Aminophosphonic acids and aminobis(phosphonic acids) as potential inhibitors of penicillin-binding proteins

Article abstract of DOI:10.1002/ejoc.200800812

Aminophosphonic acids and aminobis(phosphonic acids) have been prepared by the alkylation of Schiff bases with methyl bromoacetate or ethyl acrylate. Other pathways, like the modified Pudovik reaction and Kabachnik-Fields reaction, have been considered for the synthesis of the α-phosphonic bioisoster of aminocitrate. Partial or complete deprotection of the phosphonate ester have been realised by either acidic hydrolysis or by treatment with trimethylsilyl bromide. Evaluation against penicillin-binding proteins has shown that our compounds are modest inhibitors of class A β-lactamases, but have an interesting activity against R39 (D,D-peptidase/carboxypeptidase). Wiley-VCH Verlag GmbH & Co. KGaA, 2009.

Full text of DOI:10.1002/ejoc.200800812

FULL PAPER
DOI: 10.1002/ejoc.200800812
Aminophosphonic Acids and Aminobis(phosphonic acids) as Potential Inhibitors
of Penicillin-Binding Proteins
Joséphine Beck,[a] Sonia Gharbi,[a] Adriana Herteg-Fernea,[b] Lionel Vercheval,[b]
Carine Bebrone,[b] Patricia Lassaux,[b] Astrid Zervosen,[c] and
Jacqueline Marchand-Brynaert*[a]
Keywords: Phosphonic acids / Amines / Schiff bases / Alkylation / Pudovik reaction / Inhibitors
Aminophosphonic acids and aminobis(phosphonic acids)
have been prepared by the alkylation of Schiff bases with
methyl bromoacetate or ethyl acrylate. Other pathways, like
the modified Pudovik reaction and Kabachnik–Fields reac-
tion, have been considered for the synthesis of the α-phos-
acidic hydrolysis or by treatment with trimethylsilyl bromide.
Evaluation against penicillin-binding proteins has shown
that our compounds are modest inhibitors of class A β-lac-
tamases, but have an interesting activity against R39 (
peptidase/carboxypeptidase).
D,D-
phonic bioisoster of aminocitrate. Partial or complete depro- (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
tection of the phosphonate ester have been realised by either
Germany, 2009)
psipeptides,[5] azadepsipeptides,[6] α-oxo heterocycles,[7]
phenaceturates,[8] boronic acids[9] and phosph(on)ates.[10]
Pratt and co-workers have studied phosphonic acids as po-
tential inhibitors that act as transition-state analogues. The
reaction involves phosphonylation of the active site Ser-OH
and formation of a stable tetrahedral phosphonyl–enzyme
intermediate. They have described the preparation and bio-
chemical evaluation of phosphonic analogues of depsipep-
tides,[11] cyclic phosphates,[12] diaroyl phosph(on)ates[13] and
oxo phosph(on)ates.[14]
Introduction
Resistance to antibiotics is currently a major health con-
cern.[1] The production of β-lactamases is the most common
mechanism of resistance to β-lactam antibiotics.[2] Several
strategies have been considered to overcome the action of
these enzymes. Among them, the use of combination ther-
apy, which involves the treatment with a β-lactam antibiotic
and a β-lactamase inhibitor, has been successfully used for
two decades for the treatment of infections.[3] Clavunic acid,
sulbactam and tazobactam are the three β-lactamase inhibi-
For several years, our laboratory has been involved in
tors currently marketed.[4] However, although these β-lac- the discovery of novel non-β-lactam inhibitors of bacterial
tam inhibitors are active against class A β-lactamases, they enzymes, β-lactamases and ,-peptidases. Our work is
are almost inactive against the other classes. The emergence based on a fortuitous observation made by Fonze et al. dur-
of novel β-lactamases belonging to classes B, C and D ing the X-ray diffraction study of Bacillus licheniformis BS3
amplified the problem. The need to discover novel non-β- β-lactamase (class A enzyme) crystallised from a citrate
lactam inhibitors is now urgent because the β-lactam cycle buffer: this revealed the unexpected role of this compound,
is a structure well “known” by bacteria, which are able to which was perfectly located in the enzyme’s cavity.[15] Cit-
evolve quickly to fight against the new β-lactam drugs. Nu- rate is an inhibitor of all class A β-lactamases at the micro-
merous non-β-lactam inhibitors or substrates of β-lactam- molar level, with a Ki value of 490 µ at pH 5 against BS3.
ases have been described in the literature, for example, de- These observations led us to consider citrate as a “hit” for
the design of new affinity inhibitors of class A β-lactamases.
Recently, we described the synthesis and biochemical evalu-
[a] Unité de Chimie Organique et Médicinale, Université Catho-
lique de Louvain,
ation of a series of amino analogues and homologues of
citrate and isocitrate.[16] The activity against class A β-lac-
tamases was confirmed. Moreover, three compounds pro-
tected as lipophilic esters were good inhibitors of OXA-10,
a class D β-lactamase. In this work, the complex formed
between aminocitrate and BS3 was analysed by X-ray dif-
fraction. The results of this study have allowed us to explain
the slightly better but still modest activity of aminocitrate
compared with citrate (Ki value of 250 µ).[17]
Bâtiment Lavoisier, Place Louis Pasteur 1, 1348 Louvain-la-
Neuve, Belgium
Fax: +32-10-474168
E-mail: jacqueline.marchand@uclouvain.be
[b] Centre d’Ingénierie des Protéines, Université de Liège,
Bâtiment B6, Allée de la chimie 17, 4000 Sart-Tilman, Liège,
Belgium
[c] Centre de Recherches du Cylotron, Université de Liège,
Bâtiment B30, 4000 Sart-Tilman, Liège, Belgium
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2009, 85–97
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As a continuation of this work, we have studied the re- the reaction of diethyl (aminomethyl)phosphonate[27] and 3-
placement of the α-carboxylic function of aminocitrate by chlorobenzaldehyde in the presence of magnesium sulfate in
the bioisoster phosphonic acid and the lengthening of the dichloromethane in 76% yield after purification by
two other acidic chains to possibly create novel interactions. chromatography (Scheme 1). Thus, imine 1 was treated with
We were also interested in the biochemistry of aminobis- 2 equiv. of lithium diisopropylamide (LDA) and 2 equiv. of
(phosphonic) compounds structurally related to citrate. The methyl bromoacetate in tetrahydrofuran at –78 °C.[28] These
target molecules are presented in Figure 1.
conditions, previously used for the synthesis of amino-
citrate,[17] led to monoalkylated product 2 in 51% isolated
yield with no detectable dialkylated product 3.[17] The best
results were obtained by treatment of imine 1 with two suc-
cessive fractions of 1 equiv. each of LDA and bromoacetate.
A mixture of mono- and dialkylated products 2 and 3 was
obtained, separable by chromatography to give 38 and 21%
yields, respectively. The addition of hexamethylphos-
phoramide (HMPA) did not improve the yield of the de-
sired product 3.
Figure 1. Aminocitrate and phosphonic bioisosters.
The key role of amino acids in life chemistry has led to
intense research into various mimetics. In particular, amino-
phosphonic acids very efficiently mimic amino acids and
compete with them for the active sites of enzymes. They
exhibit numerous activities of interest in medicinal chemis-
try, namely as inhibitors of various proteolytic enzymes, ag-
onists or antagonists of receptors involved in the central
nervous system (CNS), natural product analogues and
metal complexing agents.[18,19] Geminal bis(phosphonic ac-
ids) are metabolically stable analogues of naturally occur-
ring inorganic pyrophosphates. Fleisch et al. have shown
that they are able to impair the formation and dissolution
of calcium phosphate in vitro.[20] Bis(phosphonic acids) are
currently used for the treatment of various diseases of bone
mineral metabolism like osteoporosis.[20,21] To the best of
our knowledge, aminophosphonic compounds have never
been considered as potential inhibitors of bacterial enzymes
implicated in cell wall biosynthesis and resistance to penicil-
lin antibiotics.
Scheme 1. Alkylation of imine 1.
Therefore an alternative method was applied that pro-
duced high yields of imine 2 as the key intermediate
(Scheme 2). This was prepared in three steps from 4-acet-
oxyazetidinone by an Arbuzov reaction. The acetoxy group
was substituted by triethyl phosphite in toluene in 84%
yield.[29] Treatment of the azetidinone 5 with thionyl chlo-
ride in methanol led to opening of the β-lactam ring and
Results and Discussion
Synthesis of the α-Phosphonic Analogue of Aminocitrate
Several strategies have been reported in the literature for formation of methyl ester 6 in 90% yield.[30] Finally, the
the synthesis of α-aminophosphonic derivatives.[22] Among amine 6 was treated with 3-chlorobenzaldehyde in the pres-
them are the Kabachnik–Fields and Pudovik reactions, and ence of magnesium sulfate in dichloromethane to furnish
the alkylation of iminophosphonates or related compounds. the imine 2 in 85% yield after purification by chromatog-
The Kabachnik–Fields condensation reaction is a three- raphy.
component coupling reaction of carbonyl, amine and hy-
Several conditions have been tested for the deprotonation
drophosphoryl or phosphite compounds that can be carried and alkylation of intermediate 2, and the yields of com-
1
out with or without catalyst in a solvent or a solvent-free pound 3 have been determined on the crude mixture by H
medium.[23] The Pudovik reaction is the addition of hydro- NMR spectroscopy, as summarised in Table 1 and
phosphoryl or phosphite compounds to imines.[24] Numer- Scheme 2. In the first tests, the imine solution was added to
ous Lewis acid catalysts have been used to improve the effi- the base in solution (Entries 1–6, addition mode A). The
cacy of the reaction, for example, ZnCl2, AlCl3 and CdI2.[25] conditions tested led either to the deterioration of the start-
The last approach involves the preliminarily creation of a ing material or to the formation of at least four products
protected α-aminophosphonate and its subsequent reaction (Entries 1–4). The best result was 56% of 3, obtained by
with a base and appropriate electrophilic reagents.[26]
deprotonation with sodium hydride in tetrahydrofuran (En-
The first strategy considered for the synthesis of an α- try 5), but this reaction was carried out on a small scale
phosphonic analogue of aminocitrate was the alkylation of (200 mg). In the next tests, the base was added to imine
an α-phosphonic imine. The Schiff base 1 was prepared by 2 in solution (Entries 7–9; addition mode B). When using
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Inhibitors of Penicillin-Binding Proteins
Scheme 2. Synthesis of target molecule 7.
Table 1. Conditions and bases used for the alkylation of imine 2.
Entry
Base (equiv.)
