1H, ArH), 7.30 (dd, J = 8.29, 2.07 Hz, 2H, ArH), 7.78 (d, J = 2.07 Hz, 1H, ArH).

13

C NMR (100 MHz, CDCl3): δ ppm 18.3, 56.1, 56.5,

60.9, 63.8, 86.6, 94.8, 106.2, 111.3, 120.1, 124.9, 127.1, 131.3, 133.6, 137.2, 153.5, 165.0 (C=O). HRMS: calculated for C21H22INNaO5 [M + Na]+ 518.0440; found.518.0435. 4.1.2.25. 4-(3-Iodo-4-methoxyphenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-trimethoxyphenyl) azetidin-2-one (34): was synthesised using the general procedure II above from imine 9, and dimethylacryloyl chloride to afford the product as brown oil; yield: 46%, purity (HPLC): 100%, IRνmax (ATR): 1741.3 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 1.82 (s, 3H, H7), 3.64 - 3.67 (m, 1H, H3), 3.68 (s, 6H, OCH3), 3.71 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.67 (d, J = 2.49 Hz, 1H, H4), 4.96 (s, 1H, H6), 5.01 (s, 1H, H6), 6.50 (s, 2H, ArH), 6.78 (d, J = 8.71 Hz, 1H, ArH), 7.28 (dd, J = 8.29, 2.07 Hz, 1H, ArH), 7.77 (d, J = 2.07 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 20.5, 53.5, 56.0, 56.4, 59.2, 60.9, 66.9, 86.6, 94.7, 111.3, 114.6, 127.1, 131.6, 133.5, 137.9, 153.5, 158.4, 164.9 (C=O). HRMS: calculated for C22H25INO5 [M + H]+ 510.0777; found 510.0773. 4.1.2.26.

4-(4-Methoxy-3-methylphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (35): was synthesised using the general procedure II above from imine

27

10, and phenylacetyl chloride to afford the product as yellow solid; yield: 58%, Mp: 158 °C, purity (HPLC): 95%, IRνmax (ATR): 1738.7 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.21 (s, 3H, CH3), 3.71 (s, 6H, OCH3), 3.76 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 4.27 (d, J = 2.07 Hz, 1H, H3), 4.81 (d, J = 2.49 Hz, 1H, H4), 6.61 (s, 2H, ArH), 6.82 (d, J = 8.29 Hz, 1H, ArH), 7.15 - 7.23 (m, 2H, ArH), 7.25 - 7.32 (m, 2H, ArH), 732 7.39 (m, 3H, ArH).

13

C NMR (100 MHz, CDCl3): δ ppm 16.3, 55.4, 56.0, 60.2, 60.9,

63.9, 65.0, 94.9, 110.2, 124.7, 127.8, 128.2, 133.8, 134.9, 153.5, 158.1, 165.8 (C=O). HRMS: calculated for C26H28NO5 [M + H]+ 434.1967; found 434.1973. 4.1.2.27.

4-(4-Methoxy-3-methylphenyl)-3-phenoxy-1-(3,4,5-trimethoxy

phenyl)azetidin-2-one (36): was synthesised using the general procedure II above from imine 10, and phenoxyacetyl chloride to afford the product as pale yellow powder; yield: 62%, Mp: 162 °C, purity (HPLC): 97%, IRνmax (ATR): 1758.9 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.21 (s, 3H, CH3), 3.69 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.88 (s, 1H, H4), 5.16 (s, 1H, H3), 6.57 (s, 2H, ArH), 6.79 - 6.87 (m, 2H, ArH), 6.90 (d, J = 8.71 Hz, 2H, ArH), 6.93 - 7.04 (m, 2H, ArH), 7.18 - 7.32 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 16.3, 55.4, 56.0, 60.9, 64.2, 65.0, 87.2, 95.4, 110.3, 114.6, 115.4, 121.9, 122.2, 125.4, 126.9, 128.0, 128.5, 129.6, 133.2, 153.4, 157.0, 158.4, 162.8 (C=O). HRMS: calculated for C26H28NO6: [M + H]+ 450.1917; found 450.1914. 4.1.2.28.

3-Chloro-4-(4-methoxy-3-methylphenyl)-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (37): was synthesised using the general procedure II above from imine 10, and chloroacetyl chloride to afford the product as a brown solid; yield: 42%, Mp: 142 °C, purity (HPLC): 97%. IRνmax (ATR): 1746.4 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.20 (s, 3H, CH3), 3.70 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 4.59 (d, J = 1.66 Hz, 1H, H4), 4.86 (d, J = 1.66 Hz, 1H, H3), 6.53 (s, 2H, ArH), 6.81 (d, J = 8.29 Hz, 1H, ArH), 7.07 - 7.20 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 16.3, 55.4, 56.1, 60.9, 63.2, 66.2, 95.4, 110.3, 125.0, 126.9, 128.2, 133.04, 153.5, 158.7, 160.9 (C=O). HRMS: calculated for C20H2335ClNO5 [M + H]+ 392.1265; found 392.1257. 4.1.2.29.

3,3-Dichloro-4-(4-methoxy-3-methylphenyl)-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (38): was synthesised using the general procedure II above from imine

28

10, and dichloroacetyl chloride to afford the product as yellow solid; yield: 50%, Mp: 150 °C, purity (HPLC): 95%, IRνmax (ATR): 1766.4 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.17 (s, 3H, CH3), 3.67 (s, 6H, OCH3), 3.73 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 5.37 (s, 1H, H4), 6.52 (s, 2H, ArH), 6.80 (d, J = 8.29 Hz, 1H, ArH), 7.01 - 7.16 (m, 2H, ArH).

13

C NMR (100 MHz, CDCl3): δ ppm 16.3, 55.3, 56.1, 60.9, 84.3, 96.0,

109.9, 122.8, 126.6, 127.3, 129.9, 131.9, 135.7, 153.6, 158.4, 162.1 (C=O). HRMS: calculated for C20H2235Cl2NO5 [M + H]+ 426.0875; found 426.0863. 4.1.2.30. 4-(4-Methoxy-3-methylphenyl)-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin2-one (39): was synthesised using the general procedure II above from imine 10, and crotonyl chloride to afford the product as white solid; yield: 71%, Mp: 171° C, purity (HPLC): 97%, IRνmax (ATR): 1730.3 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.17 (s, 3H, CH3), 3.65 - 3.71 (m, 7H, OCH3 & H3), 3.73 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 4.66 (d, J = 2.49 Hz, 1 H, H4), 5.19 - 5.40 (m, 2 H, H6), 5.90 - 6.06 (m, 1 H, H5), 6.54 (s, 2H, ArH), 6.78 (d, J = 8.29 Hz, 1H, ArH), 7.06 - 7.23 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 16.3, 55.3, 56.0, 60.9, 61.4, 63.8, 94.8, 110.2, 119.6, 124.7, 127.6, 128.1, 130.7, 133.9, 153.4, 158.0, 165.4 (C=O). HRMS: calculated for C22H25NNaO5 [M + Na]+ 406.1630; found 406.1644. 4.1.2.31.

4-(4-Methoxy-3-methylphenyl)-3-(prop-1-en-2-yl)-1-(3,4,5-

trimethoxyphenyl)azetidin-2-one (40): was synthesised using the general procedure II above from imine 10, and dimethylacryloyl chloride to afford the product as yellow powder; yield: 64%, Mp: 164 °C, purity (HPLC): 96%, IRνmax (ATR): 1730.7 (C=O) cm1 1

. H NMR (400 MHz, CDCl3): δ ppm 1.84 (s, 3H, H7), 2.18 (s, 3H, CH3), 3.69 (s, 6H,

OCH3), 3.73 (s, 3H, OCH3), 3.75 - 3.79 (m, 1H, H3), 3.80 (s, 3H, OCH3), 4.69 (d, J = 2.49 Hz, 1H, H4), 4.96 (s, 1H, H6), 5.03 (s, 1H, H6), 6.55 (s, 2H, ArH), 6.79 (d, J = 8.29 Hz, 1H, ArH), 7.06 - 7.20 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 16.3, 20.6, 55.3, 60.4, 60.9, 66.8, 94.8, 110.2, 114.2, 124.7, 127.7, 129.0, 133.9, 138.2, 153.4, 157.0, 165.4 (C=O). HRMS: calculated for C23H27NNaO5 [M + Na]+ 420.1787; found 420.1773. 4.1.3. 4.1.3.

General Method III: β-Lactam preparation (Reformatsky reaction)

Zinc powder (9 mmol) was activated using trimethylchlorosilane (7 mmol) in anhydrous benzene (4 mL) by heating for 15 min at 40 °C and subsequently for 2 min at 100 °C with microwave irradiation. After cooling, the appropriate imine (2 mmol) and appropriate

29

ethyl bromoacetate (5 mmol) were added to the reaction vessel and the mixture was placed in the microwave reactor for 30 min at 100 °C. The reaction mixture was filtered through Celite to remove the zinc catalyst and then diluted with DCM (30 mL). This solution was washed with saturated ammonium chloride solution (20 mL) and 25 % ammonium hydroxide (20 mL), and then with dilute HCl (40 mL), followed by water (40 mL). The organic phase was dried over anhydrous Na2SO4 and the solvent was removed by evaporation in vacuo. The crude product was isolated by flash column chromatography over silica gel (eluent: hexane: ethyl acetate gradient). 4.1.3.1

4-(3-Fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one

(41): was prepared following the general method III from imine 6 and ethyl 2bromoacetate to afford the product as yellow oil; yield: 37%, purity (HPLC): 97%, IRνmax (ATR): 1745.8 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ 2.90 (dd, J = 15.14, 2.70 Hz, 1H, H3), 3.51 (dd, J = 15.34, 5.81 Hz, 1H, H3), 3.71 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 4.88 (dd, J = 5.39, 2.49 Hz, 1H, H4), 6.51 (s, 2H, ArH), 6.95 (t, J = 8.29 Hz, 1H, ArH), 7.10 (d, J = 9.95 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ 46.9, 53.6, 56.0, 60.9, 94.5, 113.6, 121.8, 131.1, 133.8, 153.5, 157.2, 164.1 (C=O). HRMS: calculated for C19H21FNO5 [M + H]+ 362.1404; found 362.1392. 4.1.3.2

