Synthesis and Antitumor Activity of Potentially Tumor ...

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Synthesis and Antitumor Activity of Potentially Tumor Targeting Synthesis and Antitumor Activity of Potentially Tumor Targeting

Analogues of the Tetrahydrofuran Containing Acetogenins Analogues of the Tetrahydrofuran Containing Acetogenins

Patricia Gonzalez Periche The Graduate Center, City University of New York

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i

SYNTHESIS AND ANTITUMOR ACTIVITY OF POTENTIALLY TUMOR TARGETING ANALOGUES OF

THE TETRAHYDROFURAN CONTAINING ACETOGENINS

by

PATRICIA GONZALEZ PERICHE

A dissertation submitted to the Graduate Faculty in Chemistry in partial fulfilment of the requirements for the degree of Doctor of Philosophy, The City University of New York

2020

ii

© 2020

PATRICIA GONZALEZ PERICHE

All rights reserved

iii

SYNTHESIS AND ANTITUMOR ACTIVITY OF POTENTIALLY TUMOR TARGETING ANALOGUES OF

THE TETRAHYDROFURAN CONTAINING ACETOGENINS

by

Patricia Gonzalez Periche

This manuscript has been read and accepted for the Graduate Faculty in Chemistry

In satisfaction of the dissertation requirement for the degree of

Doctor of Philosophy

________________________

Date

________________________

Date

______________________________________David R. Mootoo

Chair of Examining Committee

______________________________________Brian R. Gibney

Executive office

Supervisory Committee:

Ryan P. Murelli

Akira Kawamura

Naga Vara Kishore Pillarsetty

THE CITY UNIVERSITY OF NEW YORK

iv

ABSTRACT

Synthesis and antitumor activity of potentially tumor targeting analogues of the tetrahydrofuran containing

acetogenins

by

Patricia Gonzalez Periche

Advisor: Professor David R. Mootoo

The tetrahydrofuran containing acetogenins (THF-ACGs) are a naturally occurring class of compounds

with potent toxicity against a broad range of tumors, including multidrug resistant (MDR) strains. However,

the equally high toxicity to normal cells presents a hurdle for their clinical advancement. This study entails

synthetic and tumor targeting strategies that are relevant to their therapeutic development.

Chapter 1 presents a review of the synthesis and biological activity of the THF-ACGs. Chapter 2 covers a

modular approach for the synthesis of libraries of THF-ACGs. This strategy is illustrated in the synthesis

of C-10 epimers of 4-deoxyannonomontacin (4-DAN), one of the more potent monotetrahydrofuran

acetogenins. Towards the discovery of synthetically more accessible and tumor-selective analogues,

Chapter 3 describes the application of this methodology to analogues of 4-DAN where the THF ring is

substituted by mannose with different chain lengths and/or the butenolide is substituted by simpler

heterocycles.

To address the issue of systemic toxicity, Chapter 4 presents tumor-targeting strategies for the THF-

ACGs and their mimics. In the ‘chameleon’ approach the THF moiety was replaced with the 3-O-

carbamoyl-mannose residue, present in bleomycin, a family of clinically used anti-tumor agents and

believed to be responsible for their tumor selectivity. Therefore, the carbamoyl containing mannose

residue could confer both drug potency and selectivity. A prodrug strategy in which a naturally occurring

THF-ACG or synthetic analogue is conjugated via a cleavable linker to a tumor vector, is also described.

The guiding hypothesis is that following uptake by the tumor cell, the prodrug will be degraded to release

v

the active cytotoxic agent. As proof of principle, prodrugs comprising a sugar analogue of 4-DAN or the

naturally occurring acetogenin annonacin, 2-[3-(1,3-dicarboxypropyl)ureido] pentanedioic acid (DUPA), a

highly specific vector for prostate specific membrane antigen (PSMA) in prostate cancer (PCa), and a

traceless disulfide-based linker were synthesized.

Chapter 5 presents the cytotoxicity data for the new THF-ACGs and structure activity conclusions. Using

an MTT assay at both 16 and 48 hours, the cell viability of the THF-ACGs and their mimetics were

evaluated against five cancer cell lines: MDA-MD-231 (triple negative breast), MCF-7 (breast), HCT116

(colon), PC-3 (PSMA negative prostate cancer) and LNCaP (PSMA positive prostate). THF containing

analogues generally showed a lower IC50 (ca 0.5-5 micromolar) across the panel of cell lines than ones in

which the THF is replaced by a mannose. For the analogues with butenolide replacements, the

thiophene-2-carboxamide residue was the most active substitute and their derivatives showed

comparable or higher activity than their butenolide parents. The cytotoxicity of sugar mimetics with alkyl

ether chains of different lengths and positions was also determined. While individual analogues displayed

low micromolar potency and selectivity across the five cell lines tested, no clear structure activity trends

emerged. An ACG mimetic in which the THF and butenolide segments were replaced with 3-O-

carbamoyl-mannose and thiophene-2-carboxamide residues respectively, showed potent activity in the

0.1-10 M range against LNCaP and PC-3. Although not evaluated, the tumor selectivity of this mimetic is

particularly interesting given its structural similarity to the bleomycins.

The cytotoxicity activity of the DUPA-linked prodrugs derived from a mannose based ACG mimetic and

the naturally occurring ACG annonacin was also evaluated. In the 16 h MTT assay against LNCaP

(PSMA positive), the two prodrugs were more active than their parent drugs. However, against the PSMA

negative cell lines, MDA-MD-231, MCF-7, HCT116 and PC-3 both prodrugs and their parent drugs

showed similar activity and were appreciably less active than for LNCaP. The higher toxicity for LNCaP

over the PSMA negative cell lines, albeit modest (ca 2:1), supports the notion that this selectivity is

connected to DUPA-PSMA binding, and that PSMA is an internalizing receptor. However, in a 48 h MTT

assay, while the cytotoxicity of the prodrugs and parent drugs against MDA-MD-231, MCF-7, HCT116

was similar to that observed at 16 h, the activity of the prodrugs (but not the parent drugs), against PC-3

vi

increased markedly. This unexpected trend for PC-3 does not align with our prodrug hypothesis and may

be an indication that these DUPA-linked ACGs have a different mechanism of action than their parent

drugs. More robust studies are needed to resolve this issue, as well as to improve prodrug selectivity.

vii

“Our greatest weakness lies in giving up. The most certain

way to succeed is always to try just one more time.”

Thomas Edison

viii

ACKNOWLEDGEMENTS

First, I would like to thank my mentor, Professor Mootoo, for his constant support, patience and for

helping me grow both as a scientist and as a person. Thank you, Professor. The opportunity that you

provided for undergraduate research inspired me to pursue a Ph.D.

I would also like to thank my committee members, Professor Akira Kawamura, Professor Ryan Murelli

and Professor Naga Vara Kishore Pillarsetty for their support, guidance and stimulating conversations

over the years.

To the other members of the Mootoo lab, both past and present. Particularly, Dr. Stewart Bachan, Dr.

Ahmad Altiti and Dr. Clayton Mattis, Steven Truong, Amanda Ramdular, Dayanni Bhagwandin, Avelyn de

los Reyes, I am forever thankful for your help, encouragement and support over the years.

Thanks to everyone in the Chemistry Department at Hunter College. In particular, to Dr. Matthew Devany

for his help and patience with NMR characterization. To Professor Donna McGregor, thank you for

helping me come from Spain and for being a role model as a strong and successful woman in science.

Thank you for constant guidance. I would also like to acknowledge the academic and technical staff in the

department.

I am very grateful to Graduate Center for creating such a supportive work environment and for allowing

me the opportunity to earn a Ph.D. In particular, I would like to acknowledge my classmates for their

support.

To my friends in New York, my family away from home, thank you for the good times, the support and the

endless encouragement.

To my friends and family back home, thank you for always being there, even with an ocean in between

us.

To my mom and my sister, thank you for being there every step of the way and for helping me get where I

am today.

x

TABLE OF CONTENTS

ABSTRACT.................................................................................................................................................. iv

ACKNOWLEDGEMENTS .......................................................................................................................... viii

TABLE OF CONTENTS ............................................................................................................................... x

LIST OF SYMBOLS AND ABBREVIATIONS ........................................................................................... xiii

LIST OF CANCER CELL LINES .............................................................................................................. xvii

LIST OF FIGURES .................................................................................................................................. xviii

LIST OF SCHEMES .................................................................................................................................... xx

LIST OF TABLES ...................................................................................................................................... xxi

Chapter 1. Tetrahydrofuran containing acetogenins: structure, in vivo and in vitro activity

1.1. Introduction and background ................................................................................................ 2

1.2. Tetrahydrofuran containing acetogenins ............................................................................. 2

1.3. Synthesis of THF-ACGs .......................................................................................................... 3

1.4. Mode of action ....................................................................................................................... 13

1.5. Activity studies ...................................................................................................................... 15

1.5.1. In vitro .............................................................................................................................. 16

1.5.2. In vivo .............................................................................................................................. 17

1.6. SAR studies ........................................................................................................................... 20

Chapter 2. Synthesis of 4-Deoxyannonmontacin

2.1. Introduction and background .................................................................................................. 24

2.2. Synthetic strategy ..................................................................................................................... 26

2.3. Synthesis ................................................................................................................................... 30

2.3.1. THF alkene, 2.16.................................................................................................................. 30

2.3.2. Butenolide alkene, 2.30 ....................................................................................................... 31

2.3.3. CM of THF and butenolide alkenes ..................................................................................... 32

2.3.4. Comparison of naturally obtained 4-DAN and synthetic C-10 epimers ............................... 33

2.4. Experimental .............................................................................................................................. 36

xi

Chapter 3. Analogues of 4-Deoxyannomontacin


3.2. Butenolide substitutes ............................................................................................................. 51

3.2.1. Piperazine ............................................................................................................................ 51

3.2.2. Thiophene ............................................................................................................................ 52

3.2.3. O-butyl .................................................................................................................................. 53

3.3. Tetrahydrofuran substitutes .................................................................................................... 53

3.3.1. Mannose replacement ......................................................................................................... 53

3.4. Hybrid structures ...................................................................................................................... 55

3.5. Modifications on the hydrocarbon spacer ............................................................................. 56

3.6. Synthetic strategy ..................................................................................................................... 56

3.7. Synthesis ................................................................................................................................... 58

3.7.1. Mannose alkenes ................................................................................................................. 58

3.7.2. Butenolide replacements ..................................................................................................... 59

3.7.3. Cross metathesis and final products .................................................................................... 60

3.8. Summary ……………………………………………………………………………………………….62

3.9. Experimental .............................................................................................................................. 64

Chapter 4. Design and synthesis of potential tumor-selective cytotoxic agents


4.2. The ‘chameleon’ approach ....................................................................................................... 92

4.2.1. Design .................................................................................................................................. 93

4.2.2. Synthesis .............................................................................................................................. 94

4.3. The prodrug approach .............................................................................................................. 95

4.3.1. Design .................................................................................................................................. 95

4.3.2. Drugs .................................................................................................................................... 96

4.3.2. Receptor .............................................................................................................................. 97

4.3.3. Vector ................................................................................................................................... 97

4.3.4. Linker ................................................................................................................................... 98

4.4. C11 mannose derivative .......................................................................................................... 100

4.4.1. Sugar-butenolide Prodrug Synthesis ................................................................................. 100

4.4.2. Sugar-thiophene Prodrug Synthesis .................................................................................. 103

4.5. Annonacin derivative .............................................................................................................. 104

4.5.1. Isolation, purification and characterization of annonacin ................................................... 104

xii

4.5.2. Annonacin prodrug synthesis ............................................................................................. 108

4.6. Conclusions ............................................................................................................................. 112

4.7. Experimental ............................................................................................................................ 114

Chapter 5. Antitumor activity of 4-Deoxyannomontacin and analogues

5.1. Introduction ............................................................................................................................. 131

5.2. Results ..................................................................................................................................... 131

5.2.1. THF with butenolide or butenolide substitutes (1, 2, 3, 4, 5) ............................................. 132

5.2.2. Butenolide analogues with sugar substitutes for the THF (6, 7, 8, 9, 10) .......................... 134

5.2.3. Hybrid analogues with THF and butenolide substitutes (12, 13, 14, 15) ........................... 135

5.3. Discussion ............................................................................................................................... 137

5.4. Prodrug studies ....................................................................................................................... 138

5.4.1. Cytotoxicity data for 6 and 15 against LNCaP……………………………………………...…139

5.4.2. Cytotoxicity data for 6 and 15 against PSMA negative cells………….……………………..140

5.4.3. Annonacin prodrugs…………………………………………………………..……………….…142

5.5. Summary and future directions ............................................................................................. 143

5.6. Experimental ............................................................................................................................ 145

APPENDIX ................................................................................................................................................ 146

REFERENCES .......................................................................................................................................... 344

xiii

LIST OF SYMBOLS AND ABBREVIATIONS

Ac

Ac2O

AgOTf

ATP

Bn

brine

br

brsm

BST

Bu

oC

ca

calcd

cat

CM

13C NMR

CSA

COSY

δ

d

DEAD

DCC

DCM

DIBAL-H

DMAP

DMF

acetyl

acetic anhydride

silver trifluoromethanesulfonate

adenosine triphosphate

benzyl

saturated aqueous sodium chloride solution

broad

based on recovering started material

brine shrimp letality test

butyl

degree Celsius

about

calculated

catalytic

cross metathesis

carbon-13 nuclear magnetic resonance spectrometry

camphorsulfonic acid

correlation spectroscopy

chemical shift in ppm

doublet

diethyl azodicarboxylate

dicyclohexylcarbodiimide

dichloromethane

diisobutylaluminum hydride

4-dimethylaminopyridine

N,N-dimethylformamide

xiv

2,2-DMP

DMSO

ED50

ee

Et2O

EtOAc

EtOH

eq

EVE

FCC

g

h

1H NMR

hmn

HRMS

HSQC

Hz

IC50

J

L

m

M

mCPBA

Me

MeOH

mg

MHz

min

2,2-dimetoxypropane

dimethyl sulfoxide

effective dose

enantiomeric excess

diethyl ether

ethyl acetae

ethanol

equivalent

ethyl vinyl ether

flash column chromatography

gram

hour

proton nuclear magnetic resonance spectrometry

human

high resolution mass spectrometry

heteronuclear single quantum correlation spectroscopy

hertz

50% inhibitory concentration

coupling constant

liter

multiplet

molar

meta-chloroperoxybenzoic acid

methyl

methanol

milligram

megahertz

minute

xv

mL

mmol

MOM

MP

MTPA

MS

ND

NIS

PE

PCC

Ph

Piv

ppm

PPTS

pTSOH

q

rt

s

SAR

t

TBAF

TBAI

TBDPS

TFA

THF

THP

TLC

Tr

milliliter

millimole

chloromethyl methyl

melting point

α-methoxy-α-(trifluoromethyl)phenyl acetic acid

molecular sieves

Not determined

N-Iodosuccinimide

petroleum ether

pyridinium chlorochromate

phenol

pivaloyl or trimethylacetyl)

parts per million

para-pyridinium toluenesulfonate

para-toluene sulphonic acid

quartet

room temperature

singlet

structure activity relationship

triplet

tetra-n-butylammonium fluoride

tetra-n-butylammonium iodide

tert-butyldiphenylsilyl

trifluoroacetic acid

tetrahydrofuran

tetrahydropyran

thin layer chromatography

trityl or triphenylmethyl

xvi

Ts

vs

toluenesulfonyl

versus

xvii

LIST OF CANCER CELL LINES1

3PS

9PS

A-2780

A-549

HeLa

Hep G2

HepS

L1210

LNCaP

MCF-7

MDA-MB-231

MKN-45

OC-194

OC-222

OVCAR-3

PACA-2

PC-3

S180

SMMC-7721

mouse leukemia

mouse lymphocytic leukemia

hmn ovarian

hmn non-small lung

hmn cervical epithetial

hmn liver

liver

lymphocytic leukemia

hmn prostate

hmn breast

mn breast

hmn gastric

hmn ovarian epithelial

hmn ovarian epithelial

ovarian adenocarcinoma

hmn pancreas

hmn prostate

murine sarcoma

hmn liver

xviii

LIST OF FIGURES


Figure 1.1. Structure of uvaricin .................................................................................................................... 2

Figure 1.2. Structural features of the THF containing acetogenins .............................................................. 3 Figure 1.3. Proposed interaction of the THF-ACGs with mitochondria ....................................................... 13

Figure 1.4. Takada studies on the modification on the butenolide ............................................................. 21


Figure 2.1. Structure of 4-deoxyannomontacin .......................................................................................... 24

Figure 2.2. 4-deoxyannoreticuin (2.10 and 2.11) and 4-deoxyannomotacin (2.12) .................................... 26 Figure 2.3. Previous THF-ACGs by the Mootoo laboratory ........................................................................ 27

Figure 2.4. Konno’s CM approach to solamin type structures ................................................................... 28 Figure 2.5. CM approach to the synthesis of the C-10 epimeric mixture of 4-DAN .................................... 29

Figure 2.6. X-ray structure of compound 2.39 ............................................................................................ 30 Figure 2.7. 1H chemical shifts of 4-DAN both naturally obtained and synthesized ..................................... 34

Figure 2.8. 13C chemical shifts of 4-DAN both naturally obtained and synthesized ................................... 35


Figure 3.1. Structural characteristics of the THF-AGEs and proposed replacements ................................ 50

Figure 3.2. Piperazine analogues ............................................................................................................... 51 Figure 3.3. Thiophene derivative ................................................................................................................ 52

Figure 3.4. Simple n-alkyl derivative ........................................................................................................... 53 Figure 3.5. Activity of DAR 3.8 and mannose analog 3.9 ........................................................................... 54

Figure 3.6. THF replacement analogues .................................................................................................... 55

Figure 3.7. Hybrid compounds .................................................................................................................... 55

Figure 3.8. Deoxygenated analog ............................................................................................................... 56

Figure 3.9. Coupling alkene partners for the synthesis of analogues of 4-DAN ......................................... 57


Figure 4.1. Bleomycin and carbamoylmannose derivative 4.2 ................................................................... 93

Figure 4.2. Recognition mechanism of prodrugs ....................................................................................... 95 Figure 4.3. Example of a prodrug construct using 4-DAN as the parent drug ............................................ 95

Figure 4.4. Proposed drug and prodrug pairs 4.9-4.10, 4.11-4.12, 4.13-4.14 ............................................ 96 Figure 4.5. DUPA derived vector ................................................................................................................ 97

Figure 4.6. Structure of the linker (left) and release mechanism of the prodrug (right) .............................. 99

Figure 4.7. 1H of the C11-butenolide prodrug 4.10 ................................................................................... 102 Figure 4.8. Annona muricata ..................................................................................................................... 104 Figure 4.9. Extraction of annonacin .......................................................................................................... 105 Figure 4.10. Purification of annonacin ...................................................................................................... 105 Figure 4.11. 1H NMR of annonacin reported in the literature .................................................................... 106

Figure 4.12. 1H NMR isolated annonacin .................................................................................................. 106

Figure 4.13. Linker-annonacin purification. ............................................................................................... 108 Figure 4.14. Fraction 1.1 (4.40 or 4.41) .................................................................................................... 110 Figure 4.15. Fraction 1.2 (4.38) ................................................................................................................ 110

xix


Figure 5.1. SAR conclusion based on cytotoxicity assays ........................................................................ 137 Figure 5.2. Structure of compound 6 and its prodrug, 15 ......................................................................... 138 Figure 5.3. Cytotoxicity at 16 and 48 hours of compounds 6 and 15 in PSMA positive cells ................... 139

Figure 5.4. Cytotoxicity at 16 and 48 hours of compounds 6 and 15 in PSMA negative cells ................. 140 Figure 5.5. Structure of annonacin, 2, and annonacin prodrug, 16 .......................................................... 142 Figure 5.6. Cytotoxicity at 16 and 48 hours of compounds 2 and 16 in prostate cancer cells ................. 143

xx

LIST OF SCHEMES


Scheme 2.1. The Wu approach to 4-DAN .................................................................................................. 25

Scheme 2.2. Synthesis of the THF-alkene 2.16 ......................................................................................... 31 Scheme 2.3. Synthesis of butenolide 2.30.................................................................................................. 32

Scheme 2.4. CM reaction and synthesis of C-10 epimers of 4-DAN .......................................................... 33


Scheme 3.1. Synthesis of mannose alkenes .............................................................................................. 58

Scheme 3.2. Synthesis of butenolide replacements ................................................................................... 59 Scheme 3.3. Synthesis of the compounds with butenolide replacements .................................................. 60

Scheme 3.4. Synthesis of mannose containing compounds ...................................................................... 61 Scheme 3.5. Route to hybrid analogues ..................................................................................................... 62


Scheme 4.1. Synthesis of “chameleon” compound 4.7 .............................................................................. 94

Scheme 4.2. Synthetic route to the synthesis of the DUPA derived vector 4.15 ........................................ 98 Scheme 4.3. Synthesis of linker 4.24 .......................................................................................................... 99

Scheme 4.4. Synthesis of the C11-butenolide prodrug .............................................................................. 101 Scheme 4.5. Synthesis of the C11-thiophene prodrug .............................................................................. 103

Scheme 4.6. Synthesis of the annonacin based prodrug ........................................................................ 109

Scheme 4.7. En route to the synthesis of 4-DAN prodrug ........................................................................ 112

xxi

LIST OF TABLES


Table 1.1. Strategies for the thetrahydrofuran ring synthesis ....................................................................... 4

Table 1.2. Synthetic approaches to the butenolide ....................................................................................... 7

Table 1.3. Coupling strategies of the THF and butenolide segments ........................................................... 8

Table 1.4. Recent publications on THF-ACGs syntheses .......................................................................... 11

Table 1.5. Example of the data disparity among different cell assasys in the THF-ACGs ......................... 17

Table 1.6. Summary of in vivo studies of THF-AGCs ................................................................................. 19


Table 2.1. Naturally obtained vs synthetic 4-DAN ...................................................................................... 34


Table 4.1.13C of literature reported and isolated annonacin ..................................................................... 107


Table 5.1. Cell viability (%) over 16 h at 12.5 and 25 µM ......................................................................... 132

Table 5.2. THF with butenolide or butenolide substitutes IC50s (µM) in 16 and 48 h assays ................... 133 Table 5.3. Cell viability (%) over 16 h at 12.5 and 25 µM ......................................................................... 134

Table 5.4. Cell viability (%) over 16 h at 12.5 and 25 µM ......................................................................... 135 Table 5.5. Hybrid compounds IC50s (µM) (16 and 48 h assays) .............................................................. 136

Table 5.6. Comparison of activity (% cell viability) of 15 for LNCaP vs PSMA negative cell lines at 5 M

.................................................................................................................................................................. 141

1

TETRAHYDROFURAN CONTAINING ACETOGENINS:

STRUCTURE, IN VIVO AND IN VITRO ACTIVITY

2

1.1. Introduction and background

Cancer remains a major health challenge despite major advances in early detection, diagnosis and

treatment. The current lines of treatment involve chemotherapy, surgery, radiation or most commonly, a

combination of the three. Small organic molecules are a primary line of treatment and usually those have

been identified from natural sources. Plants have been used since ancient times to treat many diseases:

between the 1940s and 2014, 175 molecules were approved for the treatment of cancer and 49% of

those are natural products or modified natural compounds.2 To this end, the naturally occurring

tetrahydrofuran containing acetogenins (THF-ACGs) are of interest because of their high potencies

against a broad panel of human cancer cell lines, including several multidrug resistant (MDR) ones and

their relatively simple structures. However, while certain THF-ACGs show selectivity for certain cancer

cells, challenges for their clinical use are systemic toxicity and a lack of understanding of their

mechanisms of action.

1.2. Tetrahydrofuran containing acetogenins

The tetrahydrofuran containing annonaceous acetogenins (THF-ACGs) are a relatively untested family of

cytotoxic agents isolated from the Annona species. These evergreen trees are usually found in tropical

and subtropical areas. ACGs can be found in the leaves, roots, seeds, pulp of fruits like graviola, soursop,

pawpaw, guanabanana, Brazilian pawpaw and

custard apple. The first compound of this class,

uvaricin3 1.1, was isolated in 1982 via an activity-

directed fractionalization using 3PS cells in vivo

(murine leukemia) testing of the extracts. Its

unusual bioactivity attracted the attention of many,

hoping to isolate and/or synthesize the future

generation of antitumor drugs based on this new Figure 1.1. Structure of uvaricin

3

class of compounds. Since then, more than 400 compounds have been isolated from natural sources.4,5,6

Many of them have been synthesized and analogues have been designed and synthesized seeking for

the “magic bullet”. Many of them have antitumoral effects, as well as antimicrobial, antimalarial,

antifeedant, antiviral, anthelminthic, antiprotozoal, pesticidal and immunosuppressive.7,2,8 Unfortunately,

as of 2019, this class of compounds are still strong candidates for the development of new drugs, but the

lack of understanding of their mode of action makes targeting of specific tumors and designing active and

selective analogues a tremendous challenge.

The highly lipophilic THF-ACGs derive from the polyketide pathway and have a C35 or C37 aliphatic chain

that contains: (i) one, two or three THF tetrahydrofuran rings that can be adjacent or not; (ii) a terminal α,

β-unsaturated δ-lactone; (iii) a series of oxygenated groups (hydroxyls, acetoxyls, ketones, epoxides)

and/or double bonds (Figure 1.2).

Figure 1.2. Structural features of the THF containing acetogenins

1.3. Synthesis of THF-ACGs

The potent biological activities of the THF-ACGs and their stereochemical complexity have motivated

considerable interest in their synthesis. Since 1996, over 100 total syntheses have been reported.9, 10, 11

This topic has been extensively reviewed.11c,12

4

Convergent, linear and biomimetic strategies for naturally occurring THF-ACGs, derivates, as well as non-

natural analogues have been described. A major emphasis has been the synthesis of the

stereochemically complex tetrahydrofuran segment which has employed inexpensive chiral starting

materials, such as carbohydrates and amino acids, and a variety of asymmetric reactions including: (i)

oxidative cyclization of hydroxy alkenes, (ii) epoxidation-epoxide opening sequence on 4-alkene-1-ol

precursor, (iii) iodoetherification of hydroxy alkenes, (iv) ring closing metathesis, (v) Sharpless

dihydroxylation followed by acid catalyzed cyclization; (vi) base catalyzed cyclization;11d (vii)

diastereoselective Williamson reaction; (viii) chiral ligand-controlled cyclization; (ix) radical cyclization.

Table 1.1 exemplifies the more popular strategies.

Table 1.1. Strategies for the tetrahydrofuran ring synthesis

Examples Target / Group

i

cis- solamin

Brown13

(+)-cis-solamin

Donohoe14

5

Trilobacin

Sinha15

ii

Parviforin

Hoye16

iii

Trilobacin

Mootoo17

iv

Solamin

Mioskowski18

v

Corossoline

Tanaka19

6

vi

Parviflorin

Trost20

vii

Squamocin A and D

Koertf21

Viii

Uvaricin

Burke22

ix

(+)- Membranacin

Lee23

Several approaches for the butenolide segment have also been reported: ((i) ruthenium catalyzed; (ii) Pd

catalyzed coupling); (iii) sulfonium enolate-elimination or (iv) the aldol-elimination. The ruthenium

catalyzed ring formation reported by Brown24 is of special interest because it accomplishes both the

synthesis of the butenolide and the coupling of the THF and butenolide units in one step .

7

Table 1.2. Synthetic approaches to the butenolide

Examples

Target / Group

i

cis-solamin

Brown24

ii

Asimicin

Marshall25

iii

Solamin

Mioskowski18

8

iv

Longicin

Hanessian26

A number of strategies have been used for the coupling of tetrahydrofuran and butenolide subunits (Table

1.3): (i) Sonogashira coupling; (ii) Wittig olefination; (iii) cross metathesis; (iv) ring closing metathesis; (v)

addition of organometals to aldehydes; (vi) Koscienski-Julia coupling; (vii) Nozaki-Hiyama-Kishi reaction;

(viii) crotylation; (ix) epoxide opening. The Sonogashira and the Grubbs approaches have the broadest

scope.

Table 1.3. Coupling strategies of the THF and butenolide segments

Examples

Target / group

i

Murisolin

Makabe27

9

ii

Gigantetrocin A

Shi28

iii

Mucocin

Mootoo29

iv

Longicin

Hanessian26

10

v

Squamostatins D

Marshall30

vi

Jimenezin

Hoffmann31

vii

Mosin B

Tanaka32

11

viii

Trilobin

Marshall33

ix

Trilobin

Sinha and Keinan34

Table 1.4 lists the more recent publications in the field. These studies have focused on using the

foregoing methods to synthesize unnatural analogues for biological studies.

Table 1.4. Recent publications on THF-ACGs syntheses

Year Title Notes Ref

2008 Synthesis and antitumor activity of C-9 epimers of the

tetrahydrofuran containing acetogenin 4-deoxyannoreticuin Total synthesis 35

2010 Synthesis of the Annonacin isolated from Annona

densicoma Total synthesis 36

2011 Modular assembly of cytotoxic acetogenin mimetics by click

linkage with nitrogen functionalities

Synthesis of analogues

-modification of the

lipophilic linkage

37

12

2013

Critical role of a methyl group on the δ-lactones ring of

annonaceous acetogenins in the potent inhibition of

mitochondrial complex I

Development of

analogues for SAR

studies

38

2013

Structure-activity relationships of hybrid annonaceous

acetogenins: powerful growth inhibitory effects of their

connecting groups between heterocycle and hydrophobic

carbon chain bearing THF ring on human cancer cell lines

Solamin analogues

using as butenolide

replacement N-pyrazole

39

2013 Total synthesis of 14,21-diepi-squamocin-K Total synthesis for SAR

studies 40

2014

New cytotoxic annonaceous acetogenin mimetics having a

nitrogen-heterocyclic terminal and their application to cell

imaging

Analogues

development -

substitution of the

butenolide

41

2014 Thiophene-3-carboxamide analogue of annonaceous

acetogenins as antitumor drug lead

Substitution of the

butenolide by a

thiophene delivers new

potent analogues

42

2014 Total synthesis of Muricadienin, the Putative Key precursor

in the Solamin Biosynthesis Total synthesis 43

2015

Synthesis of dansyl-labeled probe of thiophene analogues of

annonaceous acetogenins for visualization of cell

distribution and growth inhibitory activity toward human

cancer cell lines

Synthesis of a probe for

imaging studies 44

2016

Total synthesis of (+)- Goniodecin Total synthesis

45

2017

Covergent synthesis of stereoisomers of THF ring moiety of

acetogenin thiophene analogue and their antiproliferative

activities against human cell lines

Synthesis of analogues

– replacement of

butenolide by a

thiophene moiety

46

2017

Total synthesis of two possible diastereomers of natural 6-

chlorotetrahydrofuran acetogenin and its stereostructural

elucidation

Total synthesis 47

2018 Convergent total synthesis of asimicin via decarbonylative

radical dimerization Total synthesis 48

2018

A general diastereoselective strategy for both cis- and trans-

2,6- disubstituted tetrahydropyrans: formal total synthesis of

(+)-muconin

Total synthesis 49

13

It is evident that organic chemists have been fascinated with this class of complex structures since their

discovery and are excited and intrigued by their wide range of biological activities and potential use in the

clinic.

