Synthesis, hydrolysis, and evaluation of 3-acylamino-3,4-dihydro-2-oxo-2H-1,3-benzoxazinecarboxylic acids and linear azadepsipeptides as potential substrates/inhibitors of β-lactam-recognizing enzymes

Article abstract of DOI:10.1002/1099-0690(200101)2001:1<141::aid-ejoc141>3.0.co;2-j

The title compounds can be considered as stabilized aza analogs of previously studied dihydrobenzopyranones and linear depsipeptides, which behave as substrates or inhibitors of β-lactamases. Treatment of substituted hydrazides 9b and 9b′ with a phosgene substitute resulted in a series of N-methylated 3-acylamino-3,4-dihydro-2-oxo-2H-1,3-benzoxazine-7-and -8-carboxylic acids 2b and 2b′. However, in the case of the corresponding free NH hydrazide 9a(m), a competitive cyclization gave instead a stable 4H-1,3,4-oxadiazol-5-one 10a. To avoid this unwanted cyclization, an N-(p-methoxy)-benzylated hydrazide 9b″ was prepared. After formation of the benzoxazinone ring with carbonyldiimidazole, the removal of this new N¹-hydrazide protecting group was achieved with methanesulfonic acid in trifluoroacetic acid to give the expected 3-phenacetamido-3,4-dihydro-2-oxo-2H-1,3-benzoxazine-7-carboxylic acid 2a(m). The corresponding linear azadepsipeptides 5 were generally obtained by reaction of a hydrazide with 3-tert-butoxycarbonylphenyl chlorocarbonate. Hydrolysis of the title compounds in buffer at neutral pH was more rapid than anticipated because of the presence of mechanisms more facile than the classical B_AC2. Hydrolysis of the cyclic azadepsipeptide 2a(m), for example, involved intramolecular nucleophilic participation by the amido side chain and a slowly hydrolyzing oxadiazolone intermediate (10a). These compounds, unlike their parent depsipeptides, were not substrates or inhibitors of β-lactamase or DD-peptidase. This result probably arises from a combination of the poor carbonyl electrophilicity and the close to planar geometry of the nitrogen atom of the oxazin-2-one ring. Wiley-VCH Verlag GmbH, 2001.

Full text of DOI:10.1002/1099-0690(200101)2001:1<141::aid-ejoc141>3.0.co;2-j

FULL PAPER
Synthesis, Hydrolysis, and Evaluation of 3-Acylamino-3,4-dihydro-2-oxo-2H-
1,3-benzoxazinecarboxylic Acids and Linear Azadepsipeptides as Potential
Substrates/Inhibitors of β-Lactam-Recognizing Enzymes
D. Cabaret,[a] M. Garcia Gonzalez,[a] M. Wakselman,*[a] S. A. Adediran,[b] and
R. F. Pratt*[b]
Keywords: Enzyme inhibitors / Enzymes / Heterocycles / Peptidomimetics / Protecting groups
The title compounds can be considered as stabilized aza ana- 1,3-benzoxazine-7-carboxylic acid 2a(m). The corresponding
logs of previously studied dihydrobenzopyranones and linear
depsipeptides, which behave as substrates or inhibitors of β- tion of a hydrazide with 3-tert-butoxycarbonylphenyl chloro-
lactamases. Treatment of substituted hydrazides 9b and 9bЈ carbonate. Hydrolysis of the title compounds in buffer at
with a phosgene substitute resulted in a series of N-methyl- neutral pH was more rapid than anticipated because of the
linear azadepsipeptides 5 were generally obtained by reac-
ated 3-acylamino-3,4-dihydro-2-oxo-2H-1,3-benzoxazine-7-
and -8-carboxylic acids 2b and 2bЈ. However, in the case of
presence of mechanisms more facile than the classical BAC2.
Hydrolysis of the cyclic azadepsipeptide 2a(m), for example,
the corresponding free NH hydrazide 9a(m), a competitive involved intramolecular nucleophilic participation by the am-
cyclization gave instead a stable 4H-1,3,4-oxadiazol-5-one
10a. To avoid this unwanted cyclization, an N-(p-methoxy)-
benzylated hydrazide 9bЈЈ was prepared. After formation of
ido side chain and a slowly hydrolyzing oxadiazolone inter-
mediate (10a). These compounds, unlike their parent depsi-
peptides, were not substrates or inhibitors of β-lactamase or
the benzoxazinone ring with carbonyldiimidazole, the re- DD-peptidase. This result probably arises from a combina-
moval of this new N1-hydrazide protecting group was
achieved with methanesulfonic acid in trifluoroacetic acid to
tion of the poor carbonyl electrophilicity and the close to
planar geometry of the nitrogen atom of the oxazin-2-one
give the expected 3-phenacetamido-3,4-dihydro-2-oxo-2H- ring.
Introduction
Non-β-lactam inhibitors of β-lactamases and DD-trans-
peptidases remain attractive targets for antibacterial
drugs.[1] Recently, we observed that some δ-lactones, such
as the dihydrobenzopyranones 1a (ortho- and meta-CO2H
isomers; Figure 1), were new substrates of β-lactamases and
inhibitors of D,D-peptidases.[2,3] However, their rates of
Figure 1. Structures of compounds 1a, 2a, 3, 4, and 5
spontaneous hydrolysis were high. To increase the stability
of the molecules, we turned our attention towards their aza
analogs:
the
3-acylamino-3,4-dihydro-2-oxo-2H-1,3-
benzoxazinecarboxylic acids 2a. Incorporation of an elec-
tron-donating nitrogen atom α to the carbonyl function
should stabilize the ground state of the molecule. Moreover,
if a β-lactamase were to be acylated by a dihydrobenzoxazi-
none, the presence of this atom should also stabilize the
acyl enzyme. A derivative of the parent heterocycle, 3,4-di-
Such a stabilizing effect due to an adjacent nitrogen atom
has previously also been observed in the case of linear aza-
depsipeptides, which behave as inhibitors or as active site
titrants of several serine proteases, such as elastase, trypsin,
or thrombin.[7] Azadepsipeptides derived from lactic acid 4
(RЈ ϭ Me) or from other hydroxy acids have been prepared
for analogous purposes, but they were not inhibitors of
class A and class C β-lactamases.[8] Since the replacement
of the alcohol leaving group with an acidic phenol should
give more reactive compounds,[7a] we also planned to syn-
thesize azadepsipeptides derived from meta-hydroxybenzoic
acids: compounds of type 5. These molecules may be con-
sidered as linear analogs of the 3-acylamino-3,4-dihydro-2-
oxo-2H-1,3-benzoxazinecarboxylic acids 2.
hydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one
3,
is
known to react with chymotrypsin,[4] whereas several of its
analogues or homologues result in the production, with
cholinesterases, of stabilized carbamoyl-bound enzymes.[5,6]
[a]
´
SIRCOB, ESA CNRS 8086, Universite de Versailles,
ˆ
Bat. Lavoisier, 45, avenue des Etats Unis, 78035 Versailles,
France
E-mail: michel.wakselman@chimie.uvsq.fr
Department of Chemistry, Wesleyan University,
Middletown, Connecticut 06459, USA
E-mail: rpratt@wesleyan.edu
[b]
Eur. J. Org. Chem. 2001, 141Ϫ149
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0101Ϫ0141 $ 17.50ϩ.50/0
141

D. Cabaret, M. G. Gonzalez, M. Wakselman, S. A. Adediran, R. F. Pratt
FULL PAPER
Results and Discussion
Syntheses
To the best of our knowledge, 3-amino- and 3-acylamino-
3,4-dihydro-2H-1,3-benzoxazin-2-ones (3-acylamino-3,4-di-
hydro-2-oxobenz[e][1,3]oxazines) are unknown compounds.
A retrosynthetic analysis (Scheme 1) showed a possible me-
ans to prepare them by treatment of substituted hydrazides
9a (o: ortho-COOH; m: meta-COOH) with phosgene or a
phosgene substitute, such as carbonyldiimidazole[9,5] or
bis(trichloromethyl)carbonate.[10] The substituted hy-
drazides 9a(o,m) could be obtained by reduction of the cor-
responding hydrazones 8a(o,m),[11] themselves prepared by
condensation of hydrazides 6a with a substituted hydroxy-
benzaldehyde 7o or 7m.
