124 Current Radiopharmaceuticals, 2012, 5, 124-141 Receptor Ligands for Targeting Breast Tumours: A Brief Outlook on Radioiodination Strategies

Maria Cristina Oliveira*,1,2, Carina Neto1,2, Lurdes Gano1,2, Fernanda Marques1,2, Isabel Santos1,2, Thies Thiemann2,3, Ana Cristina Santos2,4, Filomena Botelho2,4 and Carlos F. Oliveira2,5

1Unidade de Ciências Químicas e Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Sacavém, Portugal; 2Centro de Investigação em Meio Ambiente Genética e Oncobiologia (CIMAGO); 3Faculty of Science, United Arab Emirates University, United Arab Emirates; 4Instituto de Biofísica/Biomatemática, IBILI, FMUC, Coimbra, Portugal; 5Clínica Ginecológica, FMUC, Coimbra, Portugal

Abstract: The design and development of radiolabelled derivatives has been an important area of research due to their recognized value in breast cancer management. The (ER) is a relevant biomarker in the diagnosis, prognosis and prediction of the therapeutic response in estrogen receptor positive breast tumours. Hence, many radioligands based on estradiol derivatives have been proposed for targeted functional ER imaging. The main focus of this review is to survey the current knowledge on estradiol-based radioiodinated receptor ligands synthesis for breast tumour functional imaging. The main preclinical and clinical achievements in the field will also be briefly presented to make the manuscript more comprehensive. Keywords: Breast cancer, estradiol, estrogen receptor, radioiodination, SPECT, tumor targeting.

expressed primarily in the brain, bone and vascular 1. INTRODUCTION epithelium. Breast cancer is the most malignant type of diagnosed Given the broad clinical application in women welfare of cancer among women and still remains a major cause of ligands that modulate the ER, several classes of ER targeting death in the western world. Owing to its tendency to meta- compounds that might be useful as chemotherapeutic/ stasize, even before the disease can be clinically detected, chemopreventive agents and as radioimaging agents have early diagnosis is essential for breast cancer patients' welfare been designed and evaluated all over the years. Non-invasive and survival. A majority of breast cancers express estrogen imaging of breast tumours based on their hormonal recep- receptors (ER) in the primary tumour and in many cases tor’s status can be used for hormone therapy follow-up by respond successfully to or to a selective estrogen single photon emission computed tomography (SPECT) or receptor modulator (SERM) such as . positron emission tomography (PET) [4-7]. While PET imaging gives better resolution, SPECT still remains the The estrogen receptor (ER) is a ligand-regulated trans- more practical approach for routine diagnostics in nuclear cription factor whose activity as an inducer or repressor of medicine due to the use of medium to long-lived radionuc- gene transcription depends on the nature of the ligand with lides such as radioiodine isotopes. During the last years which it is bound, as well as the nature of the associated several efforts have been made to synthesize estradiol deri- corregulator. Estrogen action is important in many tissues vatives that could be labelled with γ-emitting radionuclides, and ER is involved in the development and function of the such as with radioisotopes of iodine, with high specific acti- reproductive system and plays a role in bone density main- vity, for imaging and monitoring estrogen-receptor (ER) tenance and brain function. Thus, ER is an important target positive breast cancers by SPECT modalities. Such a for the discovery of new drugs for treating or regulating a radiodiagnostic agent with a convenient half-life would have variety of hormone-related conditions and modulation of the advantage of being non-invasive. The search for selective estrogen receptor function is of paramount importance for a agents to image in vivo receptor densities in -sensitive variety of diseases including breast cancer and osteoporosis. tumours, as well as the need for probes to study hormone The discovery of a second ER subtype, termed ERβ [1, 2] action, has led to the synthesis and evaluation of a series of has increased the level complexity of estrogen signalling. halogenated derivatives of estradiol over the last 30 years [4, The two receptor subtypes (ERα and ERβ) show significant 7]. These include estradiol derivatives labelled with bromine sequence homology in their DNA and binding domains but [8, 9], fluorine [10-14] and iodine [15-24]. However, few of exhibit differences in their tissue distribution patterns, ligand these agents have reached the clinical stage [12, 25-28]. selectivity and transcriptional activities [3]. ERα is found Additionally, due to the growing interest in Auger electron mainly in breast and uterine tissues whereas ERβ is emitters for potential radiotherapy of ER-positive breast cancers steroid carriers are needed that exhibit high selec-

tivity and uptake in the target tissue [29, 30]. Such clinical *Address correspondence to this author at the Unidade de Ciências approach would require a receptor-ligand labelled with a Químicas e Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Estrada Nacional 10, 2686-953 Sacavém, Portugal; Tel: 351 219946195; radionuclide suitable for radiotherapy, with high affinity for Fax: 351 219946185; E-mail: [email protected]

1874-4729/12 $58.00+.00 © 2012 Bentham Science Publishers Estrogen Receptor Ligands for Targeting Breast Tumours Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 125 the estrogen receptor, low affinity for non-specific binding product 123I. Nevertheless, 123I is the most appropriate for in sites and good in vivo metabolic stability [31]. vivo diagnostic procedures because of its convenient half-life (13.2 h) and emitted photon energy of 159 keV, which is Herein, we will review the chemistry, radiochemistry and highly compatible with current detector technologies. 123I where available the in vitro/in vivo biological activity of estradiol analogues that have been studied as potential appears to be the best radiohalogen based on nuclear pro- perties and absorbed radiation to the patient. The 125I isotope probes for diagnostic purposes in breast cancer research. To with its long half-life of 60 days and the low energy (27-35 be fair, viewing the great amount of literature in this field, keV) of the emitted photons is the most commonly used for more than a few pages should be written. Therefore, we have the radiolabelling of compounds for in vitro biological and tried to focus on the area of radioiodinated estradiol-based preliminary in vivo animal studies. The 131I isotope has been ligands for ER targeting, and have inevitably done so with a radiochemist’s eye, giving special attention to the radio- routinely used for clinical applications. Its properties include an acceptable half-life of 8 days and an emitted radiation of iodination methods used in their preparation. Several radio- both 364 keV photons suitable for scintigraphic applications iodinated non-steroidal (e.g. ) [32-36] and and low energy β- particles suitable for in situ radiotherapy non-steroidal antiestrogens [37-44] have also been reported [46]. in the literature for ER imaging purposes, but will not be considered within the scope of this review. In spite of its adequate nuclear properties, the positron emitter 124I has not been yet extensively explored in PET The first part of the review covers the radioiodination of imaging because of a complex decay scheme, which includes estradiol-based ligands for ER molecular targeting. In the several high energy gamma rays. However, the favourable second part, a brief overview concerning the biological eva- 4.2 day half-life added to the ease of production on cyclo- luation of ER expression with estradiol-based ligands, trons makes this radiohalogen a promising radiotracer for the including structure-activity relationship (SAR) studies and pre-clinical/clinical studies of the most relevant radioligands, development of novel probes for molecular imaging [47]. will also be presented. The review concludes with a summary and an outlook on 3. METHODS OF RADIOIODINATION the future perspectives of the application of radioiodinated Numerous classical radio-iodination methods can be estradiol-based radioligands for ER SPECT molecular readily applied to the labelling of steroidal ligands for the imaging. estrogen receptor. Beyond the key parameters to be taken into account when dealing with any radiotracer such as half- 2. IODINE RADIONUCLIDES USED IN THE life, reaction time, radiochemical purity and specific activity, SYNTHESIS OF ESTROGEN RECEPTOR IMAGING careful attention must also be paid to the labelling site. Any AGENTS structural changes on the original molecule may have an effect on the binding affinity to the target, affecting its bio- Radioisotopes of iodine provide a nice combination of logical behaviour. Only radioiodination techniques that convenient nuclear properties and high specific activity and require minimal structural modifications to maintain the versatile chemistry. They are relatively easy to use and com- specific bioactivity exhibited by the unlabelled compounds mercially available at nominal cost. Three of the radio- should be realistically considered [48-50]. isotopes (123/125/131I) have found wide use for the labelling of The stability of the carbon-iodine bond at the particular small molecules such as steroidal ligands for the estrogen labelling site must also be considered to ensure in vivo stabi- receptor due to their physical and chemical characteristics, lity of the radiopharmaceutical. A disadvantage of iodine easy acquisition and accessible equipment needed (Table 1). comparatively to other radiohalogens is its lower stability Both 125I and 131I are reactor-produced and therefore less towards the in vivo cleavage of the carbon-iodine bond. For expensive and more readily available than the cyclotron this reason, aromatic and vinyl iodides, which are more

