Rational approaches of drug design for the development of selective estrogen receptor modulators (SERMs), implicated in breast cancer

Journal Pre-proofs Rational Approaches of Drug Design for the Development of Selective Estrogen Receptor Modulators (SERMs), Implicated in Breast Cancer Subhajit Makar, Tanmay Saha, Rayala Swetha, Gopichand Gutti, Ashok Kumar, Sushil.K. Singh PII: DOI: Reference:

S0045-2068(19)31135-6 https://doi.org/10.1016/j.bioorg.2019.103380 YBIOO 103380

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Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

17 July 2019 18 October 2019 21 October 2019

Please cite this article as: S. Makar, T. Saha, R. Swetha, G. Gutti, A. Kumar, Sushil.K. Singh, Rational Approaches of Drug Design for the Development of Selective Estrogen Receptor Modulators (SERMs), Implicated in Breast Cancer, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103380

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Rational Approaches of Drug Design for the Development of Selective Estrogen Receptor Modulators (SERMs), Implicated in Breast Cancer Subhajit Makar, Tanmay Saha, Rayala Swetha, Gopichand Gutti, Ashok Kumar, Sushil. K. Singh*

Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (BHU), Varanasi-221005, U.P, India *Corresponding Author Email: [email protected] Abstract Drug discovery and development have gained momentum due to the rational drug design by engaging computational tools and bioinformatics methodologies. Bioisosteric replacements and hybrid molecular approaches are the other inventive processes, used by medicinal chemists for the desired modifications of leads for clinical drug candidates. SERMs, ought to produce inhibitory activity in breast, uterus and agonist activity in other tissues, are beneficial for estrogen-like actions. ER subtypes α and β are hormone dependent modulators of intracellular signaling and gene expression, and development of ER selective ligands could an effective approach for treatment of breast cancer. This report has critically investigated the possible designing considerations of SERMs, their in silico interactions, and potent pharmacophore generation approaches viz. indole, restricted benzothiophene [3, 2-b] indole, carborane, xanthendione, combretastatin A-4, organometallic heterocycles ,OBHS-SAHA hybrids, benzopyranones, tetrahydroisoquinolines, Dig G derivatives and their specifications in drug design and development, to rationally improve the understanding in drug discovery. This also includes various strategies for the development of dual inhibitors for the management of antiestrogenic resistance. Keywords: Estrogen receptor, SERMs, Ligand Binding Domain (LBD), Helix-12, Pharmacophore, 17-β Estradiol and His524

Table of Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction Agonist and Antagonist Induced ER Signaling Designing Considerations of SERMs Potential Pharmacophores for SERMs Development Strategy of Dual Targeting Inhibitors for Antiestrogenic Resistance Development of estrogen receptorβ (ERβ) ligands for breast cancer treatment Future Perspective and Outlook References

1. Introduction Drug development process is challenging, expensive and time consuming. Although, it has been accelerated with the advancement in computational tools and bioinformatics methodologies. Target based drug design approach has been deficient, as most of drugs developed have shown serious side effects. To overcome the challenge, multidisciplinary approaches are essential, which are the foundation of rational drug design [1]. A drug target is basically biomolecule, involved in signaling or metabolic pathways and is specific for the disease process. Biological effects are achieved, i) either by inhibiting the functions with small molecules, whose competitive binding affinity are greater than their natural ligands that bind to the active sites (within the biomolecules), or ii) by inhibiting the bimolecular interactions (between the biomolecules) [2]. Drug discovery has been significantly boosted, due to the availability of 3D crystal or solid state NMR structures of biomolecules, molecular docking tools and development of computer aided methodologies [3]. Rational drug design approaches mainly include (i) development of drug molecules with desired properties for target biomolecules (proteins or nucleic acids) in cellular processes through 3D structural information available and (ii) study of unknown targets (genes and proteins) obtained by analyzing global gene expression data of samples (untreated and treated) with a drug using advanced computational tools. A lead compound with acceptable pharmacological activity might be associated with structural features responsible for side effects and characteristics, that limit its bioavailability, influence its metabolism and excretion from the body. Bioisosteric replacement, another rationale approach, is generally used to accomplish desired modifications in the leads to obtain safer and clinically relevant drug molecules. Bioisosteres of functional groups, based on the understanding of physicochemical properties of pharmacophore viz. electronegativity, steric size, and lipophilicity, have enhanced the prospect of successful development of new chemical entities

as drugs [4, 5]. Selective estrogen receptor modulators (SERMs) are structurally diverse molecules, which interact with intracellular estrogen receptors in various tissues as ER agonists or antagonists. An ideal SERM should produce antagonistic activity in breast, uterus and agonistic activity in other tissues, and estrogen-like actions in cardiovascular, skeletal, and central nervous systems [6]. 2. Agonist and Antagonist Induced ER Signaling Estrogen ligands interact with both ERα and ERβ and regulate the transcriptional activity on different genes. Structure activity, through mutational mapping, indicates that different residues in ligand binding domain of the receptor are involved in the recognition of structurally distinct estrogens and antiestrogens. The action of estrogen receptors is tripartite, involving the receptor, ligands, and coregulator or corepressor proteins. The important interactions include, the interaction of ligands, with the two ER subtypes and with various isoforms in both normal and tumor cells. The interactions of ligand–receptor complexes with effectors include different estrogen response elements, and important regulatory proteins that control the magnitude of transcriptional response of ER [7-9]. The nuclear estrogen receptor resides, in the nuclei of target cells, remain in an inactive form. The binding to an endogenous ligand, such as estradiol, allows the receptor dimer to interact with specific DNA sequences (estrogen response elements) within the promoters of responsive genes. DNA-bound estrogen receptor regulates the target-gene transcription either positively or negatively [10]. The mechanism(s) of tissue specific mixed agonist–antagonist action of SERMs is still partly understood, but is gradually becoming clearer. The complex pharmacology of SERMs can be explained by three interactive mechanisms a) The target cells contain diverse concentrations of homodimers and heterodimers of one or both of the ER species. Estrogen receptor α acts as an activator, whereas, estrogen receptor β can inhibit the action of estrogen

receptor α by forming a heterodimer with it. SERMs i.e. raloxifene and tamoxifen produce tissue specific response by binding to both isoforms and act as pure antagonist through ER β on genes containing estrogen response elements, but function as partial agonists when acting through estrogen receptor α [11-13]. b) Protein crystallography showed the unique binding pattern of estradiol, tamoxifen, raloxifene and estrogen antagonist ICI 164,384 in estrogen-receptor conformations of each ligand and SERM bound estrogen receptors assumed a continuum of intermediate shapes [14-16]. c) Over 20 coregulators and corepressors discovered, bind to receptors and modulate their signaling either with a positive or a negative transcriptional activity. Varying combinations of coregulator or corepressor proteins interact with the estrogen receptor to modulate its function in different ways, depending on the receptor conformation acquired by ligand binding [17]. Estrogen receptor, like other members of the nuclear receptor superfamily, consists of DNAbinding domain C (DBD), ligand-binding domain E (LBD), and activating functions (AF1 in the N-terminal A/B domain and AF2 in the LBD) which mediates the transcription and gene expression. The LBD of ER (amino acids 304– 554) consists of 12 α-helices (h1-h12) is inactive in the absence of a bound ligand, but bound to heat shock proteins (largely HSP90) as a monomer [18, 19]. LBD sheds HSP90 through dimerization of receptor and becomes stabilized in a conformation in which the last helix (h12) folds over the ligand binding pocket at the time of agonist (E2) binding. It further forms a hydrophobic groove into which coactivators can bind. When an antiestrogen like tamoxifen binds to LBD, its side chain blocks h12 to form an active AF-2 conformation, thus, h12 anchors in the AF-2 hydrophobic groove and prevents coactivator binding [20]. Agonists induced ER-regulated transcription process involves the following steps: Step 1: Conformational changes by agonist binding to estrogen receptor causing structural change

in properties, leading to hsp90 dissociation and ER dimerization. Step 2: Multi segments of coactivator complex viz. SRC-1, SRC-2, E6-AP, P68, CBP, BRG-1 and RIP 140 attach to the dimer and interact with the target DNA sequences. Step 3: Further interaction with the basal transcription factors as well as stimulation of the acetylation of histones (His), elongation of the transcription and cell division (Fig.1). The antagonist induced inhibition of ER-mediated transcription process is as follows. Step 1: Antagonist bound ER induced conformational changes, distinct from agonists, lead to hsp90 dissociation and ER dimerization. Step 2: Multicomponent co-repressor complex binds to the receptor dimer to interact with DNA sequences. Step 3: Co-repressors deacetylases of histones and gene transcription are inhibited. Co-repressors are unable to bind DNA independently, being taken directly or indirectly for binding of DNA transcription factor families to repress the expression of target genes. The co-repressors responsible for ER antagonism include repressor of estrogen receptor activity (REA), silencing mediator for retinoid and thyroid hormone receptors (SMRT), nuclear receptor co-repressor (NCoR), thyroid hormone receptor interacting protein 1 (TRIP1), nuclear receptor binding SET domain protein 1 (NSD-1), Swiindependent 3 (Sin 3), and Ssn 3 (Fig.1) [21]. [Insert Fig.1 here] Fig.1: Agonist & Antagonist Induced ER Signaling 3. Designing Considerations of SERMs Biological effects of estrogen are known to be mediated by two receptor subtypes signed as ERα and ERβ. ERs are structurally conserved (A-F), consisting of variable N and carboxy terminal ends. These consist of centrally located transactivation domain (A/ B), DNA binding domain (C), and a region involved in dimerization and binding to Hsp90 (D). The ligand binding domain (LBD)

