Novel Cannabinergic Ligands and Molecular Probes

Thesis Presented

Shashank Satish Kulkarni

The Bouvé Graduate School of Health Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Medicinal Chemistry and Drug Discovery

NORTHEASTERN UNIVERSITY

BOSTON, MASSACHUSETTS

March 20th, 2017

Signature page 1

Northeastern University Bouvé Graduate School of Health Sciences

Thesis title: Novel Cannabinergic Ligands and Molecular Probes

Author: Shashank Satish Kulkarni

Program: Medicinal Chemistry

Approval for thesis requirements of the Doctor of Philosophy in Medicinal Chemistry

Thesis Committee (Chairman) ______Date ______

______Date ______

Director of the Graduate School ______Date ______

Dean ______Date ______

Copy Deposited in Library ______Date ______

Signature page 2

Northeastern University Bouvé Graduate School of Health Sciences

Thesis title: Novel Cannabinergic Ligands and Molecular Probes

Author: Shashank Satish Kulkarni

Program: Medicinal Chemistry

Approval for thesis requirements of the Doctor of Philosophy in Medicinal Chemistry

Thesis Committee (Chairman) ______Date ______

______Date ______

Director of the Graduate School ______Date ______

Table of Contents

Page

Abstract 6

Acknowledgements 8

List of Tables 10

List of Figures 11

List of Schemes 14

List of Abbreviations 16

Chapter 1 Introduction 19

Chemical Constituents of Cannabis 19

Cannabinoid Receptors 21

Activation of Cannabinoid Receptors 22

Cannabinoid Receptor Ligands 23

Cannabinoid Based Marketed Drugs 31

Need for Novel Cannabinergic Therapies 32

References 33

Chapter 2 Controlled Deactivation Nabilone Analogs 41

Objective and Specific Aims 41

Chemistry 48

Results and Discussion 61

Pharmacological Assessment 68

Controlled Deactivation Ligands as Molecular Probes 76

Chemistry 77

Results and Discussion 79

Conclusions 82

Experimental Section 84

References 147

Chapter 3 Probing the Pharmacophoric Space at the Phenolic Hydroxyl (C1) of Nabilone

Introduction 152

Objective and Specific Aims 154

Chemistry 155

Results and Discussion 157

Pharmacological Evaluation of Lead Compounds 164

Conclusions 168

Experimental Section 169

References 191

Chapter 4 Fluorescent Cannabinergic Probes 194

Introduction 194

Objective and Specific Aims 197

Chemistry 198

Results and Discussion 202

Conclusions 209

Experimental Section 210

References 239

ABSTRACT

Part I

Classical cannabinoids including the plant derived (-)-Δ9-Tetrahydrocannabinol [(-)-Δ9-THC] and its congeners produce their biological effects by modulating the cannabinoid receptors CB1 and CB2. These two GPCR’s are at present being targeted for a variety of conditions such as glaucoma, pain, neurodegeneration, inflammation and eating disorders. Unfortunately, only a limited number of cannabinergic agents have been approved till date due to the poor pharmacokinetic/pharmacodynamics (PK/PD) properties as well as adverse psychotropic side effects associated with CB1 receptor activation. We recently reported on a controlled deactivation/detoxification approach where the “soft” drug concept of enzymatic deactivation was combined with a “depot effect” that is frequently observed with Δ9-THC and other hydrophobic cannabinoids.

Our earlier generation ligands feature a metabolically labile ester group at strategic positions within the THC structure. In an effort to develop controlled deactivation cannabinoids with faster onset/offset and shorter duration of action than the currently existing THC analogs we have now focused on cannabinergic templates with enhanced polar characteristics that are associated with the depot effect. The novel compounds reported in this thesis exhibit high CB receptor binding affinity, in vitro and in vivo potency and efficacy, and are susceptible to enzymatic hydrolysis by plasma esterases in a controllable manner. Also, their hydrolytic metabolites are inactive at CB receptors. One of our second generations analog AM10843 is found to be a remarkably potent and efficacious CB1 receptor agonist with relatively short duration of action in in vivo experiments involving rodents and non-human primates. Also, to characterize the ligand binding

6 domain of the cannabinoid receptors, covalent/irreversible probes bearing a photoactivable azido

(-N3) and electrophilic isothiocyanate (NCS) and nitrate ester (ONO2) groups were synthesized.

Part II

Following the discovery of Δ9-THC and other cannabinoids, a number of compounds have been synthesized in order to define the structure activity relationships (SAR) of THC at CB1 and CB2 receptors. Δ9-THC has four distinct pharmacophores: a phenolic hydroxyl at C1, a side chain at

C3, a northern aliphatic hydroxyl at C9 or C11, and a southern aliphatic hydroxyl. Number of analogs was synthesized to probe the pharmacophoric space at the C1 position of Nabilone. SAR studies show that there exists a unique pharmacophoric space at the C1 position of nabilone where the ester functionality is best suited for cannabinoid receptor activity.

The existence of pharmacophoric space at the C1 position of nabilone led to design and synthesis of novel fluorescently labelled cannabinergic ligands as pharmacological probes to study cellular actions of cannabinoids. A variety of fluorescent analogs were synthesized and evaluated for their activity at the CB receptors. Coumarin derivatives AM10853 and AM10854 showed high affinity at the CB1 receptor and are currently being evaluated as imaging agents in cell based experiments.

Acknowledgements

It gives me great pleasure to acknowledge all those who have guided me towards the completion of my doctorate. This has been possible mainly because of the Lord above, who has been with me throughout this journey.

I would like to express my deepest gratitude to my advisor and the Director of Center for Drug

Discovery, Dr. Alexandros Makriyannis for his guidance and support throughout this process.

The drug discovery and medicinal chemistry experience and knowledge that I gained from his lab has motivated me to continue my career in the pharmaceutical industry. I would also like to extend my gratitude to my co-advisor, Dr. Spyros Nikas who has trained me to think like a scientist and for his continuous guidance throughout PhD dissertation. I am also grateful to Dr.

Jack Bergman for his behavioral pharmacology expertise and providing me with all the in vivo data. I also wish to thank Dr. Raymond Booth for serving on my thesis committee and providing suggestions for my thesis. I really appreciate Dr. Andreas Goutopoulos for serving on my thesis committee and offering an industrial perspective on my project.

I am very thankful to Dr. Michael Malamas for his selfless help. He has helped me learn the attitude and approach towards drug discovery. I would also like to thank Dr. Kiran Vemuri for day to day help with chemistry lab instruments. I would like to extend my gratitude to Dr.

Kumar Vadivel, Dr. Vidyanand Shukla and Dr. Michael McCormack for their excellent chemistry knowledge and expertise rendered during my doctoral work.

I would like to acknowledge my colleagues from the CDD Biochemistry and Pharmacology groups for the in vitro and in vivo studies. Special thanks to Dr. Anisha Korde, Chandrashekhar

Honrao, Jimit Raghav and Chris Liu for their friendship and support throughout my doctorate. I

8 am also thankful to the entire CDD and Pharmaceutical Sciences staff for their support and help throughout this process.

Words cannot express the thanks I owe to Shruthi Ramkumar, my wife for her unconditional support, love and encouragement. I would like to thank my parents, Vijaya and Satish Kulkarni for their support and guidance from day one. I would also like to thank the Ramkumar family, my brother Shreyas and his wife, Prerna for constant encouragement. I would like to extend my gratitude to my uncle, Vishwanath Naik for his advice and encouragement. Lastly, my friends especially Girish, Anay, Samved, Ameya and Pushkar and their families for their support that has guided me through the best and worst of times, and I am forever grateful to them.

List of Tables

Page

Table 1.1: Chemical constituents of cannabis sativa 21

Table 2.1: Biochemical analysis of AM7499 63

Table 2.2: Binding affinities (Ki) of controlled deactivation nabilone analogs 63

Table 2.3: Metabolic stabilities (t1/2) of controlled deactivation analogs 66

Table 2.4: Functional evaluation of controlled deactivation analogs 68

Table 2.5: Binding (Ki) and % labeling assessment of cannabinergic probes 80

Table 3.1: Binding affinities of C1 substituted nabilone analogs 159

Table 3.2: cAMP data of C1 substituted nabilone analogs 162

Table 3.3: Metabolic stability of C1 substituted nabilone analogs 163

Table 4.1: Binding affinities of fluorescently labelled cannabinergic probes 204

Table 4.2: Fluorescence properties of cannabinergic probes 205

Table 4.3: Binding affinities of fluorescent cannabinoids tethered via linker 207

Table 4.4: Functional and plasma stability assessment of key fluorescent cannabinergic analogs 209

List of Figures

Page

Figure 1.1: Structures of phytocannabinoids 22

Figure 1.2: Cannabinoid receptors signaling 24

Figure 1.3: Endocannabinoid signaling system 25

Figure 1.4: Structures of AEA and 2-AG 26

Figure 1.5: Metabolic enzymes of endocannabinoid system 27

Figure 1.6: Examples of classical cannabinoids 28

Figure 1.7: Non-classical cannabinoids 29

Figure 1.8: Aminoalkyl indole compounds 30

Figure 1.9: Diarylpyrazole analogs 31

Figure 2.1: Soft drug controlled release approach 43

Figure 2.2: Rational drug design 44

Figure 2.3: Structure and binding affinity data of AM7499 45

Figure 2.4: Cannabinoid receptor binding affinities of Nabilone 46

Figure 2.5: SAR studies of Nabilone analogs with functionalized side chain 46

Figure 2.6: Investigation of Friedel-Crafts allylation reaction 57

Figure 2.7: Hypothermia assessment of AM7499 69

Figure 2.8: Analgesia testing in mice of AM7499 and HU210 70

Figure 2.9: CB1 discriminative stimulus effects in squirrel monkeys for HU210 and AM7499 71

Figure 2.10: Hypothermic effects of AM10806, AM10843 and AM10811 72

Figure 2.11: Tail-flick latencies in a hot water-bath (52°C) after administration of AM10806, AM10843 and AM10819 74

Figure 2.12: CB1 discriminative stimulus effects in squirrel monkeys for

AM10806 and AM10843 75

Figure 2.13: Saturation binding curves using [3H] CP-55,940 for rCB1 (left panel) and hCB1 (right panel) receptors preincubated with the AM10858 (55) and AM10859 (56) analogs 82

Figure 3.1: Four major pharmacophores of classical cannabinoids 153

Figure 3.2: Exploration of pharmacophoric space at C1 position of nabilone 155

Figure 3.3:.Hypothermia assessment of nabilone (AM10806) and AM10816 in rats 165

Figure 3.4:.Hypothermia assessment of nabilone (AM10806) and AM10846 in mice 166

Figure 3.5:.Analgesia test of nabilone (AM10806) and AM10834 in mice 168

Figure 4.1: Ligand based fluorescent probes for GPCR’s 196

Figure 4.2: SAR studies of fluorescent cannabinoids 198

List of Schemes

Page

Scheme 1: Retrosynthetic analysis of cannabinergic analogs 49

Scheme 2: Synthesis of chiral terpene 49

Scheme 3: Re-synthesis of AM 7499 with an improved methodology 51

Scheme 4: Cyano substituted controlled deactivation analog 52

Scheme 5: Improved synthesis of 21 54

Scheme 6: Synthesis of C3’ ester analog 55

Scheme 7: Synthesis of C4’ ester analog 56

Scheme 8: Synthesis of an advanced building block for parallel synthesis 59

Scheme 9: Functionalized side-chain analogs 60

Scheme 10: Side-chain carbamate and urea derivatives 61

Scheme 11: Synthesis of irreversible compounds 78

Scheme 12: Synthesis of irreversible compounds using advanced acid building block 79

Scheme 13: Synthesis of nabilone 156

Scheme 14: Modification of phenolic hydroxyl group of nabilone 157

Scheme 15: Synthesis of fluorescent probes 199

Scheme 16: Synthesis of linkers 200

Scheme 17: Synthesis of linker substituted fluorescent probes 201

List of Abbreviations

THC Tetrahydrocannabinol

Δ9-THC Δ9-Tetrahydrocannabinol

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

CB Cannabinoid

CBD Cannabidiol

CBN Cannabinol

CBG Cannabigerol

CNS Central Nervous System

CB Cannabinoid

GPCRs G-protein coupled receptors

AEA Anandamide

2-AG 2-arachidonoyl Glycerol

AC Adenyl Cyclase

TRPV1 Vanilloid Receptor-Type 1 TRPV1

MAPK Mitogen-Activated Protein Kinases

ECS Endocannabinoid System

NAPE N-acylphosphatidylethanolamine

PLD Phospholipace D

FAAH Fatty Acid Amide Hydrolase

DAG Diacylglycerol

DGL Diacylglycerol-lipase

PLC Phospholipase C

MGL Monoacyl Glycerol Lipase cyp 450 Cytochrome P450

LD50 Median Lethal Dose

AIDS Acquired Immuno Deficiency Syndrome

MS Multiple Sclerosis

PK Pharmacokinetic

PD Pharmacodynamic

USFDA US Food and Drug administration

11-OH-THC 11-Hydroxytetrahydrocannabinol

Vd Volume of Distribution

BBB Blood Brain Barrier

NMR Nuclear Magnetic Resonance

IR Infrared Spectroscopy

SNAr Nucleophilic Aromatic Substitution

DMF Dimethylformamide p-TSA p-Toluenesulfonic acid

CHCl3 Chloroform

DMSO Dimethylsulfoxide

ACN Acetonitrile

DIBAL-H Diisobutylaluminium hydride

THF Tetrahydrofuran

NaHMDS Sodium hexamethyldisilyl amide

TMSOTf Trimethylsilyl trifluoromethanesulfonate

TBDMS-Cl tert-Butyldimethylchlorosilane

DMAP Dimethylaminopyridine tPSA Total Polar Surface Area

18 cAMP Cyclic Adenosine Monophosphate cLogP Calculated Logarithm of Partition Coefficient

CHAPTER 1: INTRODUCTION

Medical and recreational use of Cannabis Sativa goes beyond centuries.1 Cannabis, commonly known as marijuana, has been used to treat multiple diseases with some of the earliest record dating back to 2600 BC, prescribing its use for cramps and pain. But it was not until mid-19th century when Dr. William O’Shaughnessy reported the medical use of marijuana.2 He utilized cannabis extracts in small doses as analgesics for rheumatoid joint pain. This report also suggested marijuana’s use for stimulating digestive organs and anti-convulsive properties. After this discovery, the therapeutic role of cannabis as an anti-spasmodic agent and muscle relaxant was explored.2 Cannabis was available in the US Pharmacopeia for almost 150 years until federal legislation made it illegal in 1942.

Over the years, therapeutic indications for cannabis have been increasing but clinical evidence exists for moderately few. In 1964, Mechoulam and Gaoni elucidated structure of (-)-Δ9-

Tetrahydrocannabinol (Δ9-THC), the psychoactive component of cannabis.3 Since then, therapeutic use of cannabis and its synthetic analogs have gained momentum. The antiemetic effects were reported in a clinical trial with patients undergoing chemotherapy.4 Noyes et al demonstrated pharmacological evidence of antinociceptive effects of THC when compared to codine.5

Chemical constituents of Cannabis

Marijuana belongs to the Cannabaceae family, within the Cannabis genus and the the sativa species.6 Around 500 compounds consisting mainly of cannabinoids and terpenoids have been isolated (Table 1.1).6 Flavonoids, Sugars, hydrocarbons, steroids, amino acids and nitrogenous compounds have also been reported in small amounts. 20

Table 1.1*: Chemical constituents of cannabis sativa

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

Cannabinoids are polyketides derived from malonyl-CoA, hexanoyl-CoA unit’s prenylated with geranyl phosphate.7-9 Tetrahydrocannabinol (THC), Cannabidiol (CBD) and Cannabinol (CBN) are pharmacologically more relevant than the other known phytocannabinoids (Figure 1.1).10

THC exists in two isomeric forms, Δ9-THC and Δ8-THC depending upon the position of the double bond in the C-ring. Both isomers display (-) (6aR, 10aR) stereochemistry and are equipotent at the cannabinoid receptors (CB) but Δ8-THC is chemically more stable. The opposite enantiomer (+) (6aS, 10aS) was synthesized in the laboratory and found to be pharmacologically inactive at the CB receptors.11 The other two phytocannabinoids, CBD and

CBN are pharmacologically relevant as they are devoid of side-effects associated with the CB receptor activation.

Figure 1.1: Structures of phytocannabinoids.

Cannabinoid Receptors

The G-protein coupled receptor (GPCR) superfamily is divided into five major classes: glutamate, rhodopsin, adhesion, frizzled and secretin.12 CB receptors are membrane bound receptors that belong to rhodopsin like family of GPCR’s. Typically, a GPCR is characterized by a seven transmembrane domains which are connected by extracellular and intracellular loops.

The G-protein consists of three subunits: α, β, and γ which upon agonist binding disassociates and couples to other cellular proteins (eg. Adenyl cyclase, protein kinase, etc) which in turn triggers downstream signaling.

Howlett et al provided the first evidence of interaction of cannabinoids with cannabinoid receptors when they saw a decrease in cAMP levels in neuroblastoma cells.13, 14 To date, two cannabinoid receptors, CB1 and CB2 have been identified, purified and cloned. CB1 receptor was cloned from cDNA generated from rat cerebral cortex15 followed by characterization of CB1 as the major GPCR in the brain.16, 17 Subsequently in 1993, CB2 receptor was discovered in promyelocytic leukemia cell line.18 Lately, it’s been discovered that cannabinoids can stimulate another orphan GPCR, GPR55 and it is currently being evaluated as a potential CB3 receptor.19

CB1 receptors20 are predominantly expressed in the brain and in other organs such as kidney, liver and lungs while CB2 receptors are expressed primarily in the immune system.21

The human CB1 and CB2 receptors share only 44% and 68% amino acid sequence homology within their transmembrane domain.18 The CB1 receptor is highly conserved across species as compared to CB2 receptors.22-24 There is 97.3% sequence homology between rat and human CB1 receptor. On the other hand, the human and rat CB2 receptors share 81% amino acid sequence homology.25

Activation of CB receptors

Both CB1 and CB2 activate various signaling pathways that include adenylyl cyclase and cAMP, mitogen-activated protein kinase (MAPK) and regulate intracellular calcium levels (Figure 1.2).

14 The CB receptors are coupled to Gi/o-protein subtype. Upon receptor activation by an agonist, there is a conformational change of the receptor and an exchange of G-protein from inactive guanine nucleotide GDP to active GTP followed by dissociation of G-protein into α and βγ subunits.26 The α subunit binds to adenylate cyclase and inhibits its activity thereby decreasing

23 the levels of secondary messenger cAMP and negatively affecting downstream signaling. The signaling mechanisms for βγ subunits are different as compared to the α subunit. They regulate the phospholipase C (PLC) isoforms and activate MAPK signaling cascade.27

Figure 1.2: Cannabinoid receptors signaling28

Cannabinoid receptor ligands

Cannabinoid receptor ligands are natural products or synthetically derived analogs that bind to the CB receptors. Cannabinergic ligands can be divided into different classes depending upon their structure and pharmacological activity. These classes include endocannabinoids, classical and non-classical cannabinoids, aminoalkylindoles and diarylpyrazoles.

Endogenous ligands and the Endocannabinoid system (ECS)

The ECS is a lipid based signaling system with important regulatory functions (Figure 1.3). It consists mainly of cannabinoid receptors, CB1 and CB2; endocannabinoids 2- arachidonylglycerol (2-AG) and anandamide (AEA); metabolic enzymes mainly fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL); and biosynthetic enzymes such as diacylglycerol lipase (DAGL) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-

PLD). The discovery of CB receptors in early 1990’s suggested the presence of endogenous ligands that bind to these receptors. This led to the identification of 2 key endocannabinoids, 2-

AG and AEA (Figure 1.4).

Figure 1.3: Endocannabinoid signaling system

Studies from numerous research groups suggest the role of ECS in physiological and pathophysiological processes such as neuromodulation, inflammation, multiple sclerosis, cancer, and appetite modulation. Devane et al isolated and characterized anandamide in 1992.29 It acts as

25 a partial agonist at the CB1 receptor with a binding affinity of 61 nM for rat CB1 and 240 nM for human CB1 receptor. The agonist effects are significantly reduced at the CB2 receptor with a binding affinity of 440 nM for rat CB2 and 1930 nM for human CB2 receptor. AEA is produced on demand and has a short half-life due to due to rapid hydrolysis by FAAH which breaks down to arachidonic acid. 2-AG was isolated and characterized from canine gut.30 It shows agonist properties with low binding affinity of 472 nM for CB1 and 1400 nM for CB2 receptor.31, 32 2-

AG is present in high levels in brain and hydrolyzed by membrane bound enzyme, MGL.

Figure 1.4: Structures of AEA and 2-AG

Inhibition of FAAH and MGL is an attractive target to potentiate endocannabinoid signaling while avoiding the negative effects of CB1 activation (Figure 1.5). These inhibitors can elevate levels of AEA and 2-AG, thus stimulating their pharmacological effect and potentially reducing psychotropic side-effects. Hydrolysis of AEA and 2-AG by FAAH and MGL results in arachidonic acid (AA) which undergoes multiple reactions to form fatty acid derivatives such as prostaglandins, etc.

32 Figure 1.5*: Metabolic enzymes of endocannabinoid system . *Source: Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: A neuroprotective therapeutic modality. Life Sci. - Page 617.

Over the years, numerous research groups have put their efforts to develop potent and metabolically stable AEA and 2-AG analogs. Extensive SAR on AEA led to the discovery of

(R)-methanandamide (AM356) that showed metabolic stability towards FAAH hydrolysis and had a higher binding affinity for CB1 receptor (Ki = 20 ± 1.6 nM).33 SAR studies on the head group of 2-AG has led to the development of stable 2-AG analogs (Nikas et al. unpublished data).

Classical Cannabinoids

Classical cannabinoids are derivatives of THC with a tricyclic benzopyran ring. These ligands include naturally occurring phytocannabinoids and synthetic cannabinoids (Figure 1.6).

Figure 1.6: Examples of classical cannabinoids

Detailed SAR on classical cannabinoids has led to the recognition of four important pharmacophores: phenolic hydroxyl, a side chain, a northern aliphatic hydroxyl at C9 or C11, and a southern aliphatic hydroxyl. The side-chain pharmacophore is critical for potency of the molecule. Compounds bearing a 7 or 8 atom side-chains with a germinal dimethyl substitution at the C1’ position has shown optimal activity at the CB receptors.34 Our group has also synthesized high affinity molecules with a cyclic moiety at the C1’.35-37 Terminal substitution of carbon atom with halogens or other electrophilic groups have resulted in potent CB ligands.38, 39

Highly potent analogs with a hydroxyl group at the C9/C11 position have also been synthesized.37 The phenolic hydroxyl is important for CB1 affinity as substitution of phenolic hydroxyl with a methoxy, hydrogen or fluorine diminishes CB1 affinity but displays some affinity towards the CB2 receptors.40

Non-Classical Cannabinoids

This class of compounds has an AC bicyclic or ACD-tricyclic chemotype and was first reported by Pfizer (Figure 1.7). From their careful SAR, bicyclic analog, CP-55940 was developed showing equal affinity to the CB1 and CB2 receptors.41 This group also developed CP-55244, a potent cannabinoid with the ACD-tricyclic ring system.42 Tritiated-[3H]-CP-55940 is the most used radio ligand to test the binding affinity of cannabinoid agonists. Cannabidiol, a phytocannabinoid with a bicyclic ring system has gained lot of attention over the years for it pharmacological use.43 It has no affinity to the CB receptors and hence does not produce THC like psychotropic effects.

Figure 1.7: Non-classical cannabinoids

Aminoalkyl Indoles

Aminoalkyl indoles as a new class of cannabinergics (Figure 1.8) were discovered in 1990’s during the development of pravadoline.44 R-(+)-WIN55212-2 is a high affinity cannabinoid analog with moderate selectivity to the CB2 receptor and behaves similar to THC in in vivo studies.45 Cannabinoid receptors in the rat brain were characterized using radio labelled [3H]-R-

(+)-WIN55212-2.46 Substantial SAR around this scaffold has led to analogs with high affinity and selectivity at the CB receptors.

Figure 1.8: Aminoalkyl indole compounds

Diarylpyrazoles

The discovery and development of this class of compounds was first reported by Sanofi pharmaceuticals (Figure 1.9).47 The analogs developed in this class are antagonist or inverse agonist at the CB receptors. From this series, SR141716A also known as Rimonabant was a potent and selective CB1 inverse agonist initially approved to treat obesity.48 It was approved in

Europe and other countries but was withdrawn from the market in 2009 due to serious side- effects such as suicidal tendencies, depression, etc. AM251, iodine substituted analog of

SR141716A displays more potency and selectivity than rimonabant.49 SR144528 is a potent and selective CB2 inverse agonist developed in this class and is currently used as an investigational drug to study CB2 receptors. Scientists at Bristol Myers Squibb developed a new series of CB1

30 antagonist with the 3, 4-diarylpyrazolines scaffold. From this SAR, Ibipinabant (SLV-319) showed CB1 selectivity by 1000 fold.50

Figure 1.9: Diarylpyrazole analogs

Cannabinoid-Based Marketed Drugs

Medicinal usage of cannabis for variety of indications was well documented in the 20th century.

But due to high abuse liability, the medical usage of cannabis suffered. In the last 30 years, lot of research has been done to develop cannabinoid based therapeutics for indications such as pain, obesity, eating disorders, cancer, and glaucoma. Till date, three cannabinergic drugs have been approved for therapeutic use. They are:

Marinol. In 1985, the United States Food and Drug Administration (USFDA) approved synthetic Δ9-THC for the treatment of anorexia associated with weight loss in AIDS patients and for nausea and vomiting for patients undergoing cancer chemotherapy. It is Schedule III drug showing low risk of physical and mental dependence. Still, Marinol use is not widely accepted due to undesirable pharmacokinetic/pharmacodynamics (PK/PD) properties.51

Nabiximols/Sativex is an oralmucosal spray containing Δ9-THC and CBD in 1:1 ratio along with specific minor cannabinoids. It is currently approved in 16 countries to treat spasticity in multiple sclerosis (MS) patients. Clinical trials showing relief from neuropathic pain,52 muscle spasms,53 and bladder related symptoms54 have also been reported. It has not been approved by

USFDA for therapeutic use.

Nabilone is approved to treat nausea and vomiting in patients undergoing cancer chemotherapy and as a supplemental analgesic for neuropathic pain.55 It was first approved by USFDA in 1985 but was withdrawn from the market in 1989 due to commercial reasons. It was reapproved by

USFDA in 2006.

Need for Novel Cannabinergic Therapies

As mentioned above, only three cannabinergic drugs have been developed to date. The development of such therapeutically useful medications is difficult owing to the undesirable side effects associated with CB1 receptor activation. This includes CNS and cardiovascular effects, abuse potential, poor oral bioavailability, and unpredictable time course of action and detoxification.56 For example, Δ9-THC undergoes oxidative metabolism through the cytochrome p450 enzymes to for 11-hydroxy- Δ9-THC, a potent and long acting psychoactive cannabinoid.

Hence, there is a need for safer THC based medications with favorable oral bioavailability, consistent efficacy and predictable duration of action. Newly synthesized cannabinergic ligands may have therapeutic utility for indications such as pain, glaucoma, anorexia, and cannabis addiction.

REFERENCES

1. Hosking, R. D.; Zajicek, J. P. Therapeutic potential of cannabis in pain medicine. Br J

Anaesth 2008, 101, 59-68.

2. O'Shaughnessy, W. B. On the Preparations of the Indian Hemp, or Gunjah: Cannabis

Indica Their Effects on the Animal System in Health, and their Utility in the Treatment of

Tetanus and other Convulsive Diseases. Provincial Medical Journal and Retrospect of the

Medical Sciences 1843, 5, 363-369.

3. Mechoulam, R.; Gaoni, Y. A TOTAL SYNTHESIS OF DL-DELTA-1-

TETRAHYDROCANNABINOL, THE ACTIVE CONSTITUENT OF HASHISH. J Am Chem

Soc 1965, 87, 3273-5.

4. Sallan, S. E.; Zinberg, N. E.; Frei, E., 3rd. Antiemetic effect of delta-9- tetrahydrocannabinol in patients receiving cancer chemotherapy. N Engl J Med 1975, 293, 795-7.

5. Noyes, R., Jr.; Brunk, S. F.; Avery, D. A.; Canter, A. C. The analgesic properties of delta-9-tetrahydrocannabinol and codeine. Clin Pharmacol Ther 1975, 18, 84-9.

6. Elsohly, M. A.; Slade, D. Chemical constituents of marijuana: the complex mixture of natural cannabinoids. Life Sci 2005, 78, 539-48.

7. Stout, J. M.; Boubakir, Z.; Ambrose, S. J.; Purves, R. W.; Page, J. E. The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes. Plant J 2012, 71, 353-65.

8. Fellermeier, M.; Zenk, M. H. Prenylation of olivetolate by a hemp transferase yields cannabigerolic acid, the precursor of tetrahydrocannabinol. FEBS Lett 1998, 427, 283-5.

9. Fellermeier, M.; Eisenreich, W.; Bacher, A.; Zenk, M. H. Biosynthesis of cannabinoids.

Incorporation experiments with (13)C-labeled glucoses. Eur J Biochem 2001, 268, 1596-604.

10. Gambaro, V.; Dell’Acqua, L.; Farè, F.; Froldi, R.; Saligari, E.; Tassoni, G. Determination of primary active constituents in Cannabis preparations by high-resolution gas chromatography/flame ionization detection and high-performance liquid chromatography/UV detection. Analytica Chimica Acta 2002, 468, 245-254.

11. Mechoulam, R.; Braun, P.; Gaoni, Y. A stereospecific synthesis of (-)-delta 1- and (-)- delta 1(6)-tetrahydrocannabinols. J Am Chem Soc 1967, 89, 4552-4.

12. Fredriksson, R.; Lagerstrom, M. C.; Lundin, L. G.; Schioth, H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol 2003, 63, 1256-72.

13. Howlett, A. C.; Fleming, R. M. Cannabinoid inhibition of adenylate cyclase.

Pharmacology of the response in neuroblastoma cell membranes. Mol Pharmacol 1984, 26, 532-

14. Howlett, A. C. Cannabinoid inhibition of adenylate cyclase. Biochemistry of the response in neuroblastoma cell membranes. Mol Pharmacol 1985, 27, 429-36.

15. Devane, W. A.; Dysarz, F. A., 3rd; Johnson, M. R.; Melvin, L. S.; Howlett, A. C.

Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol 1988,

34, 605-13.

16. Herkenham, M.; Lynn, A. B.; Johnson, M. R.; Melvin, L. S.; de Costa, B. R.; Rice, K. C.

Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J Neurosci 1991, 11, 563-83.

17. Matsuda, L. A.; Lolait, S. J.; Brownstein, M. J.; Young, A. C.; Bonner, T. I. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990, 346, 561-4.

18. Munro, S.; Thomas, K. L.; Abu-Shaar, M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993, 365, 61-5.

19. Ryberg, E.; Larsson, N.; Sjogren, S.; Hjorth, S.; Hermansson, N. O.; Leonova, J.;

Elebring, T.; Nilsson, K.; Drmota, T.; Greasley, P. J. The orphan receptor GPR55 is a novel cannabinoid receptor. Br J Pharmacol 2007, 152, 1092-101.

20. Demuth, D. G.; Molleman, A. Cannabinoid signalling. Life Sci 2006, 78, 549-63.

21. Galiegue, S.; Mary, S.; Marchand, J.; Dussossoy, D.; Carriere, D.; Carayon, P.;

Bouaboula, M.; Shire, D.; Le Fur, G.; Casellas, P. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem

1995, 232, 54-61.

22. Yamaguchi, F.; Macrae, A. D.; Brenner, S. Molecular cloning of two cannabinoid type 1- like receptor genes from the puffer fish Fugu rubripes. Genomics 1996, 35, 603-5.

23. Soderstrom, K.; Johnson, F. CB1 cannabinoid receptor expression in brain regions associated with zebra finch song control. Brain Res 2000, 857, 151-7.

24. Soderstrom, K.; Leid, M.; Moore, F. L.; Murray, T. F. Behaviroal, pharmacological, and molecular characterization of an amphibian cannabinoid receptor. J Neurochem 2000, 75, 413-

23.

25. Mukherjee, S.; Adams, M.; Whiteaker, K.; Daza, A.; Kage, K.; Cassar, S.; Meyer, M.;

Yao, B. B. Species comparison and pharmacological characterization of rat and human CB2 cannabinoid receptors. Eur J Pharmacol 2004, 505, 1-9.

26. Smith, T. H.; Sim-Selley, L. J.; Selley, D. E. Cannabinoid CB1 receptor-interacting proteins: novel targets for central nervous system drug discovery? Br J Pharmacol 2010, 160,

454-66.

27. Rajagopal, S.; Rajagopal, K.; Lefkowitz, R. J. Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat Rev Drug Discov 2010, 9, 373-86.

28. Guzmán, M.; Galve-Roperh, I.; Sánchez, C. Ceramide: a new second messenger of cannabinoid action. Trends in Pharmacological Sciences 2001, 22, 19-22.

29. Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson, L. A.; Griffin, G.;

Gibson, D.; Mandelbaum, A.; Etinger, A.; Mechoulam, R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992, 258, 1946-9.

30. Mechoulam, R.; Ben-Shabat, S.; Hanus, L.; Ligumsky, M.; Kaminski, N. E.; Schatz, A.

R.; Gopher, A.; Almog, S.; Martin, B. R.; Compton, D. R.; et al. Identification of an endogenous

2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol

1995, 50, 83-90.

31. Janero, D. R.; Vadivel, S. K.; Makriyannis, A. Pharmacotherapeutic modulation of the endocannabinoid signalling system in psychiatric disorders: drug-discovery strategies. Int Rev

Psychiatry 2009, 21, 122-33.

32. Hwang, J.; Adamson, C.; Butler, D.; Janero, D. R.; Makriyannis, A.; Bahr, B. A.

Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: A neuroprotective therapeutic modality. Life sciences 2010, 86, 615-623.

33. Abadji, V.; Lin, S.; Taha, G.; Griffin, G.; Stevenson, L. A.; Pertwee, R. G.; Makriyannis,

A. (R)-methanandamide: a chiral novel anandamide possessing higher potency and metabolic stability. J Med Chem 1994, 37, 1889-93.

34. Huffman, J. W.; Miller, J. R.; Liddle, J.; Yu, S.; Thomas, B. F.; Wiley, J. L.; Martin, B.

R. Structure-activity relationships for 1',1'-dimethylalkyl-Delta8-tetrahydrocannabinols. Bioorg

Med Chem 2003, 11, 1397-410.

35. Nadipuram, A. K.; Krishnamurthy, M.; Ferreira, A. M.; Li, W.; Moore, B. M. Synthesis and testing of novel classical cannabinoids: exploring the side chain ligand binding pocket of the

CB1 and CB2 receptors. Bioorg Med Chem 2003, 11, 3121-32.

36. Papahatjis, D. P.; Nahmias, V. R.; Nikas, S. P.; Andreou, T.; Alapafuja, S. O.; Tsotinis,

A.; Guo, J.; Fan, P.; Makriyannis, A. C1'-cycloalkyl side chain pharmacophore in tetrahydrocannabinols. J Med Chem 2007, 50, 4048-60.

37. Nikas, S. P.; Alapafuja, S. O.; Papanastasiou, I.; Paronis, C. A.; Shukla, V. G.;

Papahatjis, D. P.; Bowman, A. L.; Halikhedkar, A.; Han, X.; Makriyannis, A. Novel 1′,1′-Chain

Substituted Hexahydrocannabinols: 9β-Hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol

(AM2389) a Highly Potent Cannabinoid Receptor 1 (CB1) Agonist. Journal of medicinal chemistry 2010, 53, 6996-7010.

38. Nikas, S. P.; Grzybowska, J.; Papahatjis, D. P.; Charalambous, A.; Banijamali, A. R.;

Chari, R.; Fan, P.; Kourouli, T.; Lin, S.; Nitowski, A. J.; Marciniak, G.; Guo, Y.; Li, X.; Wang,

C.-L. J.; Makriyannis, A. The role of halogen substitution in classical cannabinoids: A CB1 pharmacophore model. The AAPS Journal 2004, 6, 23-35.

39. Tius, M. A.; Kannangara, G. S. K.; Kerr, M. A.; Grace, K. J. S. Halogenated cannabinoid synthesis. Tetrahedron 1993, 49, 3291-3304.

40. Pavlopoulos, S.; Thakur, G. A.; Nikas, S. P.; Makriyannis, A. Cannabinoid receptors as therapeutic targets. Curr Pharm Des 2006, 12, 1751-69.

41. Melvin, L. S.; Johnson, M. R.; Harbert, C. A.; Milne, G. M.; Weissman, A. A cannabinoid derived prototypical analgesic. J Med Chem 1984, 27, 67-71.

42. Griffin, G.; Wray, E. J.; Martin, B. R.; Abood, M. E. Cannabinoid agonists and antagonists discriminated by receptor binding in rat cerebellum. Br J Pharmacol 1999, 128, 684-

43. CADTH Rapid Response Reports. In Cannabinoid Buccal Spray for Chronic Non-

Cancer or Neuropathic Pain: A Review of Clinical Effectiveness, Safety, and Guidelines,

Canadian Agency for Drugs and Technologies in Health

2016.

44. Bell, M. R.; D'Ambra, T. E.; Kumar, V.; Eissenstat, M. A.; Herrmann, J. L., Jr.; Wetzel,

J. R.; Rosi, D.; Philion, R. E.; Daum, S. J.; Hlasta, D. J.; et al. Antinociceptive

(aminoalkyl)indoles. J Med Chem 1991, 34, 1099-110.

45. Griffin, G.; Atkinson, P. J.; Showalter, V. M.; Martin, B. R.; Abood, M. E. Evaluation of cannabinoid receptor agonists and antagonists using the guanosine-5'-O-(3-[35S]thio)- triphosphate binding assay in rat cerebellar membranes. J Pharmacol Exp Ther 1998, 285, 553-

60.

46. Jansen, E. M.; Haycock, D. A.; Ward, S. J.; Seybold, V. S. Distribution of cannabinoid receptors in rat brain determined with aminoalkylindoles. Brain Res 1992, 575, 93-102.

47. Rinaldi-Carmona, M.; Barth, F.; Héaulme, M.; Shire, D.; Calandra, B.; Congy, C.;

Martinez, S.; Maruani, J.; Néliat, G.; Caput, D.; Ferrara, P.; Soubrié, P.; Brelière, J. C.; Le Fur,

G. SR141716A, a potent and selective antagonist of the brain cannabinoid receptor. FEBS

Letters 1994, 350, 240-244.

48. MacLennan, S. J.; Reynen, P. H.; Kwan, J.; Bonhaus, D. W. Evidence for inverse agonism of SR141716A at human recombinant cannabinoid CB1 and CB2 receptors. Br J

Pharmacol 1998, 124, 619-22.

49. Lan, R.; Lu, Q.; Fan, P.; Gatley, J.; Volkow, N. D.; Fernando, S. R.; Pertwee, R.;

Makriyannis, A. Design and synthesis of the CB1 selective cannabinoid antagonist AM281: A potential human SPECT ligand. AAPS PharmSci 1999, 1, 39-45.

50. Lange, J. H.; Coolen, H. K.; van Stuivenberg, H. H.; Dijksman, J. A.; Herremans, A. H.;

Ronken, E.; Keizer, H. G.; Tipker, K.; McCreary, A. C.; Veerman, W.; Wals, H. C.; Stork, B.;

Verveer, P. C.; den Hartog, A. P.; de Jong, N. M.; Adolfs, T. J.; Hoogendoorn, J.; Kruse, C. G.

Synthesis, biological properties, and molecular modeling investigations of novel 3,4- diarylpyrazolines as potent and selective CB(1) cannabinoid receptor antagonists. J Med Chem

2004, 47, 627-43.

51. Stott, C. G.; Guy, G. W. Cannabinoids for the pharmaceutical industry. Euphytica 2004,

140, 83-93.

52. Notcutt, W.; Price, M.; Miller, R.; Newport, S.; Phillips, C.; Simmons, S.; Sansom, C.

Initial experiences with medicinal extracts of cannabis for chronic pain: results from 34 'N of 1' studies. Anaesthesia 2004, 59, 440-52.

53. Fontelles, M. I. M.; García, C. G. Role of Cannabinoids in the Management of

Neuropathic Pain. CNS Drugs 2008, 22, 645-653.

54. Brady, C. M.; DasGupta, R.; Dalton, C.; Wiseman, O. J.; Berkley, K. J.; Fowler, C. J. An open-label pilot study of cannabis-based extracts for bladder dysfunction in advanced multiple sclerosis. Mult Scler 2004, 10, 425-33.

55. Cunningham, D.; Bradley, C. J.; Forrest, G. J.; Hutcheon, A. W.; Adams, L.; Sneddon,

M.; Harding, M.; Kerr, D. J.; Soukop, M.; Kaye, S. B. A randomized trial of oral nabilone and prochlorperazine compared to intravenous metoclopramide and dexamethasone in the treatment of nausea and vomiting induced by chemotherapy regimens containing cisplatin or cisplatin analogues. Eur J Cancer Clin Oncol 1988, 24, 685-9.

56. Sharma, R.; Nikas, S. P.; Paronis, C. A.; Wood, J. T.; Halikhedkar, A.; Guo, J. J.; Thakur,

G. A.; Kulkarni, S.; Benchama, O.; Raghav, J. G.; Gifford, R. S.; Jarbe, T. U.; Bergman, J.;

Makriyannis, A. Controlled-deactivation cannabinergic ligands. J Med Chem 2013, 56, 10142-

57.

Chapter 2: CONTROLLED DEACTIVATION NABILONE ANALOGS

OBJECTIVE AND SPECIFIC AIMS

Modulation of two distinct cannabinoid receptors CB11 and CB22 by (-)-Δ9-

Tetrahydrocannabinol ((-)-Δ9-THC) and its synthetic analogs3 is a promising pharmacotherapeutic strategy to treat variety of indications including pain, CNS disorders, glaucoma, inflammation, cancer and eating disorders.4-10 Unfortunately, only a limited number of cannabinergic agents have been approved till date due to the poor pharmacokinetic/pharmacodynamics (PK/PD) properties as well as adverse psychotropic side effects associated with CB1 receptor activation.11

Novel cannabinergic ligands were designed using the controlled deactivation approach to improve the safety issues related to the current cannabinoid medications. Our goal was to develop cannabinergic ligands with varied duration of action with different onset and offset profile. The controlled deactivation concept which was recently developed in our group uses two important factors to determine the systemic half-life of the ligand, i.e., “soft drug” approach of enzymatic deactivation with a “depot effect” that has been periodically observed with the lipophilic cannabinergic analogues.12-16 The pharmacokinetic and pharmacodynamics (PK/PD) profiles such as poor oral bioavailability and unpredictable duration of action have been improved by the soft drug approach for a variety of drugs.17-19 In the ester group containing soft drugs which are biologically active compounds are designed to undergo a predictable and controllable deactivation or metabolism to inactive metabolites in vivo after achieving their therapeutic effect. The rate of enzymatic hydrolysis by blood esterases can be modulated by

42 incorporating an appropriate stereochemical feature in the vicinity of the hydrolysable group.

The depot effect is the extent to which the ligand is sequestered within the body before it is released for systemic circulation. This process is dependent on the compound’s physicochemical properties and can be modulated by adjusting the LogP and the polar surface area (PSA). Thus, more lipophilic compounds are slowly released in the bloodstream from the depot while more polar compounds are expected to have less of a depot effect.

As shown in Figure 2.1, compounds similar to drug A (less lipophilic) are sequestered in fatty tissues followed by release in blood stream are rapidly hydrolyzed by plasma esterases.

Compounds similar to drug B (more lipophilic) are slowly released in the bloodstream from the depot and slowly inactivated by plasma esterases. The rate of enzymatic inactivation depends on structural features in the vicinity of hydrolysable group and by incorporating features modulating these two parameters (depot effect, enzymatic action) we can obtain ligands with controllable half-lives.

(Both A and B are Soft drugs)

Figure 2.1: Soft drug controlled release approach.15

Our rational design of novel cannabinergic ligands, incorporates an ester group hydrolyzable by esterases within the key pharmacophoric sites of Nabilone and the 11-hexahydro-hydroxymethyl cannabinol prototypes in such a way that the resulting carboxylic acid metabolite formed after enzymatic hydrolysis has minimal or no activity at CB receptors (Figure 2.2).

Figure 2.2: Rational drug design.

To improve the druggability and safety of existing THC-based medications is the long term aim of this work. And this can be achieved by controlling the metabolic biotransformation through enzymatic (esterase) inactivation. These novel analogs are designed to display the pharmacological profile of a cannabinergic ligand. We have been able to design and synthesize novel cannabinoid agonists that have shown in vivo, quick onset/offset profiles and controlled deactivation resulting in inactive metabolities as compared to current cannabinoid based therapies.

AM7499 (Figure 2.3), an hexahydro hydroxymethyl ester analog was synthesized earlier in our group.20 AM7499 exhibited low nanomolar binding affinity at both CB1 and CB2 receptors and showed promise in a preliminary study involving hypothermia effects in rats. This was the motivating factor for improving the synthesis of this compound that enabled eventually a detailed in vitro and in vivo pharmacological evaluation including non-human primates.

Figure 2.3: Structure and binding affinity data of AM7499.

Our lab has developed two controlled-deactivation cannabinergic templates based on the tricyclic classical cannabinoid prototype.13-15 In the first, the C-ring in THC was replaced by a hydrolyzable seven-membered lactone, while in the second; the metabolically labile ester group was placed at the 2′-position of the side chain pharmacophore.

Nabilone is a current drug approved in the market, has a better side effect profile and is less lipophilic than the classical tricyclic cannabinoids.21 Hence, nabilone skeleton was selected for the structure activity relationship (SAR) studies as it is processed faster in the body and is expected to exhibit less depot effect as compared to other classical cannabinoids. Currently, nabilone is being evaluated for potential treatment for cannabis addiction.22 Nabilone exhibits high binding affinities at both the CB receptors (Ki values, CB1: 1.7 nM, CB2: 12 nM) (Figure

2.4).

Figure 2.4: Cannabinoid receptor binding affinities of Nabilone.

To modulate onset/offset, duration of action and to determine the optimal activity at the CB receptors, we explored the pharmacophoric limits for the length of side-chain. The terminal carbon on the side chain was also functionalized using various functional groups such as cyano, morpholino, etc to modulate analogs polarity and CNS permeability (Figure 2.5).

Figure 2.5: SAR studies of Nabilone analogs with functionalized side chain.

Another goal of this project was to make ligands with varied half-lives. Hence we decided to incorporate metabolically labile ester group two or three carbons away from the steric hindrance within the side chain (Figure 2.5). Also, as an extension to this SAR we decided to introduce different functional groups to the side chain and assess the nature of these compounds (Figure

2.5).

The Ligand Assisted Protein Structure (LAPS) approach uses cellular-molecular biology/proteomic experimental paradigm for mapping and targeting putatively critical amino acid residues in the ligand-binding domains of functional proteins. CB receptor probes are high affinity ligands to which reactive groups are attached at appropriate position in order to form irreversible interaction with active site amino acid residues. In order to study the binding motifs of Nabilone analogs to the CB receptor, analogs with a photoactivable and electrophilic functional groups on the side chain pharmacophore were synthesized (Figure 2.5). The isothiocyanate is an electrophilic group that is susceptible to nucleophilic attack from cysteine.

The azide is a photoaffinity label that when exposed to irradiation, nitrogen gas is released and a nitrene is formed. This nitrene is then able to react with nearby amino acid residues in the binding domain to form a ligand-receptor complex.

Biochemical characterizations of the compounds were determined by their in vitro binding affinities for the CB1 and CB2 receptors using membrane preparations from rat brain or HEK293 cells expressing either rCB1, mCB2 or hCB2 receptors. Ligands with good binding affinities (Ki

< 30 nM) will be selected for in vitro metabolic stability towards plasma esterases and liver microsomes. Lead analogs would also be profiled for their functional activity using cAMP functional biochemical assay. The goal was to identify novel cannabinergic ligands with sub

47 nanomolar affinity at the CB receptors, with high potency and efficacy and predictable in vitro half-life.

Two well defined rodent assays (hypothermia and analgesia) were used for initial in vivo screening of lead compounds. The analgesia assay determines the ability of the test compound to activate cannabinergic signaling and thereby reduce nociceptive pain while the hypothermia assay determines the ability of test compound to act as a CB1 agonist and induce hypothermia.

Data from rodent studies will enable the identification of a lead compound with desired pharmacological activity to be studied further for advanced preclinical studies in squirrel monkeys.

CHEMISTRY

Retrosynthetic analysis of novel cannabinergic ligands represented by 16 and 10 requires the carboxy nabilone 10, chiral terpene synthon 6 and resorcinol 5 (Scheme 1). In the synthetic direction, fragments 6 and 5 are joined by Friedel Crafts reaction.

Scheme 1: Retrosynthetic analysis of cannabinergic analogs.

Starting with the commercially available (1R)-(+)-Nopinone (6), the mixture of chiral terpene diacetates (8) was synthesized in two steps as shown in Scheme 2. (1R)-(+)-Nopinone (6) was transesterified23-27 using isopropenyl acetate to give (-)-nopinone enol acetate (7),23, 25 which was then further treated with lead tetra-acetate in refluxing benzene. Upon completion of the reaction, all the insoluble materials were removed from the reaction mixture and further treated with water to remove the black precipitate of lead oxide.27 The resulting crude gave a mixture of diacetates

(8) which was used as such in the subsequent Friedel Crafts alkylation reactions.27

Scheme 2: Synthesis of chiral terpene.

Reagents and conditions: (a) isopropenyl acetate, p-TSA, reflux, 95%; (b) benzene, Lead(IV) acetate, 80 °C, 85%.

AM7499 was synthesized earlier in 10 steps from commercially available 1. This synthetic scheme involved no protection of the phenolic hydroxyl group and resulted in low chemical efficiency providing only a few milligrams of the final compound.20 Now we have modified the synthetic approach using TBDMS protection (Scheme 3) resulting in efficient resynthesis and high overall yield.

The synthesis of hexahydro ester analog is summarized in Scheme 3. The first step involves the deprotonation of (3,5-dimethoxyphenyl)acetonitrile with sodium hydride and geminal dimethylation using iodomethane to give intermediate 2 in 97% yield. Hydrolysis of the resulting nitrile group under basic conditions, gave carboxylic acid intermediate 3 in 90% yield.

Subsequent cleavage of the aryl methyl ether groups using boron tribromide in CH2Cl2 afforded

4 in 71% yield.28 Condensation of resulting resorcinol 4 with chiral terpene diacetates 8 in the presence of catalytic amounts of p-toluenesulfonic acid (p-TSA)27 gave norpinanone 9 in 14% yield. Subsequent closure of dibenzo[b,d]pyran was initiated via catalytic amounts of TMS0Tf27 to generate the ketone 10 in 65% yield. The phenolic hydroxyl group in 10 was protected using

TBDMSCl to give intermediate 11 in 80% yield. Treatment of 11 with a ylide derived from methoxymethyl triphenyl phosphonium bromide and sodium tert-amylate in benzene produced a mixture of enol ethers 12 which were hydrolyzed using wet trichloroacetic acid to give aldehyde

(mixture of α and β epimers) 13 with 97% yield. Epimerization using potassium carbonate in ethanol gave 14 with 78% yield. Sodium borohydride reduction of aldehyde 14 led to the intermediate 15 which was further treated with TBAF to yield the desired hexahydro analog 16 in 95% yield.

Scheme 3: Re-synthesis of AM 7499 with an improved methodology

Reagents and conditions: (a) CH3I, NaH, DMF, 0 ºC to rt, 2 h, 97%; (b) NaOH, n-BuOH, H2O,

50 ºC, reflux, 90%; (c) BBr3, CH2Cl2, 0 ºC to rt, 3.5 h, 71%; (d) NaHCO3,1-Bromobutane, DMF,

165 ºC, MW, 12 min, 91%; (e) 8, p-TSA, CHCl3, 0 ºC to rt, 4 days, 14%; (f) TMSOTf, CH2Cl2,

MeNO2, 0 ºC to rt, 7 h, 65%; (g) TBDMSCl, Imidazole, DMAP, DMF, rt, 12 h, 80%; (h)

BrPh3PCH2OMe, Na tert-amylate, Benzene, 0 ºC to rt, 3 h, 60%; (i) CCl3COOH, H2O, CH2Cl2, rt, 2.5 h, 97%; (j) K2CO3, EtOH, rt, 3 h, 78%; (k) NaBH4, EtOH, 0 ºC, 30 min, 81%; (l) TBAF,

THF, -40 ºC, 30 min, 95%.

