Synthesis and Biological Evaluation of 1-(Diarylmethyl)-1H-1,2,4-triazoles and 1-(Diarylmethyl)-1H-imidazoles as a Novel Class of Anti-Mitotic Agent for Activity in Breast Cancer

We report the synthesis and biochemical evaluation of compounds that are designed as hybrids of the microtubule targeting benzophenone phenstatin and the aromatase inhibitor letrozole. A preliminary screening in estrogen receptor (ER)-positive MCF-7 breast cancer cells identified 5-((2H-1,2,3-triazol-1-yl)(3,4,5-trimethoxyphenyl)methyl)-2-methoxyphenol 24 as a potent antiproliferative compound with an IC50 value of 52 nM in MCF-7 breast cancer cells (ER+/PR+) and 74 nM in triple-negative MDA-MB-231 breast cancer cells. The compounds demonstrated significant G2/M phase cell cycle arrest and induction of apoptosis in the MCF-7 cell line, inhibited tubulin polymerisation, and were selective for cancer cells when evaluated in non-tumorigenic MCF-10A breast cells. The immunofluorescence staining of MCF-7 cells confirmed that the compounds targeted tubulin and induced multinucleation, which is a recognised sign of mitotic catastrophe. Computational docking studies of compounds 19e, 21l, and 24 in the colchicine binding site of tubulin indicated potential binding conformations for the compounds. Compounds 19e and 21l were also shown to selectively inhibit aromatase. These compounds are promising candidates for development as antiproliferative, aromatase inhibitory, and microtubule-disrupting agents for breast cancer.


Introduction
Designing single agents that act against multiple biological targets is of increasing interest and prominence in medicinal chemistry [1][2][3][4]. Dual-targeting drugs are designed with the potential to be more potent and efficient and overcome many of the disadvantages of single drugs such as low solubility, side effects [5], and multidrug resistance (MDR). While the molecular mechanisms of resistance to chemotherapeutics have been identified, MDR is known to be a key factor in the failure of breast cancer chemotherapy [6]. Traditionally, drugs have been designed to target a single biological target (protein), aiming for high selectivity and thus avoiding unwanted effects due to off-target events. The interaction of a drug with multiple target proteins has been regarded as potentially associated with adverse side effects. However, for complex diseases such as cancer, it is now recognised that a single-target drug may not achieve the optimum therapeutic effect. Molecules that are effective at more than one target protein may overcome incomplete efficacy and demonstrate an increased safety profile compared to single-targeted ones [2]. Dual-targeting strategies may offer a more favourable outcome of cancer treatment.
A possible strategy to improve the outcome for postmenopausal breast cancer patients is to design compounds with dual aromatase and tubulin targeting activities, which may offer the potential benefits of improved efficacy and fewer side effects [7,8]. The objective of our research is to investigate a new series of 1-(diarylmethyl)-1H-1,2,4-triazoles and 1-(diarylmethyl)-1H-imidazoles as a novel class of antimitotic compounds with an interesting biochemical profile particularly as tubulin-targeting agents and aromatase inhibitors for the treatment of breast cancer.
Breast cancer is the most commonly diagnosed cancer in women; it is estimated that approximately one in eight women will develop breast cancer during their lifetime, and it is the most frequent cause of death for women in the age group 35-55 [9]. There were over two million new cases in 2018 [10], and the number of cases is predicted to rise due to an ageing population [11,12]. Mortality has decreased due to improved screening and early detection together with the use of adjuvant therapy [13]. Approximately 70-80% of breast cancers are hormone-dependent; their growth is stimulated in response to the hormone estrogen, with the majority of these estrogen receptor positive (ER+) cancers also expressing the progesterone receptor (ER+/PR+ cancers). Upregulation of the gene encoding the PR is directly mediated by ER, and PR modulates ERα action in breast cancer [14].
Aromatase (CYP19A1), a member of the cytochrome P-450 enzyme superfamily, catalyses the aromatisation of C-19 androgens to C-18 estrogens in the final step in estrogen biosynthesis, and it is an attractive target for selective inhibition [15][16][17]. Estrogen deprivation is an effective therapeutic intervention for hormone-dependent breast cancer (HDBC) and has been clinically established by the inhibition of the aromatase enzyme. The aromatase inhibitors (AIs), e.g., letrozole 1 [18], anastrozole 2 [19], and exemestane [20] (Figure 1a), prevent the stimulating effects of estrogen in breast tissue [19], and they are approved in the treatment of a wide spectrum of breast cancers [21]. These AIs have demonstrated superior efficacy in postmenopausal women and have few associated risks apart from reduction in bone density [8,[21][22][23], and emerging resistance [24,25].
The selective estrogen receptor modulator (SERM) tamoxifen 3a (Figure 1a) is effective for the treatment of ER+ breast cancer [13]; however, resistance is a clinical problem [26] together with a small increase in incidences of blood clots and endometrial cancers for postmenopausal women [27,28]. The potential advantage of the tamoxifen metabolites endoxifen (3b) and norendoxifen (3c) in endocrine-refractory metastatic breast cancer is reported [29]. Breast cancers that are (ER+/PR+) are likely to respond to hormone therapy such as tamoxifen and anastrozole [23], while the prophylatic use of tamoxifen, raloxifene, or anastrozole is recommended for postmenopausal women at high risk of developing breast cancer [30,31]. Approximately 20% of breast cancers overexpress the human epidermal growth factor receptor 2 (HER2), which promotes the growth of cancer cells.
Effective treatments for HER2+ breast cancers include the monoclonal antibody trastuzumab [32], the antibody-drug conjugate ado-trastuzumab emtansine [33], and the dual tyrosine kinase inhibitor lapatinib which targets both the HER/neu and the epidermal growth factor receptor (EGFR) [34]. Breast cancers are classified as triple negative (TNBC) when their growth is not supported by estrogen and progesterone nor by the presence of HER2 receptors. The clinical options for treatment of TNBC are limited due to poor response to hormonal therapy, resulting in low 5-year survival rates [35]. There is extensive diversity among breast cancer patients, and each sub-type of breast cancer has unique characteristics. The identification of sub-type-specific network biomarkers can be useful in predicting the survivability of breast cancer patients [36]. FDA-approved drugs for breast cancer in 2019 include the antibody-drug conjugate Fam-trastuzumab deruxtecan [37] (HER2-directed antibody and topoisomerase inhibitor) for the treatment of unresectable or metastatic HER2-positive breast cancer [38], the phosphoinositide-3-kinase (PI3Kα) inhibitor alpelisib [39] for the treatment of HER2negative, PIK3CA-mutated, advanced or metastatic breast cancer [40] and in 2020, tucatinib, an orally bioavailable, small molecule tyrosine kinase inhibitor for patients with HER2-positive metastatic breast cancer [41]. The microtubule-stabilising drugs paclitaxel, docetaxel, and the epothilone ixabepilone were approved for use in patients with metastatic breast cancer (MBC), alongside the microtubule destabilising vinca alkaloid eribulin [42,43]. The FDA recently granted accelerated approval to the antibody-drug (topoisomerase inhibitor) conjugate sacituzumab govitecan (Trodelvy) for previously treated metastatic TNBC [44], while ladiratuzumab vedotin (a LIV-1-targeted antibody linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE)) is in clinical trials for locally advanced or metastatic triple-negative breast cancer [45]. The steroid sulfatase inhibitor (STS) e.g., STX64 (Irosustat) has entered clinical trials for ER+ locally advanced or metastatic breast cancer [46], while inhibitors of mutant p53, e.g., PRIMA-1 and PRIMA-1 MET , overexpressed in TNBC have been demonstrated to be effective in vitro [47].
Since the potent tubulin-inhibiting activity of the 3,4,5-trimethoxyaryl function is very well documented in colchicine-binding site inhibitors [90], the 1,2,4-triazole heterocycle was next reacted with several phenstatin-type 3,4,5-trimethoxyaryl substituted benzhydryl alcohols in order to maximise the potential tubulin activity in the scaffold structures with aromatase-inhibiting action (Series 2). It was decided to retain in most compounds the 3,4,5-trimethoxyaryl group substitution (ring A) and introduce alternative substituents on the second ring (ring B). A modified synthetic procedure allowing access to the desired benzhydryl alcohol intermediates 15a-h and 18a-f is shown in Schemes 2 and 3 (step a) [91]. Scheme 2 shows the alcohols (15a-h) obtained by treatment of the appropriate aryl bromides 14a-h with n-butyllithium followed by reaction with 3,4,5-trimethoxybenzaldehyde (A ring) to afford the alcohols 15a-h in yields of 21-89%. For the preparation of compounds (18a-d) (Scheme 3), the A ring was derived from 3,4,5-trimethoxybromobenzene followed by reaction with the appropriate aldehyde 17a-d. The nitrile-containing compounds 18e,f were similarly obtained from the aldehydes 17e,f and 4-bromobenzonitrile (Scheme 3). The benzhydryl compounds were obtained in good yield after purification via flash column chromatography and the presence of the hydroxyl group was confirmed from IR (ν 3200-3600 cm −1 ).
Then, the secondary alcohols 15a-h and 18a-f were reacted with 1,2,4-triazole to afford the hybrid phenstatin/letrozole compounds 16a-h and 19a-d,f,g as racemates, except for 19b, (Schemes 2 and 3, step b). The phenolic compounds 16i, 19e, 19h, and 19i were obtained by hydrogenolysis over palladium hydroxide of the benzyl ethers 16b, 19a, 19f, and 19g respectively. From the 1 H-NMR spectrum of compound 16c, the singlet at 6.62 ppm was assigned the tertiary aliphatic proton. The singlets at 7.91 and 8.01 ppm were assigned to the triazole H-3 and H-5. In the 13 C-NMR spectrum, the tertiary CH signal was identified at 67.4 ppm, while the triazole ring C3 and C5 signals were identified at 143.5 and 152.3 ppm, respectively. X-ray crystal structures of the triazole compounds 16e, 16f, and 19c (recrystallised from dichloromethane/n-hexane) are displayed in Figure 2, while the crystal data and structure refinement are displayed in Table 1. The length of the C-N bond between the methine carbon and the triazole N-1 for compounds 16e, 16f, and 19c was measured at 1.470, 1.471, and 1.479 Å, respectively. The N1-N2 bond length was 1.366 Å (16e), 1.363 Å (16f), and 1.365 Å (19c). The N1-C5 bond length of the triazole ring was observed as 1.334 Å (16e), 1.342 Å (16f), and 1.343 Å (19c). The angle between the methine carbon and the two aromatic rings (Ar-C1-Ar) was measured as 112.51 • , 115.08 • , and 113.53 • respectively for compounds 16e, 16f, and 19c. The corresponding value for the letrozole structure is 114.0 • , while the C-N bond between the methine carbon and the triazole N-1 was 1.46 Å [92].