(E) synergizes transactivation functions with the A/B region and carboxy-terminal (F) domain. DNA binding domain (DBD), consisting of two zinc finger structures, plays a critical role in dimerisation and binding of receptors to specific DNA sequences. LBD, a multifunctional domain, induces conformational changes in the receptor and also directs gene activation or repression [22]. The differentiation of ligand binding cavities, among the two subtypes, is only due to two the signature amino acid positions of leucine 384, and methionine 421, which are replaced by methionine 336 and isoleucine 373 respectively in ERβ. The changes are not only conservative between hydrophobic residues but also reciprocal, with each ER isoform having one methionine residue and either a leucine or isoleucine residue [23]. It is established that ER itself doesn’t control transcription, but requires an interaction with coactivators or corepressors, which act as signaling intermediates between ER and general transcriptional machinery [17]. The crystal structures of LBD-ERα, determined with estradiol and synthetic nonsteroidal estrogen diethylstilbestrol (DES) (Fig.2), represented as dimer and F region but was removed for studies, would be critical for the inhibitory role of this region. Agonist-receptor complex should have the ability of ligand to be enveloped in a hydrophobic pocket, which is closed by helix 12, an essential site of AF-2 for coactivator binding and subsequent initiation of RNA polymerase activity. Repositioning of helix 12, after ligand binding, has been proposed for estrogenic and antiestrogenic actions [15, 24, 25]. The crystal structure of natural phytoestrogen genistein, a SERM with ERβ, indicated that paraphenolic hydroxyl of genistein interacted with the side chains of Glu 305, Arg 346 in ERβ, which were equivalent to Glu 353, Arg 394 of ERα respectively (Fig.2). One hydroxyl group of genistein produced a hydrogen bond with His 475 in LBD of ERβ, that was equivalent to 524 in ERα [26]. The structure and biochemical actions of two ERs in various tissues provide opportunity to develop drug molecules to target not only ER- LBD action, but also to identify secondary targets

for the binding of harmonized coactivators and corepressors [27]. The pharmacophore of ERs consists of two hydroxyl groups that are 11Aͦ apart or at least one should be bonded to an aromatic hydrophobic core to form phenolic substructure (Fig.2). The crystal structure interpretation of ERα-LBD led to an explanation about favorable amino acid residues Glu353 and Arg394, which lined the ‘A-pocket’ of receptor, and formed strong hydrogen bonds with phenolic hydroxyl group of E2. Further, 17β-hydroxyl group of steroidal hormones produced hydrogen bonding with His524 in the ‘D-ring pocket’. The ligand binding cavity, represented by hydrophobic amino acid side chains, is favorable with hydrophobic scaffold of E2. Most of the ligands contain a phenol or phenol bioisosteres, bounded to A-ring pocket. Further, additional hydroxyl group is less important for affinity and can be replaced by a hydrogen bond acceptor, which can either bind directly to His 524 in D-ring pocket or indirectly to His 524, through bridging with water molecule [28, 29]. [Insert Fig.2 here] Fig.2: Protein-Ligand Interactions: A1 & A2: 2D and 3D interactions of 17-β Estradiol (PDB: 1A52) with ERα, B1 & B2: 2D and 3D interactions of E2 (PDB: 3OLS) with ERβ, C1 & C2: 2D and 3D crystal structures of ERα with DES (PDB: 4ZN7), D1 & D2: Cocystalised structures of genistein with ERα (PDB: 1X7R), E1 & E2: Cocystalised structures of genistein with ERβ (PDB: 1X7J). The ERα and ERβ ligand binding cavities reveal only the difference between two conservative amino acid residues. The first is on β-face of binding cavity, where Leu384 of ERα is replaced by Met336 in ERβ and second is in α-face, in this Met421 of ERα is replaced by Ile373 of ERβ. Ligand substituents oriented downwards to the direction of Met 421, like the lactone ring of 16αlactone estradiol (16α-LE2) 1 (Fig.3), tend to be better accommodated in ERα and show selectivity

for ERα. Whereas, those pointed upwards of Met 336, like 8β-vinyl-estradiol (8β-VE2) (Fig.3), show selective binding to ERβ [15]. Val 392 in ERα is replaced by Met 344 in ERβ in plane of the bound estradiol in B-ring pocket. Hydrogen bond interactions between hydroxyl groups of E2 ligands and their receptors are associated between α and β subtypes (Glu 353, Arg 394 and His 524 for ERα, and Glu 305, Arg 346 and His 475 for ERβ). ERβ selective ligand i.e. phytoestrogen heteroaromatic genistein 3, possesses a largely planar topology and many of the ERβ-selective ligands viz. liquiritigenin 4, WO-01072713 5, diarylpropionitrile 9, ERB-041 7, ERB-096 8 etc. (Fig.3) have been developed from similar planar topologies. Thus, indicating that better packing of flat aromatic ligands was more acceptable with ERβ, as compared to ERα. The interaction between Met336 and central B-ring of genistein allows greater ERβ selectivity [30, 31]. Two selective ER modulators viz. tamoxifen and raloxifene, exhibited tissue selective estrogenic effects. Grese et al. studied molecular features of raloxifene, which accounted for its unique profile. The features included (a) presence of phenolic hydroxyls, (b) properties of basic amine side chain, (c) introduction of stilbene into cyclic benzothiophene and (d) position of a carbonyl at hinge region of ER between basic amine side chain and the olefin, which may impact on conformation of ER-raloxifene complex [32]. Triphenylethylene based antiestrogens such as tamoxifen demonstrated the significance of N,N-dimethylamine side chain, which blocked the uterine hypertrophic effect of estrogen. Dialkylamine moiety of raloxifene was important for antagonistic effect, among the piperidine and pyrrolidine, which were optimal for inhibitory activity [33]. [Insert Fig.3 here] Fig.3: Structure of Various Subtypes of Selective and De Novo Generated Scaffolds as SERMs

Lloyd et al. reported de novo generation of several chemotypes and scaffolds (15-19) as selective estrogen receptor ligands by scaffold hopping technique. They applied various pharmacophoric constraints from observed ligand features to generate 446 ligands, which satisfied the volume and pharmacophoric constraints. MCS grouping among 446, identified 144 ligands at a similarity cutoff of 0.5. The representatives, within an inclusion volume described by superposed active ligands from Skelgen-generated scaffold types, were phenyl benzothiophenyl 15, triaryl ethylene 16, phenyl quinolinyl 17, phenyl benzofuranyl 18 and phenyl indolyl 19 (Fig.3). In another study, a total of 127 ligands were generated by satisfying the pharmacophoric constraints. MCS similarity grouping was further employed to identify common substructures and several core scaffolds were generated using steroidal input ligands. The majority of resulting motifs consisted of a bicyclic ring system attached to another ring via single bond, as potent selective ER ligands [34]. Rationale based drug design of ERα coactivator binding inhibitors (CBIs) was performed from crystal structure of ERα-LBD complexed with diethylstilbestrol. The interactions of ERα with ligands included 16 amino residues (of helix 3, 4, 5, and 12) L354, V355, I358, A361, K362 (helix 3), L372 (helix 3,4 turn), F367, V368 (helix 4), Q375, V376, L379, E380 (helix 5), and D538, L539, E542, M543 (helix 12) respectively. Coactivator showed zone of interactions with three leucine residues in LXXLL signature motif and produced an equilateral triangle shape. To mimic the helix backbone, hydrophobic aliphatic chains, which were outside the hydrophobic groove and proceeding inward by adding residue elements, were attached. Thiazine, pyrimidine, trithiane and cyclohexane were selected as core scaffolds of ERα-CBIs and hydrophobic substituents were attached to the alternate positions around selected scaffolds. The inside-out design of CBIs was performed by hydrophobic linker substituted on naphthalene to fill the groove position of coactivator binding pocket without specifically mimicking the shape of individual leucine

