Antibacterial Agents: 1,4-Disubstituted 1,2,3-Triazole Analogs of the Oxazolidinone a Dissertation Presented to the Faculty Of

Home , Aminocoumarin, Clorobiocin, Coumermycin A1, Eperezolid, Ranbezolid, Triazole

Antibacterial Agents: 1,4-Disubstituted 1,2,3-Triazole Analogs of the Oxazolidinone

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

George Acquaah-Harrison

June 2010

This dissertation titled

Antibacterial Agents: 1,4-Disubstituted 1,2,3-Triazole Analogs of the Oxazolidinone

GEORGE ACQUAAH-HARRISON

has been approved for

the Department of Chemistry and Biochemistry

and the College of Arts and Sciences by

Stephen C. Bergmeier

Professor of Chemistry and Biochemistry

Benjamin M. Ogles

Dean, College of Arts and Sciences 3

ABSTRACT

ACQUAAH-HARRISON, GEORGE, Ph.D., June 2010, Chemistry and Biochemistry

Antibacterial Agents: 1,4-Disubstituted 1,2,3-Triazole Analogs of the Oxazolidinone )

(235 pp.)

Director of Dissertation: Stephen C. Bergmeier

The rational design, development and synthesis of structurally diverse small molecule that target RNA is immensely important in antibacterial therapy. Utilizing rational design approach to drug discovery, two lead 4,5-disubstituted 2-oxazolidinone compounds that bind to the highly conserved region of bacterial RNA with high affinity and specificity had been previously identified. This biological target called the T box antiterminator system regulates the expression of many genes including aminoacyl synthethase genes and is found predominantly in Gram-positive pathogens.

But, owing to the moderate solubilities of these leads, the focus was directed to improving the solubility challenges without compromising biological activity. To address the solubility challenges with the intent of enhancing or maintaining biological activity, a library of one hundred eight 1,4-disubstituted 1,2,3-triazole compounds that encompasses the diversity elements of the oxazolidinones were developed. This library, which entailed the bioisosteric replacement of the oxazolidinone scaffold was afforded in high yield and purity by employing the regioselective copper(I)-catalyzed azide/alkyne cycloaddition reaction. Three lead compounds, GHB-7, GHB-9 and GHB-16 with enhanced biological activity were identified that rendered them important candidates for structural activity 4 relationship studies (SAR). Besides the SAR studies, few 1,5-regioisomers were prepared to investigate the effect of regioselectivity on biological activity.

By embarking on empirical observations, thirty-two structurally relevant analogs were prepared for the SAR and other structural elaboration studies. While biological evaluation is currently ongoing, the preliminary data of the analogs, GHB-144, GHB-

151, GHB-153, GHB-156 and GHB-157 coupled with the enhanced biological activity of GHB-7 relative to the lead oxazolidinone compounds re-inforce the plausibility of finding new 1,4-substituted 1,2,3-triazole compounds with improved antibacterial activity.

Approved: ______

Stephen C. Bergmeier

Professor of Chemistry and Biochemistry 5

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my academic advisor Dr. Stephen

C. Bergmeier for his mentorship, guidance, insightful discussions and encouragements during my study.

I would also like to thank the members of my dissertation committee Dr. Mark C.

McMills, Dr. Glen Jackson and Dr. Xiaozhuo Chen for accepting my invitation to serve on my committee. Special thanks go to the faculty of the Department of Chemistry and

Biochemistry, especially Dr Jared Butcher, Dr. Klaus Himmeldirk and Dr. Kumar

Pichumani as well as the administrative staff of the department of Chemistry and biochemistry for their suggestions and support during my study.

I am thankful to the members of Dr. Bergmeier’s research group both past (Dr.

Pulipaka Aravinda, Dr. Junfeng Huang, Dr. Ahbigit Nayek, Nova Emerald and Dr. Iwona

Maciagiewicz) and present (Dr. Crina M. Orac, Weihe Zhang, Fang Fang, Susann Krake,

Gregg Wells and Dennis Roberts) for their suggestions and constructive criticisms as well as promoting a conducive laboratory atmosphere. I am also thankful to Shu Zhou in Dr.

Hines laboratory for testing all triazole analogs.

I am greatly indebted to my family: Harris Osafo-Acquaah, Mary F. Kusi-

Appouh, Franklin Effah, Victoria Dankwa & family, Gina Peters & family, Kwabena

Appiah Peprah, Mary Appiah Peprah and the rest of Peprah family as well as Dr. Isaac

Blankson and Joseph Blankson for their incessant support, advice and prayers during my doctoral studies. 6

Finally to my wife, Vivian Acquaah-Harrison and three wonderful daughters,

Ama Afrakomah Acquaah-Harrison, Nana-Afia Acquaah-Harrison and Nana-Akua

Acquaah-Harrison, thank you for recognizing the importance of education. Your love,

Charisma, encouragement, patience and motivation is what energized me through this study. 7

Dedicated to my wife Vivian A. Acquaah-Harrison and Three wonderful daughters Ama

Afrakomah Acquaah-Harrison, Nana-Afia Antwiwaa Acquaah-Harrison and Nana-Akua

Osafoaa Acquaah-Harrisonson 8

TABLE OF CONTENTS

Page

Abstract...... 3 Acknowledgments...... 5 Dedication ...... 7 List of Tables ...... 9 List of Figures...... 10 List of Schemes...... 12 Chapter 1.Introduction...... 15 1.1 Statement of purpose ...... 15 1.2 Introduction...... 15 Chapter 2.Background...... 23 2.1 History of antibiotics ...... 23 2.1.1 Natural antibiotics...... 24 2.1.2 Synthetic antibiotics...... 32 2.1.3 Antibiotics: problems and challenges ...... 35 2.2 Antibacterial drug discovery...... 37 2.3 RNA domain as a novel target for antimicrobial therapy...... 38 2.3.1 T box antiterminator RNA System ...... 40 2.4 Identification of lead compound...... 42 2.4.1 Lead compound modification/optimization...... 44 2.5 Oxazolidinone compounds ...... 46 2.5.1 Oxazolidinone library generation ...... 47 2.6 Significance of research...... 51 Chapter 3.Synthesis and Preliminary Results of 1,4-Disubstituted 1,2,3-Triazole Library52 3.1 Introduction...... 52 3.2 Retrosynthetic analysis of the triazole library ...... 56 3.3 Synthesis of azide components...... 57 3.4 Synthesis of alkyne components...... 65 3.5 Determination of reaction condition for 1,2,3-triazole library synthesis...... 68 9

3.5.1 Model studies ...... 71 3.5.2 Determination of workup conditions ...... 72 3.5.3 Generation of 1,4-disubstituted 1,2,3-triazole library...... 74 3.6 Biological evaluation of 1,4-disubstituted 1,2,3-triazole compounds ...... 81 3.6.1 Fluorescence resonance energy transfer assay...... 81 3.6.2 Antibacterial Assay...... 87 3.7 Synthesis of 1,5-disubstituted 1,2,3-triazoles analogs...... 89 3.7.1 Biological evaluation of 1,5-analogs ...... 94 3.8 Conclusion ...... 95 Chapter 4.Structure Activity Studies and Structural Elaborations of the 1,2,3-Triazole Ring 97 4.1 Introduction...... 97 4.2 Plan for SAR studies on GHB-7...... 98 4.2.1 Synthesis of azide components ...... 103 4.2.2 Synthesis of alkyne components for GHB-7 ...... 107 4.2.3 Synthesis of GHB-7 analog ...... 107 4.3 Plan for SAR studies on GHB-9...... 109 4.3.1 Synthesis of azide components ...... 113 4.3.2 Synthesis of alkyne components...... 113 4.3.3 The synthesis of GHB-9 analogs ...... 114 4.4 Plan for SAR studies on GHB-16...... 115 4.4.1 Synthesis of azide and alkyne starting materials ...... 118 4.4.2 Synthesis of GHB-16 analogs...... 119 4.5 Enantioselective synthesis of selected 1,4-substituted 1,2,3-triazoles...... 119 4.5.1 Enzymatic resolution of Trans-2-azidocyclohexanol ...... 121 4.5.2 Determination of enantiomeric excess...... 123 4.5.3 The synthesis of enantiopure azide components...... 126 4.5.4 The synthesis of enantiopure 1,4-disubstituted 1,2,3-triazole analogs ...... 127 4.6 Biological evaluation of GHB-7, GHB-9, GHB-16 and enantiopure analogs... 128 4.7 Conclusion ...... 132 10

Chapter 5.Experimental...... 133 Chapter 6.References...... 201 11

LIST OF TABLES

Page

Table 2.1. Binding affinities for oxazolidinone model RNAs...... 49 Table 3.1. Yield and purity of 94a-i, using azide 76c and all nine alkynes...... 75 Table 3.2. Yield and purity of 95a-i, using azide 64b and all nine alkynes...... 75 Table 3.3. Yield and purity of 96a-i, using azide 76b and all nine alkynes...... 76 Table 3.4. Yield and purity of 97a-i, using azide 77a and all nine alkynes...... 76 Table 3.5. Yield and purity of 98a-i, using azide 64a and all nine alkynes...... 77 Table 3.6. Yield and purity of 99a-i, using azide 54d and all nine alkynes...... 77 Table 3.7. Yield and purity of 100a-i, using azide 54c and all nine alkynes...... 78 Table 3.8. Yield and purity of 101a-i, using azide 76a and all nine alkynes...... 78 Table 3.9. Yield and purity of 102a-i, using azide 54a and all nine alkynes...... 79 Table 3.10. Yield and purity of 103a-i, using azide 77b and all nine alkynes...... 79 Table 3.11. Yield and purity of 104a-i, using azide 77c and all nine alkynes...... 80 Table 3.12. Yield and purity of 105a-i, using azide 76d and all nine alkynes...... 80 Table 3.13. FRET data for analogs with enhanced RNA binding affinities...... 83 Table 3.14. FRET data for analogs with enhanced RNA binding specificity...... 85 Table 3.15. Minimum inhibitory concentrations (μM) of 1,2,3-triazole analogs...... 88 Table 3.16. 5'Rhd screening of 1,5-analogs for model RNA AM1A and C11U...... 95 Table 4.1. Syntheses of analogs to examine the importance of NH- group...... 108 Table 4.2. Syntheses of analogs to examine the importance of phenyl group...... 108 Table 4.3. Syntheses of GHB-7 analogs to examine the importance of amine substitution...... 109 Table 4.4. 1,4-disubstituted 1,2,3-triazole analog of GHB-9...... 114 Table 4.5. 5'Rhd screening of GHB-7 analogs for model RNA AM1A and C11U...... 129 Table 4.6. 5'Rhd screening of GHB-9 analogs for model RNA AM1A and C11U...... 130 Table 4.7. 5'Rhd screening of GHB-16 analogs for model RNA AM1A and C11U...... 131 Table 4.8. 5'Rhd screening of enantiopure analogs for model RNA AM1A and C11U 131 12

LIST OF FIGURES

Page

Figure 1.1 Structure of the oxazolidinone antibiotic, linezolid...... 18 Figure 1.2. Structures of oxazolidinone leads and the 1,4-disubstituted 1,2,3-triazole.... 19 Figure 1.3. Structure of lead 1,4-disubstituted 1,2,3-triazole compounds...... 21 Figure 1.4. Structures of 1,2,3-triazole analogs GHB-18 and GHB-60...... 22 Figure 2.1. Structure of penicillin, a -lactam antibiotic...... 24 Figure 2.2. Structure of streptomycin (2) and streptidine scaffold...... 25 Figure 2.3. Examples of aminoglycosides containing the aminocyclitol (6) ring...... 26 Figure 2.4. Examples of tetracycline antibiotics...... 27 Figure 2.5. Examples of macrolide antibiotics...... 28 Figure 2.6. Examples of glycopeptide antibiotiotics...... 30 Figure 2.7. Stucture of the lipopeptide antibiotic daptomycin...... 31 Figure 2.8. Example of a sulfonamide antibiotic...... 32 Figure 2.9. Examples of quinolone family of synthetic antibiotics...... 34 Figure 2.10. Structure of an oxazolidinone antibiotic...... 35 Figure 2.11. Structures of leads identified via metabolism and clinical observation...... 43 Figure 2.12. General outline of the reiterative cycle of rational design...... 44 Figure 2.13. Example of bioisosteric replacement of nicotine to ABT 148...... 45 Figure 2.14. Examples of compounds containing the oxazolidinone scaffold...... 47 Figure 2.15. Structures of oxazolidinone antibacterial agents...... 50 Figure 3.1. Four examples of 1,2,3-triazole drugs...... 53 Figure 3.2. Oxazolidinone leads, ideal and general structure of 1,2,3-triazole library..... 54 Figure 3.3. Oxazolidinone leads, ideal and general structure of 1,2,3-triazole library..... 55 Figure 3.4. Retrosynthethesis of amine substituted 1,2,3-triazole compound...... 57 Figure 3.5. Illustration of the conformational rigidity of compound 77a-c...... 65 Figure 3.6. The twelve requisite azide building blocks...... 65 Figure 3.7. Structures of the nine propargylamine building blocks...... 68 Figure 3.8. Structures of model RNAs AM1A and C11U...... 82 Figure 3.9. A correlation graph of 1,4-disubstituted 1,2,3-triazole analogs for model RNAs AM1A and C11U...... 84 Figure 3.10. Structures of the 1,4-disubstituted 1,2,3-triazoles that bind model RNAs... 86 Figure 3.11. Structures of viable 1,2,3-triazole analogs from antibacterial assay...... 89 Figure 3.12. Proton NMR of the 1,4-disubstituted 1,2,3-triazole analog 105b...... 93 Figure 3.13. Proton NMR of the 1,5-disubstituted 1,2,3-triazole analog 110a...... 93 Figure 3.14. Structures of the 1,5-disubstituted 1,2,3-triazole analogs...... 95 Figure 4.1. Structure of the three lead 1,4-disubstituted 1,2,3-triazoles...... 98 Figure 4.2. General structure of GHB-7 analogs for SAR studies...... 98 Figure 4.3. General structure of GHB-7 analogs for SAR studies...... 99 Figure 4.4. General structure of GHB-7 analogs for SAR studies...... 100 Figure 4.5. General structure of GHB-7 analogs for SAR studies...... 101 Figure 4.6. General structure of GHB-7 analogs for SAR studies...... 103 13

Figure 4.7. Structural comparison of GHB-7 and GHB-9...... 110 Figure 4.8. General structure of GHB-9 analogs for SAR studies...... 111 Figure 4.9. General structure of GHB-9 analogs for SAR studies...... 112 Figure 4.10. General structure of GHB-9 analogs for SAR studies...... 112 Figure 4.11. General structure of GHB-9 analogs for SAR studies...... 113 Figure 4.12. Structural comparison of GHB-9 and GHB-16...... 116 Figure 4.13. Structure of GHB-16 with highlighted key functional groups...... 116 Figure 4.14. Structure of GHB-16 analog amine substitution...... 117 Figure 4.15. Structure of GHB-16 analog devoid of hydroxy group...... 117

Figure 4.16. Structure of analog one CH2 chain shorter...... 118 Figure 4.17. Structure of 1,2,3-triazole analogs GHB-18 and GHB-60...... 120 Figure 4.18. Examples of chiral and racemate drugs...... 121 Figure 4.19. The 19F-NMR spectrum of compound 162...... 125 Figure 4.20. The 19F-NMR spectrum of compound 165...... 125 Figure 4.21. The 19F-NMR spectrum of compound 76d...... 126 14

LIST OF SCHEMES Page

Scheme 2.1. Illustration of the prodrug nature of sulfonamide...... 33 Scheme 2.2. Design and synthesis of oxazolidinone compound library...... 49 Scheme 3.1. Synthesis of 3-azido-2-hydroxypropyl esters from glycidol...... 58 Scheme 3.2. Plausible explanation of the low yields of carbamate esters...... 59 Scheme 3.3. Synthesis of carbamate azide components from allyl alcohol...... 60 Scheme 3.4. Azidohydrin synthesis by Sabitha and coworkers...... 60 Scheme 3.5. Reported azidohydrin synthesis by Onaka and coworkers...... 61 Scheme 3.6. Reported azidohydrin synthesis from oxiranes...... 61 Scheme 3.7. Regioselective synthesis of azidohydrin by Boruwa and coworkers...... 62 Scheme 3.8. Synthesis of 2-azido alcohol and azido esters from commercial epoxides..64 Scheme 3.9. Synthesis of alkynes from propargyl bromide and secondary amines...... 66 Scheme 3.10. Synthesis of alkynes from N-methyl proparylamine and alkyl bromide. .. 67 Scheme 3.11. Proposed mechanism for Cu(I)-catalyzed cycloaddition...... 70 Scheme 3.12. Model studies to study reaction conditions...... 72 Scheme 3.13. Synthesis of ruthenium catalyst for 1,5-disubstituted 1,2,3-triazole analogs...... 91 Scheme 3.14. Synthesis of the 1,5-disubstituted 1,2,3-triazole analogs...... 92 Scheme 4.1. Synthesis of the azide components from glycidol...... 104 Scheme 4.2. Alternative synthetic route for acyclic azido carbonate synthesis...... 105 Scheme 4.3. Synthesis of azide component without the hydroxy group...... 105 Scheme 4.4. Synthesis of azide component without the hydroxy group...... 106 Scheme 4.5. Synthesis of the (R)- and (S)- azide components...... 107 Scheme 4.6. Synthesis of (3-(prop-2-ynyloxy)propyl)benzene 4.27...... 107 Scheme 4.7. Synthesis of analog devoid of -OH and chiral variants of GHB-7...... 109 Scheme 4.8. The synthesis azide components for GHB-9 analogs...... 113 Scheme 4.9. Synthesis of homologous alkyne components...... 114 Scheme 4.10. Synthesis of GHB-9 (GHB-152) analog devoid of hydroxy group...... 115 Scheme 4.11. Synthesis of GHB-9 (GHB-153) analogs one homologous chain shorter...... 115 Scheme 4.12. The synthesis of alkyne component of GHB-16...... 118 Scheme 4.13. Synthesis of GHB-16 analogs for SAR studies...... 119 Scheme 4.14. Synthesis of azide precursor for enzymatic resolution...... 122 Scheme 4.15. Enzymatic resolution of trans-2-azidocyclohexanoate...... 123 Scheme 4.16. Determination of enantiomeric excess of trans-2-azidocyclohexanol. ... 124 Scheme 4.17. Synthesis of enantiopure azide building blocks...... 127 Scheme 4.18. Synthesis of 1,4-disubstituted 1,2,3-triazole enantiopure analogs...... 128 15

Chapter 1. Introduction

1.1 Statement of purpose

The purpose of this research was to develop 1,4-disubstituted 1,2,3-triazole-based

antibiotics that work by interfering with the mode of transcription of the T-box

antiterminator RNA in Gram-positive bacteria.

1.2 Introduction

The increasing public awareness of the need for a new class of antibiotics1,2 to address bacterial resistance to antibiotics provides the motivation for innovation in drug discovery.3 Historically, while the discovery of new antibiotics relied on natural sources,4-

6 this approach of drug discovery had several disadvantages in that the discovery process was very cumbersome, required tedious extractions, purifications and characterizations that spanned several years of rigorous research. In addition, discovery based on natural products often afforded limited quantities of the desired therapeutic agent.7-11 This approach of drug discovery is certainly not the desired route to access new antibiotics especially when there is an urgent need for new chemical entities for antimicrobial therapy. The market’s need for new antibiotics require an innovative, reliable, quick and efficient method that elevates a breakthrough in the identification of pharmacological agents that targets highly functionalized and conserved regions in the bacteria with minimal susceptibility to mutation.

The demand for new class of antibiotics that has a unique mechanism of action can be realized via combinatorial synthesis, where well thought through parallel 16

transformations are utilized to generate compound libraries with substituent diversities.

Such libraries, whose design and development rely on natural products and/or current

drugs coupled with high throughput screening, provide an unmatched potential of

generating pharmacological agents for a preselected target. In general, four key

therapeutic sites, namely: cell wall biosynthesis,12-21 protein synthesis machinery,22-29

DNA gyrase30-33 and RNA polymerase34-37 have been the targets for nearly all the

antibiotics on the markets. As rewarding as these antibiotics are in entering the market,

the observed and general pattern has been a gradual decline in efficacy owing to bacterial

mutations. Therefore, the intellectual inquiry into antibacterial therapy mandates the

reassessment of the site of action of the antibiotic. Identifying highly functional and

extremely conserved biological targets that are not susceptible to bacterial mutation will

increase the prospects of identifying new class of antibiotics. But the daunting challenge

dwells on how to identify a functionalized and highly conserved region in each target.

With the advances in biological target identification and characterization,8,38 the bacterial RNA has been mostly favored as the venue for antibiotic research.39-42 While the

RNA plays a critical role in the biochemical functioning of the cell, the rational

development of small molecules that targets bacterial RNA is still in its infancy.39

Conversely, the bacterial RNA, which has highly conserved with functionalized domains

that are presumably slow yielding to mutation in the bacteria has been projected a

valuable therapeutic target for antimicrobial therapy.39 This notion underlying the RNA

scheme has not been the case for families of natural antibiotics such as

aminoglycosides,43-46 macrolides47,48 as well as the tetracyclines49 and their plethora of 17

analogs that target ribosomal RNA. Similar decreases in efficacy had been the case for

the synthetic antibiotics, linezolid.50-55 While linezolid has been regarded as the substitute

for the treatment of vancomycin-resistant strains56 and others,57,58 resistance to linezolid59-

63 has increasingly begun to emerge for this antibiotics that entered clinical use in the year

2000. The decrease in efficacy and the fact that nearly 20 years will elapse before the

emergence of a new class of both natural (lipopeptide, Daptomycin)64,65 and synthetic

antibiotics (oxazolidinone, Linezolid)51 are strong and compelling indicators, of the need for new class of antibiotics.

To address this challenge is the discovery of the T box antitermination regulatory mechanism in Henkin’s laboratory.66 This RNA element is found mainly in Gram-

positive bacteria, where it functions as a modulator for the expression of many amino

acid related genes during transcription.67-69 The key features and functions of this RNA

element validates it as a potential therapeutic target. With the acquired knowledge of the

gene sequences in this highly functionalized and conserved T box region, two model

RNAs namely, AM1A (wildtype)70 and C11U70 (specificity control) have been designed to facilitate the screening of small molecules. Nonetheless, the challenging task has been the development and identification of small molecules capable of binding this RNA element with high affinity and specificity. 18

O O O N NH N O F Linezolid

Figure 1.1 Structure of the oxazolidinone antibiotic, linezolid.

The idea of meeting the goal-driven purpose of identifying new class of

pharmacological agents with improved biological profiles has motivated the generation of

large compound libraries via combinatorial synthesis by medicinal chemists.71-74 The appendage modification of the oxazolidinone ring has been a research agenda of many research laboratories.75-79 The Bergmeier research laboratory has also been interested in

developing methods to enhance the synthesis of structurally elaborate oxazolidinone

compounds that binds RNA. Well defining implications of this work was the

identification of RNA ligands for the highly conserved T box antiterminator RNA

system.80 Intriguingly, these oxazolidinones bind RNA without significant dependence on electrostatic interactions but exhibit marginal solubility.81 Further structural amplications

of the oxazolidinone ring afforded two additional 4,5-disubstituted oxazolidinone, ANB-

22 and ANB-40 (Figure 2), with enhanced biological activity.81,82 These lead oxazolidinones were also found to bind to the model RNAs at nanomolar concentration.81

Encouraged by these results, the Bergmeier laboratory was extremely interested in

preparing new analogs with the goal of optimizing the biological profile of ANB-22 and

ANB-40. While augmenting the diversity elements of ANB-22 and ANB-40 to enhanced

biological profile was a key agenda to the Bergmeier group, equally important was the

goal of finding potential replacement for the oxazolidinone nucleus. The 1,2,3-triazole 19

ring (Figure 1.2) was discerned as a suitable replacement for the oxazolidinone ring. The

rational for this choice dwelled on the fact that both rings orient appending substituents in

the same space as well as the ability of the 1,2,3-triazole to function as an amide bond

surrogate83,84 and a non-classical bioisosteric replacement for the oxazolidinone ring.

Besides, the 1,4-disubstituted 1,2,3-triazole analogs, which would be devoid of the

stereochemical complications of ANB-22 and ANB-40, were envisaged to enhance

automation and the synthesis of large compound library with the potential of exhibiting

improved solubility. The synthesis of such regioisomer of the 1,2,3-triazole is well

documented.85-89

O O O O NH H O N O NH O N O N N N O ANB-22 ANB-40

R4 R2 N N N R5 R1 N O R3

1,4-disubstituted 1,2,3-triazole

R1, R2, R3, R4, R5 = H, Alkyl, Aryl

Figure 1.2. Structures of oxazolidinone leads and the 1,4-disubstituted 1,2,3-triazole.

To date, the 1,4-disubstituted 1,2,3-triazoles are prepared following several

variants of the Cu(I)-catalyzed azide/alkyne cycloaddition (CuAAC).85-89 Yet, there are no optimal conditions87 for these CuAAC and the initial attempt using typical Sharpless conditions,85 which has found wide applications, did not afford the desired amine, 20 substituted 1,4-disubstituted 1,2,3-triazole library. Conversely, utilizing a modified version of the Cu(I)-mediated method previously reported by Sharpless and co-workers, the synthesis of the desired library was realized. A total of 108 triazole analogs of the lead oxazolidinone were prepared in good yield and purity.

Preliminary biological data signified that 49 triazole library members bind RNA but six exhibited improved RNA binding affinity. Of these, GHB-7 (Figure 1.3) showed improved RNA binding activity relative to the lead oxazolidinone ANB-22 and ANB-40.

Biological data from fluorescence anisotropy screening depicted that GHB- 7 interferes with the binding of tRNA to the antiterminator element but most likely exhibited a non- tRNA stabilization of the antiterminator as was observed for ANB-40. Enzymatic probing of GHB-7 using RNase V1 symbolized a non-covalent binding to the bulge region of the antiterminator. In addition to GHB-7, two other 1,4-disubstituted 1,2,3-triazole library members, GHB-9 and GHB-16 (Figure 1.3), were identified to be active against both

Bacillus subtilis and Staphylococcus aureus. These preliminary activities garnered for

GHB-7, GHB-9 and GHB-16 rendered them invaluable candidates for lead optimization via structure activity relationship (SAR) studies. A total of 23 analogs were prepared for the SAR studies and biological evaluation is currently ongoing. 21

O N N N N N O N N N N H N OH OH OH N N N

GHB-16 GHB-7 GHB-9

Figure 1.3. Structure of lead 1,4-disubstituted 1,2,3-triazole compounds.

There are two-regioisomeric forms, namely the 1,4- and 1,5- of the 1,2,3-triazole

that are afforded depending on the condition or the transition metal mediating the

cycloaddition reaction.85,90,91 Accordingly, for completeness few 1,5-regioisomers

91 including that of GHB-7 were prepared using [Cp*RuCl(PPh3)2] as catalyst. In general

the 1,4-regioisomers have found great therapeutic utility in medicinal chemistry84 though

few 1,5-regioisomeric compounds have emerged recently as inhibitors of

acetylcholinesterase.92,93 The focus of this endeavor was to affirm that the biological activity observed solely from the 1,4-analogs.

As observed in the oxazolidinone library,81 the cyclohexyl group appeared to be an important diversity element worth investigating. The majority of the 1,4-disubstituted

1,2,3-triazoles analogs bearing the cyclohexyl group exhibit above marginal to good biological profile. Two of these analogs namely, GHB-18 and GHB-60 (Figure 1.4) were among the analogs that showed improved RNA binding activity. As these compounds were all racemic mixtures, the focus was to prepare the enantiopure analogs of GHB-18 and GHB-60 in an attempt to enhance their initial activity as well as discerned whether chirality was required for biological activity. 22

N N N N N N N O N OH O O GHB-18 GHB-60

Figure 1.4. Structures of 1,2,3-triazole analogs GHB-18 and GHB-60.

In sum, the bioisosteric replacement of the lead oxazolidinone to generate the 1,4- disubstituted 1,2,3-triazole proved valuable as 1,4-disubstituted 1,2,3-triazole analogs with improved RNA binding affinities and specificities were identified. These analogs had simplication in their topological identity relative to the oxazolidinones. While biological evaluation of analogs for SAR and other structural modification studies are currently ongoing, there is significant biological evidence, particularly of GHB-7 that signify the amine substituted 1,4-disubstituted 1,2,3-triazole analogs as viable compounds for antimicrobial therapy. 23

Chapter 2. Background

2.1 History of antibiotics

The increase in knowledge of different bacteria and the consequent growth in the

understanding of the role each play in infectious diseases provided added motivation for

the intellectual and research inquiry into antimicrobial therapy. As a result, the discovery

of antibiotics to target specific infections caused by microorganisms in the early twentieth

century was among the greatest breakthroughs in drug-discovery.94 While there are documentations in support of the fact that the ancient Chinese were cognizant of the medicinal value of natural products and embarked on natural sources such as tree bark and herbs to treat infection of microorganisms, the therapeutic agents and mechanisms of action and the structures of the active agents were unknown.95-99 In this respect, the

discovery of antibiotics revolutionized the way bacterial infections were treated.

Antibiotics can be categorized into two families; natural occurring and synthetic.

Natural antibiotics in general are isolated from the fermentation broth of fungi, molds and

bacteria with soil microorganisms forming the most productive source for majority of the

families of antibiotics.100,101 Some common examples of families of antibiotics of natural

origin are the -lactams, aminoglycosides, tetracyclines, macrolides, glycopeptides and

lipopeptides.94 In contrast, the synthetic antibiotics were discovered via random screening

and rational design. But there have been only three families of synthetic antibiotics since

the 1930s. These are the sulfonamide (sulfa drugs), quinolones and oxazolidinone.94 24

2.1.1 Natural antibiotics

The observation by Fleming in 1928 that Penicillium notratum lysis in a culture

of bacteria101,102 coupled with the mass isolation, purification, characterization and assessment of potency by Florey, Chain and others in early 1940s undoubtedly unfolded the advances in antimicrobial therapy.3 Penicillin, cephalosporin and carbapenems are

members of the large family of antibiotics called -lactams.94 This family embraces two fused heterocyclic rings as their core structure. Though generation of analogs were carried out via the modifications of the side chains, the extent of analoging was elevated upon the isolation of 6-aminopenicillanic acids. This enhanced the possibility of preparing analogs, which were previously not accessed via the fermentation process.103-110

Penicillin 1 (Figure 2.1) is currently isolated from the mold Penicillium chysogenum111

and it was among the early drugs discovered without a lead.112 The -lactams in general are bactericidal, narrow spectrum antibiotics that interfere with the biological functioning of peptidoglycan transpeptidase,112,113 a key enzyme required for cell wall biosynthesis in

Gram-positive bacteria. Resistance to penicillin in particular and -lactams in general has been attributed to the enzyme, -lactamase that catalyzes the hydrolysis of the -lactam scaffold.112,113

H N H S O N O 1 CO2H

Figure 2.1. Structure of penicillin, a -lactam antibiotic. 25

The eagerness to find a new class of antibiotics in the mid-twentieth century

prompted the purpose-oriented search for new antimicrobial agents in soil bacteria. This

investigation pioneered by Waksman led to the discovery of aminoglycoside family of

antibiotics in 1943.8,114,115 Streptomycin 2 (Figure 2.2), was the first member of the aminoglycosides to be isolated from the soil bacteria, Streptomyces griseusan116 and it

became the first antibiotic to be effective as an anti-tuberculosis agent.117,118

H2N H2N NH2 HO N O O NH2 HO N 2 H2N N O OH 3 HN 1 4 O H2N N OH NH 5 2 HO OH 6 O OH NH2 2. HO OH 3 OH OH

Figure 2.2. Structure of streptomycin (2) and streptidine scaffold.

The aminoglycoside family of antibiotics embrace the aminocyclitol (2-

deoxystreptamine)118,119 as their core structure (Figure 2.3) with the exception of

streptomycin that had a streptidine scaffold (Figure 2.2).118 In addition to their core

structure, the aminoglycosides also have a characteristic sugar derivative (glycosylation)

attached usually at position 4- and 5- (neomycin 4) or 4- and 6- (netilmicin 5) of the

aminocyclitol ring 6 (Figure 2.3).120 As a broad spectrum, concentration dependent

bactericide family of antibiotics,118 the aminoglycosides have extensively been reviewed 26

H N H2N HO NH 2 2 HO OH O HO HN NH O 2 HO O O O OH O O N O O H2N OH H HO NH2 OH OH NH2 NH2 4 5 NH2 NH2 6 OH 1 OH 2 5 NH2 4 3 OH 6

Figure 2.3. Examples of aminoglycosides containing the aminocyclitol (6) ring.

as RNA ligands119,121 and found to bind in divalent cationic sites.121,122 Their common

targets included the viral RNA of HIV,123 16S rRNA124 and ribosome enzymes

(ribozymes).125 The aminoglycosides readily bind to 30S ribosomal subunit,118 which

plays a critical role in the translation of genetic material.118 Yet their collective in depth mechanism of inhibiting protein synthesis is unclear and mode of action is different for subclass analogs.118 Undoubtedly, their inherent ability to bind to the A site on 16S rRNA has been projected to be their cardinal mode of action.126,127 Resistance to aminoglycosides has been attributed to mutations within the 30S subunit as well as the methylation of rRNA. It is also documented that a single mutation in the A site on 16S rRNA was adequate to initiate resistance to aminoglycosides.118,126-128

Apparently, the urgent demand for a new class of antibiotics primarily to treat the infections of casualties during the Second World War era could have led to the upsurge in the discovery and use of antibiotics in the mid 1900s.94 For instance, Duggar discovered

the natural antibiotics tetracyclines in 1945.2 Aureomycin 7 (Figure 2.4), a 27

chlorotetracycline was among the first members of the tetracyclines to be isolated from

the soil bacteria Streptomyces aureofaciens.129

Cl OH NMe NMe2 NMe2 H 2 OH OH O H N CONH N CONH2 OH 2 H OH OH O O OH O OH O 7 8

Figure 2.4. Examples of tetracycline antibiotics.

As such, aureomycin became the lead compound upon which varied structural

modifications were carried out to generate thousands of new analogs. Several tetracycline

subclasses including the glycylcyclines (eg tigecycline 8),130-133 which emerged in the

1990s were semisynthetic analogs.134 As an extensively heavily or well studied family of

antibiotics,49,130,135 the tetracyclines are broad-spectra bactericidal antibiotics that are

active against both Gram-positive and Gram-negative pathogens94 as well as protozoa.136

Consequently, tetracyclines have been used widely in veterinary medicine as well as the

agricultural sector.49,130,135,137,138 In general, tetracyclines interfere with the functioning of aminoacyl-tRNA by binding irreversibly to 30S ribosomal subunit.49 In this respect, tetracyclines abrogate the elongation of peptide chain during protein biosynthesis.22,23 The

mode of resistance to tetracyclines is to date unclear but attributed to a combination of

resistance genes and is rarely owed to mutations in the 30S ribosomal subunit.49

The macrolides (Figure 2.5) are another family of natural antibiotics that were

discovered in the 1950s.139 This macrolide family of antibiotics have a common or

characterisitic 14-, 15- and 16- membered lactone ring that embody several chiral centers 28

as well as the attachment of at least two sugars.140,141 Erythromycin 9, the most recognized

member of the macrolide142 was first isolated from the fermentation broth of

Streptomyces erthraceus29 and all its structurally related analogs have the neutral sugar

cladinose at C-3 and the amino sugar desosoamine at C-5.141,143 The macrolide family of antibiotics exhibit a broad sprectrum, concentration dependent bactericidal activity against a host of microorganisms including both Gram-positive and Gram-negative pathogens. Recent analogs called the ketolide142,144-146 (eg. Telithromycin 10) developed from the 14-membered lactone have found wider spectrum of activity than their progenitor macrolide.142,146,147 The macrolides antibiotics exhibit their mode of action of

action by binding to 50S rRNA in close proximity to peptidyl transferase.27,29,148-151 In more a indepth sense, the macrolides inhibit protein biosynthesis by binding to a domain in 23S rRNA that controls protein elongation by catalyzing peptide bond formation.28,152

Resistance to macrolides in general has been attributed to mutations such as methylation

of A2058 in the 23S rRNA.153

O O N O HO OH O N N OH NMe2 HO O O O O O O O OMe O O OH O NMe2 O 9 O 10

Figure 2.5. Examples of macrolide antibiotics. 29

The structurally complex family of antibiotics known as glycopeptides evolved

around the mid 1950s154-156 as part of the intellectual inquiry into finding new class of

antibiotics with a novel mode of action and a broad spectrum of activity. Vancomycin 11

(Figure 2.6), the most well known member of the glycopeptide family was isolated from

the fermentation broth of Amycolatopsis orientalis157 by Kornfeld in 1956. Following its

discovery, several hundred members of the glycopeptides were isolated from a variety of

actinomycetes genera158 but only two, namely: vancomycin and teicoplanin 12 (Figure

2.6), found important clinical utility.159 Vancomycin was considered as the antibiotic of

last resort for treatment of serious bacterial infections of Gram positive pathogens.159 All glycopeptides have a heptapeptide core structure159 and modifications of key elements on

the core structure via semisynthetic methods160-163 led to the generation of many analogs.