Table 1. Nuclear Properties and Application Areas of Some Iodine Radionuclides [45]

Radio-nuclide T1/2 Mode of decay (%) Eβ max. (keV) γ-emission keV(%) Application

Auger SPECT imaging 123I 13.2 h EC (100) 159 (83.0) (28.4/decay) Targeted radiotherapy 603 (61.0) β+ (22) 124I 4.18 d 2140 723 (10.0) PET imaging EC (78) 1691 (10.4) In vitro bioassays Auger 125I 60 d EC (100) 35.5 (6.7) Micro SPECT (19.5/decay) Targeted radiotherapy 284 (6.1) SPECT imaging 131I 8.02 h β- (100) 606 364 (81.2) Radiotherapy 637 (7.3)

126 Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 Oliveira e tal. stable than aliphatic iodides, are most often employed to radioiodine. However, compounds with very high specific confer high stability to the compounds. radioactivity cannot be prepared by iodine exchange reaction due the competition between the radioactive and the non- Many radioiodination methods have been described in radioactive species. The low in vivo concentration of estro- the literature, but only a few allow radiopharmaceutical pre- paration with good labelling yields and high specific activity. gen receptors associated with the occasionally incisive phar- macological effects of extremely low doses of the receptor- Based on the reaction type, chemical radioiodination pro- binding drugs always requires a radiopharmaceutical with cesses can be divided principally into nucleophilic and elec- high specific radioactivity and, to our knowledge, this trophilic substitution reactions [48, 51]. method of iodo-iodo exchange has never been used for Due to the wide range of efficient electrophilic and steroid-based ER ligands. nucleophilic techniques available for radioiodination of pharmaceuticals a number of aspects also have to be taken 3.1.2. Non-Isotopic Exchange into account during the selection of the appropriate labelling Non-isotopic exchange reactions make use of the dis- method. These include the site of incorporation of the placement of a non radioactive bromo (or chloro) with radio- radioiodine, which should lead only to a minimal change in active iodide. The advantage of this non-isotopic exchange is the biological activity, the knowledge of the pharmacological that a very high specific activity can be obtained, provided and toxicological properties of the iodinated compound, its that the radioiodinated compound is efficiently separated in vivo stability, the possibility of a single step synthesis, the from its brominated precursor. necessity of high radiolabelling yields and of high specific activity of the radiolabelled compound for a successful in The 16α-position in estrogens is very tolerant to rela- vivo diagnosis in patients. tively large halogens. The bromo- and iodo- analogues actually have an equivalent or even a greater affinity than the parent ligands. By contrast 16β-haloestrogens have shown 3.1. Nucleophilic Substitution remarkably reduced affinity [4]. Due to its high specific Nucleophilic substitution reactions have not been as activity and high ER binding affinity, 16α-[125I]iodoestradiol, widely used for radioiodination as electrophilic substitution forwarded by Hochberg et al. in 1979, has been one of the methods. However, their application has provided some first examples studied as a potential estrogen receptor imag- successfully radioiodinated compounds [50]. ing agent [15]. Since then, other reports have appeared des- cribing further work on this compound [15-18, 54-57] as Halogen exchange is the most common nucleophilic method for the introduction of radioiodine into organic well as on its substituted analogues (Fig. 1) [19, 20, 58-60]. molecules. In this type of reaction good organic leaving groups, such as the triflate, or an halogen atom of a non- radioactive analogue are substituted by a radioactive iodide. The advantages of these methods are mostly related to the simplicity of the mechanism involved and to the fact that iodine, like any halogen, is generally obtained in its anionic form, i.e., as iodide. In nucleophilic substitution reactions, the attacking nucleophile (in this case the iodide anion) Fig. (1). Chemical structure of 16α-[125I]iodoestradiol: positions brings an electron pair to the substrate to form the new bond, where different substituents have been introduced. and the leaving group departs with an electron pair. Several distinct mechanisms are possible depending on the substrate These 16α-[125I]iodoestradiols have been synthesised by (aliphatic or aromatic), the leaving group and the reaction simple halide exchange reaction from the corresponding conditions (solvent, temperature, etc.). Nucleophilic substi- 16β-bromo compound 1, previously prepared by bromination tution reactions can occur in aliphatic and aromatic com- of an enol (Scheme 1) [15, 58]. pounds according to the nature of the leaving group but are most generally used to radiolabel aliphatic sites [52]. OH OH 123/125 Nucleophilic substitutions on aliphatic compounds proceed Br I faster than on aromatics. Moreover, the aromatic substrate Na125I A A must be activated by electron-withdrawing groups. To CH3CN, HO HO catalyze the nucleophilic exchange in aromatic compounds 1 2 the use of metal-assisted reactions has proven to be quite 125 successful in the case of arenes. Well-known examples Scheme 1. A general example of the synthesis of 16α- include the use of ammonium salts or copper(I)- and [125I]iodoestradiol by radioiodo-bromo exchange reaction. copper(II)-assisted radioiodinations [53]. Other approaches involve the treatment of aromatic diazonium salts or the In some cases, the additional substituents introduced into decomposition of triazenes in the presence of protic solvents the 11β-position (or others) have increased their ER binding or Lewis acids with sodium radioiodide [52]. affinity and/or reduced their lipophilicity, and these changes 3.1.1. Isotopic Exchange have generally increased their target tissue uptake [20, 60]. A series of 2/4-fluoro-16α-[125I]iodoestradiol and their 11β- The simplest way to radioiodinate a small organic mole- methoxy analogues were also prepared by Ali et al. [60], cule is to replace an already incorporated iodine atom by again by the radioiodo-bromo exchange reaction. In general, Estrogen Receptor Ligands for Targeting Breast Tumours Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 127 higher binding affinities were observed for the 4-fluoro et al. (Scheme 3). However, biological results were not analogues as compared to the 2-fluoro analogues and these reported for this compound [63]. differences were more pronounced at higher incubation temperatures. 3.2. Electrophilic Substitution Given the relatively low reactivity of nucleophilic aro- Electrophilic labelling with radioiodine is the preferred matic halogen-substitution, early attempts to use copper or synthetic route because of the ease with which radioiodide copper salts as catalysts of nucleophilic radioiodination of can be oxidized in situ to an electropositive form of iodine arenes were quite successful [53]. Nevertheless, this app- (I+/Iδ+). This type of substitution is commonly employed in roach has not been extensively explored for the radioiodi- radiolabelling of ER ligands, typically via an oxidized iodo- nation of estradiol derivatives [61]. nium species (I+) reacting as an electrophile with a system of 3.1.3. Radioiodo-Dediazotization high electron density such as an aromatic ring or a double bond. As a result, a covalent carbon-iodine bond is formed Another regiospecific, nucleophilic substitution reaction with the loss of a positively charged leaving group. Ideally, is the halogenation of aromatic rings via diazonium deriva- this leaving group should be able to easily accommodate a tives of arenes. Sandmeyer-type reactions were classically positive charge, making organometallic moieties especially used to introduce a halogen into a given position of a deacti- useful in these reactions since metallic ions are easily able to vated or non-activated aromatic ring. Starting from an amino carry positive charges. Aliphatic radioiodinated tracers that precursor the reaction is carried out via the formation of a are mainly labelled via electrophilic substitution only play a diazonium salt. The radioiodo-dediazotization then proceeds minor role. Thus, nearly all the labelling methods developed according to an aromatic SN1 mechanism in the presence of up to date are most commonly employed in the synthesis of the radioiodide [50, 51]. This method has been successfully radioiodoaryl compounds [48, 52]. Molecular radioiodine used by Enginar et al. in the synthesis of two radiolabelled (I2) was the most commonly employed labelling agent in the estrone isomers, 2- and 4-[131I]iodo-3-methoxy-estrone to former days of protein labelling. Nevertheless, its volatility, assess their potential for imaging or therapy of ER positive greatly enhancing radiation exposure to the operator asso- cancers [62]. As an example the radiosynthesis of 2- ciated to low radiochemical yields and the high commercial [131I]iodo-3-methoxy-estrone (5) is depicted in Scheme 2. costs have discouraged its use. Thus, mainly two methods, The amino precursors of estrone 3 were diazotized with direct iodo-deprotonation and iodo-demetallation, both based sodium nitrite in acidic medium and the diazonium group is on in situ oxidation of radioiodide, are more suitable for then substituted with [131I]iodide, giving 2- and 4-[131I]iodo- radioiodination at the non-carrier added (n. c. a.) level and 3-methoxy-estrone. have gained great significance. In order to avoid the in situ preparation of diazonium Electrophilic reactions can be performed simply by salts, another synthetic strategy has been described, using the adding to the precursor an oxidant such as chloramine-T, so-called Wallach triazene reaction. In this process, the iodogen, Iodobeads®, peracids or other mild oxidants. diazotized amine is trapped by the formation of a triazene with a secondary amine. The decomposition of the triazene 6 Chloramine-T, (N-chloro para-toluenesulfonylamide) one of the first oxidizing agents to be used in radioiodination in the presence of the radioiodide in acidic medium yields was originally developped by Hunter and Greenwood to the desired radiolabelled species [51]. One advantage radioiodinate antibodies with high specific activity (I, Fig. 2) associated with this method is the easy purification of the [64]. Briefly, chloramine-T releases hypochlorous acid in radiolabelled product from its precursor. The synthesis of the 125 aqueous solution that oxidizes iodine to generate the electro- I labelled 17α-(p-iodophenyl)estradiol 7 derivative was achieved successfully in 40% radiochemical yield by Wang philic iodonium ions [48]. Although chloramine-T is an