Conditions[a]
Results; ratio 2/3[b]
1
2
3
4
5
6
7
8
9
tBuOK (1.2)
tBuOK (1.2)
tBuOK (1.2)
NaH (2)
THF, TA (A)
THF, 0 °C (A)
THF, –60 °C (A)
DMF, 0 °C (A)
THF, 0 °C (A)
THF, –78 °C (A)
THF, –78 °C (B)
CH3CN, 50 °C (B)
EtOH, 80 °C (B)
THF, –78 °C (A)
THF, –78 °C (A)
degradation
2, 3 and unidentified products
2, 3 and unidentified products
degradation
NaH (2)
2/3; 44:56
KHMDS (1.4)
KHMDS (1.2)
K2CO3 (2)
EtONa (1.5)
LDA (1.4)
2, 3 and unidentified products
2/3; 62:38
2/3; 94:6
transesterification of 2
2/3; 58:42
10
11
LDA (1.4) + DMPU (1.6)
2/3; 30:70
1
[a] Addition mode A or B, see text. [b] Ratio determined by H NMR spectroscopy.
potassium hexamethyldisilazide (KHMDS) as the base (En- produced the target molecule 7, the phosphonic analogue
try 7), 38% of 3 was formed, whereas phase-transfer condi- of aminocitrate, isolated as the hydrochloride salt. Selective
tions (Entry 8) led to just traces of 3. The reaction with deprotection of the phosphonate ester was carried out with
sodium ethoxide gave mainly the transesterification product trimethylsilyl bromide in dichloromethane and led quantita-
of the starting material (Entry 9). Finally, the Schiff base 2 tively to compound 8 as the hydrobromide salt.[31] Al-
was added to a solution of LDA in tetrahydrofuran, and though successful, this method is handicapped by the mod-
methyl bromoacetate was then added after 1 h at low tem- est yields of the alkylation step, combined with the arduous
perature (Entry 10) to furnish 42% of 3; similar results were purification of the protected precursor 3.
achieved on the 0.2- and 1-g scales. Treatment with two
Thus, a third strategy based on the Kabachnik–Fields
fractions of base successively did not improve the yields of reaction was envisaged. There are many examples of this
3. However, addition of 1,3-dimethyl-3,4,5,6-tetrahydropyr- reaction in the literature, but one particular case held our
imidin-2(1H)-one (DMPU) allowed to reach 70% of the de- attention in the work of Ranu et al.[32] They reported the
sired product (Entry 11). This product was purified by one-pot synthesis of a disubstituted α-aminophosphonate
chromatography with some difficulties and isolated with 10a catalysed by indium chloride (Scheme 3 with R = H).
24% yield.
Indium chloride was added to a solution of dimethyl 1,3-
When the quantity of LDA was increased, a secondary acetonedicarboxylate (Scheme 3 with R = CO2Me), ben-
product 3Ј, resulting from deprotonation and alkylation of zylamine and diethyl phosphite in tetrahydrofuran, and the
the β-carbon atom, was isolated with 5% yield. This result mixture was heated at reflux for several hours. Instead of
may be explained by the steric bulk of the phosphonate the condensation product 10b, the enamine 9 was isolated
group.
in 65% yield. Prolonged treatment of pure 9 with diethyl
Amine 4 was easily obtained in 80% yield by acidic hy- phosphite and indium chloride in tetrahydrofuran did not
drolysis of imine 3. Further treatment of 4 with 6  HCl furnish 10b. Kabachnik et al. showed that microwave acti-
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FULL PAPER
vation in the case of hindered ketones gave better results methylsilyl phosphite prepared in situ in dichloromethane
than conventional heating,[33] but in our case it was not in the presence of triethylammonium chloride (Scheme 4).
helpful.
After stirring the mixture at room temperature or heating
at reflux for several days, the starting material 9 was reco-
vered. In fact, a proton source is necessary to allow a tauto-
meric equilibrium between enamine and imine. Triethylam-
monium chloride (using the conditions of Afarinkia et al.)
did not seem to be a sufficiently strong proton source in
our case.
Moonen et al. recently reported a one-pot tandem 1,4/
1,2-addition of diethyl trimethylsilyl phosphite to α,β-unsat-
urated imines in the presence of triethylammonium chlo-
ride, ammonium sulfate or sulfuric acid.[40] Surprisingly, the
reaction was very efficient when using sulfuric acid as the
catalyst. This result prompted us to replace triethylammo-
nium chloride by sulfuric acid. The thus-modified Pudovik
reaction of 9 allowed the aminophosphonate 10b to be ob-
tained in 30% yield. Attempts to improve the yield by in-
creasing the quantity of acid and/or nucleophile were un-
successful. The amine 4 was obtained quantitatively by the
reaction of aminophosphonate 10b with hydrogen and pal-
ladium hydroxide. This novel route to 4 (and 7, see
Scheme 2) is a valuable alternative to the alkylation of α-
aminophosphonic derivatives. Overall yields are quite sim-
ilar, but the number of steps is reduced.
Scheme 3. Kabachnik–Fields reaction described by Ranu et al.
(10a) and applied to our substrate (10b).
Finally, a Pudovik-type reaction was investigated by
using 9 as the starting material. Indeed, enamines can be
used in such a reaction, similarly to imines. For instance,
the phosphonic analogue of aspartic acid, in which the α-
carboxy group is replaced by a phosphonic group, was ob-
tained by the reaction of diethyl phosphite with (acet-
amidomethylene)malonate followed by hydrolysis and de-
carboxylation.[34] Diethyl phosphite is generally considered
Synthesis of Homologues of α-Aminophosphonocitrate
Our strategy for the synthesis of products possessing
as a poor nucleophile.[35] However, its nucleophilicity can longer acidic chains was based on the alkylation of the im-
be improved in three ways: (1) The use of strong bases per- ine intermediates 1 and 2 by Michael addition. Thus, the
mits the isolation of the lithium, sodium or potassium salts, Schiff base 2 was treated with sodium ethoxide[41] and ethyl
which are then used in the Pudovik reaction.[36] (2) The use acrylate to furnish quantitatively the adduct 11. Direct hy-
of sodium ethoxide allows the tautomer equilibrium to be drolysis of imine 11 led to the formation of a mixture of
moved towards the more nucleophilic σ3λ3 form.[35] (3) The amine 12 and pyrrolidinone 13, which can be separated by
addition of an acid catalyst to activate diethyl phosphite. chromatography and isolated in 31 and 29% yields, respec-
Numerous Lewis acids have been used for the synthesis of tively. However, pure amine 12 cyclised spontaneously to
α-aminophosphonates, for example, BF3, AlCl3, Me2AlCl, furnish pyrrolidinone 13, even when the compound was
ZnCl4, TiCl4, SnCl4 and SmI2.[37] Finally, the last way to stored at –20 °C (Scheme 5).
increase the reactivity of diethyl phosphite has been de-
Treatment of pyrrolidinone 13 with 6  HCl at reflux was
scribed by Afarinkia et al.[38] who reported its transforma- expected to give the target product 15. Actually, a mixture
tion to diethyl trimethylsilyl phosphite. This last method of cyclic 14 and open-chain 15 was obtained, even after
1
has been successfully applied to our substrate. Benzylamine prolonged heating of 13, as confirmed by H NMR spec-
was treated with dimethyl 1,3-acetonedicarboxylate in the troscopy. Oleksyszyn et al. encountered the same problem
presence of molecular sieves in methanol to give 9, as de- during the synthesis of phosphonic analogues of pyroglut-
scribed by Prugh and Deana.[39] The enamine 9 [used as amic acid: in acidic media, the α-methylpyroglutamic acid
a (Z)/(E) mixture of stereoisomers in variable ratios after analogue was in equilibrium with the open-chain com-
chromatography] was added to a solution of diethyl tri- pound.[42] Selective deprotection of the phosphonate group
Scheme 4. Addition of diethyl trimethylsilyl phosphite to enamine 9.
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Inhibitors of Penicillin-Binding Proteins
Treatment of pyrrolidinone 18 with trimethylsilyl bromide
in dichloromethane gave the phosphonic acid 20 in 75%
1
yield (determined by H NMR spectroscopy).
Synthesis of α-Aminobis(phosphonic) Derivatives
Several methods have been described for the synthesis
of 1,1-bis(phosphonates).[43] A simple way to prepare such
compounds is a multicomponent reaction between carbox-
ylic acids with phosphorous acid and phosphorus trichlo-
ride, phosphorus pentachloride or phosphorus oxychlor-
ide.[44] Another recent route exploits Michael-type ad-
ditions to vinylidenebis(phosphonates).[45] Mizrahi et al.
have reported the preparation of aminobis(phosphonates)
derived from amino acids: the key step is the reaction of an
α-(aminoacyl)phosphonate with a dialkyl phosphite in the
presence of triethylamine.[46] The synthesis of iminobis-
(phosphonic) derivatives and their subsequent reduction
has been described by Palacios et al.[47] Lastly, phosphono-
azadienes have been used as precursors of substituted α-
aminobis(phosphonates).[48]
Scheme 5. Synthesis of compound 15.
Derivatives bearing two phosphonic groups were synthe-
sised by alkylation of the imine 21. The amine precursor,
which was synthesised beforehand,[49] was condensed with
3-chlorobenzaldehyde to furnish 21 in 70% yield after puri-
fication (Scheme 7). The Schiff base 21 was treated with
potassium carbonate, triethylammonium bromide and
methyl bromoacetate to give the crude imine 22 in 91%
yield.[50] The amine 23 was isolated after the smooth acidic
hydrolysis of imine 22 in 76% yield after purification by
chromatography. The target molecule 24 (hydrochloride
salt) was obtained quantitatively after treatment with 6 
HCl.
of 13 was carried out with trimethylsilyl bromide in dichlo-
romethane followed by treatment with methanol to give
compound 16 as a white hygroscopic solid in 90% yield.
The same method was applied to the synthesis of the
bis(homologue) (Scheme 6). The reaction of imine 1 with
sodium ethoxide and excess ethyl acrylate at 80 °C in etha-
nol for 24 h led to the product 17, isolated in 44% yield
after purification by chromatography. The monoalkylated
product was obtained exclusively at 20 °C with 2 equiv. of
acrylate. The starting material 1 was recovered when using
potassium carbonate as base. The acidic hydrolysis of imine
17 led to the pyrrolidinone 18 in 53% yield. After heating
18 in 6  HCl, the cyclic acid 19 was obtained quantitatively
without a trace of the open-chain product (confirmed by
1H, 13C and 31P NMR spectroscopy and mass spectrome-
try). Thus, the bis(homologue) of 7 could not be obtained.
Scheme 7. Synthesis of α-aminobis(phosphonic acid) 24.
To prepare the homologue, imine 21 was treated with
sodium ethoxide and ethyl acrylate in ethanol at 80 °C for
2 h to furnish the crude imine 25 in 98% yield (Scheme 8).
The pyrrolidinone 26, obtained by acidic hydrolysis at room
Scheme 6. Synthesis of compound 19.
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89

J. Marchand-Brynaert et al.
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temperature, was heated with 6  HCl to give quantitatively is shielded (signal shifted by about 10 ppm; compare 4, 12
the pyrrolidinone 27. The open-chain product (homologue and 23 with 3, 11 and 22) and the signal appears at δ = 53–
of 24) was not obtained.
54 ppm as a large doublet or triplet. In the cyclic structures
(13, 18 and 26), the corresponding quaternary carbon atom
is more deshielded (signal shifted by 58–59 ppm). For all
structures the 31P NMR spectra show the signal typical of
diethyl phosphonates at δ = 16–26 ppm in CDCl3. For the
corresponding phosphonic acids the 31P atom was shielded
(signal shifted by about 4 ppm), and the signal is found at
δ = 12–22 ppm in D2O.
Table 3. NMR spectroscopic data for the aminophosphonates and
pyrrolidinones.[a]
1
Compound
C-α: δC, mult., JC,P
δP
4
53.18, d, 158.3
57.90, d, 150.8
53.74, d, 158.6
55.81, t, 145.5
57.84, d, 164.7
59.27, d, 162.2
58.75, t, 152.5
26.43
25.34
28.30
21.61
24.60
Scheme 8. Synthesis of pyrrolidinone 27.
