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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).
1H NMR (500 MHz, DMSO-d
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.
1H NMR (500 MHz, DMSO-d
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.
1H NMR (500 MHz, DMSO-d
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.
1H NMR (500 MHz, DMSO-d
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).
LR-MS (ESI): calculated for C9H10NO4S (M+H)+ : 228.03; found (M+H)+: 228.0
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.
LR-MS (ESI): calculated for C12H16NO4S (M+H)+ : 270.08; found (M+H)+: 270.1
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).
1H NMR (400 MHz, CDCl
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
by flash chromatography (40% EtOAc in hexane) to obtain pure product as a white solid (0.041
g, 45.2% yield)
1H NMR (400 MHz, CDCl
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.
LR-MS (ESI): calculated for C12H16NO4S (M+H)+ : 270.08; found (M+H)+: 270.1
4-Pentanamidophenyl ethenesulfonate (2.10g)
39
Product 2.10c 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 (0.120
g, 63% yield).
1H NMR (400 MHz, CDCl
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
by flash chromatography (40% EtOAc in hexane) to obtain pure product as a white solid (0.090
g, 80% yield).
1H NMR (400 MHz, CDCl
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
LR-MS (ESI): calculated for C16H16NO5S (M+H)+
: 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
by flash chromatography (40% EtOAc in hexane) to obtain pure product as a white solid (0.040
g, 59% yield).
1H NMR (400 MHz, DMSO-d6) δ (ppm) 10.35 (s, 1H), 7.67 – 7.61 (m, 2H), 7.23 – 7.19 (m,
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.
LR-MS (ESI): calculated for C12H14NO4S (M+H)+
: 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
1H NMR (500 MHz, DMSO-d
6) δ (ppm) 10.10 (s, 1H), 9.73 (s, 2H), 7.61 – 7.56 (m, 2H), 7.21 –
7.13 (m, 6H), 6.76 – 6.69 (m, 4H), 5.63 (d, J = 1.3 Hz, 1H), 5.43 (dd, J = 4.3, 1.3 Hz, 1H), 3.77
(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.
LR-MS (ESI): calculated for C26H24NO7S (M+H)+
: 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
preparative HPLC to obtain pure product (32% yield) as a white solid.
1H NMR (500 MHz, DMSO-d
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.
LR-MS (ESI): calculated for C25H21NO7S (M+H)+
: 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
preparative HPLC to obtain pure product (24% yield) as a white solid.
1H NMR (500 MHz, DMSO-d
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
C NMR (126 MHz, DMSO-d6) δ
(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.
LR-MS (ESI): calculated for C28H28NO7S (M+H)+
: 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
preparative HPLC to obtain pure product (5% yield) as a white solid.
43
1H NMR (500 MHz, DMSO-d6) δ (ppm) 10.05 (s, 1H), 7.66 – 7.61 (m, 2H), 7.25 – 7.10 (m,
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.
LR-MS (ESI): calculated for C28H28NO7S (M+H)+
: 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
preparative HPLC to obtain pure product (25% yield) as a white solid.
1H NMR (400 MHz, DMSO-d
6) δ (ppm) 10.04 (s, 1H), 9.74 (s, 2H), 7.64 – 7.57 (m, 2H), 7.21 –
7.11 (m, 6H), 6.78 – 6.68 (m, 4H), 5.62 (d, J = 1.1 Hz, 1H), 5.42 (dd, J = 4.2, 1.2 Hz, 1H), 3.76
(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.
LR-MS (ESI): calculated for C27H26NO7S (M+H)+
: 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
preparative HPLC to obtain pure product (5% yield) as a white solid.
1H NMR (500 MHz, DMSO-d
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
preparative HPLC to obtain pure product (32% yield) as a white solid.
45
1H NMR (500 MHz, DMSO-d
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
C NMR (126 MHz, DMSO-d6) δ
(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.
LR-MS (ESI): calculated for C27H26NO7S (M+H)+
: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
preparative HPLC to obtain pure product (6% yield) as a white solid.