Scheme 2. Radiosynthesis of 2-[131I]iodo-3-methoxy-estrone (5) via a diazonium precursor [62].

Scheme 3. Radiosynthesis of 3-O-methyl-17α-(p-[125I]iodophenylethynyl)estra-1,3,5(10),6-tetraene-17β-ol (7) via a triazene precursor [63]. 128 Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 Oliveira e tal. effective oxidizing agent, numerous by-products can result this procedure was used to synthesize 2- and 4-[125I] from the harsh conditions involved specially in the labelling iodoestradiol [70]. of peptides or proteins [51]. Despite of these limitations, the process still remains popular in the radioiodination of 3.2.1. Direct Electrophilic Radioiodination (Radioiodode- estrogens. protonation) Generally, this method is limited to arenes activated for electrophilic substitution. Direct electrophilic radioiodination is simple to perform and usually provides high radiochemical yields. One disadvantage of this method is the occurrence of isomeric mixtures that in some cases are difficult to separate. The orientation of substitution follows the well known rules based on I- and M-effects, i.e. that most activating groups are ortho and para directing. Fig. (2). Chemical structure of chloramine-T (I) and iodogen (II). The direct A-ring radioiodination of 17β-estradiol (8) by To avoid the side reactions resulting from severe oxi- the chloramine-T method was first reported by Malmquist et dizing conditions, Fraker and Speck have described a al. in 1979 [71]. Later on, the same methodology was used method that uses a milder oxidizing agent, iodogen (1,3,4,6- by Fernlund et al. in the radioiodination of estradiol for tetrachloro-3α,6α-diphenylglycouril) (II, Fig. 2) [65]. Iodo- affinity binding studies to human sex hormone binding globulin (SHBG) [72]. Three A-ring radioiodinated com- gen, also known as Pierce® Iodination Reagent, is insoluble 125 125 in water and direct contact between the reagents is mini- pounds, 2-[ I]iodoestradiol, (9a), 4-[ I]iodoestradiol (9b) 125 mized since the oxidizing agent is immobilized on the wall and 2,4-[ I]-diiodoestradiol (9c) were obtained with a high of the vessel [48, 51]. While milder oxidizing conditions are specific activity and a high degree of radiochemical purity advantageous, there are some drawbacks in using this after adequate RP-HPLC purification (Scheme 4). Their particular method. The labelling reaction relies on catalysis studies pointed out the remarkably high affinity of 2-iodo- at the interface, and therefore its efficiency is a function of estradiol for human SHBG suggesting the potential interest the active surface area which can lead to lower yields and of its γ-emitting 125I-analogue for binding studies with this much longer reaction times in some instances [50]. Enlarge- globulin. ment of the surface area of the interface in an immobilized ® Although small groups at positions C-2/C-4 of the oxidizing agent is accomplished in Iodo-beads , spherical estradiol nucleus are relatively well tolerated by the ER, the units coated with chloramine-T that are dispersed in the A ring of estradiol has not been explored very much as a reaction vial. potential labelling site for the development of radioiodinated Another family of oxidants frequently employed in radio- tracers for ER targeting and not much in vivo data is iodination of sensitive substrates are the peracids. The available for these iodoaryl compounds [4, 73]. advantage of using peracids is that they preclude the forma- tion of by-products resulting from over-oxidation and no To evaluate the effect of altering the position of radio- chlorinated by-products are formed during radioiodination. labelling in the biological behaviour of A-ring radioiodinated Generally, they are formed in situ by reacting hydrogen areno-annelated estradiols Oliveira et al. followed the same peroxide with an organic acid (e.g., formic- or acetic acid). methodology in the radioiodination of an E-ring exp- Thus, the concentration of the oxidant can be kept very low anded estrane derivative [74]. Radioiodination of 1’- conducting to mild labelling [66]. However, the radioche- methoxy-benzo[4’,3’:16,17]estra-1,3,5(10),16-tetraene-3-ol mical yields obtained with peracids are usually lower than (10/MEBE) provided after reversed-phase High Per- those obtained using N-halooxidants [48]. formance Liquid Chromatography (RP-HPLC) two radio- isomers, 2-[125I]MEBE and 4-[125I]MEBE (11a/11b) in high Apart from the above mentioned oxidants for electro- radiochemical purity and high specific activity (Scheme 5). philic radioiodination there are some less often used reagents for in situ oxidation such as iodates, metal ions, iodine 3.2.2. Demetallation Techniques (Radioiodo-Demetalla- monochloride and enzymes [48, 51]. The iodine mono- tion) chloride system developed by McFarlane [67] has also been used for the direct radiolabelling of estradiol [68]. Per- Radioiodo-demetallation reactions require organometallic oxidase enzymes, in particular lactoperoxidase, have also compounds as precursors, namely organoboranes, silicium-, been used in the enzymatic direct radioiodination of estradiol germanium-, tin-, mercury-, or thallium-containing com- [68, 69]. pounds. This methodology provides several advantages over The formation of electrophilic radioiodo species can also conventional labelling procedures, due to fast and regio- be performed by electrochemical oxidation of radioiodide. selective electrophilic attack to the organometallic precursor. The advantage associated to this method is its mildness The advantages include the control over the site of halogena- without the formation of by-products. However, due to the tion, high yields and accessibility to aromatic ring systems rather complex performance and often low radiochemical which normally cannot be functionalised [48, 52, 75]. yields associated to the high production costs the electrolytic Among the known demetallation reactions, destannyla- method has not found wide application [48, 51]. However, tion is the most favoured method for electrophilic radioiodi- Estrogen Receptor Ligands for Targeting Breast Tumours Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 129

Scheme 4. Synthesis of 2-[125I]iodoestradiol, 4-[125I]iodoestradiol and 2,4-[125I]-diiodoestradiol (9a, 9b, 9c) by direct A-ring radioiodination [72].

