Substituted aryl malonamates as new serine β-lactamase substrates: Structure-activity studies

Article abstract of DOI:10.1016/j.bmc.2009.10.056

A series of substituted aryl malonamates have been prepared. These compounds are analogues of aryl phenaceturates where the amido side chain has been replaced by a retro-amide. Like the phenaceturates, these compounds are substrates of typical class A and class C β-lactamases, particularly of the latter, and of soluble DD-peptidases. The effect of substituents α to the ester carbonyl group on turnover by these enzymes is similar to that in the phenaceturates. On the other hand, N-alkylation of the side chain amide of malonamates, but not of phenaceturates, retains the susceptibility of the compounds to hydrolysis by β-lactamases. This reactivity is not enhanced, however, by bridging the amide nitrogen and Cα atoms. A phosphonate analogue of the malonamates was found to be an irreversible inhibitor of the β-lactamases. These results, therefore, provide further evidence for the covalent access of compounds bearing retro-amide side chains to the active sites of β-lactam-recognizing enzymes.

Full text of DOI:10.1016/j.bmc.2009.10.056

Bioorganic & Medicinal Chemistry 18 (2010) 282–291
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Substituted aryl malonamates as new serine b-lactamase substrates:
Structure–activity studies
S. A. Adediran a, D. Cabaret b, J.-F. Lohier b, M. Wakselman b, R. F. Pratt a,
*
a Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA
b Institut Lavoisier, UMR CNRS 8180, Université de Versailles, Bâtiment Lavoisier, 45 Avenue des Etats Unis, F-78035 Versailles, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted aryl malonamates have been prepared. These compounds are analogues of aryl
phenaceturates where the amido side chain has been replaced by a retro-amide. Like the phenaceturates,
these compounds are substrates of typical class A and class C b-lactamases, particularly of the latter, and
Received 21 September 2009
Revised 27 October 2009
Accepted 28 October 2009
Available online 31 October 2009
of soluble DD-peptidases. The effect of substituents
a to the ester carbonyl group on turnover by these
enzymes is similar to that in the phenaceturates. On the other hand, N-alkylation of the side chain amide
of malonamates, but not of phenaceturates, retains the susceptibility of the compounds to hydrolysis by
b-lactamases. This reactivity is not enhanced, however, by bridging the amide nitrogen and Ca atoms. A
phosphonate analogue of the malonamates was found to be an irreversible inhibitor of the b-lactamases.
These results, therefore, provide further evidence for the covalent access of compounds bearing retro-
amide side chains to the active sites of b-lactam-recognizing enzymes.
Keywords:
Enzyme
b-Lactamase
Substrate specificity
Enzyme kinetics
b-Lactam antibiotics
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
cillanic acid is some 102–103 times less effective as a substrate (kcat
/
Km) of typical class A and class C b-lactamases than is benzylpenicil-
lin.10 Effective transition state analogue inhibitors, for example
phosphonates11 and boronates,12 also possess the amido side
chain.
The search for new substrates and inhibitors of b-lactamases
continues. New substrates are of interest, of course, because, in
principle, new inhibitors can be derived from them. Such inhibitors
can protect b-lactams from b-lactamases and therefore amplify ac-
cess of these antibiotics to their targets, the bacterial DD-pepti-
dases. A few of these inhibitors, clavulanic acid, sulbactam and
tazobactam, have been very successful in this role in medical prac-
tice,1 and new variants continue to be developed.2 Because of the
similarity of b-lactamase and DD-peptidase active sites3, inhibitors
of the former enzymes may also be inhibitors of the latter, and
thus, potentially, antibiotics in their own right. New chemical enti-
ties that interact with the b-lactamase active site may become the
foci of new inhibitor development, for example diazabicycloocta-
nones such as NXL1044 and aryloxycarbonyl hydroxamates.5
Most good b-lactamase substrates, of structure 1 (X = O, S), are
characterized by an amido side chain. Crystal structures of com-
plexes of 1 (X = O, N, S) with a variety of b-lactamases6,7 and DD-
peptidases8,9 show that the side chain amide donates a hydrogen
bond to a backbone carbonyl of the conserved b-strand adjacent
to the active site, and accepts a hydrogen bond from the side chain
amide of the asparagine residue of a S(Y)XN motif that is found in
most of these enzymes (Fig. 1). Molecules lacking the amido side
chain are usually poorer b-lactamase substrates. For example, peni-
O
RCONH
O
RCONH
O
RNHC
R'
X
R'
R'
R''
OAr
O
OAr
-
CO2
2
3
1: X = O, NR''',S
O
O
RHNC
RNHC
-
CO2
N
O
O
O
-
CO2
5
4
Aryl phenaceturates, 2, have been shown to be substrates of ser-
ine b-lactamases of all three classes, A, C and D.13–15 More recently,
we have shown, rather counter intuitively, that retro-amido ana-
logues, aryl malonamates, 3 (R0 = H), are also b-lactamase sub-
strates with reactivities approaching those of 2.15 Molecular
modeling suggested, not surprisingly, that hydrogen bonding of
the amido side chain of 3 with the enzyme active site would be dif-
ferent from that of 2, and may involve hydrogen bond acceptance
* Corresponding author. Tel.: +1 860 685 2629; fax: +1 860 685 2211.
E-mail address: rpratt@wesleyan.edu (R.F. Pratt).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.056

S. A. Adediran et al. / Bioorg. Med. Chem. 18 (2010) 282–291
283
pounds 10 and 11 were therefore assessed. An extension of this
idea suggested that a bridge between the retro-amide nitrogen
Asn
H2N
Asn
H2N
O
O
H3N+Lys
and the
a-carbon position of the malonamate might also be
H3N+Lys
acceptable, and even fix the molecule in a reactive conformation
(see molecular modeling below). The cyclic analogues 12 and 13
were therefore prepared and assessed. Finally, the inhibitory prop-
erties of the retro-amido phosphonate 14, an analog of the phos-
phonate 15, a known transition state analogue inhibitor,11 were
determined.
O
R
O
N
N
R
H
H
O
O
NH
retro-amide
NH
2. Results and discussion
amide
Synthesis of the linear analogues7, 8, 10 and 11 was straightfor-
ward through selectively protected malonate derivatives (Scheme
Figure 1. Possible interactions between substrate amide and retro-amide groups
and active site residues.
1). The
a-methoxy derivative 9 was similarly obtained (Scheme
2). The pyrrolidinone 12 was prepared by way of a similar se-
quence, beginning with 1-benzyl-2-pyrrolidinone (Scheme 3). A
similar sequence was employed for 13 (Scheme 4). In this case, di-
rect N-benzylation of the benzolactam 27 was not feasible. Protec-
tion of the C-3 hydrogen with a t-butoxycarbonyl group was
therefore undertaken; N-benzylation and deprotection was then
straightforward. As prepared, compounds 7–9, 12 and 13 were
racemic at the malonate central carbon atom.
The phosphonate 14 was initially sought from direct coupling of
the relevant phosphonic acid with benzyl 3-hydroxybenzoate
using either dicyclohexylcarbodiimide or trichloracetonitrile as
the condensation agent. After this route was found to be unsuc-
cessful, the Arbusov reaction (Scheme 5) was then successfully
applied.
from the protonated side chain of the lysine residue of the con-
served KXYS active site motif15 (Fig. 1). At any event, it seems that
the retroamide side chain can also, under some circumstances of
substrate structure at least, interact in a productive way with the
b-lactamase active site. This finding has been extended to the ben-
zopyranones, 4, and the b-lactam 5, which, like their normal ami-
do-substrates, are serine b-lactamase substrates.16,17
The present paper describes our ventures to further extend the
theme of 3 through elaboration of the lead compound 6 (R = H). It
is well known, for example, that 6a-substituents in penams (7a in
cephems) confer interesting inhibitory properties on the parent
molecules. The methoxy and hydroxymethyl substituents, for
example, convert b-lactamase substrates into inhibitors.18–23 In
phenaceturates, these substituents (R0 in 2) gave altered substrate
The malonamates 6–13 slowly hydrolyze in aqueous buffer. Un-
der the conditions of the enzyme kinetics experiments, pseudo-
activities.24 We have therefore examined the
a-substituted malo-
first order rate constants of hydrolysis of 6–13 were 5.4 ꢀ 10ꢁ6 s
ꢁ1 15
5.7 ꢀ 10ꢁ6 sꢁ1, 1.4 ꢀ 10ꢁ6 sꢁ1, 28 ꢀ 10ꢁ6 sꢁ1, 2.4 ꢀ 10ꢁ6 sꢁ1
,
namates 7–9 and compared their reactivity with typical b-lacta-
mases with those of the parent compound 6 (R = H) and with
their normal amide analogues.
