CYCLIC BYDROXAMATES

by

MarieClaire Wilson

BSc., McGitl University, 1996.

A THESIS SUBMITTED IN PARTIAL, FILFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Chemistry

O Marie-Claire Wilson 2001 Simon Fraser University August 9,2001

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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT

Syntheses of 5-, 6-, 7- and Srnembered rings containing a hydroxamate moiety

RCONHOR' have been examined using, as the key step, a Lewis acid-promoted cyclization of an a-arninooxyester with trimethylaluminum. The 5,6- and 7-membered cyclic hydroxamates were obtained in good yield, and a crystaï structure was secured for the Tmembered ring. However, the formation of an 8-membered cyclic hydroxamate could be estabiished oniy by GC-MS.

The aminooxyester precursors of the cyclic hydroxamates were synthesized fiom

4, 5-, 6- and 7-membered lactones by successive reaction with hydrogen bromide in acetic acid, followed by rnethanol, to fom a-brorno methyl esters, and then displacement of broxnine by N-hydroxyphthalimide followed by removal of the phthalunido protection with methyihydrazine. In the case of methyl P-bromopropionate, the displacement of bromine proceeded by an elimination (to methyl acry1ate)- Michael addition sequence.

The use of N-hydroxyphthalimide as the reagent of choice for the introduction of the aminooxy group led to better yields in both the displacement and deprotection steps than

N-hydroxy succinimide.

The 5-membered ring, an isoxazolidinone, is found in the antibiotics cycloserhe and lactivicin, which have an amino or acylamino substituent at C4 respectively, and interfere with different stages of bactenal ce11 wall synthesis. The 6-mernbered kg, an oxazinone, is found in cyclocanaline, which also has an amino group at C4 and is reported to exhibit antibacterial activity. Syntheses of Ci-butoxycarbonyl-, 4- phenylacetamido- and 4-phenoxyacetamido-substituted oxazinones were achieved starting with homoserine or homosenne lactone: in one sequence, addition of the acyl substituent, followed by saponification, esterification, mesylation, displacement by N- hydroxyphthalimide and removal of the phthdimido protection produced the required a- amulooxy esters, which were cyclized successfully. The t-butoxycarbonyl group could be removed and replaced by other acyl groups. The crystal structure of 4- phenylacetylamino-[l,2]oxazinm-3-une was determined. The a-aminooxyesters could also be obtained by direct displacement of an amino- and carboxyl- protected homoserine hydroxyl group with --hhydroxysuccinimide under Mitsunobu conditions and removal of the succinimido group,

Lactivicin has a substituent on the ring nitrogen. Several attempts were made to synthesize a cyclocanaline denvative carrying a 2-carboxypropyl substituent on the ring nitrogen. The best result was achieved by coupling of the acyclic aminooxyester with the triflate of a lactic acid ester, followed by trimethylduminum-pmoted cyclization.

Starting with homoserine and (S)-Iactic acid, the methodology just described then led to the synthesis of the ultimate target of the present work, a 4-acylamino-substihited cyclocanaline carrying a protected 2-carboxypropyl substituent on the ring nitrogen.

Biological assays indicated that some of the compounds synthesized in the present work exhibit antibacterial activity versus Micrococcus luteus. DEDICATION

To my mother and fi-iend,

Claire Deschênes

And in the loving memory of my grandparents,

Mr. & Mrs. J.A. Deschênes ACKNOWLEDGMENT

This master's project has been my life for the past three years and some months

and I would like to take this opportunity to ackmwledge the people who helped me along

the way to make this thesis a reality.

Firstly, 1 would like to thank my senior supe~sor,S. Wolfe, for allowing me to

take on this project and for his financial support throughout the years. 1 would like to

thank my CO-supervisor,A. Be~et,and the department chair, M. Pinto, for supervising

my progress and for their positive support. I would like to thank P. Wilson for the good

advise on trouble-shooting.

Secondly, 1 would like to thank G. Owen, M. Tracey and M. K. Yang for

providing mass and NMR spectra and elemental analyses, respectively. 1 would like to

thank A. Kennode for providing lab space and equipment to perform the bioassays and a

special thanks to Y. Zheng and N. Raimondi for their knowledge and guidance in the field of rnicrobiology. 1 would Iike to thank R. Batchelor and F. Einstein for providing the crystal structures. 1 would like to thank C.-K. Kim, K. Yang, N. Weinberg, and 2. Shi for the information regarding the computer generated model of the active site of PBP.

Thirdly, 1 would like to thank my lab mates past and present: Y .-H. Hsieh, for his friendship and understanding, S. Ro, for his synthetic help at the start of my degree, C.

Akuche, for welcoming me into the lab group, M.-H-Cheng, a scientist that 1 admire, 1 greatly appreciate the time she took to teach me valuable laboratory techniques, G.

Shustov, for his chemistry explanations, and A. Buckley, who was a great fiiend and was very supportive during some rough times. Fourthly, 1 greatly appreciated the time that F. Carey twk to write an amazing textbook on organic chemistry. It was my organic chemistry bible.

Finally and most importantly I wodd like to thank my fiends and family for their support during this time, without whom this wodd not have been possible: Claire D., my mother and fiend, Simmons D., my dearest grandmother and fiend, Nancy R., a long time fi-iend that 1 admire greatly and love (THANKS FOR EVERYTHING YOU'VE

DONE FOR ME!), Idalgo R., my Italian brother who was always looking out for me and is a person with the biggest heart, Joanna D., my constant phone buddy and caring fnend,

Jackie L., a Eend who brought me into the world of chemistry on a fun note, Karen W., a fi-iend who always had good advice for me, Nena G., my wild Texas ex-roommate who always entertained me with her life, Patrik P., a fiiend that brought spice into my life,

John M., for his fkiendship and for making me feel goocl about myself, Suzanne C., my cousin and fnend who is constantly giving me positive support, Carole P., my cousin and fiiend who cares with al1 her heart, Therese and Rene C., my aunt and uncle who are always sending me positive thoughts and love, Clare M., a great finend who that 1 can always rely on, Tatyana K., for her fkiendship and kindness, Irena P., a fiend who always saved a prayer for me, Stephen P., for his friendship over the years, Ronald G., for his good hearted nature, Sonja K., a new fnend who opened up her home and family to me,

John S., for his positive support, Dawne, for her smile and for always caring, Laura A., my shopping buddy, John C., Jon D., and Rasmus S., the three roommates that kept me entertained with their Çiendship.

vii TABLE OF CONTENTS .. Approval...... 11

Absîract ...... -111S..

Dedication...... v

Aclrnowledgements...... xi ... Table of Contents...... mil

List of Abbreviations and Syrnbols ...... xi

List of Figures...... xvi .. List of Schemes...... xwi

List of Tabies...... xix

1. INTRODUCTION...... 1

1.1 Antibacterial Agents...... l

1.2 Bacteria...... 2

1.2.1 Bacterial Structure and the Basis of Selective Toxicity ...... 2

1.2.2 Bacterial Cell Envelope...... 3

1.2.3 Inhibitors of Bacterial Cell Wall Synthesis...... -5

1.3 Mode of Action of P-Lactarn Agents...... 8

1.4 Mode of Action of ...... 10

1.4.1 Alanine Racemase...... 11

1-4.2 ~-Alanyl-~-Aianine...... 12

1.S Insights into the Essential Structural Features of Antibacterial Agents Targeted to ... . . Pemcillm-Bmdmg Proteins...... -13 1. 5. 1 Monocyclic Antibiotics ...... -14

1.5.2 Heteroatom Activated P-Lactam Antibacterid Agents: Mono and Bicyclic

Oxarnazins ...... 15

1.5.3 Lactivicin and Derivatives ...... -16

1 .6 Canavanine, Canaline and their Denvatives ...... 17

1.6.1 Natural Products: Canavanine and Canaline ...... -17

1.6.2 Syntheses of Canaline and Related Derivatives...... 18

1.7 Design of [1,2]0xazinan- 3-one Derivatives as Novel Antibactenal Agents ...... 23

1.7.1 Computational Approach ...... 23

1.7.2 First and Second Generation Compounds ...... 24

1.7.3 Denvatives of Second Generation Compounds...... -26

RESULTS AND DISCUSSION ...... -28

2.1 Retrosynthetic Analysis ...... -28

2.2 Syntheses of 5-, 6., 7- and 8-Membered Cyclic Hydroxamates ...... -33

2.3 Cyclocanaline and N-Acylcyclocanalines...... 40

2.4 Crystal Structures...... -45

2.5 N-Alkylation of Cyclic Hydroxamates ...... 48

2.6 Synthesis of 2-1 as a Benzyl Ester ...... -34

2.7 Biological Assays ...... 55

EXPERIMENTAL...... -56

3.1 General Methods ...... -56

3-2 Bioassays ...... 103

3.2.1 Matenals ...... -103 3-2.2 Methods...... - ...... - ...... - .. - - ...... - ...... -103 4. REFERENCES ...... a.....-...... œ...... *....1O6 AcOH acetic acid

CH3CN acetonitrile

NAG N-acetylglucosamine

NAMA N-acety lmuramic acid

ADP adenosine diphosphate

ATP adenosine triphosphate

D-Ala-D-Aia D-alany l- alai aine

ROH alcohol aq- aqueous

Ph2CH benzhydry 1

PhCH2 ben& br s broad singlet tBu0 tert-butoxy

BOC tert-butoxycarbonyl

BOC-ON [2-(tert-butoxycarbonyloxyimino)-2-phenylacetoni~~e tBu tert-butyl

13cnmr carbon- 13 nuclear magnetic resonance

CI chernical ionization

CHCL c hloro form

DBU 1,8-diazabicyclo[5 .4.0]undec-7-ene

CH2C12 dichloromethane

DCC dicyclohexylcarbodiimide DEAD diethyl azodicaboxylate

Et20 diethyl ether

Dm 2,3 -d.ihydropyran

DMF dimethyIformamide

DDM diphenyldiazomethane d doublet dd doublet of doublets

EX electron impact

Et ethyl

EtOAc ethyl acetate

GC gas chromatography

GlY glycine Hz hertz h hou(s)

H2NNH2 hydrazine

HBr hydrobromic acid

HCI hydrochloric acid

IR infiared

Me1 iodomethane

LYS lysine

MgS04 magnesium sulfate ml2 mass to charge ratio

MS mass spectnrm m.p. melting point

Ms mesyl

MsCl methanesulfonyl chloride

Me methyl

MeHNNH2 methyl hydrazine

PL microliter mL milliliter mm01 millimole

M molarity min mhute(s) m multiplet

NADPH nicotinamide adenine dinucleotide phosphate (reduced form)

N normality

Pi orthophosphate

Pd(C) palladium on activated carbon

PPm parts per million PBP -binding protein

PhOCH2COClphenoxyacetyl chloride

Ph phenyl

PhCH2COCl phenylacetyl chloride

PEP phosphoenolpyruvate

Ft phthalirnido

xiii PLC preparative layer chrornatograghy

HX proton donor

'~nmr proton nuclear magnetic resonance

KOH potassium hydroxide

Pr P~OPY~

PLP pyrîdoxal-5-phosphate quant. quantitative

quartet

revolutions per minute

room temperature

Ser senne

singlet

sodium bicarbonate

NaOMe sodium methoxide

succinimido

tetrah ydro fùran

TLC thin layer chromatography

threonine

tosyl

triethylarnine

TFA tri fluoroacetic acid (CF,S02)20 trifluorornethanesuIfonic anhydride me3 trirnethylaluminum

PPh3 triphenylphosphine t trip let

UDPNAG uridine-diphospho-N-acetylglucosamine

UDPNAMA uriduie-diphospho-N-acetyhwamicacid

Val valine

Hz0 water LIST OF FIGURES

Figure 1-1 Bacterial cell. possible targets for selective attack of antibacterial agents.. 3

Figure 1-2 Bacterial ceIl wall active antibiotics...... 4

Figure 1-3 Sites of action of inhibitors of bacterial ce11 wall synthesis...... 5

Figure 1-4 Und yl-diphosphate-N-acetyImurarnic acid pentapeptide...... -7

Figure 1-5 Transpeptidation sequence of PBP and its inhibition by penicillin ...... 9

Figure 1-6 N-Acyl-D-almyl-D-danine and its stnictural analog, penicillin ...... 9

Figure 1-7 ahni ni ne and its structurai analog, D-cycioserine...... 10

Figure 1-8 Proposed mechanism for inactivation of alanine racemase by D-

cycloserine...... 12

Figure 1-9 Proposed mechanism for D-Ala-D-Ala synthetase ...... 13

Figure 1-10 Naturally occurring antibacterial agents targeted to PBPs ...... 14

Figure 1-11 Computer generated mode1 of a peptide that contains the residues that

surround the active site serine of a PBP fiom Streptomyces Rd 1...... 23

Figure 1-12 Requirements for the design of a compound targeted to a PBP ...... 24

Figure 2-1 GC of [I ,210xazocan-3.one ...... 38

Figure 2-2A EI-MS of [1.2]oxazocan-3-one ...... 39

Figure 2-2B EI-MS of [I,2]oxazinan-3-one ...... 39

Figure 2-2C EI-MS of [1,2]oxazepan-3-one ...... 40

Figure 2-3 Fragmentation of the molecular ions of cyclic hydroxamates...... 40

Figure 2-4 The crystal structure of 4-phenylacetylamino-[1,2]oxazinan-3-one...... 46

Figure 2-5 Several views of the crystal structure of [1,2]oxazepan-3-one ...... 47

xvi LIST OF SCHEMES

Scheme 1-1 Reaction of a f3-iactam antibiotic with a penicilh-recognizing protein... 10 .. Scheme 1-2 Metabolism of ~.canavinine...... 17

Scheme 1-3 Syntheses of ~~.canaline...... 19

Scheme 1-4 Synthesis of ~.canaline...... -20

Scheme 1-5 Cyclization of halo hydroxamic acids ...... -21

Scheme 1-6 Syntheses of ~~.cyclocanahe...... 22

Scheme 1-7 Synthesis of 5-hydroxy derivatives of [ 1,2]oxazinan.3.one ...... 26

Scheme 2-1 N- and O-Acylation during coupling reactions with N-hydroxyamino

acids ...... 30

Scheme 2-2 Alkylation by alcohols under Mitsunobu conditions...... 31

Scheme 2-3 Attempt to couple 2-11 and 2-12 using DCC ...... -32

Scheme 2-4 Retrosynthesis of cyclic hydroxarnates...... 33

Scheme 2-5 Four step conversion of lactones into cyclic hydroxamates ...... 34

Scheme 2-6 Syntheses of methyl y-succinimidooxy- and y.phthalimidooxybutyrate ...3 5

Scheme 2-7 Synthesis of methyl y-aminooxybutyrate (2-20)...... -35

Scheme 2-8 Cyclization of methyl y-arninooxybutyrate (2.20) ...... 36

Scheme 2-9 Retrosynthesis of N-acylated cyclocanaluies...... 41

Scheme 2-1 0 Synthesis of 2-24 (RI = t.butoxy, Rz= benzhydryl, X = OSu) ...... -42

Scheme 2-1 1 Synthesis of 2-24 (Ri = t~butoxy,Rz = methyI, X = Br) ...... 42

Scheme 2-12 Synthesis of 2-24 (Ri = t-butoxy or benzyl, Rt = methyl, X = OMS)... 43

Scheme 2-13 Synthesis of 2-22 (RI = t-butoxy or benzyl) ...... 44

xvii Scheme 2-14 Synthesis of 2-22 (RI = PhOCH2)...... -45

Scheme 2-15 Mechanism of the Mïtsunobu reaction 1...... 49

Sctieme 2-16 Mechanism of the Mitsunobu reaction Ili ...... 50

Scheme 2-17 Synthesis of 2-34 ...... 51

Scheme 2-18 Mitsunobu couphg of 2-21 and ethyl (S)-lactate ...... -32

Scheme 2-19 Mitsunobu coupling attempt of 2-20 and ethyl (S)-lactate ...... 52

Scheme 2-20 Synthesis of 2-37 ...... -53

Scheme 2-21 Synthesis of 2-40 ...... 54

Scheme 2-22 Synthesis of 2-1 as a benzyl ester ...... 34 1 IlYTRODUCTION

1.1 Antibacterial Agents

The treatment of infectious diseases has corne a long way since ancient times, fiom bizarre concoctions of folk remedies to proven natural and synthetic antibacterial agents. Before the discovery of Prontosil (a sulfonarnide) and penicillin (a B-lactam), the onfy proven eeatments for systemic infections were resirïcted to plant extracts, notably cinchona bark, containing quinine and quinidine, for malaria, and chauhoogra oil, for

Ieprosy. '

During the twentieth century, the successes of sulfonamides, penicillin and streptomycin produced a therapeutic revolution in the pharmaceutical industry, with the creation of large scale screening programs for new antibiotics. This search provided chloramphenicol (1947),' chlortetracycline (1948)~~erythromycin (1952);

(1 9561,' lincomycin (1 962):~' and gentarnicin (1963): among others. Subsequent developments focused on the production of semisynthetic derivatives of the early antibacterial compounds with the aim of improving their properties, in particular the problem of resistance, which has increased with their use. Al1 of this work eventually led to the large number of clinically used hgsthat are available today. Approxirnately 250

' Hunter, P. A.; Darby, G.K.; Russell, N.J. F$y years of Antimicrobials: Pas2 Perspectives und Future Trends. Cambridge University Press, 1995. 'Bartz, Q.R. J. Biol. Chem. 1948,172,445. Duggar, B.M. Ann. N. Y. Acad. Sci 1948, Si, 1 77. 4 McGuire, J.M.; Bunch, R.L.; Anderson, R.C.; Boaz, H.E.; Flynn, E.H.; Powell, H.M.; Smith, J.W. Antibiotics and Chemotherapy 1952,2, 28 1. McCormick, M.H.; Stark, W.M.; Pittenger, G.F.;Pittenger, R.C.; McGuire, G.M.Antibiotics Annual 1955-56 1956, New York, 606. 6 Mason, D.J.; Dietz, A.; DeBoer, C. Antimicrobial Agents and Chemotherapy 1962, 554. 7 Herr, R.R.; Sergy, M .E. Antimicrobial Agents and Chemotherapy 1962,5650. "uedernann, J.C.; Weinstein, M.J. U.S. pat. 3,091,572, 1963. antibacterial compounds are on the world ~narket,~*'~moat of which belong to the main families of p-lactams, aminoglycosides, tetracyclines, macrolides, sulfonamides and quinolones. The challenge for the new century is to find novel classes of compounds to deal with the ever-present threat of resistance to the older agents.

1.2 Bacteria

1.2.1 Bacterial Structure and the Basis of Selective Toxicity

The main principle behind antimicrobial chemotherapy is 'selective toxicity', that is, the ability to inhibit or kill the invading organisrn, without harming the host. By differentiating between the structure or metabolism of bacterial and marnmalian cells, antibactenal agents can selectively target the invader, thereby decreasing possible side effects. Though unwanted side effects may still occur, these are generally unrelated to the primary mechanism of action. The main targets that offer selectivity for antibacterial agents are the outer membrane (Gram-negative organisms), the ce11 wall peptidoglycan, the ce11 membrane, the chromosome, the ribosomes and the cytoplasrn (Figure 1-l)."