residues. The hydrophobic linkers interacted with two remaining hydrophobic residues, I689 and L693 [35]. The crystal structure of raloxifene 20 with ERα showed that the basic side chain of ligand occupied the orthogonal position relative to basic benzothiophene core. Benzothiophene core was replaced with benzothiophene [3, 2-b] indole 21, for constraining the side chain into its active conformation. Benzothiophene [3, 2-b] indole having tetracyclic core, simultaneously restricted rotation of two phenolic groups, so that the core was more coplanar with better mimicking actions (Fig.4). The attachment of side chain readily assumed the axial position. The rigid stilbene moiety of tetracyclic core was biologically relevant in many synthetic non-steroidal estrogen antagonists. The amine containing side chain of raloxifene was also critical for binding to amino acid residue of the ERα and played important role in tissue specificity [36]. [Insert Fig.4 here] Fig.4: Design strategy of Benzothiophene [3, 2-b] indole to restrict rotation 4. Potential Pharmacophores for SERMs Indole-Benzimidazole Hybrids Indole based compounds are having enormous potential in anticancer drug development and discovery. 1-Benzyl-indole-3-carbinol 22, an analog of indole-3-carbinol (I3C), was found to be thousand times more potent than indole-3-carbinol in estrogen dependent breast cancer (Fig.5) [37]. 2-Arylindole derivative 23 showed tremendous selectivity towards ERα. Bazedoxifene, an indole-based antiestrogen, was as effective as tamoxifen and also overcame resistance. The majority of SERMs approved for targeting BC, contain OH or F functionalization, so the designing approaches might be through the concept of monovalent bioisosteres. NH serves as a monovalent bioisostere of OH, and satisfies it’s function in SERMs i.e. for interaction with essential amino

acid residues in active site of ERα. Free amino group of 2-aryl benzimidazole moiety acts as hydrogen bond donor in the active site of ER-α. Therefore, N-benzylated indole-benzimidazole hybrids 24 and NH-indole-benzimidazole hybrids 25 (Fig.5) should act as good pharmacophores of SERMs and potential scaffolds for breast cancer therapeutics [38]. [Insert Fig.5 here] Fig.5: Development of Indole-Benzimidazole Hybrids as SERMs m-Carborane Bisphenols N,N-dialkylaminoethyl group, the most significant substituent in SERMs, inhibits binding of coactivators by modulating helix-12 to an unfavorable position. Carboranes, the cluster composed of boron, carbon and hydrogen, can function as lipophilic structure of various bioactive molecules including ER ligands [39]. High-binding affinity with ER and partial estrogenic activity require a phenolic ring connecting the hydrophobic motif and spherical structure. Further, hydrophobic surface of carboranes makes it easy to interact with hydrophobic residues of ligand binding pocket of receptors. The m-carborane bisphenol analogue 26 with N,N-dimethyl ethylamino basic side chain was developed as a potent pharmacophore (Fig.6) of SERMs. In spite of having geometry similar to two hydroxyl groups and spherical structure, binding affinity of o-carborane bisphenol was hundred times weaker than that of m-carborane and the EC50 was 1000 times lower too. The difference in binding affinity and agonistic activity was mediated by hydrophobicity and acidic C– H hydrogens of o-carborane cage. Since the acidic C–H hydrogen reduces hydrophobicity, ocarborane having two acidic C–H hydrogens would be unfavorable for binding to ERα, and mcarborane cage without any bare C–H hydrogen turns suitable hydrophobic pharmacophore for ligand binding pocket of ERα [40].

Benzoxathiins In the designing of ERβ selective ligands, various scaffolds have been liberally utilized including biphenyls, tetrahydro chrysenes (THC), diarylpropionitrile (DPN), aryl benzothiophenes, isoxazole, benzothiazoles, benzoxazoles, benzimidazoles, triazines, isoquinolines, isoindolines, steroidal and phytoestrogen analogues etc. Among these, many of the ligands showed low selectivity, based on receptor binding. X-ray crystallographic studies and structure-based optimization of a series of ERβ selective ligands were performed by using ab initio quantum mechanics. Different functional groups incorporated during the lead optimization produced varied interactions with ERα-Met 421, relative to ERβ-Ile 373, on the basis of both electrostatic and dispersive effects. Quantum chemical calculations of two functional groups i.e. nitrile and vinyl incorporated at 7-position of benzofurans or benzoxazole scaffold, have successfully enhanced the ERβ selectivity over 100-fold. Similar selectivity was also observed with the functional groups capable of forming differential electronic interactions with methionine side chain, relative to a purely aliphatic side chain. Crystal structure of ERβ complexed with ERB-041 indicated that, the vinyl group fits tightly into a groove formed by the side chains of residues Ile 373, Ile 376, and Phe 377, therefore there is not much room for vinyl group to move with respect to ERβ Ile 373 [41]. ERβ subtype selective SERMs centered on isoflavanone scaffold include Genistein, daidzein, coumestrol and WS-7528. These naturally occurring leads contain a common benzopyran scaffold, and exhibit a moderate selectivity for ERβ. Inclusion of a basic side chain of raloxifene into flavanone motif, increased ERα selectivity and the cis flavanone exhibited a greater affinity for ERα over ERβ. ERβ represents steric and electronic repulsions between carbonyl oxygen atom of ligand with Met 354, which is absent in ERα, where the corresponding residue is Leu 384. The

smaller groups may enhance the binding with ERβ receptor by eliminating the proposed unfavorable steric and electronic interactions with Met 354 residue. Conversely, ERα selectivity was regained by the incorporation of larger and more polar sulfur atom, replacing carbonyl oxygen of flavanone motif. Kim et al. described SAR of benzoxathiin core 27 with basic side chain of raloxifene (Fig.6). The stereochemistry at C2,3 was investigated, and cis geometry [2S,3R] was found to be an absolute configuration for retaining optimum antagonist or agonist activity profile [42, 43]. Most positive assigns of hybrid molecules of mixed biosynthetic origin led to the idea of generating novel small heterocyclic molecular entities by rationally combining two or more different natural or synthetic compounds. The structural features of two or more bioactive substances into one molecule, may lead to synergism, enhancement and modulation of desired characteristics of individual components [44]. Indole-Xanthendione Hybrids 2-Arylindole derivatives are selective to ERα and inhibit the action of estradiol in breast cancer. Xanthones and xanthendione derivatives, isolated from natural sources as well as chemically synthesized, have been known to inhibit breast cancer proliferation [45]. Indole-xanthendione hybrids 28 (Fig.6) were developed by Singla et al. The hybrid acted as potent ERα selective ligands for the management of estrogen positive breast cancer. Indole-xanthendione hybrids have shown extensive interaction with Met 343, Leu 346, Met 421, Ile 424, Leu 525, Met 522, Leu 384, Met 388, Leu 428, Ala 350, Leu 391, Phe 404, Leu 349, Leu 387, Leu 539, Trp 383, Leu 354, Pro 535 and Val 534 (PDB: 4XI3). Indole part of hybrid was effective for anchoring the xanthendione moiety in hydrophobic region of cavity. To enable hydrophobic interactions with vital amino acids and H-bond interactions with Arg 394, Lys 529 and Asn 532 of LBD similar to bazedoxifene [46].