Scheme 2. Unwanted cyclization of the substituted hydrazide 9a(m)
to give 5(4H)-1,3,4-oxadiazolone 10a, instead of the expected 3,4-
dihydro-2-oxo-3-phenylacetamino-2H-1,3-benzoxazine-7-carboxylic
lxacid 2a(m)
The above results made it clear that the synthesis of
benzoxazinones possessing a free NH group would require
the cleavage of a suitable N-protecting group after the
formation of the oxazinone ring. While N2-protected hy-
drazides are known,[15] N1 hydrazide protecting groups have
not yet been reported, to the best of our knowledge. Using
the analogy with the protection of carboxamides,[16] we
turned our attention towards the use of a p-methoxybenzyl
substituent and its possible selective removal under acidic
conditions. The starting methoxybenzylated hydrazide 6bЈЈ
was prepared by nitrosation[17] of the corresponding N-sub-
stituted phenylacetamide 11,[18] followed by reduction of
the nitrosamide 12 by means of zinc metal in acetic acid.[19]
A partial hydrogenolysis of the NϪN bond was observed
Scheme 1. Retrosynthetic analysis for the preparation of the 3-acyl-
amino-3,4-dihydro-2-oxo-2H-1,3-benzoxazine-7- and -8-carboxylic
acids 2
The 5-carboxysalicylaldehyde 7m was prepared by a during this reduction step. Condensation of this hydrazide
ReimerϪTiemann or a modified Duff reaction.[12] Con- 6bЈЈ with substituted salicylaldehyde 7m, as above, and re-
densation of this aldehyde with phenylacetylhydrazide, fol- duction of the intermediate hydrazone 8bЈЈ (R ϭ CH2C6H5)
lowed by reduction of hydrazone 8a(m) with sodium cyano- yielded the substituted hydrazides 9bЈЈ (Scheme 3). Cycliza-
borohydride, led to the substituted hydrazide 9a(m) (R ϭ tion with carbonyldiimidazole then furnished the substi-
CH2C6H5; m-CO2H). However, treatment of this product tuted benzoxazinone 2bЈЈ in good yield. Removal of the p-
with carbonyldiimidazole or triphosgene did not give the methoxybenzyl substituent in compound 2bЈЈ, to give the
benzoxazinone, but led instead to the 1,3,4-oxadiazol- benzoxazinone 2a (m), required treatment with methanesul-
5(4H)-one 10a: an aza analog[13] of the 5(4H)-oxazolones fonic acid in trifluoroacetic acid; neat TFA was unable to
(compounds well known for their role in the epimerization effect the cleavage.[16b]
of amino acid derivatives). However, these 1,3,4-oxadiazol-
While the benzylic protons were nonequivalent in the
5(4H)-ones are not very reactive species,[8,13] and molecule NMR spectra of the series of N-substituted benzoxazinones
10a did not rearrange to the benzoxazinone 2a(m) on heat- 2b, 2bЈ, and 2bЈЈ (RЈ ϭ Me or p-MeO-bzl), those in the free
ing (Scheme 2).
NH benzoxazinone 2a appeared as a singlet.
To avoid participation of the neighboring amide group,
For the synthesis of the linear azadepsipeptides 5, the 3-
leading to the oxadiazolone, we next used N-alkylated hy- tert-butoxycarbonylphenyl chlorocarbonate 13 was pre-
drazides as starting components (Scheme 3). Treatment of pared from the corresponding phenol[20] and triphos-
methylhydrazine with phenylacetic or benzoic anhydride gene.[21] Treatment of this compound 13 with a series of free
gave the corresponding hydrazides 6b and 6bЈ.[14] Treatment or N-methylated phenacetyl or benzoyl hydrazides 6b and
of these hydrazides with substituted salicylaldehyde 7m or 6c gave substituted hydrazides 14 (Scheme 4). However,
7o and reduction of the intermediate hydrazones 8b (R ϭ substituted diacylhydrazine 14Јc was obtained in poor yield
CH2C6H5) and 8bЈ (R ϭ C6H5) resulted in the substituted using this method. In this case, a better yield was obtained
hydrazides 9b and 9bЈ. Subsequent cyclization with carbon- by first treating N-methylhydrazine with chlorocarbonate
yldiimidazole led to the N-methylated benzoxazinones 2b 13. Acylation of carbamate 15 then smoothly afforded com-
and 2bЈ in good yields.
pounds 14Јc. Subsequent cleavage with trifluoroacetic acid
142
Eur. J. Org. Chem. 2001, 141Ϫ149

Potential Substrates/Inhibitors of β-Lactam-Recognizing Enzymes
FULL PAPER
Scheme 3. Synthesis of the N-substituted 3-acylamino-3,4-dihydro-2-oxo-2H-1,3-benzoxazine-7- and -8-carboxylic acids 2b؊bЈЈ and the
free NH 3,4-dihydro-2-oxo-3-phenylacetamino-2H-1,3-benzoxazine-7-carboxylic acid 2a(m)
Scheme 4. Synthesis of the linear azadepsipeptides 5
of the tert-butyl protecting groups in the whole series of methyl group of 2b(m), so distant from the carbonyl group,
molecules 14 furnished the expected azadepsipeptides 5.
could have such an effect on reactivity.[21]
Table 1. Rate constants for spontaneous reaction of azadepsipep-
tides in aqueous buffer; 0.1  MOPS buffer, pH ϭ 7.5, 25 °C
Solution and Enzyme Studies
The hydrolysis of the azadepsipeptides (carbazates) in
aqueous buffer was monitored spectrophotometrically (re-
lease of hydroxycarbonylphenol), and pseudo-first order
rate constants were calculated from absorption vs. time
data. These rate constants are reported in Table 1, along
with those of the acyclic and cyclic depsipeptides 16 (Fig-
ure 2) and 1a(m). There are several points of interest here.
Firstly, it is noticeable and surprising that 2a(m) is more
reactive than its deaza analogue 1a(m), conflicting with the
original rationale for the preparation of the aza compounds.
Similarly, 5a is slightly more reactive than 16. One would
have expected carbazates to be several orders of magnitude
less reactive to direct nucleophilic cleavage by solvent than
Compound
kobs ϫ 105 [sϪl
]
2a(m)
2b(m)
2b(o)
5a
290
0.56
0.72
1.68
4.46
3.97
6.02
6.74
56
5b
5c
5Јa
5Јc
1a(m)[a]
16[a]
1.0
[a]
Ref.[2]
Both of the above phenomena can be rationalized in
the analogous esters. Another point, which emerges below terms of intramolecular nucleophilic participation of the
as connected with the above, is the considerably lower react- amido side chain of 2a(m) in the hydrolysis of this com-
ivity of 2b(m) relative to 2a(m). It is striking that the N- pound. The alkaline hydrolysis of aryl esters of N-acyl am-
Eur. J. Org. Chem. 2001, 141Ϫ149
143

D. Cabaret, M. G. Gonzalez, M. Wakselman, S. A. Adediran, R. F. Pratt
FULL PAPER
ated from the former compound relative to that of the ox-
azolone from the latter.
Figure 2. Structure of the depsipeptide 16
Scheme 6. Dissociative mechanism for the hydrolysis of the linear
azadepsipeptide 5b
ino acids is well known to involve such catalysis and thus
a 5(4H)-oxazolone intermediate.[22,23] N-Methylation of the
Within the constraints dictated by their lifetimes in aque-
amido side chain significantly reduces the observed rates.[24] ous buffer at pH 7.5 (Table 1), none of the carbamates ap-
Furthermore, the alkaline hydrolysis of analogous azapep- peared to be either a substrate or an inhibitor of the class
tide esters is also known to involve nucleophilic amide side C Enterobacter cloacae P99 β-lactamase or the class A
chain participation and formation of 5(4H)-1,3,4-oxadiazo- TEM β-lactamase. Compound 2a(m) also had no effect on
lones.[7j,25,26] These latter heterocycles have been found to the activity of the Streptomyces R61 DD-peptidase. The
be quite refractory towards nucleophiles, particularly under spontaneous reaction rate of 2a(m) was smaller at lower
alkaline conditions,[7e,13a,26,27] under which an inert anion is pH; e.g., the pseudo-first order rate constant for disappear-
likely to form.[7e]
ance of 2a(m) at pH 6.5 (20 m, MES) was 4.1 ϫ 10Ϫ4sϪ1
.
Accordingly, the hydrolysis of 2a(m) was examined more Despite its reduced lability at this pH, 2a(m) was neither
l
closely by H NMR spectroscopy. Approximately 1 mg of a substrate nor an inhibitor of the P99 β-lactamase under
2a(m) was dissolved in 0.6 mL of 20 m NaHCO3 in D2O. these conditions.