Levy S.B. The Antibiotic Paradox: How Miracle Dnrgs Are Destroying the Miracle. New York: Plenum Press, 1992. 'O Sheehan, J-C. The Enchanted Ring: The Untoid Sfory of Penicillin. The MIT Press, Cambridge, Massachusetts, 1982. " Baron, S. (ed.) Medical Microbiofo~.2" ed. Addison-Wesley Pubiishing Company, tnc. Men10 Park, California, 1986. - Outer membrane Cell wall peptidoglycan (Gram-negative organisms) 1

Cell membrane ~ (DNA synthesis and transcription) Cytoplasrn Ribosome L (meta bolic transformations) (protein synthesis)

Figure 1-1 Bacterial cell, possible targets for selective attack of antibacterial agents.

1.2.2 Bacterial Ce11 Envelope

By use of the Gram stain, the structures of bacterial ce11 envelopes cmbe divided into two groups, positive and negative. 11,12 Both Gram-positive and Gram-negative bacterial cells contain a peptidoglycan Iayer compnsed of altemating N- acetylglucosamine (NAG) and N-acetylmurarnic acid (NAMA) units linked to short peptides. The crosslinking of these peptides gives shape to the organism and strength to the cell wall. The peptidoglycan is a usehil target for selectively toxic agents, since this structure does not exist in mammalian cells, Compounds that act on this structure at various stages of peptidoglycan biosynthesis include , cephalosporins and other

'' Hancock, 1.; Poxton, 1. Bacterial Ce11 Sugace Techniques. John Wiley and Sons Ltd., London, 1988. p-lactams, as well as D-cycloserine, glycopeptides and (Figure 1-2).

H3Ct/P03H2RCQNH O Fosfomycin OzcyC:3 - ".V2 C02H /$"" @ antiBiactam biotic 0, tl Ci HO Cycloserine

* OH Glycopeptide OH (Vancomycin)

Figure 1-2 Bacterial ce11 wall active antibiotics.

Gram-positive bacteria (staphylococci, streptococci, clostridia, etc.) have a thick

(30 m) peptidoglycan layer, which is extensively crosslinked, and is interspersed with glycophosphates, the teichoic and lipoteichoic acids. In contrat, Gram-negative bacteria

(Escherichia coli, salmonellae, pseudomonads, meningococci, etc.) have a th, loosely crosslinked peptidoglycan layer (2-3 nm) and no teichoic or lipoteichoic acid. Gram- negative bacteria also have a membrane-like structure extemal to the peptidog ly cm, which consists of lipoproteïns and lipopolysaccharides. This outer membrane is selectively permeable and thus regulates access to the underlying structures. Porins, hydrophilic pores, are found dispersed throughout the outer membrane. Small molecules,

including many antibiotics, can enter the ce11 through the porins, depending on their molecular weight, stereochemistry and ionic charge. Therefore, it is this outer membrane that often determines the spectnim of activity of antibacterial compounds.

1.2.3 Inhibitors of Bacterial Ceii Waii Synthesis

The synthesis of the bacterial ce11 wall provides several targets for therapeutically useful antibacterial agents (Figure 1-3)."~'~

UDPNAMA L C- UDPNAMA- 4 pentapetide \ Lipa carrier + Transfer to 4'(Fosfomysin] + ,(membrane) peptidoglycan / UDPNAG UOPNAG [--antibiotics

Crosslinking

Figure 14 Sites of action of inhibitors of bacterial ce11 wall synthesis.

The fnst stage takes place in the cytoplasm. The condensation of N-acetylglucosamine 1- phosphate with uridine triphosphate affords uridine-diphospho-N-acetylglucosamine

(UDPNAG), which subsequently reacts with phosphoenolpynivate (PEP)via a specific transferase to give an en01 ether, which is reduced by a NADPH-utilizing reductase enzyme to produce uridine-diphospho-N-acetylmuramic acid (UDPNAMA).

Fos fomycin, a phosphonic acid antibiotic, inhibits the enol-pyruvy1 trans ferase that

13 Gringauz, A. Introduction to Medicinai Chemistry: How Dmgs Act and Why. New York, Wiley-VCH, 1997. catalyzes the transfer of PEP to form UDPNAG-~~O~~~V~~~.'~The UDPNAMA is then linked through its carboxyl group to five amino acids, the 1st two of which, D-alanyl-D- alanine (D-Ala-D-Ala) are added as a unit. D-Alanine is derived fiom L-alanine by alanine racemase and is coupled by D-alanyl-D-alaninesynthetase; both of these reactions are inhibited by D-cycloserine. 15,16,17

in the second stage, the final product of stage 1, uridyl-diphosphate-N- acetylmuramic acid pentapeptide (Figure 14), is linked via a pyrophosphate bond to a phospholipid membrane-bound carrier, bactoprenol (a 55-carbon isoprenyl phosphate). l8

Then UDPNAG is added by glycosidation to form a p-1,4-glycosidic bond between the two glucose-denved units. At this stage, in some organisrns, five glycines are added to the terminal arnino group of lysine to complete the ce11 wall monomenc unit.13

" Gadebusch, H.H.; Stapley, E.O.; Zimmerman, S.B. Critical Reviews in Biotechnology 1992, IZ, 225. l5 Neuhaus, F.C.; Lynch, J.L. Biochernistry 1964,3,471. l6 Badet, B.; Walsh, C.T. Biochemistry 1985,24, 1333. l7 Peisach, D.; Chipman, D.M.; Van Ophem, P.W.; Manning, J.M.;Ringe, D. J. Am. Chem. Soc. 1998, 120, 2268. in Higashi, Y.; Strominger, J.L.; Sweeley, C.C. J. Biol. Chem. 1970,245,3697. Figure 1-4 Uridy 1-diphosphate-N-acetylmuramicacid pentapeptide.

This unit is then transported across the cytoplasmic membrane to the peptidoglycan growth site and the carbohydrate backbone is polymerized via continuous P-1,4- glycosidic bond formation.

In the final stage, the adjacent peptidoglycan chah are crosslinked (see below).

This gives the wall its mechanical strength. The process is inhibited by glycopeptide antibiotics such as vancomycui and , which form hydrogen-bonded complexes to a terminal ~-Ala-~-Ala.'~"'The enzyme that catalyzes the cross-linking is the site of action of penicillins, other p-lacm antibiotics (Figure 1-3) and lactivicin (see later).

- 19 WiIliams, D.H.; Bardsley, B. Angew. Chem. Int. Ed 1999,38, 1 172. 'O Walsh, C.T. Science 1999,284,442. 13Mode of Action of PLactrm Agents

The result of inhibition of the final stage cross-linking reaction is a defective wall that fails to pmtect the ceil fiom its high intemal osmotic pressure; this ultimately le& to ce11 rupture (lysis). The penicillin-binding protein (PBP), via an active site serine, forms an acyl intermediate with the pendtirnate p da ni ne of an N-acyl-D-Ala-D-Aia peptide, and the terminal amino group of another chain then reacts with this acyl-enzyme to form the cross-link (Figure 1-5).~' Penicillin, a stnicîural analog of the natural substrate (Figure 1-6), is welcomed into the active site of the penicillin-binding protein and acylates the active site serine hydroxyl group. As depicted in Scheme 1-1, the formation of the penicilloyl-enzyme inhibits the enzyme if k2>>k3.22In addition to PBPs, there are other penicillin-recognizing enzymes, termed p-lactamases, for which the rate of the hydrolysis of the penicilloyl-enzyme (k3) is approximately equal to its rate of formation (b). This behaviour resuits in the inactivation of the antibiotic and regeneration of the enzyme. The inactivation of a p-lactam antibiotic by a B-lactamase comprises the principal mechanism of bacterial resisbnce to these compounds.

- " Tipper, D.J.; Strominger, J.L. Proc. Nat!. Ac& Sci. USA 1965,54, 1133. " Wolfe, S.; Hoz, T. Can. J. Chem. 1994, 72, 1014. RCONH

OXJ COOH

Transpeptidase

CONH- la-C-Enz / II O D-ala + / NHC0-gbNH2

Penicilloyl Enzyme / ~ncosiv*jn Cross-linked Peptidoglycan

Figure 1-5 Transpeptidation sequence of PBP and its inhibition by penicillin.

Figure 16 N-Acyl-D-alanyl-D-alanineand its structural analog, penicillin. Scheme 1-1 Reaction of a P-Iactarn antibiotic with a penicillin-recogninng protein.

1.4 Mode of Action of Cycloserine

Cycloserine (oxamycin, Seromycin) is a broad-spectnim antibiotic produced by

Streptomyces or~hidaceus.~~As mentioned earlier, this antibiotic inhibits two enzymatic reactions in the first stage of bacterial ce11 wall synthesis. D-Cycloserine, which may be viewed as a cyclic analog of D-alanine (Figure 1-7), enters bacteriai cells by active transport into the cytoplasm where it is a cornpetitive inhibitor of both alanine racemase and D-alanyl-D-alanine synthetase. l3

Figure 1-7 D-Alanine and its structural analog, D-cycloserine.

- --- Stammer. C.H.;Wilson, A.N.; Holly, F.W.;Folken, K. J. Am. C'hem. Soc. 1955, 77.2346- 1.4.1 Alanine Racemase

Alanine racemase uses pyndoxal-5-phosphate (PLP) as a cofactor. An irnine linkage formed with the amino group of alanine facilitates deprotonation at the a- position. D-Cycloserine is an inhibitor of this enzyme. For some time it was thought that

D-cycloserine formed a covalent link to the protein,t4'5 but a recent crystal structure" revealed that the antibiotic is converted to a stable aromatic species attached to the cofactor and held in place by many noncovalent interactions (Figure 1-8). It is suggested that thïs cycloserine-PLP derivative is formed fiom an intermediate on the accepted catalyiic pathway for transamïnation, which tautomerizes to a stable aromatic species.

24 Bugg, T.D.H.; Walsh, C.T. Nat. Prod. Rep. 1992,9, 199. Walsh, C.T. J. Biol. Chem. 1989,264, 2393. H extemal aldimine

stable aromatic product ketimine

Figure 1-8 Proposed mechanism for inactivation of alanine racemase by D- cycloserine. l7

1.4.2 D-Manyl-D-Alanine Synthetase

D-Alany l-D-alanine synthetase possesses two binding sites for D-alanine and requires ATP to produce the dipeptide. It is proposed that the carboxyl group of the alanine bound in the donor site is activated by ATP, which is then attacked by the amino group of the alanine bond in the acceptor site (Pigure 13). **' D-Cycloserine is a

Donor and acceptor definitions given by Neuhaus and Lynch '' '6 Greenlee, W.J.; Springer, I.P.; Patchett, A.A. J. Med Chem. 1989,32, 165. cornpetitive inhibitor of both the donor and the acceptor sites of D-Na-D-Ala synthetase.

The zwitterion of D-cycloserine is the active fom of the inhibit~r.~~

ATP ,- + ADP

balanyl phosphate

------H2N7- +, 3N/xNH2yo O 0- 0- 0- 0- 0, / 9 1 po- r10- acceptor O O site donor II site Il

di peptide

Figure 1-9 Proposed mechanism for D-Ala-D-Ala ~ynthetase.~~

1.5 Insights into the Essential Structural Features of Antibacterial Agents Targeted to Penicillin-Binding Proteins

The discovery of new antibiotics through screening prograrns has shed new light on the importance of the various subunits that comprise the penicillin nucleus, which consists of a p-lactarn ring fised to a thiazolidine ring, together termed a pena~27(Figure

'' Sheehan, J-C.; Henery-Logan, KR.;Johnson, D.A. J. Am. Chem. Soc. 1953, 75,3292. 1-10). The discovery of monobactams, nocardicins and lactivicin (Figure 1-10) eventually led to the rational design of novel antibacterial agents targeted to PBPs. These include monocyclic compounds, heteroatom activated B-lactams and non-8-lactam mimics of B-lactam antibiotics.

f3-Lactarn ring '\ Thiazolidine ring

CH3 O -

Penicillins Monobactarns

Nocardicins Lactivicin

Figure 1-10 Naturally occurring antibacterial agents targeted to PBPs.

1.5.1 Monocyclic Antibiotics

The existence of n0cardici.n~~~and monobactams,29,30,3 1 and their ability to behave

28 Hashirnoto, M.; Konori, T.; Karniya, T.A. J. Am. Chem. Soc. 1976, 98,3023. 29 Sykes, RB.; Cimawsti, CM.; Bonner, D.P.; Bush, K.; Floyd, D.M.; Georgopapadakou, N.H.; Koster, W.H.; Liu, W.C.; Parker, W.L.; Principe, P.A.; Rathnum, ML.; Slusarchyk, W.A.; Trejo, W.W.; Wells, J.S. Nature 1981,291,489. 30 Floyd, D.M.;Fritz, A. W.; Cimanisti, CM.J. Org. Chem. 1982.47, 176. " Cimamsti, C.M.; Applegate, H.E.; Chang, H.W.; Floyd, D.M.; Koster, W.H.; Slusarchyk, W.A.; Young, M.G.J. Org. Chem. 1982,47, 179. as $-lactam antibiotics, demonstrated that a bicyclic nucleus is not essential for antibacterial activity. This hding led to interest in the synthesis of novel monocyclic p- lactarns. Among these unnatural monocyclic p-lactams are the monosulfactams,32.33 which contain a heteroatom activated P-lactaxn and are found to have activity comparable to the corresponding monobactams.

Monosulfactams

1.5.2 Heteroatom Activated p-Lactam Antibacterial Agents: Mono and

Bicyclic Oxamazins

Miller et al. have published extensively on mono and bicyclic oxamazhs, heteroatom-activated ~-1actarns.~~Monocyclic oxamazins (1-1) exhibit antibacterial activity against a spectnim of Gram-negative bacteria, attributed to the electronic activation of the azetidinone ring by the directly attached oxygen atom and an appropriate fit in the enzyme active site. It is noteworthy that the carboxyl group of 1-1 is located one carbon Merremoved f?om the p-lactam nitrogen than in p-lactam antibiotics.

An attractive target of the search for new antibacterial agents has been the bicyclic [4.2.0] oxamazin (1-2), whose 6-membered ring system is a feature of cephems

3' Gordon, E.M.; Ondetti, M.A.;Pluscec, J.; Cimanisti, CM.; Bonner, D.P.; Sykes, R.B. J. Am. Chem. Soc. 1982,104,6053. 33 Miller, M.J.; Biswas, A.; Krook, M.A. Tetrahedron 19%3,39,2571. 3J Woulfe, S.R.; Miller, MJ.Te~ahedmn Leîî. 1984, 25, 3293. (1-3, X = S) oxacephems (1-3, X = 0) and carbacephems (1-3, X = CH2), but has the added advantage of the heteroatom activation effecte3' Both diastereomers at the point of atîachment of the carboxyl group of one such bicyclic oxamazin family (n = O, J& = H, RI

= various acylamino groups) (1-2) are reported to have ''good" antibacterial a~tivity.~~

Oxamazins Bicyclic oxamazins Cephems (X = S) Oxacephems (X 0) 1-1 1-2 = Carbacephems (X = CH2)

1.53 Lactivicin and Derivatives

Lactivicin, isolated fiom culture filtrates of Empedobacter hctagenus YK-25 8 and Lysobacter albus YK-422, like cycloserine, contains an isoxazolidinone ring, and exhibits antibacterial activity, but by a different mechanism. Lactivicin has affinity to a

PBP and susceptibility to B-lactama~es.~'The realization that the p-lactarn ring is not essential for binding by peniciI1in-recognizing enzymes, has led to the examination of other non- p-1acta.m compounds. 38.39,40

35 Zercher, C.K.; Miller, M.J. Tetrohedron Lett. 1989,30, 7009. 36 Kroenthal, D.; Kuester, P.; Koster, W.H. Abstr~~ctsfrornthe Tenth International Congress of Heterocyclic Chemistry, Abstract G3-25,1985. 37 Nozaki, Y.; Katayama, N.; Ono, H.; Tsubotani, S.; Harada, T.; Okazaki, H.; Nakao, Y. Nature 1987,325, 179. 3x Baldwin, J.E.; Lowe, C.; Schofield, C.J. Tetrahedron Lett. 1990,31, 22 1 1. 39 Natsugari, H.; Kawano, Y.; Morimoto, A-; Yoshioka, K.; Ochiai, M. J. Chem. Soc. Chem. Commun. 1987, 62. 40 Tamura, N.; Matsushita, Y.; Yoshioka, K-;Ochiai, M. Tetrahedron 1988,44,323 1. 1.6 Canavanine, Canaüne and their Derivatives

1.6.1 Natural Products: Canavanine and Canaline

L-Candine (L-2-amùio4aminooxybutync acid) (la), the 5-oxa isostere of L-

cmithine, is the only naturally occurring amino acid possessing an aminooxy moiety.

This non-protein amino acid is found in leguminous plants and is metabolized from L-

canavanine (1-5) (the 5 -oxa isostere of L-arginine) by arginase-mediated hydrolysis

arg inase

urea

CO2 +

Scheme 1-2

Over 500 species of leguminous plants are known to produce canavanine.

Canavanine is an important metabolite of higher plants, as it has a dual function, in chemical defense against herbivores and in nitrogen storage, where it can account for

90% or more of the seed nitrogen allocated to fkee amino acidd7The fkee amùiooxy

------Kitagawa, M.; Tomiyama, T. J. Biochem. (Tokyo) 1929,11,265. " Kitagawa, M.; Yarnaàa, H. J. Biochem. (Tokyo) 1932,16,339. 43 Kitagawa, M.; Monobe, K.4. J. Biochem. (Tokyo) 1933, 18,333. 44 Kiiagawa, M.; Takani, A. J. Biochem. (Tokyo) 1936,23, 18 1. " Kitagawa, M. J. Biochem. (Tokyo) 1936,24, 107. 46 Kiiagawa, M. J. Biochem. (Tokyo) 1937,25,23. 47 Rosenthal, G.A. Biochem. Syst. Ecol. 1977,5,2 19. group of L-canaline forms Schiff base adducts with a-keto a~ids~~and with the pyridoxal phosphate (B6)c~factor~~~~ of many Bs-dependent enzymes. These properties endow cadine with mtirnetabolite,5' anticancer5' and antirnalarial activities?

1.6.2 Syntheses of Canaline and Related Derivatives

Syntheses of canaline and some of its derivatives were reported by several research groups in the late 1950s and early 1960s (Scheme 1-3). In Scheme 1-3 i), ethyl

N-hydroxyacetimidate undergoes a Michael addition to acrolein and the product is then subjected to a Strecker synthesis. Ln Scheme 1-3 ii), addition of cyanate to the carbonyl group of the lactone, followed by intrarnolecular cyclization of the resulting acyl isocyanate, yields 5-(2-hydroxyethy1)-imidazolidine-2,4-dione. The remainder of the sequence is straightforward. In Scheme 13 üi), N-benzyl groups are removed by refluxing with strong acid.