10
12
23
13
18
26
Purification/Characterisation
26.20
19.82; 19.84
A major difficulty of the chemistry of phosphonate and
phosphonic derivatives lies in their purification by standard
chromatography owing to their high polarity. Usually, pure
phosphonates can be obtained after arduous column
chromatography on silica gel, but with loss of yield. The
deprotected compounds, phosphonic/carboxylic derivatives,
were extracted with water and recovered by lyophilisation.
[a] δ given in ppm and J given in Hz.
Biochemical Evaluation
The aminophosphonic and aminobis(phosphonic) deriv-
The deprotections were thus preferably performed on pure atives were evaluated for their activity against a series of
phosphonate precursors.
clinically representative β-lactamases of class A (TEM-1,[51]
The NMR characterisation of phosphonates is also com- BS3,[15] NMCA[52]), class C (P99[53]), class D (Oxa-10[54]
)
plicated due to supplementary short- and long-range coup- and class B (BcII,[55] VIM-4[56]). The enzymes were incu-
lings with phosphorus atoms (JH,P and JC,P). The NMR bated with the tested compounds at 37 °C and pH 7 or 5
data for the series of Schiff bases are collected in Table 2. for 30 min. Then nitrocefine (a chromogenic substrate) was
We have summarised some typical H, 13C and 31P NMR added, and the hydrolysis rate of this substrate was moni-
1
chemical shifts of structural features common to the com- tored by spectrophotometry at 482 nm. In this way, the re-
pounds described above. The imine proton is deshielded sidual activity of the β-lactamases could be determined.
(signal shifted by 0.3–0.7 ppm) after dialkylation of the α- The results shown in Table 4 are expressed as a percentage
carbon atom (compare 1 and 21 with 3, 11, 17, 22 and 25), of the β-lactamase initial activity. The compounds were
and the signal appears in the region of δ = 8.2–8.7 ppm as tested at a concentration of 100 µ; low % values indicate
a doublet or triplet due to long-range H–P coupling. The very active compounds. The limit for considering a com-
signals of the corresponding carbon atoms are found at δ ≈ pound as a possible “hit” in this screening has been fixed
160–165 ppm. The α-carbon atoms (δ ≈ 65 ppm) are easily at 80%.
identified due to their large C–P coupling constant of 140–
155 Hz.
The biochemical results are disappointing because none
of the tested compounds inhibited BcII and VIM-4 (class B,
Table 3 shows the NMR spectroscopic data for the ami- zinc β-lactamase) nor P99 (class C) and Oxa-10 (class D).
nophosphonates and pyrrolidinones. Relative to their imine Like the amino analogues of citrate, the aminophosphonic
precursors, the α-carbon atom of the aminophosphonates derivatives are modestly active against class A β-lactamases.
Table 2. NMR spectroscopic data for the Schiff bases.[a]
4
3
1
Cmpound
HC=N: δH, mult., JH,P
HC=N and C-α: δC, mult., JC,P; δC, mult., JC,P
δP
1
8.23, d, 4.9
8.33, d, 4.8
8.93, d, 5.0
8.49, d, 4.7
8.49, d, 4.2
8.29, t, 4.3
8.74, t, 3.7
8.76, t, 3.3
165.24, d, 16.4; 57.55, d, 152.9
164.44, d, 15.5; 64.93, d, 157.7
160.22, d, 12.9; 64.06, d, 155.3
160.98, d, 12.3; 64.91, d, 150.1
161.04, d, 12.6; 64.82, d, 147.4
165.92, t, 15.2; 67.97, t, 149.0
164.35, t, 13.3; 68.98, t, 150.1
164.63, t, 12.0; 68.02, t, 136.8
22.48
22.92
22.81
25.62
24.31
2
3
11
17
21
22
25
15.9; 15.92
17.38
19.25
[a] δ given in ppm and J given in Hz.
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Inhibitors of Penicillin-Binding Proteins
Table 4. Inhibition of β-lactamases by aminophosphonic and aminobis(phosphonic) derivatives at pH 5 (pH 7).[a]
Compound[b]
TEM-1
BS3
NMCA
P99
Oxa-10
Bc-II
VIM-4
4
7
83 (96)
89 (100)
92 (100)
88 (80)
87 (100)
84 (100)
94 (100)
100 (95)
92 (100)
97 (100)
100 (90)
100 (95)
93 (89)
97 (89)
97 (89)
98 (100)
100 (100)
100 (100)
100 (100)
98 (99)
100 (100)
100 (99)
97 (100)
99 (100)
100 (100)
97 (100)
100 (100)
89 (100)
83 (100)
83 (100)
76 (100)
95 (100)
99 (100)
83 (91)
98 (100)
98 (100)
97 (100)
97 (93)
100 (92)
100 (92)
91 (88)
97 (100)
96 (85)
98 (93)
92 (87)
87 (99)
92 (85)
92 (88)
96 (100)
91 (97)
100 (100)
96 (84)
100 (96)
100 (X)
100 (X)
100 (X)
95 (100)
87 (X)
X (97)
X (97)
X (100)
X (100)
X (99)
X (100)
X (95)
X (100)
X (100)
X (96)
X (91)
X (96)
X (96)
X (100)
X (89)
8
10
12
13
14/15
16
18
19
20
23
24
26
27
93 (100)
97 (100)
100 (92)
100 (100)
95 (100)
100 (95)
100 (98)
100 (100)
100 (98)
91 (100)
100 (86)
92 (X)
64 (98)
100 (X)
85 (97)
100 (X)
88 (X)
95 (100)
99 (100)
99 (100)
100 (X)
96 (100)
100 (100)
97 (100)
92 (100)
100 (100)
90 (99)
100 (100)
97 (100)
88 (95)
91 (98)
90 (100)
[a] Results are expressed as a percentage of the initial activity. [b] Compounds were tested in 50 m phosphate buffer at pH 7 and in
50 m acetate buffer at pH 5 at a concentration of 100 µ; X = not tested.
Compound 8 and the mixture 14 and 15 are inhibitors of The enzyme activity was similarly diminished in the pres-
NMCA (class A). The inhibition constants (Ki) measured ence of pyrrolidinone 19. These results seem to indicate that
at pH 5 are 800 and 360 µ, respectively.
the cyclic form in the mixture 14/15 is active against R39.
Some of these molecules were tested against R39,[57]
a
low molecular weight ,-transpeptidase/carboxypeptidase.
The enzyme was incubated with the test compounds for
16 h, and then fluorescent ampicillin was added. After
45 min, the protein was denatured and the fluorescence in-
tensity was measured to determine the residual activity of
the peptidase. The results shown in Table 5 are expressed as
percentages of the R39 initial activity. The compounds were
tested at a concentration of 500 µ; low % values indicate
a very active compound. The limit for considering a com-
pound as a possible “hit” in this screening was fixed at
70%. The compounds selected in the initial run (first col-
umn) were tested again under the same conditions (second
column) and in the presence of bovin serum albumin (BSA)
to control the selectivity of the inhibition.
Conclusion
Novel aminophosphonic acids and aminobis(phosphonic
acids), analogues or homologues of aminocitrate, have been
synthesised in acceptable yields. The alkylation of α-amino-
phosphonates and -bis(phosphonates) remains a valuable
synthetic strategy. However, our modified Pudovik reaction
is a straightforward original route towards the phosphonic
bioisoster of aminocitrate 7. This compound and its homo-
logues 14/15 and 19 showed good activity against R39 ,-
peptidase. The mixture of 14/15 and the related monophos-
phonic derivative 8 were modestly active against NMCA β-
lactamase. The mode of action of these novel “hits” is un-
der investigation by co-crystallisation experiments with the
target enzymes.
The library of (bis)phosphonic derivatives that we have
prepared in our search for bacterial enzyme inhibitors could
be of great interest in other applications, for example, as
agonists/antagonists of glutamate receptors,[19] antiprolifer-
ation agents of parasites[45] and inhibitors of tumour cell
proliferation.[58]
Table 5. Inhibition of R39 by aminophosphonic and aminobis-
(phosphonic) derivatives.[a,b]
Compounds
R39
R39
R39 + 500 µ BSA
4
7
97Ϯ2
36Ϯ3
71Ϯ5
98Ϯ7
91Ϯ5
56Ϯ5
100Ϯ15
69Ϯ6
95Ϯ9
94Ϯ10
n.d.
47Ϯ5
n.d.
n.d.
n.d.
66Ϯ4
n.d.
67Ϯ8
n.d.
n.d.
52Ϯ4
n.d.
n.d.
n.d.
73Ϯ5
n.d.
72Ϯ9
n.d.
8
12
13
14/15
18
19
26
27
Experimental Section
General: Reagents and solvents were purchased from Acros, Ald-
rich, Fluka, Sigma and Balsen. Column chromatography was car-
ried out with silica gel 60 (70–230 or 230–400 mesh ASTM) sup-
plied by Merck. The IR spectra were recorded with a Shimadzu
FTIR-8400S instrument; only the most significant adsorption
bands are reported; samples analyzed as thin films deposited on
Zn/Se crystal. The 1H and 13C NMR spectra were recorded with
Bruker spectrometer models 300 UltraShield and AM-500 (at 300
or 500 MHz for 1H and at 75 or 125 MHz for 13C). The 31P NMR
was recorded with a Bruker spectrometer (121 MHz). Chemical
shifts (δ) were measured relative to chloroform or D2O and are
n.d.
n.d.
[a] Results are expressed as a percentage of the initial activity. [b]
n.d. = not determined.
Compounds 7, 14/15 and 19 were active against R39.
They are selective, interacting with the active site that recog-
nises penicillins. The phosphonic bioisoster of aminocitrate
7 can be considered as a good inhibitor of R39 because the
activity of the enzyme decreased by more than half. The
sample 14/15 is a mixture of pyrrolidinone and the open-
chain compound and gives a residual activity of about 60%. expressed in ppm. Coupling constants (J) are expressed in Hz. The
Eur. J. Org. Chem. 2009, 85–97
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
91

J. Marchand-Brynaert et al.
FULL PAPER
mass spectra were obtained with a Finnigan MAT TSQ-70 instru-
Dimethyl 3-[(3-Chlorobenzylidene)amino]-3-(diethoxyphosphoryl)-
ment. The high-resolution mass spectra (HRMS) were performed pentane-1,5-dioate (3): The crude imine 2 (383 mg, 1.06 mmol) dis-
by Prof. Flammang at the University of Mons, Belgium.
solved in dry THF (3 mL) and cooled to –78 °C was added under
argon to a solution of LDA (commercial solution in THF, 750 µL,
1.48 mmol, 1.4 equiv.) and DMPU (205 µL, 1.69 mmol, 1.6 equiv.)
in THF (2 mL). After 1 h at –78 °C, methyl bromoacetate (110 µL,
1.11 mmol, 1.05 equiv.) was added, and the mixture was allowed to
slowly warm to 20 °C overnight whilst being stirred. After addition
of water, the solution was concentrated under vacuum. The oily
residue was dissolved in dichloromethane, and the organic layer
was washed successively with NH4Cl and brine. After drying with
MgSO4 and concentration under vacuum, the residue was purified
by chromatography on silica gel (elution: EtOAc/hexane, 8:2),
which was neutralised with Et3N before use. The imine 3 was iso-
lated as a yellow oil in 24% yield (79 mg). RF(EtOAc/hexane, 8:2)
Diethyl {[(3-Chlorobenzylidene)amino]methyl}phosphonate (1): Di-
ethyl (aminomethyl)phosphonate (1 g, 5.98 mmol), MgSO4 (1 g)
and 3-chlorobenzaldehyde (610 µL, 5.39 mmol, 0.85 equiv.) were
dissolved in dichloromethane (10 mL) and stirred for 16 h. After
filtration, the organic solution was concentrated under vacuum.