1.4. Mode of action

It has been suggested that the ACGs act on cancer cells by inhibition of the NADH-ubiquinone reductase

(complex I), a membrane-bound protein of the mitochondrial electron-transport system, and the

nicotinamide adenine dinucleotide (NADH) oxidase, characteristic of the plasma membranes in tumor

cells.11c Mechanistic studies have focused on action on Complex I. McLaughlin50 has proposed a model in

which the more lipophilic parts of the molecule, the THF rings and the alkyl chain, are anchored to the

mitochondrial membrane. Once positioned, the alkyl spacer helps directing the butenolide, or warhead, to

the active site on the enzyme (Figure 1.3).2 This results in the inhibition of oxidative phosphorylation and

ATP production. Because cancer cells are estimated to rely on mitochondrial oxidation for 40-75% of the

required ATP, complex I inhibition may contribute to some degree in the antitumor activity. Alternatively,

antitumor activity may be the result of triggering the mitochondrial apoptotic pathway. The contribution of

these different pathways to the overall toxicity could explain the difference in susceptibility of different cell

lines towards the same toxic compounds.11d

Figure 1.3. Proposed interaction of the THF-ACGs with mitochondria (adapted)51

14

The mitochondrial apoptotic pathway entails the efflux of mitochondrial contents into the cytosol as a

result of the disruption of the mitochondrial membrane, which in turn may be promoted by depletion of

mitochondrial ATP. In this context, different apoptotic mechanisms have been studied.

Alteration of the cytosol would affect the endoplasmic reticulum causing release of Ca2+. The inability of

the cell to restore Ca2+ homeostasis, will trigger caspase activation an eventually cell death. This

sequence of events that are part of the endoplasmic reticulum stress disorder (ERS) were observed by

Wu’s group52 when treating the human nasopharyngeal carcinoma (NPC) cells with THF-ACGs.

Other indicators of the apoptotic pathway are the alteration of the Bcl-2/Bax protein ratio, the activation of

caspases 3 and 9 and the production superoxide anion and hydrogen peroxide (ROS). All of which were

reported by Tormo53 when treating Hep2G cells with THF-AGEs. Tormo also reported a dramatic

decrease in the mitochondrial transmembrane potential (MTP) – a measurement of the mitochondrial

function, as it is generated by the pumping of electrons across the membrane- and in the mitochondrial

membrane permeability transition pore (MPTP) – which opening allows calcium homeostasis.

Other signals/targets that have been reported to be altered by the THF-AGEs related to the mitochondrial

apoptotic pathway are: (i) Notch signaling54. Treatment of gastric cancer cells with THF-ACGs resulted in

Notch2 up-regulation in a dose dependent manner, which resulted in cell growth inhibition; (ii) The

hypoxia-inducible factor-1 (HIF-1)55 activates the expression of more than 100 genes involved in

adaptation and cell survival under hypoxia conditions. It is suggested that hypoxia increases the levels of

ROS, which in turn inactivate HIF-1α protein, resulting in activation of HIF-1. T47D cells treated with THF-

ACGs inhibited HIF-1 activation with IC50 values from 0.02-31 µM; (iii) Estrogen receptor-α (ERα).

Annonacin decreased cell survival in ERα positive cells but not on ERα negative cells, suggesting that the

receptor may play a key role in the mode of action of THF-AGEs. Moreover, annonacin also inhibited

expression of D1 and Bcl-2 receptors. These in vitro results were confirmed in in vivo assays using cell-

grafted nude mice.

It has also been observed that ACGs act against certain multidrug resistant (MDR) tumors.8 MDR is

15

attributed to an increased expression of the P-170 glycoprotein, drug efflux pump in the plasma

membrane. ACGs may act against MDR cells by their inhibition of ATP production, which is required for

active efflux.

The inhibition of NADH oxidase in the plasma membrane of certain tumor cells, involved in ATP

production for glycolysis, may lead to both necrotic and apoptotic pathways. This may explain the

selectivity observed against certain cell lines. Another basis for selectivity, could be that the mitochondria

for certain tumor cells seems to be different from their healthy counterparts. They exhibit comprehensive

metabolic differences that makes them more sensitive to mitochondrial perturbation than normal cells.56

In this context, the ACGs are an interesting group of potential clinical agents.

1.5. Activity studies

In the early stages of extraction and isolation of THF-AGEs from seeds, pulp, leaves or other parts of the

Annonaceae plants, the potential as cytotoxic agents of this class of compounds was recognized. In order

to evaluate the extracts, each one of the partition fractions from the extractions of the naturally obtained

samples were tested using the brine shrimp lethality assay. If the fraction contained the cytotoxic

compound, shrimp would not survive, and therefore the extract would be further purified to isolate

individual compounds. That could be considered the first cytotoxicity assessment of THF-ACGs.

Some research has been focused on the biological evaluation in vitro and in vivo of the crude extracts

from the plants57,58 and other researchers have isolated compounds from the extracts and studied their

biological activities. Even though the data collected for the extracts is valuable and seems to be in

concordance with the one of individual compounds, it is beyond the scope of this discussion and we will

center our attention in studies of isolated compounds.

When first discovered, the tetrahydrofuran containing annonaceous acetogenins (THF-ACGs) showed

potent cytotoxicity against a wide range of human tumor cell lines. The first in vivo study carried out by

the Upjohn company59 showed great potential against L1210 murine leukemia and A2780 ovarian

16

xenografts. Thereafter, the interest in this family of compounds, grew exponentially, resulting in the

isolation of 400 compounds in 27 years in the search of the ‘magic bullet’ against tumor cells. Even

though in vitro studies have proven useful to identify potential cytotoxic compounds or “magic bullets”

have not been so successful when carried out in vivo. In solid tumors, other factors need to be considered

such as pH differences or gradient of nutrition concentrations. Those have a great impact in the cell

viability, the metabolism and the sensitivity to treatment60 and may be the reason why these compounds

have not yet been advanced to clinical studies.

Although these compounds showed potential in the cell studies, there has been a gradual loss of interest

in this class of compounds due to the challenges with systemic toxicity. In particular there is a concern in

neuronal toxicity. It has been reported that in regions where there is a high consumption of herbal tea and

fruits from plants of the Annonaceae family61 there is a high rate of parkinsonism. Targeting compounds

to cancer cells could address these issues.

1.5.1. In vitro

The lack of a stablished protocol towards the evaluation of the compounds, makes the makes the

reproducibility and comparison of newly acquired data challenging. Based on the comparison of different

reports, the activity seems to vary with incubation time. Holschneider62 treated different cell lines with

bullatacin for 48 and 72 h observing ED50s of 10 vs 10-7 µg/mL for OC-194 cells and 50 vs 4 µg/mL for

OVCAR-3 cells, respectively. Cytotoxicities vary with incubation time and across different cell lines. To

this date there is not an explanation as to why some cell lines are more affected by THF-ACGs than

others. Elucidation of the mode of action could bring some insight to this matter. The disparity of data

collected by different groups on bullatacin is exemplified in Table 1.5.63, 64, 65

17

Table 1.5. Example of the data disparity among different cell assays in THF-ACGs

Compound Cell line ED50 (µg/mL) IC50 (µg/mL)

48 h 72 h 5 days

Bullatacin

MCF-7 <10-12 2.3x10-2

9PS

1x10-15

A-549 1.3x10-13 3.3x10-2

OC-194 10 10-7

OC-222

5 5x10-4

A-2780

10-2 6x10-6

OVCAR-3

50 4

HeLa

3.4x10-2

HepG2

3.2x10-12

SMMC-7721

4.8 x 10-3

MKN-45 5.3x10-2

1.5.2. In vivo

Not many in vivo evaluations of the THF-ACGs have been published. The first one was the activity-

directed fractionation of P388 murine leukemia assay on uvaricin. The Upjohn company reported that

treating mice with 25 µg/kg and 1.4 mg/kg would increase their life span by 124-157%, respectively.3,59

The abovementioned data showed that bullatacin and other isolated derivatives were potential antitumor

agents based on their efficacy in normal mice with L1210 murine leukemia and immunodeficient mice

bearing A2780 ovarian cancer tumors.

18

In another study, the in vivo activity of annonacin was examined using mouse lung carcinoma (LLC)

tumor cells in BDF-1 mice.66 The difference of this study with the ones that had been published before is

that the ratio of activity towards the tumor vs activity towards the host was much higher than in previous

cases: at a dose of 10 mg/kg of annonacin no mice died while three and five out of six died in the

controls. This seemed to suggest that annonacin was less toxic towards the host than other compounds.

In a separate study also on annonacin, mice bearing MCF-7 xenografts were treated with 50 mg/kg/day

for 21 days and the tumor size was measured every 3 to 4 days. It was observed that the size of the

tumor decreased in 7 to 22 days. In 2012, Fan et al67 evaluated 5 compounds: squamostatiin A,

squamostatiin E, 4-deoxyannoreticuin, desacetyluvaricin, bullatacin were tested the against S180 and

Hep9 xenograpft bearing mice. Their findings agreed with their in vitro results: (i) the two bis-THF

compounds, showed the highest antitumor inhibition of between 64 and 71% at a dose of 60 µg/kg; (ii) out

of the two bis-THF compounds, the one with adjancent THF showed higher activity and (iii) bullatacin at

higher doses showed the highest activity but also more adverse effects.

The above examples suggest that treatment of mice with THF-ACGs in vivo reduced tumor size without

affecting the host. However, in the studies below, treatment of mice with THF-AGEs would not stop tumor

growth (at low dosages) and/or would cause host death (at high dosages).

The data published in 1994 by Montz and coworkers63 proved that using a murine ovarian

terotocarcinoma (MOT) model in C3HeB/Fej mice, the injection of bullatacin made no difference in the

MOT-related animal death at non-lethal dose ranges for both, single dose administration or if the dosage

was divided over time. At low dosages the tumor growth was not inhibited and at higher dosages

bullatacin killed the host.

In the most recent in vivo study, in 2013, Li60 injected H22 hepatoma cells bearing mice with

annosquamin B, bullatacin and annosquatin B. They established that: (i) a single injection of 100 µg/kg

did not influence healthy mice; (ii) treatment of tumor bearing mice with 5 doses of 25-50 µg/kg produced

61% reduction of the tumor size, whereas the other two compounds reduced the tumor size by 53.7 and

58.7% This study agrees with Fan’s67. Additionally, they found that bullatacin resulted in severe liver and

19

kidney tissue damage proved by an increase in calcium concentration, ROS production and Bax/Bcl-2

ratio in rats.

Table 1.6 summarizes the in vivo data published in recent years and most of the examples have been

discussed above.

Table 1.6. Summary of in vivo studies of THF-AGCs

Compound Tumor cell line Daily dose

(mg/Kg) Antitumor activity

% Increase of

life span % Tumor growth

inhibition

Annonacin65

3PS

0.95 124

Bullatacin59

L1210

0.05 138

A2780

0.1 68

HepS

0.015 63

S180

0.015 66

Squamocin

L1210

0.63 113

HepS68

0.03 78

Squamostatiin A67

HepS

0.06 31

S180

0.06 54

Squamostatiin E

HepS

0.06 51

S180

0.06 51

4-Deoxyannoreticuin

HepS

0.06 67

20

S180

0.06 48

4-Desacetyluvaricin

HepS

0.06 71

S180

0.06 64

Uvaricin3

3PS

1.4 157

Asimicin

3PS

0.025 124

Rollinone

3PS

1.4 147

Rolliniastatin

3PS

0.25 128

Both the in vivo and in vitro reports demonstrate how toxic this family of compounds is to all type of cells

and supports our claim that in order to become clinical agents, these compounds need to be targeted at

cancer cells.

1.6. SAR studies

Several studies on structure activity relationships (SARs) in THF-ACGs have been published,12, 69

establishing the structural elements required for toxicity: a cyclic ether core, a butenolide, a hydrophobic

spacer which connects one end to the cyclic ether core to the butenolide, and a hydrocarbon tail, which is

attached to the other end of the cyclic ether core (Figure 1.2). However, predicting the effects of structural

modification is challenging as SAR studies have been based on different assays, primarily mitochondrial

and cytotoxicity assays, and it is often difficult to compare data from different researchers. In addition, as

discussed in the Mode of Action section, new mechanisms of action are being elucidated and SAR data

for these new pathways are not defined.

21

On the mitochondrial and cytotoxic assays, the role of the butenolide is the more extensively studied.

However, it is still uncertain if its presence is necessary for the compounds to remain active. In an early

study, Takada and coworkers70 used a bis THF model to evaluate whether inverting the (S) configuration

of the lactone found would affect the inhibitory potency of complex I. Their study concluded that the R-

configuration analog was as potent as the natural occurring one. Based on those findings, Takada

expanded the study using a bullatacin-like structure (Figure 1.4).70 He concluded that: (i) removing the

alcohol from position 4 in bullatacin 1.67 did not affect inhibitory potency, see 1.68; (ii) removing the

methyl 1.69 had no effect in the potency; (iii) the bulkier n-butyl derivative, 1.70 retained an activity in the

same order of magnitude as the parent drug.

Figure 1.4. Takada studies on the modification of the butenolide

Based on those results, it was suggested that the recognition of the butenolide (or any butenolide

substituent) by the enzyme is weak and that could be a consequence of a large cavity binding domain in

complex I.

Hoppen71 substituted the butenolide by the natural target of the mitochondrial complex I (NADH-

ubiquinone oxidoreductase), quinone. In the inhibitory studies, it was found that the lactone could be

substituted by the quinone ring and the compound would still be active. These findings led to the idea that

both ubiquinone and the anonnaceous acetogenins are competitive inhibitors of the same binding site of

complex I. Hocquemiller72 explored the possible replacements of the butenolide by synthesizing a library

of bis THF squamocin-heterocycle analogues using known complex I inhibitors, such as pyrimidines,

22

quinazoles or benzimidazoles. Some of the modifications resulted in analogues with high activities,

however, it must be noted that alteration of the structure may result in a completely different mechanism

of action.

In more recent years, other groups have substituted the butenolide for simpler heterocycles such as

thiophene,39, 42, 44, 46 piperazine41 or N-methylpyrazole39 and have obtained analogues with maintained or

increased activities. The thiophene and piperazine cases will be discussed in Chapter 3. These studies

suggest that a butenolide-like structure may be necessary for complex I targeting.

Other SAR studies propose the middle of the hydrophobic spacer as the ideal position for conjugation for

tumor targeting or for introduction of a fluorescent group or radiolabel for imaging. Others12 have

evaluated the length of the hydrophilic spacers as well as the polarity of the tetrahydrofuran and its

surroundings and was determined that it was a fundamental feature to maintain activity.

There is controversy regarding the hydroxyl groups in the hydrophilic spacer. Some claim that activity

varies depending on the position of the hydroxyls,73 Chen67 suggested that two hydroxyls flanking the

THF and a third one somewhere else are necessary, others proposed that hydroxylation at position 4

could be involved in a unique intramolecular interaction with the butenolide,74 and Takada’s70 results (see

Figure 1.3) seem to indicate that removal of the hydroxyl at position 4 does not compromise the activity of

the compound.

Even though much progress has been made in elucidating the role of the structural elements of the THF-

ACGs there is a need for a wider library of molecules to expand SAR trends and elucidate new pathways.

To this end simple, high-throughput, synthetic strategies that can deliver novel structures are of interest.

23

SYNTHESIS OF 4-DEOXYANNOMONTACIN

24


The tetrahydrofuran containing acetogenins (THF-AGEs) exhibit high activities and selectivities for certain

tumors, but they are also too potent to normal cells. Therefore, there is an interest in developing novel

synthetic analogues that may present higher selectivity towards tumors. THF-AGEs can be obtained from

natural resources, but it is challenging because of the difficult purification that results in low yields. In

order to easily access a library of compounds for structure-activity relationship studies and clinical

development is important to develop a synthetic method. That method should be convergent to allow

synthetic modification at late stages and tolerate a broad range of functional groups.

The mono THF-AGE 4-deoxyannomontacin (4-DAN), 2.1 was selected as a model to develop new

synthetic strategies as its structure is relatively simple and it shows high potency against a wide range of

human tumor cell lines in an MTT 7-day assay (Figure 2.1).

Figure 2.1. Structure of 4-deoxyannomontacin (7-day MTT assays)

4-DAN was first isolated from Goniothalamus giganteus in 1997 by McLaughin75 and coworkers. The only

total synthesis was reported in 1999 by the Wu group.76 The key reaction in this synthesis was the

Sonogashira coupling of vinyl iodide 2.5 and alkyne 2.9, in which diimide reduction25 on the enyne

product gave 4-DAN.

Iodide 2.5 was obtained in 13 steps starting from compound 2.3 in a 22% overall yield. Aldehyde 2.3 was

obtained from D-glucose 2.2. After elongation of the side chain using a Wittig-Horner reaction and

reduction of the alkene, the resulting ester was treated with propane-1,3-dithiol in the presence of

BF3.Et2O to give the lactone-diol. Then, it was protected in situ to afford a lactone that was reduced with

DIBAL-H and treated with (bromotridecylidene)triphenylphosphorane to yield a mixture of the E and Z

25

alkenes. After photoisomerization of the alkene, only Z was obtained. The alcohol was converted to the

tosylester and the alkene was subjected to asymmetric dihydroxylation using AD-mix-β. With the diol in

hand the THF ring was constructed using an intramolecular Williamson etherification on substrate 2.4.

Scheme 2.1. The Wu approach to 4-DAN

The free alcohol was protected with MOM. After deprotection of the isopropylidene, epoxide formation

and epoxide opening using lithium trimethylsilylacetylide, the resulting alkyne was converted into the vinyl

26

iodide 2.5 using tributyltin hydride followed by iodine. Alkyne 2.9 was obtained from methyl ester 2.6 and

ethyl (S)-(-)-lactate using an aldol-cyclization tandem previously reported.77 Then, the epoxide was

introduced using the terminal alkene as substrate and upon elimination, the racemic mixture was

obtained. The CoIII-Salen catalyzed hydrolytic kinetic resolution of epoxide 2.7 delivered the resolved

oxirane that was treated with n-butyl lithium and trimethylsilylacetylene to deliver silyl 2.8. Desylation of

the alkyne yielded lactone 2.9. Overall 4-DAN was synthesized in an overall yield of 15% from over 17

steps starting from 2.3 and 2.8.

In earlier studies in our laboratory, 4-deoxyannoreticuin (4-DAR), 2.10 and 2.11, a very closely related

analog of 4-DAN, showed that the configuration at the position corresponding to C-10 in 4-DAN did not

impact on cytotoxicity against several cell lines. Therefore we decided, for synthetic convenience, to

prepare 4-DAN as a mixture of C-10 epimers 2.12.35 (Figure 2.2).

Figure 2.2. 4-deoxyannorecticuin (2.10 and 2.11) and 4-deoxyannomontacin (2.12)

2.2. Synthetic strategy

Our synthetic plan follows a modular approach, involving a cross metathesis (CM) strategy that was first

developed for the synthesis of tetrahydrofuran containing acetogenins such as bullatanocin, 2.13, both

27

(S) and (R)-deoxyannoreticuin, 2.10 and 2.11, and the mannose containing analogues of the THF-ACGs,

2.18. (Figure 2.3).78, 77, 79

Synthesis of bullatanocin (2004)80:

Synthesis of 4-deoxyannoreticuin (2008)35:

Synthesis of mannose containing analogues (2013)79:

Figure 2.3. Previous THF-ACGs by the Mootoo laboratory

28

This approach was later applied to analogues of the mono-THF solamin by Konno et al.81 (Figure 2.4).

Starting with either (+)-muricatacin or (-)-muricatacin, 2.28a and 2.28b, they were able to synthesize three

different allylic alcohols, 2.24, 2.25 and 2.16. that were then subjected to a CM with the butenolide

counterparts, 2.26 and 2.27, respectively, to deliver the three known natural compounds cis-solamin A

2.21, cis-solamin B 2.22, and reticulatacin 2.23.

Konno’s work on solamin type acetogenins (2010)81:

Figure 2.4. Konno's CM approach to solamin type structures

29

Current work:

Figure 2.5. CM approach to the synthesis of the C-10 epimeric mixture of 4-DAN

For their synthesis of the THF-alkene, 2.16, (-)-muricatacin 2.28b was converted into the α,β-unsaturated

ester and then cyclized back to yield the allylic alcohol. Then, it was converted to the epoxide, that was

subsequently converted into the diol and subjected to oxidative cleavage. The resulting aldehyde was

then treated with vinyl magnesium bromide and after removal of the protecting group, compound 2.16

was obtained in 8 steps with an overall yield of 4%.

Konno’s group obtained the butenolide partners, 2.26 and 2.27 by subjecting trans-3-pentenenitrile to an

osmium catalyzed dihydroxylation followed by acid catalyzed hydrolysis-lactonization that yielded the

racemic mixture of the hydroxylactone 2.29. The mixture was then separated by using a lipase-mediated

kinetic transesterification. Our approach to the butenolide 2.30 segment must be different because of the

presence of a hydroxyl group in the hydrophobic spacer. In both cases, the convergent nature of this CM

strategy is particularly attractive because of its synthetic efficiency and flexibility for later modification in

the synthesis of analogues.

Towards the synthesis of the epimeric mixture of 4-DAN, 2.12, we applied the same strategy developed in

our lab and then expanded by Konno’s group using the alkene partners 2.16 and 2.30 (Figure 2.5).

30

2.3. Synthesis

2.3.1. THF alkene, 2.16

The synthesis of the THF allylic alcohol 2.16 started with the known alcohol 2.31 that underwent a

Johnson Claisen rearrangement to deliver unsaturated ester 2.3224 (Scheme 2.2). LiAlH4 reduction on

2.32 provided the hydroxyalkene82 2.33, which was subjected to Sharpless dihydroxylation with AD-mix β

to give triol 2.34 in excellent yields.82 Treatment of 2.34 with 2,2-DMP and p-TsOH furnished the

isopropylidene derivative 2.35. Comparison of the NMR data of the (R)-MTPA Mosher ester of 2.35 with

the diastereomeric mixture of R-Mosher esters from racemic 2.35 (obtained via OsO4 dihydroxylation on

2.33), indicated that the enantiomeric excess of 2.34 was greater than 95%. Next, PCC oxidation on 2.35

provided aldehyde 2.36, which was reacted with methyl (triphenylphosphoranylidene) acetate to yield the

Wittig product 2.37, exclusively as the E-isomer. DIBAL-H reduction of 2.37 gave the primary alcohol

2.38. The key step in the synthesis of the THF ring was the iodoetherification reaction on 2.38. Previous

studies from our group83,84,78 have shown that the iodocyclization of related isopropylidene alkenes lead to

2,5-disubstituted THFs with high selectivity for the trans isomer.17, 78 Accordingly, exposure of 2.38 to NIS

and catalytic silver triflate afforded the tetrahydrofuran ring 2.39 as a single trans isomer, in 89% yield.78

After FCC purification, compound

2.39 formed crystalline needles

which were subjected to X-ray.

The presence of the heavy atom

allowed for the determination of

the absolute configuration of the

ring as a the enantiopure trans

form (Figure 2.6). Finally,

treatment of iodohydrin 2.39 with

dimethylsulfonium methylide

provided the tetrahydrofuran

alkene 2.16 in 60% yield, via an Figure 2.6. X-ray structure of compound 2.39

31

in situ, tandem epoxide formation-epoxide opening-elimination sequence of reactions. Compound 2.16

was obtained in 8 steps from 2.32 with a 6% overall yield. Protection of the diol using MOMCl afforded

compound 2.40.

Scheme 2.2. Synthesis of THF alkene 2.16

2.3.2. Butenolide alkene, 2.30

The synthesis of the diastereomeric mixture of butenolide 2.30 started with known compound 2.41.85 This

compound was prepared as the racemic mixture via the esterification of undecenoic acid with methanol

and the treatment of the resulting methyl 10-undecenoate ester with mCPBA. Copper (I) iodide assisted

epoxide opening with 4-pentenylmagnesium bromide and protection of the resulting alkenol as the

TBDPS ether, yielded 2.42. An stablished two step aldol-elimination sequence on 2.42 and the known

lactic acid 2.43 afforded the desired butenolide 2.3035 in 5 steps from 2.41 in 32% yield.

32

Scheme 2.3. Synthesis of butenolide 2.30

2.3.3. CM of THF and butenolide alkenes

Coupling of 2.16 (or MOM substituted 2.40) and 2.30 in DCM at rt in the presence of 10 mol% of Grubbs

second-generation catalyst gave compounds 2.44 and 2.45, respectively. Selective reduction of 2.44 and

2.45 using diamide conditions afforded 2.46 and 2.47. Subsequent removal of the silyl protecting group

with in situ generated methanolic HCl afforded 4-DAN as a mixture of C-10 epimers 2.12 and selective

deprotection of the TBDPS group in the presence of MOM using HF/pyridine delivered compound 2.48.

Compound 2.48 is relevant for the potential selective functionalization that the synthetic material can

afford and that usually proves very challenging in naturally occurring compounds.

33

Scheme 2.4. CM reaction and synthesis of C-10 epimers of 4-DAN

2.3.4. Comparison of naturally obtained 4-DAN and synthetic C-10 epimers

To confirm the authenticity of our synthetic mixture C-10 epimers, 2.12, we compared the 1H and 13C

NMR to the ones of naturally occurring 4-DAN reported in the literature,75 2.1 (Table 2.1, Figures 2.7 and

2.8). As previously observed for analogous epimers at C-9 of 4-DAR, NMR data for the C-10 epimers

showed as a single compound. Within the limits of experimental measurements, this data was essentially

identical to that of the natural product. This outcome is not surprising given the remoteness of the C-10

position from the other chiral centers.

34

Table 2.1. Naturally obtained vs synthetic 4-DAN

Position Naturally obtained (4-DAN) Synthesized (2.12)

1H (δ ppm) 13C (δ ppm) 1H (δ ppm) 13C (δ ppm)

1 - 174.00 - 174.16

2 - 134.25 - 134.47

3 2.26 t (7.0) 25.13 2.23 t (7.9) 25.36

10 3.58 m 71.88 3.55 broad s 71.88

17, 22 3.40 q (5.5) 74.01 3.37 broad q (5.5) 74.01

18, 21 3.80 q (7.5) 82.66,82.61 3.77 broad q (6.2) 82.66, 82.61

34 0.88 t (7.5) 14.07 0.85 t (6.3) 14.35

35 6.99 t (1.5) 148.88 6.96 broad s 148.88

36 5.00 qq (7.2) 77.41 4.97 broad q (6.7) 77.6

Figure 2.7. 1H chemical shifts of 4-DAN both naturally obtained and synthesized

3 10 17&22 18&21 34 35 36

Naturally obtained 2.26 3.58 3.4 3.8 3.8 0.88 6.99

Synthetic 2.23 3.55 3.37 3.77 3.77 0.85 6.96

Difference in ppm 0.03 0.03 0.03 0.03 0.03 0.03 0.03

1H chemical shifts

35

Figure 2.8. 13C chemical shifts of 4-DAN both naturally obtained and synthesized

The use of a cross coupling reaction towards the synthesis of THF-ACGs was first established in our

laboratory. CM is a versatile reaction that is compatible with a wide variety of functional groups. Since

then, other groups have adopted this strategy (see Chapter 1). Here, we established a modular strategy

for the synthesis of 4-DAN. By using different CM alkene partners, this protocol can be applied towards

the synthesis of diverse analog libraries. This will be the focus of Chapter 3.

1 2 3 10 17&22 18&21 34 35 36

Naturally obtained 174 134.25 25.13 71.88 74.01 82.66 82.61 14.07 148.88

Synthetic 174.16 134.47 25.36 71.88 74.01 82.66 82.61 14.35 148.88

Difference in ppm -0.16 -0.22 -0.23 0 0 0 0 -0.28 0

-20

0

20

40

60

80

100

120

140

160

180

200

13C chemical shifts

36

2.4. Experimental

General methods

Moisture and oxygen sensitive reactions were performed under an argon atmosphere. Solvents were

purified by standard procedures or used directly from commercial sources. Thin layer chromatography

(TLC) was done on 0.25 mm thick precoated silica gel HF254 aluminum sheets. Chromatograms were

observed under UV (short and long wavelength) light and were visualized by heating plates that were

dipped in a solution of ammonium (VI) molybdate tetrahydrate (12.5 g) and cerium (IV) sulfate

tetrahydrate (5.0 g) in 10 % aqueous sulfuric acid (500 mL). Flash column chromatography (FCC) was

performed using silica gel 60 (230-400 mesh) and employed a stepwise solvent polarity gradient,

correlated with TLC mobility. 1H and 13C NMR spectra were obtained on a Bruker 400, 500 and 600 MHz

spectrometer. Chemicals shifts are relative to the deuterated solvent peak and are in parts per million

(ppm). X-ray diffraction data were collected on a Bruker X8 Kappa Apex II diffractometer. Crystal data,

data collection and refinement parameters are summarized in Table S1-I. The structure was solved using

direct methods and standard difference map techniques and was refined by full-matrix least-squares

procedures on F2 with SHELXTL86 (Version 2014/7). The absolute configuration was established by

anomalous dispersion effects.87

THF-butenolide (2.12)

A mixture of 5% Ac2Cl in MeOH (0.5 mL) was added at rt to a solution of 2.46 (30 mg, 0.035 mmol) in

CH2Cl2 (1 mL). The mixture was stirred at this temperature for 4 h, diluted with CH2Cl2 and washed with

saturated aqueous NaHCO3. The organic layer was dried (Na2SO4), filtered, and concentrated under

reduced pressure. FCC of the residue afforded 2.12 (15 mg, 68%). Rf = 0.2 (60% EtOAc/hexanes). 1H

NMR (CDCl3, 500 MHz) δ 6.96 (s, 1H), 4.97 (m, 1H), 3.77 (m, 2H), 3.55 (m, 1H), 3.37 (m, 2H), 2.23 (m,

37

3H), 1.95 (m, 3H), 1.67 (m, 4H), 1.43-1.38 (m, 7H), 1.39 (d, partially buried, J = 6.8 Hz, 3H), 1.38-1.20 (s,

38H), 0.88 (t, J = 6.3 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 174.1, 149.1, 134.5, 82.8, 82.8, 77.7, 74.3,

74.2, 72.2, 37.7, 37.6, 33.7, 33.6, 32.1, 29.9 (4 signals), 29.8 (2 signals), 29.6, 29.5, 29.3, 29.0, 27.6,

25.8, 25.7, 25.4, 22.9, 19.4, 14.4. HRMS (ESI) calcd for C37H69O6 (M+H)+ 609.5094, found 609.5087.

THF-alkene (2.16)

To a solution of Me3SI (2.23 g, 10.4 mmol) in THF (11 mL) at -78 °C was added n-BuLi (3.6 mL, 9.1

mmol) under N2. The mixture was warmed to rt, maintained at this temperature for 1 h, then recooled to -

78 °C. A solution of compound 2.39 (0.5 g, 1.2 mmol) in THF (12 mL) was then introduced and the

mixture stirred at -78 °C for 2 h. A solution of sulfonium ylide in THF (11 mL), Me3SI (1.88 g, 9.1 mmol)

and n-BuLi (2.9 mL, 7.3 mmol) was prepared at -78 oC and then warmed to rt. Then, that portion was

added to the main reaction at -78 °C, let warm up to rt, and stirred for 2 h. The reaction was then

quenched with saturated aqueous NH4Cl and extracted with EtOAc. The organic phase was dried

(Na2SO4) filtered and evaporated under reduced pressure. The residue was subjected to FCC to give

2.16 (220 mg, 60%) as a clear oil. Rf = 0.57 (50% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 5.76 (m,

1H), 5.31 (d, J = 18.6 Hz, 1H), 5.17 (m, 1H), 3.92 (m, 1H), 3.82 (m, 2H), 3.38 (m, 1H), 2.93 (br s, 1H),

2.67 (br s, 1H), 1.93 (m, 2H), 1.64 (m, 2H), 1.34 (m, 2H), 1.21 (s, 20H), 0.74 (t, J = 6.9 Hz, 3H); 13C NMR

(CDCl3, 500 MHz) δ 136.8, 117.4, 83.1, 82.4, 75.8, 74.2, 33.5, 32.1, 29.9 (2 signals), 29.8 (4 signals),

29.5, 28.7, 28.6, 25.8, 22.9, 14.3. HRMS (ESI) calcd for C20H38O3Na (M+Na)+ 349.2719, found 349.2715.

