5-Hydroxy[1,2]oxazinan-3-ones as potential carbapenem and D-ala-D-ala surrogates

Article abstract of DOI:10.1139/v03-123

The title compounds are amino acids whose nitrogen atom is enclosed in a six-membered cyclic hydroxamate bearing a C5-hydroxyl group. They belong to a proposed new family of antibacterial agents targeted to the penicillin receptor. The glycine and alanine members of the family have been synthesized, as racemates, in seven steps from the four-carbon synthon diketene and the tert-butyl esters of N-hydroxyglycine and N-hydroxyalanine. Numerous alternatives to diketene have also been examined, but these lead mainly to five-membered cyclic hydroxamates. The theoretical considerations that have led to this synthetic programme are discussed in some detail. They include analysis of the structures of natural and unnatural penicillin surrogates, analysis of the penicillin pharmacophore, and a treatment of the chemical reaction with which penicillin blocks bacterial cell wall synthesis. The glycine derivative exhibits marginal but real activity vs. Micrococcus luteus. The alanine derivative, which more closely resembles D-ala-D-ala, is fifty times more active. Two five-membered structural isomers of the glycine derivative are inactive.

Full text of DOI:10.1139/v03-123

915
5-Hydroxy[1,2]oxazinan-3-ones as potential
carbapenem and D-ala-D-ala surrogates
Saul Wolfe, Christiana Akuche, Stephen Ro, Marie-Claire Wilson,
Chan-Kyung Kim, and Zheng Shi
Abstract: The title compounds are amino acids whose nitrogen atom is enclosed in a six-membered cyclic hydrox-
amate bearing a C5-hydroxyl group. They belong to a proposed new family of antibacterial agents targeted to the peni-
cillin receptor. The glycine and alanine members of the family have been synthesized, as racemates, in seven steps
from the four-carbon synthon diketene and the tert-butyl esters of N-hydroxyglycine and N-hydroxyalanine. Numerous
alternatives to diketene have also been examined, but these lead mainly to five-membered cyclic hydroxamates. The
theoretical considerations that have led to this synthetic programme are discussed in some detail. They include analysis
of the structures of natural and unnatural penicillin surrogates, analysis of the penicillin pharmacophore, and a treat-
ment of the chemical reaction with which penicillin blocks bacterial cell wall synthesis. The glycine derivative exhibits
marginal but real activity vs. Micrococcus luteus. The alanine derivative, which more closely resembles D-ala-D-ala, is
fifty times more active. Two five-membered structural isomers of the glycine derivative are inactive.
Key words: cyclocanaline, cycloserine, lactivicin, oxamazins, peptidoglycan, penicillin-binding proteins.
Résumé : Les composés mentionnés dans le titre sont des acides aminés dont l’atome d’azote est compris dans un hy-
droxamate cyclique à six chaînons portant un groupe hydroxyle en position 5. Ils font partie de ce qui est proposé
comme une nouvelle famille d’agents antibactériens orientés vers les récepteurs de la pénicilline. On a synthétise les
membres glycine et alanine de la famille, sous la forme de racémiques, en sept étapes à partir du dicétène, un synthon
à quatre atomes de carbone, et des esters t-butyliques de la N-hydroxyglycine et de la N-hydroxyalanine. On a examiné
la possibilité d’utiliser plusieurs autres composés à la place du dicétène, mais dans chacun des cas ces dérivés de rem-
placement ont conduit principalement à la formation d’hydroxamates cycliques à cinq chaînons. On discute en détail
des considérations théoriques qui ont conduit à ce programme de synthèse. Elles comprennent l’analyse des structures
de substituts naturels et non naturels à la pénicilline, l’analyse du pharmacophore de la pénicilline et à un traitement de
la réaction chimique par laquelle la pénicilline bloque la synthèse de la membrane de la cellule des bactéries. Le dérivé
de la glycine présente une activité faible, mais réelle vis-à-vis du Micrococcus luteus. Le dérivé de l’alanine, qui res-
semble plus au D-ala-D-ala, est cinquante fois plus actif. Deux isomères de structure à cinq chaînons du dérivé de la
glycine sont inactifs.
Mots clés : cyclocanaline, cyclosérine, lactivicine, oxamazines, peptidoglycane, protéines se liant à la pénicilline.
[Traduit par la Rédaction] Wolfe et al. 936
Introduction
isoxazolidinone ring (a cyclic hydroxamate), they have dif-
ferent configurations at C4 and they operate at different
stages of cell wall synthesis. D-Cycloserine, a cyclic analog
of D-alanine (3), is a competitive inhibitor of both alanine
racemase (3) and D-alanyl-D-alanine synthetase (4). Lacti-
vicin has the L-configuration at C4 and a 2-oxoglutarate ap-
pendage attached to the ring nitrogen. Because it exhibits
affinity to penicillin-binding proteins and susceptibility to β-
Cycloserine (Oxamycin, Seromycin, 1), produced by
Streptomyces orchidaceus (1), and lactivicin (2), isolated
from culture filtrates of Empedobacter lactagenus YK-258
and Lysobacter albus YK-422 (2), are antibiotics whose ac-
tion is directed against the biosynthesis of the bacterial cell
wall peptidoglycan. Although both antibiotics contain an
Received 19 November 2002. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 15 August 2003.
This paper is dedicated to the memory of Raymond Lemieux (1920–2000), lifelong mentor and friend. … “wherefore I say that
your chief aim in the building up of Man is not to give him learning, which serves no end if he be but a walking book, but to lift
his eyes and point him to those higher levels on which there are no more things-in-themselves, but only aspects of that divine bond
which binds things together” (Saint-Exupéry).
S. Wolfe,1 C. Akuche, S. Ro, M.-C. Wilson, C.-K. Kim, and Z. Shi. Department of Chemistry, Simon Fraser University,
Burnaby, BC V5A 1S6, Canada.
1Corresponding author (e-mail: swolfe@sfu.ca).
Can. J. Chem. 81: 915–936 (2003)
doi: 10.1139/V03-123
© 2003 NRC Canada

916
Can. J. Chem. Vol. 81, 2003
lactamases (2, 5), lactivicin can be regarded as a structural
analog of penicillin (4), which is, in turn, a structural analog
of the N-acyl-D-ala-D-ala terminus (5) of the growing
peptidoglycan (6). The isoxazolidin-3-one ring of lactivicin
thus appears to function as a β-lactam surrogate, and the 5-
oxo-tetrahydrofuran-2-carboxylic acid substituent appears to
function as a D-alanine surrogate.
+
H
NH3
H
CH3CONH
O
O
N
H
O
N
O
HO2C
2
O
O
H
1
droxylamine (17) on appropriately protected and activated
homoserine synthons.
Khomutov and co-workers have reported that DL-
+
H
NH3
H
H
H
H
O
3
O-
H
H
N
CO2H
CH3
O
O
CO2H
N
CH3
H
CH3
CH3
S
N
H
N
H
H
O
R
O
R
5
4
The 6-membered homolog of cycloserine is cyclocanaline
(6), the cyclodehydration product of canaline (7). L-
Canaline, the 5-oxa isostere of ornithine, is the only naturally
occurring amino acid that possesses an aminooxy moiety.
The compound is found in leguminous plants and is derived
from L-canavanine (8), the 5-oxa isostere of L-arginine, by
arginase-mediated hydrolysis (7). Canavanine is an impor-
tant 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 free amino acids (8). The free aminooxy group
of L-canaline forms Schiff base adducts with α-keto acids (9)
and with the pyridoxal phosphate (B6) cofactor of many B6-
dependent enzymes (10). These properties endow canaline
with antimetabolite (11), anticancer (11), and antimalarial
activities (12).
The first syntheses of [1,2]oxazinan-3-one (9) and cyclo-
canaline were accomplished by cyclization of 4-aminooxy-
butyric esters under forcing basic or acidic conditions, i.e.,
N2—C3 bond formation (13).
Attempts to secure these compounds by O1–C6 cycli-
zation of halohydroxamic acids (10→11) were frustrated by
competing N2—C6 bond formation (10→12) (14). The in-
troduction of the aminooxy substituent into canaline has
been achieved using ethyl N-hydroxyacetimidate (15), ace-
tone oxime (13a), hydroxyurethane (16), or N-benzoylhy-
cyclocanaline has activity against Mycobacterium tuberculo-
sis (18, 19).
The parent 5-membered and 6-membered cyclic hydroxa-
mates are structural isomers of the N-hydroxylactams 13 and
14, and rearrangement of an N-hydroxy-2-azetidinone into a
5-membered cyclic hydroxamate has been observed by
Hirose et al. (20a) and by Miller and co-workers (20b), who
have published extensively on oxamazins (15), heteroatom-
activated β-lactams (21). Monocyclic oxamazins exhibit sig-
nificant antibacterial activity vs. Gram negative bacteria.
Their mechanism of action has not been established with
certainty,2 but it is noteworthy that the carboxyl group of 15
is one carbon further removed from the β-lactam nitrogen
than in β-lactam antibiotics. In the case of the bicyclic
[4.2.0] oxamazins 16, both diastereomers at the point of at-
tachment of the carboxyl group are claimed to have good an-
tibacterial activity (22). In contrast, thiamazins (17) are
inactive (23).
Boyd, Miller, and co-workers (24) have discussed the dif-
ference in the activities of oxamazins and thiamazins in
terms of their putative three-dimensional relationship to the
penicillin nucleus, i.e., their ability to “fit” into the active
sites of the serine peptidase receptors of β-lactam antibiotics.
The chemical reaction that follows — namely, acylation of
the serine hydroxyl group by the ␤-lactam ring — has been
2 Personal communication from Professor M.J. Miller.
© 2003 NRC Canada

Wolfe et al.
917
Fig. 1. Methanolysis of penam, with catalysis by one water molecule. Left: 3–21G* reactant complex. Right: 3–21G* transition struc-
ture. Graphics performed using Xmol.
3.82
1.91
1.65
1.33
1.16
0.98
1.14
0.98
1.70
1.25
Table 1. Calculated “barriers”a and nitrogen pyramidalityb in var-
ious azetidinones.
Azetidinone
“Barrier” (kcal/mol)a
Pyramidalityb
Penam
Penem
29.4
28.0
30.7
29.6
336.1
333.9
360.0
337.5
357.0
348.6
352.4
N-methylazetidinone
N-methoxyazetidinone
N-methylthioazetidinone 34.2
Bicyclic oxamazin
Bicyclic thiamazin
32.5
34.2
aSee text for definition of “barrier”.
bSum of the angles at nitrogen (3–21G or 3–21G* calculations; the 3–
21G* basis set is used with sulfur-containing compounds).
It can be seen that N-methoxyazetidinone is comparable in
“reactivity” to the bicyclic penam and is substantially more
“reactive” than N-methylthioazetidinone. In addition, unlike
compounds in which carbon or sulfur is attached to the β-
lactam nitrogen, the monocyclic N-methoxyazetidinone is
substantially more “reactive” than the bicyclic structure.
These trends can be understood by consideration of the
pyramidality of the β-lactam nitrogen, as given by the sum
of the angles at this center. This sum is included in Table 1.
The geometry at nitrogen has received continuous attention
(30) since Woodward first proposed (31) that the O=C-N↔
(–)O-C=N(+) amide resonance, which contributes to the sta-
bility and planarity of an amide linkage, is suppressed in a
four-membered ring and cannot be achieved within the bi-
cyclic penam structure. An equivalent, molecular orbital in-
the subject of extensive theoretical research (25). In our lab-
oratory (26) the relative “reactivities” of different substrates
are calculated, as shown in Fig. 1, for penam, the penicillin
nucleus. The reaction is modeled theoretically as a one-step,
cooperative process (27), in which O=C···O bond formation
by methanol and N···H bond formation by a catalytic water
molecule are concerted with the cleavage of the β-lactam
bond. The structure on the left of Fig. 1 is the reactant com-
plex, optimized at the 3–21G* level (28); the optimized tran-
sition structure is shown on the right. The barrier, based on
single-point MP2/6–31G* calculations, is 29.36 kcal/mol.
This number is shown in Table 1, together with the barriers
calculated similarly for the water-assisted methanolyses of
penem, N-methylazetidinone, and mono- and bicyclic
oxamazins and thiamazins. In the latter two systems, there is
more than one conformation of the 6-membered ring; based
on a recent quantitative treatment of the Curtin–Hammett
principle (29), the number shown in the table refers to the
energy difference between the most stable reactant complex
and the most stable transition structure.
*
terpretation (32) is that np–π CO charge transfer in an amide
is most effective when the nitrogen p-orbital can conjugate
with the π-system, i.e., when the nitrogen is planar. This is
not possible when the β-lactam nitrogen is at a bridgehead
position. In N-methoxy- and N-methylthioazetidinone, mini-
mization of lone-pair – lone-pair repulsion between the p-or-
bital on nitrogen and the p-orbital on oxygen or sulfur (33)
causes the methyl group to be perpendicular to the ring, so
that the nitrogen p-orbital and the X—C bond are eclipsed.
*
If the resulting np–σ XC interaction competes with the np–
*
π CO interaction, the combined effects will produce non-
planarity at nitrogen, as is seen in N-methoxyazetidinone.
© 2003 NRC Canada

918
Can. J. Chem. Vol. 81, 2003
Fig. 2. A plot of the MP2/6–31G*//3–21G(G*) “barriers” vs. the sum of the angles at nitrogen of some β-lactam compounds (from
Table 1).
MP2/6-31G*//HF/3-21G
35
34
33
32
31
30
29
28
330
334
338
342
346
350
354
358
Sum of angles at nitrogen (°)
*
The magnitude of the np–σ XC interaction depends in-
20–22 might be expected to behave as penicillin–lactivicin–
N-acyl-D-ala-D-ala analogs and exhibit antibacterial activity.
This proposal is not new. Phenylacetylation of L- and D-
cycloserine, followed by coupling to tert-butyl (DL)-2-
bromopropionate, separation of isomers, and deprotection,
*
versely upon the energy difference between np and σ XC and
also upon the square of the overlap between the interacting
orbitals (34). Although the sulfur σ* acceptor orbital is
*
*
lower-lying than the oxygen σ acceptor orbital, the np–σ SC
interaction is not effective because of the longer nitrogen—
sulfur bond (33). Consequently, the monocyclic and bicyclic
thiamazins exhibit similar “barriers” and near planarity at ni-
trogen. In the case of the oxamazins, closing the second ring
*
increases the dihedral angle between np and σ OC and de-
*
creases the contribution of the np–σ OC interaction, resulting
in increased planarity at nitrogen and an increased “barrier”.
As seen in Fig. 2, there is a rough linear relationship
(R2 = 0.81) between the calculated sum of the angles at ni-
trogen and the calculated “barriers”.
Cyclic hydroxamates as β-lactam surrogates
The foregoing considerations suggested that the cyclic
hydroxamate 2-methyl-isoxazolidin-3-one (18), identified
earlier as a β-lactam surrogate from its presence in lactivicin,
would exhibit somewhat greater planarity at nitrogen and a
somewhat higher “barrier” than its isomer N-methoxyaze-
tidinone. The calculations on 18 and its homolog 2-methyl-
[1,2]-oxazinan-3-one (19), which are summarized in Fig. 3,
are consistent with this prediction. Nonetheless, both 18 and
19 are found to have significant “reactivity”, intermediate
between penam and N-methylazetidinone, so that 2-sub-
stituted 4-acylaminocycloserines and cyclocanalines such as
© 2003 NRC Canada