4-(3-Chloro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one

(42): was prepared following the general method III from imine 7 and ethyl 2bromoacetate to afford the product as yellow oil; yield: 22%, Mp: 92 – 93 °C, purity (HPLC): 96%, IRνmax (ATR): 1751.9 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.96 (dd, J = 15.06, 2.51 Hz, 1H, H3), 3.56 (dd, J = 15.31, 5.77 Hz, 1H, H3), 3.73 (s, 3H, OCH3), 3.82 (s, 6H, OCH3), 3.93 (s, 3H, OCH3), 4.89 - 4.96 (m, 1H, H4), 6.55 (s, 2H, ArH), 6.96 (d, J = 8.53 Hz, 1H, ArH), 7.27 (br. s., 1H, ArH), 7.44 (s, 1 H, ArH). 13C NMR (100 MHz, CDCl3): δ 46.5, 53.1, 55.6, 60.5, 94.0, 112.0, 124.9, 127.5, 130.7, 134.0, 153.1, 154.8, 163.8 (C=O). HRMS: calculated for C19H2035Cl2NO5 [M + Cl]+ 412.0719; found 412.0720. 4.1.3.3

4-(3-Bromo-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one

(43): was prepared following the general method III from imine 8, and ethyl 2bromoacetate to afford the product as a brown oil; yield: 32%, purity (HPLC): 100%, IRνmax (ATR): 1747.9 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.90 (dd, J =

30

14.93, 2.49 Hz, 1H, H3), 3.50 (dd, J = 15.14, 5.60 Hz, 1H, H3), 3.70 (s, 6 H, OCH3), 3.73 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.86 (dd, J = 5.39, 2.49 Hz, 1H, H4), 6.50 (s, 2H, ArH), 6.87 (d, J = 8.29 Hz, 1H, ArH), 7.21 - 7.32 (m, 1H, ArH), 7.56 (d, J = 2.07 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 46.9, 53.4, 56.0, 56.29, 60.9, 94.5, 112.3, 126.1, 131.1, 131.6, 133.8, 153.5, 156.1, 164.1 (C=O). HRMS: calculated for C19H2179BrNO5 [M + H]+ 422.0603; found 422.0597. 4.1.3.4 4-(3-Iodo-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl) azetidin-2-one (44): was prepared following the general method III from imine 9 and ethyl 2-bromoacetate to afford the product as brown oil; yield: 32%, purity (HPLC): 96%, IRνmax (ATR): 1747.9 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.91 (dd, J = 14.93, 2.49 Hz, 1H, H3), 3.50 (dd, J = 15.14, 5.60 Hz, 1H, H3), 3.70 (s, 6H, OCH3), 3.73 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.84 (dd, J = 5.81, 2.49 Hz, 1H, H4), 6.50 (s, 2 H, ArH), 6.78 (d, J = 8.29 Hz, 1H, ArH), 7.30 (dd, J = 8.29, 2.07 Hz, 1H, ArH), 7.79 (d, J = 2.07 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 14.2, 21.0, 46.8, 53.2, 56.4, 60.3, 86.4, 94.5, 111.2, 127.1, 132.1, 133.8, 134.5, 137.3, 153.5, 158.3, 164.2 (C=O). HRMS: calculated for C19H20INNaO5 [M + Na]+ 492.0284; found 492.0278. 4.1.3.5.

4-(3-Methyl-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one

(45): was prepared following the general method III from imine 10 and ethyl 2bromoacetate to afford the product as an oil; yield: 30%, purity (HPLC): 100%, IRνmax (ATR): 1747.9 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.18 (s, 3H, CH3), 2.92 (dd, J = 15.14, 2.70 Hz, 1H, H3), 3.48 (dd, J = 15.14, 5.60 Hz, 1H, H3), 3.70 (s, 6H, OCH3), 3.77 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 4.85 (dd, J = 5.60, 2.70 Hz, 1H, H4), 6.54 (s, 2H, ArH), 6.78 (d, J = 8.29 Hz, 1H, ArH), 7.06 - 7.21 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 16.3, 46.9, 54.2, 55.4, 60.9, 94.5, 110.1, 124.7, 127.6, 129.5, 134.2, 153.4, 157.9, 164.7 (C=O). HRMS: calculated for C20H23NNaO5 [M + Na]+ 380.1474; found 380.1479. 4.1.4. General method IV: preparation of 3-hydroxy-azetidin-2-ones The imine (5mmol) and 2-acetoxyacetyl chloride (7 mmol) were dissolved in dry toluene (50 mL), under nitrogen with stirring at 0 °C. The solution was allowed to reach room temperature and then warmed to 100 °C. Dry TEA (9 mmol) was added dropwise for 10 min and kept it at 100 °C for 5 h. The reaction mixture was washed with water (50 mL

31

2), dried over anhydrous Na2SO4 and solvent was removed in vacuo. The crude product was purified by flash chromatography over silica gel (eluent: n-hexane: ethyl acetate, 2:1). Hydrazine dichloride (5 mmol) was added to a stirred solution of the crude product in methanol (30 mL) at 0 °C and under nitrogen, then dry TEA (9 mmol) was added dropwise for 10 min. The mixture was allowed to reach room temperature and then heated at reflux atfor 2-4 h. The solvent was removed by evaporation at reduced pressure, and the residue was treated with a saturated solution of KHSO4 and extracted with ethyl acetate (3

50 mL). The organic phase was dried over Na2SO4, and the solvent after

filtration, was removed by evaporation under reduced pressure. The crude residue was purified by flash chromatography over silica gel (eluent: n-hexane: ethyl acetate, 6:1) to afford the desired product. 4.1.4.1.

4-(3-Fluoro-4-methoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (46): was synthesised using the general procedure IV above from imine 6 and afforded the product as yellow powder; yield: 29%, Mp: 135 °C, purity (HPLC): 97%, IRνmax (ATR): 3372.05 (OH), 1732.68 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ3.69 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.70 (d, J = 1.66 Hz, 1H, H4), 4.75 (d, J = 1.66 Hz, 1H, H3), 6.47 (s, 2H, ArH), 6.95 (d, J = 8.50 Hz, 1H, ArH), 7.02 - 7.07 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ 56.0, 60.9, 63.1, 82.4, 95.3, 113.8, 122.3, 127.8, 132.7, 135.2, 153.6, 161.3, 169.7 (C=O).

19

F NMR (376 MHz,

CDCl3): δ ppm: - 133.26 (ArF). HRMS: calculated for C19H20FNNaO6 [M + Na]+ 400.1172; found 400.1173. 4.1.4.2.

4-(3-Chloro-4-methoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (47): was synthesised using the general procedure IV above from imine 7 and afforded the product as yellow oil; yield: 27%, purity (HPLC): 94%, IRνmax (ATR): 3221.1 (OH), 1730.2 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.72 (s, 6H, OCH3), 3.77 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.93 (s, 1H, H4), 4.77 (m, 1H, H3), 6.51 (s, 2H, ArH), 6.95 (d, J = 8.03 Hz, 1H, ArH), 7.22 (d, J = 8.03 Hz, 1H, ArH), 7.37 - 7.43 (m, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 55.6, 60.5, 64.4, 83.1, 94.86, 112.0, 122.9, 125.2, 127.6, 128.6, 132.5, 134.5, 153.1, 154.9, 165.8 (C=O). HRMS: calculated for C19H2035Cl2NO6 [M + Cl]+ 428.0668; found 428.0670.

32

4.1.4.3.

4-(3-Bromo-4-methoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (48): was synthesised using the general procedure IV above from imine 8 and afforded the product as oil; yield 39%, purity (HPLC): 97%, IRνmax (ATR): 3249.4 (OH), 1728.5 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.67 (s, 6H, OCH3), 3.74 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 4.65 (s, 1H, H4), 4.72 (s, 1H, H3), 6.44 (s, 2H, ArH), 6.86 (d, J = 8.71 Hz, 1H, ArH), 7.20 (dd, J = 8.50, 2.28 Hz, 1H, ArH), 7.52 (d, J = 2.07 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 56.1, 60.9, 64.8, 83.5, 95.3, 112.3, 126.3, 129.5, 131.1, 132.9, 134.8, 153.5, 156.2, 166.6 (C=O). HRMS: calculated for C19H1979BrNO6 [M - H]+ 436.0402; found 436.0401. 4.1.4.4. 3-Hydroxy-4-(3-iodo-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl) azetidin2-one (49): was synthesised using the general procedure IV above from imine 9 and afforded the product as yellow oil; yield 46%, purity (HPLC): 99%, IRνmax (ATR): 3386.4 (OH), 1730.0 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 3.67 (s, 6H, OCH3), 3.74 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.20 (s, 1H, H4), 4.71 (d, J = 1.66 Hz, 1H, H3), 4.73 (s, br, 1H, OH), 6.45 (s, 2H, ArH), 6.78 (d, J = 8.71 Hz, 1H, ArH), 7.16 7.27 (m, 1H, ArH), 7.76 (d, J = 2.07 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 56.5, 61.0, 64.6, 83.3, 95.4, 111.1, 127.4, 129.9, 132.8, 134.8, 137.4, 153.4, 158.5, 167.0 (C=O). HRMS: calculated for C19H21INO6 [M + H]+ 486.0414; found 486.0408. 4.1.4.5.