Combretastatin A-4 Skeleton ER ligands are having potent structural components and act by conjugate’s targeting mechanism. Combretastatin A-4 analogue is a potent antimitotic agent. When endoxifen, a SERM, is linked with Combretastatin A-4 skeleton 29 via a covalent amide bond, it caused steric hindrance, and enhanced ER antagonistic effects of endoxifen conjugate, possibly by interfering with Helix-12 in ER positive breast cancer. Furthermore, combination of antagonistic ER-ligand and Combretastatin CA-4 related acrylic acid (Fig.6) might produce selective antiproliferative effect on ER-dependent cancers [47]. Pyrido[3,4-b]-Indoles Fulvestrant had the advantage of inhibiting ERα-driven tumor cell growth. It also degraded ERα via ubiquitination, diminished growth but was having very low oral bioavailability [48]. Lasofoxifene, a SERM, is a rare example of a phenolic ER modulator with low clearance and high oral bioavailability, a property allocated to conformational effects and structurally remote from phenolic moiety on rate of glucuronidation [49]. High lipophilicity of phenol moiety in ER modulators, is associated with high clearance and poor oral absorption. High-throughput screening to identify novel ER binding motifs resulted in 90K nonphenolic compounds evaluated at a concentration of 10 μM for ERα LBD. Nonphenolic compounds possessed low clearance and high oral bioavailability as better promising drug like compounds. 1-Aryl-2,3,4,9-tetrahydro-1Hpyrido[3,4-b]-indole motif 30 (Fig.6) was identified by chris et al. as potent and bioavailable estrogen ligand for breast cancer. The compound is a substructure of PDE5 inhibitor tadalafil, and clinical candidates NITD-60934 (antimalarial) and PTC2993 (VEGFR inhibitor). This motif can be regarded as privileged substructure for the drug discovery. Introduction of fluorine at ortho

position on pendent aryl ring and mono fluorination of N-isobutyl substituent, provided 10-fold increase in potency and down regulation and combining these structural changes yielded potent compound AZD9496 30, a clinical agent as selective ER modulator (Fig.6) [50]. [Insert Fig.6 here] Fig. 6: Recently Developed Potent Pharmacophores as SERMs Organometallic SERMs Selenium containing compounds with diverse structures viz. inorganic selenium salts, selenoamino acids, methylselenocyanate and phenyl selenium derivatives, inhibit cell proliferation [51]. Various bioactive selenaheterocycles including ebselen have been discovered in recent years. The anticancer mechanism of monomethylated selenium metabolite, methyl seleninic acid (MSA), hypothesized the involvement of estrogen receptor (ER) stress signal mediators [52]. Five to six membered selenium heterocycles designed as substitute of furan, imidazole, propyl pyrazole triol, thiophene, etc. were explored as SERMs. Diphenolic selenium interacted with helix 11 through H-bonds and mimicked phenol of D-ring of E2 and stabilized helix 12 into agonist conformation. The introduction of basic side chains i.e. pyrrolidine, piperidine into selenophene based core may result in antagonistic activity as well as influence tissue selectivity due to the nature of spatial orientation of basic side chains in SERMs. Based on the consideration, luo et al. developed novel aminoethoxy-selenophene scaffold (31,32) (Fig.6) as subtype selective antagonist for estrogen receptor via directly interacting with helix 12 [53]. 2-Napthol Based Pharmacophore Most of the SERMs generally represent two crucial structural features a) mono or dihydroxy scaffold responsible for mimicking 17β-estradiol and in case of dihydroxy, both are placed at 11 Aͦ apart and b) dialkylaminoethoxyphenyl side chain is necessary for clinical SERMs viz.

tamoxifen, afimoxifene, raloxifene. X-ray crystallographic studies stated that two hydroxyl groups produce H-bonding with Glu 353-H2O-Arg 394 triad and His 524, whereas basic amino group of dialkylaminoethoxyphenyl side chain is responsible for electrostatic interaction with Asp 351 of LBD-ERα. In silico molecular modeling studies of 2-napthol based pharmacophore 33 (Fig.6) indicated the optimum distance of 11.4 Aͦ between oxygen atoms of piperidinol -OH or -OCH3 and phenolic -OH in series 1 and 2 respectively. OBHS-SAHA Hybrid Pharmacophore Two critical structural features i.e. piperidinol hydroxyl group and piperidine N of designed piperidinoethoxyphenyl analouges exhibited expected interactions with respective amino acid residues on LBD of ERα, essential for potent selectivity [54]. Hybrid ER ligand strategy to develop SERMs involves combining two bioactive drugs into a single molecule to interact with two relevant domains of the target and might possess for enhanced therapeutic activity [55]. Estradiol and tamoxifene were combined to form conjugates viz. E2-ellipticine (intercalating agent), E2chlorambucil (alkylating agent) and tamoxifen−doxorubicin (antimitotic) and the conjugates exhibited enhanced pharmacological properties [56, 57]. Exo-5,6-bis(4-hydroxyphenyl)-7oxabicyclo[2.2.1]hept-5- ene-2-sulfonic acid phenyl ester (OBHS), a partial antagonist, exhibited ER subtype selectivity, with relative binding affinity (RBA) values of 9.3% and 1.7% for ERα and ERβ respectively [58]. X-ray crystal structure of ERα-LBD with OBHS indicated that its partial antagonistic character, was achieved by indirectly modulating critical switch helix 12 in ER ligand binding domain by interactions with helix 11 and phenyl sulfonate group of OBHS. Further, sulfonate phenol makes a strong steric clash with helix 11, displaces His524 outward and repositions helix 11 to modulate orientation of helix 12 indirectly and eventually destroys surface bound coactivator binding site. OBHS is having two, 4-hydroxyphenyl substituents, one of which

mimics phenol of E2 by forming strong hydrogen bond with E353 and the other remains in E2 11β direction. But the substituents are not long enough to interact directly with helix 12. Therefore, the second phenol group can be modified with a range of functional motifs to increase antagonistic property of oxabicyclo [2.2.1]-heptene core ligands [59]. Estrogen signaling requires displacement of certain proteins i.e. HDACs from corepressor complexes and recruitment of coactivator proteins to transcription complexes containing liganded ER. Interaction of HDAC1 with activation function 2 (AF2) and DNA binding domain (DBD) of ERα occurs in nucleus [60]. In ER positive cells, knockdown of HDACs inhibition by trichostatin A (TSA), valproic acid (VPA) and butyric acid can decrease ERα levels. The combination of SERM with HDAC inhibitor requires both the drug components to be in close proximity in nucleus. Incorporation of SAHA, a clinical HDACi, into ER ligand OBHS might produce new bifunctional hybrid agents having improved efficacy and selectivity for ER [61]. The diversity of OBHS was expanded to gain selectivity in ER ligands by equipping ligands in two ways with the histone deacetylase inhibitor SAHA. The OBHS−HDACi hybrids (34,35) (Fig.6) offer a novel approach to develop efficient ER antagonists for breast cancer therapy and the ongoing in-depth mechanistic study of these compounds will promote further understanding [62]. Benzopyranone Based SERM The early drug discovery of estrogen ligands focused on design of nonsteroidal small molecules with antagonistic property against breast cancer along with other reproductive tissues and agonistic property in normal tissues. A unique cellular screen was developed to measure the effect of small molecules on estrogen receptor by inhibiting the release of cytokine, interleukin-6 (IL-6), from an ER (-) osteosarcoma cell line (U2OS) transfected with ER-α. The inhibition of IL6 release predicts potency of a drug to reduce both bone resorption and cellular proliferation [63]. High-throughput

screening of commercially available phenolic compounds identified benzopyranone analogue 39 (Fig.7), with an IC50 value of 300 nM for inhibition of IL-6 release. Several benzopyrane molecules (Acolbifene 36) were reported with ER-modulating property. The structure activity studies of SERMs provided key information on structural requirements for selective antagonistic property. Incorporation of basic side chains and D-ring phenyl group of SERMs such as tamoxifen 38 and raloxifene 37 have been shown to be critical for selective tissue antagonist effect. By incorporating the structural features of tamoxifen and raloxifene into hit molecule 39, lead compound SP500263 was produced 40, which was more potent than parent raloxifene and tamoxifen, with IC50 value of 7.7nM in MCF-7 breast cancer cell line (Fig.6) [64]. [Insert Fig.7 here] Fig. 7: Design Strategy of Benzopyranone SP500263 as SERM 4-Hydroxy Ferocifens X-ray crystal structure of ligand-binding domain (LBD) of ERα established that the ligand can bind to it as an antagonist by creating a new pocket in a flexible area of protein allowing for the basic side chain. These conformational effects modify the position of helix 12 of receptor, prevents it from interacting with certain effectors present in target cell and therefore aid in carrying out its function as an activator of specific genes. All known SERMs bear a chain of varied length but always with p or π electron sites (e.g., -NR2, -S, -R, -SO-R, -SO2R etc.) [65, 66]. Tamoxifen exists both in Z and in E configurations. The Z isomer 41 has been shown to be most strong antiestrogen. Hydroxy group, essential in active metabolite of the molecule, confers an increased level of affinity to the estrogen receptor [67]. Unfortunately, the coordination complex containing antiestrogen is too weak and the metal is unable to reach the target, as it gets hydrolyzed quickly and is also bulky