Scheme 4: Cyano substituted controlled deactivation analog

Reagents and conditions: (a) NaHCO3, Phenoxy-propyl bromide, DMF, 165 ºC, MW, 12 min,

90%; (b) 9-I-9-BBN, Hexane, 0 ºC to rt, 3.5 h, 85%; (c) 8, p-TSA, CHCl3, 0 ºC to rt, 4 days,

22%; (d) TMSOTf, CH2Cl2, MeNO2, 0 ºC to rt, 7 h, 65%; (e) NaCN, DMSO, 20 ºC – rt, 1 h,

50%; (f) K2CO3, EtOH, rt, 2 h, 50%.

The cyano cannabinoid analogue 21 was synthesized as shown in Schemes 4 and 5. For this analogue, alkylation of carboxylic acid 3 using 3-Phenoxypropyl bromide under microwave conditions was done prior to deprotection of the methoxy groups. 9-Iodo-9-BBN was used instead of BBr3 to cleave all ether groups and introduce the iodo functionality at the terminal side-chain carbon. The yield is significantly less for the deprotection of methoxy groups in the presence of BBr3 (71%) as compared to 9-Iodo-9-BBN. The resulting resorcinol 18 was afforded in 85% yield. The resorcinol was then coupled with chiral diacetates 8 in the presence of catalytic amounts of p-toluenesulfonic acid (p-TSA)27 to give intermediate 19 in 22% yield.

Bicyclic intermediate 19 was then cyclized using TMSOTf27 to yield iodo carboxy nabilone 20 in

65% yield. The iodo group was then displaced using sodium cyanide to afford two compounds which were isolated using flash column chromatography, cyano carboxy nabilone 21 with 50% yield and cyanohydrin analogue 22 with 30% yield. Cyanohydrin was then treated with potassium carbonate to convert back to 21 with good yield. The overall yield for the cyanide displacement reaction was low (60%) resulting in unacceptable loss of material and therefore needed an improvement.

Scheme 5: Improved synthesis of 21

Reagents and conditions: (a) NaCN, DMSO, 20 ºC, 30 min, 95%; (b) TMSOTf, CH2Cl2,

MeNO2, 0 ºC to rt, 7 h, 65%.

The problem of cyanohydrin formation was solved by reversing the steps of benzopyran ring closure and cyanide displacement. Most likely, the reason for the successful modification is the steric hindrance of the carbonyl group in 19, when compared to 20, that prevents the cyanohydrin formation under the experimental conditions used. The overall yields of this reaction were 80%.

Scheme 6: Synthesis of C3’ ester analog

Reagents and conditions: (a) DIBAL-H, CH2Cl2, -78 ºC, 0.5 h, 82%; (b) Ph3PClCH2OEt,

(Me3Si)2NK, THF, 0 ºC to rt, 3 h, 80%; (c) HCl, Dioxane, rt, 2 h, 80%; (d) Oxone, DMF, rt,

90%; (e) NaHCO3, 1-Bromopropane, DMF, 165 ºC, MW, 12 min, 90%; (f) 9-I-9-BBN, Hexane,

0 ºC to rt, 3.5 h, 85%; (g) 8, p-TSA, CHCl3, 0 ºC to rt, 2 days, 22%; (h) TMSOTf, CH2Cl2,

MeNO2, 0 ºC to rt, 4 h, 65%; (i) LiOH, Dioxane/H2O, 24 h, rt, 75%.

Cannabinergic C3’ ester analog 31 was synthesized in 8 steps. The cyanide group in intermediate

2 was reduced using DIBAL-H to afford aldehyde 24 in 82% yield. Wittig olefination on 24 afforded exclusively the Z olefins with J2’H-3’H = 12 Hz to yield intermediate 25 in 80% yield followed by enol ether hydrolysis using 1M HCl to give the aldehyde 26 in 80% yield. This

55 aldehyde was then oxidized to carboxylic acid using oxone to afford intermediate 27 in 90% yield followed by alkylation using 1-Bromopropane under microwave conditions to give intermediate 28 in 90% yield. Subsequent cleavage of the aryl methyl ether by 9-Iodo-9-BBN afforded resorcinol 29 in 85% yield. The resorcinol was then coupled with chiral diacetates 8 in the presence of catalytic amounts of p-toluenesulfonic acid (p-TSA)27 to give intermediate 30 in

22% yield. Bicyclic intermediate 30 was then cyclized using TMSOTf27 to afford C3’ ester cannabinergic ligand 31 with 65% yield. Alkaline hydrolysis of ester side chain in 31 provided the acid 32.29

Scheme 7: Synthesis of C4’ ester analog

Reagents and conditions: (a) Mg, MeOH, 0 ºC to rt, 3 h, 85%; (b) 9-I-9-BBN, Hexane, 0 ºC to rt,

3.5 h, 70%; (c) 8, p-TSA, CHCl3, 0 ºC to rt, 4 days, 25%; (d) TMSOTf, CH2Cl2, MeNO2, 0 ºC to rt, 4 h, 60%.

Cannabinergic C4’ ester analog 36 was synthesized in 4 steps as shown in Scheme 8.

Intermediate 25 was reduced using magnesium in methanol30 to give the methyl ester 33 in 85% yield. Deprotection of the aryl methyl ether by 9-Iodo-9-BBN resulted in resorcinol 34 in 70%

56 yield. The resorcinol was then coupled with chiral diacetates 8 in the presence of p- toluenesulfonic acid (p-TSA)27 to give the bicyclic intermediate 35 in 25% yield which was cyclized with catalytic amounts of TMSOTf27 to afford C4’ ester analog 36 in 60% yield.

With the current synthetic approach, the side chain resorcinol is prepared first followed by coupling with the chiral synthon and construction of the tricyclic skeleton. The problem with the low yielding (14-25%) Friedel Crafts allylation reaction is an important drawback of this synthetic approach. Efforts to improve the yield using different parameters such as different temperature, catalysts, reaction time, amounts and sequence of reagents were unsuccessful. A part of the puzzle describing this reaction is described below with the side chain methyl ester resorcinol a.

Figure 2.6: Investigation of Friedel-Crafts allylation reaction

In this coupling reaction, the carbocation formed undergoes Friedel Crafts allylation through the

1st pathway to give the desired product b. A side chain having a carbonyl group, reduces the

57 electron density of the aromatic ring, slows down the Friedel-Crafts allylation reaction and decreases the yield of the coupling product. Due to the slow Friedel-Crafts allylation reaction, the carbocation decomposes to apoverbenone and acetate ion as shown in the 2nd pathway resulting into acetylated resorcinol c.

A linear synthetic approach used in all the schemes reported above led to one final compound at a time. The major issue of this approach was the Friedel Crafts Allylation reaction where the yields were 14-25%. This resulted in low chemical efficiency.

In order to overcome this issue, a new synthetic method was developed for a high yielding advanced intermediate 44 to enable parallel synthesis. This convergent synthetic approach would lead to high chemical efficiency. The new methodology was used for the syntheses of side chain ester analogs of Nabilone.

Scheme 8: Synthesis of an advanced building block for parallel synthesis.

- + Reagent and conditions: (a) Br P Ph3(CH2)4CH3, (Me3Si)2NK, THF, 0 ºC to R.T, 2 h, 93%; (b)

9-I-9-BBN, Hexane, 0 ºC to rt, 3.5 h, 85%; (c) 8, p-TSA, CHCl3, 0 ºC to rt, 4 days, 55%; (d)

TMSOTf, CH2Cl2, MeNO2, 0 ºC to rt, 7 h, 65%; (e) TBDMSCl, Imidazole, DMAP, DMF, rt, 12 h, 90%; (f) NMO, OsO4, 2,6-lutidine, Acetone, Water, rt, 3 h; (g) (Diacetoxyiodo)benzene, rt, 2 h, 80% (2 steps); (h) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH, 0 ºC to rt, 4 h, 80%; (i)

TBAF, THF, -40 ºC, 30 min, 90%.

Advanced carboxylic acid intermediate 44 was synthesized to enable parallel synthesis for multiple analogs. The acid was synthesized in ten steps (Scheme 9) from 3,5-dimethoxyphenyl acetonitrile 1. Wittig olefination on 24 afforded exclusively the Z olefins with J2’H-3’H = 12 Hz to yield intermediate 37 in 93% yield followed by a 9-I-9-BBN demethylation to afford resorcinol

38 in 85% yield. The resorcinol was then coupled with chiral diacetates 8 in the presence of catalytic amounts of p-toluenesulfonic acid (p-TSA)27 to give bicyclic intermediate 39 in 55% yield. Intermediate 39 was then cyclized using TMSOTf27 to give intermediate 40 in 65% yield.

The phenolic hydroxyl on the A ring of intermediate 40 was protected using TBDMS group to yield intermediate 41 in 90% yield followed by oxidation using N-Methylmorpholine-N-oxide

(NMO)31 to yield the resulting diol 42 which without isolation was oxidized using

(diacetoxyiodo)benzene to give aldehyde 43 in 80% yield. Pinnick oxidation32 on intermediate

43 yielded carboxylic acid intermediate 44 with 80% yield. Removal of TBDMS group by tetra- n-butylammonium fluoride (TBAF) resulted in compound 45 which was tested for its activity at the CB receptors.

Scheme 9: Functionalized side-chain analogs

Reagents and conditions: (a) EDCI, DMAP, R-OH or R-SH, or R-NH2, CH2Cl2, 0 ºC to rt, 24 h,

65-75%; (b) TBAF, THF, -40 ºC, 30 min, 84-92%.

Starting from the carboxylic acid 44, side chain cannabinergic ester analogs, 46a-g were synthesized under Steglich esterification33 conditions using the specific alcohols foloowed by deprotection of the TBS group to yield 84-90% of final compounds 47a-g. Treatment of 44 with

1-butanethiol and 1-butylamine yielded the respective thioester 47h (92% yield) and amide 47i

(91% yield) analogs (Scheme 9) in two steps.

Scheme 10: Side-chain carbamate and urea derivatives

Reagents and conditions: (a) DIPEA, DPPA, CH3(CH2)2OH or CH3(CH2)2NH2, THF, 50 ºC, 5 h,

89-90%; (b) TBAF, THF, -40 ºC, 30 min, 80-90%.

Curtius rearrangement34 of carboxylic acid 44 with 1-propanol and propylamine gave the corresponding carbamate 49a and urea 49b compounds with 80 and 90% yield respectively.

RESULTS AND DISCUSSION

BIOCHEMICAL ASSESSMENT

Our SAR study (Figure 2.5) focused on chemical synthesis of controlled deactivation nabilone analogs. In vitro profiling of the C1 substituted nabilone analogs was performed in the following assays:

 Competitive radioligand binding assay for CB1 and CB2 receptors.

 Cyclic adenosine monophosphate (cAMP) assay.

 Metabolic (Plasma and microsomal) stability assays.

The binding affinities of controlled deactivation analogs were determined for the CB1 receptor

(rat brain membranes) and membrane preparations from HEK293 cells expressing human

(hCB2) and mouse (mCB2) CB2 receptors.13, 16 Displacement of [3H]-CP-55,940 from these

3 membranes was used to determine EC50 values in competition radioligand binding assays. [ H]-

CP-55,940 was used as the competing ligand as it is nonselective and has high affinity for both

CB1 and CB2 receptors. Intrinsic efficacy of key analogs was determined using forskolin stimulated cAMP assay.15, 16, 35 Lead analogs were assessed for their stability towards plasma esterases and liver microsomes.15, 16

Table 2.1: Biochemical analysis of AM7499

(Ki, nM) rCB1 hCB2 Plasma Stability (t ) min AM 1/2 EC E EC E # rCB1 mCB2 hCB2 50 max 50 max Mouse Rat Human (nM) (%) (nM) (%) 7499 2.4±0.3 0.1±0.05 3.1±0.5 2.5±0.4 87 0.3±0.06 82 3.1 >30 >30

The ability of cannabinergic analog, AM7499 to displace radiolabeled CP-55,940 from

membranes prepared from rat brain (source of CB1 receptor) and HEK 293 cells expressing

mouse and human CB2 receptor was determined and inhibition constants (Ki) from the

respective competition binding curves are listed in Table 2.1. AM 7499 has high affinity for both

the CB1 and CB2 receptors. Functional potencies of AM7499 at rCB1 and hCB2 were

determined by measuring the decrease in forskolin-stimulated cAMP levels. AM7499 is potent

agonist at both CB1 and CB2 receptors with high efficacy values. AM7499 was measured for its

stability towards plasma esterases. Testing data shows that AM7499 had short plasma half-life in

mouse while it is stable in rat and human plasma.

Table 2.2: Binding affinities (Ki) of controlled deactivation nabilone analogs

Compd & (K ,nM) R i cLogP tPSA AM # rCB1 mCB2 hCB2 10806 1.7±0.4 11.7±1.3 8.1±2.5 6.72 46.53

47a 36.7±5.6 89.7±18.1 67.5±13.4 3.82 72.83 10865

47b 4.8±0.8 32.0±5.2 8.3±3.1 3.69 72.83 10830

10 0.5±0.02 4.4±0.9 8.0±2.3 4.22 72.83 10843

47c 0.4±0.01 6.1±1.1 11.5±1.6 4.75 72.83 10828

47d 5.9±1.2 30.0±7.3 15.1±2.2 5.28 72.83 10835

47e 3.0±0.5 54.0±10.6 12.4±2.5 5.04 72.83 10863

47f 4.0±0.7 36.4±12.4 7.0±2.1 5.04 72.83 10864

10810 0.1±0.04 1.2±0.6 5.3±0.9 4.08 72.83

21 0.7±0.03 1.1±0.4 6.2±2.7 2.65 96.62 10811

47g 17.0±3.5 91.7±20.4 18.2±4.1 3.18 85.30 10855

31 11±1.4 26±3.6 90±11.3 4.09 72.83 10819 36 270±34.6 540±46.7 560±64.3 3.56 72.81 10813

45 >1000 >1000 >1000 3.21 83.83 10826

32 >1000 >1000 >1000 3.16 83.83 10831

47h 0.70±0.03 5.0±1.4 16.0±2.4 5.02 63.60 10827

47i 115±21.6 >1000 >1000 4.05 77.70 10832

49a 140.6±20 200±32 >1000 3.36 84.86 10891

49b >1000 >1000 >1000 2.68 87.66 10892

Binding affinities for controlled deactivation nabilone analogs are reported in Table 2.2. The compounds included in this study are optimized nabilone analogues in which the side chain is functionalized with different groups. As expected, the hydrolytic metabolites of these analogs had no significant affinity for the CB receptors (32 & 45). Comparison of the binding data of nabilone (AM10806) and its corresponding ester analog 10 shows that it binds well at the CB receptors. Compound 10 has higher affinity for CB1 and equal affinity for the CB2 receptor than its prototype, nabilone. The one-carbon shorter homologue 47b has significantly reduced affinities for both CB receptors, indicating that a minimum requirement for substantial affinity for both receptors in this series is a six-atom-long side chain. Extension of the side chain from 7 to 8 atoms (AM10828) showed no change in affinities at CB receptors while the affinities were still maintained (Ki = <6 nM) when the side chain had 6 or 9 atoms (47b, & 47d). Introduction of chiral methyl group 47e & 47f still maintained high affinities to the CB1 and hCB2 receptor with loss of affinity to the mCB2 receptor. We also observed that incorporation of the iodo as well as the more polar cyano 21 and morpholino 47g substituents at the distal carbon of the side chain are well tolerated. The cyano analogue 21 exhibited remarkably high affinity for the CB1 and CB2 receptors. An examination of the binding data of the 2′-carboxyester analogue 10 and its sulfur and nitrogen congeners 47h and 47i shows that the ester moiety (−C(O)-O−) can be replaced by the respective thioester (−C(O)-S−) group but the amide (−C(O)-NH−) group lacks affinity at the CB receptors. Also, the binding affinity is significantly diminished for the carbamate 49a and urea 49b analogs. The affinity of 3′-carboxyester analogue 31 to the CB

65 receptors is significantly reduced (10 times) as compared to 10 while the 4′-carboxyester analogue 36 loses affinity for the CB receptors.

In summary, the detailed SAR reported here shows that a six- to nine-atom-long side chain with gem-dimethyl substituent at the 1′-position and an ester or thio-ester group within the 2′ or 3′ chain segment results in analogs with remarkably high affinities for both CB1 and CB2 receptors. Importantly, addition of the iodo or cyano groups as well as the bulky morpholino at the terminal carbon maintains or enhances the affinity of the ligand for the CB receptors.

Table 2.3: Metabolic stabilities (t1/2) of controlled deactivation analogs

Microsomal Stability (t ) Plasma Stability (t ) min 1/2 AM # R 1/2 min Mouse Rat Human Mouse Rat Human 10806 - - - 18 42 28

10 2.9 4.4 >30 1.6 3 2.5 10843

47e >30 >30 >30 NT NT NT 10863

47f >30 >30 >30 NT NT NT 10864

21 4.5 14.8 >30 1.8 9.2 5.6 10811

31 >30 >30 >30 7.4 8.2 5.7 10819 47h >30 >30 >30 4.2 NT 9.8 10827

Enzymatic labile moieties such as ester, thioester, carbamate, or phosphate groups that are incorporated into the parent drug molecules can be targeted by esterase enzymes expressed through body organs and other tissues as well as blood.16 Esterases are a heterogeneous group of enzymes that are classified broadly as cholinesterases (including acetylcholinesterases and butyrylcholinesterases), paraoxonases, and carboxyesterases. Human serum albumin also exhibits esterase activity toward phenyl esters such as aspirin (acetylsalicylic acid). Notably, esterase activity is higher in the blood of small rodents than in large animals and humans, while carboxyesterases are found in mice and rat but not in human plasma.16

Representative analogs within this series were assessed for their metabolic stability against plasma esterases and liver microsomes. A comparison of the plasma half-lives (t1/2, Table 2.3) of the alkyl and nitrile (10, 21) analogs show increased stability for the cyano substituted analog.

Similar data was reported in previous studies from our group in Δ8-THC controlled deactivation analogs. It was surprising to see that the less sterically hindered 3’-ester analog 31 show increased stability (t1/2 =>30 min) in all three species as compared to 2’-ester analog 10. Also, introduction of chiral methyl group (47e, 47f) α to oxygen on the side-chain led to remarkable plasma stability when compared to sterically less hindered ester analog 10. In agreement on the rates of hydrolysis36 and previous studies,13 the thioester 47h exhibits higher stability than the respective oxoester 10. Overall, our data show that the rate of enzymatic inactivation of our nabilone analogs can be controlled by (1) structural features in the vicinity of the hydrolyzable group and (2) substituents at the terminal carbon.

Table 2.4: Functional evaluation of controlled deactivation analogs

rCB1 hCB2 AM # R EC50 (nM) Emax (%) EC50 (nM) Emax (%) 10806 1.9±0.3 90 0.06±0.01 68

10 1.0±0.2 95 1.1±0.5 77 10843

47c 8.9±2.3 94 8.4±1.8 46 10828

21 0.4±0.06 87 0.1±0.03 77 10811

31 15.9±3.6 90 0.2±0.09 82 10819 47h 0.2±0.04 89 83.4±4.7 72 10827

The functional potency of C1 substituted nabilone analogs tested in cAMP assay is tabulated in

Table 2.4. In both the rat CB1 and human CB2 receptors, all the analogs showed a concentration- dependent inhibition of forskolin-induced cAMP accumulation. The functional potency of the analogs was compared to the agonist CP-55,940 in the cAMP assay. We observe that all analogs tested are potent agonists at the cannabinoid receptors while the EC50 values correlate well with their respective binding affinities (Table 2.4).

PHARMACOLOGICAL ASSESSMENT OF CONTROLLED

DEACTIVATION ANALOGS

In vivo evaluation of AM7499 a. Hypothermia testing of AM7499

Figure 2.7: Hypothermia assessment of AM7499.16

Preliminary studies from our group had shown that AM7499 produced a decrease in core body temperature in a dose dependent manner. A dose of 0.1 mg/kg produced 3-4°C drop in temperature whereas a dose of 0.3 mg/kg produced a 5-6 degree drop in body temperature in rats.

It was found that rats recovered from hypothermia within 6 h of study duration at a dose of 0.1 mg/kg. AM7499 was also found to be more efficacious than AM238935 (potent CB1 agonist developed by CDD) at both the doses of 0.1 mg/kg (Figure 2.7).

69 b. Analgesia testing in mice of AM7499 and HU210

100

%MPE 40 AM7499 0.3mg/kg AM7499 0.1mg/kg HU-210 0.3mg/kg 20 HU-210 0.1mg/kg

0 20 60 180 360 Time (min)

Figure 2.8: Tail-flick latencies in a hot water-bath (52°C) after administration of AM7499 and HU210.16

To confirm the observed pharmacokinetic differences between AM7499 and the nonhydrolyzable counterpart HU21037, we used the CB1 receptor characteristic analgesia assay.

Data shows significant differences between the two drugs (AM7499 and HU210) at 20, 60, and

360 min postinjection (Figure 2.8). These differences in onset and offset of effect for the two compounds are also reflected by comparisons within each ligand over time. Pairwise comparisons suggested less analgesia at the 360 min time point compared to the three earlier time points as well as a significant difference between the recordings at 60 and 180 min postinjection for AM7499. Slow onset of effect for compound HU210 is suggested by significant

70 differences in analgesia scores at 20 as well as 60 min postinjection and the recordings at the 180 and 360 time points.

c. CB1 discriminative stimulus effects in squirrel monkeys for AM7499 and HU210

Figure 2.9: Time course CB1 discriminative stimulus effects in squirrel monkeys for HU210 and AM7499.16

AM7499 was assessed in drug discrimination studies in squirrel monkeys (Saimiri sciureus) trained to discriminate 0.01 mg/kg AM4054 (Potent CB1 agonist developed at CDD) from saline. It should be noted that studies with non-human primates are key to this project because metabolic processes and volumes of drug distribution vary between small rodent and primate species.16

In these experiments, the time courses of the lowest doses of AM7499 (0.001 mg/kg) and the nonhydrolyzable cannabinoid HU210 (0.003 mg/kg) that fully substituted for the training dose of the CB1 agonist AM4054 were compared directly in the same group of subjects. AM7499 produced evidence of CB1-related discriminative-stimulus effects within 15 min (t1/2 = 48 min) and was fully effective in three of four subjects 1 h after injection. HU210 was similar in its onset of action (t1/2 = 66 min) but did not achieve a full effect in the group of subjects until 2 h

71 after injection. Notwithstanding the generally comparable onset of behavioral effects, the two compounds varied considerably in offset of action. Thus, AM7499 began losing its CB1 discriminative-stimulus effects 4 h after injection and was completely without effect 16 h following treatment. On the other hand, HU210 fully substituted for the training drug stimulus

AM4054 24 h after injection and was completely without CB1discriminative-stimulus effects at the 36 h time point. On the basis of calculated values for loss of 50% of full effect, the duration of action for AM4799 was approximately 4 times shorter than for HU210. Neither compound had appreciable effects on response rates throughout the present time course studies.

In vivo evaluation of controlled deactivation nabilone analogs a. Hypothermia assessment

Figure 2.10: Hypothermic effects of AM10806, AM10843 and AM10811.

The hypothermic effects of the side chain carboxyester analogs AM10843 and AM10811 were compared in rats. Rectal body temperature was measured in isolated rats over a 6 h period following drug injection (detailed procedures are given in Experimental Section). In agreement with our in vitro functional characterization, compounds 10 and 21 decreased core body temperature in a dose-dependent manner, reducing body temperature by 2-4.5°C at the highest doses tested (Figure 2.7). For comparison, the effects of nabilone (AM10806) are also shown.

Both compounds had relatively fast onsets of drug effect. Significant decreases in body temperature typically occurred within 60 min after injection, although peak effects were not obtained until 2−3 h after injection. Administration of 3 mg/kg of AM10843 and 1.0 mg/kg of

AM10811 resulted in temperature decreases of 4-4.5°C, with significant recovery toward baseline within the 6 h test period. By comparison, AM10806 at a dose of 1.0 mg/kg produced a significantly higher decrease in rectal temperature (6°C), producing peak effects at 5 h post drug administration and had effects that persisted for more than 6 h.

In conclusion, our hypothermia data in rats indicate that controlled deactivation analogs have shorter duration of action than the non-hydrolysable congener, nabilone. b. Analgesia testing of key compounds

To confirm the observed pharmacokinetic differences between the side chain carboxyester analogs (10, 31) and the nonhydrolyzable counterpart AM10806, we used the CB1receptor characteristic analgesia assay. Post hoc comparisons suggested significant differences between the controlled deactivation analogs (AM10843 & AM10811) and nabilone (AM10806) at 20, 60, and 360 min post injection. These differences in onset and offset of effect for the two compounds are also reflected by comparisons within each ligand over time. Compounds 10 and 31 showed no analgesia at the 360 min time point compared to the three earlier time points as well as a

73 significant difference between the recordings at 60 and 180 min post injection. Slow onset of effect for AM10806 is suggested by significant differences in analgesia scores at 20 as well as 60 min post injection and the recordings at the 180 and 360 time points. AM10843 at a dose of 10 mg/kg has fast onset to action, showing maximal effects at 60 min post drug administration.

Similar results were seen for AM10819 but at an administered dose of 30 mg/kg. Our results show that AM10819 is slightly less potent than AM10843 but has a shorter duration of action.

Overall, analgesia testing shows that controlled deactivation analogs have fast onset and short duration of action than nabilone.

100

AM10819 30mg/kg 40 AM10819 10mg/kg AM10806 1mg/kg AM 10806 3mg/kg

% Max. Max. Possible % Efffect 20 AM 10843 3mg/kg i.p. AM 10843 10mg/kg i.p.

0 0 20 60 180 360

Time (min) Figure 2.11: Tail-flick latencies in a hot water-bath (52°C) after administration of AM10806, AM10843 and AM10819.

74 c. CB1 discriminative stimulus effects in squirrel monkeys for AM10843 and AM10806

Figure 2.12: CB1 discriminative stimulus effects in squirrel monkeys for AM10806 and AM10843.

Figure 2.12 shows results from drug discrimination experiments in monkeys using our lead controlled deactivation analog AM10843 and nabilone (AM10806). Figure 2.12a shows the ability of both drugs to substitute for a CB1 agonist in CB1 drug discrimination procedures in a group of four squirrel monkeys. We see that the lowest cumulative dose of AM10806, 0.001 mg/kg, does not produce lever-pressing on the CB1-associated lever but only on the vehicle- associated lever. As the dose is increased, however, the distribution of behavior shifts and, after the highest cumulative dose, 0.01 mg/kg, all subjects press 100% on the CB1-associated lever.

These results show that this dose of i.m. AM10806 is fully identified as a CB1 agonist.

Similarly, the open triangles show that AM10843, carboxynabilone, also has dose-related CB1-

75 like effects. It is a little more potent than AM10806, and produces full CB1-like effects after a test dose of 0.003 mg/kg.

Figure 2.12b shows the rate of responding on the two levers after drug treatment. Data shows that the response rates, given as responses or lever presses per second, in this group of monkeys is unchanged by doses of nabilone or carboxynabilone that are fully identified as the training drug, the CB1 ligand AM4054. This shows that doses of both nabilone and carboxynabilone do not alter motor performance that produce CB1 discriminative-stimulus effects, which are thought to be analogous to the subjective effects of such drugs in man.

Figure 2.12c shows the time course of action for the ability of drugs to produce CB1 discriminative-stimulus effects. Here, equivalent doses of AM10806 and AM10843, each producing a full CB1 effect, are given at different treatment times before the session. The percentage of responses on the CB1-associated lever is shown in the Y-axis, whereas the time since injection is shown along the X-axis. Results show that, for both drugs, the CB1-like discriminative-stimulus effect decreases over time. However, AM10843 has duration of action for about 2.5 to 3 hours while nabilone lasts for more than 16 hours. These data very convincingly indicate that the modification to nabilone to produce carboxynabilone does not change the CB1-like effects of nabilone but does greatly shorten its half-life, supporting our ideas for chemical modification that results in a controlled deactivation of the CB1-like effects of the nabilone molecule.

Figure 2.12d shows the corresponding response rate of the drugs, nabilone and carboxynabilone.

Data clearly shows that AM10843 has duration of action for about 2.5 to 3 hours.

CONTROLLED DEACTIVATION LIGANDS AS IRREVERSIBLE PROBES

Efforts in our lab have been focused to characterize the ligand binding domain of the cannabinoid receptors by using mutagenesis and ligand-binding docking experiments. Ligand assisted protein structure (LAPS), a technology developed at CDD allows the identification of important amino acid residues in the binding domain of cannabinoid receptors. The LAPS approach involves four important steps: a) Interaction of high affinity irreversible/covalent probes with the specific amino acids at or near the binding site of the receptors; b) Introduce point mutations in the receptor and study the effects of probe binding with the mutated receptor; c) Molecular modelling using computation techniques to understand probe-receptor interactions; and d) MS-based proteomics to identify sites of covalent attachment. Information gained from these experiments has led to the identification of a high affinity NCS probe, AM841 with the cysteine (C6.47 (355)) in helix 6 of human CB2 receptor. This probe activates the hCB2 receptor with a 40 fold greater potency as compared to the non-NCS analog of AM841.

It is not known how controlled deactivation analogs interact with the cannabinoid receptors to activate it. Information of the amino acid-ligand interaction site(s) of the binding regions of cannabinoid receptors can enhance the design and development of more potent and selective ligands. In order to pursue this goal, irreversible probes bearing a photoactivable and electrophilic group were designed. Our design incorporates the electrophilic isothiocyanate

(NCS) and nitrate ester (ONO2) groups and photoactivable azido (N3) group on the terminal end of the side-chain. This strategy will help determine the amino acid residues and the transmembrane helix(es) involved in the binding for these class of compounds.

CHEMISTRY

As shown in Scheme 11, the azido and isothiocyanate analogs at the 7’ position of the side-chain were synthesized. Compound 50 with an azide group at the terminal end of the side chain was afforded by treating 20 with tetrabutylammonium azide. The iodo group is substituted by an azide by undergoing a SN2 reaction yeilding 75% of 50. The azide was then treated with triphenylphosphine and carbon disulfide to yield isothiocyanate 51 with 78% yield.

Scheme 11: Synthesis of irreversible compounds

+ - Reagents and conditions: (a) n-Bu4N N3 , CH2Cl2, CH3NO2, 70 ºC, 24 h, 75%; (b) PPh3, CS2,

THF, rt, 12 h, 78%.

Compounds containing 7 atoms plus the reactive probes, NCS, N3 and ONO2 on the side-chain were synthesized as shown in Scheme 12. Advanced intermediate 44 was esterified under

Steglich esterification conditions using 4-bromo-1-butanol to afford the bromo substituted analog

52 with 77% yield. The TBS group in 52 was deported using TFA to afford 53 with 95% yield.

The azido analog 54 was synthesized in 80% yield by reacting bromide 53 with tetrabutylammonium azide. The representative isothiocyanate compound 55 was synthesized

(71% yield) by reacting the azide 54 with triphenylphosphine and carbon disulfide. The nitrate ester probe 56 was afforded by treatment of 53 with silver nitrate in 75% yield.

Scheme 12: Synthesis of irreversible compounds using advanced acid building block

Reagents and conditions: (a) DMAP, EDCI, 4-bromo-1-butanol, CH2Cl2, 0 ⁰C- rt, 24 h, 77%; (b)

+ - TBAF, THF, -50 ⁰C, 95%; (c) n-Bu4N N3 , CH2Cl2, CH3NO2, 70 ºC, 24 h, 80%; (d) PPh3, CS2,

THF, rt, 12 h, 71%; (e) Silver nitrate, CH3CN, 70 ºC, 24 h, 75%.

RESULTS AND DISCUSSION

Binding affinities were determined for the covalent ligands by radioligand displacement assay using the CB1 receptor (rat brain membranes) and membrane preparations from HEK293 cells expressing human (hCB2) and mouse (mCB2) CB2 receptors. Compounds were then evaluated for their abilities to label irreversibly the rCB1, hCB1 and hCB2 receptors. The membranes prepared from rat and human were equilibrated with concentration (10 fold of the Ki’s) of the respective ligand for 1 hour followed by quenching of the reaction by centrifugation. Ligands containing an azido group were photoirradiated with UV light (254 nm) followed by saturation binding assay to determine the ligand occupancy on the receptor. Control experiments were performed by irradiating the membranes in absence of the ligands. For the electrophilic covalent labelling experiments, specific binding was assessed by testing the ability of the ligand to label the receptor irreversibly and was compared to the specific binding of the control ligand ([3H]

CP-55940) (See experimental procedures for assay protocol). The binding and covalent data for the cannabinergic probes is summarized in Table 2.5.

Table 2.5: Binding (Ki) and % labeling assessment of cannabinergic probes

(K ,nM) % labeling AM # R i rCB1 mCB2 hCB2 rCB1 hCB1 hCB2 50 0.1±0.2 4.0±1.1 3.1±1.8 0 - 0 10814

51 7.9±2.3 20±3.4 22±4.1 0 - 0 10815

54 1.7±0.8 4.5±1.6 1.7±0.4 0 0 0 10857

55 4.9±2.4 21.4±4.3 5.2±1.3 0 72 62 10858

56 0.2±0.07 2.2±0.6 1.3±0.3 32 72 0 10859

Controlled deactivation probes with a side-chain bearing 6 and 7 atoms with azido, isothiocyanate, or nitrate ester at the terminal carbon were synthesized. Binding affinity values of novel probes shown in Table 2.5 had apparent Ki values below 8 nM for rCB1 receptor and 22 nM for the mouse and human CB2 receptor.

Our results show that substitution of an azido group the terminal carbon of the side-chain led to ligands displaying high affinity, but no selectivity towards the receptors tested. Both ligands 50 and 54 show no covalent binding towards the cannabinoid receptors. The 7 atom isothiocyanate probe 55 displayed covalent binding towards the hCB1 and hCB2 receptor as compared to 51 that did not show any covalent labelling. It was surprising to see that 55 did not show any covalent binding to the rCB1 receptor even though both, rat and human display 96% sequence homology for the CB1 receptor (Figure 2.13). Nitrate ester substituted analog 56, showed covalent binding towards the CB1 receptor. The percent labelling was significantly less for rCB1

(32%) as compared to hCB1 (72%) (Figure 2.13).

AM10858 (rCB1) AM10858 (hCB1)

AM10859 (rCB1) AM10859 (hCB1)

Figure 2.13: Saturation binding curves using [3H] CP-55,940 for rCB1 (left panel) and hCB1 (right panel) receptors preincubated with the AM10858 (55) and AM10859 (56) analogs.

CONCLUSIONS

The main goal of this project was to develop an optimized synthetic procedure to synthesize and develop a side-chain SAR for novel controlled deactivation nabilone analogs. Earlier reports from our group show that the pharmacological half-lives of these analogs can be controlled by joint modulation of their metabolic stabilities for plasma esterases as well as altering the polar characteristics and depot effects. We probed the side-chain pharmacophore of nabilone by introducing metabolically vulnerable groups at the 2’, 3’, and 4’-position in an effort to produce ligands with high binding affinity, in vitro and in vivo potency and efficacy, as well as its rate of enzymatic deactivation. We also introduced polar features at the terminal carbon that can modulate the depot effect and produce ligands with fast onset/offset and short duration of action.

This thesis reports the synthetic procedure for the synthesis of novel controlled deactivation nabilone analogs. Advanced acid intermediate 44 was synthesized in 10 steps to enable parallel synthesis of side-chain analogs. Novel cannabinergic analogs in which the 5-9 atom long side- chain with or without substituents, carries a metabolically liable group at the 2’, 3’, or 4’-position were synthesized and reported. Following the synthesis of novel controlled deactivation analogs, the binding affinities were determined. Among the analogs synthesized, ester and thioester substituted analogs showed high affinity to the cannabinoid receptors as compared to the amide, carbamate and urea analogs. Compounds AM10843 and AM10811 showed a short half-life than in plasma and microsomal stability assays than the other analogs tested. Functional assessment of all analogs tested showed that they were potent agonists at the cannabinoid receptors.

The results from our irreversible binding experiments demonstrate that the photoactivable azido probes AM10814 and AM10857 did not label the receptors. Isothiocyanate probe AM10858

83 labelled the hCB1 and hCB2 receptor but did not label the rCB1 receptor while the nitrate ester analog AM10859 showed covalent labelling only to the CB1 receptor. AM10858 and AM10859 are currently being evaluated for further receptor mapping studies.

Among the lead analogs that were selected and tested in vivo, AM10843, AM10811, and

AM10819 showed potent analgesic and hypothermic effects in mice and rats. To explore the validity of our controlled deactivation approach, our lead analog, AM10843 was further assessed for drug discrimination in non-human primates. Testing results show high in vivo potency with a short duration of action.

Overall, through this careful and detailed SAR, we have identified AM10843 as a short acting

CB1 agonist with duration of action of 2-3 hours.

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 recorded in CDCl3, unless otherwise stated, on a Bruker Ultra

Shield 400 WB plus (1H 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 relative to base

= 100). Purities of the tested compounds were determined by elemental analysis or by LC/MS analysis 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] and were > 95%.

2-(3,5-Dimethoxyphenyl)-2-methylpropanenitrile (2).To a stirred suspension of sodium hydride (6.7 g, 169.0 mmol) in dry DMF (40 mL) at 0 °C under an argon atmosphere was added dropwise a solution of 3,5-dimethoxyphenyl)acetonitrile (10.0 g, 56.4 mmol) and iodomethane

(10.5 mL, 169.0 mmol) in dry DMF (40 mL). The reaction temperature rose to 25 °C over a 15 min period and stirring was continued for 2 h. The reaction mixture was quenched with saturated aqueous NH4Cl solution and extracted 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 (11.0 g,

95% yield) as colorless oil. 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).

2-(3,5-Dimethoxyphenyl)-2-methylpropanoic Acid (3). A stirred mixture of (3,5- dimethoxyphenyl)-2-methylpropanenitrile (2, 5.7 g, 32.2 mmol) and NaOH (3.2 g, 80 mmol) in n-butanol/water (5 mL, 2:1 ratio) was refluxed for 4 h under argon. Volatiles were removed under reduced pressure, and the residue was acidified with 2 N HCl 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 (30% ethyl acetate in hexane) gave 3 (5.61 g, 89% yield) as a white solid, mp 97−99 °C. IR (neat) 2926, 1695 (>C=O),

1598, 1454, 1288, 1204, 1068 cm−1; 1H NMR (500 MHz, CDCl3) δ 6.54 (d, J = 2.5 Hz, 2H,

ArH), 6.37 (t, J = 2.5 Hz, 1 H, ArH), 3.79 (s, 6 H, -OCH3), 1.57 (s, 6 H, -C(CH3)2-); mass spectrum (ESI) m/z (relative intensity) 225 (M+ + H, 10), 190 (7), 179 (33), 149 (100).

2-(3,5-Dihydroxyphenyl)-2-methylpropanoic Acid (4). To a stirred solution of 3 (3.5 g, 17.8 mmol) in dry CH2Cl2 (85 mL) at −78 °C, under an argon atmosphere, was added boron tribromide (62.3 mL, 62.3 mmol, 1 M solution in CH2Cl2). Following this addition, the reaction temperature was gradually raised over a period of 3 h to 25 °C, and the stirring was continued at that temperature until the reaction was completed (4 h). Unreacted boron tribromide was destroyed by the addition of methanol and ice at 0 °C. The resulting mixture was warmed to room temperature, and volatiles were removed in vacuo. The residue was dissolved in ethyl acetate and washed with water and brine and dried (MgSO4). Solvent evaporation and purification by flash column chromatography on silica gel (40% ethyl acetate in hexane) gave 4

(2.64 g, 88% yield) as a white solid, mp 174−176 °C. IR (neat) 3180, 1688, 1601 cm−1; 1H

NMR (500 MHz, CD3OD) δ 6.33 (d, J = 2.5 Hz, 2 H, ArH), 6.15 (t, J = 2.5 Hz, 1 H, ArH), 4.91

(br s, 2H, -OH), 1.48 (s, 6 H, -C(CH3)2-); mass spectrum (ESI) m/z (relative intensity) 197 (M+

+ H, 100); exact mass (ESI) calculated for C10H13O4 (M+ + 1), 197.0814; found 197.0806.

Butyl-2-(3,5-dihydroxyphenyl)-2-methylpropanoate (5). A stirred mixture of 4 (2.0 g, 10.20 mmol), bromobutane (2.1 g, 15.30 mmol) and sodium bicarbonate (1.05 g, 12.24 mmol) in anhydrous DMF (3 mL) was heated at 165 oC for 12 min using microwave irradiation. The reaction mixture was cooled to room temperature and diluted with water and ethyl acetate. The organic layer was washed with brine, dried (MgSO4), and concentrated in vacuo. The residue was chromatographed on silica gel (50% ethyl acetate in hexane) to give 5 (2.5 g, 92% yield) as a light brown viscous oil. IR (neat): 3370, 2963, 1698, 1600, 1465, 1271, 1142 cm-1; 1H NMR

(500 MHz, CDCl3) δ 6.40 (d, J = 2.0 Hz, 2H, ArH), 6.23 (t, J = 2.0 Hz, 1H, ArH), 5.10 (s, 2H,

OH), 4.06 (t, J = 7.0 Hz, 2H, 4-H), 1.55 (quintet, J = 7.5 Hz, 2H, 5'-H), 1.51 (s, 6 H, -C(CH3)2-),

13 1.28 (sextet, J = 7.0 Hz, 2H, 6'-H), 0.87 (t, J = 7.5 Hz, 3H, 7'-H). C NMR (100 MHz CDCl3) δ

177.7 (-C(O)O-), 156.9 (ArC-3 and ArC-5), 147.4 (ArC-1), 105.5 (ArC-4 and ArC-6), 101.5

(ArC-2), 65.3 (-OCH2-), 46.5, 30.4, 26.1, 18.9, 13.5.

Butyl-2-(4-((1R,2R,5R)-6,6-dimethyl-4-oxobicyclo[3.1.1]heptan-2-yl)-3,5-dihydroxyphenyl)-

2-methylpropanoate (9). To a degassed solution of 5 (2.2 g, 8.73 mmol) and diacetates 8 (8.55

1 g, ca. 85% pure by H NMR, 30.55 mmol) in CHCl3 (88 mL) at 0 °C, under an argon atmosphere, was added p-toluenesulfonic acid monohydrate (2.32 g, 12.22 mmol). 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

(43% diethyl ether in hexane) to give 9 as a white crystalline solid (914 mg, 27% yield); Rf = 0.4

(40% diethyl ether in hexane); mp = 70-71 oC. IR (neat): 3362, 2959, 2873, 1727, 1683, 1619,

-1 1 1589, 1421, 1265, 1143 cm ; H NMR (500 MHz, CDCl3) δ 6.31 (s, 2H, ArH), 5.50 (s, 2H,

OH), 4.06 (t, J = 7.0 Hz, 2H, 4'-H), 3.95 (t, J = 7.5 Hz, 1H, 4-H), 3.50 (dd, J = 19.0 Hz, J = 8.0

Hz, 1H, 3α-H), 2.59 (dd, J = 19.0 Hz, J = 8.5 Hz, 1H, 3β-H), 2.58 (t, J = 5.0 Hz, 1H, 1-H), 2.50

(m, 1H, 7α-H), 2.46 (d, J = 10.5 Hz, 1H, 7β-H), 2.27 (t, J = 5.5 Hz, 1H, 5-H), 1.56 (quintet, J =

7.5 Hz, 2H, 5'-H), 1.50 (s, 6H, -C(CH3)2-), 1.36 (s, 3H, 6-Me), 1.28 (sextet, J = 7.0 Hz, 2H, 6'-

13 H), 0.99 (s, 3H, 6-Me), 0.87 (t, J = 7.5 Hz, 3H, 7'H). C NMR (100 MHz CDCl3) δ 213.3

(>C=O), 177.1 (-C(O)O-), 155.4 (ArC-3 and ArC-5), 143.9 (tertiary aromatic), 115.0 (tertiary aromatic), 106.1 (ArC-2 and ArC-6), 65.1 (-OCH2-), 58.0, 46.8, 46.0, 42.1, 37.6, 30.4, 29.4,

26.1, 26.0, 24.4, 22.1, 19.0, 13.6. Mass spectrum (ESI) m/z (relative intensity) 389 (M++H, 100).

Mass spectrum (EI) m/z (relative intensity) 388 (M+, 25), 305 (32), 287 (20), 243 (19), 177 (21),

+ 84 (100). Exact mass (EI) calculated for C23H32O5 (M ), 388.2250; found 388.2252. HPLC

(4.6mm x 250mm, Supelco Discovery column, acetonitrile/water) showed purity of 99.7% and retention time 8.9 min.

Butyl-2-[(6aR,10aS)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H-benzo

[c]chromen-3-yl]-2-methylpropanoate (10). To a stirred solution of 9 (850 mg, 2.20 mmol) in anhydrous CH2Cl2/CH3NO2 (3:1 mixture, 44 mL) at 0 °C, under an argon atmosphere was added trimethylsilyl trifluoromethanesulfonate (2.3 mL, 0.3 M solution in CH3NO2, 0.66 mmol).

Stirring was continued for 3 h while the temperature was allowed to rise to 25 oC. The reaction mixture was quenched using 1:1 solution of saturated NaHCO3 and brine and extracted with diethyl ether. The organic layer was washed with brine, dried (MgSO4), and concentrated under reduced pressure. Purification by flash column chromatography on silica gel (30% acetone in hexane) gave 10 as white foam (570 mg, 67% yield). mp = 35-36 oC. IR (neat): 3356, 2958,

-1 1 1727, 1697, 1620, 1579, 1419, 1259, 1142 cm ; H NMR (500 MHz,CDCl3) δ 6.41 (d, J = 2.0

Hz, 1H, Ar-H), 6.30 (br d, J = 2.0 Hz, 1H, Ar-H), 6.23 (br s, 1H, OH), 4.06 (m as td, J = 7.0 Hz,

J = 2.0 Hz, 2H, 4'-H), 3.96 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.87 (m as td,

J = 12.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.64-2.57 (m, 1H, 8eq-H), 2.50-2.40 (m, 1H, 8ax-H), 2.20-

2.08 (m, 2H, 10ax-H, 7eq-H), 1.95 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.59-1.51 (m,

3H, 7ax-H, 5'-H), 1.50 (s, 6H, -C(CH3)2-), 1.47 (s, 3H, 6-Me), 1.26 (sextet, J = 7.5 Hz, 2H, 6'-H),

13 1.11 (s, 3H, 6-Me), 0.86 (t, J = 7.5 Hz, 3H, 7'-H). C NMR (125 MHz CDCl3) δ 213.3 (>C=O),

177.1 (-C(O)O-), 155.4 (ArC-1 or ArC-5), 154.8 (ArC-5 or ArC-1), 145.4 (tertiary aromatic),

109.4 (tertiary aromatic), 107.2 (ArC-2 or ArC-4), 105.6 (ArC-4 or ArC-2), 77.1 (C-6), 64.9 (-

OCH2-), 47.6, 46.4, 45.2, 41.0, 34.9, 30.7, 28.1, 27.1, 26.5, 26.3, 19.2, 19.1, 13.9. Mass spectrum

(EI) m/z (relative intensity) 388 (M+, 79), 305 (43), 287 (100), 245 (98), 177 (94). Exact mass

+ (EI) calculated for C23H32O5 (M ), 388.2250; found 388.2246. HPLC (4.6mm x 250mm, Supelco

Discovery column, acetonitrile/water) showed purity of 98.3% and retention time 10.8 min for

10. Anal. (C23H32O5) C, H.

Butyl-2-{(6aR,10aR)-1-[(tert-butyldimethylsilyl)oxy]-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl}-2-methylpropanoate (11). To a solution of 10 (550 mg, 1.41 mmol) in anhydrous DMF (9.5 mL) under an argon atmosphere were added sequentially, imidazole (675 mg, 9.92 mmol), DMAP (172 mg, 1.41 mmol) and TBDMSCl (1.46 g, 9.72 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 (20% ethyl acetate in hexane) afforded 620 mg (87% yield) of 11 as a colorless oil. IR (neat):

-1 1 2959, 1715, 1612, 1567, 1415, 1253, 1096 cm ; H NMR (500 MHz,CDCl3) δ 6.46 (d, J = 2.0

Hz, 1H, Ar-H), 6.35 (br d, J = 2.0 Hz, 1H, Ar-H), 6.24 (br s, 1H, OH), 4.04 (m as td, J = 7.0 Hz,

J = 2.0 Hz, 2H, 4'-H), 3.76 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71 (m as td,

J = 12.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.59-2.40 (m, 1H, 8eq-H), 2.45-2.36 (m, 1H, 8ax-H), 2.17-

2.11 (m, 2H, 10ax-H, 7eq-H), 1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.56-1.51 (m,

3H, 7ax-H, 5'-H), 1.49 (s, 6H, -C(CH3)2-), 1.46 (s, 3H, 6-Me), 1.25 (sextet, J = 7.5 Hz, 2H, 6'-H),

1.09 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.85 (t, J = 7.5 Hz, 3H, 7'-H), 0.23 (s, 3H,

13 Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.1 (>C=O), 176.4

(-C(O)O-), 154.5 (ArC-1 or ArC-5), 154.4 (ArC-5 or ArC-1), 144.8 (tertiary aromatic), 113.2

(tertiary aromatic), 109.6 (ArC-2 or ArC-4), 108.1 (ArC-4 or ArC-2), 64.6 (-OCH2-), 47.7, 46.1,

45.4, 40.7, 35.0, 30.5, 27.7, 26.8, 26.2, 26.1, 25.9, 25.8, 19.0, 18.6, 18.3, 13.6, -3.7, -4.1. Mass spectrum (ESI) m/z (relative intensity) 503 (M++H, 100). Exact mass (ESI) calculated for

+ C29H47O5Si (M +H), 503.3193; found 503.3195. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.2% and retention time 6.2 min for the title compound.

Butyl-2-{(6aR,10aR)-1-[(tert-butyldimethylsilyl)oxy]-9-(methoxymethylene)-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl}-2-methylpropanoate (12).