48 Cooper, A.J.L. Arch. Biochem. Biophys. 1984,233,603. 49 Rahiala, E.-L.; Kekomaki, M.; Janne, J.; Raina, A.; Raiha, N.C.R. Biochim. Biophys. Acta 1971, 227, 337. Rahiala, E.L. Acta Chern. Scand 1973,27,386 1. 5 1 Rosenthai, G.A. Life Sciences 1997,60, 1635. 52 Berger, B.J. Antimicrobiaf Agents and Chemotherapy 2000,2540. 1.2eq KOHIEtOH 2eq HONHC0&HS Retlux 3 h 2. Dilm HCI 55% 1.2eq KOH/EtOH 2eq HONHC&C2H5 Reflux 3 h 2. Dilute HCI 47% H

NaOEtlEtOH PhCH2NHoH ) PhCH2NH0 iii)55 OEt 55-65oC for 12 h SS-6o0r6 +OEt NHCH2Ph

1. 0.5% NaOmo 1.12% HCI Reflux 4 h 2. Et3N/EtOH 85% O II

Scheme 1-353,54,55

- 53 Kaipeiskii, M-Ya.; Khomutov, R.M.;Severi& S. Zh. Obshch. Khimii 1962,32, 1357. 54 Nyberg, D-D;Christensen, B.E.J. Amer. Chem. Soc. 1957, 79, 1222. 55 KnobIer, K.; Frankel, M. J. Chem. Soc. 1958, 1632. Some years later, a synthetic route to enantiomericaiiy pure L-canaline was descrïïed by

Ozinskas and Rosenthal (Scbeme 1-44)? Noteworthy features of Scherne 14 are: (i) the

i) R=COÎCH2Ph, 82% homose serine ii) R=t-BûC, 86%

ii) R=tBOC, 84% R'o2C-W

i) R=COSH2Ph, R'=CH2Ph, 80-92% ii) R=tBOC, Re=CH2Ph,80% iii) R=C02CHzPh, R'=n-Pr, 92%

i) R=COSH2Ph1R'=CH2Ph1 80°h i) R=C02CH2Ph, R'=CHzPh, 8f0h iii) R=CO2CH2Ph, R'=n-Pr, 82% iii) R=CO2CH2Ph, R'=n-Pr, 85%

Scheme 1-4 Reagents and conditions: a) i) C1COOCH2Ph, NaHC03, H20; ii) (Me3COzC)zO, NaOH, H20/THF; b) C1COOCH2Ph, NaHC03, H20, citric acid/&O/pH 2/A; c) DCC or H'; d) ICH2COW, EtOWH20, cihic aciciD&O/pH 2/A; e) NaOH, EtOH/H20, evaporate, R'B~,DMF; f) p-TsC1, Et3N, THF; g) HONHCOPh, NaH, DMF;h) HCI, EtOH (19%, w/w)/A; i) 4N HCl, H20/A.

56 Ozinskas, A.J.; Rosenthal, G.A. J, Org. Chem. 1986,51,5047. use of iodoacetamïde to activate the sulfiir atom of methionine towards intramolecular

displacement to produce a substituted butyrolactone; (ii) the use of tosyl chloride to

activate the hydroxyl group of homoserine; (iii) the use of N-benzoyl hydroxylamine to

introduce the aminooxy function; (iv) the vigorous conditions employed to remove the

protecting groups.

After the discovery of the antibiotic closeri ri ne,^^ cyclocanahe became a target

as a potential antibacterial agent. However, attempts to synthesize cyclocanaline by

cyclization of the corresponding halo hydroxamic acids resulted in the formation of a

different heterocyclic system, 1-hydroxy-2-pyrrolidinone (Scheme 1-5). 58-59

Scheme 1-5

Khomutov et al. then found that the six-membered heterocycle, dihydro-2H-1,3-

oxazin-3(4H)-one, is formed fiom 4-(aminooxy)butyric esters under basic condition^^^,

57 Kuehl, F.A.; Wolf, F.J.; Trenner, N.R.; Peck, R.L.; Howe, E.; Hunnewell, B.D.; Downing, G.; Newstead, E.; Buhs, R.P.; Putter, 1.; Ormond, R.; Lyons, J.E.; Chaiet, L.; Folkers, K. J. Am. Chem. Soc. 1955, 77, 2344. 58 Smrî, J.; Beranek, J.; Horak, M. Collect. Czechosl. Chem. Commun. 1959,24, 1672. j9 Kochetkov, N.K.; Khomutov, R.M.; Severin, ES.;Karpeislùi, M.Ya; Budovskii, E.I.; Erashko, V.I. Zh. Obshch, Khimii 1959,29,34 17. M} Khornutov, R.M.; Karpeiskii, M-Ya.; Severin, ES. Inverst. Akd Nauk S.S.S.R. Ser. Khim. 1962, 1074. and used this reaction to synthesize cyclocanaline (Scheme 1-6i)).~'A synthesis of cyclocanaline was later achieved under acidic conditions (Scheme 1-6~)).~~

Scheme 166'.62

Yoshioka and Miwa have claimed, without experimental details, that homolactivicins (1-

6, R = COzH), which are derivatives of cyclocanaline, have antimicrobial activit~t.~~

6 1 Khornutov, R.M.; Karpeiskii, M.Ya.; Severin, ES. Inverst. Akad. Nauk S.S.S.R. Ser. Khim. 1962,2 161. 6' 6' Frankel, M.; Knobler, Y.; Bonni, E.; Bimier, S.; Zvilichovsky, O. J. Chem. Soc. (C)1969, 1 746. 63 Yoshioka, K.; Miwa, T. (Takeda Chem. Ind Ltd.) Jpn. Kokai Tokkyo Koho .lP 622 15585 18721 55851 (CI. C07D413/04), 22 Sept. 1987, Appl. 86/57922,14 Mar. 1986. Chem. Absir. 198%, 108, P 1867532. 1.7 Design of [lJ]Oxnzinan-3-one Denvitives as Novel Antibacterial Agents

1.7.1 Computational Approach

Resistance to conventional antibiotics creates an increased demand for novel

antibacterial agents. A strategy for the rational design of such compounds has been

applied to a model of the active site of a penicillin-binding protein fiom Streptomyces

R61 (Figure 1-11).~~This computational approach has been a major focus of the Wolfe

research group over the past decade.

Figure 1-11 Computer generated model of a peptide that contains the residues that surround the active site serine of a PBP fiom Streptomyces R6 1."

The computational model of the active site of the Streptomyces R61 PBP suggests

that the following requirements must be met in order to obtain potentially active

M Wolfe, S.; Jin, H.; Yang, K.; Kim, C.-K.; McEachern, E. Can. J. Chem. 1994, 72, 105 1. compounds (Figure 1-12)? i) The compound must be able to form a complex with the enzyme at the active site,

achieved through hydrogen bonding of Cl (a carboxyl group) and G2 (a

hydrogen-bond donor) of the antibacterial agent to the complementary iysine

arnino group (Lys6, Figure 1-11) and valine end group (Val 1, Figure 1-11) of the

enzyme. ii) The enzyme-substrate comprex should allow the hydroxyl group of the se~e

residue to the attack a functional group P of the antibacterial agent at a rate

comparable to that exhibited by a penicillin or cephalosporin. iii) The acyl-enzyme must not participate in the k3 step of Scheme 1-1.

Figure 1-12 Requirements for the design of a compound targeted to a PBP (see text)?

1.7.2 First and Second Generation Compounds

The first generation of compounds (1-7) designed by the Wolfe goup exhibited antibacterial activity but were found to be ~nstable.~~The second generation, N- substituted, 5-hydroxy derkatives of [1,2]oxazinan-3-ones (1-8) were synthesized

" Wolfe, S.; Zhang, C.;Johnston, B.D.; Kim C.-K. Cm. J. Chem. 1994, 72, 1066. (Scheme 1-7) and exhibited antibacterial activity and greater stabiIity than the first generation.66.67.68

" Wolfe, S.; Akuche CI.; Ro, S. US. Ser. No. 09/102,285.June 22, 1998. Allowed, November 2000. 67 Akuche, C. 1. M.Sc. Thesis, Simon Fraser University, 1997. a Ro, S. Ph.D. Thesis, Simon Fraser University, 1998. CH2Clt DHP I pTsOH, a

J (ii) NaHC03 "a,,",?

Scheme 1-7

1.7.3 Derivatives of Second Generation Compounds

The oxazinone ring has an advantage over its penicillin bioisostere, the P-lactam, since, as seen in 13, it contains several additional sites for stmcturaI manipulation. A potentially closer structural analog of N-acyl-D-alanyl-D-alanineand of penicillin would have an acylamino group at C4 (1-IO), where R is the side chah of a hown peoicillin, e.g. penicillin G (R = PhCi%), penicillin V (R = PhOCH?), (R = PhCHNK) etc., dong with the propionyl side chah attached to the nitrogen of the ring system. The syntheses of 1-10 and some its derivatives are the objectives of the present work

Wacyl-~Ala-~Ala Penicillin 1-8 1-1 O 2 RESULTS AND DISCUSSION

2.1 Retrosynthetic Analysis

The ultimate target of the present work is 2R-(3-oxo4phenylacetylamino-

[1,2]oxazïnan-2-y1)-propionic acid (2-1) which, according to the penicillin-binding protein cornputational modei, possesses the necessary groups required for the fit to and the reaction with the active site serine of a penicillin-binding protein (Figure 1-12). The oxazinone ring provides the functional group P, and the ring nitrogen and C4 substituents, G1 and G2, respectively, allow hydrogen bonding to the receptor to be achieved.

2-11

Retrosynthetic examination of the oxazinone nucleus of 2-1 suggested that a halohydroxamic acid, for example 4-bromobutyrohydroxamic acid (2-2) might serve as a precursor of the ring. However, treatment of this compound with sodium hydride had been found to lead mainly to 1-hydroxypyrrolidin-2-one (2-3).69 Similar results were

69 Motorina, I.A.; Fowler, F.W.; Grierson, D.S. J. Org. Chem. 1997,62,2098. 2-2 2-3 obtained with other halohydroxamic acids.58.59 On the other hand, in this laboratory, S.

Ro and C.I. Akuche had found that only the 6-membered ring was produced with N- substituted hydroxylamuies (Scheme 1-7).67.68 Nevertheless, a possible problem ernerged during the coupling of their key intermediate (2-4) with an N-hydroxyamino acid (2-S), using DCC. In the case of N-hydroxyglycine (R ' = H) only N-acylation was observeci, but

N-hydroxyalanine (R ' = CH,) gave products of both N- and O-acylation, (Scheme 2-1).

An additional problem with this synthetic strategy is that N-hydroxyamino acids are not particularly stable and racemize readily.34,70

Hirose, T.; Chiba, K.; Misho, S.; Nakano, J.; Ono, H. Heterocycles 1982, 19, 1 019.

Scheme 2-1 Reagents and conditions: a) DCC, CH2C12,O OC to rt, 3 h. To overcomeJ. the problem of competing O-acylation during coupling, O-protected or NO-diprotected hydroxamic acids were considered. ~iller~'.~'and ~tewart'~had reported that, under Mitsunobu conditions, the O-protected hydroxamates 2-6,2-7, and 2-

8 can be N-allcylated by alcohols, with O-alkylation of the urethane carbonyl group a

Maurer, P.J.; Miller, M. J. Am. Chem. Soc. 1982, 104, 3096. 72 Lee, B.H.; Miller, M. Org. Ch.1983,48,24. 73 Stewarî, A.O.; Brooks D.W. J. Org. Chem. 1992,57,5020. K O OPh I H2N-0CH2Ph BOCNH-OBOC HN~oph

competing reaction in the case of 2-7 (Scheme 2-2).

HN OPh Mitsunobu R-OH + I A

K =%O R,N I OPh N + 1 AOPh

Scheme 2-2

Very recently a number of additional protecting groups were examined in a study of the synthesis of N-alkylhydroxylamines using the Mitsunobu reaction (2-9+2-IO)?

Prirnary alcohols reacted smoothly, but hindered secondary alcohols gave much poorer yields .

'' Knight, D.W.; Leese, M.P. Terrahedron Lm2001,42,2593. In the present work, an attempt to alkylate the nitrogen of N.0- bis(benzyloxycarbony1) hydroxylamine (2-11) prepared fiom hydroxylamine hydrochloride and excess benyl chloroformate, with ethyl (S)-lactate under Mitsunobu conditions, was unsuccessful. An attempt to couple 2-11 with the acid 2-12 using DCC also failed (Scheme 2-3).

Scheme 2-3

Following these unsuccessful experiments an alternative strategy was examined based on the retrosynthetic sequence 2-14 2-15 2-16 (Scheme 2-4). 2-14 2-1 5 2-1 6

Scheme 2-4

As noted in Chapter 1 (Scheme loti), ring closure to fom the 6-membered cyclic hydroxamate and some of its derivatives had originally been achieved by thermal cyclization of an amuiooxyester under acidic or basic conditions.60.6 1,62 Subsequently,

Weinreb and coworkers found that the aminolysis of esters proceeds smoothly in the presence of trirnethylaluminum.75 Pirrung has employed these conditions to prepare acyclic hydroxamates76 and Yamamoto has achieved syntheses of 5, 6-, 7- and 8- rnernbered lactarns7'. In the present work, trimethylaluminum was found to be a useful reagent for the formation of the C-N bonds of unsubstituted and substituted oxazinones and other cyclic hydroxamates.

2.2 Syntheses of 5-, 6-,7- and 8-Membered Cyclic Hydroxamates

A four-step synthesis of 5-, 6-, 7- and 8-membered cyclic hydroxamates fiom 4-,

5, 6- and 7-mernbered lactones was devised (Scheme 26), consisting of conversion to wbromo methyl esters, displacement of brornine with N-hydroxyphthalimide, removal of the phthalimido protecting group, and cyclization using trimethylaluminum.

75 Levin, J.I.; Turos, E.; Weinreb, S.M. Synrh. Commun. 1982, 12,989. Pimg, MC; Chau, J, H.-L. J. Org. Chem. 1995,60,8084. 77 Yamamoto, Y.; Fumta, T. Chem. Letters 1989,797. 0

Scheme 2-5

This sequence was developeci initially using y-butyrolactone (2-17) as the starting

material. Heating of 2-17 at 75 OC with a 45% solution of hydrogen brornide in acetic

a~id,'~followed by addition of methanol, afforded methyl y-branobutyrate (2-18) in 86%

yield79. A protected amuiooxy group was then introduced successfùlly by displacement

of bromine with N-hydroxysuccinimide80-8 1,82,83 or whydroxyphthalùnides2. Both products wzre crystalline, with m.p. 69 - 71.5 OC and 79 - 81 OC, respectively, but, after

several attempts, the yield of methyl y-succinimidooxybutyrate (2-19a) could not be

increased above 28%. In contrast, the yield of methyl y-phthalimidooxybutyrate (2-19b)

was much higher (78%), (Scheme 2-6).

78 Jost, K.; Rundinger, J. Coll. Czech. Chem. Commun. 1967,32,2485. 79 Manchand, P.S.; Luk, K.-C.; Belica, P.S.; Choudhry, S.C., Wei, C.C. J. Org. Chem. 1988,53, 5507. " Edafiogho, 1.0.;Scott, K.R.; Moore, J.A.; Farrar, V.A.; Nicholson, J.M. J. Med. Chem. 1991, 34,387. 81 Ludwig, B.J.; Reisner, D.B.;Meyer, M.; Powell, L.S.; Sirnet, L.; Stiefel, F.J. J. Med. Chem. 2970, I3,60. 82 Harnor, G.H.; Rubessa, F. J. Med Chem. 1972, I5,470. 83 Bauer, L.; Ghosh, B.K. J. Org. Chem. 1965,30,4298. Scheme 2-6 Reagents and conditions: a) SuOH, Et3N, THF, rt, 4 h; SuOH, NaOMe, DMF, 100 OC, 6 h; SuOH, Et3N (2 eq), CH3CN, reflux, 3 h; SuOH, Et3N, DMF, rt, 24 h; b) FtOH, Et3N (2 eq), CH3CN, reflux, 3 h.

Removal of the succinimido protecting group of 2-19i required a 12 h treatment with four equivalents of hydrazine hydrate in methanol at room temperature to give 2-20 in 45% yield.8t In contrast, methyl y-phthalimidooxybutyrate (2-19b) gave a quantitative yield of 2-20 using 1.5 equivalents of methyihydrazïne in dichloromethane for 1.5 h at

-10 OC to O OC (Scheme 2-17)?

Scheme 2-7 Reagents and conditions: a) H2NNH2-HzO (4 eq), MeOH, rt, 12 h; b) MeHNNHz (1 -5 eq), CH2C12, -10 OC to O OC, 1.5 h.

The cyclization of 2-20 was achieved initially using the basic conditions reported by Khomutov (Scheme 2-8).60 RefluMg with potassium hydroxide in methanol for 2 h, followed by stirring at room temperature ovemight, gave the oxazinone (2-21) in 24% yield. The '~nrnrspectrum showed peaks at 8.89 (IH, br s, Mi), 4.03 (2H, t, 6.5 Hz,

w Bhat, B.; Swayze, E.E.; Wheeler, P.; Dimock, S.; Perbost, M.; Sanghvi, YS. J. Org. Chem. 1996, 61, 8 186. CfiO), 2.52 (2H, t, 7.2 Hz, COCI&) and 2.10 (2H, quintet, 6.9 Hz, COCH2C&) ppm.

The 13cnmr spectrum showed peaks at 172.9, 69.0, 27.4 and 21.6 ppm. The idiared absorptions at 3403 and 1667 cm-' were assigned to the N-H and carbonyl group. The CI

mass spectrum showed an M+l peak at m/z 102.

Trimethylaluminum gave better results.76*77 With two equivalents in

tetrahydrofuran or toIuene, and at room temperature or reflux temperature, yields of 65 -

79% of 2-21 were obtained.

a-c OMe 2479% CL

Scheme 2-8 Reagents and conditions: a) me3(2 eq), toluene, reflux, 2 h (79%); b) KOH, MeOH, reflux, 2 h, rt, (24%); c) Me3(2 eq), THF, O OC to rt, 2 h (65%).

Following these exploratory experiments the 5-, 7- and 8-membered cyclic

hydroxamates were prepared, as summarized in Table 2-1. Table 2-1 Conversion of lactones into cyclic hydroxamates.

Y ield Y ield Yield Y ield (%) (%) (%) (%)

quant.

'This reaction was found to proceed via methyl acrylate. The compound was therefore prepared more conveniently by addition of N-hydroxyphthalimide to methyl acrylate in re fluxing me thanol containhg triethylamine. b~he7-membered ring ([1,2]oxazepan-3-one) crystallized as colourless plates, m-p. 84.5 - 85.8 OC, and its X-ray structure was determined (see later). 'Sec text.

In the case of the 8-membered ring, [1,2]oxazocan-3-one, cyclization of the aminooxyester was attempted initially using 1.5% w/v solutions containing: i) hvo equivalents of trimethylaluminum in tetrahydrofuran at O - 20 OC for 2 hours; ii) two equivalents of trimethylaluminurn in tetrahydrofiuan at O - 20 OC for 21 hours; iii) two equivalents of trimethylaluminum in tetrahydrofùran at reflux temperature for 2 hours; iv) two equivalents of triniethylaluminum in tetrahydrofuran at 4 OC for 72 hours; V) two equivaients of trimethylaluminum in tetrahyàrofuran at 20 OC for six days. None of these reactions, after quenching with water, gave a promising result. In a skth experiment, the reaction was performed by dropwise treatment of four equivalents of a 0.18 M solution of trimethylalufninum, in heptane (4.96 mL) and toluene (50 mL), with 50 mL of a 0.4% w/vsolution of the ester in toluene, followed by stimng at room temperature for six days.