Wolfe et al.
919
Fig. 3. Left: 3–21G optimized structures and the sum of the angles at nitrogen in 18 and 19. Centre: 3–21G reactant complexes for the
methanolyses of 18 and 19, with catalysis by one water molecule. Right: 3–21G transition structures. The “barriers” are based on sin-
gle point MP2/6–31G* calculations.
afforded all four stereoisomers of 20 (R = phenylacetyl, R′ =
OH
H
H
N
Me) (35). However, none of these compounds exhibited ac-
tivity vs. Staphylococcus aureus or Escherichia coli. Like-
wise, phenoxyacetylation of L-cycloserine, followed by
coupling to allyl 2-chloro-2-ethoxyacetate, deprotection, and
separation of isomers yielded 20 (R = phenoxyacetyl, R′ =
OEt) (36), which were inactive against S. aureus and E. coli
at concentrations up to 1000 µg/mL. Although 21 has been
claimed to have antibacterial activity (37), this claim cannot
be confirmed (38); 22 is also inactive (38).
RCONH
O
S
SR
R1
N
CO2H
O
CO2H
23
24
HO
O
O
N
The penicillin pharmacophore consists of a reactive β-
lactam ring with an ionizable acidic group located near the
azetidinone nitrogen (24, 39, 40) and, unless the β-lactam
ring is exceptionally reactive (41), a hydrogen-bond donor
located near the azetidinone carbonyl group (42). In penicil-
lins and cephalosporins (23), the hydrogen-bond donor is the
N–H of the acylamino side chain, geminal to the carbonyl
carbon. In the carbapenems (24) (43), the hydrogen-bond
donor is the exocyclic hydroxyl group of the side chain, two
atoms removed from the carbonyl carbon. A possible five-
membered cyclic hydroxamate surrogate of a carbapenem
could have an exocyclic beta hydroxyl group, as in 25, or an
endocyclic beta hydroxyl group, as in the hemiacetal 26. The
corresponding six-membered cyclic hydroxamates are 27
and 28.
N
CO2H
CO2H
OH
O
R
O
R
25
26
H
HO
O
O
CO2H
H
CO2H
N
N
R
O
OH
O
R
27
28
Figure 4 shows the PM3-optimized (44) global minimum
of penicillin G and a PM3-optimized conformation of the β-
lactam and carbapenem surrogate 2R-(5R-hydroxy-3-oxo-
[1,2]oxazinan-2-yl)-propionic acid (29, R = Me). This com-
pound is an amino acid (D-alanine) whose nitrogen atom is
enclosed in the [1,2]oxazinan-3-one ring. Despite the (as yet
unexplained) absence of activity in 16–18, we decided to
pursue syntheses related to 29. This paper reports non-
stereospecific routes to 29, R = H (glycine) and R = Me
(alanine), and evidence of antibacterial activity in these
compounds, with significantly greater activity in the alanine
derivative.
Results and discussion
Syntheses in the glycine series
The O-benzyl ethyl ester of N-hydroxyglycine (30, R1 =
Et, R2 = Bn) was synthesized by reaction of O-benzylhy-
© 2003 NRC Canada

920
Can. J. Chem. Vol. 81, 2003
Fig. 4. Left: Carboxyl carbon to side chain nitrogen and carboxyl carbon to β-lactam oxygen distances in PM3-optimized penicillin G.
Right: Carboxyl carbon to side chain oxygen and carboxyl carbon to C3 carbonyl oxygen distances in one of the conformations of 2R-
(5R-hydroxy-3-oxo-[1,2]oxazinan-2-yl)-propionic acid.
5.68
4.47
5.90
4.34
reaction with bromoacetic acid in ethanol, esterification with
benzyl bromide in dimethylformamide, and treatment with
hydroxylamine. The tert-butyl ester (30, R1= t-Bu, R2 = H)
was obtained by sequential treatment of the sodium salt of
34 with tert-butyl bromoacetate in 2-propanol and hydroxy-
lamine.
HO
OR2
H
N
O
N
CO2H
OR1
O
R
O
29
30
Vinylacetic acid as a synthon
droxylamine hydrochloride with glyoxylic acid in refluxing
ethanol (45) and reduction of the oxime with pyridine–
borane (46).
Dicyclohexylcarbodiimide coupling of vinylacetic acid
with 30 (R1 = Et, R2 = H) gave 36, which, like 32, exhibited
infrared absorption at 1625 cm–1. Bromination of 36 in chlo-
roform at room temperature afforded a dibromide, which,
upon exposure to sodium bicarbonate, yielded a colourless
oil whose elemental analysis and mass, infrared, and proton
NMR spectra were consistent with the isoxazolidinone struc-
ture 37, X = Br. The infrared absorptions at 1710 and
1755 cm–1 were assigned to the carbonyl groups of the ester
and isoxazolidinone ring. The proton NMR spectrum
showed a downfield shift of the tertiary hydrogen from
4.55 ppm in the dibromide to 4.80 ppm in 37. The EI mass
spectrum exhibited peaks at m/z 85, 93, and 95, which were
assigned to 38 and the bromomethyl side chain of 37. The
presence of a peak at m/z 85 was used subsequently to rec-
ognize the presence of a 5-substituted isoxazolidinone ring.
The bromide 37 (X = Br) was stable to the action of silver
acetate in refluxing acetic acid, sodium formate in dimethyl-
formamide (51), potassium phthalimide in refluxing toluene
(52), sodium azide and tetrabutylammonium bromide in
refluxing benzene (53), and triethylamine and formic acid in
acetonitrile (54). The preference for cyclization to a five-
membered ring (55) and the stability of the Br-C-C-ON moi-
ety to nucleophilic displacement (56) suggested a synthesis
of 39, a structural isomer of the target compound 29 (R =
H). Coupling of 30, R1 = Bn, R2 = H, with vinylacetic acid
and iodination with iodine and sodium bicarbonate in
acetonitrile (57) gave 37 (X = I) as the benzyl ester. Peracid
oxidation (58), followed by hydrogenolysis, yielded 39.
Reaction of the alkene 36 with N-bromosuccinimide or N-
bromoacetamide in methanol, followed by a bicarbonate
wash, afforded three products, the bromide 37 and a 1:1
mixture of 40 and 41. The GC–MS of this mixture exhibited
Benzoylation yielded 31, which was debenzylated with
aluminium chloride and anisole in nitromethane (47). Since
the same compound (32) was obtained by direct benzoy-
lation of N-hydroxyglycine ethyl ester (30, R1 = Et, R2 = H),
it was concluded that this ester undergoes N- and not O-
acylation. This permitted an approach to 29, R = H, based
on N-hydroxyglycine or one of its esters and a four-carbon
synthon. A large number of these were examined, including
vinylacetic acid, cyclic anhydrides, 3,4-dihydroxybutanoic
acid, γ-butyrolactone, diketene, and the diketene–acetone
adduct 2,2,6-trimethyl-4H-1,3-dioxin-4-one (33).
Scheme 1 (48–50) summarizes the syntheses of the ethyl,
benzyl, and tert-butyl esters of N-hydroxyglycine (30, R1 =
Et, Bn, t-Bu, R2 = H). E-Benzaldoxime was isomerized to
the Z-isomer 34 with hydrogen chloride in refluxing toluene.
Reaction of the sodium salt with ethyl bromoacetate in etha-
nol afforded N-benzylideneglycine N-oxide ethyl ester 35,
and 30 (R1= Et, R2 = H) was obtained in 80% yield when 35
was treated with hydroxylamine. The benzyl ester (30, R1 =
Bn, R2 = H) was synthesized from the sodium salt of 34 by
© 2003 NRC Canada

Wolfe et al.
921
Scheme 1. Reagents and conditions: (a) HCl, toluene; then NaOH, NH4Cl, Et2O; (b) ROH, Na; then BrCH2CO2Et; (c) NH2OH;
(d) BrCH2CO2H; (e) BnBr, Et3N, DMF.
peaks at 8.07 min, with an EI-MS fragmentation at m/z 99,
assigned to 42, and at 8.43 min, with the EI-MS fragmenta-
tion at m/z 85 previously assigned to 38. The H NMR spec-
tra of 40 and 41 and related compounds are summarized in
Tables 2 and 3.
3,4-Dihydroxybutanoic acid as a synthon
Reaction of methyl 3,4-dihydroxybutanoate (46) (60, 61)
with benzaldehyde dimethyl acetal and a catalytic amount of
p-toluenesulfonic acid monohydrate in dimethylformamide
(62), followed by alkaline hydrolysis, yielded the acid 47.
This was coupled to the tert-butyl ester of N-hydroxyglycine
to give 48, and hydrogenolysis yielded 49. The reaction of
49 with triphenylphosphine and diethyl azodicarboxylate in
refluxing tetrahydrofuran (63) gave the isoxazolidinone 50.
Deprotection with trifluoroacetic acid produced the
hydroxyacid 39. Alternative cyclization – Mitsunobu condi-
tions (63) did not give promising results.
1
Cyclic anhydrides as synthons
Following Procter et al. (59), reaction of the benzyl ester
30 (R1 = Bn, R2 = H) with a cyclic anhydride, followed by
cyclization with carbodiimide, afforded the 3,6-diones 43–45
from, respectively, succinic anhydride, acetoxysuccinic an-
hydride, and maleic anhydride.
OH
N
X
O
N
MeO
MeO
O
N
O
N
CO2Et
CO2Et
CO2Et
CO2Et
O
O
36
O
O
37
40
41
.
.
+
+
O
N
HO
O
N
O
N
CO2H
H
H
O
O
O
38
42
39
© 2003 NRC Canada

922
Can. J. Chem. Vol. 81, 2003
O
O
AcO
O
N
O
N
CO2Bn
CO2Bn
O
O
44
43
O
O
N
CO2Bn
O
45
Ph
O
O
OH
O
O
HO
OH
OMe
47
46
Ph
O
O
O
t-
N
CO2 Bu
OH
48
O
HO
HO
t-
N
CO2 Bu
OH
49
HO
O
N
t-
CO2 Bu
O
50
Butyrolactone as a synthon
Attempts to prepare compounds of type 51 by a direct re-
action of N-hydroxyglycine esters with butyrolactone, using
trimethylaluminium (64) or aluminium trichloride (65) as
catalysts, were not successful. Therefore, using the method
of Larcheveque and Henrot (66), butyrolactone was con-
verted to the iodo acid 51, X = I, with trimethylsilyl iodide
© 2003 NRC Canada

Wolfe et al.
923
1
Table 3. H NMR spectra of 1,2-oxazin-3-ones.
R = OMea; R′ = Me
R = OMea; R′ = Et
R = OMea; R′ = t-Bu
R = OHb; R′ = Na
δ (ppm)
J (Hz)
δ (ppm)
J (Hz)
δ (ppm)
J (Hz)
δ (ppm)
J (Hz)
Ha
Hb
Hc
Hd
3.99
4.51
4.22
4.32
3.99
4.51
4.23
4.33
4.01
4.43
4.13
4.34
4.49
4.28
4.09
4.29
17.8
17.8
11.9
17.8
17.8
11.9
17.7
17.7
11.9
17.4
17.4
12.0
6.0
6.0
6.0
4.6
He
Hf
Hg
4.07
2.79
2.67
11.9
4.07
2.80
2.68
11.9
4.07
2.90
2.75
11.9
4.02
2.92
2.52
12.0
3.2
16.0
3.2
16.0
3.6
15.6
3.2
16.8
5.8
16.0
5.8
16.0
5.9
15.6
5.9
16.8
4.7
4.7
4.8
3.2
aIn CDCl3.
bIn D2O.
in refluxing dichloromethane, and this acid was coupled to
the tert-butyl ester of N-hydroxyglycine to give 52. Cycli-
zation to 53 was accomplished using 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU).
1
Table 4. H NMR spectra of 5-hydroxy-5-methylisoxazolidinones.
56, R = Ha
54, R = Etb
58, R = Bnb
δ (ppm) J (Hz) δ (ppm) J (Hz) δ (ppm) J (Hz)
X
Hb
Hc
Hd
He
4.39
4.20
3.17
2.77
17.7
17.7
17.2
17.2
4.51
4.12
3.02
2.76
18.2
18.2
16.8
16.8
4.53
4.19
3.00
2.76
18.1
18.1
16.8
16.8
X
OH
N
t-
CO2 Bu
OH
aIn D2O.
bIn CDCl3.
O
O
51
52
The preparation of the acid 56 (which, like 39, is a struc-
tural isomer of the target compound 29, R = H) was accom-
plished using diketene in place of the diketene–acetone
adduct (68). Reaction with the benzyl ester of N-
hydroxyglycine afforded a product, which, in chloroform-d,
exists as an 83:17 tautomeric mixture of benzyl esters 58
and 59. Hydrogenolysis produced 56 and 57 as a 2:1 tauto-
O
N
1
meric mixture in water solvent. Table 4 summarizes the H
NMR spectra of 54, 56, and 58.
t-
CO2 Bu
O
53
2,2,6-Trimethyl-4H-1,3-dioxin-4-one and diketene as
synthons
The reaction of the O-benzyl ethyl ester 30 (R1 = Et, R2 =
Bn) with the diketene–acetone adduct 33 in refluxing tolu-
ene (67), followed by hydrogenolysis, yielded the hemiketal
54, which, in chloroform-d, exists as an 85:15 tautomeric
mixture of esters 54 and 55.
OH
O
OH
N
O
N
CO2Et
CO2Et
The synthesis of 29, R = H, is summarized in Scheme 2.
Bromination of diketene at –25 °C in the presence of benzyl
alcohol afforded benzyl bromoacetoacetate 60 in quantitative
yield via the acyl bromide. Reduction with sodium boro-
O
O
55
54
© 2003 NRC Canada