(Methoxymethyl)triphenyl phosphonium chloride (2.18 g, 6.36 mmol) was suspended in 32 mL of dry benzene. Sodium tert-amylate (688 mg, 6.25 mmol) was added, and the reaction mixture was stirred for 5 min at 0 °C. Intermediate 11 (532 mg, 1.06 mmol) was dissolved in minimum amount of dry benzene and transfered to the solution of the orange ylide. The reaction mixture was stirred at room temperature for 3 h and then quenched with saturated aqueous NH4Cl solution and diluted using 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 (20% acetone in hexane) to give two geometrical isomers 12a and 12b (353 mg, 63% yield) as a colorless oil in the ratio of 2:3 respectivelly as determined by 1H NMR analysis. IR (neat): 2957,

-1 1 1728 (>C=O), 1611, 1565, 1414, 1251, 1138, 1097 cm ; H NMR (500 MHz, CDCl3) δ 6.43 (d,

J = 2.0 Hz, 1H, Ar-H, 12b), 6.41 (d, J = 2.0 Hz, 1H, Ar-H, 12a), 6.34 (d, J = 2.0 Hz, 1H, Ar-H,

12a), 6.32 (d, J = 2.0 Hz, 1H, Ar-H, 12b), 5.85 (s, 1H, =CHOMe, 12b), 5.82 (s, 1H, =CHOMe,

12a), 4.19-4.14 (m as dd, J = 13.5 Hz, J = 3.5 Hz, 1H, C-ring, 12a), 4.10-3.96 (m, 2H, 4'-H, 12a and 2H, 4'-H, 12b), 3.56 (s, 3H, OMe, 12b), 3.53 (s, 3H, OMe, 12a), 3.45-3.39 (m as dd, J =

13.5 Hz, J = 3.5 Hz, 1H, C-ring, 12b), 2.96-2.88 (m as br d, J = 14.0 Hz, 1H, C-ring, 12b), 2.34-

2.27 (m, 1H, C-ring of 12a and 1H, C-ring of 12b), 2.22-2.16 (m, 1H, C-ring, 12a), 2.09-2.00

(m, 1H, C-ring, 12a), 1.91-1.84 (m, 1H, C-ring of 12a and 1H, C-ring of 12b), 1.82-1.74 (m, 1H,

C-ring, 12b), 1.67-1.51 (m, 2H, C-ring of 12a, 2H, C-ring of 12b, 2H, 5'-H of 12a and 2H, 5'-H

91 of 12b), 1.49 (s, 6H, -C(CH3)2-, 12a), 1.48 (s, 6H, -C(CH3)2-, 12b), 1.38 (s, 3H, 6-Me, 12b), 1.37

(s, 3H, 6-Me, 12a), 1.30-1.22 (sextet and sextet overlapping, J = 7.5 Hz, 2H, 6'-H, 12a and 2H,

6'-H, 12b), 1.12-0.96 (m, s, s, s and s, overlapping, 1H, C-ring of 12a, 1H, C-ring of 12b, 3H, 6-

Me of 12a, 3H, 6-Me of 12b, 9H, -Si(Me)2CMe3, of 12a, 9H, -Si(Me)2CMe3, of 12b especially

1.02, s, -Si(Me)2CMe3, of 12b and 0.99, s, -Si(Me)2CMe3, of 12a), 0.85 (t, J = 7.5 Hz, 3H, 7'-H of 12a and 3H, 7'-H of 12b), 0.24 (s, 3H, -Si(Me)2CMe3, 12b), 0.23 (s, 3H, -Si(Me)2CMe3, 12a),

13 0.12 (s, 3H, -Si(Me)2CMe3, 12a), 0.15 (s, 3H, -Si(Me)2CMe3, 12b). C NMR (100 MHz CDCl3)

δ 176.7 (-C(O)O-), 176.6 (-C(O)O-), 155.2 (ArC), 154.8 (ArC), 154.7 (ArC), 154.6 (ArC), 144.0

(ArC), 143.9 (ArC), 140.7 (ArC), 140.2 (ArC), 116.5 (ArC), 115.7 (ArC), 114.9 (ArC), 114.7

(ArC), 109.7 (ArC), 105.5 (ArC), 108.0 (ArC), 107.8 (ArC), 64.5 (-OCH2-), 59.3 (-OMe), 59.0 (-

OMe), 49.6, 49.5, 46.0, 37.1, 35.9, 34.1, 30.5, 30.3, 29.0, 27.8, 27.7, 27.6, 26.2, 26.1, 26.0, 25.0,

19.0, 12.8, 12.5, 12.3, 13.6, -3.6, -3.8, -3.9, -4.0. Mass spectrum (ESI) m/z (relative intensity)

+ + 531 (M +H, 100). Exact mass (ESI) calculated for C31H51O5Si (M +H), 531.3506; found

531.3499. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.9% and retention time 8.4 min for the title compound.

Butyl-2-{(6aR,10aR)-1-[(tert-butyldimethylsilyl)oxy]-9-formyl-6,6-dimethyl-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl}-2-methylpropanoate (13). To the stirred solution of 12

(255 mg, 0.48 mmol) in CH2Cl2 (16 mL) under an argon atmosphere, was added wet trichloroacetic acid (391 mg, 2.40 mmol). The reaction mixture was stirred at room temperature for 2.5 h 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 13a and 13b (241 mg, 97% yield) in

92 the ratio of 5:2 respectivelly as determined by 1H NMR analysis, and it was used into the next step as such. IR (neat): 2932, 2711 (CHO), 1726 (>C=O), 1611, 1566, 1414, 1253, 1140, 1064

-1 1 cm ; H NMR (500 MHz, CDCl3) δ 9.89 (s, 1H, 9α-CHO, 13b), 9.62 (s, 1H, 9β-CHO, 13a),

6.44 (d, J = 2.0 Hz, 1H, Ar-H, 13a), 6.41 (d, J = 2.0 Hz, 1H, Ar-H, 13b), 6.34 (d, J = 2.0 Hz, 1H,

Ar-H, 13b), 6.33 (d, J = 2.0 Hz, 1H, Ar-H, 13a), 4.10-3.97 (m, 2H, 4'-H, 13a and 2H, 4'-H, 13b),

3.68-3.62 (m as br d, J = 14.0 Hz, 1H, C-ring, 13b), 3.50-3.44 (m as br d, J = 13.5 Hz, 1H, C- ring, 13a), 2.66-2.61 (m, 1H, C-ring, 13b), 2.46-2.33 (m, 2H, C-ring, 13a and 2H, C-ring, 13b),

2.31-2.24 (m, 1H, C-ring, 13b), 2.14-2.08 (m, 1H, C-ring, 13a), 2.02-1.96 (m, 1H, C-ring, 13a),

1.78-1.71 (m, 1H, C-ring, 13b), 1.58-1.41 (m, s, and s, overlapping, 2H, 5'-H of 13a, 2H, 5'-H of

13b, 3H, C-ring of 13a, 2H, C-ring of 13b, 6H, -C(CH3)2- of 13a, 6H, -C(CH3)2- of 13b), 1.38

(s, 3H, 6-Me, 13a), 1.35 (s, 3H, 6-Me, 13b), 1.29-1.21 (sextet and sextet overlapping, J = 7.5 Hz,

2H, 6'-H of 13a and 2H, 6'-H of 13b), 1.20-1.11 (m, 1H, C-ring of 13a, 1H, C-ring of 13b), 1.06

(s, 3H, 6-Me, 13a), 1.00 (s and s overlapping, 3H, 6-Me of 13b and 9H, -Si(Me)2CMe3 of 13a),

0.97 (s, 9H, -Si(Me)2CMe3, 13b), 0.86 (t, J = 7.5 Hz, 3H, 7'-H, of 13a and 3H, 7'-H, of 13b),

0.27 (s, 3H, -Si(Me)2CMe3, 13b), 0.25 (s, 3H, Si(Me)2CMe3, 13a), 0.24 (s, 3H, Si(Me)2CMe3,

+ 13b), 0.14 (s, 3H, Si(Me)2CMe3, 13a). Mass spectrum (ESI) m/z (relative intensity) 517 (M +H,

100) for both peaks at 6.2 and 6.5 min. LC/MS analysis (Waters MicroMass ZQ system) showed purity 97.8% and retention time 6.2 and 6.5 min for the two epimeric aldehydes in the ratio of

2:5 respectively.

Butyl-2-{(6aR,9R,10aR)-1-[(tert-butyldimethylsilyl)oxy]-9-formyl-6,6-dimethyl-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl}-2-methylpropanoate (14). To a solution of 13 (200 mg, 0.39 mmol) in ethanol (9.5 mL) under an argon atmosphere, was added potassium carbonate powder (269 mg, 1.95 mmol) and the mixture was stirred at room

93 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 (15% diethyl ether in hexane) gave 14 (156 mg, 78% yield) as a colorless oil. IR (neat): 2932, 2711 (CHO), 1726 (>C=O),

-1 1 1611, 1566, 1414, 1253, 1140, 1064 cm ; H NMR (500 MHz, CDCl3) δ 9.62 (s, 1H, 9β-CHO),

6.44 (d, J = 2.0 Hz, 1H, Ar-H), 6.33 (d, J = 2.0 Hz, 1H, Ar-H), 4.10-3.98 (m, 2H, 4'-H), 3.50-

3.44 (m as br d, J = 13.5 Hz, 1H, C-ring), 2.46-2.36 (m, 2H, C-ring), 2.14-2.08 (m, 1H, C-ring),

2.02-1.96 (m, 1H, C-ring), 1.58-1.42 (m, s, and s, overlapping, 2H, 5'-H, 3H, C-ring, 6H, -

C(CH3)2-, especially 1.50, s, -C(CH3)2- and 1.49, s, -C(CH3)2-), 1.38 (s, 3H, 6-Me), 1.26 (sextet,

J = 7.5 Hz, 2H, 6'-H), 1.20-1.12 (m, 1H, C-ring), 1.06 (s, 3H, 6-Me), 1.00 (s, 9H, -

Si(Me)2CMe3), 0.86 (t, J = 7.5 Hz, 3H, 7'-H), 0.25 (s, 3H, Si(Me)2CMe3), 0.14 (s, 3H,

13 Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 203.4 (>C=O), 176.5 (-C(O)O-), 154.7 (ArC-1 or

ArC-5), 154.6 (ArC-5 or ArC-1), 144.5 (tertiary aromatic), 113.8 (tertiary aromatic), 109.4 (ArC-

2 or ArC-4), 108.1 (ArC-4 or ArC-2), 64.5 (-OCH2-), 50.5, 49.0, 46.0, 35.4, 30.5, 30.1, 27.5,

26.9, 26.2, 26.1, 25.9, 25.8, 13.0, 12.8, 12.1, 13.6, -3.6, -4.2. Mass spectrum (ESI) m/z (relative

+ + intensity) 517 (M +H, 100). Exact mass (ESI) calculated for C30H49O5Si (M +H), 517.3349; found 517.3351. LC/MS analysis (Waters MicroMass ZQ system) showed purity 96.7% and retention time 6.5 min for the title compound.

Butyl-2-{(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}-2-methylpropanoate (15). Sodium borohydride (57 mg, 1.55 mmol) was added to a stirred solution of aldehyde 14 (100 mg, 0.13 mmol) in ethanol (4.8 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

94 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

(30% diethyl ether in hexane) gave 15 (84 mg, 85% yield) as a colorless viscous oil. IR (neat):

3430, 2932, 1729 (>C=O), 1611, 1566, 1414, 1253, 1140, 1064 cm-1; 1H NMR (500 MHz,

CDCl3) δ 6.43 (d, J = 2.0 Hz, 1H, Ar-H), 6.31 (d, J = 2.0 Hz, 1H, Ar-H), 4.09-3.97 (m, 2H, 4'-

H), 3.54 (dd, J = 10.0 Hz, J = 5.5 Hz, half of an AB system, 1H, -CH2OH), 3.45 (dd, J = 10.0

Hz, J = 7.0 Hz, half of an AB system, 1H, -CH2OH), 3.12-3.12 (m as br d, J = 13.0 Hz, 1H, C- ring), 2.40-2.32 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H, C-ring), 2.04-1.98 (m, 1H, C-ring), 2.94-

1.88 (m, 1H, C-ring), 1.76-1.68 (m, 1H, C-ring), 1.58-1.43 (m, s, and s, overlapping, 9H, -

C(CH3)2-, 5'-H, C-ring, especially 1.49, s, 3H, -C(CH3)2-, and 1.48, s, 3H, -C(CH3)2-), 1.37 (s,

3H, 6-Me), 1.29-1.22 (m as sextet, J = 7.0 Hz, 2H, 6'-H), 1.12-1.10 (m, 2H, C-ring), 1.05 (s, 3H,

6-Me), 0.99 (s, 9H, -Si(Me)2CMe3), 0.86 (t, J = 7.5 Hz, 3H, 7'-H), 0.76 (m as q, J = 11.0 Hz, 1H,

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

176.6 (-C(O)O-), 154.7 (ArC-1 or ArC-5), 154.6 (ArC-5 or ArC-1), 144.0 (tertiary aromatic),

114.8 (tertiary aromatic), 109.5 (ArC-2 or ArC-4), 108.0 (ArC-4 or ArC-2), 68.4 (-CH2OH), 64.5

(-OCH2-), 49.6, 46.0, 40.5, 35.5, 33.1, 30.5, 29.7, 27.6, 27.5, 26.1, 25.9, 13.0, 12.8, 12.2, 13.6, -

3.6, -4.3. Mass spectrum (ESI) m/z (relative intensity) 513 (M++H, 100). Exact mass (ESI)

+ calculated for C30H51O5Si (M +H), 513.3506; found 513.3507. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 98.3% and retention time 6.2 min for the title compound.

Butyl-2-[(6aR,9R,10aR)-1-hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (16). To a solution of 15 (75 mg,

0.15 mmol) in anhydrous THF (3.6 mL) at -40 °C, under an argon atmosphere, was added tetra-

95 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 (20% ethyl acetate in hexane) gave 16 (52 mg, 90% yield) as a white solid. mp = 59-60 oC. IR (neat): 3380, 2971, 2869, 1727, 1703, 1620, 1577, 1417, 1268,

-1 1 1141 cm ; H NMR (500 MHz, CDCl3) δ 6.39 (d, J = 2.0 Hz, 1H, Ar-H), 6.22 (d, J = 2.0 Hz,

1H, Ar-H), 5.33 (br s, 1H, ArOH), 4.05 (d, J = 6.0 Hz, 2H, 4'-H), 3.56-3.49 (m, 2H, -CH2OH),

3.23-3.18 (m as br d, J = 13.0 Hz, 1H, C-ring), 2.51-2.44 (m as td, J = 11.0 Hz, J = 2.5 Hz, 1H,

C-ring), 2.00-1.94 (m, 1H, C-ring), 1.94-1.88 (m, 1H, C-ring), 1.82-1.72 (m, 1H, C-ring), 1.58-

1.44 (quintet, s, and m, overlapping, 9H, -C(CH3)2-, 5'-H, C-ring, especially 1.49, s, 6H, -

C(CH3)2-), 1.38 (s, 3H, 6-Me), 1.29-1.22 (m as sextet, J = 7.0 Hz, 2H, 6'-H), 1.18-1.10 (m, 2H,

C-ring), 1.07 (s, 3H, 6-Me), 0.86 (t, J = 7.5 Hz, 3H, 7'-H), 0.76 (m as q, J = 11.0 Hz, 1H, C-ring).

13 C NMR (100 MHz CDCl3) δ 177.1 (-C(O)O-), 155.2 (ArC-1 or ArC-5), 154.9 (ArC-5 or ArC-

1), 144.2 (tertiary aromatic), 111.0 (tertiary aromatic), 107.2 (ArC-2 or ArC-4), 105.3 (ArC-4 or

ArC-2), 68.4 (-CH2OH), 64.7 (-OCH2-), 49.3, 46.0, 40.4, 35.0, 33.0, 30.5, 29.7, 27.7, 27.4, 26.2,

26.0, 19.0, 13.6. Mass spectum (ESI) m/z (relative intensity) 405 (M++H, 100). Mass spectum

(EI) m/z (relative intensity) 404 (M+, 100), 361 (41), 303 (65), 265 (41). Exact mass (ESI)

+ calculated for C24H3705 (M +H), 405.2641; found 405.2642. Exact mass (EI) calculated for

+ C24H3605 (M ), 404.2563; found 404.2559. HPLC (4.6 x 250 mm, Supelco discovery column, acetonitrile/water) showed purity 99.5% and retention time 10.8 min for the title compound.

3-phenoxypropyl 2-(3,5-dimethoxyphenyl)-2-methylpropanoate (17). The synthesis was carried out as described for 5 starting from 3 (1 g, 4.46 mmol) in anhydrous DMF (4.65 ml), 3- phenoxypropyl bromide (1.44 g, 6.70 mmol) and sodium bicarbonate (450 mg, 5.35 mmol) to

96 give 17 as a colorless oil (1.4 g, 90% yield). IR (neat): 2935, 1725, 1595, 1496, 1456, 1241, 1047

-1 1 cm . H NMR (500 MHz, CDCl3) δ 7.25 (tt, J = 8.0 Hz, J = 6.0 Hz, 2H, O-Ph), 6.92 (tt, J = 7.5

Hz, J = 6.0 Hz, 1H, O-Ph), 6.80 (dt, J = 8.0 Hz, J = 2 Hz, 2H, O-Ph), 6.47 (d, J = 2.0 Hz, 2H,

ArH), 6.31 (t, J = 2.5 Hz, 1H, ArH), 4.25 (t, J = 6.5 Hz, 2H, 4’-H), 3.84 (t, J = 6.5 Hz, 2H, 6’-

13 H), 3.73 (s, 6H, (OCH3)2), 2.03 (q, 2H, 5’-H), 1.54 (s, 6H, -C(CH3)2-). C NMR (100 MHz

CDCl3) δ 176.6 (-C(O)O-), 160.8, 158.9, 147.3, 129.6, 120.9, 114.6, 104.4, 98.3, 66.0, 64.1,

61.7, 55.4, 46.8, 28.7, 26.4. LC/MS analysis (Waters MicroMass ZQ system) showed purity

99.5% and retention time 4.0 min for the title compound.

3-iodopropyl 2-(3,5-dihydroxyphenyl)-2-methylpropanoate (18). To a solution of 17 (4.4 g,

12.30 mmol) in dry Hexane (245 mL) at 0 °C under an argon atmosphere was added 9-I-9-BBN

(52 ml, 52 mmol). Following the addition, the reaction temperature was gradually raised to room temperature. Stirring was continued at that temperature until completion of the reaction (3 h).

The reaction mixture was evaporated and then anhydrous Et2O (50 ml) was added. Then ethanolamine (3.4 mg, 8.9 mmol) in anhydrous THF (20 ml) was added to the solution and stirred vigorously for 30 min. The white precipitate formed was filtered out and the filtrate was concentrated and purified. Purification by flash column chromatography (25% ethyl acetate- petroleum ether) afforded 3.8 g (85% yield) of the compound 18 as a colorless oil. IR (neat):

-1 1 3368, 2975, 1699, 1598, 1463, 1439, 1265, 1110 cm . H NMR (500 MHz, CDCl3) δ 6.39 (d, J

= 2.0 Hz, 2H, ArH), 6.24 (t, J = 2.5 Hz, 1H, ArH), 5.08 (s, 1H, OH), 4.14 (t, J = 6.5 Hz, 2H, 4’-

13 H), 3.05 (t, J = 6.5 Hz, 2H, 6’-H), 2.05 (m, 2H, 5’-H), 1.52 (s, 6H, -C(CH3)2-). C NMR (100

MHz CDCl3) δ 176.9 (-C(O)O-), 156.4 (ArC-3 and ArC-5), 146.8 (ArC-1), 105.1 (ArC-4 and

ArC-6), 101.1 (ArC-2), 65.3 (-OCH2-), 46.1, 31.4, 25.6, 14.7. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 98.7% and retention time 4.2 min for the title compound.

3-iodopropyl-2-(4-((1R,2R,5R)-6,6-dimethyl-4-oxobicyclo[3.1.1]heptan-2-yl)-3,5- dihydroxyphenyl)-2-methylpropanoate (19). The synthesis was carried out as described for 9 starting from 18 (2.9 g, 7.96 mmol), diacetates 8 (6.6 g, ca. 80% pure by 1H NMR, 27.88 mmol) and p-toluenesulfonic acid monohydrate (2.12 g, 11.15 mmol) in CHCl3 (80 ml) and gave 900 mg (22% yield) of 19. mp = 68-69oC. IR (neat): 3374, 2969, 1728, 1682, 1589, 1421, 1264, 1027

-1 1 cm . H NMR (500 MHz, CDCl3) δ 6.31 (s, 2H, ArH), 5.50 (s, 2H, OH), 4.14 (t, J = 7.5 Hz, 1H,

4’-H), 3.95 (t, J = 7.0 Hz, 1H, 4-H), 3.51 (dd, J = 11.0 Hz, J = 8.0 Hz, 1H, 3α-H), 3.05 (t, J = 6.5

Hz, 2H, 6’-H), 2.64-2.57 (m, 2H, 3β-H, 1-H), 2.51 (m, 1H, 7α-H), 2.46 (d, J = 10.5 Hz, 1H, 7β-

H), 2.29 (t, J = 4.5 Hz, 1H, 5-H), 2.06 (m, 2H, 5’-H), 1.50 (s, 6H, -C(CH3)2-), 1.36 (s, 3H, 6β-

13 Me), 1.00 (s, 3H, 6α-Me). C NMR (100 MHz CDCl3) δ 217.4 (>C=O), 176.8 (-C(O)O-), 155.4

(ArC-3 and ArC-5), 143.8 (tertiary aromatic), 115.2 (tertiary aromatic), 106.0 (ArC-2 and ArC-

6), 64.6 (-OCH2-), 58.0, 46.8, 46.0, 42.1, 37.6, 31.9, 29.5, 26.1, 26.0, 25.9, 24.9, 24.5, 22.2. Mass spectrum (ESI) m/z (relative intensity) 501 (M+ + H, 100). Exact mass (ESI) calculated for

+ C22H30O5I (M + H), 501.1138; found 501.1139. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.5% and retention time 4.7 min for the title compound.

3-iodopropyl-2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanoate (20). The synthesis was carried out as described for 10 starting from 19 (300 mg, 0.6 mmol), trimethylsilyl trifluoromethanesulfonate (0.6 mL,

0.3 M solution in CH3NO2, 0.18 mmol) in anhydrous CH2Cl2/CH3NO2 (3:1 mixture, 12 mL) and

98 gave 200 mg (65% yield) of 20 as a white crystalline solid. mp = 38-39oC. IR (neat): 3180, 2971,

-1 1 1718, 1696, 1574, 1418, 1260, 1095 cm . H NMR (500 MHz, CDCl3) δ 6.41 (d, J = 2.0 Hz, 1H,

Ar-H), 6.29 (d, J = 2.0 Hz, 1H, Ar-H), 6.25 (s, 1H, OH), 4.12 (t, J = 7.5 Hz, 1H, 4’-H), 3.96

(ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.02 (t, J = 7.0 Hz, 2H, 6’-H), 2.88 (td, J

= 11.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.64-2.58 (m, 1H, 8eq-H), 2.50-2.41 (m, 1H, 8ax-H), 2.19-

2.13 (m, 2H, 10ax-H, 7eq-H), 2.06 (m, 2H, 5’-H), 2.00-1.93 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, 6a-

H), 1.55-1.53 (m, 1H, 7ax-H), 1.51 (s, 6H, -C(CH3)2-), 1.48 (s, 3H, 6β-Me), 1.12 (s, 3H, 6α-Me).

13 C NMR (100 MHz CDCl3) δ 213.9 (>C=O), 176.5 (-C(O)O-), 155.5 (ArC-1 or ArC-5), 154.6

(ArC-5 or ArC-1), 144.9 (tertiary aromatic), 109.3 (tertiary aromatic), 106.7 (ArC-2 or ArC-4),

105.2 (ArC-4 or ArC-2), 76.9 (C-6), 64.3 (-OCH2-), 47.3, 46.1, 44.9, 40.8, 34.7, 32.2, 27.8, 26.8,

26.1, 26.0, 18.9, 16.7. Mass spectrum (ESI) m/z (relative intensity) 501 (M+ + H, 100). Exact

+ mass (ESI) calculated for C22H30O5I (M + H), 501.1138; found 501.1141. LC/MS analysis

(Waters MicroMass ZQ system) showed purity 99.2% and retention time 4.9 min for the title compound.

Mixture of 3-cyanopropyl-2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (21) and 3-cyanopropyl-2-

((6aR,10aR)-9-cyano-1,9-dihydroxy-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanoate (22). To a stirred solution of 20 (27 mg, 0.054 mmol) in anhydrous DMSO (1.1 ml) at room temperature, sodium cyanide (13 mg, 0.27 mmol) was added under an argon atmosphere. The reaction mixture was stirred at the same temperature for 16 h. The reaction was quenched using H2O and extracted using Et2O. The organic layer was concentrated under vacuum and the residue was chromatographed on silica gel (35% ethyl

99 acetate in hexane) to give a mixture of 21 (11 mg, 52% yield) and 22 (4 mg, 18% yield). 22 (20 mg, 0.04 mmol) was then treated with potassium carbonate (100 mg, 0.72 mmol) in ethanol (1.2 ml) at room temperature for 2 h to yield 21 (15 mg, 75%) with a purity of 93%. mp = 43-44oC.

-1 1 IR (neat): 3248, 2971, 1720, 1685, 1572, 1417, 1260, 1093 cm . H NMR (500 MHz, CDCl3) of

21 δ 6.22 (d, J = 2.0 Hz, 1H, Ar-H), 6.31 (d, J = 2.0 Hz, 1H, Ar-H), 6.19 (s, 1H, OH), 4.17 (t, J

= 7.5 Hz, 1H, 4’-H), 3.94 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.88 (td, J =

11.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.64-2.58 (m, 1H, 8eq-H), 2.50-2.41 (m, 1H, 8ax-H), 2.24 (t, J

= 7.0 Hz, 2H, 6’-H), 2.19-2.13 (m, 2H, 10ax-H, 7eq-H), 2.00-1.90 (m, 3H, 5’-H, 6a-H), 1.55-

1.53 (m, 1H, 7ax-H), 1.51 (d, J = 1.5 Hz, 6H, -C(CH3)2-), 1.48 (s, 3H, 6β-Me), 1.12 (s, 3H, 6α-

13 Me). C NMR (100 MHz CDCl3) δ 214.0 (>C=O), 176.3 (-C(O)O-), 155.6 (ArC-1 or ArC-5),

154.7 (ArC-5 or ArC-1), 144.7 (tertiary aromatic), 118.9 (tertiary aromatic), 109.4 (ArC-2 or

ArC-4), 106.6 (ArC-4 or ArC-2), 105.2 (CN), 62.3 (-OCH2-), 47.2, 46.2, 44.8, 40.8, 34.7, 27.8,

26.8, 26.0, 24.7, 18.9, 13.9. Mass spectrum (ESI) m/z (relative intensity) 400 (M+ + H, 100).

+ Exact mass (ESI) calculated for C23H30NO5 (M + H), 400.2124; found 400.2117. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.6% and retention time 4.5 min for the title compound. 1H NMR analysis, 22 is a mixture of two isomers in a ratio of 1:0.7 respectively.

1 H NMR (500 MHz, CDCl3) of 22 δ 6.42 (d, J = 2.5 Hz, 2H, Ar-H, 22a), 6.21 (d, J = 2.5 Hz,

2H, Ar-H, 22b), 6.33 (t, J = 2.0 Hz, 2H, Ar-H, 22a), 6.29 (t, J = 2.0 Hz, 2H, Ar-H, 22b), 6.21 (s,

1H, OH, 22a), 6.17 (s, 1H, OH, 22b), 4.22-4.14 (m, 4H, 4’-H, 22a&b), 3.79 (ddd, J = 15.0 Hz, J

= 3.5 Hz, J = 2.0 Hz, 1H, C-ring, 22a), 3.64 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, C-ring, 22b), 2.97-

2.77 (m, 2H, C-ring, 22a&b), 2.42-2.10 (m, overlapping, 8H, C-ring, 6’H, 22a&b), 2.01-1.87

(m, 4H, C-ring, 22a&b), 1.76-1.61 (m, overlapping, 5H, C-ring, 5’H, 22a&b), 1.58-1.42 (m, s, s, overlapping, 14H, -C(CH3)2-, C-ring, especially 1.52, s, 6H, -C(CH3)2-, 22a and 1.51, s, 6H, -

100

C(CH3)2-, 22b), 1.41 (s, 3H, 6β-Me, 22a), 1.21 (s, 3H, 6β-Me, 22b), 1.12 (s, 3H, 6α-Me, 22a),

1.10 (s, 3H, 6α-Me, 22b), 1.07-1.01 (m, 1H, C-ring, 22b). Mass spectrum (ESI) m/z (relative

+ + intensity) 427 (M + H, 100). Exact mass (ESI) calculated for C24H31N2O5 (M + H), 427.2233; found 427.2223. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.1% and retention time 4.5 min for the title compound.

3-cyanopropyl-2-(4-((1R,2R,5R)-6,6-dimethyl-4-oxobicyclo[3.1.1]heptan-2-yl)-3,5- dihydroxyphenyl)-2-methylpropanoate (23). To a stirred solution of 19 (40 mg, 0.08 mmol) in anhydrous DMSO (4 ml) at 20°C under an argon atmosphere, sodium cyanide (20 mg, 0.4 mmol) was added. The reaction mixture was stirred vigorously at the same temperature for 30 min. The reaction was quenched using H2O and extracted using Et2O. The organic layer was concentrated under vacuum and the residue was chromatographed on silica gel (35% ethyl acetate in hexane) to give 23 as a white solid (31 mg, 95% yield). mp = 66-67oC. IR (neat): 3363,

-1 1 2971, 1728, 1682, 1588, 1421, 1262, 1026 cm . H NMR (500 MHz, CDCl3) δ 6.35 (s, 2H,

ArH), 5.77 (s, 2H, OH), 4.19 (t, J = 7.5 Hz, 1H, 4’-H), 3.96 (t, J = 7.0 Hz, 1H, 4-H), 3.51 (dd, J

= 11.0 Hz, J = 8.0 Hz, 1H, 3α-H), 2.64-2.56 (m, 2H, 3β-H, 1-H), 2.50 (m, 1H, 7α-H), 2.46 (d, J =

10.5 Hz, 1H, 7β-H), 2.32 (t, J = 6.5 Hz, 2H, 6’-H), 2.28 (t, J = 4.5 Hz, 1H, 5-H), 1.96 (m, 2H,

13 5’-H), 1.51 (s, 6H, -C(CH3)2-), 1.36 (s, 3H, 6β-Me), 0.99 (s, 3H, 6α-Me). C NMR (100 MHz

CDCl3) δ 217.2 (>C=O), 176.6 (-C(O)O-), 155.5 (ArC-1 and ArC-5), 143.6 (tertiary aromatic),

119.1 (tertiary aromatic), 115.3 (ArC-2 and ArC-4), 106.1 (CN), 62.7 (-OCH2-), 58.03, 46.8,

46.0, 42.1, 37.6, 29.5, 26.1, 26.0, 25.9, 24.4, 24.3, 22.1, 14.1. Mass spectrum (ESI) m/z (relative

+ + intensity) 400 (M + H, 100). Exact mass (ESI) calculated for C23H30NO5 (M + H), 400.2124;

101 found 400.2119. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.0% and retention time 4.3 min for the title compound.

2-(3,5-dimethoxyphenyl)-2-methylpropanal (24). To a solution of 2 (4.0 g, 17.2 mmol), in dry

CH2Cl2 (174 mL) at -78 °C under an argon atmosphere, was added diisobutylaluminum hydride

(43.4 mL, 1 M solution in hexanes) over a period of 15 min. The reaction mixture was stirred at the same temperature for 1 h and then quenched by dropwise addition of potassium sodium tartrate (10% solution in water). The resulting mixture was warmed to room temperature, stirred vigorously for 40 min, and then diluted with EtOAc.

The organic phase was separated and the aqueous phase extracted with EtOAc. The combined organic layer was washed with brine and dried over MgSO4, and the solvent was evaporated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using 15% diethyl ether-petroleum ether as eluent to give compound 24 as a white solid in 82% yield (3.5 g). IR (neat): 3425, 2960, 1591, 1455, 1422, 1202, 1110, 1044 cm-1. 1H NMR

(500 MHz, CDCl3) δ 9.46 (s, 1H), 6.40 (d, J = 1.7 Hz, 2H, ArH), 6.38 (t, J = 1.7 Hz, 1H, ArH),

3.79 (s, 6H), 1.71 (s, 6 H, -C(CH3)2-). LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.2% and retention time 4.4 min for the title compound.

Ethyl-4-(3,5-dimethoxyphenyl)-4-methylpent-2-enoate (25). To a suspension of methoxymethyltriphenylphosphonium bromide (23.8 g, 57.7 mmol) in dry THF (193 mL) at 0

°C, under an argon atmosphere, was added potassium bis(trimethylsilyl)amide (11.25 g, 56.56 mmol). The mixture was stirred for 15 min to ensure complete formation of the orange ylide. To the resulting slurry, at the same temperature, was added dropwise a solution of 24 (2.4 g, 11.54

102 mmol) in dry THF (17 mL). The reaction was stirred for 90 min 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 over MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified through a column of silica gel using 5% diethyl ether-petroleum ether as eluent to afford the compound 25 as a colorless liquid in 80% yield (2.2 g). IR (neat): 2968,

-1 1 1714, 1648, 1594, 1456, 1423, 1293, 1048 cm . H NMR (500 MHz,CDCl3) δ 7.09 (d, J = 15.5

Hz, 1H, 2’-H), 6.44 (d, J = 2.5 Hz, 2H, Ar-H), 6.33 (t, J = 2.0 Hz, 2H, Ar-H), 5.81 (d, J = 13 Hz,

1H, 3’-H), 4.18 (q, J = 7 Hz, 2H, 6’-H), 3.78 (s, 3H, OMe), 1.43 (s, 6H, -C(CH3)2-), 1.28 (t, J = 7

13 Hz, 3H, 7’-H) . C NMR (100 MHz CDCl3) δ 167.2 (-C(O)O-), 160.9 (ArC-1 and ArC-5), 157.0

(2’-H), 149.2 (tertiary aromatic), 118.3 (3’-H), 105.0 (ArC-2 and ArC-4), 97.8 (tertiary aromatic), 60.5 (-OCH2-), 55.4, 41.3, 27.9, 14.4. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.3% and retention time 4.9 min for the title compound.

3-(3,5-dimethoxyphenyl)-3-methylbutanal (26). To a stirred solution of 25 (1.1 g, 3.96 mmol) in dioxane (10 ml) was added 6.5 ml of 1 M HCl under an argon atmosphere. The reaction mixture was stirred vigorously at room temperature for 16 h. The reaction was quenched using

H2O and extracted using Et2O. The organic layer was concentrated under vacuum and then purified by flash column chromatography using (10% ethyl acetate in hexane) to give 26 (750 mg) as a colorless oil with 80% yield. IR (neat): 2963, 1717, 1592, 1455, 1422, 1203, 1049 cm-1.

1 H NMR (500 MHz,CDCl3) δ 9.50 (t, J = 3.0 Hz, 1H, CHO), 6.52 (d, J = 1.5 Hz, 2H, Ar-H),

6.33 (t, J = 2.5 Hz, 1H, Ar-H), 3.79 (s, 6H, (OMe)2), 2.63 (d, J = 2.5 Hz, 2H, 2’-H), 1.42 (s, 6H,

13 -C(CH3)2-). C NMR (100 MHz CDCl3) δ 202.7 (-CHO-), 160.7 (ArC-1 and ArC-5), 149.8

103

(tertiary aromatic), 104.2 (ArC-2 and ArC-4), 97.8 (tertiary aromatic), 56.2, 55.0, 36.7, 29.1.

LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.5% and retention time 4.4 min for the title compound.

3-(3,5-dimethoxyphenyl)-3-methylbutanoic acid (27). To a solution of 26 (300 mg, 1.35 mmol) in anhydrous DMF (13.5 ml) at room temperature under an argon atmosphere was added oxone (1.67 g, 2.70 mmol). The reaction was complete in 4 h. The reaction was quenched using

H2O and extracted using Et2O. The organic layer was concentrated under vacuum and then purified by flash column chromatography using (30% ethyl acetate in hexane) to give 27 (375 mg) as a colorless oil with 90% yield. IR (neat): 2963, 1702, 1593, 1455, 1422, 1298, 1049 cm-1.

1 H NMR (500 MHz,CDCl3) δ 6.52 (d, J = 1.5 Hz, 2H, Ar-H), 6.32 (t, J = 2.5 Hz, 1H, Ar-H),

13 3.78 (s, 6H, (OMe)2), 2.63 (s, 2H, 2’-H), 1.43 (s, 6H, -C(CH3)2-). C NMR (100 MHz CDCl3) δ

177.9 (-C(O)O-), 161.1 (ArC-1 and ArC-5) , 151.3 (tertiary aromatic), 104.7 (ArC-2 and ArC-4),

97.9 (tertiary aromatic), 55.7, 48.3, 37.7, 29.2. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.5% and retention time 4.1 min for the title compound.

Propyl-3-(3,5-dimethoxyphenyl)-3-methylbutanoate (28). To a stirred suspension of 27 (370 mg, 1.55 mmol) in anhydrous DMF (4 ml), bromoproane (287 mg, 2.33 mmol) and sodium bicarbonate (156 mg, 1.86 mmol) was added and heated at 165°C for 12 min using microwave irradiation. The reaction mixture was cooled to room temperature and diluted with water and ethyl acetate. The organic layer was separated and the aqueous layer was extraced with ethyl acetate (3 x 5 mL). The combined organic layer was washed with water and brine, dried

(MgSO4) and concentrated under vacuo. The residue was chromatographed on silica gel to give

104

28 (330 mg, 90% yield) as a colorless oil. IR (neat): 2965, 1728, 1594, 1456, 1422, 1203, 1053

-1 1 cm . H NMR (500 MHz,CDCl3) δ 6.52 (d, J = 1.5 Hz, 2H, Ar-H), 6.31 (t, J = 2.5 Hz, 1H, Ar-

H), 3.91 (t, J = 6.5 Hz, 2H, 5’-H), 3.78 (s, 6H, (OMe)2), 2.59 (s, 2H, 2’-H), 1.52 (q, 2H, 6’H),

13 1.42 (s, 6H, -C(CH3)2-), 0.84 (t, J = 7.0 Hz, 3H, 7’H). C NMR (100 MHz CDCl3) δ 171.8 (-

C(O)O-), 160.7 (ArC-1 and ArC-5) , 151.2 (tertiary aromatic), 104.5 (ArC-2 and ArC-4), 97.5

(tertiary aromatic), 65.9, 55.4, 48.5, 37.6, 29.9, 29.0, 22.1, 10.5. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 99.6% and retention time 4.9 min for the title compound.

Propyl-3-(3,5-dihydroxyphenyl)-3-methylbutanoate (29). To a solution of 28 (280 mg, 1 mmol) in dry Hexane (20 mL) at 0 °C under an argon atmosphere was added 9-I-9-BBN (3.2 mL, 1 M solution in Hexane). Following the addition, the reaction temperature was gradually raised to room temperature. Stirring was continued at that temperature until completion of the reaction (3 h). The reaction mixture was evaporated and then anhydrous Et2O (20 ml) was added.

Then ethanolamine (213 mg, 3.5 mmol) in anhydrous THF (8 ml) was added to the solution and stirred vigorously for 30 min. The white precipitate formed was filtered out and the filtrate was concentrated and purified. Purification by flash column chromatography (25% ethyl acetate- petroleum ether) afforded 190 mg (85% yield) of the compound 29 as a colorless oil. IR (neat):

-1 1 3352, 2971, 1699, 1600, 1440, 1331, 1214, 1045 cm . H NMR (500 MHz, CDCl3) δ 6.43 (d, J

= 1.5 Hz, 2H, Ar-H), 6.16 (t, J = 2.0 Hz, 1H, Ar-H), 5.99 (s, 2H, OH), 3.91 (t, J = 6.5 Hz, 2H, 5’-

H), 2.59 (s, 2H, 2’-H), 1.52 (sextet, J = 7.0 Hz, 2H, 6'-H), 1.38 (s, 6H, -C(CH3)2-), 0.84 (t, J =

13 7.0 Hz, 3H, 7’H). C NMR (100 MHz CDCl3) δ 172.5 (-C(O)O-), 156.7 (ArC-3 and ArC-5),

150.9 (ArC-1), 105.2 (ArC-4 and ArC-6), 100.6 (ArC-2), 66.0 (-OCH2-), 48.3, 37.0, 28.7, 18.9,

105

15.0. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.8% and retention time

4.0 min for the title compound.

Propyl-3-(4-((1R,2R,5R)-6,6-dimethyl-4-oxobicyclo[3.1.1]heptan-2-yl)-3,5- dihydroxyphenyl)-3-methylbutanoate (30). The synthesis was carried out as described for 9 starting from 29 (75 mg, 0.30 mmol), diacetates 8 (188 mg, ca. 80% pure by 1H NMR, 0.75 mmol) and p-toluenesulfonic acid monohydrate (80 mg, 0.42 mmol) in CHCl3 (3 ml) and gave

28 mg (22% yield) of 30 as a white crystalline solid. mp = 65-66oC. IR (neat): 3354, 2965, 1680,

-1 1 1589, 1421, 1371, 1267, 1029 cm . H NMR (500 MHz, CDCl3) δ 6.31 (s, 2H, ArH), 4.98 (s,

2H, OH), 3.94 (t, J = 7.5 Hz, 1H, 4-H), 3.91 (t, J = 7.0 Hz, 2H, 5’-H), 3.49 (dd, J = 11.0 Hz, J =

8.0 Hz, 1H, 3α-H), 2.60-2.55 (m, 2H, 3β-H, 1-H), 2.53 (s, 2H, 2’-H), 2.49 (m, 1H, 7α-H), 2.46

(d, J = 10.0 Hz, 1H, 7β-H), 2.26 (t, J = 4.5 Hz, 1H, 5-H), 1.51 (quintet, J = 7.5 Hz, 2H, 6’-H),

1.37 (s, 6H, -C(CH3)2-), 1.36 (s, 3H, 6β-Me), 0.99 (s, 3H, 6α-Me), 0.83 (t, J = 7.5 Hz, 3H, 7’H).

13 C NMR (100 MHz CDCl3) δ 217.3 (>C=O), 172.4 (-C(O)O-), 155.3 (ArC-3 and ArC-5), 148.3

(tertiary aromatic), 114.4 (tertiary aromatic), 106.3 (ArC-2 and ArC-6), 66.2 (-OCH2-), 58.2,

48.6, 47.1, 42.3, 37.9, 36.9, 29.6, 28.8, 26.3, 24.6, 22.4, 22.0, 10.6. Mass spectrum (ESI) m/z

+ + (relative intensity) 389 (M + H, 100). Exact mass (ESI) calculated for C23H33O5 (M + H),

389.2328; found 389.2321. LC/MS analysis (Waters MicroMass ZQ system) showed purity

99.0% and retention time 4.6 min for the title compound.

Propyl-3-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-3-methylbutanoate (31). The synthesis was carried out as described for

10 starting from 31 (26 mg, 0.07 mmol), trimethylsilyl trifluoromethanesulfonate (0.07 mL, 0.3

M solution in CH3NO2, 0.02 mmol) in anhydrous CH2Cl2/CH3NO2 (3:1 mixture, 1.4 mL) gave

106

16 mg (65% yield) of 31 as a white crystalline solid. mp = 33-34oC. IR (neat): 3304, 2969, 1696,

-1 1 1577, 1425, 1335, 1269, 1093 cm . H NMR (500 MHz, CDCl3) δ 6.42 (d, J = 2.0 Hz, 1H, Ar-

H), 6.32 (d, J = 2.0 Hz, 1H, Ar-H), 6.03 (s, 1H, OH), 3.95 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0

Hz, 1H, 10eq-H), 3.91 (t, J = 7.0 Hz, 2H, 5’-H), 2.87 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, 10a-H),

2.63-2.57 (m, 1H, 8eq-H), 2.54 (s, 2H, 2’-H), 2.48-2.39 (m, 1H, 8ax-H), 2.19-2.08 (m, 2H, 10ax-

H, 7eq-H), 1.98-1.91 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, 6a-H), 1.55-1.48 (m, 3H, 7ax-H, 6’-H),

1.47 (s, 3H, 6β-Me), 1.38 (s, 6 H, -C(CH3)2-), 1.11 (s, 3H, 6α-Me), 0.83 (t, J = 7.5 Hz, 3H, 7’H).

13 C NMR (100 MHz CDCl3) δ 213.6 (>C=O), 172.4 (-C(O)O-), 155.2 (ArC-1 or ArC-5), 154.6

(ArC-5 or ArC-1), 149.3 (tertiary aromatic), 108.6 (tertiary aromatic), 107.0 (ArC-2 or ArC-4),

105.4 (ArC-4 or ArC-2), 65.9 (-OCH2-), 48.5, 47.6, 45.2, 41.0, 37.1, 34.9, 28.8, 27.0, 22.11,

19.1, 13.9. Mass spectrum (ESI) m/z (relative intensity) 389 (M+ + H, 100). Exact mass (ESI)

+ calculated for C23H33O5 (M + H), 389.2328; found 389.2322. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 99.5% and retention time 4.8 min for the title compound.

3-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-3-methylbutanoic acid (32) To a stirred solution of 31 (20 mg, 0.05 mmol) in dioxane/H2O (1:1 ratio, 3 mL) at room temperature, under an argon atmosphere, was added lithium hydroxide (12 mg, 0.5 mmol). Stirring was continued for 8 h and then the reaction mixture was quenched with 1N HCl and diluted with ethyl acetate. 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. The crude product was chromatographed on silica gel (48% acetone in hexane) to give 32 (14 mg, 80% yield) as a white solid. mp 148-149°C; IR (neat): 3340, 2978, 1695, 1620, 1417 cm-1; 1H NMR

107

(500 MHz, CDCl3 + CD3OD) δ 6.40 (d, J = 2.0 Hz, 1H, ArH), 6.33 (d, J = 2.0 Hz, 1H, ArH),

6.30 (br s, 1H, OH), 4.00 (ddd, J = 15.0, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.88 (m as td, J =

12.5, J = 3.5 Hz, 1H, 10a-H), 2.65 - 2.60 (m, 1H, 8eq-H), 2.56 (s, 1H, 2’-H), 2.52 - 2.42 (m, 1H,

8ax-H), 2.19 - 2.10 (m, 2H, 10ax-H, 7eq-H), 1.96 (m as td, J = 12.2, J = 3.0 Hz, 1H, 6a-H), 1.58-

13 1.50 (m, 1H, 7ax-H), 1.47 (s, 3H, 6β-Me), 1.39 (s, 6H, -C(CH3)2-), 1.21 (s, 3H, 6α-Me). C

NMR (100 MHz CDCl3) δ 213.2 (>C=O), 176.3 (-C(O)O-), 154.8 (ArC-1 or ArC-5), 154.3

(ArC-5 or ArC-1), 148.6 (tertiary aromatic), 108.6 (tertiary aromatic), 106.8 (ArC-2 or ArC-4),

105.1 (ArC-4 or ArC-2), 47.6, 47.2, 45.0, 40.6, 37.0, 36.6, 34.6, 30.3, 28.4, 27.7, 26.8, 18.9.

Mass spectrum (ESI) m/z (relative intensity) 347 (M+ + H, 100). Exact mass (ESI) calculated for

+ C20H27O5 (M + H), 347.1858; found 347.1851. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.7% and retention time 4.3 min for the title compound.

Methyl-4-(3,5-dimethoxyphenyl)-4-methylpentanoate (33). To a stirred solution of 25 (480 mg, 2.16 mmol) in dry MeOH (22 ml) at 0°C, under an argon atmosphere was added Magnesium

(800 mg, 32.4 mmol). Following the addition, the reaction temperature was gradually raised to room temperature. Stirring was continued at that temperature until completion of the reaction (15 h). The reaction mixture was first evaporated and then dissolved in H2O to dissolve the white precipitate. To this mixture, Et2O was added and stirred vigorously. 5% HCl was added drop wise to make a clear solution and then extraction was done using Et2O. The organic layer was concentrated and the residue was chromatographed on silica gel (30% Et2O in hexane) to yield

33 with 85% yield (300 mg) as a colorless oil. IR (neat): 2954, 1734, 1593, 1455, 1422, 1296,

-1 1050 cm . 1H NMR (500 MHz,CDCl3) δ 6.47 (d, J = 1.5 Hz, 2H, Ar-H), 6.30 (t, J = 2.5 Hz, 1H,

Ar-H), 3.79 (s, 6H, (OMe)2), 3.60 (s, 3H, 6’-H), 2.10-2.05 (m, 2H, 3’-H), 1.96-1.91 (m, 2H,

108

13 2’H), 1.28 (s, 6H, -C(CH3)2-). C NMR (100 MHz CDCl3) δ 174.6 (-C(O)O-), 160.8 (ArC-1 and

ArC-5), 150.9 (tertiary aromatic), 104.8 (ArC-2 and ArC-4), 97.2 (tertiary aromatic), 55.6 (-

OCH3-), 51.6, 39.0, 37.7, 30.2, 28.9. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.4% and retention time 4.7 min for the title compound.

Methyl-4-(3,5-dihydroxyphenyl)-4-methylpentanoate (34). The synthesis was carried out as described for 25 starting from 33 (250 mg, 0.90 mmol), 9-I-9-BBN (2.7 ml, 2.7 mmol) in anhydrous hexane (15 ml) and gave 170 mg (85%yield) of 34 as a colorless oil. IR (neat): 3339,

-1 1 2958, 1710, 1595, 1439, 1239, 1161, 1026 cm . H NMR (500 MHz, CDCl3) δ 6.37 (d, J = 2.0

Hz, 2H, Ar-H), 6.20 (t, J = 2.0 Hz, 1H, Ar-H), 5.27 (s, 2H, OH), 3.61 (s, 3H, 6’-H), 2.12-2.06

13 (m, 2H, 3’-H), 1.94-1.88 (m, 2H, 2’H), 1.25 (s, 6H, -C(CH3)2-). C NMR (100 MHz CDCl3) δ

176.0 (-C(O)O-), 156.8 (ArC-1 and ArC-5), 151.4 (tertiary aromatic), 106.0 (ArC-2 and ArC-4),

100.7 (tertiary aromatic), 52.0 (-OCH3-), 38.9, 37.5, 30.2, 28.9. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 99.0% and retention time 3.7 min for the title compound.

Methyl-4-(4-((1R,2R,5R)-6,6-dimethyl-4-oxobicyclo[3.1.1]heptan-2-yl)-3,5- dihydroxyphenyl)-4-methylpentanoate (35). The synthesis was carried out as described for 9 starting from 34 (150 mg, 0.80 mmol), diacetates 8 (505 mg, ca. 80% pure by 1H NMR, 1.28 mmol) and p-toluenesulfonic acid monohydrate (243 mg, 1.28 mmol) in CHCl3 (8 ml) and gave

90 mg (25% yield) of 35 as a white crystalline solid. mp = 62-63oC. IR (neat): 3429, 2961, 1713,

-1 1 1587, 1419, 1296, 1162, 1050 cm . H NMR (500 MHz, CDCl3) δ 6.26 (s, 2H, ArH), 5.30 (s,

1H, OH), 3.95 (t, J = 7.5 Hz, 1H, 4-H), 3.62 (s, 3H, 6’-H), 3.50 (dd, J = 12.0 Hz, J = 7.5 Hz, 1H,

3α-H), 2.64-2.57 (m, 2H, 3β-H, 1-H), 2.54-2.44 (m, 2H, 7α-H, 7β-H), 2.28 (t, J = 4.5 Hz, 1H, 5-

109

H), 2.12-2.07 (m, 2H, 3’-H), 1.91-1.85 (m, 2H, 2’H), 1.36 (s, 3H, 6β-Me), 1.22 (s, 6H, -C(CH3)2-

13 ), 1.00 (s, 3H, 6α-Me). C NMR (100 MHz CDCl3) δ 216.6 (C=O), 176.4 (-C(O)O-), 159.6

(ArC-1 and ArC-5), 149.6 (tertiary aromatic), 115.9 (ArC-2 and ArC-4), 107.5 (tertiary aromatic), 60.1, 54.0, 49.8, 44.2, 43.0, 42.8, 42.6, 42.3, 42.1, 41.9, 41.7, 41.2, 39.7, 39.2, 32.4,

31.3, 31.1, 28.7, 26.4, 24.7. Mass spectrum (ESI) m/z (relative intensity) 375 (M+ + H, 100).

+ Exact mass (ESI) calculated for C22H31O5 (M + H), 375.2171; found 375.2166. LC/MS analysis

(Waters MicroMass ZQ system) showed purity 99.2% and retention time 4.5 min for the title compound.

Methyl-4-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-4-methylpentanoate (36). The synthesis was carried out as described for 10 starting from 35 (20 mg, 0.05), trimethylsilyl trifluoromethanesulfonate (0.06 mL, 0.3 M solution in CH3NO2, 0.016 mmol) in anhydrous CH2Cl2/CH3NO2 (3:1 mixture, 1 mL) and gave

12 mg (60% yield) of 36 as a white crystalline solid. mp = 36-37oC. IR (neat): 3377, 2925, 1750,

-1 1 1620, 1577, 1416, 1286, 1184, 1091 cm . H NMR (500 MHz, CDCl3) δ 6.36 (d, J = 2.0 Hz, 1H,

Ar-H), 6.28 (d, J = 2.0 Hz, 1H, Ar-H), 6.25 (s, 1H, OH), 3.98 (ddd, J = 15.0 Hz, J = 3.5 Hz, J =

2.0 Hz, 1H, 10eq-H), 3.61 (s, 3H, 6’-H), 2.88 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.64-2.58

(m, 1H, 8eq-H), 2.50-2.41 (m, 1H, 8ax-H), 2.19-2.06 (m, 4H, 3’-H, 10ax-H, 7eq-H), 1.99-1.92

(td, J = 11.5 Hz, J = 4.0 Hz, 1H, 6a-H), 1.91-1.85 (m, 2H, 2’H), 1.56-1.49 (m, 1H, 7ax-H), 1.47

13 (s, 3H, 6β-Me), 1.24 (s, 6 H, -C(CH3)2-), 1.12 (s, 3H, 6α-Me). C NMR (100 MHz CDCl3) δ

212.9 (>C=O), 174.8 (-C(O)O-), 155.1 (ArC-1 or ArC-5), 154.7 (ArC-5 or ArC-1), 149.0

(tertiary aromatic), 108.6 (tertiary aromatic), 107.7 (ArC-2 or ArC-4), 105.6 (ArC-4 or ArC-2),

51.7 (-OCH3-), 47.5, 45.4, 41.0, 37.2, 34.8, 28.6, 28.0, 27.0, 19.1. Mass spectrum (ESI) m/z

110

+ + (relative intensity) 375 (M + H, 100). Exact mass (ESI) calculated for C22H31O5 (M + H),

375.2171; found 375.2166. LC/MS analysis (Waters MicroMass ZQ system) showed purity

99.5% and retention time 4.6 min for the title compound.