The residue was purified by chromatography on silica gel (elution:
hexane/EtOAc, 8:2) to give 1 as a yellow oil (1.32 g, 76%). RF(hex-
ane/EtOAc, 8:2) = 0.30. IR: ν = 2981 (CHAr), 1639 (C=N), 1242
˜
[P(O)(OEt)2], 1026 [P(O)(OEt)2] cm–1. 1H NMR (CDCl3,
500 MHz): δ = 1.31 (t, J = 7.0 Hz, 6 H, CH3), 4.08 (d, JH,P
=
17.8 Hz, 2 H, CH2), 4.14 (q, J = 7.0 Hz, 4 H, CH2), 7.31 (t, J =
7.7 Hz, 1 H, CHAr), 7.37 (d, J = 8.0 Hz, 1 H, CHAr), 7.55 (d, J
= 7.6 Hz, 1 H, CHAr), 7.73 (s, 1 H, CHAr), 8.23 (d, JH,P = 4.9 Hz,
1 H, N=CH) ppm. 13C NMR (CDCl3, 125 MHz): δ = 16.56 (d,
JC,P = 5.8 Hz, CH3), 57.55 (d, JC,P = 152.9 Hz, CH2), 62.71 (d, JC,P
= 6.6 Hz, CH2), 126.80 (CHAr), 127.98 (CHAr), 129.98 (CHAr),
131.17 (CHAr), 134.88 (CAr), 137.49 (d, JC,P = 3.5 Hz, CAr),
165.24 (d, JC,P = 16.4 Hz, N=CH) ppm. 31P NMR (CDCl3,
121 MHz): δ = 22.48 ppm. MS (ESI): m/z = 290.11 [M + H]+,
312.09 [M + Na]+, 600.75 [2 M + Na]+. HRMS (ESI): calcd. for
C12H17ClNO3P + Na 312.0532; found 312.0526.
= 0.55. IR: ν = 2985 (CHAr), 1732 (CO Me), 1643 (C=N), 1242
˜
2
[P(O)(OEt)2], 1095 [P(O)(OEt)2] cm–1. 1H NMR (CDCl3,
500 MHz): δ = 1.30 (t, J = 7.0 Hz, 6 H, CH3), 3.36 (m, 4 H, CH2),
3.66 (s, 6 H, CH3), 4.14 (m, 4 H, CH2), 7.34 (dd, J = 8, J = 7.5 Hz,
1 H, CHAr), 7.38 (d, J = 8.1 Hz 1 H, CHAr), 7.60 (d, J = 7.6 Hz,
1 H, CHAr), 7.78 (s, 1 H, CHAr), 8.39 (d, JH,P = 5.0 Hz, 1 H,
N=CH) ppm. 13C NMR (CDCl3, 125 MHz): δ = 16.59 (JC,P
=
5.6 Hz, CH3), 36.41 (CH2), 51.84 (CH3), 64.06 (d, JC,P = 155.3 Hz,
1 C), 63.72 (JC,P = 7.1 Hz, CH2), 127.22 (CHAr), 128.13 (CHAr),
130.03 (CHAr), 131.28 (CHAr), 134.98 (CAr), 137.93 (CAr),
160.22 (JC,P = 12.9 Hz, N=CH), 170.80 (JC,P = 12.8 Hz, CO2Me)
ppm. 31P NMR (CDCl3, 121 MHz): δ = 22.81 ppm. MS (ESI): m/z
= 434.16 [M + H]+, 456.20 [M + Na]+, 888.68 [2 M + Na]+, 296.10
[M – P(O)(OEt)2]+. HRMS (ESI): calcd. for C18H25ClNO7P + Na
456.0955; found 456.0947.
Methyl 3-Amino-3-(diethoxyphosphoryl)propanoate (6): Thionyl
chloride (850 µL, 11.61 mmol, 3 equiv.) was added dropwise to a
stirred and cooled (0 °C) solution of azetidinone 5 (800 mg,
3.87 mmol) in methanol (10 mL). The mixture was stirred for 24 h
and concentrated under vacuum. The residue was taken up in
dichloromethane, and NaHCO3 (5.6 g) was added. The mixture
was stirred for 30 min, filtered and concentrated under vacuum to
obtain 5 as an orange oil (824 mg, 90%). 1H NMR (CDCl3,
300 MHz): δ = 1.36 (t, J = 6.9 Hz, 6 H, CH3), 2.59 (m, 1 H, CH2),
2.86 (m, 1 H, CH2), 3.60 (m, 1 H, CH), 3.74 (s, 3 H, CH3), 4.19
(q, J = 6.9 Hz, 4 H, CH2) ppm. 13C NMR (CDCl3, 75 MHz): δ =
16.6 (JC,P = 5.5 Hz, CH3), 36.87 (JC,P = 2.3 Hz, CH2), 45.82 (d,
JC,P = 154.8 Hz, CH), 52.1 (CH3), 62.60 (JC,P = 6.4 Hz, CH2),
171.82 (JC,P = 20.5 Hz, CO2Me) ppm. MS (APCI): m/z = 240.31
[M + H]+, 102.22 [M – P(O)(OEt)2]+.
Characterisation of the Side-Product Dimethyl 2-{[(3-Chlorobenzyl-
idene)amino](diethoxyphosphoryl)methyl}succinate (3Ј): This prod-
uct was obtained as above from imine 2 (415 mg, 1.14 mmol), LDA
(1.1 mL, 2.28 mmol, 2 equiv.), DMPU (220 µL, 1.82 mmol,
1.6 equiv.) and methyl bromoacetate (115 µL, 1.19 mmol,
1.05 equiv.) in THF (6 mL). After workup, the brown residue was
purified by chromatography on silica gel (elution: EtOAc/hexane,
8:2) and isolated as a yellow oil in 5% yield (10 mg). RF(EtOAc/
1
hexane, 8:2) = 0.50. H NMR (CDCl3, 500 MHz): δ = 1.28 (t, J =
7 Hz, 3 H, CH3), 1.34 (t, J = 7 Hz, 3 H, CH3), 3.01 (dd, J = 17.1,
J = 9.5 Hz, 1 H, CH2A), 3.11 (dd, J = 17, J = 4 Hz, 1 H, CH2A),
3.34–3.38 (m, 1 H, CH), 3.67 (s, 3 H, CH3), 3.68 (s, 3 H, CH3),
4.05–4.21 (m, 4 H, CH2), 4.27 (dd, JH,P = 16, J = 3.9 Hz, 1 H,
CH), 7.35 (dd, J = 8, J = 7 Hz, 1 H, CHAr), 7.42 (d, J = 8 Hz, 1
H, CHAr), 7.58 (d, J = 7 Hz, 1 H, CHAr), 7.76 (s, 1 H, CHAr),
8.28 (d, JH,P = 5.0 Hz, 1 H, N=CH) ppm. 13C NMR (CDCl3,
Methyl 3-[(3-Chlorobenzylidene)amino]-3-(diethoxyphosphoryl)pro-
panoate (2): Methyl 3-amino-3-(diethoxyphosphoryl)propanoate 6
(814 mg, 3.41 mmol), MgSO4 (1 g) and 3-chlorobenzaldehyde
(328 µL, 2.89 mmol, 0.85 equiv.) were dissolved in dichloromethane
(10 mL) and stirred for 16 h. After filtration, the solution was con-
centrated under vacuum. The residue was purified by chromatog-
raphy on silica gel with EtOAc as eluent to obtain 2 as a white
125 MHz): δ = 16.67 (t, JC,P = 2.61 Hz, CH3), 32.45 (d, JC,P
=
4.19 Hz, CH2), 42.74 (d, JC,P = 5.74 Hz, CH), 51.96 (CH3), 52.48
(CH3), 63.27 (d, JC,P = 6.98 Hz, CH2), 63.37 (d, JC,P = 6.92 Hz,
CH2), 68.36 (d, JC,P = 151.52 Hz, CH), 127.24 (CHAr), 128.14
(CHAr), 130.05 (CHAr), 132.17 (CHAr), 135.05 (CAr), 137.36 (d,
JC,P = 3.34 Hz, CAr), 160.77 (JC,P = 13.36 Hz, N=CH), 172.81
(CO2Me), 172.91 (d, JC,P = 17.21 Hz, CO2Me) ppm. 31P NMR
(CDCl3, 121 MHz): δ = 21.56 ppm. MS (ESI): m/z = 434.10 [M +
H]+, 456.15 [M + Na]+, 296.08 [M – P(O)(OEt)2]+.
solid (1.06 g, 85%). R (EtOAc) = 0.54. IR: ν = 2986 (CHAr), 1740
˜
F
(CO2Me), 1639 (C=N), 1250 [P(O)(OEt)2], 1049 {[P(O)(OEt)2]}
1
cm–1. H NMR (CDCl3, 500 MHz): δ = 1.30 (t, J = 7.0 Hz, 3 H,
CH3), 1.2 (t, J = 7.0 Hz, 3 H, CH3), 3.00 (m, 2 H, CH2), 3.62 (s, 3
H, CH3), 4.13 (m, 5 H, CH2, CH), 7.32 (dd, J = 8, J = 7.6 Hz, 1
H, CHAr), 7.40 (d, J = 8.05 Hz, 1 H, CHAr), 7.57 (d, J = 7.6 Hz,
1 H, CHAr), 7.76 (s, 1 H, CHAr), 8.33 (d, JH,P = 4.8 Hz, 1 H,
N=CH) ppm. 13C NMR (CDCl3, 125 MHz): δ = 16.64 (JC,P
=
5.7 Hz, CH3), 35.41 (JC,P = 2.4 Hz, CH2), 53.02 (CH3), 63.17 (JC,P Dimethyl 3-Amino-3-(diethoxyphosphoryl)pentanedioate (4): A 1 
= 6.9 Hz, CH2), 64.93 (d, JC,P = 157.7 Hz, CH), 127.21 (CHAr),
128.15 (CHAr), 129.99 (CHAr), 131.31 (CHAr), 134.94 (CAr),
HCl solution (1 mL, 1 mmol, 2.5 equiv.) was added to imine 3
(158 mg, 0.37 mmol) dissolved in acetonitrile (0.5 mL). After 10–
15 min at 20 °C, the solution was concentrated under vacuum, and
the aqueous phase was extracted three times with diethyl ether. The
aqueous phase was then neutralised with NaHCO3 and basified
with 1  NaOH to pH 10. This was extracted five times with
CH2Cl2. The organic layers were collected and washed with brine.
137.53 (CAr), 164.44 (JC,P = 15.5 Hz, N=CH), 171.26 (JC,P
=
=
21.1 Hz, CO2Me) ppm. 31P NMR (CDCl3, 121 MHz):
δ
22.92 ppm. MS (ESI): m/z = 362.19 [M + H]+, 384.14 [M + Na]+,
744.66 [2 M + Na]+, 224.12 [M – P(O)(OEt)2]+. HRMS (ESI):
calcd. for C15H21ClNO5P + Na 384.0744; found 384.0731.