924
Can. J. Chem. Vol. 81, 2003
diastereomers 67 in 54% yield and also, in contrast to the
observations made previously with N-hydroxyglycine esters,
the O-acylated compounds 68. Cyclization of 67 using DBU
yielded the oxazinone 69 in 65% yield. Deprotection with
trifluoroacetic acid or formic acid, followed by extraction
with sodium bicarbonate and lyophilization produced the so-
dium salt as a mixture of diastereomers.
Bioassay results
Bioassays were performed on agar plates seeded with
M. luteus (see Experimental). Figure 5 shows the zones of
inhibition obtained with 29, R = H, and its two structural
isomers 39 and 56 compared with the results for penicillin G
and desacetoxycephalosporin G. It can be seen that the
racemate 29 exhibits some activity but 39 and 56 are inac-
tive at 1000 µg. Table 5 summarizes semiquantitative test
data relative to penicillin G and desacetoxycephalosporin G,
expressed as (diameter of the zone in mm)/(µM of test mate-
rial) for 29, R = H, and 29, R = Me. Although both com-
pounds are only weakly active, the alanine surrogate, as a
mixture of stereoisomers, is ca. 50 times more active than
the glycine surrogate. These findings have encouraged us to
continue with this programme and to attempt to devise
stereospecific routes to compounds having the general struc-
ture 70. These studies are reported elsewhere (38).
Experimental
All solvents were dried and distilled before use according
to standard literature procedures (70). Except where indi-
cated, chemicals were of the highest available purity from
Aldrich. All reactions were performed under dry nitrogen
using oven-dried (140 °C, 24 h) glassware. The glassware
was allowed to cool in a desiccator under vacuum and as-
sembled cold, capped with rubber septa, and evacuated with
1
dry nitrogen gas. Both H and 13C nuclear magnetic reso-
nance (NMR) spectra were recorded on a Bruker Model
AMX 400 Spectrometer operating at 400.1 and 100.6 MHz,
respectively. Chemical shifts (δ) are reported in parts per
million (ppm) relative to tetramethylsilane (TMS) in an ap-
hydride in tetrahydrofuran–methanol produced the bromohy-
drin ester 61 in 80% yield. Protection of the hydroxyl group
with dihydropyran and one crystal of para-toluenesulfonic
acid in dichloromethane (69) yielded the tetrahydropyranyl
derivative 62 as a mixture of diastereomers in an overall
yield of 64% from diketene. The benzyl ester 62 was
deprotected by hydrogenolysis to give 63, and this was cou-
pled to the tert-butyl ester of N-hydroxyglycine. Cyclization
with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and removal
of both protecting groups with trifluoroacetic acid produced
the target compound.
1
propriate deuterated solvent. H NMR data are reported in
the following manner: chemical shift, integration, peak mul-
tiplicity, coupling constants, and identification. The peak
multiplicities are abbreviated as s (singlet), d (doublet), t
(triplet), q (quartet), dd (doublet of doublets), ddd (doublet
of doublets of doublets), m (multiplet), etc. 13CMR data are
reported as δ values in ppm downfield from tetramethyl-
silane. Infrared (IR) spectra were recorded on a Bomen–
Hartmann–Braun spectrometer (1% KBr or 1% solution).
High-resolution mass spectra refer to either direct inlet elec-
tron impact (EI) measurements or chemical ionization (CI)
measurements, using isobutane, and were provided by Dr. G.
Eigendorf at the University of British Columbia. Low-
resolution mass spectra refer to direct inlet EI measurements
or CI measurements, using isobutane, or fast-atom bombard-
ment (FAB), employing xenon gas, on a Kratos MS50 Spec-
trometer operated at 70 eV by G. Owens at Simon Fraser
University. Melting points (mp) were determined on a
Fisher-Johns apparatus and are uncorrected. Analytical thin-
layer chromatography was carried out on precoated Merck
Silica Gel 60 F-254 plates with aluminium backing. Spots
The sodium salt, which is chemically stable, was isolated
by successive treatment of 64 with trifluoroacetic acid and
1
sodium bicarbonate. The H NMR spectrum is summarized
in Table 3.
Synthesis of 29, R = Me
For the synthesis of N-hydroxy-α-alanine, the nitrone 65
was prepared in 40% yield by the reaction of the sodium salt
of 34 with tert-butyl α-bromopropionate in isopropanol. Hy-
drolysis with hydroxylamine hydrochloride in the presence
of sodium methoxide then gave the racemic N-hydro-
xyalanine ester 66 in 71% yield. Coupling of 66 to 63 using
dicyclohexylcarbodiimide produced a mixture of N-acylated
© 2003 NRC Canada

Wolfe et al.
925
Scheme 2. Reagents and conditions: (a) Br2, PhCH2OH, CH2Cl2, –25 °C, 15 min; (b) NaBH4, THF–CH3OH, 0 °C, 15 min; (c) DHP,
CH2Cl2, p-TsOH; (d) H2–Pd(10%)–C, THF, 45 min; (e) 30(R1 = t-Bu, R2 = H), DCC; (f) DBU; (g) TFA, NaHCO3.
Fig. 5. Bioassay results showing zones of inhibition vs. M. luteus.
HO
HO
O
N
O
600 µg
N
CO2H
CO2H
1000 µg
O
O
1000 µg
control
100 µg
O
HO
N
CO2H
O
1000 µg
PhCH2CONH
S
PhCH2CONH
0.1 µg
5 µg
S
N
N
O
O
CO2H
CO2H
© 2003 NRC Canada

926
Can. J. Chem. Vol. 81, 2003
Table 5. Zones of inhibition vs. M. luteus.
4.71 (2H, s), 4.26 (2H, q) 1.29 (3H, t). EI-MS (m/z): 121
([M]+). Anal. calcd. for C7H7NO: C 69.42, H 5.79, N 11.57;
found: C 69.44, H 5.96, N 10.99.
Weight
(µg)
Moles
(µM)
Zone size
(mm)
Ratio
(mm/µM)
Compound
Pen Ga
0.1
5
600
12
2.99 × 10–4
1.51 × 10–2
3.05
25
38
21
20
83 600
2 520
7
N-Benzylideneglycine N-oxide ethyl ester (35)
DACGb
Ethyl bromoacetate (0.244 mL, 0.367 g, 2.2 mmol) was
added to a stirred solution of sodium (0.46 g, 0.02 g-atom)
and Z-benzaldoxime (0.242 g, 2.0 mmol) in ethanol
(15 mL). Stirring was continued until a wet pH 7 was
reached. The reaction mixture was poured into water
(20 mL), extracted with ether (2 × 20 mL), dried, and con-
centrated under reduced pressure. The golden yellow oil was
purified by flash chromatography on silica gel (4.5 g) (40%
ethyl acetate – hexane) and subsequently crystallized from
ethyl acetate – hexane. The pale yellow crystals weighed
0.298 g (73%); mp 53 °C. IR (film, cm–1): 1743 (C=O),
1584 (C=N). 1H NMR (CDCl3) δ: 8.19 (1H, s, (CH=N), 7.60
(2H, m, ArH), 7.45 (3H, m, ArH), 4.71 (2H, s), 4.26 (2H, q),
1.29 (3H, t). EI-MS (m/z): 207 ([M]+). Anal. calcd. for
C11H13NO3: C 63.76, H 6.32, N 6.76; found: C 64.04, H
6.41, N 6.60.
29 (R = H)
29 (R = Me)
0.0569
350
aPenicillin G (4, R = benzyl).
bDesacetoxycephalosporin G (23, R = benzyl, R′ = Me).
were observed under short-wavelength ultraviolet light or
were visualized with iodine vapour, ninhydrin, or ceric sul-
fate. Flash column chromatography was carried out on
Merck Silica Gel 60 (230–400 Mesh), according to the
method of Still et al. (71) unless stated otherwise, using only
distilled solvents under dry nitrogen gas. Elemental analyses
were performed by M.K. Yang on a Carlo Erba model 1106
elemental analyzer at Simon Fraser University.
Z-Benzaldoxime
Benzaldehyde (5.3 g, 0.05 mol) (Fisher) and hydroxyl-
amine hydrochloride (3.6 g, 0.052 mol) (Mallinckrodt) were
added successively at room temperature to a stirred solution
of sodium hydroxide (5 g, 0.125 mol). Stirring was contin-
ued until a clear solution was obtained and the temperature
had increased to 70 °C. The reaction mixture was cooled to
35 °C, and carbon dioxide was passed in until the pH had
decreased from 14 to 10. The two layers were then sepa-
rated. The aqueous layer was extracted with toluene (4 ×
25 mL), and the combined organic phase was dried over an-
hydrous magnesium sulfate, filtered, and poured into a
250 mL three-necked flask equipped with a large-bore gas
inlet tube, stirrer, thermometer, and condenser. The flask was
heated on an oil bath, and 20 mL of toluene was removed by
distillation. The remaining solution was reheated to boiling,
the oil bath was removed, and a strong flow of anhydrous
hydrogen chloride was sparged through the vigorously
stirred solution. Addition of the gas was continued until the
oil that first separated had solidified and the temperature had
fallen to 50 °C. The resulting white slurry was cooled to
10 °C in an ice-water bath and filtered. The white crystalline
solid was washed with toluene (2 × 25 mL) and hexane and
stored in a desiccator over potassium hydroxide pellets. Di-
ethyl ether (50 mL) was added to a stirred solution of so-
dium hydroxide (4 g, 0.1 mol) in water (40 mL). The
mixture was cooled to 10 °C and the crude Z-benzaldoxime
hydrochloride was added rapidly with stirring until all of the
solid had dissolved. A solution of ammonium chloride (10 g,
0.187 mol) (Fisher) in water (40 mL) was added, and the
mixture was stirred until a clear solution was obtained
(1 min). The two layers were separated, and the aqueous
layer was extracted with ether (2 × 25 mL). The combined
ethereal extracts were dried over anhydrous magnesium sul-
fate and concentrated. Hexane (40 mL) was added to the
white slurry and the mixture concentrated again. Another
40 mL of hexane were added, the mixture was filtered, and
the solid was air dried until a constant weight was obtained.
The Z-benzaldoxime was obtained as white needles (4.26 g,
70%); mp 131 °C (lit. (45) mp 130 °C). 1H NMR (CDCl3) δ:
8.19 (1H, s, CH=N), 7.60 (2H, m, ArH), 7.45 (3H, m, ArH).
N-Hydroxyglycine ethyl ester
Method A
The nitrone 35 (180 mg, 0.87 mmol) was added to a
stirred suspension of sodium methoxide (64.8 mg,
1.2 mmol) (Anachemia) and hydroxylamine hydrochloride
(83.4 mg, 1.2 mmol) (Mallinckrodt) in methanol (3 mL).
The reaction mixture was heated on a water bath (40 °C)
with stirring until the wet pH was slightly acidic (2 min) and
then concentrated under reduced pressure. The residue was
triturated with toluene (2 × 10 mL), concentrated, and puri-
fied by flash chromatography (5 g silica gel and 40% ethyl
acetate – hexane) to give a pale yellow oil (82.7 mg, 80%).
IR (film, cm–1): 3319 (O-H, br), 3267 (N-H, m), 1735
1
(C=O). H NMR (CDCl3) δ: 5.89 (2H, br, NH-OH), 4.23
(2H, q, OCH2CH3), 3.67 (2H, s), 1.29 (3H, t, OCH2CH3).
Method B
A mixture of glyoxylic acid (1.094 g, 0.014 mol) (East-
man), hydroxylamine hydrochloride (1.00 g, 14 mmol)
(Mallinckrodt), and p-toluenesulfonic acid (0.273 g,
1 mmol) (Fisher) in dry ethanol (8 mL) was heated under re-
flux for 5 h with removal of water using a Dean-Stark trap.
The reaction mixture was concentrated, ether (100 mL) and
saturated sodium bicarbonate (100 mL) were added, and the
mixture was stirred until the phases became clear. The or-
ganic layer was separated, washed with saturated ammonium
chloride, dried over anhydrous magnesium sulfate, and con-
centrated under reduced pressure to yield ethyl glyoxylate
oxime (1.11 g, 66%). IR (film, cm–1): 3347 (OH, br), 1725
(C=O, s), 1623 (C=N, m). 1H NMR (CDCl3) δ: 9.55 (1H, b,
=NOH), 7.56 (1H, s, HC=NOH), 4.31 (2H, q, CO2CH2CH3),
1.33 (3H, t, CO2CH2CH3). CI-MS (m/z): 118 ([M + 1]).
Anal. calcd. for C4H7NO3: C 41.03, H 5.98, N 11.96; found:
C 41.41, H 6.12, N 11.43.
Ethanolic hydrogen chloride (6.76 mL of a 7 N solution
prepared by diluting 30 mL of 36% HCl to 50 mL with etha-
nol) was added to a stirred solution of the oxime ethyl ester
(1.0 g, 8.5 mmol), and pyridine–borane complex (2.16 mL
© 2003 NRC Canada

