Novel Selective Estrogen Receptor Ligand Conjugates Incorporating Endoxifen-Combretastatin and Cyclofenil-Combretastatin Hybrid Scaffolds: Synthesis and Biochemical Evaluation

Nuclear receptors such as the estrogen receptors (ERα and ERβ) modulate the effects of the estrogen hormones and are important targets for design of innovative chemotherapeutic agents for diseases such as breast cancer and osteoporosis. Conjugate and bifunctional compounds which incorporate an ER ligand offer a useful method of delivering cytotoxic drugs to tissue sites such as breast cancers which express ERs. A series of novel conjugate molecules incorporating both the ER ligands endoxifen and cyclofenil-endoxifen hybrids covalently linked to the antimitotic and tubulin targeting agent combretastatin A-4 were synthesised and evaluated as ER ligands. A number of these compounds demonstrated pro-apoptotic effects, with potent antiproliferative activity in ER-positive MCF-7 breast cancer cell lines and low cytotoxicity. These conjugates displayed binding affinity towards ERα and ERβ isoforms at nanomolar concentrations e.g., the cyclofenil-amide compound 13e is a promising lead compound of a clinically relevant ER conjugate with IC50 in MCF-7 cells of 187 nM, and binding affinity to ERα (IC50 = 19 nM) and ERβ (IC50 = 229 nM) while the endoxifen conjugate 16b demonstrates antiproliferative activity in MCF-7 cells (IC50 = 5.7 nM) and binding affinity to ERα (IC50 = 15 nM) and ERβ (IC50 = 115 nM). The ER binding effects are rationalised in a molecular modelling study in which the disruption of the ER helix-12 in the presence of compounds 11e, 13e and 16b is presented These conjugate compounds have potential application for further development as antineoplastic agents in the treatment of ER positive breast cancers.


Introduction
Breast cancer is the most common cancer in women worldwide, affecting one in eight women and representing a significant cause of cancer death in women. Incidence rates are increasing steadily, with nearly 1.7 million new cases diagnosed in 2012 worldwide [1]. The majority of early stage breast cancers are hormone-dependent and patient prognosis is good. However, when the breast cancer is or becomes hormone-independent then prognosis is poor. About 5% of breast cancers, denoted BRCA-1 and BRCA-2, are considered hereditary [2]. The two nuclear estrogen receptors (ERα and ERβ) Typical conjugates are bifunctional molecules containing covalently linked ligands or pharmacophores which are designed to produce selectivity in targeting the intracellular ER [35,36]. The objectives of the conjugate design investigated in the current research are to produce conjugates capable of the delivery of cytotoxic agents to the ER positive breast cancer tumour cell, and to increase the selectivity of these cytotoxic agents which should result in less toxicity and increased efficacy. We also wished to produce ER antagonists through inclusion of the additional, bulky linker-cytotoxic agent moiety of the conjugate structure and with the possibility of achieving a dual-action activity i.e., ER antagonism and antimitotic activity.
ER ligand conjugates of cytotoxic agents, photodynamic therapeutic agents and radioligands which deliver cytotoxic agents have been reported [31,37]. We have previously reported stable conjugates of endoxifen (a tamoxifen metabolite and ER antagonist) with DNA alkylating agents, aromatase inhibitors, COX2 inhibitors and antitubulin compounds which demonstrate antiproliferative and ER binding effects [38]. We have also reported related conjugates based on the 2-arylindole scaffold structure which selectively target the ER in hormone dependent breast cancers [39]. Many previously reported ER conjugates have included ER agonist ligands such as estradiol as the ER ligand component [37,40]. However, in the present work we have incorporated ER antagonistic ligands such as endoxifen [27] and the novel cyclofenil based ER antagonist in the designed structures.
Combretastatin A-4 (CA-4) (Figure 1), a potent antimitotic agent isolated from the bark of the South African tree Combretum caffrum. It exhibits potent antitubulin effects by binding to tubulin at the colchicine binding site. CA-4 demonstrates cytotoxicity against a wide range of human cancer cell Typical conjugates are bifunctional molecules containing covalently linked ligands or pharmacophores which are designed to produce selectivity in targeting the intracellular ER [35,36]. The objectives of the conjugate design investigated in the current research are to produce conjugates capable of the delivery of cytotoxic agents to the ER positive breast cancer tumour cell, and to increase the selectivity of these cytotoxic agents which should result in less toxicity and increased efficacy. We also wished to produce ER antagonists through inclusion of the additional, bulky linker-cytotoxic agent moiety of the conjugate structure and with the possibility of achieving a dual-action activity i.e., ER antagonism and antimitotic activity.
ER ligand conjugates of cytotoxic agents, photodynamic therapeutic agents and radioligands which deliver cytotoxic agents have been reported [31,37]. We have previously reported stable conjugates of endoxifen (a tamoxifen metabolite and ER antagonist) with DNA alkylating agents, aromatase inhibitors, COX2 inhibitors and antitubulin compounds which demonstrate antiproliferative and ER binding effects [38]. We have also reported related conjugates based on the 2-arylindole scaffold structure which selectively target the ER in hormone dependent breast cancers [39]. Many previously reported ER conjugates have included ER agonist ligands such as estradiol as the ER ligand component [37,40]. However, in the present work we have incorporated ER antagonistic ligands such as endoxifen [27] and the novel cyclofenil based ER antagonist in the designed structures. Combretastatin A-4 (CA-4) (Figure 1), a potent antimitotic agent isolated from the bark of the South African tree Combretum caffrum. It exhibits potent antitubulin effects by binding to tubulin at the colchicine binding site. CA-4 demonstrates cytotoxicity against a wide range of human cancer cell lines, including those that are multi-drug resistant [41][42][43]. However, because of poor water solubility of CA-4, a more water-soluble combretastatin A4-phosphate CA-4P (fosbretabulin, Figure 1) was synthesized and is currently under investigation in combination with pazopanib in a Phase 1b and Phase II study for the treatment of advanced recurrent ovarian cancer [44]. Many synthetic analogues of combretastatin A4 have been developed [45]. Among the various hybrids and conjugates of combretastatin A-4 which have been reported are hydrophobic combretastatin A-4 conjugated with hydrophilic irinotecan to form amphiphilic molecules (in which an azobenzene bond imparts hypoxia sensitivity) which can self assembly into nanoparticles for breast cancer synergistic therapy [46]. High-molecular weight conjugates of combretastatins with polyethylene glycol have been reported (comprising a block copolymer of a polyethylene glycol moiety and a polymer moiety having two or more carboxylic acid groups), in which a carboxylic acid group of the polymer moiety is linked to a hydroxyl group of combretastatins via an ester [47]. Combretastatin A-4 conjugated anti-angiogenic micellar drug delivery systems using dendron-polymer conjugates are also reported [48]. CA-4 analogues have been introduced onto steroid scaffolds to explore proapoptotic effects [49].
In the present work, ER antagonistic ligands such as endoxifen and cyclofenil related compounds are chosen as the targeting mechanism for the conjugate, with the objective of designing effective antiproliferative compounds for in vitro evaluation. The introduction of steric hindrance provided by the combretastatin CA-4 amide fragment is now investigated to determine if this modification enhances the ER antagonistic effects of the endoxifen and cyclofenil conjugates in the ER positive MCF-7 cells, possibly by interference with Helix-12 of the ER. To determine the influence of ER ligand structural modifications of the conjugate on antiproliferative activity in ER positive MCF-7 breast cancer cells, the following ER ligand conjugate structural types will be investigated:

Synthesis of Conjugates
For this study the required combretastatin related acrylic acids 1a-1r (Table 1) were prepared using the Perkin condensation reaction which is an efficient synthetic route for novel combretastatin acrylic acid analogues from a variety of aldehyde and phenylacetic acid starting materials as shown in Scheme 1 [50]. The carboxylic acid located on the ethylene products will facilitate the conjugate formation with the ER ligands via coupling reactions to afford the required ester or amide linking system. Selection of the series of acrylic acids for synthesis was initially based on the requirement for the 3,4,5-trimethoxyphenyl substitution for ring A of CA4; however, other structurally related substitutions were also used as shown in Scheme 1. The panel of novel and previously reported acrylic acid combretastatin analogues 1a-1r were synthesised via the Perkin reaction using both the reflux and microwave methods [51]. In all cases studied the yields for the microwave method were superior to those of the conventional technique method with shorter reaction time (<30 min) and with exclusive formation of the desired cis isomer. 2-(3,5-Dimethoxyphenyl)acetic acid and 2-(3-hydroxy-4-methoxyphenyl)acetic acid were prepared as described in the Supplementary information. Combretastatin CA-4 was used as a standard reference and was prepared by the Wittig reaction sequence [52] or by decarboxylation of 1l [50].
Endoxifen was chosen as a suitable ER-ligand scaffold due to its high affinity ER-binding properties. Additionally, the secondary amine group present on the basic side chain can undergo amide type coupling reactions to synthesise the prototype conjugated compounds. The OTBDMS protected endoxifen ligand 2a was prepared in a multistep route as we previously reported via the McMurry reaction which is a low valent titanium mediated crossed coupling of substituted benzophenones and ketones [31].  The McMurry reaction has been the route of choice for the preparation of the triarylethylene scaffold as it commonly leads to favourable E:Z isomer ratios. [53] The E:Z isomeric ratio for 2a is calculated as 1:1.3 based on the integral of the signals of the chemical shifts assigned to the OCH2 and NCH2 signals for the protons of the basic side chain in the isomeric mixtures [54]. However, 4-hydroxytamoxifen and endoxifen and related 4-hydroxysubstituted triarylethylenes undergo E/Z isomerisation under physiological conditions, and have little effect on ER activity [55][56][57]. Therefore, the E/Z isomer mixture of 2a obtained in the present work was used without further separation in the formation of the subsequent conjugates. Deprotection of 2a affords endoxifen.
The acrylic acid combretastatin analogues 1a-1j, 1m, 1p, 1q were directly coupled to the silylprotected endoxifen analogue 2a to afford the conjugates 3a-3m (Scheme 2, Table 2). The initial coupling procedure investigated DCC as the coupling agent for the synthesis of this series of conjugates. Equimolar amounts of the acrylic acid, amine 2a DCC and HOBt were reacted and the reaction was monitored via TLC. The resulting silyl-protected conjugates were treated with TBAF to afford the direct amide conjugates 3a-3m in high yields as ~1:1 (E/Z)-isomeric mixtures, Table 2. The isomeric ratios were calculated based on those of the endoxifen starting material and confirmed by integration of appropriate signals in the 1 H-NMR spectra. The presence of rotamers also resulted in complex spectra. EDC was also used as the coupling reagent for the synthesis of conjugates 3a-3k. The related amide compounds 3n, 3o, 3p and 3q were prepared for biochemical evaluation by reaction of the acrylic acids 1l, 1n and 1p with ammonium acetate and diethylamine respectively (Scheme 3 The McMurry reaction has been the route of choice for the preparation of the triarylethylene scaffold as it commonly leads to favourable E:Z isomer ratios. [53] The E:Z isomeric ratio for 2a is calculated as 1:1.3 based on the integral of the signals of the chemical shifts assigned to the OCH 2 and NCH 2 signals for the protons of the basic side chain in the isomeric mixtures [54]. However, 4-hydroxytamoxifen and endoxifen and related 4-hydroxysubstituted triarylethylenes undergo E/Z isomerisation under physiological conditions, and have little effect on ER activity [55][56][57]. Therefore, the E/Z isomer mixture of 2a obtained in the present work was used without further separation in the formation of the subsequent conjugates. Deprotection of 2a affords endoxifen. The acrylic acid combretastatin analogues 1a-1j, 1m, 1p, 1q were directly coupled to the silyl-protected endoxifen analogue 2a to afford the conjugates 3a-3m (Scheme 2, Table 2). The initial coupling procedure investigated DCC as the coupling agent for the synthesis of this series of conjugates. Equimolar amounts of the acrylic acid, amine 2a DCC and HOBt were reacted and the reaction was monitored via TLC. The resulting silyl-protected conjugates were treated with TBAF to afford the direct amide conjugates 3a-3m in high yields as~1:1 (E/Z)-isomeric mixtures, Table 2. The isomeric ratios were calculated based on those of the endoxifen starting material and confirmed by integration of appropriate signals in the 1 H-NMR spectra. The presence of rotamers also resulted in complex spectra. EDC was also used as the coupling reagent for the synthesis of conjugates 3a-3k. The related amide compounds 3n, 3o, 3p and 3q were prepared for biochemical evaluation by reaction of the acrylic acids 1l, 1n and 1p with ammonium acetate and diethylamine respectively (Scheme 3).   A further objective of this project was to prepare conjugates of endoxifen with cinnamic and phenylpropanoic acids related in structure to A and B rings of CA4. For this modified structure the substituent ring would now be either the 3,4,5-trimethoxy ring (A ring) or the 3-hydroxy-4-methoxyring (B ring). Both rings (A and B) have been shown to be relevant for the antitubulin CA4 activity [45]. The cinnamic acids 4a and 4c were prepared by reaction of the appropriate aryl aldehyde with malonic acid under microwave conditions [58]; subsequent reduction via a palladium/C hydrogenation afforded the 3-phenylpropanoic acids 4b and 4d [59] with yields in excess of 80%. (See Supplementary information). The compounds 4a-4d were then used to synthesise the required endoxifen conjugates  A further objective of this project was to prepare conjugates of endoxifen with cinnamic and phenylpropanoic acids related in structure to A and B rings of CA4. For this modified structure the substituent ring would now be either the 3,4,5-trimethoxy ring (A ring) or the 3-hydroxy-4-methoxyring (B ring). Both rings (A and B) have been shown to be relevant for the antitubulin CA4 activity [45]. The cinnamic acids 4a and 4c were prepared by reaction of the appropriate aryl aldehyde with malonic acid under microwave conditions [58]; subsequent reduction via a palladium/C hydrogenation afforded the 3-phenylpropanoic acids 4b and 4d [59] with yields in excess of 80%. (See Supplementary information). The compounds 4a-4d were then used to synthesise the required endoxifen conjugates A further objective of this project was to prepare conjugates of endoxifen with cinnamic and phenylpropanoic acids related in structure to A and B rings of CA4. For this modified structure the substituent ring would now be either the 3,4,5-trimethoxy ring (A ring) or the 3-hydroxy-4-methoxy-ring (B ring). Both rings (A and B) have been shown to be relevant for the antitubulin CA4 activity [45]. The cinnamic acids 4a and 4c were prepared by reaction of the appropriate aryl aldehyde with malonic acid under microwave conditions [58]; subsequent reduction via a palladium/C hydrogenation afforded the 3-phenylpropanoic acids 4b and 4d [59] with yields In the present study, the cyclofenil analogues chosen for synthesis retain the cycloalkyl group of the cyclofenil parent structure, while in addition, they include the basic side chain moiety of the endoxifen parent structure. Unlike the previous synthesis of the triarylethylene scaffold for the endoxifen analogues, the use of a cyclic ketone eliminates any issue of E-and Z-isomers in the products. It was envisioned that the elimination of the E/Z isomers could lead to a simpler purification step and would result in a less complex NMR spectra.
Compounds 8a-8e then underwent an ethylbromination reaction to afford 9a-9e. Following an amination reaction step, analogues 10a-10e were used in the formation of novel conjugates. Compounds 10a-10e were subsequently deprotected to afford the endoxifen-type cyclofenil analogues 11a-11e. These novel ER ligands containing a basic side chain ether similar to that of endoxifen, were subsequently used for the novel linkage to the CA4-type cytotoxic agent. The acrylic acid combretastatin analogue 1l was directly coupled to the silyl-protected cyclofenil-based analogues 10a-10e to afford the protected conjugates 12a-12e. This procedure employed EDC as the coupling reagent. The synthesis of this series of silyl-protected conjugates was similar to that for the endoxifen conjugates synthesis 3a-3k the reagent ratio was optimised: 1.2 eq. of acrylic acid, 1.4 eq. DCC, 1.4 eq. HOBt and 1 eq of amine were reacted and the reaction was monitored via TLC. The silyl-protected conjugates 12a-12e were isolated and fully characterised and then treated with TBAF to afford the direct amide conjugates 13a-13e in high yields (Scheme 5). In the present study, the cyclofenil analogues chosen for synthesis retain the cycloalkyl group of the cyclofenil parent structure, while in addition, they include the basic side chain moiety of the endoxifen parent structure. Unlike the previous synthesis of the triarylethylene scaffold for the endoxifen analogues, the use of a cyclic ketone eliminates any issue of Eand Zisomers in the products. It was envisioned that the elimination of the E/Z isomers could lead to a simpler purification step and would result in a less complex NMR spectra.
Compounds 8a-8e then underwent an ethylbromination reaction to afford 9a-9e. Following an amination reaction step, analogues 10a-10e were used in the formation of novel conjugates. Compounds 10a-10e were subsequently deprotected to afford the endoxifen-type cyclofenil analogues 11a-11e. These novel ER ligands containing a basic side chain ether similar to that of endoxifen, were subsequently used for the novel linkage to the CA4-type cytotoxic agent. The acrylic acid combretastatin analogue 1l was directly coupled to the silyl-protected cyclofenil-based analogues 10a-10e to afford the protected conjugates 12a-12e. This procedure employed EDC as the coupling reagent. The synthesis of this series of silyl-protected conjugates was similar to that for the endoxifen conjugates synthesis 3a-3k the reagent ratio was optimised: 1.2 eq. of acrylic acid, 1.4 eq. DCC, 1.4 eq. HOBt and 1 eq of amine were reacted and the reaction was monitored via TLC. The silyl-protected conjugates 12a-12e were isolated and fully characterised and then treated with TBAF to afford the direct amide conjugates 13a-13e in high yields (Scheme 5). With all of the conjugate prototypes investigated to date, the synthesis of the conjugates required a coupling reaction between a carboxylic acid group with an amine forming an amide linkage. Coupling of the phenolic functionality of CA-4 with the free carboxylic acid group of diacid linker compounds, forming ester linkages was also investigated. A diacid type linker was chosen to allow for the formation of ester and/or amide bonds with any available phenol and/or amine groups present on the conjugate component-fragments. Therefore, it is envisioned that these diacid fragments can be metabolised easily in vivo thus releasing the conjugate component ligands and possibly exerting a dual action effect.
Desmethyltamoxifen 2b was initially used as the prototype ER-ligand for the DCC coupling reaction with the dicarboxylic acid (Scheme 6). Succinic acid, DCC and HOBt were reacted with 2b and an isomeric mixture (E:Z = 1:2.3) of the product 14a was afforded (90% yield) (see Scheme 6). With all of the conjugate prototypes investigated to date, the synthesis of the conjugates required a coupling reaction between a carboxylic acid group with an amine forming an amide linkage. Coupling of the phenolic functionality of CA-4 with the free carboxylic acid group of diacid linker compounds, forming ester linkages was also investigated. A diacid type linker was chosen to allow for the formation of ester and/or amide bonds with any available phenol and/or amine groups present on the conjugate component-fragments. Therefore, it is envisioned that these diacid fragments can be metabolised easily in vivo thus releasing the conjugate component ligands and possibly exerting a dual action effect.
Desmethyltamoxifen 2b was initially used as the prototype ER-ligand for the DCC coupling reaction with the dicarboxylic acid (Scheme 6). Succinic acid, DCC and HOBt were reacted with 2b and an isomeric mixture (E:Z = 1:2.3) of the product 14a was afforded (90% yield) (see Scheme 6). It was found that the reactions involving the formation of the diacid linker compounds from secondary amine, triarylethylenes with acid anhydrides were successful without the need for any additional reagents such as DCC, HOBt or DMAP. Succinic anhydride was reacted successfully with desmethyltamoxifen 2b to afford the diacid-linker compounds 14a in 97% yield. It was found that the reactions involving the formation of the diacid linker compounds from secondary amine, triarylethylenes with acid anhydrides were successful without the need for any additional reagents such as DCC, HOBt or DMAP. Succinic anhydride was reacted successfully with desmethyltamoxifen 2b to afford the diacid-linker compounds 14a in 97% yield. In addition to the preliminary diacid reactions carried out on desmethyltamoxifen 2b, succinic anhydride was next reacted with the silyl-protected endoxifen analogue 2a to afford 14b. The phenol group present on the endoxifen ligand plays an important role in ER-binding and is a desirable functionality on the proposed conjugate structures for improved ER-binding and overall bioactivity. Initially, it was decided to carry out the conjugate prototype reactions using a single linker-ligand 14b. The succinic linker was chosen as it is a flexible fragment and allows the conjugate some   In addition to the preliminary diacid reactions carried out on desmethyltamoxifen 2b, succinic anhydride was next reacted with the silyl-protected endoxifen analogue 2a to afford 14b. The phenol group present on the endoxifen ligand plays an important role in ER-binding and is a desirable functionality on the proposed conjugate structures for improved ER-binding and overall bioactivity. Initially, it was decided to carry out the conjugate prototype reactions using a single linker-ligand 14b. The succinic linker was chosen as it is a flexible fragment and allows the conjugate some flexibility. Therefore, the prototype succinic-conjugates would not be overly restricted and adopt a configuration for optimal binding.
The use of DCC with HOBt was effective for the majority of the coupling reactions above; however coupling of CA-4 15 with the diacid linker compound 14a to afford the conjugate 16a gave very low yields (Scheme 5). It was then decided to first react the diacid linker component succinic acid with the CA4 structure and then to react the resulting CA4-linker product with the desmethyltamoxifen analogue 2b forming the amide linkage of the conjugate 16a. However, initial attempts to couple the succinic acid/anhydride linker directly to CA4 were unsuccessful using a variety of reaction conditions (e.g., DCC/HOBt, Et 3 N, or Mitsunobu conditions, PPh 3 /DIAD). Pettit et al. reported the synthesis of succinic acid esters of combretastatin analogues as CA4 prodrugs and water soluble derivatives [41]. In the present study CA4 15 was treated with 14b using DCC coupling conditions with DMAP as a base. The coupling reaction was successful (Scheme 5) and the intermediate protected conjugate was treated with TBAF to give the target compound 16b as an isomeric mixture (E:Z = 1:1) in a high yield (88%). Similarly, 16a was obtained in high yield (83%) by reaction of 15 with the desmethyltamoxifen 2b. The related conjugate 16c was prepared by coupling of the acrylic acid 14a with the phenolic ester 1s, to afford a product with additional ester functionality on the acrylic alkene (Scheme 6).
The stability of the target conjugate compounds 16a, 16b and 16c, was evaluated in phosphate buffer at pH values in the range 4-9 also in plasma and the half-life was determined to be greater than 20 h for each compound at these pH values. For the most potent conjugate compound 16b, >90% remained over pH range 4-9 at 12 h with half life >48 h; and 77% remained intact in plasma at 12 h, with a half life >48 h. We observed no significant degradation of the conjugate. This result indicates that the combretastatin stilbene moiety of the endoxifen conjugate structure is required for optimum interaction with helix 12 of the ER LBD, and contributes to the observed ER antagonistic effects.