1-(Diarylmethyl)-1H-imidazoles (Series 3 and 4)
A series of related imidazole-containing compounds were also prepared 20a-l (Series 3) and 21a-k (Series 4). The secondary alcohols 12a-h, j, k, and m were coupled to imidazole using CDI (carbonyldiimidazole) [93] to afford products 20a-k, Series 3, (Scheme 4, step a). The associated carbamate derivatives were not isolated in our reactions [94]. The hydrolysis of 20i afforded the amine 20l in 50% yield (Scheme 4, step b). Structures were optimised with variations in electron-releasing and electron-withdrawing substituents on the aryl rings. A further series of compounds containing the ring A type 3,4,5-trimethoxyaryl substituents was prepared by reacting alcohols 15a,c-h, and 18a-d with CDI to afford imidazole products 21a-k, Series 4, (Scheme 5, step a). The benzyl ether 21h was treated with Pd(OH) 2 to afford the phenol 21l as a racemate in 93% yield (Scheme 5, step b). In the 1 H NMR spectrum of compound 21i, the imidazole H4 was observed as a singlet at 6.88 ppm, while the H2 and H5 were observed at 7.44, and 7.12 ppm, respectively. The singlet at 6.38 ppm was assigned to the tertiary aliphatic CH. From the 13 C-NMR spectrum, the aliphatic tertiary CH was identified at 65.2, while the signals at 138.0, 129.4, and 119.4 ppm were assigned to the imidazole C2, C4, and C5, respectively.
Single crystal X-ray analysis was obtained for compound 21i (recrystallised from dichloromethane/n-hexane), and the crystal structure is shown in Figure 3. The crystal data and structure refinement for compound 21i are displayed in Table 1. The angle between the methine carbon and the aryl rings (114.16 • ) and also the bond length between the methine carbon and the N-1 imidazole nitrogen (1.471 Å) were similar to the corresponding values obtained for the triazole compounds 16e, 16f, and 19c ( Table 1). The bond angles between the aryl rings and the imidazole ring were determined as 111.36 • and 111.80 • , also similar to the corresponding values of 109.99 • and 112.6 • reported for letrozole [92].
An alternative approach for the preparation of phenstatin and related azole compounds using a Friedel-Crafts acylation with Eaton's reagent was also investigated (Scheme 6) [67]. 3,4,5-Trimethoxybenzoic acid was reacted with anisole (22a), 1,2-dimethoxybenzene (22b), or compound 22c (prepared by the protection of 2-methoxyphenol with chloroacetyl chloride) using Eaton's reagent (readily prepared from phosphorus pentoxide and methanesulfonic acid) to afford respectively benzophenones 23a, 23b, and 23c (Scheme 6, Step a). Then, these benzophenones were reduced to the benzhydryl alcohols 15c, 15d, and 15i, respectively with sodium borohydride (Scheme 6, step a), with the concomitant removal of the chloroacetyl protecting group of 23c. Although requiring an additional step, this method was followed after the reaction of the aryl bromide with the aldehyde to afford the alcohol as shown in Schemes 2 and 3 was not successful or did not afford a sufficient quantity of product for the next step e.g., for compound 15d, the overall yield increased to 51% compared with 30%. Then, compounds 15c and 15d were treated with CDI azole to afford the imidazole-containing products 21b and 21c (Scheme 6, step e). The phenol 15i was also reacted with 1,2,3-triazole to afford the product 24 in 77% yield, Series 4, (Scheme 6, step d). Compound 24 is the only phenstatin derivative substituted with 1,2,3-triazole synthesised in this project and was investigated for comparison with the 1,3,4-triazole compound series. In the 1 H NMR spectrum of 24, the signal at 6.99 ppm was assigned to the tertiary CH. Interestingly, the two protons of the 1,2,3-triazole ring were observed as a singlet with an integration of 2H at 7.83 ppm, while the signal at 134.9 ppm in the 13 C-NMR spectrum of 24 was assigned to the C4 and C5 of the triazole ring, indicating that alkylation occurred at N2 of the 1,2,3-triazole [95]. The alkylation of 1,2,3-triazoles may result in the formation of regioisomers depending on the reaction conditions e.g., solvent, temperature, and catalyst used [96]. The signal for the tertiary CH was observed at 71.0 ppm. The benzophenone 23c was also used in the preparation of phenstatin 7a [67]; the deprotection of 23c by reaction with sodium acetate afforded 7a in 89% yield (Scheme 6, step b), which was used as a positive control in the cell viability tests. The preparation of a series of benzhydryl derivatives substituted on the tertiary carbon with the heterocycles pyrrolidine, piperidine, and piperazine was next investigated (Series 5, Schemes 7 and 8). These products allow a comparison of biochemical activity with the related imidazole and triazole compounds from Series 1-4. The advantages of incorporating such heterocyclic rings into drugs are well known; i.e., they can increase the lipophilicity, polarity, and aqueous solubility of the drug [97]. In particular, piperazine is ranked 3 rd among the 25 most common heterocycles contained in FDA-approved drugs [98]. In the present work, the corresponding secondary benzhydryl chloride was prepared from the secondary alcohols 12b-12g, 15c, and 18a using thionyl chloride (Schemes 7 and 8, step a) [93]. The intermediate alkyl chlorides were reacted with piperidine to afford products 26a-c (Scheme 7, step c), while reaction with pyrrolidine yielded derivatives 25a-g (Scheme 7, step b).
An alternative synthesis of 1-(diarylmethyl)piperidines is reported using a copper(I)catalysed coupling reaction of aryl boronic acids with N,O-acetals and N,N-aminals [99]. All compounds are racemates apart from compound 25e and were obtained in moderate yields (23-93%). In the 1 H-NMR spectrum of compound 25b, the multiplets at 1.71-1.80 and 2.35-2.43 ppm were assigned to the pyrrolidine methylene protons at H-3,4 and H-2,5 respectively, while the tertiary CH was observed as a singlet at 4.11 ppm. In the 13 C-NMR spectrum, the pyrrolidine C-3 and C-2 signals were at 23.5 and 53.6 ppm, respectively. The signal at 75.7 ppm was assigned to the tertiary carbon. Single crystal X-ray analysis for compound 26a is shown below in Figure 3 (obtained by crystallisation in dichloromethane/n-hexane). The crystal data and structure refinement for compound 26a are displayed in Table 1. In 26a, the disordered fluorine was modelled in two positions with occupancies of 84% and 16%. The C1-N bond length was observed as 1.473 Å and the central C 14 -C 1 -C 8 and C 14 -C 1 -N 2 angles were observed as 109.28 • and 112.09 • , respectively. The piperidine ring bond lengths were 1.471 Å (N2-C3), 1.474 Å (N2-C7), and 1.514 Å (C3-C4), which differ from the N1-C bond length of the triazole ring 1.334 Å due to unsaturation.
As a further extension of this research, a related series of piperazine-containing compounds was prepared by coupling selected secondary alcohols with the appropriate piperazine derivative (Series 5, Scheme 8). The preparation of diarylmethylamines has been reported by Le Gall et al. by reaction of the aldehyde and piperidine derivative to a solution of the organozinc reagent in acetonitrile in a Mannich-type reaction [100,101]. The secondary alcohols 12e, 15c, and 18a were treated with thionyl chloride (Scheme 8, step a), and the resulting alkyl chloride was used immediately for the next reaction step (step b) by addition of the appropriate piperazine (N-phenylpiperazine, N-benzylpiperazine, pmethoxyphenylpiperazine, or N-Boc-piperazine) to afford the products 27a-g in yields up to 80%. For the preparation of compound 27e, Boc-protected piperazine was used to avoid the possible formation of the dimer. In the 1 H-NMR spectrum of compound 27d, the broad signal at 2.47 ppm is assigned to piperazine methylene protons; the singlet at 3.50 ppm is assigned to the benzyl methylene, while the singlet at 4.09 ppm corresponds to the tertiary C-1 proton. The 13 C-NMR spectrum of compound 27d further confirms the proposed structure. The signals at 51.8 and 53.3 ppm were characteristic of the piperazine ring protons, the signals at 63.0 ppm and 75.6 ppm are assigned to the benzyl methylene and tertiary CH, respectively. The deprotection of compound 27e with TFA afforded compound 27h as a yellow oil (42%), (Scheme 8, step c). A palladium-catalysed hydrogenolysis of 27g afforded the phenolic compound 27i in 45% yield (step d), which is the phenylpiperazine derivative of phenstatin. Its formation was confirmed by IR spectroscopy (3475 cm −1 ).
When the secondary alcohol 15c was treated with thionyl chloride followed by an excess of piperazine (5 equivalents), the product obtained was a piperazine dimer 28 (Scheme 8). In the 1 H-NMR spectrum of the dimer 28, the broad signal (2.40 ppm) is characteristic of the piperazine methylene protons, while the signal at 4.08 ppm integrating for 2H was assigned to the two tertiary CH protons. In the 13 C-NMR spectrum, the signal at 52.0 ppm was assigned to the piperazine ring carbons; the signal at 75.7 ppm was assigned to the CH, while the duplication of the aromatic signals confirmed the formation of the product.