Butenolide-alkene (2.30)

38

To a solution of diisopropylamine (23.2 mL, 0.16 mmol) in anhydrous THF (290 mL) at -78 oC was added

n-BuLi (46.3 mL, 2.5 M in hexane, 0.12 mol). The mixture was warmed to 0 oC and stirred at this

temperature for 10 min, then re-cooled to -78 oC. A solution of 2.42 (11.6 g, 0.02 mol) in anhydrous THF

(115 mL) was then added and the mixture maintained at -78 oC for 1 h, at which time a solution of 2.43

(11.07 g, 0.07 mmol) in anhydrous THF (115 mL) was slowly added. After 30 min, the reaction was

diluted with saturated aqueous NH4Cl and extracted with ethyl ether. The organic phase was dried

(Na2SO4) and concentrated under reduced pressure. FCC of the residue (20% EtOAc/hexanes) afforded

mixture of products (11.3 g). To the solution of this material in a mixture of MeOH (28 mL) and 2-propanol

(280 mL) was added TsOH.H2O (0.34 g, 1.79 mmol) at 0 oC. The reaction mixture was stirred at rt for 20

h, then concentrated under reduced pressure. The residue was purified by FCC (15-20 %

EtOAc/hexanes) to afford the derived lactone 2.30 as a yellow oil (6.4 g). To a solution of the material

obtained in the previous step (6.4 g) in CH2Cl2 (280 mL) was added Et3N (8.46 mL, 0.06 mol) and MsCl (2

mL, 0.02 mol) at 0 oC. After stirring at rt for 14 h, the reaction was diluted with saturated aqueous NH4Cl

and extracted with ethyl ether. The organic phase was dried (Na2SO4) and concentrated under reduced

pressure. The residue was purified by FCC to afford 2.30 (3.21 g, 52% after two steps from 2.42). 1H

NMR (CDCl3, 500 MHz) δ 7.65-7.63 (m, 4H), 7.42-7.31 (m, 6H), 6.94 (s, 1H), 5.76-5.67 (m, 1H), 4.96 (m,

1H), 4.87 (m, 1H), 4.83 (m, 1H), 3.67 (m, 1H), 2.22 (m, 2H), 1.91 (m, 2H), 1.16 (m, 3H), 1.15-1.07 (m,

18H), 1.03 (s, (H); 13C NMR (CDCl3, 500 MHz) δ 149.0, 139.2, 136.1, 135.0, 130.0, 129.5, 127.9, 127.5,

114.3, 77.3, 36.4, 36.3, 33.9, 29.7, 29.4, 29.3, 29.1, 27.5, 27.3, 26.7, 25.3, 25.0, 24.6, 19.6, 19.4. HRMS

(ESI) calcd C35H51O3Si for (M+H)+ 546.3529, found 546.3529.

Ethyl (E)-heptadec-4-enoate (2.32)

A mixture of compound 2.3182 (7.7 g, 34.0 mmol) in toluene (80 mL), triethyl orthoacetate (56.09 mL, 0.3

mol) and propionic acid (0.51 mL, 6.8 mmol), was heated at 125 °C under nitrogen for 4 h. The reaction

was then quenched with saturated aqueous NaHCO3 and extracted with EtOAc. The organic phase was

dried (Na2SO4) and evaporated under reduced pressure. The residue was subjected to FCC (2%

39

EtOAc/hexanes) to give product 2.32 (9.86 g, 98%) as a yellow oil. 1H NMR (CDCl3, 500 MHz) δ 5.37-

5.21 (m, 1H), 5.46-5.38 (m, 1H), 4.12-4.08 (m, 2H), 2.34-2.29 (d, J = 25.0 Hz, 2H), 2.29-2.27 (t, J = 11.6

Hz, 2H), 1.95-1.91 (q, J = 20.4 Hz, 2H), 1.22 (s, 20H), 0.86-0.84 (t, J = 13.7 Hz, 3H); 13C NMR (CDCl3,

500 MHz) δ 59.2, 33.4, 31.5, 30.9, 28.7, 28.1, 26.9, 21.7. HRMS (ESI) calcd for C19H37O2 (M+H)+

297.2794, found 297.2787. The NMR data was essentially identical to that reported in the literature.24

(E)-heptadec-4-en-1-ol (2.33)

LAH (2.63 g, 69.3 mmol) was added over 30 min to a solution of 2.32 (10.3 g, 34.7 mmol) in THF (400

mL) at 0 °C. The mixture was stirred at 0 °C under nitrogen for 1.5 h. Then, water (20 mL) and 1 M

aqueous NaOH (20 mL) was slowly added, sequentially, to the reaction mixture, after which anhydrous

Na2SO4 was added until a granular precipitate was obtained. The suspension was filtered, and the filtrate

was concentrated under reduced pressure. The residue was subjected to FCC (5% EtOAc/hexanes) to

give 2.33 (8.08 g, 91%) as a white solid (MP: 31-33⁰C). Rf = 0.4 (10% EtOAc/hexanes). 1H NMR (CDCl3,

500 MHz) δ 5.45-5.34 (m, 2H), 3.64 (m, 2H), 2.05 (m, 2H), 1.95 (m, 2H), 1.62 (m, 2H), 1.30-1.23 (br s,

20H), 0.86 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 131.5, 129.5, 62.8, 32.8, 32.7, 32.1, 29.9 (3

signals), 29.8, 29.7, 29.6, 29.4, 29.2, 22.9, 14.4. HRMS (ESI) calcd for C17H35O3 (M+H)+ 255.2688, found

255.2676.

(4R,5R)-heptadecane-1,4,5-triol (2.34)

Methanesulfonamide (2.0 g, 45.3 mmol), AD-mix β (18.5 g, 0.023 mol) in 1:1 (v/v) water:t-BuOH (100 mL)

was stirred for 1 h until the mixture was homogeneous. The mixture was cooled to 0 °C for 30 min at

which time an orange precipitate was formed. A solution of compound 2.33 (6.16 g, 0.17 mmol) in t-BuOH

(17 mL) was next added and the mixture was stirred at 0 °C for 48 h. The reaction was then quenched

40

with Na2SO3, filtered, extracted with EtOAc. The organic phase was concentrated in vacuo and the solid

residue recrystallized from ethyl acetate to give 2.34 (6.71 g, 95%) as white crystals (MP: 66-69⁰C). Rf =

0.15 (75% EtOAc/hexanes). 𝛼𝐷25 = 15.3. 1H NMR (CDCl3, 500 MHz) δ 3.72 (m, 2H), 3.47 (m, 2H), 3.08 (br

s, 1H), 2.33 (br s, 1H), 2.30 (br s, 1H), 1.77 (m, 2H), 1.58-1.42 (m, 4H), 1.28 (br s, 20H), 0.90 (t, J = 7.1

Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 74.9, 74.6, 63.2, 33.8, 32.1, 31.1, 29.9 (2 signals), 29.8 (2

signals), 29.6, 29.2, 25.9, 22.9, 14.3. HRMS (ESI) calcd for C17H36O3Na (M+Na)+ 311.2555, found

311.2562. The enantiomeric purity of 2.34 was determined to be greater than 95% by Mosher ester

analysis of its isopropylidene derivative 2.35 (vide infra).

(4R,5R)-4,5-O-isopropylidene-heptadecan-1-ol (2.35)

To a solution of 2.34 (8.55 g, 29.3 mmol) in CH2Cl2 (58 mL) was added 2,2-dimethoxypropane (4.32 mL,

35.1 mmol) and p-TsOH (0.56 g, 2.93 mmol). The mixture was stirred for 1 h, then quenched with

saturated aqueous NaHCO3 and extracted with CH2Cl2. The organic phase was dried (Na2SO4), filtered

and concentrated under reduced pressure. FCC of the residue (10-15% EtOAc/hexanes) afforded 2.35

(9.23 g, 96%) as a clear oil. Rf = 0.36 (35% acetone/hexanes). 1H NMR (CDCl3, 500 MHz) δ 3.65 (m, 2H),

3.59 (m, 2H), 2.24 (br s, 1H), 1.71 (m, 2H), 1.56-1.20 (m, 22H), 1.37 (s, partially buried, 6H), 0.86 (t, J =

7.1 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 108.2, 81.2, 81.1, 62.9, 32.9, 32.1, 29.9 (2 signals), 29.8 (3

signals), 29.7 (2 signals), 29.5, 27.5, 27.4, 26.3, 22.8, 14.3. HRMS (ESI) calcd for C20H40O3Na (M+Na)+

351.2875, found 351.2869.

41

(R)-MTPA ester of 2.35 (2.35a)

To a stirred solution of 2.35 (50 mg, 0.15 mmol) and (R)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl

chloride (77 mg, 0.30 mmol) in CH2Cl2 (10 mL) at 0 oC, DMAP (ca 2 mg) and DCC (63 mg, 0.30 mmol)

were added. The reaction was brought to rt and stirred for an additional 1 h at which time the mixture was

diluted with ethyl ether. The resulting suspension was filtered over Celite and the filtrate was washed with

brine and water. The organic phase was dried (Na2SO4), filtered and concentrated in vacuo. The crude

mixture was purified by FCC to give the R-MTPA-ester 2.35a as a white solid. Rf = 0.78 (30%

acetone/hexanes). 1H NMR (C6D6, 500 MHz) δ 7.68 (m, 2H), 7.09-6.96 (m, 3H), 4.10 (m, 1H), 4.05 (m,

1H), 3.49 (m 1H), 3.42 (s, 3H), 3.41 (partially buried m, 1H), 1.65-1.55 (m, 6H), 1.29 (s, 3H), 1.28 (s, 3H),

1.26 (m, 20H), 0.91 (t, J = 5.7 Hz, 3H); 13C NMR (C6D6, 500 MHz) δ 166.9, 133.5, 130.1, 130.0, 129.2,

129.0, 128.8, 128.7, 127.9, 125.6, 123.7, 108.5, 81.6, 81.0, 66.4, 55.7, 33.5, 32.7, 30.6, 30.5 (2 signals),

30.4 (2 signals), 30.2, 29.5, 28.0, 27.9, 27.1, 26.1 (2 signals), 26.0, 23.5, 14.7, 1.7. HRMS (ESI) calcd for

C30H47F3O5Na (M+Na)+ 567.3268, found 567.3249.

(R)-MTPA Ester of (+/-) 2.35 (2.35b)

2.33 (0.1 g, 0.3 mmol) was dissolved in a mixture of water and acetone (1:1, 2 mL). The reaction was

cooled down to 0 oC and osmium tetroxide in t-butanol (2.5% wt, 0.7 mL, 0.06 mmol) was added. Then,

N-methylmorpholine (0.04 mL, 0.4 mmol) was added and the reaction was stirred for 2 h at 0 oC. The

reaction was quenched with Na2SO3 (17 mg, 0.06 mmol) and stirred for 2 h. The product was extracted

using ethyl acetate and purified over FCC (70% EtOAc/hexanes) to afford a racemic mixture of the diol.

42

Rf = 0.2 (70% EtOAc/hexanes). The isopropylidene was installed using the same conditions as for the

chiral compound 2.35 and the primary alcohol was then converted to the R-MTPA-ester, 2.35b, was

purified using FCC (30-40% EtOAc/hexanes). Rf = 0.8 (35% acetone/hexanes). 1H NMR (C6D6, 400 MHz)

δ 7.66 (m, 2H), 7.05 (m, 3H), 4.07 (m, 2H), 3.89 (m, 1H), 3.50 (m, 1H), 3.47 (m, 1H), 3.38 (m, 3H), 3.33

(m, 3H), 1.55 (m, 2H), 1.40 (br s, 16H), 0.8 (m, 2H), 0.7 (m, 2H), 0.4 (m, 3H); 13C NMR (C6D6, 400 MHz) δ

165.4, 165.3, 132.0, 128.6, 128.5, 128.5, 127.7, 127.5, 127.4, 127.1, 127.0, 126.9, 126.7, 126.6, 126.1,

124.5, 121.7, 107.0, 84.1, 83.8, 80.1, 79.5, 64.9, 64.9, 60.9, 55.7, 54.1 (2 signals), 51.1, 48.9, 32.0, 31.3,

31.2, 30.2, 29.0 (2 signals), 28.9 (3 signals), 28.6, 28.0, 26.5, 26.4, 25.5, 25.3, 25.2, 24.6 (3 signals),

24.5, 23.6 (2 signals), 21.9, 13.2, 12.6.

(4R,5R)-4,5-O-isopropylidene-heptadecanal (2.36)

A suspension of powdered, freshly activated molecular sieves (7 g), Fluorosil (7 g), Celite (7 g),

CH3COONa (2.54 g, 31.2 mmol), PCC (6.61 g, 31 mmol) and 2.35 (6.84 g, 20.8 mmol) in CH2Cl2 (19 mL)

was stirred under nitrogen for 1 h. At that time the mixture was diluted with ether and filtered over

Fluorosil. The filtrate was concentrated under reduced pressure and the residue subjected to FCC to give

2.36 (4.42 g, 64%) as a yellow oil. Rf = 0.45 (15% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 9.74 (s,

1H), 3.54 (m, 2H), 2.57 (m, 2H), 1.90 (m, 1H), 1.68 (m, 1H), 1.46 (m, 2H), 1.34-1.19 (m, 20H), 1.34 (s,

partially buried, 6H), 0.86 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 202.1, 108.4, 81.0, 80.0, 40.7,

33.0, 32.1, 29.9 (2 signals), 29.8, 29.7, 27.5, 27.4, 26.3, 25.3, 22.9, 14.3.

Methyl (6R,7R,E)-6,7-O-isopropylidenenonadec-2-enoate (2.37)

43

To a solution of compound 2.36 (5.06 g, 15.5 mmol) in CH3CN (150 mL) was added methyl

(triphenylphosphoranylidene) acetate (15.5 g, 46.5 mmol). The mixture was heated at reflux for 1.5 h,

then filtered over Celite. The filtrate was concentrated under reduced pressure and the residue purified

by FCC to give 2.37 (4.67 g, 76%) as a clear oil. Rf = 0.85 (15% EtOAc/hexanes). 1H NMR (CDCl3, 500

MHz) δ 6.94 (m, 1H), 5.81 (m, 1H), 3.68 (s, 3H), 3.55 (m, 2H), 2.36 (m, 1H), 2.25 (m, 1H), 1.76 (m, 2H),

1.45 (m, 2H), 1.32-1.10 (m, 20H), 1.32 (s, partially buried 6H), 0.82 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3,

500 MHz) δ 167.2, 148.8, 121.4, 108.2, 81.0, 80.2, 51.6, 33.1, 32.1, 31.4, 29.9, 29.8 (3 signals), 29.7,

29.5, 29.0, 27.5, 27.4, 26.3, 22.9, 14.3. HRMS (ESI) calcd for C23H42O4Na (M+Na)+ 405.2981, found

405.2985.

(6R,7R,E)-6,7-O-isopropylidenenonadec-2-en-1-ol (2.38)

To a solution of compound 2.37 (1.4 g, 3.54 mmol) in CH2Cl2 (70 mL) was added a solution of 1 M

DIBAL-H in hexane (2.16 mL, 10.6 mmol) at -78 °C under nitrogen. The mixture was warmed to rt and

stirred at this temperature for an additional 1.5 h. Then water (10 mL) and 1 M aqueous NaOH (1 mL)

was slowly added, sequentially, to the reaction mixture after which anhydrous Na2SO4 was added until a

granular precipitate was obtained. The mixture was filtered over Celite and the filtrate was concentrated

under reduced pressure. FCC of the residue (10% EtOAc/hexanes) afforded 2.38 (0.7 g, 56%) as a clear

oil. Rf = 0.53 (25% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 5.66 (m, 2H), 4.06 (m, 2H), 3.57 (m,

2H), 2.23 (m, 1H), 2.10 (m, 1H), 1.69 (s, 1H), 1.56 (m, 4H), 1.32 (s, partially buried, 6H), 1.18 (m, 20H),

0.81 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 132.5, 129.6, 108.1, 81.2, 81.1, 63.9, 33.1, 32.6,

32.1, 30.0, 29.9, 29.8 (2 signals), 29.7, 29.5, 29.0, 27.5 (2 signals), 26.4, 22.9, 14.3. HRMS (ESI) calcd

for C22H42O3Na (M+Na)+ 377.3032, found 377.3026.

44

Iodohydrin (2.39)

To a solution of compound 2.38 (390 mg, 1.10 mmol) in CH3CN (100 mL) was added NIS (100 mg, 4.40

mmol) and AgOTf (6 mg, 0.02 mmol). The mixture was stirred at rt for 3 h, then diluted with a saturated

solution of Na2S2O3 and extracted with EtOAc. The organic phase was dried (Na2SO4), filtered and

evaporated in vacuo. FCC of the residue gave 2.39 (430 mg, 89%) as a white solid (MP: 47-49⁰C). Rf =

0.33 (25% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 4.23 (m, 1H), 4.11 (m, 1H), 4.00 (m, 1H), 3.95

(m, 2H), 3.46 (m, 1H), 2.85 (t, J = 13.1 Hz, 1H), 2.41 (m, 1H), 2.10 (m, 2H), 1.99-1.73 (m, 2H), 1.42 (m,

2H), 1.28 (s, 20H), 0.85 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 84.1, 83.1, 74.0, 68.0, 39.3,

34.3, 33.9, 32.1, 29.9 (2 signals), 29.8 (2 signals), 29.6, 28.2, 25.8, 22.9, 14.4 HRMS (ESI) calcd for

C19H37IO3Na (M+Na)+ 463.1685, found 463.1681.

THF alkene MOM protected (2.40)

2.16 (0.150 g, 0.4 mmol) were dissolved in 8 mL CH2Cl2. Then, MOMCl (0.14 mL,1.84 mmol) and DIPEA

(0.41 mL, 2.3 mmol) were added at 0 oC and the solution was stirred overnight. The reaction was then

quenched with water and extracted with DCM (3x). The organic phase was dried (NaSO4), concentrated

in vacuo and purified via FCC (10% EtOAc/hexanes) to afford compound 2.40 (0.18 g, quant) as a white

solid. Rf = 0.5 (20% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 5.71 (m, 1H), 5.28 (m, 1H), 5.24 (m,

1H), 4.82 (m, 1H), 4.65 (m, 4H), 3.99 (m, 3H), 3.37 (d, J = 2.8 Hz, 6H), 1.89 (m, 2H), 1.66 (m, 2H), 1.39

(m, 4H), 1.23 (m, 20H), 0.86 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 135.0, 118.9, 96.9, 94.2,

81.8, 81.1, 79.7, 79.4, 55.9, 55.5, 32.1, 31.3, 30.0, 29.9 (2 signals), 29.8, 29.6, 28.3, 28.2, 25.8, 22.9,

14.3. HRMS (ESI) calcd for C24H46O5Na (M+Na)+ 437.3243, found 437.3238.

45

Methyl 10-hydroxyhexadec-15-enoate (2.41a)

To a suspension of CuBr (I) (3.70 g, 0.26 mol) in THF (300 mL) at 0 oC was added 4-pentenylmagnesium

bromide (24 mL, 1M, 0.13 mol) over 5 min. A solution of 2.4185 (10.6 g, 43 mmol) in anhydrous THF (60

mL) was then introduced, dropwise over 5 min. The reaction was stirred 0 oC for 10 min and saturated

aqueous NH4Cl was added and the mixture extracted with ethyl ether. The organic phase was washed

with brine, dried (Na2SO4), filtered and evaporated under reduced pressure. The residue was purified to

give the derived alcohol 2.41a (10.2 g, 70%). Rf = 0.15 (10% EtOAc /hexanes). 1H NMR (CDCl3, 500

MHz) δ 5.77 (m, 1H), 4.92 (m, 1H), 4.91 (m, 1H), 3.63 (s, 3H), 3.53 (br s, 1H), 2.26 (m, 2H), 2.03 (m, 2H),

1.34-1.26 (m, 2H); 13C NMR (CDCl3, 500 MHz) δ 174.6, 139.1, 114.6, 72.1, 51.7, 37.6, 37.5, 34.3, 29.8,

29.6, 29.4, 29.3 (2 signals), 29.1, 25.8, 25.3, 25.1.

Methyl 10-((tert-butyldiphenylsilyl)oxy)hexadec-15-enoate (2.42)

A portion of the material from the previous step (7.31 g, 0.024 mol), TBDPSCl (6.24 mL, 0.024 mol) and

imidazole (8.33 g, 0.12 mol) in anhydrous THF (200 mL) were heated at reflux under nitrogen for 4 h. The

mixture was then diluted with water, extracted with ethyl ether and washed with brine. The organic phase

was dried (Na2SO4), filtered, and evaporated under reduced pressure. The residue was purified by FCC

to afford 2.42 (9.37 g, 89%) as a clear oil. Rf = 0.75 (10% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ

7.64 (m, J = 7.6 Hz, 4H), 7.36 (m, 6H), 5.72 (m, 1H), 4.89 (m, 1H), 4.87 (m, 1H), 3.67 (m, partially buried,

1H), 3.64 (s, 3H), 2.27 (t, J = 7.5 Hz, 2H), 1.91 (m, 2H), 1.08-1.25 (m, 20H), 1.04 (s, 9H); 13C NMR

(CDCl3, 500 MHz) δ 174.4, 139.1, 136.0, 134.8, 129.4, 127.4, 114.2, 73.2, 51.5, 36.3, 36.1, 34.1, 33.7,

29.6, 29.4, 29.2, 29.1, 29.0, 27.1, 25.0, 24.8, 24.4, 19.4. HRMS (ESI) calcd for C33H51O3Si (M+H)+

523.3602, found 523.3585.

46

THF-butenolide alkene (2.44)

A mixture of 2.16 (0.1 g, 0.3 mmol) and 2.30 (0.28 g, 0.51 mmol) in CH2Cl2 (2 mL) was purged with

nitrogen for 30 min. Grubbs 2nd generation catalyst (25 mg, 0.03 mmol) was then introduced and the

reaction stirred for 16 h at rt, after which DMSO (0.05 mL) was added and stirring continued for 30 min.

The mixture was then concentrated in vacuo. FCC of the residue (40% EtOAc/hexanes) afforded 2.44

(136 mg, 54%) as a clear oil. Rf = 0.65 (40% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 7.63 (m, 4H),

7.40-7.30 (m, 6H), 6.94 (s, 1H), 5.67 (m, 1H), 5.30 (m, 1H) 4.96 (m, 1H), 3.84-3.77 (m, 3H), 3.80 (m, 1H),

3.38 (m, 1H), 2.47 (br s, 1H), 2.28 (br s, partially buried, 1H), 2.21 (m, 2H), 1.90 (m, 4H), 1.63 (m, 2H),

1.45 (m, 3H), 1.33 (m, 10H), 1.22-1.14 (m, 30H), 1.00 (s, 9H), 0.85 (t, J = 6.6 Hz, 3H); 13C NMR (CDCl3,

500 MHz) δ 174.1, 149.0, 136.1, 134.9, 134.5, 129.6, 128.3, 127.6, 82.9, 82.7, 77.7, 75.9, 74.2, 73.3,

36.4, 36.3, 33.8, 32.5, 32.5, 32.1, 29.9 (4 signals), 29.8 (3 signals), 29.6, 29.4, 29.3, 28.7, 27.6, 27.3,

25.8, 25.4, 25.0, 24.6, 22.9, 19.6, 19.4, 14.3. HRMS (ESI) calcd C53H84O6SiNa (M+Na)+ 867.5935, found

867.5909.

THF-MOM-butenolide-alkene (2.45)

A mixture of 2.40 (0.180 g, 0.45 mmol) and 2.30 (0.4 g, 0.73 mmol) in CH2Cl2 (4 mL) was purged with

nitrogen for 30 min. Grubbs 2nd generation catalyst (31 mg, 0.04 mmol) was then introduced and the

reaction stirred for 16 h at rt, after which DMSO (0.05 mL) was added and stirring continued for 30 min.

The mixture was then concentrated in vacuo. FCC of the residue in 40% EtOAc/hexanes afforded 2.45

(0.26 g, 62%). Rf = 0.4 (20% EtOAc/hexanes).

47

THF-butenolide (2.46)

A solution of sodium acetate (0.62 g, 3 mmol) in water (12 mL) was added via a syringe pump, over 4 h,

to a mixture of 2.44 (85 mg, 0.10 mmol), p-toluenesulfonylhydrazide (1.25 g, 3 mmol) and DME (12 mL) at

reflux. After cooling to rt, the reaction mixture was poured into water and extracted with EtOAc. The

organic extract was washed with 2M HCl, water and brine, dried (Na2SO4), filtered and concentrated

under reduced pressure. FCC of the residue (1-5% acetone/DCM) gave 2.46 (0.04 g, 47%). Rf = 0.68 (2%

acetone/dichloromethane). 1H NMR (CDCl3, 500 MHz) δ 7.63 (m, 4H), 7.45-7.29 (m, 6H), 6.94 (s, 1H),

4.95 (m, 1H), 3.76 (m, 2H), 3.66 (m, 1H), 3.36 (m, 2H), 2.21 (m, 2H), 1.96 (m, 4H), 1.85 (br s, 2H), 1.36

(d, partially buried, J = 6.8 Hz, 3H), 1.34 (m, 10H), 1.25 (m, 36H), 1.00 (s, 9H), 0.85 (t, J = 7.1, 3H); 13C

NMR (CDCl3, 500 MHz) δ 174.1, 149.0, 136.1, 135.0, 134.5, 129.5, 127.5, 82.8, 74.2, 73.4, 36.5, 36.4,

33.7 (2 signals), 32.1, 31.8, 29.9 (2 signals), 29.8 (3 signals), 29.6, 29.4, 29.3, 28.9, 27.5, 27.3, 25.8,

25.8, 25.4, 25.0, 22.9 (2 signals), 19.6, 19.4, 14.3, 11.7. HRMS (ESI) calcd for C53H86O6SiNa (M+Na)+

869.6091, found 869.6068.

THF-MOM-butenolide(2.47)

A solution of sodium acetate (1.89 g, 0.023 mol) in water (57 mL) was added via a syringe pump, over 4

h, to a mixture of 2.45 (0.2 g, 0.21 mmol), p-toluenesulfonylhydrazide (1.7 g, 9.28 mmol) and DME (57

mL) at reflux. After cooling to rt, the reaction mixture was poured into water and extracted with EtOAc.

The organic extract was washed with 2 M HCl, water and brine, dried (Na2SO4), filtered and concentrated

under reduced pressure. FCC of the residue gave 2.47 (4 mg, 47%). Rf = 0.4 (20% EtOAc/hexanes). 1H

NMR (CDCl3, 500 MHz) δ 7.68 (m, 4H), 7.45-7.28 (m, 6H), 7.01 (s, 1H), 4.99 (m, 1H), 4.86 (m, 2H), 4.68

48

(m, 2H), 3.99 (m, 2H), 3.70 (m, 1H), 3.51 (m, 2H), 3.39 (m, 6H), 2.20 (m, 2H), 1.94 (m, 2H), 1.45 (m, 6H),

1.41 (m, 9H), 1.38 (m, 36H), 1.06 (s, 9H), 0.90 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C57H94O8SiNa

(M+Na)+ 957.6616, found 957.6601.

49

ANALOGUES OF 4-DEOXYANNOMONTACIN

50


THF-AGCs show potent activity against human cancer cell lines and therapeutic potential in animal

studies. However, their equally high toxicity to normal cells has limited their clinical development. Towards

more clinically viable analogues, we are interested in THF-ACGs that are easily accessed and more

selective to tumor cells. Accordingly, we designed a series of compounds based on the SAR trends for

the THF-ACGs discussed in Chapter 1. The potently active mono-THF-ACG 4-DAN was used as a

template for these structures. The modifications that we propose are: (i) replacement of the butenolide by

simpler non-chiral heterocycles or alkyl chains; (ii) substitution of the THF ring with a carbohydrate

moiety; (iii) variations in the hydrophobic spacer that connects the THF core or its replacement to the

butenolide or butenolide substitute (Figure 3.1). While certain of these individual modifications have been

previously shown to deliver compounds with similar activity to naturally occurring THF-ACGs, hybrid

structures that combine two or more such changes have not been widely tested. For instance, we have

shown that the THF core could be replaced with a cyclic sugar and other researchers have replaced the

butenolide with simple heterocycles, but the activity of analogues that combine both of these

modifications is unknown. Such hybrids are of interest in that they may be more easily prepared than less

modified analogues.

Figure 3.1. Structural characteristics of THF-AGEs and proposed replacements

51

As discussed earlier, previous studies from this laboratory on 4-deoxyannoreticuin,35 showed that both C-

10 epimers were similarly active. Based on this result, for synthetic simplicity we decided to prepare our

new analogues of DAN as an epimeric mixture at the corresponding C-9 carbinol in 4-DAN.

3.2. Butenolide substitutes

3.2.1. Piperazine

Zen and others88 have evaluated ACG mimetics like AA005 3.1, in which the bis-THF ring is replaced with

an ethylene glycol ether moiety (Figure 3.2). These simplified mimetics presented activities comparable to

the natural products. More recently, the same group examined even simpler mimics in which the

butenolide in these acyclic analogues was replaced with simple achiral heterocycles (cf 3.2).41 These

compounds still showed low micromolar activity. We are particularly interested in the analog containing a

terminal piperazine, 3.3, not just because it exhibited the most potent antiproliferation activity but also

because the secondary amine provides a handle for prodrugs.

Figure 3.2. Piperazine analogues

52

3.2.2. Thiophene

Kojima also investigated small heterocycles as butenolide substitutes. In the initial study, an amide linked

N-methylpyrazole, showed selectivity towards the same cell lines as the alkyl-linked equivalent, but with

higher potencies (Figure 3.3).39 In a later study, other amide-linked heterocycles, including furan or

thiophene were evaluated.42, 44, 46 Both the thiophene-3-carboxamide and thiophene-2-carboxamide

showed high potencies across a panel of 39 cell lines. The 3-carboxamide derivative was used in vivo

studies and inhibited tumor growth of the NCI-H23 xenografts implanted in mice. Following this study, we

decided to replace the butenolide in 4-DAN with a thiophene-2-carboxamide moiety. Our analogues differ

from Kojima’s with respect to the length of the hydrophobic chain that connects the THF and the

heterocycle, and the presence of the hydroxyl group in the middle of the chain.

Figure 3.3. Thiophene derivative

53

3.2.3. O-butyl

Other groups have examined simpler replacements, such as a n-butyl ester in the case of squamocin

(Figure 3.4).89 This analog was several orders of magnitude less active than squamocin but was still in

the low micromolar range. Interestingly, its activity against complex I was comparable to the natural

product. To probe whether the carbonyl group of the ester was important for activity, we targeted the n-

butyl ether derivative of 4-DAN, 3.7.