1
A H NMR spectrum after ca. 10 min [2a(m) did not dis-
Although the depsipeptides la(m) and 16 have been
solve quickly] showed the presence of only one molecular shown to be substrates of class A and C β-lactamases,[2,3,29]
species in solution [δ ϭ 3.75 (s), 3.92 (s), 7.2Ϫ7.7 (m)]. No their aza analogs 2a(m) and 5 showed no sign of interaction
change was observed over a 4 h period. After 24 h, however, with the active sites of these enzymes, nor with that of the
there was evidence of reaction, with the appearance of small Streptomyces R61 DD-peptidase. Thus, the better leaving
additional peaks. This reaction was then accelerated by group present in 5 did not appear to improve the reactivity
heating the sample at 40 °C for 4 days, at which point the of these compounds with the β-lactamases, relative to those
1
reaction was almost complete. The H NMR spectrum of compounds previously examined.[8] One reason for this
the final product contained singlets at δ ϭ 3.42 and 3.97, negative result could still be the intrinsically much smaller
and an aromatic multiplet further downfield. A comparison carbonyl electrophilicity of these compounds (carbazates)
with spectra of authentic samples under the same condi- relative to that of their deaza analogs (esters); this differ-
tions showed that the initially observed product was the ence is perhaps indicated most directly by comparison of
oxadiazolone 10a and the slowly formed material was 9a, the hydrolysis rate of 2b(m) with that of la(m) (Table 1). A
the expected hydrolysis product. This experiment proved similar difference in the reactivities of azapeptide and pep-
that the rapid reaction of 2a(m) in neutral buffer occurs tide nitrophenyl esters towards the nucleophile hy-
according to Scheme 5, yielding an oxadiazolone, which hy- droxylamine has been reported.[7i] Certainly, peptides are
drolyses only very slowly.
much poorer β-lactamase substrates than are the analogous
It seems possible from the hydrolysis rates that 5a also esters.[30,31] This is not true of the R61 DD-peptidase, how-
hydrolyses by such a mechanism. The reactivity of 5b, how- ever, with which amides and esters are comparable as sub-
ever, suggests that the alternative mechanism of Scheme 6 strates.[30] In this case, subtle features of molecular shape
might also contribute in these acyclic compounds.[7f,7j,28] must account for the low reactivity of la(m),[2] and now 2a
The greater rate of hydrolysis of 2a(m) relative to that of and 5, with the enzyme. The lack of reactivity of 5a and 5c
5c presumably reflects the greater reactivity of the (cis) 6- with the DD-peptidase, contrasting with the reactivity of 16
membered ring carbamate over that of the acyclic (trans) and its -alanine analog,[30] must also reflect such differ-
species. The greater reactivity of 2(a)m over 1a(m) (and 5a ences. In particular, the probably close to planar arrange-
over 16), despite the fact that the former is a carbazate, ment of the α-nitrogen atom, in contrast with the tetrahed-
must reflect the more facile intramolecular participation of ral carbon geometry of a substrate or inhibitor (β-lactam)
the side chain in the former case. This may be a con- at this position, is likely to be important. AMI calculations
sequence of the greater stability of the oxadiazolone gener- indicated that the sum of angles about the carbamate nitro-
Scheme 5. Mechanism of hydrolysis of the 3,4-dihydro-2-oxo-3-phenylacetamino-2H-1,3-benzoxazine-7-carboxylic acid 2a(m): participa-
tion of the amide side chain, leading to a relatively stable 5(4H)-1,3,4-oxadiazolone 10a intermediate
144
Eur. J. Org. Chem. 2001, 141Ϫ149

Potential Substrates/Inhibitors of β-Lactam-Recognizing Enzymes
FULL PAPER
quantitatively, the corresponding acid 5a, which was recrystallized
from methanol-dichloromethane, m.p. 199 °C. Ϫ Rf ϭ 0.41
(CH2Cl2/MeOH, 8:2). Ϫ H NMR ([D4] methanol): δ ϭ 3.58 (s, 2
H, CH2), 7.24 to 7.49 (m, 7 H, Ar), 7.80 (s, 1 H, H2), 7.89 (d, J ϭ
7.6 Hz, 1 H, H4). Ϫ 13C NMR ([D4]methanol): δ ϭ 40.0 (CH2),
122.4 to 134.4 (Ar), 150.7 (ArϪO), 155.0, 167.3 and 172.1 (CO).
Ϫ C16H14N2O5 (314.3): calcd. C 61.14, H 4.49, N 8.91; found C
60.81, H 4.75, N 8.77.
gen atom in the lowest energy conformation of 2a(m) was
348°. A close to planar α-nitrogen atom has been observed
in other carbazates, both free[32] and enzyme-bound.[7h]
Unlike serine proteases, serine β-lactamases and DD-
peptidases seem unable to cope at all with aza(depsi)pep-
tides. Acylation of the active site of the former enzymes
does occur although deacylation is slow. In contrast, the
latter enzymes are not acylated by these compounds. Bey-
ond the factors mentioned above, this result may reflect
stringent selection by β-lactamases and DD-peptidases,
against and for Ϫ respectively Ϫ a -methyl substituent at
the position α to the carbonyl group.[30]
1
3-{[2-Methyl-2-(phenylacetyl)hydrazino]carbonyloxy}benzoic Acid
(5b): Treatment of 1-methyl-1-phenylacetylhydrazine[14b] (164 mg,
1 mmol) with 3-(tert-butoxycarbonyl)phenyl chlorocarbonate 13 as
above gave a product which was chromatographed on silica gel
(pentane/ethyl acetate, 1:1) to give tert-butyl 3-{[2-methyl-2-
(phenylacetyl)hydrazino]carbonyloxy}benzoate 14b: 157 mg (82%).
1
Ϫ Rf ϭ 0.55. Ϫ H NMR (CDCl3): δ ϭ (2 isomers, Z and E) 1.58
Experimental Section
and 1.60 (2s, 9 H, tBu), 3.17 (s, 3 H, NCH3), 3.57 and 3.76 (2s, 2
H, CH2), 7.23 to 7.32 (m, 7 H, Ar), 7.74 to 7.89 (m, 2 H, H2 and
H4), 8.36 and 8.43 (2s, 1 H, NH). Ϫ 13C NMR (CDCl3): δ ϭ 28.3
(tBu), 35.8 and 36.5 (NCH3), 40.1 and 40.5 (CH2), 81.9 (tBu), 122.4
to 134.6 (Ar), 150.3 and 151.1 (ArϪO), 153.2Ϫ153.7, 164.9 and
173.6Ϫ173.9 (CO).
The sources of the enzymes employed, and the analytical and kin-
etic methods used, were essentially as described earlier.[2] Azadepsi-
peptide concentrations of up to 1 m were employed in the experi-
ments designed to test for enzyme inhibition. Ϫ Melting points
1
were determined with a Mettler FP61 and are uncorrected. Ϫ H
and 13C NMR spectra were recorded with a Bruker AC300 instru-
ment, at 300 MHz and 75.5 MHz, respectively. Chemical shifts are
reported in ppm; internal standard was tetramethylsilane. Ϫ TLC
was carried out on Merck 60F-254 precoated silica gel plates
(0.25 mm) and column chromatography was performed with Merck
silica gel (70Ϫ230 mesh), using various solvents as eluent. The solv-
ents used are indicated in the procedures. Benzoylhydrazine was
from Fluka, phenylacetylhydrazine and 3-formyl-2-hydroxybenzoic
acid were from Acros.
The tert-butyl ester function of this diacylated hydrazine 14b was
quantitatively cleaved as above with trifluoroacetic acid. The prod-
uct was purified by chromatography (CH2Cl2/MeOH, 85:15) to give
the crystalline acid 5b, m.p. 137 °C. Ϫ Rf ϭ 0.58. Ϫ 1H NMR
([D4]methanol): δ ϭ 3.37 (s, 3 H, NCH3), 3.97 (s, 2 H, CH2), 7.44
to 7.96 (m, 8 H, Ar). Ϫ 13C NMR ([D4]methanol): δ ϭ 36.0
(NCH3), 40.8 (CH2), 123.9 to 136.0 (Ar), 152.1Ϫ152.6 (ArϪO),
155.5Ϫ156.2, 168.8 and 175.8Ϫ176.2 (CO). Ϫ C17H16N2O5 (328.3):
calcd. C 62.18, H 4.91, N 8.53; found C 62.44, H 5.26, N 8.70.