,
5.6 ꢀ 10ꢁ6 sꢁ1
,
6.4 ꢀ 10ꢁ6 sꢁ1
,
and 22 ꢀ 10ꢁ6 sꢁ1
, respectively.
The phosphonate 14 was stable to hydrolysis under these
conditions.
O
The
a-substituted phenaceturates 7–9 are, in general, like the
PhCH2NHC
R
O
O
parent compound 6,15 substrates of the class C P99 b-lactamase
(Table 1). The methyl, benzyl and methoxy substituents do not,
however, yield a better substrate. This result resembles that for
PhCH2NC
R
O
O
O
classical amido-depsipeptides, 2, where, in general, simple a-sub-
-
CO2
stitution does not, in general, significantly enhance reactivity.24,25
In the present case, it is noticeable that the larger benzyl substitu-
ent does induce tighter apparent binding and markedly slower
turnover. Further development of this lead could result in quite
effective inhibitory substrates. It should be noted that all of the
new compounds, like the original depsipeptides, 1, are poorer
b-lactamase substrates than optimally-structured b-lactams.
Successive application of the P99 b-lactamase and the R61 DD-
peptidase to 7, a stratagem employed previously,24 shows that the
former enzyme prefers the enantiomer of 7 not favored by the
latter enzyme, although both enantiomers are substrates of the for-
mer. If it is assumed that the DD-peptidase is specific for the S-
6: R = H
7: R = Me
8: R = CH2Ph
9: R = OMe
-
CO2
10: R = Me
11: R = CH2Ph
PhCH2
N
PhCH2N
O
O
O
O
O
O
-
CO2
13
-
12
CO2
enantiomer (corresponding to its D-amino acid preference in amide
substrates24,26) then the R-enantiomer of 7 must be preferred by
the P99 b-lactamase. This result also resembles that for the classi-
O
PhCH2NHC
PhCH2CONH
cal phenaceturate 2 where the L-alanyl substrate is preferred,
P
P
although, as here, both enantiomers are substrates. The structural
O
O
O
O
basis for the latter preference has been discussed.24
O-
O-
14
15
In contrast to the above result, successive application of the P99
and R61 enzymes to 9 shows that both enzymes have the same
enantiomeric preference, presumably S. As for 7, both enantiomers
of 9 are P99 b-lactamase substrates. Such a change in stereospeci-
ficity for the P99 enzyme has been observed previously27,28 and
probably reflects the general lack of structural specificity for either
-
-
CO2
CO2
Inspection of the molecular model referred to above,15 sug-
gested that the retro- amide NH moiety may not be hydrogen-
bonded to the active site and thus, unlike in 2, N-substitution
might be accepted and perhaps lead to new interactions. Com-

284
S. A. Adediran et al. / Bioorg. Med. Chem. 18 (2010) 282–291
R3
R3
R3
R2
N
R3
HO2C
CO2R
CO2R
R2
N
a,b
e
O
O
CO2Bn
R1
CO2H
17
R1
c
Cl
O
O
O
O
O
O
18
f
O
O
R3
R2
N
d
R3
O
CO2H
17,18 a: R1=Bn, R2=H, R3=Me
b: R1=Bn, R2=H, R3=Bn
c: R1=Bn, R2=Me, R3=H
d: R1=Bn, R2=Bn, R3=H
O
R1
7,8,10,11
Scheme 1. Reagents and conditions: (a) R1R2NH/DCC/CH2Cl2/12 h; (b) KOH, 1 equiv/ROH/25 °C/1–12 h, then 20% HCl/25 °C; (c) R1R2NH/CH2Cl2/25 °C/3 h, then 10% HCl/25 °C;
(d) R1R2NH, toluene/105 °C/16 h; (e) 3-HOC6H4CO2Bn/DCC or DCC/DMAP/THF or CH2Cl2/25 °C/12 h; (f) H2/10% Pd/C/AcOEt/25 °C/2–12 h.
OMe
CO2Me
19
OMe
CO2Me
OMe
CO2Me
OMe
a
c
b
Cl
PhCH2NH
MeO2C
K+ -O2C
CO2Me
O
O
O
20
d
OMe
O
OMe
O
OMe
CO2H
PhCH2NH
22
CO2Bn
PhCH2NH
O
CO2H
PhCH2NH
f
e
21
9
O
O
O
Scheme 2. Reagents and conditions: (a) aq KOH; (b) SOCl2; (c) PhCH2NH2/CH2Cl2/1 h; (d) 1 M aq NaOH/MeOH, 1/1, 25 °C/16 h; (e) HOC6H4CO2Bn/DCC/CH2Cl2/25 °C/16 h; (f)
H2/10% Pd/C/AcOEt/25 °C/16 h.
Bn
Bn
Bn
b
a
23
N
N
N
24
O
CO2H
O
O
CO2Me
c
Bn
Bn
N
N
O
O
d
O
O
O
O
25
12
CO2H
CO2Bn
Scheme 3. Reagents and conditions: (a) LDA (1.3 equiv)/THF/ꢁ78 °C/30 min, then ClCO2Me/30 min; (b) 1 M NaOH/MeOH/25 °C/12 h, then aq HCl; (c) 3-HOC6H4CO2Bn/DCC/
CH2Cl2/25 °C/12 h; (d) H2/10% Pd/C/AcOEt/25 °C/2 h.
a
b
c
d
30
HN
O
O2N
MeO2C
BocN
O
HN
O
BnN
O
CO2tBu
CO2Me
CO2tBu
CO2Me
CO2tBu
CO2Me
CO2Me
CO2Me
29
32
26
28
27
e
g
f
h
BnN
O
BnN
O
BnN
O
BnN
O
O
O
CO2H
CO2Me
O
O
31
13
33
CO2H
CO2Bn
Scheme 4. Reagents and conditions: (a) H2/10% Pd/C/AcOEt/25 °C/3 h; (b) Boc2O (2 equiv)/DMAP/MeCN/25 °C/12 h; (c) TFA/CH2Cl2/0 °C/1 h; (d) BnCl/K2CO3/BnEt3N+Clꢁ/
MeCN/60 °C/4 h; (e) TFA/CH2Cl2/25 °C/1 h; (f) 10% NaOH/MeOH/25 °C/1 h, then aq HCl; (g) 3-HOC6H4CO2Bn/DCC/CH2Cl2/25 °C/4 h; (h) H2/10% Pd/C/AcOEt/25 °C/2 h.

S. A. Adediran et al. / Bioorg. Med. Chem. 18 (2010) 282–291
285
O
O
a
Br
Br
+
(MeO)2PO
34
HO
BnNH
CO2Me
b
O
O
P
O
O
P
c
BnNH
O
BnNH
O
O
O
14
35
H
Me
CO2H
CO2Me
Scheme 5. Reagents and conditions: (a) PhCH2NH2/DCC/HOBT/CH2Cl2/16 h; (b) 110 °C/16 h; (c) Me3SiI/50 °C/16 h.
enantiomer.24 The stereochemical assignments for
achieved as discussed above, are indicated with the relevant rate
constants in Table 1.
7
and 9,
to yield molecules that only poorly interact with the DD-pepti-
dases (Table 1).
The phosphonate 14 was found to be an inhibitor of the P99
b-lactamase (ki = 45 sꢁ1 Mꢁ1) but is considerably less effective
against the TEM enzyme (ki <1 sꢁ1 Mꢁ1), again reflecting the rela-
tive reactivities with 15. The effectiveness of 14 against the P99 en-
zyme, however, is only around 10-fold less than that of 15,11 and
may be, as in the latter case, enhanced to a considerable degree
with suitable substituents.32 Neither 14 nor 15 inhibited the DD-
peptidases.
Molecular modeling15 suggested that the N-alkylated amide
species 10 and 11 may be substrates of the P99 b-lactamase be-
cause the N–H of 6 did not appear to hydrogen-bond with an
enzyme functional group (Fig. 1). This is now found to be true
(Table 1), although the substrates generated are poorer than 6.