After addition of water and concentration to near dryness, the gelathous residue was dissolved in 200 mL of 1: 4 dichloromethane:tetrahydro furan, filtered through Celite and evaporated to dryness. The residue was subjected to preparative layer chrornatography

(PLC) on silica gel. Elution with ethyl acetate afforded three bands. One of these bands, following extraction with ethyl acetate, gave 84 mg of material. The GC of this material is shown in Figure 2-1. Only the peak at 9.8 min, which compnsed 2% of the total, gave an EI-MS (Figure 2-2A) consistent with the desired structure. Figure 2-2 shows, in addition to the MS just mentioned, the EI-MS of the 6- (Figure 2-2B) and 7-membered

(Figure 2-2C) cyclic hydroxamates.

Abundance

40G0000 I

Figure 2-1 GC of [ 1,2]oxazocan-3-one. Abundance Average 02-9.839 to 9.8Ba an. 12000 f

Figure 2-2A EI-MS of [ 1,2]oxazocan-3-one.

Abundance 60000 / l t soooo :

I

Figure 2-2B EI-MS of [ 1,2]oxazinan-3-one. Abundance

Figure 2-2C EI-MS of [ 1,210xazepan-3-one.

In each case, there is a peak at M-32 corresponding to loss of HONH fkom the molecular ion. This process is show schematically in Figure 2-3. It is noteworthy that in

Yamamoto's lacm ~yntheses~~,a 7% yield of the 8-membered ring was obtained using trimethy laluminum. This was increased to 43 % with triethy lgalliurn.

Figure 2-3 Fragmentation of the molecular ions of cyclic hydroxamates.

2.3 Cyclocanaline and N-Acylcyclocanalines

With trirnethylaluminum-promoted cyclization of an oarninooxyester established as a viable reaction, a synthesis of N-acylated cyclocanalines from homoserine could be planned, as shown retrosynthetically in 2-22, 2-23, 2-24 and 2-25, (Scheme 2-9). The

Scheme 2-9 best choices for Riand Rz were t-butoxy and methyl, respectively, but Ri = benzyl and

Rz = benzhydryl were also examineci. Three variants of X were studied, namely, X =

OH, X = OMS and X = Br.

Scheme 2-10 summarizes one sequence leading to 2-24 (Ri = t-butoxy, R2 = benzhydryl, X = OSu). Treatment of homoserine successively with BOC anhydride

2-25

Ri= OtBu RI= Ot6u RpCHPh2 R2= CHPh2 X= OH x= osu

Scheme 2-10 Reagents and conditions: a) i) (E30C)20, Et3N, 50% aq. acetone, rt, 14 h; ii) 5% citric acid (74%); iii) DDM, 1: 1 CECI2/ CH3CN, rt, 2h (70%); b) SuOH, PPh3, DEAD, THF, 15°C to rt, (47%). and diphenyldiazomethane gave 2-24 (Ri = t-butoxy, R2 = benzhydryl, X = OH), which was subjected to a Mitsunobu reaction with N-hydroxysuccinimide, diethyl azodicarboxylate and triphenylphosphine to introduce X = OSu in 47% yield. This sequence was not pursued further because better yields were achieved by following a different route.

Scheme 2-11 summarizes a second sequence leading to 2-24 (RI= t-butoxy, R2=

Me, X = Br). In this sequence, homoserine was cyclized to homoserine lactone (2-26) (a substituted butyrolactone), this compound was converted to the brorno methyl ester 2-27, as described in Section 2.2, and 2-27 was acylated with BOC anhydride. This sequence was also not pursued fixther, because milder conditions than HBrIHOAc at 75 OC were found in a third route.

Scheme 2-11 Reagents and conditions: a) 2.4 M HBr, reflux, 3 h/ rt, (81%); b) i) 45% HBr/ AcOH, 75 OC, 4 h (quant.); ii) MeOH, 2 h (65%); c) (BOC)20, Et3N, 50% aq. acetone, 3 h (75%). The third sequence, leading to 2-24 (Ri = t-butoxy or benzyl, R2 = Me, X = OMS), is summarized in Scheme 2-12- Homoserine or homoserine lactone was converted to an

N-acylated methyl ester (2-24, X = OH) and thence to the mesylate by reaction with methanesulfonyl chloride.

p 2-24 2-24

RI= OtBu, CHzPh Ri= OtBu, CHzPh RF Me RF Me O X= OH X= OMS

Scheme 2-12 Reagents and conditions: Ri= OtBu, a) i) (BOC)îO, Et3N, 50% aq. acetone, rt, 14 h; ii) 5% citric acid (74%); iii) MeI, Et3N, DMF, O OC to rt, (94%); b) i) (BOC)20, Et3N, CH2C12, O OC to rt, 96 h (90%); ii) KOH, MeOH, rt, 24 h; iii) Amberlyst 15 (H? (97%); iv) MeI, Et3N, DMF, O OC to rt, (94%); c) MsCl, Et3N, CH2C12, -15 OC to O OC, 1 h (97%). Ri= CH2Ph, b) i) PhCH2COCl, Et3N, CH2CI2, -5 OC to rt, 3 h (95%); ii) KOH, MeOH, rt, 24 h; iii) Amberlyst 15 (~3(96%); iv) Mer, Et3N, DMF, rt, (60%); c) MsCl, Et3N, CH2C12, -15 OC to O OC, 1 h (90%).

The conversion of 2-24 (X = OMS) to 2-24 (X = OFt) proceeded in 64% yield with Ri = t-butoxy, using N-hydroxyphthalimide and DBU in dimethylformamide, and in

84% yield with RI = benzyl using N-hydroxyphthalimide and triethylamine in acetonitrile. In both cases the removal of the phthalhido group and the subsequent cyclization of 2-23 to 2-22 proceeded without incident (Scheme 2-13). The phenylacetyl derivative crystallized as colourless plates, m.p. 164 - 165 OC, of acceptable quality for an

X-ray structure determination (see below). R2= Me X= OFt

Scheme 2-13 Reagents and conditions: RI=OtBu, a) FtOH, DBU, DMF, 10 OC to rt, 48 h (64%); b) MeNHNHz (1.5 eq), CH2C12, -10 OC to rt, 2 h (99%); c) ALMe3 (2 eq), THF, O OC to rt, 4 h (98%). RI= CH2Ph, a) FtOH, Et3N, CH3CN, 10 OC to rt, 24 h (84%); b) MeNHNHz (1.5 eq), CHîC12, -10 OC to rt, 3 h (99%); c) AMe3 (2 eq), toluene, O "C to rt, 4 h (35%).

The deprotection of 2-22, RI = t-butoxy, to cyclocanaline and the acylation of cyclocanaline with phenoxyacetyt chloride is summarized in Scheme 2-14. 2-22 TFA salt of cyclocanaljne Ri= OtBu

Scheme 2-14 Keagents and conditions: a) TFA, -5 OC to rt, 1 h (98%); b) PhOCH2COCI, Et3N, CH2C12,-10 OC to rt, (39%).

Starting from the lactone, 2-26, the overall yield of 2-22, (RI= CH2Ph) is 14% and the overall yield of 2-22, (Ri= PhOCH2) via 2-22, (Ri= OtBu), is 19%.

2.4 Crystal Structures

4-Pheny lacety lamuio-[ l ,210xazinan-3-one crystallizes in the P&/a space group, and is centrosymmetnc, with four molecules (two enantiomeric pairs) in the unit cell.

Figure 2-4 shows one to the enantiomeric pairs. The 6-membered ring exists in a boat conformation. O

Figure 2-4 The crystal structure of 4-phenylacety lamino-[ 1,2]oxazinan-3-one.'

The 7-membered cyclic hydroxamate crystallizes in the PZ,/a space group, and is centrosymmetric, with four molecules, consisting of two hydrogen-bonded dirners, in the unit cell. Figure 2-5 shows several views of the crystal structure. As seen in the center of this Figure, the dirner is held together by linear NH--'-'O=Chydrogen bonds. This causes the two monomeric units to be offset with respect to each other, unlike the structure drawn at the bottom of the Figure, which would have non-linear hydrogen bonds. As cm be seen at the top of Figure 2-5, the monomeric structure exists in a chair-like conformation.

Personal communication from Dr. R. Batchelor. Figure 2-5 Several views of the crystal structure of [1,2]oxazepan-3-one.'

Personal communication from Dr. R. Batchelor. 2.5 N-Alkylation of Cyclic Hydroxamates

With routes to Cacylamùio oxazinones established, attention was now tumed to the introduction of substituents on the ring nitrogen of a cyclic hydroxamate. The initial exploratory experiments were performed using BOC-protected (R)-cycloserine (2-28) and ethyl (S)-lactate under Mitsunobu conditions.

2-28

A Mitsunobu reaction is thought to prweed in three stages (Scheme 2-l~).~~In the fmt stage, triphenylphosphine, diethyl azodicarboxylate (DEAD) and a proton donor

(HX)react to form the phosphonium salt 2-29. In the second stage, this adduct reacts with an alcohol (ROH) to form an alkoxytriphenyIphosphonium salt (230). In the third stage, X- displaces R fiom the phosphonium salt, usually with inversion of configuration, to form RX (2-31) and triphenylphosphine oxide. This mechanism suggests that alcohol addition should be delayed until after the in situ formation of 2-29.

XS Mitsunobu, O. Synthesis 1981, 1. ROH P~~P-N-NHCO~E~ - P~~P'-OR + EtOzCHNNHCOzEt

Scheme 2-15

Ln an alternative mechanism (Seheme 2-16)*~,the reaction of the alcohol with the zwitterion 2-32 would lead to the allcoxytriphenylphosphoniurn cation 2-30 and a hydrazinyl anion 2-33. Reaction of 2-30 with HX would then afford RX (2-31) and tripheny lphosphine oxide. This suggests that HX addition should be delayed unt il adter the in situ formation of 2-30.

86 von Itzstein, M; Jenkines, I.D. J. Chem. Soc. Perkins Tram. 1. 1986,437. Scheme 2-16

Both modes of mkhg of the reactants were examined, using HX = BOC- cycloserine (2-28) and ROH = ethyl (S)-lactate. With tetrahydrofiuan as the solvent, both procedures led to the same mixture of products that could be partially separated by preparative layer or column chromatography. The 'Hnrnr spectnim of the mixture features methyl doublets at 1.53 and 1.58 ppm and methine quartets at 4.80 and 4.96 ppm. The CI mass spectrum shows an M+l peak at dz303 and a prominent peak at

247 corresponding to loss of C4Hs. The two isomers are tentatively considered to be diastereomers with structures corresponding to nonspecific attachment of a 2- carboxypropyl side chah (ethyl 2-(4R-tert-butoxycarbonylamino-[1,2]oxazolidin-3-one)- propionate), (2-34) (Scheme 2-17). Such a loss of stereochernical integrity has been seen previously in Mitsunobu reactions of a-hydroxyesters.87

n7 Pellegata, R.; Dosi, 1.; Vilia, M.; Lesma, G.; Palmisano, G. TetraL1edron 1985, 41, 5607. EmC CH3 Mitsunobu

Hx, OH

Scheme 2-17

Following these pretiminary experiments with the five-membered cyclic hydroxamate, the Mitsunobu coupling of [1,2]oxazinan-3-one (2-21) with ethyl (S)- lactate was exatnined (Scheme 2-18). In this case, because of the insolubility of the oxazinone in tetrahydrofuran, dicldoromethane was employed as the solvent and the azodicarboxylate was added lastaa8These conditions led to a product which could be separated by preparative layer chromatography into two isomeric adducts. The major adduct, isolated in 5 1% yield is assigned the N-alkylated structure 2-35 on the basis of an

M+l peak at 202 in the CI-MS, and the presence of a methine quartet at 5.03 ppm in the

'Hnrnr spectnim. The minor adduct, isolated in 7% yield, is assigned the O-allqlated structure 2-36 on the bais of a downfield shifi of the methine quartet to 5.20 ppm. As already noted in Scheme 2-2, O-alkylation is a known reaction of N-acylated hydroxy lamines.

Xi? Smith III, A.B.; Hale, K.f .; Rivero, R.A. Tetrahedron Lett. 1986,27, 58 13. 2-35

Scheme 2-18

The loss of stereochemicd integrity in the attachment of the carboxypropyl moiety to the ring nitrogen in the case of BOC (R)-cycloserine and the competing O- ablation in the reaction of the oxazinone suggested that attachment of a carboxypropyl chah to nitrogen be performed prior to cyclization, e-g., 2-20 + 2-37, (Scheme 2-19).

However, 2-20 was found to be unreactive under Mitsunobu conditions.

Scheme 2-19

It was then discovered that 2-37 could be synthesized very eficiently by alkylation of the amino group of 2-20 with the triflate of benzyl (S)-lactate (2-38), prepared fiom (S)-lactic acid, as show in Scheme 2-20. This alkylation reaction is reported to proceed with inversion of co~~fïguratiun,~~a finding that has been conhed

in this laboratory ."

Scheme 2-20 Reagents and conditions: a) i) DBU, MeOH; ii) PhCH2Br, DMF, rt, 30 h (76%); b) i) (CF3SO&0, 2,6-lutidine, CH2C12, -78 OC; ii) 2,6-lutidine, CHîCl2, -78 OC to rt, 18 h (74%). The cyclization of 2-37 using trimethylaluminum proceeded in 84% yield to give

the benzyl ester 2-39 which was successfully deprotected, by hydrogenolysis, to the acid

2-40 (Scheme 2-21).

X9 Kogan, T.P.; Rawson, T.E. Teh-ahedron Len. 1992,33,7089. Cheng, M.H. personal communication. Scheme 2-21 Reagents and conditions: a) AlMe3 (2 eq), toluene, O OC to rt, (84%); b) 1oY0 Pd (C)/ Hz,EtOAc, a, 24 h (99%).

2.6 Synthesis of 2-1 as the Benzyl Ester

The triflate route was then employed to akylate racemic 2-23 (Ri = CH2Ph, R2 =

Me) with benzyl (S)-lactate to give 2-41 as a 1: 1 mixture of diastereomers. This mixture was subjected to trimethylaluminum-promotedcyclization and one of the isomers of 2-42 was isolated.

Scheme 2-22 Reagents and conditions: a) i) (CF, S0&0,2,6-lutidine, CH2C12,-78 OC; ii) 2,6-lutidine, CH2C12,-78 OC to rt, 18 h (29%); b) AlMe3 (2 eq), toluene, O OC to rt, (36%). 2.7 Biological Assays

The products of the synthetic methodology just described were tested on agar plates for antibacterial activity against Micrococcus luteus. Only cyclocanaline and 2-40 exhibited activity against this organism (Table 2-2).

Table 2-2 Bioassay results.

Compounds Weight (pg) Moles (PM) Zone size (mm) mrn/pM

solvent controi 10 0.555 O O

desacetoxy cephalosporin G 10 0.O30 1 39 1300

c yclocanaline 600 5.17 58 11

cyc locanaline 400 3 -44 48 14

2-40 400 2.3 1 23 10 3 EXPERLMENTAL

3.1 General Methods

Al1 reactions were perfomed under dry nitrogen using oven-dried (140 OC, 24 h)

glassware, which was allowed to cool in a desiccator under vacuum. Solvents were

distilled pnor to use and dried according to standard literature procedures? Both 'H and

"C nuclear magnetic resonance (NMR) spectra were acquired on a Bruker Mode1 AMX

400 Spectrometer operating at 4OO. 1 MHz and 100.6 MHz, respectively. Chemical shifts

(6)are reported in parts per miilion (ppm) downfield from tetramethylsilane (TMS) in an

appropriate deuterated solvent. Infkired (IR) spectra were obtained on a Perkin-Elmer

5998 spectrometer (neat film, 1 - 2% KBr pellet or 1% solution). Low-resolution mass spectra, refer to direct inlet electron impact (EI) rneasurements or chemical ionization

(CI) measurements, were recorded on a Hewlett-Packard 5985 GC/MS/IS system.

Elemental analyses were perfomed on a Carlo Erba mode1 1 106 elemental analyzer.

Melting points (m-p.) were obtained on a Fisher-Johns apparatus, and are uncorrected.

Preparative layer chromatography (PLC) was carried out on precoated Merck Silica Gel

60 F-254 plates with aluminum backing. Spots were observed under short-wavelength ultraviolet light, and were visualized with a solution of 1% ceric sulfate, or 2% molybdic acid in 10% sulfuric acid. Flash column chromatography was carried out on Merck Silica

Gel 60 (230-400 Mesh). Micrococcus Zutezrs was supplied by Professor Susan Jensen,

University of Alberta, and the bioassay plates were prepared using aseptic techniques.

91 Pemn, D.D.; Amarego, W.L.F. Purifcation ofLuboratory Chemicals. Pergamon Press, 3", Edition, 1988. N,O-Bis(Benzyloxycarbony1) Hydroxylamine (2-11)

Hydroxylamine hydrochloride (0.102 g, 1.47 rnmol) was added cautiously to a cooled (O

OC) solution of sodium bicarbonate (0.246 g, 2.93 mol) in water (1.7 mL). The mixture was stirred for 30 min and benzyl chloroformate (0.618 mL, 0.74 g, 4.33 mol) and a solution of sodium bicarbonate (0.366 g, 4.36 mol) in water (3.38 mL) were then added successively. Stirring was continued for 30 min at O OC and then for 2 h after the ice-bath was removed. The product was collected by filtration, washed with water, and then suspended in hexanes and again collected. This process was repeated twice and the resulting soiid was dissolved in ether (8 mL) and washed with saturated sodium chlonde

(8 mL), dried over anhydrous magnesium sulfate and evaporated to give O. 180 g (4 1%) of the product as a solid, m-p. 67.5 - 68.5 OC. '~nrnr(CDC13, 6): 7.96 (lH, br s, NH), 7.36

(10H, m, Ph), 5.26 (2H, s, PhCfi0C02), 5.22 (2H, s, PhC&OCON). IR (KBr): 3222,

1806,1717 cm-'. a-Amino-l)cButyroIactone Hydrobromide (2-26)

A solution of homoserine (0.31 g, 2.6 mrnol) in 2.4 M hydrobromic acid (5.0 mL, 12 rnrnol, 4.6 eq) was refluxed for 3 h and then stirred overnight. Removal of the solvent afforded a white solid, which was dissolved in ethanol and cooled to give the product as a solid which was collected by filtration and washed with cold ethanol to give 0.384 g

(8 1%) of the product (2-26), rn-p. 117 - 1 19 OC. Mass spectrum (CI, m/z): 102 (M+l).