3-{[1-Methyl-2-(phenylacetyl)hydrazino]carbonyloxy}benzoic Acid
(5c): The same experimental procedure was used with 1-methyl-2-
phenylacetylhydrazine[14b] (164 mg, 1 mmol). The product was
chromatographed on silica gel (pentane/ethyl acetate, 1:1) to give
tert-butyl 3-{[1-methyl-2-(phenylacetyl)hydrazino]carbonyloxy}-
Procedures
3-tert-Butoxycarbonylphenyl Chlorocarbonate 13: A solution of tert-
butyl 3-hydroxybenzoate[20] (327 mg, 1.69 mmol) and dimethylanil-
ine (225 mg, 1.69 mmol) in THF (3 mL) was added slowly with
cooling to bis(trichloromethyl) carbonate (184 mg, 0.62 mmol) in
THF (3 mL). The mixture was stirred at 0 °C for 10 min and then
at room temperature for 3 h. The solution containing the crude 3-
tert-butoxycarbonylphenyl chlorocarbonate 13 was used directly in
the next step of the reaction sequence.
1
benzoate 14c: 127 mg (66%). Ϫ Rf ϭ 0.58. Ϫ H NMR (CDCl3):
δ ϭ (2 isomers, Z and E) 1.58 (s, 9 H, tBu), 3.18 and 3.33 (2s, 3
H, NCH3), 3.59 (s, 2 H, CH2), 7.26 to 7.28 (m, 6 H, Ar), 7.36 (t,
J ϭ 7.9 Hz, 1 H, H5), 7.64 and 7.72 (2s, 1 H, H2), 7.84 (d, J ϭ
7.4 Hz, 1 H, H4), 7.93 and 8.07 (2s, 1 H, NH). Ϫ 13C NMR
(CDCl3): δ ϭ 28.1 (tBu), 37.7 and 38.6 (NCH3), 41.4 (CH2), 81.5
(tBu), 121.5 to 133.7 (Ar), 150.7 and 150.9 (ArϪO), 154.5,
164.8Ϫ164.9 and 169.7Ϫ170.2 (CO).
3-{[2-(Phenylacetyl)hydrazino]carbonyloxy}benzoic Acid 5a (typical
procedure):[33] At 0 °C, a solution of phenylacetylhydrazine
(150 mg, 1 mmol) and dimethylaniline (133 mg, 1.1 mmol) in THF
(3 mL) was added with stirring to a solution of chlorocarbonate 13
obtained from 94 mg (0.5 mmol) of tert-butyl 3-hydroxybenzoate.
The mixture was stirred for 3 h at room temperature. Then 10%
HCl (10 mL) was added and the product was extracted with ethyl
acetate (20 mL). The organic layer was dried (Na2SO4) and evapor-
ated, and the residue was chromatographed on silica gel (CH2Cl2/
MeOH, 97.5:2.5) to give 151 mg (81%) of tert-butyl 3-{[2-
(phenylacetyl)hydrazino]carbonyloxy}benzoate 14a. Ϫ Rf ϭ 0.28.
The tert-butyl ester function of compound 14c was then cleaved
with trifluoroacetic acid to give the crystalline title acid 5c, m.p.
1
162 °C. Ϫ Rf 0.63 (CH2Cl2/MeOH, 8:2). Ϫ H NMR ([D4] meth-
anol): δ ϭ 3.18 and 3.31 (2s, 3 H, NCH3), 3.57 (s, 2 H, CH2), 7.22
to 7.34 (m, 6 H, Ar), 7.43 (t, J ϭ 8.0 Hz, 1 H, H5), 7.65 and 7.81
(2s, 1 H, H2), 7.87 (d, J ϭ 7.7 Hz, 1 H, H4). Ϫ 13C NMR ([D4]
methanol): δ ϭ δ 35.1 and 36.1 (NCH3), 38.7 and 38.9 (CH2), 121.2
to 133.1 (Ar), 149.8 (ArϪO), 153.3Ϫ153.4, 166.0 and 170.1Ϫ170.2
(CO). Ϫ C17H16N2O5 (328.3): calcd. C 62.18, H 4.91, N 8.53; found
C 61.96, H 5.15, N 8.33.
1
Ϫ H NMR (CDCl3): δ ϭ 1.56 (s, 9 H, tBu), 3.58 (s, 2 H, CH2),
7.26 (m, 5 H, Ar), 7.28 (d, J ϭ 7.3 Hz, 1 H, H6), 7.34 (t, J ϭ
7.9 Hz, 1 H, H5), 7.70 (d, J ϭ 1.4 Hz, 1 H, H2), 7.83 (dd, J ϭ 6.3
and 1.4 Hz, 1 H, H4), 7.85 (s, 1 H, NH), 8.23 (s, 1 H, NH). Ϫ 13C
NMR (CDCl3): δ ϭ 28.1 (tBu), 41.1 (CH2), 81.6 (tBu), 122.4 to
133.6 (Ar), 150.3 (ArϪO), 154.6, 164.8 and 171.2 (CO).
3-{[2-(Benzoyl)hydrazino]carbonyloxy}benzoic
Acid
(5Јa):
Benzoylhydrazine (163 mg, 1.2 mmol) was treated with 3-tert-bu-
toxycarbonylphenyl chlorocarbonate 13 (0.6 mmol). The product
was chromatographed on silica gel (dichloromethane/methanol,
This diacylated hydrazine 14a was next dissolved in dichlorome-
thane (1 mL) and the solution cooled to 0 °C. Trifluoroacetic acid
(0.5 mL) was added and the mixture was stirred for 3 h at room
temperature. The solvents were removed by evaporation to give,
95:5)
to
give
169 mg
(79%)
of
tert-butyl
3-{[2-
(benzoyl)hydrazino]carbonyloxy}benzoate 14Јa. Ϫ Rf 0.68. Ϫ 1H
NMR (CDCl3): δ ϭ 1.56 (s, 9 H, tBu), 7.20 to 7.50 (m, 5 H, Ar),
7.77 to 7.86 (m, 4 H, Ar), 8.32 (s, 1 H, NH), 9.24 (s, 1 H, NH). Ϫ
Eur. J. Org. Chem. 2001, 141Ϫ149
145

D. Cabaret, M. G. Gonzalez, M. Wakselman, S. A. Adediran, R. F. Pratt
FULL PAPER
1
13C NMR (CDCl3): δ ϭ 28.1 (tBu), 81.6 (tBu), 123.8 to 133.4 (Ar),
150.5 (ArϪO), 155.4, 164.9 and 167.6 (CO).
MeOH, 9:1). Ϫ H NMR ([D4]methanol): δ ϭ 3.28 and 3.43 (2s, 3
H, NCH3), 7.27 to 7.86 (m, 9 H, Ar). Ϫ 13C NMR ([D4]methanol):
δ ϭ δ 37.7 and 38.7 (NCH3), 123.3 to 132.7 (Ar), 155.0 and 155.3
The tert-butyl ester function of compound 14Јa was cleaved with
trifluoroacetic acid to give the title acid 5Јa, which was recrystal-
lized from methanol, m.p. 193 °C. Ϫ Rf ϭ 0.29 (CH2Cl2/MeOH/
AcOH, 90:10:1). Ϫ 1H NMR ([D4]methanol): δ ϭ 7.27 to 7.41 (m,
5 H, Ar), 7.48 to 7.51 (m, 4 H, Ar). Ϫ 13C NMR ([D4]methanol):
δ ϭ 123.8 to 133.4 (Ar), 152.1 (ArϪO), 156.5, 168.8 and 169.7
(CO). Ϫ C15H12N2O5 (300.3): calcd. C 59.99, H 4.03, N 9.33; found
C 59.85, H 4.04, N 9.43.
(ArϪO), 150.7Ϫ150.9, 167.1Ϫ167.3 and 170.0 (CO).
C16H14N2O5 · 1/3 H2O (320.3): calcd. C 60.00, H 4.42, N 8.75;
found C 60.04, H 4.37, N 8.76.
Ϫ
3-Hydroxy-4-{[2-(phenylacetyl)hydrazino]methyl}benzoic Acid 9a(m)
(typical procedure): A solution of 4-formyl-3-hydroxybenzoic acid
7m[12a,12b] (166 mg. 1 mmol) and phenylacetylhydrazine 6a (164 mg,
1.2 mmol) in methanol (25 mL) was refluxed for 16 h. The reaction
mixture was cooled and the crystalline product was collected. After
drying, 3-hydroxy-4-{[2-(phenylacetyl)hydrazono]methyl}benzoic
acid 8a(m) was obtained: 232 mg (78%), m.p. Ͼ 300 °C. Ϫ Rf ϭ
0.59 (CH2Cl2/MeOH/AcOH, 90:10:1). Ϫ 1H NMR ([D4]methanol):
δ ϭ (2 isomers, Z and E) 3.64 and 4.03 (2s, 2 H, CH2), 7.25 to 7.57
(m, 8 H, Ar), 8.26 and 8.39 (2s, 1 H, HCϭN). Ϫ 13C NMR ([D6]-
DMSO): δ ϭ 41.2 (CH2), 116.9 to 144.8 (Ar and CϭN), 156.1 and
156.9 (ArϪO), 166.7, 166.9, 167.1, and 172.4 (CO).