Antiproliferative Activity in MCF-7 Breast Cancer Cells
The antiproliferative activity of the conjugate compounds synthesised was first evaluated using the ER-expressing (ER-dependent) MCF-7 human breast cancer cell line. Alamar Blue dye was used to quantify the cell viability. Cytotoxicity in MCF-7 cells was determined using the LDH assay [60]. Tamoxifen (IC 50  The conjugate compounds synthesised incorporating the ER-binding triphenylethylene moiety of the SERM endoxifen 3a-3k were initially evaluated for their ability to reduce the viability of the MCF-7 cell line. In the present study, the terminal methylamine basic substituent was designed to mimic the endoxifen basic side chain structure. It was hypothesised that by incorporating a basic side chain linker of the endoxifen structure onto the combretastatin acrylic acid moiety that high affinity ER ligands with potential antagonist activity may be achieved. A range of novel substituted acrylic acids were evaluated in the MCF-7 cell line. The biochemical data observed is presented in Table 2. The most active of the series of conjugate compounds 3i and 3k demonstrated micromolar IC 50 activity (Table 3) with IC 50 value of 4.2 µM and 1.47 µM respectively. The lead compound 3i which incorporated the 3,4,5-trimethoxyphenyl Ring A and 3-hydroxy4-methoxyphenyl Ring B of CA4 in the structure of the acrylic acid moiety remains the key compound from this series.
We had previously demonstrated the activity of the reverse CA4 conjugate [31]. The diversly substituted acrylic acid analogues were further investigated to enhance the activity seen for 3i. Compound 3j containing the 3,5-dimethoxy-4-hydroxy substitution pattern (Ring B of the acrylic acid) displayed weak antiproliferative activity (IC 50 = 11.8 µM), as did the furan containing compound 3m (IC 50 = 36.5 µM). However, the majority of compounds 3a-3m investigated did not exhibit any antiproliferative activity below IC 50 of 50 µM. Therefore, this indicates that the substituent on the acrylic acid analogues play a key role in determining the effects of these conjugates on cell proliferation. Compounds 3a-3m all have cLogP values greater than 5 and are predicted to have poor oral absorption. The amides 3n-3q demonstrated low potency when evaluated against the MCF-7 cell line. Compounds 5a-5d were next investigated to determine the effect of the core structure of the conjugate scaffold on the antiproliferative activity. It was decided to determine if both aryl rings (A and B) of the combretastatin structure were required for optimum antiproliferative activity containing cinnamic acids and 3-phenylpropanoic acids which were subsequently coupled to form the desired conjugates 5a-5d. The biochemical data for compounds 5a-5d is presented in Table 3. The four compounds evaluated for this investigation showed reduced activity compared with 3i and 3k. Compounds 5a and 5b which have the p-methoxy and 3-OH substituent (Ring B CA4) showed weak activity, with IC 50 values of 51.3 and 46.7 µM respectively. Compounds 5c and 5d which contain the 3,4,5-trimethoxy substituent (Ring A) did not show any antiproliferative activity below 100 µM. Therefore from the biochemical data for this series it is apparent that the two aryl rings, A and B, of the combretastatin structure with the CA4 type substituents were essential for optimum antiproliferative activity of the conjugate compounds.
The cyclofenil-based analogues 11a-11e were synthesised based on the reported cyclofenil structure which demonstrated impressive ERα and ERβ binding affinities. It was hypothesised that by incorporating a basic side chain onto the cyclofenil structures that high affinity ER ligands with potential antagonist activity may be achieved. The cyclofenil-based analogues were designed to be used as the potential ER targeting ligands for this series of conjugates. As these cyclofenil-based analogues were novel compounds it was decided that they should be first evaluated individually for antiproliferative activity. This data would be beneficial as it would allow the cyclofenil conjugates 13a-13e to be compared directly to ER-ligands 11a-11e for reduced/enhanced activity.
The biochemical data for compounds 11a-11e is presented in Table 3. The cyclofenil-based analogues 11a-11e all showed low micromolar antiproliferative activity in MCF-7 cells with IC 50 values ranging from 1.38 to 4.78 µM. This activity allowed for the rationalization of the next series of conjugates that incorporated the cyclofenil-based analogues. In this series only 11a had a CLogP less than 5, and this compound would be expected to demonstrate reasonable oral absorption properties. The conjugates 13a-13e incorporated the novel ER-ligands 11a-11e and were obtained via a direct amide linkage to the acrylic acid analogue 1l. The biochemical data for conjugates 13a-13e is presented in Table 3. This series of conjugates 13a-13e showed mostly low micromolar activity (IC 50 range of 1.38-2.5 µM) with two compounds exhibiting sub-micromolar activity (IC 50 values of 0.182 µM for 13e and 0.346 µM for 13d). The activity of the conjugates varies depending on the nature of the cycloalkane ring attached and the most active conjugates, 13d and 13e contains the cyclooctane and cyclohexane rings respectively. The compounds 13a-13e are predicted to have poor oral absorption with CLogP greater than 5.
Of the ester linked conjugates, the combretastatin containing compounds 16a and 16b were the most potent conjugates in this series with antiproliferative activity in MCF-7 cell line of IC 50 = 90 nM and 5.7 nM respectively. The cytotxicity of compounds 16a and 16b was determined in the lactate dehydrogenase (LDH) assay to be 13.2% and 4.1% respectively, and compares favourably with the cytoxicity of endoxifen (23%) and CA-4 (13%) when evaluated in the same assay.