Anal. Calcd. for CJ&N02Br: C 26.40; H 4.44; N 7.70. Found: C 26.35; H 4.25; N

7.5 1.

a-Amino-)~BromobutyricAcid Hydrobromide

A suspension of a-arnino-y-butyrolactone hydrobromide (2-26) (97 mg, 0.53 mrnol) in

45% hydrogen bromide in acetic acid (3 mL) was stirred for 4 h at 75 OC, cooled to room temperature and concentrated under reduced pressure to give a white solid, (140 mg,

100%), which was used in the next step without Merpurification. '~nmr(40, 6):

4-10 (lH, t, 6.8 Hz, CHa),3.59 (ZH,t, 7.0 Hz, CH2y),2.50 (IH, m, Ce),2.38 (IH, m,

Cm). IR (KBr): 345 1,1720 cm-'. ~-'~utoxycarbooylu-Amin~Bromobut@Acid (2-12)

01-Amino-y-bromobutyric acid hydrobromide (1 30 mg, 0.50 mmol) was dissolved in 50%

aqueous acetone (1 -8 mL), and triethylamine (0.14 mL, 0.102 g, I .O mmol) and BOC-ON

(141 mg, 0.55 mmol) were added, and the solution was stirred for 3 h at room

temperature. The solvent was then removed under reduced pressure and the residue was

dissolved in water (3 mL), washed with ethyl acetate (3 mL), acidified to pH 3 with 5%

citric acid, and again extracted with ethyl acetate (3 x 3 mL). The latter extracts were

combined, washed successively with water (2 x 3 mL) and sanirated sodium chloride (3

mL), dried over anhydrous magnesiun sulfate, and evaporated to give 29.5 mg (21%) of

the product. '~nmr(CDC13, 6):5.56 (lH, br s, NH), 4.50 (1 H, m, CHa),3.82 (2H, rn,

CHzy), 2.18 (lH, m, Cm), 1.78 (lH, m, Cm), 1.48 (9H, s, C(CH3),).

Methyl y-Bromobutyrate (2-18)

A solution of y-butyrolactone (2-17) (25 mL, 28.0 g, 0.325 mol) in a 45% solution of

hydrogen bromide in acetic acid (75 mL) was heated for 4 h at 75 OC, cooled to room

temperature, treated with methanol (150 mL), and stirred ovemight. Evaporation of the solvent gave a dark oil, which was dissolved in ethyl acetate (150 mL) and washed successively with saturated sodium bicarbonate (2 x 150 mL), saturated sodium chloride

(150 mL), dried over anhydrous magnesium sulfate, and evaporated to yield a clear oil

(48.8 g, 86%). 1Hnmr (CDCb, 6): 3.68 (3H, s, OC&), 3.44 (2H, t, 6.4 HZ, CHly), 2.51

(2H, t, 7.2 HZ, CHza), 2.17 (2H, quintet, 6.8 Hz, CH$). 13cnrnr (CDC13, 6): 172.96,

5 l.70,32.66, 32.19,27.70. IR (neat): 1738 cm-'. Mass spectrum (CI, dz): 181 (M+l),

183 (M+3). Anal. Calcd. for CsH90aBr:C 33.17; H 5.02. Found: C 32.62; H 4.85.

Metbyl ~Suecinimidooxybutyrate(2-19a)

Method A

Methyl pbromobutyrate (2-18) (0.5 g, 2.9 mmol) was added dropwise to a solution of N- hydroxysuccinimide (0.308 g, 2.6 mmol) and triethylamine (0.38 mL, 0.276 g, 2.6 mmol) in tetrahydrofuran (20 mL). The reaction mixture was stirred for 4 h at room temperature and then filtered. The filtrate was evaporated under reduced pressure to give white crystals (47 mg, 1O%), m.p. 69 - 7 1.5 OC. '~nmr(CDCl3,6): 4.15 (2H, t, 6.1 Hz, CH2y),

3.69 (3H, s, Ocfi), 2.70 (4H, s, succinimido ring), 2.60 (2H, t, 7.3 Hz, Cfia), 2.03 (2H, t, t, 7.3 Hz, 6.8 Hz, CH$). 13cnrnr (CDCl,, 6): 173.35, 171.10, 76.18, 5 1-67, 29.87,

25.44, 23-41. IR (KBr): 1727, 17 10 cm". Mass spectrum (CI, rn/z): 2 16 (M+l). Anal.

Calcd. for C9H13N05:C 50.22; H 6.10; N 6.5 1. Found: C 50.24; H 6.14; N 6.52. Method B

A solution of rnethyl y-bromobutyrate (2-18) (1 -01 g, 5.5 mmol), N-hydroxysuccinimide

(0.576 g, 5.5 mmol) and sodium methoxide (0.282 g, 5.5 md)In dimethylformamide

(1 1 mL) was heated at 100 OC for 6 h. The solvent was then removed under reduced pressure and the residue was dissolved in ethyl acetate (100 mL) and washed successively with water (2 x 100 mL) and saturated sodium chloride (100 mL), dried over anhydrous magnesiun sulfate, and evaporated to give a solid, which was crystallized fiom ethanol to yield 0.33 1 g (28%) of the product, which was identical with the product of Method A.

Method C

A solution of methyl y-brornobutyrate (2-18) (1.O 1 g, 5.5 mmol), N-hydroxysuccinunide

(0.587 g, 5.5 mmol) and triethylarnine (1.54 mL, 1.12 g, 11.1 mmol), in acetonitrile (10 mL) was refluxed for 3 h, then cooled and filtered. The precipitate was washed with water and recrystallized fkom ethanol to give 0.180 g (15%) of the product, which was identical with the product of Method A.

Method D

A solution of methyl y-bromobutyrate (2-18) (1 .O 1 g, 5.50 mmol), N-hydroxysuccinirnide

(0.584 g, 5.50 mmol) and triethylarnine (0.77 mL, 0.559 g, 5.50 mmol), in dimethylforrnamide (1 1 mL) was stirred at room temperature for 24 h, then poured into water (80 mL), and extracted with ethyl acetate (2 x 60 mL). The combined extracts were washed successively with water (2 x 100 mL) and saturatted sodium chlonde (100 mL), dried over anhydrous magnesium sulfate, and evaporated to give a solid which was crystallized fiom ethanol to yield 0.175 g (15%) of the product, which was identical with the product made using Method A.

Methyl y Phthalimidooxybutyrate (2-19b)

A mixture of methy 1y-bromobutyrate (2-18)(1 4.7 g, 8 1.5 rnmol), N-hydroxyphthalimide

(1 3.3 g, 8 1.5 rnmol) and triethylamine (22.7 mL, 16.5 g, 0.163 mol), in acetonitrile (1 10 mL) was refluxed for 3 h. The insoluble solid was removed by filtration and the filtrate was evaporated. The residue was diluted with ethyl acetate (100 mL) and this solution was washed successively with water (3 x 100 rd,)and saturated sodium chloride (100 mL), dned over anhydrous magnesium sulfate and evaporated. The solid was recrystallized fkom hot ethanol to give 16.8 g (78%) of white crystals, m.p. 79 - 81 OC.

1Hnmr (CDCS, 6):7.79 (4H, m, phthalimido ring), 4.26 (2H, t, 6.1 Hz, CHg), 3.70 (3H, s, 0CH3),2.66 (2H, t, 7.3 Hz, CH2a),2.09 (2H, quintet, 6.7 Hz, CH$). "~nrnr(CDC13,

6): 174.50, 163.63, 134.43, 128.94, 123.48, 78.20, 51.50, 33.85, 25.14. IR (KBr): 1735,

1727 cm-'. Mass spectrum (CI, dz): 264 (M+l). Anal. Calcd. for Ci3Hi3N05: C

59.30; H 4.99; N 5.32. Found: C 59.17; H 4.97; N 5.49. Methyl yAmhooxybutyrate (2-20)

Method A

A solution of methyl y-phthalimidooxybutyrate (2-19b) (2.75 g, 10.45 mmol) in dichloromethane (50 mL) was cooled to -10 OC and treated dropwise, with stimng, with methylhydrazine (0.83 mL, 0.72 g, 15.7 mmol). Stirring was continued for 1.5 h at -10 to O OC and the mixture was then filtered. The filtrate was concentrated and the residue, in ethyl acetate (50 mL), was washed with I:l sahirated sodium chloride: saturated sodium bicarbonate (22 mL). The aqueous layer was washed with ethyl acetate (50 mL) and the combined organic extracts were drïed over anhydrous magnesium sulfate and evaporated to give a yellow oil(1.39 g, 100%). 1Hnmr (CDCL, 6): 5.25 (2H, br s, NI&),

3.68 (2H, t, 6.2 HZ, Gay), 3.68 (3H, s, OC&), 2.38 (2H, t, 7.3 Hz, C&a), 1.92 (SH, quintet, 6.7 Hz, CH$). I3cnrnr (CDCla, 6): 173.95, 74.78, 51.58, 30.71, 23.65. IR

(neat): 3322, 1734 cm-'. Mass spectrum (CI, m/z): 134 @I+l). Anal. Calcd. for

C5H11NO3:C 45.09; H 8.34; N 10.52. Found: C 45.02; H 8.36; N 10.49.

Method B

Hydrazine hydrate (0.07 mLy74.6 mg, 1.49 rnrnol) was added, with stirring, to a solution of methyl y-succinimidooxybutyrate (2-19a) (80 mg, 0.37 mmol) in methanol (3 mL).

StUring was continued at room temperature for 12 h and the solvent was then removed.

The solid residue was triturated with ethyl acetate (2 x 8 mL, 1 x 4 mL) and the ethyl acetate was evaporated to give the product as an oil (22.3 mg, 45%), identical with the

product of Method A.

Method A

A solution of methyl y-aminooxybutyrate (2-20) (0.5 1 g, 3.75 mmol) in toluene (37.5 mL) was treated with a 2.0 M solution of trimethylaluminum in hexanes (3.75 rnL, 7.5

mmol). The resulting mixture was refluxed for 2 h, cooled to room temperature, and poured onto a mixture of 5% hydrochloric acid and ice. The aqueous layer was extracted with chloroform and the organic layer was evaporated to dryness. The residue was purified by flash chromatography on silica gel, using 7:l ethyl acetate:hexanes as the eluent, to yield a colourless oil(0.30 g, 79%). '~nmr(CDCI,, 6): 8.89 (lH, br s, NH),

4.03 (2H, t, 6.5 Hz, CH2O), 2.52 (2H, t, 7.2 Hz, COCH2), 2.10 (2H, quintet, 6.9 Hz,

COCH2CH2). 13cnrnr (CDCG, 6): 172.93, 69.04, 27.42, 21 .55. IR (CH2Clz):3403, 1667 cm-'. Mass spectnim (CI, dz): 102 (M+1). Anal. Calcd. for C&17N02*0.10E20:C

46.68; H 6.97; N 13-61. Found: C 46.73; H 7.01; N 13-21.

Method B

Potassium hydroxide (0.042 g, 0.75 mol) in methanol(4 mL) was added to a solution of methyl y-aminooxybutyrate (2-20) (0.10 g, 0.75 mmol) in methanol (10 rnL) and the mixture was refluxed for 2 h, cooled to roorn temperature, stirred overnight, neutralized with 5% hydrochloric acid and evaporated. The residue was purified by preparative Iayer chromatography on silica gel using ethyl acetate as the eluent to yield an oil (18 mg,

24%), identical with the product of Method A.

Method C

A solution of methyl y-aminooxybutyrate (2-20) (3.03 g, 22.8 mmol) in tetrahydrofuran

(40 mL) was cooled to O OC and a 2.0 M solution of trimethylaliiminum in hexanes (22.8 mL, 45.6 mmc31) was added in portions. The reaction mixture was allowed to wann to room temperature, with stirring, during 2 h, and quenched with acetone (1 5mL), with sturing for 30 min, and then by slow addition of water (50 mL). Most of the solvent was rernoved under reduced pressure, and 1:4 dich1oromethane:tetrahydrofuran (300 mL) was added. Filtration through Celite and evaporation of the filtrate gave an oil (1.51 g, 65%), identical with the product of Method A.

Methyi p-Bromopropionate

A solution of P-propiolactone (2 mL, 2.29 g, 30.8 mmol) in 30% hydrogen bromide in acetic acid (10 mL) was stirred at room temperature for 20 h. Then methanol (20 rnL) was added and stirring was continued for an additional 15 h, Evaporation of the solvent gave a dark oïl, which was dissolved in ethyl acetate (50 mL) and washed successively

with saturated sodium bicarbonate (2 x 50 mL) and saturated sodium chlonde (50 mL),

dried over anhydrous rnagnesium sulfate, and evaporated to yield a clear oil (2.88 g,

56%). '~nmr(CDC13, 6): 3.77 (3H, s, 0CH3),3.62 (2H, f 6.8 Hz, CHzJ3),2.96 (2H,t,

6.8 HZCHta). IR (neat): 1742 cm-'. Mass spectnim (CI, dz): 167 (M+1), 169 (M+3).

Methyl B-Phthalimidooxypropionate

Method A

A solution of N-hydroxyphthalimide (18.1 g, 0.11 1 mol) and triethylaaiine (15.5 mL,

11.3 g, 0.1 11 mol) in methanol (100 mL) was stirred at room temperature for 10 min.

Methyl acrylate (10 mL, 9.56 g, 0.1 11 mol) was then added dropwise and the solution was heated to reflux for 18 h and then cooled to room temperature. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (150 d) and washed successively with water (50 d),saturated sodium bicarbonate (5 x 50 mL), water (50 mL), 5% citric acid (50 mL), water (50 mL) and saturated sodium chloride (50 mL), dried over anhydrous rnagnesium sulfate, and evaporated. The solid residue was recrystallized £kom hot ethanol to give white crystals, 5.88 g (21%), m.p. 76 - 77 OC. t Hnmr (CDCls, 6): 7.8 1 (4H,m, phthdimido ring), 4.51 (2H, t, 6.5 Hz, CH$), 3.71 (3H, s, 0CH3), 2.85 (2H, t, 6.5 Hz, CH2a). 13cnrnr (CDC13, 6): 170.58, 163.44, 134.53,

128.90, 123.58, 73.40, 51.95, 33.65. IR (KE3r): 1745, 1727, 1657 cd. Mass spectrum (CI, mk): 250 (M+l). Anal. Catcd. for Ci2Hl~N1O5:C 57.82; H 4.46; N 5.62. Found: C

57.70; H 4.50; N 5.46.

Method B

A mixture of methyl P-bromopropionate (0.50 g, 2.99 mmol), whydroxyphthalimide

(0.49 g, 2.99 mol) and triethylamine (0.64 mL, 0.45 g, 4.49 mmol), in acetonitrile (7 mL), was refluxed for 3 h, cooled and filtered. The filtrate was evaporated under reduced pressure and the residue was dissolved in ethyi acetate (20 mL). This solution was washed successively with water (3 x 20 mL) and saturated sodium chloride (20 mL), dried over anhydrous magnesium sulfate and evaporated. Recrystallization fkom hot ethanol gave 48.9 mg (6%) of white crystals, identical with the product of Method A.

Methyl p-Aminooxypropionate

A solution of methyl B-phthaIimidooxypropionate (1.50 g, 6.02 mrnol) in dichloromethane (50 mL) was cooled to -10 OC and methylhydrazine (0.5 mL, 0.433 g,

9.03 mol) was added dropwise with stimng. St-g was continued for 1.5 h at -10 to

O" C. The mixture was then filtered and the filtrate was concentrated. The residue was dissolved in ethyl acetate (30 mL) and this solution was washed with 1:1 saturated sodium chloride: saturated sodium bicarbonate (8 mL). The aqueous layer was washed with ethyI acetate (30 mL) and the combined organic layers were dried over anhydrous magnesium sulfate and evaporated. Purification by short column chromatography on silica gel, using a gradient solvent system, hexanes to ethyl acetate, gave 0.359 g (500/0) of an oil. '~nmr(CDC13, 6): 5.85 (2H, s, NI&), 3.73 (3H, s, CH3), 3.26 (2H, t, 6.2 Hz,

CH$), 2.67 (2H, t, 6.2 Hz, Cfia).Mass spectrum (CI, m/z): 120 (M+l).

A solution of methyl P-aminooxypropionate (0.251 g, 2.10 mmol) in tetrahydrofüran (25 mL) was cooled to O OC and a 2.0 M solution of trirnethylduniinum in hexanes (2.1 mL,

4.20 mmol) was added in portions. The reaction mixture was allowed to warm tc room temperature, with stirring, during 4 h, and then by slow addition of water (2 mL), with stirring for 30 min. Most of the solvent was removed under reduced pressure, and chloroform (300 mL) was added. Filtration through Celite and evaporation of the filtrate gave a colourless oil (83.0 mg, 45%). 1Hnmr (CDC13, 6): 9.21 (lH, br s, NH), 4.41

(2H, t, 8.2 Hz, CH$), 2.79 (2H, t, 8.2 Hz, CH2a).Mass spectrum (CI, dz): 88 (M+l).

Anal. Cakd. for C3H5N02: C 41.38; H 5.79; N 16.09. Found: C 40.89; H 5.84; N

15.55. Methyl &Bromopentanoate

A solution of &valeroiactone (5 .O mL, 5.39 g, 53.8 mmol) in 30 % hydrogen bromide in acetic acid (15 mL) was heated at 75 OC for 5 h, cooled to room temperature and mehano1 (20 rnL) was added. The mixture was stirred ovemight and the solvent was evaporated to give a dark oil, which was dissolved in ethyl acetate (50 mL) and washed successively with saturated sodium bicarbonate (2 x 50 mL) and saturated sodium chloride (50 mL), dried over anhydrous magnesiurn sulfate, and evaporated to yield a clear oil (9.16 g, 87%). 1Hnmr (CDC13, 6): 3 -67 (3H, s, 0CH3), 3.4 1 (2H, t, 6.4 Hz,

CH*@, 2.35 (ZH, t, 7.3 Hz, CHza), 1.90 (2H, m, CHg), 1.79 (2H, m, CH$). l3cnrnr

(CDCI,, 6): 173.55, 51.58, 33.00, 31.97, 23.47. IR (neat): 1737 cm-'. Mass specmim

(CI, dz): 195 (M+l), 197 (M+3).

Methyl &Phthalimidooxypentanoate

A mixture of methyl Gbromopentanoate (6.01 g, 30.8 mmol), N-hydroxyphthalimide

(5.03 g, 30.8 mmol) and triethylarnine (8.6 mL, 6.24 g, 61.5 mmol), in acetonitrile (40 mL), was refluxed for 3 h, cooled and filtered The filtrate was evaporated under reduced pressure and the residue was dissolved in ethyl acetate (75 mL). This solution was washed successively with water (3 x 75 mL) and saturated sodium chlonde (75 d), dried over anhydrous magnesium sulfate and evaporated. Recrystallization f?om hot ethanol gave 6.33 g (74%), m.p. 41 - 42 OC. %nmr (CDCI,, 6): 7.81 (4H, m, phthalirnido ring), 4.21 (2H, t, 6.1 Hz, CH2@, 3.68 (3H, s, OCH3), 2.43 (2H, t, 7.1 Hz,

CH2a), 1.86 (4H, m, CH2PCH2y). l3cnrnr (CDCl,, 6): 173.74, 163.60, 131.45, 128.93,

123.49, 77.88, 51 -55, 33.45, 27.51, 21.1 1. IR (KBr): 1727, 1710 cm-'. Mass spectrum

(CI, dz):278 (M+l). Anal. Calcd. for C1&a05: C 60.63; H 5.46; N 5.05. Found: C

60.54; H 5.43; N 5.06.

