Controlled Activation and Deactivation of Cannabinergic Ligands and Novel Mono- and Bi- Functional Classical Cannabinoid Probes

Controlled activation and deactivation of cannabinergic ligands and Novel Mono- and Bi- functional Classical Cannabinoid Probes

by Shan Jiang

B.S. in Chemistry, Nankai University M.S. in Chemistry, Nankai University

A dissertation submitted to

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

January 11th, 2019

Dissertation directed by

Alexandros Makriyannis Spyros Nikas Professor of Chemistry and Chemical Biology

Acknowledgements

I would like to express the deepest appreciation to all those who make efforts to help me complete my dissertation.

First of all, I would like to advisor Dr. Alexandros Makriyannis, who also served my dissertation defense as the chair of committee members. I believe without his gracious support, guidance and encouragement, I could not finally complete my research work and the dissertation. He has the attitude and the substance of a genius: he continually and convincingly conveyed a spirit of adventure in regard to research and scholarship, and an excitement in regard to teaching.

I also would like to thank the members of my committee for their extreme patience in the face of numerous obstacles. Dr. Spyros Nikas served as my co-advisor. He teached me a lot of the organic synthetic skills. We also have valuable discussion during patent application, paper writing and proof reading the experimental section of my thesis draft. Dr. Sunny Zhou is another my committee member. I know him for a long time. Before I joined the school, he was the graduate coordinator of the chemistry department. It was him who finally accepted me as a PhD student in this department. Last but not least, Dr. George O'Doherty is a very good organic chemist who also served as my committee member. He gave a lot of great suggestions for my dissertation. I am really grateful to them for their support and patience.

Finally, I would like to express my gratitude to my parents. Even though they are far away in

China, they always make me feel warm. I believe they are the best parents in the world. I know there is no way of repaying them, however no one will ever know the strength of my love for them. After all, they are the only people who know what my heart sounds like from the inside.

Abstract of Dissertation

Part I

We report the design, synthesis, and biochemical characterization of novel cannabinergic ligands with remarkably high binding affinities for cannabinoid receptors that has tight/irreversible binding characteristics. These molecular probes are currently being used in studies that aim to uncover the binding motifs of classical cannabinoids with CB1 and CB2 receptors by using two approaches: receptor crystallography and the Ligand Assisted Protein Structure (LAPS) approach, which combines the use of receptor mutants and mass spectrometric proteomic analysis. Our ligand design relies on the incorporation of reactive groups at judiciously chosen positions within the classical cannabinoid structure, including the aliphatic chain at C3 and the substituents at C11.

Reactive groups included the electrophilic isothiocyanate and photoactivatable azido moiety as well as the polar nitrate ester and cyano groups, all of which are capable of tight/irreversible interactions with the target protein. Incorporation of one reactive group results in mono functional probes, while incorporation of two reactive groups leads to bifunctional ligands that can carry either a single reactive group (homo-bifunctional) or two different reactive groups (hetero- bifunctional). The novel probes behave as potent CB1 agonists as evidenced by functional data while a representative nitrate ester probe is a potent analgesic in mice.

Part II

The interaction of plant derived (-)-Δ9-THC with CB1 and CB2 receptors may affect diverse features of mammalian (patho)physiology. Modulation the activity of these two receptors is therefore a very exciting pharmacotherapeutic approach for the treatment of an array of CNS and/or PNS related indications. Unfortunately, only a limited number of cannabinergic agents have

3 been approved till date due to the poor pharmacokinetic/pharmacodynamic (PK/PD) properties as well as adverse psychotropic side effects associated with CB1 receptor activation. In our work, methods for obtaining novel cannabinoid analogues with controllable-deactivation or controllable- activation and improved druggability have been developed. As one part of the design of controlled deactivation cannabinoids project, a new chemotype of cannabinol (CBN) analogues containing a seven-member lactone ring in the B-ring position have been disclosed. Our results show that certain lactone bearing compounds are good binders towards both CB1 and CB2 receptors, while their hydrolysis products are all inactive. Another approach of improving drug delivery or pharmacokinetics is the design of prodrugs. Our novel gamma-hydroxybutyric acetic acid sodium salt AM11253 is inactive towards both CB receptors. However, it can undergo a spontaneous ring- closure reaction to form the active lactone analogue under an acidic pH. This property can be utilized to design pH mediated water-soluble prodrug.

Table of Contents

Acknowledgements 2

Abstract of Dissertation 3

Table of Contents 5

List of Tables 6

List of Schemes 8

List of Figures 10

List of Abbreviations 15

Chapter 1: Introduction 19

Chapter 2: Novel Mono and Bifunctional Cannabinoid Receptor Probes 44

Chapter 3: Controlling Deactivation of Cannabinergic Ligands 131

Chapter 4: Controlling Activation of Water Soluble Cannabinergic Ligands 200

Chapter 5: Examination of Chemical and Enantiomeric Purity of Synthetic

Cannabidiol 232

Chapter 6: Design and Synthesis of Carbonate and Carbamate Modified

Nabilone Analogues 255

Reference 275

List of Tables

Table 1.1: Chemical constituents of cannabis sativa

Table 2.1: Affinities (Ki) of mono- and bi-functional classical cannabinoid probes for CB1 and

CB2 cannabinoid receptors

Table 2.2: Reductions in the specific binding of [3H]CP-55,940 after irreversible ligand

pretreatment

Table 2.3: CB1 Functional potencies (EC50) of selected probes for the CB1 and CB2 receptors

Table 2.4: ED50 for the analgesic effects of 2.18a in mice

Table 3.1: Binding affinities (Ki) of controlled deactivation CBN analogs

Table 3.2: Functional evaluation of CBN analogs

Table 3.3: Metabolic stabilities (t1/2) of controlled deactivation analogs

Table 3.4: ED50 for the analgesic effects of AM11216 and AM11225 in mice

Table 4.1: Binding affinities (Ki) of controlled deactivation CBN analogs

Table 4.2: Functional characterization of the active drug AM11208

Table 4.3: In vitro microsomal stability of AM11208

Table 4.4: ED50 for the analgesic effects of AM11208 in mice

Table 4.5: ED50 for the analgesic effects of AM11208 and AM11253 in mice (oral administration)

Table 6.1: Binding affinities of C1 substituted nabilone analogs

Table 6.2: Functional characterization of AM11200, AM11203 and AM11202

Table 6.3: Half-lives for plasma esterase of AM11200 and AM11202

List of Schemes

Scheme 2.1: Synthesis of advanced intermediate bromides

Scheme 2.2: Synthesis of monofunctional probes

Scheme 2.3: Synthesis of homo-bifunctional probes

Scheme 2.4: Synthesis of hetero-bifunctional probes

Scheme 3.1: Synthesis of AM11216 and its analogues

Scheme 3.2: Synthesis of AM11225

Scheme 3.3: Synthesis of AM11227

Scheme 3.4: Synthesis of gem dimethyl substituted B-ring lactone analog

Scheme 3.5: Synthesis of terminal functionalized B-ring lactone analogs

Scheme 4.1: Synthesis of AM11208

Scheme 4.2: Synthesis of prodrug 4.12 and 4.13

Scheme 5.1: Synthesis of (R)-MTPA-(-)-Δ8-THC and (S)-MTPA-(-)-Δ8-THC

Scheme 5.2: Synthesis of (+)-Δ8-THC

Scheme 5.3: Synthesis of (R)-MTPA-(+)-Δ8-THC and (S)-MTPA-(+)-Δ8-THC

Scheme 6.1: Synthesis of nabilone and nabilone analogues

Scheme 6.2: Synthesis of carbamate analogs

Scheme 6.3: Synthesis of carbamate analogs

List of Figures

Figure 1.1: The chemical structures of Tetrahydrocannabinol, Cannabidiol, Cannabinol

Figure 1.2: The chemical structures of four isomers of THC

Figure 1.3: CB2 receptor downstream signal pathway

Figure 1.4: Selected phyto-cannabinoids/xenocannabinoids

Figure 1.5: Selected endocannabinoids

Figure 1.6: Selected classical cannabinoids

Figure 1.7: Selected non-classical cannabinoids

Figure 1.8: Selected CB receptor agonists

Figure 1.9: Selected CB receptor antagonists

Figure 1.10: Diagrammatic representation of endocannabinoid system and major metabolic

pathway

Figure 1.11: FAAH and MGL inhibitors

Figure 1.12: Diagrammatic representation of design of chiral AEA probes

Figure 1.13: Diagrammatic representation of Ligand-Assisted Protein Structure(LAPS) procedure

Figure 1.14: Diagrammatic representation of A work flow of biochemical binding assays

Figure 1.15: Amino acid sequence of the hCB2 receptor: cysteine-to-serine/alanine mutant library

are highlight in red

Figure 1.16: Bmax reduction caused by covalent labeling of hCB2 receptors.

Figure 2.1: Functional probes with high binding affinities for CB receptors, agonist properties and

irreversible binding profiles

Figure 2.2: Design of the novel mono and bifunctional hexahydrocannabinol (HHC) probes and

structures of the natural product (-)-Δ9-THC and the marketed HHC drug nabilone.

Figure 3.1: Diagrammatic representation of design of soft drugs

Figure 3.2: Diagrammatic representation of depot effect

Figure 3.3: Design and THC-based template optimization of the first generation of controlled

deactivation ligands

Figure 3.4: SAR study on the side chain of the first generation of controlled deactivation ligands

Figure 3.5: Second generation of controlled deactivation ligands

Figure 3.6: Two generation of controlled deactivation cannabinergic analogues

Figure 3.7: SAR studies of B-ring lactone template

Figure 3.8: Plasma stability test for AM11216

Figure 3.9: Plasma stability test for AM11225

Figure 3.10: Hypothermic effects of selected doses of AM11216

Figure 3.11: Hypothermic effects of selected doses of AM11225

Figure 3.12: Tail-flick latencies in a hot water bath after administration of three doses of

compound AM11216

Figure 3.13: Tail-flick latencies in a hot water bath after administration of three doses of

compound AM11225

Figure 4.1: Structures of Δ9-THC analogs

Figure 4.2: The synthesis of THC-HG prodrug

Figure 4.3: General design and Synthesis of Carboxylic Ester Prodrugs

Figure 4.4: Chemical structures of (A) (THC), (B) THC-Val, (C) THC-Val-Val, and (D) THC-

Val-HS

Figure 4.5: An unusual water soluble and orally active unimolecular prodrug

Figure 4.6: Concentration-dependent inhibition of forskolin-stimulated cAMP accumulation in

HEK293 cells expressing rCB1 receptors by representative agonist

Figure 4.7: Concentration-dependent inhibition of forskolin-stimulated cAMP accumulation in

HEK293 cells expressing hCB2 receptors by representative inverse agonist.

Figure 4.8: Plasma stability test for AM11208

Figure 4.9: Plasma stability test for AM11209

Figure 4.10: Gastric Juice stability test for AM11253

Figure 4.11: Tail-flick latencies in a hot water bath after administration of three doses of

compound AM11208

Figure 4.12: Tail-flick latencies in a hot water bath after administration of three doses of

compound AM11208 (oral administration)

Figure 4.13: Tail-flick latencies in a hot water bath after administration of three doses of

compound AM11253 (oral administration)

Figure 4.14: Tail-flick latencies in a hot water bath after administration of three doses of

compound Δ9-THC (oral administration)

Figure 5.1: Two pairs of diastereomers of Monster ester-THC analogues

Figure 5.2: HPLC retention time for synthetic CBD, Δ8-THC and Δ9-THC

Figure 5.3: Expansion of HPLC retention time for synthetic CBD

Figure 5.4: HPLC retention time for the mixture of synthetic CBD, Δ8-THC and Δ9-THC

Figure 5.5: NMR peak assignment for Δ8-THC

Figure 5.6: NMR peak assignment for (R)-MTPA-(-)-Δ8-THC

Figure 5.7: NMR peak assignment for (R)-MTPA-(+)-Δ8-THC

Figure 5.8: Expansion of the comparison of specific NMR peaks of (-)-Δ8-THC, (R)-MTPA-(+)-

Δ8-THC and (R)-MTPA-(-)-Δ8-THC (1)

Figure 5.9: Expansion of the comparison of specific NMR peaks of (-)-Δ8-THC, (R)-MTPA-(+)-

Δ8-THC and (R)-MTPA-(-)-Δ8-THC (2)

Figure 5.10: NMR peak assignment for (S)-MTPA-(+)-Δ8-THC

Figure 5.11: NMR peak assignment for (S)-MTPA-(-)-Δ8-THC

Figure 5.12: Expansion of the comparison of specific NMR peaks of (-)-Δ8-THC, (S)-MTPA-(+)-

Δ8-THC and (S)-MTPA-(-)-Δ8-THC (1)

Figure 5.13: Expansion of the comparison of specific NMR peaks of (-)-Δ8-THC, (S)-MTPA-(+)-

Δ8-THC and (S)-MTPA-(-)-Δ8-THC (2)

Figure 6.1: Four major pharmacophores of classical cannabinoids

Figure 6.2: Exploration of pharmacophoric space at C1 position of nabilone

List of Abbreviations

AC Adenyl Cyclase

AEA Anandamide

ACN Acetonitrile

BBB Blood Brain Barrier

Bmax maximal binding capacity

BSA bovine serum albumin

CB Cannabinoid

CBD Cannabidiol

CBN Cannabinol

CNS Central Nervous System

CHCl3 Chloroform cAMP Cyclic Adenosine Monophosphate cLogP Calculated Logarithm of Partition Coefficient

CB1 cannabinoid receptor 1

CB2 cannabinoid receptor 2

CP55940 (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3- hydroxypropyl)cyclohexan-1-ol

C-terminus carboxyl-terminus

CYP 450 Cytochrome P450

DMSO Dimethylsulfoxide

DIBAL-H Diisobutylaluminium hydride

DMH-Δ9-THC Dimethylheptyl-Delta-9-tetrahydrocannabinol

DAG Diacylglycerol

DGL Diacylglycerol-lipase

DMF Dimethylformamide

DMAP Dimethylaminopyridine

DME Dimethoxyethane

EC extracellular loop

EC50 half maximal effective concentration

Emax maximum efficacy

ECS Endocannabinoid System

FAAH Fatty Acid Amide Hydrolase

GPCRs G-protein coupled receptors hCB1 human cannabinoid receptor 1 hCB2 human cannabinoid receptor 2

HBSS Hank’s balanced salt solution

HEK293 human embryonic kidney 293

HHC 9-nor-9β-hydroxyhexahydrocannabinol

IC50 half maximal inhibitory concentration

IL intracellular loop

IR Infrared Spectroscopy

KHMDS Potassium hexamethyldisilazane

Ki inhibition constant

LAPS ligand-assisted protein structure

LC liquid chromatography

LC/MS/MS liquid chromatography-tandem mass spectrometry mCB2 mouse cannabinoid receptor 2

MS mass spectrometry

MS/MS tandem mass spectrometry

MAPK Mitogen-Activated Protein Kinases

MGL Monoacyl Glycerol Lipase

NAPE N-acylphosphatidylethanolamine

NMR Nuclear Magnetic Resonance

NaHMDS Sodium hexamethyldisilyl amide

N3 azido functional group

NCS isothiocyanato functional group

N-terminus amine-terminus

PLD Phospholipace D

PLC Phospholipase C

PK Pharmacokinetic

PD Pharmacodynamic pH power of hydrogen pKa acid dissociation constant p-TSA p-Toluenesulfonic acid rCB1 rat cannabinoid receptor 1

RPM revolutions per minute

R state inactive state

R* state active state

SAR structure activity relationship

SEM standard error of measurement

THC Tetrahydrocannabinol

TRPV1 Vanilloid Receptor-Type 1 TRPV1

THF Tetrahydrofuran

TMSOTf Trimethylsilyl trifluoromethanesulfonate

TBDMS-Cl tert-Butyldimethylchlorosilane tPSA Total Polar Surface Area

TME Tris/MgCl2/EDTA

TMH transmembrane helix

USFDA US Food and Drug administration

UV ultra violet

Vd Volume of Distribution

WT wild type

Δ9-THC Δ9-Tetrahydrocannabinol

Δ8-THC Δ8-Tetrahydrocannabinol

2-AG 2-arachidonoyl Glycerol

11-OH-THC 11-Hydroxytetrahydrocannabinol

CHAPTER 1: INTRODUCTION

The Herbal Medicine: Cannabis

Archaeologists have proved that the psychoactive use of cannabis date back to prehistoric societies in Africa and Eurasia.1 Cannabis which belongs to the cannabaceae family is also famous by its synonym: marijuana or hemp.2 Cannabis is one of the oldest flowering herb which is used as food, hemp fiber medicine, and recreational drugs. the landmark discovery of cannabis was the finding of its therapeutic ability to treat variety of indications. The first finding has been documented by Dr. William O’Shaughnessy in mid-

19th century.3 In his work, a small dose of cannabis extracts worked as analgesics to relieve rheumatoid joint pain. Furthermore, the extracts also been found to successfully stimulate digestive organs as anticonvulsives in their reports. Encouraged by the discovery, numerous talented American and British scientists spurred the research to the therapeutic applications of cannabis as anti-spasmodic agents and muscle relaxants.3 To date, after it was made illegal in 1942 by fedral legislation, cannabis existed in US

Pharmacopeia for more than 70 years, needless to say that marijuana have been used in the United States for almost one and half centuries.4

The ingredients: What is Cannabis from a chemical perspective

The question of why cannabis can be utilized as medical therapeutics could be answered by looking into the constituents through a chemical view.5 The leaves of the plant

Cannabis sativa L contain an array of different types of compounds6: the psychoactive compound cannabinoids, the biomacromolecule like proteins, glycoproteins and enzymes,

19 amino acids, sugar and related molecules, small molecules, such as fatty acids, terpenes, and also a few types of steroids, as well as vitamins and pigments (details showed in

Table 1.1). It is crucial to realize that different batches of marijuana may have different percentage composition for each specific ingredient due to varying subspecies, method for preparation (i.e., harvest time), drying conditions, etc.7

Table 1.1*: Chemical constituents of cannabis sativa

*Source: Chemical constituents of marijuana: The complex mixture of natural cannabinoids- Life Sciences, Page 544.

As depicted from Table 1.1, the majority of the constituents of cannabis are cannabinoids and terpenes. Amongst the two, from a medicinal chemists’ view, cannabinoids should be emphasized. Because they are the active ingredients among all the components.8-10

When concerning about pharmacological application, the three active ingredients became predominant – tetrahydrocannabinol (THC)11, cannabidiol (CBD)12 and cannabinol

(CBN)13. The structures of these small molecules are illustrated in Figure 1.1. Typically,

CBD is a two-ring compound while the other two, THC and CBN have three-ring structure with the benzene ring holding the A-ring position. Even though CBD accounts for 40% of cannabinoids among cannabis, this molecule, as well as CBN, are not recognized by the cannabinoid receptors. In turn, they cannot trigger central neural system effects. Thus, among the three pharmacological important compounds, THC is the one that could show the primary psychoactive effect.14

Figure 1.1: The chemical structures of Tetrahydrocannabinol, Cannabidiol, Cannabinol.

Two sorts of active THCs have been identified, namely (-)-Δ9-THC and (-)-Δ8-THC. They are regioisomers and the nomenclature based on the position of the C-C double bound.

(Figure 1.2). The two regioisomers share the (-) (6aR, 10aR) stereochemistry so that they have equal affinities when binding to CB receptors.15 However, if the stereochemistry changes, the binding affinity would be lost. For example, both (+) -Δ8- (6aS, 10aS)-THC and (+) -Δ9- (6aS, 10aS)-THC are no longer good binders to CB receptors.16

Figure 1.2: The chemical structures of four isomers of THC.

Cannabinergic Related Drug Discovery

Medical and recreational use of cannabis goes beyond centuries. However, it was not until 1964 that the psychoactive component of cannabis (-)-Δ9-THC was discovered and was isolated.17-18 Since then, the desire of understanding psychotropic effect induced by phytocannabinoids derivatives led to the discovery and characterization of CB receptors.19 As a result, modulation of CB receptors by the plant-derived (-)-Δ9-THC (or

(-)-Δ8-THC) and synthetic analogues is a promising therapeutic approach to treat an array of indications such as pain, inflammation, CNS disorder, and cancer.20 There are two main cannabinoid receptors named CB1 receptors21 and CB222 receptors. Recently a new type of CB receptors, GPR55 has been identified.23 However, whether to categorize

22 it as CB3 receptor is still under controversy. Both CB1 and CB2 receptors are membrane- based receptors which belongs to class A rhodopsin-like family of the G-protein coupled receptor (GPCR) superfamily. GPCRs are the larger class of receptors located on cell membranes and are targeted by more than one third of the modern drugs.24 The two receptors exhibit 44% sequence homology in the entire amino acid sequence and 68% homology in the transmembrane domain, and as other GPCRs, their structures are built by a seven-transmembrane domain that links each other with intracellular loops and extracellular loops while coupling with G-protein.25 However, CB1 receptor are commonly found in the central neural system while CB2 receptors usually locate at the immune system (i.e., spleen). Also, other research results also proved in lower amounts, CB1 exsit in peripheral neural system, for instance, liver, kidney and other organs.26

Drug behavior is usually illustrated by the ligand-receptor interaction mechanism. Howlett and her co-workers were the first group of scientists who identified the cannabinoids-CB receptors’ interaction.27 Upon binding to CB receptors, the three heterogenous subunits, alpha, beta and gamma (which together compose the G-protein) dissociates from each other by the activation of the ligand receptors complex. Then, the G-alpha subunit tether to other cellular proteins and trigger following downstream signal pathway.28 Numerous downstream signaling are activated by the interaction between CB receptors and

29 cannabinergic ligands. The signaling includes but not limits to Gi inhibiting adenylyl

2+ cyclase, Gs stimulating adenylyl cyclase, mediating Ca fluxes and activation of phospholipases A and C, phosphorylation and activation of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, and Jun N-terminal kinase (JNK)(Figure 1.3). Take

Gi inhibiting adenylyl cyclase pathway for example. It has been proved by a number of

23 scientific work that the CB receptors are negatively coupled GPCRs, which means CB

30 agonist negatively activates the Gi/o coupled CB receptors. Cannabinoids therefore reduce the cellular cAMP level by inhibiting the adenyl cyclase (AC) upon their interaction with CB receptors.

Figure 1.3: CB2 receptor downstream signal pathway.

Source: CB2 Cannabinoid Receptors as a Therapeutic Target—What Does the Future Hold? Molecular Pharmacology

2014, 86 (4) 430-437

There are several simple ways to classify the cannabinoid receptor ligand. Based on the origin (plant-derived or synthetic ligands), The different types are phyto- or

24 xenocannabinoids (Figure 1.4) and lipid like molecules: endocannabinoids that naturally synthesized by humans or animals (Figure 1.5). Based on the structure: the derivatives of THC which share the three-ring scaffold are classical cannabinoids (Figure 1.6); other synthetic cannabinergic compounds are non-classical cannabinoids (Figure 1.7). Based on the function: those that can reduce the cellular cAMP level are CB receptor agonist

(Figure 1.8); those that can increase the cellular cAMP level are CB receptor inverse agonist; those that themselves have no effect toward controlling cAMP level, nevertheless, can oppose the action of a certain CB receptor agonist are CB receptor antagonist (Figure

1.9).

Figure 1.4: Selected phyto-cannabinoids/xenocannabinoids.

Figure 1.5: Selected endocannabinoids.

Figure 1.6: Selected classical cannabinoids.

Figure 1.7: Selected non-classical cannabinoids.

Figure 1.8: Selected CB receptor agonists.

Figure 1.9: Selected CB receptor antagonists.

Cannabinergic ligand, when applied in the pharmaceutical and medicinal field, is a double-edged sword based on their nature of modulating CNS mediated psychotropic effects. On one hand, it achieved a lot of success on developing novel therapeutic medications aiming at many indications such as neuropathic pain, emesis, and obesity.31

On the other hand, due to the high risk of addiction, the holdback of the development of cannabinergic therapeutic approach also happened in the past few decades. For example, the rimonabant as a CB1 receptor antagonist was first approved by European Medicines

Agency, however then was withdrawn due to the severe CNS side effect. Nevertheless, since the isolation and identification of the plant-derived psychoactive component (-)-Δ9-

THC of Cannabis sativa L. and the discovery and cloning of CB receptors, a great amount of research work based on interpreting the therapeutic nature of cannabinoid and their synthetic analogues have finally led to FDA-approved cannabinergic medicines available on market. To date, four of these drugs7 are:

1) Marinol (dronabinol: THC): Dronabinol is a synthetic Δ9-THC which was approved by the United States Food and Drug Administration (USFDA) in 1985. It is for the treatment of vomiting and nausea as side effects of cancer chemotherapy. Dronabinol is indicated as an alternative therapy to be utilized when the first and second line of therapy fails to effectively control patients’ symptoms of nausea and vomiting. Another function of dronabinol is to treat weight loss caused by loss of appetite, especially for the patients with human immunodeficiency virus (HIV) infection. The major side effects include confusion, dizziness, somnolence, an exaggerated sense of happiness, and lightheadedness. There may also be stomach pain as a side effect as the body is trying to adapt to the subtle change by the medication. It is rare, however, to see very serious allergic reaction caused by the drug.

2) Syndros (liquified dronabinol): Because of the unaccepted pharmacokinetic (PK) and pharmacodynamic (PD) profile, poor solubility induced absorption problems in Marinol, a new dronabinol-based drug Syndros was approved by USFDA the same year right after the approval of MARINOL. Syndros is the first and the only liquid dronabinol approved by the FDA. It is designed for the treatment of anorexia among people who have acquired immune deficiency syndrome(AIDS) and have lost weight. It is also for the treatment of nausea and vomiting cause by anticancer chemotherapy among the patients who has not been successfully treated with normal antiemetic drugs. Syndros has fast absorption and flexible dosing feature.

3) Cesamet (nabilone): The structure of nabilone is showed on Figure 1.6. nabilone is a synthetic classical cannabinoid mimicking THC that went through the structure activation relationship (SAR) optimization in terms of in vitro and in vivo efficacy and potency. As an

29 antiemetic medication to be used after anti-cancer drug, nabilone is only indicated in patients who fail other lines of therapy in suppressing symptoms of nausea and vomiting due to its unpredictable side effect, including but not limited to hallucinations, confusion, anxiety, panic, paranoia, and tachycardia.

4) Sativex (nabiximols as a United States adopted name): Different from dronabinol, sativex contains not only a 1:1 ratio of the principal cannabinoids THC and cannabidiol

CBD, but also certain minority of classical cannabinoids and other non-cannabinoid ingredients. It was developed by GW Pharmaceuticals as an oromucosal spray. Such an administration method means the drug is absorbed only inside the mouth, for instance, inside the cheek or under the tongue. Sativex was also approved as a medicine for treatment of spasticity in numerous countries outside the United States.

Endocannabinoid System (ECS)

The endocannabinoid system consists of receptors including CB1 receptors and CB2 receptors, endogenous cannabinergic ligands that served as retrograde neurotransmitters,32 and the related enzymes. The endocannabinoid signaling system is widespread in the mammalian central nervous system (including brain, spinal cord) and the peripheral nervous system.33 It is an unparallel information delivering network platform which involves modulating various physiological and cognitive processes such as appetite, emotion, memory, incentive salience, and associative learning. (Figure 1.10)34

How CB1 and CB2 G-protein coupled receptors are activated and leads to the downstream signaling have been interpreted above. Nevertheless, right after the 30 discovery, isolation, cloning and expression of the two receptors, the assumption of existence of endocannabinoids have been proposed since early 1990s. endocannabinoids are generated inside the human body which are also able to bind and active the CB receptors. Motivated by the brilliant assumption, the first endogenous ligand for CB1 receptors, anandamide was identified in 1992.35 The name anandamide was taken from an ancient Indic language of India, means “super joy”. Closely following it was the discovery of the second endogenous ligand, 2-arachidonoylglycerol (2-AG)36 which has been approved as a more abundant type of ligand in the CNS than the first discovered ligand anandamide. Both AEA and 2-AG are neurotransmitters, but different from other neurotransmitters, they are retrograde neurotransmitters, which means they are synthesized and released into a postsynaptic cell and travel back to a presynaptic neuron.

Also, these two endogenous ligand are synthesized on demand, which means they exist at a very low level in the postsynaptic cell body and have a comparatively short half-life.

The role of different types of enzymes is involving in endocannabinoids synthesis and metabolism. The synthetic enzymes mainly include N-acylphosphatidylethanolamide- phospholipase D (NAPE-PLD) which mediates the pathway of biosynthesis of AEA;37 phosphatidic acid phsophohydrolase, especially diacylglycerol lipase (DAGL), phosphoinositide-specific PLC (PI-PLC) and lyso-PLC that take charge of the generation of the 2-AG; some membrane transporters that play an important role in cellular uptaking or releasing of endocannabinoids. Degradation enzymes predominately include fatty acid amidohydrolase (FAAH) and monoacylglycerol lipase (MAGL)38. Generally, AEA is degraded by FAAH only while the second endocannabinoid 2-AG is always metabolized by both FAAH and MAGL.

Figure 1.10*: Diagrammatic representation of endocannabinoid system and major metabolic pathway. Source: 2012 Division of Medicinal Chemistry Award Address. Trekking the Cannabinoid

Road: A Personal Perspective. J. Med. Chem. 2014, 57, 3891−3911.

As a result, fully understanding the endocannabinoid system signaling will help medicinal chemists or biologists to design and develop novel therapeutic approach for a variety of indications and addiction. For instance, Inhibition of FAAH and MGL is an attractive approach to enhance endocannabinoid signaling by the same time to minimize the side effects of CB1 activation (Figure 1.11).

Figure 1.11: FAAH and MGL inhibitors.

Another perspective is to design and synthesize metabolically stable endocannabinoid analogs without losing potency and efficacy. Led by Drs. Spyros Nikas and Alexandros

Makriyannis, our group’s own involvement within this field started decades ago. Recently, the first stable endocannabinoid analog also with strong potency has been reported by

Drs. Nikas and Makriyannis. It is a chiral arachidonoyl ethanolamide (AEA) analogue, namely, (13S,1’R)-dimethylanandamide (AMG315, 3a). The Ki value of this compound for

CB1 receptor is as high as 7.8 ± 1.4 nM and behaves as a very potent in vitro CB1 agonist with the EC50 value of 0.6 ± 0.2 nM. The most incredible nature of this ligand is that it is almost comparatively resistant toward almost every sort of endocannabinoid metabolic protein such as hydrolytic and oxidative enzymes. Yingpeng Liu, Lipin Ji, et. al, have also observed analgesia when they examined the compound in the CFA-induced inflammatory pain model in the in vivo study. The in vivo potency is comparable with endogenous AEA or its hydrolytically stable analogue AM356.39 (Figure 1.12)

Figure 1.12*: Diagrammatic representation of the design of chiral AEA probes.

*Source: (R)‑N‑(1-Methyl-2-hydroxyethyl)-13‑(S)‑methyl-arachidonamide (AMG315): A Novel Chiral Potent

Endocannabinoid Ligand with Stability to Metabolizing Enzymes. J. Med. Chem. 2018, 61, 8639−8657

Ligand-Assisted Protein Structure (LAPS)

Activating or deactivating CB1 and/or CB2 receptors plays an important role in various physiological and cognitive processes which are mediated by smoothly continuous endocannabinoid system signaling. Understanding how interactions between classical cannabinoids and specific amino acids within the CB receptor binding domain occur and obtaining experimental information on the functional properties of ligand-receptor binding state40 is therefore expected to play a therapeutic role in an array of indications throughout cardiometabolic syndromes,41 overweight or obesity, drug addiction and abuse, and inflammation.42 As a result, for the purpose of obtaining insight knowledge about the structure-function relationship of the two CB receptors, we and many other research groups have developed various approach over the past few decades. These approaches include: in silico methods which are based on computational techniques, such as

34 molecular modeling,43 molecular dynamics simulation, agonist/antagonist docking; radiolabeled-ligand-binding technique44 which are used to interrogate the ligand-receptor interaction like binding affinity, competition binding and irreversible labeling ability; mutational experiment45 which are used to determine the molecular (agonist, inverse agonist, antagonist and neutral antagonist) based pharmacological. Crystallography46 is used to generate the crystal structure of the GPCRs. Basically, crystallization of CB receptors in complex with distinct ligands, although severely limited by practical difficulties, is one of the most direct biophysical approaches to provide insight into their conformation of distinct bound form (such as agonist-binding induced active state and antagonist- binding stabilized inactive state). Thermostabilizing construct optimization procedures are required to modify the wild type CB receptors. These procedures are include identifying stabilized fusion partner, truncating on both the N and C termini and mutations on specific amino acid residues. However, even such laborious processes could not guarantee a successful structural determination. To facilitate the crystal structures of CB receptors in ligand-bind form, one key is optimized CB receptor probes. A qualified candidate probe for crystallographic investigation must be a good binder with high affinity and wash- resistant binding to CB receptors. Recently, two human CB1 receptor crystal structures facilitated by small agonist and antagonist probe molecules has been determined respectively. Nevertheless, an alternative approach named “ligand-assisted protein structure” (LAPS) is currently under development in our laboratories. This experimental method requires the development of tight binding ligands capable of forming an irreversible bond with a specific amino acid residue within the receptor binding domain or immediately connect to it. The identity of the labeled amino acid residue is then uncovered

35 through purification of the ligand-receptor complex followed by digestion of the entire protein and analysis of the individual peptide fragments using sequencing or mass spectral analysis such as peptide mapping.47 (Figure 1.13)

Figure 1.13: Diagrammatic representation of Ligand-Assisted Protein Structure(LAPS) procedure.

LAPS study depend on utilization of high affinity and wash-resistant binding probes. To realize the wash-resistant binding nature, functional “tips” are usually employed to the optimized classical/nonclassical template. To date, two types of widely used functional groups are electrophilic functional groups in the representative of isothiocyanato (NCS)48 and photoactivatable groups in the representative of azido (N3). The idea of electrophilic

36 functional groups generates from their capacity of nucleophilic attacking to the electro- rich part of a specific amino acids. Take cysteine as a paradigm, upon the interaction between the NCS moiety and the nucleophilic thiol (-SH) group which belongs to the cysteine scaffold, a new covalent bond forms between the NCS modified ligand and the binding domain of the CB receptors.49 From our previous experiment, the result shows that NCS moiety can selectively recognize the SH group among other nucleophilic groups.

Photoactivatable functional groups can irreversibly or covalently connect to the receptors when they are activated by the ultraviolet (UV) light source. Over the past three decades,

50 the predominantly used photoactivatable functional group is the azido group (N3). Upon the exposure to the UV light, N3 would convert to nitrene that acts as carbene. As a very reactive electrophile, carbene can easily insert into a non-specific carbon-hydrogen covalent bond or carbon-carbon covalent bond so as to form a new covalent connection.

As a result, unlike the NCS group which can specifically bind to cysteine, the N3 group has no selectivity towards specific amino acid residue. Instead, N3 group only insets to a proximal amino acid residue.

Besides the N3 group, another type of photoactivatable functional groups has also been reported. They are the small molecular probes with benzophenones51 built into their structures. The obvious difference between these two chemotype is that N3 probes are activated by 245-nm UV light while the others are activated by a UV light source with comparatively longer wavelength, 365nm for instance.

Like such, numerous irreversible binding probes have been designed and synthesized over the past two decades. AM841 and AM3677 as the representatives of isothiocyanates functionalized probes both have the NCS group built into their terminal of alky side chain.

(Figure 1.8). These two molecules are designed and made by our labs and have been identified to have irreversible labeling ability for CB1 receptors from our earlier LAPS study. Also, within our primary research, a cannabinergic CB1 ligand analogue carrying a NCS electrophilic motif while bearing an iodine atom that can be treated as a radiolabel have been reported in 2002. In another work, 3’-fuctionalized adamantyl CB receptor probes have been synthesized. Two endogenous anandamide analogues with high affinity have also been reported in 2005. However, most physiological and psychological effects modulated by CB receptors are based on their interaction with the main psychoactive constituent of marijuana, (-)-Δ9-tetrahydrocannabinol (Δ9-THC). Thus, Δ9-

THC structure based cannabinergic probes are required. Also, in general, majorly reported cannabinoid probes are only either bearing an electrophilic group or a photoactivatable group.

In order to reveal more information on CB receptors ligand-binding and functional site(s), more diverse reactive groups which can be incorporated into tight binding prototypic ligands need to be explored. Thus, a series of novel mono-functional, homo- and hetero- bifunctional classical cannabinoid probes based on the classical Δ9-THC template have been designed and synthesized in this work. These ligands all bear functional groups (i.e.

-NCS, -CN, -N3, -ONO2). All obtained probes were tested for their respective affinities and irreversible labeling ability for CB1 and CB2 receptors. Our results show that these molecular probes with high affinities for the cannabinoid receptors, present agonist properties in the cAMP assay while at the same time exhibiting the desirable tight binding/irreversible interaction profile. The details can be found in chapter 2.

One of the most successful probes synthesized in this work is (-)-7'-Isothiocyanato-11- hydroxy-1',1'-dimethylheptylhexahydrocannabinol (AM841). It was shown to irreversibly interact with specific amino acid residues within the CB1 and CB2 receptor binding domain, which served as an indispensable starting point for the subsequent experimental characterization of ligand-binding form of the two receptor-complexes utilizing LAPS approach. Furthermore, the agonist-bound crystal structures of human CB1 in complex with AM841 were determined recently. In this effort, AM841 is one of the crucial factor to facilitate the CB1 crystallization.

Figure 1.14: Diagrammatic representation of A work flow of biochemical binding

assays.

It is only the first step for LAPS approach of obtain high binding affinity and irreversible labeling probes. (Figure 1.14) Typically, LAPS is an interdisciplinary technology composed by three principal parts: medicinal chemistry in charge of the probe design and synthesis, molecular biology and cellular biology, and bioanalytical chemistry. The

39 irreversible labeling or tight binding probes is a technical tool52 for us to learn how the small molecule (including agonist and antagonist) communicates with the biomacromolecule such as GPCRs and enzymes. To achieve this goal, design and construction of mutant library that includes every possible combination of binding sites is necessary.53 Take CB2 receptor for instance, the cysteine mutation of human CB2 receptors could be a brilliant paradigm when applying the LAPS to endocannabinoid system GPCRs study. The crucial role of the cysteine residues to GPCRs’ structure- functional correlation has been emphasized on the nucleophilic attractivity under physiological pH.54 Among all 20 types of natural amino acids, cysteine is the most reactive residue when participating in a reaction with an electrophilic probe such as NCS or nitrene moiety. This is due to the electro-rich and comparatively more electronegativity feature of thiol group under the physiological environment. A total of five cysteine residues are spread on four transmembrane helices (TMH) of human CB2 receptor: TMH1 THM2,

TMH6 and TMH7 (Figure 1.15). Two cysteines residues are on TMH7.55 Between the two cysteine, there are four amino acid residues through one single helical turn. Based on isosteric analogs principle, serine was selected as the destination of cysteine mutation.

The five cysteines have all been replaced by serine from our previous work, the three cysteine on the TMH1 THM2, TMH656 are followed by the single cysteine-to-serine mutants while the two on TMH7 are under a double cysteine-to-serine mutant because of the proximity (two cysteine in one helical turn). As an alternative, cysteine-to-alanine mutant is able to avoid the untoward effects, which generated from cysteine-to-serine mutation. Alanine is a small amino acid without polar and nucleophilic feature so the mutation is expected to minimize the conformational change of the human CB2 receptor.

The nomenclature of the mutants is generated from Ballesteros-Weinstein nomenclature.

The number 50 is gained by the most conserved amino acid residue in each certain helix.57 Take mutant C2.59(89)S as a paradigm: C is cysteine as it appeared in the wild- type amino acid; 2 stands for helix 2; the number 59 for the same cysteine is counted from the most conserved residue set as 50 on helix 2, 89 is the number counted for the cysteine through the entire amino acid sequence of the CB2 receptor, and S is serine after the mutation.

Figure 1.15*: Amino acid sequence of the hCB2 receptor: cysteine-to-serine/alanine mutant library are highlight in red.

*source: Ligand-Assisted Protein Structure (LAPS): An Experimental Paradigm for Characterizing Cannabinoid-

Receptor Ligand-Binding Domains. Methods Enzymol. 2017, 593, 217-235

After the specific probe successfully irreversible or tightly bind to the wild type CB2 receptor, the mutant human CB2 receptor will be examined by interacting with identical probe under the same reaction condition. The principle to decide the irreversible labeling or tight binding character is the ability of reducing Bmax value collected from saturation assays. The huge difference between the reduction of Bmax induced by the mutant recptor- ligand and by the wild type complex is a solid evidence to prove the target residue(s) play an important role as a component of the binding motif.58 See figure 1.16 for a paradigm.

Figure 1.16*: Bmax reduction caused by covalent labeling of hCB2 receptors. Up-left panel:

AM1336 reduced nealy 60% of hot ligand [3H]-CP55940 binding to wilde type hCB2 receptor. Up-right panel: AM1336 reduced only around 15% of hot ligand [3H]-CP55940 binding to C7.38(284)S hCB2 receptor mutant. Bottom panel: no reduction is observed

42 by AM1336 for [3H]-CP55940 binding to double mutated on hCB2 receptor

(C7.38(284)7.42(288)S).

*Reprinted from Mercier, R. W., Pei, Y., Pandarinathan, L., Janero, D. R., Zhang, J., Makriyannis, A. (2010). hCB2 ligand-interaction landscape: Cysteine residues critical to biarylpyrazole antagonist binding motif and receptor modulation. Chemistry & Biology, 17, 1132–1142; copyright 2010, with permission from Elsevier.

Besides the mutant library from site-directed mutagenesis methodology, mass spectrometry-based characterization served as a bioanalytical chemistry method has already gained its fame in the field of analysis of the structure-based ligand-receptor complex.59 As the same paradigm mentioned above, AM841-hCB2 receptor complex has been identified through a bottom-up proteomic peptide mapping analysis utilizing the liquid chromatography tandem mass spectrometry (LC-MS/MS). In this LC-MS/MS approach, sample preparation is needed. Usually, the purified ligand receptor complex undergoes a trypsin digestion procedure to convert themselves to small pieces of peptides before being injected to the mass spectrometry instruments.