NCI 60 Cell Line
The activity of conjugate compound 16a was evaluated using a 60-cell line screen facility of different cancer cell lines of diverse tumour origin in the National Cancer Institute (NCI, Bethesda, MD, USA) Division of Cancer Treatment and Diagnosis (DCTD)/Developmental Therapeutics Programme (DTP). In the one-dose screen, compound 16a displayed very high growth inhibition in the cell lines of colon cancer HCC-2998 (95%), HCT-116 (98%), HCT-15 (99%) and HT29 (93%); breast cancer BT-549 (94%), MCF-7 (90%) and MDA-MB-468 (99%); melanoma M14 (99%); CNS cancer SF-295 (94%) and U251 (90%) when evaluated at 10 µM concentration. The compound caused between 80-89% growth inhibition in a further 10 cell lines. The compound 16a displayed GI 50 (IC 50 ) values within the range 10-72 nM for most of the 60 cancer cell lines. Compound 16a displayed a GI 50 (IC 50 ) value of 36 nM and a LC 50 value greater than 100 µM in the MCF-7 breast cancer cell line, (Table 4), indicating a significant therapeutic window between the concentration required for inhibition of cancer cell growth, and the concentration that is determined to be toxic to MCF-7 breast cancer cells. As 16a does not display selectivity and enhanced activity towards the ER-positive cell line, it suggests that 16a may be exerting its potent activity by other biological mechanisms (i.e., inhibition of tubulin polymerisation) other than through the ER alone. Again, this is a promising result and highlights the possible therapeutic applications for the prototype ER-conjugates. Future research is being undertaken to determine what other mechnisms this compounds may inhibit in cancer cells. For 16a the COMPARE analysis was run on a database of common anti-cancer agents (JobID: 37888) and the larger more comprehensive database including natural products and other submitted agents (JobID: 37889). The highest correlation coefficient achieved was 0.629 in relation to the synthetic nucleoside antitumour, DNA and RNA synthesis inhibitor tiazofurin [66]. CA-4 15 was listed as a high-ranking hit with a correlation coefficient of 0.658. A correlation coefficient of above 0.6 is considered a positive correlation. This result supports the suggestion that the combretastatin moiety plays an important role in the activity profile of this ester-conjugate as it has a similar pattern or mechanism of action.