3-Hydroxy-4-(4-methoxy-3-methylphenyl)-1-(3,4,5-trimethoxyphenyl)

azetidin-2-one (50): was synthesised using the general procedure IV above from imine 10 and afforded the product as grey soild; yield: 40%, Mp: 146 °C, purity (HPLC): 96%, IRνmax (ATR): 3271.31 (OH), 1724.32 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 2.17 (s, 3H, CH3), 3.65 (s, 6H, OCH3), 3.73 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 4.71 (s, 2H, H4), 4.79 (s, 2H, H3), 6.48 (s, 2H, ArH), 6.77 (d, J = 8.29 Hz, 1H, ArH), 7.03 - 7.14 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 16.3, 55.4, 60.9, 65.8, 83.5, 95.4, 110.1, 124.9, 127.4, 128.3, 133.2, 134.7, 153.3, 167.2 (C=O). HRMS: calculated for C20H23NNaO6 [M + Na]+ 396.1423; found 396.1417. 4.1.5. General method V: preparation of 3-amino β-lactams To a stirring refluxing solution of N-phthaloylglycine (2 mmol) and Mukaiyama’s reagent (2-chloro-1-methylpyridinium iodide) (6mmol) in anhydrous DCM (40 mL), a solution of TEA (6 mmol) in anhydrous DCM (10 mL) was added over 30 minutes under

33

nitrogen at 0 °C. The reaction was kept at reflux for 2 hours at 0 °C. The solution of imine in dry DCM (10 mL) was added dropwise and was kept at reflux for another two hours at 0°C and at room temperature overnight, continuously under nitrogen, until the starting material had disappeared as monitored by TLC in 50:50, hexane: ethylacetate. The reaction was transferred to a separating funnel and washed with water (2

100 mL),

with the organic layer being retained each time. The reaction was dried over anhydrous sodium sulfate before the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography over silica gel eluted with 5:1 hexane: ethylactate. To a stirring solution of this protected β-lactam (5 mmol) under nitrogen dry ethanol was added dropwise (2 equivalents) of ethylenediamine solution in ethanol. The resulting solution was left to stir at room temperature until reaction was complete as seen on TLC. The reaction mixture was diluted with ethylacetate (50 mL) and washed with 0.1 M HCL (100 mL). The aqueous layer was further extracted with ethylacetate (2

25 mL). All organic layers were collected and washed with water (100

mL), and saturated brine (100 mL) before being dried over anhydrous sodium sulfate. Solvent was removed under reduced pressure to yield amino compound which further purified by flash chromatography over silica gel eluted with 1:1 hexane: ethylacetate. 4.1.5.1. 3-Amino-4-(3-fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl) azetidin2-one (51): was synthesised as described in the general method V of β-lactam synthesis above from imine 6, and N-phthaloylglycine to afford the 3-amino compound as yellow solid; yield: 12%, Mp: 125 ˚C, purity (HPLC): 100%, IRνmax (ATR): 3394.3, 3404.9 (NH2), 1743.93 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm: 3.70 (s, 6H, OCH3), 3.75 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 4.01 (d, J = 2.07 Hz, 1H, H3), 4.54 (d, J = 2.07 Hz, 1H, H4), 6.50 (s, 2H, ArH), 6.93 - 6.96 (m, 1H, ArH), 7.06 -7.08 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm: 56.1, 60.9, 66.01, 69.8, 95.1, 113.8, 121.9, 129.7, 133.3, 148.0, 153.5, 167.6 (C=O). 19F NMR (376 MHz, CDCl3): δ ppm: - 133.43 (ArF). HRMS: calculated for C19H22FN2O5 [M + H]+ 377.1513; found 377.1505. 4.1.5.2. 3-Amino-4-(3-chloro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)azetidin2-one (52): was synthesised as described in the general method V of β-lactam synthesis above from imine 7, and N-phthaloylglycine to afford the 3-amino compound as grey solid; yield: 28%, purity (HPLC): 96%, Mp: 111 – 112 °C, IRνmax (ATR): 3360.2, 3395.4

34

(NH2), 1751.98 (C=O) cm-1. 1H NMR (400 MHz, CDCl3): δ ppm 1.82 (br. s., 2H, NH2), 3.68 (s, 6H, OCH3), 3.73 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.99 (s, 1H, H3), 4.51 (d, J = 2.07 Hz, 1H, H4), 6.47 (s, 2H, ArH), 6.90 (d, J = 8.29 Hz, 1H, ArH), 7.18 (dd, J = 8.50, 2.28 Hz, 1H, ArH), 7.35 (d, J = 2.07 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ ppm 56.1, 56.2, 60.9, 65.9, 69.8, 95.0, 112.4, 123.3, 125.3, 127.8, 129.9, 133.4, 153.5, 155.2, 167.7 (C=O). HRMS: calculated for C19H2135Cl2N2NaO5 [M + Na]+ 415.1037; found 415.1015. 4.1.6. Stability study of compounds 18, 29 and 52
 Stability studies for compounds 18, 29 and 52
were performed by analytical HPLC using a Symmetry® column (C18, 5 mm, 4.6 x150 mm), a Waters 2487 Dual Wavelength Absorbance detector, a Waters 1525 binary HPLC pump and a Waters 717 plus Autosampler (Waters Corporation, Milford, MA, USA). Samples were detected at λ 254 nm using acetonitrile (70%): water (20%) as the mobile phase over 15 min and a flow rate of 1 mL/min. Stock solutions of the compounds are prepared using 10 mg of compounds 18, 29 and 52 in 10 mL of mobile phase (1 mg/mL). Phosphate buffers at the desired pH values (4, 7.4 and 9) were prepared following the British Pharmacopoeia monograph 2015. 30 µL of stock solution was diluted with 1 mL of appropriate buffer, shaken and injected immediately. Samples were withdrawn and analysed at time intervals of t = 0 min, 5 min, 30 min, 60 min and after every hour for 24 h.

4.2 Biochemical Evaluation of Activity: All biochemical assays were performed in triplicate on at least three independent occasions for the determination of mean values reported. 4.2.1 Cell culture: The human breast tumour cell line MCF-7 was cultured in Eagles minimum essential medium with 10% fetal bovine serum, 2 mM L-glutamine and 100 µg/mL penicillin/streptomycin. The medium was supplemented with 1% non-essential amino acids. HT-29 cells originate from a human adenocarcinoma of the colon and were originally obtained from the European Collection of Cell Cultures and were grown in DMEM Glutamax media. HT-29 media were supplemented with 10% foetal bovine serum (FBS). HEK-293T normal epithelial embryonic kidney cells were cultured in

35

Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAXTM-I in the absence of non-essential amino acids. All media contained 100 U/ml penicillin and 100 µg/mL streptomycin. Cells were maintained at 37˚C in 5% CO2 in a humidified incubator. All cells were sub-cultured 3 times/week by trypsinisation using TrypLE Express (1X). 4.2.2 Cell viability assay: Cells were seeded at a density of 5 x 103 cells/well (MCF-7) and 1x104 cells/well (HT-29 and HEK-293T) in 96-well plates. After 24 h, cells were then treated with either medium alone, vehicle [1% ethanol (v/v)] or with serial dilutions of CA-4 or β-lactam analogue (0.001-100 µM) in triplicate. Cell proliferation for MCF-7 cells was analysed using the Alamar Blue assay (Invitrogen Corp.) according to the manufacturer's instructions. After 72 h, Alamar Blue [10% (v/v)] was added to each well and plates were incubated for 3-5 h at 37˚C in the dark. Fluorescence was read using a 96-well fluorimeter with excitation at 530 nm and emission at 590 nm. Results were expressed as percentage viability relative to vehicle control (100%). Dose response curves were plotted and IC50 values (concentration of drug resulting in 50% reduction in cell survival) were obtained using the commercial software package Prism (GraphPad Software, Inc., La Jolla, CA, USA). 4.2.3 Cell cycle analysis and apoptosis: For cell cycle analysis, cells were seeded at a density of 1x105 cells/well in 6-well plates and treated with compound 46 (1 µM) for 24, 48 and 72 hr. The cells were collected by trypsinization and centrifuged at 800x g for 15 min. Cells were washed twice with ice-cold PBS and fixed in ice-cold 70% ethanol overnight at −20 °C. Fixed cells were centrifuged at 800 x g for 15 min and stained with 50 µg/mL of PI, containing 50 µg/mL of DNase-free RNase A, at 37 °C for 30 min. The DNA content of cells (10,000 cells/experimental group) was analysed by flow cytometer at 488 nm using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and all data were recorded and analysed using the CellQuest Software (Becton-Dickinson). For Annexin V/PI Apoptotic Assay, apoptotic cell death was detected by flow cytometry using Annexin V and propidium iodide (PI). MCF-7 Cells were seeded in 6 well plated at density of 1

105 cells/mL and treated with either vehicle (0.1 % (v/v) EtOH), CA-4 or

β-lactam compound 46 at different concentrations for selected time. Cells were then harvested and prepared for flow cytometric analysis. Cells were washed in 1X binding

36

buffer (20X binding buffer: 0.1M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl2 diluted in dH2O) and incubated in the dark for 30 minutes on ice in Annexin V-containing binding buffer [1:100]. Cells were then washed once in binding buffer and then re-suspended in PI-containing binding buffer [1:1000]. Samples were analysed immediately using the BD accuri flow cytometer and prism software for analysis the data. Four populations are produced during the assay Annexin V and PI negative (Q4, healthy cells), Annexin V positive and PI negative (Q3, early apoptosis), Annexin V and PI positive (Q2, late apoptosis) and Annexin V negative and PI positive (Q1, necrosis). 4.2.4 Immunofluorescence Microscopy: Confocal microscopy was used to study the effects of drug treatment on MCF-7 cytoskeleton. For immunofluorescence, MCF-7 cells were seeded at 1 × 105 cells/mL on eight chamber glass slides (BD Biosciences). Cells were treated with vehicle [1 % ethanol (v/v)], CA-4 (0.01 µM) and 46, (0.01 µM, 0.05 µM and 0.1 µM) for 16 h. Following treatment, cells were gently washed in PBS, fixed for 20 min with 4 % paraformaldehyde in PBS and permeabilised in 0.5 % Triton X-100. Following washes in PBS containing 0.1 % Tween (PBST), cells were blocked in 5 % bovine serum albumin diluted in PBST. Cells were then incubated with mouse monoclonal anti-α-tubulin−FITC antibody (clone DM1A) (Sigma) (1:100) for 2 h at rt. Following washes in PBST, cells were incubated with Alexa Fluor 488 dye (1:500) for 1 h at rt. Following washes in PBST, the cells were mounted in Ultra Cruz Mounting Media (Santa Cruz Biotechnology, Santa Cruz, CA) containing 4,6-diamino-2phenolindol dihydrochloride (DAPI). Images were captured by Leica SP8 confocal microscopy with Leica application suite X software. All images in each experiment were collected on the same day using identical parameters. Experiments were performed on three independent occasions. 4.2.5 Evaluation of Expression Levels of Anti-Apoptotic Proteins Mcl-1, proApoptotic Proteins Bax and PARP cleavage: MCF-7 cells were seeded at a density of 1 × 105 cells/flask in T25 flasks. After 48 h, whole cell lysates were prepared from untreated cells or cells treated with vehicle control (EtOH, 0.1% v/v) or compound 46 (0.05 µM, 0.1 µM and 0.5 µM). MCF-7 cells were harvested in RIPA buffer supplemented with protease inhibitors (Roche Diagnostics), phosphatase inhibitor