Figure 3.4. Simple n-alkyl derivative

3.3. Tetrahydrofuran substitutes

3.3.1. Mannose replacement

Several reviews have been published on the modification of the THF ring.90,91,92 Our group has showed

that replacing the THF core with a sugar residue delivered compounds with low micromolar level activity

that was comparable to the mono-THF-ACG, (9S)-4-deoxyannonereticuin (DAR), 3.8 (Figure 3.5).79

54

Replacement of the THF ring with a carbohydrate is attractive as stereochemically diverse sugars are

readily available, the sugar motif may act as a vector for tumor targeting, and their heavily hydroxylated

structures may increase hydrophilicity, which may be important for drug pharmacokinetics.

Figure 3.5. Activity of DAR 3.8 and mannose analog 3.9

Mannose was chosen as a THF replacement because its framework can be relatively easily

functionalized, and is a potential tumor vector, for example to glucose transporters (GLUT), which are

overexpressed on tumor cells, or other mannose specific receptors.93 In our initial study on sugar

analogues of DAR, the alkyl chain was attached at the O6 position of mannose. To further develop these

mannose-derived ACG mimetics, we were interested in the effect of altering the length and position of the

alkyl chain. This is important as related modifications in the hydrophobic chains in the natural THF-ACGs

Cell Line Jurkat (µM) HeLa (µM) MDA-MB-231 (µM) PC-3 (µM)

DAR (3.8) 17.79 20.99 34.16 43.05

Mannose analog of

DAR (3.9) 10.11 15.17 21.18 32.56

55

are known to affect both potency and selectivity. Accordingly, the new sugar analogues of 4-DAN 3.10 -

3.14 were targeted (Figure 3.6).

Figure 3.6. THF replacement analogues

3.4. Hybrid structures

An important question is whether the aforementioned SARs regarding THF and butenolide replacements

are additive. With that in mind, hybrid compounds which both the THF ring and the butenolide are

replaced with mimicking entities, were of interest (Figure 3.7). To probe this question, we targeted hybrid

structures comprising the 6-O-undecyl mannose residue and piperazine or thiophene moieties as THF

and butenolide replacements, respectively (cf 3.15 and 3.16).

Figure 3.7. Hybrid compounds

56

3.5. Modification of the hydrocarbon spacer

It has been proposed that the hydrophobic spacer connecting the THF and the butenolide controls the

active spatial arrangement between the THF and the butenolide.91 Consistent with this hypothesis, 14-

and 12- carbon chains appear to be optimal for the mono and bis-THF ACGs, respectively.11c The position

and number of hydroxyl groups also impacts on activity. Some studies suggest that having too many

alcohols in the middle chain can lower the activity of the compounds as an increase in hydrophilicity may

decrease mitochondrial uptake. Against this background we targeted the deoxygenated sugar-piperazine

hybrid structure 3.17 (Figure 3.8).

Figure 3.8. Deoxygenated analog

3.6. Synthetic strategy

The overall synthetic plan for these new analogues follows the CM approach described in Chapter 2 for 4-

DAN. Thus, the THF-alkene 3.18 (2.16 in Chapter 2), or one of its mannose substitutes 3.19-3.23, were

combined with either the butenolide alkene 3.24 (2.32 in Chapter 2) or its replacements 3.25-3.28.

57

THF and replacements Butenolide and replacements

Figure 3.9. Coupling alkene partners for the synthesis of analogues of 4-DAN

58

3.7. Synthesis

3.7.1. Mannose alkenes

For efficiency, the synthesis of the different mannose alkenes was conducted in a divergent fashion from

a known precursor, 1-propenyl 2,3-O-isopropylidene mannopyranoside 3.32. This material can be

obtained in multi-gram quantity, in a straightforward fashion, over three steps from D-mannose: (i) acid

promoted glycosidayion of mannose with allyl alcohol, (ii) isopropylidenation of the product 3.30 to give

the bis-O-isopropylidene 3.31, and (iii) selective removal of the 4,6-O-isopropylidene (Scheme 3.1). The

yield over these three steps was 35%.

Scheme 3.1. Synthesis of mannose alkenes

59

Alkylation of 3.32 using one equivalent of base in THF provided a separable mixture of 3.19 (27%) and

3.20 (13%). For a streamlined synthesis of 3.19, the primary alcohol on 3.32 was first selectively

protected as the its TBDPS ether. Then, protection of the secondary alcohol by treatment with ethyl vinyl

ether and PPTS and desilylation of the resulting product provided 3.33. Alkylation of 3.33 with undecyl

bromide and treatment of the product under mildly acidic conditions yielded 3.19 (65%). Similarly,

butylation on 3.33 afforded 3.21 (58%) and methylation on 3.20 and 3.33 afforded 3.23 (64%) and 3.22

(90%), respectively.

3.7.2. Butenolide replacements

The precursor for the butenolide replacements was alkenol 3.35, which was obtained from the LAH

reduction of ester 3.34 (Scheme 3.2). Alkenes 3.25 and 3.26, the CM precursors for the piperazine and

butyl ether analogues, were obtained from butylation and mesylation on 3.35, respectively.

Scheme 3.2. Synthesis of butenolide replacements

60

Reaction of 3.26 with ethanolic ammonia and amidation of the resulting amine 3.36 with thiophene 2-

carboxylic acid provided 3.27, the CM precursor for the thiophene analogues.

3.7.3. Cross metathesis and final products

The CM reactions for the THF analogues were performed under similar conditions to those used in the

synthesis of 4-DAN in Chapter 2. 77, 80 Thus, reaction of THF alkene 3.18 with 2 equivalents of alkenes

3.25-2.37, in the presence of 10 mol% of 2nd generation Grubbs catalyst, in DCM at room temperature

for 20 h afforded CM products 3.37, 3.38 and 3.39 in 40, 27, 23% yields, respectively. Hydrogenation of

these products provided the reduced derivatives, which were transformed via straightforward substitution,

alkylation and desilylation procedures to give the final products 3.3, 3.4 and 3.7 respectively (Scheme

3.3).

Scheme 3.3. Synthesis of the compounds with butenolide replacements

61

For the synthesis of the mannose containing analogs, 3.10-3.14, modified CM conditions using Grubbs I

catalyst at 40 oC were needed (Scheme 3.4).94 The mannose butenolide metathesis products 3.40-3.44

were obtained in 25-96% yields. Subsequently, the alkene in the CM product was selectively reduced with

diimide as described in the earlier synthesis of 4-DAN, and the isopropylidene and silyl protecting groups

on the sugar were removed using acidic conditions to afford 3.10-3.14. The yields for the deprotection

steps were low as these reactions were not optimized. In the later compounds, after optimization, these

yields were higher.

Scheme 3.4. Synthesis of mannose containing compounds

CM between the 6-O-undecenyl-mannose alkene 3.19 and the mesylate containing alkenes, 3.26 and

3.28, and the thiophene alkene 3.27 provided 3.45, 3.46 and 3.47 respectively (Scheme 3.5)

Hydrogenation of 3.45 and 3.46, treatment of the reduced derivatives with piperazine, and acid promoted

removal of isopropylidene and TBDPS protecting groups provided the mannose-piperazine hybrid

compounds 3.15 and 3.17 respectively. Similarly, hydrogenation of 3.47 and removal of protecting groups

afforded the mannose-thiophene hybrid 3.16.

62

Scheme 3.5. Route to hybrid analogues

3.8 Summary

In Chapter 3, the CM methodology that was used for the synthesis of the 4-DAN in Chapter 2, was

applied to THF analogues of 4-DAN with butenolide substitutes, 3.3, 3.4 and 3.7. It has been shown that

the butenolide residue in other ACGs or mimetics thereof, can be replaced with heterocycles such as

thiophene or piperazine without loss in activity. Biological evaluation of such heterocyclic analogues of

DAN is needed to determine the generality of this trend. The CM methodology was also applied to ACG

analogues in which the THF moiety is replaced with a mannose residue, and the butenolide is preserved

or replaced with piperazine or thiophene residues, i.e. 3.10-3.17. These carbohydrate-derived structures

which follow from earlier studies in this laboratory and the aforementioned observations on butenolide

replacement, are attractive for their relatively easy accessibility and for the potential of the sugar motif to

act as a vector for tumor targeting. Biological evaluation of this wider set of sugar-derived analogues

63

would expand the SARs for these less well-known carbohydrate analogues and pave the way to their

possible development as therapeutic agents. The development of potentially more tumor selective ACGs

and antitumor evaluation are the foci of Chapters 4 and 5.

64

3.9. Experimental

THF-piperazine (3.3)

Compound 3.37b (29 mg, 0.033 mmol) was dissolved in CH2Cl2 (2 mL) and treated with 5% AcCl in

MeOH (0.5 mL). The reaction was stirred for 24 h and then quenched with a saturated solution of

NaHCO3 in MeOH. The mixture was filtered and concentrated in vacuo. FCC of the residue (10%

MeOH/DCM) afforded 3.3 (3 mg, 14%). Rf = 0.6 (10% MeOH/DCM). 1H NMR (CD3OD, 600 MHz) δ 3.72


1.50-1.28 (m, 50H), 0.98 (t, J = 8.7 Hz, 3H); 13C NMR (CD3OD, 600 MHz) δ 84.0, 75.3, 72.6, 59.4, 50.3,

45.0, 38.6, 34.4, 33.3, 31.0, 30.8, 30.7 (2 signals), 29.8, 26.9, 23.9, 14.6. HRMS (ESI) calcd for

C38H77N2O4 (M+H)+ 625.5883, found 625.5874.

THF-thiophene (3.4)




MeOH/DCM) afforded 3.4 (7 mg, 33%). Rf = 0.4 (60% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ

7.40 (m, 1H), 7.38 (m, 1H), 7.00 (m, 1H), 5.88 (br s, 1H), 3.73 (m, 2H), 3.60 (m, 1H), 3.34 (m, 4H), 3.28

(m, 2H), 2.24 (m, 2H), 1.91 (m, 3H), 1.55-1.00 (m, 50H), 0.95 (t, J = 6.6 Hz, 3H); 13C NMR (CDCl3, 600

MHz) δ 161.8, 139.1, 129.6, 127.8, 127.6, 82.6, 74.0 (2 signals), 72.0 (2 signals), 59.0, 40.0, 37.5, 37.1,

33.5 (2 signals), 32.8, 31.9, 30.0, 29.7 (2 signals), 29.6 (2 signals), 29.5, 29.4 (2 signals), 29.2, 28.7,

65

28.0, 27.1, 26.9, 25.6 (3 signals), 25.5, 24.9, 24.5, 24.1, 22.7, 21.9, 19.8, 14.1, 13.6, 12.6, 11.4. HRMS

(ESI) calcd for C39H72NO5S (M+H)+ 666.5131, found 666.5127.

THF-butyl ether (3.7)

Compound 3.39 (50 mg, 0.047 mmol) was dissolved in EtOAc (4 mL). The solution was degassed for 30

min and Pd/C (10%, 5 mg) was introduced. Then, it was stirred under H2 atmosphere for 20 h. The

solution was filtered over Celite, concentrated in vacuo. FCC of the residue (30-35% EtOAc/hexanes)

yielded 3.39a (42 mg, 84%). Rf = 0.4 (40% EtOAc/hexanes).

Compound 3.39a (42 mg, 0.049 mmol) was dissolved in CH2Cl2 (2 mL) and treated with 5% AcCl in

MeOH (0.5 mL). The reaction was stirred overnight and then quenched with a saturated solution of


MeOH/DCM) afforded 3.7 (15 mg, 51%). Rf = 0.4 (60% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ

3.76 (m, 2H), 3.37 (m, 1H), 3.37 (m, 6H), 1.95 (m, 2H), 1.66 (m, 2H), 1.56-1.39 (m, 54H), 0.88 (t, J = 7.4

Hz, 3H), 0.84 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 82.6, 74.0 (2 signals), 72.0, 71.0, 70.6 (2

signals), 37.5 (3 signals), 33.5 (2 signals), 31.9 (2 signals), 29.8, 29.7 (4 signals), 29.6 (3 signals), 29.5 (2

signals), 29.4, 28.7, 26.2, 25.7, 25.6 (3 signals), 25.5, 22.7, 19.4, 19.2, 14.1. HRMS (ESI) calcd for

C38H77O5 (M+H)+ 613.5771, found 613.5760.

6-O-Undecyl mannoside-butenolide (3.10)

66


MeOH (0.5 mL). The reaction was stirred for 24 h and then quenched with a saturated solution NaHCO3

in MeOH. The mixture was filtered and concentrated in vacuo. FCC of the residue (5-10% MeOH/DCM)

afforded 3.10 (5 mg, 20%). Rf = 0.5 (5% MeOH/DCM). 1H NMR (CDCl3, 600 MHz) δ 5.96 (s, 1H), 4.97 (m,

1H), 4.80 (s, 1H), 3.90 (s, 1H), 3.81 (m, 1H), 3.76-3.36 (m, 13H), 2.24 (m, 2H), 1.45-1.20 (m, 42H), 1.37

(partially buried d, J = 6.8 Hz, 3H), 0.86 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 600 MHz) 148.9, 134.3, 99.5,

72.2, 71.9, 71.8, 71.7, 70.8, 70.5, 69.1, 67.8, 37.5, 31.9, 29.7, 29.6 (2 signals), 29.5 (2 signals), 29.3 (3

signals), 29.2, 29.1, 27.4, 26.1, 26.0, 25.6, 25.5, 25.2, 22.7, 19.2, 14.1. HRMS (ESI) calcd for C37H69O9

(M+H)+ 657.4942, found 657.4930.

4-O-Undecyl mannoside-butenolide (3.11)

Compound 3.41 (60 mg, 0.064 mmol) was transformed to 3.41a following the reduction protocol

described for preparation of 3.40a. FCC in 1-5% (acetone/DCM) delivered 3.41a (35 mg, 58%). Rf = 0.3

(5% acetone/DCM). 1H NMR (CDCl3, 600 MHz) δ 7.59 (m, 4H), 7.31 (m, 6H), 6.90 (s, 1H), 4.99 (m, 2H),

4.18 (m, 1H), 4.10 (m, 1H), 3.77 (m, 2H), 3.65-3.42 (m, 4H), 3.31 (m, 1H), 2.17 (m, 2H), 2.03 (br s, 1H),

1.61-1.03 (m, 51H), 0.97 (s, 9H), 3.43 (t, J = 6.7 Hz, 3H.


MeOH (0.5 mL). The reaction was stirred overnight and then quenched with saturated solution of

NaHCO3 in MeOH. The mixture was filtered and concentrated in vacuo. FCC of the residue (5-10%

MeOH/DCM) afforded 3.11 (4 mg, 17%). Rf = 0.5 (5% MeOH/DCM). 1H NMR (CDCl3, 600 MHz) δ 6.95 (s,

1H), 5.00 (m, 1H), 4.78 (s, 1H), 3.90 (m, 1H), 3.80 (m, 1H), 3.75-3.48 (m, 8H) 3.37 (m, 1H), 2.23 (m, 2H),

1.54-1.22 (m, 42H), 1.28 (partially buried d, J = 5.0 Hz, 3H), 0.85 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3,

600 MHz) δ 174.1, 148.8, 134.4, 99.6, 76.2, 73.6, 72.2, 71.8, 71.6, 71.3, 68.0, 62.4, 37.6, 32.1, 30.6,

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29.9, 29.8 (2 signals), 29.7 (2 signals), 29.6, 29.5, 29.4, 29.3, 27.6, 26.3, 26.2, 25.8, 25.7, 25.4, 22.9,

19.4, 14.3. HRMS (ESI) calcd for C37H68O9Na (M+Na)+ 679.4761, found 679.4754.

6-O-Butyl mannoside-butenolide (3.12)

Compound 3.42a (60 mg, 0.07 mmol) was dissolved in CH2Cl2 (5 mL) and treated with 5% AcCl in MeOH

(1 mL). The reaction was stirred for 24 h and then quenched with a saturated solution of NaHCO3 in

MeOH. The mixture was filtered and concentrated in vacuo. FCC of the residue (5-10% MeOH/DCM)

afforded 3.12 (5 mg, 14%). Rf = 0.6 (5% MeOH/DCM). 1H NMR (CDCl3, 600 MHz) δ 6.95 (s, 1H), 4.97 (m,

1H), 4.78 (s, 1H), 3.87 (m, 1H), 3.90-3.60 (m, 5H), 3.50 (m, 4H), 3.38 (m, 1H), 2.35 (m, 2H), 2.22 (m, 3H),

1.65-1.10 (m, 28H), 0.87 (m, 3H); 13C NMR (CDCl3, 600 MHz) δ 174.1, 149.1, 136.1, 134.4, 99.7, 72.1,

72.0, 71.9, 71.6 (2 signals), 70.7, 70.3, 69.7, 67.9, 37.6, 32.1, 31.7, 29.8 (2 signals), 29.6, 29.5, 29.4, 29.3

(2 signals), 29.2, 29.0, 28.7, 27.5 (2 signals), 27.2, 27.1, 26.1 (2 signals), 25.7, 25.6, 25.3, 25.0, 23.9 (2

signals), 22.8 (2 signals), 19.4, 19.3, 14.0. HRMS (ESI) calcd C30H55O9 for (M+H)+ 559.3846, found

559.3837.

6-O-Methyl mannoside-butenolide (3.13)

Compound 3.43a (54 mg, 0.06 mmol) was dissolved in CH2Cl2 (2 mL) and treated with 5% AcCl in MeOH

(0.5 mL). The reaction was stirred for 24 h and then quenched with a saturated solution of NaHCO3 in

MeOH. The mixture was filtered and concentrated in vacuo. FCC of the residue (5-10% MeOH/DCM)

afforded 3.13 (3 mg, 7%). Rf = 0.7 (5% MeOH/DCM). 1H NMR (CD3OD, 500 MHz) δ 7.79 (s, 1H), 7.14 (s,

68

1H), 4.94 (m, 1H), 4.57 (s, 1H), 3.64-3.18 (m, 11H), 3.28 (partially buried s, 3H), 2.10 (m, 2H), 1.26 (m,

27H). HRMS (ESI) calcd for C27H48O9Na (M+Na)+ 539.3196, found 539.3190.

6-O-Methyl-4-O-undecyl mannoside-butenolide (3.14)



NaHCO3 in MeOH. The mixture was filtered and concentrated in vacuo. FCC of the residue (60-80%

EtOAc/hexanes) afforded 3.14 (3 mg, 16%). Rf = 0.5 (60% EtOAc/hexanes). 1H NMR (CD3OD, 500 MHz)

δ 7.13 (s, 1H), 4.93 (m, 1H), 4.54 (s, 1H), 3.70 (m, 1H), 3.55 (m, 3H), 3.50-3.30 (m, 5H), 3.25 (m, 2H),

3.24 (partially buried s, 3H), 2.11 (m, 2H), 1.44 (m, 6H), 1.16 (m, 42H), 0.77 (t, J = 7.5 Hz, 3H). HRMS

(ESI) calcd for C38H70O9Na (M+Na)+ 693.4918, found 693.4910.

6-O-Undecyl mannoside-piperazine (3.15)


MeOH (1 mL). The reaction was stirred for 24 h and then quenched with a saturated solution of NaHCO3

in MeOH. The mixture was filtered and concentrated in vacuo. FCC of the residue (10% MeOH/DCM)

afforded 3.15 (7 mg, 18%). Rf = 0.3 (20% MeOH/DCM). 1H NMR (CD3OD, 600 MHz) δ 4.58 (s, 1H), 3.65-

3.24 (m, 11H), 3.18 (m, 4H), 2.88 (m, 4H), 2.48 (m, 2H), 1.64-1.04 (m, 46H), 0.88 (m, J = 7.5 Hz, 3H); 13C

NMR (CD3OD, 600 MHz) δ 101.7, 73.5, 72.9, 72.8, 72.6, 72.3, 71.9, 69.1, 68.8, 59.3, 51.4, 45.0, 38.6 (2

69

signals), 33.2, 31.0 (2 signals), 30.9 (3 signals), 30.8, 30.7 (2 signals), 30.6, 27.5 (2 signals), 26.9 (2

signals), 23.9, 14.6. HRMS (ESI) calcd for C38H77N2O7 (M+H)+ 673.5731, found 673.5724.

6-O-Undecyl mannoside-thiophene (3.16)

3.46 (70 mg, 0.07 mmol) was dissolved in EtOH (2 mL). Then 10% wt (0.7 mg) Pd/C was added and the

mixture was maintained under H2 for 16 h (2x). The mixture was filtered over Celite and the filtrate

evaporated in vacuo. The residue was dissolved in DCM (2 mL) and treated with 5% AcCl in MeOH (0.4

mL) for 4 h. Additional 5% AcCl in MeOH (0.2 mL) was added over 9 h. The mixture was neutralized with

K2CO3 in MeOH, filtered through Celite and purified by FCC (60-100% EtOAC/hexanes) to yield 3.16 (14

mg, 65%).1H NMR (CDCl3, 600 MHz) δ 7.46 (d, J = 3.6 Hz, 1H), 7.42 (d, J = 4.9 Hz, 1H), 7.04 (dd, J1 =

4.9 Hz, J2 = 3.8 Hz, 1H), 5.99 (br s, 1H), 4.79 (s, 1H), 3.88 (br s, 1H), 3.79-3.63 (m, 6H), 3.54 (m, 1H),

3.45 (m, 2H), 3.37 (m, 3H), 1.56 (m, 6H), 1.36 (m, 6H), 1.38-1.22 (m, 37H), 0.85 (t, J = 6.8 Hz, 3H); 13C

NMR (CDCl3, 600 MHz) δ 162.1, 139.3, 129.8, 128.1, 127.8, 99.7, 72.3, 72.1, 71.9, 70.7, 67.9, 40.2, 37.6

(2 signals), 32.1, 29.8 (4 signals), 29.7 (2 signals), 29.6 (2 signals), 29.5, 29.4 (2 signals), 27.1, 26.2,

25.8, 25.6, 22.9, 14.3. HRMS (ESI) calcd for C39H71NO8SNa (M+Na)+ 736.4793, found 736.4782.

6-O-Undecyl mannoside-deoxypiperazine (3.17)


MeOH (0.5 mL). The reaction was stirred for 2 h and then quenched with a solution of methanol and

70

NaHCO3. The mixture was filtered and concentrated in vacuo. FCC of the residue (5% MeOH/DCM)

afforded 3.17 (15 mg, 38%). Rf = 0.2 (10% MeOH/DCM). 1H NMR (CD3OD, 600 MHz) δ 4.62 (s, 1H), 3.66

(m, 3H), 3.58 (m, 3H), 3.44 (m, 3H), 3.33 (m, 1H), 3.12 (m, 4H), 2.65 (br s, 4H), 2.41 (br s, 2H), 1.53-1.44

(m, 6H), 1.21 (m, 42H), 0.82 (t, J = 6.8 Hz, 3H); 13C NMR (CD3OD, 600 MHz) δ 101.7, 73.5, 72.9, 72.8,

72.3, 72.0, 69.0, 68.8, 59.3, 51.2, 44.9, 33.3, 31.0, 31.0 (3 signals), 30.9 (4 signals), 30.8, 30.7 (3

signals), 28.5, 27.6, 27.5, 23.9, 14.6. HRMS (ESI) calcd for C38H77N2O6 (M+H)+ 657.5782, found

657.5775.

Propan-1-yl-2,3-O-isopropylidene-6-O-undecyl-α-D-mannopyranoside (3.19)

3.32 (1.30 g, 5 mmol) and TBAI (0.32 g, 1 mmol) were dissolved in THF (36 mL) under a N2 atmosphere.

The reaction was cooled to 0 oC and NaH (0.22 g, 5.5 mmol) was added. The reaction was warmed

slowly to rt, then recooled to 0 oC and bromoundecane (1.23 mL, 5.5 mmol) added. The mixture was

stirred for 2 h, quenched and purified via FCC (40-50% EtOAc/hexanes), to give: 0.2 g of dialkylated

material, 3.19 (0.16 g, 13%), 3.20 (0.34 g, 26%), 3.32 (0.50 g, 38% bsrm) and 4,6-di-O-undecenyl

derivative (0.20 g, 7%).

Compound 3.19 was also prepared from 3.33. Compound 3.33 (1.51 g, 4.5 mmol) and TBAI (0.29 g, 0.9

mmol) were dissolved in DMF (22 mL) under a N2 atmosphere. The mixture was cooled to 0 oC and NaH

(0.2 g, 5.0 mmol) was added. The reaction was warmed to rt over 30 min. Then, it was cooled down to 0

oC and undecenyl bromide (1.18 mL, 5 mmol) was added. The reaction was stirred overnight at rt, then

cooled to 0 oC and quenched slowly with water. The mixture was extracted with ethyl ether and the

organic phase washed with brine, dried (NaSO4) and evaporated in vacuo. The residue was resuspended

in MeOH and treated with PPTS (ca 0.5 g, pH 3-4). The mixture was stirred for 1 h, then, quenched with

saturated NaHCO3 in MeOH. The solution was filtrated and concentrated under pressure. FCC of the

residue (40-50% EtOAc/hexanes) afforded 3.19 (1.22 g, 65%). 1H NMR (CDCl3, 500 MHz) δ 5.89 (m, 1H),

71

5.28 (m, 1H), 5.20 (m, 1H), 5.03 (s, 1H), 4.20-4.10 (m, 3H), 3.99 (m, 1H), 3.73-3.60 (m, 4H), 3.47 (m, 2H),

2.93 (br s, 1H), 1.57 (m, 2H), 1.50 (s, 3H), 1.33 (s, 3H), 1.33-1.23 (m, 16 H), 0.85 (t, J = 6.0 Hz, 3H); 13C

NMR (CDCl3, 500 MHz) δ 133.7, 118.2, 109.7, 96.5, 78.2, 75.5, 72.3, 71.8, 71.7, 68.3, 68.0, 32.1, 29.8 (3

signals), 29.7, 29.5, 28.1, 26.3 (2 signals), 22.9, 14.34. HRMS (ESI) calcd for C23H42O6Na (M+Na)+

437.2879, found 437.2879.

Propan-1-yl-2,3-O-isopropylidene-4-O-undecyl-α-D-mannopyranoside (3.20)

1H NMR (CDCl3, 500 MHz) δ 5.86 (m, 1H), 5.26 (m, 1H), 5.19 (m, 1H), 5.04 (s, 1H), 4.20-4.11 (m, 3H),

3.94 (m, 1H), 3.74 (m, 2H), 3.71 (m, 1H), 3.60 (m, 1H), 3.44 (m, 1H), 3.36 (m, 1H), 1.52 (m, 5H), 1.51

(partially buried s, 3H), 1.33 (s, 3H), 1.23 (m, 14H), 0.85 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ

133.6, 118.2, 109.4, 96.4, 78.8 (2 signals), 75.9, 71.8, 68.6, 68.3, 62.9, 32.1, 30.3, 29.8, 29.7, 29.6, 28.2,

26.5, 26.3, 22.9, 14.3. HRMS (ESI) calcd for C23H42O6Na (M+Na)+ 437.2862, found 437.2874.

Propan-1-yl-2,3-O-isopropylidene-6-O-butyl-α-D-mannopyranoside (3.21)

Compound 3.21 was obtained from compound 3.33 (0.33 g, 0.99 mmol) following the procedure for 3.19

with 1-bromobutane (0.16 mL, 1.48 mmol) as the alkylating agent. After purification via FCC (20-30%

EtOAc/hexanes) 3.21 (0.18 g, 58%) was obtained. Rf = 0.75 (20% EtOAc/hexanes). 1H NMR (CDCl3, 500

MHz) δ 5.88 (m, 1H), 5.28 (m, 1H), 5.19 (m, 1H), 5.03 (s, 1H), 4.18 (m, 1H), 4.13 (s, 2H), 3.98 (m, 1H),

3.72-3.63 (m, 3H), 3.61 (m, 1H), 3.46 (m, 2H), 3.00 (br s, 1H), 1.54 (m, 2H), 1.50 (s, 3H), 1.34 (m, 2H),

72

1.32 (partially buried s, 3H), 0.89 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 133.6, 118.1, 109.7,

96.5, 78.2, 75.5, 71.9, 71.7, 71.6, 68.3, 68.0, 31.8, 28.1 (2 signals), 26.3, 19.4, 14.1. HRMS (ESI) calcd

for C16H28O6Na (M+Na)+ 339.1784, found 339.1780.

Propan-1-yl-2,3-O-isopropylidene-6-O-methyl-α-D-mannopyranoside (3.22)

3.33 (0.74 g, 2.2 mmol) and TBAI (0.24 g, 0.74 mmol) were dissolved in DMF (20 mL) under a N2

atmosphere. The reaction was cooled to 0 oC and NaH (0.13 g, 0.01 mmol) was added. The reaction was

warmed up slowly over 30 min. Then, it was cooled down to 0 oC and CH3I (0.3 mL, 4.8 mmol) was

added. The mixture was stirred overnight. The solution was brought to 0 oC, quenched slowly with water.

Then, it was extracted using ethyl ether (3x100 mL) and washed with brine. The crude residue was

resuspended in MeOH and treated with PPTS (ca 0.5 g, pH 3-4). The mixture was stirred for 1 h. Then,

quenched with saturated NaHCO3 in methanol and the solvent was evaporated in vacuo. FCC yielded

3.22 (0.55 g, 90%). Rf = 0.38 (20% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 5.88 (m, 1H), 5.28 (m,

1H), 5.19 (m, 1H), 5.04 (s, 1H), 4.19 (m, 1H), 4.12 (m, 2H), 3.67 (m, 3H), 3.59 (m, 1H), 3.38 (s, 3H), 2.68

(br s, 1H), 1.49 (s, 3H), 1.32 (s, 3H); 13C NMR (CDCl3, 500 MHz) δ 133.5, 118.0, 96.5, 78.0, 75.4, 72.9,

70.6, 68.3, 68.1, 59.6, 27.9, 26.1. HRMS (ESI) calcd for C13H23O6 (M+H)+ 275.1489, found 275.1470.

Propan-1-yl-2,3-O-isopropylidene-6-O-methyl-4-O-undecyl-α-D-mannopyranoside (3.23)

73

NaH (58 mg, 1.45 mmol) was added at 0 oC to a solution of 3.20 (0.28 g, 0.9 mmol), TBAI (66 mg, 0.18

mmol) in DMF (15 mL). The mixture was stirred for 30 min, and CH3I (0.09 mL, 1.45 mmol) was added.

The reaction was stirred for 3-4 h at rt, then cooled to 0 oC and quenched with water. The mixture was

extracted with ethyl ether (3x25 mL) and the organic phase was dried (Na2SO4) and evaporated under

reduced pressure. The residue was purified via FCC (10-15% EtOAc/hexanes) to give 3.23 (0.27 g, 64%).

Rf = 0.9 (20% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 5.86 (m, 1H), 5.25 (m, 1H), 5.16 (m, 1H),

5.05 (s, 1H), 4.17 (m, 2H), 4.10 (m, 1H), 3.96 (m, 1H), 3.76 (m, 1H), 3.64-3.53 (m, 3H), 3.44 (m, 1H), 3.36

(partially buried s, 3H), 3.34 (m, 1H), 1.52 (m, 2H), 1.50 (partially buried s, 3H), 1.31 (partially buried s,

3H), 1.22 (br s, 16H), 0.84 (t, J = 6.7 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 133.8, 117.8, 109.3, 96.6,

79.0, 76.3, 76.0, 71.7 (2 signals), 68.4, 68.0 (2 signals), 67.9, 59.4, 32.1, 30.2, 29.8, 29.6, 29.5, 28.2,

26.4, 26.3, 22.8, 14.3. HRMS (ESI) calcd for C24H44O6Na (M+Na)+ 451.3036, found 451.3028.