Nonetheless, these substrates are more reactive than N-alkylated
analogs of 2, where the amide N–H is believed to strongly inter-
act with a protein backbone carbonyl oxygen. The present result,
therefore, does support the structural model. Modeling (Fig. 2)
also suggested that extension of N-alkylation to cyclic com-
pounds such as 12 and 13 would also be acceptable to the active
site. As shown in Table 1, however, this extension did not pro-
duce better substrates and does not suggest that the freezing
out of a reactive conformer was achieved. Although the molecu-
lar modeling (Fig. 2) did suggest that a deacylation tetrahedral
intermediate, 16, derived from 12 would fit well into the active
site, it may be that flexibility around the C2–C3 bond, restricted
in the cyclic compound, is needed to achieve this structure. A
comparison of the conformation of 16 in Figure 2 with that of
the acyclic analogue 6,15 shows C1C2C3N dihedral angles of
115° and 128°, respectively. Flexibility about this bond may be
required in the acylation step.
3. Conclusions
The results described above affirm the viability of the retro-
amide side chain platform as a source of covalent access to the
active sites of b-lactam-recognizing enzymes. For a particular com-
pound, however, that access may only be available for a narrow
range of enzymes. Of particular interest are the reaction of 8 with
the P99 b-lactamase, where a slowly hydrolyzing acyl-enzyme is
probably generated, and the very positive influence of the methoxy
substitution in 9 on both DD-peptidases examined. Further inves-
tigation of these leads may produce novel inhibitors of specific en-
zymes. The retro-amide phosphonates may also be worthy of
closer scrutiny.
4. Experimental
4.1. Syntheses
PhCH2
N
3
16
O
2
4.1.1. 3-(2-Benzylcarbamoyl-propionyloxy)-benzoic acid (7)
(Scheme 1)
4.1.1.1. N-Benzyl 2-methylmalonamic acid 17a. Benzylamine
1
OH
SerO
O-
(545 lL, 5.0 mmol) and 2,2,5-trimethyl-1,3-dioxan-4,6-dione
The modeled structure includes the interesting hydrogen bond
between the substrate amido-carbonyl group and the terminal
ammonium ion of Lys 67. This interaction was first seen as a pos-
sibility in modeling of complexes of 6 (R = H).15 It is not found in
complexes of normal amido-substrates and may enforce a Tyr
150 general base mechanism of acylation.29–31All of the new dep-
sipeptides 6–13 are considerably poorer substrates of the class A
TEM b-lactamase than of the P99 enzyme. This response is found
in the classical phenaceturates also13 but the difference seems
(790 mg, 5.0 mmol) were partially dissolved in dry toluene (10 mL)
in a round-bottomed flask fitted with a condenser and a CaCl2 trap.
The mixture was heated at 105 °C for 16 h. After cooling at room
temperature, a white precipitate was collected by filtration and
recrystallized from ethyl acetate and cyclohexane to afford the title
product 17a as colorless needles (610 mg, 2.95 mmol, 59% yield).
Mp 138 °C; Rf 0.35 (ethyl acetate/acetic acid: 99/1); 1H NMR: d
(CD3COCD3) 1.37 (d, 3H, J = 7.2 Hz), 3.27 (q, 1H, J = 7.2 Hz), 4.37
(dd, 2H, J = 5.8, 14.9 Hz,), 7.13–7.29 (m, 5H), 7.79 (br s, 1H). 13C
NMR: d (CD3COCD3) 15.08, 43.66, 43.78, 46.67, 46.72, 127.87,
128.33, 129.28, 140.16, 171.29, 172.44. Anal. Calcd for
C11H13NO3: C, 63.75; H, 6.32; N, 6.76. Found: C, 63.93; H, 6.65;
N, 6.57.
heightened by a-substitution.
On the basis of their stereospecificity towards the classical dep-
sipeptides and to peptide substrates, one would expect specific (S)
substitution to enhance the reactivity (kcat/Km) of DD-peptidases
towards 3. This result is certainly seen in 7 and 9 with both the
R61 and R39 DD-peptidases (Table 1). With both enzymes, one
enantiomer of 7 and 9, which can be assumed to be S, is consider-
ably more reactive than the other (R), a preference, as noted above,
presumably rooted in the strong specificity of DD-peptidases for
4.1.1.2. 3-(2-Benzylcarbamoyl-propionyloxy)-benzoic acid ben-
zyl ester 18a. N-Benzyl 2-methylmalonamic acid 17a (232 mg,
1.12 mmol) was dissolved in
a mixture of dichloromethane
(15 mL) and DMF (few drops), then was added 3-hydroxybenzoic
acid benzyl ester (269 mg, 1.18 mmol) followed by dicyclohexyl-
natural D-alanyl peptide substrates. The N-alkylation of 6 seems

286
S. A. Adediran et al. / Bioorg. Med. Chem. 18 (2010) 282–291
carbodiimide (243 mg, 1.18 mmol). The reaction mixture was stir-
red overnight and DCU was removed by filtration. The organic
solution was washed with saturated aqueous NaHCO3 (15 mL),
brine (15 mL) and dried. The solvent was evaporated under vac-
uum to give a colorless solid which was recrystallized from ethyl
acetate/cyclohexane to give the title product 18a (230 mg,
0.55 mmol, 49% yield). Mp 117 °C. Rf 0.41 (ethyl acetate/cyclohex-
ane 40/60); 1H NMR: d (CD3COCD3) 1.46 (d, 3H, J = 7.2 Hz), 3.83 (q,
1H, J = 7.2 Hz), 4.48 (dd, 2H, J = 6.2, 14.9 Hz), 5.39 (s, 2H), 7.18–7.61
(m, 12H), 7.76 (m, 1H), 7.94 (m, 1H). 13C NMR: d (CD3COCD3) 14.28,
43.86, 47.80, 67.57, 123.48, 127.48, 127.79, 127.90, 128.33, 129.16,
129.21, 129.31, 129.50, 130.74, 132.69, 137.30, 140.28, 152.08,
165.91, 169.80, 170.31. Anal. Calcd for C25H23NO5: C, 71.93; H,
5.55; N, 3.36. Found: C, 71.72; H, 5.67; N, 3.33.
4.1.1.3. 3-(2-Benzylcarbamoyl-propionyloxy)-benzoic acid 7.
3-(2-Benzylcarbamoyl-propionyloxy)-benzoic acid benzyl ester
18a (163 mg, 0.39 mmol) was dissolved in ethyl acetate (15 mL),
palladium on charcoa1 (49 mg, 30%/w) was added and the mixture
was stirred under a hydrogen atmosphere for 24 h. The reaction
mixture was filtered through Celite and the solvent was removed
under vacuum. The residue was recrystallized from ethyl acetate/
cyclohexane to afford the title product 7 as a cotton-like colorless
solid (105 mg, 0.32 mmol, 82% yield). Mp 170 °C; Rf 0.67 (metha-
nol/ethyl acetate: 30/70); 1H NMR (300 MHz): d (CD3COCD3) 1.48
(d, 3H, J = 7.0 Hz), 3.84 (q, 1H, J = 7.0 Hz), 4.49 (dd, 2H, J = 6.0,
14.1 Hz), 7.20–7.38 (m, 6H), 7.56 (t, 1H, J = 8.2 Hz), 7.77 (m, 1H),
7.94 (m, 1H), 7.99 (br s, 1H). 13C NMR (300 MHz): d (CD3COCD3)
14.19, 14.30, 43.75, 43.87, 47.76, 47.81, 123.72, 127.19, 127.90,
127.91, 128.34, 128.35, 129.32, 130.60, 133.05, 140.27, 152.04,
166.82, 169.81, 169.89, 170.33. Anal. Calcd for C18H17NO5: C,
66.04; H, 5.24; N, 4.28. Found: C, 65.92; H, 5.33; N, 4.14.
4.1.2. 3-(2-Benzylcarbamoyl-3-phenyl-propionyloxy)-benzoic
acid (8) (Scheme 1)
4.1.2.1. 2-Benzylmalonamic acid ethyl ester N-benzylamide
17b. Benzylamine (865 lL, 7.94 mmol) and benzylmalonic acid
monoethyl ester33 (1.6 g, 7.21 mmol) were dissolved in dry dichlo-
romethane (20 mL) under argon, dicyclohexylcarbodiimide (1.78 g,
8.64 mmol) was added, and the reaction mixture was stirred over-
night. DCU was removed by filtration and the solvent was evapo-
rated under vacuum to give a oil which was chromatographed on
silica gel with cyclohexane/ethyl acetate (4/1) as eluent. The title
product 17b was isolated as a colorless solid (475 mg, 21% yield).