CHAPTER 2: Novel Mono and Bifunctional Cannabinoid

Receptor Probes

OBJECTIVE AND SPECIFIC AIMS

The two Gi/o-protein-coupled cannabinoid receptors (CBRs) CB1 and CB2 have been recognized as important therapeutic targets for CNS and cardiometabolic disorders, glaucoma, pain, cancer, as well as conditions related to the immune system.60-63 An interesting feature of these two GPCRs is their ability to be modulated by a number of structurally distinct classes of compounds including endogenous lipid like substances

(e.g., anandamide and 2-arachidonoyl glycerol), as well as exogenous synthetic (e.g., nabilone and rimonabant) and plant derived molecules [e.g., (-)-Δ9- tetrahydrocannabinol].64 For the CB1 receptor, such ability to respond to a plethora of ligands with considerably different sizes and shapes is consistent with the remarkable plasticity of the orthosteric binding pocked of this GPCR as revealed from studies on the agonist- and antagonist-bound crystal structures.46, 65

Furthermore, it is becoming increasingly evident that the ligand-dependent activation of

CB1 and CB2 is multifactorial and can activate certain signal transduction pathways relative to other signal transduction pathways [e.g., G proteins (canonical Gαi/o, or non-

66-67 canonical Gαs, Gα16, and Gαq/11) versus arrestins]. This phenomenon is more general in GPCRs and it is referred to as “functional selectivity” or “ligand bias”.68-69 Functional selectivity is the result of a ligand-dependent shift in a receptor’s conformation that favors interaction with one effector protein at the expense of other possible effector protein(s) within the continuum of possible active receptor conformations. Additionally, different

44 ligands for a GPCR may have different association and dissociation kinetics which determine the residence time of the ligand on the receptor. In the case of an agonist, the duration of the ligand-receptor complex may also influence the preferential signaling mechanisms.70 As with other GPCRs, it is now well appreciated that functional selectivity at CBRs offers the opportunity to refine cannabinoid based therapeutic approaches to improve beneficial properties and reduce side-effect liability.66-69 Thus, there is an urgent need for a better understanding of the molecular basis of the ligand-CBR binding motif(s) and the complex mechanisms and kinetics through which these binding motif(s) block or enable signaling events and intracellular processes.

Figure 2.1. Functional probes with high binding affinities for CB receptors, agonist properties and irreversible binding profiles.

Toward this goal, our laboratory has developed the Ligand Assisted Protein Structure

(LAPS), a successful approach that is currently being used to explore the binding motif(s) of cannabinergic ligands with their respective native receptors with a focus on investigating how these binding motifs are associated with distinct signaling profiles.34, 47

LAPS is a powerful approach based on the combined use of CBR mutants and mass spectrometric proteomic analysis and allows characterization of critical receptor residues that interact with chemically and functionally diverse set of ligands (including lipids) at the

45 level of specific amino acid residues.71-73 This experimental method utilizes purpose designed, biologically active, cannabinergic ligands exhibiting tight/irreversible binding characteristics for the CB1 and CB2 receptors.73-76 Design of these ligands relies on the incorporation of reactive groups at judiciously chosen positions within the cannabinoid chemical structure of interest (Figure 2.1). Over the past two decades our laboratory has developed such ligands that are representatives of different classes of cannabinergic compounds including endogenous, plant derived, and synthetic agonists and/or antagonists at CB1 and CB2.34, 47, 55, 71-78 Notwithstanding that these ligands/chemical reporters can help identifying amino acid residues within the binding pocket of the native receptor that are critical for ligand engagement and receptor function, they are also great utility in developing crystals of the mutant CB receptor-ligand complex for structural analysis using x-ray crystallography.46, 65

A considerable amount of our efforts is centered around the tricyclic hexahydrocannabinol

(HHC) prototype, a chemical scaffold that: a) exhibits the highest binding affinities for

CB1/CB2 and the most potent in vitro and in vivo agonist properties, b) encompasses three chiral centers, thus securing selectivity for CBRs over other proteins, and c) resembles the chemical structures of the marketed drugs THC and nabilone.64, 79 One of the analogs developed in this project, namely AM841,71-72, 78, 80 has distinguished itself by being both a valuable probe for LAPS studies and a unique ligand which facilitated the formation of the first crystals of the hCB1-agonist bound complex.46, 65 We report here a full account on the design, synthesis and biochemical characterization of novel HHC probes for CBRs. Incorporation of one reactive group within the HHC scaffold results in monofunctional ligands capable of interacting with one amino acid residue at the binding

46 pocket, while incorporation of two reactive groups will leads to HHC bifunctional ligands capable of interacting at two sites of the receptor to obtain more precise footprinting information. The rational design of these ligands is discussed below (Figure 2.2). All ligands synthesized were tested for binding affinities at cannabinoid CB1 and CB2 receptors and for tight/irreversible binding profiles. Representative analogs were evaluated for their functional properties at both CB1 and CB2 while an analog carrying the newly introduced nitrate ester functional group was found to behave as a potent CB1 agonists in vivo.

Figure 2.2. Design of the novel mono and bifunctional hexahydrocannabinol (HHC) probes and structures of the natural product (-)-Δ9-THC and the marketed HHC drug nabilone.

Structure-activity-relationship (SAR) studies from our own and other laboratories, has suggested that presence of gem-dimethyl or cyclopentyl groups at the C1'-position of the classical cannabinoids (HHC, or Δ9/Δ8-THC, 2, 1, Figure 2.2) enhances the ligand’s potency and efficacy.64, 81-83 It has also been demonstrated that incorporation of functional

47 groups or heterocyclic rings at the omega-position of the side chain can maintain, or even increase, the affinity of the compound for CBRs.83-86 Additionally, in the classical cannabinoids, side chains consisting of six to eight linear methylene groups are optimal for activity.64, 82, 85 Taken together all the above, our probe design incorporates the reactive group at the terminal atom of a seven or eight carbons long side chain carrying a gem-dimethyl or a cyclopentyl substituent at the C1' (3, Figure 2.2).

It has also been shown that in both HHCs and THCs presence of a hydroxyl or a keto group at the northern region of the tricyclic prototype can have a predominant effect on the ligand’s ability to bind to CBRs.64, 83, 87-88 With this in perspective, SAR work on the

HHC scaffold from our laboratory suggested that an equatorial hydroxymethyl group at

C9 is optimal for activity.89-90 Thus, our design adopts the 11-hydroxyl-HHC scaffold (2) for the side chain monofunctional probes (3) and incorporates a second reactive group at the C11 to generate the bifunctional ligands (4). The choice of the reactive group is critical as it is expected to engage the ligand with the amino acid residue(s) within the receptor’s binding domain. Such reactive groups encompass the previously reported electrophilic isothiocyanato and the photoactivatable azido groups,47, 64, 76 as well as the newly recognized nitrate moiety.46-47 We also explored the potential of the cyano group for irreversible labeling as this polar group is eight times smaller than a methyl group and thus, has the ability to develop polar interactions or hydrogen bonds with polar amino acids (e.g. serine or arginine) in sterically congested environments.91

CHEMISTRY

The mono-functional as well as the respective homo- and hetero-bifunctional probes for each series of analogs were prepared from a common advanced intermediate bromide

(2.13a or 2.13b or 2.13c, Scheme 2.1). Worthy of note is the robustness of the multistep stereoselective approach that produces the final compounds in multi milligram quantities as required for subsequent in vitro, in vivo and crystallography studies.

Scheme 2.1: Synthesis of advanced intermediate bromides

Reagents and Conditions: (a) PPh3, benzene, reflux, 72h, 98%-99%; (b) isopropenyl acetate, p-Toluenesulfonic acid, reflux, 6h, 98% (c) Pb(OAc)4, benzene, reflux, 3h; 90%(d)

- + CH3I, NaH, DMF, 0 ºC to r t, 2 h, 95%; (e) (Me3Si)2N K , Br(CH2)4Br, THF, 0 ºC to r t, 2 h,

- + 95%; (f) DIBAL-H, CH2Cl2, -78 ºC, 0.5 h, 87%; (g) Br P Ph3(CH2)5OPh, (Me3Si)2NK, THF,

0-10 ºC, 30 min, then addition to 2.3a or 2.3b, 0 ºC to r t, 2 h, 96% for 2.4a or 97% for

- + 2.4b; (h) Br P Ph3(CH2)6OPh, (Me3Si)2NK, THF, 0-10 ºC, 30 min, then addition to 2.3b, 0

ºC to r t, 2 h, 96% for 2.4c; (i) H2, 10% Pd on C, AcOEt, r t, 2.5 h, 89-98%; (j) BBr3, CH2Cl2,

-78 ºC to r t, 6 h, 85-98%; (k) 10, p-TSA, CHCl3, 0º C to r t, 4 days, 59-64%; (l) TMSOTf,

CH2Cl2/MeNO2, 0º C to r t, 3 h, 70-71%; (m) TBDMSCl, imidazole, DMAP, DMF, r t, 12 h,

- + 82-85%; (n) Cl Ph3P CH2OMe, (Me3Si)2NK, THF, 0º C to r t, 1 h, then addition to 9, 0 ºC to r t, 1.5 h, 70-73%; (o) Cl3CCOOH, CH2Cl2, r t, 50 min, 90-95%; (p) K2CO3, EtOH, r t, 3 h, 80-84%; (q) NaBH4, EtOH, 0 ºC, 30 min, 97-98%.

The optimized syntheses of the key intermediate bromides 2.13a-c are summarized in

Scheme 2.1. A short description of the synthesis for the C1'-gem-dimethyl-bromide 2.13a was presented in our recent work.65 Thus, deprotonation of 2.1 with sodium hydride followed by geminal dimethylation using methyl iodide gave nitrile 2.2a (95% yield).92

Sequential deprotonation of 2.1 with potassium bis(trimethylsilyl)amide (KHMDS) and cyclobisalkylation using 1,4-dibromobutane afforded (3,5-dimethoxyphenyl) cyclopentane carbonitrile 2.2b in very good yield (95%).81 Reduction of the cyano group in 2.2a and 2.2b with diisobutylalaminum hydride led to the respective aldehydes 2.3a and 2.3b (87-97%) which upon Wittig reaction with the ylide derived from (5- phenoxypentyl)triphenylphosphonium bromide (7a) or (6- phenoxyhexyl)triphenylphosphonium bromide (7b) and KHMDS, afforded exclusively the

81, 83 Z olefins 2.4a-c (JH2'-H3' = 11.1 Hz) in excellent yields (92-95%). In turn, the required phosphonium salts 7a or 7b were synthesized from commercially available 5- phenoxypentylbromide (6a) or 6-phenoxyhexylbromide (6b) respectively and triphenylphosphine in refluxing benzene.83 Catalytic hydrogenation of the double bond in

2.4a-c proceeded in excellent yields (89-98%) by using 10% Pd/C in ethyl acetate. This was followed by exposure of the intermediate alkanes 2.5a-c to boron tribromide which cleaved all three ether groups and introduced the C7' and C8' bromo group for resorcinols

2.6a-c (85-98% yields).83, 85 Following our modifications of an original procedure, the mixture of the chiral terpene diacetates 10 was produced in two steps from commercially available (1R)-(+)-nopinone (8).90, 93-94 Condensation of 10 with resorcinols 2.6a-c led to norpinanones 2.7a-c (59-64% yields) which upon treatment with catalytic trimethylsilyl triflate gave the 9-keto-hexahydrocannabinols 2.8a-c (70-71%) with the required (6aR,

10aR) stereochemistry.83, 94 The free phenolic hydroxyl in 2.8a-c was protected as the tert-butyldimethylsilyl (TBS) ether 2.9a-c in very good yields (82-85%).87 Treatment of commercially available (methoxymethyl)triphenylphosphonium chloride with KHMDS and coupling of the in situ formed ylide with the 9-keto-cannabinoids 2.9a-c gave the respective enol ethers 2.10a-c (70-73% yields) as 2:1 mixtures of two geometric isomers

(see experimental section) based on 1H NMR analysis.87, 89 Use of the sterically hindered base KHMDS and mild reaction conditions were necessary to optimize the yield of this

Wittig reaction. Subsequently, the methyl vinyl ethers 2.10a-c were hydrolyzed with wet trichloroacetic acid (90-95% yields) and the resulted diastereomeric mixture of C9 aldehydes 2.11a-c (2:1 ratio by 1H NMR) was epimerized to give the β-equatorial isomers

2.12a-c in 80-84% yields.87, 89 Finally, reduction with sodium borohydride afforded the key

11-hydroxy-bromides 2.13a-c in high yields (97-98%).

Synthesis of the side chain mono-functional probes was accomplished as shown in

Scheme 2.2. Thus, 11-hydroxy-intermediates 2.13a-c were treated with tetra-n- butylammonium fluoride to give the C7'/ C8'-bromo-cannabinoids 2.14a-c in high yields

(95-98%).87, 89 Displacement of the bromide in HHCs 2.14a-c by tetramethylguanidinium azide led to mono-functional C7'/C8' azide probes 2.15a-c (84-88% yields).75 Exposure of these analogs first to triphenylphosphine and then to carbon disulfide converted the azide to isothiocyanate leading to the respective probes 2.16a-c (76-87 yields).75, 95

Scheme 2.2: Synthesis of monofunctional probes

Reagents and conditions: (a) n-Bu4NF, CH2Cl2, -50 ºC, 40 min, 95-98%; (b) TMG-N3,

CH3Cl/CH3NO2, 70 ºC, 30 h, 84-88%; (c) CS2, PPh3, THF, r t, 18h, 76-87%; (d) AgNO3,

MeCN, 87º C reflux, 48 h, 84-86%; (e) n-Bu4NF, CH2Cl2, -50 ºC, 40 min, 90-92%; (f) NaCN,

DMSO, r t, 18h, 67-71%.

Reaction of bromides 2.13a-c with silver nitrate in refluxing acetonitrile46 led to C7' or C8' nitrate esters 2.17a-c (84-86% yields) from which the final side chain nitrate ester probes

2.18a-c were obtained by fluorodesilylation using tetra-n-butylammonium fluoride (90-92% yields). Treatment of bromides 2.14a-c with sodium cyanide in dimethyl sulfoxide afforded the respective C7' and C8' cyano-probes 2.19a-c in high yields (67-71%).83

Scheme 2.3: Synthesis of homo-bifunctional probes

o Reagents and conditions: (a) I2, imidazole, PPh3, benzene, 50 C, 1 h, 95%; (b) (n-

+ - Bu)4N N3 , CH2Cl2, r t, 24 h, 83%; (c) TMG-N3, CHCl3/CH3NO2, 70 ºC, 30 h, 74%; (d) n-

Bu4NF, CH2Cl2, -50 ºC, 40 min, 92%; (e) CS2, PPh3, THF r t, 18h, 86%.

Scheme 2.4: Synthesis of hetero-bifunctional probes

Reagents and conditions: (a) AgNO3, MeCN, 87º C reflux, 48 h, 90%; (b) n-Bu4NF, CH2Cl2,

-50 ºC, 40 min, 90%; (c) CS2, PPh3, THF, r t, 18h, 71%.

The bifunctional cannabinoid ligands and especially the hetero-bifunctional probes represent a much greater challenge to the synthetic chemist than the mono-functional analogs. This challenge is imposed by the need to introduce multiple reactive groups within the molecule. We have found an efficient approach to accomplish this task which takes advantage from the different reactivities of the alkyl bromides and iodides under conditions of nucleophilic substitution. Thus, the synthesis of both homo- and hetero- bifunctional probes proceeded in high overall yields from the common key intermediate

8'-bromo-11-iodo-HHC 2.20c with complete control of the halogen substitution under the experimental conditions used. Synthesis of the homo-bifunctional azido and isothiocyanato probes carrying a cyclopentyl ring at the C1' is depicted in Scheme 2.3.

The 11-hydroxy-HHC bromide 2.13c was converted to the requisite 8'-bromo-11-iodo-

HHC analog 2.20c in 95% yield by using the triphenylphosphine, iodine, imidazole method.85 The first azide displacement reaction occurs exclusively at C11 and in high yield (83%) upon exposure of 2.20c to tetra-n-butylammonium azide at room temperature.

The second azide displacement at C8' was accomplished by treatment of the 8'-bromo-

11-azido-HHC analog 2.21c with tetramethylguanidinium azide in chloroform/nitromethane at 70oC to give 2.22c (74% yield). This was followed by cleavage of the silyl ether at C1 using TBAF to produce the di-azido-probe 2.23c in 92% yield. The subsequent Staudinger reactions worked well at both the C11 and the C8' azido groups and afforded the homo-bifunctional isothiocyanato ligand 2.24c in a single operation and in high yield (86%) (Scheme 2.3). The intermediate C11 azide 2.21c served as the starting point for the synthesis of the hetero-bifunctional probes 2.26c and 2.27c (Scheme 2.4).

Thus, treatment of bromide 2.21c with silver nitrate gave the precursor compound 2.25c

(90% yield) from which the 11-azido-8'-nitrate ester probe 2.26c was prepared in 90% yield through a fluorodesilylation reaction in low temperature. The most reactive isothiocyanato group seen in the heterobifunctional probe 2.27c was introduced in the last synthetic step by exposure of the azide 2.26c to Staudinger conditions (71% yield).

RESULTS AND DISCUSSION

Cannabinoid receptor affinities

The abilities of the new compounds 2.15a-c, 2.16a-c, 2.17a-c, 2.18a-c, 2.19a-c, 2.23c,

2.24c, 2.26c and 2.27c to displace the radiolabeled CB1/CB2 agonist CP-55,940 from membranes prepared from rat brain (source of CB1) and HEK 293 cells expressing either mouse CB2 or human CB2 were determined as described earlier,83-84 and inhibition constant values (Ki) from the respective competition binding curves are listed in Table 1.

The use of two CB2 receptor preparations was aimed at addressing species differences that we observed earlier.96 In addition, because of the covalent nature of the binding of the isothiocyanates 2.16a-c, 2.24c and 2.27c, the affinities for CB receptors are presented as "apparent Ki" (Ki*) values and these are expected to be dependent on the time during which the preparation is pretreated with the ligand.76 The compounds included in this study are hexahydrocnnabinol analogs (HHCs) with one reactive group at the omega position (C7'/C8') of the C3 side chain (mono-functional probes), or two reactive groups (bifunctional probes) at both the C8' and the C11 of the HHC structure. The seven or eight carbon long side chains of the mono-functional ligands carry a gem-dimethyl or a cyclopentyl substituent at C1' while the bifunctional ligands bear a cyclopentyloctyl chain

55 at C3. In agreement with our rational design our data indicate that the mono-functional

11-OH-HHCs have remarkably high binding affinities for both the CB1 and the CB2 receptors with most of their Ki values in the picomolar range and also, they do not exhibit species differences. This suggests that within this structural motif: a) both seven and eight carbon long chain analogs substituted at C1' with the gem-dimethyl or with the bulkier cyclopentyl groups can fit well within the binding domain of CB1 and CB2, and b) presence of the reactive group (i.e., N3, NCS, ONO2, and CN) at the omega position of the chain is well tolerated.

A cursory examination of the binding affinity data of the side chain monofunctional ligands

2.15c and 2.18c with their bifunctional counterparts 2.23c and 2.26c respectively indicates that replacement of the 11-hydroxy group with an azido moiety maintains the high affinity of the ligand for both the CB1 and CB2 receptors. However, comparisons of the data of 2.18c and 2.27c suggests that presence of the electrophilic isothiocyanate at

C11 results in a reduction in the affinity of the probe for both receptors which is more accentuated in CB2. Thus, the heterobifunctional ligand 2.27c exhibits a 2- to 6-fold preference for CB1 over CB2. Interestingly, transformation of the C8'-nitrate ester moiety of this ligand (2.27c) to an isothiocyanate group (analog 2.24c) reverses the receptor preference trend and now the di-isothiocyanato probe 2.24c has a slight preference for

CB2 (3- to 10-fold).

Table 2.1. Affinities (Ki) of mono- and bi-functional classical cannabinoid probes for CB1 and CB2 cannabinoid receptors (± 95% confidence limits).

a compd X1 X2 n R1, R2 (Ki, nM) rCB1 mCB2 hCB2

2.15a -N3 -OH 2 0.41  0.05 0.8  0.1 1.40 0.06

2.15b -N3 -OH 2 0.4  0.1 0.8  0.1 0.8  0.4

2.15c -N3 -OH 3 0.5  0.2 1.6  0.1 1.5  0.3

2.16a -NCS -OH 2 0.39  0.04 0.8  0.1 3.150.04

2.16b -NCS -OH 2 1.1  0.1 0.9  0.2 1.3  0.5

2.16c -NCS -OH 3 0.4  0.1 1.1 ± 0.1 1.0  0.2

2.18a -ONO2 -OH 2 0.20  0.05 0.50  0.06 0.8  0.1

2.18b -ONO2 -OH 2 0.41  0.08 0.40  0.05 0.7  0.1

2.18c -ONO2 -OH 3 0.20  0.08 1.0  0.1 0.7  1.1

2.19a -CN -OH 2 0.40  0.05 0.8  0.1 0.4  0.1

2.19b -CN -OH 2 0.8  0.2 1.0  0.1 1.4  0.2

2.19c -CN -OH 3 0.5  0.1 0.9  0.1 0.40 0.05

33c -N3 -N3 3 0.7  0.1 1.2 0.2 1.5  0.2

2.24c -NCS -NCS 3 37.6  0.6 14.6  2.1 4.2  0.3

2.26c -ONO2 -N3 3 0.9  0.2 1.4  0.2 0.90.2

2.27c -ONO2 -NCS 3 5.5  0.3 10.9 ± 0.9 28.7  0.7

aAffinities for CB1 and CB2 were determined using rat brain (CB1) or membranes from

HEK293 cells expressing mouse or human CB2 and [3H]CP-55,940 as the radioligand following previously described procedures.83, 89 Data were analyzed using nonlinear regression analysis. Ki values were obtained from three independent experiments performed in triplicate and are expressed as the mean of the three values.

Irreversible labeling of the CB1 and CB2 receptors

The most prominent feature of the irreversible or tight binding probes which differentiates them from other high affinity binding ligands is their wash-resistant binding ability to each of the two receptors.46, 65 The ability of the mono- and bi-functional probes carrying irreversibly reacting groups to label each of the two receptors was determined by measuring reductions in the binding for the radioligand [3H]CP-55,940 when the preparation was pretreated with the irreversible ligand, and comparing it to the untreated

58 sample (details under Experimental). Following earlier work from our laboratory,76 the experiment was conducted by pretreating the sample with a concentration equal to 10- fold the compound’s Ki value for the receptor in question and subsequently measuring

3 the decrease of Bmax obtained from a saturation curve using [ H]CP-55,940. Testing results are summarized in Table 2.1, while saturation binding curves for all analogs are provided under Supporting Information. Our data clearly show that all side chain monofunctional probes (2.15a-c, 2.16a-c, 2.18a-c, and 2.19a-c) are capable of irreversibly labeling the CB1 receptor. As a general trend, the best results are obtained with the ligands carrying the seven carbons long side chains. To this extent, the C1'-gem- dimethyl substituted isothiocyanato (NCS, 2.16a), nitrate ester (ONO2, 2.18a), and cyano

(CN, 2.19a) probes exhibit optimal ability to irreversible label while the C1'-cyclopentyl substituted azido (N3, 2.15b) ligand seems to be the best probe for photoirradiation experiments.

Moreover, a cursory examination of the data on Table 2.2 reveals that, in general, the synthesized probes exhibit slightly reduced abilities to irreversibly label the CB2 receptor when compared to CB1. The preferences of the CB2 receptor for optimal side chains parallel those of CB1 for the azido (N3, 2.15b), isothiocyanato (NCS, 2.16a) and cyano

(CN, 2.19a) probes, while the C1'-cyclopentyl chains are optimal for the nitrate ester substituted ligands (ONO2, 2.18b). Comparisons of the side chain cyclopentyloctyl di- azido 2.23c and di-isothiocyanato 2.24c ligands with their mono-functional counterparts

2.15c and 2.16c, respectively, shows a reduction in the labeling ability of these homo- bifunctional probes. Within the cyclopentyloctyl side chain structural motif, the hetero- bifunctional probes 2.26c and 2.27c exhibited no ability to engage the receptor

59 irreversibly. This indicates that the stereoelectronic requirements for irreversible labeling are more stringent for the bifunctional probes than the side chain mono-functional ligands.

We postulate that this might be due to the availability of the 11-OH group in all mono- functional probes which is not the case in the bifunctional ligands. To this extend, the recently released agonist-bound crystal structure of human CB1 in complex with 2.16a shows that the 11-OH group forms a hydrogen bond with the Ile267 of the ECL2.65

Table 2.2. Reductions in the specific binding of [3H]CP-55,940 after irreversible ligand pretreatment.

compd CB1 CB2

irreversible labeling irreversible labeling

(%)* (%)*

2.15a 75  7 63  4 2.15b 87  8 65  6 2.15c 48  4 37  2 2.16a 65  5 75  5 2.16b 39  3 34  3 2.16c 51  4 19  2 2.18a 70  5 29  2 2.18b 69  4 54  3 2.18c 32  3 44  4 2.19a 76  5 60  4 2.19b 68  5 45  3 2.19c 60  4 40  3 2.23c 28  2 21  1 2.24c 37  3 15  1

2.26c No labeling No labeling 2.27c No labeling No labeling

*The receptor membranes were pretreated with a concentration equal to 10-fold the

38 compound’s Ki value. Percent labeling was calculated as : F x 100. Bmax(ligand) was

3 calculated as described under Experimental. The Bmax(control) for [ H]-CP-55,940 was determined by conducting the assay as described, but in the absence of the test ligand.

Confidence intervals (95%) are shown in parentheses.

We have already reported negative control experiments using two analogs of the C7'- isothiocyanato probe 2.16a that both lack the irreversibly reacting group at the C7' position (i.e., C7'-H and C7'-Br).78, 80 In these experiments we observed no reductions in

3 the levels of specific binding (Bmax) of [ H]CP-55,940 under conditions identical to those used for the covalent labeling. Optimal irreversible probes reported here are in continuous use in our laboratory for studying the structures of the ligand-receptor binding complexes using the LAPS approach as well as for receptor crystallography work.

Functional characterization

The long-term goal of this project was to develop mono- and bi-functional classical cannabinoid probes exhibiting irreversible/tight binding characteristics and agonist properties. Thus, the monofunctional probes 2.15a, 2.16a, 2.16b, 2.16c, 2.18a, 2.18b,

2.19a and 2.19b were selected for further evaluation in the adenylyl cyclase assay by measuring the changes in forskolin-stimulated cAMP, as described in the Experimental

Section.76, 83 We focused on these key analogs not only because they possess high CB1 receptor binding affinities and predominant ability to label this receptor subsite, but also because these representative ligands cover all of the structural motifs we have adopted in our studies. The testing results are listed in Table 2.3. We observe that all representative probes behave as potent and effective agonists at both the CB1 and CB2 receptor under this signaling pathway as they all potently decreased the levels of cAMP.

Table 2.3. CB1 Functional potencies (EC50) of selected probes for the CB1 and CB2 receptors.

rCB1 hCB2

compd a b a b EC50 (nM) E(max) (%) EC50 (nM) E(max) (%)

2.15a pending pending 16.4  5.2 53  3.9, agonist 2.16a 35.0  9.2 77  4.2, agonist 25.5  6.3 65  3.9, agonist 2.16b 35.6  10.1 87  4.7, agonist 17.5  5.6 69  3.8, agonist 2.16c pending pending 381.6  17.3 89  5.0, agonist 2.18a pending pending 6.9  1.8 39  2.5, agonist 2.18b 1.7  0.3 74  3.7, agonist 10.9  2.3 42  4.0, agonist 2.19a pending pending 2.19b pending pending 25.3  6.5 61  4.2, agonist

aFunctional potencies at rCB1 and hCB2 receptor were determined by measuring the decrease in forskolin-stimulated cAMP levels.27, 28 EC50 values were calculated using nonlinear regression analysis. Data are the average of two independent experiments run in triplicate, and 95% confidence intervals for the EC50 values are given in parentheses.

62 b Forskolin stimulated cAMP levels were normalized to 100%. E(max) is the maximum inhibition of forskolin stimulated cAMP levels and is presented as the percentage of CP-

55,940 response at 500 nM.

In vivo behavioral characterization

In earlier work we have shown that classical cannabinoids bearing cyano or isothiocyanato groups at the terminal carbon atom of the side chain exhibit in vivo effects in rodents that are consistent with CB1 activation and this is in agreement with the data obtained from the cyclase assay.84, 97 However, the nitrate ester group was introduced recently in the cannabinoid literature from our laboratory on studies with novel CB1 antagonists46, 98 and the effects of this group on the agonist cannabinoid structure have not been studied in vivo. Thus, the nitrate ester probe 2.18a was studied in the CB1 receptor-characteristic analgesia test in mice and the results are provided bellow.

Antinociception was measured using the tail flick procedure over a 6-hour period following drug injection. Prior to drug administration, the average baseline tail-flick latency was 1.6

±0.1s. Doses of 0.3-3.0 mg/kg 2.18a had significant antinociceptive effects with a mean

(± 95% CL) ED50 value of 0.56 mg/kg (0.34-0.93) (Table 2.4). The compound had fast onset of action (20 min) with peak antinociceptive effects observed at 180 mins and these effects were persisted for a 6-hour period.

Table 2.4. ED50 for the analgesic effects of 2.18a in mice.

compd Structure ED50 Values 95% CL

mg/kg mg/kg

2.18a 0.56 0.34 to 0.93

CONCLUSIONS

We report the design, synthesis, and biochemical characterization of novel cannabinergic ligands with remarkably high binding affinities for the CB1 and CB2 cannabinoid receptors, that are CB1/CB2 agonists with irreversible binding profiles. Our ligand design relies on the incorporation of a reactive group at judiciously chosen positions within the classical cannabinoid structure including the aliphatic chain at C3 and the substituents at C11

(monofunctional probes). Such groups encompass the electrophilic isothiocyanato and cyano groups, the photoactivatable aliphatic azido moiety as well as the polar nitrate ester group. Our experiments indicated that all of the groups we utilized are capable of tight/irreversible interactions with the CBRs. These groups can also be combined on the same molecule to produce bifunctional ligands potentially capable of interacting at two distinct sites within the CB1/CB2 binding domains. All probes reported here represent high affinity ligands acting as potent CB1 agonists. These molecular probes are currently being used in studies aimed at uncovering the binding motifs of classical cannabinoids

64 with the CB1 and CB2 receptors through the Ligand Assisted Protein Structure (LAPS) approach which combines the use of receptor mutants and mass spectrometric proteomic analysis. These ligands are also of great utility in developing crystals of the CB receptor- ligand complex for structural analysis using x-ray crystallography.

EXPERIMENTAL SECTION

Materials. All reagents and solvents were purchased from Aldrich Chemical Company, unless otherwise specified, and used without further purification. All anhydrous reactions were performed under a static argon atmosphere in flame-dried glassware using scrupulously dry solvents. Flash column chromatography employed silica gel 60 (230-

400 mesh). All compounds were demonstrated to be homogeneous by analytical TLC on pre-coated silica gel TLC plates (Merck, 60 F245 on glass, layer thickness 250 m), and chromatograms were visualized by phosphomolybdic acid staining. Melting points were determined on a micro-melting point apparatus and are uncorrected. IR spectra were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer. NMR spectra were

1 recorded in CDCl3, unless otherwise stated, on a Bruker Ultra Shield 400 WB plus ( H at

400 MHz, 13C at 100 MHz) or on a Varian INOVA-500 (1H at 500 MHz, 13C at 125 MHz) spectrometers and chemical shifts are reported in units of  relative to internal TMS.

Multiplicities are indicated as br (broadened), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and coupling constants (J) are reported in hertz (Hz). Low and high- resolution mass spectra were performed in School of Chemical Sciences, University of

Illinois at Urbana-Champaign. Mass spectral data are reported in the form of m/z (intensity

65 relative to base = 100). LC/MS analysis was performed by using a Waters MicroMass ZQ system [electrospray-ionization (ESI) with Waters-2525 binary gradient module coupled to a Photodiode Array Detector (Waters-2996) and ELS detector (Waters-2424) using a

XTerra MS C18, 5 µm, 4.6 mm x 50 mm column and acetonitrile/water.

2-(3,5-Dimethoxyphenyl)-2-methylpropanenitrile (2.2a)65. To a stirred suspension of sodium hydride (5.1 g, 213.4 mmol) in dry DMF (133.4 mL) at 0 °C under an argon atmosphere was added dropwise a solution of 2.1 (11.8 g, 66.7 mmol) and iodomethane

(13.2 mL, 213.4 mmol) in dry DMF (6.8 mL). The reaction temperature was rose to 25 oC over a 15 min period and stirring was continued for 2 h. The reaction mixture was quenched with saturated aqueous NH4Cl solution and diluted with diethyl ether. The organic layer was separated and the aqueous layer was extracted with diethyl ether. The combined organic layer was washed with water and brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (25% ethyl acetate in hexane) gave the title compound (13.0 g, 95% yield) as a colorless oil. IR

(neat) 2950, 2840, 2234 (w, CN), 1532, 1438, 1319, 1204, 788 cm-1. 1H NMR (500 MHz,

CDCl3) δ 6.61 (d, J = 2.0 Hz, 2H, ArH), 6.40 (t, J = 2.0 Hz, 1 H, ArH), 3.81 (s, 6 H, -OCH3),

+ 1.71 (s, 6 H, -C(CH3)2-); mass spectrum (ESI) m/z (relative intensity) 206 (M +H, 100).

HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 4.5 min for the title compound.

1-(3,5-Dimethoxyphenyl)cyclopentane-1-carbonitrile (2.2b)81. To a stirred solution of

2.1 (12 g, 67.7 mmol) in dry THF was added dropwise a suspension of 1,4-dibromobutane

(16.1 g, 74.5mmol) in dry THF (564 mL) and a solution of potassium bis(trimethylsilyl)amide in dry THF (33.7 g, 169 mmol) at r t, under an argon atmosphere.

The reaction stirring was continued for 2 h. The reaction mixture was quenched with saturated aqueous NH4Cl solution and diluted with diethyl ether. The organic layer was separated, and the aqueous layer was extracted with diethyl ether. The combined organic layer was washed with water and brine, dried (MgSO4), and concentrated in vacuo.

Purification by flash column chromatography on silica gel (25% ethyl acetate in hexane) gave the title compound (14.9 g, 95% yield) as a colorless oil. IR (neat) 2957, 2839, 2234

-1 1 (w, CN), 1594, 1456, 1425, 1321, 1204, 1154, 1063, 835 cm . H NMR (500 MHz, CDCl3)

δ 6.59 (d, J = 2.0 Hz, 2H, ArH), 6.39 (t, J = 2.0 Hz, 1 H, ArH), 3.81 (s, 6 H, -OCH3), 2.51-

2.40 (m, 2 H of the cyclopentyl ring), 2.10-1.95 (m, 4 H of the cyclopentyl ring), 1.95-1.89

13 (m, 2 H of the cyclopentyl ring). C NMR (100 MHz CDCl3) δ 161.0 (ArC), 142.0 (ArC),

124.2 (-CN), 104.4 (ArC), 99.0 (ArC), 55.2 (-OCH3), 47.8, 40.1, 24.1. mass spectrum (ESI) m/z (relative intensity) 232 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 4.7 min for the title compound.

2-(3,5-Dimethoxyphenyl)-2-methylpropanal (2.3a)65. To a solution of 2.2a (12.9 g, 63.0 mmol) in anhydrous CH2Cl2 (441 mL) at –78 °C, under an argon atmosphere was added

1M solution of DIBAL-H in Hexane (189 mL). The reaction mixture was stirred for 30 min and then quenched by dropwise addition of potassium sodium tartrate (10% solution in

67 water) at – 78 °C. Following the addition, the mixture was warmed to room temperature, stirred for an additional 50 minutes and then diluted with ethyl acetate. The organic phase was separated, and the aqueous phase extracted with ethyl acetate. The combined organic layer was washed with brine, dried (MgSO4), and the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel (10-35% ethyl acetate in hexane) to give 11.4 g of 2.3a as viscous oil in 87% yield. IR (neat) 2938, 2838, 2705 (w, CHO), 1725 (s, >C=O), 1595, 1457, 1424, 1314,

-1 1 1205, 1156, 1066, 835 cm . H NMR (500 MHz, CDCl3) δ 9.46 (s, 1H, -CHO), 6.40 (d, J

= 2.0 Hz, 2H, 2-H, 6-H, ArH), 6.39 (t, J = 2.0 Hz, 1H, 4-H, ArH), 3.78 (s, 6H, OMe), 1.43

13 (s, 6H, -C(CH3)2-). C NMR (100 MHz CDCl3) δ 201.6 (-CHO), 161.1 (ArC), 143.6 (ArC),

105.1 (ArC), 98.6 (ArC), 65.7 (>(C)CHO), 55.2 (-OCH3), 50.5, 22.3. Mass spectrum (EI) m/z (relative intensity) 208 (M+, 25), 196 (16), 179 (M+-CHO), 165 (25), 151 (14), 139 (39),

+ 91 (20), 77 (20). Exact mass (EI) calculated for C12H16O3 (M ), 208.10995; found,

208.11077. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 4.4 min for the title compound.

1-(3,5-Dimethoxyphenyl)cyclopentane-1-carbaldehyde (2.3b)81. The synthesis was carried out as described for 2.3a using 2.2b (14.5 g, 62.7 mmol) and solution of 1M

DIBAL-H in Hexane (188 mL) in anhydrous CH2Cl2 (439 mL) and gave 2.3b (13.5 mg 92% yield) as viscous oil. IR (neat) 2938, 2838, 2702 (w, CHO), 1700 (s, >C=O), 1556, 1443,

-1 1 1410, 1295, 1201, 1156, 1043, 831 cm . H NMR (500 MHz, CDCl3) δ 9.37 (s, 1H, -CHO),

6.40 (d, J = 2.0 Hz, 2H, ArH), 6.37 (t, J = 2.0 Hz, 1 H, ArH), 3.79 (s, 6 H, -OCH3), 2.49-

2.45 (m, 2 H of the cyclopentyl ring), 1.90-1.84 (m, 2 H of the cyclopentyl ring), 1.76-1.73

(m, 2 H of the cyclopentyl ring), 1.65-1.60 (m, 2 H of the cyclopentyl ring); mass spectrum

(ESI) m/z (relative intensity) 235 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco

Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 4.6 min for the title compound.

(Z)-3,5-Dimethoxy-1-(2-methyl-8-phenoxyoct-3-en-2-yl)benzene (2.4a)65. To a stirred suspension of (5-phenoxypentyl) triphenylphosphonium bromide (74.5 g, 147.4 mmol) in dry THF (39 mL) at 0 ºC, under an argon atmosphere was added potassium bis(trimethylsilyl)amide (29.0 g, 145.2 mmol). The mixture was stirred for 30 minutes at

10 ºC to ensure complete formation of the orange phosphorane. A solution of aldehyde

2.3a (11.4 g, 54.6 mmol) in 8.6 mL THF was added dropwise to the resulting slurry, at 0

ºC. The reaction was stirred for 2 hours at room temperature and upon completion was quenched by the addition of saturated aqueous NH4Cl. The organic layer was separated and the aqueous phase was extracted with diethyl ether. The combined organic layer was washed with brine and dried (MgSO4) and the solvent was evaporated under reduced pressure. The residue was purified on a silica gel (5-15 % diethyl ether in hexanes) to give 18.0 g compound 2.4a as colorless oil in 93% yield. IR (neat) 2958, 2835, 1594,

-1 1 1422, 1243, 1204, 1153, 1052, 753 cm . H NMR (500 MHz, CDCl3)  7.26 (m as t, J =

7.5 Hz, 2H, 3-H, 5-H, OPh), 6.91 (m as t, J = 7.5 Hz, 1H, 4-H, OPh), 6.83 (m as d, J =

7.5 Hz, 2H, 2-H, 6-H, OPh), 6.55 (d, J = 2.5 Hz, 2H, 2-H, 6-H, ArH), 6.27 (t, J = 2.5 Hz,

1H, 4-H, ArH), 5.65 (d t, J = 11.1 Hz, J = 1.5 Hz, 1H, 2′-H), 5.29 (dt, J = 11.1 Hz, J = 7.8

Hz, 1H, 3′-H), 3.79-3.73 (t and s overlapping, 8H, OMe and 7′-H), 1.71 (dtd, 2H, 4′-H),

13 1.56 (qt, 2H, 5′-H), 1.39 (s, 6H, -C(CH3)2-), 1.31 (qt, 2H, 6′-H). C NMR (100 MHz CDCl3)

δ 160.4 (ArC), 159.0 (ArC), 153.2 (ArC), 139.7 (>C=C<), 130.8 (>C=C<), 129.3 (ArC),

120.4 (ArC), 114.4 (ArC), 104.8 (ArC), 97.0 (ArC), 67.5 (-CH2OPh), 55.2 (-OCH3), 40.3,

31.3, 28.8, 27.9, 25.6. Mass spectrum (ESI) m/z (relative intensity) 377 (M++Na, 5), 356

(M++H+1, 30), 355 (M++H, 100), 261 (M++H-OPh, 30), 205 (20), 191 (8).165 (10), 115 (8).

HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.8 min for the title compound.

(Z)-1,3-Dimethoxy-5-[1-(6-phenoxyhex-1-en-1-yl)cyclopentyl]benzene (2.4b)83. The synthesis was carried out as described for 2.4a using 2.3b (6.8g, 29.0 mmol), (5- phenoxypentyl) triphenylphosphonium bromide (44.0 g, 87.1 mmol) and potassium bis(trimethylsilyl)amide (17.3 g, 86.7 mmol) in dry THF (529 mL) and give 2.4b (10.1 g,

92% yield) as colorless oil. IR (neat) 2956, 2813, 1587, 1410, 1217, 153, 1052, 753 cm-

1 1 . H NMR (500 MHz, CDCl3)  7.26 (m as t, J = 7.5 Hz, 2H, 3-H, 5-H, OPh), 6.91 (m as t, J = 7.5 Hz, 1H, 4-H, OPh), 6.84 (m as d, J = 7.5 Hz, 2H, 2-H, 6-H, OPh), 6.52 (d, J =

2.5 Hz, 2H, 2-H, 6-H, ArH), 6.26 (t, J = 2.5 Hz, 1H, 4-H, ArH), 5.74 (d t, J = 11.1 Hz, J =

1.5 Hz, 1H, 2′-H), 5.29 (dt, J = 11.1 Hz, J = 7.8 Hz, 1H, 3′-H), 3.78-3.73 (t and s overlapping, 8H, OMe and 7′-H), 2.04-1.97 (m, 2 H of the cyclopentyl ring), 1.94-1.87 (m,

2 H of the cyclopentyl ring),1.80 (dtd, 2H, 4′-H), 1.76-1.65 (m, 2H of the cyclopentyl ring),

1.56 (qt, 2H, 5′-H), 1.29 (s, 6H, -C(CH3)2-), 1.31 (qt, 2H, 6′-H). Mass spectrum (ESI) m/z

(relative intensity) 381 (M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.1 min for the title compound.

