Synthetic Studies on Molecules Related to the Azinothricin Family and Allopumiliotoxin 339A
A Thesis Presented to the University of London in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy
Amandine Andree Huguette LEFRANC
Christopher Ingold Laboratories Department of Chemistry University College London London WC1H OAJ May 2008 UMI Number: U591613
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT
The Azinothricin family of compounds are based on a cyclodepsipeptide core and were first encountered in the late 1980s. Most of the members exhibit potent antitumour and antibiotic activities. In 1997, the Hale group synthesised A83586C through a chemoselective coupling strategy between an unprotected cyclohexadepsipeptide and a fully elaborated pyran activated ester. In this thesis, the asymmetric synthesis of two cyclodepsipeptides analogues are investigated, the L-proline analogue of GE3 cyclodepsipeptide and the (3S,5S)-
5-hydroxypiperazic acid analogue of A83586C cyclodepsipeptide. The synthesis of analogues may be of value for elucidating the mode of action of these natural products. Furthermore, it
might allow the identification of a considerably simplified structure for industrial purposes.
In a second project, a new approach to the synthesis of (+)-allopumiliotoxin 339A was
studied. The pumiliotoxin and allopumiliotoxin class of amphibian alkaloids displays significant
cardiotonic activity. Allopumiliotoxin 339A is one of the most potent compounds of the family; its activity is due to an interaction with a modulatory site on the voltage-dependent sodium channel. Our strategy to (+)-allopumiliotoxin 339A was based on the synthesis of two main fragments, an a-alkoxyaldehyde and a functionalised side chain fragment. Our initial research to the a-alkoxyaldehyde involved a Sharpless Asymmetric Aminohydroxylation reaction. However, this reaction proved not to be feasible on the trisubstituted alkene precursor. Eventually the a- alkoxyaldehyde was successfully prepared using a Trost's opening of an epoxide followed by an asymmetric induction of chiral sulfinimine to access the desired stereochemistry. The synthesis of the side chain segment was achieved via an O-directed hydrostannation strategy developed
in the Hale group. This strategy allowed the stereoselective synthesis of the trisubstituted alkene moiety of the side chain.
1 ACKNOWLEDGMENTS
First of all, I would like to thank my supervisor, Professor Karl Hale for his supervision and guidance throughout my PhD studies.
I am grateful to Novartis for providing me a fully-funded studentship.
I would like also to acknowledge my secondary supervisor, Professor Charles Marson, for his valuable advice and his help during the submission process.
I am very grateful to Dr. Abil E. Aliev for all the time and patience he accorded me for the run and interpretation of my NMR spectra and to Dr Lisa Harris for performing mass- spectroscopy analysis.
I would like to thank Dr. Soraya Manaviazar for her support in the lab and the members of the Hale group, Mathias, Sandrine, Marcus, Pascalis, Jon, Guillaume, Claire, Yi, Mernoosh and Russel for their advice and their help. I am also grateful to my colleague in the Chemistry
Department for their support and their friendship, especially Pascal, S6bastien, Sarah, Greg,
Sandra, Laure.
Many thanks to my friends here and abroad, you have patiently listened to me complain about my work, I look forward spending more time with all of you when I finish this achievement.
I wish to thank Julien for helping me get through the difficult times, and for all the emotional support he provided.
I cannot end without thanking my family, especially my parents, on whose constant encouragement and love I have relied throughout my time at UCL.
2 ABBREVIATIONS
Ac acetyl acac acetylacetonate
AIBN azobisisobutyronitrile
All allyl
Ar aryl
B' base
BAIB [bis(acetoxy)iodo]benzene
Bn benzyl
Boc tert-butoxycarbonyl
BOM benzyloxymethyl
BOP reagent benzotriazole-1 -yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate
BOPCI A/-A/-bis(2-oxo-3-oxazolidinyl)phosphinic chloride br broad r?-Bu n-butyl f-Bu f-butyl
Bz benzoyl
Cl chemical ionisation m-CPBA mete-chloroperbenzoic acid
CSA camphorsulfonic acid d doublet dd doublet of doublet ddd doublet of doublet of doublet dt doublet of triplet dba dibenzilideneacetone
DBAD di-fert-butylazodicarboxylate
DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene
DCC 1-2-dicyclohexylcarbodiimide
3 DCM dichloromethane
DDQ dichlorodicyanoquinone
DEPC diethylphosphorocyanidate
(DHQD)2PHAL 1,4-bis(9-0-dihydroquinidinyl)-phthalazine
(DHQ)2PHAL 1,4-bis(9-0-dihydroquininyl)-phthalazine
DIEA A/,A/-diisopropylethylamine (HOnig’s base)
DIBAL-H diisobutylaluminium hydride
DMAP 4-(dimethylamino)pyridine
DMF dimethylformamide
DMP Dess-Martin periodinane
DMPU 1,3-dimethyl-3-4-5-6-tetrahydro-2(1 H)-pyrimidinone
DMSO dimethylsulfoxide
DNP dinitrophenyl dppf 1,1 '-bis(diphenylphosphino)ferrocene dr diastereoisomeric ratio
E+ electrophile ee enantiomeric excess
ESI electrospray ionisation eq equivalent
Et ethyl
FAB fast atomic bombardment
Fmoc fluorenylmethyloxycarbonyl h hour
HATU A/-[(dimethylamino)-1/-/-1l2,3-triazolo[4l5,b]pyridin-1-ylmethylene]-A/-
methylmethanaminium hexafluorophosphate
HOBt 1-hydroxybenzotriazole
HMBC heteronuclear multiple bond connectivity
HMPA hexamethylphosphoramide
HMQC heteronuclear multiple quantum coherence
HRMS high resolution mass spectroscopy
4 Hz Hertz
IR infra red
J coupling constant
KHMDS potassium hexamethyldisilazide
L ligand
LDA lithium diisopropylamide
LiDBB lithium di-ferf-butylbiphenyl m meta
M molar m/z mass to charge ratio m-CPBA mefa-chloroperbenzoic acid
Me methyl min minute mM milimolar
MOM methoxymethyl
Ms methylsulfonyl
MS molecular sieves
MTPA methoxy(trifluoromethyl)phenylacetyl)
NBS A/-bromosuccinimide
NEM A/-ethylmorpholine
NMO A/-methylmorpholine-A/-oxide
NMR nuclear magnetic resonance
Nu' nucleophile o ortho ox. oxidation p para
PCC pyridinium chlorochromate
PG protecting group
Ph phenyl
Piz piperazic
5 PMB para-methoxybenzyl
PPTS pyridinium para-toluenesulfonate i-Pr isopropyl
PTX pumiliotoxin pyr pyridine
R alkyl
RedAI-H sodium bis(2-methoxyethoxy)aluminum hydride rt room temperature s singlet
SAA Sharpless Asymmetric Aminohydroxylation
SEM [2-(trimethylsilyl)ethoxy]methyl
SM starting material t triplet
TBAF tetra r?-butylammonium fluoride
TBDMS ferf-butyldimethylsilyl
TBDPS fe/t-butyldiphenylsilyl
TEMPO 2,2,6,6-tetramethylpiperidine-1 -oxyl
Tf trifluoromethanesulfonyl
TFA trifluoroacetic acid
TFAA trifluoroacetamide
THF tetrahydrofuran
TIPS triisopropylsilyl
TLC thin layer chromatography
TMS trimethylsilyl
TPAP tetra-n-propylammonium perruthenate
Troc trichloroethoxycarbonyl
Ts p-toluenesulfonyl
Z benzyloxycarbonyl
6 TABLE OF CONTENTS
PART A: SYNTHETIC STUDIES ON MOLECULES RELATED TO THE AZINOTHRICIN FAMILY...... 9
1. T he A zinothricin fam ily of antibiotics ...... 9 1.1. Biological introduction...... 9 1.2. Isolation and Biological Activity of Some Member of the Family ...... 10
2. P r evio us w o r k on the A zinothricin Fa m il y ...... 16 2.1. Past Syntheses of Some Related Natural Products ...... 17
2.1.1. Total Synthesis of L-152,602 ...... 17 2.1.2. Total Synthesis of A83586C ...... 23 2.1.3. Synthesis of Verucopeptin Cyclodepsipeptide Core ...... 34
2.1.4. Synthesis of GE3 Cyclodepsipeptide Core ...... 37 2.2. Previous Syntheses of Analogues of the Azinothricin Family of Antibiotics ...... 39
3. Synth etic Stu d ies T o w ards A nalogues of the A zinothricin Fa m il y ...... 43 3.1. Synthesis of an L-proline analogue of GE3 cyclodepsipeptide ...... 46 3.2. Toward the synthesis of an (3S, 5S)-5-hydroxypiperazic acid modified mimetic of A83586C...... 53 4. Conclusion...... 57
PART B: SYNTHETIC STUDIES TOWARDS THE SYNTHESIS OF (+)- ALLOPUMILIOTOXIN 339A...... 59
5. Introduction ...... 59 5.1. Isolation of the Pumiliotoxin A and the Allopumiliotoxin alkaloids...... 59 5.2. Biological Properties of the Pumiliotoxins ...... 60
5.2.1. Ion Channels and the Electrical Properties of Membranes ...... 61
5.2.2. Phosphoinositide Breakdown ...... 62 5.2.3. Biological Activity of Pumiliotoxin Family of Alkaloids ...... 64 5.2.4. Structure-Activity Relationship ...... 64 5.3. Past syntheses of Some Members of the Pumiliotoxin Family of Alkaloids ...... 66
5.3.1. First Synthesis of a Pumiliotoxin A Alkaloid ...... 66 5.3.2. Overman Aldol Attachment of the Alkylidene Side Chain: First Entry to an Allopumiliotoxin
A lkaloid ...... 69 5.3.3. Total Syntheses Using Iodide-Promoted Iminium lon-Alkyne Cyclizations ...... 72 5.3.3.1. Total synthesis of Pumiliotoxin B ...... 72 5.3.3.2. First Enantioselective Total Synthesis of (+)-Allopumiliotoxin 339A ...... 74 5.3.4. Alternative Strategies for (+)-Allopumioliotoxin 339A Synthesis ...... 76
5.3.4.1. Nozaki-Kishi cyclisation ...... 76 5.3.4.2. Nickel Catalysed Synthesis ...... 79
7 5.4. Others Approaches to Pumiliotoxins and Allopumiliotoxins Indolizines Core ...... 81
5.4.1. Trost’s Total Synthesis of Allopumiliotoxin 339B ...... 81 5.4.2. Holmes’ Total Synthesis of Allopumiliotoxin 323B’ ...... 83 5.4.3. Comins’ Total Synthesis of Allopumilotoxin 267A ...... 85
5.4.4. Lin's Approach to the Indolizidine Core of Pumiliotoxins and Allopumiliotoxins ...... 87 5.4.5. Stevenson’s Rapid Synthesis of the Indolizidine Core of Pumiliotoxins and Allopumiliotoxins
...... 89
6. Synth etic effo rts to w a r d s (+)-A llopumiliotoxin 3 39 A ...... 90 6.1. Retrosynthetic Analysis of (+)-Allopumiliotoxin 339A ...... 90 6.2. Synthetic Studies Towards a-Alkoxyaldehyde 339 ...... 92
6.2.1. First Generation Strategy for a-Alkoxyaldehyde 339 ...... 92 6.2.1.1. Retrosynthetic analysis ...... 92 6.2.1.2. Attempted Implementation of the Sharpless Asymmetric Aminohydroxylation Strategy for a-Alkoxyaldehyde 339...... 92 6.2.2. New route for the synthesis of the a-alkoxyaldehyde ...... 103 6.3. Synthetic Studies Towards The Side Chain Segment ...... 110
6.3.1. The O-Directed Free-Radical Hydrostannation of Propargyl Ethers, Acetals and Alcohols
110
6.3.2. Retrosynthetic plan ...... 112 6.3.2. Implementation of the Retrosynthetic Strategy for the Side Chain Segment of (+)-Allopumiliotoxin 339A ...... 113
7. Co n c lu s io n and future w o r k ...... 118
PART C: EXPERIMENTAL...... 121
8. Sy n th etic stu d ies on M olecules R elated to the A zinothricin Fa m il y ... 122
9. T o w a r d the Sy n th esis of (+)-A llopumiliotoxin 3 3 9 A ...... 148
REFERENCES...... 184
APPENDIX...... 188
8 PART A: SYNTHETIC STUDIES ON MOLECULES RELATED TO
THE AZINOTHRICIN FAMILY
In recent years, interest in phamacologically active natural products has occupied a central position in organic chemistry. Cyclodepsipeptides are cyclic peptides possessing at least one ester linkage and their chemical syntheses represent a considerable challenge. Indeed, their unusual architecture stimulated many synthetic chemists leading them to develop numerous new synthetic methodologies. These molecules also exhibit potent biological activities. Thus, the chemical synthesis of cyclodepsipeptides can provide leads for the development of novel pharmaceutical agents.
1. The Azinothricin family of antibiotics
1.1. Biological introduction
Figure 1. The cell cycle
To understand the basis of cancer biology, it is necessary to understand the mechanisms that control cell growth and cell cycle. In fact, most eukaryotic cells reproduce
9 through a sequence of four phases: 2 gap phases (G , and G2) where RNA and protein are synthesised, one synthesis phase (S) where DNA synthesis or replication occurs, and mitosis
(M), in which the cell's chromosomes are divided between two daughter cells. The cell can also temporarily or reversibly exit cell cycle and enter a state of quiescence called G0 phase. A molecular surveillance system monitors the progress of cell cycle through various check points of which the two most important are between G-i and S and between G2 and M.
Proto-oncogenes are genes which promote cell growth and mitosis, and tumor suppressor genes discourage cell growth, or temporarily halt cell division in order to carry out DNA repair.
Typically, a series of several mutations to these genes are required before a normal cell transforms into a cancer cell. e 2F transcription factors1"4 regulate the expression of genes that control cell proliferation during the Gi/S transition. The mechanisms of action of E2Fs are very complex, and involve a network of interactions with various molecules. A pathway in which E2F is regulated by its interaction with the retinoblastoma protein (pRB), a tumor suppressor protein, had been identified.5 It is known that virtually all human cancers exhibit alterations in this pRb/E2F pathway.6 In this regard, targeting E2Fs could be a promising approach to treat cancer and molecules of the Azinothricin family that are known to inhibit, directly or indirectly, E2F transcription factors might serve as antiproliferative drugs.
1.2. Isolation and Biological Activity of Some Member of the Family
In 1986, Maehr and co-workers at Hoffman La-Roche isolated the first member of a growing family of antitumour antibiotics from Streptomyces sp. X14950. 7 They named the new molecule azinothricin 1 and found it to be one of the most potent antibiotics ever discovered in the Roche natural product screening assay. Its MIC values1 ranged from 0.008 to 0.063 pg/mL against a variety of Gram-positive strains of bacteria. The structure of azinothricin 1 is presented in Figure 2 along with other members of the family. It was determined by X-ray crystallography and chemical degradation.
f Minimum Inhibitory Concentration: the lowest concentration of drugs that prevents visible bacterial growth
10 Two years later, the second member of this family was discovered by Smitka et a !8 It was named A83586C 2 and was isolated from fermentation broths of the Guam soil microorganism Streptomyces Karnatakensis. A83586C was highly toxic to mice at doses of 9.3 mg/kg and so its clinical development was never considered viable. However, it exhibited pronounced antitumour properties in vitro against a large number of cancer cells; it was particularly efficient at inhibiting growth of a CCRF-CEM human T-cell leukaemia line, its IC50* value being 0.0135 pg/ml. Recent work has shown that A83586C 2 exhibits activity against various other mouse and human tumour cell line HCT-116 human colon cancer cells (IC50 = 40
± 10 nm), HT-29 cancer cells (IC50 = 60 ± 30 nm), MDA-MB-435 cancer cells (IC50 = 90 ± 10 nm), MCF-7 human breast cancer cells (IC50 = 90 ± 30 nm), A549 human lung cancer cells
(IC50 = 30 ± 10 nm), PC-3M human prostate cancer cells (IC50 =160 ± 30 nm) and U20S human ulterine cancer cells (IC50 = 80 ± 10 nm). A83586C 2 also possesses strong antibiotic properties against Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermis,
Streptococcus pyogenes, and Streptococcus pneumoniae.
Other isolated molecules of this class include citropeptin9 3 in 1990, GE310,11 4 in 1997, and Kettapeptin12 5 in 2006 (Figure 2).
* Inhibitory Concentration: Concentration of a drug required to observe 50% inhibition of tumour growth
11 Me Me
HO Me Me HO HO Me Me Me Me Me NH NH Me NH NH Me Me Me Me Me MeO ^ -Me u N o Me Me HN M . h n - N . . . . 4 Me HN
Azinothricin 1 A83586C 2
Me Me HO HO Me HO Me HO Me Me Me Me JMH NH ,n h n h Me Me Me Me. Me MeO
Me Me Me HN Me HN
HN HN Me Me
Citropeptin 3 GE3 4
Me
HO Me HO NH NH Me Me MeO' .M e Me HN Me HN
Kettapeptin 5
Figure 2. Structure of the A83586C/Azinothricin Family of Antibiotics
Citropeptin 3 shows in vitro and in vivo activity against P388 leukaemia cells. Its IC50 value is 0.02 pg/mL and it provides 120% life extension when given to mice with P388 lymphocytic leukaemia at the non-toxic dose of 2 mg/kg/day.
The producing strain GE3 4 was isolated from a soil sample collected in Shimane prefecture, Japan. GE3 4 possesses antibacterial and antitumour activities against human pancreatic carcinoma, PSN-1 both in vitro and in vivo. It exhibits high in vitro cytoxicity against human tumour cell lines HeLa S3, A431 and Saos-2 with IC50 value of 6nM, 16 nM, and 3.6 nM
12 respectively. The in vivo activity was examined in the human xenograft mouse tumour model: a
47% reduction in tumour size was observed when GE3 4 was given to mice at the non-toxic dose of 2 mg/kg/day. Contrary to A83586C 2, it showed only a weak activity against Gram- positive strains of bacteria. It is noteworthy that only two structural diffferences between GE3 4 and A83586C 2 are responsible for divergent profiles against bacteria.
More recently, Kettapeptin 5 was isolated by Maskey and co-workers from the ethyl acetate extract of the Streptomyces sp. Isolate GW99/1572.12 It exhibits antibacterial activity against Bacillus subtilis with a MIC value of 3.75 pg/mL, and also against Streptomyces viridochromogenes, Staphylococcus aureus and Escherichia coli. Furthermore, Kettapeptin 5 shows anticancer activity against human cell lines LXFA 629L, LXFL 529L, MAXF 401NL,
MEXF 462NL, RXF944L and UXF 1138L with IC50 values of < 0.6 jg/mL.
Other cyclodepsipeptides that are considered to belong to the Azinothricin family include the molecules shown in Figure 3: Verucopeptin 6,13,14 Variapeptin 7,9 L-156,602 815 and
Polyoxypeptins A, 9 and B 10,16IC101 11,17 and the recently discovered Pipalamycin 12.18
13 Me Me Me Me HO Me Me HO HO Me HO NH NH Me NH NH Me Me Me. Me NH OH O HN
Verucopeptin 6 Variapeptin 7
Me Me Me Me Me HO Me HO Me HO HO Me NH NH NH NH Me Me Me Me. Me Me.
OH OH Me Me HN
HN Me
9 Polyoxypeptin A : R = OH L-156,602 8 10 Polyoxypeptin B : R = H
Me Me
Me Me Me Me HO Me HO 1 Me HO HO NH NH NH NH Me Me
Me. Me Me. Me
OH Me HN Me
HN HN
IC101 11 Pipalamycin 12
Figure
Verucopeptin 6 was isolated in 1993 from the soil microorganism Actinomadura verrucospora, and exhibits specific in vitro toxicity against mouse B16-F10 cells with IC50 value of 0.004 pg/mL. Verucopeptin presents a weaker activity against P388 leukaemia and HCT-116
Human Colon Cancer with IC50 value of 0.08 pg/mL and 0.04 pg/mL respectively. In vivo activity was tested in the experimental mouse tumor system; Verucopeptin 6 significantly prolongs the
14 life expectancy of mice with B16 melanoma, conferring a 162% life extension when given at the dosage of 2 mg/kg/day.
Variapeptin 7, was isolated from Streptomyces variabilis, that was taken from a soil sample collected in Bosque, Brazil. It showed a potent activity in vitro against Gram-positive strains of bacteria and demonstrated potent cytotoxicity against P388 leukaemia cells (IC50 =
0.01 pg/mL).
L-156,602 8, isolated from cultures of Streptomyces spp. MA6348, was found to be a
C5a antagonist. Such molecules might function as anti-inflammatory agents or be useful for treating allergic disease states. Its total synthesis was achieved in 1990 by Durette et a/.19
Polyoxypeptin A, 9 and polyoxypeptin B 10 were isolated in 1999 by Umezawa and coworkers16 from the culture broth of Streptomyces MK498-98F14 and are known to exhibit potent apoptosis against human pancreatic adenocarcinoma AsPC-1 cells, an apoptosis- resistant cell line. Polyoxypeptins A, 9 and B, 10 decreased the viability in AsPC-1 cells with
ED50 values of 0.08 and 0.17 pg/mL.
IC101 1117,20 and Pipalamycin 1218were isolated from the same strain; the producing strain, MJ202-73F3, was extracted with EtOAc, concentrated to dryness and purified by chromatography with CHCIVMeOH (50:1). IC101 11 was eluted first as the major compound followed by Pipalamycin. IC101 inhibited MLCR (Mixed Lymphocyte Culture Response) with an
IC50 value of 0.009 pg/mL and showed its strongest activity against P388D! cells (IC50 = 0.006 pg/mL). Pipalamycin 12 induced apoptosis in human pancreatic adenocarcinoma AsPC-1 cells at 0.3 pg/mL in 24-48 hours.
Almost all molecules of the cyclic hexadepsipeptide class have been found to show potent cytotoxicity. However mechanisms for cytotoxicity have been little described. The synthesis of analogues following by the comparison of their biological activities should give a
15 path to determine these mechanisms. Additionally, it might prove possible to find analogues that will possess interesting biological properties.
In this first part of my PhD, I worked on the synthesis of two modified cyclodepsipeptide analogues shown in Figure 4: the L-proline modified mimetic of GE3 cyclodepsipeptide 13 and the (3S, 5S)-5-hydroxypiperazic acid modified mimetic of A83586C cyclodepsipeptide 14.
,NHNH3+CI
Me . N - / Y : Me ( O O I o ° / ° .M e ° ; .Me 0=:f ON HN : M« oh A OH hn y Me S V O
OH 13 14 L-proline modified mimetic (3S, 5S)-5-hydroxypiperazic acid of GE3 cycbdepsipeptide modified mimetic of A83586C cyclodepsipeptide
Figure 4. Two cyclodepsipeptide analogues
2. Previous work on the Azinothricin Family
Prior to our group’s research, only one group has achieved a total synthesis of a molecule of the Azinothricin family. In this part, the synthesis of L-152,602 by Durette and coworkers will be reviewed along with the work published by our group on the Azinothricin family. This work includes the total synthesis of A83586C and the synthesis of the verucopeptin and GE3 cyclodepsipeptides. Additionally, two cyclodepsipeptides analogues were also prepared.
16 2.1. Past Syntheses of Some Related Natural Products
2.1.1. Total Synthesis of L-152,602
Durette and coworkers achieved the first total synthesis of L-152,602 8 in 199019. Their original plan was to complete the synthesis with a coupling between cyclodepsipeptide 15 and the HOBt activated ester 16 (Scheme 1). Activated ester 16 would be accessed via a Seebach enantioselective Claisen condensation reaction.21 Cyclodepsipeptide 15 would originate from the linear hexapeptide 19. The linkage selected to achieve the ring closure was the bond between the glycine and /V-benzyloxy-L-alanine residues. The assembly strategy for hexapeptide 19 employed a [2 + 2 + 2] fragment condensation of dipeptides 20, 21 and 22.
17 18 NZ NHTroc
AllocHN
AllocHN^Y^
22
Scheme 1. Retrosynthetic plan to L-156,602 8
17 The synthesis of ester 16 started with a diastereoselective Frater-Seebach alkylation reaction of the enolate derived from methyl (ft)-3-hydroxybutanoate 23 with (S)-1-iodo-2- methylbutane 24 to afford alcohol 25 in 46% yield. Subsequent protection of the hydroxyl group and reduction of the methyl ester gave alcohol 26 in 52% yield. After Swern oxidation, the product aldehyde 27 was condensed with carboxymethylphosphorane to give (E)-olefin 28 in
70% yield. Hydrogenation over Pd/C and O-desilylation led to concomitant ring closure affording lactone 18. After a diastereoselective Claisen condensation between lactone 18 and the lithium enolate derived from dioxolane 17, compound 29 was obtained as a single isomer in 75% yield.
Compound 29 was then converted into the methyl pyranoside by treatment with methanolic HCI in 89% yield. Transesterification with excess sodium methoxide afforded methyl ester 30 which was converted into a potassium salt using potassium hydroxide. Finally, the N- hydroxybenzotriazole active ester 16 was prepared by reaction with the BOP reagent and NMO in DMF.
/Pr2NLit THF, -50 X ; cool to -7 8 X then add 24 (1) Et3SiOTf, CH2CI2. warm to rt (46%) M e 0 2C ^ ,o " 2,6-lutidine, -4 0 X to rt MeO 2 C Me HO Me T Me (2) DIBALH, PhMe Me HO Me -7 0 X to -40°C HO X Me Me Et3SiO Me (52%, 2 steps) 23 Me 25 26 24 Me2SO, (COCI)2 CH2CI2, -70°C, Et3N -20 X (98%) M e02C~ (1) H2, Pd/C OHC. Me EtOAc (98%) 'Me Ph3P=CHC02Me *'Y ' ' M e Me OXX O Me Me THF, A,70% Et3SiO MeM® (2) n-Bu4NF, THF Et3SiO Me 18 28 27
n-A'Me 17, i-Pr2NLi, THF, -70°C f-Bu»»< I add 18 warm to -2 5 X (75%) 17 ° A C
(1) HCI, MeOH HOxJ (1) aq. KOH, EtOH O'C to rt (89%) HQ M
(2) NaOMe L O (2) BtOP+(NMe 2 )3PF6- MeOH (78%) M eO ^O NMO, 4Asieves, DMFjj^^J'’’ 30 16
Scheme 2. Synthetic route to activated ester 16
18 The synthesis of fragments 21 and 22 required the preparation of Z-protected piperazic acids 34 (3R) and 39 (3S). It was achieved via a Diels-Alder cycloaddition between 2,4- pentadienoate 31 and di-te/t-butylazodicarboxylate 32 in hot CCI4 as shown in Scheme 3. After hydrogenation of the double bond and saponification, racemic acid 33 was obtained in 93% yield over 2 steps. The Boc groups were then cleaved with TFA and subsequent Z group protection of the N( 1) atom afforded racemic A/(1)-Z-piperazic acid that was resolved with (+) and (-) ephedrine. (3ft)-Z-piperazic acid 34 was obtained in 18% yield and (3S)-Z-piperazic acid
39 in 15% yield. To complete the synthesis of dipeptide 21, (3R)-Z-piperazic acid 34 was protected with an Fmoc group and converted to acid chloride 36 with oxalyl chloride. A Carpino biphasic coupling with protected hydroxamic acid ester 37 yielded compound 38. Fmoc- deprotection furnished the desired dipeptide 21 in 62% yield over 3 steps.
(1) CCI4, reflux (1 )T F A , CH2Cl2, (10%) 36 h (65%) (2) ZCI, aq.NaOH, (70%) HO n -NB oc HO .NZ ',NBoc (3) Optical Resolution M e 0 2c BocN (2) H2. Pd/C, MeOH Boc (3) aq.KOH, MeOH with (-) and (+) 31 32 (93%, 2 steps) 33 ephedrines, (18%) 34
Me3SiCI, /-Pr2NEt 0°C, CH2CI2 FmocCI (92%)
(j)Bn 10% aq.NaHC03 (COCI)2 ? cat DMF Me,„^N 'NZ Et2NH, MeCN Me<„ ..N ^ NZ CH2C|2 Cl HO n 'nz Scheme 3. Synthesis of dipeptide 21 (3S)-Z-piperazic acid 39 was protected as a fert-butyl ester and the latter condensed with acid chloride 40 using the Carpino two phase aq. NaHCO^acid chloride coupling conditions (Scheme 4). Finally acid 22 was obtained in 98 % yield after deprotection with TFA. 19 H 9 (1) Isobutone, cat.H25 0 4 AllocHN^~'''f^° O A llo cH N ^ ' f^ 0 Q m H 1,4-dioxane ,, II CF3CC> 2 H I n ZN'NT •'OH ------ZN OBu-f > ZN'N y \ > H k k (21 a 1IocHn " ' Y ° <98%> l ^ J 39 40 Cl 41 10% aq.NaHC03, 22 CH2CI2 (97%) Scheme 4. Synthesis of fragment 22 To achieve the synthesis of fragment 20, Caldwell and Bondy developed a synthesis of (2S, 3S)-3-hydroxyleucine 46 (Scheme 5).22 It began with a Sharpless asymmetric epoxidation on allylic alcohol 42 to give epoxide 43. After oxidation of the hydroxyl group, the resulting acid 44 was treated with benzylamine and sodium hydroxide. Regioselective opening of the epoxide occurred in 66% yield to give compound 45. Finally hydrogenolysis of the A/-benzyl group afforded (2S, 3S)-3-hydroxyleucine 46 in 89% yield. f-BuOOH, Ti(OPr-/)4 H5I0 6, R u CI3.3H20 Diethyl L-tartrate c h 3c n . c c i 4, h2q Me CH2CI2, isoctane -5°C to 0°C (78%) Me 42 -20*C (82%) 44 BnNH2, NaOH H20, reflux (66%) H2, 20% Pd(OH)2/C MeOH, AcOH,25°C HO Me O OH (89%) OH 46 45 Scheme 5. Caldwell and Bondy’s route to (2S, 3S)-3-hydroxyleucine 46 The synthesis of fragment 20 was achieved as shown in Scheme 6. (2S, 3S)-3- hydroxyleucine 46 was protected in 2 steps to afford compound 47 in 68% yield. The latter was condensed with the A/-hydroxybenzyl-(R)-Ala residue 49 in presence of CDI to afford dipeptide 20 in 67% yield. 20 NH2 Me (1) TrocCI, Troc. ■ 1 aq. N aH C 03, CH2CI2 | V® HO M® '-BuO^ ^ Troc'NH O OH (2) APrN=C(OBu )NPr-i, q A h V e 46 (63%, 2 steps) 47 CDI, CH2CI2 U y'-J (i) Tf20, CH2CI2, (67%) . O’ C, then BnONH2 „ _u 0=^r~~\Me V 2,6-lutidine ° Y ° H \ OBn .>"k . _ BnOx . Y HO Me (ii) aq.NaOH, THF N Me 4 g (58%, 3 steps) H 20 49 Scheme 6. Synthesis of fragment 20 After conversion of acid 22 to chloride 50, the peptide linkage between dipeptide acid chloride 50 and depsipeptide 20 was formed by AgCN-assisted amidation (Scheme 7). The resulting tetrapeptide 51 was then deprotected with TFA and converted into acid chloride 52 using oxalyl chloride. Finally, linear hexapeptide 19 was obtained after coupling with dipeptide 21 using 10% aq. NaHC03 in 69% over 3 steps. T roc-* NH Me f-BuO^/*\^k Troc'K,u Me NH Me ° ' ° NBU° ------Me -20 0=3C AIIoc H N ' Y ^ ° OH AII oc H N ^ Y 0 Cl HN M® Q O=Y° (C0CI^ ZN'NV‘\> OB^_ k Y cat DMF k Y A9C9Nd.chMe ZN'NV"^ (77%, 2 steps) k Y ° 22 60 S1 (1) c f 3c o 2h (2) (COCI)2 Troc^ ^NHTroc^e NH Me ?Bn>—N (j)Bn Cl Me Me Me,, .N .NZ M e ,,,/* 1 O O o ^ o Y r J \ 0 ” o= r 0 OAII o 0B nY 0 OAII 21 N IMe / - Y N Me AllocHN AllocHN 10% aq.NaHC03 CH2CI2 “ O ' 4 ' ■ U ° (69%, 3 steps) 19 52 Scheme 7. Synthetic route to linear hexapeptide 19 21 Removal of both the Alloc protecting group and the ally) ester from hexapeptide 19 was achieved in a single step by palladium-catalysed hydrostannolysis (Scheme 8). Cyclisation of the crude resulting hexapeptide was then performed by the mixed phosphonic anhydride method to give cyclodepsipeptide 53. The Troc group was then cleaved and the resulting free amine 15 coupled with the HOBt activated ester 16. Unfortunately, the amidation did not succeed, and Cadwell and co-workers isolated the cyclic peptide alcohol 54 resulting from 0,N- acyl shift. \ /^Z NHTroc -NZNH Me ^znh 2 Me (j)Bn \ — N ^ ^ JL Me Y Bny — n OBn V- n M e -,,/ N—^ n : Me Me-,, / Me M e - „ /N _ ^ jl : Me ° £ > (1) Bu3SnH, H20 I O O o X ° V CH2CI2, (Ph3P)2PdCI2 Zn ^ 0 NH o ° ? nJ v (2) [n-PrP(0)01j 0 Me AcOH N Me AlocHN ,N ^ CH2CI2, DMAP 43% (2 steps) O HN V X ) ‘ O ° 15 19 53 Me Me HO. Me 6iJPe Me DMF Me Me NH o OBn Me HN 54 Scheme 8. Formation of cyclodepsipeptide 15 and attempted coupling with activated ester 16 The authors revised their retrosynthetic plan and decided to attach the side chain at the linear hexapeptide stage (Scheme 9). The Troc protecting group was cleaved from hexapeptide 19 and the resuting crude amine was reacted with HOBt ester 16 to afford the desired hexapeptide 55 in 56% yield. Deprotection of the Alloc and allyl protective groups by palladium catalysed hydrostannolysis was accompanied by conversion of the methyl pyranoside to the hemiketal. Ring closure was achieved by means of the mixed phosphonic anhydride method to 22 afford cyclic hexapeptide 56 in 56% yield from linear hexapeptide 55. Finally, hydrogenolysis of the Z and Bn protective groups gave L-156,602 8 in 53% yield. Me Me HO. NZ NHTroc Me .Me 6n£ Me Me Me (1) Zn, AcOH NZ NH Me (2) DMF, 16 Me Me. Me : Me Me Me Y d tP e Me OAII Me AllocHN ZN (56%, 2 steps) (1) Bu3SnH, H20 CH2CI2, (Ph3P)4PdCI2 (2) [n-PrP(0)0]3 CH2CI2, DMAP (57%, 2 steps) Me Me Me Me HO Me Me Me HO Me HO HO NH NH ^IZ NH Me (2) H2, Pd/C, MeOH Me Me Me. Me Me (53%) N\Y hk'nA L-156,602 8 56 Scheme 9. Completion of the synthesis of L-156,602 8 2.1.2. Total Synthesis of A83586C A first retrosynthetic plan of A83586C 2 was elaborated; the C(1)-C(47) 57 sequence was synthesised (Scheme 10).23 Unfortunately difficulties occurred in the macrolactamisation and new ways of synthesis were considered. In 1997, our group achieved the first total synthesis of A83586C through an endgame that exploited a highly chemoselective coupling between the fully elaborated cyclodepsipeptide 59 and the activated pyran ester 60.24 The last two steps were the union of an activated ester 60 with a pre-assembled, unprotected cyclodepsipeptide 59 followed by a chemoselective hydration of the resulting glycal 58 under 23 very mild acidic conditions with wet deuterochloroform. A [2+2+2] condensation strategy was used to prepare cyclodepsipeptide 59 from peptides 64, 65 and 66. The synthesis required a total of 95 individual steps, but because it was highly convergent, the longest linear sequence was only of 28 steps. HO-** Me Me Me NH O Mo Mo Mo / —N NH NH 0 9 H / —N Me/,. / ° u O Me.,, / .M e ° ^ \ .M e u M. h^ X A 0 °H Me' OH A83586C 2 C(1)-C(47) sequence 57 Me Me Me NH NH ° 9 / N Me/,, o NH NH3*CI m /hn'n> A q ° H QPMB f-BuPh2SiO 59 V Mex PMBO^CHO Ph02S (J)SiPh2f-Bu "Me MeO X 0 NFmoc Me Me Me. 62 NHTroc Me 64 Me Mes Fmoc OH •O Me' Me BocHN 2N OBn 65 Scheme 10. Retrosynthetic analysis of A83586C 24 Synthetic planning for the pyran sector was centred on the preparation of intermediates 61, 62 and 63. Sulfone 63 was obtained from compound 67 as shown in Scheme 11. An Evans asymmetric aldol reaction between oxazolidinone 67 and aldehyde 68 afforded compound 69.25 The chiral auxiliary was cleaved from compound 69 with sodium methoxide. The resulting methyl ester was reduced with DIBAL-H to afford diol 70. Selective thioesterification of the primary alcohol with tributylphosphine and phenyldisulfide provided 71 and after O-silylation and Trost-Curran oxidation with oxone, compound 63 was obtained in a 50% overall yield. OHC )H OH Me (1) NaOMe. MeOH,CH2CI2 1 68 jt Me Me -15°C, 10 min (85%) ° \__/N Me Me n-Bu2BOTf, Et3N Me Me (2) DIBAL-H. CH2CI2 Me Me Ph Me CH2CI2. -5'C to-10°C Ph Me -78°C to -15°C (88%) 70 67 cool to -78*C, add 66 69 warm to rt (85%) (PhS)2, Bu 3P DMF, rt (82%) (1) f-BuPh2SiCI, DMF PhO^ 'SiPh2Bu-t imid, 85°C (96%) PhS OH Me (2) oxone, THF. MeOH W"- Me Me H20 , rt (99%) Me Me 63 71 Scheme 11. Synthesis of sulfone 63 Two key reactions were used to install the anti-relationship of the two adjacent stereocentres in compound 61 (Scheme 12). First, a Sharpless asymmetric epoxidation was used to prepare the chiral 2,3-epoxy alcohol 74. Then, a chelation-controlled epoxide ring- opening reaction with trimethylaluminium was performed. It proceeded with 20:1 selectivity in favour of the C(3)-ring opened product. Protection of the diol as p-methoxybenzylidene, cleavage of the obtained acetal and Swern oxidation of the resulting primary alcohol gave aldehyde 61. 25 (1) f-BuPhjSiCI, (1) RedAI-H, Et20 DMF, imidazole, rt (76%) 0*0 (73%) f-BuPh^i f-BuPh2 Si( (2) n-BuLi, THF, (2) f-Bu02 H, Ti(0-i-Pr) 4 -30'C, (HCHO)n, CH 2 CI2, 4A MS, -20°C 74 warm to rt (73%) 72 73 (-)-DET (84%, 93% ee) (1) M6 3 AI, Hex, 0°C (75%) (2) p-MeOC 6 H4 CH(OMe )2 DMF, p-TsOH, 55°C (84%) (1) DIBAL-H, CH2 CI2 f-BuPh2SiO ,C6 H 40 Me-p OPMB -78*C to rt (78%) f-BuPh2SiO (2) Me 2 SO, (COCI) 2 CH 2 CI2, -78°C, Et3N warm to rt (83%) Scheme 12. Synthesis of aldehyde 61 Unification of sulfone 63 and aldehyde 61 was successfully performed with n-BuLi (Scheme 13). The resulting p-hydroxysulfone 76 was oxidised with trifluoroacetic anhydride and DMSO. The resulting p-ketosulfone was reduced with AIBN / tri-n-butylstannane to give ketone 77. Alkene 78 was formed from compound 77 via a Grignard addition followed by POCI3- pyridine mediated dehydration. Compound 78 was obtained as the major isomer in a 2.6:1 mixture with the 1,1-disubstituted alkene. Cleavage of the primary silyl group and sulfonation of the obtained alcohol provided compound 80. P h$02 9SiPh2Bu-f n-BuLi, THF,-78*C PMBO PhS0 2 9SiPh2 f-Bu then add 61 f-BuPh^iO- ‘ Me "Me Me Me (64-75%) Me OH Me Me 76 (1 )(C F 3C 0 2 )0, Me 2 SO, CH2 CI2, -78°C, Et3N warm to rt (92%) (2)B u 3 SnH, AIBN, PhMe reflux (82%) f-BuPh2SiOv ..M e (1) MeMgBr, THF f-BuPh2SiOv^/\..Me OSiPh2f“Bu -78 °C to rt (98%) SiPh2/-Bu Me PMBO (2) POCI3, Py PMBO Me Me Me 55°C (97%) O Me Me 78 77 HF-pyridine THF (8 6 %) Ph02S ^ ^ \ . >Me SiPh2 f-Bu (1) Bu3 P, DMF, (PhS)2 (94%) SiPhjf-Bu PMBO Me (2) oxone, THF, MeOH PMBO Me H20 (96%) Me Me Me Me Me Me 80 Scheme 13. Synthesis of compound 80 26 The synthesis of fragment 62 is shown in Scheme 14. The stereochemistry of the chiral diol 82 was obtained from alkene 81 by a Sharpless catalytic asymmetric dihydroxylation reaction. A strategy involving formation of a p-methoxybenzylidene followed by acetal reduction was used to position a PMB group preferentially on the more hindered hydroxyl. Alcohols 84 and 85 were readily separated by flash chromatography. Alcohol 84 was oxidised and esterified to deliver the methyl ester 86. The last steps involved deprotection of the silyl group and oxidation of the resulting alcohol under Swern conditions. le OSiPh2Bu-f AD-Mix-JJ Me SiPh2Bu-f p-MeOC6H4CH(OMe)2 SiPh2Bu-f /-B u OH-H20 catPPTS. DMF HO P M P ~ ( 0*C (70-99% 55*C (98%) 92% ee) HO 81 82 /-B u 2AIH, CH2CI2. -78°C, 2 h then rt 5 min (63-69%) regbselectivity 84:85 = 2:1 PMBo4^CHO (1) TBAF, THF (74%) \Q SiPh2Bu t (1)P D C ,D M F y e Me 0 SiPh2Bu-f M e O ^ n (2) Me2SO, (COCI)2 MeQ- \ (2) CH2N2. E^O PMB0 J + H 0 Me0 O CH2CI2, -78*C MeO^Q (64% 2 stepi) HO^ 84 PMBO 85 62 Et3N, 0“C (82%) Scheme 14. Synthesis of p-aldehydo ester 62 The last steps of the preparation of activated ester 60 are illustrated in Scheme 15. The p-hydroxysulfone 87 formed by condensation of sulfone 80 and aldehyde 62 was subjected to Swern oxidation. Reduction of the resulting p-ketosulfone proceeded efficiently with aluminium amalgam in aqueous THF to give compound 88. A regioselective O-debenzylation was next attempted with DDQ in aqueous CH2CI2 at 0° C. It furnished a mixture of the p-hydroxyketone and the two a- and p-ring-closed hemiketals, which was treated with PPTS and methanol to give the glycal 89. Deprotection of the alcohol followed by conversion of the methyl ester to the acid afforded compound 90. Construction of the activated ester 60 was thereafter accomplished by treatment with Castro's BOP reagent, Swern oxidation, and deprotection of the tertiary PMB ether with DDQ in wet chloroform. 27 PhS02 Ph02S n-BuLi, THF, -7 8 X then OSiPh2f-Bu SiPh2f-Bu add sulfone anbn to 62 (82%) PMBO PMBO 'oh i y i r Me Me \ PMBO Me Me Me 87 Me Me PMBO LXHO (1) Me2SO, (CF3C 0 )20 MeO X 'O CH2CI2, -7 8 X , then Et3N, 62 0°C (93%) (2) Al-Hg, 10% aq. THF reflux (96%) . .Me SiPh2Bu-f (1) DDQ, C H ^ b OSiPh2Bu-f PMBO H2Q, 0X,3h PMBO (2) PPTS, MeOH, 6 0 X PMBO Me 1 h (77%, 2 steps) (1) TBAF, DMF rt, 48 h (84%) (2) EtSLi, HMPA THF, rt. 2 h .Me (1)/-Pr2NEt, BtOP(NMe2) 3 PF6 CH2CI2 (99%, 2 steps) PMBO (2) (CF3C0)20, MejSO, CH2CI2” -7 8 X ; Et3N, O X (97%) (3) DDQ, wet CHCI3, rt, 3 h (71%) Scheme 15. Synthesis of the pyran sector of A83586C During the A83586C project, the Hale group had to find new methods for the synthesis of many of the key units of A83586C. Indeed the first short enantioselective synthesis of both (3R)- and (3S>piperazic acids was developed.26 The (3R)-piperazic acid and (3S,)-piperazic acid were synthesised via tandem electrophilic hydrazination-asymmetric nucleophilic cyclisation.26 The synthesis of A/(1)-Z-(3R)-piperazic acid 34 is illustrated in Scheme 16. The route commenced with a regioselective deprotonation of (4R)-phenylmethyl-2-oxazolidinone 91 and subsequent A/-acylation with bromovaleryl chloride 92. The resulting bromide 93 was treated with LDA to produce an internally-coordinated enolate that underwent a stereoselective hydrazination with di-fe/t-butylazodicarboxylate (DBAD). Tandem cyclisation occured after addition of DMPU to afford compound 94. A/(1)-Z-(3R)-piperazic acid 34 was obtained after removal of the auxiliary using lithium hydroxide, cleavage of both Boc groups with TFA and Z group protection of the A/(1)-atom. Dipeptide 64 was obtained via a silver cyanide coupling between the acid chloride 36 derived from A/(1)-Z-/V(2)-Fmoc-(3R)-piperazic acid 35 with the glycine derivative 96. Deprotection of the acid with TFA yielded dipeptide 64. 28 /-Pr2 NH, n-BuLi n-BuLi, THF, 15 min 1 -78*C, then add 1 THF, -78°C, 35 min \ BocN. Cl Br then add DBAD in CH 2 CI2 N / stir for 30 min, add DMPU / Boc Ph O 92 Br Ph warm to rt (55-63%) Ph 93 94 (4f?)-phenylmethyl- (80-91 %) 2 -oxazolidinone LiOH, THF:H20 91 (2:1),-5 to 0°C 2 h (89%) Me 3 SiCI, (1) TFA, CH2 CI2 DIEA,CH2 CI2, reflux n HO rt, 1.5 h (94%) HO , NZ h o 2c N FmocCI then O'C to rt, HN. BocN Fmoc (2) ZCI, 1M NaOH N 18h (85%) PhMe, 0°C (93%) Boc 35 34 95 (COCI)2 c 6 h 6 60°C, 1.5 h AflCN C 6 H6, 80°C CF 3C 0 2H / NZ (81%, 2 steps) O* CH2 CI2 BnOv y— NFmoc Cl , NZ 9 Bn> — NFmoc N Fmoc 9Bn (99%) Me’"( o 36 Me/, / NH 0I ° O r 96 0 OCHPh2 HO 0 2 97 OCHPh 64 Scheme 16. Syntheses of A/(1)-Z-(3R)-piperazic acid 34 and dipeptide 64 The same silver cyanide assisted amidation methodology was used to couple the (3S)- piperazic acid derivative 99 to the acid chloride of Fmoc-A/(Me)-D-Ala 98 to yield compound 65 (Scheme 17). For both these procedures, the amino-acid chloride was used in its Fmoc- protected form because first, these Fmoc amino-acid chorides are very stable and easy to prepare but also because they are highly reactive and able to couple to donors with low nucleophilicity with minimum epimerisation at the a-stereocentre. However, difficulties were later encountered upon removal the Fmoc protecting group from 65 leading to the formation of the diketopiperazine. Replacement of the diphenyl methyl ester group from compound 65 with a t- butylcarbazide function avoided that problem. Peptidic coupling of the resulting amine 100 with acid 64 activated with BOP-CI/Et3N afforded compound 101. The Boc group was removed with TFA and the resulting acyl hydrazine oxidised to the acid with NBS.27 Esterification with diphenyldiazomethane followed by cleavage of the Fmoc group with diethylamine gave compound 102. 29 M®v ^Fmoc (1) CF 3 C 0 2 H, CH 2 CI2, PhOH Me, Fmoc^ -Me (1)(COCI)2. c 6h6 N (2) AgCN, PhMe, 70°C ^ (2) B ocNHNH 2, DCC, HOBt NH Me' Me H ■V. ------(93%, 2 steps) 'V. 'N N il.C 0 2 CHPh2 .A (3) Et2NH / MeCN (1: 1 ) ,A OH OCH2Ph NHNHBoc ‘O' 99 O 0 98 100 (92%, 2 steps) 65 / ^NZ Et3 N, -10°C BnOv y— NFmoc then BOPCI warm to 0°C, 4h (75%, 2 steps) S=o° HO 64 NZ (1) TFA, CH2 CI2, O'C (2) NBS.THF, H20 Me. Me. O'C to rt (3) Ph2 C=N2 Me2CO rt. 1 2 h Me (6 8 %, 3 steps) Me* (4) Et2 NH, MeCN ZN OCHPh. ZN NHNHBoc 102 101 Scheme 17. Formation of compound 102 Dipeptide 66 contains the (2S,3S,)-3-hydroxyleucine moiety and is common to the five antibiotics described at the beginning of this section. A novel large scale synthesis of (2S.3SJ-3- hydroxyleucine 46 was developed by the Hale group for the synthesis of A83586C.28 This new method, depicted in Scheme 18, requires only 6 steps and begins with a Wittig condensation between isobutyraldehyde 103 and carboethoxymethylene triphenylphosphorane 104 to afford alkene 105. An efficient entry into the chiral anti-amino alcohol motif was achieved by use of the Sharpless asymmetric dihydroxylation reaction.29 The resulting diol 106 was converted with thionyl chloride to its 2,3-cyclic sulfite that was oxidised in situ into sulfate 107 with RuCh/NalO^ A ring opening strategy using NaN3 afforded azido ester 108 in 92% yield. Hydrolysis of the ester with aqueous sodium hydroxide followed by hydrogenation afforded (2S.3SJ-3- hydroxyleucine 46. 30 AD-mix a, M eS0 2 NH2 ✓COjEt Me ph3p = /-Bu0H/H20 1:1 V -C H O — — 104 EtO EtO Me Me CH 2 CI2, O'C to rt O'C, 96h (96%) 103 105 (95%, 97% ee) SOCI2, CCI4, reflux, 2 h RuCI3 xH 2 0, N al0 4 MeCN, H20 (92%) (1) aq.NaOH (1M) NaN3, H 2 0-M e 2 C 0 NH2 Me Me QSO2 Me THF, O'C (93%) (1:10),1.5h HO EtO EtO Me Me (2) H2, Pd(OH) 2 20% aq. H 2 S 0 4, O OH MeOH (93%) O OH EtzO, 24 h (92%) 46 108 107 Scheme 18. Synthesis of (2S,3S>3-hydroxyleucine 46 Compound 112 was then obtained via a DMAP-assisted DCC coupling of the protected derivative 109 with acid 111. O-Deallylation of 112 with Pd(0) and morpholine followed by treatment with oxalyl chloride provided the depsipeptide acid chloride 66 (Scheme 19). ( 1 ) Troc-CI aq.NaOH Troc^ NH2 Me THF, rt(90%) ^ V® Tree. Trocs H° Y ^ A Me ( 2 vNaHC0* A"°Y^^Me jn T ( 1 )(P P h 3)4 Pd. y H lV'e ll i l L amb i Jl A lj DCC, DMAP A I I O ^ ^ ^ ^ A morpholine, THF C I s ^ A A O OH DMF, AIIBr, rt O OH CH2 CI2 : Me O'C tort (59%) [f : Me (92%) ^ 0 o ► O O 4 6 O'C to rt 0 = f^ (2) (COCI)2, C6 H6 O (1) Boc2 0, (85%) I rt 2 . 