Design, Synthesis and in Vitro Investigations of Novel Fluorescently Labeled Steroids

Design, Synthesis and in vitro investigations of Novel Fluorescently Labeled Steroids

by Nisal U. Gajadeera

B.S. in Chemistry, Northeastern University

A thesis submitted to

The Faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Master of Science

December 2018

Thesis directed by

Robert Hanson Matthews Distinguished University Professor of Chemistry and Chemical Biology

Acknowledgements

Firstly, I would like to thank my advisor, Dr Robert Hanson for providing me the opportunity to work in his lab. His guidance and support has been incredible for me throughout my time at

Northeastern. I would also like to take this opportunity to thank all the past Hanson lab members,

Dr Emily Corcoran, Dr James Teh and Kelton Barnsely.

I would also like to thank Dr Vladimir Torchilin and Dr Tatiana Levchenko for the opportunity to conduct cell studies in their lab. My heartfelt gratitude goes to Dr Livia Mendez for conducting all the incubation studies and competitive binding studies. It was under her guidance that I learned the techniques such as passaging cells, FACS and fluorescence microscopy. She played a huge role the in vivo data gathering for my compounds.

I would also like to take this opportunity to thank Dr Michael Pollastri and the members of his group, especially Dr Dana Klug, Dr Lori Ferrins and Dr Baljinder Singh for the support they’ve shown in my research by allowing me to use their analytical instruments ( LC-MS). Without their support, my research would not have been possible.

A special thanks goes to - John Bottomy and Brian D’Amico for the tremendous support they’ve given in helping me with my TA duties. Especially Brian and Dr Jason Guo for helping me with NMR. I would also like to mention Jeff Peterson and Alex Henrikson for their support in making the UV-Vis spectrometer available. Also, I would like to thank Richard Pumphry,

Cara Shockley and Tara Loschiavo for their support in my graduate studies.

To all the professors who I met during my undergraduate and graduate studies at Northeastern

University, I offer you my most sincere gratitude. I am the person who I am because of the knowledge you imparted. I would especially like to thank Dr Raymond Booth and Dr George

O’Doherty for their role in reviewing my thesis. On similar note, I would like to sincerely thank

Northeastern University community. I am very grateful for the opportunities given to me at this institution.

Finally, I would like to thank my parents for their unconditional support in throughout their life.

Without you, I wouldn’t have been able to come this far. You stood beside me during good times and bad.

Abstract of Thesis

Breast cancer is the most common cancer among women, accounting for nearly third of all the diagnosed cancers. According to the American cancer society, over 41,000 patients died from the disease in 2018. Due to the high importance of the role played by estrogen and progesterone, agents that target the estrogen receptor (ER) and progesterone receptor (PR) play a major role in breast cancer therapy. Therapeutic strategies include the use of selective estrogen receptor modulators, selective estrogen receptor down regulators and aromatase inhibitors. Treatment success of breast cancer therapy is assessed by tumor estrogen receptor status which employs immunohistochemistry among other modalities. Although suitable for assaying ER expression in primary breast tumors, the accuracy of immunohistochemistry is lower in metastases.

Fluorescent imaging methods with high ER affinity and selectivity offer the potential for noninvasive, clinical imaging for primary and metastatic tumors. My thesis describes the background, synthesis and in vitro investigations of a series of fluorescently labeled steroids.

Chapter 1 of this thesis provides an extensive review of the fluorescently labeled estrogens prepared since 1995 with an emphasis on their synthesis and their efficacy as imaging agents, with our rationale for developing the ‘next generation’ fluorescent steroidal ER imaging agents.

Chapter 2 provides our synthetic approach for the fluorescent steroidal imaging agents and their controls. Our approached utilized the 11β-(4-azidoethoxyphenyl) estradiol scaffold with small, neutral fluorophores appended Sharpless “click” chemistry.

Chapter 3 reports in vitro evaluation of our steroidal imaging agents. The results strongly suggest that our steroidal fluorophores show selectivity towards ER. Potential improvements for the steroidal-fluorophore conjugates is also discussed.

Table of Contents

Acknowledgements ...... 2

Abstract of Thesis ...... 4

Table of Contents…………………………………………..…………….………………………………6

List of Figures ...... 9

List of Schemes and Tables ...... 13

List of Abbreviations ...... 14

Chapter 1 – A review of fluorescently labeled steroids ...... 19

1.1.1 Introduction ...... 20

1.1.1 The target- Estrogen receptor ...... 21

1.1.2 Estradiol- The native ligand ...... 22

1.1.2.1 Structure-Activity Relationships ...... 22

1.1.2.2 Evaluation of ligand-ER-LBD complexes ...... 24

1.2 Fluorescent properties ...... 25

1.3 Conjugation properties ...... 27

1.3.1 Survey of fluorescent probes - representative examples...... 28

1.3.2 Fluorescent probes post-1995 ...... 29

1.4 B-ring derivatives...... 30

1.5 D-Ring Derivatives -17α-Substitution ...... 35

1.6 C-Ring substitution using 11β-subsitution ...... 49

1.7 Proposed Approach to High Affinity Fluorescent Steroidal ER Probes

- Synthesis and future directions ...... 55

1.8 References ...... 56

Chapter 2 - Design and synthesis of fluorescently labeled antiestrogens .....74

2.1 Introduction ...... 75

2.2 Synthesis ...... 79

2.3 Fluorescent properties ...... 86

2.4 Future work – alternative synthesis routes...... 87

2.4 Summary ...... 89

2.5 References...... 90

Chapter 3 – In vitro investigations of fluorescently labeled antiestrogens ...96

3.1 Introduction – previous research ...... 97

3.2 In vivo studies introduction – Estrogen receptor ...... 99

3.3 General Experimental Setup ...... 100

3.4 Time point study – NBD-E2 (12b) and Dansyl-E2 (12a)...... 103

3.5 Time point study – NBD-E2 (12b) at 1µM ...... 106

3.6 NBD-E2 (12b) competitive binding study at 1µM ...... 109

3.7 NBD-E2 (12b), NBD-propargyl (9), competitive binding and

temperature dependence in receptor localization at 1µM ...... 113

3.8 Competitive binding – NBD control compound (10b) and NBD-E2

(12b) at 1µM ...... 115

3.9 Discussion of results ...... 118

3.10 Conclusion and future work ...... 125

3.11 References ...... 126

Appendix ...... 130

List of Figures

Figure 1-1. Basic structure activity relationships for substituted estradiol derivatives...... 23

Figure 1-2. Representative examples of parent fluorescent dyes described in the review...... 27

Figure 1-3 Examples of estradiol-fluorophore conjugates prior to 1995

...... 29

Figure 1-4. Transformations of estrone to fluorescent derivatives ...... 31

Figure 1-5. 6-Carboxymethyloximino derivatives with fluorophores conjugated through oligoethylene glycol linkers ...... 32

Figure 1-6. Synthesis of 6β-and 7α-fluorophore substituted estradiols 34

Figure 1-7. Preparation of 17α-alkynyl estradiol-dye conjugates without the phenyl group ...... 36

Figure 1-8. Fluorescent ER-targeted agents derived from the 4-(amino methyl)phenyl ethynyl estradiol intermediate...... 38

Figure 1-9. Preparation of Re (I) estradiol complexes with a terminal pyridine group ...... 39

Figure 1-10. Preparation of Ir (III) estradiol polypyridine complexes .. 40

Figure 1-11. Preparation of Ruthenium (II) estradiol polypyridine complexes ...... 41 9

Figure 1-12. Preparation of Pt(II) terpyridine ethynyl estradiol

complexes...... 42

Figure 1-13. Preparation of 17α-hydrazonopyridyl ethynyl estradiol-

metallated conjugates...... 43

Figure 1-14. Preparation of 17α-BODIPY-ethynyl estradiol conjugates

using Sonogashira reaction ...... 44

Figure 1-15. Preparation of 7α/11β-substituted 17α-BODIPY-ethynyl

estradiol conjugates using Sonogashira reaction...... 45

Figure 1-16. Preparation of 17α-substituted estradiols conjugates using

copper assisted azide alkyne cyclization (CuAAC) reaction ...... 47

Figure 1-17. Preparation of 11β-methoxy-17α-substituted estradiols

conjugates using copper assisted azide alkyne cyclization (CuAAC)

reaction ...... 48

Figure 1-18. General synthetic approach to the preparation of 11β-

substituted estradiols ...... 50

Figure 1-19. Modular, convergent synthesis of fluorescent anti-cancer

agent steroidal antiestrogen conjugate ...... 54

Figure 1-20. Representative structures of 11β-substituted estradiols and

complementary substituted fluorophores ...... 56

Chapter 2 - Design and synthesis of fluorescently labeled antiestrogens .....74

Figure 2-1. Overall strategy for design and synthesis of initial series of

fluorescent steroidal antiestrogens ...... 79

Figure 2-2. Alternative synthesis pathway- alkynyl steroid and azedo

flurophore ...... 87

Chapter 3 – In vitro investigations of fluorescently labeled antiestrogens….96

Figure 3-1. Previous investigations into fluorescent steroids ...... 98

Figure 3-2. 11β-(4-azidoethoxyphenyl) derivatized fluorescent

antiestrogens and controls ...... 99

Figure 3-3. Fluorescence and brightfield images of 12a/b at 20µM ... 103

Figure 3-4. Fluorescence microscopy data for 12b and 12a - intensity vs

incubation time, 20µM ...... 104

Figure 3-5. Fluorescence and brightfield images of 12b at 1µM ...... 107

Figure 3-6. – Fluorescence microscopy data- Intensity vs. incubation

time 12b at 1µM...... 107

Figure 3-7. – FACS data for Fluorescence intensity vs. incubation time,

12b at 1µM...... 108

Figure 3-8. Fluorescence microscopy images of 12b, competitive

binding studies. Comparison with brightfield images ...... 109

Figure 3-9. Competitive binding study for 12b - fluorescence

microscopy data ...... 110

Figure 3-10. 1µM Competitive binding study for 12b, FACS data ..... 112

Figure 3-11. – Temperature dependence in competitive binding for 12b

and 9 – FACS data ...... 113

Figure 3-12. – Fluorescence microscopy data for competitive binding for compounds 10b and 12b ...... 115

Figure 3-13. Fluorescence microscopy data for competitive binding – for compounds 12b and 10b ...... 116

Figure 3-14. – FACS data for competitive binding for compounds 12b and 10b...... 117

Figure 3-15. –Accumulation of 12b in MCF-7 cells – Fluoresent,

Hoechst stain and overlay ...... 124

List of Schemes and Tables

Scheme 2-1. Synthesis of functionalized steroid derivatives 6 and....……….….81

Scheme 2-2. Synthesis of fluorescent conjugating groups 8 and 9.....……....….82

Scheme 2-3. Synthesis of fluorescently labeled steroidal antiestrogen

and steroidal control..………………………..……….…………..83

Scheme 2-4. Synthesis of functionalized steroid derivative 8R...... ….………..84

Scheme 2-5. Synthesis of functionalized steroid derivative 8r

via an alternative pathway…………………………………..……85

Scheme 2-6. General method of synthesis of Mitsunobu reaction.……..………88

Table 2-1. Excitation and Emission maxima for fluorescently labeled

Steroids…………………………………………………………86

List of Abbreviations

ANOVA Analysis of variance

Arg Arginine

BODIPY Boron-dipyrromethene

13C NMR Carbon 13 nuclear magnetic resonance

Cbz Carboxybenzy

CF3COCF3 Hexafluoroacetone

CH2Cl2 Dichloromethane

CH3CN Acetonitrile

CH3COBr Acetyl bromide

CH3COOH Acetic acid

CH3OH Methanol

CO2 Carbon dioxide

Cs2CO3 Cesium carbonate

Cu Copper

CuAAC Copper catalyzed alkyne-azide click reaction

Cy3 Cyanine dye

Dansyl 8-dimethylaminonaphthylsulfonyl 14

DBD DNA-binding domain

DCC N,N'-Dicyclohexylcarbodiimide

DCM Dichloromethane

DEAD Diethyl azodicarboxylate

DMEM Dulbecco's modified Eagle's medium

DMF Dimethyl formamide

DNA Deoxyribonucleic acid

E2 Estradiol / 17 estradiol

EE2 7-α-Ethynylestradiol

ER Estrogen receptor

FACS Fluorescence activated cell sorting

FITC Fluorescein isothiocyanate

Glu Glutamic acid

H1 NMR Proton nuclear magnetic resonance

H2O Water

H2O2 Hydrogen peroxide

HEK 293 Human embryonic kidney 293

His Histidine

IC50 Half maximal inhibitory concentration

IHC immunohistochemistry

IR Iridium

Kd Dissociation constant

KOH Potassium hydroxide

L/H-BD Ligand/hormone binding domain

LC-MS Liquid chromatography- mass spectrometry

LMW Low molecular weight

MCF-7 Michigan cancer foundation -7

Mg Magnesium

MLCT Metal to ligand charge transfer

MRI Magnetic resonance imaging

NaBH4 Sodium borohydride

NaN3 Sodium azide

NBD Nitrobenzodiazoxole

NH4Cl Ammonium chloride

NHS N-hydroxysuccinamide

NIR Near infra-red

NIR-vis-UV Near infra-red- visible- ultra violet

OH Hydroxy

OHT Hydroxytamoxifen

PBI Perylenebisimide

PBS Phosphate buffered saline

Pd Palladium

PET Positron emission tomography

PFA Paraformaldehyde

PgR Progesterone receptor

Ps-PPh3 Polymer supported triphenyphosphine

RBA Relative binding affinities

Re Rhenium

RT Room temperature

SAR Structure activity relationship

SERD Selective estrogen receptor down-regulator

SERM Selective estrogen receptor modulators

SPECT Single-photon emission computed tomography

TAMRA Carboxytetramethylrhodamin

17 t-Boc tert-butyloxycarbonyl t-BuOH tertiary butanol

TEA Triethylamine

THF Tetrahydrofuran

TsOCH2CH2OTs Ethylene glycol di tosylate

Chapter 1 – A Review of Fluorescently Labeled Steroids

1.1.1 Introduction

Largely adapted from the review article that will be published in the journal of steroids, the first chapter of the thesis examines the historical precedence of fluorescently labelled steroids. This chapter summarizes studies that have been done to develop and evaluate fluorescently labeled steroidal estrogens since 1995, the date of the last major review1. This chapter will cover the synthetic approaches used to prepare those agents and their potential as molecular imaging agents in detection and characterization of estrogen receptors in a cellular setting.

The rationale for developing fluorescent and other imaging agents for the estrogen receptor lies in its close association with hormone responsive diseases, particularly breast cancer2. According

to the American Cancer Society, over 230,000 cases of breast cancer were diagnosed in 2015,

and over 40,000 patients died from the disease3. It is the most common cancer among women,

accounting for 29% of all diagnosed cancers. Due to the high dependency of estrogen and

progesterone, agents that target the estrogen receptor (ER) play a major role in breast cancer therapy. Such strategies include the use of selective estrogen receptor modulators (SERMs), such

as tamoxifen, and selective estrogen receptor down-regulators (SERDs), such as fulvestrant4-6 additionally, aromatase inhibitors that suppress estrogen biosynthesis plays a significant role in therapy7. Diagnosis and treatment success are assessed by tumor estrogen receptor status, which

employs immunohistochemistry (IHC) among other modalities. Although IHC is suitable for assaying the ER expression in biopsy tissues obtained from primary breast tumors, its accuracy becomes lower in metastases8. As a diagnostic tool, the usage of radiolabeled ER-directed tracers

offer the potential for non-invasive, clinical imaging of both primary and metastatic tumors,

however, my review of this area of research indicates the need for additional improvements

before it can be achieved2,10,11. At the cellular and molecular level, high spatial resolution is

possible using fluorescence imaging methods, if high estrogen receptor (ER) affinity and selectivity is incorporated. The last major review on this topic, with material through 19951, has

provided the theoretical and practical bases for this area of research. At that time, an imaging

agent that met all of the criteria had not been prepared and evaluated. This chapter, as a background for our research, focuses on fluorescently labeled steroidal estrogens prepared since

1995, with attention to synthetic approaches, evaluation as potential molecular imaging agents,

and a brief discussion of advantages and disadvantages. Finally, we will describe our current

approach to develop the “next-generation’ fluorescent steroidal ER imaging agent.