92
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 85–97

Inhibitors of Penicillin-Binding Proteins
After drying (MgSO4) and concentration under vacuum, the resi-
due was purified by column chromatography on silica gel (elution:
EtOAc/iPrOH, 9:1). Amine 4 was recovered as a colourless oil
Z), 169.14 (CO2Me, E), 169.31 (CO2Me, Z), 170.94 (CO2Me, E +
Z) ppm. MS (ESI): m/z = 264.24 [M + H]+, 286.12 [M + Na]+.
Dimethyl
3-(Benzylamino)-3-(diethoxyphosphoryl)pentanedioate
(82 mg, 80%). R (EtOAc/iPrOH, 9:1) = 0.44. IR: ν = 3442 (NH ),
˜
F
2
(10b): Diethyl trimethylsilyl phosphite (90 mg, 0.205 mmol,
1.05 equiv.) and sulfuric acid (11 µL, 0.21 mmol, 0.5 equiv.) were
added to a stirred solution of enamine 9 (108 mg, 0.41 mmol) in
dichloromethane (2 mL). The mixture was stirred for 3 h and then
heated for 24 h. The organic phase was washed with NaHCO3 and
then brine, and dried with MgSO4. After concentration under vac-
uum, the product was purified by chromatography on silica gel
(elution: hexane/EtOAc, 7:3) to give 10 as a colourless oil (49.6 mg,
1735 (CO2Me), 1236 [P(O)(OEt)2], 1020 [P(O)(OEt)2] cm–1. 1H
NMR (CDCl3, 500 MHz): δ = 1.31 (t, J = 7.0 Hz, 6 H, CH3), 2.19
(br. s, 2 H, NH2), 2.81 (d, J = 12.0 Hz, 4 H, CH2), 3.67 (s, 6 H,
CH3), 4.15 (q, J = 7.0 Hz, 4 H, CH2) ppm. 13C NMR (CDCl3,
125 MHz): δ = 16.62 (JC,P = 5.5 Hz, CH3), 39.85 (JC,P = 1.7 Hz,
CH2), 51.92 (CH3), 53.18 (d, JC,P = 158.3 Hz, 1 C), 63.36 (JC,P
=
7.5 Hz, CH2), 171.08 (JC,P = 12.7 Hz, CO2Me) ppm. 31P NMR
(CDCl3, 121 MHz): δ = 26.43 ppm. MS (ESI): m/z = 312.02 [M +
H]+, 334.14 [M + Na]+, 174.07 [M – P(O)(OEt)2]+. HRMS (ESI):
calcd. for C11H22NO7P + Na 334.1032; found 334.1038.
30%). R (hexane/EtOAc, 7:3) = 0.47. IR: ν = 3471 (NH), 2985
˜
F
(CHAr), 1736 (CO2Me), 1207 [P(O)(OEt)2], 1018 [P(O)(OEt)2]
1
cm–1. H NMR (CDCl3, 500 MHz): δ = 1.34 (t, J = 7.0 Hz, 6 H,
CH3), 2.97 (d, J = 15.0 Hz, 2 H, CH2), 3.00 (d, J = 15.0 Hz, 2 H,
CH2), 3.68 (s, 6 H, CH3), 4.04 (s, 2 H, CH2), 4.19 (q, J = 7.1 Hz,
4 H, CH2), 7.28 (m, 5 H, CHAr) ppm. 13C NMR (CDCl3,
125 MHz): δ = 16.71 (d, JC,P = 5.7 Hz, CH3), 37.63 (d, JC,P = 6 Hz,
3-Amino-3-phosphonopentane-1,5-dioic Acid Hydrochloride (7):
Amine 4 (47 mg) was treated with 6  HCl (10 mL) at reflux over-
night. After extraction with CH2Cl2, the aqueous phase was con-
centrated under vacuum, and the residue was dried under high vac-
CH2), 47.89 (d, JC,P = 1.7 Hz, CH2), 51.90 (CH3), 57.90 (d, JC,P
=
uum to furnish 7 as a white solid (40 mg, quantitative yield). IR: ν
˜
= 3221 (CO2H, C–OH), 2781 (PO3H, P–OH), 2318 (NH3+), 1651
150.8 Hz, 1 C), 62.89 (JC,P = 8.1 Hz, CH2), 127.08 (CHAr), 128.33
(CHAr), 128.53 (CHAr), 140.68 (CHAr), 171.08 (JC,P = 12.6 Hz,
CO2Me) ppm. 31P NMR (CDCl3, 121 MHz): δ = 25.34 ppm. MS
(ESI): m/z = 424.05 [M + Na]+, 264.12 [M – P(O)(OEt)2]+. HRMS
(ESI): calcd. for C18H28NO7P + Na 424.1501; found 424.1516.
(NH3+), 1141 (PO3H) cm–1. H NMR (D2O, 500 MHz): δ = 2.94
1
(br. s, 2 H, CH2), 2.97 (br. s, 2 H, CH2) ppm. 13C NMR (D2O,
125 MHz): δ = 36.91 (CH2), 53.73 (d, JC,P = 141.7 Hz, 1 C), 173.67
(JC,P = 10.1 Hz, CO2H) ppm. 31P NMR (D2O, 121 MHz): δ =
12.45 ppm. MS (ESI): m/z = 226.08 [M – H]–, 208.02 [M – H2O]–,
147.10 [M – PO3H2]–. HRMS (ESI): calcd. for C5H10NO7P – H
226.0117; found 226.0118.
Treatment of 10b (25 mg, 0.062 mmol) by catalytic hydrogenation
[Pd(OH)2/C, 1 atm, 25 °C, 2 h] in EtOAc solution (1 mL) furnished
4 (19 mg, 100% yield), identical to the sample previously prepared.
[3-Amino-1,5-bis(methoxycarbonyl)pent-3-yl]phosphonic Acid Hy-
drobromide (8): BrSiMe3 (103 µL, 0.78 mmol, 4 equiv.) was added
dropwise to amine 4 (61 mg, 0.19 mmol) dissolved in dichlorometh-
ane (3 mL). The mixture was stirred at room temperature for 24 h.
After concentration under vacuum, the residue was taken up in
methanol (10 mL). The solution was heated at 50 °C for 2 h and
concentrated under high vacuum to obtain 8 as a yellow solid
Diethyl
3-[(3-Chlorobenzylidene)amino]-3-(diethoxyphosphoryl)-
hexanedioate (11): A solution of sodium ethoxide in ethanol (c =
2.68 mol/L, 104 µL, 0.28 mmol, 0.2 equiv.) was added to a stirred
solution of imine 2 (504 mg, 1.4 mmol) in ethanol (2 mL), and the
mixture was stirred for 10 min. Then ethyl acrylate (300 µL,
2.78 mmol, 2 equiv.) was added, and the mixture was heated at re-
flux for 2 h. After concentration under vacuum, the residue was
taken up in dichloromethane, washed with a saturated solution of
NH4Cl and dried with MgSO4. After concentration under vacuum,
imine 11 was obtained as a purple oil (613 mg, 92%). RF(EtOAc)
(64 mg, quantitative yield). IR: ν = 2792 (PO H, P–OH), 2360
˜
3
(NH3+), 1713 (CO2Me), 1608 (NH3+), 1180 (PO3H) cm–1. 1H
NMR (D2O, 500 MHz): δ = 2.96 (d, J = 8.0 Hz, 4 H, CH2), 3.67
(s, 6 H, CH3) ppm. 13C NMR (D2O, 125 MHz): δ = 37.07 (CH2),
53.11 (CH3), 53.89 (d, JC,P = 141.4 Hz, C), 172.33 (JC,P = 15.1 Hz,
CO2Me) ppm. 31P NMR (D2O, 121 MHz): δ = 12.38 ppm. MS
(ESI): m/z = 254.08 [M – 1]–. HRMS (ESI): calcd. for C7H14NO7P
+ H 256.0586; found 256.0589.
= 0.68. IR: ν = 2981 (CHAr), 2931–2908 (CH ), 1732 (CO Me),
˜
2
2
1639 (C=N), 1242 [P(O)(OEt)2], 1022 [P(O)(OEt)2] cm–1. 1H NMR
(CDCl3, 500 MHz): δ = 1.20 (t, J = 7 Hz, 3 H, CH3), 1.21 (t, J =
7.1 Hz, 3 H, CH3), 1.32 (t, J = 7.0 Hz, 6 H, CH3), 2.52 (m, 3 H,
CH2), 2.74 (d, J = 11 Hz, 1 H, CH2), 2.93 (dd, J = 14.6, J = 7.7 Hz,
1 H, CH2), 3.12 (dd, J = 14.6, J = 13.6 Hz, 1 H, CH2), 4.14 (m, 8
H, CH2), 7.35 (dd, J = 8, J = 7.6 Hz, 1 H, CHAr), 7.41 (d, J =
8.0 Hz, 1 H, CHAr), 7.61 (d, J = 7.5 Hz, 1 H, CHAr), 7.79 (s, 1
H, CHAr), 8.49 (d, JH,P = 4.6 Hz, 1 H, N=CH) ppm. 13C NMR
Dimethyl 3-(Benzylamino)pent-2-enedioate (9): Dimethyl acetone-
dicarboxylate (1 mL, 6.77 mmol) and benzylamine (740 µL,
6.77 mmol, 1 equiv.) were allowed to react in the presence of molec-
ular sieves (3 Å) in methanol (15 mL). The mixture was stirred at
room temperature overnight and filtered through Celite. The prod-
uct was purified by chromatography on silica gel (elution: hexane/
EtOAc, 7:3) to give 9 as a white solid (1.78 g, 67%). RF(hexane/
(CDCl3, 125 MHz): δ = 14.33 (CH3), 14.36 (CH3), 16.61 (d, JC,P
=
5.0 Hz, CH3), 29.11 (CH2), 29.59 (d, JC,P = 4.5 Hz, CH2), 38.03
(CH2), 60.57 (CH2), 60.80 (CH2), 63.44 (d, JC,P = 7.3 Hz, CH2),
63.46 (d, JC,P = 7.1 Hz, CH2), 64.91 (d, JC,P = 150.1 Hz, 1 C),
127.19 (CHAr), 128.11 (CHAr), 130.00 (CHAr), 131.28 (CHAr),
EtOAc, 7:3) = 0.47. IR: ν = 3375 (NH), 2950 (CHAr), 1740
˜
(CO2Me), 1654 (C=C), 1601 (C=C) cm–1. 1H NMR (CDCl3,
500 MHz): δ = 3.22 (s, 2 H, CH2, Z), 3.62 (s, 3 H, CH3, E), 3.65
(s, 3 H, CH3, Z), 3.68 (s, 3 H, CH3, Z), 3.74 (s, 3 H, CH3, E), 3.96
(s, 2 H, CH2, E), 4.24 (d, J = 5.4 Hz, 2 H, CH2-Ph, E), 4.45 (d, J
= 6.4 Hz, 2 H, CH2-Ph, Z), 4.62 (s, 1 H, CH, Z), 4.79 (s, 1 H, CH,
E), 5.17 (br. s, 1 H, NH, E), 7.32 (m, 5 H, CHAr), 8.92 (br. s, 1 H,
NH, Z) ppm. 13C NMR (CDCl3, 125 MHz): δ = 36.90 (CH2, Z),
38.82 (CH2, E), 47.24 (CH2-Ph, Z), 47.98 (CH2-Ph, E), 50.46 (CH3,
Z), 50.57 (CH3, E), 52.47 (CH3, E), 52.69 (CH3, Z), 85.35 (CH,
134.94 (CAr), 138.04 (d, JC,P = 3.4 Hz, CAr), 160.98 (JC,P
=
12.3 Hz, N=CH), 169.69 (JC,P = 16.1 Hz, CO2Et), 173.41 (CO2Et)
ppm. 31P NMR (CDCl3, 121 MHz): δ = 24.31 ppm. MS (ESI): m/z
= 476.16 [M + H]+, 498.19 [M + Na]+, 972.81 [2 M + Na]+, 338.16
[M – P(O)(OEt)2]+. HRMS (ESI): calcd. for C21H31ClNO7P + Na
498.1424; found 498.1439.