37

cocktail 2 (Sigma-Aldrich), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich). Equal quantities of protein (as determined by a BCA assay) were resolved by SDS-PAGE (12%) followed by transfer to PVDF membranes. Membranes were blocked in 5% bovine serum albumin/TBST for 1 h. Membranes were incubated in the relevant primary antibodies at 4 °C overnight, washed with TBST, and incubated in horseradish peroxidase conjugated secondary antibody for 1 h at rt and washed again. Western blot analysis was performed as described above using antibodies directed against Mcl-1 [1:1000] (Millipore), survivin [1:1000] (Millipore) and Bcl-2 [1:500] (Millipore) followed by incubation with a horseradish peroxidase-conjugated anti-mouse antibody [1:2000] (Promega, Madison, WI, USA). All blots were probed with anti-GAPDH antibody [1:5000] (Millipore) to confirm equal loading. Proteins were detected using enhanced chemiluminescent Western blot detection (Clarity Western ECL substrate) (Bio Rad) on the ChemiDoc MP System (Bio Rad). Experiments were performed on three independent occasions. 4.2.6 Tubulin polymerization assay: The assembly of purified bovine tubulin was monitored using a kit, BK006, purchased from Cytoskeleton Inc., (Denver, CO, USA). The assay was carried out in accordance with the manufacturer's instructions using the standard assay conditions [86]. Briefly, purified (>99%) bovine brain tubulin (3 mg/mL) in a buffer consisting of 80 mM PIPES (pH 6.9), 0.5 mM EGTA, 2 mM MgCl2, 1 mM GTP and 10% glycerol was incubated at 37 °C in the presence of either vehicle (2% (v/v) ddH2O) or Paclitaxel, CA-4, 46, 47, 48 and 50 (all at 10 µM). Light is scattered proportionally to the concentration of polymerised microtubules in the assay. Therefore, tubulin assembly was monitored turbidimetrically at 340 nm in a Spectramax 340 PC spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The absorbance was measured at 30 s intervals for 60 min. 4.2.7 Colchicine-binding Site Assay: N,N’-Ethylene-bis(iodoacetamide) (EBI) (Santa Cruz Biotechnology) dissolved in ethanol (100 mM). MCF-7 cells were seeded at a density of 5 × 104 cells/well in 6-well plates and incubated overnight. Cells were treated with vehicle control [ethanol (0.1 % v/v)], CA-4 and compound 46 (all 10 µM) for 2 h. After this time, selected wells were treated with EBI (100 µM) for 1.5 h. Following

38

treatment, cells were twice washed with ice-cold PBS and lysed by addition of Laemmli buffer. Samples were separated by SDS-PAGE, transferred to polyvinylidenedifluoride membranes, and probed with β-tubulin antibodies (Sigma Aldrich) [64]. 4.2.8. Angiogenic, vascular injury, inflammatory cytokine and chemokine ELISA assay: HT-29 cells were plated in a 6-well plate at a density of 5 x 104 cells to achieve 80-90% confluence. The cells were cultured in media (1 mL) for 24 h and then treated with compounds 5a, 41, 46, 50 and Phenstatin (5 µM). Media was removed (on ice) and stored at -80 °C. The cells were washed on ice with cold PBS and washing discarded. Cold PBS (1 mL) was added to the cells in the plate and the cells were then scraped into PBS. The contents (cells and PBS) were pipetted into an Eppendorf tube. The plate wells were rinsed with cold PBS (0.5 mL) and transferred to the Eppendorf which was centrifuged at 14000 rpm for 10 mins, at 4 °C. The supernatent (PBS) was removed and discarded and the cell pellet retained. The cell pellet was resuspended in cell lysis buffer (100 µL) [RIPA buffer (880 µL) + protease inhibitor (PI) solution (100 µL) which was obtained from the protease inhibitor tablets and phosphatase inhibitor cocktail 2 (10 µL) with phosphatase inhibitor cocktail 3 (10 µL)]. The pellet plus buffer was retained on ice for 5 min to complete cell lysis, then centrifuged at 14000 rpm for 10 min at 4 °C. The supernatant was removed to fresh Eppendorf tubes for protein quantification of lysate using Pierce BCA Assay Kit (Thermo Scientific Cat No: 23225). The quantified cell lysates were stored at -80 °C. Media were processed according to MSD (Meso Scale Discovery) multiplex protocol. To assess angiogenic, vascular injury, inflammatory cytokine and chemokine secretions from CCM (cell conditioned media), a 54-plex ELISA kit separated across 7 plates was used (Meso Scale Diagnostics, USA). The multiplex kit was used to quantify the secretions of CRP, Eotaxin, Eotaxin-3, FGF(basic), Flt-1, GM-CSF, ICAM-1, IFN-γ, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17B, IL-17C, IL-17D, IL-1RA, IL-1α, IL-1β, IL-2, IL-21, IL-22, IL-23, IL-27, IL-3, IL-31, IL-4, IL-5, IL-6, IL-7, IL-8, IL-8 (HA), IL-9, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, MIP-3α, PlGF, SAA, TARC, Tie-2, TNF-α, TNF-β, TSLP, VCAM-1, VEGF-A, VEGF-C and VEGF-D from CCM. Assays were run as per manufacturer’s recommendation, an overnight supernatant incubation protocol was used

39

for all assays except Angiogenesis Panel 1 and Vascular Injury Panel 2 which were run on the same day. CCM was run undiluted on all assays except Vascular Injury Panel 2, where a one in four dilution was used, as per previous optimisation experiments. Secretion data for all factors was normalised to cell lysate protein content by using a BCA assay (Pierce). 4.2.9. Microsomal metabolic stability To determine the stability of the test compounds CA-4, 11 and 46 in the presence of liver microsomes, test compound (3 µM) is incubated with pooled liver microsomes supplemented with alamethicin. This assay was performed by Cyprotex Discovery Ltd, UK. Test compound is incubated at 5 time points over the course of a 45 min experiment and the test compound is analysed by LC-MS/MS. Pooled human liver microsomes are purchased from a reputable commercial supplier. Microsomes are stored at -80 °C prior to use. Microsomes (final protein concentration 0.5 mg/mL), alamethicin (25 µg/mg), 0.1 M phosphate buffer pH 7.4 and test compound (final substrate concentration 3 µM; final DMSO concentration 0.25 %) are pre-incubated at 37 °C prior to the addition of UDPGA (final concentration 1 mM) to initiate the reaction. The final incubation volume is 50 µL. A minus cofactor control incubation is included for each compound tested where 0.1 M phosphate buffer pH 7.4 is added instead of cofactor (minus UDPGA). Control compounds are included with each species. All incubations are performed singularly for each test compound. Each compound is incubated for 0, 5, 15, 30 and 45 min. The control (minus UDPGA) is incubated for 45 min only. The reactions are stopped by transferring 20 µL of incubate to 60 µL methanol at the appropriate time points. The termination plates are centrifuged at 2,500 rpm for 20 min at 4 °C to precipitate the protein. Following protein precipitation, the sample supernatants are combined in cassettes of up to 4 compounds, internal standard is added and samples analysed using Cyprotex generic LC-MS/MS conditions. Data analysis of a plot of ln peak area ratio (compound peak area/internal standard peak area) against time is used to determine the gradient of the line. Subsequently, half-life and intrinsic clearance are calculated using the following equations: Elimination rate constant (k) = (- gradient);

40

Half-life (t½) (min) = 0.693 k; Intrinsic clearance (CLint) (µL/min/mg protein) = V 0.693 t½; where V = Incubation volume (µL)/Microsomal protein (mg). 4.3 Computational Procedure: The 1SA0 X-ray structure of bovine tubulin cocrystallised with N-deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-colchicine) was downloaded from the PDB website (entry 1SA0) [56]. A UniProt Align analysis confirmed a 100% sequence identity between human and bovine beta tubulin. The crystal structure was prepared using QuickPrep (minimised to a gradient of 0.001 kcal/mol/Å), Protonate 3D, Residue pKa and Partial Charges protocols in MOE 2016 with the MMFF94x force field. For β-lactam compounds (11, 17, 23, 29, 35 and 46) with metasubstituted ring B, the same protocol was followed for the 5GON X-ray crystal structure [51]. A missing loop from Leu275 to Gln282 was rebuilt and missing atoms on Arg284 were added. These omissions were not close to the binding site. Both cis enantiomers of the compounds 11, 17, 23, 29, 35 and 46 were drawn in ChemBioDraw 13.0, saved as mol files and opened in MOE. For both enantiomers of each compound, MMFF94x partial charges were calculated and each was minimised to a gradient of 0.001 kcal/mol/Å. Default parameters were used for Docking except that 500 poses were sampled for each enantiomer and the top 50 docked poses were retained for subsequent analysis [56]. 4.4 X-ray Crystallography: Data for samples 11, 13, 46 were collected on a Bruker D8 Quest ECO; data for 12cis, and 21trans were collected on an APEX DUO using Mo Kα and Cu Kα (12trans) radiation (λ = 0.71073 and 1.54178 Å). Each sample was mounted on a MiTeGen cryoloop and data collected at 100(2) K (Oxford Cryostream and Cobra cryosystems). Bruker APEX [87] software was used to collect and reduce data, determine the space group, solve and refine the structures. Absorption corrections were applied using SADABS 2014.[88] Structures were solved with the XT structure solution program[89] using Intrinsic Phasing and refined with the XL refinement package [90] using Least Squares minimisation in Olex2.[91] All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were assigned to calculated positions using a riding model with appropriately fixed isotropic thermal parameters. In 12trans the solvent

41

CH2Cl2 molecule was disordered over the symmetry position and was modelled with 25% occupancy. Restraints were used in the model to ensure convergence. Crystallographic data for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos. 1537934 1537938. 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:[email protected]). Acknowledgment: A postgraduate research scholarship from King Abdulaziz University (KAU), Ministry of Higher Education, Saudi Arabia (Ref. IRKA1001; AMM) is gratefully acknowledged. We thank Dr. Gavin McManus for assistance with confocal microscopy. The Trinity Biomedical Sciences Institute (TBSI) is supported by a capital infrastructure investment from Cycle 5 of the Irish Higher Education Authority’s Programme for Research in Third Level Institutions (PRTLI). We thank Dr. John O’Brien and Dr. Manuel Ruether for NMR spectra. DF thanks the software vendors for their continuing support of academic research efforts, in particular the contributions of the Chemical Computing Group, Biovia and OpenEye Scientific. The support and provisions of Dell Ireland, the Trinity Centre for High Performance Computing (TCHPC) and the Irish Centre for High-End Computing (ICHEC) are also gratefully acknowledged. Supporting Information Crystallographic Data for β-lactams 11, 12cis, 12trans, 13 and 46; Tier-1 Profiling Screen of Selected β-Lactams; Lipinski-Properties for Selected β-Lactams; Antiproliferative Evaluation of Compounds 12, 19, 29, 37 in the NCI60 cell line in vitro primary screen; 1H and 13C NMR spectra for Selected β-Lactams; Cell cycle arrest in MCF-7 cells induced by compound 46; Cell cycle arrest in HT-29 cells induced by compound 46; Cell apoptosis and necrosis in MCF-7 cells induced by compound 46; Cell apoptosis and necrosis in HT-29 cells induced by compound 46; Overlay of the X-ray structure of tubulin co-crystallised with DAMA-colchicine (PDB entry 1SA0) and cocrystallised with cis 3-methyl-β-lactam compound (3R/4R) (PDB entry 5GON); Secreted

42

Levels of IL-1 , IL-1 IL-1Ra and VEGF in cell conditioned media showed significant alterations following treatment with compounds Phenstatin, 5a, 41, 46 and 50 when assessed by ELISA.