3-[(2-Benzoyl-2-methylhydrazino)carbonyloxy]benzoic Acid (5Јb): As
above, 1-methyl-1-benzoylhydrazine[14a] (261 mg, 1.74 mmol) was
treated with 3-(tert-butoxycarbonyl)phenyl chlorocarbonate 13
(0.87 mmol). The product was chromatographed on silica gel (pent-
ane/ethyl acetate, 1:1) to give tert-butyl 3-[(2-benzoyl-2-methylhy-
drazino)carbonyloxy]benzoate 14Јb: 232 mg (72%), m.p. 51 °C. Ϫ
1
Rf ϭ 0.64 (dichloromethane/methanol, 1:1). Ϫ H NMR (CDCl3):
δ ϭ 1.56 (s, 9 H, tBu), 3.26 (s, 3 H, CH3), 7.27 to 7.81 (m, 9 H,
Ar), 8.93 (s, 1 H, NH). Ϫ 13C NMR (CDCl3): δ ϭ 28.1 (tBu),
81.6 (tBu), 122.2 to 134.5 (Ar), 150.2 (ArϪO), 153.5, 164.9, and
171.5 (CO).
To a solution of this hydrazone 8a(m) (95 mg, 0.32 mmol) in eth-
anol (10 mL) was added sodium cyanoborohydride (37.7 mg
0.6 mmol), and the reaction mixture was stirred for 18 h. The solu-
tion was acidified to pH ഠ 3 by addition of trifluoroacetic acid
and stirred at room temperature for a further 18 h. The solvent was
evaporated and excess hydride was destroyed by addition of 10%
HCl (10 mL). A precipitate was removed by filtration and the fil-
trate was evaporated to dryness. The residue was purified by chro-
matography (CH2Cl2/MeOH/AcOH, 90:10:1) to give the title acid
9a(m) as a white solid, 68 mg (68%), m.p. Ͼ 300 °C. Ϫ Rf ϭ 0.49.
Then, the tert-butyl ester function of compound 14Јb was cleaved
with trifluoroacetic acid as above to give the crystalline title acid
5Јb, m.p. 164 °C. Ϫ Rf ϭ 0.49 (CH2Cl2/MeOH/AcOH, 90:10:1). Ϫ
1H NMR ([D4]methanol): δ ϭ 3.32 (s, 3 H, CH3), 7.42 to 7.55 (m,
8 H, Ar), 7.86 (d, J ϭ 7.5 Hz, 1 H, H4). Ϫ 13C NMR ([D4] meth-
anol): δ ϭ 36.8 (CH3), 123.7 to 136.1 (Ar), 152.1 (ArϪO), 155.5,
168.7 and 175.8 (CO). Ϫ C16H14N2O5 (314.3): calcd. C 61.14, H
4.49, N 8.91; found C 61.24, H 4.27, N 8.57.
1
Ϫ H NMR ([D4]methanol): δ ϭ 3.40 (s, 2 H, CH2), 3.99 (s, 2 H,
CH2N), 7.13 to 7.43 (m, 8 H, Ar). Ϫ 13C NMR ([D6]DMSO): δ ϭ
40.4 (CH2), 50.1 (CH2N), 115.5 to 136.0 (Ar), 155.6 (ArϪO), 167.5
and 169.1 (CO). Ϫ C16H16N2O4 · 1/2 H2O (309.3): calcd. C 62.12,
H 5.54, N 9.06; found C 61.96, H 5.41 N 8.96.
3-[(2-Benzoyl-1-methylhydrazino)carbonyloxy]benzoic Acid 5Јc:
Firstly,
1-[3-(tert-butoxycarbonyl)phenoxycarbonyl]-1-methylhy-
drazine 15 was prepared by a slow addition of a solution of methyl-
hydrazine (190 mg, 4.12 mmol) in dichloromethane (2 mL) to 3-
tert-butoxycarbonylphenyl chlorocarbonate 13 (1.03 mmol) in
dichloromethane (4 mL). The reaction mixture was stirred for 3 h
at room temperature, then washed with water and dried (Na2SO4).
Evaporation of the solvent and chromatography on silica gel (pent-
ane/ethyl acetate, 6:4) gave product 15, 183 mg (67%). Ϫ Rf ϭ 0.27.
3-Hydroxy-4-(2-Oxo-5-benzyl-3H-1,3,4-oxadiazol-3-yl)benzoic Acid
(10a):
3-Hydroxy-4-{[2-(phenylacetyl)hydrazino]methyl}benzoic
acid 9a(m) (81 mg, 0.27 mmol) was dissolved in anhydrous tetrahy-
drofuran (25 mL). The solution was cooled to 0 °C before the addi-
tion of triethylamine (0.11 mL, 0.7 mmol) and bis(trichloromethyl)
carbonate (54 mg, 0. 18 mmol). The reaction mixture was stirred
for 24 h at room temperature; then the solvent was evaporated. The
residue was dissolved in ethyl acetate, washed with 10% HCl, and
dried. Evaporation of the solvent gave the title oxadiazolone 10a,
82 mg (94%), m.p. 190 °C. Ϫ Rf ϭ 0.48 (CH2Cl2/MeOH/AcOH,
1
Ϫ H NMR (CDCl3): δ ϭ 1.57 (s, 9 H, tBu), 3.29 (s, 3 H, CH3),
4.30 (s, 2 H, NH2), 7.28 (dt, J ϭ 7.9 and 1.8 Hz, 1 H, H6), 7.41 (t,
J ϭ 7.9 Hz, 1 H, H5), 7.70 (t, J ϭ 1.8 Hz, 1 H, H2), 7.85 (dt, J ϭ
7.7 and 1.7 Hz, 1H, H4).
1
95:5:1). Ϫ H NMR ([D6]acetone): δ ϭ 3.90 (s, 2 H, CH2), 4.88 (s,
Secondly, this hydrazide 15 (183 mg, 0.69 mmol) was dissolved in
dichloromethane (3 mL). Then, dimethylaniline (92 mg,
0.76 mmol) and benzoyl chloride (107 mg, 0.76 mmol) in dichloro-
methane (3 mL) were added. The mixture was stirred for 18 h at
room temperature. 10% HCl (10 mL) was added and the product
extracted with dichloromethane (20 mL). The organic layer was
washed with a NaHCO3 solution, then dried (Na2SO4). Evapora-
tion and chromatography on silica gel (pentane/ethyl acetate, 7:3)
gave tert-butyl 3-[(2-benzoyl-1-methylhydrazino)carbonyloxy]ben-
2 H, CH2N), 7.25 to 7.59 (m, 8 H, Ar). Ϫ 13C NMR ([D6] acetone):
δ ϭ 33.1 (CH2), 44.9 (CH2N), 116.7 to 134.7 (Ar), 154.6 (ArϪO),
155.7, 155.9 and 167.2 (CO). Ϫ C17H14N2O5 · 1/2 H2O (335.3):
calcd. C 61.07, H 4.52, N 8.38; found C 61.35, H 4.53, N 8.31.
3-Hydroxy-4-{[2-methyl-2-(phenylacetyl)hydrazino]methyl}benzoic
Acid 9b(m): A solution of 4-formyl-3-hydroxybenzoic acid (83 mg,
0.5. mmol) and N-methyl-N-phenylacetylhydrazine (98 mg,
0.6 mmol) in methanol (25 mL) was refluxed for 18 h to give 98 mg
(63%) of the 3-hydroxy-4-{[2-methyl-2-(phenylacetyl)hydra-
zono]methyl}benzoic acid 8b(m). Ϫ Rf ϭ 0.61 (CH2Cl2/MeOH/
AcOH, 90:10:1). Ϫ 1H NMR (CDCl3 ϩ ε [D4]methanol): δ ϭ 3.39
(s, 3 H, CH3), 4.06 (s, 2 H, CH2Ph), 7.20Ϫ7.50 (m, 8 H, Ar), 7.89
(s, 1 H, HCϭN). Ϫ 13C NMR (CDCl3ϩ ε [D4]methanol): δ ϭ
27.8 (CH3), 40.1 (CH2Ph), 117.6 to 134.1 (Ar), 140.9 (CϭN), 156.4
(ArϪO), 168.0 and 172.4 (CO).