A solution of methyl &phthalimidooxypentanoate (2.01 g, 72.4 mmd) in dichloromethane (50 mL) was cooled to -10 OC and methykydrazine (0.58 rnL, 0.50 g,

10.8 mmol) was added dropwise, with stirring. Stirrïng was continued for 1.5 h and the mixture was then filtered and the filtrate evaporated. The residue was dissolved in ethyl acetate (50 mL) and thîs solution was washed with 1: 1 saturated sodium chloride: saturated sodium bicarbonate (30 mL). The aqueous layer was extracted with ethyl acetate (50 mL) and the combined organic layers were dried over anhydrous rnagnesium sulfate and evaporated to give an oil (1 .O3 g, 97%)). '~nrnr(CDC13, 6): 4.50 (ZH,br s,

NI&), 3.67 (3H, s, 0CH3), 3.66 (2H, t, 6.2 Hz, CH26), 2.34 (2H, t, 7.0 Hz, CH2a), 1.66

(4H, m, CH$CHu). l3cnrnr (CDC13, 6): 173.97, 75.37, 5 1SO, 33.76, 27.43, 2 1.49. IR

(neat): 3320, 1732 cm-'. Mass spectrum (CI, dz): 148 (M+l).

A solution of methyl Gamhooxypentanoate (96.6 mg, 0.656 mmol) in tetrahydrofüran (3 rd) was cooled to O OC and a 2.0 M solution of ûimethylaluminum in hexanes (0.68 mL,

1.36 mmol) was added dropwise. The resulting solution was allowed to warm to room temperature and was stirred for 5.5 h. Acetone (0.8 mL) was added and, after 20 min, water (2 mL) was added dropwise. Most of the solvent was removed under reduced pressure, 1:4 dichloromethane:tetrahydrofuran (50 mL) was added and the mixture was filtered through Celite. Evaporation of the filtrate gave a white solid, which was recrystallized from hot ethyl acetate (61.7 mg, 82%), m.p. 84.5 - 85.8 OC. '~nmr

(CDCl3,6): 8.27 (lH, br s, NH), 4.07 (2H, m, Cl&@, 2.63 (2H, m, CHza), 1.91 (2H, m,

CHzy), 1.81 (2H, m, CH2f3). %nmr (CDC13, 6): 179.36, 78.37, 35.79, 30.94, 21.85. IR

(KBr): 3419, 1642 cm-'. Mass spectrum (CI, dz): 116 (M+l). Anal. Calcd. for

C5HgN02:C 52.15; H 7.89; N 12.17. Found: C 52.12; H 7.91; N 12.18. Methyl E-Bromohexanoate

A solution of E-caprolactone (5.2 mL, 5.36 g, 46.9 mmol) in 30% hydrogen brornide in acetic acid (12.5 d)was heated for 6 h at 75 OC, cooled to room temperature, treated with methanol (20 mL) and stirred overnight. Evaporation of the solvent gave a dark oil, whicli was dissoived in ethyl acetate (50 mL) and washed successively with saturated sodium bicarbonate (2 x 50 mL), and saturated sodium chloride (50 mL), dried over anhydrous magnesium sulfate, and evaporated to yield a clear oi1 (9.31 g, 95%). '~nmr

(CDCi3, 6): 3.67 (3H, s, OC&), 3.40 (2H, t, 6.8 Hz, Cm),2.33 (2H, t, 7.5 Hz, Caa),

1 -87 (2H, m, Cl&@, 1.66 (2H, m, CH$), 1.47 (2H, m, Gay). Mass spectnim (CI, m/z):

209 (M+I), 2 1 1 (M+3).

Methyl E-Phthaiimidooxyhexanoate

A mixture of methyl E-bromohexanoate (6.44 g, 30.8 mmol), N-hydroxyphthalimide

(5.02 g, 30.8 mrnoles), and triethylamine (8.6 mL, 6.24 g, 61-5 mmol), in acetonitrile (40 rnL), was refluxed for 3 h, cooled and filtered. The filtrate was concentrated and the residue diluted with ethyl acetate (50 mL). This solution was washed successively with water (3 x 50 mL) and saturated sodium chloride (50 mL), dried over anhydrous

magnesium sulfate and evaporated Recrystallization fiorn hot ethanol gave 8.89 g (99%)

as white cxystals, m.p. 73 - 74 OC. 'Hnmr (CDC13, 6): 7.80 (43,m, phthalimido ring),

4.20 (2H, t, 6.6 Hz, Cm), 3.67 (3H, s, 0CH3), 2.35 (2H, t, 7.4 Hz, Cas), 1.80 (2H, m,

CH&), 1.72 (2H, m, CH$), 1.55 (2H, m, CH2y). I3cnrnr (CDCI,, 6): 173.06, 163.58,

134.49, 128.90, 123.53, 77.28, 5168, 29.96, 23.52. IR (KBr): 1787, 1731 cm-'. Mass

spectrum (CI, dz): 292 @l+l). Anal. Calcd. for Ci&7NOs: C 61.84; H 5.89; N 4.81.

Found: C 6 1-49; H 5-92; N 4.75.

Methyl &-Aminooxyhexanoate

A solution of methyl E-phthalimidooxyhexanoate (1 -05 g, 3.60 rnmol) in dichloromethane (1 9 mL) was cooled to -1 0 OC, methylhydrazine (0.29 mL, 0.25 1 g, 5 -4 mol) was added dropwise with stimng, and stirring was continued for 1.5 h at -10 to O

OC. The mixture was then filtered and the filtrate was evaporated. The residue was dissolved in ethyl acetate (50 mL) and washed with 1:l saturated sodium chloride: saturated sodium. bicarbonate (30 mL). The aqueous extract was washed with ethyl acetate (50 mL) and the combined organic layers were dried over anhydrous magnesium sulfate and evaporated to give an oïl (0.567 g, 98%). '~nmr(CDC13, 6): 5.10 (2H, br s,

NF&), 3.66 (3H, s, OCH,), 3.64 (2H, t, 6.6 Hz, Cf&), 2.30 (2H, t, 7.4 Hz, C&a), 1.64

(2H, m, CH&), 1.58 (2H, m, CH$), 1.35 (2H, m, CHzy). A 2.0 M solution of trimethylduminum in heptane (4.96 mL, 9.92 mmol) was added to toluene (50 d),cooled to O OC, and a solution of methyl-&-aminooxyhexanoate (0.400 g,

2.48 mmol) in toluene (50mL)was added dropwise. The resulting solution was allowed to warm to room temperature and was stirred for 6 &YS. Water (20 mL) was added dropwise and the mixture was stirred for 30 min. Most of the solvent was removed under reduced pressure, and 1 :4 dichloromethane:tetrahydrofiiran (50 mL) was added.

Filtration through Celite and evaporation of the filtrate gave a trace amount of product.

Mass spectrum (CI, dz): 130 (M+l).

A solution of trïethylamine (7.75 mL, 5.63 g, 56.0 rnmol) in àichloromethane (10 mL) was added slowly to a suspension of a-amino-y-butyrolactone hydrobromide (2-26) (10 g, 55.0 mmol) in dichloromethane (100 mL). The mixture was cooled to O OC and a solution of BOC anhydride (13.2 g, 60.4 mmol) in dichloromethane (25 mL) was added during 5 min. The resulting suspension was allowed to warm to room temperature, stirred for 96 h, and then washed successively with 5% citric acid (50 mL), water (50 mL) and

saturated sodium chloride (50 mL), dried over anhydrous magnesium sulfate and

evaporated to give a white solid. This was recrystallized fiom toluene to yield 9.93 g,

(go%), m-p. 124 - 126 OC. '~nrnr(CDCls, 6): 5.05 (1 H, br s, NH), 4.44 (lH, m, CHy),

4.34 (IH, m, CHa), 4.24 (IH,m, CHy), 2.76 (IH, m, CE@), 2.18 (IH, m, Cm), 1.47

(9H, s, C(CH3)3). '3~nmr(CDC13, 6): 175.68, 155.72, 80.55, 65.83, 50.21, 30.34, 28.30.

IR (KBr): 3359, 1787, 1684, 1528 cm-'. Mass spectrum (CI, dz): 202 (M+l). Anal.

Caicd. for C9H1a04:C 53.71; H 7.53; N 6.96. Found: C 53.98; H 7.58; N 7.19.

Method A

To a solution of homoserine (2-25) (5.00 g, 42.0 mmol) in 50% aqueous acetone (50 niL) were added, with s tirring, triethylamine (8.8 rnL, 6.34 g, 63 .O mmol) and BOC anhydride

(1 0.1 g, 46.2 mmol). AAer 14 h, the solvent was removed under reduced pressure and the residue was dissolved in water (30 mL). This solution was acidified to pH 3-4 with 5% citric acid (1 30 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were washed with saturated sodium chloride (50 mL), dried over anhydrous magnesium sulfate, and evaporated to give a yellow oil (6.79 g, 74%). 1Hnmr (CDC13,

6): 5.49 (IH, br s, NH), 4.50 (lH, m, CHa), 3.79 (2H, m, CHfl), 2.21 (IH, m, CE@), 1.77

(IH, m, Cm), 1.41 (9H, s, C(CH3)3).l3cnrnr (CDC13, 6): 176.82, 156.40, 80.14, 58.33, 51-50, 35.14, 28.30. Mass specfillm (CI, dz): 220 (Mfl). Anal. Calcd. for

C9HrtNOj0.20H20:C 48-52; H 7.71; N 6.29. Found: C 48-72; H 7.67; N 6.04.

Method B

To a solution of 2-tert-butoxycarbonylamino-y-butyrolactone(7.00 g, 34.8 mmol) in dry methanol (80 mL), was added in portions a solution of potassium hydroxide (3.44 g, 52.2 mrnol) in methanoL(20 mL). The mixture was stirred at room temperature for 24 h and

Amberlyst 15 (~3(14.0 g, 62.6 mmol), presoaked in methanol, was added. The resin was then removed by filtration and the solution was evaporated under reduced pressure to an oil(7.39 g, 97%), identical with the product of method A.

Benzophenone Hydrazone

Ph2C=N-NH2

A mixture of benzophenone (6.02 g, 33.1 mmol) and 98% hydrazine hydrate (1.60 mL,

33.1 mol) in 95% ethano1 (200 mL) was refluxed for 9 h. The product was collected by filtration and was recrystallized fiom 95% ethanol to give 4.48 g (69%) of long white needles, m.p. 97 - 98 OC (lit.92m.p. 98 OC).

Diphenyldiazomethane

Ph2CN2

A mixture of benzophenone hydrazone (4.49 g, 22.8 mol) and anhydrous magnesiwn sulfate (2.75 g, 22.8 mmol) in dichloromethane (90 mL) was stirred for 10 min in an ice- bath, and activated manganese dioxide (6.95 g, 80.0 mmol) was then added in one portion. Stimng was continued at O OC for 2 h and then at room temperature for 1 h.