OMe OMe

X

chloramine-T HO HO 125 10 11 11a) X= I; Y=H Y 11b) X=H; Y=125I 125 125 11c) X= I; Y= I Scheme 5. Synthesis of radioiodinated areno-annelated estra-1,3,5(10),16-tetraene derivatives (11a, 11b, 11c) [74]. nations. The radioiodo-destannylation proceeds well under trated by Nakatsuka et al. in the preparation of (17α,20E)- fairly mild reaction conditions, affording the radioiodinated 21-[125I]iodo-19-norpregna-1,3,5 (10),20-tetraene-3,17β-diol product in high radiochemical yields and high regioselec- and its 11β-methoxy derivative as potential radiopharma- tivity, even when using only very small quantities of ceuticals of high specific activity (Scheme 6) [22]. organotin precursors. 3.2.2.2. Radioiodo-Demetallation with Organothallium- 3.2.2.1. Radioiodo-Deboronation and Organomercury substrates Organoboranes react with molecular iodine under basic Thallium and mercury have also been used for the syn- conditions to produce alkyl or vinyl iodides with retention of thesis of organometallic precursors for radioiodination [48, configuration [75]. However, the use of [125I]iodine mono- 52, 75]. However, the use of these metal compounds has not chloride for radioiodo-deboronation of (17α,20E)-21-[125I] yet been explored extensively in the synthesis of radio- iodo-19-norpregna-l,3,5(10),20-tetraene-3,17-diol gave a iodinated estradiols. One versatile and regioselective route compound of very low specific activity, making receptor proceeding via an organothallium reagent has been deve- studies extremely difficult [76]. In addition, the reported loped by Ali et al. in 1987 for the radioiodination of estradiol method failed to provide a pure compound even in its non- diacetate [77]. Treatment of estradiol diacetate (15) with radioactive form. Nevertheless, n. c. a. labelled vinyl iodides thallium trifluoroacetate in TFA and subsequent reaction (14) can be prepared from the appropriate alkynes (12) via with [125I]NaI followed by alkaline hydrolysis gave exclu- vinylboronic acid intermediates (13) by radioiodination with sively the radiolabelled 2-iodo isomer 17 (Scheme 7). [125I]NaI and using chloramine-T as oxidant, as demons-

Scheme 6. Synthesis of (17α,20E)-21-[125I]iodo-19-norpregna-1,3,5 (10),20-tetraene-3,17β-diol (14a) and its 11β-methoxy derivative (14b) via radioiodo-deboronation [22].

Scheme 7. Radiosynthesis of 2-[125I]iodoestradiol (17) via an arylthalium precursor [77]. 130 Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 Oliveira e tal.

3.2.2.3. Radioiodo-Destannylation alkyne in THF with a 1:1 ratio of tributyltin hydride to ethynylestradiol gives predominantely the Z-isomer, while Radioiodination via organotin chemistry has been the elevated temperature with excess tributyltin hydride and most extensively used procedure in the labelling of 17 - α AIBN or UV light, as radical initiators, gives the E-isomer as substituted estrogen derivatives [6, 78, 79]. The selection of the major product [23, 83]. this position for radioiodination was based on its ability to rapidly incorporate iodo-radionuclides (123/125/131I) and on the ER tolerance towards vinyl groups in this position [73]. The 17α-iodovinyl moiety acts similar to the ethynyl group in that it blocks D-ring oxidation and consequently decreases Scheme 10. Hydrostannylation of an alkyne with tributyltin hydride the metabolism of the hormone. Moreover, the C-I bond in [23]. this vinyl moiety is much more stable than a normal aliphatic C-I bond and hence the radiolabelled species is less prone to The isomeric tributylstannyl intermediates are converted in vivo deiodination. with retention of configuration to the corresponding 17α- 125I iodovinylestradiols. They are usually obtained in good The radiolabelled 17α-iodovinyl derivatives of estradiol [ ] radiochemical yields, by destannylation of the corresponding reported so far have been prepared via destannylation of the 125 corresponding tributylstannyl precursors. This electrophilic tributylstannyl precursors with [ I]NaI in the presence of an substitution reaction has also been described for the prepara- oxidant, followed by RP-HPLC purification. The use of a non-acidic medium has been reported as being particularly tion of a series of substituted (17α,20E)- and (17α,20Z)- 125 important for the labelling because of the sensitivity of the [ I]iodovinylestradiol derivatives and has been shown to be stereospecific, affording radioiodinated compounds in good Z-tributylstannylvinylestradiol intermediate towards the radiochemical yields and high specific activities [23, 78, 80]. reaction medium [78]. The generation in situ of the electro- A general example for the radioiodo-destannylation of a philic species of the radiohalide can be accomplished at the non carrier level with several oxidants that include hydrogen 17α-vinylestradiol derivative is depicted in Scheme 8. peroxide-glacial acetic acid, N-chlorosuccinimide and chloramine- T, among others [23, 80, 84-86]. As the iodo-tin exchange is the last step of the reaction sequence, it is important to check whether there are still small remnants of stannanes, in whatever form. Thus, in order to obtain a ”clean”, "metal-free" radiolabelled com- pound at the very end, purification of the reaction mixture by RP-HPLC is performed carefully. When the tributylstannyl group of the starting material is replaced by iodine, the difference in lipophilicity of starting material and product is significant, which greatly facilitates the separation of the two. The radioiodinated compounds obtained by destannyla- tion are more polar and have lower retention times in RP- HPLC than the stannanes. Scheme 8. General example for the radioiodo-destannylation of a 17α-vinyltributylstannylestradiol derivative. Extensive studies have been devoted to the preparation of γ-emitting estradiol derivatives for the detection of ER- positive breast tumours based on this methodology. Thus, in These vinyltributylstannyl intermediates have been pre- 125 pared by Hanson et al. in two different approaches [78]. The the last three decades several 17α-[ I]iodovinylestradiols first strategy involves the addition of (E)-2-(tributyl-stannyl) have been synthesised by radioiodo-destannylation and bio- vinyllithium reagent to the corresponding estrone derivative logically evaluated. In order to improve the binding pro- and, despite the low reaction yields (40-50%), the isolated perties and the metabolic characteristics of these compounds product have a well defined chemistry. A general example is several derivatives substituted with either 11β-methoxy, depicted in Scheme 9. The Z-tributylstannyl estradiol deriva- 11β-chloromethyl, 11β-ethyl, 7α-cyano or 7α-methyl toge- tives could not be prepared by this route [80, 81]. ther with different groups at the 11β-position, as well as A- ring-fluorinated derivatives have been studied [22, 25-28, 80, 87-90] (Fig. 3).

Scheme 9. Addition of (E)-2-(tributylstannyl)vinyllithium to a carbonyl group [78].