1, 3-dimethoxy-5-(2-methyldec-3-en-2-yl)benzene (37). To a suspension of pentyltriphenylphosphonium bromide (15.0 g, 36.3 mmol) in dry THF (200 mL) at 0 °C, under an argon atmosphere, was added potassium bis(trimethylsilyl)amide (7.01 g, 35.6 mmol). The mixture was stirred for 15 min to ensure complete formation of the orange

(butylmethylene)triphenylphosphorane. To the resulting slurry, at the same temperature, was added dropwise a solution of 24 (1.7 g, 7.26 mmol) in dry THF (17 mL). The reaction was stirred for 90 min 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 over MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified through a short column of silica gel using 5% diethyl ether-petroleum ether as eluent to afford the compound 37 as a colorless liquid in 93% yield (2.0 g). IR (neat): 2957, 1593, 1455, 1434, 1289, 1120, 1052 cm-1. 1H NMR (500

MHz, CDCl3) δ 6.55 (d, J = 2.0 Hz, 2H, ArH), 6.28 (t, J = 2.0 Hz, 1H, ArH), 5.60 (dt, J = 11.5

Hz, J = 2.0 Hz, 1H, 2’H), 5.28 (dt, J = 11.5 Hz, J = 7.5 Hz, 1H, 3’H), 3.78 (s, 6H, OMe), 1.65

(ddt, J = 2.0 Hz, J = 7.5 Hz, J = 7.5 Hz, 2H, 4’H), 1.39 (s, 6 H, -C(CH3)2-), 1.15-1.07 (m, 4H,

13 5’H, 6’H), 0.74 (t, J = 7.5 Hz, 3H, 7’H). C NMR (100 MHz CDCl3) δ 160.3 (ArC-1 and ArC-

5), 153.2 (2’-H), 138.9 (tertiary aromatic), 131.3 (3’-H), 104.7 (ArC-2 and ArC-4), 96.8 (tertiary aromatic), 55.1, 40.2, 31.2, 27.9, 22.2, 14.4. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.3% and retention time 5.6 min for the title compound.

111

5-(2-methyldec-3-en-2-yl)benzene-1,3-diol (38). To a solution of 37 (8 g, 30.5 mmol) in dry

CH2Cl2 (305 mL) at 0 °C under an argon atmosphere was added 9-I-9-BBN (85 mL, 1 M solution in CH2Cl2). Following the addition, the reaction temperature was gradually raised to room temperature. Stirring was continued at that temperature until completion of the reaction (3 h). The reaction mixture was evaporated and then anhydrous Et2O (100 ml) was added. Then ethanolamine (8.4 g, 137.4 mmol) in anhydrous THF (45 ml) was added to the solution and stirred vigorously for 30 min. The white precipitate formed was filtered out and the filtrate was concentrated and purified. Purification by flash column chromatography (25% ethyl acetate- petroleum ether) afforded 6.5 g (85% yield) of the compound 38 as a slightly brown oil. IR

-1 1 (neat): 3301, 2960, 1596, 1464, 1210, 1165, 1044 cm . H NMR (500 MHz, CDCl3) δ 6.44 (d, J

= 2.0 Hz, 2H, ArH), 6.16 (t, J = 2.0 Hz, 1H, ArH), 5.58 (dt, J = 11.5 Hz, 1H, 2’H), 5.27 (dt, J =

11.5 Hz, J = 7.5 Hz, 1H, 3’H), 4.73 (s, 1H, OH), 1.65 (ddt, J = 2.0 Hz, J = 7.5 Hz, J = 7.5 Hz,

2H, 4’H), 1.36 (s, 6 H, -C(CH3)2-), 1.16-1.09 (m, 4H, 5’H, 6’H), 0.76 (t, J = 7.5 Hz, 3H, 7’H).

13 C NMR (100 MHz CDCl3) δ 156.6 (ArC-1 and ArC-5), 153.2 (2’-H), 139.1 (tertiary aromatic),

131.3 (3’-H), 106.2 (ArC-2 and ArC-4), 100.1 (tertiary aromatic), 66.1, 58.8, 40.2, 31.6, 31.3,

28.2, 22.5, 18.3, 15.2, 14.0. LC/MS analysis (Waters MicroMass ZQ system) showed purity

99.6% and retention time 4.6 min for the title compound.

(1R,4R,5R)-4-(2,6-dihydroxy-4-(2-methyloct-3-en-2-yl)phenyl)-6,6- dimethylbicyclo[3.1.1]heptan-2-one (39). To a degassed solution of 38 (1.25 g, 4.81 mmol) and

1 diacetates (2.14 g, ca. 80% pure by H NMR, 8.99 mmol) in CHCl3 (48 mL) at 0°C, under an argon atmosphere, was added p-toluenesulfonic acid monohydrate (1.28 g, 6.73 mmol). The

112 mixture was warmed to room temperature and stirred for 3 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 removed under reduced pressure. The residue was chromatographed on silica gel (43% diethyl ether in hexane), and fractions containing almost pure product (TLC) were combined and evaporated. Further purification by recrystallization from CHCl3 and hexane gave 39 as a white crystalline solid (1.01 g, 53% yield). mp = 60-61oC. IR (neat): 3335, 2957, 1739, 1678, 1579,

-1 1 1415, 1265, 1188, 1009 cm . H NMR (500 MHz, CDCl3) δ 6.33 (s, 2H, ArH), 5.55 (dt, J = 11.0

Hz, J = 1.5 Hz, 1H, 2’-H), 5.27 (dt, J = 11.5 Hz, J = 7.0 Hz, 1H, 3’-H), 4.93 (s, 2H, OH), 3.95 (t,

J = 8.5 Hz, 1H, 4-H), 3.49 (dd, J = 18.8 Hz, J = 7.8 Hz, 1H, 3α-H), 2.62-2.55 (m, 2H, 3β-H, 1-

H), 2.54-2.45 (m, 2H, 7α-H, 7β-H), 2.27 (t, J = 5.5 Hz, 1H, 5-H), 1.70-1.63 (m, 2H, 4’-H), 1.58

(s, 3H, 6-Me), 1.36 (d, J = 6.5 Hz, 6H, -C(CH3)2-), 1.17-1.06 (m, 4H, 5’-H, 6’-H), 1.00 (s, 3H, 6-

13 Me), 0.74 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz CDCl3) δ 217.5 (C=O), 154.7 (ArC-1 and ArC-5), 154.1 (2’-H), 150.4 (tertiary aromatic), 138.7 (ArC-2 and ArC-4), 131.0 (3’-H),

106.7 (tertiary aromatic), 57.8, 46.7, 42.1, 39.4, 37.8, 31.3, 30.9, 29.3, 27.9, 26.0, 24.3, 22.2,

22.0, 13.7. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.4% and retention time 5.1 min for the title compound.

(6aR,10aR)-1-hydroxy-6,6-dimethyl-3-(2-methyloct-3-en-2-yl)-6,6a,7,8,10,10a-hexahydro-

9H-benzo[c]chromen-9-one (40). To a stirred solution of 39 (467 mg, 1.18 mmol) in anhydrous

CH2Cl2/CH3NO2 (3:1 mixture, 24 mL) at 0°C, under an argon atmosphere was added trimethylsilyl trifluoromethanesulfonate (1.2 mL, 0.3 M solution in CH3NO2, 0.36mmol). The reaction mixture was stirred at 0°C for 1 h and then gradually brought to room temperature till

113 the reaction was complete (3 h). The reaction mixture was quenched using 1:1 solution of sat.

NaHCO3 and Brine and extracted using Et2O. The organic layer was concentrated and the residue was chromatographed on silica gel (30% acetone in hexane) to yield 40 as a white solid with 61% yield (284 mg). mp = 45-46oC. IR (neat): 3282, 2956, 1739, 1693, 1577, 1414, 1210,

-1 1 1090 cm . H NMR (500 MHz,CDCl3) δ 6.45 (d, J = 2.0 Hz, 1H, Ar-H), 6.35 (d, J = 2.0 Hz, 1H,

Ar-H), 6.10 (s, 1H, OH), 5.56 (dt, J = 11.0 Hz, J = 1.5 Hz, 1H, 2’H), 5.24 (dt, J = 11.0 Hz, J =

7.5 Hz, 1H, 3’H), 3.99 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.88 (td, J = 11.5

Hz, J = 4.0 Hz, 1H, 10a-H), 2.65-2.58 (m, 1H, 8eq-H), 2.50-2.41 (m, 1H, 8ax-H), 2.20-2.10 (m,

2H, 10ax-H, 7eq-H), 2.00-1.92 (td, J = 11.5 Hz, J = 2.5 Hz, 1H, 6a-H), 1.69-1.63 (m, 2H, 4’H),

1.54-1.49 (m, 1H, 7ax-H), 1.47 (s, 3H, 6-Me), 1.35 (d, J = 2.0 Hz, 6 H, -C(CH3)2-), 1.14-1.05

(m, 7H, 5’H, 6’H, 6-Me, especially 1.11, s, 6-Me), 0.72 (t, J = 7.0 Hz, 3H, 7’H). 13C NMR (100

MHz CDCl3) δ 213.7 (C=O), 154.7 (ArC-1 and ArC-5), 154.1 (2’-H), 151.3 (tertiary aromatic),

138.7 (ArC-2 and ArC-4), 131.0 (3’-H), 106.0 (tertiary aromatic), 47.4, 45.0, 40.7, 39.6, 34.7,

31.2, 30.6, 27.7, 26.8, 22.2, 18.6, 13.7. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.1% and retention time 5.4 min for the title compound.

(6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-3-(2-methyloct-3-en-2-yl)-

6,6a,7,8,10,10a-hexahydro-9H-benzo[c]chromen-9-one (41). To a solution of 40 (1.1 g, 3.10 mmol) in anhydrous DMF (21 ml) under an argon atmosphere, imidazole (1 g, 15.54 mmol),

DMAP (189 mg, 1.55 mmol) and TBDMSCl (2.28 g, 15.2 mmol) was added. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was quenched using brine and extracted using Et2O. The organic layer was evaporated and the residue was chromatographed on silica gel (20% Et2O in hexane) to afford 1.190 g of 41 as a colorless oil with 80% yield. IR (neat): 2957, 1714, 1612, 1566, 1412, 1222, 1095 cm-1. 1H NMR (500

114

MHz,CDCl3) δ 6.49 (d, J = 2.0 Hz, 1H, Ar-H), 6.40 (d, J = 1.5 Hz, 1H, Ar-H), 5.55 (dt, J = 11.5

Hz, J = 2.0 Hz, 1H, 2’H), 5.24 (dt, J = 11.5 Hz, J = 7.5 Hz, 1H, 3’H), 3.77 (ddd, J = 15.0 Hz, J =

3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.60-2.52 (m,

1H, 8eq-H), 2.46-2.35 (m, 1H, 8ax-H), 2.18-2.05 (m, 2H, 10ax-H, 7eq-H), 1.99-1.90 (td, J =

11.5 Hz, J = 4.0 Hz, 1H, 6a-H), 1.68-1.62 (m, 2H, 4’H), 1.53-1.48 (m, 1H, 7ax-H), 1.46 (s, 3H,

6-Me), 1.35 (d, J = 4.0 Hz, 6 H, -C(CH3)2-), 1.14-1.06 (m, 7H, 5’H, 6’H, 6-Me, especially 1.08, s, 6-Me), 1.00 (s, 9H, Si(Me)2CMe3), 0.74 (t, J = 7.5 Hz, 3H, 7’H), 0.23 (s, 3H, Si(Me)2CMe3),

13 0.14 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.5 (C=O), 154.3 (ArC-1 and ArC-

5), 154.2 (2’-H), 151.1 (tertiary aromatic), 139.3 (ArC-2 and ArC-4), 131.2 (3’-H), 108.8

(tertiary aromatic), 48.1, 45.8, 41.0, 39.9, 35.3, 31.7, 31.6, 30.6, 28.1, 27.9, 27.0, 26.2, 25.8,

22.6, 18.7, 18.5, 14.1. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.0% and retention time 6.9 min for the title compound.

2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanal (43). To a stirred solution of 41

(1.14 g, 2.35 mmol) in Acetone:H2O (10:1) (28 ml) under an argon atmosphere, NMO (504 mg,

4.70 mmol), 2,6-lutidine (413 mg, 3.52 mmol) and OsO4 (75 mg, 0.28 mmol) was added at room temperature. The reaction was complete in 4 h to form 1,2-diol 42 (Rf = 0.05). This diol without isolation was treated with (diacetoxyiodo)benzene (1.14 g, 3.52 mmol) at room temperature for

30 min. The reaction was quenched using Na2S2O3 and extraction was done using Et2O. The organic layer was washed with saturated CuSO4 and then concentrated. Purification by flash column chromatography (15% ethyl acetate in hexane) afforded 700 mg (80% yield) of 43 as a colorless oil. IR (neat): 2932, 1712, 1609, 1565, 1413, 1224, 1057 cm-1. 1H NMR (500

MHz,CDCl3) δ 9.43 (s, 1H, CHO), 6.42 (d, J = 2.0 Hz, 1H, Ar-H), 6.23 (d, J = 2.0 Hz, 1H, Ar-

115

H), 3.75 (ddd, J = 13.5 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.72 (td, J = 11.5 Hz, J = 4.0

Hz, 1H, 10a-H), 2.60-2.52 (m, 1H, 8eq-H), 2.46-2.36 (m, 1H, 8ax-H), 2.19-2.07 (m, 2H, 10ax-H,

7eq-H), 1.98-1.89 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, 6a-H), 1.55-1.48 (m, 1H, 7ax-H), 1.47 (s, 3H,

6-Me), 1.38 (d, J = 2.0 Hz, 6 H, -C(CH3)2-), 1.10 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.24

13 (s, 3H, Si(Me)2CMe3), 0.16 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 209.7 (C=O),

201.6 (CHO), 154.9 (ArC-1 or ArC-5), 154.8 (ArC5 or ArC1), 141.0 (tertiary aromatic), 113.8

(ArC-2 or ArC-4), 110.2 (ArC-4 or ArC-2), 108.9 (tertiary aromatic), 49.8, 47.6, 45.2, 40.6,

34.9, 27.5, 26.6, 25.8, 22.0, 22.0, 18.5, 18.2. Mass spectrum (ESI) m/z (relative intensity) 431

+ + (M + H, 100). Exact mass (ESI) calculated for C25H39O4Si (M + H), 431.2618; found

431.2611. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.2% and retention time 5.2 min for the title compound.

2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoic acid (44). To a stirred solution of

13 (700 mg, 1.63 mmol) in t-BuOH (33 ml) and 2-methyl-2-propene (52 ml) under an argon atmosphere was added a solution of NaClO2 (2.4 g, 21.16) and NaH2PO4 (2.14 g, 17.93 mmol) in

33 ml of H2O. The reaction mixture was stirred at 0°C for 45 min and then gradually brought to room temperature till the reaction was complete (2 h). The reaction mixture was quenched using

Na2SO3 and extracted using Et2O. The organic layer was quickly passed through a pad of silica equilibrated using 20% ethyl acetate in hexane. The organic layer was then concentrated under vacuum and then purified by flash column chromatography (20% ethyl acetate in hexane) to afford 550 mg (70% yield) of 44 as a white crystalline solid. mp = 40-41oC. IR (neat): 2930,

-1 1 1699, 1567, 1414, 1254, 1095, 1057 cm . H NMR (500 MHz,CDCl3) δ 6.50 (d, J = 2.0 Hz, 1H,

Ar-H), 6.41 (d, J = 2.0 Hz, 1H, Ar-H), 3.76 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-

116

H), 2.72 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.60-2.39 (m, 1H, 8eq-H), 2.46-2.36 (m, 1H,

8ax-H), 2.18-2.06 (m, 2H, 10ax-H, 7eq-H), 1.97-1.88 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, 6a-H),

1.54-1.47 (m, 7H, 7ax-H, -C(CH3)2-, especially,1.52, d, J = 2.0 Hz, -C(CH3)2-), 1.46 (s, 3H, 6-

Me), 1.09 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.23 (s, 3H, Si(Me)2CMe3), 0.16 (s, 3H,

13 Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.3 (C=O), 182.1 (-C(O)O), 154.8 (ArC-1 or

ArC-5), 154.7 (ArC5 or ArC1), 144.1 (tertiary aromatic), 113.8 (ArC-2 or ArC-4), 109.8 (ArC-4 or ArC-2), 108.4 (tertiary aromatic), 47.9, 46.0, 45.6, 40.9, 35.2, 27.9, 27.0, 26.3, 26.2, 26.0,

18.9, 18.5. Mass spectrum (ESI) m/z (relative intensity) 447 (M+ + H, 100). Exact mass (ESI)

+ calculated for C25H39O4Si (M + H), 447.2567; found 447.2556. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 99.5% and retention time 5.2 min for the title compound.

2-[(6aR,10aR)-6a,7,8,9,10,10a-Hexahydro-1-hydroxy-6,6-dimethyl-9-oxo-6H-benzo[c] chromen-3-yl]-2-methylpropanoic acid (45). To a stirred solution of 44 (25 mg, 0.05 mmol) in dry THF at -50⁰C under an argon atmosphere, tetra-butyl ammonium fluoride (0.1 ml, 0.1 mmol) was added. Stirring was continued for 30 min and then the reaction mixture was quenched with saturated solution of ammonium chloride and diluted with diethyl ether. The organic layer was separated and the aqueous layer was extracted with diethyl ether. The crude product was chromatographed on silica gel (20% ethyl acetate in hexane) to give 45 (232 mg, 90% yield) as a white solid. mp 153-154°C; IR (neat): 3340, 2978, 1695, 1620, 1417 cm-1; 1H NMR (500 MHz,

CDCl3 + CD3OD) δ 6.39 (d, J = 1.5 Hz, 1H, ArH), 6.34 (d, J = 1.5, 1H, ArH), 4.35 (br s, 1H,

OH), 3.94 (ddd, J = 15.0, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.86 (m as td, J = 12.5, J = 3.5

Hz, 1H, 10a-H), 2.62 - 2.58 (m, 1H, 8eq-H), 2.48 - 2.41 (m, 1H, 8ax-H), 2.19 - 2.04 (m, 2H,

10ax-H, 7eq-H), 1.94 (m as td, J = 12.2, J = 3.0 Hz, 1H, 6a-H), 1.57-1.48 (m, s and s, overlapping, 7H, 7ax-H, -C(CH3)2-, especially 1.50, s, and 1.49, s, -C(CH3)2-), 1.47 (s, 3H, 6-

117

13 CH3), 1.11 (s, 3H, 6-CH3). C NMR (125 MHz CDCl3) δ 213.2 (>C=O), 180.1 (-C(O)O-), 155.1

(ArC-1 or ArC-5), 154.7 (ArC-5 or ArC-1), 144.3 (tertiary aromatic), 109.7 (tertiary aromatic),

107.1 (ArC-2 or ArC-4), 105.6 (ArC-4 or ArC-2), 65.9, 47.2, 45.7, 44.8, 40.7, 34.6, 30.3, 27.8,

26.8, 25.7, 18.9. Mass spectum (ESI) m/z (relative intensity) 333 (M++H, 100). Exact mass (ESI)

+ calculated for C19H2505 (M +H), 333.1702; found 333.1700. HPLC (4.6 x 250 mm, Supelco discovery column, acetonitrile/water) showed purity 97.5% and retention time 6.5 min for the title compound.

Ethyl 2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (46a) To a solution of 44 (50 mg,

0.11 mmol) in dry CH2Cl2 (1ml) at 0⁰C under an argon atmosphere, DMAP (33 mg, 0.27 mmol) and EDCI (39 mg, 0.2 mmol) was added. The reaction was stirred for 20 min at 0⁰C and then ethanol (8.5 mg, 0.11 mmol) was added. The mixture was warmed to room temperature and stirred for 24 h to ensure complete formation of the product. The reaction mixture was diluted with diethyl ether and washed sequentially with 5% HCl, saturated aqueous NaHCO3, and brine.

The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure.

The crude product was chromatographed on silica gel (20% ethyl acetate in hexane) to give 46a

(35 mg, 75% yield) as a colorless oil. IR (neat): 2931, 1723, 1612, 1567, 1415, 1254, 1140, 1095

-1 1 cm . H NMR (500 MHz,CDCl3) δ 6.46 (d, J = 1.5 Hz, 1H, Ar-H), 6.34 (br d, J = 2.0 Hz, 1H,

Ar-H), 4.16-4.06 (m 2H, 4'-H), 3.76 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71

(m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.60-2.52 (m, 1H, 8eq-H), 2.45-2.36 (m, 1H, 8ax-

H), 2.18-2.06 (m, 2H, 10ax-H, 7eq-H), 1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.52-

1.48 (m, 7H, 7ax-H, -C(CH3)2-, especially, 1.50, s, 3H, -C(CH3)2- and 1.48, s, 3H, -C(CH3)2-),

1.46 (s, 3H, 6-Me), 1.19 (t, J = 7.0 Hz, 3H, 5'-H), 1.09 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3),

118

13 0.23 (s, 3H, Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.6

(>C=O), 186.9 (-C(O)O-), 155.0 (ArC-1 or ArC-5), 154.9 (ArC-5 or ArC-1), 145.4 (tertiary aromatic), 113.8 (tertiary aromatic), 110.2 (ArC-2 or ArC-4), 108.5 (ArC-4 or ArC-2), 61.3,

48.2, 46.6, 45.9, 41.3, 35.6, 28.2, 27.3, 26.9, 26.7, 26.5, 19.2, 18.8, 14.6. LC/MS analysis

(Waters MicroMass ZQ system) showed purity 99.3% and retention time 5.9 min for the title compound.

Propyl 2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (46b) The synthesis was carried out as described for 46a using 44 (40 mg, 0.10 mmol), DMAP (73 mg, 0.60 mmol), EDCI (76 mg, 0.40 mmol), propanol (25 mg, 0.40 mmol) and dry CH2Cl2 (1 ml). Purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) gave title compound 46b (35 mg, 80% yield) as a colorless oil. IR (neat): 2932, 1723, 1612, 1567, 1415, 1254, 1140, 1095 cm-

1 1 . H NMR (500 MHz,CDCl3) δ 6.47 (d, J = 2.0 Hz, 1H, Ar-H), 6.35 (d, J = 1.5 Hz, 1H, Ar-H),

4.06-3.94 (m, 2H, 4'-H), 3.75 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.60-2.52 (m, 1H, 8eq-H), 2.45-2.36 (m, 1H, 8ax-H),

2.18-2.04 (m, 2H, 10ax-H, 7eq-H), 1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.62-1.57

(m, 3H, 7ax-H, 5’-H), 1.50 (s, 3H, -C(CH3)2-), 1.49 (s, 3H, -C(CH3)2-), 1.46 (s, 3H, 6-Me), 1.08

(s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.83 (t, J = 7.0 Hz, 3H, 6'-H), 0.23 (s, 3H,

13 Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.3 (>C=O), 176.6

(-C(O)O-), 154.7 (ArC-1 or ArC-5), 154.6 (ArC-5 or ArC-1), 145.1 (tertiary aromatic), 113.5

(tertiary aromatic), 109.8 (ArC-2 or ArC-4), 108.3 (ArC-4 or ArC-2), 66.5, 47.9, 46.3, 45.6,

41.0, 35.2, 27.9, 27.0, 26.4, 26.2, 22.1, 18.8, 18.5, 10.5. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.2% and retention time 6.0 min for the title compound.

119

Pentyl 2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (46c) The reaction was carried out as described for 46a using 44 (70 mg, 0.18 mmol), DMAP (132 mg, 1.07 mmol), EDCI (138 mg, 0.72 mmol), pentanol (70 mg, 0.40 mmol) and dry CH2Cl2 (1.4 ml). Purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) gave title compound 46c (48 mg, 60% yield) as a colorless oil. IR (neat): 2931, 1724, 1612, 1567, 1415, 1254, 1140, 1095 cm-

1 1 . H NMR (500 MHz,CDCl3) δ 6.46 (d, J = 1.5 Hz, 1H, Ar-H), 6.35 (d, J = 2.0 Hz, 1H, Ar-H),

4.09-3.97 (m, 2H, 4'-H), 3.76 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.60-2.52 (m, 1H, 8eq-H), 2.45-2.36 (m, 1H, 8ax-H),

2.18-2.04 (m, 2H, 10ax-H, 7eq-H), 1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.60-1.52

(m, 3H, 7ax-H, 5’-H), 1.50 (s, 3H, -C(CH3)2-), 1.49 (s, 3H, -C(CH3)2-), 1.46 (s, 3H, 6-Me), 1.31-

1.20 (m, 4H, 6’-H, 7’-H), 1.09 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.85 (t, J = 7.5 Hz, 3H,

13 8'-H), 0.23 (s, 3H, Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ

210.0 (>C=O), 176.3 (-C(O)O-), 154.3 (ArC-1 or ArC-5), 153.2 (ArC-5 or ArC-1), 144.7

(tertiary aromatic), 113.1 (tertiary aromatic), 109.5 (ArC-2 or ArC-4), 107.9 (ArC-4 or ArC-2),

64.8, 58.4, 47.5, 46.1, 45.5, 40.8, 34.8, 28.8, 27.9, 27.2, 26.3, 26.9, 25.7, 22.1, 18.7, 18.2, 13.5.

LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.3% and retention time 6.3 min for the title compound.

Hexyl 2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (46d) The synthesis was carried out as described for 46a using 44 (70 mg, 0.18 mmol), DMAP (132 mg, 1.07 mmol), EDCI (138 mg, 0.72 mmol), pentanol (81 mg, 0.40 mmol) and dry CH2Cl2 (1.4 ml). Purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) gave title compound 46d (44

120 mg, 58% yield) as a colorless oil. IR (neat): 2929, 1725, 1612, 1567, 1415, 1254, 1141, 1057 cm-

1 1 . H NMR (500 MHz,CDCl3) δ 6.46 (d, J = 2.0 Hz, 1H, Ar-H), 6.34 (d, J = 2.0 Hz, 1H, Ar-H),

4.09-3.98 (m, 2H, 4'-H), 3.76 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.60-2.52 (m, 1H, 8eq-H), 2.46-2.36 (m, 1H, 8ax-H),

2.18-2.04 (m, 2H, 10ax-H, 7eq-H), 1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.57-1.51

(m, 3H, 7ax-H, 5’-H), 1.50 (s, 3H, -C(CH3)2-), 1.49 (s, 3H, -C(CH3)2-), 1.46 (s, 3H, 6-Me), 1.30-

1.20 (m, 6H, 6’-H, 7’-H, 8’-H), 1.09 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.87 (t, J = 7.0

13 Hz, 3H, 9'-H), 0.23 (s, 3H, Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz

CDCl3) δ 210.0 (>C=O), 176.3 (-C(O)O-), 166.3 (ArC-1 or ArC-5), 154.3 (ArC-5 or ArC-1),

144.7 (tertiary aromatic), 113.1 (tertiary aromatic), 109.5 (ArC-2 or ArC-4), 107.9 (ArC-4 or

ArC-2), 64.8, 58.5, 47.5, 46.0, 45.5, 40.9, 34.5, 31.3, 29.0, 28.4, 27.3, 26.3, 26.0, 25.7, 25.7,

22.7, 18.8, 18.9, 13.7. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.1% and retention time 6.5 min for the title compound.

(S)-pentan-2-yl 2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (46e) The synthesis was carried out as described for 46a using 44 (50 mg, 0.11 mmol), DMAP (88 mg,

0.72 mmol), EDCI (92 mg, 0.48 mmol), (S)-2-pentanol (43 mg, 0.48 mmol) and dry CH2Cl2 (1 ml). Purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) gave title compound 46e (40 mg, 70% yield) as a colorless oil. IR (neat): 2932, 1718, 1612,

-1 1 1567, 1415, 1254, 1150, 1056 cm . H NMR (500 MHz,CDCl3) δ 6.48 (d, J = 1.5 Hz, 1H, Ar-

H), 6.37 (d, J = 2.0 Hz, 1H, Ar-H), 4.88-4.79 (m, 1H, 4'-H), 3.75 (ddd, J = 15.0 Hz, J = 3.5 Hz, J

= 2.0 Hz, 1H, 10eq-H), 2.71 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.60-2.52 (m, 1H,

8eq-H), 2.46-2.36 (m, 1H, 8ax-H), 2.18-2.04 (m, 2H, 10ax-H, 7eq-H), 1.93 (m as td, J = 12.0 Hz,

121

J = 3.0 Hz, 1H, 6a-H), 1.54-1.44 (m, 12H, 7ax-H, 5’-H, -C(CH3)2-, 6-Me, especially, 1.49, s, 3H,

-C(CH3)2- and 1.48, s, 3H, -C(CH3)2- and 1.46, s, 3H, 6-Me), 1.22-1.160 (m, 2H, 6’-H), 1.10 (s,

3H, -O-CH-CH3), 1.07 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.81 (t, J = 7.0 Hz, 3H, 6'-H),

13 0.23 (s, 3H, Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.3

(>C=O), 176.1 (-C(O)O-), 154.7 (ArC-1 or ArC-5), 154.6 (ArC-5 or ArC-1), 145.1 (tertiary aromatic), 113.4 (tertiary aromatic), 109.9 (ArC-2 or ArC-4), 108.5 (ArC-4 or ArC-2), 66.0,

48.0, 46.2, 45.7, 41.0, 38.1, 35.3, 27.9, 27.0, 26.4, 26.2, 26.0, 19.9, 18.8, 18.7, 18.5, 15.5, 14.1.

LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.3% and retention time 6.3 min for the title compound.

(R)-pentan-2-yl 2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (46f) The synthesis was carried out as described for 46a using 44 (50 mg, 0.11 mmol), DMAP (88 mg,

0.72 mmol), EDCI (92 mg, 0.48 mmol), (R)-2-pentanol (43 mg, 0.48 mmol) and dry CH2Cl2 (1 ml). Purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) gave title compound 46f (40 mg, 70% yield) as a colorless oil. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 99.0% and retention time 6.3 min for the title compound.

3-morpholinopropyl 2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (46g) The synthesis was carried out as described for 46a using 44 (100 mg, 0.11 mmol), DMAP (164 mg,

1.35 mmol), EDCI (168 mg, 0.88 mmol), 4-(3-Hydroxypropyl)morpholine (128 mg, 0.88 mmol) and dry CH2Cl2 (2 ml). Purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) gave title compound 46g (90 mg, 70% yield) as a colorless oil. IR (neat):

-1 1 2932, 1718, 1612, 1567, 1415, 1254, 1150, 1056 cm . H NMR (500 MHz,CDCl3) δ 6.45 (d, J =

122

1.5 Hz, 1H, Ar-H), 6.33 (d, J = 2.0 Hz, 1H, Ar-H), 4.20-4.06 (m, 1H, 4'-H), 3.74 (ddd, J = 15.0

Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.67 (t, J = 5.0 Hz, 4H, morpholine -CH2-O-CH2-),

2.71 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.61-2.52 (m, 1H, 8eq-H), 2.46-2.32 (m, 5H,

8ax-H, morpholine -CH2-N-CH2), 2.28 (t, J = 7.5 Hz, 2H, 6’-H), 2.16-2.02 (m, 2H, 10ax-H, 7eq-

H), 1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.75 (p, J = 7.5 Hz, 2H, 5’-H) 1.54-1.44

(m, 10H, 7ax-H, -C(CH3)2-, 6-Me, especially, 1.50, s, 3H, -C(CH3)2- and 1.49, s, 3H, -C(CH3)2- and 1.46, s, 3H, 6-Me), 1.09 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.23 (s, 3H,

13 Si(Me)2CMe3), 0.16 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 212.2 (>C=O), 175.7

(-C(O)O-), 155.4 (ArC-1 or ArC-5), 154.6 (ArC-5 or ArC-1), 145.6 (tertiary aromatic), 109.6

(tertiary aromatic), 106.4 (ArC-2 or ArC-4), 105.6 (ArC-4 or ArC-2), 66.4, 62.6, 55.1, 53.3,

47.1, 45.9, 44.9, 40.6, 34.5, 27.6, 26.5, 26.0, 25.9, 25.4, 18.8. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 98.3% and retention time 6.7 min for the title compound.

S-butyl 2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanethioate (46h) The synthesis was carried out as described for 46a using 44 (80 mg, 0.18 mmol), DMAP (132 mg, 1.07 mmol),

EDCI (138 mg, 0.72 mmol), butanethiol (72 mg, 0.40 mmol) and dry CH2Cl2 (1.6 ml).

Purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) gave title compound 46h (75 mg, 80% yield) as a colorless oil. IR (neat): 2931, 1713, 1611, 1567,

-1 1 1413, 1254, 1183, 1057 cm . H NMR (500 MHz,CDCl3) δ 6.49 (d, J = 1.5 Hz, 1H, Ar-H), 6.34

(d, J = 2.0 Hz, 1H, Ar-H), 3.76 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.79 (t, J

= 7.5 Hz, 2H, 4’-H), 2.72 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.60-2.52 (m, 1H, 8eq-

H), 2.46-2.36 (m, 1H, 8ax-H), 2.18-2.04 (m, 2H, 10ax-H, 7eq-H), 1.94 (m as td, J = 12.0 Hz, J =

123

3.0 Hz, 1H, 6a-H), 1.54-1.45 (m, 12H, 7ax-H, 5’-H, -C(CH3)2-, 6-Me, especially, 1.53, s, 3H, -

C(CH3)2- and 1.52, s, 3H, -C(CH3)2- and 1.46, s, 3H, 6-Me), 1.33 (sextet, J = 7.5 Hz, 2H, 6’-H),

1.10 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.88 (t, J = 7.0 Hz, 3H, 7'-H), 0.23 (s, 3H,

13 Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.6 (>C=O), 205.6

(-C(O)O-), 155.0 (ArC-1 or ArC-5), 154.9 (ArC-5 or ArC-1), 144.5 (tertiary aromatic), 114.2

(tertiary aromatic), 111.1 (ArC-2 or ArC-4), 109.4 (ArC-4 or ArC-2), 53.6, 48.2, 45.9, 41.3,

35.6, 32.0, 29.3, 28.2, 27.3, 27.0, 26.8, 26.5, 22.5, 19.2, 18.8, 14.1. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 98.7% and retention time 6.4 min for the title compound.

N-butyl-2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanamide (46i) To a stirred solution of 44

(54 mg, 0.12 mmol) in dry THF (1 ml), CDI (77 mg, 0.48 mmol) was added. The reaction was stirred at room temperature for 2 h followed by addition of butylamine (44 mg, 0.60 mmol) and the reaction continued for additional 4 h. Upon completion, the reaction mixture was diluted using ethyl acetate and quenched using 5% HCl. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The crude product was chromatographed on silica gel (30% ethyl acetate in hexane) to give 46i (45 mg, 75% yield) as a colorless oil. IR

-1 1 (neat): 3387, 2930, 1710, 1657, 1566, 1414, 1254, 1182, 1095 cm . H NMR (500 MHz,CDCl3)

δ 6.50 (d, J = 2.0 Hz, 1H, Ar-H), 6.34 (d, J = 1.5 Hz, 1H, Ar-H), 3.76 (ddd, J = 15.0 Hz, J = 3.5

Hz, J = 2.0 Hz, 1H, 10eq-H), 3.19 (sextet, J = 7.0 Hz, 1H, 4’-H), 3.10 (sextet, J = 7.0 Hz, 1H, 4’-

H), 2.73 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.61-2.54 (m, 1H, 8eq-H), 2.47-2.37 (m,

1H, 8ax-H), 2.20-2.05 (m, 2H, 10ax-H, 7eq-H), 1.94 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-

H), 1.54-1.50 (m, 1H, 7ax-H), 1.49 (s, 6H, -C(CH3)2-), 1.48 (s, 3H, 6-Me), 1.40-1.31 (m, 2H, 5’-

H), 1.21 (sextet, J = 7.5 Hz, 2H, 6’-H), 1.11 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.85 (t, J

124

13 = 7.5 Hz, 3H, 7'-H), 0.23 (s, 3H, Si(Me)2CMe3), 0.16 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz

CDCl3) δ 210.1 (>C=O), 177.1 (-C(O)O-), 155.0 (ArC-1 or ArC-5), 154.8 (ArC-5 or ArC-1),

145.5 (tertiary aromatic), 113.9 (tertiary aromatic), 110.6 (ArC-2 or ArC-4), 108.9 (ArC-4 or

ArC-2), 58.6, 47.9, 46.7, 45.6, 40.9, 39.6, 35.2, 31.7, 27.9, 26.9, 26.1, 20.1, 18.8, 18.6, 18.5,

13.9. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.2% and retention time

5.6 min for the title compound.

Ethyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanoate (47a) The synthesis was carried out as described for 45 using 46a (40 mg, 0.08 mmol), tetrabutylammonium fluoride (0.13 ml, 0.13 mmol) and dry THF (2 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 47a (25 mg, 84% yield) as a white solid. mp = 37-38oC. IR (neat):

-1 1 3278, 2930, 1752, 1653, 1566, 1418, 1252, 1188, 1075 cm . H NMR (500 MHz,CDCl3) δ 6.41

(d, J = 2.0 Hz, 1H, Ar-H), 6.29 (d, J = 2.0 Hz, 1H, Ar-H), 4.06 (q, J = 7.5 Hz, 2H, 4'-H), 3.94

(ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.87 (m as td, J = 12.5 Hz, J = 4.0 Hz,

1H, 10a-H), 2.64-2.57 (m, 1H, 8eq-H), 2.49-2.39 (m, 1H, 8ax-H), 2.20-2.09 (m, 2H, 10ax-H,

7eq-H), 1.95 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.53-1.46 (m, 10H, 7ax-H, -C(CH3)2-,

6-Me, especially, 1.49, s, -C(CH3)2- and 1.47, s, 6-Me), 1.20 (t, J = 7.0 Hz, 3H, 5'-H), 1.12 (s,

13 3H, 6-Me). C NMR (100 MHz CDCl3) δ 214.0 (>C=O), 177.3 (-C(O)O-), 155.8 (ArC-1 or

ArC-5), 155.1 (ArC-5 or ArC-1), 145.7 (tertiary aromatic), 109.7 (tertiary aromatic), 107.3 (ArC-

2 or ArC-4), 105.8 (ArC-4 or ArC-2), 61.4, 47.8, 46.6, 45.4, 41.2, 35.2, 28.3, 27.3, 26.8, 26.7,

19.4, 14.5. Mass spectrum (ESI) m/z (relative intensity) 361 (M+ + H, 100). Exact mass (ESI)

+ calculated for C21H29O5 (M + H), 361.2015; found 361.2005. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 98.8% and retention time 4.6 min for the title compound.

125

Propyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanoate (47b) The synthesis was carried out as described for 45 using 46b (25 mg, 0.06 mmol), tetrabutylammonium fluoride (0.10 ml, 0.10 mmol) and dry THF (1.5 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 47b (15 mg, 82% yield) as a white solid. mp = 36-37oC. IR

-1 1 (neat): 3293, 2973, 1722, 1694, 1577, 1417, 1257, 1183, 1093 cm . H NMR (500 MHz,CDCl3)

δ 6.69 (s, 1H, OH), 6.41 (d, J = 1.5 Hz, 1H, Ar-H), 6.33 (d, J = 2.0 Hz, 1H, Ar-H), 4.05-3.97 (m,

3H, 10eq-H, 4'-H), 2.87 (m as td, J = 12.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.65-2.58 (m, 1H, 8eq-H),

2.50-2.40 (m, 1H, 8ax-H), 2.20-2.09 (m, 2H, 10ax-H, 7eq-H), 1.95 (m as td, J = 12.0 Hz, J = 3.0

Hz, 1H, 6a-H), 1.70-1.56 (m, 3H, 7ax-H, 5’-H), 1.50 (s, 6H, -C(CH3)2-), 1.47 (s, 3H, 6-Me), 1.16

13 (s, 3H, 6-Me), 0.84 (t, J = 7.0 Hz, 3H, 6'-H). C NMR (100 MHz CDCl3) δ 212.9 (>C=O), 176.7

(-C(O)O-), 155.1 (ArC-1 or ArC-5), 154.5 (ArC-5 or ArC-1), 145.1 (tertiary aromatic), 109.1

(tertiary aromatic), 106.9 (ArC-2 or ArC-4), 105.2 (ArC-4 or ArC-2), 66.3, 47.2, 46.1, 44.9,

40.6, 34.5, 27.7, 26.7, 26.2, 26.0, 21.7, 18.7, 10.1. Mass spectrum (ESI) m/z (relative intensity)

+ + 375 (M + H, 100). Exact mass (ESI) calculated for C22H31O5 (M + H), 375.2171; found

375.2162. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.0% and retention time 4.8 min for the title compound.

Pentyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanoate (47c) The synthesis was carried out as described for 45 using 46c (30 mg, 0.06 mmol), tetrabutylammonium fluoride (0.10 ml, 0.10 mmol) and dry THF (1.5 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 47c (15 mg, 90% yield) as a white solid. mp = 34-35oC. IR

-1 1 (neat): 3266, 2956, 1719, 1693, 1578, 1417, 1257, 1183, 1093 cm . H NMR (500 MHz,CDCl3)

126

δ 6.99 (s, 1H, OH), 6.40 (d, J = 1.5 Hz, 1H, Ar-H), 6.34 (d, J = 1.5 Hz, 1H, Ar-H), 4.09-3.99 (m,

3H, 10eq-H, 4'-H), 2.88 (m as td, J = 12.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.65-2.57 (m, 1H, 8eq-H),

2.51-2.41 (m, 1H, 8ax-H), 2.20-2.09 (m, 2H, 10ax-H, 7eq-H), 1.95 (m as td, J = 12.0 Hz, J = 3.0

Hz, 1H, 6a-H), 1.61-1.51 (m, 3H, 7ax-H, 5’-H), 1.50 (s, 6H, -C(CH3)2-), 1.47 (s, 3H, 6-Me),

1.30-1.19 (m, 4H, 6’-H, 7’-H), 1.12 (s, 3H, 6-Me), 0.85 (t, J = 7.0 Hz, 3H, 8'-H). 13C NMR (100

MHz CDCl3) δ 213.4 (>C=O), 177.3 (-C(O)O-), 155.6 (ArC-1 or ArC-5), 155.1 (ArC-5 or ArC-

1), 145.7 (tertiary aromatic), 109.8 (tertiary aromatic), 107.6 (ArC-2 or ArC-4), 105.9 (ArC-4 or

ArC-2), 65.5, 47.8, 46.6, 45.5, 41.3, 35.2, 28.6, 28.5, 28.3, 27.3, 26.8, 26.7, 22.7, 19.4, 14.5.

Mass spectrum (ESI) m/z (relative intensity) 403 (M+ + H, 100). Exact mass (ESI) calculated for

+ C24H35O5 (M + H), 403.2484; found 402.2476. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.5% and retention time 5.1 min for the title compound.

Hexyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanoate (47d) The synthesis was carried out as described for 45 using 46d (32 mg, 0.06 mmol), tetrabutylammonium fluoride (0.10 ml, 0.10 mmol) and dry THF (1.5 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 47d (20 mg, 90% yield) as a white solid. mp = 36-37oC. IR

-1 1 (neat): 3282, 2926, 1724, 1694, 1578, 1417, 1255, 1183, 1093 cm . H NMR (500 MHz,CDCl3)

δ 6.39 (d, J = 1.5 Hz, 1H, Ar-H), 6.35 (d, J = 2.0 Hz, 1H, Ar-H), 4.08-4.00 (m, 3H, 10eq-H, 4'-

H), 2.88 (m as td, J = 12.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.67-2.58 (m, 1H, 8eq-H), 2.51-2.40 (m,

1H, 8ax-H), 2.22-2.09 (m, 2H, 10ax-H, 7eq-H), 1.95 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-

H), 1.60-1.52 (m, 3H, 7ax-H, 5’-H), 1.50 (s, 6H, -C(CH3)2-), 1.47 (s, 3H, 6-Me), 1.32-1.18 (m,

6H, 6’-H, 7’-H, 8’-H), 1.11 (s, 3H, 6-Me), 0.85 (t, J = 7.5 Hz, 3H, 9'-H). 13C NMR (100 MHz

CDCl3) δ 212.6 (>C=O), 176.9 (-C(O)O-), 155.2 (ArC-1 or ArC-5), 154.9 (ArC-5 or ArC-1),

127

145.4 (tertiary aromatic), 109.5 (tertiary aromatic), 107.4 (ArC-2 or ArC-4), 105.6 (ArC-4 or

ArC-2), 65.2, 47.5, 46.3, 45.3, 41.0, 34.8, 31.5, 29.9, 28.6, 28.0, 27.0, 26.4, 25.6, 22.7, 19.1,

14.2. Mass spectrum (ESI) m/z (relative intensity) 417 (M+ + H, 100). Exact mass (ESI)

+ calculated for C25H37O5 (M + H), 417.2641; found 417.2634. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 99.1% and retention time 5.2 min for the title compound.

(S)-pentan-2-yl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-

6H-benzo[c]chromen-3-yl)-2-methylpropanoate (47e) The synthesis was carried out as described for 45 using 46e (30 mg, 0.06 mmol), tetrabutylammonium fluoride (0.10 ml, 0.10 mmol) and dry THF (1.4 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 47e (20 mg, 86% yield) as a white solid. mp = 39-

40oC. IR (neat): 3279, 2973, 1716, 1694, 1578, 1417, 1257, 1184, 1043 cm-1. 1H NMR (500

MHz,CDCl3) δ 7.24 (s, 1H, OH), 6.41 (d, J = 2.0 Hz, 1H, Ar-H), 6.35 (d, J = 2.0 Hz, 1H, Ar-H),

4.91-4.83 (m, 3H, 4'-H), 4.04 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.88 (m as td, J = 12.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.65-2.57 (m, 1H, 8eq-H), 2.48-2.40 (m, 1H, 8ax-H),

2.20-2.11 (m, 2H, 10ax-H, 7eq-H), 1.95 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.55-1.45

(m, 9H, 7ax-H, 5’-H, 6-Me, -C(CH3)2-, especially 1.49, s, -C(CH3)2- and 1.46, s, 6-Me), 1.42-

1.34 (m, 1H, 6’-H), 1.26-1.09 (m, 7H, 6’-H, -O-CH-CH3, especially 1.13, s, -O-CH-CH3, and

13 1.11, s, 6-Me), 0.81 (t, J = 7.5 Hz, 3H, 7'-H). C NMR (100 MHz CDCl3) δ 213.1 (>C=O),

176.3 (-C(O)O-), 155.1 (ArC-1 or ArC-5), 154.4 (ArC-5 or ArC-1), 145.2 (tertiary aromatic),

109.0 (tertiary aromatic), 106.8 (ArC-2 or ArC-4), 105.2 (ArC-4 or ArC-2), 71.0, 47.2, 46.0,

44.9, 40.6, 37.7, 34.6, 27.7, 26.7, 26.2, 25.8, 19.6, 18.7, 18.2, 13.7. Mass spectrum (ESI) m/z

+ + (relative intensity) 403 (M + H, 100). Exact mass (ESI) calculated for C24H35O5 (M + H),

128

403.2484; found 403.2470. LC/MS analysis (Waters MicroMass ZQ system) showed purity

99.5% and retention time 5.0 min for the title compound.

(R)-pentan-2-yl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-

6H-benzo[c]chromen-3-yl)-2-methylpropanoate (47f) The synthesis was carried out as described for 45 using 46f (30 mg, 0.06 mmol), tetrabutylammonium fluoride (0.10 ml, 0.10 mmol) and dry THF (1.4 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 47f (20 mg, 86% yield) as a white solid. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.0% and retention time 5.0 min for the title compound.

3-morpholinopropyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (47g) The synthesis was carried out as described for 45 using 46g (45 mg, 0.08 mmol), tetrabutylammonium fluoride (0.11 ml,

0.11 mmol) and dry THF (1.5 ml). Purification by flash column chromatography on silica gel

(30% ethyl acetate in hexane) gave title compound 47g (32 mg, 90% yield) as a white solid. mp

= 32-33oC. IR (neat): 3647, 2925, 1723, 1619, 1508, 1459, 1256, 1183, 1043 cm-1. 1H NMR (500

MHz,CDCl3) δ 6.40 (d, J = 1.5 Hz, 1H, Ar-H), 6.24 (d, J = 2.0 Hz, 1H, Ar-H), 4.17 (t, J = 6.0

Hz, 2H, 4'-H), 3.90 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.71 (t, J = 5.0 Hz,

4H, morpholine -CH2-O-CH2-), 2.86 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.62-2.56 (m,

1H, 8eq-H), 2.49-2.34 (m, 7H, 8ax-H, 6’-H, morpholine -CH2-N-CH2), 2.18-2.08 (m, 2H, 10ax-

H, 7eq-H), 1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.78 (p, J = 7.5 Hz, 2H, 5’-H)

1.55-1.45 (m, 10H, 7ax-H, -C(CH3)2-, 6-Me, especially, 1.50, s, 3H, -C(CH3)2- and 1.49, s, 3H, -

13 C(CH3)2- and 1.47, s, 3H, 6-Me), 1.12 (s, 3H, 6-Me). C NMR (100 MHz CDCl3) δ 212.4

(>C=O), 176.5 (-C(O)O-), 155.5 (ArC-1 or ArC-5), 154.5 (ArC-5 or ArC-1), 144.8 (tertiary

129 aromatic), 109.3 (tertiary aromatic), 106.2 (ArC-2 or ArC-4), 105.2 (ArC-4 or ArC-2), 66.4,

62.6, 55.1, 53.3, 47.1, 45.9, 44.9, 40.6, 34.5, 27.6, 26.5, 26.0, 25.9, 25.4, 18.8. Mass spectrum

+ (ESI) m/z (relative intensity) 460 (M + H, 100). Exact mass (ESI) calculated for C26H38NO6

(M+ + H), 460.2699; found 460.2692. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.7% and retention time 3.9 min for the title compound.

S-butyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanethioate (47h) The synthesis was carried out as described for 45 using 46h (32 mg, 0.06 mmol), tetrabutylammonium fluoride (0.10 ml, 0.10 mmol) and dry THF (1.5 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 47h (22 mg, 92% yield) as a colorless oil. IR (neat):

-1 1 3379, 2928, 1707, 1620, 1510, 1417, 1270, 1182, 1013 cm . H NMR (500 MHz,CDCl3) δ 6.44

(d, J = 1.5 Hz, 1H, Ar-H), 6.28 (d, J = 1.5 Hz, 1H, Ar-H), 5.82 (s, 1H, OH), 3.93 (ddd, J = 15.0

Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.88 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.80

(t, J = 7.5 Hz, 2H, 4’-H), 2.64-2.56 (m, 1H, 8eq-H), 2.50-2.39 (m, 1H, 8ax-H), 2.20-2.10 (m, 2H,

10ax-H, 7eq-H), 1.96 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.56 (s, 3H, -C(CH3)2-), 1.54

(s, 3H, -C(CH3)2-), 1.52-1.45 (m, 5H, 7ax-H, 5’-H, 6-Me, 1.47, s, 3H, 6-Me), 1.34 (sextet, J =

7.5 Hz, 2H, 6’-H), 1.12 (s, 3H, 6-Me), 0.88 (t, J = 7.0 Hz, 3H, 7'-H). 13C NMR (100 MHz

CDCl3) δ 213.8 (>C=O), 205.9 (-C(O)O-), 155.6 (ArC-1 or ArC-5), 155.1 (ArC-5 or ArC-1),

144.9 (tertiary aromatic), 110.1 (tertiary aromatic), 108.5 (ArC-2 or ArC-4), 106.7 (ArC-4 or

ArC-2), 53.6, 47.8, 45.5, 41.3, 35.2, 31.9, 29.4, 28.3, 27.3, 26.9, 22.5, 19.4, 14.1. Mass spectrum

+ + (ESI) m/z (relative intensity) 405 (M + H, 100). Exact mass (ESI) calculated for C23H33O4S (M

+ H), 405.2100; found 405.2089. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.2% and retention time 5.1 min for the title compound.