43

Abbreviations CA-4

Combretastatin A-4

CA-4P

Combretastatin A-4 phosphate (phosbretabulin)

DCM

Dichoromethane

DCTD

Division of Cancer Treatment and Diagnosis

DMF

N,N-Dimethylformamide

DTP

Development Therapeutics Program

EBI

N,N′-ethylene-bis(iodoacetamide)

EI

Electron Impact

GTP

Guanidine triphosphate

HB

Hydrogen bond

HBA

Hydrogen bond acceptor

HPLC

High-Performance Liquid Chromatography

HRMS

High resolution molecular mass spectrometry

IC

Inhibitory concentration

IR

Infra-Red

MAPK

Mitogen-activated protein kinases 44

MEK

Mitogen-activated protein kinase kinase

MS

Mass spectrometry

MTD

Maximum tolerated dose

NCI

National Cancer Institute

NIH

National Institute of Health

NMR

Nuclear Magnetic Resonance

PBS

Phosphate buffer saline

SAR

Structure-activity relationship

TEA

Triethylamine

TLC

Thin Layer Cromatography

UGT

Uridine 5'-diphosphoglucuronosyltransferase (UDP-glucuronosyl

transferase) UV

Ultraviolet

VDA

Vascular Disrupting Agent

45

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14.

15.

16. 17. 18.

19.

Jordan, M.A. and L. Wilson, Microtubules as a target for anticancer drugs. Nat Rev Cancer, 2004. 4(4): p. 253-65. Lu, Y., et al., An overview of tubulin inhibitors that interact with the colchicine binding site. Pharm Res, 2012. 29(11): p. 2943-71. Pellegrini, F. and D.R. Budman, Review: tubulin function, action of antitubulin drugs, and new drug development. Cancer Invest, 2005. 23(3): p. 264-73. Tron, G.C., et al., Medicinal chemistry of combretastatin A4: present and future directions. J Med Chem, 2006. 49(11): p. 3033-44. Pettit, G.R., et al., Antineoplastic agents 322. synthesis of combretastatin A-4 prodrugs. Anticancer Drug Des, 1995. 10(4): p. 299-309. Dark, G.G., et al., Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature. Cancer Res, 1997. 57(10): p. 1829-34. Tarade, D., S. Pandey, and J. McNulty, Review of Cytotoxic CA4 Analogues that Do Not Target Microtubules: Implications for CA4 Development. Mini Rev Med Chem, 2017. Tozer, G.M., et al., The biology of the combretastatins as tumour vascular targeting agents. Int J Exp Pathol, 2002. 83(1): p. 21-38. Pettit, G.R., et al., Antineoplastic agents. 291. Isolation and synthesis of combretastatins A-4, A-5, and A-6(1A). J Med Chem, 1995. 38(10): p. 1666-72. Nam, N.H., Combretastatin A-4 analogues as antimitotic antitumor agents. Curr Med Chem, 2003. 10(17): p. 1697-722. Chaudhary, A., et al., Combretastatin a-4 analogs as anticancer agents. Mini Rev Med Chem, 2007. 7(12): p. 1186-205. Aprile, S., E. Del Grosso, and G. Grosa, Identification of the human UDPglucuronosyltransferases involved in the glucuronidation of combretastatin A-4. Drug Metab Dispos, 2010. 38(7): p. 1141-6. Aprile, S., et al., In vitro metabolism study of combretastatin A-4 in rat and human liver microsomes. Drug Metab Dispos, 2007. 35(12): p. 2252-61. Malebari, A.M., et al., beta-Lactam analogues of combretastatin A-4 prevent metabolic inactivation by glucuronidation in chemoresistant HT-29 colon cancer cells. Eur J Med Chem, 2017. 130: p. 261-285. Cai, D., et al., YSL-12, a novel microtubule-destabilizing agent, exerts potent anti-tumor activity against colon cancer in vitro and in vivo. Cancer chemotherapy and pharmacology, 2016. 77(6): p. 1217-1229. Perez-Perez, M.J., et al., Blocking Blood Flow to Solid Tumors by Destabilizing Tubulin: An Approach to Targeting Tumor Growth. J Med Chem, 2016. 59(19): p. 8685-8711. Greene, L.M., M.J. Meegan, and D.M. Zisterer, Combretastatins: more than just vascular targeting agents? J Pharmacol Exp Ther, 2015. 355(2): p. 212-27. Grisham, R., et al., Clinical trial experience with CA4P anticancer therapy: focus on efficacy, cardiovascular adverse events, and hypertension management. Gynecol Oncol Res Pract, 2018. 5: p. 1. Garon, E.B., et al., A randomized Phase II trial of the tumor vascular disrupting agent CA4P (fosbretabulin tromethamine) with carboplatin, paclitaxel, and bevacizumab in advanced nonsquamous non-small-cell lung cancer. Onco Targets Ther, 2016. 9: p. 72757283.

46

20.

21.

22.

23. 24.

25. 26.

27. 28.

29. 30.

31.

32.

33. 34.

35.

36.

Patterson, D.M., et al., Phase I clinical and pharmacokinetic evaluation of the vasculardisrupting agent OXi4503 in patients with advanced solid tumors. Clin Cancer Res, 2012. 18(5): p. 1415-25. Blay, J.Y., et al., Ombrabulin plus cisplatin versus placebo plus cisplatin in patients with advanced soft-tissue sarcomas after failure of anthracycline and ifosfamide chemotherapy: a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol, 2015. 16(5): p. 531-40. Cushman, M., et al., Synthesis and evaluation of stilbene and dihydrostilbene derivatives as potential anticancer agents that inhibit tubulin polymerization. J Med Chem, 1991. 34(8): p. 2579-88. Wu, Y.S., et al., Synthesis and evaluation of 3-aroylindoles as anticancer agents: metabolite approach. J Med Chem, 2009. 52(15): p. 4941-5. Mahal, K., et al., Combretastatin A-4 derived imidazoles show cytotoxic, antivascular, and antimetastatic effects based on cytoskeletal reorganisation. Invest New Drugs, 2015. 33(3): p. 541-54. Gaukroger, K., et al., Structural requirements for the interaction of combretastatins with tubulin: how important is the trimethoxy unit? Org Biomol Chem, 2003. 1(17): p. 3033-7. Romagnoli, R., et al., Design and Synthesis of Potent in Vitro and in Vivo Anticancer Agents Based on 1-(3',4',5'-Trimethoxyphenyl)-2-Aryl-1H-Imidazole. Sci Rep, 2016. 6: p. 26602. Beale, T.M., et al., A-ring dihalogenation increases the cellular activity of combretastatin-templated tetrazoles. ACS Med Chem Lett, 2012. 3(3): p. 177-81. Lawrence, N.J.H., Lucy A.; Rennison, David; McGown, Alan T.; Hadfield, John A., Synthesis and anticancer activity of fluorinated analogues of combretastatin A-4. Journal of Fluorine Chemistry 2003. 123(1): p. 101-108. Pettit, G.R., et al., Antineoplastic agents. 379. Synthesis of phenstatin phosphate. J Med Chem, 1998. 41(10): p. 1688-95. Chen, J., et al., Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents. J Med Chem, 2012. 55(16): p. 7285-9. Canela, M.D., et al., Novel colchicine-site binders with a cyclohexanedione scaffold identified through a ligand-based virtual screening approach. J Med Chem, 2014. 57(10): p. 3924-38. Prota, A.E., et al., The novel microtubule-destabilizing drug BAL27862 binds to the colchicine site of tubulin with distinct effects on microtubule organization. J Mol Biol, 2014. 426(8): p. 1848-60. Xu, F., et al., Design, synthesis and biological evaluation of pyridine-chalcone derivatives as novel microtubule-destabilizing agents. Eur J Med Chem, 2019. 173: p. 1-14. Li, W., et al., Discovery of Novel Quinoline-Chalcone Derivatives as Potent Antitumor Agents with Microtubule Polymerization Inhibitory Activity. J Med Chem, 2019. 62(2): p. 993-1013. Kuroiwa, K., et al., Synthesis and Structure-Activity Relationship Study of 1-Phenyl-1(quinazolin-4-yl)ethanols as Anticancer Agents. ACS Med Chem Lett, 2015. 6(3): p. 28791. Rajak, H., et al., Design of combretastatin A-4 analogs as tubulin targeted vascular disrupting agent with special emphasis on their cis-restricted isomers. Curr Pharm Des, 2013. 19(10): p. 1923-55.

47

37. 38. 39.

40. 41.

42.

43. 44.

45. 46.

47.

48. 49.

50.

51.

52.

53.

54.