1H NMR (500 MHz, DMSO-d
6) δ (ppm) 10.02 (s, 1H), 9.81 (s, 1H), 7.64 – 7.60 (m, 2H), 7.20 –
7.14 (m, 6H), 6.76 – 6.70 (m, 4H), 5.63 (d, J = 2.4 Hz, 1H), 5.43 (d, J = 4.0 Hz, 1H), 3.77 (dd, J
= 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.
LR-MS (ESI): calculated for C27H26NO7S (M+H)+
: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
preparative HPLC to obtain pure product (33% yield) as a white solid.
1H NMR (500 MHz, DMSO-d
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.
LR-MS (ESI): calculated for C32H28NO8S (M+H)+
: 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
preparative HPLC to obtain pure product (4% yield) as a white solid.
47
1H NMR (500 MHz, DMSO-d
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
C NMR (126 MHz, DMSO-d6) δ
(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.
LR-MS (ESI): calculated for C32H28NO8S (M+H)+: 586.16; found: 586.5
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
preparative HPLC to obtain pure product (35% yield) as a white solid.
1H NMR (500 MHz, DMSO-d
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.
LR-MS (ESI): calculated for C29H30NO7S (M+H)+
: 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
preparative HPLC to obtain pure product (11% yield) as a white solid.
1H NMR (500 MHz, DMSO-d
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.
LR-MS (ESI): calculated for C29H30NO7S (M+H)+: 536.18; found: 536.4
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
preparative HPLC to obtain pure product (47% yield) as a white solid.
49
1H NMR (500 MHz, DMSO-d
6) δ (ppm) 10.02 (s, 1H), 9.81 (s, 1H), 7.64 – 7.60 (m, 2H), 7.20 –
7.14 (m, 6H), 6.76 – 6.70 (m, 4H), 5.63 (d, J = 2.4 Hz, 1H), 5.43 (d, J = 4.0 Hz, 1H), 3.77 (dd, J
= 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.
LR-MS (ESI): calculated for C28H26NO7S (M+H)+
: 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.
1H NMR (500 MHz, DMSO-d
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.
LR-MS (ESI): calculated for C28H26NO7S (M+H)+
: 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
2 BF3OEt2 THF reflux 0.032 M Dimer
3 PTSA, BF3.OEt2 THF reflux 0.032 M Dimer
4 BF3OEt2 THF reflux 0.0064 M Dimer
5 BF3OEt2 THF reflux 0.0032 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.
1H NMR (400 MHz, CDCl
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
1H NMR (400 MHz, CDCl
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
1H NMR (400 MHz, CDCl
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
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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 of compound 2.2
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 of compound 2.3
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 of compound 2.4
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 of compound 2.5
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
13C NMR (126 MHz, DMSO-d6) δ C10 = 157.9, C17 =
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 of compound 2.8
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 of compound 2.9
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 of compound 2.7a
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 of compound 2.11a
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
1H NMR (500 MHz, DMSO-d6) δ H27 = 10.05 (s, 1H),
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
1H NMR (500 MHz, DMSO-d6) δ H27 = 10.01 (s, 1H),
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
1H NMR (500 MHz, DMSO-d6) δ H27 = 10.02 (s, 1H),
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
1H NMR (500 MHz, DMSO-d6) δ H27 = 10.04 (s, 1H),
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
13C NMR (126 MHz, DMSO-d6) δ C28 = 175.8, C10, =
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
1H NMR (500 MHz, DMSO-d6) δ H27 = 10.05 (s, 1H),
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
1H NMR (500 MHz, DMSO-d6) δ H27 = 10.29 (s, 1H),
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
13C NMR (126 MHz, DMSO-d6) δ C28 = 167.3, C131, =
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
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).
121
13C NMR of compound 2.11f endo
13C NMR (126 MHz, DMSO-d6) δ C27 = 171.8, C10=
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
13C NMR (126 MHz, DMSO-d6) δ C27 = 171.8, C10=
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
1H NMR (500 MHz, DMSO-d6) δ H27 = 10.02 (s, 1H),
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
13C NMR (126 MHz, DMSO-d6) δ C27 = 170.1, C10 =
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 of compound 3.4
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 of compound 3.8
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 of compound 3.10
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 of compound 3.14
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