Diethyl 3-Amino-3-(diethoxyphosphoryl)hexanedioate (12) and 5-
(Diethoxyphosphoryl)-5-[(ethoxycarbonyl)methyl]pyrrolidin-2-one (13):
E), 85.37 (CH, Z), 127.01 (CHAr, E), 127.03 (CHAr, E), 127.73 Imine 11 (676 mg, 1.42 mmol) was dissolved in acetonitrile
(CHAr, Z), 127.84 (CHAr, Z), 129.07 (CHAr, E), 129.08 (CHAr,
Z), 136.94 (CAr, E), 138.45 (CAr, Z), 154.32 (CAr, E), 156.94 (CAr,
(1.5 mL), and 1  HCl (2.2 mL, 2.2 mmol, 1.5 equiv.) was added
(1 mL, 1 mmol, 2.5 equiv.). After 30 min at room temperature, the
Eur. J. Org. Chem. 2009, 85–97
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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93

J. Marchand-Brynaert et al.
FULL PAPER
solution was concentrated under vacuum, and the aqueous phase
was extracted three times with diethyl ether. The aqueous phase
was then neutralised with NaHCO3 and basified with 1  NaOH
to pH 10. This was extracted five times with CH2Cl2. The organic
layers were collected and washed with brine. After drying (MgSO4)
and concentration under vacuum, the oily residue was purified by
column chromatography on silica gel (elution: EtOAc/iPrOH,
14:1). Two products were recovered as a colourless oil: amine 12
(154 mg, 31%) and pyrrolidinone 13 (125 mg, 29%). 12: RF(EtOAc/
After concentration under vacuum, the residue was taken up in
water and freeze-dried to obtain 16 as a white hygroscopic solid
(34 mg, 90%). IR: ν = 3282 (NH, PO H, P–OH), 1712 (CO Et),
˜
3
2
1651 (NHCO), 1207 (PO3H) cm–1. 1H NMR (D2O, 500 MHz): δ =
1.25 (t, J = 7.1 Hz, 3 H, CH3), 2.42 (m, 4 H, CH2), 2.78 (ddd, J =
14.9, J = 6.9, J = 5.1 Hz, 1 H, CH2), 2.91 (ddd, J = 15.4, J = 8, J
= 3.1 Hz, 1 H, CH2), 4.19 (q, J = 7.1 Hz, 2 H, CH2) ppm. 13C
NMR (D2O, 125 MHz): δ = 14.80 (CH3), 27.83 (CH2), 31.53
(CH2), 40.97 (d, JC,P = 8.7 Hz, CH2), 60.27 (d, JC,P = 161 Hz, C),
1
iPrOH, 20:1) = 0.47. H NMR (CDCl3, 500 MHz): δ = 1.27 (t, J
= 7.1 Hz, 6 H, CH3), 1.35 (t, J = 7.1 Hz, 6 H, CH3), 1.85 (br. s, 2
63.58 (CH2), 173.50 (d, JC,P = 15.6 Hz, CO2Et), 182.65 (d, JC,P
=
5 Hz, NHCO) ppm. 31P NMR (CDCl3, 121 MHz): δ = 22.3 ppm.
H, NH2), 2.09 (td, J = 16.3, J = 8.2 Hz, 2 H, CH2), 2.61 (m, 4 H, MS (ESI): m/z = 252.06 [M + H]+, 170.13 [M – PO3H2]. HRMS
CH2), 4.16 (m, 8 H, CH2) ppm. 13C NMR (CDCl3, 75 MHz): δ = (ESI): calcd. for C8H14NO6P + H 252.0637; found 252.0632.
14.29 (CH3), 14.36 (CH3), 16.61 (JC,P = 1.0 Hz, CH3), 29.06 (CH2),
Diethyl 4-[(3-Chlorobenzylidene)amino]-4-(diethoxyphosphoryl)hep-
31.53 (CH2), 39.7 (CH2), 53.74 (d, JC,P = 158.6 Hz, C), 60.66
tanedioate (17): A solution of sodium ethoxide in ethanol (c =
(CH2), 60.91 (CH2), 62.85 (d, JC,P = 9.1 Hz, CH2), 63.24 (d, JC,P
2.68 mol/L, 94 µL, 0.25 mmol, 0.25 equiv.) was added to a stirred
= 7.8 Hz, CH2), 170.64 (JC,P = 13 Hz, CO2Et), 173.54 (CO2Et)
solution of imine 1 (290 mg, 0.97 mmol) in ethanol (3 mL), and
ppm. 31P NMR (CDCl3, 121 MHz): δ = 28.30 ppm. MS (ESI): m/z
the mixture was stirred for 15 min. Then ethyl acrylate (422 µL,
3.9 mmol, 4 equiv.) was added, and the mixture was heated at re-
flux for 24 h. After concentration under vacuum, the residue was
taken up in ethyl acetate, washed with a saturated solution of
NH4Cl and dried with MgSO4. After concentration under vacuum,
the crude brown oil was purified by chromatography on silica gel
(elution: CH2Cl2/EtOAc, 7:3). Imine 17 was recovered as a yellow
= 354.06 [M + H]+, 376.14 [M + Na]+, 216.18 [M – P(O)(OEt)2]+.
HRMS (ESI): calcd. for C14H28NO7P + Na 376.1501; found
376.1508. 13: R (EtOAc/iPrOH, 20:1) = 0.15. IR: ν = 3213 (NH),
˜
F
2981 (CH2), 1732 (CO2Et), 1701 (NHCO), 1238 [P(O)(OEt)2], 1022
1
[P(O)(OEt)2] cm–1. H NMR (CDCl3, 300 MHz): δ = 1.28 (t, J =
7.1 Hz, 3 H, CH3), 1.35 (td, J = 7.1, JH,P = 2.7 Hz, 6 H, CH3),
2.22 (m, 1 H, CH2), 2.36 (m, 1 H, CH2), 2.57 (m, 3 H, CH2), 2.90
(dd, J = 15.1, 7.3 Hz, 1 H, CH2), 4.18 (m, 6 H, CH2), 6.21 (br. s,
NH) ppm. 13C NMR (CDCl3, 75 MHz): δ = 14.24 (CH3), 16.66
oil (206 mg, 44% yield). R (CH Cl /EtOAc, 7:3) = 0.49. IR: ν =
˜
F
2
2
2981 (CHAr), 2931–2908 (CH2), 1732 (CO2Me), 1639 (C=N), 1242
[P(O)(OEt)2], 1022 [P(O)(OEt)2] cm–1. 1H NMR (CDCl3,
500 MHz): δ = 1.26 (t, J = 7.0 Hz, 6 H, CH3), 1.32 (t, J = 7.1 Hz,
6 H, CH3), 2.29 (m, 4 H, CH2), 2.45 (m, 2 H, CH2), 2.58 (m, 2 H,
CH2), 4.2 (m, 8 H, CH2), 7.36 (dd, J = 8.1, J = 7.5 Hz, 1 H, CHAr),
7.42 (d, J = 8.1 Hz, 1 H, CHAr), 7.61 (d, J = 7.5 Hz, 1 H, CHAr),
(JC,P = 5.62 Hz, CH3), 28.47 (CH2), 29.71 (CH2), 40.74 (JC,P
=
9.4 Hz, CH2), 57.84 (d, JC,P = 164.7 Hz, C), 61.26 (CH2), 63.33 (d,
JC,P = 7.6 Hz, CH2), 63.76 (d, JC,P = 7.4 Hz, CH2), 169.61 (JC,P
=
11 Hz, CO2Et), 177.35 (JC,P = 3.7 Hz, NHCO) ppm. 31P NMR
(CDCl3, 121 MHz): δ = 24.60 ppm. MS (ESI): m/z = 308.18 [M +
H]+, 330.20 [M + Na]+, 636.90 [2 M + Na]+, 170.16 [M –
7.79 (s, 1 H, CHAr), 8.49 (d, JH,P = 4.2 Hz, 1 H, N=CH) ppm. 13
NMR (CDCl3, 125 MHz): δ = 14.37 (CH3), 16.68 (d, JC,P = 7.2 Hz,
CH3), 29.04 (d, JC,P = 6.5 Hz, CH2), 29.42 (CH2), 64.82 (d, JC,P
147.4 Hz, 1 C), 60.71 (CH2), 63.12 (JC,P = 7.4 Hz, CH2), 127.15
C
P(O)(OEt)2]+. HRMS (ESI): calcd. for C12H22NO6P
330.1082; found 330.1087.
+ Na
=
Mixture of 2-(5-Oxo-2-phosphonopyrrolidin-2-yl)acetic Acid (14) (CHAr), 127.85 (CHAr), 130.04 (CHAr), 131.25 (CHAr), 135.01
and 3-Amino-3-phosphonohexanedioic Acid Hydrochloride (15): Pyr-
rolidone 13 (100 mg) was treated with 6  HCl (10 mL) at reflux
for 20 h. After extraction with CH2Cl2, the aqueous phase was con-
centrated under vacuum, and the residue was dried under high vac-
uum to furnish a white solid (74 mg, quantitative yield), which was
a mixture in equilibrium of cyclic 14 and the noncyclic product 15.
(CAr), 138.15 (d, JC,P = 4.8 Hz, CAr), 161.04 (JC,P = 12.5 Hz,
N=CH), 173.50 (CO2Et) ppm. 31P NMR (CDCl3, 121 MHz): δ =
25.62 ppm. MS (ESI): m/z = 490.03 [M + H]+, 512.08 [M + Na]+,
352.09 [M – P(O)(OEt)2]+. HRMS (ESI): calcd. for C22H33ClNO7P
+ Na 512.1581; found 512.1595.