(Z)-1,3-Dimethoxy-5-[1-(7-phenoxyhept-1-en-1-yl)cyclopentyl]benzene (2.4c). The synthesis was carried out as described for 2.4a using 2.3b (6.5 g, 27.7 mmol), (6- phenoxyhexyl) triphenylphosphonium bromide (43.2 g, 83.2 mmol) and potassium bis(trimethylsilyl)amide (16.5 g, 82.8 mmol) in dry THF (525 mL) and give 2.4c (10.4 g,

95% yield) as colorless oil. IR (neat) 2949, 2809, 1594, 1422, 1243, 1153, 1049, 753 cm-

1 1 . H NMR (500 MHz, CDCl3)  7.27 (m as dd, J = 8.5 Hz, J = 7.5 Hz, 2H, 3-H, 5-H, OPh),

6.91 (m as t, J = 7.5 Hz, 1H, 4-H, OPh), 6.87 (m as d, J = 8.5 Hz, 2H, 2-H, 6-H, OPh),

6.52 (d, J = 2.0 Hz, 2H, 2-H, 6-H, ArH), 6.26 (t, J = 2.0 Hz, 1H, 4-H, ArH), 5.72 (d t, J =

11.0 Hz, J = 2.0 Hz, 1H, 2′-H), 5.29 (dt, J = 11.0 Hz, J = 7.5 Hz, 1H, 3′-H), 3.84 (t, 2H, 8′-

H), 3.76 (s, 6H, OMe), 2.01-1.97 (m, 2 H of the cyclopentyl ring), 1.94-1.89 (m, 2 H of the cyclopentyl ring), 1.79-1.67 (m, 6H, 4 H of the cyclopentyl ring and 4′-H), 1.60 (qt, 2H, 5′-

H), 1.21 (m, 4H, 6′-H, 7′-H). Mass spectrum (ESI) m/z (relative intensity) 395 (M++H).

HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.2 min for the title compound.

3,5-Dimethoxy-1-(2-methyl-8-phenoxyoctan-2-yl)benzene (2.5a)65. To a solution of

2.4a (17.9g, 50.4 1mmol) in ethyl acetate (504 mL) was added Pd/C (2.7 g, 15% w/w) and the resulting suspension stirred vigorously for 2.5 hours under hydrogen atmosphere at room temperature. The catalyst was removed by filtration through celite, and the filtrate was evaporated under reduced pressure to afford 16.0 g of the crude product 2.5a as colorless oil, in 89% yield which was used in the next step without further purification. IR

(neat) 2954, 2853, 1567, 1480, 1241, 1213, 1153, 831, 753 cm-1. 1H NMR (500 MHz,

CDCl3)  7.25 (m as t, J = 8.0 Hz, 2H, 3-H, 5-H, OPh), 6.92 (m as t, J = 8.0 Hz, 1H, 4-H,

OPh), 6.86 (m as d, J = 8.0Hz, 2H, 2-H, 6-H, OPh), 6.49 (d, J = 1.5 Hz, 2H, 2-H, 6-H,

ArH), 6.30 (t, J = 1.5 Hz, 1H, 4-H, ArH), 3.90 (t, J = 6.0 Hz, 2H, 7′-H), 3.79 (s, 6H, OMe),

1.75-1.68 (m, 2H, 2′H), 1.59-1.53 (m, 2H, 3′-H), 1.43-1.35 (m, 2H, 4′-H), 1.30-1.22 (m and s overlapping, 8H, 5′-H and -C(CH3)2-), 1.12-0.60 (m, 2H, 6′-H); Mass spectrum (ESI) m/z

(relative intensity) 358 (M++H+1, 30), 357 (M++H, 100), 281 (8), 263 (M++H-OPh, 5).

+ Exact mass (ESI) calculated for C23H33O3 (M +H), 357.2430; found, 357.2427. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.9 min for the title compound.

1,3-Dimethoxy-5-[1-(6-phenoxyhexyl)cyclopentyl]benzene (2.5b)83. The synthesis was carried out as described for 2.5a using 2.4b (10.0 g, 26.3 mmol) and Pd/C (1.0 g,

10% w/w) in ethyl acetate (240.1 mL) and give 2.5b (9.7 g, 97% yield) as colorless oil. IR

-1 1 (neat) 2956, 2820, 1563, 1415, 1231, 1109, 1062, 753 cm . H NMR (500 MHz, CDCl3)

 7.26 (m as dd, J = 9.0 Hz, J = 8.0 Hz, 2H, 3-H, 5-H, OPh), 6.92 (m as t, J = 8.0 Hz, 1H,

4-H, OPh), 6.86 (m as d, J = 8.0Hz, 2H, 2-H, 6-H, OPh), 6.43 (d, J = 1.5 Hz, 2H, 2-H, 6-

H, ArH), 6.29 (t, J = 1.5 Hz, 1H, 4-H, ArH), 3.89 (t, J = 6.0 Hz, 2H, 7′-H), 3.79 (s, 6H,

OMe), 1.92-1.85 (m, 2 H of the cyclopentyl ring), 1.82-1.73 (m, 2H, 2′H), 1.72-1.59 (m, 6

H of the cyclopentyl ring), 1.59-1.51 (m, 2H, 3′-H), 1.41-1.32 (m, 2H, 4′-H), 1.26-1.18 (m

2H, 5′-H), 1.07-0.96 (m, 2H, 6′-H); Mass spectrum (ESI) m/z (relative intensity) 383

(M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.1 min for the title compound.

1,3-Dimethoxy-5-[1-(7-phenoxyheptyl)cyclopentyl]benzene (2.5c). The synthesis was carried out as described for 2.5a using 2.4c (10.0g, 25.3 mmol) and10% Pd/C (1.0 g, 10% w/w) in ethyl acetate (230 mL) and give 2.5c (9.8 g, 98% yield) as colorless oil.

IR (neat) 2930, 2857, 1594, 1496, 1455, 1244, 1203, 1153, 1062, 831, 753 cm-1. 1H NMR

(500 MHz, CDCl3)  7.27 (m as dd, J = 9.0 Hz, J = 8.0 Hz, 2H, 3-H, 5-H, OPh), 6.92 (m as t, J = 8.0 Hz, 1H, 4-H, OPh), 6.87 (m as d, J = 8.0Hz, 2H, 2-H, 6-H, OPh), 6.43 (d, J =

1.5 Hz, 2H, 2-H, 6-H, ArH), 6.29 (t, J = 1.5 Hz, 1H, 4-H, ArH), 3.90 (t, J = 6.0 Hz, 2H, 8′-

H), 3.79 (s, 6H, OMe), 1.92-1.85 (m, 2 H of the cyclopentyl ring), 1.81-1.58 (m, 8H 6 H of the cyclopentyl ring and 2′H), 1.59-1.51 (m, 2H, 3′-H), 1.41-1.32 (m, 2H, 4′-H), 1.30-1.22

13 (m 2H, 5′-H), 1.20-1.13 (m, 2H, 6′-H), 1.12-0.94 (m, 2H, 7′-H); C NMR (100 MHz CDCl3)

δ 160.3 (ArC), 159.1 (ArC), 151.8 (ArC), 129.4 (ArC), 120.4 (ArC), 114.5 (ArC), 105.6

(ArC), 96.6 (ArC), 67.8 (-CH2OPh), 55.2 (-OCH3), 51.3, 41.9, 37.7, 30.2, 29.2, 26.0, 25.1

23.3. Mass spectrum (ESI) m/z (relative intensity) 397 (M++H). HPLC (4.6 mm × 250 mm,

Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.3 min for the title compound.

5-(8-Bromo-2-methyloct-2-yl)resorcinol (2.6a)65. To a stirred solution of 2.5a (15.9 g,

o 44.8 mmol) in dry CH2Cl2 448 mL), at -78 C, under an argon atmosphere, was added boron tribromide (197.1 mL, 197.1 mmol). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water

73 and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (10%-35% ethyl acetate in hexanes) afforded 12.0 g of 2.6a as a white foam in 85% yield. IR (neat)

3343 (br, OH), 2931, 2834, 1601, 1412, 1345, 1235, 1146, 993, 750 cm-1; 1H NMR (500

MHz, CDCl3)  6.38 (d, J = 2.0 Hz, 2H, 2-H, 6-H, ArH) 6.18 (t, J = 2.0 Hz, 1H, 4-H, ArH),

4.69 (br s, 2H, -OH), 3.35 (t, J = 7.0 Hz, 2H, 7′-H), 1.82-1.75 (m, 2H, 2′-H), 1.55-1.50 (m,

2H, 3′H), 1.40-1.33 (m, 2H, 4′-H), 1.23 (s and m overlapping, 8H, -C(CH3)2-, -CH2- of the side chain, especially 1.23, s, 6H, -C(CH3)2-), 1.10-1.02 (m, 2H, -CH2- of the side chain);

Mass spectrum (ESI) m/z (relative intensity) 317 (M++H+2, 100), 315 (M++H, 100), 235

+ (10), 207 (8). Exact mass (ESI) calculated for C15H24O2Br (M +H), 315.0960; found,

315.0952. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 4.5 min for the title compound.

5-[1-(6-Bromohexyl) cyclopentyl]benzene-1,3-diol (2.6b)83. The synthesis was carried out as described for 2.6a using 2.5b (9.5 g, 24.8 mmol) and boron tribromide (109.2 mL,

109.2 mmol) in CH2Cl2 (248 mL) and give 2.6b (8.0 g, 95% yield) as a white foam. IR

(neat) 3340 (br, OH), 2933, 2859, 1598, 1462, 1345, 1275, 1150, 993, 750 cm-1; 1H NMR

(500 MHz, CDCl3)  6.32 (d, J = 2.0 Hz, 2H, 2-H, 6-H, ArH) 6.17 (t, J = 2.0 Hz, 1H, 4-H,

ArH), 4.87 (br s, 2H, -OH), 3.35 (t, J = 7.0 Hz, 2H, 7′-H), 1.88-1.80 (m, 2H of the cyclopentyl ring), 1.78-1.62 (m, 8H, 6H of the cyclopentyl ring and 2′-H), 1.53-1.48 (m,

2H, 3′H), 1.36-1.29 (m, 2H, 4′-H), 1.18-1.13 (m, 2H, 5′-H), 1.12-0.94 (m, 2H, 6′-H); 13C

NMR (100 MHz CDCl3) δ 156.2 (ArC), 152.4 (ArC), 106.7 (ArC), 99.8 (ArC), 51.0, 41.6,

37.6, 34.0, 32.7, 29.3, 28.0, 25.0, 23.2. Mass spectrum (ESI) m/z (relative intensity) 341

(M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.0 min for the title compound.

5-[1-(7-Bromoheptyl)cyclopentyl]benzene-1,3-diol (2.6c). The synthesis was carried out as described for 2.6a using 2.5c (9.5 g, 24.0 mmol) and boron tribromide (105 mL,

105 mmol) in CH2Cl2 (240 mL) and give 2.6c (8.5g, 98% yield) as a white foam. IR (neat)

-1 1 3340 (br, OH), 2834, 1576, 1412, 1345, 1275, 1113, 750 cm ; H NMR (500 MHz, CDCl3)

 6.33 (d, J = 2.0 Hz, 2H, 2-H, 6-H, ArH) 6.17 (t, J = 2.0 Hz, 1H, 4-H, ArH), 4.82 (br s, 2H,

-OH), 3.37 (t, J = 7.0 Hz, 2H, 8′-H), 1.87-1.58 (m, 10H, 8H of the cyclopentyl ring and 2′-

H), 1.53-1.47 (m, 2H, 3′H), 1.37-1.29 (m, 2H, 4′-H), 1.23-1.11 (m, 4H, 5′-H, 6′-H), 1.12-

0.94 (m, 2H, 7′-H); Mass spectrum (ESI) m/z (relative intensity) 355 (M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.1 min for the title compound.

(1R,4R,5R)-4-[4-(8-Bromo-2-methyloctan-2-yl)-2,6-dihydroxyphenyl]-6,6- dimethylbicyclo [3.1.1] heptan-2-one (2.7a)65. To a degassed solution of 2.6a (11.8 g,

37.4 mmol) and p-toluenesulfonic acid monohydrate (11.4 g, 59.8 mmol) in wet CHCl3

(18 mL) diacetates 10 (15.5 g, ca. 90% pure by 1H NMR, 59.8 mmol) was added at 0C, under an argon atmosphere. The mixture was warmed to room temperature and stirred for 4 days to ensure complete formation of the product. The reaction mixture was diluted with diethyl ether and washed sequentially with water, saturated aqueous NaHCO3, and brine. The organic phase was dried over MgSO4 and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (15%-50% diethyl

75 ether in hexane) and fractions containing almost pure product (TLC) were combined and evaporated. Further purification by recrystallization from CHCl3 and hexane gave 2.7a as a white crystalline solid (10.8 g, 64% yield). m p 83-85C; IR (neat) 3349 (br, OH), 2932,

2861, 1683 (s, >C=O), 1621, 1586, 1420, 1267, 1022, 838, 733 cm-1. 1H NMR (500 MHz,

CDCl3)  6.22 (s, 2H, ArH), 4.93 (br s, 2H, OH), 3.95 (t, J = 8.5 Hz, 1H, 4-H), 3.49 (dd, J

= 18.5 Hz, J = 6.5 Hz, 1H, 3α-H), 3.38 (t, J = 7 Hz, 2H, 7′-H), 2.62 (dd, J = 18.5 Hz, J = 8

Hz, 1H, 3β-H), 2.59 (t, J = 5.0 Hz, 1H, 1-H), 2.52 (m, 1H, 7α-H), 2.46 (d, J = 10.5 Hz, 1H,

7β-H), 2.32, (t, J = 5.5 Hz, 1H, 5-H), 1.85-1.74 (m, 2H, -CH2- of the side chain group),

1.53-1.48 (m, 2H, -CH2- the side chain group), 1.41-1.34 (s and m overlapping, 5H, -CH2- of the side chain, 6-Me, especially 1.36, s, 3H, 6-Me), 1.28-1.18 (s and m overlapping,

8H, -C(CH3)2-, -CH2- of the side chain, especially 1.21, s, 6H, -C(CH3)2-), 1.12-1.04 (m,

13 2H, -CH2- of the side chain), 1.0 (s, 3H, 6-Me). C NMR (100 MHz CDCl3) δ 217.6

(>C=O), 154.8 (ArC), 149.6 (ArC), 113.4 (ArC), 106.4 (ArC), 46.7, 44.1, 42.2, 42.1, 37.9,

37.1, 34.1, 32.7, 29.5, 29.4, 28.7, 28.0, 26.2, 26.1, 24.4, 22.2. Mass spectrum (ESI) m/z

(relative intensity) 451 (M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.2 min for the title compound.

(1R,4R,5R)-4-{4-[1-(6-Bromohexyl)cyclopentyl]-2,6-dihydroxyphenyl}-6,6- dimethylbicyclo[3.1.1]heptan-2-one (2.7b)83. The synthesis was carried out as described for 2.7a using 2.6b (7.8 g, 22.9mmol), p-toluenesulfonic acid monohydrate (6.9 g, 36.7 mmol), and diacetates 10 (9.5 g, ca. 90% pure by 1H NMR, 36.7 mmol), in wet

CHCl3 (229 mL) and give 2.7b (6.7 g, 62% yield) as a white crystalline solid. IR (neat)

3347 (br, OH), 2912, 2856, 1681 (s, >C=O), 1572, 1419, 1201, 1056, 733 cm-1. 1H NMR

(500 MHz, CDCl3)  6.22 (s, 2H, ArH), 5.23 (br s, 2H, OH), 3.95 (t, J = 8.5 Hz, 1H, 4-H),

3.50 (dd, J = 18.5 Hz, J = 6.5 Hz, 1H, 3α-H), 3.36 (t, J = 7 Hz, 2H, 7′-H), 2.62 (dd, J =

18.5 Hz, J = 8 Hz, 1H, 3β-H), 2.59 (t, J = 5.0 Hz, 1H, 1-H), 2.51 (m, 1H, 7α-H), 2.48 (d, J

= 10.5 Hz, 1H, 7β-H), 2.32, (t, J = 5.5 Hz, 1H, 5-H), 1.83-1.73 (m, 4H, 2H of -CH2- of the side chain group and 2H of the cyclopentyl ring ), 1.72-1.58 (m, 6H of the cyclopentyl ring),

1.53-1.46 (m, 2H, -CH2- the side chain group), 1.38-1.30 (s and m overlapping, 5H, -CH2- of the side chain, 6-Me, especially 1.36, s, 3H, 6-Me), 1.19-1.12 (m 2H, -CH2- of the side chain), 1.03-0.96 (s and m overlapping, 5H, 6-Me and -CH2- of the side chain, especially

1.00, s, 3H, 6-Me). Mass spectrum (ESI) m/z (relative intensity) 477 (M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.5 min for the title compound.

(1R,4R,5R)-4-{4-[1-(7-Bromoheptyl)cyclopentyl]-2,6-dihydroxyphenyl}-6,6- dimethylbicyclo[3.1.1]heptan-2-one (2.7c). The synthesis was carried out as described for 2.7a using 2.6c (8.2 g, 23.1 mmol), p-toluenesulfonic acid monohydrate (7.0 g, 37.0

1 mmol), and diacetates 10 (9.8 g, ca. 90% pure by H NMR, 37.0 mmol), in wet CHCl3

(231 mL) and give 2.7c (6.7 g, 59% yield) as a white crystalline solid. IR (neat) 3348 (br,

OH), 2953, 2871, 1654 (s, >C=O), 1552, 1407, 1275, 1056, 733 cm-1. 1H NMR (500 MHz,

CDCl3)  6.22 (s, 2H, ArH), 4.83 (br s, 2H, OH), 3.94 (t, J = 8.5 Hz, 1H, 4-H), 3.49 (dd, J

= 18.5 Hz, J = 6.5 Hz, 1H, 3α-H), 3.38 (t, J = 7 Hz, 2H, 8′-H), 2.62 (dd, J = 18.5 Hz, J = 8

Hz, 1H, 3β-H), 2.59 (t, J = 5.0 Hz, 1H, 1-H), 2.52 (m, 1H, 7α-H), 2.46 (d, J = 10.5 Hz, 1H,

7β-H), 2.30, (t, J = 5.5 Hz, 1H, 5-H), 1.82-1.74 (m, 4H, 2H of -CH2- of the side chain group

77 and 2H of the cyclopentyl ring ), 1.73-1.61 (m, 6H of the cyclopentyl ring), 1.53-1.47 (m,

2H, -CH2- the side chain group), 1.39-1.31 (s and m overlapping, 5H, -CH2- of the side chain, 6-Me, especially 1.36, s, 3H, 6-Me), 1.24-1.12 (m 4H, -CH2- of the side chain),

1.03-0.95 (s and m overlapping, 5H, 6-Me and -CH2- of the side chain, especially 1.00, s,

3H, 6-Me). Mass spectrum (ESI) m/z (relative intensity) 491 (M++H). HPLC (4.6 mm ×

250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.5 min for the title compound.

(6aR,10aR)-3-(8-Bromo-2-methyloctan-2-yl)-1-hydroxy-6,6-dimethyl-

6,6a,7,8,10,10a-hexahydro-9H-benzo[c]chromen-9-one (2.8a)65. To a stirred solution of 2.7a (10.6 g, 23.4 mmol) in anhydrous CH2Cl2/CH3NO2 (3:1 mixture, 780 mL) at 0C, under an argon atmosphere was added trimethylsilyl trifluoromethanesulfonate (23.4 mL,

0.3M solution in CH3NO2, 7.0 mmol). Stirring was continued for 3 hours after the temperature allowed to rise to 25oC. The reaction was quenched with saturated aqueous

NaHCO3/brine (1:1), and diethyl ether was added. The organic phase was separated, the aqueous phase was extracted with diethyl ether, and the combined organic phase was washed with brine and dried over MgSO4. Solvent evaporation and purification by flash column chromatography on silica gel (15%-30% ethyl acetate-hexane) afforded 7.5 g (71% yield) of the title compound 2.8a as white foam. IR (neat) 3298 (br, OH), 2954, 2870, 1696

(s, >C=O), 1575, 1443, 1352, 1134, 1089, 1018, 838, 731 cm-1. 1H NMR (500 MHz,

CDCl3)  6.38 (d, J = 2 Hz, 1H, ArH), 6.25 (d, J = 2 Hz, 1H, ArH), 5.49 (br s, 1H, OH),

3.93 (m as br d, J = 15.5 Hz, 1H, 10eq-H), 3.37 (t, J = 6.5 Hz, 2H, 7′-H), 2.88 (m as td, J

= 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.62-2.58 (m, 1H, 8eq-H), 2.48-2.41 (m, 1H, 8ax-H),

2.2-2.11 (m, 2H, 10ax-H, 7eq-H), 1.97 (m as td, J = 12.0 Hz, J = 1.6 Hz, 1H, 6a-H), 1.80-

1.74 (m, 2H of -CH2- of the side chain), 1.59-1.45 (m and s overlapping, 6H, 7ax-H, 2-

H, 6-Me, especially 1.47, s, 3H, 6-Me), 1.36 (qt, J = 7.5 Hz, 2H, -CH2- of the side chain),

1.28-1.16 (m and s overlapping, 8H, -C(CH3)2-, -CH2- of the side chain, especially, 1.21, s, 6H, -C(CH3)2-), 1.13 (s, 3H, 6-Me), 1.1-1.02(m, 2H, -CH2- of the side chain). Mass spectrum (ESI) m/z (relative intensity) 451 (M++H). HPLC (4.6 mm × 250 mm, Supelco

Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.4 min for the title compound.

(6aR,10aR)-3-[1-(6-Bromohexyl)cyclopentyl]-1-hydroxy-6,6-dimethyl-

6,6a,7,8,10,10a-hexahydro-9H-benzo[c]chromen-9-one (2.8b)83. The synthesis was carried out as described for 2.8a using 2.7b (6.7 g, 14.0 mmol) and trimethylsilyl trifluoromethanesulfonate (14.0 mL, 0.3M solution in CH3NO2, 4.2 mmol), in anhydrous

CH2Cl2/CH3NO2 (3:1 mixture, 37 mL) and give 2.8b (4.7 g, 70% yield) as white foam. IR

(neat) 3299 (br, OH), 2933, 2871, 1696 (s, >C=O), 1621, 1575, 1415, 1355, 1263, 1184,

-1 1 1093, 1038, 838, 731 cm . H NMR (500 MHz, CDCl3)  6.33 (d, J = 2 Hz, 1H, ArH), 6.20

(d, J = 2 Hz, 1H, ArH), 5.63 (br s, 1H, OH), 3.95 (m as br d, J = 14.5 Hz, 1H, 10eq-H),

3.35 (t, J = 6.5 Hz, 2H, 7′-H), 2.88 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.66-

2.57 (m, 1H, 8eq-H), 2.49-2.41 (m, 1H, 8ax-H), 2.21-2.12 (m, 2H, 10ax-H, 7eq-H), 1.98

(m as td, J = 12.0 Hz, J = 1.6 Hz, 1H, 6a-H), 1.87-1.79 (m, 2H, the cyclopentyl ring ), 1.78-

1.59 (m, 8H, 2H of -CH2- the side chain group and 6H of the cyclopentyl ring), 1.53-1.46

(s and m overlapping, 5H, -CH2- of the side chain, 6-Me, especially 1.48, s, 3H, 6-Me -

CH2- the side chain group), 1.35-1.28 (m, 2H, -CH2- of the side chain), 1.19-1.11 (s and

79 m overlapping, 5H, 6-Me and -CH2- of the side chain, especially 1.13, s, 3H, 6-Me), 1.03-

13 0.95 (m, 2H, -CH2- of the side chain). C NMR (100 MHz CDCl3) δ 213.9 (>C=O), 155.1

(ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 149.8 (tertiary aromatic), 108.1 (tertiary aromatic), 107.8 (ArC-2 or ArC-4), 106.5 (ArC-4 or ArC-2), 50.6, 47.4, 45.0, 41.5, 40.8,

37.6, 37.4, 34.8, 34.0, 32.8, 29.3, 28.0, 27.8, 26.9, 24.9, 23.3, 18.9. Mass spectrum (ESI) m/z (relative intensity) 477 (M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.7 min for the title compound.

(6aR,10aR)-3-[1-(7-Bromoheptyl)cyclopentyl]-1-hydroxy-6,6-dimethyl-

6,6a,7,8,10,10a-hexahydro-9H-benzo[c]chromen-9-one (2.8c) The synthesis was carried out as described for 2.8a using 2.7c (6.5 g, 13.2 mmol) and trimethylsilyl trifluoromethanesulfonate (13.2 mL, 0.3M solution in CH3NO2, 3.9 mmol), in anhydrous

CH2Cl2/CH3NO2 (3:1 mixture, 37 mL) and give 2.8c (4.5g, 70% yield) as white foam. IR

(neat) 3294 (br, OH), 2930, 2858, 1695 (s, >C=O), 1620, 1574, 1414, 1345, 1257, 1184,

-1 1 1093, 1038, 839, 731 cm . H NMR (500 MHz, CDCl3)  6.33 (d, J = 1.5 Hz, 1H, ArH),

6.21 (d, J = 1.5 Hz, 1H, ArH), 5.79 (br s, 1H, OH), 3.97 (m as br d, J = 14.5 Hz, 1H, 10eq-

H), 3.37 (t, J = 6.5 Hz, 2H, 8′-H), 2.88 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.65-

2.58 (m, 1H, 8eq-H), 2.49-2.41 (m, 1H, 8ax-H), 2.21-2.13 (m, 2H, 10ax-H, 7eq-H), 1.98

(m as td, J = 12.0 Hz, J = 1.6 Hz, 1H, 6a-H), 1.87-1.75 (m, 4H, 2H of the cyclopentyl ring and 2H of -CH2- the side chain group), 1.74-1.58 (m, 6H, the cyclopentyl ring), 1.51-1.46

(s and m overlapping, 5H, -CH2- of the side chain, 6-Me, especially 1.48, s, 3H, 6-Me -

CH2- the side chain group), 1.35-1.29 (m, 2H, -CH2- of the side chain), 1.23-1.11 (s and

80 m overlapping, 7H, 6-Me and -CH2- of the side chain, especially 1.13, s, 3H, 6-Me), 1.02-

13 0.95 (m, 2H, -CH2- of the side chain). C NMR (100 MHz CDCl3) δ 214.7 (>C=O), 154.9

(ArC-1 or ArC-5), 154.0 (ArC-5 or ArC-1), 149.8 (tertiary aromatic), 107.9 (tertiary aromatic), 107.7 (ArC-2 or ArC-4), 106.5 (ArC-4 or ArC-2), 50.6, 47.4, 45.0, 41.6, 40.8,

37.6, 37.4, 34.8, 34.0, 32.7, 29.9, 28.5, 28.0, 27.8, 26.9, 25.0, 23.3, 18.8. Mass spectrum

(ESI) m/z (relative intensity) 491 (M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.8 min for the title compound.

(6aR,10aR)-3-(8-Bromo-2-methyloctan-2-yl)-1-[(tert-butyldimethylsilyl)oxy]-6,6- dimethyl-6,6a,7,8,10,10a-hexahydro-9H-benzo[c]chromen-9-one (2.9a)65. To a solution of 2.8a (7.4 g, 16.5 mmol) in anhydrous DMF (110 mL) under an argon atmosphere were added sequentially, imidazole (5.6 g, 82.3 mmol), DMAP (1.0 g, 8.2 mmol) and TBDMSCl (12.1 g, 80.6 mmol). The reaction mixture was stirred at room temperature for 12 h and then quenched by the addition of brine and extracted with diethyl ether. The organic phase was dried (MgSO4) and evaporated under reduced pressure.

Purification by flash column chromatography on silica gel (10%-30% diethyl ether in hexane) afforded 7.9 g (85% yield) of 2.9a as a colorless oil. IR (neat) 2931, 2859,

1713(s, >C=O), 1613, 1564, 1412, 1332, 1254, 1137, 1096, 1055, 980, 839 cm-1; 1H NMR

(500 MHz,CDCl3) δ 6.41 (d, J = 1.5 Hz, 1H, 4-H), 6.33 (br d, J = 1.5 Hz, 1H, 2-H), 3.76

(ddd, J = 15.0 Hz, J = 3.0 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.48 (t, J = 6.1 Hz, 2H, 7′-H), 2.71

(m as td, J = 14.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.59-2.52 (m, 1H, 8eq-H), 2.45-2.36 (m,

1H, 8ax-H), 2.2-2.07 (m, 2H, 10ax-H, 7eq-H), 1.95 (m as td, J = 12.5 Hz, J = 3.0 Hz, 1H,

6a-H), 1.72-1.64 (m, 2H of -CH2- of the side chain), 1.54-1.47 (m, 3H, 7ax-H, 2H of -CH2- of the side chain -H), 1.47 (s, 3H, 6-Me), 1.38-1.31 (m, 2H, -CH2- of the side chain), 1.26-

1.17(m and s overlapping, 8H, -C(CH3)2-, -CH2- of the side chain, especially, 1.21, s, 6H,

-C(CH3)2-), 1.1 (s, 3H, 6-Me), 1.09-1.04 (m, 2H, -CH2- of the side chain), 1.0 (s, 9H,

13 Si(Me)2CMe3), 0.24 (s, 3H, Si(Me)2CMe3), 0.16 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz

CDCl3) δ 210.6 (>C=O), 154.2 (ArC-1 or ArC-5), 154.0 (ArC-5 or ArC-1), 149.8 (tertiary aromatic), 111.9 (tertiary aromatic), 109.8 (ArC-2 or ArC-4), 108.3 (ArC-4 or ArC-2), 47.4,

45.5, 45.0, 44.3, 40.7, 37.3, 35.1, 32.5, 29.5, 28.8, 28.6, 27.7, 26.8, 26.7, 25.6, 24.5, 18.6,

18.3, -3.7, -4.1. Mass spectrum (ESI) m/z (relative intensity) 565 (M++H). HPLC (4.6 mm

× 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98% and retention time of 6.7 min for the title compound.

(6aR,10aR)-3-[1-(6-Bromohexyl)cyclopentyl]-1-[(tert-butyldimethylsilyl)oxy]-6,6- dimethyl-6,6a,7,8,10,10a-hexahydro-9H-benzo[c]chromen-9-one (2.9b) The synthesis was carried out as described for 2.9a using 2.8b (4.6 g, 9.7 mmol), imidazole

(3.2 g, 49.0 mmol), DMAP (597.0 mg, 4.8 mmol) and TBDMSCl (7.1 g, 47.9 mmol), in anhydrous DMF (65 mL) and give 2.9b (4.9 g, 85% yield) as a colorless oil. IR (neat)

2929, 2859, 1715 (s, >C=O), 1613, 1565, 1472, 1412, 1355, 1255, 1184, 1096, 1056,

-1 1 838, 780 cm . H NMR (500 MHz,CDCl3) δ 6.37 (d, J = 1.5 Hz, 1H, 4-H), 6.28 (br d, J =

1.5 Hz, 1H, 2-H), 3.77 (ddd, J = 15.0 Hz, J = 3.0 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.48 (t, J =

6.1 Hz, 2H, 7′-H), 2.72 (m as td, J = 14.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.60-2.52 (m, 1H,

8eq-H), 2.46-2.37 (m, 1H, 8ax-H), 2.18-2.06 (m, 2H, 10ax-H, 7eq-H), 1.96 (m as td, J =

12.5 Hz, J = 3.0 Hz, 1H, 6a-H), 1.88-1.78 (m, 2H of the cyclopentyl ring), 1.74-1.59 (m,

8H, 2H of-CH2- of the side chain group and 6H of the cyclopentyl ring) 1.54-1.44 (m, 6H,

7ax-H, 2H of-CH2- of the side chain group, 6-Me, especially, 1.47 3H, 6-Me), 1.36-1.28

(m, 2H, -CH2- of the side chain), 1.19- 1.09 (m and s overlapping, 5H, 2H of -CH2- of the side chain, 6-Me, especially, 1.10, s, 3H, 6-Me), 1.04-0.94 (s and m overlapping, 11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3), 0.24 (s,

13 3H, Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.3 (>C=O),

154.1 (ArC-1 or ArC-5), 154.0 (ArC-5 or ArC-1), 149.1 (tertiary aromatic), 112.0 (tertiary aromatic), 110.8 (ArC-2 or ArC-4), 109.2 (ArC-4 or ArC-2), 50.7, 47.8, 45.6, 45.0, 41.7,

40.8, 37.6, 37.5, 35.1, 32.6, 29.4, 27.7, 26.9, 26.7, 26.0, 25.1, 23.2, 18.6, 18.3, -3.7, -4.1.

Mass spectrum (ESI) m/z (relative intensity) 591 (M++H). HPLC (4.6 mm × 250 mm,

Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 7.0 min for the title compound.

(6aR,10aR)-3-[1-(7-Bromoheptyl)cyclopentyl]-1-[(tert-butyldimethylsilyl)oxy]-6,6- dimethyl-6,6a,7,8,10,10a-hexahydro-9H-benzo[c]chromen-9-one (2.9c) The synthesis was carried out as described for 2.9a using 2.8c (4.2 g, 8.5 mmol), imidazole

(2.9 g, 42.7 mmol), DMAP (520.1 mg, 4.3 mmol) and TBDMSCl (6.2 g, 41.7 mmol), in anhydrous DMF (56 mL) and give 2.9c (4.2g, 82% yield) as a colorless oil. IR (neat) 2910,

2859, 1715 (s, >C=O), 1615, 1563, 1412, 1345, 1215, 1056, 838, 778 cm-1. 1H NMR (500

MHz,CDCl3) δ 6.37 (d, J = 1.5 Hz, 1H, 4-H), 6.28 (br d, J = 1.5 Hz, 1H, 2-H), 3.77 (ddd, J

= 15.0 Hz, J = 3.0 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.48 (t, J = 6.1 Hz, 2H, 8′-H), 2.72 (m as td, J = 14.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.56-2.52 (m, 1H, 8eq-H), 2.45-2.37 (m, 1H, 8ax-

H), 2.18-2.07 (m, 2H, 10ax-H, 7eq-H), 1.96 (m as td, J = 12.5 Hz, J = 3.0 Hz, 1H, 6a-H),

1.88-1.76 (m, 2H of the cyclopentyl ring), 1.74-1.58 (m, 8H, 2H of-CH2- of the side chain group and 6H of the cyclopentyl ring) 1.54-1.42 (m, 6H, 7ax-H, 2H of-CH2- of the side chain group, 6-Me, especially, 1.47 3H, 6-Me), 1.36-1.29 (m, 2H, -CH2- of the side chain),

1.21- 1.08 (m and s overlapping, 7H, 4H of -CH2- of the side chain, 6-Me, especially, 1.11, s, 3H, 6-Me), 1.02-0.97 (s and m overlapping, 11H, 2H of -CH2- of the side chain,

Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3), 0.24 (s, 3H, Si(Me)2CMe3), 0.15 (s,

+ 3H, Si(Me)2CMe3). Mass spectrum (ESI) m/z (relative intensity) 605 (M +H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98% and retention time of 7.1 min for the title compound.

{[(6aR,10aR)-3-(8-Bromo-2-methyloctan-2-yl)-9-(methoxymethylene)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-yl]oxy}(tert- butyl)dimethylsilane (2.10a)65. (Methoxymethyl)triphenyl phosphonium chloride (28.5 g,

83.4 mmol) was suspended in 14 mL of dry THF. Potassium bis(trimethylsilyl)amide (16.3 g, 82.1 mmol) was then added at 0 °C, and the reaction mixture was stirred for 35 min at

0 °C and for 10 min at room temperature. Intermediate 2.9a (7.8 mg, 13.9 mmol) was dissolved in the minimum amount of dry THF and added dropwise to the solution of the orange ylide. The reaction mixture was stirred at 0oC to room temperature for 1.5 h, and then quenched with saturated aqueous NH4Cl solution at 0 °C and diluted with ethyl acetate. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with brine, dried (MgSO4), and concentrated in vacuo. The residue was chromatographed on silica gel (0%-7% diehtyl ether in hexane) to give a mixture (2.10a) of two geometrical isomers 2.10a1 and 2.10a2

(6.0 g, 73% yield) as a colorless oil in the ratio of 2:1 respectivelly as determined by 1H

NMR analysis. IR (neat) 2930, 2857, 1674, 1612, 1571, 1418, 1231, 1095, 1049, 835,

-1 1 743 cm . H NMR (500 MHz,CDCl3) 6.38 (d, J = 2.0 Hz, 1H, Ar-H, 2.10a1), 6.36 (d, J =

2.0 Hz, 1H, Ar-H, 2.10a2), 6.33 (d, J = 2.0 Hz, 1H, Ar-H, 2.10a2), 6.31 (d, J = 2.0 Hz, 1H,

Ar-H, 2.10a1), 5.86 (brs, 1H, =CHOMe, 2.10a1), 5.82 (brs, 1H, =CHOMe, 2.10a2), 4.20-

4.15 (m as dd, J = 13.5 Hz, J = 3.5 Hz, 1H, C-ring, 2.10a2), 3.56 (s, 3H, OMe, 2.10a1),

3.53 (s, 3H, OMe, 2.10a2), 3.48 (t, J = 6.5 Hz, 2H, -CH2Br for 2.10a1 and 2H, -CH2Br for

2.10a2), 3.46-3.42 (m as dd, J = 13.5 Hz, J = 3.5 Hz, 1H, C-ring, 2.10a1), 2.95-2.89 (m as br d, J = 14.0 Hz, 1H, C-ring, 2.10a1), 2.35-2.26 (m, 1H, C-ring of 2.10a1 and 1H, C-ring of 2.10a2), 2.22-2.16 (m, 1H, C-ring, 2.10a2), 2.09-2.00 (m, 1H, C-ring, 2.10a2), 1.91-1.85

(m, 1H, C-ring of 2.10a1 and 1H, C-ring of 2.10a2), 1.82-1.74 (m, 1H, C-ring, 2.10a1), 1.73-

1.53(m, 2H, 6'-H of 2.10a1, 2H, 6'-H of 2.10a2, 2H, C-ring of 2.10a1, and 2H, C-ring of

2.10a2), 1.52-1.48 (m, 2H, 2'-H of 2.10a1, 2H, 2'-H of 2.10a2), 1.39 (s, 3H, 6-Me, 2.10a1),

1.38 (s, 3H, 6-Me, 2.10a2), 1.38-1.30 (m, 2H, -CH2- of the side chain of 2.10a1, 2H, -CH2- of the side chain of 2.10a2), 1.24-1.16 (m, s and s, overlapping, 2H, -CH2- of the side chain of 2.10a1, 2H, -CH2- of the side chain of 2.10a2, 6H, -C(CH3)2- of 2.10a1, 6H, -

C(CH3)2- of 2.10a2), 1.11-0.98 (m, s, s, s and s, overlapping, 1H, C-ring of 2.10a1, 1H, C- ring of 2.10a2, 2H, -CH2- of the side chain of 2.10a1, 2H, -CH2- of the side chain of 2.10a2,

3H, 6-Me of 2.10a1, 3H, 6-Me of 2.10a2, 9H, -Si(Me)2CMe3, of 2.10a1, 9H, -Si(Me)2CMe3, of 2.10a2, especially 1.03, s, -Si(Me)2CMe3, of 2.10a1 and 1.00, s, -Si(Me)2CMe3, of

2.10a2), 0.24 (s, 3H, -Si(Me)2CMe3, 2.10a1), 0.23 (s, 3H, -Si(Me)2CMe3, 2.10a2), 0.19 (s,

3H, -Si(Me)2CMe3, 2.10a2), 0.15 (s, 3H, -Si(Me)2CMe3, 2.10a1). Mass spectrum (ESI) m/z

(relative intensity) 593 (M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column,

85 acetonitrile/water) showed purity of 98% and retention time of 7.4 min for the title compound.

{[(6aR,10aR)-3-[1-(6-Bromohexyl)cyclopentyl]-9-(methoxymethylene)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-yl]oxy}(tert- butyl)dimethylsilane (2.10b) The synthesis was carried out as described for 2.10a using

2.9b (4.5 g, 7.6 mmol), (Methoxymethyl)triphenyl phosphonium chloride (15.6 g, 45.6 mmol) and Potassium bis(trimethylsilyl)amide (8.9 g, 44.8 mmol) in dry THF and give

2.10b (3.3 g, 71% yield) as a colorless oil. IR (neat) 2929, 2858, 1684, 1611, 1561, 1410,

-1 1 1345, 1251, 1181, 1097, 1051, 835, 743 cm . H NMR (500 MHz,CDCl3) 6.33 (d, J = 2.0

Hz, 1H, Ar-H, 2.10b1), 6.31 (d, J = 2.0 Hz, 1H, Ar-H, 2.10b2), 6.29 (d, J = 2.0 Hz, 1H, Ar-

H, 2.10b2), 6.27 (d, J = 2.0 Hz, 1H, Ar-H, 2.10b1), 5.86 (brs, 1H, =CHOMe, 2.10b1), 5.81

(brs, 1H, =CHOMe, 2.10b2), 4.21- 4.16 (m as dd, J = 13.5 Hz, J = 3.5 Hz, 1H, C-ring,

2.10b2), 3.57 (s, 3H, OMe, 2.10b1), 3.53 (s, 3H, OMe, 2.10b2), 3.48 (t, J = 6.5 Hz, 2H, -

CH2Br for 2.10b1 and 2H, -CH2Br for 2.10b2), 3.46-3.41 (m as dd, J = 13.5 Hz, J = 3.5 Hz,

1H, C-ring, 2.10b1), 2.94-2.88 (m as br d, J = 14.0 Hz, 1H, C-ring, 2.10b1), 2.34-2.25 (m,

1H, C-ring of 2.10b1 and 1H, C-ring of 2.10b2), 2.22-2.16 (m, 1H, C-ring, 2.10b2), 2.09-

2.00 (m, 1H, C-ring, 2.10b2), 1.91-1.85 (m, 1H, C-ring of 2.10b1 and 1H, C-ring of 2.10b2),

1.92-1.74 (m, 2H, the cyclopentyl ring, 2.10b1, 2H, the cyclopentyl ring, 2.10b2 and 1H of

C-ring, 2.10b1), 1.73-1.58 (m, 2H, -CH2- of the side chain, 2.10b1, 2H, -CH2- of side chain,

2.10b2, 6H, the cyclopentyl ring, 2.10b1, 6H, the cyclopentyl ring, 2.10b2, 2H, C-ring of

2.10b1, and 2H, C-ring of 2.10b2), 1.52-1.48 (m, 2H, 2'-H of 2.10b1, 2H, 2'-H of 2.10b2),

1.39 (s, 3H, 6-Me, 2.10b1), 1.38 (s, 3H, 6-Me, 2.10b2), 1.34-1.28 (m, 2H, -CH2- of the side

86 chain, 2.10b1 and 2H, -CH2- of the side chain, 2.10b2), 1.24-1.16 (m, 2H, -CH2- of the side chain of 2.10b1 and 2H, -CH2- of the side chain of 2.10b2), 1.11-0.98 (m, s, s, s and s, overlapping, 1H, C-ring of 2.10b1, 1H, C-ring of 2.10b2, 2H, -CH2- of the side chain of

2.10b1, 2H, -CH2- of the side chain of 2.10b2, 3H, 6-Me of 2.10b1, 3H, 6-Me of 2.10b2,

9H, -Si(Me)2CMe3, of 2.10b1, 9H, -Si(Me)2CMe3, of 2.10b2, especially 1.03, s, -

Si(Me)2CMe3, of 2.10b1 and 1.00, s, -Si(Me)2CMe3, of 2.10b2), 0.24 (s, 3H, -Si(Me)2CMe3,

2.10b1), 0.23 (s, 3H, -Si(Me)2CMe3, 2.10b2), 0.18 (s, 3H, -Si(Me)2CMe3, 2.10b2), 0.14 (s,

+ 3H, -Si(Me)2CMe3, 2.10b1). Mass spectrum (ESI) m/z (relative intensity) 619 (M +H).

HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 7.7 min for the title compound.

{[(6aR,10aR)-3-[1-(7-Bromoheptyl)cyclopentyl]-9-(methoxymethylene)-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-yl]oxy}(tert- butyl)dimethylsilane (2.10c) The synthesis was carried out as described for 2.10a using

2.9c (4.0 g, 6.9 mmol), (Methoxymethyl)triphenyl phosphonium chloride (14.2 g, 41.6 mmol) and Potassium bis(trimethylsilyl)amide (8.1 g, 40.7 mmol) in dry THF and give

2.10c (3.0 g, 70% yield) as a colorless oil. IR (neat) 2932, 2858, 1634, 1599, 1410, 1375,

-1 1 1250, 1097, 1052, 835, 743 cm . H NMR (500 MHz,CDCl3) 6.33 (d, J = 2.0 Hz, 1H, Ar-

H, 2.10c1), 6.31 (d, J = 2.0 Hz, 1H, Ar-H, 2.10c2), 6.29 (d, J = 2.0 Hz, 1H, Ar-H, 2.10c2),

6.27 (d, J = 2.0 Hz, 1H, Ar-H, 2.10c1), 5.86 (brs, 1H, =CHOMe, 2.10c1), 5.81 (brs, 1H,

=CHOMe, 2.10c2), 4.21-4.16 (m as dd, J = 13.5 Hz, J = 3.5 Hz, 1H, C-ring, 2.10c2), 3.57

(s, 3H, OMe, 2.10c1), 3.53 (s, 3H, OMe, 2.10c2), 3.48 (t, J = 6.5 Hz, 2H, -CH2Br for 2.10c1 and 2H, -CH2Br for 2.10c2), 3.46-3.41 (m as dd, J = 13.5 Hz, J = 3.5 Hz, 1H, C-ring,

2.10c1), 2.94-2.88 (m as br d, J = 14.0 Hz, 1H, C-ring, 2.10c1), 2.34-2.25 (m, 1H, C-ring of 2.10c1 and 1H, C-ring of 2.10c2), 2.22-2.16 (m, 1H, C-ring, 2.10c2), 2.09-2.00 (m, 1H,

C-ring, 2.10c2), 1.91-1.85 (m, 1H, C-ring of 2.10c1 and 1H, C-ring of 2.10c2), 1.92-1.74

(m, 2H, the cyclopentyl ring, 2.10c1, 2H, the cyclopentyl ring, 2.10c2 and 1H of C-ring,

2.10c1), 1.73-1.58 (m, 2H, -CH2- of the side chain, 2.10c1, 2H, -CH2- of side chain, 2.10c2,

6H, the cyclopentyl ring, 2.10c1, 6H, the cyclopentyl ring, 2.10c2, 2H, C-ring of 2.10c1, and 2H, C-ring of 2.10c2), 1.52-1.48 (m, 2H, 2'-H of 2.10c1, 2H, 2'-H of 2.10c2), 1.39 (s,

3H, 6-Me, 2.10c1), 1.38 (s, 3H, 6-Me, 2.10c2), 1.34-1.28 (m, 2H, -CH2- of the side chain,

2.10c1 and 2H, -CH2- of the side chain, 2.10c2), 1.24-1.16 (m, 4H, -CH2- of the side chain of 2.10c1 and 4H, -CH2- of the side chain of 2.10c2), 1.11-0.98 (m, s, s, s and s, overlapping, 1H, C-ring of 2.10b1, 1H, C-ring of 2.10b2, 2H, -CH2- of the side chain of

2.10c1, 2H, -CH2- of the side chain of 2.10c2, 3H, 6-Me of 2.10c1, 3H, 6-Me of 2.10c2, 9H,

-Si(Me)2CMe3, of 2.10c1, 9H, -Si(Me)2CMe3, of 2.10c2, especially 1.03, s, -Si(Me)2CMe3, of 2.10c1 and 1.00, s, -Si(Me)2CMe3, of 2.10c2), 0.24 (s, 3H, -Si(Me)2CMe3, 2.10c1), 0.23

(s, 3H, -Si(Me)2CMe3, 2.10c2), 0.18 (s, 3H, -Si(Me)2CMe3, 2.10c2), 0.14 (s, 3H, -

+ Si(Me)2CMe3, 2.10c1). Mass spectrum (ESI) m/z (relative intensity) 633 (M +H). HPLC

(4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 7.8 min for the title compound.

(6aR,10aR)-3-(8-Bromo-2-methyloctan-2-yl)-1-[(tert-butyldimethylsilyl)oxy]-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-9-carbaldehyde

65 (2.11a) . To a stirred solution of 2.10a (5.7 g, 9.6 mmol) in CH2Cl2 (320 mL) under an argon atmosphere, was added wet trichloroacetic acid (7.8 g, 48 mmol). The reaction

88 mixture was stirred at room temperature for 50 min and then quenched with saturated aqueous NaHCO3 solution and diluted with diethyl ether. The organic layer was separated and the aqueous layer was extracted with diethyl ether. The combined organic phase was washed with water and brine, dried (MgSO4) and evaporated. The residue consisted of a mixture of epimeric aldehydes 2.12a and 2.11a1 5.3 g, 95% yield) in the ratio of 2:1 respectivelly as determined by 1H NMR analysis, and it was used into the next step as

1 such. H NMR (500 MHz, CDCl3) δ 9.89 (d, J = 1.5 Hz, 1H, 9α-CHO, 2.11a1), 9.63 (d, J

= 1.5 Hz, 1H, 9β-CHO, 2.12a), 6.38 (d, J = 2.0 Hz, 1H, Ar-H, 2.12a), 6.36 (d, J = 2.0 Hz,

1H, Ar-H, 2.11a1), 6.33 (d, J = 2.0 Hz, 1H, Ar-H, 2.11a1), 6.32 (d, J = 2.0 Hz, 1H, Ar-H,

2.12a), 3.69-3.63 (m as br d, J = 14.0 Hz, 1H, C-ring, 2.11a1), 3.52-3.46 (t and m as br d overlapping, t, J = 6.5 Hz, 2H, -CH2Br for 2.12a and 2H, -CH2Br for 2.11a1, m as br d, J =

13.5 Hz, 1H, C-ring, 2.12a), 2.66-2.61 (m, 1H, C-ring, 2.11a1), 2.46-2.33 (m, 2H, C-ring,

2.12a and 2H, C-ring, 2.11a1), 2.31-2.24 (m, 1H, C-ring, 2.11a1), 2.14-2.06 (m, 1H, C-ring,

2.12a), 2.02-1.96 (m, 1H, C-ring, 2.12a), 1.78-1.73 (m, 1H, C-ring, 2.11a1), 1.68 (sextet,

J = 6.7 Hz, 2H, 6'-H of 2.12a and 2H, 6'-H of 2.11a1), 1.58-1.42 (m, 2H, 2'-H of 2.12a, 2H,

2'-H of 2.11b, 2H, C-ring of 2.12a, 2H, C-ring of 2.11a1), 1.42-130 (m, s, and s, overlapping, 2H, -CH2- of the side chain of 2.12a, 2H, -CH2- of the side chain of 2.11a1,

1.39, s, 3H, 6-Me of 2.12a, 1.36, s, 3H, 6-Me of 2.11a1), 1.26-1.10 (m, 2H, -CH2- of the side chain of 2.12a, 2H, -CH2- of the side chain of 2.11a1, 2H, C-ring of 2.12a, 1H, C-ring of 2.11a1, 6H, -C(CH3)2- of 2.11a1, 6H, -C(CH3)2- of 2.12a), 1.09-1.02 (m and s, overlapping, 2H, -CH2- of the side chain of 2.12a, 2H, -CH2- of the side chain of 2.11a1,

1.08, s, 3H, 6-Me, 2.12a), 1.01-1.00 (s and s overlapping, 3H, 6-Me of 2.11a1 and 9H, -

Si(Me)2CMe3 of 2.12a), 0.97 (s, 9H, -Si(Me)2CMe3, 2.11a1), 0.28 (s, 3H, -Si(Me)2CMe3,

2.11a1), 0.26 (s, 3H, Si(Me)2CMe3, 2.12a), 0.25 (s, 3H, Si(Me)2CMe3, 2.11a1), 0.15 (s, 3H,

Si(Me)2CMe3, 2.12a).

(6aR,10aR)-3-[1-(6-Bromohexyl)cyclopentyl]-1-[(tert-butyldimethylsilyl)oxy]-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-9-carbaldehyde (2.11b)

The synthesis was carried out as described for 2.11a using 2.10b (3.0 g, 4.8 mmol) and wet trichloroacetic acid (4.7 g, 29.0 mmol) in CH2Cl2 (369 mL) and give 2.11b1 and 2.12b

1 (2.7 g, 95% yield) as a colorless oil. H NMR (500 MHz, CDCl3) δ 9.89 (d, J = 1.5 Hz, 1H,

9α-CHO, 2.11b1), 9.63 (d, J = 1.5 Hz, 1H, 9β-CHO, 2.12b), 6.34 (d, J = 2.0 Hz, 1H, Ar-H,

2.12b), 6.31 (d, J = 2.0 Hz, 1H, Ar-H, 2.11b1), 6.28 (d, J = 2.0 Hz, 1H, Ar-H, 2.11b1), 6.27

(d, J = 2.0 Hz, 1H, Ar-H, 2.12b), 3.69-3.63 (m as br d, J = 14.0 Hz, 1H, C-ring, 2.11b1),

3.52-3.45 (t and m as br d overlapping, t, J = 6.5 Hz, 2H, -CH2Br for 2.12b and 2H, -CH2Br for 2.11b1, m as br d, J = 13.5 Hz, 1H, C-ring, 2.12b), 2.65-2.60 (m, 1H, C-ring, 2.11b1),

2.46-2.37 (m, 2H, C-ring, 2.12b and 2H, C-ring, 2.11b1), 2.31-2.23 (m, 1H, C-ring, 2.11b1),

2.14-2.06 (m, 1H, C-ring, 2.12b), 2.02-1.95 (m, 1H, C-ring, 2.12b), 1.88-1.78 (m, 2H of the cyclopentyl ring, 2.12b and 2H of the cyclopentyl ring, 2.11b1), 1.74-1.58 (m and sextet overlapping, sextet, J = 6.7 Hz, 2H, 6'-H of 2.12b and 2H, 6'-H of 2.11b1, 6H of the cyclopentyl ring, 2.12b, 6H of the cyclopentyl ring, 2.11b1, 1H, C-ring, 2.11b1), 1.52-1.42

(m, 2H, 2'-H of 2.12b, 2H, 2'-H of 2.11b1, 2H, C-ring of 2.12b and 2H, C-ring of 2.11b1),

1.42-130 (m, s, and s, overlapping, 2H, -CH2- of the side chain of 2.12b, 2H, -CH2- of the side chain of 2.11b1, 1.39, s, 3H, 6-Me of 2.12b, 1.36, s, 3H, 6-Me of 2.11b1), 1.19-1.09

(m, 2H, -CH2- of the side chain of 2.12b, 2H, -CH2- of the side chain of 2.11b1, 2H, C-ring of 2.12b, 1H, C-ring of 2.11b1), 1.09-1.02 (m and s, overlapping, 2H, -CH2- of the side

90 chain of 2.12b, 2H, -CH2- of the side chain of 2.11b1, 1.08, s, 3H, 6-Me, 2.12b), 1.01-1.00

(s and s overlapping, 3H, 6-Me of 2.11b1 and 9H, -Si(Me)2CMe3 of 2.12b), 0.97 (s, 9H, -

Si(Me)2CMe3, 2.11b1), 0.28 (s, 3H, -Si(Me)2CMe3, 2.11b1), 0.26 (s, 3H, Si(Me)2CMe3,

2.12b), 0.25 (s, 3H, Si(Me)2CMe3, 2.11b1), 0.15 (s, 3H, Si(Me)2CMe3, 2.12b).

(6aR,10aR)-3-[1-(7-Bromoheptyl)cyclopentyl]-1-[(tert-butyldimethylsilyl)oxy]-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-9-carbaldehyde (2.11c)

The synthesis was carried out as described for 2.11a using 2.10c (2.5 g, 3.9 mmol) and wet trichloroacetic acid (3.2 g, 19.7 mmol) in CH2Cl2 (300 mL) and give 2.11c1 and 2.12c

1 (2.1 g, 90% yield) as a colorless oil. H NMR (500 MHz, CDCl3) δ 9.89 (d, J = 1.5 Hz, 1H,

9α-CHO, 2.11c1), 9.63 (d, J = 1.5 Hz, 1H, 9β-CHO, 2.12c), 6.34 (d, J = 2.0 Hz, 1H, Ar-H,

2.12c), 6.31 (d, J = 2.0 Hz, 1H, Ar-H, 2.11c1), 6.28 (d, J = 2.0 Hz, 1H, Ar-H, 2.11c1), 6.27

(d, J = 2.0 Hz, 1H, Ar-H, 2.12c), 3.69-3.63 (m as br d, J = 14.0 Hz, 1H, C-ring, 2.11c1),

3.52-3.45 (t and m as br d overlapping, t, J = 6.5 Hz, 2H, -CH2Br for 2.12c and 2H, -CH2Br for 2.11c1, m as br d, J = 13.5 Hz, 1H, C-ring, 2.12c), 2.65-2.60 (m, 1H, C-ring, 2.11c1),

2.46-2.37 (m, 2H, C-ring, 2.12c and 2H, C-ring, 2.11c1), 2.31-2.23 (m, 1H, C-ring, 2.11c1),

2.14-2.06 (m, 1H, C-ring, 2.12c), 2.02-1.95 (m, 1H, C-ring, 2.12c), 1.88-1.78 (m, 2H of the cyclopentyl ring, 2.12c and 2H of the cyclopentyl ring, 2.11c1), 1.74-1.58 (m and sextet overlapping, sextet, J = 6.7 Hz, 2H, 6'-H of 2.12c and 2H, 6'-H of 2.11c1, 6H of the cyclopentyl ring, 2.12c, 6H of the cyclopentyl ring, 2.11c1, 1H, C-ring, 2.11c1), 1.52-1.42

(m, 2H, 2'-H of 2.12c, 2H, 2'-H of 2.11c1, 2H, C-ring of 2.12c and 2H, C-ring of 2.11c1),

1.42-130 (m, s, and s, overlapping, 2H, -CH2- of the side chain of 2.12c, 2H, -CH2- of the side chain of 2.11c1, 1.39, s, 3H, 6-Me of 2.12c, 1.36, s, 3H, 6-Me of 2.11c1), 1.24-1.10

(m, 4H, -CH2- of the side chain of 2.12c, 4H, -CH2- of the side chain of 2.11c1, 2H, C-ring of 2.12c, 1H, C-ring of 2.11c1), 1.09-1.02 (m and s, overlapping, 2H, -CH2- of the side chain of 2.12c, 2H, -CH2- of the side chain of 2.11c1, 1.08, s, 3H, 6-Me, 2.12c), 1.01-1.00

(s and s overlapping, 3H, 6-Me of 2.11c1 and 9H, -Si(Me)2CMe3 of 2.12c), 0.97 (s, 9H, -

Si(Me)2CMe3, 2.11c1), 0.28 (s, 3H, -Si(Me)2CMe3, 2.11c1), 0.26 (s, 3H, Si(Me)2CMe3,

2.12c), 0.25 (s, 3H, Si(Me)2CMe3, 2.11c1), 0.15 (s, 3H, Si(Me)2CMe3, 2.12c).

(6aR,9R,10aR)-3-(8-Bromo-2-methyloctan-2-yl)-1-[(tert-butyldimethylsilyl)oxy]-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-9-carbaldehyde

(2.12a)65. To a solution of 2.11a (5.1 g, 8.8 mmol) in ethanol (176.5 mL) under an argon atmosphere, was added potassium carbonate powder (6.1 g, 44.1 mmol) and the mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with diethyl ether and solid materials were filtered off. The filtrate was washed with brine, dried

(MgSO4), and concentrated under reduced presure. Purification by flash column chromatography on silica gel (5%-25% diethyl ether in hexane) gave 2.12a (4.3 g, 84% yield) as a colorless oil. IR (neat) 2931, 2859, 2713 (w, CHO), 1727 (s, >C=O), 1613,

-1 1 1564, 1412, 1331, 1254, 1064, 841, 780 cm . H NMR (500 MHz, CDCl3) δ 9.63 (d, J =

1.5 Hz, 1H, 9β-CHO), 6.38 (d, J = 2.0 Hz, 1H, Ar-H), 6.32 (d, J = 2.0 Hz, 1H, Ar-H), 3.52-

3.46 (t and m as br d overlapping, t, J = 6.5 Hz, 2H, -CH2Br, m as br d, J = 13.5 Hz, 1H,

C-ring), 2.46-2.33 (m, 2H, C-ring), 2.14-2.06 (m, 1H, C-ring), 2.02-1.96 (m, 1H, C-ring),

1.69 (sextet, J = 6.7 Hz, 2H, 6'-H), 1.52-1.42 (m, 4H, 2'-H, C-ring), 1.42-130 (m and s, overlapping, 5H, -CH2- of the side chain and 1.39, s, 6-Me), 1.26-1.10 (m, 10H, -CH2- of the side chain, C-ring and -C(CH3)2-), 1.09-1.00 (m, s and s, overlapping, 14H as follows:

2H, -CH2- of the side chain, 1.08, s, 3H, 6-Me, 1.01, s, 9H, -Si(Me)2CMe3), 0.26 (s, 3H,

13 Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 203.5 (-CHO),

154.5 (ArC-1 or ArC-5), 154.2 (ArC-5 or ArC-1), 149.3 (tertiary aromatic), 111.4 (tertiary aromatic), 109.5 (ArC-2 or ArC-4), 108.4 (ArC-4 or ArC-2), 50.5, 49.0, 45.6, 45.0, 44.4,

37.3, 37.2, 35.4, 32.6, 30.2, 29.5, 28.8, 28.6, 27.6, 26.9, 26.8, 25.9, 24.5, 18.8, 18.2, -3.6,

-4.2. Mass spectrum (ESI) m/z (relative intensity) 579 (M++H). HPLC (4.6 mm × 250 mm,

Supelco Discovery column, acetonitrile/water) showed purity of 96.5% and retention time of 7.0 min for the title compound.

(6aR,9R,10aR)-3-[1-(6-Bromohexyl)cyclopentyl]-1-[(tert-butyldimethylsilyl)oxy]-

6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-9-carbaldehyde

(2.12b) The synthesis was carried out as described for 2.12a using 2.11b (2.4 g, 3.9 mmol) and potassium carbonate powder (2.7 g, 19.8 mmol) in ethanol (390 mL) and give 2.12b

(1.9 g, 81% yield) as a colorless oil. IR (neat) 2930, 2858, 2712 (w, CHO), 1727 (s, >C=O),

-1 1 1589, 1564, 1410, 1321, 1221, 1063, 841, 780 cm . H NMR (500 MHz, CDCl3) δ 9.63

(br s, 1H, 9β-CHO), 6.34 (d, J = 1.5 Hz, 1H, Ar-H), 6.27 (d, J = 1.5 Hz, 1H, Ar-H), 3.52-

3.45 (t and m as br d overlapping, t, J = 4.0 Hz, 2H, -CH2Br, m as br d, J = 13.5 Hz, 1H,

C-ring), 2.46-2.37 (m, 2H, C-ring), 2.13-2.07 (m, 1H, C-ring), 2.02-1.96 (m, 1H, C-ring),

1.88-1.78 (m, 2H of the cyclopentyl ring), 1.74-1.58 (m, 8H, 2H of-CH2- of the side chain group and 6H of the cyclopentyl ring) 1.54-1.45 (m, 4H, 2H of-CH2- of the side chain group,

2H of C-ring), 1.39 (s, 3H, 6-Me), 1.34-1.28 (m, 2H, -CH2- of the side chain), 1.24-1.19

(m, 2H, C-ring), 1.18- 1.12 (m 2H, -CH2- of the side chain), 1.07 (s, 3H, 6-Me), 1.03-0.95

(s and m overlapping, 11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.00,

93 s, 9H, Si(Me)2CMe3), 0.25 (s, 3H, Si(Me)2CMe3), 0.14 (s, 3H, Si(Me)2CMe3). Mass spectrum (ESI) m/z (relative intensity) 605 (M++H). HPLC (4.6 mm × 250 mm, Supelco

Discovery column, acetonitrile/water) showed purity of 96.5% and retention time of 7.3 min for the title compound.

(6aR,9R,10aR)-3-[1-(7-Bromoheptyl)cyclopentyl]-1-[(tert-butyldimethylsilyl)oxy]-

6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-9-carbaldehyde

(2.12c) The synthesis was carried out as described for 2.12a using 2.11c (1.9 g, 3.1 mmol) and potassium carbonate powder (2.1 g, 15.3 mmol) in ethanol (310 mL) and give 2.12c

(1.5 g, 80% yield) as a colorless oil. IR (neat) 2933, 2843, 2710 (w, CHO), 1727 (s, >C=O),

-1 1 1613, 1562, 1412, 1325, 1141, 1067, 837, 780 cm . H NMR (500 MHz, CDCl3) δ 9.63

(d, J = 1.5 Hz, 1H, 9β-CHO), 6.34 (d, J = 2.0 Hz, 1H, Ar-H), 6.27 (d, J = 2.0 Hz, 1H, Ar-

H), 3.52-3.45 (t and m as br d overlapping, t, J = 6.5 Hz, 2H, -CH2Br, m as br d, J = 13.5

Hz, 1H, C-ring), 2.44-2.37 (m, 2H, C-ring), 2.13-2.07 (m, 1H, C-ring), 2.02-1.96 (m, 1H,

C-ring), 1.88-1.78 (m, 2H of the cyclopentyl ring), 1.74-1.57 (m, 8H, 2H of-CH2- of the side chain group and 6H of the cyclopentyl ring) 1.52-1.44 (m, 4H, 2H of-CH2- of the side chain group, 2H of C-ring), 1.39 (s, 3H, 6-Me), 1.34-1.28 (m, 2H, -CH2- of the side chain),

1.24-1.10 (m, 6H, 4H of -CH2- of the side chain, 2H of C-ring), 1.07 (s, 3H, 6-Me), 1.03-

0.94 (s and m overlapping, 11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially,

13 1.00, s, 9H, Si(Me)2CMe3), 0.25 (s, 3H, Si(Me)2CMe3), 0.14 (s, 3H, Si(Me)2CMe3). C

NMR (100 MHz CDCl3) δ 203.6 (-CHO), 154.3 (ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1),

148.8 (tertiary aromatic), 112.5 (tertiary aromatic), 110.6 (ArC-2 or ArC-4), 109.3 (ArC-4 or ArC-2), 50.6, 49.0, 45.1, 41.8, 37.6, 37.5, 35.5, 32.6, 30.2 30.0, 28.7, 27.6, 26.9, 26.8,

26.7, 25.9, 25.8, 25.1, 23.3, 18.7, 18.2, -3.6, -4.1. Mass spectrum (ESI) m/z (relative intensity) 619 (M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 7.4 min for the title compound.

{(6aR,9R,10aR)-3-(8-Bromo-2-methyloctan-2-yl)-1-[(tert-butyldimethylsilyl)oxy]-

6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-9-yl}methanol

(2.13a)65. Sodium borohydride (2.1 g, 56.1 mmol) was added to a stirred solution of aldehyde 2.12a (4.1 g, 7.0 mmol) in ethanol (175 mL) at 0 oC under argon. After 30 min, the reaction was quenched with saturated ammonium chloride solution and volatiles were removed in vacuo. The residue was dissolved in ethyl acetate and water was adeed. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic phase was washed with water and brine, dried (MgSO4), and evaporated. Purification by flash column chromatography on silica gel (15%-50% diethyl ether in hexane) gave 2.13a (4.0 g, 98% yield) as a colorless viscous oil. IR (neat) 3354

(br, OH), 2930, 2858, 1612, 1562, 1464, 1411, 1253, 1140, 1064, 1047, 835,779 cm-1; 1H

NMR (500 MHz, CDCl3) δ 6.37 (d, J = 2.0 Hz, 1H, Ar-H), 6.30 (d, J = 2.0 Hz, 1H, Ar-H),

3.54 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.50-3.43 (dd and t overlapping, especially, 3.48, t, J = 6.5, 7'-H, dd, J = 10.0 Hz, J = 6.5 Hz, half of an AB system, 1H, -CH2OH), 3.18-3.16 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.40-2.32 (m as td,

J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.04-1.97 (m, 1H, C-ring), 1.94-1.88 (m, 1H, C-ring),

1.8-1.64 (m, 3H, 1H of C-ring, 2H, 6'-H ), 1.52-1.44 (m, 3H, 2'-H, C-ring), 1.4-1.3 (m and s overlapping, 5H, -CH2- of the side chain, 6-Me, especially 1.25, s, 3H, 6-Me), 1.24-1.1

(m, s, and s overlapping, 10H, -C(CH3)2-, -CH2- of the side chain, C-ring, especially, 1.20, s, 3H, -C(CH3)2-, and 1.19, s, 3H, -C(CH3)2-), 1.09-1.02 (s and m overlapping, 5H, 6-Me,

-CH2- of the side chain, especially, 1.06, s, 3H, 6-Me), 1.0 (s, 9H, Si(Me)2CMe3), 0.82-0.7

13 (m, 1H, C-ring), 0.23 (s, 3H, Si(Me)2CMe3), 0.12 (s, 3H, Si(Me)2CMe3). C NMR (100

MHz CDCl3) δ 154.5 (ArC-1 or ArC-5), 154.3 (ArC-5 or ArC-1), 149.0 (tertiary aromatic),

113.5 (tertiary aromatic), 109.7 (ArC-2 or ArC-4), 108.4 (ArC-4 or ArC-2), 68.5 (-CH2OH),

49.6, 45.1, 44.4, 41.8, 40.5, 37.2, 35.5, 33.2, 32.6, 29.8, 29.5, 28.8, 28.6, 27.6, 27.5, 26.8,

25.9, 24.5, 18,8, 18.2, -3.6, -4.3. Mass spectrum (ESI) m/z (relative intensity) 581 (M++H,

100). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.7 min for the title compound.

{(6aR,9R,10aR)-3-[1-(6-Bromohexyl)cyclopentyl]-1-[(tert-butyldimethylsilyl)oxy]-

6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-9-yl}methanol (2.13b)

The synthesis was carried out as described for 2.13a using 2.12b (1.5 g, 2.4 mmol) and

Sodium borohydride (732 mg, 19.8 mmol) in ethanol (60 mL) and give 2.13b (1.4 g, 98% yield) as a colorless viscous oil. IR (neat) 3353 (br, OH), 2930, 2858, 1612, 1562, 1463,

-1 1 1410, 1342, 1252, 1065, 1046, 835,779 cm ; H NMR (500 MHz, CDCl3) δ 6.33 (d, J =

1.5 Hz, 1H, Ar-H), 6.25 (d, J = 1.5 Hz, 1H, Ar-H), 3.54 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.50-3.43 (dd and t overlapping, especially, 3.48, t, J =

6.5, 7'-H, dd, J = 10.0 Hz, J = 6.5 Hz, half of an AB system, 1H, -CH2OH), 3.18-3.13 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.39-2.32 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring),

2.04-1.97 (m, 1H, C-ring), 1.94-1.88 (m, 1H, C-ring), 1.87-1.78 (m, 2H, the cyclopentyl ring), 1.76-1.58 (m, 9H, 6H of the cyclopentyl ring, 2H of t-CH2- of the side chain group,

1H of C-ring),1.52-1.44 (m, 3H, 2'-H, C-ring), 1.38 (s, 3H, 6Me), 1.34-1.24 (m 3H, 2H of -

CH2- of the side chain, 1H of C-ring), 1.24-1.10 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.06 (s, 3H, 6-Me), 1.04-0.94(s and m overlapping, 11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3), 0.82-0.73 (m, 1H, C-ring),

13 0.23 (s, 3H, Si(Me)2CMe3), 0.12 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 154.3

(ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 148.3 (tertiary aromatic), 113.5 (tertiary aromatic), 110.7 (ArC-2 or ArC-4), 109.3 (ArC-4 or ArC-2), 68.5 (-CH2OH), 50.6, 49.6,

45.1, 41.8, 40.5, 37.6, 37.5, 35.5, 33.2, 32.6, 29.8, 29.4, 27.6, 27.5, 26.7, 25.9, 25.1, 23.2,

18,8, 18.2, -3.6, -4.3. Mass spectrum (ESI) m/z (relative intensity) 607 (M++H, 100). HPLC

(4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 97.5% and retention time of 7.0 min for the title compound.

{(6aR,9R,10aR)-3-[1-(7-Bromoheptyl)cyclopentyl]-1-[(tert-butyldimethylsilyl)oxy]-

6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-9-yl}methanol (2.13c)

The synthesis was carried out as described for 2.13a using 2.12c (1.3 g, 2.1 mmol) and

Sodium borohydride (621 mg, 16.8 mmol) in ethanol (52.5 mL) and give 2.13c (1.2 g, 97% yield) as a colorless viscous oil. IR (neat) 3353 (br, OH), 2930, 2858, 1612, 1562, 1463,

-1 1 1410, 1341, 1253, 1063, 1047, 835,779 cm ; H NMR (500 MHz, CDCl3) δ 6.33 (d, J =

1.5 Hz, 1H, Ar-H), 6.25 (d, J = 1.5 Hz, 1H, Ar-H), 3.54 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.50-3.43 (dd and t overlapping, especially, 3.48, t, J =

6.5, 7'-H, dd, J = 10.0 Hz, J = 6.5 Hz, half of an AB system, 1H, -CH2OH), 3.18-3.13 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.39-2.32 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring),

2.04-1.98 (m, 1H, C-ring), 1.94-1.88 (m, 1H, C-ring), 1.87-1.78 (m, 2H, the cyclopentyl

97 ring), 1.76-1.58 (m, 9H, 6H of the cyclopentyl ring, 2H of -CH2- of the side chain group,

1H of C-ring), 1.52-1.44 (m, 3H, 2'-H, C-ring), 1.38 (s, 3H, 6Me), 1.34-1.24 (m 3H, 2H of

-CH2- of the side chain, 1H of C-ring), 1.24-1.10 (m, 5H, 4H of -CH2- of the side chain, 1H of C-ring), 1.06 (s, 3H, 6-Me), 1.04-0.94(s and m overlapping, 11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3), 0.82-0.73 (m, 1H, C-ring),

13 0.23 (s, 3H, Si(Me)2CMe3), 0.12 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 154.3

(ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 148.4 (tertiary aromatic), 113.5 (tertiary aromatic), 110.8 (ArC-2 or ArC-4), 109.3 (ArC-4 or ArC-2), 68.5 (-CH2OH), 50.6, 49.6,

45.1, 41.8, 40.5, 37.5, 35.5, 33.2, 32.6, 30.0, 29.8, 28.7, 27.6, 27.5, 26.8, 25.9, 25.1, 23.3,

18.8, 18.2, -3.6, -4.3. Mass spectrum (ESI) m/z (relative intensity) 621 (M++H, 100). HPLC

(4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 96.5% and retention time of 7.1 min for the title compound.

(6aR,9R,10aR)-3-(8-Bromo-2-methyloctan-2-yl)-9-(hydroxymethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.14a)65. To a solution of 2.13a

(420 mg, 0.72 mmol) in anhydrous THF (18 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (1.4 mL, 1.4 mmol, 1M solution in anhydrous

THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (20%-50% ethyl acetate in hexane) gave 2.14a (328 mg, 96% yield) as a white solid. mp = 68-70 oC. IR

(neat) 3320 (br, OH), 2932, 2863, 2368 (w, CN), 1622, 1573, 1460, 1414, 1331, 1274,

-1 1 1138, 1038, 750 cm ; H NMR (500 MHz, CDCl3) δ 6.35 (d, J = 1.0 Hz, 1H, Ar-H), 6.18

(d, J = 1.5 Hz, 1H, Ar-H), 5.18 (br s, 1H, ArOH), 3.61-3.42 (m and t overlapping, 4H, -

CH2OH, 7'-H, especially, 3.49, t, J = 6.5 Hz, 2H, -CH2OH), 3.23-3.16 (m as br d, J = 13.0

Hz, 1H, C-ring), 2.52-2.44 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, C-ring), 2.02-1.91 (m,

2H, C-ring), 1.82-1.74 (m, 1H, C-ring), 1.72-1.64 (m, 2H, -CH2- of the side chain group),

1.54-1.46 (m, 3H, 2'-H, 1H of C-ring), 1.44-1.31 (s and m overlapping, 5H, 6-Me, -CH2- of the side chain, especially, 1.39, s, 3H, 6-Me), 1.29-1.15 (s, and m overlapping, 10H, -

C(CH3)2-, -CH2- of the side chain, C-ring, especially, 1.20, s, 6H, -C(CH3)2-), 1.10-1.01 (s and m overlapping, 5H, 6-Me, -CH2- of the side chain, especially, 1.09, s, 3H, 6-Me), 0.89-

13 0.78 ((m as q, J = 11.5Hz, 1H, C-ring). C NMR (100 MHz CDCl3) δ 154.7 (ArC-1 or ArC-

5), 154.4 (ArC-5 or ArC-1), 149.7 (tertiary aromatic), 109.6 (tertiary aromatic), 107.9 (ArC-

2 or ArC-4), 105.4 (ArC-4 or ArC-2), 68.5 (-CH2OH), 49.3, 45.2, 44.2, 40.5, 37.2, 34.9,

33.1, 32.6, 29.7, 29.5, 28.7, 28.6, 27.7 ,27.4, 26.7, 24.4, 19.0. Mass spectrum (ESI) m/z

(relative intensity) 467 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.4 min for the title compound.

(6aR,9R,10aR)-3-[1-(6-Bromohexyl)cyclopentyl]-9-(hydroxymethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.14b) The synthesis was carried out as described for 2.14a using 2.13b (500 mg, 0.8 mmol) and tetra-n- butylammonium fluoride (1.6 mL, 1.6 mmol, 1M solution in anhydrous THF) in THF (28 mL) and give 2.14b (384 mg, 95% yield) as white solid. IR (neat) 3363 (br, OH), 2929,

2854, 2217 (w, CN), 1621, 1573, 1455, 1413, 1328, 1273, 1136, 1040, 750 cm-1; 1H NMR

(500 MHz, CDCl3) δ 6.29 (d, J = 1.5 Hz, 1H, Ar-H), 6.14 (d, J = 1.5 Hz, 1H, Ar-H), 5.12

(br s, 1H, ArOH), 3.54-3.46 (m and t overlapping, 4H, -CH2OH, 7'-H, especially, 3.49, t, J

= 6.5 Hz, 2H, -CH2OH), 3.24-3.16 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.52-2.44 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, C-ring), 2.00-1.90 (m, 2H, C-ring), 1.87-1.78 (m, 2H, the cyclopentyl ring), 1.72-1.58 (m, 9H, 6H of the cyclopentyl ring, 2H of -CH2- of the side chain group, 1H of C-ring), 1.52-1.44 (m, 3H, 2H of -CH2- of the side chain group, 1H of

C-ring), 1.38 (s, 3H, 6Me), 1.34-1.24 (m 3H, 2H of -CH2- of the side chain, 1H of C-ring),

1.24-1.10 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.08 (s, 3H, 6-Me), 1.03-

13 0.94(m, 2H of -CH2- of the side chain), 0.88-0.78 (m, 1H, C-ring). C NMR (100 MHz

CDCl3) δ 154.4 (ArC-1 or ArC-5), 154.2 (ArC-5 or ArC-1), 149.1 (tertiary aromatic), 109.2

(tertiary aromatic), 108.8 (ArC-2 or ArC-4), 106.3 (ArC-4 or ArC-2), 68.5 (-CH2OH), 50.5,

49.5, 45.1, 41.7, 40.5, 37.6, 37.5, 35.3, 33.2, 32.6, 29.7, 29.4, 27.6, 27.5, 26.8, 25.1, 23.2,

19.0. Mass spectrum (ESI) m/z (relative intensity) 493 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.6 min for the title compound.

(6aR,9R,10aR)-3-[1-(7-Bromoheptyl)cyclopentyl]-9-(hydroxymethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.14c) The synthesis was carried out as described for 2.14a using 2.13c (520 mg, 0.84 mmol) and tetra-n- butylammonium fluoride (1.68 mL, 1.68 mmol, 1M solution in anhydrous THF) in THF (28 ml) and give 2.14c (416 mg, 98% yield) as white solid. IR (neat) 3365 (br, OH), 2927,

2857, 2256 (w, CN), 1621, 1573, 1455, 1414, 1343, 1274, 1138, 1036, 750 cm-1; 1H NMR

(500 MHz, CDCl3) δ 6.29 (d, J = 1.5 Hz, 1H, Ar-H), 6.14 (d, J = 1.5 Hz, 1H, Ar-H), 5.12

(br s, 1H, ArOH), 3.54-3.46 (m and t overlapping, 4H, -CH2OH, 7'-H, especially, 3.49, t, J

100

= 6.5 Hz, 2H, -CH2OH), 3.24-3.16 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.52-2. .44 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, C-ring), 2.00-1.90 (m, 2H, C-ring), 1.87-1.78 (m, 2H, the cyclopentyl ring), 1.72-1.58 (m, 9H, 6H of the cyclopentyl ring, 2H of -CH2- of the side chain group, 1H of C-ring), 1.52-1.44 (m, 3H, 2H of -CH2- of the side chain group, 1H of

C-ring), 1.38 (s, 3H, 6Me), 1.34-1.24 (m 3H, 2H of -CH2- of the side chain, 1H of C-ring),

1.24-1.10 (m, 5H, 4H of -CH2- of the side chain, 1H of C-ring), 1.08 (s, 3H, 6-Me), 1.03-

13 0.94(m, 2H of -CH2- of the side chain), 0.88-0.78 (m, 1H, C-ring). C NMR (100 MHz

CDCl3) δ 154.5 (ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 149.2 (tertiary aromatic), 109.5

(tertiary aromatic), 108.9 (ArC-2 or ArC-4), 106.4 (ArC-4 or ArC-2), 68.5 (-CH2OH), 50.6,

49.4, 45.1, 41.6, 40.5, 37.6, 37.4, 34.9, 33.2, 32.6, 29.9, 29.7, 28.7, 27.7, 27.5, 26.8, 25.0,

23.3, 19.0. Mass spectrum (ESI) m/z (relative intensity) 507 (M++H, 100). HPLC (4.6 mm

× 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.7 min for the title compound.

(6aR,9R,10aR)-3-(8-Azido-2-methyloctan-2-yl)-9-(hydroxymethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.15a)65. To a stirred solution of

2.14a (160 mg, 0.34 mmol) in anhydrous CH3Cl/CH3NO2 (1:1 mixture, 6 mL) at room temperature, under an argon atmosphere was added N, N, N', N'-tetramethylguanidinium azide (1.6 g, 10.2 mmol) and stirring was continued for 1 day. On completion, the reaction was quenched with water and diluted with CH2Cl2. The organic phase was washed with brine, dried over MgSO4 and concentrated in vacuo. Purification by flash column chromatography on silica gel (50-80% diethyl ether in hexanes) gave 124 mg of 2.15a as a white solid in 84% yield. mp = 59-61 oC; IR (neat) 3343 (br, OH), 2931, 2860, 2093 (s,

101

-1 1 N3), 1713, 1621, 1537, 1413, 1331, 1268, 1138, 1011, 967, 839 cm ; H NMR (500 MHz,

CDCl3) δ 6.35 (d, J = 1.0 Hz, 1H, Ar-H), 6.19 (d, J = 1.5 Hz, 1H, Ar-H), 4.81 (br s, 1H,

ArOH), 3.57-3.47 (m, 2H, -CH2OH), 3.23-3.17 (m and t overlapping 3H, C-ring, 7'-H, especially, 3.21, t, J = 6.5 Hz, 2H, 7'-H), 2.52-2.44 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H,

C-ring), 2.02-1.91 (m, 2H, C-ring), 1.82-1.74 (m, 1H, C-ring), 1.56-1.46 (m 5H, 6'-H, 2'-H,

C-ring), 1.38 (s, 3H, 6-Me), 1.35-1.26 (m, 2H, -CH2- of the side chain), 1.25-1.11 (s and m overlapping, 10H, -C(CH3)2-, -CH2- of the side chain, C-ring, especially, 1.20, s, 6H, -

C(CH3)2-), 1.10-1.02 (s and m overlapping, 5H, 6-Me, -CH2- of the side chain, especially,

13 1.09, s, 3H, 6-Me), 0.87-0.78 ((m as q, J = 12 Hz,, 1H, C-ring). C NMR (100 MHz CDCl3)

δ 154.7 (ArC-1 or ArC-5), 154.4 (ArC-5 or ArC-1), 149.7 (tertiary aromatic), 109.6 (tertiary aromatic), 107.9 (ArC-2 or ArC-4), 105.3 (ArC-4 or ArC-2), 68.5 (-CH2OH), 51.5, 49.3,

44.2, 40.5, 37.2, 34.9, 33.1, 29.7, 29.6, 28.8, 28.7, 27.7, 27.4, 26.5, 24.4, 19.0. Mass spectrum (ESI) m/z (relative intensity) 430 (M++H, 100). Exact mass (ESI) calculated for

+ C25H40N3O3 (M +H), 430.3070; found, 430.3065. HPLC (4.6 mm × 250 mm, Supelco

Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.2 min for the title compound.