1
zoate 14Јc, 160 mg (74%), m.p. 54 °C. Ϫ Rf ϭ 0.51. Ϫ H NMR
(CDCl3): δ ϭ (2 isomers, Z and E) 1.56 and 1.58 (2s, 9 H, tBu),
3.31 and 3.47 (2s, 3 H, NCH3), 7.24 to 7.87 (m, 9 H, Ar), 8.90 and
9.05 (2s, 1 H, NH). Ϫ 13C NMR (CDCl3): δ ϭ 28.1 (tBu), 37.8
and 38.8 (NCH3), 81.6 (tBu), 122.5 to 133.5 (Ar), 150.8 and 151.0
(ArϪO), 155.1, 164.8Ϫ165.0, and 166.3Ϫ166.6 (CO).
Finally, the tert-butyl ester group of compound 14Јc was cleaved
with trifluoroacetic acid to give the title acid 5Јc, which was recrys-
Then, this hydrazone 8b(m) (65 mg, 0.2 mmol) was reduced with
tallized from dichloromethane, m.p. 172 °C. Ϫ Rf ϭ 0.43 (CH2Cl2/ sodium cyanoborohydride (25 mg, 0.4 mmol) as above. Purification
146 Eur. J. Org. Chem. 2001, 141Ϫ149

Potential Substrates/Inhibitors of β-Lactam-Recognizing Enzymes
FULL PAPER
by chromatography gave 45 mg (68%) of the title substituted hydra-
1.2 mmol), N-methyl-N-phenylacetylhydrazine (164 mg, 1.2 mmol),
zine 9b(m) as a white solid, m.p. 210 °C. Ϫ Rf ϭ 0.47 (CH2Cl2/ and triethylamine (0.17 mL, 1.22 mmol) in methanol (25 mL) was
1
MeOH/AcOH, 90:10:1). Ϫ H NMR ([D4]methanol): δ ϭ (2 iso-
mers Z and E) 3.16Ϫ3.17 (2s, 3 H, CH3), 3.69Ϫ3.76 (2s, 2 H, salt
ArCH2), 3.97Ϫ4.02 (2s, 2 H, CH2Ph), 7.12Ϫ7.58 (m, 8 H, Ar). Ϫ
zono]methyl}benzoic acid 8b(o), m.p. 152 °C. Ϫ Rf ϭ 0.50
13C NMR ([D4]methanol): δ ϭ 31.9 (CH3), 38.3 (CH2Ph), 46.4 (CH2Cl2/MeOH/AcOH, 90:10:1). Ϫ 1H NMR ([D4]methanol): δ ϭ
(CH2N), 115.6 to 136.9 (Ar), 155.7 (ArϪO), 167.4 and 172.6 (CO).
1.28 (s, 9 H, CH3), 3.16 (s, 6 H, CH2Nϩ), 3.37 (s, 3 H, CH3), 4.21
refluxed for 18 h to give 395 mg (79%) of the triethylammonium
of the 2-hydroxy-3-{[2-methyl-2-(phenylacetyl)hydra-
Ϫ C17H18N2O4 · 1/2 H2O (323.3): calcd. C 63.14, H 5.93, N 8.66; (s, 2 H, CH2Ph), 6.83 to 7.92 (m, 8 H, Ar), 8.26 (s, 1 H, HCϭ
found C 62.96, H 5.78, N 8.63.
N). Ϫ 13C NMR ([D4]methanol): δ ϭ 9.3 (CH3), 28.4 (CH3), 41.3
(CH2Ph), 47.8 (CH2Nϩ), 118.8 to 138.3 (Ar), 151.5 (ArϪO), 162.4
(CϭN), 175.2 and 175.6 (CO).
3-[Methyl(phenylacetyl)amino]-3,4-dihydro-2-oxo-2H-1,3-
benzoxazine-7-carboxylic Acid 2b(m): A solution of 3-hydroxy-4-
{[2-methyl-2-(phenylacetyl)hydrazino]methyl}benzoic acid 9b(m)
(62 mg, 0.20 mmol), triethylamine (0.03 mL, 0.2 mmol), and car-
bonyldiimidazole (42 mg, 0.4 mmol) in anhydrous THF (15 mL)
was refluxed for 4 h. The solvent was evaporated and the residue
dissolved in dichloromethane. The organic solution was washed
with 10% HCl, then with brine. Evaporation of the dried solvent
gave a product which was chromatographed to give the title benzox-
azinone 2b(m), 40 mg (59%), m.p. 264 °C. Ϫ Rf ϭ 0.50 (CH2Cl2/
Then, this hydrazone 8b(o) (206 mg, 0.5 mmol) was reduced with
sodium cyanoborohydride (38 mg, 0.6 mmol), as above. Chromato-
graphy on silica gel (CH2Cl2/MeOH/AcOH, 90:10:1) gave 120 mg
(76%) of the title hydrazine 9b(o) as a white solid, m.p. 153 °C. Ϫ
Rf ϭ 0.19. Ϫ 1H NMR ([D4]methanol): δ ϭ 3.18 (s, 3 H, CH3),
3.75 (s, 2 H, ArCH2), 3.96 (s, 2 H, CH2Ph), 6.87 to 7.84 (m, 8 H,
Ar). Ϫ 13C NMR ([D4]methanol): δ ϭ 33.4 (CH3), 40.4 (CH2Ph),
47.8 (CH2N), 119.8 to 138.0 (Ar), 161.8 (ArϪO), 175.2 and 176.7
(CO). Ϫ C17H18N2O4 · 1/4 H2O (318.8): calcd. C 64.04, H 5.85, N
8.79; found C 63.92, H 5.81, N 8.87.
1
MeOH/AcOH, 97:3:0.5). Ϫ H NMR (CDCl3 ϩ ε [D4]methanol):
δ ϭ (2 isomers) 3.06 (s, 3 H, CH3), 3.47 and 3.68 (2d, J ϭ 15.8 Hz,
2 H, CH2Ph), 3.62Ϫ4.64 and 4.46Ϫ4.82 (2dd, J ϭ 14.3 and
14.7 Hz, 2 H, CH2Ar), 7.73 to 7.82 (m, 8 H, Ar). Ϫ 13C NMR
(CDCl3 ϩ ε [D4]methanol): δ ϭ 31.3 (CH3), 40.6 (CH2Ph), 48.7
(CH2Ar), 117.6 to 135.6 (Ar), 148.2 and 149.3 (ArϪO and NCϭ
O), 166.9 and 173.6 (CO). Ϫ C18H16N2O5 · H2O (358.4): calcd. C
60.33, H 5.06, N 7.82; found C 60.46, H 4.92, N 7.68.
3,4-Dihydro-3-[methyl(phenylacetyl)amino]-2-oxo-2H-1,3-
benzoxazine-8-carboxylic Acid 2b(o): A solution of 2-hydroxy-3-{[2-
methyl-2-(phenylacetyl)hydrazino]methyl}benzoic
acid
9b(o)
(121 mg, 0.39 mmol), triethylamine (0.06 mL, 0.39 mmol), and car-
bonyldiimidazole (132 mg, 0.81 mmol) in anhydrous THF (15 mL)
was refluxed for 4 h. The title benzoxazinone 2b(o) was purified by
chromatography (CH2Cl2/MeOH/AcOH, 95:5:0.5), 70 mg (53%),
m.p. 63 °C. Ϫ Rf ϭ 0.44. Ϫ 1H NMR (CDCl3): δ ϭ (2 isomers)
3.23 and 3.33 (2s, 3 H, CH3), 3.64 to 3.78 (dd, J ϭ 14.8 Hz, 2 H,
CH2Ph), 3.71Ϫ4.74 and 4.51Ϫ5.04 (2dd, J ϭ 13.7 and 14.0 Hz, 2
H, CH2Ar), 7.02 to 7.96 (m, 8 H, Ar). Ϫ 13C NMR (CDCl3): δ ϭ
31.8 (CH3), 40.8 and 40.9 (CH2Ph), 48.7 and 49.1 (CH2Ar), 118.6
to 134.3 (Ar), 148.0 and 149.0 (ArϪO and NCϭO), 167.9 and
173.4 (CO). Ϫ C18H16N2O5 · 1/3 H2O (346.3): calcd. C 62.42, H
4.83, N 8.08; found C 62.41, H 4.79, N 7.78.