in size. This is the limiting factor in the coupling of a transporter group to a coordination complex to increase its cytotoxicity. Instead of using the compounds with coordination bonds, the use of species with strong metal-carbon covalent bonds may be a better option. Metallocenes like cisplatin are known for their own antitumor properties but based on a different mechanism. Ferrocene (n5 -C5H5)2Fe is also an archetypal metallocene and it can be used to increase the cytotoxicity of hydroxytamoxifen by attaching ferrocene to the key skeleton of diphenylethylene bearing the crucial hydroxy and dimethylamino groups. Replacement of phenyl nucleus of 4hydroxy-tamoxifen with ferrocene, which is inherently more aromatic than benzene, would be well justified. It is chemically stable in various non-oxidizing media and have antitumor activity due to metabolic formation of ferricinium ions in situ (Fig.8) [68]. A series of ferrocene derivatives, based the structure of the antiestrogenic drug tamoxifen or of its active metabolite hydroxytamoxifen, were prepared by McMurry cross-coupling reaction of the appropriate ketones. Compound 42 of the series showed greater activity, binding affinity and less toxicity than tamoxifen. Further, the Z form showed better binding affinity for the receptor, as logically visualized by a molecular modelling study of hydroxyferrocifen in the active site of the receptor (Fig.8) [69]. A series of ruthenocene organometallic selective estrogen receptor modulators 42, with various chain length (n=2-5), have also been prepared as a mixture of Z and E isomers by the same process. The relative binding affinity of ERα for n=2 and 3 were very high (85 and 53% respectively), even higher than that of hydroxytamoxifen (38.5%). Ruthenocene derivatives act as anti-estrogens as effective (n=2) or slightly more effective (n=3-5) than hydroxytamoxifen on ERα-positive breast cancer cell lines [70]. [Insert Fig.8 here] Fig 8. Development of 4-OH Ferocifene Derivatives as SERMs

Tetrahydroisoquinoline Derivatives The spatial orientation of 2-aminoethoxyphenyl side chain relative to central core of molecules has been postulated to influence endometrial properties of SERMs in women. Tamoxifen, which is having a relatively flat structure, is estrogenic in uterus and increases the risk of endometrial cancer. Basic side chain of raloxifene, oriented orthogonally to the plane of benzothiophene framework, is not associated with uterine side effects in humans [71]. The side chain has significant impact on tissue-specific agonistic and antagonistic property, and influences the pharmacological

profile

of

compound.

Renaud

et

al.

designed

and

synthesized

tetrahydroisoquinoline based estrogen receptor α (ERα) selective estrogen receptor modulators by replacing the aminoethoxy residue of conventional SERMs with novel conformationally restricted side chains. The side chain of SERMs is having strong impact on the tissue-specific agonistantagonistic properties and thus on the pharmacological profile of compounds. The group further explored the novel side chains containing SERMs and high-throughput screening of compounds provided the fluorophenyl derivative as their starting hit. The replacement of fluorine by a piperidinylethoxy side chain afforded the lead compound 43, which was conformationally restricted as compared by the Molecular modeling studies used in conjunction with the X-ray crystal structure of the ERα ligand binding domain (LBD) with raloxifene. The study also showed that diazadecaline side chain containing compound 44 mimicked the action of the SERMs (Fig.9). The compound exhibited reduced agonistic behavior in the MCF-7 cell assay in the absence of 17β-estradiol, and potential antagonist activity against breast cancer prevention with 49% bioavailability. The group identified 1-(4-fluorophenyl)-2-phenyl-1,2,3,4-tetrahydroisoquinoline6-ol (45) as a lead molecule by high throughput screening which was as an antiimplantation agent [72]. The further modification by replacement of the p-F substituent of 45 by an aminoethoxy side

chain and 1-H of the tetrahydroisoquinoline core by a 1-Me group, provided the most potent compounds 46 and 47. The compounds produced IC50 values 1.8 and 2.6 nM respectively against ERα, where as compound 43 showed IC50 value of 285 nM. Since the binding pocket of ERα is more hydrophobic, so they increased the lipophilicity was increased by incorporating alkyl substituents at 1-position of the tetrahydroisoquinoline nucleus [73, 74]. [Insert Fig.9 Here] Fig. 9: Design of Tetrahydroisoquinoline Derivatives as SERMs Conformationally Restricted Benzopyranes Researchers have developed a series of benzopyran-containing molecules viz. EM-800 48, CDRI85/287 49, which also exhibited tissue specific selective estrogen receptor modulatory activity (Fig.10) [75, 76]. It has been suggested that the difference in activity may be related to specific structural property and also due to the orientation of the basic amine-containing side chain similar to stilbene plane. While the side chain of tamoxifen is coplanar with stilbene moiety, both molecular modeling and X-ray crystallographic studies predict an orthogonal orientation of the side chain in raloxifene 50. Raloxifene derivatives, which are subjected to adopt basic chain orientation of tamoxifen, exhibit a tamoxifen like biological profile. Although, the derivatives are conformationally locked in a orientation, but maintain the raloxifene like biological profile [71]. To gain the optimal orientation of ER selective ligands, Grese et al. synthesized conformationally restricted raloxifene analogues by incorporating structural elements of both raloxifene and aforementioned benzopyrans. In particular, LY357489 (51) was obtained as an extremely potent SERM, with in vivo efficacy on bone and cholesterol metabolism, at a low dose of 0.01 mg/kg.

The significant shifting from benzothiophene to naphthalene containing nucleus 51, resulted in a remarkable boost in the in vivo potency of both agonist and antagonist activities (Fig.10) [77]. [Insert Fig.10 here] Fig. 10: Design Strategy of Conformationally Restricted Benzopyrane Derivatives as SERMs Dip-G Derived SERM Diptoindonesin G (Dip G) 52 is having tetracyclic core with A–D rings bearing a ketone, three phenolic OH groups and an additional E-ring with one more phenolic OH group (Fig.11). Dip G showed anti-proliferation effect in murine leukemia P-388 cells and immunosuppressant activity in a concanavalin A induced proliferation of mouse splenic lymphocytes (T cells) assay [78, 79]. It is also reported that Dip G regulate the stability of estrogen receptor α (ERα) and estrogen receptor β (ERβ), the members of steroid nuclear receptor superfamily with opposite effects on cell proliferation. Interestingly, Dip G decreased the stability of oncogenic ERα and increased the stability of ERβ, a tumor suppressor in breast cancer. Therefore, the natural product Dip G or its analogues may be developed as novel tissue selective estrogen receptor modulators [80]. Tang et al. developed a versatile synthetic route for the synthesis of natural product Dip G and its analogues. The synthetic strategy involved a regioselective dehydrative cyclization of aryl acetals, a regioselective bromination of benzofurans, a sequential cross-coupling of bromo-benzofurans with aryl boronic acids and BBr3- mediated tandem cyclization and demethylation. In Hs578TERβLuc Dox-inducible cell lines, ERβ was strongly stabilized by Dip G and compound 53 and was moderately stabilized by compound 54. Surprisingly, both compounds 55 and 56 destabilized ERβ, suggesting that the para hydroxyl group in place of ketone in D-ring of Dip G was critical

for stabilizing ERβ. The ortho hydroxyl group respect to the ketone as in compound 53, also produced better ERα and ERβ stabilizing effects than Dip G (Fig.11) [81]. [Insert Fig.11 here] Fig. 11: Structural Skeleton of Dip G Analogues as SERMs 3,4-Dihydroisoquinolins as SERM Development of new SERMs is still in progress to minimise the side effects. STS converts inactive sulfated

steroids

like

pregnenolone

sulfate

(PREGS),

estrone

sulfate

(E1S)

and

dehydroepiandrosterone sulfate (DHEAS) into corresponding unconjugated hormones [82]. The inhibition of STS may prevent estrogen sensitive carcinomas from transforming sulfated steroids into potent estrogens such as E1, E2, 5-androstene-3β and 17β-diol (Fig.12). The dual targeting of ERα and STS is a novel therapeutic strategy for the treatment of ER+ breast cancer by gaining maximum estrogen blockade to induce an estrogen depletion condition, but that could lead to undesirable side effects like osteoporosis [83, 84]. STS inhibitors were found to be active as SERMs in phenolic form, which required hydrolysis of sulfamate group by STS in order to interact with ERα to provide desired SERM properties. Although, it is considered that very small quantity of phenol should be released in vivo, from the hydrolysis of sulfamate group by irreversible STS inhibition, but the approach has the disadvantage of maintaining sufficient physiological concentration of phenol to exert relevant SERM action [85, 86]. Ouellet et al. synthesized a library of tetrahydroisoquinoline-N-substituted derivatives (phenolic compounds) by solid-phase chemistry, using a multi detachable sulfamate linker (Fig.12). Among the derivatives, three phenolic compounds devoid of estrogenic activity and toxicity emerged. Further, the sulfamate analogues (57, 58 and 59) of the phenolics were synthesized and tested in STS-transfected HEK-