Among these, telavancin 13, dalbavancin 14 and oritavancin 15 (Figure 2.6) have made it

as far as advanced clinical stage.159 Since all glycopeptides have similar binding pockets,160,164 they are projected to exhibit the same mechanism of action by inhibiting

transpeptidation as well as transglycosylation steps during cell wall biosynthesis.165,166

Resistance to glycopeptides in general was attributed to modifications in the bacterial cell

wall such as the thickening of peptidoglycan layer.159 With the first identification of

bacteria resistance to the glycopeptide vancomycin surfacing in 1978167,168 and the

continuous emergence of other resistance strains to vancomycin (viewed as the antibiotic

of last resort) are compelling evidence for a new class of antibiotics. 30

OH HO HO RN OH OH H OH H HO O O Cl O O O O O HO HO H2N O O O O Cl O HO H Cl OH NHO N O O O O O N N O O O HN HO H H H H H NH N N NHMe HO NH O 2 N N N O H H H O O O HO O HN H2NOC OH HO2C HO O HO OH HO 12 OH 11 O HO HO OH OH

R = 4 4 6 4 5 O O O O O NH OH NH H H OH N HO Cl N O O HN O NH O O O HN O O HO O O HN O NH OH OH HO O O NH2 O Cl HO OH Cl O NH HO HN O HO O NHO O OH OH O O HO HN NH O HO OH HO O CO2H O OH NH OH O N O O O H H OH N P Cl N OH H O OH O 13 OH 14 HN N

O OH O OH OH Cl O O Cl O O O O OH HO O O H N H H H 2 O N N N N NH N H O H NH O H2N O HO O O OH HO OH 15

Figure 2.6. Examples of glycopeptide antibiotiotics. 31

While soil microorganisms have been a rich basin for the discovery of nearly all

the families of natural antibiotics, the identification of a new class of antibiotic with

novel mode of action would span over forty years before the discovery of daptomycin 16

(natural antibitiocs).94,169,170 Daptomycin (Figure 2.7) is a cyclic lipopeptide antibiotic

isolated from the fermentation extracts of the soil bacteria Streptomyces roseosporus.171-

173 This lipopeptide antibiotic exhibits a concentration dependent bactericide efficacy173

against a broad class of multi-drug resistant Gram-positive pathogens. Daptomycin

exhibits high efficacy in treating glycopeptide-resistant bacteria and other susceptible

strains.174-184 However, the emergence of resistance strains have been encountered in case studies174,185 but the occurrence so far has been less than 0.2%.172 The mechanism of action of daptomycin is currently unclear not elucidated. But has been attributed to the calcium-dependent binding of the lipophilic tail of daptomycin to the plasma membrane and the triggering of efflux of potassium ions as a result of the depolarization of the membrane.186-190

CO H O 2 O H N N NH H2N H NH O O O CO H NH 2 HO2C HN HN O H H N O O N HN N O OH HN H H O H O O N CO NH N O 2 2 O O O CO2H NH2 16

Figure 2.7. Stucture of the lipopeptide antibiotic daptomycin. 32

2.1.2 Synthetic antibiotics

In contrast to the natural antibiotics, which were primarily isolated from the

fermentation extracts of soil bacteria, the synthetic antibiotics were generally identified

via rational design and random screening.112,191 The discovery of the sulfonamide (sulfa

drugs) antibiotics, example prontosil 17 (Figure 2.8), by Domagk in 1932 marked the

inception of the target-oriented endeavor of synthetic antibiotics.192 The identification of

prontosil was a

H2N N N

NH2 SO2NH2 17

Figure 2.8. Example of a sulfonamide antibiotic.

combination of knowledge garnered from the vital observations of Ehrlich112 pertaining to bacteria as well as the rigorous efforts by Domagk to find antibacterial agents that were biologically active against Streptococci. The advantage of prontosil’s identification dwelled on the fact that both animal and human subjects were included in the initial screening process. By carefully examining the present methods of screening compounds, prontosil would most likely have eluded modern screening techniques, which usually began with an initial in vitro cell base assays. This azo dye compound belongs to the large family of antibiotics called sulfonamide (sulfa drugs)94 and while prontosil had activity in vivo, it showed no activity during in vitro assay. This discrepancy in biological activity was attributed to the prodrug nature of prontosil.112 According to Trefousel et 33

al.,193 prontosil underwent degradation in vivo to generate sulfanilamide 18 (p- aminobenzenesulfonamide) as the active compound and triaminobenzene 19 (Scheme

2.1). Prontosil is a broad-spectrum194 bacteriostatic112 antibiotic that competed with the

metabolite p-aminobenzoic acid for the active site of a key enzyme during folic acid

biosynthesis.195-200 The importance of prontosil as an antibacterial agent did not extend

past the post second world war era because of the upsurge of more potent antibiotics with

relative safe profile but it still has limited usage.94 Conversely, the structural modification

studies that were carried out on prontosil in attempt to identify more potent agents

marked the beginning of modern day structural activity relationship studies,112 which to date has great utility in medicinal chemistry. Resistance to prontosil was attributed to modifications in the biosynthetic pathway of folic acid.201-207

NH [4H] 2 H N N N SO2NH2 2 H2N SO2NH2 + Biotransformation H2N NH2 (Azoreductase enzymes) NH2 18 19 17

Scheme 2.1. Illustration of the prodrug nature of sulfonamide.

The quinolones are another class of antibiotics that were developed synthetically

by Lesher in 1962.208,209 Nalidixic acid 20 (Figure 2.9) generated in the early 1960s was the first member of the class of synthetic antibiotics and it became the lead compound for many structural modification studies.208-210 The quinolones have been extensively studied

and currently second, third and fourth generation analogs have been developed.210 A subclass of this quinolones has a fluorine substitution typically at C-6 of the ring and is 34

called the fluoroquinolones.212 This subclass exemplified by ciprofloxacin 21 (Figure 2.6)

is the modern day analog of the quinolones class. While the fluoroquinolones were found

to be more active relative to their progenitor compounds, extensive modification of their

core structure occasionally led to analogs with poor safety profiles.212 These quinolones are broad spectra and exhibit a bactericidal activity against their target.212 This family of

synthetic antibiotics target bacterial DNA212 and interfere with the biological functioning

of DNA gyrase and topoisomerase II. This action of quinolones disrupts the unwinding

and replication of DNA.33,213-215 While quinolones are reported to have low tolerance to resistance, resistances to quinolones in general are a result of drug efflux as well as modifications in key enzymes.113,216,217

O NH CO H 8 2 2 N 7 N 1 6 4 N N HO 3 5 F O O 20 21

Figure 2.9. Examples of quinolone family of synthetic antibiotics.

Intriguingly, nearly forty years would elapse before the discovery of the third family of synthetic antibiotics. This family of antibiotics, exemplified by linezolid 22

(Figure 2.10) is called the oxazolidinone antibiotics.94,218 Linezolid is a bacteriostatic

antibiotic, which has been found to be extremely potent in treating serious infections

caused by Gram-positive pathogens such as glycopeptide resistance strains (vancomycin-

resistant strains)56 and others.57,58 In view of this, linezolid has been reserved as a suitable 35

substitute for the glycopeptide antibiotic, vancomycin. While all the antibiotics that target

protein biosynthesis often interfered with key elements during the later stages of protein

synthesis thereby inhibiting for most part polypeptide chain elongation, linezolid targets

the early stages of protein biosynthesis.219,220 Linezolid targets domain V of 23S rRNA52-

55,221 and disrupting the fusion of 30S and 50S subunits to 70S initial complex. Like the other antibiotics discussed earlier, resistance to the oxazolidinone antibiotic linezolid has began to emerge after barely ten years of linezolid on the market.59-63 While there is

limited documentation that rationalizes a plausible mechanism of bacteria resistant to

linezolid,222-224 the exact causes of resistance is unclear. Nonetheless, the emergence of

resistance to linezolid that is barely ten years on the market, coupled with the increasing

public awareness that antibiotics available may not be suitable for treating serious

bacterial infection are strong indicators of the need for a new class of antibiotics.

F O O O N N H N 22 O

Figure 2.10. Structure of an oxazolidinone antibiotic.

2.1.3 Antibiotics: problems and challenges

The incremental frequency of bacterial resistance to antibiotics has elicited the

growing public concern that antibiotics currently available would be insufficient in

treating bacteria infections. It is apparent that the adaptative abilities of bacteria

pathogens has contradicted Stewarts (U. S. Surgeon General) testimony to congress in 36

1969.8 For example, penicillin was introduced for clinical usage in 1940 and by 1960s about 80% of staphylococcus aureus were resistant to penicillin in the United States.225

By the year 2002, methicillin resistant staphylococcus aureus (MRSA) acquired from the

hospital had risen from 2% in 1974 to 57%.226 It is also worth noting that the type and

severity of the bacterial strains vary by geographical location. While from the year 2000-

2005, there were three isolates of vancomycin resistant staphylococcus aureus (VRSA) in

the United States,227-230 an European study encompassing 25 university hospitals and

3,051 isolates of staphylococcus aureus depicted that 25% were caused by MRSA.231 By

grouping the occurrence by country: over 50% was observed in Italy and Portugal, 25%

in England, France and Greece while Switzerland and Netherlands had the lowest

incidence of 2% among the European countries.231 These varied incidence and frequency of bacteria resistance to antibiotics are indications that the rigorous intellectual inquiry into antimicrobial therapy should not dwell solely on resistant management but also on the development of antibiotics with novel targets as well as mode of action.

Though nature in general and soil microorganisms in particular have served as an invaluable source for the discovery of many families of antibiotics, the thousands of analogs of each family currently in clinical use were generated either through semisynthetic or synthetic methods. These analogs in most situations were found to be more potent with enhanced safety profile relative to their progenitor natural compound.

Even though the procedure for isolating natural antibiotics from their fermentation extracts may be relatively cheap, the processes are very cumbersome. It also involves the daunting task of identifying a suitable natural source with the desired pharmacological 37

effect. On the other hand, rational design to drug discovery using combinatorial synthesis

provides an unmatched means of generating large compound libraries that exhibit both

structural diversity and complexity with the potential of modulating diverse biological

pathways.232-235 Structural diversity in its entirety is vital for hit to lead identification.236,237

In addition, complexity in the topography of library members compounds the effect of

diversity by providing analogs that are more globular and well suited for the somewhat

concave nature of the active site of enzymes or biological targets.236,238

Though generating large compound libraries does not guarantee the identification

of a lead,239 the advances in medicine coupled with the large number of reliable and innovative techniques239 as well as biological assays for evaluating desired compound

libraries for a preselected target,94 augments the prospects of lead identification.

Therefore, rational approach to drug discovery via combinatorial library synthesis71-74

provides the most convenient and suitable method of meeting the increasing demand for

antibiotics and the innovation is required in antibacterial therapy.

2.2 Antibacterial drug discovery

In general, antimicrobial drug discovery continues to be a sophisticated and

challenging intellectual endeavor in spite of all the advances in medicine and genomics.

This is evident from the limited number of new antibiotics with novel modes of action

that entered clinical use to date. Since 1998, the food and drug administration (FDA)

have approved only ten antibiotics for clinical usage. Of these, only two, namely linezolid

and daptomycin had a novel mode of action and target site.240 Undoubtedly, the majority 38 of the family of antibiotics were also discovered in the 1930s to 1960s241 and in over forty years the generation of new antibacterial agents has been mainly through semisynthetic or synthetic modifications of existing drugs.242 There has also been a steady decrease in the number of antibiotics in the past twenty years226,241 with resistance management forming the primary and inevitable agenda in the design of new antibacterial agents.8

Considering the growing concerns associated with bacterial resistance and plausible ways to address existing issues, synthetic antibiotics with novel targets have been projected as well suited to meet the challenges in antibacterial agent discovery.242

The advantages of embarking on synthetically developed small molecules dwell on the ease of implementing structure modifications as well as the identification of highly functionalized RNA domains.66,67 Bacterial RNA regulates the communication of information from DNA to amino acids on proteins.243 As such, highly conserved regions of RNA provides a platform for target validations as well as a therapeutic site for the rational development of small molecules for antibacterial discovery.244,119

2.3 RNA domain as a novel target for antimicrobial therapy

The daunting challenge in drug discovery and for this matter antibacterial drug discovery dwelled on the identification and characterization of biological targets.

Bacterial RNA increasingly receives attention as a new plaform for the rational development of new antibiotics. Though the chemistry of RNA binding antibiotics is well known, all except the oxazolidinone antibiotic linezolid were natural products with complex topographies. In addition, the families of antibiotics that bind RNA specifically 39

bind ribosomal RNA (rRNA). The aminoglycosides are the most studied family of

antibiotics but the prospects of readily generating analogs are hampered by their

structural complexities.

RNA dynamics such as its flexibility and rapid conformational changes pose a

barrier for targeting specific RNA regions.245 Conversely, the increase in the understanding of RNA recognition principles246-249 promotes the opportunity for rational approach such as the design of unique scaffolds capable of binding and modulating RNA functions. RNA binding ligands are well known, but the challenge in identifying small molecules for antimicrobial therapy dwell in the ability to discern new chemical entities that bind RNA with high affinity and specificity.

The RNA scheme plays a pivotal role in the catalytic and regulation activity of the cell. It has also been involved in vital processes that pertain to the communication of informational as well as the structural morphology of the cell.250-254 These functions

render RNA as a therapeutic target for the biological evaluation of small molecules.

Besides, the identification of key RNA-RNA interactions situated in the noncoding

regions of mRNA that regulates the mechanisms associated with transcription and

translation such as initiation, elongation and termination further validate bacterial RNA

as an important site for antimicrobial therapy.255,256 One such element that has been thoroughly studied and characterized is the T-box antitermination RNA system discovered by the Henkin research group.66 40

2.3.1 T box antiterminator RNA System

The interactions that occur at the 5'-untranslated region of the mRNA has been

thoroughly investigated to be important for the assessment of the efficiency of

translation.251,252,257 These interactions in which specific RNA, namely transfer RNA

(tRNA) plays a key role in modulating the transcription regulation.251,258 This 5'-

untranslated leader region of mRNA embody the structurally conserved T box RNA

element that is essential for the regulation of several amino acid related genes such as

aminoacyl-tRNA synthetase.67-69 This structured and highly conserved RNA element is also responsible for the regulation of genes associated with amino acid biosynthesis as well as transport genes.67-69 The T box element consisting of a 14 nucleotide259,260

sequence is found mainly in Gram-positive bacteria. Examples of the bacteria that have

this RNA element are Streptococcus, Staphylococcus, Enterococcus, Deinococcus,

Bacillius, Mycobacterium, Corynebacterium, Streptomyces, Listeria and Clostridium.

This RNA structural element is also present in a limited number of Gram-negative

bacteria including Geobacter.67,261 Since approximately 67% of bacterial pathogens

identified in the hospital setting are Gram-positive pathogens, it has been demonstrated

that the disruption of T box functions was adequate in inhibiting cell proliferation.

The leader region consisting of highly structured primary and secondary elements

embodies two competing alternate RNA structures known as the terminator and

antiterminator.259,260,262 The antiterminator element consists of two helices that encapsulate

a seven-nucleotide bulge.259,262 As the two elements are competing alternating secondary 41

structures, the formation of one element abrogates the other. According to Grundy and

others, the interactions that occur between the cognate tRNA and the nascent leader RNA

leading to the terminator or antiterminator modulates the expression of genes in the T box

element and dictate the progression of transcription.67-69,263 Thorough investigation of the

T box antiterminator mechanism revealed that the regulation of genes expression in that region was owing to a limitation of a specific amino acid starvation. Therefore, in response to the starvation, two competing independent mechanisms are initiated (charged and uncharged tRNA).264,265 In the situation of a charged tRNA, the attachment of amino acid at the 2'- or 3'- position of adenosine265,266 has been documented to prevents the

interaction of the anticodon of tRNA with specifier sequence (a critical initiation stage of

the antitermination mechanism).263,267 As a result, the base pairing of the acceptor end of

the tRNA is prevented primarily owing to steric factors. The inhibition of base pairing

leads to the formation of the terminator structure.268 Consequently, the pause signal is recognized by polymerase, which causes it to fall off resulting in the premature termination of transcription.269 In contrast, the interaction of the anticodon of tRNA and

the specifier sequence is stimulated in the presence of uncharged tRNA. In addition, the

base pairing of the acceptor end of the tRNA and the bulge of the antiterminator, which is

the most conserved structure of the T box system is also stimulated. The aforementioned

base pairing stabilizes the antiterminator and propagate the read-through of the whole or

complete structural gene.270,271 A model of the antiterminator has been reproduced based

on genetic, biochemical and structural biology assessments70,259,262 and determined to

function in the absence of cellular factors.272 42

It is worth noting that the presence of the intrinsic T box elements in many genera

of Gram-positive bacteria validates it as a therapeutic site for the rational design and

development of small molecules as novel class of antibiotics. In addition, the absence of

this highly structured RNA element in eukaryotics further buttresses the biological utility

of the T box and the antiterminator structure in particular as an unmatched target for new

chemical entity.

2.4 Identification of lead compound

The most common method for lead identification until the 1980s was through

random screening of compounds without regards to their functionality or scaffold.112 This method of evaluation for a lead compound played a vital role in the discovery of the sulfa drug, prontosil in the 1930s when the urgent need for antibiotics compelled the random screening of thousands of azo dyes. Random screening is also a critical component of diversity-oriented synthesis, where divergent synthetic methods provide large libraries exhibiting both structural diversity and complexity.74,273,274 An alternative but more of a

supplemental approach to random screening is target screening, where compounds,

having similarites in their topography are evaluated for a preselected biological target.112

In a trivial sense, mishaps in drug metabolism studies and clinical observation also

contribute to the identification of a lead compound. While the sulindac 23 was developed as an anti-inflammatory drug, metabolism studies depicted the active agent to be the drug degradation product 24 (Figure).275 The drug sildenafil citrate 25 is a classical example of the contributions of clinical observations in lead identification. Sildenafil citrate an 43

antianginal drug was observed during clinical observation to exhibit prolong vasodilation

effect owing to its ability to inhibit phosphodiesterase.276,277

COOH COOH O F F OEtHN N N N

O S 2 N O S S N 23 24 25

Figure 2.11. Structures of leads identified via metabolism and clinical observation.

The modern day method of lead identification is via rational design and

development of compound libraries. The focus in this approach dwells on the ability to

develop pharmacological agents via combinatorial synthesis that are capable of

modulating a preselected target.112 Rational design approach in most situations rely on the natural products as well as existing drugs with the goal-driven purpose of targeting a specfic biological target. As highlighted in Figure 2.12, rational design approach to lead identification involves reiterative cycle of generating and screening new chemical entities or analogs until the compound with the desired pharmacological activity is afforded. This method of lead identification has been extremely beneficial especially with all the advances in medicine. Yet, a lead may exhibit undesired physiological qualities and lead optimization without compromising activity becomes inevitable.112 44

Library synthesis Purification

Rational library design Analysis

Biological evaluation

Lead compound

Figure 2.12. General outline of the reiterative cycle of rational design.

2.4.1 Lead compound modification/optimization

Structural modification of a lead compound is a frequent synthetic approach

undertaken by medicinal chemist in the attempt to optimize the biological activity as well

as the safety profile of a lead.278 The most widely adopted method for lead modification is

through structure activity relationship (SAR) studies.278 This requires a systematic

alteration of the lead compound using innovations in combinatorial chemistry.112,279-285

While small modifications of a lead have the potential of enhancing or attenuating the

biological activity of a lead, functionalization of complex compounds is seemingly challenging.112 In this respect, the empirical analytical data compiled by Andrews286,287

becomes a vital tool in ascertaining if certain functionalizations, particularly of complex

compounds are critical.

The generation of analogs is the hallmark of SAR studies and the large number of 45

analogs generated for SAR studies are often accessed using structural modification

techniques, which includes homologation.112 The advantages of exploring homologation is exemplified by Richardson288 and Dohme et al.289 Analogs for SAR studies are also obtained by other lead modification techniques that include ring chain transformation, chain branching and bioisosterism.112 Homologation together with chain branching and

ring chain transformation are critical lead approaches often embarked on to improve upon

the biological activity as well as the pharmacokinetic properties such as solubility or

lipophilicity of a lead.112

Bioisosterism is an important and well-recognized lead modification approach

undertaken to enhance the biological activity of a lead compound. Bioisoteres, which are

groups, substituents or scaffolds that exhibit comparable or similar biological activity aid

in shredding off unwanted side effects and improve pharmacokinetics as well as

pharmacodynamics of a lead.112,290 This lead modification method has been extensively

employed by medicinal chemist with the goal-driven agenda of enhancing activity. An

example of a bioisosteric replacement of a lead is the substitution of pyridine moiety in

nicotine with isoxazole to generate new nicotinic agonist such as ABT 418 27 (Figure

2.13).291,292

N N N O N 26 27

Figure 2.13. Example of bioisosteric replacement of nicotine to ABT 148. 46

Lead modifications in an attempt to optimize the biological activity of a lead

usually require reiterative cycles of structure modifications until the chemical entity with

the desired biological activity is afforded. Lead modification has been part of the ongoing

research agenda of the rational design and development of oxazolidinone compounds that

bind RNA in the Bergmeier laboratory. By carrying out several structural alterations of

the oxazolidinone scaffold paved way for the identification of 4,5-disubstituted 2-

oxazolidinone analogs with enhanced biological activity and improved solubility.81, 82

2.5 Oxazolidinone compounds

There are documentations that depict the signifcant biological utility of the

oxazolidinone scaffold, which is found in both natural and synthetically derived

compounds. For example, cyctoxazone 28 (Figure 2.14), the 4,5-disubstituted 2-

oxazolidinone isolated from Streptomyces species is an immunomudulator that disrupts

the signaling between Th1 and Th2 macrophages.293,294 The oxazolidinone analogue of epilocarpine 29, which is primarily synthesized from histine is actively used to treat glaucoma and xerostomia.295 The oxazolidinone ring is also the core structure of the synthetic antibiotics such as linezolid (Figure 2.10) and Dup 721 30, which are both active in treating multi-drug resistant Gram-positive pathogens.296 Owing to the synthetic

and biological relevance of the oxazolidinone scaffold, it has been the center of several

research agendas including an ongoing one in the Bergmeier laboratory. 47

O O O O NH NH O N O N O HO N HO N O 28 OMe 29 30

Figure 2.14. Examples of compounds containing the oxazolidinone scaffold.

2.5.1 Oxazolidinone library generation

The Bergmeier laboratory had previously developed an acylnitrene method that enhances the parallel synthesis of di- or trisubstituted 2-oxazolidinones compounds.80,297,298 The substitution patterns of this class of oxazolidinone compounds were very different from that reported for the 3,5-disubstituted oxazolidinone antibacterial agents such as linezolid that has great cinical utitlity.54,55,299 As outlined in

Scheme 2.2, the formation of the bicyclic aziridine 34 was critical for the generation of the oxazolidinone analog via parallel synthesis. The synthesis began with the monotritylation of 3,4-dihydroxy butane 31. The allylic alcohol 32 was acylated with p- nitrophenyl chloroformate following by azidolysis to generate the azidoformate 33, which was subjected to thermolysis to provide the bicyclic aziridine 34 (key intermediates) as a single diastereomer in good yield. The bicyclic aziridine 34 was opened with the requisite organometallic reagents to afford the 4,5-disubstituted 2- oxazolidinone 35. The N-H of the oxazolidinone 35 was alkylated or acylated

(compound 36) followed by detritylation and acylation of the resulting alcohol to provide the SK library of tricyclic oxazolidinone 37. This library consisted of 27 members of which SK-4 (R1 = Ph, R2 = 4-(OMe)Ph, R3 = Bn) was identified as the lead. 48

This lead compound exhibited good binding affinity for antiterminator though its

solubility was marginal.81 Structural modifications of SK-4 led to the identification of

SB-1 (R1 = Ph, R2 = H, R3 = Bn) as the second lead compound from compound 38.81

This lead compound showed good binding affinity and enhanced specificity (based on

FRET assay). But as observed in SK-4, SB-1 also had marginal solubility.

Based on the affinities and specificities of SK-4 and SB-1, the group was interested in improving upon the solubility without compromising RNA binding affinity.

The approach taken by the group was to replace R1 with a variety of amine substitutions

(NR2) 39. Amines such as N-methyl aniline, N-methyl-phenethylamine, morpholine, N- phenyl piperazine etc., were employed to enhance the diversity of the amine

82 3 substitutions. On the other hand, R = PhCH2 was maintained in compound 40 for

majority of the analogs, since it was discerned on as the optimal ester for previous

library.82 The group also sought to prepare some analogs where R3 was substituted with a

carbamate group. This was done to improve upon the biological activity and solubility

challenges as well as the potential of availing additional non-covalent interaction towards

the preselected RNA target.82 The preparation of the amine substituted oxazolidinone led

3 2 to the identification of two additional leads, ANB-22 (R = Ph, NR = N(CH2CH2)2NPh)

3 2 and ANB-40 (R = 4-(CH3CO)C6H4NH, NR = N(CH2CH2)2NPh) that bind at nanomolar

concentations with moderate solubilities. The Kd values for SK-4, SB-1, ANB-22 and

ANB-40 are depicted in Table 2.1. 49

Table 2.1. Binding affinities for oxazolidinone model RNAs

Oxazolidinone analogs AM1A Kd (μM) AM1A (C11U) Kd (μM) SK-4 3.4 ± 1.9 25 ± 10 SB-1 9 ± 4.5 125 ± 9 ANB-22 13 ± 4 100 ± 30 ANB-40 0.9 ± 0.4 < 1a 81,82 Kd values (μM) based on FRET assay with 100 nM labeled RNA a Kd observed to be below the assay’s detection limits.

O O OH OH TrCl 1. ClC(O)O(4-NO2Ph O HO TrO O N3 N 2. NaN 3 TrO TrO 31 32 33 34 H

O O O O 1 O R3-COCl O 2 2 NH R -MgX N R2 N R R -Br R3 O TrO KF/Al O CuCN TrO 2 3 R1 R1 35 O 37 R1 36

O O NH 3 R3 O R -COCl

O 38 R1

O O R3-COCl or O HNR4, CH Cl NH O NH 2 2 R3 O 1. TFA 3 TrO 2 2. R -NCO 2 O 40 NR 39 NR

R1, R2, R3, R4 = alkyl, aryl diversity elements

Scheme 2.2. Design and synthesis of oxazolidinone compound library.

Intrigued by the enhanced RNA binding affinities and specificities for ANB-22 and ANB-40, the group embarked on enzymatic cleavage assay to determine the binding

modes of ANB-22 and ANB-40 using RNase T1 and RNase A. The data garnered from 50 the enzymatic probing coupled with other in vitro assessments signified that ANB-22 exhibited inhibiting activity that obviates the in vitro antitermination. On the contrary, the data for ANB-40 supported a non-tRNA induce stabilization of antiterminator.82

Beside the RNA binding evaluation, library members were also evaluated for their ability to inhibit bacteria growth using alamar blue dye reduction assay. Three oxazolidinones from the ANB-series (ANB-22, ANB-24 and ANB-27) showed good antibacterial activity in the preliminary screening. The compounds were further subjected

O O O NH O O O NH Ph O Ph ANB-24 N O N ANB-27

Figure 2.15. Structures of oxazolidinone antibacterial agents.

to quantitative assay to determine their IC50 values. The compounds ANB-24 (320 μM) and ANB-27 (60 μM) showed very good inhibitory activity against Bacillus subtilis while ANB-22 showed only marginal activity. The structure of oxazolidinone antibacterial leads ANB-24 and ANB-27 is depicted Figure 2.15. These results from the oxazolidinone library clearly indicate that rational design approach to drug discovery elevates the prospects of identifying small molecules with a novel targets for antimicrobial therapy. 51

2.6 Significance of research

Multi-drug resistance continues to be a worldwide threat in the public environments and hospitals.300,301 Consequently, nearly all antibiotics present on the market are at least thwarted by mutation by one of the mechanism documented by

Davis302 and Poole.303 The development of resistance to vancomycin, which for many years clinicians have resorted to as the last treatment option for acute bacterial infections and linezolid that is barely ten years on the market are indicators of the urgent need for a new class of antibiotics. With advances in RNA research and identification of RNA-RNA regulatory interactions, the T box antitermination element67 provides unprecedented platform for the rigorous investigation of new antibiotics. But the challenge resides on discerning on small molecules via rational design and development that are capable of binding RNA with high specificity. The identification of oxazolidinone leads ANB-22 and ANB-40 that interferes with T box functioning and ANB-24 and ANB-27 that exhibit inhibitory activity against bacterial growth provide a platform for the development of small molecules with the potential of binding RNA. Therefore, the exploration of lead optimization approaches that incorporates both structure activity relationship (SAR) and structure property relationaship (SPR) on the oxazolidinone scaffold would elevate the potential of identifying new leads. In addition, the generation of analogs via bioisosteric replacements of the oxazolidinone scaffold would provide innovation in antimicrobial therapy to address the current issues of bacterial resistance. 52

Chapter 3. Synthesis and Preliminary Results of 1,4-Disubstituted 1,2,3-Triazole Library

3.1 Introduction

Library generation utilizing combinatorial synthesis of the 4,5-disubstituted 2-

oxazolidinone80 has been a paramount part of the research agenda in the Bergmeier laboratory. Utilizing rational design and synthesis, the Bergmeier group had previously identified two oxazolidinones ANB-22 and ANB-40 that bind to a preselected target (T box antiterminator RNA) at nanomolar concentrations (Figure 3.2).82,83 While the group is currently exploring structural modifications to enhance the activity of ANB-22 and ANB-

40, the group is also interested in finding suitable replacements for the oxazolidinone

scaffold. The 1,2,3-triazole ring had been reported to exhibit tendencies of mimicking

peptide bonds (amide bond surrogate),304,83 thus a suitable non-classic biosiosteric

replacement for the oxazolidinone scaffold. The thought of exploring the 1,2,3-triazole

ring as a substitute for the oxazolidinone added more excitement to the inquiry into

antibacterial therapy due to the synthetic and biological benefits of the triazole

scaffold.305-317 Utilizing the 1,2,3-triazole as a bioisostere for the amide bond has several advantages,87,318,319 which include the potential for enhancement of pharmacokinetic properities such as solubility (owing to the increased diplole of the 1,2,3-triazole ring relative to amide) as well as biological activity.304 This lead modification technique has been exemplified by Brik et al, in their investigation into finding HIV-1 protease inhibitors.83

The 1,2,3-triazoles in general are aromatic heterocyclic compounds that have been

found to exhibit varied biological activity depending on the nature and position of 53

attachment of diversity elements. In general, there are two possible regioisomeric forms,

namely the 1,4 and 1,5-regioisomers that are afforded selectively85,320 or as a regioisomeric mixture90 depending on the synthetic methodology. However, the 1,4- regioisomer (1,4-disubstituted 1,2,3-triazoles) in particular has found extensive synthetic and biological applications.321-323 For example, the 1,2,3-triazole compound tazobactam

41 is a -lactamase inhibitor used in combination with the -lactam antibiotic piperacillin.324,325 The cephalosporin analog, cefatrizine 42 is also an antibiotic used in the treatment of bacterial infections of the urinary tract, liver and gallbladder etc,304,326

The 1,4-disubstituted 1,2,3-triazoles rufinamide 43 exhibits anticonvulsant activity and

has been used to treat childhood mental impairment of the Lennox-syndrome.327 The

triazole derivative carboxyamidotriazole 44 is an angiogenesis inhibitor useful in cancer

therapy.328

O O HO O F S HN N N N N N O N N N S N N O O COOH OH F S 41 N 43 NH2 H 42 O H2N

O O Cl NH2 H2N

N N Cl Cl N 44

Figure 3.1. Four examples of 1,2,3-triazole drugs.

Based on these biological utility of the 1,2,3-triazole scaffold, it was inferred that

the substitution of the oxazolidinone ring with the 1,2,3-triazole scaffold would avail the 54 potential of mitigating the solubility challenges associated with ANB-22 and ANB-40 as well as afford RNA ligands whose synthesis are enhanced via automation. In furtherance of this notion, the plan was to prepare a library of 1,4-disubstituted 1,2,3-triazole analogs of the oxazolidinone leads. Ideal 1,2,3-triazole analogs of ANB-22 and ANB-40 would be structure 45 (Figure 3.2), where R1 represents the acyl or carbamates groups and R2, R3 =

(CH2CH2)NPh. Instead the focus was to develop the library having the general structure

46 (Figure 3.2). This library provides the prospects of having 1,2,3-triazole analogs of

ANB-22 and ANB-40 as well as the opportunity of incorporating other acyl/carbamate and amine groups to enhance the diversity of the library.

O O O O NH H O N O NH

O N O N N N O ANB-22 ANB-40

R3 2 R1 O N R N N N N N R1, R2, R3, R4, R5 = H, Alkyl, Aryl O O R5 R1 R5 45 N 46 N R4 R4

Figure 3.2. Oxazolidinone leads, ideal and general structure of 1,2,3-triazole library.

Since the plan for the 1,4-disubstituted 1,2,3-triazole library was to maintain the same diversity elements as those used in the oxazolinone library, the three dimensional representation of both libraries were examined. The minimum energy conformation of the oxazolidinone ring was overlayed on the 1,2,3-triazole scaffold and found that both rings 55 orient the same appending diversity elements in the same space (Figure 3.3). The distance between the amine and acyl substitution on both rings were also determined to be comparable. The distance from the basic nitrogen to the carbonyl oxygen in the oxazolidinone measured 8.47Å as compared to 7.96 Å for the 1,4-regioisomer of the

1,2,3-triazole.

Figure 3.3. Oxazolidinone leads, ideal and general structure of 1,2,3-triazole library.

A library consisting of one hundred eight (108) members was projected as a good size for this “hit to lead” approach of discerning on potential analogs and also accessing 56

the viability of the 1,4-disubstituted 1,2,3-triazole as antibacterial agents and RNA

ligands in particular. The synthesis of the 1,4-disubstituted 1,2,3-triazole is well

documented. Consequently, considering the prospects of analoging and the feasibility of

accessing the desired library, it was envisaged that the regioselective synthesis of the 1,4-

disubstituted 1,2,3-triazole would be enhanced via Cu(I)-catalyzed cycloaddition

(CuAAC) method reported by Sharpless and coworkers.85

3.2 Retrosynthetic analysis of the triazole library

The 1,4-disubstituted 1,2,3-triazole compounds are regioselectively prepared from the Cu(I)-catalyzed 1,3-dipolar cycloaddition reactions of an organic azide and alkyne.85

As a result, functionalizations of the azide and alkyne component are extremely

important in enhancing the diversity in the desired compound library. The general

structure of the 1,4-disubstituted 1,2,3-triazole 46 and key azide 47 and alkyne 48

intermediates are highlighted in Figure 3.3. The azide components can be prepared from

glycidol 49 or other commercially available epoxides 50. The alkyne components, which are mainly propargylamine-derived alkynes can also be accessed via N-alkylation of propargyl bromide 51 or N-methylpropargylamine 52 with the requisite alkyl halides. 57

R3 2 O R HO or 3 N O R 3 R2 R3 2 1 O 49 R R 50 N N 47 N 1 O R R5 + N 46 R5 R4 or N 4 Br NH R 51 52 48

Figure 3.4. Retrosynthethesis of amine substituted 1,2,3-triazole compound.

3.3 Synthesis of azide components

The library generation began with the synthesis of the azide components. The azide components were not commercially available and had to be prepared from glycidol and four other commercially available epoxides, namely, styrene oxide, cyclohexene oxide, hexene oxide and benzyl glycidyl ether. The initial plan was to prepare all the desired glycidyl esters and glycidyl carbamates intermediates 53a-e from the same method but that proved futile (Scheme 3.1). The reaction proceeded with very low yields of the desired glycidyl esters and carbamates coupled with the challenging task of optimizing the reaction conditions. Conducting a series of reactions in which the conditions were varied indicated that the epoxide ring was sensitive to the aqueous workup conditions employed. A TLC analysis of the reaction mixture after 4 h, indicated completion of the acylation reaction and a stain of a product less polar relative to glycidol was observed. But, after workup a product whose polarity was close to that of glycidol was observed on TLC. Proton NMR analysis depicted compound to be the ring opening product (alcohol) instead of the epoxide. To circumvent this challenging situation, the acylation condition reported by Stamatov,329 which was devoid of aqueous workup, was 58

O O O N3 RCOCl or RNCO, DCM NaN3, CH3OH/H2O R O R O HO DMAP, rt, 24 h O NH4Cl, rt, 20-24 h 49 53a-e 54a-c OH 53a: R = CH (CH ) , 91% 3 2 6 54a: R = CH (CH ) , 77% 53b: R = Ph, 90% 3 2 6 54b: R = Ph, 81% 53c: R = Cyclohexyl, 93% 54c: R = Cyclohexyl, 80% 53d: R= PhCH2NH, - 53e: R= CH3(CH2)3NH, -

Scheme 3.1. Synthesis of 3-azido-2-hydroxypropyl esters from glycidol.

adopted. Glycidol 49 was then acylated with three different acid chlorides to afford the

glycidyl esters 53a-c in 90-93% yield. Reacting glycidol 49 with both benzyl and butyl isocyanate under the same reaction conditions provided very low yields of the expected carbamates 53d and 53e. A TLC of the reaction mixture did not leave any stain of the

starting materials in 5% PMA solution. This observation was indicative of the reaction

proceeding to completion in favor of byproducts. Two plausible explanations for the low

yield of the glycidyl carbamates were proposed and each attributed the low yield of the

glycidyl carbamates to intramolecular ring opening as outlined in Scheme 3.2. As

reported by Langlois and coworker in their synthesis of enantiopure oxazolidinone using

ring opening of epoxide,340 ring opening of the epoxide could be intiated by the

isocyanate oxygen of compound 55 leading to the formation of the cyclic carbonate 58

and amine 59 after hydrolysis. Alternatively, the intramolecular ring opening of the

epoxide could have been initiated by the isocyanate nitrogen of compound 60 to generate

the oxazolidinone 61, which reacts with water to produce the 4-hydroxy oxazolidinone

derivative 62. 59

R R N N O O H2O hydrolysis R O O O + N O O O OH RNH2 55 O O HO HO 56 57 58 59

O O + C Et N OH N 3 R 49 -H

O O : O: R R O N N H2O N R O O O HO O 60 61 62

Scheme 3.2. Plausible explanation of the low yields of carbamate esters.

This low yield of the carbamates warranted an alternative synthetic route.

Therefore, an alternative method that utilized the allyl alcohol 63 as starting material was developed (Scheme 3.3) to prepare the required glycidyl carbamates. Selection of the acyl substitutions were based on the acyl substituents of the oxazolidinone library members that showed good RNA binding activity. Benzyl substitution was observed as the optimal for oxazolidinone analogs, but for this study, a phenyl subsituent was included to enhance the diversity in the acyl substitution. 60

1 O O R NCO, DCM mCPBA, DCM R1 R1 HO N O N O Et3N, rt, 24 h H rt, 24 h H O 63 64a-b 65a-b 1 64a: R = PhCH2, 97% 1 1 65a: R = PhCH2, 80% 64b: R = CH3(CH2)3, 95% 1 65b: R = CH3(CH2)3, 82% NaN3, CH3OH/H2O NH4Cl, rt, 20-24 h O N3 R1 N O H OH 66a-b

1 66a: R = PhCH2, 77% 1 66b: R = CH3(CH2)3, 81%

Scheme 3.3. Synthesis of carbamate azide components from allyl alcohol.

Having the glycidyl esters 53a-c in hand, the task ahead was to find a suitable method to prepare the azidohydrins 54a-c. Methods for azidolysis have been well researched and azidohydrins in general have been prepared from the ring opening of epoxides with a variety of azide compounds including sodium azide. Sabitha and coworkers had reported on a regioselective ring opening reaction of epoxides such as compound 67 that were mediated by Ce(III) salt and carried out in acetonitrile/water mixture. This method provided their desired azidohydrins 68 in very good yields

(Scheme 3.4).330

CeCl •7H O/NaN OH O 3 2 3 1 2 1 N3 R = alkyl, aryl, alkoxy and R =H CH CN,H O 9:1, 3h reflux R 1 2 1 2 3 2 R , R = Alkyl R R R2 67 68

Scheme 3.4. Azidohydrin synthesis by Sabitha and coworkers. 61

Onaka and coworkers had also reported on a high yielding synthesis of azidohydrins

using different solid supports that were activated with sodium azide. This epoxide ring

opening method, which utilized solid supports such as silica gel, zeolite and alumina was

reported to provide the expected azidohydrin 70 from the epoxide 69 in quantitative yields (Scheme 3.5).331

OH O NaN3/ solid support 3 4 3 N3 R = alkyl, aryl, alkoxy and R =H Benzene, 80 °C R 3 4 3 R , R = Alkyl R R4 R4 69 70

Scheme 3.5. Reported azidohydrin synthesis by Onaka and coworkers.

Kiasat and coworkers had reported on a solvent free condition for the regioselective

synthesis of azidohydrin 72 from the epoxide 71. According to Kiasat and coworkers, the solvent free ring opening reaction of epoxide 71 using activated silica gel afforded the requisited azidohdrin in very good yields (Scheme 3.6).332

OH O NaN3, SiO2 R5 = alkyl, aryl, alkoxy and R6 =H, 80 °C 5 N3 R R5, R6 = Alkyl R5 R6 R6 71 72

Scheme 3.6. Reported azidohydrin synthesis from oxiranes.

Furthermore, Boruwa and coworkers had reported on a short and highly efficient ring

opening method of epoxides such as compound 73 using sodium azide in acetonitrile at 62 room temperature to provide the desired azidohydrin 74 in very good to excellent yields

(Scheme 3.7).333

OH O NaN , CH CN 3 3 N 4Å molecular sieves R7 3 R7 = alkyl, aryl, alkoxy and R8 =H, ester 7 R R8 rt, 1.6 h R8 73 74

Scheme 3.7. Regioselective synthesis of azidohydrin by Boruwa and coworkers.

Thus, a test reaction was carried out using glycidyl benzoate 53b but surprisingly, none of the four methods highlighted above provide an optimal condition for opening the glycidyl ester. All four methods provided either very low yields of the desired azidohydrin or afforded complexes mixtures. A careful scrutiny of the substrate of the epoxides of all four reactions indicated that none had employed an epoxide of structure similar to compound 53b. Two other epoxide-opening reactions334-335 were attempted in an effort to find an optimal condition for preparing the desired azidohydrins but both reactions provided complex reaction mixtures with unreasonable low yields of the expected azidohydrins. Eventually, the classical protocol,336-339 which entails the use of sodium azide in the presence of ammonium chloride, was employed to afford the desired azide components 54a-c in 77-81% yield. This synthesis provides a set of azide building blocks suitable for preparing 1,4-disubstituted 1,2,3-triazole analogs of ANB-22.

The focus of this part of the synthesis of azide building blocks was to prepare two carbamates that are suitable for preparing analogs of 1,4-disubstituted 1,2,3-triazole analogs whose topography resemble that of ANB-40. The benzyl and butyl diversity 63

elements were selected to examine the effect that aromatic and saturated aliphatic

substituents would have on biological activity of their corresponding 1,4-disubstituted

1,2,3-triazole analogs. Thus, having rationalized and attributed the low yield of benzyl

and butyl intramolecular cyclization,339 the projection was to start the synthesis from allylic alcohol 63, then generate the epoxide 65a-b after the formation of allyl carbamate

64a-b. (Scheme 3.3). Allyl alcohol 63 was treated with benzyl and butyl isocyanate to provide the corresponding allyl carbamates 64a-b in 95-97% yield (Scheme 3.3).

Epoxidation of the olefinic bond of 64a-b using mCPBA336 afforded the epoxide 65a-b in

80-82% yield. This was followed by azide generation using the classical protocol to provide the carbomyl azide 66a-b in 77-81% yield (Scheme 3.3). This provides a second set of azide building blocks suitable for preparing 1,4-disubstituted 1,2,3-triazole analogs of ANB-40.

To enhance the diversity in the azide building blocks, four commercially available epoxides 3.6a-d (Scheme 3.8) were subjected to the method of azodolysis adopted. This generated four additional azidohydrins 3.15a-d in good yields. Of these, the azidohydrin

3.15d was acylated with three acid chlorides to provide the azido esters 3.16a-c (Scheme

3.8). 64

OH O NaN , CH OH/H O N N 3 3 2 R3 3 RCOCl, DCM 3O R2 2 NH Cl, rt, 20-24 h R R3 4 DMAP, Et N, N3 OH 3 O R 75a-d rt, 24 h 76a-d 76d 77a-c

76a: R2 = Ph, R3 = H, 92% 77a R = Ph, 92% 2 3 76b: R = CH3(CH2)3, R = H, 94% 77b R = PhCH2, 88% 2 3 76c: R = PhCH2OCH2, R = H, 93% 77c R = PhOCH2, 91% 2 3 76d: R , R = -(CH2)4-, 98%

Scheme 3.8. Synthesis of 2-azido alcohol and azido esters from commercial epoxides.

These azidoesters added complexity and conformational rigidity to the azide components

as the conformation in which the bulky ester group is at the equatorial position

(compound 78) will predominate over the other 79 (Figure 3.4). In addition, the synthesis

of the azidoesters 77a-c would provide building blocks suitable for preparing 1,4-

disubstituted 1,2,3-triazole analogs of ANB-22 (oxazolidinone lead compound). Worth

noting, the lead oxazolidinone compound ANB-22 incorporated PhCH2 as its diversity

element on the ester group. By examining the structures of compounds 77a-c, it is

apparent that the azidoester 77b (R = PhCH2) provides the direct 1,4-disubstituted 1,2,3-

triazole analog of ANB-22. The azidoester 77a with R = Ph avail a means of

investigating the effect of homologation (useful lead modification technique). As the lead

oxazolidinone, ANB-22 binds model RNAs without significant dependence on

electrostatics. Compound 77c (R = PhOCH2) could provide additional non-covalent binding to the target RNA. It also provides a means of examining an optimal position of the phenyl group for the anticipated 1,4-disubstituted 1,2,3-triazole analogs. Further, these azidoesters signified the untapped potential of functionalizing the azide components. 65

N3 R 77a: R = COPh O N 77b: R = COCH2Ph 3 77c: R = COCH OPh 78 79 O 2 R

Figure 3.5. Illustration of the conformational rigidity of compound 77a-c.

In sum, the twelve-azide components depicted below were prepared as building blocks for the synthesis of the 1,4-disubstituted 1,2,3-triazole library (Figure 3.5). Based on the assessment of the oxazolidinone library, these azide components span the topographical identity of the diversity elements employed for acyl functionalization of the oxazolidinone compounds.

O O O O

O N3 O N O N N O N3 3 3 H OH OH OH 54a OH 54c 54b 66a O OH N O N3 N O N3 N 3 H 3 OH OH OH 76c 66b 76a 76b OH O O O O O N3 N O O 3 76d 77b N3 N3 77a 77c

Figure 3.6. The twelve requisite azide building blocks.

3.4 Synthesis of alkyne components

The lead oxazolidinones ANB-22, ANB-40, ANB-24 and ANB-27 all utilized

amine diversity elements. Thus the 1,2,3-triazole analogs were designed to emulate the

substitution patterns of the oxazolidinone library. But the requisite propargylamine-

derived alkyne building bolcks were also not commercially available and had to be 66

prepared. It was envisaged that N-alkylation reactions using the requisite secondary

amine and alkyl halide would enable the synthesis of the desired propargylamine derived

alkynes. The syntheses of either the same or structurally similar alkynes341,342 have been reported. Typically, the requisite alkyl halide and secondary amines are reacted in either methanol or tetrahydrofuran to provide the amine-derived alkynes. The syntheses of the alkyne precursors were prepared in one step by refluxing propargyl bromide 51 with six

different secondary amines 80a-f (Scheme 3.8). These amines were carefully selected to

match those used in the synthesis of the oxazolidinone library. For example, the

secondary amines 80a-e were among the amine diversity elements for the oxazolidinone

library. The amine 80f was included to enhance the diversity of the propargylamine

building blocks. This alkylation method provided six of the alkyne components required

for the generation of the triazole library. These six propargylamine-derived alkynes were

accessed in moderate to good yields.

R2 R2 K CO , THF, reflux, 6-24 h Br + R1 N 2 3 N H R1 51 80a-f 48a-f

48a R1= nBu, R2 = nBu, 72% 1 2 48b R = Me, R = CH2CH2Ph, 75% 1 2 48c R , R = (CH2CH2)2O, 84% 1 2 48d R , R = (CH2CH2)2CHPh, 80% 1 2 48e R , R = (CH2CH2)2NPh, 78% 1 2 48f R , R = CH2CH2CH2(OCOCH2CH3)CHCH2, 69%

Scheme 3.9. Synthesis of alkynes from propargyl bromide and secondary amines.

The first N-alkylation method provided six of the nine alkynes needed for the synthesis of the 1,2,3-triazole analogs. The three remaining precursors had to be prepared 67 via a different route because the starting materials (alkyl halides) were either not commercially available or very expensive. The alkyne derivatives were prepared by following a similar N-alkylation method as in Scheme 3.9. The commercially available

N-methyl propargylamine 52 was refluxed with three different alkyl halides 81a-c to afford the remaining propargylamine derived alkyne components, 82a-c, in good yields

(Scheme 3.9). It is worth noting that, the alkyne components were accessed in one step following the two different synthetic routes.

R H K CO , CH OH, reflux, 24 h R Br 2 3 3 N + N 52 81a-c 82a-c

82a: R= C6H11, 74% 82b: R= CH2CH2(CH3)2, 77%

82c: R = CH2CH2Ph, 80%

Scheme 3.10. Synthesis of alkynes from N-methyl proparylamine and alkyl bromide.

Figure 3.5, depicts the structures of all nine-alkyne building blocks needed for the synthesis of 1,2,3-triazole analogs of the oxazolidinone. As observed in the azide components, these structurally diverse alkyne components provide means of increasing the diversity of the appendage decoration of the amine substitution. 68

N N N N N Bu Me Ph O N 48a Ph 48b 48d Ph 48c 48e

N O N N Me N Me Me Me O Me Ph 48f 82b 82c 82a

Figure 3.7. Structures of the nine propargylamine building blocks.

3.5 Determination of reaction condition for 1,2,3-triazole library synthesis.

To recap, the 1,4-disubstituted 1,2,3-triazole compounds are regioselectively and

efficiently prepared via Cu(l)-mediated azide and terminal alkyne 1,3-dipolar

cycloaddition. This discovery by Sharpless85 and Melda320 groups in 2002 has had varied

applications in medicinal chemistry for the synthesis of 1,4-disubstituted 1,2,3-triazole

derivatives in high yields and purity. Though there are no known optimal reaction

conditions for the Cu(I)-mediated cycloaddition, the method reported by Sharpless and

coworkers was adopted for this study. This is because the in-situ generation of Cu(l) ions

from Cu(ll) salts utilized relatively simple reaction conditions that do not require the use

of nitrogenous base as in Cu(I) salts applications.85 The reaction time is relatively short

and reaction is carried out in water using tert-butyl alcohol as co-solvent. Further,

considering the reaction conditions employed, workup procedure is also relatively simple.

Having the twelve azide components and nine propargylamine-derived alkynes in

hand, synthesis of the 1,4-disubstituted 1,2,3-triazole compound library began. A test

cycloaddition reaction following typical Sharpless conditions and using the azide 54b and phenylacetylene provided the expected 1,4-disubstituted 1,2,3-triazole in 98% yield. 69

Based on these results, the inclination was to run a few cycloaddition reactions using selected building blocks for the 1,2,3-triazole analogs. This was a measure taken to ascertain if the previously reported Cu(I) catalyzed azide/alkyne cycloaddition reaction will be suitable for generating the desired amine substituted 1,2,3-triazole library. In this initial endeavor, the synthesis of sixteen 1,4-disubstituted 1,2,3-triazoles were attempted using four azides [54b, 54c, 76c and 76d] and four alkynes [48a, 48d, 48e and 82c] an array of 4x4=16. Though the literature reported yields on the order of 90% or more and completion of cycloaddition reaction in 8 hours by following Sharpless conditions, this was not the case for the attempted cycloadditions. Approximately 20% product and substantial amount of starting material was recovered after stirring the reaction mixture at room temperature for 48 hours. Heating the reaction mixture at 50 °C for 6 hours did not alter the yield of the reaction. These discouragingly low yields prompted further investigation and scrutiny of the reaction conditions. The mechanism proposed by

Sharpless and coworkers is highlighted in scheme 3.10. According to the mechanism, the reaction is initiated by the oxidative insertion of Cu(I) into the terminal alkyne 84 to generate the reactive copper acetylenide 85. This reactive intermediate reacts with the azide 86 to generate the intermediate 87, which cyclized to provide the six membered intermediate 88. The intermediate rearranges to the copper substituted 1,2,3-triazole 83.

Since the reaction is carried out in protic solvents, the Cu(I)-catalyzed is generated and the desired 1,4-disubstituted 1,2,3-triazole prepared. 70

2 R CuLn R2 N N 1 N R 2 R N N 1 CuLn 83 N R N 89 N N R1 88

+ [LnCu]

R2 H 84 2 R CuLn

N 1 N N R 87 R2 CuLn 85

N N N R1 86

Scheme 3.11. Proposed mechanism for Cu(I)-catalyzed cycloaddition.

Though copper (I)-catalyzed azide/alkyne cycloaddition (CuAAC) has been extensively utilized in synthesizing diverse 1,2,3-triazole compounds, the presence of basic amine elements in its applications appears to be limited.343-347 Considering the fact that the progression of the cycloaddition dwelled on the formation of the copper acetylenide, two plausible explanations were put forth to account for the low yields observed. The first rational projection was that copper has high affinity for amines and that majority of the catalytic amount of Cu(II) used most likely coordinated to the nitrogen. Thus, presenting limited amount of Cu(I) ions in solution to effect the formation of the reactive copper acetylenide that is required to initiate the cycloaddition. The second reason was that, since the reaction was conducted in water, the Cu(I) generated from the one electron reduction of Cu(II) underwent simultaneous disproportionation regenerating Cu(II) and Cu(0). Both explanations lead to a situation where Cu(I) ion is 71

drastically reduced in solution. If these advanced reasoning hold true for the low yield of

the attempted cycloaddition, then two hypotheses could be investigated.

1. Copper is engaged in coordinate bonding to the amine of the propargylamine

derived alkynes and stoichiometric amount (instead of catalytic) of Cu(II) is

required to drive the cycloaddition reaction to completion.

2. Water enhances the disproportionation of Cu(I) to Cu(II) and Cu(0) and

organic solvent devoid of water is required to avail more Cu(I) in solution for

the manifestation of the copper acetylenide.

3.5.1 Model studies

To circumvent the challenges encountered in the earlier cycloaddition reactions,

two hypotheses were examined. Three model studies were designed and carried out in an

attempt to discern suitable reaction conditions for the cycloaddition. Reaction 1 and 2

were put forth to examine the first hypothesis and reaction 3 for the second hypothesis.

With the notion that the poor reactivity was owing to the nature of the alkyne used, the

same azidohydrin 76d was used in all three-model reactions (Scheme 3.11).

Utilizing phenylacetylene (without an amine group) as the alkyne components in

reaction 1 and following the same conditions adopted for the attempted cycloadditions,

led to a quantitative yield 96% of the 1,2,3-triazole 91. In constrast, using

propargylamine derived alkyne 82c in reaction 2 and stoichiometric amount of Cu(II) salt also led to 92% yield of the desired 1,4-disubstituted 1,2,3-triazole compound 92. These two observations suggested the azide and alkyne decide the amount of copper(I). Further, employing reactions conditions that utilizes the direct use of Cu(I) salt (catalytic amount) 72

in dry organic solvent such as acetonitrile in reaction 3, also led to 93% yield of the

product 92. Though workup for reaction 3, was relatively lengthy, it signified that avoiding water probably minimized the rate of disproportionation, thereby availing more

Cu(I) ion in solution to effect cycloaddition. These results from the model studies clearly indicated that a stoichiometric amount of Cu(II) salts were needed to drive the cycloaddition to completion for the triazole library, which utilized propargylamine- derived alkynes.

N N N3 CuSO4• 5H2O (5 mol%) N ...... 1 + Sodium ascorbate (50 mol%) OH H O/tBuOH, r.t OH 76d 90 2 96% 91

N N N N CuSO4• 5H2O (1 equiv) N 3 N ...... 2 + Sodium ascorbate (2 equiv) t OH H2O/ BuOH, r.t OH 82c 76d 92% 92

CuI (0.1 equiv), CH CN N N N N 3 ...... 3 3 N N + 2,6-lutidine OH 93% 76d 82c OH 92

Scheme 3.12. Model studies to study reaction conditions.

3.5.2 Determination of workup conditions

A Typical workup procedure for CuAAC entails concentrating the reaction

mixture, diluting the residue with dichloromethane, washing consecutively with a

mixture of 1:1 NH4OH/H2O, H2O, brine, drying over MgSO4, filtering and concentrating 73

the filtrate. This seemed challenging considering the projected library size. This

prompted our inquiry into identifying a workup procedure that would facilitate the clean

up of the desired 1,4-disubstituted 1,2,3-triazole library members in a short time. The

following three-workup procedures were carried out in an attempt to discern a relatively

simple and quick workup condition for subsequent library:

1. Simple filtration using polyvinylpyridine as scavenger for copper ions.

2. Adding polyvinylpyridine as scavenger to the reaction mixture and

filtering through a phase separator.

3. Adding a mixture of 1:1 NH4OH/H2O to the reaction mixture and filtering

through a phase separator.

All three-workup procedures required the reaction mixture to be concentrated to at least

half its initial volume prior to workup. Simple filtration using polyvinylpyridine as

scavenger was efficient in cleaning up library members when catalytic amount of Cu(II)

salts were used. As the amount of Cu(II) salts was increased to stoichiometric equivalent,

generic purification using polyvinyl pyridine as scavenger became inefficient method in

removing copper residues from the mixture. This was apparent from the paramagnetic

effect of copper in the proton NMR. Consequently, aqueous workup was required to

completely remove copper ions. This was time-consuming considering the size of the

library. Adding polyvinylpyridine as a scavenger and filtering the mixture through a

phase separator produced similar results as generic purification. Dissolving the reaction

mixture in 1:1 NH4OH/H2O and using the phase separator provided a quick, efficient and simple workup procedure. The products were afforded in high yields and purity. As a 74 result, the third workup procedure was adopted for the workup of the 1,2,3-triazole library.

3.5.3 Generation of 1,4-disubstituted 1,2,3-triazole library

Having identified a suitable reaction condition to effect cycloaddition and an efficient workup procedure, the library generation began. The twelve azide (Figure 3.3) and nine alkynes (Figure 3.4) building blocks were subjected to the modified reaction conditions of the previously reported CuAAC. A total of one hundred eight (array of 12 x 9 = 108) 1,4-disubstituted 1,2,3-triazole compounds were prepared by reacting a selected azide component with all nine alkyne in a batch (Table1-12). This 1,4- disubstituted 1,2,3-triazole synthesis was expedited by the robot (automation), which dispensed the required amounts of the starting materials into the designated vials. The yields of individual members of the 1,2,3-triazole are depicted in the tables 1-12 below.

An average yield of 88-96% and purities in the range of 80-95% (HPLC) were obtained for the compound library. The mass of each member was also verified using LC-MS analysis. 75

Table 3.1. Yield and purity of 94a-i, using azide 76c and all nine alkynes

CuSO •5H O, H O/tBuOH N O N3 + 4 2 2 O N N OH R sodium ascorbate, rt, 18-24 h OH 76c 93a-i 94a-i R

Entry Comp ID R % Purity % Yield N 94a GHB-111 Me 81 95 Ph N 94b GHB-113 nBu 82 89 N Me 94c GHB-95 Me 90 93 Me 94d GHB-114 N 98 95 Ph 94e GHB-66 N 94 92 O 94f GHB-25 N O 98 91 O N 94g GHB-24 N 81 96 Ph N 94h GHB-65 Me 98 93

N 94i GHB-112 Me Ph 84 91

Table 3.2. Yield and purity of 95a-i, using azide 64b and all nine alkynes.

O O t N O N3 + CuSO4•5H2O, H2O/ BuOH N H N O N OH R sodium ascorbate, rt, 18-24 h H N OH 64b 93a-i 95a-i R

Entry Compd ID R % Purity % Yield N 95a GHB-94 Me 99 91 Ph N 95b GHB-67 nBu 96 94 N Me 95c GHB-69 Me 84 92 Me 95d GHB-93 N 100 94 Ph 95e GHB-42 N 100 92 O 95f GHB-68 N O 92 92 O N 95g GHB-40 N 80 87 Ph N 95h GHB-70 Me 85 90

N 95i GHB-102 Me Ph 94 95 76

Table 3.3. Yield and purity of 96a-i, using azide 76b and all nine alkynes

N CuSO •5H O, H O/tBuOH N N3 + 4 2 2 N OH R sodium ascorbate, rt, 18-24 h OH 76b 93a-i 96a-i R

Entry Compd ID R % Purity % Yield N 96a GHB-9 Me 86 92 Ph N 96b GHB-108 nBu 100 92 N Me 96c GHB-16 Me 86 89 Me 96d GHB-19 N 90 93 Ph 96e GHB-63 N 90 92 O 96f GHB-11 N O 99 91 O N 96g GHB-13 N 84 88 Ph N 96h GHB-12 Me 98 96

N 96i GHB-107 Me Ph 99 93

Table 3.4. Yield and purity of 97a-i, using azide 77a and all nine alkynes

N O t CuSO4•5H2O, H2O/ BuOH N + N O R sodium ascorbate, rt, 18-24 h N3 O 77a 93a-i O 97a-i

Entry Compd ID R % Purity % Yield N 97a GHB-54 Me 100 91 Ph N 97b GHB-55 nBu 81 93 N Me 97c GHB-51 Me 100 94 Me 97d GHB-31 N 100 94 Ph 97e GHB-83 N 84 92 O 97f GHB-49 N O 94 91 O N 97g GHB-58 N 82 92 Ph N 97h GHB-32 Me 83 93

N 97i GHB-56 Me Ph 84 92 77

Table 3.5. Yield and purity of 98a-i, using azide 64a and all nine alkynes

O O CuSO •5H O, H O/tBuOH N O N3 + 4 2 2 N H R sodium ascorbate, rt, 18-24 h N O N OH H N 64a 93a-i OH 98a-i R

Entry Compd ID R % Purity % Yield N 98a GHB-7 Me 98 92 Ph N 98b GHB-27 nBu 100 92 N Me 98c GHB-29 Me 81 92 Me 98d GHB-61 N 100 92 Ph 98e GHB-52 N 100 90 O 98f GHB-28 N O 97 96 O N 98g GHB-57 N 80 88 Ph N 98h GHB-30 Me 84 95

N 98i GHB-26 Me Ph 95 93

Table 3.6. Yield and purity of 99a-i, using azide 54d and all nine alkynes

O O N O N CuSO •5H O, H O/tBuOH O N 3 + 4 2 2 N OH R sodium ascorbate, rt, 18-24 h OH 54b 93a-i 99a-i R

Entry Compd ID R % Purity % Yield N 99a GHB-77 Me 83 91 Ph N 99b GHB-79 nBu 81 92 N Me 99c GHB-81 Me 84 92 Me 99d GHB-104 N 84 94 Ph 99e GHB-43 N 88 90 O 99f GHB-80 N O 84 93 O N 99g GHB-41 N 81 92 Ph N 99h GHB-82 Me 82 95

N 99i GHB-78 Me Ph 82 93 78

Table 3.7. Yield and purity of 100a-i, using azide 54c and all nine alkynes

O O

CuSO •5H O, H O/tBuOH N O N3 + 4 2 2 O N N OH R sodium ascorbate, rt, 18-24 h OH 54c 93a-i 100a-i R

Entry Compd ID R % Purity % Yield N 100a GHB-97 Me 100 94 Ph N 100b GHB-71 nBu 83 91 N Me 100c GHB-73 Me 85 92 Me 100d GHB-101 N 90 97 Ph 100e GHB-46 N 91 90 O 100f GHB-72 N O 96 96 O N 100g GHB-2 N 82 90 Ph N 100h GHB-74 Me 84 94

N 100i GHB-103 Me Ph 100 94

Table 3.8. Yield and purity of 101a-i, using azide 76a and all nine alkynes

t N + CuSO4•5H2O, H2O/ BuOH N N3 sodium ascorbate, rt, 18-24 h N R OH OH 93a-i R 76a 101a-i

Entry Compd ID R % Purity % Yield N 101a GHB-92 Me 84 96 Ph N 101b GHB-53 nBu 94 92 N Me 101c GHB-23 Me 95 89 Me 101d GHB-4 N 89 93 Ph 101e GHB-64 N 100 93 O 101f GHB-109 N O 98 93 O N 101g GHB-22 N 80 90 Ph N 101h GHB-110 Me 94 95

N 101i GHB-105 Me Ph 84 91 79

Table 3.9. Yield and purity of 102a-i, using azide 54a and all nine alkynes

O O

t N O N + CuSO4•5H2O, H2O/ BuOH O N 5 3 5 N OH R sodium ascorbate, rt, 18-24 h OH 102a-i 54a 93a-i R

Entry Compd ID R % Purity % Yield N 102a GHB-75 Me 81 92 Ph N 102b GHB-76 nBu 93 94 N Me 102c GHB-100 Me 84 89 Me 102d GHB-106 N 100 94 Ph 102e GHB-85 N 87 96 O 102f GHB-99 N O 97 92 O N 102g GHB-59 N 98 91 Ph N 102h GHB-96 Me 95 93

N 102i GHB-98 Me Ph 91 93

Table 3.10. Yield and purity of 103a-i, using azide 77b and all nine alkynes

R O O N CuSO •5H O, H O/tBuOH N + 4 2 2 O O R sodium ascorbate, rt, 18-24 h N N3 93a-i 103a-i 77b

Entry Compd ID R % Purity % Yield N 103a GHB-86 Me 96 91 Ph N 103b GHB-44 nBu 86 93 N Me 103c GHB-88 Me 95 94 Me 103d GHB-89 N 98 94 Ph 103e GHB-84 N 86 92 O 103f GHB-48 N O 100 91 O N 103g GHB-90 N 81 92 Ph N 103h GHB-45 Me 95 93

N 103i GHB-87 Me Ph 86 92 80

Table 3.11. Yield and purity of 104a-i, using azide 77c and all nine alkynes

O R t N O + CuSO4•5H2O, H2O/ BuOH N O O O R sodium ascorbate, rt, 18-24 h N O N3 77c 93a-i 104a-i

Entry Compd ID R % Purity % Yield N 104a GHB-33 Me 97 95 Ph N 104b GHB-37 nBu 81 91 N Me 104c GHB-60 Me 82 88 Me 104d GHB-34 N 98 91 Ph 104e GHB-47 N 81 92 O 104f GHB-38 N O 96 92 O N 104g GHB-35 N 84 89 Ph N 104h GHB-39 Me 84 93

N 104i GHB-36 Me Ph 93 92

Table 3.12. Yield and purity of 105a-i, using azide 76d and all nine alkynes

OH t OH + CuSO4•5H2O, H2O/ BuOH R sodium ascorbate, rt, 18-24 h N3 N N N 93a-i 76d 105a-i R

Entry Compd ID R % Purity % Yield N 105a GHB-1 Me 89 92 Ph N 105b GHB-18 nBu 100 92 N Me 105c GHB-15 Me 100 91 Me 105d GHB-20 N 84 94 Ph 105e GHB-62 N 82 92 O 105f GHB-14 N O 98 92 O N 105g GHB-10 N 90 88 Ph N 105h GHB-21 Me 81 96

N 105i GHB-17 Me Ph 100 93 81

3.6 Biological evaluation of 1,4-disubstituted 1,2,3-triazole compounds

Prior to the compounds being sent to collaborators for biological evaluation, the

hydrochloride salts were prepared. The compounds were then dissolved in 99% DMSO to

afford a concentration of 50 mM stock solution, out of which the required amounts were

drawn and submitted to the Hines’ group, Ohio University, for RNA binding affinity and

specificity studies. Aliquot samples were also sent to the Priestley group, Montana

University, to be evaluated for activity towards specific bacteria strains.

3.6.1 Fluorescence resonance energy transfer assay

The 1,4-disubstituted 1,2,3-triazole compounds were evaluated in floresecence

resonance energy transfer (FRET) assay.348 Shu Zhou conducted FRET assay in Hines

laboratory. FRET is a result of the transfer of energy between donor and acceptor

fluorophores and is a widely used method for studying the interaction of small molecules

with macromolecules. As FRET349-352 is capable of measuring the distance between two fluorophores, it has been instrumental in providing important information on interactions between small molecules and RNA and RNA-RNA-interactions. As such, this method has been extensively employed in antimicrobial therapy to evaluate the binding of antibiotics preselected targets such as RNA (AM1A).66,67

Based on the insightful information FRET provides, it was used to assess the

ability of the 1,2,3-triazole analogs to bind RNA. Two model RNAs, namely AM1A

(wildtype)70 and C11U (specificity control)70 were employed for the in vitro screening.

The two model RNAs AM1A and C11U were labeled in a manner to enhance FRET 82

studies. The donor, namely fluorescein was attached on the 3 end, while the acceptor end

located on U18 in the UUCG loop was labelled with rhodamine as previously described

(Figure 3.8).81

U U18 U U18 11 C C C C C C G U G A C G G A C C G 9A G C 9 G CG G A G G U A2 G U A2 G6 UGC G6UGC GC A1 GC A1 GC GC AU AU 5'GC3' 5'GC3' AM1A C11U

Figure 3.8. Structures of model RNAs AM1A and C11U.

This change in relative percentage fluorescence intensity F, which had been previously matched with binding affinities348 was employed to evaluate the RNA binding affinities of the 1,4-disubstituted 1,2,3-triazoles (Table 3.13). Worth noting, of the one hundred eight 1,4-disubstituted 1,2,3-triazole that were subjected to the FRET assay, fifty increased the florescence intensity more than that of the lead oxazolidinones ANB-22

(8.53%) and ANB-40 (7.67%).82 83

Table 3.13. FRET data for analogs with enhanced RNA binding affinities.

a Compd F A(%) FC(%) Compd FA(%) FC(%) Compd FA(%) FC(%)

b ANB-22 8.53 19.53 GHB-46 11.70 18.05 GHB-71 9.07 9.63

ANB-40 7.67 13.38 GHB-47 13.47 22.72 GHB-72 28.48 26.13

GHB-9 9.39 14.36 GHB-48 17.95 18.22 GHB-76 11.66 15.17

GHB-14 9.17 8.65 GHB-49 23.00 28.49 GHB-79 14.12 13.46

GHB-21 12.83 16.15 GHB-50 14.94 20.05 GHB-80 18.92 23.73

GHB-24 9.89 10.52 GHB-57 9.94 14.39 GHB-81 15.23 16.01

GHB-25 16.07 20.17 GHB-58 20.05 24.05 GHB-82 15.65 17.37

GHB-26 19.92 27.38 GHB-59 32.93 34.73 GHB-83 22.60 24.76

GHB-27 20.43 24.49 GHB-60 12.25 19.17 GHB-84 16.09 20.19

GHB-28 25.51 22.32 GHB-61 13.01 25.07 GHB-107 14.18 13.82

GHB-29 16.21 21.38 GHB-62 13.95 23.19 GHB-108 10.68 11.57

GHB-30 16.43 22.17 GHB-63 11.74 23.19 GHB-109 12.75 13.34

GHB-35 10.16 13.94 GHB-64 11.73 15.73 GHB-110 18.21 14.55

GHB-37 9.64 16.31 GHB-65 19.60 23.21 GHB-111 15.07 17.68

GHB-38 25.78 26.08 GHB-66 14.54 28.75 GHB-112 18.43 19.55

GHB-40 11.30 12.74 GHB-67 21.65 32.76 GHB-113 24.09 29.09

GHB-42 16.79 21.12 GHB-68 29.72 25.65 GHB-7 2.5 9.7

GHB-43 19.50 25.03 GHB-70 14.51 22.54 a The relative fluorescence intensity change F was calculated by F=[(F-F0)/F0]*100, where F is the fluorescence intensity with ligand and F0 is without ligand at 585 nm upon excitation at 467 nm. b All the compounds were tested at a final concentration of 10 μM. A = AM1A, C = C11U Shu Zhou conducted FRET assay in Hines laboratory. 84

A correlation graph of AM1A and C11U gave a straight line, which did not go through the origin (zero) but cut above the AM1A axis (Figure 3.9). This signified the high specificity of the 1,4-disubstituted 1,2,3-analogs for the wildtype RNA model (AM1A) relative to the control (C11U).

Figure 3.9. A correlation graph of 1,4-disubstituted 1,2,3-triazole analogs for model RNAs AM1A and C11U.

Because a key part of this work was to identify 1,4-disubstituted 1,2,3-triazole analogs that bind model RNAs with high specificity, Table 3.13 was re-sorted to reflect the specificities of the 1,4-disubstituted 1,2,3-triazole analogs relative to the lead oxazolidinones ANB-22 and ANB-40. A larger difference between the fluorescence

intensity of AM1A (FAM1A(%)) and C11U (FC11U(%)) signified higher specificity. For this comparison, the specificity of ANB-40 was taken as the cut-off for evaluating the

1,4-disubstituted 1,2,3-triazole analogs (Table 3.14). Intriguingly, thirteen 1,4- disubstituted 1,2,3-triazole analogs showed enhance specificity relative to ANB-40. 85

Table 3.14. FRET data for analogs with enhanced RNA binding specificity

Compound FAM1A(%) FC11U(%) F C11U(%)-FAM1A(%) ANb-22 8.53 19.53 11.00 ANB-40 7.67 13.38 5.71 GHB-66 14.54 28.75 14.21 GHB-61 13.01 25.07 12.06 GHB-63 11.74 23.19 11.45 GHB-67 21.65 32.76 10.51 GHB-47 13.47 22.75 9.28 GHB-62 13.95 23.19 9.24 GHB-70 14.51 22.54 8.03 GHB-26 19.92 27.38 7.46 GHB-7 2.50 9.70 7.20 GHB-60 12.25 19.17 6.92 GHB-37 9.64 16.31 6.67 GHB-46 11.70 18.05 6.35 GHB-30 16.43 22.17 5.74

The relative fluorescence intensity change F was calculated by F=[(F-F0)/F0]*100, where F is the fluorescence intensity with ligand and F0 is without ligand at 585 nm upon excitation at 467 nm. b All the compounds were tested at a final concentration of 10 μM. Only analogs with specificities greater than 5.71 are indicated on the table.

FC11U(%)-FAM1A(%) = Specificity

Of these, GHB-61, GHB-63 and GHB-66 showed improved specificities relative to

ANB-22, with GHB-66 exhibiting the largest F value of 14.21. The structures of

structures of all thirteen 1,4-disubstituted 1,2,3-triazole analogs that exhibited good RNA

binding affinities and specificities are shown in Figure 3.10. By closely examining the

structures of all analogs (Figure 3.10), it is apparent that the five analogs, GHB-46,

GHB-47, GHB-62, GHB-63 and GHB-66 had the same amine diversity element

(morpholine) but different substituent diversity at position 1 of the 1,2,3-triazole ring.

But clearly, the analogs GHB-7 and GHB-26 demonstrated the effect of homologation of

biological activity with GHB-26 (one homologous chain shorter in amine substitution)

exhibiting enhanced biological activity. While the FRET data for GHB-7 and GHB-26 86 may suggest a decrease in homologous chain as essential for RNA binding activity, more data is required to generalize this effect and it would interesting to investigate the activity of an analog that is two methylene chains shorter than GHB-7. The effect of ring chain transformation is also evident from the structures of GHB-7 and GHB-61 with GHB-61 having a relatively hindered rotation owing to the piperidine ring having improved RNA affinity and specificity. Furthermore, it is possible from the structures of GHB-37, GHB-

47, GHB-60 and GHB-62 that the cyclohexyl group, which confers conformational rigidity to those analogs, may be key for biological activity.

O N O N N N N O N N OH H N N N OH OH GHB-66 N O GHB-61 N GHB-63 N O

O N N O N N O N N N H N N N O N OH H N GHB-67 O OH N O O GHB-7 N O GHB-47 O N N N O N O N N N H N N OH N O N H N GHB-70 N OH OH GHB-26 O N GHB-62 N N N N N O O N N N N O N N O N N O N N O OH H O OH O O O GHB-46 GHB-30 GHB-60 N GHB-37 N O

Figure 3.10. Structures of the 1,4-disubstituted 1,2,3-triazoles that bind model RNAs. 87

3.6.2 Antibacterial Assay

In addition to the RNA binding evaluation, the 1,4-disubstituted 1,2,3-triazole

analogs were examined in the antibacterial assay to determine their ability to inhibit the

growth of selected Gram-positive pathogens (Bacillus subtilis and Staphylococcus aureus). Antibacterial assay was carried out in Priestley laboratory. All the I,4- disubstituted 1,2,3-triazole analogs were evaluated using 10% Alamar Blue dye in a 2- fold dilution series containing the test organism (1104 to 1105 cells/mL) in a 96 well

plate. An analog was considered to be active if at the tested concentration less than 1%

reduction of the blue resazurin (lmax 570 nm) to the pink (lmax 600 nm) was observed. The lowest concentration that inhibited the Alamar Blue reduction was taken as the minimun inhibitory concentration (MIC).

Nine 1,4-disubstituted 1,2,3-triazole analogs exhibited moderate to good activity towards one or both bacterial strains with GHB-7 and GHB-9 having the best antibacterial activity as depicted in Table 3.16. The analog GHB-16 was found to have

weak activity against Bacillis subtilis and Staphylococcus aureus relative to GHB-7 and

GHB-9. Nonetheless, this analog was identified to be very active against Methicilin resistance staphylococcus aureus (MRSA) strains with a minimum inhibitory concentration of less than 8 μM (MIC < 8 μM). 88

Table 3.15. Minimum inhibitory concentrations (μM) of 1,2,3-triazole analogs

1,2,3-Triazole analogs Bacillis subtilis Staphylococcus aureus GHB-7 250 500 GHB-9 250 500 GHB-16 1000 - GHB-19 1000 - GHB-107 500 500 GHB-108 1000 - GHB-110 1000 - GHB-111 500 1000 GHB-112 500 1000 Only 1,4-disubstituted 1,2,3-triazole compounds that showed moderate to good activity based on Alamar Blue reduction were included in Table 3.15. – indicated no activity was detected within the assay’s limits. Antibacterial assay were carried out in Priestley laboratory.

By thoroughly examining the topology of all nine analogs, it was apparent from the analogs GHB-7, GHB-9, GHB-19 and GHB-111, with a common amine substitution that the nature of the diversity element attached to the nitrogen of the 1,2,3-triazole ring may be critical for the inhibitory activity exhibited. This notion is supported by the structures of the analogs GHB-7 and GHB-111. While GHB-7 and GHB-111 have the same amine substitution, the appending of hydroxy ether on the analog GHB-111 instead of the hydroxy carbamate on GHB-7 resulted in a two-fold weaker activity against both

Bacillus subtilis and staphylococcus aureus. On the contrary, the effect of homologation in the amine substitution of GHB-111 and GHB-112 did not alter the biological activity of the two analogs. The structures of the analogs GHB-9, GHB-16, GHB-19, GHB-107 and GHB-108 signified the importance of the 2-hydroxylhexyl group for biological activity, yet the enhancement in activity may reside in the nature of the amine substitution. The analogs GHB-9 and GHB-107 (having amine substitution CH2- shorter) portray the effect of homologation as an inhibitory activity two-fold weaker for Bacillus 89

subtilis was observed for GHB-9. The effect of ring chain transformation of GHB-19

(restricted rotation) relative to GHB-9 also led to a fourfold weaker antibacterial activity

against Bacillus subtilis. Furthermore, the variations in the amine substitution of GHB-9

and GHB-16 (lack of aromatic substitution) led to a fourfold decrease in antibacterial

activity of GHB-16. As empirical observations have been utilized to give highlight on the

effect of diversity elements on antibacterial activity, stucture activity relationship studies

would be require to definitely tie structure to biological activity. The structures of GHB-9

and GHB-16 are interesting and will be explore further in attempt to enhance their initial antibacterial activity.

O N N N O N N N H N N N Ph OH Ph N OH OH GHB-7 N GHB-9 N GHB-16 N

N N N N N N N N N OH OH Ph OH N GHB-19 N Ph GHB-107 N GHB-108

N N N O N O N N N N N OH Ph OH Ph OH N N GHB-110 N GHB-111 GHB-112

Figure 3.11. Structures of viable 1,2,3-triazole analogs from antibacterial assay.

3.7 Synthesis of 1,5-disubstituted 1,2,3-triazoles analogs

Prior to the discovery of the CuAAc,85,319 the 1,2,3-triazole compounds were prepared as a mixture of two regioisomeric compounds, namely the 1,4- and 1,5-.90 Thus, 90

for completeness, few 1,5-variants of the 1,4-disubstituted 1,2,3-triazole analogs that

have shown good biological profile were prepared. Both the 1,4 and 1,5-regioisomer of

the 1,2,3-triazole have been used as amide bond surrogates in different capacities in the

synthesis of biologically active compounds.84 Therefore, to ascertain that the biological profile exhibited was not owe to residual 1,5-analogs, five 1,5-analogs particularly that of

GHB-7 were prepared.

In contrast to the 1,4-regioisomers that were regioselectively synthesis from the

Cu(I)-catalyzed azide/alkyne cycloaddition,85,319 the 1,5-regioisomers are prepared using

either magnesium353,354 or ruthenium91,355 catalyst. Magnesium catalyst in the form of

Grignard reagent (EtMgCl) had been used mainly for the cycloaddition of terminal alkynes (formation of the reactive magnesium acetylenide) and azide. The use of selected

* ruthenium(II) catalyst such as Cp Ru(PPh3)2Cl has been extensively used in cycloadditions involving both terminal and internal alkynes in its applications.91

By carefully examining the nature of both precursors for the 1,5-analogs, the

* 91 decision was made to use the Cp Ru(PPh3)2Cl as the ruthenium catalyst. Conversely,

this catalyst was not commercially available and had to be prepared. Chinn et al356 had

* reported on the synthesis of this ruthenium catalyst from the polymer [Cp Ru(Cl)2]n.

* According to Chinn et al, by refluxing, the ruthenium polymer [Cp Ru(Cl)2]n and

triphenylphosphine in ethanol produce the desired catalyst in good yield. In view of this,

* attention was directed towards the synthesis of the ruthenium catalyst (Cp Ru(PPh3)2Cl).

The polymer 106, was refluxed in absolute ethanol together with triphenylphosphine for

28 hours to give the orange crystalline solid 107 in very good yield (Scheme 3.13). 91

CH3CH2OH, PPh3, reflux 87% Ru Ru Cl Cl Cl PPh3 Ph3P 106 107

Scheme 3.13. Synthesis of ruthenium catalyst for 1,5-disubstituted 1,2,3-triazole analogs.

Having the ruthenium catalyst, the azide and alkyne components in hand, the synthesis of the 1,5-analogs began. Typically 1-5 mol% catalyst loading are employed in ruthenium-mediated cycloaddition reaction of an azide and alkyne to produce the requisite 1,5-disubstituted 1,2,3-triazole.91 But reflecting on the challenges encountered during the synthesis of the scouting library of 1,4-disubstituted 1,2,3-triazole compounds and the notion that transition metals may exhibit tendencies of coordinating to amines

(propargylamine derivatives used), a higher catalyst loading of 20 mol% was used to offset any amount of the catalyst that may complex to the propargylamine derived- alkynes used. As outlined in Scheme 3.14, the cycloaddition reactions proceeded with an average yield of 80%, but chromatographing the desired compounds became a bit challenging owing to the formation of triphenyphosphine oxide byproduct, whose polarity was very close to that of the 1,5-analogs. 92

N N 107 N3 R1 N + N Benzene, Reflux 1 R 2 R O R 4 h O N 2 R R 108a-b 109a-b 110a-b

1 2 110a R = H, R = CH3(CH2)3, R = CH3(CH2)3, 83% 1 2 110b R = PhOCH2CO, R , R = (CH2CH2)2O, 80%

O O 107 R3 N N O N + 3 N N O N N H 4 Benzene, Reflux H OH R 4 h OH 66a 111a-c 112a-c N 3 R4 R

3 4 112a R = CH3, R = PhCH2CH2CH2, 81% 3 4 112b R = CH3 R = CH2CH2CH(CH2)3, 81% 3 4 112c R , R = CH2CH2CH2(OCOCH2CH3)CHCH2, 78%

Scheme 3.14. Synthesis of the 1,5-disubstituted 1,2,3-triazole analogs.

The 1,5-disubstituted analogs were verified and differentiated from their 1,4- counterparts using proton (1H) NMR as reported by Alvarez and coworkers.316 According to Alvarez and coworkers, the chemical shifts of the 1,4-regioisomers were shifted more downfield relative to their 1,5-counterparts.316 To illustrate, the chemical shift of the H-5

(7.61 ppm) of the 1,4-disubstituted 1.2.3-triazole (Figure 3.12) and the H-4 (7.50 ppm) of

the 1,5-regioisomer (Figure 3.13) were examined to differentiate between the two

regioisomers. A difference of 0.10 ppm was observed between the H-5 of the 1,4-

regioisomer and H-4 of the 1,5- regioisomers. The salts and requisite concentrations of

the 1,2,3-triazole in DMSO were prepared prior to compounds being dispatched to

collaborators for biological evaluation. 93

N N

5 N

H OH

105b

Figure 3.12. Proton NMR of the 1,4-disubstituted 1,2,3-triazole analog 105b.

N 4 H N

OH N

110a

Figure 3.13. Proton NMR of the 1,5-disubstituted 1,2,3-triazole analog 110a. 94

3.7.1 Biological evaluation of 1,5-analogs

The 1,5-disubstituted 1,2,3-triazoles were evaluated in the 5' rhodamine (5'Rhd) assay to assess their binding affinities and specficities for model RNAs. Preliminary biological data from 5' rhodamine (5'Rhd) screening indicate that the 1,5-disubstituted analogs exhibit weaker RNA binding affinities relative to their 1,4-counterparts. The binding specificities obtained also indicated that all 1,4-analogs except GHB-18 exhibited significant enhancement in binding specificities relative to their 1,5– counterpart (Table 3.16). Since the objective for the library generation was to identify analogs that bind with high specificity, the 1,5-analogs were excluded from further biological evaluation. 95

Table 3.16. 5'Rhd screening of 1,5-analogs for model RNA AM1A and C11U

Entry 1,5-Analogs FAM1A (%) FC11U (%) Specificity FAM (%) 105b GHB-18 14.7 6.9 -7.8 -5.9 110a GHB-130 -6.3 -8.7 -2.4 -6.1 110b GHB-47 -0.8 0.7 1.5 -2.9 104f GHB-131 -5.2 -9.0 -3.8 -2.1 98f GHB-28 -11.8 1.0 12.8 -4.4 112c GHB-132 -10 -12.2 -2.2 -3.4 98c GHB-29 -7.0 -2.9 4.1 -4.3 112b GHB-133 -9.6 -10.7 -1.1 -5.4 98a GHB-7 -14.2 -16.7 -2.5 -6.9 112a GHB-134 -7.5 -14.0 -6.5 -4.0

The relative fluorescence intensity change F was calculated by F=[(F-F0)/F0]*100, where F is the fluorescence intensity with ligand and F0 is without ligand at 585 nm upon excitation at 467 nm. All the compounds were tested at a final concentration of 10 μM.

FC11U(%)-FAM1A(%) = Specificity. Analogs labeled in red are the progenitor compounds.

N N O N N N N N N O N H N N OH O OH N O O N O GHB-132 GHB-130 O GHB-131 O O O N N N O N N O N H N H N OH OH GHB-134 N GHB-133 N

Figure 3.14. Structures of the 1,5-disubstituted 1,2,3-triazole analogs.

3.8 Conclusion

A suitable and efficient condition for generating amine substituted 1,4-

disubstituted 1,2,3-triazole library using stoichiometric amount of Cu(II) salts have been

developed as part of the ongoing efforts to identify potential analogs of the oxazolidinone 96

scaffold. These regioisomer of the 1,2,3-triazole have served as an effective bioisosteric

replacement of the oxazolidinone ring and eight compounds have exhibited good RNA

binding activity. Of these, GHB-7 showed improved RNA binding activity relative to

that of the lead oxazolidinone compounds ANB-22 and ANB-40. In addition, two, 1,4- disubstituted 1,2,3-triazole analogs (GHB-7 and GHB-9) were identified to be viable in the antibacterial activity. The analogs exhibit growth inhibitory activity against Bacillus subtilis and Staphylococcus aureus. Besides, GHB-16 exhibited activity against

methicillin resistance Staphylococcus aureus (MRSA) strains. These encouraging

biological results of the 1,4-disubstituted 1,2,3-triazole compounds reinforced the need

for structural activity relationship studies. However, the preliminary activity of the 1,2,3-

triazole analogs provde a platform that elevate the plausibility of finding lead 1,4-

disubstituted 1,2,3-triazole analogs with enhanced antibacterial activity in general and

RNA binding affinities in particular. 97

Chapter 4. Structure Activity Studies and Structural Elaborations of the 1,2,3-

Triazole Ring

4.1 Introduction

The research endeavors to find 1,4-disubstituted 1,2,3-triazole antibacterial agents led to the identification of three lead compounds, namely GHB-7, GHB-9 and

GHB-16. To recap, these 1,2,3-triazole analogs exhibited biological profiles that rendered them important candidates for lead optimization via structure activity relationship (SAR). Intriguingly, the structures of GHB-9 and GHB-16, which were

pharmacologically active in the antibacterial assay overlapped, as both were prepared

from the same azide building block. On the other hand, the structure of the lead RNA

binding ligand, GHB-7, also overlapped with GHB-9, as both were prepared from the

same alkyne building block. Therefore, considering the biological profile of each analog

and the similarities in their functional components, it was projected that SAR studies

would enhance understanding of how each structural component was related to its

activity and as such add more dimensions to the design of subsequent libraries. This

study was also projected as a useful research endeavor of identifying potential leads with

enhanced antibacterial activity in general and improved RNA binding affinity and

specificity in particular. 98

O N N N N N O N N N N H N OH OH OH N N N

GHB-16 GHB-7 GHB-9

Figure 4.1. Structure of the three lead 1,4-disubstituted 1,2,3-triazoles.

4.2 Plan for SAR studies on GHB-7

Utilizing empirical assessment, the focus of the SAR studies on GHB-7 was to strategically alter key functional components with the goal-driven purpose of identifying new leads with enhanced activity as well as the pharmacophoric groups. The three key structural elements highlighted on GHB-7 (Figure 4.2) were of keen interest to this study and each was carefully examined to prepare structurally relevant analogs.

O N N O N H N OH N GHB-7

Evaluate importance Examine the biological of NH-/Phenyl ring relevance of hydroxyl Assess the biological on biological activity group relevance of amine substitution

Figure 4.2. General structure of GHB-7 analogs for SAR studies.

The plan for this study was to first examinine if the NH-group is needed for activity and also to determine if the position of the phenyl ring is optimal. To move 99

forward with this inquiry, CH2NH- was removed to prepare the analog 113a (Figure 4.3).

The NH- group was removed to prepare the analog 113b, which incorporate the same

acyl substitution as ANB-22. Two isosteric replacements, where NH- was substituted

with CH2- and O- were carried out to generate the analogs 113c and 113e respectively.

Compound 113c should provide the needed opportunity of evaluating if the position of

the phenyl ring in GHB-7 is optimal. As part of the modification of the NH-group to

evaluate its biological utility, CH2NH- was replaced with O- to generate the analogs

113d.

O O N N O O N O O N N N OH OH 113d 113e N N

O N N O N H N OH

GHB-7 N

O N O O N N N OH O O N N 113a N OH O N N N 113c OH N 113b N

Figure 4.3. General structure of GHB-7 analogs for SAR studies. 100

Essential to this study was the need to investigate if the phenyl ring was necessary

for biological activity. To move forward with this objective, the phenyl ring was

functionalized and also replaced a heterocyclic ring. Two substitutions, which entail the

replacement of para H- of aromatic ring with methyl (CH3-) and methoxy (CH3O-) were carried out to provide the analogs 114b and 114c respectively (Figure 4.4). The phenyl ring was also replaced with the heterocyclic compound furan to generate the analog 114d.

Further, the role of the benzyl group (PhCH2-) was examined by replacing CH2- with -

CHCH3 to produce the analog 114a.

O O N O N N O N N N O N H H N OH OH O 114c 114d N N

O N N O N H N OH

GHB-7 N

O O N N N O N N N O N H H N OH OH 114a 114b N N

Figure 4.4. General structure of GHB-7 analogs for SAR studies.

While the placement of the the -OH group in GHB-7 may have been critical for pharmacokinetics such as solubility, important aspect of this study was to investigate if 101

the hydroxy group was needed for biological activity. To meet this objective, the analog

115a in which –OH is substituted with H- was generated (Figure 4.5). In addition, the

effect of chirality on biological activity was also axamined by fostering the development

of the two enantiomers 115b and 115c.

O O N N N O N N O N H N H N OH OH 115b 115c N N

O N N O N H N OH GHB-7 N

O N N O N H N 115a N

Figure 4.5. General structure of GHB-7 analogs for SAR studies.

An integral part of this SAR study was the interest of determining if the amine

substitution was necessary for biological activity. For this purpose, Ph(CH2)3N(CH3)CH2-

was replaced with Ph- and CH3(CH2)2CH2- to provide the analogs 116a and 116b respectively (Figure 4.6). In addition, CH3N- was replaced with O- to generate 116c.

Equally important to this study was the need to evaluate if the position of the phenyl 102

group was optimal. The objective was accomplished by utilizing the effect of

homologation to vary the chain length of the amine substitution. Homologation is a lead

optimization approach often embarked on to improve upon the pharmacokinetics as well

as the biological profile of a lead. This lead modification technique has been exemplified

by Richard288 and Dohme et al289 in their investigations of the effect of side chain length

on biological activity. In view of this, the plan was to incorporate propargylamine-

derived alkyne that was two homologous chains shorter than that of the progenitor

compound, GHB-7. This decision was based on the fact that an analog one methylene shorter in the amine substitution had been prepared during the primary library that binds

RNA better than GHB-7. Consequently, the commercially available alkyne component that was two homologous chains shorter (N-benzyl-N-methylprop-2-yn-1-amine) was

utilized to generate the analog 116d. In all, the plan for the SAR on GHB-7 was to

prepare 16 analogs that promote the assessment of key structural features of GHB-7. 103

O O N N N O N N O N N H N H OH OH 116c 116d O N

O N N O N H N OH GHB-7 N

O O N N N O N N O N N H N H OH OH 116a 116b

Figure 4.6. General structure of GHB-7 analogs for SAR studies.

4.2.1 Synthesis of azide components

Several azide building blocks required for the synthesis of GHB-7 analogs were not commercially available and had to be prepared starting from the requisite glycidol

(Scheme 4.1). The commercially available glycidol was treated with three acid chlorides and two chloroformates to provide the three glycidyl esters 118a-c and two glycidyl formates 118d-e in 92-95% yield (Scheme 4.1). These epoxides 118a-e were subjected to the previously adopted azide opening reaction. The gylcidyl esters 117a-c gave the corresponding azides 119a-c in good yields. The completion of this synthesis provided a set of the azides building blocks required for the diversification of the NH-group. 104

O O O DMAP, DCM O NaN , NH Cl HO + 3 4 R Cl 0 °C, r.t, 4 h R O CH OH : H O(8 : 1) R O N3 O 3 2 OH 49 117a-e 118a-e r.t, 24 h 119a-e

118a R = Ph, 92% 119a R = Ph, 75% 118b R = PhCH2, 91% 119b R = PhCH2, 72% 118c R = PhCH2CH2, 94% 119c R = PhCH2CH2, 75% 118d R = PhO, 94% 119d R = PhO, - 118e R = PhCH O, 95% 2 119e R = PhCH2O, -

Scheme 4.1. Synthesis of the azide components from glycidol.

Surprisingly, the ring opening of the two-glycidyl formates became very

challenging and led to the formation of a cyclic carbonate in quantitative yield. Varying

the reaction conditions did not provide the desired acyclic azidoformates but rather the

cyclic azide byproduct. Since all the attempts to prepare the azido carbonates from the

glycidyl formates resulted in the formation of a cyclic five membered azide compound

121 (Scheme 4.2), an alternative synthetic route was developed in the reverse fashion.

Glycidol 49 was opened with sodium azide to provide the azide compound 120.

Attempted acylation using the requisite chloroformates again afforded the cyclic azido

carbonate 121. It became apparent, after several attempts that compound 121 was afforded irrespective of the method adopted. 105

O OH OH O R Cl NaN3, NH4Cl R O N HO HO N3 3 CH3OH : H2O (8:1) 120 DMAP, Et N, 49 3 O 119d-e r.t, 24 h, 83% 0 °C, 2-3 h R Cl R = PhO, PhCH2O O

DMAP, Et3N, 0 °C, 2-3 h

O O + ROH

N3 121

Scheme 4.2. Alternative synthetic route for acyclic azido carbonate synthesis.

To synthesize the remaining azide building blocks required for preparing GHB-7 analogs, glycidol 49 was treated with four different isocyanates 122a-d to give the glycidyl carbamates 123a-d in 83-90% yield (Scheme 4.3). These carbamates were subjected to the previously adopted azide opening reaction to provide another set of the azide building blocks 124a-d in good yield.

O O O DMAP, DCM NaN , NH Cl HO + R-N=C=O R 3 4 N O R 0 °C, r.t, 4 h CH OH : H O(8 : 1) N O N3 H O 3 2 H 49 122a-d 123a-d r.t, 24 h 124a-d OH

123a R = PhCH(CH3), 83% 124a R = PhCH(CH3), 73% 123b R = 4-(CH3)PhCH2, 90% 124b R = 4-(CH3)PhCH2, 82% 123c R = 4-(CH3O)PhCH2, 84% 124c R = 4-(CH3O)PhCH2, 79 123d R = (Furan-2-yl)methyl, 87% 124d R = (Furan-2-yl)methyl, 77%

Scheme 4.3. Synthesis of azide component without the hydroxy group. 106

The third set of azide component required for preparing the analog 115a was

prepared from 3-bromo-1-propanol 125 (Scheme 4.4). The alcohol 125 was subjected to

azidolysis using NaN3 to afford the azido alcohol 126 in 70% yield. This azide 126 was then treated with benzyl isocyanate to provide the azide component 128 devoid of the hydroxyl group in good yield.

O DMF, NaN3 DMAP, Et N HO Br HO N3 + N=C=O 3 r.t, 8 h N O N3 r.t, 8 h H 125 70% 126 127 128 85%

Scheme 4.4. Synthesis of azide component without the hydroxy group.

An important aspect of the preparation of GHB-7 analogs was the kindled desire of preparing the chiral analogs of GHB-7. To move forward with this synthesis, (R)- and

(S)- azide components had to be prepared. The (R)-enantiomer of glycidol 129 was treated with benzyl isocyanate 127 at 0 °C and chromatographed without workup to give the glycidyl carbamate 130 in 70% yield (Scheme 4.5). Carrying out the same reaction at room temperature led to a very low yield of the desired compounds. The epoxide 130 was then subjected to azidolysis to afford the (R)-enantiomer of the azide component 131 in

74% yield. In a similar manner, the (S)-enantiomer of glycidol 132 was treated with benzyl isocyanate 127 followed by azidolysis to afford the (S)-enantiomer of the azide component 134. The completion of these syntheses brought the total number of azide components for GHB-7 analogs to 14, two less than that proposed. 107

O O O N=C=O + DMAP, DCM O NaN3, NH4Cl HO N O N O N3 r.t, 4 h H CH OH:H O (8 :1) H 3 2 OH 127 129 70% 130 r.t, 24 h, 74% 131

O O O DMAP, DCM O NaN3, NH4Cl N=C=O + HO N O N O N3 H CH OH:H O(8 :1) H r.t, 4 h 3 2 OH 127 132 134 72% 133 r.t, 24 h, 73%

Scheme 4.5. Synthesis of the (R)- and (S)- azide components.

4.2.2 Synthesis of alkyne components for GHB-7

All the alkyne building blocks with the exception of 3-(prop-2- ynyloxy)propyl)benzene 137 were either commercially available or had been prepared earlier. The alkyne 3-(prop-2-ynyloxy)propyl)benzene 137 was prepared in 68% yield from the O-alkylation of propargyl alcohol 135 and 1-bromo-3-phenylpropane 136

(Scheme 4.6). Beside this alkyne, three commercially available alkynes were carefully selected with the purpose-oriented goal of enhancing biological activity.

+ Br THF, K2CO3, reflux, 24 h O OH 135 136 68% 137

Scheme 4.6. Synthesis of (3-(prop-2-ynyloxy)propyl)benzene 4.27.

4.2.3 Synthesis of GHB-7 analog

Having prepared the azide components 119a-c, 124a-d, 128, 131 and 134 and alkyne components 82c and 138a-d, it was time to genetate the requisite GHB-7 analogs.

The syntheses of GHB-7 analogs were carried out by following the modified reaction 108

conditions of the previously adopted CuAAC. The GHB-7 analogs were obtained in

quantitative yields and purity. In general, yields in the range of 93-95% were afforded by

passing the crude products through silica plug. A total of fourteen 1,4-disubstituted 1,2,3-

triazole analogs of GHB-7 were prepared from these four synthetic routes (Table 4.1,

Table 4.2, Table 4.3 and Scheme 4.7).

Table 4.1. Syntheses of analogs to examine the importance of NH- group

O O t CuSO4• 5H2O, H2O/ BuOH N N R O N N R O N3 + Sodium ascorbate OH OH Ph r.t, 24 h 119a-c 82c 113a-c N Ph

Entry R %Yield

113a Ph 95

113b PhCH2 93 113c PhCH2CH2 92

Table 4.2. Syntheses of analogs to examine the importance of phenyl group.

O O R N CuSO • 5H O, H O/tBuOH N O N R N 4 2 2 H N N O N3 + OH H Sodium ascorbate OH r.t, 24 h Ph 114a-d N Ph 124a-d 82c Entry R %yield

114a PhCH(CH3)NH 93 114b 4-(CH3)PhCH2NH 95 114c 4-(CH3O)PhCH2NH 95 114d (Furan-2-yl)methylamino 93

109

O O CUSO4• 5H2O N N N O N3 + N O N H Sodium ascorbate H N t 128 82c Ph H2O/ BuOH(1:1) 115a r.t, 24 h N Ph 97%

O O CUSO • 5H O 4 2 N N O N3 + N N O N H Sodium ascorbate H N OH t OH H2O/ BuOH(1:1) 82c 115b 131 Ph r.t, 24 h N Ph 91%

O O CUSO • 5H O N 4 2 N O N N Sodium ascorbate H N N O N3 + t OH H H2O/ BuOH(1:1) OH 82c Ph 115c 134 r.t, 24 h N Ph 90%

Scheme 4.7. Synthesis of analog devoid of -OH and chiral variants of GHB-7.

Table 4.3. Syntheses of GHB-7 analogs to examine the importance of amine substitution.

O O CuSO • 5H O, H O/tBuOH N N O N3 + R 4 2 2 N O N H H N OH Sodium ascorbate OH 64a 138a-d r.t, 24 h 116a-d R

Entry R %Yield 116a Ph 98

116b CH3CH2CH2CH2 97

116c PhCH2(CH2)2OCH2 95 116d PhCH2N(CH3)CH2 95

4.3 Plan for SAR studies on GHB-9

The second lead compound identified from the primary library was GHB-9.

Structurally, both GHB-7 and GHB-9 have the same tertiary amine substitution of

(Ph(CH2)3N(CH3)CH2- (Figure 4.7). In addition, they both possessed a hydroxyl group on the -carbon of position 1, with the only observable difference being the tether adjoining 110

the -carbon. The lead GHB-9 did not bind RNA as in the case of GHB-7, instead it

exhibited good inhibitory activity against Bacillus subtilis and Staphylococcus aureus

bacterial strain. While GHB-7 and GHB-9 exhibited comparable antibacterial activity, it can be inferred from this empirical scrutiny of both structures that the enhanced RNA binding activity of GHB-7 was precipitated in the polar carbamate group of GHB-7. This

observation arouses curiosity if preparing more polar analogs of GHB-9 would enhance

its RNA binding affinity. On the other hand, since GHB-7 and GHB-9 had the same

tertiary amine substitution and it was possible the antibacterial activity was precipitated

by the nature of the amine substitution. This relentless investigation of the topology of

GHB-9 rendered it a useful synthetic candidate for SAR evaluation.

O 2 2 1 N 1 N N O N 3 N 3 H N N OH 5 4 OH 5 4 GHB-7 N N GHB-9

Figure 4.7. Structural comparison of GHB-7 and GHB-9.

Based on this assessment, the focus was to carry out structural elaborations on

GHB-9 in attempt to enhance its initial biological profile. The plan for the SAR studies

was to focus on the structural modifications of the highlighted functional groups of

GHB-9 (Figure 4.8). 111

N N N OH N GHB-9 Evaluate effect of lipophilicity on Examine the biological biological activity relevance of hydroxyl group Assess the biological relevance of amine substitution

Figure 4.8. General structure of GHB-9 analogs for SAR studies.

As a pattern established for compound that exhibited antibacterial activity in

Chapter 3 (Figure 3.11), the amine substitution appeared to either enhance or attenuate

activity of analogs with the same substitution at N-1 as in GHB-9. For this reason, a

primary focus was to investigate the significance of the amine substitution. As discussed

earlier, homologation has been a critical lead modification technique used to modify the

biological profile of a lead. Thus, the projection was to incorporate the synthesis of

compounds that have both one and two homologous chains shorter and longer, 139a-c in

this study. But a compound one methylene group shorter (Ph(CH2)2N(CH3)CH2CCH) had been prepared in the primary library and found to exhibit marginal activity in both RNA

binding and antibacterial evaluation. Based on these, the analogs having two CH2 group

shorter (139c) as well as that having one (139a) and two (139b) homologous chain longer

were generated to investigate the optimal position of the phenyl ring. In addition,

determining the role the amine substitution plays in the biological activity of GHB-9 was

also of keen importance to this study. To move forward with this, the ether variant 139

(N- is substituted with O-) was developed. 112

N N N N N N OH OH 139d 139c N O

N N N OH N GHB-9 N N N OH 139b N N N N OH

139a N

Figure 4.9. General structure of GHB-9 analogs for SAR studies.

To expand the inquiry into identifying GHB-9 analogs with improved activity, the role of the hydroxyl group was also examined. In this sense, it was presumed that if lipophicity was key in improving activity of GHB-9, then preparing analog 140 (Figure

4.10), devoid of the hydroxyl group should enhance activity.

N N N N N N OH 140 N N GHB-9

Figure 4.10. General structure of GHB-9 analogs for SAR studies.

Further, the analog 141 also provides a means of assessing the effect of homologation and increased polarity on biological activity. For this purpose, a compound 113

two CH2- group shorter than the progenitor compound was developed. Longer or lengthy chain compounds were excluded from this study, as these did not exhibit any useful biological profile during the preliminary screening of the primary library.

N N N N N N OH OH GHB-9 141 N N

Figure 4.11. General structure of GHB-9 analogs for SAR studies.

4.3.1 Synthesis of azide components

To facilitate the synthesis of GHB-9 analogs, the azide building blocks 143 and

145 required for the synthesis of the analogs 140 and 141 had to be prepared. Butene

oxide was subjected to azidolysis using the previously adopted method to afford the azide

component 143 in 93% yield (Scheme 4.8). The azide 145 was also provided in very

good yield by treating 1-bromohexane 144 with sodium azide in DMF (Scheme 4.8).

NaN3, NH4Cl NaN3, DMF N3 Br N3 O CH OH : H O(8:1) OH r.t, 8 h 142 3 2 r.t, 24 h, 92% 143 144 94% 145

Scheme 4.8. The synthesis azide components for GHB-9 analogs.

4.3.2 Synthesis of alkyne components

The requisite alkyne components were prepared via N-alkylation of N-methyl

propargyl amine 52 with two alkyl halides 146 a-b. This reaction proceeded at a very

slow rate and the starting alkyne was not completely consumed even after refluxing for 114

two days. The yield of the reaction also appeared to decrease with increase in

homologous chain between the amine and phenyl ring.

R + N H Br K2CO3, THF, Reflux, 48 h N 52 146a-b 147a-b R 147a: R = Ph, 67% 147b R = PhCH2, 60%

Scheme 4.9. Synthesis of homologous alkyne components.

4.3.3 The synthesis of GHB-9 analogs

The GHB-9 analogs were prepared by following the modified reaction conditions for the previously reported CuAAC (Table 4.4, Scheme 4.10 and Scheme 4.11). The analogs were afforded in quantitative yields and purity. In general, yields in the range of

91-94% were obtained after passing the crude products through silica plug. A total of six

1,4-disubstituted 1,2,3-triazole analogs of GHB-9 were prepared.

Table 4.4. 1,4-disubstiuted 1,2,3-triazole analog of GHB-9

t N3 + CuSO4•5H2O, H2O/ BuOH(1:1) N R N N OH Sodium ascorbate, r.t, 24 h, OH 76b 148a-d 139a-d R

Entry R % Yield

139a Ph(CH2)4NCH3 92 139b Ph(CH2)5NCH3 91 139c PhCH2NCH3 95 139d Ph(CH2)3O 94 115

CuSO •5H O, H O/tBuOH(1:1) N 4 2 2 N N N 3 + N Sodium ascorbate, r.t, 24 h, 145 140 82c Ph 94% N Ph Scheme 4.10. Synthesis of GHB-9 (GHB-152) analog devoid of hydroxy group.

N CuSO •5H O, H O/tBuOH(1:1) N + 4 2 2 N N 3 N OH Sodium ascorbate, r.t, 24 h, OH 143 82c Ph 93% 141 N Ph Scheme 4.11. Synthesis of GHB-9 (GHB-153) analogs one homologous chain shorter.

4.4 Plan for SAR studies on GHB-16

The third lead identified from the primary library was GHB-16. Though GHB-16

and GHB-7 have different substituent diversities, the structures of GHB-9 and GHB-16

overlap (Figure 4.11). These leads, which were prepared from the same azide

components, exhibited marginal RNA binding activity. On the contrary, both leads

showed bacterial growth inhibitory activity, with GHB-9 exhibiting enhanced activity.

The substitution patterns of GHB-16 and GHB-9 are different from that of the lead

oxazolidinone antibacterial agents ANB-24 and ANB-27.

By thoroughly matching the appendage decoration of the 1,2,3-triazole analogs to

antibacterial activity, it was apparent that the 1,2,3-triazole analogs having the esters,

carbamates (except GHB-7) substitutions and longer alkyl chain did not orient analogs in

the same space as the lead oxazolidinone ANB-24 and ANB-27. Intriguingly, of the nine

compounds that exhibited inhibitory activity, five (GHB-9, GHB-16, GHB-19, GHB-

107 and GHB-108) had the same tether (CH3CH2CH2CH2CHOHCH2) attached to

position 1 of the 1,2,3-triazole ring. Heuristically, it can be projected that the nature of 116 the tether attched to position 1 of the 1,2,3-triazole scaffold to a greater extent was responsible for the biological activity and that the enhancement of activity of GHB-9 relative to GHB-16 was owing to the nature of the tertiary amine substituent incorporated.

2 2 1 N 1 N N N N3 N3 OH 5 4 OH 5 4 N N GHB-9 GHB-16

Figure 4.12. Structural comparison of GHB-9 and GHB-16.

Considering the structure of GHB-16, the notion was to prepare few analogs that target the alteration of key elements or functional groups with the goal of identifying the pharmacophoric group and also improve upon the preliminary activity. The plan for the

SAR studies on GHB-16 was to prepare one analog each of the highlighted functional groups (Figure 4.12).

N N N OH N GHB-16 Evaluate effect of lipophilicity on Examine the biological biological activity relevance of hydroxyl group Assess the biological relevance of amine substitution

Figure 4.13. Structure of GHB-16 with highlighted key functional groups. 117

The primary focus of this SAR studies on GHB-16 was to investigate whether the amine substitution was necessary for the biological profile exhibited. In this respect, the

GHB-16 analog, 148 (Figure 4.13) having its amine substituent (CH3N-) replaced with ether (O-) was developed.

N N N N N N OH OH N GHB-16 148 O

Figure 4.14. Structure of GHB-16 analog amine substitution.

In furtherance of this study, the importance of the hydroxyl group was examined.

Consequently, it was perceived that if lipophilicity was vital for activity then compound

149 (Figure 4.14), devoid of the hydroxyl group would have the potential of enhancing the initial activity of GHB-16.

N N N N N N OH 149 N GHB-16 N

Figure 4.15. Structure of GHB-16 analog devoid of hydroxy group.

In addition, the effect of homologation was utilized to accesss if the lipophilic aliphatic chain was critical for activity. Accordingly, considering the prospects of generating analogs befitting this criterion, there was precedence of longer chains 118 attenuating activity (from preliminary data of primary library). As such, only the analog

150 (Figure 4.15), which is a homolgous chain shorter, was considered in the study.

N N N N OH N N OH 150 N GHB-16 N

Figure 4.16. Structure of analog one CH2 chain shorter.

4.4.1 Synthesis of azide and alkyne starting materials

The two lead compounds, GHB-16 and GHB-9 were prepared from the same azide compound. Owing to this, the two azide precusors required for the synthesis of

GHB-16 analogs had been synthesized earlier while preparing GHB-9 analogs.

Nonetheless, the ether derivative 152 required for the synthesis of GHB-16 analog 148 had to be prepared in order to move forward with the synthesis of new analogs. This ether variant 152 was accessed via O-alkylation of propargyl alcohol 135 and 1-bromo-3- methylbutane 151 in 70% yield in one step.

Acetone OH + Br O 135 151 Reflux, 48 h, 70% 152

Scheme 4.12. The synthesis of alkyne component of GHB-16. 119

4.4.2 Synthesis of GHB-16 analogs

The analogs of GHB-16 were prepared in quantitative yield and purity via

CuAAC. The synthesis of the analogs is outlined in Scheme 4.13. The requisite concentration of analogs in DMSO were prepared prior to sending compounds to collaborators for biological evaluation.

t N N + CuSO4•5H2O, H2O/ BuOH(1:1) N 3 O N OH Sodium ascorbate, r.t, 24 h, OH 93% 76b 152 148 O

t N + CuSO4•5H2O, H2O/ BuOH(1:1) N 3 N N N Sodium ascorbate, r.t, 24 h, 97% 149 145 82b N

N CuSO •5H O, H O/tBuOH(1:1) N + 4 2 2 N N 3 N OH Sodium ascorbate, r.t, 24 h, OH 91% 143 82b 150 N

Scheme 4.13. Synthesis of GHB-16 analogs for SAR studies.

4.5 Enantioselective synthesis of selected 1,4-substituted 1,2,3-triazoles

Based on the empirical observation of the structures of the 1,4-disubstituted 1,2,3- triazole compounds that showed good biological activity, it was apparent that the cyclohexy group was present in majority of the compounds that exhibited good RNA binding activity. For example, the 1,4-disubstituted analogs, GHB-18 and GHB-60 embodying the cyclohexyl group showed enhanced RNA binding activity (Figure 4.6).

While these analogs were not the best compound, they depicted the cyclohexyl group as a potential diversity element worth exploring. Beside the thought of carrying out SAR 120

studies to optimize the activity of GHB-18 and GHB-60, there was the kindled interest of

resolving the racemic 2-azidocyclohexanol into their enantiomers and then preparing the

chiral analogs of GHB-18 and GHB-60.

N N N N N N N O N OH O O GHB-18 GHB-60

Figure 4.17. Structure of 1,2,3-triazole analogs GHB-18 and GHB-60.

The endeavor of resolving GHB-18 and GHB-60 into their enantiomers was seen as an instrumental way of discerning whether the biological activity exhibited was a result of a single enantiomer or the racemic mixture. There is precedence of enantiomers as well as their racemate eliciting the same or different physiological effects. In the latter, one enantiomer could impair the activity of the other. For example, while the (S)- enantiomer of the citalopram 153 and the racemate 155 are comparatively potent drugs for the treatment of anxiety disorders the (S)-enantiomer are preferred based on clinical considerations (Figure 4.7).357-362 The (R)-enantiomer 154 is completely inactive in this regard. The (S,S)-enantiomer of the drug ethambutol 156 is potent against tuberculosis, while the racemate 158 exhibits roughly one tenth of the anti-mycobacterial activity. The

(R,R)-enantiomer 157 was found to exhibit no anti-mycobacterial activity.363,364 In view of this, the projection was that by preparing the chiral analogs of both GHB-18 and

GHB-60 would have the potential of enhancing the activity of the progenitor compounds. 121

NC NC NC

O O O

NMe2 NMe 2 NMe2

F F F

(S)-Citalopram 153 (R)-Citalopram 154 (RS)-Citalopram 155

CH OH CH OH CH OH H 2 H 2 H 2 N N N N N N H H H CH2OH CH2OH CH2OH (R,S)-Ethambutol 158 (S,S)-Ethambutol 156 (R,R)-Ethambutol 157

Figure 4.18. Examples of chiral and racemate drugs.

4.5.1 Enzymatic resolution of Trans-2-azidocyclohexanol

In order to carry out the enantioselective hydrolysis, roughly 10 g of trans-2- azidocyclohexanol had to be prepared. The synthesis of trans-2-azidocyclohexanol 76d began with the previously adopted ring opening of cyclohexene oxide 75d using sodium azide (Scheme 4.14). This method provided the racemic trans-2-azidocyclohexanol 76d in quantitative yield of 98%. Next, the acylating reagent, butyryl chloride 160 was prepared by heating butyryl acid 159 in thionyl chloride for three-hours. The azido alcohol 76d was then acylated with the acid chloride 160 to afford trans-2- azidocyclohexanoate 161, required for the enzymatic resolution in good yield. 122

NaN3, NH4Cl N3 O MeOH/H O (8:1) N 2 OH 3 75d 98% 76d DCM, DMAP O + Et3N, r.t, 12 h O SOCl2, DMF O 92% O 50 °C OH Cl (±)-161 159 160

Scheme 4.14. Synthesis of azide precursor for enzymatic resolution.

Faber et al365 and Govindaraju et al366 have reported a procedure for enzymatic resolution of racemic trans-2-azidocyclohexanoate 161. Typically, the azido ester was treated with lipase in a solution of sodium phosphate buffer and hydrolysis monitored at room temperature. By following the reported procedure, compound 161 was subjected to enantioselective hydrolysis mediated by lipase (Amano-PS) in a solution of sodium phosphate buffer for 3 hours. This led to the recovery of 41% yield of the optically pure azido alcohol 162 and the mixture 163 (containing little 162) after chromatography. The optically enriched compound 163 was further subjected to enzymatic resolution for 5 hours to afford the azido ester 164. Methanolysis of 164 afforded the optically pure compound 165 in 30% yield (Scheme 4.15). The optical rotations of compounds 162 (-

66.8 °) and 165 (+66.4 °) were obtained and found to match that reported.365 Next, the

enantiomeric excess of the two enantiomers 162 and 165 were determined using Mosher

chloride. 123

41% N3

OH N 3 162 (R, R) Lipase (Amano-PS) O 0.02 N Na3PO4 buffer O pH = 7.2, 1N NaOH r.t, 3 h N ( ± )-161 3

O 163 --Major; (1R, 2R) --Minor Lipase (Amano-PS) 0.02 N Na3PO4 buffer pH = 7.2, 1N NaOH r.t, 5 h

37%

N3 N3 N NaOMe 3 MeOH OH O 30% OH 165 (S, S) O 162 (1R, 2R) 164

Scheme 4.15. Enzymatic resolution of trans-2-azidocyclohexanoate.

4.5.2 Determination of enantiomeric excess

Methods for determining the enantiomeric excess have been reported.367-371 In general the enantiomers in question were treated with Moshers acid.367-369 As shown in

Scheme 4.16, the optically pure enantiomers, 162, 165 and the racemic 76d were treated with Mosher’s acid chloride, (S)-(+)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride (MTPA-Cl) to provide the corresponding diasteromers, which were found not to be discernable by 1H-NMR. 124

O N3 N3 CF3 DCM, DMAP, Et N, r.t, + Cl 3 O OH OMe CF Ph 96%, O 3 162 166 OMe 167 Ph

N O 3 N + CF DCM, DMAP, Et N, r.t, 3O Cl 3 3 OH OMe CF Ph 95%, O 3 165 166 OMe 168 Ph

N3 O DCM, DMAP, Et N, r.t, N3 + CF 3 O Cl 3 95% CF3 OH OMe O Ph OMe 76d 166 169 Ph

Scheme 4.16. Determination of enantiomeric excess of trans-2-azidocyclohexanol.

On the contrary, these diasteromers were explicitly resolved in 19F-NMR and the enantiopurities were determined by using the integrals of the peaks. The 19F-NMR of the two enantiomers 162, 165 and racemate 76d are shown in Figure 4.18. Compound 162 was found to have an enantiomeric excess of > 99% ee, while the other enantiomer 165 was determined to have a 96% ee. 125

OH 162

Figure 4.19. The 19F-NMR spectrum of compound 162.

OH 165

Figure 4.20. The 19F-NMR spectrum of compound 165. 126

OH 76d

Figure 4.21. The 19F-NMR spectrum of compound 76d.

4.5.3 The synthesis of enantiopure azide components

To enable the synthesis of the enantiopure analogs, the optically pure azide components 171 and 172 were synthesized. The previously resolved (R,R)-azido alcohol

162 was acylated with phenoxyacetyl chloride 170 to give the optically pure enantiomer

171. In a similar manner, the enantiomerically pure azide component 172 was prepared by acylating the azidoester 165 with the acid chloride 170. Both azido esters 171 and 172 were synthesized in one step in very good yields. 127

O N3 O N DMAP, Et N, DCM 3 + Cl 3 O r.t, 24 h, 91% O OH O 162 170 171 O N 3 O DMAP, Et N, DCM N3 + Cl 3 O r.t, 24 h, 91% O OH O 165 170 172

Scheme 4.17. Synthesis of enantiopure azide building blocks.

4.5.4 The synthesis of enantiopure 1,4-disubstituted 1,2,3-triazole analogs

Having the four-azide precursors 162, 165, 171 and 172 as well as the two-alkyne components 48a and 82b in hand, the CuAAC was applied to generate the optically pure analogs of the progenitor compounds, GHB-18 and GHB-60 (Scheme 4.17). Compounds were sent to collaborators for biological testing after amassing analytical data and preparing the requisite concentrations in DMSO. A total of four enantiomerically pure analogs were prepared. 128

N N N N3 t CuSO4•5H2O, H2O/ BuOH N + N sodium ascorbate, rt, 18-24 h OH 95% OH 173 162 48a

N N N N3 N O t + CuSO4•5H2O, H2O/ BuOH O N O sodium ascorbate, rt, 18-24 h O 171 82b 93% O O 174

N N N N3 t N + CuSO4•5H2O, H2O/ BuOH N OH sodium ascorbate, rt, 18-24 h 175 48a OH 165 94%

N N N N 3O t N O + CuSO4•5H2O, H2O/ BuOH O N sodium ascorbate, rt, 18-24 h O 172 82a 94% O O 176

Scheme 4.18. Synthesis of 1,4-disubstituted 1,2,3-triazole enantiopure analogs.

4.6 Biological evaluation of GHB-7, GHB-9, GHB-16 and enantiopure analogs

The analogs of GHB-7, GHB-9, GHB-16 and enantiopure analogs are currently undergoing biological evaluation to determine their RNA binding affinities and specificities in both Hines. Aliquot amount of each analog was also sent to Priestley laboratory for antibacterial evaluation. The 5'Rhd data from Hines indicated that few analogs exhibited improved binding to model RNAs relative to the progenitor compounds. 129

All GHB-7 analogs exhibited improved RNA binding towards model RNA AM1A (Table

4.5). But only two GHB-7 and GHB-144 showed enhanced specificity. Of these, GHB-

144 much improved specificity relative to GHB-7. The structure of GHB-144 coupled

that of GHB-26 indicated that a dcreased in the homologous chain of the amine

substitution was critical for specificity.

Table 4.5. 5'Rhd screening of GHB-7 analogs for model RNA AM1A and C11U

Entry Analogs FAM1A (%) FC11U (%) Specificity FAM (%) 98a GHB-7 -14.2 -16.7 -2.5 -6.8 113c GHB-135 -7.8 -16.3 -8.5 -7.3 113b GHB-136 -11.3 -11.7 -0.4 -3.5 113a GHB-137 -2.9 -7.8 -4.9 -1.8 115b GHB-138 -6.7 -12.9 -6.2 -8.0 115c GHB-139 -2.8 -13.0 -10.2 -9.0 114c GHB-140 -1.7 -7.6 -5.9 -3.0 114d GHB-141 -5.3 -12.2 -6.9 -3.5 114b GHB-142 -8.9 -11.9 -3.0 -4.1 114a GHB-143 -7.8 -13.9 -6.1 -2.9 116d GHB-144 -5.4 13.0 18.4 -2.8 116b GHB-145 0.1 -4.6 -4.5 3.6 116a GHB-146 7.1 -3.3 -10.4 4.6 116c GHB-147 -3.9 -12.9 -9.0 -1.48 115a GHB-148 -1.0 -8.4 -7.4 5.5

The relative fluorescence intensity change F was calculated by F=[(F-F0)/F0]*100, where F is the fluorescence intensity with ligand and F0 is without ligand at 585 nm upon excitation at 467 nm. All the compounds were tested at a final concentration of 10 μM.

FC11U(%)-FAM1A(%) = Specificity

All analogs of GHB-9 showed improved specificity with GHB-151 having the best

specificity (Table 4.6). Though the analogs GHB-149 (having one CH2- longer amine substitution) and GHB-150 (having two CH2- longer amine substitution) showed good 130 specificities, the specificity obtained for GHB-151 clearly signified that a decrease in the homologous chain between the amine and phenyl ring was required increased specificity.

Table 4.6. 5'Rhd screening of GHB-9 analogs for model RNA AM1A and C11U

Entry Analogs FAM1A (%) FC11U (%) Specificity FAM (%) 96a GHB-9 -7.24 -13.71 -6.47 -1.49 139b GHB-149 -2.2 -8.0 -5.8 -1.8 139a GHB-150 -1.6 -3.8 -2.2 -3.1 139c GHB-151 -6.8 -5.2 1.6 -1.8 140 GHB-152 -4.2 -8.9 -4.7 -4.1 141 GHB-153 -1.9 -1.4 0.5 -0.5 139d GHB-161 -3.7 -7.9 -4.2 -2.3

The relative fluorescence intensity change F was calculated by F=[(F-F0)/F0]*100, where F is the fluorescence intensity with ligand and F0 is without ligand at 585 nm upon excitation at 467 nm. All the compounds were tested at a final concentration of 10 μM. FC11U(%)-FAM1A(%) = Specificity

While the progenitor compound GHB-16 exhibited significant binding affinity for model RNA AM1A, the three analogs GHB-154, GHB-155 and GHB-156 had improved specificities (Table 4.7) with GHB-156 being the best analog. This implied that decreasing the lipophilic chain appending to N-1 of the 1,2,3-triazole ring from 6 to 4 was necessary for improved activity. The analog GHB-155 also revealed that importance of other heteroatom substitution as the replacement of CH3N- with O- led to increase in specificity. 131

Table 4.7. 5'Rhd screening of GHB-16 analogs for model RNA AM1A and C11U

Entry Analogs FAM1A (%) FC11U (%) Specificity FAM (%) 96c GHB-16 11.68 6.60 -5.1 -2.47 149 GHB-154 0.9 -4.1 -5.0 -2.6 148 GHB-155 -6.1 -5.3 0.8 -3.3 150 GHB-156 -3.5 -1.8 1.7 -0.2

The relative fluorescence intensity change F was calculated by F=[(F-F0)/F0]*100, where F is the fluorescence intensity with ligand and F0 is without ligand at 585 nm upon excitation at 467 nm. All the compounds were tested at a final concentration of 10 μM.

FC11U(%)-FAM1A(%) = Specificity

Although GHB-18 showed high RNA binding affinities relative to the chiral analogs GHB-157 and GHB-158 (Table 4.8), the improved specificities of the chiral analogs clearly indicated that enantioselectivity was key for specificity. But more biological data would be needed to assess the effects the enantiomers have on each other.

Table 4.8. 5'Rhd screening of enantiopure analogs for model RNA AM1A and C11U

Entry Analogs FAM1A (%) FC11U (%) Specificity FAM (%) 105b GHB-18 14.7 6.9 -7.8 -5.90 173 GHB-157 -1.4 -2.9 -1.5 -1.7 175 GHB-158 -0.6 -5.3 -4.7 -0.1 104c GHB-60 -10.4 5.8 1.5 -1.3 174 GHB-159 -8.0 -9.2 -1.2 -5.4 176 GHB-160 -9.2 -8.4 0.8 -3.7

The relative fluorescence intensity change F was calculated by F=[(F-F0)/F0]*100, where F is the fluorescence intensity with ligand and F0 is without ligand at 585 nm upon excitation at 467 nm. All the compounds were tested at a final concentration of 10 μM.

FC11U(%)-FAM1A(%) = Specificity 132

4.7 Conclusion

Relying on empirical observations of the lead compound identified in the primary library, a total of twenty-seven 1,4-disubstitututed 1,2,3-triazole analogs were carefully designed and synthesized for SAR studies. Homologation and isosteric replacement techniques were utilized to develop analogs that elevate the potential of identifying new leads with enhanced activity. Preliminary biological evaluation depicted enhancement of florescence intensity for new analog during 5'Rhd screening. The data also indicated that their binding pattern emulated that of their progenitor compounds.

The data on GHB-7 analogs explicitly revealed that the position of the phenyl group was not optimal and that a decrease in homologous chain to the N-methyl N- methylphenyl substitution was optimal for both GHB-7 and GHB-9 analogs. The effect of homologation was identified as critical for also improving the activity of GHB-16 analog, GHB-156. The hdroxyl group of all progenitor compounds and the carbamate group of GHB-7 were necessary for biological activity. Chirality was determined to be important but not necessary for biological activity. The analogs GHB-144, GHB-151,

GHB-153, GHB-156 and GHB-157 were identified to bind model RNA with significant improvement in specificity. 133

Chapter 5. Experimental

All reagents and starting materials were purchased from commercial suppliers.

All reactions except epoxide ring opening reaction with sodium azide were conducted

under an atmosphere of argon unless otherwise stated. TLC was performed on Whatman

precoated silica gel F234 or dynamic adsorbent inc. precoated silica gel F254 aluminium

plate. Visualization of all compounds having a chromophore were carried out with a UV

light. Non-UV active compounds were stained using phosphomolybdic acid in ethanol or

ninhydrin in ethanol solution. Purification of desired products were carried out using

flash chromatography on Silica gel (230-400 mesh) purchased from Silicycle.

Triethylamine was distilled from calcium hydride prior to use. DMF was dried over BaO

and distilled under reduced pressure and stored over 4Å molecular sieves and stored

under argon atmosphere. DCM and THF were dried over a column of packed alumina.

Rotation data were obtained on a Rudolph research analytical, Autopol® IV automatic

polarimeter using the sodium D line. 1H-NMR spectra were obtained on a Bruker

300MHz spectrometer and referenced to TMS. 13C-NMR spectra were obtained on a

1 13 Bruker 300(75 MHz) spectrometer and referenced to CDCl3. Both H-NMR and C-

NMR spectra were taken in deuterated chloroform solution. Adopted NMR abbreviations

were as follows: s = singlet, bs =broad singlet, d = doublet, bd = broad doublet, dd

=doublet of doublet, t = triplet, dt = doublet of triplet, q = quartet, m = multiplet. HPLC

data were obtained on Shimadzu using Supelco discovery C8 column (15cm x 4.6 mm, 5

μm), eluting at 1 mL/min with a gradient elution starting at 30% of 1% AcOH in MeOH-

H2O going to 90% over 26 minutes as the mobile phase eluant unless otherwise stated. 134

Retention times are reported in minutes. Mass verifications were carried out on a

Shimadzu 2010A LC/MS using APCI probe. IR data were acquired on a Shimadzu

Advantage FTIR-8400. Melting points were obtained on a Mel-temp , from laboratory devices, USA.

General procedure A for the synthesis of glycidyl ester 53a-e. To a solution of

glycidol (1 equiv) in CH2Cl2 (1.35 M) at 0 °C was added DMAP (1.2 equiv). The reaction mixture was stirred for 30 minutes and acid chloride (1.2 equiv) was added. The reaction was warmed to r.t and stirring was continued at r.t for 4 h under an argon atmosphere.

The reaction mixture was then poured through silica gel pad and washed with CH2Cl2

(100 mL). The filtrate was concentrated and chromatographed (25% EtOAc in hexanes) to provide the desired glycidyl esters 53a-e.

General procedure B for allyl carbamates 64a-b. To a solution of allyl alcohol 63 (1

equiv) in CH2Cl2 (1.72 M) at 0 °C was added Et3N (3 equiv) and DMAP (0.2 equiv). The reaction mixture was stirred at 0 ° C for 30 minutes and the desired isocyanate (1.2 equiv) was added. The reaction mixture was warmed to r.t and stirring was continued at r.t for 4 h under an argon atmosphere. The reaction mixture was then washed with 1M HCl (3x),

saturated NaHCO3 (3x), H2O (3x), brine (2x), dried over MgSO4, filtered, concentrated and chromatographed (50% EtOAc in hexanes) to provide the allyl carbamates 64a and

64b. 135

General procedure C for glycidyl carbamate 65a-b. To a solution of allyl carbamate

64 (1 equiv) in CH2Cl2 (0.53 M) at 0 °C was added mCPBA (1.2 equiv). The reaction

mixture was warmed to room temperature and stirred at r.t for 24 h under an atmosphere

of argon. The reaction mixture was then diluted with Et2O, washed with 1 M NaOH (3x), dried over MgSO4, filtered, concentrated, and chromatographed (50% EtOAc in hexanes) to provide the glycidyl carbamates 65a and 65b.

O O 53a

Oxiran-2-ylmethyl octanoate 53a. Glycidol 49 (1.1 g, 15 mmol) was reacted with octanoyl chloride (3.1 mL, 2.9 g, 18 mmol) following the general procedure A for glycidyl esters to afford 2.7 g (91%) of the ester 53a as a pale yellow oil that matched

372 1 analytical data previously reported. H NMR (CDCl3, 300 MHz) 3.92 (dd, J = 3.1,

12.3, 1H), 3.42 (dd, J = 6.3, 12.3, 1H), 2.72-2.69 (m, 1H), 2.33 (t, J = 4.9, 1H), 2.15 (dd,

J = 2.6, 5.0, 1H), 1.87 (t, J = 7.4, 2H), 1.23-1.09 (m, 2H), 0.93-0.73 (m, 8H), 0.42 (t, J =

6.8, 3H).

O O 53b

Oxiran-2-ylmethyl benzoate 53b. Glycidol 49 (1.1 g, 15 mmol) was reacted with benzoyl chloride (2.5 g, 18 mmol) following the general procedure A for glycidyl esters to afford 2.4 g (90%) of the ester 53b as a yellow oil that matched analytical data 136

373 1 previously reported. H NMR (CDCl3, 300 MHz) 8.07 (d, J = 7.2, 2H), 7.57 (t, J =

7.2, 1H), 7.45 (t, J = 7.1, 2H), 4.65 (dd, J = 1.0, 3.1, 1H), 4.19 (dd, J = 6.2, 12.3, 1H),

13 3.37-3.31 (m, 1H), 2.98 (t, J = 4.9, 1H), 2.74-2.72 (m, 1H). C NMR (CDCl3, 75 MHz)

166.3, 133.2, 129.7, 128.4, 65.4, 49.5, 44.7.

O O 53c

Oxiran-2-ylmethyl cyclohexanecarboxylate 53c. Glycidol 49 (1.1 g, 15 mmol) was reacted with cyclohexanecarbonyl chloride (2.6 g, 18 mmol) following the general procedure A for glycidyl esters to afford 2.6 g (93%) of the ester 53c as a colorless oil

374 1 that matched analytical data previously reported. H NMR (CDCl3, 300 MHz) 4.21

(dd, J = 3, 12.3, 1H), 3.74 (dd, J = 6.1, 12.3, 1H), 3.02-2.99 (m, 1H), 2.64 (t, J = 4.6, 1H),

2.46 (dd, J = 2.6, 4.9, 1H), 1.74 (d, J = 12.4, 2H), 1.67-1.42 (m, 3H), 1.40-1.21 (m, 2H),

13 1.21-0.99 (m, 3H). C NMR (CDCl3, 75 MHz) 175.2, 64.3, 49.1, 44.2, 42.8, 28.8,

25.57, 25.19.

N O H 64a

Allyl benzylcarbamate 64a. Allyl alcohol 63 (0.81 g, 15 mmol) was reacted with benzyl isocyanate (2.4 g, 18 mmol) following the general procedure B for allyl carbamates to afford 2.7 g (97%) of the allyl carbamate 64a as a yellow oil that matched analytical data

375 1 previously reported. H NMR (CDCl3, 300 MHz) 7.31-7.24 (m, 5H); 5.97-584 (m, 137

13 1H), 5.31-4.95 (m, 3H), 4.57 (d, J = 5.4, 2H), 4.34 (d, J = 6.0, 2H). C NMR (CDCl3, 75

MHz) 156.3, 138.5, 132.9, 128.6, 127.4, 117.6, 65.7, 45.1.

N O H 64b

Allyl butylcarbamate 64b. Allyl alcohol 63 (0.81 g, 15 mmol) was reacted with butyl

isocyanate (1.76 g, 18 mmol) following the general procedure B for allyl carbamates to

afford 2.2 g (95%) of the carbamate 64b as a yellow oil that matched analytical data

376 1 previously reported. H NMR (CDCl3, 300 MHz) 5.99-5.85 (m, 1H), 5.29 (d, J =

17.2, 1H), 5.20 (d, J = 10.4, 1H). 4.85 (s, 1H), 4.56 (d, J = 5.0, 2H), 3.18 (q, J = 6.6, 2H),

13 1.54-1.44 (m, 2H), 1.41-1.29 (m, 2H), 0.92 (t, J = 7.0, 3H). C NMR (CDCl3, 75 MHz)

156.4, 133.3, 117.5, 65.4, 40.8, 32.1, 19.9, 13.7.

N O H O 65a

Oxiran-2-ylmethyl benzylcarbamate 65a. Allyl benzylcarbamate 64a (1.13 g, 6.1 mmol) was reacted with mCPBA (1.26 g, 7.3 mmol) following the general procedure C for glycidyl carbamates to afford 1.0 g (80%) of the carbamate 65a as a yellow oil that

377 1 matched analytical data previously reported. H NMR (CDCl3, 300 MHz) 7.36-7.26

(m, 5H); 5.20 (s, 1H), 4.45 (dd, J = 2.9, 12.2, 1H), 4.36 (d, J = 6.0, 2H), 3.90 (dd, J = 6.3,

12.2, 1H), 3.21-3.17 (m, 1H), 2.82 (t, J = 4.6, 1H), 2.62 (dd, J = 2.4, 4.4, 1H). 13C NMR

(CDCl3, 75 MHz) 156.1, 138.3, 128.7, 127.6, 127.5, 65.6, 49.8, 45.2, 44.6. 138

N O H O 65b

Oxiran-2-ylmethyl butylcarbamate 65b. Allyl butylcarbamate 64b (1.60 g, 10.5

mmol) was reacted with mCPBA (2.17 g, 12.6 mmol) following the general procedure C

for glycidyl carbamates to afford 1.5 g (82%) of the carbamate 65b as a yellow oil.1H

NMR (CDCl3, 300 MHz) 5.00 (s, 1H); 4.42 (dd, J = 2.7, 12.2, 1H), 3.87(dd, J = 6.3,

12.2, 1H), 3.21-3.14 (m, 3H), 2.83 (t, J = 4.7, 1H), 2.64 (dd, J = 2.6, 4.8, 1H), 1.54-1.44

13 (m, 2H), 1.41-1.28 (m, 2H), 0.92 (t, J = 7.2, 3H). C NMR (CDCl3, 75 MHz) 156.2,

65.3, 49.9, 44.6, 40.8, 32.0, 19.9, 13.7.

General procedure A for the synthesis of azide components 54a-c, 66a-b and 76a-d.

To a mixture of epoxide (1 equiv), NH4Cl (2 equiv) in CH3OH and H2O mixture (0.28 M,

8:1) was added NaN3 (10 equiv). The reaction mixture was degassed and stirred at room

temperature for 24 h. The reaction mixture was then concentrated to 1/10 its volume,

diluted with water (20 mL) and extracted with EtOAc (3x). The combined organic layer

was washed with brine (2x), dried over MgSO4, filtered, concentrated and chromatographed (35% EtOAc in hexanes) to provide the azide components 54a-c, 66a-b and 76a-d.

General procedure B for the synthesis of azide components 77a-c. To a solution of

trans 2-azidocyclohexanol 76d (1 equiv) in CH2Cl2 (0.71 M) at 0 °C was added Et3N (3

equiv) and DMAP (0.02 equiv). The reaction mixture was stirred for 30 minutes and acid 139

chloride (1.2 equiv) was added. The reaction mixture was gradually warmed to r.t and

stirring was continued at r.t for 24 h under an argon atmosphere. The reaction mixture

was then washed with 1 M HCl (3x), saturated NaHCO3 (3x), H2O (3x), brine (2x), dried

over MgSO4, filtered, concentrated and chromatographed (25% EtOAc in hexanes) to provide compound 77a-c.

O N3 54a OH

3-azido-2-hydroxypropyl octanoate 54a. Glycidyl octanoate 53a (1.13 g, 5.6 mmol)

was reacted with NaN3 (3.6 g, 56 mmol) following the general procedure A for azide

compounds syntheses to afford 1.1 g (77%) of the azide 54a as a pale yellow oil. 1H

NMR (CDCl3, 300 MHz) 4.15 (dd, J = 5.2, 12.2, 1H), 4.10 (dd, J = 5.2, 12.2, 1H), 4.04-

3.97 (m, 1H), 3.41 (dd, J = 5.9, 12.8, 1H), 3.35 (dd, J = 5.9, 12.8, 1H), 3.03 (d, J = 5.0,

1H), 2.33 (t, J = 7.5, 2H), 1.66-1.56 (m, 2H), 1.35-1.21 (m, 8H), 0.86 (t, J = 6.9, 3H). 13C

NMR (CDCl3, 75 MHz) 174.8, 69.6, 66.1, 54.1, 34.7, 32.3, 29.7, 29.5, 25.5, 23.2, 14.7.

-1 IR (CDCl3) 2100 cm . 140

O N3 OH 54b

3-azido-2-hydroxypropyl benzoate 54b. Glycidyl benzoate 53b (1.1 g, 6.2 mmol) was

reacted with NaN3 (4.0 g, 62 mmol) following the general procedure A for azide compounds syntheses to afford 1.0 g (81%) of the azide 54b as an orange-yellow oil that

378 1 matched analytical data previously reported. H NMR (CDCl3, 300 MHz) 8.04 (d, J =

7.3, 2H), 7.58 (t, J = 7.2, 1H), 7.44 (t, J = 7.9, 2H), 4.44 (dd, J = 5.3, 20.5, 1H), 4.36 (dd,

J = 5.3, 20.5, 1H), 4.21-4.12 (m, 1H), 3.51 (dd, J = 5.5, 13.0, 1H), 3.46 (dd, J = 5.5, 13.0,

13 1H), 3.22 (d, J = 4.4, 1H). C NMR (CDCl3, 75 MHz) 166.8, 133.5, 129.7, 129.4,

-1 128.5, 69.1, 66.1, 53.56. IR (CDCl3) 2106 cm .

O N3 OH 54c

3-azido-2-hydroxypropyl cyclohexanecarboxylate 54c. Glycidyl

cyclohexanecarboxylate 53c (1.13 g, 6.1 mmol) was reacted with NaN3 (4 g, 61 mmol)

following the general procedure A for azide compounds syntheses to afford 1.12 g (81%)

1 of the azide 54c as a pale yellow oil. H NMR (CDCl3, 300 MHz) 4.11 (dd, J = 5.4,

20.4, 1H), 4.03 (dd, J = 5.4, 20.4, 1H), 3.99-3.89 (m, 1H); 3.36 (dd, J = 5.0, 12.7, 1H),

3.29 (dd, J = 5.0, 12.7, 1H), 3.00 (d, J = 5.0, 1H), 2.28 (tt, J = 3.6, 11.2, 1H), 1.90-1.79

13 (m, 2H), 1.75-1.53 (m, 3H), 1.45-1.10 (m, 5H), C NMR (CDCl3, 75 MHz) 176.3,

-1 69.0, 65.3, 53.5, 43.0, 29.0, 25.6, 25.3. IR (CDCl3) 2100 cm . 141

N O N3 H OH 66a

3-azido-2-hydroxypropyl benzylcarbamate 66a. Glycidyl benzylcarbamate 65a (0.97

g, 3.8 mmol) was reacted with NaN3 (2.5 g, 38 mmol) following the general procedure A

for azide compounds syntheses to afford 0.87 g (77%) of the azide 66a as an orange-

1 yellow oil. H NMR (CDCl3, 300 MHz) 7.40-7.23 (m, 5H), 5.27 (s, 1H), 4.36 (d, J =

5.2, 2H), 4.19 (dd, J = 4.3, 12.1, 1H), 4.14 (dd, J = 4.3, 12.1, 1H), 4.02-3.89 (m, 1H),

13 3.37-3.20 (m, 3H). C NMR (CDCl3, 75 MHz) 156.5, 137.7, 128.5, 127.4, 127.3, 69.3,

-1 66.4, 53.1, 44.95. IR (CDCl3) 2100 cm .

N O N3 H OH 66b

3-azido-2-hydroxypropyl butylcarbamate 66b. Glycidyl butylcarbamate 65b (0.93 g,

4.3 mmol) was reacted with NaN3 (2.8 g, 43 mmol) following the general procedure A

for azide compounds syntheses to afford 0.91 g (81%) of the carbamate 66b as an orange-

1 yellow oil. H NMR (CDCl3, 300 MHz) 4.90 (bs, 1H), 4.20-4.09 (m, 2H), 4.04-3.95 (m,

1H), 3.43-3.29 (m, 3H), 3.18 (q, J = 6.5, 2H), 1.50-1.43 (m, 2H), 1.42-1.28 (m, 2H), 0.93

13 (t, J = 7.3, 3H), C NMR (CDCl3, 75 MHz) 155.3, 68.1, 64.9, 51.9, 39.4, 30.4, 18.3,

-1 12.1. IR (CDCl3) 2100 cm . 142

N3 76a

2-azido-1-phenylethanol 76a. The styrene oxide 75a (1.2 g, 10 mmol) was reacted with

NaN3 (6.5 g, 100 mmol) following the general procedure A for azide compounds syntheses to afford 1.5 g (92%) of the azide 76a as a yellow oil that matched analytical

379 1 data previously reported. H NMR (CDCl3, 300 MHz) 7.43-7.31 (m, 5H), 4.66 (t, J =

13 6.5, 1H), 3.75 (t, J = 6.0, 2H), 2.08 (t, J = 6.0, 1H); C NMR (CDCl3, 75 MHz) 135.5,

-1 128.1, 127.9, 126.4, 67.1, 65.7. IR (CDCl3) 2100 cm

N3 OH 76b

1-Azidohexan-2-ol 76b. 2-Butyloxirane 75b (1.0 g, 10 mmol) was reacted with NaN3

(6.5 g, 100 mmol) following the general procedure A for azide compounds syntheses to

afford 1.35 g (94%) of the azide 76b as a colorless oil that matched analytical data

380 1 previously reported. H NMR (CDCl3, 300 MHz) 3.84-3.68 (m, 1H), 3.38 (dd, J =

3.4, 12.4, 1H), 3.25 (dd, J = 7.4, 12.4, 1H), 2.00 (s, 1H), 1.56-1.25 (m, 6H), 0.92 (t, J =

13 6.9, 3H). C NMR (CDCl3, 75 MHz) 70.3, 56.6, 33.5, 27.1, 22.1, 13.4. IR (CDCl3)

2100 cm-1. 143

O N3 OH 76c

1-azido-3-(benzyloxy)propan-2-ol 76c. Benzyl glycidylether 75c (1.6 g, 10 mmol) was

reacted with NaN3 (6.5 g, 100 mmol) following the general procedure A for azide

compounds syntheses to afford 1.95 g (93%) of the azide 76c as a pale yellow oil that

381 1 matched analytical data previously reported. H NMR (CDCl3, 300 MHz) 7.39-7.27

(m, 5H), 4.55 (s, 2H), 4.01-3.89 (m, 1H), 3.53 (dd, J = 4.4, 9.6, 1H), 3.48 (dd, J = 4.4,

9.6, 1H), 3.39 (dd, J = 6.0, 12.9, 1H), 3.34 (dd, J = 6.0, 12.9, 1H), 2.56 (d, J = 3.9, 1H).

13 C NMR (CDCl3, 75 MHz) 136.1, 127.1, 126.5, 126.5, 72.1, 69.8, 68.2, 52.0. IR

-1 (CDCl3) 2096 cm .

N3 76d

Trans-2-azidocyclohexanol 76d. Cyclohexene oxide 75d (1.0 g, 10 mmol) was reacted

with NaN3 (6.5 g, 10 mmol) following the general procedure A for azide compounds

syntheses to afford 1.38 g (98%) of the azide 76d as a white solid that matched analytical

382 1 data previously reported. H NMR (CDCl3, 300 MHz) 3.37-3.26 (m, 1H), 3.18-3.05

(m, 1H), 2.22 (d, J = 3.5, 1H), 2.05-1.90 (m, 2H), 1.74-1.60 (m, 2H); 1.36-1.11 (m, 4H).

13 -1 C NMR (CDCl3, 75 MHz) 72.4, 65.9, 31.9, 28.6, 23.1, 22.7. IR (CDCl3) 2096 cm . 144

O N3 77a

(1R,2R)-2-azidocyclohexyl benzoate 77a. 2-Azido cyclohexanol 76d (1.1 g, 11.3 mmol)

was acylated with benzoyl chloride (2.0 g, 13.6 mmol) following the general procedure B

for azide compounds syntheses to afford 2.1 g (92%) of the azide 77a as a white solid

383 1 that matched analytical data previously reported. H NMR (CDCl3, 300 MHz) 8.10

(d, J = 7.9, 2H), 7.58 (t, J = 7.2, 1H), 7.45 (t, J = 7.5, 2H), 5.00-4.89 (m, 1H), 3.63-3.51

13 (m, 1H), 2.29-2.05 (m, 2H), 1.83-1.72 (m, 2H), 1.54-1.27 (m, 4H). C NMR (CDCl3, 75

MHz) 165.8, 133.1, 130.1, 129.7, 128.4, 76.0, 63.4, 30.5, 30.4, 23.8, 23.5. IR (CDCl3)

2100 cm-1. m.p 112-115 °C.

O N3 77b

2-azidocyclohexyl 2-phenylacetate 77b. 2-Azido cyclohexanol 76d (0.5 g, 3.5 mmol)

was acylated with phenylacetyl chloride (0.65 g, 4.2 mmol) following the general

procedure B for azide compounds syntheses to afford 0.80 g, (88%) of the azide 77b as a

1 colorless oil. H NMR (CDCl3, 300 MHz) 7.32-7.23 (m, 5H), 4.73-4.65 (m, 1H), 3.65

(s, 2H), 3.41-3.32 (m, 1H), 2.05-1.98 (m, 2H), 1.73-1.67 (m, 2H), 1.41-1.21 (m, 4H). 13C

NMR (CDCl3, 75 MHz) 170.9, 133.9, 129.3, 128.6, 127.1, 75.8, 63.1, 41.5, 30.4, 30.3,

-1 23.7, 23.4. IR (CDCl3) 2100 cm 145

O O O N 3 77c

2-azidocyclohexyl 2-phenoxyacetate 77c. 2-Azido cyclohexanol 76d (0.51 g, 3.6 mmol) was acylated with phenoxyacetyl chloride (0.75 g, 4.4 mmol) following the general procedure B for azide compounds syntheses to afford 0.90 g (91%) of the azide 77a as a

1 colorless oil. H NMR (CDCl3, 300 MHz) 7.31 (t, J = 7.6, 2H), 7.01 (t, J = 7.4, 1H),

6.94 (d, J = 8.0, 2H), 4.86-4.78 (m, 1H), 4.70 (d, J = 16.2, 1H), 4.64 (d, J = 16.2, 1H),

3.45-3.34 (m, 1H), 2.11-1.98 (m, 2H), 1.79-1.65 (m, 2H); 1.45-1.18 (m, 4H). 13C NMR

(CDCl3, 75 MHz) 168.3, 157.8, 129.6, 121.7, 114.7, 76.4, 65.3, 63.0, 30.4, 30.2, 23.7,

-1 23.4. IR (CDCl3) 2096 cm .

General procedure A for the synthesis of propargylamine derivatives 48a-f. To a

solution of propargyl bromide 51 (1.2 equiv) in THF (0.84 M) was added a secondary

amine 80 (1 equiv). The reaction mixture was stirred for 5 minutes and K2CO3 (2 equiv)

was added. The reaction mixture was reflux for 24 h under an atmosphere of argon. The

reaction mixture was then filtered, concentrated and chromatographed (50% EtOAc in

hexanes) to provide the propargylamine-derived alkynes 48a-f.

General procedure B for the synthesis of propargylamine derivatives 82a-c. To a

solution of N-methylprop-2-yn-1-amine 52 (1 equiv) in CH3OH (1.45 M) was added

alkyl bromide 81 (1.2 equiv). The reaction mixture was stirred for 5 minutes and K2CO3

(2 equiv) was added. The reaction mixture was refluxed for 24 h under an argon 146

atmosphere, then filtered, concentrated and chromatographed (50% EtOAc in hexane) to

afford propargylamine-derived alkynes 82a-c.

48a

N-butyl-N-(prop-2-ynyl)butan-1-amine 48a. Propargyl bromide 51 (1.1 g, 9.29 mmol)

was reacted with dibutylamine 80a (1.0 g, 7.74 mmol) following general procedure A for

propargylamine synthesis to afford 0.93 g (72%) of the alkyne 48a as an orange-yellow

384 1 oil that matched analytical data previously reported. H NMR (CDCl3, 300 MHz)

3.40 (d, J = 2.3, 2H), 2.45 (t, J = 7.1, 4H), 2.15 (t, J = 2.3, 1H), 1.49-1.26 (m, 8H), 0.92

13 (t, J = 7.1, 6H). C NMR (CDCl3, 75 MHz) 78.2, 71.7, 52.7, 41.0, 29.0, 19.9, 13.3.

N Me 48c

N-methyl-N-phenethylprop-2-yn-1-amine 48b. Propargyl bromide 51 (1.1 g, 8.88

mmol) was reacted with N-methyl-2-phenylethanamine 80b (1.0 g, 7.40 mmol) following

general procedure A for propargylamine synthesis to afford 0.96 g, (75%) of the alkyne

1 48b as a reddish brown oil. H NMR (CDCl3, 300 MHz) 7.28-7.14 (m, 5H), 3.37 (d, J =

2.4, 2H), 2.78-2.72 (m, 2H), 2.69-2.63 (m, 2H), 2.35 (s, 3H), 2.21 (t, J = 2.4, 1H). 13C

NMR (CDCl3, 75 MHz) 140.2, 128.7, 128.4, 126.1, 78.5, 73.3, 57.4, 45.6, 41.8, 34.3. 147

N 48c O

4-(prop-2-ynyl)morpholine 48c. Compound 48c was prepared by following the method

of Verron et al1. Propargyl bromide 51 (1.6 g, 13.8 mmol) was dissolved in THF (10 mL).

To this reaction mixture was added K2CO3 (3.2 g, 23.0 mmol) followed by morpholine

80c (1.0 g, 11.5 mmol). The reaction mixture was refluxed for 6 h under an atmosphere

of argon then extracted with CH3OH (15 mL, 3x). The combined organic layer was concentrated to afford a white solid. The solid was suspended in CH2Cl2 (30 mL) for 20 mins then filtered, concentrated and distilled (Kugelrohr distillation, 50 °C) to afforded

1.2 g, (84%) of 4-prop-2-ynyl-morpholine 48c as a colorless oil that matched analytical

342 1 data previously reported. H NMR (CDCl3, 300 MHz) 3.72 (t, J = 4.8, 4H), 3.27 (d, J

13 = 2.4, 2H), 2.55 (t, J = 4.9, 4H), 2.26 (t, J = 2.4, 1H). C NMR (CDCl3, 75 MHz) 78.4,

73.4, 66.8, 52.2, 47.2.

48d

4-phenyl-1-(prop-2-ynyl)piperidine 48d. Propargyl bromide 51 (1.0 g, 6.2 mmol) was reacted with 4-phenylpiperidine 80d (0.89 g, 7.44 mmol) following general procedure A for propargylamine synthesis to afford 0.98 g, (80%) of the alkyne 48d as a white solid

385 1 that matched analytical data previously reported. H NMR (CDCl3, 300 MHz) 7.36-

7.19 (m, 5H), 3.38 (d, J = 2.8, 2H), 3.08-3.00 (m, 2H), 2.59-2.47 (m, 1H), 2.37 (dt, J = 148

13 3.5, 11.5, 2H), 2.29 (t, J = 3.5, 1H), 1.95-1.80 (m, 4H). C NMR (CDCl3, 75 MHz)

145.8, 128.0, 126.5, 125.8, 78.7, 72.6, 52.6, 46.9, 41.8, 33.0. m.p 62-65 °C.

N N 48e

1-phenyl-4-(prop-2-ynyl)piperazine 48e. Propargyl bromide 51 (0.88 g, 7.4 mmol) was

reacted with 4-phenylpiperazine 80e (1.0 g, 6.2 mmol) following general procedure A

for propargylamine synthesis to afford 0.97 g (78%) of the alkyne 48e as a yellow solid

386 1 that matched analytical data previously reported. H NMR (CDCl3, 300 MHz) 7.29-

7.34 (m, 2H), 6.93 (d, J = 8.8, 2H), 6.86 (t, J = 7.3, 1H), 3.36 (d, J = 2.5, 2H), 3.24 (t, J =

13 5.0, 4H), 2.74 (t, J = 5.2, 4H), 2.27 (t, J = 2.4, 1H). C NMR (CDCl3, 75 MHz) 150.9,

128.8, 119.4, 115.8, 78.3, 73.0, 51.6, 48.7, 46.6. m.p 45-47 °C.

N O

O 48f

Ethyl 1-(prop-2-ynyl)piperidine-3-carboxylate 48f. Propargyl bromide 51 (0.91 g, 7.7 mmol) was reacted with ethyl nipecotate 80f (1.0 g, 6.4 mmol) following general procedure A for propargylamine synthesis to afford 0.86 g (69%) of the alkyne 48f as a

1 yellow oil. H NMR (CDCl3, 300 MHz) 4.13 (q, J = 7.1, 2H), 3.33 (d, J = 2.4, 2H), 3.01

(db, 1H), 2.81-2.74 (m, 1H), 2.64-2.24 (m, 1H), 2.78 (t, J = 10.7, 1H), 2.26-2.17 (m, 2H),

1.97-1.90 (m, 1H), 1.81-1.72 (m, 1H), 1.68-1.52 (m, 1H), 1.50-1.41 (m, 1H), 1.26 (t, J = 149

13 7.1, 3H). C NMR (CDCl3, 75 MHz) 173.5, 78.3, 72.7, 59.9, 53.9, 51.8, 46.8, 41.4,

26.0, 24.0, 13.7.

N Me

82a

N-(cyclohexylmethyl)-N-methylprop-2-yn-1-amine 82a. N-methyl propargylamine 52

(1.0 g, 14.5 mmol) was reacted with (bromomethyl)cyclohexane 81a (3.1 g, 17.4 mmol) following general procedure B for propargylamine synthesis to afford 1.74 g, (74%) of

1 the alkyne 82a as a reddish brown oil. H NMR (CDCl3, 300 MHz) 2.97 (d, J = 3.0,

2H), 1.93 (s, 3H), 1.89-1.85 (m, 3H), 1.49 (m, 5H), 1.11-1.04 (m, 1H), 0.98-0.76 (m,

13 3H), 0.61-0.44 (m, 2H). C NMR (CDCl3, 75 MHz) 78.7, 72.8, 62.5, 45.8, 42.0, 35.5,

31.6, 26.7, 26.0.

N Me Me Me 82b

N,3-dimethyl-N-(prop-2-ynyl)butan-1-amine 82b. N-methyl propargylamine 52 (1.0 g,

14.5 mmol) was reacted with 1-bromo-3-methylbutane 81b (2.6 g, 17.4 mmol) following general procedure B for propargylamine synthesis to afford 1.54 g (77%) of the alkyne

1 82b as a redish brown oil. H NMR (CDCl3, 300 MHz) 3.34 (d, J = 2.4, 2H), 2.42 (t, J

= 7.6, 2H), 2.30 (s, 3H), 2.20 (t, J = 2.4, 1H), 1.68-1.53 (m, 1H), 1.39-1.30 (m, 2H), 0.91

13 (d, J = 6.6, 6H). C NMR (CDCl3, 75 MHz) 77.8, 71.9, 53.0, 44.6, 40.9, 35.7, 25.4,

21.7. 150

82c

N-methyl-N-(3-phenylpropyl)prop-2-yn-1-amine 82c. N-methyl propargylamine 52

(1.0 g, 14.5 mmol) was reacted with 1-bromo-3-phenylpropane 81c (3.4 g, 17.4 mmol) following general procedure A for propargylamine synthesis to afford 2.2 g, (80%) of the

1 alkyne 82c as a redish brown oil. H NMR (CDCl3, 300 MHz) 7.29-7.24 (m, 2H), 7.19-

7.14 (m, 3H), 3.34 (d, J = 2.4, 2H), 2.64 (t, J = 7.6, 2H), 2.44 (t, J = 7.2, 2H), 2.31 (s,

13 3H), 2.19 (t, J = 2.4, 1H), 1.84-1.74 (m, 2H). C NMR (CDCl3, 75 MHz) 139.9, 126.2,

126.1, 123.5, 76.4, 70.7, 52.9, 43.3, 39.5, 31.3, 27.0.

General method for 1,4-disubstituted 1,2,3-triazole synthesis

t To a solution of azide compound (1.0 equiv) in BuOH/H2O mixture (0.2 M, 1:1) at 25 °C was added propargylamine derived alkyne (1.1 equiv). To this reaction mixture was added CuSO4•5H2O (1.0 M in H2O, 1.0 equiv) followed by sodium ascorbate (1.0 M in

H2O, 2.0 equiv). The reaction mixture was stirred at room temperature for 24 h, then

concentrated to a fourth its volume and diluted with CH2Cl2 (2 mL). NH4OH/H2O (1:1= 2

mL) was added and the mixture separated using a biotage phase separator. The CH2Cl2

filtrate was concentrated, charged onto silica plug and washed with 50% EtOAc in

hexanes as forerun eluant. This was followed by 5% CH3OH in CH2Cl2 to afford the

desired 1,4-disubstituted 1,2,3-triazole. 151

O N N O N H N OH 98a N

2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl

benzylcarbamate 98a. Azide 64a (25 mg, 0.100 mmol) and the alkyne 82c (19 mg,

0.106 mmol) were reacted following the general method for triazole synthesis to afford

1 41 mg (94%) of the 1,2,3-triazole 98a. H NMR (CDCl3, 300 MHz) 7.49 (s, 1H); 7.21-

7.11 (m, 7H), 7.06-7.01(m, 3H), 5.72 (t, J = 6.9, 1H), 4.50 (dd, J = 3, 13.4, 1H), 4.40-

4.28 (m, 3H), 4.27-4.16 (m, 2H), 4.16-4.03 (m, 2H), 3.62 (s, 2H), 2.61 (t, J = 7.5, 2H),

2.42 (t, J = 7.5, 2H), 2.21 (s, 3H), 1.90-1.72 (m, 2H). HPLC (1% AcOH in CH3OH :

H2O) RT 2.92 (97%), MS (APCI) : M+H expected 437.53, obtained 438.95.

OH N N N N 105g

2-(4-((4-phenylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)cyclohexanol 105g.

Azide 76d (15 mg, 0.106 mmol) and the alkyne 48e (21 mg, 0.106 mmol) were reacted following the general method for triazole synthesis to provide 34 mg (95%) of the 1,2,3-

1 triazole 105g. H NMR (CDCl3, 300MHz) 7.60 (s, 1H), 7.29-7.24 (m, 2H), 6.93-6.84

(m, 3H), 5.31 (s, 1H), 4.18-4.14 (m, 1H), 4.01-3.98 (m, 1H), 3.72 (s, 2H), 3.20 (t, J = 4.7,

4H), 2.70 (t, J = 5.0, 4H), 2.22-2.18 (m, 2H), 1.94-1.86 (m, 3H), 1.48-1.42 (m, 3H).

HPLC (1% AcOH in CH3OH : H2O) RT 2.51 (84%), MS (APCI) : M+H expected 306.45, obtained 307.30. 152

N O N N OH O 96f N

Ethyl 1-((1-(2-hydroxyhexyl)-1H-1,2,3-triazol-4-yl)methyl)piperidine-3-carboxylate

96f. Azide 76b (15 mg, 0.105 mmol) and the alkyne 48f (20 mg, 0.106 mmol) were

reacted following the general method for triazole synthesis to provide 32 mg (91%) of the

1 1,2,3-triazole 96f. H NMR (CDCl3, 300MHz) 7.68 (s, 1H), 4.48-4.41 (dt, J = 3.1, 13.8,

1H), 4.27-4.02 (m, 5H), 3.76 (d, J = 14.2, 1H), 3.70 (d, J = 14.2, 1H), 3.03 (d, J = 10.9,

1H), 2.86 (d, J = 10.4, 1H), 2.62-2.57 (m, 1H), 2.32 (t, J = 10.7, 1H), 2.18 (t, J = 10.7,

1H), 1.98-1.91 (m, 1H), 1.72-1.69 (m, 1H), 1.68-1.58 (m, 1H), 1.50-1.43 (m, 4H), 1.42-

1.37 (m, 3H), 1.23 (t, J = 7.2, 3H), 0.90 (t, J = 6.9, 3H). HPLC (CH3OH : H2O) RT 14.25

(99%), gradient elution 55% to 95% CH3OH over 26 mins. MS (APCI) : M+H expected

338.45, obtained 339.30.

N N N OH 96h N

1-(4-(((cyclohexylmethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)hexan-2-ol

96h. Azide 76b (15 mg, 0.105 mmol) and the alkyne 82a (17 mg, 0.105 mmol) were

reacted following the general method for triazole synthesis to provide 30 mg (93%) of the

1,2,3-triazole 96h. 1H NMR (CDCl3, 300MHz) 7.47 (s, 1H), 4.24-4.17 (m, 1H), 4.01

(dd, J = 7.8, 13.8, 1H), 3.89-3.74 (m, 1H), 3.52 (s, 2H), 2.11-2.05 (bs, 5H), 1.73 (s, 1H),

1.55-1.43 (m, 5H), 3.00-2.00 (m, 3H), 1.13-1.08 (m, 3H), 1.03-0.88 (m, 3H), 0.69-0.62 153

(m, 5H). HPLC (CH3OH : H2O) RT 16.53 (95%), gradient elution 55% to 95% CH3OH over 26 mins. MS (APCI) : M+H expected 308.26, obtained 309.25.

N N N OH 96g N N

1-(4-((4-phenylpiperazin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)hexan-2-ol 96g.

Azide 76b (15 mg, 0.105 mmol) and the alkyne 48e (21 mg, 0.105 mmol) were reacted following the general method for triazole synthesis to provide 35 mg (96%) of the 1,2,3-

1 triazole 96g. H NMR (CDCl3, 300MHz) 7.70 (s, 1H), 7.29-7.24 (m, 2H), 6.93-6.84 (m,

3H), 4.46 (dd, J = 2.5, 14.0, 1H), 4.24 (dd, J = 8, 14, 1H), 4.10-4.00 (m, 1H), 3.76 (s,

2H), 3.42 (s, 2H), 3.23-3.17 (bs, 4H), 2.67-1.80 (bs, 4H), 1.53-1.28 (m, 6H), 0.92 (t, J =

7, 3H). HPLC (1% AcOH in CH3OH : H2O) RT 9.79 (84%). MS (APCI) : M+H expected

343.47 obtained 344.90.

OH N N N N O 105f O

Ethyl 1-((1-(2-hydroxycyclohexyl)-1H-1,2,3-triazol-4-yl)methyl)piperidine-3- carboxylate 105f.

Azide 76d (15 mg, 0.106 mmol) and the alkyne 48f (21 mg, 0.106 mmol) were reacted

following the general method for triazole synthesis to provide 33 mg (92%) of the 1,2,3-

1 triazole 105f. H NMR (CDCl3, 300MHz) 7.60 (s, 1H), 4.20-4.07 (m, 3H), 3.99-3.91

(m, 1H), 3.79(s, 1H), 3.69-3.66 (m, 2H), 3.01 (d, J = 10.8, 1H), 2.82 (d, J = 11.0, 1H), 154

2.63-2.54 (m, 1H), 2.34-2.25 (m, 1H), 2.23-2.07 (m, 3H), 1.94-1.80 (m, 4H), 1.75-1.68

(m, 1H), 1.66-1.53 (m, 1H), 1.51-1.34 (m, 4H), 1.23 (t, J= 7.0, 3H). HPLC (1% AcOH in

CH3OH : H2O) RT 12.84 (98%). MS (APCI) : M+H expected 336.43, obtained 337.90.

OH N N N N 105h 2-(4-(((cyclohexylmethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexanol 105h.

Azide 76d (15 mg, 0.106 mmol) and the alkyne 82a (18 mg, 0.106 mmol) were reacted

following the general method for triazole synthesis to provide 30 mg (92%) of the 1,2,3-

1 triazole 105h. H NMR (CDCl3, 300MHz) 7.61 (s, 1H), 4.25-4.10 (m, 3H), 4.02-3.90

(m, 1H), 3.69 (s, 2H) 2.26 (s, 3H), 2.22-2.15 (m, 4H), 1.95-1.82 (m, 3H), 1.80-1.60 (m,

5H), 1.53-1.35 (m, 4H), 1.30-1.10 (m, 3H), 0.92-0.76 (m, 2H). HPLC (CH3OH : H2O) RT

18.94 (81%), gradient elution 55% to 95% CH3OH over 26 mins. MS (APCI) : M+H expected 306.24, obtained 307.90.

N O O N N OH O 94f N

Ethyl 1-((1-(3-(benzyloxy)-2-hydroxypropyl)-1H-1,2,3-triazol-4- yl)methyl)piperidine-3-carboxylate 94f.

Azide 76c (15 mg, 0.072 mmol) and the alkyne 48f (14 mg, 0.072 mmol) were reacted following the general method for triazole synthesis to provide 26 mg (91%) of the 1,2,3-

1 triazole 94f. H NMR (CDCl3, 300MHz) 7.56 (s, 1H), 7.32-7.22 (m, 5H), 4.49-4.47 (m, 155

3H), 4.37-4.29 (m, 1H), 4.20-4.10 (m, 1H), 4.08-4.05 (q, J = 4.5, 2H), 3.70-3.60 (s, 2H),

3.48-3.30 (m, 3H), 2.95 (d, J = 9.6, 1H), 2.75 (d, J = 9.6, 1H), 2.54 (t, J = 10.4, 1H),

2.27-2.21 (m, 1H), 2.18-2.03 (m, 2H), 1.86 (d, J = 11.1, 1H), 1.69-1.37 (m, 3H), 1.16 (t, J

= 7.1, 4H). HPLC (CH3OH : H2O) RT 14.56 (98%), gradient elution 55% to 95% CH3OH over 26 mins. MS (APCI) : M+H expected 402.49, obtained 403.25.

O N N O N H N OH 98b N

3-(4-((dibutylamino)methyl)-1H-1,2,3-triazol-1-yl)-2-hydroxypropyl benzylcarbamate 98b.

Azide 64a (20 mg, 0.080 mmol) and the alkyne 48a (13 mg, 0.080 mmol) were reacted following the general method for triazole synthesis to provide 31 mg (92%) of the 1,2,3-

1 triazole 98b. H NMR (CDCl3, 300MHz) 7.59 (s, 1H), 7.35-7.28 (m, 5H), 5.56 (t, J =

5.6, 1H), 4.41 (dd, J = 3.3, 13.6, 1H), 4.40-4.30 (m, 3H), 4.28-4.10 (m, 4H), 3.72 (s, 2H),

2.41 (t, J = 7.2, 4H), 1.48-1.43 (m, 4H), 1.41-1.27 (m, 4H), 0.90 (t, J = 7.2, 6H). HPLC

(1% AcOH in CH3OH : H2O) RT 2.50 (100%). MS (APCI) : M+H expected 417.55,

obtained 418.35. 156

O N N O N O H N OH O 98f N

Ethyl 1-((1-(3-(benzylcarbamoyloxy)-2-hydroxypropyl)-1H-1,2,3-triazol-4- yl)methyl)piperidine-3-carboxylate 98f.

Azide 64a (20 mg, 0.080 mmol) and the alkyne 48f (16 mg, 0.080 mmol) were reacted following the general method for triazole synthesis to provide 34 mg (96%) of the 1,2,3-

1 triazole 98f. H NMR (CDCl3, 300MHz) 7.66 (s, 1H), 7.33-7.27 (m, 5H), 5.79 (t, J =

5.4, 1H), 4.50 (dt, J = 2.8, 13.7, 1H), 4.40-4.30 (m, 3H), 4.28-4.18 (m, 2H), 4.16-4.06 (m,

4H), 3.64 (d, J = 14.2, 1H), 3.60 (d, J = 14.2, 1H), 2.95 (d, J = 10.8, 1H), 2.75 (d, J =

11.1, 1H), 2.59-2.50 (m, 1H), 2.25 (t, J = 10.2, 1H), 2.09 (t, J = 10.2, 1H), 1.92-1.87 (m,

1H), 1.73-1.67 (m, 1H), 1.51-1.37 (m, 2H), 1.23 (t, J = 7.2, 3H). HPLC (CH3OH : H2O)

RT 13.88 (97%), gradient elution 55% to 95% CH3OH over 26 mins. MS (APCI) : M+H

expected 445.51, obtained 446.25.

O N N O N H N OH 98c N

2-Hydroxy-3-(4-((isopentyl(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl benzylcarbamate 98c.

Azide 64a (20 mg, 0.080 mmol) and the alkyne 82b (11 mg, 0.080 mmol) were reacted

following the general method for triazole synthesis to provide 29 mg (92%) of the 1,2,3-

1 triazole 98c. H NMR (CDCl3, 300MHz) 7.81 (s, 1H), 7.35-7.28 (m, 6H), 5.50 (t, J =

5.4, 1H), 4.54 (dd, J = 3.0, 13.6, 1H), 4.40-4.36 (m, 3H), 4.30-4.24 (m, 1H), 4.20 (d, J =

4.1, 1H), 4.12 (dd, J = 5.2, 11.6, 1H), 3.79 (s, 2H), 2.57-2.51 (m, 2H), 2.32 (s, 3H), 1.65- 157

1.55 (m, 1H), 151-1.43 (m, 2H), 0.91 (t, J = 6.6, 6H). HPLC (CH3OH : H2O) RT 3.25

(82%), gradient elution 55% to 95% CH3OH over 26 mins. MS (APCI) : M+H expected

389.49, obtained 390.30.

O N N O N H N OH 98h N

3-(4-(((cyclohexylmethyl)(methyl) amino)methyl)-1H-1,2,3-triazol-1-yl)-2- hydroxypropyl benzylcarbamate 98h.

Azide 64a (20 mg, 0.080 mmol) and the alkyne 82a (13 mg, 0.080 mmol) were reacted following the general method for triazole synthesis to provide 30 mg (90%) of the 1,2,3-

1 triazole 98h. H NMR (CDCl3, 300MHz) 7.51 (s, 1H), 7.26-7.19 (m, 5H), 5.39 (t, J =

5.4, 1H), 4.43 (dd, J = 3.3, 13.7, 1H), 4.34 (m, 3H), 4.18-4.13 (m, 1H), 4.11 (d, J = 3.7,

1H), 4.07 (dd, J = 4.0, 13.3, 1H), 3.53 (s, 2H), 2.11 (s, 3H), 2.07 (d, J = 7.2, 2H), 1.70-

1.59 (m, 5H), 1.46-1.36 (m, 1H), 1.18-1.09 (m, 4H), 0.79-0.68 (m, 2H). HPLC (CH3OH :

H2O) RT 11.89 (84%). MS (APCI) : M+H expected 415.53, obtained 416.95.

N N N N O

O 97d 2-(4-((4-phenylpiperidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)cyclohexyl benzoate 97d.

Azide 77a (20 mg, 0.082 mmol) and the alkyne 48d (16 mg, 0.082 mmol) were reacted

following the general method for triazole synthesis to provide 33 mg (90%) of the 1,2,3- 158

1 triazole 97d. H NMR (CDCl3, 300MHz) 7.88-785 (m, 2H), 7.49 (tt, J = 0.9, 7.5, 1H),

7.39-7.27 (m, 4H), 7.23-7.15 (m, 3H), 7.58 (s, 1H), 5.38-5.29 (m, 1H), 4.79-4.70 (m,

1H), 3.68 (s, 2H), 2.89-2.84 (m, 2H), 2.39-2.23 (m, 3H), 2.09-1.94 (m, 5H), 1.72-1.51

(m, 7H). HPLC (1% AcOH in CH3OH : H2O) RT 2.52 (100%). MS (APCI) : M+H

expected 444.25, obtained 445.30.

N N N N O O O 104h 2-(4-(((cyclohexylmethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexyl 2- phenoxyacetate 104h.

Azide 77c (20 mg, 0.073 mmol) and the alkyne 82a (12 mg, 0.073 mmol) were reacted following the general method for triazole synthesis to provide 30 mg (93%) of the 1,2,3-

1 triazole 104h. H NMR (CDCl3, 300MHZ) 7.46 (s, 1H), 7.29-7.23 (m, 2H), 6.97 (tt, J =

0.9, 7.4, 1H), 6.75-6.71 (m, 2H), 5.27-5.18 (m, 1H), 4.60-4.51 (m, 1H), 4.48 (d, J = 16.3,

1H), 4.39 (d, J = 16.3, 1H), 3.63 (m, 2H), 2.29-2.21 (m, 2H), 2.16 (s, 3H), 2.12 (d, J =

7.1, 2H), 2.00-1.91 (m, 3H), 1.79-1.60 (m, 5H), 1.59-1.40 (m, 4H), 1.29-1.11 (m, 3H),

0.89-0.74 (m, 2H). HPLC (CH3OH : H2O) RT 13.92 (84%), gradient elution 55% to 95%

CH3OH over 26 mins. MS (APCI) : M+H expected 440.28, obtained 441.90. 159

N N N N O O O 104a 2-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexyl 2- phenoxyacetate 104a.

Azide 77c (20 mg, 0.073 mmol) and the alkyne 82c (14 mg, 0.073 mmol) were reacted

following the general method for triazole synthesis to provide 32 mg (95%) of the 1,2,3-

1 triazole 104a. H NMR (CDCl3, 300MHz) 7.44 (s, 1H), 7.28-7.17 (m, 7H), 6.97 (t, J =

7.2, 1H), 6.73 (d, J = 8.1, 2H), 5.28-5.17 (m, 1H), 4.52-4.42 (m, 4H), 3.68 (s, 2H), 2.62

(t, J = 7.8, 2H), 2.41 (t, J = 7.2, 2H), 2.21 (s, 3H), 1.94-1.83 (m, 7H), 1.53-1.47 (m, 4H).

HPLC (CH3OH : H2O) RT 20.29 (97%), gradient elution 55% to 95% CH3OH over 26 mins. MS (APCI) : M+H expected 462.58, obtained 463.00.

N N N N O O O 104d 2-(4-((4-phenylpiperidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)cyclohexyl 2- phenoxyacetate 104d.

Azide 77c (20 mg, 0.073 mmol) and the alkyne 48d (15 mg, 0.073 mmol) were reacted following the general method for triazole synthesis to provide 32 mg (91%) of the 1,2,3-

1 triazole 104d. H NMR (CDCl3, 300MHz) 7.53 (s, 1H), 7.32-7.17 (m, 7H), 6.97 (tt, J =

0.9, 7.5, 1H), 6.76-6.72 (m, 2H), 5.29-5.20 (m, 1H), 4.62-4.53 (m, 1H), 4.49 (d, J = 16.3,

1H), 4.40 (d, J = 16.3, 1H), 3.70 (s, 2H), 3.04-2.99 (m, 2H), 2.49-2.41 (m, 1H), 2.32-2.22 160

(m, 2H), 2.19-2.09 (m, 2H), 2.03-1.09 (m, 3H), 1.82-1.76 (m, 4H), 1.60-1.48 (m, 3H).

HPLC (1% AcOH in CH3OH : H2O) RT 14.48 (98%). MS (APCI) : M+H expected

474.59, obtained 475.85.

N N N N O O O 104b 2-(4-((dibutylamino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexyl 2-phenoxyacetate 104b.

Azide 77c (20 mg, 0.073 mmol) and the alkyne 48a (12 mg, 0.073 mmol) were reacted following the general method for triazole synthesis to provide 30 mg (91%) of the 1,2,3-

1 triazole 104b. H NMR (CDCl3, 300MHz) 7.46 (s, 1H), 7.26-7.21 (m, 2H), 6.98-6.90

(m, 1H), 6.72 (d, J = 8.3, 2H), 5.24-5.16 (m, 1H), 4.67-4.49 (m, 1H), 4.45 (d, J = 16.2,

1H), 4.36 (d, J = 16.2, 1H), 3.72 (s, 2H), 2.38 (t, J = 7.2, 4H), 2.25-2.21 (m, 2H), 1.98-

1.89 (m, 3H), 1.51-1.38 (m, 7H), 1.31-1.19 (m, 5H), 0.86 (t, J = 7.2, 6H). HPLC (CH3OH

: H2O) RT 20.38 (81%), gradient elution 55% to 95% CH3OH over 26 mins. MS (APCI) :

M+H expected 442.59 obtained 444.30. 161

O O

N N N N O O O 104f Ethyl 1-((1-(2-(2-phenoxyacetoxy)cyclohexyl)-1H-1,2,3-triazol-4- yl)methyl)piperidine-3-carboxylate 104f.

Azide 77c (20 mg, 0.073 mmol) and the alkyne 48f (14 mg, 0.073 mmol) were reacted following the general method for triazole synthesis to provide 31 mg (92%) of the 1,2,3-

1 triazole 104f. H NMR (CDCl3, 300MHz) 7.49 (d, J = 6, 1H), 7.29-7.23 (m, 2H), 6.98

(tt, J = 0.9, 6.4, 1H), 6.73 (dd, J = 0.8, 7.9, 2H), 5.23 (m, 1H), 4.58-4.53 (m, 1H), 4.48 (d,

J = 16.3, 1H), 4.39 (d, J = 16.3, 1H), 4.16-4.06 (m, 2H), 3.70 (d, J = 14.2, 1H), 3.63 (d, J

= 14.2, 1H), 2.95 (t, J = 8.1, 1H), 2.76-2.70 (m, 1H), 2.58-2.50 (m, 1H), 2.30-2.22 (m,

3H), 2.19-1.81 (m, 6H), 1.71-1.65 (m, 1H), 1.60-1.37 (m, 5H), 1.23 (t, J = 7.2, 3H).

HPLC (CH3OH : H2O) RT 15.25 (96%), gradient elution 55% to 95% CH3OH over 26 mins. MS (APCI) : M+H expected 470.56 obtained 470.80.

N N N N O

O 103b 2-(4-((dibutylamino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexyl 2-phenylacetate 103b.

Azide 77b (25 mg, 0.096 mmol) and the alkyne 48a (16 mg, 0.096 mmol) were reacted

following the general method for triazole synthesis to provide to provide 38 mg (93%) of 162

1 the 1,2,3-triazole 103b. H NMR (CDCl3, 300MHz) 7.10 (s, 1H), 7.07-6.99 (m, 3H),

6.86-6.82 (m, 2H), 4.92-4.83 (m, 1H), 4.35-4.24 (m, 1H), 3.47 (s, 2H), 3.20 (s, 2H), 2.14

(t, J = 7.2, 3H), 2.10-1.92 (m, 2H), 1.72-1.64 (m, 3H), 1.29-1.16 (m, 7H), 1.11-0.99 (m,

5H), 0.69 (t, J = 7.2, 6H). HPLC (CH3OH : H2O) RT 16.80 (86%), gradient elution 55% to

95% CH3OH over 26 mins. MS (APCI) : M+H expected 426.59 obtained 430.00.

N N N N O

O 103h 2-(4-(((cyclohexylmethyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexyl 2- phenylacetate 103h.

Azide 77b (25 mg, 0.0.096 mmol) and the alkyne 82a (16 mg, 0.096 mmol) were reacted

following the general method for triazole synthesis to provide 38 mg (93%) of the 1,2,3-

1 triazole 103h. H NMR (CDCl3, 300MHz) 7.56 (s, 1H), 7.09-7.00 (m, 3H), 6.90-6.84

(m, 2H), 4.93-4.84 (m, 1H), 4.36-4.27 (m, 1H), 3.39 (s, 2H), 3.22 (s, 2H), 2.10-1.90 (m,

7H), 1.79-1.65 (m, 3H), 1.64-1.40 (m, 6H), 1.33-1.21 (m, 4H), 1.10-0.90 (m, 4H), 0.68-

0.54 (m, 2H). HPLC (CH3OH : H2O) RT 19.88 (95%). MS (APCI) : M+H expected

424.58 obtained 423.40. 163

O N N O N H N OH 98d N

2-hydroxy-3-(4-((4-phenylpiperidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propyl benzylcarbamate 98d.

Azide 64a (20 mg, 0.080 mmol) and the alkyne 48d (16 mg, 0.080 mmol) were reacted

following the general method for triazole synthesis to provide 33 mg (92%) of the 1,2,3-

1 triazole 98d. H NMR (CDCl3, 300MHz) 7.69 (s, 1H), 7.33-7.24 (m, 7H), 7.22-7.19 (m,

3H), 5.71 (t, J = 6.8, 1H), 4.53 (dd, J = 3.0, 13.6, 1H), 4.39-4.21 (m, 5H), 4.20-4.10 (m,

2H), 3.03 (d, J = 11.4, 2H), 2.53-2.43 (m, 1H), 2.20-2.10 (m, 2H), 1.83-1.72 (m, 4H).

HPLC (1% AcOH in CH3OH : H2O) RT 2.33 (100%). MS (APCI) : M+H expected

449.55, obtained 450.35.

Ru Cl PPh3 Ph3P 107

Chloropentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) 107. To a

solution of Ph3P (2.3 g, 0.9 mmol) in absolute ethanol (50 mL) was added [Cp*RuCl]n

(0.50 g). The reaction mixture was refluxed under an argon atmosphere for 28 h. The

formation of orange microcrystal signified completion of reaction. The reaction mixture

was then cooled to -20 °C and filtered. The crystals were washed with cold pentane (10

1 mL, 3x) to give the 1.13 g (87%) of Cp*Ru(PPh)3Cl 107 as orange solid. H NMR 164

(CDCl3, 300 MHz) 8.00-7.00 (m, 28H), 1.57 (bs, 3H), 1.35 (bs, 12H), Resonance 6.84 ppm, 4.62 ppm.

General procedure for 1,5-disubstituted 1,2,3-triazole 4.61a-b and 4.63a-c synthesis components To a solution of azide component (1 equiv) and alkyne component (1.2

equiv) in dried benzene (0.04 M) was added Cp*Ru(PPh)2Cl 4.58 (20 mol%). The reaction mixture was refluxed under an argon atmosphere for 3 h. The reaction mixture

was then concentrated and chromatographed (50% EtOAc in Hexanes to 5% CH3OH in

CH2Cl2 to provide the 1,5-disubstituted 1,2,3-triazole derivative.

N N N

OH N 110a

Trans-2-(5-((dibutylamino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexanol 110a. Trans-2- azidocyclohexanol 76d (13 mg, 0.09 mmol) and N-butyl-N-(prop-2-ynyl)butan-1-amine

48b (23 mg, 0.14 mmol) were reacted following the general method for 1,5-disubstituted

1 1,2,3-triazole synthesis to afford 23 mg (83%) of 110a. H NMR (CDCl3, 300 MHz)

7.50 (s, 1H), 4.85 (bs, 1H), 4.15-4.06 (m, 2H), 3.98-3.90 (m, 2H), 3.59 (s, 2H), 2.54-2.45

13 (m, 2H), 1.93-1.80 (m, 2H), 1.54-1.22 (m, 11H), 0.91 (t, J = 7.2, 6H). C NMR (CDCl3,

75 MHz) 134.3, 133.8, 73.2, 64.1, 53.1, 46.2, 35.6, 31.5, 28.1, 25.2, 24.1, 20.5, 13.9.

HPLC (1% AcOH in CH3OH : H2O) RT 4.00 (86%). MS (APCI) : M+H expected 308.46, obtained 308.95. 165

N N N

N O O O O 110b

Trans-2-(5-(morpholinomethyl)-1H-1,2,3-triazol-1-yl)cyclohexyl 2-phenoxyacetate

110b. Trans-2-azidocyclohexyl 2-phenoxyacetate 77c (25 mg, 0.10 mmol) and the

alkyne, 4-(prop-2-ynyl)morpholine 48c (15 mg, 0.12 mmol) were reacted following the

general method for 1,5-disubstituted 1,2,3-triazole synthesis to afford 30 mg (80%) of

1 110b. H NMR (CDCl3, 300 MHz) 7.36 (s, 1H), 7.27-7.20 (m, 2H), 6.96 (t, J = 7.4,

1H), 6.63 (d, J = 7.8, 2H), 5.49-5.40 (m, 1H), 4.69-4.60 (m, 1H), 4.38 (s, 2H), 3.69-3.59

(m, 4H), 3.51 (d, J = 13.9, 1H), 3.27 (d, J = 13.9, 1H), 2.41-2.28 (m, 5H), 2.19 (dt, J =

3.7, 12.9, 1H), 2.08-2.02 (m, 1H), 1.96-1.84 (m, 2H), 1.65-1.32 (m, 3H). 13C NMR

(CDCl3, 75 MHz) 166.1, 155.8, 132.0, 131.3, 127.9, 119.9, 112.3, 74.5, 65.1, 63.5,

58.7, 51.5, 48.8, 30.2, 29.4, 23.0, 21.9. HPLC (1% AcOH in CH3OH : H2O) RT 3.00

(98%).

O N N O N H N OH 112a N

2-hydroxy-3-(5-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl benzylcarbamate 112a. 3-Azido-2-hydroxypropyl benzylcarbamate 64a (25 mg, 0.10 mmol) and N-methyl-N-(3-phenylpropyl)prop-2-yn-1-amine 82c (28 mg, 0.15 mmol) were reacted following the general method for 1,5-disubstituted 1,2,3-triazole synthesis 166

1 to afford 36 mg, (81%) of 112a. H NMR (CDCl3, 300 MHz) 7.47 (s, 1H), 7.29-7.18

(m, 7H), 7.13-7.06 (m, 3H), 5.05 (bs, 1H), 4.61 (d, J = 14.3, 1H), 4.27 (d, J = 6.0, 1H),

4.23-4.13 (m, 1H), 4.11 (bs, 3H), 3.51 (d, J = 13.8, 1H), 3.49 (d, J = 13.8, 1H), 2.52 (d, J

13 = 7.4, 2H), 2.47-2.35 (m, 2H), 2.21 (s, 3H), 1.84-1.73 (m, 2H). C NMR (CDCl3, 75

MHz) 155.2, 140.2, 137.2, 133.8, 132.6, 127.7, 127.5, 127.3, 126.5, 125.1, 68.3, 65.4,

55.8, 51.2, 48.0, 44.2, 40.3, 32.5, 27.0. HPLC (1% AcOH in CH3OH : H2O) RT 3.30

(94%). MS (APCI) : M+H expected 437.30, obtained 438.00.

O N N O N H N OH 112b N

2-hydroxy-3-(5-((isopentyl(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl

benzylcarbamate 112b. 3-Azido-2-hydroxypropyl benzylcarbamate 64a (25 mg, 0.10 mmol) and N,3-dimethyl-N-(prop-2-ynyl)butan-1-amine 82b (21 mg, 0.15 mmol) were reacted following the general method for 1,5-disubstituted 1,2,3-triazole synthesis to

1 afford 32 mg (81%) of 112b. H NMR (CDCl3, 300 MHz) 7.59 (s, 1H), 7.37-7.27 (m,

5H), 5.16 (bs, 1H), 4.70 (d, J = 14.2, 1H), 4.38 (d, J = 6.0, 2H), 4.32-4.23 (m, 1H), 4.18

(bs, 3H), 3.59 (d, J = 13.8, 1H), 3.72 (d, J = 13.7, 1H), 2.55-2.37 (m, 2H), 2.19 (s, 3H),

1.61-1.47 (m, 1H), 1.45-1.36 (m, 2H), 1.25 (bs, 2H), 0.88 (d, J = 6.4, 2H). 13C NMR

(CDCl3, 75 MHz) 156.2, 138.2, 134.9, 133.7, 128.7, 127.6, 69.3, 66.5, 55.6, 52.4, 49.0,

41.2, 35.0, 29.7, 26.5, 22.6, 22.5. HPLC (1% AcOH in CH3OH : H2O) RT 3.70 (87%). MS

(APCI) : M+H expected 389.25 obtained 389.95 167

O N N O N H N OH 112c N O O

Ethyl 1-((1-(3-(benzylcarbamoyloxy)-2-hydroxypropyl)-1H-1,2,3-triazol-

5yl)methyl)piperidine-3-carboxylate 112c. 3-Azido-2-hydroxypropyl benzylcarbamate

64a (25 mg, 0.10 mmol) and ethyl 1-(prop-2-ynyl)piperidine-3-carboxylate 48f (30 mg,

0.15 mmol) were reacted following the general method for 1,5-disubstituted 1,2,3-triazole

1 synthesis to afford 35 mg (78%) of 112c. H NMR (CDCl3, 300 MHz) 7.58 (s, 1H),

7.37-7.24 (m, 5H), 5.50 (bs, 1H), 4.67 (d, J = 13.9, 1H), 4.38 (d, J = 6.0, 2H), 4.31-4.05

(m, 6H), 3.51 (m, 2H), 3.02 (d, J = 10.3, 1H), 2.76-2.14 (m, 4H), 2.12-1.76 (m, 2H),

13 1.75-1.42 (m, 4H), 1.26-1.19 (m, 4H). C NMR (CDCl3, 75 MHz) 166.1, 155.8, 132.0,

131.3, 127.9, 119.9, 112.3, 74.5, 65.1, 63.5, 58.7, 51.5, 48.8, 30.2, 29.4, 23.0, 21.9.

HPLC (1% AcOH in CH3OH : H2O) RT 3.10 (98%). MS (APCI) : M+H expected 445.23, obtained 445.95.

General method for the synthesis of azide components. To a solution of glycidol (1

equiv) in CH2Cl2 (1.35 M) at 0 °C was added DMAP (1.2 equiv). The reaction mixture was stirred for 30 minutes and acid chloride (1.2 equiv) was added. The reaction was warmed to r.t and stirring continued at r.t for 4 h under an argon atmosphere. The

reaction mixture was poured through silica gel pad and washed with CH2Cl2 (100 mL)

then concentrated to afford the crude epoxide 118b-e. Without further purification, the 168

crude epoxide was used in the next step of the synthesis. To a mixture of epoxide (1

equiv), NH4Cl (2 equiv) in CH3OH and H2O (0.10 M, 8:1) was added NaN3 (10 equiv).

The reaction mixture was degassed and stirred at r.t for 24 h. The reaction mixture was

then concentrated to 1/10 its volume, diluted with H2O and extracted with EtOAc (3x).

The combined organic layer was washed with brine (2x), dried over MgSO4, filtered,

concentrated and chromatographed (35% EtOAc in hexanes) to provide the azide

component 119b-c.

O N3 119b OH

3-azido-2-hydroxypropyl 2-phenylacetate 119b. Glycidol 49 (220 mg, 3.0 mmol) and

phenylacetyl chloride 117b (560 mg, 3.6 mmol) were reacted to provide glycidyl ester

118b. Glycidyl ester 118b (100 mg, 0.52 mmol) was reacted with NaN3 (338 mg, 5.2 mmol) following the general method for the synthesis of azide components to afford 100

1 mg (72%) of the azide 119b as a yellow oil. H NMR (CDCl3, 300 MHz) 7.40-7.27 (m,

5H), 4.26-4.12 (m, 2H), 4.05-3.95 (m, 1H), 3.69 (d, J = 2.8, 2H), 3.40-3.27 (m, 2H),

13 2.40-2.35 (m, 1H). C NMR (CDCl3, 75 MHz) 171.6, 133.5, 129.2, 128.7, 127.4, 69.0,

-1 65.9, 53.3, 41.2. IR (CDCl3) 2102 cm . 169

O N3 119c OH

3-azido-2-hydroxypropyl 3-phenylpropanoate 119c. Glycidol 49 (220 mg, 3.0 mmol)

and hydrocinnamoyl chloride 117c (608 mg, 3.6 mmol) were reacted to provide glycidyl

ester 118c. Glycidyl ester 118c (150 mg, 0.73 mmol) was reacted with NaN3 (470 mg, 7.3

mmol) following the general method for the synthesis of azide components to afford 0.14

1 g (75%) of the azide 119c as a yellow oil. H NMR (CDCl3, 300 MHz) 7.37-7.21 (m,

5H), 4.17 (dd, J = 5.0, 11.6, 1H), 4.11 (dd, J = 5.0, 11.6, 1H), 3.98-3.89 (m, 1H), 3.32

(dd, J = 5.4, 12.6, 1H), 3.27 (dd, J = 5.4, 12.6, 1H), 2.99 (t, J = 7.5, 2H), 2.72 (t, J = 7.6,

13 2H), 2.29 (d, J = 5.0, 1H). C NMR (CDCl3, 75 MHz) 172.9, 140.1, 128.6, 128.3,

-1 126.5, 69.0, 65.7, 53.3, 35.6, 30.9. IR (CDCl3) 2102 cm .

General method for the synthesis of carbamoyl azides components 124a-d, 131 and

134. To a solution of the requisite glycidol (1 equiv) in CH2Cl2 (1.35 M) at 0 °C was added DMAP (1.2 equiv). The reaction mixture was stirred for 30 minutes and the requisite isocyanate (1.2 equiv) was added. The reaction mixture was stirred at 0 °C for 3 h under an argon atmosphere. The reaction mixture was then poured through silica gel

pad and washed with CH2Cl2 (100 mL) to afford the crude epoxides 123a-d, 130, and 133

(solvent was removed at at 0 °C on the pump). Without further purification, the crude epoxides were used in the next step of the synthesis. To a mixture of epoxide (1 equiv),

-2 NH4Cl (2 equiv) in CH3OH and H2O mixture (9.48x10 M, 8:1) was added NaN3 (10

equiv). The reaction mixture was degassed and stirred at room temperature for 24 h. The 170

reaction mixture was concentrated to 1/10 its volume, diluted with water and extracted

with EtOAc (3x). The combined organic layer was washed with brine (2x), dried over

MgSO4, filtered, concentrated and chromatographed (50% EtOAc in hexanes) to provide

the azide components 124a-d, 131, and 134.

N O N3 H OH 124a

3-azido-2-hydroxypropyl (R)-1-phenylethylcarbamate 124a. Glycidol 49 (220 mg, 3.0

mmol) was reacted with (R)-(+)- methylbenzyl isocyanate 122a (530 mg, 3.6 mmol) to

provide glycidyl carbamate 123a. Glycidyl carbamate 123a (35 mg, 0.16 mmol) was

reacted with NaN3 (105 mg, 1.6 mmol) following the general method for the synthesis of

carbamoyl azides to afford 30 mg (73%) of the azide 124a as a yellow oil. 1H NMR

(CDCl3, 300 MHz) 7.31-7.21 (m, 5H), 5.81 (d, J = 7.6, 1H), 4.79-4.63 (m, 1H), 4.11-

13 3.72 (m, 4H), 3.24 (m, 2H), 1.41 (d, J = 7.1, 3H). C NMR (CDCl3, 75 MHz) 171.6,

155.9, 143.5, 128.7, 127.3, 125.9, 69.1, 66.2, 60.6, 53.3, 50.8, 22.3, 21.0, 14.1. IR

-1 (CDCl3) 2104cm .

N O N3 H OH 124b

3-azido-2-hydroxypropyl 4-methylbenzylcarbamate 124b. Glycidol 49 (220 mg, 3.0

mmol) was reacted with 4-methylbenzyl isocyanate 122b (530 mg, 3.6 mmol) to provide

glycidyl carbamate 123b. Glycidyl carbamate 123b (35 mg, 0.15 mmol) was reacted with 171

NaN3 (100 mg, 1.5 mmol) following the general method for the synthesis of carbamoyl

1 azides to afford 35 mg (82%) of the azide 124b as a yellow oil. H NMR (CDCl3, 300

MHz) 7.17 (d, J = 8.3, 2H), 7.13 (d, J = 8.2, 2H), 5.24 (bs, 1H), 4.30 (d, J = 5.9, 2H),

13 4.21-4.08 (m, 2H), 4.00-3.93 (m, 1H), 3.36-3.26 (m, 3H), 2.33 (s, 3H). C NMR (CDCl3,

75 MHz) 155.9, 136.5, 134.1, 128.5, 126.7, 68.7, 65.8, 52.5, 44.1, 20.2. IR (CDCl3)

2104 cm-1.

N O N3 H OH O 124c

3-azido-2-hydroxypropyl 4-methoxybenzylcarbamate 124c. Glycidol 49 (220 mg, 3.0 mmol) was reacted with 4-methoxybenzyl isocyanate 122c (588 mg, 3.6 mmol) to provide glycidyl carbamate 123c. Glycidyl carbamate 123c (25 mg, 0.11 mmol) was

reacted with NaN3 (70 mg, 1.1 mmol) following the general method for the synthesis of carbamoyl azides to afford 25 mg (79%) of the azide 124c as a yellow oil. 1H NMR

(CDCl3, 300 MHz) 7.21 (d, J = 8.5, 2H), 6.86 (d, J = 8.8, 2H), 5.32 (bs, 1H), 4.27 (d, J

= 5.7, 2H), 4.17 (dd, J = 4.8, 11.7, 1H), 4.11 (dd, J = 5.3, 11.7, 1H), 4.03-3.93 (m, 1H),

13 3.78 (s, 3H), 3.42-3.24 (m, 3H). C NMR (CDCl3, 75 MHz) 158.5, 156.1, 129.5, 128.3,

-1 113.5, 68.9, 65.9, 54.7, 52.7, 44.1. IR (CDCl3) 2104 cm . 172

O O N O N3 H OH 124b

3-azido-2-hydroxypropyl furan-2-ylmethylcarbamate 124d. Glycidol 49 (220 mg, 3.0

mmol) was reacted with furfuryl isocyanate 122d (530 mg, 3.6 mmol) to provide glycidyl

carbamate 123d. Glycidyl carbamate 123d (35 g, 0.18 mmol) was reacted with NaN3

(113 mg, 1.8 mmol) following the general method for the synthesis of carbamoyl azides

1 to afford 30 mg (77%) of the azide 124d as a yellow oil. H NMR (CDCl3, 300 MHz)

7.36 (d, J = 1.5, 1H), 6.32 (t, J = 2.4, 1H), 6.24 (t, J = 3.0, 1H), 5.21 (bs, 1H), 4.36 (d, J =

5.8, 2H), 4.21 (dd, J = 4.7, 11.8, 1H), 4.14 (dd, J = 4.7, 11.8, 1H), 4.03-3.94 (m, 1H),

13 3.40 (dd, J = 5.0, 12.8, 1H), 3.02 (d, J = 4.7, 1H). C NMR (CDCl3, 75 MHz) 155.9,

-1 150.6, 141.9, 109.9, 107.0, 69.1, 66.3, 52.8, 37.7. IR (CDCl3) 2106 cm .

N O N3 H 128

3-azidopropyl benzylcarbamate 128. To a solution of 3-bromopropanol 125 (3 g, 21.5

mmol) in DMF (30 mL) was added NaN3 (13.9 g, 215 mmol). The reaction mixture was stirred under an argon atmosphere for 6 h then diluted EtOAc (80 mL) and transferred into a separatory funnel. The mixture was washed with water (4 x 30 mL), brine (2 x 20

mL), dried over MgSO4, filtered and concentrated to afford the crude azido alcohol 4.20

-1 (IR (CDCl3) 2097 cm ). Without further purification, the crude azide compound was used

in the step of the reaction. To a solution of 3-azidopropanol 126 (100 mg, 0.99 mmol) in

CH2Cl2 (1.5 mL) at 0 °C was added DMAP (150 mg, 1.2 mmol). The reaction mixture 173

was stirred at 0 °C for 30 minutes and benzyl isocyanate 127 (160 mg, 1.19 mmol) was

added. Stirring was continued at 0 °C for 3 h under an argon atmosphere. The reaction

mixture was then poured through silica gel pad and washed with CH2Cl2 (20 mL). The

filtrate was concentrated and chromatographed (50% EtOAc in hexanes) to afford 0.2 g

1 (85%) of 3-azidopropyl benzylcarbamate 128 as a yellow oil. H NMR (CDCl3, 300

MHz) 7.37-7.24 (m, 5H), 5.06 (bs, 1H), 4.36 (d, J = 5.7, 2H), 4.18 (t, J = 6.1, 2H), 3.37

13 (t, J = 6.2, 2H), 1.94-1.85 (m, 2H). C NMR (CDCl3, 75 MHz) 155.7, 137.7, 128.0,

-1 126.9, 61.3, 47.6, 44.4, 27.9. IR (CDCl3) 2098 cm .

N O N3 H OH 131

(R)-3-azido-2-hydroxypropyl benzylcarbamate 131. (R)-(+)-glycidol 129 (110 mg,

1.5 mmol) was reacted with benzyl isocyanate 127 (240 mg, 1.8 mmol) to provide the

glycidyl carbamate 130. Glycidyl carbamate 130 (25 mg, 0.12 mmol) was reacted with

NaN3 (78 mg, 1.2 mmol) following the method for the synthesis of glycidyl carbamates to provide 20 mg (74%) of (R)-3-azido-2-hydroxypropyl benzylcarbamate 131 as a

1 yellow oil. H NMR (CDCl3, 300 MHz) 7.40-7.26 (m, 5H), 5.23 (bs, 1H), 4.38 (d, J =

5.9, 2H), 4.23 (dd, J = 4.5, 11.8, 1H), 4.17 (dd, J = 4.5, 11.8, 1H), 4.02-3.94 (m, 1H),

13 3.44-3.11 (m, 3H). C NMR (CDCl3, 75 MHz) 156.8, 138.0, 128.8, 127.7, 127.6, 69.6,

-1 25 66.7, 53.4, 45.3. IR (CDCl3) 2103 cm . []D = + 5.8 ° (c 1.1, CH2Cl2). 174

N O N3 H OH 134

(S)-3-azido-2-hydroxypropyl benzylcarbamate 134. (S)-(-)-glycidol 132 (110 mg, 1.5 mmol) was reacted with benzyl isocyanate 127 (240 g, 1.8 mmol) to provide glycidyl

carbamate 133. Glycidyl carbamate 133 (25 mg, 0.12 mmol) was reacted with NaN3 (78

mg, 1.2 mmol) following the general method for the synthesis of glycidyl carbamates to

provide 20 mg (73%) of (S)-3-azido-2-hydroxypropyl benzylcarbamate 134 as a yellow

1 oil. H NMR (CDCl3, 300 MHz) 7.31-7.19 (m, 5H), 5.15 (bs, 1H), 4.29 (d, J = 5.9, 2H),

4.13 (dd, J = 5.0, 11.8, 1H), 4.07 (dd, J = 5.0, 11.8, 1H), 3.95-3.86 (m, 1H), 3.35-3.04 (m,

13 3H). C NMR (CDCl3, 75 MHz) 156.8, 138.0, 128.8, 127.7, 127.6, 69.6, 66.7, 53.4,

-1 25 45.3. IR (CDCl3) 2104 cm . []D = -6.0 ° (c 1.2, CH2Cl2).

O 137

(3-(prop-2-ynyloxy)propyl)benzene 137. To a mixture of propargyl alcohol 135 (0.2 g,

3.57 mmol) and K2CO3 (0.99 g, 7.14 mmol) in acetone (2 mL) was added 1-bromo-3- phenylpropane 136 (0.78 g, 3.92 mmol). The reaction mixture was refluxed under an argon atmosphere for 24 h. The reaction mixture was filtered, concentrated and chromatographed to give 0.42 g (68%) of (3-(prop-2-ynyloxy)propyl)benzene 137 as a

1 pale yellow oil. H NMR (CDCl3, 300 MHz) 7.28-7.12 (m, 5H), 4.09 (d, J = 2.3, 2H),

3.49 (t, J = 6.4, 2H), 2.68 (t, J = 7.7, 2H), 2.38 (t, J = 2.3, 1H), 1,93-1.84 (m, 2H). 13C

NMR (CDCl3, 75 MHz) 141.8, 128.3, 125.8, 80.0, 74.2, 69.2, 58.0, 32.2, 31.1. 175

O N O N N OH 113a N

2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl benzoate 113a. 3-Azido-2-hydroxypropyl benzoate (13 mg, 0.07 mmol) and N-methyl-

N-(3-phenylpropyl)prop-2-yn-1-amine (20 mg, 0.11 mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford 25 mg (95%) of GHB-7 analog

1 113a. H NMR (CDCl3, 300 MHz) 8.05 (d, J = 7.4, 2H), 7.63-7.54 (m, 2H), 7.46 (t, J =

7.8, 2H), 7.29-7.12 (m, 6H), 4.64-4.56 (m, 1H), 4.47-4.37 (m, 3H), 3.67 (s, 2H), 2.61 (t, J

= 7.6, 2H), 2.42 (t, J = 7.2, 2H), 2.24 (s, 3H), 1.88-1.77 (m, 2H), 1.25 (bs, 1H). 13C NMR

(CDCl3, 75 MHz) 166.1, 144.4, 141.6, 133.1, 129.3, 128.9, 128.1, 128.0, 127.9, 125.3,

123.7, 68.4, 65.5, 55.9, 52.6, 51.9, 41.6, 33.0, 29.3, 28.4. HPLC (CH3OH : H2O) RT 13.32

(83%). MS (APCI) : M+H expected 408.49, obtained 408.90.

O N O N N 113b OH N

2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl

2-phenylacetate 113b. 3-Azido-2-hydroxypropyl 2-phenylacetate (13 mg, 0.07 mmol) and N-methyl-N-(3-phenylpropyl)prop-2-yn-1-amine (15 mg, 0.09 mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford 23 mg (93%) of GHB-

1 7 analog 113b. H NMR (CDCl3, 300 MHz) 7.40 (s, 1H), 7.37-7.24 (m, 7H), 7.20-7.14

(m, 3H), 5.13 (bs, 1H), 4.44-4.35 (m, 4H), 4.12 (t, J = 5.9, 2H), 3.67 (s, 2H), 2.62 (t, J = 176

13 7.6, 2H), 2.42 (t, J = 7.2, 2H), 2.28-2.18 (m, 5H), 1.88-1.78 (m, 2H). C NMR (CDCl3,

75 MHz) 155.2, 144.2, 141.2, 137.3, 127.7, 127.4, 127.3, 126.6, 126.5, 124.6, 121.7,

60.5, 55.4, 51.4, 46.1, 44.1, 41.1, 32.5, 28.9, 28.0. HPLC (1% AcOH in CH3OH : H2O)

RT 3.06 (99%). MS (APCI) : M+H expected 422.52, obtained 422.95.

O N O N N OH 113c N

2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl

3-phenylpropanoate 113c. 3-Azido-2-hydroxypropyl 3-phenylpropanoate (13 mg, 0.05 mmol) and N-methyl-N-(3-phenylpropyl)prop-2-yn-1-amine (15 mg, 0.08 mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford 20 mg (92%)

1 of GHB-7 analog 113c. H NMR (CDCl3, 300 MHz) 7.48 (s, 1H), 7.32-7.14 (m, 11H),

4.42-4.34 (m, 1H), 4.23-4.04 (m, 3H), 3.68 (s, 2H), 3.54-3.47 (m, 1H), 2.97 (t, J = 7.7,

2H), 2.69 (t, J = 7.6, 2H), 2.61 (t, J = 7.5, 2H), 2.44 (t, J = 7.3, 2H), 2.25 (s, 3H), 1.89-

13 1.78 (m, 2H), 1.25 (bs, 1H). C NMR (CDCl3, 75 MHz) 171.2, 142.8, 140.8, 138.5,

127.0, 126.8, 126.7, 126.6, 124.8, 124.2, 122.6, 66.9, 63.8, 54.7, 51.1, 50.5, 40.2, 33.9,

31.8, 29.6, 27.0. HPLC (1% AcOH in CH3OH : H2O) RT 2.22 (100%). MS (APCI) :

M+H expected 436.55, obtained 436.95. 177

O N N O N H N OH 114a N

2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl

(R)-1-phenylethylcarbamate 114d. 3-Azido-2-hydroxypropyl (R)-1- phenylethylcarbamate (13 mg, 0.05 mmol) and N-methyl-N-(3-phenylpropyl)prop-2-yn-

1-amine (12 mg, 0.06 mmol) were reacted following the general method for 1,2,3-triazole

1 synthesis to afford of 20 mg (93%) of GHB-7 analog 114d. H NMR (CDCl3, 300 MHz)

7.58 (s, 1H), 7.37-7.21 (m, 8H), 7.20-7.13 (t, J = 7.7, 3H), 5.48 (d, J = 7.4, 1H), 4.80 (t,

J = 7.0, 1H), 4.52-4.01 (m, 6H), 3.67 (s, 2H), 2.60 (t, J = 7.6, 2H), 2.44 (t, J = 7.5, 2H),

13 2.23 (s, 3H), 1.88-1.78 (m, 2H), 1.48 (d, J = 6.8, 3H). C NMR (CDCl3, 75 MHz)

153.2, 141.6, 140.7, 139.4, 126.3, 126.0, 125.9, 125.0, 123.5, 123.4, 122.9, 122.1, 66.4,

63.7, 53.8, 50.5, 49.6, 48.5, 39.3, 31.0, 27.3, 26.0, 19.9. HPLC (1% AcOH in CH3OH :

H2O) RT 2.25 (99%). MS (APCI) : M+H expected 451.56, obtained 451.95.

O N N O N H N OH 114b N

2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl

4-methylbenzylcarbamate 114b. 3-Azido-2-hydroxypropyl 4-methylbenzylcarbamate

(13 mg, 0.05 mmol) and N-methyl-N-(3-phenylpropyl)prop-2-yn-1-amine (12 mg, 0.06 mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford 20

1 mg (95%) of GHB-7 analog 114b. H NMR (CDCl3, 300 MHz) 7.55 (s, 1H), 7.29-7.24 178

(m, 3H), 7.19-7.11 (m, 7H), 5.35 (t, J = 5.3, 1H), 4.48 (dd, J = 2.4, 13.3, 1H), 4.37-4.06

(m, 6H), 3.65 (s, 1H), 3.58-3.34 (bs, 1H), 2.61 (t, J = 7.5, 2H), 2.42 (t, J = 7.2, 2H), 2.33

13 (s, 3H), 2.23 (s, 3H), 1.87-1.77 (m, 2H). C NMR (CDCl3, 75 MHz) 156.3, 144.4,

141.8, 137.2, 129.2, 128.2, 128.1, 127.3, 125.5, 123.9, 68.9, 66.1, 56.1, 52.6, 52.0, 44.8,

41.7, 32.2, 28.5, 20.8. HPLC (1% AcOH in CH3OH : H2O) RT 2.22 (93%). MS (APCI) :

M+H expected 451.56 obtained 451.95.

O N N O N H N OH O 114c N

2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl

4-methoxybenzylcarbamate 114c. 3-Azido-2-hydroxypropyl 4- methoxybenzylcarbamate (13 mg, 0.05 mmol) and N-methyl-N-(3-phenylpropyl)prop-2- yn-1-amine (12 mg, 0.09 mmol) were reacted following the general method for 1,2,3-

1 triazole synthesis to afford 22 mg (95%) of GHB-7 analog 114c. H NMR (CDCl3, 300

MHz) 7.57 (s, 1H), 7.29-7.14 (m, 7H), 6.84 (d, J = 8.6, 7H), 5.51 (t, J = 5.6, 1H), 4.66-

4.54 (bs, 1H), 4.47 (dd, J = 3.2, 13.7, 1H), 4.34-4.05 (m, 6H), 3.77 (s, 3H), 3.64 (s, 2H),

2.59 (t, J = 7.6, 2H), 2.42 (t, J = 7.3, 2H), 2.21 (s, 3H), 1.86-1.77 (m, 2H). 13C NMR

(CDCl3, 75 MHz) 159.1, 156.5, 144.3, 141.9, 130.1, 128.9, 128.4, 128.3, 125.8, 124.4,

114.1, 68.9, 66.2, 56.3, 55.3, 52.9, 52.1, 44.7, 41.8, 33.5, 29.7, 28.6. HPLC (1% AcOH in

CH3OH : H2O) RT 2.20 (99%). MS (APCI) : M+H expected 467.56, obtained 467.95. 179

O O N N O N H N OH 114d N

2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl furan-2-ylmethylcarbamate 114d. 3-Azido-2-hydroxypropyl furan-2- ylmethylcarbamate (13 mg, 0.06 mmol) and N-methyl-N-(3-phenylpropyl)prop-2-yn-1- amine (13 mg, 0.07 mmol) were reacted following the general method for 1,2,3-triazole

1 synthesis to afford 22 mg (93%) of GHB-7 analog, 114d. H NMR (CDCl3, 300 MHz)

7.57 (s, 1H), 7.33-7.32 (bs, 1H), 7.26 (t, J = 6.7, 2H), 7.16 (t, J = 5.8, 3H), 6.29 (t, J =

2.6, 1H), 6.21 (d, J = 3.2, 1H), 5.70 (t, J = 5.6, 1H), 4.48 (dd, J = 3.4, 13.8, 1H), 4.35-

4.06 (m, 6H), 3.64 (s, 2H), 2.59 (t, J = 7.6, 2H), 2.42 (t, J = 6.7, 2H), 2.21 (s, 3H), 1.88-

13 1.76 (m, 2H). C NMR (CDCl3, 75 MHz) 154.9, 149.9, 142.9, 140.9, 140.6, 127.0,

126.9, 124.4, 123.0, 109.1, 106.1, 67.4, 64.9, 54.9, 51.5, 50.8, 40.4, 36.7, 32.1, 27.2.

HPLC (1% AcOH in CH3OH : H2O) RT 2.22 (99%). MS (APCI) : M+H expected 427.50, obtained 427.90.

O N N O N H N 115a N

3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propyl benzylcarbamate 115a. 3-Azidopropyl benzylcarbamate (13 mg, 0.06mmol) and N- methyl-N-(3-phenylpropyl)prop-2-yn-1-amine (12 mg, 0.07 mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford 22 mg (97%) of GHB- 180

1 7 analog 115a. H NMR (CDCl3, 300 MHz) 7.40 (s, 1H), 7.37-7.24 (m, 7H), 7.20-7.14

(m, 3H), 5.13 (bs, 1H), 4.44-4.35 (m, 4H), 4.12 (t, J = 5.9, 6H), 3.67 (s, 2H), 2.62 (t, J =

13 7.6, 2H), 2.42 (t, J = 7.2, 2H), 2.28-2.18 (m, 5H), 1.88-1.78 (m, 2H). C NMR (CDCl3,

75 MHz) 155.2, 144.2, 141.2, 137.3, 127.7, 127.4, 127.3, 126.6, 126.5, 124.6, 121.7,

60.5, 55.4, 51.4, 46.1, 44.1, 41.1, 32.5, 28.9, 28.0. HPLC (1% AcOH in CH3OH : H2O)

RT 2.21 (98%). MS (APCI) : M+H expected 421.54 obtained 422.00.

O N N O N H N OH 116a

2-hydroxy-3-(4-phenyl-1H-1,2,3-triazol-1-yl)propyl benzylcarbamate 116a. 3-Azido-

2-hydroxypropyl benzylcarbamate (13 mg, 0.05 mmol) and phenylacetylene (8 mg, 0.08

mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford 19

1 mg (98%) of GHB-7 analog 116a. H NMR (CDCl3, 300 MHz) 7.89 (s, 1H), 7.78 (d, J

= 7.1, 3H), 7.44-7.11 (m, 8H), 5.26-5.16 (bs, 1H), 4.59 (dd, J = 2.7, 13.5, 1H), 4.45-4.26

13 (m, 5H), 4.17 (dd, J = 5.1, 11.7, 1H), 3.81 (d, J = 4.0, 1H). C NMR (CDCl3, 75 MHz)

147.8, 130.4, 128.9, 128.8 128.2, 127.2, 127.8, 127.6, 125.7, 121.2, 69.4, 66.6, 56.6, 53.0,

45.3. HPLC (CH3OH : H2O) RT 16.67 (91%). MS (APCI) : M+H expected 352.39 obtained 352.80. 181

O N N O N H N OH 116b

3-(4-butyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropyl benzylcarbamate 116b. 3-Azido-2-

hydroxypropyl benzylcarbamate (25 mg, 0.10 mmol) and hexyne (12 mg, 0.15 mmol)

were reacted following the general method for 1,2,3-triazole synthesis to afford 32 mg

1 (97%) of GHB-7 analog 116b. H NMR (CDCl3, 300 MHz) 7.40 (s, 1H), 7.36-7.24 (m,

5H), 5.70 (t, J = 5.3, 1H), 4.49-4.10 (m, 8H), 2.62 (t, J = 7.6, 2H), 1.65-1.54 (m, 2H),

13 1.41-1.31 (m, 2H), 0.91 (t, J = 7.3, 3H). C NMR (CDCl3, 75 MHz) 156.7, 148.3,

138.1, 128.7, 127.6, 127.5, 122.2, 69.1, 66.3, 52.8, 45.2, 31.5, 25.2, 22.3, 13.8. HPLC

(CH3OH : H2O) RT 17.32 (87%). MS (APCI) : M+H expected 332.40, obtained 332.90.

O N N O N H N OH 116c N

3-(4-((benzyl(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)-2-hydroxypropyl benzylcarbamate 116c. 3-Azido-2-hydroxypropyl benzylcarbamate (13 mg, 0.05 mmol) and N-benzyl-N-methylprop-2-yn-1-amine (12 mg, 0.08mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford 20 mg (95%) of GHB-7 analog

1 116c. H NMR (CDCl3, 300 MHz) 7.62 (s, 1H), 7.37-7.24 (m, 10H), 5.48 (t, J = 5.5,

1H), 4.50 (dd, J = 2.8, 13.5, 1H), 4.38-4.30 (m, 3H), 4.28-4.15 (m, 2H), 4.12 (dd, J = 5.2,

13 11.6, 1H), 3.66 (s, 2H), 3.53 (s, 2H), 2.20 (s, 3H), 1.25 (bs, 1H). C NMR (CDCl3, 75 182

MHz) 156.6, 144.9, 138.1, 129.2, 128.7, 128.3, 127.6, 127.5, 127.2, 124.3, 69, 66.3,

61.3, 52.9, 51.8, 45.2, 41.9. HPLC (CH3OH : H2O) RT 15.75 (90%).

O N N O N H N OH 116d O

2-hydroxy-3-(4-((3-phenylpropoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl benzylcarbamate 116d. 3-Azido-2-hydroxypropyl benzylcarbamate (13 mg, 0.05 mmol) and alkyne (3-(prop-2-ynyloxy)propyl)benzene 138d (14 mg, 0.08mmol) were reacted

following the general method for 1,2,3-triazole synthesis to afford 20 mg (95%) of GHB-

1 7 analog 116d. H NMR (CDCl3, 300 MHz) 7.64 (s, 1H), 7.30-7.12 (m, 10H), 5.87 (t, J

= 5.8, 1H), 4.57-4.03 (m, 10H), 3.45 (t, J = 6.5, 2H), 2.63 (t, J = 7.4, 2H), 1.90-1.81 (m,

13 2H). C NMR (CDCl3, 75 MHz) 156.6, 145.2, 141.8, 138.0, 128.7, 128.4, 128.3, 127.7,

127.5, 125.8, 124.0, 69.9, 69.1, 66.3, 64.2 52.9, 45.2, 32.2, 31.1. HPLC (1% AcOH in

CH3OH : H2O) RT 15.26 (99%). MS (APCI) : M+H expected 424.49, obtained 424.90.