5 h I O^OH aq.NaOH. dioxane O^OH B o c H N ^ T ^ B o c H N ^ V Me X ______°°C (90%) r T OBn OBn H3C e (2) NaH, BnBr BocHN : 112 66 OH DMF (32%) 0Bn 110 111 Scheme 19. Synthesis of depsipeptide 66 The final steps of the synthesis of A83586C are shown in Scheme 20. The coupling of chloride 66 to amine 102 was achieved efficiently (86% yield) with silver cyanide at 60 °C to afford peptide 113. The reaction needed to be conducted for 2 min otherwise decomposition quickly ensued. The Troc group was then detached with Zn dust and replaced with a Z group. A mild acidolysis with TFA and phenol was used to cleave the Boc and the diphenylmethyl groups and generate compound 114. The phenol trapped out the diphenylmethyl cation and prevented it from causing unwanted side reactions. After screening many activation reagents unsuccessfully, macrolactamisation of compound 114 was eventually accomplished with HATU and /V-ethylmorpholine in CH2CI2 under conditions of very high dilution30 in 25-40% yield. The 31 benzyl protecting groups were then cleanly deprotected by catalytic hydrogenation over a 10% Pd on carbon catalyst in methanol containing 1% eq of HCI. This acidic medium helped prevent O- to A/-acyl shift from occuring in the /3-hydroxyleucine residue. The last steps of the synthesis consisted in the union of the crude cyclodepsipeptide core 59 with the activated ester 60. Both were suspended in CH2CI2 and after cooling that mixture to -78 °C, Et3N was added and the reaction mixture warmed to room temperature and stirred for 10 minutes. Compound 58 was obtained in 31% yield after chromatography purification. Finally compound 58 was hydrated with wet deuterochloroform to deliver A83586C 2 in a quantitative yield. 32 rv Trocx NZ NHTroc 9BnV-NH NH Me Cl Me II Aj /Me ° AgCN, C6 H6 f O o O N 0 P 60*C for 2 min A\,-M e C °i O ' N Me Me'' A^-Me (73-86%) BocHN BocHN : OBn OBn ZN T OCHPh 2 ZN^J''' OCHPh2 66 113 102 (1) Zn (70 eq), AcOH/H20 (10:1), rt, 3h (2) ZCI, 10% aq NaH C 0 3 CH 2 CI2, rt, 2h, (78%, 2 steps) (3) TFA /C H 2 CI2 (2:1) PhOH, 0°C, 1h (100%) /vIZ NHZ HATU, CH2 CI2, 0*C, then slow addition of 114 and NEM in CH2 CI2, then 0°C for 2h and rt for 3Oh (25-40%) ^ Y ° O C^COz^ 114 10% Pd/C MeOH, HCI H2, rt, 24h Me Me -Me HO Me } N o Me BtO Me ^ HN.N "I 4 mix together in CH 2 CI2, ^ ' C , add Et3N (9.3 eq), rt 10 min (31%, 2 steps) Me. Me HO HO Me Me OH /MH NH /JHNH CDCI3 wet Me Me 72 h, 0°C Me Me Me Me 100% s=k -Me ,Me u N o N o Me y f hn Me OH Me HN.N A Me HN Y A83586C 2 Scheme 20. Total synthesis of A83586C 2 33 2.1.3. Synthesis of Verucopeptin Cyclodepsipeptide Core In 2001, our group reported a synthesis of the cyclodepsipeptide core of verucopeptin 6.31 This antitumour antibiotic incorporates a more functionalised pyran sector than other members of the azinothricin family but a greatly simplified cyclodepsipeptide core. Achieving its synthesis could pave the way for the synthesis of simplified analogues. The relative and absolute configurations of verucopeptin remain currently unknown. Hence it was decided to synthesise the diastereoisomer that was closest in structure to the other members of the family i.e. the isomer containing a (3Rj-piperazic acid unit linked to a (2S,3S>)-3-hydroxyleucine. With the experience gained during the A83586C synthesis, a [2+2+2] fragment condensation strategy was planned to link the linear hexapeptide domain. This strategy would also involved a union between fragments 116 and 117. Fragment 117 could be obtained by successive coupling between compounds 118 and 120, removal of the Fmoc group from the resulting tetrapeptide and union with fragment 119 using acid chloride coupling technology (Scheme 21). Verucopeptin 6 o NZ NFmoc BocN 118 Me NHNHBoc O 120 Scheme 21. Retrosynthetic plan of verucopeptin 6 34 The synthesis of peptide 118 commenced from the protected hydroxamic acid 121, the synthesis of which was reported by Kolasa and Chimiak.32 Compound 121 was esterified with diphenyldiazomethane 122 to afford compound 123. Silver cyanide assisted coupling with acid chloride 36, followed by removal of the diphenylmethyl ester group of 124 gave acid 118 (Scheme 22). 36 122 n2 NZ BnO BnO JJZ cioc BnO, NFmoc NH V 9)Bry BrOo —n NFmoc f Ph Fmoc H-% CF3C 0 2H, PhOH O Me2CO V=o AgCN, C6H6 CH2CI2, O'C, 2 h 70*C, 40 min HO rt, 20 min Ph2HCO X'OCHPh2 (96%) (58-70%) (96%) 121 123 124 118 Scheme 22. Synthesis of peptide 116 The peptide linkage of tetrapeptide 127 was built by a DMAP-assisted DCC coupling between compounds 125 and 126 (Scheme 23). Removal of the Z group followed by coupling of the resulting amine 120 with compound 118 using BOP-CI and Et3N afforded compound 128. Subsequent cleavage of the Fmoc group gave tetrapeptide 129 in 87% yield. OH Me' DCC, DMAP 125 • N ^ A n ^ N H N H B oc H2- P c*(OH)2 NHNHBoc Me' N^ V NHNHBoc CH2CI2 MeOH, rt H 'X 120 H2N i f (73-85%) 127 (100%) O 126 O z BOP-CI, Et3N, BnON y — NFmoc CH2CI2, -20°C (20min) then warm to 0°C, 3h (82%) K HO 118 ( ^NZ 9 Bn V -N H 9Bn yr— v NFmoc Et2NH, MeCN \ o U ° rt, 20 min (87%) r ® Me~N\ yjP Me— N NH a NH a />—NHNHBoc NHNHBoc 129 128 J / 1 Scheme 23. Synthesis of tetrapeptide 129 35 To complete the preparation of cyclodepsipeptide 117, the synthesis of acid chloride 119 still had to be accomplished (Scheme 24). A DCC-DMAP mediated O-esterification between alcohol 109 and acid 130 provided ester 131, which was submitted to deallylation with Pd(0)/morpholine and chlorination with oxalyl chloride to give acid chloride 119. The coupling between compounds 119 and 129 took place efficiently (80% yield) when silver cyanide was used as the promoter and the mixture was heated at 80 °C for 2-3 min. Hexapeptide 132 was then converted to the amino acid 133 by TFA treatment followed by chemoselective oxidation of the Gly acyl hydrazide group with NBS.27 Contrary to the A83586C synthesis, the macrolactamisation had to be carried out before A/-Troc to N-Z group interconversion otherwise degradation occurred under the basic Z-protection conditions. The cyclisation was performed using Carpino’s HATU protocol30 and cyclodepsipetide 134 was obtained in 67% yield over 3 steps. The Troc-urethane was then detached with Zn dust and successfully replaced with a Z group using benzylchloroformate and 10% aqueous NaHC03. Catalytic hydrogenation in methanol in the presence of one equivalent of HCI afforded cyclodepsipeptide 117. 36 Trocv Trocv » (1)(P P h3)4Pd Troc' nw AIIO morpholne, THF Cl DCC, DMAP rt 30 min Me O 119 ■I " * Q/vMBocN. ° ^ > CH2Cl2 , rt, 24 h (2) (COCOa. C6H6 C OH Me (83-87%) H rt, 2.5 h BocN. (66-75%, 2 steps) BocN, 130 109 Me Me 131 129, AgCN, CeHe 80°C, 2-3 min (78-81 %) NZ NZ NHTroc / NHTroc BnO (1) CF3C 02H, CH2CI2, rt, 2 h )=0C 0 ° 0^ < C M e -N M e -NN. .. 0 _ / (2) NBS, THF/HjO N— ^ Boc- Nn rt, 2 h Me Me HN NHNHBoc CF 3 CO2 133 132 HATU, CH2CI2, O’C then slow addition of 133 and NEM (over 8 h) in CH2CI2 rt for 48 h, c = 0.0004 (67%, 3 steps) (1) Zn dust, A c OH/H20 (10:1), rt 1.5 h ^ H r . NHTroc Me (2) ZCI,10% aq. N aH C 03 H9 n | 3 M e CH2CI2, rt, 1 h (78-84%, 2 steps) ( q M® i i u _ ^ (3 )H 2.10% Pd/C(0.1 eq) MeN. , 0 n \ MeOH (0.01M), HCI MeN in MeOH (100%) J 11 134 Scheme 24. Synthesis of Verucopeptin cyclodepsipeptide core 117 2.1.4. Synthesis of GE3 Cyclodepsipeptide Core In 2002, our group achieved the synthesis of the GE3 cyclodepsipeptide. The retrosynthetic plan which was based on the synthesis of A83586C, is shown in scheme 25. It also involved a [2+2+2] fragment condensation strategy and utilised two of the three dipeptide components that were used for the synthesis of A83586C. 37 Me Me Me Me HO BtO O Me HO Me Me Me NHNH Me :> Me ci- Me, /MH NH: Me Me Me Me Me HN ,Me HN Me Me Me HN Me HN GE3 4 136 NHTroc Me Me ,NZ O ciy S ^ Me CJ>Bn\—Bn>—NFmoc NFmoc n A Me, ° s° Me X 0 ^\^MX e ZN NHNHBoc O^OH BocHN : OBn 137 64 66 v J fragments common to A83586C Scheme 25. Retrosynthetic analysis of the GE3 cyclodepsipeptide The synthesis of tetrapeptide 137 is depicted in Scheme 26. It started from the (3S)- A/(1)-Z-A/(2)-Fmoc-piperazic acid 138 which was protected with a f-butylcarbazide function using DCC as the condensing agent. The Fmoc group was then detached to allow 139 to be coupled with acid chloride 140 in presence of silver cyanide. Tetrapeptide 137 was obtained after cleavage of the Fmoc group. All efforts to couple tetrapeptide 137 to acid 64 using the BOP- CI/NEt3 system which was so efficient in the A83586C synthesis, failed. After having tried a wide range of conditions, it was discovered that the combination of BOP-CI and collidine could yield the desired tetrapeptide 141 in 66% yield. The Fmoc group was then cleaved from 141, and the third fragment, 66, was attached using silver cyanide to give peptide 142. After the cleavage of the two Boc group, the A/-acyl hydrazine was oxidised with NBS/water to give the acid. The macrolactamisation was achieved using the same conditions as for A83586C, i.e. with HATU in very high dilution in CH2CI2, as developed by Carpino. It proceeded in a 40% yield over three steps from compound 142. The last steps involved replacing the Troc group with a Z group and 38 deprotecting the three Z-groups by hydrogenolysis under mildly acidic conditions to give the cyclodepsipeptide 136. Fmocn. .Me (1) N 140 H . .Me (1) DCC, BocNHNHj, Me N Fmoc THF, 0"C, 1 h -N . ,.C 02f Me then rt 20 h (91%) .K.X ZN ZN NHNHBoc Me' Cl Me (2) EtzNH, MeCN, AgCN,C6H6, 8OX x NHNHBoc rt, 15 min (99%) 50 min (90%) 138 139 (2) Et2NH, MeCN, zO 137 rt, 15 min (84%) BOP-CI, CH2CI2, -2 0 X add collidine (1.1 eq) stir for 20 min, add 64 and coUidine (1.1 eq) 16 h at rt (66%) NHTroc Me ,NZ 9)Bry Bny>—o— n NFmoc f Me (1) TFA, CH2Cl2, O X, 2 h Me. (1) Et2NH, MeCN, Me/. (2) NBS, THF/H20 (1:1), rt 2 h rt, 15 min (83%) .M e J .Me ° (3) Addition to HATU and N EM in Me N BocHN (2) 66, AgCN, C6H6 CH2CI2 (0.00086 M) over 6 h at Me 8 0 X , 2-3 min (65%) Me O X , 2 h at O X , rt for 60 h OBn (40%, 3 steps) Me Me ZN NHNHBoc 6 . i NHNHBoc 141 141 HZ NHTroc (1) Zn dust A cOH/H 20 Me (10:1), rt, 25 min. (2) ZCI, 10% aq NaHC03 Me Me rt, 1 h (55%, 2 steps) (3) H2, 10% Pd/C Me MeOH, HCI (100%) Me. HN Me HN 143 136 Scheme 26. Preparation of the GE3 cyclodepsipeptide 136 2.2. Previous Syntheses of Analogues of the Azinothricin Family of Antibiotics The synthesis of analogues of A83586C is of interest as these may be of value for elucidating the mode of antitumour action of these natural products. In fact, by testing analogues, it should be possible to determine which part of the molecule is essential for potent antitumour activity. Analogue work might also allow a considerably simplified structure to be identified that has good antitumour properties and which can be more readily synthesised industrially. Some cyclodepsipeptides analogues have already been synthesised: the A-epi- analogue33 144 is one such compound, as is the L-proline modified A83586C 145. 39 The 4-ep/'-analogue 144 has a (3ft)-piperazic acid component replacing the (3S)- piperazic acid unit (Figure 5). Making this change improved the yield of the macrolactamisation from 25% to 70% but the 4-ep/'-analogue 144 was much less active as an antitumour drug. It is believed that in that analogue, the C(8)-carbonyl adopts a conformation cis to the C(7), whereas the relationship between these two bonds is trans in A83586C. Figure 5. 4-ep/-A83586C 144 For this reason, it was decided to synthesise an analogue that had similar conformational properties than A83586C. The L-proline analogue was selected, because, first, the (3S)-piperazic acid has previously functioned as a very effective mimic of L-proline in ACE- inhibitor such as cilazaprill34 and secondly, cyclisation at an activated proline residue is usually free of racemisation risk. The synthesis of the L-proline modified mimetic of the A83586C cyclodepsipeptide 145 was thus performed in our laboratory in 2002.35 The strategy involved coupling of fragments 146, 64, and 66 as shown in Scheme 27. cr NHTroc NFmoc NHNHBoc BocHN 146 146 64 66 Scheme 27. Retrosynthetic plan to the L-proline modified mimetic of the A83586C cyclodepsipeptide 145 40 Units 64 and 66 of A83586C were used for the synthesis of cyclodepsipeptide 145. Dipeptide 146 was prepared from /V-Z-L-proline 147 as shown in Scheme 28. The latter was converted to the acyl hydrazide 148 by treatment with BocNHNH2/DCC followed by catalytic hydrogenation to cleave the Z group. A peptide bond was formed by silver cyanide assisted coupling between Fmoc-A/-methyl-D-alanyl-chloride 149 and compound 148 to afford compound 150. Fmoc cleavage afforded amine 146 which was purified and readily coupled to acid 64 by treatment with BOPCI and Et3N at low temperature. Fmoc deprotection followed by condensation of tetrapeptide 152 with acid chloride 66, using silver cyanide as a promoter, gave hexapeptide 153 in 73% yield. The last stages of the synthesis were performed in the same order as for verucopeptin and GE3 cyclodepsipeptides: the two Boc groups were cleaved with TFA, the liberated A/-acylhydrazine was converted to the acid with NBS, and the macrolactamisation was carried out at very high dilution utilising HATU as the carboxyl activating reagent. Macrolactam 154 was obtained in 48% yield over 3 steps. Acquisition of cyclodepsipeptide 147 was accomplished after Z replacement of the Troc group and hydrogenation over Pd/C in the presence of one equivalent of HCI in MeOH. 41 , (1) DCC, NH2NHBoc AgCN, C6 H6, Fmocx .Me ^ N ^ ,..C 0 2H THF, O'C to rt, 24h, (84%) ,.CO NHNHBoc 80”C, 50 min (93%) N (2) Pd(OH)2/C 20%. H2 O' Fmocv .Me Me MeOH, rt 12h, (100%) r s 147 148 NHNHBoc O' ,4,« .V 160 Et2NH (40 eq) MeCN (94%) NZ NFmoc H, .Me Me. Me. 64, BOP-CI, Et3N Et2NH (40 eq) .Me CH2 CI2 at-20°C Me MeCN, rt stir for 2 0 min NHNHBoc (100%) Me warm to 0°C for 1 .5 h Me (89%) NHNHBoc 146 NHNHBoc 162 161 6 6 , AgCN, C6 H6 80”C, 8 min (73%) NHTroc ; n z NZ NHTroc ^Bn Me Me, Me M e - ,,/N ( 1 ) CF3CO 2 H, CH2CI2 , 0 °C, 2 h (2) NBS, THF/H2 0, rt, 2 h X ° ^ .M e 0 n,M® r, , ^ ^ M e Me , BocHN : ( 3 ) mjx with NEM then add over 6 h to u NH OBn HATU very diluted in CH 2 CI2 at 0”C Me Me r V stir at rt for 72 h (48%, 3 steps) ^ NHNHBoc 164 153 (1) Zn dust (85 eq) AcOH /H20 (10:1), rt, 20 min (2) ZCI, 10% aq. N aH C 0 3 V Cr CH2 CI2, rt, 1 h, (80%, 2 steps) n h n h 3 (3) H2, 10% Pd/C, MeOH, HCI Me rt, 24 h (100%) Me, 'Me Me Me 145 Scheme 28. Synthesis of the L-proline modified mimetic of the A83586C cyclodepsipeptide 145 42 3. Synthetic Studies Towards Analogues of the Azinothricin Family As outlined in the introduction, our group had developed a large knowledge concerning the synthesis of molecules of the Azinothricin family at the outset of this project and so was now in a very good position to exploit this knowledge for constructing more analogues. As a member of this research program, I worked on the synthesis of two modified cyclodepsipeptides. The l- proline modified mimetic of GE3 cyclodepsipeptide 13 was chosen because: (a) GE3 was the most potent E2F inhibitor to have been discovered so far and we considered that its proline congener is likely to have promising properties, (b) comparing its biological activity with the already synthesised L-proline modified mimetic of A83586C would help to uncover which features of these molecules were essential for activity and; (c) the structure being simpler, the synthesis could potentially be more readily achieved on a large scale as was required for possible industrial applications. We also selected the (3S, 5S)-5-hydroxypiperazic acid modified mimetic of A83586C, 14, in order to analyse how the biological and pharmacokinetic properties of a more hydrophilic molecule would vary; a key problem with molecules of the A83586C class is their poor water solubility. Adding an extra hydroxyl might improve this situation and give a more readily administered drug. The retrosynthetic analyses of these two cyclodepsipeptides are depicted in Scheme 29. 43 NHTroc NFmoc Me Me Me Me 64 OH Me Me BocHN HN Me OBn A oH Me Me' Me NHNHBoc L-proline modified mimetic of GE3 cyclodepsipeptide /JHNH3 Me NHTroc NFmoc Cl Me Me I 0 ° 0 .Me 0 ;=f^ Fmoc Me BocHN 0H OBn V NHNHBoc 66 HO 14 (3S, 5S)-5-hydroxypiperazic acid modified mimetic of A83586C cyclodepsipeptide Scheme 29. Retrosynthetic plan to two cyclodepsipeptides Again, a [2+2+2]-fragment condensation strategy would be adopted. Two of the three fragments are common to both cyclodepsipeptides and their syntheses had already be developed for the A83586C synthesis. Thus, a large part of this project is dedicated to large scale synthesis. To access dipeptide 66, the 6 step synthesis of hydroxyleucine 46 was performed on a multigram scale without purification by chromatography (Scheme 30). Hydroxyleucine 46 was obtained in 45% yield from isobutyraldehyde 107. The detail of each step was described earlier in the introduction. Then, the DMAP-assisted DCC coupling yielded dipeptide 60, which was deprotected using Pd(0) and morpholine to afford acid 157 in 89% yield. Acid 157 would be converted into chloride 66 just before use. The spectral data obtained for acid 157 corresponded to that published by our group during the GE3 cyclodepsipeptide synthesis.36 40 g of the protected hydroxyleucine 109 was synthesised. 44 (1) AD-mix a, M eS 02NH2 Ph3P^5t^ c 0 2Et f-Bu0H/H20 1:1 Me. 104 ______O’C. 96h -CHO EtO EtO Me c h 2c i 2 Me NaN3, (2) SOCI2, CCI4t reflux H20-M e 2C 0 2 h; add RuCI3 xH20 103 105 107 (1:10),1.5h N al04, MeCN, H20 20% aq. H2S 0 4 Et20, 24 h O^/OH TrocN (1) Troc-CI, aq. (1) aq.NaOH (1M) NaOH, THF, rt NH2 Me THF, O'C AIIO HO EtO BocHN Me (2) NaHC03 Me (2) H2, Pd(OH)2 Me OBn O OH DMF, AIIBr, rt O OH MeOH, O OH (67%, 2 steps) (45%, 6 steps) 111 109 46 108 Trocx DCC, DMAP Trocs NH Me CH2CI2 O'C to rt AIIO ^A^A.. (PPh3)4Pd, HO Me (80%) ]f : Me morpholine, THF O .0 ------O D 0 = ^ O'C tort 0= / (89%) >\^-Me Me BocHN : BocHN OBn OBn 112 157 Scheme 30. Synthesis of acid 157 Compound 64 was prepared using the route developed in the A83586C synthesis (Scheme 31). Compounds 34 and 96 were already prepared by L. Lazarides of this group.36 Protection of 34 was achieved in a good yield using FmocCI in presence of diisopropylethylamine and trimethylsilyl chloride. The acid was converted to its chloride and combined with 96 in a silver cyanide mediated coupling. The acid group of compound 97 was then deprotected with TFA to afford acid 64. Me3SiCI, DIEA CH2CI2 reflux (COCI)2 (20 eq) NZ .NZ Cl h o2c FmocCI then O'C H 0 2C N n ' n z Fmoc C6H6 60'C, 1.5 h Fmoc to rt,18 h (81%) 34 35 26 (J)Bn AgCN (1.5 eq) M6YNH C6H6, 8 0 'C (78%, 2 steps) 96 o OCHPh2 ( ^NZ ( ^NZ (j)B n \— NFmoc CF3C 0 2H (36 eq) 9 BnX “ NFmoc MPhOH (1.4 eq) ( o - ' - CH2CI2, O'C, OH 3 h (91%) OCHPh2 64 97 Scheme 31. Synthetic route to compound 64 45 3.1.Synthesis of an L-proline analogue ofGE3 cyclodepsipeptide The synthetic route to dipeptide 155 is depicted in Scheme 32. It started from commercially available D-leucine 158. 37 An Fmoc group was attached to amine 158 with Fmoc CI and sodium carbonate in dioxane. Addition of water and ether to the reaction mixture, acidification of aqueous layers and extraction with diethyl ether allowed isolation of acid 159. The latter was cyclised using paraformaldehyde in presence of phenol in toluene at 120°C using a Dean-Stark apparatus. Treatment of crude oxazolidinone 160 with TFA / Et3SiH in chloroform liberated the Fmoc protected A/-methylamine 161 which was obtained in 74% yield over 2 steps after crystallisation. The 500 MHz 1H NMR spectrum of compound 161 in CDCI3 indicated the presence of two rotamers. The formation of the peptide linkage between amine 148 and the acid chloride of 161 was carried out using silver cyanide at 80 °C in benzene for 30 min to provide dipeptide 155 in 90% yield over 2 steps. Fmoc aq N a H C 0 3 (2 eq) M e N H 2 FmocCI (1.09eq) Me NHNHFmoc (CH20)n,TsOH 1e N—"\ X a ,0 160 Me/ ^ ^ ^ C 0 2H dioxane, 0°C to rt Me C 0 2H PhMe, 120°C Me (100%) 158 159 TFA, Et3SiH CHCI3l rt, 2 days (74%, 2 steps) Mev Mev (1) (COCI)2,CH2CI2, rt, 2 h Me NFmoc NFmoc Me O o (2 ) Me Me A 161 NHNHBoc \ ] NHNHBoc o 148 AgCN (1.5 eq), C6H6 155 reflux at 80°C for 30 min. (90% , 2 steps) Scheme 32. Synthesis of fragment 155 Fmoc deprotection of dipeptide 155 was achieved with diethylamine (35 eq) in MeCN at room temperature for 35 minutes. After purification of the crude residue by silica gel chromatography, deprotected dipeptide 162 could not be isolated pure. TLC analysis showed that a faster moving product had formed during the purification. The presence of 10% of 46 diketopiperazine 163 was detected by 1H NMR analysis of the mixture obtained after flash chromatography (Scheme 33). Me M0'NFmoc Et2NH H'N'M® Me, X U o . MeCN - J k / . O O Me / N n A rt, 25 min / N x X Z NM® ^ J' NHNHBoc (87%) ^ J' NHNHBoc 155 162 163 Scheme 33. Removal of the Fmoc group from compound 155 A second purification by Si02 flash chromatography of the mixture allowed isolation of pure diketopiperazine 163. However, dipeptide 162 could never been separated from some diketopiperazine 163. 500 MHz 1H NMR spectrum of the mixture of 162 and 163 in CDCI3 is shown in Figure 6 and 500 MHz 1H NMR spectrum of diketopiperazine 163 in CDCI3 is shown in Figure 7. In the 1H NMR spectrum of diketopiperazine 163 in CDCI3l the lack of singlet around 5 1.40 ppm indicated the absence of the tert -butyl group. Additionally, the signal corresponding to the N-Me group in diketopiperazine 163 is shifted downfield compared to 162 due to the proximity of the carbonyl group. 47 IM . 65 CIKl'3 - 2 38 k Me] Me NHN Me Me NMe 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm Figure 6 . 1H NMR spectrum of the mixture containing dipeptide 162 with 10% of diketopiperazine 163 in CDCI3 at 298K cyclisation produ< Me CDC13 - 298K Me NMe 4.0 3.5 3.0 2.0 1.5 1.0 0 . 5 ppm Figure 7 .1H NMR spectrum of 163 in CDCI3 at 298K 48 It is believed that the formation of diketopiperazine 163 is triggered by the acidity of the silica as shown in Scheme 34. M e^ .Me M0'NH + Y ” f* Me Mev O _ - -•n — H+ transfer ^ \ 9 ~ ° v ? ------/ ?Me Me .N. A I U NHNHBoc ( Y NHNHBoc .N \ 7Y> W O ^ - H + 162 162 163 Scheme 34. Mechanism of formation of diketopiperazine 163 A diketopiperazine formation was observed at the same stage of the synthesis of A83586C24 (Scheme 35). However, the acid was protected with the more labile Ph2CH ester group and the diketopiperazine formation was avoided by replacement of the Ph2CH ester group with a BocNHNH group. MV FmoC Me MeA (* 0 v0 Et2NH ^ ^ ° Y ^ NMe , N ^ A MeCN - 'N ' ZN V OCHPhj Z^' 'T "O 66 164 Me Fmoc (1 )C F 3C 02H, CH2CI2. PhOH Mev (2) B ocNHNH 2, DCC, HOBt NH Me 65 100 Scheme 35. Formation of diketopiperazine 164 observed in the A83586C synthesis Attempts to couple the purified dipeptide 162 (with 10% of diketopiperazine 163) to acid 64 remained unsuccessful. This difficulty was overcome by coupling crude dipeptide 162 and acid 64 with BOPCI and NEt3 at -10°C in dichloromethane (Scheme 36). Tetrapeptide 165 was obtained in 73% yield over 2 steps; its formation was easily followed on the TLC plate with the appearance of a yellow spot with anisaldehyde. The formation of tetrapeptide 165 was confirmed by FAB HRMS analysis which contained an (M+Na)+ peak at m/e 1024.48548 (Calcd (M+Na)+ for CssHeyNyNaOn 1024.47960). Tetrapeptide 165 was then deprotected using 49 diethylamine (40 eq) in MeCN at room temperature for 25 minutes to afford 166 in quantitative yield. NFmoc NH FmoCs .Me Me Me N (1) Et2NH (40 eq) (3) Et2NH (40 eq) MeCN, rt, 15 min Me. X/jO MeCN. rt. 25 min .Me .Me Me' I ft (2) ( 100%) { NZ Me Me )B no \— fsjF O " NHNHB0C 9 V -N F m o e Me Me' NHNHBoc NHNHBoc 161 M° Y 0 . 64 O OH 165 BOP-CI, Et3N, CH2CI2 -10*C, 3 h (73%. 2 steps) Scheme 36. Synthesis of tetrapeptide 166 The final steps of the synthesis of cyclodepsipeptide 13 are shown in Scheme 37. Acid 157 was first converted to chloride 66. The [4+2] condensation was achieved by heating chloride 66 with tetrapeptide 166 in the presence of AgCN in C6H6 at 80°C. It is important to stop heating this reaction after 5 min, otherwise decomposition might ensue. The structure of the resulting compound 167 was confirmed by FAB HRMS analysis which contained an (M+Na)+ peak at m/e 1396.54502 (Calcd (M+Na)+ for CesHgoNgNaO^ 1396.54176). In fact, the 1H NMR and 13C NMR spectra of 167 were complex due to the presence of rotamers. The conversion of hexapeptide 167 to cyclodepsipeptide 168 was performed in 70% yield using the 3-step protocol developed in this research program. The Boc groups were cleaved by treatment with excess trifluoroacetic acid, the acyl hydrazine was oxidised with NBS in THF/H20 to give the acid and high dilution macrolatamisation with HATU/NEM provided compound 168. In this macrolactamisation, a solution of hexapeptide 167 (1 eq) and NEM (13.5 eq) in dry CH2CI2 (0.0008 M) was added dropwise to an ice-cold solution of HATU in dry CH2CI2 (0.0008 M). After the addition, the reaction mixture was warmed to rt, stirred for 48 h and concentrated in vacuo. Cyclodepsipeptide 168 was obtained after an acid/base work-up and purification by Si02 flash chromatography. The product appeared as 2 inseparable spots. The only way to confirm the structure of the cyclodepsipeptide was to use mass spectroscopy. The FAB HRMS spectrum of 168 contained the appropriate (M+Na)+ peak at m/e 1164.39772 (Calcd for CssCbHyoN/NaOn (M+Na)+ 1164.39946). The Troc group was then detached with Zn dust in aqueous acetic acid, 50 and the crude amine immediately capped with a Z-group using benzylchloroformate and 10% aqueous NaHC03. The product appeared again as 2 inseparable spots after purification by silica gel flash chromatography and the structure was confirmed by mass spectroscopy analysis. Cyclodepsipeptide 169 shows the appropriate (M+Na)+ peak at m/e 1124.52750 (Calcd for C6oH75N7Na013 (M+Na)+ 1124.53203). Hydrogenation over Pd/C in the presence of one equivalent of HCI in MeOH provided the final cyclodepsipeptide 13 as a yellow powder. NHTroc (1 )C 0 C I2 TroCv (2) AgCN,C6H6 NH Me HO- Me NZ O ,0 BocNH Me, Me BocHN .Me NHNHBoc OBn Me. 157 167 Me' (1) CF3C02H. CH2CI2, OX, 2 h NHNHBoc (2) NBS, THF/H20 , rt, 2 h (3) NEM and hexapeptide added (67%) over 6 h to HATU very diluted in 166 CH2CI2 at O X , stir at rt for 72 h (70%, 3 steps) NZ NHZ NHTroc >Bn Bn Me Me Me, Me (1) Zn dust Me A cOH/H20 (10:1) (2) ZCI, aq N aH C 03 Me Me CH2CI2 (57%, 2 steps) NH Me NH Me. OBn OBn Me Me 169 168 10% Pd/C, MeOH, H2, rt, 24 h (94%) Me Me, Me ,Me Me Me. NH OH Me 13 Scheme 37. Synthesis of an L-proline analogue of GE3 cyclodepsipeptide 13 51 1H and 13C NMR spectra of cyclodepsipeptide 13 in MeOD are shown in Figure 8 and 9. 6.5 6.0 5.55.0 4.5 4.0 3.5 3.0 2.5 2.0 1 .5 1.0 0.5 ppm Figure 8 .1H NMR spectrum of cyclodepsipeptide 13 in MeOD ri-AL-124nc MeOD 2 90 k 190 180 170 16050 140 130 120 110 ICO 90 80 70 60 50 40 30 20 10 ppm Figure 9 .13C NMR spectrum of cyclodepsipeptide 13 in MeOD These NMR spectra hinted that the formation of the desired cyclodepsipeptide occured. However 13C NMR spectrum shows that the product 13 is not pure. As it is very difficult to purify 52 a salt, a more careful purification will have to be carried out at an earlier stage. Indeed all effort to purify the mixture obtained for compound 169 by S i02 flash chromatography and preparative chromatography failed. 3.2. Toward the synthesis of an (3S, 5S)-5-hydroxypiperazic acid modified mimetic ofA83586C The retrosynthetic plan of the cyclodepsipetide analogue of A83586C incorporating a (3S, 5S)-5-hydroxypiperazic acid 14 was described earlier in this section (Scheme 29). Since dipeptides 64 and 66 has already been synthesised, our attention focused on the formation of compound 156 which contains the (3S, 5S)-5-hydroxypiperazic acid moiety. The desired protected form of hydroxypiperazic acid was synthesised following a procedure developed in our lab38 (Scheme 38 and 39). The synthesis commenced with a regioselective protection of d - mannitol 170 as shown in Scheme 38.39 First, protection of 1,3-diols occurred with benzaldehyde and conc. H2S04 in dry DMF for 4 days. The reaction had to be quenched very slowly with saturated aqueous NaHC03 because of liberation of gas. The resulting diol was protected with benzylbromide and sodium hydride in dry DMF to give 171. Compound 171 was then hydrolysed with conc. HCI in MeOH and water. After neutralisation of the reaction mixture with 20% aq. NaHC03 and extractive work-up with EtOAc, the resulting tetrol 172 was selectively protected with TBDPSCI and imidazole. Diol 172 was obtained as the single product and was cleaved with Pb(OAc)4 in CH2CI2 to give aldehyde 173. 53 Ph (1) PhCHO, H2S 0 4 ^ (1) Et0H/H20 , HCI OH OH TBDPSO OH OBn rt, 3 days (51%) 80°C, 18 h (67%) HO' OH OH (2) NaH (4 eq), DMF OBn O . 0 <2> Imidazole (4.5 eq) OBn OH OTBDPS BnBr (2.3 eq), rt Y TBDPSCI (2.2 eq) 18 h (53%) Ph 0°C, rt, 48 h (99%) 172 D-mannitol 170 171 Pb(OAc)4 (1.1 eq) Na2C 0 3 (2 eq) CH2CI2 rt, 0.5 h OBn OHC'^s '0TBDPS 173 Scheme 38. Formation of aldehyde 173 Aldehyde 173 was condensed with known phosphonate 17440 in a Wittig-Horner olefination to furnish alkene 175. This reaction took place under Roush-Masamune conditions41, i.e. in the presence of lithium chloride and diisopropylamine; Li+ form a complex with the carbanion derived from the phosphonate. Desilylation was next performed with 40% aq. HF to afford alcohol 173 in 93% yield. A carefully monitored hydrogenation allowed chemoselective reduction of the the olefin. The hydrogenated product 177 move slightly slower than alkene 176 on TLC. The more polar product visible on TLC is the debenzylated product. The resulting alcohol 177 was converted to bromide 178 using PPh3 and CBr4. The piperazic unit was then elaborated by delivery of di-ferf-butylazodicarboxylate to the lithium enolate of compound 178. This highly stereoselective hydrazination took place only when DMPU was added to the reaction mixture and proceeded in 58% yield. The chiral auxiliary was cleaved with sodium methoxide in 93% yield to give methyl ester 180. The Boc protective group was removed with TFA in CH2CI2, and the crude TFA salt was selectively protected using benzylchloroformate in aq. NaHC03. Hydrolysis of ester 181 with Li0H-H20 produced hydroxy-piperazic acid derivative 182 in 98% yield. It is noteworthy that using this procedure, we were able to produce 20 g of intermediate 182. 54 LiCI (2 eq), /-Pr2NEt (4 eq) o O O o 40% aq. HF O stir 15 min at rt, then add IJ A ,X _ P -0 E t .OBn THF/ MeCN ...OBn 0 N ------O N OEt OBn A OTBDPS rt- 48 h (93%) .A ^O TB D P S OH OHC 174 Ph 175 18 h at rt Ph 176 (74%, 2 steps) H2, 10% Pd/C (wet) EtOAc (89%) LDA (1.1 eq), THF at O O PPh3 (2 eq) O -78°C,1h, then add DBAD ..•OBn .'OBn CBr4 (2 eq) .A. OBn (1.2 eq) in THF, 1 h A O N add DMPU (16eq), 20 min Br THF, 0°C to rt OH Boc at -78°C, 50 min at rt 1.5 h (82%) Ph Ph Ph 179 178 177 NaOMe, MeOH CH2CI2 -30°C (93%) (1 )C F 3C 0 2H, c h 2c i 2 ...OBn Li0H-H20, MeO (2) ZCI, NaHC03 THF/H20 ...OBn ..OBn BocN. MeO (88%, 2 steps) (98%) 180 182 Scheme 39. Synthetic route to hydroxypiperazic derivative 182 Acid 182 was converted to compound 183 using f-butylcarbazate in the presence of DCC and HOBt (Scheme 40). Subsequent coupling of acyl hydrazine 183 with Fmoc-A/-methyl- D-alanoyl chloride 184 in benzene at 80 °C using silver cyanide as a promoter proceeded in 92% yield. After cleavage of the Fmoc group under standard conditions, the [2+2] coupling was conducted at -20 °C using BOPCI as a coupling reagent and collidine as a base. Tetrapeptide 185 was obtained as a white foam in 68% yield over 2 steps. The structure of compound 185 was confirmed by FAB HRMS analysis which showed a (M+Na)+ peak at m/e 1237.52395 (Calcd for C67H74N8NaOi4 (M+Na)+ 1237.52219). The 1H NMR and 13C NMR spectra displayed very broad peaks due to the presence of rotamers. Eventually cleavage of the Fmoc group provided compound 186 in 99% yield. 55 Mes ,Fmoc B ocNHNH 2 AgCN, PhMe,70°C N >.OBn DCC, HOBt (1) Et2NH, MeCN NZ \ ,NZ (2) Collidine, -20°C, 9Bny—NFmoc then BOPCI Me, Me, Et2NH, MeCN ( ^NZ ? Bry — NFmoc (99%) Me,, Me' Me‘ J O warm to rt overnight ZN NHNHBoc ZN NHNHBoc u u n (75%, 2 steps) 64 OBn OBn 186 185 Scheme 40. Formation of tetrapeptide 186 Coupling of the deprotected tetrapeptide 186 with acid chloride 66 was performed using silver cyanide as shown in Scheme 41. Trocs NH Cl NZ Me NZ NHTroc ( p Bo n \_ N'H )B n \— Nh 0 yO / X ^ 186 187 Scheme 41. Formation of hexapeptide 187 and attempt of macrolactamisation TLC analysis of the crude mixture showed the formation of 2 compounds. After separation by silica gel flash chromatography, two compounds were isolated in 43% and 48% yield respectively. FAB HRMS analysis showed a (M+Na)+ peak at m/e 1609.59520 and a (M+Na)+ peak at m/e 1609.59929. These two compounds showed a (M+Na)+ peak that matched the theoretical mass of hexapeptide 186 (Calcd (M+Na)+for CyyClaHgyN^NaC^o : 1609.58436). 56 It can be speculated that the [4 + 2] condensation gave rise to diastereoisomers; some epimerisation had occured. Nevertheless, we decided to carry out the 3 steps macrolactamisation procedure on both compounds separately. For both reactions, TLC analysis showed the formation of at least five compounds, and no pure product could be isolated from the reaction mixtures. Unfortunately hexapeptide 186 was all used up in this last reaction. Therefore, the macrolactamisation could not be repeated. 4. Conclusion Our initial aim of this work was to achieve the total synthesis of two new cyclodepsipeptide rings; the proline analogue cyclodepsipeptide of GE3 13 and the (3S,5S)-5-hydroxypiperazic acid analogue cyclodepsipeptide of A83586C 14. These syntheses were based on a [2+2+2] coupling of three dipeptides (Scheme 42). Dipeptide 64 and depsipeptide 66 were synthesised using procedures previously developed in our lab and large amount (40g) of protected (2S,3S)-3-hydroxyleucine 109, precursor of 66 were prepared. Furthermore, intermediates 155 and 156 were successly prepared. Intermediate 155 was prepared in 8 steps and 36% overall yield from L-proline and D-leucine. Intermediate 156 was synthesised in 24 steps and <0.04% yield from commercially available starting materials. 20 g of protected (3S,5S)-5-hydroxypiperazic acid 182 were prepared. 57 O z NHTroc ? Bn> — NFmoc ciy Trocv Me, Me V < :: 0 ,0 AIIO X . . ° •« Me Me O OH Me O OH Me F m o c ^ ^ e BocHN HN Me OBn 109 Me Me' Me XI 66 NHNHBoc 13 O" 166 NHTroc ( PZ Cl Me NFmoc Me Me-, 1 ° CT'OH BocHN E L s P ^ Mes ,Fmoc ®Bn Me 64 N 66 - o K Me -N ..1 ,,CBn HO ZN Y NHNHBoc HO 14 OBn 182 156 Scheme 42. Cyclodepsipeptide 13 was prepared but it could not be purified. Further investigation would have been needed to confirm the formation of 13 and develop a procedure to access the pure form of 13. Furthermore, degradation occured at the macrolactamisation step of the synthesis of cyclodepsipeptide 14. Again further work would have been required to find a way to close the cyclodepsipeptide ring. However, we learnt at that time that Novartis no longer wished to test the proposed analogues. We thus decided that it was not worth starting again the multistep synthesis of some precursors that were missing to pursue the investigation. We therefore changed programme and decided to start investigating the total synthesis of (+)-allopumiliotoxin 339A. 58 PART B: SYNTHETIC STUDIES TOWARDS THE SYNTHESIS OF (+)-ALLOPUMILIOTOXIN 339A 5. Introduction 5.1. Isolation of the Pumiliotoxin A and the Allopumiliotoxin alkaloids A wide range of lipophilic alkaloids are present in the skin of amphibians.42 The pumiliotoxin A and allopumiliotoxin classes of alkaloids are a major group of alkaloids of general structure 187 (Figure 10). The pumiliotoxin A alkaloids have R, = R2 = H whereas the allopumiliotoxins have a 7-hydroxy substituent on the indolizidine ring (R! or R2 = OH). The allopumiliotoxins are the most complex members of this class of alkaloids and are present in Dendrobatid frogs in a small quantity. pumiliotoxin A alkaloids : = R 2 = H allopumiliotoxin alkaloids : R 1 = H, R2 = OH or R, = O H ,R 2 = H Me OH Figure 10. General structure of the pumiliotoxin family of alkaloids The pumiliotoxin alkaloids were originally believed to be unique to Dendrobatid frogs, however they are now known to be present in virtually all anurans that are chemically defended by the presence of lipophilic alkaloids.43'44 The pumiliotoxins appear to be derived from dietary sources45, such as ants and arthropods. Recently the pumiliotoxins 307A and 323A were detected in extracts of formicine ants46, other pumiliotoxins (237A and 251D) were found in oribatid mites.47 Furthermore, it has been recently shown that dendrobatid frogs have an 59 enzyme, pumiliotoxin 7-hydroxylase that can convert a dietary pumiliotoxin to a more toxic allopumiliotoxin.48 In 1967, pumilotoxin A (327A) and pumiliotoxin B (323A) were the first molecules of the family to be isolated by Daly and co-workers, from the brightly colored Panamanian poison frog Dendrobates pumilio49 The structures of these pumiliotoxins remained unknown until 1980 when X-ray analysis of the crystalline hydrochloride salt of pumiliotoxin 251D established the structure and absolute configuration of this alkaloid.50 Analysis by mass spectroscopy and NMR allowed the elucidation of the structures of other members of the family.50,51 Figure 11 shows the structure of some pumiliotoxin alkaloids. Allopumiliotoxin 339A 191 was isolated, along with its isomer, allopumiliotoxin 339B, in skin extracts of Dendrobates auratus in 1984.51 Allopumiliotoxin 339A differs from 339B in the stereochemistry of the hydroxyl group at C(7). Allopumiliotoxin 339A and 339B had been first detected in 1978 and had been incorrectly classified as the alkaloid 395, due to the fact that they were converted to a dimethylsilanate during gas chromatography analysis.52 They represent minor alkaloids in these Dendrobatid frogs. The allopumiliotoxins 339 are the hydroxyl congeners of pumiliotoxin B. Me q h Me q h Me OH Me OH Me OH Me OH Pumiliotoxin A 188 Pumiliotoxin B 189 Pumiliotoxin 251D 190 Allopumiliotoxin 339A 191 Figure 11 : Structure of some pumiliotoxin alkaloids 5.2. Biological Properties of the Pumiliotoxins The dendrobatids frogs are notoriously toxic; the native people of Western Columbia use skin secretions from three species of these frogs to poison the darts used in hunting. The pumiliotoxin family of alkaloids exhibit potent cardiotonic and myotonic activities. 60 5.2.1. Ion Channels and the Electrical Properties of Membranes Ion channels are proteins that form hydrophilic pores across plasma membranes of cells (Figure 12). Most of the ion channels exist in either an open or a closed conformation, and are said to be gated. The main types of stimuli that are known to cause ion channels to open are the binding of a ligand (ligand-gated channels) or a change of the voltage across the membrane (voltage-gated channels). Na+ outside Cell membrane inside Figure 12. Voltage gated sodium channels A voltage gradient, also called an electric potential exists across the plasma membrane of all cells, the inside of the cell is negative relative to the outside. This potential across the membrane at rest is called the resting potential and is between -30mV and -70 mV. When the cell is at rest, anions give it a negative charge, with sodium ions outside and potassium ions inside the cell. In most animal cells, this potential does not vary with time. However, neurons and muscles cells, which are electrically active cells, undergo changes in their membrane potential. When changes occur in the membranes, the voltage-gated sodium channels respond to allow sodium ions to enter thereby, balancing the charge. When this occurs the membrane potential becomes less negative and more sodium channels open, causing an even greater influx of sodium ions. When the membrane potential reaches around +30 mV, the gates of the sodium channels close and the voltage-gated potassium channels begin to open. The sodium channel remains unactivated for a few milliseconds, such that no further sodium ions can enter the cell from that gate. 61 This occurs at one little segment of the axon at a time: sodium ions go in at section one; which triggers the efflux of potassium ions to start at section one and the influx of sodium ions at section two. That, in turn, triggers potassium ions to exit at section two and sodium ions to enter at section three; and so on along the membrane (Figure 13). More Sodium Channels Open Sodium Channels Close Sodium Channels Potassium Channels Open Open Potassium Channels Close ♦30 mV 0 mV -70 mV Time (ms) 1 ms Figure 13. An action potential An action potential results from the sequential opening and closing of voltage-gated cation channels and propagates along the cell. In conclusion, voltage-gated sodium channels are responsible for the generation and conduction of action potentials along the membrane and a transmission of a signal from one end of the cell to the other. 5.2.2. Phosphoinositide Breakdown A variety of natural products that can affect voltage-dependent sodium channels were found to stimulate the phosphoinositide breakdown in brain synaptoneurosomes. Daly et al. have shown that modulation of sodium ion or sodium channel causes phosphatidylinositol turnover. However, the mechanism involved in this process is still unclear.53 Phosphatidylinositol is a class of phospholipid, made up of glycerol, fatty acids and a hexahydric alcohol, inositol (Figure 14). 