130

N-butyl-2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanamide (47i) The synthesis was carried out as described for 45 using 46i (32 mg, 0.07 mmol), tetrabutylammonium fluoride (0.10 ml, 0.10 mmol) and dry THF (1.5 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 47i (22 mg, 91% yield) as a colorless oil. IR (neat): 3146, 2965,

-1 1 1700, 1633, 1531, 1410, 1271, 1181, 1093 cm . H NMR (500 MHz,CDCl3) δ 6.43 (d, J = 2.0

Hz, 1H, Ar-H), 6.28 (d, J = 1.5 Hz, 1H, Ar-H), 3.97 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz,

1H, 10eq-H), 3.19-3.09 (m, 2H, 4’-H), 2.90 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.64-

2.56 (m, 1H, 8eq-H), 2.49-2.39 (m, 1H, 8ax-H), 2.16-2.07 (m, 2H, 10ax-H, 7eq-H), 1.96 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.58-1.44 (m, 10H, 7ax-H, -C(CH3)2-, 6-Me, especially

1.49, s, -C(CH3)2- and 1.48, s, -C(CH3)2- and 1.47, s, 6-Me), 1.40-1.31 (m, 2H, 5’-H), 1.20

(sextet, J = 7.5 Hz, 2H, 6’-H), 1.14 (s, 3H, 6-Me), 0.82 (t, J = 7.5 Hz, 3H, 7'-H). ). 13C NMR

(100 MHz CDCl3) δ 212.3 (>C=O), 178.7 (-C(O)O-), 157.2 (ArC-1 or ArC-5), 155.2 (ArC-5 or

ArC-1), 145.8 (tertiary aromatic), 110.4 (tertiary aromatic), 106.8 (ArC-2 or ArC-4), 106.5 (ArC-

4 or ArC-2), 47.91, 47.09, 45.61, 41.22, 40.17, 35.08, 31.82, 28.38, 27.24, 27.19, 27.10, 20.38,

19.41, 14.17. Mass spectrum (ESI) m/z (relative intensity) 388 (M+ + H, 100). Exact mass (ESI)

+ calculated for C23H34NO4 (M + H), 388.2428; found 388.2480. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 98.9% and retention time 4.5 min for the title compound.

Propyl (2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)propan-2-yl)carbamate (48a) To a solution of 44 (25 mg, 0.05 mmol) in dry THF (1ml), DPPA (16 mg, 0.05 mmol), DIPEA (8 mg, 0.05 mmol) and propanol (3 mg, 0.05 mmol) was added. The reaction was refluxed for 15 h. Upon complete formation of the product, the reaction mixture was quenched using saturated solution of NaHCO3

131 and extracted using ethyl acetate. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The crude product was chromatographed on silica gel (25% ethyl acetate in hexane) to give 48a (25 mg, 89% yield) as a colorless oil. IR (neat): 3319, 2969,

-1 1 1710, 1614, 1569, 1414, 1253, 1189, 1021 cm . H NMR (500 MHz,CDCl3) δ 6.49 (d, J = 2.0

Hz, 1H, Ar-H), 6.42 (br d, J = 2.0 Hz, 1H, Ar-H), 4.97 (br s, 1H, NH), 3.92 (t, J = 7.0 Hz, 2H,

5’-H), 3.76 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71 (m as td, J = 12.5 Hz, J

= 3.5 Hz, 1H, 10a-H), 2.60-2.52 (m, 1H, 8eq-H), 2.46-2.36 (m, 1H, 8ax-H), 2.18-2.06 (m, 2H,

10ax-H, 7eq-H), 1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.74 (sextet, J = 7.0 Hz, 2H,

6’-H), 1.60 (s, 3H, -C(CH3)2-), 1.59 (s, 3H, -C(CH3)2-), 1.52-1.47 (m, 1H, 7ax-H), 1.46 (s, 3H, 6-

Me), 1.09 (s, 3H, 6-Me), 1.00 (s, 9H, Si(Me)2CMe3), 0.94 (t, J = 7.5 Hz, 3H, 7'-H), 0.24 (s, 3H,

13 Si(Me)2CMe3), 0.15 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.3 (>C=O), 154.7

(-C(O)O-), 150.8 (ArC-1 or ArC-5), 129.9 (ArC-5 or ArC-1), 125.4 (tertiary aromatic), 120.2

(tertiary aromatic), 108.7 (ArC-2 or ArC-4), 107.6 (ArC-4 or ArC-2), 71.1, 55.0, 47.9, 45.6,

40.9, 35.2, 27.9, 27.0, 26.1, 23.8, 23.7, 18.9, 18.4, 10.5, 10.1. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 98.0% and retention time 5.6 min for the title compound.

1-(2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)propan-2-yl)-3-propylurea (48b) The reaction was performed as described for 48a using 44 ((25 mg, 0.05 mmol), dry THF (1ml), DPPA (16 mg,

0.05 mmol), DIPEA (8 mg, 0.05 mmol) and propylamine (3 mg, 0.05 mmol). Purification by flash column chromatography on silica gel (25% ethyl acetate in hexane) gave title compound

48b (25 mg, 90% yield) as a colorless oil. IR (neat): 3350, 2926, 1711, 1639, 1567, 1414, 1254,

-1 1 1183, 1095 cm . H NMR (500 MHz,CDCl3) δ 6.58 (d, J = 1.5 Hz, 1H, Ar-H), 6.53 (br d, J =

1.5 Hz, 1H, Ar-H), 4.67 (s, 1H, NH), 4.03 (t, J = 5.5 Hz, 1H, NH), 3.75 (ddd, J = 15.0 Hz, J =

132

3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.07 (sextet, J = 6.5 Hz, 1H, 5’-H), 2.94 (sextet, J = 6.5 Hz, 1H,

5’-H), 2.72 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.61-2.53 (m, 1H, 8eq-H), 2.47-2.36

(m, 1H, 8ax-H), 2.19-2.11 (m, 1H, 10ax-H), 2.04 (t, J = 14.5 Hz, 1H, 7eq-H), 1.93 (m as td, J =

12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.56 (s, 3H, -C(CH3)2-), 1.53 (s, 3H, -C(CH3)2-), 1.52-1.48 (m,

1H, 7ax-H), 1.47 (s, 3H, 6-Me), 1.28 (sextet, J = 7.5 Hz, 2H, 6’-H), 1.09 (s, 3H, 6-Me), 1.00 (s,

9H, Si(Me)2CMe3), 0.68 (t, J = 7.5 Hz, 3H, 7'-H), 0.24 (s, 3H, Si(Me)2CMe3), 0.16 (s, 3H,

13 Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.3 (>C=O), 163.2 (-C(O)NH-), 155.7 (ArC-1 or ArC-5), 155.4 (ArC-5 or ArC-1), 146.9 (tertiary aromatic), 114.6 (tertiary aromatic), 109.6

(ArC-2 or ArC-4), 108.4 (ArC-4 or ArC-2), 67.5, 62.1, 54.8, 48.3, 45.9, 42.4, 41.2, 35.5, 30.6,

30.5, 30.2, 28.2, 27.3, 26.4, 23.5, 19.1, 18.8, 11.7. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98.8% and retention time 5.3 min for the title compound.

Propyl (2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)propan-2-yl)carbamate (49a) The synthesis was carried out as described for 45 using 48a (25 mg, 0.05 mmol), tetrabutylammonium fluoride (0.10 ml, 0.10 mmol) and dry THF (1 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 49a (15 mg, 79% yield) as a white solid. mp = 44-

45oC. IR (neat): 3225, 2969, 1760, 1623, 1537, 1417, 1262, 1180, 1089 cm-1. 1H NMR (500

MHz,CDCl3) δ 6.83 (br s, 1H, OH), 6.43 (d, J = 2.0 Hz, 1H, Ar-H), 6.40 (br d, J = 2.0 Hz, 1H,

Ar-H), 5.03 (s, 1H, NH), 4.03-3.90 (m, 3H, 10eq-H, 5’-H), 2.86 (m as td, J = 12.5 Hz, J = 3.5

Hz, 1H, 10a-H), 2.64-2.56 (m, 1H, 8eq-H), 2.50-2.40 (m, 1H, 8ax-H), 2.20-2.07 (m, 2H, 10ax-H,

7eq-H), 1.95 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.68-1.49 (m, 9H, 7ax-H, 6’-H, -

C(CH3)2-, especially, 1.60, s, -C(CH3)2-), 1.47 (s, 3H, 6-Me), 1.15 (s, 3H, 6-Me), 0.91 (t, J =

13 7.5 Hz, 3H, 7'-H). C NMR (100 MHz CDCl3) δ 212.6 (>C=O), 155.6 (ArC-1 or ArC-5), 155.2

133

(ArC-5 or ArC-1), 148.4 (-C(O)O-), 126.0 (tertiary aromatic), 109.8 (tertiary aromatic), 106.9

(ArC-2 or ArC-4), 105.1 (ArC-4 or ArC-2), 55.4, 47.8, 45.7, 41.3, 35.2, 30.8, 28.3, 27.3, 22.8,

19.4, 10.8. Mass spectrum (ESI) m/z (relative intensity) 390 (M+ + H, 100). Exact mass (ESI)

+ calculated for C22H32NO5 (M + H), 390.2280; found 390.2270. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 99.3% and retention time 4.5 min for the title compound.

1-(2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)propan-2-yl)-3-propylurea (49b) The synthesis was carried out as described for 45 using 48b (23 mg, 0.04 mmol), tetrabutylammonium fluoride (0.10 ml, 0.10 mmol) and dry THF (1 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 49b (12 mg, 71% yield) as a white solid. mp = 40-

41oC. IR (neat): 3404, 2969, 1698, 1622, 1555, 1420, 1269, 1182, 1043 cm-1. 1H NMR (500

MHz,CDCl3) δ 6.66 (d, J = 1.5 Hz, 1H, Ar-H), 6.39 (d, J = 1.5 Hz, 1H, Ar-H), 5.12 (s, 1H, NH),

4.13 (t, J = 5.5 Hz, 1H, NH), 3.96 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.07

(q, J = 7.0 Hz, 2H, 5’-H), 2.90 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.63-2.55 (m, 1H,

8eq-H), 2.47-2.36 (m, 1H, 8ax-H), 2.19-2.10 (m, 1H, 10ax-H), 2.04 (t, J = 14.5 Hz, 1H, 7eq-H),

1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.60-1.50 (m, 7H, 7ax-H, -C(CH3)2-, especially, 1.54, s, -C(CH3)2 and 1.52, s, -C(CH3)2-), 1.48 (s, 3H, 6-Me), 1.20 (sextet, J = 7.0 Hz,

13 2H, 6’-H), 1.12 (s, 3H, 6-Me), 0.60 (t, J = 7.5 Hz, 3H, 7'-H). C NMR (100 MHz CDCl3) δ

211.2 (>C=O), 165.4 (-C(O)NH-), 155.3 (ArC-1 or ArC-5), 155.1 (ArC-5 or ArC-1), 146.5

(tertiary aromatic), 114.2 (tertiary aromatic), 109.8 (ArC-2 or ArC-4), 108.2 (ArC-4 or ArC-2),

56.2, 46.3, 45.2, 42.4, 34.2, 31.4, 28.6, 27.9, 23.5, 19.8, 11.4. Mass spectrum (ESI) m/z (relative

+ + intensity) 389 (M + H, 100). Exact mass (ESI) calculated for C23H33O5 (M + H), 389.2440;

134 found 389.2435. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.1% and retention time 4.2 min for the title compound.

3-azidopropyl-2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanoate (50) To a stirred solution of 20 (50 mg, 0.10 mmol) in anhydrous CH2Cl2/CH3NO2 (3:1 mixture, 2 mL) at 70ºC, tetrabutylammonium azide

(850 mg, 3.0 mmol) was added. The reaction was stirred at same temperature for 24 h. The reaction mixture was quenched by brine and extraction was done using Et2O. The organic layer was concentrated under vacuum and the residue was chromatographed on silica gel (40% ethyl acetate in hexane) to give 50 (35 mg, 75% yield). IR (neat): 3327, 2975, 2096, 1726, 1694, 1577,

-1 1 1417, 1254, 1184 cm . H NMR (500 MHz, CDCl3) δ 6.56 (s, 1H, OH), 6.40 (d, J = 2.0 Hz, 1H,

Ar-H), 6.31 (d, J = 2.0 Hz, 1H, Ar-H), 4.15 (t, J = 7.5 Hz, 1H, 4’-H), 3.99 (ddd, J = 15.0 Hz, J =

3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.20 (t, J = 7.0 Hz, 1H, 6’-H), 2.88 (td, J = 11.5 Hz, J = 4.0 Hz,

1H, 10a-H), 2.64-2.58 (m, 1H, 8eq-H), 2.50-2.41 (m, 1H, 8ax-H), 2.20-2.12 (m, 2H, 10ax-H,

7eq-H), 2.00-1.92 (m, 1H, 6a-H), 1.84 (m, 2H, 5’-H), 1.56-1.52 (m, 1H, 7ax-H), 1.51 (d, J = 1.5

13 Hz, 6H, -C(CH3)2-), 1.47 (s, 3H, 6β-Me), 1.12 (s, 3H, 6α-Me). C NMR (100 MHz CDCl3) δ

213.7 (>C=O), 176.4 (-C(O)O-), 155.3 (ArC-1 or ArC-5), 154.5 (ArC-5 or ArC-1), 144.7

(tertiary aromatic), 109.2 (tertiary aromatic), 106.7 (ArC-2 or ArC-4), 105.1 (ArC-4 or ArC-2),

61.45, 47.90, 47.19, 46.04, 44.74, 40.66, 34.63, 27.92, 27.69, 26.75, 25.97, 18.78. Mass spectrum (ESI) m/z (relative intensity) 416 (M+ + H, 100). Exact mass (ESI) calculated for

+ C22H30N3O5 (M + H), 416.2185; found 416.2179. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.5% and retention time 4.8 min for the title compound.

135

3-isothiocyanatopropyl-2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (51) To a stirred solution of 24

(20 mg, 0.05 mmol) in anhydrous THF (0.5 ml) at room temperature, triphenhyl phosphine (63 mg, 0.12 mmol) and carbon disulfide (110 mg, 0.75 mmol) was added. The reaction was stirred continiously for 12 h. The reaction mixture was diluted with Et2O and then evaporated under vacuum. The crude was chromatographed on silica gel (35% ethyl acetate in hexane) to give 51

(14 mg, 78% yield). IR (neat): 3292, 2971, 2105, 1725, 1693, 1577, 1417, 1254, 1183 cm-1. 1H

NMR (500 MHz, CDCl3) δ 6.40 (d, J = 2.0 Hz, 1H, Ar-H), 6.35 (s, 1H, OH), 6.29 (d, J = 2.0 Hz,

1H, Ar-H), 4.17 (t, J = 7.5 Hz, 1H, 4’-H), 3.98 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H,

10eq-H), 3.40 (t, J = 7.0 Hz, 1H, 6’-H), 2.89 (td, J = 11.5 Hz, J = 4.0 Hz, 1H, 10a-H), 2.64-2.58

(m, 1H, 8eq-H), 2.51-2.42 (m, 1H, 8ax-H), 2.20-2.12 (m, 2H, 10ax-H, 7eq-H), 2.00-1.91 (m, 3H,

5’-H, 6a-H), 1.55-1.53 (m, 1H, 7ax-H), 1.51 (d, J = 1.5 Hz, 6H, -C(CH3)2-), 1.48 (s, 3H, 6β-Me),

13 1.12 (s, 3H, 6α-Me). C NMR (100 MHz CDCl3) δ 211.4 (>C=O), 176.3 (-C(O)O-), 155.1

(ArC-1 or ArC-5), 155.0 (ArC-5 or ArC-1), 145.1 (tertiary aromatic), 109.9 (tertiary aromatic),

107.5 (ArC-2 or ArC-4), 105.4 (ArC-4 or ArC-2), 61.08, 47.50, 46.38, 45.42, 41.85, 40.96,

34.78, 29.11, 28.03, 26.94, 26.29, 26.19, 19.13. Mass spectrum (ESI) m/z (relative intensity) 432

+ + (M + H, 100). Exact mass (ESI) calculated for C23H30NO5S (M + H), 432.1845; found

432.1838. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.2% and retention time 4.9 min for the title compound.

4-bromobutyl 2-((6aR,10aR)-1-((tert-butyldimethylsilyl)oxy)-6,6-dimethyl-9-oxo-

6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (52) The synthesis was carried out as described for 46a using 44 (200 mg, 0.45 mmol), DMAP (330 mg,

136

2.70 mmol), EDCI (345 mg, 1.80 mmol), 4-bromo-1-butanol (275 mg, 1.80 mmol) and dry

CH2Cl2 (6.5 ml). Purification by flash column chromatography on silica gel (20% ethyl acetate in hexane) gave title compound 52 (200 mg, 77% yield) as a colorless oil. IR (neat): 2931, 1726,

-1 1 1611, 1567, 1414, 1253, 1139 cm . H NMR (500 MHz,CDCl3) δ 6.45 (d, J = 2.0 Hz, 1H, Ar-

H), 6.33 (d, J = 2.0 Hz, 1H, Ar-H), 4.14-4.01 (m, 2H, 4'-H), 3.75 (ddd, J = 15.0 Hz, J = 3.5 Hz, J

= 2.0 Hz, 1H, 10eq-H), 3.31 (t, J = 7.0 Hz, 3H, 7’-H), 2.71 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H,

10a-H), 2.60-2.52 (m, 1H, 8eq-H), 2.46-2.36 (m, 1H, 8ax-H), 2.18-2.04 (m, 2H, 10ax-H, 7eq-H),

1.93 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.84-1.79 (m, 2H, 5’-H), 1.78-1.68 (m, 2H, 6’-

H), 1.52-1.48 (m, 7H, 7ax-H, -C(CH3)2-, especially, 1.50, s, -C(CH3)2- and 1.49, s, -C(CH3)2-),

1.46 (s, 3H, 6-Me), 1.09 (s, 3H, 6-Me), 0.99 (s, 9H, Si(Me)2CMe3), 0.24 (s, 3H, Si(Me)2CMe3),

13 0.16 (s, 3H, Si(Me)2CMe3). C NMR (100 MHz CDCl3) δ 210.6 (>C=O), 176.8 (-C(O)O-),

155.1 (ArC-1 or ArC-5), 155.0 (ArC-5 or ArC-1), 145.1 (tertiary aromatic), 113.9 (tertiary aromatic), 110.0 (ArC-2 or ArC-4), 108.5 (ArC-4 or ArC-2), 64.30, 48.25, 46.61, 45.96, 41.32,

35.58, 33.49, 29.81, 28.26, 27.72, 27.35, 26.65, 26.51, 19.25, 18.85. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 98.9% and retention time 6.1 min for the title compound.

4-bromobutyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanoate (53) The synthesis was carried out as described for 45 using 52 (100 mg, 0.17 mmol), tetrabutylammonium fluoride (0.26 ml, 0.26 mmol) and dry THF (4.3 ml). Purification by flash column chromatography on silica gel (30% ethyl acetate in hexane) gave title compound 53 (78 mg, 95% yield) as a white solid. IR (neat): 3293, 2973,

-1 1 1724, 1694, 1577, 1459, 1254, 1183 cm . H NMR (500 MHz,CDCl3) δ 6.52 (s, 1H, OH), 6.40

(d, J = 1.5 Hz, 1H, Ar-H), 6.31 (d, J = 2.0 Hz, 1H, Ar-H), 4.09 (t, J = 6.0 Hz, 2H, 4'-H), 3.99

137

(ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.32 (t, J = 6.5 Hz, 3H, 7’-H), 2.88 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.66-2.58 (m, 1H, 8eq-H), 2.52-2.41 (m, 1H, 8ax-H),

2.21-2.11 (m, 2H, 10ax-H, 7eq-H), 1.97 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.84-1.77

(m, 2H, 5’-H), 1.76-1.70 (m, 2H, 6’-H), 1.53-1.49 (m, 7H, 7ax-H, -C(CH3)2-, especially, 1.51, s,

13 -C(CH3)2- and 1.50, s, -C(CH3)2-), 1.47 (s, 3H, 6-Me), 1.12 (s, 3H, 6-Me). C NMR (100 MHz

CDCl3) δ 213.7 (>C=O), 176.9 (-C(O)O-), 155.6 (ArC-1 or ArC-5), 154.8 (ArC-5 or ArC-1),

145.2 (tertiary aromatic), 109.5 (tertiary aromatic), 107.0 (ArC-2 or ArC-4), 105.4 (ArC-4 or

ArC-2), 66.07, 64.05, 47.52, 46.35, 45.14, 41.00, 34.94, 33.32, 29.91, 29.47, 28.04, 27.35, 27.06,

26.35, 19.14, 15.45. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.6% and retention time 4.9 min for the title compound.

4-azidobutyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-3-yl)-2-methylpropanoate (54) The synthesis was carried out as described for 50 using 53 (40 mg, 0.11 mmol), CH2Cl2/CH3NO2 (3:1 mixture, 1.8 ml) and tetrabutylammonium azide (915 mg, 3.21 mmol). Purification by flash column chromatography on silica gel (40% ethyl acetate in hexane) gave title compound 54 (28 mg, 80% yield) as a white solid. IR (neat): 3361, 2974, 2094, 1723, 1694, 1577, 1460, 1254, 1093 cm-1. 1H NMR (500

MHz,CDCl3) δ 6.41 (d, J = 2.0 Hz, 1H, Ar-H), 6.28 (d, J = 2.0 Hz, 1H, Ar-H), 4.10 (t, J = 6.0

Hz, 2H, 4'-H), 3.95 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.23 (t, J = 7.0 Hz,

3H, 7’-H), 2.88 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H, 10a-H), 2.65-2.57 (m, 1H, 8eq-H), 2.51-

2.40 (m, 1H, 8ax-H), 2.21-2.10 (m, 2H, 10ax-H, 7eq-H), 1.96 (m as td, J = 12.0 Hz, J = 3.0 Hz,

1H, 6a-H), 1.72-1.64 (m, 2H, 5’-H), 1.57-1.48 (m, 9H, 7ax-H, 6’-H, -C(CH3)2-, especially, 1.51,

13 s, -C(CH3)2- and 1.50, s, -C(CH3)2-), 1.47 (s, 3H, 6-Me), 1.12 (s, 3H, 6-Me). C NMR (100

138

MHz CDCl3) δ 213.2 (>C=O), 176.6 (-C(O)O-), 155.2 (ArC-1 or ArC-5), 154.5 (ArC-5 or ArC-

1), 144.9 (tertiary aromatic), 109.2 (tertiary aromatic), 106.8 (ArC-2 or ArC-4), 105.2 (ArC-4 or

ArC-2), 64.01, 50.85, 47.20, 46.06, 44.84, 40.67, 34.60, 27.72, 26.73, 26.10, 26.05, 25.66, 25.35,

18.79. Mass spectrum (ESI) m/z (relative intensity) 430 (M+ + H, 100). Exact mass (ESI)

+ calculated for C23H32N3O5 (M + H), 430.2342; found 430.2330. LC/MS analysis (Waters

MicroMass ZQ system) showed purity 99.1% and retention time 4.9 min for the title compound.

4-isothiocyanatobutyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a- hexahydro-6H-benzo[c]chromen-3-yl)-2-methylpropanoate (55) The synthesis was carried out as described for 51 using 54 (28 mg, 0.06 mmol), triphenhyl phosphine (86 mg, 0.32 mmol), carbon disulfide (137 mg, 1.80 mmol) and dry THF (1.2 ml). Purification by flash column chromatography on silica gel (35% ethyl acetate in hexane) gave title compound 55 (20 mg, 71% yield) as a white solid. IR (neat): 3264, 2928, 2106, 1725, 1694, 1577, 1417, 1254, 1093 cm-1. 1H

NMR (500 MHz,CDCl3) δ 7.11 (s, 1H, OH), 6.39 (d, J = 2.0 Hz, 1H, Ar-H), 6.34 (d, J = 2.0 Hz,

1H, Ar-H), 4.10 (m as td, J = 6.5 Hz, J = 2.0 Hz, 2H, 4'-H), 4.05 (ddd, J = 15.0 Hz, J = 3.5 Hz, J

= 2.0 Hz, 1H, 10eq-H), 3.40 (t, J = 7.0 Hz, 3H, 7’-H), 2.90 (m as td, J = 12.5 Hz, J = 3.5 Hz, 1H,

10a-H), 2.67-2.58 (m, 1H, 8eq-H), 2.55-2.43 (m, 1H, 8ax-H), 2.23-2.10 (m, 2H, 10ax-H, 7eq-H),

1.99 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.75-1.67 (m, 2H, 5’-H), 1.66-1.59 (m, 2H, 6’-

H), 1.58-1.49 (m, 7H, 7ax-H, -C(CH3)2-, especially, 1.52, s, -C(CH3)2- and 1.51, s, -C(CH3)2-),

13 1.48 (s, 3H, 6-Me), 1.12 (s, 3H, 6-Me). C NMR (100 MHz CDCl3) δ 213.5 (>C=O), 176.5 (-

C(O)O-), 155.3 (ArC-1 or ArC-5), 154.6 (ArC-5 or ArC-1), 144.8 (tertiary aromatic), 109.2

(tertiary aromatic), 106.7 (ArC-2 or ArC-4), 105.1 (ArC-4 or ArC-2), 63.69, 47.16, 46.01, 44.79,

44.41, 40.69, 34.63, 27.71, 26.76, 26.69, 26.05, 25.92, 25.45, 18.80. Mass spectrum (ESI) m/z

139

+ + (relative intensity) 446 (M + H, 100). Exact mass (ESI) calculated for C24H32NO5S (M + H),

446.2001; found 446.1992. LC/MS analysis (Waters MicroMass ZQ system) showed purity

98.8% and retention time 5.0 min for the title compound.

4-(nitrooxy)butyl 2-((6aR,10aR)-1-hydroxy-6,6-dimethyl-9-oxo-6a,7,8,9,10,10a-hexahydro-

6H-benzo[c]chromen-3-yl)-2-methylpropanoate (56) To a stirred solution of 53 (22 mg, 0.05 mmol) in acetonitrile (1.25 ml) at 70⁰C, silver nitrate (120 mg, 0.70 mmol) was added. The reaction was stirred continiously for 24 h at the same temperature. Upon completion, the reaction mixture was diluted with acetonitrile and filtered. The solute was concentrated under vaccum followed by addition of ethyl acetate and water. The organic layer was concentrated under vaccum and the residue was chromatographed on silica gel (40% ethyl acetate in hexane) to give

56 (16 mg, 75% yield) as a white solid. IR (neat): 3448, 2930, 1713, 1693, 1626, 1575, 1417,

-1 1 1205, 1095 cm . H NMR (500 MHz,CDCl3) δ 6.40 (d, J = 1.5 Hz, 1H, Ar-H), 6.27 (d, J = 1.5

Hz, 1H, Ar-H), 6.00 (s, 1H, OH), 4.36 (t, J = 6.0 Hz, 2H, 4'-H), 4.13-4.08 (m, 2H, 7’-H), 3.94

(ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.88 (m as td, J = 12.5 Hz, J = 3.5 Hz,

1H, 10a-H), 2.64-2.56 (m, 1H, 8eq-H), 2.50-2.40 (m, 1H, 8ax-H), 2.20-2.11 (m, 2H, 10ax-H,

7eq-H), 1.95 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.73-1.63 (m, 4H, 5’-H, 6’-H), 1.54-

1.49 (m, 7H, 7ax-H, -C(CH3)2-, especially, 1.51, s, -C(CH3)2- and 1.50, s, -C(CH3)2-), 1.47 (s,

13 3H, 6-Me), 1.11 (s, 3H, 6-Me). C NMR (100 MHz CDCl3) δ 213.5 (>C=O), 176.5 (-C(O)O-),

155.2 (ArC-1 or ArC-5), 154.6 (ArC-5 or ArC-1), 144.8 (tertiary aromatic), 109.2 (tertiary aromatic), 106.8 (ArC-2 or ArC-4), 105.1 (ArC-4 or ArC-2), 63.71, 47.18, 46.03, 44.76, 40.67,

34.63, 27.70, 26.76, 26.07, 25.93, 24.69, 23.41, 18.77. Mass spectrum (ESI) m/z (relative

+ + intensity) 450 (M + H, 100). Exact mass (ESI) calculated for C23H32NO8 (M + H), 450.2128;

140 found 450.2122. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99.3% and retention time 4.9 min for the title compound.

13, 35 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. Results from the competition assays

38 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.39 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

141

EC50 values were determined by non-linear regression analysis using GraphPad Prism software

(GraphPad Software, Inc., San Diego, CA).

Plasma stability.13 Compounds or their proposed metabolites were diluted (200 µM) in mouse or rat plasma and incubated at 37 C, 100 rpm. At various time points, samples were taken, diluted 1:4 in acetonitrile and centrifuged to precipitate the proteins. The resulting supernatant was analyzed by HPLC. 4-Nitrophenyl butyrate was used as a control in each experiment. In vitro plasma half-lives were determined using exponential decay calculations in Prism

(GraphPad).

HPLC Analysis: Chromatographic separation was achieved using a Supelco Discovery C18 (4.6 x 250 mm) column on a Waters Alliance HPLC system. Mobile phase consisted of acetonitrile

(A) and a mixture of 60% water (acidified with 8.5% o-phosphoric acid) and 40% acetonitrile

(B). Gradient elution started with 5% A, transitioning to 95% A over ten minutes and holding for five minutes before returning to starting conditions; run time was 15 minutes, the flow rate was 1 mL/min and UV detection was used at each compound’s maximal absorbance (204 & 230 nM).

Methods for characterization of in vivo effects13, 16, 40

a. Rodents.

Subjects. For hypothermia testing, female Sprague-Dawley rats (n = 6/group), weighing between

142 unrestricted access to food and water. For tail-flick withdrawal (analgesia) testing, male CD-1 mice (n = 6/group), weighting between 30 and 35 g (Charles River, Wilmington MA), were used.

Mice were housed 4/cage in a climate controlled vivarium with unrestricted access to food and water and acclimated to these conditions for at least a week before any experimental manipulations occurred. Analgesia testing took place between 11:00 am and 7:00 pm. Mice were used once.

Procedures. Temperature was recorded using a thermistor probe (Model 401, Measurement

Specialties, Inc., Dayton, OH) inserted into the rectum at a depth of 6 cm and secured to the tail with micropore tape. Rats were minimally restrained and isolated in 38x50x10cm plastic stalls.

Temperature was read to the nearest 0.01 oC using a Thermometer (Model 4000A, Measurement

Specialties, Inc.).

Two base-line temperature measures were recorded at 15 min intervals, and drugs were injected immediately after the second baseline was recorded. After injection, temperature was recorded every 30 min for three hours and every hour thereafter for a total of 6 hours. The change in temperature was determined for each rat by subtracting temperature readings from the average of the two baseline measures. Analgesia testing utilized a thermostatically controlled 2L water bath commercially available from VWR International® where the water temperature was set at 52 oC

(± 0.5 oC). The tail was immersed into the water at a depth of 2 cm and the withdrawal latency recorded by a commercially available stopwatch (Fisher Scientific), allowing measurements in seconds and 1/100 s. Cut-off was set at 10 s to minimize the risk of tissue damage. A test session consisted of 5 recordings, the first of which constituted the base-line recording.

Injections occurred immediately after the base-line recording and the remaining recordings took place 20, 60, 180, and 360 min post administration. Prior to this testing, the animals had been

143 accustomed to the procedure for three consecutive “mock” sessions where the water was held at

38oC, i.e., average body temperature of mice; no tail-flicks are elicited by this water temperature.

The 3rd “mock” session also included an i.p. injection of vehicle (10 mL/kg). The tail-flick withdrawal latencies are expressed as a percentage of maximum possible effect (%MPE), according to the formula: %MPE = [(test latency minus base-line latency) divided by (10 minus base-line latency)] times 100.

Drugs. For hypothermia testing, compounds 59, 61 and 68 were initially dissolved in a solution of 20% ethanol, 20% alkamuls, and 60% saline, and were further diluted with saline. Injections were administered s.c. in a volume of 1.0 mL/kg. For tail-flick withdrawal (analgesia) testing, 59 and 77 were initially dissolved in 2% dimethyl sulfoxide, 4% Tween-80 and 4% propylene glycol before saline was slowly added just prior to the 10 mL/kg i.p. administration. All suspensions were freshly prepared for analgesia testing.

b. Nonhuman primates.

Subjects. Four adult male squirrel monkeys (Saimiri sciureus) that were trained in the behavioral and pharmacological procedures used here served as subjects (see below). Experimental sessions were conducted 5 days a week (Monday–Friday). Each subject had previously received acute intramuscular (i.m.) injections of 0.01 mg/kg of the cannabinoid 28 in training sessions at least twice weekly and other cannabinergic compounds in test sessions conducted no more than once weekly; no test compounds were administered for at least 7 days prior to the present studies. The experimental protocol for the present studies was approved by the Institutional

Animal Care and Use Committee at McLean Hospital. Subjects were maintained in a facility licensed by the U.S. Department of Agriculture and in accordance with the Guidelines for the

144

Care and Use of Mammals in Neuroscience and Behavioral Research (National Research

Council, 2003).

Apparatus. During experimental sessions, subjects were seated in a Plexiglas chair within a ventilated sound- and light-attenuating chamber.41 The front panel of the chair was outfitted with two response levers that were positioned 6 cm left and right of center. Each lever-press with a force of at least 0.25 N closed a microswitch, produced an audible click, and was recorded as a response. Red stimulus lights were mounted behind the transparent front panel of the chair, approximately 10 cm above each response lever. Before each session, a shaved portion of each subject’s tail was coated with electrode paste and placed under brass electrodes for the delivery of brief, low-intensity current (see below). Experimental events and data collection were controlled by Med Associates (St. Albans, VT) interfacing equipment and operating software.

Behavioral procedure. The subjects were previously trained to discriminate the pre-session administration of 0.01 mg/kg of the cannabinoid agonist AM4054 or its vehicle (20% ethanol/20% emulphor/60% saline) by responding on one of two levers. Briefly, subjects initially were trained to terminate visual stimuli associated with the delivery of brief, low- intensity current (200 ms; 3 mA) across the electrodes by depressing one of the two response levers; the inactive lever was removed to facilitate early training. The active lever varied until subjects reliably terminated visual stimuli by responding on either lever. Subsequently, both levers were present in all sessions and the active lever was signaled only by a pre-session injection: one lever was active only following the i.m. injection of the cannabinoid CB1 agonist

AM4054 (0.01 mg/kg, i.m.), whereas the other lever was active only following i.m. injection of vehicle; right and left lever assignments were counterbalanced among subjects. Under terminal conditions, each training session began with a 10-minute timeout period during which all lights

145 were extinguished and responding had no programmed consequences. After the timeout period, two red stimulus lights above each lever were illuminated and completion of 10 consecutive responses [fixed ratio (FR) 10] on the active lever extinguished all stimulus lights and initiated a

50-second timeout. Responses on the inactive lever reset the FR requirement. Current delivery was scheduled for delivery every 10 seconds until either the FR 10 was completed on the correct lever or 30 seconds elapsed, whichever came first.

Drug testing. Testing was conducted to determine the extent to which different doses of drugs substituted for the training compound, i.e., produced responding on the training drug-associated response lever. Tests for the time course of substitution of compounds AM7499, AM10843,

AM10806 and HU210 to the training stimulus were conducted when a subject’s discrimination performance was at least 90% accurate for four of the last five training sessions and on the immediately preceding session. Procedurally, test sessions differed from training sessions in two ways. First, 10 consecutive responses on either lever extinguished the stimulus lights and associated program of current delivery, and initiated the 50-second timeout. Second, no current deliveries were scheduled during test sessions so as to preclude possible stimulus-induced enhancement of responding. Other schedule contingencies were unchanged. In initial experiments, dose-ranging procedures were employed to quickly establish the lowest dose that fully substituted for (≥ 90% responding on the CB1 lever) the 0.01 mg/kg of the CB1 agonist

AM4054. In these procedures, each subject received one of several doses of each drug (0.0001 –

0.003 mg/kg) 60 min prior to the test session; doses were chosen based on data from receptor binding studies and previous work in rodents or monkeys. Next, the lowest effective dose of each drug was studied in all subjects. To assess behavioral onset and time course of action of the

146 doses of the four drugs, experiments with each drug were arranged to include multiple sequential test sessions that began 15, 30, 60, 120, 240, 480, and 960 minutes after injection.

147

REFERENCES

1. Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson, L. A.; Griffin, G.;

Gibson, D.; Mandelbaum, A.; Etinger, A.; Mechoulam, R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992, 258, 1946-9.

2. Munro, S.; Thomas, K. L.; Abu-Shaar, M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993, 365, 61-5.

3. Pavlopoulos, S.; Thakur, G. A.; Nikas, S. P.; Makriyannis, A. Cannabinoid receptors as therapeutic targets. Curr Pharm Des 2006, 12, 1751-69.

4. Han, S.; Thatte, J.; Buzard, D. J.; Jones, R. M. Therapeutic utility of cannabinoid receptor type 2 (CB(2)) selective agonists. J Med Chem 2013, 56, 8224-56.

5. Hwang, J.; Adamson, C.; Butler, D.; Janero, D. R.; Makriyannis, A.; Bahr, B. A.

Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: a neuroprotective therapeutic modality. Life Sci 2010, 86, 615-23.

6. Karst, M.; Wippermann, S.; Ahrens, J. Role of cannabinoids in the treatment of pain and

(painful) spasticity. Drugs 2010, 70, 2409-38.

7. Lu, D.; Vemuri, V. K.; Duclos, R. I., Jr.; Makriyannis, A. The cannabinergic system as a target for anti-inflammatory therapies. Curr Top Med Chem 2006, 6, 1401-26.

8. Pacher, P.; Batkai, S.; Kunos, G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev 2006, 58, 389-462.

9. Pertwee, R. G. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: delta9-tetrahydrocannabinol, cannabidiol and delta9-tetrahydrocannabivarin. Br J

Pharmacol 2008, 153, 199-215.

148

10. Vemuri, V. K.; Makriyannis, A. Medicinal chemistry of cannabinoids. Clin Pharmacol

Ther 2015, 97, 553-8.

11. Grotenhermen, F. Pharmacokinetics and pharmacodynamics of cannabinoids. Clin

Pharmacokinet 2003, 42, 327-60.

12. Makriyannis, A. 2012 Division of medicinal chemistry award address. Trekking the cannabinoid road: a personal perspective. J Med Chem 2014, 57, 3891-911.

13. Nikas, S. P.; Sharma, R.; Paronis, C. A.; Kulkarni, S.; Thakur, G. A.; Hurst, D.; Wood, J.

T.; Gifford, R. S.; Rajarshi, G.; Liu, Y.; Raghav, J. G.; Guo, J. J.; Jarbe, T. U.; Reggio, P. H.;

Bergman, J.; Makriyannis, A. Probing the carboxyester side chain in controlled deactivation (-)- delta(8)-tetrahydrocannabinols. J Med Chem 2015, 58, 665-81.

14. Sharma, R.; Nikas, S. P.; Guo, J. J.; Mallipeddi, S.; Wood, J. T.; Makriyannis, A. C-ring cannabinoid lactones: a novel cannabinergic chemotype. ACS Med Chem Lett 2014, 5, 400-4.

15. Sharma, R.; Nikas, S. P.; Paronis, C. A.; Wood, J. T.; Halikhedkar, A.; Guo, J. J.; Thakur,

G. A.; Kulkarni, S.; Benchama, O.; Raghav, J. G.; Gifford, R. S.; Jarbe, T. U.; Bergman, J.;

Makriyannis, A. Controlled-deactivation cannabinergic ligands. J Med Chem 2013, 56, 10142-

57.

16. Kulkarni, S.; Nikas, S. P.; Sharma, R.; Jiang, S.; Paronis, C. A.; Leonard, M. Z.; Zhang,

B.; Honrao, C.; Mallipeddi, S.; Raghav, J. G.; Benchama, O.; Jarbe, T. U.; Bergman, J.;

Makriyannis, A. Novel C-Ring-Hydroxy-Substituted Controlled Deactivation Cannabinergic

Analogues. J Med Chem 2016, 59, 6903-19.

17. Bodor, N.; Buchwald, P. Soft drug design: general principles and recent applications.

Med Res Rev 2000, 20, 58-101.

149

18. Bodor, N.; Buchwald, P. Ophthalmic drug design based on the metabolic activity of the eye: soft drugs and chemical delivery systems. Aaps j 2005, 7, E820-33.

19. Bodor, N.; Buchwald, P. Recent advances in retrometabolic drug design (RMDD) and development. Pharmazie 2010, 65, 395-403.

20. R., S. Design and synthesis of novel cannabinoids with controlled detoxification. PhD

Thesis 2011.

21. Ware, M. A.; Daeninck, P.; Maida, V. A review of nabilone in the treatment of chemotherapy-induced nausea and vomiting. Ther Clin Risk Manag 2008, 4, 99-107.

22. Haney, M.; Cooper, Z. D.; Bedi, G.; Vosburg, S. K.; Comer, S. D.; Foltin, R. W.

Nabilone decreases marijuana withdrawal and a laboratory measure of marijuana relapse.

Neuropsychopharmacology 2013, 38, 1557-65.

23. Archer, R. A.; Blanchard, W. B.; Day, W. A.; Johnson, D. W.; Lavagnino, E. R.; Ryan,

C. W.; Baldwin, J. E. Cannabinoids. 3. Synthetic approaches to 9-ketocannabinoids. Total synthesis of nabilone. J Org Chem 1977, 42, 2277-84.

24. (1R)-(+)-Nopinone of high optical purity is commercially available or it can be synthesized by ozonolysis of commercially available (-)-B-pinene.

25. Coxon, J. M. G., R. P.; Hartshorn, M. P. Derivatives of nopinone. Aust. J. Chem. 1970,

23, 1069-1071.

26. Grimshaw, J.; Grimshaw, J. T.; Juneja, H. R. Apoverbenone (6,6-dimethylnorpin-3-en-2- one). An investigation into its preparation by dehydrobromination of a sterically hindered bromo-ketone. Journal of the Chemical Society, Perkin Transactions 1 1972, 50-52.

27. Nikas, S. P. T., G. A.; Parrish, D.; Alapafuja, S. O.; Huestis, M. A.; Makriyannis, A.

Aconcise methodology for the synthesis of (-)-Δ9-tetrahydrocannabinol and (-)-Δ9-

150 tetrahydrocannabivarin metabolites and their regiospecifically deuterated analogs. Tetrahedron

2007, 63, 8112-8123.

28. Nikas, S. P.; Grzybowska, J.; Papahatjis, D. P.; Charalambous, A.; Banijamali, A. R.;

Chari, R.; Fan, P.; Kourouli, T.; Lin, S.; Nitowski, A. J.; Marciniak, G.; Guo, Y.; Li, X.; Wang,

C. L.; Makriyannis, A. The role of halogen substitution in classical cannabinoids: a CB1 pharmacophore model. Aaps j 2004, 6, e30.

29. Nikas, S. P.; D'Souza, M.; Makriyannis, A. Enantioselective synthesis of (10S)- and

(10R)-methyl-anandamides. Tetrahedron 2012, 68.

30. Clase, J., Money, T. An enentiospecific route to C, D ring synthons for steroid synthesis

Canadian Journal of Chemistry-Revue Canadienne De Chimie 1992, 70, 1537-1544.

31. Schroeder, M. Osmium tetraoxide cis hydroxylation of unsaturated substrates. Chemical

Reviews 1980, 80, 187-213.

32. Lindgren, B. O. N., Torsten; Husebye, Steinar; Mikalsen, Yvind; Leander, Kurt; Swahn,

Carl-Gunnar. Preparation of Carboxylic Acids from Aldehydes (Including Hydroxylated

Benzaldehydes) by Oxidation with Chlorite. Acta Chemica Scandinavica 1973, 27.

33. Neises, B.; Steglich, W. Simple Method for the Esterification of Carboxylic Acids.

Angewandte Chemie International Edition in English 1978, 17, 522-524.

34. Curtius, T. "Ueber Stickstoffwasserstoffsäure (Azoimid) N3H" [On hydrazoic acid

(azoimide) N3H]. Berichte der Deutschen chemischen Gesellschaft zu Berlin. 1890, 23, 3023–

3033.

35. Nikas, S. P.; Alapafuja, S. O.; Papanastasiou, I.; Paronis, C. A.; Shukla, V. G.;

Papahatjis, D. P.; Bowman, A. L.; Halikhedkar, A.; Han, X.; Makriyannis, A. Novel 1',1'-chain substituted hexahydrocannabinols: 9beta-hydroxy-3-(1-hexyl-cyclobut-1-yl)-

151 hexahydrocannabinol (AM2389) a highly potent cannabinoid receptor 1 (CB1) agonist. J Med

Chem 2010, 53, 6996-7010.

36. Um, P.-J.; Drueckhammer, D. G. Dynamic Enzymatic Resolution of Thioesters. Journal of the American Chemical Society 1998, 120, 5605-5610.

37. Mechoulam, R.; Lander, N.; University, A.; Zahalka, J. Synthesis of the individual, pharmacologically distinct, enantiomers of a tetrahydrocannabinol derivative. Tetrahedron:

Asymmetry 1990, 1, 315-318.

38. Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.

Biochem Pharmacol 1973, 22, 3099-108.

39. Ogawa, G.; Tius, M. A.; Zhou, H.; Nikas, S. P.; Halikhedkar, A.; Mallipeddi, S.;

Makriyannis, A. 3'-functionalized adamantyl cannabinoid receptor probes. J Med Chem 2015,

58, 3104-16.

40. Paronis, C. A.; Nikas, S. P.; Shukla, V. G.; Makriyannis, A. Delta(9)-

Tetrahydrocannabinol acts as a partial agonist/antagonist in mice. Behav Pharmacol 2012, 23,

802-5.

41. Kangas, B. D.; Delatte, M. S.; Vemuri, V. K.; Thakur, G. A.; Nikas, S. P.; Subramanian,

K. V.; Shukla, V. G.; Makriyannis, A.; Bergman, J. Cannabinoid discrimination and antagonism by CB(1) neutral and inverse agonist antagonists. J. Pharmacol. Exp. Ther. 2013, 344, 561-7.

152

CHAPTER 3: PROBING THE PHARMACOPHORIC SPACE AT THE PHENOLIC HYDROXYL (C1) OF NABILONE

INTRODUCTION

Following the discovery of Δ9-THC and other cannabinoids, a number of analogs and modifications have been synthesized in order to define the structure activity relationships (SAR) of THC at CB1 and CB2 receptors. Δ9-THC has four distinct pharmacophores: a phenolic hydroxyl at C1, a side chain at C3, a northern aliphatic hydroxyl at C9 or C11, and a southern aliphatic hydroxyl.1 (Figure 3.1)

Figure 3.1: Four major pharmacophores of classical cannabinoids.

The early SAR studies of classical cannabinoids comprised of C3 side chain modifications.

Majority of the analogs reported contain saturated, straight or branched alkyl chains. C3 side chains featuring heteroatoms, unsaturated alkyl side chain, and esters, carboxylic acids, ethers, nitriles and heterocycles as functional groups have been reported.2 These substitutions have shown variable effects on CB1 and CB2 selectivity and potency. There is a direct correlation

153 between the length of the C3 side chain of THC and the binding affinity of CB1 and CB2; the increase in chain length results in increase in binding affinity at both the receptors.3 Of the major structural features defined for CB SAR, the C3 side chain has the biggest effect on the binding affinity for CB receptors. Having examined different lengths of alkyl chains a requirement of at least 3 carbons with 5-8 carbon length chains being ideal has been observed.4, 5 Also, it has been noticed that an enhancement of binding affinity at the CB1 receptor is facilitated by adding methyl groups on the alkyl chain preferably at the 1’ and 2’ positions.6

Minor structural changes to the C9/C11 pharmacophore can modulate receptor binding.7 Binding affinity is greatly enhanced when substitutions occur at this position. Retention of activity at both receptors at sub nano-molar levels is observed when C11 group is hydroxylated. Conversion of a methylene group to carbonyl at the C9 position resulted in Nabilone, a marketed CB drug.8

Modification of C1 phenol on the THC scaffold can result in drastic changes in the pharmacological activity. This includes analogs modified with minor changes to the phenolic group or those who lack the phenolic hydroxyl group.9 Another observation was that removal of phenol or etherification produced compounds that showed a considerable CB2 selectivity. In Δ8-

THC-DMH, a phenol to methyl ether conversion produced an approximate 800 fold selectivity for CB2 over CB1.10 Also, a series of water-soluble Δ8-THC analogs were synthesized by esterifying the phenol.11

154

OBJECTIVE AND SPECIFIC AIMS

The objective of this project was to probe the pharmacophoric space at the C1 position of

Nabilone (Figure 3.2). The starting point of this SAR was to explore pharmacophoric limits of chain length required for optimal activity at CB receptors. We sought to explore the pharmacophoric space by replacing methyl groups with small size alkyl chains and carbocyclic rings. In addition to probe the van der Walls interactions, weak electrostatic interactions and hydrogen bonding interactions we are replacing the methyl group with small size oxygen containing heterocyclic rings. Novel analogs were assessed for their binding affinity for rat CB1, mouse CB2 and human CB2. Compounds possessing high binding affinity were then selected for their functional assessment using the cAMP functional biochemical assay followed by metabolic stability towards plasma esterases and liver microsomes. Lead analogs were then pharmacologically evaluated in hypothermia and analgesia rodant assays.

Figure 3.2: Exploration of pharmacophoric space at C1 position of nabilone.

155

CHEMISTRY

Scheme 13: Synthesis of nabilone

Reagents and conditions: (a) 8, p-TSA, CHCl3, 0 ºC to rt, 4 days, 55%; (b) TMSOTf, CH2Cl2,

MeNO2, 0 ºC to rt, 7 h, 65%.

Nabilone was synthesized in 2 steps as shown in Scheme 13. Comercially available resorcinal 57 was coupled with chiral diacetates 812, 13 in the presence of catalytic amounts of p- toluenesulfonic acid (p-TSA) to give bicyclic intermediate 58 in 55% yield followed by benzopyran ring closure using TMSOTf12 to afford nabilone 59 with 65% yield.

In order to explore the SAR of nabilone on ring A, analogs bearing ether and ester groups in place of phenolic hydroxyl were synthesized as shown in Scheme 14. Modification of the phenolic hydroxyl with ether group was accomplished by reacting 59 with 1-bromopentane and potassium carbonate in acetone to yield 60 in 50% yield.14 Acetylated nabilone 61 was

14 synthesized by treatment of 59 with DMAP and acetic anhydride in CH2Cl2 to yield 80% of 61.

Ester substituted analogs 62a-76 afforded by treatment of 59 with EDCI, DMAP and respective carboxylic acid.15 Acetonide substituted analogs 65-67 were treated with TFA to yield the respective 1,2-diols 77&78 and 1,3-diol 79.

156

Scheme 14: Modification of phenolic hydroxyl group of nabilone

Reagents and conditions: (a) 1-bromopentane, potassium carbonate, acetone, 45 ºC, 15 h, 50%;

(b) DMAP, acetic anhydride, CH2Cl2, rt, 30 min, 80%; (c) EDCI, DMAP, R-OH, CH2Cl2, 0 ºC to rt, 24 h, 58-91%; (d) TFA, CH2Cl2, rt, 1 h, 90-95%.

157

RESULTS AND DISCUSSION

BIOCHEMICAL ASSESSMENT

Our SAR study (Figure 3.2) focused on chemical synthesis to explore the pharmacophoric chemical space at the C1 position of nabilone. In vitro profiling of the C1 substituted nabilone analogs was performed in the following assays:

 Competitive radioligand binding assay for CB1 and CB2 receptors.

 Cyclic adenosine monophosphate (cAMP) assay.

 Metabolic (Plasma and microsomal) stability assays.