Wolfe et al.
927
of 8 mol/L, 17.28 mmol) in ethanol (10 mL) was added at
such a rate that the reaction temperature remained below
40 °C. Stirring was continued for 8 h, while the reaction was
monitored by thin layer chromatography (TLC). The reac-
tion mixture was then concentrated, and the pale yellow syr-
upy residue was dissolved in dichloromethane (50 mL).
Sodium carbonate (4 g) was added, and the mixture was
stirred at room temperature for 30 h, filtered, and the filtrate
concentrated under reduced pressure. The crude oil was pu-
rified by flash chromatography (60% ethyl acetate – hexane)
to give a pale yellow oil (0.673 g, 67%). CI-MS (m/z): 120
([M + 1]). Anal. calcd. for C4H9NO3: C 40.34, H 7.56, N
11.76; found: C 40.21, H 7.55, N 11.54.
moved. A suspension of the residue in water (10 mL) was
cooled, acidified with 0.1 N hydrochloric acid (9.5 mL), and
filtered. The white crystals were collected and dried at 1 torr
(1 torr = 133.322 Pa) to give 1.21 g (66%) of product; mp
175–177 °C (lit. (47) mp 178–179 °C). IR (KBr, cm–1):
3069, 1724, 1617. EI-MS (m/z): 179 ([M]+).
N-Benzylidene-glycine N-oxide benzyl ester
To a cloudy solution of N-benzylideneglycine N-oxide
(0.181 g, 1.01 mmol) in dimethylformamide (2 mL) was
added triethylamine (0.141 mL, 103 mg, 1.01 mmol), fol-
lowed by dropwise addition of benzyl bromide (0.120 mL,
0.173 g, 1.01 mmol) (Sigma). The reaction mixture was
stirred for 13 h, poured into water (10 mL), and extracted
with chloroform (2 × 10 mL). The chloroform extract was
dried over anhydrous sodium sulfate, concentrated, and the
residue was purified by column chromatography (60% ethyl
acetate – hexanes) to give 0.191 g (70%) of product. IR
(CH2Cl2, cm–1): 3054, 1753, 1587. 1H NMR (CDCl3) δ: 8.25
(2H, m, Ar + =CH), 7.45–7.34 (9H, m, Ar), 5.27 (2H, s,
CH2Ph), 4.76 (2H, s, CH2). EI-MS (m/z): 269 ([M]+). Anal.
calcd. for C16H15NO3: C 71.37, H 5.58, N 5.20; found: C
71.13, H 5.80, N 5.40.
N-Benzyloxyoxime of ethyl glyoxylate
A mixture of glyoxylic acid (0.476 g, 6.26 mmol) (East-
man), O-benzylhydroxylamine hydrochloride (1.00 g,
6.26 mmol), and p-toluenesulfonic acid (0.12 g,
0.626 mmol) (Fisher) in dry ethanol (8 mL) was refluxed for
6 h with removal of water using a Dean-Stark trap. The reac-
tion mixture was concentrated and dichloromethane (50 mL)
was added to the yellow oily residue. The solution was
washed successively with water (30 mL), saturated sodium
bicarbonate (30 mL), and saturated sodium chloride
(30 mL), dried over anhydrous magnesium sulfate, and con-
centrated under reduced pressure to give a pale yellow oil
(1.08 g, 83%). IR (CDCl3, cm–1): 1727 (C=O), 1600 (C=N).
1H NMR (CDCl3) δ: 7.57 (1H, s, N=CH), 7.39 (5H, s,
OCH2Ph), 5.32 (2H, s, OCH2Ph), 4.33 (2H, q, OCH2CH3),
1.36 (3H, t, OCH2CH3). CI-MS (m/z): 208 ([M + 1]). Anal.
calcd. for C11H13NO3: C 63.77, H 6.28, N 6.76; found: C
63.06, H 6.33, N 7.34.
N-Hydroxyglycine benzyl ester
A suspension of N-benzylideneglycine N-oxide benzyl es-
ter (1.35 g, 5.02 mmol) and hydroxylamine hydrochloride
(352 mg, 5.02 mmol) (Mallinckrodt) in ethanol (10 mL) was
brought into solution by heating. The solution was concen-
trated, and the residue was taken up in ether (50 mL) and
washed with saturated sodium bicarbonate (20 mL). The
ether layer was separated, dried over anhydrous magnesium
sulfate, and the solvent was removed under reduced pres-
sure. The product was purified by flash chromatography
(60% ethyl acetate – hexanes) to give 772 mg (85%) of
N-Benzyloxyglycine ethyl ester (30, R1 = Et, R2 = Bn)
Ethanolic hydrogen chloride (6.76 mL of a 7 N solution
prepared by diluting 30 mL of 36% HCl to 50 mL with etha-
nol) was added to a stirred solution of the oxime (3.5 g,
16.91 mmol), and pyridine–borane (8.54 mL of 8 mol/L,
68.32 mmol) in ethanol (10 mL) was added at such a rate
that the reaction temperature did not exceed 40 °C. Stirring
was continued for 13.5 h with monitoring by TLC, and the
solvent was then removed. The pale yellow syrup was dis-
solved in dichloromethane (50 mL), and the solution was
washed with N-sodium hydroxide, dried over anhydrous so-
dium sulfate, and concentrated under reduced pressure. The
crude oil was purified by flash chromatography using 2%
ethanol in dichloromethane as the eluent. The pale yellow oil
thereby obtained weighed 3.0 g (85%). IR (CDCl3, cm–1):
1
white solid. IR (CH2Cl2, cm–1): 3290, 3055, 1741. H NMR
(CDCl3) δ: 7.36 (5H, s, Ar), 5.22 (2H, s, PhCH2), 3.73
(2H, s, NCH2). EI-MS (m/z): 181 ([M]+). Anal. calcd. for
C8H11NO3: C 59.67, H 6.08, N 7.73; found: C 59.74, H
6.07, N 7.11.
tert-Butyl bromoacetate
A solution of oxalyl chloride (6.18 mL, 8.99 g,
0.071 mol) in dichloromethane (10 mL) was added to a
cooled (0 °C) solution of bromoacetic acid (8.21 g,
0.059 mol) (Lancaster) and dimethylformamide (3 drops) in
dichloromethane (40 mL). The pale yellow solution was
stirred for 1 h at 0 °C, then at room temperature for 3 h, con-
centrated to about 10 mL, and slowly added to a cooled so-
lution of tert-butanol (5.255 g, 0.071 mol) and triethylamine
(10.71 mL, 7.78 g, 0.077 mol) in dichloromethane (45 mL).
The dark brown suspension was stirred for 45 min at 0 °C,
then for 3 h at room temperature, and poured into water
(60 mL). The organic layer was separated and the aqueous
layer extracted with dichloromethane (2 × 15 mL). The or-
ganic extract was washed with saturated sodium bicarbonate
(50 mL), dried over anhydrous magnesium sulfate, and con-
1
3273 (N-H, w), 1739 (C=O, s). H NMR (CDCl3) δ: 7.36
(5H, s, OCH2Ph), 6.13 (1H, t, N-H), 4.75 (2H, s, OCH2Ph),
4.24 (2H, q, OCH2CH3), 3.62 (2H, d, NCH2C(O)), 1.30 (3H,
t, OCH2CH3). CI-MS (m/z): 210 ([M + 1]). Anal. calcd. for
C11H15NO3N: C 63.16, H 7.18, N 6.70; found: C 63.37, H
7.01, N 7.50.
N-Benzylidene glycine N-oxide
Sodium (0.46 g, 0.02 g-atom) and Z-benzaldoxime
(1.21 g, 10 mmol) in ethanol (40 mL) were stirred until the
sodium dissolved. Then bromoacetic acid (1.53 g, 11 mmol)
(Lancaster) was added in one portion, with stirring. Stirring
was continued for 1 h at 70 °C, and the solvent was re-
1
centrated to give 4.48 g (39%) of product. H NMR (CDCl3)
δ: 3.74 (2H, s), 1.47 (9H, s). CI-MS (m/z): 197:195 (1:1).
© 2003 NRC Canada

928
Can. J. Chem. Vol. 81, 2003
(m/z): 250 ([M + 1]). Anal. calcd. for C14H19NO3: C 67.45,
N-Benzylidene glycine N-oxide tert-butyl ester
H 7.68, N 5.62; found: C 67.10, H 7.59, N 5.90.
Z-Benzaldoxime (5.64 g, 46.61 mmol) and tert-butyl
bromoacetate (7.58 mL, 10 g, 51.27 mmol) were added suc-
cessively to a solution prepared by dissolving 1.07 g
(0.047 g-atom) of sodium metal in 2-propanol (120 mL).
The dark brown suspension was stirred for 2 h at room tem-
perature, poured into water (100 mL), and extracted with di-
chloromethane (5 × 20 mL). The combined organic extracts
were dried over anhydrous magnesium sulfate and concen-
trated. The crude product was purified by flash chromatogra-
phy (60% ethyl acetate – hexanes) to give 8.74 g (80%) of
an oil. IR (CH2Cl2, cm–1): 3054, 1746, 1580, 1260. 1H NMR
(CDCl3) δ: 8.24 (2H, m, Ar + =CH), 7.42 (4H, m, Ar), 4.62
(2H, s, CH2), 1.51 (9H, s, t-Bu). CI-MS (m/z): 236 ([M +
1]). Anal. calcd. for C13H17NO3: C66.38, H 7.23, N 5.96;
found: C 66.78, H 7.10, N 6.01.
tert-Butyl N-hydroxyalanine
The N-oxide (460 mg, 185 mmol) was added to a stirred
suspension of sodium methoxide (141 mg, 2.61 mmol) and
hydroxylamine hydrochloride (182 mg, 2.62 mmol) in dry
methanol (5.5 mL). This mixture was stirred at 40 °C until
the solid dissolved, and the solution was then concentrated.
The residue was shaken with ethyl acetate (10 mL), filtered,
and the filtrate was concentrated. The residue was
chromatographed on silica gel. Elution with ethyl acetate –
hexanes (2:3) afforded 211 mg (71%) of N-hydroxyalanine
tert-butyl ester; mp 73–74 °C. IR (KBr, cm–1): 3158, 1746.
1H NMR (CDCl3) δ: 5.61 (2H, br s, NH + OH), 3.60 (1H, q,
7.1 Hz, α-CH), 1.48 (9H, s, CO2C(CH3)3), 1.22 (3H, d,
7.1 Hz, CH3). 13C NMR (CDCl3) δ: 173.37 (ester C=O),
81.54 (CO2C(CH3)3), 60.97 (α-CH), 28.06 (CO2C(CH3)3),
14.65 (CH3). CI-MS (m/z): 162 ([M + 1]). Anal. calcd. for
C7H15NO3: C 52.16, H 9.38, N 8.69; found: C 51.98, H
9.30, N 8.55.
N-Hydroxyglycine tert-butyl ester
The nitrone (1.3 g, 5.53 mmol) was added to a stirred sus-
pension of sodium methoxide (0.418 g, 7.74 mmol)
(Anachemia) and hydroxylamine hydrochloride (0.538 g,
7.74 mmol) (Mallinckrodt) in methanol (6 mL). The reaction
mixture was placed in a water bath (50 °C), stirred until all
of the nitrone had dissolved, and was then concentrated. The
residue was dissolved in dichloromethane (50 mL), filtered,
and concentrated. The resultant oil was purified by flash
chromatography (60% ethyl acetate – hexanes) to give
0.650 g (80%) of the product. IR (film, cm–1): 3279, 1735.
1H NMR (CDCl3) δ: 5.46 (2H, br, HNOH), 3.62 (2H, s,
CH2), 1.49 (9H, s, t-Bu). CI-MS (m/z): 148 ([M + 1]). Anal.
calcd. for C6H13NO3: C 48.98, H 8.84, N 9.52; found: C
49.13, H 8.65, N 9.44.
N-Benzoyl, N-benzyloxyglycine ethyl ester (31)
Benzoyl chloride (0.028 mL, 0.034 g, 0.23 mmol) (Fluka)
was added dropwise to a cooled (0 °C) solution of N-benzyl-
oxyglycine ethyl ester (48 mg, 0.23 mmol) and triethylamine
(0.032 mL, 0.023 g, 0.023 mmol) in dichloromethane
(0.5 mL). The cloudy solution was stirred for 20 min, di-
luted with dichloromethane (25 mL), and washed succes-
sively with saturated sodium bicarbonate (10 mL) and
saturated sodium chloride (10 mL), dried over anhydrous
magnesium sulfate, and concentrated to a pale yellow oil
(70.7 mg, 98%). IR (film, cm–1): 1746 (C=O, s), 1626. H
NMR (CDCl3) δ: 7.71–7.07 (10H, m, Ar), 4.71 (2H, s,
OCH2Ar), 4.42 (2H, s, CH2), 4.24 (2H, q, CO2CH2CH3),
1.29 (3H, t, CO2CH2CH3). CI-MS (m/z): 314 ([M + 1]).
1
tert-Butyl 2-bromopropionate
Isobutylene (2.4 g, 42.8 mmol) was condensed into a pres-
sure bottle at –15 °C. Dioxane (6 mL) and 2-bromopropionic
acid (3.5 mL, 38.9 mmol) were added, and the mixture was
stirred for 5 min, warmed to –10 °C, and concentrated sulfu-
ric acid (250 µL) was added. The bottle was sealed, the reac-
tion mixture was stirred overnight at room temperature, and
the bottle was then opened and the contents poured into di-
chloromethane (50 mL). The solution was washed with 20%
potassium carbonate (50 mL) and water (50 mL), dried over
anhydrous magnesium sulfate, and evaporated to give tert-
Synthesis of 32
Method A
A solution of 31 (38.2 mg, 0.12 mmol) and anisole
(0.053 mL, 0.49 mmol) (BDH) in dichloromethane (1 mL)
was added dropwise at 0 °C to a solution of aluminium
trichloride (48.8 mg, 0.37 mmol) (Anachemia) in nitro-
methane (1 mL). The resultant pink solution was stirred at
0 °C for 90 min and then at room temperature for 10 min.
The reaction mixture was diluted with ethyl acetate (30 mL)
and washed successively with N-hydrochloric acid (10 mL)
and 4% sodium bicarbonate (40 mL). The organic layer was
dried over anhydrous magnesium sulfate and concentrated to
give a bright orange oil, which was purified by preparative
layer chromatography (PLC) (40% ethyl acetate – hexane).
The product (12.5 mg, 45%) was identical to that obtained
from Method B.
1
butyl bromopropionate (2.17 g, 27%). H NMR (CDCl3) δ:
4.31 (1H, q, 7.1 Hz), 1.81 (3H, d, 7.1 Hz), 1.52 (9H, s).
tert-Butyl N-benzylidenealanine N-oxide
Z-Benzaldoxime (580 mg, 4.79 mmol) and tert-butyl 2-
bromopropionate (980 mg, 4.69 mmol) were added succes-
sively to a solution of sodium hydride (200 mg of a 60% dis-
persion in mineral oil, 5.00 mmol) in 2-propanol (20 mL).
The suspension was stirred at room temperature for 3 h, then
poured into water, and extracted with ethyl acetate (2 ×
20 mL). The organic extract was dried over anhydrous mag-
nesium sulfate and evaporated to give a white solid.
Trituration with anhydrous ether (8 mL) afforded the nitrone
(464 mg, 40%); mp 115–117 °C. IR (KBr, cm–1): 1736,
1582. 1H NMR (CDCl3) δ: 8.26–8.23 (2H, m, Ar + HC=N+),
7.43–7.41 (4H, m, Ar), 4.65 (1H, q, 7.0 Hz, α-CH), 1.73
(3H, d, 7.0 Hz, CH3), 1.47 (9H, s, CO2C(CH3)3). CI-MS
Method B
Benzoyl chloride (0.016 mL, 0.019 g, 0.14 mmol) (Fluka)
was added dropwise to a cooled (0 °C) solution of N-
hydroxyglycine ethyl ester (16.4 mg, 0.14 mmol) and
triethylamine (0.019 mL, 0.014 g, 0.014 mmol) in dichloro-
methane (0.5 mL). The suspension was stirred for 1 h, and
© 2003 NRC Canada