Pettit, G.R., et al., Antineoplastic agents 393. Synthesis of the trans-isomer of combretastatin A-4 prodrug. Anticancer Drug Des, 1998. 13(8): p. 981-93. Liu, Y.M., et al., Tubulin inhibitors: a patent review. Expert Opin Ther Pat, 2014. 24(1): p. 69-88. O Patil, P., et al., Recent Advancement in Discovery and Development of Natural Product Combretastatin-inspired Anticancer Agents. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents), 2015. 15(8): p. 955-969. Seddigi, Z.S., et al., Recent advances in combretastatin based derivatives and prodrugs as antimitotic agents. Medchemcomm, 2017. 8(8): p. 1592-1603. Greene, T.F., et al., Synthesis and Biochemical Evaluation of 3-Phenoxy-1,4diarylazetidin-2-ones as Tubulin-Targeting Antitumor Agents. J Med Chem, 2016. 59(1): p. 90-113. Carr, M., et al., Lead identification of conformationally restricted beta-lactam type combretastatin analogues: synthesis, antiproliferative activity and tubulin targeting effects. Eur J Med Chem, 2010. 45(12): p. 5752-66. O'Boyle, N.M., et al., Synthesis and evaluation of azetidinone analogues of combretastatin A-4 as tubulin targeting agents. J Med Chem, 2010. 53(24): p. 8569-84. O’Boyle, N.M., et al., Synthesis, biochemical and molecular modelling studies of antiproliferative azetidinones causing microtubule disruption and mitotic catastrophe. European journal of medicinal chemistry, 2011. 46(9): p. 4595-4607. O'Boyle, N.M., et al., Synthesis, evaluation and structural studies of antiproliferative tubulin-targeting azetidin-2-ones. Bioorg Med Chem, 2011. 19(7): p. 2306-25. Sun, L., et al., Examination of the 1,4-disubstituted azetidinone ring system as a template for combretastatin A-4 conformationally restricted analogue design. Bioorg Med Chem Lett, 2004. 14(9): p. 2041-6. Tripodi, F., et al., Synthesis and biological evaluation of 1,4-diaryl-2-azetidinones as specific anticancer agents: activation of adenosine monophosphate activated protein kinase and induction of apoptosis. J Med Chem, 2012. 55(5): p. 2112-24. Tripodi, F., et al., Synthesis and biological evaluation of new 3-amino-2-azetidinone derivatives as anti-colorectal cancer agents. Medchemcomm, 2018. 9(5): p. 843-852. Fu, D.J., et al., Structure-Activity Relationship Studies of beta-Lactam-azide Analogues as Orally Active Antitumor Agents Targeting the Tubulin Colchicine Site. Sci Rep, 2017. 7(1): p. 12788. Zhou, P., et al., Design, synthesis, biological evaluation and cocrystal structures with tubulin of chiral β-lactam bridged combretastatin A-4 analogues as potent antitumor agents. European Journal of Medicinal Chemistry, 2018. 144: p. 817-842. Zhou, P., et al., Potent antitumor activities and structure basis of the chiral β-lactam bridged analogue of combretastatin A-4 binding to tubulin. Journal of Medicinal Chemistry, 2016. 59(22): p. 10329-10334. Nathwani, S.M., et al., Novel cis-restricted beta-lactam combretastatin A-4 analogues display anti-vascular and anti-metastatic properties in vitro. Oncol Rep, 2013. 29(2): p. 585-94. Romagnoli, R., et al., Synthesis and evaluation of 1,5-disubstituted tetrazoles as rigid analogues of combretastatin A-4 with potent antiproliferative and antitumor activity. J Med Chem, 2012. 55(1): p. 475-88. Chen, J., et al., Recent development and SAR analysis of colchicine binding site inhibitors. Mini Rev Med Chem, 2009. 9(10): p. 1174-90.

48

55.

56. 57. 58. 59. 60. 61. 62. 63. 64.

65. 66.

67. 68.

69. 70.

71. 72. 73.

74.

Walsh, O.M., et al., Synthesis of 3-acetoxyazetidin-2-ones and 3-hydroxyazetidin-2-ones with antifungal and antibacterial activity. European Journal of Medicinal Chemistry, 1996. 31(12): p. 989-1000. Ravelli, R.B., et al., Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature, 2004. 428(6979): p. 198-202. Lara-Ochoa, F. and G. Espinosa-Pérez, A new synthesis of combretastatins A-4 and AVE8062A. Tetrahedron Letters, 2007. 48(39): p. 7007-7010. Combes, S., et al., Synthesis and biological evaluation of 4-arylcoumarin analogues of combretastatins. Part 2. J Med Chem, 2011. 54(9): p. 3153-62. Gillis, E.P., et al., Applications of fluorine in medicinal chemistry. Journal of medicinal chemistry, 2015. 58(21): p. 8315-8359. Swallow, S., Chapter Two - Fluorine in Medicinal Chemistry, in Progress in Medicinal Chemistry, G. Lawton and D.R. Witty, Editors. 2015, Elsevier. p. 65-133. Böhm, H.J., et al., Fluorine in medicinal chemistry. ChemBioChem, 2004. 5(5): p. 637643. National Cancer Institute Biological Testing Branch; National Cancer Institute; Bethesda, MD; https://dtp.nci.nih.gov/branches/btb/hfa.html (accessed 16th January 2019). 2019. Dumontet, C. and M.A. Jordan, Microtubule-binding agents: a dynamic field of cancer therapeutics. Nature reviews Drug discovery, 2010. 9(10): p. 790-803. Fortin, S., et al., Quick and simple detection technique to assess the binding of antimicrotubule agents to the colchicine-binding site. Biol Proced Online, 2010. 12(1): p. 113-7. Canela, M.-D., et al., Targeting the colchicine site in tubulin through cyclohexanedione derivatives. RSC Advances, 2016. 6(23): p. 19492-19506. Kanthou, C., et al., The tubulin-binding agent combretastatin A-4-phosphate arrests endothelial cells in mitosis and induces mitotic cell death. The American journal of pathology, 2004. 165(4): p. 1401-1411. Perez-Perez, M.J., et al., Blocking Blood Flow to Solid Tumors by Destabilizing Tubulin: An Approach to Targeting Tumor Growth. J Med Chem, 2016. Radogna, F., M. Dicato, and M. Diederich, Cancer-type-specific crosstalk between autophagy, necroptosis and apoptosis as a pharmacological target. Biochem Pharmacol, 2015. 94(1): p. 1-11. Han, W., et al., Shikonin circumvents cancer drug resistance by induction of a necroptotic death. Mol Cancer Ther, 2007. 6(5): p. 1641-9. Pasupuleti, N., et al., 5-Benzylglycinyl-amiloride kills proliferating and nonproliferating malignant glioma cells through caspase-independent necroptosis mediated by apoptosis-inducing factor. J Pharmacol Exp Ther, 2013. 344(3): p. 600-15. Li, N., et al., D-galactose induces necroptotic cell death in neuroblastoma cell lines. J Cell Biochem, 2011. 112(12): p. 3834-44. Bezerra, D.P., et al., Piplartine induces inhibition of leukemia cell proliferation triggering both apoptosis and necrosis pathways. Toxicology in vitro, 2007. 21(1): p. 1-8. Greene, L.M., et al., Combretazet-3 a novel synthetic cis-stable combretastatin A-4azetidinone hybrid with enhanced stability and therapeutic efficacy in colon cancer. Oncol Rep, 2013. 29(6): p. 2451-8. Greene, L.M., et al., The vascular targeting agent Combretastatin-A4 directly induces autophagy in adenocarcinoma-derived colon cancer cells. Biochemical pharmacology, 2012. 84(5): p. 612-624.

49

75. 76. 77. 78. 79.

80. 81. 82.

83. 84. 85. 86.

87. 88. 89. 90. 91. 92.

93.

94.

Su, Z., et al., Apoptosis, autophagy, necroptosis, and cancer metastasis. Mol Cancer, 2015. 14: p. 48. Nikoletopoulou, V., et al., Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta, 2013. 1833(12): p. 3448-59. Mori, A., et al., Expression of the antiapoptosis gene survivin in human leukemia. International journal of hematology, 2002. 75(2): p. 161-165. Jha, K., M. Shukla, and M. Pandey, Survivin expression and targeting in breast cancer. Surgical oncology, 2012. 21(2): p. 125-131. de Bruin, E.C. and J.P. Medema, Apoptosis and non-apoptotic deaths in cancer development and treatment response. Cancer treatment reviews, 2008. 34(8): p. 737749. Duffy, M.J., et al., Survivin: a promising tumor biomarker. Cancer letters, 2007. 249(1): p. 49-60. Voronov, E., Y. Carmi, and R.N. Apte, The role IL-1 in tumor-mediated angiogenesis. Front Physiol, 2014. 5: p. 114. Nasu, K., et al., Interleukin-1beta regulates vascular endothelial growth factor and soluble fms-like tyrosine kinase-1 secretion by human oviductal epithelial cells and stromal fibroblasts. Gynecol Endocrinol, 2006. 22(9): p. 495-500. Gaspari, R., et al., Structural Basis of cis- and trans-Combretastatin Binding to Tubulin. Chem, 2017. 2(1): p. 102-113. Wang, Y., et al., Structures of a diverse set of colchicine binding site inhibitors in complex with tubulin provide a rationale for drug discovery. Febs j, 2016. 283(1): p. 102-11. Molecular Operating Environment (MOE), C.C.G.I., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2016. Wienecke, A. and G. Bacher, Indibulin, a novel microtubule inhibitor, discriminates between mature neuronal and nonneuronal tubulin. Cancer research, 2009. 69(1): p. 171-177. Bruker APEX2 v2012.12-0, Bruker AXS Inc., Madison, Wisconsin, USA. 2012. SADABS, Bruker AXS Inc., Madison, Wisconsin, USA; Sheldrick, G. M. University of Göttingen, Germany. 2014. Sheldrick, G.M., SHELXT - integrated space-group and crystal-structure determination. Acta Crystallogr A Found Adv, 2015. 71(Pt 1): p. 3-8. Sheldrick, G.M., A short history of SHELX. Acta Crystallogr A, 2008. 64(Pt 1): p. 112-22. Dolomanov, O.V., et al., OLEX2: a complete structure solution, refinement and analysis program. Journal of Applied Crystallography, 2009. 42: p. 339-341. Cushman, M., et al., Synthesis and evaluation of analogs of (Z)-1-(4-methoxyphenyl)-2(3,4,5-trimethoxyphenyl)ethene as potential cytotoxic and antimitotic agents. Journal of Medicinal Chemistry, 1992. 35(12): p. 2293-2306. Flynn, B.L., et al., The synthesis and tubulin binding activity of thiophene-based analogues of combretastatin A-4. Bioorganic & Medicinal Chemistry Letters, 2001. 11(17): p. 2341-2343. Valtorta, S., et al., A novel AMPK activator reduces glucose uptake and inhibits tumor progression in a mouse xenograft model of colorectal cancer. Invest New Drugs, 2014. 32(6): p. 1123-33.