Some solid was removed by filtration through Celite, and the filtrate was cmcentrated under reduced pressure to a dark, purple oil which crystallized upon cooling to give 4.42 g (99%) of red needles, rn-p. 33 - 34 OC (lit.93m.p. 35 OC).

~~~utoxycarbonylHomoserine Benzhydryl Ester (2-24, RI = OtBu, R2= CEl?hn X

= OH)

A solution of N- 'butoxy~arbon~lhomoserine (0.803 g, 3.66 mmol) in a mixture of dichloromethane (20 mL) and acetonitrile (20 mL) was treated dropwise, with stimng, with a solution of diphenyldiazomethane (0.713 g, 3.66 mmol) in dichloromethane (3 d).Stirring was continued for 2h afier the addition was complete, and the solvent was then removed under reduced pressure to give a yellow oil. This was dissolved in ethyl acetate (50 mL) and washed successively with water (2 x 50 rnL) and saturated sodium chloride (50 mL), dried over anhydrous inagnesium sulfate, and evaporated. Purification by flash chromatography on silica gel, using ethyl acetate-hexanes (1: 1) as the eluent, gave the product as an oil, (0.992 g, 70%). '~nmr(CDCls, 6): 7.31 (lOH, m, Ar), 6.90

(ZH,s, CHO), 5.38 (lH, br d, 7.9 Hz, NH), 4.62 (lH, m, CHor), 3.65 (2H, m, Cm),3.80

(1 H, br s, OH), 2.24 (1 H, m, Cm), 1S8 (1 H, m, Cl@), 1.44 (9H, s, C(CH3),). IR (neat):

92 Miller, J.B. J. Org. C'hem. 1959,24, 560. 3499, 3321, 1736, 1688 cm-'. Mass spectrum (CI, m/z): 386 (M+l). Anal. Calcd. for

C22Hz7N05'0.25H20:C 67.78; H 7.13; N 3.60. Pound: C 67.68; H 7.14; N 3.61.

Benzhydryl- N-'butoxycarbonyl - a-Amino~uccinimidoorybutyr.te(2-24, RI =

OtBu, R2 = CHPh2, X = OSu)

A solution of benzhydryl N-'butoxycarbonyl homoserine (2-24,RI = OtBu, RI = CHPh2,

X = Oa) (1 .O1 g, 2.62 mmol) and N-hydroxysuccinimide (0.3 13 g, 2.72 mmol) in tetrahydrofuran (12 mL) was cooled to 15 OC and triphenylphosphine (0.702 g, 2.67 mol) and diethyl azodicarboxylate (0.45 mL, 0.498 g, 2.86 mrnol) were added successively. The reaction mixture was allowed to warm to room temperature, stirred ovemight, and the solvent was then removed under reduced pressure. Purification by flash chrornatography on silica gel, using 3: 1 hexanes:ethyl acetate as the eluent, gave a white solid (0.589 g, 47%). '~nrnr(CDC13, 6): 7.33 (lOH, m, Ar), 6.92 (lH, s, CHO),

5.81 (lH, br d, 8.2 Hz, NH), 4.62 (IH, m, CH@, 4.13 (2H, m, CHU), 2.65 (4H, s, succinimido ring), 2.27 (2H, m, CH&, 1.44 (9H,s, C(CH&). Mass spectrum (CI, dz):

383 ((M+l)-(BOC)). Anal. Calcd. for C2&0N207'0.75H20: C 62.89; H 6.41. Found: C

62.83; H 6.60.

93 Reirnlinger, H. Ber. 1954, 97, 3493. Methyl a-Amiao~BromobutyrateHydrochloride (2-27)

Hydrogen chloride was bubbled into a suspension of a-amino-y-bromobutyrate hydrobrornide (0.1 65 g, 0.586 mmol) in methanol (5 mL) for 2 h at such a rate that the temperature of the reaction mixture did not exceed 40 OC. The solvent was then removed under reduced pressure, and the residue was dissolved in methanol and the solution was reevaporated. This procedure was repeated once more and the residual oil was dried in vacuo in a desiccator over sodium hydroxide pellets. The resulting crystals were trîturated with ether, collected and washed with ether to give 89 mg (65%), m.p. 96 - 98

OC. '~nmr@20, 8): 4.33 (lH, t, 6.67 Hz, CHa), 3.83 (3H, s, OCH,), 3.59 (2H, m,

CH2y), 2.56 (lH, m, Cm), 2.39 (lH, m, CE@). Mass spectrum (CI, mh): 196 (M+l),

198 (M+3).

Methyl N-t~utoxycarbonyls-~mi~romobutyrate(2-24, RI = OtBu, R2= Me, X

= Br)

To a solution of methyl a-amino-y-bromobutyrate hydrochloride (2-27) (29.7 mg, 0.129 rnmol) in 50% aqueous acetone (1 mL), were added, with stirring, triethylamine (0.039 mL, 28.3 mg, 0.258 mol) and BOC anhydride (30.1 mg, 0.142 mmol). Stimng was continued for 3 h at roorn temperature and the solvent was then removed under reduced pressure. The residue was purified by preparative layer chromatography on silica gel, using 2: 1 hexanes:ethyl acetate as the eluent, to give 28.4 mg (75%) of the product. t Hnmr (CDC13, 6): 5.05 (IH, br d, 7.5 Hz, NH), 4.41 (lH, m, Clria), 3.73 (3H, s, 0CH3),

3.40 (2H, t, 6.8 Hz, Gay), 2.37 (2H, m, CH$), 1.42 (9H, S, C(CH3)3).

Triethylamine Salt of ~-'~utoxycarbon~lHomoserine

A solution of homoserine (5.00 g, 42.0 mmol) in 50% aqueous acetone (50 mL), was treated with triethylamine (8.8 mL, 6.39 g, 63.0 mmole) and BOC anhydride (10.1 g, 46.2 mol) and stirred ovemight at room temperature. Removal of the solvent gave the salt

(13.5 g, 100%). 1Hnmr pro, 6): 4.02 (lH, m, CHU), 3.64 (2H, m, CH29,3.18 (2H, q,

7.3 Hz, CH2CH3) 1.98 (lH, m, Cm),1.82 (lH, m, Cm), 1.41 (9H,s, C(CH3)3), 1.26

(3H, t, 7.3 Hz, CH3).l3cnrnr (D20, 6): 181.68, 160.33, 83.76, 61.09, 55.48,49.30, 36.57,

30.28, 10.85. IR (CH2Cl2):3413, 1709, 1693 cm-'. Mass spectrum (CI, m/z): 164 (M+l-

BOC). Methyl N-'~utoxycarbonylHomoserinate (2-24, Ri = OtBu, R2= Me, X = OH)

Method A

A solution of the tnethylamine salt of IV-'butoxycarbonyl homoserine (7.49 g, 23.4 mmol)

in dimethylformamide (50 mL) was cooled to O - 10 OC, stirred, iodomethane (1.6 rnL,

3.65 g, 25.7 mmol) was added, the cooling bath was removed, and stimng was continued

overnight. Most of the solvent was removed under reduced pressure and the residue was

diluted with ethyl acetate (80 mL) and washed with water (20 mL). The aqueous extract

was washed with ethyl acetate (2 x 25 mL), and the combined organic extracts were

washed successively with 5% citric acid (2 x 15 mL), water (15 mL), satwated sodium

sulfite (1 5 mL), saturated sodium bicarbonate (2 x 15 II&) and saturated sodium chloride

(2 x 15 mL), dried over anhydrous magnesium sulfate and evaporated to give a colourless

oil(5.15 g, 94%). '~nmr(CDCli, 6): 5.43 (1H, br s, NH), 4.52 (1 H, m, CHa), 3.80 (3H,

s, Cao), 3.73 (ZH,m, CHU), 3.26 (lH, br s, ON), 2.1 1 (1 H, m, Cm), 1.64 (1 H, m,

Cm), 1.49 (9H, s, C(CH3)& 13cnrnr (CDC13, 6): 1 73.62, 156.80, 80.16, 58.65, 52.50,

51.04,36.18,28.66. Mass spectrum (CI, m/z): 234 (M+l).

Method B

To a solution of ~-~butox~carbon~lhomoserine (7.00 g, 31.9 mmol) in dimethylformamide (40 mL) was added, during 30 min, a solution of triethylamine (4.50 mL, 3.27 g, 32.2 mmol) in dimethylformamide (15 mL). Iodornethane (4.00 rnL, 9.1 1 g, 64.2 mol) -was then added in one portion and the reaction mixture was stirred overnight, and then concentrated under reduced pressure to remove most of the solvent. The residue was diluted with ethyl acetate (100 mL) and water (20 mL), the aqueous layer was washed with ethyl acetate (2 x 25 mL), and the combined organic layers were washed successively with 5% citric acid (2 x 15 mL), water (1 5 mL), saturated sodium sulfite (1 5 d),saturated sodium bicarbonate (2 x 15 mL) and saturated sodium chloride (2 x 15 mL), dned over anhydrous magnesium sulfate and evaporated to give a colourless oil

(5.79 g, 78%), identical with the product of Method A.

Methyl 2-t~utoxycarbonylamino~ethanesulf~~y~~~~yb~tyrate(2-24, RI = OtBu,

R2= Me, X = OMS)

Methanesulfonyl chloride (2.14 mL, 3.16 g, 27.6 mrnol) was added dropwise at -15 to -

10 OC to a solution of methyl N-*butoxycarbonyl homoserinate (2-24, Ri = OtBu, Rz =

Me, X = OH) (5.6 g, 24.0 mmol) and triethylamine (4.0 mL, 2.90 g, 28.8 mmol) in dichlorornethane (80 mL). The reaction mixture was allowed to warm to O OC and was stirred at that temperature for 1 h. Cold 10% potassium bisulfate (25 mL) was added, and the aqueous layer was separated and washed with dichloromethane (2 x 25 mL). The combined organic layers were washed with sodium bicarbonate (2 x 35 rnL), dried over anhydrous magnesium sulfate, and evaporated to give a pale yellow oil (7.25 g, 97%), which was characterized filly after the next step. '~nrnr(CDC13, 6): 5.25 (lH, br s, NH), Phenylacetyl Chloride

A solution of phenylacetic acid (1.55 g, 11.4 mmol) in thionyl chloride (5 mL, 8.16 g,

70.0 mmol) was refluxed for 1 h, cooled to room temperature, concentrated and distilled to give 1.59 g (90%) of the acid chlonde, b.p. 45 - 46O/ 1 torr. '~nmr(CDCI,, 6): 7.41

(5H,m, Ar), 4.19 (2H, s, CH2). IR (mat): 3034, 1800 cm-'.

Triethylamine (7.7 mL, 5.6 g, 55.0 mmol) was added slowly to a suspension of a-amino-

'y-butyrolactone hydrobromide (2-26) (5.0 g, 27.5 mrnol) in dichloromethane (50 rd).

The mixture was stirred for 10 min, cooled to -5 OC and treated, during 30 min, with a solution of phenylacetyl chloride (3.3 mL, 3.83 g, 24.8 mmol) in dichloromethane (20 mL). The mixture was allowed to warm to room temperature, stirred for 3 h, and then washed successively with water (25 mL), 1N hydrochloric acid (15 mL), water (15 mL) and 1 :1 water:saturated sodium bicarbonate (20 mL), dried over anhydrous magnesium sulfate and evaporated to give a white solid (5.14 g, 95%), m.p. 124 - 126 OC. '~nmr (CDC13, 6): 7.33 (SH,m, Ar), 5.97 (lH, br s, NH), 4.50 (lH, m, CHy), 4.43 (lH, f 9.1

Hz, CHa),4.25 (1H7 m, CHy), 3.63 (2H, s, PhCH*), 2.79 (lH, m, Cm), 2.08 (lH, m,

Cm). I3cnrnr (CDCl,, 6): 174.98, 171.49, 134.06, 129.39, 129.13, 127.59, 65.95,

49.37, 43.31, 30.32. IR (KBr): 3305, 1779, 1652 cm-'. Mass spectrum (CI, dz): 220

(M+l). Anal. Calcd. for C12H13N03: C 65.73; H 5.99; N 6.39. Found: C 65.69; H 5.97;

N 6.12.

N-Phenylacetyl Homoserine

A solution of potassium hydroxide (4.51 g, 68.8 mmoi) in methanol(20 mL) was added

in portions to a suspension of a-phenylacetamido-y-butyrolactone(1 0.1 g, 45.9 mmol) in dry methanol (100 mL). The mixture was stirred at room temperature for 24 h and

Arnberlyst 15 (H? (1 8.5 g, 82.5 mmol), presoaked in methanol, was added. The mixture was filtered and the filtrate was evaporated to an oil (10.9 g, 96%), which was used directly in the next step. N-Phenylacetyl Homoserine Methyl Ester (2-24, RI = 0CH2Ph, R2= Me, X = 08)

A solution of N-pheny lacety1 homoserine (3.1 9 g, 13 -5 mmol) in dimethylformamide (30

mL) was treated, in portions, with a solution of triethylamine (1 -88 mL, 1.36 g, 13.5

mmol) in dimethylfomiamde (1 0 mL). Then iodomethane (1.68 mL, 3.82 g, 26.9 mmol)

was added in one portion and stirring was continued overnight. Most of the solvent was

removed under reduced pressure and the residue was diluted with ethyl acetate (100 mL)

and water (20 mL). The aqueous layer was washed with ethyl acetate (2 x 25 rnL) and

the combined organic layers were washed successively with 5% citric acid (2 x 15 mL),

water (1 5 mL), saturated sodium sulfite (15 mL), saturated sodium bicarbonate (2 x 15 mL) and saturated sodium chloride (2 x 15 mL), dried over anhydrous magnesium sulfate and evaporated to give a pale yellow oil(2.02 g, 60%). '~nmr(CDC13, 6): 7.32 (SH,m,

Ar), 6.38 (1H,br d, 6.8 Hz, NH), 4.73 (lH, m, CHa), 3.73 (3H, s, Cl&), 3.64 (lH, m,

CHy), 3.63 (2H, s, PhCHZ), 3.49(1H, rn, CHy), 2.33 (lH, brs, OH), 2.13 (1H, m,Cm),

1.56 (IH, m, Cm). '3~nmr(CDCl3, 6): 172.94, 172.15, 134.21, 129.32, 129.09, 127.56,

58.09, 52.64,49.66,43.45, 35.63. IR (CHzClz): 3297, 1743, 1658 cm-'. Mass spectrum

(CI, dz): 252 (M+l). N-Pheny1acetyl-O-MethanesuKonyl Homoserine Methyl Ester (2-24, R1 = OCHtPh,

Rz= Me, X = OMS)

A solution of N-phenylacetyl homoserine methyl ester (2-24, RI = 0CH2Ph, R2= Me, X

= OH) (1.90 g, 7.56 mmol) and triethylamine (1.26 mL, 0.91 g, 9.07 mmol), in dichloromethane (30 mL), was cooled to -15 to -10 "C and methanesulfonyl chloride

(0.67 mL, 1.00 g, 8.70 mol) was added dropwise. The mixture was allowed to warrn to

O OC and was stirred at that temperature for 1 h. Cold 1Wpotassium bisulfate (15 mL) was added and the aqueous layer was separated and washed with dichloromethane (2 x 15 d).The combined organic layers were washed with sodium bicarbonate (2 x 25 mL), dried over mhydrous magnesium sulfate, and evaporated to give a yellow oil (2.23 g,

90%). 1Hnmr (CDCb 6): 7.38 (5H,m, Ar), 6.19 (1 H, br d, 7.1 Hz, NH), 4.70 (1 H, m,

CHa),4.17 (2H, t, 6.2 Hz, Cm),3.76 (3H, s, COCfi), 3.64 (2H, s, PhCH2), 2.90 (3H, s,

S02CH3),2.34 (1H, m, Cm), 2.12 (lH, m, CE@). l3cnrnr (CDCI,, 6): 1il -73, 171.02,

134.36, 129.34, 129.12, 127.55,65.68,52.77,49.34,43.63, 37.16, 32.46. Mass spectmm

(CI, rnlz): 330 (M+l). Methyl ~-'~utorycarbonylu-~minqphthrrlimidoo- (2-24, RI = OtBu,

Rz= Me, X = OFt)

1,8-Diazabicycl0[5.4.0]undec-7-ene (3-2 mL, 3.18 g, 20.9 mmol) was added dropwise to a solution of N-hydroxyphthalunide (3.40 g, 20.9 mmol) in dimethylformamide (30 mL).

The solution was stirred for 30 min, cooled to 10 - 15 OC and a solution of methyl 2- t butoxycarbonylamino-4-methanesulfonylooxybuate (2-24, Ri = OtBu, R2 = Me, X =

OMS) (6.5 g, 20.9 mmol) in dirnethylformamide (10 mL) was added dropwise. The mixture was then allowed to warm to room temperature and stirred for 48 h.

Approximately 30 mL of solvent were removed under reduced pressure and ethyl acetate

(100 mL) and water (30 mL) were added. The layers were separated and the aqueous layer was washed with ethyl acetate (2 x 50 mL). The combuied organic layers were washed successively with saturated sodium bicarbonate (5 x 30 mL), water (30 mL), 5% citric acid (30 mL), water (30 mL) and saturated sodium chloride (30 mL), dried over anhydrous magnesium sulfate, and evaporated to yield 8.58 g of a solid. Recrystallization from hot ethanol gave white crystals, 5.07 g (640/0), m.p. 135 - 136 OC. '~nmr(CDC13,

8): 7.81 (4H, rn, phthalimido ring), 5.68 (1 H, d 7.6 Hz, NH), 4.55 (lH, m, CHa),4.3 1

(2H, t, 6.1 HZ, CHfl), 3.76 (3H, S, CH3), 2.30 (2H, m, CH$), 1.45 (9H, S, C(CH3)3). IR

(Ur): 3365, 1746, 1725, 1680 cm". Mass spectrum (CI, dz): 379 (M+l). Anal.

Calcd. for C18H22NZ07:C 57.13; H 5.87; N 7.40. Found: C 57.21; H 5.93; N 7.33. Methyl ~~~utoxycarboaylu-~minwp~minooxybutyrate(2-23, RI = OtBu, & =

Me)

O

OMe

A solution of methyl N-'butoxycarbonyl-a-amino-y-phthalimidooxybute(2-24, RI =

OtBu, RI = Me, X = OFt) (0.50 g, 1.32 mmol) in dichloromethane (20 mL) was cooled to -10 OC and rnethylhydrazine (0.10 mL, 91.1 mg, 1.98 mmol) was added dropwise, with stimng. Stïrring was continued for 2 h at -10 to O OC and the mixture was then filtered. The filtrate was concentrated and the residue, in ethyl acetate (15 mL), was washed with 1: 1 saturated sodium chloride: saturated sodium bicarbonate (10 mL). The aqueous layer was washed with ethyl acetate (15 mL) and the combined organic layers were dried over anhydrous magnesium sulfate and evaporated to give a white solid (0.325 g, 100%). '~nrnr(CDCh, 6): 5.62 (2H, br s, 0NH2), 5.29 (lH, d, 6.0 Hz, NH), 4.41

(lH, m, CHa), 3.75 (3H, s, CH3),3.75 (2H, t, 6.2 Hz, CHzy), 2.07 (2H, m, CH$), 1.45

(9H, s, C(CN3)& l3cnrnr (CDCb, 6): IR (KBr): 3336, 1738, 1698 cm-'. Mass spectrum (CI, dz): 249 (M+l). Anal. Calcd. for CI0H20N205:C 48.37; H 8.13; N

11-28. Found: C 48.77; H 7.96; N 1 1.36. 4-'~utoxycarbonylamin0-[1,2]0~azinan-3-0ne (2-22, Ri = OtBu)

A solution of methyl N-'butoxycarbonyl-a-amino-y-amuiooxybuty(2-23, Ri = OtBu,

Rr = Me) (0.150 g, 0.607 mmol) in tetrahydrofuran (15 mL) was cooled to O OC and a 2.0

M solution of trimethylalumuiurn in heptane (0.60 mL, 1.21 mol) was added dropwise.

The reaction mixture was allowed to warm to room temperature and was stirred for 4 h.

Water (2 mL) was added dropwise and, after 15 min, the solution was concentrated to near dryness, diluted with 1:4 dichloromethme:tetrahydro£uran (50 d)and filtered through Celite. The filtrate was concentrated to give a white solid (128 mg, 98%), m.p.

1 19 - 120 OC. '~nmr(CDCls, 8): 8.65 (1 H, br s, ONH), 5.43 (1 H, br s, NH), 4.54 (1 H, m, CHa),4.21 (lH, m, CHy), 4.08 (lH, m, Cm),2.88 (1H, m, Cm), 1.74 (lH, m,

Cq), 1-46 (9H, s, C(CH3),). I3cnmr (CDCI,, 6): 174.1 1, 155.35, 80.03, 69.77, 47.78,

29.74, 28.09. IR (KBr): 3364, 1696, 1679, 1528 cm-'. Mass spectrum (CI, m/z): 21 7

(M+l). Anal. CaIcd. for C9Hl6N204: C 49.98; H 7.47; N 12.96. Found: C 50.17; H

7.47; N 12.74. Methyl a-Phenylacetylamin0-~Phthalimidooxyb~~ate(2-24, Ri = 0CH2PL, R2 =

Me, X = OFt)

Triethylamine (0.95 mL, 0.69 g, 6.78 mmol) was added slowly to a solution of N- hydroxyphthalimide (1.1 1 g, 6.78 mmol) in acetonitrile (15 d).The solution was stirred for 20 min, cooled to 10 to 15 OC, and a solution of N-phenylacetyl-O- methanesulfonyl homoserine methyl ester (2-24, Ri = 0CH2Ph, R2 = Me, X = OMS)

(2.23 g, 6.78 mol) in acetonitrile (5 mL) was added dropwise. The reaction mixture was allowed to warm to room temperature and stimng was continued for 24 h. The solvent was removed and the residue was dissolved in ethyl acetate (50 mL). This solution was washed successively with water (2 x 15 mL), saturated sodium bicarbonate

(8 x 15 mL), water (15 mL), 5% citric acid (15 mL), water (15 mL) and saturated sodium chioride (15 d),dried over anhydrous magnesium sulfate, and evaporated. The resulting solid was recrystallized fkom hot ethanol to give 2.25 g (84%) as white crysials, m.p. 138 - 139 OC. 'Hnmr (CDC13, 6): 7.8 1 (4H,m, phthalirnido ring), 7.24 (5H,m, Ph),

6.15 (lH, br s, NH), 4.87 (lH, m, CHa),4.21 (2H, t, 5.8 Hz, CHly), 3.73 (3H, s, CH3),

3.67 (ZH, s, PhCH?), 2.30 (2H, m, CH$). IR (KBr): 3348, 1792, 1735, 1664 cm-'.

Mass spectrum (CI, dz): 397 (M+l). Anal. Calcd. for Cll&&O6: C 63.62; H 5.10;

N 7.07. Found: C 63.97; H 5.19; N 7.12. Methyl Zlheaylacetylamin0-4-Aminoorybutyrote (2-23, RI= OCHtPh, R2= Me)

PhCH2CONH\IJ OMe

Methylhydrazine (0.20 mL, 0.174 g, 3.78 mmol) was added dropwise at -10 OC to a

solution of methy 1 a-pheny lacety lamino-y-phthalunidooxybutan (2-24, Ri =

OCH2Ph, R2 = Me, X = OFt) (1.00 g, 2.52 mol) in dichloromethane (40 mL). The cooling bath was removed and stirring was continued for 3 h. The mixture was then filtered and the filtrate was evaporated. The residue, in ethyl acetate (30 mL), was washed with I: 1 saturated sodium chloride: saturated sodium bicarbonate (6 mL). The aqueous layer was extracted with ethyl acetate (30 mL) and the combined organic extract was dried over anhydrous rnagnesium sulfate aïid evaporated to give a pale yellow oil