Estrogen Receptor Binding Studies
It was necessary to test the binding efficiency of each compound for both ER isoforms (ERα and ERβ), to determine how effectively the ligand binds to the receptor and whether the compound is acting as an antagonist or agonist. The competitive binding experiments were conducted using a fluorescence polarization assay which consists of purified baculovirus expressed human ERα and ERβ and fluoromone, a fluorescein labelled estrogen ligand [67,68]. The most active SERM type compounds and conjugates incorporating an ER type ligand from the antiproliferative assay were selected for ER binding study. The selectivity value reported is the ratio of relative binding affinity (RBA) values ERα relative to ERβ, for each of the compounds. For selectivity values greater than 1, the compounds have a more pronounced affinity for ERα binding site, while for values less than 1, the compounds have a more pronounced affinity for the ERβ binding site.
The most potent compounds evaluated for both ERα and ERβ affinity exhibit impressive binding activity. Compound 3i displayed ER binding activity in the nanomolar region for both ER isoforms (IC 50 ERα 182 nM and IC 50 ERβ 436 nM), with selectivity (3.13) for ERα thus demonstrating that the incorporation of a combretastatin-endoxifen hybrid scaffold structure results in a compound with potential SERM properties. The cyclofenil type compounds 11c and 11e display nanomolar activity in ERβ (IC 50 = 199 nM and IC 50 = 67 nM respectively) and low micromolar activity in ERα (IC 50 = 1.738 µM and IC 50 = 3.162 µM respectively), with selectivity for ERβ in both cases. Compound 11e which was a cyclofenil type ligand, exhibited the greatest selectivity (46 fold) towards the ERβ isoform while compound 13e which was a direct conjugate of 11e displayed the greatest selectivity (12.3 fold) towards ERα. The corresponding cyclofenil conjugate 13e demonstrated potent nanomolar binding activity for ERα (IC 50 = 19.0 nM) and ERβ (IC 50 = 229 nM).
The CA4 conjugate 16b displays significant ER binding affinities which help explain the antiproliferative activity of the conjugate. Interestingly the succinic-endoxifen linker compound 14c displays potent binding affinities in both ERα and ERβ with competitive binding IC 50 values of 11.3 nM (ERα) and 6.7 nM (ERβ). These values represent an approximately 3-fold increase in binding affinity in both ER isoforms when compared to 4-hydroxytamoxifen. The carboxylic acid group may interact favourably with a residue in the binding site. Interestingly, the carboxylic acids such as 14a (Table 3) closely related (differing only in the hydroxy moiety on the triarylethylene aryl ring scaffold) displayed no significant antiproliferative activity.
As in the antiproliferative assays, the conjugate 16b demonstrated impressive ER binding results and is suitable as a possible lead compound for further development. The conjugate displays nanomolar binding to the ERα and ERβ, [IC 50 ERα 52.1 nM and IC 50 ERβ 115 nM]. The phenolic hydroxy functionality present on the endoxifen fragment can interact favourably with the residues Glu353, Arg394 and His524 in the ER binding site through hydrogen bonding which can explain the high binding affinity. The combretastatin fragment may also form favourable interactions in the binding site as there is comparable ERα binding affinity with endoxifen. Presumably, the hydroxy functionality present on the combretastatin fragment is involved in hydrogen bonding.
Often the relative binding affinity (RBA) of estrogen receptor ligands is reported. Estradiol is typically used as the reference ligand and is taken as the 100% binding value. Using the reference IC 50 values obtained from the literature for estradiol in the ERα (5.7 nM) and ERβ (5.6 nM), the RBAs of the selected conjugates were calculated (see Table 5). All of the conjugates investigated in the ER competitive binding assays had RBA values greater than or equal to 1%. The RBA values between of 5-16% would be considered moderate binding while the RBA value for 14c demonstrates strong RBA ERα = 50.44, ERβ = 83.6. The lead conjugates e.g., 13e and 16b display very impressive RBA values of ERα = 30 and ERβ 2.44 for 13e and ERα = 10.94, ERβ 4.87 for 16b.

Effects of Selected Active Compounds on Cell Cycle and Cell Death.
Based on the cell viability assay results, a selected cohort of the most active compounds from each series (IC 50  Results showed that all compounds tested increased apoptosis when compared to vehicle-treated cells. Of the active compounds, the cyclofenil based analogues 11c and 11e proved significantly more active than the positive control tamoxifen (p value < 0.001). The most potent compound tested, 11c induced 79.7 ± 5.2% apoptosis at 10 µM while compound 11e induced 76% apoptosis. Conjugates 13c, 13d and 13e demonstrated pro-apoptotic effects of 20%, 36% and 48% respectively.
Interestingly, in addition to inducing apoptosis, the CA4 direct amide cyclofenil based conjugate 13d (p value < 0.01) also induced a significant G2/M-arrest (27%) in MCF-7 cells when compared to vehicle-treated cells, with 13e causing a lesser effect on G2M arrest of 18%. A G2/M arrest typically, though not necessarily always, precedes apoptosis. Future studies at earlier time-points (24 h and 48 h) will be undertaken to establish if the other active compounds in this cohort also induce a G2/M arrest preceding apoptosis. A G2/M arrest is commonly observed with tubulin-targeting compounds and so this data suggests that the tubulin-targeting ability of combretastatins is maintained when complexed to various ligands with induction of significant apoptosis.
Next, the same cohort of compounds was tested on peripheral blood mononuclear cells (PBMCs) isolated from the blood of healthy donors. Cells were treated with 10 µM of each compound for 72 h and subjected to flow cytometry. Results showed that only two of the compounds, (11c and 11e), the most active compounds in the MCF-7 cells), induced apoptosis in PBMCs, with no toxic effects observed with the other compounds. None of the compounds caused a G2/M arrest in PBMCs. The potent activity of 13d, 13e and 3i of this cohort of compounds in MCF-7 breast cancer cells and their lack of toxicity in normal PBMCs make them ideal candidates for further anti-cancer studies.
of the cell cycle and the number of apoptotic or dead cells. Samples were treated for 72 h at 10 μM.
Results showed that all compounds tested increased apoptosis when compared to vehicle-treated cells. Of the active compounds, the cyclofenil based analogues 11c and 11e proved significantly more active than the positive control tamoxifen (p value < 0.001). The most potent compound tested, 11c induced 79.7 ± 5.2% apoptosis at 10 μM while compound 11e induced 76% apoptosis. Conjugates 13c, 13d and 13e demonstrated pro-apoptotic effects of 20%, 36% and 48% respectively. Interestingly, in addition to inducing apoptosis, the CA4 direct amide cyclofenil based conjugate 13d (p value < 0.01) also induced a significant G2/M-arrest (27%) in MCF-7 cells when compared to vehicle-treated cells, with 13e causing a lesser effect on G2M arrest of 18%. A G2/M arrest typically, though not necessarily always, precedes apoptosis. Future studies at earlier time-points (24 h and 48 h) will be undertaken to establish if the other active compounds in this cohort also induce a G2/M arrest