62 o Phosphatidylinositol Figure 14. Phosphatidylinositol (PI) Phosphatidylinositol is especially abundant in brain tissue, where it can amount to 10% of the phospholipids, but it is present in all tissues and cell types. Phosphatidylinositol is phosphorylated by a number of different kinases that place the phosphate moiety mainly on positions 4 and 5 of the inositol ring (PIP2). Cleavage of phosphatidylinositol phosphates leads to generation of secondary messengers, 1,2-diacylglycerol (DAG), a lipophilic molecule that remains linked to the membrane, and free phosphorylated inositols (IP3), which can diffuse into the cytosol (Scheme 43). Diacyglycerols regulate the activity of a group of at least a dozen related phosphorylating enzymes known as protein kinase C which, in turn, control many key cellular functions, including differentiation, proliferation, metabolism and apoptosis. The various inositol phosphates appear to be involved in the control of cellular events in very specific ways; for example, inositol 1,4,5-trisphosphate is an important cellular messenger stimulating calcium ion release from the endoplasmic reticulum. o Phosphatidylinositol 4,5-bisphosphate (PIP2) O O 1,2-diacylglycerol (DAG) Inositol 1,4,5-triphosphate (IP3) Scheme 43. Phosphoinositide breakdown 63 5.2.3. Biological Activity of Pumiliotoxin Family of Alkaloids Pumiliotoxin B (PTX-B) and some of its congeners have marked myotonic and cardiotonic activities. In nerve-striated muscle preparations, PTX-B increases direct and indirect elicited twitch. In guinea pig ileum segments, PTX-B caused rhythmic contractures which were suppressed by tetrodotoxin, a specific antagonist of the sodium potential channel.54 This activity is due to interaction of PTX-B with a site on the voltage dependant sodium channel in synaptoneurosomes.55 It results in a delay in inactivation of the sodium channel and repetitive firing in nerve and muscle. The resultant influx of sodium will cause activation of phosphatidylinositols thereby generating two second messengers, the inositols phosphates (which can mobilize calcium ions) and the diacylglycerols (which activate protein kinase C).53 In neuroblastoma cells, PTX-B and active congeners had no effect on sodium flux unless synergized by a scorpion venom.56 The pumiliotoxin binding site on voltage-dependant sodium channel appears to be allosterically coupled to the scorpion venom site.55 Biological activity of various pumiliotoxins was initially evaluated by measuring the force and the rate of spontaneous contractions in guinea pig atrial preparations,57 and later, the phosphoinositide breakdown and the sodium influx in synaptoneurosomes.58,59 5.2.4. Structure-Activity Relationship The structures of the pumiliotoxin alkaloids discussed are drawn in Figure 15 64 Me OH Me OH Me OH Pumiliotoxin A 188 Pumiliotoxin B 189 Pumiliotoxin 251D 190 15 Me X ^ O H ^ " ' O H OH y c ^ o w Me OH Me OH Me OH Me OH Allopumiliotoxin 339A 191 Allopumiliotoxin 339B 192 Allopumiliotoxin 323B' 193 Alopumilbtoxin 267A 194 Figure 15. Members of the pumiliotoxin family of alkaloids Pumiliotoxin B 189 is the most potent and efficacious of the compounds tested with respect to its chronotropic (rate) and inotropic (force) effects in guinea pig atrial preparations. It causes at 6 pM a 3 fold increase in force and a 2-fold increase in the rate of contractions. The nature of the side chain is critical for the biological activities of pumiliotoxins. In pumiliotoxin A 188, the lack of the side chain C16-hydroxy group results in a reduction in activity. The absence of both C15 and C16 hydroxy groups yields cardiodepressant compounds. However, in allopumiliotoxins, the number of hydroxy groups on the side chain does not change the activity. Allopumiliotoxin 323B’ 193, the allo(C-7-hydroxy)pumiliotoxin of pumiliotoxin A, is as active as allopumiliotoxin 339A 191. In conclusion, at least three hydroxyl groups appear to be required for high cardiotonic activity. The 7-hydroxy group on the indolizidine ring must also be axial for high cardiotonic activity; allopumiliotoxin 339B 192 is significantly less active than allopumiliotoxin 339A 191 and pumiliotoxin B 189. Allopumiliotoxin 339A is as potent as PTX-B, with respect to rate of contractions (150% of control at 6 pM) but less efficacious with respect to the force (160% of control at 6 pM).57,58 The structure-activity profile for stimulation of sodium flux and phosphoinositide breakdown by pumiliotoxins is very similar to the cardiotonic activity.56 Allopumiliotoxin 339A is the only alkaloid of the pumiliotoxin family to be more effective than pumiliotoxin B in stimulating 65 sodium influx (120% of response to PTX-B) and phosphoinositide breakdown (110% of response to PTX-B). Indeed, in guinea pig cerebral cortical synaptoneurosomes, the percent of control of phosphoinositide breakdown is 280% for PTX-B at 10 pM compared to 304% for Allo- PTX 339A at the same concentration.58 5.3. Past syntheses of Some Members of the Pumiliotoxin Family of Alkaloids 5.3.1. First Synthesis of a Pumiliotoxin A Alkaloid The first total synthesis of a pumiliotoxin A alkaloid was reported by Overman and Bell in 1981.60 They achieved an enantiospecific synthesis of pumiliotoxin 251D 190 using an iminium ion-vinylsilane cyclisation to stereospecifically assemble the (Z)-6-alkylideneindolizidine ring system (Scheme 44). The cyclisation precursor 195 would be obtained from the L-proline- derived epoxide 196 and the vinyl nucleophile 197. N C 02Bn MesSi- / — n-Bu H ' "O m' Me Me OH 197 Me 196 (+)-pumiliotoxin 251D 190 195 \y (I L-proline Me3S r n-Bu 198 Scheme 44. Overman’s first retrosynthetic plan to pumiliotoxin 251D A non-stereoselective route was developed to access epoxide 196 from L-proline. Reaction of A/-carbobenzyloxy-L-proline methyl ester 199 with MeMgl, followed by dehydration of the resulting tertiary alcohol with thionyl chloride afforded the propenyl derivative 200 in 54% yield. Epoxidation with m-CPBA gave in quantitative yield a 1:1 mixture of epoxides 196 and 201 that could be easily separated on a large scale (Scheme 45). 66 N- C 0 2Bn ( 1 ) MeMgl (2.1 eq) ^ N <-C02Bn m. CPBA A - N' c ° 2 Bn ^ N.C 0 2Bn CK o 2M. <2>s° ^ r ,5'c ’ M - r * H iSiA) « ;e H J.0 H =eo 199 200 196 201 Scheme 45. Preparation of epoxides 196 and 201 Elaboration of the side chain of (+)-pumiliotoxin 251D started with the reduction of 1- heptyne-3-one with B-3-pinanyl-9-borabicyclo[3.3.1]nonane into (S)-1-heptyn-3-ol 202. Conversion into silyl carbonate 203 followed by organocuprate coupling provided stereospecifically the (R)-silyl akyne 198 in 56% yield from alcohol 202. After treatment of alkyne 198 with DIBAL-H, the resulting vinylalanate 204 was heated at reflux with epoxide 196 to afford bicyclic carbonate 205 in 38% yield. Compound 205 was hydrolysed into the aminoalcohol, which was directly converted to the cyclopentaoxazolidine 206 with paraformaldehyde. The cyclisation was achieved by heating 206 in ethanol in the presence of camphorsulfonic acid (Scheme 46). Me 0 C0 2 >s / /7 -Bu Me,,. n-Bu M e-,,^n-Bu H O v^n-B u (1) MeLi, TMSCI MeMgBr DIBAL-H M e 3 Si- (2) MeOCOCI Cul (80%) MeLi (70%) SiMe3 SiMe3 AI/-Bu2Me 202 204 203 198 C 0 2Bn EtOH reflux (38%) H ) ' ''O 196 Me Me. SiMe: 77-Bu CSA, EtOH (60% from 205) ,0 SiMe 3 Me reflux H ~ n-Bu Me OH Me 205 X = O (1) 3M KOH, reflux (2) (CH2 0)„, EtOH (+)-pumiliotoxin 251D 190 207 206 X (80%) Scheme 46. Overman’s route to (+)-pumiliotoxin 251D 190 This first total synthesis of a pumiliotoxin A alkaloid proceeded in 13% yield from (S)-1-heptyn-3-ol and 4.7% yield from A/-carbobenzyloxy-L-proline methyl ester. Iminium ion- 67 vinylsilane cyclisation was developed in this synthesis. This methodology was used in the total syntheses of pumiliotoxin B 189 in 198461 and pumiliotoxin A 188 in 1985.62 The synthesis of pumiliotoxin B 189 is summarised in Scheme 47. At the time this retrosynthetic plan was designed, the stereochemistry of the allylic diol of (+)-pumiliotoxin B 189 was still unknown. The strategy adopted allowed access to any of the four possible diastereoisomers. Iminium ion-vinylsilane cyclisation was used to obtain alkylideneindolizidine aldehyde 209 which was condensed with stabilised ylide 210 to provide (+)-pumiliotoxin B 189. e QSiPh 2 f-Bu C 0 2Bn (+)-Pumiliotoxin B 189 Scheme 47. Preparation of (+)-pumiliotoxin B 189 using iminium ion-vinylsilane cyclisation Using the same methodology, (+)-pumiliotoxin A was synthesised from epoxide 196 and vinylalanate of 211 (Scheme 48). C 0 2Bn Me3Si H 1 'O Me Me OH 196 (+)-pumiliotoxin A 188 Scheme 48. Preparation of (+)-pumiliotoxin A 188 using iminium ion-vinylsilane cyclisation In theses 3 syntheses, low yields were obtained in the epoxide-alanate coupling step requiring optimisation for each side chain nucleophile. A more efficient strategy had to be designed in order to synthesise larger amounts of the compounds necessary for investigation of the biological activities of the pumiliotoxin family of alkaloids. 6 8 5.3.2. Overman Aldol Attachment of the Alkylidene Side Chain: First Entry to an Allopumiliotoxin Alkaloid The first synthesis of an allopumiliotoxin A alkaloid was reported in 1984 by Overman and Goldstein.63,64 Their method was based on an aldol attachment of the alkylidene side chain 214 to indolizidinone 213 as illustrated in Scheme 49 and was used in the synthesis of allopumiliotoxin 267A and allopumiliotoxin 339B. Me-,, H . Me OH Me OH Me OH Allopumiliotoxins 213 214 Scheme 49. Overman Aldol Attachment of the Alkylidene Side Chain The synthesis of indolizidinone 213 started from A/-Boc-L-proline 215 which was first converted to ketone 216 in 2 steps (Scheme 50). After deprotection with TFA, the labile 2-acetopyrrolidine salt 217 was treated with 5 equivalent of 1-lithio-1-methoxyallene 218 in THF at -78 °C to afford the allenyl pyrrolidine 219 as a single diastereoisomer. Crude 219 was reacted with slightly less than one equivalent of p-toluenesulfonic acid in acetonitrile to provide the bicyclic enol ether 220 in 25-45% yield from compound 216. Conversion of enol ether 220 to the 7-indolizidinone 213 was then achieved in 76% yield by hydrolysis with 5% aqueous HCI. 69 X 218 (1) 2-pySH, DCC /"-nboc CF3 CQ2H Li OMe / NBoc CF 3 CO2' (2) Me2CuLi PhOMe OK 0 -78°C OMe M, c o 2h M- H, (70%) Y r ds > 97:3 Me OH ' Me Me 219 215 216 217 TsOH, CH3CN (25% - 45% from 216) Scheme 50. Synthesis of intermediate 213 The final steps of the synthesis of (+)-allopumiliotoxin 267A 194 are shown in Scheme 51. The best result for the aldol condensation was obtained with 2 equivalent of trityllithium in ether 0°C. The aldol adduct 222 was directly dehydrated with trifluoroacetic anhydride and DBU to yield compound 223, which was selectively reduced with Me4NBH(OAc)3 to afford (+)-allopumiliotoxin 267A 194 in 73% yield. n-Pr (CF3 C 0 )20 DBU Me 4 NBH(OAc) 3 Acetone-HOAc (73%), ds> 25 : 1 M e\ / Y Y ' Me __ Me OH (+)-allopumiSotoxin 267A 194 Scheme 51. Synthesis of (+)-allopumiliotoxin 267A 194 Similar chemistry was utilised for the preparation of (+)-allopumiliotoxin 339B 192 as illustrated in Scheme 52. Reduction of compound 226 under Luche conditions furnished only the equatorial alcohol 227 in 58% yield. Silylation of alcohol 227 followed by debenzylation with 70 sodium in ammonia and Swern oxidation afforded aldehyde 228. Wittig olefination of aldehyde 228 with enantiomerically pure ylide 210 provided the a’-silyloxy (£)-enone 229 in 54% yield. Threo-selective reduction with LiAIH4 was accompanied by desilylation to afford (+)-allopumiliotoxin 339B 192 in 44% yield. •OBn OBn 3 2 (1) Ph CLi, Et 0 , -78°C (CF 3 C 0)20 OH DBU Me OH Me OH Me OH 213 H 224 22S 226 NaBH 4 C6CI3 (58%) Me-/, Me-,, -OBn CHO (1) n-BuLi, HMPA f-BuMe2SiCI (2) Na, NH 3 OTBDMS (3) Swem ox. Me OH (23%) Me OH 227 228 Me QTBDPS (55%) Ph3P'i ]f^ ' Me O 210 e QTBDPS LiAIH* (44%) ''OTBDMS Me OH Me OH 229 (+)-allopumiliotoxin 339B 192 Scheme 52. Overman’s route to (+)-allopumiliotoxin 339B 192 The overall yields of these syntheses are quite low; (+)-allopumiliotoxin 267A was prepared in 9 steps and 5.6% overall yield from A/-Boc-L-proline and (+)-allopumiliotoxin 339B was prepared in 14 steps and an overall yield <1% from A/-Boc-L-proline. However, these syntheses allowed confirmation of the absolute configurations of these alkaloids. Moreover, this aldol dehydration strategy was also employed by Gallagher and coworkers in their synthesis of pumiliotoxin 251D.65 71 5.3.3. Total Syntheses Using Iodide-Promoted Iminium lon-Alkyne Cyclizations Overman et al. reported that alkynes can react intramolecularly with iminium ions in the presence of a nucleophile.66 This strategy, illustrated in scheme 53, was first utilized to prepare pumiliotoxin A in 198867 and has later been successful for the preparation of pumiliotoxin B68 and allopumiliotoxins alkaloids59,69 as well as many structural analogues.70 Scheme 53. Preparation of pumiliotoxins using iodide-promoted ion-alkyne cyclisation 5.3.3.1. Total synthesis of Pumiliotoxin B (+)-Pumiliotoxin B 189 was prepared using iodide-promoted iminium ion-alkyne cyclisation from epoxide 196 and alkyne 233 (Scheme 54). Me (+)-pumiliotoxin B 189 Scheme 54. Retrosynthetic analysis of (+)-pumiliotoxin B The synthesis of alkyne 233 is depicted in Scheme 55 and began with Evans allylation of propionyl oxazolidinone 234. Removal of the auxiliary with hydrogen peroxide and LiAIH4 reduction afforded (ft)-2-methyl-4-pentenol 236. Dibromide 237 was obtained after Swern oxidation of alcohol 236 followed by dibromomethylenation with PPh3 and CBr4. Wittig 72 olefination of aldehyde 238 with phosphorane 210 provided a-siloxyenone 239 which was selectively reduced with triisobutylaluminium. Deprotection of the silyl group afforded the syn diol 240 which was converted to the acetonide with 2,2-dimethoxypropane. Conversion of the dibromoalkene to the terminal alkyne 233 was achieved under standard conditions. The synthesis of alkyne 233 was achieved in 8 steps from pentanol 236 and 42% overall yield. ( 1 ) LHMDS (1) H2O2 , LiOH (1) Swern oxid. Me. 'V 1J M L 1 . 9 /N (2 ) c h 2 c h c h 2i 9 /N (2) UAIH4 (2)P Ph 3 ,CBr4‘r / — < (85%) ) — < Me (100%) Me (67%) Br Me Ph Me Ph Me 237 234 235 236 Me OTBDPS O s04, NMO N al0 4 Ph3 P ^ fT^ Me (94%) Me QH e QTBDPS jj (1)(7-Bu)aAI Br 210 Br Me (2) TBAF CHO CH2 CI2, 40 (1) Me 2 C(OMe )2 p-TsOH (2) n-BuLi, NH4 CI (79%) 233 Me Scheme 55. Synthesis of the side chain segment of pumiliotoxin B Coupling of pyrrolidine epoxide 196 with 2 equivalents of the diethylaluminium derivative of alkyne 233 proceeded efficiently to afford intermediate 241 in 95% yield (Scheme 56). In contrast to the vinylsilane route, the unreacted alkyne 233 was easily recovered allowing this key coupling step to proceed in excellent yield with net use of stoichiometric amounts of the coupling partners. The same conditions were used in the synthesis of pumiliotoxin A and analogues. Hydrolytic removal of the carbamate group with Ba(OH)2 provided alkynylamine 242 in 73% yield. After optimisation, the iodide-promoted cyclisation was performed with 1.5 equiv of PPTS which resulted in both cyclisation and isopropylidene cleavage to afford compound 243 as a single isomer in 65% yield. Deiodination of 243 followed by protonolysis provided (+)- pumiliotoxin B 189 in 89% yield. 73 Me Me-,, (1) n-BuLi, EtjAICI ) Nal, (CH 2 0)n Me PPTS, H20 M e ______z__, OH NR Me 100"C, (6 8 %) Me ■C02Bn 233 Me Me 196 (0.5 eq) Me OH Me OH 243 241 R = Z Me Toluene-hexane, O'C 242 R = H Me OH Me OH (+)-Pumiliotoxin B 189 Scheme 56. Preparation of (+)-pumiliotoxin B using iodide-promoted ion-alkyne cyclisation This synthesis allowed preparation of 500 mg of pumiliotoxin B for biological and structural investigations. The overall yield was 8% from /V-carbobenzyloxy-L-proline and 10% from (4S, 5S)-4-methyl-5-phenyl-2-oxazolidinone, the commercially available precursor of acyloxazolidinone 234. 5.3.3.2. First Enantioselective Total Synthesis of (+)-Allopumiliotoxin 339A The first total synthesis of (+)-allopumiliotoxin 339A was reported in 1992 by Overman et al. The general approach is outlined in retrosynthetic format in Scheme 57 and involves the coupling of the proline-derived aldehyde 245 with the terminal alkyne 233.69 The key issue of the synthesis was the viability of the pivotal nucleophile-promoted iminium ion-alkyne cyclisation step with a substrate that contained a potentially labile and inductively deactivating C(7) allylic hydroxyl group. This was achieved using the electron-withdrawing cyanomethyl protecting group which would disfavour competitive chelation with the pyrrolidine nitrogen during the carbonyl addition step. 74 Me OH (+)-allopumiliotoxin 339A 191 Scheme 57. First retrosynthetic plan to (+)-allopumiliotoxin 339A 191 Aldehyde 245 was obtained from propenyl derivative 246 through an efficient 9 step- sequence which is depicted in Scheme 58.71 lodocyclisation of 246 afforded a bicyclic iodo carbamate 247 which was converted to alcohol 248 after conversion into a nitrate ester and reduction. After protection of the alcohol, the cyclic carbamate was hydrolysed with KOH and the secondary amine was protected with a cyanomethyl group to give compound 249. Following protection of the tertiary alcohol with benzyl bromide, the primary alcohol was deprotected and oxidised under Swern conditions to afford aldehyde 245 in 45% overall yield from 246. / ' - N - C 0 2Bn (1) A gN 0 3 ,MeCN, 80°C ^.MeCNr (2) Zn, NH4 OAc, 0°C o l H Me <84% > (95%, 2 steps) H Me 246 247 248 (1) SEM-CI, /-Pr2NEt (2) KOH, EtOH-HzO, 80°C (3) ICH2 CN, Et3 N, THF (1) BnBr, KH, THF, rtto reflux (2) LiBF4, MeCN-HjO, 70°C CHO "OSEM (3) Swern oxidation Me OBn Me OH (59%, 6 steps) 245 249 Scheme 58. Synthesis of aldehyde 245 Addition of compound 250, the alkynyl lithium derivative of 233, to aldehyde 245 occured in 68% yield with 4:1 diastereoselectivity in favour of 251. A first iminium ion cyclisation by internal attack of oxygen nucleophile yielded cyclopentaoxazine 252. Then iodide-promoted cyclisation occured cleanly at 100 °C with loss of isopropylidene group to afford 75 alkylideneindolizidine 253 in 76% overall yield from 251. Deiodination followed by removal of the benzyl ether provided (+)-allopumiolliotoxin 339A 191 in 62% yield (Scheme 59). Li Me Me Me AgOTf 250 THF CN (68 %) (94%) ^ e OBn Me Me Me Me 251 252 Nal, (CH 2 0)n, CSA H2 0-acetone, 100°C (81%) OH OH Me (1) n-BuLi, MeOH Me (2) Li, NH3, -78°C OH OH (62%) H A 0H OH Me OH Me OBn (+)-allopumiiotoxin 339A 191 253 Scheme 59. First total synthesis of (+)-allopumiliotoxin 339A This first synthesis of (+)-allopumiolliotoxin 339A proceeded in 17 steps and 7.5% overall yield from A/-(benzyloxycarbonyl)-L-proline and 16 steps and 6% overall yield from the commercially available precursor of acyloxazolidinone 234. 5.3.4. Alternative Strategies for (+)-Allopumioliotoxin 339A Synthesis 5.3.4.1. Nozaki-Kishi cyclisation In 1992, Kibayashi and co-workers utilised an intramolecular Cr(ll)-mediated coupling reaction72,73 to build the indolizine framework of the allopumiliotoxin alkaloids.74,75 The strategy employed is summarized in Scheme 60, the cyclisation proceeded via an alkenylchromium (III) species 254. The cyclisation precursor 255 came from the combination of the protected pyrrolidine fragment 256 and allylic bromide 257. 76 Cr(lll) . . . OR' R OH CHO H • OBn ° Me I Me OH Me OH (+)-allopumiliotoxin 339A 254 255 256 257 191 Scheme 60. Kibayashi’s retrosynthetic analysis of allopumiliotoxins The synthesis of pyrrolidine 256 commenced with the preparation of ketone 259 with a thiol esterification-Grignard reaction of A/-Boc-L-proline 215 (Scheme 61). Deprotection of 259 with trifluoroacetic acid afforded the pyrrolidine trifluoroacetate salt, which was immediately treated with 3-lithio-1,3-dithiane to produce the tertiary alcohol 260 as a single diastereoisomer consistent with a Cram chelation-controlled transition state. The cyclic dithioacetal group of 260 was converted to the dimethyl acetal with methanol and Hg(CI04)2. Protection the amino group with a cyanomethyl group afforded compound 261. O-benzylation of the tertiary alcohol was then achieved using benzyl bromide and potassium hydride. Removal of the cyanomethyl group with AgN03 and subsequent A/-protection by the Z group afforded carbamate 262. Acetal hydrolysis and NaBH4 reduction of the resulting aldehyde proceeded in very good yield to provide alcohol 263. Silylation of alcohol 263 and hydrogenolytic removal of the Z group resulted in 256. Pyrrolidine 256 was prepared in 13 steps and 14 % yield from A/-Boc-L-proline. 258 a j o (1) PPh3) MeCN (1 )C F 3 C 0 2H N S -S N (1)H g(C I0 4 )2 3 H20 T reflux (98%) /-" N B o c CH2 CI2, rt MeOH, rt ( 6 8 %) /" " N OMe v f r Me (2) MeMgBr, (2) 1,3-dithiane (2) ICH2 CN, Et3N OMe / NBoc • i ■ THF, 0°C, (85%) H o o-BuLi, hexane/THF THF, rt (89%) H Me0H 215 v 4 „, -78°C (54%) C 0 2H 259 261 (1) BnBr, KH THF, reflux (2) AgN 03, EtOH, rt (3) ZCI, Et3 N, CH2 CI2 0°C to rt (1)TBDMSCI, imidazole (6 8 %) (1) 3N HCI, THF DMAP, CH2 CI2, rt (94%) (2) NaBH4, MeOH, rt / ^ N Z OMe OTBDMS (2) H2, 10% Pd-C . OH (97%) OMe »/'0H MeOH (85%) H 1 OBn ■ V\Q.OBn Me Me H Me' 256 263 262 Scheme 61. Synthesis of 256 77 The side chain segment was elaborated from the known D-4-deoxythreose derivative (Scheme 62). Aldehyde 264 was first subjected to Grignard reaction with MeMgBr followed by PCC oxidation to afford the methyl ketone 265 which was converted to the (E)-olefin 266 by Horner-Emmons condensation. The ester group was reduced to the alcohol that was transformed to the bromide 267 under standard conditions. Evans alkylation with propionyl oxazolidinone 268 was used to install the R stereogenic center of compound 269. The Evans auxiliary was cleaved with LiAIH4 and the resulting alcohol oxidised under Swern conditions to give aldehyde 270. Treatment of aldehyde 270 with CBr4 and PPh3 provided a dibromide, which was converted to the propargylic alcohol 271 by reaction with butyllithium and paraformaldehyde. Synthesis of 271 was performed in 10 steps and 30% overall yield from aldehyde 264. Palladium-catalysed hydrostannation of 266 furnished the (E)-2-(tributylstannyl)alkene 272 in 93% yield along with a minor amount (3.8%) of its regioisomer. lododestannylation gave the (E)-iodoalkene, which was then transformed to the desired allylic bromide 257 in 96% yield. e Me (1) DIBAL-H, CH 2 CI2 (1) MeMgBr ft Y® (/-Pr0) 2 P(0)CH 2 C 0 2Et hexane,-78X JU THF, O X NaH, benzene, rt EtO?C JC ------► Me n - c ------► Br P (2)CBr4 ,PPh 3 p c c ,c h 2c i2 (84%> Me' M® (73%) / Me T * Me CH 2 CI2 2 6 7 IMe Me Me (92%) Me 266 264 265 O N LDA, THF -78X to 0°C (83%) Me Me 268 (1) CBr4, PPh3 e Me CH2 CI2, OX OHC (1) LiAIH4, THF, O X (2) n-BuLi, THF (2) Swern oxidation Me (HCHO)n (84%) Me (77 %) 270 269 Bu3SnH PdCI2 (PPh3 ) 2 (93%) Me Me e Me (1) l2, CH 2 CI2 Bu3Sn . O (2) CBr4, PPh3 ° ' 7 C , 1 CH 2 CI2. 0»C M / Me (96%) 257 Scheme 62. Preparation of allylic bromide 257 78 The final route that led to (+)-allopumiliotoxin 339A is shown in scheme 63. Coupling of bromide 257 and amine 256 in the presence of the Hunig’s base provided compound 273 in 70% yield. Desilylation of 273 followed by Swern oxidation afforded aldehyde 274. Cyclisation was performed smoothly in 79% yield by treatment with nickel(ll)/chromium(ll) and with complete stereoselectivity to afford compound 275. Cleavage of the isopropylidene group followed by debenzylation gave (+)-allopumiliotoxin 339A 191 in 71% yield. This synthesis of (+)-allopumiliotoxin 339A was achieved in 19 steps and 4.5% yield from /V-(te/?-butoxycarbonyl)-L-proline and 24 steps and <4% yield from L-threonine. Me Me-,, Me Me + Me (1)TBAF, THF Me Me Me NH (70%) OTBDMS (2) Swern ox. Me OBn (81%) Co h o t b d m s 273 274 Me 266 CrCI2 (3 equiv.) N iC t (2.5 mol %) DMF, rt (79%) Me Me Me OH (1) 3N HCI, THF, rt Me Me (2) Li, NH3, THF,-78°C OH H A 0H (71%) Me OH Me OBn 276 (+)-allopumiliotoxin 339A 191 Scheme 63. Preparation of (+)-allopumiliotoxin 339A by Kibayashi et al. 5.3.4.2. Nickel Catalysed Synthesis In 1999, Montgomery and Tang reported a nickel catalyzed synthesis of (+)- allopumiliotoxin 339A as summarised in Scheme 64.76 Their synthesis involved a triethylsilane- promoted cyclisation of ynal 276. 79 Me Me Me Me OH r A OS EM OH Me OH Me Me OBn (+)-allopumiliotoxin 339A 191 276 277 278 Scheme 64. Montgomery and Tang’s retrosynthetic plan to (+)-allopumiliotoxin 339A Synthesis of the alkylideneindolizidine ring began with the coupling of compound 277 with bromide 278 in the presence of Hunig’s base in 92% yield (Scheme 65). Oxazolidinone 277 was prepared from L-proline methyl ester following Overman’s procedure71 as illustrated in Scheme 58 and propargyl bromide 278 by bromination of the corresponding alcohol 271 used in Kibayashi total synthesis of allopumiliotoxin 339A.75 Benzylation of the tertiary alcohol and desilylation of the primary alcohol led to alcohol 280 in 75% yield. Alcohol 280 was then oxidised under Swern conditions to yield the cyclisation substrate 281. Aldehyde 281 was then treated with triethylsilane (5 eq), Ni(COD)2 (0.2 eq) and tributylphosphine (0.8 equ) in THF at 0°C for 18 h to afford bicycle 282 as a single diastereoisomer in 93% yield. After removal of all the protecting groups, (+)-allopumiliotoxin 339A 191 was obtained in 64% yield. Me Me Me Me Me Me (1) KOH, EtOH 'Me O7S Me Me (1) BnBr, KH, THF Me (2) 278, /-Pr 2 NEt, THF Me Me 278 Me (2) TBAF, THF (74%, 2 steps) (75%) OSEM ‘OH Me OH Me OBn -OSEM 279 280 Me Swern Ox. 277 (89%) (1) HF.pyridine, THF Et3SiH (5 eq) (2) 3N HCI/THF Ni(COD) 2 Me OH (+)-allopumiiotoxin 339A 192 Scheme 65. Synthesis of (+)-allopumiliotoxin 339A by Montgomery et al. 80 It is noteworthy that this route also led to (+)-allopumiliotoxin 339B: after removal of the silyl group from compound 282, the alcohol was oxidised under Swern conditions. The resulting ketone 283 was stereoselectively reduced with CeCI3/NaBH4 in methanol in 95% yield. Deprotection with 3N HCI and then Li/NH3 afforded (+)-allopumiliotoxin 339B 192 (Scheme 66). Me-,, (1) HF .pyridine (1) CeCI 3 .7H 2 0, THF NaBH4, MeOH 0"/-Me Me (2) Swern Ox. Me (2) 3N HCI/THF (75%) (3) Li. NH3 ’OTES (71%) Me OBn Me OBn M eO H 282 283 (+)-allopumiliotoxin 339B 192 Scheme 66. Synthesis of (+)-allopumiliotoxin 339B by Montgomery et al. 5.4. Others Approaches to Pumiliotoxins and Allopumiliotoxins Indolizines Core 5.4.1. Trost’s Total Synthesis of Allopumiliotoxin 339B Trost and Scanlan reported a synthesis of allopumiliotoxin 339B using Pd(0) chemistry.77 A Pd(0)-mediated allylic alkylation of epoxide 284 was used to transfer the chirality from the indolizine ring to the side chain and a Pd(0)-mediated cyclisation of vinyl epoxide 286 was employed to forge the piperidine ring (Scheme 67). HA "'OH MeOH (+)-allopumiliotoxin 339B 192 Scheme 67. Trost’s retrosynthetic plan to (+)-allopumiliotoxin 339B 81 Once again, A/-Boc-L-proline was the starting material of choice as shown in Scheme 68. Cyclisation precursor 286 was generated by chelation-controlled addition of allyltitanium intermediate 287 to the unprotected 2-acetylpyrrolidine generated from ketone 216. S- Methylation and subsequent base treatment yielded epoxide 286. The best result for the cyclisation step was obtained using Rd2(dba)3.CHCI3 as the catalyst and compound 289 as the chiral ligand. Formation of epoxide 284 was achieved using CF3C03H. Palladium(0)-catalysed condensation of vinyl epoxide 284 and allyl sulfone 290 proceeded with a perfect chirality transfer from C(6) to C(11) using Pd2(dba)3.CHCI3 and dppf in the presence of water. Direct reductive desulfonylation of the crude product provided ketone 291 in 24% yield from epoxide 284. Reduction-desilylation of compound 291 yielded (+)-allopumiliotoxin 339B 192. The total synthesis was accomplished in 11 steps and <3% yield from A/-(terf-butoxycarbonyl)-L-proline. (1) CF 3 C 02H Me Me (1 )(C 0 C I) 2 287 rS /^NBoc MeNH(OMe).HCI /~"N B oc (/-PrQ 4 )Ti~- SEt CF. NH < 4 (2) MeMgBr M y O (60-81%) c o 2h (CF3 C 0)20 SEt (74%) (49-72%) Me HMe OH 286 215 216 288 1.5% Pd2 (dba) 3 .CHCI3 H2 0, THF, 65°C /fS (66-73%) 0 'p'0 (1) 5% Pd^dbak.CHCU 20% dppf, H20 289 QTBDPS PhS0 2 Me QTBDPS 290 Me CF3C 0 3H (2) Na(Hg) (24%) Me OH Me OH Me OH 284 285 LiAIH4 (68 %) H A " o h Me OH (+)-allopumiiotoxin 339B 192 Scheme 68. Trost’s total synthesis of (+)-allopumiliotoxin 339B 82 5.4.2. Holmes’ Total Synthesis of Allopumiliotoxin 323B’ Holmes and Tan reported the first synthesis of an allopumiliotoxin alkaloid that did not use a proline derivative as a starting material.78,79 Their strategy was based on an aldol condensation of aldehyde 292 with the potassium enolate of the indolizine core 213 (Scheme 69). The indolizine core 213 was synthesised from the isoxazolidine 293, which can be derived from an intramolecular [3 + 2] cycloaddition reaction of the (Z)-A/-alkenylnitrone 294. Me OH OBOM OH H Me OH Me OH 213 Allopumiliotoxin 323B' 193 TBDMS OBz 0 . + -. N QTBDMS BzO 293 294 Scheme 69. Retrosynthetic analysis to (+)-allopumiliotoxin 323B’ by Holmes et al. The synthesis began with (ft)-te/t-butyl-3-hydroxy-pent-4-enoate 295 which was obtained by enzymatic resolution with Amino PS lipase (Scheme 70). After silylation of the alcohol, the ester group was reduced with DIBAL-H into the aldehyde 296. Treatment with hydroxylamine gave oxime 297 which was subsequently subjected to a 2 steps reduction-condensation procedure to provide the (Z)-/V-alkenylnitrone 294. The intramolecular [3 + 2] cycloaddition reaction was carried out by heating a dilute solution of the crude nitrone 294 in toluene for 18 h at 70°C to give four oxazolidine cycloadducts. Isoxazolidine 293 was obtained in 32% yield and was separated by flash chromatography. 83 (1) TBDMSCI H2NOH.HCI HO. NaBH3CN HO. J JH ^ imidazole, CH2CI2 jj>TBDMS H2o/EtOH [j1 OTBDMS O* 299 TBDMSO c h 2 ci2 OBz BzO JV 301 8% OBz 293 32% 0.+ ^ N OTBDMS TBDMSO t b d m s o - ^ P n 294 -OBz 302 8% BzO 300 5% Scheme 70. Synthesis of oxazolidine 293 Hydrogenolysis of isoxazolidine 293 followed by Z protection of the resulting free amino group provided the hydoxymethyl Z-protected piperidine 304 in 90% yield (Scheme 71). The dehydration the primary alcohol to form exocyclic alkene 305 was achieved by formation of a selenide followed by oxidation with one equivalent of m-chloroperoxybenzoic acid. Ozonolysis of alkene 305 gave a ketone that was subjected to a diastereofacial selective nucleophilic addition with an excess MeMgBr. Concomitant removal of the benzoyl group occured at this stage of the synthesis and provided alcohol 306. Selective tosylation of the primary alcohol and removal of the benzyloxycarbonyl group triggered an intramolecular ring closure that yielded the indolizidine 307. Desilylation and Swern oxidation gave the desired indolizidone core 213 in 50% yield. (1) H2. MeOH (1) n-Bu3 P, THF TBDMS BzO. 1 0 % Pd/C ZN p-NOzPhSeCN BzO (2) ZCI, Et2 0, OTBDMS (2) rn-CPBA, CH 2 CI2 OTBDMS aq. NaH C 0 3 (64%) BzO (90%) OH 305 293 304 (1) 0 3, CH 2 CI2, PPh3 (2) MeMgBr, THF (80%) (1) TsCI, DMAP (1) TBAF, THF HO. Et3 N, CH 2 CI2 zr (2) Swern ox OTBDMS OTBDMS Me OH (50%) (2) 10% Pd/C Me OH Me OH NH4 +HC 02-, MeOH (93%) 307 306 Scheme 71. Holmes’s route to Overman intermediate 213 84 The synthesis of the side chain aldehyde 292 was achieved in 8 steps and 30% overall yield.79 The aldol condensation between aldehyde 292 and the indolizidone core 213 was carried out using KHMDS and a mixed solvent (HMPA/THF)(Scheme 72). Direct dehydration afforded 308 in 42% yield. Reduction using tetramethylammonium triacetoxyborohydride and a catalytic amount of acetic acid in acetone gave the anf/'-diol as the only observable product. Finally, removal of the BOM protecting group using lithium di-fert-butylbiphenyl provided (+)-allopumiliotoxin 323B’ 193. This synthesis was achieved in 20 steps and 1.7% overall yield from the /3-hydroxyester 295 and was significantly different from other previous syntheses by contructing the chiral azabicyclic core in avoidance of using L-proline or its derivative. (1) KHMDS, THF/HMPA Me Me Me (1) Me4NHH(OAc )3 Me 292 OBOM OBOM acetone, AcOH (2) DBU, TFAA. DMAP (2) LiDBB, THF, -78°C Me OH (78%) -50*C, CH2CI2 Me OH (42%) Me OH 213 308 (+)-allopumiliotoxin 323B' 193 Scheme 72. Total synthesis of (+)-allopumiliotoxin 323B’ by Holmes et al. 5.4.3. Comins’ Total Synthesis of Allopumilotoxin 267A More recently, Comins and co-workers devised a route to (+)-allopumiliotoxin 267A using an enantiopure dihydropyridone building block.80 Once again, an aldol condensation strategy was chosen and the synthesis of Overman’s intermediate 213 was the key issue of the synthesis (Scheme 73). The synthesis commenced by lithiation of pyridine 309 with mesityl lithium81 followed by treatment with methyl iodide to give pyridine 310. To a 1-acylpyridium salt, prepared in situ from pyridine 310, and (+)-TCC chloroformate, was added lithiated ethyl propiolate. An acidic work-up provided dihydropyridone 311 in 70% yield and 96% de. Catalytic hydrogenation of the triple bond afforded compound 312 and did not affect the enone system protected with the TIPS group. Removal of the chiral auxiliary with lithium methoxide resulted in cyclisation and provided indolizidinone 313 as an 8:1 mixture of diastereoisomers. This mixture 85 was subjected to an acetoxylation in a stereocontrolled manner using lead acetate in refluxing AcOH/m-hexafluoroxylene to give compound 314. Protodesilylation of 314 using formic acid afforded compound 315, which was reduced using K-Selectride followed by lithium aluminium hydride to afford diol 316 in 83% yield. Finally, oxidation under Swern conditions provided Overman’s intermediate 213. (1) R OCOCI ^ u OMir -TIPS (1) MesLi ^ ^ y T I P S (2) y -^ -C O ;E, RO.C- n ^ ' T I P S ^ P d C „ O ^ - ^ T I P S , (2) Mel r m H,n+ r-mn(100%) o/.^ Et02C‘ OMe (66%) OMe W H3 ° + (70%) E t02C Me Me 309 Me 310 311 312 R* = (+)-trans-2-(a-cumyl)cyclohexyl (+)-TCC LiOMe, MeOH (80%) Swern (1) K-Selectrine 9 Formic TIPS Pb(OAc)4 oxidation (2) LiAIH4 > 'OH (85%> Me OH Me OH Me OH Me OH 316 Scheme 73. Comins’ route to Overman intermediate 213 Overman’s intermediate 213 was converted to (+)-allopumiliotoxin 267A in 48% yield using a modified literature procedure (Scheme 74). 64 Me<„ ‘ Me Me4NBH(OAc)3 M e % / \ (1) Ph3CLi, THF, -78°C Acetone-HOAc ^ M e ,_ 2 Me (95%) / ^ n Me OH Me OH Me OH 213 (3) TFAA, DBU, DMAP 317 (+)-allopumJiotoxin 267A 194 one pot (51%) Scheme 74. Final steps to (+)-allopumiliotoxin 267A 194 8 6 5.4.4. Lin’s Approach to the Indolizidine Core of Pumiliotoxins and Allopumiliotoxins In 2003, Lin and co-workers developed a method to construct the chiral azabicyclic core without using proline and its homologue.82 As shown in retrosynthetic analysis depicted in Scheme 75, a common key intermediate was used to access both pumiliotoxins and allopumiliotoxins. Their strategy was based on an intramolecular aminolysis of epoxide. Pumiliotoxins Me OH 213 Allopumiliotoxins Scheme 75. Lin’s retrosynthetic analysis of pumiliotoxin alkaloids from 319 The synthesis started from a Sharpless asymmetric dihydroxylation of the trisubstituted olefin 320 that afforded diol 321 in 98% yield and 95% ee (Scheme 76). A monomesylation of the secondary alcohol followed by treatment with K2C03 yielded epoxide 322. After hydrogenolytic cleavage of the benzyl group, the alcohol was activated into mesylate 323. A stepwise substitution-ring-opening sequence took place gently after treatment of benzylamine in refluxing MeCN to provide the pyrrolidine derivative 324 as a single isomer. Protection of the tertiary alcohol, followed by selective removal of A/-benzyl and acetylation afforded acetamide 325 in 75% yield. A Claisen condensation was then successfully achieved using potassium hydride and gave the desired intermediate 319. 87 AD-mix-a , f-BuOH HO Me (1) MsCI, pyr, CH2CI2, 0°C H20 , MsNH2, 0*C (2) K2C 0 3, EtOH, rt BnO BnO C 0 2Et BnO C 0 2Et (98%) (95%) 322 320 H2, 10% Pd/C EtOH, MsCI Et3N, CH2CI2 (95%) (1) BnBr, KH,reflux 30 min BnNH2, MeCN (2) H2, 10% Pd/C, EtOH THF, rt reflux, 48 h / N^^Me C 0 2Et (3) AcjO, Et3N, CH2CI2 (97%) O (75%) Me OH e OBn HMe OBn 323 325 Scheme 76. Preparation of intermediate 319 Sodium borohydride reduction of ketone 319 furnished alcohol 326 that was converted to its mesylate and subjected to elimination under basic conditions to provide compound 327 in 90% yield (Scheme 77). The pumiliotoxin intermediate 318 was obtained after saturation of the double bond and debenzylation in quantitative yield. The allopumiliotoxin intermediate 213 was obtained after reduction with LiAIH4 of lactam 319, followed by benzyl deprotection and Swern oxidation. H2, 10% Pd/C NaBH4, MeOH EtOH 0°C, (100%) (2) DBU.tol, reflux (100%) (90%) LiAIH4, THF reflux, (75%) (2) Swern ox. (75%) Scheme 77. Preparation of the indolizidine core of pumiliotoxins and allopumiliotoxins from 319 8 8 5.4.5. Stevenson’s Rapid Synthesis of the Indolizidine Core of Pumiliotoxins and Allopumiliotoxins More recently, Stevenson et al. reported a rapid synthesis of the two key intermediates 318 and 213 in the synthesis of pumiliotoxins and allopumiliotoxins.83 Their retrosynthesis analysis is summarised in Scheme 78. Pumiliotoxins Allopumiliotoxins Scheme 78. Stevenson’s retrosynthetic analysis of pumiliotoxin alkaloids from alkene 329 These 2 key indolizidines were prepared from a common alkene precursor 329 whose synthesis is depicted in Scheme 79. The synthesis commenced from the known carbamate61 330 that was deprotected and coupled in situ with 3-butenoic acid 331 to afford alkene 332. A ring closing metathesis using second generation Grubbs’ catalyst provided alkene 329 in 88% yield. NBoc 2nd gen Grubbs' catalyst (88%) Scheme 79. Preparation of key intermediate 329 The synthesis of the pumiliotoxin intermediate 318 started with epoxidation of alkene 329 with m-CPBA. An in situ ring opening reaction using strongly basic ion-exchange resin Amberlite 89 IRA-400-(OH) afforded alkene 333 in 85% yield. Hydrogenation over a palladium catalyst provided the desired intermediate 318. To obtain the allopumiliotoxin intermediate 213, osmium tetroxide catalysed dihydroxylation was achieved on alkene 329. An acetylation was performed to isolate the polar water-soluble product 334. After reduction of amide 334 and Swern oxidation of alcohol 335, the key allopumiliotoxin intermediate was isolated in 34% yield from alkene 329. An X-ray structure of diol 335 confirmed the stereochemistry of the intermediate. (1) m-CPBA (2) Amberlite IRA-40O(OH) resin (100%) (85%) Me Me OH Me OH 329 333 318 Swern ox. (84%) OAc (78%) OH (44%) Me Me OH Me OH Me OH 329 334 335 213 Scheme 80. Synthesis of Overman’s intermediate by Stevenson et al. 6. Synthetic efforts towards (+)-Allopumiliotoxin 339A 6.1. Retrosynthetic Analysis of (+)-Allopumiliotoxin 339A There were two main aims guiding our retrosynthetic planning for (+)-allopumiliotoxin 339A. First, we wished to demonstrate the utility of the recently developed O-directed free radical hydrostannation of disubstituted acetylenes with Ph3SnH/Et3B/air in total synthesis. Secondly, we wished to develop a very flexible route capable of being readily modified to create novel analogues for future biological testing. 90 (+)-Allopumiliotoxin 339A would be synthesised from its protected form 275 as shows in Scheme 81. The last steps of the synthesis would involve acidic deprotection of the isopropylidene, and O-debenzylation with Li/ammonia as previously described.74 Indolizidine 275 would be prepared from ketone 336 via a hydroxyl directed reduction using Me4NBH(OAc)3 analogously to that used in the synthesis of (+)-allopumiliotoxin 267A (Scheme 74).64 Enone 336 would be constructed by an aldol condensation of ketone 213 with aldehyde 270 followed by dehydration; the geometry would arise from the desire to minimise steric repulsions in the product. Although, this aldol methodology has been widely used in the total synthesis of various pumiliotoxins and allopumiliotoxins, it has never been applied in the synthesis of (+)- allopumiliotoxin 339A. Intermediate 213 appeared derivable from enone 338 by a base-inducted intramolecular conjugate addition. Enone 338 would itself be prepared from a vinyl stannane addition to a-alkoxyaldehyde 339 followed by oxidation of the resulting alcohol and deprotection of the Boc-group. Aldehyde 270 would be made by removal of the auxiliary from intermediate 337 followed by reduction. Deprotection Selective reduction M6 QH \ Me OBn Me OBn 336 (+)-allopumiliotoxin 339A 191 276 eiriol condensation Vinyl stannane Intramolecular corrugate addition addition r\ar\ rntanf inn NTFAf^ 339 338 213 270 337 Scheme 81. Retrosynthetic plan for (+)-Allopumiliotoxin 339A 191 91 6.2. Synthetic Studies Towards a-Alkoxyaldehyde 339 6.2.1. First Generation Strategy for a-Alkoxyaldehyde 339 6.2.1.1. Retrosynthetic analysis Sharp!oss asymmetric Stabilised Wlttlg amlnohydroxylatlon Oleflnatlon 339 340 341 342 343 Scheme 82. Initial retrosynthetic plan for a-alkoxyaldehyde 339 Our approach to a-alkoxyaldehyde 339 would avoid the use of L-proline as a starting material, to allow for flexibility in ring substitution patterns and different ring sizes in analogues. a-Alkoxyaldehyde 339 appeared derivable from compound 340 via intramolecular SN2 cyclisation. According to Baldwin’s Rules, a 5 -exo-tet ring closure of this sort should be favourable. Reduction of the ester to the aldehyde would complete the synthesis. The key step to introduce the 2,3-syr?-aminoalcohol motif would be a regioselective Sharpless asymmetric aminohydroxylation (SAA) performed on alkene 341. The trisubstituted alkene would be prepared by a Wittig condensation between aldehyde 242 and the Marshall phosphorane 343, an ylide well known to favour this stereochemical outcome. 6.2.1.2. Attempted Implementation of the Sharpless Asymmetric Aminohydroxylation Strategy for a-Alkoxyaldehyde 339 The synthesis of a suitable precursor for the Sharpless asymmetric aminohydroxylation was first investigated; we explored a variety of procedures to form the monoprotected alcohol 345 from 1,4-butanediol 344. The use of silver oxide and potassium iodide described by Bouzide and Sauve was too expensive to be applied for the first step of our total synthesis.84 Choudary et al. have reported a monotosylation of 1,4-butanediol mediated by metal-exchanged 92 Montmorillonite clay catalyst.85 The preparation of the catalyst was not straightforward and thus we decided to use Ahlberg and Wu’s method.86 The tosylation was achieved in absence of solvent employing p-toluenesulfonyl chloride as tosylating agent in presence of DMAP (0.04 eq) and triethylamine (1.05 eq) in 1,4-butanediol (7.7 eq). The original authors found that the use of solvent favoured the formation of the ditosylate and recommended that the extractive work-up with dichloromethane be conducted as quickly as possible. Two spots appeared on TLC with anisaldehyde, the more polar one was the mono-protected alcohol 345 and the faster moving the diprotected alcohol. The product 345 could not be stored as it decomposed into the diprotected alcohol. Furthermore, the mono-protected alcohol 345 could not be isolated as it decomposed into the diprotected alcohol during purification by Si02 flash chromatography. As a consequence, the PCC oxidation was performed on the crude mixture in dichloromethane at room temperature. After a quick purification on a pad of silica gel, the unstable aldehyde 342 was reacted with carbethoxyethylidene triphenylphosphorane 343 in dichloromethane at room temperature overnight. In this Wittig olefination, the use of phosphorane 343 gave rise to the E-alkene selectively. Evaporation and purification by Si02 chromatography led to alkene 341 in 21% yield over 3 steps (Scheme 83). 343 Ph3f\\ /P TsCI. DMAP PCC(1eq) Me OEt ° H NEt3 rt 2h °H CH2CI2 , 2 h 0 CH2 CI2, rt 20h Ts° x ^ ' v^ ' 5^ r ^ ^ O E t (21%, 3 steps) 1,4-butanediol 344 345 342 3 4 1 Scheme 83. Synthesis of alkene 341 Evidence for the formation of this alkene was given by the presence of a tq at 5 6.54 ppm on the 500 MHz spectrum of 341 in CDCI3. The chemical shift of an alkene proton is influenced by other alkene substituents and can be predicted using the formula 5 = 5.2 + Zgem + Zeis + Ztrans.87 In our case, we should have 6 6.6 ppm for the E-alkene and 6 6.3 ppm for the Z- alkene. Our chemical shift of 5 6.54 ppm is in favour of the E-isomer. The low yield obtained for the synthesis of alkene 341 led us to develop a new intermediate for the Sharpless asymmetric aminohydroxylation. It was thought that the p-toluenesulfonyl leaving group might be beneficially replaced by a bromide, and the synthesis 93 of bromide 348 was therefore investigated. The developed route, depicted in Scheme 84, commenced with the reduction of bromobutyronitrile 346 with DIBAL-H in ether at 0°C.88 This furnished aldehyde 347 after hydrolysis of the intermediary imine. Aldehyde 347 was volatile, and so the crude product was reacted directly with carboethoxyethylidene triphenylphosphorane 343 to afford olefin 348 as a yellow oil in 61% yield over 2 steps. Analysis by 500 MHz 1H NMR confirmed the formation of the E-alkene with the presence of a tq at 6.68 ppm. Again, the olefinic proton is deshielded because of the presence of the cis-carbonyl, the geometry is thus confirmed. 