(0.664 g, 99%). '~nmr(CDC13, 6): 7.35 (5H,m, Ph), 6.50 (1H,br s, NH), 4.68 (1 H, m,

CHa), 3.75 (3H, s, COCH3), 3.65 (2H, t, 5.8 Hz, CH~.I),3.65 (2H, s, PhCHz), 2.03 (ZH, m, CH$). Mass spectrum (CI, dz): 267 (M+l).

A 2.0 M solution of trimethylaluminum in hexanes (1.13 mL, 2.25 mmol) was added dropwise, at O OC, to the canaline derivative (2-23, RI = 0CH2Ph, R2 = Me) (0.300 g, 1.13 mmol) in toluene (20 mL). The ice-bath was removed and stimng was contïnued

for 4 h. The reaction mixture was then treated dropwise with water (1 mL). After an

additional 15 min, the solvent was removed under reduced pressure to near-mess, 1:4

dich1oromethane:tetrahydrofuran (150 mi,) was added, and the mixture was filtered

through Celite. The filtrate was concentrated and the residue was purified by short

column chromatography on silica gel with a gradient solvent system, hexanes to ethyl

acetate, to give a white solid (91 -2 mg, 35%), m-p. 164 - 165 OC. '~amr(CDC13, 6):

8.17 (lH, brs, ONH), 7.32 (SH, m, Ar), 6.41 (lH, brs,NH), 4.72 (lH, m, CHa), 4.23

(1H, m, Cm),4.04 (lH, m, CHy), 3.63 (2H, s, PhCa), 2.98 (lH, m, Cm), 1.63 (lH, rn,

Cm). I3cnrnr (CDClî, 6): 173.52, 171.O8, 134.45, 129.30, 128.96, 127.37, 69.84,47.02,

43.56, 28.99. IR wr): 3307, 1694, 1660 cm-'. Mass spectrum (CI, mh): 235 (M+l).

Anal. Calcd. for C12H1&03:C 61S2; H 6.04; N 1 1.96. Found: C 61.32; H 6.04; N

11.92.

TFA Salt of Cyclocanaline

4-f~utoxycarbonylamino-[1,2]oxazin-3-one(2-22, Ri = 0th) (50.9 mg, 0.235 mrnol) was added, with stirring, to a cooled (O to -5 OC) solution of trifluoroacetic acid (0.75 mL). The mixture was allowed to wam to room temperature and was stirred for 1 h.

The solvent was then removed under reduced pressure and the residue was triturated with ether to give a white solid (53.0 mg, 98%). 'Hnmr @O, 6): 4.29 (lH, m, Cm),4.09

(IH, m,CHy),4.08 (lH,m,CHa),2-77(1H,m,C~),2.06(lH,m, Cm).

Triethylamine (0.2 mL, 0.145 g, 1.43 mmol) was added slowly to a suspension of the trifluoroacetic acid salt of cyclocanaline (0.150 g, 0.63 mmol) in dichloromethane (5 mL), the mixture was cooled to -10 OC, and a solution of phenoxyacetyl chloride (0.079 mL, 97.2 mg, 0.57 mmol) in dichloromethane (2 rnL) was added dropwise. The reaction mixture was allowed to warm to room temperature and was stirred ovemight and then washed successively with water (2 mL), 5% citric acid (2 mL), water (2 mL) and 1: 1 saturated sodium chl0ride:saturated sodium bicarbonate (2 d),dried over anhydrous magnesiurn sulfate, and evaporated. The residue was purified by short column chromatography on silica gel, using hexanes to ethyl acetate gradient solvent system, to give a white solid (29.8 mg, 38%). 1Hnmr (CDC13, 6): 7.53 (Ili, br s, NH), 7.15 (5H, m,

Ar), 4.84 (lH, m, CHa),4.55 (2H, s, PhOCH2), 4.28 (lH, m, CKy), 4.1 1 (lH, m, CHy),

3.03 (1 H, m, Cm), 1.78 (lH, m, Cm).l3cnrnr (CDC13, 6): 173.06, 168.47, 157.20,

129.75, 122.17, 1 14.79,69.89,67.29,46.66,28.94. IR Pr):33 14, 1686, 1655 cm-'.

Mass spectrum (CI, dz):251 (M+l). To a suspension of @)-cycloserine (2.00 g, 19.6 mmol) in 50% aqueous acetone (25 mL) were added triethylamine (4.1 mL, 2.98 g, 29.4 mmol) and BOC-ON (5.3 1 g, 2 1.5 mmol). The reaction mixture was stirred ovemight at room temperature, most of the solvect was removed under reduced pressure, and ethyl acetate (70 mL) was added. The aqueous layer was separated, acidified to pH 3-4 with 5% citric acid (75 mL) and extracted with ethyl acetate (90 mL). The latter layer was washed successively with water (3 x 90 mL) and saturated sodium chloride (90 mL), dned over anhydrous magnesium sulfate, and evaporated. Purification by fiash chromatography on silica gel, using ethyl aceîate-hexanes (2:l) as the eiuent, gave 2.41 g (61%) of the product (2-28), m.p. 140 - 142 OC. '~nmr(CDCI,, 6): 9.88 (1 H, br s, NH), 5.50 (1H, br s, BOCNH), 4.79

(lH, m, Cm), 4.58 (lH, m, CHa),4.10 (lH, m, CE@), 1.45 (9H, s, C(CH3)).I3cnrnr

(CDCls, 6): 170.8 1, 155.73, 80.78, 74.80, 52.77, 28.24. IR (CH2C12): 17 11, 1689 cm-'.

M27s spectrum (CI, dz): 203 (M+1). Anal. Calcd. for CSH1&O4: C 47.52; H 6.98; N

13.85. Found: C 47.67; H 7.07; N 13.10. To a solution of triphenylphosphine (0.103 g, 0.393 mmol) in tetrahydrofuran (3.2 mL), cooled to -56 to -50 OC, was added diethyl azodicarboxylate (0.058 mL, 64.1 mg, 0.371 mmol). The solution was stirred for 10 min, ethyl (S)-lactate (0.031 mL, 32.3 mg, 0.272 mmol) was added, and stimng was continued for an additional 10 min at -50 OC, at which time N-htoxycarbonyl-(R)-cycloserine (2-28) (50.0 mg, 0.247 mmol) was added.

Stirrir~gwas continued while the resulting mixture was allowed to wmto room temperature during 2.5 h. The solvent was removed under reduced pressure. Purification of the residue by flash chromatography on silica gel, using 2:l hexanes:ethyl acetate as the eluent, yielded a 1: 1 mixture of diastereomenc products (64.7 mg, 87%). '~nmr

(CDCI3, 6): 5.25 (lH, br s, BOCNH, both isomers), 4.96 (lH, q, 6.9 Hz, CHCH3, one isorner), 4.80 (1 H, q, 7.5 Hz, CHCH3, second isomer), 4.71 (1H, m, Cm,both isomers),

4.57 (1 H, m, CH- both isomers), 4.22 (2H, q, 7.1 Hz, C&CH3, one isomer), 4.2 1 (2H, q, 7.1 Hz, C&CH3, second isomer), 4.21 (lH, m, Cm, both isomers), 1.58 (3H, d, 6.7

Hz, CHCH3, one isorner), 1.53 (3H, d, 7.4 Hz, CHCH3, second isomer), 1.45 (9H, s,

C(CH3), one isomer), 1.44 (9H, s, C(CH3), second isomer), 1.29 (3H, t, 7.1 Hz, CHîCH3, one isomer), 1.28 (3H, t, 7.1 Hz, CH2CH3, second isomer). Mass spectrum (CI, dz):

303 (M+l) (both isomers). 2-35 2-36

Ethyl (S)-lactate (0.062 mL, 64.6 mg, 0.544 rnrnol) was added at room temperature to a stirred solution of [l,2]oxazinan-3-one (2-21) (45.4 mg, 0.449 mol) in dichloromethane

(2.8 mL). Triphenylphosphine (0.157 g, 0.597 mmol) was then added, stirring was continued for 10 min at room temperature and the reaction mixture was then cooled in an ice-bath and diethyl uodicarboxylate (0.093 mL, 0.103 g, 0.593 mmol) was added dropwise. The solution was aflowed to warm to room temperature overnight, with stimng. The solvent was removed under reduced pressure and the residue was purified by preparative layer chromatography on silica gel, using 7:4 hexanes:ethyl acetate as the eluent, to give 46.4 mg (5 1%) of the N-alkylated adduct and 6.7 mg (7%) of the 0- allcylated adduct. N-Alkylated adduct: '~nmr(CDC13, 6):5 -03 (1 H, q, 6.9 Hz, CH), 4.23

(2H, q, 7.1 Hz, CH2CH3), 3.87 (2H, rn, CH2y), 2.34 (ZH,rn, CHta), 2.03 (2H, m, CH$),

1.5 1 (3H, d, 7.0 Hz, CHCfi), 1.3 1 (3H, t, 7.1 Hz, CHrC&). O-Allcylated adduct: '~nmr

(CDCl3,6): 5.20 (lH, q, 7.3 Hz, CH), 4.24 (2H, q, 7.1 Hz, CHZCH~),4.17 (2H, m, CHly),

2.56 (2H, m, Cfia), 2.14 (2H, m, CH$), 1.50 (3H, d, 7.3 Hz, CHCH3), 1.29 (3H, t, 7.2

HZ, CH2CH7). 1,8-Diazabicyclo[5.4~O]undec-7-ene(1 8 mL, 18.3 g, 0.12 mol) was added slowly, with

stirring, to a solution of 85% (S)-Iactic acid (12.7 g, 0.12 mol) in methanol (50 mL). The

solvent was removed under reduced pressure at 70 - 80 OC, and the resulting oil in

dimethylformamide (50 mL), was cooled to 15 OC. Benzyl bromide (1 1.9 mL, 17.1 g,

0.10 mol) was added dropwise and the reaction mixture was stirred at room temperature for 30 h. AAer removal of approximately 40 mL of solvent by vacuum distillation, ethyl acetate (100 mL) was added, followed by water (30 d).The aqueous Iayer was washed with ethyl acetate (2 x 30 mL) and the combined organic extracts were washed successively with water (30 mL), 5% citric acid (30 mL), water (30 mL), saturated sodium bicarbonate (3 x 30 mL) and saturated sodium chloride (2 x 30 mL), dried over anhydrous magnesium sulfate, and evaporated. The crude product was distilled, to give a colourless oi1 (13.8 g, 76%), b.p. 1 19 - 123 O/ 1 torr. '~nmr(CDCl,, 6): 7.37 (SH, rn,

Ar), 5.21 (2H,s,PhC&), 4.32 (lH, q, 6.9 Hz,CH),2.38 (lH, br s, OH), 1.43 (3H,d, 6.9

Hz, CH3). ). 13cnrnr (CDCl3, 6): 175.52, 135.23, 128.66, 128.54, 128.22, 67.3 1, 66.84,

20.37. IR (neat): 3483, 1738 cm-'. Mass spectrum (CI, dz): 18 1 (M+l). ). Anal. Calcd. for CroHiz03:C 66.64; H 6.73. Found: C 66.30; H 6.75. Methyl v[N4l-Benzyioxycarbonyl-Ethyi)Aminooxyl-Butyrate (2-37)

A solution of 2,6-lutidine (0.35 mL, 0.32 g, 3.0 mrnol) in dichloromethane (3 mL) was added at -78 OC to a solution of benzyl @)-lactate (0.271 g, 1.5 mmol) in dichloromethane (15 mL). The solution was stirred for 10 min, trifluorometbanesulfonic anhydride (0.25 mL, 0.42 g, 1.5 mrnol) was added dropwise and stirring was continued for 30 min at -78 to -50 OC. A solution of methyl y-aminooxybutyrate (2-20) (0.170 g,

1.28 mrnol) and 2,6-lutidine (0.175 mL, 0.16 g, 1.5 mmol) in dichloromethane (6 mL) was then added dropwise at -78 OC. The reaction mixture was allowed to warm to room temperature, stirred for 18 h, and then washed with water (2 x10 mL). The organic phase was Mer washed with saturated sodium chloride (10 mL), dried over anhydrous magnesium sulfate and evaporated. Purification by flash chromatography on silica gel using a gradient system, hexanes to ethyl acetate, gave 1.63 g, (74%) of the product.

1Hnmr (CDC13, 8): 7.34 (5H, m, Ar), 5.19 (2H, s, PhCHz), 5.14 (IH,br d, ONH), 3.75

(lH, q, 7.0 Hz, CH), 3.69 (2H, t, 6.3 Hz, CH2y), 3.65 (3H, s, Oc&), 2.33 (2H, t, 7.4 Hz,

CHza), 1.87 (2H, m, CH$), 1.23 (3H, d, 7.0 Hz, CHCH3).13cnrnr (CDCb, 6): 174.1 1,

173.89, 135.67, 128.55, 128.28, 128.11, 73.01, 66.55, 58.88, 51.54, 30.68, 23.91, 14.75.

IR (neat): 3264, 174 1, 1736 cm-'. Mass spectrum (CI, dz): 296 (M+1). A solution of the benzyl ester (2-37) (1.63 g, 5.5 mmole) in toluene (80 mL) was cooled to O OC and a 2.0 M solution of trimethylalumhum in toluene (5.4 mL, 10.8 mol) was added dropwise. The reaction mixture was allowed to warm to room temperature, stined overnight, and then cooled to O OC, treated dropwise with methanol (16 mL) and stirred for 20 min. Water (10 mL) was added, the ice-bath was removed and stirring was continued for 15 min. The solution was concentrated to near-dryness, 1:4 dich1oromethane:tetrahydrofiiran (200 mL) was added and the mixture was filtered through Celite. The filtrate was evaporated to give the crude product, 1.19 g, together with some unreacted starting material. Purification by flash chromatography on silica gel using an ethyl acetate-hexanes gradient solvent system, 10% to 40%, gave a clear oil

(0.99 g, 84%). '~nmr(CDCI,, 6): 7.35 (SH,m, Ar), 5.24 (lH, q, 7.3 Hz, CH), 5.20 (1H, d, 13.0 Hz, PhCH), 5.14 (lH, d, 13.0 Hz, PhCH), 4.14 (lH, m, CHy), 3.98 (lH, m, Cm),

2.51 (2H, m, CH2@, 2.08 (ZH,m, CH$), 1.50 (3H, d, 7.3 Hz, CHCH3). 'bmr(CDC13,

6): 170.90, 170.13, 135.45, 128.53, 128.30, 128.07, 69.32, 67.09, 53.17, 28.32, 22.11,

13.76. IR (neat): 1 744, 1 660 cm-'. Mass spectrum (CI,mh): 264 (M+l). ~R-(~-OXO-[I,~JOxazinan-2-yl)-Propionie Acid (2-40)

To ethyl acetate (1 5 mL) was added 1OYO palladium on activated carbon (83 .O mg, 0.078 mol) and the suspension was stirred in an atmosphere of hydrogen for 1 h. The benzyl ester (2-39) (0.206 g, 0.78 mol) in ethyl acetate (15 mL) was then added and stimog was continued for 24 h. The mixture was fiitered through Celite, which was washed with ethyl acetate (2 x 4 mL), and the combined filtrates were evaporated to give a white solid

(0.13 g, 99%). 1Hnmr (CDC13, 6): 5.22 (lH, q, 7.3 Hz, CH), 4.23 (lH, m, CHy), 4.05

(1 H, m, CHy), 2.56 (ZH,m, CH2a), 2.13 (2H, m, CH$), 1-52 (3H, d, 7.3 Hz, CHCH3).

"cnrnr (CDC13, 6): 173.78, 171.18, 69.61, 53.19, 28.26, 22.09, 13.58. IR (KBr): 1744,

1621 cm-'. Mass spectnim (CI, dz): 174 (M+l). Anal. Calcd. for CIH~~NO~:C 49.54;

H 6.61; N 8.09. Found: C 49.36; H 6.52; N 7.86.

Methyl y[N-(1-Benzyloxycarbonyl-Ethyl) Aminooxyj-2-Phenylacetylamino

Butyrate (2-41)

A solution of 2,6-lutidine (0.60 mL, 0.54 g, 5.0 mmol) in dichloromethane (3 mL) was added, with stirring, to a cooled (-78 OC) solution of benzyl (S)-lactate (0.450 g, 2.5 mmol) in dichioromethane (15 rnL). Mer 10 min, trifluoromethanesulfonic anhydride

(0.42 mL, 0.7 1 g, 2.5 mmol) was added dropwise and stirring was continued at -78 to -

50 OC for an additional 30 min. A solution of methyl 2-phenylacetylamino4 aminooxybutyrate (2-23) (0.678 g, 2.5 mmol) and 2,6-lutidine (0-30 mL, 0.27 g, 2.5 mmol) in dichloromethane (15 mL) was then added dropwise. When the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred for

18 h. Water (2 x10 mL) was added and the separated organic phase was washed with saturated sodium chloride (10 mL), dried over anhydrous magnes& sulfate and evaporated. The residue was purified by flash chromatography on silica gel using a gradient system, hexanes to ethyl acetate, to give an oil (0.304 g, 29%). 4-

Phenylacetylamino-[1,2]oxazinan-3-one(2-22, Ri = PhOCH*) was also found as a side product (0.136 g, 23%). '~nmr(CDCb, 6): 7.34 (lOH, m, PhCHtCO, PhCH20, both isomers), 6.35 (IH, d, 7.9 Hz, CONH, one isomer, exchanges in CD30D), 6.32 (lH, d,

7.5 Hz, CONH, second isomer, exchanges in CD30D), 5.77 (1H, d, 9.5 Hz, ONH, one isomer, exchanges in CD30D), 5.74 (IH, d, 8.9 Hz, ONH, second isomer, exchanges in

CD30D), 5.16 (2H, d, 4.1 Hz, PhCH20, one isomer), 5.15 (2H, d, 4.1 Hz, PhCH20, second isomer), 4.62 (lH, m, CH& both isomers), 3.69 (2H, t, 6.3 Hz, Gay, both isomers), 3.68 (3H, s, 0CH3, one isomer), 3.67 (3H, s, OC&, second isomer), 3.68-3.66

(IH, m, CHCH3, both isomers), 3.59 (2H, rn, PhCaCo, both isomers), 2.01 (2H, m,

CH$, both isomers), 1.19 (3H, d, 7.2 Hz, CHCH3, one isomer) 1.18 (3H, d, 7.2 Hz,

CHCH3, second isomer). I3cnrnr (CDCI,, 6): 173.56, 172.32, 170.82, 135.55, 134.74,

129.37, 128.90, 128.87, 128.59, 128.21, S27.25, 70.30 (PhCH20, one isomer), 70.09

(PhCH20, second isomer), 66.76 (CH2y, both isomers), 58.63 (CHCH3, one isomer), 58.57 (CHCH3, second isomer), 52.36 (PhCH$X, both isomers), 50.23 (OC&, both isomers), 43.50 (CHa, both isomers), 30.45 (CH$, both isomers), 14.72 (CHCH3, one isomer), 14.62 (CHCH3, second isomer). IR (CHîC13: 3286, 1742, 1660 cd' Mass spectrum (CI, mh): 429 (M+l). Anal. Calcd. for CuH2&Oa'0.75Hz0: C 62.51; H

6.68; N 6.34. Found: C 62.36; H 6.35; N 6.38.

Benzyl Ester of 2R-(3-0x0- 4-Phenylacetylamino-[1,2]0xazinan3-yl)-Propionic

Acid (2-42)

A 2.0 M solution of trimethyldufninum in hexanes (0.20 mL, 0.4 mmol) was added dropwise, at O OC, to a solution of (2-41) (42.0 mg, 0.10 mmol) in toluene (10 mL). The cooling bath was removed and the reaction mixture was stimed overnight. Water (1 mL) was then added slowly and stirring was continued for 15 min. The solvent was evaporated to near-dryness under reduced pressure, 1:4 dich1oromethane:tetrahydrohan

(100 mL) was added, and the mixture wâs filtered through Celite. The filtrate was evaporated to give an oil (0.0 18 g). Purification by short column chromatography, using a hexanes to ethyl acetate gradient solvent system, gave 3.9 mg of one isomer as a white solid, m-p. 106 - 107 OC. '~nmr(CD30D, 6): 7.31 (10H, m, PhCH2C0, PhCH20 ), 6.44

(IH, d, 5.6 Hz, CONH, 5.14 (2H, q, 8.8 Hz, PhCH20), 5.10 (IH, q, 7.2 Hz, CHCH3),

4.66 (lH, m, CHa),4.22 (lH, m, CNy), 4.06 (lH, m, Cm), 3.61 (2H,s, PhCfi), 2.85

(1H, m, CW), 1.60 (lH, m, CE@), 1.48 (3H, d, 7.3 Hz, CH3). 13cnrnr (CD30D, 6): 3.2 BioAssays

3.2.1 Materials

Agar Medium f 1%w/v)

Yeast extract (1.5 g), peptone (2.5 g), glucose (0.5 g) and agar (5.0 g) were dissolved in deionized water, the volume was diluted to 500 mL and the solution was stenlized by autoclaving at 121 OC for 20 min.

Pre~arationof Agar Plates

Under sterile conditions, the agar medium (150 mL) was heated to 100 OC, cooled to 50

OC and distributed into ten petri dishes. Once the medium had hardened, the plates were inverted and stored at 4 OC.

Liauid Medium

Yeast extract (0.3 g), peptone (0.5 g) and glu cose (0.1 g) were dissolved in deioniz water, the volume was diluted to 100 mL and the solution was sterilized by autoclaving at

121 OC for 20 min.

3.22 Methods

Ste~l

Under sterile conditions, fieeze-dried Micrococcus Zuteus was dissolved in liquid medium

(10 rnL) and incubated at 250 rpm for 12 h at 30 OC. Ste~2

Liquid bacterial culture fiom step 1 (0.5 mL) and 40 % glycerol in deionized water (0.5

mL) were vortexed under sterile conditions, in a tube fitted with a screw cap and an air-

tight gasket. The resulting 20% glycerol bacterial stock solution was stored at -80 OC.

Ster, 3

An inoculating needle was dipped into 20 % glycerol bacterial stock solution fiom step 2 and, under sterile conditions, streaked onto the surface of an agar plate. The inverted plate was incubated at 30 OC for 12 h and then stored at 4 OC.

Step 4

Under sterile conditions, a bacterial colony of Micrococnrs luteus was lifted fkom the surface of the agar plate fiom step 3 using a toothpick, added to liquid medium (10 mL), and incubated at 250 rpm for 12 h at 30 OC.

Step 5

Under sterile conditions, the agar medium (1 50 mL) was heated to 100 OC, cooled to 50

OC, liquid bacterial culture from step 4 (4 mL) was added with swirling, and the medium was distributed into ten petri dishes. Once the medium had hardened, the plates were inverted and stored at 4 OC.

Ste~6

A sterile filter disc was treated with an aliquot of a known concentration of the test compound and the solvent was removed by air drying under sterile conditions. This disc, a solvent control disc and a disc containing a known weight of a penicillin or cephalosporin, were placed on an agar plate seeded with Mirnococcus Zuteus fkom step 5.

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