Mp 74 °C; Rf 0.17 (ethyl acetate/cyclohexane 10/90); 1H NMR: d
(CDCl3) 1.15 (t, 3H, J = 7.0 Hz), 3.26 (dd, 2H, J = 6.6, 8.1 Hz), 3.52
(dd, 1H, J = 6.6, 8.1 Hz), 4.11 (q, 2H, J = 7.0 Hz), 4.43 (dd, 2H,
J = 5.5,14.7 Hz), 6.71 (t, 1H, J = 5.5 Hz), 7.15–7.34 (m, 10H). 13C
NMR: d (CDCl3) 14.15, 36.65, 43.86, 55.11, 61.73, 127.01, 127.68,
127.82, 128.14, 128.72, 128.86, 129.13, 138.01, 167.79, 171.34.
Anal. Calcd for C19H21NO3: C, 73.29; H, 6.80; N, 4.50. Found: C,
72.96; H, 7.02; N, 4.67.
4.1.2.2. 3-(2-Benzylcarbamoyl-3-phenyl-propionyloxy)-benzoic
acid benzyl ester 18b. 2-Benzylmalonamic acid ethyl ester N-
benzylamide 18a (350 mg, 1.13 mmol) was dissolved in warm eth-
anol (10 ml). After cooling, potassium hydroxide (63 mg,
1.13 mmol, 45% w/w in water) was added dropwise and stirring
was maintained for 1 h. Ethanol and water were removed under
vacuum and the residue was dissolved in water (15 mL). The aque-
ous layer was acidified to pH 5 using dropwise addition of 20% HCl
at 0 °C and extracted with ethyl acetate (2 ꢀ 15 mL). The organic
layer was washed with brine (15 mL), dried over MgSO4 and evap-
orated to dryness to afford crude 2-benzylmalonamic acid N-ben-
zylamide as a white solid. This acid (303 mg, 1.07 mmol) and
3-hydroxybenzoic acid benzyl ester (245 mg, 1.07 mmol) were

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287
Figure 2. Stereoview of an energy-minimized tetrahedral intermediate structure, 16, formed on reaction of the P99 b-lactamase with the cyclic retro-amide 12. Only heavy
atoms are shown.
dissolved in dry tetrahydrofuran (15 mL), and DMAP (6 mg,
0.05 mmol) and dicyclohexylcarbodiimide (231 mg, 1.12 mmol)
were added. The reaction mixture was stirred overnight. DCU
was removed by filtration and the solvent was evaporated under
vacuum to give a colorless solid which was recrystallized from
ethyl acetate/cyclohexane to give the title product 18b (268 mg,
51% yield). Mp 132 °C; Rf 0.28 (ethyl acetate/cyclohexane 30/70);
1H NMR: d (CDCl3) 3.37 (dd, 2H, J = 6.9, 8.5 Hz), 3.78 (dd, 1H,
J = 6.9, 8.5 Hz), 4.46 (dd, 2H, J = 5.9,14.7 Hz), 5.36 (s, 2H), 6.64 (t,
1H, J = 5.9 Hz), 7.03 (m, 1H), 7.16–7.45 (m, 16H), 7.54 (m, 1H),
7.94 (m, 1H). 13C NMR: d (CDCl3) 36.84, 44.06, 55.16, 67.28,
122.79, 126.32, 127.37, 127.81, 127.87, 128.13, 128.56, 128.63,
128.86, 128.94, 128.96, 129.21, 129.70, 131.96, 135.92, 137.44,
137.77, 150.28, 165.49, 167.09, 169.89. Anal. Calcd for
C19H21NO3ꢂ1/2H2O: C, 74.09; H, 5.62; N, 2.79. Found: C, 74.11; H,
5.67; N, 2.87.
with a 10% aqueous HCl solution. The organic layer was dried over
MgSO4, the solvent evaporated, and the product purified by silica
gel chromatography (pentane/AcOEt 1/1). The title product 20,
783 mg (67%), was thus obtained. Mp 94 °C. Rf 0.35. 1H NMR
(CDCl3): d = 3.46 (s, 3H), 3.85 (s, 3H), 4.37 (s, 1H), 4.48 (m, 2H),
6.90 (m, 1H), 7.26–7.34 (m, 5H). 13C NMR (CDCl3): d = 43.45,
53.08, 58.66, 81.63, 127.87–137.66, 165.49, 168.11.
4.1.3.2. N-Benzyl-2-methoxymalonamic acid 21. Methyl N-ben-
zyl-2-methoxymalonamate 20 (421 mg, 1.78 mmol) was dissolved
in methanol (5 ml). NaOH solution (1 M, 5 ml) was added and the
mixture stirred at room temperature overnight. The methanol was
evaporated. The aqueous phase was washed with ethyl acetate,
acidified and the product extracted with AcOEt. The organic layer
was dried over MgSO4 and the solvent evaporated to give 355 mg
(86%) of the title product 21. Mp 106 °C. 1H NMR (CDCl3):
d = 3.63 (s, 3H), 4.36 (s, 1H), 4.50 (m, 2H), 7.26–7.39 (m, 6H),
8.87 (s, 1H). 13C NMR (CDCl3): d = 43.90, 60.09, 79.18, 128.08–
136.76, 167.89, 168.45.
4.1.2.3. 3-(2-Benzylcarbamoyl-3-phenyl-propionyloxy)-benzoic
acid 8. 3-(2-Benzylcarbamoyl-3-phenyl-propionyloxy)-benzoic
acid benzyl ester 18b (127 mg, 0.26 mmol) was dissolved in warm
ethyl acetate (30 mL) and the solution was slowly cooled to room
temperature. Palladium on charcoa1 (38 mg, 30%/w) was added
and the mixture was stirred under a hydrogen atmosphere for
24 h. The reaction mixture was filtered through Celite and the sol-
vent was removed under vacuum. The residue was recrystallized
from ethyl acetate/cyclohexane to afford the title product 8 as a
white solid (73 mg, 70% yield). Mp 174 °C; Rf 0.53 (methanol/
ethyl acetate 10/90); 1H NMR: d (CDCl3+MeOD) 3.24 (d, 2H,
J = 11.8 Hz), 3.74 (t, 1H, J = 11.8 Hz), 4.30 (s, 2H), 6.98–7.05 (m,
3H), 7.11–7.24 (m, 8H), 7.32 (t, 1H, J = 11.8 Hz), 7.54 (m, 1H),
7.81 (m, 1H). 13C NMR: d (CDCl3+MeOD) 35.52, 43.60, 54.76,
122.75, 125.90, 126.97, 127.37, 127.51, 127.59, 128.58, 128.67,
129.03, 129.44, 137.54, 137.67, 147.67, 150.27, 167.85, 169.05,
170.52. Anal. Calcd for C24H21NO5: C, 71.45; H, 5.25; N, 3.55.
Found: C, 71.41; H, 5.35; N, 3.55.
4.1.3.3. 3-Benzyloxycarbonylphenyl N-benzyl-2-methoxymalo-
namate 22. To a solution of 223 mg (1 mmol) of N-benzyl-2-
methoxy-malonamic acid 21 in dichloromethane (2 ml) were
added 228 mg (1 mmol) of benzyl 3-hydroxybenzoate35 and
206 mg (1 mmol) of DCC. The reaction mixture was stirred at room
temperature for 16 h and then filtered to remove DCU. The solvent
was evaporated and the residue recrystallized from diethyl ether to
give 236 mg (54%) of the title product 22. Mp 86 °C. Rf 0.68 (pen-
tane/AcOEt 1/1). 1H NMR (CDCl3): d = 3.59 (s, 3H), 4.54 (m, 2H),
4.61 (s, 1H), 5.38 (s, 2H), 7.01 (s, 1H), 7.27–7.50 (m, 12H), 7.87 (t,
1H), 7.99 (dt, 1H). 13C NMR (CDCl3): d = 43.55, 58.92, 67.24,
81.64, 122.77–137.56, 150.44, 165,10, 165,56, 166,15. Anal. Calcd
for C25H23NO6: C, 69.27; H, 5.35; N, 3.23. Found: C, 69.26; H,
5.37; N, 3.27.