The alternative way is based on the hydrostannylation of

17α-ethynylestradiol. A general example for the hydro- Fig. (3). (17α,20E/Z)-[123/125I]iodovinylestradiol: positions where stannylation of an alkyne with tributyltin hydride is illus- different substituents have been introduced. trated in Scheme 10. Changing the reaction conditions, e.g., stoichiometry, temperature and time, provide either the E- or The introduction of a 17α-iodovinyl as well as the intro- Z-isomers as the major product [23]. Thermal addition to the duction of a 7α-alkyl group in the estradiol framework has Estrogen Receptor Ligands for Targeting Breast Tumours Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 131 shown to increase the ER binding affinity, and the combina- differentiate the target receptor from other receptors and tion of these two substituents has also been investigated [80]. non-specific proteins and also appropriate metabolic and Although non-natural 7α-substituted estranes have been pharmacokinetic profile [78, 94]. regarded as compounds of considerable pharmaceutical There are several comprehensive reviews on the applica- interest, studies on 7β-derivatives have not been reported in tion of SPECT and PET imaging agents for estrogen receptor the literature as in most instances the 7 -stereoisomers are α in breast cancer detection and management [5-7, 104]. formed predominantly. Therefore, only a brief outline concerning SAR studies and The effect of the absence of a stereocenter at the C7- the reported pre-clinical/clinical data for the most relevant position of the estradiol molecule, as compared to the satu- estradiol-based radioligands will be provided here. rated analogues, was investigated, also (Fig. 4). The intro- duction of a 6,7-double bond in the B-ring of estradiol, 4.1. Structure Affinity Relationship (SAR) Studies known to be well tolerated by the estrogen receptor, gives the steroid a different conformation that was reflected in its The biological response of a cell to a steroid hormone is biological behaviour [91]. associated to the binding of the steroid to the receptor since without binding no response will be elicited. It is also known that a reasonable correlation exists between the in vivo bio- logical behavior of a steroid and its in vitro receptor affinity. Receptor binding affinity is a function of both temperature and equilibrium time, among other factors. Most ER binding assays have been performed between 0 and 4ºC, since the receptor protein is more stable at low temperatures. How- ever, complete equilibrium might not be attained at this temperature especially, for high affinity ligands [105]. The Fig. (4). Chemical structures of (17 ,20E/Z)- 125I iodovinyl-6- α [ ] incubation conditions may influence the kinetics of the dehydroestradiol [91]. formation and dissociation of the hormone-receptor com- plex. This can explain the differences between the relative A series of 7-substituted 6,7-dehydroestradiols was also binding affinity (RBA) values observed in the literature for studied by Neto et al. to understand how the effect of the same isomer [83, 89]. RBA values measured at 4ºC altering the chemical structure of the chain might influence the ability for molecular ER recognition (Fig. 5). Some reflect the association rates, while those measured at 25ºC reflect more closely equilibrium conditions [89]. radioiodinated 17α-iodovinyl-6,7-dehydroestradiol analo- gues with cyanoalkyl and carboxamidoalkyl substituents on Other parameters such as different sources of receptor the C-7 position (different lengths of alkyl chains and (tissue minces and cytosol homogenate preparations) used in different substituents at the end of the spacer chain) were the assays can affect the values of relative affinities encoun- synthesized successfully and biologically evaluated [92, 93]. tered in the literature. Also, the ER binding specificity may differ from one animal species to another therefore the RBA values of estrogens from studies involving different animal species, could lead to equivocal conclusions about the effect of a substituent on human ER binding [94]. Beyond the steroid receptor non-specific binding, such as to albumin, estrogens also bind with very high affinity to the sex human binding globulin (SHBG). The binding specificity for these Fig. (5). Chemical structures of radioiodinated 17α-iodovinyl-6,7- proteins is, however, quite different from that of the receptor, dehydroestradiol analogues with different substituents on the C-7 so it seems possible to design receptor ligands with high position [92, 93]. affinity for the specific receptor and low affinity for the serum binders. It is also important that the metabolic reac- 4. BIOLOGICAL EVALUATION OF ESTROGEN tions which can occur with the receptor ligand should not RECEPTOR EXPRESSION WITH ESTRADIOL- lead to the accumulation of circulating labeled metabolites BASED LIGANDS that enter tissues but no longer bind to the receptor. Estrogen receptors provide a system with favorable There is a considerable structural diversity among the binding characteristics for the uptake and selective retention estradiol derivatives that bind to the ER. For the develop- of imaging radiopharmaceuticals [94, 95]. Radiohalogenated ment of ER-based radiopharmaceuticals, the selection of the estrogens with suitable binding properties, such as high substituent groups and their site of attachment to the binding affinity and moderate to low non-specific binding, estradiol molecule, have been based on substitutions that are have been shown to be taken up by target sites with high known from previous work to increase the in vivo biological efficiency and great selectivity [8, 9, 10-14, 20-25, 54-58, activity [105-107] or to improve the uptake selectivity [31, 80, 81, 86, 87, 95-103]. An effective breast tumor-imaging 58, 81, 100] of estrogens, either by enhancing ER binding, ligand should present high specific activity, high binding reducing non-specific or serum binding, or altering the route selectivity which is associated to the ligand’s ability to or reducing the rate of metabolism.

132 Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 Oliveira e tal.

The introduction of a substituent in the ligand’s molecule Estradiols bearing a large group attached covalently can affect the binding to the receptor in two distinct ways. through a long “spacer” may significantly bind to the The substituent can alter the binding by favourable or estrogen receptor [123]. The 11β-position of estrogens is a unfavourable interaction with the receptor and/or alter the site where relatively large, non-polar substituents, such as binding by a conformational effect, i.e. by changing the ethyl or chloromethyl groups, are well tolerated by the overall shape of the ligand and accordingly, the positions of receptor [10, 105, 124]. By contrast, polar substituents at the other groups in the molecule relative to the receptor 11β-position not sufficiently far-away from the ligand core [108]. The position of a substituent in the steroid molecule reduce receptor binding [10, 124]. A 7α-methyl group is well will determine the extent to which it will interact directly tolerated by the estrogen receptor [20, 125] but the combined with the receptor and/or modify the shape of the steroid effect of both 7α-methyl and 11β-methoxy substituents leads [109]. These two substituent effects can deal in antagonism to compounds that have lost virtually all the ER affinity [20]. to each other; a group may induce favourable conformational A 17α-ethynyl substituent enhances receptor-binding changes in the molecule while its nature (e.g. electronega- affinity and would appear to act in synergy with certain other tivity, selective binding via hydrogen bonding, etc.) can substituents, such as the 11β-methoxy or 11β-ethoxy group negatively affect the binding affinity. [126]. Substituents larger than the ethynyl group and/or the The effects of diverse substituents on the binding of extension of the 17α-moiety to a substituent containing three estradiol to the ER, obtained from in vitro competitive or more carbons are usually poorly tolerated by the ER. binding assays, have been extensively reported in the However, despite the apparent steric intolerance at the 17α- literature. These effects have been only described in terms of position, several organometallic 17α-substituted derivatives increasing or decreasing binding affinity relatively to have shown to retain considerable ER affinity suggesting the estradiol but these relative values have been used very often presence of a binding site in this region that can accept spe- to anticipate the in vivo biological behaviour of the radio- cies of hydrophobic character [127-129]. Also, groups linked active analogues of the substituted estradiols. They have also to an ethynyl or vinyl group are well tolerated. The 17α- been useful, together with data from affinity-labelling and iodoethynyl estradiol, despite of the larger volume of the site-directed mutagenesis studies in predicting the charac- iodine relatively to a proton, show quite favourable binding teristics for the binding site of estradiol in the estrogen probably due to the greater polarizability of the iodine [34, receptor whilst the X-ray crystallographic structure of the ER 130]. 17α-vinyl estradiols have also been investigated in ligand-binding domain was not yet known [73, 110]. considerable detail [23, 83, 131, 132]. The presence of the Estradiol is a non-polar and hydrophobic molecule, iodovinyl group does not significantly alter the ER binding except at its molecular termini. Most of its skeletal flexibility affinity [22, 100, 133]. Nevertheless, with iodovinylestra- resides in the B-ring and binds to the ER in a low-energy diols, the binding behaviour is strongly dependent on the conformation. Hydroxyl groups at C-3 and C-17 of the stereochemistry of the vinyl group. Preliminary binding estradiol molecule are necessary for effective binding to the assay studies from Ali et al. with (17α,20E/Z)-iodovinyl- estrogen receptor [73, 108]. The hydrocarbon structure that estradiol have shown that the Z isomer possesses a slightly separates the two oxygen functions confers an appropriate higher ER binding affinity than the E isomer [23]. Further configuration to these functionalities, allowing for their work on the effect of stereochemistry at C20, in the series of interaction with amino acid residues of the ER ligand- 17α-halovinyl estradiols, has shown a marked difference binding domain. The hydrocarbon structural framework between the E and Z isomer. The Z-isomer showed much itself also plays a very important role in the binding of the higher binding affinity than the E isomers, with bulky ligand to the receptor [73]. Modifications on the spatial substituents enhancing affinity at this position. The opposite configuration of the ligands hydrocarbon structure usually pattern was observed in the E series [83]. The behaviour of lead to different interactions resulting in a decreased binding the related phenyl, phenylthio, and phenylseleno vinyl estra- affinity to the estrogen receptor [108, 109]. diols has been shown to follow the same pattern suggesting that the orientation of the substituent on the vinyl group 4.1.1. Estradiol Ring Structural Substitution Effects on ER towards the receptor differs considerably depending on the Binding configuration of the double bond E and Z [131, 134]. Analysis of the effect of A-ring substituents on the ER The lower affinity of the 17α-E-vinyl isomers has been binding affinity of estradiol suggests a negative influence of explained as being attributable to their structural similarity to groups of any size at C-1 [73, 111]. Small groups are reason- the 17α-ethynyl series that is sensitive to small changes in ably well tolerated at C-2 and C-4 whereas large groups and molecular volume [83]. For the E isomers and 17α-iodoethy- those that might involve an intramolecular hydrogen bond nyl derivatives the iodo-group is oriented in a similar way, substantially reduce binding affinity [60, 73, 89, 112-115]. outward from below the α-face of the steroid and qualita- The ER is intolerant to both polar and non-polar groups tively similar binding affinities are observed. The Z isomers at C-6 [73, 116]. Groups at the 7α-position are well tolerated, have the substituent directed towards a different portion of even if they are of considerable length [115, 117-121]. Polar the receptor, inward toward the α-face, and this could exp- functions are not well tolerated unless they are positioned lain the observed different values for the binding affinity away from the core of the steroid structure [117, 119, 120, suggesting an additional hydrophobic pocket that can accom- 122]. modate small to moderately sized lipophilic moieties in this region. Estrogen Receptor Ligands for Targeting Breast Tumours Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 133