343 Ph3 Dibal in Hex V/ . . Mg ° E t Br Ether, O'C to rt wO CH r u2 nCI2, . rt,rt 2 onh 0 h OEt 61% over 2 steps Me Bromobutyronitrile 346 3 4 7 3 4 g Scheme 84. Synthesis of alkene 348 With precursors 341 and 348 in hand, we investigated the Sharpless asymmetric aminohydroxylation, which provides protected vicinal aminoalcohols enantioselectively in a single step from alkenes as illustrated in Scheme 85. ROCONH2 NiwrnnR om (DHQ)2PHAL NHCOOR o h K20 s 02(QH)4 ^ R i ^ \ ^ R 2 NaOH, /-BuOCI OH NHCOOR 349 R'0H/H20 3 5 0 3 5 1 Scheme 85. The Sharpless Asymmetric Aminohydroxylation The SAA was first reported in 1996 as a process in which the nitrogen source was the chloramine salt of a sulphonamide 352.89 Since then, various alternative nitrogen sources have been investigated and the sulfonamides can be replaced by alkyl carbamates 353 or by amides 354 (Figure 16). Therefore, a large number of aminoalcohol products can be obtained depending on which protecting group is desired. In each case, the reactive species is the alkali metal salt made in situ from the /V-halogenated compound derived from the sulfonamides, carbamates and amides. 94 CK qs? CK 1 BK 1 NR ^ N OR NR © © © © © © Na Na Li 352 353 364 Figure 16. The nitrogen sources used in the SAA The enantioselectivity of the reaction results from the presence of chiral ligands that favour addition to one enantiopic face of the alkene. The asymmetric induction follows the rules described for the Sharpless Asymmetric Dihydroxylation (AD);29 (DHQD)2PHAL directs addition to the /3-face and (DHQ)2PHAL to the cr-face (Figure 17). (DHQD)2PHAL (DHQ)2PHAL Figure 17. The Sharpless model for the asymmetric hydroxylation One of the new challenges of the SAA is the control of the regioselectivity which can be influenced by many factors. However, it has been found that, generally, the nitrogen prefers to add on the less substituted end of the alkene. In the case of a trisubstituted alkene, the steric hindrance at one end is so high compared to the other that the SAA usually only gives the less hindered amine product. Finally, K2Os02(OH)4 is generally used as a catalyst. Returning to our particular case, (DHQ)2PHAL would give rise to the desired enantioselectivity according to the AD mnemonic proposed by Sharpless depicted in Figure 17. 95 A carbamate-based nitrogen source was90 the more attractive nitrogen source in our system 341 or 348 as it would allow to fix directly the Boc group onto the nitrogen. The sodium salt of the A/-chlorocarbamate was made in situ from tert- butylcarbamate, tert- butylhypochlorite,91 and aqueous NaOH following Sharpless’ procedure.92 A quantitative investigation of the solvent revealed that a mixture of acetonitrile/water was the solvent of choice leading to a partial consumption of the starting material. No reaction occurred with alkenes 341 and 348 according to TLC analysis when r?-PrOH/water 2:1 was used, and the use of n-PrOH/water 1:1 led to degradation products. Difficulties were also encountered in removing unreacted tert -butylcarbamate and we did not manage to isolate any of the products that were formed. Nevertheless, we could see on TLC with anisaldehyde that at least 5 products had formed. Therefore, we decided to use benzylcarbamate as the nitrogen source in 1:1 MeCN/H20 in the hope that its separation from the desired compound might be easier. Again five new spots appeared on the TLC along with the starting material that was not consumed completely. An extractive work-up with sodium sulfite and EtOAc afforded a complex mixture of products which was purified using Si02 flash chromatography. Unfortunately, it was not possible to completely purify the main product from the excess benzylcarbamate. TLC analysis showed that the aminohydroxylation of alkenes 341 and 348 led to the same main compound, hinting that a cyclisation might have occurred in situ. In fact, four different compounds can arise from the Sharpless Asymmetric Aminohydroxylation step depending on the regioselectivity of both the aminohydroxylation and the ring closure. The formation of these four compounds is shown in Scheme 86. 96 1 OH BnO NCINa Br OEt OEt OEt 4% K2 0 s 0 2 (0 H ) 4 Me OH Me NHZ Me 5% (DHQ)2PHAL MeCN/H20 1:1 rt, 2 0 h 345 356 .NZ NHZ OH O OEt 'OEt OEt NHZ OH OEt NHZ 357 358 359 360 Scheme 86. Formation of the four possible products after the SAA To establish the structure of the major product, hoped to be 357, the Z group was hydrogenated with Pd(OH)2 in ethanol and the resulting free amine was protected with a dinitrophenyl group as shown in Scheme 87. .1 H2, Pd(OH) 2 BnO NCINa O NaHCO 3 , 2,4-DNPF N 0 2 OEt Me 4% K2 0s02(0H)4 Me OH OEt Et0H’ rt- 5 h OEt CH2 CI2, H2 0 , EtOH 5% (DHQ)2PHAL overnight rt, overnight OH OEt MeCN/H20 1:1 rt, 2 0 h 357 361 362 Scheme 87. Formation of the DNP-derivative 362 The four different compounds that could be obtained after the 2 step sequence described in Scheme 87 are depicted in Figure 18. 97 362 363 364 365 Figure 18. The peaks of the proton and carbon spectra of the 2,4-dinitrophenylated product were first assigned using 500 MHz 1H NMR, 13C NMR, COSY, HMQC and DEPT experiments in CDCI3. In fact, the CH close to the nitrogen was found much deshielded at 5 4.60 ppm. The use of COSY and HMQC experiments allowed attribution of the different CH2 peaks. The protons on C3 are obviously the more deshielded at 5 3.58 and 2.73 ppm. Furthermore, a 1H-1H coupling was visible on the 500 MHz cosy spectrum between the protons on C3 and the one on C2. The protons of C2 were thus detected at 5 2.05 and 1.71 ppm and the one on C1 at 6 2.20 ppm. Finally, analysis of the HMBC spectrum demonstrated that the cyclisation had given rise to a nitrogen-containing 5-membered ring (Figure 19). In the HMBC spectrum, a long-range 1H-13C coupling between the protons on C3 and the quaternary carbon of the DNP at 6 147.4 ppm was observed; hence compounds 363 and 364 could be ruled out. Additionally, there was a 1H-13C coupling between the hydrogen of the only CH of the ring with C3 at 6 62.3 ppm. A reciprocal 1H-13C coupling between the protons on C3 and the carbon of CH at 6 68.1 ppm was also observed. Thus structure 365 could be discarded. NMR studies confirmed the desired structure of 362 and as a consequence, the structure of compound 357. oh OEt Figure 19. The long-range 1H-13C coupling in 362 98 We were thus pleased to find that the formation of 357 occurred in one step. We now had to optimize the reaction using bromide 345 in order to increase the yield and solve the purification issues. The amount of catalyst K2Os02(OH)4 was increased from 5 to 8 mol% and the amount of ligand (DHQ)2PHAL from 6 to 10%. We were pleased to observe complete disappearance of the starting material according to TLC analysis. Again, a complex mixture of compounds was obtained and it was not possible to isolate all of them. Unfortunately, after purification on silica gel, the main blue spot visible on the TLC with anisaldehyde was found to be the tetrahydrofuran 366 arising from the cyclisation of the dihydroxylated product (Scheme 88 ). Br BnQX NCINa NZ O OEt + byproducts OEt Me 8% K20 s 0 2 (0 H )4 OEt 10%(DHQD)2PHAL MeCN/H20 1:1 345 rt, 2 0 h Scheme 88. The Sharpless Asymmetric Aminohydroxylation of alkene 366 Tetrahydrofuran 366 was contaminated with the excess benzylcarbamate and extensive purification by Si02 flash chromatography allowed isolation of a small amount of nearly pure tetrahydrofuran 366 in order to carry out analyses. Its structure was confirmed by its FAB HRMS which contained an (M+H)+ peak at m/e 189.11286 (Calcd (M+H)+ 189.11268 for C9H1704). Significantly, we increased the amounts of K20s 0 2(0H)4 and ligand in order to drive the reaction to completion, we instead favoured the formation of the dihydroxylated product which cyclised into tetrahydrofuran 366. The formation of the diol by-products during SAA reactions is a common issue. The main recommendations to avoid its formation are to decrease both the amount of water and of the osmium catalyst in the reaction mixture. The proportion of water was thus reduced to a 3:2 MeCN/water ratio. We could not reduce the proportion of water any further as the starting material remained untouched when a 2:1 MeCN/water mixture was used. To understand the role of water in the SAA, it is instructive to examine the proposed mechanism for the aminohydroxylation (Scheme 89). 99 high selectivity hydrolysis addition NHR OH o V R' R 370 primary cycle secondary cycle 367 R-N 369 addition hydrolysis R1 368 , , ^ r . SAD RHN OH low selectivity L = Ligand 372 373 Scheme 89. A proposed mechanism for the SAA This mechanism involves two catalytic cycles, each giving different selectivity pattern. In the primary cycle the alkene addition to the imidotrioosmium (VIII) species 367 is mediated by the ligand to give the osmium azaglycolate 368. Species 370 is then formed after re-oxidation of 368 by the nitrogen source 369. Azaglycolate 370 can then either be hydrolysed by water to afford the product with high selectivity or be attacked by the alkene and enter the secondary cycle. The limiting step in each cycle is the hydrolysis of the azaglycolate complexes by water. High water concentration is thus needed for the reaction to occur.93 Moreover, if the concentration of the osmium catalyst is too high, the alkene can react directly with the catalyst and undergo a ligand-mediated asymmetric dihydroxylation reaction, to form an osmium (VI) glycolate complex 372, analogous to the azaglycolate complex 368. Formation of the diol 373 will ensue. After much effort at optimizing the process, bromide 345 was used as the substrate and only 5% mol of the osmium catalyst was used. Furthermore, K20s 0 2(0H)4 was added at 0 °C, by small portions over 20 min. A quick purification by Si02 flash chromatography was achieved 100 to remove most by-products, and hydrogenation of the partially purified mixture in presence of Boc-anhydride94 led to a-alkoxyester 375. Decomposition of the excess benzylcarbamate 374 occurred under these reductive conditions and a-alkoxyester 375 could be purified successfully after Si02 flash chromatography and was obtained as a white foam in 15% yield from olefin 345 (Scheme 90). (DHQ)2PHAL ( 6 mol %) K2 0 s 0 2 (0 H ) 4 (5mol %) 9 BnOCONH2 (3.1 eq) ' Br + BnOCONH2 OEt 'OEt OEt NaOH (3 eq) Me Me OH Me OH 374 f-BuOCI (3 eq) 345 MeCN / H20 (3:2) 357 H2, 5% Pd/C B o c 20 NBoc 15% yield from 345 OEt Me OH Scheme 90. Improved conditions for the Sharpless Asymmetric Aminohydroxylation of 345 We finally developed a reproducible procedure for the synthesis of the a-alkoxyester 375. Even though the yield was quite low, this method did allow us to install 2 stereocenters, to carry out the cyclisation and to protect the nitrogen in just 2 steps. The difficulties we met are probably due to the fact that the substrate is a trisubstituted alkene. In fact, there are very few examples in the literature in which trisubstituted alkenes have been used as substrates for the SAA. Barboni et al. could not apply the SAA in their synthesis of Paclitaxel, and used a SAD instead.95 Clark et al. succeeded in 2001 using Chloramine-T as the nitrogen source.96 Wang et al. did not try to use the aminohydroxylation in their work towards the Pumiliotoxin synthesis and prepared a-alkoxyester 324 from alkene 320 in five steps (Scheme 76).82 We next investigated the protection of alcohol 375. Most of the conditions we tried left the alcohol untouched (Table 1, entry 2,4,5,6). 101 Table 1. Protection of alcohol 304 Entry Conditions Yield 1 NaH (1 eq), BnBr (1 eq), DMF, rt, 3 h Mixture 2 Benzyltrichloroacetimidate (1.2 eq), PPTS (0.1 eq), Et20, 50 °C,18 h No reaction 3 NaH (1 eq), BnBr (1 eq), n-Bu4NI, CH2CI2, rt By-products 4 MOMCI (2 eq), /-Pr2NEt (2 eq), THF, 80°C, 3 days No reaction 5 TBDPSCI (2 eq), imidazole (1.1 eq), DMF, rt, 24 h No reaction 6 TBDMSCI (2 eq), imidazole (1.1 eq), DMF, rt, 24 h No reaction Treatment of alcohol 375 with NaH and BnBr (entry 1) led to a complex mixture of unidentified compounds. However when n-Bu4NI was added to the reaction mixture (entry 3), we isolated an inseparable mixture of carbamates 376 and 377 as shown in Scheme 91. NBoc I l NaH, BnBr / N O n-(Bu)4 NI, CH2 CI2 ^ ^ C 0 2Et ^ = C 0 2Bn Me OH n Me H Me 375 376 377 Scheme 91. Protection of alcohol 375 The carbamate formation could be easily explained by nucleophilic addition of the alkoxide to the carbonyl group of the Boc as illustrated in Scheme 92. O NaH k~\0 ^ A C 0 2Et -o- CH. Me OH Me C 0 2Et H tie 0 0 * ' 375 378 376 Scheme 92. Formation of carbamate 376 102 Formation of the transesterification product was much more surprising as we are under basic conditions. Saponification of the ester might occur if the medium is not completely dry, the resulting carboxylate would then attack BnBr to form 377. I I 1 ^ ^ 'O H O-H 0 o 375 379 380 377 Scheme 93. Formation of carbamate 377 In light of the collective problems we experienced in this route, we eventually decided to investigate an alternative method for securing the a-alkoxyaldehyde. 6.2.2. New route for the synthesis of the a-alkoxyaldehyde In light of these difficulties, we then explored a completely new strategy to synthesise the targeted a-alkoxyaldehyde, and again, our goal was to avoid the use of L-proline. This strategy would install one stereocenter at a time as depicted in Scheme 94. Aldehyde 381 would originate from linear alkene 382 via a 3-step sequence. We would utilise the chiral induction of tert -butyl sulfinimine 383 to introduce the (C8a) stereocenter of 382 through an addition of an allyl magnesium halide. The sulfinyl group not only would play the role of a chiral auxiliary, but would then be used as a protecting group for the nitrogen. In fact, the sulfinyl group can be removed with TFA like a Boc protecting group. The C(8) stereocenter would be delivered at the first step of the synthesis using a Trost opening of epoxide 385. With due modification, these disconnections would potentially give us the flexibility to synthesise the a-alkoxyaldehyde with different substituents on the 5 membered-ring. 103 Hydroboratlon, m esylatlon Allyl magnesium halide Condensation with Trost opening and cycllsatlon addition tert-butanesulflnamlde of epoxide r-Bu s / A ? f-Bu ' NH 0 i —S f-Bu ♦ N ' y : 'OMe 8 a X 0Me OPMB OPMB OPMB 382 383 384 385 Scheme 94. New retrosynthetic plan to a-alkoxyaldehyde The synthesis started with a chemo-, regio- and enantioselective addition of p-methoxybenzylalcohol to the commercially available racemic isoprene oxide 385. This deracemization reaction, originally described by Trost and co-workers, was achieved using 0.01 equiv of Pd2(dba)3.CHCl3, 0.01 equiv of triethyl borate, 0.01 equiv of chiral ligand 386 and 1 equiv of PMB-OH97 (Scheme 95). Pd2 (dba) 3 CHCI3 Et3B, PMBOH Q, HN I* CH2 CI2, rt, 1 2 h (\ ph2p_\ = / 6 8 %, 82%ee d 385 384 386 Ligand (R,R)-L1 Scheme 95. The Trost opening of epoxide 385 Many factors come into play for the addition to be chemo-, regio- and enantioselective as summarised in Scheme 96. In the absence of Et3B, attack of a nucleophile would occur at the less hindered end of the TT-allylpalladium intermediate. The first role of the triethylborane is to control the regioselectivity. Secondly, Et3B enhances the nucleophilicity of the alcohol, which is normally a poor nucleophile, and finally, Et3B controls the chemoselectivity of the reaction. In fact, the product primary alcohol does not react after its formation. Finally, for the reaction to be enantioselective, the diastereomeric interconversion of the TT-allylpalladium intermediates must be faster than the alkoxide attack. This issue is controlled by the palladium catalyst and its ligand. 104 Scheme 96. Mechanism of Trost’s reaction In our case, the addition of p-methoxybenzylalcohol proceeded in 68% yield and 82% ee. The enantiomeric excess was determined from the 300 MHz 19F NMR analysis of the Mosher’s ester 391. Alcohol 384 was reacted with the Mosher acid (R)-(+)-MTPA in presence of DCC and DMAP in dichloromethane to afford ester 391 (Scheme 97). The diastereoisomeric excess was then easily calculated by integrating the signals from the trifluoromethyl group in the 19F NMR spectrum.98 (,Kj-<+)-MTPA DCC, DMAP 384 391 Scheme 97. Synthesis of the Mosher’s ester 391 Alcohol 384 was next oxidised under Swern conditions into the unstable aldehyde 392 (Scheme 98). After rapid purification by Si02 flash chromatography, the structure of aldehyde 392 was confirmed by its 500 MHz 1H NMR spectrum that showed a singlet at 6 9.49 ppm due to the aldehyde proton. On normal runs, aldehyde 392 was used crude and converted to the carboxylic acid 393 by Pinnick addition using sodium chlorite in the presence of NaH2P04 and 2-methyl-2-butene to act as a chlorine scavenger.99 Again, acid 393 was purified only for analytical purpose as it could be used crude for the next step. Evidence for the success of this reaction was given by the presence of a very broad OH band that overlapped with the CH band around 3250 cm'1 in the infra-red spectrum of acid 393. Furthermore, 500 MHz 1H NMR analysis in CDCb showed the disappearance of the aldehyde hydrogen previously at 5 9.49 ppm. Treatment of the crude acid with Mel and K20O3 in DMF afforded ester 394 which was purified by Si02 flash chromatography. The combined three-step sequence was easily implemented on a large scale since it only required purification at the last step, ester 394 being obtained pure in a very good yield (80%) over the 3 steps. Ozonolysis of the double bond led to aldehyde 395 in 92% yield, as evidenced by the presence of a singlet at 5 9.64 ppm in the 500 MHz 1H NMR spectrum of the isolated product. Aldehyde 395 was condensed in dichloromethane with chiral fR^-fe/t-butylsulfinamide 396 in presence of anhydrous copper sulfate to afford (7?J-terf-butanesulfinyl imine 383 in a very good yield.100 Copper sulfate is an excellent promoter of this reaction; it acts both as a Lewis acid and as a drying agent. Importantly, the work-up of this reaction was very easy; the reaction mixture was filtered through a pad of silica, the filtrate was concentrated in vacuo to afford the pure sulfinyl imine 383. The success of this conversion was apparent from the presence of the imine proton at 6 8.17 ppm in the 500 MHz 1H NMR spectrum of 383 in CDCI3. DMSO, (COCI) 2 NaCI02, NaHP0 4 NEt3, CH 2 CI2 f-BuOH, H20 'O -78° C, to rt Me' OPMB 2 -methyl- 2 -butene OH rt, 2 h Me OPMB 393 384 392 Mel, K 2 C 0 3 DMF, rt (80%, 3 steps) 396 Y i) 0 3, CH2 CI2 f-Bu^+ NH2 MeOH OMe f-Bu + N OMe O ' y . OMe C uS 04, THF Me OPMB Me OPMB Me OPMB ii) Me2S 4 days (97%) (92 %) 383 395 394 Scheme 98. Synthesis of sulfinylimine 383 Chiral (RJ-terf-butanesulfinamide 396 is commercially available. However, it is very expensive and can be prepared easily on a large scale following Ellman’s procedure101(Scheme 99). First, chiral ligand 399 is made by mixing 1 equivalent of (1S,2R)-(-)-cis-1-amino-2- indanol 397 with 1 equivalent of 3,5-di-tert-butyl salicylaldehyde 398 in EtOH. The ligand is obtained as a yellow solid after evaporation of EtOH. (RJ-fe/t-butanesulfinamide is then prepared in two steps from tert -butyl disulfide 400. A catalytic asymmetric oxidation of the tert -butyl disulfide 106 using hydrogen peroxide and catalytic amount of vanadium(IV) acetylacetonate in the presence of chiral ligand 399 affords the (S)-f-butyl-f-butanethiosulfinate 401. Hydrogen peroxide must be added in a very slow, steady stream at 0 °C. The course of the reaction was conveniently followed by 500 MHz 1H NMR. Following extraction with dichloromethane, crude 401 is added to a solution of lithium amide in liquid ammonia to afford ('RJ-terf-butanesulfinamide 396. We were able to conduct this procedure on 20 g scale. EtOH n 0 LiNHo, NH3, THF n X VO(acac)2, acetone v / Fe(N 0 3 )3 9 H20 W / x 0°C, 30% H 2 0 2 (aq) / \ ii) ice, CICH 2 C 0 2H / \ 3 9 9 (70% from 400) 400 401 396 Scheme 99. Synthesis of (7^-terf-butanesulfinamide 396 With imine 383 in hand, our initial plan was to synthesise the homoallylic sulfinamide 382 using the addition of an allylmagnesium halide. Despite this reaction being widely described in the literature,102 when we added allylmagnesium bromide to fe/t-butanesulfinylimine 383, a very low yield of 382 was obtained. After some literature searching, we found a very interesting paper from Foubelo and Yus who reported the indium-mediated addition of allyl bromides to fe/f-butanesulfinylimine.103 Their conditions were applied to our substrate and to our delight the indium-mediated addition of allyl bromide to sulfinyl imime 383 took place smoothly at 60°C, using 1.3 equiv of In and 1.3 equiv of AIIBr in THF. The homoallylic sulfinamide 382 was obtained in 85% after purification by Si02 flash chromatography. The diastereomeric ratio of the reaction was found to be 80:20, as determined by 500 MHz 1H NMR spectroscopy in CDCI3. It can be explained by a chair like transition state. The reactivity of the electrophilic centre of the imine is increased by coordination of the indium to nitrogen while coordination to the oxygen of the sulfinyl group is responsible for the face selectivity (Scheme 100). 107 383 382 402 Scheme 100. Indium-mediated addition of allyl bromide The diastereoisomers could not be separated at this stage and the hydroboration reaction was next investigated on the mixture enriched in olefin 382. The use of pinacol borane, catechol borane and 9 BBN was unsuccessful. We believe that this lack of reactivity is attributable to the steric hindrance of the (R)-A/-tert-butanesulfinamide group. Therefore, the reaction was achieved using the less hindered BH3-THF complex (Scheme 101). A complex mixture of compounds was formed, from which alcohol 403 was isolated in 40% yield for the two steps from sulfinyl imine 383. Evidence for the success of this reaction was provided by the appearance of an OH stretching band at 3333 cm'1 in the infra-red spectrum of alcohol 403. Moreover, 500 MHz 1H NMR analysis in CDCI3 showed disappearance of the olefinic protons at 5 5.71 and 4.97 ppm. O-Mesylation was then accomplished in a nearly quantitative yield using MsCI and NEt3. The structure of mesylate 404 was confirmed by the appearance of a singlet at 5 2.93 ppm in the 500 MHz 1H NMR spectrum of 404 in CDCI3. i) BH3-THF THF, 2h f-Bu MsCI, NEt3 ii) H2 0 2 ,Et0H OMe ^ ^ 2 ^ 2 . 99% MsO Buffer 7, 1h OMe Me OPMB (49%) Me OPMB Me OPMB 404 382 403 NaH imidazole THF, (70%) f-Buv / TEMPO ,S Dibal-H BAIB THF, rt CH2 CI2 (89%) OMe (50%) Me OPMB Me OPMB Me' OPMB 381 406 405 Scheme 101. Synthesis of a-alkoxyaldehyde 381 108 Cyclisation did occur in the presence of NaH/imidazole which permitted internal nucleophilic displacement in 72% yield. Proof of the cyclisation was provided by the 125 MHz 2D NMR-HMBC experiment in CDCI3 that showed the long range 1H-13C couplings (Figure 20). Specifically, a coupling was observed between carbon 3 and the proton a to the nitrogen. t-B ik / ° HMBC OMe OPMB 405 Figure 20. The long range 1H-13C couplings in 405 Finally, ester 405 was reduced with DIBAL-H to alcohol 406. Success was confirmed by the OH stretching band at 3391 cm'1 in its infra-red spectrum. Attempts to oxidise alcohol 405 into the desired aldehyde failed using PCC; TPAP/NMO gave an incomplete reaction according to TLC. Eventually, the oxidation was achieved using [bis(acetoxy)iodo]benzene (BAIB) in presence of catalytic amount of TEMPO;104 it provided the desired a-alkoxyaldehyde 381 in 50% yield. The oxidising agent is the oxaammonium salt 407 resulting from the dismutation of TEMPO 411 in the presence of acetic acid (Scheme 102). The catalytic amount of acetic acid needed for the dismutation to occur arises from a ligand exchange around the iodide atom. TEMPO 411 is regenerated from hydroxylamine 410 by action of BAIB. 109 Phl(OAc)2 + ROH Phl(OAc)(OR) + AcOH + OH O OH 407 408 409 410 AcOH Phl(OAc) 2 O' TEMPO 411 Scheme 102. Mechanism of alcohol oxidation using TEMPO/BAIB The structure of aldehyde 381 was confirmed by its 500 MHz 1H NMR spectrum in CDCI3 that showed a singlet at 6 9.68 ppm. 6.3. Synthetic Studies Towards The Side Chain Segment 6.3.1. The O-Directed Free-Radical Hydrostannation of Propargyl Ethers, Acetals and Alcohols Our synthesis of the side chain aldehyde segment 270 would utilise the recently developed O-directed free-radical hydrostannation of disubstituted alkynes for stereodefined alkene construction105,106 (Scheme 103). In this methodology, vinyl triphenylstannanes 413 are obtained in a regio- and stereoselective manner from disubstituted propargylic allyl oxygenated alkylacetylenes 412. The vinyl triphenylstannanes 413 are usually readily manipulated into trisubstituted alkenes with complete retention of olefin geometry following a tin-iodide exchange and transition metal catalysed cross-coupling.107 110 R 3 Coupling R2' SnPh; Scheme 103. The O-directed hydrostanation and the subsequent elaboration of trisubstituted alkenes The procedure utilises 1.5 eq of Ph3SnH and 0.1 eq of Et3B in toluene at 0.1 M concentration with respect to the starting alkene. Coordination of Ph3SnH to the O-atom of the acetylene and subsequent H-atom abstraction and radical attack of the alkyne lead to two possible transition invertomers 416 and 417. Scheme 104 shows that great steric hindrance would operate in the transition state 417 in the vinyl radical H-atom abstraction step. In the transition state 416, however, the minimized steric repulsions preferentially lead to the formation of 413. / Rl q H Rs Ph3S n '~ 0 > ^ - R y ~ Ph3Sn-H_y ^ Ph3Sn SnPh3 412 417 418 disfavored transition state / R1 ph3Sn ^ ^ R 2 3 H-SnPh 3 SnPtv 416 413 Favored transition state Scheme 104. Transition states of the O-directed free radical hydrostannation The previously reported O-directed free-radical hydrostannation employing Bu3SnH is much less effective. This can be explained by two reasons. First, the electron-withdrawing phenyl group enhances the Lewis acidity of the tin atom, and secondly, greater steric effects favour the 416 transition state. Our group has demonstrated that the vinyltriphenylstannane products 413 can readily undergo tin-halogen exchange with retention of configuration.107 The reaction outcome was not as predictable as it might at first sight appear. In fact, past literature shows many examples 111 where the iododemetallation of p-vinyltriphenylstannanes with an allylic O-substituent (that could coordinate) could lead to replacement of one the phenyl groups by the iodide atom. However, X-ray crystallography of many vinyltriphenylstannanes of structure 414 later showed that O- coordination does not occur in these systems; hence the “normal” outcome. Finally, the resulting vinyl iodides can readily be converted into trisubstituted alkenes through a range of Pd (0) catalyzed cross-coupling techniques. 6.3.2. Retrosynthetic plan Returning to our allopumiliotoxin 339A synthesis, aldehyde 270 would be prepared from oxazolidinone 419. Trisubstituted alkene 419 appeared derivable from alkyne 337 using the previously described methodology. This would give us great flexibility regarding the substituent on the trisubstituted alkene. The C(11) stereocenter would be installed in an asymmetric alkylation with bromide 420 using the induction of a chiral auxiliary. Bromide 420 would itself be prepared from ester 421 through reduction into the aldehyde followed by a modified Corey- Fuchs alkynylation (Scheme 105). 112 Removal of the auxiliary O-directed free radical hydrostannation Reduction T,n halogen exchange Stllle coupling Reduction Asym m etric Corey-Fuchs alkylation alkynylatlon Scheme 105. Our retrosynthetic analysis of the side chain segment of (+)-allopumiliotoxin 339A 6.3.2. Implementation of the Retrosynthetic Strategy for the Side Chain Segment of (+)-Allopumiliotoxin 339A Despite ester 421 being commercially available, it is very expensive. It was thus prepared 10fi on a large scale from L-threonine in 41% yield according to Servi procedure. Kirschning and coworkers published a synthesis of dibromide 422 from ester 421.109 Unfortunately, very low yields were obtained when we followed this protocol. This is probably because reduction of ester 421 led to a mixture of aldehyde 264 and its hydrate.110 We modified their procedure by adding a dehydration step. The crude mixture obtained from the DIBAL-H reduction of ester 421 was dehydrated in CH2CI2 at reflux using a Sohxlet extractor containing 4A activated molecular sieves for water removal. The resulting solution was then added to a mixture of CBr4 (2 eq), PPh3 (2 eq) and Zn (2 eq) in dichloromethane. After removal of the triphenylphosphine oxide, dibromide 422 was obtained in 41% yield from ester 421 after purification by Si02 flash chromatography. Analysis by IR and NMR spectroscopy was in accordance with the data published by Kirschning. Dibromide 422 was then converted to the propargyl alcohol 423 using n-butyllithium and paraformaldehyde in THF at -78 °C. TLC analysis showed complete disappearance of starting material and the appearance of a single spot. As the alcohol was very unstable, it was not possible to carry out all the characterisation. It was usually used straight 113 after purification. Nevertheless, evidence for its formation was given by the disappearance of the ethylenic proton at 6 6.49 ppm in the 500 MHz 1H NMR spectrum in CDCI3. Subsequent treatment with CBr4 and PPh3 in THF at 0°C provided bromide 424 in 63% yield. Again, because of the instability of the bromide, a full characterisation could not be achieved. It is noteworthy that dibromide 422, propargylic alcohol 423 and bromide 424 are all very sensitive and can not be stored without appreciable losses. Their immediate use is therefore recommended. Me (1) DIBAL-H CBr4, PPh3 -78*C, THF, 45 min Zn, CH 2 CI2 Br MeO JU H JUC 422 ( 2 ) soxhlet, CH 2 CI2 rt, 24 h Br Me powdered 4A MS Me (41%, 3 steps) ° / r Me Me Me Me 421 2 0 h 264 (1) n-Buli, THF -78°C to rt 2.5 h (2) HCHO, -78°C 30 min, (92%) CBr4, PPh3, THF 0°C, 20 min Scheme 106. Synthesis of bromide 420 We next explored a variety of conditions to achieve the Evans alkylation between bromide 420 and oxazolidinone 424 (Table 2). Table 2. Entry Conditions Yield 1 KHMDS (1.3 eq), THF, -78 °C, 1 h 45% 2 KHMDS (1.3 eq), HMPA (5 eq), THF, -78 °C to 0 °C, 2 h degradation 3 LDA (1.1 eq), THF, -78 °C overnight 15% 4 n-BuLi (1.1 eq), THF, -78 °C overnight No reaction Use of lithium hexamethyldisilazide as a base (entry 1) gave the best result providing alkyne 337 in a very moderate 45% yield (Scheme 107). When HMPA was added to the 114 reaction mixture, degradation occurred (entry 2). Using LDA as a base (entry 3) afforded 337 in a very poor yield (15%), and n-BuLi left the starting material untouched (entry 4). Me KHMDS, THF I^Iq -78° C to 0°C O' Ph then Me Me 0 ^ 0 Ph 424 337 Scheme 107. Evans alkylation of oxazolidinone 424 with bromide 420 We next investigated the feasibility of stereoselectively transforming acetylene 337 into trisubstituted olefin 41 9.105,107 The O-directed free-radical hydrostannation was achieved successfully in 75% yield using Ph3SnH and Et3B in toluene (Scheme 108). 500 MHz NMR analysis of the product in CDCI3 showed that a mixture of isomers in ratio 95:5 was obtained. The structure of the major isomer 425 was confirmed by an apparent triplet at 6 6.60 ppm, showing that stannation had occurred at the carbon a to the oxygen. The minor isomer was believed to be 426 even though no characterisation was possible. The iododestannation proceeded with complete preservation of olefin geometry in 80% yield. Finally, a Stille coupling with Me4Sn afforded the trisubstituted alkene 419 in 72% yield. Evidence for the success of this reaction was given by the appearance of a singlet at 6 1.63 ppm, integrating for 3 protons in the 500 MHz 1H NMR spectrum in CDCI3. Moreover, the olefinic proton was deshielded from 6 6.14 ppm for the vinyl iodide to 6 5.53 ppm for the trisubstituted alkene. 115 Me Ph3SnH Et3B ^ Ph Ph + PhMe, rt Me Me Me (75%) Me Me Me 337 425 426 l2, c h 2c i 2 rt 4 2 5 /4 2 6 95 : 5 (80%) Me Me4Sn, Cul, Ph3As PdCI2(MeCN)2 Ph Ph Me DMF, NEt3, 140°C Me Me (72%) Me 419 427 Scheme 108. Synthesis of trisubstituted alkene 419 These last 3 steps showed us the feasibility of this methodology on this kind of substrates but the low yield for the alkylation reaction led us to abandon the use of the Evans auxiliary. Instead we investigated the alkylation reaction using Oppolzer's camphorsultam auxiliary. This strategy turned out to be successful as treatment of A/-propionylsultam 428 with n-butyllithium followed by bromide 420 in HMPA proceeded in 90% yield.111 The camphor sultam auxiliary was then removed under very mild conditions by means of a titanium-mediated transesterification.112,113 Exposure of adduct 429 to 10 equiv of Ti(OEt)4 in ethanol at reflux led to ester 430 in a quantitative yield along with 96% of the recovered sultam. This reaction was extremely convenient as it led directly to the ester. Indeed the other methods to remove the sultam auxiliary led either to the carboxylic acid (LiOH, H202) or to the secondary alcohol (LiAIH4). Moreover, it was very easy to separate the auxiliary from the ester because of the difference of polarity of these two molecules. In fact, on TLC, the ester was the fast moving product and the auxiliary stayed close to the baseline. When comparing the infra-red spectra of the starting material and of the product ester, it was clear that the stretching band of the C=0 moved from 1697 cm'1 for the amide to 1736 cm'1 for the ester (Scheme 109). 116 in HMPA / Me Me/ 'm 0 Me 429 Scheme 109. Synthesis of propargyl acetal 430 We next addressed the O-directed free-radical hydrostannation of propargyl acetal 430. We were very pleased to find out that the reaction proceeded in a very good yield using 2 eq of Ph3SnH and 3 eq of Et3B in toluene (Scheme 110). Vinylstannane 431 was obtained as a single diastereoisomer in 92% yield. Confirmation that the stannation occurred at the carbon a to the oxygen was assessed by 500 MHz 1H NMR analysis of 431 in CDCI3, which showed the olefinic proton as a ddd at 5 6.55 ppm. Furthermore, the vinylic proton (H13) showed a 1H-117Sn coupling constant of 150 Hz which is the expected value for this geometry. Ph 3SnH Et3 B, PhMe Me Me 4 Sn, Cul Ph3 As, NEt3 PdCI2 (MeCN ) 2 DMF, 130°C 2 h (80%) ' Phl(OAc) 2 DIBAL-H TEMPO THF, rt, 1 h Me Scheme 110. Synthesis of aldehyde 270 Subsequent tin-halogen exchange furnished iodide 432 in 77% yield. The success of this conversion was apparent from the absence of any aromatic protons in the 500 MHz NMR spectrum of 432 in CDCI3. Once again, the formation of the iodo(diphenyl)stannylidene was not detected. A Pd(0)-mediated Stille coupling was successfully performed in the presence of copper(l) iodide and triphenylarsine in DMF at 140°C and trisubstituted alkene 433 was obtained in 72% yield. Evidence for the success of this reaction was given by the appearance of a singlet at 5 1.63 ppm integrating for 3 protons in the 500 NHz 1H NMR spectrum of 433 in CDCI3. Moreover, the olefinic proton was deshielded from 6 6.07 ppm for the vinyl iodide to 5 5.45 ppm for the trisubstituted alkene. DIBAL-H reduction afforded alcohol 434 in 75% yield as evidenced by the appearance of a broad OH stretching band at 3433 cm'1 in the infra-red spectrum of 434. Oxidation of the alcohol into aldehyde 370 was achieved in 50% yield using Phl(OAc)2/TEMPO oxidation.104 The success of the oxidation was apparent from the presence of a singlet at 6 9.64 ppm in the 500 MHz 1H NMR spectrum in CDCI3. 7. Conclusion and future work The initial goal of this work was to achieve the total synthesis of (+)-allopumiliotoxin 339A. The latter shows marked cardiotonic and myotonic activity. Our retrosynthetic plan was based on the synthesis of a-alkoxyaldehyde 339 and a side chain aldehyde 270 containing a trisubsituted alkene as summarised in Scheme 111. (+)-allopumiliotoxin 339A 191 \ ✓ V ,Boc H Me OBn 339 Scheme 111. Retrosynthetic analysis of (+)-allopumiliotoxin 339A Our initial efforts focused on the synthesis of a-alkoxyaldehyde 339 using a Sharpless Asymmetric Aminohydroxylation (SAA). A four-step route was developed to a-alkoxyester 375 118 from bromobutyronitrile 346 (Scheme 112). The key reaction was the SAA which allowed to install two stereocenters and to close the nitrogen 5 membered ring in only one step. However a very low yield was obtained and difficulties were encountered to protect alcohol 375. (1) DIBAL-H in Hex < 2 ) p h 3 p ^ p (1) SAA (2 ) H2 5% Pd/C NBoc O Me OEt Boc20 OEt OEt CH2CI2i rt, 20h (15%) Me OH Bromobutyronitrile 346 (61%, 2 steps) 345 Scheme 112. Synthesis of a-alkoxyester 375 The second approach to a-alkoxyaldehyde 381 was based on two key steps (Scheme 113). First a Trost's opening of racemic aldehyde 385 afforded alcohol 384 which was converted in 4 steps to aldehyde 395. Secondly, attachment of chiral (R)-tert-butanesulfinamide 396 to aldehyde 395 using copper sulfate afforded chiral sulfinylimine 382. It allowed to install the second stereocenter via an asymmetric indium-mediated addition of allylbromide. Compound 382 was converted to ester 405 after hydroboration, activation of the resulting alcohol and base mediated cyclisation. Eventually ester 405 was converted to aldehyde 381. a-Alkoxyaldehyde 381 was synthesised in 6% overall yield over 12 steps. 396 Pd2(dba)3CHCl 3 0 Et3B, PMBOH 1 (R,R)-Li 386 /- Bu + NH2 O' X OMe f-Bu" + 'N OMe CH2 a 2, rt, 1 2 h C uS 04, THF Me OPMB Me OPMB Me OPMB (6 8 %, 82% ee) 4 days (97%) 385 395 383 In, AIIBr THF, 55°C (85%, 60%ee) f-Bux / f-Bu v / /-Bu OMe Me OPMB Me OPMB Me OPMB 405 382 Scheme 113. . Synthesis of a-alkoxyaldehyde 381 The synthesis of the side chain aldehyde 270 is summarised in Scheme 114 and started from methyl ester 421 which was converted into bromide 420 in 4 steps. An asymmetric 119 alkylation of bromide 420 with compound 428 afforded alkyne 429. After removal of the sultam auxiliary, alkyne 430 underwent the O-directed free-radical hydrostannation reaction in very good yield to afford stannane 431 as a single isomer. Conversion to trisubstituted alkene 434 was achieved via tin halogen exchange followed by Stille coupling. Finally ester 434 was converted to aldehyde 270 in 2 steps. The side chain aldehyde 270 was prepared in 4.2% overall yield over 12 steps from ester 421. Me. .Me Me. Me JU 4 steps 428 0 2S-/ Me Me Me / M e 421 420 Me 429 Ti(OEt)4, EtOH 150°C, 3 days (100%) Ph3Sn Ph3SnH ( 1 ) l2, c h 2 ci2 Et3 B, PhMe (2) Me 4 Sn, Cul rt, 2 0 h / M e Ph 3 As, NEt3 EtO (92%) PdCI2 (MeCN ) 2 434 431 430 Scheme 114. Synthesis of side chain aldehyde 270 We have developed syntheses of two precursors needed in our designed route to (+)-allopumiliotoxin 339A. The strength of these routes is their flexibility. In fact, the avoidance of L-proline in the a-alkoxyaldehyde 381 synthesis would allow us to anchor substituents on the 5 membered-ring or even to build a 6 membered-ring. Furthermore, we proved the great utility of the O-directed free-radical hydrostannation reaction of disubstituted alkynes with Ph3SnH and Et3B; using this method, we could easily modify the substituent on the trisubstituted alkene. 120 PART C: EXPERIMENTAL All starting materials were obtained commercially from Aldrich, Acros, Avocado, Lancaster or BDH. Reactions were carried out under a nitrogen atmosphere with freshly distilled solvents unless otherwise stated. All solvents were reagent grade. Dichloromethane, benzene and acetonitrile were distilled from calcium hydride under nitrogen. Diethyl ether and THF were distilled from sodium under nitrogen. Where petrol is specified this refers to the fraction that boils in the range 40-60 °C. 4-Toluensulfonyl chloride was recrystallised from chloroform/petrol prior to use. All other reagents were used as supplied from the manufacturer. Reactions carried out at - 78 °C were cooled by means of an acetone/dry ice bath, those at -10 °C by means of an ice/salt/water bath and those at 0 °C by means of an ice/water bath. Flash column column chromatography was carried out on Kieselgel 60 40/60A (220-240 mesh) silica gel. TLC was carried out on pre-coated glass-backed plates (Merck Kieselgel 60 F254), visualised at 254 nm and stained with either of anisaldehyde, iodine, or PMA. Infrared spectra (IR) were recorded on a SHIMADZU FT-IR 8700 using potassium bromide (neat) disc. The wave number is given in cm '1 with intensity strong = s, medium = m, weak = w. Optical rotations were measured on an Optical Activity, Polaar 2000 automatic polarimeter. Mass measurements were recorded by Mr John Hill or Dr Lisa Harris of the Christopher Ingold Laboratories on a VG70-SE (Cl+, El+, FAB+). Proton Nuclear Magnetic Resonance spectra (1H NMR) were recorded on a Bruker AMX- 500 NMR Spectrometer at 500 MHz or on a Bruker AMX-400 NMR Spectrometer at 400 MHz. 13C NMR were recorded at 125 MHz. The NMR spectra were recorded with reference to the solvent peak (CHCI 3 in CDCI 3 at 7.24 ppm for 1H NMR and 77.0 ppm for 13C NMR). 13C DEPT (CH and CH 3 positive and CH 2 negative) was also used to assign 13C NMR. HMQC (proton- carbon coupling) and HMBC (proton-carbon long range coupling) were used to determined structures. The signals are noted as s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of doublets, t = triplet,q = quadruplet, dt = doublet of triplet, tt = triplet of triplet, tq = triplet of quadruplet, m = multiplet and br = broad. Coupling constants J are reported in Hz 121 8. Synthetic studies on Molecules Related to the Azinothricin Family Acid 64 NFmoc 9 ® ° / — NFmoc TFA, PhOH Me Me. OCHPh2 OH 64 To ester 97114 (11.43 g, 0.0138 mol) in dry CH2CI2 (150 mL) at 0 °C under N2 was added PhOH (1.85 g, 0.197 mol) and TFA (38 mL, 0.496 mol). The reaction mixture was stirred at 0 °C for 3 h and then concentrated in vacuo. The residue was co-evaporated from toluene (50 mL x 3). The crude residue was purified by Si02 flash chromatography (gradient elution 5:1 to 2:1 petrol:EtOAc) to give acid 64 as a white foam (8.34 g, 91%). IR (neat) 3160 (br), 3065 (w), 2951 (m), 1715 (s, br), 1450 (s), 1412 (s), 1358 (m), 1296 (s), 1257 (s), 1196 (s), 1124 (m), 1090 (m), 1051 (w), 970 (w), 912 (w), 883 (w), 741 (s), 700 (m). 1H-NMR (500 MHz, CDCI3, 298K) 6 7.74-7.13 (m, br, Ar), 6.0 (s, br), 5.47 (m, br), 5.22-4.77 (m, br), 4.73-4.46 (m, br), 4.31 (s, br), 4.21-3.96 (m, br), 2.86 (m, br), 2.33 (s, br), 2.20 (s, br), 1.84- 1.61 (m, br), 1.50-1.38 (m, br), 1.25 (s, br). 