4.1.3.4. 3-Hydroxycarbonylphenyl N-benzyl-2-methoxymalona-
mate 9. To a solution of 95 mg of ester 22 in ethyl acetate (30 ml)
was added 24 mg of 10% Pd/C. The hydrogenolysis was performed
in a Parr apparatus for 16 h. The catalyst was removed by filtration
and the solvent evaporated to give 60 mg (80%) of the title product
9. Mp 152 °C. Rf 0.19 (pentane/AcOEt 1/1). 1H NMR (CD3COCD3):
d = 3.58 (s, 3H), 4.51 (m, 2H), 4.75 (s, 1H), 7.23–7.42 (m, 12H),
7.60 (t, 1H), 7.80 (s, 1H), 7.96 (d, 1H), 8.15 (s, 1H). 13C NMR
4.1.3. 3-Hydroxycarbonylphenyl N-benzyl-2-methoxymalona-
mate (9) (Scheme 2)
4.1.3.1. N-Benzyl-2-methoxymalonamic acid methyl ester 20.
To a solution of methyl chlorocarbonylmethoxyacetate 1934
(825 mg, 5 mmol) in dichloromethane (10 ml) at 0 °C was added
a solution of benzylamine (1.07 g, 10 mmol) in dichloromethane
(10 ml). The reaction mixture was stirred for 1 h and then washed

288
S. A. Adediran et al. / Bioorg. Med. Chem. 18 (2010) 282–291
(CD3COCD3): d = 43.39, 58.87, 82.52, 117.05–140.07, 151.64,
166.20, 166.74, 167.42. Anal. Calcd for C18H17NO6: C, 62.97; H,
4.99; N, 4.08. Found: C, 62.86; H, 4.91; N, 4.35.
4.1.5. 3-[2-(Benzylaminocarbonyl)-ethanoyl]-oxybenzoic acid
11
4.1.5.1. Methyl N,N-dibenzyl malonamate. To a solution of
methyl 3-chloro-3-oxopropionate (255 mg, 1.86 mmol) in dichlo-
romethane (5 mL) at 0 °C was added dropwise a solution of diben-
zylamine (736 mg, 3.73 mmol) in dichloromethane (2 mL). The
reaction mixture was stirred at room temperature for 3 h. The solu-
tion was washed with 10% aqueous HCl, dried over MgSO4, the sol-
vent evaporated under vacuum, and the residue purified by silica
gel chromatography (eluent: cyclohexane/ethyl acetate 7/3). The
title compound (538 mg, 97%) was obtained as a liquid. Rf 0.34
(cyclohexane/ethyl acetate: 7/3). 1H NMR (CDCl3) 3.56 (s, 2H),
3.76 (s, 3H), 4.45 and 4.65 (2s, 2H), 7.16–7.42 (m, 10H). 13C NMR
(CDCl3) d 48.6, 50.8, 52.6, 126.6–136.8, 166.9, 168.2.
4.1.4. 3-[2-(N-Methyl-benzylaminocarbonyl)-ethanoyl]-
oxybenzoic acid 10
4.1.4.1. Methyl N-methyl-N-benzylmalonamate. To a solution
of methyl 3-chloro-3-oxopropionate 1.273 g (9.33 mmol) in
dichloromethane (10 mL) at 0 °C, was added dropwise a solution
of N-methylbenzylamine (2.26 g, 18.6 mmol) in dichloromethane
(5 mL). The reaction mixture was stirred at 25 °C for 3 h. The solu-
tion was washed with 10% aqueous HCl, dried over MgSO4, the sol-
vent evaporated, and the residue purified by silica gel
chromatography (eluent: cyclohexane/ethyl acetate 1/1). The title
compound (1.72 g, 83%) was obtained as a liquid. Rf 0.51 (cyclohex-
ane/ethyl acetate: 1/3). 1H NMR (CDCl3) (2 conformers) d 2.91 and
2.97 (2s, 3H), 3.49 and 3.53 (2s, 2H), 3.71 and 3.76 (2s, 3H), 4.53
and 4.61 (2s, 2H), 7.16–7.37 (m, 5H). 13C NMR (CDCl3) (2 conform-
ers) d 34.2 and 35.5, 41.2 and 41.4, 51.1 and 54.1, 52.6, 126.5–
136.9, 166.3 and 168.1.
4.1.5.2. N,N-Dibenzylmalonamic acid 17d. Methyl N,N-dib-
enzylmalonamate (411 mg, 1.53 mmol) was dissolved in methanol
(10 mL). NaOH solution (1 M, 10 mL) was added and the mixture
was stirred at room temperature overnight. The methanol was re-
moved by evaporation and the residual aqueous solution acidified
with 20% aqueous HCl. The reaction mixture was extracted with
ethyl acetate. The organic phase was dried (MgSO4) and evapo-
rated under vacuum to give 305 mg (78% yield) of the title product
17d. Rf 0.53 (acetic acid/ethyl acetate: 2.5/97.5). 1H NMR
(CD3COCD3) d 3.63 (s, 2H), 4.62 and 4.65 (2s, 2H), 7.27 to 7.41
(m, 10H). 13C NMR (CDCl3) d 39.0, 49.2, 51.2, 127.8–137.9, 168.7,
170.1.
4.1.4.2. N-Methyl-N-benzyl-malonamic acid 17c. Methyl N-
methyl-N-benzylmalonamate (1.1 g, 5 mmol) was dissolved in
methanol (15 mL). NaOH solution (1 M, 15 mL) was added and
the mixture was stirred at room temperature overnight. After
the methanol was evaporated, the solution was acidified with
20% aqueous HCl and extracted with ethyl acetate. The organic
solution was dried (MgSO4) and the solvent evaporated under
vacuum to give quantitatively the title product 17c. Rf 0.39 (acetic
acid/ethyl acetate: 2.5/97.5). 1H NMR (CD3COCD3) (2 conformers)
d 2.95 and 3.05 (2s, 3H), 3.57 and 3.60 (2s, 2H), 4.66 and 4.69 (2s,
2H), 7.29–7.40 (m, 5H). 13C NMR (CDCl3) (2 conformers) d 34.1
and 35.4, 37.8 and 38.0, 51.4 and 53.8, 127.6–137.9, 168.5
and170.3.
4.1.5.3. Benzyl 3-[2-(N,N-dibenzylaminocarbonyl)-ethanoyl]-oxy-
benzoate 18d. To a solution of 267 mg (0.94 mmol) of N,N-dib-
enzylmalonamic acid, 17d, in dichloromethane (6 mL) were added
194 mg (0.94 mmol) of DCC and 214 mg (0.94 mmol) of benzyl
3-hydroxybenzoate. The mixture was stirred at room temperature
for 2 h. DCU was filtered off. The solvent was evaporated and the res-
idue purified by chromatography (eluents: cyclohexane/ethyl ace-
tate: 9/1, then 8/2) to give 242 mg (52% yield) of the title product
18d as a liquid. Rf 0.29 (cyclohexane/ethyl acetate: 8/2). 1H NMR
(CDCl3) 3.80 (s, 2H), 4.52 and 4.71 (2s, 2H), 5.39 (s, 2H), 7.21–7.48
(m, 17H), 7.84 (t, 1H), 8.00 (dt, 1H). 13C NMR (CDCl3) d 41.4, 48.7,
50.8, 67.2, 122.8–136.6, 150.6, 165.6, 166.2, 166.4.
4.1.4.3. Benzyl 3-[2-(N-methyl-benzylaminocarbonyl)-ethanoyl]-
oxybenzoate 18c. To a solution of 310 mg (1.5 mmol) of N-methyl-
N-benzyl-malonamic acid 17c in dichloromethane (10 mL) were
added 309 mg (1.5 mmol) of DCC and 340 mg (1.5 mmol) of benzyl
3-hydroxybenzoate. The mixture was stirred at room temperature
overnight. DCU was removed by filtration. The solvent was evapo-
rated and the residue purified by silica gel chromatography (eluent:
cyclohexane/ethyl acetate: 7/3) to give 445 mg (71% yield) of title
product 18c as a liquid. Rf 0.43 (cyclohexane/ethyl acetate: 1/1). 1H
NMR (CDCl3) (2 conformers) d 2.99 and 3.04 (2s, 3H), 3.74 and
3.78 (2s, 2H), 4.59 and 4.67 (2s, 2H), 5.37 and 5.38 (2s, 2H), 7.21–
7.51 (m, 12H), 7.82 and 7.85 (2t, 1H), 7.97 and 7.99 (2dt, 1H). 13C
NMR (CDCl3) (2 conformers) d 34.4 and 35.5, 41.2 and 42.6, 51.2
and 54.2, 67.1 and 67.2, 122.9–136.7, 150.6 and 152.0, 165.5,
165.8, 166.1.