The binding affinities of C1 substituted nabilone analogs were determined for the CB1 receptor

(rat brain membranes) and membrane preparations from HEK293 cells expressing human

(hCB2) and mouse (mCB2) CB2 receptors. Displacement of [3H]-CP-55,940 from these

3 membranes was used to determine EC50 values in competition radioligand binding assays. [ H]-

CP-55,940 was used as the competing ligand as it is nonselective and has high affinity for both

CB1 and CB2 receptors. Intrinsic efficacy of key analogs was determined using forskolin stimulated cAMP assay. Lead analogs were assessed for their stability towards plasma esterases and liver microsomes.

Results from previous SAR studies at the C1 position of Δ8-THC analogs have shown that etherification or removal of phenol led to analogs showing selectivity for CB2. Also, in an effort to produce water soluble cannabinoids, the phenolic hydroxyl of Δ8-THC was converted to hydrochloride salts of butyl esters. Therefore, in order to explore the effects of substituents at the

C1 position of nabilone, analogs reported in Table 3.1 were synthesized.

158

Table 3.1: Binding affinities of C1 substituted nabilone analogs

Compd & (K ,nM) R i AM # rCB1 mCB2 hCB2 60 >530 - >1000 10861 61 28.7±8.5 321±45 1000 10816

62a 17.7±4.3 - 62.9±9.6 10841

62b 28.3±6.3 - 48.0±8.3 10842

63 30.7±5.1 - 126±34 10852

64 11.7±3.7 - 74-191 10870

65 15.3±2.4 19.8±3.3 8.0±2.8 10833

77 4.3±1.1 16.8±2.7 15.1±3.5 10834

66 2.5±0.9 13.7±1.6 15.0±2.4 10836

78 3.6±0.7 28.5±4.1 51.0±8.9 10837

159

67 91.3±20.2 >1000 261±37 10839

79 222.3±47.0 >1000 425±42 10840

68 6.8±2.5 39.6±4.6 10.9±3.3 10846

69 2.2±0.4 50-100 37.9±7.9 10889

70 5.3±1.8 - 10.6±2.9 10890

71 64.1±9.9 - >530 10844

72 215.9±28.7 - >1000 10877

73 12.9±2.2 - 48.0±7.2 10878

74 >530 - >530 10875

75 52.1±6.9 - 287.6 10876

76 195±26 - >530 10845

160

Most of the analogs exhibited high affinity for the CB1 receptor while few analogs showed good affinity to the CB2 receptor. Conversion of phenol to ether in AM10861 (60) showed significant loss of CB1 activity with a complete loss of affinity at the CB2 receptor. Ester substituted analogs containing acetonides (65 & 66), 1,2-diols (77 & 78), and the tetrahydrofuran ring (68-

70) showed the highest binding affinity to both CB1 and CB2 receptors. Alkyl esters, AM10816

(61), AM10841 (62a) and AM10842 (62b) showed similar binding affinity to CB1 receptor. It is surprising to see that the acetylated nabilone (61) showed complete loss of activity at the CB2 receptor as compared to analogs 62a and 62b. Alkyl esters with a Bromo (63) and azido (64) substitution at the terminal end showed similar binding affinity to CB1 and CB2 receptors. It is interesting to see that the 1,3-diol analog, AM10840 (79) loose affinity to both CB1 and CB2 receptor as compared to 1,2-diols, AM10834 (77) and AM10837 (78). 3-methoxyoxolane analog, AM10878 (73) showed significantly higher binding affinity to both the receptors as compared to 3-methoxyoxane analog, AM10876 (75). Cyclopentyl analog AM10844 (71), 1, 3- dioxane analog AM10839 (67) and methoxyphenyl substituted analog AM10845 (76) showed reduced affinities while AM10875 (74) and AM10877 (72) showed complete loss of affinity to the CB1 and CB2 receptors. It is interesting to note that analogs with carbocyclic rings containing oxygen show high binding affinities to both the receptors.

In summary, ester substituted analogs at C1 position of nabilone appear to exhibit no significant preference for either of the two receptors. In general, analogs containing the acetonides (65 &

66), 1,2-diols (77 & 78) and carbocyclic rings containing oxygen showed high affinity to the

CB1 receptor. Thus comparison of binding data of analogs reported in Table 3.1 suggests that there exists a unique pharmacophoric chemical space at the C1 position of nabilone.

161

Table 3.2: cAMP data of C1 substituted nabilone analogs

rCB1 hCB2 AM # R EC50 (nM) Emax (%) EC50 (nM) Emax (%)

65 1.7±0.8 86 13.8±1.7 69 10833

77 6.1±1.2 78 - - 10834

68 1.6±0.5 90 56.0±6.6 69 10846

The functional potency of C1 substituted nabilone analogs tested in cAMP assay is tabulated in

Table 3.2. In both the rat CB1 and human CB2 receptors, all the analogs showed a concentration- dependent inhibition of forskolin-induced cAMP accumulation. The functional potency of the analogs was compared to the agonist CP-55,940 in the cAMP assay. The ability of compounds

AM10833, AM10834 and AM10846 to activate the rat CB1 and human CB2 receptors was assessed in the functional cAMP assay using HEK-293 cells expressing recombinant cannabinoid receptors. According to the cAMP results Table 3.2, AM10833 (65) showed a concentration-dependent inhibition of forskolin-induced cAMP accumulation with an EC50 value of 1.7 nM (Emax of 86%) at the CB1 receptor and an EC50 value of 13.8 nM (Emax of 69%) at hCB2 recpetor. Thus AM10833 is equipotent at both receptors and acts as a full agonist at the

162 rCB1 and hCB2 receptor. Similarly, AM10834 (77) exhibited full agonism at the CB1 receptor with an EC50 value of 6.1 nM (Emax of 78%). Full agonism was also observed in AM10846 (68) which displayed an EC50 value of 1.6 nM (Emax of 90%) at the CB1 receptor and an EC50 value of 56.0 nM (Emax of 69%) at hCB2 recpetor.

Table 3.3: Metabolic stability of C1 substituted nabilone analogs

Microsomal Stability (t ) Plasma Stability (t ) min 1/2 AM # R 1/2 min Mouse Rat Human Mouse Rat Human 61 <5 5 >30 - - - 10816

77 8 4.5 >30 4.4 - 7.8 10834

79 >30 >30 >30 2.6 >30 6.3 10840

68 >30 >30 >30 - - - 10846

Selected compounds with low nanomolar binding affinities were assessed for their stability towards plasma esterases and liver microsomes. A comparison of the plasma half-lives (t1/2,

Table 3.3) of the C1 substituted nabilone analogs shows that AM10816 (61) and AM10834 (77) have short half-life in both mouse and rat plasma (t1/2 = <8 min) and are stable in human plasma.

163

AM10840 (79) and AM10846 (68) show stability of more than 30 min in mouse, rat and human plasma. The lack of carboxyesterases in human plasma tends to compounds displaying stability of >30 min. Microsomal stability data shows that AM10834 (77) and AM10840 (79) have a t1/2 =

<8 min in mouse and humans. It is surprising to see that 79 is stable (>30 min) in rat microsomes.

164

PHARMACOLOGICAL EVALUATION OF LEAD COMPOUNDS

Successful compounds resulting from in vitro screening were assessed in vivo using two well defined rodent assays, hypothermia and analgesia. Hypothermic effects were assessed by measuring the rectal body temperature in isolated mice and rats over 24 h periods. Compounds were also tested in the CB1 receptor characteristic analgesia test in mice using the well-defined tail flick test.

Three lead analogs were selected for pharmacological evaluation based on the binding affinities to the cannabinoid receptor, functional assessment and their metabolic stability.

Evaluation of AM10816 (61)

Binding data shows that AM10816 (61) has a good affinity at the rCB1 receptor while it lacks affinity to the CB2 receptor. In vitro plasma (rat) stability showed that the compound had a half- life of 5 min.

Figure 3.3:.Hypothermia assessment of nabilone (AM10806) and AM10816 in rats.

In vivo activity of nabilone (59) and compound 61 were determined by assessing their effects on body temperature. Body temperature was measured in isolated mice over a 6 h period following drug injection. Both the compounds significantly reduced temperature (4-5°C) at a dose of 1

165 mg/kg. The offset of nabilone was around 360 min while there was a slow recovery for 61 towards baseline. Compound 61 had a faster onset of drug effect at a dose of 3 mg/kg, as significant effects were observed 60 min after injection. The body temperature was still reduced at 6 h after injection.

In vivo data shows that compound 61 produces hypothermia activity and is able to produce similar effects as other cannabionoid agonists. The effect of compound 57 at the highest dose tested (3 mg/kg), had a faster onset and longer duration of action than nabilone.

Evaluation of AM10846 (68)

AM10846 displayed high affinity to rCB1 and hCB2 receptor (Table 3.1) and was found to be a full agonist at the CB1 and CB2 receptor in the cAMP assay (Table 3.2). Metabolic stability data also shows that this compound is stable (t1/2 = <30 min) in mouse, rat and human plasma.

C) C) -2

-4

-6

-8 AM10806/Nabilone 3mg/kg AM10846 5.6mg/kg

Change in Temperature ( -10 AM10846 3mg/kg

-12 0 20 60 180 360 720 1440 Time (min)

Figure 3.4:.Hypothermia assessment of nabilone (AM10806) and AM10846 in mice.

166

The in vivo potency of the tetrahydrofuran substituted analog AM10846 (68) was explored in the hypothermia test (Figure 3.4) in mice. In this assay, body temperature was measured in rats over a 24 h period following drug injection. AM10846 at a dose of 3 mg/kg did not significantly decrease core body temperature. At a higher dose of 5.6 mg/kg, the onset of drug occurred within

60-180 minutes after injection (10ºC drop in temperature) and the hypothermic effect was still seen at 24 hours (7ºC drop in temperature). Nabilone, AM10806 (59) at a dose of 3 mg/kg had a similar onset to action, showing peak effects between 60-180 minutes after injection (10ºC drop in temperature) and the effect was still seen at 6 hours (7ºC drop in temperature). We did not assess nabilone over 24 hours but previous studies show that nabilone has duration of action of

24 hours.

This experiment shows that compound 68 produced significant hypothermia at the highest dose tested (5.6 mg/kg). Results show that AM10846 produces similar cannabinergic effect like

AM10806 but has a significantly longer duration of action that nabilone.

Evaluation of AM108434 (77)

Compound 77 showed good in vitro profile with high binding affinities to the CB receptors, potent agonist at the CB1 receptor and metabolic stability of <8 minutes in mouse plasma and microsomes.

The in vivo potency of the compound 77 was assessed in the CB1 receptor characteristic analgesia assay (Figure 3.5) in mice. Antinociception was measured using a tail-flick procedure over a 6 hour period after drug injection. Both, AM10834 and AM10806 did not produce significant analgesia at a dose of 3 mg/kg and 1 mg/kg respectively. AM10806 at a dose of 10 mg/kg, had an onset to action within 60-180 minutes after injection while at a similar dose, 167

AM10834 had a faster onset to action producing peak effects within 20-60 minutes. Both the compounds showed similar offset profiles at 6 hours. This suggests that even though AM10834 has faster onset than AM10806, both compounds have a similar duration of action.

100

%MPE 40

AM10834 3mg/kg AM10834 10mg/kg 20 AM10806 1mg/kg AM10806 10mg/kg

0 0 20 60 180 360

Time (min)

Figure 3.5:.Analgesia test of nabilone (AM10806) and AM10834 in mice.

168

CONCLUSIONS

The main goal of this project was to explore the pharmacophoric space at the C1 position of nabilone. The goal was achieved with the generation of ether and ester substituted analogs that resulted from the modification of the phenol. Ester analogs maintained high affinity at the cannabinoid receptors as compared to the etherified analog 60. Among the compounds synthesized, acetonides (65 & 66), 1,2-diols (77 & 78) and oxygen containing carbocyclic (68,

69, 70 & 73) derivatives displayed high affinity to the CB1 receptor. The in vitro data of the analogs tested showed full agonist activity at the cannabinoid receptors. Metabolic stability data showed that compounds 68 & 79 have higher plasma stability as compared to 61 & 77. As these molecules displayed characteristic CB1 agonist profile in vitro, we tested these analogs in rodents. AM10846 showed potent hypothermic effects in rats and studies illustrate that the duration of action can be significantly extended as compared to nabilone (AM10806). Analgesic assessment of AM10834 displayed faster onset but a similar duration of action as nabilone.

Overall our studies show that there exists a unique pharmacophoric space at the C1 position of nabilone where the ester functionality is best suited for cannabinoid receptor activity.

169

EXPERIMENTAL SECTION

Shield 400 WB plus (1H 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 relative to base

(Waters-2424) using a XTerra MS C18, 5 µm, 4.6 mm x 50 mm column and acetonitrile/water] and were > 95%.

Standard Procedure for the Steglich esterification:

170

To a stirred solution of 4-Dimethylaminopyridine (DMAP) (6 equivalence) and N-(3-

Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) (4 equivalence) in dry

CH2Cl2 at 0°C, carboxylic acid (4 equivalence) was added. The reaction was stirred for 20 min at

0°C and then nabilone (59) (1 equivalence) was added under argon atmosphere. The mixture was warmed to room temperature and stirred for 24 h to ensure complete formation of the product.

The reaction mixture was diluted with diethyl ether and washed sequentially with 5% HCl, saturated aqueous NaHCO3, and brine. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-1-(pentyloxy)-6,6a,7,8,10,10a-hexahydro-

9H-benzo[c]chromen-9-one (60).. To a stirred solution of 59 (25 mg, 0.06 mmol) in dry acetone

(0.4 ml) at 45 °C, dry potassium carbonate ( 25 mg, 0.18 mmol) and bromopentane (12 mg, 0.07 mmol) was added under argon atmosphere. The reaction mixture was refluxed for 15 h. Upon completion, the reaction mixture was diluted with acetone and then filtered. The filtrate was then concentrated under vacuum and diethyl ether was added to the residue and then washed with saturated ammonium chloride. The organic layer was concentrated under vacuum and the residue was chromatographed on silica gel (30% ethyl acetate in hexane) to give 60 as a colorless oil (14 mg, 50% yield). IR (neat): 2925, 1712, 1611, 1567, 1414, 1235, 1111, 1064 cm−1. 1H NMR (500

MHz, CDCl3) δ 6.41 (d, J = 1.5 Hz, 1H, ArH), 6.35 (d, J = 1.4 Hz, 1H, ArH), 3.36 (m, 2H, 2’’-

H), 3.80 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.82 (m as td, J = 12.4 Hz, J =

3.4 Hz, 1H, 10a-H), 2.61-2.54 (m, 1H, 8eq-H), 2.46-2.36 (m, 1H, 8ax-H), 2.17-2.05 (m, 2H,

10ax-H, 7eq-H), 1.98-1.90 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.82 (p, J = 7.5 Hz, 2H, 3’’-

H), 1.55-1.36 (m, 10H, 7ax-H, 2’-H, 6-Me, 4’’-H, 5’’-H especially 1.46, s, 6-Me), 1.24-1.16 (m,

12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.23, s, -C(CH3)2-), 1.11 (s, 3H, 6-Me), 1.07

171

(sextet, J = 7.5 Hz, 2H, 6’-H), 0.95 (t, J = 7.5 Hz, 3H, 7’-H), 0.84 (t, J = 7.0 Hz, 3H, 7’-H). 13C

NMR (100 MHz, CDCl3) δ 209.6 (>C=O), 157.6 (ArC-1 or ArC-5), 153.9 (ArC-5 or ArC-1),

150.4 (tertiary aromatic), 109.5 (tertiary aromatic), 107.9 (ArC-2 or ArC-4), 101.5 (ArC-4 or

ArC-2), 68.0, 47.6, 45.9, 44.4, 40.7, 37.7, 34.6, 31.7, 30.0, 29.0, 28.7, 28.3, 27.8, 26.6, 24.6,

22.6, 22.4, 18.8, 14.0. Mass spectrum (ESI) m/z (relative intensity) 443 (M+ + H, 100). Exact

+ mass (ESI) calculated for C29H47O3 (M + H), 443.3525; found 443.3517. LC/MS analysis

(Waters MicroMass ZQ system) showed purity of 99.4% and retention time of 6.8 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl acetate (61). To a stirred solution of 59 (50 mg, 0.13 mmol) in dry

CH2Cl2 at 0 °C, DMAP (25 mg, 0.20 mmol) was added under argon atmosphere. To that mixture, acetic anhydride (20.5 mg, 0.20 mmol) was added and stirred for 30 min. Upon completion, the reaction mixture was diluted using diethyl ether and quenched using water. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (20% AcOEt in hexane) to give 61 as a colorless oil

(44 mg, 80% yield). IR (neat): 2930, 1712, 1623, 1623, 1563, 1459, 1258, 1198, 1133 cm−1. 1H

NMR (500 MHz, CDCl3) δ 6.70 (d, J = 1.5 Hz, 1H, ArH), 6.51 (d, J = 1.4 Hz, 1H, ArH), 3.27

(ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.72 (m as td, J = 12.4 Hz, J = 3.4 Hz,

1H, 10a-H), 2.57-2.53 (m, 1H, 8eq-H), 2.44-2.37 (m, 1H, 8ax-H), 2.32 (s, 3H, O-CH3), 2.25-2.13

(m, 2H, 10ax-H, 7eq-H), 2.10-1.97 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.51-1.46 (m, 6H,

7ax-H, 2’-H, 6-Me, especially 1.48, s, 6-Me), 1.24-1.15 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J

13 = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.5 (>C=O), 168.8 (-C(O)-O), 153.9

172

(ArC-1 or ArC-5), 150.9 (ArC-5 or ArC-1), 149.0 (tertiary aromatic), 113.6 (tertiary aromatic),

113.0 (ArC-2 or ArC-4), 112.3 (ArC4 or ArC-2), 47.4, 45.7, 44.2, 40.5, 37.4, 34.8, 31.6, 29.8,

28.4, 27.6, 26.6, 24.4, 22.5, 21.1, 18.8, 13.9. Mass spectrum (ESI) m/z (relative intensity) 415

+ + (M + H, 100). Exact mass (ESI) calculated for C26H39O4 (M + H), 415.2848; found 415.2841.

LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.5% and retention time of

5.8 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl pentanoate (62a). The reaction was performed using the standard procedure reported above using DMAP (37 mg, 0.30 mmol), EDCI (38 mg, 0.20 mmol), dry

CH2Cl2 (0.8 ml), valeric acid (16 mg, 0.15 mmol) and 59 (20 mg, 0.13 mmol). The residue was chromatographed on silica gel (20% AcOEt in hexane) to give 62a as a colorless oil (20 mg,

70% yield). IR (neat): 2929, 1757, 1713, 1623, 1563, 1413, 1257, 1120, 1032 cm−1. 1H NMR

(500 MHz, CDCl3) δ 6.69 (d, J = 1.5 Hz, 1H, ArH), 6.49 (d, J = 1.4 Hz, 1H, ArH), 3.27 (ddd, J =

15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H),

2.61-2.53 (m, 3H, 8eq-H, 3’’-H), 2.45-2.36 (m, 1H, 8ax-H), 2.25-2.11 (m, 2H, 10ax-H, 7eq-H),

1.99-1.92 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.78-1.70 (m, 2H, 4’’-H), 1.53-1.41 (m, 8H,

7ax-H, 2’-H, 6-Me, 5’’-H especially 1.48, s, 6-Me), 1.26-1.16 (m, 12H, 3’-H, 4’-H, 5’-H, -

C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H),

13 0.97 (t, J = 7.0 Hz, 3H, 6’’-H), 0.84 (t, J = 7.0 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ

209.5 (>C=O), 171.7 (-C(O)-O-), 154.0 (ArC-1 or ArC-5), 151.2 (ArC-5 or ArC-1), 149.2

(tertiary aromatic), 113.8 (tertiary aromatic), 113.0 (ArC-2 or ArC-4), 112.4 (ArC-4 or ArC-2),

48.9, 47.5, 45.9, 44.3, 40.6, 37.5, 34.9, 34.2, 31.7, 29.9, 28.5, 27.7, 26.8, 26.7, 24.5, 22.6, 22.3,

18.9, 14.0, 13.7. Mass spectrum (ESI) m/z (relative intensity) 457 (M+ + H, 100). Exact mass

173

+ (ESI) calculated for C29H45O4 (M + H), 457.3318; found 457.3306. LC/MS analysis (Waters

MicroMass ZQ system) showed purity of 98.9% and retention time of 6.3 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl heptanoate (62b). The reaction was performed using the standard procedure reported above using DMAP (37 mg, 0.30 mmol), EDCI (38 mg, 0.20 mmol), dry

CH2Cl2 (0.8 ml), heptanoic acid (16 mg, 0.15 mmol) and 59 (20 mg, 0.13 mmol). The residue was chromatographed on silica gel (20% AcOEt in hexane) to give 62b as a colorless oil (20 mg,

70% yield). IR (neat): 2929, 1757, 1714, 1623, 1563, 1413, 1257, 1197, 1032 cm−1. 1H NMR

(500 MHz, CDCl3) δ 6.69 (d, J = 1.5 Hz, 1H, ArH), 6.49 (d, J = 1.4 Hz, 1H, ArH), 3.27 (ddd, J =

15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.71 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H),

2.61-2.53 (m, 3H, 8eq-H, 3’’-H), 2.45-2.36 (m, 1H, 8ax-H), 2.26-2.11 (m, 2H, 10ax-H, 7eq-H),

1.99-1.92 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.78-1.71 (m, 2H, 4’’-H), 1.53-1.38 (m, 8H,

7ax-H, 2’-H, 6-Me, 5’’-H especially 1.48, s, 6-Me), 1.36-1.31 (m, 4H, 6’’-H, 7’’-H), 1.26-1.16

(m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s, 3H, 6-Me), 1.07

(sextet, J = 7.5 Hz, 2H, 6’-H), 0.90 (t, J = 7.0 Hz, 3H, 8’’-H), 0.84 (t, J = 7.0 Hz, 3H, 7’-H). 13C

NMR (100 MHz, CDCl3) δ 209.5 (>C=O), 171.8 (-C(O)-O-), 154.0 (ArC-1 or ArC-5), 150.9

(ArC-5 or ArC-1), 149.2 (tertiary aromatic), 113.8 (tertiary aromatic), 113.0 (ArC-2 or ArC-4),

112.4 (ArC-4 or ArC-2), 50.8, 47.5, 45.9, 44.3, 40.6, 37.5, 34.9, 34.5, 31.7, 31.4, 29.9, 28.8,

28.5, 27.7, 26.7, 24.7, 24.5, 22.6, 22.5, 19.7, 18.9, 14.0, 13.6. LC/MS analysis (Waters

MicroMass ZQ system) showed purity of 99.2% and retention time of 6.6 min for the title compound.

174

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 5-bromopentanoate (63). The reaction was performed using the standard procedure reported above using DMAP (37 mg, 0.30 mmol), EDCI (38 mg, 0.20 mmol), dry CH2Cl2 (0.8 ml), 5-Bromovaleric acid (28 mg, 0.15 mmol) and 59 (25 mg, 0.05 mmol). The residue was chromatographed on silica gel (20% AcOEt in hexane) to give 63 as a colorless oil (27 mg, 85% yield). IR (neat): 2928, 1757, 1712, 1623, 1562, 1413, 1256, 1137,

−1 1 1031 cm . H NMR (500 MHz, CDCl3) δ 6.70 (d, J = 1.5 Hz, 1H, ArH), 6.50 (d, J = 1.4 Hz, 1H,

ArH), 3.47 (t, J = 7.0 Hz, 2H, 6’’-H), 3.23 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-

H), 2.69 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.64 (td, J = 1.5 Hz, J = 7.5 Hz, 2H, 3’’-

H), 2.59-2.53 (m, 1H, 8eq-H), 2.46-2.37 (m, 1H, 8ax-H), 2.26-2.11 (m, 2H, 10ax-H, 7eq-H),

2.01-1.88 (m, 5H, 6a-H, 4’’-H, 5’’-H), 1.51-1.47 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.48, s,

6-Me), 1.25-1.16 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s,

3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J = 7.0 Hz, 3H, 7’-H). 13C NMR (100

MHz, CDCl3) δ 209.5 (>C=O), 171.0 (-C(O)-O-), 154.0 (ArC-1 or ArC-5), 151.0 (ArC-5 or

ArC-1), 149.1 (tertiary aromatic), 113.6 (tertiary aromatic), 113.1 (ArC-2 or ArC-4), 112.3 (ArC-

4 or ArC-2), 47.5, 45.9, 44.3, 40.6, 37.5, 35.0, 33.4, 32.9, 32.0, 31.7, 29.9, 29.6, 28.5, 27.7, 26.7,

24.5, 23.3, 22.6, 18.9, 15.2, 14.0. Mass spectrum (ESI) m/z (relative intensity) 535 (M+ + H,

+ 100). Exact mass (ESI) calculated for C29H44O4Br (M + H), 535.2423; found 535.2426. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99% and retention time of 6.2 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 5-azidopentanoate (64). To a stirred solution of 63 (10 mg, 0.02 mmol) in anhydrous CH2Cl2/CH3NO2 (3:1 mixture, 0.4 mL) at room temperature, tetrabutylammonium

175 azide (53 mg, 0.2 mmol) was added. The reaction was stirred at same temperature for 1 h. The reaction mixture was quenched by brine and extraction was done using Et2O. The organic layer was concentrated under vacuum and the residue was chromatographed on silica gel (40% ethyl acetate in hexane) to give 64 as a colorless oil (4 mg, 60% yield). IR (neat): 2929, 2095, 1758,

−1 1 1712, 1623, 1563, 1413, 1256, 1197, 1031 cm . H NMR (500 MHz, CDCl3) δ 6.70 (d, J = 1.5

Hz, 1H, ArH), 6.50 (d, J = 1.4 Hz, 1H, ArH), 3.36 (t, J = 7.0 Hz, 2H, 6’’-H), 3.23 (ddd, J = 15.0

Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.69 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.64

(td, J = 1.5 Hz, J = 7.5 Hz, 2H, 3’’-H), 2.59-2.53 (m, 1H, 8eq-H), 2.46-2.37 (m, 1H, 8ax-H),

2.26-2.11 (m, 2H, 10ax-H, 7eq-H), 2.01-1.88 (m, 5H, 6a-H, 4’’-H, 5’’-H), 1.51-1.47 (m, 6H,

7ax-H, 2’-H, 6-Me, especially 1.48, s, 6-Me), 1.25-1.16 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J

13 = 7.0 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.6 (>C=O), 171.0 (-C(O)-O-), 154.0

(ArC-1 or ArC-5), 151.0 (ArC-5 or ArC-1), 149.1 (tertiary aromatic), 113.6 (tertiary aromatic),

113.1 (ArC-2 or ArC-4), 112.3 (ArC-4 or ArC-2), 47.5, 45.9, 44.3, 40.6, 37.5, 35.0, 33.4, 32.9,

32.0, 31.7, 29.9, 29.6, 28.5, 27.7, 26.7, 24.5, 23.3, 22.6, 18.9, 15.2, 14.0. Mass spectrum (ESI)

+ + m/z (relative intensity) 498 (M + H, 100). Exact mass (ESI) calculated for C29H44N3O4 (M +

H), 498.3332; found 498.3331. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.7% and retention time of 6.2 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl (R)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (65). The reaction was performed using the standard procedure reported above using DMAP (100 mg, 0.8 mmol), EDCI

(108 mg, 0.56 mmol), dry CH2Cl2 (2 ml), (R)-2,2-Dimethyl-1,3-dioxolane-4-carboxylic acid (60 mg, 0.40 mmol) and 59 (50 mg, 0.14 mmol). The residue was chromatographed on silica gel

176

(30% AcOEt in hexane) to give 65 as a colorless oil (61 mg, 91% yield). IR (neat): 2930, 1779,

−1 1 1712, 1623, 1563, 1413, 1257, 1155, 1068 cm . H NMR (500 MHz, CDCl3) δ 6.72 (d, J = 1.5

Hz, 1H, ArH), 6.50 (d, J = 1.4 Hz, 1H, ArH), 4.86 (dd, J = 5.5 Hz, J = 7.0 Hz, 1H, 3’’-H), 4.21

(dd, J = 5.5 Hz, J = 7.0 Hz, 1H, 4’’-H), 4.34 (dd, J = 5.5 Hz, J = 7.0 Hz, 1H, 4’’-H), 3.23 (ddd, J

= 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.74 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-

H), 2.59-2.53 (m, 1H, 8eq-H), 2.44-2.35 (m, 1H, 8ax-H), 2.25-2.10 (m, 2H, 10ax-H, 7eq-H),

2.00-1.91 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.51-1.45 (m, 12H, 7ax-H, 2’-H, 6-Me, 5’’-H especially 1.48, s, 6-Me, and 1.46, s, 5’’-H), 1.23-1.16 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J

13 = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.2 (>C=O), 169.1 (-C(O)-O-), 154.1

(ArC-1 or ArC-5), 151.1 (ArC-5 or ArC-1), 148.7 (tertiary aromatic), 113.8 (tertiary aromatic),

113.5 (ArC-2 or ArC-4), 111.9 (ArC-4 or ArC-2), 111.8 (O-C-O), 74.1 (-CH-O-), 67.1 (CH2-O-

), 58.4, 47.5, 45.9, 44.3, 40.5, 37.5, 34.6, 31.7, 29.9, 28.5, 27.6, 26.4, 25.9, 25.4, 24.5, 22.6, 18.8,

14.0. Mass spectrum (ESI) m/z (relative intensity) 501 (M+ + H, 100). Exact mass (ESI)

+ calculated for C30H45O6 (M + H), 501.3216; found 501.3206. LC/MS analysis (Waters

MicroMass ZQ system) showed purity of 99% and retention time of 6.0 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (66). The reaction was performed using the standard procedure reported above using DMAP (148 mg, 1.20 mmol),

EDCI (158 mg, 0.80 mmol), dry CH2Cl2 (3 ml), (S)-2,2-Dimethyl-1,3-dioxolane-4-carboxylic acid (88 mg, 0.60 mmol) and 59 (75 mg, 0.20 mmol). The residue was chromatographed on silica gel (30% AcOEt in hexane) to give 66 as a colorless oil (100 mg, 85% yield). LC/MS

177 analysis (Waters MicroMass ZQ system) showed purity of 98.5% and retention time of 6.0 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 2,2,5-trimethyl-1,3-dioxane-5-carboxylate (67). The reaction was performed using the standard procedure reported above using DMAP (98 mg, 0.80 mmol), EDCI

(103 mg, 0.53 mmol), dry CH2Cl2 (2 ml), 2,2,5-Trimethyl-1,3-dioxane-5-carboxylic acid (70 mg,

0.40 mmol) and 59 (50 mg, 0.13 mmol). The residue was chromatographed on silica gel (20%

AcOEt in hexane) to give 67 as a colorless oil (70 mg, 80% yield). IR (neat): 2930, 1750, 1712,

−1 1 1623, 1562, 1413, 1271, 1197, 1100 cm . H NMR (500 MHz, CDCl3) δ 6.73 (d, J = 1.5 Hz,

1H, ArH), 6.49 (d, J = 1.4 Hz, 1H, ArH), 4.37-4.33 (m as dd, J = 12.0 Hz, J = 7.0 Hz, 2H, -CH2-

O-), 3.83-3.77 (m as dd, J = 12.0 Hz, J = 7.0 Hz, 2H, -CH2-O-) 3.28 (ddd, J = 15.0 Hz, J = 3.5

Hz, J = 2.0 Hz, 1H, 10eq-H), 2.76 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.60-2.54 (m,

1H, 8eq-H), 2.45-2.38 (m, 1H, 8ax-H), 2.28-2.13 (m, 2H, 10ax-H, 7eq-H), 2.02-1.94 (m as td, J

= 12 Hz, J = 3 Hz, 1H, 6a-H), 1.56-1.49 (m, 9H, 7ax-H, 2’-H, -O-C(CH3)2-O-, especially 1.50, s,

-O-C(CH3)2-O-), 1.38 (s, 3H, C(CH3)), 1.46 (s, 3H, 6-Me), 1.24-1.15 (m, 12H, 3’-H, 4’-H, 5’-

H, -C(CH3)2-, especially 1.26, s, -C(CH3)2-), 1.15 (s, 3H, 6-Me), 1.08 (sextet, J = 7.5 Hz, 2H, 6’-

13 H), 0.86 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.3 (>C=O), 172.6 (-C(O)-

O-), 154.0 (ArC-1 or ArC-5), 151.0 (ArC-5 or ArC-1), 149.3 (tertiary aromatic), 114.2 (tertiary aromatic), 113.2 (ArC-2 or ArC-4), 112.2 (ArC-4 or ArC-2), 98.2 (O-C-O), 66.2 (-CH-O-), 66.1

(CH-O-), 47.7, 46.2, 44.3, 42.4, 40.5, 37.5, 34.7, 31.7, 29.9, 28.6, 28.4, 27.6, 26.6, 25.5, 24.5,

22.6, 22.0, 18.7, 18.2, 14.0. Mass spectrum (ESI) m/z (relative intensity) 529 (M+ + H, 100).

+ Exact mass (ESI) calculated for C32H49O6 (M + H), 529.3529; found 529.3524. LC/MS analysis

178

(Waters MicroMass ZQ system) showed purity of 99.2% and retention time of 6.1 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl tetrahydrofuran-2-carboxylate (68). The reaction was performed using the standard procedure reported above using DMAP (37 mg, 0.30 mmol), EDCI (38 mg, 0.20 mmol), dry CH2Cl2 (0.8 ml), tetrahydro-2-furoic acid (18 mg, 0.15 mmol) and 59 (25 mg, 0.05 mmol). The residue was chromatographed on silica gel (20% AcOEt in hexane) to give 68 as a colorless oil (25 mg, 80% yield).LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.7% and retention time of 5.9 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl (R)-tetrahydrofuran-2-carboxylate (69). The reaction was performed using the standard procedure reported above using DMAP (37 mg, 0.30 mmol), EDCI (38 mg,

0.20 mmol), dry CH2Cl2 (0.8 ml), tetrahydro-2-furoic acid (18 mg, 0.15 mmol) and 59 (25 mg,

0.05 mmol). The residue was chromatographed on silica gel (20% AcOEt in hexane) to give 69 as a colorless oil (22 mg, 78% yield).LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.2% and retention time of 5.9 min for the title compound. IR (neat): 2928, 1754,

−1 1 1712, 1623, 1562, 1413, 1256, 1183, 1030 cm . H NMR (500 MHz, CDCl3) δ 6.71 (d, J = 1.5

Hz, 1H, ArH), 6.52 (d, J = 1.4 Hz, 1H, ArH), 4.20-4.15 (dd, J = 2.5 Hz, J = 9.0 Hz, 1H, furan ring), 4.16-4.10 (m, 1H, furan ring), 4.04-3.98 (m, 1H, furan ring), 3.29 (ddd, J = 15.0 Hz, J =

3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.73 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.58-2.52

(m, 1H, 8eq-H), 2.46-2.36 (m, 1H, 8ax-H), 2.27-1.93 (m, 7H, 10ax-H, 7eq-H, 6a-H, furan ring),

1.52-1.47 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.48, s, 6-Me), 1.25-1.15 (m, 12H, 3’-H, 4’-H,

5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s, 3H, 6-Me), 1.05 (sextet, J = 7.5 Hz, 2H,

179

13 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.5 (>C=O), 171.3 (-

C(O)-O-), 154.0 (ArC-1 or ArC-5), 151.0 (ArC-5 or ArC-1), 148.9 (tertiary aromatic), 113.6

(tertiary aromatic), 113.2 (ArC-2 or ArC-4), 112.4 (ArC-4 or ArC-2), 69.6, 47.6, 45.9, 44.3,

40.7, 37.5, 34.9, 31.7, 30.1, 29.9, 28.5, 27.6, 26.7, 25.6, 24.5, 22.6, 18.8, 14.0. Mass spectrum

+ + (ESI) m/z (relative intensity) 471 (M + H, 100). Exact mass (ESI) calculated for C29H43O5 (M

+ H), 471.3110; found 471.3102. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.0% and retention time of 5.9 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl (S)-tetrahydrofuran-2-carboxylate (70). The reaction was performed using the standard procedure reported above using DMAP (37 mg, 0.30 mmol), EDCI (38 mg,

0.20 mmol), dry CH2Cl2 (0.8 ml), tetrahydro-2-furoic acid (18 mg, 0.15 mmol) and 59 (25 mg,

0.05 mmol). The residue was chromatographed on silica gel (20% AcOEt in hexane) to give 70 as a colorless oil (23 mg, 79% yield). LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.9% and retention time of 5.9 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl cyclopentanecarboxylate (71). The reaction was performed using the standard procedure reported above using DMAP (37 mg, 0.30 mmol), EDCI (38 mg, 0.20 mmol), dry CH2Cl2 (0.8 ml), cyclopentane acid (17 mg, 0.15 mmol) and 59 (25 mg, 0.05 mmol).

The residue was chromatographed on silica gel (20% AcOEt in hexane) to give 71 as a colorless oil (25 mg, 78% yield). IR (neat): 2929, 1752, 1713, 1622, 1563, 1413, 1257, 1182, 1086 cm−1.

1 H NMR (500 MHz, CDCl3) δ 6.69 (d, J = 1.5 Hz, 1H, ArH), 6.48 (d, J = 1.4 Hz, 1H, ArH), 3.33

(ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.99 (q, J = 8.0 Hz, 1H, cyclopentane ring) 2.70 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.58-2.52 (m, 1H, 8eq-H), 2.45-2.36

180

(m, 1H, 8ax-H), 2.25-2.03 (m, 4H, 10ax-H, 7eq-H, cyclopentane ring), 1.99-1.85 (m, 3H, 6a-H, cyclopentane ring), 1.83-1.75 (m, 2H, cyclopentane ring), 1.71-1.62 (m, 2H, cyclopentane ring),

1.53-1.46 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.48, s, 6-Me), 1.24-1.15 (m, 12H, 3’-H, 4’-H,

5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2- ), 1.12 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H,

13 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.5 (>C=O), 174.7 (-

C(O)-O-), 154.0 (ArC-1 or ArC-5), 150.9 (ArC-5 or ArC-1), 149.4 (tertiary aromatic), 113.9

(tertiary aromatic), 112.9 (ArC-2 or ArC-4), 112.3 (ArC-4 or ArC-2), 47.6, 45.9, 44.3, 44.0,

40.7, 37.5, 34.9, 31.7, 30.1, 30.0, 29.9, 28.5, 27.6, 26.7, 25.9, 25.8, 24.5, 22.6, 18.8, 14.0. Mass spectrum (ESI) m/z (relative intensity) 469 (M+ + H, 100). Exact mass (ESI) calculated for

+ C30H45O4 (M + H), 469.3318; found 469.3310. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.4% and retention time of 6.4 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 1-methoxycyclopentane-1-carboxylate (72). The reaction was performed using the standard procedure reported above using DMAP (41 mg, 0.34 mmol), EDCI

(43 mg, 0.28 mmol), dry CH2Cl2 (1 ml), 1-methoxycyclopentane-1-carboxylic acid (41 mg, 0.28 mmol) and 59 (25 mg, 0.05 mmol). The residue was chromatographed on silica gel (30% AcOEt in hexane) to give 72 as a colorless oil (20 mg, 60% yield). IR (neat): 2929, 1749, 1712, 1622,

1561, 1412, 1277, 1199, 1026 cm−1. 1H NMR (500 MHz, CDCl3) δ 6.71 (d, J = 1.5 Hz, 1H,

ArH), 6.47 (d, J = 1.4 Hz, 1H, ArH), 3.37 (s, 3H, O-CH3), 3.33 (ddd, J = 15.0 Hz, J = 3.5 Hz, J =

2.0 Hz, 1H, 10eq-H), 2.71 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.57-2.51 (m, 1H, 8eq-

H), 2.44-2.36 (m, 1H, 8ax-H), 2.24-2.07 (m, 6H, 10ax-H, 7eq-H, cyclopentane ring), 2.00-1.94

(m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.88-1.82 (m, 4H, cyclohexane ring), 1.52-1.47 (m, 6H,

7ax-H, 2’-H, 6-Me, especially 1.48, s, 6-Me), 1.25-1.16 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-,

181 especially 1.22, s, -C(CH3)2-), 1.13 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J

13 = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.2 (>C=O), 172.7 (-C(O)-O-), 154.1

(ArC-1 or ArC-5), 151.0 (ArC-5 or ArC-1), 149.3 (tertiary aromatic), 114.0 (tertiary aromatic),

113.1 (ArC-2 or ArC-4), 112.0 (ArC-4 or ArC-2), 88.6, 52.9, 47.7, 45.9, 44.3, 40.7, 37.5, 36.0,

35.0, 31.7, 29.9, 29.7, 28.6, 28.5, 27.6, 26.8, 24.5, 24.1, 24.0, 22.6, 18.8, 14.0. Mass spectrum

+ + (ESI) m/z (relative intensity) 499 (M + H, 100). Exact mass (ESI) calculated for C31H47O5 (M

+ H), 499.3423; found 499.3421. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.5% and retention time of 6.2 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 3-methoxytetrahydrofuran-3-carboxylate (73). The reaction was performed using the standard procedure reported above using DMAP (41 mg, 0.34 mmol), EDCI

(43 mg, 0.28 mmol), dry CH2Cl2 (1 ml), 3-methoxytetrahydrofuran-3-carboxylic acid (42 mg,

0.28 mmol) and 59 (25 mg, 0.05 mmol). The residue was chromatographed on silica gel (30%

AcOEt in hexane) to give 73 as a colorless oil (18 mg, 58% yield). IR (neat): 2929, 1750, 1713,

−1 1 1622, 1562, 1413, 1227, 1137, 1030 cm . H NMR (500 MHz, CDCl3) δ 6.73 (d, J = 1.5 Hz,

1H, ArH), 6.48 (d, J = 1.4 Hz, 1H, ArH), 4.20-4.15 (m, 2H, furan ring), 4.11-4.05 (m, 2H, furan ring), 3.46 (s, 3H, O-CH3), 3.27 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.69 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.60-2.38 (m, 4H, 8eq-H, 8ax-H, furan ring), 2.27-

2.14 (m, 4H, 10ax-H, 7eq-H), 2.01-1.94 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.54-1.48 (m,

6H, 7ax-H, 2’-H, 6-Me, especially 1.49, s, 6-Me), 1.25-1.17 (m, 12H, 3’-H, 4’-H, 5’-H, -

C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.13 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H),

13 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.1 (>C=O), 170.3 (-C(O)-O-),

154.2 (ArC-1 or ArC-5), 151.1 (ArC-5 or ArC-1), 149.0 (tertiary aromatic), 113.8 (tertiary

182 aromatic), 113.5 (ArC-2 or ArC-4), 111.8 (ArC-4 or ArC-2), 88.0, 68.2, 53.7, 53.6, 47.7, 45.9,

44.3, 40.7, 37.5, 36.1, 35.0, 31.7, 29.9, 28.5, 28.5, 27.6, 26.8, 24.5, 22.6, 18.8, 14.0. Mass spectrum (ESI) m/z (relative intensity) 501 (M+ + H, 100). Exact mass (ESI) calculated for

+ C30H45O6 (M + H), 501.3216; found 501.3211. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.9% and retention time of 5.9 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 1-methoxycyclohexane-1-carboxylate (74). The reaction was performed using the standard procedure reported above using DMAP (50 mg, 0.40 mmol), EDCI (54 mg,

0.28 mmol), dry CH2Cl2 (1 ml), 1-methoxycyclohexanecarboxylic acid (45 mg, 0.28 mmol) and

59 (25 mg, 0.05 mmol). The residue was chromatographed on silica gel (30% AcOEt in hexane) to give 74 as a colorless oil (25 mg, 73% yield). IR (neat): 2930, 1748, 1713, 1622, 1562, 1412,

1261, 1120, 1007 cm−1. 1H NMR (500 MHz, CDCl3) δ 6.70 (d, J = 1.5 Hz, 1H, ArH), 6.44 (d, J

= 1.4 Hz, 1H, ArH), 3.37 (s, 3H, O-CH3), 3.34 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H,

10eq-H), 2.71 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.57-2.51 (m, 1H, 8eq-H), 2.44-

2.35 (m, 1H, 8ax-H), 2.24-2.07 (m, 4H, 10ax-H, 7eq-H, cyclohexane ring), 1.99-1.86 (m, 4H, 6a-

H, cyclohexane ring), 1.70-1.60 (m, 6H, cyclohexane ring), 1.52-1.47 (m, 6H, 7ax-H, 2’-H, 6-

Me, especially 1.48, s, 6-Me), 1.25-1.16 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s,

-C(CH3)2-), 1.13 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-

13 H). C NMR (100 MHz, CDCl3) δ 209.2 (>C=O), 172.6 (-C(O)-O-), 154.0 (ArC-1 or ArC-5),

150.9 (ArC-5 or ArC-1), 149.4 (tertiary aromatic), 114.1 (tertiary aromatic), 113.1 (ArC-2 or

ArC-4), 112.0 (ArC-4 or ArC-2), 79.5, 51.9, 47.7, 45.9, 44.2, 40.7, 37.5, 34.9, 32.0, 31.7, 31.3,

29.9, 28.6, 28.5, 27.6, 26.7, 25.2, 24.5, 22.6, 21.3, 21.2, 18.8, 14.0. Mass spectrum (ESI) m/z

+ + (relative intensity) 513 (M + H, 100). Exact mass (ESI) calculated for C32H49O5 (M + H),

183

513.3580; found 513.3577. LC/MS analysis (Waters MicroMass ZQ system) showed purity of

99.5% and retention time of 6.4 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 4-methoxytetrahydro-2H-pyran-4-carboxylate (75).. The reaction was performed using the standard procedure reported above using DMAP (50 mg, 0.40 mmol), EDCI

(54 mg, 0.28 mmol), dry CH2Cl2 (1 ml), 4-methoxyoxane-4-carboxylic acid (47 mg, 0.28 mmol) and 59 (25 mg, 0.05 mmol). The residue was chromatographed on silica gel (30% AcOEt in

1 hexane) to give 75 as a colorless oil (22 mg, 70% yield). H NMR (500 MHz, CDCl3) δ 6.72 (d,

J = 1.5 Hz, 1H, ArH), 6.44 (d, J = 1.4 Hz, 1H, ArH), 3.84-3.79 (m, 4H, pyran ring), 3.40 (s, 3H,

O-CH3), 3.30 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.69 (m as td, J = 12.4 Hz,

J = 3.4 Hz, 1H, 10a-H), 2.57-2.51 (m, 1H, 8eq-H), 2.45-2.36 (m, 1H, 8ax-H), 2.28-2.12 (m, 4H,

10ax-H, 7eq-H, pyran ring), 2.09-2.01 (m, 2H, pyran ring), 2.00-1.94 (m as td, J = 12 Hz, J = 3

Hz, 1H, 6a-H), 1.52-1.48 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.49, s, 6-Me), 1.26-1.17 (m,

12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.14 (s, 3H, 6-Me), 1.06

13 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ

209.1 (>C=O), 171.2 (-C(O)-O-), 154.1 (ArC-1 or ArC-5), 151.1 (ArC-5 or ArC-1), 149.2

(tertiary aromatic), 114.0 (tertiary aromatic), 113.4 (ArC-2 or ArC-4), 111.8 (ArC-4 or ArC-2),

63.3, 52.1, 47.7, 45.9, 44.2, 40.7, 37.5, 35.0, 32.3, 31.7, 31.4, 29.9, 28.6, 28.5, 27.6, 26.8, 24.5,

22.6, 18.8, 14.0. Mass spectrum (ESI) m/z (relative intensity) 515 (M+ + H, 100). Exact mass

+ (ESI) calculated for C31H47O6 (M + H), 515.3373; found 515.3372. LC/MS analysis (Waters

MicroMass ZQ system) showed purity of 99.1% and retention time of 5.9 min for the title compound.

184

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 3-(4-methoxyphenyl)propanoate (76) The reaction was performed using the standard procedure reported above using DMAP (37 mg, 0.30 mmol), EDCI (38 mg,

0.20 mmol), dry CH2Cl2 (0.8 ml), 3-(4-methoxyphenyl) propionic acid (27 mg, 0.15 mmol) and

59 (25 mg, 0.05 mmol). The residue was chromatographed on silica gel (20% AcOEt in hexane) to give 76 as a colorless oil (25 mg, 70% yield). IR (neat): 2929, 1757, 1711, 1623, 1563, 1413,

−1 1 1246, 1179, 1035 cm . H NMR (500 MHz, CDCl3) δ 7.20 (d, J = 2.5 Hz, 2H, Phenyl ring),

6.84 (d, J = 8.0 Hz, 2H, Phenyl ring), 6.67 (d, J = 1.5 Hz, 1H, ArH), 6.39 (d, J = 1.4 Hz, 1H,

ArH), 3.70 (s, 3H, OMe), 3.21 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.20-2.96

(m, 2H, -C(O)-CH2), 2.91-2.85 (m, 2H, -C(O)-CH2-CH2-), 2.58-2.44 (m, 2H, 10a-H, 8eq-H),

2.44-2.33 (m, 1H, 8ax-H), 2.21-2.07 (m, 2H, 10ax-H, 7eq-H), 1.94-1.86 (m as td, J = 12 Hz, J =

3 Hz, 1H, 6a-H), 1.52-1.44 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.49, s, 6-Me), 1.28-1.18 (m,

12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.20, s, -C(CH3)2-), 1.08 (s, 3H, 6-Me), 1.05

13 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ

209.3 (>C=O), 170.8 (-C(O)-O-), 158.0 (ArH, Phenyl ring), 153.8 (ArC-1 or ArC-5), 150.8

(ArC-5 or ArC-1), 149.0 (tertiary aromatic), 132.0 (ArH, Phenyl ring), 129.5 (ArH, Phenyl ring),

113.8 (tertiary aromatic), 113.6 (ArC-2 or ArC-4), 112.9 (ArH, Phenyl ring), 112.3 (ArC-4 or

ArC-2), 55.1, 47.3, 45.8, 44.2, 40.4, 37.4, 36.2, 34.5, 31.6, 29.9, 29.8, 28.4, 28.3, 27.5, 26.3,

24.4, 22.5, 18.7, 13.9. Mass spectrum (ESI) m/z (relative intensity) 535 (M+ + H, 100). Exact

+ mass (ESI) calculated for C34H47O5 (M + H), 535.3423; found 535.3419. LC/MS analysis

(Waters MicroMass ZQ system) showed purity of 99.2% and retention time of 6.2 min for the title compound.

185

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl (R)-2,3-dihydroxypropanoate (77). To a stirred solution of 66 (80 mg,

0.16 mmol) in CH2Cl2 (3.5 ml) at room temperature was added TFA (1 ml) under argon atmosphere. The reaction was stirred for 15 min to ensure complete formation of the product.

The reaction was quenched using saturated aqueous NaHCO3 and extracted using AcOEt. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (40% AcOEt in hexane) to give 77 as colorless oil (70 mg, 95% yield). IR (neat): 3384, 2955, 1761, 1703, 1622, 1563, 1414, 1267, 1183, 1111 cm−1.

1 H NMR (500 MHz, CDCl3) δ 6.73 (d, J = 1.5 Hz, 1H, ArH), 6.57 (d, J = 1.4 Hz, 1H, ArH), 4.53

(dd, J = 5.5 Hz, J = 7.0 Hz, 1H, -CH-OH), 4.14 (dd, J = 5.5 Hz, J = 7.0 Hz, 1H, -C(CH2)-OH),

4.08 (dd, J = 5.5 Hz, J = 7.0 Hz, 1H, -C(CH2)-OH), 3.29 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0

Hz, 1H, 10eq-H), 2.73 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.60-2.53 (m, 1H, 8eq-H),

2.47-2.40 (m, 1H, 8ax-H), 2.26-2.15 (m, 2H, 10ax-H, 7eq-H), 2.03-1.96 (m as td, J = 12 Hz, J =

3 Hz, 1H, 6a-H), 1.53-1.48 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.49, s, 6-Me), 1.23-1.16 (m,

12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s, 3H, 6-Me), 1.06

13 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ

212.1 (>C=O), 171.6 (-C(O)-O-), 154.2 (ArC-1 or ArC-5), 151.3 (ArC-5 or ArC-1), 148.9

(tertiary aromatic), 113.9 (tertiary aromatic), 113.7 (ArC-2 or ArC-4), 112.1 (ArC-4 or ArC-2),

72.2 (-CH-OH), 63.2 (CH2-O-), 48.0, 46.5, 44.3, 40.7, 37.6, 35.3, 31.7, 29.9, 28.5, 27.6, 27.1,

24.5, 22.6, 18.7, 14.0. Mass spectrum (ESI) m/z (relative intensity) 461 (M+ + H, 100). Exact

+ mass (ESI) calculated for C27H41O6 (M + H), 461.2903; found 461.2895. LC/MS analysis

(Waters MicroMass ZQ system) showed purity of 98.6% and retention time of 5.2 min for the title compound.