Stability Studies
HPLC stability studies were performed on representative compounds 21l and 24 to establish their stability at different pH systems, which mimic in vivo conditions. Compound 21l was chosen among these imidazole compounds for HPLC stability studies at three different pH systems; acidic pH 4, pH 7.4, and basic pH 9 (acid pH found in the stomach, basic found in the intestine, and pH 7.4 in the plasma). The degradation of compound 21l was minimal with 80% of 23l remaining at both pH 7.4 and pH 9 and 90% at pH 4 after 24 h. The 1,2,3-triazole compound 24 was observed to be most stable at pH 4 with 65% remaining after 24 h compared to 60% at pH 9 and 50% at pH 7.4.

In Vitro Antiproliferative Activity in MCF-7 Breast Cancer Cells
The antiproliferative activity of the panel of hybrid compounds 1-(diarylmethyl)-1H-1,2,4-triazoles (Series 1 and 2) and 1-(diarylmethyl)-1H-imidazoles (Series 3 and 4) was initially evaluated in the MCF-7 human breast cancer cell using the standard alamarBlue assay. In addition, a number of related compounds containing the aliphatic amines pyrrolidine, piperidine, and piperazine were investigated (Series 5). The MCF-7 human breast cancer cell line is estrogen receptor (ER)-positive, progesterone receptor (PR)-positive, and HER2 negative. Compounds were initially screened at two concentrations (1 and 0.1 µM) for antiproliferative activity in MCF-7 cells to determine the structure-activity relationship for these hybrid compounds and to identify the most potent compounds for further investigation. Compounds that were synthetic intermediates for the final compound were not screened, as they were not considered as potential actives in the study. The results obtained from this preliminary screen are displayed in Figures 4-6. Then, those compounds showing potential activity (cell viability <50%) were selected for further evaluation at different concentrations and in other cell lines. CA-4 (4a) (24% viable cells at 1 µM) and phenstatin (7a) (30% viable cells at 1 µM) induced a potent antiproliferative effect and were used as positive controls. Ethanol (1% v/v) was used as the vehicle control (with 99% cell viability). The preliminary results obtained for these novel compounds (Series 1-5) are discussed by structural type.
3.1.1. Series 1: 1-(Diarylmethyl)-1H-1,2,4-triazoles 13b-g, l-o The first class of compounds tested 1-(diarylmethyl)-1H-1,2,4-triazoles (13b-g, 13l-o, Figure 4A) were weakly active, with 68-90% viability for the two concentrations tested (1 µM and 0.1 µM). These compounds carry a single substituent at the para position on one or both aryl rings (Cl, F, Br, OH, OCH 3 , CH 3 , etc.) indicating that the triazole ring alone is not sufficient for the induction of antiproliferative activity in MCF-7 cells. The most active compounds were the diphenolic derivative 13o with 68% viability (1 µM) and the amino compound 13m (72% viability 1 µM). It appears that specific substituents are required on both the A and B rings of the benzophenone for activity, as also observed for phenstatin and analogues [67]. Since the potent tubulin inhibiting activity of the 3,4,5-trimethoxyaryl function is very well documented [90], the preliminary screening in MCF-7 cells of the panel of 1,2,4-triazole containing compounds (16a, c-i, 19b-e) synthesised having the 3,4,5-trimethoxyphenyl motif (A ring) together with various substituents on the B ring was next investigated ( Figure 4B, two concentrations of 1 µM and 0.1 µM). The most potent compound was identified as 19e having the characteristic 3-hydroxy-4-methoxyaryl B ring as in phenstatin and CA-4 (29% viability at 1 µM), while the ethanol control (1% v/v) resulted in 99% viability. Two compounds with moderate activity were identified as 16c (4-methoxy group in the B ring) with 75% cell viability at 1 µM and 16g (4-fluoro in B ring) with 77% viable cells at 1 µM. The remaining 3,4,5-trimethoxyphenylmethyl-1H-1,2,4-triazole compounds investigated having various substituents on the B ring e.g., 4-F, 4-CN, 4-OH, 4-CH 3 were not as potent as the lead compound with viability >80% at 1 µM, while compounds 19h and 19i were found to be inactive with half maximal inhibitory concentration (IC 50 ) values greater than 100 µM. This result demonstrated that even small changes to the phenstatin scaffold were unfavourable for antiproliferative activity. From the initial screening results, it was concluded that the 1,2,4-triazole heterocycle alone was not sufficient to improve activity in the benzhydryl compounds compared to phenstatin.  The IC 50 value for the most potent triazole-phenstatin hybrid compound 19e was determined in MCF-7 as 0.42 ± 0.07 µM at 72 h (Table 2). 19e is a hybrid of phenstatin with the 3,4,5-trimethoxyaryl motif (ring A) and the 3-hydroxy-4-methoxyaryl B ring, but it is also related to the aromatase inhibitor letrozole due to the 1,2,4-triazole heterocycle. The hybrid structure suggests a potential for dual tubulin/aromatase activity, and therefore, this compound was selected for aromatase inhibition assay.  Compound 24 is the only example synthesised containing the 1,2,3-triazole heterocycle and is also a direct analogue of phenstatin because of the presence of the 3,4,5trimethoxyaryl motif (ring A) and the 3-hydroxy-4-methoxyaryl B ring. This structure showed excellent activity in MCF-7 cells with 27% cell viability at 1 µM and the IC 50 value for the compound was determined as 52 nM (Table 2), which compares with CA-4 (IC 50 = 3.9 nM) [102,103]. 24 was selected for further studies on different cell lines and for cell cycle analysis. Phenstatin (7a) was synthesised in our laboratory for use as a positive control (IC 50 = 1.61 ± 2.7 nM) [104].
The results obtained from the preliminary screening of the benzhydryl imidazole derivatives, 20b-k and l are shown in Figure 5A. These compounds carry a single substituent at the para position on one or both aryl rings (Cl, F, Br, OH, OCH 3 , CH 3 , etc). This library of compounds did not show any significant activity, with cell viability of 67-90% at concentrations of 1 and 0.1 µM, as observed for the Series 1 1,2,4-triazole derivatives 13b-g and l-o, indicating that the imidazole ring alone is not sufficient for antiproliferative activity. The most active compounds in this panel were identified as the 4-nitro derivative 20b and the 4-fluoro substituted compound 20d (73% and 67% cell viability respectively at 1 µM).