((1-Butoxydec-9-en-4-yl)oxy)(tert-butyl)diphenylsilane (3.25)

Compound 3.25 was prepared following the procedure described for 3.21. For 3.25: 1H NMR (CDCl3, 500

MHz) δ 7.67 (m, 4H), 7.42-7.34 (m, 6H), 5.73 (m, 1H), 4.93 (m, 1H), 4.90 (m, 1H), 3.69 (m, 1H), 3.40 (m,

4H), 1.94 (m, 2H), 1.56 (m, 4H), 1.40-1.07 (m, 22H), 1.05 (br s, 9H), 0.90 (t, J = 7.4 Hz, 3H); 13C NMR

(CDCl3, 500 MHz) δ 139.3, 136.2, 135.8, 135.0, 129.7, 129.6, 127.8, 127.6, 114.4, 73.4, 71.2, 70.9, 36.5,

36.3, 33.9, 32.1, 30.0, 29.9, 29.7 (3 signals), 29.2, 27.3, 27.1, 26.4, 25.1, 24.6, 19.6 (2 signals), 14.2.

HRMS (ESI) calcd for C36H59O2Si (M+H)+ 551.4279, found 551.4263.

10-((tert-Butyldiphenylsilyl)oxy)hexadec-15-en-1-yl methanesulfonate (3.26)

To a solution of compound 3.35 (1.0 g, 2.14 mmol) at 0 oC in CH2Cl2 (30 mL) was added Et3N (0.35 mL,

2.57 mmol) and MsCl (0.19 mL, 2.57 mmol). The reaction was warmed to rt and stirred overnight. The

74

reaction was diluted with H2O and extracted with ethyl ether (3x100 mL). The organic phase was washed

with brine, dried (Na2SO4) and evaporated in vacuo. FCC of the residue afforded 3.26 (0.84 g, 69%) as a

yellow oil. Rf = 0.5 (20% EtOAc/hexanes).1H NMR (CDCl3, 500 MHz) δ 7.65 (m, 4H), 7.41-7.32 (m, 6H),

5.72 (m, 1H), 4.91 (m, 1H), 4.88 (m, 1H), 4.20 (t, J = 6.6 Hz, 2H), 3.67 (m, 1H), 2.98 (s, 3H), 1.91 (m, 2H),

1.72 (m, 2H), 1.40-1.06 (m, 20H), 1.02 (br s, 9H); 13C NMR (CDCl3, 500 MHz) δ 139.3, 136.1, 129.6,

127.6, 114.4, 73.4, 70.4, 37.6, 36.5, 36.3, 34.0, 29.9, 29.8, 29.6, 29.5, 29.3, 29.2, 29.1, 27.3, 25.6, 25.1,

24.6, 19.6. HRMS (ESI) calcd for C33H52O4SSiNa (M+Na)+ 595.3253, found 595.3246.

10-Hydroxyhexadec-15-en-1-yl methanesulfonate (3.26a)

A 5% solution of acetyl chloride in MeOH (1.7 mL) was added to a mixture of 3.26 (1.0 g, 1.75 mmol) and

DCM (17 mL). The mixture was stirred for 16 h at rt, then neutralized with methanolic sodium bicarbonate.

The mixture was filtered and concentrated in vacuo. FCC of the residue (20-30% EtOAc/hexanes) yielded

3.26a (0.39 g, 66%). Rf = 0.5 (20% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 5.77 (m, 1H), 4.97 (m,

1H), 4.93 (m, 1H), 4.20 (t, J = 6.0 Hz, 2H), 3.56 (m, 1H), 2.99 (s, 3H), 2.06 (m, 2H), 1.70 (m, 2H), 1.39-

1.34 (m, 20H); 13C NMR (CDCl3, 500 MHz) δ 139.1, 114.5, 72.0, 70.4, 37.6, 37.5, 37.4, 33.9, 29.7, 29.6,

29.5, 29.2, 29.1 (2 signals), 25.7, 25.5, 25.3. HRMS (ESI) calcd for C17H34O4SiNa (M+Na)+ 357.2070,

found 357.2064.

10-Iodohexadec-15-en-1-yl methanesulfonate (3.26b)

A mixture of 3.26a (0.38 g, 1.15 mmol), Ph3P (0.33 g, 1.26 mmol) and imidazole (90 mg, 1.38 mmol)

benzene (12 mL) was stirred for 15 min at rt. Then, iodine (0.15 g, 1.26 mmol) was introduced and stirring

continued for 2 h. The reaction was quenched with aqueous Na2S2O3 and extracted with ethyl acetate.

75

The organic phase was dried (Na2SO4) and evaporated in vacuo. The residue was purified via FCC (15%

EtOAc/hexanes) to yield 3.26b (0.31 g, 61%). Rf = 0.6 (20% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz)

δ 5.78 (m, 1H), 4.98 (m, 1H), 4.92 (m, 1H), 4.20 (t, J = 6.0 Hz, 2H), 4.08 (m, 1H), 2.98 (s, 3H), 2.05 (m,

2H), 1.84 (m, 2H), 1.75-1.63 (m, 4H), 1.52 (s, 2H), 1.43 (m, 6H), 1.31 (m, 8H); 13C NMR (CDCl3, 600

MHz) δ 138.7, 114.6, 70.2, 40.6, 40.5, 37.4, 33.6, 29.5, 29.3, 29.1, 29.0 (2 signals), 28.8, 28.1 25.4.

N-(10-((tert-Butyldiphenylsilyl)oxy)hexadec-15-en-1-yl)thiophene-2-carboxamide (3.27)

Amine 3.36 (0.26 g, 0.52 mmol) and 2-thiophene carboxylic acid (0.10 g, 0.78 mmol) were azeotroped

and dissolved in DCM (26 mL) Then, EDCI (0.30 g, 1.58 mmol), dry Et3N (0.29 mL, 2.10 mmol) and

catalytic DMAP (13 mg, 0.10 mmol) were added. The reaction was stirred for 16 h. Then, it was extracted

using more DCM (3x10 mL) and it was washed with water (3x). The organic phase was dried (Na2SO4)

and evaporated and FCC of the residue yielded 3.27 (0.22 g, 69%). Rf = 0.7 (20% EtOAc/hexanes). 1H

NMR (CDCl3, 600 MHz) δ 7.65 (m, 4H), 7.45 (m, 2H), 7.42 (m, 6H), 7.04 (d, J = 6.0 Hz, 1H) 5.90 (br s,

1H), 5.71 (m, 1H), 4.90 (m, 1H), 4.88 (m, 1H), 3.67 (m, 1H), 3.40 (m, 2H), 1.90 (m, 2H), 1.57 (m, 4H),

1.36 (m, 6H), 1.15 (m, 12H), 1.08 (s, 9H); 13C NMR (CDCl3, 500 MHz) δ 139.3, 136.2, 135.0, 129.8,

129.6, 128.0, 127.8, 127.6, 114.4, 73.4, 40.3, 36.5, 36.3, 33.9, 30.0, 29.9, 29.7, 29.7, 29.5, 29.2, 27.3,

27.2, 25.0, 24.6, 19.6.

Hexadec-15-en-1-yl methanesulfonate (3.28)

Compound 3.26b (0.31 g, 0.07 mmol) was stirred with zinc (0.26 g, 4.05 mmol) and methanol (15 mL).

The reaction was quenched with water, extracted with ethyl ether (3x10 mL) and purified via FCC to

obtain 3.28 (0.25 g, quantitative yield). Rf = 0.46 (20% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ

76

5.80 (m, 1H), 4.98 (m, 1H), 4.91 (m, 1H), 4.21 (t, J = 6.0 Hz, 2H), 2.99 (s, 3H), 2.02 (m, 2H), 1.73 (m, 2H),

1.39-1.30 (m, 4H), (br s, 18H). 13C NMR (CDCl3, 600 MHz) δ 139.5, 114.3, 70.4, 37.6, 34.0, 29.8, 29.8,

29.7, 29.6, 29.4, 29.3, 29.2 (2 signals), 25.6.

Propen-1-yl-2,3-O-isopropylidene-α-D-mannopyranoside (3.32)

To a solution of 3.34 (3.3 g, 0.01 mmol) in MeOH (50 mL) was added PPTS (0.24 g, 0.96 mmol). The

reaction mixture was stirred for 4 h and then quenched with a saturated solution of NaHCO3 in MeOH.

The mixture was concentrated in vacuo. FCC of the residue in 50-60% EtOAc/hexanes gave 3.35 (2.19 g,

95% brsm). Rf = 0.34 (50% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 5.86 (m, 1H), 5.30 (m, 1H),

5.22 (m, 1H), 5.05 (s, 1H), 4.17 (m, 3H), 3.98 (m, 1H), 3.83 (m, 2H), 3.74 (m, 1H), 3.64 (m 1H), 2.51 (br s,

1H), 2.04 (m, 1H), 1.51 (s, 3H), 1.34 (s, 3H); 13C NMR (CDCl3, 500 MHz) δ 133.5, 128.6, 118.4, 109.9,

96.7, 75.7, 70.1, 69.8, 68.5, 62.8, 28.1, 26.3.

Propen-1-yl-6-O-tert-butyldiphenylsilyl-2,3-O-isopropylidene-α-D-mannopyranoside (3.32a)

3.32 (2.19 g, 8.41 mmol), TBDPSCl (2.37 mL, 9.25 mmol) and imidazole (1.14 g, 16.82 mmol) were

dissolved in THF (84 mL) and stirred overnight. The solvent was evaporated in vacuo and the mixture

was redissolved in ethyl ether and water and extracted (3x100 mL). The combined organic phase was

washed with brine, dried (Na2SO4) and the solvent was evaporated in vacuo. FCC of the residue afforded

product 3.32a (1.7 g, 56% brsm). Rf = 0.4 (20% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 7.72 (m,

4H), 7.44 (m, 6H), 5.90 (m, 1H), 5.28 (m, 1H), 5.22 (m, 1H), 5.06 (s, 1H), 4.18 (m, 3H), 3.99 (m, 1H), 3.95

(m, 2H), 3.91 (m, 1H), 3.88 (m, 1H), 2.75 (m, 1H), 1.53 (s, 3H), 1.27 (s, 3H), 1.08 (s, 9H); 13C NMR

77

(CDCl3, 500 MHz) δ 135.8, 135.7, 133.6, 133.0, 130.0 (2 signals), 127.9 (2 signals), 118.1, 109.7, 96.3,

78.3, 75.5, 71.0, 69.6, 68.0, 64.8, 28.1, 27.0, 26.3, 19.4. HRMS (ESI) calcd for C28H38O6SiNa (M+Na)+

521.2335, found 521.2329.

Propen-1-yl-6-O-tert-butyldiphenylsilyl-4-O-[1-ethoxyethyl]-2,3-O-isopropylidene-α-D-

mannopyranoside (3.32b)

PPTS (ca 1 g) was added to a mixture of 3.32a (1.7 g, 3.4 mmol), ethyl vinyl ether and DCM solution (1:1,

100 mL) until a pH of 4. The reaction was stirred for 2 h and quenched with methanolic NaHCO3. The

solution was filtered and evaporated to give crude 3.32b (ca 1.42 g). Rf = 0.69 (20% EtOAc/hexanes). 1H

NMR (CDCl3, 500 MHz) δ 7.71 (m, 4H), 7.38 (m, 6H), 5.90 (m, 1H), 5.26 (m, 1H), 5.21 (m, 1H), 5.08 (m,

1H), 4.90 (m, 1H), 4.24-3.99 (m, 3H), 3.96-3.75 (m, 3H), 3.74 (m, 2H), 3.74-3.68 (m, 2H), 1.52 (s, 3H),

1.26 (s, 3H), 1.20 (m, 4H), 1.04 (s, 9H), 0.80 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 136.1,

135.8 (2 signals), 134.0, 133.9, 133.8 (2 signals), 133.5, 133.3, 129.8 (2 signals), 129.7 (2 signals), 127.8

(2 signals), 127.7, 118.1, 118.0, 109.4, 109.3, 100.9, 100.0, 96.1, 95.9, 79.1, 78.7, 76.0 (2 signals), 73.3,

73.2, 69.8 (2 signals), 67.7, 67.6, 63.6, 62.9, 61.8, 61.0, 28.2, 28.1, 27.0, 26.6 (2 signals), 22.9, 21.2,

20.4, 19.5, 19.4 (2 signals), 15.3, 14.3.

Propen-1-yl-4-O-[1-ethoxyethyl]-2,3-O-isopropylidene-α-D-mannopyranoside (3.33)

A mixture of crude 3.35b (ca 1.42 g), TBAF (0.1 M in THF, 50 mL, 5 mmol) and THF (100 mL) was stirred

overnight at rt. The solvent was then evaporated in vacuo. FCC of the residue yielded 3.36 (0.7 g, 85%).

Rf = 0.28 (20% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 5.98 (m, 1H), 5.34 (m, 1H), 5.22 (m, 1H),

78

5.10 (m, 1H), 4.95 (m, 1H), 4.29 (m, 1H), 4.14 (m, 2H), 3.95 (m, 1H), 3.78 (m, 3H), 3.64 (m, 3H), 1.47 (s,

3H), 1.26 (m, 6H), 1.23 (m, 3H); 13C NMR (CDCl3, 500 MHz) δ 133.7, 133.6, 118.2 (2 signals), 109.5,

109.4, 101.3, 100.9, 78.9, 78.5, 76.3, 76.0, 73.5, 72.8, 69.0, 68.7, 68.3, 68.2, 62.2, 62.1, 62.0, 28.3, 28.2,

26.5 (2 signals), 20.7, 20.4, 15.4, 15.3.

10-((tert-Butyldiphenylsilyl)oxy)hexadec-15-en-1-ol (3.35)

LAH (0.1 g, 2.7 mmol) was slowly added to a solution of compound 3.34 (0.92 g, 1.8 mmol) in CH2Cl2 (18

mL) at 0 oC. The mixture was warmed to rt and stirred for an additional 1 h. Then, water (10 mL) and

NaOH (1 mL, 1M), were added, subsequently. Na2SO4 was added to dry the H2O and the mixture was

filtered over Celite. The filtrated was concentrated in vacuo and then was subjected to FCC to afford

compound 3.35 (0.71 g, 85%). Rf = 0.2 (10% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ 7.65 (m,


1.92 (m, 2H), 1.54 (m, 4H), 1.38-1.17 (m, 8H), 1.02 (s, 9H). HRMS (ESI) calcd for C32H50O2SiNa (M+Na)+

517.3478, found 517.3472.

10-((tert-Butyldiphenylsilyl)oxy)hexadec-15-en-1-amine (3.36)

Ethanol (ca 200 mL) was saturated with ammonia gas at 0 oC by bubbling for 30 minutes. 3.26 (0.65 g,

1.13 mmol) was placed in a high-pressure tube and the saturated ammonia solution (150 mL) was added.

The vessel was closed, and the reaction was heated at approximately 80 oC for 4 h. The reaction was

brought to rt and the solvent was concentrated in vacuo. The residue was purified via FCC (10%

MeOH/DCM) to afford 3.36 (0.48 g, 94%). Rf = 0.32 (20% MeOH/DCM). 1H NMR (CDCl3, 600 MHz) δ

7.63 (m, 4H), 7.36 (m, 6H), 5.70 (m, 1H), 4.88 (m, 1H), 4.85 (m, 1H), 4.10 (m, 1H), 3.10 (m, 2H), 1.90 (m,

2H), 1.45 (m, 2H), 1.35 (m, 4H), 1.16 (m, 16H), 1.06 (s, 9H); 13C NMR (CDCl3, 500 MHz) δ 139.2, 136.1,

79

134.9, 129.5, 127.5, 114.3, 73.3, 40.1, 39.4, 36.4, 36.3, 33.9, 29.8, 29.7, 29.6, 29.2, 29.1, 27.8, 27.2,

26.7, 25.0, 24.5, 19.6.

THF-mesylate-alkene (3.37)

Nitrogen was bubbled through a solution of 3.18 (96 mg, 0.29 mmol) and 3.26 (0.33 g, 0.58 mmol) in dry

CH2Cl2 (3 mL) for 30 min and at rt. At that point Grubbs 2nd generation catalyst (25 mg, 10 mol%) was

added and the mixture stirred for 20 h. Then, the reaction was quenched with DMSO (0.05 mL). After

stirring for an additional 30 in the solution was evaporated in vacuo. FCC of the residue afforded 3.37 (45

mg, 40%). Rf = 0.35 (40% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 7.64 (m, 4H), 7.34 (m, 6H),

5.67 (m, 1H), 5.30 (m, 1H), 4.19 (t, J = 6.7 Hz, 2H), 3.79 (m, 3H), 3.66 (m, 1H), 3.38 (m, 1H), 2.97 (s, 3H),

2.40 (br s, 1H), 2.24 (br s, 1H), 1.90 (m, 4H), 1.72-1.06 (m, 46H), 1.06 (s, partially buried, 9H), 0.85 (t, J =

7.1 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 135.9, 129.4, 127.4, 82.7, 82.5, 74.0, 73.1, 70.2, 37.4, 36.3,

33.6, 31.9, 29.6 (3 signals), 29.4, 29.3 (2 signals), 29.1, 29.0, 28.4, 27.1, 25.6, 25.4, 24.8, 22.7,19.4, 14.1.

HRMS (ESI) calcd for C51H86O7SSiNa (M+Na)+ 893.5761, found 893.5743.

THF-mesylate (3.37a)

A solution of compound 3.37 (45 mg, 0.05 mmol) in EtOAc (2 mL) and Pd/C (20 mg, 10% wt) was purged

with N2 for 30 min. Then, the mixture was stirred under a hydrogen atmosphere (balloon) for 20 h. The

reaction was then purged with N2 and filtered through a bed of Celite to yield 3.37a (45 mg, quantitative).

1H NMR (CDCl3, 600 MHz) δ 7.64 (m, 4H), 7.36 (m, 6H), 4.19 (t, J = 6.6 Hz, 2H), 3.76 (m, 2H), 3.67 (m,

1H), 3.36 (m, 2H), 2.98 (s, 3H), 1.95 (m, 2H), 1.74–1.07 (m, 22H), 1.01 (s, 9H), 0.85 (t, J = 6.5 Hz, 3H);

80

13C NMR (CDCl3, 600 MHz) δ 136.1, 135.0, 129.5, 127.5, 82.8, 74.2 (2 signals), 73.4, 70.4, 37.5, 36.5,

36.4, 33.7, 33.6, 32.1, 29.9 (2 signals), 29.8 (3 signals), 29.6 (2 signals), 29.5, 29.3, 29.2, 28.9, 27.3, 25.8

(2 signals), 25.6, 25.0, 22.9, 19.6, 14.3. HRMS (ESI) calcd for C51H88O7SSiNa (M+Na)+ 895.5918, found

895.5904.

THF-piperazine (3.37b)

To a solution of compound 3.37a (45 mg, 0.05 mmol) in dry CH3CN (2 mL) was added piperazine (0.22 g,

0.26 mmol) and the mixture was refluxed for 4 h. The mixture was evaporated under reduced pressure.

FCC of the residue yielded 3.37b (29 mg, 64%). Rf = 0.57 (10% MeOH/DCM). 1H NMR (CDCl3, 500 MHz)

δ 7.63 (m, 4 H), 7.34 (m, 6H), 3.76 (m, 2H), 3.66 (m, 1H), 3.36 (m, 2H), 3.11 (br s, 4H), 2.64 (br s, 4H),

2.39 (m, 2H), 1.96 (m, 2H), 1.62-1.22 (m, 18H), 1.01 (br s, partially buried), 1.00 (s, partially buried, 9H),

0.84 (t, J = 6.7 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 136.1, 135.0, 129.6, 127.6, 82.8, 74.4, 73.4, 58.5,

50.7, 44.0, 36.7, 36.4, 33.6, 32.1, 29.9, 29.9, 29.9, 29.8, 29.7, 29.7, 29.7, 29.6, 29.6, 29.1, 29.0, 27.5,

27.3, 26.6, 25.8, 25.7, 25.0, 24.9, 22.9, 19.6, 14.4. HRMS (ESI) calcd for C54H95N2O4Si (M+H)+ 862.6983,

found 862.6940.

THF-thiophene-alkene (3.38)

Following the procedure described for 3.37, CM of 3.18 (60 mg, 0.18 mmol) and 3.27 (0.22 g, 0.36 mmol)

yielded 3.38 (43 mg, 27%). Rf = 0.47 (40% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ 7.42 (m, 4H),

7.39 (m, 1H), 7.37 (m, 1H), 7.32 (m, 6H), 7.04 (m, 1H), 6,02 (br s, 1H), 5.67 (m, 1H), 5.30 (m, 1H), 3.80

(m, 3H), 3.66 (m, 1H), 3.39 (m, 3H), 2.59 (br s, 1H), 2.43 (br s, 1H), 1.90 (m, 4H), 1.54-1.15 (m, 46H),

0.86 (t, J = 6.7 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 161.9, 139.3, 136.0, 134.9 (2 signals), 129.7,

81

129.5, 128.1, 127.9, 127.7, 127.5, 82.8, 82.6, 74.1, 73.2, 40.2, 36.4, 36.2, 33.62, 32.4 (2 signals), 32.0,

29.8 (5 signals), 29.7 (2 signals), 29.6, 29.5 (2 signals), 29.4, 29.2, 28.6, 27.2, 27.1, 25.8, 25.0, 24.5,

22.8, 19.5, 14.2.

THF-thiophene (3.38a)

Compound 3.38 (43 mg, 0.047 mmol) was dissolved in EtOAc (2 mL). The solution was degassed for 30

min and Pd/C (40 mg, 10%) was introduced. Then, the reaction was stirred under H2 atmosphere for 20 h.

The solution was filtered over Celite and concentrated in vacuo. NMR indicated the presence of alkene,

so the hydrogenation procedure was repeated. Then, FCC (40-50% EtOAc/hexanes) of the crude product

gave 3.38a (31 mg, 72%). Rf = 0.5 (40% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ 7.64 (m, 4H),

7.46 (d, J = 1.1 Hz, 1H), 7.45 (d, J = 1.1 Hz, 1H), 7.35 (m, 6H), 7.04 (d, J = 1.3 Hz ,1H), 5.98 (br s, 1H),

3.76 (m, 2H), 3.66 (m, 1H), 3.39 (m, 4H), 1.96 (m, 3H), 1.65 (m, 3H), 1.56 (m, 3H), 1.35-1.12 (m, 47H),

1.09 (s, 9H), 0.86 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 162.0, 139.3, 136.1, 135.0, 134.9 (2

signals), 129.8, 129.6, 129.5, 128.2, 128.0, 127.7, 127.6, 127.5, 82.9, 72.2 (2 signals), 73.4, 73.3, 40.2,

36.4, 32.1, 29.9 (2 signals), 29.8 (2 signals), 29.6 (3 signals), 29.5, 28.9, 28.6, 27.3, 27.1, 25.8, 25.0,

22.9, 19.6, 14.3.

THF-butyl ether-alkene (3.39)

Following the procedure for 3.37, CM of alkenes 3.18 (86 mg, 0.26 mmol) and 3.25 (0.13 g, 0.52 mmol)

yielded 3.39 (50 mg, 23%). Rf = 0.51 (40% EtOAc/hexanes). 1H NMR (CDCl3, 400 MHz) δ 7.63 (m, 4H),

7.35 (m, 6H), 5.65 (m, 1H), 5.30 (m, 1H), 3.80 (m, 3H), 3.67 (m, 1H), 3.37 (m, 4H),1.8 (m, 5H), 1.32 (m,

82

8H), 1.23 (br s, 44H), 1.01 (s, 9H), 0.89 (t, J = 7.3 Hz, 3H), 0.84 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 500

MHz) δ 139.3, 136.1, 135.0, 129.5, 127.6, 114.3, 73.4, 71.2, 70.9, 36.5, 36.3, 33.9, 32.1, 30.0, 29.9, 29.7

(3 signals), 29.1, 27.3, 26.4, 25.0, 24.7, 19.6 (2 signals), 14.2.

6-O-Undecyl mannoside-butenolide-alkene (3.40)

Nitrogen was bubbled through a solution of 3.19 (57 mg, 0.14 mmol) and butenolide 3.24 (39 mg g, 0.07

mmol) in dry CH2Cl2 (1 mL) for 30 min and at rt. At that point Grubbs 1st generation catalyst (25 mg, 10

mol%) was added and the mixture refluxed for 6 h. Then, the reaction was quenched with DMSO (0.05

mL). After stirring for an additional 30 min the solution was evaporated in vacuo. FCC of the residue

afforded 3.40 (54 mg, 96%) as an E/Z mixture (E/Z~3/1). Rf = 0.38 (20% EtOAc/hexanes x 2). 1H NMR

(CDCl3, 500 MHz) δ 7.59 (m, 4H), 7.31 (m, 6H), 6.90 (m, 1H), 5.60-5.41 (m, 2H), 4.97 (s, 1H), 4.92 (m,

1H), 4.10-3.84 (m, 4H), 3.61-3.56 (m, 5H), 3.39 (m, 2H), 2.93 (m, 1H), 2.17 (t, J = 7.7 Hz, 2H), 1.85 (m,

2H), 1.52-1.18 (bm, 45H), (s, 9H), 0.80 (t, J = 6.7 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 174.1, 149.0,

136.3 (2 signals), 136.1, 135.1, 134.9 (2 signals), 134.5, 129.6, 127.6, 124.9, 124.6, 109.7, 96.3, 96.0,

75.6, 75.5, 73.3, 72.3, 71.8, 71.7, 71.6, 68.0 (2 signals), 36.5, 36.3, 32.4, 32.1, 29.9, 29.8 (2 signals), 29.7

(2 signals), 29.5, 29.4, 29.3, 29.2, 28.1 (2 signals), 27.5, 27.3, 26.3, 25.4, 25.0, 24.7 (2 signals), 22.9,

19.6, 19.4, 14.3. HRMS (ESI) calcd for C56H88O9SiNa (M+Na)+ 955.6090, found 955.6088.

83

6-O-Undecyl mannoside-butenolide (3.40a)

To a solution of 3.40 (70 mg, 0.078 mmol) and TsNHNH2 (0.98 g, 5.26 mmol) in DME (7.8 mL) at reflux,

was added a solution of NaOAc (0.53 g, 6.51 mmol) in H2O (10 mL) over 4 h. The mixture was then

cooled, diluted with EtOAc, and washed with water. The organic phase was dried (Na2SO4), filtered and

concentrated in vacuo. FCC in 1-5% (acetone/DCM) delivered 3.40a (35 mg, 48%). Rf = 0.42 (5%

acetone/DCM). 1H NMR (CDCl3, 500 MHz) δ 7.64 (m, 4H), 7.34 (m, 6H), 6.94 (s, 1H), 4.96 (m, 2H), 4.12

(m, 2H), 3.66 (m, 6H), 3.37 (m, 3H), 2.93 (m, 1H), 2.21 (m, 2H), 1.49-1.09 (bm, 51H), 1.08 (s, 9H), 0.85

(t, J = 7.5 Hz, 3H). HRMS (ESI) calcd for C56H90O9SiNa (M+Na)+ 957.6252, found 957.6225.

4-O-Undecyl mannoside-butenolide-alkene (3.41)

Following the procedure described for 3.40, CM for 3.20 (0.21 g, 0.52 mmol) and 3.24 (0.150 g, 0.26

mmol) yielded 3.41 (6 mg, 25%) as an E/Z mixture (E/Z~4/1). Rf = 0.4 (30% EtOAc/hexanes). 1H NMR

(CDCl3, 500 MHz) δ 7.59 (m, 4H), 7.28 (m, 6H), 6.90 (s, 1H), 5.56-5.40 (m, 2H), 5.40 (m, 1H), 4.99 (s,

1H), 4.92 (m, 1H), 4.16 (m, 1H), 4.05 (m, 2H), 3.82-3.75 (m, 5H), 3.56 (m, 1H), 3.43 (m, 1H), 3.31 (m,

1H), 2.16 (m, 2H), 2.05-1.80 (m, 2H), 1.60-1.10 (bm, 45H), 1.10 (s, 9H), 0.82 (t, J = 6.7 Hz, 3H); 13C NMR

(CDCl3, 500 MHz) δ 174.1, 149.0, 136.3 (2 signals), 136.1, 134.9 (2 signals), 134.4, 129.5, 127.5, 124.8,

109.3, 96,2, 95.9, 78.8, 76.0, 71.7, 68.5, 68.0, 62.9, 36.4, 36.3, 32.4, 32.1, 30.2, 28.8, 29.7 (2 signals),

29.6, 29.5, 29.4, 29.3, 29.1, 28.2, 27.5, 27.2, 26.5, 26.2, 25.3, 25.0, 22.8, 19.6, 19.4.

84

6-O-Butyl mannoside-butenolide-alkene (3.42)

Following the procedure described for 3.40, the CM of 3.21 (0.12 g, 0.37 mmol) and 3.24 (80 mg, 0.18

mmol) yielded 3.42 (60 mg, 80%) as an E/Z mixture (E/Z~4/1). Rf = 0.2 (30% EtOAc/hexanes). 1H NMR

(CDCl3, 500 MHz) δ 7.63 (m, 4H), 7.37 (m, 6H), 6.94 (s, 1H), 5.61-5.44 (m, 2H), 5.00 (s, 1H), 4.96 (m,

1H), 4.12-3.88 (m, 4H), 3.71-3.60 (m, 5H), 3.50 (m, 2H), 2.91 (br s, 1H), 2.20 (m, 2H), 1.90 (m, 2H), 1.55-

1.10 (m, 33H), 1.00 (s, 9H), 0.86 (m, 3H); 13C NMR (CDCl3, 500 MHz) 149.1, 136.1, 129.6, 127.6, 124.9,

109.7, 96.1, 75.6, 73.3, 72.0, 71.9, 71.8, 68.1, 68.0, 36.5, 36.3, 32.5, 31.9, 29.8, 29.5, 29.4, 29.2, 28.1,

27.6, 27.4, 27.3, 26.3, 25.4, 25.1, 24.8 (2 signals), 19.7, 19.5 (2 signals), 14.1.

6-O-Butyl mannoside-butenolide (3.42a)

Compound 3.42 (0.1 g, 0.12 mmol) was transformed to 3.42a following the reduction protocol described

for preparation of 3.40a. FCC in 1-5% (acetone/DCM) delivered 3.42a (60 mg, 61%). Rf = 0.42 (5%

acetone/DCM). 1H NMR (CDCl3, 500 MHz) δ 7.68 (m, 4H), 7.28 (m, 6H), 6.90 (s, 1H), 4.91 (m, 2H), 4.08

(m, 2H), 3.63 (m, 6H), 3.45 (m, 2H), 3.35 (m, 1H), 3.00 (br s, 1H), 2.15 (m, 2H), 1.49-1.10 (m, 37H), 1.02

(s, 9H), 0.79 (t, J = 7.7 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ 174.1, 149.1, 136.1, 135.0, 134.9, 134.5,

129.9, 129.6, 127.5, 126.7, 109.7, 97.3, 78.1, 75.6, 73.4, 71.9, 71.8, 71.5,68.0, 36.5 (2 signals), 31.8,

29.8 (2 signals), 29.5, 29.4, 29.3, 28.1, 27.5, 27.3, 26.3 (2 signals), 25.4, 25.0, 19.6, 19.5, 19.4, 14.1.