4.1.5.4. 3-[2-(Benzylaminocarbonyl)-ethanoyl]-oxybenzoic acid
11. To a solution of the benzyl ester 18d (131 mg, 0.27 mmol) in
ethyl acetate (10 mL) was added 22 mg of Pd/C 10% and the mix-
ture was hydrogenated at rt for 2 h. The reaction mixture was fil-
tered and the solvent evaporated. The solid residue was washed
with pentane to give 101 mg (94%) of title product 11, which was
recrystallized from acetone. Mp 137 °C Rf 0.59 (pentane/acetone:
1/1). 1H NMR (acetone) d 3.95 (s, 2H), 4.66 (s, 4H), 7.29–7.44 (m,
11H), 7.58 (t, 1H), 7.85 (t, 1H), 7.96 (dt, 1H). 13C NMR (CD3COCD3)
d 41.8, 49.1, 51.5, 123.9–138.4, 152.1, 166.9, 167.3, 167.5. Anal.
Calcd for C24H21NO5: C, 71.45; H, 5.25; N, 3.47; Found: C, 71.38;
H, 5.33; N, 3.32.
4.1.4.4. 3-[2-(N-Methyl-benzylaminocarbonyl)-ethanoyl]-oxyben-
zoic acid 10. The benzyl ester 18c (140 mg, 0.33 mmol) was dis-
solved in ethyl acetate (10 mL). 20 mg of 10% Pd/C was added and
the solution was hydrogenated at room temperature for 2 h. The
reaction mixture was filtered and the solvent evaporated. The solid
residue was washed with pentane to give 96 mg (88% yield) of title
product 10, which was recrystallized from acetone. Mp 147 °C. Rf
0.48 (acetone). 1H NMR (CD3COCD3) (2 conformers) d 2.95 and 3.08
(2s, 3H), 3.89 and 3.93 (2s, 2H), 4.66 and 4.71 (2s, 2H), 7.26–7.47
(m, 6H), 7.57 and 7.58 (2t, 1H), 7.83 and 7.85 (2t, 1H), 7.94 and
7.95 (2dt, 1H). 13C NMR (CD3COCD3) (2 conformers) d 33.9 and
35.7, 41.7 and 41.9, 51.2 and 54.2, 123.9–138.5, 152.0 and 152.1,
166.8, 166.9, 167.3. Anal. Calcd for C18H17NO5: C, 66.04; H, 5.24; N,
4.28. Found: C, 65.92; H, 5.32; N, 4.12.
4.1.6. 3-Carboxylphenyl 1-benzyl-2-oxo-pyrrolidine-3-carboxy-
late 12 (Scheme 3)
4.1.6.1. Methyl 1-benzyl-2-oxo-pyrrolidine-3-carboxylate 23.
1-Benzyl-2-pyrrolidinone (262 mg, 1.5 mmol) was dissolved in
anhydrous THF (3 mL). The solution was cooled to ꢁ78 °C under
argon, and a 2 N solution of LDA (1 mL, 1.3 equiv) was added drop-
wise. After a further 30 min stirring at ꢁ78 °C, methyl chlorofor-
mate (107 mg, 1.12 mmol) was added. After 30 min at ꢁ78 °C,
the temperature was allowed to rise to 25 °C and the mixture
was stirred for 1 h. The reaction mixture was then poured into
aqueous 1 M HCl and extracted with ethyl acetate. The organic
phase was dried over MgSO4, the solvent was removed by

S. A. Adediran et al. / Bioorg. Med. Chem. 18 (2010) 282–291
289
evaporation, and the residue chromatographed on silica gel
(eluent: cyclohexane/AcOEt 25/75) to give 91 mg (35% yield) of
the title compound 23 as an oil. Rf 0.35. 1H NMR (CDCl3) d 2.24
and 2.39 (2 m, 2H), 3.23 and 3.39 (2 m, 2H,), 3.52 (dd, J = 9.5,
4.8 Hz, 1H), 3.80 (s, 3H), 4.48 (s, 2H), 7.23–7.37 (m, 5H). 13C NMR
Methyl 2-nitrobenzylmalonate 26: 1H NMR (CDCl3) d 3.52 (d,
J = 7.7 Hz, 2H), 3.71 (s, 6H), 3.93 (t, 1H), 7.35 (d, 1H), 7.40–7.55
(m, 2H), 8.01 (d, 1H). 13C NMR (CDCl3) d 32.43, 52.33, 52.90,
169.11.
(CDCl3)
169.70, 170.71.
d
22.17, 45.08, 46.97, 48.31, 52.63, 127.68–135.88,
4.1.7.2. Methyl 2-oxo-1,2,3,4-tetrahydroquinoline-3-carboxyl-
ate 27. To a solution of compound 26 (240 mg, 0.9 mmol) in ethyl
acetate (15 mL) was added 20 mg of Pd/C and the mixture stirred
for 3 h at 25 °C under an H2 atmosphere. Filtration, evaporation
of the solvent and column chromatography of the residue (eluent:
cyclohexane/AcOEt 50/50) afforded 155 mg (84%) of the title prod-
uct 27. Mp 166.5 °C. Rf 0.38. 1H NMR (CDCl3) d 3.13 (dd, J = 6.2,
15.8 Hz, 1H), 3.41 (dd, J = 9.0, 15.8 Hz, 1H), 3.66 (dd, 1H), 3.78 (s,
3H), 6.85 (d, 1H), 7.00 (t, 1H), 7.15–7.23 (m, 2H), 9.09 (s, 1H). 13C
4.1.6.2. 1-Benzyl-2-oxo-pyrrolidine-3-carboxylic acid 24. To a
solution of methyl 1-benzyl-2-oxo-pyrrolidine-3-carboxylate 23
(178 mg, 0.76 mmol) in methanol (5 mL) was added NaOH solution
(1 M, 5 mL) and the reaction mixture was stirred at 25 °C over-
night. After evaporation of the methanol and extraction of the
residual aqueous solution with ethyl acetate, the aqueous phase
was acidified, extracted with ethyl acetate, and the extract dried
over MgSO4. Evaporation of the solvent gave quantitatively the ti-
tle acid 24. Rf 0.42 (AcOEt/AcOH 95/5). 1H NMR (CD3COCD3) d 2.31
(m, 2H), 3.36 (m, 2H), 3.55 (t, J = 8.7 Hz, 1H), 4.49 (dd, 2H), 7.27–
7.34 (m, 5H). 13C NMR (CD3COCD3) d 22.75, 45.69, 47.16, 48.28,
128.27–137.43, 171.28, 171.68.
NMR (CDCl3)
d 29.03, 47.56, 52.94, 115.88–136.71, 167.65,
169.80. Anal. Calcd for C11H11NO3: C, 64.38; H, 5.40; N, 6.83.
Found: C, 64.27; H, 5.53; N, 6.64.
4.1.7.3. Methyl N-3-di-tert-butoxycarbonyl-2-oxo-1,2,3,4-tetra-
hydroquinoline-3-carboxylate 28. To a solution of ester 27
(95 mg, 0.5 mmol) in acetonitrile (5 mL) was added 218 mg
(1.0 mmol) of Boc2O and 61 mg of DMAP and the mixture was stir-
red at 25 °C overnight. Evaporation of the solvent and silica gel
chromatography (eluent: cyclohexane/AcOEt 70/30) gave 155 mg
(76%) of the title product 28. Mp 108.4 °C. Rf 0.47. (24 mg of methyl
3-tert-butoxycarbonyl-2-oxo-1,2,3,4-tetrahydroquinoline-3-car-
boxylate 28 were also isolated). 1H NMR (CDCl3) d 1.28 (s, 9H), 1.62
(s, 9H), 3.40 (d, J = 15.6 Hz, 1H), 3.60 (d, 1H), 3.81 (s, 3H), 6.91 (d,
1H), 7.09 (t, 1H), 7.09–7.24 (m, 2H). 13C NMR (CDCl3) d 27.56,
27.81, 33.36, 53.57, 63.57, 83.95, 85.75, 116.85–136.38, 151.26,
163.98, 165.16, 167.23.