1.1.1 The target- Estrogen receptor

The estrogen receptor is one member of family of structurally homologous nuclear transcription factors12,13. The receptors consist of 12 helices and 2 beta sheets that form 6 subdomains. While the terminal domains are primarily involved with co-factor recruitment, the interior domains are more highly conserved, providing the ligand (hormone) binding domain

(L/H-BD) and the DNA-binding domain (DBD). Within the steroid hormone receptor family, the estrogen receptors consist of two major sub-types- ERα and ERβ- which mediate different physiological functions, with ERα being the more closely associated with endocrine activity and endocrine disorders, such as breast cancer12-14. The amino acid sequence of the ERs is now well established and expression of the proteins led initially to x-ray crystal structures of the liganded hormone binding domain15-17. X-ray crystal structures, using steroidal and nonsteroidal agonists

21 and antagonists as ligands, have provided the rationale for interpreting the structure activity relationships (SARs) that were generated in previous decades. Such information about the interactions between the small molecules and the cognate receptors is critical for the design, synthesis and evaluation of potential molecular imaging agents.

1.1.2 Estradiol- The native ligand

Although ligands for the estrogen receptor may be either steroidal or nonsteroidal, we considered retaining the default steroidal structure to be particularly crucial. For therapeutic agents, nonsteroidal agents are acceptable as long as the off-target interactions are minimal. For imaging purposes, any non-target interaction is undesirable and therefore maintenance of the steroidal core was a major concern. Information regarding interactions between the steroid and the receptor has largely been derived from two primary sources, structure activity relationships

(competitive binding and functional assays) 18-22 and crystallographic studies with the liganded hormone binding domain15-17, 23-25. Both are necessary as they provide complementary views of the interactions. Being the native ligand for the estrogen receptor, a compound with an estradiol

(E2) core is considered an ideal vector for imaging agents due to its ability of localizing in tissues that express ER. All the imaging agents that are under discussion in the review are molecules that have an E2 core with a fluorescent group appended by a short linker.

1.1.2.1 Structure-Activity Relationships

The essential structure activity relationships prior to the initial x-ray crystal structure of estradiol-ER-LBD were reviewed by Ojasoo and Anstead19, 20. (Figure 1-1) The 3-and 17β-

22 hydroxy groups were essential to binding as any substitution at those positions was detrimental to binding. Removal of the hydroxyl group (3/17β-deoxy) or replacement by amino-isostere was also detrimental. Halogenation on the carbon adjacent to the 3/17훽-hydroxyls reduced binding

>C5 or aryl groups to 4-alkoxyphenyl retained binding affinity but converted the compounds to

ER antagonists, suggesting changes in conformation. Virtually all other positions on the estradiol scaffold are synthetically inaccessible, except by total synthesis, and therefore have not been extensively examined.

Figure 1-1. Basic structure activity relationships for substituted estradiol derivatives.

1.1.2.2 Evaluation of ligand-ER-LBD complexes

The understanding of interactions between the ER and ligands has been largely derived from examination of complexes between agonists and antagonists with the estrogen receptor ligand binding domain (ER-LBD). As this statement suggests, the proteins used in these studies were not the intact receptor and in most cases, did not include complementary co-regulatory proteins/peptides often present in physiological processes15-17, 23-25. Nevertheless, these complexes provide a basis for understanding the earlier SAR studies (binding and efficacy). The key feature present in x-ray crystal structures of steroidal estrogen complexes is the critical hydrogen bonding interaction between the 3-OH and Glu- and Arg and a molecule of water within the binding site. This is present in essentially all the complexes. The 17β-OH also forms a hydrogen bond (although not as strong) with the N-H of His-524 in helix-12. The rest of the steroid scaffold is enclosed within a hydrophobic envelope which tolerates few substituents. This observation rationalizes the low steric tolerance within the steroid skeleton as well as those sites adjacent to the key hydrogen bonding interactions. Non-steroidal estrogens also fit within this binding cavity and generally are subject to the same constraints19. Non-steroidal triarylethylene antagonists (and their structural analogs) which contain the dialkylaminoethoxy group generate a different conformation of the receptor17. In this conformation helix-12, which normally encloses the ligand and exposes residues that interact with co-activator proteins, rotates and prevents co- activator protein binding. It is this conformation that is generated by the 11β-/7α-substituted estradiol derivatives expressing antagonist activity. The X-ray crystal structures for the 7α- substituted antagonist ICI 164384 demonstrates that the steroidal scaffold has rotated around the

C3-C17 axis to project the substituent into the 11β-pocket with the terminal component extending into the solvent exposed region of the complex24. Another complex of interest was reported in which estradiol was substituted at the 17α-position with a bulky ethynyl arene group terminally substituted with a metal chelate25. In this complex, the metal chelating moiety extends beyond the surface of the ER-LBD and distorts the helices. As a result, it appears that those two regions of the receptor accommodate greater substituent diversity and steric flexibility than other component of the ligand binding domain. As the subsequent sections of the review will describe, most of the derivatives target those two regions and the receptor-related properties (affinity, efficacy) can be rationalized based on those crystal structures.

In addition to the site for incorporation or conjugation onto the steroidal scaffold, other criteria associated with the properties of the fluorescent moiety itself must be considered. Among the factors specifically related to the fluorophore are excitation-emission characteristics, intensity, quantum yield, stability/quenching, and physical-chemical properties. Aspects that are involved with conjugation include the functional groups present on the steroidal ligand and those on the fluorophore, and which therefore influence the ligation strategy.

1.2 Fluorescent properties

The ultimate biological application influences the choice of fluorophore, as in vitro imaging of cells or tissues, can tolerate a wide range of fluorescence properties26-28. In vivo imaging, in which the excitation radiation must penetrate up to several cm of tissue, requires near IR (NIR) fluorophores29,30. (Figure 1-2) Among the common NIR dyes are the cyanine-based compounds whose excitation-emission properties span a broad band of the near infra-red- visible- ultra violet

(NIR-vis-UV) spectrum29-33. Non-IR fluorophores commonly used for imaging include nitrobenzodiazoxole (NBD) 34, 35, 8-dimethylaminonaphthylsulfonyl (dansyl)34, 36-38 boron-

25 dipyrromethene (BODIPY)39-43, fluorescein44-46, rhodamine derivatives47, 48, Alexa dyes49,50, and perylene bisimide dyes51,52, among others. There are an ample number of reviews that have described syntheses, fluorescent properties, and stability in solution of the various methods.

Although the cyanine dyes have highly desirable fluorescent properties, their physicochemical properties tend to compromise receptor binding properties of low molecular weight (LMW) ligands. Dansyl, BODIPY and NBD tend to be more tolerated by LMW ligands due to their small size and neutrality, however, the fluorescence properties tend to be suboptimal.

Fluorescein and rhodamine derivatives tend to be a reasonable compromise for imaging and size, however, they are both inherently charged molecules which tends to compromise binding properties. As a result, the incorporation of any of these fluorophores needs to be considered carefully when designing a conjugate that targets an intracellular receptor, such as ER.

Figure 1-2. Representative examples of parent fluorescent dyes described in the review.

1.3 Conjugation properties

The ligation chemistries depend upon the functional groups present on the steroid and fluorophore moieties53, 54. Because the 3- hydroxyl group of estradiol is required for binding, no conjugations are permitted at that site. While the 17β-hydroxyl is not as intimately hydrogen bonded to histidine-524 (His-524), ether, ester or carbamate derivatives are much weaker, suggesting that fluorophore conjugation at that position is also detrimental to binding. As a result, new functional groups capable of undergoing conjugation must be introduced at positions that do not seriously compromise binding properties. As the previous section indicated, these would typically be at the B-ring (positions 6/7) or the D-ring (16/17α). Typical functional groups would include carbonyl (ketone) or carboxylic acids at the B-ring or extended

(substituted aryl) ethynyl groups at the D-ring. Substitution in this case could be amino or carboxylic acids. As the results will show, there are limitations to the size of the groups, but these functional groups would accommodate the functional groups present on the fluorophore.

Most of the studies employ commercially available fluorophores in which a functional group, such as an amine or carboxylic acid, capable of facile conjugation is present. Direct conjugation between the two components followed by chromatographic separation yields the desired product.

Other variations include carbamate/urea/thiourea formation between alcohol/amine groups and iso(thio)cyanate derivatives. More recent use of “click” reactions has also been described 55-58. If the two components are not directly compatible, a bifunctional linker can be used to bridge them by undergoing conjugation with one component first followed by the second. The choice is usually dictated by availability, cost and ease of separation.

Ultimately, the steroid-fluorophore conjugate needs to retain ER-binding affinity, selectivity, stability and appropriate fluorescence properties. In the following sections, we will examine the studies over the past 20 years in which investigators have attempted to prepare reagents capable of delineating ER residence. We will review the design and synthetic strategies and evaluate the success of their approaches. Ultimately we will suggest alternate approaches to addressing the deficiencies observed in the studies to date.

1.3.1 Survey of fluorescent probes - representative examples before 1995.

The studies described in prior reviews were predicated on the need to image ER both in vitro and in vivo. Because of the interest in developing new breast cancer diagnostic techniques, significant research efforts focused on fluorescently labeled estrogens, starting from late 1970s.

Many of these compounds were derivatives of estradiol to which activated fluorescein derivatives or other fluorophore, for example, were conjugated59-61. Typical sites for conjugation were at C6, C7 or C17 positions, because as these positions were synthetically accessible. As can now be appreciated via X-ray crystal structures, the relative binding affinities (RBA) compared to the estradiol (RBA = 100%) were typically very low (<5%). Examples of molecular probes as described in the reviews are shown in (Figure 1-3).

Figure 1-3 Examples of estradiol-fluorophore conjugates prior to 1995

1.3.2 Fluorescent probes post-1995

Most of the studies published since 1995 have focused on two regions of the steroid scaffold, namely the B- and D-rings. As previously noted, X-ray crystal structures of the ligand-ER-LBD complexes had suggested steric tolerance in those regions. Major variations involved the introduction of different functional groups for conjugation to support the newer families of fluorophores.

1.4 B-ring derivatives

Introduction of fluorophores at the B-ring has been rationalized based on synthetic accessibility and retention of receptor binding affinity. Studies led by Adamczyck, prepared a series of fluorescent derivatives possessing sp3 hybridization at the C-6 position62-66, (figure 1-4)

Reduction of the 6-oximinoestradiol gave a mixture of 6α – and 6β-amino estradiols which were separated. Both isomers were converted to their fluorescent derivatives by conjugation to the appropriate fluoresceinyl or acridinium reagent. The 6α-amino isomer was selected for initial conjugation based on its higher yield and also because the substituent was on the less substituted

(alpha) face of the steroid scaffold. The amino estradiol was conjugated with N- hydroxysuccinamide (NHS) esters of fluorescent dyes. Again, prior conjugation with an amino- terminated linker provided the extended version of the estradiol-fluorescent probe. In a further development of probes, this group prepared and evaluated 6β-carbon-based substituents on the

B-ring, presumably to minimize the effect that electron withdrawing groups may have on the phenolic A-ring. Two conjugative approaches were explored. No data were reported for either fluorescent or receptor/antibody binding properties.

In a separate study the fluorophore was modified to incorporate the hydroxylamine moiety which would undergo the condensation with 6-oxoestradiol (as well as other steroidal mono- ketones). 64-66 the syntheses of the 6- of (5-/6-fluoresceinylmethyl)-oximes of estradiol proceeded in 49-56% yields, no biological properties of these derivatives were reported. Conjugation of the carboxymethyloxime with mono tert-butyloxycarbonyl (t-Boc) ethylenedimine followed by amidation of the NHS ester of the luminescent acridinium dye gave a new probe. The resultant

30 product was evaluated as a ligand for an anti-estradiol antibody using surface plasmon resonance.

Figure 1-4 Transformations of estrone to fluorescent derivatives

In studies that further demonstrated the absence of ERα binding capacity, Cristophe and

Meyer-Almes prepared and evaluated a series of 6-carboxymethyloximino estradiol derivatives.

The carboxylic acid was conjugated to the fluorescent dyes by a variety of linkers67. (Figure 1-5)

None of the conjugates demonstrated appreciable affinity for ERα (RBA<1%), however, they retained significant affinity for the anti-ER antibody. Reported dissociation constants (Kd) were in the subnanomolar range, comparable to those values previously observed.

Figure 1-5 6-Carboxymethyloximino derivatives with fluorophores conjugated through oligoethylene glycol linkers

Use of the Reformatsky reaction on the 6-oxoestradiol gave in four steps the 6β-carboxymethyl estradiol, which could be further elaborated with t-Boc ethylene diamine68. Reaction with activated fluorescent dyes gave the desired probes. (Figure 1-4) Alternatively, C- alkylation of protected 6-oxoestradiol with 5-bromo-1-pentene, followed by ozonolysis, oxidation of the aldehyde, esterification, reduction of the 6-oxo group and ultimate xanthate deoxygenation gave the 7α-carboxypropyl estradiol69. N,N'-Dicyclohexylcarbodiimide (DCC) coupling of the carboxylic acid with the ethylene diamine derivative of the luminescent dye gave the corresponding probe. All of these compounds were evaluated using anti-estradiol antibodies generated against the 6-carboxymethyloximino estradiol antigen. While these probes demonstrated significant affinity for the antibody, with equilibrium dissociation constants in the low nanomolar to subnanomolar range, their affinity for the estrogen receptor or its ligand binding domain was not reported. Based upon established SAR, they would presumed to have low RBA values (<5%).

In a separate study designed to develop ER targeted fluorescent imaging agents, Okamoto, et al., prepared a 7α-alkenyl boron-dipyrromethane estradiol derivative using Grubbs olefin cross metathesis chemistry70. As opposed to the 6β/7α-derivatives reported by Adamczyck, et al., 68,69 their product demonstrated high ER binding affinity (RBA = 76%) and significant in vivo uterotrophic activity. Furthermore, co-localization of the fluorescent derivative with Hoechst

33342 in uterine cells was also observed. These findings suggest that the nature of the fluorophore and site of its incorporation into the overall steroidal structure strongly influences the binding properties and pharmacological responses.

Nevertheless, the overall impression from these studies remains that, while these fluorescent derivatives of estradiol are readily accessible from both the steroidal and fluorescent reagents, most of the resultant products have low binding affinity for the estrogen receptor. Of the two positions, the 7α-position has potential advantages in terms of receptor binding due to its use with the fulvestrant derivatives, however, incorporation of the sterically demanding and highly polar fluorophore appears to compromise interactions at the receptor level24.

Figure 1-6 Synthesis of 6β-and 7α-fluorophore substituted estradiols

1.5 D-Ring Derivatives -17α-Substitution

Although some interest has continued with the 17β-derivatives which have marginal ER- properties,71 the position on the estradiol scaffold that received the most attention was the 17α- site. These studies were largely premised on the observation that 17α-ethynyl estradiol and its congeners retained high ER binding affinity and pharmacological (estrogenic) activity19, 20.

Because of the alkynyl group, this position become synthetically accessible to a variety of functional groups, and therefore has been widely exploited for potential therapeutic applications.

It has also provided the basis for developing imaging agents, e.g., SPECT, PET and MRI72-78. In this review, the focus will only cover the studies related to the fluorescent probes, some of which are structurally similar to the radiolabeled derivatives.

A summary of the initial efforts is shown in (Figure 1-7). The seminal work in this area was performed by the Adamcyzk group which had previously prepare several B-ring derivatives79.

Addition of dilithio-5-hexyn-1-ol to protected estrone, followed by mesylation, conversion to the terminal azide and reduction gave the amino-hexynyl estradiol. Conjugation with NHS esters of

5-carboxyfluorescein and carboxypropyl acridinium dyes gave the fluorescent probes. The compounds displayed modest affinity for an anti-estradiol-carboxymethyloxime antibody (1/40th of estradiol) but no data for the ER were provided. A similar approach was employed by Ray in an effort to develop estrogen conjugates for photodynamic therapy. The initial synthesis generated a 17α-alkynyl estradiol conjugated to chlorin e6 which possessed 0.1% the binding affinity of estradiol for the ER80. In the second approach, reaction of the THP-protected 3-butyn-

1-ol with protected estrone gave, after deprotection, the terminal alcohol. Esterification with the carboxyporphyrin dye gave the estradiol-pyropheophorbide conjugate that was evaluated in

MCF-7 cells, a breast cancer cell line that over-expresses ER. Incubation of the conjugate at

10μM provided internalization whereas the dye itself was excluded from the cells. Binding data for the conjugate, however, was not provided81. Based upon earlier work82 ethynyl estradiol was terminally carboxylated using methyl lithium and dry ice (CO2). The resultant carboxylic acid was converted to a series of carboxybenzyl (Cbz) protected amino-amides using typical coupling chemistry. Deprotection followed by conjugation with the NHS ester of 5(6)-carboxyfluorescein gave the fluorescent probe.

Figure 1-7 Preparation of 17α-alkynyl estradiol-dye conjugates 79-84

Although the resultant probes demonstrated competitive binding to the estrogen receptor, the

IC50 values across the series were in the 190-12000 nM range. These are much lower affinities

36 than estradiol which is typically in low nMf (1-3nM) range83. These results were consistent with

SAR established in the 1970’s84.

The majority of the D-ring substituted estradiol derivatives were prepared using variations of the 17α-arylethynyl group. These compounds are readily accessible via Sonogashira chemistry and permit introduction of a wide variety of fluorophores, including organic dyes, metal chelates, and fluorescent nanoparticles. The most direct method for preparing these conjugates involve the synthesis of the 4-aminomethylphenyl derivative of 17α-ethynyl estradiol. Once prepared, it could simply be conjugated using the activated ester of the appropriate organic dye85-90.(Figure

1-8) All of the derivatives demonstrated good fluorescent properties and were evaluated in assays that evaluated ER binding properties. The estradiol-fluorescein isothiocyanate (FITC) conjugate was reported to have a RBA value of 14% compared to estradiol, in a study that compared it to hydroxytamoxifen (OHT) and raloxifene conjugates91. The estradiol- carboxyfluorescein and Alexa Fluor conjugates possessed RBA values in the 15-25% range and were used to evaluate rapid estrogen signaling mechanisms in vitro88-90. The estradiol- perylenebisimide (PBI) conjugate was characterized for its fluorescence properties (emission at

592 nm) and quantum yield (0.36 in water), however, its ER-targeting properties were determined only by its localization in MCF-7 vs HEK 293 cell lines. While higher uptake was observed with the ER-positive MCF-7 cells compared to the ER-negative cells, no specific values were provided for ER relative binding affinity.

Figure 1-8 Fluorescent ER-targeted agents derived from the 4-(amino methyl)phenyl ethynyl estradiol intermediate.

A second approach based upon the Sonogashira coupling reaction used a terminal pyridine group as an anchor for metal-based fluorophores. Lo, et al, looked at proximal and distal rhenium and iridium complexes83-86. In their initial series a pyridine ligand linked to 17α-ethynyl estradiol provided the last coordination site on the Rhenium (I) tricarbonyl phenanthroline complex where the phenanthroline was unsubstituted or had methyl/phenyl substitution. (Figure 1-9) The ligand was prepared by a Sonogashira coupling of ethynyl estradiol and 4-bromopyridine for the proximal ligand and 4-(N-(6-(4-iodobenzoylamino) hexanoyl)aminomethyl)-pyridine of the distal ligand. The products, isolated in 35-45% yields, demonstrated long-lived green to orange- yellow luminescence. The unsubstituted proximal complex had a LogP comparable to estradiol

(3.07 vs 3.20) however, the distal complex and the substitute phenanthroline complexes had 38

LogP values of 3.9-6.5, indicating significantly more lipophilicity. The binding affinity of the complexes was characterized as similar to that of other estradiol derivatives substituted at the

17α-position with bulky substituents, indicating low binding affinity.

Figure 1-9 Preparation of Re (I) estradiol complexes with a terminal pyridine group The second series employed a carboxy substituted cyclometalated iridium (III)-polypyridine complex generated by coordination of the preformed4-(N-(6-(4-17α-

39 ethynylestradiolyl)benzoylamino)hexyl)aminocarbonyl)-4’-methyl-2,2’-bipyridine94.(Figure 1-

10)

Figure 1-10 Preparation of Ir (III) estradiol polypyridine complexes

A variant in which the Ir(III) was complexed as part of a linear phenyl-bipyridyl array was also evaluated. Both variations were used to prepare the corresponding Ruthenium(II) complexes95.

(Figure 1-11)The emission intensities of the Ir (III) complexes were 5-10-fold greater than the

Re(I) complexes. Emission was observed in the presence of ERα whereas it was quenched in the absence, suggesting the potential of these complexes for imaging the receptor in vitro. No binding data for the receptor were explicitly provided.

Figure 1-11 Preparation of Ruthenium (II) estradiol polypyridine complexes

A variation of this approach was used by Hannon, et al., in preparing a series of terpyridineplatinum (II) derivatives of ethynyl estradiol96,97. (Figure 1-12) It was based upon the ability of terpyridineplatinum (II) chloride to react with terminal alkynes forming stable complexes which fluorescence via metal to ligand charge transfer (MLCT).

Figure 1-12. Preparation of Pt(II) terpyridine ethynyl estradiol complexes.

The initial synthesis involved the preparation on the terpyridine ethynyl estradiol followed by complexation with bis(benzonitrile)platinium (II) chloride. Reaction of the resulting complex with phenylacetylene in the presence of Cu(I) iodide gave the first complex. Alternatively, preparation of terpyridineplatinum (II) chloride followed by reaction with ethynyl estradiol in the presence of Cu(I) iodide gave the inverse complex. ER binding affinities for the terpyridine ethynyl estradiol intermediate and the platinum (II) chloride complex were <2% and <0.3% respectively, suggesting a low RBA for the final phenylacetylene complex and its inverse analog.

Fluorescent properties of the complexes were similar to terpyridineplatinum (II) complexes lacking the ethynyl estradiol group. However, it was noted that fluorescence generated by

42 excitation of the MLCT area was observed only in aprotic solvents and not in protic solvents, presumable due to O-H vibrational quenching.

A third approach, developed by Arterburn, et al, and also based upon the Sonogashira coupling reaction, used the fluorescent properties of (pyridine-2-yl) hydrazone rhenium (I) tricarbonyl complexes98-102 (Figure 1-13). In this approach, preparation of the appropriate (5-halo-pyridin-2- yl) hydrazonyl/hydrazinyl Re(I) tricarbonyl complex, followed by coupling to ethynyl estradiol provided the requisite ER-targeted product.

Figure 1-13. Preparation of 17α-hydrazonopyridyl ethynyl estradiol- metallated conjugates.

In one instance, excitation at 420 nm provided emission at approximately 550 nm. The authors noted that the excitation occurred at longer wavelength than that observed for the Ir(III) complexes (370 nm)84. Receptor binding data were not provided for the hydrazone complex

43 suggesting that like other highly substitute (sterically demanding) 17α-ethynylaryl estradiols, the binding affinity was low. In their prior studies with Re(I) tricarbonyl complexes of pyridine-2-y hydrazines, high RBA values (RBA – 38%) were observed. However, those complexes were not fluorescent.

More recently, van Lier, et al. used the Sonogashira coupling reaction to append an aryl derivative of the BODIPY fluorophore103 (Figure 1-14, 1-15). This study utilized ethynyl estradiol as well as A-ring (2-/4-F) and C-ring (11β-OCH3) derivatives as coupling partners. All of the products demonstrated excellent absorption-emission properties, consistent with the

BODIPY moiety.

Figure 1-14 Preparation of 17α-BODIPY-ethynyl estradiol conjugates using Sonogashira reaction

However, introduction of the fluorophore at the 17α-position essentially eliminated ER binding affinity. Extending the fluorophore to a more distal positon by introducing an alkyl chain between the ethynyl group and the fluorophore had a minimal effect on fluorescence but in this instance significant receptor binding affinity (RBA = 12-16%) was retained. This approach was also extended to substituted estradiol derivatives as well as more functionalized BODIPY derivatives104 (Figure1-15).

Figure 1-15 Preparation of 7α/11β-substituted 17α-BODIPY-ethynyl estradiol conjugates using Sonogashira reaction.

The fourth approach to developing fluorescent derivatives of estradiol via the 17α-ethynyl group involved the use of Sharpless’ [3+2]-cycloaddition “click” chemistry105,106. (Figure 1-16,

1-17) In a study designed to steroid receptors in target cells, Chen, et al, coupled an azido derivative of the cyanine dye (Cy3) to ethynyl estradiol using the copper catalyzed alkyne-azide click reaction (CuAAC) 107. Estrogen receptor selectivity was evaluated by the uptake of the conjugate in MCF-7 (ER-positive) cell versus MDA-MB-231 (ER-negative) cells. Preferential uptake by the MCF-7 cells was observed, but no binding data were provided. Van Lier also used the CuAAC reaction to generate two conjugates with the phenyl-BODIPY fluorophore, one proximal to the steroid nucleus and one more distal. While fluorescent properties of the fluorophore were retained, virtually all of the ER binding was lost103, 104.

Figure 1-16 Preparation of 17α-substituted estradiols conjugates using copper assisted azide alkyne cyclization (CuAAC) reaction

Figure 1-17 Preparation of 11β-methoxy-17α-substituted estradiols conjugates using copper assisted azide alkyne cyclization (CuAAC) reaction

The results from these studies suggest that fluorescent groups can readily be appended to the

17α-position of the steroid nucleus using a variety of conjugation reactions. While almost all of these conjugates retain the fluorescent properties of the fluorophore, receptor binding properties, where measured, were largely lost. Three key factors must be considered in evaluating this class of conjugates as the parent ethynyl estradiol has essentially the same receptor binding affinity as estradiol. As demonstrated by the crystal structure for the 17α-substituted estradiol derivative with a metal chelating group25, bulky substituents, such as the fluorophores used in these studies are poorly tolerated and result in significant distortions of the ligand-receptor complex, leading to reduced binding affinity. Secondly, many of the fluorophores either have a net charge, are zwitterionic, or are ionized under binding conditions, which may also reduce binding affinity to the estrogen receptor. Third, the pendant fluorophores are large, polycyclic structures with

48 dimensions and masses greater than the parent estradiol moiety, which may impart binding and cell localization characteristics independent of the estradiol group. For example, the terpyridineplatinum (II) complexes possess significant DNA intercalating activity96, 97. As a result, it is not surprising the binding assays and cell localization studies do not demonstrate high levels of ER activity or ER-targeting for these conjugates.

1.6 C-Ring substitution using 11β-subsitution

Virtually all of the fluorescent derivatives of estradiol have used substituents on the A-. B-, and

D-rings to provide access to conjugation. That is almost exclusively due to the relative ease with which those substituted estradiols can be prepared. 6-Oxo-estradiol, readily accessible by benzylic oxidation, generates both 6- and 7-substituted estradiols. Ethynylation (alkynylation) of estrone provides intermediates for both Pd(0) coupling reactions (Sonogashira) and “click” reactions. SAR studies, however, indicated that affinity and activity decline precipitously with increased substituent size, which diminishes the attractiveness of these approaches for developing fluorescent estradiols. The 11β-position on estradiol, however, is a site that has been shown to tolerate a wide variety of functional groups while retaining both ER-affinity and biological activity19,20. Small alkyl, alkoxy and unsubstituted aryl groups provided high affinity agonists while longer alkyl groups or substituted 4-alkoxyphenyl groups are high affinity antagonists108-110. One significant challenge for exploiting these properties has been that the requisite intermediates are not readily accessible by simple synthetic methods.

The typical method for preparing such derivatives is a multi-step process beginning with estradiol/estrone 3-O-methyl ether (Figure 1-18). Birch reduction, bromination-

49 didehydrobromination provides the di-ene. Ketalization deconjugates the system to generate the

5(10), 9(11)-diene which undergoes selective α-epoxidation at the 5(10)-double bond. The key step in the process is the Cu(I)-assisted 1,4-addition of a Grignard reagent at the 11β-position, followed by hydrolysis of the ketal and elimination of water to yield the 11β-substituted estra-

4,9-dien-3-one. Aromatization is achieved using acetic anhydride-acetyl bromide, and reduction followed by saponification gives the final 11β-substituted estradiol109-111. Although the sequence requires 9-10 steps, most of the individual reactions proceed in good yields. The process also tolerates a wide variety of alkyl, alkenyl and aryl groups at the 11β-position of estradiol.

Figure 1-18 General synthetic approach to the preparation of 11β-substituted estradiols 110-112

Due to the more extensive synthetic transformations involved, only a few fluorescent 11β- substituted steroids have been reported. The first example was described by Ray using the 3,17β- dibenzyl ether of 11β-(2-hydroxyethyl)estradiol as the starting material113. The steroid was conjugated with a carboxylic acid derivative of porphyrin to give the corresponding ester which

50 was then de-benzylated using catalytic hydrogenation. The product exhibited good fluorescence properties (λ Excitation = 413 nm, λ Emission = 650 nm), although quantum yields were slightly lower

(25%) compared to the parent porphyrin. This suggested that introduction of the steroidal component onto the fluorophore was well tolerated. Binding studies, however, indicated that relative binding affinity compared to estradiol was approximately 0.1%. Given that the parent steroid had an affinity greater than estradiol, the effect of attaching the sterically demanding porphyrin moiety, even at the terminus of a linear tether, was detrimental to receptor binding.

Functional assays were not reported and therefore it was not known whether the derivative retained agonist properties similar to the parent steroid, or antagonist properties similar to long chain 11β-substituted estradiols.

A solution to this issue was presented in the development of fluorescent derivatives of the steroidal antiprogestin mefipristone RU-486114-116. Synthesis of the derivatives was simplified by using RU-486 as the starting material. The initial study generated the N-NBD derivative which has suboptimal fluorescence properties when incubated with the receptor114. Subsequently, N- demethylation gave the nor-derivative which was initial alkylated with 6-bromohexan-1-ol, followed by conjugation with FITC. The product retained the fluorescence characteristics of the fluorophore, however, receptor binding properties were significantly poorer compared to RU-486

(relative potency 3-4%). This loss in receptor binding could be attributed either to charge properties of the fluorophore (anionic) or steric hindrance as the hexanol intermediate was essentially equipotent with RU-486115. In the subsequent study, the N-demethylated intermediate was alkylated instead with 6-bromohexanoic acid and the carboxylic acid was conjugated with

N-t-Boc-3,6-dioxaoctan-1,8-diamine. Deprotection of the terminal amine followed by conjugation with the appropriate rhodamine (TAMRA) or BODIPY dyes gave the final products.

Both derivatives retained the fluorescence properties of the parent dyes and the Progesterone receptor (PgR).PgR binding properties of the parent steroid116. Molecular modeling studies indicated that the derivatives expressed similar binding modes within the binding pocket and that the tether-fluorophore component exited the binding pocket with the fluorophore distinctly outside the protein shell (solvent exposed). Subsequent studies demonstrated that the derivatives were selective for PgR expressing cells and that the localization was receptor mediated.

Therefore, the results supported the application of this approach for developing similar estrogen receptor directed fluorescent derivatives.

Further support for this approach was provided by studies in which nonsteroidal antiestrogens were substituted at the amino group. It has been well established that 11β-(4- aminoalkoxyphenyl) substituents on estradiol occupy the same binding site as the aminoalkoxyphenyl group of the nonsteroidal antagonists tamoxifen and raloxifene19, 20. Crystal structures indicate that the amino group is at the protein-solvent interface and that linker groups would project into the solvent shell19, 20. Although fluorescent derivatives of tamoxifen have been reported, the low affinity of the nonsteroidal scaffold led to poor receptor localizing properties116, 117. This aspect has been addressed using N-fluorophore substituted of OHT

(hydroxytamoxifen) and raloxifene89,91. In an effort to track estrogen receptor localization in cells, Weatherman, et al, prepared three derivatives of HOT based on the N-(6-aminohexyl) hydroxytamoxifen (OHT-6C)89.Conjugation using the NHS ester of the fluorophore

(carboxyfluorescein, BODIPY-FL, and Alexa Fluor 546) gave the final products. The conjugates retained their fluorescent properties, essentially identical to the parent fluorophore, although the quantum yields were somewhat reduced. Receptor binding properties for the fluorescent derivatives were also reduced compared to both estradiol and the non-conjugated OHT-6C

52 intermediate. Relative affinities ranged from approximately 10% for the fluorescein derivative to

<2% for the Alexa Fluor 546 derivative. Whether the loss of binding affinity was due to electronic or steric factors was not determined at the time.

A more recent study by Seitz, et al, evaluated two series of fluorophore-tagged non-steroidal antiestrogens91. Based upon previous work, they reduced azidoethoxy derivatives of tamoxifen and raloxifene to the corresponding aminoethoxy intermediates. Conjugation with the NHS esters of cyanine dyes of the MiDye series gave two parallel series of fluorescent probes. As seen in other studies, conjugation of the dye to the ER ligand had essentially no effect on the fluorescent properties of the dye. In contrast to previous studies, the resultant conjugates retained high affinity for ER, with relative binding affinities (RBA) in the 21-33% range for the hydroxytamoxifen agents and 68-122% range for the raloxifene derivatives. These were the highest values observed at the time. Cell localization studies were performed using ER+ and ER- cell lines with inconclusive results, suggesting that further studies were necessary.

Our approach to develop ER targeted fluorescent probes combined elements used by both

Tsien and Seitz. Our earlier studies provided the expertise to efficiently access steroidal derivatives with a wide variety of 11β-substituents, including the (4-azidoethoxyphenyl) group.

These compounds possessed good ER binding affinity (RBA = 20-40%) and were potent antagonists118. We also had prepared variations on the side chains and conjugated them to potent chemotherapeutic agents, such as mitomycin C and geldanamycin, however, in vitro receptor selectivity (greater potency at ER+ vs ER- cell lines or estradiol displacement/reversal) was not demonstrated119, 120. Subsequently we developed a novel doxorubicin- steroidal antiestrogen conjugate in which the two components were assembled independently and conjugated in the final step using CuAAC chemistry121 (Figure 1-19).The final compound demonstrated both

Figure 1-19 Modular, convergent synthesis of fluorescent anti-cancer agent steroidal antiestrogen conjugate

selective cytotoxicity for ER+ cancer cells (MCF-7) vs ER- cells (MB-MDA-231), and, the effect was reversed by administration of estradiol. These results strongly implicated an estrogen receptor-mediated process. Because of the inherent fluorescent properties of doxorubicin, uptake and localization of the conjugate was also studied in these to cell lines. This was the first study in which a steroidal estrogen receptor targeted compound displayed these properties and provided a rational basis for designing and preparing newer fluorescent antiestrogen conjugates with better imaging properties.

1.7 Proposed Approach to High Affinity Fluorescent Steroidal ER Probes - Synthesis and future directions

Our proposed approach to the development of a new generation of fluorescent estrogen receptor imaging agents employs many of the elements present in the doxorubicin-antiestrogen project. The steroidal component would incorporate the 11β-(4-oxyphenyl) estradiol scaffold associated with high ER affinity, anti-estrogenic properties and metabolic stability. Projecting from the 4-oxyphenyl group, one could append variety of tether lengths and terminal functional groups. One could then identify fluorophores possessing a variety of imaging properties as well as physico-chemical properties. These could then be modified with tethers of various lengths and functionalized with terminal groups complementary to those on the steroidal component.

Conjugation using a “click” ligation or other coupling reaction in the final step provides a series of fluorescent agents which should be able to image the presence of ER in a variety of settings, ranging from cells to whole animals. Representative fluorophores, steroids and tethers (linkers) are illustrated in Figure 1-20. The following chapters discusses the synthesis strategies, fluorescent properties and in vitro cell localization studies of our target compounds.

Figure 1-20 Representative structures of 11β-substituted estradiols and complementary substituted fluorophores.

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Chapter 2 - Design and Synthesis of Fluorescently Labeled Antiestrogens

2.1 Introduction

This chapter outlines our strategy, methodology and results in our synthesis of high affinity steroidal antagonists and controls, and is adapted from the article “Design and Synthesis of

Fluorescently Labeled Steroidal Antiestrogens” which will be published in Steroids.

The rationale for developing fluorescent imaging agents for the estrogen receptor lies in its close association with hormone responsive diseases, particularly breast cancer1, 2. According to the American Cancer Society, over 268,000 cases of breast cancer were diagnosed in 2017, and over 41,000 patients died from the disease3. It is the most common cancer among women, accounting for 29% of all diagnosed cancers. Because approximately two thirds of breast cancers are dependent on estrogen and/or progesterone, agents that target the ER play a major role in breast cancer therapy. Such strategies include the use of SERMs, such as tamoxifen, and SERDs, such as fulvestrant4-6. At the cellular and molecular level, high spatial resolution is possible using fluorescence imaging methods, if high ER affinity and selectivity were incorporated within the ligand. The last major review on this topic, with material through 19951, provided the theoretical and practical bases for this area of research. At that time, an imaging agent that met all of the criteria had not been prepared and evaluated. We have recently reviewed the development of fluorescently labeled steroidal estrogens prepared since 1995, particularly the synthetic approaches, their evaluation as potential molecular imaging agents, and a brief discussion of their advantages/disadvantages7.In this study we describe our current approach to develop the

“next-generation’ fluorescent steroidal ER imaging agent in which we use a steroidal antiestrogen as the scaffold.

Although ligands for the estrogen receptor may be either steroidal or nonsteroidal, we considered retention of the default steroidal structure to be particularly crucial. Information regarding interactions between the steroid and the receptor has largely been derived from two primary sources, structure activity relationships (competitive binding and functional assays) 8-12 and crystallographic studies with the liganded hormone binding domain13-18. Both are necessary as they provide complementary views of the interactions. The essential structure activity relationships prior to the initial x-ray crystal structure of estradiol-ER-LBD were reviewed by

Ojasoo and Anstead9, 10. (Figure 1-1) The understanding of interactions between the ER and ligands has been largely derived from examination of complexes between agonists and antagonists with the ER-LBD. These complexes provide a rational basis for understanding the earlier SAR studies on binding and efficacy. The key feature present in x-ray crystal structures of steroidal estrogen complexes is the critical hydrogen bonding interaction between the 3-OH and

Glu-353 and Arg-394 and a molecule of water within the binding site. This is present in essentially all the complexes. The 17β-OH also forms a hydrogen bond (although not as strong) with the N-H of His-524 in helix-12. The rest of the steroid scaffold is enclosed within a hydrophobic envelope which tolerates few substituents. The X-ray crystal structures for the 7α- substituted antagonist ICI 164384 demonstrates that the steroidal scaffold has rotated around the

C3-C17 axis to project the substituent into the 11β-pocket with the terminal component extending into the solvent exposed region of the complex17.

76 intensity, quantum yield, stability/quenching, and physic-chemical properties. Aspects that are involved with conjugation include the functional groups present on the steroidal ligand and those on the fluorophore, and which may influence the ligation strategy. Non-IR fluorophores commonly used for imaging include NBD,19, 20, dansyl 21-23, BODIPY25-28, among others. An overview of the flouorophores was given in figure 1-2.

Success of the ligation chemistries depends upon the functional groups present on the steroid and fluorophore moieties29, 30. Because the 3- hydroxyl and 17β-hydroxyl groups of estradiol are required for binding, no conjugations are permitted at that site. As a result, new functional groups capable of undergoing conjugation must be introduced at positions that do not seriously compromise binding properties. As our review7indicated, those potential sites were on the B-ring

(positions 6/7) or the D-ring (16/17α) and typically included a carbonyl (ketone) or carboxylic acids at the B-ring sites or extended (substituted aryl) ethynyl groups at the D-ring. In virtually every case, although the fluorescent derivatives were synthetically accessible, maintenance of high ER affinity and selectivity were lost.

Our approach was influenced by the development of fluorescent derivatives of the steroidal anti-progestin mefipristone (RU-486)31-33 Sequential modification of the terminal group on the

11β substituent gave fluorescent derivatives with increasingly higher PgR affinity. In the final study, deprotection of the terminal amine followed by conjugation with the appropriate rhodamine or BODIPY dyes gave the final products that retained the fluorescence properties of the parent dyes and the PgR binding properties of the parent steroid33. Molecular modeling studies indicated that the derivatives expressed similar binding modes within the binding pocket and that the tether-fluorophore component exited the binding pocket with the fluorophore distinctly outside the protein shell (solvent exposed). Therefore, the results supported the

77 application of this approach for developing similar fluorescent derivatives using 11β-substituted steroidal antiestrogens.

Our approach to develop ER targeted fluorescent probes combined elements used by both

Tsien and Seitz33, 34.Our earlier studies provided synthetic access to steroidal derivatives with a wide variety of 11β-substituents, including the (4-azidoethoxyphenyl) group. These compounds possessed good ER binding affinity (RBA = 20-40%) and were potent antagonists35.We also had prepared variations on the side chains and conjugated them to potent chemotherapeutic agents, such as mitomycin C and geldanamycin36, 37. Subsequently we developed a novel doxorubicin- steroidal antiestrogen conjugate in which the two components were assembled independently and conjugated in the final step using CuAAC chemistry38 (Figure 1-19). The final compound, which was fluorescent due to the doxorubicin chromophore, demonstrated an estrogen receptor- mediated intracellular localization. This was the first study in which a steroidal ER targeted compound displayed such properties and therefore provided a rational basis for the design and synthesis of antiestrogen conjugates with better fluorescent properties.

In this current study we chose to incorporate the 11β-(4-azidoethoxyphenyl) estradiol scaffold which we previously described as an effective ER-targeting group. To ultimately evaluate the effect of specific functional groups on intracellular localization (affinity and selectivity) we included the estrone derivative (17-keto) which would have reduced ER affinity, and the estra-

4,9-dien-3,17-dione analog which would lack affinity for the ER as a control agent. As fluorescent coupling partners we selected the propargylamine derivatives of NBD- and dansyl fluorophores, components that have been used by others for cellular imaging39, 40. Coupling of the two components could then be achieved using the [3 + 2] copper-assisted azide- alkyne cyclization (CuAAC) reaction41-43 as shown in figure 2-1.

Figure 2-1 Overall strategy for design and synthesis of initial series of fluorescent steroidal antiestrogens.

2.2 Synthesis

Our method for preparation for the final compounds (Figure 2-4) also consisted of synthesizing structurally related compounds to test the influence of functional groups on intracellular by an order of magnitude, and the de-aromatization of the A-ring essentially eliminated ER-binding.

The use of the 2-azidoethyl linker would provide three properties to the final products. First, the azidoethyl group confers antagonist properties to the steroidal ligand which would generate a novel probe for interrogating ER-containing cells. Second, the azide provides a facile click method for conjugating the steroidal component to a complementary alkynylated partner, in this

79 case a fluorescent component. Third, the clicked product would have the fluorescent group extended into the solvent space in the ligand-receptor complex as the triazole would occupy the same site as the tertiary amine of the aminoethoxy nonsteroidal antiestrogens. Because this initial study was to demonstrate proof of concept, physicochemical properties were more important than imaging characteristics for the fluorescent component. Both the dansyl and NBD fluorophores are relatively low molecular weight and neutral groups. More compatible with the chemistries of the steroid component than fluorescein, rhodamine or cyanine agents. Also, conversion to simple propargylated derivatives was simple and the products were easy to purify and store. Conjugation with the complementary azido reagents using the Copper(I)-assisted cycloaddition method is robust and would give the final products in good yields. If the initial set of fluorescent probes were to successfully demonstrate selective ER binding, then subsequent studies could extend the methods to include variation on the linker attached to the (4-oxyphenyl) group, different fluorophores as well as different conjugation chemistries.

The synthesis of the steroidal scaffold was accomplished using our previously described method. (Scheme 2-1) Starting with commercially available diene-dione monoketal 1, epoxidation with hydrogen peroxide under basic conditions gave the 5,10-α-epoxide as the major product, (5.5:1α:β isomers) which was isolated by flash chromatography in a 68% yield. Cu(I)- mediated 1,4-Grignard addition of the silylated phenolic reagent, followed by acidic hydrolysis, deprotection and elimination gave the 11β-(4-hydroxyphenyl)-estra-4,9-diene-3,17-dione 4 in a

47% isolated yield for the combined two-steps. Alkylation with ethylene glycol ditosylate provided the monotosylate intermediate 5 in a 53% isolated yield and which was converted to the corresponding azidoethoxyphenyl derivative 6 in a 93% yield. This intermediate would

80 constitute the steroidal scaffold for the control derivatives which would lack affinity for the estrogen receptor. Aromatization with acetic anhydride-acetyl bromide in dichloromethane smoothly converted the diene-dione intermdiate to the corresponding aromatic estrone 7 in a

74% isolated yield.

Scheme 2-1 Synthesis of functionalized steroid derivatives 6 and 7. Reagents and conditions: (i)

CF3COCF3, 50% H2O2, pyridine, CH2Cl2, 0°C, 18h, 68% yield α-isomer; (ii) a. 4-bromophenoxy trimethylsilane 3, Mg; b.Cu(I)I, THF, -10°C to RT; c. aq. NH4Cl ; (iii) CH3CO2H-H2O (7:3),

60°C, 1.5 h; (iv) TsOCH2CH2OTs, Cs2CO3, CH3CN, 120°C, 20 h, sealed tube; (v) NaN3, ethanol, reflux, 18h; (vi) acetic anhydride, CH3COBr, CH2Cl2, R.T., 18h.

As previously noted, the criteria for the fluorescent components primarily focused on their being low molecular weight, stable, neutral molecules. Preparation of the fluorescent coupling partners was achieved using relatively simple coupling reactions. (Scheme 2-3) Reaction of propargyl amine with dansyl chloride under basic conditions gave a 65% isolated yield of

81 dansylated amine 8. Displacement of the chloro-substituent of NBD-Cl by propargyl amine in methanol provided the corresponding NBD-propargyl amine 9 in a 65% isolated yield.

Scheme 2-2 Synthesis of fluorescent conjugating groups 8 and 9. Reagents and conditions: (i) propargyl amine, TEA, CH2Cl2, R.T., 18h; (ii) propargyl amine, Cs2CO3, THF, R.T., 18h

Coupling Synthesis of the control compounds 10a and 10b was accomplished in one step using the Cu(I)-assisted azide-alkyne coupling reaction with the azido-steroidal diene-dione 6 and the propargylated fluorophores 8 and 9. Flash chromatography of the crude product gave the pure fluorescent derivatives 10a and 10b in 66 % and 46% yields, repectively. Coupling of the acetylated steroidal ketone 7 with the propargylated fluorophores, followed by saponification of the 3-acetate gave, after chromatographic purification, the final products 11a and 11b in 75% and 90% yields. Repetition of the first step, followed by reduction of the ketone and saponification of the ester, gave after chromatographic purification, the fluorescent high affinity steroidal antagonists 12a and 12b in 99% and 74% yields. All products were characterized by

LC-MS, 1H-and 13C-NMR for purity and identity.

Scheme 2-3. Synthesis of fluorescently labeled steroidal antiestrogen and steroidal control.

Reagents and conditions: (i) CuSO4, sodium ascorbate, 8/9, t-BuOH-H2O, 90°C, 18h; (ii) a.

KOH, CH3OH; b. CH3CO2H; (iii) NaBH4, MeOH

The originally planned synthetic route for obtaining 12a and 12b was to convert acetylated ketone 7 to an alcohol by borohydride reduction36, 37.This pathway had been extensively used in our prior research to obtain the azido estradiol derivative before appending substituents at the

11β-(4-oxyphenyl) position. But, this synthetic route was proven unsuccessful despite numerous tries utilizing different conditions, compound 7 possibly undergoing a reduction reaction that is

83 similar to what had been reported by Edgar et.al.44 We devised an alternate reaction pathway by reducing the diene-dione monoketal 1 with sodium borohydride in order to obtain the alcohol at

17β position. Our plan was to continue rest of the transformations while keeping the 17β alcohol intact in order to obtain 8r.

Scheme 2-4 Synthesis of functionalized steroid derivative 8R. Reagents and conditions: (i)

o NaBH4, MeOH, 0 C (ii) CF3COCF3, 50% H2O2, pyridine, CH2Cl2, 0°C, 18h, 68% yield α- isomer; (ii) a. 4-bromophenoxy trimethylsilane 3, Mg; b.Cu(I)I, THF, -10°C to RT; c. aq. NH4Cl

; (iii) CH3CO2H-H2O (7:3), 60°C, 1.5 h; (iv) TsOCH2CH2OTs, Cs2CO3, CH3CN, 120°C, 20 h, sealed tube; (v) NaN3, ethanol, reflux, 18h; (vi) acetic anhydride, CH3COBr, CH2Cl2, RT., 18h;

(vii) KOH, MeOH, RT, 18h.

This pathway was proven feasible, although it typically yielded low yields (50-60%) a mixture of mono and di acetyl compound was observed. The usage of excess KOH to hydrolyze the di

84 acetyl group and the resultant acidification of the workup as well as column chromatography made this pathway non trivial. This approach was not optimized.

Another approach we undertook consisted of aromatizing 5, followed by reduction and saponification while having the ethynyl di-tosylate at the 11β-(4-oxyphenyl) position.

Scheme 2-5 Synthesis of functionalized steroid derivative 8r via an alternative pathway.

Reagents and conditions (i) acetic anhydride, CH3COBr, CH2Cl2, rt., 18h; (ii) NaBH4, KOH,

o ; 4 C, 4h , (iii) NaN3, DMF, reflux, 18h.

In this approach, the reduction and hydrolysis followed by chromatography yielded a mixture of product. The product mixture was subjected to azidation. Although the 8R was characterized, factors such as low yields, non-trivial workups prevented us from adopting this synthetic route.

2.3 Fluorescent properties

The six final fluorescent derivatives were evaluated for their fluorescent characteristics. Table

2-1. In all cases, the compounds retained the excitation and emission properties associated with their respective dansyl- or NBD-fluorophores. This suggests that the presence of the steroidal component, whether aromatic or conjugated diene-one system, did not produce any quenching or alteration of the fluorophore, and that the compounds can be used as probes to evaluate estrogen receptors in a cellular context.

Table 2-1 Excitation and Emission maxima for fluorescently labeled steroids

Compound Excitation maximum (nm) Emission maximum (nm)

10a Control-dansyl 340 680

10b Control-NBD 470 520

11a Estrone-dansyl 340 680

11b Estrone-NBD 470 520

12a Estradiol-dansyl 360 680

12b Estradiol-NBD 470 520

Spectra determined in methanol at concentrations from 0.0015 µM to 0.003 µM. The excitation- emission charts are included in the appendix.

2.4 Future work – alternative synthesis routes

Our synthetic route consisted of using an azedo steroid and an alkynyl fluorophore.

Alternatively, an alkynyl steroid and an azedo fluorophore can be used to circumvent the synthetic issues we faced.

Figure 2-2 Alternative synthesis pathway- alkynyl steroid and azedo flurophore

Previously, our group had synthesized the N-methyl-porpargylamine derivative of 6 by reacting

2-(N-methyl-N-prop-2-yn-1-yl amino) ethanol with 4 using Mitsunobu reaction in good yield38.

This derivative could be easily converted to its alkynyl estradiol derivative. Structurally similar to the steroidal antiestrogen RU 39411, which demonstrated good RBA. (39%). Identical to the compounds shown in our series, the steroidal core of 8x holds high affinity towards the

Scheme 2-6 General method of synthesis of Mitsunobu reaction

Fluorophores appended to these compounds contain the same two carbon tether length as in our series, and would not have an adverse effect on binding. The triazole formation upon the click reaction would happen in the same proximal region in the ER binding pocket as well.

Therefore, these fluorophores would have similar physical-chemical properties as the compounds we synthesized. Furthermore, the corresponding azedo fluorphores for NBD and dansyl have been synthesized in good yield,45making the alternative reaction pathway viable. Starting from

6x, this method can be utilized to make the necessary control steroid and fluorescent antiestrogens.

Figure 2-3 – Fluorophores and controls with alkynyl steroid and azedo fluorophore

2.4 Summary

We have described the design and successful synthesis of a new class of fluorescently labeled estrogen receptor targeted compounds. Starting from the steroidal estrogenic core, we have introduced a substituent at the 11β-position such that it can be readily modified to accept a large fluorophore without compromising receptor binding affinity. The fact that the substituent also changes the pharmacology from estrogen to antiestrogen provides an intriguing aspect as it is largely unknown what effect that modification has upon receptor localization. The synthetic chemistry used to introduce the substituent also permits the generation of analogs or derivatives with lower affinity for the receptor, thereby providing for internal controls. Although the initial fluorophores-NBD and dansyl-are not considered optimal for receptor imaging, the fact that they are small and neutral groups allows them to serve as proof-of-concept agents. Because the site of ligation is at the receptor-solvent interface, if these agents can successfully image estrogen receptor density within the cells, other fluorophores with better imaging properties, but which are either larger or charged, can be evaluated in future studies. Alternate synthetic routes conferring better yields would also be explored.

The evaluation of this series of fluorescently labeled steroid to image estrogen receptors in various cell lines is currently in progress and the results of that study will be presented in future publications.

Addendum

1H NMR of 12b was updated, as per the suggestion of a reviewer of this thesis.

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Chapter 3 – In vitro Investigations of Fluorescently Labeled Antiestrogens

3.1 Introduction – previous research

Our group has been researching fluorescent 11β-substituted steroids, the first compounds synthesized by Ray et.al with a tetraphenylporphyrin appended via a linker1. Despite exhibiting good fluorescence properties, the compound had poor binding properties, due to by attaching a bulky porphyrin group via a linker. As a remedy for the poor binding properties conferred from the bulky porphyrin group, we took inspiration from the design of fluorescent derivatives of the antiprogestin mefipristone RU-486234 that had been used to target the PgR, where the binding orientation of the ligand is similar to that of the ER, where a tethered fluophore exiting the binding pocket and orienting outside the protein shell. This was further confirmed by the initial synthesis of RU 45196 by Philibert et. al2, demonstrating good binding properties towards the

PgR (figure 3-1). Yet the binding properties of the fluorescein isocyante (FITC) derivative of

Ru-486, the fluorophore appended by a hexanol group at 11β position demonstrated a loss of binding affinity towards the PgR 3. This loss of receptor binding could be attributed to the ionic character of the FITC fluorophore or the steric hindrance caused by the tether, highlighting the need for a neutral fluorophore and a sterically feasible tether for an initial study. Similarly, studies conducted on non-steroidal anti estrogen fluorophores by Seitz et al. indicated that the conjugation of the fluorophore to the nonsteroidal ligand had no effect on the fluorescent properties of the fluorophore. Furthermore, these conjugates retained high affinity to the ER5.

Our successful development of a novel doxorubicin-steroidal antiestrogen by appending a two independently synthesized components using CuAAC chemistry6 served as a precursor for our research. The doxorubicin-antiestrogen compound was fluorescent and demonstrated selective

97 cytotoxicity towards ER+ cancer cells ( MCF-7).This effect was reversed by administration of estradiol, implicating an ER mediated process. Due to the inherent fluorescence of the doxorubicin-antiestrogen compound its uptake and distribution in ER+ cells could be assessed by fluorescent techniques. This served as a rational basis for our research for the synthesis of a fluorescent antiestrogen with better imaging properties.

Figure 3-1 Previous investigations into fluorescent steroids

Recent work by Van Lier, et.al (figure 1-14, 1-15) involved using a similar strategy, by appending a BODIPY fluorophore at the 17α-position in ethynyl estradiol and in a modified estradiol derivative using Sonogoshira coupling and click chemistry. Despite its excellent fluorescent properties conferred by the BODIPY moiety, the compounds lacked the RBA towards ER due to the position of the fluorophore. In compounds where the fluorophore was appended by a linker, higher RBA was observed7,8. Our synthesis work, extensively described in chapter 2 of this thesis, we incorporated the 11β-(4-azidoethoxyphenyl) estradiol scaffold as the

98 core steroidal platform where the fluorophores are appended by a short linker. As for the flurophores, we selected the propargylamine derivatives of NBD- and dansyl for their low molecular weight and neutral charge9,10,11,12,13. The coupling of the fluorophore and the steroidal core was achieved copper assisted ‘click’ reaction (figure 2-1). We synthesized six fluorescent derivatives – two fluorescent estradiol derivatives 12 a/b, two fluorescent estrone derivatives with partial affinity 11 a/b and two fluorescent estra-4,9-dien-3,17-dione analogs 10a/b which lack the affinity for the ER, to be used as control steroids for in vivo studies. Our compounds retained the excitation and emission properties associated with their fluorophores, suggesting that these compounds can be used as probes to evaluate estrogen receptors in a cellular context.

Figure 3-2 11β-(4-azidoethoxyphenyl) derivatized fluorescent antiestrogens and controls

3.2 In vivo studies introduction – Estrogen receptor

Cellular uptake and distribution of 17β estradiol (E2), the endogenous ligand for ER happens through passive diffusion or binding to the cognate estrogen receptors that are embedded within the cell membrane. This binding of estradiol with membrane estrogen results in endosome formation. Once within the cell, E2 binds to the ligand binding domain of the ER and undergoes

99 a conformational change, permitting ER dimerization. This conformational change induced by the ligand-ER interaction drives the transcriptional process. For the synthetic ligands that target the ER the process of uptake, binding, coregulatory recruitment and transcriptional activity is altered 14, yet this general internalization mechanism for ER can be utilized in the uptake of our high affinity fluorescent steroids. Previously, our work on doxorubicin- antiestrogen compounds served as a rational basis for our fluorescent steroids. In the in vitro experiments, we expect to assess the uptake of our fluorescent derivatives in MCF-7 ( ER + cells) and in MDA-MB-231 (

ER- cells). Also, we expect to confirm that the uptake of fluorescent steroids is estrogen receptor mediated.

3.3 General Experimental Setup

20 µM time point study

MDA-MB-231 (ER-) and MCF-7 (ER+) breast cancer cells were used. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%, 50 U/mL penicillin and 50 µg/mL streptomycin. Cells were kept at 37°C, 5% CO2 atmosphere at high humidity for optimum growth conditions and during exposure to the compounds. Cells were seeded on 8-well chambers and allowed to grow until reaching 70-80% confluence prior to the treatment. Treatment was done in duplicate (2 wells/compound/cell line). Control groups were treated only with DMEM. The compounds were solubilized in methanol later diluted with

DMEM for treatment. They were added to the wells at 20 µM and cells were incubated for 30 min, 2h and 24h. After treatment time was completed, the cells were washed twice to remove the non-internalized compound. Cells were fixed with paraformaldehyde at 2% for 20 min at room

100 temperature. Hoechst was added for nuclei staining, followed by incubation for 10 minutes. The chamber was removed from the slides. Mounting medium was added on top of the cells, followed by the addition of coverslips. The slides were allowed to rest at room temperature for

1h and transferred to the freezer (-20°C) for further microscopy analysis.

1µM time point study – General procedure

The cells were treated at a steroid concentration of 1µM. MCF7 and MDA-MB-231 cells seeded at 80,000 cells per well 24h prior to the experiment. Cells were treated at different time points (1 plate per time point, 8 wells in each plate at 0.5h, 2h and 24h. Cells were washed 2 times with

Phosphate buffered saline (PBS), fixed with paraformaldehyde (PFA) 2% (in PBS solution) for

20 min, followed by addition of Hoechst at 10 µg/mL in PBS (cells were stored in this solution at

4oC until fluorescence microscopy analysis.

1µM competitive binding assay – General procedure

MCF7 and MDA-MB-231 cells seeded at 80,000 cells per well 24h prior to the experiment. The proper groups were pre incubated with17-α-Ethynylestradiol (EE2) at 50 µM for 1h. NBD-

Estradiol 12b was added and was incubated for 2h. Cells were washed twice with PBS, fixed with PFA 2% (in PBS solution) for 20 minutes. Hoechst was added at 10µg/mL in PBS. The plates were stored at 4oC until fluorescence microscopy analysis.

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Fluorescence-activated cell sorting (FACS) – General procedure

The media is removed, 200µL of trypsin is added and incubated for 5 min. 500µL of complete medium is added, centrifuged and washed twice with PBS. The cells were re suspended in

300µL of PBS for analysis.

Fluorescence microscopy

Fluorescence microscopy studies were conducted on Keyence BZ-X-710 fluorescence microscope. Green filter was used for fluorescence images, and an exposure time of 1/20 seconds at 40X magnification was used. Four snapshots consisting of an image of the brightfield, (a black and white image of the plate) blue filter (hoechst stain), green filter (fluorescent image) and an overlay of the all the images was taken. Three snapshots were taken of each plate, and three plates was used for each experiment. Each snapshot contained at least five digitalized images of cells for data analysis. The data was analyzed using Imagej version 1.52a.

Statistical analysis

Statistical analysis was based on FACS data. The data was analyzed using Graphpad using

ANOVA and Turkeys multiple comparisons test. Differences were considered significant at p ≤

0.05. **** signifies p < 0.0001

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3.4 Time point study – NBD-E2 (12b) and Dansyl-E2 (12a)

This experiment was carried out to assess the cell uptake of dansyl-estradiol and NBD-estradiol

(12a and 12b) at 20µM in MCF-7 and MBD-MB-231 cells

MCF-7 Bright field MCF-7 Green filter MDA-MB-231 MDA-MB-231

Brightfield Green filter 0.5h

12b

0.5h

12a

12b

12a

24h

12b

24h

12a

Figure 3-3 Fluorescence and brightfield images of 12a/b at 20µM

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Fluorescent microscopy data, NBD-E2 (12b) andd Dansyl-E2 (12a) Fluorescent intensity vs time- 20µM

25000.00

20000.00

15000.00

10000.00 Fluoresence Intensity Fluoresence Intensity

5000.00

0.00 MCF Control MCF Dansyl MCF NBD MDA control MDA Dansyl MDA NBD

0.5h 2hr 24hr

Figure 3-4 Fluorescence microscopy data for 12b and 12a - intensity vs incubation time, 20µM

The fluorescent steroids were incubated in MCF-7 (ER +) and MDA-MB-231 (ER-) cells for

0.5h, 2h and 24h, and the fluorophore uptake was assessed by fluorescence microscopy. A clear increase in fluorescent intensity was observed in both MCF-7 and MDA-MB-231 cells, strongly implying nonspecific uptake on both cell lines (Figure 3-3, 3-4).

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At all time points, the intensity of NBD-E2 (12b) was higher than the intensity of dansyl- estradiol (12a) by an average of 3.2 times on MCF-7 cells. Due to the high concentration of steroid fluorophore of this experiment, subsequent fluorescence microscopy studies were conducted at lower concentrations. It was discovered that the dansyl-estradiol (12a) fluorophore was undetectable at concentrations lower than 2µM. For the NBD-estradiol (12b) compound, experiments were conducted at concentrations of 10-100ηM in order to establish the limit of detection. NBD-estradiol at 1µM was chosen as the steroidal fluorophore and the threshold of detection for later experiments.

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3.5 Time point study – NBD-E2 (12b) at 1µM

This experiment was conducted to measure the fluorescence intensity of NBD-estradiol (12b) at 1µM in MCF-7 vs MDA-MB-231 Cells at different time intervals

MCF –Brightfield MCF - Fluorescent MDA-MB-231 MDA-MB-231

Brightfield Green filter 0.5h ctrl

0.5h

12b

2h ctrl

12b

24h ctrl

24h

12b

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Figure 3-5 Fluorescence and brightfield images of control and 12b at 1µM

Fluorescence microscopy data- 1µM incubation of NBD-E2 (12b) timepoint 1600

1400

1200

1000

800

600 Fluorescence Intensity Intensity Fluorescence 400

200

0 MCF Control MDA control MCF NBD MDA NBD

0.5h 2h 24h

Figure 3-6 – Fluorescence microscopy data- Fluorescence intensity vs. incubation time (12b) at 1µM.

FACS - 1µM Timepoint Study, NBD-E2(12b) 14

12 ****

10 MDA-MB-231 MCF7 8 Control

4 Fluorescene Fluorescene fold intensity

0 0.5 2 24

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Figure 3-7 – FACS data for Fluorescence intensity vs. incubation time of 12b at 1µM.

According to fluorescence microscopy data, the fluorescent steroid indicates a higher uptake versus the controls (on an average of 14 times higher on MCF-7 cell line). For the MCF-7 cell line, data indicates a 19.5% decrease between 0.5h and 2h and a slight increase (3%) in intensity between 2h and 24h. As for the MDA-MB-231 cell line, the fluorescence intensity steadily increases between time points.

FACS data indicates a similar trend, yet for the MCF-7 cell line, the decrease in intensity between the 0.5h and 2h timepoints is 2.7%. Conversely, FACS data indicates a 54.7% decrease of intensity between 2h timepoint and 24h timepoint. Fluorophore incubated in MDA-MB-231 shows a 16% increase in intensity between the 2h and 24h time point. Unlike the steady increase shown in the fluorescence microscopy study, it shows a 55.2% decrease between 2h and 24h timepoint for MDA-MB-231 cells.

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3.6 NBD-E2 (12b) competitive binding study at 1µM

This experiment was conducted to evaluate if the cellular uptake is estrogen receptor mediated or nonspecific.

Control NBD-E2(12b) cont. NBD-E2 (12b)

incubated with EE2 MCF-7

Brightfield

MCF-7

Green filter

MDA-MB-231

Brightfield

MDA-MB-231

Green filter

Figure 3-8 Fluorescence microscopy images of 12b, competitive binding studies. Comparison with brightfield images

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Fluorescence microscopy, Competitive binding study, 1µM NBD-E2 (12b) 1200.00

1000.00

800.00 Incubation with estradiol (EE2), removed after 1h 600.00 Continuous incubation with estradiol (EE2)

Fluorescence Intensity Intensity Fluorescence 400.00

200.00

0.00

Figure 3-9 Competitive binding study for 12b - fluorescence microscopy data

In figures 3-8 and 3-9, the NBD-E2 (12b) compound shows a 15 fold increase compared to the control in fluorescence microscopy and a 50 fold increase of intensity in FACS. Both the fluorescence microscopy and FACS data show a reduction of the fluorescence intensity of NBD-

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E2 (12b) on continuous incubation with estradiol in the MCF-7 cell line. In the fluorescence microscopy study, this shows a 77% decrease. For the FACS study, the decrease is 42%, implying the blocking of estrogen receptors upon addition of estradiol, strongly suggesting that the uptake is estrogen receptor mediated.

A similar quenching of intensity is shown for the ER- MDA-MB-231 cell line as well. In the fluorescence microscopy study, the NBD-E2 (12b) compound shows a 45% reduction in intensity when continuously incubated with estradiol. In the FACS study, it shows a 32% reduction in upon incubated with estradiol. The trend of NBD-E2 (12b) showing uptake in

MDA-MB-231 (shown by a 2.7 fold increase of intensity in fluorescence microscopy and 8.8 fold increase in FACS) is observed in previous experiments as well. But, the quenching of the fluorescence intensity upon adding estradiol potentially implies that a low level of ER expression in ER- cells such as MDA-MB-23115.

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FACS- Competitive binding study, 1µM NBD-E2 (12b) 60 **** **** 50

20 Fluorescence Fluorescence fold intensity 10

Figure 3-10 1µM Competitive binding study for 12b, FACS data

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3.7 NBD-E2 (12b), NBD-propargyl (9), competitive binding and temperature dependence in receptor localization at 1µM

In this set of experiments, we assessed the effect temperature has on cell internalization as well as the role played by the fluorescent moiety in cellular uptake.

FACS data - NBD-E2 (12b), NBD-propargyl (9) - Competitive binding at 37oC and 4oC 80 **** **** 70

60 **** 50

30 37C 4C

Fluorescene fold intensity (FFI) fold intensity Fluorescene 20

Figure 3-11 – Temperature dependence in competitive binding for 12b and 9 – FACS data

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Compared to the control, the NBD-E2 (12b) showed a 70 fold increase in MCF7, and a 20 fold increase in MDA-MB-231 at 37oC. The intensity decreased by 74% at 4oC in MCF-7, and by

47% in MDA-MB-231. Yet, at 4oC, an 18 fold fluorescence intensity was seen, suggesting an energy-independent process where the fluorophore is directly translocated through cell membrane.

A 3.5 fold higher uptake was observed for NBD-E2 (12b) at 37oC in MCF-7 cells. However, this ratio decreased to 1.8 fold higher uptake, signifying the predominantly estrogen receptor mediated uptake mechanism. Additionally, the NBD-propargyl (9) did not show significant internalization in cells at either temperature, NBD-E2 showing a 45 fold increase at 37oC and a

16.5 fold increase at 4oC

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3.8 Competitive binding – NBD control compound (10b) and NBD-E2 (12b) at 1µM The goal of this experiment was to compare the cellular uptake of NBD-control (10b) compound to NBD-E2 (12b), and to conduct competitive binding studies in the presence and absence of

EE2.

MCF-7 Brightfield MCF-7 green filter MDA-MB-231 MDA-MB-

Brightfield 231green filter Ctrl

NBD-

Ctrl

(10b)

NBD-

Ctrl

(10b)

+ EE2

NBD(co. - incub.)E2

(12b)

NBD-

(12b)

+ EE2

(co. incub.) Figure 3-12 – Fluorescence microscopy data for competitive binding for compounds 10b and 12b

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Fluorescence microscopy, Competitive binding of NBD-E2(12b) and NBD- control (10b), 1µM 800

700

600

500

400

300 Fluorescence Intensity Intensity Fluorescence 200

100

Figure 3-13 Fluorescence microscopy data for competitive binding – for compounds 12b and 10b

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FACS - Competitive binding of NBD-E2 (12b) and NBD-control (10b), 1µM 60 **** ****

20 Fluorescence fold intensity FFI) ( fold Fluorescence intensity 10

Figure 3-14 – FACS data for competitive binding for compounds 12b and 10b

Compared with the control, the NBD-Control (10b) compound shows a 2.7 fold increase in the fluorescent intensity, whereas the NBD-E2 (12b) compound shows a 9.3 fold increase in fluorescence microscopy data. As expected, in figures 3-12 and 3-13, both fluorescence microscopy and FACS data indicate a reduction of fluorescence intensity for NBD-Control (10b) compound, compared with NBD-E2 (12b). In fluorescence microscopy, this corresponds to a

71% reduction of fluorescence (for MCF-7 cells). Competitive binding with estradiol indicates a

79% reduction of fluorescence for NBD-E2 (12b) and a reduction of 32% for the NBD-Control

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(10b) compound (for MCF-7). For the MDA-MB-231 cells, NBD-Control (10b) this indicates a

45% reduction of intensity when compared with NBD-E2 (12b). Competitive binding assays carried out in the MDA-MB-231 cell line indicates a decrease of 40% for NBD-E2 (12b) and a reduction of 5% for NBD-Control (10b).

The trend shown in fluorescence microscopy studies continues in FACS data as well. NBD-

Control (10b) showing a 17 fold increase and NBD-E2 (12b) showing a 50 fold increase. For

MCF-7 cells, the NBD-Control (10b) shows a 66% decrease in fluorescent intensity compared with the NBD-E2 (12b). Upon incubating with estradiol, the NBD-E2 (12b) shows a 37% decrease in intensity, and the NBD-control (10b) compound shows a 31% decrease. A similar trend is shown in MDA-MB-231 cell line as well, showing a 64.5% reduction in intensity for

NBD-Control (10b) in comparison with NBD-E2 (12b). The incubation with estradiol further reduces the fluorescence intensity of NBD-E2 (12b) by 30% and the intensity of the control compound (10b) by 15%.

3.9 Discussion of results

20µM timepoint study

The first experiment was designed to assess the cellular uptake of our fluorescent estradiols

(12a/12b) as a function of three time points, at 0.5h, 2h and 24h. A high concentration of the fluorophore (20µM) was used for the initial study. Visually, there was a stark contrast between the images of NBD and dansyl at all time points, NBD having a much higher intensity than

118 dansyl. NBD-E2 (12b) displayed a higher contrast against the background for MCF-7 cells for

0.5 and 2h timepoints (figure 3-3). Data from fluorescent microscopy indicated a linear increase of the fluorescent intensity as a function of time for both the NBD and dansyl fluorophores

(figure 3-4). Interestingly, such an increase was also observed in the MDA-MB-231 cell line as well as in the controls. The concentration that was used for this experiment, 20µM was approximately 20,000 greater than that of the binding affinity of estradiol for the ER16. The linear increase in all cell lines as a function of time strongly implies saturation of receptors

(specific and nonspecific) with the fluorophore.

Due to the high concentration used in the initial experiment, subsequent experiments were conducted at lower fluorophore concentrations to establish the lowest threshold of detection.

Incubation studies were carried out at 1-100ηM for the NBD fluorophore at 1µM for the dansyl fluorophore. Neither of these ranges of concentration gave visible fluorescent images and subsequent fluorescent microscopy/ FACS studies were carried out at a concentration 1µM using the NBD-E2 (12b) fluorophore.

1µM timepoint study using NBD-E2 (12b)

For the 1µM timepoint study for NBD-E2 (12b), a greater contrast between the background and the cells was observed for MCF-7 cells (figure 3-5). This phenomena was especially visible at 0.5 h and at 2h timepoints. Fluorescence microscopy data indicated a considerable higher uptake - approximately 36 fold increase compared to the controls for MCF-7 at all timepoints

(figure 3-6). The uptake, highest at 0.5h is reduced at 2h and is slightly increased for the 24h timepoint for MCF-7. For the MDA-MB-231 cell line, a linear increase was observed. For FACS

119 data, an average of a 24 fold increase in intensity (compared with controls) was observed for

MCF-7, and an average of 7.8 fold increase in intensity was observed for MDA-MB-231 cells.

Statistical analysis of the FACS data indicated the difference between MCF-7 and MDA-MB-

231 statistically significant for 0.5h, p ≤ 0.0001 (figure 3-7).

For this study, fluorescent microscopy and FACS data show divergent trends. Unlike the considerable decrease observed after 0.5h for fluorescent microscopy, the fluorescent fold increase only shows a slight decrease between 0.5 and 2h. However, the FACS data shows a considerable decrease in the intensity between 2h and 24 for both MCF-7 and MDA-MB-231.

This divergence between FACS and fluorescent microscopy could have been caused by the poor condition of MCF-7 cells that were used in the preliminary studies due to repeated passages.

1µM NBD-E2 (12b) competitive binding study

The purpose of the competitive binding study was to demonstrate the ER uptake of NBD-E2

(12b) was estrogen receptor mediated. This study was modeled after the competitive binding study for our investigation of the ER-Dox compound6.

Initial experimental setup for the competitive binding study consisted of incubating MCF-7 and

MDA-MB-231 cells with 50µM EE2 for 1h, followed by the removal of media containing unbound EE2 prior to adding NBD-E2 (12b) followed by an incubation of 2h with NBD-E2 and the remaining EE2. While the addition of EE2 lead to a marked decrease of the fluorescence intensity of NBD-E2, it was observed that the removal of media containing EE2 lead to an artificially high fluorescent intensity.

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For later experiments, the cells were incubated with 50 µM of EE2 NBD-E2(12b) was continuously incubated with EE2 for 2h. The continuous incubation lead to an even lower fluorescence intensity than what was observed previously. For fluorescent microscopy, this reduction was 77%, and for FACS it was 42%. A similar reduction was observed in MDA-MB-

231 cell line as well. In the fluorescent microscopy images, a reduction of brightness was observed for the cells that were continuously incubated with EE2 (in both cell lines). For the

MCF-7 (12b), this reduction of intensity was considered statistically significant, p ≤ 0.0001

(figure 3-8, 3-9). The reduction in fluorescent intensity, observed in both FACS and fluorescent microscopy corresponds to saturation of ER by the addition of EE2, strongly suggesting a predominantly estrogen receptor mediated uptake mechanism.

NBD-E2 (12b), NBD-propargyl (9), competitive binding and temperature dependence in receptor localization at 1µM

The purpose of this study was the assessment of the role played by the steroidal core in the receptor localization of the fluorophore. Additionally, the effect of temperature on fluorophore internalization as well as competitive binding at different temperature was also investigated.

NBD-propargyl (9) recorded a drastically low fluorescence intensity compared to NBD-E2

(12b) in MCF-7 (45 and 9 fold) and in MDA-MB-231 (15 and 10 fold), FACS data. This reduction in the intensity implies that the steroidal core plays a major role in the internalization process17 (figure 3-11).

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For the temperature dependence studies, the results indicated a great reduction of the overall intensity (74% decrease for MCF-7 and a 47% decrease in MDA-MB-231 for NBD-E2. 4oC signifies a temperature at which the cellular metabolism is attenuated, mostly inhibiting receptor mediated ( energy dependent) cellular uptake. Interestingly, at 4oC, and 18 fold fluorescence intensity was observed in MCF-7 and a 10 fold intensity was observed in MDA-MB-231. The 18 fold intensity was decreased by 49% at the addition of EE2. This observation implies that while the lower temperatures greatly reduces the receptor mediated uptake, it doesn’t completely eliminate it. The 9 fold increase of intensity signifies that the internalization is also achieved by energy independent routes such as direct translocation through the cell membrane. The fluorescent intensity differences for the steroid was considered statistically significant between

37oC and 4oC, p < 0.0001

Competitive binding – NBD control compound (10b) and NBD-E2 (12b) at 1µM

The purpose of this experiment was to investigate the binding ability of the NBD-control compound (10b), a compound that lacks the characteristic structural features that are involved in binding to the ER. Additionally, both the NBD-control (10b) and NBD-E2 (12b) were subjected to competitive binding studies in the presence and absence of EE2. In the NBD-control (10b) compound, the ketone at the 3 position lacks the ability to hydrogen bond with Glu- and Arg- amino acids in the binding site, and the ketone at the 17β position lacks the ability to form a hydrogen bond with the N-H of the His-524 in helix 12. The lack of aromaticity of the A ring also contributes detrimentally to ER binding 18,16. Therefore, we hypothesized that the control compound should confer a reduced intensity due to the lack of ability to bind for the ER.

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In the fluorescence microscopy images, a reduction in brightness is observed for NBD-control

(10b) in comparison with NBD-E2 (12b) in the MCF-7 cell line (figure 3-12). Fluorescence microscopy and FACS data show a notably reduced intensity for NBD-control (10b) in comparison with NBD-E2 (12b); a 71% reduction in MCF-7 cells and a 45% reduction in MDA-

MB-231 cells (according to fluorescence microscopy data), 66% reduction in MCF-7 and 64% reduction in FACS (figure 3-13). These intensities were further reduced upon adding EE2. The reduction of the fluorescent intensity for the control compound proves the predicted outcome of a reduced intensity for the NBD-control compound, signifying a greatly reduced cellular uptake.

However the NBD-control (10b) compound shows an appreciable fluorescent intensity in comparison with the controls, suggesting a cellular uptake promoted by the lipophilic steroidal core17. The differences in the uptake between NBD-E2 (12b), NBD-control (10b) and NBD-E2 incubated with EE2 is considered statistically significant with a p ≤ 0.0001.

Nonspecific binding and cytoplasmic accumulation of fluorescent steroids

For all our in vitro studies, the accumulation of our ER specific fluorescent compounds in

MDA-MB-231 cells was routinely observed. This accumulation was observed as a linear increase as a function of time in the early timepoint studies. In the NBD-Control / NBD-E2 study

(figure 3-13, 3-14), it was observed that the fluorescent intensity of the NBD-control (10b) was less than that of NBD-E2 (12b) ( a 55% reduction in fluorescent microscopy, a 65% reduction in

FACS). The fluorescence intensity was further reduced upon co-incubation with EE2, lending credence to the assumption of MDA-MB-231 cells expressing a low level of ER15. In addition to the existence of ER in MDA-MB-231 cell line, passive diffusion through cell membrane, promoted by the lipophilic steroidal core could have contributed for the accumulation in MDA-

MB-231.

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In the fluorescence microscopy images, only a negligibly low level of fluorescence in the nucleus was observed (fig 3-15). This was somewhat of an unexpected observation for a cell line that expresses nuclear/ intracellular estrogen receptors abundantly. However, this phenomena has been observed in other investigation as well19,20,21.

MCF-7 NBD-E2 (12b) 1µM NBD- E2(12b) Hoechst Overlay

(nuclear stain)

Figure 3-15 –Accumulation of 12b in MCF-7 cells – Fluoresent, Hoechst stain and overlay

As evident in the fig (3-15) the fluorescent accumulation was greater in the cytoplasm, rather than the nuclear compartment. The experiment for the temperature dependence of the receptor localization (figure 3-11) revealed that a considerable amount of cellular localization happening through energy independent routes such as direct translocation through the cell membrane. This factor, as well as the possible quenching of the fluorophore in the nucleus22 could have caused the increased fluorescent intensity in the cytoplasmic, rather than in the nuclear region of the cell.

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3.10 Conclusion and future work

In this investigation, we have demonstrated the ability to synthesize a series of fluorescent antiestrogesn based on 11-β substituted steroids. The in vitro investigations revealed greatly enhanced fluorescence intensity for the NBD-E2 (12b) in MCF-7 cell line, in comparison the the

MDA-MB-231 cell line. This enhanced fluorescence intensity was quenched upon the addition of EE2, strongly suggesting an ER mediated cellular uptake.

This series of fluorescent derivatives serve as a proof of concept for fluorescent imaging agents based on 11-β substituted steroids. Due to the sub optimal fluorophore characteristics, a high concentration of fluorophore was used for the threshold of detection in fluorescent images. The concentration used in this study, 1µM of three orders of magnitude higher than the binding affinity of E2, potentially causing undesirable non-specific accumulations observed in our investigations. Therefore, future investigations would focus on appending a more efficient fluorophore such as BODIPY 23 that is potentially capable of producing fluorescent microscopy/

FACS images at nanomolar levels. Additionally, instead of the neutral fluorophore, our investigations would focus on appending charged fluorophores with different linker lengths and assessing the in vitro effects of appending a charged fluorophore to the 11-β steroid. The synthesis of a fluorescent steroid that is capable of imaging ER+ cells at nanomolar levels would allow us to extend our investigations for the imaging of other nuclear receptors.

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Appendix

130

Experimental methods

All reactions were carried out in dry glassware unless otherwise noted. Dry glassware was obtained by heating in a laboratory oven at 113oC overnight (minimum of 12h).

After removal, all glassware was sealed with a rubber septum and placed under a steady stream of dry N2 via a 20 gauge 1.5 inch needle and cooled to ambient temperature.

All reactions were carried out under an inert (argon) atmosphere with freshly distilled solvents unless otherwise noted. THF was distilled from sodium-benzophenone immediately prior to use. Dichloromethane was distilled from calcium hydride prior to use and methanol was dried by heating at reflux with magnesium turnings and then distilled onto activated, crushed 4Å molecular sieves. Other anhydrous solvents were purchased as extra dry (< 10 ppm water) and stored over molecular sieves. The starting material, estra-5(10), 9(11)-diene-3,17-dione 3-ethylenedioxy ketal, was purchase from

Shanghai Richem International Co and analyzed prior to use. All other reagents were obtained from Sigma-Aldrich Chemical Company or Fisher Scientific and used as provided.

1H-NMR and 13C spectra were obtained on a Varian mercury 400 ( 400 MHz) and are reported in parts per million (ppm). All coupling values (J) are reported in Hz. The NMR spectra were processed using MestReNova version 6.1.0-6224. LC-MS were obtained on

131

Waters e2795 separations module ( LC) using a Sunfire C18 column and Waters micromass 7Q ( MS). IR spectra were obtained on a Bruker alpha –e ATR spectrometer.

Analytical thin layer chromatography (TLC) was performed using silica gel 60A precoated sheets (Sigma-Aldrich) and were visualized using 254 nm/ 366 nm UV lamp, in p-anisaldehyde stain where indicated. All column (flash) chromatography was performed on silica gel unless otherwise indicated.

Experimental information

Synthesis of 3,3-ethylenedioxy-5(10)-α-epoxy-estra-9(11)-ene-17-one 2a and 3,3- ethylenedioxy-5(10)-β-epoxy-estra-9(11)-ene-17-one (2b).

3,3-Ethylenedioxy -estra-5(10),9(11)-diene-17-one 1 (3.0g, 9.6 mmol) was dissolved in

8mL of dichloromethane. Hexafluoroacetone (175µL (1.4 mmol), pyridine (77 µL, 0.96 mmol), and hydrogen peroxide (50%, 1.5 mL, 49.1 mmol) were added at 00C. After 18 hrs. aqueous sodium thiosulfate (2g in 50mL) was added to quench the reaction. The aqueous layer was extracted with dichloromethane (3 X 100 mL) Organic fractions were combined, dried over magnesium sulfate (anhydrous), filtered and evaporated to dryness

The colorless residue was triturated with ether to give a white solid that was collected by filtration and identified by 1H-NMR as the α-epoxide. The filtrate contained a mixture of the two epoxides which was separated using flash chromatography. The desired product

2a eluted first with hexane-ethyl acetate (7:3). This product was combined with the initial

132 precipitate to give an overall isolated yield of 2.2 g (6.6 mmol, 68%). The β-epoxide was isolated in subsequent fractions to give an isolated yield of 0.4g (1.2 mmol, 13%).

1H NMR (400 MHz, CDCl3) δ 6.00 (s, 1H), 3.86 (m, 4H), 2.41 (m, 2H), 2.15 – 1.98 (m,

6H), 1.93 – 1.81 (m, 3H), 1.77 – 1.56 (m, 2H), 1.50 (d, J = 11.0 Hz, 4H), 1.18 (qd, J =

12.4, 3.2 Hz, 1H), 0.82 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 220.10, 136.43, 125.54,

106.76 , 64.15, 63.95, 61.44 , 59.90 , 46.50 , 46.15 , 40.09 , 36.92 , 35.77 , 33.45 , 31.42 ,

27.88 , 24.92 , 22.04 , 21.75 , 14.59

LC -MS – m/z - observed M+1 – 331.42

Synthesis of 11β-(4-hydroxyphenyl)-estra-4,9-diene-3,17-dione (4)

Freshly distilled THF (50 mL) was added to a round bottom flask containing magnesium turnings (2.5g, 104 mmol, oven-dried overnight). A granule of iodine was added to effect a deep reddish brown color. At this point, of 4-bromophenoxy trimethylsilane 3 (10mL,

51 mmol) was added dropwise, 1mL initially, and subsequently 0.5mL aliquots every

15min. After addition of 5mL of 3, the reaction mixture was gently warmed to 60oC. The color of the reaction changed from the initial reddish brown to yellow and eventually to a metallic grey. 3,3-Ethylenedioxy-5(10)-α-epoxy-estra-9(11)-ene-17-one 2a (2.1 g, 6.3 mmol) was dissolved in 15 mL of anhydrous THF. Cu (I) iodide (0.16g, 0,84 mmol) was added to the solution and the mixture was cooled to -10° C. The Grignard reagent was added dropwise and the resultant mixture was warmed to ambient temperature. After 18 h the reaction was quenched by the addition of aqueous ammonium chloride and ethyl

133 acetate (total 70mL). The organic layer was separated, washed twice with water (40mL), dried over magnesium sulfate (anhyd), filtered and evaporated to dryness to give a crude oil. The oil was dissolved in acetic acid- water (20 mL, 7:3 by volume) and the solution was heated at 60° C for 1.5 h. The reaction was diluted with ethyl acetate (20 mL) and neutralized by the addition of aqueous sodium bicarbonate (saturated). The organic phase was separated and the aqueous phase was extracted with ethyl acetate (2 X 20 mL). The organic layers were combined, washed with brine, dried over magnesium sulfate, filtered and evaporated to dryness. The crude product was purified by flash chromatography (7:3 hexane: ethyl acetate) to give 11β-(4-hydroxyphenyl)-estra-4, 9-diene-3,17-dione 4 (1.07 g, 3.0 mmol) in a 47% yield for two steps.

Rf = 0.2 (hexane : ethyl acetate 1:1)

1 H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H), 6.93 (d, J = 8.5 Hz, 2H), 6.73 (d, J = 8.5 Hz,

2H), 5.76 (s, 1H), 2.75-2.64 (m, 1H), 2.62-2.54 (m, 3H), 2.48-2.21 (m, 6H), 2.14 – 2.02

(m, 2H), 1.97 – 1.94 (m, 1H), 1.89-1.84 (dd, J = 7.1 Hz, 1H), 1.61-1.43 (m, 3H), 0.51 (s,

13 3H). C NMR (100 MHz, CDCl3) δ 220.81, 200.59, 157.46 , 154.66 , 146.15, 134.72 ,

129.65 , 127.78, 122.81 , 115.59, 50.43 , 47.67 , 39.53 , 37.90 , 37.75, 36.50, 35.42 ,

30.82, 26.54 , 25.63 , 21.78 , 14.31. LC -MS – m/z - observed M+1 – 363.24.

Retention time – 1.92min.

Synthesis of 11β-[4-(2-tosyloxyethoxy)phenyl]-estra-4,9-diene-3,17-dione (5)

To 11β-(4-hydroxyphenyl)-estra-4a,9-diene-3,17-dione 4 (510 mg, 1.41mmol) dissolved in acetonitrile (6 mL) was added cesium carbonate (0.71 g, 2.1 mmol). The reaction mixture was stirred for 30 min followed by the addition of ethylene glycol ditosylate

134

(0.35 g, 0.94 mmol). The reaction vessel was sealed and heated at 120° C for 20 h. The reaction mixture was cooled to ambient temperature and poured into a mixture of ethyl acetate and water (100 mL, 1:1 by volume) The organic layer was separated, washed with water (2 x 50mL), dried over magnesium sulfate (anhyd), filtered and evaporated to dryness. The crude product was purified by flash chromatography (ethyl acetate: hexane,

1:1) to afford afforded 5 (0.40 g, 0.7 mmol) in 53% isolated yield.

Rf = 0.13 (hexane : ethyl acetate 1:1)

1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 7.05 (d,

J = 8.4 Hz, 2H), 6.70 (d, J = 8.5 Hz, 2H), 5.77 (s, 1H), 4.34-4.30 (m, 2H), 4.12 – 4.08 (m,

2H), 2.77 - 2.67(m, 1H), 2.66 – 2.55 (m, 3H), 2.43 (s, 3H), 2.50 – 2.19 (m, 6H), 2.18 –

1.96 (m, 3H), 1.93 – 1.85 (dd, 1H), 1.66 – 1.46 (m, 3H), 0.52 (s, 3H). 13C NMR (100

MHz, CDCl3) δ219.96, 218.96 , 199.34 , 156.25 , 156.02 , 145.07 , 144.83 , 136.72 ,

132.80 , 130.14 , 129.94 , 128.02, 123.46 , 114.72 , 68.20 , 65.47, 50.62 , 47.69 , 39.58 ,

37.90, 36.85 , 35.47 , 30.93 , 29.75 , 26.80 , 25.92 , 21.83, 21.14, 14.36.

LC -MS – m/z - observed M+1 – 561.42

Retention time – 3.04min

Synthesis of 11β-[4-(2-azidoethoxy)phenyl]-estra-4,9-diene-3,17-dione (6)

11β-[4-(2-tosyloxyethoxy)phenyl]-estra-4,9-diene-3,17-dione 5 (120 mg, 0.2 mmol) was dissolved in 8mL of hot ethanol. Sodium azide (55mg, 0.84 mmol) was added and the reaction was heated at reflux for 18 hrs. The reaction mixture was cooled to ambient temperature poured into 50mL ethyl acetate. The organic phase was washed with water (3

X 50mL), dried over magnesium sulfate, filtered and evaporated to dryness The crude

135 product was purified by flash chromatography (hexane: ethyl acetate 1:1) to afford 83mg

( 0.19 mmol) of 6, corresponding to isolated yield of 93%.

Rf = 0.29 ( hexane : ethyl acetate 1:1)

1H NMR (400 MHz, CDCl3) δ 7.10 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 5.79 (s,

1H), 4.11 (t, J = 4.8 Hz, 2H), 3.59 (t, J = 4.7 Hz, 2H), 2.80 – 2.70 (m, 1H), 2.63 (m, 3H),

2.55 – 2.24 (m, 6H), 2.20 – 2.07 (m, 2H), 2.05 – 1.98 (m, 1H), 1.92 (dd, 1H), 1.66 – 1.50

13 (m, 3H), 0.56 (s, 3H). C NMR (100 MHz, CDCl3) δ 230.13, 199.56 , 156.60 , 156.20 ,

145.02 , 136.80 , 130.32 , 128.18 , 123.64 , 114.91 , 67.13 , 50.81 , 50.40 , 47.88 , 39.76 ,

38.07, 37.98, 37.02 , 35.64 , 31.10 , 26.98 , 26.11 , 22.08 , 14.65. LC -MS – m/z - observed M+1 – 432.26. Retention time – 2.77min IR 2109.39 cm-1 (azide)

Synthesis of 3-hydroxy-11β-[4-(2-azidoethoxy)phenyl]-estra-1,3,5(10)-triene-17-one acetate (7)

To 11β-[4-(2-azidoethoxy)phenyl]-estra-4,9-diene-3,17-dione 6 (83mg, 0.19 mmol) dissolved in dichloromethane (5 mL) were added acetic anhydride (18μL, 0.2mmol) and acetyl bromide (35μL, 0.47mmol). The reaction solution was stirred at ambient temperature for 18 h and then quenched by the addition of aqueous sodium bicarbonate

(20 mL). The organic layer was washed with water, dried over magnesium sulfate

(anhyd), filtered and evaporated to dryness. All the aqueous fractions were back extracted with ethyl acetate. The organic fractions were collected and were dried with

MgSO4. The crude product was purified using flash chromatography (7:3 hexane: ethyl acetate) to give 67 mg (0.14 mmol) of 7, corresponding to 74% yield.

136

Rf = 0.18 (7:3 Hexane: Ethyl Acetate)

1 H NMR (400 MHz, CDCl3) δ 7.00 – 6.91 (m, 3H), 6.86 (s, 1H), 6.65 (d, J = 8.3 Hz,

2H), 5.29 (s, 1H), 4.07 – 3.96 (m, 2H), 3.61 – 3.46 (m, 2H), 3.07- 3.01 (m, 1H), 3.01 –

2.88 (m, 2H), 2.56 – 2.39 (m, 2H), 2.38 – 2.35 (m, 1H), 2.24 (s, 3H), 2.20 – 2.00 (m, 3H),

1.96 (dd, 1H), 1.72 – 1.43 (m, 3H), 0.45 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 220.20,

169.74 , 155.51, 148.19 , 137.53, 135.66, 130.58 , 127.57 ,121.73 , 119.24 , 113.89 ,

66.74 , 52.22 , 50.28 , 48.19 , 47.69 , 40.03 , 38.12 , 35.39 , 34.94 , 30.04 , 27.18 , 21.33 ,

15.25.

LC -MS – m/z - observed M+1 – 474.30 ; M+18 – 491.32. Retention time – 3.44min.

Synthesis of N-Dansyl propargylamine (8).

To dansyl chloride (55mg, 0.21mmol) dissolved in dichloromethane (4mL) was added sequentially triethyl amine (20µL, 0.21mmol) and propargyl amine (40µL, 0.63mmol).

The reaction was stirred at ambient temperature for 16 h and then evaporated to dryness.

The resultant crude material was purified by flash chromatography (hexane:ethyl acetate

3:2) to yield 40mg ( 0.14 mmol, 65%) of the final product 8.

Rf = 0.5 (1:1 hexane: ethyl acetate)

1 H NMR (400 MHz, CDCl3) δ 8.56 (d, J = 8.5 Hz, 1H), 8.26 (t, J = 8.3 Hz, 2H), 7.55

(dd, J = 14.8, 7.2 Hz, 2H), 7.19 (d, J = 7.5 Hz, 1H), 4.87 (t, J = 5.9 Hz, 1H), 3.77 (dd, J =

13 6.0, 2.5 Hz, 2H), 2.89 (s, 6H), 1.91 (s, 1H) C NMR (100 MHz, CDCl3) δ 152.19,

137

134.24, 130.97, 130.10, 129.98, 129.89, 128.73, 123.31, 118.61, 115.34, 77.86 72.84,

45.55, 33.16

LC -MS – m/z - observed M+1 – 289.17

Retention time 2.13 min

Synthesis of 7-nitro-N-(prop-2-yn-1-yl)benzo[c][1,2,5] oxadiazol-4-amine (9)

To 4-chloro-7-nitrobenzo[c][1,2,5] oxadiazole, (75mg 0.37mmol) dissolved in 3mL

THF, were added propargyl amine (26μL, 0.40mmol), cesium carbonate (136mg,

0.42mmol). The reaction mixture was stirred at ambient temperature for 18h. The reaction mixture was poured into ethyl acetate (50mL) and washed with water (2 x

25mL), dried over magnesium sulfate (anhydrous), filtered and evaporated to dryness.

The crude material was purified by flash chromatography (hexane: ethyl acetate 3:2) to give 53mg ( 0.24 mmol, 65% yield) of 9.

Rf = 0.17 ( hexane: ethyl acetate 7:3)

1 H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 8.5 Hz, 1H), 6.37 (t, J = 12.5 Hz, 2H), 4.31

(d, J = 3.7 Hz, 3H), 2.43 (s, 1H), 1.62 (s, 1H)

Synthesis of 11β-(4-(2-(4-(aminomethyl)-1H-1,2,3- triazol-1-yl)-ethoxy)phenyl) estra

4,9-diene-3,17-dione (10a)

11β-[4-(2-azidoethyoxy)phenyl]-estra-4,9-diene-3,17-dione, 6, (13mg, 0.03 mmol) was dissolved in tert-butanol : water ( 1:1, 4mL). N-Dansyl propargylamine, 8, (10mg, 0.03 mmol), sodium ascorbate (350µL, 0.035 mmol in a 0.1mmol/mL solution) and copper

(II) sulfate, (70µL, 0.007 mmol in a 0.1M solution) were added. The reaction vessel was

138 sealed and heated at 80oC for 16 hrs. The reaction was cooled and partitioned between ethyl acetate and water. The organic phase was washed sequentially with water and brine, dried over magnesium sulfate (anhydrous), filtered and evaporated to dryness. The crude material was purified by flash chromatography (ethyl acetate: hexane 9:1) to obtain 14mg

( 0.019 mmol, 66% yield) of 11β-(4-(2-(4-(aminomethyl)-1H-1,2,3- triazol-1-yl)- ethoxy)phenyl) estra-4,9-diene-3,17-dione 10a.

Rf = 0.13 (9:1 ethyl acetate: hexane)

1 H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 8.5 Hz, 1H), 7.85 (s, 1H), 7.08 (d, J = 8.3 Hz,

2H), 6.97 (s, 1H), 6.74 (d, J = 8.4 Hz, 2H), 6.34 (d, J = 8.5Hz, 1H), 5.79 (s, 1H), 4.83 -

4.78 (d, 2H), 4.40 – 4.27 (m, 2H), 3.34 (s, 1H), 2.77 - 2.67 (m, 1H), 2.66 – 2.57 (m, 3H),

2.53 – 2.32 (m, 6H), 2.31- 2.20 (m, 2H), 2.19 – 2.12 (m, 2H), 1.96 – 1.88 (dd, 1H), 0.95 –

13 0.80 (m, 3H), 0.51 (s, 3H). C NMR (100 MHz, CDCl3) δ 218.88, 199.22 , 155.85,

151.96 , 144.56 , 143.72, 137.06, 134.30, 130.12 , 130.12, 129.80, 129.59, 129.49,

128.61, 128.03 , 123.37, 123.07, 118.48, 115.28, 114.62, 66.04, 50.52, 49.64, 47.59,

45.39, 39.48, 38.79, 37.88, 37.82, 36.74, 35.36, 30.82, 26.70, 25.84, 21.80, 14.43.

LC -MS – m/z - observed M+1 – 720.27. Retention time 2.66 min.

IR 1735.46 cm-1 (17 C=O), 1658.35 cm-1 (3 C=O)

Synthesis of 11β-(4-(2-4(-((( 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)methyl-1H-

1,2,3-triazol-1-yl)ethoxy)phenyl)-estra-4,9-diene-3,17-dione (10b)

To 11β-[4-(2-azidoethyoxy) phenyl]-estra-4,9-diene-3,17-dione, 6, (16mg 0.04 mmol) of was dissolved in a solution of tert butanol-water (1:1, 5mL v/v). 7-nitro-N-(prop-2-yn-1- yl) benzo[c][1,2,5] oxadiazol-4-amine 9 (15mg, 0.06mmol) sodium ascorbate (350µL

139

0.035 mmol in a solution of 0.1mmol/mL) , copper (II) sulfate, (70µL, 0.007 mmol in a solution of 0.1mmol/mL) were added and the reaction vial was sealed. The reaction was heated at 80oC with stirring for 18h, cooled to ambient temperature and partitioned between ethyl acetate and water. The organic phase was washed with water (2 x 20mL), brine, dried over magnesium sulfate (anhydrous), filtered and evaporated to dryness. The crude solid was purified by flash chromatography (ethyl acetate : hexane, 9:1) to give the pure product 10b (12mg, 0.018mmol) in 46% isolated yield.

Rf = 0.04 (9:1 ethyl acetate: hexane)

1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 8.5 Hz, 1H), 7.85 (s, 1H), 7.26 (s, 1H), 7.08

(d, J = 8.3 Hz, 2H), 6.97 (s, 1H), 6.74 (d, J = 8.4 Hz, 2H), 6.35 (d, J = 8.5 Hz, 1H), 4.81

(d, J = 18.5 Hz, 2H), 4.44 – 4.25 (m, 2H), 3.34 (s, 1H), 2.77 – 2.68 (m, 1H), 2.54 – 2.0

(m, 9H), 1.96 – 1.88 (dd, 1H), 1.92 (dd, J = 13.7, 7.2 Hz, 1H), 1.61 – 1.50 (m, 3H), 0.96 –

0.78 (m, 3H), 0.51 (s, 3H).

LC -MS – m/z - observed M+1 – 650.21. Retention time 2.34 min.

IR= 1733.58 cm-1 (17 C=O), 1657.42 cm-1 (3 C=O)

Synthesis of 3-hydroxy-11β-(4-(2-(4-(((5-dimethylamino) naphthalene)-1-sulfonamido)- methyl)-1H-1,2,3-triazol-1-yl)ethoxy)phenyl)-estra-1,3,5(10)-triene-17-one-3-acetate

(11a)

3-Hydroxy-11β-[-4-(2-azidoethoxy)-phenyl]-estra-1,3,5(10)-triene-17-one acetate 7

(36mg , 0.08 mmol) was dissolved in tert-butanol – water (6mL, 1:1 solution). N-Dansyl propargylamine, 8a, (33mg, 0.1mmol), sodium ascorbate (350µL, 0.035 mmol in a solution of 0.1M), copper (II) sulfate (70µL ,0.007 mmol in a solution of 0.1M) were

140 added to the reaction vial, sealed and the reaction was heated at 90oC for 18 h. The reaction was cooled and partitioned between ethyl acetate and water. The organic phase was washed sequentially with water and brine, dried over magnesium sulfate (anhyd), filtered and evaporated to dryness. The crude material was purified by flash chromatography (step gradient from 1:1 to 4:1 ethyl acetate: hexane) to give the pure acetylated intermediate (40mg, 0.052 mmol, 75%).

The acetylated intermediate (20mg, 0.03mmol) was dissolved in methanol (4mL) and saponified by the addition of 4.2mg (0.07mmol) of potassium hydroxide. Isolation of the product yielded 11a (18mg, 99%).

Rf = 0.16 (7:3 hexane: ethyl acetate)

1 H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 8.5 Hz, 1H), 8.17 (t, J = 7.8 Hz, 2H), 7.44 (t, J

= 8.0 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.32 (s, 1H), 7.11 (d, J = 7.5 Hz, 1H), 6.98 (d, J =

8.1 Hz, 2H), 6.75 (d, J = 8.4 Hz, 1H), 6.62 (s, 1H), 6.54 (d, J = 8.2 Hz, 2H), 6.38 (d, J =

7.9 Hz, 1H), 5.74 (s, 1H), 5.29 (s, 1H), 5.11 (s, 1H), 4.43 (s, 2H), 4.15 (d, J = 5.5 Hz,

2H), 4.05 (d, J = 4.3 Hz, 2H), 3.97 (s, 1H), 2.85 (s, 6H), 2.47 (d, J = 14.9 Hz, 2H), 2.30

(d, J = 11.2 Hz, 2H), 2.18 – 2.03 (m, 3H), 2.00 – 1.90 (m, 1H), 1.67 – 1.40 (m, 3H), 0.43

(s, 3H).

13 C NMR (100 MHz, CDCl3) 154.84 , 153.54 , 151.80 , 137.65 , 136.76 , 134.60 ,

130.84 , 130.62, 129.75, 129.65 , 129.55 , 128.69, 127.63, 123.27, 118.82 , 115.59,

115.41 , 113.79 , 113.65, 65.81 , 52.15 , 49.78 , 48.34 , 47.41 , 45.55 , 39.98 , 38.53 ,

38.24 , 35.48 , 35.18 , 30.11 , 29.83, 27.34, 21.48 , 15.37 , -29.25.

LC -MS – m/z - observed M+1 – 720.28. Retention time 2.83min.

141

IR = 1735.46 cm-1 (17 C=O)

Synthesis of 11 β – (4-(2-(4-(((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)methyl)-1H-

1,2,3-triazol-1-yl-ethoxy)phenyl)-estra-1,3,5(10)trien-17-one acetate (11b)

3-Hydroxy-11β-[-4-(2-azidoethoxy)-phenyl]-estra-1,3,5(10)-triene-17-one acetate (7)

(20mg , 0.04 mmol) was dissolved tert- butanol – water ( 5mL, 1:1 v/v). 7-nitro-N-(prop-

2-yn-1-yl)benzo[c][1,2,5] oxadiazol-4-amine, 9a, (18mg, 0.08 mmol), sodium ascorbate,

(350µL , 0.035 mmol – 0.1M solution), copper (II) sulfate (70µL , 0.007 mmol – in a

0.1M solution) were added and the reaction vessel was sealed. The reaction was heated to

80oC and stirred for 18h. The reaction was cooled to ambient temperature and partitioned between ethyl acetate and water. The organic phase was washed with water (2 x 20 mL), brine, dried over magnesium sulfate (anhydrous), filtered and evaporated to dryness. The crude solid was purified by flash chromatography (ethyl acetate: hexane, 4:1) to give

21mg of the pure intermediate. 10mg (0.01mmol) of the intermediate was dissolved in methanol (4mL) and saponified by the addition of potassium hydroxide (4.2mg,

0.07mmol). After stirring for 2h, the reaction was quenched by the addition of glacial acetic acid and partitioned between ethyl acetate and water. The organic phase was washed with water (2 x 20mL), brine, dried over magnesium sulfate (anhydrous), filtered and evaporated to dryness. The crude solid was purified by flash chromatography (ethyl acetate: hexane, 4:1) to give 18mg of the pure product in 90% isolated yield.

Rf = 0.28, 100 % Ethyl acetate

1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 8.5Hz, 1H), 7.81 (s, 1H), 7.09 – 7.01(m, 1H),

6.97 (s, 1H), 6.73 (d, J = 8.5 Hz, 2H), 6.60 (s, 1H), 6.53 (d, J = 8.2 Hz, 1H), 6.37 (d, J =

142

8.2 Hz, 1H), 6.33 (d, J = 8.6 Hz, 1H), 4.79 (d, J = 5.1 Hz, 2H), 4.71 (s, 2H), 4.21 (s, 2H),

3.37 (s, 1H), 2.93 – 2.81 (m, 1H), 2.55 – 1.9 (m, 9H), 1.70 – 1.42 (m, 3H), 0.92 - 0.78 (m,

2H), 0.40 (s, 3H)

LC -MS – m/z - observed M+1 – 650.18. Retention time 2.57 min.

IR = 1733.29 cm-1 (17 C=O)

Synthesis of 11β-((-(4-(2-(4-(((5-dimethylamino) naphthalene)-1-sulfonamido)-methyl)-

1H-1,2,3-triazol-1-yl)ethoxy)phenyl)-estra-1,3,5(10)-triene-3,17β-diol (12a)

To 3-hydroxy-11β-(4-(2-(4-(((5-dimethylamino) naphthalene)-1-sulfonamido)-methyl)-

1H-1,2,3-triazol-1-yl)ethoxy)phenyl)-estra-1,3,5(10)-triene-17-one-3-acetate 10 (20mg

0.03mmol) dissolved in methanol (4mL) was added sodium borohydride (1.8mg,

0.05mmol) at 0oC. The reaction stirred for 4h, warming to ambient temperature.

Potassium hydroxide in methanol (4.2mg, 2mL) was added and the reaction was stirred for an additional 16h. The reaction was quenched by the addition of glacial acetic acid and partitioned between ethyl acetate and water. The organic fraction was washed with water (2 x 10mL), dried over magnesium sulfate (anhydrous), filtered and evaporated to dryness. The product was isolated in essentially quantitative yield (14 mg, 0.019mmol,

99%).

Rf = 0.18 (7:3 ethyl acetate: hexane)

1 H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 8.5 Hz, 1H), 8.17 (t, J = 8.0 Hz, 2H), 7.40 (dt,

7.9 Hz, 2H), 7.28 (s, 1H),7.09 (d, J = 7.5 Hz, 1H), 6.95 (d, J = 8.0 Hz, 2H), 6.72 (d, J =

8.5 Hz, 1H), 6.59 (s, 1H), 6.50 (d, J = 8.1 Hz, 2H), 6.36 (d, J = 7.9 Hz, 1H), 5.76 (s, 1H),

5.11 (s, 1H), 4.39 (s, 2H), 4.12 (dd, , 6.8 Hz, 4H), 4.01 (s, 2H), 3.88 (s, 1H), 3.66 (m,

143

1H), 3.34 (s, 1H), 2.84 (s, 6H), 2.46 (d, J = 12.7 Hz, 1H), 2.18 – 1.95 (m, 5H), 1.80 –

13 1.63 (m, 1H), 0.94 – 0.77 (m, 3H), 0.30 (s, 3H). C NMR (100 MHz, CDCl3) δ 154.64,

153.37, 151.88, 137.87, 137.26, 134.56, 130.92, 130.65, 130.34, 129.78, 129.69, 129.57,

128.70, 127.64, 123.28, 118.78, 115.52, 115.39, 113.61, 113.48, 65.83, 60.57, 51.79,

49.76, 47.32, 45.54, 43.76, 38.65, 38.45, 35.55, 30.26, 29.84, 28.03, 23.28, 21.21, 14.33.

LC -MS – m/z - observed M+1 – 722.27. Retention time – 2.61min.

IR = 3296.03 cm-1, 3265.35 cm-1

Synthesis of 11 β – (4-(2-(4-(((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)methyl)-1H-

1,2,3-triazol-1-yl-ethoxy)phenyl)-estra-1,3,5(10)trien-3,17β-diol (12b)

To 11 β – (4-(2-(4-(((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)methyl)-1H-1,2,3- triazol-1-yl-ethoxy)phenyl)-estra-1,3,5(10)trien-17-one acetate 11b (10mg , 0.03mmol) dissolved in methanol (4mL) at 0oC, was added sodium borohydride (1mg, 0.03 mmol) and the reaction was warmed to ambient temperature and stirred for an additional 2h.

Potassium hydroxide (5mg, 0.07mmol) was added and the reaction was stirred for an additional16h. The reaction was quenched by the addition of glacial acetic acid and partitioned between ethyl acetate and water. The organic phase was washed with water (2 x 20mL), brine, dried over magnesium sulfate (anhydrous), filtered and evaporated to dryness. The crude solid was purified by flash chromatography (ethyl acetate: hexane

9:1) to give the pure product 12b (7mg, 0.01mmol) in 74% yield.

Rf = 0.22, 100% ethyl acetate

1 H NMR (400 MHz, CDCl3) δ 8.43 (t, J = 8.6 Hz, 1H), 7.77 (s, 1H), 6.95 (d, J = 8.1 Hz,

1H), 6.86 (s, 1H), 6.76 (d, J = 8.5 Hz, 2H), 6.58 (s, 1H), 6.53 (d, J = 8.3 Hz, 1H), 6.35

144

(dd, J = 14.5, 8.5 Hz, 2H), 4.79 (s, 2H), 4.72 (s, 2H), 4.23 (s, 2H), 3.04 - 2.92 (m, 1H),

2.90 – 2.74 (m, 2H), 2.57 (s, 1H), 2.45 (dd, 1H), 2.37 – 2.29 (m, 1H), 2.21 (dd, 1H), 2.11,

(d, 1H), 2.00 (m, 1H), 1.82 – 1.48 (m, 7H), 0.88 (m, 2H), 0.28 (s, 3H).

LC -MS – m/z - observed M+1 – 652.15. Retention time 2.41 min IR = 3321.91 cm -1

145

1H NMR of 2a

13 C NMR of 2a

146

Mass spectrum of 2a

147

1H NMR of 4

13C NMR of 4

148

LC Chromatogram of 4

Mass Spectrum of 4

149

1H NMR of 5

13C NMR of 5

150

LC Chromatogram of 5

Mass Spectrum of 5

151

1H NMR of 6

13C NMR of 6

152

LC chromatogram of 6

Mass spectrum of 6

153

1H NMR of 7

13C NMR of 7

154

LC Chromatogram of 7

Mass spectrum of 7 M+1 value =474. M+18 = 491. M-32 = 446.

155

1H NMR of 8a

13C NMR of 8a

156

LC Chromatogram of 8a

Mass Spectrum of 8a

157

1H NMR of 9a

158

1H NMR of 10a

13C NMR of 10a

159

LC Chromatogram of 10a UV peak at 1.01 is an artifact of the solvent, Methanol

Mass Spectrum of 10a

160

IR spectrum of 10a

161

1H NMR of 11a

13C NMR of 11a

162

LC Chromatogram of 11a

Mass Spectrum of 11a

163

IR spectrum of 11a

164

1H NMR of 12a

13C NMR of 12a

165

LC Chromatogram of 12a

Mass spectrum of 12a

166

IR spectrum of 12a

1H NMR of 10b

167

LC Chromatogram of 10b

Mass spectrum of 10b M+1 = 650

168

IR Spectrum of 10b

1H NMR of 11b

169

LC Chromatogram of 11b

Mass spectrum of 11b

170

IR spectrum of 11b

171

1 H NMR of 12b in CDCl3

1H NMR of 12b in CD3OD

172

LC Chromatogram of 12b

Mass spectrum of 12b

173

IR spectrum 12b

174

Fluorescence spectra – Absorption and emission, normalized

10b Control-NBD 10a Control-Dansyl 1.2 1.2 Normalized Emission Spectrum Normalized Absorbance 1 Normalized Emission 1 Normalized Absorbance

0.8 0.8

0.6 0.6

0.4 0.4

Normalized Intensity Normalized Normalized Intensity Normalized 0.2 0.2

0 0 260 460 660 300 400 500 600 700 800 -0.2 -0.2 Wavelength (nm) Wavelength (nm) 11a Estrone-Dansyl 11b Estrone-NBD 1.2 1.2 Normalized Absorbance 1 Normalized Absorbance 1 Normalized Emission 0.8 Normalized Emission 0.8 0.6 0.6 0.4 0.4 Normalized Intensity Normalized 0.2 0.2 0 INtensity Normalized 300 400 500 600 700 800 0 -0.2 Wavelength ( nm) 300 400 500 600 700 800 -0.2 Wavelength (nm)

12a Estradiol-Dansyl 12b Estradiol-NBD 1.2 1.2 Normalized Absorbance Normalized Absorption Normalized Emission 1 1 Normalized Emission 0.8 0.8

0.6 0.6

0.4 0.4 Normalized Intensity Normalized

0.2 0.2 Normalized Intensity Normalized

0 0 300 400 500 600 700 800 300 400 500 600 700 800 -0.2 -0.2 Wavelength (nm) Wavelength (nm)

175

176