85

6-O-Methyl mannoside-butenolide-alkene (3.43)

Following the procedure described for 3.40, the CM of 3.22 (91 mg, 0.33 mmol) and 3.24 (90 mg, 0.17

mmol) yielded 3.43 (0.12 g, 92%) as an E/Z mixture (E/Z~3/1). Rf = 0.2 (30% EtOAc/hexanes). 1H NMR

(CDCl3, 500 MHz) δ 7.64 (m, 4H), 7.34 (m, 6H), 6.94 (s, 1H), 5.65-5.45 (m, 2H), 5.10 (s, 1H), 5.02 (m,

1H), 4.08-3.91 (m, 4H), 3.68 (m, 4H), 3.58 (m, 1H), 3.39 (m, 3H), 2.67 (br s, 1H), 2.22 (m, 2H), 1.89-1.13

(m, 27H), 1.01 (s, 9H); 13C NMR (CDCl3, 600 MHz) δ 174.1, 149.0, 136.3, 136.2 (3 signals), 136.1, 134.9

(2 signals), 134.5, 129.6, 129.5, 127.6, 127.5 (2 signals), 125.2, 125.0, 109.4, 98.3, 96.4, 96.0. 73.3, 72.0

(2 signals), 70.4, 70.3, 69.9, 69.8, 69.5, 68.3, 68.0, 67.8, 56.1, 36.5, 36.3, 32.4 (2 signals), 32.1, 31.1,

29.9 (2 signals), 29.8, 29.7 (2 signals), 29.5, 29.4, 29.3, 29.2, 28.0, 27.5, 27.2, 26.6, 26.3 (2 signals),

25.3, 25.0, 24.7, 22.9, 19.6, 19.4, 14.3. HRMS (ESI) calcd for C46H68O9SiNa (M+Na)+ 815.4525, found

815.4514.

6-O-Methyl mannoside-butenolide (3.43a)



(5% acetone/DCM). 1H NMR (CDCl3, 500 MHz) δ 7.59 (m, 4H), 7.30 (m, 6H), 6.90 (s, 1H), 4.92 (m, 2H),

4.09 (m, 2H), 3.61 (m, 6H), 3.33 (m, 1H), 3.32 ( partially buried s, 3H), 2.16 (m, 2H), 1.46-1.10 (m, 33H),

1.03 (s, 9H); 13C NMR (CDCl3, 500 MHz) δ 174.1, 149, 136.1, 134.9 (2 signals), 134.5, 129.5, 127.5,

109.7, 97.3, 78.2, 75.6, 73.3, 73.0, 70.5, 68.5, 68.0, 59.7, 36.4, 29.8, 29.7, 29.5, 29.4, 29.3, 28.0, 27.5,

27.2, 26.2 (2 signals), 25.3, 25.0, 19.6, 19.4.

86

6-O-Methyl-4-O-undecyl mannoside-butenolide-alkene (3.44)

Following the procedure described for 3.40, CM of 3.23 (0.12 g, 0.29 mmol) and 3.24 (80 g, 0.14 mmol)

yielded 3.44 (60 mg, 44%) as an E/Z mixture (E/Z~3/1). Rf = 0.8 (20% EtOAc/hexanes). 1H NMR (CDCl3,

500 MHz) δ 7.63 (m, 4H), 7.35 (m, 6H), 6.94 (s, 1H), 5.60-5.45 (m, 2H), 5.03 (s, 1H), 4.96 (m, 1H), 4.15

(m, 1H), 4.08 (m, 2H), 3.86 (m, 1H), 3.78 (m, 1H), 3.59 (m, 4H), 3.46 (1H), 3.41 (s, 3H), 3.36 (m, 4H),

2.22 (m, 2H), 1.89 (m, 2H), 1.57-1.49 (m, 45H), 1.08 (s, 9H), 0.86 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3,

600 MHz) δ 171.2, 149.1, 146.6 (2 signals), 139.3, 136.7, 136.2, 135.0, 134.8, 129.6, 129.0, 127.6, 124.9,

114.4, 109.3, 104.2, 96.7, 96.6, 96.2, 79.1, 73.4, 73.3, 71.8, 62.8, 59.5, 36.5, 36.4, 33.9, 32.1, 30.2, 29.9,

29.7 (2 signals), 29.6, 29.4, 29.3 (2 signals), 29.2, 28.2, 27.4, 27.3, 26.5, 26.3, 25.1 (2 signals), 25.0,

24.6, 22.9, 19.6, 19.4, 14.3. HRMS (ESI) calcd for C57H90O9SiNa (M+Na)+ 969.6246, found 969.6237.

6-O-Methyl-4-O-undecyl mannoside-butenolide (3.44a)



(2% acetone/DCM). 1H NMR (CDCl3, 500 MHz) δ 7.64 (m, 4H), 7.34 (m, 6H), 6.94 (s, 1H), 4.98 (s, 1H),

4.97 (m, 1H), 4.15 (m, 1H), 4.07 (m, 1H), 3.78 (m, 1H), 3.62 (m, 5H), 3.46 (m, 1H), 3.36 (partially buried s,

3H), 3.34 (m, 2H), 2.22 (m, 2H), 1.50-1.08 (m, 51H), 1.01 (s, 9H), 0.85 (t, J = 7.5 Hz, 3H); 13C NMR

(CDCl3, 500 MHz) δ 174.1, 149.0, 136.1, 135.0 (2 signals), 130.0 (2 signals), 128.0, 109.3, 97.3, 79.1,

77.6, 76.4, 76.2, 73.4, 71.8, 71.7, 68.2, 67.7, 59.5, 36.5 (2 signals), 32.1, 30.3, 29.9, 29.8 (3 signals),

29.7, 29.6, 29.4, 29.3, 28.2, 27.5, 27.3, 26.5, 26.3 (2 signals), 25.4, 25.1, 25.0, 22.9, 19.6.

87

6-O-Undecyl mannoside-mesylate-alkene (3.45)

Following the procedure described for 3.40, CM of 3.19 (0.3 g, 0.72 mmol) and 3.26 (0.2 g, 0.35 mmol)

yielded 3.45 (0.160 g, 48%) as an E/Z mixture (E/Z~4/1). 1H NMR (CDCl3, 600 MHz) δ 7.65 (m, 4H), 7.35

(m, 6H), 5.65-5.46 (m, 2H), 5.02 (s, 1H), 4.21 (t, J = 5.0 Hz, 2H), 4.13 (m, 3H), 3.90 (m, 1H), 3.69 (m, 4H),

3.63 (m, 1H), 3.49 (m, 2H), 2.99 (s, 3H), 2.91 (br s, 1H), 1.9 (m, 2H), 1.73 (m, 2H), 1.57-1.24 (m, 46H),

1.05 (s, 9H), 0.87 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 136.3, 136.2, 135.0 (2 signals), 129.6,

128.6, 127.6 (2 signals), 125.0, 109.7, 96.2, 75.6, 73.4, 72.3, 71.9, 71.7, 70.4, 68.1, 37.6, 36.6, 36.4,

32.5, 32.1, 31.2, 29.8, 29.7(2 signals), 29.6, 29.4, 29.3, 29.2, 28.1, 27.3, 26.3, 25.6, 25.1, 24.8, 22.9,

19.7, 14.3; HRMS (ESI) calcd for C54H90O10SSiNa (M+Na)+ 958.5916, found 958.5917.

6-O-Undecyl mannoside-mesylate (3.45a)

Compound 3.45 (0.16 g, 1 mmol) was transformed to 3.45a following the reduction protocol described for

preparation of 3.37a (0.16 g, quant). 1H NMR (CDCl3, 600 MHz) δ 7.65 (m, 4H), 7.38 (m, 6H), 4.96 (s,

1H), 4.21 (t, J = 6.0 Hz, 2H), 4.13 (m, 2H), 3.68 (m, 6H), 3.45 (m, 3H), 2.99 (s, 3H), 2.94 (s, 1H), 1.73 (m,

2H), 1.55-1.13 (m, 50H), 1.01 (s, 9H), 0.88 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 136.1, 135.0,

129.6, 127.6, 109.7, 97.3, 78.1, 75.6,73.4, 72.3,71.9, 71.6, 70.4, 68.1, 68.0, 37.6, 36.5, 32.1, 29.8 (3

signals), 29.7, 29.6 (2 signals), 29.5, 29.3, 29.2, 28.1, 27.3, 26.3, 25.6, 25.1, 22.9, 19.6, 14.3.

88

6-O-Undecyl mannoside-piperazine (3.45b)

Following the procedure for the synthesis of 3.37b, 3.45a (0.16 g, 0.16 mmol) was converted to

piperazine 3.45b (62 mg, 40%). 1H NMR (CDCl3, 600 MHz) δ 7.64 (m, 4H), 7.35 (m, 6H), 4.95 (s, 1H),

4.10 (m, 2H), 3.65 (m, 5H), 3.47 (m, 4H), 3.19 (br s, 4H), 2.72 (br s, 4H), 2.39 (m, 2H), 1.50-1.09 (m,

52H), 1.01 (s, 9H), 0.86 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 136.1, 135.0 (2 signals), 129.6,

127.6, 109.7, 97.3, 78.2, 75.6, 73.4, 72.3, 71.7, 71.4, 68.1, 68.0, 58.4, 50.3, 43.8, 36.5 (2 signals), 32.1,

29.9 (2 signals), 29.8 (2 signals), 29.7 (2 signals), 29.6, 28.1, 27.5, 27.30, 26.6, 26.3 (2 signals), 25.1 (2

signals), 22.9, 19.6, 14.4.

6-O-Undecyl mannoside-thiophene-alkene (3.46)

Following the procedure described for 3.40, CM for 3.19 (0.1 g, 0.24 mmol) and 3.27 (0.29 g, 0.48 mmol)

yielded 3.41 (6 mg, 25%). FCC gave a mixture of CM product and thiophene dimer (~300 mg), which was

treated with 5% AcCl in MeOH (1.5 mL) in DCM (15 mL). The reaction was stirred for 1 h at rt and then

quenched with a solution of methanolic NaHCO3. The mixture was filtered and concentrated in vacuo.

FCC of the residue (5% MeOH/DCM) afforded 3.46 (100 mg, 44% after two steps). 1H NMR (CDCl3, 600

MHz) δ 7.59 (m, 4H), 7.41 (d, J = 3.7 Hz, 1H), 7.38 (d, J = 0.9 Hz, 1H), 7.33 (m, 2H), 7.28 (m, 4H), 6.99

(m, 1H), 5.88 (br s, 1H), 5.59-5.36 (m, 2H), 4.91 (m, 1H), 4.03 (m, 1H), 3.94-3.53 (m, 8H), 3.46-3.29 (m,

4H), 3.10 (m, 1H), 2.78 (br s, 1H), 2.50 (br s, 1H), 1.87 (m, 2H), 1.33-1.03 (m, 40H), 0.97 (s, 9H), 0.81 (t,

J = 7.5 Hz, 3H).

89

6-O-Undecyl mannoside-10-deoxy-mesylate-alkene (3.47)

Following the procedure described for 3.40, CM for 3.19 (0.28 g, 0.67 mmol) and 3.28 (0.11 g, 0.33

mmol) yielded 3.47 (0.12 g, 48%) as an E/Z mixture (E/Z~4/1). Rf = 0.4 (20% EtOAc/hexanes). 1H NMR

(CDCl3, 600 MHz) δ 5.71-5.68 (m, 2H), 5.01 (m, 1H), 4.19 (t, J = 7.5 Hz, 2H), 4.12 (m, 3H), 3.92 (m, 1H),

3.68 (m, 3H), 3.60 (m, 1H), 3.45 (m, 2H), 2.97 (s, 3H), 2.95 (m, 1H), 2.01 (m, 2H), 1.72 (m, 2H), 1.59-1.26

(m, 44H), 0.85 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 136.5, 124.9, 109.7, 96.1, 78.2, 75.6,

72.3, 71.8, 71.7, 70.4, 68.1, 68.0, 37.6, 32.5, 32.1, 29.9 (2 signals), 29.8 (2 signals), 29.7, 29.7 (2

signals), 29.6, 29.5 (2 signals), 29.3, 29.4, 29.2, 28.1, 26.3, 25.6, 22.9, 14.3.

6-O-Undecyl mannoside-10-deoxy-mesylate (3.47a)

3.47 (0.117 g, 0.16 mmol) was dissolved in ethyl acetate (3 mL) and degassed for 30 minutes. At that

time, Pd/C (11 mg, 10% wt) was added and the reaction was stirred overnight under hydrogen

atmosphere. The solution was filtered over Celite and evaporated to deliver 3.47a (85 mg, 48%).

90

6-O-Undecylmannoside-10-deoxy-piperazine (3.47b)

Following the procedure for 3.37b, compound 3.47a (85 mg, 0.011 mmol) was converted into piperazine

3.47b (44 mg, 30%). 1H NMR (CDCl3, 500 MHz) δ 4.96 (s, 1H), 4.12 (m, 1H), 4.10 (s, 1H), 3.66 (m, 4H),

3.61 (m, 1H), 3.47 (m, 2H), 3.40 (m, 1H), 2.89 (t, J = 5.0 Hz, 4H), 2.40 (br s, 4H), 2.28 (m, 1H), 1.70 (br s,

4H), 1.55 (m, 4H), 1.50 (s, 3H), 1.33 (s, 3H), 1.23 (br s, 33H), 0.85 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3,

500 MHz) δ 109.6, 97.3, 78.2, 75.6, 72.3, 71.8, 71.5, 68.1, 68.0, 59.7, 54.7, 46.2, 32.1, 29.9 (2 signals),

29.8 (4 signals), 29.7, 29.6 (3 signals), 28.1, 27.8, 26.8, 26.4, 26.3, 22.9, 14.3.

91

DESIGN AND SYNTHESIS OF POTENTIAL

TUMOR-SELECTIVE CYTOTOXIC AGENTS

92


Traditional cancer therapies are designed to interfere with cell pathways involved in proliferation or cell

survival.95 Those therapies are effective against cancer cells, but invariably. Therefore, tumor-specific

cytotoxic agents are desirable.96 Herein, we describe targeting strategies for THF-ACGs. For reasons

stated earlier, 4-DAN was used for proof of principle.

Two studies on the targeting of THF-AGEs to cancer cells have been reported. In one, the authors

explore folate targeted, β-cyclodextrin derived, nano-suspensions, (a biphasic system of pure drug

particles of less than 1 nanometer in an aqueous vehicle) in vivo.97 The conjugates showed enhanced

cytotoxicity and cell uptake for folate receptor-positive 4T1 cells. In 4T1 tumor bearing mice, the

acetogenin nanosuspensions aggregated in tumors and enhanced drug uptake only in the tumor site. In

the other study, biotin-conjugated acetogenins showed higher potency and selectivity in a cellular assay,

than the parent drugs.68

We have designed two strategies for tumor-targeted THF-ACGs. The first is a prodrug approach in which

an ACG or an ACG mimetic is attached to a vector that binds to a tumor specific receptor. Upon cellular

internalization the cytotoxic agent will be released. The other strategy is a ‘chameleon’ approach, in which

the sugar ring of a sugar containing ACG mimetic is “disguised” as a tumor vector. Advantages of the

chameleon strategy over the prodrug approach are (i) simpler synthetic chemistry; (ii) the drug and the

vector entity are one in the same and potential problems with release of the active drug before delivery to

the cellular target do not arise. However, designing a chameleon molecule can be more challenging than

a prodrug as precise site modification may be needed (vide infra).

4.2. The ‘chameleon’ approach

The mannose structures described in earlier chapters are relevant to the chameleon approach. As

discussed previously, mannose replacements are not only of interest for their easy synthetic accessibility,

but also because they are potentially tumor vectors. For example, certain GLUTs that are overexpressed

on tumor cells also recognize mannose motifs and could help internalize mannose containing

93

molecules.98 In addition, the 3-O-carbamoyl mannose subunit of the bleomycin family of clinical agents,

has been shown to be primarily responsible for the tumor selectivity of the bleomycins (Figure 4.1).

93c,99,100

4.2.1. Design

The design of our chameleon analogues is based on the following hypothesis: replacement of the THF

moiety in 4-DAN with a 3-O-carbamoylmannose residue will confer tumor selectivity but will not abrogate

potency. This design is challenging because the domain responsible for selectivity essentially overlaps

with a segment of the molecule that impacts on potency. In this regard, questions are: (i) will the presence

of the 3-O-carbomyl moiety reduce cytotoxicity; (ii) will the 6-O-alkyl chain on the mannose residue lower

tumor selectivity. In comparison, the prodrug constructs, while involving more complex synthetic

chemistry, are simpler to design as the domains for potency and selectively are remote and therefore

likely to function independently .

Figure 4.1. Bleomicyn and carbamoylmannose derivative 4.2

94

4.2.2. Synthesis

Regioselective benzoylation on the triol 4.3 using dibutyltin oxide and 4-methoxybenzyl isocyanate

afforded the PMB protected carbamate, 4.4 (Scheme 4.1). Treatment of 4.4 with ceric ammonium nitrate

(CAN) afforded the deprotected carbamate 4.5. Cross metathesis of 4.5 and thiophene alkene 4.6 (3.27

in Chapter 3) provided the heterodimer in 41% yield. Hydrogenation and removal of the silyl protecting

group delivered 4.7 in 67% after two steps.

Scheme 4.1. Synthesis of ‘chameleon’ compound 4.7

95

4.3. The prodrug approach

This strategy uses a prodrug that is

comprised of an ACG or an ACG mimetic

that is conjugated via a cleavable linker to a

vector for a tumor-specific cell surface

receptor (Figure 4.2). Upon recognition by

the tumor cell, the prodrug is internalized and

the drug is released by an intracellular

mechanism.

4.3.1. Design

The underlying hypothesis is that the tumor-specific vector on the prodrug will facilitate delivery to tumor

but not to normal cells. Once internalized, the linkage will be cleaved, and the liberated drug will kill the

cell. The mechanism for drug release is critical as this should be a pathway that is tumor specific to

prevent drug release before delivery to the drug target. The use of a traceless linker is important as the

released drug entity is identical to the parent drug, thus assuring activity.

Figure 4.3. Example of a prodrug construct using 4-DAN as the parent drug

Figure 4.2. Recognition mechanism of prodrugs

96

For proof of principle, our vector target is PSMA which is overexpressed on prostate cancer cells and the

linker is a previously validated disulfide-based construct (Figure 4.3). Details of the choice and synthesis

of the specific drug, tumor receptor, vector and linker are presented in the following sections.

4.3.2. Drugs

One of the analogues of 4-DAN, 4.9 (3.10 in Chapter 3) was selected as the drug entity as it is easily

prepared and was active against several important tumor cell lines (Figure 4.4). The synthesis of the

derived prodrug 4.10 lays the groundwork for prodrugs from other drugs like ACG mimetic 4.11 (3.16 in

Chapter 3), and the naturally occurring ACG annonacin 4.13 (Figure 4.4).

Figure 4.4. Proposed drug and prodrug pairs 4.9-4.10, 4.11-4.12, 4.13-4.14

97

Figure 4.5. DUPA derived vector

4.3.2. Receptor

Prostate-specific membrane antigen (PSMA) is a type II cell surface glycoprotein and is the most well

established highly restricted prostate epithelial cell membrane antigen.101 This highly restricted prostate-

related antigen is an integral cell membrane protein. Moreover, the expression level of PSMA has been

shown to increase as the stage and the grade of the tumor progresses. There is a high interest in PSMA

as a target for prostate-specific imaging and antitumor agents.102 Furthermore, recent studies have

demonstrated that PSMA is also expressed in nonprostatic tumor neovasculature and it is almost absent

in the vasculature of healthy tissues.101 Accordingly much effort has been made in developing PSMA-

targeted agents.103

4.3.3. Vector

2-[3-(1,3-dicarboxypropyl)ureido] pentanedioic acid (DUPA), is a

PSMA-specific ligand and enters PSMA expressing cells via

endocytosis.103b DUPA has shown promise for targeting human

lymph node prostate cancer (LNCaP) cells with many different

therapeutic warheads.103a The specificity of DUPA conjugates

for prostate cancers was evaluated by studying the uptake of

DUPA-targeted 99mTc radioimaging agents by human prostate

cancer (LNCaP) tumors in athymic nude mice. It was found that the retention of the agent was limited to

PCa xenograft and kidneys.104 Unlike murine kidneys, human kidneys express low levels of PSMA, These

data suggested that DUPA might constitute an ideal ligand for prostate cancer-targeted therapy.103a

Following a known procedure,105 glutamate salts 4.16 and 4.17 were coupled using triphosgene to deliver

urea 4.18. Selective removal of the benzyl protecting group provided 4.19, which was treated with 4.20 to

deliver 4.21. Chloride present under these conditions apparently led to substitution of the tosylate.

Treatment of 4.21 with potassium thioacetate yielded 4.22. Exposure of 4.22 to trifluoroacetic acid in the

98

presence of triisobutylsilane gave the tris-carboxylic acid 4.23.106 The material was stored and

deprotected only as needed, under nitrogen, to avoid the formation of the disulfide derivative.

Scheme 4.2. Synthetic route to the synthesis of the DUPA derived vector 4.15

4.3.4. Linker

The linker should be such that the drug is only released after the prodrug has been internalized, and not

before. In addition, a traceless linker is attractive as this allows for release of a drug entity that is identical

to the parent drug. The linker used in our prodrug 4.24 is susceptible to cleavage by thiolates, which are

in much higher concentration in cells than in plasma. The proposed release mechanism107 is shown in

Figure 4.6.

99

Figure 4.6. Structure of the linker 4.24 (left) and release mechanism of the prodrug (right)

Our initial synthesis of 4.24 followed the Wender protocol.108 This synthesis started with the reaction of 2-

mercaptoethanol 4.25 and 2,2’-dipyridyl disulfide 4.26 to give the mixed disulfide 4.27. As reported by

Satyam, we found the isolation of the pure product to be tedious, especially in a large scale.107 Therefore,

4.27 was prepared by first treating 2-mercapthopyridine 4.28 with sulfuryl chloride and reacting the crude

material with 4.25. This provided much improved yields (40% vs 10%). Reaction of 4.27 with p-

nitrophenylchloroformate 4.29 afforded 4.24.

Scheme 4.3. Synthesis of linker 4.24

100

4.4. C11 mannose derivatives

4.4.1. Sugar-butenolide Prodrug Synthesis

The free alcohol in alkene 4.30 was protected as the MOM derivative 4.31, which was then subjected to

CM with butenolide alkene 4.32 under the previously described conditions to afford 4.33. Reduction of the

double bond and selective deprotection of the TBDPS ether, provided alcohol 4.34 for vector conjugation.

Treatment of 4.34 with nitrophenylcarbonate 4.24 in the presence of DMAP gave 4.35. Next, removal of

the isopropylidene and MOM protecting groups by treatment of 4.35 with methanolic HCl in

dichloromethane yielded the derived triol. Disulfide exchange on this material with in situ generated

DUPA-derived thiol 4.15 delivered prodrug 4.10 in 80% yield from the sugar disulfide precursor (Scheme

4.4). This product was characterized by NMR and HRMS (Figure 4.7).

101

Scheme 4.4. Synthesis of the C11-butenolide prodrug, 4.10

102

Figure 4.7. 1H of C11-butenolide prodrug 4.10

11’

m, o q, k

l, p

20

103

4.4.2. Sugar-thiophene Prodrug Synthesis

The methodology developed for the synthesis of compound 4.10 was applied to 4.11 as the parent drug,

towards the synthesis of prodrug 4.12. Cross metathesis of alkenes 4.31 and 4.36 (3.27 in Chapter 3)

delivered 4.37. After reduction of the double bond, selective deprotection of the silyl group, linker

conjugation and removal of acetal protecting groups afforded protecting group afforded the pyridyl

disulfide precursor 4.37b to prodrug 4.12.

Scheme 4.5. Synthesis of the C11-thiophene prodrug

104

4.5. Annonacin prodrugs

Using the naturally obtained compound avoids a challenging

synthesis. However, the purification of the natural extract may not be

trivial. Furthermore, executed complex reactions on the natural

product may also be problematic. In this context, total synthesis is

advantageous as such reactions can be more straightforwardly

performed on synthetic precursors.

A crude sample of annonacin from Annona muricata was provided by

Professor Mohindra Seepersaud of the University of The West Indies, St Augustine. Details for extraction

of this material and our protocol for the further purification of annonacin are described in the following

section.

4.5.1. Isolation, purification and characterization of annonacin

Approximately 1.5 kg of mature and semi-mature fruits’ seeds were blended and extracted with ethanol

(Figure 4.9). The extract was then partitioned in DCM and water and using the brine lethality test (BST)

the organic extract was found to be more active.

The extraction process was repeated two more times on the crude initial extract and crude compound

was extracted every time. Each of the extracts were purified using dichloromethane and increasing

amounts of methanol. We performed two subsequent purifications on 25 g of this sample (Figure 4.10).

The first purification was done using 60-100% EtOAc/hexanes to 20% methanol/EtOAc. The second

column involved only increasing amounts of methanol (up to 6%) in dichloromethane. This sequence

provided 1.22 g of pure annonacin, 4.13, whose NMR data was essentially identical to that reported

(Figure 4.9, 4.10, Table 4.1).109

Figure 4.8. Annona muricata

105

Figure 4.9. Extraction of annonacin .

TLC 1 semipurified TLC 2 purified

10% MeOH/DCM (2x) 10% MeOH/DCM (2x)

Figure 4.10. Purification of annonacin

106

Figure 4.11. 1H NMR of annonacin reported in the literature

.

Figure 4.12. 1H NMR isolated annonacin

107

The 13C of the extracted annonacin and the one reported in the literature are comparable. See table

below:

Table 4.1. 13C of literature reported

and isolated annonacin

Carbon Literature annonacin

Isolated annonacin

1 174.6 174.32

2 131.1 131.05

3 22-38 22.58-37.18

4 71.6 70.25

5 29.5 29.77-29.12

6 29.5 29.77-29.13

7 29.5 29.77-29.14

8 29.5 29.77-29.15

9 29.5 29.77-29.16

10 69.8 69.85

11 29.5 29.77-29.12

12 29.5 29.77-29.13

13 29.5 29.77-29.14

14 29.5 29.77-29.15

15 73.9 74.17

16 82.6 82.72

17 22-38 22.58-37.18

18 22-38 22.58-37.18

19 82.7 82.63

20 74.1 74.85

21 29.5 29.77-29.12

22-29 29.5 29.77-29.13

30 29.5 29.77-29.14

31 29.5 29.77-29.15

32 14.1 14.13

33 151.8 152.09

34 77.9 78.14

35 19.0 19.05

108

4.5.2. Annonacin prodrug synthesis

With pure annonacin in hand we followed the same route used for the mannose-derived prodrug.

Annonacin, 4.13, was reacted with linker 4.24. For solubility reasons, pyridine was used as a co-solvent

with dichloromethane. This reaction provided a complex mixture comprising mono- and di-substituted

carbonates and unreacted annonacin (Figure 4.13). To purify the complex mixture a first purification was

needed to remove the impurities from the linker and di-substituted annonacins. The mixture of

monosubstituted derivatives was separated into two fractions using methanol/dichloromethane. These

fractions were each further separated into two fractions using acetone/hexane to give four fractions (1.1,

1.2, 2.1 and 2.2).

Figure 4.13. Linker-annonacin purification

109

Fractions 1.2 and 2.2 were assigned the structures 4.38 and 4.39, respectively from the 1HNMR and

HRMS data. Similarly, the compounds in fractions 1.1 and 2.1 were determined to be the other two mono-

carbonate regioisomers, 4.40 and 4.41. Unfortunately, it was not possible to distinguish between these

two structures. Nevertheless, as fraction 1.1 was the only material that was obtained in significant

amounts, it was subjected to the disulfide exchange reaction with 4.15. NMR analysis indicated that the

product was either 4.14 which is derived from 4.41, or the regioisomer that comes from 4.40 (Figures

4.14-4.16 and Appendix).

Scheme 4.6. Synthesis of the annonacin based prodrug

110

Figure 4.14. Fraction 1.1 (4.40 or 4.41)

Figure 4.15. Fraction 1.2 (4.38)

111

Figure 4.16. Fraction 2.2 (4.39)

Using the naturally obtained annonacin as the drug of choice for the prodrug strategy was challenging

because of the chemistry per se, but because selective installation of the linker in annonacin was poor

and purification of the product mixture was extremely difficult. In this context use of a suitably protected

drug entity such as 4.42 (cf 2.48 in Chapter 2), makes a case for total synthesis as a solution to these

problems.

112

Scheme 4.7. En route to the synthesis of 4-DAN prodrug

4.6. Conclusions

The syntheses of two classes of potentially tumor-selective molecules were developed, sugar-derived

agents (termed “chameleon: analogues), in which a mannose based ACG mimetic was modified to

resemble the sugar recognition element in the bleomycin family of antitumor agents, and prodrugs

derived from ACGs or ACG mimetics. The chameleon molecule is appealing because it does not require

complex vector conjugation steps and it avoids a potential problem with prodrugs, i.e.drug release before

delivery to the tumor target. However, the potency of the modified sugar residue or its effectiveness as a

tumor selective ligand remains to be tested. The prodrug synthesis was applied to two synthetic

analogues of 4-DAN and the naturally obtained annonacin. This use of a traceless linker is noteworthy

113

because it facilitates release of the original unmodified drug. Therefore, retention of drug potency is very

likely, but as mentioned above, drug release before delivery to the tumor target could be problematic. For

both classes of these ACG-based drugs, their high degree of hydrophobicity could compromise selectivity

because of non-specific cellular uptake. The cytotoxic properties of these potential tumor selective

molecules and their forerunners from Chapter 3 will be discussed in Chapter 5.

114

4.7. Experimental

Propen-1-yl-4-O-(4-methoxybenzyl)carbamoyl-6-O-undecyl-α-D-mannopyrannoside (4.4)

Triol 4.3 (53 mg, 0.14 mmol) and Bu2SnO (45 mg, 0.18 mmol) were heated in toluene at reflux for 1 h,

then the solvent was evaporated. The residue was dissolved in DMF (1 mL) and PMBNCO (22 µL, 0.15

mmol) was added. The reaction mixture was stirred at rt for 1 h and quenched with MeOH (0.05 mL) and

stirred for additional 30 min. The solvent was removed in vacuo and the residue was purified via FCC to

deliver 4.4 (57 mg, 75%). Rf = 0.55 (5% MeOH/DCM). 1H NMR (CDCl3, 600 MHz) δ 7.23 (m, 2H), 6.88

(m, 2H), 5.91 (m, 1H), 5.33 (m, 1H), 5.23 (m, 1H), 5.03 (m, 1H), 4.87 (br s, 1H), 4.33 (m, 2H), 4.22 (m,


0.91 (t, J = 7.5 Hz, 3H).

Propen-1-yl-4-O-carbamoyl-6-O-undecyl-α-D-mannopyranoside (4.5)

Compound 4.4 (57 mg, 0.11 mmol) and CAN (0.27 g, 0.49 mmol) were dissolved in a 3:1 mixture of

CH3CN:H2O (3 mL) and stirred for 30 min. Then, it was diluted with saturated NaHCO3 and stirred for

additional 30 min. The mixture was then filtered through Celite, extracted with EtOAc and dried (Na2SO4).