13C-NMR (125 MHz, CDCI3, 298K) 6 174.0, 172.6, 155.4, 143.8, 143.1, 141.3, 136.2, 134.0, 129.6-126.4 (Ar), 125.1, 120.0, 68.7, 68.3, 67.8, 50.5, 49.9, 47.0, 46.9, 45.6, 44.0, 29.7, 23.9, 23.1, 19.3, 18.6, 14.2. FAB (+) HRMS: Calcd. for C38H38N208: (M+H)+: m/e 664.26588; Found: mle 664.26409. The spectral data for this molecule corresponded with that in literature.36 122 Acid 157 Trocv NH Me NH AIIO- HO Me (Ph3 P)4Pd Me morpholine THF (89%) Me Me BocHN BocHN OBn OBn 112 157 To a solution of ester 11224 (7.64 g, 11.7 mmol) in THF (46 mL) at rt, under N2, was added morpholine (8.0 mL, 93.5 mmol) and tetrakis(triphenylphosphine) palladium (1.35 g, 1.17 mmol). The reaction mixture was stirred at rt for 30 min, diluted with ether (40 mL) and washed with 1 M aq. KHS04 (2 x 60 mL) and brine (40 mL). The organic layer was dried (MgS04), filtered and concentrated in vacuo. The product was purified by Si02 flash chromatography (gradient elution 4:1 to 1:2 petrol:EtOAc) affording the title compound 157 as a yellow foam (6.37 g, 89%). IR (neat) 3327 (br), 2979 (m), 1728 (s), 1508 (m), 1163 (m), 1095 (w), 816 (w), 737 (w), 698 (w) 1H-NMR (500 MHz, CDCI3, 298K) 5 7.34-7.22 (m, 5 H, Ar), 6.53 (d, J = 8.7 Hz, 1 H), 5.41 (d, J = 9.4 Hz, 1 H), 4.99 (dd, J = 8.5, 3.9 Hz, 1 H), 4.71 (d, J = 12.2 Hz, 1 H, CH20), 4.69 (m, 1 H) 4.62 (d, J = 12.2 Hz, 1 H, CH20), 4.56 (d, J = 11.5 Hz, 1 H, CH20), 4.42 (dd, J = 9.5, 2.4 Hz, 1 H), 4.37 (d, J = 11.5 Hz, 1 H, CH20), 4.13 (m, 1 H), 2.21 (m, 1 H), 1.44 (s, 9 H, (CH3)3), 1-25 (d, J = 6.2 Hz, 3 H, CH(CH3)), 1.05 (d, J = 6.9 Hz, 3 H, CH(CH3)2) , 0.87 (d, J = 6.9 Hz, 3 H, CH(CH3)2). 13C-NMR (125 MHz, CDCI3, 298K) 5 171.0, 170.5, 156.9, 154.1, 137.5, 128.3, 127.7, 127.4, 95.3, 81.0, 80.9, 74.7, 74.2, 70.4, 58.6, 55.1, 29.3, 28.3, 19.1, 18.7, 16.2. FAB (+) HRMS: Calcd. for C25CI3H35N2Na09: (M+Na)+: mle 635.13057; Found: m/e 635.12828. The spectral data for this molecule corresponded with that in the literature.36 123 Acid 159 Me NH2 FmocCI, Dioxane Me NHFmoc 10% aq.Na2C03 M e^'^C C ^H D-leudne 158 159 To D-leucine 158 (10 g, 0.076 mol) in dioxane (75 mL) at 0° C was added 10 % aq. Na2C03 (162 mL, 0.152 mol). A solution of Fmoc-CI (21.4 g, 0.083 mol) in dioxane (115 mL) was added dropwise over 30 min. The reaction mixture was stirred at 0 °C for 30 min and at rt for 2 h. H20 (200 mL) was added to the reaction mixture that was extracted with ether (3 x 250 mL). The aqueous layer was acidified to pH 2 with conc. HCI and extracted with EtOAc (3 x 250 mL). The combined organic layers were washed with brine (200 mL), dried (MgS04) and filtered. The solvent was then removed in vacuo. The resulting oil was used for the next step and was not purified any further. Acid 110 Fmoc TFA, Et3SiH Me NHFmoc (CH 2 0 )n , Me N \ C H C I3 r Me NFmoc Me i/ ^ ^ ^ C C02H0 2H TsOH, TsOH, PhMe PhMe Mf}' (74%, (74%, 3 3 steps)steps) Me X ' ^ C 0 2H 159 160 0 161 To the crude /V-Fmoc-D-Leucine 159 (0.076 mol) in dry toluene (330 mL) under N2 at rt was added paraformaldehyde (2.28 g, 0.076 mol) and TsOH (1.16 g, 6.1 mmol). The mixture was heated at reflux for 1 h and water was removed with a dean-stark apparatus. The reaction mixture was cooled to rt, washed with 10% aq. NaHC03 (200 mL), dried (MgS04) and filtered. The solvent was then removed in vacuo and compound 160, which was not characterised any further, was used immediately for the next step as described below. To crude 160 (25.4 g, 0.0696 mol) in CHCI3 (140 mL) at rt under N2 was added TFA (134 mL, 1.809 mol) and Et3SiH (33 mL, 0.209 mol). The reaction mixture was stirred at rt for 2 days and then concentrated in vacuo. The residue was co-evaporated from toluene (3 x 100 mL). The crude product crystallised following trituration of the bulk syrup with EtOAc and petrol. Acid 161 was obtained as a white solid (19 g, 74% over 3 steps). [a]D + 24.7° (c 1.03, CH2CI2). 124 IR (Neat) 2941 (m), 1753 (s), 1653 (s), 1448 (w), 1327 (w), 1167 (m), 1113 (m), 1038 (w), 806 (w), 762 (m), 739 (m), 650 (w), 606 (w), 542 (w). 1H-NMR (500 MHz, C6D6, 298K) Major rotamer 6 7.50 (m, Ph.), 7.40 (m, Ph.), 7.19 (m, Ph.), 5.14 (dd, 1 H, J = 11.2, 4.6 Hz, CHC02H ), 4.42 (m, 2 H, COOChh), 3.90 (t, J = 6.6 Hz, C 02CH2CH), 2.63 (s, 3 H, NCH3 ), 1.66 (m, 1 H, ChbCHCOzH), 1.56 (m, 1 H, ChhCHCC^H), 1.36 (m, 1 H, CH(CH3)2), 0.84 (d, J = 6.5 Hz, 3 H, (CH3)2), 0.77 (d, J = 6.5 Hz, 3 H, (CH3)2); Minor rotamer 6 7.50 (m, Ph.), 7.40 (m, Ph.), 7.19 (m, Ph.), 4. 59 (dd, 2 H, J = 10.5, 5.3 Hz, COOCh^), 4.42 (m, 1 H, CHC02H), 4.03 (t, J = 6.6 Hz, C 02CH2CH), 2.76 (s, 3 H, NCH3 ), 1.47 (m, 1 H, ChkCHCOzH), 1.36 (m, 1 H, CH(CH3)2), 1.26 (m, 1 H, ChbCHCOzH), 0.66 (d, J = 6.5 Hz, 3 H, (CH3)2), 0.59 (d, J = 6.5 Hz, 3 H, (CH3)2). 13C-NMR (125 MHz, C6D6, 298K) Major rotamer : 5 177.5 (NCOO), 157.3 (C02H), 144.6 (Ar), 144.3 (Ar),141.8 (Ar),128.5 (Ar),125.3 (Ar),125.2 (Ar),120.1 (Ar), 67.7 (COOCH2), 57.1 (CHC02H), 47.7 (C02CH2CH), 37.4 (CH2CHC02H), 30.2 (NCH3), 25.0 (CH(CH3)2), 23.2 ((CH3)2), 21.2 ((CH3)2). Minor rotam er: 6 177.7 (NCOO), 157.3 (C02H), 144.5 (Ar), 144.4 (Ar), 141.8 (Ar),127.3 (Ar),125.0 (Ar), 124.9 (Ar),120.1 (Ar), 67.4 (COOCH2), 56.8 (CHC02H), 47.6 (C02CH2CH), 37.7 (CH2CHC02H, m), 30.7 (NCH3), 24.8 (CH(CH3)2), 23.0 ((CH3)2), 21.0 ((CH3)2). FAB (+) HRMS: Calcd. for C22H25NaN04: (M+Na)+: mle 390.16812; Found: m/e 390.16842. The spectral data for this molecule corresponded with that in the literature.36 125 Amine 94 H2, MeOH H .N 0CONHNHBoc Pd(OH)2/C^ N s -'C O NH NH Boc V J 435 08 %) " {-J 148 To compound 43535 (16.7 g, 46 mmol) in dry MeOH (90 mL) at rt was added 10% Pd(OH)2 on carbon (0.490 g, 4.65 mmol). The reaction vessel was sequentially purged with H2 gas (3 times) before being allowed to stir vigorously under H2 at rt overnight. The suspension was then filtered through a pad of Celite® and the solvent concentrated in vacuo. The resulting white foam (10.28 g) was sufficiently pure for use in the next step and was not purified any further. Therefore, the yield was assumed to be ca. 98%. IR (neat) 3250 (s), 3051 (w), 2974 (s), 2933 (w), 2870 (w), 1743 (s), 1670 (s), 1545 (m), 1394 (w), 1365 (m), 1302 (w), 1248 (s), 1161 (s), 1090 (w), 1045 (w), 874 (w), 762 (w), 627 (w). 1H-NMR (500 MHz, CDCI3, 298K) 5 3.82 (dd, J = 9.2, 5.2 Hz, 1 H, CHCO), 2.98 (m, 1 H, NCHz), 2.91 (m, 1 H, NCH2), 2.11 (m, 1 H, CFhCHCO), 1.95 (m, 1 H, CI^CHCO), 1.75 (m, 1 H, CHbCHzCHCO), 1.68 (m, 1 H, ChbCHzCHCO), 1.44 (s, 9 H, C(CH3)3). 13C-NMR (125 MHz, CDCI3, 298K) 5 174.0 (NCOOf-Bu), 155.1 (CHC=ONH), 81.5 (OC(CH3)3), 59.8 (CH), 47.2 (CH2N), 30.5 (CH2CHCO), 28.1 (C(CH3)3), 26.0 (CH2CH2CHCO). FAB (+) HRMS: Calcd. for C10H10NaN3O3: (M+H)+: m/e 230.15046; Found: m/e 230.15177. The spectral data for this molecule corresponded with that in the literature.35 126 Dipeptide 111 .N. ^CONHNHBoc Me\ ir- M < y NFmoc Me NFmoc (COCI)2^ M© 'NFmoc 1 4 8 Mey _ y ^ V ° g M e'^C02H CH^ m/^C O C . AflCN'C6He ^ J. X^NHBoc 161 140 ' H 155 To acid 161 (13.4 g, 0.0366 mol) at rt under N2 was added dry CH2CI2(100 mL) followed by (COCI)2 (61 mL, 7.32 mol). The mixture was stirred at rt for 2 h, it was concentrated in vacuo and evaporated from C6H6 (2 x 50 mL). To the residue was added a solution of the amine 148 (8.4 g, 0.0366 mol) in dry C6H6 (120 mL) over 10 min under N2 at rt. Then AgCN (7.36 g, 0.549 mmol) was added and the reaction mixture was heated at reflux for 30 min. The reaction mixture was cooled to rt and EtOAc (200 mL) was added. The suspension was then filtered through a pad of Celite®, the solvent concentrated in vacuo, and the crude residue purified by Si02 flash chromatography (gradient elution 2:1 to 1:2 petrol:EtOAc) to give dipeptide 155 as a white foam (19.1 g, 90%). [a]D + 3.78° (c 0.56, CH2CI2). IR (neat) 3288 (br), 2958 (m), 1707 (s), 1697 (s), 1639 (m), 1448 (m), 1396 (w), 1309 (w), 1250 (w), 1159 (m), 1041 (w), 744 (m). 1H-NMR (500 MHz, CDCI3, 298K) 6 8.63 (br), 8.44 (br), 7.72 (m), 7.66 (m), 7.37 (m), 7.28 (m), 6.52 (m, br), 4.85 (m 1 H), 4.77 (dd, J= 11.1, 3.7 Hz, 1 H), 3.51 (m, br), 4.42 (m), 4.31, 4.22 (t), 4.15 (m, br), 4.05 (t) 3.51 (m, br), 2.83 (s, 3 H, NCH3), 2.67 (br), 2.45 (m, br), 2.26-1.73 (m, br), 1.59 (m, br), 1.42 (m, br), 1.39 (s, 9 H, C(CH3)3), 0.91 (dd, J = 6.6, 2.3 Hz), 0.51 (dd, 3 H, CH- CH3), 0.49 (d, 3 H, CH-CH3). 13C-NMR (125 MHz, CDCI3, 298K) 5 170.9, 170.0, 156.7, 155.5, 154.9, 143.7, 141.2, 127.6, 127.0, 124.9, 124.1, 119.9, 81.1, 67.5, 65.6, 58.7, 58.4, 55.1, 54.4, 47.2, 47.0, 46.0, 37.2, 29.6, 29.4, 28.0, 25.3, 25.0, 24.4, 24.3, 23.1, 22.3. FAB (+) HRMS: Calcd. for C32H42N4Na06: (M+Na)+: m/e 601.30019; Found: m/e 601.29959. 127 Dipeptide 162 NFmoc NH Me. Et2NH, MeCN Me 5 NMe Me NHBoc(87%) Me NHBoc ’O " 1 2 3 2 3 156 162 163 To dipeptide 155 (19.1 g, 33 mmol) in dry MeCN (240 mL) at rt and under N2 was added Et2NH (120 mL, 1.15 mol). The reaction mixture was stirred at rt for 30 min before it was diluted with EtOAc (150 mL) and concentrated in vacuo. The crude residue was purified by Si02 flash chromatography (gradient elution 1:1 to 0:10 petrol:EtOAc and 1:5 EtOAc:MeOH) to afford a mixture of free amine 162 and diketopiperazine 163 as a white foam (10.22 g, 87%). Data for 162 1H-NMR (500 MHz, CDCI3, 298K) 6 4.58 (d, J = 6.1 Hz, 1 H, H4 ), 3.77 (td, J = 8.9, 6.1 Hz, 1 H, H5 ), 3.44 (m, 1 H, H1), 3.30 (dd, J = 8.8, 4.9 Hz, 1 H, CHNMe), 2.42 (m, 1 H, H1 ), 2.32 (s, 3 H, NCH3), 2.15 (m, 1 H, H3), 1.98 (m, 2 H, H3, H2), 1.91 (m, 1 H, H2), 1.79 (m, 1 H, H7), 1.42 (s, 9 H, C(CH3)3), 0.91 (dd, J = 6.7 Hz, 3 H, CH-CH3), 0.90 (d, J = 6.64 Hz, 3 H, CH-CH3). 13C-NMR (125 MHz, CDCI3, 298K) 5 175.6 (C=0), 166.0 (C=0), 154.0 (C=0), 81.3 (OC(CH3)3), 60.0 (CHNMe), 58.4 (CHCONHNHBoc), 46.9 (CH2), 42.1 (CH2), 34.8 (NCH3), 28.1 ((CH3)3), 27.1 (C7), 24.8 (CH2CH2N), 24.9 (CH(CH3)2), 23.8 (CH-CH3), 22.0 (CH-CH3). FAB (+) HRMS: Calcd. for C17H33N404: (M+H)+: mle 225.16029; Found: m/e 225.16071. 128 After purification by Si02 flash chromatography diketopiperazine 163 (0.95 g) was isolated: Me 5 NMe 113 [a]D- 124.7° (c 0.59, CH2CI2) IR (neat film) 2952 (m), 2879 (w), 1872 (s), 1443 (m), 1404 (w), 1304 (w), 1240 (w), 1169 (w). 1H-NMR (500 MHz, CDCI3, 298K) 6 4.03 (dd, J = 9.4, 6.9 Hz, 1 H, H4 ), 3.77 (dd, J = 8.6, 6.3 Hz, 1 H, H5 ), 3.54 (m, 1 H, H1), 3.46 (m, 1 H, H1), 2.92 (s, 3 H, NCH3), 2.35 (m, 1 H, H3), 1.96 (m, 2 H, H3, H2), 1.83 (m, 1 H, H2), 1.75 (m, 1 H, H7), 1.59 (m, 2 H, H6), 1.55 (m, 2 H, H6), 0.91 (d, J = 6.6 Hz, 3 H, CH-CH3), 0.87 (d, J = 6.6 Hz, 3 H, CH-CH3). 13C-NMR (125 MHz, CDCI3, 298K) 5 167.5 (C=0), 165.9 (C=0), 63.4 (C5), 58.2 (C4), 45.5 (C1), 40.4 (C6), 32.9 (NCH3), 29.2 (C3), 24.5 (C7), 22.8 (CH-CH3), 22.5 (C2), 22.1 (CH-CH3). FAB (+) HRMS: Calcd. for C12H21N20 2: (M+H)+: mle 225.16029; Found: m/e 225.16071. Tetrapeptide 165 OBn NFmoc (1) Et2NH (40 eq), M eC N, rt 25 min Me. Me NHNHB oc \ ,NZ Me 9 Bn> — NFmoc in C H 2C I2 at -10°C Me' 155 add BOP-CI and Et3N NHNHBoc stir for 3 h OH 64 (73%, 2 steps) 165 To dipeptide 155 (2.0 g, 3.46 mmol) in dry MeCN (20 mL) was added at rt under N2, Et2NH (10.8 mL, 10.4 mmol). The reaction mixture was stirred at rt under N2 for 30 min and concentrated in vacuo. The crude residue was co-evaporated from benzene (3 x 20 mL) and a 129 solution of dipeptide 8 (2.3 g, 3.46 mmol) in dry CH2CI2 (18.6 mL) was added at rt under N2 over 15 min. The reaction mixture was cooled to -10 °C and dry Et3N (1.06 mL, 7.60 mmol) was added dropwise followed by BOP-CI (1.06 g, 4.14 mmol). The reactants were allowed to stir at - 10 °C for 20 min and at 0 °C for 2.5 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with 0.5 M aq. HCI (2 x 60 mL), 5% aq. NaHC03 (2 x 60 mL) and brine (60 mL). The organic layer was dried (MgS04), filtered and concentrated in vacuo. The product was purified by Si02 flash chromatography (gradient elution 2:1 to 1:2 petrol:EtOAc) to afford tetrapeptide 165 as a white foam (2.55 g, 74%). [a]D - 45.0° (c 0.74, CH2CI2). IR (neat) 3304 (br), 2955 (m), 1707 (s), 1649 (s), 1450 (m), 1365 (w), 1296 (w), 1250 (m), 1161 (w), 1090 (w), 1049 (w), 741 (m), 698 (m). 1H-NMR (500 MHz, CDCI3, 298K) 8.65-8.41 (m, br), 7.71-7.01 (m, br), 6.79-6.13 (m, br), 5.54- 2.80 (m, br), 2.23 (m, br), 1.80-1.57 (m, br), 1.50-1.11 (m, br), 0.95-0.80 (m, br). 13C-NMR (125 MHz, CDCI3, 298K) 6 171.4, 170.6, 170.3, 155.7, 155.4, 155.0, 154.9, 143.7, 143.0, 141.2, 136.3, 136.0, 134.1, 125.4-124.8 (Ar), 119.8, 80.6, 78.6, 68.6, 67.6, 58.6, 53.4, 50.0, 47.3, 46.7, 45.1, 43.8, 37.1, 30.5, 29.6, 28.0, 24.8, 24.5, 23.7, 23.1, 22.8, 22.3, 18.9, 18.5, 14.2. FAB (+) HRMS: Calcd. for CssHeyNyNaOn: (M+Na)+: m/e 1024.47960; Found: m/e 1024.48548. 130 Tetrapeptide 166 NZ NFmoc Me, ,Me MeCN, rt, 15 min (99%) Me Me Me Me NHNHBoc NHNHBoc 165 166 To tetrapeptide 165 (2.45 g, 2.55 mmol) in dry MeCN (21 mL) under N2 was added Et2NH (9.3 mL, 89 mmol) and the reaction mixture was stirred at rt for 25 min and concentrated in vacuo. The crude oil was purified by Si02 flash chromatography (gradient elution 4:1 to 0:1 petrol:EtOAc) to afford tetrapeptide 166 as a white foam (1.90 g, 99%). [a]D + 5.71° (c 0.59, CH2CI2). IR (neat) 3292 (br), 2930 (m), 1699 (m), 1645 (s), 1452 (m), 1404 (w), 1367 (w), 1261 (m), 1159 (m), 754 (w), 700 (w). 1H-NMR (500 MHz, CDCI3, 298K) 5 8.66 (br), 7.35-7.19 (m, br), 6.77 (br), 5.37 (m, br), 5.27 (m, br), 5.18 (d, J = 12.3 Hz), 5.07 (d, J = 12.3 Hz), 5.11-4.95 (m, br), 4.55 (d, J = 8.2 Hz), 4.20 (br), 3.89 (d, J = 9.8 Hz), 3.77 (br), 3.42 (br), 3.08 (s, 3 H, NCH3), 2.97 (br), 2.21-1.58 (m, br), 1.49 (d, J = 6.9 Hz), 1.39 (s, 9 H), 1.22 (s), 0.94 (d, J = 7.4 Hz), 0.93 (d, J = 6.5 Hz), 0.85 (m, br). 13C-NMR (125 MHz, CDCI3, 298K) 6 172.5, 171.6, 170.1, 155.8, 155.5, 136.7, 136.4, 129.0, 128.9, 128.7, 128.4, 128.0, 127.9, 80.8, 80.1, 77.2, 67.2, 59.1, 57.4, 56.6, 54.5, 52.7, 47.6, 44.6, 37.0, 31.7, 30.7, 30.1, 28.1, 26.9, 24.9, 24.5, 23.3, 23.0, 22.4, 13.5. FAB (+) HRMS: Calcd. for C^HsyNyNaOg: (M+Na)+: m/e 802.41153; Found: m/e 802.41531. 131 Depsipeptide 167 NHTroc Me Me NH Me HO Me NH Me Me, BocNH Me. OBn NHNHBoc O" (71 %, 2 steps) To a stirred solution of acid 157 (480 mg, 0.78 mmol) in dry C6H6 (2.4 mL) at rt under N2 was added (COCI)2 (2.4 mL, 27.3 mmol). The reaction mixture was stirred at rt under N2 for 2.5 h and then concentrated in vacuo. The resulting oil was co-evaporated from benzene (3 x 10 mL). To the residue was added a solution of tetrapeptide 166 (610 mg, 0.78 mmol) in dry C6H6 (7.2 mL) at rt under N2. AgCN (167 mg, 1.25 mmol) was then added in one portion. The reaction vessel was fitted with a reflux condenser and immersed for 10 min in an oil bath pre heated to 80 °C. The reaction mixture was then cooled, diluted with EtOAC (10 mL) and filtered through Celite®, the Celite® was then washed with EtOAc. The filtrate was concentrated in vacuo and the product purified by Si02 flash chromatography (gradient elution 4:1 to 1:1 petrol:EtOAc) to afford depsipeptide 167 as a white foam (764 mg, 71%). [a]D- 57.1° (c 0.41, CH2CI2). IR (neat) 3306 (br), 2876 (w), 2930 (m), 1716 (s), 1643 (s), 1698 (m), 1508 (m), 1452 (m), 1390 (m), 1367 (w), 1240 (m), 1161 (s), 1045 (w), 735(w), 698 (w). 1H NMR (500 MHz, CDCI3, 298K) 5 8.82 (br), 8.54 (br), 8.00 (br), 7.67 (br), 7.49-7.11 (m, br), 6.74 (br), 5.86 (br), 5.59 (br), 5.60 (m, br), 4.47 (m, br), 4.32 (m, br), 4.12 (m, br), 3.73 (m, br), 3.49 (m, br), 3.31 (m, br), 3.07 (m, br), 2.93 (m, br), 2.80 (m, br), 2.60-2.45 (m, br), 2.26 (m, br), 2.10-1.75 (m, br), 1.49-1.25 (m, br), 1.21 (d, J = 6.2 Hz), 0.95-0.81 (m, br). 13C-NMR (125 MHz, CDCI3, 298K) 5 174.0, 172.4, 171.3, 170.6, 170.4, 169.7, 156.0, 155.5, 155.1, 154.0, 153.3, 137.6, 135.7, 133.5, 129.2, 128.3, 128.6, 128.2, 127.5, 127.4, 126.0, 132 125.7, 95.3, 81.5, 81.1, 79.8, 79.1, 78.5, 74.4, 70.7, 70.3, 68.6, 68.0, 58.4, 58.1, 57.8, 52.8, 52.4, 51.4, 50.8, 50.6, 48.2, 47.6, 47.1, 45.5, 37.0, 28.6, 28.2, 28.0, 24.8, 24.3, 22.9, 22.7, 22.4, 19.8, 19.6, 17.9, 17.1, 16.3, 16.1, 15.9, 13.9. FAB (+) HRMS: Calcd. for CegClaHgoNgNaO^: (M+Na)+: m/e 1396.54176; Found: m/e 1396.54502. Cyclodepsipeptide 168 NHTroc NZ NHTroc (j)Bn (1) CF3C 0 2 H, CH2CI2i 0 °C, 2 h (2) NBS, THF/H2 0, rt, 2 h oX *Me °X^Me (3) NEM and deprotected 167 added N BocNH : over 6 h to HATU very diluted in Mev > 7 ^ 0 O Bn CH 2 CI2 at 0°C, stir at rt for 72 h, (70%, 3 steps) Me Me 7 1 \ NHNHBoc 167 To depsipeptide 167 (1.19 g, 0.865 mmol) in dry CH2CI2 (13.4 mL) at 0 °C under N2 was added in one portion CF3C02H (13.4 mL, 0.17 mol). The reaction mixture was stirred for 2 h at 0 °C, concentrated in vacuo and co-evaporated from toluene (2x10 mL) to remove the excess CF3C02H. To the residue in THF (9.7 mL) and H20 (9.7 mL) was added NBS (0.31 g, 1.7 mmol) portionwise over 10 min. The reaction mixture was stirred at rt for 2 h, diluted with EtOAc (50mL) and washed with brine (50 mL). The organic layer was dried (MgS04), filtered and concentrated in vacuo affording a white foam. To a suspension of HATU (3.28 g, 8.65 mmol) in dry CH2CI2 (1 L) at 0 °C and under N2 was added dropwise over 5 h, a solution of the above crude product and A/-ethylmorpholine (1.48 mL, 11.6 mmol) in dry CH2CI2 (1 L). The reaction mixture was stirred at rt for 60 h. The reaction mixture was concentrated in vacuo and the yellow residue dissolved in EtOAc (300 mL), washed with 1 M aq. HCI (2 x 70 mL), 5% aq. NaHC03 (2 x 70 mL) and brine (70 mL). The organic layer was dried (MgS04), filtered and concentrated in vacuo. The product was purified by Si02 flash chromatography (gradient elution 4:1 to 2:1 petrol:EtOAc) to afford cyclodepsipeptide 168 as a white foam (698 mg, 70% over 3 steps). [a]D + 3.90° (c 0.59, CH2CI2). 133 IR (neat) 3290 (br), 2876 (w), 1739 (s), 1676 (s), 1641 (s), 1508 (m), 1452 (w), 1391 (m), 1234 (m), 1196 (w), 1153 (w), 1090 (w), 1040 (w), 999 (w), 746 (m), 700 (m). 1H-NMR (500 MHz, CDCI3l 298K) 6 7.44-7.17 (m, br), 5.86-5.39 (m, br), 5.24-4.49 (m, br), 4.21- 4.01 (m, br), 3.91 (m, br), 3.84 (m, br), 3.78 (m, br), 3.42 (m, br), 3.44 (m, br), 3.26, 3.21 (m, br), 2.94 (m, br), 2.83 (m, br), 2.07-1.65 (m, br), 1.56 (m, br), 1.50-1.40 (m, br), 1.23 (br), 1.17 (d, J = 6.4 Hz), 1.15 (d, J = 6.4 Hz), 0.98-0.73 (m, br). 13C-NMR (125 MHz, CDCI3, 298K) 5 173.1, 172.3, 171.2, 170.1, 169.5, 168.7, 165.7, 157.8, 155.8, 153.9, 153.1, 137.8, 135.3, 134.0, 129.5, 128.9, 128.5, 128.2, 127.9, 127.5, 95.1, 79.5, 74.9, 70.7, 69.0, 60.4, 59.0, 58.7, 57.2, 55.9, 55.6, 53.5, 53.0, 51.1, 49.6, 48.2, 47.8, 46.9, 45.7, 37.7, 37.4, 30.3, 29.7, 29.6, 28.4, 28.0, 27.7, 26.4, 24.9, 24.7, 23.2, 22.9, 22.7, 22.2, 19.5, 17.1, 15.8, 15.5, 14.9, 13.8. FAB (+) HRMS: Calcd. for C55CI3H7oN7Na013: (M+Na)+: m/e 1164.39946; Found: m/e 1164.39772. Cyclodepsipeptide 169 NZ NHTroc NZ NHZ OBn Me OBn Me Me (1) Zn dust MeMe Me Ac OH/H20 (10:1) ,Me Me (2) ZCI, aq NaHC0 3 Me Me. NH CH2CI2 Me. (56%, 2 steps) OBn Me"Me' OBn 168 169 To cyclodepsipeptide 168 (337 mg, 0.296 mmol) in a solution of 10 :1 AcOH:H20 ( 7.7 mL) at rt was added Zn dust (387 mg, 5.90 mmol). The reaction mixture was left to stir at rt for 1 h and was filtered through Celite®. The Celite® pad was washed with THF (50 mL) and the filtrate was concentrated in vacuo and the residue co-evaporated from toluene (2x6 mL). To the resulting residue was added dropwise and simultaneously, over 10 min, a solution of Z-CI (0.13 mL, 0.888 mmol) in CH2CI2 (1.55 mL) and 10% aq. NaHC03 (1.55 mL). The reaction mixture was diluted with EtOAc (20 mL), washed with brine (10 mL), dried over MgS04, filtered 134 and concentrated in vacuo. The product was purified by Si02 flash chromatography (gradient elution 4:1 to 2:1 petrol:EtOAc) to afford 169 as a white foam (184 mg, 56% over 2 steps). IR (neat) 3302 (m), 2959 (m), 1728 (s), 1676 (s), 1634 (s), 1506 (m), 1452 (w), 1394 (m), 1257 (m), 1236 (m), 1194 (w), 1151 (w), 1035 (w), 1001 (w), 741 (m), 700 (m). 1H-NMR (500 MHz, CDCI3, 298K) 5 7.45-7.09 (m, br), 6.73 (m, br), 5.76-5.49 (m, br), 5.42(m, br), 5.27-5.81 (m, br), 4.70 (m, br), 4.63-4.47 (m, br), 4.15 (m, br), 4.07 (m, br), 3.91 (m,br), 3.78 (m, br), 3.69 (m, br), 3.43 (m, br), 3.25 (m, br), 3.23 (m, br), 2.94 (m, br), 2.81 (m, br), 2.01- 1.40 (m, br), 1.23 (s), 1.16 (m), 0.96-0.70 (m, br). 13C-NMR (125 MHz, CDCI3, 298K) 5 173.0, 171.1, 170.4, 170.1, 169.8, 169.5, 168.7, 165.8, 157.8, 155.4, 154.8, 137.8, 136.2, 135.8, 135.4, 134.0, 129.6, 128.9, 128.6, 128.5, 128.3, 128.2, 128.1, 128.0, 127.8, 127.5, 127.4, 79.1, 77.9, 75.3, 74.6, 77.2, 70.8, 70.5, 68.9, 68.7, 67.0, 59.0, 57.1, 55.6, 52.8, 52.4, 51.1, 50.8, 49.3, 48.2, 47.8, 46.8, 45.7, 45.1, 37.6, 37.3, 30.3, 29.8, 28.4, 27.9, 26.4, 25.1, 24.9, 24.6, 23.2, 23.0, 22.7, 22.1, 19.6, 17.2, 15.6, 15.0, 13.6. FAB (+) HRMS: Calcd. for CeoHysNyNaOia: (M+Na)+: m/e 1124.53203; Found: m/e 1124.52750. Cyclodepsipeptide 13 jMZ NHZ ci- Bn Me Me H2 , 10% Pd/C Me, Me Me, Me MeOH, HCI (100%) Me Me NH Me Me NH OBn OH Me Me c r 169 To cyclodepsipeptide 169 (0.210 g, 0.19 mmol), in dry methanolic HCI (0.01 M, 19 mL, 0.19 mmol) was added 10% wet Pd/C (0.14 g) and the system purged with H2. The reaction mixture was left to stir vigorously at room temperature for 24 h, diluted with MeOH (20 mL) and filtered through Celite®. The Celite® was washed well with MeOH (100 mL) and the filtrate concentrated in vacuo to yield 13 as a yellow solid (0.14 g, 94%). 135 1H-NMR (500 MHz, CDCI3, 298K) 5 6.17 (m), 6.06 (br), 5.80, 5.64, 5.58, 5.49 (m, br), 5.08 (s), 5.03 (br), 4.72 (m, br), 4.65 (s, br), 4.59 (m, br), 4.56-4.47 (m, br), 4.43 (m, br), 4.34 (m, br), 4.10-3.38 (m, br), 3.22 (m, br),3.13 (m, br),3.02 (m, br), 2.97 (m, br), 2.93 (s, 3 H, NCH3), 2.31- 1.44 (m, br), 1.36 (d), 1.33 (m, br), 1.21 (d), 1.17 (m, br), 1.07 (m, br), 0.95 (s, br). 13C-NMR (125 MHz, CDCI3, 298K) 5 174.9, 174.4, 173.1, 172.9, 172.3, 171.7, 171.4, 171.2, 170.9, 170.1, 169.8, 168.4,168.1, 156.7, 79.4, 79.0, 75.4, 70.6, 70.1, 69.4, 68.7, 67.8, 66.5, 63.2, 62.1, 61.6, 61.3, 58.8,58.3, 56.9, 56.5, 55.1, 54.5, 54.1, 53.2, 52.5, 51.8, 50.4, 49.8, 46.3, 38.3, 31.5, 31.0, 30.8, 29.6,29.4, 28.4, 27.9, 26.5, 26.0, 25.9, 25.8, 25.7, 23.4, 23.3, 23.1, 22.9, 22.4, 22.1, 21.5, 21.1, 20.4, 20.2, 20.1, 19.8, 19.4, 18.2, 16.1, 15.1, 14.9, 14.5. The NMR analysis showed that the salt is not pure. Oxazolidinone 179 DB A D , D M P U (58%) Boc To a stirred solution of diisopropylamine (0.93 mL, 6.65 mmol) in THF (5.7 mL) at -78 °C was added dropwise n-BuLi (2.5 M in hexanes, 2.7 mL, 6.65 mol). The reaction mixture was stirred at -78 °C for 35 min. A solution of the bromide 17838 (2.7 g, 6.04 mmol) in THF (6 mL) was added dropwise to the mixture. The mixture was stirred at -78 °C for 75 min and a solution of DBAD (1.67 g, 7.26 mmol) in CH2CI2 (7 mL) was added. After 1h 40 min at -78 °C, DMPU (11.7 mL, 96.8 mmol) was added dropwise and the mixture was maintained at -78 °C for 5 min, then allowed to warm to rt and stirred at rt for 50 min. The mixture was added to a flask containing Et02 (170 mL) and sat. aq. KH2P04 (100 mL). The organic layer was washed with water (100 mL), brine (100 mL), dried (MgS04) and filtered. The solvent was then removed in vacuo and the crude residue was purified by Si02 flash chromatography (graduent elution 8:1 to 136 5:1 petrol:EtOAc) and recrystallised with petrol and ether to afford compound 179 as a white solid (2.10 g, 58%). [a]D + 50.7 (c 0.49, CH2CI2). 1H-NMR (400 MHz, DMSO, 353K) 5 7.26 (m, Ar), 5.73 (m br, 1 H, H5), 4.56 (d, J = 11.9 Hz, 1 H, OCH^Ar), 4.51 (br, 1 H, CHNH), 4.47 (d, J = 11.9 Hz, 1 H, OCHzAr), 4.22 (br, 1 H, H2), 4.14 (m, 2 H, CH^O), 3.72 (m, br, 1 H, H3), 3.14 (m br, 1 H, H2), 3.09 (m, br, 1 H, ChLAr), 2.92 (m, br, 1 H, ChhAr), 2.13 (ddd, J = 3.3, 6.9, 14.1 Hz 1 H, H4), 1.97 (m, br, 1 H, H4), 1.46 (s, CH3), 1.48 (s, CH3). 13C-NMR (125 MHz, C6H6, 353K) 5 168.8, 167.6 (br), 153.3 (0=C(OfBu)), 152.5, 138.1, 135.2, 128.8, 128.0, 127.5, 126.9, 126.3, 80.6, 78.9, 69.2, 69.1, 66.3, 54.8, 52.5 (br), 36.3, 29.6, 27.4 (C(CH3)3), 27.5 (C(CH3)3). OQ The spectral data for this molecule corresponded with that in literature. Ester 180 NaOMe CH2CI2 BocN BocN To a solution of 179 (11.8 g, 19.8 mmol) in CH2CI2 (120 mL) was added a solution of NaOMe (0.4 M, 55 mL, 21.8 mmol) at - 30 °C. The reaction mixture was stirred at -30 °C for 15 min and then was neutralised to pH 6 with 10% aq. KH2P04. The mixture was then extracted with CH2CI2 (3 x 100 mL). The combined organic layers were dried (MgS04) and filtered. The solvent was then removed in vacuo and the crude residue was purified by Si02 flash chromatography with 4:1 petrol:EtOAc as eluent to afford the title compound 180 as a yellow oil (8.40 g, 94 %). [a]D - 8.45° (c 0.72, CH2CI2). 137 IR (neat) 2978 (m), 2932 (w), 1763 (m), 1732 (s), 1697 (s). 1450 (w), 1416 (s), 1392 (s), 1365 (s), 1342 (m), 1296 (m), 1250 (m), 1171 (s), 1132 (m), 1094 (s), 858 (w), 752 (w), 735 (w). 1H-NMR (400 MHz, DMSO, 353K) 5 7.28 (m, Ar), 4.78 (dd, J = 2.8, 6.9 Hz, 1 H, BocNCHCOOMe), 4.52 (d, J = 11.6 Hz, 1 H, OCJiAr), 4.43 (d, J= 11.6 Hz, 1 H, OChhAr), 4.22 (d br, J = 13.0 Hz, ChhNBoc), 3.68 (m, br, 1 H, CHOBn), 3.48 (s, 3 H, OCH3), 3.0 (m, 1 H, CH2NB0C), 2.32 (d, br, J = 2.3 Hz, 1 H, CH-CH^CH), 1.95 (ddd, J = 2.9, 6.9, 14.1 Hz 1 H, CH-Chb-CH), 1.43 (s, 9 H, C(CH3)3), 1.42 (s, 9 H, C(CH3)3). 13C-NMR (125 MHz, C6H6, 333K) 6 169.4 (Me0C=0), 153.2 (C=0), 138.1, 127.4, 126.6, 80.6 , 78.8 (C(CH3)3), 78.6 (C(CH3)3), 68.9 (OCH2Ar), 68.6 (CHOBn), 52.1 (br, CHNBoc), 50.8 (OCH3), 44.6 (br, CH2NBoc), 28.9 (CH-CH2-CH), 27.5 (C(CH3)3), 27.4 (C(CH3)3). FAB (+) HRMS: Calcd. for C23H38N204: (M+H)+: m/e 451.24441; Found: m/e 451.24565. Ester 181 (1)TFA,CH 2 CI2 ..OBn (2) NaHC03, ZCI MeO' I' CH2 CI2 MeO' (94%, 2 steps) Boc 180 181 To a solution of 180 (8.40 g, 18.6 mmol) in CH2CI2 (50 mL) was added TFA (13.8 mL, 186 mmol). The reaction mixture was stirred at rt for 2 h and evaporated in vacuo. The oil was co-evaporated from CH2CI2 and used directly for the next step. To a solution of crude TFA salt (18.6 mmol) in CH2CI2 (122 mL) was added at 0°C, 10% aq. NaHC03 (18.6 mL). Then a solution of ZCI in CH2CI2 (1 M, 18.6 mL, 18.6 mmol) was added dropwise via pipette over 15 min. After stirring 20 min at rt, the layers were separated and the aqueous layer extracted with EtOAc (3 x 100 mL). The combined organic layers were dried (MgS04) and filtered. The solvent was then removed in vacuo and the crude residue was purified by Si02 flash chromatography with 2:1 petrol:EtOAc as eluent to afford compound 181 as a yellow oil (6.8 g, 94 % over 2 steps). 138 [a]D + 20.2° (c 0.28, CH2CI2). IR (neat) 3283 (w), 3032 (w), 2951 (m), 2870 (m), 1742 (s), 1705 (s), 1605 (w), 1497 (w), 1448 (m), 1410 (m), 1350 (m), 1269 (m), 1111 (m), 1169 (s), 1092 (m), 1028 (w), 744 (m), 700 (m). 1H-NMR (500 MHz, C6H6, 333K) 6 7.31 (d, J= 7.3 Hz , Ar), 7.31 (m, Ar), 5.18 (d, J= 12.3 Hz, 1 H, COOChbAr), 5.09 (d, J = 12.3 Hz, 1 H, COOCHsAr), 4.12 (d, J = 12.3 Hz, 1 H, OCh^Ar), 4.06 (d, J= 12.3 Hz, 1 H, OCh^Ar), 3.73 (m, br, 1 H, HNCHCOOH), 3.38 (s, 3 H, OCH3), 3.10 (m, br, 2 H, CF^NZ), 3.14 (m, 1 H, CHOBn), 2.49 (d, br, J = 18.0 Hz, 1 H, CH-Chh-CH), 2.09 (ddd, J = 2.2, 4.6, 18.8 Hz 1 H, CH-CI^-CH). 13C-NMR (125 MHz, C6H6, 333K) 6 165.0 (MeOC=0), 154.5 (C=0), 138.3 (Ar), 136.7 (Ar), 128.7-127.6 (Ar), 70.2 (OCH2Ar), 68.3 (CHOBn), 65.4 (COOCH2Ar), 51.7 (OCH3), 51.6 (HNCHCOOH), 44.5 (CH2NZ), 38.4 (CH-CH2-CH). FAB (+) HRMS: Calcd. for C2iH24N2Na05: (M+Na)+: m/e 407.15828; Found: m/e 407.15878. The spectral data for this molecule corresponded with that in literature.38 Acid 182 LiOH-H20 O'0Bn THF:H2Q (2:1) MeO' HO' (98%) 181 182 To a mixture of ester 181 (3.75 g, 9.7 mmol) in THF/H20 (2:1, 74 mL:37 mL) at 0 °C was added LiOH-H20 (0.409 g, 9.7 mmol). The resulting mixture was stirred 15 min at 0 °C. 10% aq. HCI was added until pH 2 was attained and the mixture was extracted with EtOAc (3 x 80 mL). The combined organic layers were washed with brine, dried (MgS04) and filtered. The solvent was then removed in vacuo and the resulting oil (3.54 g) was sufficiently pure for use in 139 the next step and was not purified any further. Therefore, the yield of the formation of 182 was assumed to be ca. 98%. IR (neat) 3210 (br), 3032 (w), 2920 (br), 1703 (s), 1497 (w), 1452 (w), 1410 (m), 1354 (w), 1257 (m), 1178 (m), 1122 (m), 1092 (m), 1153 (w), 742 (m), 700 (m). 1H-NMR (400 MHz, C6H6, 333K) 6 7.22-7.03 (m, 10 H, Ar), 6.76 (br, 2 H, NH, OH), 5.05 (d, J = 12.5 Hz, 1 H, COOCHsAr), 5.00 (d, J = 12.5 Hz, 1 H, COOCFbAr), 4.37 (d, J = 12.1 Hz, 1 H, O C h h A r ) , 4.20 (d, J = 12.1 Hz, 1 H, OCh^Ar), 3.44 (dd, J = 13.1, 6.2 Hz, 1 H, HNCHCOOH), 3.23 (t, J= 5.7 Hz, 2 H, CFbNZ), 3.14 (m, 1 H, CHOBn), 2.17 (m, 1 H, CH-CFb-CH), 1.67 (m, 1 H, CH-CFb-CH). 13C-NMR (125 MHz, C6H6, 333K) 6 172.7 (C=0), 157.1 (C=0), 138.4 (Ar), 136.6 (Ar), 128.7- 127.6 (Ar), 70.5 (OCH2Ar), 70.3 (CHOBn), 68.3 (COOCH2Ar), 55.7 (HNCHCOOH), 48.8 (CH2NZ), 30.8 (CH-CH2-CH). FAB (+) HRMS: Calcd. for C20H22N2NaO5: (M+H)+: m/e 371.16069; Found: m/e 371.16179. 140 Compound 183 NH 2NHB oc , d c c HOB t, THF BocHNHN To acid 182 (9.7 mmol) in dry THF (55 mL), was added at 0 °C under N2, HOBt (1.42 g, 10.5 mmol), DCC (2.17 g, 10.5 mmol) and BocNHNH2 (1.39 g, 10.5 mmol). The reaction mixture was stirred at 0 °C for 30 min and at rt overnight. The suspension was then filtered through a pad of Celite®, the filtrate was washed with 1 M aq. HCI (50 mL), 5% aq. NaHC03 (50 mL) and brine (50 mL). The organic layer was dried on MgS04 and concentrated in vacuo, the crude residue was purified by Si02 flash chromatography (gradient elution 2:1 to 1:2 petrol:EtOAc) to give 183 as a white foam (4.3 g, 93%). [a]D - 40.8° (c 0.51, CH2CI2). IR (neat) 3277 (br), 2977 (w), 2930 (w), 1695 (s), 1499 (w), 1454 (w), 1367 (w), 1344 (m), 1163 (s), 1124 (m), 1091 (w), 1026 (w), 737 (w), 698 (w). 1H-NMR (400 MHz, C6H6, 333K) 5 9.47 (s, 1 H, CONH) 7.25-7.06 (m, 10 H, Ar), 6.76 (br, 2 H, NH, OH), 5.11 (d, J = 12.5 Hz, 1 H, COOCjiAr), 5.07 (d, J = 12.5 Hz, 1 H, COOCFbAr), 4.42 (d, J = 12.1 Hz, 1 H, OCFbAr), 4.27 (d, J = 12.1 Hz, 1 H, OCHzAr), 3.56 (dd, J = 13.2, 6.0 Hz, 1 H, CFbNZ), 3.43 (t, J = 5.7 Hz, 1 H, HNCHCOOH), 3.32 (dd, J = 13.2, 1.6 Hz, 1 H, C H ^Z ), 3.28 (m, 1 H, CHOBn), 2.30 (m, 1 H, CH-CFb-CH), 1.79 (m, 1 H, CH-CFb-CH), 1.36 (s, 9 H, C(CH3)3). 13C-NMR (125 MHz, C6H6, 333K) 5 170.5 (C=0), 156.7 (C=0), 155.8 (C=0), 139.3 (Ar), 137.1 (Ar), 128.7-127.5 (Ar), 80.6 (C(CH3), 70.4 (OCH2Ar), 70.2 (CHOBn), 67.9 (COOCH2Ar), 56.0 (HNCHCOOH), 49.0 (CH2NZ), 30.3 (CH-CH2-CH), 28.3 (C(CH3)). FAB (+) HRMS: Calcd. for C25H33N406: (M+H)+: m/e 485.24000; Found: m/e 485.23909. 141 Dipeptide 156 M eN ^Fmoc (1) (COCI)2, c h 2c i 2 N F m txx .M e (2) AgCN, PhMe, 70°C N Me Me NHNHBoc OH BocHNHN 184 183 "N OBn 156 Z (81 %, 2 steps) To acid 184 (2.0 g, 6.34 mmol, dried from C6H6) at rt under N2 was added dry CH2CI2 (15 mL) followed by (COCI)2 (11.06 mL, 127 mmol). The mixture was stirred at rt for 2 h, concentrated in vacuo and dried from C6H6. To the residue was added a solution of the amine 183 (2.9 g, 6.02 mmol, dried from C6H6) in C6H6 (20 mL) at rt under N2. Then AgCN (1.27 g, 9.51 mmol) was added and the reaction mixture was heated at reflux for 40 min. The reaction mixture was cooled to rt and EtOAc was added (30 mL). The suspension was then filtered through a pad of Celite®, the solvent concentrated in vacuo, and the crude residue was purified by Si02 flash chromatography (gradient elution 3:1 to 1:1 petrol:EtOAc) to give 156 as a white foam (3.95 g, 81%, 2 steps). [a]o + 3.97° (c 0.57, CH2CI2). IR (neat) 3284 (br), 2977 (w), 1703 (s), 1450 (m), 1403 (m), 1365 (w), 1310 (w), 1241 (m), 1159 (m), 1120 (w), 1153 (w), 741 (m), 698 (m). 1H-NMR (500 MHz, CDCI3, 298K) 6 9.98 (m, br), 7.75-7.65 (m, br), 7.42-7.04 (m, br), 6.12 (m, br), 5.34-4.68 (m, br), 4.50 (d, J = 12.3 Hz), 4.33 (d, J = 12.3 Hz), 4.44-4.08 (m, br), 4.57 (m, br), 3.37 (m, br), 3.16 (d), 2.93 (br), 2.84-2.50 (m, br), 2.32 (br), 1.87 (br), 1.72 (br), 1.45 (s) 1.41 (s), 1.30 (m, br), 0.86 (m, br). 13C-NMR (125 MHz, CDCI3, 298K) 6 174.0, 168.8, 157.8, 155.8,154.5, 143.7,141.1, 137.8, 134.8, 128.6, 128.5, 128.3,128.2, 127.9, 127.6, 127.4, 127.1, 127.0, 125.5, 124.9, 124.4, 124.2, 119.8, 80.8, 69.7, 69.0, 67.7, 67.5, 66.4 52.1, 51.6, 50.9, 50.0, 47.0, 29.6, 29.1, 28.0, 27.2, 27.0, 14.9, 14.1. 142 FAB (+) HRMS: Calcd. for C44H49N5Na09: (M+Na)+: m/e 814.34278; Found: m/e 814.34649. Tetrapeptide 185 (1) Et2NH, MeCN (2) Collidine, -20°C NFmoc then BOPCI Me Me' NFmoc ZN NHNHBoc Me Me' 64 ZN' NHNHBoc OBn OH 156 (67%, 2 steps) To dipeptide 156 (2.0 g, 2.52 mmol) in dry MeCN (20 mL) was added at rt under N2l Et2NH (9.2 mL, 88.4 mmol). The reaction mixture was stirred at rt under N2 for 30 min and concentrated in vacuo. The crude residue was co-evaporated from benzene. Dipeptide 64 (1.6 g, 2.41 mmol) in dry CH2CI2 (4.2 mL) at rt under N2 was cooled at -10 °C and dry collidine (0.62 mL, 5.55 mmol) followed by BOP-CI (0.737 g, 3.03 mmol) were added. The reaction mixture was stirred at -10 °C for 20 min and a solution of the deprotected amine in dry CH2CI2 (5.0 mL) was added at -10 °C. The reactants were allowed to stir at -10 °C for 20 min and at 0 °C for 2.5 h. The reaction mixture was diluted with EtOAc (80 mL), washed with 0.5 M aq. HCI (2 x 40 mL), 5% aq. NaHC03 (2 x 40 mL) and brine (40 mL). The organic layer was dried over MgS04, filtered and concentrated in vacuo. The product was purified by Si02 flash chromatography (gradient elution 3:1 to 1:2 petrol:EtOAc) to afford tetrapeptide 185 as a white foam (2.07 g, 67%, 2 steps). [a]D - 25.8° (c 0.69, CH2CI2). IR (neat) 3290 (br), 2974 (m), 1716 (S), 1666 (s), 1499 (m), 1456 (m), 1392 (m), 1242 (s), 1161 (s), 1124 (w), 1078 (w), 999 (w), 735 (m), 700 (m). 1H-NMR (500 MHz, CDCI3, 298K) 5 9.84 (br), 9.62 (br), 7.76-7.10 (m, br), 6.73 (m, br), 5.60- 4.70 (m, br), 4.58-3.86 (m, br), 3.70-2.59 (m, br), 2.28 (m, br), 2.12 (m, br), 2.02 (m, br), 1.88- 1.68 (m, br), 1.42 (s), 1.39-1.32 (m, br), 1.24 (m, br). 143 13C-NMR (125 MHz, CDCI3i 298K) 5 172.3, 169.8, 168.7, 164.0, 158.7, 155.7, 154.5, 154.1, 143.7, 143.1, 141.2, 137.9,136.4, 135.9, 135.2, 134.7, 128.4, 128.2, 127.7, 127.4, 127.2, 127.0, 125.0, 119.9, 80.9, 78.5, 70.5, 69.7, 68.5, 67.4, 57.5, 57.0, 56.0, 52.8, 52.1, 49.7, 46.7, 45.2, 43.9, 35.0, 32.1, 29.8, 28.0, 26.9, 24.0, 23.7, 18.8, 18.4, 15.6, 13.9. FAB (+) HRMS: Calcd. for CeyH^NaNaOu: (M+Na)+: m/e 1237.52219; Found: m/e 1237.52395. Tetrapeptide 186 NFmoc NH Me Me Et2NH. MeCN rt, 15 min Me' Me’ (97%) ZN NHNHBoc ZN NHNHBoc OBn OBn 185 186 To tetrapeptide 185 (1.90 g, 1.56 mmol) in dry MeCN (15 mL) under N2 was added Et2NH (5.7 mL, 54.7 mmol) and the reaction mixture was stirred at rt for 25 min and concentrated in vacuo. The crude residue was purified by Si02 flash chromatography (gradient elution 4:1 to 0:1 petrol:EtOAc) to afford tetrapeptide 186 as a white foam (1.5 g, 97%). [a]D - 35.3° (c 0.43, CH2CI2). IR (neat) 3284 (br), 2935 (w), 1701 (s), 1653 (s), 1452 (m), 1406 (m), 1364 (w), 1310 (w), 1259 (m), 1161 (m), 1122 (w), 1083 (w), 744 (m), 700 (m). 1H-NMR (500 MHz, CDCI3, 298K) 5 9.80 (br), 9.61 (br), 8.58 (br), 7.36-7.15 (m, br), 6.18 (br), 6.0 (br), 6.73 (m, br), 5.45-4.85 (m, br), 4.50-3.80 (m, br), 3.70-2.59 (m, br), 2.30-1.67 (m, br), 1.52-1.20 (m, br). 144 13C-NMR (125 MHz, CDCI3) 298K) 6 174.0, 173.2, 172.6, 169.7, 169.2, 168.8, 158.7, 155.7, 155.0, 154.6, 137.9, 137.6, 136.5, 136.2, 135.2, 134.2, 133.5, 129.4, 129.0, 128.6, 128.4, 128.2, 127.9, 127.4, 127.2, 80.9, 78.7, 69.7, 67.2, 56.5, 52.5, 51.2, 46.8, 45.0, 31.6, 30.0, 29.5, 28.0, 26.8, 23.0, 15.8, 14.5, 13.9. FAB (+) HRMS: Calcd. for C52H64N8Na012: (M+Na)+: m/e 1015.45412; Found: m/e 1015.46087. Depsipeptide 131 (1 )(C 0 C I)2 NZ NHTroc (2) AgCN,C6H( Bn< NH if = Me HO Me NZ Me Me Me ,0 BocHN Me Me OBn BocHN -Me 186 OBn ZN NHNHBoc 157 Me' OBn ZN NHNHBoc 187 OBn (81 %, 2 steps) To acid 157 (303 mg, 0.49 mmol) in dry C6H6 (1.5 mL) at rt under N2 was added (COCI)2 (1.5 mL, 17.2 mmol). The reaction mixture was stirred at rt under N2 for 2.5 h and concentrated in vacuo. The resulting oil was co-evaporated from benzene. To the residue was added a solution of tetrapeptide 186 (600 mg, 0.49 mmol) in dry C6H6 (4.6 mL) at rt under N2. AgCN (106 mg, 0.79 mmol) was then added in one portion. The reaction vessel was fitted with a reflux condenser and immersed for 10 min in an oil bath pre-heated to 80°C. The reaction mixture was then cooled, diluted with EtOAC and filtered through Celite®, the Celite® was then washed with EtOAc. The filtrate was concentrated in vacuo and the product purified by Si02 flash chromatography (gradient elution 4:1 to 1:3 petrol:EtOAc) to afford depsipeptide 187 in the form of two isomers 187a (339 mg, 43%) and 187b (375 mg, 48%). [a]D - 42.5° (c 0.59, CH2CI2) [a]D -12.3° (c 0.60, CH2CI2) 145 IR (neat) 3288 (br), 2974 (m), 1716 (S), 1666 (s), 1499 (m), 1456 (m), 1392 (m), 1242 (s), 1161 (s), 1124 (w), 1078 (w), 999 (w), 735 (m), 700 (m). 1H-NMR (500 MHz, CDCI3, 298K) 5 7.43-7.15 (m, br), 5.69 (m, br), 5.43-4.91 (m, br), 4.76-4.07 (m, br), 3.64 (m, br), 3.54 (m, br), 3.20-3.03 (m, br), 3.02 (s), 2.95-2.70 (m, br), 2.03 (m, br), 1.65 (d, br, J = 6.9 Hz), 1.40 (s), 1.39 (s), 1.34 (s), 1.25-1.12 (m, br), 0.88 (m). 13C-NMR (125 MHz, CDCI3, 298K) 5 171.9, 171.0, 170.0, 168.6, 157.9, 156.3, 156.0, 155.7, 155.2, 154.6, 154.0, 153.3, 138.0, 137.7, 135.8, 134.4, 128.4, 128.3, 127.5, 127.3, 81.2, 79.9, 78.8, 74.7, 70.6, 69.8, 68.8, 68.4, 58.2, 52.4, 51.1, 50.1, 48.4, 47.2, 45.6, 30.5, 28.7, 28.1, 24.5, 19.7, 17.3, 16.7, 16.5, 16.2, 15.9, 15.7, 13.9. The two compounds obtained were analysed separately by Mass Spectroscopy FAB (+) HRMS: Calcd. for CyyClsHgyN^NaC^o: (M+Na)+: m/e 1609.58436; Found: m/e 1609.59520. FAB (+) HRMS: Calcd. for CyyClaHgyN^NaOzo: (M+Na)+: m/e 1609.58436; Found: m/e 1609.59929. 146 Cyclodepsipeptide 436 NZ NHTroc NZ NHTroc (1 )C F 3 C 02H ,C H 2 Cl2, 0°C, 2 h YVCe (2) NBS, THF/H 2 0 , rt, 2 h Me-, / N 1 ,M ec OBn OBn 187 436 To depsipeptide 187 (1.45 g, 0.912 mmol) in dry CH2CI2 (14.1 mL) at 0 °C under N2 was added in one portion CF3C02H (14.1 mL, 182 mmol). The reaction mixture was stirred for 2 h at 0 °C, concentrated in vacuo, co-evaporated from toluene (2 x 10 mL) to remove the excess CF3C02H. T o the residue in THF (10.2 mL) and H20 (10.2 mL) was added NBS (0.32 g, 1.82 mmol) portionwise over 10 min. The reaction mixture was stirred at rt for 2 h, diluted with EtOAc (50mL) and washed with brine (50 mL). The organic layer was dried over MgS04, filtered and concentrated in vacuo to give a white foam. To a suspension of HATU (3.47 g, 9.12 mmol) in dry CH2CI2 (1 L) at 0°C and under N2 was added dropwise over 5 h, a solution of the above crude product and A/-ethylmorpholine (1.56 mL, 12.3 mmol) in dry CH2CI2 (1 L). The reaction mixture was stirred at rt for 60 h. The reaction mixture was concentrated in vacuo and the yellow residue dissolved in EtOAc (500 mL), washed with 1M aq. HCI (2 x 150 mL), 5% aq. NaHC03 (2 x 150 mL) and brine (150 mL). The organic layer was dried over MgS04, filtered and concentrated in vacuo. The crude residue was purified by Si02 flash chromatography (gradient elution 4:1 to 1:2 petrol:EtOAc) but no product could be isolated. 147 9. Toward the Synthesis of (+)-Allopumiliotoxin 339A Aldehyde 342 TsCI, DMAP PCC NEt3, CH2CI2 c h 2c i 2 1,4-butanediol 344 345 342 To a 250-mL round-bottomed flask were added 1,4-butanediol (20 g, 0.222 mol, 7.7 eq), NEt3 (4.2 mL, 0.0303 mol, 1.05 eq), DMAP (128 mg, 0.04 eq) and recrystallised TsCI (5.50 g, 0.0288 mol, 1 eq). The reaction mixture was stirred at rt for 1.5 h before being added to a separating funnel containing CH2CI2 (160 mL) and HCI (8 mL of 37% HCI and 112 mL of H20). The aqueous layer was extracted with CH2CI2 (100mL), combined organic layers were washed with water, dried (MgS04), and concentrated in vacuo. To the crude oil in CH2CI2 (600 mL) was added PCC (6.21 g, 0.0288 mol, 1 eq). The reaction mixture was stirred at rt for 2.5 h and concentrated in vacuo. The obtained slurry was very rapidly purified by Si02 flash chromatography (gradient elution 6:1 to 3:1 petrol:EtOAc); the unstable aldehyde 342 was obtained as a yellow oil. Aldehyde 342, which was not characterised any further, was used immediately for the next step as described below.86 Alkene 341 OEt O TsO- C H 2CI2 OEt Me (21 %, 3 steps) 342 341 To aldehyde 342 in CH2CI2 (30 mL) was added carbethoxyethylidene triphenylphosphorane (10.44 g, 0.0288 mol, 1 eq). The reaction mixture was stirred at rt for 20 h and concentrated in vacuo. The crude residue was purified by Si02 flash chromatography (gradient elution 10:1 to 6:1 petrol:EtOAc) to afford alkene 341 as a yellow oil (1.95 g, 21 % over 3 steps). 148 IR (neat film) 2982 (m), 1707 (s), 1649 (m), 1599 (m), 1447 (m), 1364 (s), 1267 (s), 1176 (s), 1134 (m), 1097 (m), 1007 (w), 932 (m), 816 (m), 744 (m) 663 (s) 577 (m), 555 (s). 1H-NMR (500 MHz, CDCI3, 298K) 5 7.71 (ddd, J = 8.4, 3.7, 2.0 Hz, 2 H, Ph ), 7.28 (d, J = 8.5 Hz, 2 H, Ph ), 6.54 (tq, J = 7.5, 1.3 Hz 1 H, HC=C), 4.10 (q, J = 7.2 Hz, 2 H, OChh), 3.97 (t, J = 6.2 Hz, 2 H, H3), 2.38 (s, 3 H, CH3-Ar), 2.14 (dd, J = 14.2, 7.4 Hz, 2 H, H1), 1.73 (m, 2 H, H2), 1.71 (s, 3 H, CH=CCH3), 1.21 (s, 3 H, OCH2CH3). 13C-NMR (125 MHz, CDCI3, 298K) 6 167.6 (C=0), 144.7 (C Ar), 139.3 (CH=C), 132.5 (C Ar), 129.7 (CH Ar), 129.0 (C(CH3)), 127.7 (CH Ar), 69.5 (C3), 60.3 (OCH2CH3), 27.6 (C1), 24.3 (C2), 21.4 (CH3-Ar), 14.1 (C=C(CH3)), 12.2 (OCH2CH3). FAB (+) HRMS: Calcd. for Ci6H22Na05S: (M+Na)+: mle 349.10856; Found: m/e 349.10794. Alkene 348 Ph* W DIBAL-H ------Me OEt i OEt Et20, 0°C to rt CH2CI2 346 (61 %, 2 steps) 4-bromobutyronitrile 347 348 To a three-necked, round-bottomed flask, equipped with an addition funnel and containing 4-bromobutyronitrile 346 (20 g, 13.51 mL, 0.135 mol) and ether (135 mL) was added dropwise over 0.5 h at 0 °C a solution of DIBAL-H (1 M in hexane, 175 mL, 0.175 mol). The mixture was stirred at 0 °C for 2 h and at rt for 0.5 h, and then was slowly added to a precooled (0 °C) 10% aq. H2S04 (250 mL). The resulting solution was stirred for 1 h and extracted with Et20 (2 x 250 mL). The combined organic layers were washed with water and brine, dried (MgS04), and concentrated in vacuo (water bath at 28 °C, aldehyde 347 is volatile). To the crude oil in CH2CI2 (135 mL) was added carbethoxyethylidene triphenylphosphorane (48.9 g, 0.135 mol, 1 eq). The reaction mixture was stirred at rt for 20 h, concentrated in vacuo and the crude residue was purified by Si02 flash chromatography (gradient elution 30:1 to 10:1 petrol:EtOAc) affording the title compound 348 as a yellow oil (19.29 g, 61 % over 2 steps).88 IR (neat film) 3402 (w), 2978 (m), 1713 (s), 1651 (m), 1443 (m), 1365 (w), 1280 (br), 1111 (m). 149 1H-NMR (500 MHz, CDCI3, 298K) 5 6.68 (tq. J = 7.4, 1.3 Hz, 1 H, HC=C), 4.17 (q, J = 7.2 Hz, 2 H, CHjO), 3.40 (t, J = 6.5 Hz, 2 H, H3), 2.33 (ddd, J = 14.7, 7.5, 0.6 Hz, 2 H, H1), 1.99 (m, 2 H, H2), 1.84 (s, 3 H, CH=CCH3), 1.30 (t, J = 7.2 Hz, 3 H, CH3CH20). 