Molecular Modelling of 11e, 16b and 13e in ERα and ERβ
A retrospective molecular modelling study of 11e, 16b and 13e was undertaken to rationalise the selectivity profile differences of the compounds for ERα and ERβ. Two amino acid differences within the ligand binding pocket of the ER isoforms are replacement of Met412 and Leu384 in ERα with Ile373 and Met336 respectively in ERβ which have an impact on the following results.
The 3ERT X-ray structure of hERα co-crystallised with 4-hydroxytamoxifen (4-OHT) [69] was downloaded from the PDB website. For ERβ the 1NDE X-ray structure co-crystallised with a triazine modulator was used [70]. An in-depth binding poses analysis on compounds 11e, 16b and 13e was performed. The top five docked poses of each compound in each isoform were analysed and mapping to the X-ray structure ligands binding poses was considered. Amino acid numbering corresponds to AA(#ERα)/(#ERβ) unless specified in the text.
The 3ERT X-ray structure of hERα co-crystallised with 4-hydroxytamoxifen (4-OHT) [69] was downloaded from the PDB website. For ERβ the 1NDE X-ray structure co-crystallised with a triazine modulator was used [70]. An in-depth binding poses analysis on compounds 11e, 16b and 13e was performed. The top five docked poses of each compound in each isoform were analysed and mapping to the X-ray structure ligands binding poses was considered. Amino acid numbering corresponds to AA(#ERα)/(#ERβ) unless specified in the text. 11e docked well into both ER isoforms and recapitulated the interactions formed by 4-OHT in the 3ERT X-ray structure. The 4-methylcyclohexylidene moiety occupies a hydrophobic pocket delineated by Leu525, Met343, Met421 (Ile373 in ERβ) and Leu384 (Met336 in ERβ). In ERα each of 11e docked well into both ER isoforms and recapitulated the interactions formed by 4-OHT in the 3ERT X-ray structure. The 4-methylcyclohexylidene moiety occupies a hydrophobic pocket delineated by Leu525, Met343, Met421 (Ile373 in ERβ) and Leu384 (Met336 in ERβ). In ERα each of these amino acids are closer to the ligand and form a tighter binding sub-pocket than for ERβ. In 4-OHT the D-ring phenyl group occupies this sub-pocket and is a planar conformationally rigid structure but the corresponding moiety in 11e is 4-methylcyclohexylidene which is bulkier and converts between multiple conformational states. This results in clashing with the more tightly packed ERα amino acids. Additionally, in ERβ Leu384 is mutated to Met336 which, in the 1NDE X-ray structure, forms a hydrogen bond acceptor interaction (HBA) with a cocrystallised water molecule, thereby decreasing the hydrophobicity of the sub-pocket. Both of these factors result in a 47-fold binding affinity preference for ERβ over ERα of this compound. The similarities in overlays between 11e docked in ERα and ERβ is illustrated in Figure 3.
Docking of compound 13e in ERα retained the same 4-OHT mapping binding mode as observed for 11e. Analogous to the situation with 16b, the methylamide moiety of 13e would clash with Leu476 of ERβ so the ligand is significantly shifted upwards thereby unable to make optimal interactions within the binding pocket. This is reflected in the 12-fold selectivity of the ligand for ERα over ERβ and is shown in Figure 4. docked in ERα and ERβ is illustrated in Figure 3.
Docking of compound 13e in ERα retained the same 4-OHT mapping binding mode as observed for 11e. Analogous to the situation with 16b, the methylamide moiety of 13e would clash with Leu476 of ERβ so the ligand is significantly shifted upwards thereby unable to make optimal interactions within the binding pocket. This is reflected in the 12-fold selectivity of the ligand for ERα over ERβ and is shown in Figure 4. . Ranked poses of 13e in ERα and ERβ overlaid on 4-OHT X-ray structure. The potential clashing interaction between Leu525/476 (rendered in tube style) of ERβ with the methylamide of 13e is present. Carbon atoms of 13e are illustrated in dark green in ERα, dark blue in ERβ and grey in 4-OHT (oxygen atoms are red; nitrogen in dark blue; sulphur in yellow). ERα and associated water molecules are in light green, ERβ and associated water molecules are in light blue. Amino acid numbering is for ERα except when different in ERβ where both labels are used. PDB structures 3ERT [69] and 1NDE [70] were used for molecular docking. . Ranked poses of 13e in ERα and ERβ overlaid on 4-OHT X-ray structure. The potential clashing interaction between Leu525/476 (rendered in tube style) of ERβ with the methylamide of 13e is present. Carbon atoms of 13e are illustrated in dark green in ERα, dark blue in ERβ and grey in 4-OHT (oxygen atoms are red; nitrogen in dark blue; sulphur in yellow). ERα and associated water molecules are in light green, ERβ and associated water molecules are in light blue. Amino acid numbering is for ERα except when different in ERβ where both labels are used. PDB structures 3ERT [69] and 1NDE [70] were used for molecular docking.
16b docked with a perfect overlay on the ERα OHT X-ray. The corresponding docked pose in ERβ is capable of making the same hydrogen bond donor (HBD) interaction as the 4-OHT A-ring phenolic oxygen atom but the ligand is shifted upwards in the binding site. A contributing factor to this is the repositioning of Leu525/476 which is adjacent to the 4-OHT C-ring in ERα but flipped 180 • upwards towards the binding pocket entrance in ERβ.
As highlighted with a red circle in Figure 5, this repositioned Leu now clashes with the methylamide of the side-chain of 16b in ERβ so the ligand is shifted away and twisted so as to avoid electrostatic repulsion between the amide carbonyl oxygen atom and Asp351/303. This is revealed in a 7-fold binding affinity preference for ERα over ERβ.
this is the repositioning of Leu525/476 which is adjacent to the 4-OHT C-ring in ERα but flipped 180° upwards towards the binding pocket entrance in ERβ.
As highlighted with a red circle in Figure 5, this repositioned Leu now clashes with the methylamide of the side-chain of 16b in ERβ so the ligand is shifted away and twisted so as to avoid electrostatic repulsion between the amide carbonyl oxygen atom and Asp351/303. This is revealed in a 7-fold binding affinity preference for ERα over ERβ. The potential clashing interaction between Leu525/476 (rendered in tube style) of ERβ with the methylamide of 16b is circled in red. Carbon atoms of 16b are illustrated in dark green in ERα, dark blue in ERβ and grey in 4-OHT (oxygen atoms are red; nitrogen in dark blue; sulphur in yellow). ERα and associated water molecules are in light green, ERβ and associated water molecules are in light blue. Amino acid numbering is for ERα except when different in ERβ where both labels are used. PDB structures 3ERT [69] and 1NDE [70] were used for molecular docking.

General Information
All reagents were commercially available and were used without further purification unless otherwise indicated [19]. Tetrahydrofuran (THF) was distilled immediately prior to use from Na/Benzophenone under a slight positive pressure of nitrogen, toluene was dried by distillation from Figure 5. Ranked poses of 16b in ERα and ERβ overlaid on 4-OHT X-ray structure. The potential clashing interaction between Leu525/476 (rendered in tube style) of ERβ with the methylamide of 16b is circled in red. Carbon atoms of 16b are illustrated in dark green in ERα, dark blue in ERβ and grey in 4-OHT (oxygen atoms are red; nitrogen in dark blue; sulphur in yellow). ERα and associated water molecules are in light green, ERβ and associated water molecules are in light blue. Amino acid numbering is for ERα except when different in ERβ where both labels are used. PDB structures 3ERT [69] and 1NDE [70] were used for molecular docking.

General Information
All reagents were commercially available and were used without further purification unless otherwise indicated [19]. Tetrahydrofuran (THF) was distilled immediately prior to use from Na/Benzophenone under a slight positive pressure of nitrogen, toluene was dried by distillation from sodium and stored on activated molecular sieves (4 Å) and dichloromethane was dried by distillation from calcium hydride prior to use. Uncorrected melting points were measured on a Gallenkamp apparatus. Infra-red (IR) spectra were recorded as thin film on NaCl plates, or as potassium bromide discs on a Perkin Elmer FT-IR Specrtum 100 spectrometer (Perkin Elmer, Waltham, MA, USA). 1 H-and 13 C-nuclear magnetic resonance (NMR) spectra were recorded at 27 • C on a Bruker Avance DPX 400 spectrometer (400.13 MHz, 1 H; 100.61 MHz, 13 C) (Bruker, Billerica, MA, USA) at 20 • C in CDCl 3 (internal standard tetramethylsilane (TMS)), CD 3 OD or DMSO-d 6

General Method for Synthesis of Acrylic Acids 1a-1r
Method A: A mixture of the appropriate the appropriate phenylacetic acid (0.50 g, 1 equivalent), benzaldehyde (1 equivalent), acetic anhydride (2 mL) and triethylamine (1 mL) were heated under reflux for 3-5 h. After acidification with concentrated hydrochloric acid (5 mL), the resulting solid was filtered off and recrystallised to yield the required acrylic acid. Method B: A mixture of the appropriate benzaldehyde (1 eq.), the appropriate phenylacetic acid (700 mg, 1 eq.), acetic anhydride (2 mL) and triethylamine (1 mL) were reacted in the microwave reactor at a 120 • C and for 30 min. After acidification with concentrated hydrochloric acid (5 mL), the resulting solid was filtered and recrystallised to afford the required acrylic acid.       The organic phases were combined, dried over sodium sulfate and the solvent evaporated in vacuo to afford a crude product which was then purified via flash chromatography (DCM:MeOH) to afford the product as a brown oil (0.28 g, 77%, E/Z = 1:1.1). 1

General Method for the Synthesis of Endoxifen-Acrylic Acid Conjugates 3a-3m
Step (i): A mixture of the required acrylic acid (1 eq., 0.154 mmol), DCC (1 eq., 0.154 mmol, 32 mg), and HOBt (1 eq., 0.154 mmol, 21 mg) was suspended in 3 mL of anhydrous DCM and stirred for 10 min under a nitrogen atmosphere. The endoxifen derivative 2a (75 mg, 1 eq., 0.154 mmol) was dissolved in 3 mL of anhydrous DCM and slowly added to the mixture via syringe. Reaction was allowed stir for 24-48 h. Reaction was monitored via TLC. The reaction mixture was diluted to 15 mL with anhydrous DCM and filtered to remove DCU. The filtrate was evaporated to dryness under reduced pressure. Purification was not required at this step. Silyl deprotection: The residue was dissolved in THF (3 mL) and stirred under nitrogen. A solution of 0.1 M TBAF (2 eq.) was added to the mixture and allowed to stir for 24 h. The mixture was evaporated to dryness under reduced pressure. The residue was dissolved in DCM and was washed with 10% HCl solution. The resulting organic phase was dried over sodium sulphate and evaporated to dryness under vacuum. The material was purified via flash chromatography on silica gel.

General Method for the Synthesis of Endoxifen-Cinnamic Acid/3-Phenylpropanoic Acid Conjugates 5a-5d
Step (A): A mixture of the appropriate cinnamic acid/3-phenylpropanoic acid (1 eq., 0.154 mmol), DCC (1 eq., 0.154 mmol, 32 mg), and HOBt (1 eq., 0.154 mmol, 21 mg) were suspended in anhydrous dichloromethane (3 mL) and stirred for 10 min under a nitrogen. The silyl-protected endoxifen derivative 2a (75 mg, 1 eq., 0.154 mmol) was dissolved in anhydrous DCM (3 mL) and slowly added to the mixture via syringe. The reaction was allowed stir for 24-48 h and monitored via TLC. The reaction mixture was diluted to 15 mL with anhydrous DCM and filtered to remove DCU. The filtrate was evaporated to dryness under reduced pressure and the residue was dissolved in THF (3 mL) and stirred under nitrogen atmosphere. A solution of 0.1 M TBAF (2 eq.) was added to the mixture and allowed to stir for 24 h. The solvent was evaporated to dryness under reduced pressure. The residue was dissolved in DCM and was washed with 10% HCl solution. The resulting organic phase was dried over sodium sulphate and evaporated to dryness. The material was purified via flash chromatography on silica gel, (eluent: DCM:EtOAc).
Step (B): A mixture of the appropriate cinnamic acid/3-phenylpropanoic acid (1.2 eq), EDC (1.4 eq.), and HOBt (1.4 eq.) were suspended in anhydrous dichloromethane (3 mL) and stirred for 10 min under a nitrogen atmosphere. The protected endoxifen (1 eq.) 2a was dissolved in anhydrous dichloromethane (3 mL) and slowly added to the mixture via syringe and the reaction was stirred for 16 h and monitored via TLC. The reaction mixture was diluted to 15 mL with anhydrous dichloromethane. To this mixture, water (20 mL) was added. The aqueous phase was extracted with DCM (20 mL × 3), brine (50 mL), dried over Na 2 SO 4 and evaporated to dryness in vacuo to yield the crude product. The material was purified via flash chromatography on silica gel. (eluent: DCM:EtOAc). The above residue was dissolved in THF (3 mL) and stirred under nitrogen atmosphere. A solution of TBAF (1 M in THF, 1 eq.) was added to the mixture and allowed to stir for 1 h. The mixture was evaporated to dryness under reduced pressure. The material was purified via flash chromatography over silica gel (eluent: DCM:EtOAc) to afford the product. (i) Following the general method above, the cinnamic acid/3-phenylpropanoic acid analogue 4d was reacted with endoxifen derivative 2a. The crude mixture was then taken to the next step without further purification. The final product was purified via flash chromatography over silica gel (DCM:EtOAc, gradient 6:1 to 3:1) to afford the product as a brown resin (29%). (ii) As per general method, the 3-phenylpropanoic acid 4d (1.2 eq., 118 mg, 0.492 mmol), EDC (1.4 eq., 110 mg, 0.572 mmol), and HOBt (1.4 eq., 77.6 mg, 0.572 mmol) was reacted with endoxifen 2a (1 eq., 200 mg, 0.41 mmol). The crude product was afforded as a brown resin. The material was purified via flash chromatography over silica gel (DCM:EtOAc, gradient 20:1 to 10:1) to afford the product as a white solid.  [4-(tert-Butyldimethylsilanyloxy)phenyl]-(4-hydroxyphenyl)methanone (7a). 4,4 -Dihydroxybenzo-phenone (6, 6.00 g, 28 mmol) and imidazole (2.094 g, 31 mmol) were dissolved in DMF (20 mL) with stirring. A solution of tert-butyldimethylsilyl chloride (4.22 g, 28 mmol) in DMF (20 mL) was added to the mixture over 1 h. The reaction mixture was allowed to stir at room temperature for 16 h followed by the addition of ethyl acetate (100 mL) and 10% hydrochloric acid (50 mL). The organic layer was washed with water (100 mL), brine (100 mL), dried over sodium sulphate and evaporated to dryness in vacuo to yield crude product. The material was purified via flash chromatography on silica gel (n-hexane:DCM, 5:1) to afford the product 7a as a light yellow oil.

General Method for Cyclofenil Derivatives 8a-8e via McMurry Coupling
Titanium tetrachloride (4.5 eq., 5.2 g, 37.4 mmol, 3 mL) was added via a syringe dropwise to zinc dust (9 eq., 3.58 g, 54.81 mmol) in dry THF (50 mL) and the mixture was refluxed for 2 h in darkness and under nitrogen. The appropriate phenolic ketone 7a (1 eq.) and cyclic ketone (3 eq.) were dissolved in dry THF (40 mL). This solution was added to the titanium tetrachloride/zinc mixture carefully via a syringe. The reaction mixture was then refluxed for a further 3 h. and then cooled and then diluted with EtOAc (75 mL) and 10% K 2 CO 3 solution. The mixture was filtered under vacuum and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with 10% K 2 CO 3 solution (20 mL), water (50 mL) and brine (50 mL) then dried over Na 2 SO 4 , filtered and the solvent was evaporated under reduced pressure to afford the crude product. The material was purified via flash chromatography over silica gel to afford the product.

General Method for the Preparation of Bromoethyl Ethers 9a-9e
The appropriate phenol 8a-8e (1 eq.) was dissolved in 1,2-dibromoethane (~50 eq.) with stirring. Tetrabutylammonium hydrogen sulfate (5 mmol) was added followed by 1 M NaOH solution (50 mL). The biphasic mixture was stirred vigorously at room temperature for 16 h. followed by addition of DCM (100 mL) and NaHCO 3 solution (100 mL). The aqueous layer was extracted with DCM (2 × 100 mL) and the organic extracts were combined and washed with water (50 mL), brine (50 mL) and dried over Na 2 SO 4 . The solvent was evaporated under reduced pressure, and the residue was purified via flash chromatography over silica gel (eluent: DCM:n-hexane) to afford the product.

Cell Culture of MCF-7 Cell Line
The human breast carcinoma cell line, MCF-7, was purchased from the European Collection of Animal Cell Cultures (ECACC, Public Health England, Porton Down, UK). The cells were maintained in MCF-7 complete medium; consisting of Eagle's Minimum Essential Medium (MEM) supplemented with 10% (v/v) Foetal Bovine Serum (FBS), 2 mM L-glutamine, 100 µg/mL penicillin/streptomycin and 1% (v/v) non-essential amino acids. Cell cultures were maintained at 37 • C under a humidified atmosphere of 5% CO 2 /95% O 2 . The MTT assay was performed according to the reported protocol. The tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is taken up only by metabolically active cells and cleaved to form a formazan dye by mitochondrial dehydrogenases [30]. Assays were repeated in three experiments performed in triplicate (unless otherwise stated) and reported results represent the mean value ± standard error mean. Graphs of percentage cell viability versus concentration of the subject compound were processed using PRISM [78].

Alamar Blue Assay for Measurement of Antiproliferative Effects
The biochemical assay was performed in triplicate on at least three independent occasions for the determinationof the mean values reported. MCF-7 cells were seeded in triplicate in 96-well plates at a density of 2.5 × 10 4 cells/mL in a total volume of 200 µL per well, and incubated at 37 • C for 24 h. Cells were treated with varying concentrations of the appropriate compounds to yield the desired final concentration. Ethanol was used as a vehicle and cells were treated with 1% ethanol (v/v) in all experiments. Plates were incubated for 72 h at 37 • C + 5% CO 2 after which Alamar Blue (Invitrogen, Carlsbad, CA, USA) was added to each well. The plates were incubated for a further 4 h without exposure to light. EMEM medium with the addition of Alamar Blue was used as a blank. Vehicle treated cells were considered to be 100% viable and the viabilities of each compound was calculated accordingly. Results were calculated using transformed data [Final Concentration = Log (Final Concentration)] to plot a non-linear, sigmoidal dose response curve from which the relative IC 50 values were determined.

Lactate Dehydrogenase Assay for Measurement of Cytotoxicity
In this assay, the release of cytoplasmic lactate dehydrogenase (LDH) is used as a measure of cell lysis. MCF-7 cells were seeded at a density of 1 × 10 4 cells/well in a 96-well plate and incubated for 24 h. The cells were then dosed with 2 µL volumes of the test compounds, over the concentration range 1 nM-50 µM. Cytotoxicity was determined using the CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI, USA) following the manufacturer's protocol [79].