The ER is moderately intolerant of polarity in D-ring Dawley rats by Oliveira et al. Both 2-[125I]MEBE (11a) and area, except for the specific 17α-position. Small, non-polar 4-[125I] MEBE (11b) (Scheme 5) are stable in vivo and are substituents are well tolerated at the 16α-position, whereas mainly excreted through the hepatobiliary pathway. Both large substituents result in poor affinity, especially those localize in the uterus and ovaries via a receptor mediated with increased polarity [54, 135, 136]. In contrast, the 16β- process, but the 2-[125I]MEBE isomer (11a) has the higher position is sterically less permissive [112, 125]. The 14- and specific ER binding and uterus selectivity. The favourable in the 15-position can accept small, non-polar groups without vitro/in vivo stability and biodistribution profiles of these appreciable loss of binding affinity [137]. compounds suggest a need for further exploration of their potential clinical application [74]. A complete recounting of all the ER binding affinity data reported in the literature would be voluminous therefore only 4.2.2. Radiolabelled 16α-Iodoestradiols representative RBA values from selected compounds will be referred throughout the text. The ER binding affinity data The high ER binding affinity found for 16α-iodoestradiol when available from the literature will be presented as (RBA=56%) led to its study as a potential estrogen receptor relative binding affinity values (RBA = 100 X IC estradiol/ imaging agent [15-18]. In biodistribution studies with 16α- 50 [125I]iodoestradiol, Hochberg et al. observed a high uptake in IC50 for tested compound, where IC50 is the concentration which inhibits [3H]estradiol binding by 50%). the uteri of female rats and a high target tissue to blood ratio [15]. However, in subsequent studies with rats bearing mam- mary tumours, Gatley et al. observed a rapid metabolism and 4.2. Pre-Clinical and Clinical Studies of Radioiodinated high excretion of the steroid, indicating that these parameters Ligands also have to be taken into account in the design of a Research on specific tumor receptor imaging has mainly radiopharmaceutical [16]. been directed to the measurement of the estrogen receptor Additional substituents introduced into 11β- or 7α-posi- status since breast cancer patients in whom these receptors tions, well tolerated by ER, have shown to decrease metabo- are present may benefit from hormonal therapy. Several lism and non-specific binding of the 16α-iodoestradiol estradiol derivatives have been radioiodinated at an aryl derivative, while increasing receptor specificity [106]. The position or at the 16β- position and vinylestradiols have been subtle modifications sometimes increase the ER binding radioiodinated at the 17α- position, in either the cis (Z)- or affinity and the target tissue uptake [19, 20, 60]. Studies the trans- (E) position. Although few of them have reached 125 from Zielinski et al. have shown that 16α-[ I]iodo-11β- the clinical stage, imaging of breast cancer in humans has methoxy-17β-estradiol binds to ER with an affinity equal to been accomplished with the most promising radioiodinated that of estradiol (RBA=100%). In vivo it concentrates in an 16α-[123I]iodoestradiol and 17α-[123I]iodovinylestradiols. estrogen receptor-dependent manner in the uterus and shows 4.2.1. Radiolabelled Iodoarylestradiols a remarkable, prolonged and much higher uterus to blood ratios than 16α-[125I]iodoestradiol, suggesting is ability to Not many biological data are available for the iodoaryl target estrogen receptor positive tumours [19, 58]. Later compounds since the introduction of an iodo group in the A- research led to the synthesis of other labelled 16α-iodo- ring of estradiol (2-iodo, 4-iodoestradiol) results in a estradiol derivatives, 11β-ethoxy- and 7α-methyl-16α-[125I] decrease in binding affinity (RBA<0.1%) as compared to 125 iodoestradiol (RBA=16.5%; 55.2%, respectively). Both com- estradiol [114]. 2-[ I]Iodoestradiol (Scheme 4) was the first pounds show receptor-mediated tissue uptake in the uteri of compound prepared by electrophilic aromatic substitution on immature rats. However, uterus to blood and uterus to non- the A ring, but a low uterus-to-blood ratio was found in bio- target ratios were more favourable for 7α-methyl-16α-[125I]- distribution studies, suggesting a lack of ability to concen- iodoestradiol (26d), pointing to the potential interest of its trate in estrogen-sensitive tissues by a receptor-mediated 123I analogue for SPECT imaging of breast tumours [20]. mechanism. Moreover, the reported low in vitro receptor The chemical structures of 16α-[125I]iodoestradiol and the binding affinity values have suggested that iodination in the most relevant derivatives that have been explored as ortho position of the A-ring of estradiol interferes with potential ER imaging radioligands are depicted in Fig. (6). SHBG binding [138, 139]. Two further derivatives of estrone, 2- and 4-[131I]iodo-3- methoxy-estrone (Scheme 2), were prepared by Enginar et al., who found for both radioligands receptor-mediated uptake in target organs such as uterus and ovaries in female Albino Wistar rats. However, significant differentiations in stomach, kidneys and intestines were encountered between the two radiolabelled isomers suggesting that beyond lipo- philicity, the molecular structure and the position of the Fig. (6) Chemical structures of 16α-[125I]iodoestradiol (26a) and its attached radionuclide are also important for the selective most relevant derivatives 26b-d [16, 19, 20]. binding to the receptor [62]. The influence of the site of radioiodination (C-2 vs. C-4) The main metabolic pathway of estradiol derivatives on the biological behaviour of two radioiodinated areno- involves modification of the A and D rings [140]. In the annelated estra-1,3,5(10),16-tetraene isomers was evaluated design of biologically active , substitution of through biodistribution studies in immature female Sprague- hydrogen for fluorine atoms has been an important strategy. 134 Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 Oliveira e tal.

Introduction of fluorine at 2- or 4-position of estradiol 133, 146]. Comparative studies with the compounds in framework does not affect hormonal activity or binding transplanted tumours containing various concentrations of affinity towards the estrogen receptor, but instead influences receptor suggest that the tissue uptake level is qualitatively A ring catabolic rates [141-143]. A series of 2/4-fluoro-16α- related to the ER concentration [24, 133]. 125 [ I]iodoestradiols and their 11β-methoxy analogues were 125 125 (17α,20E/Z)-[ I]iodovinylestradiols ( IVE2, Fig. 8) prepared by Ali et al. 60 . In general, higher binding [ ] have been studied extensively since the first established affinities were observed for the 4-fluoro analogues as synthetic procedures made the compounds readily available compared to the 2-fluoro analogues (23% vs. 17%) and these [21, 22, 78]. In immature female rats, the compounds have differences were more pronounced at higher incubation shown greater uterine uptake and similar uterus-to-blood temperatures. Tissue distribution in immature female rats 125 ratios when compared to 16α-[ I]-iodoestradiol [21]. Ali showed that the highest ER-mediated uterus uptake and the et al. prepared both the (17α,20E)- and (17α,20Z)- most favourable uterus to blood and uterus to non-target 125 [ I]iodovinylestradiol isomers and compared their ER tissue uptake ratios can be observed with 4-fluoro-11 - β binding affinity and in vivo target tissue uptake in immature methoxy-16 - 125I iodoestradiol (27d), indicating the poten- α [ ] female rats (Fig. 8). Their results suggested that the 20Z tial value of its 123I analogue as a radiopharmaceutical for configuration provides the better receptor interaction (RBA receptor imaging (Fig. 7). = 46.7% vs 32.8% for the 20E isomer) and the higher receptor-mediated target tissue uptake. However, a higher in vivo instability was found for the Z-configurated compound [23].