5-(Diethoxyphosphoryl)-5-[2-(ethoxycarbonyl)ethyl]pyrrolidin-2-one
(18): Imine 17 (206 mg, 0.42 mmol) was dissolved in acetonitrile
(0.5 mL), and 1  HCl (1 mL, 1 mmol, 2.5 equiv.) was added. After
30 min at room temperature, the solution was concentrated under
vacuum, and the aqueous phase was extracted three times with
diethyl ether. The aqueous phase was then neutralised with
NaHCO3 and basified with 1  NaOH to pH 10. This was ex-
tracted five times with CH2Cl2. The organic layers were collected
and washed with brine. After drying (MgSO4) and concentration
under vacuum, the oily residue was purified by column chromatog-
raphy on silica gel (elution: CH2Cl2/EtOAc, 5:5). Pyrrolidinone 18
was isolated as a pale-yellow oil (70 mg, 53% yield). RF(CH2Cl2/
14: IR: ν = 3333 (NH), 2923 (CO H, PO H, C–OH, P–OH), 1647
˜
2
3
(CO2H, NHCO), 1207 (PO3H) cm–1. 1H NMR (D2O, 500 MHz): δ
= 2.25–2.28 (m, 1 H, CH2), 2.45–2.47 (m, 3 H, CH2), 2.74 (dd, J
= 15.5, 7.5 Hz, 1 H, CH2), 2.84 (dd, J = 15.5, 8.1 Hz, 1 H, CH2)
ppm. 13C NMR (D2O, 125 MHz): δ = 26.91 (CH2), 30.51 (CH2),
39.74 (JC,P = 8.5 Hz, CH2), 59.28 (d, JC,P = 156.5 Hz, 1 C), 174.53
(JC,P = 14.8 Hz, CO2H), 181.68 (JC,P = 4.9 Hz, NHCO) ppm. 31P
NMR (CDCl3, 121 MHz): δ = 20.81 ppm. MS (ESI): m/z = 224.04
[M + H]+, 447.00 [2 M + H]+. HRMS (ESI): calcd. for C6H10NO6P
1
+ H 224.0324; found 224.0316. 15: H NMR (D2O, 500 MHz): δ
= 2.12 (m, 2 H, CH2), 2.53 (m, 1 H, CH2), 2.58 (m, 1 H, CH2),
2.76 (m, 1 H, CH2), 2.87 (dd, J = 17.0, 7.3 Hz, 1 H, CH2) ppm.
13C NMR (D2O, 125 MHz): δ = 28.53 (CH2), 29.10 (JC,P = 2.8 Hz,
CH2), 35.93 (CH2), 55.29 (d, JC,P = 143.6 Hz, 1 C), 173.80 (d, JC,P
= 13.5 Hz, CO2H), 176.91 (CO2H) ppm. MS (ESI): m/z = 242.05
[M + H]+. HRMS (ESI): calcd. for C6H12NO7P + H 242.0430;
found 242.0441.
EtOAc, 5:5) = 0.18. IR: ν = 3321 (NH), 2982–2847 (CH ), 1704
˜
2
(NHCO, CO2Et), 1242 [P(O)(OEt)2], 1022 [P(O)(OEt)2] cm–1. 1H
NMR (CDCl3, 300 MHz): δ = 1.25 (t, J = 7.1 Hz, 3 H, CH3), 1.34
(td, J = 7.1, JH,P = 2.3 Hz, 6 H, CH3), 1.97 (m, 2 H, CH2), 2.36
(m, 6 H, CH2), 4.16 (m, 6 H, CH2), 6.24 (br. s, 1 H, NH) ppm. 13
C
NMR (CDCl3, 125 MHz): δ = 14.35 (CH3), 16.73 (t, JC,P = 5.6 Hz,
CH3), 27.32 (CH2), 28.54 (d, JC,P = 7.5 Hz, CH2), 30.02 (CH2),
30.74 (d, JC,P = 9.2 Hz, CH2), 59.27 (d, JC,P = 162.2 Hz, 1 C), 61.01
(CH2), 63.08 (d, JC,P = 7.8 Hz, CH2), 63.68 (d, JC,P = 7.4 Hz, CH2),
172.97 (CO2Et), 177.97 (d, JC,P = 7.4 Hz, NHCO) ppm. 31P NMR
[2-(Ethoxycarbonylmethyl)-5-oxopyrrolidin-2-yl]phosphonic
Acid
(16): BrSiMe3 (77 µL, 0.58 mmol, 4 equiv.) was added dropwise to
the pyrrolidone 13 (45 mg, 0.15 mmol) dissolved in dichlorometh-
ane (3 mL). The mixture was stirred at room temperature for 24 h.
94
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Eur. J. Org. Chem. 2009, 85–97

Inhibitors of Penicillin-Binding Proteins
(CDCl3, 121 MHz): δ = 26.20 ppm. MS (ESI): m/z = 322.06 [M +
washed with brine and dried with MgSO4. Crude 22 was recovered
H]+, 344.18 [M + Na]+, 184.05 [M – P(O)(OEt)2]+. HRMS (ESI): as a yellow oil after concentration under vacuum (73 mg, 91%).
calcd. for C13H24NO6P + Na 344.1239; found 344.1236.
R (EtOAc/iPrOH, 9:1) = 0.48. IR: ν = 2981 (CHAr), 1740
˜
F
(CO2Me), 1639 (C=N), 1250 [P(O)(OEt)2], 1022 [P(O)(OEt)2] cm–1.
1H NMR (CDCl3, 500 MHz): δ = 1.29 (t, J = 7.1 Hz, 6 H, CH3),
1.30 (t, J = 7.1 Hz, 6 H, CH3), 3.31 (dd, J = 14.4, J = 11.1 Hz, 2
H, CH2), 3.61 (s, 3 H, CH3), 4.18 (m, 8 H, CH2), 7.33 (dd, J = 7.5,
J = 8 Hz, 1 H, CHAr), 7.39 (d, J = 8 Hz, 1 H, CHAr), 7.64 (d, J
= 7.5 Hz, 1 H, CHAr), 7.79 (s, 1 H, CHAr), 8.74 (t, JH,P = 3.7 Hz,
1 H, N=CH) ppm. 13C NMR (CDCl3, 125 MHz): δ = 16.55 (d,
3-(5-Oxo-2-phosphonopyrrolidin-2-yl)propanoic Acid (19): Pyrrol-
idone 18 (70 mg) was treated with 6  HCl (10 mL) at reflux for
48 h. After extraction with CH2Cl2, the aqueous phase was freeze-
dried to furnish 19 as a white hygroscopic solid (56 mg, quantitative
yield). IR: ν = 3198 (NH), 3313 (CO H, C–OH), 2955 (CH ), 2851
˜
2
2
(PO3H, P–OH), 1732 (CO2H), 1701 (NHCO), 1207 (PO3H2) cm–1.
1H NMR (D2O, 300 MHz): δ = 1.98 (m, 4 H, CH2), 2.35 (m, 4 H,
CH2) ppm. 13C NMR (D2O, 75 MHz): δ = 25.89 (CH2), 28.23 (JC,P
= 9 Hz, CH2), 29.68 (JC,P = 7.7 Hz, CH2), 30.42 (CH2), 60.35 (d,
JC,P = 156 Hz, C), 177.77 (CO2H), 181.44 (NHCO) ppm. 31P NMR
(CDCl3, 121 MHz): δ = 22.46 ppm. MS (ESI): m/z = 236.16 [M +
H]+, 218.01 [M – H2O]+. HRMS (ESI): calcd. for C7H12NO6P +
Na 238.0481; found 238.0483.
JC,P = 6 Hz, CH3), 35.66 (CH2), 51.95 (CH3), 64.02 (d, JC,P
7.5 Hz, CH2), 64.14 (d, JC,P = 6.9 Hz, CH2), 68.98 (t, JC,P
=
=
144 Hz, 1 C), 127.11 (CHAr), 128.20 (CHAr), 130.05 (CHAr),
131.37 (CHAr), 134.95 (CAr), 137.90 (t, JC,P = 3 Hz, CAr), 164.35
(t, JC,P = 13.3 Hz, N=CH), 169.65 (t, JC,P = 11.6 Hz, CO2Me) ppm.
31P NMR (CDCl3, 121 MHz): δ = 17.38 ppm. MS (ESI): m/z =
498.12 [M + H]+, 520.16 [M + Na]+, 1016.68 [2 M + Na]+. HRMS
(ESI): calcd. for C19H30ClNO8P2 + H 498.1213; found 498.1221.
2-[2-(Ethoxycarbonyl)ethyl]-5-oxopyrrolidin-2-ylphosphonic Acid
(20): BrSiMe3 (78 µL, 0.59 mmol, 4 equiv.) was added dropwise to
the pyrrolidinone 18 (48 mg, 0.15 mmol) dissolved in dichlorometh-
ane (2 mL). The mixture was stirred at room temperature for 24 h.
After concentration under vacuum, the residue was taken up in
Methyl 3-Amino-3,3-bis(diethoxyphosphoryl)propanoate (23): Imine
22 (152 mg, 0.31 mmol) was dissolved in acetonitrile (0.5 mL), and
1  HCl (1 mL, 1 mmol, 2.5 equiv.) was added. After 30 min at
room temperature, the solution was concentrated under vacuum,
and the aqueous phase was extracted three times with diethyl ether.
The aqueous phase was then neutralised with NaHCO3 and basi-
fied with 1  NaOH to pH 10. This was extracted five times with
CH2Cl2. The organic layers were collected and washed with brine.
After drying (MgSO4) and concentration under vacuum, the oily
residue was purified by column chromatography on silica gel (elu-
tion: EtOAc/iPrOH, 9:1). Amine 23 was isolated as a colourless oil
water and freeze-dried to obtain 20 as a white solid (38 mg, 75%
1
yield determined by H NMR). IR: ν = 3282 (NH), 2935 (PO H,
˜
3
P–OH), 2981 (CH2), 1716 (CO2Et), 1666 (NHCO), 1196 (PO3H2)
cm–1. 1H NMR (D2O, 500 MHz): δ = 1.09 (t, J = 7.1 Hz, 3 H,
CH3), 1.98 (m, 3 H, CH2), 2.33 (m, 5 H, CH2), 4.00 (q, J = 7.1 Hz,
2 H, CH2) ppm. 13C NMR (D2O, 125 MHz): δ = 13.62 (CH3), 26.08
(CH2), 28.59 (JC,P = 8.9 Hz, CH2), 29.92 (JC,P = 8.4 Hz, CH2), 30.50
(CH2), 60.67 (d, JC,P = 158 Hz, 1 C), 62.37 (CH2), 176.23 (CO2Et),
181.52 (NHCO) ppm. 31 P NMR (CDCl3, 121 MHz): δ =
24.63 ppm. MS (ESI): m/z = 288.10 [M + Na]+, 530.99 [2 M +
H]+, 552.96 [2 M + Na]+, 184.04 [M – PO3H2]+. HRMS (ESI):
calcd. for C9H16NO6P + Na 288.0613; found 287.9703.
(87 mg, 76%). R (EtOAc/iPrOH, 9:1) = 0.21. IR: ν = 3464 (NH ),
˜
F
2
1H
1736 (CO2Me), 1242 [P(O)(OEt)2], 1022 [P(O)(OEt)2] cm–1
.
NMR (CDCl3, 300 MHz): δ = 1.33 (t, J = 7.1 Hz, 12 H, CH3),
2.30 (br. s, 2 H, NH2), 2.86 (d, J = 14.1 Hz, 1 H, CH2), 2.91 (d, J
= 14.1 Hz, 1 H, CH2), 3.69 (s, 3 H, CH3), 4.18 (m, 8 H, CH2) ppm.
13C NMR (CDCl3, 75 MHz): δ = 16.58 (d, JC,P = 2.9 Hz, CH3),
16.66 (d, JC,P = 3 Hz, CH3), 36.85 (t, JC,P = 2.4 Hz, CH2), 52.07
(CH3), 55.81 (t, JC,P = 145.5 Hz, 1 C), 63.68 (t, JC,P = 3.6 Hz,
CH2), 64.09 (t, JC,P = 3.4 Hz, CH2), 170.81 (t, JC,P = 12.9 Hz,
CO2Me) ppm. 31P NMR (CDCl3, 121 MHz): δ = 21.61 ppm. MS
(ESI): m/z = 376.00 [M + H]+, 398.16 [M + Na]+, 750.79 [2 M +
H]+, 772.74 [2 M + Na]+, 238.11 [M – P(O)(OEt)2]+. HRMS (ESI):
calcd. for C12H27NO8P2 + Na 398.1110; found 398.1126.