50

51

52

53

Table 1: ORTEP representation of the X-ray crystal structures of meta-fluoro ring B βlactams 11, 12cis, 12trans, 13 and 46 with 50% thermal ellipsoids (all hydrogen atoms omitted for clarity) Compound

Structure

X-ray representation

11

12trans

12cis

54

13

46

55

Table 2: X-ray Crystallographic Data for m-fluorophenyl ring B β-lactams 11, 12cis, 12trans, 13 and 46

Structure

Ring plane normal

Ring plane normal

Ring A to central

Ring B to central a

b

RingAB

RingBC

Torsion (°)c

Torsion (°)d

AB angle(°)

BC angle(°)

Torsion (°)

91.77(5)

97.50(5)

-166.14(17)

-144.50(13)

69.3(2)

-121.93(15)

88.13(5)

29.68(5)

152.92(18)

148.08(13)

-68.9(2)

116.97(16)

12cis

76.88(5)

102.93(5)

174.72(15)

131.88(14)

-57.21(18) 9.77(18)

12trans

84.38(18)

112.1(2)

-176.8(5)

-129.6(5)

65.1(7)

-123.6(5)

13

95.46(4)

-

-152.68(12)

-165.35(11)

62.00(16)

-114.43(9)

46

66.74(5)

-

179.57(14)

166.43(13)

-63.0(2)

117.01(13)

11

a

C18-C13-N1-C2, C26-C21-N1-C2, C26-C21-N1-C2, C14-C13-N1-C2, C14-C13-N1-C2;

b c

Torsion (°)

C10-C5-C4-N1, C7-C6-C5-N1, C11-C6-C5-N1, C6-C5-C4-N1, C6-C5-C4-N1;

C13-N1-C4-C5, C21-N1-C5-C6, C21-N1-C5-C6, C13-N1-C4-C5, C13-N1-C4-C5;

d

C5-C4-C3-C26, C6-C5-C4-O14, C6-C5-C4-O14, C5-C4-C3-Cl1, C5-C4-C3-O26.

* = 2 independent molecules in the asymmetric unit. Each angle given but only the first atom numbering scheme is outlined above.

56

Table 3: Antiproliferative effects of β-lactams CA-4, 5a, 5b, 11-52 in MCF-7 and HT-29 cells IC50 value (µM)a, b

clogPc

MCF-7

HT-29

0.009 ± 0.001 0.155 ± 0.030 0.037 ± 0.005 0.117 ± 0.010 0.015 ± 0.002 0.678 ± 0.018 0.024 ± 0.008 0.314 ± 0.009 0.144 ± 0.070 0.154 ± 0.070 0.293 ± 0.050 0.480 ± 0.090 0.325 ± 0.030 0.577 ± 0.090 0.194 ± 0.017 1.541 ± 0.100 0.091 ± 0.050 0.333 ± 0.040 0.355± 0.010 0.232 ± 0.050 0.190 ± 0.080 0.251 ± 0.040 0.306 ± 0.050 1.052 ± 0.300 0.153 ± 0.010 0.313 ± 0.020 0.222 ± 0.010 0.319 ± 0.010 0.197 ± 0.080

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

X F F F F F F Cl Cl Cl Cl Cl Cl Br Br Br Br Br

R1 C6H5 OC6H5 Cl Cl C2H3 C3H5 C6H5 OC6H5 Cl Cl C2H3 C3H5 C6H5 OC6H5 Cl Cl C2H3

R2 H H H Cl H H H H H Cl H H H H H Cl H

3.96 4.43 3.49 4.67 3.17 3.57 4.50 4.79 4.03 5.21 3.71 4.11 4.65 4.94 4.18 5.36 3.86

0.017 ± 0.003 0.073 ± 0.002 0.040 ± 0.003 0.173 ± 0.010 0.038 ± 0.018 0.323 ± 0.060 0.230 ± 0.010 0.430 ± 0.040 0.091 ± 0.007 0.140 ± 0.030 0.120 ± 0.010 0.270 ± 0.010 0.651 ± 0.009 0.818 ± 0.090 0.603 ± 0.080 1.120 ± 0.200 0.088 ± 0.020

28 29 30 31 32 33 34 35 36 37 38 39

Br I I I I I I CH3 CH3 CH3 CH3 CH3

C3H5 C6H5 OC6H5 Cl Cl C2H3 C3H5 C6H5 OC6H5 Cl Cl C2H3

H H H H Cl H H H H H Cl H

4.26 4.86 5.15 4.39 5.57 4.07 4.47 4.37 4.66 3.90 5.09 3.58

0.284 ± 0.020 0.648 ± 0.090 0.208 ± 0.020 0.421 ± 0.030 0.885 ± 0.070 0.215 ± 0.090 1.354 ± 0.090 0.462 ± 0.070 2.235 ± 0.600 0.433 ± 0.050 0.747 ± 0.060 0.328 ± 0.090

57

Relative potency IC50 value MCF-7/ IC50 value HT-29

1.88 0.47 1.08 1.48 2.53 0.48 9.58 1.37 0.63 0.91 0.41 0.56 2.00 1.42 3.12 0.73 0.97 0.83 1.83 0.90 2.22 3.53 0.70 1.29 3.02 7.14 1.95 2.34 1.67

40 41 42 43 44 45 46 47 48 49 50 51 52 5ad

CH3 F Cl Br I CH3 F Cl Br I CH3 F Cl OH

C3H5 H H H H H OH OH OH OH OH NH2 NH2 C6H5

H H H H H H H H H H H H H H

3.98 2.40 2.94 3.09 3.30 2.82 2.14 3.49 3.63 4.02 2.92 2.03 2.57 3.14

5bd CA-4e

H

C6H5

H

3.88 3.32

0.773 ± 0.090 0.022 ± 0.001 0.140 ± 0.030 0.060 ± 0.010 0.821 ± 0.030 0.317 ± 0.090 0.022 ± 0.006 0.012 ± 0.002 0.044 ± 0.006 0.100 ± 0.007 0.005 ± 0.001 0.483 ± 0.003 0.141 ± 0.030 0.004 ± 0.0006 0.015 ± 0.002 0.004 ± 0.0005

0.358 ± 0.020 0.015 ± 0.002 0.154 ± 0.070 0.064 ± 0.010 0.260 ± 0.010 0.189 ± 0.090 0.003 ± 0.001 0.018 ± 0.005 0.009 ± 0.001 0.021 ±0.001 0.007 ± 0.002 0.238 ± 0.010 0.167 ± 0.070 0.430 ± 0.060

2.16 1.47 0.91 0.94 3.16 1.68 7.33 0.67 4.89 4.77 0.71 2.03 0.84 0.01

0.022 ± 0.003 0.68 4.165 ± 0.100 0.001

a

IC50 values are half maximal inhibitory concentrations required to block the growth stimulation of cells. Values represent the mean for three experiments performed in triplicate.

b

c

All compounds evaluated as trans isomers.

CLogP values calculated from CS ChemBioDraw 13.0.

d

References. [43, 52]

The IC50 value obtained for CA-4 (0.0035 µM for MCF-7 and 0.474 µM for HT-29 µM) are in good agreement with reported values [53, 92-94].

e

58

Table 4: Antiproliferative evaluation of compound 11 in the NCI60 cell line in vitro screena Panel

Cell line

Compound 11

Panel

Cell line

GI50 (µM)b Leukaemia

CCRF-CEM

0.0361

HL-60 (TB)

GI50 (µM)b LOX IMVI

0.0296

0.0223

MALME-3M

> 100

K-562

0.0321

M14

0.0226

MOLT-4

0.0381

MDA-MB435

0.0187

RPMI-8226

0.0413

SK-MEL-2

0.0354

SR

0.0373

SK-MEL-28

1.37

0.0368

SK-MEL-5

0.0271

EKVX

0.0776

UACC-257

ntc

HOP-62

0.0379

UACC-62

0.0543

HOP-92

2.84

IGROV1

0.0428

NCI-H226

0.0350

OVCAR-3

0.0280

NCI-H23

0.0489

OVCAR-4

0.0871

NCI-H332M

0.0687

OVCAR-5

0.0517

NCI-H460

0.0344

OVCAR-8

0.0344

NCI-H552

0.0186

NCI/ADRRES

0.0394

COLO 205

0.0194

SK-OV-3

0.0348

HCT-2998

0.0394

786-0

0.0314

Non-Small A549 Cell Lung Cancer

Colon Cancer

Compound 11

Melanoma

Ovarian cancer

Renal cancer

59

CNS Cancer

Prostate cancer

a

HCT-116

0.0324

A498

0.0270

HCT-15

0.0336

ACHN

0.0440

HT-29

0.0260

CAKI-1

0.0332

KM12

0.0331

RXF393

nt c

SW-620

0.0419

SN12C

0.0561

SF-268

0.0443

TK-10

1.68

SF295

0.0290

UO-31

0.0771

SF539

0.0234

MCF-7

0.0296

SNB-19

0.0499

MDA-MB231

0.0452

SNB-75

0.0314

HS578T

0.0434

U251

0.0359

BT-549

0.0266

PC-3

0.0403

T-47D

> 100

DU-145

0.0354

MDA-MB468

0.0287

Breast cancer

Data obtained from NCI in vitro human tumour cell screen. b GI50 is the molar concentration of the compound causing c

50% inhibition of growth of the tumour cells. nt = not tested.

60

Table 5: Summary of NCI 60 cell line in vitro primary screening result for compounds 11, 12, 19, 29 and 37 GI50a (µM)

TGIa (µM)

LC50a (µM)

CA-4

0.099

10.3

85.5

D-788819

11

0.057

20.8

95.4

D-791048

12

0.190

39.8

89.1

D-791049

19

0.112

23.4

89.1

NCI ref No.

Compound

D-613729

Structure

61

D-792957

29

0.676

45.7

95.4

D-788820

37

0.190

19.0

89.1

a

The GI50, TGI and LC50 values are determined, which represent the molar drug concentration required to cause 50% growth inhibition, total growth inhibition and the concentration that kills 50% of the cells, respectively. The compounds were evaluated using five different concentrations (100 µM, 10 µM, 1 µM, 0.1 µM and 0.01 µM) and incubations were carried out over 48 h exposures to the drug [63].