Series 4: 1-(Aryl-(3,4,5-Trimethoxyphenyl)Methyl)-1H-Imidazoles 21a-g, i-l
The results obtained from the preliminary screening of the panel of phenstatin hybrid compounds carrying imidazole as the heterocyclic ring (21a-g, i-l) in MCF-7 cells are shown in Figure 5B. From the library of 3,4,5-trimethoxydiphenylmethyl-1H-imidazole derivatives (21a-g, i-l), compound 21l was significantly the most active (31% viable cells at 1 µM), confirming the observation that the phenstatin scaffold is required for optimum activity. The remaining compounds in the series demonstrated weak activity, with viability >80% at 1 µM. The IC 50 value of the most potent imidazole containing compound 21l was determined as 0.132 ± 0.007 µM in MCF-7 cells (Table 2) The results of preliminary evaluation of the panel of pyrrolidine and piperidine derivatives 25a-g and 26a-c in MCF-7 cells are shown Figure 6A. These compounds were not sufficiently active when compared to the positive controls CA-4 and phenstatin (7a). The most potent examples were identified as the piperidine derivative 26b, showing the lowest percentage of viable cells (78%) at 1 µM and containing the 3,4,5trimethoxyphenyl (ring A) and 4-methoxyphenyl (Ring B), together with the corresponding pyrrolidine containing compounds 25g and 25d (82% and 80% viability at 1 µM). The (3,4,5-trimethoxyphenyl)(methyl)piperazine derivatives (27c,d,f,h,i) were screened at three concentrations (10, 1, 0.1 µM) ( Figure 6B). Compound 27f was identified as the most active, with a percentage of viable cells of 42% at 10 µM, 76% at 1 µM and 84% at 0.1 µM. Benzylpiperazine 27i, which is more closely related in structure to phenstatin, displayed promising antiproliferative activity at 10 µM (48% cell viability).
From the results obtained above, it is interesting to see that inclusion of the triazole heterocycle on the phenstatin scaffold (as in compounds 21l and 24) results in greater antiproliferative effects in the MCF-7 cell line than the corresponding imidazole compound (19e). By comparison, replacement of the azole with pyrrolidine, piperidine, or piperazine resulted in decreased antiproliferative activity. The antiproliferative activity of the most potent azole compounds 19e, 21l, and 24 may be correlated to the logP values (see Supplementary Information). The imidazole compound 19e has a lower logP (2.41) when compared to the 1,2,4-triazole compound 21l (logP of 2.91) and the 1,2,3-triazole compound 24 (logP 3.50); the antiproliferative activity of the compounds 19e, 21l, and 24 in MCF-7 cells are determined as IC 50 = 0.42, 0.13, and 0.052 µM, respectively. In addition, the total polar surface (TPSA) area for these compounds is in the range 74.22-87.86 Å 2 < 140 Å 2 . However, compounds with higher logP values e.g., the piperazine compounds 27f (5.50) and 27d (4.67) display poor activity.

Antiproliferative Activity of Selected Analogues in MDA-MB-231 and HL60 Cell Lines
A number of the more potent compounds synthesised were evaluated in the triplenegative MDA-MB-231 cell line with 72 h incubation time (see Table 2). For the triazole compound 19e, an IC 50 value of 0.98 µM was obtained in MDA-MB-231 cells, although this is not as potent as observed in the MCF-7 cells (IC 50 = 0.42 µM, Table 2). The lower IC 50 value for the imidazole compound 21l (0.237 µM) indicates that the imidazole heterocycle in 21l contributes to the antiproliferative activity more effectively than the 1,2,4-triazole ring in compound 19e. The novel 1,2,3-triazole compound 24 was the best of all analogues tested in MCF-7 cells (IC 50 = 0.052 µM). The result obtained for 24 in the MDA-MB-231 cell line was also very promising (IC 50 = 0.074 µM), Table 2, and compares very favourably with the reported activity of phenstatin in MDA-MB-231 cells (IC 50 = 1.5 µM [105]), indicating that the 1,2,3-triazole has very potent antiproliferative effects compared to imidazole or 1,2,4-triazole present in the related compounds 21l and 19e. Since the antiproliferative effects of 1,2,3-triazole-phenstatin hybrid compounds has not previously been investigated, this heterocycle is especially interesting for further development.
In a further study, the antiproliferative effects of the novel imidazole compound 21l, 1,3,4-triazole compound 19e, and the 1,2,3-triazole compound 24 (structurally related to letrozole and phenstatin) in HL-60 leukaemia cells was also investigated. HL-60 leukaemia cells were used as an in vitro model for acute myeloid leukaemia. Both MCF-7 and HL-60 cell lines are CA-4 sensitive are highly susceptible to the effects of tubulin-targeting compounds [102]. The IC 50 value of 0.156 µM obtained for imidazole compound 21l identifies it as a lead compound for future development. The 1,2,3-triazole compound 24 was also potent in the leukaemia HL-60 cell line with an IC 50 value of 0.173 µM, while 19e was less potent, IC 50 = 261 µM. (IC 50 value for phenstatin = 0.031 µM [106]). This experiment demonstrated the selective effect of interchanging the imidazole, 1,3,4-triazole, and 1,2,3-triazole heterocycles on cell viability in HL-60 cells.