Wolfe et al.
929
the solvent was then removed. The residue was redissolved
in ether (10 mL), filtered, and the filtrate was concentrated.
The oil was subjected to PLC (60% ethyl acetate – hexanes)
to give 14.8 mg (41%) of the product. IR (film, cm–1): 1743
(C=O, s), 1672. H NMR (CDCl3) δ: 7.72 (5H, m, Ar), 4.47
(2H, s), 4.25 (2H, q), 1.30 (3H, t). CI-MS (m/z): 224 ([M +
(30 mL), dried over anhydrous magnesium sulfate, and
evaporated under reduced pressure. The product was puri-
fied by flash chromatography (60% ethyl acetate – hexanes)
to give an oil 93 mg (28%). IR (film, cm–1): 1718, 1750
(C=O). 1H NMR (CDCl3) δ: 4.70–4.63 (1H, m), 4.26 (1H, d,
CHH, 17.88 Hz), 4.24 (1H, d, CHH, 17.88 Hz), 4.22 (2H,
q), 3.39 (1H, dd, 10.40, 5.52 Hz), 3.35 (1H, dd, 10.40,
7.44 Hz), 3.0 (1H, dd, 16.78, 8.0 Hz), 2.76 (1H, dd, 16.78,
7.24 Hz), 1.28 (3H, t).
1
1]).
Vinylacetamido N-hydroxyglycine ethyl ester (36)
To a stirred solution of N-hydroxyglycine ethyl ester
(117 mg, 0.98 mmol) in dichloromethane (2 mL) was added
vinylacetic acid (0.088 mL, 0.089 g, 1.03 mmol) in one por-
tion. The solution was cooled to 0 °C, and a solution of
dicyclohexylcarbodiimide (213 mg, 1.03 mmol) (BDH) in
dichloromethane (2 mL) was added dropwise during 10 min.
Stirring was continued for 11 h while the mixture warmed to
room temperature. The mixture was then concentrated under
reduced pressure, and the residue was extracted with ether
(2 × 10 mL). The combined organic extracts were dried,
concentrated, and the crude product was purified by flash
chromatography (30 g silica gel and 40% ethyl acetate –
hexane) to give a pale yellow oil (94 mg, 51%). IR
(film, cm–1): 3318 (O-H, br), 3083 (C=C, w), 1742 (C=O, s),
Oxidation of 37, X = I
m-Chloroperbenzoic acid (45 mg of 85%, 0.22 mmol) was
added to a solution of the iodide (46 mg, 0.15 mmol) in di-
chloromethane (2 mL). The purple solution was stirred for
5 h, then diluted with dichloromethane (10 mL), washed
with saturated sodium thiosulfate (10 mL), and dried over
anhydrous magnesium sulfate. The residue was purified by
PLC (80% ethyl acetate – hexanes) to give 8 mg of product
(25%). IR (CHCl3, cm–1): 1747, 1703 (C=O). 1H NMR
(CDCl3) δ: 4.69–4.63 (1H, m), 4.57 (1H, d, CHH, 18 Hz),
4.24 (2H, q), 4.06 (1H, d, CHH, 18 Hz), 3.91 (1H, dd,
12.80, 2.40 Hz), 3.67 (1H, dd, 12.80, 4.0 Hz), 2.96 (1H, dd,
16.60, 8.70 Hz), 2.90 (1H, dd, 16.60, 6.20 Hz), 1.30 (3H, t).
CI-MS (m/z): 204 ([M + 1]). EI-MS (m/z): 203 ([M]+).
1
1625 (N-C(O), s). H NMR (CDCl3) δ: 5.98–5.91 (1H, m,
CH=CH2), 5.21–5.18 (2H, d, CH=CH2), 4.48 (2H, s), 4.22
(2H, q), 3.36 (2H, d), 1.29 (3H, t). EI-MS (m/z): 187 ([M]+).
Coupling of N-hydroxyglycine benzyl ester and
vinylacetic acid
Bromination of 36
A solution of bromine (0.041 mL, 0.128 g, 0.80 mmol)
(Fisher) in chloroform (1 mL) was added dropwise, under
nitrogen, at room temperature, to a stirred solution of
vinylacetamido N-hydroxyglycine ethyl ester (0.125 g,
0.66 mmol) in chloroform (0.6 mL). Stirring was continued
for 2 h, and the solvent was then removed to give a deep or-
ange oil (0.148 g, 100%). IR (film, cm–1): 3211 (OH, br),
Dicyclohexylcarbodiimide (1.38 g, 6.68 mmol) (BDH)
was added to a cooled solution of N-hydroxyglycine benzyl
ester (1.15 g, 6.36 mmol) and vinylacetic acid (0.57 mL,
575 mg, 6.68 mmol) in dichloromethane (30 mL). The
cloudy mixture was stirred for 0.5 h at 0 °C and then for
3.5 h at room temperature, filtered, and concentrated. The
resulting oil was purified by flash chromatography (60%
ethyl acetate – hexane) to give 912 mg (58%) of the product.
IR (film, cm–1): 3200 (OH, s), 1749 (C=O, s), 1633 (C=O,
amide, s). 1H NMR (CDCl3) δ: 7.38–7.33 (5H, m, Ar), 6.02–
5.89 (1H, m), 5.21–5.17 (2H, m, =CH2), 5.19 (2H, s,
CH2Ph), 4.52 (2H, s), 3.34 (2H, d, CH2C(O). CI-MS (m/z):
250 ([M + 1]). Anal. calcd. for C13H15NO4: C 62.65, H 6.02;
N, 5.62; found: C 62.88, H 6.22, N 6.00.
1
1745 (C=O), 1650 (N-C(O)). H NMR (CDCl3) δ: 7.87 (1H,
OH, br), 4.55 (1H, m), 4.50 (2H, s), 4.33 (2H, q), 3.86 (2H,
dd), 3.38 (2H, dd), 1.30 (3H, t). EI-MS (m/z): 345 : 347 :
349 (1:2:1). The oil was dissolved in dichloromethane
(2 mL), and saturated sodium bicarbonate (3 mL) was added.
The mixture was stirred for 2 h, washed with saturated so-
dium chloride (5 mL), dried, and concentrated. The crude
product was purified by PLC (60% ethyl acetate – hexane)
to give a colourless oil (0.077g, 50%). IR (film, cm–1):
1
Iodocyclization of the benzyl ester
1718–1750 (C=O). H NMR (CDCl3) δ: 4.80–4.75 (1H, m),
4.29 (1H, d, CHH, 17 Hz), 4.28 (1H, d, CHH, 17Hz), 3.58
(1H, dd, 10.78, 5.52 Hz), 3.53 (1H, dd, 10.78, 6.76 Hz), 3.02
(1H, dd, 16.84, 8.24 Hz), 2.83 (1H, dd, 16.84, 7.04 Hz), 4.23
(2H, q, CO2CH2CH3), 1.29 (3H, t, CO2CH2CH3). EI-MS
(m/z) (relative intensity): 267 (12.1), 265 (12.6), 194 (78.4),
192 (68.3), 149 (58.2), 147 (57.4), 95 (7.4), 93 (8.7), 85
(17.8). Anal. calcd. for C8H12NO4Br: C 36.11, H 4.55, N
5.26; found: C 35.75, H 4.58, N 5.25.
Iodine (1.23 g, 4.83 mmol) and sodium bicarbonate
(0.41 g, 8.83 mmol) were added to a solution of the benzyl
ester (0.40 g, 1.61 mmol) in acetonitrile (10 mL). The solu-
tion was stirred for 3 h at 0 °C and then diluted with ether
(20 mL) and washed successively with saturated sodium
thiosulfate (20 mL) and saturated sodium chloride (20 mL),
dried over anhydrous magnesium sulfate, and evaporated un-
der reduced pressure. The product was purified by flash
chromatography (60% ethyl acetate – hexanes) to give an oil
0.345 g (57%). IR (film, cm–1): 1718, 1750 (C=O). 1H NMR
(CDCl3) δ: 7.38–7.33 (5H, m, Ar), 5.21 (1H, d, CHHPh,
12.08 Hz), 5.19 (1H, d, CHHPh, 12.08 Hz), 4.61–4.58
(1H, m), 4.33 (1H, d, CHH, 17.84 Hz), 4.30 (1H, d, CHH,
17.84 Hz), 3.33 (1H, dd, 10.36, 5.52 Hz), 3.28 (1H, dd,
10.36, 7.56 Hz), 2.98 (1H, dd, 16.80, 8.04 Hz), 2.73 (1H, dd,
16.80, 7.24 Hz). EI-MS (m/z): 375 ([M]+).
Iodination of 36
Iodine (041 g, 1.60 mmol) and sodium bicarbonate
(0.27 g, 3.21 mmol) were added to a solution of 36 (0.20 g,
1.07 mmol) in acetonitrile (5 mL). The solution was stirred
for 7 h at 0 °C and then diluted with ether (50 mL) and
washed successively with saturated sodium thiosulfate
(30 mL), water (30 mL), and saturated sodium chloride
© 2003 NRC Canada

930
Can. J. Chem. Vol. 81, 2003
(0.5 mL) was added with stirring. Stirring was continued for
16 h. The suspension was then filtered, and the white solid
was dried at 1 torr to give 183 mg (65%) of the product. H
Oxidation of the iodomethyl benzyl ester
m-Chloroperbenzoic acid (68%) (351 mg, 1.38 mmol) was
added to a solution of the ester (345 mg, 0.92 mmol) in di-
chloromethane (6 mL). The purple solution was stirred for
24 h, then diluted with ether (50 mL), washed with saturated
sodium thiosulfate (10 mL), and dried over anhydrous mag-
nesium sulfate. The residue was purified by PLC (80% ethyl
acetate – hexanes) to give 66 mg of product (27%). IR
(CHCl3, cm–1): 1718, 1750 (C=O). 1H NMR (CDCl3) δ:
7.38–7.33 (5H, m, Ar), 5.21 (1H, d, CHHPh, 12.08 Hz),
5.19 (1H, d, CHHPh, 12.08 Hz), 4.66–4.60 (1 H, m), 4.57
(1H, d, CHH, 18 Hz), 4.13 (1H, d, CHH, 18 Hz), 3.87 (1H,
dd, 12.80, 2.48 Hz), 3.66 (1H, dd, 12.80, 4.24 Hz), 2.92 (1H,
dd, 16.66, 8.56 Hz), 2.87 (1H, dd, 16.66, 6.68 Hz). CI-MS
(m/z): 266 ([M + 1]).
1
NMR ((CD3)2CO) δ: 7.41–7.33 (5H, m, Ar), 5.17 (2H, s,
CH2Ph), 4.41 (2H, s), 2.82–2.55 (4H. m, (CH2)2). The
hydroxysuccinamic acid (175.6 mg, 0.62 mmol) was sus-
pended in dichloromethane (0.65 mL) under nitrogen at
0 °C, and dicyclohexylcarbodiimide (128 mg, 0.62 mmol)
was added in one portion. Stirring was continued for 4 h at
0 °C. The mixture was then filtered through Celite and the
filtrate concentrated under reduced pressure to afford an oily
residue. Purification by PLC (40% ethyl acetate – hexanes)
gave 105 mg (64%) of the product as an oil. 1H NMR
(CDCl3) δ: 7.41–7.33 (5H, m, Ar), 5.21 (2H, s, CH2Ph),
4.49 (2H, s), 2.91–2.87 (2H, m), 2.77–2.74 (2H, m). CI-MS
(m/z): 264 ([M + 1]).
Synthesis of 39
Palladium (10% on carbon) (250 mg) was added to a solu-
tion of the benzyl ester (190.5 mg, 0.72 mmol) in ethanol
(10 mL). The resulting suspension was fitted with a balloon,
flushed thrice with hydrogen, and then stirred under hydro-
gen for 1 h at room temperature. After filtration through
Celite and evaporation of the filtrate, the oil was redissolved
in water (10 mL), filtered, and the filtrate was lyophilized to
5-Acetoxy-tetrahydro-1,2-oxazin-3,6-dione (44)
N-Hydroxyglycine benzyl ester (362 mg, 2 mmol) in di-
chloromethane (1 mL) was added with stirring to a solution
of acetoxysuccinic anhydride (4.11 mg, 2.6 mmol) in di-
chloromethane (1 mL). Stirring was continued for 16 h. The
suspension was then filtered, and the white solid was col-
lected and dried at 1 torr to give 518.2 mg (80%) of the
1
1
give a pale green viscous oil 120 mg (95%). H NMR (D2O)
product. H NMR ((CD3)2CO) δ: 7.41–7.33 (5H, m, Ar),
δ: 4.74 (1H,m), 4.36 (1H, d, 18.16 Hz), 4.34 (1H, d,
18.16 Hz), 3.78 (1H, dd, 12.88, 3.2 Hz), 3.74 (1H, dd, 12.88,
5.64 Hz), 3.02 (1H, dd, 17.04, 8.76 Hz), 2.79 (1H, dd, 17.04,
7.68 Hz). CI-MS (m/z): 176 ([M + 1]).
5.87 (1H, dd, 2.68, 2.76 Hz), 5.20 (2H, s, CH2Ph), 4.66 (1H,
d, 17.6 Hz), 4.18 (1H, d, 17.6 Hz), 3.10 (1H, dd, 17,
2.72 Hz), 2.59 (1H, dd, 17.10 Hz). CI-MS (m/z): 340 ([M +
1]). This product (500 mg, 1.55 mmol) was suspended under
nitrogen in dichloromethane (1.5 mL), the suspension was
cooled to 0 °C, and dicyclohexylcarbodiimide (319 mg,
1.55 mmol) was added in one portion. Stirring was contin-
ued at 0 °C for 4 h, and the slurry was then filtered through
Celite and the filtrate concentrated under reduced pressure.
The oily residue was purified by PLC (40% ethyl acetate –
hexanes) to give 417.3 mg (88%) of the product as an oil. 1H
NMR (CDCl3) δ: 7.41–7.32 (5H, m, Ar), 5.61 (1H, dd, 5.28,
11.16 Hz), 5.20 (2H, s, CH2Ph), 4.58 (1H, d, 18.12 Hz),
4.49 (1H, d, 18.12 Hz), 3.22 (1H, dd, 16.72, 11.16 Hz), 3.34
(1H, dd, 16.72, 5.28 Hz). CI-MS (m/z): 322 ([M + 1]).
Reaction of 36 with N-bromosuccinimide in methanol
A solution of 36 (100 mg, 0.53 mmol) in dry methanol
(5 mL) was cooled in an ice-water bath, and N-
bromosuccinimide (95 mg, 0.53 mmol) (BDH) was added in
one portion. Stirring was continued at 0 °C for 15 min and
then at room temperature for 45 min. The reaction mixture
was concentrated, and the residue was subjected to flash
chromatography (60% ethyl acetate -hexanes). Two products
were obtained. These were dissolved in dichloromethane
(3 mL), washed with saturated sodium bicarbonate (2 mL),
dried over anhydrous sodium sulfate, and concentrated to
give 37 (X = Br) (9.6 mg) and a mixture of 40 and 41
(25 mg). IR (film, cm–1): 1793, 1751, 1700. 1H NMR
(CDCl3) δ: 41: 4.51 (1H, d, 17.8 Hz, CHH), 4.23 (1H, d,
17.8 Hz, CHH), 4.33 (1H, dd, 11.9, 6.0 Hz, CHH), 4.23 (2H,
q, OCH2CH3), 4.07 (1H, dd, 11.9, 3.6 Hz, CHH), 4.02–3.98
(1H, m, CH), 2.80 (1H, dd, 16.0 Hz, 5.8 Hz, CHH), 2.68
(1H, dd, 16.0 Hz, 4.7 Hz, CHH), 1.30 (3H, t, OCH2CH3).
40: 4.75–4.73 (1H, m, CH), 4.28 (1H, d, 17.8 Hz, CHH),
4.23 (2H, q, OCH2CH3), 4.22 (1H, d, 17.8 Hz, CHH), 3.62
(1H, dd, 10.9, 6.2 Hz, CHH), 3.54 (H, dd, 10.9, 3.6 Hz,
CHH), 2.81 (1H, dd, 16.6, 8.2 Hz, CHH), 2.75 (1H, dd,
16.6, 8.8 Hz, CHH), 1.30 (3H, t, OCH2CH3). EI-MS (m/z):
217 ([M]+), 144, 112, 99, 85. CI-MS (m/z): 218 ([M + 1]).
GC–CI-MS (m/z): 218 ([M + 1]), 99 (RT = 9.4 min), 218
([M + 1]), 85 (RT = 9.7 min).
Synthesis of 45
Maleic anhydride (98 mg, 1 mmol) was suspended in di-
chloromethane (0.5 mL), and
a
solution of N-
hydroxyglycine benzyl ester (181 mg, 1 mmol) in dichloro-
methane (0.5 mL) was added with stirring. Stirring was con-
tinued for 16 h. The suspension was then filtered, and the
white solid was dried at 1 torr to give 178 mg (64%) of a
1
solid. H NMR (CDCl3) δ: 7.41–7.32 (5H, m, Ar), 7.11 (1H,
d, 12.88 Hz), 6.35 (1H, d, 12.88 Hz), 5.22 (2H, s, CH2Ph),
4.59 (2H, s). The solid (171 mg, 0.61 mmol) was suspended
in dichloromethane (1 mL) under nitrogen; the suspension
was cooled 0 °C, and dicyclohexylcarbodiimide (126.5 mg,
0.61 mmol) was added in one portion. Stirring was contin-
ued at 0 °C for 4 h. The slurry was then filtered through
Celite and the filtrate concentrated under reduced pressure to
afford an oily residue. Purification by PLC (40% ethyl ace-
tate – hexanes) gave 154 mg (96%) of the product as a yel-
Synthesis of 43
1
Succinic anhydride (100 mg, 1 mmol) was suspended in
dichloromethane (0.5 mL), and a solution of N-hydroxy-
glycine benzyl ester (181 mg, 1 mmol) in dichloromethane
low solid. H NMR (CDCl3) δ: 7.39–7.32 (5H, m, Ar), 7.01
(1H, d, 10.2 Hz), 6.78 (1H, d, 10.2 Hz), 5.20 (2H, s,
CH2Ph), 4.66 (2H, s). CI-MS (m/z): 262 ([M + 1]).
© 2003 NRC Canada

Wolfe et al.
931
6.72 Hz), 3.99 (1H, dd, 8.32, 5.48 Hz), 3.75 (1H, dd, 8.50,
6.60 Hz), 2.88 (1H, dd, 16.36, 6.60 Hz), 2.71 (1H, dd, 16.36,
6.96 Hz), 2.68 (1H, dd, 16.36, 6.80 Hz). CI-MS (m/z): 209
([M + 1]).
Methyl 3,4-dihydroxybutanoate
DL-Malic acid (20 g, 0.15 mol) was dissolved in a solution
prepared by addition of acetyl chloride (6.7 mL) (Fisher) to
methanol (134 mL). The solution was stirred at room tem-
perature for 13 h and then concentrated. The product was
purified by flash chromatography (60% ethyl acetate – hex-
anes) to give a colourless oil (18.5 g, 77%). IR (film, cm–1):
3470, 1732. 1H NMR (CDCl3) δ: 4.44 (1H, br), 3.75 (3H, s),
3.65 (3H, s), 3.18 (1H, br), 2.81 (1H, dd, 16.48, 4.36 Hz),
2.74 (1H, dd, 16.48, 6.12 Hz). CI-MS (m/z):163 ([M + 1]).
Anal. calcd. for C6H10O5: C44.44, H 6.17; found: C 44.20,
H 6.13.
Coupling of 47 and N-hydroxyglycine tert-butyl ester
Dicyclohexylcarbodiimide (0.859 g, 4.16 mmol) (BDH)
was added to a cooled solution of 47 (0.87 g, 4.16 mmol)
and N-hydroxyglycine tert-butyl ester (0.60 g, 4.09 mmol) in
dichloromethane (20 mL). The cloudy mixture was stirred at
0 °C for 0.5 h and at room temperature for 4 h and then fil-
tered and the filtrate concentrated. Purification of the oil by
flash chromatography (60% ethyl acetate – hexane) gave
0.650 g (47%) of the product 48. IR (film, cm–1): 3249,
Borane–dimethylsulfide (11 mL of 10 mol/L, 0.11 mol)
was added dropwise to a solution of dimethylmalate (17.80 g,
0.11 mol) in tetrahydrofuran (220 mL). The solution was
stirred at 20 °C for 30 min, and sodium borohydride (0.208 g,
5.5 mmol) (BDH) was added in one portion. Stirring was
continued for 30 min, methanol (70 mL) was added
dropwise, and after an additional 30 min the solvent was re-
moved under reduced pressure. The product was purified by
flash chromatography (60% ethyl acetate – hexanes) to give
a colourless viscous oil (12.27 g, 83%). IR (film, cm–1):
1
1739, 1641. H NMR (CDCl3) δ: 7.49–7.35 (5H, m, Ar),
5.96 (1H, s), 5.86 (1H, s), 4.59–4.62 (1H, m), 4.45 (1H, d,
17.72 Hz), 4.39 (1H, dd, 8.56, 6.08 Hz), 4.38 (1H, d,
17.76 Hz), 4.31 (1H, d, 17.76 Hz), 4.29 (1H, d, 17.72 Hz),
4.25 (1H, dd, 8.20, 6.80 Hz.), 3.89 (1H, dd, 8.20, 5.84 Hz),
3.79 (1H, dd, 8.56, 7.12 Hz), 3.20 (1H, dd, 15.92, 6.12 Hz),
3.02 (1H, dd, 15.54,5.60 Hz), 2.92 (1H, dd, 15.54, 7 Hz),
2.81 (1H, dd, 15.92, 7.16 Hz), 1.48 (9H, s, tert-butyl). CI-
MS (m/z): 338 ([M + 1]). Anal. calcd. for C17H23NO6:
C60.53, H 6.82, N 4.15; found: C 60.84, H 7.07, N 4.36.
1
3414, 1738. H NMR (CDCl3) δ: 4.07 (1H, m), 3.66 (3H, s),
3.61 (1H, dd, 11.36, 3.52 Hz), 3.46 (1H, dd, 11.36, 6.2 Hz),
2.80 (2H, br), 2.50 (1H, dd, 16.64, 8.52 Hz), 2.44 (1H, dd,
16.64, 4.16 Hz). CI-MS (m/z): 135 ([M + 1]). Anal. calcd.
for C5H10O4: C 44.78, H 7.46; found: C 44.12, H 7.55.
Hydrogenolysis of 48
Palladium on carbon (10%) (0.400 g) was added to a solu-
tion of 48 (0.400 g, 1.19 mmol) in tetrahydrofuran (15 mL).
The suspension was flushed thrice with hydrogen, then
stirred under hydrogen for 3 h, filtered through Celite, and
the filtrate concentrated. The residue was purified by PLC
(80% ethyl acetate – hexanes) to give 49 as a viscous golden
Benzylidene derivative of methyl 3,4-
dihydroxybutanoate
A solution of methyl 3,4-dihydroxybutanoate (11.44 g,
85.36 mmol), benzaldehyde dimethyl acetal (11 mL,
11.15 g, 3.16 mmol), and p-toluenesulfonic acid mono-
hydrate (812 mg, 4.27 mmol) (Fisher) in dimethylformamide
(20 mL) was stirred at room temperature for 24 h and then
poured into ice-water (50 mL) and extracted with ethyl ace-
tate (5 × 40 mL). The organic extract was washed with satu-
rated sodium chloride (30 mL), dried over anhydrous sodium
sulfate, and concentrated. Purification by column chroma-
tography (60% ethyl acetate – hexanes) gave 18.5 g (77%)
1
oil (0.150 g, 51%). IR (film, cm–1): 3362, 1737, 1635. H
NMR (D2O) δ: 4.37 (1H, d, 17.72 Hz), 4.30 (1H, d,
17.72 Hz), 4.13 (1H, m), 3.62 (1H, dd, 11.76, 4 Hz), 3.53
(1H, dd, 11.76, 6.88 Hz), 2.78 (1H, dd, 15.15, 8.24 Hz), 2.71
(1H, dd, 15.15, 4.96 Hz), 1.49 (9H, s). CI-MS (m/z): 250
([M + 1]). Anal. calcd. for C10H19NO6.1.5H2O: C43.47, H
7.37, N 5.07; found: C 43.45, H 7.29, N 5.26.
of a colourless oil. IR (film, cm–1): 3034, 1737 (C=O). H
1
NMR (CDCl3) δ: 7.49–7.35 (5H, m, Ar), 5.96 (1H, s), 5.86
(1H, s), 4.68–4.60 (1H, m), 4.21 (1H, dd, 8.28, 6.72 Hz),
4.12 (1H, dd, 8.44, 6.16 Hz), 3.98 (1H, dd, 8.44, 6.60 Hz),
3.86 (1H, dd, 8.28, 5.68 Hz), 2.85 (1H, dd, 16.06, 6.36 Hz),
2.82 (1H, dd, 16.06, 6.36 Hz), 2.66 (1H, dd, 16.06, 7.20 Hz),
2.65 (1H, dd, 16.06, 7.32 Hz). CI-MS (m/z): 223 ([M + 1]).
Cyclization of 49
A mixture of the diol 49 (68.8 mg, 0.28 mmol), triphenyl-
phosphine (78.3 mg, 0.30 mmol), and diethylazodicar-
boxylate (65 µL, 72 mg, 0.41 mmol) in tetrahydrofuran
(9 mL) was stirred for 24 h at room temperature and then
concentrated under reduced pressure. The residue was puri-
fied by PLC (60% ethyl acetate – hexanes) to give 50
1
Preparation of 47
(68 mg). IR (film, cm–1): 3279, 1735. H NMR (CDCl3) δ:
Sodium hydroxide (25 mL of 1 mol/L, 25 mmol) was
added to a solution of the benzylidene methyl ester (5.75 g,
25.9 mmol) in methanol (40 mL). The reaction mixture was
stirred for 30 min, and the solvent was then removed. The
residue was dissolved in ethyl acetate (50 mL) and water
(30 mL). The aqueous phase was acidified with N-hydro-
chloric acid and extracted with dichloromethane (4 ×
30 mL). This extract was dried over anhydrous sodium sul-
fate and concentrated to give 3.16 g (59%) of product. IR
4.64–4.59 (1H, m), 4.37 (1H, d, 17.84 Hz), 3.98 (1H, d,
17.84 Hz), 3.86 (1H, dd, 12.64, 2.68 Hz), 3.65 (1H, dd,
12.64, 4.16 Hz), 2.89 (1H, dd, 16.56, 8.52 Hz), 2.85 (1H, dd,
16.56, 6.64 Hz), 1.47 (9H, s). EI-MS (m/z): 231 ([M]+). CI-
MS (m/z): 232. This compound was dissolved in trifluoro-
acetic acid (0.5 mL) and stirred under a nitrogen atmosphere
for 1.5 h. The solution was then freeze-dried to give 28 mg
1
(57%) of 39. H NMR (D2O) δ: 4.74 (1H, m), 4.36 (1H, d,
18.16 Hz), 4.34 (1H, d, 18.16 Hz), 3.78 (1H, dd, 12.88,
3.2 Hz), 3.74 (1H, dd, 12.88, 5.64 Hz), 3.02 (1H, dd, 17.0,
8.76 Hz), 2.79 (1H, dd, 17.0, 7.68 Hz). CI-MS (m/z): 176
([M + 1]).
1
(film, cm–1): 3037, 1713 (C=O). H NMR (CDCl3) δ: 7.49–
7.36 (5H, m, Ar), 5.96 (1H, s), 5.82 (1H, s), 4.68–4.60
(1H, m), 4.37 (1H, dd, 8.50, 6.20 Hz), 4.11 (1H, dd, 8.32,
© 2003 NRC Canada

932
Can. J. Chem. Vol. 81, 2003
4-Iodobutanoic acid
Hydrogenolysis of acetoacetamido N-benzyloxyglycine
ethyl ester
Trimethylsilyl chloride (5.0 mL, 4.28 g, 33.4 mmol) was
added dropwise under nitrogen to a suspension of sodium io-
dide (5.84 g, 34.0 mmol) in dichloromethane (60 mL). The
mixture was stirred for 15 min, and γ-butyrolactone (2.0 mL,
2.24 g, 26.0 mmol) was then added dropwise (15 min).
Stirring was continued at room temperature for 1.5 h and
then at reflux for 42 h. Water (50 mL) and ether (100 mL)
were added, and the organic layer was separated, washed
with 10% sodium thiosulfate (50 mL), and then extracted
with sodium bicarbonate (50 mL). The bicarbonate extract
was acidified to pH 2–3 with 1 mol/L hydrochloric acid and
extracted with ether (2 × 25 mL). The ether extract was
washed with saturated sodium chloride (50 mL), dried over
anhydrous sodium sulfate, and evaporated under reduced
Palladium on carbon (10%) (97 mg) was added to a solu-
tion of acetoacetamido N-benzyloxyglycine ethyl ester
(104 mg, 0.35 mmol) in ethanol (2 mL). The resulting
suspension was fitted with a balloon, flushed thrice with hy-
drogen, and then stirred under hydrogen for 1 h at room
temperature. After filtration through Celite and evaporation
of the filtrate, the oil was purified by chromatography
through a short column of silica gel using 60% ethyl acetate-
hexanes. The colourless oil (65.2 mg, 91%) was identified as
an 85:15 mixture of 54 and 55. IR (film, cm–1): 3390, 1746,
1
1687. H NMR (CDCl3) δ: 54: 4.52 (1H, d, 18.16 Hz), 4.24
(2H, q), 4.12 (1H, d, 18.16 Hz), 3.03 (1H, d, 16.76 Hz), 2.75
(1H, d, 16.76 Hz), 1.60 (3H, s), 1.29 (3H, t); 55: 4.54
(2H, s), 4.24 (2H, q), 3.75 (2H, s), 2.29 (3H, s), 1.29 (3H, s).
CI-MS (m/z): 204 ([M + 1]). Anal. calcd. for C8H13NO5: C
47.29, H 6.70, N 6.20; found: C 47.57, H 6.40, N 6.90.
1
pressure to give 814 mg (15%) of a colourless oil. H NMR
(CDCl3) δ: 3.25 (2H, t, 6.72 Hz), 2.52 (2H, t, 7.28 Hz), 2.13
(2H, q).
Reaction of 4-iodobutanoic acid with N-hydroxyglycine
tert-butyl ester
Synthesis of 56
Diketene (0.13 mL, 0.12 g, 1.67 mmol) was added to a so-
lution of N-hydroxyglycine benzyl ester (0.303 g, 1.67 mmol)
in chloroform (3 mL). The solution was stirred for 3 h at
room temperature, concentrated under reduced preesure, and
purified by PLC (60% EtOAc–hexanes). The product was a
Dicyclohexylcarbodiimide (51 mg, 0.25 mmol) (BDH)
was added under nitrogen to a cooled (0 °C) solution of the
acid (65 mg, 0.30 mmol) in dichloromethane (2 mL), and a
solution of N-hydroxyglycine tert-butyl ester (34 mg,
0.23 mmol) in dichloromethane (2 mL) was added dropwise,
with stirring. The cloudy mixture was stirred for 12 h at
0 °C – room temperature and was then filtered. The filtrate
was evaporated, and the residue was purified by flash chro-
matography (40% ethyl acetate – hexane) to give 16.8 mg
(21%) of 52 as a yellow oil.
1
yellow solid (0.16 g, 36%). H NMR (CDCl3) δ: 7.37–7.32
(5H, m, Ar), 5.18 (2H, s, CH2Ph), 4.53 (1H, d, 18.12 Hz),
4.19 (1H, d, 18.12 Hz), 3 (1H, d, 16.84 Hz), 2.76 (1H, d,
16.84 Hz), 1.60 (3H, s, CH3). CI-MS (m/z): 266 ([M + 1]).
The solid (150 mg, 0.57 mmol) was dissolved in ethanol,
and palladium on carbon (150 mg, 10%) was added. The
suspension was fitted with a balloon, flushed thrice with hy-
drogen, and then stirred under hydrogen for 1 h at room
temperature, filtered through Celite, and evaporated. The oil
was redissolved in water (10 mL), filtered, and the filtrate
was lyophilized to give a brown solid (0 mg, 81%) identified
Preparation of tert-butyl 3-oxo-[1,2]oxazinan-2-yl-
acetate
The oil 52 (4 mg, 0.012 mmol) was dissolved in dichloro-
methane (5 mL); triethylamine (13 µL, 9.44 mg,
0.093 mmol) was added, and the solution was stirred for 3 h.
Then DBU (4 µL, 4 1 mg, 0.027 mmol) was added, and stir-
ring was continued for 3.5 h. The solvent was then removed.
The residue was dissolved in ethyl acetate (5 mL), washed
with saturated sodium chloride, dried over anhydrous so-
1
as a 2:1 mixture of 56 and 57. H NMR (D2O) δ: 56: 4.39
(1H, d, 17.72 Hz), 4.20 (1H, d, 17.72 Hz), 3.17 (1H, d,
17.16 Hz), 2.77 (1H, d, 17.16 Hz), 1.60 (3H, s); 57: 4.38
(2H, s), 3.38 (2H, s), 2.29 (3H, s). CI-MS (m/z): 176 ([M +
1]).
1
dium sulfate, and concentrated to give 3 mg of 53. H NMR
(CDCl3) δ: 4.28 (2H, s, CH2), 4.16 (2H, t, 6.84 Hz, OCH2),
2.55 (2H, t, 7.2 Hz, CH2CO), 2.14 (2H, m), 1.47 (9H, s)
Benzyl-γ-bromoacetoacetate
Diketene (500 µL, 545 mg, 6.5 mmol) was dissolved in
dichloromethane (2 mL), and the solution was cooled to
–25 °C and treated dropwise with a solution of bromine
(333 µL, 1.02 g, 6.5 mmol) in dichloromethane (2 mL). Af-
ter the addition was complete, the solution was stirred at
–25 °C for 15 min, and benzyl alcohol (700 µL, 732 mg,
6.8 mmol) was added dropwise. Stirring was continued for
15 min, and the solution was warmed to room temperature
and evaporated. The residue was dissolved in diethyl ether
(20 mL), washed successively with saturated sodium bicar-
bonate (2 × 20 mL), water (20 mL), and saturated sodium
chloride (20 mL), dried over anhydrous magnesium sulfate,
and evaporated to give benzyl γ-bromoacetoacetate as a pale
yellow oil (1.70 g, 97%). IR (neat, cm–1): 3033, 1734, 1654.
1H NMR (CDCl3) δ: 7.36 (5H, m), 5.19 (2H, s), 4.02
(2H, s), 3.75 (2H, s). CI-MS (m/z): 271, 273 ([M + 1], [M +
Acetoacetamido N-benzyloxyglycine ethyl ester
A solution of N-benzyloxyglycine ethyl ester (0.159 g,
0.76 mmol) and 2,2,6-trimethyl-4H-1,3-dioxin-4-one (0.1 mL,
0.76 mmol) in toluene (0.76 mL) was immersed in a pre-
heated (110 °C) oil bath and stirred vigorously for 4 h and
monitored by TLC. The reaction mixture was then concen-
trated under reduced pressure and further dried at 1 torr for
2 h to give a viscous orange oil (0.222 g, 100%). IR
(CDCl3, cm–1): 1749 (C=O, s), 1670 (NC(O)). 1H NMR
(CDCl3) δ: 7.40 (5H, s, OCH2Ph), 4.88 (2H, s, OCH2Ph),
4.32 (2H, s, (O)CCH2C(O)), 4.22 (2H, q, OCH2CH3), 3.55
(2H, s, NCH2CO2Et), 2.25 (3H, s, CH3C(O)), 1.29 (3H, t,
OCH2CH3). CI-MS (m/z): 294 ([M + 1]). Anal. calcd. for
C15H19NO5: C 61.43, H 6.48, N 4.78; found: C 61.21, H
6.74, N 5.14.
© 2003 NRC Canada

Wolfe et al.
933
3]). Anal. calcd. for C11H11BrO3: C 48.69, H 4.05; found: C
48.55, H 3.94.
gel. Elution with 30:70 ethyl acetate:hexane gave 81 mg
(64%) of a viscous oil.
Protected 2-carboxymethyl-5-hydroxy-1,2-oxazine-3-one
A portion of the above oil (37 mg, 0.093 mmol) was dis-
solved in methylene chloride (2 mL), and triethylamine
(14 µL, 10.2 mg, 0.10 mmol) was added. The solution was
left under nitrogen for 1 h, and DBU (7.0 µL, 0.047 mmol)
was added, followed after 2.5 h by an additional 5.0 µL
(033 mmol) of DBU. Stirring was continued under nitrogen
for 3.2 h, and the reaction mixture was then diluted with
methylene chloride (10 mL), washed with water (10 mL),
dried over anhydrous sodium sulfate, and evaporated. Chro-
matography on silica gel and elution with 45:55 ethyl ace-
tate:hexane gave the protected oxazinone as a semisolid and
Benzyl-3-hydroxy-4-bromobutanoate
Benzyl bromoacetoacetate (500 mg, 1.84 mmol) was dis-
solved in a mixture of tetrahydrofuran (9 mL) and methanol
(1 mL), the solution was cooled in an ice-bath, and sodium
borohydride (72 mg, 1.90 mmol) was added in one portion.
Stirring was continued in the ice-bath for 15 min and ethyl
acetate (50 mL) and 1 mol/L hydrochloric acid (1.5 mL)
were added. The aqueous layer was separated, extracted with
ethyl acetate (20 mL), and the combined organic layers were
washed with saturated sodium bicarbonate (40 mL), dried
over anhydrous magnesium sulfate, and evaporated to give
benzyl 3-hydroxy-4-bromobutanoate 433 mg, 86%). IR
1
(neat, cm–1): 3443, 3064, 1731, 1624. H NMR (CDCl3) δ:
1
as a 1:1 mixture of diastereomers. H NMR (CDCl3) δ: 4.66
(1H, m, THP proton, one isomer), 4.41 (1H, m, THP proton,
second isomer), 4.32–4.45 (2H, m, both isomers), 3.83
(1H, m, 5-H of one isomer), 2.94–2.62 (2H, both isomers,
two ddd), 1.8 (2H, m), 1.7 (2H, m), 1.55 (2H, m), 1.47
(9H, s).
7.36 (5H, s), 5.17 (2H, s), 4.26 (1H, m), 3.50 (1H, dd, 5.0,
10.5 Hz), 3.47 (1H, dd, 5.6, 10.5 Hz), 3.06 (1H, d, 5.1 Hz),
2.72 (1H, dd, 5.0, 16.6 Hz), 2.69 (1H, dd, 7.3, 16.6 Hz). CI-
MS (m/z): 273, 275 ([M + 1], [M + 3]). Anal. calcd. for
C11H13BrO3·0.5H2O: C 53.75, H 5.88; found: C 53.65, H
5.60.
5-Hydroxy-3-oxo-[1,2]oxazinan-2-yl-acetic acid
The protected oxazinone (11 mg, 0.35 mmol) was dis-
solved in methylene chloride (1 mL), cooled in an ice-bath,
and trifluoroacetic acid (500 µL) was added. Additional
trifluoroacetic acid (3 µL) was added after 15 min, after an
additional 20 min (500 µL), and after an additional 30 min
(300 µL). After an additional 55 min, the solvent was re-
moved. The residue was redissolved in trifluoroacetic acid
(500 µL), evaporated after 30 min, and the residue shaken
with ethyl acetate (2 mL), water (1 mL), and sodium bicar-
bonate (3 mg). The aqueous phase was separated, washed
with ethyl acetate, and lyophilized to give 29, R = H, as the
Benzyl 3-[2-tetrahydropyranyloxy]-4-bromobutanoate
The bromohydrin (845 mg, 3.09 mmol) was dissolved in
dichloromethane (10 mL), and dihydropyran (300 µL,
277 mg, 3.29 mmol) and p-toluenesulfonic acid monohy-
drate (3 crystals) were added. The solution was stirred at
room temperature for 1.5 h. Diethyl ether (40 mL) was then
added, and the solution was washed with saturated sodium
bicarbonate (2 × 40 mL), dried over anhydrous sodium sul-
fate, and evaporated. Purification by column chromatogra-
phy using ethyl acetate – hexanes (3:7) gave the product 62
(1.07 g, 97%) as a 1:1 mixture of diastereomers. IR
(neat, cm–1): 3033, 1737. CI-MS (m/z): 357, 359 ([M + 1],
[M + 3]). Calcd. for C16H21BrO4: C 53.75, H 5.88; found: C
53.65, H 5.60.
1
sodium salt. H NMR (D2O) δ: 4.49 (1H, m), 4.29 (1H, dd,
12.1, 4.6 Hz), 4.28 (1H, d, 18.2 Hz), 4.15 (1H, d, 18.2 Hz),
4.02 (1H, ddd, 12.1, 3.24, 0.8 Hz), 2.92 (1H, dd, 16.8,
5.9 Hz), 2.52 (1H, ddd, 16.8, 3.16 Hz).
3-[2-Tetrahydropyranyloxy]-4-bromobutanoic acid
The protected bromohydrin 62 (73 mg, 0.20 mmol) was
dissolved in tetrahydrofuran (3 mL) and 10% palladium on
charcoal (65 mg) was added. The mixture was flushed thrice
with nitrogen, thrice with hydrogen, and then stirred under
hydrogen for 45 min. The mixture was filtered through
Celite, the Celite was rinsed with ethyl acetate (10 mL), and
the combined filtrates were evaporated to give the product
63 as a colourless oil, 54 mg (98%). IR (neat, cm–1): 1715.
CI-MS (m/z): 267, 269 ([M + 1], [M + 3]).
Coupling of 63 to the tert-butyl ester of N-hydroxyalanine
The acid 63 (54 mg, 0.20 mmol) was dissolved in di-
chloromethane (3 mL), the solution was cooled in an ice-
bath, stirred, and dicyclohexylcarbodiimide (41 mg,
0.20 mmol) was added, followed by a solution of the tert-
butyl ester in dichloromethane (2 mL). The mixture was
stirred in the ice-bath for 5 min; the ice-bath was then re-
moved, and stirring was continued for 3 h. The mixture was
filtered, and the filtrate was evaporated. The residue was
chromatographed on silica gel. Elution with ethyl acetate –
hexanes (2:3) gave 67 (74 mg, 54%) and products of O-
acylation. IR (CH2Cl2, cm–1): 3432, 1736, 1657. CI-MS
(m/z): 410, 412 ([M + 1], [M + 3]).
Coupling of 63 with tert-butyl N-hydroxyglycine
The acid 63 (85 mg, 0.32 mmol) was dissolved in methy-
lene chloride (3 mL), the solution was cooled in an ice-bath,
stirred, and dicyclohexylcarbodiimide (70 mg, 0.34 mmol)
was added, followed by a solution of the tert-butyl ester
(46.8 mg, 0.32 mmol) in methylene chloride (3 mL). The
cloudy mixture was stirred, initially at 0 °C, and allowed to
warm to room temperature. After 12 h, ether (20mL) was
added, the mixture was filtered, and the filtrate was evapo-
rated. The product was purified by chromatography on silica
Cyclization of 67
The N-acylated compound 67 (36 mg, 0.088 mmol) was
dissolved in dichloromethane (10 mL), cooled in an ice-bath,
and 1,8-diazabicyclo[5.4.0]undec-7-ene (15 µL, 0.1 mmol)
was added. The solvent was removed after 1 h. The residue
was dissolved in ethyl acetate (25 mL), and the solution was
washed with water (2 × 25 mL), dried over anhydrous mag-
© 2003 NRC Canada

934
Can. J. Chem. Vol. 81, 2003
nesium sulfate, and evaporated to give the protected
oxazinone 69 (25 mg, 85%). IR (CH2Cl2, cm–1): 1736, 1678.
CI-MS (m/z): 330 ([M + 1]). HR-MS-CI calcd. for
C16H27NO6: 330.1917 ([M + 1]); found: 330.1916.
Step 3: An inoculating needle was dipped into 20% glyc-
erol bacterial stock solution from Step 2 and, under sterile
conditions, streaked onto the surface of an agar plate. The
inverted plate was incubated at 30 °C for 12 h and then
stored at 4 °C.
Step 4: Under sterile conditions, a bacterial colony of
M. luteus was lifted from the surface of the agar plate from
Step 3 using a toothpick, added to liquid medium (10 mL),
and incubated at 250 rpm for 12 h at 30 °C.
Step 5: Under sterile conditions, the agar medium
(150 mL) was heated to 100 °C, cooled to 50 °C; liquid bac-
terial 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 °C.
Step 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 M. luteus from Step 5. The plate was
inverted and incubated for 12 h at 30 °C.
2-(5-Hydroxy-3-oxo-[1,2]oxazinan-2-yl-propionic acid
(29, R = Me)
The protected oxazinone 69 (10.1 mg, 0.031 mmol) was
dissolved in dichloromethane; the solution was cooled in an
ice-bath, and trifluoroacetic acid (1 mL, 13.0 mmol) was
added. The solution was stirred for 30 min, and the solvent
was then removed. The residue was dissolved in trifluoro-
acetic acid (500 µL), and the solution was stirred at room
temperature for 15 min and evaporated. The residue was
shaken with ethyl acetate (3 mL) and a solution of sodium
bicarbonate (2.8 mg) in water (2 mL). The aqueous phase
was lyophilized to give the sodium salt of 29, R = Me as an
approximately 1:1 mixture of RR–SS and RS–SR
diastereomers. 1H NMR (D2O) δ: 4.89 (1H, q, 7.3 Hz, α-CH,
one isomer), 4.88 (1H, q, 7.3 Hz, α-CH, second isomer),
4.72–4.69 (2H, m, H5, both isomers), 4.54 (1H, d, 10.5 Hz,
H6, one isomer), 4.53 (1H, d, 10.5 Hz, H6, second isomer),
4.35 (2H, d, 10.5 Hz, H6, both isomers), 2.97 (1H, d,
18.3 Hz, H4, one isomer), 2.95 (1H, d, 18.3 Hz, H4, second
isomer), 2.51 (2H, m, 18.3 Hz, H4, both isomers), 1.45 (3H,
d, 7.3 Hz, CH3, one isomer, 1.42 (3H, d, 7.3 Hz, CH3, sec-
ond isomer). CI-MS (m/z, for the acid): 190 ([M + 1]), 172
([M + 1 – H2O]). HR-MS-CI calcd. for C7H12NO5: 190.0716
([M + 1]); found: 190.0712.
Acknowledgement
This research was supported financially by an Individual
Operating Grant from the Natural Sciences and Engineering
Research Council of Canada (NSERC).
References
Bioassays
1. C.H. Stammer, A.N. Wilson, F.W. Holly, and K. Folkers. J.
Am. Chem. Soc. 77, 2346 (1955).
Materials
2. S. Harada, S. Tsubotani, T. Hida, H. Ono, and H. Okazaki.
Tetrahedron Lett. 27, 6229 (1986); Y. Nozaki, N. Katayama,
H. Ono, S. Tsubotani, T. Harada, H. Okazaki, and Y. Nakao.
Nature (London), 325, 179 (1987).
Agar medium (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 sterilized by
autoclaving at 121 °C for 20 min.
3. D. Peisach, D.M. Chipman, P.W. Van Ophem, J.M. Manning,
and D. Ringe. J. Am. Chem. Soc. 120, 2268 (1998).
4. F.C. Neuhaus and J.L. Lynch. Biochemistry, 3, 471 (1964); B.
Badet and C.T. Walsh. Biochemistry, 24, 1333 (1985).
5. S. Harada, S. Tsubotani, T. Hida, K. Koyama, M. Kondo, and
H. Ono. Tetrahedron, 44, 6589 (1988); Y. Nakao. Spec. Publ.
R. Soc. Chem. 70, 119 (1989); Y. Nozaki, N. Katayama, S.
Harada, H. Ono, and H. Okazaki. J. Antibiot. 42, 84 (1989).
6. D.J. Tipper and J.L. Strominger. Proc. Natl. Acad. Sci. U.S.A.
54, 1133 (1965).
Preparation of agar plates
Under sterile conditions, the agar medium (150 mL) was
heated to 100 °C, cooled to 50 °C, and distributed into ten
petri dishes. Once the medium had hardened, the plates were
inverted and stored at 4 °C.
Liquid medium
7. M. Kitagawa and T. Tomiyama. J. Biochem. (Tokyo), 11, 265
(1929); M. Kitagawa and H. Yamada. J. Biochem (Tokyo), 16,
339 (1932); M. Kitagawa and K.-I. Monobe. J. Biochem (To-
kyo), 18, 333 (1933); M. Kitagawa and A. Takani. J. Biochem
(Tokyo), 23, 181 (1936); M. Kitagawa. J. Biochem (Tokyo),
24, 197 (1936); 25, 23 (1937).
8. G.A. Rosenthal. Biochem. Syst. Ecol. 5, 219 (1977).
9. A.J.L. Cooper. Arch. Biochem. Biophys. 233, 603 (1984).
10. E.-L. Rahiala, M. Kekomaki, J. Janne, A. Raina, and N.C.R.
Raiha. Biochim. Biophys. Acta, 227, 337 (1971); E.-L.
Rahiala. Acta. Chem. Scand. 27, 3861 (1973).
Yeast extract (0.3 g), peptone (0.5 g), and glucose (0.1 g)
were dissolved in deionized water; the volume was diluted
to 100 mL, and the solution was sterilized by autoclaving at
121 °C for 20 min.
Methods
Step 1: Under sterile conditions, freeze-dried M. luteus
was dissolved in liquid medium (10 mL) and incubated at
250 rpm for 12 h at 30 °C.
Step 2: Liquid bacterial culture from step 1 (0.5 mL) and
40% glycerol in deionized water (0.5 mL) were vortexed un-
der 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 °C.
11. G.A. Rosenthal. Life Sci. 60, 1635 (1997).
12. B.J. Berger. Antimicrob. Agents Chemother. 44, 2540 (2000).
13. (a) R.M. Khomutov, M.Ya. Karpeiskii, and E.S. Severin. Izv.
Akad. Nauk. SSSR Ser. Khim. 1074 (1962); (b) M. Frankel, Y.
© 2003 NRC Canada

Wolfe et al.
Knobler, E. Bonni, S. Bittner, and G. Zvilichovsky. J. Chem.
935
26. S. Wolfe. Can. J. Chem. 72, 1014 (1994); S. Wolfe, C.-K.
Kim, and K. Yang. Can. J. Chem. 72, 1033 (1994).
27. S. Wolfe, C.-K. Kim, K. Yang, N. Weinberg, and Z. Shi. J.
Am. Chem. Soc. 117, 4240 (1995); S. Wolfe, Z. Shi, K. Yang,
S. Ro, N. Weinberg, and C.-K. Kim. Can. J. Chem. 76, 114
(1998).
28. W.J. Hehre, L. Radom, P. von R. Schleyer, and J.A. Pople. Ab
initio molecular orbital theory. Wiley-Interscience, New York.
1986.
29. S. Wolfe, A.V. Buckley, and N. Weinberg. Can. J. Chem. 79,
1284 (2001).
30. R.M. Sweet and L.F. Dahl. J. Am. Chem. Soc. 92, 5489 (1970.
31. R.B. Woodward. In The chemistry of penicillin. Edited by H.T.
Clarke, J.R. Johnson, and R. Robinson. Princeton University
Press, Princeon, N.J. 1949. p. 440.
Soc. C, 1746 (1969).
14. J. Smrt, J. Beranek, and M. Horak. Coll. Czech. Chem.
Commun. 24, 1672 (1959); N.K. Kotchetkov, R.M. Khomutov,
E.S. Severin, M.Ya. Karpeiskii, E.I. Budovskii, and V.I.
Erashko. Zh. Obsch. Khim. 29, 3417 (1959).
15. M.Ya. Karpeiskii, R.M. Khomutov, and S. Severin. Zh. Obsch.
Khim. 32, 1357 (1962).
16. D.D. Nyberg and B.E. Christensen. J. Am. Chem. Soc. 79,
1222 (1957).
17. A.J. Ozinskas and G.A. Rosenthal. J. Org. Chem. 51, 5047
(1986).
18. R.M. Khomutov, M. Karpeiskii, M.A. Breger, and E.S.
Severin. Vopr. Med. Khim. 8, 389 (1962).
19. R.M. Khomutov, M.Y. Karpeiskii, M.A. Breger, and E.S.
Severin. Fed. Proc. 22, 637 (1963).
32. A. Rauk. Orbital interaction theory of organic chemistry. John
Wiley, New York, N.Y. 1994.
20. (a). T. Hirose, K. Chiba, S. Mishio, J. Nakano, and H. Uno.
Heterocycles, 19, 1019 (1982); (b) C.K. Zercher and M.J.
Miller. Tetrahedron Lett. 30, 7009 (1989); (c) A. Biswas, M.J.
Miller, M.A. Crook, and S.R. Woulfe. Spec. Publ. R. Soc.
Chem. 52, 305 (1985).
21. S.R. Woulfe and M.J. Miller. Tetrahedron Lett. 25, 3293
(1984); S.R. Woulfe and M.J. Miller. J. Med. Chem. 28, 1447
(1985); M.J. Miller. Acc. Chem. Res. 19, 49 (1986).
22. D. Kroenthal, P. Kuester, and W.H. Koster. Intl. Congr.
Heterocycl. Chem. 10th, Abstract G3–25, 1985.
33. T.A. Albright, J.K. Burdett, and M.-H. Whangbo. Orbital in-
teractions in chemistry. John Wiley, New York, N.Y. 1985.
34. S. Wolfe and Z. Shi. Isr. J. Chem. 40, 343 (2000).
35. A. Tsuji, T. Yamana, S. Matsutani, and N. Tsuji. Heterocycles,
8, 153 (1977). See also footnote 18 of J. Marchand-Brynert
and L. Ghosez. Recent Prog. Chem. Synth. Antibiot. 727
(1990).
36. J.E. Baldwin, C. Lowe, and C.J. Schofield. Tetrahedron Lett.
31, 2211 (1990).
37. K. Yoshioka and T. Miwa. Jpn Kokai Tokkyo Koho JP
62215585. Sept 22, 1987. Chem. Abstr. 108, P186753z (1988).
38. S. Wolfe, M.-C. Wilson, M.-H. Cheng, G.V. Shustov, and C.I.
Akuche. Can. J. Chem. This issue.
39. J. Lamotte, G. Dive, and J.-M. Ghuysen. Eur. J. Med. Chem.
26, 43 (1991).
40. N.C. Cohen. J. Med. Chem. 26, 259 (1983).
41. H.R. Pfaendler, J. Gosteli, R.B. Woodward, and G. Rihs. J.
Am. Chem. Soc. 103, 4526 (1981).
42. S. Wolfe, K. Yang, and M. Khalil. Can. J. Chem. 66, 2733
(1988).
43. T.N. Salzmann, R.W. Ratcliffe, B.G. Christensen, and F.A.
Bouffard. J. Am. Chem. Soc. 102, 6161 (1980).
44. J.J.P. Stewart. J. Comput. Chem. 10, 209 (1989).
45. E.F. Schoenewaldt, R.B. Kinnel, and P. Davis. J. Org. Chem.
33, 4270 (1968).
46. T. Polinski and A. Chimiak. J. Org. Chem. 41, 2092 (1976).
47. E. Buehler and G.B. Brown. J. Org. Chem. 32, 265 (1967).
48. J.D.M. Herscheid and H.C.J. Ottenheijm. Tetrahedron Lett. 51,
5143 (1978).
23. S.R. Woulfe, H. Iwagami, and M.J. Miller. Tetrahedron Lett.
26, 3891 (1985); S.R. Woulfe and M.J. Miller. J. Org. Chem.
51, 3133 (1986).
24. D.B. Boyd, C. Eigenbrot, J.M. Indelicato, M.J. Miller, C.E.
Pasini, and S.R. Woulfe. J. Med. Chem. 30, 528 (1987).
25. D.B. Boyd. In Amide linkage. Edited by A. Greenberg, C.M.
Breneman, and J.F. Liebman. John Wiley, New York. 2000.
p. 337; J. Frau, J. Donosa, F. Munoz, and F.G. Blanco. Helv.
Chim. Acta, 77, 1557 (1994); G. Dive, D. Dehareng, and D.
Peeters. Int. J. Quantum Chem. 58, 85 (1996); J. Frau, J.
Donoso, F. Munoz, and F. Garcia-Blanco. Helv. Chim. Acta,
79, 353 (1996); J. Pitarch, M.F. Ruiz-Lopez, J.-L Pascual-
Ahuir, E. Silla, and I. Tunon. J. Phys. Chem. B, 101, 3581
(1997); J. Frau, J. Donoso, F. Munoz, B. Vilanova, and F. Gar-
cia-Blanco. Helv. Chim. Acta. 80, 739 (1997); M. Coll, J.
Frau, J. Donoso, and F. Munoz. THEOCHEM. 426, 323
(1998); J. Frau, J. Donoso, F. Munoz, B. Vilanova, and F. Gar-
cia-Blanco. THEOCHEM. 426, 313 (1998); J. Pitarch, M.F.
Ruiz-Lopez, E. Silla, J.-L. Pascual-Ahuir, and I. Tunon. J. Am.
Chem. Soc. 120, 2146 (1998); N. Diaz, D. Suarez, and T.L.
Sordo. Chem. Eur. J. 5, 1045 (1999); N. Diaz, D. Suarez, and
T.L. Sordo. J. Org. Chem. 64, 3281 (1999); I. Massova and
P.A. Kollman. J. Phys. Chem. B, 103, 8628 (1999); G. Dive
and D. Dehareng. Int. J. Quantum Chem. 73, 161 (1999); J.
Pitarch, J.-L. Pascal-Ahuir, E. Silla, I. Tunon, and M.F. Ruiz-
Lopez. J. Comput. Chem. 20, 1401 (1999); J. Pitarch, J.-L.
Pascual-Ahuir, E. Silla, I. Tunon, and V. Moliner. J. Chem.
Soc. Perkin Trans. 2, 1351 (1999); J. Pitarch, J.-L. Pascual-
Ahuir, E. Silla, I. Tunon, M.F. Ruiz-Lopez, C. Millot, and J.
Bertran. Theor. Chem. Acc. 101, 336 (1999); R. Lopez, M.I.
Menendez, N. Diaz, D. Suarez, P. Campomanes, and T.L.
Sordo. Rec. Res. Dev. in Phys. Chem. 4, 157 (2000); J.
Pitarch, J.-L. Pascual-Ahuir, E. Silla, and I. Tunon. Perkin 2.
761 (2000); N. Diaz, D. Suarez, T.L. Sordo, and K.M. Merz,
Jr. J. Phys. Chem. B, 105, 11302 (2001); N. Diaz, D. Suarez,
T.L. Sordo, I. Tunon, and E. Silla. Chem. Eur. J. 8, 859
(2002); N. Diaz, T.L. Sordo, K.M. Merz, Jr., and D. Suarez. J.
Am. Chem. Soc. 125, 672 (2003).
49. M.W. Tijhuis, J.D.M. Herscheid, and H.C.J. Ottenheijm. Syn-
thesis, 890 (1980).
50. T. Tsuji, T. Kataoka, M. Yoshioka, Y. Sendo, S. Hirai, T.
Maeda, and W. Nagata. Tetrahedron Lett. 30, 2793 (1979).
51. M. Sato, J. Sakaki, K. Takayama, S. Kobayashi, M. Suzuki,
and C. Kaneko. Chem. Pharm. Bull. 38, 94 (1990).
52. J. Zhu, S. Robin, N. Goasdoue, A. Loupy, and H. Galons.
Synlett, 97 (1995).
53. A. Koziara, K. Osowska-Pacewicka, S. Zawadzki, and A.
Zwierzak. Synthesis, 202 (1985).
54. J. Alexander, M.L. Renyer, and H. Veerapanane. Synth.
Commun. 25, 3875 (1995).
55. J.E. Baldwin. J. Chem. Soc. Chem. Commun. 734 (1976).
56. A. Streitwieser, Jr. Chem. Rev. 56, 571 (1956).
57. G. Cardillo and M. Orena. Tetrahedron, 46, 3321 (1990); J.M.
Barks and D.W. Knight. Tetrahedron Lett. 35, 7259 (1994).
58. H.J. Reich and S.L. Peake. J. Am. Chem. Soc. 100, 4888
(1978).
© 2003 NRC Canada

936
Can. J. Chem. Vol. 81, 2003
59. G. Procter, J. Nally, and N.H.R. Ordsmith. Tetrahedron, 51,
12 837 (1995).
66. M. Larcheveque and S. Henrot. Tetrahedron, 46, 4277 (1990).
67. R.J. Clemens and J.A. Hyatt. J. Org. Chem. 50, 2431 (1985).
68. R.J. Clemens. Chem. Rev. 86, 241 (1986).
69. K.F. Bernady, M.B. Floyd, J.F. Poletto, and M.J. Weiss. J.
Org.Chem. 44, 1438 (1979).
60. T. Takigawa and T. Matsuo. Tetrahedron, 35, 933 (1979).
61. S. Saito, T. Hasegawa, M. Inaba, R. Nishida, T. Fujii, S.
Nomizu, and T. Moriwake. Chem. Lett. 1389 (1984).
62. C. Brockway, P. Kocienski, and C. Pant. J. Chem. Soc. Perkin
Trans. 1, 875 (1984).
70. D.D. Perrin and W.L.F. Armarego. In Purification of labora-
tory chemicals. 3rd ed. Pergamon. 1988.
63. D.I. Highes. Org. React. (New York), 42, 335 (1992).
64. M.F. Lipton, A. Basha, and S.A. Weinreb. Org. Synth. 59, 49
(1979).
71. W.C. Still, M. Kahn, and A. Mitra. J. Org. Chem. 43, 2923
(1978).
65. D.C. Harrowven and R.F. Dainty. Tetrahedron Lett. 36, 6739
(1995).
© 2003 NRC Canada