O N N O N H N OH 115b N

(R)-2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-

yl)propyl benzylcarbamate 115b. (R)-3-azido-2-hydroxypropyl benzylcarbamate (13

mg, 0.05 mmol) and N-methyl-N-(3-phenylpropyl)prop-2-yn-1-amine (12 mg, 0.08

mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford 20 183

1 mg (91%) of GHB-7 analog 115b. H NMR (CDCl3, 300 MHz) 7.55 (s, 1H), 7.30-7.22

(m, 7H), 7.16 (t, J = 7.6, 3H), 5.69 (t, J = 5.4, 1H), 4.45 (dd, J = 3.0, 13.7, 1H), 4.35-4.25

(m, 3H), 4.23-4.13 (m, 2H), 4.12-4.04 (m, 2H), 3.60 (s, 2H), 3.59 (t, J = 7.6, 2H), 2.39 (t,

13 J = 7.2, 3H), 2.18 (s, 3H), 1.85-1.73 (m, 2H). C NMR (CDCl3, 75 MHz) 156.6, 144.6,

142.0, 138.2, 128.7, 128.4, 128.3, 127.6, 127.5, 125.8, 124.3, 68.8, 66.2, 56.1, 52.9 52.2,

45.2, 41.9, 33.5, 28.7. HPLC (1% AcOH in CH3OH : H2O) RT 2.23 (100%). MS (APCI) :

M+H expected 437.53 obtained 437.90.

O N N O N H N OH 115c N

(S)-2-hydroxy-3-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1- yl)propyl benzylcarbamate 115c. (S)-3-azido-2-hydroxypropyl benzylcarbamate (13 mg, 0.05 mmol) and the alkyne N-methyl-N-(3-phenylpropyl)prop-2-yn-1-amine (12 mg,

0.08 mmol) were reacted following the general method for 1,2,3-triazole synthesis to

1 afford 20 mg (90%) of GHB-7 analog 115c. H NMR (CDCl3, 300 MHz) 7.55 (s, 1H),

7.30-7.22 (m, 7H), 7.16 (t, J = 7.6, 3H), 5.69 (t, J = 5.4, 1H), 4.45 (dd, J = 3.0, 13.7, 1H),

4.35-4.25 (m, 3H), 4.23-4.13 (m, 2H), 4.12-4.04 (m, 2H), 3.60 (s, 2H), 3.59 (t, J = 7.6,

13 2H), 2.39 (t, J = 7.2, 3H), 2.18 (s, 3H), 1.85-1.73 (m, 2H). C NMR (CDCl3, 75 MHz)

156.6, 144.6, 142.0, 138.2, 128.7, 128.4, 128.3, 127.6, 127.5, 125.8, 124.3, 68.8, 66.2,

56.1, 52.9 52.2, 45.2, 41.9, 33.5, 28.7. HPLC (1% AcOH in CH3OH : H2O) RT 2.29

(100%). MS (APCI) : M+H expected 437.53, obtained 437.90. 184

N3 OH 143

1-azidobutan-2-ol 143. To a mixture of butene oxide (5.0 g, 69.3 mmol), NH4Cl (7.42 g,

138.7 mmol) in methanol and water mixture (8:1 = 270 mL) was added NaN3 (36.0 g,

554.4 mmol). The reaction mixture was degassed and stirred at room temperature for 24

h. The reaction mixture was then concentrated to 1/10 its volume, diluted with water (100

mL) and extracted with EtOAc (60 mL, 3x). The combined organic layer was washed

with brine (60 mL, 2x), dried over MgSO4, filtered, concentrated and distilled (69-72 °C)

1 to provide 6.63 g (83%) of 1-azidobutan-2-ol 143 as a pale yellow oil. H NMR (CDCl3,

300 MHz) 3.74-3.65 (m, 1H), 3.38 (dd, J = 3.6, 12.5, 1H), 3.26 (dd, J = 7.4, 12.5, 1H),

13 2.25 (s, 1H), 1.58-1.48 (m, 2H), 0.98 (t, J = 7.4, 3H). C NMR (CDCl3, 75 MHz) 70.3,

-1 54.8, 25.4, 7.9. IR (CDCl3) 2098 cm .

N3 145

1-azidohexane 145. To a solution of 1-bromohexane (3 g, 18.2 mmol) in DMF (30 mL),

was added NaN3 (11.8 g, 182 mmol). The reaction mixture was stirred under an argon

atmosphere for 12 h. The reaction mixture was then diluted with hexane (70 mL), washed

with water (50 mL, 6x), brine (50 mL, 2x), dried over MgSO4, filtered, concentrated and distilled (71-74 °C) to provide 1.7g (80%) of 1-azidohexane 145 as a colorless oil. 1H

NMR (CDCl3, 300 MHz) 3.25 (t, J = 6.9, 2H), 1.64-1.54 (m, 2H), 1.42-1.25 (m, 1H), 185

13 0.90 (t, J = 6.8, 3H). C NMR (CDCl3, 75 MHz) 51.6, 31.4, 28.9, 26.5, 22.6, 14.0. IR

-1 (CDCl3) 2096cm .

147a

N-methyl-4-phenyl-N-(prop-2-ynyl)butan-1-amine 147a. N-methyl propargylamine

(130 mg, 1.8 mmol) was dissolved in THF (5 mL). To this reaction mixture was added

potassium carbonate (220 mg, 1.9 mmol) and 1-chloro-5-phenylpentane (220 g, 1.3

mmol). The reaction mixture was refluxed for 24 h under an atmosphere of argon. The

reaction mixture was then filtered, concentrated and chromatographed to provide 180 g

(67%) of N-methyl-4-phenyl-N-(prop-2-ynyl)butan-1-amine 147a as a orange-yellow oil.

1 H NMR (CDCl3, 300 MHz) 7.20-7.05 (m, 5H), 3.23 (d, J = 2.4, 2H), 2.54 (t, J = 7.4,

2H), 2.34 (t, J = 7.4, 2H), 2.20 (s, 3H), 2.11 (t, J = 2.4, 1H), 1.61-1.51 (m, 2H), 1.46-1.35

13 (m, 2H). C NMR (CDCl3, 75 MHz) 141.6, 127.6, 127.4, 124.8, 77.9, 72.1, 54.7, 44.7,

40.9, 34.9, 28.4, 26.4.

147b

N-methyl-5-phenyl-N-(prop-2-ynyl)pentan-1-amine 147b. To a solution of N-methyl

propargylamine (130 mg, 1.9 mmol) in THF (5 mL) was added potassium carbonate (380

mg, 3.4 mmol) followed by 1-chloro-5-phenylpentane (250 mg, 1.4 mmol). The reaction

mixture was refluxed for 24 h under an argon atmosphere then filtered, concentrated and

chromatographed to provide 210 mg (60%) of N-methyl-5-phenyl-N-(prop-2- 186

1 ynyl)pentan-1-amine 147b as a orange-yellow oil. H NMR (CDCl3, 300 MHz) 7.22-

7.07 (m, 5H), 3.26 (d, J = 2.4, 2H), 2.56 (t, J = 7.6, 2H), 2.32 (t, J = 7.2, 2H), 2.22 (s,

3H), 2.12 (t, J = 2.4, 1H), 1.62-1.51 (m, 2H), 1.47-1.36 (m, 2H), 1.34-1.25 (m, 2H). 13C

NMR (CDCl3, 75 MHz) 141.6, 127.3, 127.1, 124.5, 77.6, 71.8, 54.5, 44.4, 40.7, 34.8,

30.3, 26.4, 25.9.

N N N OH 139a N

1-(4-((methyl(4-phenylbutyl)amino)methyl)-1H-1,2,3-triazol-1-yl)hexan-2-ol 139a.

1-Azidohexan-2-ol (12 mg, 0.09 mmol) and N-methyl-4-phenyl-N-(prop-2-ynyl)butan-1-

amine (20 mg, 0.10 mmol) were reacted following the general method for 1,2,3-triazole

synthesis to afford 26 mg (92%) of GHB-9 analog 139a as a reddish brown oil.1H NMR

(CDCl3, 300 MHz) 7.58 (s, 2H), 7.26-7.24 (m, 2H), 7.17-7.14 (m, 3H), 4.43 (dd, J =

2.8, 13.9, 1H), 4.22 (dd, J = 7.8, 13.8, 1H), 4.06-3.99 (m, 1H), 3.69 (s, 2H), 2.93 (bs,

1H), 2.61 (t, J = 7.0, 2H), 2.43 (t, J = 7.4, 2H), 2.25 (s, 3H), 1.63-1.32 (m, 10H), 0.91 (t, J

13 = 6.9, 3H). C NMR (CDCl3, 75 MHz) 140.9, 126.9, 126.7, 124.2, 122.6, 68.9, 55.3,

54.4, 50.8, 40.5, 34.2, 32.6, 28.2, 27.6, 25.9, 25.2, 20.9, 12.4. HPLC (1% AcOH in

CH3OH : H2O) RT 10.97 (88%). MS (APCI) : M+H expected 344.49, obtained 344.95. 187

N N N OH 139b N

1-(4-((methyl(5-phenylpentyl)amino)methyl)-1H-1,2,3-triazol-1-yl)hexan-2-ol 139b.

1-Azidohexan-2-ol (100 mg, 0.07 mmol) and N-methyl-5-phenyl-N-(prop-2-ynyl)pentan-

1-amine (20 mg, 0.08 mmol) were reacted following the general method for 1,2,3-triazole

synthesis to afford 22 mg (91%) of GHB-9 analog, 139b as a reddish brown oil. 1H NMR

(CDCl3, 300 MHz) 7.59 (s, 2H), 7.27-7.24 (m, 2H), 7.18-7.14 (m, 3H), 4.43 (dd, J =

2.7, 13.8, 1H), 4.22 (dd, J = 7.9, 13.8, 1H), 4.06-3.99 (m, 1H), 3.68 (s, 2H), 3.14 (bs,

1H), 2.60 (t, J = 7.0, 2H), 2.40 (t, J = 7.4, 2H), 2.25 (s, 3H), 1.65-1.28 (m, 12H), 0.91 (t, J

13 = 6.9, 3H). C NMR (CDCl3, 75 MHz) 140.3, 126.1, 125.9, 123.3, 121.8 68.2, 54.7,

53.7, 50.0, 39.7, 35.6, 31.9, 29.0, 27.4, 25.2, 24.8, 24.6, 20.2, 11.7. HPLC (1% AcOH in

CH3OH : H2O) RT 11.04 (87%). MS (APCI) : M+H expected 358.52, obtained 359.00.

N N N OH 139d O

1-(4-((3-phenylpropoxy)methyl)-1H-1,2,3-triazol-1-yl)hexan-2-ol 139d. 1-azidohexan-

2-ol 76b (13 mg, 0.09 mmol) and (3-(prop-2-ynyloxy)propyl)benzene (18 mg, 0.11

mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford

1 26 mg (94%) of GHB-9 analog 139d. H NMR (CDCl3, 300 MHz) 7.63 (s, 1H), 7.29-

7.24 (m, 2H), 7.20-7.14 (m, 3H), 4.58 (s, 2H), 4.43 (dd, J = 2.8, 13.8, 1H), 4.20 (dd, J =

7.9, 13.8, 1H), 4.06-4.01 (m, 1H), 3.52 (t, J = 6.5, 2H), 3.09 (d, J = 4.7, 1H), 2.67 (t, J =

13 7.4, 2H), 1.97-1.86 (m, 2H), 1.53-1.30 (m, 6H), 0.91 (t, J = 6.8, 3H). C NMR (CDCl3, 188

75 MHz) 144.4, 141.2, 127.9, 127.7, 125.2, 125.2, 123.3, 69.8, 69.3, 63.7, 55.5, 33.5,

31.7, 30.6, 26.9, 21.9, 13.4. HPLC (CH3OH : H2O) RT 15.79 (94%). MS (APCI) : M+H

expected 317.43 obtained 317.90.

N N N 140 N

N-((1-hexyl-1H-1,2,3-triazol-4-yl)methyl)-N-methyl-3-phenylpropan-1-amine 140. 1-

Azidohexane 145 (23 mg, 0.20 mmol) and N-methyl-N-(3-phenylpropyl)prop-2-yn-1-

amine 82c (25 mg, 0.14 mmol) were reacted following the general method for 1,2,3-

1 triazole synthesis to afford 38 mg (94%) 0f GHB-9 analog 140. H NMR (CDCl3, 300

MHz) 7.36 (s, 1H), 7.30-7.25 (m, 2H), 7.19-7.17 (m, 3H), 4.31 (t, J = 7.3, 2H), 3.69 (s,

2H), 2.62 (t, J = 7.6, 2H), 2.43 (t, J = 7.2, 2H), 2.25 (s, 3H), 1.89-179 (m, 4H), 1.33-1.25

13 (m, 6H), 0.88 (t, J = 6.7, 3H). C NMR (CDCl3, 75 MHz) 143.9, 141.1, 127.2, 127.1,

124.6, 120.9, 55.2, 51.3, 49.1, 40.9, 32.4, 29.9, 29.1, 27.9, 24.9, 21.3, 12.8. HPLC (1%

AcOH in CH3OH : H2O) RT 15.78 (89%). MS (APCI) : M+H expected 314.47, obtained

314.95.

N N N OH 141 N

1-(4-((methyl(3-phenylpropyl)amino)methyl)-1H-1,2,3-triazol-1-yl)butan-2-ol 141. 1-

azidobutan-2-ol 143 (23 mg, 0.20 mmol) and N-methyl-N-(3-phenylpropyl)prop-2-yn-1-

amine 82c (25 mg, 0.14 mmol) were reacted following the general method for 1,2,3- 189

1 triazole synthesis to afford 37 mg (93%) of GHB-9 analog 141. H NMR (CDCl3, 300

MHz) 7.52 (s, 1H), 7.28-7.25 (m, 2H), 7.20-7.16 (m, 3H), 4.44 (dd, J = 2.8, 13.9, 1H),

4.21 (dd, J = 7.9, 13.9, 1H), 3.99-3.93 (m, 1H), 3.67 (s, 2H), 3.04 (bs, 1H), 2.62 (t, J =

7.6, 2H), 2.43 (t, J = 7.3, 2H), 2.24 (s, 3H), 1.89-1.78 (m, 2H), 1.61-1.42 (m, 2H), 1.02 (t,

13 J = 7.4, 3H). C NMR (CDCl3, 75 MHz) 143.6, 141.1, 127.5, 127.4, 124.8, 123.1, 70.8,

55.4, 51.4, 41.1, 32.5, 27.9, 26.5, 8.9. HPLC (1% AcOH in CH3OH : H2O) RT 11.16

(87%). MS (APCI) : M+H expected 302.41, obtained 302.95.

N N N OH 139c N

1-(4-((benzyl(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)hexan-2-ol 139c. 1-

Azidohexan-2-ol 76b (13 mg, 0.09 mmol) and N-benzyl-N-methylprop-2-yn-1-amine

148c (17 mg, 0.11 mmol) were reacted following the general method for 1,2,3-triazole

1 synthesis to afford 25 mg (95%) of GHB-9 analog 139c. H NMR (CDCl3, 300 MHz)

7.60 (s, 1H), 7.31-7.22 (m, 5H), 4.43 (dd, J = 2.8, 13.8, 1H), 4.21 (dd, J = 7.9, 13.8, 1H),

4.06-4.02 (m, 1H), 3.71 (s, 2H), 3.53 (s, 2H), 3.30 (bs, 1H), 2.21 (s, 3H), 1.48-1.31 (m,

13 6H), 0.90 (t, J = 6.8, 3H). C NMR (CDCl3, 75 MHz) 144.2, 137.7, 128.3, 127.5,

126.3, 123.3, 69.6, 60.6, 55.3, 51.3, 41.4, 33.4, 26.8, 21.8, 13.2. HPLC (1% AcOH in

CH3OH : H2O) RT 11.04 (90%). MS (APCI) : M+H expected 302.41 obtained 302.95. 190

O 152

3-methyl-1-(prop-2-ynyloxy)butane 152. To a solution of propargyl alcohol 135 (2 g,

35 mmol) in acetone (20 mL) was added K2CO3 (9.9 g, 70 mmol). The reaction mixture

was stirred for 5 minutes under an argon atmosphere and 1-bromo-3-methylbutane 151

(6.5 g, 43 mmol) was added. The reaction mixture was refluxed for 24 h then the reaction

mixture was filtered and concentrated. The residue was distilled to afford 2.96 g (70%) of

1 3-methyl-1-(prop-2-ynyloxy)butane 152 as pale yellow oil. H NMR (CDCl3, 300 MHz)

4.15 (d, J = 2.4, 2H), 3.53 (t, J = 6.7, 2H), 2.41 (t, J = 2.3, 1H), 1.76-1.65 (m, 1H), 0.91

13 (d, J = 6.7, 6H). C NMR (CDCl3, 75 MHz) 79.9, 73.9, 68.5, 57.9, 38.2, 24.9, 22.5.

N N N OH 148 O

1-(4-(isopentyloxymethyl)-1H-1,2,3-triazol-1-yl)hexan-2-ol 148. 1-azidohexan-2-ol (17 mg, 0.12 mmol) and 3-methyl-1-(prop-2-ynyloxy)butane (13 mg, 0.10 mmol) were reacted following the general method for 1,2,3-triazole synthesis to afford 25 mg (93%)

1 of GHB-16 analog, 148. H NMR (CDCl3, 300 MHz) 7.66 (s, 1H), 4.57 (s, 2H), 4.44

(dd, J = 2.9, 13.8, 1H), 4.22 (dd, J = 7.9, 13.8, 1H), 4.06-4.03 (m, 1H), 3.53 (t, J = 6.8,

13 2H), 1.71-1.64 (m, 1H), 1.52-1.26 (m, 8H), 0.97-0.87 (m, 9H). C NMR (CDCl3, 75

MHz) 142.7, 67.9, 66.9, 61.8, 53.7, 36.0, 31.7, 25.2, 22.7, 20.2, 20.1, 11.6. HPLC (1%

AcOH in CH3OH : H2O) RT 10.28 (96%). MS (APCI) : M+H expected 269.38 obtained

269.95. 191

N N N 149 N

N-((1-hexyl-1H-1,2,3-triazol-4-yl)methyl)-N,3-dimethylbutan-1-amine 149. 1-

azidohexane (17 mg, 0.14 mmol) and N,3-dimethyl-N-(prop-2-ynyl)butan-1-amine (15

mg, 0.11 mmol) were reacted following the general method for 1,2,3-triazole synthesis to

1 afford 28 mg (97%) of GHB-16 analog 149. H NMR (CDCl3, 300 MHz) 7.47 (s, 1H),

4.34 (t, J = 7.2, 2H), 3.68 (s, 2H), 2.39 (t, J = 7.6, 2H), 2.24 (s, 3H), 1.87 (t, J = 7.0, 2H),

13 1.62-1.54 (m, 1H), 1.43-1.25 (m, 8H), 0.89-0.82 (m, 9H). C NMR (CDCl3, 75 MHz)

143.6, 120.6, 53.8, 51.1, 48.7, 40.6, 34.8, 29.6, 28.7, 24.8, 24.6, 21.2, 20.8, 12.4. HPLC

(1% AcOH in CH3OH : H2O) RT 2.21 (97%). MS (APCI) : M+H expected 266.43,

obtained 269.95

N N N OH

150 N

1-(4-((isopentyl(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)butan-2-ol 150. 1-

Azidobutan-2-ol (16 mg, 0.14 mmol) and the alkyne N,3-dimethyl-N-(prop-2-ynyl)butan-

1-amine (13 mg, 0.09 mmol) were reacted following the general method for 1,2,3-triazole

1 synthesis to afford 21 mg, (91%) of GHB-16 analog 150. H NMR (CDCl3, 300 MHz)

7.62 (s, 1H), 4.46 (dd, J = 2.8, 13.9, 1H), 4.24 (dd, J = 7.9, 13.9, 1H), 4.02-3.94 (m, 1H),

3.69 (s, 2H), 2.97 (bs, 1H), 2.42 (t, J = 8.0, 2H), 2.24 (s, 3H), 1.62-1.48 (m, 3H), 1.44-

13 1.37 (m, 2H), 1.03 (t, J = 7.4, 3H), 0.88 (d, J = 6.6, 6H). C NMR (CDCl3, 75 MHz) 192

143.4, 122.8, 70.6, 54.3, 54.1, 51.2, 40.8, 34.9, 28.5, 26.2, 25.2, 21.5, 8.6. HPLC (1%

AcOH in CH3OH : H2O) RT 2.48 (92%). MS (APCI) : M+H expected 254.37 obtained

254.90

O ( ± )-161

Trans-2-azidocyclohexyl butyrate 161. To a solution of thionyl chloride (11 g, 125

mmol) was added catalytic amount of DMF (2.4 mL). The mixture was heated gently to

50 °C and butyryl acid (18 g, 150 mmol) was added slowly over a period of 30 minutes.

The reaction mixture was then refluxed for 2 h and concentrated to afford the crude

butyryl chloride 160. Compound 160 was used in the next step of the synthesis without

further purification. To a solution of trans-2-azidocyclohexanol 76d (10.0 g, 70 mmol), in

CH2Cl2 (120 mL) at 0 °C was added Et3N (35.8 g, 354 mmol) followed by DMAP (0.86 g, 7.08 mmol). To this stirring reaction mixture was added butyryl chloride (11 g, 106.3 mmol) then the reaction mixture was warmed to r.t and stirred for 12 h under an atmosphere of argon. The reaction mixture was poured through silica gel pad and washed

with CH2Cl2 (300 mL). The filtrate was transferred into a separatory funnel and washed with HCl (50 mL, 3x), saturated NaHCO3 (50 mL, 3x), H2O (100 mL, 2x), brine (50 mL,

2x), dried over MgSO4, filtered, concentrated and chromatographed (5% EtOAc in hexanes) to provide 8.0 g (82%) of trans-2-azidocyclohexyl butyrate 161 as a yellow oil.

1 H NMR (CDCl3, 300 MHz) 4.60-4.52 (m, 1H), 3.29-3.21 (m, 1H), 2.19 (t, J = 7.3,

2H), 1.94-1.89 (m, 2H), 1.63-1.49 (m, 4H), 1.26-1.13 (m, 4H), 0.84 (t, J = 7.3, 3H). 13C 193

NMR (CDCl3, 75 MHz) 172.9, 75.2, 63.2, 36.3, 30.6, 23.8, 23.5, 18.4, 13.5. IR (CDCl3)

1728cm-1, 2092cm-1.

OH 162

(1R,2R)-2-azidocyclohexanol 162. To a mixture of Amano lipase PS (500 mg) in

sodium phosphate buffer (150 mL, PH 7.2) was added the racemic trans-2-

azidocyclohexyl butyrate (5.0 g, 23.7 mmol). The reaction mixture was stirred for 3 h

(until 40% of starting material was consumed). Drops of 1 M NaOH were used to offset

the changes in PH of the reaction mixture during the 3 h span. The reaction mixture was

then filtered and the filtrate extracted with CH2Cl2 (60 mL, 5x). The combined organic layer was dried over MgSO4, filtered, concentrated and chromatographed (15% EtOAc in hexane) to afford 1.2 g (37%) of (1R,2R)-2-azidocyclohexanol 162 as pale yellow oil. 1H

NMR (CDCl3, 300 MHz) 3.45-3.33 (m, 1H), 3.23-3.13 (m, 1H), 2.66 (s, 1H), 2.11-1.96

13 (m, 2H), 1.81-1.67 (m, 2H), 1.41-1.19 (m, 4H). C NMR (CDCl3, 75 MHz) 72.4, 65.9,

-1 25 31.9, 28.6, 23.1, 22.7. IR (CDCl3) 2092 cm . []D = -66.8 ° (c 1.53, CH2Cl2), 99% ee using compound 167. 194

OH 165

(1S,2S)-2-azidocyclohexanol 165. To a mixture of Amano lipase PS (0.50g) in sodium phosphate buffer (150 mL, PH 7.2) was added the optically enriched (1S,2S)-2- azidocyclohexyl butyrate (2.8 g, 13.3 mmol). The reaction mixture was stirred for 5 h

(until starting material was completely consumed). Drops of 1M NaOH were used to offset the changes in PH of the reaction mixture. The reaction mixture was then filtered

and the filtrate extracted with CH2Cl2 (60 mL, 5x). The combined organic layer was dried

over MgSO4, filtered, concentrated to give compound 164, which without further purification was used in the step of the synthesis. Compound 164 (2.3 g, 10.9 mmol) was treated with catalytic amount of NaOMe in MeOH (25 mL). The reaction mixture was

concentrated and the residue diluted with CH2Cl2 (100 mL), washed with 1M HCl (30

mL, 2x), H2O (30 mL, 3x), dried over MgSO4, filtered, concentrated and chromatographed (25% EtOAc in hexanes) to afford 0.95 g (31%) of 165 as a pale yellow

1 oil. H NMR (CDCl3, 300 MHz) 3.42-3.34 (m, 1H), 3.22-3.14 (m, 1H), 2.32 (s, 1H),

13 2.09-2.00 (m, 2H), 1.78-1.73 (m, 2H), 1.37-1.23 (m, 4H). C NMR (CDCl3, 75 MHz)

-1 25 73.6, 67.2, 33.1, 29.8, 24.3, 23.9. IR (CDCl3) 2096 cm . []D = +66.4 ° (c 1.70,

CH2Cl2), 96% ee using compound 168.

General procedure for preparing Mosher’s salts 167-169. To a solution of the

requisite trans-2-azidocyclohexanol (1 equiv) in CH2Cl2 (0.55 M) at 0 °C was added Et3N

(3.0 equiv) and DMAP (0.12 equiv) consecutively. The reaction mixture was stirred for 195

30 minutes and (S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl chloride 166 (1.2

equiv) was added. The reaction mixture was warmed to r.t and stirring continued at r.t for

4 h under an argon atmosphere. The reaction mixture was then washed with 1M HCl (0.5

mL, 3x), NaHCO3 (0.5 mL, 3x), H2O (1 mL, 2x), dried over MgSO4, filtered,

concentrated and chromatographed (15% EtOAc in hexanes) to provide the desired

mosher’s salt.

N 3O CF O 3 OMe 167 Ph

(R)-((1R,2R)-2-azidocyclohexyl) 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate 167.

Compound 162 (39 mg, 0.27 mmol) was acylated with (S)-3,3,3-trifluoro-2-methoxy-2-

phenylpropanoyl chloride 166 (83 mg, 0.33 mmol) following the general method for

preparing mosher’s salt to provide 93 mg (96%) of (R)-((1R,2R)-2-azidocyclohexyl)

1 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate 167 as a yellow oil. H NMR (CDCl3,

300 MHz) 7.58-7.55 (m, 2H), 7.42-7.37 (m, 3H), 4.86-4.79 (m, 1H), 3.60 (s, 3H), 3.43-

3.35 (m, 1H), 2.20-2.04 (m, 2H), 1.80-1.71 (m, 2H), 1.53-1.23 (m, 4H). 19F NMR

(CDCl3, 470 MHz) -156.8.

N 3O CF O 3 OMe 168 Ph

(R)-((1S,2S)-2-azidocyclohexyl) 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate 168.

Compound 165 (39 mg, 0.27 mmol) was acylated with (S)-3,3,3-trifluoro-2-methoxy-2- 196

phenylpropanoyl chloride 166 (83 mg, 0.33 mmol) to provide 92 mg (95%) of (R)-

((1S,2S)-2-azidocyclohexyl) 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate 168 as a

1 yellow oil. H NMR (CDCl3, 300 MHz) 7.57-7.55 (m, 2H), 7.45-7.38 (m, 3H), 4.91-

4.83 (m, 1H), 3.56 (s, 3H), 3.40-3.32 (m, 1H), 2.18-2.06 (m, 2H), 1.79-1.75 (m, 2H),

19 1.55-1.30 (m, 4H). F NMR (CDCl3, 470 MHz) -156.8, -157.1.

N 3O CF O 3 OMe 169 Ph

(R)-((1R,2R)-2-azidocyclohexyl) 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate 169.

Compound 76d (39 mg, 0.27 mmol) was acylated with (S)-3,3,3-trifluoro-2-methoxy-2- phenylpropanoyl chloride 166 (83 mg, 0.33 mmol) following the general method for preparing mosher’s salt to provide 92 mg (95%) of (R)-((1S,2S)-2-azidocyclohexyl)

1 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate 169 as a yellow oil. H NMR (CDCl3, 300

MHz) 7.58-7.55 (m, 2H), 7.42-7.37 (m, 3H), 4.91-4.79 (m, 1H), 3.58 (d, J = 9.8, 3H),

3.44-3.32 (m, 1H), 2.20-2.06 (m, 2H), 1.80-1.70 (m, 2H), 1.53-1.23 (m, 4H). 19F NMR

(CDCl3, 470 MHz) -156.8, -157.1.

N 3O O O 171

(1R,2R)-2-azidocyclohexyl 2-phenoxyacetate 171. To a solution of 2-

azidocyclohexanol 162 (0.51 g, 3.6 mmol) in CH2Cl2 (5 mL) at 0 °C was added DMAP

(0.54 g, 4.4 mmol). The reaction mixture was stirred for 30 minutes and phenoxyacetyl 197

chloride 170 (0.75 g, 4.4 mmol) was added. The reaction mixture was warmed to r.t and

stirring continued for 6 h. The reaction mixture was then poured through silica gel pad

and washed with CH2Cl2 (50 mL). The filtrate was concentrated and chromatographed

(45% EtOAc in hexanes) to provide 0.92 g (91%) of 2-azidocyclohexyl 2-phenoxyacetate

1 as yellow oil. H NMR (CDCl3, 300 MHz) 7.33-7.25 (m, 2H) 7.02-6.89 (m, 3H), 4,69

(d, J = 16.1, 1H), 4.63 (d, J = 16.1, 1H), 3.43-3.35 (m, 1H), 2.12-2.04 (m, 2H), 1.77-1.69

13 (m, 2H), 1.47-1.24 (m, 4H). C NMR (CDCl3, 300 MHz) 167.5, 157.0, 128.8, 121.0,

-1 25 113.9, 64.6, 62.3, 29.6, 29.4, 22.9, 22.6. IR (CHCl3) 2098 cm . []D = -0.92 ° (c 2.5,

CH2Cl2).

N 3O O O 172

(1S,2S)-2-azidocyclohexyl 2-phenoxyacetate 172. To a solution of 2-azidocyclohexanol

165 (0.51 g, 3.6 mmol) in CH2Cl2 (5 mL) at 0 °C was added DMAP (0.54 g, 4.4 mmol).

The reaction mixture was stirred for 30 minutes and phenoxyacetyl chloride 170 (0.75 g,

4.4 mmol) was added. The reaction mixture was warmed to r.t and stirring continued for

6 h. The reaction mixture was then poured through silica gel pad and washed with CH2Cl2

(50 mL). The filtrate was concentrated and chromatographed (45% EtOAc in hexanes) to

provide 0.92 g (91%) of 2-azidocyclohexyl 2-phenoxyacetate 172 as a yellow oil. 1H

NMR (CDCl3, 300 MHz) 7.33-7.25 (m, 2H) 7.02-6.89 (m, 3H), 4.69 (d, J = 16.2, 1H),

4.62 (d, J = 16.2, 1H), 3.42-3.35 (m, 1H), 2.12-1.99 (m, 2H), 1.80-1.69 (m, 2H), 1.46- 198

13 1.22 (m, 4H). C NMR (CDCl3, 300 MHz) 167.0, 156.5, 128.2, 120.5, 113.4, 64.1,

-1 25 61.7, 29.1, 28.9, 22.4, 22.1. IR (CHCl3) 2098 cm . []D = +0.93 ° (c 2.4, CH2Cl2).

N N N N

OH 173

(1R,2R)-2-(4-((dibutylamino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexanol 173.

(1R,2R)-2-Azidocyclohexanol 162 (13 mg, 0.09 mmol) and N-butyl-N-(prop-2-

ynyl)butan-1-amine 48a (23 mg, 0.14 mmol) were reacted following the general method

1 for 1,2,3-triazole synthesis to afford 25 mg (95%) of 173. H NMR (CDCl3, 300 MHz)

7.48 (s, 1H) 4.17-4.08 (m, 1H), 4.02-3.94 (m, 1H), 3.70 (bs, 3H), 2.40 (t, J = 7.3, 4H),

2.20-2.15 (m, 2H), 1.97-1.84 (m, 3H), 1.50-1.21 (m, 12H), 0.88 (t, J = 7.3, 6H). 13C NMR

(CDCl3, 75 MHz) 143.8, 121.0, 71.3, 65.5, 52.3, 47.7, 32,7, 30.5, 27.9, 23.6, 22.9, 19.4,

25 12.9. []D = -8.8 ° (c 1.1, CH2Cl2). HPLC (1% AcOH in CH3OH : H2O) RT 2.20 (99%).

MS (APCI) : M+H expected 308.46, obtained 309.00.

N N N N

O O O 174

(1R,2R)-2-(4-((isopentyl(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexyl 2-

phenoxyacetate 174. (1R,2R)-2-Azidocyclohexyl 2-phenoxyacetate 171 (18 mg, 0.06

mmol) and N,3-dimethyl-N-(prop-2-ynyl)butan-1-amine 82b (13 mg, 0.10 mmol) were 199

reacted following the general method for 1,2,3-triazole synthesis to afford 23 mg (93%)

1 of 174. H NMR (CDCl3, 300 MHz) 7.70 (s, 1H), 7.28-7.22 (m, 2H), 6.96 (t, J = 7.4,

1H), 6.74 (d, J = 7.9, 2H), 5.24-5.15 (m, 1H), 4.57-4.40 (m, 3H), 3.78 (s, 2H), 2.49 (t, J =

7.7, 2H), 2.28-2.19 (m, 5H), 2.08-1.91 (m, 3H), 1.62-1.36 (m, 6H), 0.87 (d, J = 6.5, 6H).

13 C NMR (CDCl3, 75 MHz) 167.7, 157.3, 129.3, 121.9, 121.4, 114.3, 74.7, 64.6, 62.9,

25 54.9, 51.8, 41.0, 35.1, 31.5, 30.9, 26.1, 24.2, 23.4, 22.3. []D = -12.8 ° (c 1.78, CH2Cl2).

HPLC (1% AcOH in CH3OH : H2O) RT 11.14 (86%). MS (APCI) : M+H expected

414.54, obtained 415.00.

N N N N

OH 175

(1S,2S)-2-(4-((dibutylamino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexanol 175. (1S,2S)-

2-azidocyclohexanol 165 (13 mg, 0.09mmol) and N-butyl-N-(prop-2-ynyl)butan-1-amine

48a (23 mg, 0.13 mmol) were reacted following the general method for 1,2,3-triazole

1 synthesis to afford 25 mg (94%) of 175. H NMR (CDCl3, 300 MHz) 7.51 (s, 1H) 4.32-

4.22 (bs, 1H), 4.18-4.09 (m, 1H), 4.01-3.93 (m, 1H), 3.73 (s, 2H), 2.44 (t, J = 7.4, 4H),

2.20-2.16 (m, 2H), 1.92-1.84 (m, 3H), 1.51-1.22 (m, 12H), 0.89 (t, J = 7.3, 6H). 13C NMR

(CDCl3, 75 MHz) 144.1, 121.8, 71.9, 66.1, 52.7, 48.2, 33.3, 31.1, 28.3, 24.2, 23.5, 20.0,

25 13.5. []D = +9.1 ° (c 1.24, CH2Cl2). HPLC (1% AcOH in CH3OH : H2O) RT 2.48 (92%).

MS (APCI) : M+H expected 308.46, obtained 309.00. 200

N N N N

O O O 176

(1S,2S)-2-(4-((isopentyl(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)cyclohexyl 2- phenoxyacetate 176. (1S,2S)-2-azidocyclohexyl 2-phenoxyacetate 165 (31 mg, 0.11

mmol) and N,3-dimethyl-N-(prop-2-ynyl)butan-1-amine 82b (13 mg, 0.093 mmol) were

reacted following the general method for 1,2,3-triazole synthesis to afford 35 mg (94%)

1 of 176. H NMR (CDCl3, 300 MHz) 7.51 (s, 1H), 7.27-7.22 (m, 2H), 6.96 (t, J = 7.4,

1H), 6.73 (d, J = 8.0, 2H), 5.25-5.16 (m, 1H), 4.58-4.49 (m, 1H), 4.47 (d, J = 16.3, 1H),

4.39 (d, J = 16.3, 1H), 3.66 (s, 2H), 2.38 (t, J = 7.7, 2H), 2.26-2.19 (m, 5H), 2.03-1.90

13 (m, 3H), 1.60-1.34 (m, 6H), 0.87 (d, J = 6.5, 6H). C NMR (CDCl3, 75 MHz) 167.9,

157.6, 129.5, 121.7, 121.0, 114.5, 74.9, 64.8, 62.9, 55.4, 52.5, 41.9, 36.2, 31.9, 31.2, 26.4,

25 24.4, 23.7, 22.7. []D = +12.9 ° (c 1.9, CH2Cl2). HPLC (1% AcOH in CH3OH : H2O) RT

15.27 (99%). MS (APCI) : M+H expected 414.54, obtained 415.00. 201

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