FCC of the residue afforded 4.5 (33 mg, 75%). Rf = 0.2 (5% MeOH/DCM). 1H NMR (CDCl3, 600 MHz) δ

5.83 (m, 1H), 5.23 (m, 1H), 5.13 (m, 1H), 4.85 (m, 1H), 4.78 (br s, 1H), 4.12 (m, 1H), 3.91 (m, 3H), 3.69

(m, 3H), 3.41 (m, 2H), 1.51 (m, 2H), 1.19 (m, 16H), 0.81 (t, J = 6.7 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ

157.6, 133.5, 117.8, 98.9, 75.3, 72.1, 71.1, 70.4, 69.5, 68.2, 66.9, 31.9, 29.6, 29.5, 29.5, 29.4, 26.0, 22.7,

14.1.

115

4-O-Carbamoyl-6-O-undecylmannose-thiophene-alkene (4.5a)

Nitrogen was bubbled through a solution of 4.5 (33 mg, 0.08 mmol) and 4.6 (0.1 g, 0.17 mmol) in dry

CH2Cl2 (3 mL) for 30 min and at rt. At that point Grubbs 1st generation catalyst (12 mg, 0.015 mmol) was

added and the mixture heated for 2.5 h at 40 oC. Then, the reaction was quenched with DMSO (0.05 mL).

After stirring for an additional 30 min the solution was evaporated in vacuo. FCC of the residue afforded

0.312 g Rf = 0.25 (30% EtOAc/hexanes). FCC afforded heterodimer 4.5a (0.44 g, 41%). Rf = 0.15 (5%

MeOH/DCM). 1H NMR (CDCl3, 600 MHz) δ 7.58 (m, 4H), 7.40 (m, 1H), 7.32 (m, 1H), 7.28 (m, 6H), 6.99

(m, 1H), 5.94 (br s, 1H), 5.55-5.36 (m, 2H), 4.91 (m, 1H), 4.78 (br s, 1H), 4.03 (m, 1H), 3.94-3.82 (m, 2H),

3.63 (m, 4H), 3.43 (m, 3H), 3.35 (m, 2H), 1.86 (m, 2H), 1.51 (m, 4H), 1.30 (m, 4H), 1.29-1.26 (m, 34H),

0.97 (s, 9H), 0.81 (t, J = 6.9 Hz, 3H).

4-O-Carbamoyl-6-O-undecylmannose-8’-tert-butyldiphenylsiloxy-thiophene (4.5b)

A mixture of 4.5a (0.44 g, 0.44 mmol) and 10% Pd/C (0.05 g) in EtOH (2 mL) was stirred for 16 h under a

H2 atmosphere, then filtered over Celite. The filtrate was then evaporated in vacuo and the residue

subjected to FCC to give 4.5b (0.44 g, quantitative). Rf = 0.15 (5% MeOH/DCM). 1H NMR (CDCl3, 600

MHz) δ 7.69 (m, 4H), 7.50 (m, 1H), 7.47 (m, 1H), 7.38 (m, 6H), 7.09 (m, 1H), 6.01 (br s, 1H), 4.98 (m,

1H), 4.83 (s, 1H), 4.04 (br s, 1H), 3.99 (t, 1H), 3.77-3.67 (m, 4H), 3.50 (m, 3H), 3.44 (m, 3H), 1.61 (m,

4H), 1.33-1.10 (m, 38H), 1.07 (s, 9H), 0.89 (t, J = 6.8 Hz, 3H).

116

8’-Hydroxy-17’-(thiophene-2-carboxamido)heptadecyl-3-O-carbamoyl-6-O-undecyl-α-D-

mannopyranoside (4.7)

A crude sample of 4.5b (40 mg, 0.052 mmol) was dissolved in DCM (2 mL) and treated with 5% AcCl in

MeOH (0.6 mL) for 4 h. Additional 5% AcCl in MeOH (0.3 mL) was then introduced and stirring continued

for 4 h The reaction was then quenched with saturated K2CO3 in methanol, filtered and evaporated in

vacuo. FCC of the residue (80-100% EtOAc/hexanes and 5-10% MeOH/EtOAc) afforded 4.7 (20 mg,

67%). Rf = 0.3 (5% MeOH/DCM). 1H NMR (CDCl3, 600 MHz) δ 7.51 (dd, J1 = 3.66 Hz , J2 = 1.08 Hz, 1H),

7.47 (dd, J1 = 4.98 Hz, J2 = 1.14 Hz, 1H), 7.08 (dd, J1 = 4.98 Hz , J2 = 3.72 Hz, 1H), 6.04 (br s, 1H), 4.98

(m, 1H), 4.83 (s, 1H), 4.03 (br s, 1H), 3.96 (t, J = 2.58 Hz, 1H), 3.79-3.69 (m, 4H), 3.59 (m, 1H), 3.45 (m,

2H), 3.41 (m, 3H), 1.60 (m, 6H), 1.30-1.10 (m, 40H), 0.88 (t. J = 6.8 Hz, 3H); 13C NMR (CDCl3, 600 MHz)

162.1, 157.5, 139.3, 136.1, 129.8, 128.1, 127.8, 99.8, 75.8, 72.3, 72.1, 70.9, 69.9, 68.1, 40.3, 37.6 (3

signals), 37.5, 32.1, 29.8 (3 signals), 29.7 (2 signals), 29.6 (2 signals), 29.4 (2 signals), 29.3, 29.2, 27.1,

26.3, 26.2, 26.1, 25.8, 25.6 (2 signals), 22.9, 14.3. HRMS (ESI) calcd for C40H72N2O9SNa (M+Na)+

779.4851, found 779.4841.

6-O-Undecyl mannose-butenolide-DUPA conjugate (4.10)

Under a N2 atmosphere, 4.35a (16 mg, 0.021 mmol) was dissolved in MeOH (0.5 mL) and NaOMe (0.1

mL, 0.83 M) was added until a pH 8-10. After 10 min, 5 M HCl in MeOH was added until pH 6-7. Next, a

117

solution of 4.35a (18 mg, 0.031 mmol) in methanol (0.5 mL) was added to the reaction mixture. The

reaction was stirred for 18 h, then, the solvent was removed in vacuo. FCC of the residue (15-20%

MeOH/DCM) afforded 4.10 (20 mg, 80%). 1H NMR (MeOD, 600 MHz) δ 7.20 (s, 1H), 4.97 (m, 1H), 4.61

(partially buried s, 1H), 4.60 (m, 1H), 4.25 (t, J = 6.3 Hz, 2H), 4.06 (m, 2H), 3.67-3.20 (m, 9H), 3.11 (m,

3H), 2.84 (t, J = 6.3 Hz, 2H), 2.64 (t, J = 7.3 Hz, 2H), 2.26 (m, 4H), 2.15 (m, 2H), 2.13 (m, 2H), 1.88 (m,

2H), 1.40 (m, 14H), 1.28 (partially buried d, J = 6.7 Hz, 3H), 1.24 (m, 18H), 1.31-1.19 (m, 25H), 0.80 (t, J

= 6.7 Hz, 3H); 13C NMR (MeOD, 600 MHz) δ 176.4, 156.7, 152.4, 134.7, 106.8, 101.7, 80.2, 70.8, 73.5,

72.9, 72.8, 72.3, 71.9, 69.1, 68.8, 66.7, 40.5, 39.8, 38.4, 35.4 (2 signals), 33.3, 31.5, 31.0 (2 signals),

30.8, 30.7 (2 signals), 30.6, 30.5, 30.4 (2 signals), 30.2 (2 signals), 29.3, 28.7, 27.7, 27.5, 27.4, 26.5,

26.4, 23.9, 23.1, 19.4, 14.6. HRMS (ESI) calcd for C57H100N3O19S2 (M+H)+ 1194.6387, found 1194.6299.

Annonacin-DUPA conjugate (4.14)

Following the procedure for the synthesis of 4.10, 4.40/4.41 (9 mg, 0.01 mmol) was transformed to 4.14

(5 mg, 41%). 1H NMR (CDCl3, 600 MHz) δ 7.16 (s, 1H), 4.86 (m, 1H), 4.45 (m, 3H), 4.15 (t, J = 6.2 Hz,

2H), 3.93 (m, 5H), 3.59 (m, 2H), 3.45 (m, 2H), 3.21 (m, 2H), 3.03 (m, 8H), 2.75 (t, J = 6.2 Hz, 2H), 2.54 (t,

J = 7.1 Hz, 2H), 2.49 (m, 2H), 2.34 (m, 4H), 2.23-2.10 (m, 16H), 1.87-1.06 (m, 44H), 0.71 (t, J = 6.8 Hz,

3H). HRMS (ESI) calcd for C57H100N3O17S2 (M+H)+ 1162.6489, found 1162.6495.

5-Benzyl 1-(tert-butyl) (((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate (4.18)

118

A solution of L-glutamic acid di-tertbutyl ester hydrochloride 4.16 (1.0 g, 3.38 mmol) and Et3N (1.54 mL,

11.09 mmol) in DCM (30 mL) was cooled to -78 oC. A solution of triphosgene (0.34 g, 0.34 mmol) in DCM

(10 mL) was added dropwise via a syringe over 30 min. The reaction was stirred and allowed to warm to

rt for 1 h. Then, 4.17 (0.7 g, 2.03 mmol) and Et3N (3 mL, 2.03 mmol) were succesively added and the

reaction was stirred at rt for 16 h. The reaction mixture was then diluted with DCM and washed with H2O,

dried (Na2SO4) and concentrated under reduced pressure. FCC (80% EtOAc/hexanes) delivered 4.18

(1.0 g, 51%). Rf = 0.67 (50% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ 7.33 (br s, 5H), 5.23 (s, 2H),

5.09 (m, 1H), 4.98 (m, 1H), 4.43 (m, 1H), 4.26 (m, 1H), 2.24 (m, 4H), 2.11 (m, 2H), 1.80 (m, 2H), 1.51 (s,

9H), 1.40 (s, 9H), 1.36 (s, 9H); 13C NMR (CDCl3, 600 MHz) δ 172.9, 172.7, 172.5, 172.1, 156.9, 135.6,

128.8, 128.6, 128.5, 82.3, 82.3, 80.9, 80.8 (2 signals), 67.4, 53.3, 52.9, 31.8, 31.7, 28.3, 28.3, 28.2.

HRMS (ESI) calcd for C30H46N2O9Na (M+Na)+ 601.3096, found 601.3091. The NMR data was essentially

identical to that reported in the literature.110

(S)-5-(tert-butoxy)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-5-oxopentanoic acid

(4.19)

Compound 4.18 (1.0 g, 1.7 mmol) was dissolved in ethyl acetate (30 mL). The mixture was purged with

N2 and Pd/C catalyst (0.1 g, 10%) was added. Then, the reaction was stirred under H2 overnight. The

mixture was then purged with N2 and filtered over Celite, and the filtrate concentrated under reduced

pressure. FCC of the residue yielded 4.19 (0.7 g, 82%). Compound did not show well on TLC, so the

reaction was followed by NMR. 1H NMR (CDCl3, 600 MHz) δ 5.29 (br s, 1H), 5.22 (br s, 1H), 4.43 (m, 1H),

4.29 (m, 1H), 2.28 (m, 4H), 2.02 (m, 2H), 1.84 (m, 2H), 1.43 (s, 9H), 1.40 (m, 18H); 13C NMR (CDCl3, 500

MHz) δ 172.5, 172.0, 156.8, 82.0, 80.5, 53.0, 31.6, 28.4, 28.1, 28.0. HRMS (ESI) calcd for C23H40N2O9Na

(M+Na)+ 511.2626, found 511.2619. The NMR data was essentially identical to that reported in the

literature.110

119

6-Aminohexyl-4-methylbenzenesulfonate hydrochloride (4.20)

6-((tert-butoxycarbonyl)amino)hexyl 4-methylbenzenesulfonate111 (5.0 g, 0.013 mol) was stirred in a

solution of 4 M HCl in dioxane at rt for 16 h. After that, it was concentrated in vacuo to provide 4.20 as a

solid (4.13 g, quantitative). 1H NMR (C5H5N, 500 MHz) δ 8.01 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H),

4.07 (t, J = 6.4 Hz, 2H), 3.18 (t, J = 7.5 Hz, 2H), 2.24 (s. 3H), 1.90 (m, 2H), 1.50 (m, 2H), 1.23 (m, 2H),

1.17 (m, 2H); 13C NMR (C5H5N, 500 MHz) δ 145.6, 134.2, 129.5, 128.8, 71.5, 40.0, 29.3, 28.4, 26.7, 25.5,

21.8.

Di-tert-butyl (((S)-1-(tert-butoxy)-5-((6-chlorohexyl)amino)-1,5-dioxopentan-2-yl)carbamoyl)-L-

glutamate (4.21)

To a mixture of 4.19 (0.7 g, 1.43 mmol) and DCM (70 mL) at 0 oC under N2, 4.20 (0.64 g, 2.11 mmol),

EDCI (0.81 g, 4.24 mmol), Et3N (0.73 mL, 5.72 mmol) and DMAP (5 mg, 0.043 mmol) were added. The

reaction was warmed slowly to rt and then stirred for 16 h. FCC (70% EtOAc/hexanes) delivered 4.19

(0.38 g, 44%). Rf = 0.3 (30 % EtOAC/hexanes). 1H NMR (CDCl3, 500 MHz) δ 6.61 (br s, 1H), 5.90 (m,


1.98 (m, 2H), 1.80 (m, 2H), 1.70-1.27 (m, 33H); 13C NMR (CDCl3, 500 MHz) δ 173.5, 172.6, 172.3, 172.1,

157.3, 82.2, 81.1, 80.8, 53.7, 53.2, 45.2, 39.6, 32.6, 32.1, 31.7, 29.5, 28.5, 26.7, 26.3.

Tri-tert-butyl (14S,18S)-2,11,16-trioxo-3-thia-10,15,17-triazaicosane-14,18,20-tricarboxylate (4.22)

120

Compound 4.21 (0.38 g, 0.65 mmol) was dissolved in dry DMSO (2 mL). KSAc (0.22 g, 1.9 mmol) and

18-crown-6 ether (17 mg, 0.064 mmol) were added and the mixture stirred at rt for 2 h. The solvent was

evaporated in vacuo. FCC of the residue provided 4.22 (0.13 g, 31%). Rf = 0.3 (20 % EtOAC/hexanes).

1H NMR (CDCl3, 600 MHz) δ 6.48 (m, 1H), 5.50 (br s, 1H), 5.05 (m, 1H), 4.30 (m, 1H), 4.19 (m, 1H), 3.22

(m, 2H), 2.82 (t, J = 7.3 Hz, 2H), 2.40 (m, 1H), 2.29 (s, 3H), 2.40 (m, 1H), 2.24 (m, 1H), 2.03 (m, 1H), 1.84

(m, 1H), 1.48-1.20 (m, 35H); 13C NMR (CDCl3, 600 MHz) δ 195.5, 173.5, 172.6, 172.2, 172.1, 157.3, 82.2,

81.1 (2 signals), 53.7, 53.2, 39.5, 32.1, 31.7, 30.9, 29.5, 29.4, 29.1, 28.5, 28.4 (2 signals), 28.3 (2

signals), 28.2, 26.4. HRMS (ESI) calcd for C31H55N3O9SNa (M+Na)+ 668.3551, found 668.3543.

(14S,18S)-2,11,16-Trioxo-3-thia-10,15,17-triazaicosane-14,18,20-tricarboxylic acid (4.23)

4.22 (25 mg, 0.037 mmol) was stirred in TFA (0.25 mL), DCM (0.25 mL) and TIBS (0.02 mL) at rt for 1.5

h. The solution was then concentrated in vacuo. FCC of the residue (25% MeOH/DCM to

HOAc:MeOH:DCM, 0.1:2:8) yielded 4.23 (20 mg, quantitatve). Rf = 0.3 (30% MeOH/DCM). 1H NMR

(MeOD, 400 MHz) δ 4.30 (m, 1H), 4.20 (m, 1H), 3.21 (m, 2H), 2.88 (t, J = 7.2 Hz, 2H), 2.38 (m, 4H), 2.32

(s, 3H), 2.16 (m, 1H), 2.04 (m, 1H), 1.81 (m, 1H), 1.77 (m, 2H), 1.52 (m, 4H), 1.39 (m, 4H); 13C NMR

(CDCl3, 400 MHz) δ 197.8, 176.8, 174.7, 160.0, 54.7, 54.0, 40.4, 31.4, 30.8, 30.6, 30.3, 29.9, 29.6, 29.5,

29.0, 27.5. HRMS (ESI) calcd for C19H32N3O9S (M+H)+ 478.1854, found 478.1843.

4-Nitrophenyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate (4.24)

To a solution of 4.27 (0.50 g, 2.7 mmol), pyridine (0.3 mL, 4 mmol) and DCM (10 mL) at rt was added

dropwise over 20 min a solution of 4-nitrophenylchloroformate (0.75 g, 4.0 mmol) in DCM (10 mL). The

121

mixture was stirred overnight, then was washed with water (3x). The organic phase was dried (Na2SO4),

filtered and evaporated under reduced pressure. FCC (15% EtOAc/hexanes) of the residue yielded 4.24

(0.71 g, 76%). Rf = 0.45 (20% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 8.47 (m, 1H), 8.34 (m, 2H),

8.14 (m, 2H), 7.55 (m, 2H), 7.32 (m, 2H), 7.14 (m, 1H), 4.58 (t, J = 1.7 Hz, 2H), 3.10 (t, J = 6.2 Hz, 2H);

13C NMR (CDCl3, 500 MHz) δ 159.1, 155.4, 152.2, 149.9, 137.2, 126.3, 125.0, 123.2, 122.1, 120.2, 115.7,

66.7, 36.7. HRMS (ESI) calcd for C14H13N2O5S2 (M+H)+ 352.0188, found 352.0187. The NMR data

reported is essentially identical to the one found in literature.96

2-(Pyridin-2-yldisulfaneyl)ethan-1-ol (4.27)

Sulfuryl chloride (50 mL, 1M solution in DCM) was added over a period of 20 min to a stirred solution of 2-

mercaptopyridine, 4.28 (5.0 g, 0.045 mol) in dry DCM (50 mL) at 0-5 oC under a nitrogen atmosphere.

The mixture was stirred for 2 h and concentrated in vacuo. The yellow granular solid was taken up in dry

DCM (200 mL) and cooled to 0 oC. To this stirred suspension at 0-5 oC was added a solution of 2-

mercaptoethanol 4.25 (3.4 mL, 0.048 mol) in DCM (10 mL) dropwise over 5 min. Within 15-20 min a

yellow solid started to separate. The mixture was stirred at rt overnight. The product was filtered, washed

with DCM, and dried under high vacuum. The residue was resuspended in DCM and DMAP (5.50 g,

0.045 mmol) was added. The suspension was filtered and concentrated in vacuo. FCC of the residue

(10% EtOAc/hexanes) yielded 4.27 (3.4 g, 40%). Rf = 0.16 (20% EtOAc/hexanes). 1H NMR (CDCl3,

500MHz) δ 8.53 (d, J = 3.8 Hz, 1H), 7.41 (m, 1H), 7.32 (m, 1H), 7.19 (m, 1H), 3.78 (m, 2H), 2.93 (m, 2H);

13C NMR (CDCl3, 500MHz) δ 159.1, 149.8, 136.9, 122.0, 121.6, 58.2, 42.8; HRMS (ESI) calcd for

C7H10NOS2 (M+H)+ 187.0126, found 187.0130.

122

Propen-1-yl-3,4-O-isopropyledene-4-O-methoxymethyl-6-O-undecyl-α-D-mannopyranoside (4.31)

Alcohol 4.30 (0.40 g, 0.96 mmol) was dissolved in DCM (16 mL), MOMCl (0.29 mL, 3.81 mmol), DIPEA

(0.86 mL, 0.006 mmol) were added at 0 oC and the solution was stirred overnight. The reaction was then

quenched with water and extracted with DCM. The organic phase was dried Na2SO4, concentrated in

vacuo and purified via FCC (25% EtOAc/hexanes) to afford compound 4.31 (0.3 g, 68%). Rf = 0.62 (20%

EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ 5.87 (m, 1H), 5.26 (m, 1H), 5.17 (m, 1H), 5.65 (s, 1H),

4.86 (d, J = 5.3 Hz, 1H), 4.65 (d, J = 5.2 Hz, 1H), 4.19 (m, 1H), 4.11 (m, 1H), 3.98 (m, 1H), 3.66 (m, 3H),

3.57 (m, 1H), 3.48 (m, 1H), 3.42 (m, 4H), 1.55 (m, 4H), 1.50 (s, 3H), 1.31 (partially buried s, 3H), 1.30-

1.22 (m, 21H), 0.86 (t, J = 5.8 Hz, 3H).; 13C NMR δ 133.6, 117.8, 109.3, 96.3, 96.2, 78.4, 75.9, 73.2, 71.8,

69.7, 68.3, 67.9, 56.0, 31.9, 29.8, 29.7, 29.6, 29.5, 29.3, 27.9, 26.4, 26.2, 22.7, 14.2. HRMS (ESI) calcd

for C25H47O7 (M+H)+ 458.3244, found 458.3243.

4-O-Methoxymethyl-6-O-undecyl mannoside-butenolide-alkene (4.33)

A mixture of 4.31 (0.3 g, 0.65 mmol) and 4.32 (0.18 g, 0.33 mmol) in dry CH2Cl2 (3.3 mL) was purged with

nitrogen. Grubbs I catalyst (26 mg, 0.03 mmol) was then added and the mixture purged with nitrogen,

then heated at reflux for 6 h. At that time the mixture was cooled to rt and DMSO (0.05 mL) was added.

After 1 h the mixture was concentrated in vacuo. FCC (30% EtOAc/hexanes) of the residue afforded 4.33

(0.27 g, 42%). Rf = 0.53 (20% EtOAc/hexanes). 1H NMR (CDCl3, 500 MHz) δ 7.37 (m, 4H), 7.33 (m, 6H),

6.95 (s, 1H), 5.62 (m, 1H), 5.48 (m, 1H), 5.05 (s, 1H), 4.97 (m, 1H), 4.86 (m, 1H), 4.66 (d, J = 6.3 Hz, 1H),

4.18-4.10 (m, 3H), 3.80 (m, 1H), 3.67 (m, 4H), 3.57 (m, 1H), 3.50 (m, 1H), 3.38 (m, 4H), 2.22 (m, 2H),

123

1.90 (m, 2H), 1.50-1.20 (m, 45H), 1.00 (s, 9H), 0.85 (t, J = 7.2 Hz, 3H). HRMS (ESI) calcd for C58H93O10SI

(M+H)+ 976.6460, found 976.6454.

4-O-Methoxymethyl-6-O-undecyl mannoside-butenolide (4.33a)

Compound 4.33 (0.27 g, 0.27 mmol) and TsNHNH2 (1.8 g, 9.67 mmol) were dissolved in DME (60 mL). At

reflux, a solution of NaOAc (2.1 g, 25.6 mmol) in H2O (63 mL) was added dropwise over a 4 h period. The

mixture was then cooled, diluted with ethyl acetate, and washed with water. The organic phase was dried

(Na2SO4), filtered and concentrated in vacuo. FCC in 1-5% (acetone/DCM) delivered 4.33a. Rf = 0.5 (2%

acetone/DCM). 1H NMR (CDCl3, 600 MHz) δ 7.64 (m, 4H), 7.33 (m, 6H), 6.93 (m, 1H), 4.99 (s, 1H), 4.97

(m, 1H), 4.86 (d, J = 6.2 Hz, 1H), 4.66 (d, J = 6.2 Hz, 1H), 4.17 (m, 1H), 4.07 (m, 1H), 3.66 (m, 5H), 3.58

(m, 1H), 3.46 (m, 1H), 3.40 (m, 2H), 3.38 (s, 3H), 2.40 (s, 1H), 2.22 (m, 2H), 1.50 (m, 17H), 1.24 (m, 6H),

1.21 (m, 18H), 1.19 (m, 8H), 1.01 (s, 9H), 0.87 (m, 3H). HRMS (ESI) calcd for C58H92O10Si (M+Na)+

1001.6514, found 1001.6500.

10-Hydroxy-4-O-methoxymethyl-6-O-undecylmannoside-butenolide-alkene (4.34)

Compound 4.33a was then dissolved in a mixture of pyridine (1 mL) and HF/pyridine (1 mL) at 0 oC. The

mixture was stirred for 16 h, then diluted with water and extracted with ethyl ether. FCC of the residue

afforded 4.33b (85 mg, 43% after two steps). 1H NMR (CDCl3, 500 MHz) δ 6.96 (s, 1H), 4.99 (s, 1H), 4.97

124

(m, 1H), 4.86 (d, J = 6.2 Hz, 1H), 4.66 (d, J = 6.3 Hz, 1H), 4.17 (m, 1H), 4.07 (d, J = 5.6 Hz, 1H), 3.68 (m,

5H), 3.56 (m, 2H), 3.47 (m, 1H), 3.38 (m, 4H), 2.41 (m, 1H), 1.60-1.32 (m, 51H), 0.86 (t, J = 6.9 Hz, 3H);

13C NMR (CDCl3, 600 MHz) δ 174.1, 149.1, 134.5, 109.4, 97.1, 95.5, 78.7, 76.2, 73.4, 72.1, 72.0, 69.9,

68.4, 67.6, 56.2, 37.7 (2 signals), 32.1, 29.9, 29.8, 29.7 (2 signals), 29.6, 29.5 ( 2 signals), 29.3, 28.0,

27.6, 26.6, 26.3 (2 signals), 25.8, 25.3, 22.9, 19.4, 14.3.

[1-(5-Methyl-2-oxo-2,5-dihydrofuran-3-yl)-pentadecan-8-yl-(2-(pyridin-2-yldisulfaneyl)ethyl)

carbonate]-15-yl-2,3-O-isopropylidene-4-O-methoxymethyl-6-O-undecyl-α-D-mannopyranoside

(4.35)

DMAP (12 mg, 0.11 mmol) was added to a solution of 4.34 (50 mg, 0.07 mmol) and linker 4.24 (40 mg,

0.11 mmol) in DCM (1 mL). The reaction was monitored by TLC, and quenched with water after 16 h, on

disappearance of 4.24. The solvent was evaporated and FCC of the residue (20% EtOAc/hexanes)

yielded 4.35 (30 mg, 50%). Rf = 0.3 (20% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ 8.45 (d, J = 4.7

Hz, 1H), 7.63 m, 2H), 7.07 (m, 1H), 6.95 (m, 1H), 4.98 (s, 1H), 4.96 (m, 1H), 4.85 (d, J = 6.2 Hz, 2H), 4.35

(m, 2H), 4.15 (t, J = 6.1 Hz, 1H), 4.10 (m, 1H), 4.05 (m, 1H), 3.56 (m, 4H), 3.55 (m, 1H), 3.48 (m, 1H),

3.45 (m, 1H), 3.37 (m, 4H), 3.04 (t, J = 6.1 Hz, 2H), 2.22 (m, 2H), 1.52 (m, 10H), 1.49 (s, 3H), 1.37 (d, J =

6.8 Hz, 3H), 1.29 (s, 3H), 1.27-1.21 (m, 26H), 0.84 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 500 MHz) δ

174.1, 159.7, 155.0, 149.9, 149.1, 137.3, 134.4, 121.1, 120.0, 109.4, 97.1, 96.5, 79.6, 78.6, 76.2, 73.3,

72.0, 69.8, 68.3, 67.6, 65.2, 56.2, 37.2, 34.2, 32.1, 29.9, 29.8, 29.7, 29.5, 29.4, 29.3 (2 signals), 28.0,

27.5, 26.6, 26.3, 25.3, 22.9, 19.4, 14.3.

125

[1-(5-Methyl-2-oxo-2,5-dihydrofuran-3-yl)-pentadecan-8-yl-(2-(pyridin-2-

yldisulfaneyl)ethyl)carbonate]-15-yl-6-O-undecyl-α-D-mannopyranoside (4.35a)

Compound 4.35 (30 mg, 0.03 mmol) was dissolved in CH2Cl2 (2 mL) and treated with 5% AcCl in MeOH

(0.5 mL). The reaction was stirred for 16 h and then quenched with a saturated solution of NaHCO3 in

methanol. The mixture was filtered, and the filtrate concentrated in vacuo. FCC of the residue (5%

MeOH/DCM) afforded 4.35a (18 mg, 69%). 1H NMR (CDCl3, 500 MHz) δ 8.51 (d, J = 4.5 Hz, 1H), 7.69

(m, 2H), 7.14 (m, 1H), 7.01 (s, 1H), 5.02 (m, 1H), 4.85 (s, 1H), 4.60 (m, 1H), 4.41 (t, J = 6.5 Hz, 2H), 3.94

(br s, 1H), 3.85-3.45 (m, 6H), 3.54 (m, 2H), 3.43 (m, 1H), 3.10 (t, J = 6.5 Hz, 2H), 2.28 (t, J = 8.2 Hz, 2H),

1.59 (m, 18H), 1.40 (d, J = 5.8 Hz, 3H), 1.28 (m, 27H), 0.90 (t, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 500

MHz) δ 174.2, 160.0, 155.0, 149.9, 149.2, 137.3, 134.4, 121.1, 120.1, 99.7 (2 signals), 79.6, 72.3, 71.9,

71.8, 70.8, 70.6, 69.0 (2 signals), 68.0 (2 signals), 65.2, 53.6, 51.1, 37.2, 34.2, 32.1, 29.9, 29.8, 29.7,

29.6, 29.5 (2 signals), 29.4 (2 signals), 29.3 (2 signals), 27.5, 26.2 (3 signals), 25.3 (2 signals), 22.9, 19.4,

14.3. HRMS (ESI) calcd for C45H76NO11S2 (M+H)+ 870.4860, found 870.4850.

4-O-Methoxymethyl-6-O-undecyl mannoside-thiophene alkene (4.37)

A mixture of 4.31 (160 mg, 0.35 mmol) and 4.36 (0.56 g, 0.93 mmol) in dry CH2Cl2 (10 mL) was purged

with nitrogen. Grubbs I catalyst (43 mg, 0.05 mmol) was then introduced and the mixture heated at reflux

for 6 h. At that time the mixture was cooled to rt and DMSO (0.05 mL) was added. After 1 h the mixture

126

was concentrated in vacuo. FCC (40% EtOAc/hexanes) of the residue afforded compound 4.37 (245 mg,

68%). Rf = 0.53 (20% EtOAc/hexanes). 1H NMR (CDCl3, 600 MHz) δ 7.64 (m, 4H), 7.46 (m, 1H), 7.44 (m,

1H), 7.34 (m, 6H), 7.04 (m, 1H), 5.05 (br s, 1H), 4.86 (d, J = 6.3 Hz, 1H), 4.66 (d, J = 6.2 Hz, 1H), 4.18


1.55 (m, 6H), 1.37 (s, 3H), 1.37-1.12 (m, 41H), 1.02 (s, 9H), 0.86 (t, J = 5.1 Hz, 3H); 13C NMR (CDCl3,

600 MHz) δ 162.0, 139.3, 136.1 (2 signals), 135.0, 134.9, 129.8, 129.6 (2 signals), 128.0, 127.7, 127.6,

125.1, 109.4, 96.5, 96.0, 78.6, 76.2, 73.4, 73.3, 72.0, 69.9, 68.4, 67.8, 56.1, 40.2, 36.5, 35.3, 32.4, 32.1,

29.9 (2 signals), 29.8, 29.7 (2 signals), 29.6, 29.5 (2 signals), 29.2, 28.0, 27.3, 27.1, 26.6, 26.4, 25.0,

24.7, 22.9, 19.6, 14.3. HRMS (ESI) calcd for C60H95NO9SSiNa (M+Na)+ 1056.6389, found 1056.6389.

4-O-Methoxymethyl-6-O-undecyl mannoside-thiophene (4.37a)

A solution of compound 4.37 (245 mg, 0.23 mmol) in EtOH (10 mL) and Pd/C (150 mg, 10% wt) was

purged with N2 for 30 min. Then, the mixture was stirred under a hydrogen atmosphere (balloon) for 12 h.

Additional Pd/C was then added (50 mg, 10% wt) and stirring continued for additional 24 h. The reaction

was then purged with N2 and filtered through a bed of Celite to yield 4.37a (0.2 g, 82 %). 1H NMR (CDCl3,

600 MHz) δ 7.64 (m, 4H), 7.43 (m, 1H), 7.38 (m, 1H), 7.34 (m, 6H), 7.04 (m, 1H), 5.92 (br s, 1H), 5.00 (s,

1H), 4.86 (d, J = 6.3 Hz, 1H), 4.66 (d, J = 6.2 Hz, 1H), 4.18 (m, 1H), 4.07 (m, 1H), 3.68 (m, 6H), 3.64 (m,

1H), 3.47 (m, 1H), 3.37 (m, 7H), 1.56 (m, 6H), 1.55 (s, 3H), 1.37 (m, 4H), 1.32 (partially buried s, 3H),

1.09-1.26 (m, 35H), 1.02 (s, 9H), 0.85 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 162.0, 139.4,

136.2, 135.0, 129.9, 129.8, 129.6, 128.0, 127.9, 127.8, 127.6, 109.5, 97.1, 96.5, 78.7, 76.3, 73.4, 72.0,

69.9, 68.4, 67.8, 56.2, 40.3, 36.5, 32.1, 30.0, 29.9 (2 signals), 29.8 (2 signals), 29.7 (2 signals), 29.6 (2

signals), 29.5, 28.1, 27.3, 27.2, 26.8, 26.6, 26.4 (2 signals), 25.1, 22.9, 22.6, 19.6, 14.3.

127

6-O-undecyl mannoside-2-(pyridin-2-yldisulfaneyl)ethyl)carbonate-thiophene (4.37b)

4.37a (200 mg, 0.19 mmol) was dissolved in pyridine (2 mL) and HF/pyridine (10%, 1 mL) was added.

The reaction was stirred at 45 oC for 24 h and then at rt for additional 24 h. The product was isolated and

purified following the procedure used for 4.33b to afford the derived alcohol (91 mg, 65%). Rf = 0.4 (40%

EtOAc/hexanes). A mixture of this material (90 mg, 0.11 mmol), linker 4.24 (80 mg, 0.23 mmol) and

DMAP (30 mg, 0.25 mmol) in DCM (1 mL) was stirred at rt for 16 h. The solvent was then evaporated,

and the residue subject to FCC. The partially purified product (Rf = 0.4 (40% EtOAc/hexanes). was

dissolved in DCM (4 mL) and 5% AcCl in MeOH (0.6 mL) was added. The reaction mixture was then

evaporated under reduced pressure at rt. FCC of the residue afforded 4.37b (76 mg, 73% after two

steps) Rf = 0.42 (8% MeOH/DCM). 1H NMR (CDCl3, 600 MHz) δ 8.41 (m, 1H), 7.61 (m, 1H), 7.59 (m,

1H), 7.41 (m, 1H), 7.38 (m, 1H), 7.19 (m, 1H), 7.05 (m, 1H), 5.99 (br s, 1H), 4.75 (s, 1H), 4.62 (m, 1H),

4.31 (t, J = 6.5 Hz, 2H), 3.84-3.58 (m, 7H), 3.44 (m, 2H), 3.33 (m, 3H), 3.01 (t, J = 6.3 Hz, 2H), 1.56 (m,

12H), 1.27 (m, 37H), 0.81 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 600 MHz) δ 162.1, 159.7, 155.0, 149.9,

139.3, 137.3, 129.8, 128.0, 127.7, 121.1, 120.1 (2 signals), 99.7, 79.6, 72.3, 71.9, 71.8 (2 signals), 70.7

(2 signals), 69.4 (2 signals), 68.0, 67.9, 65.2, 40.2, 37.3, 34.1 (2 signals), 32.1, 29.8 (3 signals), 29.7,

29.6, 29.5 (3 signals), 29.4 (2 signals), 27.1, 26.2 (2 signals), 25.3, 22.9, 14.3. HRMS (ESI) calcd for

C47H79N2O10S3 (M+H)+ 927.4890, found 927.4891.

128

Annonacin 2-(pyridin-2-yldisulfaneyl)ethyl)carbonates

Annonacin 4.13 (0.25 g, 0.412 mmol), was dissolved in 6 mL of a DCM: pyridine mixture (9:1) under N2.

Nitrophenylcarbonate 4.24 (0.13 g, 0.37 mmol) and DMAP (50 mg, 0.41 mmol) were added and the

reaction monitored by TLC. After 16 h, methanol (0.05 mL) was added and after 30 min stirring the

mixture was concentrated in vacuo. The residue was purified by multiple FCC (see text for details), to give

4.38 (6 mg, 2% yield), 4.39 (3 mg, 1% yield), 4.40 (or 4.41) (9 mg, 3%), an unseparated mixture of 4.40

and 4.41 (45 mg, 12%) and recovered 4.13 (0.1, 29%). For 4.38: Rf = 0.6 (8% MeOH/DCM). 1H NMR

(CDCl3, 500 MHz) δ 8.46 (m, 1H), 7.64 (m, 2H), 7.17 (br s, 1H), 7.09 (m, 1H), 5.04 (m, 1H), 4.67 (m, 1H),

4.36 (t, J = 6.5 Hz, 2H), 3.80 (m, 3H), 3.37 (m, 2H), 3.05 (t, J = 6.5 Hz, 2H), 2.52 (m, 1H), 2.32 (m, 5H),

1.96 (m, 6H), 1.45-1.00 (m, 36H), 1.41 (partially buried d, J = 6.9 Hz, 3H), 0.83 (m, 3H). HRMS (ESI)

129

calcd for C45H76NO9S2 (M+H)+ 838.4956, found 838.4927. For 4.39: Rf = 0.5 (8% MeOH/DCM). 1H NMR

(CDCl3, 500 MHz) δ 8.46 (m, 1H), 7.64 (m, 2H), 7.11 (br s, 1H), 7.03 (m, 1H), 4.99 (m, 1H), 4.91 (m. 1H),

4.37 (t , J = 6.7 Hz, 2H), 3.78 (m, 2H), 3.56 (br s, 1H), 3.38 (br s, 2H), 3.04 (t, J = 6.5 Hz, 2H), 2.57 (m,

2H), 2.31 (m, 1H), 2.27 (m, 1H), 1.97 (m 4H), 1.54-1.36 (m, 8H), 1.37 (partially buried d, J = 6.9 Hz, 3H),

0.34 (m, 3H). For 4.40 (or 4.41): Rf = 0.6 (8% MeOH/DCM). 1H NMR (CDCl3, 500 MHz) δ 8.40 (m, 1H),

7.60 (m, 2H), 7.12 (s, 1H), 7.06 (m, 1H), 4.99 (m, 1H), 4.6 (m, 1H), 4.32 (m, 2H), 3.92 (m, 1H), 3.75 (m,

2H), 3.51 (br s, 1H), 3.28 (m, 1H), 2.99 (m, 2H), 2.47 (m, 1H), 2.34 (m, 2H), 2.28 (m ,1H), 1.91 (m, 3H),

1.45-1.29 (m, 10H), 1.39 (partially buried d, J = 6.9 Hz, 3H), 1.19 (m, 34H), 0.82 (m, 3H).

130

ANTITUMOR ACTIVITY OF 4-DEOXYANNOMONTACIN AND ANALOGUES

131

5.1. Introduction

The cytotoxicity of naturally occurring THF-ACGs and unnatural analogues against a broad range of

human tumor cell lines has been widely studied.12, 63 The MTT assay is the most common assay used.

However, because studies have been performed at different timelines and against different cell lines, it is

often difficult to compare the data. This is particularly important as the activity of certain analogues has

been shown to vary with the length of time that the cells are incubated with the drug.62 Herein, the activity

of our synthetic compounds in the MTT assay at 16 and 48 h is presented.

The cell lines evaluated were the prostate cancer (PCa) cells, PC-3 and LNCaP, MCF-7 (MDR breast),

MDA-MB-231 (breast) and HTC116 (colon). This panel was chosen to test activity against different tumor

types and to evaluate activity of parent drugs and their DUPA-linked conjugates against PSMA (prostate

specific membrane antigen) positive and negative cells. LNCaP is known to overexpress PSMA which

binds DUPA with high specificity and affinity, whereas wild-type PC-3, and the non-PCa lines do not

present PSMA.103a, b, 110 Therefore, the DUPA-drug conjugate of a given parent drug is expected to be

more active than the parent drug against LNCaP cells, and other PSMA positive cells compared to PSMA

negative cells.

5.2. Results

Activities are reported as the half maximal inhibitory concentration (IC50). As it was not possible to

calculate an IC50 for analogues with low activity, the % cell viability at 12.5 µM and 25 µM is also reported

to give a measure of the relative activity of all test compounds. Compounds were first assayed at 16 h at

1-25 µM against MCF-7, MDA-MB-231, HTC116, PC-3 and LNCaP. Analogues with significant activity or

of structure activity significance were advanced to a 48 h assay starting at a wider range of

concentrations.

For comparison of the data, the test compounds are grouped according to structural similarity: (a) THF

with butenolide or butenolide substitutes (1, 2, 3, 4, 5); (b) Sugar analogues with butenolide (6, 7, 8, 9,

132

10); (c) Hybrid analogues with both THF and butenolide substitutes (11, 12, 13, 14), including the

‘chameleon’ analog, 14. The results for each subgroup are discussed in the following sections.

5.2.1. THF with butenolide or butenolide substitutes (1, 2, 3, 4, 5)

This group comprises the naturally occurring THF-butenolides 4-DAN, 1 and annonacin, 2, and 3, 4 and

5, THF analogues in which the butenolide is replaced with O-n-butyl, N-linked piperazine and thiophene-

2-carboxamide moieties, respectively (Table 5.1). In the 16 h MTT assay, all analogues except 3 showed

less than 50% cell viability at concentrations lower than 25 M against at least four of the five cell lines.

The four active analogues 1, 2, 4 and 5 were further evaluated at lower concentrations over 48 h (Table

5.2, Figure S5-I).

Table 5.1. Cell viability (%) over 16 h at 12.5 and 25 µM (ND = Not determined, *data collected in an independent study)

Compound

MBA-MD-231

HTC116 LNCaP PC-3 MCF-7

12.5 25 12.5 25 12.5 25 12.5 25 12.5 25

75 60 40 30 10 10 45 40 45 35

80 70 55 45 30 30 40 30 50 50

80 75 ND ND 70 55 80 75 ND ND

75 60 60 40 20 20 40 30 35 25

30 25 30 20 10 0 30 30 45 40

133

Table 5.2. THF with butenolide or butenolide substitutes IC50s (µM) in 16 and 48 h assays (ND = Not determined)

Compound

MBA-MD-231


16 48 16 48 16 48 16 48 16 48

ND 1.5 <0.1 0.2 <0.1 0.01 4 0.2 9 0.8

ND ND 14 ND 7 5 15 4 24 ND

ND ND ND ND <0.1 5 ND ND ND ND

ND ND 17 1.5 <0.1 0.1 3 3 4 ND

<0.1 2 6 0.2 <0.1 0.6 <0.1 0.2 <0.1 0.4

As discussed earlier, and noted in other studies, in certain cases the IC50s values at 16 and 48 h were

noticeably different, but no clear trends emerged for 1, 2, 4 and 5. For example, in MCF-7, the IC50s for 1

at 16 and 48 h are 9 µM and 0.8 µM respectively. In contrast, compound 5 was more active at 16 than at

48 h (IC50s: 0.1 vs 2 µM).

The epimeric mixture of 4-DAN 1, and thiophene 5, showed similar activity across the five cell lines and

were the most potent compounds, at both 16 and 48 h. Compound 2, annonacin, structurally very similar

to 1 except for the presence of an additional hydroxyl group at C-4, was somewhat lower in potency than

1. The piperazine analog 4, was not as active as 1, 2 and 5, but of comparable potency.

134

5.2.2. Butenolide analogues with sugar substitutes for the THF (6, 7, 8, 9, 10)

Table 5.3. Cell viability (%) over 16 h at 12.5 and 25 µM (ND = Not determined, *data collected in an independent study)

Compound

MBA-MD-231

HTC116 PC-3 MCF-7

12.5 25 12.5 25 12.5 25 12.5 25

95 80 65 60 55 55 80 60

75 50 ND ND 75 55 ND ND

80 80 65 65 60 60 60 60

70 65 60 40 55 50 75 65

60 55 80 75 60 50 65 55

Analogues 6, 7, 8, 9 and 10 probed the effect of replacing the THF with a sugar residue and the length

and position of a hydrocarbon chain on the sugar. An initial evaluation was performed at 16 h against four

cell lines (MBA-MD-231, HTC116, PC-3 and MCF-7). Several cases of close to 50% inhibition in the 10-

25 M range were observed, but as a group these compounds were noticeably less active than the THF

containing compounds against the four cell lines. Specific instances of high potency and selectivity were

observed but no clear SAR trends regarding the effect of alkyl chain length and position emerged. In

particular, the 6-O-undecyl and 6-O-butyl analogues 6 and 8 respectively, were selective for PC-3 and

135

MCF-7 with close to 50% inhibition at less than 5 M. Of these, 6 was selected for further examination at

48 h and at lower concentrations (cell viability at 5 µM: MBA-MD-231: 90%; HTC116: 60%; LNCaP: 60%;

PC-3: 75%; MCF-7: 80%) as it showed broader activity across the different cell lines than the others. As

discussed for the THF-butenolide and butenolide replacement compounds in Section 5.2.1, for the case

of compound 6, there is no clear trend in increase or decrease in activity at 16 vs 48 h.

5.2.3. Hybrid analogues with THF and butenolide substitutes (11, 12, 13, 14)

These compounds were investigated to evaluate effects of substituting the THF and butenolide moieties

in the natural compounds with sugar residues and other heterocycles respectively.

Table 5.4. Cell viability (%) over 16 h at 12.5 and 25 µM (ND = Not determined)

Compound

MBA-MD-231


12.5 25 12.5 25 12.5 25 12.5 25 12.5 25

80 55 50 40 75 55 50 40 40 10

55 40 65 55 75 50 40 45 70 70

ND ND ND ND 45 40 40 35 ND ND


The sugar-piperazine analogues 11 and 12 were evaluated at both 16 and 48 h against the five cell lines.

The 6-O-undecyl derivative 11, and the C-10 deoxy analog 12, showed low micromolar activity, with the

136

latter being the more active and comparable in activity to 1. Both compounds were more active at 16 h

than 48 h (Table 5.5, S5-III). The thiophene analogues 13 and 14 were only evaluated against PCa cell

lines. The 6-O-undecyl derivative 13 exhibited IC50s of less than 1 M and was more active than the

parent THF-butenolide, 1. This compound remains to be further examined at lower concentrations and

longer incubation times (Table 5.5, Figure S5-III). Compound 14, the 2-carbamoyl analog of 13, preserves

most of the activity despite the structural modification.

Table 5.5. Hybrid compounds IC50s (µM) (16 and 48 h assays)

Compound

MBA-MD-231

HTC116 LNCaP PC-3 MCF-

7

16 16 16 48 16 48 16

ND 25 ND ND 4 ND 3

25 ND 24 ND <0.1 ND ND

ND ND ND <0.1 2 0.1 ND

ND ND ND <0.1 12 6 ND

Based on the limited data for these hybrid molecules replacement of the THF and butenolide segments of

the natural occurring ACGs with sugar and N-linked piperazine or thiophene-2-carboxamide residues

respectively, delivered analogues with comparable potency to the parent THF-butenolide. Of all the

analogues tested the approximate order of activity appears to be: THF-thiophene/piperazine ~ THF-

butenolide > sugar-thiophene/piperazine > sugar-butenolide.

137

5.3. Discussion

As determination of IC50s was not possible for the entire panel of test compounds, to allow for a more

complete comparison of activities, Table S5-II summarizes activities at 12.5 and 25 mM for all analogues.

Our SAR observations are generally consistent with previous studies (Figure 5.1).12,69 Thus, the

observation that replacement of the butenolide with thiophene or a piperazine leads to analogues with

comparable activity to the THF-butenolide parent, agrees with earlier results by Kojima42, 44, 56, 112 and

Yao.41 However, in contrast to other studies, substitution of the butenolide with a simple n-butyl chain led

to considerable loss in activity.113

Figure 5.1. SAR conclusion based on cytotoxicity assays

Substitution of the THF ring by a mannose delivered compounds with activity in the 25 µM range, which

was somewhat lower than the THF-butenolide parent, and in line with earlier results from this laboratory.79

No clear trend emerged regarding the effects of the length and location of the hydrocarbon chain on the

sugar, as individual analogues with specific chain lengths and positions all displayed significant activity.

However, these SAR effects merit closer evaluation as several notable cases of selectivity and potency

were observed.

138

There have been contradicting reports of the effect that the hydroxyl group in the hydrocarbon spacer has

on activity.67, 70,70 In this context, 11, the C6-undecyl-mannose-piperazine and 12, its C10-deoxygenated

counterpart both showed high potency, but their relative toxicity was dependent on the cell line studied. In

a similar vein, we also found that annonacin 2, was less active than 4-DAN, 1, the 4-deoxy congener of 2.

The most interesting compounds resulted from replacing both the THF and butenolide segments of the

naturally occurring ACGs with sugar and N-piperazine or thiophene-2-carboxamide residues. These

hybrid structures are appealing in that they are of comparable potency to the parent THF-butenolide

parents but are considerably more synthetically accessible. The piperazine analogues are of additional

interest in that the secondary amine allows for straightforward conjugation to imaging entities or tumor

vectors. In the latter context, sugar hybrids such as the 3-O-carbamoyl derivative, 14 have additional

appeal in that the sugar residue, following from the tumor selectivity of the bleomycins, is an “in-built”

potential tumor vector.93c, 99-100

5.4. Prodrug studies

Figure 5.2. Structure of compound 6 and its prodrug 15

Our hypothesis is that conjugation of a cytotoxic entity to a tumor-specific vector leads to a drug that is

more toxic to tumor cells that overexpress the receptor than cells that do not. To test this hypothesis for

our ACG mimetics, the sugar-butenolide 6 and its DUPA prodrug analog 15, were assayed against the

139

PSMA positive prostate cell line, LNCaP, and the PSMA negative cells, PC-3 (prostate), MCF-7 (MDR

breast), MDA-MB-231 (breast) and HTC116 (colon). Cytotoxicity data was collected over 16 and 48 h.

5.4.1. Cytotoxicity data for 6 and 15 against LNCaP

Over 16 h the prodrug 15 was an order of magnitude more active against LNCaP than the parent drug 6

(IC50: 2.5 vs 24 µM, Figure 5.3) This increase in potency of 15 is consistent with the presence of PSMA on

LNCaP and the notion that PSMA is an internalizing receptor than facilitates uptake of DUPA conjugates,

thereby leading to increase drug uptake and potency.103a, b Similar trends in activity for 6 and 15 were

observed over 48 hours.

16 h 48 h

LN

CaP

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

Figure 5.3. Cytotoxicity at 16 and 48 hours of compounds 6 and 15 in PSMA positive cells

140

5.4.2 Cytotoxicity data for 6 and 15 against PSMA negative cells

16 h 48 h

MB

A-M

B-2

31

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

HT

C116

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

MC

F-7

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

PC

-3

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

Figure 5.4. Cytotoxicity at 16 and 48 hours of compounds 6 and 15 in PSMA negative cells

141

Compounds 6 and 15 showed a similar activity profile against the PSMA negative cells MBA-MB-231,

HTC116 and MCF-7 (Figure 5.4). At 16 h, whereas the prodrug 15 was significantly more active than 6

against LNCaP (ca 20 vs 50% cell viability at 25 M), the activity of 6 and 15 against the three PSMA

negative cell lines was similar (ca 70-90% cell viability at 25 M).

As a crude measure of selectivity, the activity at 5 M of 15 at 16 h against LNCaP vs the PSMA negative

cell lines are compared in Table 5.6. The relatively higher toxicity against LNCaP vs the PSMA negative

cells, albeit modest, supports our hypothesis that the DUPA conjugated prodrug 15 should be more

selective for PSMA than PSMA negative cell lines.

Table 5.6. Comparison of activity (% cell viability) of 15 for LNCaP vs PSMA negative cell lines at 5 M

MBA-MB-231 HTC116 PC-3 LNCaP MCF-7

% viability at

5 M and 16 h 80 80 80 40 80

% viability PSMA (–)

% viability LNCaP 2 2 2 1 2

It is also noteworthy that 15 was subtly but consistently lower in activity than 6 against the three PSMA

negative cell lines (ca 90 vs 70% cell viability at 5 M). This may be an indication that in the absence of

the PSMA transporter, the more polar DUPA-conjugate 15 is internalized more slowly than 6. For the 48 h

assay, the activities of 6 and 15 against these three PSMA negative cell lines, were essentially the same

compared to the data at 16 h.

In contrast to data for the three previously discussed PSMA negative cell lines, the 16 and 48 h data

against PC-3, which is also PSMA negative, is conflicting. The 16 h data for PC-3 is in line with our

hypothesis and the observations for the other PSMA negative cell lines in that both 6 and 15 show

similarly modest activity (ca 70 - 90% cell viability at 5 M). However, the activity of 6 and 15 in the 48 h

assay seemingly disagrees with our hypothesis in that the prodrug 15 is significantly more active than the

parent drug 6. This was unexpected as PC-3 is PSMA negative, and therefore the activity of 6 and 15

142

was predicted to be similar, as was the case for the other three PSMA negative cell lines at 48 h. In

comparison, the activity of 6 in the 16 and 48 h assay was relatively unchanged.

5.4.3. Annonacin prodrugs

To evaluate the generality of the behavior of ACGs and their DUPA conjugates against LNCaP and PC-3

cells, the cytotoxicity of annonacin 2 and its prodrug 16 was evaluated (Figure 5.5). These data indicate

similar trends in activity against LNCaP and PC-3 as for 6 and 15.

Figure 5.5. Structure of annonacin, 2, and annonacin prodrug, 16

At 16 h, the prodrug 16 was more active against LNCaP than PC-3 (40 vs 60% viability at 5 M, Figure

5.6). However, it should be noted that the difference in selectivity against the two cell lines is somewhat

lower than for 6 and 15, which may be a consequence of the higher intrinsic toxicity of parent drug 2

compared to 6. Similar to the behavior of 6 and 15, at 48 h, prodrug 16 is significantly more active than 2

against PC-3. This unexpected activity of DUPA-conjugated prodrugs 15 and 16 against PC-3 suggest

that they may have a different mechanism of action against PC-3 compared to the other cell lines.

143

Figure 5.6. Cytotoxicity at 16 and 48 hours of compounds 2 and 16 in prostate cancer cells

5.5. Summary and future directions

These cytotoxicity studies have revealed that ACG mimetics in which the THF and butenolide residues

are replaced with sugar and thiophene or piperazine moieties respectively, have comparable or more

potent activities than their THF-butenolide parents. The relatively easy synthetic access to these novel

mimetics makes them attractive as leads for clinical development. In this context, studies were performed

on prodrug constructs comprising a sugar-butenolide and DUPA, a validated vector to PSMA expressing

tumors. Our preliminary assessment of prodrug selectivity albeit modest is consistent with the notion that

selectivity is connected to DUPA-PSMA recognition. In an alternative tumor targeting approach, a sugar-

thiophene analog containing the 3-O-carbamoyl residue that has been implicated in the selectivity of the

16 h 48 h

PC

-3

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

LN

CaP

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

144

bleomycin family of antitumor agents, was also synthesized. This molecule showed activities in the 0.1-10

M range against the prostate tumor cell lines LNCaP and PC-3; its tumor selectivity remains to be

investigated.

More detailed studies will be performed to probe the selectivity observed for our ACG-DUPA prodrugs.

First, to confirm that the toxicity of the prodrug is due primarily to the drug entity and not the DUPA vector,

activity of the disulfide of the vector (i.e the DUPA ligand), and prodrugs with inactive “drug” entities To

improve selectivity prodrugs with drug entities of varying potencies such as the parent THF-butenolide,

THF-thiophene and sugar-thiophene analogues will be screened. Along these lines, it will be also

interesting to evaluate sugar residues with shorter hydrocarbon chains, as this may decrease non-specific

drug uptake. To determine whether the selectivity of these conjugates is in fact due to binding to PSMA,

competitive inhibitory experiments using 2-(phosphonomethyl)pentanedioic acid (PMPA) - an inhibitor of

DUPA – can be performed.103a These studies will lay the groundwork for ACG derived prodrugs with other

established tumor vectors, such as folate and less explored entities as the 3-O-carbamoyl subunit of the

bleomycins.

The activity against a wider range of tumor cells will further inform on selectivity. The prostate cell lines

MDA-PCA-2b and RWPE-1, are of particular interest. The first is highly prevalent among African

Americans and is relevant to health disparities; the second is a normal prostate epithelial cell line and is

needed for evaluating selectivity. More detailed mechanistic studies will require fluorescently labeled

analogues to determine surface binding, and the uptake and intracellular distribution of the compounds. In

this context, the C-10 alcohol, the amine in piperazine analogues and the 6-O-alkyl chain are practical

positions for incorporation of these labels. Animal experiments using xenografts will be the prelude to

more long-term drug development.

145

5.6. Experimental

The MTT assay was used to determine metabolic activity of cells growing in monolayers by measuring

using a microplate reader (PowerWave HT Microplate Spectrophotometer) their ability to reduce the

yellow- colored MTT to a colored formazan. The data is expressed in percentage of control (i.e. optical

density of formazan from control cells).

HTC-116 colon cancer cell line, MCF-7 breast cancer, MDA-MB-231 triple negative breast cancer, PC-3,

and LNCaP prostate cancer were obtained from the American Type Culture Collection (ATCC, Manassas,

VA, USA). All batches of culture media were supplemented with 10% (v/v) of fetal bovine serum (Life

Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL

amphotericin B (Sigma). HCT116 colon cancer cells, MCF-7, and MDA-MB-231 breast cancers were

cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma), LNCaP was cultured in RPMI-1640,

and PC-3 in F-12K Medium (Kaighn's Modification of Ham's F-12 Medium). All cells were maintained at

37 oC in a 5% CO2 humidified atmosphere. Briefly, 20000 cells were plated on sterile 96-well plates (100

µL of medium per well) and left undisturbed for 48 h. Then, cells were treated with different

concentrations of compounds and incubated for 16 and 48 hours, respectively. After the incubation, cell

viability was evaluated and quantified.

The values of percentage of cell viability presented correspond to the mean of 3 independent experiments

with error bars corresponding to the standard deviation of the mean. The IC50s were estimated using

GraphPad Prism v8.3.0.

146

APPENDIX

184

C19H37IO3

lattice Monoclinic

formula C19H37IO3

formula weight 440.38

space group P21

a/Å 11.9231(4)

b/Å 4.8429(2)

c/Å 18.6632(7)

/˚ 90

/˚ 106.4017(18)

/˚ 90

V/Å3 1033.80(7)

Z 2

temperature (K) 130(2)

radiation (, Å) 0.71073

(calcd.) g cm-3 1.415

(Mo K), mm-1 1.561

max, deg. 39.510

no. of data collected 37955

no. of data 12055

no. of parameters 213

R1 [I > 2 (I)] 0.0292

wR2 [I > 2 (I)] 0.0639

R1 [all data] 0.0391

wR2 [all data] 0.0669

GOF

1.031

Rint 0.0281

Table S1.1. Crystal, intensity collection, and refinement data of compound 2.39

185

185

186

186

187

187

188

188

189

189

190

190

191

191

192

192

193

193

194

194

195

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196

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197

198

198

199

199

200

200

201

201

202

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203

203

204

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207

207

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211

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212

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214

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216

216

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217

218

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219

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220

221

221

222

222

223

223

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231

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233

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234

235

235

236

236

237

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239

239

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241

241

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249

250

250

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251

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253

254

254

255

255

256

256

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257

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258

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263

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278

279

279

280

280

281

281

282

282

283

283

284

284

285

285

286

286

287

287

288

288

COSY of compound 4.10

289

289

COSY of compound 4.10

290

290

HSQC of compound 4.10

291

291


292

292


293

293

294

294

`

295

295

296

296

297

297

298

298

299

299

300

300

301

301

302

302

303

303

304

304

305

305

306

306

307

307

.

308

308

309

309

310

310

311

311

312

312

313

313

314

314

315

315

316

316

317

317

CDCl3

318

318

CD3OD

319

319

320

320

321

321

322

322

323

323

324

324

CDCl3

325

325

CD3OD

326

326

CDCl3

327

327

CD3OD

328

328

COSY of 4.37b

329

329

HSQC of 4.37b

330

330

331

331

COSY of isolated fraction 1.2 – Annonacin prodrug

332

332

333

333

COSY of fraction 2.2 – Annonacin prodrug

334

334

335

335

COSY of fraction 1.1 – Annonacin prodrug

336

336

Table S5-I. List of compounds evaluated in Chapter 5

THF-butenolide or butenolide substitutes

Sugar analogues with butenolide Hybrid analogues with THF and

butenolide substitutes

337

16 h 48 h

MB

A-M

B-2

31

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

HT

C116

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

LN

CaP

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

338

Figure S5-I. Cytotoxicity at 16 and 48 h of THF containing compounds 1, 2, 3, 4 and 5 (Compound 3 at

16 h was evaluated in separate study)

PC

-3

0 5 10 15 20 25

0

50

100

Concentration (M)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

MC

F-7

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

339

16 h (0.1-25 µM) M

BA

-MB

-231

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

PC

-3

0 5 10 15 20 25

0

50

100

Concentration (M)

% C

ell V

iab

ilit

y

HT

C116

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

M

CF

-7

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

1

70

75

80

85

90

95

100

Data 1

6 8 9 10

Figure S5-II. Cytotoxicity at 16 h of sugar analogues with butenolide 6, 8, 9, 10

340

16 h 48 h M

BA

-MB

-231

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

HT

C116

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

LN

CaP

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

341

PC

-3

0 5 10 15 20 25

0

50

100

Concentration (M)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

MC

F-7

0 5 10 15 20 25

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

50

100

Concentration (µM)

% C

ell V

iab

ilit

y

Figure S5-III. Cytotoxicity of hybrid analogues with THF and butenolide substitutes (11, 12, 13, 14)

342

Table S5-II. Cell viability (%) over 16 h at 12.5 and 25 µM for all compounds

Compound

MBA-MD-231


12.5 25 12.5 25 12.5 25 12.5

25 12.5 25

75 60 40 30 10 10 45 40 45 35

80 70 55 45 30 30 40 30 50 50

80 75 ND ND 70 55 80 75 ND ND

75 60 60 40 20 20 40 30 35 25

30 25 30 20 10 0 30 30 45 40

95 80 65 60 55 55 ND ND 80 60

75 50 ND ND 75 55 ND ND ND ND

80 80 65 65 60 60 ND ND 60 60

343

70 65 60 40 55 50 ND ND 75 65

60 55 80 75 60 50 ND ND 65 55

80 55 50 40 75 55 50 40 40 10

55 40 65 55 75 50 40 45 70 70



344

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345

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