4-[(2-Benzoyl-2-methylhydrazino)methyl]-3-hydroxybenzoic Acid
9bЈ(m): A solution of 4-formyl-3-hydroxybenzoic acid (250 mg, 1.5.
mmol) and N-methyl-N-benzoylhydrazine (225 mg, 1.5 mmol) in
methanol (25 mL) was refluxed for 16 h to give 324 mg (74%) of 4-
[(2-benzoyl-2-methylhydrazono)methyl]-3-hydroxybenzoic
acid
1
8bЈ(m). Ϫ Rf ϭ 0.54 (CH2Cl2/MeOH/AcOH, 95:5:1). Ϫ H NMR
(CDCl3 ϩ ε [D4]methanol): δ ϭ 3.43 (s, 3 H, CH3), 7.18Ϫ7.39
(m, 8 H, Ar), 7.84 (s, 1 H, HCϭN). Ϫ 13C NMR (CDCl3 ϩ ε
[D4]methanol): δ ϭ 28.1 (CH3), 117.8 to 134.3 (Ar), 142.1 (CϭN),
156.3 (ArϪO), 167.8 and 171.6 (CO).
3-[(2-Benzoyl-2-methylhydrazino)methyl]-2-hydroxybenzoic Acid
9bЈ(o): A solution of 3-formyl-2-hydroxybenzoic acid (122 mg, 0.73
mmol) and N-methyl-N-benzoylhydrazine (110 mg, 0.73 mmol) was
refluxed in methanol (15 mL) to give 174 mg (74%) of the 3-[(2-
benzoyl-2-methylhydrazino)methyl]-2-hydroxybenzoic acid 8bЈ(o).
Ϫ Rf ϭ 0.42 (CH2Cl2/MeOH/AcOH, 90:10:1). Ϫ 1H NMR
([D6]DMSO): δ ϭ 3.50 (s, 3 H, CH3), 7.87Ϫ7.80 (m, 8 H, Ar), 8.13
(s, 1 H, HCϭN). Ϫ 13C NMR ([D4] methanol): δ ϭ 28.6 (CH3),
114.0 to 135.5 (Ar), 147.1 (CϭN), 159.7 (ArϪO), 170.0 and 171.9
(CO).
Then, this hydrazone 8bЈ(m) (284 mg, 1.05 mmol) was reduced with
sodium cyanoborohydride (125 mg, 2 mmol) as above to give
157 mg (55%) of the title hydrazine 9bЈ(m) as a white solid, m.p.
191 °C. Ϫ Rf 0.28 (CH2Cl2/MeOH/AcOH, 95:5/1). Ϫ 1H NMR
([D4]methanol): δ ϭ 3.12 (s, 3 H, CH3), 3.98 (s, 2 H, ArCH2), 6.90
to 7.58 (m, 8 H, Ar). Ϫ 13C NMR ([D4]methanol): δ ϭ 32.7 (CH3),
37.3 (CH2N), 115.5 to 130.1 (Ar), 153.3 (ArϪO), 168.2 and 169.9
(CO). Ϫ C16H16N2O4 (300.3): calcd. C 63.99, H 5.37, N 9.33; found
C 64.11, H 5.39, N 9.01.
3-[Benzoyl(methyl)amino]-3,4-dihydro-2-oxo-2H-1,3-benzoxazine-7-
carboxylic Acid 2bЈ(m): A solution of 4-[(2-benzoyl-2-methylhy-
Then, this hydrazone 8bЈ(o) (148 mg, 0.5 mmol) was reduced with
sodium cyanoborohydride (63 mg, 1 mmol) to give 124 mg (83%)
of the title hydrazine 9bЈ(o) as a white solid, m.p. 168 °C. Ϫ Rf ϭ
0.42 (CH2Cl2/MeOH/AcOH, 90:10:1). Ϫ 1H NMR ([D6]DMSO):
δ ϭ 3.19 (s, 3 H, CH3), 3.89 (s, 2 H, ArCH2), 6.73 to 7.67 (m, 8
H, Ar). Ϫ 13C NMR ([D4]methanol): δ ϭ 32.9 (CH3), 40.4 (CH2N),
112.4 to 136.1 (Ar), 159.8 (ArϪO), 171.2 and 172.4 (CO). Ϫ
C16H16N2O4 · 1/4 H2O (304.8): calcd. C 63.04, H 5.45, N 9.19;
found C 63.16, H 5.29, N 9.01.
drazino)methyl]-3-hydroxybenzoic
acid
9bЈ(m)
(110 mg,
0.39 mmol), triethylamine (0.12 mL, 0.8 mmol), and carbonyldiimi-
dazole (117 mg, 0.72 mmol) in anhydrous THF (20 mL) was re-
fluxed for 4 h. The solvent was evaporated and the title benzoxazi-
none 2bЈ(m) was obtained by chromatography (CH2Cl2/MeOH/
AcOH, 97:3:0.5), 82 mg (68%), m.p. 252 °C. Ϫ Rf ϭ 0.45. Ϫ 1H
NMR ([D6]DMSO): δ ϭ 3.26 (s, 3 H, CH3), 4.64 to 5.14 (m, 2 H,
CH2Ar), 7.30 to 7.71 (m, 8 H, Ar). Ϫ 13C NMR ([D6]DMSO): δ ϭ
31.5 (CH3), 48.8 (CH2Ar), 116.0 to 132.0 (Ar), 147.9 and 148.6
(ArϪO and NCϭO), 166.1 and 172.6 (CO). Ϫ C17H14N2O5 ·
1/3 H2O (332.3): calcd. C 61.44, H 4.42, N 8.42; found C 61.48, H
4.48, N 8.23.
3-[(Benzoyl(methyl)amino]-3,4-dihydro-2-oxo-2H-1,3-benzoxazine-
8-carboxylic Acid 2bЈ(o): A solution of 3-[(2-benzoyl-2-methylhy-
drazino)methyl]-2-hydroxybenzoic acid 9bЈ(o) (83 mg, 0.29 mmol),
triethylamine (0.07 mL, 0.29 mmol), and carbonyldiimidazole
(94 mg, 0.58 mmol) in anhydrous THF (20 mL) was refluxed for
4 h. The title benzoxazinone 2bЈ(o) was obtained as above, 55 mg
2-Hydroxy-3-{[2-methyl-2-(phenylacetyl)hydrazino]methyl}benzoic
Acid 9b(o): A solution of 3-formyl-2-hydroxybenzoic acid (200 mg,
Eur. J. Org. Chem. 2001, 141Ϫ149
147

D. Cabaret, M. G. Gonzalez, M. Wakselman, S. A. Adediran, R. F. Pratt
FULL PAPER
(66%), m.p. 179 °C. Ϫ Rf ϭ 0.36 (CH2Cl2/MeOH/AcOH, 97:3:0.5).
Ϫ H NMR ([D4]methanol): δ ϭ 3.30 (s, 3 H, CH3), 3.71 (s, 2 H,
(CH2), 55.9 (CH3), 115.5 to 137.0 (Ar), 139.9 (CϭN), 157.7 and
160.4 (ArϪO), 169.1 and 174.6 (CO).
1
CH2Ar), 7.12 to 7.76 (m, 8 H, Ar). Ϫ 13C NMR ([D4]methanol):
δ ϭ 32.3 (CH3), 38.1 (CH2Ar), 123.8 to 133.4 (Ar), 152.1 and 156.4
(ArϪO and NCϭO), 168.8 and 169.7 (CO). Ϫ C17H14N2O5
(326.3): calcd. C 62.57, H 4.32, N 8.58; found C 62.32, H 4.46,
N 8.44.
Then, this hydrazone 8bЈЈ(m) (191 mg, 0.46 mmol) was reduced
with sodium cyanoborohydride (50 mg, 0.8 mmol) in methanol
(10 mL), at pH 3 (addition of trifluoroacetic acid). The reaction
was stirred for 16 h at room temperature, the solvent evaporated,
and the residue chromatographed (CH2Cl2/MeOH, 9:1) to give
131 mg (68%) of the title hydrazine 9bЈЈ(m) as a white solid, m.p.
N-(4-Methoxybenzyl)phenylacetamide 11: To a 1  solution of so-
dium hydroxide (5 mL) were added, successively at 0 °C, 4-methoxy-
benzylamine (0.41 g, 3 mmol) and phenylacetyl chloride (0.46 g,
3 mmol). The mixture was then stirred at room temperature for 3 h.
The product was extracted with dichloromethane and the organic
layer dried over sodium sulfate. The solvent was evaporated and
the residue purified by chromatography (pentane/AcOEt, 1:1), to
give 0.56 g (73%) of the title amide 11, m.p. 143 °C. Ϫ Rf ϭ 0.49.