(6aR,9R,10aR)-3-[1-(6-Azidohexyl)cyclopentyl]-9-(hydroxymethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.15b). The synthesis was carried out as described for 2.15a using 2.14b (180 mg, 0.36 mmol) and N, N, N', N' - tetramethylguanidinium azide (1.7 g, 10.8 mmol) in anhydrous CH3Cl/CH3NO2 (1:1 mixture, 6 mL) and give 2.15b (141 mg, 86% yield) as a colorless oil. IR (neat) 3343 (br,

OH), 2934, 2850, 2094 (s, N3), 1723, 1601, 1571, 1413, 1341, 1275, 1145, 1035, 976,

102

-1 1 789 cm ; H NMR (500 MHz, CDCl3) δ 6.30 (d, J = 1.0 Hz, 1H, Ar-H), 6.14 (d, J = 1.5 Hz,

1H, Ar-H), 4.78 (br s, 1H, ArOH), 3.56-3.46 (m, 2H, -CH2OH), 3.30-3.18 (m and t overlapping 3H, 1H of C-ring, 7'-H, especially, 3.21, t, J = 6.5 Hz, 2H, 7'-H), 2.52-2.44 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, C-ring), 1.98-1.88 (m, 2H, C-ring), 1.87-1.78 (m, 3H,

2H of the cyclopentyl ring, 1H of C-ring), 1.72-1.58 (m, 6H, cyclopentyl ring), 1.56-1.44

(m 5H, 6'-H, 2'-H, C-ring), 1.39 (s, 3H, 6Me), 1.30-1.11 (m, 6H, 4H of -CH2- of the side chain, 2H of C-ring), 1.09 (s, 3H, 6-Me), 1.04-0.93 (m, 2H of -CH2- of the side chain),

13 0.88-0.78 (m, 1H, C-ring). C NMR (100 MHz CDCl3) δ 154.5 (ArC-1 or ArC-5), 154.2

(ArC-5 or ArC-1), 148.9 (tertiary aromatic), 109.6 (tertiary aromatic), 108.8 (ArC-2 or ArC-

4), 106.3 (ArC-4 or ArC-2), 68.5 (-CH2OH), 51.4, 50.5, 49.3, 41.6, 40.6, 37.6, 37.4, 34.9,

33.2, 29.7, 29.6, 28.7, 27.7, 27.5, 26.5, 24.9, 23.3, 19.0. Mass spectum (ESI) m/z (relative

+ + intensity) 456 (M + H, 100). Exact mass (ESI) calculated for C27H42N3O3 (M + H),

456.3226; found 456.3226. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.5 min for the title compound.

(6aR,9R,10aR)-3-[1-(7-Azidoheptyl)cyclopentyl]-9-(hydroxymethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.15c). The synthesis was carried out as described for 2.15a using 2.14c (190 mg, 0.37 mmol) and N, N, N', N' - tetramethylguanidinium azide (1.7 g, 11.2 mmol) in anhydrous CH3Cl/CH3NO2 (1:1 mixture, 6 mL) and give 2.15c (152 mg, 88% yield) as a colorless oil. IR (neat) 3328 (br,

- OH), 2930, 2861, 2096 (s, N3), 1621, 1537, 1456, 1414, 1345, 1270, 1085, 1035, 835 cm

1 1 ; H NMR (500 MHz, CDCl3) δ 6.28 (d, J = 1.0 Hz, 1H, Ar-H), 6.19 (d, J = 1.5 Hz, 1H, Ar-

103

H), 4.45 (br s, 1H, ArOH), 3.56-3.46 (m, 2H, -CH2OH), 3.30-3.18 (m and t overlapping 3H,

C-ring, 8'-H, especially, 3.21, t, J = 6.5 Hz, 2H, 8'-H), 2.52-2.44 (m as td, J = 11.0 Hz, J =

2.5 Hz, 1H, C-ring), 1.98-1.88 (m, 2H, C-ring), 1.87-1.78 (m, 3H, 2H of the cyclopentyl ring, 1H of C-ring), 1.72-1.58 (m, 6H, cyclopentyl ring), 1.56-1.44 (m 5H, 6'-H, 2'-H, C- ring), 1.38 (s, 3H, 6Me), 1.30-1.09 (m, 8H, 6H of -CH2- of the side chain, 2H of C-ring),

1.07 (s, 3H, 6-Me), 1.01-0.93 (m, 2H of -CH2- of the side chain), 0.88-0.78 (m, 1H, C-

13 ring). C NMR (100 MHz CDCl3) δ 154.4 (ArC-1 or ArC-5), 154.2 (ArC-5 or ArC-1), 149.0

(tertiary aromatic), 109.6 (tertiary aromatic), 108.8 (ArC-2 or ArC-4), 106.4 (ArC-4 or ArC-

2), 68.5 (-CH2OH), 51.4, 50.6, 49.4, 41.7, 40.5, 37.6, 37.4, 34.9, 33.2, 29.9, 29.7, 28.9,

28.7, 27.7, 27.5, 26.6, 25.0, 23.3, 19.0. Mass spectum (ESI) m/z (relative intensity) 470

+ + (M + H, 100). Exact mass (ESI) calculated for C28H44N3O3 (M + H), 470.3383; found

470.3382. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.7 min for the title compound.

(6aR,9R,10aR)-9-(Hydroxymethyl)-3-(8-isothiocyanato-2-methyloctan-2-yl)-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.16a)65. To a solution of 2.15a (120 mg, 0.28 mmol), in anhydrous THF (5.6 ml) at room temperature, was added triphenyl phosphine (365 mg, 1.4 mmol). Carbon disulfide (0.55 mL, 8.4 mmol) was then added dropwise and the reaction mixture was stirred for an additional 10 hours at the same temperature. Upon completion, the reaction mixture was concentrated under reduced pressure and purified by flash column chromatography on silica gel (50-80% diethyl ether in hexanes) to give 95 mg of 2.16a as white solid in 76 % yield. mp = 63-65 oC. IR (neat) 3332 (br, OH), 2931, 2860, 2093 (s, NCS), 1620, 1537, 1451, 1413, 1331,

104

-1 1 1269, 1137, 1037, 966, 838 cm ; H NMR (500 MHz, CDCl3) δ 6.35 (d, J = 1.5 Hz, 1H,

Ar-H), 6.19 (d, J = 2.0 Hz, 1H, Ar-H), 4.76 (br s, 1H, ArOH), 3.52 (m, 2H, -CH2OH), 3.46

(t, J = 6.5 Hz, 2H, 7'-H), 3.22-3.16 (m as d, J = 13 Hz, 1H, C-ring), 2.51-2.44 (m as td, J

= 11.0 Hz, J = 2.5 Hz, 1H, C-ring), 2.02-1.91 (m, 2H, C-ring), 1.82-1.74 (m, 1H, C-ring),

1.65-1.56 (m, 2H, 6'-H), 1.54-1.46 (m, 3H, 2'-H, C-ring), 1.39 (s, 3H, 6-Me), 1.37-1.29 (m,

2H, -CH2- of the side chain group), 1.26-1.11 (s and m overlapping, 10H, -C(CH3)2-, -CH2- of the side chain, C-ring, especially, 1.20, s, 6H, -C(CH3)2-), 1.10-1.03 (s and m overlapping, 5H, 6-Me, -CH2- of the side chain, especially, 1.09, s, 3H, 6-Me), 0.87-0.7

13 (m as q, J = 12 Hz, 1H, C-ring); C NMR (100 MHz CDCl3) δ 154.6 (ArC-1 or ArC-5),

154.5 (ArC-5 or ArC-1), 149.6 (tertiary aromatic), 130.1 (NCS), 109.7 (tertiary aromatic),

107.8 (ArC-2 or ArC-4), 105.3 (ArC-4 or ArC-2), 68.5 (-CH2OH), 49.3, 45.0, 44.1, 40.5,

37.2, 35.0, 33.1, 29.8, 29.7, 29.3, 28.7, 28.6, 27.7, 27.4, 26.3, 24.3, 19.0. Mass spectrum

(ESI) m/z (relative intensity) 446 (M++H, 100). Exact mass (ESI) calculated for

+ C26H40NO3S (M +H), 446.2729; found, 446.2726. HPLC (4.6 mm × 250 mm, Supelco

Discovery column, acetonitrile/water) showed purity of 99.5% and retention time of 5.4 min for the title compound.

(6aR,9R,10aR)-9-(Hydroxymethyl)-3-[1-(6-isothiocyanatohexyl)cyclopentyl]-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.16b). The synthesis was carried out as described for 2.16a using 2.15b (90 mg, 0.19 mmol) triphenyl phosphine (258 mg, 0.98 mmol). Carbon disulfide (0.37 mL, 5.7 mmol) in THF (3.8 ml) and give 2.16b (78 mg, 87% yield) as white solid. IR (neat) 3365 (br, OH), 2932, 2865,

2104 (s, NCS), 1621, 1572, 1451, 1452, 1413, 1345, 1273, 1137, 1035, 839 cm-1; 1H

105

NMR (500 MHz, CDCl3) δ 6.29 (d, J = 2.0 Hz, 1H, Ar-H), 6.16 (d, J = 2.0 Hz, 1H, Ar-H),

5.16 (br s, 1H, ArOH), 3.53 (m as d, J = 5.5Hz, 2H, -CH2OH), 3.44 (t, J = 6.5 Hz, 2H, 7'-

H), 3.25-3.19 (m as d, J = 13 Hz, 1H, C-ring), 2.51-2.44 (m as td, J = 11.0 Hz, J = 2.5 Hz,

1H, C-ring), 1.98-1.90 (m, 2H, C-ring), 1.87-1.75 (m, 3H, 2H of the cyclopentyl ring, 1H of

C-ring), 1.72-1.54 (m, 8H, 6H of cyclopentyl ring, 2H of -CH2- of the side chain ), 1.54-

1.44 (m 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.39 (s, 3H, 6Me), 1.34-1.22 (m,

3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.19-1.1.10 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.08 (s, 3H, 6-Me), 1.04-0.94 (m, 2H of -CH2- of the side chain),

13 0.86-0.78 (m, 1H, C-ring). C NMR (100 MHz CDCl3) δ 154.5 (ArC-1 or ArC-5), 154.3

(ArC-5 or ArC-1), 148.9 (tertiary aromatic), 129.6 (NCS), 109.6 (tertiary aromati.c), 108.7

(ArC-2 or ArC-4), 106.3 (ArC-4 or ArC-2), 68.5 (-CH2OH), 50.5, 49.4, 44.6, 41.5, 40.5,

37.6, 37.4, 35.0, 33.2, 29.8, 29.7, 29.2, 27.7, 27.5, 26.3, 24.7, 23.3, 19.0. Mass spectrum

(ESI) m/z (relative intensity) 472 (M++H, 100). Exact mass (ESI) calculated for

+ C28H42NO3S (M + H), 472.2885; found 472.2882. HPLC (4.6 mm × 250 mm, Supelco

Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.6 min for the title compound.

(6aR,9R,10aR)-9-(Hydroxymethyl)-3-[1-(7-isothiocyanatoheptyl)cyclopentyl]-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.16c). The synthesis was carried out as described for 2.16a using 2.15c (140 mg, 0.30 mmol), triphenyl phosphine (390 mg, 1.5 mmol) and carbon disulfide (0.6 mL, 9.0 mmol) in THF

(6ml) and give 2.16c (123 mg, 85% yield) as white solid. IR (neat) 3336 (br, OH), 2932,

2865, 2094 (s, NCS), 1622, 1508, 1414, 1309, 1278, 1098, 1037, 835 cm-1;1H NMR (500

106

MHz, CDCl3) δ 6.30 (d, J = 1.5 Hz, 1H, Ar-H), 6.16 (d, J = 1.5 Hz, 1H, Ar-H), 4.85 (br s,

1H, ArOH), 3.55-3.45 (m, 4H, -CH2OH, 8'-especially, 3.47, t, J = 6.5 Hz, 2H, 8'-H), 3.24-

3.18 (m as d, J = 13 Hz, 1H, C-ring), 2.51-2.44 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, C- ring), 1.98-1.90 (m, 2H, C-ring), 1.87-1.73 (m, 3H, 2H of the cyclopentyl ring, 1H of C- ring), 1.72-1.58 (m, 8H, 6H of cyclopentyl ring, 2H of -CH2- of the side chain ), 1.53-1.42

(m 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.42-1.38 (s and m, 4H, 6Me, 1H of

C-ring, especially, 1.39, s, 6Me), 1.34-1.26 (m, 2H, -CH2- of the side chain), 1.23-1.1.10

(m, 5H, 4H of -CH2- of the side chain, 1H of C-ring), 1.09 (s, 3H, 6-Me), 1.04-0.94 (m, 2H

13 of -CH2- of the side chain), 0.86-0.78 (m, 1H, C-ring). C NMR (100 MHz CDCl3) δ 154.5

(ArC-1 or ArC-5), 154.2 (ArC-5 or ArC-1), 148.9 (tertiary aromatic), 129.8 (NCS), 109.6

(tertiary aromatic), 108.8 (ArC-2 or ArC-4), 106.4 (ArC-4 or ArC-2), 68.5 (-CH2OH), 50.6,

49.4, 45.0, 41.7, 40.5, 37.6, 37.4, 35.0, 33.2, 29.9, 29.8, 29.7, 28.5, 27.7, 27.5, 26.4, 24.9,

23.3, 19.0. Mass spectrum (ESI) m/z (relative intensity) 486 (M++H, 100). Exact mass

+ (ESI) calculated for C29H44NO3S (M + H), 486.3042; found 486.3040. HPLC (4.6 mm ×

250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.8 min for the title compound.

7-{(6aR,9R,10aR)-1-[(tert-Butyldimethylsilyl)oxy]-9-(hydroxymethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl}-7-methyloctyl nitrate (2.17a).

To a stirred solution of 2.13a (120 mg, 0.2 mmol) in 4 ml anhydrous MeCN under an argon atmosphere was added silver nitrate (272 mg, 1.6 mmol). The reaction mixture was refluxed for 46 h. Solid materials were filtered off. The filtrate was concentrated under reduced pressure and purification by flash column chromatography on silica gel (15%-50%

107 diethyl ether-hexane) afforded 355 mg (71% yield) of the title compound 2.17a (94 mg,

84% yield) as white foam. IR (neat) 3355 (br, OH), 2931, 2860, 1628 (s, ONO2), 1562,

-1 1 1471, 1411, 1329, 1278 (s, ONO2), 1140, 1063, 976, 844, 779 cm ; H NMR (500 MHz,

CDCl3) δ 6.37 (d, J = 2.0 Hz, 1H, Ar-H), 6.30 (d, J = 2.0 Hz, 1H, Ar-H), 4.39 (t, J = 6.5,

2H, 7'-H)3.54 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.46 (dd,

J = 10.0 Hz, J = 6.5 Hz, 1H, half of an AB system, -CH2OH), 3.19-3.13 (m as br d, J =

13.0 Hz, 1H, C-ring), 2.40-2.32 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.04-1.97

(m, 1H, C-ring), 1.94-1.88 (m, 1H, C-ring), 1.78-1.68 (m, 1H, C-ring), 1.67-1.60 (m, 2H,

6'-H ), 1.52-1.44 (m, 3H, 2'-H, C-ring), 1.38(s, 3H, 6Me), 1.34-1.24(m, 4H, -CH2- of the side chain), 1.24-1.17 (s and s overlapping, 6H, -C(CH3)2-, especially, 1.20, s, 3H, -

C(CH3)2-, and 1.19, s, 3H, -C(CH3)2-), 1.16-1.10 (m, 2H, C-ring), 1.09-1.02 (s and m overlapping, 5H, 6-Me, -CH2- of the side chain, especially, 1.06, s, 3H, 6-Me), 1.00 (s, 9H,

Si(Me)2CMe3), 0.82-0.7 (m, 1H, C-ring), 0.23 (s, 3H, Si(Me)2CMe3), 0.12 (s, 3H,

13 Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 154.5 (ArC-1 or ArC-5), 154.3 (ArC-5 or ArC-

1), 148.9 (tertiary aromatic), 113.6 (tertiary aromatic), 109.7 (ArC-2 or ArC-4), 108.4 (ArC-

4 or ArC-2), 73.4 (-CH2ONO2), 68.5 (-CH2OH), 49.6, 44.3, 40.5, 37.2, 35.5, 33.2, 29.8,

29.7, 28.8, 28.7, 27.6, 27.5, 26.6, 25.9, 25.5, 24.4, 18,8, 18.2, -3.6, -4.3. Mass spectrum

(ESI) m/z (relative intensity) 564 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco

Discovery column, acetonitrile/water) showed purity of 96.5% and retention time of 6.5 min for the title compound.

6-{1-[(6aR,9R,10aR)-1-[(tert-Butyldimethylsilyl)oxy]-9-(hydroxymethyl)-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]cyclopentyl}hexyl

108 nitrate (2.17b). The synthesis was carried out as described for 2.17a using 2.13b (100 mg, 0.16 mmol), and silver nitrate (170 mg, 1.0 mmol) in anhydrous MeCN (2.6 mL) and give 2.17b (80 mg, 85% yield) as white foam. IR (neat) 3368 (br, OH), 2931, 2860, 1628

-1 1 (s, ONO2), 1562, 1464, 1411, 1342, 1278 (s, ONO2), 1140, 1063, 979, 849, 779 cm ; H

NMR (500 MHz, CDCl3) δ 6.33 (d, J = 2.0 Hz, 1H, Ar-H), 6.25 (d, J = 2.0 Hz, 1H, Ar-H),

4.37 (t, J = 6.5, 2H, 7'-H), 3.53 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H, -

CH2OH), 3.46 (dd, J = 10.0 Hz, J = 6.5 Hz, 1H, half of an AB system, -CH2OH), 3.19-3.13

(m as br d, J = 13.0 Hz, 1H, C-ring), 2.40-2.32 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C- ring), 2.04-1.97 (m, 1H, C-ring), 1.94-1.88 (m, 1H, C-ring), 1.87-1.78 (m, 2H, the cyclopentyl ring), 1.72-1.54 (m, 9H, 6H of cyclopentyl ring, 2H of -CH2- of the side chain,

1H of the C-ring ), 1.52-1.44 (m 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.38 (s,

3H, 6Me), 1.34-1.24 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.19-1.1.10 (m,

3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.06 (s, 3H, 6-Me), 1.04-0.92 (s and m,

11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3),

13 0.82-0.72 (m, 1H, C-ring), 0.23 (s, 3H, Si(Me)2CMe3), 0.12 (s, 3H, Si(Me)2CMe3). C

NMR (100 MHz CDCl3) δ 154.3 (ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 148.2 (tertiary aromatic), 113.6 (tertiary aromatic), 110.7 (ArC-2 or ArC-4), 109.3 (ArC-4 or ArC-2), 73.4

(-CH2ONO2), 68.5 (-CH2OH), 50.6, 49.6, 41.7, 40.5, 37.5, 35.5, 33.2, 29.8, 29.6, 27.6,

27.5, 26.6, 25.9, 25.4, 25.0, 23.2, 18,8, 18.2, -3.6, -4.3. Mass spectrum (ESI) m/z (relative intensity) 590 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.7 min for the title compound.

109

7-{1-[(6aR,9R,10aR)-1-[(tert-Butyldimethylsilyl)oxy]-9-(hydroxymethyl)-6,6- dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]cyclopentyl}heptyl nitrate (2.17c). The synthesis was carried out as described for 2.17a using 2.13c (100 mg, 0.16 ml), and silver nitrate (217.6 mg, 1.3 mmol) in anhydrous MeCN (3.2mL) and give 2.17c (83 mg, 86% yield) as white foam. IR (neat) 3364 (br, OH), 29321, 2859, 1628

-1 1 (s, ONO2), 1573, 1461, 1409, 1338, 1278 (s, ONO2), 1147, 1063, 945, 850, 780 cm ; H

NMR (500 MHz, CDCl3) δ 6.33 (d, J = 2.0 Hz, 1H, Ar-H), 6.25 (d, J = 2.0 Hz, 1H, Ar-H),

4.39 (t, J = 6.5, 2H, 8'-H), 3.53 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H, -

CH2OH), 3.46 (dd, J = 10.0 Hz, J = 6.5 Hz, 1H, half of an AB system, -CH2OH), 3.19-3.13

1H of the C-ring ), 1.52-1.44 (m 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.38 (s,

3H, 6Me), 1.34-1.24 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.21-1.1.10 (m,

5H, 4H of -CH2- of the side chain, 1H of C-ring), 1.06 (s, 3H, 6-Me), 1.04-0.92 (s and m,

11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3),

13 0.82-0.72 (m, 1H, C-ring), 0.23 (s, 3H, Si(Me)2CMe3), 0.12 (s, 3H, Si(Me)2CMe3). C

NMR (100 MHz CDCl3) δ 154.4 (ArC-1 or ArC-5), 1542 (ArC-5 or ArC-1), 148.1 (tertiary aromatic), 113.6 (tertiary aromatic), 110.6 (ArC-2 or ArC-4), 109.3 (ArC-4 or ArC-2), 73.4

(-CH2ONO2), 68.5 (-CH2OH), 50.6, 49.6, 41.7, 40.5, 37.6, 35.5, 33.2, 297, 29.4, 28.6,

27.6, 27.5, 26.6, 25.8, 25.3, 25.1, 23.2, 18,7, 18.2, -3.6, -4.3. Mass spectrum (ESI) m/z

(relative intensity) 604 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco Discovery column,

110 acetonitrile/water) showed purity of 98.5% and retention time of 6.9 min for the title compound.

7-[(6aR,9R,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl]-7-methyloctyl nitrate (2.18a). To a solution of

2.17a (92 mg, 0.16 mmol) in anhydrous THF (4 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (0.19 mL, 0.19 mmol, 1M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (15%-50% ethyl acetate in hexane) gave 2.18a (65 mg, 90% yield) as a white solid. IR (neat) 3334

(br, OH), 2928, 2860, 1624 (s, ONO2), 1573, 1461, 1414, 1332, 1227 (s, ONO2), 1138,

-1 1 1040, 968, 865 cm ; H NMR (500 MHz, CDCl3) δ 6.35 (d, J = 1.5 Hz, 1H, Ar-H), 6.19 (d,

J = 1.5 Hz, 1H, Ar-H), 4.85 (br s, 1H, OH), 4.40 (t, J = 6.5, 2H, 7'-H), 3.54 (dd, J = 10.5

Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.52 (dd, J = 10.0 Hz, J = 6.5 Hz, 1H, half of an AB system, -CH2OH), 3.23-3.17 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.52-2.44

(m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.04-1.97 (m, 1H, C-ring), 1.94-1.90 (m,

1H, C-ring), 1.82-1.72 (m, 1H, C-ring), 1.67-1.60 (m, 2H, 6'-H ), 1.52-1.44 (m, 3H, 2'-H,

C-ring), 1.38(s, 3H, 6Me), 1.34-1.24(m, 2H, -CH2- of the side chain), 1.24-1.17 (s and m overlapping, 8H, -C(CH3)2-, 2H of -CH2- of the side chain, especially, 1.20, s, 6H, -

C(CH3)2-), 1.17-1.09 (m, 2H, C-ring), 1.09-1.02 (s and m overlapping, 5H, 6-Me, -CH2- of the side chain, especially, 1.06, s, 3H, 6-Me), 0.82-0.7 (m, 1H, C-ring). 13C NMR (100

MHz CDCl3) δ 154.7 (ArC-1 or ArC-5), 154.5 (ArC-5 or ArC-1), 149.6 (tertiary aromatic),

111

109.7 (tertiary aromatic), 107.9 (ArC-2 or ArC-4), 105.3 (ArC-4 or ArC-2), 73.4 (-

CH2ONO2), 68.5 (-CH2OH), 49.3, 44.2, 40.5, 37.2, 34.9, 33.1, 29.7, 29.6, 28.7, 28.6, 27.7,

27.4, 26.6, 25.4, 24.3, 19.0. Mass spectrum (ESI) m/z (relative intensity) 450 (M++H, 100).

+ Exact mass (ESI) calculated for C25H40NO6 (M + H), 450.2856; found 450.2853. HPLC

(4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.3 min for the title compound.

6-{1-[(6aR,9R,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl] cyclopentyl} hexyl nitrate (2.18b). The synthesis was carried out as described for 2.18a using 2.17b (75 mg, 0.13 mmol) and tetra-n-butylammonium fluoride (0.15 mL, 0.15 mmol, 1M solution in anhydrous THF) in

THF (3.2) and give 2.18b (57 mg, 92% yield) as white solid. IR (neat) 3354 (br, OH), 2933,

-1 2863, 1626 (s, ONO2), 1573, 1414, 1367, 1228 (s, ONO2), 1137, 1086, 1035, 863 cm ;

1 H NMR (500 MHz, CDCl3) δ 6.30 (d, J = 2.0 Hz, 1H, Ar-H), 6.13 (d, J = 2.0 Hz, 1H, Ar-

H), 5.81 (br s, 1H, OH), 4.38 (t, J = 6.5, 2H, 7'-H), 3.53 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.46 (dd, J = 10.0 Hz, J = 6.5 Hz, 1H, half of an AB system, -CH2OH), 3.22-3.16 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.52-2.44 (m as td, J =

11.0 Hz, J = 3.0 Hz, 1H, C-ring), 1.99-1.90 (m, 2H, C-ring), 1.87-1.72 (m, 2H, the cyclopentyl ring), 1.72-1.54 (m, 9H, 6H of cyclopentyl ring, 2H of -CH2- of the side chain,

1H of the C-ring ), 1.52-1.43 (m 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.38 (s,

3H, 6Me), 1.32-1.24 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.20-1.1.11 (m,

2H, 2H of -CH2- of the side chain, 1H of C-ring), 1.07 (s, 3H, 6-Me), 1.04-0.92 (m, 2H, -

13 CH2- of the side chain,), 0.82-0.72 (m, 1H, C-ring). C NMR (100 MHz CDCl3) δ 154.5

112

(ArC-1 or ArC-5), 154.3 (ArC-5 or ArC-1), 148.9 (tertiary aromatic), 109.6 (tertiary aromatic), 108.7 (ArC-2 or ArC-4), 106.4 (ArC-4 or ArC-2), 73.4 (-CH2ONO2), 68.5 (-

CH2OH), 50.5, 49.3, 41.5, 40.5, 37.6, 37.4, 34.9, 33.2, 29.7, 29.5, 27.7, 27.5, 26.6, 25.4,

24.8, 23.3, 19.0. Mass spectrum (ESI) m/z (relative intensity) 476 (M++H, 100). Exact

+ mass (ESI) calculated for C27H42NO6 (M + H), 476.3012; found 476.3008. HPLC (4.6 mm

× 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.5 min for the title compound.

7-{1-[(6aR,9R,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl]cyclopentyl}heptyl nitrate (2.18c). The synthesis was carried out as described for 2.18a using 2.17c (80 mg, 0.13 mmol) and tetra-n-butylammonium fluoride (0.15 mL, 0.15 mmol, 1M solution in anhydrous THF) in

THF (3.2mL) and give 2.18c (57 mg, 90% yield) as white solid. IR (neat) 3336 (br, OH),

2932, 2861, 1626 (s, ONO2), 1572, 1414, 1337, 1228 (s, ONO2), 1137, 1036, 967, 863

-1 1 cm ; H NMR (500 MHz, CDCl3) δ 6.29 (d, J = 1.5 Hz, 1H, Ar-H), 6.14 (d, J = 1.0 Hz, 1H,

Ar-H), 5.17 (br s, 1H, OH), 4.40 (t, J = 6.5, 2H, 8'-H), 3.53 (dd, J = 10.5 Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.46 (dd, J = 10.0 Hz, J = 6.5 Hz, 1H, half of an AB system, -CH2OH), 3.26-3.20 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.51-2.44 (m as td, J =

11.0 Hz, J = 3.0 Hz, 1H, C-ring), 1.99-1.88 (m, 2H, C-ring), 1.87-1.74 (m, 2H, the cyclopentyl ring), 1.72-1.54 (m, 9H, 6H of cyclopentyl ring, 2H of -CH2- of the side chain,

1H of the C-ring ), 1.52-1.43 (m 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.38 (s,

3H, 6Me), 1.32-1.24 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.22-1.1.10 (m,

5H, 4H of -CH2- of the side chain, 1H of C-ring), 1.07 (s, 3H, 6-Me), 1.04-0.92 (m, 2H, -

113

13 CH2- of the side chain,), 0.82-0.72 (m, 1H, C-ring). C NMR (100 MHz CDCl3) δ 154.5

(ArC-1 or ArC-5), 154.3 (ArC-5 or ArC-1), 149.0 (tertiary aromatic), 109.6 (tertiary aromatic), 108.8 (ArC-2 or ArC-4), 106.4 (ArC-4 or ArC-2), 73.4 (-CH2ONO2), 68.5 (-

CH2OH), 50.6, 49.4, 41.7, 40.5, 37.6, 37.4, 35.0, 33.2, 29.9, 29.7, 28.9, 27.7, 27.5, 26.6,

25.5, 24.9, 23.3, 19.0. Mass spectrum (ESI) m/z (relative intensity) 490 (M++H, 100).

+ Exact mass (ESI) calculated for C28H44NO6 (M + H), 490.3169; found 490.3165. HPLC

(4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.6 min for the title compound.

8-[(6aR,9R,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl]-8-methylnonanenitrile (2.19a). To a solution of

2.14a (100 mg, 0.21 mmol) in anhydrous DMSO (4.2 mL) at 5 °C , under an argon atmosphere, was added NaCN (51.5 mg, 1.1 mmol). The reaction mixture was stirred for

18h at r t, and then quenched using ice. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (20%-50% ethyl acetate in hexane) gave 2.19a (60 mg, 70% yield) as a white solid. IR (neat) 3384 (br, OH), 2929,

2862, 2367 (s, CN), 1621, 1572, 1415, 1337, 1341, 1278, 1040, 840 cm-1; 1H NMR (500

MHz, CDCl3) δ 6.34 (d, J = 1.5 Hz, 1H, Ar-H), 6.21 (d, J = 1.5 Hz, 1H, Ar-H), 4.95 (br s,

1H, OH), 3.53 (m, 2H, -CH2OH), 3.21-3.17 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.52-

2.44 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.29 (t, J = 6.5, 2H, 7'-H), 2.04-1.90

(m, 2H, C-ring), 1.82-1.72 (m, 1H, C-ring), 1.60-1.55 (m, 2H, 6'-H ), 1.52-1.46 (m, 3H, 2'-

H, C-ring), 1.40-1.32(s and m overlapping, 6Me, 2H of -CH2- of the side chain, especially,

1.38, s, 3H, 6Me), 1.24-1.17 (s and m overlapping, 8H, -C(CH3)2-, 2H of -CH2- of the side

114 chain, especially, 1.20, s, 6H, -C(CH3)2-), 1.18-1.11 (m, 2H, C-ring), 1.09-1.02 (s and m overlapping, 5H, 6-Me, -CH2- of the side chain, especially, 1.06, s, 3H, 6-Me), 0.82-0.7

13 (m, 1H, C-ring). C NMR (100 MHz CDCl3) δ 154.6 (ArC-1 or ArC-5), 154.5 (ArC-5 or

ArC-1), 149.5 (tertiary aromatic), 119.9 (-CN), 109.7 (tertiary aromatic), 107.8 (ArC-2 or

ArC-4), 105.3 (ArC-4 or ArC-2), 68.5 (-CH2OH), 49.4, 44.5, 40.5, 37.2, 34.9, 33.1, 29.7,

29.1, 28.7, 28.6, 28.3, 27.7, 27.4, 25.1, 24.1, 19.05, 17.1 (-CH2CN). Mass spectrum (ESI)

+ + m/z (relative intensity) 414 (M +H, 100). Exact mass (ESI) calculated for C26H40NO3 (M

+ H), 414.3008; found 414.3003. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.0 min for the title compound.

7-{1-[(6aR,9R,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl]cyclopentyl}heptanenitrile (2.19b). The synthesis was carried out as described for 2.19a using 2.14b (100 mg, 0.20 mmol) and

NaCN (49 mg, 1.0 mmol) in DMSO (4.0 mL) and give 2.19b (59 mg, 67% yield) as white solid. IR (neat) 3389 (br, OH), 2933, 2865, 2305 (s, CN), 1621, 1573, 1453, 1414, 1342,

-1 1 1139, 1036, 839 cm ; H NMR (500 MHz, CDCl3) δ 6.29 (d, J = 1.5 Hz, 1H, Ar-H), 6.14

(d, J = 1.5 Hz, 1H, Ar-H), 4.86 (br s, 1H, OH), 3.53 (m, 2H, -CH2OH), 3.24-3.17 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.52-2.44 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.27

(t, J = 6.5, 2H, 7'-H), 2.04-1.90 (m, 2H, C-ring), 1.87-1.74 (m, 3H, 2H of the cyclopentyl ring, 1H of C-ring), 1.72-1.59 (m, 6H, cyclopentyl ring), 1.58-1.43 (m 5H, 4H of -CH2- of the side chain, 1H of C-ring), 1.39 (s, 3H, 6Me), 1.36-1.24 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.20-1.1.10 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.08

115

13 (s, 3H, 6-Me), 1.04-0.92 (m, 2H, -CH2- of the side chain,), 0.89-0.81 (m, 1H, C-ring). C

NMR (100 MHz CDCl3) δ 154.4 (ArC-1 or ArC-5), 154.2 (ArC-5 or ArC-1), 148.8 (tertiary aromatic), 119.9 (CN), 109.7 (tertiary aromatic), 108.7 (ArC-2 or ArC-4), 106.4 (ArC-4 or

ArC-2), 68.5 (-CH2OH), 50.5, 49.4, 41.4, 40.5, 37.6, 37.4, 35.0, 33.2, 29.7, 29.1, 28.3,

27.7, 27.5, 25.2, 24.7, 23.3, 19.0, 17.0 (-CH2CN). Mass spectrum (ESI) m/z (relative

+ + intensity) 440 (M +H, 100). Exact mass (ESI) calculated for C28H42NO3 (M + H),

440.3165; found 440.3164. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.2 min for the title compound.

8-{1-[(6aR,9R,10aR)-1-Hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl]cyclopentyl}octanenitrile (2.19c). The synthesis was carried out as described for 2.19a using 2.14c (100 mg, 0.19 mmol) and

NaCN (47.5 mg, 0.95mmol) in DMSO (3.8 mL) and give 2.19c (61 mg, 71% yield) as white solid. IR (neat) 3382 (br, OH), 2934, 2867, 2295 (s, CN), 1621, 1573, 1415, 1308,

-1 1 1273, 1135, 1039, 839 cm ; H NMR (500 MHz, CDCl3) δ 6.28 (d, J = 1.5 Hz, 1H, Ar-H),

6.17 (d, J = 1.5 Hz, 1H, Ar-H), 5.45 (br s, 1H, OH), 3.53 (m, 2H, -CH2OH), 3.25-3.19 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.51-2.44 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring),

2.30 (t, J = 6.5, 2H, 8'-H), 2.00-1.89 (m, 2H, C-ring), 1.87-1.74 (m, 3H, 2H of the cyclopentyl ring, 1H of C-ring), 1.72-1.56 (m, 8H, 2H of -CH2- of the side chain, 6H of cyclopentyl ring), 1.52-1.43 (m 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.39 (s,

3H, 6Me), 1.37-1.25 (m, 3H, 2H of -CH2- of the side chain, 1H of C-ring), 1.19-1.1.11 (m,

5H, 4H of -CH2- of the side chain, 1H of C-ring), 1.09 (s, 3H, 6-Me), 1.04-0.92 (m, 2H, -

116

13 CH2- of the side chain,), 0.87-0.79 (m, 1H, C-ring). C NMR (100 MHz CDCl3) δ 154.4

(ArC-1 or ArC-5), 154.3 (ArC-5 or ArC-1), 148.9 (tertiary aromatic), 119.9 (CN), 109.6

(tertiary aromatic), 108.7 (ArC-2 or ArC-4), 106.4 (ArC-4 or ArC-2), 68.5 (-CH2OH), 50.5,

49.4, 41.5, 40.5, 37.7, 37.5, 35.0, 33.2, 29.7, 29.6, 28.4, 28.3, 27.7, 27.5, 25.2, 24.8, 23.3,

+ 19.0, 17.0 (-CH2CN). Mass spectrum (ESI) m/z (relative intensity) 454 (M +H, 100). Exact

+ mass (ESI) calculated for C29H44NO3 (M + H), 454.3321; found 454.3319. HPLC (4.6 mm

× 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.3 min for the title compound.

{[(6aR,9R,10aR)-3-[1-(7-Bromoheptyl)cyclopentyl)]-9-(iodomethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-yl]oxy}(tert- butyl)dimethylsilane (2.20c). To a solution of 2.13c (500 mg, 0.8 mmol) in anhydrous benzene (16 mL), under an argon atmosphere, was added imidazole (217 mg, 3.2 mmol), triphenylphosphine (419.7 mg, 1.6 mmol), then the reaction mixture was heated to 50 °C, followed by a dropwise addition of a solution of iodine (406 mg, 1.6 mmol) in benzene.

The reaction mixture was stirred for 30 min at 50 °C. The reaction was quenched by an aqueous sodium sulfite solution. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (0%-15% diethyl ether in hexane) gave 2.20c

(564 mg, 95% yield) as a white oil. IR (neat) 2929, 2857, 1612, 1562, 1411, 1361, 1324,

-1 1 1253, 1058, 835, 779 cm ; H NMR (500 MHz, CDCl3) δ 6.33 (d, J = 2.0 Hz, 1H, Ar-H),

6.26 (d, J = 2.0 Hz, 1H, Ar-H), 3.48(t, J = 6.5 Hz, 8'-H) 3.26-3.19 (dd and m as d overlapping, 2H, 1H of C-ring, 1H of -CH2I, especially, m as br d, J = 13.0 Hz, 1H, C-ring,

3.21, dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2I) 3.08 (dd, J = 10.0 Hz,

117

J = 5.0 Hz, half of an AB system, 1H, -CH2I), 2.40-2.33 (m as td, J = 11.0 Hz, J = 3.0 Hz,

1H, C-ring), 2.12-2.05 (m, 1H, C-ring), 1.92-1.88 (m, 1H, C-ring), 1.87-1.78 (m, 2H, the cyclopentyl ring), 1.76-1.58 (m, 9H, 6H of the cyclopentyl ring, 2H of -CH2- of the side chain group, 1H of C-ring), 1.52-1.44 (m, 3H, 2'-H, C-ring), 1.38 (s, 3H, 6Me), 1.36-1.29

(m, 2H, -CH2- of the side chain), 1.22-1.10 (m, 6H, 4H of -CH2- of the side chain, 2H of

C-ring), 1.05 (s, 3H, 6-Me), 1.02-0.93 (s and m overlapping, 11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3), 0.89-0.82 (m, 1H, C-ring),

13 0.22 (s, 3H, Si(Me)2CMe3), 0.13 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 154.3

(ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 148.5 (tertiary aromatic), 113.0 (tertiary aromatic), 110.8 (ArC-2 or ArC-4), 109.4 (ArC-4 or ArC-2), 50.6, 49.2, 45.1, 41.8, 49.7,

37.6, 37.0, 35.5, 33.5, 32.6, 30.1, 29.9, 28.7, 27.6, 27.5, 26.8, 26.0, 25.2, 23.3, 18.7, 18.3,

+ 15.1 (-CH2I), -3.8, -4.6. Mass spectrum (ESI) m/z (relative intensity) 731 (M +H, 100).

HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 8.0 min for the title compound.

{[(6aR,9R,10aR)-9-(Azidomethyl)-3-[1-(7-bromoheptyl)cyclopentyl]-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-yl]oxy}(tert- butyl)dimethylsilane. (2.21c) To a solution of 2.20c (500 mg, 0.77 mmol) in anhydrous

CH2Cl2 (15.5 mL) at r t, under an argon atmosphere, was added tetrabutylammonium azide (1.6 g, 7.7 mmol). The reaction mixture was stirred for 30h at the same temperature, and then quenched by brine. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (0%-10% diethyl ether in hexane) gave 2.21c

(413 mg, 83% yield) as a white foam. IR (neat) 2929, 2858, 2097 (s, N3), 1612, 1563,

118

-1 1 1463, 1411, 1342, 1254 1138, 1062, 873 cm ; H NMR (500 MHz, CDCl3) δ 6.33 (d, J =

2.0 Hz, 1H, Ar-H), 6.26 (d, J = 2.0 Hz, 1H, Ar-H), 3.48(t, J = 6.5 Hz, 8'-H) 3.23-3.17 (dd and m as d overlapping, 2H, 1H of C-ring, 1H of -CH2N3, especially, m as br d, J = 13.0

Hz, 1H, C-ring, 3.21, dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2N3) 3.14

(dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2N3), 2.38-2.31 (m as td, J =

11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.02-1.97 (m, 1H, C-ring), 1.92-1.88 (m, 1H, C-ring),

1.87-1.73 (m, 3H, 2H of the cyclopentyl ring, 1H of C-ring), 1.76-1.58 (m, 8H, 6H of the cyclopentyl ring, 2H of -CH2- of the side chain group), 1.52-1.44 (m, 3H, 2'-H, C-ring),

1.38 (s, 3H, 6Me), 1.35-1.28 (m, 2H, -CH2- of the side chain), 1.24-1.10 (m, 6H, 4H of -

CH2- of the side chain, 2H of C-ring), 1.05 (s, 3H, 6-Me), 1.02-0.91 (s and m overlapping,

11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3),

13 0.86-0.79 (m, 1H, C-ring), 0.22 (s, 3H, Si(Me)2CMe3), 0.13 (s, 3H, Si(Me)2CMe3). C

NMR (100 MHz CDCl3) δ 154.4 (ArC-1 or ArC-5), 154.2 (ArC-5 or ArC-1), 149.3 (tertiary aromatic), 113.5 (tertiary aromatic), 1108 (ArC-2 or ArC-4), 109.5 (ArC-4 or ArC-2), 57.8,

51.5, 50.4, 49.1, 41.6, 40.5, 37.7, 37.5, 34.9, 34.1, 30.6, 29.9, 28.8, 28.7, 27.6, 27.3, 26.6,

25.9, 25.0, 23.2, 18.6, 18.1, -3.6, -4.3. Mass spectrum (ESI) m/z (relative intensity) 646

(M++H, 100). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 9.1 min for the title compound.