Fig. (7). Chemical structures of 2/4-fluoro-16α-[125I]iodoestradiol (27a/b) and their 11β-methoxy analogues (27c/d) [60].

Owing to the similar in vivo behaviour in the animal 125 77 123/125 Fig. (8). Chemical structures of (17α,20E/Z)-[ I]iodovinyl- model of 16α-[ Br]bromoestradiol and 16α-[ I]iodo- estradiols 28 and 29 [23]. estradiol, as reported by McElvany et al., and the good imaging characteristics of 123I, several clinical trials were Structure activity relationship data show that substituents undertaken with 16α-[123I]iodoestradiol (26a, Fig. 6) [97]. at either the 11β- or 7α-position are well tolerated [109, 147] In a SPECT study with 28 breast cancer patients, Preston and radiolabeled (17α,20E/Z)-[125I]iodovinylestradiol (IVE) et al. observed uptake in eight of nine proven breast cancers, has been prepared with a number of different substituents at four of which were ER+, three ER− and two of undeter- the 11β-position and biological evaluation has been reported mined ER status. There was one false negative of unknown [6]. ER status. All the metastases and axillary lymph nodes were Based on an earlier studies reporting that 11 -alkyl detected [98]. In another study conducted by Schober et al. β substituents enhance the binding affinity of the compounds with a series of 21 female patients, 14 of whom with breast to ER Hanson et al. prepared a set of novel 11 -substituted carcinoma and 7 suffering from benign breast tumors, 77.7% β estradiol derivatives and evaluated their receptor binding (7/9) of ER+ tumors were visualized whereas all the 12 ER− properties [124, 148-150]. In this series, the 11 -ethylestra- tumors and benign breast tumors were not [99]. Scheidhauer β diol analogue (30a) demonstrated a high ER affinity (RBA = et al. reported a sensitivity of 67% (20/30) and specificity of 43%) that, together with the relative ease of synthesis, led to 92% (11/12), using 16α-[123I] iodoestra-17 -diol (26a) in a β the preparation of (17 ,20E)- 125I iodovinyl-11 -ethyl- multicentre trial involving 42 breast cancer patients with α [ ] β estradiol (30a, Fig. 9). Tissue biodistribution data of 30a in known ER status [144]. Kenady et al., in a set of 29 patients immature female rats revealed a high uterine uptake, a long with breast cancer, injected 16α-[123I] iodoestra-17 -diol β retention and a significant degree of selectivity in target (26a) and found that ER functional imaging was most note- tissues [88]. worthy in patients with chest wall tumours and inflammatory breast cancer [145]. In conclusion, SPECT imaging with 16α-123I-estradiol (26a) can localize ER positive breast tumors, including some that may be difficult to detect using conventional diagnostic imaging. 4.2.3. Radiolabelled 17α-Iodovinylestradiols

Radiolabelled 17α-iodovinylestradiol derivatives are viewed as particularly promising breast cancer imaging Fig. (9). Chemical structures of 11β-substituted-(17α,20E/Z)- agents due to their high ER binding affinity and adequate 123/125 biological properties [22-24, 80, 81, 84, 85, 96, 100-102, [ I]iodovinylestradiols 30 and 31 [80, 88, 101].

Estrogen Receptor Ligands for Targeting Breast Tumours Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 135

Following previous observations that the presence of the Based on previous observations that the addition of a 2- 11β-methoxy group in the steroid frame, like in or 4-fluoro atom to the A ring of estradiol improves the (11β-methoxy derivative of 17α-ethynylestradiol) could biological behaviour of the steroid, Ali et al. evaluated a reduce the nonspecific binding to SHBG while retaining series of 2- and 4-fluoro derivatives of the isomeric receptor affinity [98, 112] the moxestrol analogues (17α, (17α,20E)- and (17α,20Z)-[125I]iodovinylestradiol as well as 20E/Z)-[125I]iodovinyl-11β-methoxy-estradiol (E/Z-[125I] analogues substituted with either 7α-methyl or 11β-methoxy MIVE, 30b/31b) have also been studied [22, 25, 81, 100, groups (Fig. 10). In general, the highest ER binding affinities 133]. were found for the 4-fluoro derivatives with Z configuration. 125 However, the combination of a fluoro substituent on the A- In the first reported studies the E isomer of 17α-[ I] 125 ring and 7α- or 11β-substitution was found to decrease the iodovinyl-11β-methoxy-estradiol (E-[ I]MIVE, 30b) demonstrated both high selectivity and uterine uptake in affinity for the receptor. The most favourable biological behaviour within this series as studied in immature female biodistribution assays together with a reduced nonspecific rats was demonstrated for the 4-fluoro-(17 ,20Z)- binding [22, 81, 100, 133, 151]. α [123I]iodovinylestradiol (33b) [89]. In comparative studies between both E and Z isomers Addition of a 7α-methyl substituent to estrogens has been because of the higher in vivo stability and the more favour- 125 reported to enhance the binding affinity for the ER as well as able uterus-to-blood ratio observed for E-[ I]MIVE (30b) 125 uterus uptake [153]. However, more bulky groups are known as compared to Z-[ I]MIVE (31b) in immature female rats, 125 to reduce the affinity for the ER [154]. (17α,20E/Z)-[ I] Ali et al. suggested its potential radiopharmaceutical applica- iodovinylestradiols derivatives featuring an electron-with- tion for ER imaging of breast cancer [80]. In a following drawing nitrile group at the 7α-position were also evaluated report, Foulon et al. have also selected the E isomer on the [90]. In spite of the relative high uterus uptake values basis of its much higher uterus-to-blood ratio at 2h p.i. in immature mice, as compared to that of the Z isomer [84]. In obtained for both isomers, the unfavourable contrast ratios render the potential usefulness of these 7α-cyano radiolabel- subsequent biodistribution studies conducted by Rijks et al. led estrogens for SPECT imaging less likely. Radioactive with both isomers 30b/31b in immature female rats the 123 substituents at the 7α-position that have been reported authors also identified the E-[ I]MIVE (30b) as the better include a long chain 7α-undecyl group labeled with radio- agent on the basis of the uterus-to-blood uptake ratio [96]. iodine [119]. However, taking into account that the target tissue-to-fat ratio could also be an important issue to consider, especially Many of the compounds with high ER affinity were still at longer time periods after injection, the authors concluded found to be unsuitable for in vivo imaging by a number of that the Z isomer of 17α-[123I]iodovinyl-11β-methoxy-estra- reasons, such as nonspecific binding, rapid metabolism, and diol (Z-[123I]MIVE) should be selected over E-[123I]MIVE high in vivo instability [10, 140]. In general, one important [96, 103]. A most promising biodistribution profile was reason for the nonspecific binding of these estrogens is their confirmed for Z-[123I]MIVE (31b) in normal and tumour- lipophilic nature. bearing female rats, and here the Z-isomer (31b) showed a The influence of the introduction of a C6-C7 double bond higher ER-mediated uptake and retention in uterus, ovaries, on the biological properties of the estradiol molecule was pituitary, hypothalamus and mammary tumours than the E- studied for the above mentioned radiolabelled estrogen isomer (30b). The uterus- and tumour-to-non-target tissue 125 125 derivatives, [ I](E)- and [ I](Z)-3-methoxy-17α-iodo- uptake ratios were also highest for Z- 123I MIVE (31b) [102]. [ ] vinylestra-1,3,5(10),6-tetraen-17β-ol, 23 and 24 (Fig. 4) [91]. The in vivo stability of the 20Z isomer has been questioned Once again, the Z isomer (24) showed the highest target despite its higher affinity for ER [96, 152]. Interconversion tissue uptake. However, the C6/C7 double bond on the to the 20E isomer has been reported to occur in the uterus of estradiol frame did not enhance the uterus to non-target immature female rats [152]. However, chromatographic tissue ratios when compared to the most suitable tracer analysis of plasma samples of healthy volunteers and cancer 123 reported in the literature, [ I](Z)-11β-methoxy-17α-iodo- patients, as well as in vitro plasma incubations, confirmed vinylestradiol (31b) [26]. the human in vivo stability of the 20Z isomer [28]. Long alkyl chains can also be accommodated in the 7α- Following the observation that linkage of a strong elec- position of the estradiol molecule without a substantial tron-negative chloromethyl group at the 11 -position of 17 - β β reduction of affinity for the estrogen receptor as found for estradiol significantly increases the ER binding, Quivy and , a new pure ER antagonist recently approved by Zeicher synthesized E- and Z- isomers of 123I iodovinyl- [ ] the FDA for the treatment of advanced or metastatic breast 11β-chloromethyl-estradiol (E-CMIV, 30c and Z-CMIV, cancer [118, 155]. Thus, the preclinical evaluation of a series 31c). Both derivatives showed higher ER binding affinity of 7-alkyl-substituted 6,7-dehydroestradiols was recently and selectivity than estradiol itself. On the basis of X-ray 6,7 reported by Neto et al. Three novel 17α-ethynyl-Δ -estra- crystallographic data for Z-CMIV, the authors hypothesized 125 diols and their trans 17α-[ I]-iodovinyl analogues (Fig. 5), that the Z-configuration might have a better interaction with containing different C7-cyanoalkyl chains, were studied to the ligand binding domain of ER 87 . Biodistribution [ ] understand how the length of such alkyl chains, attached to studies and scintigraphic images of human breast carcinoma the 6,7-dehydro estradiol framework, might affect the xenografts demonstrated receptor-mediated and selective receptor binding affinity and the specific uptake of such accumulation of Z- 123I CMIV (31c) in ER positive tumours, [ ] compounds in a ER expressing tumor cell line. with a significantly high target to non-target ratio [101]. 136 Current Radiopharmaceuticals, 2012, Vol. 5, No. 2 Oliveira e tal.