NCI Cell Line Screening for 19e, 21l, 25g, 26b, and 27d
Five novel substituted phenstatin compounds from the present work ((19e, (Series 2) 21l, (Series 4), 25g, 26b and 27d (Series 5)) were selected for evaluation in the NCI 60 cell line screen [107] following initial analysis of the Lipinski (drug-like) properties from the Tier-1 profiling screen, together with predictions of the relevant absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties e.g., metabolic stability, permeability, blood-brain barrier partition, plasma protein binding, and human intestinal absorption properties (see Tables S1 and S2 Supplementary Information). The compounds are predicted to be moderately lipophilic-hydrophilic, revealing their drug-like pharmacokinetic profiles and are potentially suitable candidates for further investigation.
The results obtained for the triazole compound 19e in the NCI 60 cancer cell line screening (GI 50 values, five doses) [107] are shown in Table 3. (GI 50 is defined as the concentration for 50% of maximal inhibition of cell proliferation). In general, 19e showed good activity on most of the cell lines with GI 50 values in the sub-micromolar range. The activity was particularly potent in all of the leukaemia, CNS, and prostate cancer cell lines. The activity in MCF-7 cells (GI 50 = 0.347 µM) was in close agreement with the value obtained from our in-house viability assay of 0.424 µM. The compound displayed significant activity in the TNBC cell lines HS-578T (GI 50 = 0.548 µM) and MDA-MB-468 (GI 50 = 0.371 µM) and in the BT-549 invasive ductal carcinoma cell line (GI 50 = 0.618 µM). Potent anti-cancer activity was also observed against the ovarian cancer cells, e.g., OVOCAR-3 cell line (0.323 µM) and colon cancer, e.g., chemoresistant HT-29 cells (GI 50 = 0.330 µM). The best activity for 19e among all of the 60 cell lines tested was the melanoma cell line MDA-MB-435 in which the GI 50 value was 0.181 µM. The MID GI 50 was calculated as 0.243 µM over all 60 cell lines. The MID value obtained for TGI (total growth inhibition) was 53.7 µM, and for LC 50 , it was 97.7 µM, indicating that the lethal concentration of the drug is very high and well above the GI 50 value, indicating that 19e has low toxicity. The results of the NCI COMPARE analysis for compound 19e are shown in Table 4. Based on the GI 50 mean graph and on the TGI mean graph, the compound with the highest rank was vinblastine sulphate with r values of 0.586 and 0.737, respectively. Correlation values (r) are Pearson correlation coefficients. The National Cancer Institute (NCI) screening of imidazole compound 21l also demonstrated very good results showing that the compound not only is active against breast cancer cells but also against other types of cancer (see Table 2). Compound 21l proved active against all of the leukaemia cell lines; in particular, very promising activity was measured in SR cells (GI 50 Table 3, it was observed that based on the mean GI 50 value, the activity of our 21l is most closely related to paclitaxel (r = 0.587). Based on TGI values, the compound with the highest ranking was maytansine (r = 0.775); both are tubulin-targeting agents. Correlation values (r) are Pearson correlation coefficients and LC 50 values all >0.1 mM. Compounds 25g, 26b, and 27d were also selected for evaluation in the NCI 60 cell line one-dose screen (see Table S4, Supplementary Information). The mean growth percentages for 25g, 26b, and 27d were 73.1%, 34.2%, and 65.5% over the 60 cell line panel at 10 µM. Interestingly, the piperidine compound 26b displayed significant potency in the breast cancer panel, with mean growth of 30.3% over this cell panel, with notable potency in the triple negative breast cancer cell lines HS587T (16.6% growth) and MDA-MB-468 (9.3% growth). In the leukaemia panel, the mean growth obtained is 23.5% over this 60 cell panel and significantly 4.36% growth for the acute myeloid leukaemia HL-60 cell line. Compound 26b also displayed notable potency in the CA-4 resistant colon cancer cell line HT-29 with 7.93% growth recorded.

Evaluation of Toxicity in MCF-10A Cells
The potent phenstatin derivatives 19e, 21l, and 24 were selected for toxicity evaluation in the non-tumorigenic MCF-10A epithelial breast cancer cell line. The human mammary epithelial cell line MCF10A is widely used as an in vitro model for normal breast cell function and transformation [108]. The viability of the MCF-10A cells was determined after treatment with compounds 19e and 21l at four different concentrations of 10, 1, 0.5, and 0.4 µM for 24 h ( Figure 7A,B). It was observed that at the highest concentration (10 µM), compounds 19e and 21l show a cell death of approximately 50%. At 1 µM concentration, compound 19e does not show any loss in cell viability (99% viability), while compound 21l resulted in 73% cell viability, still above the IC 50 values of 0.42 µM (19e) and 0.13 µM (21l) in MCF-7 cells. When the experiment was repeated with an increased incubation time of 48 h, it was observed that the percentage of viable cells at 10 µM concentration decreased for compounds 19e and 21l to approximately 30% ( Figure 7A,B). The percentage of viable cells at 1 µM decreased to 64% for compound 21l, while it did not change significantly for 19e (>94%). For both compounds, viability at 0.5 µM and 0.4 µM is close to 100%, which means that the compounds are not toxic toward healthy cells at lower concentrations corresponding to their IC 50 values. The third screening for 19e and 21l was performed at 72 h, which is the incubation time used through all the screenings in MCF-7 ( Figure 7A,B). It is interesting to note that as the concentration of the drug decreases from 1 to 0.5 and 0.4 µM, the percentage of viable cells increases significantly, with viable cells percentage >80% at 0.4 µM for all compounds tested. This demonstrates that even at concentrations that are toxic to the MCF-7 cancer cells, the MCF-10A are not killed by the drug. Therefore, the compounds selected demonstrate good antiproliferative activity and additionally show good selectivity and low cytotoxicity to normal cells. Compound 24 was evaluated in MCF-10A cells at three different concentrations: 10, 1, and 0.1 µM over 72 h ( Figure 7C). The percentage of viable cells at the three different concentrations was 61%, 71%, and 96%, respectively, with higher percentage of cells alive at the lower concentration. Compound 24 demonstrates good selectivity for cancer cells and low cytotoxicity even if the percentage of viable cells at 1 µM was slightly lower than the value observed previously for compounds 19e and 21l (>80%) at 72 h. These results are also supported by the low toxicity of the compounds determined from the NCI evaluation. Tubulin-targeting drugs such as taxanes and vinca alkaloids are among the most effective anti-cancer therapeutics in the treatment of castration-resistant prostate cancer and triple-negative breast cancer. However, their use is limited by toxicities including neutropenia and neurotoxicity; additionally, tumour cells can develop resistance to these drugs [109]. Our results demonstrate that azoles 19e, 21l, and 24 were less toxic to normal human breast cells than to breast cancer cells, providing a potential window of selectivity.