{[(6aR,9R,10aR)-3-[1-(7-Azidoheptyl)cyclopentyl]-9-(azidomethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-yl]oxy}(tert- butyl)dimethylsilane (2.22c) To a solution of 2.21c (200 mg, 0.31 mmol) in anhydrous

CH2Cl2 (5.2 mL) at r t, under an argon atmosphere, was added N, N, N', N' -

119 tetramethylguanidinium azide (1.4 g, 9.1 mmol) and stirring was continued for 1 day. On completion, the reaction was quenched with water and diluted with CH2Cl2. The organic phase was washed with brine, dried over MgSO4 and concentrated in vacuo. Purification by flash column chromatography on silica gel (3-8% diethyl ether in hexanes) gave 151 mg of 2.22c in 80% yield as a white foam in 74% yield. IR (neat) 2928, 2857, 2097 (s,

N3), 1756, 1612, 1411, 1342, 1254, 1138, 1062, 839 cm-1; 1H NMR (500 MHz, CDCl3)

δ 6.33 (d, J = 1.5 Hz, 1H, Ar-H), 6.26 (d, J = 2.0 Hz, 1H, Ar-H), 3.24-3.15 (dd, t and m as d overlapping, 3H, C-ring, -CH2N3, 8'-H, especially, 3.21, t, J = 6.5 Hz, 8'-H, m as br d, J

= 13.0 Hz, 1H, C-ring, 3.21, dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -

CH2N3) 3.14 (dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2N3), 2.38-2.31

(m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.02-1.97 (m, 1H, C-ring), 1.92-1.88 (m,

1H, C-ring), 1.87-1.73 (m, 3H, 2H of the cyclopentyl ring, 1H of C-ring), 1.71-1.58 (m, 6H, cyclopentyl ring), 1.54-1.43 (m, 5H, 4H of -CH2- of the side chain group, 1H of C-ring),

1.38 (s, 3H, 6Me), 1.31-1.24 (m, 2H, -CH2- of the side chain), 1.21-1.10 (m, 6H, 4H of -

CH2- of the side chain, 2H of C-ring), 1.06 (s, 3H, 6-Me), 1.02-0.91 (s and m overlapping,

11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.00, s, 9H, Si(Me)2CMe3),

0.84-0.76 (m, 1H, C-ring), 0.24 (s, 3H, Si(Me)2CMe3), 0.12 (s, 3H, Si(Me)2CMe3). 13C

NMR (100 MHz CDCl3) δ 154.3 (ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 149.1 (tertiary aromatic), 113.6 (tertiary aromatic), 110.8 (ArC-2 or ArC-4), 109.5 (ArC-4 or ArC-2), 57.8,

51.5, 50.5, 49.0, 41.6, 38.2, 37.7, 37.5, 34.9, 34.1, 30.6, 29.8, 28.9, 28.7, 27.7, 27.4, 26.6,

25.9, 25.0, 23.2, 18.6, 18.1, -3.6, -4.3. Mass spectrum (ESI) m/z (relative intensity) 609

(M++H, 100). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 8.8 min for the title compound.

120

(6aR,9R,10aR)-3-[1-(7-Azidoheptyl)cyclopentyl]-9-(azidomethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (2.23c). To a solution of 2.22c

(150 mg, 0.24 mmol) in anhydrous THF (6.1 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (0.3 mL, 0.3 mmol, 1M solution in anhydrous

THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (8%-25% ethyl acetate in hexane) gave 2.23c (114 mg, 92% yield) as a white solid. IR (neat) 2931, 2861,

2097 (s, N3), 1756, 1622, 1413, 1331, 1251, 1138, 1039, 839 cm-1; 1H NMR (500 MHz,

CDCl3) δ 6.29 (d, J = 1.5 Hz, 1H, Ar-H), 6.14 (d, J = 2.0 Hz, 1H, Ar-H), 5.12 (br s, 1H,

OH) 3.27-3.19 (dd, t and m as d overlapping, 3H, C-ring, -CH2N3, 8'-H, especially, 3.21, t, J = 6.5 Hz, 8'-H, m as br d, J = 13.0 Hz, 1H, C-ring, 3.21, dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2N3) 3.15 (dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system,

1H, -CH2N3), 2.51-2.43 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.02-1.97 (m, 1H,

C-ring), 1.95-1.88 (m, 1H, C-ring), 1.87-1.75 (m, 3H, 2H of the cyclopentyl ring, 1H of C- ring), 1.73-1.58 (m, 6H, cyclopentyl ring), 1.55-1.42 (m, 5H, 4H of -CH2- of the side chain group, 1H of C-ring), 1.38 (s, 3H, 6Me), 1.31-1.24 (m, 2H, -CH2- of the side chain), 1.21-

1.10 (m, 6H, 4H of -CH2- of the side chain, 2H of C-ring), 1.08 (s, 3H, 6-Me), 1.04-0.93

(m, 2H, -CH2- of the side chain), 0.85-0.81 (m, 1H, C-ring). 13C NMR (100 MHz CDCl3)

δ 154.4 (ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 149.1 (tertiary aromatic), 109.2 (tertiary aromatic), 108.8 (ArC-2 or ArC-4), 106.3 (ArC-4 or ArC-2), 57.8, 51.4, 50.6, 49.1, 41.7,

38.1, 37.5, 37.4, 35.0, 34.2, 30.8, 30.0, 28.9, 28.7, 27.7, 27.3, 26.6, 25.0, 23.3, 19.0.

121

Mass spectrum (ESI) m/z (relative intensity) 495 (M++H, 100). Exact mass (ESI) calculated for C28H43N6O2 (M+ + H), 495.3447; found 495.3440. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.3 min for the title compound.

(6aR,9R,10aR)-3-[1-(7-Isothiocyanatoheptyl)cyclopentyl]-9-

(isothiocyanatomethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-ol (2.24c). To a solution of 2.23c (65 mg, 0.13 mmol), in anhydrous

THF (4.3 ml) at room temperature, was added triphenyl phosphine (340 mg, 1.3 mmol).

Carbon disulfide (0.48 mL, 8.0 mmol) was then added dropwise and the reaction mixture was stirred for an additional 10 hours at the same temperature. Upon completion, the reaction mixture was concentrated under reduced pressure and purified by flash column chromatography on silica gel (50-80% diethyl ether in hexanes) to give 59 mg of 2.24c in

86% yield as white solid. IR (neat) 2923, 2859, 2106 (s, NCS), 1728, 1618, 1574, 1413,

1372, 1339, 1251, 1139, 1036, 863 cm-1; 1H NMR (500 MHz, CDCl3) δ 6.30 (d, J = 1.5

Hz, 1H, Ar-H), 6.14 (d, J = 1.0 Hz, 1H, Ar-H), 4.90 (br s, 1H, OH) 3.51-3.44 (dd and t, 2H,

-CH2NCS, 8'-H, especially, 3.49, t, J = 6.5 Hz, 8'-H, m as br d, J = 13.0 Hz, 1H, C-ring,

3.21, dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2NCS), 3.41 (dd, J =

10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2NCS), 3.29-3.23(m as br d, J = 12.5

Hz, 1H, C-ring), 2.53-2.45 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.02-1.90 (m,

2H, C-ring), 1.86-1.78 (m, 2H, the cyclopentyl ring), 1.73-1.58 (m, 9H, 6H of cyclopentyl ring, 1H of C-ring, 2H of -CH2- of the side chain group), 1.53-1.44 (m, 3H, 2H of -CH2- of the side chain group, 1H of C-ring), 1.39 (s, 3H, 6Me), 1.34-1.26 (m, 2H, -CH2- of the

122 side chain), 1.23-1.11 (m, 6H, 4H of -CH2- of the side chain, 2H of C-ring), 1.09 (s, 3H,

6-Me), 1.04-0.93 (m, 2H, -CH2- of the side chain), 0.92-0.89 (m, 1H, C-ring). 13C NMR

(100 MHz CDCl3) δ 154.4 (ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 149.2 (tertiary aromatic), 130.1 (NCS), 108.9 (tertiary aromatic), 108.7 (ArC-2 or ArC-4), 106.4 (ArC-4 or ArC-2), 50.9, 50.6, 48.8, 45.0, 41.6, 38.5, 37.5, 37.4, 34.9, 33.8, 30.4, 29.9, 29.8, 28.5,

27.7, 27.2, 26.4, 24.9, 23.3, 19.0. Mass spectrum (ESI) m/z (relative intensity) 527 (M++H,

100). Exact mass (ESI) calculated for C30H43N2O2S2 (M+ + H), 527.2766; found

527.2776. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.5 min for the title compound.

7-{1-[(6aR,9R,10aR)-9-(Azidomethyl)-1-[(tert-butyldimethylsilyl)oxy]-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]cyclopentyl}heptyl nitrate

(2.25c). To a stirred solution of 2.21c (200 mg, 0.3 mmol) in anhydrous MeCN (6.1 mL) under an argon atmosphere was added silver nitrate (527 mg, 3.1 mmol). The reaction mixture was refluxed for 46 h. Solid materials were filtered off. The filtrate was concentrated under reduced pressure and purification by flash column chromatography on silica gel (15%-50% diethyl ether-hexane) afforded 169 mg (90% yield) of the title compound 2.25c as white foam. IR (neat) 2929, 2858, 2097 (s, N3), 1788, 1631 (s,

ONO2), 1563, 1464, 1411, 1342, 1268 (s, ONO2), 1138, 1062, 836 cm-1; 1H NMR (500

MHz, CDCl3) δ 6.33 (d, J = 2.0 Hz, 1H, Ar-H), 6.26 (d, J = 2.0 Hz, 1H, Ar-H), 4.39 (t, J =

7.0 Hz, 8'-H), 3.23-3.17 (dd and m as d overlapping, 2H, 1H of C-ring, 1H of -CH2N3, especially, m as br d, J = 13.0 Hz, 1H, C-ring, 3.21, dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2N3) 3.14 (dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H,

123

-CH2N3), 2.38-2.31 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.03-1.97 (m, 1H, C- ring), 1.92-1.88 (m, 1H, C-ring), 1.87-1.71 (m, 3H, 2H of the cyclopentyl ring, 1H of C- ring), 1.71-1.58 (m, 8H, 6H of the cyclopentyl ring, 2H of -CH2- of the side chain group),

1.52-1.44 (m, 3H, 2'-H, C-ring), 1.38 (s, 3H, 6Me), 1.33-1.24 (m, 2H, -CH2- of the side chain), 1.24-1.10 (m, 6H, 4H of -CH2- of the side chain, 2H of C-ring), 1.05 (s, 3H, 6-Me),

1.04-0.91 (s and m overlapping, 11H, 2H of -CH2- of the side chain, Si(Me)2CMe3, especially, 1.01, s, 9H, Si(Me)2CMe3), 0.84-0.76 (m, 1H, C-ring), 0.24 (s, 3H,

Si(Me)2CMe3), 0.12 (s, 3H, Si(Me)2CMe3). 13C NMR (100 MHz CDCl3) δ 154.5 (ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 148.5 (tertiary aromatic), 113.6 (tertiary aromatic),

110.7 (ArC-2 or ArC-4), 109.3 (ArC-4 or ArC-2), 73.4 (-CH2ONO2), 57.8, 50.6, 49.1, 41.7,

38.1, 37.5, 37.3, 35.0, 34.2, 30.8, 29.9, 28.8, 27.7, 27.4, 26.6, 25.9, 25.5, 25.0, 23.3, 18.8,

18.2, -3.6, -4.3. Mass spectrum (ESI) m/z (relative intensity) 629 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.0% and retention time of 8.5 min for the title compound.

7-{1-[(6aR,9R,10aR)-9-(Azidomethyl)-1-hydroxy-6,6-dimethyl-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl]cyclopentyl}heptyl nitrate (2.26c). To a solution of 2.25c (160 mg, 0.25 mmol) in anhydrous THF (6.4 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (0.3 mL, 0.3 mmol, 1M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel

(8%-25% ethyl acetate in hexane) gave 2.26c (113 mg, 90% yield) as a white solid. IR

124

(neat) 2929, 2858, 2097 (s, N3), 1788, 1631 (s, ONO2), 1563, 1411, 1337, 1342, 1277 (s,

-1 1 ONO2), 1138, 1045, 836 cm ; H NMR (500 MHz, CDCl3) δ 6.30 (d, J = 1.5 Hz, 1H, Ar-

H), 6.13 (d, J = 1.5 Hz, 1H, Ar-H), 4.70 (s, 1H, OH), 4.40 (t, J = 7.0 Hz, 8'-H), 3.25 (dd, J

= 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2N3), 3.23-3.17 (m as d, J = 13.0 Hz,

1H, C-ring), 3.16 (dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2N3), 2.52-

2.43 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.03-1.97 (m, 1H, C-ring), 1.94-1.88

(m, 1H, C-ring), 1.87-1.78 (m, 3H, 2H of the cyclopentyl ring, 1H of C-ring), 1.71-1.58 (m,

8H, 6H of the cyclopentyl ring, 2H of -CH2- of the side chain group), 1.52-1.44 (m, 3H, 2'-

H, C-ring), 1.38 (s, 3H, 6Me), 1.33-1.24 (m, 2H, -CH2- of the side chain), 1.24-1.10 (m,

6H, 4H of -CH2- of the side chain, 2H of C-ring), 1.09 (s, 3H, 6-Me), 1.02-0.92 (m, 2H, -

13 CH2- of the side chain), 0.89-0.81 (m, 1H, C-ring). C NMR (100 MHz CDCl3) δ 154.4

(ArC-1 or ArC-5), 154.2 (ArC-5 or ArC-1), 149.1 (tertiary aromatic), 109.2 (tertiary aromatic), 108.9 (ArC-2 or ArC-4), 106.4 (ArC-4 or ArC-2), 73.4 (-CH2ONO2), 57.8, 50.5,

49.0, 41.6, 38.1, 37.6, 37.4, 34.9, 34.2, 30.8, 29.9, 28.9, 27.7, 27.4, 26.6, 25.5, 24.9, 23.3,

19.0. Mass spectrum (ESI) m/z (relative intensity) 515 (M++H, 100). Exact mass (ESI)

+ calculated for C28H43N4O5 (M + H), 515.3233; found 515.3231. HPLC (4.6 mm × 250 mm,

Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.2 min for the title compound.

7-{1-[(6aR,9R,10aR)-1-Hydroxy-9-(isothiocyanatomethyl)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]cyclopentyl}heptyl nitrate

(2.27c). To a solution of 2.26c (65 mg, 0.13 mmol), in anhydrous THF (2.5 ml) at room temperature, was added triphenyl phosphine (170 mg, 0.65 mmol). Carbon disulfide (0.24

125 mL, 3.9 mmol) was then added dropwise and the reaction mixture was stirred for an additional 10 hours at the same temperature. Upon completion, the reaction mixture was concentrated under reduced pressure and purified by flash column chromatography on silica gel (50-80% diethyl ether in hexanes) to give 50 mg of 2.27c in 71% yield as white solid. IR (neat) 2934, 2855, 2097 (s, NCS), 1787, 1626 (s, ONO2), 1573, 1461, 1413,

-1 1 1376, 1278 (s, ONO2), 1139, 1034, 863 cm ; H NMR (500 MHz, CDCl3) δ 6.30 (d, J =

1.5 Hz, 1H, Ar-H), 6.13 (d, J = 1.5 Hz, 1H, Ar-H), 4.72 (s, 1H, OH), 4.40 (t, J = 7.0 Hz, 8'-

H), 3.47 (dd, J = 10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2NCS), 3.41 (dd, J =

10.0 Hz, J = 5.0 Hz, half of an AB system, 1H, -CH2NCS), 3.27-3.22 (m as d, J = 13.0 Hz,

1H, C-ring), 2.52-2.43 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, C-ring), 2.03-1.91 (m, 3H, C- ring), 1.86-1.78 (m, 2H, the cyclopentyl ring, 1H of C-ring), 1.74-1.58 (m, 8H, 6H of the cyclopentyl ring, 2H of -CH2- of the side chain group), 1.52-1.44 (m, 3H, 2'-H, C-ring),

1.39 (s, 3H, 6Me), 1.33-1.10 (m, 8H, 6H of -CH2- of the side chain, 2H of C-ring), 1.08 (s,

13 3H, 6-Me), 1.02-0.92 (m, 2H, -CH2- of the side chain), 0.91-0.87 (m, 1H, C-ring). C NMR

(100 MHz CDCl3) δ 154.4 (ArC-1 or ArC-5), 154.1 (ArC-5 or ArC-1), 149.2 (tertiary aromatic), 129.7 (NCS), 109.2 (tertiary aromatic), 108.9 (ArC-2 or ArC-4), 106.3 (ArC-4 or ArC-2), 73.4 (-CH2ONO2), 50.9, 50.5, 48.8, 41.6, 38.5, 37.6, 37.4, 34.9, 33.8, 30.6,

29.9, 28.5, 27.7, 27.2, 26.6, 25.5, 24.9, 23.9, 19.0. Mass spectrum (ESI) m/z (relative

+ + intensity) 531 (M +H, 100). Exact mass (ESI) calculated for C29H43N2O5S (M + H),

531.2893; found 531.2890. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.3 min for the title compound.

126

Radioligand Binding Assays. The affinities (Ki) of the new compounds for rat CB1 receptor as well as for mouse and human CB2 receptors were obtained by using membrane preparations from rat brain or HEK293 cells expressing either mCB2 or hCB2 receptors, respectively, and [3H]CP-55,940 as the radioligand, as previously described.83-

84 Results from the competition assays were analyzed using nonlinear regression to determine the IC50 values for the ligand; Ki values were calculated from the IC50 (Prism by GraphPad Software, Inc.). Each experiment was performed in triplicate and Ki values determined from three independent experiments and are expressed as the mean of the three values.

cAMP Assay.76, 89 HEK293 cells stably expressing rCB1 or hCB2 receptors were used for the studies. The cAMP assay was carried out using PerkinElmer’s Lance ultra cAMP kit following the protocol of the manufacturer. Briefly, the assays were carried out in 384- well plates using 1000-1500 cells/well. The cells were harvested with non-enzymatic cell dissociation reagent Versene, washed once with HBSS and resuspended in the stimulation buffer. The various concentrations of the test compound (5 L) in forskolin (2

M final concentration) containing stimulation buffer were added to the plate followed by the cell suspension (5 L). Cells were stimulated for 30 min at room temperature. Eu- cAMP tracer working solution (5 L) and Ulight-anti-cAMP working solution (5 L) were then added to the plate and incubated at room temperature for 60 minutes. The data were collected on a Perkin-Elmer Envision instrument. The EC50 values were determined by non-linear regression analysis using GraphPad Prism software (GraphPad Software, Inc.,

San Diego, CA).

127

Photoaffinity Covalent Labeling. Rat brain membranes (rCB1, Pel-Freez Biologicals,

Rogers, AR) were prepared following the previously described and appropriately modified procedures.76 Membranes from human CB2 receptors (hCB2) expressed in HEK29376 were incubated with the azido ligands in concentrations of 10-fold their Ki values for 30 min at 37 °C in a water bath with gentle agitation and then exposed to UV (254 nm) for 1 min to activate the ligand.99 Unbound excess ligand was washed out twice with 1% BSA in TME. This was followed by an additional washing to remove residual BSA, and the membranes were isolated by centrifugation (Beckman Coulter, JA 20, 17 000 rpm, 10 min, 25 °C). A blank membrane sample was treated in parallel using the same procedure and used as a control.

Electrophilic Covalent Labeling. CB1 and CB2 membranes were incubated with the test ligand in a concentration of 10-fold their Ki values at 37 °C in a water bath with gentle agitation for 1 h and treated as above without the photoirradiation step. Unbound excess ligand was washed out twice with 1% BSA in TME and once with TME alone to remove

BSA, and the membranes were isolated by centrifugation.

Saturation Binding Assay. Protein concentrations were determined by using a Bio-Red

Bradford protein assay kit,100 and saturation binding assays were performed in a 96-well format.80 Membrane pellets were resuspended in TME containing 0.1% BSA. A total of

25 μg of protein was added to each well, and [3H]CP-55,940 was diluted in 0.1%

BSA/TME buffer to yield ligand concentrations ranging from 0.5 to 23.8 nM. Nonspecific

128 binding was determined in the presence of 4 μM unlabeled CP-55,940. The assay plates were incubated at 30 °C with gentle agitation for 1 h. The resultant mixture was then transferred to Unifilter GF/B filter plates, and the bound ligand was separated from unbound using a Packard Filtermate-96 cell harvester (PerkinElmer Packard, Shelton,

CT). Filter plates were washed five times with ice-cold wash buffer (50 mM Tris-base, 5 mM magnesium chloride with 0.5% BSA, pH 7.4). Bound radioactivity was quantitated in a Packard TopCount scintillation counter. Nonspecific binding was subtracted from the total bound radioactivity to calculate the specific binding of [3H]CP-55,940 (measured as pmol/g in saturation curves). Saturation assays were performed in triplicate, and data points were presented as the mean ± SEM. Bmax and Kd values were calculated by nonlinear regression using GraphPad Prism 4.0 (one-site binding analysis equation Y =

BmaxX/(Kd + X), GraphPad Software, San Diego, CA).

Methods for Characterization of in vivo Effects.83, 89

Subjects: Male CD-1 mice (n=6/dose) weighing between 25-30 g (Charles River,

Wilmington, MA) were used. Mice were housed 4/cage in a climate-controlled vivarium with unrestricted access to food and water. Mice were acclimatized to these vivarium conditions for at least one week before any experimental procedure. Analgesia testing took place at same time between 9am and 4 pm. Mice were used once.

Drugs: For tail-flick analgesia testing, compound 2.18a was initially dissolved in 2% dimethyl sulfoxide, 4% Tween-80 and 4% propylene glycol before saline was slowly added just prior to the 10ml/kg i.p. administration. All dug suspensions were prepared just prior to analgesia testing.

129

Analgesia procedure: Tail-flick latency test was performed using a thermostatically controlled 2L water bath commercially available from VWR international. Temperature of water bath for test day was set at 52oC. For testing the analgesic effects of compound

2.18a, tip of the tail (2cm) was immersed in water bath and withdrawal latency was recorded using a commercially available stopwatch (Fischer Scientific) permitting

1 measurement within /100 s. Injections occurred immediately following baseline recording, five recordings occurred after drug administration at 20, 60, 180 and 360 mins respectively to complete the analgesia testing. Animals were acclimatized for three days prior to the test day (4th day), where tails were immersed in water bath held at room temperature. The tail-flick withdrawal latencies are expressed as a percentage of maximum possible effect (%MPE), based on the formula %MPE = [(test latency minus baseline latency) divided by (10 minus baseline latency)] times 100.

Data analysis: Non-linear regression analysis of %MPE data were performed after log-X transformation were performed using Prism Software (v. 5, GraphPad Software, San

Diego, CA) to provide estimates of the independent variable when the co-ordinates of X intersected with Y=50 and their 95% confidence limits (ED50 ±95% CL; regression model: log dose or log time vs. response- variable slope with the top and bottom of the curves constrained to 100 and 0). Results are presented as the mean (±SEM).

130

CHAPTER 3: Controlling deactivation of cannabinergic

ligands

OBJECTIVE AND SPECIFIC AIMS

The diverse feature of mammalian (patho)physiology is related to the interaction of plant derived (-)-Δ9-THC with CB1 and CB2 receptors.101 Modulation the activity of these two receptors is therefore a very exciting pharmacotherapeutic therapeutics for the treatment of various CNS and/or PNS related indications.61, 102 However only few of Δ9-THC based medications have been approved by the USFDA. The main reason for the limited number of such drugs is undesired pharmacokinetic/pharmacodynamic profiles and unpredictable

CNS related side effect such as confusion, dizziness, sleepiness, an exaggerated sense of happiness.103 Medicinal chemists, biologists, and pharmacologists have continuously sought for approches to develop novel THC-based analogues with improved druggability and safety. Among all the diligent and brilliant effort, one of the most outstanding outcome is the development of soft drugs.104-105 A soft drug can be designed by incorporating a controllable deactivated part into the scaffold of a opimized lead compound or drug molecule as their bioactive congeners to improve PK/PD profiles. After the acceptable pharmacological/biological goal has been realized, the drug undergoes a predictable and controllable metapolitical procedure and finally convert to inactive components (Figure

3.1). Drug remifentanil is a representative paradigm.106 This medication is a potent and ultra-short time acting opioid agonist. A limited number of soft cannabinergic drugs have been reported. One of the examples is N-benzyl-benzopyrone esters modified “nabitan” analogues. These compounds show activity on regulating the intraocular pressure.107-108

131

Figure 3.1: Diagrammatic representation of design of soft drugs.

The involvement of our group’s research work with soft drug development led to the combination of the “soft” ligand/drug strategy (based on enzymatic degradation) with

“depot effect”84. Depot effect is determined by molecular lipophilicity as well as its tissue distribution and retention. Compounds with depot effect therefore become “sticky” to the fatty tissue so that it is difficult for them to join blood stream circulation. Log P and PSA values, as two characteristics of compound’s polarity, regulate the depot effect. For example, the more lipophilic molecule has a higher chance to stay in fatty tissue for longer duration than the more lipophilic molecule. This is the main reason the cannabinergic molecules exhibit a very long and unpredictable half-life. The lipophilic nature of a compound is mediated by its polarity. Consequently, increasing the polarity of a certain compound is expected to reduce the depot effect (Figure 3.2).

132

Figure 3.2: Diagrammatic representation of depot effect.

Source: Controlled-Deactivation Cannabinergic Ligands. J. Med. Chem. 2013, 56, 10142−10157.

The main purpose of the controlled deactivation cannabinoids project is to design and synthesize novel fast-onset and fast-offset cannabinergic ligands with short duration of action. The first chemotype of our design was the incorporation of a hydrolysable active ester group with the (−)-Δ9-THC template. The ester group was built up to the side chain of this template. The carboxylic acid metabolite without the side chain scaffold was no longer able to activate the CB1 and CB2 receptors92 (Figure 3.3). Initial SAR study included adding the steric hindered groups (such as: methyl group, gem dimethyl group and cyclo-butyl ring) at the α position of the ester group and extending the side chain to seven carbon length. The goal of this study was to examine the dynamic rate of the hydrolyzation. The selected (−)-Δ9-THC template had been approved nearly equipotent

133 with the plant derived isomer (−)-Δ8-THC. Such design and structure optimization maintained the potency and efficacy of the lead compound while favoring hydrolytic control of the pharmacological half-life.

Figure 3.3: Design and THC-based template optimization of the first generation of controlled deactivation ligands.

Source: Controlled-Deactivation Cannabinergic Ligands. J. Med. Chem. 2013, 56, 10142−10157.

Efforts were also placed on how to minimize the depot effect that discussed above as an alternative in facilitating the short duration of activation. Based on the same prototype, the side chain was further interrogated. The ester group (−C(O)-O−) at the 2′-position was replaced respectively by reverse ester (−O−C(O)−), the corresponding thioester (−C(O)-

S−) and the hydrolytically more stable amide group (−C(O)-NH−) (Figure 3.4). The side chain length that covered a range from four to nine atom-long were made and investigated.

To obtain ligands with different polarity, cyano-, bromo-, and imidazolyl-groups were built up at the terminal of the side chain tail. Under such interrogation, we were seeking for the optimized structure chemotype which enhanced both in vitro and in vivo potency and

134 efficacy meanwhile maintaining the capability of regulation the half-life of the enzymatic- based inactivation (Figure 3.4).84

Figure 3.4: SAR study on the side chain of the first generation of controlled deactivation ligands.

Source: Probing the Carboxyester Side Chain in Controlled Deactivation (−)‑Δ8‑Tetrahydrocannabinols. J. Med.

Chem. 2015, 58, 665−681.

From the initial study above, the result shows that when incorporating polar groups like imidazolyl ring or cyano group on the side chain of the THC based template, the ligand resistance to the esterase was enhanced. This implies that synergistical effect of reducing depot effect and shorten enzymatic deactivation time cannot be achieved, especially when both the soft components and the polar spots are designed in the same side chain.

We are therefore looking for solutions to enhance the polarity so as to reduce the depot effect while maintaining the controllable deactivation feature of THC analogues. In our newly designed second generation controlled-deactivation ligands, the metabolically labile ester group at the 2′-position of the side chain remains unmodified. However, a hydroxyl group as a polar spot were introduced at the C9 or C11 positions of the tricyclic

135 cannabinoid structure (Figure 3.5). The polar hydroxyl group with the stereochemistry at the C9 position is suggested by our earlier work on pharmacophore refinement and lead optimization in the classical cannabinoid templates. Some of the most potent agonists were also treated to rodents and monkeysin in hypothermia testing, analgesia testing and drug discrimination studies for further work on clinical development.89

Figure 3.5: Second generation of controlled deactivation ligands.

Source: Novel C‑Ring-Hydroxy-Substituted Controlled Deactivation Cannabinergic Analogues J. Med. Chem. 2016,

59, 6903−6919.

As a summery, an array of (−)-Δ8-THCs or (−)-Δ9-THCs controllable deactivation analogues have been reported by our group. However, the metabolically labile ester group was only limited into the side chain pharmacophore. In order to develop other creative prototype of controllable cannabinoids, we sought to build labile ester group to

B-ring on the classical cannabinoid template (Figure 3.6). There are several advantages of converting linear ester group to a cyclic lactone ring: (1) Differing from ester group, the 136 metabolization of lactone ring may be facilitated by distinct hydrolase other than esterase.

Paraoxonase is a paradigm. Only human paraoxonase are lactoneases which can efficiently metabolize 1, 5-lactone analogues.109 Thus, investigation of the new chemotype lactone analogues provides a possible approach to address enzymatic species differences.110 (2) As a unimolecular soft drug, only the inactive hydrolytic product will form under specific metabolic condition without any other side products. The risk of side effect and toxicity are therefore notably reduced.111

Figure 3.6: Two generation of controlled deactivation cannabinergic analogues.

According to this concept, my own involvements on this project are the design, synthesis and pharmacologic evaluation of the new chemotype controlled-deactivation cannabinol

(CBN) analogues containing a seven-member lactone ring in the B-ring position (Figure

3.6). CBN is also a plant derived cannabinergic constituent that offers a unique profile of effects and benefits including: pain relief, anti-convulsive and anti-insomnia.112-114 Based

137 on our previous study, the cannabinol lactones are expected to be susceptible to hydrolytic metabolism by plasma hydrolase.115 Our results also show that certain lactone bearing compounds are good binders towards both CB1 and CB2 receptors, while the hydrolysis products are all inactive. It implicates that the disruption of the tricyclic pharmacophore of cannabinol analogues results in the loss of affinity and pharmacological activity on CB receptors. Further SAR studies led to the discovery of the

(S)-isomer in this family of analogues: (7S)-1,10-dihydroxy-7-methyl-3-(2-methyloctan-2- yl)dibenzo[b,d]oxepin-6(7H)-one. This compound exhibits remarkably high in vitro and in vivo potency. The synthetic steps are showed in Scheme 3.1-3.5.

CHEMISTRY

Synthesis of the B-ring lactone 3.9 and its congeners are depicted in Scheme 3.1. The A- ring was built up through three steps: methyl esterification of the carbonic acid (90%); demethylation to obtain the phenolic hydroxyl group (78%), and the protection of the phenolic hydroxyl group through triisopropylsilyl ether (TIPS) in 92% yield. Compound 3.5 was converted to the boronic acid 3.6 by treatment with trimethyl borate in 72% yield. The scaffold of the biphenol analog could be reached by the Suzuki coupling of compound 3.4 and 3.6 under alkaline condition, catalyzed by tetrakis(triphenylphosphine) palladium(0) in 1,2-dimethoxyethane/water (5:1) solution in 70% yield. When we treated the biphenyl ring analog 3.7 with BBr3, three products can be afforded under different conditions. This is the most attractive part of this synthesis. For example, if the reaction was allowed to reach room temperature and treated with excess of boron tribromide, the product was mainly ring closed compound 3.8 with the A-rinng phenolic hydroxyl unprotected in 60% yield. If the reaction occurred in the lower temperature at -78 oC two equivalent boron

138 tribromide, the predominate product was the ring-closed compound 3.10 (15%) with phenolic hydroxyl protected by methyl group and the biphenyl compound 3.12 (15%). The three compounds were successfully separated and purified by the flash column chromatography on silica gel. Compounds 3.8, 3.10, 3.12 were further deprotected by tetrabutylammonium fluoride to provide the final compound 3.9 (AM11216) in 91% yield,

3.11 in 92% yield and 3.13 in 90% yield respectively. Compound 3.14 was obtained directly from reaction of 3.7 with tetrabutylammonium fluoride in 95% yield.

Scheme 3.1: Synthesis of AM11216 and its analogues

139

The stereoselective synthesis of AM11216 analogues were achieved through asymmetric reaction mediated by Evans-type oxazolidinethione auxiliaries (Scheme 3.2 and 3.3). The two isomers AM11225 and AM11227 can be afforded through the similar synthetic step pattern. Thus, synthesis of AM11225 described here served as a paradigm. Starting from

2-(2-bromo-4-methoxyphenyl)acetic acid, the Evens reagent, (S)-4-benzyloxazolidin-2- one was introduced to the template of 3.15 in 85% yield in THF under n-BuLi deprotonation after convert the acetic acid of 3.15 to acyl chloride analog 3.16. Then chiral methyl group was introduced at the benzylic position of 3.17 by the reaction of 3.17 with methyl iodide in THF upon the deprotonation by sodium bis(trimethylsilyl)amide.

Benzyloxazolidin group was then removed by treating 3.18 (81%) with lithium hydroxide in THF/H2O (5:1) solution and provided 3.19 in 89% yield. Methyl ester 3.20 (90%) which formed through methyl-esterification with (trimethylsilyl) diazomethane solution was treated with boron tribromide to give the corresponding phenol hydroxyl analog 3.21 in

79%, and the resulting 3.21 was protected by TIPS triflate to give 3.22 in 88% yield. The final compound AM11225 was then prepared stepwise from 3.22 through three reactions:

Suzuki coupling (60%), BBr3 demethylation (62%) and n-Bu4NF deprotection (94%). The other enantiomer AM11227 was obtained under the same procedure using (R)-4- benzyloxazolidin-2-one as the Evans-type oxazolidinethione auxiliaries (Scheme 3.3).

Scheme 3.2: Synthesis of AM11225

140

Scheme 3.3: Synthesis of AM11227

141

Synthesis of the gem dimethyl substitution of B-ring lactone 3.46 is depicted in Scheme

3.4. It represents a greater challenge to us than the non-substitution and the mono-methyl substitution analogs. This challenge was imposed by the difficulties of introducing steric hindrance. Our strategy was to add the two methyl groups stepwise after the Suzuki coupling because the steric hindrance groups also affect the efficiency of the coupling reaction. Typically, the exposure of 3.37 to NaCN in DMSO solvent produced the cyano- analog 3.38 (85%), which was then converted to 3.39 in 78% yield by BBr3 demethylation.

The phenolic hydroxyl group was immediately protected by TIPS as 3.40 in 88% yield as described earlier. The resulting compound 3.40 was directly coupled with the boronic acid

3.6 catalyzed by Pd(PPh3)4 under alkaline condition in mix solvent DME/H2O (5:1) to

142 afford compound 3.41 (70%). Treatment of 3.41 with excess sodium hydride and methyl iodide three times stepwise finally gave 3.42 in 35% yield. The gem-dimethyl substituted cyano analog was hydrolyzed by sodium hydroxide in the solution of n-BuOH/water (3:1) to give 3.43 in 73% yield. Acetic acid 3.43 was exposed to BBr3 in dichloromethane to produce demethylated compound 3.44 (76%). Ring cyclization of 3.44 took place under the treatment of p-Toluenesulfonic acid to give the three-ring analogue 3.45 (61%). The final compound 3.46 was obtained by the deprotection of 3.45 through the treatment of tera-n-butylammonium fluoride in high yield (96%).

Scheme 3.4: Synthesis of gem dimethyl substituted B-ring lactone analog

143

Synthesis of the gem dimethyl substitution of B-ring lactone 3.46 was depicted in Scheme

3.5. The synthesis method of resorcinol-like building block 3.57 has been described in chapter 2. Thus, deprotonation of 3.53 with sodium hydride followed by geminal dimethylation using methyl iodide gave nitrile 3.54 (95% yield). Reduction of the cyano group in 3.54 with diisobutylalaminum hydride led to the aldehydes 3.55 (87%) which upon Wittig reaction with the ylide derived from (5-phenoxypentyl)triphenylphosphonium bromide by KHMDS, afforded exclusively the Z olefin 3.56 (JH2'-H3' = 11.1 Hz) in excellent yields (96%). In turn, the required phosphonium salts were synthesized from commercially available 5-phenoxypentylbromide and triphenylphosphine in refluxing benzene. Catalytic hydrogenation of the double bond in 3.56 proceeded in excellent yield

(89 %). This was followed by exposure of the intermediate alkanes 3.57 to trimethyl borate, which builds the boronic acid group into the scaffold to give 3.58 (85-98% yields).

Coupling 3.58 with 3.52 gave the Suzuki coupling product 3.59 in 68% yield. This was followed by exposure of the 3.59 to boron tribromide to cleave all three ether groups, introduce the C7' bromo group at the terminal of side chain and simultaneously cyclize the lactone ring in (68% yields). Subsequent treatment 3.60 with tetra-butylammonium fluoride gave the key intermediate 3.61 (86%). Treatment of 3.61 with sodium cyanide resulted in cyano-functionalized compound 3.62 in 81% yield while treatment of 3.61 with silver nitrate gave nitrate ester compound 3.63 in 81% yield.

Scheme 3.5: Synthesis of terminal functionalized B-ring lactone analogs

144

RESULTS AND DISCUSSION

CB receptor affinities

Based on our previous study, the CBN scaffold have been optimized by a seven-atom long side chain with 1′-gem dimethyl substitution. Thus, the SAR study here mainly

145 focused on the terminal of the side chain and the α-substitution of the lactone ring (see

Figure 3.7). Modification of the terminal of the side chain with specific functional groups, such as azido group or nitrate ester, was expected to enhance both in vitro and in vivo potency and efficacy, while α-substitution of the lactone ring might assist in increasing the selectivity toward specific CB receptors (Figure 3.7).

The abilities of the AM compounds (list in Table 3.1) to displace the radiolabeled CB1/CB2 agonist CP-55,940 from membranes prepared from rat brain (source of CB1) and HEK

293 cells expressing human CB2 were determined as described earlier. Inhibition constant values (Ki) from the respective competition binding curves are listed in Table 3.1.

These compounds include: (1) bi-phenol analogues; (2) CBN lactone analogues; (3) metabolic acid products. In agreement with our rational design, the hydrolytic metabolites

AM11221, AM11251 and AM11261 have no significant affinities for CB1 and CB2 receptors, minimizing the possibility of undesirable cannabinoid receptor related side effects. The bi-phenol analogues which usually present non-specific binding towards an array of proteins, however, also shows no significant affinities for CB1 and CB2 receptors.

The Ki value of AM11215 and AM11219 imply that modification of either of the two phenol hydroxyl groups would lead to the loss of CB receptors binding affinity. On the other hand, all the CBN analogues with unmodified phenol hydroxyl group exhibit desirable binding affinity.

146

Figure 3.7: SAR studies of B-ring lactone template.

Table 3.1: Binding affinities (Ki) of controlled deactivation CBN analogs.

AM Sturcture cLogP tPSA Number rCB1 hCB2

AM11218 372 ± 20.5 145 ±12.1 6.72 46.53

AM11220 124 ± 5.6 47.7 ± 3.4 3.82 72.83

AM11279 135 ± 6.8 84.2 ± 9.1 3.69 72.83

AM11222 >1000 >1000

AM11214 >1000 >1000

AM11215 234 ± 14.7 >1000

147

AM11216 30.5 ± 3.7 22.1 ± 2.3 4.22 72.83

AM11219 521.3 ± 43.1 81.5 ±12.2 4.75 72.83

AM11225 5.3 ± 1.2 0.6 ± 0.02 5.28 72.83

AM11227 32.2 ± 6.5 15.7 ± 2.5 5.04 72.83

AM11259 1.7 ± 0.3 3.3 ± 0.4 5.04 72.83

AM11269 NT NT 4.08 72.83

AM11271 NT NT 2.65 96.62

AM11272 NT NT 3.18 85.30

AM11221 660 ± 30.8 >1000 4.09 72.83

AM11251 >1000 >1000 3.56 72.81

148

AM11261 >1000 >1000 3.21 83.83

According to the binding affinities of the three compounds, AM11216, AM11225 and

AM11227, introduction of (R)-mono-methyl substituents at the α position of the lactone ring (AM11225) induces incredible enhancement of binding affinities toward CB receptors.

Especially for CB2 receptors, AM11225 exhibits almost 40-fold higher affinity than

AM11216. However, introduction of (S)-mono-methyl substituents at the α position of the lactone ring (AM11227) seems to help maintain the same Ki value as AM11216 (without any substitution) does. Introduction of gem di-methyl substituents at the α position of the lactone ring (AM11259) also leads to comparatively higher binding affinities toward CB receptors than AM11216, 20-fold and 8-fold enhancement in CB1 and CB2 receptor affinities, respectively.

Functional characterization

AM11216, AM11225 and AM11227 were selected for further functional characterization.

Experiments were carried out by measuring changes in forskolin-stimulated cAMP, as detailed earlier. Our testing results (Table 3.2) show that all three compounds, however in different degrees, decreased the levels of cAMP. It indicates that within this signaling mechanism these compounds behaves as potent agonists at both the CB1 and CB2 receptors. Of the three lactone analogues reported here, the most interesting phenomenon is that the R-isomer, AM11225 predominantly enhanced the potency while

149 the S-isomer incredibly reduced potency by compared with their respective non- substitution analog AM11216.

Table 3.2: Functional evaluation of CBN analogs.

AM rCB1 hCB2 Structure number EC50 (nM) Emax (%) EC50 (nM) Emax (%)

AM11216 57.2 ± 9.3 44 155.7±10.1 82

AM11225 4.6 ± 0.5 59 1.2 ± 0.2 69

AM11227 376 ± 32.1 89 NT NT

In vitro plasma stability studies

AM11216, and AM11225 were also evaluated for their in vitro plasma stability. They were diluted in various plasma solutions: mouse plasma, rat plasma, and human plasma. After incubated in 37 °C, 100 rpm., samples were taken at various time points and was further diluted and centrifuged to get rid of the proteins. The resulting supernatant was analyzed by HPLC. Agreed with our logical design, AM11216 and AM11225 were hydrolyzed by esterases in rat and mouse plasma to form the metabolite AM11221 and AM11251 respectively while they remain stable in various buffers (Figure 3.8 and Figure 3.9). The mouse and rat plasma esterase stabilities of the two compounds correlates well with our

150 previous studies. The non-substitution analog AM11216 is less stable than the (αR)- mono-methyl substitution analog AM11225 in mouse and rat plasma (Table 3.3).

However, the most exciting discovery is that AM11225 is hydrolyzed in human plasma

(Figure 3.9). This is the first time we observed that the ester modified cannabinergic compounds metabolite in human plasma. Carboxyesterases are typically found in mice and rats, but not in human plasma. Esterase activity is also higher in the blood of small rodents than in large animals and humans. All of our first generation of linear carboxy-Δ8-

THC analogues are therefore stable in human plasma. Esterases are a heterogeneous group of enzymes that are classified broadly as cholinesterases (including acetylcholinesterases and butyrylcholinesterases), paraoxonases, and carboxyesterases.

Paraoxonases 2, other than carboxyesterases, have been found in human plasma, specifically metabolize 1,5-lactone ring analogues. Thus, our novel prototype controlled- deactivation cannabinergic analogue bearing a lactone ring is able to be deactivated in human plasma. Therefore, investigating the stability of these analogues provides an alternative to address enzymatic species differences.

Table 3.3: Metabolic stabilities (t1/2) of controlled deactivation analogs.

Plasma Stability (t1/2) AM Structure min Number Mouse Rat Human

AM11216 1.7 1.7 52.2

151

AM11225 19.6 9.2 15.0

AM-11216 stability 125 Human Plasma 100 Mouse Plasma Rat Plasma 75 Buffer Gastric Juice 50 Acetonitrile

25 Percent Percent Remaining

0 0 10 20 30 40 Time (minutes)

Figure 3.8: Plasma stability test for AM11216.

AM-11225 Stability at 210 nm 125 Human Plasma 100 Mouse Plasma Rat Plasma 75 Buffer Gastric Juice 50 Acetonitrile

25 Percent Percent Remaining

0 0 10 20 30 40 Time (minutes)

Figure 3.9: Plasma stability test for AM11225.

In vivo behavioral characterization: Hypothermia Testing

152

The hypothermic effects of AM11216 and its αR-methyl substitution analog AM11225 were compared in mice. Rectal temperature was measured in isolated mice over a 360 minute period following drug injection (detailed procedures are given in Experimental

Section). In agreement with our in vitro functional characterization, AM11216 and

AM11225 both decreased core body temperature in a dose-dependent manner, reducing body temperature by 7.9 and 9.2 °C respectively at the highest doses tested (Figure 3.10 and Figure 3.11). Both compounds had relatively fast onsets of drug effect. With the treatment of AM11216, significant temperature decreases typically occurred within 20 minutes after the injections, and peak effects were not obtained until 60 minutes after injections. The significant recovery toward baseline were achieved within the 6 h test period (Figure 3.10). Significant decreases with the treatment of AM11225 in body temperature also occurred within 20 min after injection with significant recovery toward baseline within the 6 h test period. However the peak effects were not obtained until 180 min after the injections (Figure 3.11). The comparison of the duration of significant body temperature decreases triggered by these two compounds indicates that the design of steric and isomeric substitution at the α position of lactone ring maintains their ability of control over the pharmacological half-life.

153

AM11216 Hypothermia IP

-2

-4

-6

Temp (C)

-8

-10

-12 0 20 60 180 360 Time (min)

5.6mg/kg 10.0mg/kg 30.0 mg/kg

Figure 3.10: Hypothermic effects of selected doses of AM11216.

154

AM11225 Hypothermia IP

-2

-4

-6

Temp (C)

-8

-10

-12 0 20 60 180 360 Time (min)

3.0 mg/kg 5.6mg/kg 18.0 mg/kg

Figure 3.11: Hypothermic effects of selected doses of AM11225.

In vivo behavioral characterization: Analgesia Testing

To confirm the observed pharmacokinetic differences between AM11216 and its methyl- substituted congener AM11225, we used the CB1 receptor characteristic analgesia assay.

Antinociception was measured using the tail flick procedure over a 6-hour period following drug injection. Prior to drug administration, the average baseline tail-flick latency was 1.6

±0.1s. Doses of 10.0-30.0 mg/kg AM11216 had significant antinociceptive effects (Figure

3.12) with a mean (± 95% CL) ED50 value of 28.64 mg/kg (23.61 to 34.76) at 180 min

(Table 3.4). Doses of 5.6-18 mg/kg AM11225 had equal significant antinociceptive effects

(Figure 3.13) which led to a lower ED50 value of 12.89 mg/kg (10.90 to 15.23) with a mean

(± 95% CL) at 180 min (Table 3.4).

155

AM11216 Analgesia IP

100

%MPE

20 60 180 360 Time (min)

10.0 mg/kg 18.0 mg/kg 30.0 mg/kg

Figure 3.12: Tail-flick latencies in a hot water bath after administration of three doses of compound AM11216.

156

AM11225 Analgesia IP

100

%MPE 40

0 20 60 180 360 Time (min)

5.6 mg/kg 10.0 mg/kg 18.0 mg/kg

Figure 3.13: Tail-flick latencies in a hot water bath after administration of three doses of compound AM11225.

Table 3.4: ED50 for the analgesic effects of AM11216 and AM11225 in mice.

AM ED50 Value (95 %) Structure Number mg/kg mg/kg

AM11216 28.64 (23.61 to 34.76)

AM11225 12.89 (10.90 to 15.23)

CONCLUSIONS

157

An array of bi-phenol ring canabinergic analogues, CBN lactone built-in analogues and their metabolites have successfully been synthesized and evaluated in terms of in vitro binding affinities, functional characterization, plasma stability, and in vivo behavioral characterization. Collectively, our design of the new chemotype controlled-deactivation cannbinergic lactone congeners maintains their nature of control over the pharmacological half-life. In addition, for the first time we observed the hydrolyzation of ester based cannabinoid soft drug in human plasma.

EXPERIMENTAL SECTION

1 recorded in CDCl3, unless otherwise stated, on a Bruker Ultra Shield 400 WB plus ( H at

400 MHz, 13C at 100 MHz) or on a Varian INOVA-500 (1H at 500 MHz, 13C at 125 MHz) spectrometers and chemical shifts are reported in units of  relative to internal TMS.

Multiplicities are indicated as br (broadened), s (singlet), d (doublet), t (triplet), q (quartet),

158 m (multiplet) and coupling constants (J) are reported in hertz (Hz). Low and high- resolution mass spectra were performed in School of Chemical Sciences, University of

Illinois at Urbana-Champaign. Mass spectral data are reported in the form of m/z (intensity relative to base = 100). LC/MS analysis was performed by using a Waters MicroMass ZQ system [electrospray-ionization (ESI) with Waters-2525 binary gradient module coupled to a Photodiode Array Detector (Waters-2996) and ELS detector (Waters-2424) using a

XTerra MS C18, 5 µm, 4.6 mm x 50 mm column and acetonitrile/water. methyl 2-(2-bromo-4-methoxyphenyl)acetate (3.2)65. 2-(2-bromo-4-methoxyphenyl) acetic acid (10.0 g, 40.8 mmol) was dissolved and stirred in wet Et2O/MeOH (2:1 mixture,

680.1 mL) at room temperature under an argon atmosphere, (diazomethyl)trimethylsilane was added dropwise (81.6 mL, 2M in hexane) to the stirring solution. The color of the solution turned to light yellow upon the addition. After stirring for 2 h, the reaction mixture was quenched with saturated aqueous NaHCO3 solution and then diluted by Et2O. The organic layer was separated and the aqueous layer was extracted with diethyl ether. The combined organic layer was washed with water and brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (25% diethyl ether in hexane) gave the title compound (8.2 g, 78% yield) as a colorless oil. 1H

NMR (500 MHz, CDCl3) δ 7.19 (d, J = 9.0 Hz, 1H, ArH), 7.12 (d, J = 2.5 Hz, 1 H, ArH),

6.83 (dd, J = 9.0 Hz, J = 2.5 Hz, 1 H, ArH ), 3.79 (s, 3 H, -OCH3), 3.73 (s, 2 H, -CH2C(O)-),

+ 3.71 (s, 3 H, CH3C(O)O-); mass spectrum (ESI) m/z (relative intensity) 258 (M +H, 100).

methyl 2-(2-bromo-4-hydroxyphenyl)acetate (3.3)81. To a stirred solution of 3.2 (8.2 g,

o 31.8 mmol) in dry CH2Cl2 (212.2 mL), at -78 C, under an argon atmosphere, was added

159 boron tribromide (47.7 mL, 1M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 6.1 g of 3.3 as a white foam in 78% yield. IR (neat) 3343 (br,

OH), 2931, 2834, 1601, 1412, 1345, 1235, 1146, 993, 750 cm-1; 1H NMR (500 MHz,

CDCl3) δ 7.05 (d, J = 8.0 Hz, 1H, ArH), 6.93 (d, J = 2.5 Hz, 1 H, ArH), 6.63 (dd, J = 8.0

Hz, J = 2.5 Hz, 1 H, ArH ), 3.76 (s, 3 H, CH3C(O)O-), 3.73 (s, 2 H, -CH2C(O)-); Mass spectrum (ESI) m/z (relative intensity) 244 (M++H, 100) methyl 2-(2-bromo-4-((triisopropylsilyl)oxy)phenyl)acetate (3.4)65. To a solution of

3.3 (6.0 g, 24.5 mmol) in anhydrous CH2Cl2 (40.8 mL) under an argon atmosphere were added sequentially, 2,6-lutidine (11.8 g, 110.25 mmol), and triisopropylsilyl trifluoromethanesulfonate (22.5 g, 73.5 mmol) at 0 oC. Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature for 2 h and then quenched by the addition of NH4Cl and extracted with diethyl ether. The organic phase was dried (MgSO4) and evaporated under reduced pressure.

Purification by flash column chromatography on silica gel (10%-20% diethyl ether in hexane) afforded 9.6 g (98% yield) of 3.4 as a colorless oil. IR (neat) 2946, 1743, 1600,

-1 1 1490, 1293, 1273, 1250, 1160, 937, 882, 683 cm ; H NMR (500 MHz, CDCl3) δ 7.12 (d,

J = 9.0 Hz, 1H, ArH), 7.11 (d, J = 2.5 Hz, 1 H, ArH), 6.79 (dd, J = 9.0 Hz, J = 2.5 Hz, 1 H,

160

ArH ), 3.72 (s, 3 H, CH3C(O)O-), 3.71 (s, 2 H, -CH2C(O)-), 1.28-1.23 (m, 3 H, -

13 Si(CH(Me)2)3, 1.09 (d, J = 7.0 Hz, 18 H, -Si(CH(Me)2)3); C NMR (100 MHz CDCl3) δ

190.6 (>C=O), 157.4 (ArC), 132.8 (ArC),128.7 (ArC), 125.6 (ArC), 124.9 (ArC), 122.1

(ArC), 51.9, 38.6, 18.3, 12.7.

(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)boronic acid (3.6)65 To a solution of 1,3- dimethoxy-5-(2-methyloctan-2-yl) benzene (10.0 g, 37.8 mmol) in anhydrous THF (110 mL) under an argon atmosphere was added n-BuLi (21.2 ml, 53.0 mmol, 2.5 M in hexane) at -78 oC. The mixture was stirred for 45 min followed by warmed to 10 oC and stirred for

1.5h, then cooled again to -78 oC and stirred for 30 min. Trimethyl borate was added at this temperature. Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature overnight and then quenched by the addition of HCl until the pH value reach to around 3~4 at 0 oC. The mixture was then extracted with dichloromethane. The organic phase was dried (MgSO4) and evaporated under reduced pressure. Purification by flash column chromatography on silica gel (10%-20% diethyl ether in hexane) afforded 8.7 g (75% yield) of 3.6 as a

1 colorless oil. H NMR (500 MHz, CDCl3) δ 7.19 (s, 2H, OH), 6.58 (s, 2 H, ArH), 3.92 (s, 6

H, -OCH3), 1.62-1.57 (m, 2 H, 2′-H), 1.29 (s, 6 H, -C(CH3)2-), 1.27-1.18 (m, 6 H, -CH2- of the side chain), 1.08-1.02 (m, 2H, -CH2- of the side chain), 0.85 (t, J = 6.5 Hz, 7′-H); Mass spectrum (ESI) m/z (relative intensity) 309.2 (M++H). HPLC (4.6 mm × 250 mm, Supelco

Discovery column, acetonitrile/water) showed purity of 98% and retention time of 5.5 min for the title compound. methyl 2-(2',6'-dimethoxy-4'-(2-methyloctan-2-yl)-5-((triisopropylsilyl)oxy)-[1,1'- biphenyl]-2-yl)acetate (3.7)65 To a solution of the mixture of 3.4 (9.2 g, 23.0 mmol) and

161

3.6 (8.5 g, 27.6 mmol) in DME/H2O (5:1 mixture, 328.6 mL) in microwave vessel under an argon atmosphere were added sequentially CsCO3 (30.0 g, 92.0 mmol) and Pd(PPh3)4

(5.3 g, 4.6 mmol). After the addition, the vessel was sealed. The resulting mixture was irradiated using the open vessel mode at 110 oC for 15 min. The reaction mixture was quenched with water. The organic phase was dried (MgSO4) and evaporated under reduced pressure. Purification by flash column chromatography on silica gel (10%-25% diethyl ether in hexane) afforded 9.1 g (68% yield) of 3.7 as a colorless oil. IR (neat) 2987,

2810, 1698(s, >C=O), 1547, 1512, 1389, 1330, 1212, 1086, 1023, 1004, 932, 815 cm-1;

1 H NMR (500 MHz,CDCl3) δ 7.21 (d, J = 8.5 Hz, 1H, ArH), 6.82 ( dd, J = 8.5 Hz, J = 2.5

Hz, 1H, ArH), 6.73 (d, J = 2.5 Hz, 1H, ArH), 6.66, (s, 2H, ArH), 3.65 (s, 6H, -OCH3), 3.54

(s, 3H, CH3OC(O)-), 3.34 (s, 2H, -CH2C(O)-), 1.65-1.59 (m, 2H, 2′-H), 1.33 (s, 6H, -

C(CH3)-), 1.30-1.20 (m, 11H, 8H of -CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.1 (d,

13 J = 7 Hz, 18H, -Si(CH(Me)2)3), 0.86(t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ

172.6 (>C=O), 157.2 (ArC), 154.3 (ArC), 151.5 (ArC), 135.5 (ArC), 130.5 (ArC), 126.3

(ArC), 123.1 (ArC), 118.8 (ArC), 115.2 (ArC), 55.6, 51.5, 44.6, 38.3, 38.0, 31.8, 30.0, 29.0,

24.6, 22.6, 17.9, 14.0, 12.7. Mass spectrum (ESI) m/z (relative intensity) 585 (M++H).

HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98% and retention time of 7.5 min for the title compound.

1-hydroxy-3-(2-methyloctan-2-yl)-10-((triisopropylsilyl)oxy)dibenzo[b,d]oxepin-

65 6(7H)-one (3.8) . To a stirred solution of 3.7 (5.0 g, 8.5 mmol) in dry CH2Cl2 (57.0 mL), at -78 oC, under an argon atmosphere, was added boron tribromide (36.6 mL, 36.6 mmol,

1 M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of

162 the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 2.7 g of 3.8 as a white foam in 60% yield. IR (neat) 3343 (br, OH), 2931, 2834, 1601, 1412,

-1 1 1345, 1235, 1146, 993, 750 cm ; H NMR (500 MHz,CDCl3) δ 7.29 (d, J = 8.0 Hz, 1H,

ArH), 7.25 ( d, J = 2.5, 1H, ArH), 6.94 (dd, J = 8.0 Hz, J = 2.5 Hz, 1H, ArH), 6.86, (dd, J =

8.5 Hz, J = 1.5 Hz 2H, ArH), 5.62 (s, 1 H, -OH) 3.57 (dd, J = 43.5 Hz, J = 13 Hz, 2H, -

CH2-C(O)-), 1.62-1.55 (m, 2H, 2′-H), 1.33 (s, 6H, -C(CH3)-), 1.30-1.20 (m, 11H, 8H of -

CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.1 (d, J = 7 Hz, 18H, -Si(CH(Me)2)3), 0.86

13 (t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 169.9 (>C=O), 156.1 (ArC), 153.0 (ArC),

152.4 (ArC), 150.5(ArC), 131.1 (ArC), 130.5 (ArC), 125.2 (ArC), 120.7 (ArC), 119.1 (ArC),

113.8 (ArC), 111.0 (ArC), 110.9 (ArC), 44.2, 39.2, 37.9, 31.7, 29.9, 28.8, 28.5, 24.6, 22.6,

17.9, 14.0, 12.6. Mass spectrum (ESI) m/z (relative intensity) 525 (M++H); found, HPLC

(4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 6.9 min for the title compound.

1,10-dihydroxy-3-(2-methyloctan-2-yl)dibenzo[b,d]oxepin-6(7H)-one (3.9)65 To a solution of 3.8 (2.6 g, 5.0 mmol) in anhydrous THF (148.6 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (5.9 mL, 5.9 mmol, 1M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (20%-50%

163 ethyl acetate in hexane) gave 3.9 (1.7 g, 91% yield) as a white solid. mp = 68-70 oC. IR

(neat) 3320 (br, OH), 2932, 2863, 1622, 1573, 1460, 1414, 1331, 1274, 1138, 1038, 750

-1 1 cm ; H NMR (500 MHz,CDCl3) δ 7.31 (d, J = 8.0 Hz, 1H, ArH), 7.23 ( d, J = 2.5, 1H,

ArH), 6.89 (dd, J = 8.0 Hz, J = 2.5 Hz, 1H, ArH), 6.86, (dd, J = 8.5 Hz, J = 1.5 Hz 2H,

ArH), 5.58 (s, 1 H, -OH), 5.30 (s, 1 H, -OH), 3.58 (dd, J = 43.5 Hz, J = 13 Hz, 2H, -CH2-

C(O)-), 1.62-1.55 (m, 2H, 2′-H), 1.33 (s, 6H, -C(CH3)-), 1.30-1.20 (m, 8H, -CH2- of the

13 side chain), 0.86 (t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 170.5 (>C=O), 156.1

(ArC), 153.2 (ArC), 152.5 (ArC), 150.6 (ArC), 131.6 (ArC), 129.8 (ArC), 123.6 (ArC), 116.1

(ArC), 115.9 (ArC), 114.2 (ArC), 110.9 (ArC), 110.5 (ArC), 55.6, 44.2, 39.1, 37.8, 31.7,

29.9, 28.8, 28.5, 24.6, 22.6. Mass spectrum (ESI) m/z (relative intensity) 369 (M++H); found, HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.3 min for the title compound.

1-methoxy-3-(2-methyloctan-2-yl)-10-((triisopropylsilyl)oxy)dibenzo[b,d]oxepin-

65 6(7H)-one (3.10) . To a stirred solution of 3.7 (1.0 g, 1.7 mmol) in dry CH2Cl2 (11.4 mL), at -78 oC, under an argon atmosphere, was added boron tribromide (3.4 mL, 3.4 mmol, 1

M in CH2Cl2). Following the addition, the reaction mixture was stirred for 1h at the same temperature. The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 137 mg of 3.10 as a white foam in 15% yield. IR (neat) 2900, 2645, 1590, 1367, 1303, 1225,

-1 1 1095, 956, 690 cm ; H NMR (500 MHz,CDCl3) δ 7.35 (d, J = 2.5 Hz, 1H, ArH), 7.20 ( d,

164

J = 8.5, 1H, ArH), 6.90 (dd, J = 8.0 Hz, J = 2.5 Hz, 1H, ArH), 6.87, (d, J = 1.5 Hz, 1H, ArH)

6.82, (d, J = 1.5 Hz, 1H, ArH), 3.83 (s, 3H, -CH3) 3.53 (dd, J = 43.5 Hz, J = 13 Hz, 2H, -

CH2-C(O)-), 1.65-1.58 (m, 2H, 2′-H), 1.33 (s, 6H, -C(CH3)-), 1.30-1.20 (m, 11H, 8H of -

CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.1 (d, J = 7 Hz, 18H, -Si(CH(Me)2)3), 0.85

13 (t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 170.2 (>C=O), 156.5 (ArC), 154.9 (ArC),

152.18 (ArC),150.7 (ArC), 131.5 (ArC), 130.5 (ArC), 124.4 (ArC), 122.1 (ArC), 120.5 (ArC),

116.1 (ArC), 111.3 (ArC), 106.0 (ArC), 60.4, 55.8, 44.3, 39.3, 38.2, 31.7, 29.9, 28.9, 28.6,

24.6, 22.6, 21.0, 17.9, 17.8, 15.2, 14.2, 14.0, 12.6. Mass spectrum (ESI) m/z (relative intensity) 539 (M++H). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 7.45 min for the title compound.

1,10-dihydroxy-3-(2-methyloctan-2-yl)dibenzo[b,d]oxepin-6(7H)-one (3.11)65. To a solution of 3.10 (120 mg, 0.22 mmol) in anhydrous THF (5.6 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (0.26 mL, 0.26 mmol, 1M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel

(20%-50% ethyl acetate in hexane) gave 3.11 (77.4 mg, 92% yield) as a white solid. mp

= 68-70 oC. IR (neat) 3315 (br, OH), 2984, 2816, 1604, 1553, 1379, 1331, 1252, 1107,

-1 1 1021, 783 cm ; H NMR (500 MHz,CDCl3) δ 7.31 (d, J = 2.5 Hz, 1H, ArH), 7.22 ( d, J =

8.5, 1H, ArH), 6.88 (s, 1H, ArH), 6.85 (dd, J = 8.0 Hz, J = 2.5 Hz, 1H, ArH), 6.83, (s, 1H,

ArH), 3.86 (s, 3H, -CH3) 3.53 (dd, J = 43.5 Hz, J = 13 Hz, 2H, -CH2-C(O)-), 1.65-1.58 (m,

2H, 2′-H), 1.33 (s, 6H, -C(CH3)-), 1.30-1.20 (m, 8H, -CH2- of the side chain), 0.85 (t, J =

165

13 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 170.1 (>C=O), 156.5 (ArC), 154.3 (ArC), 152.5

(ArC), 150.8 (ArC), 132.0 (ArC), 129.1 (ArC), 124.3 (ArC), 117.7 (ArC), 115.8 (ArC), 115.7

(ArC), 111.4 (ArC), 106.1 (ArC), 56.0, 44.3, 39.2, 38.2, 31.7, 29.9, 28.8, 28.6, 24.6, 22.6,

14.0. Mass spectrum (ESI) m/z (relative intensity) 383 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.6 min for the title compound. methyl 2-(2',6'-dihydroxy-4'-(2-methyloctan-2-yl)-5-((triisopropylsilyl)oxy)-[1,1'- biphenyl]-2-yl)acetate (3.12)65. To a stirred solution of 3.7 (1.0 g, 1.7 mmol) in dry

o CH2Cl2 (11.4 mL), at -78 C, under an argon atmosphere, was added boron tribromide

(3.4 mL, 3.4 mmol, 1 M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to 0 oC and the stirring was continued at that temperature until completion of the reaction (2 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine.

The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 141.8 mg of 3.12 as a white foam in 15% yield. IR (neat) 3313 (br, OH),

-1 1 2897, 2806, 1578, 1432, 1312, 1248, 1106, 983, 721 cm ; H NMR (500 MHz,CDCl3) δ

7.25 (d, J = 8.5 Hz, 1H, ArH), 6.96 ( dd, J = 8.5 Hz, J = 2.5 Hz, 1H, ArH), 6.85 (d, J = 2.5

Hz, 1H, ArH), 6.57, (s, 2H, ArH), 4.85 (s, 1H, -OH), 3.59 (s, 3H, CH3OC(O)-), 3.47 (s, 2H,

-CH2C(O)-), 1.65-1.59 (m, 2H, 2′-H), 1.33 (s, 6H, -C(CH3)-), 1.30-1.20 (m, 11H, 8H of -

CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.1 (d, J = 7 Hz, 18H, -Si(CH(Me)2)3), 0.86(t,

13 J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 173.2 (>C=O), 156.4 (ArC), 153.0 (ArC),

166

152.8 (ArC), 132.4 (ArC), 132.0 (ArC), 128.1 (ArC), 123.5 (ArC), 121.3 (ArC), 111.5 (ArC),

106.0 (ArC), 52.2, 44.4, 37.8, 37.7, 31.8, 30.0, 28.7, 24.6, 22.6, 17.9, 14.0, 12.6. Mass spectrum (ESI) m/z (relative intensity) 557 (M++H, 100). HPLC (4.6 mm × 250 mm,

Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.5 min for the title compound. methyl 2-(2',5,6'-trihydroxy-4'-(2-methyloctan-2-yl)-[1,1'-biphenyl]-2-yl)acetate

(3.13)65. To a solution of 3.12 (130 mg, 0.23 mmol) in anhydrous THF (5.8 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (0.27 mL, 0.27 mmol, 1M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution.

Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (20%-50% ethyl acetate in hexane) gave 3.13 (88.4 mg, 96% yield) as a white solid. mp = 68-70 oC. IR (neat) 3296 (br, OH), 2957, 2831, 1640, 1521, 1443, 1397,

-1 1 1302, 1263, 1203, 1007, 776 cm ; H NMR (500 MHz,CDCl3) δ 7.22 (d, J = 8.0 Hz, 1H,

ArH), 6.82 ( dd, J = 8.0 Hz, J = 2.5 Hz, 1H, ArH), 6.75 (d, J = 2.5 Hz, 1H, ArH), 6.55, (s,

2H, ArH), 5.98 (s, 1H, -OH), 5.11 (s, 1H, -OH), 3.60 (s, 3H, CH3OC(O)-), 3.49 (s, 2H, -

CH2C(O)-), 1.65-1.59 (m, 2H, 2′-H), 1.33 (s, 6H, -C(CH3)-), 1.30-1.20 (m, 8H, -CH2- of the

13 side chain), 0.86(t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 173.6 (>C=O), 155.7

(ArC), 153.0 (ArC), 152.9 (ArC), 132.6 (ArC), 132.5(ArC), 127.6 (ArC), 118.8 (ArC), 116.9

(ArC), 111.3 (ArC), 106.2 (ArC), 52.3, 44.4, 37.8, 37.7, 31.8, 30.0, 28.7, 24.6, 22.6, 14.0.

Mass spectrum (ESI) m/z (relative intensity) 401 (M++H, 100). HPLC (4.6 mm × 250 mm,

Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 4.4 min for the title compound.

167

1,10-dihydroxy-3-(2-methyloctan-2-yl)dibenzo[b,d]oxepin-6(7H)-one (3.14)65. To a solution of 3.7 (500 mg, 0.85 mmol) in anhydrous THF (21.3 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (1.0 mL, 1.0 mmol, 1M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel

(20%-50% ethyl acetate in hexane) gave 3.14 (346 mg, 95% yield) as a white solid. mp

= 68-70 oC. IR (neat) 3334 (br, OH), 2945, 2809, 1627, 1590, 1436, 1399, 1301, 1215,

-1 1 1087, 1014, 693 cm ; H NMR (500 MHz,CDCl3) δ 7.22 (d, J = 8.5 Hz, 1H, ArH), 6.75

( dd, J = 8.5 Hz, J = 2.5 Hz, 1H, ArH), 6.66 (d, J = 2.5 Hz, 1H, ArH), 6.56, (s, 2H, ArH),

3.68 (s, 6H, -OCH3), 3.54 (s, 3H, CH3OC(O)-), 3.35 (s, 2H, -CH2C(O)-), 1.65-1.59 (m, 2H,

2′-H), 1.33 (s, 6H, -C(CH3)-), 1.30-1.20 (m, 8H, -CH2- of the side chain), 0.86(t, J = 6.5

13 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 172.6 (>C=O), 157.0 (ArC), 154.1 (ArC), 151.6

(ArC), 135.8 (ArC), 131.0 (ArC), 126.0 (ArC), 118.2 (ArC), 114.6 (ArC), 114.5 (ArC), 101.9

(ArC), 55.6, 51.5, 44.6, 38.3, 37.9, 31.7, 30.0, 29.0, 24.6, 22.6, 14.0. Mass spectrum

(ESI) m/z (relative intensity) 429 (M++H, 100). HPLC (4.6 mm × 250 mm, Supelco

Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 5.7 min for the title compound.

2-(2-bromo-4-methoxyphenyl)acetyl chloride (3.16)65. To a solution of 2-(2-bromo-4- methoxyphenyl) acetic acid (7.0 g, 28.6 mmol) in anhydrous benzene (95.2 mL) at room temperature, under an argon atmosphere, were added sequentially oxalyl dichloride

(100.1 mL, 200.2 mmol, 2M solution in anhydrous CH2Cl2), 5 drops of anhydrous DMF.

168

The reaction mixture was stirred for overnight at the same temperature. The reaction was quenched by evaporating volatiles under reduces pressure and further dried under high reduced pressure for 2 hours. The crude (7..4 g)was used for next recation without any further purification.

(S)-4-benzyl-3-(2-(2-bromo-4-methoxyphenyl)acetyl)oxazolidin-2-one (3.17) To a stirred solution of (S)-4-benzyloxazolidin-2-one (11.4 g, 29.2 mmol) in dry THF (448 mL) at -78 oC, under an argon atmosphere, was added n-BuLi(12.7 mL, 31.8 mmol, 2.5 M in hexane) dropwise. Following the addition, the reaction mixture was gradually warmed to

-20 oC during an hour period. Then the resulting mixture was cooled again to -78 oC, followed by the slowly addition of 3.16 (7.0 g 26.5 mmol) in THF (ml). The reaction mixture was allowed to stir at 0 oC for 2h before quenched with 1M HCl and diluted with ethyl acetate. The organic phase was washed with brine, dried over MgSO4 and concentrated in vacuo. Purification by flash column chromatography on silica gel (10-45% ethyl acetate in hexanes) gave 9.1 g of 3.17 as a white solid in 85% yield. IR (neat) 2925, 1772, 1702,

-1 1 1605, 1494, 1363, 1250, 1196, 1028, 702 cm ; H NMR (500 MHz, CDCl3) δ 7.33 (t, J =

7.0 Hz, 2H, Ar-H), 7.27 (t, J = 7.5 Hz, 1H, Ar-H), 7.22 (d, J = 8 Hz, 2H, ArH), 7.17 (q, J =

2.5 Hz, 2H, ArH), 6.87 (dd, J = 8.5 Hz, J = 2.5 Hz, 2H, ArH), 4.73-4.67 (m, 1H, -NCH-),

4.36 (dd, J = 58.5 Hz, J = 18 Hz, 2H, -CH2C(O)-) 4.29-4.21 (m, 2H, -CH2O-), 3.81 (s, 1H,

-OCH3), 3.35 (dd, J = 13.0 Hz, J = 3.5 Hz, 1H, -CH2CH(CH2)N-), 2.80 (dd, J = 13.0 Hz, J

13 = 9.5 Hz, 1H, -CH2CH(CH2)N-); C NMR (100 MHz CDCl3) δ 170.4, 159.5, 153.6 (ArC),

135.3(ArC), 132.1(ArC), 129.5 (ArC), 129.0 (ArC), 127.4 (ArC), 126.0 (ArC), 125.5 (ArC),

118.2 (ArC), 113.7 (ArC), 66.5, 55.6, 55.5, 42.2, 37.9.

169

(S)-4-benzyl-3-((S)-2-(2-bromo-4-methoxyphenyl)propanoyl)oxazolidin-2-one (3.18)

To a stirred solution of 3.17 (8.0 g, 19.8 mmol) in dry THF (141.3 mL) at -78 oC, under an argon atmosphere, was added NaHMDS (21.8 mL, 1M in THF) dropwise over a period of 30 min. Iodomethane (13.5 g, 95.0 mmol)was added to the resulting mixture dropwise after stirred for 1h at -78 oC. Then the resulting mixture was allowed to warm to -30 oC and stirred for another 1h. The reaction mixture was quenched with saturated NH4Cl and diluted with ethyl acetate. The organic phase was washed with brine, dried over MgSO4 and concentrated in vacuo. Purification by flash column chromatography on silica gel (10-

40% diethyl ether in hexanes) gave 6.7 g of 3.18 as a white solid in 84% yield. IR (neat)

2838, 1778, 1697, 1604, 1493, 1358, 1233, 1027, 855, 702 cm-1; 1H NMR (500 MHz,

CDCl3) δ 7.34 (t, J = 7.0 Hz, 2H, Ar-H), 7.28 (t, J = 7.5 Hz, 1H, Ar-H), 7.23 (d, J = 8 Hz,

2H, ArH), 7.13 (q, J = 2.5 Hz, 2H, ArH), 6.87 (dd, J = 8.5 Hz, J = 2.5 Hz, 2H, ArH), 5.31-

5.24 (m, 1H, -CH(CH3)-), 4.72-4.64 (m, 1H, -NCH-), 4.18-4.6 (d, J = 5 Hz, 2H, -CH2O-),

3.79 (s, 1H, -OCH3), 3.35 (dd, J = 13.0 Hz, J = 3.5 Hz, 1H, -CH2CH(CH2)N-), 2.80 (dd, J

13 = 13.0 Hz, J = 9.5 Hz, 1H, -CH2CH(CH2)N-), 1.36 (d, J = 2.0 Hz, -CH3); C NMR (100

MHz CDCl3) δ 174.6, 159.1, 152.7 (ArC), 135.3(ArC), 131.7(ArC), 129.4 (ArC), 129.0

(ArC), 128.6 (ArC), 127.4 (ArC), 125.1 (ArC), 118.3 (ArC), 113.7 (ArC), 66.2, 55.7, 55.5,

43.0, 38.0, 17.4.

(S)-2-(2-bromo-4-methoxyphenyl)propanoic acid (3.19) To a stirred solution of 3.18

o (6.0 g, 14.3 mmol) in THF/H2O (3:1 in mixture, 119.5 ml) at 0 C, under an argon atmosphere, was added lithium hydroxide in H2O (15.0 g, 358.5 mmol) dropwise. Stirring was continued for 1h and then the reaction mixture was quenched with 0.5N NaHCO3

170 and diluted with dichloromethane. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic phase was washed with water and brine, dried (MgSO4), and concentrated under vacuo to give 3.3 g of 3.19 as

1 a pure white solid in 89 % yield. H NMR (500 MHz, CDCl3) δ 7.25 (t, J = 8.5 Hz, 1H, Ar-

H), 7.12 (t, J = 3.0 Hz, 1H, Ar-H), 6.86 (dd, J = 8.5 Hz, J = 3.0 Hz, 1H, ArH), 4.24-4.18 (m,

1H, -CH(CH3)-), 3.78 (s, 1H, -OCH3), 1.49 (d, J = 2.0 Hz, -CH3);

methyl (S)-2-(2-bromo-4-methoxyphenyl)propanoate (3.20)65. Compound 3.19 (3.0 g,

11.6 mmol) was dissolved and stirred in wet Et2O/MeOH (2:1 mixture, 232.5mL) at room temperature under an argon atmosphere, (diazomethyl)trimethylsilane was added dropwise (23.2 mL, 46.4 mmol 2 M in hexane) to the stirring solution. The color of the solution turned to light yellow upon the addition. After stirring for 2 h, the reaction mixture was quenched with saturated aqueous NaHCO3 solution and then diluted by Et2O. The organic layer was separated and the aqueous layer was extracted with diethyl ether. The combined organic layer was washed with water and brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (25% diethyl ether in hexane) gave the title compound (2.9 g, 90 % yield) as a colorless oil. 1H

NMR (500 MHz, CDCl3) δ 7.21 (t, J = 8.5 Hz, 1H, Ar-H), 7.11 (t, J = 3.0 Hz, 1H, Ar-H),

6.85 (dd, J = 8.5 Hz, J = 3.0 Hz, 1H, ArH), 4.18-4.13 (m, 1H, -CH(CH3)-), 3.78 (s, 1H, -

OCH3), 3.68 (s, 1H, -C(O)OCH3), 1.46 (d, J = 2.0 Hz, -CH3).

methyl (S)-2-(2-bromo-4-hydroxyphenyl)propanoate (3.21)81. To a stirred solution of

o 3.20 (2.6 g, 9.5 mmol) in dry CH2Cl2 (448 mL), at -78 C, under an argon atmosphere,

171 was added boron tribromide (14.3 mL, 14.3 mmol, 1 M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 1.9 g of 3.21 as a white foam in 79%

1 yield. H NMR (500 MHz, CDCl3) δ 7.16 (t, J = 8.5 Hz, 1H, Ar-H), 7.04 (t, J = 3.0 Hz, 1H,

Ar-H), 6.75 (dd, J = 8.5 Hz, J = 3.0 Hz, 1H, ArH), 5.16 (s, 1H, -OH), 4.17-4.12 (m, 1H, -

CH(CH3)-), 3.69 (s, 1H, -C(O)OCH3), 1.46 (d, J = 2.0 Hz, -CH3). methyl (S)-2-(2-bromo-4-((triisopropylsilyl)oxy)phenyl)propanoate (3.22)65. To a solution of 3.21 (1.8 g, 6.9 mmol) in anhydrous CH2Cl2 (11.5 mL) under an argon atmosphere were added sequentially, 2,6-lutidine (6.6 g, 62.1 mmol), and triisopropylsilyl trifluoromethanesulfonate (12.7 g, 41.4 mmol) at 0 oC. Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature for 2 h and then quenched by the addition of NH4Cl and extracted with diethyl ether. The organic phase was dried (MgSO4) and evaporated under reduced pressure.

Purification by flash column chromatography on silica gel (10%-20% diethyl ether in hexane) afforded 2.5 g (88% yield) of 3.22 as a colorless oil. IR (neat) 2946, 1740, 1598,

-1 1 1489, 1286, 1166, 933, 882, 686 cm ; H NMR (500 MHz, CDCl3) δ 7.16 (t, J = 8.5 Hz,

1H, Ar-H), 7.04 (t, J = 3.0 Hz, 1H, Ar-H), 6.75 (dd, J = 8.5 Hz, J = 3.0 Hz, 1H, ArH), 4.17-

4.12 (m, 1H, -CH(CH3)-), 3.69 (s, 1H, -C(O)OCH3), 1.46 (d, J = 2.0 Hz, -CH3), 1.28-1.23

172

13 (m, 3 H, -Si(CH(Me)2)3, 1.09 (d, J = 7.0 Hz, 18 H, -Si(CH(Me)2)3); C NMR (100 MHz

CDCl3) δ 174.8, 155.5 (ArC), 132.4(ArC), 128.5 (ArC), 124.1 (ArC), 119.3 (ArC), 52.1,

43.8, 18.1, 17.9, 12.6. methyl (S)-2-(2',6'-dimethoxy-4'-(2-methyloctan-2-yl)-5-((triisopropylsilyl)oxy)-[1,1'- biphenyl]-2-yl)propanoate (3.23)65. To a solution of the mixture of 3.22 (2.3 g, 5.5 mmol) and 3.6 (2.0 g, 6.6 mmol) in DME/H2O (5:1 mixture, 183.3 mL) in microwave vessel under an argon atmosphere were added sequentially CsCO3 (7.1 g, 22.0 mmol) and Pd(PPh3)4

(635.5 mg, 0.6 mmol). After the addition, the vessel was sealed. The resulting mixture was irradiated using the open vessel mode at 110 oC for 15 min. The reaction mixture was quenched with water. The organic phase was dried (MgSO4) and evaporated under reduced pressure. Purification by flash column chromatography on silica gel (10%-25% diethyl ether in hexane) afforded 2.0 g (60% yield) of 3.23 as a colorless oil. IR (neat)

2931, 1736, 1572, 1462, 1297, 1241, 1205, 1126, 942, 882, 671 cm-1; 1H NMR (500

MHz,CDCl3) δ 7.24 (d, J = 8.5 Hz, 1H, ArH), 6.82 ( dd, J = 8.5 Hz, J = 2.0 Hz, 1H, ArH),

6.66 (d, J = 2.0 Hz, 1H, ArH), 6.57, (d, J = 3.0 Hz, 2H, ArH), 3.66 (s, 3H, -OCH3), 3.65 (s,

3H, -OCH3), 3.55 (s, 3H, CH3OC(O)-), 3.49-3.40 (m, 1H, -CH(CH3)C(O)-), 1.65-1.59 (m,

2H, 2′-H), 1.33 (s, 6H, -C(CH3)-), 1.30 (d, J = 7.5 Hz, 3H, -CH3), 1.27-1.20 (m, 11H, 8H of

-CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.1 (d, J = 7 Hz, 18H, -Si(CH(Me)2)3), 0.86(t,

13 J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 176.2, 157.5 (ArC), 157.3 (ArC), 154.2

(ArC), 151.4 (ArC), 135.1 (ArC), 132.7 (ArC), 127.0 (ArC), 122.8 (ArC), 119.0 (ArC), 115.2

(ArC), 102.3 (ArC), 102.0 (ArC), 55.7, 55.6, 51.5, 44.6, 41.1, 38.3, 31.7, 30.0, 28.9, 24.6,

22.6, 19.1, 17.9, 14.1, 12.7.

173

(7S)-1-hydroxy-7-methyl-3-(2-methyloctan-2-yl)-10-

((triisopropylsilyl)oxy)dibenzo[b,d]oxepin-6(7H)-one (3.24)65. To a stirred solution of

o 3.23 (1.8 g, 3.0 mmol) in dry CH2Cl2 (20.0 mL), at -78 C, under an argon atmosphere, was added boron tribromide (12.9 mL, 12.9 mmol, 1 M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 1.0 g of 3.24 as a white foam in 62% yield. IR (neat) 2928, 1716, 1532, 1471, 1123, 1066, 1005, 902, 736, 662 cm-1; 1H NMR

(500 MHz,CDCl3) δ 7.33 (d, J = 9.0 Hz, 1H, ArH), 7.23 (d, J = 2.5 Hz, 1H, ArH), 6.99 ( dd,

J = 9.0 Hz, J = 2.5 Hz, 1H, ArH), 6.86 (dd, J = 15.0 Hz, J = 2.0 Hz, 2H, ArH), 5.64 (s, 1H,

-OH), 3.64-3.58 (m, 1H, -CH(CH3)C(O)-), 1.63 (d, J = 7.0 Hz, 3H, -CH3), 1.60-1.57 (m,

2H, 2′-H), 1.29 (s, 6H, -C(CH3)-), 1.27-1.20 (m, 11H, 8H of -CH2- of the side chain, 3H of

13 -Si(CH(Me)2)3), 1.1 (d, J = 7 Hz, 18H, -Si(CH(Me)2)3), 0.86 (t, J = 6.5 Hz, 7′-H); C NMR

(100 MHz CDCl3) δ 177.3, 156.2 (ArC), 155.5 (ArC), 153.7 (ArC), 151.8 (ArC), 134.9

(ArC), 132.2 (ArC), 125.6 (ArC), 122.3 (ArC), 118.3 (ArC), 114.9 (ArC), 101.9 (ArC), 101.6

(ArC), 55.4, 55.3, 49.5, 43.4, 39.4, 38.8, 30.7, 30.0, 28.2, 25.3, 23.7, 18.4, 17.8, 14.0,

12.3.

(7S)-1,10-dihydroxy-7-methyl-3-(2-methyloctan-2-yl)dibenzo[b,d]oxepin-6(7H)-one

(3.25)65. To a solution of 3.24 (900 mg, 1.7 mmol) in anhydrous THF (41.8 mL) at -40 °C,

174 under an argon atmosphere, was added tetra-n-butylammonium fluoride (2.0 mL, 2.0 mmol, 1M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution.

Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (20%-50% ethyl acetate in hexane) gave 3.25 (611 mg, 94% yield) as a white solid. mp = 68-70 oC. IR (neat) 3357, 2928, 1729, 1621, 1408, 1289, 1200, 1154, 1134,

-1 1 1045, 1022, 828, 722 cm ; H NMR (500 MHz,CDCl3) δ 7.33 (d, J = 9.0 Hz, 1H, ArH),

7.21 (d, J = 2.5 Hz, 1H, ArH), 6.95 ( dd, J = 9.0 Hz, J = 2.5 Hz, 1H, ArH), 6.85 (dd, J =

15.0 Hz, J = 2.0 Hz, 2H, ArH), 5.70 (s, 1H, -OH), 5.57 (s, 1H, -OH), 3.63-3.58 (m, 1H, -

CH(CH3)C(O)-), 1.63 (d, J = 7.0 Hz, 3H, -CH3), 1.61-1.56 (m, 2H, 2′-H), 1.29 (s, 6H, -

13 C(CH3)-), 1.27-1.18 (m, 8H, -CH2- of the side chain), 0.86 (t, J = 6.5 Hz, 7′-H); C NMR

(100 MHz CDCl3) δ 172.6, 155.3 (ArC), 153.1 (ArC), 152.5 (ArC), 150.1 (ArC), 131.5

(ArC), 128.3 (ArC), 126.3 (ArC), 116.3 (ArC), 115.1 (ArC), 113.8 (ArC), 111.0 (ArC), 110.5

(ArC), 38.4, 37.9, 31.7, 29.9, 28.7, 24.6, 22.6, 15.1, 14.0, 12.3.

(R)-4-benzyl-3-(2-(2-bromo-4-methoxyphenyl)acetyl)oxazolidin-2-one (3.28) To a stirred solution of (R)-4-benzyloxazolidin-2-one (1.5 g, 8.3 mmol) in dry THF (15.2 mL) at -78 oC, under an argon atmosphere, was added n-BuLi (3.6 mL, 9.1 mmol, 2.5 M in hexane) dropwise. Following the addition, the reaction mixture was gradually warmed to

-20 oC during an hour period. Then the resulting mixture was cooled again to -78 oC, followed by the slowly addition of 3.27 (2.0 g, 7.6 mmol) in THF (ml). The reaction mixture was allowed to stir at 0 oC for 2h before quenched with 1M HCl and diluted with ethyl acetate. The organic phase was washed with brine, dried over MgSO4 and concentrated

175 in vacuo. Purification by flash column chromatography on silica gel (10-45% ethyl acetate in hexanes) gave 2.6 g of 3.28 as a white solid in 84% yield. IR (neat) 2925, 1772, 1702,

-1 1 1605, 1494, 1363, 1250, 1196, 1028, 702 cm ; H NMR (500 MHz, CDCl3) δ 7.33 (t, J =

7.0 Hz, 2H, Ar-H), 7.27 (t, J = 7.5 Hz, 1H, Ar-H), 7.22 (d, J = 8 Hz, 2H, ArH), 7.17 (q, J =

2.5 Hz, 2H, ArH), 6.87 (dd, J = 8.5 Hz, J = 2.5 Hz, 2H, ArH), 4.73-4.67 (m, 1H, -NCH-),

4.36 (dd, J = 58.5 Hz, J = 18 Hz, 2H, -CH2C(O)-) 4.29-4.21 (m, 2H, -CH2O-), 3.81 (s, 1H,

-OCH3), 3.35 (dd, J = 13.0 Hz, J = 3.5 Hz, 1H, -CH2CH(CH2)N-), 2.80 (dd, J = 13.0 Hz, J

13 = 9.5 Hz, 1H, -CH2CH(CH2)N-); C NMR (100 MHz CDCl3) δ 170.4, 159.5, 153.6 (ArC),

135.3(ArC), 132.1(ArC), 129.5 (ArC), 129.0 (ArC), 127.4 (ArC), 126.0 (ArC), 125.5 (ArC),

118.2 (ArC), 113.7 (ArC) 66.5, 55.6, 55.5, 42.2, 37.9.

(R)-4-benzyl-3-((R)-2-(2-bromo-4-methoxyphenyl)propanoyl)oxazolidin-2-one (3.29)

To a stirred solution of 3.28 (2.0 g, 4.9 mmol) in dry THF (35 mL) at -78 oC, under an argon atmosphere, was added NaHMDS (5.4 mL, 5.4 mmol, 1 M in THF) dropwise over a period of 30 min. Iodomethane (3.3 g, 23.5 mmol)was added to the resulting mixture dropwise after stirred for 1h at -78 oC. Then the resulting mixture was allowed to warm to

-30 oC and stirred for another 1h. The reaction mixture was quenched with saturated

NH4Cl and diluted with ethyl acetate. The organic phase was washed with brine, dried over MgSO4 and concentrated in vacuo. Purification by flash column chromatography on silica gel (10-40% diethyl ether in hexanes) gave 1.6 g of 3.29 as a white solid in 79% yield. IR (neat) 2838, 1778, 1697, 1604, 1493, 1358, 1233, 1027, 855, 702 cm-1; 1H NMR

(500 MHz, CDCl3) δ 7.34 (t, J = 7.0 Hz, 2H, Ar-H), 7.28 (t, J = 7.5 Hz, 1H, Ar-H), 7.23 (d,

J = 8 Hz, 2H, ArH), 7.13 (q, J = 2.5 Hz, 2H, ArH), 6.87 (dd, J = 8.5 Hz, J = 2.5 Hz, 2H,

ArH), 5.31-5.24 (m, 1H, -CH(CH3)-), 4.72-4.64 (m, 1H, -NCH-), 4.18-4.6 (d, J = 5 Hz, 2H,

176

-CH2O-), 3.79 (s, 1H, -OCH3), 3.35 (dd, J = 13.0 Hz, J = 3.5 Hz, 1H, -CH2CH(CH2)N-),

13 2.80 (dd, J = 13.0 Hz, J = 9.5 Hz, 1H, -CH2CH(CH2)N-), 1.36 (d, J = 2.0 Hz, -CH3); C

NMR (100 MHz CDCl3) δ 174.6, 159.1, 152.7 (ArC), 135.3(ArC), 131.7(ArC), 129.4 (ArC),

129.0 (ArC), 128.6 (ArC), 127.4 (ArC), 125.1 (ArC), 118.3 (ArC), 113.7 (ArC), 66.2, 55.7,

55.5, 43.0, 38.0, 17.4.

(R)-2-(2-bromo-4-methoxyphenyl)propanoic acid (3.30) To a stirred solution of 3.29

o (1.4 g, 3.3 mmol) in THF/H2O (3:1 in mixture, 27.9 ml) at 0 C, under an argon atmosphere, was added lithium hydroxide in H2O (415 mg, 9.9 mmol) dropwise. Stirring was continued for 1h and then the reaction mixture was quenched with 0.5N NaHCO3 and diluted with dichloromethane. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic phase was washed with water and brine, dried

(MgSO4), and concentrated under vacuo to give 775 mg of 3.30 as a pure white solid in

1 91% yield. H NMR (500 MHz, CDCl3) δ 7.25 (t, J = 8.5 Hz, 1H, Ar-H), 7.12 (t, J = 3.0 Hz,

1H, Ar-H), 6.86 (dd, J = 8.5 Hz, J = 3.0 Hz, 1H, ArH), 4.24-4.18 (m, 1H, -CH(CH3)-), 3.78

(s, 1H, -OCH3), 1.49 (d, J = 2.0 Hz, -CH3);

methyl (R)-2-(2-bromo-4-methoxyphenyl)propanoate (3.31)65. Compound 3.30 (600 mg, 2.3 mmol) was dissolved and stirred in wet Et2O/MeOH (2:1 mixture, 46.5 mL) at room temperature under an argon atmosphere, (diazomethyl)trimethylsilane was added dropwise (4.6 mL, 9.2 mmol, 2 M in hexane) to the stirring solution. The color of the solution turned to light yellow upon the addition. After stirring for 2 h, the reaction mixture was quenched with saturated aqueous NaHCO3 solution and then diluted by Et2O. The

177 organic layer was separated and the aqueous layer was extracted with diethyl ether. The combined organic layer was washed with water and brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (25% diethyl ether in hexane) gave the title compound (546 mg, 87% yield) as a colorless oil.

1 H NMR (500 MHz, CDCl3) δ 7.21 (t, J = 8.5 Hz, 1H, Ar-H), 7.11 (t, J = 3.0 Hz, 1H, Ar-H),

6.85 (dd, J = 8.5 Hz, J = 3.0 Hz, 1H, ArH), 4.18-4.13 (m, 1H, -CH(CH3)-), 3.78 (s, 1H, -

OCH3), 3.68 (s, 1H, -C(O)OCH3), 1.46 (d, J = 2.0 Hz, -CH3).

methyl (R)-2-(2-bromo-4-hydroxyphenyl)propanoate (3.32)81. To a stirred solution of

o 3.31 (400 mg, 1.5 mmol) in dry CH2Cl2 (9.8 mL), at -78 C, under an argon atmosphere, was added boron tribromide (2.3 mL, 2.3 mmol, 1 M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 299 mg of 3.32 as a white foam in 77%

1 yield. H NMR (500 MHz, CDCl3) δ 7.16 (t, J = 8.5 Hz, 1H, Ar-H), 7.04 (t, J = 3.0 Hz, 1H,

Ar-H), 6.75 (dd, J = 8.5 Hz, J = 3.0 Hz, 1H, ArH), 5.16 (s, 1H, -OH), 4.17-4.12 (m, 1H, -

CH(CH3)-), 3.69 (s, 1H, -C(O)OCH3), 1.46 (d, J = 2.0 Hz, -CH3). methyl (R)-2-(2-bromo-4-((triisopropylsilyl)oxy)phenyl)propanoate (3.33)65. To a solution of 3.32 (200 mg, 0.77 mmol) in anhydrous CH2Cl2 (1.2 mL) under an argon

178 atmosphere were added sequentially, 2,6-lutidine (743 mg, 6.9 mmol), and triisopropylsilyl trifluoromethanesulfonate (1.4 g, 4.6 mmol) at 0 oC. Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature for 2 h and then quenched by the addition of NH4Cl and extracted with diethyl ether. The organic phase was dried (MgSO4) and evaporated under reduced pressure. Purification by flash column chromatography on silica gel (10%-20% diethyl ether in hexane) afforded 278 mg (87% yield) of 3.33 as a colorless oil. IR (neat)

-1 1 2946, 1740, 1598, 1489, 1286, 1166, 933, 882, 686 cm ; H NMR (500 MHz, CDCl3) δ

7.16 (t, J = 8.5 Hz, 1H, Ar-H), 7.04 (t, J = 3.0 Hz, 1H, Ar-H), 6.75 (dd, J = 8.5 Hz, J = 3.0

Hz, 1H, ArH), 4.17-4.12 (m, 1H, -CH(CH3)-), 3.69 (s, 1H, -C(O)OCH3), 1.46 (d, J = 2.0 Hz,

13 -CH3), 1.28-1.23 (m, 3 H, -Si(CH(Me)2)3, 1.09 (d, J = 7.0 Hz, 18 H, -Si(CH(Me)2)3); C

NMR (100 MHz CDCl3) δ 174.8, 155.5 (ArC), 132.4(ArC), 128.5 (ArC), 124.1 (ArC), 119.3

(ArC), 52.1, 43.8, 18.1, 17.9, 12.6. methyl (R)-2-(2',6'-dimethoxy-4'-(2-methyloctan-2-yl)-5-((triisopropylsilyl)oxy)-[1,1'- biphenyl]-2-yl)propanoate (3.34)65. To a solution of the mixture of 3.33 (200 mg, 0.48 mmol) and 3.6 (180 mg, 0.57 mmol) in DME/H2O (5:1 mixture, 1.6 mL) in microwave vessel under an argon atmosphere were added sequentially CsCO3 (626 mg, 1.9 mmol) and Pd(PPh3)4 (55 mg, 0.05 mmol). After the addition, the vessel was sealed. The resulting mixture was irradiated using the open vessel mode at 110 oC for 15 min. The reaction mixture was quenched with water. The organic phase was dried (MgSO4) and evaporated under reduced pressure. Purification by flash column chromatography on silica gel (10%-25% diethyl ether in hexane) afforded 150 mg (52% yield) of 3.34 as a colorless oil. IR (neat) 2931, 1736, 1572, 1462, 1297, 1241, 1205, 1126, 942, 882, 671

179

-1 1 cm ; H NMR (500 MHz,CDCl3) δ 7.24 (d, J = 8.5 Hz, 1H, ArH), 6.82 ( dd, J = 8.5 Hz, J

= 2.0 Hz, 1H, ArH), 6.66 (d, J = 2.0 Hz, 1H, ArH), 6.57, (d, J = 3.0 Hz, 2H, ArH), 3.66 (s,

3H, -OCH3), 3.65 (s, 3H, -OCH3), 3.55 (s, 3H, CH3OC(O)-), 3.49-3.40 (m, 1H, -

CH(CH3)C(O)-), 1.65-1.59 (m, 2H, 2′-H), 1.33 (s, 6H, -C(CH3)-), 1.30 (d, J = 7.5 Hz, 3H, -

CH3), 1.27-1.20 (m, 11H, 8H of -CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.1 (d, J =

13 7 Hz, 18H, -Si(CH(Me)2)3), 0.86(t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 176.2,

157.5 (ArC), 157.3 (ArC), 154.2 (ArC), 151.4 (ArC), 135.1 (ArC), 132.7 (ArC), 127.0 (ArC),

122.8 (ArC), 119.0 (ArC), 115.2 (ArC), 102.3 (ArC), 102.0 (ArC), 55.7, 55.6, 51.5, 44.6,

41.1, 38.3, 31.7, 30.0, 28.9, 24.6, 22.6, 19.1, 17.9, 14.1, 12.7.

(7R)-1-hydroxy-7-methyl-3-(2-methyloctan-2-yl)-10-

((triisopropylsilyl)oxy)dibenzo[b,d]oxepin-6(7H)-one (3.35)65. To a stirred solution of

o 3.34 (140 g, 0.23 mmol) in dry CH2Cl2 (1.6 mL), at -78 C, under an argon atmosphere, was added boron tribromide (0.92 mL, 0.92 mmol, 1 M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 87 mg of 3.35 as a white foam in 70 % yield. IR (neat) 2928, 1716, 1532, 1471, 1123, 1066, 1005, 902, 736, 662 cm-1; 1H NMR

(500 MHz,CDCl3) δ 7.33 (d, J = 9.0 Hz, 1H, ArH), 7.23 (d, J = 2.5 Hz, 1H, ArH), 6.99 ( dd,

180

J = 9.0 Hz, J = 2.5 Hz, 1H, ArH), 6.86 (dd, J = 15.0 Hz, J = 2.0 Hz, 2H, ArH), 5.64 (s, 1H,

-OH), 3.64-3.58 (m, 1H, -CH(CH3)C(O)-), 1.63 (d, J = 7.0 Hz, 3H, -CH3), 1.60-1.57 (m,

2H, 2′-H), 1.29 (s, 6H, -C(CH3)-), 1.27-1.20 (m, 11H, 8H of -CH2- of the side chain, 3H of

13 -Si(CH(Me)2)3), 1.1 (d, J = 7 Hz, 18H, -Si(CH(Me)2)3), 0.86 (t, J = 6.5 Hz, 7′-H); C NMR

(100 MHz CDCl3) δ 177.3, 156.2 (ArC), 155.5 (ArC), 153.7 (ArC), 151.8 (ArC), 134.9

(ArC), 132.2 (ArC), 125.6 (ArC), 122.3 (ArC), 118.3 (ArC), 114.9 (ArC), 101.9 (ArC), 101.6

(ArC), 55.4, 55.3, 49.5, 43.4, 39.4, 38.8, 30.7, 30.0, 28.2, 25.3, 23.7, 18.4, 17.8, 14.0,

12.3.

(7R)-1,10-dihydroxy-7-methyl-3-(2-methyloctan-2-yl)dibenzo[b,d]oxepin-6(7H)-one

(3.36)65. To a solution of 3.35 (70 mg, 0.13 mmol) in anhydrous THF (3.2 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (0.16 mL, 0.16 mmol, 1M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution.

Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (20%-50% ethyl acetate in hexane) gave 3.36 (48 mg, 96% yield) as a white solid. mp = 68-70 oC. IR (neat) 3357, 2937, 1729, 1621, 1408, 1303, 1256, 1154, 1045,

-1 1 1022, 828, 722 cm ; H NMR (500 MHz,CDCl3) δ 7.33 (d, J = 9.0 Hz, 1H, ArH), 7.21 (d,

J = 2.5 Hz, 1H, ArH), 6.95 ( dd, J = 9.0 Hz, J = 2.5 Hz, 1H, ArH), 6.85 (dd, J = 15.0 Hz, J

= 2.0 Hz, 2H, ArH), 5.70 (s, 1H, -OH), 5.57 (s, 1H, -OH), 3.63-3.58 (m, 1H, -

CH(CH3)C(O)-), 1.63 (d, J = 7.0 Hz, 3H, -CH3), 1.61-1.56 (m, 2H, 2′-H), 1.29 (s, 6H, -

13 C(CH3)-), 1.27-1.18 (m, 8H, -CH2- of the side chain), 0.86 (t, J = 6.5 Hz, 7′-H); C NMR

(100 MHz CDCl3) δ 172.6, 155.3 (ArC), 153.1 (ArC), 152.5 (ArC), 150.1 (ArC), 131.5

181

(ArC), 128.3 (ArC), 126.3 (ArC), 116.3 (ArC), 115.1 (ArC), 113.8 (ArC), 111.0 (ArC), 110.5

(ArC), 38.4, 37.9, 31.7, 29.9, 28.7, 24.6, 22.6, 15.1, 14.0, 12.3.

2-(2-bromo-4-methoxyphenyl)acetonitrile (3.38)65. To a solution of 3.37 (1.0 g, 3.6 mmol) in anhydrous DMSO (71 mL) at 5 °C , under an argon atmosphere, was added

NaCN (882 mg, 18 mmol). The reaction mixture was allowed to warm to room temperature and stirred overnight at the same temperature, and then quenched by ice. Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel

(5%-15% ethyl acetate in hexane) gave 3.38 (691 mg, 85% yield) as a white solid. mp =

68-70 oC. IR (neat) 2943, 2250 (w, CN), 1737, 1603, 1491, 1287, 1237, 1026, 861, 810,

-1 1 677 cm ; H NMR (500 MHz, CDCl3) δ 7.41 (d, J = 8.5 Hz, 1H, ArH), 7.18 (d, J = 2.5 Hz,

1 H, ArH), 6.91 (dd, J = 8.5 Hz, J = 2.5 Hz, 1 H, ArH ), 3.83 (s, 3 H, -OCH3), 3.79 (s, 2 H,

13 -CH2CN); C NMR (100 MHz CDCl3) δ 160.1 (CN), 128.8 (ArC), 122.5 (ArC), 118.4

(ArC), 115.6 (ArC), 113.9 (ArC), 110.8 (ArC), 55.7, 22.9.

2-(2-bromo-4-hydroxyphenyl)acetonitrile (3.39). To a stirred solution of 3.38 (680 mg,

o 3.0 mmol) in dry CH2Cl2 (20 mL), at -78 C, under an argon atmosphere, was added boron tribromide (4.5 mL, 4.5 mmol, 1 M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under

182 reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 496 mg of 3.30 as a white foam in 78% yield. IR (neat) 2893,

2275 (w, CN), 1702, 1578, 1432, 1269, 1134, 1085, 832, 643 cm-1; 1H NMR (500 MHz,

CDCl3) δ 7.38 (d, J = 8.5 Hz, 1H, ArH), 7.12 (d, J = 2.5 Hz, 1 H, ArH), 6.85 (dd, J = 8.5

13 Hz, J = 2.5 Hz, 1 H, ArH ), 5.43 (s, 1H, -OH), 3.79 (s, 2 H, -CH2CN); C NMR (100 MHz

CDCl3) δ 162.2 (CN), 128.3 (ArC), 121.8 (ArC), 120.0 (ArC), 117.3 (ArC), 114.9 (ArC),

112.2 (ArC), 22.5.

2-(2-bromo-4-((triisopropylsilyl)oxy)phenyl)acetonitrile (3.40)65. To a solution of 3.39

(440 mg, 2.1 mmol) in anhydrous CH2Cl2 (3.5 mL) under an argon atmosphere were added sequentially, 2,6-lutidine (1.9 g, 18 mmol), and triisopropylsilyl trifluoromethanesulfonate (3.7 g, 12 mmol) at 0 oC. Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature for 2 h and then quenched by the addition of NH4Cl and extracted with diethyl ether. The organic phase was dried (MgSO4) and evaporated under reduced pressure.

Purification by flash column chromatography on silica gel (10%-20% diethyl ether in hexane) afforded 648 mg (88% yield) of 3.40 as a colorless oil. IR (neat) 2889, 2312 (w,

-1 1 CN), 1688, 1532, 1411, 1253, 1101, 1096, 847, 641 cm ; H NMR (500 MHz, CDCl3) δ

7.38 (d, J = 8.5 Hz, 1H, ArH), 7.12 (d, J = 2.5 Hz, 1 H, ArH), 6.85 (dd, J = 8.5 Hz, J = 2.5

Hz, 1 H, ArH ), 3.79 (s, 2 H, -CH2CN); 1.28-1.23 (m, 3H, -Si(CH(Me)2)3, 1.09 (d, J = 7.0

13 Hz, 18H, -Si(CH(Me)2)3); C NMR (100 MHz CDCl3) δ 161.4 (CN), 129.0 (ArC), 121.2

(ArC), 119.3 (ArC), 117.8 (ArC), 114.6(ArC), 111.2 (ArC), 23.1, 17.7, 12.5.

2-(2',6'-dimethoxy-4'-(2-methyloctan-2-yl)-5-((triisopropylsilyl)oxy)-[1,1'-biphenyl]-

2-yl)acetonitrile (3.41)65. To a solution of the mixture of 3.40 (600 mg, 1.6 mmol) and 3.6

183

(602 mg, 2.0 mmol) in DME/H2O (5:1 mixture, 5.3 mL) in microwave vessel under an argon atmosphere were added sequentially CsCO3 (2.1 g, 6.4 mmol) and Pd(PPh3)4 (295 mg, 0.16 mmol). After the addition, the vessel was sealed. The resulting mixture was irradiated using the open vessel mode at 110 oC for 15 min. The reaction mixture was quenched with water. The organic phase was dried (MgSO4) and evaporated under reduced pressure. Purification by flash column chromatography on silica gel (10%-25% diethyl ether in hexane) afforded 618 mg (70% yield) of 3.41 as a colorless oil. IR (neat)

2929, 2163, 1738(s, >C=O), 1607, 1569, 1462, 1406, 1241, 1123, 1037, 1020, 918, 831,

-1 1 672 cm ; H NMR (500 MHz,CDCl3) δ 7.42 (d, J = 8.5 Hz, 1H, ArH), 6.91 ( dd, J = 8.5 Hz,

J = 2.5 Hz, 1H, ArH), 6.78 (d, J = 2.5 Hz, 1H, ArH), 6.60, (s, 2H, ArH), 3.73 (s, 6H, -OCH3),

3.41 (s, 2H, -CH2CN), 1.65-1.59 (m, 2H, 2′-H), 1.34 (s, 6H, -C(CH3)-), 1.30-1.20 (m, 11H,

8H of -CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.1 (d, J = 7 Hz, 18H, -Si(CH(Me)2)3),

13 0.86(t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 158.7, 155.3 (ArC), 137.5 (ArC),

129.3 (ArC), 120.0 (ArC), 119.5 (ArC), 116.5 (ArC), 113.4 (ArC), 113.1 (ArC), 102.4 (ArC),

58.5, 44.6, 38.7, 34.5, 33.2, 29.2, 28.7, 23.5, 21.2, 17.8, 12.5, 11.2.

2-(2',6'-dimethoxy-4'-(2-methyloctan-2-yl)-5-((triisopropylsilyl)oxy)-[1,1'-biphenyl]-

2-yl)-2-methylpropanenitrile (3.42)65. To a stirred suspension of sodium hydride (82 mg,

3.4 mmol) in dry DMF (7.6 mL) at 0 °C under an argon atmosphere was added dropwise a solution of 3.41 (590 g, 1.07 mmol) and iodomethane (482.8 mg, 3.4 mmol) in dry DMF

(7.6 mL). The reaction temperature was rose to 25 oC over a 15 min period and stirring was continued for 2 h. The reaction mixture was quenched with saturated aqueous NH4Cl solution and diluted with diethyl ether. The organic layer was separated and the aqueous

184 layer was extracted with diethyl ether. The combined organic layer was washed with water and brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (25% ethyl acetate in hexane) gave the title compound (217 mg, 35% yield) as a colorless oil. IR (neat) 2930, 2232 (w, CN), 1608, 1570, 1408, 1242,

-1 1 1123, 1037, 831, 673 cm . H NMR (500 MHz,CDCl3) δ 7.58 (d, J = 8.5 Hz, 1H, ArH),

6.90 ( dd, J = 8.5 Hz, J = 2.5 Hz, 1H, ArH), 6.63 (d, J = 2.5 Hz, 1H, ArH), 6.56, (s, 2H,

ArH), 3.73 (s, 6H, -OCH3), 1.65-1.59 (m, 2H, 2′-H), 1.52 (s, 6H, -C(CH3)2CN), 1.34 (s,

6H, -C(CH3)-), 1.30-1.20 (m, 11H, 8H of -CH2- of the side chain, 3H of -Si(CH(Me)2)3),

13 1.1 (d, J = 7 Hz, 18H, -Si(CH(Me)2)3), 0.86(t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3)

δ 158.4, 157.4 (ArC), 152.2(ArC), 134.9 (ArC), 131.4 (ArC), 128.0 (ArC), 125.4 (ArC),

118.5 (ArC), 115.9 (ArC), 112.9 (ArC), 101.5 (ArC), 55.3, 55.2, 44.7, 38.4, 37.4, 31.7,

29.9, 29.0, 28.8, 24.6, 22.6, 17.8, 14.0, 12.6.

2-(2',6'-dimethoxy-4'-(2-methyloctan-2-yl)-5-((triisopropylsilyl)oxy)-[1,1'-biphenyl]-

2-yl)-2-methylpropanoic acid (3.43)65. To a stirred solution of 3.42 (190 mg, 0.32 mmol) in n-BuOH:H2O (1:1 in mixture, 0.3 mL) at room temperature under an argon atmosphere was added sodium hydroxide (64 mg, 1.6 mmol). The resulting reaction mixture was stirring for 2 h. Then quenched with saturated aqueous 1 M HCl solution and diluted with diethyl ether. The organic layer was separated and the aqueous layer was extracted with diethyl ether. The combined organic layer was washed with water and brine, dried

(MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (70% ethyl acetate in hexane) gave the title compound (140 mg, 73% yield) as a colorless oil. IR (neat) 3354, 2928, 1739, 1682, 1608, 1571, 1463, 1408, 1242, 1123,

185

-1 1 1037, 830, 672 cm . H NMR (500 MHz,CDCl3) δ 7.34 (d, J = 8.5 Hz, 1H, ArH), 6.88 ( dd,

J = 8.5 Hz, J = 2.5 Hz, 1H, ArH), 6.63 (d, J = 2.5 Hz, 1H, ArH), 6.58, (s, 2H, ArH), 3.72 (s,

6H, -OCH3), 1.68-1.59 (m, 2H, 2′-H), 1.36 (s, 6H, -C(CH3)2COOH), 1.28 (s, 6H, -C(CH3)-),

1.28-1.20 (m, 11H, 8H of -CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.1 (d, J = 7 Hz,

13 18H, -Si(CH(Me)2)3), 0.86(t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 181.3, 157.6

(ArC), 157.1(ArC), 151.6 (ArC), 134.4 (ArC), 130.6 (ArC), 117.4 (ArC), 113.7 (ArC),

101.8(ArC), 55.6, 55.1, 48.4, 44.7, 38.3, 31.7, 29.9, 29.0, 27.8, 24.6, 22.6, 17.9, 14.0,

12.6.

2-(2',6'-dihydroxy-4'-(2-methyloctan-2-yl)-5-((triisopropylsilyl)oxy)-[1,1'-biphenyl]-

2-yl)-2-methylpropanoic acid (3.44)65. To a stirred solution of 3.43 (130 mg, 0.2 mmol)

o in dry CH2Cl2 (1.5 mL), at -78 C, under an argon atmosphere, was added boron tribromide (0.86 mL, 0.86 mmol, 1 M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 87 mg of 3.44 as a white foam in 76% yield. IR (neat) 2913,

1705, 1658, 1612, 1534, 1428, 1400, 1216, 1154, 1007, 839, 657 cm-1. 1H NMR (500

MHz,CDCl3) δ 7.34 (d, J = 8.5 Hz, 1H, ArH), 6.88 ( dd, J = 8.5 Hz, J = 2.5 Hz, 1H, ArH),

6.63 (d, J = 2.5 Hz, 1H, ArH), 6.58, (s, 2H, ArH), 5.35 (s, 2H, -OH), 1.68-1.59 (m, 2H, 2′-

186

H), 1.36 (s, 6H, -C(CH3)2COOH), 1.28 (s, 6H, -C(CH3)-), 1.28-1.20 (m, 11H, 8H of -CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.1 (d, J = 7 Hz, 18H, -Si(CH(Me)2)3), 0.86(t, J =

13 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 181.6, 156.9 (ArC), 156.3(ArC), 151.3 (ArC),

132.3 (ArC), 130.2 (ArC), 116.9 (ArC), 114.2 (ArC), 101.9 (ArC), 48.3, 44.5, 37.3, 33.2,

29.9, 28.8, 27.3, 24.6, 22.3, 17.5, 14.0, 12.6.

1-hydroxy-7,7-dimethyl-3-(2-methyloctan-2-yl)-10-

((triisopropylsilyl)oxy)dibenzo[b,d]oxepin-6(7H)-one (3.45)65. To a stirred solution of

3.44 (70 mg, 0.12 mmol) in dry toluene (1.2 mL) at room temperature, under an argon atmosphere, was added p-toluenesulfonic acid (2.3 mg, 0.012 mmol). Following the addition, the reaction mixture was gradually heated to 110 oC and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was warmed to room temperature and quench by NH4Cl. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure.

Purification by flash column chromatography on silica gel (20% ethyl acetate in hexanes) afforded 40 mg of 3.45 as a white foam in 61% yield. IR (neat) 2929, 1736, 1627, 1609,

-1 1 1524, 1478, 1435, 1237, 1106, 1083, 856, 643 cm . H NMR (500 MHz,CDCl3) δ 7.33 (d,

J = 9.0 Hz, 1H, ArH), 7.23 (d, J = 2.5 Hz, 1H, ArH), 6.99 ( dd, J = 9.0 Hz, J = 2.5 Hz, 1H,

ArH), 6.86 (dd, J = 15.0 Hz, J = 2.0 Hz, 2H, ArH), 5.64 (s, 1H, -OH), 1.79 (s, 3H, -

C(CH3)2C(O)O-), 1.60-1.57 (m, 2H, 2′-H), 1.29 (s, 6H, -C(CH3)-), 1.27-1.20 (m, 11H, 8H of -CH2- of the side chain, 3H of -Si(CH(Me)2)3), 1.12 (s, 3H, -C(CH3)2C(O)O-), 1.1 (d, J

13 = 7 Hz, 18H, -Si(CH(Me)2)3), 0.86 (t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ 173.5,

187

155.8 (ArC), 153.5(ArC), 152.1 (ArC), 150.9 (ArC), 133.7 (ArC), 131.6 (ArC), 129.1 (ArC),

116.4 (ArC), 115.7 (ArC), 114.4 (ArC), 110.5 (ArC), 109.8(ArC), 46.4, 44.8, 37.6, 31.5,

29.6, 28.3, 26.8, 24.7, 22.9, 22.7, 17.8, 15.8, 14.2, 12.4.

1,10-dihydroxy-7,7-dimethyl-3-(2-methyloctan-2-yl)dibenzo[b,d]oxepin-6(7H)-one

(3.46)65. To a solution of 3.45 (30 mg, 0.05 mmol) in anhydrous THF (1.25 mL) at -40 °C, under an argon atmosphere, was added tetra-n-butylammonium fluoride (0.06 mL, 0.06 mmol, 1M solution in anhydrous THF). The reaction mixture was stirred for 30 min at the same temperature, and then quenched using a saturated aqueous NH4Cl solution.

Extractive isolation with diethyl ether, and purification by flash column chromatography on silica gel (20%-50% ethyl acetate in hexane) gave 3.46 (19 mg, 96% yield) as a white solid. mp = 68-70 oC. IR (neat) 3358, 2927, 1717, 1582, 1408, 1216, 1139, 1049, 1021,

-1 1 855, 735 cm ; H NMR (500 MHz,CDCl3) δ 7.48 (d, J = 9.0 Hz, 1H, ArH), 7.23 (d, J = 2.5

Hz, 1H, ArH), 6.92 ( dd, J = 9.0 Hz, J = 2.5 Hz, 1H, ArH), 6.83 (dd, J = 15.0 Hz, J = 2.0

Hz, 2H, ArH), 5.66 (s, 1H, -OH), 5.44 (s, 1H, -OH), 1.79 (s, 3H, -C(CH3)2C(O)O-), 1.60-

1.57 (m, 2H, 2′-H), 1.29 (s, 6H, -C(CH3)-), 1.27-1.20 (m, 8H, -CH2- of the side chain), 1.12

13 (s, 3H, -C(CH3)2C(O)O-), 0.86 (t, J = 6.5 Hz, 7′-H); C NMR (100 MHz CDCl3) δ173.3,

155.3 (ArC), 153.2 (ArC), 151.9 (ArC), 150.4 (ArC), 133.1 (ArC), 131.5 (ArC), 128.4 (ArC),

116.0 (ArC), 115.7 (ArC), 114.9 (ArC), 110.8 (ArC), 109.9 (ArC), 46.2, 44.3, 37.9, 31.7,

29.8, 28.6, 26.9, 24.6, 22.5, 22.3, 15.2, 14.0. methyl 2-(2-bromo-4-methoxyphenyl)acetate (3.50)65. 2-(2-bromo-4-methoxyphenyl) acetic acid (5.0 g, 20.4 mmol) was dissolved and stirred in wet Et2O/MeOH (2:1 mixture,

340 mL) at room temperature under an argon atmosphere, (diazomethyl)trimethylsilane

188 was added dropwise (40.8 mL, 2M in hexane) to the stirring solution. The color of the solution turned to light yellow upon the addition. After stirring for 2 h, the reaction mixture was quenched with saturated aqueous NaHCO3 solution and then diluted by Et2O. The organic layer was separated and the aqueous layer was extracted with diethyl ether. The combined organic layer was washed with water and brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (25% diethyl ether in hexane) gave the title compound (4.1 g, 78% yield) as a colorless oil. 1H

NMR (500 MHz, CDCl3) δ 7.19 (d, J = 9.0 Hz, 1H, ArH), 7.12 (d, J = 2.5 Hz, 1 H, ArH),

6.83 (dd, J = 9.0 Hz, J = 2.5 Hz, 1 H, ArH ), 3.79 (s, 3 H, -OCH3), 3.73 (s, 2 H, -CH2C(O)-),

+ 3.71 (s, 3 H, CH3C(O)O-); mass spectrum (ESI) m/z (relative intensity) 258 (M +H, 100).

methyl 2-(2-bromo-4-hydroxyphenyl)acetate (3.51)81. To a stirred solution of 3.50 (4.1

o g, 15.9 mmol) in dry CH2Cl2 (106 mL), at -78 C, under an argon atmosphere, was added boron tribromide (23.8 mL, 1M in CH2Cl2). Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature until completion of the reaction (6 h). The reaction mixture was then poured into ice-water, warmed to room temperature and volatiles were removed in vacuo. The residue was diluted with ethyl acetate and washed with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (50% ethyl acetate in hexanes) afforded 3.0 g of 3.51 as a white foam in 78% yield. IR (neat) 3343

(br, OH), 2931, 2834, 1601, 1412, 1345, 1235, 1146, 993, 750 cm-1; 1H NMR (500 MHz,

CDCl3) δ 7.05 (d, J = 8.0 Hz, 1H, ArH), 6.93 (d, J = 2.5 Hz, 1 H, ArH), 6.63 (dd, J = 8.0

189

3.51 (3.0 g, 12.3 mmol) in anhydrous CH2Cl2 (20.4 mL) under an argon atmosphere were added sequentially, 2,6-lutidine (5.9 g, 55.1 mmol), and triisopropylsilyl trifluoromethanesulfonate (11.3 g, 36.7 mmol) at 0 oC. Following the addition, the reaction mixture was gradually warmed to room temperature and the stirring was continued at that temperature for 2 h and then quenched by the addition of NH4Cl and extracted with diethyl ether. The organic phase was dried (MgSO4) and evaporated under reduced pressure.

Purification by flash column chromatography on silica gel (10%-20% diethyl ether in hexane) afforded 4.8 g (98% yield) of 3.52 as a colorless oil. IR (neat) 2867, 1743, 1601,

-1 1 1490, 1273, 1160, 997, 937, 882, 683 cm ; H NMR (500 MHz, CDCl3) δ 7.12 (d, J = 9.0

Hz, 1H, ArH), 7.11 (d, J = 2.5 Hz, 1 H, ArH), 6.79 (dd, J = 9.0 Hz, J = 2.5 Hz, 1 H, ArH ),

3.72 (s, 3 H, CH3C(O)O-), 3.71 (s, 2 H, -CH2C(O)-), 1.28-1.23 (m, 3 H, -Si(CH(Me)2)3,

13 1.09 (d, J = 7.0 Hz, 18 H, -Si(CH(Me)2)3); C NMR (100 MHz CDCl3) δ 190.6 (>C=O),

157.4 (ArC), 132.8 (ArC),128.7 (ArC), 125.6 (ArC), 124.9 (ArC), 122.1 (ArC), 51.9, 38.6,

18.3, 12.7.

2-(3,5-Dimethoxyphenyl)-2-methylpropanenitrile (3.54)65. To a stirred suspension of sodium hydride (5.1 g, 213.4 mmol) in dry DMF (133.4 mL) at 0 °C under an argon atmosphere was added dropwise a solution of 3.53 (11.8 g, 66.7 mmol) and iodomethane

(13.2 mL, 213.4 mmol) in dry DMF (6.8 mL). The reaction temperature was rose to 25 oC over a 15 min period and stirring was continued for 2 h. The reaction mixture was

190 quenched with saturated aqueous NH4Cl solution and diluted with diethyl ether. The organic layer was separated and the aqueous layer was extracted with diethyl ether. The combined organic layer was washed with water and brine, dried (MgSO4), and concentrated in vacuo. Purification by flash column chromatography on silica gel (25% ethyl acetate in hexane) gave the title compound (13.0 g, 95% yield) as a colorless oil. IR

(neat) 2950, 2840, 2234 (w, CN), 1532, 1438, 1319, 1204, 788 cm-1. 1H NMR (500 MHz,

CDCl3) δ 6.61 (d, J = 2.0 Hz, 2H, ArH), 6.40 (t, J = 2.0 Hz, 1 H, ArH), 3.81 (s, 6 H, -OCH3),

+ 1.71 (s, 6 H, -C(CH3)2-); mass spectrum (ESI) m/z (relative intensity) 206 (M +H, 100).

HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 4.5 min for the title compound.

2-(3,5-Dimethoxyphenyl)-2-methylpropanal (3.55)65. To a solution of 3.54 (12.9 g, 63.0 mmol) in anhydrous CH2Cl2 (441 mL) at –78 °C, under an argon atmosphere was added

1M solution of DIBAL-H in Hexane (189 mL). The reaction mixture was stirred for 30 min and then quenched by dropwise addition of potassium sodium tartrate (10% solution in water) at – 78 °C. Following the addition, the mixture was warmed to room temperature, stirred for an additional 50 minutes and then diluted with ethyl acetate. The organic phase was separated, and the aqueous phase extracted with ethyl acetate. The combined organic layer was washed with brine, dried (MgSO4), and the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel (10-35% ethyl acetate in hexane) to give 11.4 g of 3.55 as viscous oil in 87% yield. IR (neat) 2938, 2838, 2705 (w, CHO), 1725 (s, >C=O), 1595, 1457, 1424, 1314,

-1 1 1205, 1156, 1066, 835 cm . H NMR (500 MHz, CDCl3) δ 9.46 (s, 1H, -CHO), 6.40 (d, J

191

= 2.0 Hz, 2H, 2-H, 6-H, ArH), 6.39 (t, J = 2.0 Hz, 1H, 4-H, ArH), 3.78 (s, 6H, OMe), 1.43

13 (s, 6H, -C(CH3)2-). C NMR (100 MHz CDCl3) δ 201.6 (-CHO), 161.1 (ArC), 143.6 (ArC),

105.1 (ArC), 98.6 (ArC), 65.7 (>(C)CHO), 55.2 (-OCH3), 50.5, 22.3. Mass spectrum (EI) m/z (relative intensity) 208 (M+, 25), 196 (16), 179 (M+-CHO), 165 (25), 151 (14), 139 (39),

+ 91 (20), 77 (20). Exact mass (EI) calculated for C12H16O3 (M ), 208.10995; found,

208.11077. HPLC (4.6 mm × 250 mm, Supelco Discovery column, acetonitrile/water) showed purity of 98.5% and retention time of 4.4 min for the title compound.

(Z)-3,5-Dimethoxy-1-(2-methyl-8-phenoxyoct-3-en-2-yl)benzene (3.56)65. To a stirred suspension of (5-phenoxypentyl) triphenylphosphonium bromide (74.5 g, 147.4 mmol) in dry THF (39 mL) at 0 ºC, under an argon atmosphere was added potassium bis(trimethylsilyl)amide (29.0 g, 145.2 mmol). The mixture was stirred for 30 minutes at

10 ºC to ensure complete formation of the orange phosphorane. A solution of aldehyde

3.55 (11.4 g, 54.6 mmol) in 8.6 mL THF was added dropwise to the resulting slurry, at 0

ºC. The reaction was stirred for 2 hours at room temperature and upon completion was quenched by the addition of saturated aqueous NH4Cl. The organic layer was separated and the aqueous phase was extracted with diethyl ether. The combined organic layer was washed with brine and dried (MgSO4) and the solvent was evaporated under reduced pressure. The residue was purified on a silica gel (5-15 % diethyl ether in hexanes) to give 18.0 g compound 3.56 as colorless oil in 93% yield. IR (neat) 2958, 2835, 1594,

-1 1 1422, 1243, 1204, 1153, 1052, 753 cm . H NMR (500 MHz, CDCl3)  7.26 (m as t, J =

7.5 Hz, 2H, 3-H, 5-H, OPh), 6.91 (m as t, J = 7.5 Hz, 1H, 4-H, OPh), 6.83 (m as d, J =

7.5 Hz, 2H, 2-H, 6-H, OPh), 6.55 (d, J = 2.5 Hz, 2H, 2-H, 6-H, ArH), 6.27 (t, J = 2.5 Hz,

192