OH OH R1 I R1

X X I

R R HO 2 HO 2 32a/33a) X=F; Y=H; R =H; R =H Y 32 Y 1 2 33 32b/33b)X=H;Y=F;R1=H; R2=H 32c/33c) X=F; Y=H; R1=H; R2=CH3 32d/33d)X=H;Y=F;R=H; R =CH E-isomer Z-isomer 1 2 3 32e/33e) X=F; Y=H; R1=OCH3;R2=H 32f/33f) X=H; Y=F; R1=OCH3;R2=H Fig. (10). Chemical structures of 2/4-fluoro-(17α, 20E/Z)-[125I]iodovinylestradiol 32a,b/33a,b and their 11β-methoxy and 7α-methyl analogues 32c-f,33c-f [89].

The introduction of a ω-cyanoalkyl substituent at C7 of the remaining one planar scintigraphy negative patient. the 6,7-dehydroestradiol led to a decrease of binding affinity Furthermore, planar scintigraphy was positive in 69% (9/13) to ER. The length of the alkyl chain affects the ER binding of the patients with metastatic breast disease. This diagnostic affinity, which clearly enhances with an increase of chain modality also allowed the differentiation of metastases from length, following the trend butyl < pentyl < hexyl. Cellular non-malignant abnormalities on the bone scan. Rijks et al. binding studies with breast carcinoma ER positive cells reported high sensitivity for Z-[123I]MIVE imaging and suggested a non ER mediated uptake mechanism [92]. The established its high specificity tumor uptake by complete introduction of a hexyl chain with either a cyano or an amido blockade of uptake with tamoxifen. terminal group at the 7-position of a 6,7-dehydroestradiol Nachar et al. studied 13 patients with abnormal mammo- as a means to enhance and/or alter the receptor response graphy or a suspected breast mass on physical examination was also evaluated by the same group. Thus, (E)-7- 123 and demonstrated that the uptake of Z-[ I]MIVE on (carboxamidopentyl)-17α-[125I]iodovinylestra-1,3,5(10),6- 125 scintigraphy correlated well with in vitro ER quantification. tetraene-3,17β-diol and (E)-7-(6’-cyanohexyl)-17α-[ I] 123 Z-[ I]MIVE uptake was demonstrated in 50% (5/10) of the iodovinylestra-1,3,5(10),6-tetraene-3,17β-diol were stu-died. patients with histologically proven breast cancer. The While the ethynyl precursor of the steroidal amide showed a authors reported false positive results in two patients; how- very low in vitro ER binding affinity and the radio-iodinated ever, in eight patients with breast cancer, good agreement analogue exhibited an unsatisfactory biodistribution in 123 between Z-[ I]MIVE scintigraphy and in vitro ER immature female rats, the ethynyl precursor of the steroidal determination was found [27, 28]. cyano derivative possessed a comparatively better in vitro binding affinity to the receptor and the corresponding On the basis of results obtained in a series of 22 patients radiolabelled analogue showed high specific uptake and high with primary breast cancer, Bennink et al. also concluded selectivity for target tissues but no competitive binding to the that Z-[123I]MIVE scintigraphy is a sensitive and non- receptor in vivo when estradiol is coinjected [93]. invasive tool for ER detection in breast cancer patients [156]. In a subsequent study designed to predict in vivo response to The potential of 123I-radiolabelled MIVE for ER imaging antiestrogen treatment in ER+ breast cancer, Bennink et al. was investigated in several independent clinical trials. In a 123 showed that patients with high initial uptake of Z-[ I]MIVE earlier study Ribeiro-Barras and colleagues demonstrated and complete blockage of Z-[123I]MIVE incorporation after that human scintigraphy with E-[123I]iodovinyl-11β-meth- treatment with tamoxifen had a significantly longer progres- oxyestradiol (E-[123I]MIVE)) (Fig. 1) could be used to assess sion-free interval than those with low uptake of the tracer the presence of the estrogen receptor in primary and and partial or incomplete blockade under therapy [157]. This metastatic breast cancers [25]. In a study with 19 women 123 study clearly demonstrates that Z-[ I]MIVE targeted imag- with carcinoma of the breast, nine of whom had bone ing of functional ERs can distinguish responding from non- metastases, the primary breast cancer was visualized in two responding breast cancer patients treated with an antiestro- of four patients with high ER concentrations in breast tissue, gen drug. While both primary and metastatic tumours could whereas four breast tumors with a low ER concentration be detected with good sensitivity and selectivity in these could not be detected suggesting a poor correlation between studies, extensive correlations between imaging and clinical the presence of ER and tumour uptake. They could also outcome have not been done so far. image axillary lymph node metastases in two patients and bone metastases in 44% (4/9) of the patients. Using the more selective cis-stereoisomer, Z-11β- 5. SUMMARY AND FUTURE PERSPECTIVES 123 methoxy-17α-iodovinylestradiol (Z-[ I]MIVE) (Fig. 6), The SPECT imaging of estrogen receptors started three Rijks et al. reported the first clinical study for imaging both decades ago and since then several radioiodinated estradiol primary and metastatic ER+ breast cancers [102, 103]. In a derivatives have been synthesised to effectively assess ER trial with 12 primary breast cancer patients and 13 patients status and predict prognosis and therapeutic responsiveness with metastatic breast disease, planar imaging showed Z- of breast tumours. Many radioiodinated estradiol derivatives 123 [ I]MIVE uptake in 11 patients with primary breast cancer, have been widely explored as potential SPECT agents due to of whom nine were found to be ER+ on immunohistoche- several radioiodination approaches available for the syn- mistry (IHC). Interestingly, ER IHC assay was negative for thesis of different estradiol-based compounds. 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Received: October 28, 2010 Revised: January 11, 2011 Accepted: July 07, 2011