Effects of Compounds 21l and 24 on Cell Cycle Arrest and Apoptosis
To investigate further the mechanism of action of the novel azole compounds synthesised, the effect of selected potent compounds 21l and 24 was investigated in MCF-7 cells by flow cytometry and propidium iodide (PI) staining, allowing the percentage of cells in each phase of the cell cycle to be quantified ( Figure 8). For the imidazole compound 21l, three time points were analysed (24, 48, and 72 h), and the values obtained for apoptosis and the G 2 /M phase of the cell cycle were quantified (concentration 1 µM), as shown in Figure 8A. It was observed that the percentage of cells undergoing apoptosis (sub-G 1 ) increases significantly at all three time points to 15%, 31%, and 37% respectively compared to the background level of apoptosis with the vehicle ethanol (2%, 4%, and 2%) at the corresponding time points. It is also interesting to notice how the percentage of cells in the G 2 /M phase for the treated sample (47%, 43%, and 40%) is statistically higher than the cells in the same phase for the control sample treated with the vehicle (26%, 25%, 25%) at the corresponding time points. G 2 /M cell cycle arrest is strongly associated with an inhibition of tubulin polymerisation. CA-4 and related tubulin targeting compounds cause G2/M arrest. Hence, the higher percentage of cells observed in cells treated with 21l may suggest that the mechanism of action is indeed the inhibition of tubulin polymerisation. (v/v)) and was determined by alamarBlue assay (average ± SEM of three independent experiments). Statistical analysis was performed using two-way ANOVA (***, p < 0.001). The 1,2,3-triazole compound 24, which was the most potent compound evaluated in the viability assay, demonstrated the same effects on the relative percentages of cells in apoptosis and the G 2 /M phase, as shown in Figure 8B. Apoptosis increased with time, with a statistically significant difference compared to vehicle control at 72 h. A high percentage of cells were arrested in the G 2 /M phase (52%, 56%, and 59%) at time points 24, 48, and 72 h respectively, following treatment with compound 24 with a much lower percentage of cells in the G 2 /M phase for the sample treated with the vehicle (28%, 23%, and 24%) at the same time points.
Phenstatin 7a was used as a positive control through all the biological experiments. Cell cycle analysis of MCF-7 cells treated with phenstatin at time points 24, 48, and 72 h and a concentration of 1 µM showed a very low percentage of cells undergoing apoptosis at 24 and 48 h, as shown in Figure 8C. Apoptosis increased to 18% between 48 and 72 h, while the percentage of cells in the G 2 /M phase was correspondingly high (65%, 49%, and 51% at 24, 48, and 72 h, respectively). This pattern was also observed in the compounds 21l and 24 tested, but the percentage of cells in apoptosis was always higher than for phenstatin, possibly suggesting differences in the effects of these compounds on tubulin arising from the presence of the azole in the modified structures.
The  Figure 9A). When MCF-7 cells were treated with 21l (0.1, 0.5, and 1 µM), the average proportion of Annexin V-stained positive cells (total apoptotic cells) increased from 0.9% in control cells to 14.1%, 17.5%, and 19.3%, respectively. These results suggested that compound 21l induces the apoptosis of MCF-7 cells in a dose-dependent manner. In MDA-MB-231 cells, the percentage of cells observed in apoptosis following treatment with 21l was significantly lower with 3.6%, 5.9%, and 6.9% at 0.1, 0.5, and 1.0 µM respectively, as shown in Figure 9B. In contrast, for phenstatin, the Annexin V-stained positive cells (total apoptotic) cells were determined as 36.1% and 46% in MCF-7 cells at 0.1 µM and 0.5 µM, respectively, as shown in Figure 9A. The total apoptotic MDA-MB-231 cells were determined as 16.6% and 17.9% following treatment with phenstatin (0.1 and 0.5 µM), respectively, as shown in Figure 9B.

Tubulin Polymerisation
Compound 21l was selected for further analysis using a tubulin polymerisation assay. Its promising antiproliferative activity (IC 50 = 0.237 µM in MCF-7 cells) combined with structural features related to phenstatin 7a and CA-4 indicate that the mechanism of action of this compound could be the inhibition of tubulin polymerisation. The assay is based on the capacity of microtubules to scatter light proportionally to their concentration. The imidazole compound 21l (red) showed good inhibition of tubulin polymerisation after 60 min (V max value 2.84 ± 0.10 mOD/min at 10 µM), corresponding to a 1.34-fold reduction of the polymer mass compared to the vehicle, as shown in Figure 10A. Paclitaxel (in green) was used as a positive control as it stabilises polymerised tubulin, as shown in Figure 10A. Phenstatin 7a is a potent inhibitor of tubulin polymerisation comparable to CA-4 [66], as shown in Figure 10B. Incubation with either imidazole 21l or phenstatin resulted in a significant inhibition of tubulin polymerisation and assembly.  Following the experiment above, the in vitro effects of compounds 19e and 21l were examined on the microtubule structure of MCF-7 breast cancer cells with confocal microscopy using anti-tubulin antibodies. Paclitaxel and phenstatin, a known polymeriser and depolymeriser of tubulin respectively, were used as controls. In Figure 11A, a wellorganised microtubule network (stained green) is clearly seen for the vehicle control, together with the MCF-7 cell nuclei (stained blue). Hyperpolymerisation of tubulin was demonstrated in the paclitaxel-treated sample ( Figure 11B), whereas the phenstatin-treated sample Figure 11C shows an extensive depolymerisation of tubulin. Cells treated with the azoles 19e ( Figure 11D) and 21l ( Figure 11E) displayed disorganised microtubule networks with similar effects to phenstatin, together with multinucleation (formation of multiple micronuclei), which is a recognised sign of mitotic catastrophe [110] previously observed by us and others upon treatment with tubulin-targeting agents such as CA-4 and related compounds in non-small cell lung cancer cells and breast cancer MCF-7 cells [111,112].

Effects of Compounds 21l and 24 on Expression Levels of Apoptosis-Associated Proteins
Some of the novel compounds synthesised during the project were selected for further investigation of their mechanism of action as pro-apoptotic agents based on their effect on the expression of proteins that can regulate apoptosis or proteins involved in the regulation of DNA repair. The effects of compounds 21l and 24 on apoptosis were evaluated by Western blotting. Apoptosis regulating proteins Bcl-2 and Mcl-1 were investigated along with PARP. PARP (poly ADP-ribose polymerase) is involved in the repair of DNA singlestrand breaks in response to environmental stress [113]; and PARP cleavage is considered a hallmark of apoptosis. Bcl-2 is an anti-apoptotic protein that prevents apoptosis by sequestering caspases (apoptosis promoters) or by preventing the release of pro-apoptotic cytochrome c and AIF (apoptosis inducing factor) from the mitochondria into the cytoplasm [114]. The Mcl-1 protein belongs to the Bcl-2 family; it is also an anti-apoptotic protein localised in the mitochondrial outer membrane that acts at a very early stage in the cascade, leading to the release of the cytochrome c [115]. Pro-and anti-apoptotic members of the Bcl-2 family can heterodimerise and titrate each other's functions. If the expression levels of Mcl-1 and Bcl-2 are reduced (by drug treatment), apoptosis may be triggered.
From the results obtained, no change in the expression levels of two anti-apoptotic proteins was observed, indicating that Bcl-2 and Mcl-1 may not play a critical role in the pro-apoptotic mechanism of action of the compounds (Figure 12). A significant reduction in the expression of full-length PARP (116 kDa) between the vehicle and the treated MCF-7 cells was observed (Figure 12), suggesting that 21l and 24 cause PARP cleavage. PARP enzymes play a crucial role in the DNA repair, and PARP cleavage is affected by caspase 3 activity. PARP enzymes are found in the cell nucleus and are activated by damage of the DNA single strand; therefore, the inhibition of DNA repair in cancer cells represents an attractive strategy in cancer therapy [116]. In conclusion, the proposed mechanism of action of these compounds as pro-apoptotic drugs is supported by the observed increase in the percentage of cell in subG1 in the cell cycle profile, the flow cytometric analysis of Annexin V/PI-stained cells, and also by PARP cleavage.