186

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl (S)-2,3-dihydroxypropanoate (78). To a stirred solution of 67 (15 mg,

0.03 mmol) in CH2Cl2 (0.5 ml) at room temperature was added TFA (0.1 ml). The reaction was stirred for 15 min to ensure complete formation of the product. The reaction was quenched using saturated aqueous NaHCO3 and extracted using AcOEt. The organic phase was dried over

MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (40% AcOEt in hexane) to give 78 as colorless oil (10 mg, 92% yield). LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.9% and retention time of 5.2 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate (79). To a stirred solution of 68 (15 mg, 0.03 mmol) in CH2Cl2 (0.5 ml) at room temperature was added TFA (0.1 ml). The reaction was stirred for 15 min to ensure complete formation of the product. The reaction was quenched using saturated aqueous NaHCO3 and extracted using AcOEt. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (40% AcOEt in hexane) to give 79 as colorless oil (10 mg, 90% yield). IR (neat): 3394, 2928, 1746, 1705, 1623, 1562, 1412, 1259, 1198, 1099 cm−1.

1 H NMR (500 MHz, CDCl3) δ 6.72 (d, J = 1.5 Hz, 1H, ArH), 6.46 (d, J = 1.4 Hz, 1H, ArH), 4.09

(d, J = 10.5 Hz, 1H, -CH2-O-), 4.00 (d, J = 11.0 Hz, 1H, -CH2-O-), 3.89-3.82 (m as dd, J = 11.0

Hz, J = 6.5 Hz, 2H, -CH2-O-) 3.32 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.75

(m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.58-2.52 (m, 1H, 8eq-H), 2.49-2.40 (m, 1H, 8ax-

H), 2.27-2.14 (m, 2H, 10ax-H, 7eq-H), 2.04-1.97 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.53-

1.46 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.48, s, 6-Me), 1.33 (s, 3H, C(CH3)), 1.24-1.15 (m,

187

12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s, 3H, 6-Me), 1.08

13 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ

211.7 (>C=O), 174.2 (-C(O)-O-), 154.0 (ArC-1 or ArC-5), 151.2 (ArC-5 or ArC-1), 149.1

(tertiary aromatic), 114.0 (tertiary aromatic), 113.3 (ArC-2 or ArC-4), 112.2 (ArC-4 or ArC-2),

67.4 (-CH-OH), 67.3 (CH-OH), 50.2, 48.0, 46.5, 44.3, 40.8, 37.5, 35.2, 31.7, 29.9, 29.7, 28.6,

28.5, 27.6, 27.1, 24.5, 22.6, 18.8, 17.3, 14.0. Mass spectrum (ESI) m/z (relative intensity) 489

+ + (M + H, 100). Exact mass (ESI) calculated for C29H45O6 (M + H), 489.3216; found 489.3208.

LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.4% and retention time of

5.3 min for the title compound.

13, 16 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. Results from the competition assays

17 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.18 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

188 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).

Plasma stability.16 Compounds or their proposed metabolites were diluted (200 µM) in mouse or rat plasma and incubated at 37 C, 100 rpm. At various time points, samples were taken, diluted 1:4 in acetonitrile and centrifuged to precipitate the proteins. The resulting supernatant was analyzed by HPLC. 4-Nitrophenyl butyrate was used as a control in each experiment. In vitro plasma half-lives were determined using exponential decay calculations in Prism

(GraphPad).

HPLC Analysis: Chromatographic separation was achieved using a Supelco Discovery C18 (4.6 x 250 mm) column on a Waters Alliance HPLC system. Mobile phase consisted of acetonitrile

(A) and a mixture of 60% water (acidified with 8.5% o-phosphoric acid) and 40% acetonitrile

189

Methods for characterization of in vivo effects in rodents.16, 19

Subjects. For hypothermia testing, female Sprague-Dawley rats (n = 6/group), weighing between

250 and 350 g (Charles River, Wilmington MA) were used. Rats were tested repeatedly with at least five days intervening between drug sessions. Experiments occurred at approximately the same time (10:00 am - 5:00 pm) during the light portion of the daily light/dark cycle. Outside of experimental sessions, rats were pair housed (2/cage) in a climate controlled vivarium with unrestricted access to food and water. For tail-flick withdrawal (analgesia) testing, male CD-1 mice (n = 6/group), weighting between 30 and 35 g (Charles River, Wilmington MA), were used.

Procedures. Temperature was recorded using a thermistor probe (Model 401, Measurement

Specialties, Inc., Dayton, OH) inserted into the rectum at a depth of 6 cm and secured to the tail with micropore tape. Rats were minimally restrained and isolated in 38x50x10cm plastic stalls.

Temperature was read to the nearest 0.01 oC using a Thermometer (Model 4000A, Measurement

Specialties, Inc.).

190 commercially available from VWR International® where the water temperature was set at 52 oC

Injections occurred immediately after the base-line recording and the remaining recordings took place 20, 60, 180, and 360 min post administration. Prior to this testing, the animals had been accustomed to the procedure for three consecutive “mock” sessions where the water was held at

38oC, i.e., average body temperature of mice; no tail-flicks are elicited by this water temperature.

191

REFERENCES

1. Bow, E. W.; Rimoldi, J. M. The Structure-Function Relationships of Classical

Cannabinoids: CB1/CB2 Modulation. Perspect Medicin Chem 2016, 8, 17-39.

2. Console-Bram, L.; Marcu, J.; Abood, M. E. Cannabinoid receptors: nomenclature and pharmacological principles. Prog Neuropsychopharmacol Biol Psychiatry 2012, 38, 4-15.

3. Andersson, D. A.; Gentry, C.; Alenmyr, L.; Killander, D.; Lewis, S. E.; Andersson, A.;

Bucher, B.; Galzi, J. L.; Sterner, O.; Bevan, S.; Hogestatt, E. D.; Zygmunt, P. M. TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Delta(9)- tetrahydrocannabiorcol. Nat Commun 2011, 2, 551.

4. Thomas, A.; Stevenson, L. A.; Wease, K. N.; Price, M. R.; Baillie, G.; Ross, R. A.;

Pertwee, R. G. Evidence that the plant cannabinoid Delta9-tetrahydrocannabivarin is a cannabinoid CB1 and CB2 receptor antagonist. Br J Pharmacol 2005, 146, 917-26.

5. Martin, B. R.; Jefferson, R.; Winckler, R.; Wiley, J. L.; Huffman, J. W.; Crocker, P. J.;

Saha, B.; Razdan, R. K. Manipulation of the tetrahydrocannabinol side chain delineates agonists, partial agonists, and antagonists. J Pharmacol Exp Ther 1999, 290, 1065-79.

6. Johnson M.R., M. L. S. The discovery of nonclassical cannabinoid analgetics. In:

Mechoulam R, ed. Cannabinoids as Therapeutic Agents. Boca Raton, FL. CRC Press 1986, 121-

145.

7. Svizenska, I.; Dubovy, P.; Sulcova, A. Cannabinoid receptors 1 and 2 (CB1 and CB2), their distribution, ligands and functional involvement in nervous system structures--a short review. Pharmacol Biochem Behav 2008, 90, 501-11.

192

8. Archer, R. A.; Blanchard, W. B.; Day, W. A.; Johnson, D. W.; Lavagnino, E. R.; Ryan,

C. W.; Baldwin, J. E. Cannabinoids. 3. Synthetic approaches to 9-ketocannabinoids. Total synthesis of nabilone. J Org Chem 1977, 42, 2277-84.

9. Huffman, J. W.; Liddle, J.; Yu, S.; Aung, M. M.; Abood, M. E.; Wiley, J. L.; Martin, B.

R. 3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC and related compounds: synthesis of selective ligands for the CB2 receptor. Bioorg Med Chem 1999, 7, 2905-14.

10. Gareau, Y.; Dufresne, C.; Gallant, M.; Rochette, C.; Sawyer, N.; Slipetz, D. M.;

Tremblay, N.; Weech, P. K.; Metters, K. M.; Labelle, M. Structure activity relationships of tetrahydrocannabinol analogues on human cannabinoid receptors. Bioorganic & Medicinal

Chemistry Letters 1996, 6, 189-194.

11. Pertwee, R. G.; Gibson, T. M.; Stevenson, L. A.; Ross, R. A.; Banner, W. K.; Saha, B.;

Razdan, R. K.; Martin, B. R. O-1057, a potent water-soluble cannabinoid receptor agonist with antinociceptive properties. Br J Pharmacol 2000, 129, 1577-84.

12. Nikas, S. P.; Thakur, G. A.; Parrish, D.; Alapafuja, S. O.; Huestis, M. A.; Makriyannis,

A. A concise methodology for the synthesis of (−)-Δ9-tetrahydrocannabinol and (−)-Δ9- tetrahydrocannabivarin metabolites and their regiospecifically deuterated analogs. Tetrahedron

2007, 63, 8112-8123.

13. Nikas, S. P.; Alapafuja, S. O.; Papanastasiou, I.; Paronis, C. A.; Shukla, V. G.;

Papahatjis, D. P.; Bowman, A. L.; Halikhedkar, A.; Han, X.; Makriyannis, A. Novel 1',1'-chain substituted hexahydrocannabinols: 9beta-hydroxy-3-(1-hexyl-cyclobut-1-yl)- hexahydrocannabinol (AM2389) a highly potent cannabinoid receptor 1 (CB1) agonist. J Med

Chem 2010, 53, 6996-7010.

193

14. Meshram, G. A.; Patil, V. D. Simple and Efficient Method for Acetylation of Alcohols,

Phenols, Amines, and Thiols Using Anhydrous NiCl2 Under Solvent-Free Conditions. Synthetic

Communications 2009, 39, 2516-2528.

15. Neises, B.; Steglich, W. Simple Method for the Esterification of Carboxylic Acids.

Angewandte Chemie International Edition in English 1978, 17, 522-524.

16. Nikas, S. P.; Sharma, R.; Paronis, C. A.; Kulkarni, S.; Thakur, G. A.; Hurst, D.; Wood, J.

T.; Gifford, R. S.; Rajarshi, G.; Liu, Y.; Raghav, J. G.; Guo, J. J.; Jarbe, T. U.; Reggio, P. H.;

Bergman, J.; Makriyannis, A. Probing the carboxyester side chain in controlled deactivation (-)- delta(8)-tetrahydrocannabinols. J Med Chem 2015, 58, 665-81.

17. Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.

Biochem Pharmacol 1973, 22, 3099-108.

18. Ogawa, G.; Tius, M. A.; Zhou, H.; Nikas, S. P.; Halikhedkar, A.; Mallipeddi, S.;

Makriyannis, A. 3'-functionalized adamantyl cannabinoid receptor probes. J Med Chem 2015,

58, 3104-16.

19. Paronis, C. A.; Nikas, S. P.; Shukla, V. G.; Makriyannis, A. Delta(9)-

Tetrahydrocannabinol acts as a partial agonist/antagonist in mice. Behav Pharmacol 2012, 23,

802-5.

194

CHAPTER 4: FLUORESCENT CANNABINERGIC PROBES

INTRODUCTION

To better understand the mechanism of GPCR signaling, structural biology tools such as crystallography, mutagenesis, and biophysical techniques have been used in the recent past.1 In order to enable deeper understanding at the molecular level, innovations in real-time observations of ligand−receptor and receptor−receptor interactions, both at the cellular level and and/or in vivo are needed.2

Analysis of structure and dynamics of proteins can be studied with the help of fluorescence techniques.3 This approach to observe receptors in living cells is chosen over isotope-labeled methods as it is more biocompatible, affordable and feasible. Fluorescent probes in the form of fluorescent-labeled ligands, antibodies, proteins, and amino acids, are being used in the fields of medicine, chemistry, biology, and genomics.4 These fluorescent-labeled ligands help in real-time observations of ligand−receptor interactions and also to visualize and detect the location of

GPCRs. Fluorescent ligands are also widely used to also evaluate major drug candidates.5

Fluorescence microscopes with nanoscale spatial resolution are used for single particle analysis which utilizes the fluorescent-ligands for detection.5 Presently, fluorescent ligands are made by conjugating an agonist or an antagonist of the GPCRs to a variety of fluorophores. This method has resulted in GPCR fluorescent ligands with selectivity and high affinity.6

To design a probe, the physicochemical properties and the pharmacological activities of the probe have to be considered.7 As fluorophores have a large mass, its conjugation to ligands of

195 pharmacophores that are targeted towards GPCRs might have affect the final product’s affinity and selectivity to the receptor. If these properties decrease, the fluorescent conjugate is of no use.

The hydrophobic nature of the fluorophors may cause non-specific binding and this can affect the physicochemical properties of the fluorescent conjugate.2 Hence, the choice of a fluorophore is based on retention of affinity of the ligand to the receptor and the position of attachment of the fluorophore to the ligand structure. This selection will help in minimizing the influence on receptor binding affinity.

Figure 4.1: Ligand based fluorescent probes for GPCR’s.2

Structurally, a classical fluorescent ligand is generally made up of three segments: the pharmacophore, the fluorophore, (which are both the essential components of the fluoroligand) and the linker, (between the pharmacophore and fluorophore) provides the adequate space to prevent the loss of pharmacological activity of the desired receptor (Figure 4.1).5 Generally, the linker is a carbon chain and ends with heteroatoms like nitrogen atoms. The terminal heteroatom groups are couple with the fluorophores or pharmacophores. Research suggests that the nonspecific binding of a fluoroligand would increase if there is high lipophilicity.2

196

Previous attempts to make fluorescent cannabinoids

Daly et al attempted to develop a fluorescent cannabinoid targeting the CB1 receptor. AM251, a well know CB1 antagonist (rCB1 Ki = 0.8 nM) was conjugated with tetra methyl rhodamine group to obtain T1117.8 This molecule showed complete loss of affinity to the CB1 receptor

(rCB1 Ki = 500 nM). Endocannabinoid based probes were developed by conjugating biotin to the head and tail groups of Anandamide and 2-AG. These compounds showed binding affinity of

(84.7-450 nM) to CB1 and CB2 receptors.9 Recently, biotinylated probes targeting the cannabinoid receptors were reported. Conjugation of HU210 with biotin showed equal affinity to both CB1 and CB2 receptors while conjugation of biotin to HU308 resulted in a selective CB2 probe.10

The first CB2 fluorescent probe was reported by Yates et al when they conjugated a fluorescent dye, NBD-F to a selective CB2 agonist, JWH-015 but this resulted in complete loss of affinity for the CB2 receptor.11 Since then there have been multiple reports of fluorescent CB2 ligands.

Mbc94 was developed by attaching CB2 inverse agonist, SR144528 (hCB2 Ki = 15 nM) with a near infrared dye, IR800 but this compound showed 17 fold reduced affinity to CB2 receptor.12

Fluorescent CB2 antagonist with a fluorophore incorporated in the 6-methoxy-N-alkyl isatin scaffold has also been reported.13 This compound was selective to hCB2 (Ki = 387 nM) and fluorescently labelled cells overexpressed with hCB2 receptor. Recently Ling et al reported a fluorescent imaging probe (NIR760-XLP6) that showed selectivity towards CB2 receptor.14 This probe bound to hCB2 with nanomolar affinity (Ki = 169.1 nM) and fluorescently labelled DBT-

CB2 cells. This compound also showed promise in mouse tumor models.

197

OBJECTIVE AND SPECIFIC AIMS

The objective of this project was to design and synthesize novel, high affinity fluorescently labelled cannabinergic ligands as pharmacological probes to study cellular actions of cannabinoids. Fluorescent cannabinoids were developed by conjugating a well-known CB1 agonist, Nabilone to a fluorophore (e.g. Coumarin, Rhodamine, etc) (Figure 4.2). To develop a

SAR at the C1 pharmacophore of nabilone, fluorescently labelled probes were achieved by incorporating a linker between the fluorophore and nabilone (Figure 4.2). While exploring the pharmacophoric space at C1 position of nabilone, the ester group was well tolerated while the ether functionality lacked affinity at the CB receptors. Hence, linkers containing the ester group at C1 position were considered. Novel fluorescent probes were evaluated by assessing their binding affinity for the rat CB1, mouse CB2, and human CB2. Fluorescence excitation and emission values were determined for analogs possessing high binding affinity. Lead analogs were further assessed for their functional activity using the cAMP biochemical assay followed by in vitro metabolic stability towards plasma esterases.

Figure 4.2: SAR studies of fluorescent cannabinoids.

198

CHEMISTRY

Scheme 15: Synthesis of fluorescent probes

Reagents and conditions: (a) R-COOH, EDCI, DMAP, CH2Cl2 or DMF, 0 ºC to rt, 24 h, 52-73%

(b) R-Cl, Et3N, CH2Cl2, rt, 20 h, 63%.

Fluorescently labelled cannabinergic ligands bearing an ester group in place of phenolic hydroxyl were synthesized as shown in Scheme 15. Biotinylated probe (80) was synthesized under Steglich esterification15 conditions by reacting biotin in the presence of EDCI, DMAP and

59 to yield 80 in 73% yield. Similarly, coumarin derivatives 81 & 82 were synthesized with 52

199 and 66% respectively. The polar Rhodamine B fluorophore was also esterified to afford 70% of

84. Dansylated analog (83) was obtained in 63% yield by treatment of dansyl chloride with Et3N and 59.

Scheme 16: Synthesis of linkers

Reagents and conditions: (a) CsCO3, CH3I, DMF, rt, 20 h, 90% (b) LDA, CH3I, THF, -78 ºC, 6 h, 70%, (c) MeOH, NaOH, 50 ºC, 3 h, 90%.

In order to explore the SAR of fluorescent cannabinoids at the C1 position of nabilone, linkers containing sterically hindered acids were synthesized (Scheme 16). These acids were esterified to form a linker as shown in Scheme 17. The labile ester group is prone to hydrolysis hence germinal dimethyl group was added alpha to the carbonyl group to prevent easy hydrolysis.

Starting from boc-protected amino acids, germinal dimethyl substituted linkers were synthesized in 3 steps as shown in Scheme 16. The 1st step involves treatment of the boc-protected amino acid (85 & 86) with CsCO3 and methyl iodide to afford methyl ester analogs 87 & 88 in 90% yield.16 Compounds 90 & 91 were obtained in 70% yield by deprotonation of intermediate 87 &

200

88 with LDA and germinal dimethylation using iodomethane. The final step involved hydrolysis of the methyl ester by sodium hydroxide to afford analogs 91 &92 in 90% yield.

Scheme 17: Synthesis of linker substituted fluorescent probes

Reagents and conditions: (a) X-COOH, HBTU, DIPEA, DMF, 50 ºC, 24 h, 80-82% (b) TFA,

CH2Cl2, 1 h, 89-92%, (c) CDI, R1 or R2, DMF, 0 ºC – rt, 21 h, 78-81%.

Fluorescently labelled cannabinergic probes bearing a linker were synthesized to develop a SAR at the C1 position of nabilone and the synthesis is depicted in Scheme 17. Treatment of sterically

201 hindered boc-protected amino acids with DIPEA, HBTU17 and 59 resulted in ester intermediates

(93-96) in 80-82% yield followed by removal of the boc group to afford primary amine analogs

97-100 in 89-92% yield. Coumarin 343 and 7-methoxycoumarin-3-carboxylic acid were then coupled to intermediates 97-100 via amide functionality to give the desired final compounds

101-108 with 78-81% yield.18

202

RESULTS AND DISCUSSION

Our SAR study (Figure 4.2) focused on chemical synthesis to investigate the effects of fluorophores at the C1 position of nabilone. In vitro profiling of the C1 substituted nabilone analogs was performed in the following assays:

 Competitive radioligand binding assay for CB1 and CB2 receptors.

 Cyclic adenosine monophosphate (cAMP) assay.

 Metabolic (Plasma) stability assay.

 Fluorescence excitation and emission spectra

The binding affinities of fluorescently labelled cannabinergic probes were determined for the

CB1 receptor (rat brain membranes) and membrane preparations from HEK293 cells expressing human (hCB2) and mouse (mCB2) CB2 receptors.19, 20 Displacement of [3H]-CP-55,940 from

21 these membranes was used to determine IC50 values in competition radioligand binding assays.

[3H]-CP-55,940 was used as the competing ligand as it is nonselective and has high affinity for both CB1 and CB2 receptors. Fluorescence measurements were carried out on compounds showing high binding affinity followed by evaluation of functional response in the cAMP assay.20, 22 Key analogs were also assessed for their stability towards plasma esterases.19

203

Table 4.1: Binding affinities of fluorescently labelled cannabinergic probes

Compd & (K ,nM) R i AM # rCB1 mCB2 hCB2 80 10849 57.1±9.3 - 369

81 80.4±7.8 >1000 669 10853

82 50.0±7.2 207 556 10854

83 484 - >1000 10868

84 165.0±12.4 ~375 369 10880

In order to explore the effects of fluorophores at the C1 position of nabilone, analogs reported in

Table 4.1 were synthesized. During the exploration of pharmacophoric space (shown before,

Table 3.1), conversion of the phenol to ester functionality resulted in high affinity ligands.

204

Hence, fluorophores were conjugated with nabilone by an ester group with the exception of dansyl fluorophore which was attached via sulfone moiety. Most of the analogs synthesized showed good binding affinity to the CB1 receptor but the affinity to the CB2 receptor was diminished significantly. 7-methoxycoumarin substituted analog AM10854 (82) showed highest binding affinity (Ki = 50 nM) to the CB1 receptor while conjugation of dansyl fluorophore,

AM10868 (83) showed significant loss of affinity (Ki = 484 nM) to the CB1 receptor.

Biotinylated, AM10849 (80) and coumarin 343 substituted analog, AM10853 (81) showed modest binding affinities (Ki = 57 & 80 nM) respectively to the CB1 receptor. It was surprising to see that the bulky rhodamine substituted compound, AM10880 (84) still maintained affinity

(Ki = 165 nM) at the CB1 receptor.

Table 4.2: Fluorescence properties of cannabinergic probes

Ethanol Acetonitrile AM # R Ex λmax Em λmax Ex λmax Em λmax

80 310 645 313 647 10849

81 330 489 334 488 10853

82 452 479 452 480 10854

205

83 450 479 444 470 10868

84 451 601 452 606 10880

The fluorescence properties of novel fluorescently labelled cannabinergic probes were investigated in two different organic solvents, ethanol and acetonitrile and their excitation and emission values are reported in Table 4.2. All the analogs reported exhibited fluorescence in both the solvents. Dansyl (83) and coumarin derivatives 81 & 82 exhibited emission wavelength at

479, 489 and 479 nm in ethanol near the blue region. Similar emission wavelength was seen when these compounds were tested in acetonitrile. Rhodamine conjugated probe (84) showed emission maxima at longer wavelength (601 and 606 nm) near to red light in ethanol and acetonitrile. It was surprising to see that the biotinylated analog (80) exhibited emission wavelength (645 and 647 nm) near red light region.

From the 1st generation fluorescent cannabinoids (Table 4.1), the coumarin substituted analogs

81 & 82 showed good binding affinities (Ki = 80 and 50 nM) to the CB1 receptor. Our goal was to develop high affinity fluorescently labelled probes targeting the CB1 receptor. Hence to further explore the SAR around the C1 pharmacophore of nabilone, linkers were incorporated between the fluorophores and nabilone. The binding affinities of these analogs are reported in

Table 4.3.

206

Table 4.3: Binding affinities of fluorescent cannabinoids tethered via linker

Compd (K ,nM) X R i & AM # rCB1 mCB2 hCB2

105 >1000 - >1000 10871

101 377 - 200 10882

106 150.2±13.6 - 159±12.7 10895

102 >1000 - >1000 10898

107 >1000 - >1000 10893

103 >1000 - >1000 10896

108 >1000 - 551.9 10894

104 >1000 - >1000 10897

207

AM10882 (101), a conjugate of nabilone and coumarin 343 tethered through a dimethyl- propionic amide linker displayed significant loss of affinity to the CB1 receptor (Ki = 377 nM) while increasing affinity to CB2 receptor as compared to AM10853 (81), a non-tethered analog.

Extension of chain length of the linker group of 101 to higher alkyl homologues led to complete loss of affinity to the CB1 and CB2 receptor (102-104, Ki = >1000 nM). It is surprising to see that replacing the coumarin fluorophore in AM10871 (105) led to complete loss of affinity to

CB1 and CB2 receptor (Ki = >1000 nM) even though it has a similar linker length as compound

101. Conjugation of nabilone and 7-methoxycoumarin via dimethyl-butyric amide linker afforded AM10895 (106) which showed equal affinity to both CB1 and CB2 receptor. Chain length extension of linker group of 106 to dimethyl-pentanoic amide and dimethyl-hexanoic amide led to compounds AM10893 (107) and AM10894 (108) showing binding affinity to hCB2

(Ki = 372 and 250 nM) with a complete loss of affinity to the CB1 receptor (Ki = >1000 nM).

The binding data from Table 4.1 and 4.3 describes our SAR studies of fluorescently labelled cannabinergic ligands. The compounds were prepared by attaching a fluorophore directly to the phenolic hydroxyl of nabilone or by conjugating a linker between the fluorophore and nabilone.

Binding data shows that fluorescent analogs linked directly to the C1 position of nabilone via ester group show high binding affinity to the CB1 receptor while the affinity is diminished for the CB2 receptors. Fluorescent analogs tethered via linker showed significant loss of affinity to the CB1 receptor while some analogs (101, 106, 107 and 108) showed affinity to the CB2 receptor.

208

Table 4.4: Functional and plasma stability assessment of key fluorescent cannabinergic analogs

rCB1 Plasma Stability (t ) min AM # R 1/2 EC50 (nM) Emax (%) Mouse Rat Human

80 - - 1.5 5.3 >30 10849

81 217±16.2 68 >30 >30 >30 10853

82 42±3.6 78 >30 >30 >30 10854

Compounds 81 & 82 were tested in the cAMP assay for their functional potency (Table 4.4).

Both analogs showed agonist profile at the CB1 receptor (EC50 = 217 and 42 nM).

AM10849 (80), AM10853 (81) and AM10854 (82) were tested for their stability towards plasma

esterases (Table 4.4). Data shows that coumarin derivatives 81 & 82 are stable in mouse, rat and

human plasma while biotinylated probe 80 showed a short plasma half-life (t1/2 = <5.3 min) in

mouse and rat plasma.

209

CONCLUSIONS

In this project, we have described a series of cannabinergic probes comprising of bulky fluorescent functional groups linked directly or indirectly to the phenolic hydroxyl of nabilone. A variety of fluorescent analogs were synthesized and evaluated for their activity at the CB receptors. The SAR study revealed that fluorophores tethered directly to the C1 position of nabilone via ester group showed good affinity to the CB1 receptor as compared to the linker substituted fluorescent analogs. Coumarin derivatives AM10853 and AM10854 afforded the best

CB1 activity profile and are currently being evaluated as imaging agents in cell based experiments.

210

EXPERIMENTAL SECTION

Shield 400 WB plus (1H 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 relative to base

(Waters-2424) using a XTerra MS C18, 5 µm, 4.6 mm x 50 mm column and acetonitrile/water] and were > 95%.

211

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4- yl)pentanoate (80). To a stirred solution of DMAP (37 mg, 0.30 mmol) and EDCI (38 mg, 0.20 mmol) in dry DMF (0.8 ml) at 0 °C under an argon atmosphere, Biotin (37 mg, 0.15 mmol) was added. The reaction was stirred for 20 min at 0 °C and then 59 (50 mg, 0.134 mmol) was added.

The mixture was warmed to room temperature and stirred for 24 h to ensure complete formation of the product. The reaction mixture was diluted with diethyl ether and washed sequentially with

5% HCl, 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

(50% AcOEt in hexane) to give 80 as a white crystalline solid (30 mg, 73% yield). mp = 120-

121oC. IR (neat): 3233, 2929, 1757, 1695, 1623, 1563, 1459, 1258, 1198, 1133 cm−1. 1H NMR

(500 MHz, CDCl3) δ 6.70 (d, J = 2.0 Hz, 1H, ArH), 6.50 (d, J = 2.0 Hz, 1H, ArH), 5.18 (s, 1H,

NH), 4.77 (s, 1H, NH), 4.53-4.50 (m, 1H, -NH-CH-), 4.39-4.34 (m, 1H, -NH-CH-), 3.25 (ddd, J

= 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.23-3.18 (m, 1H, -S-CH-), 2.95 (dd, J = 13.5

Hz, J = 4.5 Hz, 1H, -S-CH2-), 2.75 (d, J = 13.5 Hz, 1H, -S-CH2-), 2.68 (m as td, J = 12.4 Hz, J =

3.4 Hz, 1H, 10a-H), 2.62 (t, J = 7.0 Hz, 2H, C(O)O-CH2-), 2.58-2.52 (m, 1H, 8eq-H), 2.46-2.37

(m, 1H, 8ax-H), 2.26-2.11 (m, 2H, 10ax-H, 7eq-H), 2.00-1.92 (m as td, J = 12.5 Hz, J = 3 Hz,

1H, 6a-H), 1.85-1.68 (m, 5H, C(O)O-CH2-CH2-CH2-), 1.58-1.47 (m, 7H, 7ax-H, 2’-H, C(O)O-

CH2-CH2-CH2-,6-Me, especially 1.48, s, 6-Me), 1.27-1.17 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.12 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J

13 = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 210.09 (>C=O), 171.80 (-C(O)-O), 163.74

(>C=O), 154.22 (ArC-1 or ArC-5), 151.22 (ArC-5 or ArC-1), 149.38 (tertiary aromatic), 113.93

(tertiary aromatic), 113.28 (ArC-2 or ArC-4), 112.60 (ArC4 or ArC-2), 62.23, 60.31, 55.62,

212

47.76, 46.16, 44.55, 40.92, 40.74, 37.75, 35.16, 34.28, 31.93, 30.14, 28.75, 28.61, 28.49, 27.90,

26.92, 24.80, 24.73, 22.85, 19.11, 14.29. Mass spectrum (ESI) m/z (relative intensity) 599 (M+ +

+ H, 100). Exact mass (ESI) calculated for C34H51N2O5S (M + H), 599.3519; found 509.3513.

LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.5% and retention time of

5.5 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1- ij]quinoline-10-carboxylate (81). To a stirred solution of DMAP (99 mg, 0.80 mmol) and EDCI

(103 mg, 0.53 mmol) in dry CH2Cl2 (2 ml) at 0 °C under an argon atmosphere, Coumarin 343

(151 mg, 0.53 mmol) was added. The reaction was stirred for 20 min at 0°C and then 59 (50 mg,

0.134 mmol) was added. The mixture was warmed to room temperature and stirred for 24 h to ensure complete formation of the product. The reaction mixture was diluted with diethyl ether and washed sequentially with 5% HCl, 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 (50% AcOEt in hexane) to give 81 as a yellow crystalline solid

(45 mg, 52% yield). mp = 132-133 °C. IR (neat): 2927, 1762, 1710, 1619, 1588, 1442, 1366,

−1 1 1235, 1197, 1108 cm . H NMR (500 MHz, CDCl3) δ 8.51 (s, 1H, ArH-Coumarin), 7.01 (s, 1H,

ArH-Coumarin), 6.70 (d, J = 1.5 Hz, 1H, ArH), 6.65 (d, J = 2.0 Hz, 1H, ArH), 3.42-3.33

(overlapping multiples, 5H, 10eq-H, Coumarin -N-CH2-), 2.92-2.85 (Overlapping multiples, 3H,

10a-H, Coumarin -N-CH2-CH2-CH2-), 2.77 (t, J = 6.0 Hz, Coumarin N-CH2-CH2-CH2-), 2.55-

2.49 (m, 1H, 8eq-H), 2.41-2.33 (m, 1H, 8ax-H), 2.26-2.17 (m, 1H, 10ax-H), 2.13-2.09 (m, 1H,

7eq-H), 1.99-1.91 (overlapping multiples, 5H, Coumarin N-CH2-CH2-CH2-, 6a-H), 1.53-1.46

(m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.48, s, 6-Me), 1.25-1.15 (m, 12H, 3’-H, 4’-H, 5’-H, -

213

C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.16 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H),

13 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.53 (>C=O), 161.99 (-C(O)-O),

158.31 (-C(O)-O), 153.78 (ArC-1 or ArC-5), 153.73 (ArC-5 or ArC-1), 150.59 (tertiary aromatic), 149.96 (ArC-Coumarin), 149.30 (ArC-Coumarin), 149.00 (ArC-Coumarin), 127.33

(ArC-Coumarin), 119.28 (ArC-Coumarin), 114.28 (tertiary aromatic), 112.74 (ArC-2 and ArC-

4), 107.68 (ArC-Coumarin), 105.63 (ArC-Coumarin), 105.50 (ArC-Coumarin), 50.23, 49.86,

47.33, 45.84, 44.28, 40.33, 37.44, 34.44, 31.62, 30.20, 29.85, 28.47, 27.60, 27.30, 26.19, 24.44,

22.54, 21.01, 20.03, 19.93, 18.86, 13.96. Mass spectrum (ESI) m/z (relative intensity) 640 (M+ +

+ H, 100). Exact mass (ESI) calculated for C40H50NO6 (M + H), 640.3638; found 640.3631.

LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.7% and retention time of

6.3 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 7-methoxy-2-oxo-2H-chromene-3-carboxylate (82). To a stirred solution of DMAP (37 mg, 0.30 mmol) and EDCI (38 mg, 0.20 mmol) in dry CH2Cl2 (0.8 ml) at

0 °C under an argon atmosphere, 7-Methoxycoumarin-3-carboxylic acid (33 mg, 0.15 mmol) was added. The reaction was stirred for 20 min at 0 °C and then 59 (50 mg, 0.134 mmol) was added. The mixture was warmed to room temperature and stirred for 24 h to ensure complete formation of the product. The reaction mixture was diluted with diethyl ether and washed sequentially with 5% HCl, 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 (50% AcOEt in hexane) to give 82 as a white crystalline solid (25 mg, 66% yield). mp = 122-123 °C. IR (neat): 2929, 1746, 1713, 1604, 1556, 1412, 1373, 1200,

−1 1 1100, 1026 cm . H NMR (500 MHz, CDCl3) δ 8.74 (s, 1H, ArH-Coumarin), 7.63 (d, J = 9.0

214

Hz, 1H, ArH-Coumarin), 6.93 (dd, J = 9.0 Hz, J = 2.5 Hz, 1H, ArH-Coumarin), 6.85 (d, J = 2.5

Hz, 1H, ArH-Coumarin), 6.74 (d, J = 1.5 Hz, 1H, ArH), 6.67 (d, J = 2.0 Hz, 1H, ArH), 3.93 (s,

3H, O-CH3), 3.35 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.86 (m as td, J = 12.4

Hz, J = 3.4 Hz, 1H, 10a-H), 2.56-2.50 (m, 1H, 8eq-H), 2.44-2.35 (m, 1H, 8ax-H), 2.31-2.24 (m,

1H, 10ax-H), 2.16-2.10 (m, 1H, 7eq-H), 2.02-1.94 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H),

1.55-1.47 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.49, s, 6-Me), 1.27-1.17 (m, 12H, 3’-H, 4’-H,

5’-H, -C(CH3)2-, especially 1.24, s, -C(CH3)2-), 1.16 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H,

13 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.43 (>C=O), 165.63 (-

C(O)-O), 161.08 (-C(O)-O), 157.88 (ArC-Coumarin), 156.73 (ArC-Coumarin), 153.94 (ArC-1 or

ArC-5), 150.88 (ArC-5 or ArC-1), 148.90 (tertiary aromatic), 131.14 (ArC-Coumarin), 113.93

(tertiary aromatic), 113.83 (ArC-Coumarin), 113.21 (ArC-Coumarin), 112.58 (ArC-2 or ArC-4),

112.43 (ArC-4 or ArC-2), 111.60 (ArC-Coumarin), 100.27 (ArC-Coumarin), 77.04, 55.96,

47.42, 46.00, 44.26, 40.42, 37.50, 34.60, 31.62, 29.83, 28.46, 27.58, 26.38, 24.44, 22.53, 18.81,

13.96. Mass spectrum (ESI) m/z (relative intensity) 575 (M+ + H, 100). Exact mass (ESI)

+ calculated for C35H43O7 (M + H), 575.3009; found 575.3007. LC/MS analysis (Waters

MicroMass ZQ system) showed purity of 99.3% and retention time of 6.0 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 5-(dimethylamino)naphthalene-1-sulfonate (83). To a stirred solution of 59 (25 mg, 0.07 mmol) in dry CH2Cl2 (2 ml) at room temperature under an argon atmosphere, a solution of Et3N and Dansyl Chloride in CH2Cl2 (1 ml) was added. The reaction was stirred for

20 h to ensure complete formation of the product. Upon completion, the solvent was evaporated and the crude mixture was chromatographed on silica gel, 30% AcOEt in hexane to yield 83 as a

215 white solid (35 mg, 63%). mp = 72-73 °C. IR (neat): 2928, 1712, 1621, 1562, 1457, 1370, 1325,

−1 1 1202, 1023 cm . H NMR (500 MHz, CDCl3) δ 8.59 (d, J = 8.0 Hz, 1H, ArH-Dansyl), 8.47 (d, J

= 8.0 Hz, 1H, ArH-Dansyl), 8.03 (d, J = 7.0 Hz, 1H, ArH-Dansyl), 7.62 (td, J = 8.0 Hz, J = 1.5

Hz, 1H, ArH-Dansyl), 7.42 (td, J = 8.0 Hz, J = 1.5 Hz, 1H, ArH-Dansyl), 7.21 (d, J = 7.0 Hz,

1H, ArH-Dansyl), 6.58 (d, J = 1.5 Hz, 1H, ArH), 6.17 (d, J = 2.0 Hz, 1H, ArH), 3.67 (ddd, J =

15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.88 (s, 6H, (CH3)2), 2.55 (m as td, J = 12.5 Hz, J

= 4.0 Hz, 1H, 10a-H), 2.52-2.45 (m, 1H, 8eq-H), 2.35-2.26 (m, 1H, 8ax-H), 2.11-1.96 (m, 2H,

10ax-H, 7eq-H), 1.78-1.71 (m as td, J = 11.5 Hz, J = 3 Hz, 1H, 6a-H), 1.37 (s, 3H, 6-Me), 1.29-

1.07 (m, 11H, 7ax-H, 10 side-chain protons), 0.95 (s, 3H, C(CH3)-), 0.92 (s, 3H, C(CH3)-), 0.85

13 (t, J = 6.5 Hz, 3H, 7’-H), 0.76 (s, 3H, 6-Me). C NMR (100 MHz, CDCl3) δ 208.95 (>C=O),

153.87 (ArC-1 or ArC-5), 151.75 (ArC-Dansyl), 150.38 (ArC-5 or ArC-1), 148.12 (tertiary aromatic), 131.74 (ArC-Dansyl), 131.46 (ArC-Dansyl), 131.16 (ArC-Dansyl), 130.03 (ArC-

Dansyl), 129.68 (ArC-Dansyl), 129.13 (ArC-Dansyl), 122.86 (ArC-Dansyl), 119.52 (ArC-

Dansyl), 115.56 (ArC-Dansyl), 115.09 (tertiary aromatic), 113.86 (ArC-2 or ArC-4), 112.82

(ArC4 or ArC-2), 46.65, 45.31, 45.26, 43.90, 40.03, 37.22, 33.64, 31.64, 29.73, 28.42, 27.97,

27.39, 25.74, 24.37, 22.53, 18.26, 13.98. Mass spectrum (ESI) m/z (relative intensity) 606 (M+ +

+ H, 100). Exact mass (ESI) calculated for C36H48NO5S (M + H), 606.3253; found 606.3255.

LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.5% and retention time of

6.6 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 2-(3-(diethyl-l4-azanylidene)-6-(diethylamino)-3H-xanthen-9- yl)benzoate (84). To a stirred solution of DMAP (25 mg, 0.20 mmol) and EDCI (27 mg, 0.14 mmol) in dry CH2Cl2 (1 ml) at 0 °C under an argon atmosphere, Rhodamine B (68 mg, 0.14

216 mmol) was added. The reaction was stirred for 20 min at 0°C and then 59 (25 mg, 0.07 mmol) was added. The mixture was warmed to room temperature and stirred for 24 h to ensure complete formation of the product. The reaction mixture was diluted with CH2Cl2 and quenched using water. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (10% MeOH in CH2Cl2) to give 84 as a dark red crystalline solid (35 mg, 70% yield). mp = 150-151 °C. IR (neat): 2927,

−1 1 1737, 1635, 1466, 1466, 1413, 1338, 1247, 1180, 1074 cm . H NMR (500 MHz, CDCl3) δ 8.50

(d, J = 7.5 Hz, 1H, ArH-Rhodamine), 7.93 (td, J = 7.5 Hz, J = 1.5 Hz, 1H, ArH-Rhodamine),

7.87 (td, J = 7.5 Hz, J = 1.5 Hz, 1H, ArH-Rhodamine), 7.42 (d, J = 7.5 Hz, 1H, ArH-

Rhodamine), 7.22 (d, J = 9.5 Hz, 1H, ArH-Rhodamine), 7.14 (d, J = 10.0 Hz, 1H, ArH-

Rhodamine), 6.92 (d, J = 9.5 Hz, 1H, ArH-Rhodamine), 6.91 (d, J = 9.5 Hz, 1H, ArH-

Rhodamine), 6.85 (d, J = 2.5 Hz, 1H, ArH-Rhodamine), 6.81 (d, J = 2.5 Hz, 1H, ArH-

Rhodamine), 6.62 (d, J = 1.5 Hz, 1H, ArH), 6.03 (d, J = 2.0 Hz, 1H, ArH), 3.71-3.59 (m, 8H, N-

CH2-CH3-), 2.92 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.48-2.42 (m, 2H, 10a-

H, 8eq-H), 2.38-2.29 (m, 1H, 8ax-H), 2.11-1.98 (m, 2H, 10ax-H, 7eq-H), 1.87-1.80 (m as td, J =

14.5 Hz, J = 2.5 Hz, 1H, 6a-H), 1.42 (s, 3H, 6-Me), 1.40-1.28 (m, 15H, 7ax-H, 2’-H, (CH3)4),

1.26-1.11 (m, 6H, side-chain protons), 1.08 (s, 6H, -C(CH3)2-), 0.98-0,93 (m, 5H, side-chain

13 protons, especially 0.97, s, 6-Me), 0.82 (t, J = 7.0 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ

209.11 (>C=O), 163.11 (-C(O)-O), 158.10 (ArC-Rhodamine), 157.74 (ArC-Rhodamine), 157.61

(ArC-Rhodamine), 155.56 (ArC-Rhodamine), 155.31 (ArC-Rhodamine), 154.00 (ArC-1 or ArC-

5), 151.03 (ArC-5 or ArC-1), 148.43 (tertiary aromatic), 134.14 (ArC-Rhodamine), 133.89 (ArC-

Rhodamine), 131.81 (ArC-Rhodamine), 131.44 (ArC-Rhodamine), 131.22 (ArC-Rhodamine),

130.72 (ArC-Rhodamine), 130.52 (ArC-Rhodamine), 128.92 (ArC-Rhodamine), 114.50 (ArC-

217

Rhodamine), 114.19 (tertiary aromatic), 113.46 (ArC-2 or ArC-4), 111.38 (ArC-4 or ArC-2),

96.40 (ArC-Rhodamine), 46.88, 46.11, 45.99, 45.61, 44.16, 40.61, 37.36, 34.50, 31.57, 29.76,

29.59, 28.31, 27.54, 26.37, 24.33, 22.49, 18.71, 13.97, 12.56. Mass spectrum (ESI) m/z (relative

+ + intensity) 797 (M + H, 100). Exact mass (ESI) calculated for C34H51N2O5S (M + H), 797.4893; found 797.4886. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.8% and retention time of 9.4 min for the title compound.

Methyl 5-((tert-butoxycarbonyl)amino)pentanoate (87). To a stirred solution of 85 (600 mg,

2.76 mmol) in dry DMF (15 ml) at room temperature, CsCO3 (900 mg, 2.76 mmol) and CH3I

(400 mg, 2.76 mmol) were added under an argon atmosphere. The mixture was stirred for 20 h to ensure complete formation of the product. The reaction mixture was diluted with ethyl acetate and quenched using water. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (40% ethyl acetate in hexane) to give 87 as a colorless oil (500 mg, 78% yield). IR (neat): 3362, 2935, 1737,

−1 1 1690, 1516, 1452, 1438, 1365, 1246, 1160, 1098 cm . H NMR (500 MHz, CDCl3) δ 4.28 (s,

1H, NH), 3.67 (s, 3H, O-CH3), 3.16-3.10 (m, 2H, -NH-CH2-), 2.33 (t, J = 7.0 Hz, 2H, -C(O)O-

CH2-), 1.70-1.62 (m, 2H, -C(O)O-CH2-CH2-), 1.56-1.46 (m, 2H, -C(O)O-CH2-CH2-CH2-), 1.43

13 (s, 9H, t-Butyl). C NMR (100 MHz, CDCl3) δ 173.71 (>C=O), 155.81 (>C=O), 78.87, 77.22,

76.90, 76.58, 51.32, 39.92, 33.38, 29.30, 28.21, 21.87. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.6% and retention time of 4.0 min for the title compound.

Methyl 6-((tert-butoxycarbonyl)amino)hexanoate (88). The reaction was performed similar to as described for 87 using 86 (600 mg, 2.6 mmol), dry DMF (15 ml), CsCO3 (845 mg, 2.6 mmol) and CH3I (370 mg, 2.6 mmol). The residue was chromatographed on silica gel (40% ethyl acetate in hexane) to give 88 as a colorless oil (500 mg, 78% yield). IR (neat): 3364, 2934, 1736,

218

−1 1 1692, 1517, 1454, 1437, 1365, 1247, 1155, 1092 cm . H NMR (500 MHz, CDCl3) δ 4.58 (s,

1H, NH), 3.66 (s, 3H, O-CH3), 3.15-3.07 (m, 2H, -NH-CH2-), 2.31 (t, J = 7.5 Hz, 2H, -C(O)O-

CH2-), 1.69-1.60 (m, 2H, -C(O)O-(CH2)3-CH2-), 1.54-1.43 (m, 11H, -C(O)O-CH2-CH2-,

13 especially 1.44 s, t-Butyl), 1.39-1.30 (m, 2H, -C(O)O-(CH2)2-CH2-). C NMR (100 MHz,

CDCl3) δ 173.48 (>C=O), 155.46 (>C=O), 78.41, 76.87, 76.55, 76.23, 50.88, 39.77, 33.33,

29.14, 27.82, 25.69, 23.97. LC/MS analysis (Waters MicroMass ZQ system) showed purity of

99.2% and retention time of 4.3 min for the title compound.

Methyl 5-((tert-butoxycarbonyl)amino)-2,2-dimethylpentanoate (89). To a stirred solution of

87 (500 mg, 2.16 mmol) in dry THF (30 ml) at -78 ºC, LDA (930 mg, 8.6 mmol) was added under an argon atmosphere. The reaction mixture was stirred at the same temperature for 15 min followed by addition of CH3I (3.0 g, 21.6 mmol). The mixture was stirred for additional 30 min to ensure complete formation of the product. The reaction mixture was diluted with diethyl ether and quenched using saturated solution of ammonium chloride. The organic phase was dried over

MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (30% ethyl acetate in hexane) to give 89 as brown oil (300 mg, 70% yield). IR

(neat): 3378, 2951, 1712, 1693, 1517, 1474, 1452, 1365, 1247, 1165, 984 cm−1. 1H NMR (500

MHz, CDCl3) δ 4.28 (s, 1H, NH), 3.66 (s, 3H, O-CH3), 3.13-3.05 (m, 2H, -NH-CH2-), 1.56-1.50

(m, 2H, -NH-CH2-CH2-CH2-), 1.45-1.38 (m, 11H, -NH-CH2-CH2-, especially 1.44, s, t-Butyl),

13 1.17 (s, 6H, -C(CH3)2-). C NMR (100 MHz, CDCl3) δ 178.12 (>C=O), 155.81 (>C=O), 78.98,

51.63, 41.93, 40.72, 37.52, 28.31, 25.54, 25.04. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.5% and retention time of 4.5 min for the title compound.

Methyl 6-((tert-butoxycarbonyl)amino)-2,2-dimethylhexanoate (90). The synthesis was carried out as described for 89 using 88 (500 mg, 2.04 mmol), dry THF (15 ml), LDA (873 mg,

219

8.16 mmol) and CH3I (2.9 g, 20.4 mmol). The residue was chromatographed on silica gel (30% ethyl acetate in hexane) to give 90 as brown oil (300 mg, 70% yield). IR (neat): 3377, 2974,

−1 1 1720, 1680, 1521, 1474, 1456, 1365, 1250, 1173, 1082 cm . H NMR (500 MHz, CDCl3) δ 4.28

(s, 1H, NH), 3.65 (s, 3H, O-CH3), 3.22-3.13 (m, 2H, -NH-CH2-), 1.56-1.42 (m, 13H, -NH-CH2-

CH2-CH2-,-NH-CH2-CH2-, especially 1.45, s, t-Butyl), 1.24-1.13 (m, 8H, -NH-(CH2)3-CH2-,

13 especially 1.16, s, -C(CH3)2-). C NMR (100 MHz, CDCl3) δ 178.27 (>C=O), 155.82 (>C=O),

78.92, 51.56, 42.13, 40.21, 40.15, 30.29, 28.30, 25.04, 22.06. LC/MS analysis (Waters

MicroMass ZQ system) showed purity of 99.4% and retention time of 4.7 min for the title compound.

5-((tert-butoxycarbonyl)amino)-2,2-dimethylpentanoic acid (91). To a stirred solution of 89

(300 mg, 1.10 mmol) in dry methanol (12 ml) at 50 ºC, NaOH (88 mg, 2.2 mmol) was added under an argon atmosphere. The mixture was stirred for 4 h to ensure complete formation of the product. Upon completion, the mixture was diluted with diethyl ether and quenched using water.

The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure.

The residue was chromatographed on silica gel (30% ethyl acetate in hexane) to give 91 as colorless oil (250 mg, 85% yield). IR (neat): 3312, 2941, 1730, 1646, 1477, 1457, 1409, 1371,

−1 1 1283, 1153, 1059 cm . H NMR (500 MHz, CDCl3) δ 5.78 (s, 1H, OH), 4.62 (s, 1H, NH), 3.08-

3.00 (m, 2H, -NH-CH2-), 1.58-1.51 (m, 2H, -NH-CH2-CH2-CH2-), 1.50-1.42 (m, 11H, -NH-CH2-

13 CH2-, especially 1.44, s, t-Butyl), 1.20 (s, 6H, -C(CH3)2). C NMR (100 MHz, CDCl3) δ 183.65

(>C=O), 155.89 (>C=O), 79.08, 41.74, 40.77, 37.33, 28.31, 25.49, 24.90. LC/MS analysis

(Waters MicroMass ZQ system) showed purity of 98.8% and retention time of 4.0 min for the title compound.

220

6-((tert-butoxycarbonyl)amino)-2,2-dimethylhexanoic acid (92). The reaction was performed similar to as described for 91 using 90 (400 mg, 1.54 mmol), dry methanol (10 ml) and NaOH

(123 mg, 3.0 mmol). The residue was chromatographed on silica gel (30% ethyl acetate in hexane) to give 92 as colorless oil (350 mg, 88% yield). IR (neat): 3133, 2945, 1722, 1659, 1472,

−1 1 1449, 1403, 1367, 1228, 1149, 1066 cm . H NMR (500 MHz, CDCl3) δ 5.65 (s, 1H, OH), 4.58

(s, 1H, NH), 3.10-3.00 (m, 2H, -NH-CH2-), 1.58-1.51 (m, 2H, -NH-CH2-CH2-CH2-), 1.50-1.41

(m, 11H, -NH-(CH2)2-CH2-, especially 1.44, s, t-Butyl), 1.33-1.16 (m, 8H, -NH-(CH2)3-CH2-,

13 especially 1.18, s, -C(CH3)2). C NMR (100 MHz, CDCl3) δ 183.69 (>C=O), 155.90 (>C=O),

79.02, 41.95, 40.24, 39.94, 30.33, 28.32, 24.90, 22.02. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.6% and retention time of 4.5 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropanoate (93). To a stirred suspension of 3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropanoic acid (250 mg, 1.2 mmol) in dry DMF (6 ml) at 50 ºC, DIPEA (416 mg, 3.22 mmol) and HBTU (522 mg, 1.37 mmol) was added under an argon atmosphere. The mixture was stirred for 3 h followed by the addition of 59 (200 mg, 0.53 mmol) and stirring continued for additional 21 h. Upon completion the reaction was diluted with ethyl acetate and quenched using water. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (25% ethyl acetate in hexane) to give 93 as colorless oil (300 mg,

82% yield). IR (neat): 3179, 2930, 1710, 1623, 1506, 1459, 1413, 1388, 1365, 1248, 1168, 1044

−1 1 cm . H NMR (500 MHz, CDCl3) δ 6.71 (d, J = 2.0 Hz, 1H, ArH), 6.43 (d, J = 1.5 Hz, 1H,

ArH), 5.07 (t, J = 4.5 Hz, 1H, NH), 3.44-3.29 (m, 2H, -NH-CH2-), 3.23 (ddd, J = 15.0 Hz, J =

3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.68 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.59-2.52

221

(m, 1H, 8eq-H), 2.46-2.36 (m, 1H, 8ax-H), 2.24-2.12 (m, 2H, 10ax-H, 7eq-H), 2.10-1.93 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.54-1.46 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.48, s, 6-

Me), 1.43 (s, 9H, t-Butyl), 1.41 (s, 3H, -C(O)O-C(CH3)2-), 1.39 (s, 3H, -C(O)O-C(CH3)2-), 1.24-

1.16 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.13 (s, 3H, 6-Me),

13 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J = 7.0 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3)

δ 209.51 (>C=O), 202.07 (-C(O)-O), 183.66 (>C=O), 154.17 (ArC-1 or ArC-5), 151.20 (ArC-5 or ArC-1), 149.39 (tertiary aromatic), 114.20 (tertiary aromatic), 113.34 (ArC-2 or ArC-4),

112.16 (ArC4 or ArC-2), 79.37, 48.52, 48.01, 46.32, 44.43, 44.20, 40.82, 37.67, 35.07, 31.83,

30.03, 28.63, 28.49, 27.74, 26.96, 24.64, 23.16, 22.75, 18.92, 14.18. LC/MS analysis (Waters

MicroMass ZQ system) showed purity of 99.2% and retention time of 6.3 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 4-((tert-butoxycarbonyl)amino)-2,2-dimethylbutanoate (94). The synthesis was performed similar to as described for 93 using 4-((tert-butoxycarbonyl)amino)-2,2- dimethylbutanoic acid (250 mg, 1.06 mmol), dry DMF (6 ml), DIPEA (416 mg, 3.22 mmol),

HBTU (522 mg, 1.37 mmol) and 59 (200 mg, 0.53 mmol). The residue was chromatographed on silica gel (25% ethyl acetate in hexane) to give 94 as colorless oil (290 mg, 80% yield). IR

(neat): 3185, 2931, 1710, 1623, 1512, 1457, 1413, 1366, 1228, 1136, 1027 cm−1. 1H NMR (500

MHz, CDCl3) δ 6.70 (d, J = 1.5 Hz, 1H, ArH), 6.46 (d, J = 1.5 Hz, 1H, ArH), 5.10 (s, 1H, NH),

3.27 (multiplet overlapping, 2H, 10eq-H, -NH-CH2-), 3.12-3.02 (m, 1H, -NH-CH2-), 2.69 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.60-2.52 (m, 1H, 8eq-H), 2.47-2.33 (m, 1H, 8ax-H),

2.26-2.11 (m, 2H, 10ax-H, 7eq-H), 2.10-1.90 (multiplets overlapping, 3H, 6a-H, -NH-CH2-CH2-

), 1.53-1.47 (m, 6H, 7ax-H, 2’-H, 6-Me, especially 1.48, s, 6-Me), 1.45 (s, 9H, t-Butyl), 1.39 (s,

222

3H, -C(O)O-C(CH3)2-), 1.38 (s, 3H, -C(O)O-C(CH3)2-), 1.24-1.18 (m, 12H, 3’-H, 4’-H, 5’-H, -

C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.13 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H),

13 0.84 (t, J = 7.0 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 210.47 (>C=O), 208.51 (-C(O)-O),

179.45 (>C=O), 154.55 (ArC-1 or ArC-5), 151.63 (ArC-5 or ArC-1), 150.18 (tertiary aromatic),

114.58 (tertiary aromatic), 113.59 (ArC-2 or ArC-4), 112.71 (ArC4 or ArC-2), 83.29, 48.50,

46.88, 44.82, 43.28, 42.78, 41.36, 40.68, 38.09, 35.60, 33.41, 32.26, 30.46, 29.14, 28.99, 28.59,

28.19, 27.41, 26.10, 25.07, 24.87, 23.18, 19.35, 14.61. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.2% and retention time of 6.4 min for the title compound.

(6aS,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 5-((tert-butoxycarbonyl)amino)-2,2-dimethylpentanoate (95). The synthesis was performed similar to as described for 93 using 91 (250 mg, 1.06 mmol), dry DMF

(6 ml), DIPEA (416 mg, 3.22 mmol), HBTU (522 mg, 1.37 mmol) and 59 (200 mg, 0.53 mmol).

The residue was chromatographed on silica gel (25% ethyl acetate in hexane) to give 95 as colorless oil (292 mg, 81% yield). IR (neat): 3403, 2930, 1709, 1622, 1510, 1457, 1413, 1364,

−1 1 1247, 1120, 1027 cm . H NMR (500 MHz, CDCl3) δ 6.72 (d, J = 2.0 Hz, 1H, ArH), 6.42 (d, J

= 1.5 Hz, 1H, ArH), 4.92 (s, 1H, NH), 3.31 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-

H), 3.23-3.14 (m, 2H, -NH-CH2-), 2.71 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.62-2.54

(m, 1H, 8eq-H), 2.48-2.38 (m, 1H, 8ax-H), 2.26-2.13 (m, 2H, 10ax-H, 7eq-H), 2.04-1.95 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.79-1.71 (m, 2H, -NH-CH2-CH2-), 1.57-1.50 (m, 8H, 7ax-H,

2’-H, -NH-CH2-CH2-CH2-, 6-Me, especially 1.51, s, 6-Me), 1.46 (s, 9H, t-Butyl), 1.38 (s, 6H, -

C(O)O-C(CH3)2-), 1.27-1.18 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.24, s, -C(CH3)2-

), 1.15 (s, 3H, 6-Me), 1.09 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.87 (t, J = 7.0 Hz, 3H, 7’-H). 13C

NMR (100 MHz, CDCl3) δ 209.58 (>C=O), 175.97 (-C(O)-O), 155.92 (-C(O)-NH-), 153.92

223

(ArC-1 or ArC-5), 150.87 (ArC-5 or ArC-1), 149.55 (tertiary aromatic), 114.13 (tertiary aromatic), 112.93 (ArC-2 or ArC-4), 111.94 (ArC4 or ArC-2), 47.85, 46.15, 44.22, 42.37, 40.72,

37.41, 34.87, 31.63, 29.84, 28.54, 28.34, 27.55, 26.73, 25.56, 25.01, 24.88, 24.43, 22.56, 18.72,

13.99. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.7% and retention time of 6.4 min for the title compound.

(6aS,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 6-((tert-butoxycarbonyl)amino)-2,2-dimethylhexanoate (96). The synthesis was performed similar to as described for 93 using 92 (250 mg, 1.06 mmol), dry DMF

(6 ml), DIPEA (416 mg, 3.22 mmol), HBTU (522 mg, 1.37 mmol) and 59 (200 mg, 0.53 mmol).

The residue was chromatographed on silica gel (25% ethyl acetate in hexane) to give 96 as colorless oil (300 mg, 82% yield). IR (neat): 3394, 2930, 1710, 1622, 1512, 1457, 1413, 1364,

−1 1 1250, 1171, 1085 cm . H NMR (500 MHz, CDCl3) δ 6.69 (d, J = 1.5 Hz, 1H, ArH), 6.39 (d, J

= 1.5 Hz, 1H, ArH), 4.64 (s, 1H, NH), 3.29 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-

H), 3.20-3.08 (m, 2H, -NH-CH2-), 2.68 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.59-2.51

(m, 1H, 8eq-H), 2.45-2.34 (m, 1H, 8ax-H), 2.23-2.10 (m, 2H, 10ax-H, 7eq-H), 2.01-1.92 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.78-1.62 (m, 2H, -NH-CH2-CH2-), 1.57-1.46 (m, 10H, 7ax-

H, 2’-H, -NH-(CH2)2-CH2-, -NH-(CH2)3-CH2-, 6-Me, especially 1.48, s, 6-Me), 1.43 (s, 9H, t-

Butyl), 1.36 (s, 3H, -C(O)O-C(CH3)2-), 1.34 (s, 3H, -C(O)O-C(CH3)2-), 1.24-1.15 (m, 12H, 3’-H,

4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.13 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5

13 Hz, 2H, 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.42 (>C=O),

175.99 (-C(O)-O), 155.87 (-C(O)-NH-), 153.91 (ArC-1 or ArC-5), 150.81 (ArC-5 or ArC-1),

149.54 (tertiary aromatic), 114.14 (tertiary aromatic), 112.88 (ArC-2 or ArC-4), 111.95 (ArC4 or

ArC-2), 47.79, 46.08, 44.23, 42.57, 40.67, 40.38, 40.14, 37.40, 34.88, 31.62, 30.35, 29.84, 28.53,

224

28.41, 28.34, 27.54, 26.73, 25.21, 24.82, 24.42, 22.56, 22.24, 18.72, 13.99. LC/MS analysis

(Waters MicroMass ZQ system) showed purity of 99.1% and retention time of 6.5 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 3-amino-2,2-dimethylpropanoate (97). To a stirred solution of 93 (150 mg, 0.26 mmol) in CH2Cl2 (5 ml) at room temperature under an argon atmosphere was added

TFA (2.5 ml). The reaction was stirred for 1 h to ensure complete formation of the product. The reaction was quenched using saturated aqueous NaHCO3 and extracted using AcOEt. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (10% MeOH in CH2Cl2) to give 97 as white solid

(110 mg, 95% yield). mp = 68-69 °C. IR (neat): 2929, 1737, 1672, 1526, 1413, 1365, 1245, 1135,

−1 1 1110 cm . H NMR (500 MHz, CDCl3) δ 6.75 (d, J = 1.5 Hz, 1H, ArH), 6.41 (d, J = 2.0 Hz, 1H,

ArH), 3.16-3.00 (multiplet overlapping, 3H, 10eq-H, -NH2-CH2-), 2.67 (m as td, J = 12.4 Hz, J =

3.4 Hz, 1H, 10a-H), 2.58-2.50 (m, 1H, 8eq-H), 2.47-2.37 (m, 1H, 8ax-H), 2.28 (t, J = 9.0 Hz,

1H, 10ax-H), 2.16-2.12 (m, 1H, 7eq-H), 2.10-1.94 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.58

(s, 3H, -C(O)O-C(CH3)2-), 1.54 (s, 3H, -C(O)O-C(CH3)2-), 1.53-1.46 (m, 6H, 7ax-H, 2’-H, 6-

Me, especially 1.48, s, 6-Me), 1.29-1.14 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s,

-C(CH3)2-), 1.11 (s, 3H, 6-Me), 1.05 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J = 7.0 Hz, 3H, 7’-

13 H). C NMR (100 MHz, CDCl3) δ 210.68 (>C=O), 175.18 (>C=O), 154.65 (ArC-1 or ArC-5),

151.77 (ArC-5 or ArC-1), 149.26 (tertiary aromatic), 114.38 (tertiary aromatic), 114.10 (ArC-2 or ArC-4), 112.19 (ArC4 or ArC-2), 66.30, 48.34, 47.38, 46.81, 44.75, 41.66, 41.07, 38.04,

35.15, 32.17, 30.35, 30.16, 28.95, 28.05, 27.10, 24.97, 23.84, 23.23, 23.09, 19.15, 14.51. LC/MS

225 analysis (Waters MicroMass ZQ system) showed purity of 98.3% and retention time of 5.5 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 4-amino-2,2-dimethylbutanoate (98). The reaction was performed similar to as described for 97 using 94 (200 mg, 0.34 mmol), CH2Cl2 (6 ml) and TFA (3 ml). The residue was chromatographed on silica gel (10% MeOH in CH2Cl2) to give 98 as white solid

(155 mg, 92% yield). mp = 69-70 °C. IR (neat): 2929, 1678, 1624, 1563, 1456, 1414, 1365, 1226,

−1 1 1140, 1029 cm . H NMR (500 MHz, CDCl3) δ 6.69 (d, J = 1.5 Hz, 1H, ArH), 6.35 (d, J = 2.0

Hz, 1H, ArH), 3.20-3.00 (multiplet overlapping, 3H, 10eq-H, -NH2-CH2-), 2.65 (m, 1H, 10a-H,

8eq-H), 2.44-2.30 (m, 1H, 8ax-H), 2.31-2.23 (m, 1H, 10ax-H), 2.15-2.03 (m, 3H, 7eq-H, -NH2-

CH2-CH2-), 2.00-1.95 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.51-1.38 (m, 9H, 7ax-H, 2’-H, -

C(O)O-C(CH3)2-), 1.30 (s, 3H, 6-Me), 1.27-1.16 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.20, s, -C(CH3)2-), 1.09 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.83 (t, J

13 = 7.0 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 211.72 (>C=O), 175.61 (>C=O), 154.64

(ArC-1 or ArC-5), 149.62 (ArC-5 or ArC-1), 147.66 (tertiary aromatic), 115.01 (tertiary aromatic), 114.65 (ArC-2 or ArC-4), 111.18 (ArC4 or ArC-2), 48.35, 47.61, 47.24, 44.70, 41.57,

39.84, 31.90, 31.52, 30.10, 29.92, 28.77, 27.78, 25.89, 24.74, 23.01, 22.83, 21.69, 18.79, 14..65.

LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.8% and retention time of

5.6 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 5-amino-2,2-dimethylpentanoate (99). The reaction was performed similar to as described for 97 using 95 (260 mg, 0.42 mmol), CH2Cl2 (7 ml) and TFA (3.5 ml).

The residue was chromatographed on silica gel (10% MeOH in CH2Cl2) to give 99 as white solid

226

(195 mg, 91% yield). mp = 68-69 °C. IR (neat): 2930, 1747, 1675, 1566, 1457, 1413, 1365, 1222,

−1 1 1113, 1032 cm . H NMR (500 MHz, CDCl3) δ 6.69 (d, J = 1.5 Hz, 1H, ArH), 6.35 (d, J = 2.0

Hz, 1H, ArH), 3.16 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 3.12-3.00 (m, 2H, -

NH2-CH2-), 2.70 (m as td, J = 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 2.65-2.52 (m, 1H, 8eq-H), 2.46-

2.34 (m, 1H, 8ax-H), 2.33-2.21 (m, 1H, 10ax-H), 2.18-2.08 (m, 1H, 7eq-H), 2.01-1.92 (m as td, J

= 12 Hz, J = 3 Hz, 1H, 6a-H),1.86-1.76 (m, 4H, -NH2-CH2-CH2-CH2-, -NH2-CH2-CH2-), 1.54-

1.44 (m, 9H, 7ax-H, 2’-H, -C(O)O-C(CH3)2-), 1.38 (s, 3H, 6-Me), 1.25-1.15 (m, 12H, 3’-H, 4’-

H, 5’-H, -C(CH3)2-, especially 1.20, s, -C(CH3)2-), 1.11 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz,

13 2H, 6’-H), 0.83 (t, J = 7.0 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 213.66 (>C=O), 176.38

(>C=O), 154.63 (ArC-1 or ArC-5), 151.67 (ArC-5 or ArC-1), 150.32 (tertiary aromatic), 115.20

(tertiary aromatic), 114.62 (ArC-2 or ArC-4), 112.35 (ArC4 or ArC-2), 48.36, 46.81, 44.79,

42.97, 41.20, 41.06, 38.03, 36.67, 35.65, 32.23, 30.44, 30.24, 29.16, 28.99, 28.13, 27.29, 27.14,

25.86, 25.03, 24.70, 23.56, 23.17, 19.22, 14.59. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.6% and retention time of 5.5 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 6-amino-2,2-dimethylhexanoate (100). The reaction was performed similar to as described for 97 using 96 (240 mg, 0.40 mmol), CH2Cl2 (7 ml) and TFA (3.5 ml).

The residue was chromatographed on silica gel (10% MeOH in CH2Cl2) to give 100 as white solid (180 mg, 89% yield). mp = 67-68 °C. IR (neat): 2933, 1671, 1563, 1458, 1413, 1389, 1326,

−1 1 1187, 1100, 1029 cm . H NMR (500 MHz, CDCl3) δ 6.73 (d, J = 1.5 Hz, 1H, ArH), 6.34 (d, J

= 2.0 Hz, 1H, ArH), 3.92-3.72 (multiplet overlapping, 3H, 10eq-H, -NH2-CH2-), 3.37 (m as td, J

= 12.4 Hz, J = 3.4 Hz, 1H, 10a-H), 3.12-2.86 (m, 3H, 8eq-H, -NH2-CH2-CH2-), 2.74-2.51 (m,

2H, 10ax-H, 8ax-H), 2.31-2.06 (m, 5H, 7eq-H, -NH2-CH2-CH2-CH2-, -NH2-CH2-CH2-CH2-CH2-

227

), 2.00-1.86 (m as td, J = 12 Hz, J = 3 Hz, 1H, 6a-H), 1.55-1.42 (m, 9H, 7ax-H, 2’-H, -C(O)O-

C(CH3)2-), 1.27-1.14 (m, 15H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, 6-Me, especially 1.22, s, -C(CH3)2-),

1.11 (s, 3H, 6-Me), 1.05 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.93 (t, J = 7.0 Hz, 3H, 7’-H). 13C NMR

(100 MHz, CDCl3) δ 212.06 (>C=O), 175.94 (>C=O), 154.59 (ArC-1 or ArC-5), 151.46 (ArC-5 or ArC-1), 150.24 (tertiary aromatic), 118.12 (tertiary aromatic), 114.58 (ArC-2 or ArC-4),

112.32 (ArC4 or ArC-2), 48.42, 44.77, 43.11, 38.14, 38.02, 35.93, 35.52, 32.22, 30.41, 29.31,

29.15, 29.00, 28.14, 27.71, 27.10, 25.59, 25.38, 25.03, 23.16, 22.88, 20.33, 19.25, 19.01, 14.59.

LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.9% and retention time of

5.5 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 2,2-dimethyl-3-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3- f]pyrido[3,2,1-ij]quinoline-10-carboxamido)propanoate (101). To a stirred solution of coumarin 343 (10mg, 0.03 mmol) in dry DMF (1 ml) at 50 ºC, CDI (17 mg, 010 mmol) was added under an argon atmosphere. The reaction mixture was stirred at same temperature for 15 h followed by addition of 97 (28 mg, 0.06 mmol) and stirring continued for additional 6 h. Upon completion, the mixture was diluted with diethyl ether and quenched using water. The organic phase was dried over MgSO4, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel (30% ethyl acetate in hexane) to give 101 as yellow solid (15 mg, 75% yield). mp = 131-132 ºC. IR (neat): 3854, 2931, 1704, 1618, 1522, 1447, 1376, 1309,

−1 1 1184, 1120 cm . H NMR (500 MHz, CDCl3) δ 9.23 (t, J = 4.5 Hz, 1H, NH), 8.61 (s, 1H, ArH-

Coumarin), 7.00 (s, 1H, ArH-Coumarin), 6.69 (d, J = 2.0 Hz, 1H, ArH), 6.55 (d, J = 2.0 Hz, 1H,

ArH), 3.78-3.69 (m, 2H, -CH2-N-C(O)-), 3.33-3.28 (m, 4H, Coumarin N-CH2-), 3.22 (ddd, J =

15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H), 2.87-2.73 (Overlapping multiples, 5H, 10a-H,

228

Coumarin N-CH2-CH2-CH2-), 2.52-2.44 (m, 1H, 8eq-H), 2.39-2.28 (m, 1H, 8ax-H), 2.21-2.08

(m, 2H, 7eq-H, 10ax-H), 2.02-1.90 (overlapping multiples, 5H, Coumarin N-CH2-CH2-CH2-, 6a-

H), 1.54-1.37 (m, 14H, 7ax-H, 2’-H, -C(CH3)2-CH2-NH-, -C(O)O-C(CH3)2-, 6-Me, especially

1.48, s, 6-Me), 1.28-1.20 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-),

1.17 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.84 (t, J = 6.5 Hz, 3H, 7’-H). 13C NMR

(100 MHz, CDCl3) δ 209.33 (>C=O), 175.10 (-C(O)-O), 164.09 (-C(O)-NH), 163.10 (-C(O)-O),

152.89 (ArC-1 or ArC-5), 151.20 (ArC-5 or ArC-1), 149.73 (tertiary aromatic), 148.43 (ArC-

Coumarin), 127.28 (ArC-Coumarin), 119.71 (ArC-Coumarin), 114.72 (ArC-Coumarin), 113.26

(ArC-Coumarin), 112.57 (tertiary aromatic), 109.45 (ArC-2 and ArC-4), 108.55 (ArC-

Coumarin), 105.79 (ArC-Coumarin), 50.44, 50.02, 48.18, 47.28, 46.32, 44.67, 44.55, 40.91,

37.78, 34.79, 31.96, 30.16, 28.74, 27.88, 27.69, 26.68, 24.76, 24.01, 22.99, 22.88, 21.41, 20.50,

20.27, 19.07, 14.30. Mass spectrum (ESI) m/z (relative intensity) 739 (M+ + H, 100). Exact mass

+ (ESI) calculated for C45H49N2O7 (M + H), 739.4322; found 739.4314. LC/MS analysis (Waters

MicroMass ZQ system) showed purity of 99.3% and retention time of 6.5 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 2,2-dimethyl-4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3- f]pyrido[3,2,1-ij]quinoline-10-carboxamido)butanoate (102). The reaction was performed similar to as described for 101 using coumarin 343 (10 mg, 0.03 mmol), CDI (17 mg, 0.10 mmol), 98 (29 mg, 0.06 mmol) in dry DMF (1 ml) and gave 14 mg (78% yield) of 102 as a yellow solid. mp = 132-133 ºC. IR (neat): 3728, 2931, 1747, 1706, 1618, 1588, 1510, 1444,

−1 1 1366, 1309, 1175, 1110 cm . H NMR (500 MHz, CDCl3) δ 8.92 (t, J = 4.5 Hz, 1H, NH), 8.59

(s, 1H, ArH-Coumarin), 7.00 (s, 1H, ArH-Coumarin), 6.70 (d, J = 2.0 Hz, 1H, ArH), 6.52 (d, J =

229

2.0 Hz, 1H, ArH), 3.61-3.45 (m, 2H, -CH2-NH-C(O)-), 3.37-3.25 (overlapping multiples, 5H,

10eq-H, Coumarin -N-CH2-), 2.88 (t, J = 6.5 Hz, 2H, Coumarin -N-CH2-CH2-CH2-), 2.80-2.68

(Overlapping multiples, 3H, 10a-H, Coumarin -N-CH2-CH2-CH2-), 2.45-2.33 (m, 1H, 8eq-H),

2.45-2.20 (m, 5H, 8ax-H, 7eq-H, 10ax-H, -NH-CH2-CH2-), 2.10-1.93 (overlapping multiples,

5H, Coumarin N-CH2-CH2-CH2-, 6a-H), 1.55-1.46 (m, 9H, 7ax-H, 2’-H, -C(O)O-C(CH3)2-),

1.42 (s, 3H, 6-Me), 1.26-1.17 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -

C(CH3)2-), 1.14 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.83 (t, J = 6.5 Hz, 3H, 7’-

13 H). C NMR (100 MHz, CDCl3) δ 209.14 (>C=O), 175.50 (-C(O)-O), 163.33 (-C(O)-NH),

162.90 (-C(O)-O), 152.56 (ArC-1 or ArC-5), 150.94 (ArC-5 or ArC-1), 149.53 (tertiary aromatic), 147.86 (ArC-Coumarin), 126.88 (ArC-Coumarin), 119.42 (ArC-Coumarin), 114.14

(ArC-Coumarin), 112.89 (ArC-Coumarin), 112.24 (tertiary aromatic), 109.22 (ArC-2 and ArC-

4), 108.20 (ArC-Coumarin), 105.63 (ArC-Coumarin), 50.13, 49.73, 47.88, 46.19, 44.23, 41.73,

40.63, 39.64, 37.47, 35.97, 34.78, 31.65, 30.24, 29.86, 28.52, 28.45, 27.56, 27.38, 26.67, 25.21,

24.97, 24.46, 22.57, 21.10, 20.16, 20.05, 18.75, 14.00. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.7% and retention time of 6.7 min for the title compound.

(6aS,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 2,2-dimethyl-5-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3- f]pyrido[3,2,1-ij]quinoline-10-carboxamido)pentanoate (103). The reaction was performed similar to as described for 101 using coumarin 343 (10 mg, 0.03 mmol), CDI (17 mg, 0.10 mmol), 99 (30 mg, 0.06 mmol) in dry DMF (1 ml) and gave 15 mg (80% yield) of 103 as a yellow solid. mp = 130-131 ºC. IR (neat): 3747, 2930, 1693, 1617, 1587, 1505, 1476, 1366,

−1 1 1308, 1174, 1109 cm . H NMR (500 MHz, CDCl3) δ 8.93 (t, J = 4.5 Hz, 1H, NH), 8.60 (s, 1H,

ArH-Coumarin), 7.00 (s, 1H, ArH-Coumarin), 6.68 (d, J = 2.0 Hz, 1H, ArH), 6.42 (d, J = 2.0 Hz,

230

1H, ArH), 3.54-3.42 (m, 2H, -CH2-NH-C(O)-), 3.36-3.25 (overlapping multiples, 5H, 10eq-H,

Coumarin -N-CH2-), 2.88 (t, J = 7.0 Hz, 2H, Coumarin -N-CH2-CH2-CH2-), 2.77 (t, J = 7.0 Hz,

2H, Coumarin -N-CH2-CH2-CH2-), 2.70 (m as td, J = 12.4 Hz, J = 3.0 Hz, 1H, 10a-H), 2.59-

2.53 (m, 1H, 8eq-H), 2.44-2.34 (m, 1H, 8ax-H), 2.24-2.10 (m, 2H, 10ax-H, 7eq-H), 2.10-1.92

(overlapping multiples, 5H, Coumarin -N-CH2-CH2-CH2-, 6a-H), 1.85-1.65 (m, 4H, -NH-CH2-

CH2-CH2-), 1.54-1.47 (m, 6H, 7ax-H, 2’-H, especially 1.48, s, 6-Me), 1.39 (s, 3H, -C(O)O-

C(CH3)2-), 1.35 (s, 3H, -C(O)O-C(CH3)2-), 1.24-1.14 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.13 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.83 (t, J

13 = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 210.33 (>C=O), 176.60 (-C(O)-O), 163.75

(-C(O)-NH), 162.59 (-C(O)-O), 153.31 (ArC-1 or ArC-5), 150.79 (ArC-5 or ArC-1), 151.68

(tertiary aromatic), 148.75 (ArC-Coumarin), 127.66 (ArC-Coumarin), 119.81 (ArC-Coumarin),

114.79 (ArC-Coumarin), 113.20 (ArC-Coumarin), 112.50 (tertiary aromatic), 110.10 (ArC-2 and

ArC-4), 109.05 (ArC-Coumarin), 106.23 (ArC-Coumarin), 50.97, 50.56, 48.70, 46.96, 45.08,

43.24, 41.49, 40.55, 38.66, 38.26, 35.65, 32.48, 30.69, 29.36, 29.25, 28.40, 28.22, 27.51, 26.00,

25.65, 25.28, 23.40, 21.94, 21.00, 20.89, 19.59, 14.83. Mass spectrum (ESI) m/z (relative

+ + intensity) 767 (M + H, 100). Exact mass (ESI) calculated for C47H63N2O7 (M + H), 767.4635; found 767.4636. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.3% and retention time of 6.7 min for the title compound.

(6aS,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 2,2-dimethyl-6-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3- f]pyrido[3,2,1-ij]quinoline-10-carboxamido)hexanoate (104). The reaction was performed similar to as described for 101 using coumarin 343 (10 mg, 0.03 mmol), CDI (17 mg, 0.10 mmol), 100 (31 mg, 0.06 mmol) in dry DMF (1 ml) and gave 14 mg (78% yield) of 104 as a

231 yellow solid. mp = 133-134 ºC. IR (neat): 3723, 2961, 1709, 1619, 1517, 1418, 1260, 1141 cm−1.

1 H NMR (500 MHz, CDCl3) δ 8.87 (t, J = 4.5 Hz, 1H, NH), 8.60 (s, 1H, ArH-Coumarin), 7.00

(s, 1H, ArH-Coumarin), 6.68 (d, J = 2.0 Hz, 1H, ArH), 6.41 (d, J = 2.0 Hz, 1H, ArH), 3.45 (q, J

= 7.0 Hz, 2H, -CH2-NH-C(O)-), 3.36-3.25 (overlapping multiples, 5H, 10eq-H, Coumarin -N-

CH2-), 2.88 (t, J = 6.0 Hz, 2H, Coumarin -N-CH2-CH2-CH2-), 2.77 (t, J = 6.0 Hz, 2H, Coumarin

-N-CH2-CH2-CH2-), 2.69 (m as td, J = 12.0 Hz, J = 3.5 Hz, 1H, 10a-H), 2.59-2.52 (m, 1H, 8eq-

H), 2.44-2.36 (m, 1H, 8ax-H), 2.24-2.10 (m, 2H, 10ax-H, 7eq-H), 2.10-1.92 (overlapping multiples, 5H, Coumarin -N-CH2-CH2-CH2-, 6a-H), 1.80-1.63 (m, 4H, -NH-CH2-CH2-CH2-

CH2), 1.52-1.46 (m, 6H, 7ax-H, 2’-H, especially 1.47, s, 6-Me), 1.38 (s, 3H, -C(O)O-C(CH3)2-),

1.33 (s, 3H, -C(O)O-C(CH3)2-), 1.23-1.14 (m, 14H, 3’-H, 4’-H, 5’-H, -NH-CH2-CH2-CH2-CH2, -

C(CH3)2-, especially 1.21, s, -C(CH3)2-), 1.13 (s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H),

13 0.83 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.17 (>C=O), 176.01 (-C(O)-O),

163.34 (-C(O)-NH), 162.92 (-C(O)-O), 153.88 (ArC-1 or ArC-5), 150.79 (ArC-5 or ArC-1),

149.58 (tertiary aromatic), 147.88 (ArC-Coumarin), 126.87 (ArC-Coumarin), 119.43 (ArC-

Coumarin), 114.18 (ArC-Coumarin), 112.81 (ArC-Coumarin), 112.09 (tertiary aromatic), 109.25

(ArC-2 and ArC-4), 108.20 (ArC-Coumarin), 105.58 (ArC-Coumarin), 50.13, 49.72, 47.81,

46.09, 44.24, 42.56, 40.63, 40.17, 39.37, 37.40, 34.81, 31.63, 30.23, 29.99, 29.85, 28.52, 28.42,

27.56, 27.38, 26.66, 25.15, 24.71, 24.43, 22.56, 22.35, 21.10, 20.16, 20.05, 18.74, 13.99. Mass spectrum (ESI) m/z (relative intensity) 781 (M+ + H, 100). Exact mass (ESI) calculated for

+ C34H51N2O5S (M + H), 781.4792; found 781.4772. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 98.5% and retention time of 6.6 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 3-(7-methoxy-2-oxo-2H-chromene-3-carboxamido)-2,2-

232 dimethylpropanoate (105). The reaction was performed similar to as described for 101 using 7- methoxycoumarin-3-carboxylic acid (10 mg, 0.04 mmol), CDI (22 mg, 0.13 mmol), 97 (38 mg,

0.08 mmol) in dry DMF (1 ml) and gave 14 mg (78% yield) of 105 as a white crystalline solid. mp = 119-120 ºC. IR (neat): 3358, 2930, 1725, 1618, 1537, 1413, 1372, 1219, 1118, 1026 cm−1.

1 H NMR (500 MHz, CDCl3) δ 9.11 (t, J = 3.0 Hz, 1H, NH), 8.85 (s, 1H, ArH-Coumarin), 7.59

(d, J = 9.0 Hz, 1H, ArH-Coumarin), 6.94 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H, ArH-Coumarin), 6.85

(d, J = 2.5 Hz, 1H, ArH-Coumarin), 6.71 (d, J = 1.5 Hz, 1H, ArH), 6.52 (d, J = 2.0 Hz, 1H,

ArH), 3.91 (s, 3H, O-CH3), 3.82 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H, -NH-CH2-), 3.72 (dd, J = 8.5

Hz, J = 2.5 Hz, 1H, -NH-CH2-), 3.20 (ddd, J = 15.0 Hz, J = 3.5 Hz, J = 2.0 Hz, 1H, 10eq-H),

2.80 (m as td, J = 12.0 Hz, J = 2.5 Hz, 1H, 10a-H), 2.49-2.43 (m, 1H, 8eq-H), 2.38-2.29 (m, 1H,

8ax-H), 2.20-2.09 (m, 2H, 7eq-H, 10ax-H), 1.99-1.90 (m as td, J = 12.0 Hz, J = 3.0 Hz, 1H, 6a-

H), 1.55-1.47 (m, 12H, 7ax-H, 2’-H, -C(O)O-C(CH3)2-, 6-Me, especially 1.49, s, 6-Me), 1.24-

1.15 (m, 15H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.23, s, -C(CH3)2-, and 1.19, s, 6-Me),

13 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.83 (t, J = 6.5 Hz, 3H, 7’-H). C NMR (100 MHz, CDCl3)

δ 209.06 (>C=O), 174.71 (C(O)-O), 164.64 (-C(O)-NH-), 162.33 (-C(O)-O), 161.59 (ArC-

Coumarin), 156.60 (ArC-Coumarin), 153.90 (ArC-Coumarin), 150.90 (ArC-1 or ArC-5), 149.29

(ArC-5 or ArC-1), 148.26 (tertiary aromatic), 130.85 (ArC-Coumarin), 114.82 (tertiary aromatic), 114.36 (ArC-Coumarin), 113.74 (ArC-Coumarin), 113.08 (ArC-Coumarin), 112.42

(ArC-2 or ArC-4), 112.00 (ArC-4 or ArC-2), 100.16 (ArC-Coumarin), 55.88, 47.86, 47.02,

45.97, 44.22, 40.63, 37.46, 34.57, 31.62, 30.23, 29.83, 29.58, 28.47, 28.42, 27.55, 26.47, 24.43,

23.82, 22.58, 22.55, 18.72, 13.98. Mass spectrum (ESI) m/z (relative intensity) 674 (M+ + H,

+ 100). Exact mass (ESI) calculated for C40H50NO8 (M + H), 674.3693; found 674.3685. LC/MS

233 analysis (Waters MicroMass ZQ system) showed purity of 99.2% and retention time of 6.2 min for the title compound.

(6aR,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 4-(6-methoxy-2-oxo-2H-chromene-3-carboxamido)-2,2- dimethylbutanoate (106). The reaction was performed similar to as described for 101 using 7- methoxycoumarin-3-carboxylic acid (10 mg, 0.04 mmol), CDI (22 mg, 0.13 mmol), 98 (39 mg,

0.08 mmol) in dry DMF (1 ml) and gave 15 mg (80% yield) of 106 as a white crystalline solid. mp = 118-119 ºC. IR (neat): 3363, 2930, 1740, 1619, 1539, 1413, 1372, 1223, 1142, 1114, 1027

−1 1 cm . H NMR (500 MHz, CDCl3) δ 8.84 (t, J = 5.0 Hz, 1H, NH), 8.82 (s, 1H, ArH-Coumarin),

7.58 (d, J = 9.0 Hz, 1H, ArH-Coumarin), 6.94 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H, ArH-Coumarin),

6.86 (d, J = 2.5 Hz, 1H, ArH-Coumarin), 6.70 (d, J = 2.0 Hz, 1H, ArH), 6.51 (d, J = 2.0 Hz, 1H,

ArH), 3.91 (s, 3H, O-CH3), 3.62-3.48 (m, 2H, -NH-CH2-), 3.30 (ddd, J = 15.0 Hz, J = 3.5 Hz, J

= 2.0 Hz, 1H, 10eq-H), 2.72 (m as td, J = 11.0 Hz, J = 3.0 Hz, 1H, 10a-H), 2.59-2.52 (m, 1H,

8eq-H), 2.45-2.32 (m, 1H, 8ax-H), 2.25-2.10 (m, 2H, 7eq-H, 10ax-H), 2.09-1.92 (multiplet overlapping, 3H, -NH-CH2-CH2-, 6a-H), 1.54-1.46 (m, 9H, 7ax-H, 2’-H, -C(O)O-C(CH3)2-),

1.44 (s, 3H, 6-Me), 1.24-1.11 (m, 15H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.23, s, -

C(CH3)2-, and 1.14, s, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.83 (t, J = 6.5 Hz, 3H, 7’-H).

13 C NMR (100 MHz, CDCl3) δ 209.20 (>C=O), 175.38 (C(O)-O), 164.68 (-C(O)-NH-), 161.82

(-C(O)-O), 161.66 (ArC-Coumarin), 156.56 (ArC-Coumarin), 153.90 (ArC-Coumarin), 150.95

(ArC-1 or ArC-5), 149.48 (ArC-5 or ArC-1), 148.01 (tertiary aromatic), 130.79 (ArC-Coumarin),

114.88 (tertiary aromatic), 114.11 (ArC-Coumarin), 113.86 (ArC-Coumarin), 112.96 (ArC-2 or

ArC-4), 112.37 (ArC-4 or ArC-2), 112.16 (ArC-Coumarin), 100.22 (ArC-Coumarin), 55.91,

47.89, 46.22, 44.22, 41.71, 40.66, 39.49, 37.47, 36.23, 34.84, 32.13, 31.83, 31.64, 30.24, 29.86,

234

29.60, 29.27, 28.53, 28.44, 27.56, 26.71, 26.31, 25.22, 25.02, 24.46, 23.33, 22.57, 18.75, 13.99.

Mass spectrum (ESI) m/z (relative intensity) 688 (M+ + H, 100). Exact mass (ESI) calculated for

+ C41H54NO8 (M + H), 688.3849; found 688.3842. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.5% and retention time of 6.3 min for the title compound.

(6aS,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 5-(7-methoxy-2-oxo-2H-chromene-3-carboxamido)-2,2- dimethylpentanoate (107). The reaction was performed similar to as described for 101 using 7- methoxycoumarin-3-carboxylic acid (10 mg, 0.04 mmol), CDI (22 mg, 0.13 mmol), 99 (40 mg,

0.08 mmol) in dry DMF (1 ml) and gave 14 mg (78% yield) of 107 as a white crystalline solid. mp = 120-121 ºC. IR (neat): 3737, 2930, 1746, 1618, 1540, 1413, 1370, 1218, 1116, 1028 cm−1.

1 H NMR (500 MHz, CDCl3) δ 8.85 (t, J = 5.5 Hz, 1H, NH), 8.83 (s, 1H, ArH-Coumarin), 7.58

(d, J = 9.0 Hz, 1H, ArH-Coumarin), 6.94 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H, ArH-Coumarin), 6.86

(d, J = 2.5 Hz, 1H, ArH-Coumarin), 6.69 (d, J = 2.0 Hz, 1H, ArH), 6.41 (d, J = 2.0 Hz, 1H,

ArH), 3.91 (s, 3H, O-CH3), 3.57-3.46 (m, 2H, -NH-CH2-), 3.30 (ddd, J = 15.0 Hz, J = 3.5 Hz, J

= 2.0 Hz, 1H, 10eq-H), 2.70 (m as td, J = 13.0 Hz, J = 2.5 Hz, 1H, 10a-H), 2.59-2.53 (m, 1H,

8eq-H), 2.44-2.33 (m, 1H, 8ax-H), 2.27-2.10 (m, 2H, 7eq-H, 10ax-H), 2.01-1.93 (m as td, J =

12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.84-1.66 (m, 4H, -NH-CH2-CH2-CH2-), 1.55-1.46 (m, 6H, 7ax-

H, 2’-H, especially 1.48, s, 6-Me), 1.39 (s, 3H, -C(O)O-C(CH3)2-), 1.36 (s, 3H, -C(O)O-

C(CH3)2-), 1.23-1.15 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially 1.22, s, -C(CH3)2-), 1.13

(s, 3H, 6-Me), 1.06 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.83 (t, J = 6.5 Hz, 3H, 7’-H). 13C NMR (100

MHz, CDCl3) δ 209.30 (>C=O), 175.81 (C(O)-O), 164.64 (-C(O)-NH-), 161.88 (-C(O)-O),

161.88 (ArC-Coumarin), 161.67 (ArC-Coumarin), 156.54 (ArC-Coumarin), 153.91 (ArC-

Coumarin), 150.84 (ArC-1 or ArC-5), 149.55 (ArC-5 or ArC-1), 148.04 (tertiary aromatic),

235

130.78 (ArC-Coumarin), 114.19 (tertiary aromatic), 113.82 (ArC-Coumarin), 112.89 (ArC-

Coumarin), 112.38 (ArC-2 or ArC-4), 112.04 (ArC-4 or ArC-2), 100.21 (ArC-Coumarin), 55.90,

47.87, 46.15, 44.23, 42.39, 40.67, 39.94, 37.70, 37.42, 34.86, 31.63, 30.24, 29.84, 28.52, 28.41,

27.56, 26.71, 25.12, 24.88, 24.44, 22.56, 18.74, 13.99. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.0% and retention time of 6.4 min for the title compound.

(6aS,10aR)-6,6-dimethyl-3-(2-methyloctan-2-yl)-9-oxo-6a,7,8,9,10,10a-hexahydro-6H- benzo[c]chromen-1-yl 6-(7-methoxy-2-oxo-2H-chromene-3-carboxamido)-2,2- dimethylhexanoate (108). The reaction was performed similar to as described for 101 using 7- methoxycoumarin-3-carboxylic acid (10 mg, 0.04 mmol), CDI (22 mg, 0.13 mmol), 97 (41 mg,

0.08 mmol) in dry DMF (1 ml) and gave 16 mg (81% yield) of 108 as a white crystalline solid. mp = 121-122 ºC. IR (neat): 3352, 2927, 1731, 1619, 1540, 1463, 1372, 1224, 1141, 1115, 1026

−1 1 cm . H NMR (500 MHz, CDCl3) δ 8.83 (s, 1H, ArH-Coumarin), 8.78 (t, J = 5.5 Hz, 1H, NH),

7.59 (d, J = 9.0 Hz, 1H, ArH-Coumarin), 6.94 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H, ArH-Coumarin),

6.86 (d, J = 2.5 Hz, 1H, ArH-Coumarin), 6.69 (d, J = 2.0 Hz, 1H, ArH), 6.40 (d, J = 2.0 Hz, 1H,

ArH), 3.91 (s, 3H, O-CH3), 3.51-3.44 (m, 2H, -NH-CH2-), 3.30 (ddd, J = 15.0 Hz, J = 3.5 Hz, J

= 2.0 Hz, 1H, 10eq-H), 2.69 (m as td, J = 14.0 Hz, J = 3.5 Hz, 1H, 10a-H), 2.59-2.52 (m, 1H,

8eq-H), 2.45-2.35 (m, 1H, 8ax-H), 2.22-2.10 (m, 2H, 7eq-H, 10ax-H), 2.01-1.93 (m as td, J =

12.0 Hz, J = 3.0 Hz, 1H, 6a-H), 1.82-1.66 (m, 4H, -NH-CH2-CH2-CH2-CH2-), 1.54-1.44 (m, 8H,

7ax-H, 2’-H, -NH-CH2-CH2-CH2-CH2-, especially 1.48, s, 6-Me), 1.38 (s, 3H, -C(O)O-C(CH3)2-

), 1.34 (s, 3H, -C(O)O-C(CH3)2-), 1.24-1.11 (m, 12H, 3’-H, 4’-H, 5’-H, -C(CH3)2-, especially

1.21, s, -C(CH3)2-), 1.13 (s, 3H, 6-Me), 1.07 (sextet, J = 7.5 Hz, 2H, 6’-H), 0.83 (t, J = 6.5 Hz,

13 3H, 7’-H). C NMR (100 MHz, CDCl3) δ 209.18 (>C=O), 176.23 (C(O)-O), 164.64 (-C(O)-NH-

), 161.82 (-C(O)-O), 156.52 (ArC-Coumarin), 153.91 (ArC-Coumarin), 150.79 (ArC-1 or ArC-

236

5), 149.56 (ArC-5 or ArC-1), 148.01 (tertiary aromatic), 130.78 (ArC-Coumarin), 114.91

(tertiary aromatic), 114.16 (ArC-Coumarin), 113.83 (ArC-Coumarin), 112.85 (ArC-2 or ArC-4),

112.04 (ArC-4 or ArC-2), 100.21 (ArC-Coumarin), 55.90, 47.81, 46.09, 44.23, 42.56, 40.64,

40.09, 39.59, 37.41, 34.84, 31.63, 29.85, 28.52, 28.41, 27.56, 26.70, 25.14, 24.75, 24.43, 22.56,

22.34, 18.74, 13.98. LC/MS analysis (Waters MicroMass ZQ system) showed purity of 99.6% and retention time of 6.2 min for the title compound.

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.19, 20 Results from the competition assays were analyzed using nonlinear regression to determine the IC50 values for the ligand; Ki

21 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.20, 22 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

237 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).

Plasma stability.19 Compounds or their proposed metabolites were diluted (200 µM) in mouse or rat plasma and incubated at 37 C, 100 rpm. At various time points, samples were taken, diluted 1:4 in acetonitrile and centrifuged to precipitate the proteins. The resulting supernatant was analyzed by HPLC. 4-Nitrophenyl butyrate was used as a control in each experiment. In vitro plasma half-lives were determined using exponential decay calculations in Prism

(GraphPad).

HPLC Analysis: Chromatographic separation was achieved using a Supelco Discovery C18 (4.6 x 250 mm) column on a Waters Alliance HPLC system. Mobile phase consisted of acetonitrile

(A) and a mixture of 60% water (acidified with 8.5% o-phosphoric acid) and 40% acetonitrile

Spectral measurements. The fluorescence spectra were recorded on Hitachi F-7000 spectrofluorometer in ethanol and acetonitrile at a concentration of 1 mg mL–1. The fluorescence spectra were recorded using excitation into the maximum of the longest wavelength absorption band program. The fluorescence of the solution was measured in a 1 cm3 cuvette in the right

238 angle arrangement. The fluorescence spectra were taken by the excitation into the maximum of the longest wavelength absorption band.

239

REFERENCES

1. Granier, S.; Kobilka, B. A new era of GPCR structural and chemical biology. Nat Chem

Biol 2012, 8, 670-3.

2. Ma, Z.; Du, L.; Li, M. Toward fluorescent probes for G-protein-coupled receptors

(GPCRs). J Med Chem 2014, 57, 8187-203.

3. Lakowicz, J. R. Principles of Fluorescence Spectroscopy. 2006.

4. Filmore, D. It's a GPCR world. Mod. Drug Discovery 2004, 7, 24-28.

5. Leopoldo, M.; Lacivita, E.; Berardi, F.; Perrone, R. Developments in fluorescent probes for receptor research. Drug Discov Today 2009, 14, 706-12.

6. Vernall, A. J.; Hill, S. J.; Kellam, B. The evolving small-molecule fluorescent-conjugate toolbox for Class A GPCRs. Br J Pharmacol 2014, 171, 1073-84.

7. Daly, C. J.; McGrath, J. C. Fluorescent ligands, antibodies, and proteins for the study of receptors. Pharmacol Ther 2003, 100, 101-18.

8. Daly, C. J.; Ross, R. A.; Whyte, J.; Henstridge, C. M.; Irving, A. J.; McGrath, J. C.

Fluorescent ligand binding reveals heterogeneous distribution of adrenoceptors and 'cannabinoid- like' receptors in small arteries. Br J Pharmacol 2010, 159, 787-96.

9. Martin-Couce, L.; Martin-Fontecha, M.; Capolicchio, S.; Lopez-Rodriguez, M. L.;

Ortega-Gutierrez, S. Development of endocannabinoid-based chemical probes for the study of cannabinoid receptors. J Med Chem 2011, 54, 5265-9.

10. Martin-Couce, L.; Martin-Fontecha, M.; Palomares, O.; Mestre, L.; Cordomi, A.;

Hernangomez, M.; Palma, S.; Pardo, L.; Guaza, C.; Lopez-Rodriguez, M. L.; Ortega-Gutierrez,

S. Chemical probes for the recognition of cannabinoid receptors in native systems. Angew Chem

Int Ed Engl 2012, 51, 6896-9.

240

11. Yates, A. S.; Doughty, S. W.; Kendall, D. A.; Kellam, B. Chemical modification of the naphthoyl 3-position of JWH-015: in search of a fluorescent probe to the cannabinoid CB2 receptor. Bioorg Med Chem Lett 2005, 15, 3758-62.

12. Bai, M.; Sexton, M.; Stella, N.; Bornhop, D. J. MBC94, a conjugable ligand for cannabinoid CB 2 receptor imaging. Bioconjug Chem 2008, 19, 988-92.

13. Petrov, R. R.; Ferrini, M. E.; Jaffar, Z.; Thompson, C. M.; Roberts, K.; Diaz, P. Design and evaluation of a novel fluorescent CB2 ligand as probe for receptor visualization in immune cells. Bioorg Med Chem Lett 2011, 21, 5859-62.

14. Ling, X.; Zhang, S.; Shao, P.; Li, W.; Yang, L.; Ding, Y.; Xu, C.; Stella, N.; Bai, M. A novel near-infrared fluorescence imaging probe that preferentially binds to cannabinoid receptors

CB2R over CB1R. Biomaterials 2015, 57, 169-78.

15. Neises, B.; Steglich, W. Simple Method for the Esterification of Carboxylic Acids.

Angewandte Chemie International Edition in English 1978, 17, 522-524.

16. Castillo, J.-C.; Orrego-Hernández, J.; Portilla, J. Cs2CO3-Promoted Direct N-Alkylation:

Highly Chemoselective Synthesis of N-Alkylated Benzylamines and Anilines. European Journal of Organic Chemistry 2016, 2016, 3824-3835.

17. Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J.; Zhang, C.; Lee, Y.; Foxman, B.

M.; Henklein, P.; Hanay, C.; Mugge, C.; Wenschuh, H.; Klose, J.; Beyermann, M.; Bienert, M.

The uronium/guanidinium Peptide coupling reagents: finally the true uronium salts. Angew Chem

Int Ed Engl 2002, 41, 441-5.

18. Staab, H. A. New Methods of Preparative Organic Chmistry IV. Syntheses Using

Heterocyclic Amides (Azolides). Angewandte Chemie International Edition in English 1962, 1,

351-367.

241

19. Nikas, S. P.; Sharma, R.; Paronis, C. A.; Kulkarni, S.; Thakur, G. A.; Hurst, D.; Wood, J.

T.; Gifford, R. S.; Rajarshi, G.; Liu, Y.; Raghav, J. G.; Guo, J. J.; Jarbe, T. U.; Reggio, P. H.;

Bergman, J.; Makriyannis, A. Probing the carboxyester side chain in controlled deactivation (-)- delta(8)-tetrahydrocannabinols. J Med Chem 2015, 58, 665-81.

20. Nikas, S. P.; Alapafuja, S. O.; Papanastasiou, I.; Paronis, C. A.; Shukla, V. G.;

Chem 2010, 53, 6996-7010.

21. Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.

Biochem Pharmacol 1973, 22, 3099-108.

22. Ogawa, G.; Tius, M. A.; Zhou, H.; Nikas, S. P.; Halikhedkar, A.; Mallipeddi, S.;

Makriyannis, A. 3'-functionalized adamantyl cannabinoid receptor probes. J Med Chem 2015,

58, 3104-16.

242