293 cells. The analogues were found to be potent inhibitors (IC50 values of 3.9, 8.9, and 16.6 nM respectively) [87]. However, in T-47D cells, the estrogenic activity was absent and only moderate antiestrogenic activity was observed, but the significant stimulation of proliferation and alkaline phosphatase activity in osteoblast-like Saos-2 cells, indicated SERM activity (Fig.12) [87]. [Insert Fig.12 here] Fig. 12: Design Strategy of Dual Inhibitors of STS and ERα: A) conversion of sulfamate compounds into phenolic compounds by STS or by chemical hydrolysis. B) the sulfamate compound (I) binds to active site of STS and inactivate enzyme. the sulfamate compound (I), via releasing phenolic group, binds to ERα and act as a SERMs.

2-Phenyl Benzothiophene Based Pharmacophore Fulvestrant 62, the only SERD currently approved for treatment of ER positive breast cancer, may overcome mechanisms of resistance to AIs and sulphatase inhibitors. Its efficacy is limited due to poor oral bioavailability and other physicochemical properties [88, 89]. The nonsteroidal estrogen antagonist, GW-5638 60 and its more active metabolite GW-7604 61 were developed to address tissue dependent antagonism acquired by tamoxifen [90]. It may be hypothesized to combine a nonsteroidal core viz. benzothiophene, found in arzoxifene, with an appropriately selected side chain like carboxylic acid moiety found in GW-7604 61, to obtain a SERM with desired orally bioavailable tissue selective profile. Similar approach was undertaken in the identification of clinical candidate GDC-0810 63 [91]. By applying the hybrid pharmacophore-based approach, Tri et al. designed and synthesized a series of potent benzothiophene-containing compounds. The group identified LSZ102 (65), a clinical candidate in Phase I/Ib trials for treatment of ERα positive breast cancer. The compound is having with bioavailability and preclinical activity as SERMs

(Fig.13). The substitution at ortho position of 2-aryl ring is proved to be crucial for high potency and substitution at para position is favorable for the pharmacokinetic properties [92]. [Insert Fig.13 here] Fig. 13: Development Strategy of Clinical Candidate LSZ102 5. Development Strategy of Dual Targeting Inhibitors for Antiestrogenic Resistance The antihormonal strategy based therapeutic applications with tamoxifen, AIs and sulfatase inhibitors have certainly achieved zenith in clinic, but the drug resistance has opened new challenges and emerging research era in estrogen signaling. Two different approaches have been considered for developing novel clinical SERMs. The first approach is suitable modification(s) of existing chemical entities to produce new molecules with improved therapeutic efficacy by retaining the same basic mechanism of action. The second approach consisted of investigation of other pharmacological pathways through different molecular mechanisms of ER signaling. The combinations of endocrine therapies are under investigation to improve treatment strategies against resistance in postmenopausal patients with advanced or metastatic breast cancer. Combination of faslodex and arimidex is in phase 3 studies, anastrozole with anastrozole plus fulvestrant is being tried, in post-menopausal women with ER+ breast cancer. Both the combinations have shown acceptable clinical results after primary treatment of localized tumor. Crosstalk between ER and growth factor pathways viz. VEGF, HER-2, IGF-1 and phosphatidylinositol 3-kinase (PI3K) is critical for the development of resistance in hormonal therapy [93, 94]. This section of review includes different dual targeting ligands i.e. ER and EGFR/ HER2/ cdk1 for the management of acquired endocrine resistance. 2-Anilino-3-Aryl-Quinolines

VEGFR-2 inhibitors prevent angiogenesis as well as SERMs resistance in breast cancer through the regulation of Ras/MAPK pathway. A drug simultaneously targeting ERα and VEGFR-2 is considered to display better effect for the treatment of breast cancer. Sunitinib, a VEGFR-2 inhibitor contains an aromatic scaffold, flexible side chain with tertiary amine substituent, bears structural similarities with SERMs. A series of 2, 3-diaryl-isoquinolinone and 6-aryl-indenoisoquinolone derivatives were reported to establish the dual targeting hypothesis as multiple ligands of ERα and VEGFR-2. Quinoline containing VEGFR-2 inhibitor like carbozatinib drew great attention with similar structure i.e. two oxygen substituents at C-6, 7 and flexible long chain at C-4 of quinoline nucleus. Additionally, quinoline has also been reported to be an excellent ERα ligand [95]. Xiang et al. designed and synthesized a series of 3-aryl-quinoline derivatives with various basic side chains at the end. The 3aryl quinoline scaffold mimicked the structural features of SERMs. Amide side chains, with same length as in SERMs were tried with RTK inhibitors. The amides were used to link the side chain improved the inhibition of VEGFR-2 and methoxyl at C-6 or C-7 was used to mimic the estradiol to strengthen the affinity with ERα. The group identified compounds 68 and 69, with high ERα binding affinity and VEGFR-2 inhibitory activity. Moreover, two compounds exhibited excellent antiproliferative activity against MCF-7 and HUVEC cell lines with low micromolar IC50 (1-8μM) (Fig.14) [96]. [Insert Fig.14 here] Fig.14: Rational design of 2-anilino-3-aryl-quinolines targeting ERα & VEGFR-2 Diaryl Isoquinolinones The cross-talk between ER and tyrosine kinase receptor signaling has drawn interest in advance research for novel and effective anti-breast cancer agents. VEGFR-2 inhibitors not only inhibit

angiogenesis in breast cancer, but also retard acquired resistance by SERMs through inhibition of Ras/MAPK pathway. Tang et al. reported that compounds with an anti-estrogen as well as VEGFR-2 inhibitory property may produce better effect in breast cancer. It is further reported that VEGFR-2 inhibitors with indol-2-one scaffold, such as Sunitinib 72 and YM231146 73, have some structural similarities with SERMs in terms of aromatic scaffold and flexible side chain with tertiary amine substituent in end. 2,3-Diaryl isoquinolinone derivatives were prepared with the characteristics of both SERMs (70,71) and VEGFR-2 inhibitors, and the carbonyl function of isoquinolinone was expected to act as indol-2- one. The length of side chain was also taken into consideration to evaluate its influence on activity. Biological profiling of compounds 74, 75 and 76 exhibited marked anti-proliferative and anti-angiogenesis activities through ERα and VEGFR-2 dependent mechanisms (Fig. 15) [97]. [Insert Fig.15 here] Fig. 15: Chemical Structures of SERMs, VEGFR-2 Inhibitors and Designed compounds Chromen-2-Ones Coumarins, a versatile class of pharmacologically active scaffold, are known to exert significant biological activities including anticancer [98], anti-HIV [99], anti-microbial [100] and antiinflammatory [101]. Among the diverse properties, anticancer property was extensively investigated. The anticancer mechanism of coumarin analogues include anti-angiogenesis and induction of apoptosis, independently [102, 103]. A series of novel SERMs based on 3-aryl-4anilino-2H-chromen-2-one core have been reported. Piperidyl substituted analogue 77 (with OH at C-7 position of the coumarin nucleus) exhibited excellent ERα binding affinity and antiproliferative activity against MCF-7 and Ishikawa cells. The hydrogen binding of the lead

compound 77 indicated that the interactions between 7-OH with Glu353 and Arg394 and 4’-OMe with His524 played critical role in stabilizing its binding mode with ERα [104]. NH linker and bioisosteric replacement of O atom were also introduced and subsequently, two more groups (F and OH) were placed at C-4’ position of 3-phenyl substituent. The introduction of fluorine to a bioactive molecule caused minimal steric alterations and facilitated the interaction with enzyme active site [105, 106]. Additionally, hydroxyl containing analogues function as hydrogen bond donors and thus could retain essential hydrogen bonds with amino acid residues of ERα receptor and VEGFR-2 enzyme. Finally, various tertiary amines including dimethyl amine, diethyl amine, pyrrolidinyl and piperidyl were incorporated to terminal basic side chain to obtain altered antiestrogenic activity. Potent VEGFR inhibitors, Vandetanib 78 and Cabotantinib 79 were in clinical trials for the treatment of angiogenesis related diseases [107, 108]. The essential structural skeleton of VEGFR-2 inhibitors include fused aromatic system and substituted aniline or aryloxy moiety, to target the kinase domain of VEGFR. Several coumarin analogues, developed to prevent angiogenesis by inhibiting endothelial cell growth, attracted marked attention in anticancer drug discovery [109, 110]. 3, 4-Disubstituted-2H-chromen-2-one containing fused aromatic ring and basic side chains have emerged as an interesting scaffold for designing ERα as well as VEGFR inhibitors. The structural optimization has also been guided by molecular docking of a putative compound 80 (R1= R2= OH) into the VEGFR-2 ligand binding domain (PDB: 1YWN). The molecular docking studies represented hydrogen bond interactions and Pi-Pi interactions of coumarin moiety in the binding site formed by the amino acid residues phe916, Cys917 and Lys866, for VEGFR-2 inhibition. Among all the synthesized compounds, compound 81 exhibited an IC50 value of 2.19 μM for ERα binding affinity, retained excellent inhibition on VGFR-2 and also suppressed the growth of angiogenesis related cells (Fig.16) [111].

[Insert Fig.16 here] Fig. 16: Design of 3-Aryl-4-Anilino/Aryloxy-2H-Chromen-2-One Scaffold as ERα and VEGFR2 Inhibitors Pyrimidines Pyrimidine, a versatile druglike scaffold of enormous medicinal value, has the ability to interact with diverse targets including enzymes, receptors, DNA, protein targets, etc [112]. The ring has been broadly used as a common structural skeleton in various kinases including VEGFR-2 inhibitors (82-84) and exhibited potent anticancer activity [113, 114]. With a view to explore different scaffolds as multiple ligands of ERα and VEGFR-2, Luo et al. designed and synthesized basic side chain containing 2, 4-disubstituted pyrimidine derivatives for breast cancer therapeutics. Firstly, pyrimidine motif was used as a core structure to initiate the interactions with ERα ligandbinding domain (LBD) similar to m-carborane and pyrazole and showing interactions with VEGFR-2 kinase as well [115]. A substituted aromatic ring was then introduced at 2- or 5-position to occupy the hydrophobic pocket of ER and VEGFR-2 kinase receptors [116]. Further, N, Ndialkylamino containing side chains were introduced at para-position of 4-phenyl substituent, that inhibited binding of co-activators by changing conformation of helix-12 of ER. Finally, the bridging atoms between phenyl and pyrimidine moieties were explored and bioisosteric group O atom was also employed against established NH linker. The most potent compound 85, an analog of 2-(4- hydroxylphenyl) pyrimidine, was 19-fold more efficacious than tamoxifen in MCF-7 cancer cells and exhibited excellent ERα binding affinity (IC50 =1.64 μM) with marked VEGFR2 inhibition (IC50 = 0.085 mM) [117] (Fig.17). [Insert Fig.17 here]

Fig. 17: Development of pyrimidine-based ERα/VEGFR-2 Ligands β-Lactams Tubulin is an αβ heterodimeric protein and main constituent of microtubules, which is essential for mitotic cell division. Tubulin inhibitors such as paclitaxel and vinblastine are in clinical use for the treatment of various type of cancers. The β-lactam scaffold is template for variety of drugs and preclinical compounds including antibiotics, tubulin-targeting agents, SERMs, cholesterol absorption inhibitors and anti-asthmatics etc. [118]. The antiproliferative activity of SERM-type compounds containing β-lactam (azetidinone) scaffold with a basic side-chain, demonstrated antiestrogenic effect in MCF-7 cells (Fig.18) [119]. Boyle et al. explored a series of β-lactams to identify potential lead compounds for further development as ERα ligands. The group identified a common scaffold for the development of designed multiple ligands targeting both the ER and tubulin. The synthesized β-Lactams, 86 and 87 showed strong affinity for ERα (IC50 values 40 & 8 nM, respectively) and ERβ (IC50 values 19 and 15 nM). Compound 87 was most potent in antiproliferative screening on MCF-7 breast cancer cells and caused further accumulation of cells in G2/M phase (mitotic blockade) and depolymerization of tubulin in MCF-7 cells. It also induced apoptosis and downregulated pro-survival proteins Bcl-2 and Mcl-1 (Fig.18) [120]. [Insert Fig.18 here] Fig. 18: β-lactams as ER-tubulin ligands for anti-estrogenic resistance Norendoxifen-Imidazole Hybrids Aromatase (CYP19), a member of general class of cytochrome P450 enzymes, catalyzes conversion of androgens to estrogens, a crucial step of biosynthesis of estrogens [121]. Currently,

imidazole containing AIs viz. letrozole and anastrozole, have been approved by FDA [122]. The antagonistic blockade of ERs by a dual aromatase and selective estrogen ligand might act synergistically with decreased estrogen concentration due to aromatase inhibition. Estrogen agonist effect of SERMs would be beneficial for normal cells, while antiestrogen effect of dual AI/SERM would be beneficial for breast cancer cells, by blocking the effect of residual estrogen resulting from incomplete aromatase inhibition. Combination of aromatase inhibitors (89,90) and pure estrogen antagonist/down regulator fulvestrant was more effective than either letrozole or fulvestrant alone in suppressing breast tumor growth and delaying in the development of tumor resistance [123, 124]. Norendoxifen 88 is a metabolite of tamoxifen (Fig.19), is also a potent SERM as well as aromatase inhibitor. To optimize efficacy and selectivity of CYP19, a series of Norendoxifen analogues were subsequently designed and prepared by structure-based drug design approach. This led to the synthesis of 4′-hydroxynorendoxifen, that produced elevated potency against aromatase and higher affinity for ER-α and ER-β. Most of Norendoxifen analogues undergo facile E/Z isomerization in solution and complicate the pharmacological profile to limit its use. The analouges, as expected, have different biological activities against aromatase, ERs and other CYPs [125, 126]. To overcome the problem associated with E/Z isomerization, a series of triphenylethylene bisphenol analogues containing imidazole and tetrazole were designed by eliminating the aminoethoxy side chain of norendoxifen, and placing two identical substituents on one end of double bond and eliminating the possibility of E/Z isomers. These compounds produced enhanced aromatase inhibitory activity, ER-α and ER-β binding affinity and antagonized βestradiol-stimulated transcriptional activity in MCF-7 human breast cancer cells [127]. The findings of studies may facilitate the development of a new generation of dual AI/SERM agents. Further, imidazole and tetrazole containing compounds 91 & 92 showed excellent aromatase

inhibitory activity (16.9 & 4.77 nM) respectively and ERα binding affinity both (300 nM) (Fig.19) [128]. [Insert Fig.19 here] Fig. 19: Development of Dual Action Ligands of Estrogen and Aromatase 6. Development of Estrogen Receptorβ (ERβ) Ligands for Breast Cancer Treatment The identification of ERβ led to the re-evaluation of estrogenic action in all target tissues and estrogen associated diseases, including breast cancer. However, the exact role of ERβ in breast cancer still remains elusive, after two decades of research [129, 130]. The small pocket of ERβ may contribute to identify selective molecules. Among the ERβ selective ligands, diarylpropionitrile (DPN) has been studied exhaustively [131]. Hypermethylation of ESR2 promoter produced a significant decrease in ERβ-mRNA expression in breast cancer cells, as compared to normal epithelial cells. An interesting future therapy for ERα-positive and negative breast cancers could be the combination of epigenetic modifier i.e. tamoxifen with ERβ agonists [132]. Two ERβ agonists, WAY-20070 and the novel estrogen analogue (L17), produced excellent reduction in G2-M phase correlated with effects on cyclin D1 and cyclin E expression in a SERM/SERD-resistant breast cancer cell lines. ERβ agonists engaged both ERα and ERβ to Bcl2 response element strongly, reducing Bcl-2mRNA and protein in an ERβ-dependent manner. L17 introduced RIP140 to Bcl-2 promoter in cells overexpresses ERβ. The exposure to ERβ ligands also resulted in increased processing of LC3-I to LC3-II, an indicative of enhanced autophagic flux. It is suggested that combined therapy with ERβ agonist and autophagy inhibitor may offer a novel approach of treatment for SERM and SERD-resistant breast cancers [132].

