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Chemical and biological studies of novel ligands of the human androgen receptor

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Chemical and biological studies of novel ligands of the human androgen receptor

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Content CHEMICAL AND BIOLOGICAL STUDIES OF NOVEL LIGANDS OF
THE HUMAN ANDROGEN RECEPTOR

by

Yuanye Sun

___________________________________________________

A Dissertation Presented to the
Faculty of the Graduate School
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
(CHEMISTRY)

May 2022

Copyright 2022 Yuanye Sun
ii

Acknowledgements
The work presented in this dissertation would not have been possible without my mentors,
colleagues, family, and friends. I am grateful to all of you.
First, I would like to thank Prof. McKenna. Thank you for giving me the opportunity to
work in your group and for providing invaluable guidance and support.
I would like to thank Boris and Inah, who helped me in the lab with scientific and
administrative work. I would like to thank USC Graduate School and the Chemistry department
for admission. I would also like to thank the Chemistry department, Dornsife T32 Chemistry-
Biology Interface Training Fellowship, Dornsife College and USC Graduate School Ph.D.
Fellowship in the Sciences, and the Lawrence J. Ellison Institute for Transformative Medicine of
USC for funding.
I would like to thank my colleague Chao for being such a good mentor. I would like to
thank our collaborators at the Keck School of Medicine and the Ellison Institute: Katherin, Greg,
Harish, Prof. Ruderman, Prof. Agus, Prof. Lee, and everyone else who contributed to the project.
I would also like to thank Ray, Fariborz, Jeffrey, and everyone in the Stevens group and the
Cherezov group at the Bridge Institute, where I learned about protein crystallography and
conducted crystallographic work.
I would like to thank all past and current members in the McKenna group, whom I spent a
lot of time with every day. Everyone is kind, helpful, and fun.
I would like to thank my friends. I feel extremely fortunate to have you in my life.
Finally, I would like to thank my parents, for their unconditional and unlimited love.

iii

Table of Contents
ACKNOWLEDGEMENTS ............................................................................................................ ii
LIST OF TABLES .......................................................................................................................... v
LIST OF FIGURES ....................................................................................................................... vi
LIST OF SCHEMES.................................................................................................................... viii
ABSTRACT ................................................................................................................................... ix
CHAPTER 1 INTRODUCTION .............................................................................................. 1
1.1 Prostate cancer ................................................................................................................ 1
1.2 The role of androgen receptor (AR) in prostate cancer .................................................. 2
1.2.1 AR structure and function ....................................................................................... 2
1.2.2 AR pharmacology ................................................................................................... 5
1.2.3 AR in prostate cancer .............................................................................................. 7
1.3 Antiandrogens and other treatments for prostate cancer................................................. 8
1.4 Our approach to new antiandrogens ............................................................................. 10
1.4.1 Clinical need for new antiandrogens..................................................................... 10
1.4.2 Our starting point: BMS-641988 .......................................................................... 11
1.4.3 Improving BMS-641988 ....................................................................................... 13
Chapter 1 references ................................................................................................................. 15
CHAPTER 2 AR REGULATION BY ENANTIOMERIC LIGANDS .................................. 18
2.1 Chiral molecules in drug discovery .............................................................................. 18
2.2 AR agonist-antagonist pairs of enantiomeric ligands ................................................... 19
2.2.1 AR agonist-antagonist pairs of enantiomeric compounds .................................... 20
2.2.2 Impact of chiral impurities .................................................................................... 23
2.3 In silico models of novel AR ligands bound to AR ...................................................... 25
2.3.1 Induced fit docking models ................................................................................... 25
2.3.2 State of the art of AR molecular dynamics ........................................................... 27
2.4 Efforts to obtain experimental models of AR LBD-agonist binary complexes ............ 29
2.5 Chiral purity in large-scale manufacturing ................................................................... 35
2.6 Experimental section ..................................................................................................... 38
Chapter 2 references ................................................................................................................. 42
iv

CHAPTER 3 EVALUATION OF NOVEL AR ANTAGONISTS ........................................ 45
3.1 Design and synthesis of novel AR antagonists ............................................................. 45
3.1.1 Design rationale .................................................................................................... 45
3.1.2 Synthesis ............................................................................................................... 46
3.2 In vitro screening of novel AR antagonists................................................................... 48
3.2.1 Agonistic activities of BMS-641988 .................................................................... 48
3.2.2 Structure-activity relationship (SAR) studies ....................................................... 48
3.3 In vitro evaluation of lead compounds.......................................................................... 54
3.3.1 In vitro efficacy studies ......................................................................................... 54
3.3.2 In vitro and in silico safety studies ....................................................................... 55
3.4 Gram scale synthesis and purification of key intermediate .......................................... 57
3.5 Experimental section ..................................................................................................... 60
Chapter 3 references ................................................................................................................. 74
CHAPTER 4 SELECTIVE AR DEGRADERS BASED ON NOVEL SCAFFOLD ............. 76
4.1 Induced protein degradation and selective AR degraders ............................................. 76
4.1.1 Induced protein degradation ................................................................................. 76
4.1.2 Design principles of selective AR degraders (SARDs) ........................................ 77
4.1.3 Exemplary SARDs in clinical and preclinical studies .......................................... 80
4.2 SARDs based on our scaffold ....................................................................................... 81
4.2.1 Design rationale .................................................................................................... 81
4.2.2 Synthesis ............................................................................................................... 82
4.3 Future directions ........................................................................................................... 85
4.4 Experimental section ..................................................................................................... 86
Chapter 4 references ................................................................................................................. 93
CHAPTER 5 CONCLUSIONS AND OUTLOOK................................................................. 95
Chapter 5 references ................................................................................................................. 98
BIBLIOGRAPHY ......................................................................................................................... 99
APPENDICES ............................................................................................................................ 107

v

List of Tables
Table 1.1 Treatments for prostate cancer ...................................................................................... 10
Table 1.2 Our goal of improving BMS-641988............................................................................ 14
Table 2.1 Crystallization conditions screened for AR LBD-DHT binary complex. .................... 30
Table 2.2 Crystallization conditions screened for AR LBD-agonist binary complexes. .............. 32
Table 2.3 Refolding buffers screened for AR LBD refolding. ..................................................... 33
Table 3.1 SAR studies of novel AR antagonists. .......................................................................... 49
Table 3.2 In vitro efficacy of lead compounds. ............................................................................ 55

vi

List of Figures
Figure 1.1 Incidence and mortality rate of prostate cancer. ............................................................ 1
Figure 1.2 The domains of AR ....................................................................................................... 3
Figure 1.3 Structure of AR LBD in complex with DHT (PDB: 2AMA). ...................................... 3
Figure 1.4 Interactome of AR on STRING interaction network. ................................................... 5
Figure 1.5 Natural and synthetic androgens. .................................................................................. 6
Figure 1.6 Second generation antiandrogens. ................................................................................. 9
Figure 1.7 Scaffolds and lead compounds of AR antagonists from BMS. ................................... 12
Figure 1.8 In vivo metabolism of BMS-641988. .......................................................................... 13
Figure 2.1 Structures of Thalidomide and Ketamine. ................................................................... 19
Figure 2.2 Structure of the intermediate I. .................................................................................... 20
Figure 2.3 Hyperspeckling of GFP-AR under treatments of BMS-641988 and S-BMS. ............. 21
Figure 2.4 Hyperspeckling and luciferase expression under treatments of enantiomeric
compounds. ................................................................................................................................... 21
Figure 2.5 Gene expression profile in VCaP cells treated by compound 06R/S. ......................... 22
Figure 2.6 Luciferase expression and viability of VCaP cells when treated with contaminated R-
enantiomer drugs. .......................................................................................................................... 24
Figure 2.7 Potency and efficacy of enantiomers. .......................................................................... 24
Figure 2.8 In silico models of AR LBD bound to 02S and 02R. .................................................. 26
Figure 2.9 Mutation studies of key residues in the AR LBD binding pocket. .............................. 26
Figure 2.10 Structures of bicalutamide, R-3, S-1. ........................................................................ 28
Figure 2.11 Experimental model of AR LBD in complex with DHT obtained in our group. ...... 30
Figure 2.12 Size Exclusion Column chromatography of AR LBD after ligand exchange. .......... 34
Figure 2.13 Mass spectrometry of extracted ligands of protein sample after ligand exchange .... 34
Figure 2.14 Study of mobile phase for chiral separation of intermediate I and its enantiomer. ... 35
vii

Figure 2.15 Study of loading for chiral separation of intermediate I and its enantiomer. ............ 36
Figure 2.16 Study of enantiopurity of intermediate I and its enantiomer after one round of chiral
purification. ................................................................................................................................... 37
Figure 2.17 Study of enantiopurity of intermediate I and its enantiomer after two rounds of chiral
purification. ................................................................................................................................... 37
Figure 3.1 Predicted metabolism of representative compounds ................................................... 46
Figure 3.2 BMS-641988 is an AR agonist in LNCaP cells expressing AR T878A. .................... 48
Figure 3.3 Fragment approach and summary of SAR derived from our compounds. .................. 49
Figure 3.4 Atropisomer separation of Compound 11R. ................................................................ 52
Figure 3.5 Structure of proxalutamide (GT-0918). ....................................................................... 54
Figure 3.6 Calculated logBB of representative clinical compounds and lead compounds. .......... 56
Figure 3.7 Predicted SOM for lead compounds............................................................................ 56
Figure 3.8 Extracted ion chromatograms of microsomal metabolites of representative
compounds. ................................................................................................................................... 57
Figure 3.9 Purification of Diels-Alder cycloaddition reaction. .................................................... 59
Figure 3.10 Chiral separation of intermediate I and its enantiomer. ............................................ 60
Figure 4.1 Examples of hydrophobic tagging. .............................................................................. 76
Figure 4.2 Examples of ligands of E3 ligases ............................................................................... 77
Figure 4.3 Strategies to combat AR antagonist-resistant prostate cancer ..................................... 78
Figure 4.4 Examples of AR modulators as SARDs ...................................................................... 78
Figure 4.5 Structures of two rationally designed SARDs. ............................................................ 79
Figure 4.6 Structure of ARV-110. ................................................................................................ 80
Figure 4.7 Structure of MTX-23 ................................................................................................... 81
Figure 4.8 Potential tethering point for SARD. ............................................................................ 82
Figure 4.9 Testing of proof-of-concept SARD molecules ............................................................ 85
Figure 4.10 Dosing of PROTAC .................................................................................................. 86
viii

List of Schemes
Scheme 3.1 Synthesis and chiral separation of derivatives of BMS-641988. .............................. 47
Scheme 3.2 Synthesis and chiral separation of Compound 05R/S. .............................................. 54
Scheme 4.1 Convergent synthesis of SARDs based on our scaffold ............................................ 83
Scheme 4.2 Synthesis of Compound 20. ...................................................................................... 83
Scheme 4.3 Attempted ester deprotection of methyl 6-(3-(2-(2-(adamantan-1-
yl)acetamido)ethoxy)propanamido)-1H-indole-3-carboxylate. .................................................... 84
Scheme 4.4 Synthesis of Compound 21. ...................................................................................... 84

.
ix

Abstract
Prostate cancer accounts for around 13% of all male cancer diagnoses and 7% of all cancer
diagnoses worldwide. The androgen receptor (AR) is the master regulator in the androgen
signaling pathway, and it is a critical factor in all stages of prostate cancer including castration
resistant prostate cancer (CRPC). Second generation antiandrogen drugs such as enzalutamide
exhibit limited efficacy profiles due to drug resistance and unfavorable safety profiles due to
neurotoxicity. Thus, there is an urgent clinical need for novel antiandrogens that can overcome
drug resistance. In a medicinal chemistry campaign to develop novel antiandrogens, we addressed
these issues by modifying BMS-641988, a known AR antagonist. We discovered that the
enantiomer of BMS-641988 is a potent AR agonist, and this chirality-dependent agonist-antagonist
pairing is present consistently in a series of AR ligands based on the BMS-641988 scaffold.
Additionally, we conducted extensive structure-activity relationship (SAR) studies and identified
several promising lead compounds that exhibited excellent in vitro safety and efficacy profiles.
Furthermore, we developed novel selective AR degraders (SARDs) based on our compounds. Our
research provides new insights into the molecular dynamics of AR and a cautionary tale in drug
discovery. Our compounds could lead to new treatments for drug resistant prostate cancer and
other AR-related diseases.

1

Chapter 1 Introduction
1.1 Prostate cancer
The prostate is a male reproductive organ that produces seminal fluid.
1
Prostate cancer is
one of the most common cancers affecting male worldwide, and it is especially prominent in
developed countries (Figure 1.1).
1
Globally, Prostate cancer accounts for around 13% of all male
cancer diagnoses and 7% of all cancer diagnoses.
1
Approximately 4% of all cancer deaths in the
world can be attributed to prostate cancer.
1
In the US, one in seven men will be diagnosed with
prostate cancer within his lifetime.
1

Several risk factors are associated with prostate cancer diagnosis. Prostate cancer
unproportionally affects men over the age of 65 years.
1, 2
It is most prevalent in Western countries
and has an extremely high mortality rate among patients of African descent (Figure 1.1).
1, 2

Additionally, genetic mutations, family history, as well as unhealthy lifestyles all predispose men
to prostate cancer.
2
Routine prostate specific antigen (PSA) testing is the most common way to
detect prostate cancer.
1
Since its introduction in the 1980’s, PSA testing has enabled early
diagnosis and improved prostate cancer survival rates.
1, 2

Figure 1.1 Incidence and mortality rate of prostate cancer.
2

Prostate cancer generally has a high 5-year survival rate, especially for low-grade
diseases.
1, 2
In the US, the 5-year survival rate is well above 95%.
1, 2
Despite the high survival rate,
prostate cancer is still the second leading cause of cancer mortality among males in the US.
2

1.2 The role of androgen receptor (AR) in prostate cancer
The androgen receptor (AR) belongs to the steroid hormone group of nuclear receptors,
together with the progesterone receptor, the glucocorticoid receptor, the mineralocorticoid
receptor, and the estrogen receptor.
3, 4
AR is expressed in multiple organs including the prostate,
adrenal gland, skeletal muscle, and the central nervous system (CNS), with the highest expression
level in the prostate.
4
AR is a ligand-dependent transcription factor that controls the expression of
specific genes.
4, 5

1.2.1 AR structure and function
Like other nuclear receptors, AR consists of three domains (Figure 1.2): the N-terminal
domain (NTD), the DNA binding domain (DBD), and the C-terminal ligand binding domain
(LBD).
3-6
DBD tethers AR to androgen response element (ARE), which is a consensus sequence
5’-TGTTCT-3’ recognized by the AR DBD.
3, 4
ARE is generally located in the promoter or
enhancer region of AR-targeting genes.
3, 4
It has been shown that non-coding AR binding sites are
frequently mutated in prostate cancer, which contributes to elevated AR activities.
7
The activation
function 1 (AF1) domain in the NTD is constitutively active, and can recruit transcription
machinery independent of AR ligands.
3, 4
The activation function 2 (AF2) domain in the LBD is
ligand dependent.
3, 4
There is no experimental structure of full-length AR, but the structure of
agonist-bound LBD have been solved (Figure 1.3, PDB:2AMA).
8
LBDs of steroid receptors share
3

a similar structural feature, the “ 𝛼 -helical sandwich”, including AR LBD (Figure 1.3).
4
All the
available AR LBD structures are solved in complex with an agonist, and there is no structure
available for apo-state or antagonist-bound structures.
8
The state of art of AR structure and
molecular dynamics will be further discussed in Chapter 2.

Figure 1.2 The domains of AR
NTD: N-terminal domain; DBD: DNA binding domain; H: hinge; LBD: ligand binding domain.
AF1 and AF2 are located within NTD and LBD, respectively.

Figure 1.3 Structure of AR LBD in complex with DHT (PDB: 2AMA).
The AR LBD has a “helical sandwich” structural feature. The helices are colored in rainbow. DHT
is colored in pink.

In the cytosol, AR is associated various heat shock proteins (HSPs).
4
These HSPs work
together to stabilize AR and to prevent AR degradation.
4
Upon agonist binding, AR dissociates
4

from HSPs, dimerizes, binds to importin-α, and enters the nucleus.
4
This process is called
translocation.
4
Nuclear AR dimers then bind to the ARE, recruit co-regulators and the
transcriptional machineries.
4
The process of initiating or inhibiting transcription of target genes is
called transactivation.
4
Translocation and transactivation are distinct cellular processes that can be
studied by different assays such as fluorescence microscopy and the luciferase transcription assay.
9

On the other hand, AR bound to antagonist does not recruit transcriptional machineries because of
their structural conformations.
4

In addition to the genomic pathway, the non-genomic pathway of AR has also been found
in multiple organs including the prostate.
3
Rather than translocating to the nucleus, AR activates
other cytosolic proteins such as kinases, and thus initiates signaling cascades without any
transcriptional activity.
3
For example, AR is involved in the phosphoinositide 3-kinase – protein
kinase B (PI3K-AKT) pathway, which encompasses many well-known oncogenes such as mTOR,
BCL, and p53.
5
A search for proteome-wide AR interaction on STRING interaction network
resulted in more than 100 direct interactors (highest confidence, interaction score > 0.9) (Figure
1.4).
10
As we continue to study the biology of AR, it is certain that this interactome network will
become larger.
5

Figure 1.4 Interactome of AR on STRING interaction network.
AR is in the center and labeled in a red circle. All lines represent direct protein-protein interactions.

1.2.2 AR pharmacology
The two most prominent endogenous androgens are testosterone (Figure 1.5) and 5α-
dihydrotestosterone (DHT, Figure 1.5).
3
The biosynthesis of testosterone is regulated by
luteinizing hormone, which is in turn regulated by gonadotropin-releasing hormone.
3
Testosterone
can be converted into the more potent DHT by intracellular 5α-reductase.
3
Androgens have both
androgenic and anabolic effects.
3, 4
They are responsible for male sexual differentiation in puberty
as well as spermatogenesis in adult male.
3, 4
They also regulate physiological conditions such as
6

muscle mass and bone mineral density.
4
Because of this, androgens are used in a variety of clinical
applications, such as male hypogonadism, chronic renal failure, and trauma.
4

Besides testosterone and DHT, many steroidal AR agonists have been synthesized. Most
notably, R1881 (methyltrienolone, Figure 1.5) is a synthetic steroid with Kd below 1 nM, much
lower than that of either testosterone or DHT.
4
Even though R1881 is not approved for any medical
use in the US, it is regularly used in research settings as a potent AR agonist. Anabolic steroids,
such as oxandrolone (Figure 1.5), are a class of synthetic androgens which possess stronger
anabolic than androgenic activities.
4
They are medically used for various indications including
chronic infections and trauma.
4

Some synthetic steroids antagonize the androgenic effects of androgens, and thus these
steroids are antiandrogens. Steroidal antiandrogens such as cyproterone acetate (Figure 1.5) have
therapeutic potential in the treatment of acne and for male contraception.
4
Since the clinical
application of steroidal AR ligands is often limited by low oral bioavailability and hepatotoxicity,
the development of AR antagonists has been largely focused on nonsteroidal antiandrogens.
4

Currently, all the approved antiandrogens for the treatment of prostate cancer are nonsteroidal
antiandrogens.
3, 4

Figure 1.5 Natural and synthetic androgens.

7

To separate the androgenic effects of AR from its anabolic effects, selective AR modulators
have been developed.
4
These molecules are weakly agonistic or even antagonistic in the prostate
but strongly agonistic in the pituitary and muscle.
4
Due to their tissue selectivity, AR modulators
can be used to treat muscle-related conditions without stimulating the prostate.
3, 4

1.2.3 AR in prostate cancer
Prostate cells require AR and androgens to survive and proliferate, and the rates of cell
proliferation and cell death are controlled by AR signaling pathway.
4
In prostate cancer cells,
perturbation of the androgen signaling pathway leads to uncontrolled cell growth.
4

Both the initiation and the progression of prostate cancer can usually be attributed to
activation or dysregulation of androgen-dependent growth-promoting pathways.
3
For instance, AR
mutations and variants are highly correlated with predisposition of prostate cancer in several meta-
analyses.
3
Additionally, increased serum levels of the prostate specific antigen, which suggests
increased activity of AR, is an indirect biomarker in prostate cancer patients.
3
Furthermore, AR is
involved in various androgen-dependent biochemical pathways, including the RAS/RAF pathway,
the Wnt β–catenin pathway, the PI3K–AKT–mTOR pathway, and many others indicated in DNA
repair and cell cycle regulation.
3, 11
The underlying biology of prostate cancer provided the
rationale for the use of androgen deprivation as treatments. Androgen deprivation was first shown
to be an effective treatment for prostate cancer by the performance of orchiectomy.
3

Although prostate cancer in general has an extremely high 5-year survival rate, castration-
resistant prostate cancer (CRPC) is associated with poor prognosis.
12
Patients on androgen
deprivation therapy inevitably relapse after long-term remission.
3
CRPC patients no longer
respond to traditional androgen deprivation therapy.
12
Additionally, patients with CRPC often have
metastatic tumors which lead to high mortality rates.
12
In the early 2000’s, it was found that AR
8

itself is a key driver in CRPC, despite the therapeutic resistance of CRPC.
11, 12
The androgen
signaling axis is activated via various mechanisms, such as increased sensitivity to endogenous
androgens, drug resistant mutations, and ligand-independent AR activation.
3
The expression of
AR variants such as AR-V7 is also prominent in some CRPC patients.
13
AR-V7 lacks the AR LBD
and is thus constitutively active.
13
AR-V7 can drive progression of CRPC via complex
mechanisms and can be used as a biomarker to predict treatment outcome.
13

There is enough evidence that AR plays a critical role in advanced CRPC and remains the
key driver of disease even after the failure of hormone therapies or chemotherapies.
3
As the master
regulator of the androgenic pathway and the conductor of numerous other pathways, AR is still
intensely researched as the primary therapeutic target for prostate cancer.

1.3 Antiandrogens and other treatments for prostate cancer
In 1995, bicalutamide was introduced to the market and became the standard antiandrogen
treatment for prostate cancer, replacing flutamide.
4, 12
However, in the late 90’s and early 2000’s,
there was a consensus that bicalutamide was not good enough for a variety of reasons.
14
Limited
efficacy in CRPC was one of the most prominent concerns surrounding bicalutamide. After more
than a decade of research, enzalutamide (Figure 1.6) was developed.
14

Enzalutamide gained approval from the United States Food and Drug Administration
(FDA) in 2012 for both metastatic and non-metastatic CRPC.
15
In chemotherapy-naïve patients,
65% of patients on enzalutamide achieved one year of radiographic progression-free survival,
versus only 14% among patients receiving placebo.
16
In patients who had previously received
chemotherapy, enzalutamide prolonged both overall survival and radiographic progression-free
survival by around 5 months.
16
It also improved the quality of life in patients.
17
However, a small
9

number of patients experience seizure as a significant side effect of enzalutamide.
17
Around the
same time, abiraterone acetate was approved for both castration-resistant and -sensitive metastatic
prostate cancer.
15
It acts as an androgen synthesis inhibitor, rather than a direct anti-androgen. As
a steroidal drug, it has many disadvantages compared with enzalutamide and must be used in
combination with a corticosteroid.
15

The success of enzalutamide led to the development of other second-generation
antiandrogens. Apalutamide (Figure 1.6) is extremely similar to enzalutamide but displayed
greater efficacy in murine xenograft model.
18
Darolutamide (Figure 1.6) is structurally distinct
from enzalutamide and apalutamide and has some notable advantages.
19
It is efficacious towards
all clinically relevant AR mutants, including the F877L (nomenclature of GenBank mRNA
sequence M20132.1) mutant that implies resistance to both enzalutamide and apalutamide.
19

Furthermore, it decreases the protein expression of AR and the constitutively active mutant AR-
V7 at the mRNA level, in addition to antagonizing AR.
20
Darolutamide also has negligible blood–
brain barrier (BBB) penetration and thus low risk of neurotoxicity.
20

Figure 1.6 Second generation antiandrogens.

During non-metastatic stages of prostate cancer, life-extending therapies such as
antiandrogens can serve as effective treatments.
21
In metastatic CRPC, hormone therapies
including abiraterone and enzalutamide, as well as chemotherapy docetaxel, are used as treatments
10

(Table 1.1).
22, 23
Cabazitaxel (Table 1.1) has been proved to significantly improve survival in
patients with metastatic CRPC who have previously been treated with docetaxel, abiraterone, or
enzalutamide.
21
Recently, PARP inhibitors Rucaparib and Olaparib (Table 1.1) were approved for
metastatic CRPC with deficient homologues DNA repair.
24
Patients with advanced CRPC can also
benefit from approved immunotherapies (Table 1.1).
25
Keytruda is a PD-1 inhibitor approved for
advanced prostate cancer with specific genetic markers.
26
With many more investigational
therapies currently in clinical trials, we can expect a more diverse therapeutic landscape, including
combination therapies, in the near future.

Table 1.1 Treatments for prostate cancer
Type of therapy Examples Mechanism of Action
Hormone therapies Leuprolide (Lupron) Luteinizing hormone-releasing
hormone agonists
Relugolix (Orgovyx) Luteinizing hormone-releasing
hormone antagonists
Abiraterone (Zytiga) CYP17A1 inhibitor
Enzalutamide (Xtandi); Apalutamide
(Erleada); Darolutamide (Nubeqa)
Antiandrogen
Immunotherapies Sipuleucel-T (Provenge) T-cell based vaccine
Pembrolizumab (Keytruda) PD-1 inhibitor
Targeted cancer
therapies
Rucaparib (Rubraca);
Olaparib (Lynparza)
Poly-ADP ribose polymerase-1
(PARP-1) inhibitor
Chemotherapies Docetaxel (Taxotere)
Cabazitaxel (Jevtana)
Microtubule stabilizer
Removal of tumor Surgery, Radiation, Cryotherapy Physical removal of tumor

1.4 Our approach to new antiandrogens
1.4.1 Clinical need for new antiandrogens
Although antiandrogens have benefited millions of prostate cancer patients, 20–40% of
patients still do not respond to them.
15
For some patients who initially respond to antiandrogens,
11

their survival is prolonged for no more than a few months.
6
Furthermore, enzalutamide is still the
only second-generation antiandrogen approved for metastatic CRPC.
15
The need for new
antiandrogens is further exacerbated by drug resistance.
11
Point mutations in AR LBD consist the
most prominent resistance mechanism. For example, T878A (bicalutamide resistance),
3
W742L
(bicalutamide resistance),
3
F877L (enzalutamide and apalutamide resistance),
3, 27
and other point
mutations have been discovered in clinical samples. Resistance can also occur via ligand
promiscuity, where AR mutants become less specific for androgens and are promiscuously
activated by other steroidal ligands such as glucocorticoids.
3
Additionally, resistance can occur
beyond the AR signaling pathway.
3
For example, a recent study showed that both apalutamide and
darolutamide induced AKR1C3 expression in vitro.
28
AKR1C3 is an aldo-keto reductase involved
in de novo androgen production.
28
It is a major driver of cancer cell proliferation in CRPC and
overexpression of AKR1C3 would intuitively counter the effect of antiandrogens.
28

Our understanding of AR biology, coupled with clinical limitations of current
antiandrogens, demonstrates that there is clearly an urgent need for new antiandrogens with
outstanding safety and efficacy profiles.
1.4.2 Our starting point: BMS-641988
In the late 1990s, Bristol Myers Squibb (BMS) joined many other pharmaceutical
companies in a race to develop a new antiandrogen for the treatment of prostate cancer.
29, 30
Via
extensive screening, BMS identified bicyclic imides and bicyclic hydantoins as novel scaffolds
(Figure 1.7)
31
that bound to the AR LBD within the same binding pocket as the endogenous ligand
DHT.
32, 33

Extensive structure-activity relationship (SAR) studies were carried out to optimize these
scaffolds.
29, 30, 34-37
Chemical modifications were tested on both the aromatic ring and the bicyclic
12

structure (Figure 1.7). It is the bicyclic structure that distinguished BMS compounds from other
competitors and it is where BMS dedicated most of their efforts.
29
A variety of bicyclic structures
and derivatives were tested, including bicyclic sultam, diazatricylic, and bicyclohydantoin (Figure
1.7). The oxabicyclic compound BMS-501949 (Figure 1.7) showed great promise as an AR
antagonist with satisfactory potency, high oral bioavailability, and long half-life in mouse
models.
29
However, it readily penetrated the blood-brain barrier (BBB) and inhibited the GABAA
receptors which resulted in convulsions in animal models.
29, 30
BMS pivoted to focus on the safety
profile of their lead compound BMS-501949.
30
BMS-779333 (Figure 1.7) was developed in this
optimization process. Structurally, it differs from BMS-501949 in that it contains an additional 6-
membered ring fused to the oxabicyclic core. BMS-779333 was an AR pan-antagonist against all
AR mutations available at that time, but it also displayed significant neurotoxicity in animal
models.
29, 30

Figure 1.7 Scaffolds and lead compounds of AR antagonists from BMS.
13

BMS-641988 (Figure 1.8) was later developed from an SAR screen of C-5 endo-
substituted analogues of BMS-501949.
29, 30, 33
BMS-641988 was pushed into clinical trial despite
its unfavorable properties.
30
Preclinical studies showed that BMS-641988 agonized some AR
mutants in vitro, which foretold its limited efficacy.
29, 30
Later on, it was found that BMS-641988
was extensively metabolized to BMS-501949 via CYP3A4 and reductase in vivo (Figure 1.8).
29,
30
In-human pharmacokinetics studies revealed that the steady-state concentrations of its
metabolite BMS-501949 exceeded those of the parent drug BMS-641988 with a ratio of 2.5:1.
30

The high serum concentration of BMS-501949 was particularly concerning.
30
BMS-641988 failed
Phase I clinical trial and BMS decided to terminate the development of this family of
antiandrogens.
30, 33

Figure 1.8 In vivo metabolism of BMS-641988.
BMS-641988 is metabolized by CYP 3A4 to BMS-570511, and then rapidly reduced to BMS-
501949. BMS-501949 is itself an AR antagonist but has significant neurotoxicity due to BBB
penetration.

1.4.3 Improving BMS-641988
In our medicinal chemistry campaign to develop a novel treatment for prostate cancer,
BMS-641988 serves as a suitable starting point. Chemically, it contains an interesting oxabicyclic
core structure distinct from any other AR ligand. Biologically, much knowledge has been obtained
on this family of compounds in terms of their in vivo ADMET (Absorption, Distribution,
14

Metabolism, Elimination, Toxicity) and PK/PD. As attractive as it might be, BMS-641988 still
needs a lot of imporvements before it becomes a safe and efficacious treatment.
To improve BMS-641988 as a clinical drug for prostate cancer, the paramount task is to
improve its safety profile, i.e. to avoid the production of BMS-501949 as well as penentration of
the parent compound itself through BBB (Table 1.2). Secondly, since BMS-641988 is agonistic
in some AR mutants, we need to make modifictaions such that our compounds are AR pan-
antagonistic (Table 1.2). Finally, since enzalutamide, apaludamide and darolutamide have been
approved for prostate cancer and CRPC after the discvoery of BMS-641988, we aim to develop
antiandrogens that remain efficacious in enzalutamide-resistant prostate cancer (Table 1.2). In
summary, our goal is to develop a best-in-class antiandrogen.

Table 1.2 Our goal of improving BMS-641988.
Efficacy Safety
BMS-641988 Has limited efficacy in wt AR; no
efficacy in drug-resistant AR
mutants.
Produces neurotoxic metabolite
BMS-501949.
Enzalutamide Has no efficacy in enzalutamide-
resistant prostate cancer.
Causes seizures in some cases due to
BBB penetration.
Our goal Improved efficacy in wt AR;
maintained efficacy in
enzalutamide-resistant AR mutants.
Low probability of BBB penetration,
improved metabolism to avoid
neurotoxic metabolite.

15

Chapter 1 references
1. Barsouk, A.; Padala, S. A.; Vakiti, A.; Mohammed, A.; Saginala, K.; Thandra, K. C.;
Rawla, P.; Barsouk, A., Epidemiology, Staging and Management of Prostate Cancer. Medical
Sciences 2020, 8 (3), 28.
2. Rawla, P., Epidemiology of Prostate Cancer. World Journal of Oncology 2019, 10 (2), 63-89.
3. Tan, M. H. E.; Li, J.; Xu, H. E.; Melcher, K.; Yong, E.-l., Androgen receptor: structure, role
in prostate cancer and drug discovery. Acta Pharmacologica Sinica 2015, 36 (1), 3-23.
4. Gao, W.; Bohl, C. E.; Dalton, J. T., Chemistry and Structural Biology of Androgen Receptor.
Chemical Reviews 2005, 105 (9), 3352-3370.
5. Yap, T. A.; Smith, A. D.; Ferraldeschi, R.; Al-Lazikani, B.; Workman, P.; de Bono, J. S.,
Drug discovery in advanced prostate cancer: translating biology into therapy. Nature Reviews
Drug Discovery 2016, 15 (10), 699-718.
6. Centenera, M. M.; Selth, L. A.; Ebrahimie, E.; Butler, L. M.; Tilley, W. D., New
opportunities for targeting the androgen receptor in prostate cancer. Cold Spring Harbor
Perspectives in Medicine 2018, 8 (12), a030478.
7. Morova, T.; McNeill, D. R.; Lallous, N.; Gönen, M.; Dalal, K.; Wilson, D. M.; Gürsoy, A.;
Keskin, Ö.; Lack, N. A., Androgen receptor-binding sites are highly mutated in prostate cancer.
Nature Communications 2020, 11 (1), 1-10.
8. Pereira de Jésus-Tran, K.; Côté, P.-L.; Cantin, L.; Blanchet, J.; Labrie, F.; Breton, R.,
Comparison of crystal structures of human androgen receptor ligand-binding domain complexed
with various agonists reveals molecular determinants responsible for binding affinity. Protein
Science 2006, 15 (5), 987-999.
9. Patsch, K.; Liu, C.; Zapotoczny, G.; Sun, Y.; Sura, H.; Ung, N.; Sun, R. X.; Haliday, B.;
Yu, C.; Aljehani, M.; Lee, J. S. H.; Kashemirov, B. A.; Agus, D. B.; McKenna, C. E.;
Ruderman, D., Paradoxical androgen receptor regulation by small molecule enantiomers.
Proceedings of the National Academy of Sciences 2021, 118 (12), e2100918118.
10. Szklarczyk, D.; Gable, A. L.; Nastou, K. C.; Lyon, D.; Kirsch, R.; Pyysalo, S.; Doncheva,
N. T.; Legeay, M.; Fang, T.; Bork, P.; Jensen, L. J.; von Mering, C., The STRING database in
2021: customizable protein–protein networks, and functional characterization of user-uploaded
gene/measurement sets. Nucleic Acids Research 2020, 49 (D1), D605-D612.
11. Schmidt, K. T.; Huitema, A. D.; Chau, C. H.; Figg, W. D., Resistance to second-generation
androgen receptor antagonists in prostate cancer. Nature Reviews Urology 2021, 1-18.
12. Scher, H. I.; Sawyers, C. L., Biology of Progressive, Castration-Resistant Prostate Cancer:
Directed Therapies Targeting the Androgen-Receptor Signaling Axis. Journal of Clinical
Oncology 2005, 23 (32), 8253-8261.
13. Uo, T.; Plymate, S. R.; Sprenger, C. C., The potential of AR-V7 as a therapeutic target.
Expert Opinion on Therapeutic Targets 2018, 22 (3), 201-216.
14. Ha, Y.-S.; Kim, I. Y., Enzalutamide: looking back at its preclinical discovery. Expert Opinion
on Drug Discovery 2014, 9 (7), 837-845.
15. Rice, M. A.; Malhotra, S. V.; Stoyanova, T., Second-generation antiandrogens: from
discovery to standard of care in castration resistant prostate cancer. Frontiers in Oncology 2019,
9, 801.
16. Beer, T. M.; Armstrong, A. J.; Rathkopf, D. E.; Loriot, Y.; Sternberg, C. N.; Higano, C.
S.; Iversen, P.; Bhattacharya, S.; Carles, J.; Chowdhury, S.; Davis, I. D.; de Bono, J. S.;
Evans, C. P.; Fizazi, K.; Joshua, A. M.; Kim, C.-S.; Kimura, G.; Mainwaring, P.; Mansbach,
16

H.; Miller, K.; Noonberg, S. B.; Perabo, F.; Phung, D.; Saad, F.; Scher, H. I.; Taplin, M.-E.;
Venner, P. M.; Tombal, B., Enzalutamide in metastatic prostate cancer before chemotherapy.
New England Journal of Medicine 2014, 371 (5), 424-433.
17. Scher, H. I.; Fizazi, K.; Saad, F.; Taplin, M.-E.; Sternberg, C. N.; Miller, K.; De Wit, R.;
Mulders, P.; Chi, K. N.; Shore, N. D.; Armstrong, A. J.; Flaig, T. W.; Fléchon, A.;
Mainwaring, P.; Fleming, M.; Hainsworth, J. D.; Hirmand, M.; Selby, B.; Seely, L.; de Bono,
J. S., Increased survival with enzalutamide in prostate cancer after chemotherapy. New England
Journal of Medicine 2012, 367 (13), 1187-1197.
18. Clegg, N. J.; Wongvipat, J.; Joseph, J. D.; Tran, C.; Ouk, S.; Dilhas, A.; Chen, Y.;
Grillot, K.; Bischoff, E. D.; Cai, L.; Aparicio, A.; Dorow, S.; Arora, V.; Shao, G.; Qian, J.;
Zhao, H.; Yang, G.; Cao, C.; Sensintaffar, J.; Wasielewska, T.; Herbert, M. R.; Bonnefous,
C.; Darimont, B.; Scher, H. I.; Smith-Jones, P.; Klang, M.; Smith, N. D.; De Stanchina, E.;
Wu, N.; Ouerfelli, O.; Rix, P. J.; Heyman, R. A.; Jung, M. E.; Sawyers, C. L.; Hager, J. H.,
ARN-509: a novel antiandrogen for prostate cancer treatment. Cancer Research 2012, 72 (6),
1494-1503.
19. Moilanen, A.-M.; Riikonen, R.; Oksala, R.; Ravanti, L.; Aho, E.; Wohlfahrt, G.;
Nykänen, P. S.; Törmäkangas, O. P.; Palvimo, J. J.; Kallio, P. J., Discovery of ODM-201, a
new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen
signaling-directed prostate cancer therapies. Scientific Reports 2015, 5 (1), 1-11.
20. Yu, J.; Zhou, P.; Hu, M.; Yang, L.; Yan, G.; Xu, R.; Deng, Y.; Li, X.; Chen, Y.,
Discovery and biological evaluation of darolutamide derivatives as inhibitors and down-
regulators of wild-type AR and the mutants. European Journal of Medicinal Chemistry 2019,
182, 111608.
21. de Wit, R.; de Bono, J.; Sternberg, C. N.; Fizazi, K.; Tombal, B.; Wülfing, C.; Kramer,
G.; Eymard, J.-C.; Bamias, A.; Carles, J.; Iacovelli, R.; Melichar, B.; Sverrisdóttir, Á.;
Theodore, C.; Feyerabend, S.; Helissey, C.; Ozatilgan, A.; Geffriaud-Ricouard, C.; Castellano,
D., Cabazitaxel versus Abiraterone or Enzalutamide in Metastatic Prostate Cancer. New England
Journal of Medicine 2019, 381 (26), 2506-2518.
22. Sartor, O.; Pal, S. K., Abiraterone and its place in the treatment of metastatic CRPC. Nature
Reviews Clinical Oncology 2013, 10 (1), 6-8.
23. Schweizer, M. T.; Zhou, X. C.; Wang, H.; Bassi, S.; Carducci, M. A.; Eisenberger, M. A.;
Antonarakis, E. S., The Influence of Prior Abiraterone Treatment on the Clinical Activity of
Docetaxel in Men with Metastatic Castration-resistant Prostate Cancer. European Urology 2014,
66 (4), 646-652.
24. Wolford, J. E.; Bai, J.; Moore, K. N.; Kristeleit, R.; Monk, B. J.; Tewari, K. S., Cost-
effectiveness of niraparib, rucaparib, and olaparib for treatment of platinum-resistant, recurrent
ovarian carcinoma. Gynecologic Oncology 2020, 157 (2), 500-507.
25. Hadley, J.; Yabroff, K. R.; Barrett, M. J.; Penson, D. F.; Saigal, C. S.; Potosky, A. L.,
Comparative effectiveness of prostate cancer treatments: evaluating statistical adjustments for
confounding in observational data. Journal of the National Cancer Institute 2010, 102 (23),
1780-1793.
26. Cha, H.-R.; Lee, J. H.; Ponnazhagan, S., Revisiting immunotherapy: a focus on prostate
cancer. Cancer Research 2020, 80 (8), 1615-1623.
27. Korpal, M.; Korn, J. M.; Gao, X.; Rakiec, D. P.; Ruddy, D. A.; Doshi, S.; Yuan, J.;
Kovats, S. G.; Kim, S.; Cooke, V. G.; Monahan, J. E.; Stegmeier, F.; Roberts, T. M.; Sellers,
17

W. R.; Zhou, W.; Zhu, P., An F876L mutation in androgen receptor confers genetic and
phenotypic resistance to MDV3100 (enzalutamide). Cancer Discovery 2013, 3 (9), 1030-1043.
28. Morsy, A.; Trippier, P. C., Reversal of apalutamide and darolutamide aldo-keto reductase
1C3-mediated resistance by a small molecule inhibitor. ACS Chemical Biology 2020, 15 (3),
646-650.
29. Gavai, A. V.; Foster, W. R.; Balog, A.; Vite, G. D., Novel Androgen Receptor Antagonists
for the Treatment of Prostate Cancer. In Accounts in Drug Discovery, 2010; pp 120-141.
30. Foster, W. R.; Car, B. D.; Shi, H.; Levesque, P. C.; Obermeier, M. T.; Gan, J.; Arezzo, J.
C.; Powlin, S. S.; Dinchuk, J. E.; Balog, A., Drug safety is a barrier to the discovery and
development of new androgen receptor antagonists. The Prostate 2011, 71 (5), 480-488.
31. Balog, A.; Salvati, M. E.; Shan, W.; Mathur, A.; Leith, L. W.; Wei, D. D.; Attar, R. M.;
Geng, J.; Rizzo, C. A.; Wang, C.; Krystek, S. R.; Tokarski, J. S.; Hunt, J. T.; Gottardis, M.;
Weinmann, R., The synthesis and evaluation of [2.2. 1]-bicycloazahydantoins as androgen
receptor antagonists. Bioorganic & Medicinal Chemistry Letters 2004, 14 (24), 6107-6111.
32. Sack, J. S.; Kish, K. F.; Wang, C.; Attar, R. M.; Kiefer, S. E.; An, Y.; Wu, G. Y.;
Scheffler, J. E.; Salvati, M. E.; Krystek, S. R.; Weinmann, R.; Einspahr, H. M.,
Crystallographic structures of the ligand-binding domains of the androgen receptor and its
T877A mutant complexed with the natural agonist dihydrotestosterone. Proceedings of the
National Academy of Sciences 2001, 98 (9), 4904-4909.
33. Balog, A.; Rampulla, R.; Martin, G. S.; Krystek, S. R.; Attar, R.; Dell-John, J.; DiMarco,
J. D.; Fairfax, D.; Gougoutas, J.; Holst, C. L.; Nation, A.; Rizzo, C.; Rossiter, L. M.;
Schweizer, L.; Shan, W.; Spergel, S.; Spires, T.; Cornelius, G.; Gottardis, M.; Trainor, G.;
Vite, G. D.; Salvati, M. E., Discovery of BMS-641988, a novel androgen receptor antagonist for
the treatment of prostate cancer. ACS Medicinal Chemistry Letters 2015, 6 (8), 908-912.
34. Sun, C.; Robl, J. A.; Wang, T. C.; Huang, Y.; Kuhns, J. E.; Lupisella, J. A.; Beehler, B.
C.; Golla, R.; Sleph, P. G.; Seethala, R.; Fura, A.; Krystek, S. R.; An, Y.; Malley, M. F.;
Sack, J. S.; Salvati, M. E.; Grover, G. J.; Ostrowski, J.; Hamann, L. G., Discovery of potent,
orally-active, and muscle-selective androgen receptor modulators based on an N-aryl-
hydroxybicyclohydantoin scaffold. Journal of Medicinal Chemistry 2006, 49 (26), 7596-7599.
35. Salvati, M. E.; Balog, A.; Shan, W.; Rampulla, R.; Giese, S.; Mitt, T.; Furch, J. A.; Vite,
G. D.; Attar, R. M.; Jure-Kunkel, M.; Geng, J.; Rizzo, C. A.; Gottardis, M. M.; Krystek, S.
R.; Gougoutas, J.; Galella, M. A.; Obermeier, M.; Fura, A.; Chandrasena, G., Identification
and optimization of a novel series of [2.2.1]-oxabicyclo imide-based androgen receptor
antagonists. Bioorganic & Medicinal Chemistry Letters 2008, 18 (6), 1910-1915.
36. Xiao, H.-Y.; Balog, A.; Attar, R. M.; Fairfax, D.; Fleming, L. B.; Holst, C. L.; Martin, G.
S.; Rossiter, L. M.; Chen, J.; Cvjic, M.-E.; Dell-John, J.; Geng, J.; Gottardis, M. M.; Han,
W.-C.; Nation, A.; Obermeier, M.; Rizzo, C. A.; Schweizer, L.; Spires Jr., T.; Shan, W.;
Gavai, A. V.; Salvati, M. E.; Vitea, G., Design and synthesis of 4-[3,5-dioxo-11-oxa-4,9-
diazatricyclo[5.3.1.02,6]undec-4-yl]-2-trifluoromethyl-benzonitriles as androgen receptor
antagonists. Bioorganic & Medicinal Chemistry Letters 2010, 20 (15), 4491-4495.
37. Shan, W.; Balog, A.; Nation, A.; Zhu, X.; Chen, J.; Cvijic, M. E.; Geng, J.; Rizzo, C. A.;
Spires Jr, T.; Attar, R. M.; Obermeier, M.; Traeger, S.; Dai, J.; Zhang, Y.; Galella, M.;
Trainor, G.; Vite, G. D.; Gavai, A. V., [2.2.1]-Bicyclic sultams as potent androgen receptor
antagonists. Bioorganic & Medicinal Chemistry Letters 2016, 26 (23), 5707-5711.

18
Chapter 2 AR regulation by enantiomeric ligands
2.1 Chiral molecules in drug discovery
In 1992, Milton et al. published an elegant paper on Science titled “Total chemical
synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate
specificity”.
1
This paper demonstrated that HIV-1 protease is a chiral macromolecule which
recognizes chiral substrates, and that enantiomeric proteins should display reciprocal chiral
specificity to their substrates.
1
Needlessly to say, chirality is abundant in biological systems and
chiral specificity would apply to any biomolecules that has chirality itself.
In the same year, the FDA published a guide document known as “Development of New
Stereoisomeric Drugs”.
2
This document focused on “issues relating to the study and
pharmaceutical development of individual enantiomers and racemates”, as it used to be common
practice to develop racemate drugs.
2
The FDA discussed issues on manufacturing, characterizing
and testing of individual enantiomers.
2

Although enantiomers share common physical properties (excepting chiral interactions),
their biological effects are usually distinguishable.
2
The FDA now requires drug manufacturers to
study the full profiles of each enantiomer in the case of chiral drugs.
2
The most famous case of
chiral drugs is probably the thalidomide (Figure 2.1) tragedy.
3
In the late 1950s and early 1960s,
racemic thalidomide was used in some countries to relieve nausea during pregnancy.
3
However, it
soon became evident that this drug could cause birth defects.
3
We now know that the (R)-
thalidomide is responsible for sedative effects whereas the (S)-thalidomide is teratogenic.
3
A more
recent example is ketamine (Figure 2.1): esketamine and arketamine are two enantiomers of
ketamine that both bind to the NMDA receptor.
4, 5
However, esketamine is much more potent and
has been developed into an antidepressant.
5

19
As demonstrated in these examples, the biological effects of enantiomers can be either
unrelated or highly related. There are rare cases where two enantiomers act on the same biological
target and initiate opposite functional effects. For example, both the sodium channel membrane
proteins and the calcium channel membrane proteins are able to accommodate enantiomeric
compounds which have opposite functional effects, but the binding sites seem to be different and
stereoselective.
6-8

Figure 2.1 Structures of Thalidomide and Ketamine.

2.2 AR agonist-antagonist pairs of enantiomeric ligands
All of the aforementioned BMS preclinical and clinical candidates are chiral molecules.
9,
10
BMS-641988 is the 3aR,4R,5R,7R,7aS stereoisomer (“R” at the C5 position).
10
BMS postulated
that both BMS-641988 and its enantiomer (hereafter named “S-BMS”) would be AR antagonists,
based on their in silico model (futher discussed in Chapter 2.3).
10
However, the chemical and
biological properties of S-BMS were not reported.
10
It remains unknown whether BMS actually
tested the S-BMS as AR ligands.
Due to a Diels-Alder addition in the synthesis of BMS-641988, racemic intermediates are
produced and the individual enantiomers were separated using chiral columns (synthesis detailed
in Chapter 3).
10
In the manufacturing process described by BMS, the racemic intermediate was
separated by preparative chiral column to obtain the enantiopure intermediate I (Figure 2.2).The
20
enantiopure intermediate I was subsequently used in the synthesis to obtain BMS-641988. Due to
limited capacity for chiral HPLC purification, we chose to conduct the chiral resolution at the end
of the synthesis (synthesis detailed in Chapter 3). Thus, we obtain pairs of enantiomers. For the
purpose of this discussion, the 3aR,4R,5R,7R,7aS enantiomer is hereafter named “Compound ##R”
or “the R enantiomer”, and the 3aS,4S,5S,7S,7aR enantiomer is hereafter named “Compound ##S”
or “the S enantiomer”.

Figure 2.2 Structure of the intermediate I.

2.2.1 AR agonist-antagonist pairs of enantiomeric compounds
We first synthesized and tested BMS-641988 and S-BMS. As expected, BMS-641988
inhibited hyperspeckling of GFP-AR caused by R1881 in PC3 cells (Figure 2.3A). To our surprise,
S-BMS displayed AR agonistic activity.
11
S-BMS caused substantial hyperspeckling of GFP-AR
in PC3 cells (Figure 2.3B), indicative of nuclear translocation of GFP-AR upon binding of
agonists.
11
This prompted us to investigate the agonist-antagonist relationship for all our
subsequent compounds.
We designed and synthesized a series of amide analogues of BMS-641988, namely
compound 01, 02, 03 and 06 (design, synthesis and structures detailed in Chapter 3). All of those
enantiomeric pairs displayed the agonist-antagonist relationship, with the R-enantiomer being the
antagonist and the S-enantiomer the agonist.
11
In PC3 prostate cancer cells expressing GFP-AR,
S-enantiomers alone and R1881 caused hyperspeckling of GFP-AR, while R-enantiomers and
21
enzalutamide inhibited R1881-induced hyperspeckling (Figure 2.4A).
11
In cells expressing ARE-
luciferase, S-enantiomers alone and R1881 activated ARE-luciferase expression, while R-
enantiomers and enzalutamide significantly inhibited R1881-induced ARE-luciferase expression
(Figure 2.4B).
11
In most cases, the S-enantiomers generated more than 100% luciferase signal
(100% normalized to 1 nM R1881), indicating that they are extremely effective agonists.

Figure 2.3 Hyperspeckling of GFP-AR under treatments of BMS-641988 and S-BMS.
Confocal microscopy of PC3 cells expressing GFP-AR, after treatment with 10 μM purified BMS-
641988 and S-BMS (180 min) with or without 1 nM R1881 (90 min).

Figure 2.4 Hyperspeckling and luciferase expression under treatments of enantiomeric
compounds.
(A) Nuclear speckling quantification of compound pairs and (B) ARE-luciferase assays in cells
treated with 10 μM drug + 1 nM R1881. Data (n ≥ 3) are mean ±SD. ENZ = enzalutamide. *P <
0.05, **P < 0.01, ***P < 0.001, n.s., not significant.

22
In order to study how our compounds affect gene expression, we examined genome-wide
gene expression in VCaP cells, a hormone-responsive but -independent model of CRPC (Figure
2.5).
11
Using the RT
2
Profiler PCR Array Human Androgen Receptor Signaling Targets RT-qPCR
kit, we found that compound 06S alone activated 82 AR target genes, similar to the effect of
DHT.
11
On the other hand, 06R inhibited DHT-induced gene expression, similar to the effect of
enzalutamide.
11

As we continued our exploration of compounds with the same scaffold, we consistently
observed paired activities of agonists-antagonists. The list of compounds is detailed in Chapter 3.
We not only developed novel antiandrogens, but also discovered novel non-steroidal androgens.
To our knowledge, this is the first reported example of this agonist-antagonist relationship of
enantiomeric compounds for AR.

Figure 2.5 Gene expression profile in VCaP cells treated by compound 06R/S.
RT-qPCR is performed with the commercial “RT
2
Profiler PCR Array Human Androgen Receptor
Signaling Targets” kit. Gene expressions were projected along the expression change vector
between NTC (0%) and DHT (100%). Data (n = 3) are mean ± SD. ENZ = enzalutamide. *P <
0.05, **P < 0.01, ***P < 0.001, n.s., not significant.

23
2.2.2 Impact of chiral impurities
This phenomenon of paired activities of agonists-antagonists provoked us to examine how
chiral contamination could interfere with biological activity of these compound. During
preparation of these enantiomeric compounds, each enantiomer is purified multiple times to ensure
their purity. Before biological testing, each individual enantiomer was analyzed for their chiral
purity on HPLC.
To measure how chiral impurities could impact the outcome of biological activities of these
compounds, we spiked highly purified R-enantiomers with their S-isomers and treated cells with
the compounds of various enantiopurity. In the presence of 1 nM R1881, ARE-luciferase signals
were inhibited by pure R-enantiomers (Figure 2.6A), but the signals rapidly increased with below
1% S-enantiomer contamination.
11
Less than 3% S-enantiomers contamination halved the
antagonistic effect of R-enantiomers, and 9% canceled out its antagonist effect entirely (Figure
2.6A).
11
The S-isomer contamination was even more detrimental in cell viability assays. After 6-
day treatment with 10 μM of the compounds with various enantiopurity, pure R-enantiomers
inhibited R1881-induced cancer cell growth as expected (Figure 2.6B).
11
As little as 0.1% S-
enantiomer contamination caused statistically significant increase in cell viability, and around 2%
S-enantiomer contamination completely rescued the viability phenotype (Figure 2.6B).
11

In order to study why such small amounts of contamination could cause such significant
differences in our in vitro assays, we determined the binding affinity of both enantiomers. Cell-
free binding assay showed that S-enantiomers have much higher binding affinity (single digit nM)
than their respective R-enantiomer counterparts (Figure 2.7A).
11
Enzalutamide also displayed a
relatively lower binding affinity. This is consistent with literature reports that AR antagonists
usually have low binding affinity in cell-free assays, possibly due to unstable protein configuration
24
upon antagonist binding.
12, 13
Furthermore, EC50 of agonists obtained by ARE-luciferase assays
were consistently lower than their respective R-enantiomer counterparts (Figure 2.7B).
11
The high
binding affinity and high potency of the S-enantiomer agonists explained why less than 1% S-
enantiomer contamination in R-enantiomer can cause drastic difference in our biological test.

Figure 2.6 Luciferase expression and viability of VCaP cells when treated with contaminated
R-enantiomer drugs.
(A) ARE-luciferase was measured after 24 h. (B) VCaP cell viability after 6 d of treatment using
CellTiter-Glo. Triangles represent compound 06; circles represent compound 01. Data (n = 3) are
mean ± SD. Dashed lines represent EC50. Luciferase expression and viability are scaled to NTC
(0%) and 1 nM R1881 (100%). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.

Figure 2.7 Potency and efficacy of enantiomers.
(A) Binding affinity obtained via competition binding measured by fluorescence polarization. (B)
Efficacy obtained by ARE-luciferase dose–response curves run in antagonist mode (1 nM R1881)
with (R)-drugs and in agonist mode ( −R1881) with their respective (S)-isomers. Data (n ≥ 3) are
mean ± SD. ENZ = enzalutamide. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.

25
2.3 In silico models of novel AR ligands bound to AR
2.3.1 Induced fit docking models
To further explore this agonist-antagonist relationship of our enantiomeric compounds, we
performed in silico modeling experiments. Induced fit docking of compound 02S in AR ligand
binding domain (LBD) (PDB: 1E3G) confirmed a closed conformation of Helix 12 (H12) of the
AR LBD, consistent with an agonist conformation.
11
Due to the absence of crystal structures of
antagonist-bound AR LBD, antagonists cannot be directly docked to available AR LBD
structures.
11
Thus, we built a homology model of open-conformation AR LBD based on
progesterone receptor (PR) LBD. The PR is one of the most similar nuclear hormone receptors to
AR among all the hormone receptors,
14
and there are available structures for antagonist-bound
PR.
15, 16
An open-conformation AR LBD was built with an open-conformation PR structure (PDB:
2OVM).
15
Induced fit docking of compound 02R on this AR LBD homology model showed that
the R-enantiomer obstructs H12 and prevents a closed conformation of H12.
11

As shown in Figure 2.8, the two enantiomers possess different orientations in the binding
pocket of AR LBD.
11
In the agonist-bound AR LBD model, the ligand is oriented so that H12 can
close to form a stable conformation. In the antagonist-bound AR LBD model, the ligand protrudes
out of the binding pocket and H12 cannot close as in the agonist-bound conformation. In both
agonist and antagonist, hydrogen bonds are observed between the ligands and the residues R752
and N705, analogous to the binding modes of DHT.
11

In order to further validate our in silico models, we performed mutagenesis studies with
AR mutants N705S and R752Q. In both mutants, the S enantiomers, as well as R1881, failed to
generate any ARE-luciferase signals, suggesting no agonistic activities. Furthermore, the R
enantiomers also did not induce any ARE-luciferase signals in these mutants. Overall, this
26
experiment confirmed these critical binding sites in cells expressing point mutations that
significantly reduced functions of our compounds in ARE luciferase assay (Figure 2.9).
11

Figure 2.8 In silico models of AR LBD bound to 02S and 02R.
Overlay of compound 02S (blue) docked onto AR-LBD in closed conformation (turquoise), and
02R (gold) in the open AR homology model (grey).

Figure 2.9 Mutation studies of key residues in the AR LBD binding pocket.
Cells expressing GFP-AR with point mutations are treated with 10 μM drug + 1 nM R1881. Data
(n ≥ 3) are mean ± SD and scaled to NTC (0%). NTC = no treatment control, ENZ: enzalutamide.
*P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.

27
2.3.2 State of the art of AR molecular dynamics
As mentioned previously, BMS conducted an in silico modelling study which led to the
conclusion that both BMS-641988 and S-BMS would be AR antagonists.
10
At that time, only
DHT-bound AR LBD structure was available, and homology modeling of antagonist-bound AR
LBD was not possible due to the lack of analogous structures. Based on our work, we can clearly
state the importance of correct in silico AR models for antagonist-bound AR modelling.
Although we were, to our knowledge, the first group to report such chirality-dependent AR
agonist-antagonist pairs, there have been substantial literature reports where subtle changes in AR
ligand structures led to dramatic change in their functional activities.
17
Here we discuss a few
compounds in hope of illustrating the intriguing dynamics of AR.
Bicalutamide serves well as an example. It is sold as a racemic mixture,
18
even though only
the R-isomer is responsible for the AR antagonism.
18-20
The S-isomer is nontoxic and thus the co-
administration of both enantiomers was allowed and well tolerated by patients.
18
Interestingly, the
two enantiomers have different PK profiles in human and the metabolic processes of the two
enantiomers are different.
18, 21

R-3 and S-1 (Figure 2.10) are two AR agonists structurally related to bicalutamide.
22
Bohl
et al. demonstrated the molecular interactions between AR LBD and these ligands, although no
particular explanation was given to explain why bicalutamide is an AR antagonist whereas R-3
and S-1 are AR agonists.
22
Even though many AR LBD binary complexes structures have been
solved since then, we are still not able to understand what structural features differentiate AR
antagonists from AR agonists solely based on protein structures. Significant drawbacks of AR
protein crystallography contribute to this limitation in our understanding. First, the solved
structures do not represent the full length AR.
17
Due to the flexible nature of the hinge domain,
28
current crystallographic technologies cannot solve the structure of AR as a whole.
17
Second, the
AR LBD must be in agonist-stabilized structures.
22
During protein purification, the AR LBD can
only be extracted as soluble proteins when expressed in the presence of an agonist in E. coli.
expression system.
19, 22, 23
Third, we still know very little about how AR interact with other
proteins, especially their chaperone HSPs.
24

Figure 2.10 Structures of bicalutamide, R-3, S-1.

To advance our understanding on AR conformations and dynamics when bound to different
ligands, many computational methods have been developed to specifically study AR protein-
ligand interactions. Recent studies using molecular dynamics modelling and machine learning
algorithms have provided some additional insights into the actions of AR ligands. A study used
unbiased molecular dynamics to illustrate an allosteric pathway of AR LBD H12 movement upon
ligand binding.
25
The authors also constructed the free energy profiles of transition process
between the antagonistic form and agonistic form of the AR LBD.
25
In another study, molecular
dynamics of the bicalutamide-bound AR LBD showed that the AF2 site is key to AR co-factor
interactions upon antagonist binding.
13
Several machine learning algorithms for the AR have been
29
developed and extensively compared.
26, 27
These algorithms deepened our understanding of the
differentiating factors of AR agonist and antagonist, such as the number of aliphatic carbons and
the degree of saturation.

2.4 Efforts to obtain experimental models of AR LBD-agonist binary complexes
To further study the binding mode of our compounds, we attempted to obtain experimental
models of our S agonists-bound AR (antagonist-bound AR cannot be crystalized as
aforementioned). Dr. Fariborz Nasertorabi mentored crystallization work and obtained the X-ray
structure of AR LBD-DHT binary complex.
First, we successfully obtained the X-ray structure of AR LBD in complex with the
endogenous ligand DHT. Plasmid of AR LBD with a poly-His tag was obtained commercially
from Addgene. His-tagged AR LBD was overexpressed in E. coli BL21DE3 cells in the presence
of 30 μM DHT.
16, 23, 28-31
AR LBD-DHT binary complex was purified by immobilized metal
affinity chromatography (IMAC Ni selection) and size exclusion column (SEC). The His tag was
removed at a thrombin cleavage site, and the AR LBD-DHT binary complex was purified again
through reverse Ni selection. The sample was concentrated to 3.2 mg/mL in a buffer containing
25 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT, 5 μM DHT. Crystallization
drops were prepared by a Mosquito robot (TTP Labtech) in 1:1 (v/v) sitting drop of purified protein
and well solution. We screened crystallization conditions based on literature reports (Table 2.1).
16,
19, 22, 23, 28-31
Crystals appeared in precipitant solution containing MgSO4 at pH 8 and grew to their
final size within two weeks. Crystals diffracted at 2.5-3.0 Å, where the ligand was clearly visible
with strong density (Figure 2.11).
28

30
Table 2.1 Crystallization conditions screened for AR LBD-DHT binary complex.
Concentrations of precipitants were varied under each and every pH listed in the table, to give
matrices of screening conditions.
Precipitant Concentration pH
MgSO4 1.4 – 1.8 M (incremental
increase of 0.1 M)
7 (50 mM HEPES), 7.5 (50 mM
HEPES), 8 (50 mM Tris), 8.5 (50
mM Tris)
Li2SO4 0.6 – 1.2 M (incremental
increase of 0.2 M)
7 (50 mM HEPES), 8 (50 mM Tris)
Sodium potassium tartrate 0.5 – 1 M (incremental
increase of 0.1 M)
7 (50 mM HEPES), 7.5 (50 mM
HEPES), 8 (50 mM Tris), 8.5 (50
mM Tris)
Disodium tartrate 0.5 – 1 M (incremental
increase of 0.1 M)
7 (50 mM HEPES), 7.5 (50 mM
HEPES), 8 (50 mM Tris), 8.5 (50
mM Tris)
Na2HPO4 with 5% PEG400 0.4 – 0.8 M (incremental
increase of 0.2 M)
8 (50 mM Tris), 8.5 (50 mM Tris)
Na2HPO4with 5% PEG400
and 0.1 M (NH4)2HPO4
0.4 – 0.8 M (incremental
increase of 0.2 M)
8 (50 mM Tris), 8.5 (50 mM Tris)

Figure 2.11 Experimental model of AR LBD in complex with DHT obtained in our group.
AR LBD is colored in orange, and DHT in pink. A sulfate molecule is also seen on diffraction
pattern. The structure was not refined further, since similar structures already exist in PDB. The
statistics are based on a few rounds of refinement.

31
Next, we tried to obtain AR LBD structures bound to S-isomer agonists. Even though we
were not able to obtain crystals via the following protocols, our experience is valuable as a
reference for future endeavors.
Protocol 1: We opted to use racemates as our ligands during protein expression because at
that time it was not feasible for us to prepare enantiopure S-isomers at the scale needed for protein
expression (around 30 mg ligand needed for every 5 g harvested cells). We rationalized that the
proteins should be occupied solely by S-isomer agonists even if we used racemates, because (1)
our S-isomer agonists have much higher binding affinity than their R-isomers in vitro (see Figure
2.7), (2) antagonist-bound AR LBD is unstable in E. coli expression system. We selected 02S and
10S (See Chapter 3) as our ligands of interest because they showed the highest binding affinity at
the time. As described above, we overexpressed His-tagged AR LBD in the presence of racemate
02 or racemate 10. All purification buffers were supplemented with racemate 02 or racemate 10.
Pure AR LBD in complex with either compound 02S or 10S was successfully obtained and
concentrated to 2.3 mg/mL and 3.2 mg/mL, respectively. Due to limited amount of available
protein, we decided to only screen crystallization conditions which either gave crystals or resulted
in significant amount of precipitation for AR LBD-DHT binary complex in our previous
experiments.
22, 23, 28
We prepared a matrix using “pH vs precipitant concentration” for selected
crystallization conditions (Table 2.2). Unfortunately, we were not able to obtain any crystals under
these conditions, and the limited supply of ligands discouraged us to conduct more screenings.

32
Table 2.2 Crystallization conditions screened for AR LBD-agonist binary complexes.
For each binary complex, Concentrations of precipitants were varied under each and every pH
listed in the table, to give matrices of screening conditions.
Precipitant Concentration pH
MgSO4 1.5 – 2.5 M (incremental
increase of 0.2 M)
7 (50 mM HEPES), 7.5 (50
mM HEPES), 8 (50 mM Tris),
8.5 (50 mM Tris)
Sodium potassium tartrate 0.5 – 1 M (incremental
increase of 0.1 M)
7 (50 mM HEPES), 7.5 (50
mM HEPES), 8 (50 mM Tris),
8.5 (50 mM Tris)
Sodium potassium tartrate
and MgSO4
0.5 – 1 M for both precipitants
(incremental increase of 0.1
M)
7 (50 mM HEPES), 7.5 (50
mM HEPES), 8 (50 mM Tris),
8.5 (50 mM Tris)
Sodium citrate 0.6 – 0.8 M (incremental
increase of 0.1 M)
7 (50 mM HEPES), 7.5 (50
mM HEPES), 8 (50 mM Tris),
8.5 (50 mM Tris)

Protocol 2:We also attempted to obtain AR LBD-ligand complex using a denaturing-
refolding protocol reported in literature.
32
Since no ligand was necessary during protein
expression, this method only required a small amount of the ligand of interest. To test the feasibility
of this method, we expressed His-tagged AR LBD without any ligand, and obtained AR LBD as
precipitated inclusion bodies after cell lysis. We dissolved the inclusion bodies in a 6 M
guanidinium chloride denaturing buffer.
32
After sufficient solubilization at room temperature, the
supernatant was then added dropwise to a refolding buffer with a final dilution factor of 30x. After
the addition was complete, the solution was stirred for additional time and insoluble materials were
removed by centrifugation. The supernatant was incubated with Ni beads to select His-tagged AR
LBD. Several refolding buffers were tested (Table 2.3), but none yielded a significant amount of
refolded protein.

33
Table 2.3 Refolding buffers screened for AR LBD refolding.
Buffer Refolding buffer components (containing 10 μM DHT)
1 50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 0.1 M Arginine, 2 mM DTT
2 50 mM HEPES pH 7.5, 10% glycerol, 4% DMSO, 0.2% octyl beta-D-
glucopyranoside (BOG), 0.1 M Arginine, 2 mM DTT
3 50 mM HEPES pH 7.5, 10% glycerol, 4% DMSO, 0.2% octyl beta-D-
glucopyranoside (BOG), 0.1 M Proline, 2 mM DTT

Protocol 3:We attempted to use a ligand exchange protocol reported in literature.
33
This
method only required small amount of the ligand of interest during protein purification. We opted
to test the ligand exchange between DHT and compound 02S. We expressed His-tagged AR LBD
in the presence of DHT as described above. During purification, buffers were amended with 1 μM
racemic compound 02. After Ni selection, an additional 20x molar excess of racemic compound
02 (10x molar excess of 02S) was added to the protein solution and the solution was incubated at
4
⁰
C overnight. EDTA was also added to chelate trace amount of Ni in solution. After overnight
incubation, precipitation was observed in the samples, and SEC chromatography showed
significant heterogeneity in the sample (Figure 2.12). Additionally, mass spectrometry revealed
that the protein sample after ligand exchange still contains DHT (Figure 2.13), which suggested
the ligand exchange was not complete. These observations discouraged us from attempting
crystallization with this protein sample.
In conclusion, we found that the crystallization condition for AR LBD-agonist binary
complex is highly ligand-dependent. Overexpression of AR LBD in the presence of the ligand of
interest is still the most reliable way to obtain pure AR LBD-agonist binary complex and further
attempts should focus on this protocol. Additionally, co-expression of HSPs with AR LBD could
be attempted to enhance the solubility of apo-state AR LBD.
34
Finally, cryo-EM methods could
circumvent the necessity for obtaining crystals either full-length AR LBD or AR variants such as
AR-V7.
35

34

Figure 2.12 Size Exclusion Column chromatography of AR LBD after ligand exchange.
Heterogeneous AR LBD was eluted at 17 – 22 mL elution volume.

Figure 2.13 Mass spectrometry of extracted ligands of protein sample after ligand exchange
MS was performed in ESI negative mode. A peak of m/z 325.3 corresponds to [DHT + Cl]
-
.

35
2.5 Chiral purity in large-scale manufacturing
BMS reported the chiral separation of intermediate I (Figure 2.2) in Chiralcel OD (50 x
500 mm) column, with at least 99% enantiomeric excess (ee).
10
Based on our findings, we
concluded that our AR antagonists must have a chiral purity of at least 99.8% ee (99.9% R
enantiomer m/m) for reliable in vitro or in vivo study results.
11
Thus, we studied the chiral HPLC
separation of the intermediate in ChiralCel OD-H (4.6 x 250 mm, 5 μm) analytical column in
preparation for future large-scale synthesis.
First, we studied the chiral separation under different mobile phases. We injected 400 μg
racemate sample to avoid bias associated with overloading. The mobile phase 50% EtOH/Hexane
resulted in sharper peak shapes than the 50% isopropanol (IPA)/Hexane (Figure 2.14). The cycle
time was much shorter in 50% EtOH/Hexane (20 min) than in 50% IPA/Hexane (35 min). Thus,
EtOH/Hexane should be used for this racemic compound.

Figure 2.14 Study of mobile phase for chiral separation of intermediate I and its enantiomer.
400 μg racemate samples were injected into ChiralCel OD-H (4.6 x 250 mm, 5 μm).

Datafile Name:CL6-EtOH-400ug1.lcd
Sample Name:CL6-EtOH-400ug
Sample ID:CL6-EtOH-400ug
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
2750
3000
3250
mV
Detector A 254nm
Datafile Name:CL6-400ug-1.lcd
Sample Name:CL6
Sample ID:CL6
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
2750
3000
mV
Detector A 254nm
50% IPA/Hexane
50% EtOH/Hexane
36
Next, we studied the loading capacity in 50% EtOH/Hexane. Due to excellent separation
of the two enantiomers, 5 mg of the racemate sample could be loaded with good resolution of two
isomer peaks (Figure 2.15). This loading capacity indicates that gram scale separation could be
easily carried out with preparative columns.

Figure 2.15 Study of loading for chiral separation of intermediate I and its enantiomer.
1 – 5 mg racemate samples were injected into ChiralCel OD-H (4.6 x 250 mm, 5 μm). Mobile
phase was 50% EtOH/Hexane

Finally, we studied the required iterations of chiral purification. After one round of
separation in either 50% EtOH/Hexane or 50% IPA/Hexane, the purity of the individual
enantiomers was not above 99.8% ee (Figure 2.16), even though the peaks are well separated.
After two rounds of separation, no chiral contaminations were detected at 254 nm UV (Figure
2.17), and the purity of the individual enantiomers reached 99.8% ee.
Datafile Name:CL6-EtOH-3000ug-1.lcd
Sample Name:CL6-EtOH
Sample ID:CL6-EtOH
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
500
1000
1500
2000
2500
3000
3500
mV
Detector A 254nm
Datafile Name:CL6-EtOH-5000ug-1.lcd
Sample Name:CL6-EtOH
Sample ID:CL6-EtOH
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
500
1000
1500
2000
2500
3000
3500
mV
Detector A 254nm
Datafile Name:CL6-EtOH-1000ug1.lcd
Sample Name:CL6-EtOH-1000ug
Sample ID:CL6-EtOH-1000ug
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
500
1000
1500
2000
2500
3000
3500
mV
Detector A 254nm
1 mg racemate
3 mg racemate
5 mg racemate
37

Figure 2.16 Study of enantiopurity of intermediate I and its enantiomer after one round of
chiral purification.
After one round of purification in ChiralCel OD-H (4.6 x 250 mm, 5 μm)., 200 μg – 400 μg of
enantiomers were injected individually. Blank samples were injected prior to each run to ensure
that no contamination was present in the system which could interfere with the purity analysis.

Figure 2.17 Study of enantiopurity of intermediate I and its enantiomer after two rounds of
chiral purification.
After two rounds of purification in ChiralCel OD-H (4.6 x 250 mm, 5 μm), 200 μg – 400 μg of
enantiomers were injected individually.

Datafile Name:CL6A-puri twice-purity1.lcd
Sample Name:CL6A-puri twice-purity
Sample ID:CL6A-puri twice-purity
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
50
100
150
200
250
300
350
400
mV
Detector A 254nm
Datafile Name:CL6B-puri twice-purity1.lcd
Sample Name:CL6B-puri twice-purity
Sample ID:CL6B-puri twice-purity
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
50
100
150
200
250
300
350
400
450
500
550
mV
Detector A 254nm
Datafile Name:CL6A-purity-2.lcd
Sample Name:CL6A-purity
Sample ID:CL6A-purity
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 min
0
100
200
300
400
500
600
700
800
mV
Detector A 254nm
Datafile Name:null-7.lcd
Sample Name:null
Sample ID:null
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
10
20
30
40
50
60
70
80
90
100
mV
Detector A 254nm
Datafile Name:null-6.lcd
Sample Name:null
Sample ID:null
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min
0
10
20
30
40
50
60
70
80
90
100
110
120
130
mV
Detector A 254nm
Datafile Name:CL6B-purity-3.lcd
Sample Name:CL6B-purity
Sample ID:CL6B-purity
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 min
0
100
200
300
400
500
600
700
mV
Detector A 254nm
50% IPA/Hexane
50% EtOH/Hexane
Datafile Name:EtOH-null-1.lcd
Sample Name:null
Sample ID:null
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
50
100
150
200
250
300
350
400
450
500
550
mV
Detector A 254nm
Datafile Name:CL6A-EtOH-purity-1.lcd
Sample Name:CL6A-EtOH-purity
Sample ID:CL6A-EtOH-purity
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 min
0
50
100
150
200
250
300
350
400
mV
Detector A 254nm
Datafile Name:EtOH-null-3.lcd
Sample Name:null
Sample ID:null
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
-50
0
50
100
150
200
250
300
350
400
450
500
550
mV
Detector A 254nm
Datafile Name:CL6B-EtOH-purity-1.lcd
Sample Name:CL6B-EtOH-purity
Sample ID:CL6B-EtOH-purity
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
50
100
150
200
250
300
350
400
mV
Detector A 254nm
Blank
Blank
ll injection
Blank

Blank

38
2.6 Experimental section
In silico modeling: Agonist docking was performed with the wt AR-LBD (PDB: 1E3G) in
complex with R1881 using Schrodinger Suite 2018-3 (Glide, Prime). For antagonists, a homology
model of wt AR-LBD in open conformation was built with Schrodinger Prime using the
antagonist-bound progesterone receptor (PDB ID: 2OVM) as a template. Ligands were prepared
with LigPrep. For induced fit docking, each ligand was docked (Glide module) with the standard
precision (SP). Side chains within a 5.0 A radius of each ligand pose were searched using Prime’s
side-chain sampling algorithms. Defined regions of the protein-ligand complexes were minimized
using OPLS3. Refined protein-ligand complexes were re-docked using Extra Precision (XP)
scoring in Glide. Top scoring docked poses (based on GlideScore and Prime energy) were analyzed
and compared. Predicted residues with hydrogen bonds were reported according to Genbank
mRNA sequence M20132.1 to mirror the AR crystal structure (PDB ID: 1E3G) used in this study.
AR LBD plasmid extraction and transformation: Plasmid encoding human AR LBD cDNA
is obtained from Addgene (#89083, deposited by E. Wilson). The human AR LBD amino acid
residues 663-919 with a histidine tag is cloned into pET-15b vector with ampicillin resistance, and
the plasmid is kept at DH5α strain. The DH5α cells are grown in TB media at 37⁰C overnight,
centrifuged, and the pellets collected. Plasmid is extracted with QIAprep® Spin Miniprep Kit and
stored in Elution Buffer at -80⁰C. 20 ng of plasmid is added to 50 μL of E. coli BL21DE3 strain
and incubated on ice for 5 min. The cells are plated on agar plate with Carbenicillin and grown at
37⁰C overnight. A single colony is picked and grown in TB media at 37⁰C for 5-7 hours. 600 μL
cell culture is added to 400 μL glycerol to make a 40% glycerol stock. The glycerol stock was
stored at -80⁰C.
39
AR LBD protein expression and purification: E. coli BL21DE3 containing the desired
plasmid is grown in LB media at 37⁰C overnight and then expanded to 2 L cell culture in LB media
supplemented with 30 μM DHT (or the ligand of interest). The cell culture was grown at 20⁰C until
OD reached 0.4, and then induced with 100 nM isopropyl-b-D-thiogalactoside (IPTG). Protein
expression was carried out at 20⁰C overnight. Harvested cells (around 5-7 g) were lysed with
sonication in a buffer containing 25 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 30 μM
DHT (or the ligand of interest). The broken cells were centrifuged for 45 min at 50,000x g at 4⁰C.
The supernatant was incubated with 0.5 mL Ni beads at 4⁰C for 1 h. Ni beads were then washed
and eluted dropwise with 800 uL elution buffer containing 25 mM HEPES pH 7.5, 300 mM NaCl,
300 mM imidazole, 10% glycerol, 10 μM DHT (or the ligand of interest). The eluted AR LBD
was further purified on a Superdex 75 size exclusion column at 4⁰C with a buffer containing 25
mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT, 5 μM DHT (or the ligand of
interest). The purified AR LBD-DHT binary complex was then incubated with 10 NIH unit
thrombin at room temperature for 4 h, before it was passed through 20 μL Ni beads for reverse
IMAC selection. The eluent was concentrated in centrifugal concentrators to 3.2 mg/mL.
Protein denaturing and refolding of AR LBD: The human AR LBD was expressed as
described above without ligand of interest in growth media. Harvested cells (around 5-7 g) were
lysed as described above without ligand of interest in any buffer. The pellet obtained after
centrifugation containing the inclusion body was washed with the same lysis buffer amended with
1% Triton X-100, and then dissolved in a denaturing buffer containing 25 mM HEPES pH 7.5, 6
M GdmCl (adjusted in buffer to final pH 7.5), 5 mM DTT to a protein concentration of 3 mg/mL.
The denaturing solution was stirred at room temperature for 2 h, and then centrifuged at room temp
at 30,000x g for 15 min to remove insoluble materials. The supernatant was then added dropwise
40
at 1 mL/min at room temperature to 30x volume of refolding buffers containing 10 μM of the
ligand of interest. After the addition was complete, the solution was allowed to sit for an additional
1 h at room temperature and then centrifuged at room temp at 30,000x g for 15 min to remove
insoluble materials. The supernatant was incubated with 0.5 mL Ni beads at 4⁰C and eluted as
described above.
Ligand exchange of AR LBD: The human AR LBD was overexpressed in the presence of
DHT as described above. Harvested cells (around 5-7 g) were lysed and AR LBD was purified as
described above, except for that the buffers were amended with 1 μM ligand of interest. After Ni
selection, the protein sample was incubated with 10x molar excess of ligand of interest at 4⁰C
overnight. 1 mM of EDTA was added to chelate trace Ni in the solution. After overnight
incubation, precipitate was observed and removed by centrifugation at 4⁰C. The protein sample
was then purified on a Superdex 75 size exclusion column as described above. Mass spectrometry
analysis of ligand exchange sample was performed on a fraction of the sample after overnight
ligand exchange. The unbound ligands in the buffer were first filtered out by three cycles of
concentrating the sample to 1/10 the volume and then diluting it back with the same buffer
containing no ligands. The sample was then denatured with 6x volume acetone at -20⁰C, vortexed
briefly, and incubated at -20⁰C for 20 min. The precipitated protein was removed by centrifugation
and the supernatant was dried and analyzed in mass spectrometry (ESI).
Crystallization, diffraction, and structure refinement: Crystallization drops were prepared
in Mosquito robot (TTP Labtech). Crystals were formed in the sitting drop vapor diffusion method
at room temperature. Crystals appeared within 7 d in drops of a 1:1 (v/v) ratio of purified protein
and precipitant. The crystals of AR LBD-DHT complexes appeared in the following conditions:
1.4 M - 1.6 M MgSO4, pH = 8 (50 mM Tris). Crystals were allowed to grow for 2 wk. Crystals are
41
soaked with glycerol prior to flash-freezing in liquid N2. X-ray diffraction data was collected at
100K at the Stanford Synchrotron Radiation Lightsource. The collected data were indexed and
integrated with X-ray Detector Software (XDS) and scaled using Scala, a part of the CCP4 suite.
Initial phase information of the DHT-bound AR LBD was obtained by molecular replacement
using Phaser with the previously solved structure (PDB:2AMA) as the search model. Waters were
added using ArpWarp during the initial round of the refinement. The structure was improved by
iterative rounds of model building and refinement using the programs Coot and Refmac5.
Chiral purity analysis: Chiral HPLC was performed on Shimadzu Prominence with
Chiralcel OD-H (5μm, 250 mm×4.6 mm); eluents hexane/isopropanol or hexane/ethanol with
detection at 254 nm; column temperature of 20°C.

42
Chapter 2 references
1. Milton, R. d.; Milton, S.; Kent, S., Total chemical synthesis of a D-enzyme: the enantiomers
of HIV-1 protease show reciprocal chiral substrate specificity [corrected]. Science 1992, 256
(5062), 1445-1448.
2. Tomaszewski, J.; Rumore, M. M., Stereoisomeric drugs: FDA's policy statement and the
impact on drug development. Drug Development and Industrial Pharmacy 1994, 20 (2), 119-
139.
3. Kim, J. H.; Scialli, A. R., Thalidomide: the tragedy of birth defects and the effective treatment
of disease. Toxicological Sciences 2011, 122 (1), 1-6.
4. Hirota, K.; Lambert, D., Ketamine: its mechanism(s) of action and unusual clinical uses.
British Journal of Anaesthesia 1996, 77 (4), 441-444.
5. Kim, J.; Farchione, T.; Potter, A.; Chen, Q.; Temple, R., Esketamine for Treatment-Resistant
Depression — First FDA-Approved Antidepressant in a New Class. New England Journal of
Medicine 2019, 381 (1), 1-4.
6. Wang, G.; Dugas, M.; Armah, I. B.; Honerjäger, P., Interaction between DPI 201-106
enantiomers at the cardiac sodium channel. Molecular Pharmacology 1990, 37 (1), 17-24.
7. Romey, G.; Quast, U.; Pauron, D.; Frelin, C.; Renaud, J.; Lazdunski, M., Na+ channels as
sites of action of the cardioactive agent DPI 201-106 with agonist and antagonist enantiomers.
Proceedings of the National Academy of Sciences 1987, 84 (3), 896-900.
8. Hof, R.; Rüegg, U.; Hof, A.; Vogel, A., Stereoselectivity at the calcium channel: opposite
action of the enantiomers of a 1, 4-dihydropyridine. Journal of cardiovascular pharmacology
1985, 7 (4), 689-693.
9. Foster, W. R.; Car, B. D.; Shi, H.; Levesque, P. C.; Obermeier, M. T.; Gan, J.; Arezzo, J.
C.; Powlin, S. S.; Dinchuk, J. E.; Balog, A., Drug safety is a barrier to the discovery and
development of new androgen receptor antagonists. The Prostate 2011, 71 (5), 480-488.
10. Balog, A.; Rampulla, R.; Martin, G. S.; Krystek, S. R.; Attar, R.; Dell-John, J.; DiMarco,
J. D.; Fairfax, D.; Gougoutas, J.; Holst, C. L.; Nation, A.; Rizzo, C.; Rossiter, L. M.;
Schweizer, L.; Shan, W.; Spergel, S.; Spires, T.; Cornelius, G.; Gottardis, M.; Trainor, G.;
Vite, G. D.; Salvati, M. E., Discovery of BMS-641988, a novel androgen receptor antagonist for
the treatment of prostate cancer. ACS Medicinal Chemistry Letters 2015, 6 (8), 908-912.
11. Patsch, K.; Liu, C.; Zapotoczny, G.; Sun, Y.; Sura, H.; Ung, N.; Sun, R. X.; Haliday, B.;
Yu, C.; Aljehani, M.; Lee, J. S. H.; Kashemirov, B. A.; Agus, D. B.; McKenna, C. E.;
Ruderman, D., Paradoxical androgen receptor regulation by small molecule enantiomers.
Proceedings of the National Academy of Sciences 2021, 118 (12), e2100918118.
12. Marhefka, C. A.; Gao, W.; Chung, K.; Kim, J.; He, Y.; Yin, D.; Bohl, C.; Dalton, J. T.;
Miller, D. D., Design, synthesis, and biological characterization of metabolically stable selective
androgen receptor modulators. Journal of Medicinal Chemistry 2004, 47 (4), 993-998.
13. Sakkiah, S.; Kusko, R.; Pan, B.; Guo, W.; Ge, W.; Tong, W.; Hong, H., Structural changes
due to antagonist binding in ligand binding pocket of androgen receptor elucidated through
molecular dynamics simulations. Frontiers in Pharmacology 2018, 9, 492.
14. Laudet, V.; Hänni, C.; Coll, J.; Catzeflis, F.; Stéhelin, D., Evolution of the nuclear receptor
gene superfamily. The EMBO Journal 1992, 11 (3), 1003-1013.
15. Madauss, K. P.; Grygielko, E. T.; Deng, S.-J.; Sulpizio, A. C.; Stanley, T. B.; Wu, C.;
Short, S. A.; Thompson, S. K.; Stewart, E. L.; Laping, N. J.; Williams, S. P.; Bray, J. D., A
43
Structural and in Vitro Characterization of Asoprisnil: A Selective Progesterone Receptor
Modulator. Molecular Endocrinology 2007, 21 (5), 1066-1081.
16. Matias, P. M.; Donner, P.; Coelho, R.; Thomaz, M.; Peixoto, C.; Macedo, S.; Otto, N.;
Joschko, S.; Scholz, P.; Wegg, A.; Bä sler, S.; Schä fer, M.; Egner, U.; Carrondo, M. A.,
Structural evidence for ligand specificity in the binding domain of the human androgen receptor:
implications for pathogenic gene mutations. Journal of Biological Chemistry 2000, 275 (34),
26164-26171.
17. Gao, W.; Bohl, C. E.; Dalton, J. T., Chemistry and Structural Biology of Androgen Receptor.
Chemical Reviews 2005, 105 (9), 3352-3370.
18. Cockshott, I. D., Bicalutamide. Clinical Pharmacokinetics 2004, 43 (13), 855-878.
19. Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T., Structural basis for
antagonism and resistance of bicalutamide in prostate cancer. Proceedings of the National
Academy of Sciences 2005, 102 (17), 6201-6206.
20. Liu, H.; Han, R.; Li, J.; Liu, H.; Zheng, L., Molecular mechanism of R-bicalutamide
switching from androgen receptor antagonist to agonist induced by amino acid mutations using
molecular dynamics simulations and free energy calculation. Journal of Computer-Aided
Molecular Design 2016, 30 (12), 1189-1200.
21. Grosse, L.; Campeau, A. S.; Caron, S.; Morin, F. A.; Meunier, K.; Trottier, J.; Caron, P.;
Verreault, M.; Barbier, O., Enantiomer selective glucuronidation of the non ‐steroidal pure
anti ‐androgen bicalutamide by human liver and kidney: Role of the human UDP ‐
glucuronosyltransferase (UGT) 1A9 enzyme. Basic & Clinical Pharmacology & Toxicology
2013, 113 (2), 92-102.
22. Bohl, C. E.; Miller, D. D.; Chen, J.; Bell, C. E.; Dalton, J. T., Structural basis for
accommodation of nonsteroidal ligands in the androgen receptor. Journal of Biological
Chemistry 2005, 280 (45), 37747-37754.
23. Sack, J. S.; Kish, K. F.; Wang, C.; Attar, R. M.; Kiefer, S. E.; An, Y.; Wu, G. Y.;
Scheffler, J. E.; Salvati, M. E.; Krystek, S. R.; Weinmann, R.; Einspahr, H. M.,
Crystallographic structures of the ligand-binding domains of the androgen receptor and its
T877A mutant complexed with the natural agonist dihydrotestosterone. Proceedings of the
National Academy of Sciences 2001, 98 (9), 4904-4909.
24. Zoubeidi, A.; Zardan, A.; Beraldi, E.; Fazli, L.; Sowery, R.; Rennie, P.; Nelson, C.;
Gleave, M., Cooperative interactions between androgen receptor (AR) and heat-shock protein 27
facilitate AR transcriptional activity. Cancer Research 2007, 67 (21), 10455-10465.
25. Duan, M.; Liu, N.; Zhou, W.; Li, D.; Yang, M.; Hou, T., Structural diversity of ligand-
binding androgen receptors revealed by microsecond long molecular dynamics simulations and
enhanced sampling. Journal of Chemical Theory and Computation 2016, 12 (9), 4611-4619.
26. Zorn, K. M.; Foil, D. H.; Lane, T. R.; Hillwalker, W.; Feifarek, D. J.; Jones, F.; Klaren,
W. D.; Brinkman, A. M.; Ekins, S., Comparison of Machine Learning Models for the Androgen
Receptor. Environmental Science & Technology 2020, 54 (21), 13690-13700.
27. Grisoni, F.; Consonni, V.; Ballabio, D., Machine learning consensus to predict the binding to
the androgen receptor within the CoMPARA project. Journal of Chemical Information and
Modeling 2019, 59 (5), 1839-1848.
28. Pereira de Jésus-Tran, K.; Côté, P.-L.; Cantin, L.; Blanchet, J.; Labrie, F.; Breton, R.,
Comparison of crystal structures of human androgen receptor ligand-binding domain complexed
with various agonists reveals molecular determinants responsible for binding affinity. Protein
Science 2006, 15 (5), 987-999.
44
29. Askew, E. B.; Gampe, R. T.; Stanley, T. B.; Faggart, J. L.; Wilson, E. M., Modulation of
androgen receptor activation function 2 by testosterone and dihydrotestosterone. Journal of
Biological Chemistry 2007, 282 (35), 25801-25816.
30. He, B.; Gampe Jr, R. T.; Kole, A. J.; Hnat, A. T.; Stanley, T. B.; An, G.; Stewart, E. L.;
Kalman, R. I.; Minges, J. T.; Wilson, E. M., Structural basis for androgen receptor interdomain
and coactivator interactions suggests a transition in nuclear receptor activation function
dominance. Molecular Cell 2004, 16 (3), 425-438.
31. Wang, F.; Liu, X.-q.; Li, H.; Liang, K.-n.; Miner, J. N.; Hong, M.; Kallel, E. A.; Van
Oeveren, A.; Zhi, L.; Jiang, T., Structure of the ligand-binding domain (LBD) of human
androgen receptor in complex with a selective modulator LGD2226. Acta Crystallographica
Section F: Structural Biology and Crystallization Communications 2006, 62 (11), 1067-1071.
32. Saeed, A.; Vaught, G. M.; Gavardinas, K.; Matthews, D.; Green, J. E.; Losada, P. G.;
Bullock, H. A.; Calvert, N. A.; Patel, N. J.; Sweetana, S. A.; Krishnan, V.; Henck, J. W.;
Luz, J. G.; Wang, Y.; Jadhav, P., 2-Chloro-4-[[(1R, 2R)-2-hydroxy-2-methyl-
cyclopentyl]amino]-3-methyl-benzonitrile: A Transdermal Selective Androgen Receptor
Modulator (SARM) for Muscle Atrophy. Journal of Medicinal Chemistry 2016, 59 (2), 750-755.
33. Unwalla, R.; Mousseau, J. J.; Fadeyi, O. O.; Choi, C.; Parris, K.; Hu, B.; Kenney, T.;
Chippari, S.; McNally, C.; Vishwanathan, K.; Kilbourne, E.; Thompson, C.; Nagpal, S.;
Wrobel, J.; Yudt, M.; Morris, C. A.; Powell, D.; Gilbert, A. M.; Chekler, E. L. P., Structure-
Based Approach To Identify 5-[4-Hydroxyphenyl]pyrrole-2-carbonitrile Derivatives as Potent
and Tissue Selective Androgen Receptor Modulators. Journal of Medicinal Chemistry 2017, 60
(14), 6451-6457.
34. Peng, S.; Chu, Z.; Lu, J.; Li, D.; Wang, Y.; Yang, S.; Zhang, Y., Co-expression of
chaperones from P. furiosus enhanced the soluble expression of the recombinant
hyperthermophilic α-amylase in E. coli. Cell Stress and Chaperones 2016, 21 (3), 477-484.
35. Bai, X.-c.; McMullan, G.; Scheres, S. H. W., How cryo-EM is revolutionizing structural
biology. Trends in Biochemical Sciences 2015, 40 (1), 49-57.

45
Chapter 3 Evaluation of novel AR antagonists
3.1 Design and synthesis of novel AR antagonists
3.1.1 Design rationale
To design AR antagonists based on the BMS-641988 scaffold, we started by addressing
prominent safety problem of BMS-641988.
1
As described in Chapter 1, BMS-641988 is
extensively metabolized in vivo to BMS-501949.
1
BMS-501949 readily crosses the blood-brain
barrier (BBB) inhibiting GABAA receptors, and likely triggered seizures observed in clinical
trials.
1
To prevent in vivo generation of BMS-501949, we performed metabolic predictions in silico
using the Schrödinger P450 Site of Metabolism (SOM) module.
2
As expected, the C5-N bond in
BMS-641988 has the highest intrinsic P450 3A4 reactivity (Figure 3.1), suggesting that it is the
most susceptible bond towards P450 3A4 metabolism.
Inspired by the unique structure of darolutamide
3
(Figure 1.6), we designed three amide
derivatives of BMS-641988, namely compound 01R, 02R, and 03R. Schrödinger SOM
2

calculation showed that the C5-N bonds in these molecules are no longer the most susceptible
bond towards P450 3A4 metabolism (Figure 3.1). This result prompted us to synthesize and test
a series of novel amide analogues of the oxabicyclic scaffold.
Moreover, previous AR modeling studies from others and us (See Chapter 2.3 for detailed
discussion),
4-7
as well as the study published by Balog et al.,
8
suggested that the C-5 substituent in
AR antagonists is critical to the final conformation of H11 and H12 and subsequent destabilization
of the AR-LBD. Taking these findings into account, we designed a series of C-5 (R)-stereoisomer
substituents for our new drug series. To extend the SAR, we also investigated substitutions on the
aniline ring.
46

Figure 3.1 Predicted metabolism of representative compounds
SOM calculation was done in Schrödinger P450 Site of Metabolism (SOM) module. The size of
the green dot is positively correlated with the probability of metabolism by P450 3A4 enzyme.

3.1.2 Synthesis
Compounds were synthesized as reported by Balog, et al, using the general route shown in
Scheme 3.1.
8
A symmetrical Diels−Alder cycloaddition of the alkene 2 and the diene MEM 2,5-
dimethyl-3-furoate yielded a racemate (3.3a and 3.3b). Hydrogenation under Pd/C catalysis led to
the formation of the endo-substituted esters 3.4a (intermediate I) and 3.4b, which were deprotected
to give 3.5a and 3.5b. Curtius rearrangement and subsequent TFA-promoted cleavage converted
the acids to amines 3.7a and 3.7b. Coupling of amines 3.7 with various carboxylic acids 3.8
afforded the target amide-pharmacophores. In the original synthesis of BMS-641988, 3.4a
(intermediate I) and 3.4b were separated using semi-preparative chiral HPLC and only the
intermediate I was carried forward to produce the target compound. In our syntheses, 3.4a and
3.4b were carried forward en route to the final target C-5 (R/S)-mixtures, which were then resolved
chirally to afford both individual enantiomers. Each individual enantiomer underwent additional
chiral purification to ensure the enantiopurity.
47
Anilines 3.1 and carboxylic acids 3.8 were either obtained from commercial sources or
synthesized by known procedures (See Appendix).
9-11
It is noteworthy exposure to methanol
should be avoided during purification to prevent ring opening of the pyrrolidine-dione.
The stereochemistry of individual enantiomers was extrapolated from their optical rotation.
According to the multiple publications by BMS (See Chapter 1.4), R-enantiomers of these family
of compounds have negative specific rotations. Since the specific rotation of BMS-641988 was
reported,
8
we determined it to be [α]
24
D = −28.1° (MeOH). The specific rotation of S-BMS was
measured to be [α]
24
D = +26.1° (MeOH). The absolute structures of both BMS-641988 and S-BMS
were confirmed by x-ray crystallography (See Appendix).

Scheme 3.1 Synthesis and chiral separation of derivatives of BMS-641988.

Reagents and conditions: (a) maleic anhydride, glacial acetic acid, reflux overnight, 90%; (b) 2-
MEM 2,5-dimethylfuran-3-carboxylate, 125°C, 1.5 h, then rt overnight, 25%; (c) H2, Pd/C,
EtOAc, rt overnight, 75%; (d) 3 N HCl, THF, rt for 16 h, 99%; (e) 2-trimethylsilylethanol, DPPA,
Et3N, 4 Å MS, 1,4-dioxane, 75°C, 53%; (f) TFA, CH2Cl2, rt for 2 h, quantitative yield; (g)
carboxylic acid, DIPEA, HATU, DMF, rt overnight; (h) chiral HPLC separation.

48
3.2 In vitro screening of novel AR antagonists
3.2.1 Agonistic activities of BMS-641988
A previous report identified BMS-641988 as an antagonist in LNCaP cells expressing AR
T878A, a mutation that reduces ligand specificity frequently reported in prostate cancer patients.
5,
12
However, treatment with the drug promoted LNCaP proliferation.
12
To investigate these reports,
we treated LNCaP cells with BMS-641988. We found that BMS-641988 alone induced ARE-
luciferase expression in LNCaP cells (Figure 3.2A). Increasing doses confirmed the drug is indeed
agonistic in AR T878A mutation (Figure 3.2B). This agonism may explain the limited efficacy of
BMS-641988 in clinical studies.
1

Figure 3.2 BMS-641988 is an AR agonist in LNCaP cells expressing AR T878A.
(A) ARE-luciferase assays in cells treated with 10 µM drug + 1 nM R1881. Signals are presented as mean
with standard deviation (n = 2). (B) Individual agonist mode dose-response curve used to estimate absolute
IC 50. Signals are normalized to NTC (0%) and 1 nM R1881 (100%).

3.2.2 Structure-activity relationship (SAR) studies
In order to confirm the design of our compounds and to study the SAR of these AR
antagonists, we employed a fragment approach (Figure 3.3). Some of the novel compounds
described here were designed and synthesized by Dr. Chao Liu.
49
We screened the compounds with ARE-luciferase assays in LNCaP cells treated with
R1881. As expected, enzalutamide effectively antagonized LNCaP AR (Table 3.1). We first
designed and tested compounds 01R, 02R, and 03R (see Chapter 2). In contrast to BMS-6431988,
all three compounds reduced ARE-luciferase in a dose-dependent manner (Table 3.1), supporting
our design rationale.
To study the role of the oxabicyclic core, we tested compound 07. Compound 07 did not
elicit a measurable response in ARE-luciferase assay (Table 3.1), demonstrating that the
oxabicyclic core is essential for the antagonist activity for this family of compounds.

Figure 3.3 Fragment approach and summary of SAR derived from our compounds.

Table 3.1 SAR studies of novel AR antagonists.
IC50 value is an average of n = 1-7 replicates. Emax is normalized to 1 nM R1881 as 100%.
BMS-641988 and compound 04R were agonistic and their IC50 values were not obtained.
Compound 07 did not display AR activity and its value was not obtained.
Compound X R
LNCaP ARE-luciferase
IC50 (nM) Emax (%)
Enzalutamide - - 234 4
BMS-641988 H

NA 91
01R H

842 9
50
02R H

446 11
03R H

3559 24
04R F

NA 102
05R H

2129 18
06R F

1107 15
07

NA 92
08R H

2522 33
09R H

1255 22
10R H

416 16
11R Me

2202 42
12R H (3’ Cl)

870 8
13R H

4325 44
14R H

2456 25
15R F

NA (data pending) NA (data pending)
16R H

521 10
17R H

NA (data pending) NA (data pending)
18R H

NA (data pending) NA (data pending)
19R H

NA (data pending) NA (data pending)

51
Then, to study the SAR of substitution on the aniline ring, we compared 04R, 06R, 11R,
and 15R (results pending). Compound 04R did not lower ARE-luciferase signals as an AR
antagonist (Table 3.1). Comparing BMS-641988 with 04R, we conclude that the C-5 substituent,
but not the 2’-aniline substituent, plays a crucial role in defining the antagonistic activity in this
family of compounds. 12R, which possesses a 3’-chloro instead of 3’-CF3 on the anilinering,
displayed better efficacy than 03R, suggesting that 3’-chloro could be a better substituent than 3’-
CF3. Compound 06R antagonized LNCaP AR better than 11R. Comparing 06R and 11R with
03R, we conclude that 2’-fluoro-substitution did not significantly impact activity, whereas 2’-
methyl-substitution significantly lowered efficacy. This observation could be explained by the
existence of two atropisomers in 11R, only one of which effectively inhibits AR (Figure 3.4).
13

Evidence to support the existence of these quasi-stable atropisomers is provided by the observation
that enantiopure 11R separated into two peaks on reversed-phase HPLC (Figure 3.4A). These
species both re-equilibrated after standing overnight in a solution of MeCN/H2O (Figure 3.4B, C).
Hartree-Fock 3-21G calculations

in Spartan 14 (Wavefunction Inc.) estimated the N-Ar bond
rotational energy barriers (in vacuum) to be 58 kJ/mol and 86 kJ/mol for 06R and 11R, respectively
(Figure 3.4D).
13, 14
The rotational barrier 2-Me-N-phenylmaleimide (structurally similar to 11R)
was experimentally measured to be 87 kJ/mol by
1
H NMR.
14
We postulate that oxygen in the
oxabicyclic ring forms additional H-bonds with protic solvents and increased the rotational barriers
for 11R. Since complete separation of atropisomers requires as least a 93.3 kJ/mol rotational
energy barrier at 300 K (half-life of at least 1000 s),
13
atropisomers of 11R are unlikely to be well
separated at room temperature.

52

Figure 3.4 Atropisomer separation of Compound 11R.
(A) Enantiopure 11R separates into two peaks on reserve-phase HPLC (40%-60% MeCN/H2O). (B) and
(C) Samples from both peaks equilibrate after standing overnight in a solution of MeCN/H2O. Hartree-
Fock 3-21G calculations

in Spartan 14 (Wavefunction Inc.) show a 58 kJ/mol rotational energy barrier for
Compound 06R, and an 86 kJ/mol for Compound 11R.

Finally, to study the SAR of C-5 endo substitution (Table 3.1), we designed and tested
compounds 05R, 08R, 09R, 10R, 13R, 14R, 16R, 17R, 18R (results pending), and 19R (results
pending). Compounds 09R and 10R have connected aromatic rings substituents, while 08R and
16R have fused aromatic ring substituents at C-5 endo position. Compound 10R is a better
antagonist than 09R, which shows that heteroatom species (oxygen or sulfur) also influences
D
53
antagonist activity. Compound 16R is both more potent and more effective than 08R, despite their
identical ligand shape. This demonstrates that subtle change of heteroatom (nitrogen) position
drastically influences antagonist activity. Compounds 13R and 14R have non-aromatic ring
substituents, and they weakly antagonized AR, showing that non-aromatic ring substituents
resulted in much lower activities than their counterparts with aromatic ring substituents.
Compound 05R (synthesis in Scheme 3.2
15
) has a carboxamide isosteric triazole moiety at C-5
endo position. Compound 05R significantly decreased luciferase transcription even though it has
low binding affinity, showing that the AR-LBD binding pocket can accommodate isosteric
moieties of carboxamide at the C-5 endo position. The design of Compound 18R was inspired by
the clinical candidate proxalutamide (also known as GT-0918, Figure 3.5). Proxalutamide is an
AR antagonist that also decreases AR protein expression level in vivo, although the exact
mechanism of this protein expression regulation is unknown.
16

In summary, these results imply that the binding pocket in the AR-LBD is able to
accommodate a wide range of C-5 endo substituents. Our SAR studies are summarized in Figure
3.3. We greatly expanded the SAR to include multiple carboxamide analogues, which were not
well studied by Balog et al.
8

54
Scheme 3.2 Synthesis and chiral separation of Compound 05R/S.

Reagents and conditions: (a) 2,5-dimethylfuran, neat, 60
o
C, overnight, 75%; (b) BH3/THF at 0
o
C, 30 min; then 0.5 M Na2HPO4/NaH2PO4 buffer until pH 7.2, 0
o
C; then H2O2, for 30 min , 71%;
(c) Tf2O, pyridine, anhydrous DCM, 0
o
C, 1h, 61%; (d) NaN3, DMF, rt overnight, 79%; (e) 3-
butynol, copper (II) sulfate pentahydrate, and sodium ascorbate, 1:1 tBuOH:water, 40
o
C, 2 d.,
30%; (f) chiral HPLC separation.

Figure 3.5 Structure of proxalutamide (GT-0918).

3.3 In vitro evaluation of lead compounds
3.3.1 In vitro efficacy studies
To evaluate our compounds as potential preclinical candidates for treatment of prostate
cancer, especially CRPC, we measured their effect on VCaP cells, a hormone-responsive but -
independent model of CRPC expressing wildtype AR. We tested structurally representative drugs
that showed promising efficacy and potency in ARE-luciferase assays, namely 01R, 06R, 08R,
55
10R and 16R (Table 3.2). Compounds 01R, 06R and 16R potently and effectively reduced cell
viability in VCaP cells, while 08R and 10R did not induce a significant response (Emax > 50%). In
addition, preliminary data showed that these compounds remain effective antagonists in the
enzalutamide-resistant AR F877L/T878A mutant (Table 3.2).
17
These data support 01R, 06R, and
16R as promising preclinical candidates.

Table 3.2 In vitro efficacy of lead compounds.
BMS-641988 did not reach IC50 in VCaP viability assay.
Compound VCaP viability
IC50 (nM)
Effective against AR
F877L/T878A mutant
Enzalutamide 63 No
BMS-641988 N/A No
01R 342 Yes
06R 151 Yes
16R 131 Yes

3.3.2 In vitro and in silico safety studies
To estimate the safety profiles of our lead compounds, we predicted their BBB penetration
relative to known androgen antagonists using the Schrödinger QikProp module.
2
Setting logBB <
−1 as a practical threshold,
18
we obtained computational predictions with good correlations with
available in vivo data (Figure 3.6).
1
For example, flutamide, enzalutamide and BMS-501949 were
predicted to readily penetrate the BBB, whereas bicalutamide, darolutamide, BMS-641988, as well
as our lead compounds all had logBB values well below –1. We also performed Schrodinger SOM
56
calculations
2
on our lead compounds. Unlike the C5-N bond in BMS-641988, the C5-N bonds in
our lead compounds are not susceptible to P450 3A4 enzyme cleavage (Figure 3.7). These results
showed that our lead compounds are unlikely to be metabolized to the neurotoxic BMS-501949.

Figure 3.6 Calculated logBB of representative clinical compounds and lead compounds.
Compounds with logBB values lower than -1 are shown in blue, and values higher than -1 in orange.

Figure 3.7 Predicted SOM for lead compounds.
The C5-N bond is not the most susceptible bond to P450 metabolism in any of the lead compounds.
-2.5 -2 -1.5 -1 -0.5 0
Componud 16R
Compound 06R
Compound 01R
BMS-641988
BMS-501949
Darolutamide
Enzalutamide
Bicalutamide
Flutamide
Predicted logBB
57
Our in silico calculations were confirmed by in vitro liver microsome metabolism assays.
Toxic metabolite that accumulated in the BMS-641988 sample (BMS-501949) were not detected
in 01R (BMS-501949) or 06R (o-fluoro-BMS-501949) after 8 h incubation (Figure 3.8). The rates
of intrinsic clearance were comparable across 01R (15.3 µl/min/mg), 06R (10.3 µl/min/mg), BMS-
641988 (10.1 µl/min/mg). Taken together, these results suggest improved safety profiles of our
lead compounds compared to BMS-641988 and enzalutamide.

Figure 3.8 Extracted ion chromatograms of microsomal metabolites of representative compounds.
Rates of intrinsic clearance were established by measuring levels of intact compound after increasing
incubation times using mass spectrometry.

3.4 Gram scale synthesis and purification of key intermediate
To prepare for in vivo studies of our lead compounds, we conducted the scale-up of
synthesis. Instead of separating the enantiomers at the end of the synthetic route as described in
Chapter 3.1, we opted to separate the key compound intermediate I (refer to Chapter 2.2 and
Chapter 3.1) as described by BMS.
58
First, 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(trifluoromethyl)benzonitrile was
synthesized in 30-gram scale reactions. 4-amino-2-(trifluoromethyl)benzonitrile (20.5 g, 110
mmol) and maleic anhydride (12.9 g, 132 mmol) were dissolved in glacial acetic acid (110 mL)
and refluxed overnight. Acetic acid was removed in vacuum after the reaction was completed, and
the product was extracted with EtOAc and washed with water. A yellow to brown solid was
obtained (22.6 g, 77% yield) without further purification.
In parallel, MEM 2,5-dimethylfuran-3-carboxylate was synthesized in 30-gram scale
reactions. 2,5-dimethylfuran-3-carboxylic acid (14.0 g, 100 mmol) and K2CO3 (16.6 g, 120 mmol)
were dissolved in dry DMF (100 mL). MEM chloride was added dropwise to the solution at 0⁰C
and the reaction was stirred at room temp for overnight. After the reaction was completed, the
solvent DMF was removed by washing the crude with brine (5x volume of DMF), and the crude
was extracted with EtOAc (300 mL). A yellow oil was obtained (19.3 g, 85% yield) without further
purification.
Then, 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-(trifluoromethyl)benzonitrile (10.6 g,
40 mmol) and MEM 2,5-dimethylfuran-3-carboxylate (13.7 g, 60 mmol) were heated at 125⁰C for
2 h and stirred at room temp overnight in a Diels-Alder cycloaddition reaction. The crude was
loaded with celite (3 g celite per gram crude), and purified by flash chromatography in 0%-100%
EtOAc in Hexane (Figure 3.9). Due to the limited capacity of purification instrument CombiFlash
RF Teledyne (max 80 g silica column), each purification could not exceed 8 g sample loading.
Thus, multiple purifications were necessary for each 20-gram scale reaction. Due to low yield
(4.1g, 21%), the reaction was repeated to obtain enough product as an orange oil.

59

Figure 3.9 Purification of Diels-Alder cycloaddition reaction.
6 g crude dry-loaded with 18 g celite. Column: RediSep Silica 80g. Mobile phase: A: Hexane, B:
EtOAc.

Next, hydrogenation was performed in a three-necked flask. The racemic Diels-Alder
cycloaddition product (10.9 g, 22 mmol) and Pd/C (600 mg, 10% Pd, 50% wet) were dissolved in
EtOAc (120 mL). The flask was flushed with nitrogen and then hydrogen. The reaction was stirred
at room temp overnight under hydrogen (pressurized in balloons). After the reaction was
completed, the Pd/C was filtered out on a layer of celite, and the crude was purified in silica column
with 35% EtOAc/Hexane. The reaction (6.8 g, 62% yield) was repeated to obtain enough racemic
product (intermediate I and its enantiomer) as a yellow solid.
Intermediate I and its enantiomer were then chirally resolved in a preparative ChiralCel
OD-H column (30 x 250 mm, 5 μm) in tandem with a ChiralCel OD-H guard column (30 x 50
mm, 5 μm). The racemate was loaded as a MeOH solution (40 mg/mL), and the mobile phase was
60
in 50% EtOH/Hexane (refer to Chapter 2.5). 400 mg of racemate sample could be separated with
satisfactory peak resolution (Figure 3.10).

Figure 3.10 Chiral separation of intermediate I and its enantiomer.
400 mg racemate sample was injected as a MeOH solution (40 mg/mL) to ChiralCel OD-H column
(30 x 250 mm, 5 μm) in tandem with a ChiralCel OD-H guard column (30 x 50 mm, 5 μm). The
mobile phase was in 50% EtOH/Hexane. The S isomer has a retention time of 9.7 min, and the R
isomer 16.5 min.

3.5 Experimental section
In silico calculation of logBB and site of metabolism: Calculations were performed in
Schrodinger Maestro. Compounds were prepared with LigPrep. LogBB was calculated with the
QikProp module. Site of metabolism was generated with the P450 Site of Metabolism (SOM)
module.
Chemical synthesis: Unless otherwise noted, all reagents and solvents were commercially
available and used as received. The progress of all reactions was monitored on precoated silica gel
plates (with fluorescence indicator UV254) using ethyl acetate/hexane or
dichloromethane/methanol as solvent systems. Column chromatography was performed with
61
ISCO CombiFlashRf+ Lumen flash chromatography with the solvent mixtures specified in the
corresponding experiment. NMR spectra were recorded on either a Varian 500 or 600 at room
temperature. NMR samples are prepared in 1-2 mg/mL concentration. Data is reported as follows:
chemical shift (ppm, δ relative to residual solvent peak for
1
H and
13
C), multiplicity (s = singlet, d
= doublet, t = triplet, q = quartet, m = multiplet, and br = broad), coupling constant (Hz), and
integration.
19
F NMR spectra were recorded with proton decoupling. Optical rotation was
determined by a Jasco P2000 polarimeter, and specific rotation [α]
20
D for a sample solution is given
in deg cm
3
g
−1
dm
−1
. Measurement accuracy for the Jasco P2000 polarimeter is ±0.002⁰ (optical
rotation up to 1⁰) or ±0.2% (optical rotation larger than 1⁰), and repeatability is ±0.002⁰. Time-of-
flight high resolution mass spectrometry (TOF-HRMS) was performed on a Water Synapt G2-Si
ESI spectrometer (performed at the School of Chemical Sciences Mass Spectrometry Laboratory
(MSL) at the University of Illinois). Low-resolution mass spectrometry (LRMS) analysis was
performed on Advion Expression or Finnigan LCQ Deca XP Max equipped with an ESI source
and an APCI source. Advion Expression was calibrated weekly for both ESI source and APCI
source with five standards (m/z ranging from 100 to 2000). Chiral HPLC separation was performed
on Shimadzu Prominence: column, Chiralcel OD-H (5μm, 250 mm×4.6 mm) or ProntoSIL AX
QN (5μm, 150 mm×8.0 mm) or ProntoSIL Chiral AX QD-1 (5 µm, 150 mm×4.0 mm) or Regis
Reflect C-Amylose A (5μm, 250 mm×4.6 mm); eluents hexane/isopropanol or hexane/ethanol
with detection at 254 nm; column temperature of 20°C. All final compounds were further purified
by reverse phase HPLC with the same instrument on Phenomenex Luna C18 column with 50%
acetonitrile in water.
General synthetic method of amide coupling: The amine intermediates were obtained as
described in Chapter 3 or as described in Appendix. The carboxylic acids were obtained
62
commercially or synthesized as described in Appendix. The amide coupling was performed as
follows: 1.5 equiv. acid, 1.5 equiv. Et3N, and 1.5 equiv. HATU were stirred in dry DMF for 20
min before added dropwise to a solution of 1 equiv. amine and 1 equiv. Et3N in dry DMF. The
reaction was stirred at room temperature overnight. The reaction mixture was extracted with ethyl
acetate and washed with brine. The crude was purified by either flash chromatography
(DCM/MeOH) or reverse phase HPLC (MeCN/H2O), before the enantiomers were separated by
chiral HPLC.
4-((3aR,4R,5R,7R,7aS)-5-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)-4,7-dimethyl-1,3-
dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-2-(trifluoromethyl)benzonitrile, (Compound
05R). Compound 05R was prepared as described in Chapter 3. A mixture of the azide
intermediate (36 mg, 0.09 mmol), 3-butynol (9.5mg, 0.135 mmol), copper (II) sulfate pentahydrate
(6.7 mg, 0.027 mmol), and sodium ascorbate (10.7 mg, 0.054 mmol) in 1:1 tert-butanol:water (1
mL) was stirred at 40
o
C for 2 days. The volatiles were removed under reduced pressure and the
residue was partitioned between ethyl acetate and water. The organic layer was washed with brine,
dried over Na2SO4, filtered and concentrated. The residue was purified by flash column
chromatography (0-10% MeOH in DCM) to give the racemic product as a white solid (6.4 mg,
30%). Further separation of the two enantiomers was achieved by chiral HPLC using a Chiralcel
OD-H column (250 × 4.6 mm, 5 µm) eluting with 50% isopropanol in hexane at 1 mL/min and
254 nm detection. Compound 05R had a retention time of 14.5 min. [α]
23
D = -5.3° (c = 0.15,
MeOH).
1
H NMR (600 MHz, CDCl3) δ 7.95 (d, J = 8.1 Hz, 1H), 7.84 (s, 1H), 7.73 (d, J = 8.3 Hz,
1H), 7.51 (s, 1H), 4.68 (m, 1H), 4.01 (q, J = 6.0 Hz, 2H), 3.53 (d, J = 7.3 Hz, 1H), 3.12 (d, J = 7.3
Hz, 1H), 3.03 – 2.99 (m, 2H), 2.97 (dd, J = 13.4, 4.7 Hz, 1H), 2.49 (t, J = 12.5 Hz, 1H), 2.28 (t, J
= 5.8 Hz, 1H), 1.74 (s, 3H), 1.70 (s, 3H).
13
C NMR (151 MHz, CDCl3) δ 173.77, 173.31, 135.69,
63
135.39, 134.03, 133.70, 129.49, 124.39 (q, J = 4.8 Hz), 123.13, 120.41, 114.72, 109.71, 87.08,
86.56, 67.66, 61.42, 53.12, 48.61, 42.31, 28.61, 18.29, 16.71.
19
F NMR (564 MHz, CDCl3): δ -
62.07. ESI-MS: [2M+Na]
+
calcd for C44H40F6N10O8Na
+
, 973.3; found, 973.1.
4-((3aS,4S,5S,7S,7aR)-5-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)-4,7-dimethyl-1,3-
dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-2-(trifluoromethyl)benzonitrile, (Compound
05S). Compound 05S was prepared via the same reaction as Compound 05R, and separated as an
enantiopure compound as described above. Compound 05S had a retention time of 9.5 min. [α]
22
D
= +7.0° (c = 0.1, MeOH).
1
H NMR (600 MHz, CDCl3) δ 7.95 (d, J = 8.1 Hz, 1H), 7.84 (s, 1H),
7.73 (d, J = 8.3 Hz, 1H), 7.51 (s, 1H), 4.68 (m, 1H), 4.01 (q, J = 6.0 Hz, 2H), 3.53 (d, J = 7.3 Hz,
1H), 3.12 (d, J = 7.3 Hz, 1H), 3.03 – 2.99 (m, 2H), 2.97 (dd, J = 13.4, 4.7 Hz, 1H), 2.49 (t, J = 12.5
Hz, 1H), 2.28 (t, J = 5.8 Hz, 1H), 1.74 (s, 3H), 1.70 (s, 3H).
13
C NMR (151 MHz, CDCl3) δ 173.77,
173.31, 135.69, 135.39, 134.03, 133.70, 129.49, 124.39 (q, J = 4.8 Hz), 123.13, 120.41, 114.72,
109.71, 87.08, 86.56, 67.66, 61.42, 53.12, 48.61, 42.31, 28.61, 18.29, 16.71.
19
F NMR (564 MHz,
CDCl3): δ -62.07. ESI-MS: [2M+Na]
+
calcd for C44H40F6N10O8Na
+
, 973.2832; found, 973.0.
N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide
(Compound 06R). Compound 06R was prepared from amine intermediate as described in general
synthetic method. Racemic mixture obtained from amine intermediate (20 mg, 0.053 mmol) as a
white solid, yield 26% (7 mg). Separation of the two enantiomers was achieved by chiral HPLC
using a ProntoSIL Chiral AX QN-1 column (150 × 8.0 mm, 5 µm) eluting with 50% isopropanol
in hexane at 3 mL/min and 254 nm detection. Compound 06R had a retention time of 20.1 min.
[α]
20
D = −37.2° (c = 0.5, EtOAc).
1
H NMR (600 MHz, CD3CN) δ 11.43 (s, 1H), 7.89 (d, J = 8.4
Hz, 1H), 7.78 (s, 1H), 7.39 (d, J = 7.4 Hz, 1H), 6.58 (s, 1H), 4.91 (q, J = 6.6 Hz, 1H), 4.44–4.39
64
(m, 1H), 3.64 (s, 1H), 3.45 (s, 1H), 3.39 (s, 1H), 2.26–2.22 (m, 1H), 1.82 (dd, J = 13.1, 5.2 Hz,
1H), 1.52 (s, 3H), 1.50 (s, 3H), 1.46 (d, J = 6.6 Hz, 3H).
13
C NMR (151 MHz, CD3CN) δ 175.10,
174.60, 134.80, 132.58, 122.35 (q, J = 273.8 Hz), 115.56, 112.44, 102.77, 88.73, 86.27, 62.84,
57.48, 55.26, 50.15, 43.39, 23.83, 18.62, 17.72.
19
F NMR (564 MHz, CD3CN) δ −58.62, -116.25.
ESI-MS: [M - H]
-
calcd for C24H20F4N5O5
-
, 534.1; found, 534.2.
N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide
(Compound 06S). Compound 06S was prepared from amine intermediate as described in general
synthetic method, and separated as an enantiopure compound as described above. Compound 06S
had a retention time of 8.7 min. [α]
20
D = +39.0° (c = 0.5, EtOAc).
1
H NMR (600 MHz, Acetonitrile-
d3) δ 11.37 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 6.58 (s, 1H),
4.91 (q, J = 6.6 Hz, 1H), 4.44–4.39 (m, 1H), 3.56 (s, 1H), 3.45 (s, 1H), 3.39 (s, 1H), 2.27–2.22 (m,
1H), 1.82 (dd, J = 13.1, 5.2 Hz, 1H), 1.52 (s, 3H), 1.50 (s, 3H), 1.46 (d, J = 6.1 Hz, 3H).
13
C NMR
(151 MHz, Acetonitrile-d3) δ 175.10, 174.60, 134.95, 132.60, 122.35 (q, J = 274.4 Hz), 115.57,
112.45, 102.76, 88.78, 86.30, 63.11, 57.55, 55.30, 50.19, 43.47, 23.87, 18.64, 17.74.
19
F NMR
(564 MHz, Acetonitrile-d3) δ −58.63. ESI-MS: [M - H]
-
calcd for C24H20F4N5O5
-
, 534.1; found,
534.2.
3-(4-Acetylpiperazin-1-yl)-N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-
(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-
yl)propenamide (Compound 13R). Compound 13R was prepared from amine intermediate as
described in general synthetic method. Racemic mixture obtained from amine intermediate (19
mg, 0.05 mmol) as a white solid, yield 7% (1.9 mg). Separation of the two enantiomers was
achieved by chiral HPLC using a Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 50%
65
isopropanol in hexane 1 mL/min and 254 nm detection. Compound 13R had a retention time of
11.5 min. [α]
20
D = -37.0° (c = 0.1, MeOH).
1
H NMR (600 MHz, CD3CN) δ 8.10 (d, J = 8.3 Hz,
1H), 7.88 (s, 1H), 7.81 (dd, J = 8.3, 2.0 Hz, 1H), 7.68 (broad, 1H), 4.26 – 4.22 (m, 1H), 3.55 – 3.50
(m, 2H), 3.48 (t, J = 6., 2H), 3.41 (dd, J = 7.2, 1.7 Hz, 1H), 3.24 (d, J = 7.2 Hz, 1H), 2.78 – 2.55
(m, 2H), 2.55 – 2.42 (m, 3H), 2.41 – 2.27 (m, 3H), 2.22 (t, J = 13.1 Hz, 1H), 2.00 (s, 3H), 1.59
(dd, J = 13.1, 5.3 Hz, 1H), 1.54 (s, 3H), 1.49 (s, 3H).
13
C NMR (151 MHz, CD3CN) δ 175.11,
174.41, 172.02, 168.57, 136.63, 136.06, 132.64, 132.37, 130.38, 124.63 (q, J = 5.1 Hz), 115.11,
108.91, 87.60, 85.24, 56.32, 54.00, 53.78, 52.72, 52.23, 48.53, 45.98, 43.25, 41.07, 32.79, 20.53,
17.65, 16.63.
19
F NMR (564 MHz, CD3CN): δ −62.64. ESI-MS: [M+H]
+
calcd for C27H31F3N5O5
+
,
562.2; found, 561.4.
3-(4-Acetylpiperazin-1-yl)-N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-
4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)propenamide (Compound 13S).
Compound 13S prepared from amine intermediate as described in general synthetic method, and
separated as an enantiopure compound as described above. Compound 13S had a retention time of
17.5 min. [α]
22
D = +7.5° (c = 0.073, MeCN).
1
H NMR (600 MHz, CD3CN) δ 8.10 (d, J = 8.3 Hz,
1H), 7.88 (s, 1H), 7.81 (dd, J = 8.3, 2.0 Hz, 1H), 7.68 (broad, 1H), 4.26 – 4.22 (m, 1H), 3.55 – 3.50
(m, 2H), 3.48 (t, J = 6., 2H), 3.41 (dd, J = 7.2, 1.7 Hz, 1H), 3.24 (d, J = 7.2 Hz, 1H), 2.78 – 2.55
(m, 2H), 2.55 – 2.42 (m, 3H), 2.41 – 2.27 (m, 3H), 2.22 (t, J = 13.1 Hz, 1H), 2.00 (s, 3H), 1.59
(dd, J = 13.1, 5.3 Hz, 1H), 1.54 (s, 3H), 1.49 (s, 3H).
13
C NMR (151 MHz, CD3CN) δ 175.11,
174.41, 172.02, 168.57, 136.63, 136.06, 132.64, 132.37, 130.38, 124.63 (q, J = 5.1 Hz), 115.11,
108.91, 87.60, 85.24, 56.32, 54.00, 53.78, 52.72, 52.23, 48.53, 45.98, 43.25, 41.07, 32.79, 20.53,
17.65, 16.63.
19
F NMR (564 MHz, CD3CN): δ −62.64. ESI-MS: [M+H]
+
calcd for C27H31F3N5O5
+
,
562.2; found, 561.4.
66
1-Acetyl-N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)piperidine-4-carboxamide (Compound 14R).
Compound 14R was prepared from amine intermediate as described in general synthetic method.
Racemic mixture obtained from amine intermediate (19 mg, 0.05 mmol) as a white solid, yield
36% (4.9 mg). Separation of the two enantiomers was achieved by chiral HPLC using a Chiralcel
OD-H column (250 × 4.6 mm, 5 µm) eluting with 40%-90% isopropanol in hexane at 1 mL/min
over 20 min and 254 nm detection. Compound 14R had a retention time of 13.0 min. [α]
23
D = −8.0°
(c = 0.2, MeCN).
1
H NMR (600 MHz, CDCl3) δ 7.95 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.74 (d, J
= 8.0 Hz, 1H), 5.81 (dd, J = 22.3, 7.2 Hz, 1H), 4.59 (d, J = 13.2 Hz, 1H), 4.31 (d, J = 6.1 Hz, 1H),
3.90 (d, J = 13.8 Hz, 1H), 3.42 – 3.22 (m, 1H), 3.15 – 3.10 (m, 2H), 2.67 (t, J = 12.7 Hz, 1H), 2.31-
2.37 (m, 2H), 2.11 (s, 3H), 1.97 – 1.81 (m, 2H), 1.79 – 1.66 (m, 2H), 1.63 (s, 3H), 1.60 (s, 3H),
13
C NMR (151 MHz, CDCl3) δ 174.37, 174.05, 173.33, 168.97, 135.72, 135.38, 129.44, 124.32
(q, J = 5.1 Hz), 122.69, 120.87, 114.72, 109.66, 88.03, 85.56, 56.99, 53.64, 48.14, 45.77, 43.82,
43.23, 40.91, 29.03, 28.91, 28.65, 28.42, 21.45, 18.27, 16.97.
19
F NMR (564 MHz, CDCl3): δ
−62.08. ESI-MS: [M-H]
-
calcd for C26H26 F3N4O5
-
, 531.2; found, 531.2.
1-Acetyl-N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)piperidine-4-carboxamide (Compound 14S).
Compound 14S was prepared from amine intermediate as described in general synthetic method,
and separated as an enantiopure compound as described above. Compound 14S had a retention
time of 17.5 min. [α]
22
D = +8.5° (c = 0.2, MeCN).
1
H NMR (600 MHz, CDCl3) δ 7.95 (d, J = 8.4
Hz, 1H), 7.86 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 5.81 (dd, J = 22.3, 7.2 Hz, 1H), 4.59 (d, J = 13.2
Hz, 1H), 4.31 (d, J = 6.1 Hz, 1H), 3.90 (d, J = 13.8 Hz, 1H), 3.42 – 3.22 (m, 1H), 3.15 – 3.10 (m,
2H), 2.67 (t, J = 12.7 Hz, 1H), 2.31- 2.37 (m, 2H), 2.11 (s, 3H), 1.97 – 1.81 (m, 2H), 1.79 – 1.66
67
(m, 2H), 1.63 (s, 3H), 1.60 (s, 3H),
13
C NMR (151 MHz, CDCl3) δ 174.37, 174.05, 173.33, 168.97,
135.72, 135.38, 129.44, 124.32 (q, J = 5.1 Hz), 122.69, 120.87, 114.72, 109.66, 88.03, 85.56,
56.99, 53.64, 48.14, 45.77, 43.82, 43.23, 40.91, 29.03, 28.91, 28.65, 28.42, 21.45, 18.27, 16.97.
19
F
NMR (564 MHz, CDCl3): δ −62.08. ESI-MS: [M-H]
-
calcd for C26H26 F3N4O5
-
, 531.2; found,
531.2.
N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-pyrazole-3-carboxamide (Compound 15R).
Compound 15R was prepared from amine intermediate as described in general synthetic method.
Racemic mixture obtained from amine intermediate (19 mg, 0.05 mmol) as a white solid, yield
34% (8.4 mg). Separation of the two enantiomers was achieved by chiral HPLC using a Chiralcel
OD-H column (250 × 4.6 mm, 5 µm) eluting with 40%-80% isopropanol in hexane at 1 mL/min
over 20 min and 254 nm detection. Compound 15R had a retention time of 8.2 min. [α]
23
D = −36.5°
(c = 0.2, MeCN).
1
H NMR (600 MHz, CD3CN) δ 7.89 (d, J = 8.3 Hz, 1H), 7.79 (s, 1H), 7.70 (s,
1H), 7.42 (s, 1H), 6.77 (s, 1H), 4.43 (m, 1H), 3.46 (d, J = 7.3 Hz, 1H), 3.40 (d, J = 7.3 Hz, 1H),
2.25 (dd, J = 13.2, 11.8 Hz, 1H), 1.83 (dd, J = 13.1, 5.3 Hz, 1H),
13
C NMR (151 MHz, CD3CN) δ
174.08, 173.59, 161.99, 155.62, 153.84, 133.88, 131.60 (q, J = 4.4 Hz), 126.35, 124.06, 122.24,
120.42, 114.54, 111.46, 105.42, 87.75, 85.30, 56.56, 49.18, 17.63, 16.72.
19
F NMR (564 MHz,
CD3CN): δ −58.63 (d, J = 20.9 Hz), -116.24 (d, J = 21.9 Hz). ESI-MS: [M-H]
-
calcd for
C 22H 17F4N5O4
-
, 490.1; found 489.6.
N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-2-fluoro-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-pyrazole-3-carboxamide (Compound 15S).
Compound 15S was prepared from amine intermediate as described in general synthetic method,
and separated as an enantiopure compound as described above. Compound 15S had a retention
68
time of 12.7 min. [α]
22
D = +35.0° (c = 0.2, MeCN).
1
H NMR (600 MHz, CD3CN) δ 7.89 (d, J =
8.3 Hz, 1H), 7.79 (s, 1H), 7.70 (s, 1H), 7.42 (s, 1H), 6.77 (s, 1H), 4.43 (m, 1H), 3.46 (d, J = 7.3
Hz, 1H), 3.40 (d, J = 7.3 Hz, 1H), 2.25 (dd, J = 13.2, 11.8 Hz, 1H), 1.83 (dd, J = 13.1, 5.3 Hz, 1H),
13
C NMR (151 MHz, CD3CN) δ 174.08, 173.59, 161.99, 155.62, 153.84, 133.88, 131.60 (q, J =
4.4 Hz), 126.35, 124.06, 122.24, 120.42, 114.54, 111.46, 105.42, 87.75, 85.30, 56.56, 49.18, 17.63,
16.72.
19
F NMR (564 MHz, CD3CN): δ −58.63 (d, J = 20.9 Hz), -116.24 (d, J = 21.9 Hz). ESI-MS:
[M-H]
-
calcd for C22H17F4N5O4
-
, 490.1; found 489.6.
N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide
(Compound 16R). Compound 16R was prepared from amine intermediate as described in general
synthetic method. Racemic mixture obtained from amine intermediate (15 mg, 0.04 mmol) as a
white solid, yield 0.9 mg (10%). Separation of the two enantiomers was achieved by chiral HPLC
using a ChiralCel OD-H column (250 × 4.6 mm, 5 µm) eluting with 35%-55% isopropanol in
hexane at 1 mL/min over 20 min and 254 nm detection. Compound 16R had a retention time of
7.8 min. [α]
22
D = -30.6
o
(c = 0.05, MeCN).
1
H NMR (600 MHz, CD3CN) δ 9.98 (br s, 1H), 8.50
(s, 1H), 8.33 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H), 8.04 (s, 1H), 7.90 (s, 1H), 7.82 (dd, J = 8.4 Hz, 2.0
Hz, 1H), 7.32 (m, 1H), 6.94 (s, 1H), 4.49–4.53 (m, 1H), 3.55 (d, J = 7.2 Hz, 1H), 3.36 (d, J = 7.2
Hz, 1H), 2.27–2.31 (m, 1H), 1.82 (dd, J = 13.1, 5.2 Hz, 1H), 1.58 (s, 3H), 1.57 (s, 3H).
13
C NMR
(151 MHz, CD3CN) δ 175.19, 174.53, 164.54, 148.46, 144.20, 136.67, 136.05, 130.51, 129.57,
127.43, 124.68 (q, J = 273.8 Hz), 123.24, 118.59, 115.12, 109.77, 108.87, 87.80, 85.26, 56.59,
53.91, 48.65, 42.80, 17.72, 16.81.
19
F NMR (564 MHz, CD3CN) δ −62.64 (s). ESI-MS: [M - H]
-

calcd for C26H19F3N5O4
-
, 522.1; found, 522.4.
69
N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide
(Compound 16S). Compound 16S was prepared from amine intermediate as described in general
synthetic method, and separated as an enantiopure compound as described above. Compound 16S
had a retention time of 13.0 min. [α]
22
D = +29.3
o
(c = 0.05, MeCN).
1
H NMR (600 MHz, CD3CN)
δ δ 9.98 (br s, 1H), 8.50 (s, 1H), 8.33 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H), 8.04 (s, 1H), 7.90 (s, 1H),
7.82 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.32 (m, 1H), 6.94 (s, 1H), 4.49–4.53 (m, 1H), 3.55 (d, J = 7.2
Hz, 1H), 3.36 (d, J = 7.2 Hz, 1H), 2.27–2.31 (m, 1H), 1.82 (dd, J = 13.1, 5.2 Hz, 1H), 1.58 (s, 3H),
1.57 (s, 3H).
13
C NMR (151 MHz, CD3CN) δ 175.19, 174.53, 164.65, 148.38, 144.20, 136.67,
136.04, 130.51, 129.39, 127.44, 124.68 (q, J = 273.8 Hz), 123.24, 118.59, 115.12, 109.86, 108.88,
87.80, 85.26, 56.59, 53.91, 48.65, 42.80, 17.72, 16.81.
19
F NMR (564 MHz, CD3CN) δ −62.64 (s).
ESI-MS: [M - H]
-
calcd for C26H19 F3N5O4
-
, 522.1; found, 522.4.
N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-indole-3-carboxamide (Compound 17R).
Compound 17R was prepared from amine intermediate as described in general synthetic method.
Racemic mixture obtained from amine intermediate (9.5 mg, 0.025 mmol) as a white solid, yield
2.9 mg (44%). Separation of the two enantiomers was achieved by chiral HPLC using a Regis
Whelk-O1 column (250 × 4.6 mm, 5 µm) eluting with 60% isopropanol in hexane at 1.5 mL/min
over 30 min and 254 nm detection. Compound 17R had a retention time of 17.5 min. [α]
22
D = -
27.0
o
(c = 0.2, MeCN).
1
H NMR (600 MHz, CD3CN) δ 9.68 (br s, 1H), 8.16 (d, J = 8.0 Hz, 1H),
8.06 (d, J = 8.0 Hz, 1H), 7.91 (s, 1H), 7.88 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz,
1H), 7.23-7.18 (m, 2H), 6.85 (d, J = 7.8 Hz, 1H), 4.52–4.48 (m, 1H), 3.52 (d, J = 7.3 Hz, 1H),
3.34 (d, J = 7.3 Hz, 1H), 2.29–2.25 (m, 1H), 1.80 (dd, J = 13.0, 5.3 Hz, 1H), 1.56 (s, 3H), 1.55 (s,
70
3H).
13
C NMR (151 MHz, CD3CN) δ 175.23, 174.57, 165.18, 136.69, 136.32, 136.04, 130.50,
127.41, 127.25, 126.25, 124.68 (q), 122.52, 121.13, 120.95, 115.12, 111.76, 110.77, 108.86, 87.87,
85.24, 56.56, 53.93, 48.69, 42.85, 17.73, 16.82.
19
F NMR (564 MHz, CD3CN) δ −62.64 (s). ESI-
MS: [M - H]
-
calcd for C27H20N3O4F4
-
, 521.14; found, 521.3.
N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-indole-3-carboxamide (Compound 17S).
Compound 17S was prepared from amine intermediate as described in general synthetic method,
and separated as an enantiopure compound as described above. Compound 17S had a retention
time of 22.5 min. Yield 3.0 mg (46%). [α]
22
D = +25.5
o
(c = 0.2, MeCN). 1H NMR (600 MHz,
CD3CN) δ 9.68 (br s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.91 (s, 1H), 7.88
(s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.23-7.18 (m, 2H), 6.85 (d, J = 7.8 Hz,
1H), 4.52–4.48 (m, 1H), 3.52 (d, J = 7.3 Hz, 1H), 3.34 (d, J = 7.3 Hz, 1H), 2.29–2.25 (m, 1H),
1.80 (dd, J = 13.0, 5.3 Hz, 1H), 1.56 (s, 3H), 1.55 (s, 3H).
13
C NMR (151 MHz, CD3CN) δ 175.23,
174.57, 165.18, 136.69, 136.32, 136.04, 130.50, 127.41, 127.25, 126.25, 124.68 (q), 122.52,
121.13, 120.95, 115.12, 111.76, 110.77, 108.86, 87.87, 85.24, 56.56, 53.93, 48.69, 42.85, 17.73,
16.82.
19
F NMR (564 MHz, CD3CN) δ −62.64 (s). ESI-MS: [M - H]
-
calcd for C27H20N3O4F4
-
,
521.14; found, 521.3.
N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-3-(oxazol-2-yl)propanamide (Compound 18R).
Compound 18R was prepared from amine intermediate as described in general synthetic method,
and separated as an enantiopure compound as described above. Compound 18R had a retention
time of 6.2 min. Yield 17.5 mg (76%) [α]
22
D = -2.5
o
(c = 0.2, MeCN).
1
H NMR (600 MHz, CD3CN)
δ 8.12 (d, J = 7.7 Hz, 1H), 7.90 (d, J = 2.0 Hz 1H), 7.83 (dd, J = 8.6 Hz, 1.7 Hz, 1H), 7.74 (d, J =
71
0.9 Hz, 1H), 7.05 (t, J = 0.9 Hz, 1H), 6.84 (br s, 1H), 4.26–4.23 (m, 1H), 3.49 (d, J = 7.2 Hz, 1H),
3.23 (d, J = 7.2 Hz, 1H), 3.11-3.00 (m, 2H), 2.70-2.60 (m, 2H), 2.23–2.19 (m, 1H), 1.604 (dd, J =
13.1, 5.1 Hz, 1H), 1.54 (s, 3H), 1.45 (s, 3H).
13
C NMR (151 MHz, CD3CN) δ 175.19, 174.45,
171.37, 163.91, 138.86, 136.68, 136.04, 132.63, 132.41, 130.54, 126.86, 124.70 (q, J = 5.0 Hz),
123.24, 121.43, 115.12, 108.89, 87.67, 85.18, 56.52, 53.69, 48.44, 42.90, 32.19, 23.37, 17.65,
16.58.
19
F NMR (564 MHz, CD3CN) δ −62.63 (s). ESI-MS: [M + Na]
+
calcd for C24H21F3N4O5Na
+
,
525.1; found, 525.3.
N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-3-(oxazol-2-yl)propanamide (Compound 18S).
Compound 18S was prepared from amine intermediate as described in general synthetic method.
Racemic mixture obtained from amine intermediate (35.0mg, 0.092 mmol) as a white solid, yield
17.5 mg (76%). Separation of the two enantiomers was achieved by chiral HPLC using a Regis C-
Amylose column (250 × 4.6 mm, 5 µm) eluting with 25% isopropanol in hexane at 1.5 mL/min
over 30 min and 254 nm detection. Compound 18S had a retention time of 5.0 min. [α]
22
D = +3.0
o

(c = 0.2, MeCN).
1
H NMR (600 MHz, CD3CN) δ 8.12 (d, J = 7.7 Hz, 1H), 7.90 (d, J = 2.0 Hz
1H), 7.83 (dd, J = 8.6 Hz, 1.7 Hz, 1H), 7.74 (d, J = 0.9 Hz, 1H), 7.05 (t, J = 0.9 Hz, 1H), 6.84 (br
s, 1H), 4.26–4.23 (m, 1H), 3.49 (d, J = 7.2 Hz, 1H), 3.23 (d, J = 7.2 Hz, 1H), 3.11-3.00 (m, 2H),
2.70-2.60 (m, 2H), 2.23–2.19 (m, 1H), 1.604 (dd, J = 13.1, 5.1 Hz, 1H), 1.54 (s, 3H), 1.45 (s, 3H).
13
C NMR (151 MHz, CD3CN) δ 175.19, 174.45, 171.37, 163.91, 138.86, 136.68, 136.04, 132.63,
132.41, 130.54, 126.86, 124.70 (q, J = 5.0 Hz), 123.24, 121.43, 115.12, 108.89, 87.67, 85.18,
56.52, 53.69, 48.44, 42.90, 32.19, 23.37, 17.65, 16.58.
19
F NMR (564 MHz, CD3CN) δ −62.63 (s).
ESI-MS: [M + Na]
+
calcd for C24H21F3N4O5Na
+
, 525.1; found, 525.4.
72
N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)benzofuran-3-sulfonamide (Compound 19R).
Compound 19R was prepared from amine intermediate as described in general synthetic method.
Racemic mixture obtained from amine intermediate (30.0 mg, 0.079 mmol) as a white solid, yield
14.5 mg (66%). Separation of the two enantiomers was achieved by chiral HPLC using a Regis C-
Amylose column (250 × 4.6 mm, 5 µm) eluting with 25% isopropanol in hexane at 1.5 mL/min
over 30 min and 254 nm detection. Compound 19R had a retention time of 6.0 min. [α]
22
D = -59.3
o

(c = 0.2, MeCN).
1
H NMR (600 MHz, CD3CN) δ 8.08 (d, J = 8.3 Hz, 1H), 7.85 (s, 1H), 7.79-7.77
(m, 2H), 7.65 (d, J = 8.4 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.49 (s, 1H), 7.41 (t, J = 7.5 Hz, 1H),
6.59 (br s, 1H), 3.70–3.67 (m, 1H), 3.42 (d, J = 7.3 Hz, 1H), 3.18 (d, J = 7.3 Hz, 1H), 2.03–1.99
(m, 1H), 1.54 (dd, J = 13.2, 4.9 Hz, 1H), 1.46 (s, 3H), 1.44 (s, 3H).
13
C NMR (151 MHz, CD3CN)
δ 174.57, 174.14, 155.64, 150.40, 136.55, 136.06, 132.43, 130.51, 127.96, 126.13, 124.66 (q, J =
5.1 Hz), 124.46, 123.23, 121.40, 115.09, 112.32, 112.06, 108.98, 86.52, 85.24, 60.72, 53.50, 48.37,
42.88, 17.49, 15.56.
19
F NMR (564 MHz, CD3CN) δ −62.65 (s). ESI-MS: [M - H]
-
calcd for
C26H19F3N3O6S
-
, 588.1; found, 588.2.
N-((3aS,4S,5S,7S,7aR)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)benzofuran-3-sulfonamide (Compound 19S).
Compound 19S was prepared from amine intermediate as described in general synthetic method,
and separated as an enantiopure compound as described above. Compound 19S had a retention
time of 7.5 min. Yield 14.5 mg (66%). [α]
22
D = +63.1
o
(c = 0.2, MeCN).
1
H NMR (600 MHz,
CD3CN) δ 8.08 (d, J = 8.3 Hz, 1H), 7.85 (s, 1H), 7.79-7.77 (m, 2H), 7.65 (d, J = 8.4 Hz, 1H), 7.55
(t, J = 7.5 Hz, 1H), 7.49 (s, 1H), 7.41 (t, J = 7.5 Hz, 1H), 6.59 (br s, 1H), 3.70–3.67 (m, 1H), 3.42
(d, J = 7.3 Hz, 1H), 3.18 (d, J = 7.3 Hz, 1H), 2.03–1.99 (m, 1H), 1.54 (dd, J = 13.2, 4.9 Hz, 1H),
73
1.46 (s, 3H), 1.44 (s, 3H).
13
C NMR (151 MHz, CD3CN) δ 174.57, 174.14, 155.64, 150.40, 136.55,
136.06, 132.43, 130.51, 127.96, 126.13, 124.66 (q, J = 5.1 Hz), 124.46, 123.23, 121.40, 115.09,
112.32, 112.06, 108.98, 86.52, 85.24, 60.72, 53.50, 48.37, 42.88, 17.49, 15.56.
19
F NMR (564
MHz, CD3CN) δ −62.65 (s). ESI-MS: [M - H]
-
calcd for C26H19F3N3O6S
-
, 588.1; found, 588.3.

74
Chapter 3 references
1. Foster, W. R.; Car, B. D.; Shi, H.; Levesque, P. C.; Obermeier, M. T.; Gan, J.; Arezzo, J.
C.; Powlin, S. S.; Dinchuk, J. E.; Balog, A., Drug safety is a barrier to the discovery and
development of new androgen receptor antagonists. The Prostate 2011, 71 (5), 480-488.
2. Schrödinger Release 2021-3; New York, NY, 2021.
3. Moilanen, A.-M.; Riikonen, R.; Oksala, R.; Ravanti, L.; Aho, E.; Wohlfahrt, G.; Nykänen,
P. S.; Törmäkangas, O. P.; Palvimo, J. J.; Kallio, P. J., Discovery of ODM-201, a new-
generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-
directed prostate cancer therapies. Scientific Reports 2015, 5 (1), 1-11.
4. Patsch, K.; Liu, C.; Zapotoczny, G.; Sun, Y.; Sura, H.; Ung, N.; Sun, R. X.; Haliday, B.;
Yu, C.; Aljehani, M.; Lee, J. S. H.; Kashemirov, B. A.; Agus, D. B.; McKenna, C. E.;
Ruderman, D., Paradoxical androgen receptor regulation by small molecule enantiomers.
Proceedings of the National Academy of Sciences 2021, 118 (12), e2100918118.
5. Sack, J. S.; Kish, K. F.; Wang, C.; Attar, R. M.; Kiefer, S. E.; An, Y.; Wu, G. Y.;
Scheffler, J. E.; Salvati, M. E.; Krystek, S. R.; Weinmann, R.; Einspahr, H. M.,
Crystallographic structures of the ligand-binding domains of the androgen receptor and its
T877A mutant complexed with the natural agonist dihydrotestosterone. Proceedings of the
National Academy of Sciences 2001, 98 (9), 4904-4909.
6. Wang, F.; Liu, X.-q.; Li, H.; Liang, K.-n.; Miner, J. N.; Hong, M.; Kallel, E. A.; Van
Oeveren, A.; Zhi, L.; Jiang, T., Structure of the ligand-binding domain (LBD) of human
androgen receptor in complex with a selective modulator LGD2226. Acta Crystallographica
Section F: Structural Biology and Crystallization Communications 2006, 62 (11), 1067-1071.
7. Bohl, C. E.; Miller, D. D.; Chen, J.; Bell, C. E.; Dalton, J. T., Structural basis for
accommodation of nonsteroidal ligands in the androgen receptor. Journal of Biological
Chemistry 2005, 280 (45), 37747-37754.
8. Balog, A.; Rampulla, R.; Martin, G. S.; Krystek, S. R.; Attar, R.; Dell-John, J.; DiMarco, J.
D.; Fairfax, D.; Gougoutas, J.; Holst, C. L.; Nation, A.; Rizzo, C.; Rossiter, L. M.;
Schweizer, L.; Shan, W.; Spergel, S.; Spires, T.; Cornelius, G.; Gottardis, M.; Trainor, G.;
Vite, G. D.; Salvati, M. E., Discovery of BMS-641988, a novel androgen receptor antagonist for
the treatment of prostate cancer. ACS Medicinal Chemistry Letters 2015, 6 (8), 908-912.
9. Labrie, F.; Singh, S. M.; Labrecque, R.; Ciobanu, L. C. Non-steroidal antiandrogens and
selective androgen receptor modulators with a pyridyl moiety. US 2015/0175576A1, June. 25,
2015.
10. Johns, B. A.; Shotwell, J. B. Compounds. WO 2012/067664 Al, May 24, 2012.
11. Lai, K. W.; Liang, J.; Zhang, B.; Labadie, S.; Ortwine, D.; Dragovich, P.; Kiefer, J.;
Gehling, V. S.; Harmange, J.-C. Pyrrolidine Amide Compounds as Histone Demethylase
Inhibitors. WO 2016/057924 Al.
12. Rathkopf, D.; Liu, G.; Carducci, M. A.; Eisenberger, M. A.; Anand, A.; Morris, M. J.;
Slovin, S. F.; Sasaki, Y.; Takahashi, S.; Ozono, S.; Fung, N. K. E.; Cheng, S.; Gan, J.;
Gottardis, M.; Obermeier, M. T.; Reddy, J.; Zhang, S.; Vakkalagadda, B. J.; Alland, L.;
Wilding, G.; Scher, H. I., Phase I Dose-Escalation Study of the Novel Antiandrogen BMS-
641988 in Patients with Castration-Resistant Prostate Cancer. Clinical Cancer Research 2011,
17 (4), 880-887.
13. Bonne, D.; Rodriguez, J., A Bird's Eye View of Atropisomers Featuring a Five-Membered
Ring. European Journal of Organic Chemistry 2018, 2018 (20-21), 2417 - 2431.
75
14. Keiki, K.; Isao, T.; Shigeo, K.; Makoto, Y.; Kazutoshi, Y., Stereoselective Synthesis of 2-
Alkylamino-N-(2 ′-alkylphenyl)succinimide Conformers. Chemistry Letters 1994, 23 (9), 1605-
1606.
15. Salvati, M. E.; Balog, J.; Pickering, D.; Giese, S.; Fura, A.; Li, W.; Patel, R.; Hanson, R.;
Mitt, T.; Roberge, J.; Corte, J.; Spergel, S.; Rampulla, R.; Misra, R.; Xiao, H.-Y. Fused
Heterocyclic succinimide compounds and analogs thereof, modulators of nuclear hormone
receptor function. US20050272799A1, December 08, 2005.
16. Zhou, T.; Xu, W.; Zhang, W.; Sun, Y.; Yan, H.; Gao, X.; Wang, F.; Zhou, Q.; Hou, J.;
Ren, S.; Yang, Q.; Yang, B.; Xu, C.; Zhou, Q.; Wang, M.; Chen, C.; Sun, Y., Preclinical
profile and phase I clinical trial of a novel androgen receptor antagonist GT0918 in castration-
resistant prostate cancer. European Journal of Cancer 2020, 134, 29-40.
17. Korpal, M.; Korn, J. M.; Gao, X.; Rakiec, D. P.; Ruddy, D. A.; Doshi, S.; Yuan, J.;
Kovats, S. G.; Kim, S.; Cooke, V. G.; Monahan, J. E.; Stegmeier, F.; Roberts, T. M.; Sellers,
W. R.; Zhou, W.; Zhu, P., An F876L mutation in androgen receptor confers genetic and
phenotypic resistance to MDV3100 (enzalutamide). Cancer Discovery 2013, 3 (9), 1030-1043.
18. Kunwittaya, S.; Nantasenamat, C.; Treeratanapiboon, L.; Srisarin, A.; Isarankura-Na-
Ayudhya, C.; Prachayasittikul, V., Influence of logBB cut-off on the prediction of blood-brain
barrier permeability. Biomedical and Applied Technology Journal 2013, 1, 16-34.

76
Chapter 4 Selective AR degraders based on novel scaffold
4.1 Induced protein degradation and selective AR degraders
4.1.1 Induced protein degradation
As we enter a new era of drug discovery, the amount of drug modalities is rapidly
expanding. Induced biomolecular degradation is one of such emerging modalities. Instead of
inhibiting a target, it aims to degrade the target by cellular machineries via catalytic events.
1
The
shift from “occupancy-driven pharmacology” to “event-driven pharmacology” is gaining
significant attention in recent years.
1
Both proteins and nucleic acids have been targets for selective
degradation, and protein degradation is mediated by either hydrophobic tagging (HyT) or a class
of molecules called “PROteolysis TArgeting Chimera (PROTAC)”.
1, 2
HyT and PROTAC both
engage cellular degradation machineries but they work via different mechanisms.
HyT is a hydrophobic chemical moiety (Figure 4.1) covalently attached to a ligand of the
protein target.
1, 3
Since proteins usually do not expose hydrophobic regions to its milieu, the cell
would be tricked to recognize HyT as an unfolded or misfolded protein due for degradation.
1, 3
On
the other hand, PROTAC uses specific molecules to recruit E3 ubiquitin ligases (Figure 4.2),
which in turn ubiquitinates the target protein and sends it off for degradation.
1, 4
Despite the large
number of known E3 ligases, only a few have been successfully utilized in PROTAC.
4

Figure 4.1 Examples of hydrophobic tagging.
77

Figure 4.2 Examples of ligands of E3 ligases

Since HyT and PROTAC molecules merely act as magnets between the target protein and
the degrading enzymes, it is possible to resurrect abandoned binders that could not adequately
inhibit protein activities.
1, 5
Because of their catalytic nature, they bring a variety of advantages:
1,
5
(1) they possess sub-stoichiometric activities; (2) they can potentially target “undruggable”
proteins such as kinases; (3) they can overcome resistance issues common in cancer therapies; (4)
they can bind to any available binding site on the target protein. However, the medicinal
development of these molecules also faces considerable challenges and limitations.
1, 5
First and
foremost, PROTAC molecules usually have large molecular weight which is unsuitable for oral
delivery.
1, 5
Additionally, the dosing regimen is difficult to determine because too much PROTAC
can result in the reformation of binary complex instead of ternary complex.
1, 5
Furthermore, the in
vivo toxicity profiles of these molecules have been disappointing so far.
1, 5

Based on the principles of induced protein degradation, many selective hormone receptor
degraders have been developed in recent years. The estrogen receptor (ER) and the AR are two
prominent examples where researchers have found success in combating drug resistance.
1

4.1.2 Design principles of selective AR degraders (SARDs)
To combat drug resistance in prostate cancer, especially the resistance rising from point
mutations in AR LBD, selective AR degraders (SARDs) were developed.
6, 7
Although there are
78
other strategies to treat drug resistant prostate cancer (Figure 4.3),
8, 9
such as inhibiting other
domains of AR, or inhibiting the AR interactome, we will focus our discuss on SARDs. Since
SARDs are broadly defined as any molecules that selectively degrade AR, there are mainly two
types of SARDs:
6

Figure 4.3 Strategies to combat AR antagonist-resistant prostate cancer

The first type of SARDs are AR modulators as degraders themselves. These SARDs are
usually discovered via extensive screening for degradation phenotype and there is little rationale
for initial design. UT-34 (Figure 4.4) is a molecule with such property.
10
Discovered by library
screening, it is an AR pan-antagonist which also reduces AR expression level. Importantly, it
reduced growth of tumors in enzalutamide-resistant prostate cancer xenografts. Structurally, it is
derived from bicalutamide. AZD3514 (Figure 4.4) is another SARD in this category.
11

Structurally, it is unrelated to any other AR ligands. It both modulates AR activities and degrades
AR, but it failed clinical trial due to toxicity.
11

Figure 4.4 Examples of AR modulators as SARDs
Selective AR Degraders
(SARDs).
AR ligand as degrader itself
(discovered via screening)
AR ligand linked to a degron
(rationally designed from a known ligand)
Inhibitors that bind to
other domains of AR
LBD inhibitors efficacious
on mutant AR
79
The second type of SARDs are AR ligands covalently linked to degrons (HyT or E3 ligase
ligands). These SARDs are rationally designed and optimized based on a known AR ligand, as
shown in Figure 4.5.
6
Based on previous literature, we can deduce the key elements that should
guide our design. First and foremost, it is paramount to choose a high-affinity ligand, since the
chemical modification of SARD will generally decrease the affinity of ligand.
6, 7
The tethering
point should be chosen so that the addition of the linker would not greatly affect the binding of the
ligand. In addition, the linker should be optimized in terms of both its length and its structure.
Finally, the type of degron drastically impacts the degrading effect of the SARD, even though the
binding affinity of degron to degrading machinery is not important.
6

Figure 4.5 Structures of two rationally designed SARDs.

80
4.1.3 Exemplary SARDs in clinical and preclinical studies
ARV-110 (Figure 4.6) is a rationally designed SARD currently in Phase 2 clinical trial.
2

It is an orally bioavailable molecule.
12
It contains an AR ligand, a structurally rigid linker, and a
thalidomide-based Cereblon ligand (See Chapter 4.1.1).
12
In preclinical studies, ARV-110
displayed a degradation concentration 50% (DC50) of 1 nM in VCaP cells.
12
Additionally, it only
degraded AR in nearly 4,000 proteins measured in VCaP cells.
12
In Phase I clinical trial, ARV-
110 showed an acceptable safety profile and a promising efficacy profile.
12

There are many other SARDs currently in preclinical development.
13-15
Recently, Kim et
al. developed MTX-23 (Figure 4.7), which degraded both full length AR and AR-V7.
16
AR-V7 is
a constitutively active AR mutant that lacks the AR LBD.
16
They used a ligand for the AR DBD
rather than AR LBD, and thus the SARD can bind to the DBD of both full length AR and AR-
V7.
16
In preclinical studies, MTX-23 displayed DC50 of 0.37 μM for AR-V7 and 2 μM for full
length AR.
16

As illustrated in these examples, the design of SARDs is flexible and can be easily modified
according to specific goals for the molecules. Hopefully, we will see many SARDs enter clinical
trials and eventually get FDA approval.

Figure 4.6 Structure of ARV-110.
81

Figure 4.7 Structure of MTX-23

4.2 SARDs based on our scaffold
4.2.1 Design rationale
Since our compounds are highly potent AR ligands, they provide the unique opportunity
to be modified into SARDs. As a proof of concept, we chose the Compound 08 scaffold as our
AR ligand because both the antagonist and agonist showed good EC50.
To find a reasonable tethering point, we tested several modifications on the indazole ring
in silico. Ideally, modifications such as methylation or acylation on the tethering point would not
drastically decrease binding affinity of the ligand. Using Molsoft substituent screening, we
identified 5’ position and 6’ position as promising tethering points (Figure 4.8). Next, we designed
the linker to be the classic polyethylene glycol (PEG).
14
To determine the suitable length of the
PEG linker, we turned to reported SARDs with similar linear linkers.
13, 14, 17, 18
Based on these
reported SARD molecules, we estimate our linker to be between 6 and 10 atoms long. Finally, we
chose adamantane as a HyT degron. We synthesized racemic SARDs as a proof of concept.
82

Figure 4.8 Potential tethering point for SARD.

4.2.2 Synthesis
The general synthetic route of our SARDs follows a convergent synthesis approach
(Scheme 4.1).
To synthesize the 5’-substituted indazole, 5-(3-(2-(2-(adamantan-1-
yl)acetamido)ethoxy)propanamido)-1H-indazole-3-carboxylic acid, we started with the
commercially available 5-amino-1H-indazole-3-carboxylic acid (Scheme 4.2). After protection of
the carboxylic acid by a methyl group, the linker and the adamantane are attached stepwise to the
indazole moiety via amide coupling reactions. Notably, intermediate compound 4 could not be
extracted by organic solvent in the presence of water or saturated NaHCO3 or Na2CO3 solutions,
due to the high aqueous solubility of this compound. Thus, intermediate compound 4 was used in
situ after removal of excess solvent and TFA. The final deprotection of methyl ester is performed
under a mild condition with Et3N and LiBr.
19
Hydrolysis using NaOH in either aqueous or organic
solution led to either no reaction or decomposition of the methyl ester. The moiety containing the
degron was then coupled to the core structure to give Compound 20 (Scheme 4.2).

83
Scheme 4.1 Convergent synthesis of SARDs based on our scaffold

Scheme 4.2 Synthesis of Compound 20.

Reagents and conditions: (a) MeOH, conc. H2SO4, reflux, overnight, 56%; (b) HATU, DIPEA,
DMF, rt, 2h, 51%; (c) TFA, DCM, rt, 2h, quant; (d) HATU, DIPEA, DMF, rt, 2h, 61%; (e) Et3N,
LiBr, MeCN/H2O, 50
o
C, overnight, quant; (f) HOSu, EDC-HCl, THF/DMF, rt, overnight, 36%.

84
We attempted the same synthetic route to prepare the 6’-substituted indole, 6-(3-(2-(2-
(adamantan-1-yl)acetamido)ethoxy)propanamido)-1H-indole-3-carboxylic acid. However, the
methyl ester deprotection proved to be unfeasible under various conditions (Scheme 4.3).
19-22

Thus, we carried out the synthesis without protecting carboxylic acid (Scheme 4.4). Each
intermediate was purified by reverse-phase HPLC. The moiety containing the degron was then
coupled to the core structure to give Compound 21 (Scheme 4.2). We plan to test these proof-of-
concept SARDs and develop them in further studies beyond the scope of this dissertation.

Scheme 4.3 Attempted ester deprotection of methyl 6-(3-(2-(2-(adamantan-1-
yl)acetamido)ethoxy)propanamido)-1H-indole-3-carboxylate.

Scheme 4.4 Synthesis of Compound 21.

Reagents and conditions: (a) HATU, DIPEA, DMF, rt, 2h,, 54%; (b) TFA, DCM, rt, 2h, 75%; (c)
HATU, DIPEA, DMF, rt, 2h,, 37%; (d) TBTU, Et3N, THF/DMF, rt, overnight, 36%.
85
4.3 Future directions
To test our proof-of-concept SARDs, we need to systematically determine its binding
affinity to AR as well as its degrading capabilities (Figure 4.9).
Due to modifications to the AR ligands, we expect SARDs to have lower binding affinity
to AR than their parent ligands.
7, 14
Problematic tethering points, as well as short linkers, can cause
significant decrease or even loss in binding affinity. Additionally, linker lengths can drastically
influence the degrading capabilities of the SARDs, because an optimal distance between AR and
the degrading machinery must be achieved for efficient protein degradation.
23
To optimize the
SARDs, we can rigidify the linkers to make the molecules more “drug-like”.
14
In addition, we can
also screen different degrons to reach optimal degradation. Last but not least, the dosing of SARD
is critical to its success, because excess amount of SARDs leads to the “hook effect” where there
is no degradation (Figure 4.10).
In summary, there is many opportunities for screening and optimization of our SARDs
molecules in the future.

Figure 4.9 Testing of proof-of-concept SARD molecules
86

Figure 4.10 Dosing of PROTAC
As the concentration of PROTAC molecule increases, target degradation decreases due to excess
formation of binary complex. Figure is modified from Moreau et al., British Journal of
Pharmacology, 2020; 177, 1709–1718.

4.4 Experimental section
Chemical synthesis: Unless otherwise noted, all reagents and solvents were commercially
available and used as received. The progress of all reactions was monitored on precoated silica gel
plates (with fluorescence indicator UV254) using ethyl acetate/hexane or
dichloromethane/methanol as solvent systems. Column chromatography was performed with
ISCO CombiFlashRf+ Lumen flash chromatography with the solvent mixtures specified in the
corresponding experiment. NMR spectra were recorded on either a Varian 400, 500 or 600 at room
temperature. NMR samples are prepared in 1-2 mg/mL concentration. Data is reported as follows:
chemical shift (ppm, δ relative to residual solvent peak for
1
H and
13
C), multiplicity (s = singlet, d
= doublet, t = triplet, q = quartet, m = multiplet, and br = broad), coupling constant (Hz), and
integration.
19
F NMR spectra were recorded with proton decoupling. Low-resolution mass
87
spectrometry (LRMS) analysis was performed on Finnigan LCQ Deca XP Max equipped with an
ESI source and an APCI source. HPLC purification was performed on Shimadzu Prominence with
Phenomenex Luna C18 column (10 x 250 mm) with 50% acetonitrile in water.
Methyl 5-amino-1H-indazole-3-carboxylate. 5-amino-1H-indazole-3-carboxylic acid (142
mg, 0.8 mmol) was dissolved in 10 mL MeOH. 1.5 mL concentrated H2SO4 was added and the
reaction mixture was refluxed overnight. The volatiles were removed under reduced pressure and
the residue was partitioned between ethyl acetate and saturated NaHCO3 solution. The product was
obtained as a brown solid without purification. Yield: 85 mg (56%).
1
H NMR (500 MHz, CD3CN):
δ 7.42 (dd, J = 8.87 Hz, 0.78 Hz, 1H), 7.28 (dd, J = 2.16 Hz, 0.78 Hz, 1H), 6.91 (dd, J = 8.87 Hz,
2.16 Hz, 1H), 3.93 (s, 3H).
Methyl 5-(3-(2-((tert-butoxycarbonyl)amino)ethoxy)propanamido)-1H-indazole-3-
carboxylate. 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propanoic acid (28 mg, 0.12 mmol) and
triethylamine (0.03 mL, 0.24 mmol) were dissolved in anhydrous DMF and HATU (46 mg, 0.12
mmol) was added. After 30 minutes, the activated acid solution was added dropwise to amine in
anhydrous DMF. The reaction was stirred at room temperature for 2 hours. After completion, the
residue was partitioned between ethyl acetate and brine. The crude mixture was purified by flash
chromatography in 15% MeOH/DCM. The product was obtained as a white solid.Yield: 21 mg
(51%).
1
H NMR (500 MHz, CDCl3) δ 8.49 (s, 1H), 8.15 (s, 1H), 7.48 (br s, 1H), 7.44 (br s, 1H),
3.94 (s, 3H), 3.84 (t, J = 5.73 Hz, 2H), 3.60 (t, J = 5.73 Hz, 2H), 3.37 (br s, 2H), 2.69 (t, J = 5.73
Hz, 2H), 1.39 (s, 9H).
2-(3-((3-(Methoxycarbonyl)-1H-indazol-5-yl)amino)-3-oxopropoxy)ethan-1-aminium
trifluoroacetate. The Boc-protected amine (30 mg, 0.075 mmol) and trifluoroacetic acid (0.5 mL)
were dissolved in 1.5 mL DCM. The reaction was stirred at room temperature for 1 hour. The
88
volatiles were removed under reduced pressure and the residue was partitioned between ethyl
acetate and water. The aqueous phase was collected and water was removed under reduced
pressure. The product was obtained as a white solid without purification.Yield: 30 mg (quant.).
1
H
NMR (500 MHz, CD3CN) δ 8.90 (s, 1H), 8.30 (s, 1H), 7.51 (m, 2H), 7.22 (br s, 3H), 3.92 (s, 3H),
3.81 (t, J = 5.57 Hz, 2H), 3.72 (t, J = 5.57 Hz, 2H), 3.19 (br s, 2H), 2.65 (t, J = 5.57 Hz, 2H).
Methyl 5-(3-(2-(2-(adamantan-1-yl)acetamido)ethoxy)propanamido)-1H-indazole-3-
carboxylate. Adamantane acetic acid (3.6 mg, 0.19 mmol) and triethylamine (0.007 mL, 0.051
mmol) were dissolved in anhydrous DMF and HATU (7.1 mg, 0.019 mmol) was added. After 30
minutes, the activated acid solution was added dropwise to a DMF solution of amine (5.2 mg,
0.017 mmol) and triethylamine (0.007 mL, 0.051 mmol). The reaction was stirred at room
temperature for 2 hours. After completion, the residue was partitioned between ethyl acetate and
brine. The crude mixture was purified by reverse phase HPLC in isocratic MeCN/H 2O. The
product was obtained as a white solid. Yield: 5.0 mg (61%).
1
H NMR (500 MHz, CDCl3) δ 8.25
(s, 1H), 8.03 (s, 1H), 7.80 (d, J = 8.92 1H), 7.53 (d, J = 9.06 1H), 4.04 (s, 3H), 3.85 (t, J = 5.60
Hz, 2H), 3.63 (t, J = 5.60 Hz, 2H), 3.50 (br s, 2H), 2.68 (t, J = 5.60 Hz, 2H), 1.90-1.80 (m, 6H),
1.70-1.49 (m, 11H).
5-(3-(2-(2-(Adamantan-1-yl)acetamido)ethoxy)propanamido)-1H-indazole-3-carboxylic
acid. The methyl ester (5.0 mg, 0.01 mmol) was dissolved in 0.2 mL of MeCN containing 2%
H2O. Triethylamine (0.004 mL, 0.032 mmol) and LiBr (9.1 mg, 0.10 mmol) were added to the
solution. The reaction mixture was heated at 50
o
C overnight. The volatiles were removed under
reduced pressure and the residue was partitioned between ethyl acetate and saturated NH4Cl. The
crude mixture was purified by reverse phase HPLC in isocratic MeCN/H2O. The product was
obtained as a white solid.Yield: 4.9 mg (quant.).
1
H NMR (400 MHz, CD3OD) δ 8.33 (s, 1H), 7,71
89
(s, 1H), 7.52 (d, J = 9.05, 1H), 3.82 (t, J = 5.90 Hz, 2H), 3.563 (t, J = 5.90 Hz, 2H), 3.35 (t, J =
5.90 Hz, 2H), 2.65 (t, J = 5.90 Hz, 2H), 1.83 (s, 5H), 1.70-1.60 (m, 3H), 1.58-1.48 (m, 9H).
5-(3-(2-(2-(Adamantan-1-yl)acetamido)ethoxy)propanamido)-N-((3aS,4S,5S,7S,7aR)-2-
(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-
yl)-1H-indazole-3-carboxamide and 5-(3-(2-(2-(adamantan-1-
yl)acetamido)ethoxy)propanamido)-N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-
(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-
indazole-3-carboxamide (Compound 20). A mixture of the amine intermediate (0.9 mg, 0.0024
mmol), acid intermediate (1.0 mg, 0.0022 mmol), HOSu (0.3 mg, 0.0026 mmol), were dissolved
in 1:1 THF:DMF (0.1 mL). EDCHCl (0.5 mg, 0.0026 mmol) was added to the mixture and stirred
at room temperature overnight. The volatiles were removed under reduced pressure and the residue
was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried
over Na2SO4, filtered and concentrated. The residue was purified by reverse HPLC (40-80%
MeCN in H2O) to give the racemic product as a white solid. Yield 0.7 mg (36%).
1
H NMR (600
MHz, CD3CN) δ 11.56 (s, 1H), 8.57-8.55 (m, 2H), 8.09 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 2.0 Hz,
1H), 7.81 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.62-7.51 (m, 3H), 6.37 (s, 1H), 4.56–4.51 (m, 1H), 3.79
(t, J = 5.9 Hz, 2H), 3.52 (t, J = 5.4 Hz, 2H), 3.50 (d, J = 7.3 Hz, 1H), 3.40 (d, J = 7.3 Hz, 1H), 3.30
(t, J = 5.4 Hz, 2H), 2.59 (t, J = 5.4 Hz, 2H), 2.27–2.31 (m, 1H), 1.90 (dd, J = 13.1, 5.2 Hz, 1H),
1.82 (s, 3H), 1.73 (s, 2H), 1.65-1.60 (m, 3H), 1.59 (s, 3H), 1.58 (s, 3H), 1.50-1.44 (m, 9H).
13
C
NMR (151 MHz, CD3CN) δ 175.03, 174.52, 170.42, 169.88, 162.90, 138.58, 138.35, 136.67,
136.04, 134.07, 132.63, 130.51, 124.68 (q, J = 273.8 Hz), 122.05, 121.36, 115.12, 111.26, 110.79,
108.89, 87.88, 85.36, 69.10, 66.02, 56.48, 53.77, 50.70, 48.70, 42.53, 42.10, 38.34, 37.36, 36.45,
90
32.21, 28.63, 17.68, 16.85.
19
F NMR (564 MHz, CD3CN) δ −62.64 (s). ESI-MS: [M - H]
-
calcd
for C43H45F3N7O7
-
: 828.3, found 828.5.
6-(3-(2-((Tert-butoxycarbonyl)amino)ethoxy)propanamido)-1H-indole-3-carboxylic acid.
3-(2-((tert-butoxycarbonyl)amino)ethoxy)propanoic acid (10.3 mg, 0.044 mmol) and
triethylamine (0.02 mL, 0.12 m mol) were dissolved in anhydrous DMF and HATU (16.7 mg,
0.044 mmol) was added. After 30 minutes, the activated acid solution was added dropwise to the
amine (7.1 mg, 0.040 mmol) in anhydrous DMF. The reaction was stirred at room temperature for
2 hours. After completion, the residue was partitioned between ethyl acetate and saturated NH4Cl
solution. The crude mixture was purified by reverse phase HPLC in isocratic MeCN/H 2O. The
product was obtained as a colorless oil. Yield: 9.0 mg (54%).
1
H NMR (400 MHz, CD3OD) δ 8.02-
7.98 (m, 2H), 8.15 (s, 1H), 7.88 (s, 1H), 7.13 (d, J = 8.55 Hz, 1H), 3.82 (t, J = 6.05 Hz, 2H), 3.53
(t, J = 5.61 Hz, 2H), 3.24 (t, J = 5.61 Hz, 2H), 2.69 (t, J = 6.04 Hz, 2H), 1.39 (s, 9H).
6-(3-(2-Aminoethoxy)propanamido)-1H-indole-3-carboxylic acid. The Boc-protected
amine (9.0 mg, 0.023 mmol) and trifluoroacetic acid (0.3 mL) were dissolved in 1 mL DCM. The
reaction was stirred at room temperature for 1 hour. The volatiles were removed under reduced
pressure and the residue was partitioned between ethyl acetate and water. The aqueous phase was
collected and water was removed under reduced pressure. The product was obtained as a white
solid without purification. Yield: 5 mg (75%).
1
H NMR (500 MHz, D2O) δ 7.97 (s, 1H), 7.88 (d,
J = 8.52 Hz, 1H), 7.61 (s, 2H), 7.07 (d, J = 8.47 Hz, 1H), 3.78 (t, J = 5.88 Hz, 2H), 3.66 (t, J =
5.31 Hz, 2H), 3.06 (t, J = 5.35 Hz, 2H), 2.63 (t, J = 5.87 Hz, 2H).
6-(3-(2-(2-(Adamantan-1-yl)acetamido)ethoxy)propanamido)-1H-indole-3-carboxylic
acid. Adamantane acetic acid (3.9 mg, 0.02 mmol) and triethylamine (0.007 mL, 0.051 mmol)
were dissolved in anhydrous DMF and HATU (7.6 mg, 0.02 mmol) was added. After 30 minutes,
91
the activated acid solution was added dropwise to a DMF solution of the amine (5.0 mg, 0.017
mmol) and triethylamine (0.007 mL, 0.051 mmol). The reaction was stirred at room temperature
for 2 hours. After completion, the residue was partitioned between ethyl acetate and brine. The
crude mixture was purified by reverse phase HPLC in isocratic MeCN/H2O. The product was
obtained as a white solid. Yield: 3 mg (37%).
1
H NMR (500 MHz, CD3OD) δ 8.05 (s, 1H), 8.01
(d, J = 8.63, 1H), 7.89 (s, 1H), 7.62 (br. s, 1H), 7.15 (d, J = 8.63, 1H), 3.82 (t, J = 5.93 Hz, 2H),
3.56 (t, J = 5.40 Hz, 2H), 3.35 (t, J = 5.40 Hz, 2H), 2.66 (t, J = 5.88 Hz, 2H), 1.83 (s, 2H), 1.70-
1.60 (m, 54H), 1.57-1.51 (m, 11H).
6-(3-(2-(2-(adamantan-1-yl)acetamido)ethoxy)propanamido)-N-((3aS,4S,5S,7S,7aR)-2-
(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-dioxooctahydro-1H-4,7-epoxyisoindol-5-
yl)-1H-indole-3-carboxamide and 6-(3-(2-(2-(adamantan-1-yl)acetamido)ethoxy)propanamido)-
N-((3aR,4R,5R,7R,7aS)-2-(4-cyano-3-(trifluoromethyl)phenyl)-4,7-dimethyl-1,3-
dioxooctahydro-1H-4,7-epoxyisoindol-5-yl)-1H-indole-3-carboxamide (Compound 21). A
mixture of the amine intermediate (1.4 mg, 0.0037 mmol), acid intermediate (1.6 mg, 0.0034
mmol), Et3N (0.001 mL, 0.0068 mmol), were dissolved in 1:2 THF:DMF (0.1 mL). TBTU (1.4
mg, 0.0037 mmol) was added to the mixture and stirred at room temperature overnight. The
volatiles were removed under reduced pressure and the residue was partitioned between ethyl
acetate and water. The organic layer was washed with brine, dried over Na2SO4, filtered and
concentrated. The residue was purified by reverse HPLC in isocratic MeCN in H2O to give the
racemic product as a white solid. Yield 1.0 mg (36%).
1
H NMR (600 MHz, CD3CN) δ 9.62 (s,
1H), 8.50 (s, 1H), 8.14 (s, 1H), 8.10-8.07 (m, 2H), 7.90 (s, 1H), 7.85 (d, J = 2.9 Hz, 1H), 7.85 (d,
J = 8.4 Hz, 2.0 Hz, 1H), 7.14 (dd, J = 8.6 Hz, 1.6 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H) 6.35 (s, 1H),
4.53–4.49 (m, 1H), 3.78 (t, J = 5.9 Hz, 2H), 3.53 (d, J = 7.3 Hz, 1H), 3.51 (t, J = 5.4 Hz, 2H), 3.35
92
(d, J = 7.3 Hz, 1H), 3.30 (t, J = 5.8 Hz, 2H), 2.58 (t, J = 5.9 Hz, 2H), 2.27–2.25 (m, 1H), 1.84 (s,
3H), 1.80 (dd, J = 13.1, 5.3 Hz, 1H), 1.73 (s, 2H), 1.65-1.60 (m, 3H), 1.57 (s, 3H), 1.56 (s, 3H),
1.50-1.44 (m, 9H).
13
C NMR (151 MHz, CD3CN) δ 175.19, 174.55, 170.45, 169.69, 165.07,
136.68, 136.42, 136.04, 134.43, 130.50, 127.15, 124.65 (q, J = 273.8 Hz), 122.78, 121.26, 115.12,
114.10, 110.84, 108.87, 102.63, 87.87, 85.24, 69.09, 66.11, 56.53, 53.92, 50.70, 48.66, 42.82,
38.35, 37.41, 36.47, 32.23, 28.65, 17.72, 16.81.
19
F NMR (564 MHz, CD3CN) δ −62.63 (s). ESI-
MS: [M - H]
-
calcd for C43H45F3N7O7
-
: 827.3, found 827.8.

93
Chapter 4 references
1. Lai, A. C.; Crews, C. M., Induced protein degradation: an emerging drug discovery paradigm.
Nature Reviews Drug Discovery 2017, 16 (2), 101-114.
2. Sun, X.; Gao, H.; Yang, Y.; He, M.; Wu, Y.; Song, Y.; Tong, Y.; Rao, Y., PROTACs:
great opportunities for academia and industry. Signal Transduction and Targeted Therapy 2019,
4 (1), 64.
3. Neklesa, T. K.; Tae, H. S.; Schneekloth, A. R.; Stulberg, M. J.; Corson, T. W.; Sundberg, T.
B.; Raina, K.; Holley, S. A.; Crews, C. M., Small-molecule hydrophobic tagging–induced
degradation of HaloTag fusion proteins. Nature Chemical Biology 2011, 7 (8), 538-543.
4. Bricelj, A.; Steinebach, C.; Kuchta, R.; Gütschow, M.; Sosič, I., E3 ligase ligands in
successful protacs: an overview of syntheses and linker attachment points. Frontiers in
Chemistry 2021, 9, 707317.
5. Neklesa, T. K.; Winkler, J. D.; Crews, C. M., Targeted protein degradation by PROTACs.
Pharmacology & Therapeutics 2017, 174, 138-144.
6. Luan, H.; Xu, P.; Meng, Y.; Li, Z.; Bian, J., A critical update on the strategies towards
modulators targeting androgen receptors. Bioorganic & Medicinal Chemistry 2020, 28 (13),
115554.
7. Gustafson, J. L.; Neklesa, T. K.; Cox, C. S.; Roth, A. G.; Buckley, D. L.; Tae, H. S.;
Sundberg, T. B.; Stagg, D. B.; Hines, J.; McDonnell, D. P.; Norris, J. D.; Crews, C. M., Small-
Molecule-Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging.
Angewandte Chemie International Edition 2015, 54 (33), 9659-9662.
8. Yap, T. A.; Smith, A. D.; Ferraldeschi, R.; Al-Lazikani, B.; Workman, P.; de Bono, J. S.,
Drug discovery in advanced prostate cancer: translating biology into therapy. Nature Reviews
Drug Discovery 2016, 15 (10), 699-718.
9. Centenera, M. M.; Selth, L. A.; Ebrahimie, E.; Butler, L. M.; Tilley, W. D., New
opportunities for targeting the androgen receptor in prostate cancer. Cold Spring Harbor
Perspectives in Medicine 2018, 8 (12), a030478.
10. Ponnusamy, S.; He, Y.; Hwang, D.-J.; Thiyagarajan, T.; Houtman, R.; Bocharova, V.;
Sumpter, B. G.; Fernandez, E.; Johnson, D.; Du, Z.; Pfeffer, L. M.; Getzenberg, R. H.;
McEwan, I. J.; Miller, D. D.; Narayanan, R., Orally Bioavailable Androgen Receptor Degrader,
Potential Next-Generation Therapeutic for Enzalutamide-Resistant Prostate Cancer. Clinical
Cancer Research 2019, 25 (22), 6764-6780.
11. Loddick, S. A.; Ross, S. J.; Thomason, A. G.; Robinson, D. M.; Walker, G. E.; Dunkley,
T. P. J.; Brave, S. R.; Broadbent, N.; Stratton, N. C.; Trueman, D.; Mouchet, E.; Shaheen, F.
S.; Jacobs, V. N.; Cumberbatch, M.; Wilson, J.; Jones, R. D. O.; Bradbury, R. H.; Rabow, A.;
Gaughan, L.; Womack, C.; Barry, S. T.; Robson, C. N.; Critchlow, S. E.; Wedge, S. R.;
Brooks, A. N., AZD3514: A Small Molecule That Modulates Androgen Receptor Signaling and
Function In Vitro and In Vivo. Molecular Cancer Therapeutics 2013, 12 (9), 1715-1727.
12. Snyder, L. B. In Discovery of ARV-110, a first in class androgen receptor degrading
PROTAC® for the treatment of men with metastatic castration resistant prostate cancer, April
10−15; American Association for Cancer Research: 2021.
13. Chen, L.; Han, L.; Mao, S.; Xu, P.; Xu, X.; Zhao, R.; Wu, Z.; Zhong, K.; Yu, G.; Wang,
X., Discovery of A031 as effective proteolysis targeting chimera (PROTAC) androgen receptor
(AR) degrader for the treatment of prostate cancer. European Journal of Medicinal Chemistry
2021, 216, 113307.
94
14. Han, X.; Zhao, L.; Xiang, W.; Qin, C.; Miao, B.; McEachern, D.; Wang, Y.; Metwally,
H.; Wang, L.; Matvekas, A.; Wen, B.; Sun, D.; Wang, S., Strategies toward Discovery of
Potent and Orally Bioavailable Proteolysis Targeting Chimera Degraders of Androgen Receptor
for the Treatment of Prostate Cancer. Journal of Medicinal Chemistry 2021, 64 (17), 12831-
12854.
15. Beretta, G. L.; Zaffaroni, N., Androgen Receptor-Directed Molecular Conjugates for
Targeting Prostate Cancer. Frontiers in Chemistry 2019, 7 (369).
16. Lee, G. T.; Nagaya, N.; Desantis, J.; Madura, K.; Sabaawy, H. E.; Kim, W.-J.; Vaz, R. J.;
Cruciani, G.; Kim, I. Y., Effects of MTX-23, a Novel PROTAC of Androgen Receptor Splice
Variant-7 and Androgen Receptor, on CRPC Resistant to Second-Line Antiandrogen Therapy.
Molecular Cancer Therapeutics 2021, 20 (3), 490-499.
17. Xie, H.; Liang, J.-J.; Wang, Y.-L.; Hu, T.-X.; Wang, J.-Y.; Yang, R.-H.; Yan, J.-K.;
Zhang, Q.-R.; Xu, X.; Liu, H.-M.; Ke, Y., The design, synthesis and anti-tumor mechanism
study of new androgen receptor degrader. European Journal of Medicinal Chemistry 2020, 204,
112512.
18. Shibata, N.; Nagai, K.; Morita, Y.; Ujikawa, O.; Ohoka, N.; Hattori, T.; Koyama, R.;
Sano, O.; Imaeda, Y.; Nara, H.; Cho, N.; Naito, M., Development of Protein Degradation
Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of
Apoptosis Protein Ligands. Journal of Medicinal Chemistry 2018, 61 (2), 543-575.
19. Mattsson, S.; Dahlström, M.; Karlsson, S., A mild hydrolysis of esters mediated by lithium
salts. Tetrahedron Letters 2007, 48 (14), 2497-2499.
20. Mc Murry, J., Ester Cleavages via SN2-Type Dealkylation. Organic Reactions 2011, 187-
224.
21. Beutner, G. L.; Cohen, B. M.; DelMonte, A. J.; Dixon, D. D.; Fraunhoffer, K. J.; Glace, A.
W.; Lo, E.; Stevens, J. M.; Vanyo, D.; Wilbert, C., Revisiting the Cleavage of Evans
Oxazolidinones with LiOH/H2O2. Organic Process Research & Development 2019, 23 (7),
1378-1385.
22. Laganis, E. D.; Chenard, B. L., Metal silanolates: organic soluble equivalents for O−2.
Tetrahedron Letters 1984, 25 (51), 5831-5834.
23. Moreau, K.; Coen, M.; Zhang, A. X.; Pachl, F.; Castaldi, M. P.; Dahl, G.; Boyd, H.;
Scott, C.; Newham, P., Proteolysis-targeting chimeras in drug development: A safety
perspective. British Journal of Pharmacology 2020, 177 (8), 1709-1718.

95
Chapter 5 Conclusions and outlook
This dissertation describes our medicinal campaign to develop a new generation of
antiandrogens as novel treatments for prostate cancer. Through interdisciplinary collaboration, we
discovered an unexpected phenomenon arising from chirality of our molecules,
1
which has
profound implications in drug screening and large scale manufacturing. We studied AR LBD
structure and molecular dynamics in silico and in vitro. We rationally designed and developed
promising lead compounds that could lead to new therapeutics for prostate cancer, as well as for
other diseases caused by AR dysregulation.
2, 3

In the second chapter, we described the opposite functional effects of enantiomeric AR
ligands. We observed that BMS-641988 had a previously uncharacterized enantiomer “S-BMS”
that agonized AR. We showed that this enantiomer-dependent agonist-antagonist pairing property
was consistently observed among a series of BMS-641988 derivatives. This is, to our knowledge,
the first such example with a protein target in oncology and in AR. Our work adds on to other
reported examples where subtle changes in structures of AR ligands could lead to drastic change
in their functional effects.
4
We further demonstrated the importance of molecular dynamics and
structural conformations of AR by in silico modeling. Attempts to crystalize AR LBD with our
own ligands showed that the conditions for crystal formation was ligand-dependent, and that AR
LBD was unstable during ligand exchange. Future efforts to solve the structures of AR LBD bound
with our ligands should focus on expressing AR LBD in the presence of agonists. Co-expression
with HSPs or Cryo-EM techniques could also be used for solving the structure of full-length AR
or AR variants.
5
We concluded that since a miniscule amount of chiral contamination led to
significant changes in their biological activities, extremely high purity (> 99.8% ee) is required for
accurate study of this family of compounds. This enantiopurity requirement was not discussed by
96
the inventors of BMS-641988. On the other hand, we serendipitously discovered highly potent and
effective novel AR agonists, which could potentially be useful in some diseases such as Duchenne
muscular dystrophy.
3

In the third chapter, we described our design and synthesis for novel AR antagonist based
on the oxabicyclic structure of BMS-641988. We performed SAR studies in a series of BMS-
641988 analogues. SAR studies at the C5 position indicated that the ligand binding pocket inside
the AR LBD can accommodate a wide variety of substituents at this position. Several promising
compounds were identified in this process, which showed excellent ARE-luciferase IC50 in LNCaP
cell lines and cell viability IC50 in VCaP cell lines. Furthermore, preliminary data showed that our
compounds are effective in enzalutamide-resistant prostate cancer cells expressing AR F877L
mutant. To ensure that our lead compounds have acceptable safety profiles, we performed in silico
and in vitro studies of their logBB and metabolism. We demonstrated that our lead compounds are
unlikely to penetrate the BBB and also unlikely to produce the neurotoxic metabolite BMS-
501949. Our work addressed the critical problem of drug safety in BMS-641988 and enzalutamide,
and our lead compounds showed superior in vitro efficacy and safety to BMS-641988 and
enzalutamide. Future work should focus on exploring SAR of C5 heterocycle substitutions. In vivo
studies are needed to further study the efficacy and safety of our lead compounds.
In the fourth chapter, we described the design and synthesis for proof-of-concept SARD
molecules based on our scaffold. Protein degradation is an exciting drug modality that could
address drug resistance problems. Many SARDs have been shown to be effective in enzalutamide-
resistant prostate cancer cells. The only SARD molecule currently in clinical trial, ARV-110,
showed excellent safety profiles in Phase I clinical trial.
6
Detailed biological characterization of
these molecules are necessary for future design and optimization of SARDs based on our scaffold.
97
Significant challenges and obstacles still exist for SARD molecules, including binding affinity,
toxicity, and oral bioavailability.
In conclusion, the work presented in this dissertation demonstrated the rational design and
serendipitous discovery of a series of enantiomeric AR ligands that hold promise in prostate cancer
and other AR-related diseases. Besides the strategies discussed in this dissertation, other promising
treatments have been reported for CRPC. For example, AR DBD inhibitors and SARDs that bind
to AR DBD would effectively target the AR-V7 variant observed in some patients.
7, 8

Homodimerization of AR could also be inhibited to prevent transactivation.
9
Inhibition of AR
expression at the mRNA level can also be an attractive option for patients with highly mutated
AR.
10
Inhibitors for other proteins within the androgen signaling pathway, such as mTORC, have
been studied in clinical settings.
11
Cell therapies and immunotherapies, including bispecific
antibodies, are being developed for CRPC and other types of cancers.
12
Furthermore, next-
generation sequencing will enable precision medicines to treat patients with personalized
therapies.
12
Thus, it is reasonable to envision a future where diverse therapeutics will work together
to benefit patients with CRPC and other AR-related diseases.

98
Chapter 5 references
1. Patsch, K.; Liu, C.; Zapotoczny, G.; Sun, Y.; Sura, H.; Ung, N.; Sun, R. X.; Haliday, B.;
Yu, C.; Aljehani, M.; Lee, J. S. H.; Kashemirov, B. A.; Agus, D. B.; McKenna, C. E.;
Ruderman, D., Paradoxical androgen receptor regulation by small molecule enantiomers.
Proceedings of the National Academy of Sciences 2021, 118 (12), e2100918118.
2. Schmidt, K. T.; Huitema, A. D.; Chau, C. H.; Figg, W. D., Resistance to second-generation
androgen receptor antagonists in prostate cancer. Nature Reviews Urology 2021, 1-18.
3. Matsumoto, T.; Sakari, M.; Okada, M.; Yokoyama, A.; Takahashi, S.; Kouzmenko, A.;
Kato, S., The androgen receptor in health and disease. Annual Review of Physiology 2013, 75,
201-224.
4. Bohl, C. E.; Miller, D. D.; Chen, J.; Bell, C. E.; Dalton, J. T., Structural basis for
accommodation of nonsteroidal ligands in the androgen receptor. Journal of Biological
Chemistry 2005, 280 (45), 37747-37754.
5. Bai, X.-c.; McMullan, G.; Scheres, S. H. W., How cryo-EM is revolutionizing structural
biology. Trends in Biochemical Sciences 2015, 40 (1), 49-57.
6. Snyder, L. B. In Discovery of ARV-110, a first in class androgen receptor degrading
PROTAC® for the treatment of men with metastatic castration resistant prostate cancer, April
10−15; American Association for Cancer Research: 2021.
7. Lee, G. T.; Nagaya, N.; Desantis, J.; Madura, K.; Sabaawy, H. E.; Kim, W.-J.; Vaz, R. J.;
Cruciani, G.; Kim, I. Y., Effects of MTX-23, a Novel PROTAC of Androgen Receptor Splice
Variant-7 and Androgen Receptor, on CRPC Resistant to Second-Line Antiandrogen Therapy.
Molecular Cancer Therapeutics 2021, 20 (3), 490-499.
8. Centenera, M. M.; Selth, L. A.; Ebrahimie, E.; Butler, L. M.; Tilley, W. D., New
opportunities for targeting the androgen receptor in prostate cancer. Cold Spring Harbor
Perspectives in Medicine 2018, 8 (12), a030478.
9. Nadal, M.; Prekovic, S.; Gallastegui, N.; Helsen, C.; Abella, M.; Zielinska, K.; Gay, M.;
Vilaseca, M.; Taulès, M.; Houtsmuller, A. B.; van Royen, M. E.; Claessens, F.; Fuentes-Prior,
P.; Estébanez-Perpiñá, E., Structure of the homodimeric androgen receptor ligand-binding
domain. Nature Communications 2017, 8 (1), 14388.
10. Yu, J.; Zhou, P.; Hu, M.; Yang, L.; Yan, G.; Xu, R.; Deng, Y.; Li, X.; Chen, Y.,
Discovery and biological evaluation of darolutamide derivatives as inhibitors and down-
regulators of wild-type AR and the mutants. European Journal of Medicinal Chemistry 2019,
182, 111608.
11. Yap, T. A.; Smith, A. D.; Ferraldeschi, R.; Al-Lazikani, B.; Workman, P.; de Bono, J. S.,
Drug discovery in advanced prostate cancer: translating biology into therapy. Nature Reviews
Drug Discovery 2016, 15 (10), 699-718.
12. Cha, H.-R.; Lee, J. H.; Ponnazhagan, S., Revisiting immunotherapy: a focus on prostate
cancer. Cancer Research 2020, 80 (8), 1615-1623.

99
Bibliography
1. Askew, E. B.; Gampe, R. T.; Stanley, T. B.; Faggart, J. L.; Wilson, E. M., Modulation of
androgen receptor activation function 2 by testosterone and dihydrotestosterone. Journal of
Biological Chemistry 2007, 282 (35), 25801-25816.
2. Bai, X.-c.; McMullan, G.; Scheres, S. H. W., How cryo-EM is revolutionizing structural
biology. Trends in Biochemical Sciences 2015, 40 (1), 49-57.
3. Balog, A.; Rampulla, R.; Martin, G. S.; Krystek, S. R.; Attar, R.; Dell-John, J.; DiMarco, J.
D.; Fairfax, D.; Gougoutas, J.; Holst, C. L.; Nation, A.; Rizzo, C.; Rossiter, L. M.;
Schweizer, L.; Shan, W.; Spergel, S.; Spires, T.; Cornelius, G.; Gottardis, M.; Trainor, G.;
Vite, G. D.; Salvati, M. E., Discovery of BMS-641988, a novel androgen receptor antagonist for
the treatment of prostate cancer. ACS Medicinal Chemistry Letters 2015, 6 (8), 908-912.
4. Balog, A.; Salvati, M. E.; Shan, W.; Mathur, A.; Leith, L. W.; Wei, D. D.; Attar, R. M.;
Geng, J.; Rizzo, C. A.; Wang, C.; Krystek, S. R.; Tokarski, J. S.; Hunt, J. T.; Gottardis, M.;
Weinmann, R., The synthesis and evaluation of [2.2. 1]-bicycloazahydantoins as androgen
receptor antagonists. Bioorganic & Medicinal Chemistry Letters 2004, 14 (24), 6107-6111.
5. Bambury, R.; Scher, H. In Enzalutamide: Development from bench to bedside, Urologic
Oncology: Seminars and Original Investigations, Elsevier: 2015; pp 280-288.
6. Barsouk, A.; Padala, S. A.; Vakiti, A.; Mohammed, A.; Saginala, K.; Thandra, K. C.;
Rawla, P.; Barsouk, A., Epidemiology, Staging and Management of Prostate Cancer. Medical
Sciences 2020, 8 (3), 28.
7. Beer, T. M.; Armstrong, A. J.; Rathkopf, D. E.; Loriot, Y.; Sternberg, C. N.; Higano, C. S.;
Iversen, P.; Bhattacharya, S.; Carles, J.; Chowdhury, S.; Davis, I. D.; de Bono, J. S.; Evans,
C. P.; Fizazi, K.; Joshua, A. M.; Kim, C.-S.; Kimura, G.; Mainwaring, P.; Mansbach, H.;
Miller, K.; Noonberg, S. B.; Perabo, F.; Phung, D.; Saad, F.; Scher, H. I.; Taplin, M.-E.;
Venner, P. M.; Tombal, B., Enzalutamide in metastatic prostate cancer before chemotherapy.
New England Journal of Medicine 2014, 371 (5), 424-433.
8. Beretta, G. L.; Zaffaroni, N., Androgen Receptor-Directed Molecular Conjugates for Targeting
Prostate Cancer. Frontiers in Chemistry 2019, 7 (369).
9. Beutner, G. L.; Cohen, B. M.; DelMonte, A. J.; Dixon, D. D.; Fraunhoffer, K. J.; Glace, A.
W.; Lo, E.; Stevens, J. M.; Vanyo, D.; Wilbert, C., Revisiting the Cleavage of Evans
Oxazolidinones with LiOH/H2O2. Organic Process Research & Development 2019, 23 (7),
1378-1385.
10. Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T., Structural basis for
antagonism and resistance of bicalutamide in prostate cancer. Proceedings of the National
Academy of Sciences 2005, 102 (17), 6201-6206.
11. Bohl, C. E.; Miller, D. D.; Chen, J.; Bell, C. E.; Dalton, J. T., Structural basis for
accommodation of nonsteroidal ligands in the androgen receptor. Journal of Biological
Chemistry 2005, 280 (45), 37747-37754.
12. Bonne, D.; Rodriguez, J., A Bird's Eye View of Atropisomers Featuring a Five-Membered
Ring. European Journal of Organic Chemistry 2018, 2018 (20-21), 2417 - 2431.
13. Bricelj, A.; Steinebach, C.; Kuchta, R.; Gütschow, M.; Sosič, I., E3 ligase ligands in
successful protacs: an overview of syntheses and linker attachment points. Frontiers in
Chemistry 2021, 9, 707317.
100
14. Centenera, M. M.; Selth, L. A.; Ebrahimie, E.; Butler, L. M.; Tilley, W. D., New
opportunities for targeting the androgen receptor in prostate cancer. Cold Spring Harbor
Perspectives in Medicine 2018, 8 (12), a030478.
15. Cha, H.-R.; Lee, J. H.; Ponnazhagan, S., Revisiting immunotherapy: a focus on prostate
cancer. Cancer Research 2020, 80 (8), 1615-1623.
16. Chen, L.; Han, L.; Mao, S.; Xu, P.; Xu, X.; Zhao, R.; Wu, Z.; Zhong, K.; Yu, G.; Wang,
X., Discovery of A031 as effective proteolysis targeting chimera (PROTAC) androgen receptor
(AR) degrader for the treatment of prostate cancer. European Journal of Medicinal Chemistry
2021, 216, 113307.
17. Clegg, N. J.; Wongvipat, J.; Joseph, J. D.; Tran, C.; Ouk, S.; Dilhas, A.; Chen, Y.;
Grillot, K.; Bischoff, E. D.; Cai, L.; Aparicio, A.; Dorow, S.; Arora, V.; Shao, G.; Qian, J.;
Zhao, H.; Yang, G.; Cao, C.; Sensintaffar, J.; Wasielewska, T.; Herbert, M. R.; Bonnefous,
C.; Darimont, B.; Scher, H. I.; Smith-Jones, P.; Klang, M.; Smith, N. D.; De Stanchina, E.;
Wu, N.; Ouerfelli, O.; Rix, P. J.; Heyman, R. A.; Jung, M. E.; Sawyers, C. L.; Hager, J. H.,
ARN-509: a novel antiandrogen for prostate cancer treatment. Cancer Research 2012, 72 (6),
1494-1503.
18. Cockshott, I. D., Bicalutamide. Clinical Pharmacokinetics 2004, 43 (13), 855-878.
19. de Wit, R.; de Bono, J.; Sternberg, C. N.; Fizazi, K.; Tombal, B.; Wülfing, C.; Kramer,
G.; Eymard, J.-C.; Bamias, A.; Carles, J.; Iacovelli, R.; Melichar, B.; Sverrisdóttir, Á.;
Theodore, C.; Feyerabend, S.; Helissey, C.; Ozatilgan, A.; Geffriaud-Ricouard, C.; Castellano,
D., Cabazitaxel versus Abiraterone or Enzalutamide in Metastatic Prostate Cancer. New England
Journal of Medicine 2019, 381 (26), 2506-2518.
20. Duan, M.; Liu, N.; Zhou, W.; Li, D.; Yang, M.; Hou, T., Structural diversity of ligand-
binding androgen receptors revealed by microsecond long molecular dynamics simulations and
enhanced sampling. Journal of Chemical Theory and Computation 2016, 12 (9), 4611-4619.
21. Foster, W. R.; Car, B. D.; Shi, H.; Levesque, P. C.; Obermeier, M. T.; Gan, J.; Arezzo, J.
C.; Powlin, S. S.; Dinchuk, J. E.; Balog, A., Drug safety is a barrier to the discovery and
development of new androgen receptor antagonists. The Prostate 2011, 71 (5), 480-488.
22. Gao, W.; Bohl, C. E.; Dalton, J. T., Chemistry and Structural Biology of Androgen Receptor.
Chemical Reviews 2005, 105 (9), 3352-3370.
23. Gavai, A. V.; Foster, W. R.; Balog, A.; Vite, G. D., Novel Androgen Receptor Antagonists
for the Treatment of Prostate Cancer. In Accounts in Drug Discovery, 2010; pp 120-141.
24. Grisoni, F.; Consonni, V.; Ballabio, D., Machine learning consensus to predict the binding to
the androgen receptor within the CoMPARA project. Journal of Chemical Information and
Modeling 2019, 59 (5), 1839-1848.
25. Grosse, L.; Campeau, A. S.; Caron, S.; Morin, F. A.; Meunier, K.; Trottier, J.; Caron, P.;
Verreault, M.; Barbier, O., Enantiomer selective glucuronidation of the non‐steroidal pure anti‐
androgen bicalutamide by human liver and kidney: Role of the human UDP‐
glucuronosyltransferase (UGT) 1A9 enzyme. Basic & Clinical Pharmacology & Toxicology
2013, 113 (2), 92-102.
26. Gustafson, J. L.; Neklesa, T. K.; Cox, C. S.; Roth, A. G.; Buckley, D. L.; Tae, H. S.;
Sundberg, T. B.; Stagg, D. B.; Hines, J.; McDonnell, D. P.; Norris, J. D.; Crews, C. M., Small-
Molecule-Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging.
Angewandte Chemie International Edition 2015, 54 (33), 9659-9662.
27. Ha, Y.-S.; Kim, I. Y., Enzalutamide: looking back at its preclinical discovery. Expert Opinion
on Drug Discovery 2014, 9 (7), 837-845.
101
28. Hadley, J.; Yabroff, K. R.; Barrett, M. J.; Penson, D. F.; Saigal, C. S.; Potosky, A. L.,
Comparative effectiveness of prostate cancer treatments: evaluating statistical adjustments for
confounding in observational data. Journal of the National Cancer Institute 2010, 102 (23),
1780-1793.
29. Han, X.; Zhao, L.; Xiang, W.; Qin, C.; Miao, B.; McEachern, D.; Wang, Y.; Metwally,
H.; Wang, L.; Matvekas, A.; Wen, B.; Sun, D.; Wang, S., Strategies toward Discovery of
Potent and Orally Bioavailable Proteolysis Targeting Chimera Degraders of Androgen Receptor
for the Treatment of Prostate Cancer. Journal of Medicinal Chemistry 2021, 64 (17), 12831-
12854.
30. He, B.; Gampe Jr, R. T.; Kole, A. J.; Hnat, A. T.; Stanley, T. B.; An, G.; Stewart, E. L.;
Kalman, R. I.; Minges, J. T.; Wilson, E. M., Structural basis for androgen receptor interdomain
and coactivator interactions suggests a transition in nuclear receptor activation function
dominance. Molecular Cell 2004, 16 (3), 425-438.
31. Hirota, K.; Lambert, D., Ketamine: its mechanism(s) of action and unusual clinical uses.
British Journal of Anaesthesia 1996, 77 (4), 441-444.
32. Hof, R.; Rüegg, U.; Hof, A.; Vogel, A., Stereoselectivity at the calcium channel: opposite
action of the enantiomers of a 1, 4-dihydropyridine. Journal of cardiovascular pharmacology
1985, 7 (4), 689-693.
33. Johns, B. A.; Shotwell, J. B. Compounds. WO 2012/067664 Al, May 24, 2012.
34. Jones, A.; Chen, J.; Hwang, D. J.; Miller, D. D.; Dalton, J. T., Preclinical Characterization
of a (S)-N-(4-Cyano-3-Trifluoromethyl-Phenyl)-3-(3-Fluoro, 4-Chlorophenoxy)-2-Hydroxy-2-
Methyl-Propanamide: A Selective Androgen Receptor Modulator for Hormonal Male
Contraception. Endocrinology 2009, 150 (1), 385-395.
35. Keiki, K.; Isao, T.; Shigeo, K.; Makoto, Y.; Kazutoshi, Y., Stereoselective Synthesis of 2-
Alkylamino-N-(2′-alkylphenyl)succinimide Conformers. Chemistry Letters 1994, 23 (9), 1605-
1606.
36. Kim, J.; Farchione, T.; Potter, A.; Chen, Q.; Temple, R., Esketamine for Treatment-
Resistant Depression — First FDA-Approved Antidepressant in a New Class. New England
Journal of Medicine 2019, 381 (1), 1-4.
37. Kim, J. H.; Scialli, A. R., Thalidomide: the tragedy of birth defects and the effective treatment
of disease. Toxicological Sciences 2011, 122 (1), 1-6.
38. Korpal, M.; Korn, J. M.; Gao, X.; Rakiec, D. P.; Ruddy, D. A.; Doshi, S.; Yuan, J.;
Kovats, S. G.; Kim, S.; Cooke, V. G.; Monahan, J. E.; Stegmeier, F.; Roberts, T. M.; Sellers,
W. R.; Zhou, W.; Zhu, P., An F876L mutation in androgen receptor confers genetic and
phenotypic resistance to MDV3100 (enzalutamide). Cancer Discovery 2013, 3 (9), 1030-1043.
39. Kunwittaya, S.; Nantasenamat, C.; Treeratanapiboon, L.; Srisarin, A.; Isarankura-Na-
Ayudhya, C.; Prachayasittikul, V., Influence of logBB cut-off on the prediction of blood-brain
barrier permeability. Biomedical and Applied Technology Journal 2013, 1, 16-34.
40. Labrie, F.; Singh, S. M.; Labrecque, R.; Ciobanu, L. C. Non-steroidal antiandrogens and
selective androgen receptor modulators with a pyridyl moiety. US 2015/0175576A1, June. 25,
2015.
41. Laganis, E. D.; Chenard, B. L., Metal silanolates: organic soluble equivalents for O−2.
Tetrahedron Letters 1984, 25 (51), 5831-5834.
42. Lai, A. C.; Crews, C. M., Induced protein degradation: an emerging drug discovery paradigm.
Nature Reviews Drug Discovery 2017, 16 (2), 101-114.
102
43. Lai, K. W.; Liang, J.; Zhang, B.; Labadie, S.; Ortwine, D.; Dragovich, P.; Kiefer, J.;
Gehling, V. S.; Harmange, J.-C. Pyrrolidine Amide Compounds as Histone Demethylase
Inhibitors. WO 2016/057924 Al.
44. Laudet, V.; Hänni, C.; Coll, J.; Catzeflis, F.; Stéhelin, D., Evolution of the nuclear receptor
gene superfamily. The EMBO Journal 1992, 11 (3), 1003-1013.
45. Lee, G. T.; Nagaya, N.; Desantis, J.; Madura, K.; Sabaawy, H. E.; Kim, W.-J.; Vaz, R. J.;
Cruciani, G.; Kim, I. Y., Effects of MTX-23, a Novel PROTAC of Androgen Receptor Splice
Variant-7 and Androgen Receptor, on CRPC Resistant to Second-Line Antiandrogen Therapy.
Molecular Cancer Therapeutics 2021, 20 (3), 490-499.
46. Liu, H.; Han, R.; Li, J.; Liu, H.; Zheng, L., Molecular mechanism of R-bicalutamide
switching from androgen receptor antagonist to agonist induced by amino acid mutations using
molecular dynamics simulations and free energy calculation. Journal of Computer-Aided
Molecular Design 2016, 30 (12), 1189-1200.
47. Loddick, S. A.; Ross, S. J.; Thomason, A. G.; Robinson, D. M.; Walker, G. E.; Dunkley,
T. P. J.; Brave, S. R.; Broadbent, N.; Stratton, N. C.; Trueman, D.; Mouchet, E.; Shaheen, F.
S.; Jacobs, V. N.; Cumberbatch, M.; Wilson, J.; Jones, R. D. O.; Bradbury, R. H.; Rabow, A.;
Gaughan, L.; Womack, C.; Barry, S. T.; Robson, C. N.; Critchlow, S. E.; Wedge, S. R.;
Brooks, A. N., AZD3514: A Small Molecule That Modulates Androgen Receptor Signaling and
Function In Vitro and In Vivo. Molecular Cancer Therapeutics 2013, 12 (9), 1715-1727.
48. Luan, H.; Xu, P.; Meng, Y.; Li, Z.; Bian, J., A critical update on the strategies towards
modulators targeting androgen receptors. Bioorganic & Medicinal Chemistry 2020, 28 (13),
115554.
49. Madauss, K. P.; Grygielko, E. T.; Deng, S.-J.; Sulpizio, A. C.; Stanley, T. B.; Wu, C.;
Short, S. A.; Thompson, S. K.; Stewart, E. L.; Laping, N. J.; Williams, S. P.; Bray, J. D., A
Structural and in Vitro Characterization of Asoprisnil: A Selective Progesterone Receptor
Modulator. Molecular Endocrinology 2007, 21 (5), 1066-1081.
50. Marhefka, C. A.; Gao, W.; Chung, K.; Kim, J.; He, Y.; Yin, D.; Bohl, C.; Dalton, J. T.;
Miller, D. D., Design, synthesis, and biological characterization of metabolically stable selective
androgen receptor modulators. Journal of Medicinal Chemistry 2004, 47 (4), 993-998.
51. Matias, P. M.; Donner, P.; Coelho, R.; Thomaz, M.; Peixoto, C.; Macedo, S.; Otto, N.;
Joschko, S.; Scholz, P.; Wegg, A.; Bä sler, S.; Schä fer, M.; Egner, U.; Carrondo, M. A.,
Structural evidence for ligand specificity in the binding domain of the human androgen receptor:
implications for pathogenic gene mutations. Journal of Biological Chemistry 2000, 275 (34),
26164-26171.
52. Matsumoto, T.; Sakari, M.; Okada, M.; Yokoyama, A.; Takahashi, S.; Kouzmenko, A.;
Kato, S., The androgen receptor in health and disease. Annual Review of Physiology 2013, 75,
201-224.
53. Mattsson, S.; Dahlström, M.; Karlsson, S., A mild hydrolysis of esters mediated by lithium
salts. Tetrahedron Letters 2007, 48 (14), 2497-2499.
54. Mc Murry, J., Ester Cleavages via SN2-Type Dealkylation. Organic Reactions 2011, 187-
224.
55. Milton, R. d.; Milton, S.; Kent, S., Total chemical synthesis of a D-enzyme: the enantiomers
of HIV-1 protease show reciprocal chiral substrate specificity [corrected]. Science 1992, 256
(5062), 1445-1448.
56. Moilanen, A.-M.; Riikonen, R.; Oksala, R.; Ravanti, L.; Aho, E.; Wohlfahrt, G.;
Nykänen, P. S.; Törmäkangas, O. P.; Palvimo, J. J.; Kallio, P. J., Discovery of ODM-201, a
103
new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen
signaling-directed prostate cancer therapies. Scientific Reports 2015, 5 (1), 1-11.
57. Moreau, K.; Coen, M.; Zhang, A. X.; Pachl, F.; Castaldi, M. P.; Dahl, G.; Boyd, H.;
Scott, C.; Newham, P., Proteolysis-targeting chimeras in drug development: A safety
perspective. British Journal of Pharmacology 2020, 177 (8), 1709-1718.
58. Morova, T.; McNeill, D. R.; Lallous, N.; Gönen, M.; Dalal, K.; Wilson, D. M.; Gürsoy,
A.; Keskin, Ö.; Lack, N. A., Androgen receptor-binding sites are highly mutated in prostate
cancer. Nature Communications 2020, 11 (1), 1-10.
59. Morsy, A.; Trippier, P. C., Reversal of apalutamide and darolutamide aldo-keto reductase
1C3-mediated resistance by a small molecule inhibitor. ACS Chemical Biology 2020, 15 (3),
646-650.
60. Mullard, A., Targeted degraders clear first safety hurdles. Nature Reviews Drug Discovery
2020, 19 (7), 435-436.
61. Nadal, M.; Prekovic, S.; Gallastegui, N.; Helsen, C.; Abella, M.; Zielinska, K.; Gay, M.;
Vilaseca, M.; Taulès, M.; Houtsmuller, A. B.; van Royen, M. E.; Claessens, F.; Fuentes-Prior,
P.; Estébanez-Perpiñá, E., Structure of the homodimeric androgen receptor ligand-binding
domain. Nature Communications 2017, 8 (1), 14388.
62. Neklesa, T. K.; Tae, H. S.; Schneekloth, A. R.; Stulberg, M. J.; Corson, T. W.; Sundberg,
T. B.; Raina, K.; Holley, S. A.; Crews, C. M., Small-molecule hydrophobic tagging–induced
degradation of HaloTag fusion proteins. Nature Chemical Biology 2011, 7 (8), 538-543.
63. Neklesa, T. K.; Winkler, J. D.; Crews, C. M., Targeted protein degradation by PROTACs.
Pharmacology & Therapeutics 2017, 174, 138-144.
64. Patsch, K.; Liu, C.; Zapotoczny, G.; Sun, Y.; Sura, H.; Ung, N.; Sun, R. X.; Haliday, B.;
Yu, C.; Aljehani, M.; Lee, J. S. H.; Kashemirov, B. A.; Agus, D. B.; McKenna, C. E.;
Ruderman, D., Paradoxical androgen receptor regulation by small molecule enantiomers.
Proceedings of the National Academy of Sciences 2021, 118 (12), e2100918118.
65. Peng, S.; Chu, Z.; Lu, J.; Li, D.; Wang, Y.; Yang, S.; Zhang, Y., Co-expression of
chaperones from P. furiosus enhanced the soluble expression of the recombinant
hyperthermophilic α-amylase in E. coli. Cell Stress and Chaperones 2016, 21 (3), 477-484.
66. Pereira de Jésus-Tran, K.; Côté, P.-L.; Cantin, L.; Blanchet, J.; Labrie, F.; Breton, R.,
Comparison of crystal structures of human androgen receptor ligand-binding domain complexed
with various agonists reveals molecular determinants responsible for binding affinity. Protein
Science 2006, 15 (5), 987-999.
67. Ponnusamy, S.; He, Y.; Hwang, D.-J.; Thiyagarajan, T.; Houtman, R.; Bocharova, V.;
Sumpter, B. G.; Fernandez, E.; Johnson, D.; Du, Z.; Pfeffer, L. M.; Getzenberg, R. H.;
McEwan, I. J.; Miller, D. D.; Narayanan, R., Orally Bioavailable Androgen Receptor Degrader,
Potential Next-Generation Therapeutic for Enzalutamide-Resistant Prostate Cancer. Clinical
Cancer Research 2019, 25 (22), 6764-6780.
68. Rathkopf, D.; Liu, G.; Carducci, M. A.; Eisenberger, M. A.; Anand, A.; Morris, M. J.;
Slovin, S. F.; Sasaki, Y.; Takahashi, S.; Ozono, S.; Fung, N. K. E.; Cheng, S.; Gan, J.;
Gottardis, M.; Obermeier, M. T.; Reddy, J.; Zhang, S.; Vakkalagadda, B. J.; Alland, L.;
Wilding, G.; Scher, H. I., Phase I Dose-Escalation Study of the Novel Antiandrogen BMS-
641988 in Patients with Castration-Resistant Prostate Cancer. Clinical Cancer Research 2011,
17 (4), 880-887.
69. Rawla, P., Epidemiology of Prostate Cancer. World Journal of Oncology 2019, 10 (2), 63-89.
104
70. Rice, M. A.; Malhotra, S. V.; Stoyanova, T., Second-generation antiandrogens: from
discovery to standard of care in castration resistant prostate cancer. Frontiers in Oncology 2019,
9, 801.
71. Romey, G.; Quast, U.; Pauron, D.; Frelin, C.; Renaud, J.; Lazdunski, M., Na+ channels as
sites of action of the cardioactive agent DPI 201-106 with agonist and antagonist enantiomers.
Proceedings of the National Academy of Sciences 1987, 84 (3), 896-900.
72. Sack, J. S.; Kish, K. F.; Wang, C.; Attar, R. M.; Kiefer, S. E.; An, Y.; Wu, G. Y.;
Scheffler, J. E.; Salvati, M. E.; Krystek, S. R.; Weinmann, R.; Einspahr, H. M.,
Crystallographic structures of the ligand-binding domains of the androgen receptor and its
T877A mutant complexed with the natural agonist dihydrotestosterone. Proceedings of the
National Academy of Sciences 2001, 98 (9), 4904-4909.
73. Saeed, A.; Vaught, G. M.; Gavardinas, K.; Matthews, D.; Green, J. E.; Losada, P. G.;
Bullock, H. A.; Calvert, N. A.; Patel, N. J.; Sweetana, S. A.; Krishnan, V.; Henck, J. W.;
Luz, J. G.; Wang, Y.; Jadhav, P., 2-Chloro-4-[[(1R, 2R)-2-hydroxy-2-methyl-
cyclopentyl]amino]-3-methyl-benzonitrile: A Transdermal Selective Androgen Receptor
Modulator (SARM) for Muscle Atrophy. Journal of Medicinal Chemistry 2016, 59 (2), 750-755.
74. Sakkiah, S.; Kusko, R.; Pan, B.; Guo, W.; Ge, W.; Tong, W.; Hong, H., Structural changes
due to antagonist binding in ligand binding pocket of androgen receptor elucidated through
molecular dynamics simulations. Frontiers in Pharmacology 2018, 9, 492.
75. Salvati, M. E.; Balog, A.; Shan, W.; Rampulla, R.; Giese, S.; Mitt, T.; Furch, J. A.; Vite,
G. D.; Attar, R. M.; Jure-Kunkel, M.; Geng, J.; Rizzo, C. A.; Gottardis, M. M.; Krystek, S.
R.; Gougoutas, J.; Galella, M. A.; Obermeier, M.; Fura, A.; Chandrasena, G., Identification
and optimization of a novel series of [2.2.1]-oxabicyclo imide-based androgen receptor
antagonists. Bioorganic & Medicinal Chemistry Letters 2008, 18 (6), 1910-1915.
76. Salvati, M. E.; Balog, J.; Pickering, D.; Giese, S.; Fura, A.; Li, W.; Patel, R.; Hanson, R.;
Mitt, T.; Roberge, J.; Corte, J.; Spergel, S.; Rampulla, R.; Misra, R.; Xiao, H.-Y. Fused
Heterocyclic succinimide compounds and analogs thereof, modulators of nuclear hormone
receptor function. US20050272799A1, December 08, 2005.
77. Sartor, O.; Pal, S. K., Abiraterone and its place in the treatment of metastatic CRPC. Nature
Reviews Clinical Oncology 2013, 10 (1), 6-8.
78. Scher, H. I.; Fizazi, K.; Saad, F.; Taplin, M.-E.; Sternberg, C. N.; Miller, K.; De Wit, R.;
Mulders, P.; Chi, K. N.; Shore, N. D.; Armstrong, A. J.; Flaig, T. W.; Fléchon, A.;
Mainwaring, P.; Fleming, M.; Hainsworth, J. D.; Hirmand, M.; Selby, B.; Seely, L.; de Bono,
J. S., Increased survival with enzalutamide in prostate cancer after chemotherapy. New England
Journal of Medicine 2012, 367 (13), 1187-1197.
79. Scher, H. I.; Sawyers, C. L., Biology of Progressive, Castration-Resistant Prostate Cancer:
Directed Therapies Targeting the Androgen-Receptor Signaling Axis. Journal of Clinical
Oncology 2005, 23 (32), 8253-8261.
80. Schmidt, K. T.; Huitema, A. D.; Chau, C. H.; Figg, W. D., Resistance to second-generation
androgen receptor antagonists in prostate cancer. Nature Reviews Urology 2021, 1-18.
81. Schrödinger Release 2021-3; New York, NY, 2021.
82. Schweizer, M. T.; Zhou, X. C.; Wang, H.; Bassi, S.; Carducci, M. A.; Eisenberger, M. A.;
Antonarakis, E. S., The Influence of Prior Abiraterone Treatment on the Clinical Activity of
Docetaxel in Men with Metastatic Castration-resistant Prostate Cancer. European Urology 2014,
66 (4), 646-652.
105
83. Shan, W.; Balog, A.; Nation, A.; Zhu, X.; Chen, J.; Cvijic, M. E.; Geng, J.; Rizzo, C. A.;
Spires Jr, T.; Attar, R. M.; Obermeier, M.; Traeger, S.; Dai, J.; Zhang, Y.; Galella, M.;
Trainor, G.; Vite, G. D.; Gavai, A. V., [2.2.1]-Bicyclic sultams as potent androgen receptor
antagonists. Bioorganic & Medicinal Chemistry Letters 2016, 26 (23), 5707-5711.
84. Shibata, N.; Nagai, K.; Morita, Y.; Ujikawa, O.; Ohoka, N.; Hattori, T.; Koyama, R.;
Sano, O.; Imaeda, Y.; Nara, H.; Cho, N.; Naito, M., Development of Protein Degradation
Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of
Apoptosis Protein Ligands. Journal of Medicinal Chemistry 2018, 61 (2), 543-575.
85. Snyder, L. B. In Discovery of ARV-110, a first in class androgen receptor degrading
PROTAC® for the treatment of men with metastatic castration resistant prostate cancer, April
10−15; American Association for Cancer Research: 2021.
86. Sun, C.; Robl, J. A.; Wang, T. C.; Huang, Y.; Kuhns, J. E.; Lupisella, J. A.; Beehler, B.
C.; Golla, R.; Sleph, P. G.; Seethala, R.; Fura, A.; Krystek, S. R.; An, Y.; Malley, M. F.;
Sack, J. S.; Salvati, M. E.; Grover, G. J.; Ostrowski, J.; Hamann, L. G., Discovery of potent,
orally-active, and muscle-selective androgen receptor modulators based on an N-aryl-
hydroxybicyclohydantoin scaffold. Journal of Medicinal Chemistry 2006, 49 (26), 7596-7599.
87. Sun, X.; Gao, H.; Yang, Y.; He, M.; Wu, Y.; Song, Y.; Tong, Y.; Rao, Y., PROTACs:
great opportunities for academia and industry. Signal Transduction and Targeted Therapy 2019,
4 (1), 64.
88. Szklarczyk, D.; Gable, A. L.; Nastou, K. C.; Lyon, D.; Kirsch, R.; Pyysalo, S.; Doncheva,
N. T.; Legeay, M.; Fang, T.; Bork, P.; Jensen, L. J.; von Mering, C., The STRING database in
2021: customizable protein–protein networks, and functional characterization of user-uploaded
gene/measurement sets. Nucleic Acids Research 2020, 49 (D1), D605-D612.
89. Tan, M. H. E.; Li, J.; Xu, H. E.; Melcher, K.; Yong, E.-l., Androgen receptor: structure, role
in prostate cancer and drug discovery. Acta Pharmacologica Sinica 2015, 36 (1), 3-23.
90. Tomaszewski, J.; Rumore, M. M., Stereoisomeric drugs: FDA's policy statement and the
impact on drug development. Drug Development and Industrial Pharmacy 1994, 20 (2), 119-
139.
91. Unwalla, R.; Mousseau, J. J.; Fadeyi, O. O.; Choi, C.; Parris, K.; Hu, B.; Kenney, T.;
Chippari, S.; McNally, C.; Vishwanathan, K.; Kilbourne, E.; Thompson, C.; Nagpal, S.;
Wrobel, J.; Yudt, M.; Morris, C. A.; Powell, D.; Gilbert, A. M.; Chekler, E. L. P., Structure-
Based Approach To Identify 5-[4-Hydroxyphenyl]pyrrole-2-carbonitrile Derivatives as Potent
and Tissue Selective Androgen Receptor Modulators. Journal of Medicinal Chemistry 2017, 60
(14), 6451-6457.
92. Uo, T.; Plymate, S. R.; Sprenger, C. C., The potential of AR-V7 as a therapeutic target.
Expert Opinion on Therapeutic Targets 2018, 22 (3), 201-216.
93. Wang, F.; Liu, X.-q.; Li, H.; Liang, K.-n.; Miner, J. N.; Hong, M.; Kallel, E. A.; Van
Oeveren, A.; Zhi, L.; Jiang, T., Structure of the ligand-binding domain (LBD) of human
androgen receptor in complex with a selective modulator LGD2226. Acta Crystallographica
Section F: Structural Biology and Crystallization Communications 2006, 62 (11), 1067-1071.
94. Wang, G.; Dugas, M.; Armah, I. B.; Honerjäger, P., Interaction between DPI 201-106
enantiomers at the cardiac sodium channel. Molecular Pharmacology 1990, 37 (1), 17-24.
95. Wolford, J. E.; Bai, J.; Moore, K. N.; Kristeleit, R.; Monk, B. J.; Tewari, K. S., Cost-
effectiveness of niraparib, rucaparib, and olaparib for treatment of platinum-resistant, recurrent
ovarian carcinoma. Gynecologic Oncology 2020, 157 (2), 500-507.
106
96. Xiao, H.-Y.; Balog, A.; Attar, R. M.; Fairfax, D.; Fleming, L. B.; Holst, C. L.; Martin, G.
S.; Rossiter, L. M.; Chen, J.; Cvjic, M.-E.; Dell-John, J.; Geng, J.; Gottardis, M. M.; Han,
W.-C.; Nation, A.; Obermeier, M.; Rizzo, C. A.; Schweizer, L.; Spires Jr., T.; Shan, W.;
Gavai, A. V.; Salvati, M. E.; Vitea, G., Design and synthesis of 4-[3,5-dioxo-11-oxa-4,9-
diazatricyclo[5.3.1.02,6]undec-4-yl]-2-trifluoromethyl-benzonitriles as androgen receptor
antagonists. Bioorganic & Medicinal Chemistry Letters 2010, 20 (15), 4491-4495.
97. Xie, H.; Liang, J.-J.; Wang, Y.-L.; Hu, T.-X.; Wang, J.-Y.; Yang, R.-H.; Yan, J.-K.;
Zhang, Q.-R.; Xu, X.; Liu, H.-M.; Ke, Y., The design, synthesis and anti-tumor mechanism
study of new androgen receptor degrader. European Journal of Medicinal Chemistry 2020, 204,
112512.
98. Yap, T. A.; Smith, A. D.; Ferraldeschi, R.; Al-Lazikani, B.; Workman, P.; de Bono, J. S.,
Drug discovery in advanced prostate cancer: translating biology into therapy. Nature Reviews
Drug Discovery 2016, 15 (10), 699-718.
99. Yu, J.; Zhou, P.; Hu, M.; Yang, L.; Yan, G.; Xu, R.; Deng, Y.; Li, X.; Chen, Y.,
Discovery and biological evaluation of darolutamide derivatives as inhibitors and down-
regulators of wild-type AR and the mutants. European Journal of Medicinal Chemistry 2019,
182, 111608.
100. Zhou, T.; Xu, W.; Zhang, W.; Sun, Y.; Yan, H.; Gao, X.; Wang, F.; Zhou, Q.; Hou, J.;
Ren, S.; Yang, Q.; Yang, B.; Xu, C.; Zhou, Q.; Wang, M.; Chen, C.; Sun, Y., Preclinical
profile and phase I clinical trial of a novel androgen receptor antagonist GT0918 in castration-
resistant prostate cancer. European Journal of Cancer 2020, 134, 29-40.
101. Zorn, K. M.; Foil, D. H.; Lane, T. R.; Hillwalker, W.; Feifarek, D. J.; Jones, F.; Klaren,
W. D.; Brinkman, A. M.; Ekins, S., Comparison of Machine Learning Models for the Androgen
Receptor. Environmental Science & Technology 2020, 54 (21), 13690-13700.
102. Zoubeidi, A.; Zardan, A.; Beraldi, E.; Fazli, L.; Sowery, R.; Rennie, P.; Nelson, C.;
Gleave, M., Cooperative interactions between androgen receptor (AR) and heat-shock protein 27
facilitate AR transcriptional activity. Cancer Research 2007, 67 (21), 10455-10465.

107
Appendices
Appendix A: Synthetic Schemes for intermediates
Scheme A1: Synthesis of 3-acetyl-1H-pyrazole-5-carboxylic acid and 3-(1-hydroxyethyl)-
1H-pyrazole-5-carboxylic acid.
1

Reagents and conditions: (a) 3-Butyn-2-one, toluene, rt, overnight, 76% (b) 5 N NaOH, rt, 3h,
79%, used without further purification (c) NaBH4, MeOH, 0
o
C, 3h, 89%, used without further
purification.

Scheme A2: Synthesis of 3-(4-acetylpiperazin-1-yl) propanoic acid.

Reagents and conditions: (a) 3-bromopropanoic acid, MeCN, Na2CO3, 40
o
C, 5h, quant., used
without further purification.

108
Appendix B: X-ray crystal structures of BMS-631988 and its enantiomer
Table B1: BMS-641988

The crystals for BMS-641988 were prepared in a 4 mL glass vial. To a sample of BMS-641988 (around 5 mg) was
added 1 mL of hexane, dichloromethane was then added dropwise until a clear solution was formed. The cap was
loosened slightly to allow slow evaporation of the solvents and the vial was kept at room temperature for several
days until BMS-641988 was crystallized out.
A colorless prism-like specimen of C40H42F6N6O11S2, approximate dimensions 0.072 mm x 0.111 mm
x 0.378 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured on a Bruker
APEX DUO system equipped with a fine-focus tube (MoKα, λ = 0.71073 Å) and a TRIUMPH curved-crystal
monochromator. The total exposure time was 6.67 hours. The frames were integrated with the Bruker SAINT
software package using a SAINT V8.38A (Bruker AXS, 2013) algorithm. The integration of the data using
a monoclinic unit cell yielded a total of 11628 reflections to a maximum θ angle of 30.47° (0.70 Å resolution), of
which 5780 were independent (average redundancy 2.012, completeness = 91.3%, Rint = 2.68%, Rsig = 4.87%)
and 4711 (81.51%) were greater than 2σ(F2). The final cell constants
of a = 24.155(6) Å, b = 7.1483(19) Å, c = 13.157(3) Å, β = 108.634(4)°, volume = 2152.7(10) Å3, are based upon
the refinement of the XYZ-centroids of 6077 reflections above 20 σ(I) with 5.548° < 2θ < 60.26°. Data were
corrected for absorption effects using the multi-scan method (SADABS). The ratio of minimum to maximum
apparent transmission was 0.772. The calculated minimum and maximum transmission coefficients (based on
crystal size) are 0.9230and 0.9850. The structure was solved and refined using the Bruker SHELXTL Software
Package, using the space group C 1 2 1, with Z = 2 for the formula unit, C40H42F6N6O11S2. The final anisotropic
full-matrix least-squares refinement on
F2 with 323 variables converged at R1 = 4.09%, for the observed data and wR2 = 9.33% for all data. The goodness-
of-fit was 1.041. The largest peak in the final difference electron density synthesis was 0.604 e-/Å3 and the largest
hole was -0.275 e-/Å3 with an RMS deviation of 0.050 e-/Å3. On the basis of the final model, the calculated density
was 1.482 g/cm3 and F(000), 996 e-.

Chemical formula C40H42F6N6O11S2
Formula weight 960.91 g/mol
Temperature 100(2) K
Wavelength 0.71073 Å
Crystal size 0.072 x 0.111 x 0.378 mm
Crystal habit colorless prism
Crystal system monoclinic
Space group C 1 2 1
Unit cell dimensions a = 24.155(6) Å α = 90°
b = 7.1483(19) Å β = 108.634(4)°
c = 13.157(3) Å γ = 90°
Volume 2152.7(10) Å
3

Z 2
Density (calculated) 1.482 g/cm
3

Absorption coefficient 0.217 mm
-1

F(000) 996
109
Theta range for data collection 1.63 to 30.47°
Index ranges -34<=h<=34, -10<=k<=10, -18<=l<=16
Reflections collected 11628
Independent reflections 5780 [R(int) = 0.0268]
Coverage of independent reflections 91.3%
Absorption correction multi-scan
Max. and min. transmission 0.9850 and 0.9230
Structure solution technique direct methods
Structure solution program SHELXTL XT 2014/5 (Bruker AXS, 2014)
Refinement method Full-matrix least-squares on F
2

Refinement program SHELXTL XL 2018/3 (Bruker AXS, 2018)
Function minimized Σ w(Fo
2
- Fc
2
)
2

Data / restraints / parameters 5780 / 13 / 323
Goodness-of-fit on F
2
1.041
Final R indices 4711 data; I>2σ(I) R1 = 0.0409, wR2 =
0.0867
all data R1 = 0.0575, wR2 =
0.0933
Weighting scheme
w=1/[σ
2
(Fo
2
)+(0.0481P)
2
]
where P=(Fo
2
+2Fc
2
)/3
Absolute structure parameter 0.07(4)
Largest diff. peak and hole 0.604 and -0.275 eÅ
-3

R.M.S. deviation from mean 0.050 eÅ
-3

Table B2: S-BMS

The crystals for S-BMS were prepared in the same way as BMS-641988.
A clear colorless prism-like specimen of C20H21F3N3O5.5S, approximate dimensions 0.115 mm x 0.156 mm x 0.784 mm, was
used for the X-ray crystallographic analysis. The X-ray intensity data were measured on a Bruker APEX II CCD Bruker APEX
DUO system equipped with a fine-focus tube (MoKα, λ = 0.71073 Å) and a TRIUMPH curved-crystal monochromator. The
total exposure time was 7.00 hours. The frames were integrated with the Bruker SAINT software package using a SAINT
V8.38A (Bruker AXS, 2013) algorithm. The integration of the data using a monoclinic unit cell yielded a total
of 26807 reflections to a maximum θ angle of 30.53° (0.70 Å resolution), of which 6501 were independent (average
redundancy 4.124, completeness = 99.5%, Rint = 2.84%, Rsig = 2.18%) and 6126 (94.23%) were greater than 2σ(F2). The final
cell constants of a = 24.140(4) Å, b = 7.1560(11) Å, c = 13.165(2) Å, β = 108.646(2)°, volume = 2154.8(6) Å3, are based upon
the refinement of the XYZ-centroids of 9442 reflections above 20 σ(I) with 5.549° < 2θ < 60.92°. Data were corrected for
absorption effects using the multi-scan method (SADABS). The ratio of minimum to maximum apparent transmission
was 0.903. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group C 1 2 1,
with Z = 4 for the formula unit, C20H21F3N3O5.50S. The final anisotropic full-matrix least-squares refinement on
F2 with 350 variables converged at R1 = 3.05%, for the observed data and wR2 = 8.04% for all data. The goodness-of-fit
was 1.028. The largest peak in the final difference electron density synthesis was 0.770 e-/Å3 and the largest hole was -0.200 e-
/Å3 with an RMS deviation of 0.043 e-/Å3. On the basis of the final model, the calculated density was 1.481 g/cm3 and
F(000), 996 e-.
110

Chemical formula C20H21F3N3O5.50S
Formula weight 480.46 g/mol
Temperature 100(2) K
Wavelength 0.71073 Å
Crystal size 0.115 x 0.156 x 0.784 mm
Crystal habit clear colorless prism
Crystal system monoclinic
Space group C 1 2 1
Unit cell dimensions a = 24.140(4) Å α = 90°
b = 7.1560(11) Å β = 108.646(2)°
c = 13.165(2) Å γ = 90°
Volume 2154.8(6) Å
3

Z 4
Density (calculated) 1.481 g/cm
3

Absorption coefficient 0.217 mm
-1

F(000) 996
Theta range for data collection 1.63 to 30.53°
Index ranges -34<=h<=34, -10<=k<=10, -18<=l<=18
Reflections collected 26807
Independent reflections 6501 [R(int) = 0.0284]
Coverage of independent reflections 99.5%
Absorption correction multi-scan
Structure solution technique direct methods
Structure solution program SHELXT 2014/5 (Sheldrick, 2014)
Refinement method Full-matrix least-squares on F
2

Refinement program SHELXL-2018/3 (Sheldrick, 2018)
Function minimized Σ w(Fo
2
- Fc
2
)
2

Data / restraints / parameters 6501 / 2 / 350
Goodness-of-fit on F
2
1.028
Δ/σmax 0.002
Final R indices 6126 data; I>2σ(I) R1 = 0.0305, wR2 = 0.0786
all data R1 = 0.0331, wR2 = 0.0804
Weighting scheme
w=1/[σ
2
(Fo
2
)+(0.0477P)
2
+0.7089P]
where P=(Fo
2
+2Fc
2
)/3
Absolute structure parameter -0.004(18)
Largest diff. peak and hole 0.770 and -0.200 eÅ
-3

R.M.S. deviation from mean 0.043 eÅ
-3

111
Appendix C: Characterization of final compounds
Compound 05R

Figure B1:
1
H NMR (600 MHz, CDCl3) of Compound 05R.

Figure B2:
13
C NMR (151 MHz, CDCl3) of Compound 05R/S.
112

Figure B3:
19
F NMR (564 MHz, CDCl3) of Compound 05R/S.

Figure B4: Chiral HPLC trace of Compound 05R.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 50%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs01055-20180602-hs01055B.lcd
Sample Name:hs01055-20180602-hs01055B
Sample ID:hs01055-20180602-hs01055B
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 min
0
500
1000
1500
2000
2500
3000
mAU
254nm,4nm
113
Compound 05S

Figure B5:
1
H NMR (600 MHz, CDCl3) of Compound 05S.

Figure B6:
13
C NMR (151 MHz, CDCl3) of Compound 05R/S.
114

Figure B7:
19
F NMR (564 MHz, CDCl3) of Compound 05R/S.

Figure B8: Chiral HPLC trace of Compound 05S.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 50%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs01055-20180602-hs01055A.lcd
Sample Name:hs01055-20180602-hs01055A
Sample ID:hs01055-20180602-hs01055A
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 min
0
500
1000
1500
2000
2500
3000
mAU
254nm,4nm
115
Compound 06R

Figure B9:
1
H NMR (600 MHz, CD3CN) of Compound 06R.

Figure B10:
13
C NMR (151 MHz, CD3CN) of Compound 06R.
116

Figure B11:
19
F NMR (564 MHz, CD3CN) of Compound 06R.

Figure B12: Chiral HPLC trace of Compound 06R.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 50%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

117
Compound 06S

Figure B13:
1
H NMR (600 MHz, CD3CN) of Compound 06S.

Figure B14:
13
C NMR (151 MHz, CD3CN) of Compound 06S.
118

Figure B15:
19
F NMR (564 MHz, CD3CN) of Compound 06S.

Figure B16: Chiral HPLC trace of Compound 06S.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 50%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

119
Compound 13R

Figure B17:
1
H NMR (600 MHz, CD3CN) of Compound 13R.

Figure B18:
13
C NMR (151 MHz, CD3CN) of Compound 13R.
120

Figure B19:
19
F NMR (564 MHz, CD3CN) of Compound 13R.

Figure B20: Chiral HPLC trace of Compound 13R.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 60%-90%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

121
Compound 13S

Figure B21:
1
H NMR (600 MHz, CD3CN) of Compound 13S.

Figure B22:
13
C NMR (151 MHz, CD3CN) of Compound 13S.
122

Figure B23:
19
F NMR (564 MHz, CD3CN) of Compound 13S.

Figure B24: Chiral HPLC trace of Compound 13S.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 60%-90%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Dataf ile Name:hs01076B-purity analy sis1.lcd
Sample Name:hs01076B purity analy sis
0.0 5.0 10.0 15.0 20.0 25.0 min
0
1000
2000
3000
4000
5000
6000
mV
Detector A 254nm
123
Compound 14R

Figure B25:
1
H NMR (600 MHz, CDCl3) of Compound 14R.

Figure B26:
13
C NMR (151 MHz, CDCl3) of Compound 14R.
124

Figure B27:
19
F NMR (564 MHz, CDCl3) of Compound 14R.

Figure B28: Chiral HPLC trace of Compound 14R.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 60%-90%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs01077A-purity analysis1.lcd
Sample Name:hs01077A-purity analysis
0.0 5.0 10.0 15.0 20.0 25.0 min
0
1000
2000
3000
4000
mAU
254nm,4nm
125
Compound 14S

Figure B29:
1
H NMR (600 MHz, CDCl3) of Compound 14S.

Figure B30:
13
C NMR (151 MHz, CDCl3) of Compound 14S.
126

Figure B31:
19
F NMR (564 MHz, CDCl3) of Compound 14S.

Figure B32: Chiral HPLC trace of Compound 14S.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 60%-90%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs01077B-purity analysis1.lcd
Sample Name:hs01077B-purity analysis
0.0 5.0 10.0 15.0 20.0 25.0 30.0 min
0
1000
2000
3000
4000
mAU
254nm,4nm
127
Compound 15R

Figure B33:
1
H NMR (600 MHz, CD3CN) of Compound 15R.

Figure B34:
13
C NMR (151 MHz, CD3CN) of Compound 15R.
128

Figure B35:
19
F NMR (564 MHz, CD3CN) of Compound 15R.

Figure B36: Chiral HPLC trace of Compound 15R.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with isocratic
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs01079A-purity analysis-1.lcd
Sample Name:hs01079A-purity analysis
Sample ID:hs01079A-purity analysis
0.0 5.0 10.0 15.0 20.0 25.0 min
0
500
1000
1500
mAU
254nm,4nm
129
Compound 15S

Figure B37:
1
H NMR (600 MHz, CD3CN) of Compound 15S.

Figure B38:
13
C NMR (151 MHz, CD3CN) of Compound 15S.
130

Figure B39:
19
F NMR (564 MHz, CD3CN) of Compound 15S.

Figure B40: Chiral HPLC trace of Compound 15S.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with isocratic
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs01079B-purity analysis-1.lcd
Sample Name:hs01079B-purity analysis
Sample ID:hs01079B-purity analysis
0.0 5.0 10.0 15.0 20.0 25.0 min
0
500
1000
1500
mAU
254nm,4nm
131
Compound 16R

Figure B41:
1
H NMR (600 MHz, CD3CN) of Compound 16R.

Figure B42:
13
C NMR (151 MHz, CD3CN) of Compound 16R.
132

Figure B43:
19
F NMR (564 MHz, CD3CN) of Compound 16R.

Figure B44: Chiral HPLC trace of Compound 16R.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 35%-55%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

133
Compound 16S

Figure B45:
1
H NMR (600 MHz, CD3CN) of Compound 16S.

Figure B46:
13
C NMR (151 MHz, CD3CN) of Compound 16S.
134

Figure B47:
19
F NMR (564 MHz, CD3CN) of Compound 16S.

Figure B48: Chiral HPLC trace of Compound 16S.
Chiral HPLC conditions: Chiralcel OD-H column (250 × 4.6 mm, 5 µm) eluting with 35%-55%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs01091B-purity analysis1.lcd
Sample Name:hs01091B-purity analysis
Sample ID:hs01091B-purity analysis
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min
0
250
500
750
1000
mAU
0.0
25.0
50.0
75.0
%
254nm,4nm
135
Compound 17R

Figure B49:
1
H NMR (600 MHz, CD3CN) of Compound 17R.

Figure B50:
13
C NMR (151 MHz, CD3CN) of Compound 17R.
136

Figure B51:
19
F NMR (564 MHz, CD3CN) of Compound 17R.

Figure B52: Chiral HPLC trace of Compound 17R.
Chiral HPLC conditions: Regis Whelk-O column (250 × 4.6 mm, 5 µm) eluting with 60%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs02023A-purity-1.lcd
Sample Name:hs02023A-purity
Sample ID:hs02023A-purity
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
2750
mV
Detector A 254nm
137
Compound 17S

Figure B53:
1
H NMR (600 MHz, CD3CN) of Compound 17S.

Figure B54:
13
C NMR (151 MHz, CD3CN) of Compound 17S.
138

Figure B55:
19
F NMR (564 MHz, CD3CN) of Compound 17S.

Figure B56: Chiral HPLC trace of Compound 17S.
Chiral HPLC conditions: Regis Whelk-O column (250 × 4.6 mm, 5 µm) eluting with 60%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs02023B-purity-1.lcd
Sample Name:hs02023B-purity
Sample ID:hs02023B-purity
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
mV
Detector A 254nm
139
Compound 18R

Figure B57:
1
H NMR (600 MHz, CD3CN) of Compound 18R.

140
Figure B58:
13
C NMR (151 MHz, CD3CN) of Compound 18R.
\
Figure B59:
19
F NMR (564 MHz, CD3CN) of Compound 18R.

Figure B60: Chiral HPLC trace of Compound 18R.
Chiral HPLC conditions: Regis C-Amylose column (250 × 4.6 mm, 5 µm) eluting with 25%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:CL51A-purity-1.lcd
Sample Name:CL51A-purity
Sample ID:CL51A-purity
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
mV
Detector A 254nm
141
Compound 18S

Figure B61:
1
H NMR (600 MHz, CD3CN) of Compound 18S.

142
Figure B62:
13
C NMR (151 MHz, CD3CN) of Compound 18S.

Figure B63:
19
F NMR (564 MHz, CD3CN) of Compound 18S.

Figure B64: Chiral HPLC trace of Compound 18S.
Chiral HPLC conditions: Regis C-Amylose column (250 × 4.6 mm, 5 µm) eluting with 25%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:CL51B-purity-1.lcd
Sample Name:CL51B-purity
Sample ID:CL51B-purity
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
mV
Detector A 254nm
143
Compound 19R

Figure B65:
1
H NMR (600 MHz, CD3CN) of Compound 19R.

Figure B66:
13
C NMR (151 MHz, CD3CN) of Compound 19R.
144

Figure B67:
19
F NMR (564 MHz, CD3CN) of Compound 19R.

Figure B68: Chiral HPLC trace of Compound 19R.
Chiral HPLC conditions: Regis C-Amylose column (250 × 4.6 mm, 5 µm) eluting with 25%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:CL60A--purity-1.lcd
Sample Name:CL60A-purity
Sample ID:CL60A-purity
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
0
1000
2000
3000
4000
5000
6000
7000
mV
Detector A 254nm
145
Compound 19S

Figure B69:
1
H NMR (600 MHz, CD3CN) of Compound 19S.

Figure B70:
13
C NMR (151 MHz, CD3CN) of Compound 19S.
146

Figure B71:
19
F NMR (564 MHz, CD3CN) of Compound 19S.

Figure B72: Chiral HPLC trace of Compound 19S.
Chiral HPLC conditions: Regis C-Amylose column (250 × 4.6 mm, 5 µm) eluting with 25%
isopropanol in hexane; flow rate, 1 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:CL60B-purity-2.lcd
Sample Name:CL60B-purity
Sample ID:CL60B-purity
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
0
1000
2000
3000
4000
5000
6000
7000
mV
Detector A 254nm
147
Compound 20
Racemic mixture of

Figure B73:
1
H NMR (600 MHz, CD3CN) of Compound 20.

148

Figure B74:
13
C NMR (151 MHz, CD3CN) of Compound 20.

Figure B75:
19
F NMR (564 MHz, CD3CN) of Compound 20.

149

Figure B72: HPLC trace of Compound 20.
HPLC conditions: Luna C18 (250 × 10 mm, 5 µm) eluting with 50% acetonitrile in water; flow
rate, 4 mL/min; wavelength, UV 254 nm; room temperature.

Datafile Name:hs02019-purity1.lcd
Sample Name:hs02019-purity
Sample ID:hs02019-purity
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
mV
Detector A 254nm
150
Compound 21
Racemic mixture of

Figure B77:
1
H NMR (600 MHz, CD3CN) of Compound 21.

151

Figure B78:
13
C NMR (151 MHz, CD3CN) of Compound 21.

Figure B79:
19
F NMR (564 MHz, CD3CN) of Compound 21.

152

Figure B80: HPLC trace of Compound 21.
HPLC conditions: Luna C18 (250 × 10 mm, 5 µm) eluting with 50% acetonitrile in water; flow
rate, 4 mL/min; wavelength, UV 254 nm; room temperature.

Appendix references:
1. Lai, K. W.; Liang, J.; Zhang, B.; Labadie, S.; Ortwine, D.; Dragovich, P.; Kiefer, J.;
Gehling, V. S.; Harmange, J.-C. Pyrrolidine Amide Compounds as Histone Demethylase
Inhibitors. WO 2016/057924 Al.

Datafile Name:hs02039-purity2.lcd
Sample Name:hs0203-purity
Sample ID:hs02039-purity
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
mV
Detector A 254nm

Abstract (if available)

Abstract Prostate cancer accounts for around 13% of all male cancer diagnoses and 7% of all cancer diagnoses worldwide. The androgen receptor (AR) is the master regulator in the androgen signaling pathway, and it is a critical factor in all stages of prostate cancer including castration resistant prostate cancer (CRPC). Second generation antiandrogen drugs such as enzalutamide exhibit limited efficacy profiles due to drug resistance and unfavorable safety profiles due to neurotoxicity. Thus, there is an urgent clinical need for novel antiandrogens that can overcome drug resistance. In a medicinal chemistry campaign to develop novel antiandrogens, we addressed these issues by modifying BMS-641988, a known AR antagonist. We discovered that the enantiomer of BMS-641988 is a potent AR agonist, and this chirality-dependent agonist-antagonist pairing is present consistently in a series of AR ligands based on the BMS-641988 scaffold. Additionally, we conducted extensive structure-activity relationship (SAR) studies and identified several promising lead compounds that exhibited excellent in vitro safety and efficacy profiles. Furthermore, we developed novel selective AR degraders (SARDs) based on our compounds. Our research provides new insights into the molecular dynamics of AR and a cautionary tale in drug discovery. Our compounds could lead to new treatments for drug resistant prostate cancer and other AR-related diseases.

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Asset Metadata

Creator Sun, Yuanye (author)

Core Title Chemical and biological studies of novel ligands of the human androgen receptor

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Degree Conferral Date 2022-05

Publication Date 02/03/2024

Defense Date 01/14/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag androgen receptor,antiandrogens,chirality,drug discovery,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles (committee chair), Cherezov, Vadim (committee member), Lee, Jerry (committee member)

Creator Email hammondsun95@gmail.com,yuanye.sun@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC110618320

Unique identifier UC110618320

Legacy Identifier etd-SunYuanye-10376

Document Type Dissertation

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Type texts

Source 20220207-usctheses-batch-911 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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