Estrogen Receptor Fluorescent Polarisation Assay
Competitive binding affinity experiments were carried out using purified baculovirus-expressed human ERα and ERβ and fluoromone, a fluorescein labelled estrogen ligand. ER binding ability of the selected compounds was investigated using ERα and ERβ fluorescence based ER competitive assay kits supplied by Invitrogen [67,68]. The assay was performed using a protocol described by the manufacturer. The assay allows for high throughput screening of potential ER-subtype ligands. The selected compounds were screened using both the ERα and ERβ competitive assay kits. The protocol for carrying out the assay was similar for both ER subtypes. Principally, the main difference between the kits relates to the functional receptor concentration and the specific activity of the different ERs [33,34]. The recombinant ER and the fluorescent estrogen ligand were removed from the −80 • freezer and thawed on ice (4 • C) for 1 h prior to use. Previously prepared serial dilutions of the test compounds, consisting of the concentration range: 0.1 mM, 10 µM, 1 µM, 100 nM and 10 nM, were pipetted (1 µL) into a 96-well Greiner black-bottomed multiwell plate. The final concentration in the well was diluted by two orders of magnitude. The concentration range of test compounds can be adjusted accordingly to best suit the assay. Each compound was repeated in duplicate. The ES2 screening buffer (100 mM potassium phosphate (pH 7.4), 100 µg/mL bovine gamma-globulin and 0.02% sodium azide) was pipetted (49 µL) into each well containing the test compounds. The amount of ER/Fluormone ™ ES2 complex was calculated based on a final reaction volume of 100 µL per well, thus 50 µL of complex was required per well. The complex was made up with ES2 screening buffer and was then pipetted (50 µL) to the required wells. The controls used in the assay consisted of a well containing 100 µL of screening buffer (no fluorescence expected), 50:50 complex/buffer (maximum polarisation), 1:49:50 ethanol(test sample diluent)/complex/buffer (~maximum polarisation = negative control) and 1:49:50 Estradiol(10 µM)/complex/buffer (~minimum polarisation = positive control). The plate was read on a BMG Pherastar fluorescence polarisation instrument with 485 nm excitation and 530 nm emission interference filters and processed using the Pherastar software. The plate was read over a 20 min period with polarisation readings taken every two minutes. The average of the polarisation readings was reported. Curve-fitting of the polarisation results were carried out using PRISM. The concentration of the test compound that results in a half-maximum shift in polarisation equals the IC 50 of the test compound. The IC 50 value is a measure of the relative binding affinity of the test compound for the ER.

Flow Cytometry
MCF-7 cells were seeded at a density of 5 × 10 4 cells/well in a 12-well plate and treated with the indicated compounds for 72 h. Samples were then centrifuged at 650× g for 5 min and resuspended in 100 µL ice-cold PBS. Ice-cold 70% (v/v) ethanol (1 mL) was then added to fix the samples overnight at 4 • C. Samples were subsequently centrifuged at 800× g for 10 min and resuspended in 200 µL PBS. RNase A (12.5 µL of 10 mg/mL) and propidium iodide (37.5 µL of 1 mg/ mL) were added to samples which were then incubated for 30 min at 37 • C. Cell cycle analysis was performed at 488 nm using a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The Macintosh-based application CellQuest was then used to analyse the data of 10,000 gated cells once cell debris had been excluded. Data points represent the mean ± S.E.M. of three independent experiments.

Generation of Human Peripheral Blood Mononuclear Cells
Fresh peripheral blood was collected from healthy donors and diluted 1:3 with RPMI medium and added to half the equivalent volume of Lymphoprep. Samples were centrifuged at 750× g for 30 min to form a Ficoll gradient. The white buffy layer containing the peripheral blood mononuclear cells was removed, diluted to a volume of 50 mL with medium and centrifuged again for 10 min at 650× g. Cells were then seeded at a density of 1 × 10 6 cells in 1 mL of RPMI medium supplemented with 10% FBS, 2 mM L-glutamine and 100 U/mL penicillin and 100 µg/mL streptomycin.

NCI One-Dose and Five-Dose Screen Output
The NCI one-dose screen output is reported as a mean graph of the percent growth of treated cells and is similar in appearance to mean graphs generated in the 5-dose assay [80]. The value reported for the one-dose assay is growth relative to the no-drug control and relative to the time zero number of cells. The one-dose assay allows detection of growth inhibition (values between 0 and 100) and lethality (values less than 0). For example, a value of 100 means no growth inhibition. A value of 40 would mean 60% growth inhibition. A value of zero means no net growth over the course of the experiment. A value of −40 would mean 40% lethality. A value of −100 means all cells are dead. The results from the five-dose screen for 15a were manually entered into the COMPARE analysis software via an on-line submission form [80]. The results from the COMPARE analysis are retrievable on-line by searching using the relevant JobID reference number. The COMPARE analysis was run on a database of common anti-cancer agents (JobID: 37888) and the larger more comprehensive database including natural products and other submitted agents (JobID: 37889).

Conclusions
The estrogen receptors ERα and ERβ which modulate the effects of the estrogen hormones are important targets for design of chemotherapeutic agents whch target diseases such as breast cancer and osteoporosis. A series of novel conjugate molecules incorporating the ER ligands endoxifen and cyclofenil-endoxifen hybrids covalently linked to the antimitotic and tubulin targetting agent CA-4 were synthesised. These conjugates were evaluated as ER targeting ligands. A number of these compounds demonstrated pro-apoptotic effects, with significant antiproliferative activity in ER-positive MCF-7 breast cancer cell lines. These conjugates displayed potent binding affinity towards ERα and ERβ isoforms at nanomolar concentrations as determined by FP assay. The endoxifen conjugate 16b demonstrates antiproliferative activity in ER positive MCF-7 breast cancer cells (IC 50 = 5.7 nM) and ER binding affinity to ERα (IC 50 = 52 nM) and ERβ (IC 50 = 115 nM). The conjugate 16a displayed significant growth inhibition effects on the NCI 60 cancer cell panel, suggesting that this compound may also inhibit cancer cell growth through non-ER dependent mechanisms. These conjugate compounds are identified as suitable lead compounds for future drug development based on favourable in vitro stability profile over pH range 4-9 and also in plasma. The conjugate 16a differs from 16b only by a hydroxy functionality which is known to improve the binding of the ER-ligand fragment and is presumably the major factor in the 18-fold difference in antiproliferative potency. This is reinforced by the high affinity binding of 16b to ERα with an IC 50 ERα of 52.1 nM. It remains unclear the exact role the combretastatin moiety has in the antiproliferative effect. These two conjugates 16a and 16b are covalently bound to the CA4 fragment by ester linkages which could be readily hydrolysed in vivo thus releasing the CA4. The compound 14a represents the cleaved endoxifen-linker component of conjugate 16a and displays very low antiproliferative activity. The intact conjugate may be exerting antiproliferative activity through binding in the ER resulting in the displacement of helix-12 and ER-antagonism. However, if the conjugate is cleaved the combretastatin may be exerting its antimitotic activity. Additionally, the conjugate may be working through both an ER-antagonistic pathway and antimitotic pathway i.e., through dual-action.
The cyclofenil-amide compound 13e is also identified as a promising lead compound of a clinically relevant ER conjugate with IC 50 in MCF-7 cells of 187 nM, and ER binding affinity to ERα (IC 50 = 19 nM) and ERβ (IC 50 = 229 nM), which can target ER in the breast cancer cell and can thus deliver a cytotoxic agent CA4 to the cancer cell. Conjugate and bifunctional compounds which incorporate an ER ligand offer a useful method of delivering cytotoxic drugs to tissue sites such as breast cancers which express ERs. From the results obtained in the NCI 60 cell line assay, it is clear that compounds such as 16a, although displaying nanomolar potency for MCF-7 ER positive cells and high affinity ER binding, do not display selectivity in activity towards the ER-expressing cell line MCF-7, indicating that 16a may also be modulating additional biological mechanisms (i.e., in promoting apoptosis or inhibition of tubulin polymerisation) other than via the ER alone. This result highlights possible therapeutic applications for these prototype ER-conjugates. Further research may determine the mechanism of antiproliferative action of these compounds in cancer cells. These conjugate compounds have potential application for further development as antineoplastic agents, (possibly modulating more than a single molecular target-in this case tubulin), in the treatment of both ER positive and ER negative breast cancers and these chemical classes of structures may be useful as scaffolds to be considered for multitarget drugs in future drug development studies.