4.1.6.3. 3-Benzyloxycarbonylphenyl 1-benzyl-2-oxo-pyrrolidine-
3-carboxylate 25. To a solution of the acid 24 (305 mg, 1.39 mmol)
in dichloromethane (5 mL) was added 318 mg (1.39 mmol) of ben-
zyl 3-hydroxybenzoate, and then 287 mg (1.39 mmol) of DCC. The
reaction mixture was stirred at 25 °C overnight. DCU was removed
by filtration and the solvent evaporated under vacuum. Two column
chromatographies on silica gel (eluent: hexane/AcOEt 70/30, then
dichlorométhane/acetone 97.5/2.5) afforded 525 mg (88%) of the ti-
tle ester 25 as an oil. Rf 0.48 (ether). 1H NMR (CDCl3) d 2.39, 2.52 (2 m,
2H), 3.31, 3.44 (2 m, 2H), 3.76 (dd, J = 6.9, 9.4 Hz, 1H), 4.53 (s, 2H),
5.38 (s, 2H), 7.28- 7.45 (m, 12H), 7.85 (t, 1H), 7.98 (dt, 1H). 13C
NMR (CDCl3) d 22.43, 45.31, 47.35, 48.72, 117.25–135.97, 152.32,
165.64, 168.96, 169.36. Anal. Calcd for C26H23NO5: C, 72.71; H,
5.40; N, 3.26. Found: C, 72.50; H, 5.49; N, 2.95.
4.1.7.4. Methyl 3-tert-butoxycarbonyl-2-oxo-1,2,3,4-tetrahydro-
quinoline-3-carboxylate 29. To a stirred solution of compound 28
(232 mg, 0.59 mmol) in dichloromethane (5 mL) at 0 °C was added
TFA (0.5 mL). After 1 h, toluene (5 mL) was added and the solvents
were evaporated under vacuum at room temperature. Silica gel col-
umn chromatography (eluent: cyclohexane/AcOEt 70/30) afforded
161 mg (92%) of the title product 29, as an oil. Rf 0.27. 1H NMR
(CDCl3) d 1.36 (s, 9H), 3.47 (d, J = 15.6 Hz, 1H), 3.58 (d, 1H), 3.79
(s, 3H), 6.82 (d, 1H), 7.01 (t, 1H), 7.16–7.27 (m, 2H), 9.35 (s, 1H).
13C NMR (CDCl3) d 27.70, 33.40, 53.49, 62.94, 83.76, 115.99–
136.36, 165.76, 166.60, 167.80.
4.1.6.4. 3-Carboxylphenyl 1-benzyl-2-oxo-pyrrolidine-3-carbox-
ylate 12. To a solution of ester 25 (193 mg, 0.45 mmol) in ethyl
acetate (10 mL) was added 20 mg of 10% Pd/C and the mixture stir-
red for 2 h under a hydrogen atmosphere. Filtration and evapora-
tion of the solvent afforded the title acid 12 as a syrup. Rf 0.68
(AcOEt/AcOH 99/1). 1H NMR (CD3COCD3) d 2.49 (m, 2H), 3.44 (m,
2H), 3.83 (t, J = 8.5 Hz, 1H), 4.47, 4.58 (2d, J = 14.9 Hz, 2H),
7.27–7.35 (m, 5H), 7.44 (dt, 1H), 7.59 (t,1H), 7.83 (t, 1H), 7.97
(dt, 1H). 13C NMR (CD3COCD3) d 23.21, 45.81, 47.28, 49.42,
123.79–137.77, 151.99, 166.81, 167.96, 170.10. Anal. Calcd for
C19H17NO5ꢂ1/2H2O: C, 65.50; H, 5.20; N, 4.02. Found: C, 65.31; H,
5.14; N, 4.11.
4.1.7.5. Methyl N-benzyl-3-tert-butoxycarbonyl-2-oxo-1,2,3,4-
tetrahydroquinoline-3-carboxylate 30. To a solution of com-
pound 29 (260 mg, 0.85 mmol) in acetonitrile (10 mL) was added
30 mg of benzyltriethylammonium chloride, 250 mg of K2CO3 then
benzyl chloride (0.22 mL) and the mixture was heated at 60 °C for
4 h under stirring. After cooling of the mixture and its extraction
with ethyl acetate, the organic phase was washed with water
and dried over MgSO4. Silica gel column chromatography (eluent:
cyclohexane/AcOEt 80/20) afforded 278 mg (83%) of the title prod-
uct 30, as an oil. Rf 0.36. 1H NMR (CDCl3) d 1.33 (s, 9H), 3.49 (d,
J = 15.6 Hz, 1H), 3.60 (d, 1H), 3.81 (s, 3H), 5.18 (s, 2H), 6.88 (d,
1H), 7.01 (d, 1H), 7.13 (t, 1H), 7.18–7.42 (m, 6H). 13C NMR (CDCl3)
d 27.79, 33.36, 47.86, 53.61, 63.50, 83.83, 115.98- 139.39, 165.19,
165.89, 167.97.
4.1.7. 3-Carboxylphenyl N-benzyl-2-oxo-1,2,3,4-tetrahydroqui-
noline-3-carboxylate 13 (Scheme 4)
4.1.7.1. Methyl 2-nitrobenzylmalonate 26. To a solution of di-
methyl malonate (1.056 g, 8.0 mmol) in DMF (18 mL) was slowly
added 320 mg (8.0 mmol) of sodium hydride (60% in oil) and the
mixture stirred for 15 min at 25 °C. 2-Nitrobenzyl chloride
(343 mg, 2.0 mmol) was added and the reaction mixture stirred
for a further 2 h., then poured into 10% HCl. After extraction of
the acidified solution with ethyl acetate, the organic phase was
washed three times with distilled water, brine, and then dried over
MgSO4. Dimethyl malonate was removed in a Büchi apparatus at
150 °C under vacuum (20 mm Hg). The residue (530 mg) was a
mixture of monoalkylated and dialkylated products in a ratio 83/
11 (NMR). Two spots were observed by tlc (eluent: cyclohexane/
AcOEt 70/30). Rf monoalkylated derivative: 0.46; Rf dialkylated
derivative: 0.42. These products were separated and purified by sil-
ica gel column chromatography.
4.1.7.6. Methyl N-benzyl-2-oxo-1,2,3,4-tetrahydroquinoline-3-
carboxylate 31. To
a solution of compound 30 (162 mg,
0.55 mmol) in dichloromethane (1 mL) was added TFA (1 mL)
and the mixture was stirred for 1 h at 25 °C. Silica gel column chro-
matography (eluent: cyclohexane/AcOEt 70/30) gave 113 mg (93%)
of the title product 31. Mp 107 °C. Rf 0.40. 1H NMR (CDCl3) d 3.18
(dd, J = 15.5, 5.9 Hz, 1H), 3.41 (dd, J = 15.5, 8.3 Hz, 1H), 3.80 (dd,

290
S. A. Adediran et al. / Bioorg. Med. Chem. 18 (2010) 282–291
1H), 3.76 (s, 3H), 5.07, 5.33 (2d, J = 16.2 Hz, 2H), 6.90 (d, 1H), 7.01
(t, 1H), 7.13 (t, 1H), 7.17–7.37 (m, 6H). 13C NMR (CDCl3) d 29.01,
47.81, 48.31, 52.83, 115.98–139.43, 166.75, 169.94.
4.1.8.2. 3-[(Benzylcarbamoyl-methyl)-hydroxy-phosphinoyloxy]-
benzoic acid 14. A mixture of 3-[(benzylcarbamoyl-methyl)-
methoxy-phosphinoyloxy]-benzoic acid methyl ester 35 (98 mg,
0.26 mmol) and iodotrimethylsilane (150 lL, 1.05 mmol) was
4.1.7.7. N-Benzyl-2-oxo-1,2,3,4-tetrahydroquinoline-3-carbox-
ylic acid 32. To a solution of ester 31 (108 mg, 0.37 mmol) in
methanol (5 mL) was added 10% aqueous NaOH (5 mL). After the
solution was stirred for 1 h at 25 °C, methanol was removed by
evaporation and the aqueous phase extracted with ethyl acetate.