Tetraethyl {[(3-Chlorobenzylidene)amino]methylene}diphosphonate
(21): Tetraethyl (aminomethylene)diphosphonate (546 mg,
1.8 mmol), MgSO4 (1 g) and 3-chlorobenzaldehyde (183 µL,
1.62 mmol, 0.9 equiv.) were stirred in dichloromethane (7 mL) for
16 h. After filtration, the organic solution was concentrated under
vacuum. The oily residue was purified by chromatography on silica
gel (elution: EtOAc/CH2Cl2, 7:3) to give 21 as a yellow oil (566 mg,
70% yield). R (EtOAc/CH Cl , 7:3) = 0.34. IR: ν = 2981 (CHAr),
˜
F
2
2
1631 (C=N), 1253 [P(O)(OEt)2], 1026 [P(O)(OEt)2] cm–1. 1H NMR
(CDCl3, 500 MHz): δ = 1.33 (t, J = 7.1 Hz, 6 H, CH3), 1.35 (t, J
= 7.1 Hz, 6 H, CH3), 4.25 (m, 8 H, CH2), 4.38 (t, JH,P = 18.3 Hz,
1 H, CH), 7.37 (dd, J = 8.3, J = 7.4 Hz, 1 H, CHAr), 7.44 (d, J =
8.3 Hz, 1 H, CHAr), 7.63 (d, J = 7.4 Hz, 1 H, CHAr), 7.81 (s, 1
H, CHAr), 8.29 (t, JH,P = 4.3 Hz, 1 H, N=CH) ppm. 13C NMR
3-Amino-3,3-diphosphonopropanoic Acid Hydrochloride (24): Amine
23 (54 mg) was treated with 6  HCl (10 mL) at reflux overnight.
After extraction with CH2Cl2, the aqueous phase was freeze-dried
to furnish 24 as a white solid (39 mg, quantitative yield). IR: ν
˜
= 3055 (CO2H, C–OH), 2762 (PO3H, P–OH), 2360 (NH3+), 1716
(CO2H), 1508 (NH3+), 1141(PO3H) cm–1 1H NMR (D2O,
.
(CDCl3, 75 MHz): δ = 16.30 (CH3), 16.35 (CH3), 63.37 (d, JC,P
7.2 Hz, CH2), 63.50 (d, JC,P = 7.1 Hz, CH2), 67.97 (t, JC,P
=
=
500 MHz): δ = 3.00 (t, JH,P = 11.5 Hz, 2 H, CH2) ppm. 13C NMR
(D2O, 125 MHz): δ = 35.14 (CH2), 55.19 (t, JC,P = 127 Hz, 1 C),
174.50 (JC,P = 10.3 Hz, CO2H) ppm. 31P NMR (D2O, 121 MHz):
δ = 11.59 ppm. MS (ESI): m/z = 248.05 [M – H]–. HRMS (ESI):
calcd. for C3H9NO8P2 + H 249.9882; found 249.9889.
149 Hz, CH), 126.98 (CHAr), 127.92 (CHAr), 129.90 (CHAr),
131.39 (CHAr), 134.75 (CAr), 137.10 (JC,P = 2.8 Hz, CAr), 166.00
(JC,P = 15.3 Hz, N=CH) ppm. 31P NMR (CDCl3, 121 MHz): δ =
15.90–15.92 ppm. MS (ESI): m/z = 426.13 [M + H]+. HRMS (ESI):
calcd. for C16H26ClNO6P2 + Na 448.0822; found 448.0809.
Ethyl 4-[(3-Chlorobenzylidene)amino]-4,4-bis(diethoxyphosphoryl)-
Methyl 3-[(Chlorobenzylidene)amino]-3,3-bis(diethoxyphosphoryl)- butanoate (25): A solution of sodium ethoxide in ethanol (c =
propanoate (22): Potassium carbonate (44 mg, 0.33 mmol, 2.68 mol/L, 240 µL, 0.64 mmol, 0.25 equiv.) was added to a stirred
2.0 equiv.), tetrabutylammonium bromide (5 mg, 0.016 mmol,
0.1 equiv.) and methyl bromoacetate (16 µL, 0.17 mmol,
1.05 equiv.) were added successively to imine 21 (70 mg, 0.16 mmol)
dissolved in acetonitrile (1 mL). The heterogeneous mixture was
heated at 50 °C for 18 h. The mixture was filtered and concentrated
under vacuum. The residue was taken up in dichloromethane,
solution of imine 21 (1 g, 2.5 mmol) in ethanol (8 mL), and the
mixture was stirred for 15 min. Then ethyl acrylate (555 µL,
5.12 mmol, 2 equiv.) was added, and the mixture was heated at re-
flux for 2 h. After concentration under vacuum, the residue was
taken up in ethyl acetate, washed with a saturated solution of
NH4Cl and dried with MgSO4. After concentration under vacuum,
Eur. J. Org. Chem. 2009, 85–97
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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95

J. Marchand-Brynaert et al.
FULL PAPER
crude imine 25 was recovered as a brown oil (1.33 g, 98%).
ual activity was determined by comparison with the variation of
R (EtOAc) = 0.35. IR: ν = 2981 (CHAr), 1740 (CO Me), 1639 the absorbance of the reference (sample without inhibitor) and is
˜
F
2
(C=N), 1250 [P(O)(OEt)2], 1022 [P(O)(OEt)2] cm–1. 1H NMR
(CDCl3, 500 MHz): δ = 1.25 (t, J = 7 Hz, 3 H, CH3), 1.29 (t, J = tial activities; variations of results are within an error of Ϯ3%. A
7 Hz, 6 H, CH3), 1.32 (t, J = 7 Hz, 6 H, CH3), 2.68 (m, 4 H, CH2), plot of V/VI (ratio of hydrolysis in the absence and in the presence
shown in Table 4. Results are expressed as a percentage of the ini-
4.14 (q, J = 7 Hz, 2 H, CH2), 4.26 (m, 8 H, CH2), 7.36 (t, J = of inhibitors) versus inhibitor concentration gave the inhibition
7.5 Hz, 1 H, CHAr), 7.41 (d, J = 7.5 Hz, 1 H, CHAr), 7.62 (d, J constant indicated in the text. All experiments were performed
= 7.5 Hz, 1 H, CHAr), 7.78 (s, 1 H, CHAr), 8.76 (t, JH,P = 3 Hz, three times.
1 H, N=CH) ppm. 13C NMR (CDCl3, 125 MHz): δ = 14.41 (CH3),
Assay with R39: The tested enzyme was produced and purified at
16.66 (CH3), 29.68 (t, JC,P = 6.2 Hz, CH2), 32.22 (CH2), 60.68
the University of Liège (Centre d’Ingénierie des Proteins, Prof. B.
(CH2), 63.34 (JC,P = 7.2 Hz, CH2), 63.49 (JC,P = 7 Hz, CH2), 68.02
Joris). Inhibition studies were performed at 30 °C in sodium phos-
(d, JC,P = 136.8 Hz, 1 C), 127.17 (CHAr), 127.98 (CHAr), 130.10
phate buffer (50 m, pH 7) containing 5% DMF to ensure product
(CHAr), 131.46 (CHAr), 135.05 (CAr), 138.12 (CAr), 164.63 (JC,P
solubility. Compounds were tested at a concentration of 500 µ;
= 12 Hz, N=CH), 173.53 (CO2Et) ppm. 31P NMR (CDCl3,
experiments were performed in triplicate. Inhibitor (500 µ) and
121 MHz): δ = 19.25 ppm. MS (ESI): m/z = 525.94 [M + H]+,
protein (0.8 µ) were mixed and incubated for 16 h. The residual
548.06 [M + Na]+, 1072.78 [2 M + Na]+, 388. [M – P(O)(OEt)2]+.
free protein was counterlabelled with fluorescein-labelled ampicillin
HRMS (ESI): calcd. C21H34ClNO8P2 + Na 548.1346, found
(25 µ) for 45 min. Then the reaction was stopped by the addition
548.1342.
of SDS-PAGE loading buffer and the mixture was heated at 100 °C
2,2-Bis(diethoxyphosphoryl)pyrrolidin-5-one (26): Imine 25 (758 mg,
1.6 mmol) was dissolved in acetonitrile (1.5 mL), and 1  HCl
(4.8 mL, 4.8 mmol, 1.5 equiv.) was added. After 30 min at room
temperature, the solution was concentrated under vacuum, and the
aqueous phase was extracted three times with diethyl ether. The
aqueous phase was then neutralised with NaHCO3 and basified
with 1  NaOH to pH 10. This was extracted five times with
CH2Cl2. The organic layers were collected and washed with brine.
After drying (MgSO4) and concentration under vacuum, pyrrol-
idinone 26 was recovered as a white solid (225 mg, 50% yield). IR:
for 4 min. The reaction mixture was then submitted to SDS-PAGE.
The fluorescent complexes (formed by reaction of residual PBPs
and ampicillin) were visualised by using a Molecular Imager FX
and quantified with the Quantity One Software of BIORAD.[59]
The background fluorescence was subtracted in each assay.
Supporting Information (see footnote on the first page of this arti-
cle): 13C NMR spectra of the described compounds (Figures S1–
S23).
ν = 3321 (NH), 2981–2847 (CH ), 1701 (NHCO), 1246 [P(O)-
˜
2
Acknowledgments
(OEt)2], 1161 [P(O)(OEt)2], 1018 [P(O)(OEt)2] cm–1. 1H NMR
(CDCl3, 300 MHz): δ = 1.36 (t, J = 7.1 Hz, 12 H, CH3), 2.55 (m,
4 H, CH2), 4.24 (m, 8 H, CH2), 5.84 (br. s, 1 H, NHCO) ppm. 13C
NMR (CDCl3, 125 MHz): δ = 16.55 (CH3), 24.76 (CH2), 29.20
(CH2), 58.76 (t, JC,P = 152.5 Hz, 1 C), 177.70 (NHCO) ppm. 31P
NMR (CDCl3, 121 MHz): δ = 19.82–19.94 ppm. MS (ESI): m/z =
358 [M + H]+, 380.14 [M + Na]+. HRMS (ESI): calcd. for
C12H25NO7P2 + Na 380.1004; found 380.0996.
The biochemical evaluations against β-lactamases and R39 were
performed in the laboratories of Professors M. Galleni and A.
Luxen, respectively (University of Liège, Belgium). This work was
supported by the Belgian Program on Interuniversity Poles of At-
traction (PAI 5/33 and PAI 6/19), the Fonds de la Recherche Sci-
entifique (FRS-FNRS) and the Université Catholique de Louvain.
J. M.-B. is senior research associate at the FRS-FNRS (Belgium).
5-Oxopyrrolidine-2,2-diyldiphosphonic Acid (27): Pyrrolidinone 26
(91 mg) was treated with 6  HCl (10 mL) at reflux for 48 h. After
extraction with CH2Cl2, the aqueous phase was concentrated under
vacuum, and the residue was dried under high vacuum to furnish
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Received: August 22, 2008
Published Online: November 21, 2008
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97