13C-NMR (125 MHz, CDCI3, 298K) 6 168.0 (C=0), 139.6 (HC=C), 129.3 (CH=C), 60.5 (CH20), 33.0 (C3), 31.4 (C2), 27.0 (C1), 14.3 (CH3CH20) 12.5 (CH=CCH3). FAB (+) HRMS: Calcd. for C9BrH15Na02: (M+Na)+: m/e 257.01531; Found: m/e 257.01563. Alcohol 357 (DHQ)2PHAL (5 mol %) K2 0 s0 2 ( 0 H )4 (4 mol %) biz BnOCONH2 (3.1 eq) + BnOCONH OEt 'OEt NaOH (3 eq) Me f-BuOCI (3 eq) Me OH MeCN /H 20 (1:1) 345 357 To benzylcarbamate (940 mg, 6.2 mmol) in MeCN (5 mL) at rt was added a freshly prepared solution of NaOH (0.244 g of NaOH in 10 mL of water) followed by a freshly prepared solution of terf-butylhypochlorite (0.66 mL, 6.10 mmol). Then a solution of the ligand (DHQD)2PHAL (77 mg, 0.059 mmol) in MeCN (5 mL) was added followed by olefin 347 (500 mg, 2.00 mmol) and the osmium catalyst. (29 mg, 0.079 mmol). The reaction mixture was stirred at rt for 24 h and did not go to competion according to TLC analysis. It was then quenched with saturated sodium sulfite (10 mL). The two phases were separated and the aqueous phase was extracted with EtOAc (3 x 20 mL). The combined organic extracts were washed with brine (20 mL), dried (MgS04) and concentrated. Purification by chromatography (gradient elution petrol:EtOAc 30:1 to 20:1) provided 357 contaminated by benzylcarbamate (100 mg). The mixture which was not characterised any further was used directly for the next step as described below. 150 Alcohol 362 HZ n o 2 NaHC03, 2,4-DNPF q H OEt EtOH, rt, CH2 CI2, H2 0, EtOH overnight rt, overnight OH OEt 367 361 362 To a mixture of alcohol 357 and benzylcarbamate (100 mg) in EtOH (10 mL) under N2 was added Pd(OH)2 (20% wet, 10 mg). The flask was purged and the N2 balloon was replaced by a H2 balloon. The mixture was stirred rigorously for 20 h. The suspension was filtered through a pad of Celite® and the solvent concentrated in vacuo. To the crude residue in CH2CI2 (1 mL), H20 (1 mL), and EtOH (2 mL) was added NaHC03 (250 mg, 3 mmol) and dinitrofluorobenzene (0.38 mL, 3 mmol). The reaction mixture was stirred at rt overnight and extracted with EtOAc. The combined organic layers were dried (MgS04) and filtered. The solvent was then removed in vacuo and the crude residue was purified by Si02 flash chromatography (gradient elution 20:1 to 5:1 petrol:EtOAc) affording 362 as a yellow oil (55 mg). 1H-NMR (500 MHz, CDCI3, 298K) 5 8.69 (d, J = 2.7 Hz, 1 H, Ar), 8.10 (dd, J = 9.5, 2.7 Hz, 1 H, Ar), 7.22 (d, J = 9.5 Hz, 1 H, Ar), 4.60 (t, J = 7.2 Hz, 1 H, CH-N), 3.92 (dd, J = 14.3, 7.2 Hz, 1 H, CH2-O), 3.82 (dd, J = 14.3, 7.2 Hz, 2 H, CHj-O), 3.58 (td, J = 10.4, 6.4 Hz, 1 H, H3), 2.73 (m, 1 H, H3), 2.20 (m, 2 H, H1), 2.05 (m, 1 H, H2), 1.71 (m, 1 H, H2), 1.39 (s, 3 H,CCH3), 1.13 (t, 3 H, CH3CH20). 13C-NMR (125 MHz, CDCI3, 298K) 5 174.8 (C=0), 147.4 (Ar), 137.1 (A r), 136.9 (Ar), 126.8 (Ar), 123.7 (Ar), 117.8 (Ar), 68.1 (CHN), 64.1 (CH2-0), 62.3 (C3), 25.5 (C1), 25.1 (C2), 21.9 (C(CH3)), 13.7 (CH3CH20). FAB (+) HRMS: Calcd. for C15H19NaN307: (M+Na)+: m/e 376.11206; Found: m/e 376.11254. 151 Tetrahydrofuran 366 (DHQ)2PHAL (10 mol %) 0 K2 0 s0 2 (0 H) 4 ( 8 mol %) / - " 0 O /■"■ NZ O . X BnOCONH2 (3.1 eq) 2 < B r'v ^ ^ V ^ O E t ------^ OEt Me NaOH (3 eq) 1 Me OH Me OH Me f-BuOCI (3 eq) 346 MeCN / H20 (3:2) 366 357 To benzylcarbamate (1.99 g, 13.2 mmol) in MeCN (17 mL) at rt was added a freshly prepared solution of NaOH (0.50 g of NaOH in 20 mL of water) followed by freshly prepared terf-butylhypochlorite (1.43 g, 13.0 mmol). Then a solution of the ligand (DHQD)2PHAL (0.33 g, 0.425 mmol) in MeCN (17 mL) was added followed by olefin 345 (1 g, 4.25 mmol) and the osmium catalyst (0.125 g, 0.340 mmol). The reaction mixture was stirred at rt for 20 h and quenched with saturated sodium sulfite. The two phases were separated and the aqueous phase was extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with brine (20 mL), dried (MgS04) and concentrated in vacuo. Extensive purification by Si02 flash chromatography (gradient elution 30:1 to 10:1 petrol:EtOAc) followed by preparative chromatography provided the nearly pure dihydroxylated product 366 in order to carry out analysis. Data for 366: IR (neat film) 3510 (m), 2982 (s), 2878 (m), 1736 (s), 1454 (w), 1373 (w), 1259 (m), 1203 (m), 1136 (m), 1072 (m), 1024 (w). 1H-NMR (500 MHz, CDCI3, 298K) 5 4.17 (qd, J = 10.7, 7.0 Hz, 2 H, ChbO), 4.02 (t, J = 7.0 Hz 1 H, CH-O), 3.77 (m, 1 H, H3), 3.69 (m, 1 H, H3), 3.14 (br, 1 H, OH), 1.83 (m, 4 H, H1, H2), 1.22 (s, 3 H, CCH3) 1.21 (t, J= 7.0 Hz, 3 H, CH3CH20). 13C-NMR (125 MHz, CDCI3, 298K) 6 175.6 ((OEt)-C=0), 82.9 (CH-O), 75.6 (CH3COH), 69.2 (C3), 61.6 (CH20), 26.0 (C1), 25.1 (C2), 21.4 (HO-CCH3), 13.9 (CH3CH20). FAB (+) HRMS: Calcd. for C9H1704: (M+H)+: mie 189.11268; Found: mle 189.11286. 152 Improved procedure for Sharpless Asymmetric Aminohydroxylation93 Alcohol 357 • (DHQ)2PHAL (6 mol %) n K20 s0 2(0H)4 (5mol %) r-Nz. O J I BnOCONH2 (3.1 eq) B r \ ^ \ I NaOH (3 eq) Me OH Me NBuOCI (3 eq) 345 MeCN/H20 (3:2) 357 To benzylcarbamate (11.15 g, 73.8 mmol) in MeCN (90 mL) at rt was added a freshly prepared solution of NaOH (2.9 g of NaOH in 112 mL of water, 72 mmol) followed by freshly prepared te/f-butylhypochlorite91 (7.87 g, 72 mmol). Then a solution of the ligand (DHQ)2PHAL (1.11 g, 1.4 mmol) in MeCN (90 mL) was added (It is important that the ligand be completely dissolved in MeCN) and the reaction mixture was cooled to 0 °C. Then olefin 345 was added (5.6 g, 23.8 mmol) followed by the osmium catalyst that was added by portion over 20 min (0.439 g, 1.19 mmol). The reaction mixture was stirred at 0 °C for 30 min and at rt for 2 h and quenched with saturated sodium sulfite (150 mL). The two phases were separated and the aqueous phase was extracted with EtOAc (3 x 100 mL). The combined organic phases were washed with brine (150 mL), dried (MgS04) and concentrated in vacuo. Purification by Si02 flash chromatography (gradient elution 30:1 to 20:1 petrol:EtOAc) provided alcohol 357 contaminated by some excess benzylcarbamate. Intermediate 357 was not characterised any further and was used immediately for the next step described below. Alcohol 375 NBoc O 2 OEt Me OH Me OH 357 375 To a mixture of the alcohol 357 contaminated by some benzylcarbamate (7.8 g) in EtOH (100 mL) under N2 was added Pd/C (10% wet, 2.0 g) and Boc20 (7.3 g, 35.7 mmol). The flask was purged and the N2 balloon was replaced by a H2 balloon. The mixture was stirred rigorously overnight. The suspension was filtered through a pad of Celite® and the solvent concentrated in vacuo. The resulting oil was purified by Si02 flash chromatography (gradient elution 20:1 to 10:1 petrol:EtOAc) affording the title compound 375 as a colorless oil (1.05 g, 15 % over 2 steps).94 153 [a]D- 16.8° (c 0.34, CH2CI2). IR (neat film) 3512 (m), 3342 (w), 2978 (s), 1730 (s), 1697 (s), 1454 (w), 1392 (s), 1257 (m), 1171 (s), 1119 (m), 1078 (w), 1024 (w). 1H-NMR (500 MHz, CDCI3, 298K) 6 4.54 (br, 1 H, OH), 4.19 (m, 1 H, Ch^O), 4.14 (m, 1H, N-CH), 4.09 (m, 1 H, ChhO), 3.50 (br, 1 H, H3), 3.20 (m, 1 H, H3), 1.86 (m, 3 H, H2, H1), 1.67 (m, 1 H, H2), 1.39 (s, 9 H, C(CH3)3), 1.27 (s, 3 H, CCH3) 1.20 (t, J= 7.0 Hz, 3 H, CH3CH20). 13C-NMR (125 MHz, CDCI3, 298K) 5 175.1 (COOEt), 156.3 (0=C-0(C(CH3)3), 80.0 (C(CH3)3), 77.2 (CH3COH), 63.3 (CHNH), 61.7 (CH20), 48.0 (C3), 28.3 (C(CH3)3), 26.8 (C1), 24.4 (C2), 20.6 (HO-CCH3), 14.0 (CH3CH20). FAB (+) HRMS: Calcd. for C44H49N5Na0 9: (M+Na)+: mle 310.16303; Found: mle 310.16196. Mixture of 376 and 377 NBoc NaH, BnBr . X , I OEt n-(Bu)4 NI, CH 2 CI2 ■CHC 0 2Et >CH Me OH 1 0°C, 3 h H Me H MeC° 2Bn 376 377 To a solution of 375 (100 mg, 0.311 mmol) in THF (1.5 mL) at 0 °C under N2 was added NaH (60% suspension in oil, 12.5 mg, 0.311 mmol). After 10 min at 0 °C, r?-(Bu4)NI (11.5 mg, 0.031 mmol) was added followed by BnBr (0.040 mL, 0.311 mmol). The reaction was warmed to rt and stirred for 3 h. It was then slowly quenched with water (3 mL) and extracted with EtOAc (3x10 mL). The resulting oil was purified by Si02 flash chromatography (gradient elution 10:1 to 2:1 petrol:EtOAc) affording an inseparable mixture of 376 and 377 (55 mg) in proportion 2:1. 1H-NMR (500 MHz, CDCI3, 298K) 376 5 7.37-7.31 (m, 5 H, Ar), 5.24 (d, J = 12.4 Hz, 1 H, PhCHzO ), 5.21 (d, J= 12.4 Hz, 1 H, PhCHzO), 3.96 (m, 1 H, CH-N), 3.63 (m, 1 H, H3), 3.18 (m, 1 H, H3), 2.07 (m, 1 H, H2), 2.07 (m, 2 H, H2, H1), 1.58 (s, 3 H, CCH3), 1.50 (m, 1 H, H1); 377 5 4.25 (qd, J = 7.1 Hz, 2 H, OCHzCHa), 3.96 (m, 1 H, CH-N), 3.63 (m, 1 H, H3), 3.18 (m, 1 H, H3), 154 2.07 (m, 1 H, H2), 2.07 (m, 2 H, H2, H1), 1.58 (s, 3 H, CCH3), 1.50 (m, 1 H, H1), 1.30 (t, J = 12.4 Hz, 3 H, CH3CH20). 13C-NMR (125 MHz, CDCI3, 298K) 5 172.1, 171.8 (C=0), 160.2, 160.1 (C=0), 134.9, 128.7, 126.6, 128.1 (Ar), 80.2, 67.8 (PhCH20), 66.2, 66.1 (CHN), 62.4 (0-CH2CH3), 46.0 (C3), 26.1 (C1), 24.9 (C2), 19.2 (C(CH3)), 14.0 (CH3CH20). Alcohol 384 Pd2(dba)3CHa3 Et3B, PMBOH (S.g-L, 2 2 1 2 CH CI , rt, h ^ J ) - p p h 2 Ph2 p - n f } 6 8 %, 82%ee 386 385 384 Ligand (’S.SJ-L.) To a 2 L round bottomed flask containing (dba)3Pd2.CHCI3 (0.749 g, 0.723 mmol), chiral ligand (S.S)-!^ 386 (1.5 g, 2.17 mmol) and PMB-OH (9.05 mL, 72.3 mmol) was added dry CH2CI2 (720 mL) at rt under N2. The resulting dark purple mixture was stirred at rt until it turned a deep orange color (roughly 15 min). Triethylborane (1 M solution in THF, 0.723 mL, 0.723 mmol) was added followed by 2-methyl-2-vinyloxirane 385 (8.04 mL, 72.3 mmol). The reaction mixture was stirred for 20 h. The solvent was removed in vacuo and the crude product was purified by Si02 flash chromatography (gradient elution 30:1 to 10:1 petrol:EtOAc) affording alcohol 384 as a yellow oil (11.00 g, 68 %, 82% ee).97 [a]D + 18.1° (c 0.21, CH2CI2). IR (neat film) 3440 (br), 2933 (br), 1726 (s), 1612 (m), 1514 (s), 1460 (w), 1412 (w), 1379 (w), 1248 (s), 1174 (w), 1115 (m), 1038 (m), 929 (w), 822 (m), 733 (m). 1H-NMR (500 MHz, CDCI3, 298K) 6 7.23 (d, J = 8.6 Hz, 2 H, Ar), 6.86 (d, J = 8.6 Hz, 2 H, Ar), 5.90 (dd, J = 17.6, 10.9 Hz, 1 H, CH=CH2), 5.32 (dd, J= 17.5, 1.1 Hz, 1 H, CH=C\^), 5.42 (dd, J = 11.0, 1.1 Hz, 1 H, CH=CH2), 4.32 (s, 2 H, O-Chh-Ar), 3.78 (s, 3 H, OCH3), 3.52 (dd, J = 11.1, 5.0 Hz, 1 H, CHs-OH), 3.45 (dd, J = 11.1, 6.4 Hz, 1 H, Chh-OH), 2.10 (t, J = 6.4 Hz, 1 H, OH), 1.36 (s, 3H, CH3). 155 13C-NMR (125 MHz, CDCI3i 298K) 5 159.0 (Ar), 139.5 (CH=CH2), 131.0 (Ar), 129.0 (Ar), 117.1 (CH=CH2), 113.3 (Ar), 78.3 (MeCOPMB), 69.5 (CH2-OH), 65.8 (0-CH2-Ar), 55.2 (OCH3), 18.6 (CH3). FAB (+) HRMS: Calcd. for Ci3H18Na03: (M+Na)+: mle 245.11536; Found: 245.11588. Ester 391 (RM+)-MTPA DCC. DMAP o r oAxph Me OPMB MeO bF 3 384 391 To alcohol 384 (50 mg, 0.227 mmol) and (R)-(+)-MTPA (53 mg, 0.227 mmol) was added at rt under N2, CH2CI2 (0.8 mL) followed by DCC (47 mg, 0.227 mmol) and DMAP (2.8 mg, 0.023 mmol). The reaction mixture was stirred at rt overnight but did not go to completion according to TLC analysis. The solvent was removed in vacuo and the crude product was purified by Si02 flash chromatography (10:1 petrol:EtOAc) to isolate ester 391 (55 mg, 56%), the faster moving product of the TLC as yellow oil. 19F NMR of the mixture showed a 100:10 mixture of diastereoisomers (82% ee). [a]D + 33.1° (c 0.56, CH2CI2). IR (neat film) 2947 (m), 2849 (w), 1753 (s), 1612 (w), 1512 (m), 1454 (w), 1379 (w), 1250 (s), 1175 (s), 1119(m), 1032 (s), 822 (w), 719 (w). 1H-NMR (500 MHz, CDCI3, 298K) 5 7.53 (d, J = 8.0 Hz, 2 H, Ar), 7.36 (m, 1 H, Ar), 7.29 (m, 2 H, Ar), 7.18 (d, J = 8.7 Hz, 2 H, Ar(PMB)), 6.83 (d, J = 8.7 Hz, 2 H, Ar(PMB)), 5.84 (dd, J = 17.4, 11.4 Hz, 1 H, CH=CH2), 5.32 (dd, J = 11.4, 0.9, 1 H, C H ^hb), 5.29 (dd, J = 17.4, 0.9 Hz, 1 H, CH=CH2), 4.39 (d, J= 11.2 Hz, 1 H, CHj-OCO), 4.32 (s, 2 H, OCh^Ar), 4.25 (d, J= 11.2 Hz, 1 H, CH2-OC=0 ), 3.78 (s, 3 H, ArCH3), 3.52 (s, 3 H, 3FCCOCH3), 1.38 (s, 3 H, CH3). 156 13C-NMR (125 MHz, CDCIa, 298K) 6 166.3 (C=0), 158.9 (Ar), 138.9 (CH=CH2) , 132.2 (Ar), 130.9 (Ar), 129.5 (Ar), 128.7 (Ar), 128.3 (Ar), 127.5 (Ar), 117.7 (CH=CH2), 113.7 (Ar), 84.6 (q, CF3), 76.4 (MeCOPMB), 70.8 (CH2-0C=0), 65.8 (0-CH2-Ar), 55.4 (OCH3), 55.3 (OCH3), 19.7 (CH3). FAB (+) HRMS: Calcd. for C23H25F3Na0 5: (M+Na)+: m/e 451.15517; Found: 461.15438. Aldehyde 392 Swern Oxidation Me OPMB Me OPMB 384 392 To a stirred solution of DMSO (19.9 mL, 280 mmol) in dry CH2CI2 (287 mL) at -78° C under N2 was added (COCI)2 (12.2 mL, 140 mmol) dropwise over 5 min. The reaction mixture was stirred at -78 °C under N2 for 20 min and a solution of alcohol 384 (10.38 g, 46.7 mmol) in dry CH2CI2 (97 mL) was added via canula over 15 min. The reaction mixture was stirred at - 78 °C for 30 min and freshly distilled NEt3 (98.3 mL, 700 mmol) was added dropwise, the mixture was allowed to warm to rt over 20 min and was then quenched with brine (150 mL) and extracted with CH2CI2 (3 x 100 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo to afford the crude aldehyde 392 which was used without further purification. An analytical sample was purified by Si02 flash chromatography (gradient elution 20:1 to 10:1 petrol:EtOAc) affording the title compound 392 as a yellow oil. 1H-NMR (500 MHz, CDCI3, 298K) 5 9.49 (s, 1 H, CHO), 7.28 (d, J = 8.7 Hz, 2 H, Ar), 6.87 (d, J = 8.7 Hz, 2 H, Ar), 5.81 (dd, J = 17.6, 10.8 Hz, 1 H, CH=CH2), 5.46 (dd, J = 17.6, 0.9 Hz, 1 H, CH=CH2), 5.42 (dd, J = 10.8, 0.9 Hz, 1 H, CH=CFb), 4.46 (d, J = 10.8 Hz, 1 H, CFh-O), 4.40 (d, J= 10.8 Hz, 1 H, CHj-O), 3.79 (s, 3 H, OCH3), 1.45 (s, 3 H,CH3). 13C-NMR (125 MHz, CDCI3, 298K) 6 200.7 (CHO), 159.3 (Ar), 135.5 (CH=CH2), 130.2 (Ar), 129.3 (Ar), 119.1 (CH=CH2), 113.9 (Ar), 83.4 (MeCOPMB), 65.8 (CH2-0), 55.3 (OCH3), 18.7 (CH3). 157 FAB (+) HRMS: Calcd. for C13H16Na03: (M+Na)+: mle 243.09971; Found: m/e 243.09876. Acid 393 NaCI02, NaH2P 0 4 /-BuOH, H20 OH 2-methyl-2-butene Me' OPMB rt, 2h 392 393 To the crude aldehyde 392 (46.7 mmol) in f-BuOH (140 mL) and 2-methyl-2-butene, were added simultaneously over 15 min at rt a solution of NaCI02 (12.7 g, 140 mmol) and a solution of NaH2P04 (16.79 g, 140 mmol) in water (140 mL). The reaction mixture was stirred at rt for 2 h, then diluted with sat. aq. NH4CI (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo to afford the crude carboxylic acid 393 which was used without further purification. An analytical sample was purified by Si02 flash chromatography (gradient elution 5:1 to 1:2 petrol:EtOAc) affording the title compound 393 as a colorless oil. [a]D + 11.0° (c 0.63, CH2CI2). IR (neat film) 3180 (br), 2995 (m), 2943 (m), 1718 (s), 1612 (m), 1514 (s), 1458 (w), 1381 (w), 1248 (s), 1120 (m), 1034 (m), 933 (w), 822 (m). 1H-NMR (500 MHz, CDCI3, 298K) 5 9.96 (s, broad, 1 H, COOH), 7.28 (d, J = 8.7 Hz, 2 H, Ar), 6.87 (d, J = 8.7 Hz, 2 H, Ar), 6.01 (dd, J = 17.4, 10.7 Hz, 1 H, CH=CH2), 5.49 (dd, J = 17.6, 0.9 Hz, 1 H, CH=CH2), 5.39 (dd, J = 10.8, 0.9 Hz, 1 H, C H ^hh), 4.47 (d, J= 10.4 Hz, 1 H, CHj-O), 4.43 (d, J = 10.4 Hz, 1 H, CH2-O), 3.79 (s, 3 H, OCH3), 1.64 (s, 3 H,CH3). 13C-NMR (125 MHz, CDCI3, 298K) 5 176.2 (COOH), 159.3 (Ar), 136.9 (CH=CH2),129.7 (Ar), 129.4 (Ar), 118.3 (CH=CH2), 113.9 (Ar), 80.4 (MeCOPMB), 66.5 (CH2-0), 55.3 (OCH3), 21.9 (CH3). 158 FAB (+) HRMS: Calcd. for C13H16Na04: (M+Na)+: m/e 237.11268; Found: m/e 237.11215. Ester 394 o Mel, K2C 0 3 DMF, rt OMe (80%, 3 steps) Me OPMB Me OPMB 394393 To a solution of the crude acid 393 (47.6 mmol) in dry DMF (155 mL) at rt under N2 was added K2C03 (32.2 g, 233 mmol) followed by Mel (291 mL, 467 mmol) over 10 min. The reaction mixture was stirred at rt for 3 h then quenched by addition of water (100 mL) and extracted with EtOAc (3 x 100 mL). The combined organic extracts were washed with water (3 x 100 mL), dried (MgS04) and concentrated in vacuo. Purification by Si02 flash column chromatography (gradient elution 30:1 to 20:1 petrol:EtOAc) afforded ester 394 as a yellow oil (9.39 g, 80%, 3 steps). [a]D + 9.54° (c 1.05, CH2CI2). IR (neat film) 2995 (m), 2851 (m), 2908 (w), 2874 (w), 2837 (w), 1740 (s), 1612 (m), 1514 (s), 1460 (m), 1381 (w), 1300 (w), 1250 (s), 1201 (w), 1176 (w), 1117 (s), 1034 (m), 935 (w), 824 (m ). 1H-NMR (500 MHz, CDCI3, 298K) 5 7.30 (d, J = 8.7 Hz, 2 H, Ar), 6.85 (d, J = 8.7 Hz, 2 H, Ar), 6.06 (dd, J = 17.4, 10.7 Hz, 1 H, CH=CH2), 5.41 (dd, J= 17.5, 0.9 Hz, 1 H, C H ^Fh), 5.29 (dd, J = 10.8, 0.9 Hz, 1 H, CH=CH2), 4.45 (d, J = 10.6 Hz, 1 H, CFk-O), 4.41 (d, J = 10.6 Hz, 1 H, CHj-O), 3.77 (s, 3 H, OCH3), 3.76 (s, 3 H, OCH3), 1.58 (s, 3 H,CH3). 13C-NMR (125 MHz, CDCI3, 298K) 6 173.3 (COOCH3), 159.0 (Ar), 138.2 (CH=CH2) , 130.6 (Ar), 129.1 (Ar), 116.5 (CH=CH2), 113.6 (Ar), 80.5 (MeCOPMB), 66.6 (CH2-0), 55.2 (OCH3), 52.2 (OCH3), 23.3 (CH3). FAB (+) HRMS: Calcd. for C14H1904: (M+H)+: m/e 251.12833; Found: m/e 251.12869. 159 Aldehyde 395 i) O3 , CH2CI2 MeOH OMe O 'OMe Me OPMB ii) Me2S Me OPMB (92%) 394 395 A mixture of alkene 395 (1.0 g, 4.0 mmol) in CH2CI2 (25mL) and MeOH (1.25 mL) at -78 °C was purged with 0 2 for 30 s and 0 3 was bubbled into the flask until the mixture turned blue. Residual ozone was flushed out with nitrogen and Me2S (2.22 mL, 40 mmol) was added at -78 °C. The reaction mixture was warmed to rt, stirred for 30 min and concentrated in vacuo. Purification by Si02 flash column chromatography (gradient elution 7:1 to 2:1 petrol:EtOAc) afforded aldehyde 395 as a yellow oil (0.94 g, 92%). [a]D - 3.16 0 (c 0.28, CH2CI2). IR (neat film) 3447 (br), 2955 (m), 2841 (w), 1736 (s), 1688 (w), 1606 (m), 1514 (s), 1458 (m), 1254 (s), 1157 (m, br), 1032 (m), 827 (m). 1H-NMR (500 MHz, CDCI3, 298K) 6 9.64 (s, 1 H, CHO), 7.32 (d, J = 8.7 Hz, 2 H, Ar), 6.87 (d, J = 8.7 Hz, 2 H, Ar), 4.54 (d ,J = 10.4 Hz, 1 H, CHj-O), 4.50 (d, J = 10.4 Hz, 1 H, CH2-O), 3.79 (s, 3 H, COOCH3), 3.78 (s, 3 H, OCH3), 1.58 (s, 3 H,CH3). 13C-NMR (125 MHz, CDCI3, 298K) 5 197.3 (CHO), 169.6 (COOCH3), 159.5 (Ar), 129.6 (Ar), 129.3 (Ar), 113.9 (Ar), 84.8 (MeCOPMB), 67.8 (CH2-0), 55.3 (OCH3), 52.8 (COOCH3), 18.3 (CH3). Cl HRMS: Calcd. for C5H1605 m/e : 252.09977; Found: 252.09953. 160 Sulfinimine 383 396 'p f-Bu + NH- OMe OMe Me OPMB CuS04, THF 5 days (97%) Me OPMB 395 383 To a solution of aldehyde 395 (1.87 g, 7.4 mmol) in dry CH2CI2 (15 mL) at rt under N2 was added anhydrous CuS04 (0.90 g, 7.4 mmol) followed by solid (R)-terf-butanesulfinamide (0.87 g, 7.4 mmol). The reaction mixture was stirred at rt for 5 days, then filtered through Celite® and concentrated in vacuo. The resulting oil (2.56 g) was used in the next step and was not purified any further. Therefore, the yield of the formation of sulfinimine 383 was assumed to be ca. 97%.100 [a]D - 43.0° (c 0.52, CH2CI2). IR (neat film) 2955 (m), 1745 (s), 1618 (m), 1514 (s), 1456 (m), 1369 (w), 1250 (s), 1257 (br), 1032 (w), 827 (w). 1H-NMR (500 MHz, CDCI3, 298K) 5 8.17 (s, 1 H, N=CH), 7.28 (d, J = 8.8 Hz, 2 H, Ar), 6.84 (d, J = 8.8 Hz, 2 H, Ar), 4.52 (d, J = 10.4 Hz, 1 H, CJi-O), 4.50 (d, J = 10.4 Hz, 1 H, CHj-O), 3.76 (s, 3 H, OCH3), 3.75 (s, 3 H, OCH3), 1.67 (s, 3 H, CH3), 1.18 (s, 9 H, C(CH3)3). 13C-NMR (125 MHz, CDCI3, 298K) 5 170.8 (COOCH3), 167.1 (N=CH), 159.3 (Ar), 129.6 (Ar), 129.3 (Ar), 113.7 (Ar), 82.4 (MeCOPMB), 67.6 (CH2-0), 57.7 (C(CH3)3), 55.2 (OCH3), 52.5 (OCH3), 22.4 (C(CH3)3), 21.2 (CH3). FAB (+) HRMS: Calcd. for C17H25NNa0 5S: (M+Na)+: m/e 378.13511; Found: mle 378.13362. 161 Alkene 382 - ? 9 0 AIIBr, In I ^ U THF. 55°C f-Bu"? VNH tr~0 Me / (85%) Me OPMB OMe Me OPMB 383 382 To sulfinimine 383 (200 mg, 562 mmol) in dry THF (1.9 mL) at rt under N2 was added Indium (84 mg, 731 mmol) followed by allylbromide (0.098 mL, 731 mmol) after 10 min. The reaction vessel was fitted with a reflux condenser and immersed for 40 min in an oil bath pre heated to 55 °C. The reaction mixture was then cooled to rt, quenched with water (5 mL) and extracted with EtOAc (3 x 10 mL). The combined organic extracts were dried (MgS04) and concentrated in vacuo. Purification by Si02 flash column chromatography (gradient elution 5:1 to 1:1 petrol:EtOAc) afforded alkene 382 as the major product of a mixture of 2 isomers in proportion 80/20 (189 mg, 85 %).103 IR (neat film) 3331 (w), 2951 (s), 1742 (s), 1612 (w), 1514 (m), 1462 (m), 1387 (w), 1250 (s), 1178 (w), 1119 (m), 1072 (s), 1037 (m), 912 (w), 820 (w). 1H-NMR (500 MHz, CDCI3, 298K) Major isomer fS) 6 7.30 (d, J = 8.6 Hz, 2 H, Ar), 6.85 (d, J = 8.6 Hz, 2 H, Ar), 5.71 (m, 1 H, HC=CH2), 4.97 (m, 2 H, H C ^ th ), 4.60 (d, J = 10.3 Hz, 1 H, CHa-O), 4.41 (d, J = 10.3 Hz, 1 H, CHa-O), 3.89 (d, J = 8.5 Hz, 1 H, NH), 3.77 (s, 3 H, OCH3), 3.74 (s, 3 H, COOCH3), 3.53 (dt, J = 9.1, 3.5 Hz, 1 H, CH-NH), 2.29 (m, 1 H, Ch^CH), 2.15 (m, 1 H, CH^CH), 1.68 (s, 3 H, CH3C), 1.16 (s, 9 H, C(CH3)3); Minor isomer (R) 6 7.27 (d, J = 8.6 Hz, 2 H, Ar), 6.84 (d, J = 8.6 Hz, 2 H, Ar), 5.94 (m, 1 H, IHC=CH2), 5.09 (m, 2 H, H C ^ tb ), 4.59 (d, J = 10.3 Hz, 1 H, CHa-O), 4.42 (d, J = 10.3 Hz, 1 H, CHa-O), 3.81 (d, J = 8.6 Hz, 1 H, NH), 3.77 (s, 3 H, OCH3), 3.68 (s, 3 H, COOCH3), 3.58 (m 1 H, CH-NH), 2.56 (m, 1 H, ChhCH), 2.40 (m, 1 H, ChbCH), 1.50 (s, 3 H, CH3C), 1.12 (s, 9 H, C(CH3)3). 13C-NMR (125 MHz, CDCI3, 298K) Major isomer (S) 6 173.8 (COOCH3), 159.1 (Ar), 135.4 (HC=CH2), 130.5 (Ar), 129.2 (Ar), 117.3 (HC=CH2), 113.6 (Ar), 82.1 (MeCOPMB), 66.5 (CH2- O), 63.5 (CH-NH), 56.8 (C(CH3)3), 55.2 (OCH3), 52.1 (COOCH3), 37.1 (CH3C), 22.9 (C(CH3)3), 20.0 (CH3) Minor isomer (R) 5 173.3 (COOCH3), 159.0 (Ar), 134.8 (HC=CH2), 130.6 (Ar), 128.9 162 (Ar), 118.0 (HC=CH2), 113.6 (Ar), 82.9 (MeCOPMB), 66.3 (CH2-0), 63.0 (CH-NH), 56.2 (C(CH3)3), 55.2 (OCH3), 51.8 (COOCH3), 35.8 (CH3C), 22.6 (C(CH3)3), 19.1 (CH3). FAB (+) HRMS: Calcd. for C20H31NNaO5S: (M+Na)+: m/e 420.18205; Found: m/e 420.18129. Alcohol 403 i)B H r THF THF, 2h f-Bu ii) H2 0 2 ,Et0H HO Buffer 7, 1 h Me OPMB (40%, 2 steps) Me OPMB 382 403 To alkene 382 (180 mg, 0.453 mmol) in dry THF (2.26 mL) was added a BH3-THF solution (1 M in THF, 0.22 mL, 0.226 mmol) at rt under N2 . The resulting solution was stirred at rt for 1.5 h and EtOH (0.9 mL), a pH 7 buffer solution (0.9 mL) and H20 2 (30% in water, 0.9 mL) were added. (The pH 7 buffer was made by dissolving 2 g of NaOH pellet in 1 L of water and by adding 6.8 g of KH2P04) The mixture was stirred for 1.5 h at rt, quenched slowly at 0 °C with sat. aq. Na2S203 (5 mL) and extracted with EtOAc (3x10 mL). The combined organic extracts were dried (MgS04) and concentrated in vacuo. Purification by Si02 flash column chromatography (gradient elution 5:1 to 1:3 petrol:EtOAc) afforded alcohol 403 as a white foam (88 mg, 40 %, 2 steps). [a]D + 8.5° (c 1.01, CH2CI2). IR (neat film) 3333 (br), 2947 (s), 2908 (s), 2874 (s), 2837 (s), 1740 (s), 1612 (m), 1514 (s), 1460 (w), 1381 (w), 1300 (w), 1259 (s), 1202 (w), 1177 (w), 1117 (m), 1034 (m), 933 (w), 823.5 (w). 1H-NMR (500 MHz, CDCI3, 298K) 6 7.27 (d, J = 8.7 Hz, 2 H, Ar), 6.84 (d, J = 8.7 Hz, 2 H, Ar), 4.57 (d, J = 10.3 Hz, 1 H, CH2-O), 4.40 (d, J = 10.3 Hz, 1 H, CFh-O), 3.89 (d, J = 8.5 Hz, 1 H, NH), 3.76 (s, 3 H, OCH3), 3.74 (s, 3 H, COOCH3), 3.54 (m, 2 H, H3), 3.42 (td, J= 10.9, 2.6 Hz, 1 H, CH-NH), 1.88 (s br, 1 H, OH), 1.69 (m, 1 H, H1), 1.65 (s, 3 H, CH3), 1.63 (m, 1 H, H2), 1.44 (m, 2 H, H1, H2), 1.18 (s, 9 H, C(CH3)3). 163 13C-NMR (125 MHz, CDCI3, 298K) 6 173.7 (COOCH3), 159.2 (Ar), 130.4 (Ar), 129.3 (Ar), 113.7 (Ar), 82.5 (MeCOPMB), 69.4 (C3), 66.6 (CH2-0), 62.8 (CH-NH), 56.8 (C(CH3)3), 55.2 (COOCH3), 52.2 (OCH3), 37.3 (CH3-C), 28.5 (C2), 26.0 (C1), 22.9 (C(CH3)3), 19.8 (CH3). FAB (+) HRMS: Calcd. for C2oH3iNna0 6S: (M+Na)+: mle 438.19262; Found: mle 438.23181. Mesylate 404 t-Bu + NH f-Bu * NH HO MsO OMe OMe C H 2CI2, rt Me OPMB 7 min (99%)(99%) Me OPMB 403 404 To a solution of alcohol 403 (460 mg, 1.11 mmol) in dry CH2CI2 (3.7 mL) was added dropwise CH3S02CI (0.17 mL, 0.22 mmol) and NEt3 (0.77 mL, 5.55 mL). The reaction mixture was stirred at rt for 7 min, quenched with water (10 mL) and extracted with CH2CI2 (3x10 mL). The combined organic extracts were dried (MgS04) and concentrated in vacuo. Purification by Si02 flash column chromatography (gradient elution 3:1 to 1:2 petrol:EtOAc) afforded the title compound 404 as a colorless oil (540 mg, 99%). [a]D - 27.3° (c 0.67, CH2CI2). IR (neat film) 2963 (s), 1741 (m), 1612 (w), 1514 (w), 1454 (w), 1356 (m), 1259 (s), 1173 (m), 1101 (s), 1026 (s), 930 (w), 800 (s). 1H-NMR (500 MHz, CDCI3, 298K) 5 7.27 (d, J = 8.7 Hz, 2 H, Ar), 6.84 (d, J = 8.7 Hz, 2 H, Ar), 4.59 (d, J = 10.3 Hz, 1 H, CH2-O), 4.40 (d, J = 10.3 Hz, 1 H, Chh-O), 4.13 (td, J = 6.07, 0.9 Hz, 2 H, H3), 3.86 (d, J = 8.4 Hz, 1 H, NH), 3.77 (s, 3 H, OCH3), 3.76 (s, 3 H, OCH3), 3.43 (td, J = 9.3, 2.9 Hz, 1 H, CH-NH), 2.93 (s, 3 H, CH3-S), 1.92 (m, 1 H, H2), 1.66 (s, 3 H,CH3), 1.63 (m, 2 H, H2, H1), 1.45 (m, 1 H, H1), 1.17 (s, 9 H, C(CH3)3). 164 13C-NMR (125 MHz, CDCI3i 298K) 6 173.9 (COOCH3), 159.1 (Ar), 130.5 (Ar), 129.3 (Ar), 113.7 (Ar), 82.5 (MeCOPMB), 66.5 (CH2-0), 63.2 (CH-NH), 62.1 (C3) 56.7 (C(CH3)3), 55.2 (COOCH3), 52.1 (OCH3), 29.5 (C1), 28.6 (C2), 22.9 (C(CH3)3), 19.7 (CH3). FAB (+) HRMS: Calcd. for C21H35NNa0 8S: (M+H)+: m/e 494.18822; Found: m/e 494.18907. Ester 405 t-Bu^¥ NH imidazole NaH, THF MsO 2 OMe OMe 0oC to rt Me OPMB Me OPMB 1 h (70%) 404 405 To mesylate 404 (367 mg, 0.743 mmol) in THF (2.5 mL) at 0 °C under N2 was added imidazole (5 mg, 0.074 mmol) followed by NaH (60% dispersion in oil, 60 mg, 1.490 mmol). The reaction mixture was stirred at 0 °C for 15 min and at rt for a further 1 h. The reaction mixture was quenched with MeOH (0.3 mL) and water (2 mL) and extracted with Et20 (3x5 mL). The combined organic extracts were dried (MgS04) and concentrated in vacuo. Purification by Si02 flash column chromatography (gradient elution 3:1 to 1:1 petrol:EtOAc) afforded the title compound 405 as a yellow oil (227 mg, 70%). [a]D + 38.6° (c 0.74, CH2CI2). IR (neat film) 3442 (s), 2953 (s), 1738 (s), 1612 (w), 1514 (m),1456 (m), 1383 (w), 1250 (s), 1173 (w), 1120 (m), 1078 (w), 1034 (w), 822 (w). 1H-NMR (500 MHz, CDCI3, 298K) 6 7.30 (d, J = 8.5 Hz, 2 H, Ar), 6.81 (d, J = 8.6 Hz, 2 H, Ar), 4.36 (s, 2 H, CH2-O), 4.03 (dd, J = 8.0, 2.7 Hz, 1 H, CH-N), 3.74 (s, 3 H, OCH3), 3.71 (s, 3 H, OCH3), 3.28 (m, 1 H, H3), 3.16 (m, 1 H, H3), 2.01 (m, 1 H, H2), 1.76 (m, 2 H, H2, H1), 1.68 (m, 1 H, H1), 1.56 (s, 3 H, CH3C), 1.15 (s, 9 H, C(CH3)3). 165 13C-NMR (125 MHz, CDCIa, 298K) 6 173.9 (COOCH3), 158.8 (Ar), 130.7 (Ar), 128.9 (Ar), 113.5 (Ar), 83.4 (MeCOPMB), 66.3 (CH2-0), 64.7 (CH-NH), 58.4 (C(CH3)3), 55.1 (COOCH3), 52.0 (OCH3), 50.2 (C3), 29.5 (C2), 28.6 (C1), 23.2 (C(CH3)3), 18.5 (CH3). FAB (+) HRMS: Calcd. for C17H25NNa0 5S: (M+Na)+: m/e 368.18954; Found: m/e 368.19047. Alcohol 406 - f-BuN / ° /-Buv / ° <; s DIBAL-H, THF A 0H Me OPMB Me OPMB 405 406 To ester 406 (255 mg, 0.641 mmol) in THF (2.2 mL) at -78 °C under N2 was added dropwise a solution of DIBAL-H (1.5 M in toluene, 0.94 mL, 1.28 mmol).104 The reaction mixture was stirred at -78 °C for 10 min and at rt for 1 h. A saturated aqueous solution of Rochelle’s salt (5 mL) was added followed by Et20 (10 mL). The mixture was stirred at rt for 1 h and extracted with Et20 (3x10 mL). The combined organic extracts were dried (MgS04) and concentrated in vacuo. Purification by Si02 flash column chromatography (gradient elution 4:1 to 1:1 petrohEtOAc) afforded alcohol 406 as a white foam (212 mg, 89%). [o ] d + 46.7° (c 0.51, CH2CI2). IR (neat film) 3391 (br), 2957 (s), 2874 (m), 2874 (s), 1612 (w), 1514 (s), 1462 (m), 1371 (w), 1300 (w), 1246 (s), 1173 (w), 1121 (w), 1070 (s), 1040 (s), 822 (w). 1H-NMR (500 MHz, CDCI3, 298K) 6 7.25 (d, J = 8.7 Hz, 2 H, Ar), 6.82 (d, J = 8.7 Hz, 2 H, Ar), 4.53 (d, J = 10.8 Hz, 1 H, CHj-OAr), 4.49 (d, J=10.8 Hz, 1 H, CH^O), 4.19 (dd, J = 8.4, 5.7 Hz, 1 H, CH-N), 3.80 (dd, J= 12.8 Hz, 1 H, ChbOH), 3.75 (s, 3 H, OCH3), 3.66 (dd, J= 12.8, 9.0 Hz, 1 H, ChbOH), 3.11 (m, 1 H, H3), 3.04 (m, 1 H, H3), 1.95 (m, 2 H, H1), 1.83 (m, 1 H, H2), 1.70 (m, 1 H, H2), 1.15 (s, 9 H, C(CH3)3), 1.06 (s, 3 H, CH3). 166 13C-NMR (125 MHz, CDCI3i 298K) 6 158.7 (Ar), 131.5 (Ar), 128.8 (Ar), 113.5 (Ar), 80.2 (MeCOPMB), 64.5 (CH2-OH), 64.1 (CH2-OAr), 60.1 (CHN), 58.7 (C(CH3)3), 55.2 (OCH3), 51.2 (C3), 26.5 (C1), 25.7 (C2), 22.9 (C(CH3)3), 16.7 (CH3). FAB (+) HRMS: Calcd. for C19H31NNa04S: (M+Na)+: mle 393.19496; Found: mle 393.19392. Aldehyde 381 TEMPO, BAIB A 0H CH2 CI2 (50%) Me OPMB Me OPMB To a solution of alcohol 406 (0.10 g, 0.271 mmol) in dry CH2CI2 (2.7 mL) at rt under N2 was added TEMPO (6.8 mg, 0.044 mmol) followed by [bis(acetoxy)iodo]benzene (141 mg, 0.439 mmol). The reaction mixture was stirred at rt for 5 h, quenched with sat. aq. Na2S203 (5 mL) and extracted with CH2CI2 (3x10 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude product was purified by Si02 flash chromatography (gradient elution: 5:1 to 1:1 petrol:EtOAc) to afford aldehyde 381 as a colorless oil (50 mg, 50%).104 [a]D + 39.4° (c 0.45, CH2CI2). IR (neat film) 2956 (s), 1730 (s), 1612 (w), 1514 (s), 1462 (m), 1385 (w), 1362 (w), 1302 (w), 1248 (s), 1175 (w), 1127 (w), 1088 (s), 1034 (s), 822 (w). 1H-NMR (500 MHz, CDCI3, 298K) 5 9.68 (s, 1 H, CHO), 7.24 (d, J = 8.7 Hz, 2 H, Ar), 6.84 (d, J = 8.7 Hz, 2 H, Ar), 4.46 (d, J= 10.8 Hz, 1 H, CFh-O), 4.28 (d, J = 10.8 Hz, 1H, CHj-O), 4.06 (t, J = 6.7 Hz, 1 H, CH-N), 3.78 (s, 3 H, OCH3), 3.19 (m, 1 H, H3), 3.11 (m, 1 H, H3), 1.89 (m, 2 H, H1), 1.72 (m, 2 H, H2), 1.37 (s, 3 H, CH3), 1.13 (s, 9 H, C(CH3)3). 167 13C-NMR (125 MHz, CDCI3i 298K) 5 204.1 (C=0), 159.2 (Ar), 130.2 (Ar), 129.2 (Ar), 113.7 (Ar), 84.5 (MeCOPMB), 66.1 (CH2-0), 62.2 (CH-NH), 58.5 (C(CH3)3), 55.2 (OCH3), 51.3 (C3), 29.7 (C1), 27.1 (C2), 24.9 (C(CH3)3), 14.1 (CH3). FAB (+) HRMS: Calcd. for C19H3oN04S: (M+H)+: m/e 368.18954; Found: mle 368.19047. Ester 421 ft Me i) NaN 0 2, H2 S0 4 jf V® M ») MeOH. AcCI M e O '" T o HO 1 OH ------A / - NH2 iii) 2,2-dimethoxypropane / M e . , PPTS, CH 2 CI2 Me L-threonine i i 421 (L)-Threonine (110 g, 0.92 mol) was suspended in water (250 mL) at -5 °C and was treated simultaneously while stirring with a solution of NaN02 (69 g, 1 mol) in water (100 mL) and concentrated H2S04 (27.9 mL, 0.5 mol) in water (75 mL). The two solutions were added at such a rate that the temperature remained between 0 °C and 5 °C. The solution was then stirred at rt for 20 h. The water was evaporated in vacuo and the remaining mixture treated with EtOH (150 mL). The salts were then filtered and the solution evaporated to dryness. The crude mixture was dissolved in MeOH (150 mL) and acetyl chloride (128 mL, 1.4 mol) was added very slowly at 0 °C under N2. The reaction mixture was then refluxed for 6 h. Evaporation of the solvent gave the crude dihydroxy ester which was dissolved in CH2CI2 (150 mL) and treated with 2,2-dimethoxypropane (226 mL, 1.84 mol) and PPTS (23 g, 0.092 mol). The reaction mixture was stirred at rt for 20 h, quenched with aq. sat. NaHC03 and extracted with CH2CI2 (3 x 150 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. Distillation (70-75 °C, 1 mmHg) afforded methyl ester 421 (70 g, 43%) as a colorless oil.108 [a]D -17.1° (c 1, CHCI3). 1H-NMR (500 MHz, CDCI3, 298K) 5 4.01 (dq, J = 8.0, 6.0 Hz, 1 H, CH-CH3), 3.87 (d, J = 8.0 Hz, 1 H, CH-C=0), 3.60 (s, 3 H, 0-CH3), 1.37 (s, 3 H, CH3), 1.35 (s, 3 H, CH3) 168 13C-NMR (125 MHz, CDCI3i 298K) 6 170.8 (C=0), 110.5 (C(CH3)2), 80.3 (CH-C=0), 75.0 (CH- CH3), 52.3 (0-CH3), 27.1 (C(CH3)2), 25.6 (C(CH3)2), 18.4 (CH-CH3). The spectral data for this molecule corresponded with that in the literature. Dibromide 422 i) DIBAL-H, ether, -78°C ii) Soxhlet, CH2CI2, reflux M eO o P iii) Zn, CBr4, PPh3, CH2Cl2 ... ^ M p (41%, 2 steps) Br “ " /'M e 421 Me 42 2 Me To a solution of the ester 421 (15 g, 83.1 mmol) in dry ether (90 mL) at -78 °C was added dropwise over 30 min via addition funnel, a solution of DIBAL-H in hexanes (1 M, 100 mL, 100 mmol). The reaction mixture was stirred at -78 °C for 45 min and quenched with a saturated aqueous solution of Rochelle’s salt (100 mL). The mixture was poured into a conical flask containing Et20 (200 mL), stirred at rt for 1 h and extracted with Et20 (3 x 250 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo to afford a mixture of the aldehyde and its hydrate. The crude oil was dissolved in CH2CI2 (120 mL) and the mixture was heated at reflux for 20 h in a Soxhlet apparatus containing activated 4 A powdered molecular sieves. The reaction mixture was directly added to a suspension which has been prepared as follows. To a suspension of CBr4 (55.0 g, 166 mmol) and Zn (10.85 g, 166 mmol) at 0 °C in CH2CI2 (320 mL) was added PPh3 by portions ove 20 min (43.50 g, 166 mmol). After 24 h at rt, the crude aldehyde in CH2CI2 was added via canula over 10 min at 0 °C. The reaction mixture was stirred at 0 °C for 2 h and then poured onto petrol (200 mL) and filtered through Celite®. The filtrate was concentrated in vacuo, the residue was filtered through Celite® and the Celite®was washed with petroleum ether (5 x 100 mL). The procedure was repeated until no dibromoolefin was detected by TLC. The crude oil was purified by Si02 flash chromatography (20:1 petrol:EtOAc) to afford the desired unstable product 422 as a colorless oil (10.3 g, 41% over 2 steps).109 [a]D - 3.1° (c 1, CHCI3). 169 IR (neat film) 3460 (br, w), 2984 (s), 2934 (m), 2874 (m), 1622 (m), 1452 (m), 1377 (s), 1242 (s), 1173 (s), 1092 (s), 1038 (s), 926 (m), 862 (s), 810 (m), 783 (s). 1H-NMR (500 MHz, CDCI3, 298K) 6 6.42 (d, J = 7.9 Hz, 1 H, Br2C=CH), 4.21 (t, J = 8.3 Hz, 1 H, OCHC=), 3.86 (m, 1 H, OCHCH3), 1.80 (br, OH), 1.43 (s, 3 H, CCH3), 1.37 (s, 3 H, CCH3), 1.32 (d, J = 6.2 Hz, 3 H, CHCH3). 13C-NMR (125 MHz, CDCI3, 298K) 6 135.4 (Br2C=CH), 109.3.0 (C(CH3)2), 93.8 (Br2C=), 82.1 (OCHC=), 75.8 (OCHCH3), 27.2 (CCH3), 26.7 (CCH3), 17.0 (CHCH3). The spectral data for this molecule corresponded to those reported in the literature. Alcohol 423 Me ii) HCHO (solid) To a solution of the dibromo olefin 422 (5.4 g, 18.1 mmol) in dry THF (80 mL) at -78 °C under N2 was added dropwise over 5 min a solution of n-BuLi (2.5 M in hexanes, 16 mL, 39.9 mmol). The reaction mixture was stirred at -78 °C for 1 h and then allowed to warm to rt where it was stirred for a further 1.5 h before being cooled to -78 °C. Solid paraformaldehyde (1.0 g, 36.2 mmol) was added in one portion and the reaction was held at -78 °C for 30 min before being warmed to rt where it was stirred for 1 h. Brine (100 mL) was added to the reaction mixture which was then extracted with Et20 (3 x 100 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude product was quickly purified by Si02 flash chromatography (gradient elution: 10:1 to 3:1 petrol:EtOAc) to afford the propargylic alcohol 423 as a non stable colorless oil (2.83 g, 92%). The structure of alcohol 423 was confirmed by its 500 MHz 1H-NMR spectrum in CDCI3 and was used directly without further characterisation. 170 1H-NMR (500 MHz, CDCI3, 298K) 6 4.29 (s br, 2 H, CH2OH), 4.15 (dt, J = 8.2, 1.6 Hz, 1 H, OCHCEC), 4.06 (ddd, J = 11.9, 8.2, 6.0 Hz, 1 H, OCHCH3), 1.80 (br, OH), 1.42 (s, 3 H, CCH3), I.39 (s, 3 H, CCH3), 1.32 (d, J = 6.0 Hz, 3 H, CHCH3) . Bromide 420 CBr4> PPh3 0 .0 THF 0 - 7 C 4 2 3 Me Me (63%) ______4 2 0 Me Me To the propargylic alcohol 423 (4.1g, 24 mmol) and PPh3 (12.6 g, 48 mmol) in dry THF (120 mL) at 0 °C was added by portions over 30 min, CBr4 (16 g, 48 mmol). The resulting brown mixture was stirred at 0 °C for 20 min and petrol (100 mL) and ether (100 mL) were added. Triphenylphosphine oxide was removed by filtration through Celite® and the filtered pad washed with petrol (3 x 150 mL). The procedure was repeated until no bromide was detected by TLC. The filtrate and washings were concentrated in vacuo. The crude oil was quickly purified by Si02 flash chromatography (gradient elution 40:1 to 20:1 petrol:EtOAc) to afford 420 as an unstable yellow oil (3.5 g, 63%). The structure of 420 was confirmed by its 500 MHz 1H-NMR spectrum in CDCI3 and was used directly without further characterisation. 1H-NMR (500 MHz, CDCI3) 298K) 6 4.15 (dt, J = 8.2, 1.9 Hz, 1 H, OCHCEC), 4.05 (ddd, J = II.9 , 8.2, 6.0 Hz, 1 H, OCHCH3), 4.29 (d, J = 1.8 Hz, 2 H, Ch^Br), 1.42 (s, 3 H, CCH3), 1.40 (s, 3 H, CCH3), 1.30 (d, J = 6.0 Hz, 3 H, CHCH3). 171 Alkyne 337 KHMDS, THF l^lg -TS'C to 0°C Ph 424 To a solution of 424 (0.91 g, 3.90 mmol) in THF (8 mL) at -78 °C under N2 was added dropwise over 10 min a solution of KHMDS (0.5 M in PhMe, 9.36 mL, 4.68 mmol). The reaction mixture was stirred at -78 °C for 1 h. A solution of bromide 420 (0. 91 g, 3.90 mmol) in THF (10 mL) was added dropwise over 10 min via canula. The reaction mixture was stirred at -78 °C for 40 min and at 0 °C for 2 h. The mixture was then quenched with sat. aq. NH4CI (10 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude product was purified by Si02 flash chromatography; gradient elution: 20:1 to 6:1 petrol:EtOAc to recover the unreacted starting material 424 (0.2 g, 22%) and 5:1 to 4:1 to afford alkyne 337 in the form of a colorless oil (0.685 g, 45%). [a]D + 30.2° (c 0.45, CH2CI2). IR (neat film) 2982 (w), 2922 (m), 2852 (w) 1780 (s),1699 (m), 1454 (w), 1385 (m), 1242 (m), 1213 (m), 1171 (w), 1094 (m), 1028 (m). 1H-NMR (500 MHz, CDCI3, 298K) 6 7.35-7.26 (m, 5 H, Ar), 4.66 (m, 1 H, CH-N), 4.17 (dd, J = 16.7, 9.0 Hz, 1 H, CH^O), 4.14 (dd, J = 9.0, 3.2 Hz, 1 H, ChhO), 4.09 (dt, J = 8.1, 1.9 Hz, 1 H, CEC-CH-O), 3.99 (qd, J= 8.1, 6.0 Hz, 1 H, CH3-CH-0), 3.91 (m, 1H, CH2-CH-CH3), 3.27 (dd, J = 13.4, 3.3 Hz, 1 H, CJ^Ar), 2.74 (dd, J = 13.4, 9.5 Hz, 1 H, CFbAr), 2.61 (ddd, J = 16.9, 6.9, 2.0 Hz, 1H, CH-CFh-CE), 2.53 (ddd, J = 16.9, 6.9, 2.0 Hz, 1 H, CH-CHs-CE), 1.35 (s, 3 H, CCH3), 1.34 (s, 3 H, CCH3), 1.28 (d, J = 6.0 Hz, 3 H, CH3CHO), 1.19 (d, J = 6.9 Hz, 3 H, CH3CH). 13C-NMR (125 MHz, CDCI3, 298K) 6 174.9 (CHC=0), 152.9 (0-C=0), 135.1(C, Ar), 129.3 (CH, Ar), 128.9 (CH, Ar), 127.3 (C, Ar), 109.2 (C(CH3)2), 84.1 (CH2-CEC), 77.9 (CEC-CHO), 77.5 172 (CH3-CH-0), 72.1 (CEC-CH-O), 66.1 (CH20), 55.2 (CH-N), 37.9 (CH2Ar), 37.2 (CH2CHCH3), 27.1 (C(CH3)2), 26.3 (C(CH3)2), 16.8 (CH3CHO), 16.5 (CH3CH). FAB (+) HRMS: Calcd. for C22H27NNa05: (M+Na)+: m/e 408.17868; Found: m/e 408.178770. Stannane 425 PhMe, rt (72%) 337 425 426 Ph3SnH (0.418 g, 1.19 mmol) was weighed in a glove bag and added to a round- bottomed flask containing alkyne 337 (0.230 g, 0.596 mmol). The flask was immediately purged with N2 and dry PhMe (0.6 mL) was added. Et3B (1 M solutions in hexanes, 0.21 mL, 0.21 mmol) was then added dropwise. Air was injected into the reaction vessel to initiate the reaction. The reactants were stirred at rt overnight. H20 (10 mL) was added and the mixture was extracted with EtOAc (3 x 10 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude residue was purified by Si02 flash chromatography (gradient elution: 100:1 to 25:1 petrol:EtOAc) without separation of the individual vinylstannanes 425 and 426 from each other. 500 MHz NMR analysis of this unseparated mixture in CDCI3 indicated that vinylstannanes 425 and 426 were present in 10:1 ratio. The mixture was obtained in the form of a colorless oil (0.33 g, 75%).105 The structure of the minor isomer 426 is only assigned tentatively. [a]D + 15.1° (c 0.33, CH2CI2). IR (neat film) 3061 (w), 2980 (m), 2928 (m), 2862 (w), 1780 (s), 1699 (m), 1614 (w), 1429 (m), 1383 (s), 1213 (s), 1097 (m), 1024 (m), 972 (w), 918 (w), 731 (m), 700.1 (m), 509 (w), 451 (w). 1H-NMR (500 MHz, CDCI3, 298K) 5 7.64-7.13 (m, 20 H, Ar), 6.60 (dd, J = 6.62, 8.20 Hz ( 3J 117Sn-1H = 149 Hz), 1 H, HC=CSn), 4.54 (m, 1 H, CH-N), 4.10 (dd, J= 9.0 Hz, 1 H, CFhO), 4.07 173 (ddd, J = 9.0, 3.1 Hz, 1 H, CH2O), 3.75 (m, CH3-CH-0), 3.62 (m, CH3-CH-C=0), 3.12 (dd, J = 13.4, 3.3 Hz, 1 H, C h h A r), 2.74 (d d , J = 13.4, 9.5 Hz, 1 H, CHzAr), 2.61 (m, 1 H, C ^ C H ^ , 2.17 (m, 1 H, CH2CH=), 1.25 (s, 3 H, CCH3), 1.20 (d, J = 6.1 Hz, 3 H, CH3CH-O), 0.79 (d, J = 6.9 Hz, 3 H, CH3CHC=0), 0.66 (s, 3 H, CCH3). 13C-NMR (125 MHz, CDCI3, 298K) 5 176.0 (CHC=0), 152.9 (0-C=0), 144.3 (HC=CSn), 140.1, 139.7 (CSn), 137.1 129.5, 129.3, 129.1, 128.9, 128.8, 128.5, 128.3, 127.3, 107.9 (C(CH3)2), 89.3 (CHN), 65.9 (CH2-0), 55.3 (CH-N), 38.0 (CH2Ar), 37.6 (CH3CHC=0), 37.4 (CH2CH=) 27.2 (C(CH3)2), 25.9 (C(CH3)2, 16.7 (CH3CHO), 16.4 (CH3CHC=0). FAB (+) HRMS: Calcd. for C4oH43NNa0 5Sn: (M+Na)+: mle 760.20608; Found: m/e 760.20449. Iodide 427 Me Me Ph Ph Me Me 0 °C, (80%) Me 425 427 To a stirred solution of vinyl triphenylstannane 425 (250 mg, 0.338 mmol) in dry CH2CI2 (4 mL)at 0 °C under N2 was added solid l2 (103 mg, 0.406 mmol) in one portion. The mixture was stirred at rt for 1 h. The solvent was removed in vacuo and the residue purified by Si02 flash chromatography (gradient elution: 20:1 to 6:1 petrol:EtOAc) to give vinyl iodide 427 as a pale yellow oil (140 mg, 80%).107 [a]D + 41.8° (c 0.16, CH2CI2). IR (neat film) 2982 (m), 2930 (m), 2851 (w) 1780 (s), 1724 (m), 1699 (m), 1454 (w), 1383 (m), 1350 (w), 1242 (s), 1211 (m), 1175 (w), 1099 (m), 1036 (m), 974 (w), 858 (w), 731 (m), 702 (m). 1H-NMR (500 MHz, CDCI3, 298K) 6 7.34-7.17 (m, 5 H, Ar), 6.14 (t, J = 6.8 Hz, 1 H, HC=CI), 4.66 (m, 1 H, CH-N), 4.18 (dd, J= 16.5, 9.1 Hz, 1 H, Ch^O), 4.15 (dd, J = 9.1, 3.1 Hz, 1 H, 174 ChhO), 4.0 (qd, J = 8.0, 6.0 Hz, 1 H, CH3-CH-0), 3.90 (m, 1 H, CH3-CH-C=0) 3.59 (d, J = 8.0 Hz, 1 H, IC-CH-O), 3.27 (dd, J = 13.4, 3.1 Hz, 1 H, ChbAr), 2.77 (dd, J = 13.4, 9.9 Hz, 1 H, ChbAr), 2.63 (m, 1 H, CH-CH2-CH=C), 2.54 (m, 1 H, CH-CFL-CHC), 1.48 (s, 3 H, C(CH3)2), 1.41 (s, 3 H, C(CH3)2), 1.28 (d, J = 6.1 Hz, 3 H, CH3CHO), 1.19 (d, J = 6.8 Hz, 3 H, CH3CH). 13C-NMR (125 MHz, CDCI3, 298K) 6 175.8 (CHC=0), 153.1 (0-C=0), 136.4 (HC=CI), 135.2 (Ar), 129.4 (Ar), 129.0 (Ar), 127.4 (Ar), 109.1 (C(CH3)2), 109.0 (HC=CI), 87.8 (IC-CH-O), 77.6 (CH3-CH-0), 66.1 (CH20), 55.4 (CH-N), 39.6 (CH-CH2-CH=C), 38.1 (CH2Ar), 36.8 (CH3-CH- CH2), 27.7 (C(CH3)2), 27.1 (C(CH3)2, 16.8 (CH3CHO), 16.4 (CH3CH). FAB (+) HRMS: Calcd. for C22H28INNa05: (M+Na)+: mle 536.09044; Found: mle 536.09098. Alkene 419 Me Me 4 Sn, Cul, Ph3As Me (MeCN) 2 PdCI2 Ph Ph Me DMF, NEt3, 140 'C Me Me (72%) 427 419 To a solution of vinyl iodide 427 (57 mg, 0.11 mmol) in dry DMF (1.1 ml_) and freshly distilled Et3N (1.1 mL, 7.89 mmol) at rt under N2 was added Me4Sn (0.05 ml_, 0.36 mmol), Cul (2 mg, 0.011 mmol), and Ph3As (3.3 mg, 0.011 mmol) followed by (MeCN)2PdCI2 (2.8 mg, 0.011 mmol). The reactants were heated at 130 °C for 2 h, cooled to rt, and H20 (5 mL) was added. After extraction with Et20 (3x5 mL), the combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude product was purified by Si02 flash chromatography (gradient elution: 100:1 to 6:1 petrol:EtOAc) to afford alkene 419 in the form of a colorless oil (32 mg, 72%).107 [a]D + 4.4° (c 0.20, CH2CI2). IR (neat film) 2980 (m), 2926 (m), 2876 (w) 1780 (s), 1724 (m), 1452 (w), 1383 (m), 1242 (s), 1097 (m), 1036 (w), 974 (w), 729 (m). 175 1H-NMR (500 MHz, CDCI3, 298K) 5 7.35-7.15 (m, 5 H, Ar), 5.53 (t, J = 7.2 Hz, 1 H, HC=CCH3), 4.66 (m, 1 H, CH-N), 4.15 (dd, J= 16.5, 9.1 Hz, 1 H, CH2O), 4.15 (dd, J = 9.1, 3.1 Hz, 1 H, CHzO), 3.83 (m, 3 H, CH3-CH-0, CH3-CH-CH2, IC-CH-O), 3.26 (dd, J= 13.4, 3.4 Hz, 1 H, ChbAr), 2.69 (dd, J = 13.4, 9.9 Hz, 1 H, Cj^Ar), 2.50 (m, 1 H, CH-CH2-CH=), 2.27 (m, 1 H, CH- CH2-CH=), 1.67 (s, 3 H, =C(CH3)), 1.38 (s, 3 H, C(CH3)2), 1.37 (s, 3 H, C(CH3)2), 1.20 (d, J = 5.6 Hz, 3 H, CH3CHO), 1.15 (d, J = 6.8 Hz, 3 H, CH3CH). 13C-NMR (125 MHz, CDCI3, 298K) 5 176.6 (CHC=0), 153.1 (0-C=0), 135.3 (Ar), 127.3 (Ar), 126.3 (HC=CCH3), 108.0 (C(CH3)2), 109.0 (HC=CI), 88.4 (CH3C-CH-0), 74.4 (CH3-CH-0), 66.0 (CH20), 55.4 (CH-N), 38.0 (CH2Ar), 37.4 (CH2CHCH3), 31.9 (CH-CH2-CH=), 27.5 (C(CH3)2), 26.9 (C(CH3)2, 17.0 (CH3CHO), 16.4 (C=CCH3). FAB (+) HRMS: Calcd. for C23H31NNa05: (M+Na)+: m/e 424.20998; Found: m/e 424.21073. Alkyne 429 (1) nBuLi in Hex, THF Me' 420 Me Me in HMPA 428 429 (84%) Me Me To a solution of compound 428111 (1.16 g, 4.29 mmol) in THF (21 mL) at -78 °C under N2 was added dropwise over 10 min a solution of n-BuLi (2.5 M in Hexanes, 1.7 mL, 4.29 mmol). The reaction mixture was stirred at -78 °C for 1 h and a solution of bromide 420 (1.0 g, 4.3 mmol) in HMPA (2.24 mL, 12.9 mmol) was added dropwise via canula over 10 min. The reaction mixture was stirred at -78 °C for 4 h, quenched with water (20 mL) and extracted with Et20 (3 x 20 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude product was purified by Si02 flash chromatography (gradient elution: 50:1 to 20:1 petrol:EtOAc) to afford alkyne 429 in the form of a white foam (1.53 g, 84%). [a]D + 52.2° (c1,CH2CI2). 