The extract was dried over MgSO4 and the solvent removed under
vacuum, quantitatively affording the title acid 32 as an oil. Rf 0.56
(eluent: AcOEt/AcOH 97.5/2.5). 1H NMR (CDCl3) d 3.27 (dd, J = 15.0,
6.1 Hz, 1H), 3.38 (dd, J = 15.0, 11.6 Hz, 1H), 3.70 (dd, 1H), 5.17, 5.27
(2d, J = 16.2 Hz, 2H), 6.95 (d, 1H), 7.07 (t, 1H), 7.18 (t, 1H), 7.20–
7.35 (m, 6H). 13C NMR (CDCl3) d 28.50, 45.21, 47.40, 115.07–
138.71, 169.08. 172.12. Anal. Calcd for C17H15NO3: C, 72.58; H,
5.38; N, 4.98. Found: C, 72.47; H, 5.51; N, 4.74.
stirred and heated at 50 °C for 16 h. The reaction mixture was then
allowed to cool and excess iodotrimethylsilane was removed under
vacuum. The resulting pale yellow solid was recrystallised from
methanol/dichloromethane affording the product 14 as a colorless
powder (49 mg, 54%). Mp 201 °C; Rf 0.27 (ethyl acetate/methanol
1:1); 1H NMR d (MeOD) 3.09 (d, 2H, J = 21.7 Hz), 4.41 (s, 2H),
7.22–7.46 (m, 7H) 7.83- 7.88 (m, 2H); 13C NMR d (MeOD+
CD3SOCD3) 36.92 (d, J = 133 Hz), 44.39, 123.20 (d, J = 3.9 Hz),
126.73 (d, J = 4.5 Hz), 127.21 (d, J = 1.1 Hz), 128.38, 128.72,
129.74, 131.09, 133.93 (d, J = 1.1 Hz), 140.01, 152.23 (d,
J = 7.9 Hz), 166.91 (d, J = 6.2 Hz), 168.52. Anal. Calcd for C16H16
-
NO6Pꢂ1/2H2O: C, 53.63; H, 4.78; N, 3.91. Found: C, 53.61; H, 4.76;
N, 3.69.
4.1.7.8. 3-Benzyloxycarbonylphenyl N-benzyl-2-oxo-1,2,3,4-tet-
rahydroquinoline-3-carb-oxylate 33. To a solution of acid 32
(107 mg, 0.38 mmol) in dichloromethane (3 mL) was added
88 mg (0.38 mmol) of benzyl 3-hydroxylbenzoate and 79 mg
(0.38 mmol) of DCC. After the mixture was stirred for 4 h at
25 °C, the solvent was evaporated and the residue chromato-
graphed on silica gel (eluent : dichloromethane/acetone 99/1) to
give 133 mg (71%) of the title product 33, as an oil. Rf 0.41. 1H
NMR (CDCl3) d 3.32 (dd, J = 15.8, 5.9 Hz, 1H), 3.54 (dd, J = 15.8,
8.6 Hz, 1H), 4.04 (dd, 1H), 5.13, 5.34 (2d, J = 16.2 Hz, 2H), 5.36 (s,
2H), 6.94 (d, 1H), 7.02 (t, 1H), 7.15 (t, 1H), 7.18–7.44 (m, 12H),
7.73 (t, 1H), 7.97 (dt, 1H). 13C NMR (CDCl3) d 29.16, 47.27, 48.49,
67.19, 114.97–139.51, 150.58, 165.55, 166.35, 168.12.
4.2. Enzyme kinetics
The Enterobacter cloacae P99 and TEM-2 b-lactamases were pur-
chased from the Center for Applied Microbiology and Research,
Porton Down, Wilts., UK, and used as supplied. The Streptomyces
R6136 and Actinomadura R3937 DD-peptidases were generously
supplied by Professor J. -M. Frère of the University of Liège, Bel-
gium and Dr. Paulette Charlier, also of the University of Liège,
respectively. Cephalothin was a gift from Eli Lilly and Co.
All kinetics measurements were carried out in 100 mM MOPS
buffer, pH 7.5 at 25 °C. Stock solutions of the substrates were
prepared in DMSO. These stock solutions were then diluted into
buffer such that the final DMSO concentration was less than 5%.
At this level, DMSO did not affect the measured initial rates of
the enzyme-catalyzed reactions. Steady-state kinetics parameters
were obtained from initial rate measurements (spectrophotomet-
ric; appearance of m-hydroxybenzoate at 290 or 300 nm) at a
series of substrate concentrations. Non-linear fits of the data to
the Henri-Michaelis–Menten equation were then computed to
obtain values of the kinetics parameters. In several cases, the lin-
earity of these curves allowed only values of kcat/Km to be ob-
tained, with lower limits to kcat and Km estimated. The Km
value for reaction of 8 with the P99 b-lactamase was obtained
4.1.7.9. 3-Carboxylphenyl N-benzyl-2-oxo-1,2,3,4-tetrahydro-
quinoline-3-carboxylate 13. To a solution of ester 33 (115 mg,
0.23 mmol) in ethyl acetate (10 mL) was added 10% Pd/C (20 mg)
and the mixture was stirred for 2 h under a hydrogen atmosphere.
After removal of the Pd/C by filtration and evaporation of the sol-
vent, the solid residue was recrystallised from dichloromethane
to give 89 mg (95%) of the title product 13. Mp 147 °C. Rf 0.63
(AcOEt/AcOH 99/1). 1H NMR (CD3COCD3) d 3.41 (dd, J = 15.7,
2.0 Hz, 1H), 3.51 (dd, J = 15.7, 9.7 Hz, 1H), 4.17 (dd, 1H), 5.23.
5.34 (2d, J = 16.4 Hz, 2H), 7.01–7.40 (m, 10H), 7.58 (t, 1H), 7.79
(t, 1H), 7.94 (dt, 1H). 13C NMR (CD3COCD3) d 29.34, 46.84, 48.93,
116.78–140.29, 151.88, 166.77, 167.07, 169.08. Anal. Calcd for
C24H19NO5ꢂH2O: C, 68.72; H, 5.04; N, 3.34. Found: C, 68.67; H,
4.76; N, 3.32.
as the Ki value for inhibition of cephalothin (200 lM) turnover.
Rates of inhibition of the P99 and TEM-2 b-lactamases by the
phosphonates 14 and 15 were obtained from measurements of
enzyme activity against 0.2 mM cephalothin as a function of time
of incubation of the enzyme with the inhibitor. Reaction mix-
tures contained enzyme (0.2
the P99 enzyme and 3 mM for TEM-2. The measured initial rates
decreased in first order fashion. Exponential curve fitting
yielded pseudo-first order rate constants of inhibition and thus
second order rate constants.
lM) and phosphonate, 100 lM for
4.1.8. 3-[(Benzylcarbamoyl-methyl)-hydroxy-phosphinoyloxy]-
benzoic acid 14 (Scheme 5)
a
4.1.8.1. Methyl 3-[(benzylcarbamoyl-methyl)-methoxy-phosphi-
noyloxy]-benzoate 35. The phosphite 34 (840 mg, 3.44 mmol)
and N-benzyl-2-bromoacetamide (392 mg, 1.72 mmol) were
heated together with stirring for 16 h at 110 °C. After cooling the
reaction mixture, the product was isolated by silica gel chromatog-
raphy (eluent: dichloromethane/acetone (4:1). Recrystallisation of
the product from ethyl acetate/cyclohexane afforded 290 mg (45%
yield) of 35 as a colorless solid. Mp 103 °C; Rf 0.18; 1H NMR d
(CDCl3) 3.06 (d, 2H, J = 21.1 Hz), 3.84 (d, 3H, J = 11.4 Hz), 3.91 (s,
3H), 4.44 (d, 2H, J = 5.9 Hz), 7.01 (t, 1H, J = 5.9 Hz), 7.20–7.45 (m,
7H), 7.81–7.91 (m, 2H); 13C NMR d (CDCl3) 34.75 (d, J = 132 Hz),
43.91, 52.33, 53.86 (d, J = 6.7 Hz), 121.56 (d, J = 4.5 Hz), 125.09
(d, J = 3.9 Hz), 126.66 (d, J = 1.1 Hz), 127.49, 127.61, 128.65,
129.91 (d, J = 1.1 Hz), 132.10 (d, J = 1.1 Hz), 137.66, 149.86 (d,
J = 8.4 Hz), 162.85 (d, J = 3.9 Hz), 165.86. Anal. Calcd for
C18H20NO6P: C, 57.30; H, 5.34; N, 3.71. Found: C, 57.22; H, 5.55;
N, 3.94.
4.3. Molecular modeling
The structure of Figure 2 was derived from a computational
model of the enzyme–substrate complex that was set up essen-
tially as previously described15 and run on an SGI Octane 2 com-
puter with INSIGHT II 2005 (Accelrys, San Diego, CA). In these
models of the P99 b-lactamase, Lys 67 and Lys 315 were cationic,
Tyr 150 was neutral, and the tetrahedral intermediate 16 was an-
ionic. After the the C2–N ethylene bridge was constructed, the sta-
bility of the structure was explored by molecular dynamics (200 ps
runs, where the entire protein together with solvating water mol-
ecules were unrestricted). A typical snapshot of the dominant con-
formation was selected for energy minimization.

S. A. Adediran et al. / Bioorg. Med. Chem. 18 (2010) 282–291
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