1
187 °C. Ϫ Rf ϭ 0.43. Ϫ H NMR ([D4]methanol): δ ϭ 3.76 (s, 3
H, CH3), 3.82 (s, 2 H, ArCH2), 3.92 (s, 2 H, ArCH2), 4.79 (s, 2 H,
ArCH2), 6.83 to 7.86 (m, 12 H, Ar). Ϫ 13C NMR ([D4]methanol):
δ ϭ 40.0 (CH2Ar), 45.7 and 48.2 (CH2Ar and CH2N), 55.3 (CH3),
114.2 to 136.1 (Ar), 155.7 and 159.2 (ArϪO), 169.0 and 174.9
(CO). Ϫ C24H24N2O5 · H2O (438.5): calcd. C 65.74, H 5.97, N 6.39;
found C 65.91, H 5.98, N 6.36.
1
Ϫ H NMR (CDCl3): δ ϭ 3.61 (s, 2 H, CH2), 3.79 (s, 3 H, CH3),
3,4-Dihydro-3-[(phenylacetyl)amino]-2-oxo-2H-1,3-benzoxazine-7-
carboxylic Acid 2a(m): A solution of 3-hydroxy-4-{[2-(4-methoxyb-
enzyl)-2-(phenylacetyl)hydrazino]methyl}benzoic acid 9bЈЈ(m)
(89 mg, 0.21 mmol), triethylamine (0.031 mL, 0.21 mmol), and car-
bonyldiimidazole (68 mg, 0.42 mmol) in anhydrous THF (5 mL)
was refluxed for 4 h. Then, the solvent was evaporated and the
residue dissolved in dichloromethane. The organic solution was
washed with 10% HCl, then with brine. After drying, evaporation
of the solvent gave a product which was chromatographed to give
the 3,4-dihydro-3-[4-methoxybenzyl(phenylacetyl)amino]-2-oxob-
enz[e][1,3]oxazine-7-carboxylic acid 2bЈЈ(m), 57 mg (60%), m.p. 55
4.34 (d, J ϭ 5.7 Hz, 2 H, CH2), 5.68 (s, 1 H, NH), 6.83 (d, J ϭ
8.8 Hz, 2 H, Ar), 7.11 (d, J ϭ 8.8 Hz, 2 H, Ar), 7.25 to 7.38 (m, 5
H, Ar). Ϫ 13C NMR (CDCl3): δ ϭ 43.3 and 44.0 (CH2Ar), 55.4
(CH3), 114.2 to 135.0 (Ar), 159.1 (ArϪO), 171.0 (CO). Ϫ
C16H17NO2 (255.3): calcd. C 75.27, H 6.71, N 5.49; found C 75.48,
H 6.74, N 5.41.
1-(4-Methoxybenzyl)-1-phenylacetylhydrazine 6bЈЈ: In a first step,
the nitroso derivative 12 was prepared in the following manner. N-
(4-Methoxybenzyl)phenylacetamide 11 (0.7 g, 2.74 mmol) was dis-
solved in a mixture of acetic acid (3 mL) and acetic anhydride
(18 mL). The solution was cooled to 0 °C, then sodium nitrite
(1.86 g, 27 mmol) was added slowly, with stirring. The mixture was
stirred for 1 h at room temperature, then poured onto ice. The
product was extracted with ether and the organic layer washed with
a saturated solution of sodium bicarbonate, dried over sodium sulf-
ate and the solvent evaporated. The nitroso derivative 12 was not
1
°C. Ϫ Rf ϭ 0.62 (AcOEt). Ϫ H NMR (CDCl3): δ ϭ (2 isomers)
3.75 and 3.76 (2s, 3 H, OCH3), 3.65 to 4.04 (m, 4 H, 2 CH2Ar),
4.29 to 5.44 (2dd, J ϭ 14.3 and 14.5 Hz, 2 H, NCH2Ar), 6.75 to
7.51 (m, 12 H, Ar), 8.05 (s, 1 H, COOH). Ϫ 13C NMR (CDCl3):
δ ϭ 40.8 and 41.0 (CH2Ph), 49.8 and 51.9 (CH2Ar), 55.4 (CH3),
114.4 to 133.9 (Ar), 148.5, 149.6 and 159.6 (ArϪO and NCϭO),
170.1, 170.8 and 173.5 (CO).
1
purified. Ϫ H NMR (CDCl3): δ ϭ 3.75 (s, 3 H, CH3), 4.46 (s, 2
Benzoxazinone 2bЈЈ(m) (30 mg, 0.67 mmol) was dissolved in TFA
(0.5 mL), methanesulfonic acid (0.25 mL) was added, and the mix-
ture was stirred for two days at room temperature. Water was added
to the mixture, and the aqueous solution was saturated with NaCl
and extracted with ethyl acetate. Drying over sodium sulfate and
evaporation of the solvent gave a solid which was chromatographed
to give the title compound 2a(m), 15.5 mg (71%), m.p. 180 °C. Ϫ
H, CH2), 4.83 (s, 2 H, CH2), 6.65 to 7.42 (m, 9 H, Ar).The crude
product was dissolved in ethyl acetate (5 mL); acetic acid (4 mL)
and zinc dust (1.5 g) were then slowly added. After completion of
the addition, the mixture was stirred for 3 h at room temperature.
Excess zinc was removed by filtration and the filtrate was treated
with a solution of sodium hydroxide; the alkaline solution was ex-
tracted with dichloromethane. The solvent was evaporated and the
hydrazine purified by chromatography (pentane/AcOEt, 1:1), to
give 0.48 g (65%) of the 1-(4-methoxybenzyl)-1-phenylacetylhydra-
1
Rf ϭ 0.56 (AcOEt/AcOH, 99:1). Ϫ H NMR (CDCl3 ϩ ε [D4]me-
thanol): δ ϭ 3.67 (s, 2 H, CH2Ar), 4.75 (s, 2 H, NCH2Ar), 7.11 (d,
J ϭ 8.1, 1 H, H5), 7.31 to 7.35 (m, 5 H, Ar), 7.64 (s, 1 H, H2), 7.78
(d, J ϭ 7.8 Hz, 1 H, H6). Ϫ 13C NMR (CDCl3): δ ϭ 40.9 (CH2Ph),
50.7 (CH2Ar), 117.7 to 133.7 (Ar), 148.8 and 150.3 (ArϪO and
NCϭO), 167.4 and 171.0 (CO). Ϫ C17H14N2O5 (326.3): calcd. C
62.57, H 4.32, N 8.58; found C 62.19, H 4.51, N 8.22.
1
zine 6bЈЈ, m.p. 122 °C. Ϫ Rf ϭ 0.43. Ϫ H NMR ([D4]methanol):
δ ϭ 3.52 and 3.98 (2s, 2 H, CH2), 3.76 and 3.78 (2s, 3 H, CH3),
4.28 and 4.66 (2s, 2 H, CH2), 6.82 to 7.30 (m, 9 H, Ar). Ϫ 13C
NMR ([D4]methanol): δ ϭ 40.7 (CH2Ar), 43.9 and 44.0 (CH2ArЈ),
55.3 and 55.8 (CH3), 115.0 to 132.0 (Ar), 160.5 and 161.0 (ArϪO),
173.9 and 176.2 (CO). Ϫ C16H18N2O2 (270.3): calcd. C 71.09, H
6.71, N 10.36; found C 71.25, H 6.64, N 10.48.
Acknowledgments
3-Hydroxy-4-{[2-(4-methoxybenzyl)-2-(phenylacetyl)hydrazino]-
methyl}benzoic Acid 9bЈЈ(m): A solution of 4-formyl-3-hydroxyben-
zoic acid 7m (141 mg, 0.85 mmol), triethylamine 0.13 mL
(0.93 mmol), and 1-(4-methoxybenzyl)-1-phenylacetylhydrazine
6bЈЈ (240 mg, 0.89 mmol) in methanol (15 mL) was refluxed for
16 h. The solvent was evaporated and the residue chromatographed
(CH2Cl2/MeOH, 9:1) to give 248 mg (67%) of the 3-hydroxy-4-
{[2-(4-methoxybenzyl)-2-(phenylacetyl)hydrazono]methyl}benzoic
8bЈЈ(m), m.p. 234 °C. Ϫ Rf ϭ 0.48. Ϫ 1H NMR (CDCl3 ϩ ε
[D4]methanol]): δ ϭ 3.77 (s, 3 H, CH3), 4.31 (s, 2 H, CH2), 5.26 (s,
2 H, CH2), 6.88 to 7.78 (m, 12 H, Ar), 8.17 (s, 1 H, HCϭN). Ϫ
13C NMR (CDCl3 ϩ ε [D4]methanol): δ ϭ 41.48 (CH2), 44.9
This research was supported by the National Institute of Health
(R.F.P).
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