176 IR (neat film) 2980 (s), 2887 (m), 1697 (s), 1456 (w), 1381 (m), 1331 (s), 1267 (m), 1236 (s), 1169 (m), 1128 (m), 1093 (w), 1061 (w), 1030 (m), 976 (w), 860 (w), 769 (w), 540 (m). 1H-NMR (500 MHz, CDCI3, 298K) 6 4.03 (dt, J= 8.3, 1.8 Hz, 1 H, CEC-CH-O), 3.96 (qd, J = 8.1, 5.9 Hz, 1 H, CH3-CH-O), 3.85 (dd, J = 7.5, 5.0 Hz, 1 H, CH-N), 3.44 (q, J= 13.8 Hz , 2H, 0 2S- CH2), 3.25 (m, 1 H, CH-CH3), 2.55 (ddd, J= 16.6, 6.4, 1.8 Hz, 1 H, CH-Ch^-CE), 2.49 (ddd, J = 16.6, 6.4, 1.8 Hz, 1 H, CH-Ch^-CE), 2.04 (m, 2 H, CH2-CHN), 1.85 (m, 3 H, CHz-CH-Chh, CHC(CH3)2), 1-38 (s, 3 H, 0 2C(CH3)2), 1.36 (m, 2 H, CCH2-CH2) 1.35 (s, 3 H, 0 2C(CH3)2 , 1.27 (d, J = 6.0 Hz, 3 H, CH3CHO), 1.19 (d, J = 6.6 Hz, 3 H, CH3CH), 1.14 (s, 3 H, HCC(CH3)2), 0.94 (s, 3 H, HCC(CH3)2). 13C-NMR (125 MHz, CDCI3, 298K) 6 174.0 (C=0), 109.1 (C(CH3)2), 83.6 (CH2-CEC), 78.1 (CEC-CHO), 77.5 (CH3-CH-O), 72.0 (CEC-CH-O), 65.2 (CH-N), 53.1 (CH2-S), 48.3 (CCH3 (Xc)), 47.7 (C-CH2S), 44.6 (CHC(CH3)2), 39.0 (CH-CH3), 38.4 (CH2-CHN), 32.8 (CH2), 27.1 (02C(CH3)2), 26.4 (CH2-CH2), 26.4 (HC(CH3)2), 24.3 (CH-CH2-CEC), 20.9 (HC(CH3)2), 19.8 (02C(CH3)2), 16.6 (CH3CHO), 16.0 (CH3CH). FAB (+) HRMS: Calcd. for C22H33NNa0 5S: (M+Na)+: m/e 446.19770; Found: m/e 446.19707. Alkyne 430 Ti(OEt)4. EtOH Me 130°C, 3 days (100%) To a solution of alkyne 429 (760 mg, 1.8 mmol) in absolute EtOH (36 mL) was added Ti(OEt)4 (3.77 mL, 18 mmol) in one portion. The resulting heterogeneous mixture was heated at reflux for 3 days. After being cooled to 0°C, the mixture was quenched with 1M aqueous HCI (15 mL) and extracted with Et20 (3 x 200 mL). The combined organic extracts were washed with sat. aq. NaHC03 (50 mL), dried (MgS04), filtered and concentrated in vacuo to give a 177 mixture of ethyl ester 430 and recovered sultam 436. The crude residue was purified by Si02 flash chromatography (20:1 petrol:EtOAc) to afford ethyl ester 430 as a colorless oil (455 mg, 100%) and (5:1 petrol:EtOAc) to recover the auxiliary 373 (370 mg, 96%).112 [a]D - 2.18° (c1.01,CH2CI2). IR (neat film) 2984 (s), 2933 (m), 1736 (s), 1456 (w), 1431 (m), 1377 (m), 1234 (m), 1175 (s), 1099 (m), 1028 (s), 858 (w). 1H-NMR (500 MHz, CDCI3) 298K) 6 4.08 (q, J = 7.1 Hz, 2 H, CH2-O), 4.07 (dt, J = 8.1, 1.9 Hz, 1 H, CEC-CH-O), 3.97 (qd, J = 8.1, 6.1 Hz, 1 H, CH3-CH-O), 2.58 (m, 1 H, CH-CH3), 2.45 (ddd, J = 16.7, 5.9, 1.9 Hz, 1 H, CI^-CH), 2.36 (ddd, J = 16.7, 7.6, 1.9 Hz, 1H, CH2-CH), 1.39 (s, 3 H, CCH3), 1.36 (s, 3 H, C CH3), 1.27 (d, J = 6.0 Hz, 3 H, CH3-CH-0), 1.22 (t, J = 7.1 Hz, 3 H, CH3CH20), 1.21 (d, J = 7.0 Hz, 3 H, CH3CH). 13C-NMR (125 MHz, CDCI3, 298K) 5 174.7 (C=0), 109.2 (C(CH3)2), 84.5 (CEC), 77.6 (CH3-CH- O), 72.1 (CEC-CH-O), 60.5 (CH2-0), 38.7 (CH-CH3), 27.1 (CCH3), 26.3 (CCH3), 22.9 (CH2-CH), 16.8 (CH3CH), 16.3 (CH3-CH-0), 14.2 (CH3CH20). FAB (+) HRMS: Calcd. for C14H22Na04: (M+Na)+: m/e 277.14157; Found: mle 277.14189. Stannane 431 Me Ph3SnH Ph3Sn Me X ^ Et3 B, Air EtO O Tol CD (92%) EtO O /Me 430 M e 431 Me Ph3SnH (1.77 g, 5.03 mmol) was weighed in a glove bag and added to a round- bottomed flask containing alkyne 430 (0.640 g, 2.52 mmol). The flask was immediately purged with N2 and dry PhMe (2.52 mL) was added. Et3B (1 M solutions in hexanes, 0.76 mL, 0.756 mmol) was then added dropwise. Air was injected into the reaction vessel to initiate the reaction. The reactants were stirred at rt overnight. H20 (30 mL) was added and the mixture 178 was extracted with EtOAc (3 x 30 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude product was purified by Si02 flash chromatography (gradient elution: 100:1 to 25:1 petrol:EtOAc) to afford the vinylstannane 431 as a single isomer in the form of a colorless oil (1.40 g, 92%).105 [a]D- 19.1° (c 0.42, CH2CI2). IR (neat film) 3059 (w), 2980 (s), 2931 (m), 2866 (w), 1732 (s), 1454 (w), 1431 (m), 1375 (s), 1229 (m), 1180 (s), 1095 (m), 1026 (m), 858 (w), 731 (s), 700 (s). 1H-NMR (500 MHz, CDCI3l 298K) 5 7.56 (m, 6 H, Ar), 7.32 (m, 9 H, Ar), 6.55 (ddd, J = 8.4, 6.4, 1.2 Hz ( 3J 11?Sn-1H = 150 Hz), 1 H, HC=CSn), 3.16 (dd, J = 8.4, 1.1 Hz, 1 H, SnC-CH-O), 4.01 (q, J = 7.2, 1.7 Hz, 2 H, CHj-O), 3.73 (qd, J = 8.4, 1.7 Hz, 1 H, CH3-CH-0), 2.32 (m, 1 H, CHb- CH), 2.13 (m, 2 H, CHs-CH, CH-CH3), 1.25 (s, 3 H, CCH3), 1.19 (d, J= 6.0 Hz, 3 H, CH3-CH-0), 1.16 (t, J= 7.1 Hz, 3 H, CH3CH20), 0.72 (d, J = 6.9 Hz, 3 H, CH3CH), 0.66 (s, 3 H, CCH3). 13C-NMR (125 MHz, CDCI3, 298K) 5 175.4 (C=0), 144.7 (HC=CSn), 139.6 (HC=CSn), 137.0 (Ar), 128.9 (Ar), 128.7 (Ar), 108.0 (C(CH3)2), 89.3 (SnC-CH-O), 77.1 (CH3-CH-0), 60.2 (CH20), 39.8 (CH-CH3), 37.4 (CH2CH=), 27.2 (CCH3), 25.9 (CCH3), 16.6 (CH3CH), 16.5 (CH3CHO), 14.2 (CH3CH20). FAB (+) HRMS: Calcd. for C32H38Na04Sn: (M+Na)+: m/e 629.16896; Found: m/e 629.16654. Iodide 432 Me Me 0 °C (77%) Me EtO' EtOMe Me 431 432 To a stirred solution of vinyl triphenylstannane 431 (1.2 g, 2.00 mmol) in dry CH2CI2 (20 mL) at 0 °C under N2 was added solid l2 (0.604 g, 2.38 mmol) in one portion. The mixture was stirred at rt for 1 h. The solvent was removed in vacuo and the residue purified by Si02 flash 179 chromatography (gradient elution: 40:1 to 10:1 petrol:EtOAc) to give vinyl iodide 432 as a pale yellow oil (0.600 g, 77 %).107 [a]D + 4.8° (c 1.19, CH2CI2). IR (neat film) 3439 (w), 2995 (s), 2931 (m), 2908 (s), 1732 (s), 1454 (w), 1377 (m), 1234 (m), 1178 (s), 1101 (m), 1036 (m), 860 (w). 1H-NMR (500 MHz, CDCI3) 298K) 5 6.07 (ddd, J = 7.2, 6.3, 0.9 Hz, 1 H, HC=CI), 4.10 (q, J = 7.2 Hz, 2 H, CHz-O), 3.97 (qd, J = 8.0, 6.0 Hz, 1 H, CH3-CH-0), 3.55 (d, J = 8.0 Hz, 1 H, IC-CH-O), 2.56 (m, 1 H, CH-CH3), 2.52 (m, 1 H, CHz-CH), 2.39 (m, CFh-CH), 1.48 (s, 3 H, CCH3), 1.40 (s, 3 H, CCH3), 1.26 (d, J = 6.0 Hz, 3 H, CH3CHO), 1.22 (t, J= 7.1 Hz, 3 H, CH3CH20), 1.17 (d, J = 6.9 Hz, 3 H, CH3CH). 13C-NMR (125 MHz, CDCI3, 298K) 6 175.4 (C=0), 136.6 (HC=CI), 109.1 (C(CH3)2), 108.6 (HC=CI), 87.7 (C=IC-CH-0), 76.8 (CH3-CH-0), 60.5 (CH2-0), 39.3 (CH2CH=), 38.5 (CH-CH3), 27.7 (CCH3), 27.0 (CCH3), 16.9 (CH3CH), 16.7 (CH3-CH-0), 14.2 (CH3CH20). FAB (+) HRMS: Calcd. for C14H23INa04: (M+Na)+: m/e 405.05387; Found: m/e 405.054665. Alkene 433 Me Me4Sn, Ph3As Cul, (MeCN)2PdCI2 Me/( Me CH2CI2, ISO'C, 3h Me (80%) EtO' Me EtO Me 432 433 To a solution of vinyl iodide 432 (600 mg, 1.53 mmol) in dry DMF (15.3 mL) and freshly distilled Et3N (15.3 mL, 109 mmol) at rt under N2 was added Me4Sn (0.70 mL, 5.05 mmol), Cul (29 mg, 0.153 mmol), and Ph3As (47 mg, 0.153 mmol) followed by (CH3CN)2PdCI2 (39 mg, 0.153 mmol). The reactants were heated at 130 °C for 3 h, cooled to rt, and H20 (50 mL) was added. After extraction with Et20 (3 x 100 mL), the combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude product was purified by Si02 flash 180 chromatography (gradient elution: 100:1 to 20:1 petrol:EtOAc) to afford alkene 433 in the form of a colorless oil (330 mg, 80%).107 [a]D- 8.1° (c 0.26, CH2CI2). IR (neat film) 2980 (s), 2931 (m), 2878 (w), 1734 (s), 1456 (w), 1375 (m), 1240 (m), 1173 (m), 1097 (m), 1034 (m), 864 (w). 1H-NMR (500 MHz, CDCI3, 298K) 5 5.45 (t, J = 6.9 Hz, 1 H, HC=CMe), 4.09 (q, J = 7.2 Hz, 2 H, CHz-O), 3.82 (m, 1 H, CH3-CH-0), 3.80 (m, 1 H, C=C-CH-0), 2.45 (m, 1 H, CH-CH3), 2.38 (m, 1 H, CH2-CH), 2.20 (m, 1 H, Chh-CH), 1.63 (s, 1 H, C=CCH3), 1.39 (s, 6 H, C(CH3)2), 1.22 (t, J = 7.1Hz, 3 H, CH3CH20), 1.18 (d, J= 5.6 Hz, 3 H, CH3CHO), 1.11 (d, J= 7.0 Hz, 3 H, CH3CH). 13C-NMR (125 MHz, CDCI3, 298K) 6 176.0 (C=0), 133.1 (MeC=CH), 126.4 (HC=CMe), 108.0 (C(CH3)2), 88.3 (C=C-CH-0), 74.5 (CH3-CH-0), 60.3 (CH2-0), 39.4 (CH-CH3), 31.7 (CH2CH=), 27.5 (CCH3), 26.8 (CCH3), 17.0 (CH3CH), 16.4 (CH3CHO), 14.2 (CH3CH20), 11.8 (C=CCH3). FAB (+) HRMS: Calcd. for C15H26Na04: (M+Na)+: m/e 293.17287; Found: m/e 293.17287. Alcohol 434 Me Me Me Dibal-H. THF (75%) EtO' Me HO' Me Me Me 433 434 To a solution of alkene 433 (190 mg, 0.703 mmol) in dry THF (2.34 mL) at rt under N2 was added dropwise over 5 min a solution of DIBAL-H (1.5 M in toluene, 1.16 mL, 1.75 mmol). The reaction mixture was stirred for 1 h and a saturated aqueous solution of Rochelle’s salt (10 mL) and ether (10 mL) were added. The resulting mixture was stirred at rt for 1 h and extracted with Et20 (3 x 20 mL). The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude product was purified by Si02 flash chromatography (gradient elution: 10:1 to 6:1 petrol:EtOAc) to afford alcohol 434 as a white foam (1.20 g, 75%). 181 [a]D + 7.4° (c 0.68, CH2CI2). IR (neat film) 3433 (br), 2980 (s), 2928 (m), 2878 (m), 1726 (w), 1454 (w), 1377 (m), 1238 (s), 1175 (m), 1095 (m), 1034 (s), 862 (w). 1H-NMR (500 MHz, CDCI3, 298K) 5 5.50 (qd, J = 6.9 Hz, 1 H, HC=CMe), 3.84 (m, 1 H, CH3-CH- O), 3.82 (m, 1 H, =C-CH-0), 3.47 (dd, J = 6.1, 10.6 Hz, 1 H, ChhOH), 3.41 (dd, J = 6.1, 10.6 Hz, 1 H, ChhOH), 2.16 (m, 1 H, Chh-CH), 1.89 (m, 1 H, CHh-CH), 1.68 (m, 1 H, CH-CH3), 1.62 (s, 1 H, C=CCH3), 1.39 (s, 6 H, C(CH3)2), 1.18 (d, J = 5.6 Hz, 3 H, CH3CHO), 0.88 (d, J = 6.8 Hz, 3 H, CH3CH). 13C-NMR (125 MHz, CDCI3, 298K) 5 132.0 (HC=CMe), 128.3 (HC=CMe), 107.9 (C(CH3)2), 88.7 (C=C-CH-0), 74.3 (CH3-CH-0), 67.7 (CH2-0), 36.1 (CH-CH3), 31.4 (CH2CH=), 27.4 (CCH3), 26.7 (CCH3), 17.0 (CH3CH), 16.4 (CH3CHO), 11.5 (C=CCH3). FAB (+) HRMS: Calcd. for C13H24Na03: (M+Na)+: m/e 251.16231; Found: mle 251.16182. Aldehyde 210 Me Me Me Me TEMPO, BAIB HO Me Me Me Me 434 270 To a solution of alcohol 434 (0.10 g, 0.271 mmol) in dry CH2CI2 (2.7 mL) under N2 was added TEMPO radical (6.8 mg, 0.044 mmol) followed by [bis(acetoxy)iodo]benzene (141 mg, 0.439 mmol) in one portion. The reaction mixture was stirred at rt for 5 h, quenched with sat. aq. Na2S203 (5 mL) and extracted with CH2CI2. The combined organic extracts were dried (MgS04), filtered and concentrated in vacuo. The crude product was purified by Si02 flash chromatography (gradient elution: 5:1 to 1:1 petrol:EtOAc) to afford aldehyde 270 as a colorless oil (50 mg, 50%).104 182 1H-NMR (500 MHz, CDCI3) 298K) 5 9.64 (d, J= 1.3 Hz, 1 H, CHO), 5.47 (dddd, J = 6.9 Hz, 1 H, HC=CMe), 3.83 (m, 1 H, CH3-CH-0), 3.82 (m, 1 H, C=C-CH-0), 2.43 (m, 2 H, Chh-CH), 2.17 (m, 1 H, CH-CH3), 1.65 (s, 1 H, C=CCH3), 1.40 (s, 6 H, C(CH3)2), 1.19 (d, J = 5.6 Hz, 3 H, CH3CHO), 1.08 (d, J = 7.0 Hz, 3 H, CH3CH). 13C-NMR (125 MHz, CDCI3, 298K) 6 204.3 (CHO), 133.6 (MeC=CH), 125.8 (HC=CMe), 108.0 (C(CH3)2), 88.7 (C=C-CH-0), 74.3 (CH3-CH-0), 46.3 (CH-CH3), 31.4 (CH2CH=C), 27.4 (CCH3), 26.8 (CCH3), 17.0 (CH3CHO), 13.0 (CH3CH), 11.7 (C=CCH3). FAB (+) HRMS: Calcd. for C13H22Na03: (M+Na)+: m/e 249.14666 ; Found: mle 249.14601. 183 REFERENCES (1) Bernards, R. Biochimica et Biophysics Acta, Reviews on Cancer 1997, 1333, M33. (2) Black, A. R.; Azizkhan-Clifford, J. Gene 1999, 237, 281. (3) Muller, H.; Helin, K. 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E.; Sharp, M. J. J. Am. Chem. Soc. 1988, 110, 612. (67) Overman, L. E.; Sharp, M. J. Tetrahedron Lett. 1988, 29, 901. (68) Lin, N.-H.; Overman, L. E.; Rabinowitz, M. H.; Robinson, L. A.; Sharp, M. J.; Zablocki, J. J. Am. Chem. Soc. 1996, 118, 9062. (69) Overman, L. E.; Robinson, L. A.; Zablocki, J. J. Am. Chem. Soc.1992, 114, 368. (70) Bargar, T. M.; Lett, R. M.; Johnson, P. L.; Hunter, J. E.; Chang, C. P.; Pernich, D. J.; Sabol, M. R.; Dick, M. R. J. Agric. Food Chem. 1995, 43, 1044. (71) Lett, R. M.; Overmann, L. E.; Zablocki, J. Tetrahedron Letters 1988, 29, 6541. (72) Rowley, M.; Kishi, Y. Tetrahedron Lett. 1988, 29, 4909. (73) Takai, K.; Tagashira, M.; Kuroda, T.; Oshima, K.; Utimoto, K.; Nozaki, H. J. Am. Chem. Soc. 1986, 108, 6048. (74) Aoyagi, S.; Wang, T. C.; Kibayashi, C. J. Am. Chem. Soc. 1992,114, 10653. (75) Aoyagi, S.; Wang, T. C.; Kibayashi, C. J. Am. Chem. Soc. 1993,115, 11393. (76) Tang, X.-Q.; Montgomery, J. J. Am. Chem. Soc. 2000, 122, 6950. (77) Trost, B. M.; Scanlan, T. S. J. Am. Chem. Soc. 1989, 111, 4988. (78) Tan, C.-H.; Stork, T.; Feeder, N.; Holmes, A. B. Tetrahedron Lett. 1999,40, 1397. (79) Choon-Hong Tan, A. B. H. Chemistry 2001, 7, 1845. (80) Comins, D. L.; Huang, S.; McArdle, C. L.; Ingalls, C. L. Org. Lett. 2001, 3, 469. (81) Comins, D. L.; LaMunyon, D. H. Tetrahedron Lett. 1988, 29, 773. (82) Wang, B.; Fang, K.; Lin, G.-Q. Tetrahedron Lett. 2003, 44, 7981. (83) O'Mahony, G.; Nieuwenhuyzen, M.; Armstrong, P.; Stevenson, P. J. J. Org. Chem. 2004, 69, 3968. (84) Bouzide, A.; Sauve, G. Org. Lett. 2002, 4, 2329. 186 (85) Choudary, B. M.; Chowdari, N. S.; Kantam, M. L. Tetrahedron 2000, 56, 7291. (86) Wu, Y.; Ahlberg, P. J. Org. Chem. 1994, 59, 5076. (87) Silverstein Spectrometric Identification of Organic Compounds', 5th Ed., Wiley, p215, 1991. (88) Little, R. D.; Verhe, R.; Monte, W. T.; Nugent, S.; Dawson, J. R. J. Org. Chem. 1982, 47, 362. (89) Guigen Li, H.-T. C., K. B. Sharpless, Angew. Chem.Int. Ed. Eng. 1996, 35, 451. (90) Guigen Li, H. H. A., K. B. Sharpless, Angew. Chem.Int. Ed. Eng. 1996, 35, 2813. (91) Mintz, M. J.; Walling, C. Organic Syntheses 1969, 49, 9. (92) Reddy, K. L.; Sharpless, K. B. J. Am. Chem. Soc. 1998, 120, 1207. (93) Bodkin, J. A.; McLeod, M. D. J. Chem. Soc. Perkin Trans. 1 2002, 2733. (94) Bajwa, J. S. Tetrahedron Lett. 1992, 33, 2955. (95) Barboni, L.; Lambertucci, C.; Ballini, R.; Appendino, G.; Bombardelli, E. Tetrahedron Lett. 1998, 39, 7Ml. (96) Clark, J. S.; Townsend, R. J.; Blake, A. J.; Teat, S. J.; Johns, A. Tetrahedron Lett. 2001, 42, 3235. (97) Trost, B. M.; McEachern, E. J.; Toste, F. D. J. Am. Chem. Soc. 1998, 120, 12702. (98) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543. (99) Kraus, G. A.; Taschner, M. J. J. Org. Chem. 1980, 45, 1175. (100) Liu, G.; Cogan, D. A.; Owens, T. D.; Tang, T. P.; Ellman, J. A. J. Org. Chem. 1999, 64, 1278. (101) Weix, D. J.; Ellman, J. A.; Wang, X.; Curran, D. P. Organic Syntheses 2005, 82, 157. (102) Ellman, J. A.; Owens, T. D.; Tang, T. P. Accounts of Chemical Research 2002, 35, 984. (103) Foubelo, F.; Yus, M. Tetrahedron: Asym 2004, 15, 3823. (104) De Mico, A.; Margarita, R.; Parlanti, L.; Vescovi, A.; Piancatelli, G. J. Org. Chem. 1997, 62, 6974. (105) Dimopoulos, P.; Athlan, A.; Manaviazar, S.; George, J.; Walters, M.; Lazarides, L.; Aliev, A. E.; Hale, K. J. Org. Lett. 2005, 7, 5369. (106) Dimopoulos, P.; George, J.; Tocher, D. A.; Manaviazar, S.; Hale, K. J. Org. Lett. 2005, 7, 5377. (107) Dimopoulos, P.; Athlan, A.; Manaviazar, S.; Hale, K. J. Org. Lett. 2005, 7, 5373. (108) Servi, S. J. Org. Chem. 1985, 50, 5865. (109) Kirschning, A.; Hary, U.; Ries, M. Tetrahedron 1995, 51, 2297. (110) Roush, W. R.; Harris, D. J.; Lesur, B. M. Tetrahedron Lett. 1983, 24, 2227. (111) Oppolzer, W.; Moretti, R.; Thomi, S. Tetrahedron Lett. 1989, 30, 5603. (112) Seebach, D.; Hungerbuehler, E.; Naef, R.; Schnurrenberger, P.; Weidmann, B.; Zueger, M. Synthesis 1982, 138. (113) Garner, P.; Ho, W. B.; Grandhee, S. K.; Youngs, W. J.; Kennedy, V. O. J. Org. Chem. 1991, 56, 5893. (114) Hale, K. J.; Delisser, V. M.; Yeh, L.-K.; Peak,; Manaviazar, S.; Bhatia, G.S. Tetrahedron Lett. 1994, 35, 7685. 187 APPENDIX 188 I-A L -9 2 CDC13 298K < NZ 9 ^ rV —NFmoc OH i i i i i i i i i T T T T I I I f t— , f""i— 1— 1— r i i i i i i i i i p p m I-AL-92 13C CDC13 - 298K ( NZ OBn OH 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 01221104: Scan 473 (110.17 min) - Back Base: 154.00 Int: 6.06293e+006 Sample: VG-70SE Positive Ion FAB 1 0 0 % 154 90% Sample: l-AL-92 Nz Instrument Resolution: 6000 NFmoc 80% Theoretical Mass: (M+H) 664.26588 Measured Mass: (M+H) 664.26409 Error: 2.69 ppm 70% 60% 50% - 40% 307 30% 20% 10% 17$ 460 664 341 391 5 4 Q ______61? , . [ 0% ■ III. jj.iiiii all A 100 200 300 400 500 600 700 800 m/z 85.0 %T 80.0 75.0 70.0 494.7 65.0 970.1 60.0 3 (p 3 5 .7 1051.1 2950.9 3064.7 700.1 55.0 1089.7 1124.4 50.0 1195.8 1296.1 740.6 45.0 1257.5 / Viz 1678.0 O B n \— NFm oc 1411.8 1450.4 Me. 40.0 CT'OH 1714.6 4000.0 3500.0 3000.0 2500.0 2000.0 1750 0 1500.0 1250.0 1000.0 750.0 500.0 - I-AL-92 1 / c m II-A L -1 0 2 Troc CDC13 - 2 98K NH Me H0Y X r i Me O p O: „ _ M e BocHN OBn U j ppm II-AL-102 DEPT Trocs CDC13 - 298K NH Me HO Me O O °i> \ . M e BocHN OBn "I ...... I...... I...... I...... I...... I...... I...... I...... I...... 'I...... I...... I...... I...... I...... I...... I...... I...... I...... I...... I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm II-AL-102 13C Trocs NH Me CDC13 - 298K HO Me O O O cV Me BocHN OBn T ™ i l l I l l l l i TTTTTTTTT | I I I I II I I I | I I I I I I I I I i" 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 01131006: Scan 63 (12.50 min) - Back Base: 254.00 Int: 2.10092e+006 Sample: VG 70-SE Positive Ion FAB 105% |Sample: ll-AL-102 NH Me Theoretical Mass (M+Na) 635.13057 Measured Mass: (M+Na) 635.12828 Error: 3.61 ppm X ° BocHN " 0 07.9 150 500 1000 1400 m/z 105.0 %T 100.0 95.0 90.0 569.0 85.0 /A I I \ J\,/ 811(8 700.1 , / ( ! | t 2877.6 80.0 3327.0 10029 ! 736.8 1643.(2 i 2933.5 i J i. 1053.1 75.0 2976.0 1662.51 1095.5 70.0 1508.2 65.0 Troc. NH Me 1163.0 HO Me O P 60.0 O: Me BocHN OBn 1728.1 55.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 I-A L -4 9 CDC13 - 298K H Nv .CONHNHBoc j i i k k . - i ------1 i------1------1------1------1 i------1------1------1 j i i ------1------1------1------1 i —I 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0 .5 ppm I-AL-4 9 CDC13 - 298K DEPT H N ^CONHNHBoc I-A L -4 9 CDC13 - 298K 13C H N .CONHNHBoc I I I | I I l"T I 1 I I 1 | 1 1 I 1 1 I I I I | I 1 I I I I I I I j I 1 I I I » I I I j If TT T T T'T T | T1 I I I I I I 1 | n T I I 1 I I I | I II II I I » I | » T I I 1 11 I 1 | I f I I » I I I > | I M I 1 I T'T I I T T M T » | T T TT I I M ! |"ri' r m 'TT T | IT T 'T 111 I Ij 1 I 1 1 1 1 1 1 1 j 1 T 1 T I 1 I I 1 | 1 I 1 I 1 I 1 1 1 | 1 1 I 1 1 I 1 11 | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm I-AL-4 9 CDC13 - 298K ppm H N .CONHNHBoc 5 I-A L -4 9 CDC13 - 2 98K HMQC ppm H N vCONHNHBoc -5 0 -6 0 -9 0 5.0 4 .5 4.03 .5 3.0 2 .5 2.0 1 .5 ppm 04280404: Scan 209 (38.27 min) Base: 230.00 Int: 6.49348e+006 Sample: VG 70-SE Positive Ion FAB 105% 230 90% Sample: l-AL-49 Theoretical Mass: 230.15046 (M+H) H 80% N vCONHNHBoc Measured Mass: 230.15177 (M+H) Error = 5.72 ppm 70% 174 60% 50% 40% 30% 20% 10% 503 270 307 38; [ 525 ______61Q 0 % 500 550 600 650 100.0 — r %T 90.0 80.0 — 70.0 60.0 50.0 40.0 30.0 20.0 NHNHBoc 10.0 — 5000.0 2500.02000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 1 /cm n 0.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 1 /cm I-A L -5 0 C6D6 - 298K MeN NFmoc I-AL-50 C6D6 - 298K CEPT Mes NFmoc C02H MMl 'I...... I...... I...... I...... I...... I...... I...... I...... I 80 70 60 50 40 30 20 10 ppm I-A L -5 0 C6D6 - 298K 13C Me, NFmoc I-AL-50 C6D6 - 298K < 1 0 5 ^ ppm Me. NFmoc C02H -2 -3 -4 oCj -7 8 7 6 5 4 3 2 1 p p m I-AL-50 C6D6 - 2 98K ppm Me NFmoc 20 40 60 80 100 120 140 160 p p m 01131006: Scan 134 (26.70 min) - Back Base: 176.00 Int: 1.66005e+006 Sample: VG 70-SE Positive Ion FAB 100% 176 Sample: l-AL-50 Me. Instrument Resolution: 7000 90% NFmoc Theoretical Mass (M+Na) 390.16812 Measured Mass: (M+Na) 390.16842 C 02H Error: 0.77 ppm 80% 390 70% 60% 412 50% 479 421 400 450 500 75.0 %T 70.0 — i 70.0 65.0 — %T 60.0 36V 1.5 3 6513^10 I 2941.2 I 448.4 ll326.9 3853.5 2941.2 I I 50.0 — 1 1326.9 | 2958.6 1448.4 I lj 12.9 76j- 2958.6 1112.9 61.8 45.0 731 l 66.9 1753 738.7 40.0 1166.9 1753 3 1652.9 35.0 — 3.0 2500.0 2000.0 1750.0 j 1500.0 1250.0 1000.0 750.0 500.0 1652.9 1/cm 30.0 — 7 j i i i i | i i r~i j i i i i i i \ i i | r I ! I i i 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 I-AL-50 1 /c m uidd I £ £ 17 £ I ■ i » i i l i i i I ■ ■ i i » » kill i i i i i i i i |___ |__ |__ | l |__ |__ | l |__ .1 |----1----- 1—|--1----1----1—1—|----1----L. M86£ - £1303 N 90T-OV-II e|/M Nootuj II—AL-106 Fmocx ^Me DEPT y CDCL3 - 298K \ 0 NHNHBoc m+mymy likM «KAA|Lf II-AL-10 6 Fmoc Me 13C v O CDC13 - 2 98K 9 A " ' NHNHBoc L •Mm m m n » i t i i | i i i i t i i , T .mTTTrTT1TTr...... j,,,,,,, r, 1TT11TT[m m ,...... j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm I I - A L - 1 0 6 COSY CDC13 - 298K Fmocv Me N ° 0 ' NHNHBoc II-A L -1 0 6 HMQC aA Lju ppm CDC13 - 298K Fmoc, NHNHBoc - 80 -100 -120 -140 -160 I I I I I I I IT" M I I I I 1 I I I I I'T T'T'TT ppm 01131006: Scan Avg 78-85 (15.50 - 16.90 min) - Back Base: 176.00 Int: 2.T9332e+006 Sample: VG 70-SE Positive Ion FAB 100% 17fc Sample: ll-AL-106 Me. Instrument Resolution: 7000 NFmoc 90% Theoretical Mass (M+Na) 601.30019 Measured Mass: (M+Na) 601.29959 Error: 1.00 ppm NHBoc 80% 70% 60% 50% 40% 20% 199 601 329 10% 501 150 200 250 300 350 450400 500 550 600 650 700 750 m/z 95.0 %T 92.5 5423 90.0 873. 964.3 87.5 85.0 — 744.5 1041.5 2877.6 82.5 130p.6 1369.4 ]94 2958.6 0 1 80.0 3288.4 1396.4 77.5 1448.4 1639.4 1159.1 75.0 72.5 NHNHBoc 1697.2 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 Baseline Dataset 1/cm II-AL-106 I-AL-65ws cyclisation product CDC13 - 298K NMe j l L uL » L _ 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm I-AL-65ws cyclisation product CDC13 - 298K NMe A . j u k Jli ^ . fL ______ TTT t 1 1 1 1 1 | i r— i 1 1 1 ■■"i i i 1 1 1 i 1 | 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm I-AL-65ws CDCL3 - 298K DEPT -TV^*V ’ 1 ' T -1 - t “ r - 40 35 30 25 20 15 10 5 ppm I-AL-65ws CDCL3 - 2 98K 13C ■Aw* M m i i 11 11 i 11 i i i > m i i t j i I I i r i ! i 11 1 r i i n i 11 11 t i i i i Ti i r t i i'i 11 T'T i n i r| i i n i 'n i r| 11 t t i n 11 j t i m h i i'| i i t t i p i i i ^ 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm I-AL-65ws CDCL3 - 298K COSY ppm 0 a -4.0 -4.5 5 ppm I-AL-65ws CDCL3 - 298K ppm JU___ -10 -20 a -30 r 40 -50 o r 60 r 7 0 -80 -90 5.0 4.5 4 . 0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 03290404: Scan 125 (22.87 min) - Back Base: 154.00 Int: 4.91413e+006 Sample: VG 70-SE Positive Ion FAB 105% Sample: l-AL-65ws (FAB) 90% Theoretical Mass: 225.16029 (M+H) Measured Mass: 225.16071 (M+H) N M e Error = 1.90 ppm 80% - 70% - 60% 50% - 40% 307 30% 20% 225 289 10% 166 27' 244 0% J k M . (mi... i iiill i. — .ii fllli, . ..ill 33? , 37? ^ 150 200 250 300 350 400 450 m/z II-AL-7 3 BnO 298K NFmoc CDC13 NHNHBoc 10 6 57 4 3 2 1 ppm BnO II-AL-73 NFmoc D E P T CDC13 - 298K ,Me O A NHNHBoc II-AL-73 BnO ,N Z I NFmoc 13C M e,, /N CDC13 - 298K 1 ✓Me" N o A NHNHBoc A - 1 i f —jlL IuLA i r i i i 11 i i i 11 11 i i i 11 i i i i i i i i i i i i i i i i t t i in i in i mi i “T i i n | i iTTTi t t t rm i n ip irm rrr 111 11 i 111 i j 11 1111 i 111n i n 111 i 11 in i in 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm II-A L -7 3 cosy ppm CDC13 - 298K 49 BnO ,NZ NFmoc .Me NHNHBoc - 8 I I I I I I I I I j I I I I I I I I I | I 10 9 8 II-AL-73 HMQC CDC3 - 298K BnO NFmoc ,Me NHNHBoc 03170305: Scan Avg 153-154 (35.50 - 35.73 min) - Back Base: 176.00 Int: 542989 Sample: VG-70SE Positive Ion FAB 100% 176 Sample: ll-AL-73 \ ,N Z Instrument Resolution: 7000 O Bn\_NFmoc 90% Theoretical Mass: (M+Na) 1024.47960 Measured Mass: (M+Na) 1024.48548 Error: 5.74 ppm ,Me 80% Me 70% Me' NHNHBoc 60% 50% 40% 30% 326 20% 413 1024 1064 10% 499 665 814 935 973 0% 100 500 1000 1200 95.0 %T 90.0 3857. 877. 547. 912.3 621.0 85.0 — 1008.7 698.2 2869.9 80.0 I I 1049.2 3303.8 1089.7 j 2931.6 1130.2 75.0 2954.7 93.9 740.6 ’61 1161.1 I 1365. 1423.4 70.0 — 1249.8 ( ,N Z OBnY- NFmoc 1450.4 65.0 Me 1649.0 Me 60.0 — Me' NHNHBoc 1706.9 4000.03500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 NHNHBoc /v^ a W "i i i i i i i T I I I I |"T "T 1 I 1 I I ■'“ n BnO II-AL-72 NH D E P T CDC13 - 298K ,Me NHNHBoc m m m m m nJ iL UL U p * * * T p r BnO NH >0 NHNHBoc i i i | i i i i i i i n | i i i 11i i 11i i ii 11i i ij iTTT ii i i I i Ii I i I i Ij i i I i Ii Ii Ii Ii Ii Ii Ij |i Ii Ii Ii Ii I i Ii I i Ii Ij i i iTTTTTTT i i i i i i j i Ii Ii IIi i Ii I i Ii IT11 Ii IIii ITTin i Ii I111 ii i 111i i i iiri i 11 i iI iI iI Ii iI Ii Ii 1i j i i i i i i i i i j i ■r~ni i i i11 i ttti i i j i i i i i i i i i j i i i i i i TTTJi i i j i i i i i i i i i j i i i i i i i mT j i i i j i i i i i i i i i Vj i i i i i i i i i j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm II-A L -7 2 COSY CDC13 - 298K 500 MHz NZ BnO NH £P to NHNHBoc 10 987654321 p p m II-AL-72 HMQC CDC13 - 298K 50 0 MHz ppm BnO - 20 NH ,Me 40 NHNHBoc 60 - 80 -100 120 140 160 i i i i i i i i i | i i i i i I I i i 1 p p m 02170105: Scan 23 (5.17 min) - Back Base: 176.00 Int: 3.74185e+006 Sample: VG-70SE Positive Ion FAB 105% Bn' 176 NH Samle: ll-AL-72 90% Instrument Resolution: 7000 Theoretical Mass: (M+Na) 802.41153 Me 80% Measured Mass: (M+Na) 802.41531 Error: 4.71 ppm Me NHNHBoc 70% 60% 50% 40% 30% 20% 329 10% 47< 802 0% LiLhL.B. -i.jl i l l 62? 100 200 300 400 500 600 700 800 900 1000 m/z 2869.9 80.0 1367.fi 2929.7 1404.1 j 1159.1 1261.4 77.5 1452.3 75.0 169&.2 72.5 70.0 67.5 NHNHBoc 1645.2 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 a II-AL-105 NHTroc NZ CDC13 - 2 98K OBn Me Me ,Me Me BocNH Me. OBn Me NHNHBoc II-AL-105 CDC13 - 298K NHTroc NZ Me OBn Me ,Me Me BocNH Me. OBn Me NHNHBoc J UL ■I...... I...... i...... i...... i ...... i...... i ...... i ...... J"l 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm II-AL-105 13C NHTroc CDC13 298K NZ Me OBn Me ,Me Me BocNH Me. OBn Me NHNHBoc 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm II-AL-105 J\ ppm COSY CDC13 - 298K NHTroc NZ Me OBn Me .Me - 2 Me BocNH Me. OBn Me NHNHBoc - 4 - 6 - 7 - 9 | I I' I I I I I I I |"T I I I I T~l I I j 1 TTTT I I I | |~| | 1-| I I | I I | | T I I 'I I I | I | | I I | f I 1 I I | I r 1 I 1 I I I I | I » I I I I I I I | I I I r~l I l~l I j~ I I'T I I I -10 10 987654321 ppm II-AL-105 ppm HMQC CDC13 - 2 98K NHTroc NZ Me - 20 OBn Me ,Me Me 40 BocNH Me. OBn Me NHNHBoc - 60 - 80 -100 -120 -140 -160 -180 ppm 01210205: Scan Avg 76-79 (17.53 - 18.23 min) - Back Base: 1398.00 Int: 385997 Sample: VG-70SE Positive Ion FAB NHTroc 100% NZ Me OBn 1397 Me Sample: ll-AL-105 Me Instrument Resolution: 7000 90% Theroetical Mass: (M+Na) 1396.54176 _Me Measured Mass: (M+Na) 1396.54502 O" Me BocNH Error: 2.33 ppm Me O OBn K ' NHNHBoc 3641.4 698.2 3664.5 734.8 2875.7 1311.5 1045.3 2929.7 \ 3305.8 1367.4) NHTroc 14^2.3 124 82.0 1390.6 Me N r 1161.1 80.0 BocNH 1643.2 1716.5 NHNHBoc 78.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 II-AL-105 1/cm I I - A L - 1 1 9 CDC13 298K NZ NHTroc OBn Me Me Me ,M e Me Me Me W 9 8 7 4 2 0 ppm II-AL—119 dept CDC13 - 298K NZ NHTroc OBn > i Me Me ,M e Me Me NH Me • i...... i ...... I ...... I ...... I ...... I ...... I ...... i ...... i ...... I ...... i ...... i ...... i ...... i ...... i 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm II-AL-119 13C CDC13 - 298K NZ NHTroc OBn Me Me Me Me Me NH Me r- i ii 1111 T I II I I I I I I TT T I I I TTT I I I I I IT I I I I I I I I I j T I II 1 I 1 I I I I I I I T I I I I I I I I I I I I I I I I I | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm II-A L -1 1 9 cosy ppm CDC13 - 298K NZ NHTroc OBn Me Me Me Me Me NH Me Bo o« i*9 10 9 8 7 6 5 4 3 2 1 ppm II-AL—119 HMQC CDC13 - 298K 1 NZ NHTroc OBn Me 20 Me Me # Me NH 40 Me Me 60 «r 0 * ^ . * 80 -100 -120 -140 160 ■ 11111111 j 111111111 j 1111111111111111111 j 111111111 j 111111111 j 11111 I I I I I I i" II | I I 11 I 10 9 8 7 6 5 4 ppm 01050405: Scan Avg 209-214 (48.57 - 49.73 min) - Back Base: 176.00 Int: 5.79586e+006 T Sample: 1 VG-70SE Positive ' Ion FAB 100% NZ NHTroc Sample: ll-AL-119 OBn / | Me Theoretical Mass: (M+Na) 1164.39946 Me Measured Mass: (M+Na) 1164.39772 90% Error: 1.49 ppm Me Me NH 80% OBn Me 70% 60% 50% 40% 30% 326 20% 10% 413 1166 150 500 1000 1300 100.0 95.0 — 90.0 85.0 — 999.1 80.0 3290 . 1039.6 2875.7 700.1 75.0 746.4 1089.7 1126.4 70.0 — 1153.4 2958.6 l/J 1195.8 65.0 — NZ NHTroc 150822 OBn Me 1234.4 1390.6 Me Me 60.0 ,Me Me NH 76.0 55.0 Me 1641.3 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 - II- A L - 1 1 9 1/cm II—AL-114 CDC13 - 298K NZ NHZ OBn Me Me Me Me Me NH Me “ | 1 I I 1— I 1— 1 1 I | 1— 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1— 1 1 1 1 1 1 1— 1 1 1 1 1— 1 1 1 1 1 1 1— I 1— 1 1 1 1 1 1 1 1— IT— I 1 1 1 1 1 1 1 I ■ 1 1 1 1 1 1 1 1 1 I 1 I I I | I I I I ' I l “ l l “ “ | 9 8 7 6 5 4 3 2 1 0 ppm II-AL-114 DEPT CDC13 - 298K NZ NHZ OBn Me Me Me Me Me L OBn Me '"I...... I...... I...... I...... I...... I...... I...... I...... I...... I" 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm II-AL-114 DEPT NZ NHZ CDC13 - 298K OBn Me Me Me Me Me NH Me I ' 1 ' ' I ' ' ' 1 I 1 1 1 ' l ' ' ' ' I ' ' 1 ' I ' ' ' ' I 1' 1 ' i 80 75 70 65 60 55 50 45 35 30 25 20 15 ppm II-AL-114 13C CDC13 - 298K NZ NHZ • U iA i J J L J U 'I— ,T'I rTTl" ' I I .rTTirTT1111TT1 70 60 50 40 30 20 10 ppm II-AL-114 ppm COSY CDC13 - 298K r0.5 NZ NHZ OBn Me 7 1 . 0 Me 7 1.5 Me Me Me - 2.0 Me" -2.5 -3.0 -3.5 -4.0 -4.5 -5.0 -5.5 - 6.0 76.5 -7.0 -7.5 i 1 l~i—r , r-rrn m 8 . 0 ppm 03170305: Scan Avg 102-103 (23.60 - 23.83 min) - Back Base: 176.00 Int: 2.13996e+006 Sample:C ' VG-70SE ' Positive ~ ‘ Ion FAB 100% Sample: ll-AL-114 O B n O | HZ Instrument Resolution: 7000 Me 90% Theoretical Mass: (M+Na) 1124.53203 Me Measured Mass: (M+Na) 1124.52750 Error: 4.00 ppm 80% Me Me NH OBn Me" 70% 60% 50% 40% 30% 20% 326 10% 1125 281 413 558 667 78Q 1017 150 500 1000 1400 m/z 90.0 80 .0 70.0 482.2 $62.4i 582.5 916.1 615.2 60.0 1001.0 3301. 2875.7 50.0 1035.7 1087.8 1126.4 ¥40.6 1 1 5 1 .4 i 700.1 40.0 1342 2958.6 1452.3 1 1 9 3 .9 1506.3 1236.3 Me 1257 .5 Me 1394.4 20.0 — Me V 1639.4 Me 1676.0 1728.1 I | I I I ! | I I ! I | I I i I | I i I 1 j i i i ] r 4000.0 3500.0 3000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 - II-AL-114 1/cm II-AL—124 MeOD - 298K OH Me Me Me ,Me Me Me NH Me I 1 I I I I ppm I I —AL -1 2 4 298KMeOD OH Me Me ,M e Me Me NH Me 1 T ' ' ' ' I 1 1 ' ^ I T' ’ ' | ' ' ' | ' ' ' 1 | ' 1 | | I I I I I T | 1 1 ■ . | 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm II-AL-124 DEPT MeOD - 2 98K OH Me Me ,M e Me Me NH Me rTTTT TTTrT 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm ri-A L -1 2 4 DEPT MeOD - 2 98K OH Me Me Me Me NH OH Me j mJL V ***** n>v f r I ' ’ ' ’ I ' ' ' ' I ’ ' ’ ‘ I ’ ■"■" 1 "| 11 1 '"| '-| n-i-' T-J i ■ ■ i | i ■ ■" i |- ■ i .-. | . . .- ^-r-r-j-r ,-,-r , ■, . . . | 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 ppm MeOD - 2 98K NH NH3+Cr 1 1 1 . II-AL-124 cosy ppm MeOD - 2 98K hO.5 OH Me Me 1.0 ,Me Me Me Oft p i . 5 Me - 2.0 -2.5 3.0 -3.5 -4.0 -4.5 5.0 5.5 t—i—i—r t—r t—r ■j— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |- t—r L 6.0 .0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 7 d d U L 6 5 4 L ia J 3 ul ^ a ____ 1 2 L 2 0 0 -100 -180 -160 -120 -140 ppm ppm 20 60 Me Me" Me eD 298K - MeOD OH II—AL—124 II—AL—124 HMQC NH OH Me Me Me -a i l l I i i i i I I ■p- TT 1111 i ' i i i" i i i i"i i i i i i |' i i n j TT t — i—i—i—i—r I I I I I I I I I I I I I I I I I I I I I I I I 1 1 I I I I I r r r 90 r 80 r 60 r 4 0 -30 r 20 r 70 ppm ppm 50 10 Me Me Me eD 298K - MeOD II-AL-124 II-AL-124 OH HMQC HMQC Me ,M Me Me Me I-AL-127 DMSO - 353K o o r Boc Ph I I I I 'T-| I I I' T I I I 1 ? | I I I I I 1 1 T I | l I "T"T I I r T f I I I 1 I I I I I ppm I-AL-127 DEPT DMSO - 333K BocN Boc Ph i i i i i i i i i i i ii i i i| r i i i i i i i i | i 1 i i i i | i i i i i i i i i | i i 80 70 60 50 40 30 20 10 ppm I-AL-127 13C DMSO - 353K o o BocN Boc JUoi^JuJTb-i | I I I I I I I t 1 j 1 I I T'l"TT"r i,,|' 1 "I ‘I I I I I I I | f I ITTT 1 T T 1 I I I I I I I I j i r i l'T i i i i j i i i i i i i i i jT i l T I I I I I |'i *r i i i i i i i | 11 t t i i i i i | i i ti i i i i i | r r m i r 11 j i 11 i i i i i i | i i i i i i i i T | n 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm I-AL-12 9 DMSO - 353k MeO' Boc I...... T I ...... T T T I I" I I I I I I 9 8 7 6 5 4 3 2 1 0 ppm I-AL-12 9 DMSO - 373K MeO' BocN Boc lja A J u T T T TT T T T T ~r T T T T T T T T T TT T T T TTT T T T T 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm I-AL-129 DEPT DMSO - 353K BocN '(fill i i i i i i i i i j i i i i i i i i i | i i i i i i i i i | i i i i » i i i i j i i i i i i t i t | i i i i » i i i i j i I i i i ii i » i i rrr i i i i i j i i i i i i i i i j i i i i i i i i i j i i 180 170 160 150 140 130 120 110 100 90 I-AL-129 13C DMSO - 353K O MeO BocN Boc P*W|W^^ 1 17 1 11012 90 80 70 60 50 30 2040 ppm I-AL-12 9 COSY :DC13 - 353} ■ 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ppm 02020804: Scan Avg 164-166 (38.07 - 38.53 min) - Back Base: 250.00 Int: 2.32882e+00o Sample: VG-SE 70 Positive Ion FAB 105% 250 Sample: l-AL-129 (FAB) Theoretical Mass: 451.24441 (M+H) Measured Mass: 451.24565 90% Error: 2.7 ppm 80% 295 70% 339 60% 50% 40% 30% 154 20% 191 10% 235 217 321 0% ! ill 130 200 250 300 350 m/z I-AL-162-1 C6D6 - 298K / t— i— i— r —|— i— i— i— f— i— i— r - ?—r —|—i— i— i— i— i— i— i— i— i— |“ i— i— i— i— i— i— i— i— r— |— i— i— i— i— i— i— i— i— r —| 3 2 1 0 ppm dept C6D6 - 2 98K I-AL-162-1 13 C C6D6 - 298K i n it t J I j i i » i i » i i i | i i i i i i i i > | i i i irnrii i i i -rt ji i i i i i rr rr i i i i i l j I I I I I l I I I j I I I I I I I I I j I I I I I I i i r | i i i i ii i i i irin | I'TTT, I I I | I I I m-, ' I | I I IT'ITT"! I | I...... I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm I-AL-162-1 _L«_ jlL J l ppm COSY C6D6 - 298K oOBn MeO 1 1 8 *1 i i i i iI I I I I I I I I I T TT I I I I I i i i i i r I I I I I I I TTT I I I TV TTT ppm j J l A. ppm I-AL-162-1 HMQC C6D6 - 2 98K 20 O vvOBn 40 MeO 60 80 -100 -120 -140 -160 -180 8 7 6 5 4 2 1 ppm 02131006: Scan 103 (20.50 min) - Back Base: 407.00 Int: 6.26243e+006 Sample: VG 70-SE Positive Ion FAB 1 0 0 % 4 0 f 90% Sample: l-AL-162 Instrument Resolution: 10,000 Theoretical Mass (C21H24N205): 407.15828 (M+Na) MeO 80% Measured Mass: 407.15878 Error: 1.2ppm 70% 60% 50% 40% 176 30% 20% 30' 91 199 10% 136 329 451 0%J > .ill! i....Ii ii 11.11.1 Lv— .11. ..L.. I lJiiL 110.0 i r %T \ i 100 . 0 — — 90.0 32 82 J6 80.0 81 912 .3 V 70.0 960.5 , 2869.9 995.2 3031.9 1604 1028.0 2950.9 60.0 14f f 700 .1 50.0 744 .E 1/ j O 13 5(0 40.0 I I ,vO B n 1409J9 I 1091.6 MeO I 1741.6 1448 .4 1^11.2 i 705.0 126p.1 1168.8 1 3 0.0 i l r 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 - I-AL-162 1 /cm I-AL-166 C6D6 333K 400 MHz HO HN LA. JUL ” |— i— t- t — r—i— i— r —i — i— |— i— i— i— i— i— i— i— r —i— |— i— i— r— i— i— i— i— i— i— |— i— r—i — i— i— i— i— i— i— |— r— i — i— i— i— i i i— i— |— T -1- 1 1 ' I ' 1 r_r 9 8 7 6 5 4 1 0 ppm I-AL-166 C6D6 - 333K 40 0 MHz voOBn , ______X | r T T TT 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm o dept C6D6 - 333K z I-AL-166 13C C6D6 333K HO mmmmrnmmmmmmmmmmmmmm mm 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 01050505: Scan 85 (15.53 min) - Back Base: 154.00 Int: 2.9/87e+006 Sample: VG 70-SE Positive Ion FAB 1 0 0 % - Sample: l-AL-166 90% “ Instrument Resolution: 6000 Theoretical Mass: (M+H) 371.16069 Measured Mass: (M+H) 371.16179 80% Error: 2.96 ppm 9 7 .5 %T 9 5 .0 6 0 7 .5 9 2 .5 — i 9 1 0 .3 9 0 .0 ----- 1 0 2 8 .0 87 .5 7 0 0 .1 1496.7 7 4 2 .5 1 5 9 5 .0 8 5 .0 — 2 8 9 6 .9 3 0 3 1 .9 ■2 3 | 1 0 9 1 .6 82.5 1 1 2 2 .5 1 3 5 3 1 1 7 8 .4 1 2 5 5 .6 8 0 .0 — 1 4 0 9 .9 4 0 5 . 0 7 7 .5 7 5 .0 HO i 703.0 7 2 .5 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 I-al-166 1/cm I-AL-172 C6D6 - 333K BocHNHN a M. 9 8 7 6 5 dept C6D6 - 333K O T I i I I I I I l I I I I I I I I I I I I » i I' t'l i i i i i i i i i M il i I I 1 80 70 60 50 40 30 20 10 ppm I-AL-172 13C C6D6 - 333K BocHNHN MM i i | i i i i 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 2 0 10 ppm I-AL-172 cosy C6D6 - 333K BocHNHN 4 5 6 7 8 10 ppm 01050505: Scan Avg 60-66 (10.95 -12.05 min) - Back Base: 154.00 Int: 5.43327e+006 Sample: VG /0-SE Positive Ion FAB 1 0 0 % 154 Sample: l-AL-172 xxOBn 90% Instrument Resolution: 6000 BocHNHN Theoretical Mass: (M+H) 485.24000 Measured Mass: (M+H) 485.23909 Error: 1.88 ppm 80% 70% 60% 50% 40% 30% 485 20% 429 10% 385 460 400 450 500 550 600 80.0 — 871.c 736.8 1026.1 2871.8 698.2 70.0 2929.7 2977.9 1091.6 65.0 — 1367.4 1454.2 1124.4 1498.6 3276.8 60.0 1163.0 55.0 oOBn BocHNHN 695.3 50.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 I-AL-172 1/cm Me Fmoc II-AL-38 CDC13 - 298K Me' ZN NHNHBoc OBn 107 6 5 4 3 2 1 ppm II-AL-38 Me. Fmoc D e p t CDC13 - 298K ZN NHNHBoc M es ^Fmoc N II-AL-38 13C Me' y ° o CDC13 - 2 98K z n ' N > " " '^ n h n h b o c o OBn il_ J k 190 110 100 90 80 70 60 50 40 30 20 ppm II- A L - 3 8 COSY p p m CDC13 - 298K 500MHz M ex ^Fmoc O Me’ N J l, Zrjr^|'' NHNHBoc OBn < -10 11 10 6 57 4 3 2 1 ppm II-AL-38 HMQC ppm CDC13 - 298K 20 Me 0 N NHNHBoc - 40 znO" OBn - 60 80 -100 -120 -140 160 r „i r i i » i i i r m r r r i i i i i i i i i i 3 2 1 ppm 01131006: Scan 18 (3.50 min) - Back Base: 176.00 Int: 5.91313e+006 Sample: VG 70-SE Positive Ion FAB 105% 176 Sample: ll-AL-38 Mev Fmoc Instrument Resolution: 7000 Theoretical Mass (M+Na) 814.34278 Me' 90% Measured Mass: (M+Na) 814.34649 Error: 4.56 ppm NHNHBocZN 80% OBn 70% 60% 50% 40% 30% 20% 714 217 347 10% 405 96^ 47! 654 758 150 300 400 500 600 700 800 900 1000 85.0 — %T 82.5 — 80.0 77.5 — 698.2 75.0 2334.7 1589.2 1026.1 72.5 2896.9 1083.9 740.6 70.0 I 2934.5 2976.9 >9.6 1365.5 1120.6 67.5 — 3284.5 1403.1 65.0 1450.4 124j. 1 1159.1 62.5 M e v Fm oc 60.0 Me' NHNHB oc ZN 57.5 — 703.0 OBn 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 II-AL-38 1/cm II-A L -4 2 BnO„ CDC13 298K Fmoc Me Me' ZN NHNHBoc OBn t— i— i— i— i— i— i— i— i—j —i— i— i— i— i— |— |— |— |— |— |— |— |— !— i— |— i— r—i— |— (—t— |— |— |— r—!— |— i— |— |— i— |— i— i— i— i— i— i— |— i— i— i— i— i— i— i— i— i— |— i— i— i— i— i— r ~ i— i— r - | — i— i— i i i— i— i— i— i— |— i— r - i i— r —i— i— r - i — j— i— i— i— i— i— i— i— i— i— |— i— i— i— i— r “ i— i— i- !— | 10 9 8 7 6 5 4 3 2 1 ppm II-AL-42 DEPT BnO. CDC13 - 298K Fmoc Me Me' ZN NHNHBoc OBn ,JL^. p . I ...... I " T ...... I ...... I ...... I ...... I ...... I ...... I ...... I ' 150 140 130 120 110 100 90 80 70 II-AL-42 BnC) 13C Fmoc Me-,, CDC13 - 298K Me Me* ZN NHNHBoc OBn J J - P 1 I I 1 TT I I I j I 111 I I T'TT'j I I I I I I I I r | I I I I I I I I I | I'"I I | | f | 1 | j 1 | I I I I II | j | | | | | | | | | | | | 1 I r T I I I | I I I I I I 1 1 I j I 1 II I II I I | I I I I I I I I I j I I I I I I I I I j I 1 I I I I I I I j I 1 I I I I I I I | I I I I I I I I I j I I I I I IT I T j I I I I T I I I 1 j T T 1 I I I I I T | T 1 T T I 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm II- A L - 4 2 COSY CDC13 - 298K BnC> Fmoc Me Me' ZN NHNHBoc OBn II-AL-42 HMQC CDC13 - 298K 20 NZ Fmoc Me Me' ZN" NHNHBoc OBn 80 100 120 140 160 p p m 01131204: Scan Avg 142-143 (32.93 - 33.17 min) - Back Base: 176.00 Int. 3.52667e+006 Sample: VG-70SE Positive Ion FAB 100% 17(f> Sample: IIAL42 90% Instrument Resolution: 7000 O Bn\—NFmoc Theoretical Mass: (M+Na) 1237.52219 Me Measured Mass: 1237.52395 80% Error: 1.42 ppm Me 70% Me' z n ' n ' NHNHBoc 60% OBn 50% 40% 30% 20% 326 10% 413 1237 0% 100 500 1000 1400 m/z 9 5 .0 — %T I 9 2 .5 — 6 6 9 .3 9 0 .0 86 2. 9 1 2 .3 I 8 7 .5 9 7 2 .1 8 5 .0 — 7 0 0 .1 8 2 .5 — 3298.0 2935.5 7 4 6 .4 8 0 .0 7 7 .5 — 1 0 8 7 .8 1 3 5 9 1 1 2 2 .5 11 6 1 .1 7 5 . 0 — \ ,NZ OBn V— NFmoc 1406.0 1245 9 Me, 1 6 5 6 .7 1 4 5 2 .3 7 2 .5 — 7 0 .0 Me' ZN NHNHBoc 6 7 .5 1 7 0 3 .0 OBn 6 5 .0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 II-AL-42 1/cm NZ OBn NH II-AL-48 CDC13 - 298K Me ■Vo ZN'NV"^NHNHBoc 0 OBn i i i— i— |— i i i— i i i— i— i— i— |— i— i— n — i— i— i— i— i— p i — i— i— i— i— r~i— i— i— | i i i i— i— i— i— i— r-j— i— i— i— i— i— i— i— i— i— | 4 3 2 1 0 ppm NZ II-AL-48 dept CDC13 - 2 98K Me Me M e'V, V > ° o Z N ^ J j" ' NHNHBoc OBn J J T ’I...... I...... l...... I...... I...... i,,r...... I ...... (" " T m 'Tm T ', " T " ...... I ' rn-prr TT-p-r ■T'T’T-",-" 1 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm II-AL-4 8 13C CDC13 - 2 98K Me Me’ NHNHBoc OBn — - 7j - irr^ ilLtaalr i i i |~t i i i i i i i i | i i i i i r r i i j i i i i i i i i i | i n i i i i i i j ...... r i | i i i i i i i i i i ii i i i I I1 I I I I I I I I I I 190 180 170 160 150 140 130 120 110 100 90 ppm II-AL-4 8 cosy CDC13 - 298K - 1 NZ OBn - 2 NH Me Me .0 Me o - 4 N Jl NHNHBoc - 5 OBn - 6 - 7 - 9 -10 -11 | I I I I I I I I I | I I I I I I T T I j I I I' I I I I I I j I i i i iiii|ii i i i i i i rp i i i i i i ii|iiii i iiiiIi ^ iiiiirrirrr i|iiiiiiiii 11 10 9 8 7 6 5 4 3 2 1 ppm II-AL-4 8 HMQC CDC13 - 298K Me M e' V ° O „N J|^ ZN Y NHNHBoc u 01131204: Scan 174 (40.40 min) - Back Base: 176.00 Int: 2.39606e+006 Sample: VG-70SE Positive Ion FAB 100% 17fc NZ 90% Sample: ll-AL-48 Instrument Resolution: 7000 Me,, Theoretical Mass: (M+Na) 1015.45412 '/ 80% Measured Mass: (M+Na) 1015.46087 Error: 6.64 ppm 70% Me' z n ' n "' NHNHBoc 60% OBn 50% 40% 30% 20% 326 1015 10% 413 241 47 58 895 807 1187 j i i L 690 ■L> .J., lLy.il. — j ___ 0° / * - 100 500 1000 1300 m/z 100.0 %T _ 95.0 90.0 — 2358.8 85.0 1024.1 700.1 744.5 80.0 1083.9 75.0 2935.5 1122.5 3290.3 70.0 1363 1161.1 1406.0 65.0 14523 1249.8 NZ 60.0 ~ 1652.9 55.0 Me' NHNHBoc 50.0 701.1 OBn 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 II-AL-48 1/cm II—AL—110 CDC13 - 298K NZ NHTroc BnO Me Me Me Me BocHN Me' OBn ZN NHNHBoc OBn .A_ i — |"~i— i— i— n — i— i— i— i | i— i— i— i— r —t— i— i t " |— i— i— i i i— i— i— i —i— |— i— p—i— i— i— p—i i i— |— i— i— i— i— I— i i i— i— |— i— i— i— i— i— i— r - i — i j i— i— i— i— i—t — i— i— i— |— i— r —r —r —i— i— i— i— i— p - i— r - i — i— i— i— i— i— i— | i i ” i i— i— n — i— i— |— r ~ i— i— i— i i i— i— i— | 1 0 9 8 7 6 5 4 3 2 1 0 ppm II-AL-110-1 DEPT CDC13 - 298K /NZ NHTroc i BocHN NHNHBoc OBn MtytynVyM ' ...... I...... i ...... I...... i ...... |...... j ...... , 111111111!. 11 " I " I" "I.....i.....i 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 2 0 10 ppm II-AL-110 13C CDC13 - 298K NZ NHTroc BnO Me BocHN Me' OBn ZN NHNHBoc OBn 190 180 170 160 150 140 130 120 110 100 90 70 60 50 40 30 20 10 ppm80 NZ NHTroc BocHN NHNHBoc II-AL- 1 1 0 4 a ppm HMQC CDC 13 - 2 9 8 K b 20 NZ NHTroc BnO Me Me b 40 Me Me BocHN Me* OBn b 60 ZN NHNHBoc OBn b 80 bioo b 120 b 14 0 b 160 b 180 l ?nn 01210205: Scan 93 (21.50 min) - Back Base: 176.00 Int: 3.01202e+006 Sample: VG-70SE Positive Ion FAB 105% Sample: ll-AL-110-1 NZ NHTroc Instrument Resolution: 7000 BnO Me 90% - Theroetical Mass: (M+Na) 1609.58436 Measured Mass: (M+Na) 1609.59520 Me Error: 6.73 ppm 80% - Me Me Sample: ll-AL-110-2 BocHN Instrument Resolution: 7000 70% - Me1 OBn Theroetical Mass: (M+Na) 1609.58436 Measured Mass: (M+Na) 1609.59929 ZN NHNHBoc 60% - Error: 9.28 ppm OBn 50% - 40% - 30% 1612 20% - 326 1483 405 589 10% I 689 1101 1349 o o j i i i i J..ii.i , 1 . ,.ii L| ill Jl .1 1.. ...I ill. 111L..1, AL 150 500 1000 1500 2000 m/z III-AL-15 CDC13 - 298K .. AL 1 u u "T— I— 1— I— |— I— I— I— I— |— i— i— i— I— |— I— r — I— I— |— I— I— I— I— I— I— I— I— I— I— I— I— i— I— I— I— I— i— i— |— i— I— I— I— |— I— I— i— I— |— r— 8 . 5 8.0 7 .5 7.0 6.5 6.0 5. 