Aromatase Inhibition
An objective of this research was the design of dual-acting tubulin/aromatase inhibitors. The evaluation of the aromatase inhibitory activity of the most potent compounds prepared was next investigated. Three compounds of the phenstatin hybrid panel 21l, 24 and 19e were selected for evaluation against two cytochrome members of the P450 family: CYP19 and CYP1A1. CYP19 is the aromatase cytochrome directly responsible for the synthesis of estradiol by the aromatisation of its steroid precursors testosterone and androstenedione, while CYP1A1 is involved in the metabolism of estrogen. The specificity of aromatase inhibition was evaluated by an assay carried out with xenobiotic-metabolising cytochrome P450 enzymes CYP1A1. The methodology applied in this study requires the detection of the hydrolysed dibenzylfluorescein (DBF) by the aromatase enzyme [117]. Aromatase and CYP1A1 inhibition were quantified by measuring the fluorescent intensity of fluorescein, the hydrolysis product of dibenzylfluorescein (DBF), by aromatase as previously described [118,119]. Naringenin was used as a positive control, yielding IC 50 values of 4.9 µM. The test was initially conducted at one concentration (20 µg/mL). Further experiments to determine the IC 50 value were performed if the compound caused greater than 90% inhibition at 20 µg/mL. The results are presented in Table 5. Of these, 1,2,3-triazole 24 was inactive, as it did not show any inhibition of the enzyme at 20 µg/mL, 0.05 µM (0.01% for CYP19 and 12.81% for CYP1A1), whereas imidazole 21l (0.05 µM) and 1,2,4-triazole 19e (0.05 µM) were active in the first screen against CYP19 ( Table 5). The inhibition for imidazole 21l, although potent, was not concentration-dependent, and the IC 50 could not be determined. 1,2,4-Triazole 19e inhibited aromatase in a concentrationdependent manner, and its IC 50 was determined as 29 µM. Of all the tested compounds (21l, 24, and 19e), none showed significant inhibition of CYP1A1, yielding IC 50 values above 53 µM, which is regarded as inactive [119,120]. From the results obtained, we can suggest that the 1,2,4-triazole heterocycle is required for aromatase inhibition in the phenstatin related compound 19e. Therefore, the 1,2,4-triazole compound 19e could be identified as a potential dual-acting drug for the treatment of breast cancer targeting both aromatase inhibition and tubulin polymerisation.

Molecular Docking of Phenstatin Hybrids 19e, 21l, and 24
Compounds 19e, 21l, and 24 were next examined in tubulin molecular docking experiments to rationalise the observed biochemical activities. These three molecules contain a 3-hydroxy-4-methoxy substituted aromatic ring and a 3,4,5-trimethoxyphenyl ring and differ in the nitrogen heterocycle that is substituted on the benzyhdryl linkage. The compounds phenstatin 7a and N-deacetyl-N-(2-mercaptoacetyl)colchicine (DAMA-colchicine) were used as reference compounds in the docking experiments. Since the compounds 19e, 21l, and 24 were synthesised as racemates, both R and S enantiomers of each compound were docked in the crystallised tubulin structure 1SA0 [121] and ranked based on the substituent and enantiomer giving the best binding results as illustrated in Figure  13. The co-crystallised tubulin DAMA-colchicine structure 1SA0 [121] was used for this study, as it has been demonstrated that both CA-4 4a and phenstatin 7a interact at the colchicine-binding site of tubulin. Figure 13A-C shows the binding of the S enantiomers, the ranking for the binding of the three different compounds in order: S-21l, S-24, and S-19e. All three compounds demonstrate a strong interaction with the same amino acid residue Lys352. Compound S-21l forms a hydrogen bond acceptor interaction between an imidazole nitrogen and Ser178. The imidazole also forms a π-CH interaction with Leu248. Compounds S-24 and S-19e show very similar behaviour; they do not bind Ser178 but still have the same interaction with Leu248. In the R-enantiomer series, the heterocycle is directed differently, and very different binding poses and less favourable binding interactions between the ligands and the tubulin binding site are predicted for these compounds ( Figure 13D-F). In order to maintain the A and C-ring overlays, the heterocycle would clash with binding site amino acids, so for the three R-enantiomers, the heterocycle overlays with either the A or C-ring and the 3,4,5-trimethoxyphenyl mapping is no longer possible or not as ideal.
Compound S-21l was the highest ranked compound in the series; therefore, it would be of interest to obtain in vitro results for the enantiomerically pure compound. Phenstatin 7a also maps well to the colchicine binding pose with the 3,4-5-trimethoxyaryl residues overlaying effectively and the B-ring 4-methoxy group positioned to form a hydrogen bond with Lys352 ( Figure 12G). The results provide rationalisation of the observed biochemical experiments in which cell cycle and tubulin binding was confirmed, indicating that these compounds are apoptotic and tubulin depolymerising agents. Ligands are rendered as tube and amino acids as a line. Tubulin amino acids and DAMA-colchicine are coloured by atom type; the three heterocycles are coloured 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.

Chemistry
All reagents were commercially available and were used without further purification unless otherwise indicated. Anhydrous solvents were purchased from Sigma. Uncorrected melting points were measured on a Gallenkamp apparatus. Infrared (IR) spectra were recorded on a Perkin Elmer FT-IR Paragon 1000 spectrometer. 1 H and 13 C nuclear magnetic resonance spectra (NMR) were recorded at 27 • C on a Brucker DPX 400 spectrometer (400.13 MHz, 1 H; 100.61 MHz, 13 C) in CDCl 3 (internal standard tetramethylsilane (TMS)). For CDCl 3 , 1 H NMR spectra were assigned relative to the TMS peak at 0.00 ppm, and 13 C NMR spectra were assigned relative to the middle CDCl 3 peak at 77.0 ppm. Electrospray ionisation mass spectrometry (ESI-MS) was performed in the positive ion mode on a liquid chromatography time-of-flight mass spectrometer (Micromass LCT, Waters Ltd., Manchester, UK). The samples were introduced to the ion source by an LC system (Waters Alliance 2795, Waters Corporation, Milford, MA, USA) in acetonitrile/water (60:40% v/v) at 200 µL/min. The capillary voltage of the mass spectrometer was at 3 kV. The sample cone (de-clustering) voltage was set at 40 V. For exact mass determination, the instrument was externally calibrated for the mass range m/z 100 to 1000. A lock (reference) mass (m/z 556.2771) was used. Mass measurement accuracies of < ±5 ppm were obtained. Thin-layer chromatography (TLC) was performed using Merck Silica gel 60 TLC aluminium sheets with fluorescent indicator visualising 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. 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 × 4.6 mm chromatography column. Samples were detected using a wavelength of 254 nm. All samples were analysed using acetonitrile (60%)/water (40%) over 10 min and a flow rate of 1 mL/min. Microwave experiments were carried using a Biotage Discover CEM microwave synthesiser on a standard power setting (maximum power supplied is 300 watts) unless otherwise stated. Details of the synthesis and characterisation of intermediate compounds and target azole products are available in the Supporting Information.

General Method A: Preparation of Alcohols
To a solution of the benzophenone in methanol (25 mL), NaBH 4 (1 eq) was added in small portions. The solution was stirred at 0 • C until the reaction was complete from TLC. Dilute HCl (10%) was added, and the solvent was removed with the rotary evaporator. Then, the product was dissolved in ethyl acetate (30 mL) and washed with water (20 mL) and brine (10 mL), dried over sodium sulphate, filtered, and concentrated. Purification via flash column chromatography (eluent: n-hexane/ethyl acetate 1:1) afforded the product.

General Method E for the Preparation of Diarylmethylpyrrolidines, Diarylmethylpiperidines and Diarylmethylpiperazines
The benzhydryl alcohol (1 eq) was reacted with thionyl chloride (5 eq) in dry DCM (30 mL) for 12 h. The reaction mixture was concentrated under reduced pressure, and the crude product was used in the next step without any further purification. The chlorinated benzhydryl alcohol was reacted with pyrrolidine or piperidine (5 eq) in dry ACN (30 mL) and refluxed for 12 h. The solvent was removed, and the residue was dissolved in DCM (50 mL) and washed with 1 M NaOH (30 mL). The organic phase was dried over sodium sulphate, filtered, and concentrated. Then, the crude product was purified via flash chromatography (eluent: n-hexane/ethyl acetate).

Stability Study of Compounds 21l and 24
Stability studies for compounds 21l and 24 were performed by analytical HPLC using a Symmetry ® column (C18, 5 mm, 4.6 × 150 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 (30%) 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 21l and 24 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 2020. Then, 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 hourly for 24 h.