Breast cancer (MC4-L2) cells with endogenous ERα and ERβ expression and T47D human breast cancer cells with recombinant ERβ (T47D-ERβ) were identified and used to explore the in-vitro and in-vivo effects of ERβ agonists 2,3-bis DPN 93 and WAY-20070 94. ERα agonist 4,4’,4”-(4propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) or 17β-estradiol (E2) induced mammary gland hyperplasia and MC4-L2 tumour growth, correlating higher number of mitotic and lower number of apoptotic features. ERβ agonists induced apoptosis by upregulation of p53, a tumor’s suppressor gene and apoptotic inducible factor. The agonists also increased caspase 3 activity, whereas PPT and E2 stimulated proliferation. PI3K-AKT signaling is necessary to allow proliferation stimulated by ER agonists. Whereas, inhibition of PI3K by LY294002 restrained ERβ-induced proliferation, which established the antiproliferative effect exerted by ERβ agonists in hormone dependent breast cancer [133]. Meyers et al. reported novel DPN analogues for the subtype selectivity of ERβ, in which both ligand core and aromatic rings were modified by reconstructing the phenolic hydroxy groups, alkyl substituents and nitrile groups. Other DPN analogues were also prepared by replacing the nitrile group with acetylene or polar substituents, to mimic the linear geometry of nitrile group. Meso-2,3-Bis(4-hydroxyphenyl) succinonitrile 95 and 2,3-bis(4-hydroxyphenyl)-succinonitrile 96 (Fig.20) showed excellent ERβ selective affinity and potency. The acetylene analogues showed higher binding affinity but somewhat lower selectivity than their nitrile counterparts. The study suggested that the nitrile group was critical for ERβ selectivity due to optimal combination of linear geometry and polarity. The addition of a second nitrile group in DPN or addition of methyl to an ortho position on the β-aromatic ring increased affinity as well as selectivity for ERβ [134]. Salicylaldimine derivatives were developed as selective agonists for ERβ, by bioisosteric replacement of phenol ring, with a hydrogen bonded pseudocyclic ring. Several structural modifications were applied to selected molecules of monoaryl-salicylaldoximes to improve their

ERβ selective receptor affinity and agonist property. Among all modifications, the best results were obtained by simultaneous introduction of a meta-fluorine atom into para-hydroxyphenyl substituent present at 4-position of salicylaldoxime and with a chloro group in 3-position of central scaffold. Compound 97 showed excellent affinity (Ki = 7.1 nM) and selectivity for ERβ over ERα, and represented as a selective and potent ERβ agonist with an EC50 value of 4.8 nM [135]. By structural modification of ERβ selective genistein 98, a new series of 3-arylquinazolines are prepared and evaluated for their ERα and β affinities. 5,7-Dihydroxy-3-(4-hydroxyphenyl)-4(3H)quinazolinone 99 (Fig.20) acted as an agonist on ERβ subtype with 62-fold higher binding affinity [IC50(ERβ)=179 nM] and 38-fold higher potency in a transcription assay [EC50(ERβ)=76 nM] than genistein. Transformation of C=O group into C=S group produced 5,7-dihydroxy-3-(4hydroxyphenyl)-4(3H)-quinazolinethione 100, with 56-fold higher binding affinity for ERβ over ERα [IC50(ERβ)=47 nM] and 215 fold higher potency in transcription assay with EC50(ERβ) value of 13 nM [136]. Malamas et al. developed biphenolic azoles as highly selective estrogen receptor-β agonists. The potent and selective analogues of the series had comparable binding affinities for ERβ as the natural ligand 17β-estradiol, but were >100-fold selective over ERα. The design strategy of molecules was performed by X-ray structures of ERβ co-crystallized with various ligands as well as molecular modeling studies. The design strategy enabled to take advantage of a single conservative residue substitution in the ligand-binding pocket, ERα Met421- ERβ Ile373, to optimize ERβ selectivity. Further, 7-substituted benzoxazoles were most selective ligands of both azole series, and compound 101 was 200-fold selective for ERβ (Fig.20) [137]. Fig.20: Structures of Potent ERβ Agonists for The Treatment of Breast Cancer

7. Future Perspective and Outlook Therapeutic applications of antihormonal strategy with tamoxifen, aromatase inhibitors and sulfatase inhibitors, have probably extended its to maximum in clinic, but the drug resistance has now opened a new challenge in estrogen signaling. The selective estrogen modulators have therapeutic potential in many pathophysiological conditions viz. breast cancer, osteoporosis, Alzheimer’s disease, hyperlipidemia, coronary heart disease, atherosclerosis, inflammation and ageing etc. Development approaches of tissue as well as subtypes selective estrogenic ligands may show advantage on estrogen over non-traditional target tissues, while mitigating disadvantages, particularly on estrogen positive cancers. The preliminary studies on new SERMs such as lasofoxifene, bazedoxifene, arzoxifene and ospemifene etc. are still to be established in large scale clinical trials and are currently under way. In breast carcinoma, several clinical studies have established that ERs can stimulate expression or activation of key molecules involved in estrogen signaling pathways. Further phosphorylation activates ERs in the absence of estrogenic ligands and results in loss of estrogen dependency. Several growth factors like VEGFR-2, HER-2, EGFR, PDGF-β etc., contributed a major role in development of endocrine resistance, suggesting that signal transduction growth factor inhibitors with endocrine therapy may be effective to overcome resistance [138]. Other molecular pathways viz. IGF1R, insulin like growth factor-1 receptor, MAPK and p42/44 MAPK associated with estrogen signaling are also important in tumour progression and combination therapy. Targeting the ER signaling pathways has provided many useful breast cancer therapeutics such as SERDs, SERMs, AIs and sulfatase inhibitors [139, 140]. Further understanding of complexities of ER signaling pathway will provide opportunities for the development of new and improved estrogen receptor modulators, which may replace existing ones or operate with them. Successful

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Subhajit Makar earned Master of Pharmacy (Medicinal Chemistry) from Central University of Punjab, India. Currently, he is pursuing Ph.D. from Indian Institute of Technology (Banaras Hindu

University), Varanasi, India. His research area includes computational drug design methodologies (structure-based drug design and ligand-based drug design), synthesis of designed molecules in the field of anticancer agents by targeting estrogen signaling pathway as well as HSP90 protein and synthesis of chemical dye for live cell imaging. Tanmay Saha pursued his Master of Pharmacy in Pharmaceutical Chemistry from Indian Institute of Technology (Banaras Hindu University), Varanasi. His research area included design, synthesis and evaluation of novel indole-based molecules as anti-cancer ligands. Rayala Swetha obtained Master of Pharmacy in Pharmaceutical Chemistry from Indian Institute of Technology (Banaras Hindu University), Varanasi and is currently pursuing her Ph.D. from the same Institute. Her research area includes design and synthesis of small molecules in the field of anti-Alzheimer's and anticancer agents. Gopichand Gutti received his Master of Pharmacy in Medicinal Chemistry from Nirma University, Ahmedabad. Currently he is pursuing Ph.D. from Indian Institute of Technology (Banaras Hindu University), Varanasi, India. His research area includes design, synthesis and evaluation of anti-Alzheimer's and anticancer agents. Dr. Ashok Kumar is senior research officer at Department of Pharmaceutical Engg. & Technology in Indian Institute of Technology (Banaras Hindu University), Varanasi, India. He received his Ph.D. in chemistry from department of applied chemistry, Institute of Technology, Banaras Hindu University. His research interests currently focus on the development of anticancer and anti- Alzheimer’s agents. Dr. Sushil K. Singh is Professor of Pharmaceutical Chemistry at Indian Institute of Technology (Banaras Hindu University), Varanasi, India. He received his Ph.D. in pharmaceutical chemistry

on natural products drug discovery from Institute of Technology, Banaras Hindu University. His research interests currently focus on the development of estrogen, EGFR and HSP90 inhibitors with their role in underlying mechanism associated with the progression of cancer and also working on development of novel AchE and NMDA antagonists for management of Alzheimer's diseases.

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OH group of genistein produced a hydrogen bond with His 475 in LBD of ERβ, was equivalent to 524 in ERα. The pharmacophores of ERs consists of two hydroxyl groups, are 11Aͦ apart. Val392 in ERα is replaced by Met344 in ERβ in plane of bounded estradiol in B-ring pocket. 2-Arylindole derivatives showed tremendous selectivity towards ERα. N, N-dialkylamino side chain at para-position of 4-phenyl inhibited co-activators binding by changing conformation of helix-12.

Conflict of Interest Authors do not have any conflict of interest.