5 5.0 4.5 4 .0 III-AL-15 dept CDC13 - 298K O Me * » »' i i i | i » i i 1 r11 i i | i i"i i i r"i t i j i i i i i i i i i | i i i i i r r r i 1 i i i n i i i i j TTTi i i i i i | i i i i i i""i i i j i i i i i i i i i j i i i i i i i i r j i i i i i i i i i j i i i i i i i i i j i i i i i i t i t | i i i i i i i i i | i i r i'r i i i ryii i "i i i i 'ttj 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm III-AL-15 13C CDC13 - 298K O TsO. OEt Me 1 1 1 [ 1 11 i 11 ii i j i 11 i i ii Fr | i 11 11 i i i i j i i i 11 i i 11 j m n i 11 i i j i i m i i i i i j i i r 111 i i i j i i i i i i 11 i j i i i 11 11 i 11 11 11 i i i 11 j i i i i i i i 11 j ii 11 11 11 i j i 11 i i i i r i j i i i 11 i i i i j 11 irr 11 i i j 11 i i m i 11 j 11 i nr i II j i i 11 11 i i i j i 11 i rir i i j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm III-AL-15 ppm cosy CDC13 - 298K TsO OEt r 2 r 3 r 5 -7 3 ppm III-AL-15 ppm HMQC i i * i CDC13 - 298K O 20 TsO Y "^ O E t Me 40 60 80 100 120 140 160 180 i i i ...... | i i i i n i 1 1 | 1...... i i i j i i i i i i i i i | ...... | ..... b 9 8 7 6 5 4 ppm III-AL-15 ppm HMBC CDC13 - 298K O 20 TsO Y < 3 E t Me 40 60 80 100 120 140 160 180 ppm 03250507: Scan 65 (9.65 min) - Back Base: 349.00 Int: 2.25384e+006 Sample: VG 70-SE Positive Ion FAB 1 0 0 % 346 Sample: III-AL-15 90% Instrument Resolution: 7000 Theoretical Mass: (M+Na) 349.10856 Measured Mass: (M+Na) 349.10794 Error: 1.78 ppm 80% 70% 60% 50% 40% 30% 20% 28 10% 49 67' 0 % - 1 0 0 . 0 %T 90.0 80.0 i 70.0 60.0 ! I 50.0 ! I ! (2904.6 ;9 0 . 8 i I 1598.9 40.0 H j) 2929.7 1649.0 j 2958.6 1 4 4 1 6 . 1) 123 1006(8 2981.7 076.7 30.0 1 744 i illI : 1f ! j>56.6 j 20.0 — 34.1 j 8158 i i 931.6 1097.4 TsO OEt 555.5 10.0 — I ! i 706.9 663.5 jl 176.5 1363.6 1 i r 4000.0 3500.0 3000.0 2500.0 2 0 0 0 .0 1750.0 1500.0 1250.0 100 0.0 750.0 500.0 III-AL-15 1/cm III-AL-68 CDC13 - 298K O Me j,______r i 9 8 7 6 5 4 3 2 1 0 ppm III-AL-68 DEPT CDC13 - 298K O Me III-AL-68 13C CDC13 - 298K O M e " , ! " !Tm » T^-nT-i-i-i-fxr, ...... J...... , ...... , ...... , ...... , ...... -j.. 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 2 0 10 ppm III-AL-68 L l ppm COSY CDC13 - 298K -0.5 O Me -1.5 - 2.0 - 2.5 - 3.0 - 3.5 - 4.0 - 4.5 - 5.0 - 5.5 - 6.0 -6.5 ^ ^ { ' - 7 . 0 T 11...... ” 1—i— r~i— i—r ■ i i— i- “ i— i— i i i i—i— r* ppm III-AL- 6 8 HMBC CDC13 - 298K O 03101006: Scan 248 (57.67 min) - Back Base: 235.00 Int: 526362 Sample: VG70-SE Positive Cl-Methane 100% 23k Sample: III-AL-68 90% Instrument Resolution: 7000 Theoretical Mass (M+H) 235.03336 OEt Measured Mass: (M+H) 235.03376 80% Error: 1.70 ppm 70% 1 2 7 60% 50%- 40%- 2 0 7 30%- 1 5 5 1 1 3 191 20% 10%- 99 1 6 9 221 0% 80 150 200 250 300 350 400 m/z 1 : 3402.2 1041.5 1 6 5 1 .0 1380.9 1442.7 2977.9 1 1 1 0 .9 1272.9 1712.7 3500.0 3000.0 2500.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 III-AL-68 1/cm III-AL-63-3 CDC13 - 298K Me' OH OEt t — i— i— i— i— i— i— i— i— |— i— i— i— i— i— i— i— i— i— |— i— i— i— i— i— i— i— i— i— |— i— i— i— i— i— r —i— i— i— |— i— i— i— i— i— i— i— i— i— |— i— i— i— i— i— i— i— i— i— |— I— i— i— i— i— i— i— i— i— |— i— I— i— i— i— i— i— I— i— |— i— i— i— i— i— '— I— i— i— | i i ' i i i i ' ' | 1 1 1 1 1 1 1 1— 1 | 9 8 7 6 5 4 3 2 1 0 ppm III-AL-63-3 DEPT CDC13 - 298K M e ’ OH OEt ,ir ...... i...... i...... i...... i. r ...... ,...... j. TTrT " I" ' I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm III-AL-63-3 13C CDC13 - 298K NO. Me' OH OEt III-AL-63-3 COSY CDC13 - 298K M e’ III-AL-63-3 Ik A 1 JL ppm HMQC CDC13 - 298K 20 0 2N n o 2 40 Me’ OH OEt 80 -100 -120 -140 -160 -180 I 1 I I I I I I 1 | I'T I I I I I I I j I I I I I I I I 'I ...... I I I I I I j I I I I I I I I 1 T i i | i i i i i i i i i I I I I III! I 2 1 ppm III-AL-63-3 HMBC CDC13 - 298K Me’ OH OEt 03280906: Scan 131 [26.10 min) - Back Base: 376.00 Int: 169889 Sample: VG 70-SE Positive Ion FAB 105% Sample: III-AL-63-3 Instrument Resolution: 7000 Theoretical Mass: (M+Na) 376.11206 90% Measured Mass: (M+Na) 376.11080 Error: 3.35 ppm Me' 80% OH OEt 70% 60% 173 III-AL-95-3 CDC13 - 298K r i —i—i—|—i—i—i—i—i—i—i—i—i—j—i—i—i—i—i—r— i—n —|—i—i i i i—i i i—r—j—i—i—i i i i —i—r~i—| 3 2 1 0 ppm III-AL-95-3 DEPT CDC13 - 298K O Et M e OH III-AL-95-3 13C CDC13 - 298K r~ o o Me OH I...... i...... i...... I...... j . | ...... |...... |...... |...... |...... ■ j...... |" "I...... I...... I...... I...... I...... I...... I 190 180 170 160 150 140 130 120 110 100 90 60 50 40 30 20 10 ppm III-AL-95-3 1 A L m ______\ , L. p p m c o s y CDC13 - 298K 7 0 . 5 1 1 . 0 OEt Me OH 7 1 .5 - 2.0 r 2.5 r 3.0 -3.5 r 4.0 7 4 . 5 -5.0 r 5.5 7 6 .O -6.5 7 7 . 0 7 7 . 5 - 8.0 ppm III-AL-95-3 j._ i AJL ak X —A.______ppm HMQC CDC13 - 298K o o - 20 OEt Me OH - 40 - 60 - 80 -100 -120 -140 -160 -180 I I I I I ‘I " I T ' l " 200 I I I I I I ' r 111 | i i l i i r i i i i i—i i i i ppm 01070905: Scan Avg 13-15 (2.83 - 3.30 min) Base: 89.00 Int: 5.00158e+006 Sample: VG70-SE Positive Cl-Methane 1 0 0 % - l 89 Sample: lll-AL-95-3 90% Instrument Resolution: 7000 Theoretical Mass: (M+H) 189.11268 O E t Measured Mass: (M+H) 189.11286 Me OH 80% Error: 0.95 ppm 1UU.U %T 90.0 — 80.0 70.0 929.6 60.0 13712 1454.2 50.0 — 1024.1 3510.2 j 2877.6 40.0 2949.0 1 1 3M 30.0 2981.7 1203.5 1259.4 1072.3 20.0 1735.8 10.0 OEt M e OH 0.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 Baseline Dataset 1/cm III-AL-95-3 III-AL-102 CDC13 - 298K NBoc O OEt OH 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm III-AL-102 dept CDC13 - 298K NBoc O 40 35 30 25 20 15 10 5 ppm III-AL-102 13C CDC13 - 298K NBoc O ■I...... I'1...... I...... I...... I...... I...... I...... I...... I...... I...... I...... T1 i I I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm III-AL-102 COSY jL ppm CDC13 - 298K NBoc O OEt OH 4 5 . 0 4.5 4 . 0 3 . 5 3.0 2.5 2 . 0 1.5 1 . 0 ppm III-AL-102 ppm HMQC CDC13 - 298K 5 NBoc O OEt -10 -15 -20 -25 -30 -35 -40 -45 -50 55 p 60 ii, «q Qi -65 n“ i“ i 1 “i— i— i— i- 1 1 1 r_r 1 i 1--- 1--- 1--- 1--- 1--- 1— i---- 1--- 1--- r- 5.0 1.5 1.0 ppm III-AL-102 _> A _ A ppm HMQC CDC13 - 298K 5 NBoc O 10 OEt 15 20 25 3 30 35 40 45 *Q» 50 55 60 65 1--- 1--- 1--- 1--- 1--- 1---1--- 1--- 1--- 1----1-- 1--- 1--- 1--- 1-----1-1--- 1--- 1--- 1-----1-1--- 1---1---1----- 1-1---1--- 1---1---r 5.0 4.5 4.0 3.5 3.0 2.5 2.0 ppm III-AL-102 _j / NBoc O & o - 2 0 / Jf / OH - 40 - 60 - 80 -100 -120 -140 -160 -180 1 1 1 i | i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— j— i— i— i— i— |— i i i 1--- 1--- 1--- 1--- 1 — i 1 i i 1 r 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 03021006: Scan 12(2.30min) Base: 310.00 Int: 6.5535e+006>535e+006 Sample: VG 70-SE Positive Ion FAB 100% 310 Sample: III-AL-102 90% - Instrument Resolution: 7000 Theoretical Mass: (M+Na) 310.16303 NBoc O Measured Mass: (M+Na) 310.16196 OEt 80% Error: 3.45 ppm OH 70% - 176 60% - 50% - 40% - 30% - 254 460 199 10%- 329 210 232 349 486 294 379 200 250 300 350 400 450 500 110.0 — %T loo.o — I 9 0 .0 8 0 .0 3342.4 70.0 — 4 3 5 1 2 .1 2895.0 60.0 2937.4 1666.4 1257 5 50.0 1 1 1 8 .6 2977.9 730.0 40.0 1170.7 NBoc O OEt 1392.5 OH 1697.2 30.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 III-AL-102 III-AL-177 CDC13 - 298K H (5leC° 2Bn 9 8 7 5 4 3 2 1 0 ppm I I I - A L - 1 7 7 DEPT CDC13 - 298K CC^Bn I I I - A L - 1 7 7 O o 13C U II CDC13 - 298K C l ? ♦ r V > X /J~^C02Et ^TjN x)2Bn H Me H Me * t i y m T ’- ■ T " ...... I ...... I ...... I ...... | " . ! . .TT'I | ' 1 1 TTTI I I | .... , | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm III-AL-177 ppm COSY CDC13 - 298K O A C02Et c * H Me ''UP* 0 50 8 7 6 4 3 2 1 ppm III-AL-177 A-A-Jx. ppm HMQC CDC13 - 298K O O h 20 A -n \ w ^ C 0 2Et 'K H M e H ^iec o 2Bn - 40 - 60 - 80 -100 -120 -140 -160 -180 -200 8 7 6 5 4 2 1 ppm IV-AL-11 CDC13 - 298K f OH OPMB 1 i L ^ X . ’T t T* 9 8 7 6 5 4 3 2 1 0 ppm IV - A L - 1 1 DEPT CDC13 - 298K 'OH OPMB I V —AL—11 13C CDC13 - 298K f OH OPMB 190 180 170 160 150 140 130 120 110 100 90 80 70 6050 40 30 2 0 10 ppm IV-AL-11 _t__ I_ i I _AN_ A ^ —JL ppm COSY CDC13 - 298K 7 0 . 5 f OH OPMB 7 1 . 0 7 1.5 72.0 -2.5 73.0 7 3.5 -4.0 7 4 .5 -5.0 75.5 76.0 76.5 6 0 77.0 -7.5 - 8.0 I I I I I 1 I I I I—I I I I I I I—T T " -I—I—I I I I I I I—I I r I I I I 1—I I I I I I I ppm ppm IV-AL— 11 HMQC CDC13 - 298K - 20 : o h O P MB - 40 - 60 - 80 -100 -120 -140 -160 -180 ppm IV-AL-11 HMBC CDC13 - 298K OH ' OPM B 01131006: Scan Avg 100-104 (19.90 - 20.70 min) - Back Base: 176.00 Int: 3./6819e+006 Sample: VG 70-SE Positive Ion FAB 100%-j 17j> ISample:Sample: IV-AL-11 90% InstrumentInstrument Resolution:Resolution: 70007000 f OH Theoretical Mass (M+Na) 245.11536 / Measured Mass: (M+Na) 245.11588 OPMB 80% 4 Error:Error: 2.122.12 ppmppm 70% - 245245 60% - 50% - 40% 100.0 %T A 1 f t 5 1 8 .8 9 0 .0 i n 8 § l3 ! ! I I \ / 1585 .4 ! I !!II 8 0 .0 — ll \VJ / 2 8 4 2 .9 9 2 9 . 3 4 3 8 .8 y v V 1 ^ 7 3 2 .9 2 8 6 9 . 9 j 8 2 1 .6 7 0 .0 — nTp.O 2 9 3 3 .5 1 6 1 2 .4 1 4 11 .p 1 4 6 0 .0 | 1 \ 6 0 .0 11 \ 4j.8 5 0 .0 'OH 1514.0 OPMB 1 0 3 7 .6 1 7 2 6 .2 4 0 .0 30.0 1 2 4 7 .9 j 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 iV-AL -n 1/cm O III-AL-163 CDC13 298K ^ ° y ' Ph OPMB MeO CF 2 Wl JL JuuL__ ,-A. -L—a JJ— t — i— 1— 1— 1— i— 1— | — 1— 1— 1— 1— 1— i— 1— i— 1— |— i— 1— 1— 1— 1— 1— 1— 1— 1— |— 1— 1— 1— 1— 1— i— 1— 1— 1— |— i— 1— I— i— 1— r 1---- 1---- 1—I 1---- 1---- 1---- 1---- 1---- 1---- 1---- 1---- 1---- 1---- 1---- 1 V"I 1---- 1---- 1---- 1---- 1—1---- 1---- 1---- 1---- 1---- 1---- 1 7 6 5 4 2 10 ppm III-AL-163 d e p t CDC13 - 298K O Q-" "■—-P h OPMB MeO CF3 ■ ■ l...... I...... I...... I...... I...... I...... ‘ 1' I...... i...... I...... I...... ‘ 11 190 180 170 160 150 140 130 120 110 100 90 III-AL-163 13C CDC13 - 298K O O' Ph OPMB MeO 'CF3 frA JLi 4m *«*■ J. 444MMMlM»M4« 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm I I I - A L - 1 6 3 cosy j u ULJL ppm CDC13 - 298K o O' Ph OPMB MeO 'CF3 r 2 - 3 r 4 - 5 r l 9 8 7 65 4 3 2 1 ppm III-AL-163 HMQC ppm CDC13 - 298K 20 OPMB MeO CF3 40 60 80 -100 -120 -140 -160 9 8 7 6 4 3 2 1 ppm ppm 7 9 -19 -20 -20 - .JO 2 -7 -72.05 -72.00 -71.95 90 ~73 f19cpd - 72.076 MB O CF3 C eO M B PM O h P - ^ - O 1 0 01160806: Scan Avg 205-215 (40.90 - 42.90 min) - Back Base: 461.00 Int: 4.70589e+006 Sample: VG 70-SE Positive Ion FAB 105% 461 Sample: lll-AL-163 Instrument Resolution: 8000 O 90% - Theoretical Mass: (M+Na) 461.15517 O'" Measured Mass: (M+Na) 461.15438 OPMB MeO CF3 Error: 1.71 ppm 80% - 447 477 545 571 450 500 550 600 %T 1 0 0 . 0 - 95.0 - 90.0 - M 85.0 — 1 1714^ 719.4 2848.7 821.6 137^.0 80.0 - 1612 \ 2947.0 1454.2 110714.3 75.0 i I ! 70.0 1512.1 I l l 65.0 60.0 1031.8 1753.2 55.0 OPMB MeO CF3 1249.8 1174.6 50.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 III-AL-163 1/cm IV-AL-13 CDC13 - 298K ^ ^ b P M B ppm IV-AL-13 dept CDC13 - 298K OPMB i t | i r 2 0 10 ppm 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 IV-AL-13 13C CDC13 - 298K , O OPMB 200 180 160 140 120 100 80 60 40 20 ppm IV -A L -1 3 COSY J U i_ji 1L ppm CDC13 - 298K o OPMB 10 10 9 8 7 5 4 3 2 1 ppm IV—AL—13 HMQC ppm CDC13 - 293K 0 OPMB 20 40 60 80 100 120 140 160 180 200 p p m IV-AL-13 HMBC ppm CDC13 - 298K ‘O OPMB 20 40 60 80 100 120 140 160 180 200 p p m 01270206: Scan Avg 386-387 (70.72 - 70.90 min) - Back Base: 176.00 Int: 4.33015e+006 Sample: VG 70-SE Positive Ion FAB 1 0 0 % 17(p Sample: IV-AL-13 Instrument Resolution: 7000 90% Theoretical Mass: (M+Na) 243.09971 O Measured Mass: (M+Na) 243.09876 OPMB Error: 3.91 ppm 80% 70% 60% 50% 40% 30% 20% 199 10% 242 160 214 268 0 % - 150 200 250 m/z III-AL-148 CDC13 - 298K r oh OPMB 0 ppm III-AL-148 CDC13 - 298K O OH OPMB r 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm III-AL-148 DEPT CDC13 - 298K f OH OPMB 'i...... i...... i...... i...... i...... i...... i 60 50 40 30 20 10 ppm 13C CDC13 - 298K f OH OPMB III-A L-14 8 COSY Ji I I 111 I ppm CDC13 - 298K O - 1 OH OPMB -2 3 -4 -5 -7 5 4 3 2 ppm 111-AL-14 8 HMQC CDC13 - 298K jUL ppm O 20 OH OPMB 40 60 80 -100 tyd -120 -140 -160 -180 9 8 7 6 5 4 3 2 1 ppm III-AL-148 ppm HMBC CDC13 - 298K O 20 OH OPMB 40 -100 -120 -140 -160 -180 8 7 6 5 4 3 2 ppm 03151205: Scan Avq 165-172 (30.20 - 31.48 min) - Back Base: 154.00 Int: 860049 Sample: VG 70-SE Positive Ion FAB 105% 154 Sample: lll-AL-148 Instrument Resolution: 7000 Theoretical Mass: (M+H) 237.11268 : oh 90% J Measured Mass: (M+H) 237.11215 Error: 2.24 ppm OPMB 80% - 70% - 60%- 50% ~ 236 40%- 307 30% 20% 289 259 10% 274 166 219 329 145 200 250 300 350 400 m/z I 933.4! I I ! !l 1380. 1458.1 821.6 U / 2839.0 1299V! 1612 1 78.4 2943.2 2995.2 \! 1033.8 1514.0 1120.6 O OH 1718.5 OPMB 1247.9 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0750.0 500.0 m_A L_148 1/cm III-AL-149 CDC13 - 298K O ^ V u M e ' OPMB I JL JL ppm III-AL-149 DEPT CDC13 - 298K O OPMB 111 i...... I...... | |...... ■ | ...... | ...... | ...... 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm III-AL-149 13C CDC13 - 298K O OMe OPMB 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm I I I - A L - 1 4 9 COSY PPm CDC13 - 298K O OMe OPMB 3 4 5 7 8 p p m III-AL-149 HMQC CDC13 - 298K O OMe OPMB III-AL-149 HMBC CDC13 - 298K OPMB 01191205: Scan Avg 132-133 (24.15-24.33 min) RsicR Base: 154.00 Int: 600839 Sample: VGVG 70-SE 70-SE Po Positive Ion FAB 105% 154 Sample: III-AL-149 Instrument Resolution: 7000 Theoretical Mass: (M+H) 251.12833 OMe 90% -{ Measured Mass: (M+H) 251.12869 OPMB Error: 1.43 ppm 80% 70% A 60% 250 50% H 40% A 30% 20% A 307 273 289 10% 166 189 227 341 357 389 0% 140200 250 300 350 400 m/z 110.0 %T 1 0 0 . 0 — \r \ 9 0 .0 8 0 .0 7 0 .0 — 2 8 3 7 .1 2 8 7 3 . 7 2 9 0 8 .5 8 2 3 .5 6 0 .0 2 9 5 0 .9 1 4 6 0 .0 2 9 9 5 . 2 5 0 .0 1 1 7 5 .5 1201 6 1 0 3 3 .8 4 0 .0 1 5 1 4 .0 1 1 1 6 .7 3 0 .0 1 7 3 9 .7 1 2 4 9 .8 20.0 — OMe OPMB 10.0 — 0.0 4000.0 3500.0 3000.0 2500.0 2000.01750.0 1500.0 1250.0 1000.0 750.0500.0 III-AL-149 1/cm IV-AL-105 CDC13 - 298K OPMB 1 - - *■ n Aiii 4 3 2 1 0 ppm IV-AL-105 DEPT CDC13 - 298K o O OMe OPMB '"I ...... I ...... I ...... T r T’"[-ITT-HT-T ' I | ...... | ...... | ...... ( ...... , | ...... "I ...... I ...... m'ttj i , . i i . i i i , . i rT'i'm i | ...... , ...... r " ...... | | ...... | 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-AL-105 13C 295K- CDC13 OPMB m m ********* I » I j I I » j I I I I I I 1 > I | I ! ! THM I'TI'I rim I I I I j I i i > I 1 I I I j I I I I I I I I I | I I I I I I I I I j I I I I I I I I I j > I » I l‘M I I | I I I I I 'I ‘I I I j I I I I I I I I I |" i » i i I i^ m r r n n ^ "I I'"' I ' T T T" I'"' I 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm IV—AL-105 cosy Jl JL ppm CDC13 - 298K O O OMe OPMB 10 11 11 10 9 8 6 5 4 3 2 17 ppm IV-AL-105 J] L ppm HMQC 295K- CDC13 O 20 c r y OMe OPMB 40 60 80 -100 -120 -140 -160 -180 -200 I I I I 1 T l I | | I 1 I I I M r I j 1 M T TT I I I j I T 7 T T T T T T j T 1 i 1 I I 1 t l j i n I T I 1 I I | I II I I I I II TTTTTTTTT 1 1 I II II | I I I II I I I I I 11 10 ppm IV-AL-105 HMBC JU L ppm 295K- CDC13 O Q ' Y OMe 20 OPMB 40 60 80 -100 -120 -140 -160 -180 -200 TTTTTTTTTTTTTTTTTT I 1 I I I I II I| I I I I I 1 ITT TTTTTTTTT i i i i i i i i i | 11 10 ppm 01070606: Scan 6(1.30min) Base: 121.00 Int: 6.5535e+006 Sample: VG70-SE Positive Cl-lsobutane 1 0 0 % 12t Sample: IV-AL-105 90% Instrument Resolution: 9000 O Theroetical Mass: 252.09977 Measured Mass: 252.09953 o X 0Me Error: 0.95 ppm OPMB 1 0 0 . 0 — %T J ! ! ft) 9 0 .0 i i \ \ / I i 8 0 .0 \/ 1 3 4 4 6 .6 2 8 4 1 .0 1 3 8 2 .9 8 2 7 .4 7 0 .0 1 6 8 7 2 9 5 4 .7 14 5 8 .1 6 0 . 0 1 6 0 6 1 0 3 1 .8 O 1 1 5 7 .2 5 0 .0 O OMe OPMB 1 5 1 4 .0 1 7 3 5 .8 2 5 3 .6 40.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 ------TV_AT 1/cm III-AL-180-2 CDC13 - 298K OPMB r 1 1 | r " ' f —i i i f— i— i— i— |— i— i— i— i— i— i— *— i— i— |— i— i— i— i— i— i— i— i— i— |— i— i— i— i— i— i— i— i— i— |— i— i— I— i— i— i— i— i— i— |— i— i— i— r— i— r I I I I I T r i » » | t i i i— i— r - i — i" i p 10 9 8 7 6 5 0 ppm III-AL-180-2 d e p t CDC13 - 298K O o f-Bu"S^ N ^ V < 3 M e ' OPMB "i....i....i....i....i....i....i....i....i.... i....i.... r 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm III-AL-180-2 13C CDC13 - 2 98K OPMB i i 1T " .. 1 .... I.... I.I' I...... I I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm III-AL-180-2 Jl ii 1. JL ppm COSY CDC13 - 298K 0 O - 1 1 OMe OPMB -2 -3 L 5 L 6 -7 I 1 I I ! I I I ! 7 ppm III-AL-180-2 p p m HMQC CDC13 - 298K 0 O 20 1 f-Bu^S"N OMe OPMB 40 80 -100 -120 -140 -160 -180 98 7 4 3 2 1 ppm III-AL-180-2 p p m HMBC CDC13 - 298K 0 O 20 1 f-B u ^S "N O M e OPMB 40 80 -100 -120 -140 -160 -180 98 7 4 3 2 1 ppm 01160806: Scan Avg 390-400 (77.90 - 79.90 min) - Back Base: 173.00 Int: 3.10403e+0063. Sample: VG 70-SE Positive Ion FAB 100%-| 17$ Sample: lll-AL-180-2 90% Instrument Resolution: 8000 Theoretical Mass: (M+Na) 378.13511 Measured Mass: (M+Na) 378.13362 OMe Error: 3.94 ppm OPMB 80% 199 70%- 60% - 323 50% 40% 30% 242 349 472 20%- 214 378 10%- 160 391 311 440 200 250 300 350140 400 450 500 1 0 0 . 0 %T 90.0 — 80.0 — 70.0 — 582.5 3471.6 981.7 2839.0 60.0 — 2871.8 130 50.0 ~ 1369.4 821.6 1456.2 2954.7 40.0 — 1618 1031.8 30.0 — 1514.0 1745.5 20.0 — 1089.7 1126.4 1249.8 t-Bu OMe 10.0 — OPMB 0.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 III-AL-180-2 1/cm III-AL-176 CDC13 - 2 98K t- Bu NH O OMe OPMB T ~ l~ " r ' ...... | ...... \ 'r ...... — i — ...... , ■ , - n - r ...... | ...... | ...... | ...... ■ ■ ■ ■ , ...... | 9 8 7 6 5 4 3 2 1 0 ppm I I I - A L - 1 7 6 DEPT CDC13 - 298K OPMB m Lw*l "I ...... I...... I...... ’'" 'I ...... "I ...... I...... I"’...... -I.., ...... |...... r 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 111—AL—17 6 13C CDC13 - 298K OPMB W l i..... I..... I..... I..... I""...I"'... I... ""'T.....11'' ■■I" 11 I" 1...... I | 1-r ...... i | i i i ri i i it | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm III-AL-176 Jk 1- 1 JLa. JLJL JL ppm COSY 7 CDC13 - 298K 7 0 . 5 9 f-Bu"S"NH O 7 1 .5 • OPMB ^2.0 00 (I D 72.5 73.0 73.5 74.0 74.5 75.0 75.5 76.0 76.5 77.0 r-7.5 - 8.0 8 7 6 5 4 3 2 1 ppm ppm III-A L-176 HMQC CDC13 - 298K O 20 ■ /S . f-Bu NH O fX)Me - 40 OPMB - 60 - 80 -100 -120 -140 -160 -180 I I I I I 1 TT "! ppm ------ppm III—AL—176 HMBC CDC13 - 298K O - 20 ■ f-BuxSxNH O - 40 Me ' OPMB - 60 - 80 -100 -120 -140 -160 -180 I ! I I I I T I -| ppm 01270906: Scan 42 (8.30 min) Base: 176.00 Int: 5.6423e+006 Sample: VG 70-SE Positive Ion FAB 105% 176 Sample: lll-AL-176 Instrument Resolution: 7000 Theoretical Mass: (M+Na) 420.18205 90% 121 Measured Mass: (M+Na) 420.18129 Error: 1.81 ppm OMe OPMB 110.0 i % T 100.0 9 0 .0 iY\ ! i 8 0 .0 3 0 7 2 .4 3 3 3 0 .8 2 8 4 1 . 0 7 0 .0 2 8 7 3 . 7 8 1 9 .7 \ ! 1 6 1 2|.4 9 1 2 .3 I / 6 0 .0 (/ 13 86.7 1 4 6 1 .9 5 0 .0 2 9 5 0 . 9 |V 1 5 1 4 .0 1 1 ^ 4' I / j | 1 0 3 7 .6 4 0 .0 111) 8 .6 i: t-Bu NH O ;! V ( I OMe 1 7 4 1 .6 OPMB 1 0 7 2 .3 30.0 1 2 4 9 .8 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 1/cm IV-AL-21 CDC13 - 298K OPMB JJL j l L u - j — i— i— i— i— i— i—i— r—i— |— i— i— i— i— i— i— i— i— i— |— i— i— i— i— i— i— i— r—i— j— i- !— i— i- ' i i i i | 3 2 1 0 ppm IV - A L - 2 1 DEPT CDC13 - 298K O ■ t-Bu' "NH O HO OMe OPMB "I ...... I...... I , ■ ■ r * '...... |...... " | ...... | " , . ITT . i |...... ,...... ,...... I ...... I...... I ...... 1 I I I I I I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-AL—21 13C CDC13 - 298K < OMe OPMB feUAUMok •aatwM ■aha ■UiA*Ae i r i 11 i iiiiiI I I I i I Ii i i| n I I I I I 1 I I I 1 I I i » I T ~rrTrrrn 'I "T I r™l" I...... I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm i AJL n • «• 2 1 r 7 r 5 r -2 ppm ppm D1 - 298K - CDC13 IV-AL-21 -uSN O f-Bu"S"NH COSY 0 ■ OPMB OMe IV—AL-21 HMQC CDC13 - 298K jUl ppm O ■ -S. 20 t-Bu NH O OMe OPMB -100 -120 -140 -160 -180 10 9 8 7 2 1 ppm 01270206: Scan 369 (67.60 min) - Back Base: 173.00 Int: 2.89815e+006 Sample: VG 70-SE Positive Ion FAB 1 0 0 % Sample: IV-AL-21 90% Instrument Resolution: 7000 Theoretical Mass: (M+Na) 438.19262 f-Bu NH O Measured Mass: (M+Na) 438.23181 HO. Error: 89.40 ppm OMe 80% OPMB 70% 60% 50% 40% 30% 438 323 199 20% 472 10% 242 349 499 301 391 267 140 200 250 300 350 400 450 550500 600 105.0 %T 100.0 95.0 — 817.8 90.0 — 2036.7 1612.4 85.0 1380.9 2353.0 j 1458.1 12900.7 1512.1 80.0 3332.8 2947.0 75.0 f-Bux "NH O 1735.8 HO OMe OPMB 1249.8 1041.5 4000.0 3500.0 3000.0 2500.0 2000:0 1750:0 1500:0 1250:0' 1000.0 500.00 750.0' IV-al-21 v - o i - 1/cm IV-AL-2 5 CDC13 - 2 98K OPMB lili LajlJ IV -A L -2 5 DEPT CDC13 - 298K OPMB IV-AL-25 13C CDC13 - 298K OPMB l+fa rm "T T I I I I I I I i I 11 i 11 11 i ri M II I II I I I I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm IV -A L -2 5 COSY JLJL jH A c l ppm CDC13 - 298K t- Bu NH O MsO - 1 OMe OPMB < -2 - 3 4 e» Qi - 7 8 7 2 1 ppm IV-AL-25 ppm HMQC CDC13 - 2 98K 0 1 .s 20 f-Bu NH O MsO OMe OPMB 40 80 -100 -120 -140 -160 -180 9 7 4 38 2 1 ppm IV-AL-2 5 ppm HMBC CDC13 - 298K 20 t- Bu NH O MsO OMe 40 OPMB -100 -120 -140 -160 -180 9 8 7 4 3 2 1 ppm 01130306: Scan Avg 260-266 1-7.62 - 48.72 min] - Back Base: 155.00 Int: 3.4272e+00( Sample: VG 70-SE Positive Ion FAB 100% 15k 30f Sample: IV-AL-25 90% Instrument Resolution: 8000 Theoretical Mass: (M+H) 494.18822 f-Bu NH O Measured Mass: (M+H) 494.18907 MsO 80% Error: 1.72 ppm OMe OPMB 70% 60% 50% 40% 30% 20% 494 219 10% 341 391 0%4 100200 250 300150 350 400 450 500 550 650600 700 m/z 80.0 - 636.5 %T J 75.0 - 700.1 70.0 - 2869.9 145141.2 929.0 65.0 - 1 5 1 4 . 60.0 - 1741.6 55.0 - 172.6 50.0 - 2962.5 45.0 — f-Bu NH O MsO 1101.3 OMe 800.4 40.0 — OPMB 026.1 1259.4 i I r i— | i i i i | i i i i | i r i i I r i — i i— r 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 - IV-AL-25 1/cm IV-AL-30 CDC13 - 298K OMe OPMB J JL _A_ A IV-AL-30 d ept CDC13 - 298K t- Bus_ / OMe OPMB j*. " 1...... i...... i...... I' ...... i...... i...... i ...... i ...... i ...... i...... i ■ " r " ' T 7 "T 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-AL-30 CDC13 - 298K OMe OPMB 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm a_jAm . u. J k ppm IV-AL-30 COSY CDC13 - 298K OMe OPMB 8 7 3 2 1 ppm IV-AL-30 ppm HMQC CDC13 - 298K OMe OPMB 60 80 100 120 140 160 180 ppm IV-AL-30 HMBC ppm CDC13 - 298K 20 ° C O OMe OPMB 40 80 -100 -120 -140 -160 -180 9 87 5 4 3 2 1 ppm 03080306: Scan 249 (45.60 min) - Back Base: 154.00 Int: 5.31583e+006 Sample: VG 70-SE Positive Ion FAB 105% 154 Sample: IV-AL-30 Instrument Resolution: 9000 Theoretical Mass (M+H): 398.20011 90% J Measured Mass: (M+H): 398.19931 Error: 2.01 ppm OMe OPMB 80% 70% 60%-] 50%- 398 40% i 30% 20% 307 1 0 % 289 219 341 204 235 260 42 %T 9 5 . 0 9 0 .0 8 2 1 .6 3 4 4 2 .7 8 5 .0 1 6 1 2 2 8 7 7 . 6 8 0 .0 1 4 5 6 .2 1 5 1 4 .0 1 1|7|2V6 2 9 5 2 .8 7 5 . 0 1 0 3 3 .8 I 1 0 7 8 .1 1120.6 7 0 .0 1 7 3 7 .7 OMe OPMB 1 2 4 9 .8 65.0 1 1 I TH ! I r n | r I I 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1 5 0 0 .0 1250.0 1000.0 750.0 500.0 i / _ IV-AL-3 6 CDCL3 - 2 98K f OH OPMB 9 8 7 6 5 4 3 2 1 0 ppm IV -A L -3 6 DEPT CDCL3 - 2 98K f OH OPMB .... . ■i...... i 190 180 170 160 150 140 130 120 110 10090 80 70 60 50 40 30 2010 ppm IV-AL-3 6 13C CDCL3 - 298K f OH OPMB < 11 j ■ i ■ i ■ ■ i i i j i 11 i i i 11 11 i 11 i i 11 i i j i i i 11 i i i i j i rrr it t 'i i"j r rn 11 i 11 j i i i i 11 i i i j i i i i i i i i rpi i i 11 111 i | i > ■ i i i > i i j...... j i r I T | I I I I I I I I I j I I I I I I I I I j I I I I I I I I I | I I I I I I I I I | I I 'T'T'T T I I I j I I T I I I I 1 I | I 1 1 I 1 I 1 I I j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm IV -A L -3 6 COSY CDCL3 - 298K f OH OPMB IV-AL-3 6 ppm HMQC CDCL3 - 298K t- Bu / 20 OH 40 OPMB 60 80 -100 -120 -140 160 8 7 5 4 3 2 1 ppm IV-AL-3 6 ppm HMBC J__ L JL A)L CDCL3 - 298K 20 o f OH 40 OPMB 60 80 -100 -120 -140 -160 8 7 6 5 4 3 2 1 ppm 01160806: Scan 424 (84.70 min) - Back Base: 176.00 Int. 5.75134e+006 Sample: VG 70-SE Positive Ion FAB 105% 176 jSample: IV-AL-36 f-BuN ✓ Instrument Resolution: 8000 [Theoretical Mass: (M+Na) 392.18714 90% Measured Mass: (M+Na) 392.18674 Error: 1.02 ppm OH OPMB 80% 70% 60% 199 50% 392 40% 323 30% 20% 242 349 472 10% 214 286 371 0%- 150 200 250 300 350 400 450 500 m /z %T 100.0 95.0 90.0 85.0 I ! 80.0 821.6 75.0 70.0 3390.6 2873.7 65.0 60.0 2956.7 55.0 f-Bu„ / 50.0 OH OPMB 45.0 i i i i i i i r 4000.0 3500.0 3000.0 2500.0 2000.0 750.0 IV-AL-36 IV-AL-38 CDC13 - 298K OPMB U j L T— nrT 7 5 ppm 13C CDC13 - 298K OPMB ** IV-AL-38 J__ L JUL u l ppm cosy CDC13 - 298K OPMB 4 i i i i i i TT T T I I T I TT I I I I I I I I I TTTT I II I I TTT T TT i i i in i r i in i n i 10 ppm IV-AL-38 J__L — ppm HMQC CDC13 - 298K 20 40 OPMB 60 - -100 -120 -140 -160 -180 200 j*TI"l I I I I I I | I I I I I I I I I I I I I I I I I I I I | I 1 I I I I I I 1 | I I I i i l I i I ...... I ...... 10 ppm jLJL ■1.1. JL ppm IV-AL-38 HMBC CDC13 - 298K 20 40 OPMB 60 80 -100 -120 -140 -160 -180 -200 | i i i i i i i i i i i i i i i | i i i i i i i i i | I I M I I I I I I I I I 1 I I I I I I I I I I I I i i i i I I i I I I i i i i i i i i i 10 ppm 01130306: Scan Avg 332-334 (60.82 - 61.18 min) - Back Base: 154.00 Int: 6.26878e+006 Sample: VG 70-SE Positive Ion FAB 105% 154 Sample: IV-AL-38 Instrument Resolution: 8000 90% Theoretical Mass: (M+H) 368.18954 Measured Mass: (M+H) 368.19047 Error: 2.53 ppm 80% OPMB 70% 60% 50% 40% 30% 307 20% 289 10% 219 27' 36 460 244 341 ljU... 0 % 140 200 250 300 350 400 450 500 m/z %T _ 75.0 752 . 520./ 70.0 3436.9 576.7 65.0 60.0 2837.1 1361 55.0 1384 821. 6 2871.8 50.0 1461.9 45.0 2956.7 40.0 t - Bi> / 1033.8 1730.0 35.0 1072 .3 1514.0 _p.247.p- OPMB 1087.8 3 0.0 4000.0 3500.0 3000.0 2500.02 0 0 0 . 0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 T T 7 "A T O O 1/cm IV-AL-13 8 CDC13 - 298K MeO I ■ 1 ■ 1 1 ' i ' i | ■ i i— i— i— i— i— i— i— |— i— i— i— i— i— i— i— i— i— |— i— i— i— i— i— i— i— i— i— |— i— i— i— i— i— i— i— i— i— |— i— i— i— i— i— i— i— i— i— | - I I I I I I I I I I I I I I I I I 1 I I I I I I I I I ' ' H 9 8 7 6 5 4 ppm IV —AL—138 DEPT CDC13 - 2 98K O M eO - o 5 7 ^ IV —AL—138 13C CDC13 - 298K M eO 190 180 70 50 40 20 10 ppm IV—AL-111 CDC13 - 298K Me Br Me Me i i i < I i 1 P 1 ppm IV-AL-62 CDC13 - 298K M e HO M e M e / ------ i i i i » i I ...... | ...... [ | 1 1 1 ' i-r-r-r-r-p t 1- 9 8 7 6 5 4 ppm IV-AL-122 CDC13 - 298K M e j l J w U J U U i 7 6 IV —AL—122 DEPT CDC13 - 2 98K Me Ph ■‘i ...... I...... I...... I...... '"I...... I...... '"I...... " ' I I...... I...... I...... I...... i...... I...... i...... I...... '...... i...... 1...... 1 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV —AL-122 Me 13C CDC13 - 298K Jl Jw>1 ■ k ..a. T 7 80 70 60 50 40 30 2 0 10 ppm IV-AL-122 _ J J u jk _ _ A ______L i . ppm COSY CDC13 - 298K -0.5 Me 7 1 . 0 7 1 . 5 Ph 7 2 .0 72.5 -3.0 n 73.5 74.0 74.5 -5.0 5.5 b 6 . 0 6.5 r 7.0 -7.5 ;=! 8 .0 8 7 6 4 3 25 1 ppm IV-AL-122 ppm HMQC CDC13 - 298K Ph - 60 - 80 -100 -120 -140 -160 -180 ppm 04250507: Scan 45 (6.65 min) Base: 349.00 Int: 53791 Sample: VG 70-SE Positive Ion FAB 100% 197 346 Sample: IV-AL-122 90% Instrument Resolution: 7000 Theoretical Mass: (M+Na) 760.20608 Measured Mass: (M+Na) 760.20449 Ph 80% Error: 2.09 ppm 70% 60% 50% 40% 30% 151 20% 275 760 660 10% 600 241 0%J 100 200 300 400 500 600 700 800 m/z IV-AL—70 CDC13 - 298K ppm IV -A L -7 0 DEPT CDC13 - 298K Ph ■xrprr- 190 180 170 160 150 140 130 120 110 100 90 IV -A L -7 0 13C CDC13 - 298K I V - A L - 7 0 ppm COSY CDC13 - 298K 7 0 . 5 L1.0 (D 7 3.0 -3.5 74.0 7 4 . 5 7 5.0 7 5.5 76.0 76.5 77.0 7 7 .5 : 8 . 0 ppm ______ppm IV-AL-7 0 HMQC CDC13 - 298K - 20 (D - 40 - 60 - 80 -100 -120 -140 -160 -180 ! 1 1 11 i i i I 200 ppm ppm IV-AL-70 HMBC CDC13 - 298K - 20 (D - 40 Ph 60 80 -100 120 -140 -160 -180 L 200 ppm 02131006: Scan 49 (9.70 min) - Back Base: 408.00 Int: 5.26814e+006 Sample: VG 70-SE Positive Ion FAB 100% 40& Me 90% Sample: IV-AL-70 Instrument Resolution: 10,000 Theoretical Mass (C22H27N05): 408.17868 (M+Na) Ph- 80% Measured Mass: 408.178770 Error: 0.22ppm 70%- 60%- 50%- 40%- 30%- 20%- 10% 558 178 328 536 222 370 464 584 0% ___iL_ 100 150 200 250 300 350 400 450 500 550 600 650 700 750 m/z 100.0 95.0 90.0 2852.5 85.0 211981.7 54.2 2922.0 1 1 H 0 1352 80.0 1028.0 1093.6 1213.1 1699.2 75.0 1242.1 1384.8 70.0 65.0 1780.2 i i i i r i r ii r r i | i r 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500 0 1250.0 1000.0 750.0 500.0 - IV-AL-70 1/cm IV -A L -1 2 6 CDC13 - 298K Me Ph j ------ j l i t . L_ a . i ...... i ...... I ’ ' ' ' ," 7 '' 1 ' I ...... 1 ' I ' ' 1 ' , ' 1 ' ' T 9 8 7 6 5 4 ppm IV -A L-126 DEPT CDC13 - 298K I Me Ph IV -A L-126 Me 13C CDC13 - 298K Ph ■■■i...... i...... i...... i...... r ------1"-----1...... i...... li" 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV -A L -1 2 6 ppm COSY CDC13 - 298K 7 0 . 5 Me 7 1 . 0 7 1. 5 72.0 72.5 73.0 73.5 74.0 74.5 75.0 75.5 76.0 76.5 77 .0 7 7 .5 - 8.0 ppm IV-AL-12 6 ppm HMQC CDC13 - 298K Me Ph - 60 - 80 -100 -120 -140 -160 -180 r i i i i i i i | ppm IV-AL-12 6 Jjl A li 1 m______luUL ppm HMBC CDC13 - 298K 20 Me 40 Ph 60 80 -100 -120 -140 -160 -180 8 7 6 5 4 2 1 ppm RT: 19.50 AV: 1 SB: 384 0.42-18.08 , 19.70-20.02 NL: 3.41E6 + c E 536 100 Me 95 Sample: IV-AL-126 Instrument Resolution: 6250 go- Theoretical Mass: (M+Na) 536.09044 Ph 85 Measured Mass: (M+Na) 536.09098 Error: 1.01 ppm 80- 75- 70- 65: 60: 55: 50: 45: 577 40: 35- 30: 537 25: 261 445 20: 217 281 320 383 552 15: 343 578 305 531 624 10: 456 486 280 321 418 538 5 : 246 262 384 487 553 610 625 235 282 349 377 398 430 457 473 510 195 I 247 306 322 489 575 579 604 ! 649 J..i, I ni,i .iimii lijJi .in 0- 'T™ I"' 200 250 300 350 400 450 500 550 600 650 m/z 11 0 .0 %T 100.0 "/v4•wit, "m. 90.0 974.0 858.3 702.0 80.0 2850.6 1018.3 731.0 2929.7 1035.7 70.0 2981.7 60.0 1099.3 1699.2 1211.2 Me 1724.2 1382.9 50.0 1242.1 Ph 1780.2 40.0 500.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 1/cm — — IV-AL-126 IV -A L -1 2 8 CDC13 - 298K Me Me J ______L l i jUL u IV -A L-128 DEPT Me Me CDC13 - 2 98K 80 70 60 50 40 30 20 10 ppm IV -A L-128 13C CDC13 - 298K Me Me -rTTTTT 70 60 50 40 30 2 0 10 ppm IV -A L -1 2 8 1 jlJ l ji - -k a *l. ppm COSY CDC13 - 298K 0.5 “ 1.0 Me Me p i . 5 Ph 7 2 . 0 4 4 p 2 .5 ■ 7 3 . 0 9 7 3.5 -4.0 7 4. 5 < -5.0 5.5 7 6 .O 7 6 . 5 -7.0 7 7 . 5 - t— i—i—r i i i i r r i i i TT I I I I 1 I I I TT—I—T~i— I—T I I I i i i TT ■n 8.0 ppm IV-AL-128 -it - J * t ppm HMQC CDC13 - 298K - 20 Me Me - 40 - 60 - 80 -100 -120 -140 -160 -180 I I i i I i' i i | ppm IV-AL-12 8 ppm HMBC CDC13 - 298K Me Me - 20 - 40 - 60 - 80 -100 -120 -140 -160 -180 t—i—i—i—i—i—i—r - j ppm 01131006: Scan 147 (29.30 min) - Back Base: 176.00 | Int: ~2.23748e+006 ~ ------Sample: VG 70-SE Positive Ion FAB 105% 176 Sample: IV-AL-128 Me Me Instrument Resolution: 7000 Theoretical Mass (M+Na) 424.20998 90% Measured Mass: (M+Na) 424.21073 Ph Error: 1.77 ppm 80% i 70% H 424 50% H 40% -\ 30% 20% 329 199 10% 344 256 289 312 464 200 250 300 350 400 450 500 m/z 1 0 5 . 0 - %T 1 0 0 . 0 - ■ V ' W"S« \ 9 5 . 0 - 9 0 . 0 - 2 8 7 5 . 7 7 2 9 . 0 8 5 . 0 - 2 9 2 5 . 8 1 4 5 2 . 3 2 9 7 9 . 8 7 6 5 1 0 3 5 . 7 8 0 . 0 — I 7 5 . 0 - 1 0 9 7 . 4 Me Me 7 0 . 0 Ph 1 2 4 2 .1 1 7 8 0 . 2 6 5 . 0 1 7 2 4 . 2 60.0 i | i r 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 IV-AL-128 1/cm I V - A L - 1 62 CDC13 298K ' j i ■■■■!■■■ ■ r 1 1 i 1 1 ri i 1 1 1 1 r ‘ , t ’ |-r . i i r , , , . , i i . . , . . i i ! - i . ■ , ■ ■ . i , ■ ■ ■ ■ 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm I V - A L - 1 62 CDC13 - 298K 1 . . . ! . . . . | . . .— . |— .— .— . .— |— .— . .— .— [— 4.0 3.5 3.0 2.5 2.0 1.5 IV -A L-162 DEPT CDC13 - 298K I ...... I ...... I ’ ■ ■ ' ...... I ...... I ...... I ...... , . . n r - . " T " , ...... r p m r T T T ' , . . . r-. " " | r " « . . r . | r , m . . . . | , ■ r , , . . , ...... , ...... , . . | ...... i . . , ...... | ...... , 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-A L-162 DEPT CDC13 - 298K " i ‘ 1 ■ ■ i • • ■ ■ i ■ ■ ■ ■ i ...... 1 ...... i _ r ■ ■ r i ■ ■ ■ ■ i 1 ■ 1 ■ r 1 1 1— 1 i ■ ■ ■ ' - | '■ ■ ■ > , ■ • • ■ | • ■ ■ ■ | • ■ • ■ | • ■ ■ ■ i ■ ■ ■ ■ i ■ ■ ■ ■ i ■ ■ ■ ■ i ■ • ■ i 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 ppm IV-AL-162 13C CDC13 - 298K I I I I I I I I I I I I...... I...... I'1...... ''I...... ‘I ■)" " " TT,I'T,,I" " I'...... I'" ...... |"""' "1" i...... | 190 180 170 160 150 140 130 120 110 100 90 70 60 50 40 30 20 10 ppm MJUUL A L J j Ak Jt . A a A tw J 1 l - -0.5 -2.5 -1.5 -3.5 -3.0 -4.5 -4.0 ppm ppm 2.0 1.0 5.0 c "O Xc D1 - 298K - CDC13 IV-AL-162 COSY r= N =xr OCO IV-AL-162 HMQC i k » ill. .a. a a t _ A JL t ppm CDC13 - 298K 10 -20 Xc Xr = N -30 -40 -50 -60 -70 80 -90 | i— i— i— i— |— i— i— i— I— |— I— i— i— i— |— i— i— i— i— |— 1— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— I— i— |— I— i— i— i— |— i— i— r 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm IV-AL-162 HMBC ik JL -llllJ L J L Me. _ j __ ppm CDC13 - 298K r 10 V xc = h r 20 V O r 30 0 0 -40 r 50 -60 r 7 0 r 80 r 90 4.5 3.5 3.0 2.54.0 2.0 1.5 1.0 0.5 ppm IV - A L - l62 _^L M_jl ppm HMBC CDC13 - 298K V - 20 Xc Xr= N - 40 \ S' o2 - 60 80 -100 -120 -140 -160 -180 ppm 01050906: Scan 89 (17.70 min) - Back Base: 446.00 Int: 2.00573e+006 Sample: VG 70-SE Positive Ion FAB 100%n 44& Sample: IV-AL-162 90%- Instrument Resolution: 8000 Theoretical Mass: (M+Na) 446.19770 Measured Mass: (M+Na) 446.19707 80%- Error: 1.14 ppm 70%- 60%- 50%- 40% 20% 173 472 10%- 323 622 541 242 371 596 150 200 250 300 350 400 450 500 550 600 650 700 m/z 105.0 %T 100.0 V \ 95.0 / ' T\ \ I \ 90.0 1 SI ! v 85.0 769.5 860.2 80.0 975.9 75.0 70.0 2887.2 540.0 1456.2 1060.8 65.0 1093.6 2941.2 60.0 2979.8 1267 I | 1029.9 55.0 1380.9 1128.3 1168.8 1697.2 50.0 1213.1 | 1236.3 45.0 oco 1330.8 i i i— r 4000.0 3500.0 3000.0 2500.0 2 0 0 0 .0 1750.0 1500.0 1250.0 1 0 0 0 .0 750.0 500.0 IV-AL-162 1/cm IV-AL-150 CDC13 - 298K f EtO 'O 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm IV-AL-150 DEPT CDC13 - 298K EtO 'O y¥m IV-AL-150 13C CDC13 - 298K Me,, E tO ^O 190 180 170 160 150 140 130 120 110 100 80 70 5060 40 30 20 10 ppm J u l ppm 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4 . 0 4.5 TT T 5.0 .0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm IV-AL-150 HMQC J u l .A A -A rv^ ppm CDC13 - 298K 20 40 80 -100 -120 -140 -160 -180 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm IV-AL-150 HMBC CDC13 - 298K J u l JlLJk ^JVA_i ppm 20 40 60 80 -100 -120 -140 -160 -180 | 1 1 1 ' | ' ' i— i— |— i— i— r— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— | 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 01290806: Scan Avc I 36-37 (7.10-7.30 min) - Back Base: 173.00 Int: 1.5 >3103e+006 Sample: VG 70-SE Positive Ion FAB 105% 173 Sample: IV-AL-150 Instrument Resolution: 7000 90% Theoretical Mass: (M+Na) 277.14157 Measured Mass: (M+Na) 277.14189 Error: 1.15 ppm 80% 70% 60% 50% 323 40% 199 30% 277 20% 349 242 10% 213 371 16( 0%^ UlL 150 200 250 300 350 400 m/z 80.0 70.0 60.0 50.0 2933.5 40.0 \J 1377.1 109p.3 30.0 2983.7 12 ' h * 1028.0 1174.6 20.0 Me, 1735.8 EtO 10.0 0 .0 ! I ! i I 4000.0 3500.0 3000.0 2500.0 2 0 0 0 .0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 IV-AL-156 CDC13 - 298K Eta i l ______ - r r~T' "T“T' 9 8 7 6 5 4 3 2 ppm IV-AL-15 6 CDC13 - 2 98K EtO ppm JU i i ' 1 ' 1 i ■ ■ ■ ■ i ■ ■ ■ ■ i ■ 1 1 1 i 1 1 1 1 i 1 1 1 1 i 1 1 1 ' " ' i 1 ' 1 1 i 1 ■ ■ i ■ ■ ■ ■ i ■ ■ ■ ■ i ■ ■ ■ ■ i ■ ■ ■ ■ i 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm IV-AL-156 DEPT CDC13 - 298K SnPh 3 Me Me/, EtO " 0 ■I...... i...... i...... " " | " ...... |...... |...... |...... |...... |...... |...... |...... i...... i...... i...... i...... I...... r l I I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-AL-156 13C CDC13 - 298K EtO "■i...i i i i "'I.1 i i .....i....TTi.....i "TT i i,nrT... i.....i .....r1 TTirirr'rTl i i TTT i.. . " i.... | |.....I I I 1 I '7T|.....|.....TT]..... | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-AL-15 6 W. .. U - ppm COSY CDC13 - 298K SnPh3 Me EtO "0 ■ OQo -5.5 s 7.5 8 i i i iT r i i r" i i i i i i i t — i—i—i— i—i—i— i—r IT T i i i i i I i i i i r T T T "i—i—i—i—i—\—\—i—rl—r ppm /An il______JL J L A i l _ > J v . W. - u -J 1 - ppm IV-AL-156 HMQC CDC13 - 298K 20 SnPh3 Me Me, 40 EtO "O 60 80 -100 -120 -140 -160 -180 8 7 6 34 2 1 ppm il _a J v_ ppm IV-AL-156 HMBC CDC13 - 2 98K 20 SnPh3 Me Me, - 40 EtO "O - 60 - 80 -100 -120 -140 -160 e a 0 -180 01061006: Scan 12 (2.30 min) Base: 218.00 Int: 3.51508e+006 Sample: VG 70-SE Positive Ion FAB 105% 218 Sample: IV-AL-156 Instrument Resolution: 7000 90% (Theoretical Mass: (M+Na) 629.16896 Measured Mass: (M+Na) 629.16654 Error: 3.85 ppm 80% ■] I / ' 1'- IV / ! 1 I 1618.2 1344.3 4.2 858.3 2866.0 1I454 .2 30fflS.9 449.4 14- 1.1 2931.6 ! I 1026.1 700.1 SnPh3 Me 1375.2 V 731.0 EtO O H ? 8-6 1095.5 2979.8 1732.0 1180.4 4000.0 3500.0 3000.0 2500.0 2 0 0 0 .0 1750.0 1500.0 1250.0 1 0 0 0 .0 750.0 500.0 IV -A L - 156 1/cm IV-AL-160 CDC13 - 298K I Me EtO '0 I r J L . l i J j i n -i 1------1------i------1—I—i—i—I—|—i—i—i—i—|—i—f—i—i—|—r—i ------1 i | i I—i------i—j—r—i—i—i—|—i------i—I—I—|—r— 1- 1—r—| - 1---i—i - 1 j' ■ t-t i—i j i i- i i—|—i—r ~ i ------I—|—i----1------i------i—|------1---1—r —i—j- 1—r—I—r—| 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm IV—AL-160 DEPT CDCL3 - 298K W«M "i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i.... i 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-AL—160 13C CDC13 - 298K Me,,, EtO 0 m * m m m * * # * -|-i i i'i i i i i i j i i i i i i i i i j i i i i rr i i i j i i i i i i i i i-j-i i i i i i i i i j i i i i i i i i i j i - r r r p i i i I i i rrri I i1 I I I I I IT i i j i i iiiiim j i i i i i nii ji iiri i i i i j i i iiii i i i j i i i i M iii | i i i i i i i i i j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm — : 7 JtJk. |—1— 1" 11 1 — | — 1 1 1i— " ———— 1 — 1 — 1■ 11 |1 1 1 1 1 ■ ' 1—1—1—1—i i | 1 1 v 1 1—i 1 r 1—1 1 1 1—1 1 |—1 1—1 |" —r 1 1 1 1 1—1 1—1 1 ■ 1 1 |—1 1 1 r 1 1 1 | 6 4 2 1 2 3 4 5 G 3 O Lk. JL !Q80“ —Hi. 1 | | 1 j- 2 . 0 - .1 5 '- 7 p3.5 '73.0 p2.5 7 4.5 r 0 . 4 p -7.0 76.5 6 7 7 ppm 5 .5 ppm 1.0 0 5 .5 .O . 0 D1 - 298K - CDC13 IV-AL-160 COSY Me I IV-AL-160 i______L a___ I______fUk. ppm HMQC CDC13 - 298K - 20 - 40 - 60 - 80 e -100 -120 o -140 -160 -180 |— I— i i I— i— r— i— i— i— |— i— i— i—i— i— i— i— i— i j i— i— i— i— i— i— i— i— i— |— i— i— i— i i i— i— r— i— |— i— i— i— i— i— i— I— i— r-|— i— I— i— i— i— i— i— I— I— 1~ 1 7 6 5 4 3 2 1 ppm IV-AL-16 0 HMBC ppm CDCL3 - 298K Me EtO - 40 - 60 - 80 -100 -120 -140 -160 -180 t—i—i—n —i—i—i—| ppm 01290806: Scan Avg 53-54 (10.50 -10.70 min) - Back Base: 173.00 Int: 6.50748e+006 Sample: VG 70-SE Positive Ion FAB 105% 173 Sample: IV-AL-160 Instrument Resolution: 7000 Theoretical Mass: (M+Na) 405.05387 90% Measured Mass: (M+Na) 405.05465 Error: 1.93 ppm EtO 80% 70% 60% 50% 40% 30% 199 20% 323 10% 21 242 349 0% H. ...n. 95.0 3438.8 2891.1 92.5 2931.6 90.0 87.5 2981.7 85.0 82.5 80.0 — 1178.4 EtO O 1732.0 77.5 i — i— i— r 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0500.0 IV-AL-160 IV -A L-161 CDC13 - 298K Me Me Me,,, EtO ' ' | 1 1 1 1 | ' ' ' i | i i i i | i I ■ i | i- 1--- 1--- 1--- 1---- 1- 1--- 1--- 1--- 1---- ,-- 1--- >--- 1--- 1----- 1- 1--- 1 r 1---- 1- 1--- 1--- 1--- 1---- 1-- 1--- 1--- 1--- 1 ----- 1- 1--- 1--- 1----- 1 i- i 1--- 1--- 1---- 1 i i i | i i i < | i ■ < i | 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm IV -A L -161 DEPT CDC13 - 298K " 1...... i...... I...... i.... 1" I...... 1...... r I...... I"'.... T"' ■ ■ ■ ■ ■■11 ■■ ■ 11..... i. -1 . j .....| r,r,r ...... ,...... |...... |...... | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-AL-161 13C CDC13 - 298K JO. JLa__a . L J. p p m IV-AL-161 COSY CDC13 - 298K -0.5 M e Me - 1.0 -1.5 - 2.0 QO -2.5 -3.0 -3.5 I -4.0 (D -4.5 -5.0 -5.5 - 6.0 ppm I ..... Ju u a . - ppm IV -A L -1 61 n HMQC CDC13 - 298K 20 M e Me 40 EtO ' 0 80 -100 -120 -140 -160 -180 0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm IV -A L-161 J Jl ppm HMBC CDC13 - 298K 20 M e Me 40 EtO ' 0 60 80 -100 -120 140 -160 -180 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 01050906: Scan Avg 101-102 (20.10 - 20.30 min) - Back Base: 293.00 Int: 3.27675e+006 Sample: VG 70-SE Positive Ion FAB 100% 29& Sample: IV-AL-161 Instrument Resolution: 8000 90% Theoretical Mass: (M+Na) 293.17287 Me, Measured Mass: (M+Na) 293.17223 Error: 2.18 ppm EtO 80% 70%- 60% 50% 323 40% 30% 20% 10% 307 27 348 0%- 4 ^ - 400 450 500 1 1 0 . 0 - % T 1 1 0 0 . 0 - 9 0 .0 - 8 0 .0 - 7 0 .0 - 2 8 7 7 . 6 14 5 6 0 .0 2 9 3 1 . 6 5 0 .0 2 9 7 9 . 8 4 0 .0 1 7 3 3 .9 3 0 .0 20.0 Me Me 10.0 EtO O 0.0 4000.0 3500.0 3000.0 2500.0 2000.0 1500.0 1250.0 1000.0 750.0 500.0 IV-AL-161 1/cm IV-AL-168-2 CDC13 - 298K M e Me LJ Li IV-AL-168-2 DEPT CDC13 - 298K M e M e " 1...... I...... I ' " 1....I...... I...... I...... I "T^ "T'r...... I...... I...... I...... ]"' .... I " " I...... I...... I -1-f-r rrrTI rjr-..... ,...... | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-AL-168-2 13C CDC13 - 298K M e M e i...... i I I"' ■11 ■ ■ii'1 ■ 't q i" i ■ i ...... i" 1" ■ r, ■ r ■m r^ 1 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV-AL-168-2 COSY A A A , . • j ___ ppm CDC13 - 298K -0.5 M e Me - 1.0 -1.5 ■ © - 2.0 @ 0 -2.5 -3.0 -3.5 -4.0 -4.5 -5.0 -5.5 - |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— I— i— i— |— i— i— i— i— j— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i— i— i— i— |— i ' ' i | i '-—i 1 | 1 r " 6.0 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1. 0 0.5 ppm os o on on on o (_n o 00 on oo o iso on NO o h- 1 On I-1 o o on 11 I I II I I I | I I I I I I II I [■ I I I I I I I I I I I I I ! I I I I I III I I I I I I I I I I I I I I I I I I I I I I1TII I I I I I I IITI I I I II I I I II I I II I I 11 II I I I III I I I I I I I I I I I I I I I I I I I I I I I I IKI I I I I I I Iri I I I I I I I ^d I—1 "d 3 00 NO O SO CO -J CTi on JS> oo NO ^d O o O O O O o o o o o o o 3 CTi O On On (J1 o On o Oo On 00 o fo on c-o o h-1 on o 0 5 I—1 o o on 11111111111111111m i 111 irn 11111 i i n 11111 iti 111111n 111 m 1111 n 11111 i'i i n 11 iri 1111111111 ii 111111I I111111 rn r 1111 itti i i i ii iv m 1111111111111111111111111111111 I-* g on 4^ 00 NO o 00 cn> on 4^ 00 K) M o O o O o o o o o o o o o o o 01080906: Scan Avg 75-78 (14.90 - 15.50 min) - Back Base: 173.00 Int: 977730 Sample: VG 70-SE Positive Ion FAB 100% 17b Sample: IV-AL-168-2 90% Instrument Resolution: 8000 Me 251 Theoretical Mass: (M+Na) 251.16231 Measured Mass: (M+Na) 251.16182 Error: 1.95 ppm HO 80% 70% 60%- 50%- 40% 30% 323 20% 349 10% 19 23^ 0% ^ 1 r 1 r- 145 200 250 300 350 m/z 105.0 %T 100.0 J 95.0 n /T\ 90.0 /r 1726.2 85.0 i ! I l l 80.0 i /i 862.1 \ / I I 75.0 14542 3433. 70.0 1 1 7 4 .6 65.0 2877.6 13 77.1 1095.5 j 2927.7 2979.8 60.0 1238.2 Me Me 55.0 1033.8 50.0 4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0 500.0 IV-AL-17 3 CDC13 - 298K J j L jL jL j lJ ppm IV —AL-17 3 DEPT CDC13 - 298K Me Me ‘” i...... i...... i...... i...... i...... i...... i...... i...... i...... i...... i...... r " "I...... I...... I...... n,"l ...... I...... "I ...... ""I ...... I"....."I...... I 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm IV —AL-173 13C CDC13 - 298K Me Me Me,,, 200 180 160 140 120 100 80 60 40 20 IV-AL—173 * * Ii 1 11_____ i—l ppm COSY CDC13 - 298K Me Me ep ep A E- 1 «* J! - 2 Q 0g Si* - 5 CD 9 * - 7 - 9 r 10 ------IL i 1 i i i i i r i i | i i i m r ii i | i i i i i i ii i i -L 11 10 2 1 ppm IV -A L -1 73 1 ppm HMQC CDC13 - 298K M e M e 20 40 80 -100 -120 -140 -160 -180 -200 L 220 11 10 8 7 6 5 34 2 1 ppm 04150906: Scan 19 (3.70 min) Base: 173.00 Int: 3.29535e+006 Sample: VG 70-SE Positive Ion FAB 100%-) 178 Sample: IV-AL-173 Me Me Instrument Resolution: 7000 90% Theroetical Mass: (M+Na) 249.14666 Me-,, Measured Mass: (M+Na) 249.14601 Error: 2.61 ppm 80% 70% 60% 50%H 40% 30% i 199 323 20% 249 283 34 221 x,„. , 0%4 -4411+ ill',1 1 ■ i ■ ■ ■ i111 ■'11 'V 350 400 155 200 250 300 m/z