X-ray Crystallography
Data for samples 16e, 16f, 19c, 21e, and 26a were collected on a Bruker APEX DUO using Mo Kα and Cu Kα radiation (λ = 0.71073 and 1.54178 Å). Each sample was mounted on a MiTeGen cryoloop and data were collected at 100(2) K using an Oxford Cobra cryosystem. Bruker APEX [123] software was used to collect and reduce data, determine the space group, solve, and refine the structures. Absorption corrections were applied using SADABS 2014 [124]. Structures were solved with the XT structure solution program [125] using Intrinsic Phasing and refined with the XL refinement package [126] using Least Squares minimisation. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were assigned to calculated positions using a riding model with appropriately fixed isotropic thermal parameters. Molecular graphics were generated using OLEX2 [127]. All structures are racemates. In 26a, the disordered fluorine was modelled in two positions with occupancies of 84% and 16%. Geometric restraints (SADI) were used to model the C-F bond lengths. Crystallographic data for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos. 201543, 2015432, 2015433, 2015434, and 2015435. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail:deposit@ccdc.cam.ac.uk).

Biochemical Evaluation of Activity
All biochemical assays were performed in triplicate and on at least three independent occasions for the determination of mean values reported.

Cell Cycle Analysis
Cells were seeded at a density of 1 ×

Tubulin Polymerisation 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 [128]. Briefly, purified (>99%) bovine brain tubulin (3 mg/mL) in a buffer consisting of 80 mM piperazine-N,N'bis(2-ethanesulfonic acid) (PIPES) (pH 6.9), 0.5 mM ethylene glycol tetraacetic acid (EGTA), 2 mM MgCl 2 , 1 mM guanosine-5'-triphosphate (GTP)GTP and 10% glycerol was incubated at 37 • C in the presence of either vehicle (2% (v/v) ddH 2 O) paclitaxel, phenstatin (7a), or 21l (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. 4.4.8. Cytochrome P450 Assays (CYP19 (Aromatase) and CYP1A1) The substrate DBF (dibenzylfluorescein) was obtained from Gentest Corporation (Woburn, MA). All human recombinant cytochrome P450 enzymes were purchased from BD Biosciences, San Jose, CA. Aromatase and CYP1A1 inhibition were quantified by measuring the fluorescent intensity of fluorescein, the hydrolysis product of dibenzylfluorescein (DBF), by aromatase, as previously described [118,119]. In brief, the test substance (10 µL) was pre-incubated with a NADPH regenerating system (90 µL of 2.6 mM NADP + , 7.6 mM glucose 6-phosphate, 0.8 U/mL glucose 6-phosphate dehydrogenase, 13.9 mM MgCl 2 , and 1 mg/mL albumin in 50 mM potassium phosphate, pH 7.4), for 10 min, at 37 • C, before 100 µL of the enzyme and substrate (E/S) mixture were added (4.0 pmol/well of CYP19/0.4 µM DBF; 5.0 pmol/well of CYP2C8/2.0 µM DBF; 5.0 pmol/well of CYP3A4/ 2.0 µM DBF and 0.5 pmol/well of CYP1A1/2.0 µM DBF). The reaction mixtures were incubated for 30 min (excepting CYP1A1, 25 min) at 37 • C to allow the generation of product, quenched with 75 µL of 2 N NaOH, shaken for 5 min, and incubated for 2 h at 37 • C to enhance the noise/background ratio. Finally, fluorescence was measured at 485 nm (excitation) and 530 nm (emission). Three independent experiments were performed, each one in triplicate, and the average values were used to construct dose-response curves. At least four concentrations of the test substance were used, and the IC 50 value was calculated (Tablecurve TM 2D, AISN Software, EUA, 1996). Naringenin was used as positive controls, yielding an IC 50 value of 4.9 µM. Compounds 19e, 21l, and 24 were dissolved in dimethyl sulfoxide (DMSO) and diluted to final concentrations. An equivalent volume of DMSO was added to control wells, and this had no measurable effect on cultured cells or enzymes. Compounds are considered for further experiments when showing inhibition great than 90%.

Molecular Modelling and Docking Study
The X-ray structure of bovine tubulin co-crystallised with N-deacetyl-N-(2-mercaptoacetyl)colchicine (DAMA-colchicine) 1SA0 [121] was downloaded from the PDB website. A UniProt Align analysis confirmed a 100% sequence identity between human and bovine β 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 2015 with the MMFF94x force field [129]. Both enantiomers of selected compounds 19e, 24, and 21l 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 300 poses were sampled for each enantiomer, and the top 50 docked poses were retained for subsequent analysis.

Conclusions
In this work, a novel series of heterocyclic phenstatin-based compounds have been designed and synthesised as tubulin-targeting agents. The structural modifications introduced on the phenstatin moiety included the nitrogen heterocycles 1,2,4-triazole, 1,2,3triazole, and imidazole to afford a hybrid structure of the vascular targeting agent phenstatin and the aromatase inhibitor letrozole, which contains a 1,2,4-triazole heterocycle. The introduction of aliphatic amines such as pyrrolidine, piperazine, and various piperidine derivatives was also achieved. The resulting compounds were investigated for potential dual activity as tubulin and aromatase inhibitors. All novel compounds were initially evaluated in the MCF-7 breast cancer cell line and of particular interest were compounds 19e, 21l, and 24, which displayed antiproliferative activity in the nanomolar range e.g., 19e (IC 50 = 424 nM, 21l (IC 50 = 132 nM), and 24 (IC 50 = 52 nM). They were selected for further studies to provide a better understanding of their mechanism of action in breast cancer cells.
The most potent compounds 21l and 24 were evaluated in MCF-10A cells (normal breast epithelial cells) for cytotoxicity. Minimal cell death was observed when treated at a concentration similar to the IC 50 value of the compounds in MCF-7 cells, indicating that the compounds were selective towards cancer cells. Compounds showed impressive antiproliferative activity at nanomolar levels against a range of susceptible human cancer cell lines when tested in the 60 cancer cell line panel of the NCI. Cell cycle analysis of compounds 21l and 24 resulted in an increase in G 2 /M arrest and apoptotic cell death in MCF-7 cells. Flow cytometric analysis of Annexin V/PI-stained cells indicated that compound 21l induces the apoptosis of MCF-7 cells in a dose-dependent manner. Compounds 21l and 24 were also shown to promote PARP cleavage and an inhibition of tubulin polymerisation. The tubulin effects were confirmed when MCF-7 cells treated with the azoles 19e and 21l displayed disorganised microtubule networks with similar effects to phenstatin, together with multinucleation.
The molecular docking of selected compounds indicated possible binding to the colchicine-binding site of tubulin and a preference for the S enantiomer. The results showed an efficient introduction of the azoles 1,2,4-triazole, 1,2,3-triazole, and imidazole on the phenstatin scaffold structure to retain antiproliferative effects. The selective inhibition of aromatase is an important tool to select compounds that act as chemopreventative agents for hormone-dependent cancer [130]. The aromatase inhibition of the most potent antiproliferative compounds 19e, 21l, and 24 was evaluated, and compound 19e was identified as the most potent with over 85% inhibition of CYP19 at 20 µM and an IC 50 of 29 µM. We can conclude that the 1,2,4-triazole heterocycle is essential for aromatase inhibition in these compounds, and its activity was optimised when included in a phenstatin-related scaffold such as 19e. On the basis of the structural modifications of phenstatin described in this work, e.g., introduction of the azoles 1,2,4-triazole, 1,2,3-triazole, and imidazole on the phenstatin scaffold, we have developed lead compounds that exhibit promising anti-cancer properties with potential for further development. The investigation of the stereoselective effects of the compounds together with the optimisation of the dual aromatase-antiproliferative action of compound 19e is in progress.

Acknowledgments:
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). This study was also co-funded under the European Regional Development. We thank Susan McDonnell, School of Chemical and Bioprocess Engineering, University College Dublin for the kind gift of MCF-10A cells, Gavin McManus for assistance with confocal microscopy, and Barry Moran for flow cytometry. Synthetic contributions from Rebecca Hirschberger and Ayat Sherif are also appreciated. We thank John O'Brien and 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.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript: