development of novel anti-estrogens for endocrine ... · development of novel anti-estrogens for...

Development of Novel Anti-Estrogens for Endocrine Resistant Breast Cancer

Sarika Rajalekshmi Devi

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in

partial fulfillment of the requirements for the degree of

Master of Science

In

Chemistry

Jatinder Josan, Chair

Webster Santos

David Kingston

December 8, 2015

Blacksburg, Virginia

Keywords: Estrogen receptor breast cancer, Antiestrogens, Anti-inflammatory effects

Development of Novel Anti-Estrogens for Endocrine Resistant Breast Cancer

Sarika Rajalekshmi Devi

ABSTRACT

ER+ breast cancer raises a significant diagnostic challenge since resistance invariably

develops to the current endocrine therapies. 70% of breast cancers are ER+, which results from

the overexpression of estrogen receptor. ER mediates strong anti-inflammatory signaling in ER+

tissues. Once activated with estradiol (E2), ER inhibits inflammatory gene expression via

protein-protein interactions that block NF-κB transcriptional activity. Importantly, NF-κB is a

primary mediator of resistance in many cancers, including breast cancer. All current endocrine

suppressive treatments block this palliative signaling pathway, along with the desired

proliferative pathway. Thus, there is a significant unmet clinical need for novel endocrine

treatments for breast cancer that can ameliorate patient outcome in resistant populations, be less

prone to resistance development, retain anti-inflammatory action, and cause fewer side effects.

Following the hypothesis driven approach, the work described here introduces structural

analogs of an innovative ligand scaffold, 5,6-bis-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-

ene-2-sulfonic acid phenyl ester, termed OBHS, which reduces gene activation through ligand-

induced shifts in helices 8 and 11, thereby indirectly modulating helix 12 of ER (hence, indirect

antagonists). This new class of ligands with a bicyclic hydrophobic core retains strong anti-

inflammatory effects while dialing out the proliferative effects of E2 (similar to Selective

Estrogen Receptor Modulators, SERMS), and could potentially replace the current endocrine

therapies of breast cancer. In this work, we carried out rational design and syntheses of two

series of OBHS analogs, namely OBHS-A (for acetamido derivatives), and OBHS-P (for

iii

propargyl derivatives), while we explored a synthetic methodology for a third series of OBHS

compounds.

Many analogs from the OBHS-A series exhibited high binding affinity. For example, the

exo diastereomer of 2.11a, 2.11b, 2.11c, 2.11d, and 2.11e exhibited Relative Binding Affinities

(RBAs) of 22.6%, 10.5%, 19.5%, 12.1%, and 14.4%, respectively. As observed before, endo

OBHS compounds exhibited lower binding affinities than exo compounds. The RBA values with

acetamide, and isobutyramide (i.e. short hydrophobic chains) were very comparable to each

other. However, unexpectedly the propionamide compound showed lower binding affinity than

butyramide. Nevertheless, we consider OBHS analogs with RBA values greater than 1% (Kd =

20 nM) to be very potent. This data is only the first step in a battery of assays that will be

conducted eventually on these compounds. In particular, our emphasis is in ascertaining and

improving the NF-κB mediated anti-inflammatory property, where these compounds have shown

promising activity in conjunction with their anti-proliferative activity.

iv

Dedicated to

I dedicate this thesis to three beautiful females in my life.

My Mother, Rajalekshmi Devi

Mother-in-law, Vilasini Amma

My little sunshine, Vedha Nair

v

Acknowledgement

Thank you Lord for being there with me always!!!

First and foremost, I would like to express my sincere gratitude to my advisor Prof.

Jatinder Josan for the continuous guidance and support for my research. His guidance helped me

a lot in my research and writing this thesis. I would like to thank the rest of my thesis committee:

Prof. David Kingston, Prof. Webster Santos, and Dr. Felicia Etzkorn, for their encouragement

and valuable suggestion. I thank my fellow labmates in Virginia Tech: Jasmine, Kiaya, Alex,

Phil, Jose, Ashley, Nathan, David, Brandon, Divya, and Aman for the immense support, and for

all the fun we have had. Thank you to the entire Chemistry Department at Virginia Tech, all the

faculty members and staff that I took classes from and worked with.

I am grateful to the collaborators of this project, Professor John Katzenellenbogen, UIUC

and, Professor Kendall Nettles of Scripps Research Institute, Florida. The biological evaluations

in this thesis would not have been possible without the help of Kathy Carlson and Teresa Martin

from the University of Illinois, Urbana-Champaign (UIUC) and Sathish Srinivasan from The

Scripps Research Institute, Florida.

I would like to thank my family for their immense support. Thanks to my Dad, Achuthan

Pillai, Mom, Rajalekshmi Devi and my elder brother Pramod who let my ambitions grow as

much as I wished. A special thanks to my Father-in law Bahuleyan Nair and Mother in law,

Vilasini Amma for their unconditional love and support.

My husband Mahesh, brothers, sister-in-laws, brother-in-laws, nephews and nieces

deserve my thanks as well. Last but not least, I want to thank my little daughter Veda, who

missed valuable time with her Mom through her first year.

vi

Table of Contents

Chapter 1: Estrogen receptor positive (ER+) breast cancer and status of current

endocrine therapies

1.1 Estrogen receptor and breast cancer………………………………………………….1

1.2 Structure and molecular mechanism of ER…………………………………………...2

1.3 Current endocrine therapies…………………………………………………………...3

1.4 Anti-estrogens and anti- inflammation………………………………………………..5

Chapter 2: Synthesis and biological study of OBHS structural analogs

2.1 Introduction: Discovery of a novel bicyclic SERM: OBHS…………………………8

2.2 Hypothesis…………………………………………………………………………....9

2.3 Structural analogs of OBHS with anti-inflammatory properties…………………….11

2.4 Synthesis of para-iodophenyl sulfonate Analog of OBHS (OBHS-I)……………….13

2.5 Synthesis of a library of propargyl analogs of OBHS (OBHS-P) compounds ……...15

2.6 Synthesis of a library of acetamido analogs of OBHS (OBHS-A) compound………15

2.7 Biological evaluation of OBHS analogues……………………………………..........19

2.8 Experimental section…………………………………………………………………28

Chapter 3: Design and synthetic methodology for CBHS

3.1 Introduction…………………………………………………………………………..52

3.2 Synthesis of CBHS lead..…………………………………………………………….53

3.3 Experimental section…………………………………………………………………58

Chapter 4: Conclusion and future work

4.1 CBHS series………………………………………………………………………….64

4.2 NBHS series………………………………………………………………………….65

References ……………………………………………………………………………………....67

Appendix………………………………………………………………………………………...74

vii

Abbreviations

AF, Activation Function

BR, Burgess Reagent

CBHS, 7-carboxy OBHS

DBD, DNA Binding Domain

DIBAL-H, Diisobutylaluminumhydride

DMAD, Dimethyl acetylenedicarboxylate

E1, Estrone

E2, 17β-Estradiol

ER, Estrogen Receptor

ERE, Estrogen Response Elements

IL-6, Interleukin-6

LA, Lewis Acid

LBD, Ligand Binding Domain

LR-MS, Low Resolution Mass Spectrometry

NBHS, 7-aza OBHS

NF-κB, Nuclear Factor kappa B

OBHS, 5,6-bis-(4-hydroxy-phenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester

OBHS-A, OBHS-Acetamido

OBHS-P, OBHS-Propargyl

PPTS, Pyridinium para-Toluene Sulfonic Acid

PTSA, Para-Toluene Sulfonic Acid

RAL, Raloxifene

RBA, Relative Binding Affinity

SERD, Selective Estrogen Receptor Down-regulator

SERM, Selective Estrogen Receptor Modulator

TAM, Tamoxifen

TNFα, Tumor Necrosis Factor α

TR-FRET, Time Resolved Fluorescence Resonance Energy Transfer

1

CHAPTER 1

ESTROGEN RECEPTOR POSITIVE (ER+) BREAST CANCER AND STATUS OF

CURRENT ENDOCRINE THERAPIES

1.1 Estrogen Receptor (ER) and Breast Cancer

Breast cancer is the second most common cancer in women after skin cancer.1 It can occur in

both men and women, but it is very rare in men. It affects one in eight women during their lives.

Breast cancer occurs when a cancerous tumor originates in the breast, which spreads to other

parts of the body in advanced stages. Approximately 70% of breast cancers are estrogen receptor

(ER) positive which means cancer cells grow in response to estrogen hormone.1

Estrogens are steroidal compounds that primarily function as female sex hormones. The three

hormones that make the family of estrogen are estrone, estradiol, and estriol, in which estradiol is

the most potent form. Estradiol (estra-1,3,5(10)-triene-3,17β-diol), also called E2, (Fig 1.1) is

secreted mainly by the ovaries and in small amounts by the adrenal cortex,1 and plays an

important role in the regulation of bone mass and strength.2

The pharmacological action of

estradiol is mediated by estrogen receptors. Estrogen receptors are nuclear receptor proteins that

dimerize upon binding with estrogens, such as E2. The activated ER-E2 complex translocates to

the nucleus and binds to estrogen response elements (ERE) in DNA controlling the gene

function.3-4

This allows for the recruitment of coactivators and transcriptional machinery leading

to mRNA production and protein synthesis. There are two subclasses of ER, ERα and ERβ,

which have different tissue distribution and differences in their ligand binding preferences.5 ERα

is mainly found in breast, endometrium, and ovarian stromal cells. ERβ is found in kidney, brain,

bone, and heart cells.

2

Figure 1.1 A) Estrone B) 17β-estradiol, E2. C) 2D model of ER-E2 which shows the

interactions of estradiol with Glu and Arg

1.3 Structure and Molecular Mechanism of ER

Both ERα and ERβ have six functional regions from the N-terminus to the C-terminus,

labeled as A-F (Fig. 1.2). The N-terminus A/B domain (NTD) is involved in intramolecular and

intermolecular protein-protein interactions, and encodes hormone-independent transcriptional

activation function (AF-1). The AF-1 domain has the least homology between ER subtypes. The

primary function of this domain is to modulate the phosphorylation and transcriptional function.

Domain C corresponds to the DNA-binding domain (DBD), which allows ER to dimerize and

bind to specific ERE sequences on DNA. It has 97% homology between the ER subtypes.

Region D, the hinge region, separates the DBD and the ligand-binding domain (LBD). The LBD,

region E has 53% homology between ER subtypes, which is responsible for the selectivity of ER

ligands. Regions E/F, transcriptional activation function domain (AF-2), are ligand dependent,

and responsible for the activation of gene transcription in response to agonists.5 Both ERα and

ERβ have high affinity to E2, though ERα has slightly higher affinity than ERβ.9-12

3

Fig. 1.2 Functional domains of ERα and ERβ.

1.4 Current Endocrine Therapies

1.4.1. Anti-Estrogens and Selective Estrogen Receptor Modulators (SERMs)

Initially, the hormone replacement therapy (HRT), estrogen plus progestin, was used to

alleviate menopausal symptoms and prevent osteoporosis.6, 11

It was found that the long-term use

of HRT increased the risk of breast cancer, due to an increased risk of cellular proliferation by

estrogen. This led to the development of antiestrogen drugs to block the action of estrogen and

prevent breast and uterine cancer. Anti-estrogens work mechanistically by competitive inhibition

of the estradiol-binding site, thus preventing gene transcription. Coactivators cannot bind to pure

antiestrogen-bound receptor, so no gene activation occurs.10

Although these molecules do

prevent the induction of cellular proliferation, thereby reducing the risk of cancer, they also

diminish the health benefits of estrogens such as maintenance of optimal bone density,

cytoprotective and cardio-protective activity, anti-inflammatory activity, and neuroprotection in

women. Tamoxifen, initially proposed as a method of contraception, was the first antiestrogen

4

approved by the FDA. However, Tamoxifen was later proven to have estrogenic effects in the

uterus while having antiestrogenic effects in the breast.7,12

This observation gave rise to the term

Selective Estrogen Receptor Modulators (SERMS), which defines the diverse functions ER

ligands have in different tissues, i.e. acting as agonists, partial agonists, or antagonists depending

upon the tissue type (Fig. 1.3).9,14

SERMs act by selectively modulating the receptor in a specific tissue by either activating

or suppressing its molecular function. Tamoxifen (TAM, Fig. 1.3A) acts as an antagonist in the

breast cancer cells but has agonistic properties in tissues such as bone, uterus, liver, and

cardiovascular system. Although the agonist effects of Tamoxifen in bone tissues supports bone

maintenance, its estrogenic effects in the endometrium increases the risk of endometrial cancer.

The SERM, Raloxifene (RAL, Fig. 1.3B) acts as an agonist in bone tissues and as an antagonist

in breast and uterine tissues. Further, unlike Tamoxifen, it inhibits the growth of endometrial

cancer.7 However, both Tamoxifen and Raloxifene increase the incidence of hot flashes, strokes,

and deep vein thrombosis (DVT) - a problem that seems to be inherent to all ER based therapies,

although at varying levels.11

Fig. 1.3 SERMs A) Tamoxifen B) Raloxifene (BSC: Basic Side Chain)

5

1.5 Anti-Estrogens and Inflammation

Approximately two-thirds of breast cancers are Estrogen Receptor positive (ER+).

However, only about 50% of these cases benefit from the endocrine therapies. Further, many

cases that do respond initially, relapse eventually, and thus, become resistant to front-line

therapies targeting the ER.30,14

In addition to driving proliferation in the breast and uterus, the ER

protein mediates strong anti-inflammatory signaling in ER+ tissues. The activated ER inhibits

inflammatory gene expression via protein-protein interactions that block the NF-κB

transcriptional activity. Nuclear Factor kappa B (NF-κB) is a primary mediator of resistance in

many cancers, including breast cancer, and regulates anti-apoptotic genes, and genes related to

proliferation, invasion and angiogenesis. All current endocrine suppressive treatments, however,

block this palliative signaling pathway, along with the desired proliferative pathway. Resistance

and toxicity in response to therapy is the primary cause of endocrine therapy failure today.17

In

breast cancer, NF-κB signaling is active in both ER+ and ER- breast cancer cells and

corresponds to progression of hormone-independent phenotype.

Interleukin 6 (IL-6) is a major pro-inflammatory cytokine that may contribute to the link

between inflammation and cancer. IL-6 stimulates NF-κB, which results in increase in

inflammatory activity, promoting continued tumorigenesis. In ER+ breast carcinoma, NF-κB was

reported to be a marker of high-risk subclass of tumors.11

Most genes that are targeted by IL-6

are involved in cell cycle progression and suppression of apoptosis. By influencing anti-

apoptotic pathways, IL-6 contributes to survival of DNA-damaged cells. Studies have shown that

there is a correlation between IL-6 serum levels and prognosis of metastatic breast cancer. IL-6

expression in malignant breast tissue was found to be higher than in non-malignant tissues.

Tumor necrosis factor α (TNFα) is a serum protein that has the ability to induce necrosis of

6

tumors. Exposure to low dose, chronic TNFα production is related to tumor promotion. High

TNFα levels have been found in the blood of cancer patients with various tumors, including

breast cancer patients.11

In vitro studies have shown that TNFα promotes breast cancer cell

proliferation and enhances estrogen-induced cell proliferation. The NF-κB pathway has been

found to be critical for TNFα induced tumor growth and progression by several mechanisms.18

All current endocrine treatments, including Tamoxifen and Raloxifene, block the desirable anti-

inflammatory pathway besides blocking the proliferative pathway. Thus, our research focus in

this project has been to develop a novel class of anti-estrogens that block the proliferative

pathway, but dials in the beneficial anti-inflammatory activity. Through ligand class analysis

(structural analysis with high throughput x-ray crystallography), a novel binding hotspot was

identified, that dials out the gene activation/proliferative signaling of ER ligands, while retaining

the strong anti-inflammatory properties of estradiol (E2).

Fig. 1.4. 7-oxa-bicyclo[2.2.1]heptene sulfonate (OBHS)

The Katzenellenebogen group at UIUC reported in 2005 a unique 3D-topological ligand

scaffold, termed OBHS (5,6-bis-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic

acid phenyl ester) as an anti-estrogen. Later, they identified this scaffold as a prototypical

member of a class of antiestrogens that bound to ERα preferentially, reduced gene activation

through ligand-induced shifts in helix 11, and induced modest anti-inflammatory activity (Fig.

1.5). It also reduced tumorigenic effects that arise from macrophage infiltration of tumors and

caused marked regression of both endocrine-sensitive and highly aggressive tamoxifen-resistant

7

tumors.22

Notably though, this ligand possesses anti-inflammatory activity as measured by

reduction in IL-6 secretion. From ligand class analysis, we discovered that this prototypical

ligand perturbs helix 12 indirectly by modulating the helix 8/11 first (Fig. 1.5). OBHS causes a

narrow displacement of His 524 in helix 11, which leads to slight unwinding of helix 11 end, and

perturbing the recruitment of helix 12 to define the AF-2 pocket. This prototypical OBHS forms

the basis of further structure based drug design, as will be discussed next.

Fig. 1.5. Three binding modes for ER ligands. SERMs, helix8/11such a RAL or TAM show a

bulky group exiting the pocket between helices 3 and 11 and occluding the binding of helix 12 to

form the AF2 pocket where coactivator proteins bind. The OBHS phenyl group modulates helix

8/11, which correlates to anti-inflammatory activity. (credit: Dr. Kendal Nettles)

8

CHAPTER 2

SYNTHESIS & BIOLOGICAL EVALUATION OF OBHS STRUCTURAL ANALOGS

2.1 Introduction: Discovery of a Novel Bicyclic SERM, OBHS

Traditional SERMs or nonsteroidal antiestrogens typically have planar structures, defined

by fused or stilbene type aromatic scaffolds. The studies of ligand-binding pocket of ER reveals

that there is enough unoccupied space above and below the plane of E2.14

This led to the idea

that a three dimensional central hydrophobic core structure can fill the unoccupied space in ER.

The binding pocket of ERα has a probe-accessible volume of ca. 450 Å3, whereas E2 has a

molecular volume of only 245 Å3. Though smaller than ERα, the ligand-binding pocket of ERβ

is also larger than E2. Previous studies have shown that furan core ligands have high binding

affinity and ERα subtype selectivity.17

With this information, Zhou et al. reported a new class of

ER ligands containing a 7-oxabicyclo[2.2.1]hept-5-ene core.37

It was found that a triaryl furan

has the highest binding affinity and ER subtype selectivity compared to other structural

variations. The structure-activity relationship (SAR) studies on this prototype showed that the

compound with a phenyl sulfonate group in the C-2 position of the bicyclic core had the highest

binding affinity (Fig. 1.4). The one with the highest binding affinity of all compounds tested was

exo-5,6-bis-(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid phenyl ester

(OBHS) (Fig. 1.4).20,37

A chemical feature common in nearly all synthetic ER ligands having

good binding affinity is the presence of the phenol that mimics the steroidal “A ring” present in

Note: The binding affinity assays were done by Dr. Teresa Martin and Ms. Kathy Carlson

in the research group of Prof. John A. Katzenellenbogen at the University of Illinois,

Urbana Champaign (UIUC). The cell studies were conducted by Dr. Sathish Srinivasan in

the research group of Prof. Kendall Nettles, at The Scripps Research Institute (TSRI),

Florida.

9

natural estrogens (Fig. 1.1). This phenol forms important hydrogen bonding interactions with

Glu 353 and Arg 394 residues in the ERα ligand-binding pocket (Figs. 1.5 and 2.1).

The OBHS lead was found to be the most effective estrogen antagonist studied in this

series of 3D shaped ER ligands (as opposed to planar antiestrogens).36

Assays were conducted on

human endometrial cancer cells (HEC-1) and showed that OBHS is an antagonist on both ER

subtypes.17

Thus, OBHS represented a novel structural class of ER antagonists by lacking the

canonical extended polar or basic side chains (BSCs) commonly found in ER antagonists, and by

introducing a third functionality that extends towards helices 8/11 (+). OBHS has a binding

affinity preference for ERα, but an efficacy preference for ERβ at maximal dose (the cause of

this is not entirely clear). However, the potency of OBHS remains higher on ERα in correlation

with its binding affinity profile. The relative binding affinity (RBA) of OBHS is 9.3% and 1.7%

for ERα and ERβ, respectively. (Note that these values have been revised recently when assays

were conducted with HPLC-purified OBHS fractions; vide infra).

2.2 Hypothesis

The mechanism of SERMs is to directly reposition helix 12 (Fig 1.5), whereas OBHS

compounds indirectly modulate helix 12 by regulating their docking onto helix 11, leading us to

call them indirect antagonists. Thus, we can independently dial in the inhibition of NF-κB

(typical for agonists) yet retain the antiproliferative properties (typical for SERMs) through two

distinct structural features, by distorting helix-11 and displacing helix-12. We hypothesize that

the OBHS-based ER ligands are effective for their activity in blocking ER+ breast cancers by

maintaining the anti-inflammatory effect of ER agonists that is lost in SERMs and full

antagonists, while also maintaining the antiproliferative activity of these compounds. Thus, ER

antagonists will prove effective in treating endocrine-resistant breast cancers (ERBCs).

10

In the X-ray crystallographic studies in collaboration with Prof. Kendall Nettles at Scripps,

Florida, “ligand-class analysis” was conducted by generating dozens of x-ray crystal structures in

a high-throughput mode, and the binding of ligands to ERα was studied in order to reveal the

common motifs that are responsible for anti-inflammatory activity.15

It was found that the

orientation of the phenyl sulfonate group in OBHS, and a phenyl ring from a different series -

cyclofenil compounds, were oriented along helix 8/11, and it was this orientation in ER ligands

that consistently resulted in ER-mediated anti-inflammatory activity. Further, this distortion

opened up a narrow channel along this axis leading to a wider surface-exposed sub-pocket close

to the ER dimer interface (Fig. 2.1). This subpocket is lined with both hydrophobic and

hydrophilic residues (Fig. 2.1B).

Fig. 2.1. OBHS bound ERα (A) The phenyl ring and the phenol ring B points towards helices 11

and 12, respectively. (B) Due to the slight displacement of helix 11, a narrow channel forms

towards the para position of bulky phenyl side chain. Down arrow points to the placement of

basic side chains (BSCs) seen in contemporary SERMs, which occludes the binding the helix 12

(also see Fig. 1.3). (credit: Dr. Josan)

Previously, Dr. Josan and coworkers conjectured that the para position of the phenyl

sulfonate group in OBHS could be modified with small groups that could exit this narrow

11

channel, and thus modulate the binding affinity and anti-inflammatory activity. They settled on a

propargyl group, and an acetamido group for initial testing of OBHS analogs. Both of these

ligands retained significant binding affinity, albeit somewhat lower than OBHS [RBA (Relative

Binding Affinity) for propargyl alcohol group is 2%, and that for acetamido is 3.6%; see section

2.7 for details on RBA measurement]. We rationalized that even though there was a slight

reduction in their binding affinity, perhaps due to larger distortion of the very narrow channel,

we could recover this affinity loss by incorporating hydrophobic or hydrophilic groups on the

propargyl and acetamido chains that could bind favorably to the surface exposed sub-pocket.

However, our most important aim in this context was to test the effect on anti-inflammatory

activity mediated by these ligands. If proven and successful in completely abolishing NF-κB

mediated inflammatory activity, these ER ligands would be a novel class of antiestrogens with

both antiproliferative and anti-inflammatory activity, which could be utilized in multiple ER-

mediated diseases, including ER+ breast cancer. Further, such a series of ligands would be an

innovative example of developing drugs that inhibit or address multiple hallmarks of cancer,

which likely could lead to more effective treatments of cancer. With this in mind, we planned to

develop a library of OBHS derivatives with alkyne and amide substitutions on the phenyl

sulfonate group to exploit this new binding hotspot, as discussed further.

2.3 Structural Analogs of OBHS with Anti-Inflammatory Properties.

OBHS is a new class of estrogen receptor ligands that was designed to have

the desired three dimensional topology to fit inside the wide ER ligand binding pocket.13,20

The

structural studies showed that the new hydrophobic bicyclic structure could fill the extra space in

the ER binding pocket. Thus, the OBHS compound with a bulky phenyl sulfonate group in the 2

position showed the highest binding affinity among all the compounds studied. The new class of

12

compounds reduced gene activation through ligand-induced shifts in helix 11 (Fig. 2.2A&B).20

As mentioned earlier, while SERMs directly reposition helix 12 (Fig. 2.2B), OBHS compounds

indirectly modulate helix 12 by regulating its docking onto helix 11 (indirect antagonism).21-24

Our aim was to design compounds targeting the OBHS binding site between helices 8

and 11, as well as to increase the binding affinity through potential hydrophobic or H-bonding

interactions with the residues lining the narrow channel and the exposed hotspot (Fig. 2.2A). An

alkyne substitution at the para position of the phenyl sulfonate group could allow this

modification to exit out of the pocket, or more appropriately allow the receptor to fold around

this modification without incurring heavy steric penalty (Fig. 2.2 B). An acetamido group could

participate in H-bonding with residues or backbone atoms lining the narrow channel.

Fig. 2.2 Design of OBHS analogs. (A) X-ray crystal structure of OBHS bound to ERα. (B)

OBHS with a propargyl side chain in two predominant poses – one in which the phenyl group

aligns with the crystal structure of OBHS (green) to H-bond with E339 (blue), and another where

the phenyl sulfonate group rotates to form a H-bond with S527 (purple) (Credit: Dr. Josan).

narrow channel

SERM side chain directionhelix 12

helix 11

helix 8

solventexposed

R394

E353

OS

ON

OO

O

HO

BrOBHS-2010-26

helix 11

helix 8helix 3

A B

A B

13

Fig. 2.3 left to right OBHS-A Series and OBHS-P Series

2.4 Synthesis of para-Iodophenyl Sulfonate Analog of OBHS (OBHS-I)

Following the procedure of Zhou et al.20,24

with some modifications, OBHS-I was

synthesized (Scheme 2.1). Methoxyphenylacetic acid was alkylated with phenacyl bromide

followed by cyclization with 4 eq. of the base DBU in MeCN solvent, to yield the lactone 2.1 in

a single step in 84.5% yield. The lactone was demethylated using pyridinium HCl to give

product 2.2 in quantitative yield. The lactone was reduced to furan 2.3 by initially reacting the

lactone with excess of DIBAL-H (typically 5-6 eq.) for 4-6 hours at -78 C, followed by reverse

quench in MeOH also at -78 C, and treatment with 10% H2SO4 for 1 hour. Although the yield

for the DIBAL-H reduction step was improved by performing a reverse quench in methanol, the

scale-up of this reaction was tedious due to the need to consistently maintain -78 C for longer

than 6 hours (typically 8-10 hours), and due to the large excess of MeOH needed for the reverse

quench at -78 ͦ C. Therefore, several small 1g-scale reactions were more effective in improving

the yield. The dienophile sulfonate was synthesized by a substitution elimination reaction of 2-

chloroethane sulfonyl chloride with iodophenol in the presence of a base and diethyl ether as

solvent, which yielded the product in 98% yield.23

The last step in the synthesis of OBHS-I

compound was the Diels-Alder (DA) reaction of the 3,4-substituted furan and the vinyl phenyl

sulfonate (Scheme 2.1).

14

Scheme 2.1. Synthesis of Diene 2.3, Dienophile 2.4 and OBHS-I 2.5 by Diels-Alder reaction of

2.3 and 2.4.

The DA reactions were typically carried out in a 1-10 mL reaction vial using 2-3 drops of

THF and under a N2 atmosphere at 110 C for 48 h. The product obtained was a mixture (~4:1)

of exo isomer (thermodynamic product) and endo isomer (kinetic product). Zhou et al.13

previously reported that the exo isomer of OBHS showed a higher binding affinity than the endo

isomer. It is to be noted that the exo and endo diastereomers were not separable with flash

chromatography under various solvent combinations. Thus, the mixture was directly used for

further late-stage diversification since we expected that the diastereomers could be readily

separated with preparative HPLC once derivatized with amido or propargyl chains.

15

2.5 Synthesis of a Library of Propargyl Analogs of OBHS (OBHS-P) Compounds

The OBHS-P series was synthesized using Sonogashira cross coupling27

of OBHS-I with

different propargyl compounds (Scheme 2.2) (see Table 2.1 for all synthesized analogs and

yield). The mixture of exo and endo isomers of OBHS-I were directly used for the reaction and

the isomers separated using preparative HPLC following the reaction completion.

Scheme 2.2 Synthesis of OBHS-P analogs 2.6(a-c) by Sonogashira reaction

2.6 Synthesis of a Library of Acetamido Analogs of OBHS (OBHS-A) Compounds

An initial attempt to synthesize the OBHS–A analog series by the Buchwald amidation

reaction25,26

was unsuccessful. Various catalysts–ligands–base combinations were tried (catalysts

such as Pd(OAc)2, Pd2(dba)3 with bases such as potassium tert-butoxide(KOtBu), sodium tert-

butoxide (NaOtBu), cesium carbonate, and ligands such as BINAP and Xanthphos). No reaction

occurred at room temperature whereas retro Diels Alder reaction occurred at higher temp (≥ 50

C) where furan was isolated following the reaction. We also investigated the Goldberg

amidation reaction for this purpose, however with the same results, i.e. retro-DA reaction. When

the Goldberg reaction with acetamide was tried at room temperature with a large excess of the

reagents, a limited conversion with ~10 % yield of the desired acetamido-OBHS product was

obtained. Finally, we developed a new synthetic scheme for the OBHS-A series where we first

synthesized the vinyl phenyl sulfonate dienophile with the desired amido chain, followed by DA

16

reaction. This was done by the nucleophilic substitution-elimination reaction of 2-

chloroethanesulfonylchloride with the amide-derivatized phenol (Scheme 2.3).

Scheme 2.3. Synthesis of OBHS-A analogs 11(a-c)

Table 2.1 OBHS-P Analogs with exo yield.

Entry Product Structure % Isolated Yield (Exo)

1. 2.6a

38

2. 2.6b

41

17

In cases where a suitable amide derivative of phenol was not available commercially, we

altered the dienophile synthetic route by synthesizing a Boc-protected-4-aminophenyl vinyl

sulfonate (Scheme 2.4). The Boc group was then deprotected, and the free amine reacted with a

suitable acylating reagent. The dienophiles 2.7 and 2.10 were synthesized in moderate to good

yield. Finally OBHS-A analogs 2.11 were synthesized by Diels Alder reaction under microwave

conditions at 110 C, cutting the reaction time from 48 h to 2 h, when the reaction was found to

be complete (see Table 2.2 for all synthesized analogs and yields). The DA products were again

a mixture of exo and endo (typically 4:1). The two isomers were separated by preparative HPLC

using MeCN/water/HCOOH acid as solvent system.

Scheme 2.4 Synthesis of OBHS-A analogs 11(d-g).

18

Table 2.2 OBHS-A Analogs with exo and endo yield.

Entry Product Structure % Isolated Yield

Exo Endo

1. 2.11a

47 Not Isolated

2. 2.11b

32

Not Isolated

3. 2.11c

28 5

4. 2.11d

24 5

5 2.11e

32 6

6 2.11f

33 4

7 2.11g

35 11

8 2.11h

47 9

19

2.7 Biological Evaluation of OBHS analogs

The biological work for ER activity was carried out in collaboration with Dr. John

Katzenellenbogen at the University of Illinois for binding studies, and Dr. Kendall Nettles at

Scripps, Florida for in vitro work. The relative binding affinities (RBAs) of the OBHS analogs

for ER and ER are listed in Table 2.4. The affinity of a ligand for ERα or ERβ can be

specified using two different systems43

. Typically, the ligand affinities are expressed relative to

that of E2, as a relative-binding affinity (RBA) value. These RBA values come directly from

IC50 values determined in competitive radiometric or fluorometric ligand-binding assays43

.

RBA(%) = (IC50

estradiol / IC50

ligand )´100

Thus, an RBA of 100% represents an affinity equivalent to that of E2 on either ER or ER.

Another way of expressing ligand affinity is as a Ki value, which is also calculated from the IC50

values obtained from competitive ligand-binding assays, using the Cheng–Prusoff equation.

Ki= IC50/ (1+ [tracer]/Kdtracer)

In competitive radiometric ligand-binding assays, the tracer is generally E2 (Kd values for E2 are

0.2 nM for ERα and 0.5 nM for ERβ). These E2 Kd values can be used to estimate ligand Kd

values from the RBA values43

.

Kdligand

= Kdestradiol/(RBA/100)

The RBA values of OBHS for ERα and ERβ are 27.8%, and 3.1%, which is equivalent to Kd of

0.7 nM and 22.2 nM, respectively. As compared to OBHS parent lead, many analogs from the

OBHS-A series exhibited higher binding affinity. For example, the exo diastereomer of 2.11a,

20

2.11b, 2.11c, 2.11d, and 2.11e exhibited RBAs of 22.6%, 10.5%, 19.5%, 12.1%, and 14.4%,

respectively. As observed before,20

endo OBHS compounds always exhibit lower binding

affinities than exo compounds. It is to be noted that all of the OBHS derivatives reported thus far

had binding affinity values of 1-5% or lower. Thus, it is exciting to see results from this series

with some analogs possessing higher binding affinity than OBHS (pending further validation).

Further, the addition of short hydrophobic chains did not result in any appreciable loss of binding

affinity. In fact, the RBA values with acetamide, propionamide, and isobutyramide (i.e. short

hydrophobic chains) were very comparable to each other. However, unexpectedly the

bioisosteric replacement of isobutyramide with cyclopropylcarboxamide led to somewhat

reduced binding affinity.

Finally, it is to be emphasized here that even though the RBA values of all these

compounds could suggest to a casual reader that the analogs are not so active, converting these

values to Kd makes it quite clear that all these compounds are within 1-10 nM range of binding

affinity. Once the basic SERM side chain is coupled to one of the phenols, it is expected that the

binding affinity will be slightly reduced, although by not much. Regardless, we consider any

RBA values greater than 1% (Kd = 20 nM) to be very good compounds, and this data is only the

first step in a battery of assays that will be conducted eventually on these compounds (Fig. 2.4)

(vide infra). Our major emphasis is, however, in ascertaining and improving their anti-

inflammatory properties in conjunction with their anti-proliferative properties, as explained later.

Further, even if the ligand is agonistic in breast (MCF7 cells as model line) or uterus (Ishikawa

cells as model line), we can readily dial out this activity by appending the basic SERM side chain

on one of the phenols that would direct the SERM side chain towards helix 12, thus occluding its

21

refolding onto the rest of the protein to form AF-2 pocket, and preventing AF-2 dependent

transcriptional activities.

Table 2.4 Relative Binding affinities (RBA) of the OBHS-A and OBHS-P analogs

Entry Compound Structure(R group)

Binding Affinity (RBA) α/β

ratio ERα ERβ

1 OBHS H 27.8±1.0 (=2.15 nM) 3.1± 0.4 9.1

OBHS-A Series

2 2.11b Exo

10.5 ± 0.7 0.877 ± 0.03 12

3 2.11a Exo

22.6 ± 3.5 0.919 ± 0.2 24.6

4 2.11d Exo

12.1 ± 2.2 0.691 ± 0.2 17.5

5 2.11d Endo

0.140 ± 0.01 0.039 ±0.01 3.6

6 2.11c Exo

19.5 ± 5.4 0.516 ± 0.02 37.8

7 2.11c Endo 0.147 ± 0.03 0.043 ± 0.01 3.4

8 2.11e Exo

14.4 ± 1.3 0.534 ± 0.1 26.9

9 2.11e Endo

0.153 ± 0.04 0.037 ± 0.001 4.1

10 2.11f Exo

6.67 ± 1.8 0.286 ± 0.01 23.3

11 2.11f Endo

0.203 ± 0.03 0.036 ± 0.01 5.6

12 2.11g Exo

6.36 ± 1.6 0.858 ± 0.1 7.4

22

13 2.11g Endo

0.267 ± 0.4 0.135 ± 0.02 1.9

14 2.11h Exo

2.24 ± 0.5 0.231 ± 0.06 9.7

15 2.11h Endo

0.168 ± 0.03 0.048 ± 0.002 3.5

OBHS-P Series

16 2.10 (=9.5 nM)

17 2.6a Exo

3.5 ± 0.3 1.42 ± 0.3 2.5

18 2.6b Exo

4.5 ± 1.4 2.20 ± 0.2 2.0

w`

Figure 2.4 ER structural and functional screening

23

2.7.1 Cell Studies:

Different ligands display different activities on AF1 and AF2 domains; for e.g., TAM and

RAL is agonist on AF1 whereas estradiol and DES (Diethylstilbestrol) are agonistic on AF2.44

Some tissues have predominantly AF1 while others have AF2 dependent transcriptional

activities (which in turn depends upon the cofactors involved).49

What we are looking for is ERα

antagonistic activity in breast, uterus, and endometrial tissue (to be effective as an anti-cancer

agent), while having agonistic activity, if possible, in other tissues to have cardioprotective,40

bone loss preventative, and neuroprotective effects (as chemoprevention for such indications as

Alzheimer’s disease,42

multiple sclerosis,41

inflammatory bowel disease, IBD). Further, as

pointed out earlier, the agonistic anti-inflammatory activity from these analogs is highly

desirable to prevent NF-κB dependent endocrine resistance development in breast cancer, and for

other disease indications such as IBD. The agonistic and antagonistic activities of OBHS

analogs were tested in HepG2 cells containing transient transfection of ER-luciferase construct,

in the absence or presence of estradiol (E2), respectively.

Table 2.5 Luciferase activity was measured in HepG2 cells transfected with 3 x-ERE-driven

luciferase reporter. Compound Compound + 10 nM E2

2.11a Exo

2.11b Exo

2.11d Exo

2.11d Endo

24

2.11g Exo

2.11g Endo

2.11c Exo

2.11c Endo

2.11h Exo

2.11h Endo

2.11e Exo

2.11e Endo

2.11f Exo

2.11f Endo

2.6a Exo

2.6b Exo

As seen in Table 2.5, the lead OBHS-A compound shows both agonisitic and partial

antagonistic activity (in presence of 10 nM of E2; bold curves show agonistic activity whereas

dotted curves show antagonistic activity). Some compounds such as 2.11f, 2.11h and 2.6b show

agonist activity but no antagonist activity. Ideally the agonist and antagonist curves should meet

at higher doses, which can be seen with most compounds such as 2.11a, c and d (exo) while in

order it could be expected to do so at even higher doses. However, in some compounds such as

25

2.11f, these curves do not seem to meet even at higher doses, the reasons for which are not clear.

These assays show that the lead compound OBHS-A and some of its analogs have the capability

to show both agonist and antagonist characteristics. The antagonistic activities of the OBHS

analogs compared to tamoxifen and ICI compounds are shown in Fig. 2.5.

In further assays, we will define the AF-1 and AF-2 dependence of these activities, and

characterize the different functional activities in cells lines representing various tissues. The

antiproliferative activity of these analogs will be tested in both tamoxifen-naïve MCF7 cells and

tamoxifen-resistant MCF7 cells (TamR-MCF7), both of which are ER positive and Luminal A

subtype (responsive to antiestrogen in primary disease stage), BT474, which is ER positive but

Luminal B subtype (that typically does not respond well to anti-estrogen therapies), and MDA-

MB-231, which is an ER negative cell line (and hence does not responsive to anti-estrogen

therapies at all). To mechanistically study the pharmacological profile of these ligands, the

compounds will be tested in a) MCF7 cells with endogenous ERα expression, HepG2 cells with

high transient expression of b) ERα, or c) ERα with F-domain truncated, d) HEK293 cells

containing ERα-LBD with Gal4-DBD, and e) HepG2 cells containing ERα-LBD and native

DBD, but lacking the N-terminal domain (thus the AF-1 domain). The latter two assays can

ascertain the AF-1 (part of N-terminal domain) vs AF-2 (part of LBD) activity.

26

Fig. 2.5. Antagonistic activity of OBHS analogs compared to OBHS at 10 µM. (preliminary

assay results; pending further experiments to validate results and ascertain error)

To study the anti-inflammatory activity, the compounds are tested for suppression of IL-

6 secretion in MCF7 cells, upon TNF-α stimulation that activates NF-κB response. The anti-

inflammatory activities of compounds from previous batch are shown in Fig 2.6. As expected,

E2 and particularly dexamethasone exhibit anti-inflammatory behavior while the ICI compound

(fulvestrant) has no response. OBHS and some of its analogs show anti-inflammatory activity

comparable to that of E2. The compound 2.11a exo from previous batch exhibited remarkably

higher anti-inflammatory activity than even E2. As stated earlier, we plan to retest this

compound in proliferation assay, and if found suitable, we will add SERM side chain on ring B

phenol for more potent anti-proliferative activity. Also it is to be noted that, these compounds

from previous batch were impure, the assays for the pure exo compounds are in progress.

27

Fig. 2.6 Anti-inflammatory activity of OBHS analogs compared to OBHS, E2, ICI and Dex at 10

µM. IL-6 secretion was monitored upon stimulation with TNF. (preliminary assay results;

pending further experiments to validate results and ascertain error)

Finally, in this battery of primary assays, we test for GREB1 mRNA expression in MCF7

cells. GREB1 expression level demonstrates a significant correlation with ER phenotype in a

panel of breast cancer cells.45

ER directly controls GREB1 expression. Studies in primary breast

cancers have shown that GREB1 is overexpressed in ER-positive breast cancers as compared

with ER-negative breast cancers by 3.5-fold. GREB1 is also induced by beta-estradiol in the ER-

positive endometrial cell line ECC-1.46

GREB1 induction by E2 is rapid, increasing by about 7.3

fold by 2 h in MCF-7 cells. The pattern of expression suggests a critical role for this gene in the

response of tissues and tumors to β-estradiol. Suppression of GREB1 using siRNA blocks

estrogen-induced growth in MCF-7 cells. Thus, GREB1 is likely a key regulator of estrogen-

induced breast cancer growth, and has potential as a new biomarker for predicting E2-dependent

and anti-E2-responsive breast cancers. The results from previous batch of OBHS leads are shown

28

is Fig. 2.7. OBHS-A and P leads inhibit GREB1 transcription better than OBHS parent lead

compound.

Fig. 2.7 GREB 1 mRNA inhibition of OBHS compounds. (preliminary assay results; pending

further experiments to validate results and ascertain error)

2.8 Experimental section

All the reagents were purchased from commercial suppliers and used without purification

unless specified. Dry solvents for the reactions were purchased from EMD Millipore and further

dried over molecular sieves (3Å). Glassware was oven dried prior to the experiment and all

experiments unless specified were conducted under an inert atmosphere. Reaction progress was

monitored using thin layer chromatography (TLC) on Fisher TLC plates coated on aluminum

and visualized under UV light. Purification was done with automated flash chromatography

using silica gel Redisep columns (Teledyne ISCO combiflash, Redisep column). HPLC

preparative runs were performed on a Waters HPLC system using prep C18, 5µM OBD column.

HPLC analytical runs were performed on a Waters HPLC system using C18, 5µM column. 1H

29

NMR and 13

C NMR spectra were obtained on a 400 MHz or 500 MHz Bruker instrument and

processed using Mnova software. Chemical shifts are reported in ppm with TMS as internal

standard. All NMR spectra are referenced to either TMS or residual solvents.

3,4-bis(4-Methoxyphenyl)furan-2(5H)-one (2.1)

4-methoxyphenylacetic acid (3.6 g, 21.7 mmol) was dissolved in MeCN (15 mL) and DBU (3.3

mL, 1.02 eq) was added and stirred for 20 mins in a round bottom flask. In another flask, 2-

bromo-4-methoxy acetophenone (5 g, 1 eq) was taken, MeCN (15 mL) was added and then the

mixture stirred for 20 mins. Homogenous solution from this flask was added dropwise to the

flask containing the acid using an addition funnel and the resulting mixture stirred for 1 h DBU

(9.9 mL, 3.04 eq) was then added and the solution stirred for additional 4 h. The reaction mixture

was evaporated to dryness, and partitioned between EtOAc and 1M HCl. The organic layer was

washed with water, brine, and then finally dried over anhydrous magnesium sulfate. The crude

product was obtained as a dark orange solid, which upon recrystallization with ethanol yielded

(5.4 g, 85%) bright yellow crystals.

1H NMR (400MHz, CDCl

3) δ (ppm) 7.34 – 7.28 (m, 2H), 7.26 – 7.21 (m, 2H), 6.87 – 6.83 (m,

2H), 6.81 – 6.75 (m, 2H), 5.06 (s, 2H), 3.76 (d, J = 6.1 Hz, 6H).

LR-MS (ESI): calculated for C18H17O4 (M+H)+

: 297.1; found: 297.1

3,4-bis(4-Hydroxyphenyl)furan-2(5H)-one (2.2)

30

A 250 mL round bottomed flask was charged with product 2.1 (6.5 g, 21.9 mmol), and

pyridinium hydrochloride (25 g, 4 eq) was added and the mixture stirred at 220 C for 4 h. The

crude product was partitioned with EtOAc and 1 M HCl. The organic layer was washed with

water, brine, and dried over anhydrous magnesium sulfate to obtain a light brown solid (5.8 g,

100% yield) which had >95% purity and was carried over to the next step.

1H NMR (500 MHz, DMSO-d

6) δ (ppm) 10.08 (s, 1H), 9.70 (s, 1H), 7.28 – 7.24 (m, 2H), 7.18 –

7.14 (m, 2H), 6.82 (d, J = 8.6 Hz, 2H), 6.78 – 6.74 (m, 2H), 5.25 (s, 2H).13

C NMR (126 MHz,

DMSO-d6) δ (ppm) 174.2, 160.0, 157.9, 156.0, 130.9, 129.7, 122.3, 121.9, 121.8, 116.19, 115.9,

70.6.

LR-MS (ESI): calculated for C16H13O4 (M+H)+ : 269.08; found: 269.0

4,4'-(Furan-3,4-diyl)diphenol (2.3)

A 250 mL 3-neck round bottom flask was charged with product 2.2 (1g, 3.7 mmol) and cooled to

-78 C using acetone / dry ice bath. Dry THF (40 mL) was added to the flask under N2 using an

addition funnel. DIBAL-H (1M in hexane, 25 mL, 6 eq) was added dropwise using an addition

funnel and stirred at -78 °C for 4h. After 4 h, the reaction was stopped by performing a reverse

quench by methanol at -78 °C. The reaction mixture was allowed to warm to RT, 10% HCl was

added and stirred for another 30 mins. The organic layer was extracted using ethyl acetate. The

31

combined organic layer was washed with water, brine, and dried over anhydrous magnesium

sulfate to obtain furan 2.3 as a light yellow solid (0.94 g, 96% yield).


6) δ (ppm) 9.45 (s, 2H), 7.75 (s, 2H), 7.04 – 6.98 (m, 4H), 6.74 –

6.68 (m, 4H). 13

C NMR (126 MHz, DMSO-d6) δ (ppm) 156.9, 140.6, 129.7, 125.5, 122.8, 115.7.

LR-MS (ESI): calculated for C16H13O3 (M+H)+ : 253.28; found: 253.10

4-Iodophenol ethenesulfonate (2.4)

Iodophenol (6.8 g, 30.9 mmol) was taken in diethyl ether, cooled to 0C and added triethylamine

(4.5 mL, 1 eq). The homogeneous solution from this flask was added to flask with 2-

chloroethane sulfonyl chloride (3.2 mL, 1 eq) in diethyl ether, and cooled to 0 C.

Trimethylamine (6.5 mL, 1.5 eq) was added to the reaction mixture and stirred for 2 h. The

reaction mixture was partitioned with ethyl acetate and 1M HCl and extracted. The combined

organic layer was washed with water and brine, dried over anhydrous magnesium sulfate, and

evaporated to dryness. The crude product was purified by flash chromatography (70:30 hexane:

ethyl acetate) to obtain pure product (9 g, 94% yield) as white solid.

1H NMR (400 MHz, CDCl

3) δ (ppm) 7.66 – 7.61 (m, 2H), 6.95 – 6.90 (m, 2H), 6.58 (dd, J =

16.6, 10.0 Hz, 1H), 6.31 (dd, J = 16.6, 0.7 Hz, 1H), 6.12 (dd, J = 9.9, 0.7 Hz, 1H).

32

LR-MS (ESI): calculated for C8H8IO3S (M+H)+ : 311.10; found: 311.11

4-Iodophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonate (2.5)

Furan 2.3 and dienophile 2.4 was weighed into a small vial with a screwcap, were added 2 drops

of dry THF, and purged the vial with N2. The reaction was stirred under N2 at 110 C for 48 h.

The crude product was obtained as a black sticky material, which was further purified by flash

chromatography (70:30 hexane: ethyl acetate). The product obtained was a mixture of exo and

endo isomers (82:18) as a light yellow solid in 50% yield, which was confirmed by NMR and

HPLC.


6) δ (ppm) 9.68 (d, J = 17.3 Hz, 2H), 7.77 – 7.73 (m, 2H), 7.18 –

7.09 (m, 7H), 6.77 – 6.68 (m, 5H), 5.64 (d, J = 1.2 Hz, 1H), 5.44 (dd, J = 4.3, 1.1 Hz, 1H), 3.86

(dd, J = 8.2, 4.4 Hz, 1H), 2.22 (dt, J = 11.9, 4.4 Hz, 1 H), 2.15 (dd, J = 12.1, 8.3 Hz, 1H). 13

C

NMR (126 MHz, DMSO-d6 δ (ppm) 157.8, 149.2, 141.2, 136.7, 129.5, 128.8, 125.2, C

15,19 =

123.5, 116.0, 115.9, 93.2, 84.1, 82.5, 60.9, 30.8, 21.2.

LR-MS (ESI): calculated for C26H20IO6S (M+H)+ : 563.0; found: 563.0

General Procedure A: Sonogashira Coupling

The OBHS –I compound was dissolved in dry DMF in a glass vial. The vial was closed with a

rubber septum and evacuated under a vacuum and backfilled with argon. Pd catalyst, base and

33

CuI were added to the vial which was again evacuated and backfilled with argon. Finally, the

alkyne was added and the reaction was stirred at RT overnight. The mixture quenched with H2O,

after which the organic material was extracted with EtOAc (3 × 30 mL) and dried over

anhydrous MgSO4 and the solvent was evaporated under vacuum. The crude product was

purified using prep HPLC.

Exo-4-(3-hydroxy-3-methylbut-1-yn-1-yl)phenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo

[2.2.1] hept-5-ene-2-sulfonate (2.6a)

Compound 2.6a was synthesized following general procedure A. The crude product was purified

by preparative HPLC to obtain pure product (38% yield) as a white solid.


6) δ (ppm) 9.79 (s, 2H), ), 7.43 – 7.39 (m, 2H), 7.30 – 7.25 (m,

2H), 7.19 – 7.12 (m, 4H), 6.76 – 6.70 (m, 4H), 5.65 (d, J = 1.2 Hz, 1H), 5.55 (s, 1H), 5.43 (dd, J

= 4.3, 1.2 Hz, 1H), 3.85 (dd, J = 8.2, 4.4 Hz, 1H), 2.21 (dt, J = 12.0, 4.4 Hz, 1H), 2.13 (dd, J =

12.1, 8.3 Hz, 1H), 1.46 (s, 6H). 13

C NMR (126 MHz, DMSO-d6) δ (ppm) 157.8, 157.7, 141.2,

136.6, 133.3, 129.4, 128.8, 123.5, 123.1, 122.83, 122.15, 116.0, 115.9, 97.5, 84.1, 82.5, 64.0,

31.9, 30.8.

LR-MS (ESI): calculated for C29H27O7S (M+H)+ : 519.15; found: 519.4

34

Exo-4-(4-hydroxybut-1-yn-1-yl)phenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-

ene-2-sulfonate (2.6b)

Compound 2.6b was synthesized following general procedure A. The crude product was purified

by preparative HPLC to obtain pure product (41% yield) as a white solid.


6) δ (ppm) 9.75 (s, 2H), 7.43 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 8.7

Hz, 2H), 7.18 – 7.12 (m, 4H), 6.76 – 6.69 (m, 4H), 5.65 (s, 1H), 5.43 (d, J = 4.2 Hz, 1H), 4.58 (t,

J = 6.6 Hz, 1H), 3.85 (dd, J = 8.2, 4.4 Hz, 1H), 2.21 (dt, J = 12.0, 4.4 Hz, 1H), 2.14 (dd, J = 12.1,

8.3 Hz, 1H), 1.37 (d, J = 6.6 Hz, 3H). 13

C NMR (126 MHz, DMSO-d6) δ (ppm) 157.9, 157.8,

141.2, 136.7, 133.3, 129.5, 128.8, 123.5, 123.1, , 122.8, 122.0, 116.1, 115.9, 94.8, 82.5, 58.1,

24.9.

LR-MS (ESI): calculated for C28H25O7S (M+H)+ : 505.13; found (M+H)+: 505.3

General Procedure B for the synthesis of dienophiles 2.7a-2.7c

Aniline was dissolved in MeCN, cooled to 0 C and triethylamine (1 eq) was added. The

homogeneous solution from this flask was added to the flask containing 2-chloroethane sulfonyl

chloride suspended in MeCN, and cooled to 0 C. Triethylamine (1.5 eq) was added to the

reaction mixture and stirred for 2 h. The reaction mixture was partitioned with ethyl acetate and

35

1M HCl and extracted. The combined organic layers were washed with water and brine, dried

over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was obtained

as an off-white solid.

4-Acetamidophenyl ethenesulfonate (2.7a)

Product 2.7a was synthesized following the general procedure B. The crude product was purified

using flash chromatography (60% EtOAc in DCM) to obtain pure product as white solid (0.794

g, 81% yield).

1H NMR (400 MHz, DMSO-d6) δ (ppm) 10.10 (s, 1H), 7.67 – 7.60 (m, 2H), 7.24 – 7.20 (m,

2H), 7.20 – 7.15 (m, 1H), 6.37 (dd, J = 10.0, 0.8 Hz, 1H), 6.29 – 6.24 (m, 1H), 2.04 (s, 3H). 13

C

NMR (126 MHz, DMSO-d6) δ (ppm) 168.9, 144.4, 138.9, 133.7, 132.6, 123.1, 120.4, 24.4.

LR-MS (ESI): calculated for C10H12NO4S (M+H)+ : 242.05; found (M+H)+: 242.0

4-Formamidophenyl ethenesulfonate (2.7b)

Product 2.7b was synthesized following general procedure B. The crude product was purified

using flash chromatography 40% EtOAc in DCM) to obtain pure product as white solid (0.750 g,

80% yield).

36

1H NMR (400 MHz, CDCl3) δ (ppm) 8.32 (d, J = 1.7 Hz, 1H), 7.63 – 7.31 (m, 2H), 7.19 – 7.08

(m, 2H), 6.60 (ddd, J = 16.6, 10.0, 6.5 Hz, 1H), 6.31 (ddd, J = 16.6, 10.4, 0.7 Hz, 1H), 6.13 (td, J

= 9.8, 0.7 Hz, 1H).


4-Butyramidophenyl ethenesulfonate (2.7c)

Product 2.7c was synthesized following general procedure B. The crude product was purified

using flash chromatography 40% EtOAc in DCM to obtain pure product as white solid (0.750 g,

80% yield).

1H NMR (400 MHz, CDCl3) δ (ppm) 7.57 – 7.51 (m, 2H), 7.30 (s, 1H), 7.18 – 7.13 (m, 2H), H

7

= 6.64 (dd, J = 16.6, 10.0 Hz, 1H), 6.34 (dd, J = 16.6, 0.7 Hz, 1H), 6.15 (dd, J = 10.0, 0.7 Hz,

1H), 2.38 – 2.29 (m, 2H), 1.81 – 1.67 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). 13

C NMR (126 MHz,

DMSO-d6) δ (ppm) 171.7, 144.3, 138.9, 133.7, 132.6, 123.1, 120.5, 38.7, 18.9, 14.0.


Tert-butyl-(4((vinylsulfonyl)oxy)phenyl)glycine (2.8)

4-Boc-aminophenol (1g, 1eq) was taken in diethyl ether, cooled to 0 C and triethylamine (0.6

mL, 1 eq) was added. The homogeneous solution from this flask was added to the flask with 2-

chloroethane sulfonyl chloride (0.75 mL, 1.5 eq), and cooled to 0 C. Triethylamine (1 mL, 1.5

eq) was added to the reaction mixture and stirred for 2 h. The reaction mixture was partitioned

with ethyl acetate and 1M HCl and extracted. The combined organic layer was washed with

water and brine, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude

37

product was purified using flash chromatography (60% EtOAc in DCM) to obtain pure product

as white solid (1.25 g, 89%).

1H NMR (400 MHz, CDCl3) δ (ppm) 7.36 (d, J = 9.0 Hz, 2H), 7.13 (d, J = 9.0 Hz, 2H), 6.66 –

6.57 (m, 1H), 6.32 (dd, J = 16.6, 0.7 Hz, 1H), 6.13 (dd, J = 10.0, 0.6 Hz, 1H), 1.49 (s, 9H).

4-((Vinylsulfonyl)oxy)phenyl)glycine TFA salt (2.9)

Tert-butyl-(4((vinylsulfonyl)oxy)phenyl)glycine (1.29 g) was taken in a round bottom flask and

50% TFA in DCM was added ,and the reaction mixture stirred at RT for 3 h. After 3 h DCM and

excess TFA was removed by purging with air, and dried to obtain off white solid (98% yield).

1H NMR (400 MHz, DMSO-d6) δ (ppm) 7.15 – 7.06 (m, 1H), 7.03 (d, J = 8.8 Hz, 2H), 6.76 (d, J

= 8.9 Hz, 2H), 6.31 (dd, J = 10.0, 0.7 Hz, 1H), 6.20 (dd, J = 16.6, 0.7 Hz, 1H).

LR-MS (ESI): calculated for C8H10NO3S (M-TFA)+

: 200.04; found: 200.03

General Procedure C for synthesis of dienophile (2.10d - 2.10h)

4-((vinylsulfonyl)oxy)phenyl)glycine TFA salt (1g, 3.4 mmol) was added to DCM in a round

bottom flask, and cooled to 0 C. Acyl chloride was added to the reaction mixture followed by

Hunig’s base and stirred at RT for 1h.

4-Propionamidophenyl ethenesulfonate (2.10d)

38

Product 2.10a was synthesized following general procedure C. The crude product was purified

by flash chromatography (40% EtOAc in hexane) to obtain pure product as a white solid (0.058

g, 67.5% yield).


3) δ (ppm) 7.55 (d, J = 8.9 Hz, 2H), 7.18 (d, J = 9.0 Hz, 3H), 6.69 –

6.61 (m, 1H), 6.35 (dd, J = 16.6, 0.7 Hz, 1H), 6.16 (dd, J = 9.9, 0.6 Hz, 1H), 2.40 (d, J = 7.5 Hz,

2H), 1.25 (t, J = 7.5 Hz, 3H).

4-Iosbutyramidophenyl ethenesulfonate (2.10e)

Product 2.10b was synthesized following general procedure C. The crude product was purified


g, 45.2% yield)


3) δ (ppm) 7.54 (d, J = 9.0 Hz, 2H), 7.18 – 7.12 (m, 2H), 6.68 – 6.57

(m, 1H), 6.32 (dt, J = 16.6, 0.5 Hz, 1H), 6.14 (dt, J = 10.0, 0.6 Hz, 1H), 2.49 (s, 1H), 1.23 (dd, J

= 6.8, 1.3 Hz, 6H). 13

C NMR (126 MHz, CDCl3) δ (ppm) 137.1, 131.9, 131.8, 122.8, 122.6,

120.7, 77.2, 36.6, 19.5.


4-Pentanamidophenyl ethenesulfonate (2.10g)

39

Product 2.10c was synthesized following general procedure C. The crude product was purified


g, 63% yield).


3) δ (ppm) 7.53 (s, 1H), 7.51 – 7.45 (m, 2H), 7.11 – 7.05 (m, 2H), 6.58

(dd, J = 16.6, 10.0 Hz, 1H), 6.27 (dd, J = 16.6, 0.7 Hz, 1H), 6.10 (dd, J = 10.0, 0.7 Hz, 1H), 2.32

– 2.26 (m, 2H), 1.66 – 1.58 (m, 2H), 1.36 – 1.27 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H). 13

C NMR

(126 MHz, CDCl3) δ (ppm) 171.7, 145.0, 137.2, 132.0, 131.7, 122.7, 120.8, 37.3, 27.6, 22.3,

13.8.

LR-MS (ESI): calculated for C13H18NO4S (M+H)+

: 284.1; found: 284.2

4-(2-Phenoxyacetamido)phenyl ethenesulfonate (2.10f)

Product 2.10d was synthesized following general procedure C. The crude product was purified


g, 80% yield).


3) δ (ppm) 8.29 (s, 1H), 7.60 – 7.54 (m, 2H), ), 7.33 – 7.26 (m, 2H),

7.18 – 7.13 (m, 2H), 7.05 – 6.98 (m, 1H), 6.97 – 6.89 (m, 2H), 6.59 (dd, J = 16.6, 10.0 Hz, 1H),

6.29 (dd, J = 16.6, 0.7 Hz, 1H), 6.11 (dd, J = 10.0, 0.7 Hz, 1H), 4.56 (s, 2H). 13

C NMR (126

MHz, CDCl3) δ (ppm) 166.4, 156.8, 145.7, 135.8, 131.9, 131.8, 129.9, 129.6, 123.0, 122.6, C

8 =

121.2.

40


: 334.08; found: 334.08

4-(Cyclopropanecarboxamido)phenyl ethenesulfonate (2.10h)

Product 2.10e was synthesized following general procedure C. The crude product was purified


g, 59% yield).


2H), 7.20 – 7.14 (m, 1H), 6.36 (dd, J = 10.0, 0.8 Hz, 1H), 6.26 (dd, J = 16.5, 0.8 Hz, 1H), 1.79 –

1.71 (m, 1H), 0.83 – 0.76 (m, 4H). 13

C NMR (126 MHz, DMSO-d6) δ (ppm) 172.2, 144.3, 38.8,

133.6, 132.6, 123.1, 120.4, 14.9, 7.7.


: 268.07; found: 268.1

General Procedure D: Diels Alder Reaction

Furan and dienophile were weighed into a microwave vial, and few drops of dry THF were

added. The vial was evacuated and backfilled with N2, and the reaction mixture was heated in

microwave conditions at 110 C for 2 h. The crude product was obtained as a brown sticky

material, which was further purified by preparative HPLC.

Exo-4-acetamidophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11a)

Exo-2.11a was synthesized following general procedure C. The crude product was purified by

preparative HPLC to obtain pure product (47% yield) as a white solid.

41


6) δ (ppm) 10.10 (s, 1H), 9.73 (s, 2H), 7.61 – 7.56 (m, 2H), 7.21 –


(dd, J = 8.2, 4.4 Hz, 1H), 2.24 – 2.12 (m, 2H), 2.04 (s, 3H). 13

C NMR (126 MHz, DMSO-d6) δ

(ppm) 168.8, 157.9, 157.8, 144.3, 141.2, 138.7, 136.7, 129.4, 128.8, 123.6, 123.0, 122.8, 120.5,

116.0, 115.9, 84.1, 82.5, 60.6, 30.8, 24.4.


: 494.13; found (M+H)+: 494.3

Exo-4-formamidophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11b)

Exo-2.11b was synthesized following general procedure C. The crude product was purified by



6) δ (ppm) 10.40 (s, 1H), 8.28 (s, 1H), 7.64 – 7.57 (m, 2H), 7.26 –

7.21 (m, 2H), 7.19 – 7.13 (m, 4H), 6.77 – 6.69 (m, 4H), 5.63 (d, J = 1.2 Hz, 1H), 5.43 (dd, J =

4.3, 1.2 Hz, 1H), 3.79 (dd, J = 8.1, 4.4 Hz, 1H), 2.26 – 2.12 (m, 2H). 13

C NMR (126 MHz,

DMSO-d6) δ (ppm) 166.4, 158.0, 157.9, 144.7, 141.2, 137.9, 137.6, 129.4, 128.8, 123.8, 123.5,

123.3, 122.7, 120.7, 119.0, 116.1, 115.9, 84.1, 82.5, 60.6, 30.6.


: 480.11; found (M+H)+: 480.3

42

Exo-4-butyramidophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11d)

Exo-2.11d was synthesized following general procedure C. The crude product was purified by



6) δ (ppm) 10.04 (s, 1H), 9.78 (s, 2H), 7.63 – 7.57 (m, 2H), 7.20 –

7.11 (m, 6H), 6.78 – 6.67 (m, 4H), 5.64 – 5.60 (m, 1H), 5.43 (d, J = 4.0 Hz, 1H), 3.77 (dd, J =

8.2, 4.5 Hz, 1H), 2.28 (t, J = 7.3 Hz, 2H), 2.22 (dt, J = 12.0, 4.4 Hz, 1H), 2.15 (dd, J = 12.1, 8.2

Hz, 1H), 1.60 (q, J = 7.4 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). 13


(ppm) 175.8, 157.9, 157.8, 144.27, 141.23, 138.78, 136.77 129.4, 128.8, 123.7, 123.1, 123.0,

120.5, 116.0, 115.9, 84.1, 82.6, 60.5, 39.0, 29.8, 18.9, 14.1.


: 522.16; found : 522.4

Endo-4-butyramidophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11d)

Endo-2.11d was synthesized following general procedure C. The crude product was purified by


43


6H), 6.74 – 6.60 (m, 4H), 5.64 (dd, J = 4.2, 1.2 Hz, 1H), 5.36 (dd, J = 4.7, 1.1 Hz, 1H), 4.35 (dt, J

= 9.1, 4.4 Hz, 1H), 2.57 (m, 1H), 2.28 (t, J = 7.3 Hz, 2H), 1.68 (dd, J = 11.7, 4.5 Hz, 1H), 1.60

(dt, J = 14.6, 7.4 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). 13

C NMR (126 MHz, DMSO-d6) δ (ppm)

171.7, 157.8, 157.3, 143.8,138.8, 136.77 , 129.4, 129.4, 124.2, 123.6, 123.1, 120.4, 115.9,

115.3, 84.1, 82.6, 60.5 38.7, 29.8, 18.9, 14.0.


: 522.16; found : 522.4

Exo-4-propionamidophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11c)

Exo-2.11c was synthesized following general procedure C. The crude product was purified by



6) δ (ppm) 10.04 (s, 1H), 9.74 (s, 2H), 7.64 – 7.57 (m, 2H), 7.21 –


(dd, J = 8.1, 4.5 Hz, 1H), 2.31 (m, J = 7.5 Hz, 2H), 2.25 – 2.11 (m, 2H), 1.07 (t, J = 7.5 Hz, 3H).

13C NMR (126 MHz, DMSO-d

6) δ (ppm) 172.5, 158.2, 158.1, 141.0, 138.8, 136.6, 129.4, 128.7,

123.3, 123.0, 122.5, 120.4, 116.1, 115.9, 84.1, 82.5, 60.5, 30.6, 29.9, 10.0.


: 508.15; found: 508.3

44

Endo-4-propionamido,phenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11c)

Endo-2.11c was synthesized following general procedure C. The crude product was purified by



6) δ (ppm) 10.05 (s, 1H), 9.72 (s, 2H), 7.67 – 7.57 (m, 2H), 7.25 –

7.17 (m, 4H), 7.14 – 7.11 (m, 2H), 6.74 – 6.62 (m, 4H), 5.64 (dd, J = 4.1, 1.2 Hz, 1H), 5.36 (dd,

J = 4.7, 1.2 Hz, 1H), 4.35 (dt, J = 9.1, 4.3 Hz, 1H), 2.54 (m, 1H), 2.32 (q, J = 7.5 Hz, 2H), 1.67

(dd, J = 11.7, 4.5 Hz, 1H), 1.08 (t, J = 7.5 Hz, 3H). 13

C NMR (126 MHz, DMSO-d6) δ (ppm)

172.5, 166.2, 157.8, 57.3, 140.8, 138.8, 135.7, 129.4, 129.4, 124.2, 123.6, 123.1, 122.8, 120.4,

115.9, 115.3, 83.9, 82.7, 59.4, 29.9, 10.0.

LR-MS (ESI): calculated for C27H26NO7S (M+H)+: 508.15; found: 508.3

Exo-4-isobutyramidophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11e)

Exo-2.11e was synthesized following general procedure C. The crude product was purified by


45


6) δ (ppm) 10.01 (s, 1H), 9.80 (s, 2H), 7.63 – 7.57 (m, 2H), 7.20 –

7.10 (m, 6H), 6.77 – 6.66 (m, 4H), 5.62 (d, J = 2.4 Hz, 1H), 5.42 (d, J = 4.0 Hz, 1H), 3.76 (dd, J

= 8.2, 4.5 Hz, 1H), 2.57 (ddd, J = 12.1, 8.3, 6.0 Hz, 1H), 2.21 (dt, J = 12.0, 4.3 Hz, 1H), 2.15

(dd, J = 12.0, 8.3 Hz, 1H), 1.08 (dd, J = 6.9, 2.2 Hz, 6H). 13


(ppm) 175.8, 157.9, 157.8, 144.2, 141.2, 138.8, 136.7, 129.4, 128.7, 123.6, 123.0, 122.7, 120.6,

116.0, 115.9, 84.1, 82.5, 60.5, 35.2 , 30.8, 19.


:522.16; found (M+H)+: 522.4

Endo-4-isobutyramidophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11e)

Endo- 2.11e was synthesized following general procedure C. The crude product was purified by



6) δ (ppm) 10.02 (s, 1H), 9.81 (s, 1H), 7.64 – 7.60 (m, 2H), 7.20 –


= 8.2, 4.5 Hz, 1H), 2.58 (ddd, J = 12.1, 8.3, 6.0 Hz, 1H), 2.25 – 2.12 (m, 2H), 1.09 (dd, J = 6.9,

2.2 Hz, 6H). 13

C NMR (126 MHz, DMSO-d6) δ (ppm) 175.8, 157.9, 157.7, 144.2, 141.2, 138.8,

136.7, 129.4, 128.8, 123.6, 123.0, 122.8, 120.6, 116.0, 115.9, 84.1, 82.5, 60.5, 35.3, 30.0, 19.9.


:521.15; found (M+H)+: 522.4

46

Exo-4-(2-phenoxyacetamido)phenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-

ene-2-sulfonate (2.11f)

Exo-2.11f was synthesized following general procedure C. The crude product was purified by



6) δ (ppm) 10.29 (s, 1H), 9.79 (s, 2H), 7.67 – 7.63 (m, 2H), 7.33 –

7.29 (m, 2H), 7.24 – 7.20 (m, 2H), 7.19 – 7.13 (m, 4H), 7.01 – 6.96 (m, 3H), 6.76 – 6.69 (m,

4H), 5.63 (d, J = 1.2 Hz, 1H), 5.43 (dd, J = 4.3, 1.2 Hz, 1H), 4.69 (s, 2H), 3.78 (dd, J = 8.2, 4.5

Hz, 1H), 2.23 (dt, J = 12.1, 4.4 Hz, 1H), 2.16 (dd, J = 12.1, 8.2 Hz, 1H). 13

C NMR (126 MHz,

DMSO-d6) δ (ppm) 167.3, 158.1, 157.8, 157.7, 144.8, 141.2, 137.7, 136.7, 130.0, 129.5, 128.8,

123.6, 123.1, 122.8, 121.7, 121.4, 116.0, 115.9, 115.1, 84.1, 82.5, 67.4, 60.6, 30.8.


: 586.16; found :586.5

Endo-4-(2-phenoxyacetamido)phenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-

ene-2-sulfonate (2.11f)

Endo-2.11f was synthesized following general procedure C. The crude product was purified by


47


6) δ (ppm) 10.28 (s, 1H), 9.65 (d, J = 53.2 Hz, 2H), 7.72 – 7.67

(m, 2H), 7.35 – 7.29 (m, 2H), 7.24 – 7.16 (m, 6H), 7.01 – 6.98 (m, 3H), 6.74 – 6.70 (m, 2H),

6.65 – 6.62 (m, 2H), 5.65 (dd, J = 4.2, 1.2 Hz, 1H), 5.36 (dd, J = 4.7, 1.3 Hz, 1H), 4.71 (s, 2H),

4.37 (dt, J = 9.1, 4.3 Hz, 1H), 1.68 (dd, J = 11.8, 4.5 Hz, 1H). 13


(ppm) 167.2, 158.2, 157.8, 157.3, 144.8, 140.8, 137.8, 135.7, 129.9, 129.4, 128.8, 123.6, 123.1,

122.8, 121.6, 121.2, 115.9, 115.3, 115.1, 83.9, 82.7, 67.5, 59.5, 29.9.


Exo-4-pentanamidophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11g)

Exo-2.11g was synthesized following general procedure C. The crude product was purified by



6) δ (ppm) 10.04 (s, 1H), 9.74 (s, 2H), 7.64 – 7.56 (m, 2H), 7.21 –

7.13 (m, 6H), 6.77 – 6.68 (m, 4H), 5.63 (d, J = 1.2 Hz, 1H), 5.43 (dd, J = 4.4, 1.2 Hz, 1H), H2 =

3.77 (dd, J = 8.2, 4.5 Hz, 1H), 2.30 (t, J = 7.5 Hz, 2H), 2.22 (dt, J = 12.0, 4.4 Hz, 1H), 2.16 (dd,

J = 12.1, 8.3 Hz, 1H), 1.57 (tt, J = 7.6, 6.5 Hz, 2H), 1.35 – 1.27 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H).

13C NMR (126 MHz, DMSO-d

6) δ (ppm) 171.8, 157.9, 157.7, 144.2, 141.2, 138.7, 136.7, 129.4,

128.8, 123.6, 123.0, 122.8, 120.5, 116.0, 115.9, 84.1, 82.5, 60.6, 36.5, 30.8, 27.6, 22.2, 14.1.


: 536.18; found: 536.4

48

Endo-4-pentanamidophenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-

sulfonate (2.11g)

Endo-2.11g was synthesized following general procedure C. The crude product was purified by



6) δ (ppm) 10.09 (s, 1H), 9.69 (d, J = 53.8 Hz, 2H), 7.70 – 7.66

(m, 2H), 7.30 – 7.23 (m, 4H), 7.20 – 7.16 (m, 2H), 6.79 – 6.76 (m, 2H), 6.71 – 6.67 (m, 2H),

5.70 (dd, J = 4.2, 1.2 Hz, 1H), 5.42 (dd, J = 4.7, 1.3 Hz, 1H), 4.41 (dt, J = 9.1, 4.3 Hz, 1H), 2.36

(t, J = 7.5 Hz, 2H), 1.74 (dd, J = 11.8, 4.5 Hz, 1H), 1.62 (q, J = 7.5 Hz, 2H), 1.37 (dt, J = 14.4,

7.3 Hz, 2H), 0.95 (d, J = 7.4 Hz, 3H). 13

C NMR (126 MHz, DMSO-d6) δ (ppm) 171.8, 157.7,

157.3, 143.8, 140.8, 138.8, 135.8, 129.4, 128.4, 123.6, 123.7, 123.1, 120.4, 115.9, 115.3, 83.9,

82.7, 59.4, 36.5, 29.9, 27.6, 22.2, 14.1.


Exo-4-(cyclopropanecarboxamido)phenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo[2.2.1]

hept-5-ene-2-sulfonate (2.11h)

Exo-2.11h was synthesized following general procedure C. The crude product was purified by


49


6) δ (ppm) 10.02 (s, 1H), 9.81 (s, 1H), 7.64 – 7.60 (m, 2H), 7.20 –


= 8.2, 4.5 Hz, 1H), 2.58 (ddd, J = 12.1, 8.3, 6.0 Hz, 1H), 2.25 – 2.12 (m, 3H), 1.09 (dd, J = 6.9,

2.2 Hz, 6H). 13

C NMR (126 MHz, DMSO-d6) δ (ppm) 170.1, 155.8, 155.6, 142.1, 136.6, 134.3,

127.3, 126.6, 121.5, 120.9, 120.7, 118.3, 113.9, 113.8, 82.0, 80.4, 58.6, 28.3, 12.8, 5.6.


: 520.15; found (M+H)+: 520.3

Endo-4-(cyclopropanecarboxamido)phenyl-5,6-bis(4-hydroxyphenyl)-7-oxabicyclo

[2.2.1]hept-5-ene-2-sulfonate (2.11h)

Endo- 2.11h was synthesized following general procedure C. The crude product was purified by

preparative HPLC to obtain pure product (9.7% yield) as a white solid.


6) δ (ppm) 10.35 (s, 1H), 9.65 (d, J = 57.2 Hz, 2H), 7.63 – 7.58 (m,

2H), 7.22 – 7.15 (m, 4H), 7.13 – 7.09 (m, 2H), 6.72 – 6.69 (m, 2H), 6.63 – 6.60 (m, 2H), 5.63

(dd, J = 4.2, 1.2 Hz, 1H), 5.34 (dd, J = 4.7, 1.3 Hz, 1H), 4.33 (dt, J = 9.1, 4.3 Hz, 1H), 1.77 –

1.72 (m, 1H), 1.66 (dd, J = 11.7, 4.5 Hz, 1H), 0.80 – 0.75 (m, 4H). 13

C NMR (126 MHz, DMSO-

d6) δ (ppm) 172.2, 157.8, 157.3, 143.8, 140.8, 138.8, 135.7, 129.7, 129.4, 124.2, 123.7, 123.1,

120.4, 115.9, 115.7, C4

= 83.9, 82.7, 59.4, 29.9, 14.9, 7.7.


: 520.15; found (M+H)+: 520.3

50

Estrogen Receptor Binding Affinity

The assays were conducted on full-length, purified human ERs [PanVera/Invitrogen

(Carlsbad, CA)], ERα and ERβ by a competitive radiometric binding assay, using 10 nM [3H]

estradiol as tracer ([6,7-3H]estra-1,3,5(10)-triene-3,17-β-diol, Ci/mmol, Amersham BioSciences,

Piscataway, NJ). Incubations were carried out for 18- 24 h at 0 °C. Hydroxyapatite (BioRad,

Hercules, CA) was used to absorb the receptor-ligand complexes, and free ligand was washed

away. Assays with uterine cytosol preparations were done in a related manner, as previously

described, but charcoal-coated dextran was used to adsorb free tracer. The binding affinities are

expressed as relative binding affinity (RBA) values with the RBA of estradiol set to 100%. The

values given are the average (range or SD of two to three independent determinations. Estradiol

binds to ERα and uterine cytosol ER with a Kd of 0.2 nM and to ERβ with a Kd of 0.5 nM.

Luciferase Assay

HepG2 cells were cultured in growth media containing Dulbecco’s minimum essential

medium (DMEM) (Cellgro by Mediatech, Inc., Manassas, VA) supplemented with 10% fetal

bovine serum (FBS) (Hyclone by Thermo Scientific, South Logan, UT) and 1% nonessential

amino acids (Cellgro), penicillin− streptomycin−neomycin antibiotic mixture, and Glutamax

(Gibco by Invitrogen Corp. Carlsbad, CA), and maintained at 37°C and 5% CO2.32,33

The cells

were transfected with 10.0 μg of 3X ERE-luciferase reporter plus 1.6 μg of ER expression vector

per 10 cm dish using Fugene HD reagent (Roche Applied Sciences, Indianapolis, IN). Next day,

the cells were resuspended in phenol red-free growth media containing 10% charcoal−dextran

sulfate-treated FBS, transferred to 384-well plates at a density of 20,000 cells/well, incubated

overnight at 37 ºC and 5% CO2, and treated in triplicate with increasing doses of ER ligands.

51

After 24 h, luciferase activity was measured using BriteLite reagent (Perkin-Elmer Inc., Shelton,

CT) according to the manufacturer’s protocol.

52

CHAPTER 3

SYNTHETIC METHODOLOGY FOR 7-DEOXO CARBON ANALOGS OF OBHS

(CBHS)

3.1 Introduction:

In addition to modifications to OBHS to design compounds targeting the ER ligand

binding site between helices 8 and 11, we sought to increase the affinity of OBHS through

modifications on the bicyclic core that occupies the bulk of the hydrophobic pocket. The 7-oxo-

bridge of OBHS is flanked by non-polar residues in the binding pocket, namely Leu 346 (helix

3), Phe 404 (β-sheet), Met 421 and Phe 425 (helix 8) (Fig. 3.1A). We hypothesized that a

hydrophobic group at the 7-oxo position of OBHS could increase the binding affinity without

disordering nearby helix 7 of ERα. TFMPV-E2 (trifluoromethyl-substituted phenylvinyl

estradiol), a full agonist that targets this binding hotspot possesses high affinity but also tend to

disorder helix 7 into a loop.19

Thus, replacing the oxo-bridge in OBHS with hydrophobic groups

like CF2 or CH2 (CBHS series) could increase the binding affinity to ER. With this in mind, we

devised an expedient strategy to synthesize these ligands with a Diels-Alder synthetic step. We

rationalized that a substituted cyclopentadiene as a diene for CBHS series would not be a good

choice due to rapid reversible [1,5]-hydrogen shifts that could lead to generation of multiple

regioisomers. Therefore, we attempted to synthesize CBHS analog from a potential

cyclopentadienone system, which can be readily made from benzyl/anisil analogs. However,

cyclopentadienone is very unstable and thus highly reactive due to its antiaromatic character,

leading to rapid formation of dimers. Another DA route was thus devised starting from 1,2,3,4-

tetrachloro-5,5-dimethoxycyclopenta-1,3-diene.

53

Fig 3.1 A) Oxo ring placed over a cleft of hydrophobic residues that may be further modified B)

TFMPV-E2, a flexible pocket for full agonists involves ligand induced remodeling of helix 7

into a loop, accommodating a bulky side group within the pocket.

3.2 Synthesis of CBHS Lead

We initially devised the synthesis of a limited series of CBHS analogs as shown in Fig.

3.2, starting from anisil and acetone, as shown in Scheme 3.1.

Fig. 3.2. CBHS lead and analogs.

As mentioned earlier, we expected the cyclopentadienone 3.5 to be very reactive. We

hypothesized that we might be able to trap this in situ generated reactive species with suitable

dienophiles under high concentration of reactive dienophiles such as maleimide and DMAD

(dimethyl acetylenedicarboxylate). To prepare the carbon-bridged (or norbornene) OBHS

A B

54

paralogs, a precursor to the diene, diarylhydroxycyclopentenone, was prepared from p-anisil and

acetone, and the very reactive diene precursor was generated in situ.28

The proposed synthetic

scheme for CBHS is shown in Scheme 3.1. The alcohol 3.4 was successfully synthesized in 69%

yield. Various reactions were conducted to trap the highly unstable cyclopentadienone

(dehydrated product of 3.4) with the various dienophiles, as discussed further.

Scheme 3.1 Proposed scheme for the synthesis of CBHS.

(A) Trapping Experiments with Maleimide and DMAD

The reaction of diene with maleimide was carried out with a large excess of maleimide

and Burgess reagent (Scheme 3.2A). Later we found that KHSO4 could be used as a dehydrating

agent in place of the expensive Burgess reagent. The Diels-Alder product was obtained as a

yellow solid in 48.5 % yield after purification. The product formed was exclusively endo isomer

of maleimide, as also confirmed with the crystal structure as shown in Fig. 3.3.

Trapping the diene formed in the reaction with DMAD was also carried out since we

thought the high reactivity of alkyne dienophiles could enable us to prepare OBHS derivatives by

starting with alkynyl phenyl sulfonates. A selective reduction of the double bond in conjugation

with sulfone/sulfonate can be carried out with NaBH4. However, we obtained a CO extruded

55

product upon heating leading to the formation of benzene analogs (Scheme 3.2B). Therefore, the

alkyne dienophiles were not pursued further as a synthetic strategy for OBHS analogs.

Scheme 3.2 Diels Alder reaction of alcohol 3.4 with maleimide and DMAD

Fig 3.2 Crystal structure of compound 3.9

(B) Trapping Experiments with Vinyl Phenyl Sulfonate

We carried out trapping of dienone 3.5 with phenyl vinyl sulfonate (Table 3.1). However

only dimer product was obtained as shown in Scheme 3.3. Next we attempted trapping under

various Lewis acid and temperature conditions (Table 3.1). Diels-Alder reaction in the presence

56

of excess of vinyl phenyl sulfonate and different Lewis acid also yielded dimer. Later screening

for the Diels Alder reaction was conducted at different dilutions (Table 3.2) with various Lewis

acid catalysts. The dimer formation was found to be suppressed at higher dilution. Reactions

carried out at 3.2 mM dilution showed traces of desired product, which was confirmed by mass

spectrometry. Disappointed with the results, we resorted to protection of ketone as a ketal in

order to form more stable 2,3-acylcyclopentadiene, 3.12, which cannot undergo [1,5]-hydrogen

shifts. However, this was also not successful (Scheme 3.4). Therefore, we attempted to protect

the tertiary alcohol as acetate with Ac2O (DMAP) and as silyl ethers (TMS, TIPS). However,

again no reaction was observed or dimer formation was observed when carried out at higher

temperatures. We have not yet attempted a benzyl ether protection, which we will carry out in the

future under neutral condition with alkyl pyridinium sulfonate.47

Scheme 3.3 Diels Alder reaction of alcohol 3.4 with vinyl phenyl sulfonate 2.3

Scheme 3.4 Protection of ketone 3.4

57

Table 3.1 Different reagents and solvents tried to generate 3.6.

Entry Reagents Solvent Conditions Remarks

1 TsCl / Pyridine CH2Cl2 RT No reaction

2 PTSA Glac.CH3COOH Reflux Dimer

3 Burgess Reagent CH2Cl2 RT No Reaction

4 Burgess Reagent CH2Cl2 -20 C No Reaction

5 Burgess Reagent THF reflux Dimer

6 Burgess Reagent 1,2-DCB 120 C Dimer

7 KHSO4 1,2-DCB 120 C Dimer

Table 3.2 Diels Alder reaction at different dilutions with Lewis Acid catalysts

Entry Lewis Acid Solvent Temp (C) Dilution Remarks

1 BF3OEt2 THF reflux 0.032 M Dimer


3 PTSA, BF3.OEt2 THF reflux 0.032 M Dimer



Table 3.3 Protection of ketone

Entry Reagents Solvent Temp (C) Catalyst Remarks

1 Ethylene Glycol CH2Cl2 RT PTSA No reaction

2 Ethylene Glycol CH2Cl2 RT BF3OEt2 No Reaction

3 Ethylene Glycol Toluene RT PPTS No Reaction

4 Ethylene Glycol Toluene 100 PPTS Dimer

C) Alternative Diels-Alder Route

The cyclopentadienone 3.5 is highly susceptible to dimerization because of its anti-

aromatic character. After our initial screens of Scheme 3.1, we decided to pursue a different

synthetic strategy from readily available 1,2,3,4-tetrachloro-5,5-dimethoxycyclopenta-1,3-diene

(TDCP) as shown in Scheme 3.4. The DA product 3.14 can be proto-dehalogenated with

nBu3SnH.34

Vinyl chlorides are typically unreactive in cross coupling reaction. However, some

reports have used NiCl2(dppp) as a catalyst in Kumada coupling.39

Tetrahalonorbornenes are

easily accessible via a Diels-Alder reaction.33

Thus, we carried out Diels-Alder reaction of

58

commercially available TDCP with the vinyl phenyl sulfonate, which yielded product 3.14 in

35% yield after purification. The following reactions to synthesize the CBHS analogs are

currently under development (Scheme 3.5).

Scheme 3.5 Modified synthetic scheme for CBHS.

3.3. Experimental Section

All the reagents were purchased from commercial suppliers and used without purification

unless specified. Dry solvents for the reactions were purchased from EMD Millipore and further

dried over molecular sieves (3Å). Glasswares was oven dried prior to the experiment and all

experiments unless specified is conducted under an inert atmosphere. Reaction progress was

monitored using thin layer chromatography (TLC) on Fisher TLC plates coated on aluminum

and visualized under UV light. Purification was done with automated flash chromatography

using silica gel Redisep column (Teledyne ISCO combiflash, Redisep column). 1H NMR and

13C

NMR spectra were obtained in a 400 or 500 MHz instrument and processed using Mnova

software. Chemical shifts are reported in ppm with TMS as internal standard. All NMR spectra

are referenced to either TMS or residual solvents.

4-Hydroxy-3,4-bis(4-methoxyphenyl)-2-cyclopentenone (3.4)

59

In a round bottomed flask p-anisil (1 g, 3.7 mmol) and KOH (1.04 g, 5.01 eq) were combined

with 50 mL of absolute ethanol. The reaction mixture was heated to reflux, acetone (2 mL) was

added dropwise. The reaction stirred at reflux for an additional 2 hours. After cooling to room

temperature the solvents were removed and partitioned between 3N HCl and EtOAc. The

aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with

brine, dried over anhydrous magnesium sulfate, and concentrated to yield as dark red oil. This oil

was purified by column chromatography (60:40 hexane: ethyl acetate) to yield 3.4 (0.6 g, 69%)

as a red oil, which turned into a foam upon drying under high vacuum.


3) δ (ppm) 7.53 – 7.48 (m, 2H), 7.34 – 7.29 (m, 2H), 6.85 – 6.80 (m,

2H), 6.79 – 6.75 (m, 2H), 6.55 – 6.52 (m, 1H), 3.75 (dd, J = 3.6, 1.1 Hz, 6H), 3.00 – 2.72 (m,

3H).

5,6-bis(4-Methoxyphenyl)-3,4,7,7-tetrahydro-1H-4,7-methanoisoindole-1,3,8(2H)-trione

(3.8)

Alcohol 3.4 and maleimide was combined in a vial, added THF and was stirred under N2 under

microwave conditions at 75 C for 1 h. The crude product obtained was purified by flash

chromatography (50:50 hexane: ethyl acetate) in combiflash to obtain 3.8 as yellow solid, which

upon recrystallization yielded bright orange crystals in 48 % yield.

60


3) δ (ppm) 7.14 – 7.09 (m, 4H), 6.71 – 6.66 (m, 4H), 3.75 (dd, J = 3.1,

1.7 Hz, 2H), 3.70 (s, 6H), 3.55 (dd, J = 3.2, 1.7 Hz, 2H).

LR-MS (ESI): calculated for C23H20NO5 (M+H)+

: 390.14; found: 390.3

Dimethyl-4,4''-dimethoxy-[1,1':2',1''-terphenyl]-4',5'-dicarboxylate (3.9)

Alcohol 3.4 and DMAD was combined in a vial with THF as solvent, and reaction was stirred

under N2 with microwave conditions at 75 C for 1 h. The crude product 3.9 was obtained as

yellow solid.

1H NMR (400 MHz, CDCl3) δ (ppm) 7.72 (d, J = 0.3 Hz, 2H), 7.06 – 7.03 (m, 4H), 6.77 – 6.75

(m, 4H), 3.91 (s, 6H), 3.77 (s, 6H).

3,3,5,6-tetrakis(4-methoxyphenyl)-2,3,3,4,7,7-hexahydro-1H-4,7-methanoindene-1,8-dione

(3.10)

Alcohol 3.4 and dienophile 3 was combined in a vial with THF as solvent, and reaction was

stirred under N2 with microwave conditions at 75 C for 1 h. The crude product obtained was

purified by flash chromatography (50:50 hexane: ethyl acetate) to obtain 3.10 as yellow solid.

61


3) δ (ppm) 7.38 – 7.33 (m, 2H), 7.23 – 7.18 (m, 2H), 7.05 – 7.00 (m,

2H), 6.88 – 6.81 (m, 4H), 6.79 (s, 1H), 6.74 – 6.70 (m, 2H), 6.47 – 6.43 (m, 2H), 6.41 – 6.36 (m,

2H), 4.24 (d, J = 1.7 Hz, 1H), 3.76 (d, J = 6.7 Hz, 6H), 3.70 (dd, J = 4.8, 1.7 Hz, 1H), 3.64 (d, J

= 11.2 Hz, 6H), 3.02 (d, J = 4.8 Hz, 1H).

1,2,3,4-tetrachloro-7,7-dimethoxy-5-(phenylsulfonyl)bicyclo[2.2.1]hept-2-ene (3.14)

1,2,3,4-tetrachloro-5,5-dimethoxycyclopenta-1,3-diene (1 g, 3.8 mmol) and vinyl phenyl sulfone

(0.770g, 1.2 eq) were combined in a vial, 2 drops of THF added, and reaction was stirred under

N2 with microwave conditions at 75 C for 1 h. The crude product obtained was purified by flash

chromatography (60:40 hexane: ethyl acetate) to obtain 3.14 as off-white solid (1.3 g, 79%).

1H NMR (400 MHz, Chloroform-d) δ (ppm) 7.96 – 7.90 (m, 2H), 7.70 – 7.65 (m, 1H), 7.61 –

7.53 (m, 2H), 4.07 (ddd, J = 9.5, 4.9, 0.5 Hz, 1H), 3.54 (d, J = 0.5 Hz, 3H), 3.49 (d, J = 0.5 Hz,

3H), 2.64 – 2.55 (m, 1H), 2.49 (ddd, J = 12.5, 4.9, 0.5 Hz, 1H).

62

CHAPTER 4

CONCLUSION AND FUTURE WORK

OBHS, a novel oxabicyclic ER ligand, represents an exciting therapeutic candidate for

endocrine-naïve and endocrine-resistant breast cancers. The potential of OBHS arises from its

unique ability to act as an ER antagonist while still maintaining NF-κB mediated anti-

inflammatory activity, as seen with endogenous ligand estradiol (E2). However, this activity is

only about 40-50% of what is seen with E2. It is our hypothesis that this activity that is present in

OBHS, and E2 (and another ligand from cyclofenil series), but lacking in tamoxifen, arises from

protein-protein interactions originating from the helix 8/11 interface (but may not necessarily

from a direct interaction of this interface with NF-κB complex). From a high-throughput X-ray

crystallographic screen of more than 300 ER ligands (belonging to many different series of anti-

estrogens) conducted in Nettles group at Scripps, Florida, it was ascertained that the ligands that

direct side chains towards this interface (and only few known ligands do), either slightly twist or

unwind the helix 11 end. This gives rise to or correlates with the ER-mediated NF-B inhibition

giving rise to anti-inflammatory activity. Based upon this observation, several series of OBHS

analogs were synthesized and tested previously.20

However, all these ligands resulted in reduced

binding affinity, while either not improving or reducing the anti-inflammatory activity. We

hypothesized that the narrow channel opened up by the phenyl sulfonate chain, and leading to the

solvent-exposed subpocket could be exploited for enhancing the anti-inflammatory activity.

However, the extremely narrow channel posed a significant problem in derivatization without

affecting the binding affinity. Thus, we chose two strategies: a) use an alkyne chain that could

provide the least bulkiest/narrowest group to exit this channel, and b) use an amide group to

63

possibly form H-bonds in the channel that could offset binding affinity loss from distorting the

binding pocket.

Based upon this hypothesis, we synthesized OBHS-P (propargyl) and OBHS-A (amide)

series of analogs to target the ligand binding site, with phenyl sulfonate chain pointing towards

helices 8 and 11, for possible enhancement in anti-inflammatory activity while increasing or

retaining the binding affinity (Fig. 2.1). From the compounds tested so far, exo diastereomers of

OBHS-A and OBHS-P series exhibited very good binding affinities for ERα. Interestingly, even

though the sub-pocket is surface-exposed, it is lined by multiple hydrophobic residues.

Interestingly, the compounds we tested so far with hydrophobic side chains also retained the

binding affinity of the parent OBHS-A and OBHS-P compounds. Previously, modifications with

halogens on the para position of phenyl group resulted in significant loss of binding affinity

(RBA<1%),20

which suggested a steric penalty for binding in this spot. However, from the series

tested here, most compounds retained the binding affinity, and some showed even higher activity

than OBHS. These compounds are currently being tested in mechanistic and cell studies. In the

future, we would also like to explore hydrophilic substitutions in OBHS-A series to explore this

sub-pocket, as well as to test the effect of these substitutions on anti-inflammatory activity. Also

OBHS analogs tested here lack the basic SERM side chain, and thus, it is expected that the

response from these ligands will further improve, at least for anti-proliferative activity, once the

basic side chain is added to the ring B phenol. However, its effect on agonistic activity in various

other tissues cannot be ascertained a priori as exemplified by the bone loss preventative activity

with raloxifene, but not with tamoxifen, even though both possess the SERM chain.

Finally, the work from OBHS so far suggests that we have found a novel way to not only

modulate anti-inflammatory activity, but also increase OBHS affinity through H-bond forming

64

substitutions. Interestingly, this cleft lies near the ER dimer interface. As pointed out in chapter

1, genomic activity of ER depends upon homodimerization (via direct ligand-dependent ER

dimer binding to EREs, ligand-dependent “tethered” mode binding to other tissue factors, or

ligand-independent mode where growth factors phosphorylate ER leading to its dimerization),

and possibly heterodimerization with ERβ.48

Thus, the inhibition of the dimer formation, or

reduction in the t1/2 residence time of dimer formation (in other words, faster exchange rate

between monomers), could lead to another approach of modulating ER function. In our future

detailed studies, we will test these analogs in ER dimer exchange assays with TR-FRET (ER

monomers labeled with a suitable time-resolved donor and acceptor).

4.1 CBHS

We will continue to explore the synthetic route as discussed in Chapter 3. However, for

the sake of time, we plan to utilize the successful synthesis of maleimide-DA compound 3.9 as a

surrogate to test our hypothesis that the 7-oxo replacements with hydrophobic groups will lead to

enhancement in binding affinity. An N-Me maleimide analog of OBHS with exo stereochemistry

was previously reported to have an RBA value of 0.020 %. Thus using this as a standard, we will

make an endo N-Me maleimide analogs of OBHS, CBHS (with carboxy, methylene and

difluoromethylene).

Figure 4.1 The proposed N-Me maleimide analogs of OBHS and CBHS for comparison of

binding affinities

65

We will evaluate these analogs in binding assays and in vitro studies to test our

hypothesis and make an assessment on the need to expand further resources for synthetic

strategies as discussed in Chapter 3.

4.2 NBHS series

Scheme 4.2 Proposed synthetic scheme for NBHS.

We also propose here modifications on the OBHS core to validate and further explore

structure-function of ER binding by replacing the 7-oxobridge with N-alkyl/acyl/aryl (NBHS

series) groups. The nitrogen-bridged bicyclic ligands, NBHS (Scheme 4.1A) can be prepared by

a Diels-Alder reaction with N-substituted pyrroles, using suitable conditions (strong electron

withdrawing groups35

or complexing agents that reduce pyrrole aromaticity38

). The pyrrole can

be prepared from dialkylation of tosylamide followed by McMurry coupling and oxidation of the

pyrrolidine ring. After the Diels-Alder reaction, the tosyl group can be cleaved with SmI2, and

the NBHS scaffold diversified by alkylating and acylating group. Finally the phenols can be

demethylated with BBr3 or Py.HCl as previously shown in Scheme 2.1. From our limited studies

with N-Ts/Boc and unsubstituted pyrrole (Dr. Zhong-Ke Yao), we have observed that, we can

obtain endo diastereomers with vinyl tolyl sulfone, which can be converted to exo diastereomer

66

by treating with a base. However, after Ts/Boc deprotection, the free amino analog undergoes

spontaneous retro DA reaction. Although we have yet not tested 3,4-dianisyl-N-Boc pyrrole

derivatives, we might have to derivatize amino group as acyl derivatives before we carry out DA

reaction. Hence this proposed scheme will likely require some modifications for final library

synthesis, and even then, we might be able to only obtain N-acyl derivatives.

67

REFERENCES

1. http://www.cancer.gov/types/breast

2. http://www.breastcancer.org/symptoms/diagnosis/hormone_status

3. Weitzmann, M. N.; Pacifici, R. Estrogen deficiency and bone loss: an inflammatory tale. J.

Clin. Invest. 2006, 116, 1186-1190.

4. Levin, E. R. Plasma membrane estrogen receptors. Trends Endocrinol. Metab. 2009, 20,

477-482.

5. http://www.cancer.gov/cancertopics 2014

6. Pettersson, K.; Gustafsson, J. A. Role of estrogen receptor beta in estrogen action. Annu.

Rev. Physiol. 2001, 63, 165-192.

7. Maximov, P. Y.; Lee, T. M.; Jordan, V. C. The discovery and development of selective

estrogen receptor modulators (SERMs) for clinical practice. Curr. Clin. Pharmacol. 2013, 8,

135-155.

8. Bai, Z.; Gust, R. Breast cancer, estrogen receptor and ligands. Arch. Pharm. (Weinheim)

2009, 342, 133-149.

9. Marino, M.; Galluzzo, P.; Ascenzi, P. Estrogen signaling multiple pathways to impact gene

transcription. Curr. Genomics 2006, 7, 497-508.

10. Ali, S.; Buluwela, L.; Coombes, R. C. Antiestrogens and their therapeutic applications in

breast cancer and other diseases. Annu. Rev. Med. 2011, 62, 217-232.

11. Osborne, C. K.; Zhao, H.; Fuqua, S. A. Selective estrogen receptor modulators: structure,

function, and clinical use. J. Clin. Oncol. 2000, 18, 3172-3186.

12. Banerjee, S.; Chambliss, K. L.; Mineo, C.; Shaul, P. W. Recent insights into non-nuclear

actions of estrogen receptor alpha. Steroids 2014, 81, 64-69.

http://www.breastcancer.org/symptoms/diagnosis/hormone_status

http://www.cancer.gov/cancertopics

68

13. H. W.; Perkins, R.; Tong, W.; Hong, H. Versatility or promiscuity: the estrogen receptors,

control of ligand selectivity and an update on subtype selective ligands. Int. J. Environ. Res.

Public Health 2014, 11, 8709-8742.

14. Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard, R. E.; Bonn, T.; Engstrom, O.;

Ohman, L.; Greene, G. L.; Gustafsson, J.-A.; Carlquist, M. Molecular basis of agonism and

antagonism in the oestrogen receptor. Nature 1997, 389, 753-758.

15. Zheng, Y.; Zhu, M.; Srinivasan, S.; Nwachukwu, J. C.; Cavett, V.; Min, J.; Carlson, K. E.;

Wang, P.; Dong, C.; Katzenellenbogen, J. A.; Nettles, K. W.; Zhou, H. B. Development of

selective estrogen receptor modulator (SERM)-like activity through an indirect mechanism

of estrogen receptor antagonism: defining the binding mode of 7-oxabicyclo[2.2.1]hept-5-

ene scaffold core ligands. Chem. Med. Chem. 2012, 7, 1094-1100.

16. Shoda, T.; Okuhira, K.; Kato, M.; Demizu, Y.; Inoue, H.; Naito, M.; Kurihara, M. Design

and synthesis of tamoxifen derivatives as a selective estrogen receptor down-regulator.

Bioorg. Med. Chem. Lett. 2014, 24, 87-89.

17. Mortensen, D. S.; Rodriguez, A. L.; Carlson, K. E.; Sun, J.; Katzenellenbogen, B. S.;

Katzenellenbogen, J. A. Synthesis and biological evaluation of a novel series of furans:

ligands selective for estrogen receptor α. J. Med. Chem. 2001, 44, 3838-3848.

18. Antoon, J. W.; White, M. D.; Slaughter, E. M.; Driver, J. L.; Khalili, H. S.; Elliott, S.;

Smith, C. D.; Burow, M. E.; Beckman, B. S. Targeting NF-kB mediated breast cancer

chemoresistance through selective inhibition of sphingosine kinase-2. Cancer Biol. Ther.

2011, 11, 678-689.

69

19. Nettles, K. W.; Bruning, J. B.; Gil, G.; O'Neill, E. E.; Nowak, J.; Hughs, A.; Kim, Y.;

DeSombre, E. R.; Dilis, R.; Hanson, R. N.; Joachimiak, A.; Greene, G. L. Structural

plasticity in the oestrogen receptor ligand-binding domain. EMBO Rep. 2007, 8, 563-568.

20. Zhou, H. B.; Comninos, J. S.; Stossi, F.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A.

Synthesis and evaluation of estrogen receptor ligands with bridged oxabicyclic cores

containing a diarylethylene motif: estrogen antagonists of unusual structure. J. Med. Chem.

2005, 48, 7261-7274.

21. Zhou, H. B.; Sheng, S.; Compton, D. R.; Kim, Y.; Joachimiak, A.; Sharma, S.; Carlson, K.

E.; Katzenellenbogen, B. S.; Nettles, K. W.; Greene, G. L.; Katzenellenbogen, J. A.

Structure-guided optimization of estrogen receptor binding affinity and antagonist potency

of pyrazolopyrimidines with basic side chains. J. Med. Chem. 2007, 50, 399-403.

22. Nettles, K. W.; Bruning, J. B.; Gil, G.; Nowak, J.; Sharma, S. K.; Hahm, J. B.; Kulp, K.;

Hochberg, R. B.; Zhou, H.; Katzenellenbogen, J. A.; Katzenellenbogen, B. S.; Kim, Y.;

Joachmiak, A.; Greene, G. L. NF kappa B selectivity of estrogen receptor ligands revealed

by comparative crystallographic analyses. Nat. Chem. Biol. 2008, 4, 241-247.

23. Bruning, J. B.; Parent, A. A.; Gil, G.; Zhao, M.; Nowak, J.; Pace, M. C.; Smith, C. L.;

Afonine, P. V.; Adams, P. D.; Katzenellenbogen, J. A.; Nettles, K. W. Coupling of receptor

conformation and ligand orientation determine graded activity. Nat. Chem. Biol. 2010, 6,

837-843.

24. Zheng, Y.; Zhu, M.; Srinivasan, S.; Nwachukwu, J. C.; Cavett, V.; Min, J.; Carlson, K. E.;

Wang, P.; Dong, C.; Katzenellenbogen, J. A.; Nettles, K. W.; Zhou, H. B. Development of

selective estrogen receptor modulator (SERM)-like activity through an indirect mechanism

70

of estrogen receptor antagonism: defining the binding mode of 7-oxabicyclo[2.2.1]hept-5-

ene scaffold core ligands. Chem. Med. Chem. 2012, 7, 1094-1100.

25. Bhagwanth, S.; Waterson, A. G.; Adjabeng, G. M.; Hornberger, K. R. Room-temperature

Pd-catalyzed amidation of aryl bromides using tert-butyl carbamate. J. Org. Chem. 2009,

74, 4634-4637.

26. Willis, M. C.; Chauhan, J.; Whittingham, W. G. A new reactivity pattern for vinyl

bromides: cine-substitution via palladium catalysed C-N coupling/Michael addition

reactions. Org. Biomol. Chem. 2005, 3, 3094-3095.

27. Elangovan, A.; Wang, Y.-H.; Ho, T. I. Sonogashira coupling reaction with diminished

homocoupling. Org. Lett. 2003, 5, 1841-1844.

28. Gavrin, L. K.; Lee, A.; Provencher, B. A.; Massefski, W. W.; Huhn, S. D.; Ciszewski, G.

M.; Cole, D. C.; McKew, J. C. Synthesis of pyrazolo[1,5-alpha]pyrimidinone regioisomers.

J. Org. Chem. 2007, 72, 1043-1046.

29. Yeh, W. L.; Shioda, K.; Coser, K. R.; Rivizzigno, D.; McSweeney, K. R.; Shioda, T.

Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor alpha

protein in MCF-7 cells require the CSK c-Src tyrosine kinase. PLoS One 2013, 8, 60889-

60892.

30. Wardell, S. E.; Marks, J. R.; McDonnell, D. P. The turnover of estrogen receptor alpha by

the selective estrogen receptor degrader (SERD) fulvestrant is a saturable process that is not

required for antagonist efficacy. Biochem. Pharmacol. 2011, 82, 122-130.

31. Teixeira, C.; Reed, J. C.; Pratt, M. A. Estrogen promotes chemotherapeutic drug resistance

by a mechanism involving Bcl-2 proto-oncogene expression in human breast cancer cells.

Cancer Res. 1995, 55, 3902-3907.

71

32. Sun, J.; Meyers, M. J.; Fink, B. E.; Rajendran, R.; Katzenellenbogen, J. A.;

Katzenellenbogen, B. S. Novel ligands that function as selective estrogens or antiestrogens

for estrogen receptor-alpha or estrogen receptor-beta. Endocrinol. 1999, 140, 800-804.

33. Shiau, A. K.; Barstad, D.; Radek, J. T.; Meyers, M. J.; Nettles, K. W.; Katzenellenbogen, B.

S.; Katzenellenbogen, J. A.; Agard, D. A.; Greene, G. L. Structural characterization of a

subtype-selective ligand reveals a novel mode of estrogen receptor antagonism. Nat. Struct.

Biol. 2002, 9, 359-364.

34. Khan, F. A.; Prabhudas, B. A simple and preparatively useful tributylstannane mediated

selective reduction and bridgehead functionalization of tetrahalonorbornene derivatives.

Tetrahedron Lett. 1999, 40, 9289-9292.

35. Rajasekar, S.; Anbarasan, P. Rhodium-catalyzed transannulation of 1,2,3-triazoles to

polysubstituted pyrroles. J. Org. Chem. 2014, 79 , 8428-8434.

36. Craig Jordan, V.; McDaniel, R.; Agboke, F.; Maximov, P. Y. The evolution of nonsteroidal

antiestrogens to become selective estrogen receptor modulators. Steroids 2014, 90, 3-12.

37. Wang, P.; Min, J.; Nwachukwu, J. C.; Cavett, V.; Carlson, K. E.; Guo, P.; Zhu, M.; Zheng,

Y.; Dong, C.; Katzenellenbogen, J. A.; Nettles, K. W.; Zhou, H. B. Identification and

structure-activity relationships of a novel series of estrogen receptor ligands based on 7-

thiabicyclo[2.2.1]hept-2-ene-7-oxide. J. Med. Chem. 2012, 55, 2324-2341.

38. Harman, W. D. The activation of aromatic molecules with pentaammineosmium(II). Chem.

Rev. 1997, 97, 1953-1978.

39. Liu, N.; Wang, Z. X. Kumada coupling of aryl, heteroaryl, and vinyl chlorides catalyzed by

amido pincer nickel complexes. J. Org. Chem. 2011, 76, 10031-10038.

72

40. Arnal J F., Fontaine C., Galés A B., Favre J., Laurell H., Lenfant F O., Gourdy P. Estrogen

receptors and endothelium. Arterioscler. Thromb. Vasc. Biol. 2010, 30, 1506-1512.

41. Morales, L. B.; Loo, K. K.; Liu, H. B.; Peterson, C.; Tiwari-Woodruff, S.; Voskuhl, R. R.

Treatment with an estrogen receptor alpha ligand is neuroprotective in experimental

autoimmune encephalomyelitis. J. Neurosci. 2006, 26, 6823-6833.

42. Peri, A.; Serio, M. Estrogen receptor-mediated neuroprotection: The role of the Alzheimer’s

disease-related gene seladin-1. Neuropsychiatr. Dis. Treat. 2008, 4, 817-824.

43. Minutolo, F.; Macchia, M.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Estrogen

receptor beta ligands: recent advances and biomedical applications. Med. Res. Rev. 2011,

31, 364-442.

44. Liu, J. Y.; Mooney, S. D. Characterization of ligand type of estrogen receptor by MD

simulation and mm-PBSA free energy analysis. Int. J. Biochem. Mol. Biol. 2011, 2, 190-

198.

45. Mohammed, H.; D’Santos, C.; Serandour, Aurelien A.; Ali, H. R.; Brown, Gordon D.;

Atkins, A.; Rueda, Oscar M.; Holmes, Kelly A.; Theodorou, V.; Robinson, Jessica L. L.;

Zwart, W.; Saadi, A.; Ross-Innes, Caryn S.; Chin, S.-F.; Menon, S.; Stingl, J.; Palmieri, C.;

Caldas, C.; Carroll, Jason S. Endogenous purification reveals GREB1 as a key estrogen

receptor regulatory factor. Cell Rep. 2013, 3, 342-349.

46. Pellegrini, C.; Gori, I.; Achtari, C.; Hornung, D.; Chardonnens, E.; Wunder, D.; Fiche, M.;

Canny, G. O. The expression of estrogen receptors as well as GREB1, c-MYC, and cyclin

D1, estrogen-regulated genes implicated in proliferation, is increased in peritoneal

endometriosis. Fertil. Steril. 2012, 98, 1200-1208.

73

47. Poon, K. W. C.; Dudley, G. B. Mix-and-heat benzylation of alcohols using a bench-stable

Pyridinium Salt. J. Org. Chem. 2006, 71, 3923-3927.

48. Powell, E.; Shanle, E.; Brinkman, A.; Li, J.; Keles, S.; Wisinski, K. B.;Huang W, Xu W.

Identification of estrogen receptor dimer selective ligands reveals growth-inhibitory effects

on cells that co-express ERα and ERβ. PLoS One 2012, 7, 30993-31006.

49. Smith, C. L.; O'Malley, B. W. Coregulator function: a key to understanding tissue

specificity of selective receptor modulators. Endocrinol. Rev. 2004, 25, 45-71.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Smith%20CL%5BAuthor%5D&cauthor=true&cauthor_uid=14769827

http://www.ncbi.nlm.nih.gov/pubmed/?term=O%27Malley%20BW%5BAuthor%5D&cauthor=true&cauthor_uid=14769827

74

APPENDIX

NMR Spectra List

1H NMR of Compound 2.1 ……………………………………………………………………...76

1H NMR of Compound 2.2 ……………………………………………………………………...77

13C NMR of Compound 2.2.…………………………………………………………..................78

1H NMR of Compound 2.3 ……………………………………………………………………...79

13C NMR of Compound 2.3.. …………………………………………………………................80

1H NMR of Compound 2.4 ……………………………………………………………………...81

1H NMR of Compound 2.5 ……………………………………………………………………...82

1H NMR of Compound 2.6a ………………………………………………………………….....83

13C NMR of Compound 2.6a …………………………………………………………................84

1H NMR of Compound 2.6b ………………………………………………………………….....85

13C NMR of Compound 2.6b ……………………………………………………………………86

1H NMR of Compound 2.8 ……………………………………………………………...............87

1H NMR of Compound 2.9 ……………………………………………………………………...88

1H NMR of Compound 2.7a ………………………………………………………………….....89

13C NMR of Compound 2.7a ……………………………………………………………………90

1H NMR of Compound 2.7b ………………………………………………………………….....91

13C NMR of Compound 2.7b ……………………………………………………………………92

1H NMR of Compound 2.10d……………………………………………………………………93

1H NMR of Compound 2.10e……………………………………………………………………94

1H NMR of Compound 2.7c …………………………………………………………………....95

13C NMR of Compound 2.7c ……………………………………………………………………96

1H NMR of Compound 2.10f…………………………………………………………………....97

13C NMR of Compound 2.10f …………………………………………………………………..98

1H NMR of Compound 2.10g……………………………………………………………………99

13C NMR of Compound 2.10g …………………………………………………………………100

1H NMR of Compound 2.10h…………………………………………………………………..101

13C NMR of Compound 2.10h …………………………………………………………………102

1H NMR of Compound 2.11a ………………………………………………………………….103

13C NMR of Compound 2.11a …………………………………………………………………104

1H NMR of Compound 2.11b ………………………………………………………………….105

13C NMR of Compound 2.11b …………………………………………………………………106

1H NMR of Compound 2.11c Exo ……………………………………………………………..107

13C NMR of Compound 2.11c Exo …………………………………………………………….108

1H NMR of Compound 2.11c Endo………………………………………………………….....109

13C NMR of Compound 2.11c Endo……………………………………………………………110

1H NMR of Compound 2.11e Exo …………………………………………………………......111

13C NMR of Compound 2.11e Exo …………………………………………………………….112

1H NMR of Compound 2.11e Endo…………………………………………………………….113

13C NMR of Compound 2.11e Endo……………………………………………………............114

1H NMR of Compound 2.11d Exo …………………………………………………………......115

13C NMR of Compound 2.11d Exo …………………………………………………………….116

1H NMR of Compound 2.11d Endo…………………………………………………………….117

75

13C NMR of Compound 2.11d Endo……………………………………………………...........118

1H NMR of Compound 2.11f Exo ………………………………………………………….......119

13C NMR of Compound 2.11f Exo …………………………………………………………….120

1H NMR of Compound 2.11f Endo………………………………………………………….....121

13C NMR of Compound 2.11f Endo……………………………………………………............122

1H NMR of Compound 2.11gExo ………………………………………………………….......123

13C NMR of Compound 2.11g Exo …………………………………………………………….124

1H NMR of Compound 2.11g Endo…………………………………………………………….125

13C NMR of Compound 2.11g Endo……………………………………………………...........126

1H NMR of Compound 2.11h Exo …………………………………………………………......127

13C NMR of Compound 2.11h Exo ………………………………………………………….....128

1H NMR of Compound 2.11h Endo…………………………………………………………….129

13C NMR of Compound 2.11h Endo……………………………………………………...........130

1H NMR of Compound 3.4 ……………………………………………………………………131

1H NMR of Compound 3.8 …………………………………………………………………….132

1H NMR of Compound 3.10 …………………………………………………………………...133

1H NMR of Compound 3.14 …………………………………………………………………...134

Mass Spectra List

LR-MS of Compound 2.1.……………………………………………………………...............135

LR-MS of Compound 2.2..……………………………………………………………..............136

LR-MS of Compound 2.3..……………………………………………………………..............137

LR-MS of Compound 2.6a……………………………………………………………..............138

LR-MS of Compound 2.6b……………………………………………………………..............139

LR-MS of Compound 2.7a …………………………………………………….........................140

LR-MS of Compound 2.7b …………………………………………………….........................141

LR-MS of Compound 2.7c……………………………………………………..........................142

LR-MS of Compound 2.10e……………………………………………………........................143

LR-MS of Compound 2.10g……………………………………………………........................144

LR-MS of Compound 2.11a Exo…………………………………………………….................145

LR-MS of Compound 2.11b Exo…………………………………………………….................146

LR-MS of Compound 2.11d Exo…………………………………………………….................147

LR-MS of Compound 2.11d Endo ………………………………………………......................148

LR-MS of Compound 2.11c Exo…………………………………………………….................149

LR-MS of Compound 2.11c Endo ………………………………………………......................150

LR-MS of Compound 2.11e Exo…………………………………………………….................151

LR-MS of Compound 2.11e Endo ………………………………………………......................152

LR-MS of Compound 2.11f Exo……………………………………………………..................153

LR-MS of Compound 2.11f Endo ……………………………………………….......................154

LR-MS of Compound 2.11g Exo…………………………………………………….................155

LR-MS of Compound 2.11g Endo ………………………………………………......................156

LR-MS of Compound 2.11hExo……………………………………………………..................157

LR-MS of Compound 2.11h Endo ………………………………………………......................158

LR-MS of Compound 3.4……………………………………………………………................159

1H NMR of compound 2.1

1H NMR (400MHz, CDCl3) δ H13,17 = 7.34 – 7.28 (m, 2H), H6,10 =

7.26 – 7.21 (m, 2H), H14,16 = 6.87 – 6.83 (m, 2H), H7,9 = 6.81 – 6.75

(m, 2H), H5 = 5.06 (s, 2H), H8,11 = 3.76 (d, J = 6.1 Hz, 6H).

76


1H NMR (500 MHz, DMSO-d6) δ H19= 10.08 (s, 1H), H12 = 9.70

(s, 1H), H14,18 = 7.28 – 7.24 (m, 2H), H7,11. = 7.18 – 7.14 (m,

2H), H8,10 = 6.82 (d, J = 8.6 Hz, 2H), H15,17 = 6.78 – 6.74 (m,

2H), H5 = 5.25 (s, 2H).

77

13C NMR of compound 2.2

13C NMR (126 MHz, DMSO-d6) δ C2 = 174.2, C16 = 160.0, C9 = 157.9,

C4 = 156.0, C6 = 130.9, C3 = 129.7, C3 = 122.3, C14,18= 121.9, C7,11 =

121.8, C15,17= 116.19, C8,10 = 115.9, C5 = 70.6.

78


1H NMR (500 MHz, DMSO-d6) δ H12,19 = 9.45 (s, 2H), H2,5 = 7.75 (s,

2H), H7,11,14,18 = 7.04 – 6.98 (m, 4H), H8,10,15,17 = 6.74 – 6.68 (m, 4H).

79

13C NMR of compound 2.3

13C NMR (126 MHz, DMSO-d6) δ C9,16 = 156.9, C2,5 = 140.6, C7,11,14,18

= 129.7, C3,4 = 125.5, C6,13 = 122.8, C8,10,15,17 = 115.7.

80


1H NMR (400 MHz, CDCl3) δ H3,5 = 7.66 – 7.61 (m, 2H), H2,6 =

6.95 – 6.90 (m, 2H), H7 = 6.58 (dd, J = 16.6, 10.0 Hz, 1H), H8 =

6.31 (dd, J = 16.6, 0.7 Hz, 1H), H8 = 6.12 (dd, J = 9.9, 0.7 Hz, 1H).

81


1H NMR (500 MHz, DMSO-d6) δ H13,20 = 9.68 (d, J = 17.3 Hz,

2H), H23,25 = 7.77 – 7.73 (m, 2H), H8,12,15,19,22,26 = 7.18 – 7.09 (m,

7H), H9,11,16,19 = 6.77 – 6.68 (m, 5H), H4 = 5.64 (d, J = 1.2 Hz, 1H),

H1 = 5.44 (dd, J = 4.3, 1.1 Hz, 1H), H2 = 3.86 (dd, J = 8.2, 4.4 Hz,

1H), H3 = 2.22 (dt, J = 11.9, 4.4 Hz, 1 H), H3 = 2.15 (dd, J = 12.1,

8.3 Hz, 1H).

82

1H NMR of compound 2.6a

1H NMR (500 MHz, DMSO-d6) δ H13,20 = 9.79 (s, 2H),

), H23,25 = 7.43 – 7.39 (m, 2H), H22,26 = 7.30 – 7.25 (m,

2H), H8,12,15,19, = 7.19 – 7.12 (m, 4H), H9,11,16,19 = 6.76 –

6.70 (m, 4H), H4 = 5.65 (d, J = 1.2 Hz, 1H), H32 = 5.55

(s, 1H), H1 = 5.43 (dd, J = 4.3, 1.2 Hz, 1H), H2 = 3.85

(dd, J = 8.2, 4.4 Hz, 1H), H3 = 2.21 (dt, J = 12.0, 4.4

Hz, 1H), H3 = 2.13 (dd, J = 12.1, 8.3 Hz, 1H), H30,31 =

1.46 (s, 6H).

83

13C NMR of compound 2.6a

13C NMR (126 MHz, DMSO-d6) δ C10 = 157.8, C17 =

157.7, C21 = 141.2, C23,25 = 136.6, C7,14 = 133.3, C5=

129.4, C6 = 128.8, C8,12 = 123.5, C15,19 = 123.1, C24 =

122.83, C22,26 = 122.15, C9,11 = 116.0, C16,18 = 115.9,

C24= 97.5, C28= 84.1, C27= 82.5, C29 = 64.0, C303,1 =

31.9, C3= 30.8.

84

1H NMR of compound 2.6b

1H NMR (500 MHz, DMSO-d6) δ H13,20 = 9.75 (s, 2H),

H23,25 = 7.43 (d, J = 8.7 Hz, 2H), H22,26 = 7.27 (d, J =

8.7 Hz, 2H), H8,12,15,19, = 7.18 – 7.12 (m, 4H), H9,11,16,19

= 6.76 – 6.69 (m, 4H), H4 = 5.65 (s, 1H), H31 = 5.43 (d,

J = 4.2 Hz, 1H), 4.58 (t, J = 6.6 Hz, 1H), H2 = 3.85 (dd,

J = 8.2, 4.4 Hz, 1H), H3 = 2.21 (dt, J = 12.0, 4.4 Hz,

1H), H3 = 2.14 (dd, J = 12.1, 8.3 Hz, 1H), H30, = 1.37

(d, J = 6.6 Hz, 3H).

85

13C NMR of compound 2.6b


157.8, C21 = 141.2, C23,25 = 136.7, C7,14 = 133.3, C5=

129.5, C6 = 128.8, C8,12, = 123.5, C15,19 = 123.1, , C24 =

122.8, C22,26 = 122.0, C9,11 = 116.1, C16,18 = 115.9, C28=

94.8, C27= 82.5, C29 = 58.1,C30 = 24.9.

86


1H NMR (400 MHz, CDCl3) δ H3,5 = 7.36 (d, J = 9.0 Hz,

2H), H2,6 = 7.13 (d, J = 9.0 Hz, 2H), H7 = 6.66 – 6.57

(m, 1H), H8 = 6.32 (dd, J = 16.6, 0.7 Hz, 1H), H8 = 6.13

(dd, J = 10.0, 0.6 Hz, 1H), H12,13,14 = 1.49 (s, 9H).

87


1H NMR (400 MHz, DMSO-d6) δ H7 = 7.15 – 7.06

(m, 1H), H2,6 =7.03 (d, J = 8.8 Hz, 2H), H3,5 = 6.76

(d, J = 8.9 Hz, 2H), H8 = 6.31 (dd, J = 10.0, 0.7 Hz,

1H), H8 = 6.20 (dd, J = 16.6, 0.7 Hz, 1H).

88


1H NMR (400 MHz, DMSO-d6) δ H9 = 10.10 (s, 1H), H3,5 =

7.67 – 7.60 (m, 2H), H2,6 = 7.24 – 7.20 (m, 2H), H7 = 7.20 –

7.15 (m, 1H), H8 = 6.37 (dd, J = 10.0, 0.8 Hz, 1H), H8 = 6.29 –

6.24 (m, 1H), H11 = 2.04 (s, 3H).

89

13C NMR of compound 2.7a

13C NMR (126 MHz, DMSO-d6) δ C10= 168.9, C1= 144.4, C4 = 138.9, C7 = 133.7, C3,5 = 132.6, C2,6 = 123.1, C8 = 120.4, C11 = 24.4.

90

1H NMR of compound 2.7b

1H NMR (400 MHz, CDCl3) δ H11 = 8.32 (d, J = 1.7 Hz,

1H), H3,5 = 7.63 – 7.31 (m, 2H), H2,6 = 7.19 – 7.08 (m, 2H),

H7 = 6.60 (ddd, J = 16.6, 10.0, 6.5 Hz, 1H), H8 = 6.31 (ddd,

J = 16.6, 10.4, 0.7 Hz, 1H), H8 = 6.13 (td, J = 9.8, 0.7 Hz,

1H).

91

1H NMR of compound 2.10d

1H NMR (400 MHz, CDCl3) δ H3,5 = 7.55 (d, J = 8.9 Hz, 2H), H2,6

= 7.18 (d, J = 9.0 Hz, 3H), H7 = 6.69 – 6.61 (m, 1H), H8 = 6.35 (dd,

J = 16.6, 0.7 Hz, 1H), H8 = 6.16 (dd, J = 9.9, 0.6 Hz, 1H), H11 =

2.40 (d, J = 7.5 Hz, 2H), H12 = 1.25 (t, J = 7.5 Hz, 3H).

92

1H NMR of compound 2.10e

1H NMR (400 MHz, CDCl3) δ H3,5 = 7.54 (d, J = 9.0 Hz, 2H),

H2,6 = 7.18 – 7.12 (m, 2H), H7 = 6.68 – 6.57 (m, 1H), H8 = 6.32

(dt, J = 16.6, 0.5 Hz, 1H), H8 = 6.14 (dt, J = 10.0, 0.6 Hz, 1H),

H11 = 2.49 (s, 1H), H12,13 = 1.23 (dd, J = 6.8, 1.3 Hz, 6H).

93

13C NMR of compound 2.10e

13C NMR (126 MHz, CDCl3) δ C1= 137.1, C7 = 131.9, C4

= 131.8, C3,5 = 122.8, C8 = 122.6, C2,6 = 120.7, 77.2, C11

= 36.6C12,13 = 19.5.

94

1H NMR of compound 2.7c

1H NMR (400 MHz, CDCl3) δ H3,5 = 7.57 – 7.51 (m, 2H), H7 = 7.30

(s, 1H), H2,6 = 7.18 – 7.13 (m, 2H), H7 = 6.64 (dd, J = 16.6, 10.0 Hz,

1H), H8 = 6.34 (dd, J = 16.6, 0.7 Hz, 1H), H8 = 6.15 (dd, J = 10.0, 0.7

Hz, 1H), H11 = 2.38 – 2.29 (m, 2H), H13 = 1.81 – 1.67 (m, 2H), H13 =

0.99 (t, J = 7.4 Hz, 3H).

95

13C NMR of compound 2.7c

13C NMR (126 MHz, DMSO-d6) δ C10 = 171.7, C1= 144.3, C4 = 138.9, C7 = 133.7, C2,6 = 132.6, C3,5 = 123.1, C8 = 120.5, C11 = 38.7, C12 = 18.9, C13= 14.0.

96

1H NMR of compound 2.10f

1H NMR (400 MHz, CDCl3) δ H10 = 8.29 (s, 1H), H3,5 = 7.60 –

7.54 (m, 2H), ), H14,16 = 7.33 – 7.26 (m, 2H), H2,6 = 7.18 – 7.13 (m,

2H), H15 = 7.05 – 6.98 (m, 1H), H13,17 = 6.97 – 6.89 (m, 2H), H7 =

6.59 (dd, J = 16.6, 10.0 Hz, 1H), H8 = 6.29 (dd, J = 16.6, 0.7 Hz,

1H), H8 = 6.11 (dd, J = 10.0, 0.7 Hz, 1H), 4.56 (s, 2H).

97

13C NMR of compound 2.10f

13C NMR (126 MHz, CDCl3) δ C10 = 166.4, C14,16 = 156.8,

C1 = 145.7, C4 = 135.8, C7 = 131.9, C2,6 = 131.8, C13,17 =

129.9, C15 = 129.6, C3,5 = 123.0, 122.6, C8 = 121.2.

98

1H NMR of compound 2.10g

1H NMR (400 MHz, CDCl3) δ 7.53 (s, 1H), H3,5 = 7.51 – 7.45

(m, 2H), H2,6 = 7.11 – 7.05 (m, 2H), H7 = 6.58 (dd, J = 16.6, 10.0

Hz, 1H), H8 = 6.27 (dd, J = 16.6, 0.7 Hz, 1H), H8 = 6.10 (dd, J =

10.0, 0.7 Hz, 1H), H11 = 2.32 – 2.26 (m, 2H), H12 = 1.66 – 1.58

(m, 2H), H13 = 1.36 – 1.27 (m, 2H), H14 = 0.86 (t, J = 7.4 Hz,

3H).

99

13C NMR of compound 2.10g

13C NMR (126 MHz, CDCl3) δ C10 = 171.7, C1= 145.0, C4 =

137.2, C7 = 132.0, C2,6 = 131.7, C3,5 = 122.7, C8 = 120.8 C11 =,

37.3, C12 = 27.6, C13= 22.3, C14= 13.8.

100

1H NMR of compound 2.10h

1H NMR (400 MHz, DMSO-d6) δ H10 = 10.35 (s, 1H), H3,5 =

7.67 – 7.61 (m, 2H), H2,6 = 7.23 – 7.19 (m, 2H), H7 = 7.20 –

7.14 (m, 1H), H8 = 6.36 (dd, J = 10.0, 0.8 Hz, 1H), H8 = 6.26

(dd, J = 16.5, 0.8 Hz, 1H), H11 = 1.79 – 1.71 (m, 1H), H12,13 =

0.83 – 0.76 (m, 4H).

101

13C NMR of compound 2.10h

13C NMR (126 MHz, DMSO-d6) δ C10 = 172.2, C1= 144.3, C4

= 138.8, C7 = 133.6, C2,6 = 132.6, C3,5 = 123.1, C8 = 120.4,

C11 =14.9, C12 ,13 = 7.7.

102


1H NMR (500 MHz, DMSO-d6) δ H27 = 10.10 (s,

1H), H13,20 = 9.73 (s, 2H), H23,25 = 7.61 – 7.56 (m,

2H), H22,26 ,8,12,15,18 = 7.21 – 7.13 (m, 6H), H9,11,16,19

= 6.76 – 6.69 (m, 4H), H4 = 5.63 (d, J = 1.3 Hz, 1H),

H1 = 5.43 (dd, J = 4.3, 1.3 Hz, 1H), H2 = 3.77 (dd, J

= 8.2, 4.4 Hz, 1H), H3= 2.24 – 2.12 (m, 2H), H29 =

2.04 (s, 3H).

103

13C NMR of compound 2.11a Exo

13C NMR (126 MHz, DMSO-d6) δ C28 = 168.8, δ C10, =

157.9, C17 = 157.8, C21 = 144.3, C23,25 = 141.2, ,C24 =

138.7, C7,14 = 136.7, C5 = 129.4, C6 = 128.8, C8,12, =

123.6, C15,19 = 123.0, C24 = 122.8, C22,26 = 120.5, C9,11, =

116.0, C6,18 = 115.9, C4 = 84.1, C1 = 82.5, C2 = 60.6, C29

= 30.8, C3 = 24.4.

104

1H NMR of compound 2.11b Exo

1H NMR (500 MHz, DMSO-d6) δ H27 = 10.40 (s,

1H), H13,20 = 8.28 (s, 1H), H23,25 = 7.64 – 7.57 (m,

2H), H22,26 = 7.26 – 7.21 (m, 2H), H8,12,15,18 = 7.19 –

7.13 (m, 4H), H9,11,16,19 = 6.77 – 6.69 (m, 4H), H4 =

5.63 (d, J = 1.2 Hz, 1H), H1 = 5.43 (dd, J = 4.3, 1.2

Hz, 1H), H2 = 3.79 (dd, J = 8.1, 4.4 Hz, 1H), H3=

2.26 – 2.12 (m, 2H).

105

13C NMR of compound 2.11b Exo

13C NMR (126 MHz, DMSO-d6) δ C28 = 166.4,

163.1, 160.2, C10 = 158.0, C17 = 157.9, C21 = 144.7,

C23,25 = 141.2, C23,25 = 137.9, C924 = 137.6, 136.7,

C5, = 129.4, C6 = 128.8, C8,12, = 123.8, 123.5, C15,19

= 123.3, C24 = 122.7, C22,26 = 120.7, 119.0, C9,11 =

116.1, C16,18 = 115.9, C4 = 84.1, C1 = 82.5, C2 =

60.6, C3 = 30.6.

106

1H NMR of compound 2.11c Exo

1H NMR (400 MHz, DMSO-d6) δ H27 = 10.04 (s, 1H),

H13,20 = 9.74 (s, 2H), H23,25 = 7.64 – 7.57 (m, 2H), H22,26,

8,12,15,18 = 7.21 – 7.11 (m, 6H), H9,11,16,19 = 6.78 – 6.68 (m,

4H), H4 = 5.62 (d, J = 1.1 Hz, 1H), H1 = 5.42 (dd, J = 4.2,

1.2 Hz, 1H), H2 = 3.76 (dd, J = 8.1, 4.5 Hz, 1H), H3= 2.31

(m, J = 7.5 Hz, 2H), H29 = 2.25 – 2.11 (m, 2H), H30 = 1.07

(t, J = 7.5 Hz, 3H).

107

13C NMR of compound 2.11c Exo

13C NMR (126 MHz, DMSO-d6) δ C28 = 172.5, C10, =

158.2, C17 = 158.1, C21 = 141.0, C23,25 = 138.8, C7,14 =

136.6, C5,= 129.4, C6 = 128.7, C8,12, = 123.3, C15,19 =

123.0, C24 = 122.5, C22,26 = 120.4, C9,11, = 116.1, C16,18 =

115.9, C4 = 84.1, C1 = 82.5, C2 = 60.5, C29 = 30.6, C3 =

29.9, C30 = 10.0.

108

1H NMR of compound 2.11c Endo


H13,20 = 9.72 (s, 2H), H23,25 = 7.67 – 7.57 (m, 2H), H22,26,

8,12,15,18 = 7.25 – 7.17 (m, 4H), H22,26, =7.14 – 7.11 (m,

2H), H9,11,16,19 = 6.74 – 6.62 (m, 4H), H4 = 5.64 (dd, J =

4.1, 1.2 Hz, 1H), H1 = 5.36 (dd, J = 4.7, 1.2 Hz, 1H), H2 =

4.35 (dt, J = 9.1, 4.3 Hz, 1H), H3 = 2.54 (m, 1H), H29 =

2.32 (q, J = 7.5 Hz, 2H), H3= 1.67 (dd, J = 11.7, 4.5 Hz,

1H), H30 = 1.08 (t, J = 7.5 Hz, 3H).

109

13C NMR of compound 2.11c endo

13C NMR (126 MHz, DMSO-d6) δ C28 = 172.5, 166.2,

C10, = 157.8, C17 = 57.3, C21 = 140.8, C23,25 = 138.8, C7,14

= 135.7, C5,= 129.4, C6 = 129.4, 124.2, C8,12, = 123.6,

C15,19 = 123.1, C24 = 122.8, C22,26 = 120.4, C9,11, =

115.9, C16,18 = 115.3, C4 = 83.9, C1 = 82.7, C2 = 59.4, C3

= 29.9, C30 = 10.0.

110

1H NMR of compound 2.11e exo


H13,20 = 9.80 (s, 2H), H23,25 = 7.63 – 7.57 (m, 2H), H22,26,

8,12,15,18 = 7.20 – 7.10 (m, 6H), H9,11,16,19 = 6.77 – 6.66 (m,

4H), H4 = 5.62 (d, J = 2.4 Hz, 1H), H1 = 5.42 (d, J = 4.0

Hz, 1H), H2 = 3.76 (dd, J = 8.2, 4.5 Hz, 1H), H3= 2.57

(ddd, J = 12.1, 8.3, 6.0 Hz, 1H), H3= 2.21 (dt, J = 12.0, 4.3

Hz, 1H), H29 = 2.15 (dd, J = 12.0, 8.3 Hz, 1H), H30 ,31=

1.08 (dd, J = 6.9, 2.2 Hz, 6H).

111

13C NMR of compound 2.11e exo

13C NMR (126 MHz, DMSO-d6) δ C28 = 175.8, C10,17 =

157.9, 157.8, 144.2, C21 = 141.2, C23,25 = 138.8, C7,14 =

136.7, C5,= 129.4, C6 = 128.7, C8,12, = 123.6, C15,19 =

123.0, C24 = 122.7, C22,26 = 120.6, C16,18 = 116.0, C16,18 =

115.9, C4 = 84.1, C1 = 82.5, C2 = 60.5, C29 = 35.2 , C3 =

30.8, C30,31= 19.

112

1H NMR of compound 2.11e endo


H13,20 = 9.81 (s, 1H), H23,25 = 7.64 – 7.60 (m, 2H), H22,26,

8,12,15,18 = 7.20 – 7.14 (m, 6H), H9,11,16,19 = 6.76 – 6.70 (m,

4H), H4 = 5.63 (d, J = 2.4 Hz, 1H), H1 = 5.43 (d, J = 4.0

Hz, 1H), H2 = 3.77 (dd, J = 8.2, 4.5 Hz, 1H), H3= 2.58

(ddd, J = 12.1, 8.3, 6.0 Hz, 1H), H3,29= 2.25 – 2.12 (m,

2H), H30,31= 1.09 (dd, J = 6.9, 2.2 Hz, 6H).

113

13C NMR of compound 2.11e endo

13C NMR (126 MHz, DMSO-d6) δ C28 = 175.8, C10,17 =

157.9, 157.7, 144.2, C21 = 141.2, C23,25 = 138.8, C7,14 =

136.7, C5,= 129.4, C6,= 128.8, C8,12, = 123.6, C15,19 =

123.0, C24 = 122.8, C22,26 = 120.6, C9,11,16,18 = 116.0,

C9,11,16,18 = 115.9, C4 = 84.1, C1 = 82.5, C2 = 60.5, C29 =

35.3, C2 = 30.0, C30,31= 19.9.

114

1H NMR of compound 2.11d exo


H13,20 = 9.78 (s, 2H), H23,25 = 7.63 – 7.57 (m, 2H), H22,26,

8,12,15,18 = 7.20 – 7.11 (m, 6H), H9,11,16,19 = 6.78 – 6.67 (m,

4H), H4 = 5.64 – 5.60 (m, 1H), H1= 5.43 (d, J = 4.0 Hz,

1H), H2 = 3.77 (dd, J = 8.2, 4.5 Hz, 1H), H29 = 2.28 (t, J =

7.3 Hz, 2H), H3 = 2.22 (dt, J = 12.0, 4.4 Hz, 1H), H3 =

2.15 (dd, J = 12.1, 8.2 Hz, 1H), H30 = 1.60 (q, J = 7.4 Hz,

2H), H31= 0.91 (t, J = 7.4 Hz, 3H).

115

13C NMR of compound 2.11d exo


157.9, C,17 = 157.8, 144.27, C21 = 141.23, C23,25 = 138.78,

C7,14 = 136.77 C5,= 129.4, C6 = 128.8, C8,12, = 123.7, C15,19

= 123.1, C24 = 123.0, C22,26 = 120.5, C9,11= 116.0, C9,11,16,18

= 115.9, C4 = 84.1, C1 = 82.6, C2 = 60.5, C29= 39.0, C,3=

29.8, C,30= 18.9, C,31= 14.1.

116

1H NMR of compound 2.11d endo


H23,25 = 7.66 – 7.61 (m, 2H), H22,26, 8,12,15,18 = 7.25 – 7.10

(m, 6H), H9,11,16,19 = 6.74 – 6.60 (m, 4H), H4 = 5.64 (dd, J

= 4.2, 1.2 Hz, 1H), H1= 5.36 (dd, J = 4.7, 1.1 Hz, 1H), H2

= 4.35 (dt, J = 9.1, 4.4 Hz, 1H), H3 = 2.57 (m, 1H), H29 =

2.28 (t, J = 7.3 Hz, 2H), H3 = 1.68 (dd, J = 11.7, 4.5 Hz,

1H), H30 = 1.60 (dt, J = 14.6, 7.4 Hz, 2H), H31= 0.91 (t, J

= 7.4 Hz, 3H).

117

13C NMR of compound 2.11dendo

13C NMR (126 MHz, DMSO-d6) δ C27 = 171.7, C10=

157.8, C,17 = 157.3, C21 = 143.8, , C23,25 = 138.8, C7,14 =

136.77 , C5,= 129.4, C6 = 129.4, C8,12, = 124.2, C15,19 =

123.6, C24= 123.1, C22,26 = 120.4, C9,11= 115.9, C16,18 =

115.3, C4 = 84.1, C1 = 82.6, , C2 = 60.5 C,29 = 38.7, C,30=

29.8, C30= 18.9, C,31= 14.0.

118

1H NMR of compound 2.11f exo


H13,20= 9.79 (s, 2H), H23,25 = 7.67 – 7.63 (m, 2H), H22,26 =

7.33 – 7.29 (m, 2H), H8,12, = 7.24 – 7.20 (m, 2H), H9,11,16,19

= 7.19 – 7.13 (m, 4H), H15,18,34 = 7.01 – 6.96 (m, 3H),

H32,33,35,36 = 6.76 – 6.69 (m, 4H), H4 = 5.63 (d, J = 1.2 Hz,

1H), H1= 5.43 (dd, J = 4.3, 1.2 Hz, 1H), H29 = 4.69 (s,

2H), H2 = 3.78 (dd, J = 8.2, 4.5 Hz, 1H), H3 = 2.23 (dt, J =

12.1, 4.4 Hz, 1H), H3 = 2.16 (dd, J = 12.1, 8.2 Hz, 1H).

119

13C NMR of compound 2.11f exo


158.1, C,10 = 157.8, C17, = 157.7, 144.8, C21 = 141.2, C23,25

= 137.7, C7,14 = 136.7, C33,35, = 130.0, C5,= 129.5, C6 =

128.8, C8,12, = 123.6, C15,19 = 123.1, C24 = 122.8, C34, =

121.7, C22,26 = 121.4, C9,11= 116.0, C16,18 = 115.9, C32,36, =

115.1, C4 = 84.1, C1 = 82.5, C29 = 67.4, C2 = 60.6, C2 =

30.8.

120

1H NMR of compound 2.11f endo


H13,20 = 9.74 (s, 2H), H23,25 = 7.64 – 7.56 (m, 2H),

H22,26, 8,12,15,18 = , 7.21 – 7.13 (m, 6H), H9,11,16,19 = 6.77

– 6.68 (m, 4H), H4 = 5.63 (d, J = 1.2 Hz, 1H), H1 =

5.43 (dd, J = 4.4, 1.2 Hz, 1H), H2 = 3.77 (dd, J = 8.2,

4.5 Hz, 1H), H29 = 2.30 (t, J = 7.5 Hz, 2H), H3 = 2.22

(dt, J = 12.0, 4.4 Hz, 1H), H3 = 2.16 (dd, J = 12.1, 8.3

Hz, 1H), H30 = 1.57 (tt, J = 7.6, 6.5 Hz, 2H), H31 = 1.35

– 1.27 (m, 2H), H32 = 0.90 (t, J = 7.3 Hz, 3H).

121

13C NMR of compound 2.11f endo


157.9, C,17 = 157.7, 144.2, C21 = 141.2, C23,25 = 138.7,

C7,14 = 136.7, C5,= 129.4, C6 = 128.8, C8,12, = 123.6,

C15,19 = 123.0, C24 = 122.8, C22,26 = 120.5, C9,11= 116.0,

C16,18 = 115.9, C4 = 84.1, C1 = 82.5, C2 = 60.6, C29 =

36.5, C3 = 30.8, C30 = 27.6, C31 = 22.2, C32 = 14.1.

122

1H NMR of compound 2.11g exo

1H NMR (500 MHz, DMSO-d6) δ H27 = 10.04 (s, 1H), H13,20

= 9.74 (s, 2H), H23,25 = 7.64 – 7.56 (m, 2H), H22,26, 8,12,15,18 = ,

7.21 – 7.13 (m, 6H), H9,11,16,19 = 6.77 – 6.68 (m, 4H), H4 =

5.63 (d, J = 1.2 Hz, 1H), H1 = 5.43 (dd, J = 4.4, 1.2 Hz, 1H),

H2 = 3.77 (dd, J = 8.2, 4.5 Hz, 1H), H29 = 2.30 (t, J = 7.5 Hz,

2H), H3 = 2.22 (dt, J = 12.0, 4.4 Hz, 1H), H3 = 2.16 (dd, J =

12.1, 8.3 Hz, 1H), H30 = 1.57 (tt, J = 7.6, 6.5 Hz, 2H), H31 =

1.35 – 1.27 (m, 2H), H32 = 0.90 (t, J = 7.3 Hz, 3H).

123

13C NMR of compound 2.11g exo


157.9, C,17 = 157.7, 144.2, C21 = 141.2, C23,25 = 138.7,

C7,14 = 136.7, C5,= 129.4, C6 = 128.8, C8,12, = 123.6, C15,19

= 123.0, C24 = 122.8, C22,26 = 120.5, C9,11= 116.0, C16,18 =

115.9, C4 = 84.1, C1 = 82.5, C2 = 60.6, C29 = 36.5, C3 =

30.8, C30 = 27.6, C31 = 22.2, C32 = 14.1.

124

1H NMR of compound 2.11g endo

1H NMR (500 MHz, DMSO-d6) δ H27 = 10.09 (s, 1H), H13,20

= 9.69 (d, J = 53.8 Hz, 2H), H23,25 = 7.70 – 7.66 (m, 2H),

H22,26, 8,12, = , 7.30 – 7.23 (m, 4H), H15,18 = , 7.20 – 7.16 (m,

2H), H9,11,= 6.79 – 6.76 (m, 2H), H16,19 = 6.71 – 6.67 (m,

2H), H4 = 5.70 (dd, J = 4.2, 1.2 Hz, 1H), H1 = 5.42 (dd, J =

4.7, 1.3 Hz, 1H), H2 = 4.41 (dt, J = 9.1, 4.3 Hz, 1H), H29 =

2.36 (t, J = 7.5 Hz, 2H), H3 = 1.74 (dd, J = 11.8, 4.5 Hz, 1H),

H30 = 1.62 (q, J = 7.5 Hz, 2H), H31 = 1.37 (dt, J = 14.4, 7.3

Hz, 2H), H32 = 0.95 (d, J = 7.4 Hz, 3H).

125

13C NMR of compound 2.11g endo

13C NMR (126 MHz, DMSO-d6) δ δ C27 = 171.8, C10=

157.7, C,17 = 157.3, 143.8, C21 = 140.8, , C23,25 = 138.8,

C7,14 = 135.8, C5,= 129.4, C6 = 128.4, C8,12, = 123.6, C15,19

= 123.7, C24 = 123.1, C22,26 = 120.4, C9,11= 115.9, C16,18 =

115.3, C4 = 83.9, C1 = 82.7, C2 = 59.4, C29 = 36.5, C3 =

29.9, C30 = 27.6, C31 = 22.2, C32 = 14.1.

126

1H NMR of compound 2.11h exo


H13,20 = 9.81 (s, 1H), H23,25 = 7.64 – 7.60 (m, 2H), H22,26,

8,12,15,18 = 7.20 – 7.14 (m, 6H), H9,11,16,19 = 6.76 – 6.70 (m,

4H), H4 = 5.63 (d, J = 2.4 Hz, 1H), H1 = 5.43 (d, J = 4.0

Hz, 1H), H2 = 3.77 (dd, J = 8.2, 4.5 Hz, 1H), H3= 2.58

(ddd, J = 12.1, 8.3, 6.0 Hz, 1H), H3,29= 2.25 – 2.12 (m,

3H), H30,31= 1.09 (dd, J = 6.9, 2.2 Hz, 6H).

127

13C NMR of compound 2.11h exo


155.8, C,17 = 155.6, C21 = 142.1, C23,25 = 136.6, C7,14 =

134.3, C5 = 127.3, C6 = 126.6, C8,12 = 121.5, C15,19 = 120.9,

C24 = 120.7, C22,26 = 118.3, C9,11, = 113.9, C16,18 = 113.8,

C4 = 82.0, C1 = 80.4, , C2 = 58.6, C,3 = 28.3, C,29 = 12.8,

C,30,31 = 5.6.

128

1H NMR of compound 2.11h endo

1H NMR (500 MHz, DMSO-d6) δ δ H27 = 10.35 (s, 1H),

H13,20= 9.65 (d, J = 57.2 Hz, 2H), H23,25 = 7.63 – 7.58 (m,

2H), H8,12,15,18 = 7.22 – 7.15 (m, 4H), H22,26 = 7.13 – 7.09 (m,

2H), H9,11, = 6.72 – 6.69 (m, 2H), H916,19 = 6.63 – 6.60 (m,

2H), H4 = 5.63 (dd, J = 4.2, 1.2 Hz, 1H), H1 = 5.34 (dd, J =

4.7, 1.3 Hz, 1H), H2 = 4.33 (dt, J = 9.1, 4.3 Hz, 1H), H3 =

1.77 – 1.72 (m, 1H), H3 = 1.66 (dd, J = 11.7, 4.5 Hz, 1H),

H30,31 = 0.80 – 0.75 (m, 4H).

129

13C NMR of compound 2.11h endo

13C NMR (126 MHz, DMSO-d6) δ C27 = 172.2, C10,17

= 157.8, 157.3, C21 = 143.8, 140.8, C23,25 = = 138.8,

C7,14 = 135.7, C5,= 129.7, C6 = 129.4, C8,12, = 124.2,

C15,19 = 123.7, C24 = 123.1, C22,26 = 120.4, C9,11, =

115.9, C16,18 = 115.7, C4 = 83.9, C1 = 82.7, C2 = 59.4,

C,3 = 29.9, C,29 = 14.9, C,30,31 = 7.7.

130


1H NMR (400 MHz, CDCl3) δ H15,19 = 7.53 – 7.48 (m,

2H), H8,12 = 7.34 – 7.29 (m, 2H), H9,11 = 6.85 – 6.80 (m,

2H), H16,18 = 6.79 – 6.75 (m, 2H), H2 = 6.55 – 6.52 (m,

1H), H13,20 = 3.75 (dd, J = 3.6, 1.1 Hz, 6H), H5,4 = 3.00 –

2.72 (m, 3H).

131


1H NMR (400 MHz, CDCl3) δ H8,12,15,19 = 7.14 – 7.09 (m,

4H), H9,11,16,18 = 6.71 – 6.66 (m, 4H), H1,4 = 3.75 (dd, J =

3.1, 1.7 Hz, 2H), H13,20 = 3.70 (s, 6H), H2,3 = 3.55 (dd, J =

3.2, 1.7 Hz, 2H).

132


1H NMR (400 MHz, CDCl3) δ H15,19 = 7.38 – 7.33 (m,

2H), H8,12 = 7.23 – 7.18 (m, 2H), H31,35 = 7.05 – 7.00 (m,

2H), H9,11,16,18 = 6.88 – 6.81 (m, 4H), H22 = 6.79 (s, 1H),

H25,29 = 6.74 – 6.70 (m, 2H), H26,28 = 6.47 – 6.43 (m, 2H),

H32,34 = 6.41 – 6.36 (m, 2H), H3 = 4.24 (d, J = 1.7 Hz, 1H),

H13,20 = 3.76 (d, J = 6.7 Hz, 6H), H25,29 = 3.70 (dd, J = 4.8,

1.7 Hz, 1H), H36,37 = 3.64 (d, J = 11.2 Hz, 6H), H4 = 3.02

(d, J = 4.8 Hz, 1H).

133


1H NMR (400 MHz, Chloroform-d) δ H8,12 = 7.96 – 7.90

(m, 2H), H10 = 7.70 – 7.65 (m, 1H), H9,11 = 7.61 – 7.53

(m, 2H), H2 = 4.07 (ddd, J = 9.5, 4.9, 0.5 Hz, 1H), H13 =

3.54 (d, J = 0.5 Hz, 3H), H14 = 3.49 (d, J = 0.5 Hz, 3H),

H3 = 2.64 – 2.55 (m, 1H), H3= 2.49 (ddd, J = 12.5, 4.9, 0.5

Hz, 1H).

134

135

MS for compound 2.1

136

MS for compound 2.2

137

MS for compound 2.3

138

MS for compound 2.6a Exo

139

MS for compound 2.6b Exo

140

MS for compound 2.7a

141

MS for compound 2.7b

142

MS for compound 2.7c

143

MS for compound 2.10e

144

MS for compound 2.10g

145

MS for compound 2.11a exo

146

MS for compound 2.11b exo

147

MS for compound 2.11d exo

148

MS for compound 2.11d endo

149

MS for compound 2.11c exo

150

MS for compound 2.11c endo

151

MS for compound 2.11e exo

152

MS for compound 2.11e endo

153

MS for compound 2.11f exo

154

MS for compound 2.11f endo

155

MS for compound 2.11g exo

156

MS for compound 2.11g endo

157

MS for compound 2.11hendo

158

MS for compound 2.11h exo

159

MS for compound 3.4

development of novel anti-estrogens for endocrine ... · development of novel anti-estrogens for...

Documents