62

Figure 1: Chemical structures of colchicine, CA-4 and related colchicine-binding microtubule-destabilising agents

63

% cell viability in HT-29 cells

5a 5b 11 17 23 29 35

100

50

(IC50=430 nM) (IC50=22 nM) (IC50=9 nM) (IC50=24 nM) (IC50=325 nM) (IC50=355 nM) (IC50=153 nM)

0 -9

-7

-5

Log10 concentration (M)

Figure 2: A comparison of antiproliferative activity for different 3-substituted phenyl βlactams in HT-29 cells. Cells were grown in 96-well plates and treated with β-lactam compounds (5a, 5b, 11, 17, 23, 29 and 35) at 0.01–50 µM for 72 h. Cell viability was expressed as a percentage of vehicle control [ethanol 1% (v/v)] treated cells. The values represent the mean ± S.E.M. for three independent experiments performed in triplicate.

64

80

*** ***

*** ***

10

*** ***

1

% cell viability

100

*** ***

60

*** ***

HEK-293T cells MCF-7 cells HT-29 cells

40 20

50

1 0.

0.

01

0

Conc. (µM)

Figure 3: Effect of compound 46 on viability of MCF-7 and HT-29 cells and nontumorigenic HEK-293T cells. Cells were grown in 96-well plates and treated with compound 46 at 0.01–50 µM for 72 h. Cell viability was expressed as a percentage of vehicle control [ethanol 1% (v/v)] treated cells and was measured by AlamarBlue assay (average of three independent experiments).Two-Way ANOVA (Bonferroni post-test) was used to test for statistical significance (***, P <0.05).

65

OCH 3 F

HO

46

O

OCH 3 47

OCH 3

O

OCH 3 48

N OCH3

H3 CO

CH3

HO

N OCH3

H3 CO

Br

HO

N OCH3

H3 CO

Cl

HO

N O

OCH 3

OCH 3

O

OCH 3 50

OCH3

H3 CO

OCH 3

Figure 4: Effect of β-lactam compounds 46, 47, 48 and 50, CA-4 and Paclitaxel (all at 10 µM) on in vitro tubulin polymerisation. Purified bovine tubulin and GTP were mixed in a 96-well plate. Compounds were added and the reaction was started by warming the solution from 4 to 37 °C. Ethanol (1%v/v) was used as a vehicle control. The effect on tubulin assembly was monitored in a Spectramax 340PC spectrophotometer at 340 nm at 30 s intervals for 30 min at 37 °C. The graph shows one representative experiment. Each experiment was performed in triplicate.

66

Figure 5: CA-4 and β-lactam 46 depolymerise the microtubule network of MCF-7 cells. MCF-7 cells were treated with vehicle control [1% ethanol (v/v)], paclitaxel (1 µM), CA4 (0.01 µM), or 46 (0.01, 0.05 and 0.1 µM) for 5 hr. Cells were fixed in 100% cold MeOH and stained with mouse monoclonal anti-α-tubulin-FITC antibody (clone DM1A) (green), Alexa Flour 488 dye, and counterstained with DAPI (blue). Images were captured by Leica SP8 confocal microscopy with Leica application suite X software. Representative confocal micrographs of three separate experiments are shown. Scale bar: 50 µM.

67

Figure 6: Effects of CA-4 and β-lactam 46 on the inhibition of the bisthioalkylation of Cys239 and Cys354 of β-tubulin by N,N’-ethylene-bis(iodoacetamide) (EBI) in MCF-7 cells. Cells were treated with vehicle control [ethanol 0.1% (v/v)], CA-4 or 46 (all 10 µM) for 2 h; selected samples were then treated with EBI (100 µM) for an additional 1.5 h. Cells were harvested, lysed and analysed using sedimentation and Western blotting for β-tubulin and β-tubulin-EBI adduct. Results are indicative of three separate experiments, performed independently. To confirm equal protein loading, each membrane was stripped and reprobed with GAPDH antibody.

68

A

% viable of MCF-7 cells

80

***

***

G0/G1 G2/M

***

60

Sub-G1 S

40 20

48

24

ol co nt r

72

0

Time hr

B

% viable HT-29 cells

100

G0/G1 G2/M

80 60

***

***

***

sub-G1 S

40 20

72

48

24

co nt ro l

0

Time hr

Figure 7: (A) Compound 46 induced G2/M arrest followed by apoptosis in a time dependent manner in MCF-7 cells. Cells were treated with either vehicle [0.1% ethanol (v/v)] or compound 46 (1 µM) for 24, 48 and 72 hr. (B) Compound 46 induced G2/M arrest in a time dependent manner in HT-29 cells. Cells were treated with either vehicle control [0.1% ethanol (v/v)] or compound 46 (1 µM) for 24, 48 and 72 hr. Cells were

69

then fixed, stained with PI, and analysed by flow cytometry. Cell cycle analysis was performed on histograms of gated counts per DNA area (FL2-A). The number of cells with <2N (sub-G1), 2N (G0G1), and 4N (G2/M) DNA content was determined with CellQuest software. Values represent as the mean ± SEM for three separate experiments. Statistical analysis was performed using two-way ANOVA (***, p < 0.001).

70

A

% apoptotic cells

30

*** 20

Early apoptosis Late apoptosis Necrosis

** 10

nM 00 10

10

0

nM

nM 10

C

on

tr o

l

0

Concentration (nM)

25

*** 20

Early apoptosis Late apoptosis Necrosis

15 10 5

72

48

24

0 co nt ro l

% apoptotic or necrotic cells

B

Time hr

Figure 8: (A) Compound 46 induced cell apoptosis in MCF-7 cells. Apoptotic cells of MCF-7 cells were detected using the Annexin V-FITC/PI double staining after exposure to 46 at different concentrations (10, 100, 1000 nM) for 48 h and analyzed by flow cytometry. The vehicle control was ethanol 0.1% (v/v). (B) Compound 46 induced cell apoptosis and necrosis in HT-29 cells. Apoptotic cells of HT-29 cells were detected using the Annexin V-FITC/PI double staining after exposure to 46 (1 µM) at different time points (24, 48, 72 h) and analyzed by flow cytometry. The vehicle control was 0.1%

71

ethanol (v/v). Values represent as the mean ± SEM for three separate experiments. Statistical analysis was performed using two-way ANOVA (***, p < 0.001).

72

Figure 9: β-Lactam 46 decreases the expression of anti-apoptotic proteins Bcl-2, Mcl-1 and survivin in MCF-7 cells. MCF-7 cells were treated with vehicle control (ethanol 0.1% v/v) or 46 at the indicated concentrations (0.05, 0.1 or 0.5 µM) for 48 h (left) or 72 h (right). Then, the cells were harvested for Western blot analysis to detect the level of the apoptosis related proteins. Results are indicative of three separate experiments, performed independently. To confirm equal protein loading, each membrane was stripped and reprobed with GAPDH antibody.

73

(A)

(B)

Figure 10: Overlay of the X-ray structure of tubulin cocrystallised with DAMAcolchicine (PDB entry 1SA0 [56]) on the best ranked docked poses of the 3S/4R enantiomers of (A) halogen substituted 3-phenyl β-lactam compounds 11(light green), 17 (blue) and methyl-substituted β-lactam 35 (dark green), and (B) halogen-substituted βlactams 23 (yellow) and 29 (orange). Ligands are rendered as tube and amino acids as

74

line. Tubulin amino acids and DAMA-colchicine are coloured by atom type, 11 light green, 17 blue, 23 yellow, 29 orange and 35 dark green. The atoms are coloured by element type, carbon = grey, hydrogen = white, oxygen = red, nitrogen = blue, sulphur = yellow. Key amino acid residues are labelled and multiple residues are hidden to enable a clearer view.

75

Figure 11: Overlay of the X-ray structure of tubulin cocrystallised with DAMAcolchicine (PDB entry 1SA0, [56]) on the best ranked docked pose of 3S/4S enantiomer of 46. Ligands are rendered as tube and amino acids as line. Tubulin amino acids and DAMA-colchicine are coloured by atom type, carbon atoms of 46 are dark green. Atom type: carbon = grey, hydrogen = white, oxygen = red, nitrogen = blue, sulphur = yellow. Key amino acid residues are labelled and multiple residues are hidden to enable a clearer view.

76

Graphical abstract

77

β-Lactams with Antiproliferative and Antiapoptotic Activity in Breast and Chemoresistant Colon Cancer Cells

Azizah M. Malebaria,b*, Darren Faynec, Seema M. Nathwanic, Fiona O'Connelle, Sara Nooranib, Brendan Twamleyd, Niamh M. O’Boyleb, Jacintha O'Sullivane, Daniela M. Zistererc and Mary J. Meeganb

a

Department of Pharmaceutical Chemistry, College of Pharmacy, King Abdulaziz

University, Jeddah, Saudi Arabia b

School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Trinity

Biomedical Sciences Institute, 152-160 Pearse Street, Dublin 2, Ireland c

School of Biochemistry and Immunology, Trinity College Dublin, Trinity

Biomedical Sciences Institute, 152-160 Pearse Street, Dublin 2, Ireland d e

School of Chemistry, Trinity College Dublin, Dublin 2, Ireland

Trinity Translational Medicine Institute, Department of Surgery, Trinity College

Dublin, Ireland, Dublin 2, Ireland.

Highlights: • • • • •

Ring B modified β-Lactam Combretastatin A-4 analogues synthesized Antiproliferative effects in UGT-expressing chemoresistant HT-29 colon cancer cells Compounds inhibit tubulin polymerization G2M arrest and apoptosis demonstrated in MCF-7 breast cancer cells Glucuronidation in HT-29 cells may be inhibited by modification of ring B

β-Lactam analogues of CA-4 target breast cancer and chemoresistant colon cancer cells Azizah M. Malebaria,b*, Darren Faynec, Seema M. Nathwanic, Fiona O'Connelle, Sara Nooranib, Brendan Twamleyd, Niamh M. O’Boyleb, Jacintha O'Sullivane, Daniela M. Zistererc and Mary J. Meeganb

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests

*Corresponding author: Azizah M. Malebaria E-mail: [email protected]